Mating behaviour and competitiveness of 
 male Glossina brevipalpis and Glossina austeni 
in relation to biological and operational attributes for use in the 
Sterile Insect Technique 
 
 
Chantel Janet de Beer 
 
 
Submitted in fulfilment of the requirements in respect of the Doctoral degree qualification 
in Entomology in the Department of Zoology and Entomology in the  
Faculty of Natural and Agricultural Sciences at the  
University of the Free State 
 
 
2016 
 
 
 
 
Promoter: Dr. G.J. Venter 
Co-Promoters: Dr. M.J.B. Vreysen & Dr. S.L. Brink 
 
  
 
 
DECLARATION 
 
I, Chantel Janet de Beer, declare that the Doctoral Degree research thesis that I herewith 
submit for the Doctoral Degree qualification in Entomology at the University of the Free 
State is my independent work, and that I have not previously submitted it for a qualification 
at another institution of higher education. 
 
 
I, Chantel Janet de Beer, hereby declare that I am aware that the copyright is vested in the 
University of the Free State. 
 
 
I, Chantel Janet de Beer, hereby declare that all royalties as regards intellectual property 
that was developed during the course of and/or in connection with the study at the University 
of the Free State, will accrue to the University. 
 
 
 
 
 
______________________________ 
Chantel Janet de Beer 
  
ii 
 
 
PREFACE 
 
Manuscript format 
This thesis is presented in the format required by the Medical and Veterinary Entomology 
Journal. 
http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-2915/homepage/ForAuthors.html 
 
 
Ethical considerations 
Materials used in the study posed no health risk to researchers and no vertebrate animals 
were harmed. Permission to do research in terms of Section 20 of the animal diseases act 
of, 1984 (ACT no. 35 of 1984) has been granted for tsetse fly collection and colony 
maintenance, Ref 12/11/1/1/9 and 12/11/1/1. The study was done as part of a project on 
National Assets (000773) at the Agricultural Research Council-Onderstepoort Veterinary 
Institute (ARC-OVI) in collaboration with the Joint FAO/IAEA Division of Nuclear 
Techniques in Food and Agriculture under the coordinated research project (CRP) 
12618/R0/RBF and research project 17753/R0 as well as the Department of Technical 
Cooperation of the IAEA under project RAF 5069. 
 
  
iii 
 
 
ACKNOWLEDGEMENTS 
 
I would like to thank the Agricultural Research Council-Onderstepoort Veterinary Institute 
(ARC-OVI), the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture in 
Austria and the Department of Science and Technology in South Africa for funding these 
projects and giving me the opportunity to use this project towards my studies. 
I am grateful to Marc Vreysen for his assistance, constant support and ongoing 
collaboration. Without his personal interest, unquenchable enthusiasm and sharing his 
tsetse fly expertise, this work would be difficult to accomplish. I want to thank Gert Venter 
for his contribution to my scientific development and continuous support and for anchoring 
the cloud castle. 
I also thank Gratian Mutika, Geoffrey Gimonneau, Jérémy Bouyer, James Patterson, 
Adly Abd Alla, Abdalla Latif, Luis Neves, Karin Kappmeier Green, Johan Esterhuizen and 
Danie de Klerk for advice and sharing their vast knowledge on various aspects of the tsetse 
field. A special word of thanks to Marin Carmen and Andrew Parker for their patience in 
learning the more intrigue aspects of colony maintenance during my visits to the colonies 
in Seibersdorf. 
A special thanks to Agnes Baloyi and Percy Moyaba for their hard work in day to day 
running of the tsetse fly colonies at the ARC-OVI. I also wish to thank Elize de Jager and 
Johannes Mojela who previously worked in the colony. Without a smooth running and well 
maintained colony this study would not have been possible. A special word of thanks to 
Jerome Ntshangase and Petros Gazu for the maintenance of the field station at Kuleni, 
KwaZulu-Natal and field assistance. I also thank Solly Boikanyo and Dahpney Majatladi for 
sharing the field cages with me. 
I also want to acknowledge the support of the State Veterinarians Jenny Price and 
Lundi Ntantiso from the Department of Veterinary Services KwaZulu-Natal. 
The scientific input of Sonja Brink is greatly appreciated. I want to thank Truuske 
Gerdes for editing and criticism on the final draft of this work. 
I would also like to thank my family and friends, that supported me through this difficult 
process. Finally, special thanks is given to Henry and Minke van der Westhuizen for always 
pushing me to better myself, even if this had tested their unlimited patience. 
I leave u with this very important question to consider while reading this work, as it 
was first pondered by Lewis Carrol’s Mad Hatter: Why is a Raven like a Writing desk? 
  
iv 
 
ABSTRACT 
 
In South Africa, African Animal Trypanosomosis (AAT), caused by Trypanosomae parasites 
transmitted by Glossina brevipalpis and Glossina austeni (Diptera: Glossinidae), is 
restricted to the north east of KwaZulu-Natal Province with an estimated 250 000 cattle 
being at risk. For the control of these flies an area-wide integrated pest management (AW-
IPM) strategy with a sterile insect technique (SIT) component has been proposed. 
Accurate knowledge of the distribution of target populations is fundamental to the 
success of any control programme. In the present study tsetse fly distribution was 
determined with odour baited H traps and cattle screened using the buffy coat analyses to 
produce updated tsetse fly distribution, abundance and trypanosome prevalence maps for 
north eastern KwaZulu-Natal. Glossina brevipalpis and G. austeni were collected in areas 
where they had previously not been captured. Vegetation and temperature was shown to 
influence their distribution and abundance. The fact that no significant correlation between 
tsetse fly abundance and nagana prevalence could be established underlines the complex 
interactions between these two entities. This was epitomised by the fact that despite large 
differences in the apparent densities of G. austeni and G. brevipalpis, overall trypanosome 
prevalence was similar in all districts in north eastern KwaZulu-Natal. This indicated that 
both species can play a role in transmission of AAT and need to be controlled. 
The G. brevipalpis and G. austeni populations of north eastern KwaZulu-Natal 
extends into southern Mozambique (both species) and Swaziland (G. austeni). 
Morphometrical analyses showed an absence of any significant barriers to gene flow 
between the various KwaZulu-Natal populations as well as between the South African 
populations and those of the two neighbouring countries. Tsetse fly control in a localised 
area will therefore be subjected to reinvasion from uncontrolled areas. An area-wide 
approach, i.e. against the entire tsetse fly population of South Africa, southern Mozambique 
and Swaziland will therefore be essential. 
The maintenance of colonised G. brevipalpis and G. austeni at the Agricultural 
Research Council-Onderstepoort Veterinary Institute (ARC-OVI), necessitate a high quality 
blood source. For the potential improvement of the current rearing diet various 
anticoagulants, phagostimulants and blood sources were evaluated and production 
assessed using standardised 30-day bioassays. Defibrinated bovine blood was found to be 
the most suitable. Anticoagulants such as sodium citrate, a combination of citrate and 
sodium acid, phosphate dextrose adenine and citric acid can be used to simplify blood 
collection. While G. brevipalpis preferred bovine to porcine blood, G. austeni preferred a 
mixture of equal parts bovine and porcine blood. The phagostimulants adenosine 
triphosphate, as well as tri-posphates of inosine, and the mono-posphates of guanosine 
v 
 
and cytosine improved production in both species. Attempts to colonise the local KwaZulu-
Natal strain of G. brevipalpis failed due to a reluctance of field flies to feed on the artificial 
feeding system. 
In preparation for the SIT component the irradiation sensitivity of colonised 
G. brevipalpis and G. austeni when treated as adults and late-stage pupa was determined. 
A dose of 40 Gy induced 97% sterility in G. brevipalpis males when irradiated as late-stage 
pupae and 80 Gy induced a 99% sterility in flies irradiated as adults. Higher doses were 
required for G. austeni, with 80 Gy and 100 Gy inducing higher than 97% sterility in females 
that mated with males treated as adults or late-stage pupae. 
As colonised and irradiated males must be able to compete with their wild 
counterparts the mating performance of the colonised G. brevipalpis and G. austeni was 
determined under near natural conditions in walk-in field cages. Although the mating latency 
for both species was shorter, their mating performance did not differ significantly between 
mornings and afternoons. For both species mating frequency was significantly higher in 
nine-day-old males compared to six- or three-day-old males. Age did not affect the males’ 
ability to transfer sperm, their mating duration or mating latency. There was no significant 
difference in mating performance of sterile and fertile males. 
This study indicated that AAT and tsetse flies are abundant in KwaZulu-Natal and 
tsetse fly presence seems to be a dynamic process that is influenced by a number of 
environmental factors. The earlier proposed AW-IPM strategy with a SIT component, 
although still applicable, will need to be adapted to incorporate the new distributions 
records. Initial results indicate that the colonies at the ARC-OVI will be suitable for 
programmes that have a SIT component. 
 
Keywords: 
Glossina brevipalpis, Glossina austeni, distribution, Trypanosomosis, morphometrics, 
colonies, radiation sensitivity, mating performance 
  
vi 
 
UITTREKSEL 
 
In Suid-Afrika is Afrika Trypanosomiase van diere (ATD), wat deur Trypanosomae parasiete 
veroorsaak word en deur Glossina brevipalpis en Glossina austeni (Diptera: Glossinidae), 
oorgedra word, beperk tot die noordoostelike KwaZulu-Natal Provinsie. Na beraming is 
sowat 250 000 beeste tans blootgestel aan die siekte. 'n Area-wye geïntegreerde 
plaagbestuur (AW-IPB) strategie met 'n steriele insek tegniek (SIT) komponent word vir die 
beheer van die vlieë voorgestel. 
Die sukses van ‘n beheerprogram sal afhang van akkurate inligting oor waar 
tsetsevlieё voorkom. In die huidige studie is H tipe valle saam met geurlokaas gebruik om 
tsetsevlieё te versamel. Trypanosomiase infeksiesyfers in beeste is ook gemonitor. Die 
inligting is gebruik om bestaande kaarte van tsetsevlieg verspreiding, volopheid en 
trypanosomiase voorkomsyfer in diere op te dateer. Glossina brevipalpis en G. austeni is 
versamel in gebiede waar hulle voorheen afwesig was. Daar is gevind dat plantegroei en 
temperatuur die verspreiding en volopheid van tsetsevlieё beïnvloed. Die feit dat geen 
betekenisvolle korrelasie tussen vlieg getalle en ATD voorkomsyfer bepaal kon word nie 
beklemtoon die komplekse wisselwerking tussen hierdie twee entiteite. Dit word 
beklemtoon deur die waarneming dat, ten spyte van groot verskille in die oënskynlike 
digthede van G. austeni en G. brevipalpis, die algehele trypanosomiase voorkomsyfers 
tussen die distrikte in die noordoostelike KwaZulu-Natal nie verskil het nie. Dit dui aan dat 
beide spesies 'n rol kan speel in die oordrag van die siekte en dus beheer sal moet word. 
Die verspreiding van die tsetsevliegbevolking wat in die noordooste van KwaZulu-
Natal voorkom, strek tot in Swaziland en die suide van Mosambiek. Morfometriese 
ontledings toon 'n afwesigheid van betekenisvolle grense aan en dat dat inteling tussen die 
verskillende KwaZulu-Natal bevolkings asook tussen die Suid-Afrikaanse bevolking en dié 
van die twee buurlande voorkom. Tsetsevlieg beheer in 'n gelokaliseerde afgebakende 
area sal dus onderworpe wees aan herbesmetting vanaf die onbeheerde aangrensende 
gebiede. 'n Area-wye benadering, dit wil sê teen die hele tsetsevliegbevolking van Suid-
Afrika, Swaziland en die suide van Mosambiek sal dus noodsaaklik wees. 
Die instandhouding van die kolonies van G. brevipalpis en G. austeni by die 
Landbounavorsingsraad-Onderstepoort Veeartsenykunde-Instituuut (LNR-OVI) vereis 'n 
bloedvooraad van hoë gehalte. Vir die moontlike opgradering van die huidige dieet, is ‘n 
aantal antistolmiddels, voedingstimulante en bloedbronne geëvalueer deur produksie met 
‘n gestandardiseerde 30-dag biologiese keuringsproses te bepaal. Gedefibriniseerde 
beesbloed was die mees geskikste. Antistolmiddels soos natriumsitraat, 'n kombinasie van 
sitraat en natriumsuur, fosfaat-dekstrose-adenien en sitroensuur kan gebruik word om 
vii 
 
bloed versameling te vergemaklik. Terwyl G. brevipalpis bees- bo varkbloed verkies, 
verkies G. austeni 'n mengsel van gelyke dele bees- en varkbloed. Die voedingstimulante 
adenosientrifosfaat, asook tri-fosfate van inosien, en die mono-fosfaat van guanosien en 
sitosien het verbeterde produksie in beide spesies tot gevolg gehad. Pogings om die 
plaaslike KwaZulu-Natal G. brevipalpis te koloniseer was onsuksesvol as gevolg van 'n 
onwilligheid van veldvlieё om op die kunsmatige voedingstelsel te voer. 
As ‘n voorvereiste vir SIT was die bestraling-sensitiwiteit van G. brevipalpis en 
G. austeni volwassenes en laat-stadium papies bepaal. 'n Dosis van 40 Gy het ‘n 97% 
steriliteit in G. brevipalpis tot gevolg gehad wanneer laat-stadium papies bestraal is, en 
80 Gy 'n 99% steriliteit as volwasse vlieë bestraal is. Glossina austeni het hoёr dosisse 
vereis, 80 Gy en 100 Gy veroorsaak ‘n hoёr as 97% steriliteit in wyfies wat met mannetjies 
wat as volwassenes of laat-stadium papies bestraal is, gepaar het. 
Gekoloniseerdes bestraalde mannetjies moet in staat wees om met met hul veld 
eweknieë te kan meeding. Die parings gedrag van gekoloniseerde G. brevipalpis en 
G. austeni was onder bykans natuurlike veldtoestande in instap-veldhokke bepaal. 
Alhoewel die tydsverloop voor paring het vir beide spesies korter was, was daar nie 
betekenisvolle verskille in hulle paring-prestasie soos in die oggend of middag bepaal nie. 
Vir beide spesies was die paring-frekwensie vir 9-dae-oue mannetjies aansienlik hoër as 
dié van 6 of 3-dae-oue mannetjies. Ouderdom het geen invloed op die vermoë van die 
mannetjies om sperm oor te dra nie of tydverloop voor paring gehad nie. Daar was geen 
beduidende verskil in paring-prestasie van steriele en vrugbare mannetjies nie. 
Die huidige studie dui aan dat ATD en tsetsevlieë algemeen in KwaZulu-Natal 
voorkom en dat tsetsevlieg teenwoordigheid 'n dinamiese proses is wat deur 
omgewingsfaktore beïnvloed word. Die voorgestelde AW-IPB met 'n SIT komponent, 
alhoewel steeds van toepassing, sal aangepas moet word om die ogedateerde data te 
inkorporeer. Voorlopige resultate dui aan dat die kolonies by die LNR-OVI geskik sal wees 
vir gebruik in SIT. 
 
Sleutelwoorde: 
Glossina brevipalpis, Glossina austeni, verspreiding, trypanosomiase, morfometrie, 
kolonies, bestraling sensitiwiteit, paring-mededingendheid 
  
viii 
 
TABLE OF CONTENTS 
 
DECLARATION ............................................................................................................... ii 
PREFACE ....................................................................................................................... iii 
ACKNOWLEDGEMENTS ............................................................................................... iv 
ABSTRACT ..................................................................................................................... v 
UITTREKSEL................................................................................................................. vii 
LIST OF FIGURES ........................................................................................................ xii 
LIST OF TABLES ......................................................................................................... xvi 
 
Chapter 1: Introduction 
1.1 Literature review .................................................................................................... 1 
1.1.1 The history of the tsetse fly and Trypanosomosis............................................ 1 
1.1.2 Economic impact of Trypanosomosis .............................................................. 1 
1.1.3 Tsetse fly systematics, distribution and biology ............................................... 3 
1.1.4 African Trypanosomosis management ............................................................ 4 
1.2 Study justification ................................................................................................. 11 
1.3 Aim ...................................................................................................................... 13 
1.4 Objectives ............................................................................................................ 13 
 
Chapter 2: Tsetse fly distribution and Animal Trypanosomosis prevalence 
2.1 Introduction .......................................................................................................... 15 
2.2 Materials and methods......................................................................................... 20 
2.2.1 Study area .................................................................................................... 20 
2.2.2 Tsetse fly survey ........................................................................................... 21 
2.2.3 Environmental factors affecting tsetse fly distribution .................................... 21 
2.2.4 Trypanosomosis survey ................................................................................ 23 
2.2.5 Statistical analysis......................................................................................... 23 
2.3 Results ................................................................................................................ 24 
2.3.1 Tsetse fly distribution and apparent density (AD) .......................................... 24 
2.3.2 Environmental factors affecting tsetse fly distribution .................................... 29 
2.3.3 Tsetse fly and Trypanosomosis association .................................................. 33 
2.4 Discussion ........................................................................................................... 36 
 
 
 
ix 
 
Chapter 3: Comparison of geometric morphometric markers between tsetse 
populations of South Africa, southern Mozambique and Swaziland 
3.1 Introduction .......................................................................................................... 40 
3.2 Materials and methods......................................................................................... 42 
3.2.1 Study sites and fly collection ......................................................................... 42 
3.2.2 Morphometric analysis .................................................................................. 44 
3.3 Results ................................................................................................................ 45 
3.3.1 Species differentiation ................................................................................... 45 
3.3.2 Seasonal effects and sexual dimorphism ...................................................... 46 
3.3.3 Population Isolation ....................................................................................... 53 
3.3.4 Field flies compared with colony flies ............................................................ 59 
3.4 Discussion ........................................................................................................... 61 
 
Chapter 4: Collection, processing and host source of the rearing diet for colonised 
tsetse flies 
4.1 Introduction .......................................................................................................... 64 
4.2 Materials and methods......................................................................................... 67 
4.2.1 Tsetse fly colonies ........................................................................................ 67 
4.2.2 Blood collection and processing .................................................................... 67 
4.2.3 Assessment of suitability of blood source as maintenance diet ..................... 68 
4.2.4 Phagostimulation .......................................................................................... 69 
4.2.5 Bioassay ....................................................................................................... 69 
4.2.6 The colonisation of tsetse flies ...................................................................... 70 
4.2.7 Statistical analysis......................................................................................... 71 
4.3 Results ................................................................................................................ 71 
4.3.1 Anticoagulants in the rearing diet .................................................................. 71 
4.3.2 Blood source for maintenance diet ................................................................ 75 
4.3.3 Evaluation of phagostimulants to improve production in the tsetse fly colonies 
 ..................................................................................................................... 79 
4.3.4 The colonisation of tsetse flies collected from north eastern KwaZulu-Natal ..... 
 ..................................................................................................................... 79 
4.4 Discussion ........................................................................................................... 82 
 
Chapter 5: Evaluation of radiation sensitivity of tsetse males 
5.1 Introduction .......................................................................................................... 85 
5.2 Materials and methods......................................................................................... 87 
5.2.1 Colony tsetse flies ......................................................................................... 87 
x 
 
5.2.2 Radiation evaluation procedures ................................................................... 87 
5.2.3 Statistical analysis......................................................................................... 88 
5.3 Results ................................................................................................................ 88 
5.3.1 Adult emergence rate .................................................................................... 88 
5.3.2 Reproduction in females mated with males irradiated as adults or pupae ..... 91 
5.3.3 Reproductive status of females inseminated by males irradiated as adult or 
pupa ............................................................................................................. 97 
5.3.4 Male survival ................................................................................................. 97 
5.4 Discussion ........................................................................................................... 99 
 
Chapter 6: Comparative assessment of the mating performance of tsetse males 
under field cage conditions 
6.1 Introduction ........................................................................................................ 102 
6.2 Materials and methods....................................................................................... 103 
6.2.1 Colony tsetse flies ....................................................................................... 103 
6.2.2 Walk-in field cage and environmental conditions ......................................... 103 
6.2.3 Time of peak mating activity ........................................................................ 105 
6.2.4 Optimal mating age ..................................................................................... 105 
6.2.5 Sterile versus fertile males .......................................................................... 105 
6.2.6 Mating performance indicators .................................................................... 106 
6.2.7 Statistical analysis....................................................................................... 106 
6.3 Results .............................................................................................................. 107 
6.3.1 Environmental conditions ............................................................................ 107 
6.3.2 Activity in field cage .................................................................................... 109 
6.3.3 Time of peak mating activity ........................................................................ 109 
6.3.4 Optimal mating age  .................................................................................... 111 
6.3.5 Sterile versus fertile males .......................................................................... 114 
6.4 Discussion ......................................................................................................... 117 
 
Chapter 7: Concluding remarks and recommendations 
 ................................................................................................................................... 121 
 
REFERENCES ........................................................................................................... 127 
APPENDIX ................................................................................................................. 151 
  
xi 
 
LIST OF FIGURES 
 
Chapter 1: Introduction 
1.1. Predicted distribution (in red) of Fusca (Austenina) (A), Palpalis (Nemorhina) (B) and 
Morsitans (Glossina) (C) species groups in Africa .................................................. 4 
1.2. Predicted distribution (in red) of Glossina brevipalpis (A) and Glossina austeni (B) in 
Africa ..................................................................................................................... 4 
1.3.  The efficiency of a conventional control approaches in combination with the sterile 
insect technique in relation to target population densities .................................... 10 
 
Chapter 2: Tsetse fly distribution and Animal Trypanosomosis prevalence 
2.1. The distribution of Glossina pallidipes, Glossina brevipalpis and Glossina austeni in 
KwaZulu-Natal prior to 1953 ................................................................................ 16 
2.2. Distribution of Glossina brevipalpis and Glossina austeni based on a survey carried 
out from 1993 to 1999 with odour baited XT sticky traps. Only positive trap catches 
are indicated ........................................................................................................ 19 
2.3. Location of the weather stations in relation to the tsetse H traps used for determining 
potential relationships between tsetse fly apparent density (AD) and climate variables 
in north eastern KwaZulu-Natal ............................................................................ 22 
2.4. Tsetse fly apparent density (AD: flies/trap/day) of Glossina brevipalpis (A) and 
Glossina austeni (B) from April 2005 to August 2009 ........................................... 27 
2.5. Updated distribution of Glossina brevipalpis and Glossina austeni as determined with 
odour baited XT sticky traps traps (1993 – 1999) and H traps (2005 – 2015) surveys. 
 ............................................................................................................................ 28 
2.6. Vegetation class (Bouyer & Guerrini, 2010) maps and satellite images of four tsetse 
fly collection sites in north eastern KwaZulu-Natal. A: Pelani, B: Mbazwana, C: False 
Bay Park, E: Hluhluwe-iMfolozi Park (Google earth, 2013a, b, c; 2014). The average 
AD (SD) for each species, expressed as flies/trap/day are indicated for each site ... 
 ............................................................................................................................ 31 
2.7. Inverse distance weighted interpolation of environmental data collected at weather 
stations in north eastern KwaZulu-Natal from July 2008 to August 2009 (A: Av. 
Maximum Temperature (°C), B: Av. Minimum Temperature (°C), C: Av. Maximum 
Relative Humidity (%), D: Av. Minimum Relative Humidity (%), E: Av. Rainfall (mm), 
F: Av. Relative Evapotranspiration (mm), G: Av. Radiation (MJ/m2) and H: Av. Hourly 
Wind Speed (m/s)) ............................................................................................... 32 
2.8. Inverse distance weighted interpolation of trypanosome infection rate at dip tanks in 
north eastern KwaZulu-Natal (1: Ekuhlehleni, 2: Khume, 3: Ndumo, 4: Manzibomvu, 
xii 
 
5: Nhlanjwana, 6: Thengani, 7: Ntabayengwe, 8: Ngwenyambili, 9: Phelendaba, 10: 
Pongola, 11: Makhathini, 12: Mseleni, 13: Mpini, 14: Mkhumbikazane, 15: Mbazwana, 
16: Zineshe, 17: Khipha, 18: Nibela, 19: Nthwati, 20: Ekuphindisweni, 21: Mvutshini, 
22: Qakweni, 23: Mahlambanyathi, 24: Boomerang, 25: Ocilwane) ..................... 34 
 
Chapter 3: Comparison of geometric morphometric markers between South Africa, 
southern Mozambique and Swaziland tsetse fly populations 
3.1. Sites where Glossina brevipalpis and Glossina austeni were collected with odour-
baited H traps for comparative geometric morphometrics in Swaziland (1: Mlawula 
Nature Reserve), Mozambique (2: Reserva Especial de Maputo) and South Africa (3: 
Ndumu; 4: Tembe, 5: Kosi Bay, 6: Mbazana, 7: Lower Mkhuze, 8: Phinda, 9: False 
Bay, 10: Hluhluwe-iMfolozi Park, 11: Boomerang, 12: St Lucia) ........................... 43 
3.2. Glossina austeni wing indicating the nine landmarks as defined by vein intersections
 ............................................................................................................................ 44 
3.3. Multivariate regression of the first partial warp (derived from the shape of the wing) 
on centroid size of the right wings of female Glossina brevipalpis (yellow triangles) 
and Glossina austeni (purple circles). The first partial warp represents 80% of the total 
discrimination ....................................................................................................... 46 
3.4. Centroid size variations of the wings of male and female Glossina brevipalpis (A) and 
Glossina austeni (B) according to season. Each box shows the group median 
separating the 25th and 75th quartiles, capped bars indicate maximum and minimum 
values, circles indicating the outliers. (Black: Ndumu G. brevipalpis males; Light 
purple: Ndumu G. brevipalpis females; Dark blue: St Lucia G. austeni (A) or 
G. brevipalpis (B) males; Dark pink: St Lucia G. brevipalpis (A) or G. austeni (B) 
females; Green: Phinda G. austeni males; Dark purple: Phinda G. austeni females). 
Boxes followed by a different letter indicate that the sizes were significantly different 
at the 5% level ..................................................................................................... 50 
3.5. The distribution of Glossina brevipalpis female (A, B) and male’s (C, D) wing shape 
in the morhospace defined by the first two Canonical variants, flies were collected 
from Ndumu (A, C) and St Lucia (B, D) ................................................................ 52 
3.6. The distribution of Glossina austeni female (A, B) and male’s (C, D) wing shape in 
the morhospace defined by the first two Canonical variants, flies were collected from 
Phinda (A, C) and St Lucia (B, D) ........................................................................ 52 
3.7. Centroid size variations of the right wings of female Glossina brevipalpis (A) and 
Glossina austeni (B) according to localities. Each box shows the group median 
separating the 25th and 75th quartiles, capped bars indicate maximum and minimum 
xiii 
 
values, circles indicating the outliers. Boxes followed by a different letter indicate that 
the sizes were significantly different at the 5% level ............................................. 55 
3.8. The distribution of Glossina brevipalpis (A) and Glossina austeni (B) female right wing 
shape in the morhospace defined by the first two conical variants ....................... 57 
3.9. Linear regression of the Mahalanobis distances of Glossina brevipalpis (A) and 
Glossina austeni (B) compared with geographic distances (km) between their 
collection sites (Solid red line represents the linear regression, broken red line the 
95% confidence interval) ...................................................................................... 58 
3.10. Variations in centroid size of the right wings of female Glossina brevipalpis (A) and 
Glossina austeni (B) according to localities, as well as colony reared flies. Each box 
shows the group median separating the 25th and 75th quartiles, capped bars indicate 
maximum and minimum values, circles indicating the outliers. Boxes followed by a 
different letter indicate that the sizes were significantly different at the 5% level .. 59 
3.11. The distribution of Glossina brevipalpis (A) and Glossina austeni (B) female right wing 
shape in the morhospace defined the first two Canonical variants, flies were collected 
from varies sites as well as colony reared ............................................................ 60 
 
Chapter 4: Collection, processing and host source of the rearing diet for colonised 
tsetse flies 
4.1. Quality factor (QF) values for the blood collected with anticoagulants as obtained in 
the bioassay for Glossina brevipalpis and Glossina austeni. Each box shows the 
group median separating the 25th and 75th quartiles, capped bars indicate maximum 
and minimum values. Boxes denoted by a different letter indicate that the QF values 
were significantly different for each species at the 5% level ................................. 73 
4.2. Quality factor (QF) values for different combinations of bovine/porcine blood diets 
obtained using the standard bioassay for Glossina brevipalpis and Glossina austeni. 
Each box shows the group median separating the 25th and 75th quartiles, capped bars 
indicate maximum and minimum values. Boxes denoted by a different letter indicate 
that the QF values were significantly different for each species at the 5% level. .. 77 
4.3. Quality factor (QF) values for blood mixed with different phagostimulants as obtained 
in the standard bioassay for Glossina brevipalpis and Glossina austeni. Each box 
shows the group median separating the 25th and 75th quartiles, capped bars indicate 
maximum and minimum values. Boxes denoted by a different letter indicate that the 
QF values were significantly different for each species at the 5% level ................ 80 
 
 
 
xiv 
 
Chapter 5: Evaluation of radiation sensitivity of tsetse males 
5.1. Lifespan of Glossina brevipalpis males irradiated either as adults or as pupae three- 
(group 1), five- (group 2) or seven- (group 3) days before expected emergence .. 98 
5.2. Lifespan of Glossina austeni males irradiated either as adults or as pupae three- 
(group 1), five- (group 2) or seven- (group 3) days before expected emergence .. 98 
 
Chapter 6: Comparative assessment of the mating performance of tsetse males 
under field cage conditions 
6.1. The outside (A) and inside (B) of a cylindrical walk-in field cage, made of panels of 
polyester netting joined with black nylon strips, deployed in a small forest at the ARC-
OVI, Pretoria, South Africa ................................................................................. 104 
6.2. Mean temperature (A) and relative humidity (B) recorded in field cages in the 
mornings and the afternoons during March 2012. Each box shows the group median 
separating the 25th and 75th quartiles, capped bars indicate maximum and minimum 
values, black dots indicating the outliers ............................................................ 108 
6.3. Temperature and relative humidity recorded in the field cage during March 2012, 
February to March 2013 and September 2014. Each box shows the group median 
separating the 25th and 75th quartiles, capped bars indicate maximum and minimum 
values, circles indicating the outliers .................................................................. 109 
6.4. Cumulative mating for Glossina austeni and Glossina brevipalpis in the morning and 
in the afternoon .................................................................................................. 110 
6.5. Number of males of different age groups that mated with Glossina brevipalpis and 
Glossina austeni females in the field cage. Each box shows the group median 
separating the 25th and 75th quartiles, capped bars indicate maximum and minimum 
values, circles indicating the outliers. Boxes denoted by a different letter indicate that 
the numbers were significantly different at the 5% level ..................................... 112 
6.6. Number of irradiated male (40, 80 or 100 Gy) and untreated male Glossina brevipalpis 
and Glossina austeni that mated with untreated females in a field cage. Each box 
shows the group median separating the 25th and 75th quartiles, capped bars indicate 
maximum and minimum values, circles indicating the outliers ............................ 115 
  
xv 
 
LIST OF TABLES 
 
Chapter 2: Tsetse fly distribution and Animal Trypanosomosis prevalence 
2.1. Tsetse H trap collections of Glossina brevipalpis and Glossina austeni from April 2005 
to April 2009 at 18 sites in north eastern KwaZulu-Natal ...................................... 26 
2.2. Weather station data and tsetse fly apparent density (July 2008 to August 2009) .... 
 ............................................................................................................................ 30 
2.3. Trypanosome infection rates in cattle from November 2005 to November 2009 in 
north eastern KwaZulu-Natal. The blood was collected from cattle at dip tanks and 
the infection rate was as determined by the buffy coat technique  ....................... 35 
 
Chapter 3: Comparison of geometric morphometric markers between South Africa, 
southern Mozambique and Swaziland tsetse fly populations 
3.1. Summary of number of Glossina brevipalpis and Glossina austeni wings analysed in 
the seasonal and sexual dimorphism geometric morphometric analysis, Multivariate 
regression of partial warps on size, statistical significance estimated by 10 000-runs 
permutation tests. ................................................................................................ 49 
3.2. Summary of the Mahalanobis and Procrustes distances for the Spatial and Temporal 
wing shape changes in Glossina brevipalpis and Glossina austeni ...................... 53 
3.3. Summary of Glossina brevipalpis and Glossina austeni wings used in the phenetic 
geometric morphometric analysis ........................................................................ 56 
 
Chapter 4: Collection, processing and host source of the rearing diet for colonised 
tsetse flies 
4.1. Anticoagulants tested for their potential use in blood collection, as opposed to 
defribination, for both Glossina brevipalpis and Glossina austeni rearing diets. 
Numbers followed by an * indicate significant differences between the anticoagulant 
and the defribinated blood for each species at the 5% level ................................. 74 
4.2. Bovine/porcine blood combinations tested for their potential use as rearing diet for 
Glossina brevipalpis and Glossina austeni. Numbers followed by an * indicate a 
significant difference between the bovine blood (control) and the various 
combinations for each species and each group at the 5% level ........................... 78 
4.3. Blood mixed with different phagostimulats tested for their potential use as rearing diet 
for Glossina brevipalpis and Glossina austeni ...................................................... 81 
 
 
xvi 
 
Chapter 5: Evaluation of radiation sensitivity of tsetse males 
5.1. Comparison of emergence rates of adult Glossina brevipalpis from pupae irradiated 
with different doses and on different days before expected emergence ............... 89 
5.2. Comparison of emergence rates of Glossina austeni pupae irradiated with different 
doses and on different days before expected emergence .................................... 90 
5.3. Production of Glossina brevipalpis females mated with males irradiated with different 
doses at different developmental stages .............................................................. 93 
5.4. Production of Glossina austeni females mated with males irradiated with different 
doses at different developmental stages .............................................................. 94 
5.5. Reproductive status of Glossina brevipalpis females mated with irradiated males at 
different developmental stages and radiation levels and dissected after an 
experimental period of 60 days ............................................................................ 95 
5.6. Reproductive status of Glossina austeni females mated with irradiated males at 
different developmental stages and radiation levels and dissected after an 
experimental period of 60 days ............................................................................ 96 
 
Chapter 6: Comparative assessment of the mating performance of tsetse males 
under field cage conditions 
6.1. Mating parameters for Glossina brevipalpis and Glossina austeni in field cages for 
assessing the time of peak mating activity, optimal mating age and the effect of 
radiation on male competiveness ....................................................................... 113 
6.2. Production of Glossina brevipalpis and Glossina austeni females mated with sterile 
and fertile males in field cages ........................................................................... 116 
6.3. Production of Glossina brevipalpis and Glossina austeni females mated with sterile 
and fertile males under laboratory conditions ..................................................... 116 
 
xvii 
 
Chapter 1: Introduction 
_________________________________________ 
 
Chapter 1 
 
Introduction 
 
1.1 Literature review 
1.1.1 The history of the tsetse fly and Trypanosomosis 
Blood feeding habits of insects, and therefore the potential for pathogen transmission, 
evolved between 200 to 150 to million years ago (MYA) (Grimaldi & Engel, 2005; Mans, 
2011). It has been estimated that Salivarian trypanosomes (including African 
trypanosomes) became established as gut parasites of some insects around 380 MYA 
(Steverding, 2008). Krafsur (2009) suggested that the tsetse fly origins predate continental 
separation in the cretaceous by more than 100 MYA. He based his findings on the discovery 
of a sister group of the modern tsetse fly in the Florissant shale of Colorado dating to 35 
MYA (Grimaldi, 1992; Krafsur, 2009) as well as a Glossina-like fossil from the Oligocene 
strata in Germany (Grimaldi & Engel, 2005). These findings suggest a near worldwide 
distribution of tsetse flies 30 to 40 MYA and indicate that trypanosomes have been 
transmitted by tsetse flies to mammals for more than 35 million years. 
Throughout history, from Ancient Egyptian times, through the Middle Ages up to early 
modern times diseases very similar to Human African Trypanosomosis (HAT) and African 
Animal Trypanosomosis (AAT) have been recorded (Steverding, 2008). The first medical 
report on HAT was published by John Aktins in 1734, but the nature of the illness was, 
however, still unknown (Cox, 2004). More than a century later, in 1852, David Livingston, 
after observing tsetse fly biting activity on cattle, suggested that the bites of these flies might 
be the cause of AAT (Steverding, 2008). It was, however only in 1895 that David Bruce 
showed that Trypanosoma brucei caused AAT. Six years later, in 1901, Robert Michael 
observed trypanosomes in human blood (Forde, 1902; cited in Steverding, 2008). 
In 1903 David Bruce showed that tsetse flies were transmitting HAT. Although he 
initially believed that only mechanical transmission was involved, he changed his view when 
Friedrich Karl Klein demonstrated the cyclical transmission of T. brucei in tsetse flies 
(Steverding, 2008). Today, more than a 100 years after this crucial discovery, African 
trypanosomes still have a devastating effect on humans and animals in sub-Saharan Africa. 
 
1.1.2 Economic impact of Trypanosomosis 
Africa has a surface area of 30.2 million km2 (including adjacent islands) covering 20.4% of 
the Earth's land area and 6% of its total surface, and is, after Asia, the second-largest 
continent (Sayre, 1999). With 1.1 billion inhabitants Africa is again, after Asia, the second-
1 
 
Chapter 1: Introduction 
_________________________________________ 
 
most-populous continent. In 2013 its inhabitants accounted for about 15% of the world's 
human population (Gudmastad, 2013). Africa is the poorest and most underdeveloped 
continent and in 2005 it was estimated that 80.5% of the population in sub-Saharan Africa 
was living on an income of less than USD 2.50 a day (SESRIC, 2007). Sub-Saharan Africa 
is considered to be the least successful in reducing poverty. In 2005 half of Africa’s 
population was living in poverty (<USD 1.25 per day). It has been estimated that the 
average poor person in sub-Saharan Africa survive on USD 0.70 per day, and that he or 
she was poorer in 2003 than in 1973 (Economic report on Africa, 2004). Although a number 
of factors may influence the development and economical growth in Africa, tsetse flies have 
been mentioned as one of the fundamental contributing detrimental factors (Feldmann et 
al., 2005; Alsan, 2015). 
Tsetse flies, considered as the sole cyclical vectors of African trypanosomes, infest 
about 10 million km² of sub-Saharan Africa (Leak, 1999; Rayaisse et al., 2011). These 
trypanosomae parasites cause Human African Trypanosomosis (HAT), also known as 
sleeping sickness and African Animal Trypanosomosis (AAT) or nagana (Leak, 1999; 
Vreysen et al., 2013). 
Sleeping sickness occur in two forms, an acute form, caused by Trypanosoma brucei 
rhodesiense, mainly present in East Africa and a chronic form, caused by Trypanosoma 
brucei gambiense, in West Africa. Both forms are fatal if left untreated and have an impact 
of 1.59 M DALYs (disability adjusted life years) (Esterhuizen et al., 2011). In sub-Saharan 
Africa, the disease is endemic in 36 of 48 countries with 60 million of 400 million inhabitants 
being at risk. In 1997 about 450 000 people were infected with sleeping sickness (Barrett, 
2006). This number was reduced to 70 000 cases per year in 2000 (Simarro, 2006; Aksoy, 
2011). Since 2000, as the result of intensified surveillance and treatment campaigns in 
combination with vector control (Courtin et al., 2015; Tirados et al., 2015), the number of 
cases have declined by 73% (WHO, 2014). 
African Animal Trypanosomosis (AAT) is considered by many agricultural and 
veterinary economists as the single greatest constraint to increased livestock production in 
sub-Saharan Africa (Vreysen et al., 2013). The direct annual production losses in cattle are 
estimated between USD 600 and 1200 million (Hursey & Slingenbergh, 1995; Vreysen et 
al., 2013). The overall annual lost potential in livestock and crop production can be as high 
as USD 4750 million (Budd, 1999; Vreysen et al., 2013). A second important consideration 
is that tsetse flies prevent the integration of crop farming and livestock keeping, which is 
crucial to the development of sustainable agricultural systems (Feldmann & Hendrichs, 
1995; Vreysen et al., 2013). 
2 
 
Chapter 1: Introduction 
_________________________________________ 
 
Human African Trypanosomosis (HAT) is absent in South Africa and AAT is restricted 
to the uMkhanyakude District Municipality in the north east of the KwaZulu-Natal Province. 
This is a rural district with approximately 573 000 (Statistics South Africa, 2016) inhabitants 
and is considered to be one of the most deprived districts in South Africa. Unemployment 
rates are high, access to piped water and electricity low. The area is characterised by many 
female-headed households with high numbers of children and low education levels. The 
majority of the farmers are communal farmers that follow a free roaming grazing practice. 
It is estimated that 350 000 cattle are at risk of AAT in the area (Kappmeier et al.,1998; 
Kappmeier Green et al., 2007). 
 
1.1.3 Tsetse fly systematics, distribution and biology 
Tsetse flies are Diptera classed in the family Glossinidae, which consists of only one genus 
i.e. Glossina Wiedemann 1830. The genus is divided into three subgenera namely 
Austenina, Nemorhina and Glossina that correspond to the Fusca, Palpalis and Morsitans 
species groups respectively (Leak, 1999; Krafsur, 2009). Based on habitat preferences 
these groups are also referred to as Forest (Fusca), Riverine (Palpalis) and Savannah 
(Morsitans) flies. There are 31 recognised species and subspecies at present (Leak, 1999). 
Although the family is considered to be restricted to Africa (Moloo, 1993) (Fig. 1.1), they 
have recently been recorded in south-west Arabia (Elsen et al., 1990) as well as in Gizar in 
Saudi Arabia (Phelps & Lovemore, 2004). 
Tsetse flies’ feeding behaviour and reproductive biology make them not only unusual 
but also highly successful (Krafsur, 2009). Differing from most insects that have a r-
reproductive strategy, tsetse flies are typical K-strategists. They are obligatorily 
haematophagous and both males and females feed on blood. They reproduce by 
adenotrophic viviparity, and one larva at a time is being nourished in utero by a secretion 
from the uterine gland (Saunders & Dodd, 1972; Tobe & Langley, 1978; Benoit et al., 2015). 
The adult and larval stages are depended on the same source of food i.e. vertebrate blood. 
There are three instars while the larva matures in the female fly. After 7 to 12 days, 
depending on environmental temperatures, a mature larva is deposited in the soil were it 
pupates within approximately four hours. Adult development approximately takes between 
27 to 40 days and is temperature dependant. Females are inseminated within their first 
week of adulthood and can larviposite their first larvae at the earliest, 16 days after 
emergence. The minimum time needed to produce two offspring is about 25 days, 
generation time is c. 43 days at 25 °C (Krafsur, 2009). Characteristically for K-strategists 
the high adult survival rate, which typically exceeds 97% per day (Rogers & Radolph, 
1984a, b, 1985), compensates for their slow reproduction rate. 
3 
 
Chapter 1: Introduction 
_________________________________________ 
 
 
 
Fig. 1.1. Predicted distribution (in red) of Fusca (Austenina) (A), Palpalis (Nemorhina) (B) 
and Morsitans (Glossina) (C) species groups in Africa (Wint & Rogers, 2000). 
 
 
Fig.1.2. Predicted distribution (in red) of Glossina brevipalpis (A) and Glossina austeni (B) 
in Africa (Wint & Rogers, 2000). 
 
1.1.4 African Trypanosomosis management 
There are several tools available in the African Trypanosomosis management toolbox. 
Control can be focused on the Trypanosome parasite or on the tsetse fly vector or on a 
combination of both parasite and vector. More options are available for vector control than 
for the control of the Trypanosome parasite. If the most applicable strategy for a specific 
area or situation is not selected the effective management of Trypanosomosis will remain 
problematic. 
 
1.1.4.1 Disease control 
There is no available preventive vaccine against African Trypanosomosis and treatment 
depends on continuous dosage with trypanocidal drugs. Drugs used for nagana control 
4 
 
Chapter 1: Introduction 
_________________________________________ 
 
include therapeutic Diminazene (Berenil®) or the prophylactic Isometamidium (Samorin® 
and Trypamidium®) (Kappmeier Green, 2002). Ethidium bromide (Homidium®) has also 
been commonly used since the 1950s to treat Trypanosomosis in cattle. The continuous 
use of these drugs can increase the risk of drug management errors, such as under dosing 
or excessive use, that can lead to the development of trypanosome resistance. Drug 
resistance has been reported in 11 of the 36 AAT endemic countries (Leak, 1999). 
Another means of disease control is through the promotion of trypanotolerant 
livestock in tsetse fly infested areas. These trypanotolerant breeds can to a certain degree 
control the intensity, prevalence and duration of parasitism and thereby limiting the 
pathological effect (Murray et al., 1982). A shortcoming of this approach is that 
trypanotolerance is an innate characteristic under genetic control. A limiting factor is that 
trypanotolerant livestock only comprise approximately 5% of the current cattle population 
in Africa (Dolan, 1998). These breeds are usually smaller animals that produce less milk 
and meat and have limited draught power. 
 
1.1.4.2 Vector control 
Because of the limited options for controlling the parasite, e.g. no vaccines and drug 
resistance, vector control remains to date the most effective and economical means of 
Trypanosomosis management. A number of measures, which are continuously being 
improved, are available for the control of tsetse flies. 
 
 Earlier control 
o Bush clearing 
The removal or alteration of suitable tsetse fly habitat is one of the oldest forms of tsetse fly 
control (Du Toit, 1954). Discriminative bush clearing has been used successfully in West 
and East Africa (Leak, 1999). This method has however, become environmentally 
unacceptable and is no longer practised. The expansion of the human populations had a 
very similar effect in many instances. It is evident in rural Africa, and even in South Africa, 
that due to the ever increasing need for agricultural land, more and more of the tsetse fly 
habitat outside of protected areas is becoming unsuitable for tsetse fly survival (Leak, 
1999). 
 
o Host animal reduction 
The culling of wild animals in South Africa from 1872 to 1888 resulted in a gradual 
disappearance of tsetse flies from two-thirds of the infested area. This epitomized the close 
relationship between tsetse fly presence and densities of wild animal populations. Similarly, 
5 
 
Chapter 1: Introduction 
_________________________________________ 
 
in 1897 a severe rinderpest pandemic in southern Africa decimated large numbers of 
animals in the north eastern parts of South Africa which led to the disappearance of 
Glossina morsitans morsitans from the area until today (Rossiter, 2004). The decisive 
elimination of wild animals for the control of tsetse fly started in 1914 (Jack, 1914; Leak, 
1999). This method was used in many southern African countries (Leak, 1999) and between 
1946 and 1950, more than 138 000 wild animals were culled in Zululand (north eastern 
KwaZulu-Natal) alone as part of a tsetse fly control programme (Du Toit, 1954). This 
practise has not only become environmentally unacceptable, but tsetse fly feeding habits 
that differ between tsetse groups can shift from wild to domestic animals and the removal 
of all wild potential tsetse hosts can be challenging as they can be elusive and difficult to 
eliminate (Leak, 1999). 
 
 Chemical control 
The chemical warfare on tsetse flies started in 1945 when DDT (1,1,1-trichloro-2,2-di(4-
chlorophenyl)ethane) became available. Compounds that have commonly been used for 
tsetse fly control are organochlorines (e.g. DDT, dieldrin and endosulfan), pyrethroids (e.g. 
deltamethrin, alpha-cypermethrin, natural pyrethrum) and avermectins (ivermectin) (Leak, 
1999). Insecticide spraying can be divided into ground and aerial spraying. Ground spraying 
with residual insecticides was the principal method used from 1950 to the 1970’s (Leak, 
1999). The chemicals were, depending on the selectiveness and accessibility of the area, 
dispensed to tsetse fly resting sites from knapsacks, mist blowers and unimogs. Aerial 
spraying was developed for a more effective control of tsetse flies over larger areas using 
fixed-wing aircraft and helicopters. In many instances, a combination of ground and aerial 
spraying was used to apply residual as well as non-residual insecticides (Du Toit, 1954; 
Hursey & Allsopp, 1984). 
The campaign to eradicate G. pallidipes from Zululand between 1945 and 1952, was 
the first successful widespread use of insecticides. Novel synthetic insecticides such as 
DDT and HCH (hexachlorobenzene) were used, in residual aerial spraying campaigns 
together with trapping and bush clearing resulting in a zone free of G. pallidipes in north 
eastern KwaZulu-Natal (Du Toit & Kluge, 1949; Du Toit, 1954). 
Glossina m. centralis was eradicated from the Okavango Delta and Kwando-Linyanti 
system in Botswana using aerial spraying of non-residual doses of deltamethrin (Kgori et 
al., 2006). Two successive spraying operations, with deltamethrin applied at night using 
turbo thrust fixed-wing aircraft, were implemented in the Okavango Delta in 2001 and 2002 
(Allsopp & Phillemon-Motsu, 2002; Kgori et al., 2006; 2009). A target barrier was erected 
between successive operations to prevent fly reinvasion into the cleared areas. In 2006 the 
6 
 
Chapter 1: Introduction 
_________________________________________ 
 
Kwando-Linanti systems were similarly treated (Kurugundla, 2012). As insecticides are 
deemed to be harmful to the environment an impact assessment study was conducted in 
the Okavango Delta. However, according to this study the non-residual insecticide aerial 
spraying did not inflict serious harm to terrestrial or aquatic invertebrates (Kurugundla et 
al., 2012). This campaign indicated that it is possible to eliminate G. m. centralis over 
relatively large areas. The sequential aerosol technique is, however, not always suitable for 
eradication of a tsetse fly population as was demonstrated in Ghana for the riverine tsetse 
species (Adam et al., 2013). 
 
 Bait technologies 
Instead of the large scale indiscriminative treatment of an area, insecticides can be used 
with selective methods such as traps, targets and animals. Bait technology combines visual 
and olfactory cues to attract, capture or kill tsetse flies (Vale, 1974; 1993). Tsetse flies are 
attracted to the blue/black colour combination of the trap, or animal, and when the fly alights 
on the treated cloth or animal, it absorbs the insecticide through its tarsi and dies. This 
strategy has been used in the past 30 years in many tsetse fly control campaigns, either on 
its own or as part of integrated pest management strategies. Although bait technologies 
can be labour intensive it is relatively cheap and simple to implement and is probably most 
useful as a long term suppression strategy. 
Much time and efforts have been invested in the development and refinement of the 
tsetse fly traps and targets (Lindh et al., 2009) and various designs are available for specific 
tsetse species. Kuzoe & Schofielld (2005) provide a comprehensive review on advances in 
this field and emphasise that traps and targets are species specific and that all traps are 
not equally effective for all species. Various traps and targets are available to be used 
against specific species in specific environments (Kuzoe & Schofielld, 2005). It is therefore 
important that the most appropriate trap or target be selected for monitoring or control. 
Glossina austeni and Glossina brevipalpis exist sympatrically in certain areas in South 
Africa. These two species are not equally attracted to the H trap or the sticky XT trap (cross-
shaped targets) (Kappmeier, 2000). Trapping efficiency can be improved by baiting the 
traps with odours. While this seems effective for G. brevipalpis it has no effect on G. austeni 
(Kappmeier & Nevill, 1999a). Esterhuizen et al. (2006) indicated differences in the 
effectiveness of odour-baited insecticide-treated targets (Kappmeier & Nevill, 1999b). While 
targets deployed at a density of eight per km² can suppress G. austeni this density will be 
ineffective for the control of G. brevipalpis (Esterhuizen et al., 2006). However, for the 
suppression of the same species, G. austeni, on Unguja Island, Zanzibar a much higher 
density of 50 targets per km² was needed (Vreysen et al., 2000). 
7 
 
Chapter 1: Introduction 
_________________________________________ 
 
Current vector control in HAT endemic areas is mainly with the newly developed tiny 
targets (Rayaisse et al., 2011; Torr et al., 2011). These tiny targets, based on the same 
principles as the large savannah-type cloth targets, are eight times smaller (0.25 m x 0.5 m) 
than the traditional targets (1 m x 1.5 m), and apparently have the same killing efficiency 
for Glossina fuscipes as the bigger ones, however, this point has been much debated 
(Bouyer et al., 2013a). Less cloth is required to make the tiny targets and less insecticide 
is needed for impregnation, making these targets more economical and easier to deploy 
over large areas in tropical Africa (Esterhuizen et al., 2006; Shaw et al., 2015). 
The live-bait technology involves the application of insecticides on domestic animals 
through dipping, spraying or pour-ons. This will only be effective if a large proportion of the 
tsetse fly population is feeding on domestic rather than wild animals (Leak, 1999). The 
existing extensive dipping network in KwaZulu-Natal that was established mainly for tick 
control was used in combination with insecticide-treated targets and animal drug treatment 
to control an outbreak of nagana in 1990 (Bagnall, 1993; Kappmeier et al., 1998). A 
disadvantage of using live animals as baits is that it may not prevent initial trypanosome 
transmission, as the flies can feed and potentially transmit the parasite before dying. 
A method that can be used to reduce cost in live-bait technology is the restricted 
application of insecticides to only those areas on the animal where the majority of the tsetse 
flies land and feed (Vale, 2003; Esterhuizen, 2007). In terms of cost saving, restricted 
application of insecticides can achieve a reduction of up to 80% in chemical usage (Vale, 
2003). This technique was shown to be promising for control in communal farming areas 
where livestock constitutes a major source of food for the tsetse fly population (Bouyer et 
al., 2007; Torr et al., 2007; Bouyer et al., 2009; 2011). Continuous research is needed to 
improve insecticide applications on animals (Ndeledje et al., 2013; Muhanguzi et al., 2014) 
and to reduce these disadvantages. 
 
 Biological control 
A range of natural enemies of tsetse flies have been recorded, e.g. hymenopteran and 
dipteran parasites (Fiedler, 1954; Fiedler et al., 1954; Leak, 1999), parasitic mites, helminth 
parasites (Poinar et al., 1981) and fungi (Kaaya, 1989). Although some hymenopteran and 
dipteran parasites and fungi had a marginal detrimental effect on tsetse fly populations 
(Leak, 1999) they were not as successful as anticipated in reducing them. 
 
 Insect growth regulators and juvenile hormone 
Both these techniques exploit as a weakness, the complex and low reproductive potential 
of tsetse flies (Leak, 1999). Insect growth regulators interfere with chitin synthesis thereby 
8 
 
Chapter 1: Introduction 
_________________________________________ 
 
preventing successful reproduction, whereas juvenile hormones disrupt the reproductive 
cycle resulting in abortions; both methods essentially render the females sterile (Leak, 
1999). Laboratory experiments showed that the growth regulator difubenzuron (DFB; 
Dimilin) was effective for G. m. morsitans (Jordan et al., 1979). A limitation of the method 
is the need to apply sufficient quantities of the chemicals to sterilize flies throughout their 
reproductive lives (Leak, 1999). 
The field effectiveness of the juvenile hormone pyripoxyfen was proven (Hargrove & 
Langley, 1993). A potential disadvantage of these techniques is that they have a delayed 
effect on the population as these do not kill the fly immediately. This delayed effect can, 
however, also be seen as an advantage, since the fly itself will transmit the bio agent within 
the population thereby amplifying the impact of the control (Bouyer & Lefrançois, 2014). 
 
 Genetic control methods 
o Symbiont-based methods 
Sophisticated symbiotic associations between tsetse flies and at least three endosymbiotic 
bacteria, Wigglesworthia, Sodalis and Wolbachia have been documented (Doudoumis et 
al., 2013; Snyder & Rio, 2013; Bourtzis et al., 2016). The presence of different strains of 
Wolbachia in different tsetse fly populations may lead to incompatible genetic crossings 
between these groups resulting in embryonic death of the fly. This cytoplasmic 
incompatibility (CI) (Bourtzis, 2007) can thus be used to control tsetse flies (Alam et al., 
2011). Wolbachia-induced CI is known as the incompatible insect technique (IIT) and it has 
been suggested for use in parallel with the sterile insect technique (SIT) or alone, as a 
driving system to replace the population with a strain that has a desirable genotype (e.g. 
resistant to trypanosome infections) (Bourtzis, 2007; McGraw & O’Neill, 2013; Bourtzis et 
al., 2016). Investigations into the use of the genetically modified symbiont Sodalis so that 
they carry a gene that expresses anti-trypanosomal nanobodies, and as making the tsetse 
fly refractory to trypanosome infections are underway (De Vooght et al., 2014; Bourtzis et 
al., 2016), tsetse flies that harbour the recombinant Sodalis can vertically transmit them to 
their offspring and this will enable the spread of vector incompetence within the tsetse fly 
population. Wigglesworthia glossinidia is critical for tsetse fly reproduction and plays a role 
in progeny development (Benoit et al., 2015). When Wigglesworthia are absent in tsetse 
flies their progeny is immunocompromised and infertile (Benoit et al., 2015) and this might 
lead to newly developed control methods. 
 
 
 
9 
 
Chapter 1: Introduction 
_________________________________________ 
 
o Sterile insect technique 
Although the sterilising effect of radiation on insects was already known in the 1930s 
(Runner, 1916; Muller, 1927), it was only 20 years later in the 1950s that the potential of 
this technique for the control of insects was realised (Baumhover, 2001; 2002). The sterile 
insect technique (SIT) involves the sterilisation with radiation of males from the mass-
rearing of the target species (Robinson, 2005) followed by the release of these males in 
sufficient numbers to outcompete their wild counterparts (Klassen & Curtis, 2005; Bourtzis 
et al., 2016). The mating of these sterile males with wild fertile females results in no 
progeny, which leads to a population reduction and in some cases the eradication of the 
target population (Bourtzis et al., 2016). 
For maximum effectiveness the sterile males must outnumber the fertile males and 
the SIT may therefore be less cost-effective if the target population is large. Many 
conventional control methods (e.g. insecticide spraying) are both cost and operationally 
effective when the target population is high. The application of conventional control 
methods followed by SIT may lead to eradication (Fig. 1.3) (Feldmann & Hendrichs, 2001). 
In contrast to conventional control tactics, such as insecticide spraying that are very 
effective at high insect population densities but less effective at low population densities, 
the SIT will become more effective as the targeted insect population decreases (Fig. 1.3). 
 
 
Fig.1.3. The efficiency of a conventional control approaches in combination with the sterile 
insect technique in relation to target population densities (Feldmann & Hendrichs, 2001). 
 
10 
 
Chapter 1: Introduction 
_________________________________________ 
 
The slow reproduction rate in tsetse flies will contribute to the susses of the SIT. The 
feasibility of the SIT for the eradication of entire populations has been demonstrated on 
several occasions (Vreysen et al., 2000; Feldmann et al., 2005; Vreysen et al., 2013). The 
elimination of G. austeni from the island of Unguja, Zanzibar, is probably the most 
remarkable. The last G. austeni individual was collected in September 1996, and the last 
T. vivax recorded in 1998 (Dyck et al., 2000). The SIT is species-specific and is considered 
an environmentally friendly control method by most authors (Knipling, 1959, sited in 
Bourtzis et al., 2016). 
 
1.2 Study justification 
After an epidemiological silence of nearly 33 years, a severe outbreak of nagana in north 
eastern KwaZulu-Natal in 1990, showed the devastating socio-economic impact of tsetse-
transmitted nagana on these largely rural communities of South Africa (Bagnall, 1993). 
These outbreaks led to the re-establishment of tsetse flies and trypanosome research in 
South Africa. 
Surveys conducted between 1993 and 1999, indicated two species, G. brevipalpis 
and G. austeni, to be present in an area of 16 000 km2 in the north eastern part of the 
KwaZulu-Natal Province (Kappmeier Green, 2002). The infested area stretched from St 
Lucia (-28.499639, 32.395194) in the south to the border of Mozambique (-26.8692, 
32.8342) in the north and from the coast in the east up to the Hluhluwe-Imfolozi Game 
Reserve (-28.33416, 31.691222) in the west (Kappmeier Green, 2002). 
The G. brevipalpis belt in Africa starts in Ethiopia in East Africa from where it extends 
southwards to Somalia, Uganda, Kenya, Rwanda, Burundi and Tanzania (Fig. 1.2 A). In 
southern Africa G. brevipalpis is present in Malawi, Zambia, Zimbabwe and the northern 
and central part of Mozambique (Moloo, 1993) (Fig. 1.2 A). Glossina austeni is found, in 
lower numbers than G. brevipalpis in East Africa from Somalia in the north, extending south 
into Kenya, Tanzania, Zimbabwe and the northern and central parts of Mozambique (Fig. 
1.2 B) (Moloo, 1993). 
These South African tsetse fly populations, extending to southern Mozambique (both 
species) and Swaziland (G. austeni) represent their most southern distribution (Saini & 
Simarro, 2008; Sigauque et al., 2000) they are expected to be geographically isolated from 
the main tsetse fly belt. 
Initial studies facilitated the development of an area-wide integrated pest 
management (AW-IPM) strategy that include a sterile insect technique (SIT) component to 
establish a tsetse fly free South Africa (Kappmeier Green et al., 2007). Using the tsetse fly 
presence and abundance data, as determined with odour baited XT sticky traps, this AW-
11 
 
Chapter 1: Introduction 
_________________________________________ 
 
IPM strategy suggested the division of the infested area into four zones from south to north 
with the successive implementation of four phases (pre-suppression, suppression, SIT and 
post-eradication) in each zone following the rolling carpet principle (Kappmeier Green et 
al., 2007). 
Determining the accurate geographic distribution and abundance of the tsetse fly 
population to be eradicated will be vital for the success of not only the strategy as proposed 
by Kappmeier Green et al. (2007) but for any proposed control effort. The area that needs 
to be treated will directly affect the outcome, sustainability and cost of any proposed control 
campaign. 
A basic requirement for determining the accurate distribution of a population is the 
availability of efficient sampling devices. The development of the H trap and its 
accompanying artificial odour system, which was shown to be more effective than the odour 
baited XT sticky traps for the sampling of G. brevipalpis and G. austeni (Kappmeier & Nevill, 
1999a; Kappmeier, 2000), necessitated a re-assessment of the tsetse fly distribution in the 
area. The initial surveys were conducted between 1993 and 1999 and the available data 
may not be a true reflection of the current situation. A “probability of presence” model 
(Hendrickx, 2002; Hendrickx et al., 2003) predicts a wider geographic distribution range for 
both G. brevipalpis and G. austeni than had been indicated by the 1993 to 1999 survey 
data (Hendrickx, 2002) and this needs to be validated. 
The degree of geographic isolation of a population targeted for control will determine 
to what extent reinvasion form neighbouring populations play a role in the success of any 
proposed control programme. Kappmeier Green (2002) stated that the tsetse fly infected 
area could be divided into four zones. In Zone I, in the south, only G. brevipalpis were 
present, in zone II and III mainly G. austeni were present while zone IV contained both 
species. This apparent fragmentation suggested that these populations might be 
geographical isolated from each other and that reinvasion from neighbouring areas may be 
minimal. Before designing an appropriate control strategy for the area it needs to be 
determine if the South African populations are geographically isolated from those in 
southern Mozambique and Swaziland. 
As part of the proposed strategy for an area-wide control campaign with a SIT 
component, laboratory colonies of both South African tsetse species were established at 
the Agricultural Research Council-Onderstepoort Veterinary Institute (ARC-OVI), Pretoria, 
in 2002. These colonies of G. brevipalpis and G. austeni were respectively established 
using seed material from the Tsetse and Trypanosomiasis Research Institute (TTRI) (now 
named Vector & Vector-Borne Diseases Research Institute) Tanga, Tanzania and the 
Entomology Unit of the Food and Agriculture Organization (FAO)/International Atomic 
12 
 
Chapter 1: Introduction 
_________________________________________ 
 
Energy Agency (IAEA) Laboratories in Seibersdorf, Austria (now called the FAO/IAEA 
Insect Pest Control Laboratory). The development of tsetse fly rearing capabilities, with the 
capacity to produce high quality males that would be comparable and competitive with their 
wild counterparts will be essential in the SIT component of an area-wide control campaign 
(Parker, 2005). These colonies need to be maintained at sustainable and economical 
acceptable levels. 
The SIT involves the sterilisation of males with radiation obtained from a mass-rearing 
facility of the target species (Bakri et al., 2005; Robinson, 2005). It is essential to determine 
the optimal radiation dose to induce the necessary level of sterility in the colonised males. 
A too low dose may lead to the release of fertile males in the area while a too high dose will 
negatively impact on their competitiveness (Bakri et al., 2005). The optimal age of the adult 
flies or the pupa for irradiation needs to be determined as well as any detrimental effect of 
radiation on the fitness and mating capability of these males. The sterilised males must be 
able to outcompete their fertile counterparts (Parker, 2005; Vreysen et al., 2011). 
 
1.3 Aim 
The main aim of this study was to contribute towards the development of the proposed AW-
IPM strategy. The more specific aims were (1) to determine the mating behaviour and 
performance of colonised irradiated G. brevipalpis and G. austeni flies in relation to 
biological and operational attributes for use in SIT, (2) to update the maps of tsetse fly 
abundance and trypanosome prevalence, and (3) to assess the degree of isolation between 
the different populations of the two species. 
 
1.4 Objectives 
 
To achieve these aims, the following objectives were defined. 
 
 The existing distribution maps of tsetse fly abundance and Trypanosomosis 
prevalence in South Africa were updated. The distribution and abundance of tsetse 
flies were discussed in terms of environmental factors such as vegetation and climate. 
Lastly the correlation between tsetse fly apparent densities and infection prevalence 
of the disease in livestock was investigated. 
 The applicabilty geometric morphometrics to determine the extent of potential genetic 
isolation between the various populations of G. brevipalpis and G. austeni present in 
South Africa were assessed. Subsequently the South African populations were 
compared with flies collected in neighbouring southern Mozambique and Swaziland. 
13 
 
Chapter 1: Introduction 
_________________________________________ 
 
These three wild populations was also be compared with laboratory colonies 
maintained at the ARC-OVI. Geometric morphometrics was used to assess seasonal 
and sexual dimorphism differences. 
 To optimise the blood diet provided to the colonies maintained at the ARC-OVI and 
to accecc the effect of anticoagulants, phagostimulants and hosts on the nutritional 
value of the blood. Attempts was made to establish a KwaZulu-Natal strain colony of 
G. brevipalpis. 
 The radiation dose needed to sterilise G. brevipalpis and G. austeni adults and pupae 
was determined. 
 The mating behaviour and performance of colonised G. brevipalpis and G. austeni 
under near natural conditions using walk-in field cages were assessed. 
 
These results will contribute to the understanding of the ecology and behaviour of 
G. brevipalpis and G. austeni not only in South Africa but in all areas in Africa where these 
two species are found. Information generated in this study will enable us to refine the 
proposed control strategy and determine if southern Mozambique and Swaziland need to 
be included in the proposed strategy. As very little research is conducted elsewhere on 
these two species specifically, the data generated can be applied to all areas where 
G. brevipalpis and G. austeni are present. It must be emphasised that in Africa, a 
sustainable alleviation or if possible the removal of HAT and AAT should be the aim 
irrespectively of the control method. 
 
14 
 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
Chapter 2 
 
Tsetse fly distribution and Animal Trypanosomosis prevalence1 
 
2.1 Introduction 
The discovery that Trypanosoma brucei was the cause of African animal Trypanosomosis 
(AAT), also known as nagana, can be dated back more than a century when it was recorded 
for the first time in north eastern KwaZulu-Natal (formerly Zululand), South Africa, in the 
1880’s (Bruce, 1895; Bagnall, 1993; Steverding, 2008). In 1895, Sir David Bruce stated that 
game animals were the reservoir hosts of the causative trypanosome species and that 
these protozoan parasites were transmitted between their mammalian hosts by tsetse flies 
(Diptera: Glossinidae) (Bruce, 1895). Of the four species, Glossina morsitans morsitans, 
Glossina pallidipes, Glossina brevipalpis, and Glossina austeni (Glossina brandoni) that 
have historically been recorded in South Africa, G. m. morsitans was the only one that was 
encountered in the most northern part of the country (Fuller, 1923). Glossina brandoni, 
which has its type locality in north eastern KwaZulu-Natal, is currently considered as a 
synonym of G. austeni (Phelps & Lovemore, 2004). The other three species, i.e., 
G. pallidipes, G. brevipalpis and G. austeni were restricted to north eastern KwaZulu-Natal 
Province in South Africa (Fig. 2.1) (Fuller, 1923). Glossina pallidipes was the predominant 
species. Based on its abundance, it was considered the most important vector of AAT at 
that time (Fuller & Mossop, 1929). Glossina brevipalpis and G. austeni, which were confined 
to smaller areas with mostly dense vegetation, were not considered important vectors of 
AAT. The sheer abundance of G. pallidipes was illustrated by the large numbers of flies 
being trapped on certain occasions, i.e. in 1932, two million flies were collected within a 
month after deployment of 1000 Harris traps in north eastern KwaZulu-Natal and nearly 
eight million flies over that entire year (Harris, 1932). The presence of large numbers of 
tsetse flies, and especially G. pallidipes, in north eastern KwaZulu-Natal resulted in severe 
outbreaks of nagana between 1942 and 1946 (Du Toit, 1954). This led to an insecticide-
based spraying programme with DDT and HCH and by 1953 G. pallidipes was eradicated 
from KwaZulu-Natal (Du Toit, 1954). 
The programme succeeded because it integrated several control tactics (aerial and 
ground spraying, trapping and bush clearing) and proved to be sustainable as the entire 
                                                          
1 Partially published as: 
De Beer, C.J., Venter, G.J., Kappmeier Green, K., Esterhuizen, J., De Klerk, D.G., Ntshangase, J., Vreysen, 
M.J.B., Pienaar, R., Motloang, M., Ntantiso, L. & Latif, A.A. (2016) An update of the tsetse fly (Diptera: 
Glossinidae) distribution and African animal trypanosomosis prevalence in north-eastern KwaZulu-Natal, 
South Africa. Onderstepoort Journal of Veterinary Research, 83 (1), a1172. 
15 
 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
population was targeted and KwaZulu-Natal is today still free of G. pallidipes. This 
represents one of the first tsetse fly control programmes that implemented area-wide 
integrated pest management (AW-IPM) principles successfully (Klassen, 2005; Vreysen et 
al., 2007). An additional benefit from this campaign was the apparent removal of 
G. brevipalpis from the Hluhluwe-iMfolozi Park (Du Toit, 1954). 
 
Fig. 2.1. The distribution of Glossina pallidipes, Glossina brevipalpis and Glossina austeni 
in KwaZulu-Natal prior to 1953 (Du Toit, 1954). 
 
In areas where G. brevipalpis and G. austeni were found in the absence of 
G. pallidipes (Fig. 2.1), no control was implemented, and consequently, these two species 
16 
 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
remained present in north eastern KwaZulu-Natal (Kappmeier et al., 1998). From 1955 
onwards, only sporadic cases of nagana were recorded there (Bagnall, 1993) and it was 
assumed that agricultural developmental changes such as the establishment of commercial 
pine and eucalyptus plantations and bush clearing for livestock production had rendered 
this area unsuitable for tsetse flies (Kappmeier et al.,1998). 
In 1990, a severe outbreak of nagana once again occurred in north eastern KwaZulu-
Natal and cattle mortalities during this outbreak were exacerbated by the co-occurrence of 
a severe drought (Kappmeier et al.,1998; Emslie, 2005). Emergency control measures, 
utilising the extensive dipping network used for tick control, were implemented (Kappmeier 
et al.,1998). As part of the control measures the active ingredient of the routinely used 
dipping agent, was changed from amitraz, an acaricide, to the pyrethroid cyhalothrin, a 
wider spectrum insecticide (Hall & Fischer, 1984) for two years (Bagnall, 1993). Whereas 
the pyrethroid cyhalothrin is very effective at controlling both ticks and tsetse flies (Bagnall, 
1993), the efficacy of amitraz is limited to ticks and is considered ineffective against dipteran 
flies (Hall & Fischer, 1984). The adopted cattle-dipping regime in combination with animal 
treatments using trypanocidal drugs brought the disease outbreak under control 
(Kappmeier et al., 1998). 
The 1990 outbreaks illustrated the fact that nagana had remained a serious 
debilitating problem in north eastern KwaZulu-Natal, with mixed infections of Trypanosoma 
congolense and Trypanosoma vivax (Bagnall, 1993). Periodic screening of cattle at dip 
tanks in the tsetse-infested area indicated that T. congolense was the dominant species 
(Van den Bossche et al., 2006; Mamabolo et al., 2009; Motloang et al., 2012; 2014) with 
T. vivax being less abundant (Mamabolo et al., 2009). Recently Trypanosoma theileri and 
T. brucei were detected based on the alignment of 18S rRNA gene sequences which were 
compared to trypanosome sequences available at the National Centre for Biotechnology 
Information (NCBI) (Taioe, 2014). Two sub-genotypes of T. congolense, the Savannah and 
Kilifi types, and mixed infections (Gillingwater et al., 2010) of these were identified with PCR 
in the area (Mamabolo et al., 2009). These surveys showed that trypanosome infections in 
cattle at dip tanks close to the Hluhluwe-iMfolozi Park were higher than at dip tanks further 
away from this Game Reserve (Van den Bossche et al., 2006; Motloang et al., 2014; 
Ntantiso et al., 2014). 
Long-term management of nagana by dipping and/or curative treatment of infected 
cattle with trypanocidal drugs is neither cost-effective nor sustainable (Bagnall, 1994; Shaw, 
2009) and it became evident that a long-term solution to the nagana problem in South Africa 
needed to be investigated. Initial surveys confirmed the absence of G. pallidipes and 
17 
 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
showed that G. brevipalpis and G. austeni were still the only species present in KwaZulu-
Natal (Kappmeier Green, 2002). 
Between 1993 and 1999, an extensive tsetse fly survey was conducted in an area of 
approximately 12 000 km2 using odour baited sticky XT traps (cross-shaped targets) to 
assess the distribution of each species (Kappmeier & Nevill, 1999a; Kappmeier Green & 
Venter, 2007) (Fig. 2.2). This survey indicated that G. brevipalpis was present in a southern 
and a northern band, commonly associated with game reserves and other protected areas 
(Fig. 2.2). The distribution of G. austeni was continuous from south to north but did not 
extend as far west as that of G. brevipalpis. Glossina austeni was also very common in 
communal farming areas (Fig. 2.2) (Nevill, 1993; Kappmeier Green, 2002). The 1993-1999 
survey did not establish the southernmost distribution limit of G. brevipalpis and G. austeni, 
as the sampling frame was only designed to determine broad distribution limits. It was, 
however, suggested that the southernmost limit of the tsetse fly distribution in South Africa, 
and therefore Africa, could roughly be regarded as the southernmost extent of the Umfolozi 
River (Kappmeier Green, 2002). The 1993-1999, surveys also showed that G. brevipalpis 
and to a lesser extent G. austeni, were present close to the coast (Fig. 2.2) in areas not 
sampled in the 1950’s (Du Toit, 1954). 
The 1993 to 1999 sticky trap data were used to develop a probability of presence 
model (Hendrickx, 2002; Hendrickx et al., 2003). This model, based on environmental and 
climate variables, predicted a wider geographical distribution range for both G. brevipalpis 
and G. austeni than what was indicated by the survey data (Hendrickx, 2002). However, 
Hendrickx et al. (2003) suggested that the model may have overestimated tsetse fly 
distribution. 
Determination of the accurate distribution of the target insect, as well as the 
interaction between the insect and the causative agent it transmits, is vital for the successful 
implementation of any proposed control campaign, and will directly affect its outcome, 
sustainability and cost (Vreysen et al., 2007; Shaw, 2009). Additional ongoing efforts to 
generate continental as well as national Atlases of tsetse flies and AAT (Cecchi et al., 2014; 
2015) will benefit from continuously updated data sets. 
The development of an improved trap for G. brevipalpis and G. austeni (Kappmeier, 
2000) and an enhanced artificial odour system for G. brevipalpis (Kappmeier & Nevill, 
1999a), allowed a more effective sampling of both species in South Africa. 
Although the 1993 to 1999 surveys of Kappmeier Green et al. (2007) provided 
accurate data on the distribution and abundance of the two fly species, these maps may be 
outdated. Additionally the model presented by Hendrickx (2002), predicting a wider 
distribution range for both species, reinforced the need to verify the distribution and validate 
18 
 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
the model. An update of the geographical distribution and abundance of tsetse flies as well 
as Trypanosomosis prevalence in KwaZulu-Natal was considered a prerequisite for future 
planning of control efforts. 
 
Fig. 2.2. Distribution of Glossina brevipalpis and Glossina austeni based on a survey carried 
out from 1993 to 1999 with odour baited XT sticky traps. Only positive trap catches are 
indicated. 
 
The objective of the present study was to update the distribution maps of tsetse flies 
and Trypanosomosis prevalence in South Africa. Additionally, the distribution and 
19 
 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
abundance of tsetse flies in the area were assessed in terms of some additional 
environmental factors such as vegetation and climate. Correlation between tsetse fly 
apparent densities and infection prevalence of the disease in livestock were investigated. 
 
2.2 Materials and methods 
2.2.1 Study area 
The tsetse fly infested area (± 16 000 km2) in South Africa is confined to the north eastern 
part of the KwaZulu-Natal Province. The area stretches approximately from the Umfolozi 
River (-28.5204, 32.3123) in the south to the border of Mozambique (-26.8692, 32.8342) in 
the north, and from the Indian Ocean coast in the east up to the west of the Hluhluwe-
iMfolozi Park (-28.33416, 31.691222) (Fig. 2.2) (Kappmeier Green, 2002). Although some 
commercial cattle farms are present, it is predominantly a subsistence cattle-farming area 
with numerous communal farms interspersed with a number of protected areas. According 
to the local State Vetenarian there are an estimated 250 000 cattle and 130 000 small 
ruminants in the area and cattle are treated with trypanocides on an ad hoc basis. 
The protected areas consist of provincial and private game parks and reserves. The 
area surrounding the extensive fresh water lake system, iSimangaliso Wetland Park, was 
proclaimed as South Africa’s first world heritage site in 1999. These areas contain a wide 
variety of game animals, such as birds, rodents and small primates (e.g. Cercopithecus 
mitis) but also large numbers of bigger mammals that are potential hosts for tsetse flies. 
The main game animals in the area are buffalo (Syncerus caffer), hippopotamus 
(Hippopotamus amphibius), white rhinoceros (Ceratotherium simum), elephant (Loxodonta 
africana), bush pig (Potamochoerus porcus) and warthog (Phacochoerus aethiopicus). 
Antelopes in the area include species such as red duiker (Cephalophus natalensis), blue 
duiker (Philantomba monticola), impala (Aepyceros melampus), reedbuck (Redunca 
arundinum), blue wildebeest (Connochaetes taurinus), waterbuck (Kobus ellipsiprymnus), 
bushbuck (Tragelaphus scriptus), nyala (Tragelaphus angasii) and kudu (Tragelaphus 
strepsiceros). The target area contains a number of state forests, mostly pine and 
eucalyptus plantations, and commercial sugarcane farms. 
The climate is subtropical with the average minimum temperatures ranging from 
10.52 ± 1.26 °C in July (winter) to 21.71 ± 0.67 °C in January (summer). The average 
maximum ranges from 24.65 ± 0.77 °C in July to 30.86 ± 1.38 °C in January. The average 
minimum relative humidity ranges from 39.92 ± 8.23% in July to 62.04 ± 6.39% in October 
whereas the average maximum ranges from 92.23 ± 3.73% in June to 94.94 ± 3.13% in 
February. The area receives an average maximum of 132.92 ± 92.95 mm of rain in January, 
and most of the precipitation is received in the hot season from October to March. However, 
20 
 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
it can also rain in the cold dry season from April to September with a minimum average 
9.27 ± 9.68 mm of rain in July. On average, rainfall is higher on the coast compared to the 
interior. 
 
2.2.2 Tsetse fly survey 
For the main part of the survey, tsetse flies were collected at 18 sites located in four 
magisterial districts: Ingwavuma, Ubombo, Hlabisa and the northern part of Enseleni (Fig. 
2.4). A total of 77 odour-baited H traps (Kappmeier & Nevill, 1999b; Kappmeier, 2000) were 
deployed at these 18 sites from April 2005 to April 2009. The number of trap days ranged 
from 723 at Ocilwane (northern Enseleni) to 1661 days at False Bay Park (eastern Hlabisa 
district). Two supplementary surveys were carried out in Hlabisa and the northern parts of 
Enseleni from April to May 2012 (an average of 26 ± 1.62 days) and from April to June 2015 
(an average of 63 ± 1.36 days), mainly to determine more accurately the southernmost 
occurrence of the tsetse fly populations. 
The traps were baited with odours to enhance trapping of G. brevipalpis (Kappmeier 
& Nevill, 1999a). These baits consisted of 1-octen-3-ol and 4-methylphenol at a ratio of 1:8 
that were released at 4.4 mg/h and 7.6 mg/h, respectively. The chemicals were dispensed 
from seven heat-sealed sachets (7 cm x 9 cm) made of low-density polyethylene sleeves 
(wall thickness 150 microns) placed near the entrance of the trap. A 300 mL brown glass 
bottle that dispensed acetone through a 6 mm hole in the lid at a rate of ca. 350 mg/h was 
placed next to the H trap (Esterhuizen, 2007; Kappmeier Green, 2002). Flies were collected 
in a 20% ethanol solution to which an antiseptic, Savlon® (Johnson & Johnson, Pharmedica 
Laboratories (Pty) Ltd. Rattray Road, East London, South Africa) (0.4 mL/L) and formalin 
(0.4 mL/L) had been added to preserve the sampled flies as well as to combat ant and 
spider predation. Traps were emptied and serviced every 14 days. The number of each 
species collected over this period was counted and results expressed as apparent density 
(AD), i.e. the number of flies per trap per day. 
Data from several independent surveys, done over a couple of years, was 
incorporated in this study. 
 
2.2.3 Environmental factors affecting tsetse fly distribution 
2.2.3.1 Vegetation 
An updated vegetation map for the infested area (north eastern KwaZulu-Natal, southern 
Mozambique and Swaziland) was developed in 2010 (Bouyer & Guerrini, 2010). The main 
land-cover classes that were considered relevant for the presence or absence of 
G. brevipalpis and G. austeni were indicated on the map as savannah woodland, 
21 
 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
herbaceous savannah, shrub savannah, dense dry forest, gallery forest, tree plantations, 
crops (agricultural areas), urban areas, swamps, water bodies and bare ground (Bouyer & 
Guerrini, 2010). In the present study, this vegetation map was combined with satellite 
images (Google earth) of the area to assess the potential correlation between vegetation 
and tsetse fly distribution and/or abundance. To enable comparison with the vegetation 
data produced in 2010, tsetse fly apparent densities as obtained from the surveys 
implemented between July 2008 and August 2009 were correlated to the different 
vegetation classes found at each trapping site. 
 
2.2.3.2 Climate 
Climate data obtained from seven permanent weather stations (Fig. 2.3), maintained by the 
Agricultural Research Council-Institute for Soil, Climate and Water (ARC-ISCW), were used 
to develop climate maps of the study area. Tsetse fly apparent densities as obtained from 
five of the sampling sites (Kosi Bay, Mkuzi, Kuleni, Bushlands and St Lucia) located within 
8 km from one of these seven weather stations (Fig. 2.3), were used to determine 
correlations between tsetse fly AD and certain climate variables. Similar to the vegetation 
comparisons, climate data and tsetse fly AD from July 2008 to August 2009 were used. 
 
 
Fig. 2.3. Location of the weather stations in relation to the tsetse H traps used for 
determining potential relationships between tsetse fly apparent density (AD) and climate 
variables in north eastern KwaZulu-Natal. 
22 
 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
2.2.4 Trypanosomosis survey 
A large number of communal dip tanks, constructed, maintained and operated by the 
Provincial Department of Veterinary Services, are routinely used for tick control and are 
evenly distributed throughout the tsetse-infested area. The tick control policy, as prescribed 
by the Department of Veterinary Services, consists of weekly dipping of cattle in the summer 
and fortnightly dipping during winter. Cattle in the communal farming areas as well as on 
the commercial farm Boomerang roam freely. The trypanosome infection prevalence in 
cattle was determined at 25 dip tanks from November 2005 to November 2007. All animals 
that congregated at a particular dip tank were considered as one herd as they grazed 
together and were managed using the same animal husbandry practice (Emslie, 2005; 
Ntantiso et al., 2014). Herd size was variable and ranged from 13 to 6411 animals, and the 
number of cattle owners at each dip tank was likewise very variable and ranged between 
five and 296. Cattle were screened on the day they were scheduled for routine dipping. 
These herds were divided into groups of 30 to 40 cattle and two to three animals in each 
group were, after consent from the owner, randomly sampled. Data from several 
independent surveys (published and unpublished), done over a couple of years, was 
incorporated in this study. 
Blood was collected from the tail or jugular vein of adult animals using 10 mL 
vacutainer tubes containing the anticoagulant EDTA (BD Vacutainer®; BD, Plymouth, UK). 
The blood was transferred to micro haematocrit centrifuge capillary tubes (Marienfeld-
Superior, Lauda-Königshofen, Germany) that were sealed with Cristaseal (Hawksley) and 
centrifuged in a haematocrit centrifuge for 5 minutes at 9000 rpm. The buffy coat of each 
specimen was extruded onto a microscope slide, under a cover slip and examined for motile 
trypanosomes using a compound microscope at 40 times magnification (Paris et al., 1982). 
Trypanosomosis prevalence at each dip tank was expressed as the number of cattle with 
positive trypanosomes in their buffy coat expressed as a proportion of the number of cattle 
screened. 
 
2.2.5 Statistical analysis 
All data were analysed using the statistical software GraphPadInstat (version 3.00, 2003). 
Tsetse fly relative abundance was expressed as the apparent density (AD) of each species, 
i.e. the number of flies collected per trap per day. For comparison of the relative abundance 
between G. brevipalpis and G. austeni populations, a paired test was used. Data was not 
normally distributed and the nonparametric Wilcoxon matched pairs test was used. To 
evaluate the AD within each tsetse species population a one-way analysis of variance 
(ANOVA) was used to differentiate between the mean tsetse fly AD. The data was not 
23 
 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
normally distributed so a nonparametric method (Kruskal-Wallis test) was used. 
Additionally, Dunn’s multiple comparison tests were used if the P value < 0.05. 
Proportional differences in trypanosome infection prevalence, expressed as the 
number of cattle that had positive trypanosomes in their buffy coat as a proportion of the 
number of cattle screened, were determined with Chi-square (χ2) analysis with the Yate’s 
continuity correction. 
Linear regression analysis was carried out on trypanosome infection prevalence and 
tsetse fly AD, as well as infection prevalence and distances from game reserves. All 
statistical tests were done at the 5% significance level. 
Maps were developed in ArcGIS10.1. For the trypanosome infection prevalence as 
well as the climate data an inverse distance weighted interpolation method was used with 
a power 2 function and a variable search radius setting at 10 points. 
 
2.3 Results 
2.3.1 Tsetse fly distribution and apparent density (AD) 
2.3.1.1 Apparent density (AD) 
The 77 H traps deployed at 18 sites located in the four magisterial districts i.e. Ingwavuma, 
Ubombo, Hlabisa and Enseleni, collected 216 449 G. brevipalpis (AD = 2.64 flies/trap/day) 
and 17 097 G. austeni (AD = 0.21 flies/trap/day) between 1 April 2005 and 31 August 2009 
(Table 2.1; Fig. 2.4 A, B). 
While both tsetse fly species were collected in the three northerly districts, only 
G. brevipalpis was trapped (with six H traps) at Ocilwane in the northern part of the 
southerly Enseleni district (Table 2.1; Fig. 2.4 A). The overall AD of G. brevipalpis (2.64 
flies/trap/day) was significantly higher (P < 0.01) than that of G. austeni (0.21 flies/trap/day). 
Comparison for the three northerly districts indicated that the AD of G. brevipalpis was 
significantly higher than that of G. austeni in the Ingwavuma (P = 0.01) and Hlabisa 
(P < 0.01) districts (Table 2.1; Fig. 2.4 A, B). The AD of G. brevipalpis (0.03 flies/trap/day) 
in the Ubombo district was significantly lower than that of G. austeni (0.33 flies/trap/day) 
(P < 0.01) (Table 2.1). 
Glossina brevipalpis was most abundant in the Hlabisa district (3.76 flies/trap/day), 
and the AD was significantly higher (P < 0.01) than that of the Ubombo district (0.03 
flies/trap/day); an area where previously no G. brevipalpis had been collected (Kappmeier 
Green, 2002) (Table 2.1; Fig. 2.4 A). 
Significantly higher (P = 0.03) numbers of G. austeni (AD = 0.33 flies/trap/day) were 
collected in the Ubombo district as compared to the Hlabisa (0.02 flies/trap/day) and the 
Ingwavuma districts (0.04 flies/trap/day) (Table 2.1). No G. austeni was trapped at Ocilwane 
24 
 
Chapter 2: Tsetse and Trypanosomosis 
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in the Enseleni district (Table 2.1; Fig. 2.4 B). The trapping data indicated that the Monzi 
forest in the Hlabisa district was the most southerly distribution of G. austeni in KwaZulu-
Natal (and Africa). 
The greatest variations in ADs between sites within the same magisterial district 
(Table 2.1) were observed in the Hlabisa district for both G. brevipalpis and G. austeni 
(P < 0.01 for both species). The AD of G. brevipalpis in the Hluhluwe-iMfolozi Park (10.74 
flies/trap/day) was significantly higher than that seen at Kuleni (0.17 flies/trap/day). There 
were also significant differences in AD of the G. brevipalpis populations of St Lucia (7.25 
flies/trap/day) and Kuleni (0.17 flies/trap/day). Glossina austeni was most abundant at 
Boomerang (0.78 flies/trap/day), and this AD was significantly higher than that of 0.02 
flies/trap/day obtained in the Hluhluwe-iMfolozi Park. In the Ubombo district the AD of 
G. brevipalpis at False Bay Park (1.55 flies/trap/day) was significantly higher (P = 0.04) than 
that at Phinda (0.06 flies/trap/day) and Lower Mkhuze (0.06 flies/trap/day). The AD of 
G. austeni was significantly higher at False Bay Park (0.60 flies/trap/day) (P < 0.01) in the 
Ubombo district as compared with the AD in Mkhuze (0.01 flies/trap/day). Finally, in the 
Ingwavuma district there were no significant differences in relative abundances between 
individual sites for both G. brevipalpis (P = 0.11) and G. austeni (P = 0.11). 
 
2.3.1.2 Tsetse fly distribution 
The updated distribution of G. brevipalpis and G. austeni, taking only absence and 
presence into account, as determined with H traps during the last 10 years, from 2005 to 
2015, is presented in Fig. 2.5. 
The most obvious differences in the present survey results, compared to those of the 
odour baited XT sticky traps (Kappmeier Green, 2002) (Fig. 2.2), were that G. brevipalpis 
was collected in the Ubombo district and G. austeni in the Hluhluwe-iMfolozi Park (Fig. 2.5). 
Additionally, G. brevipalpis was collected at sites south of the Umfolozi River previously 
considered the southernmost distribution of tsetse flies in South Africa (Kappmeier Green, 
2002). This is an indication that the most southern limit of G. brevipalpis is still not clearly 
defined. The present distribution data is, however, in agreement with the prediction maps 
of Hendrickx et al. (2002).
25 
 
 
Chapter 2: Tsetse and Trypanosomosis 
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Table 2.1. Tsetse H trap collections of Glossina brevipalpis and Glossina austeni from April 2005 to April 2009 at 18 sites in north eastern 
KwaZulu-Natal. 
G. brevipalpis G. austeni 
No. on Number Total no. of Total Apparent Total Apparent 
 map* Tsetse fly sample site  of traps  Start date End date trap days collected density collected density 
Ingwavuma          
 1 Ndumu 1 14-06-2006 26-08-2009 1169 6358 5.44 210 0.18 
 2 Tembe 4 14-06-2006 26-08-2009 4606 1529 0.33 135 0.03 
 3 Pelani 1 14-06-2006 26-08-2009 1169 11 0.01 0 0.00 
 4 Kosi Bay 2 19-04-2006 26-08-2009 2262 444 0.20 1 <0.01 
Total/District 8 14-06-2006 26-08-2009 9206 8342 0.91 346 0.04 
Ubombo          
 5 Tshongwe 2 04-07-2006 25-08-2009 2275 0 0.00 7 <0.01 
 6 Mbazwana 2 14-06-2006 11-08-2009 1750 0 0.00 263 0.15 
 7 Lower Mkhuze 3 14-06-2006 25-08-2009 3504 204 0.06 2022 0.58 
 8 Mkhuze 2 25-10-2006 21-08-2009 2062 1 <0.01 23 0.01 
 9 Phinda 4 04-07-2006 25-08-2009 4550 261 0.06 2284 0.50 
Total/District 13 14-06-2005 25-08-2009 14 141 466 0.03 4599 0.33 
Hlabisa          
 10 Kuleni 3 01-01-2006 21-08-2009 3984 697 0.17 856 0.21 
 11 False Bay Park 4 01-04-2005 18-08-2009 6400 9894 1.55 3849 0.60 
 12 Ekuphinidisweni 6 01-05-2006 23-08-2009 5371 8529 1.59 0 0.00 
 13 Hluhluwe-iMfolozi Park 15 01-11-2005 19-08-2009 11 201 120 313 10.74 277 0.02 
 14 Mvutshini 12 01-05-2005 18-08-2009 15 239 32451 2.13 65 <0.01 
 15 Hlambanyathi 1 01-03-2006 31-08-2009 1279 28 0.02 1 <0.01 
 16 Boomerang 6 01-04-2005 03-08-2009 8199 11 627 1.42 6424 0.78 
 17 St Lucia 3 23-10-2006 21-08-2009 3099 22 470 7.25 680 0.22 
Total/District 50 01-04-2005 31-08-2009 54 772 206 009 3.76 12 152 0.22 
Enseleni          
 18 Ocilwane 6 01-11-2007 24-08-2009 3882 1632 0.42 0 0.00 
Total/District 6 01-11-2007 24-08-2009 3882 1632 0.42 0 0.00 
Total/Study area 77 01-04-2005 31-08-2009 82 001 216 449 2.64 17 097 0.21 
*The number on map refers to Fig. 2.4. 
 
 26 
 
Chapter 2: Tsetse and Trypanosomosis 
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26 
Chapter 2: Tsetse and Trypanosomosis 
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27 
 
Fig. 2.4. Tsetse fly apparent density (AD: flies/trap/day) for Glossina brevipalpis (A) and Glossina austeni (B) from April 
2005 to August 2009. 
 
Chapter 2: Tsetse and Trypanosomosis 
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Fig. 2.5. Updated distribution of Glossina brevipalpis and Glossina austeni as 
determined with odour baited XT sticky traps (1993 - 1999) and odour baited H traps 
(2005 – 2015) surveys. 
 
 
28 
 
Chapter 2: Tsetse and Trypanosomosis 
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2.3.2 Environmental factors affecting tsetse fly distribution 
2.3.2.1 Vegetation 
Fly collections made with a subset of H traps, from July 2008 to August 2009, were used to 
correlate tsetse fly AD with vegetation classes at each trapping site. In these subsets 75 
traps collected 73 078 G. brevipalpis and 4011 G. austeni over one-year of sampling. 
The highest ADs for both G. brevipalpis (4.60 flies/trap/day) and G. austeni (0.32 
flies/trap/day) were found in the dense dry forest vegetation class. The second highest AD 
of G. brevipalpis was found in the shrub savannah (2.14 flies/trap/day) followed by the 
savannah woodland (1.99 flies/trap/day) and the herbaceous savannah (1.57 
flies/trap/day). The AD of G. brevipalpis was the lowest in the swamp (0.93 flies/trap/day) 
class. The ADs of G. brevipalpis were significantly higher (P = 0.05) for the dense dry forest 
than the savannah woodland classes. 
The AD for G. austeni was significantly different (P = 0.02) between the five 
vegetation classes, particularly between the dense dry forest (0.32 flies/trap/day) with was 
higher than the shrub savannah (0.07 flies/trap/day). After the dense dry forest, G. austeni 
was most abundant in the swamps (AD = 0.29 flies/trap/day), followed by the savannah 
woodlands (0.13) and the shrub savannah (0.07). The lowest AD (0.001 flies/trap/day) was 
recorded in the herbaceous savannah.  
These AD results seem to indicate that G. austeni is less adaptable to different 
vegetation types and that this fly was mainly restricted to denser vegetation e.g. dry forest. 
This difference in adaptation to vegetation classes of G. brevipalpis and G. austeni can be 
illustrated by the results obtained at four sites. 
At Pelani in the north, located 4 km from the Ndumo Game Reserve, tsetse fly 
abundance was low and only G. brevipalpis (AD = 0.02 flies/trap/day) was sampled (Fig. 
2.6 A). Dense dry forest was not very abundant in the area. The vegetation mainly consisted 
of swampy areas with shrub savannah, herbaceous savannah and bare ground, with 
patches of savannah woodlands (Fig. 2.6 A). 
Mbazwana (Fig. 2.6 B) was not near any game areas and its vegetation consisted 
mainly of savannah woodlands and shrub savannah with patches of dry forest and 
herbaceous savannah. Dense dry forest was, however, more abundant than at Pelani. Only 
G. austeni was collected with an AD of 0.07 flies/trap/day. 
The third site, False Bay Park (Fig. 2.6 C) is located inside the iSimangaliso Wetland 
Park. Vegetation at this site consisted mainly of savannah woodland with large patches of 
dense dry forests. Both G. brevipalpis (average AD = 1.03; SD = 0.56 flies/trap/day) and 
G. austeni (average AD = 0.53; SD = 0.16 flies/trap/day) were collected and their ADs 
where similar and relatively high. 
29 
 
Chapter 2: Tsetse and Trypanosomosis 
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The most southern site, Hluhluwe-iMfolozi Park, also in a protected area, contained 
mainly shrub savannah, with patches of savannah woodland, dense dry forest and 
herbaceous savannah. Here the average AD (11.14, SD = 8.42 flies/trap/day) of 
G. brevipalpis was much higher than the average AD (0.01, SD = 0.01 flies/trap/day) of 
G. austeni. 
 
2.3.2.2 Climate 
Climate data from the weather stations and tsetse fly ADs, obtained during the July 2008 
to August 2009 surveys, at Kosi Bay, Mkuzi, Kuleni, Bushlands, and St Lucia, are presented 
in Table 2.2. A total of 16 traps collected 15 978 G. brevipalpis and 1345 G. austeni over 
the one-year period at these five sites. 
The highest average maximum temperature (27.5 °C ± 4.2) as well as the lowest 
average minimum temperature (15.6 °C ± 4.5) during this period were recorded at Mkuzi 
located 54 km from the coast (Table 2.2). At the coastal sites, variations in both the average 
temperature and relative humidity were less pronounced (Table 2.2). At the coastal sites of 
Bushlands and St Lucia that had more moderate temperature ranges (Fig. 2.7 A, B; Table 
2.2) and higher relative humidity (Fig. 2.7 C, D; Table 2.2), ADs for both G. brevipalpis and 
G. austeni were high. In contrast, at Mkuzi a site characterised by more extreme variations 
between the average maximum (Fig. 2.7 A) and minimum (Fig. 2.7 B) temperatures and a 
lower relative humidity (Fig. 2.7 C, D) and rainfall (Fig. 2.7 E), relative abundance of 
G. brevipalpis (average AD = 0.001, SD = 0.009 flies/trap/day) was very low (Table 2.2). 
The relationships between tsetse fly AD and average evaporation (Fig. 2.7 F), radiation 
(Fig. 2.7 G) and wind speed (Fig. 2.7 H) were less obvious. 
 
Table 2.2. Weather station data and tsetse fly apparent density (July 2008 to August 2009). 
  Kosi Bay Mkuzi Kuleni Bushlands St Lucia 
Av. Maximum Temperature °C  25.67 27.46 26.82 26.75  26.45 
(SD) (3.15) (4.20) (4.04) (3.52) (3.52) 
Av. Minimum Temperature °C  17.44 15.56 17.03 16.35  17.08 
(SD) (3.86) (4.50) (3.82) (3.74) (4.30) 
Av. Maximum Relative Humidity %  94.47 85.65 89.16 91.12  99.47 
(SD) (4.16) (10.81) (4.05) (4.42) (1.71) 
Av. Minimum Relative Humidity %  61.79 37.68 49.52 51.07 69.03 
(SD) (12.31) (17.63) (14.69) (14.22) (13.09) 
Av. Rainfall mm  2.01 0.64 2.17 1.50 2.31 
(SD) (9.27) (3.51) (10.66) (4.98) (6.54) 
Av. Relative Evapotranspiration mm 3.05 2.89 3.36 2.94 2.89 
(SD) (1.10) (2.59) (1.38) (1.54) (1.31) 
Av. Hourly Wind Speed m/s  2.69 0.84 1.93 2.78 2.26 
(SD) (0.91) (0.35) (0.54) (2.22) (1.17) 
Av. Radiation MJ/m2  14.47 13.07 16.66 13.52 15.83 
(SD) (8.75) (5.20) (6.40) (7.45) (6.71) 
Av. Apparent Density Glossina 0.14 0.001 0.10 2.23 10.87 
brevipalpis (SD) (0.20) (0.009) (0.11) (2.31) (8.07) 
Av. Apparent Density Glossina 0.27 0.41 0.40 
0 0 
austeni (SD) (0.27) (0.41) (0.46) 
30 
 
Chapter 2: Tsetse and Trypanosomosis 
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31 
 
Fig. 2.6. Vegetation class (Bouyer & Guerrini, 2010) maps and satellite images of four tsetse fly collection sites in north eastern 
KwaZulu-Natal. A: Pelani, B: Mbazwana, C: False Bay Park, E: Hluhluwe-iMfolozi Park (Google earth, 2013a, b, c; 2014). The average 
AD (SD) for each species, expressed as flies/trap/day are indicated for each site. 
 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
32 
 
Fig. 2.7. Inverse distance weighted interpolation of environmental data collected at weather stations in north eastern KwaZulu-Natal 
from July 2008 to August 2009 (A: Av. Maximum Temperature (°C), B: Av. Minimum Temperature (°C), C: Av. Maximum Relative 
Humidity (%), D: Av. Minimum Relative Humidity (%), E: Av. Rainfall (mm), F: Av. Relative Evapotranspiration (mm), G: Av. Radiation 
(MJ/m2) and H: Av. Hourly Wind Speed (m/s)). 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
2.3.3 Tsetse fly and Trypanosomosis association 
2.3.3.1 Trypanosome infection at dip tanks 
Blood samples collected from 1034 cattle at 25 dip tanks in the tsetse-infested area were 
examined microscopically for trypanosomes (Table 2.2). The number of cattle screened per 
dip tank ranged from 27 at Ekuphindisweni to 54 at Boomerang (Table 2.2). 
Data of trypanosome prevalence (Table 2.2) were, as for the tsetse fly data, grouped 
per magisterial district (Table 2.1). The results showed that trypanosome infection was 
widespread in north eastern KwaZulu-Natal with infected cattle found at 21 (84.0%) of the 
25 dip tanks surveyed (Table 2.2; Fig. 2.8). Trypanosome prevalence ranged from 20% in 
the Ubombo district to 3% for the Enseleni district and did not differ significantly (P = 0.05, 
χ2 = 7.91, d.f. = 3) between the four magisterial districts. Notwithstanding the absence of 
significant differences between the four districts, the proportional representation of positive 
animals at 25 individual dip tanks differed significantly (P < 0.01, χ2 = 173.30, d.f. = 24). 
Differences in the proportion of positive animals per dip tank were also significant within the 
districts of Hlabisa (P < 0.01, χ2 = 51.462, d.f. = 6), Ingwavuma (P < 0.01, χ2 = 59.078, 
d.f. = 9) and Ubombo (P < 0.01, χ2 = 51.211, d.f. = 6). These proportional differences in 
infection prevalence between individual dip tanks seemed to depend on the distance of the 
dip tank from a protected area or game reserve. 
The highest prevalence of 48% was recorded at the Mseleni dip tank situated next to 
the northern parts of iSimangaliso Wetland Park in the Ubombo district (Table 2.2). 
Similarly, trypanosome prevalence in cattle was high at dip tanks close to protected areas 
and/or game parks (Fig. 2.6). Especially in the Hlabisa district, a positive linear regression 
(r2 = 0.62, P = 0.04) was evident between trypanosome prevalence and the distance 
between the dip tank and the protected areas/game parks. This same trend, although no 
statistically significant (Ingwavuma: r2 = 0.22, P = 0.17 and Ubombo: r2 = 0.19, P = 0.41) 
was seen in the other two districts. The correlation between infection prevalence and 
distance from the game parks was further epitomised by the absence of infected animals 
at the two dip tanks (Ntabayengwe and Makhathini) located more than 20 km from a game 
park (Table 2.2). 
 
2.3.3.2 Tsetse fly abundance and Trypanosomosis infection rate correlations 
The trypanosome prevalence in cattle, determined with the buffy coat technique was 
correlated with the tsetse fly relative abundance, as determined with odour-baited H traps. 
Only dip tanks (N = 18) that had H traps deployed within a 12 km radius were selected for 
the regression analyses (Table 2.2). No linear correlation was found between trypanosome 
prevalence and the relative abundance of either G. brevipalpis (r2 = 0.01, P = 0.68), 
33 
 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
G. austeni (r2 = 0.05, P = 0.40) or the total tsetse catches (r2 = 0.04, P = 0.43). However, at 
some of the dip tanks with high trypanosome prevalence in the cattle, e.g. Mseleni (48%) 
and Mkhumbikazana (37%) in the Ubombo district, the AD for G. austeni was relatively high 
in the absence of G. brevipalpis (Fig. 2.4 A, B). In contrast, at the Ekuphindisweni dip tank 
in Hlabisa where screened cattle showed a similarly high infection prevalence (37%), the 
relative abundance of G. brevipalpis was high, in the absence of G. austeni (Fig. 2.4 A, B). 
The relative abundance of both G. brevipalpis and G. austeni was relatively high at the dip 
tank on the commercial farm Boomerang located west of and next to the southern extent of 
the iSimangaliso Wetland Park and was also where the highest trypanosome prevalence 
(44%) in the Hlabisa district was recorded. 
 
Fig. 2.8. Inverse distance weighted interpolation of trypanosome infection rate at dip tanks 
in north eastern KwaZulu-Natal (1: Ekuhlehleni, 2: Khume, 3: Ndumo, 4: Manzibomvu, 5: 
Nhlanjwana, 6: Thengani, 7: Ntabayengwe, 8: Ngwenyambili, 9: Phelendaba, 10: Pongola, 
11: Makhathini, 12: Mseleni, 13: Mpini, 14: Mkhumbikazane, 15: Mbazwana, 16: Zineshe, 
17: Khipha, 18: Nibela, 19: Nthwati, 20: Ekuphindisweni, 21: Mvutshini, 22: Qakweni, 23: 
Mahlambanyathi, 24: Boomerang, 25: Ocilwane). 
34 
 
 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
Table 2.3. Trypanosome infection rates in cattle from November 2005 to November 2009 in north eastern KwaZulu-Natal. The blood was collected 
from cattle at dip tanks and the infection rate was determined by the buffy coat technique. 
 Distance from Game Buffy coat examination Trypanosome infection No. on map* Dip tank  Dip tank sample date 
Park (km) Positive Negative prevalence  
Ingwavuma       
 1 Ekuhlehleni 25-05-2007 11.6 1 49 0.02 
 2 Khume 01-06-2007 9.4 0 30 0.00 
 3 *Ndumo 30-05-2007 3.4 4 39 0.09 
 4 *Manzibomvu 05-09-2007 0.3 18 32 0.36 
 5 *Nhlanjwana 31-05-2007 2.6 11 31 0.26 
 6 Thengani  17-05-2007 2.1 4 35 0.10 
 7 Ntabayengwe  29-05-2007 26.9 0 38 0.00 
 8 *Ngwenyambili 14-07-2006 7.5 11 21 0.34 
 9 *Phelendaba 15-05-2007 8.2 7 31 0.18 
 10 *Pongola 06-08-2007 3.3 0 30 0.00 
Total/District    56 336 0.14 
Ubombo       
 11 Makhathini 06-07-2007 20.2 0 32 0.00 
 12 *Mseleni 05-08-2007 1.9 22 24 0.48 
 13 Mpini 05-11-2007 2 1 41 0.02 
 14 *Mkhumbikazare 05-10-2007 7 18 31 0.37 
 15 *Mbazwana 05-07-2007 4.3 4 34 0.11 
 16 *Zineshe 06-04-2007 2.2 9 33 0.21 
 17 *Nibela 27-01-2007 3 3 30 0.09 
Total/District    57 225 0.20 
Hlabisa       
 18 Khipha 23-11-2006 7.3 2 34 0.06 
 19 *Nthwati 15-02-2006 6 1 38 0.03 
 20 *Ekuphindisweni 25-10-2006 3 10 17 0.37 
 21 *Mvutshini 21-05-2007 3 7 27 0.21 
 22 *Qakweni 15-08-2006 7.4 8 83 0.09 
 23 *Mahlambanyati 30-11-2005 9.6 5 45 0.10 
 24 *Boomerang 18-08-2006 9.5 24 30 0.44 
Total/District    57 274 0.17 
Enseleni       
 25 *Ocilwane 09-07-2007 4.3 1 28 0.03 
Total/District       1 28 0.03 
Total/Study area    171 863 0.17 
*Dip tanks used for trypanosome infection and tsetse fly numbers regression analysis. The number on map refers to Fig. 2.8. 
 35 
 
Chapter 2: Tsetse and Trypanosomosis 
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35 
Chapter 2: Tsetse and Trypanosomosis 
_________________________________________ 
 
2.4 Discussion 
Baseline data on tsetse fly distribution, relative abundance and species composition, as 
well as the severity of the Trypanosomosis problem are essential in developing an 
appropriate cost effective, sustainable area-wide control strategy (Vreysen 2005; Leak et 
al., 2008; Shaw, 2009). Previous surveys, using odour baited XT sticky traps, showed that 
G. brevipalpis was restricted to two distinct bands in the north and south of the area in 
KwaZulu-Natal (Kappmeier Green, 2002) (Figs. 2.2 & 2.5). These two bands, except for a 
few patches predicted centrally, were also more or less defined in the distribution prediction 
model for this species (Hendrickx et al., 2003; Kappmeier Green et al., 2007). In the present 
survey, the H trap sampled low numbers of G. brevipalpis in at least five sites in the central 
area east of the Mkuzi Game Reserve and north of the southernmost previously defined 
distribution band (Kappmeier Green, 2002) (Fig. 2.5). Therefore, these positive catches 
could partly be used to validate the prediction model of Hendrickx (Hendrickx et al., 2003) 
showing that G. brevipalpis is also present in the central and southern parts of the Ubombo 
district, where it was previously not found. Whether this distribution is indeed patchy or 
continuous still needs to be verified. 
During the 1993-1999 surveys (Kappmeier Green, 2002), G. austeni was sampled at 
the Hluhluwe Dam east of Hluhluwe-iMfolozi Park (Figs. 2.2 & 2.5). During the present 
surveys all traps inside the Hluhluwe-iMfolozi Park adjacent to the Hluhluwe Dam found 
G. austeni at very low AD (0.02 flies/trap/day) (Table 2.1; Fig. 2.4 B). The presence of 
G. austeni along this transect confirmed the accuracy of the G. austeni distribution 
prediction model of Hendrickx et al. (2003). Glossina brevipalpis was also trapped, but this 
was expected considering that it was previously collected in and outside the Hluhluwe-
iMfolozi Park (Kappmeier Green, 2002). Taking into account that G. austeni is primarily 
restricted to and does not disperse far from dense vegetation (Esterhuizen et al., 2005; 
Esterhuizen, 2007), and that bush clearing in the communal farming areas has increased 
considerably (Kappmeier et al., 1998), it is likely that game reserves and protected areas 
will become more important in sustaining G. austeni populations in the future. 
The H traps seemed to be more effective than the XT sticky trap as both species were 
collected in areas previously considered negative. However, caution is required in 
interpreting trap efficiencies if traps are not evaluated simultaneously. In addition, land use 
and the vegetation cover might have changed considerably over a period of ten years, 
which could have significantly influenced the distribution and abundance of tsetse flies. 
The 1993 to 1999 survey indicated the southernmost extent of the Umfolozi River 
(- 28.5204, 32.3123) as the most southerly distribution of G. brevipalpis (Kappmeier Green, 
2002). Glossina austeni was not trapped south of the St Lucia estuary, where the Umfolozi 
36 
 
Chapter 2: Tsetse and Trypanosomosis 
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River enters the lake (Kappmeier Green, 2002). The tsetse fly prediction model of Hendrickx 
et al. (2003) suggested a very low probability of occurrence for G. austeni south of the 
Umfolozi River, but a much higher probability of G. brevipalpis occurring. During the survey 
conducted in 2015 G. brevipalpis was collected at a communal farm 10 km south of the 
southern border of the Hluhluwe-iMfolozi Park - an area which had not previously been 
surveyed (Fig. 2.5). This indicates that the most southern limit of G. brevipalpis remain 
undefined. Available data indicates the southern limit for G. austeni to be the Umfolozi 
River, as was previously assumed (Kappmeier Green, 2002). Similarly, the most western 
extent of the distribution of each species still needs to be verified. Available trap catches 
indicate that the western distribution limit of G. austeni was -28.10357, 32.08377 (in the 
Hluhluwe-iMfolozi Park), whereas G. brevipalpis was collected much further west at -
28.50192, 31.881, i.e. west of the Hluhluwe-iMfolozi Park (Fig. 2.5). 
In agreement with previous studies, vegetation and climate do play a noticeable role 
in determining the distribution and abundance of both G. brevipalpis and G. austeni 
(Kappmeier Green, 2002; Esterhuizen et al., 2005; Esterhuizen, 2007). Preliminary data 
presented in the current study indicates that vegetation and climate cannot always fully 
explain the distribution patterns of both species. This indicates that factors such as 
differential fly mobility as discussed for G. brevipalpis and G. austeni by Esterhuizen (2007) 
or host preference and availability may also play an important role. Despite the presence 
of suitable vegetation and climate in most parts of the area high relative abundance of 
G. brevipalpis was closely linked to the vicinity of protected game areas, where important 
hosts of G. brevipalpis such as hippopotamus, elephant and buffalo were present (Wetzel 
& Glasgow, 1956; Moloo, 1993; Clausen et al., 1998). In contrast, G. austeni, albeit only in 
low numbers, was also found in areas were only small game such as bush pig and duikers, 
which are known hosts for this species (Wetzel & Glasgow, 1956; Moloo, 1993; Clausen et 
al., 1998), were present. Notwithstanding the apparent host preferences of these two 
species, it was shown that both will also feed on cattle (Moloo, 1993) and that cattle will 
probably be able to sustain tsetse fly populations in the absence of game. 
It is, however, clear that the presence and abundance of both species will largely 
depend on environmental conditions in the area. Changes in the climate, agricultural 
practises and land use can have a significant and rapid impact on tsetse fly abundance in 
the area. To manage the Trypanosomosis problem successfully it will be essential to 
constantly monitor tsetse fly burdens at selected sites. This will be of special importance 
before the implementation of any proposed area-wide control campaign. 
Trap catches have been used to update the tsetse fly distribution prediction models 
for both G. brevipalpis and G. austeni (Hendrickx, 2007). Since only data up to 2009 are 
37 
 
Chapter 2: Tsetse and Trypanosomosis 
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presented, ongoing and future surveys will have to be processed and incorporated into 
these distribution maps and models. The available trap data have partly validated this 
prediction model, emphasizing that the value of these models as tools for planning tsetse 
fly and Trypanosomosis intervention practices should not be underestimated. Furthermore, 
the new distribution data will need to be taken into account in the tsetse fly control strategy 
proposed by Kappmeier Green et al. (2007), which needs to be modified accordingly. 
Notwithstanding the fragmentary nature of the data, consolidating data from several 
independent studies, trypanosome infections in cattle were recorded at 21 of the 25 dip 
tanks which clearly showed that the disease was still abundant and highly prevalent in north 
eastern KwaZulu-Natal at the time of these surveys (Fig. 2.8). A potential shortcoming of 
the trypanosome survey may have been that infections were not identified to species level. 
It was, however, previously indicated that T. congolense is the dominant species in the area 
(Van den Bossche et al., 2006; Mamabolo et al., 2009; Motloang et al., 2012). 
The data on the spatial distribution of the trypanosome infection in north eastern 
KwaZulu-Natal indicated that the disease was widespread in cattle. This encompasses the 
areas close to the Mozambique border in the north to a dip tank close to the Umfolozi River 
in the south, covering the entire length of the tsetse fly infested area of north eastern 
KwaZulu-Natal (Fig. 2.8). In general, higher infection rates were found in dip tanks located 
near game reserves and protected natural areas, an observation also made by Van den 
Bossche et al. (2006) and Ntantiso et al. (2014) for dip tanks near the Hluhluwe-iMfolozi 
Park. This game-livestock-tsetse fly interface poses a high risk for cattle becoming infected 
not only for the Hluhluwe-iMfolozi Park (Ntantiso et al., 2014), but possibly throughout north 
eastern KwaZulu-Natal. 
In 1994, the highest prevalence of Trypanosomosis was recorded in the Ubombo 
district (Kappmeier et al., 1998). Although in the present study the dip tank (Mseleni) with 
highest incidence of cattle infection (48%) was also found in this district, the overall 
trypanosome prevalence for the district was not significantly different from that of the 
Ingwavuma and Hlabisa districts confirming that Trypanosomosis remains a problem 
throughout the whole area. 
The apparent absence of a significant linear correlation between trypanosome 
prevalence and the relative abundance of tsetse flies in north eastern KwaZulu-Natal may 
indicate that a number of factors, such as the tsetse fly and Trypanosomosis monitoring 
regimes used (a trap placement radius of 12 km could be too large), could influence the 
outcome of such a regression. This can also partly be attributed to the co-existence of the 
two tsetse species, each with a different vectorial capacity and/or competence. Previous 
studies indicated that the vector competence of G. austeni for T. congolense, the most 
38 
 
Chapter 2: Tsetse and Trypanosomosis 
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abundant Trypanosoma species in north eastern KwaZulu-Natal, was significantly higher 
than that of G. brevipalpis (Motloang et al., 2012). Our data seems to corroborate this 
greater vector competence of G. austeni in view of the relatively high trypanosome 
prevalence recorded in cattle at the Mseleni dip tank where only G. austeni flies were 
sampled (Mbazwana AD = 0.15 flies/trap/day). However, cattle at the Ekuphindisweni dip 
tank showed a similar high trypanosome prevalence, but G. austeni was not sampled and; 
only G. brevipalpis was trapped in relatively high numbers (Ekuphindisweni AD = 1.59 
flies/trap/day) indicating that G. brevipalpis was most likely responsible for transmission in 
this area. In general, high apparent densities of G. brevipalpis in the vicinity of dip tanks 
resulted in trypanosome infection rates that were as high as those found at dip tanks where 
G. austeni was present but at low apparent densities. Hendrickx et al. (2003) also noted 
that low G. austeni densities coincided with areas of higher Trypanosomosis prevalence in 
the northern communal areas. However, the very high ADs of G. brevipalpis (on average 
12.6 times higher than that of G. austeni) and the much lower trap catches of G. austeni 
might be resulting from the intrinsic biases of the trapping system. It is known that G. austeni 
responds relatively poorly to traps (Kappmeier, 2000) and is not attracted to any of the 
odours (Kappmeier & Nevill, 1999a; Kappmeier Green, 2002) used to bait the traps. 
Essentially, mark-release-recapture studies will be needed to confirm the low efficacy of the 
sampling tool used for G. austeni. 
If the relative abundance of G. brevipalpis is indeed a reflection of actual population 
densities, it can be hypothesised that the high trypanosome infection prevalence in certain 
areas might be the result of the greater densities of G. brevipalpis despite their lower vector 
competence. The relative low vector competence of G. brevipalpis previously found in 
South Africa has probably resulted in an underestimation of the importance of G. brevipalpis 
in the epidemiology of nagana in north eastern KwaZulu-Natal. Vector competence studies 
in the laboratory have shown that the susceptibility of G. brevipalpis for T. congolense can 
be as high as 12.3% (Moloo et al., 1998). In Uganda it was shown that the infection rates 
in field collected G. brevipalpis could be 2.6% (Harley 1967a, b; Moloo et al., 1980). In 
addition, trypanosome infection rates in tsetse flies are not constant and can change over 
time and between populations. In Uganda infection rates varied considerably between 
seasons and with the age of the G. brevipalpis population (Harley 1966; 1967a, b). 
Both G. brevipalpis and G. austeni will readily feed on cattle (Moloo, 1993; Clausen 
et al., 1998) and can therefore play a role in the transmission of Trypanosomosis in 
KwaZulu-Natal. Similar average trypanosome infection rates in the three northerly districts, 
even though the relative abundance and distribution of G. austeni and G. brevipalpis were 
clearly different, seem to support this hypothesis. 
39 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
Chapter 3 
 
Comparison of geometric morphometric markers between tsetse populations of 
South Africa, southern Mozambique and Swaziland 
 
3.1 Introduction 
Due to the debilitating impact of African trypanosomoses, effective management of tsetse 
flies is gaining increased importance throughout its distribution area in Africa. It has been 
suggested that eradication of selected tsetse fly populations may in some cases be of 
greater economic benefit than long-term suppression (Kaba et al., 2012). Tsetse fly 
suppression needs to be done continuously, because tsetse fly populations may recover if 
suppression is stopped, due to surviving flies (Hargrove, 2003; Patterson & Schofield, 
2005). Knowledge of the structure of the targeted tsetse fly population and the size of the 
geographical area that needs to be covered is very important, as this will determine the 
most suitable control strategy (Patterson & Schofield, 2005). The degree of geographic and 
genetic isolation of the targeted population is extremely important as it will indicate the 
reinvasion potential from neighbouring populations into the target area (Solano et al., 2010). 
Eradication will only be feasible and sustainable if the targeted population represents 
a geographically well-defined or genetically isolated population, or if a rolling carpet 
approach is implemented (Hendrichs et al., 2005). The rolling-carpet or wave-principle 
approach is dynamic as the basic operational phases (pre-intervention, population 
reduction, release of sterile insects and maintenance of pest status) are carried out 
simultaneously in a phased manner (Hendrichs et al., 2005). 
An eradication strategy was successfully implemented on several geographically 
isolated islands off the coast of Africa e.g. Glossina pallidipes was eradicated on São Tomé 
and Príncipe in 1914 (Leak, 1999) and Glossina austeni was eradicated from Unguja Island, 
Zanzibar between 1993 and 1997 (Vreysen et al., 2000). An isolated population of 
G. pallidipes was successfully eradicated in South Africa and southern Mozambique after 
the implemantaion of an eradication strategy from 1945 to 1951 (Du Toit, 1954). It was also 
reported that Glossina tachinoides, Glossina palpalis palpalis and Glossina morsitans 
submorsitans were eradicated from an area of over 200 000 km2 in northern Nigeria in 
1955-1978 (Davies & Blasdale, 1960) using a rolling carpet approach, anecdotal data 
indicating that these riverine species might have reinvaded this, however, is not confirmed.  
On mainland Africa, determining the geographic limits of tsetse fly populations and 
the degree of genetic interactions between them remains challenging, even more so if 
abundance is low. The difficulty in determining the most southerly distribution of 
40 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
Glossina brevipalpis and G. austeni in South Africa as discussed in Chapter 2 serves as an 
example. 
In South Africa tsetse flies are distributed from the Enseleni district (G. brevipalpis) or  
the Hlabisa district (G. austeni) to the border with Mozambique (Fig. 2.4), where they extend 
into the Matutuine district of Maputo province in southern Mozambique (Fig. 3.1) (Sigauque 
et al., 2000; Mulandane, 2013). In southern Mozambique both these species are 
continuously distributed up to the northern border of the Reserva Especial de Maputo and 
no genetically isolated pockets could be identified (Mulandane, 2013). In the absence of 
G. brevipalpis, G. austeni flies were collected in the Mlawula Nature Reserve in Swaziland 
close to the Mozambique border (Fig. 3.1) (Saini & Simarro, 2008). 
Previous surveys in South Africa showed G. brevipalpis to be restricted to two distinct 
bands in the north and south of north eastern KwaZulu-Natal (Du Toit, 1954; Kappmeier 
Green, 2002). As indicated in Chapter 2, low numbers of G. brevipalpis were recently found 
in at least five sites between these two defined bands. It has now become evident that 
G. brevipalpis, as well as G. austeni, is distributed more or less continuously throughout the 
area. However, due to the relative low abundance of G. brevipalpis in the Ubombo district 
(Table 2.1; Fig. 2.5 A) it might well be that the two pockets found in the north and south of 
the tsetse fly infected area could be both geographically and genetically isolated. The very 
low abundance of G. austeni and their relatively sedentary behaviour as well as their 
association with specific vegetation types (Chapter 2) that have a patchy distribution in the 
target area (Esterhuizen, 2007) may favour the creation of genetically isolated localised 
populations. Despite the fact that G. austeni, and perhaps also G. brevipalpis, are 
distributed throughout north eastern KwaZulu-Natal, populations are restricted to pockets 
of dense vegetation (Esterhuizen, 2007). It has been shown that habitat fragmentation 
creates conditions to which tsetse fly populations respond physiologically and 
demographically thereby affecting tsetse-trypanosome interactions and hence influencing 
Trypanosomosis risk (Mweempwa et al., 2015). 
Molecular and/or morphometric markers can be used to determine possible gene flow 
between subpopulations as an indirect measure of fly dispersal (Solano et al., 2000; 
Gooding & Krafsur, 2005; Camara et al., 2006; Bouyer et al., 2007; 2009; Solano et al., 
2009). Geometric wing morphometry has proven to be a reliable and inexpensive 
alternative to the more expensive techniques of DNA-sequence analysis (Patterson & 
Schofield, 2005; Kaba et al., 2012). Using geometric wing morphometry as well as 
microsatellite markers, the Glossina palpalis gambiensis populations that inhabit the Loos 
Islands 5 km off the coast of mainland Guinea, were shown to be genetically isolated from 
two sites in mainland Guinea (Camara et al., 2006). Another example is the 
41 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
G. p. gambiensis population of the Niayes in Senegal that was shown to be isolated from 
the main tsetse fly belt 120 km away in the eastern part of the country (Solano et al., 2010). 
Geometric morphometrics is a simple and cost effective tool that can easily be used 
in any laboratory to study tsetse fly population structure (Patterson & Schofield, 2005). 
Some authors , e.g. Solano et al. (2010) have indicated that genetic analysis is more reliable 
than morphometrics, as the correlation between these two techniques is only 0.5 and that 
morphometric analysis is probably influenced by environmental factors. It is hypothesised 
that morphometrics can be used as an easy first level of screening for possible population 
fragmentation as no specialised equipment is needed for this analysis. In the present study, 
the applicability of phenetic (geometric morphometrics) markers to differentiate between 
the two tsetse species found in South Africa was determined. Geometric morphometrics 
were subsequently used to assess the extent of the potential genetic isolation between the 
different populations of G. brevipalpis and G. austeni. The possible influence of seasonal 
variation on the reliability of geometric morphometrics was also determined. In addition, the 
South African populations were compared with flies collected in the neighbouring 
Mozambique and Swaziland. Lastly, these wild populations were compared with laboratory 
colonies maintained at the Agricultural Research Council-Onderstepoort Veterinary 
Institute (ARC-OVI), established from seed material from Tanzania and Kenya respectively. 
The level of genetic isolation, indicated by the phonetic markers, between these different 
populations within South Africa and between the three countries will indicate the invasive 
potential of flies from neighbouring areas into the controlled or tsetse fly free areas and will 
facilitate in selecting the most appropriate control strategy. 
 
3.2 Materials and methods 
3.2.1 Study sites and fly collection 
Glossina brevipalpis and G. austeni were collected from July 2008 to September 2009 at 
10 sites in South Africa and at one site each in southern Mozambique and Swaziland 
(Fig. 3.1) with odour-baited H traps as described in Chapter 2. In Swaziland the site (# 1) 
was in the Mlawula Nature Reserve (Fig. 3.1) and the vegetation consists mainly of shrubs 
and herbaceous savannah with very small patches of dry forest and savannah woodland 
(Bouyer & Guerrini, 2010). In southern Mozambique, the site (# 2) was in the Reserva 
Especial de Maputo (Fig. 3.1) where the vegetation was mainly dry forest and shrub 
savannah with small patches of swamp and herbaceous savannah (Bouyer & Guerrini, 
2010). Sites three to twelve were in South Africa (Fig. 3.1) and are described in detail in 
Chapter 2. At three of these sites G. brevipalpis (Ndumu (# 3) and St Lucia (# 12)) and 
G. austeni (Phinda (# 8) and St Lucia (# 12)) was collected monthly to assess the effect of 
42 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
seasonal variation on geometric morphometrics results. Flies collected from June to August 
were grouped as the winter collection, from September to November as spring, from 
December to February as summer and from March to May as an autumn collection. 
 
 
Fig. 3.1. Sites where Glossina brevipalpis and Glossina austeni were collected with odour-
baited H traps for comparative geometric morphometrics in Swaziland (1: Mlawula Nature 
Reserve), Mozambique (2: Reserva Especial de Maputo) and South Africa (3: Ndumu; 4: 
Tembe, 5: Kosi Bay, 6: Mbazana, 7: Lower Mkhuze, 8: Phinda, 9: False Bay, 10: Hluhluwe-
iMfolozi Park, 11: Boomerang, 12: St Lucia). 
 
43 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
The geographical distances between sites ranged from 228 km (Mlawula Nature 
Reserve and St Lucia) to 35 km (Lower Mkhuze and Phinda) (Fig. 3.1). Geographic barriers 
can promote genetic isolation, e.g. the Lebombo Mountain range between Swaziland and 
Mozambique and Lake St Lucia between the sites at St Lucia and Boomerang (Fig. 3.1). 
The study area has a subtropical climate as described in Chapter 2. Most of the rain 
is received in the hot season from October to March. However, it also rains during the cold, 
dry season from April to September. The coastal areas receive on average more rain than 
the interior. 
Flies from laboratory colonies of G. brevipalpis and G. austeni were compared to field 
collected flies. The colony flies were maintained under standard colony conditions (23-
24 °C, 75-80% RH and subdued/indirect lighting, 12 h light/12 h dark) (Feldmann, 1994a; 
FAO/IAEA standard operating procedures, 2006). 
 
3.2.2 Morphometric analysis 
For morphometrics analysis tsetse fly wings were removed and dry mounted between two 
microscope slides (Patterson & Schofield, 2005). The wings were photographed using a 
Dino X Lite Digital Microscope. 
Firstly, a comparison between the two South African species was done by comparing 
the right wings of female G. brevipalpis and G. austeni. Secondly, the potential seasonal 
effects and sexual dimorphism were investigated using the right and left wings of both 
sexes. Finally, to assess the degree of isolation of the different populations, only the right 
wings of female flies were used. 
Nine landmarks (Cartesian coordinates) were defined by vein intersections (Fig. 3.2) 
and digitised using the COO program of the CLIC software package (Dujardin et al., 2010). 
All further analyses on the Cartesian coordinates were done using the MorphoJ integrated 
software package (Klingenberg, 2011). 
 
Fig. 3.2. Glossina austeni wing indicating the nine landmarks as defined by vein 
intersections. 
44 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
The Cartesian coordinates were subjected to a generalized Procrustes analysis 
(Rohlf, 1996), and variations in wing shape (partial warps) were determined by Procrustes 
superposition through generalized least squares (Rohlf, 1999). Centroid size was 
determined by the square root of the sum of the squared distances of all landmarks from 
the centroid (Bookstein, 1991). A frequently used insect body size estimator is the length 
of the wing along its largest axis. Furthermore, the relationship between centroid size and 
the traditional wing length showed good correlation (Dujardin, 2011). Centroid size was thus 
used as an estimator of tsetse fly body size. Centroid size was analysed using the statistical 
software GraphPadInstat (version 3.00, 2003). To differentiate between the centroid size of 
the two species an unpaired test was done with a Welch correction that provided a two-
tailed P-value. Additionally, ANOVA was used to assess significant differences between the 
means of the centroid size (P values < 0.05 were considered significant) of the wings from 
flies collected seasonally at the various sites in the study area. As data were normally 
distributed standard (parametric) methods were used, Tukey’s test was applied. Principal 
component analyses of the shape variables were done using MorphoJ (Klingenberg, 2011) 
and provided 14 partial warps. Multivariate regression of partial warps on size was used to 
estimate the residual allometry, and the statistical significance estimated by 10 000-runs 
permutation tests (Klingenberg, 2011). Differences in wing shape were determined by 
Canonical shape dissimilarity. Linear regression was used to determine both Mahalanobis 
distances and geographic distances. The Mahalanobis distances (Mahalanobis,1936) were 
examined for significance by the permutation test (10 000 runs). 
 
3.3 Results 
3.3.1 Species differentiation 
The right wings of 345 female G. brevipalpis and 346 female G. austeni (Table 3.3) were 
analysed for size and shape differences. There was a significant difference (P < 0.01) 
between the average wing centroid size of G. austeni (924 ± 31) and G. brevipalpis 
(1537 ± 47) (Table 3.3), the latter being much larger (Figs. 3.3, 3.4, 3.7 & 3.10). The first 
discriminant factor (shape components) accounted for 80% of the variance which clearly 
indicated a separation of the shape of the wings of G. brevipalpis and G. austeni (Fig. 3.3). 
This provided sufficient evidence that geometric wing morphometry would 
differentiate between two species one of which belonged to the fusca group (G. brevipalpis) 
and the other to the morsitans group (G. austeni). The multivariate regression of the first 
relative warp against centroid size (Fig. 3.3) (100 000 permutation rounds) was significant 
(P = 0.03), which indicates that there is an allometric effect (i.e. the size influences the 
45 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
shape in a significant way) when comparing the wing morphometric of G. brevipalpis and 
G. austeni. 
 
 
 
Fig. 3.3. Multivariate regression of the first partial warp (derived from the shape of the wing) 
on centroid size of the right wings of female Glossina brevipalpis (yellow triangles) and 
Glossina austeni (purple circles). The first partial warp represents 80% of the total 
discrimination. 
 
3.3.2 Seasonal effects and sexual dimorphism 
Seasonal variation and sexual dimorphism in centroid size and shape of the two species 
was determined at Ndumu, Phinda and St Lucia. Glossina brevipalpis was collected from 
Nduma and St Lucia and G. austeni from Phinda and St Lucia. The wings were evaluated 
separately for each species, site and sex. The allometric effect was removed so that size 
and shape could be analysed separately. Table 3.1 summarises the collection dates as well 
as the number of female/male and right/left wings analysed. 
 
3.3.2.1 Centroid size variation 
A seasonal effect on the wing centroid size of males and females of both species was 
observed. At Ndumu, on average the smallest centroid size for males were collected in 
spring (1346 ± 50) and the largest (1409 ± 23) in autumn. There were significant differences 
46 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
in the average centroid size of G. brevipalpis males collected in winter (1380 ± 37) versus 
those collected in spring (1346 ± 50) (P < 0.01) and autumn (1409 ± 23) (P < 0.05). 
Significant differences were also found in the average centroid size for males collected in 
spring (1346 ± 50) versus summer (1367 ± 35) (P < 0.05) and autumn (1409 ± 23) 
(P < 0.01). The average male centroid size was also different between summer (1367 ± 35) 
versus autumn (1409 ± 23) (P < 0.01) (Fig. 3.4 A). 
The centroid size of G. brevipalpis females at Ndumu also differed seasonally. 
Glossina brevipalpis females with the smallest average centroid size were collected in the 
spring (1461 ± 80) and the largest (1541 ± 43) in autumn. The average centroid size for 
females collected in spring were significantly smaller from those collected in all other 
seasons (P < 0.01 in all cases) (Fig. 3.4 A). 
At the second G. brevipalpis collection site, St Lucia, the males with the smallest 
average wing centroid size were from spring (1362 ± 46) and the largest (1449 ± 66) from 
winter (Table 3.1; Fig. 3.4 A). There were also significant differences in the centroid size of 
males collected in winter (1449 ± 66) versus spring (1362 ± 46), summer (1388 ± 31) and 
autumn (1397 ± 28) (P < 0.01 in all cases) as well as spring versus summer (P < 0.05) and 
spring versus autumn (P < 0.01) (Fig. 3.4 A). 
For the G. brevipalpis females collected at St Lucia the average female G. brevipalpis 
wing centroid size ranged from 1514 ± 64 in spring to 1585 ± 43 in autumn (Table 3.1; 
Fig. 3.4 A). There were significant differences in the average centroid size between autumn 
(1586 ± 43) versus winter (1558 ± 64) (P < 0.05), spring (1514 ± 64) and summer 
(1539 ± 49) (P < 0.01), as well as winter versus spring (P < 0.01) (Fig. 3.4 A). 
A similar seasonal effect on the wing centroid size was observed for G. austeni. At 
Phinda the average wing centroid size for G. austeni males ranged from 860 ± 20 in autumn 
to 892 ± 19 in winter and the males collected in winter was significantly smaller from those 
of summer (P = 0.01) (Table 3.1; Fig. 3.4 B). 
For G. austeni females the average wing centroid size ranged from 911 ± 29 in 
autumn to 941 ± 21 in spring and was significantly different in females collected in winter 
(939 ± 25) versus summer (919 ± 21) and autumn (911 ± 29) also spring (941 ± 21) versus 
summer and autumn (P < 0.01 in all cases) (Table 3.1; Fig. 3.4 B). 
At the second G. austeni collection site, St Lucia, the male wing centroid size ranged 
from 850 ± 16 in spring to 885 ± 28 in winter. (Table 3.1; Fig. 3.4 B). There were significant 
differences in the average wing centroid size of males collected in winter (885 ± 28) versus 
spring (850 ± 16) as well as those of spring versus autumn (860 ± 20) (P < 0.05 in both 
cases). 
47 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
The female wing centroid size ranged between 932 ± 20 in summer and 946 ± 29 in 
winter and there were also significant differences in the centroid size of females collected 
in  winter (946 ± 29) compared with summer (932 ± 20) and autumn (933 ± 26) (P < 0.05) 
(Fig. 3.4 B). 
There was a strong sexual dimorphism for both G. brevipalpis and G. austeni. The 
average wing centroid size of males was significantly smaller than that of females in all 
seasons for both species (P < 0.01) (Fig. 3.4 A, B). 
The average centroid size of male G. brevipalpis ranged from 1346 ± 50 at Ndumu in 
the interior to 1449 ± 65 for males at St Lucia on the coast (Table 3.1). Except for autumn 
the average centroid size of males from Ndumu were on average smaller than the males 
from St Lucia, especially in winter (P < 0.01). Similarly, the average centroid size of females 
from Ndumu (1461 ± 80) were on average significantly smaller than those from St Lucia 
(1586 ± 43) (P < 0.01) except in summer (Table 3.1; Fig. 3.4 A). 
These trends were not observed for G. austeni and for both males (P = 0.10) and 
females (P = 0.09). There were no significant size differences in the average centroid size 
between flies of Phinda and St Lucia. The average wing centroid size of males from St 
Lucia (850 ± 16) was slightly smaller than that of those collected at Phinda (892 ± 19) (Table 
3.1; Fig. 3.4 B). The G. austeni females collected at St Lucia (942 ± 29) were on average 
marginally bigger than those of Phinda especially in autumn (911 ± 29) (Table 3.1; Fig. 3.4 
B). 
For both species, the variation in centroid size between localities was similar to the 
seasonal variation in centroid size at each of the sites (Table 3.1, Fig. 3.4 B). 
 
48 
 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
Table 3.1. Summary of number of Glossina brevipalpis and Glossina austeni wings analysed in the seasonal and sexual dimorphism geometric 
morphometric analysis. Multivariate regression of partial warps on size, statistical significance estimated by 10 000-runs permutation tests. 
No. of  wings analysed Shape component variation (%) Average Centroid Size Multivariate regression 
   Female Male Female Male (± SD) (10 000 runs, P value) Collection Date 
Canonical  Canonical  Canonical  Canonical  
Left Right Left Right Variate 1 Variate 2 Variate 1 Variate 2 Female Male Female Male 
G. brevipalpis              
 Ndumu              
  Winter Jul'08 - Aug'08 30 30 30 30 1532 (42) 1380 (37) 
  Spring Oct'08  30 30 30 30 1461 (80) 1346 (50) 76 14 74 16 0.004 <0.001 
  Summer Dec'08 - Jan'09 30 30 19 20 1539 (30) 1367 (35) 
  Autumn Mar'09  10 10 40 40 1541 (43) 1409 (23) 
 St Lucia              
  Winter Jul'08  30 30 30 29 1558 (64) 1449 (66) 
  Spring Aug'08 28 29 30 30 1514 (64) 1362 (46) 75 18 64 25 0.008 0.061 
  Summer Dec'08 - Feb'09 30 29 30 30 1539 (49) 1388 (31) 
  Autumn Apr'09  29 29 30 30 1586 (43) 1397 (28) 
G. austeni              
 Phinda              
  Winter Jul'08 - Aug'08 30 30 9 8 939 (25) 892 (19) 
  Spring Sep'08 - Nov'08 30 30 29 29 941 (21) 872 (37) 64 27 55 28 0.006 0.091 
  Summer Dec'08 - Feb'09 21 20 20 20 919 (21) 864 (23) 
  Autumn Mar'09  9 9 3 3 911 (29) 860 (20) 
 St Lucia              
  Winter Jun'09 - Aug'09 30 30 30 30 946 (29) 885 (28) 
  Spring Sep'08 - Nov'08 30 30 4 4 938 (21) 850 (16) 69 17 66 29 0.625 <0.001 
  Summer Dec'08 - Feb'09 29 30 9 9 932 (20) 870 (12) 
  Autumn Mar'09 - May'09 31 30 31 31 933 (26) 883 (35) 
 
 
49 
 
 
Chapter 3: Geometric Morphometrics 
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49 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
50 
 
Fig. 3.4. Centroid size variations of the wings of male and female Glossina brevipalpis (A) and Glossina austeni (B) according to 
season. Each box shows the group median separating the 25th and 75th quartiles, capped bars indicate maximum and minimum values, 
circles indicating the outliers. (Black: Ndumu G. brevipalpis males; Light purple: Ndumu G. brevipalpis females; Dark blue: St Lucia 
G. austeni (A) or G. brevipalpis (B) males; Dark pink: St Lucia G. brevipalpis (A) or G. austeni (B) females;Green: Phinda G. austeni 
males; Dark purple: Phinda G. austeni females). Boxes followed by a different letter indicate that the sizes were significantly different 
at the 5% level. 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
3.3.2.2 Shape variation 
The first two discriminant factors (shape components) in all the analyses (species, site and 
sex) accounted for more than 83% of the variance (Table 3.1). The multivariate regression 
of the first relative warp against centroid size (100 000 permutation rounds) was significant 
(Table 3.1) in all the analyses except for G. brevipalpis males (P = 0.06) and G. austeni 
females (P = 0.62) collected at St Lucia as well as G. austeni males (P = 0.09) from Phinda 
(Table 3.1). This indicates an allometric effect which could be due to seasonal influences 
but the overall low numbers of G. brevipalpis and G. austeni collected in selected seasons 
(Table 3.1) could have amplified the allometric effect. 
Although there was no significant shape separation between sites as well as season 
for both species some tendencies were observed. The first discriminant factor for 
G. brevipalpis at Ndumu for both males (Fig. 3.5 C) and females (Fig. 3.5 A) indicated a 
shape change between flies collected in autumn versus spring; this trend was absent in the 
G. brevipalpis collected from St Lucia (Fig. 3.5 B, D). Similar shape changes of the first 
discriminant factor between flies collected in autumn versus spring were also observed for 
G. austeni males (Fig. 3.6 C) and females (Fig. 3.6 A) from Phinda as well as for males 
(Fig. 3.6 B) from St Lucia. Furthermore, a second shape change of the first discriminant 
factor of G. austeni males collected at Phinda in winter versus autumn was observed 
(Fig. 3.6 C) but the more pronounced autumn separation at Phinda was most likely due to 
the very low sample size (Table 3.1). No shape separations of both the first and second 
discriminant factor were observed for G. austeni females collected at St Lucia (Fig. 3.6 B). 
Comparisons of spatial versus temporal shape differences at these three sites 
indicated that Mahalanobis distances for G. brevipalpis wings were similar or greater 
between seasons as compared with between sites (Table 3.2). The Mahalanobis distances 
between Ndumu and St Lucia was 1.28 (Table 3.2). At Ndumu the Mahalanobis distances 
between seasons ranged from 1.01 (summer versus autumn) to 2.67 (spring versus 
autumn) (Table 3.2). At St Lucia it ranged from 0.70 (summer versus autumn) to 1.88 
(spring versus autumn). The Procrustes distances showed the same pattern (Table 3.2). 
For G. austeni similar results were seen, but, to a lesser extent. Mahalanobis distances 
were greater between seasons e.g. winter versus autumn (1.90), spring versus autumn 
(2.24) at Phinda and winter versus spring (1.82), spring versus autumn (1.76) at St Lucia 
than between Phinda versus St Lucia (1.47). The Procrustes distances had similar results 
(Table 3.2). 
51 
 
Chapter 3: Geometric Morphometrics 
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Fig. 3.5. The distribution of Glossina brevipalpis female (A, B) and male’s (C, D) wing shape 
in the morhospace defined by the first two Canonical variants, flies were collected from 
Ndumu (A, C) and St Lucia (B, D). 
 
 
Fig. 3.6. The distribution of Glossina austeni female (A, B) and male’s (C, D) wing shape in 
the morhospace defined by the first two Canonical variants, flies were collected from Phinda 
(A, C) and St Lucia (B, D). 
52 
 
Chapter 3: Geometric Morphometrics 
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Table 3.2. Summary of the Mahalanobis and Procrustes distances for the spatial and 
temporal wing shape changes in Glossina brevipalpis and Glossina austeni. 
    
Shape component variation 
Mahalanobis 
    (%) Procrustes distances 
Canonical  Canonical  distances 
(P < 0.01)    
    Variate 2 Variate 2 
G. brevipalpis     
 Spatial     
   Ndumu vs. St Lucia 100 0 1.28 0.004 
 
 Temporal    
  Ndumu 
 
   
   Winter vs. Spring 1.31 0.005 
   Winter vs. Summer 1.46 0.005 
   Winter vs. Autumn 2.20 0.007 78 17 
   Spring vs. Summer 2.13 0.009 
   Spring vs. Autumn 2.67 0.010 
   Summer vs. Autumn 1.01 0.003 
  St Lucia 
   
 
   Winter vs. Spring 1.41 0.005 
   Winter vs. Summer 1.13 0.005 
   Winter vs. Autumn 1.14 0.004 74 18 
   Spring vs. Summer 1.82 0.008 
   Spring vs. Autumn 1.88 0.007 
   Summer vs. Autumn 0.70 0.003 
G. austeni      
 Spatial     
   Phinda vs. St Lucia 100 0 1.47 0.005 
 Temporal 
 
   
  Phinda 
 
   
   Winter vs. Spring 0.94 0.003 
   Winter vs. Summer 1.02 0.004 
   Winter vs. Autumn 1.90 0.009 65 21 
   Spring vs. Summer 1.11 0.004 
   Spring vs. Autumn 2.24 0.010 
   Summer vs. Autumn 1.63 0.008 
  St Lucia 
   
 
   Winter vs. Spring 1.82 0.006 
   Winter vs. Summer 1.31 0.005 
   Winter vs. Autumn 0.89 0.004 70 18 
   Spring vs. Summer 1.13 0.002 
   Spring vs. Autumn 1.76 0.005 
   Summer vs. Autumn 1.22 0.004 
 
3.3.3 Population isolation 
Right wing size and shape of females for both species were analysed to assess the level 
of genetic isolation between populations in South Africa as well as between them and those 
populations of southern Mozambique and the G. austeni population in Swaziland. Table 3.3 
summarises the sites and dates that flies were collected and the number of female right 
wings analysed. The allometric effect was removed so that size and shape could be 
analysed independently. 
 
3.3.3.1 Centroid size 
The average female wing centroid size for G. brevipalpis ranged from 1512 ± 35 collected 
in Hluhluwe-iMfolozi Park to 1568 ± 35 from St Lucia (Table 3.3; Fig. 3.7 A). Significant 
differences were found between the populations of the Hluhluwe-iMfolozi Park (1512 ± 35) 
and southern Mozambique (1550 ± 69) (P < 0.01), Ndumu (1544 ± 38) (P < 0.05), Kosi Bay 
(1549 ± 41) (P < 0.05) and St Lucia (1568 ± 35) (P < 0.01) (Fig. 3.7 A). Significant 
53 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
differences were also seen between the G. brevipalpis population collected in southern 
Mozambique (1550 ± 69) and those of Boomerang (1518 ± 45) (P < 0.05), Tembe 
(1520 ± 40) versus St Lucia (1568 ± 35) (P < 0.01) and Boomerang versus St Lucia 
(P < 0.01) (Fig. 3.7 A). 
The average female wing centroid size of G. austeni ranged from 892 ± 37 collected 
in Swaziland to 940 ± 52 from St Lucia (Table 3.3; Fig. 3.7 A). The average wing centroid 
size for flies from Swaziland (892 ± 37) was significantly smaller (P < 0.01) from that of the 
flies from southern Mozambique as well as the flies from South Africa (Fig. 3.7 B). The only 
other significant difference (P < 0.01) in wing centroid size was observed between female 
G. austeni from Phinda (916 ± 26) and St Lucia (941 ± 52) (Table 3.3; Fig. 3.7 B). 
 
3.3.3.2 Shape variation 
The two species were evaluated separately and the first two discriminant factors (shape 
components) accounted for 71% and 69% of the variance for G. brevipalpis and G. austeni, 
respectively. 
These discriminant factors indicated that there was no clear shape separation 
between the G. brevipalpis collected from the sites in South Africa and southern 
Mozambique (Fig. 3.8 A). The multivariate regression of the first relative warp against 
centroid size (100 000 permutation rounds) was also not significant (P = 0.30) indicating 
that there was no residual allometry. 
In contrast the multivariate regression was significant (P < 0.01) for G. austeni. The 
same no shape separation was observed for G. austeni collected from the different sites in 
South Africa as well as the G. austeni collected from southern Mozambique (Fig. 3.8 B). 
The only separation within the first discriminant factors was between G. austeni from 
Swaziland and St Lucia. These two sites were 228 km apart and were the two most 
geographically distant sites (Fig. 3.1). A linear regression of Mahalanobis distances derived 
from shape variation and geographical distances for both G. brevipalpis (r2 = 0.51, P < 0.01) 
(Fig. 3.9 A) and G. austeni (r2 = 0.65, P < 0.01) (Fig. 3.9 B) was significant, which suggests 
a process of genetic isolation by geographic distance. 
54 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
 
Fig. 3.7. Centroid size variations of the right wings of female Glossina brevipalpis (A) and 
Glossina austeni (B) according to localities. Each box shows the group median separating 
the 25th and 75th quartiles, capped bars indicate maximum and minimum values, circles 
indicating the outliers. Boxes followed by a different letter indicate that the sizes were 
significantly different at the 5% level.
55 
 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
Table 3.3. Summary of Glossina brevipalpis and Glossina austeni wings used in the phenetic geometric morphometric analysis. 
   
   No. female Shape component variation (%) Multivariate Average Centroid 
Date right wings Canonical Canonical regression (10 000 
Size (± SD) 
   used Variate 1 Variate 2 runs, P value) 
G. brevipalpis  345     
 Mozambique       
  Reserva Especial de Maputo  Aug '09 – Sep '09 36 1550 (±69) 
 South Africa    
  Ndumo Nov '08 – Mar '09 40 1544 (± 38) 
  Tembe Sep '08 – Oct '08 40 1520 (± 40) 
  Kosi Bay Jul '08 – Mar '09 31 1549 (±41) 47 24 0.303 
  Lower Mkhuze Sep '08 – Apr '09 40 1542 (± 56) 
  False Bay Park Nov '08 – Mar '09 37 1540 (± 33) 
  Hluhluwe-iMfolozi Park Nov '08 – Mar '09 41 1512 (± 35) 
  Boomerang Nov '08 – Mar '09 40 1518 (± 45) 
     St Lucia  Feb '09 – Mar '09  40    1568 (± 35)  
G. austeni  346     
 Swaziland       
  Mlawula Nature Reserve Apr '09 40 892 (± 37) 
 Mozambique    
  Reserva Especial de Maputo Aug '09 – Sep '09 14 934 (± 28) 
 South Africa    
  Ndumo Sep '08 – Mar '09 40 929 (±28) 
  Mbazana Aug '08 – Mar '09 40 925 (±25) 52 17 0.001 
  Lower Mkhuze Aug '08 – Mar '09 40 926 (± 33) 
  Phinda Nov '08 – Mar '09 40 916 (± 26) 
  False Bay Park Jan '09 – Mar '09 40 924 (± 30) 
  Hluhluwe-iMfolozi Park Nov '08 – Mar '09 13 925 (± 19) 
  Boomerang Nov '08 – Feb '09 39 935 (± 26) 
  St Lucia Mar '09 – Apr '09 40 940 (± 52) 
 
56 
 
 
Chapter 3: Geometric Morphometrics 
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56 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
57 
 
Fig. 3.8. The distribution of Glossina brevipalpis (A) and Glossina austeni (B) female right wing shape in the morhospace defined 
by the first two Canonical variants. 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
 
Fig. 3.9. Linear regression of the Mahalanobis distances of Glossina brevipalpis (A) and 
Glossina austeni (B) compared with geographic distances (km) between their collection 
sites (Solid red line represents the linear regression, broken red line the 95% confidence 
interval). 
58 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
3.3.4 Field flies compared with colony flies 
Female G. brevipalpis (St Lucia (40), Lower Mkuze (40), Mozambique (36)) and G. austeni 
(St Lucia (40), Lower Mkuze (40), Mozambique (14), Swaziland (40)) from the study area 
(Table 3.3) and colonised G. brevipalpis (34) and G. austeni (39) wings were analysed for 
size and shape differences. 
Colonised female wing centroid size of G. brevipalpis (P < 0.01) and G. austeni 
(P < 0.01) were significantly smaller from those of field collected flies (Fig. 3.10 A, B). The 
colony flies were on average much smaller than the field collected flies (Fig. 3.10 B). 
The first two discriminant factors (shape components) accounted for 73% and 76% 
of the variance for G. brevipalpis and G. austeni, respectively. The multivariate regression 
of the first relative warp against centroid size (100 000 permutation rounds) was not 
significant for G. brevipalpis (P = 0.08) and was significant for G. austeni (P = 0.03). 
For G. brevipalpis the first discriminant factor indicated a shape separation between 
colony and field flies (Fig. 3.11 A). The same shape separation between colony and field 
flies was also seen in G. austeni, however, this was more pronounced. There was also a 
separation indicated by the second discriminant factor between colonised G. austeni and 
flies from Swaziland (Fig. 3.11 B). 
 
 
Fig. 3.10. Variations in centroid size of the right wings of female Glossina brevipalpis (A) 
and Glossina austeni (B) according to localities, as well as colony reared flies. Each box 
shows the group median separating the 25th and 75th quartiles, capped bars indicate 
maximum and minimum values, circles indicating the outliers. Boxes followed by a different 
letter indicate that the sizes were significantly different at the 5% level. 
 
 
 
 
59 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
60 
 
Fig. 3.11. The distribution of Glossina brevipalpis (A) and Glossina austeni (B) female right wing shape in the morhospace defined 
the first two Canonical variants, flies were collected from varies sites as well as colony reared. 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
3.4 Discussion 
We demonstrated that geometric analysis could successfully distinguish between 
G. brevipalpis and G. austeni at a species level as also revealed by results presented by 
Patterson & Schofield (2005) for G. morsitans s.l., G. pallidipes and G. swynnertoni in the 
morsitans group, G. p. gambiensis, G.  fuscipes and G. tachinoides in the palpalis group 
and G. brevipalpis in the fusca group. 
Patterson & Schofield (2005) stated that wing shape apparently represents a 
relatively neutral trait that is not heavily modulated by ecological adaptation or 
environmental constraints. It is, however, evident from this study that environmental factors 
such as humidity and temperature can influence both shape and size to a certan extend of 
G. brevipalpis and G. austeni (Fig. 3.4; Table 3.2). In tsetse flies most of the pre-imago 
development is relatively protected from external influences as the larvae develops in utero 
(females are mobile and can avoid unfavourable environments) which to a certain extent  
buffers pre-imago against factors that can lead to morphometric variations (Glasgow, 1961). 
The effect of humidity and temperature becomes more evident when pupae are in the soil. 
Glasgow (1961) showed that higher temperatures tend to result in smaller individuals, while 
Déjardin & Maillot (1964) indicated that increased humidity tends to result in larger 
individuals. This “temperature-size rule” (Atkinson, 1994) can be considered as an adaptive 
strategy that allows insects to optimize their fitness in a changing seasonal environment 
(Angilletta et al., 2004; Kingsolver & Huey, 2008; Clemmensen & Hahn, 2015). 
Seasonal climatic effects were shown to influence the size (as determined by the 
length of the “cutting edge” of the “hatchet cell” and the thorax) of G. palpalis palpalis in 
forested areas of the Ivory Coast (Sane et al., 2000). These authors indicated that the size 
of an individual during collection is directly related to the ecological conditions in which the 
“mother” lived, giving an indication of the possible physiological state of the population 
about two months prior to collection (Sane et al., 2000). These authors collected larger 
individuals in the wet season and smaller ones in the dry season, therefore larger 
individuals in the wet season could reflect a population that has a good physiological start 
in the dry season (Sane et al., 2000). 
Similar to the results obtained by Sane et al. (2000) seasonal climatic effects in size 
(as determined by wing centroid size) were seen for both G. brevipalpis and G. austeni 
collected in north eastern KwaZulu-Natal. The G. brevipalpis collected at Ndumu, located 
in the interior of the study area, were on average smaller than those collected from St Lucia 
on the coast. Temperature and humidity fluctuations were more pronounced at Ndumu than 
at St Lucia. The temperature at Ndumu ranged from 27 °C in the hot months to 15.5 °C in 
the colder months, and the relative humidity from 80% in the rainy to 50% in the dry season 
(Chapter 2). Whereas the temperature at St Lucia ranged from 26 °C to 17 °C and the 
61 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
relative humidity from 99% to 68% (Chapter 2). Additionally, for both G. brevipalpis and 
G. austeni males and females, larger individuals were collected at the end of the hot season 
in autumn. This could reflect that populations located at sites with less pronounced 
fluctuations in environmental conditions, together with those at the end of a more favourable 
season (i.e. a hot, wet summer), would be in a better physiological condition than individuals 
collected in both areas and seasons where they were subjected to increased environmental 
stress. 
Sexual dimorphism is well-known in insects and sex-related differences can occur in 
various organs such as eyes (Lau et al., 2007), legs (Tseng & Rowe, 1999) head capsules 
(Wilkinson & Dodson, 1997) and wings (Ribak et al., 2009; Tejeda et al., 2014, Virginio et 
al., 2015). For tsetse flies sexual size dimorphism was previously reported for G. palpalis 
gambiensis (Camara et al., 2006; Bouyer et al., 2007). An obvious sexual size dimorphism 
with females being larger than males for both G. brevipalpis and G. austeni was reported. 
This size difference between males and females was more pronounced in summer than in 
winter, indicating that environmental conditions can also play a role in sexual dimorphism. 
The evolution of sexual dimorphism in tsetse flies might be more influenced by natural 
selection than sexual selection. 
An objective of the present study was to assess if geometric morphometric markers 
can be used to determine whether the various tsetse fly populations in South Africa were 
genetically isolated, and whether the South African populations were isolated or not from 
those in southern Mozambique or Swaziland. This will have important consequences for 
the selection and development of control strategies. Despite the apparent size differences, 
as mentioned above, morphometric markers indicated that there were no significant 
differences in wing shape between the various tsetse fly populations in South African (SA), 
southern Mozambique (SM) or Swaziland. Based on wing shape variations the G. austeni 
from Swaziland seem to be more isolated from those in the main SA-SM tsetse fly belt. This 
may indicate that a more reliable method such as genetic analysis will be needed to confirm 
these results. Preliminary molecular work with mitochondrial DNA also indicated the 
G. austeni from Swaziland to be more isolated from the main SA-SM tsetse fly belt but that 
gene flow between these population does exist (Koekemoer, ARC-OVI, unpublished data). 
Based on our current results the Swaziland population can, however, not be treated as an 
isolated population. Clear positive linear relationships between Mahalanobis distances and 
geographical distances were seen in both species. This could indicate that it is a slow on-
going process for these populations to have less genetic exchanges due to restricted tsetse 
fly dispersal (Kaba et al., 2012). 
62 
 
Chapter 3: Geometric Morphometrics 
_________________________________________ 
 
Our data indicate the absence of any significant barriers to gene flow between the 
three countries and the whole population should therefore be considered as a homogenous 
one and treated as such. 
This means that localised control efforts against tsetse flies in a given area will be 
subjected to reinvasion from uncontrolled sites. There is therefore a need for an area-wide 
approach, i.e. the control effort should be directed against the entire tsetse fly population of 
South Africa, southern Mozambique and Swaziland. This would entail that the entire area 
should be controlled simultaneously or by a continuous progression of interventions, using 
barriers of impregnated traps and/or targets between sites following the rolling carpet 
principle (Vreysen et al., 2007). There is in addition the need to confirm that the South 
African, southern Mozambique and Swaziland population is isolated from the main tsetse 
fly belt north of Maputo (it is assumed that this belt starts approximately 500 km further 
north in central Mozambique south of the Save River) (Dias, 1961; Mulandane, 2013). 
A strategy based on area-wide integrated pest management (AW-IPM) principles that 
include a SIT component was proposed to create a sustainable tsetse fly free area in South 
Africa (Kappmeier Green et al., 2007; Vreysen et al., 2007). The proposed strategy included 
the suppression of the G. brevipalpis and G. austeni populations with the sequential aerosol 
technique (Kappmeier Green et al., 2007) followed by the release of sterile males (Vreysen 
et al., 2000) to eradicate all potential relic pockets (Kappmeier Green et al., 2007). The two 
colonies that are being maintained at the ARC-OVI will need to be up-scaled to be able to 
produce sufficient sterile flies for the campaign. However, our study has indicated that the 
colony flies are different in shape and smaller in size compared with wild flies in KwaZulu-
Natal, southern Mozambique and Swaziland. As this may lead to concerns about the sterile 
insect’s quality as well as their competitiveness, field studies are required to further 
investigate these aspects. 
 
63 
 
Chapter 4: Rearing diet for colonised tsetse flies 
_________________________________________ 
 
Chapter 4 
 
Collection, processing and host source of the rearing diet for colonised tsetse flies2 
 
4.1 Introduction 
Studying insects in the wild, although essential and rewarding, presents a wide range of 
logistical, financial and sometimes even ethical challenges. This becomes more relevant if 
these insects are involved in pathogen transmission. Laboratory colonies allow research to 
be done on pathogen-free insects of a known age and biological background. Insect 
colonies are valuable assets that bring highly equipped laboratories together with insects 
that can only be found in remote areas. A good example of this is reflected in tsetse fly 
research. Tsetse flies are only present in Africa south of the Sahara (Moloo, 1993). Although 
most tsetse populations are found in rural areas, a large amount of research has been done 
on tsetse flies as a result of colonisation. This ranges from basic research on the life cycle 
and pathogenic role of tsetse flies (Ward, 1970) to advanced genome sequencing projects 
(International Glossina Genome Initiative, 2014). Furthermore, laboratory-adapted colonies 
were instrumental in the development of sophisticated vector control strategies such as the 
sterile insect technique (SIT) for tsetse flies (Feldmann et al., 2005) and other vectors (Oliva 
et al., 2012), as well as the implementation of transgenic (Alphey, 2014) and symbiont-
based (Atyame et al., 2011) control methods for mosquitoes. 
The first successful attempts to maintain tsetse flies in the laboratory were reported 
in 1917 when E. Roubaud succeeded in maintaining Glossina morsitans submorsitans 
collected in Senegal in culture for three years at the Pasteur Institute in Paris, France 
(Roubaud, 1917). This colony, however, consisted of only 32 flies (Ward, 1970). Glossina 
palpalis was the next species to be colonised at laboratories in Belgium and England with 
varying degrees of success for the next 30 years (Mellanby & Mellanby, 1937; Rodhain & 
Van Hoof, 1944; Ward, 1970). 
The possibility of larger self-sustainable colonies only became feasible with the 
development of a blood feeding station that could simultaneously hold four guinea pigs and 
eight cages of flies for feeding (Geigy, 1948). Despite the fact that 2000 flies could be fed 
in 3 to 4 hours with this system (Geigy, 1948; Ward, 1970) the colony proved not to be self-
sustainable. The first self-sustainable colony was established from 43 Glossina morsitans 
collected in Govura, Mozambique and maintained at the Escola Nacional de Saúde 
                                                          
2 Partially published as: 
De Beer, C.J., Venter, G.J. & Vreysen, M.J.B. (accepted) Improving the diet for the rearing of Glossina 
brevipalpis Newstead and Glossina austeni Newstead: blood source and collection – processing – feeding 
procedures. PLoS ONE. 
64 
 
Chapter 4: Rearing diet for colonised tsetse flies 
_________________________________________ 
 
Pública e de Medicina Tropical, Lisbon, Portugal in 1959 (De Azevedo & Da Pinhão, 1964; 
Ward, 1970). This colony was kept in a controlled environmental chamber at 26 °C and 
70% RH with a 12-hr light/dark photoperiod. These flies were fed on the shaved flanks of 
guinea pigs (De Azevedo & Pinhão, 1964; Ward, 1970). 
The colonisation of Glossina austeni at the Tsetse Research Laboratory, School of 
Veterinary Medicine, University of Bristol, Langford, England was another noteworthy 
contribution towards the large-scale rearing of tsetse flies. This colony was established from 
pupae collected on Unguja Island, Zanzibar and the emerged flies were fed on the ears of 
lop-eared rabbits (Nash et al., 1966a). This laboratory subsequently developed protocols 
on emergence, fertilization, adult and pupae maintenance, fly feeding and host suitability 
(Nash et al., 1966a, b, c; 1967; 1968; Jordan et al., 1966; 1967; 1968; Curtis & Jordan, 
1968). This colony was self-sustaining by 1966 and in 1967 produced 88 000 pupae (Nash 
et al., 1968). 
By 1970, a variety of biological studies had been conducted on colonised tsetse 
species including but not limited to Glossina tachinoides, G. austeni, G. morsitans and 
G. m. submorsitans around the world (Ward, 1970). A colony of Glossina brevipalpis was 
established from 181 females collected in mid-1982 in the Kibwezi Forest, Kenya (Moloo & 
Kutuza, 1988). Adult flies were collected using the moving vehicle method (Bursell, 1961) 
and were fed on the ears of lop-eared rabbits under controlled laboratory conditions 
(25 ± 0.5 °C and 80-85% RH) at the Tsetse Vector Laboratory of the International 
Laboratory for Research on Animal Diseases (ILRAD) (Moloo & Kutuza, 1988). The number 
of mature females in this colony increased from 1000 in mid-1983, to 4000 in mid-1984 and 
to 5000 by December 1984 (Moloo & Kutuza, 1988). 
One of the main obstacles in tsetse fly colonisation is the supply of high quality blood 
meals. Tsetse flies are obligate blood feeders and they reproduce by adenotrophic 
viviparity, making adult and larval stages dependent on the same source of food and 
insufficient nutrition will lead to abortions (Mellanby, 1937; Ward, 1970). A high quality food 
source is essential for the growth and sustainability of tsetse fly colonies (Feldmann, 
1994a). Increased abortion rates and reduced productivity of pupae resulting from an 
inadequate diet will hamper colony growth, which is already inherently low due to the slow 
reproductive cycle of tsetse flies. The maintenance of live animals for tsetse fly feeding will 
be costly, labour intensive and come with a set of ethical considerations. The development 
of in vitro feeding techniques became essential as the need for mass-rearing of the fly for 
experimental work, as well as for the possible use in programmes that had an SIT 
component became more pressing (Mews et al., 1976). Many attempts were made to feed 
tsetse flies in vitro through membranes (Roubaud, 1917; Lester & Loyd, 1928; Rogers, 
1971; Langley, 1972; Bauer & Wetzel, 1976) and laboratories in Europe, shifted their G. m. 
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Chapter 4: Rearing diet for colonised tsetse flies 
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morsitans colonies from animal to in vitro feeding (Mews & Ruhm, 1971; Nash et al., 1971; 
Mews et al., 1977; Wetzel & Luger, 1978). In vitro feeding also proved to be suitable for the 
establishment of colonies from field collected flies and a G. austeni colony was established 
from pupae collected on Zanzibar in 1986 at the Insect Pest Control Laboratory (IPCL) 
(formerly Entomology Unit) in Seibersdorf, Austria and maintained on an in vitro feeding 
system using bovine blood (Opiyo et al., 2000). 
In any tsetse fly mass-rearing facility, the logistics of obtaining sterile, high quality 
blood remains challenging. An added complication is the variation in nutritional value of the 
collected blood which is influenced by genetic, environmental, chemical and physical 
factors of the host animal (Kabayo et al., 1988). Other factors which play a significant role 
include chemicals and microbiological contaminants which the blood was exposed to during 
collection, handling and storage (Kabayo et al., 1988). To maintain viable healthy tsetse fly 
colonies, it is important that standard blood collection procedures are strictly adhered to, as 
this will ensure the continuous supply of a product that has the same quality as fresh blood 
collected directly from donor animals. 
In 2002, laboratory colonies of G. brevipalpis and G. austeni were established at the 
Agricultural Research Council-Onderstepoort Veterinary Institute (ARC-OVI) in Pretoria, 
South Africa using seed material from the Tsetse and Trypanosomiasis Research Institute 
(TTRI) (now named Vector & Vector-Borne Diseases Research Institute) Tanga, Tanzania 
and the Entomology Unit of the Food and Agriculture Organization (FAO)/International 
Atomic Energy Agency (IAEA) Laboratories in Seibersdorf, Austria (now called the 
FAO/IAEA Insect Pest Control Laboratory (IPCL)) respectively. The original G. brevipalpis 
colony was initiated in mid-1982 from material collected in the Kibwezi Forest, Kenya as 
described above (Moloo & Kutuza, 1988). The original G. austeni colony was established 
at the TTRI in September 1982 from pupae collected in the Jozani Forest, Unguja Island of 
Zanzibar. This colony was initially maintained on rabbits before an in vitro feeding system 
was introduced in 1988 (Tarimo et al., 1988). 
In view of the importance of providing high quality blood to tsetse colonies, 
optimisation of blood collection procedures and the development and validation of methods 
that may improve colony performance should be part of any colony maintenance program. 
The process of collecting blood at the abattoir could be simplified by using anticoagulants 
as a surrogate for mechanical stirring. We therefore tested the effect of mixing various 
anticoagulants on the nutritional value of the blood. 
Ii is known that the host preference of G. brevipalpis and G. austeni in nature may 
include, in addition to bovines, smaller mammals such as bush pigs (Moloo, 1993). Porcine- 
and bovine blood, and various combinations thereof, were therefore evaluated as rearing 
diets for these two species. Finally, it is also known that phagostimulants may enhance 
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blood intake of female tsetse flies and hence, increase productivity. We therefore also 
tested the effects of some phagostimulants on the performance of the two colonies. 
 
4.2 Materials and methods 
4.2.1 Tsetse fly colonies 
The G. brevipalpis and G. austeni colony flies were maintained under standard rearing 
conditions of 23-24 °C, 75-80% RH and subdued/indirect lighting with a 12 h light/12 h dark 
photoperiod (Feldmann, 1994a; FAO/IAEA standard operating procedures 2006). Since the 
establishment of these colonies at the ARC-OVI, they have been maintained on defibrinated 
bovine blood, collected from slaughtered cows at an abattoir, using an artificial in vitro 
membrane feeding system (Wetzel & Luger, 1978; Feldmann, 1994a; FAO/IAEA standard 
operating procedures, 2006). In 2015 the G. brevipalpis colony consisted of 13 000 
reproducing females that produced 7000 pupae weekly and the G. austeni colony had a 
size of 16 000 reproducing females that produced 5000 pupae weekly. The total colony size 
including both males and females of both species was 37 000 flies at the end of 2015. 
 
4.2.2 Blood collection and processing 
4.2.2.1 Porcine and bovine blood collected from commercial abattoirs for 
routine colony maintenance 
The two tsetse colonies at the ARC-OVI are currently maintained on cattle blood collected 
at Morgan (Pty)Ltd abattoir (-26.2545, 28.4299), situated outside the tsetse fly and nagana 
infected area, where cattle are slaughtered for human consumption. Although the veterinary 
history of these animals is unknown, all animals slaughtered are accompanied by a health 
certificate stating they are healthy and of good quality. This abattoir does not slaughter for 
disease control purposes. 
Collection of blood was carried out as follows: cattle were stunned, then suspended 
by their hind legs and their throat slit. The swath of blood was directed into a bucket and 
transferred to 40 L containers where it was defibrinated for 10-15 minutes using a custom-
made stainless steel electric paddle stirrer. The clotted fibrin was removed by hand and the 
blood pooled in a 500 L container after which it was divided into 5 L canisters and stored at 
-20 °C (FAO/IAEA standard operating procedures, 2006). 
Porcine blood was obtained from pigs with a known veterinary history, kept at the 
Agricultural Research Council-Animal Improvement Institute for breeding and experimental 
purposes. The pigs were slaughtered at the Bon Accord Abattoir in Irene, south of Pretoria. 
Animals were stunned, the jugular vein exposed and severed, and the blood collected in a 
3 L sterilized glass jar containing glass beads, and defibrinated by agitating for 10 to 15 
minutes before it was dispensed into 0.3 L containers and stored at -20 °C. 
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The bovine and porcine blood collected from the abattoirs was irradiated when frozen 
with 2 kGy at Synergy Heath, a commercial irradiation facility and stored in 5 L (bovine) or 
0.3 L (porcine) containers at -20 °C until used. All collected blood was tested for bacterial 
contamination before being used for tsetse feeding. Contaminated blood samples were 
appropriately discarded (FAO/IAEA standard operating procedures, 2006). 
 
4.2.2.2 Bovine blood collected with a closed sterile system 
As the veterinary history of the cattle slaughtered at the commercial abattoir was not known, 
cattle from a closed quarantined herd with a known veterinary background was also used 
for blood collection. For this purpose, animals were bled from the jugular vein using a trocar 
and cannula. Blood was drained directly (in a closed sterile system) into a sterilized 3 L 
glass jar containing one of the following anticoagulants; acid citrate dextrose (ACD), sodium 
citrate, a combination of citric acid and sodium citrate, citrate phosphate dextrose adenine 
(CPDA) or citric acid. For the evaluation of the phagostimulants defibrinated blood was 
used. Blood was collected in 3 L glass jars containing only glass beads and agitated for 10 
to 15 minutes. The blood collected with anticoagulants and defibrination were decanted into 
20 mL containers and stored at -20 °C. 
 
4.2.3 Assessment of suitability of blood source as maintenance diet 
Bovine and porcine blood and mixtures thereof were evaluated as diets for the routine 
maintenance of G. brevipalpis and G. austeni colonies. In a first set of experiments, 
productivity of both species was assessed when fed daily on a single blood source (i.e. 
bovine or porcine) or in the following mixed combinations (75% bovine / 25% porcine, 50% 
bovine / 50% porcine, and 25% bovine / 75% porcine). Blood that was collected for colony 
maintenance form the commercial abattoirs were used. 
In a second set of experiments, productivity of both species was assessed when fed 
daily on a single blood source that was alternated between days according to the following 
schedule: 
 One day bovine followed by four days of porcine blood, 
 Three days bovine followed by three days of porcine blood, 
 Four days bovine followed by one day of porcine blood. 
For this set of experiments bovine blood that was collected using the closed sterile system 
and porcine blood from the abattoir system was used. 
 
 
 
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4.2.4 Phagostimulation 
The nucleotides adenosine triphosphate (ATP), inosine triposphates (ITP), guanosine 
monophosphate (GMP) and cytosine monophosphate (CMP) were the phagostimulants 
evaluated. An amount of 0.055 g of each compound was diluted in 1 mL of distilled water 
and the dilutions were kept at -20 °C and used within three days. Only 0.02 mL of each 
solution (concentration of 10-3 M) was used for 20 mL of bovine blood collected with the 
closed sterile system, as described above.  
 
4.2.5 Bioassay 
Female fly survival (percentage alive on day 18 and 30 post eclosion respectively), 
fecundity (number of pupae per mature female at day 18) and pupal size are three essential 
parameters routinely used for assessing colony performance (Wetzel & Luger, 1978). 
Fecundity was determined by the number of pupae produced per mature female day (Curtis, 
1968). Mature female days were calculated for each treatment by adding the number of 
flies alive each day, starting on day 18 after emergence until the end of the experiment on 
day 30 (Curtis, 1968). 
These parameters are used in a formula that calculates a Quality Factor (QF) of the 
blood as a comprehensive indicator for colony production (Feldmann, 1994a; FAO/IAEA 
standard operating procedures, 2006).  
Blood quality was evaluated using a standardised bioassay (FAO/IAEA standard 
operating procedures, 2006) whereby productivity of flies that were fed daily on the selected 
diet for 30 days using an artificial membrane feeding system was assessed. This 
standardised bioassay is also routinely used for quality control on the blood used in the 
colony. That enabled the QF of the selected diet to be calculated (Feldmann 1994b; 
FAO/IAEA standard operating procedures, 2006). A QF above 1 is considered acceptable 
and indicates that the blood diet is suitable for colony maintenance (Feldmann, 1994a; b). 
The protocol of the bioassay consisted of mating 30 three-day-old females with 30 
eight-day-old males at a 1 : 1 ratio. Males and females were kept together for four days 
under standard colony conditions (23-24 °C, 75-80% RH and subdued/indirect lighting). 
After removing the male flies, female survival, pupae production and abortions (i.e. expelled 
eggs and immature larval stages) were monitored daily for 30 days (Feldmann, 1994a; 
FAO/IAEA standard operating procedures, 2006). 
All pupae produced were mechanically sorted into one of five class sizes (A - E). The 
sorter was calibrated according to the standards established by the FAO/IAEA. For 
G. austeni the measurements ranged between 2.3 mm (A) and 3.0 mm (E), and for 
G. brevipalpis between 3.5 mm (A) and 4.3 mm (E). The weight (mg) of the different pupal 
69 
 
Chapter 4: Rearing diet for colonised tsetse flies 
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size classes were A (<16), B (16– <19), C (19– <21), D (21– <23), E (≥23) for G. austeni 
and A (<56), B (56– <68), C (68– <76), D (76– <84), E (≥84) for G. brevipalpis. 
After 30 days, all surviving females were dissected to determine their reproductive 
status (presence/absence of egg/larvae in the uterus and spermatecae fill). All bioassays 
were replicated four times. 
The QFs were calculated using the formula (Feldmann, 1994a; FAO/IAEA standard 
operating procedures, 2006): 
 
QF = FS30 + PT + 0.3(PB) + 0.4(PC) + 0.5(PD) + 0.6(PE) + 0.3(E&I) + 0.6(II&III) - 0.2(PA) -0.5(AB) - BO 
     FS30 + FS18 
 
Where: 
QF  Quality factor 
FS18  Female flies surviving on day 18 
FS30  Female flies surviving to day 30 
PA  No. of size class A pupae 
PB  No. of size class B pupae 
PC  No. of size class C pupae 
PD  No. of size class D pupae 
PE   No. of size class E pupae 
PT  Total pupae 
AB  Abortions 
E&I  Eggs or first instar larvae in utero 
II&III  Second or third instar larvae in utero 
BO  Blockage of the oviducts or other reproductive abnormality 
 
4.2.6 The colonisation of tsetse flies 
The G. brevipalpis and G. austeni colonies at the ARC-OVI have an East-African origin, 
and come from a different geographical population than those in north eastern KwaZulu-
Natal. Morphometric differences between the local KwaZulu-Natal and the ARC-OVI 
colony’s flies have been recorded (Chapter 3). Before the initiation of an operational 
programme that includes the release of sterile males, it is essential to establish colonies of 
the local strains to study their biology, mating competitiveness in field cages and in the 
target area as well as Trypanosomosis/fly interactions. These aspects need to be compared 
to those of the available established colonies with an East-African origin. This information 
will be required for the development of an appropriate control strategy to manage the 
nagana problem in north eastern KwaZulu-Natal. 
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Chapter 4: Rearing diet for colonised tsetse flies 
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Flies for potential colonization were collected from the western shores of the 
iSimangaliso Wetland Park, Phinda and Hluhluwe-iMfolozi Park (Fig. 2.5). These areas 
were previously shown to harbour large populations of G. brevipalpis and G. austeni 
(Chapter 2). Flies were collected from 11 August 2014 to 25 April 2015 with 34 odour baited 
H traps (Kappmeier & Nevill, 1999a; Kappmeier, 2000). Traps were emptied twice daily and 
the flies transferred from the collection bottles to fly holding cages. Flies were fed on the 
day of collection on abattoir collected bovine blood (as described 4.2.2.1) using an artificial 
membrane feeding system. The day after collection male and female flies were separated. 
Collected flies were kept at the KwaZulu-Natal Tsetse Research Station under standard 
colony conditions (23-24 °C, 75-80% RH and subdued/indirect lighting) (Feldmann, 1994a; 
FAO/IAEA standard operating procedures, 2006) for up to 30 days before transportation in 
Styrofoam boxes (with wet tissue paper) to the colony facility at the ARC-OVI. 
 
4.2.7 Statistical analysis 
Data was analysed using the statistical programs GraphPadInstat (version 3.00, 2003) and 
VSN International (2012). The experiments were designed as a randomised block design 
in four blocks. Differences in survival rate and QF values between treatments were 
evaluated with an analysis of variance (ANOVA). The data was shown to be normally 
distributed with homogeneous treatment variances. Treatment means were separated 
using Fishers' protected t-test least significant difference (LSD) at the 5% level of 
significance (Snedecor & Cochran, 1980), if the F-probability from the ANOVA was 
significant at 5%. 
 
4.3 Results 
4.3.1 Anticoagulants in the rearing diet 
The anticoagulant study indicated that survival of both G. brevipalpis females (98%) and 
G. austeni females (95%) was the highest for flies that had fed on defibrinated blood (Table 
4.1). 
The survival rate of G. brevipalpis females that had fed on blood collected with ACD 
(93%) (P = 0.14) and sodium citrate (95%) (P = 0.50) was not significantly different from 
that of defibrinated blood (98%). However, survival of G. brevipalpis fed on blood collected 
with the citric sodium combination (88%, P = 0.01), CPDA (84%, P < 0.01) and citric acid 
(80%, P < 0.01) was significantly lower than that of flies fed on defibrinated blood (Table 
4.1). 
Survival of G. austeni females that were fed on blood collected with ACD (86%), citric 
sodium combination (85%), citric acid (85%), sodium citrate (68%) and CPDA (80%) was 
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Chapter 4: Rearing diet for colonised tsetse flies 
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significantly lower (P < 0.02 in all cases) than that of flies fed on the defibrinated blood 
(95%) (Table 4.1). 
The highest fecundity was observed for G. brevipalpis females fed on blood collected 
with the citric sodium combination (0.059) and citric acid (0.061) both was above that of the 
defibrinated blood and these flies also produced the highest percentage of large pupae 
(Class C > 68 mg), i.e. 87% and 93%, respectively (Table 4.1). Glossina brevipalpis females 
that fed on blood collected with ACD had the lowest fecundity (0.047) and produced the 
smallest pupae. 
The fecundity of G. austeni fed on defibrinated blood as well as blood collected with 
CPDA and citric acid was above 0.060, and those that had fed on CPDA produced the 
largest pupae (88% > 19 mg). The lowest fecundity of 0.049 was observed for G. austeni 
fed on blood collected with sodium citrate. The smallest pupa was produced by females 
that fed on blood collected with ACD (Table 4.1). 
Insemination rate of all surviving females on day 30 was higher than 95% and the 
spermatecae fill was above 0.5 for most of the female flies of both species, irrespective of 
the treatment (Table 4.1). 
In the current comparison, the bioassay for both G. brevipalpis and G. austeni that 
had been fed blood collected with the anticoagulants as well as the defibrinated blood gave 
a QF above 1, except for G. brevipalpis that fed on blood collected with ACD (0.93) (Table 
4.1; Fig. 4.1). 
For the G. brevipalpis there was no significant difference (P = 0.14) in the QF values 
between defibrinated blood and the tested anticoagulants. For G. austeni a significant 
difference (P = 0.05) was seen between the defibrinated blood (1.31) and that collected 
with ACD (1.16) and sodium citrate (1.03) (Fig. 4.1). 
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Chapter 4: Rearing diet for colonised tsetse flies 
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Fig. 4.1. Quality Factor (QF) values for the blood collected with anticoagulants as obtained 
in the bioassay for Glossina brevipalpis and Glossina austeni. Each box shows the group 
median separating the 25th and 75th quartiles, capped bars indicate maximum and minimum 
values. Boxes denoted by a different letter indicate that the QF values were significantly 
different for each species at the 5% level. 
73 
 
 
Chapter 4: Rearing diet for colonised tsetse flies 
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Table 4.1. Anticoagulants tested for their potential use in blood collection, as opposed to defribination, for both Glossina brevipalpis and Glossina 
austeni rearing diets. Numbers followed by an * indicate significant differences between the anticoagulant and the defribinated blood for each 
species at the 5% level. 
No. of Uterus 
mature Pupal size classes Quality Viable instar Spermatheca fill 
Pupae 
Females Recently Empty   Fecundity factor larvae Insemination 
produced ovulated due to 
Day Day (QF) 
A B C D E egg abortion I II III 0.25 0.5 0.75 1 
 18  30           
G. brevipalpis                 
   Defibrination 117 117 80 0.053 4 6 14 21 35 1.11 13 31 29 20 24 1.00 7 49 49 12 
  Acid Citrate 
118 111 71 0.047 11 24 21 9 6 0.93 23 36 33 11 8 1.00 5 42 56 8 
Dextrose  
  Sodium citrate 116 114 82 0.055 15 16 18 23 10 1.07 30 26 25 22 10 0.95 15 42 46 4 
  Citric & Sodium 109 106* 83 0.059 1 5 17 32 28 1.12 6 33 48 12 5 1.00 0 60 39 6 
  Citrate 
Phosphate 
108 101* 68 0.050 3 7 15 21 22 1.05 21 24 31 16 9 0.98 12 52 35 2 
Dextrose 
Adenine  
  Citric ac id    98  96 *  7 7  0.0 61  4   4  1  4  2 4 3  1  1.1 7  6   2 4  4 2  1 4  9   1.0 0  0   4 8  4 3  4  
G. austeni 
  Defibrination 115 114 100 0.067 12 14 28 31 15 1.31 27 7 8 32 39 0.97 16 58 29 5 
  Acid Citrate 
111 103* 82 0.059 13 18 22 22 7 1.16 28 11 14 19 31 1.00 27 37 35 4 
Dextrose  
  Sodium citrate 98 81* 57 0.049 9 10 22 11 5 1.03 23 12 3 17 25 0.99 25 30 16 8 
  Citric & Sodium 115 102* 88 0.062 6 6 24 34 18 1.22 25 9 10 28 23 0.98 9 60 29 2 
  Citrate 
Phosphate 
108 96* 86 0.065 4 6 25 34 17 1.26 15 9 19 28 24 0.97 7 61 18 7 
Dextrose 
Adenine  
  Citric acid   106 102* 86 0.063 8 8 24 35 10 1.22 15 16 23 25 22 0.99 18 50 26 5 
 
 
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Chapter 4: Rearing diet for colonised tsetse flies 
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Chapter 4: Rearing diet for colonised tsetse flies 
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4.3.2 Blood source for maintenance diet 
Two feeding regimes, i.e. mixtures of bovine and porcine blood in different proportions or 
only bovine- or porcine blood in different combinations of days in a six-day cycle, were 
tested. 
The comparison of mixtures of bovine and porcine blood in different proportions 
showed that the survival rate of both G. brevipalpis (83%) and G. austeni (72%) was the 
highest for the 50% / 50% combination (Table 4.2). Feeding flies only with bovine (control) 
blood reduced the overall survival rate to 53% and 61% for G. brevipalpis and G. austeni, 
respectively (Table 4.2). The 25% bovine / 75% porcine combination gave the lowest 
survival rate, 35% for G. brevipalpis and 32% for G. austeni. The survival rate of 
G. brevipalpis that fed on 100% bovine (control) (53%) blood was significantly different from 
flies that had fed on all the combination diets (P < 0.01) as well as the 100% porcine (80%) 
one (P < 0.01) (Table 4.2). The survival rates of G. austeni fed on the 100% porcine (78%) 
and the 25% bovine / 75% porcine (32%) combination was significantly different (P < 0.01) 
compared to flies that had fed on the 100% bovine (61%) diet (Table 4.2). 
The overall fecundity was very low with the highest fecundity recorded for 
G. brevipalpis that had fed on the 50% / 50% combination diet (0.039) and G. austeni that 
had fed on the 75% bovine / 25% porcine combination (0.050) and the 50% / 50% 
combination (0.052). The highest percentage of large pupae (size class C and above) was 
produced by the G. brevipalpis fed blood in the 75% bovine / 25% porcine combination 
(87%), and this was also the case for G. austeni (60%) (Table 4.2). 
Insemination rate was for both species and for all treatments ≥ 0.88 and the 
spermathecal fill was in most cases between 0.5 and 0.75 (Table 4.2). 
All the QF values for the premix combination feeding regimes for G. brevipalpis were 
below 1, except for flies that fed on the 100% bovine (control) (1.11) diet (Table 4.2; Fig. 
4.2). The QF values for the G. brevipalpis flies fed on the 100% porcine (0.73), 25% bovine 
/ 75% porcine combination (0.54) and 75% bovine / 25% porcine combination (0.70) were 
significantly different (P < 0.01) from the flies that had fed on the 100% bovine diet (1.11) 
(Fig. 4.2). The QF values for G. austeni that fed on the 100% bovine (control) (0.84) diet 
were significantly higher (P < 0.01) than those that fed on the 25% bovine / 75% porcine 
combination (0.56) diet. In this specific evaluation the QF values obtained for both 
G. brevipalpis and G. austeni with the bovine blood control were lower than normal which 
may indicate that the overall quality of the bovine as well as porcine blood used in this trial 
was low. 
During the second feeding regime evaluation, blood from a single host was offered to 
the flies in different sequences during a six-day cycle. This evaluation was not done 
concurrently with the first and a different blood batch was used. 
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Chapter 4: Rearing diet for colonised tsetse flies 
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Survival of the G. brevipalpis females ranged from 98% for those fed on the bovine 
(control) as well as only the porcine blood, but survival was reduced to 88% for flies fed on 
the bovine (3 days) – porcine (3 days) combination (Table 4.2). The survival rate for 
G. brevipalpis that fed on the bovine (3 days) – porcine (3 days) (88%) combination 
(P < 0.01) were significantly lower than that compared to those fed only on bovine blood 
(97%). 
Glossina austeni females had a slightly lower survival rate ranging from 83% for flies 
that had fed on bovine blood only to 73% for flies fed on the porcine diet. There were no 
significant differences in survival rate of the G. austeni fed on bovine blood (83%) compared 
with any of the other combinations including the porcine diet (73%) (Table 4.2). 
Fecundity ranged from 0.067 for the G. brevipalpis that had fed on the bovine (3 days) 
– porcine (3 days) combination to 0.057 for flies that had fed on the porcine only diet. 
Glossina brevipalpis females that fed on pure porcine produced the smallest pupae (Table 
4.2), whereas the largest pupae were produced by flies fed on the bovine (4 days) – porcine 
(1 day) combination. These data seem to indicate a relationship between the use of bovine 
blood and the size (larger) of the G. brevipalpis pupae. 
Glossina austeni females that had fed on the bovine (4 days) – porcine (1 day) 
combination produced the highest number of pupae with a fecundity of 0.061 and those fed 
on the bovine (3 days) – porcine (3 days) produced the least (0.055). The G. austeni 
females that were offered blood in combination diets produced larger pupae than those fed 
on a single source diet (Table 4.2). 
The insemination rates for both species were above 0.95 and in the majority the 
spermatecae fill was in the 0.5 or 0.75 class (Table 4.2). All the combination diets in this 
second feeding regime resulted in QF values above 1 for both species (Table 4.2; Fig. 4.2), 
with no significant differences between treatments. The highest QF value for G. brevipalpis 
(1.21) was obtained when using the bovine (3 days) – porcine (3 days) combination while 
the bovine (1 day) – porcine (4 days) gave the highest QF for G. austeni (1.21). 
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Chapter 4: Rearing diet for colonised tsetse flies 
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Fig. 4.2. Quality Factor (QF) values for different combinations of bovine/porcine blood diets 
obtained using the standard bioassay for Glossina brevipalpis and Glossina austeni. Each 
box shows the group median separating the 25th and 75th quartiles, capped bars indicate 
maximum and minimum values. Boxes denoted by a different letter indicate that the QF 
values were significantly different for each species at the 5% level. 
 
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Chapter 4: Rearing diet for colonised tsetse flies 
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Table 4.2. Bovine/porcine blood combinations tested for their potential use as rearing diet for Glossina brevipalpis and Glossina austeni. Numbers 
followed by an * indicate a significant difference between the bovine blood (control) and the various combinations for each species and each 
group at the 5% level. 
Uterus 
No. of 
Viable 
mature Pupal size classes Quality Spermatheca fill 
Pupae Recently Empty instar 
  Females Fecundity factor Insemination 
produced ovulated due to larvae 
(QF) 
Day Day egg abortion 
A B C D E I II III 0.25 0.5 0.75 1 
18 30 
Premix 
combination diet                     
G. brevipalpis                     
   Bovine (bov) 80 63 20 0.020 2 2 5 5 6 1.11 22 18 9 5 9 0.98 10 31 20 1 
   Porcine (por) 112 96* 41 0.031 7 5 13 8 8 0.73 15 24 30 16 3 1.00 0 36 54 6 
   25%bov/75%por 60 38* 22 0.035 4 2 7 6 3 0.54 10 17 3 3 5 0.88 3 13 20 1 
   75%bov/25%por 100 85* 42 0.032 1 4 9 13 11 0.79 27 29 16 7 3 0.95 19 20 39 3 
   50%bov/ 50%por  113  99*  55  0.039 8   8  14  19  6  0.95  11  14 1 7  36  8  0.96  18  24  43  1 
G. austeni                     
   Bovine (bov) 83 73 36 0.035 6 15 9 5 1 0.84 27 20 9 11 6 0.97 22 33 23 3 
   Porcine (por) 105 93* 56 0.040 29 9 15 2 1 0.86 44 17 15 10 4 1.00 20 31 37 2 
   25%bov/75%por 60 38* 13 0.022 5 2 3 2 1 0.56 10 17 2 4 5 0.88 3 13 20 1 
   75%bov/25%por 96 85 58 0.050 13 10 17 13 5 0.93 27 28 5 18 3 0.95 19 20 39 3 
   50%bov/ 50%por  105  86  63  0.052  17 1 0 1 5 1 4  7  0.99  14  12 3 1 2 0  7  0.96  18  24  43  1 
Alternating diet                     
G. brevipalpis                     
   Bovine (bov) 118 117 91 0.060 5 6 16 34 30 1.13 30 35 24 23 5 1.00 0 57 52 8 
   Porcine (por) 118 118 88 0.057 6 14 19 27 22 1.11 43 24 28 7 7 0.99 5 62 43 6 
   bov(1#)-por(4#) 118 112 95 0.063 5 8 26 24 32 1.19 50 9 16 7 7 0.95 11 48 23 7 
   bov(4#)-por(1#) 117 116 92 0.061 5 5 24 26 32 1.17 37 16 28 12 6 0.96 26 43 28 3 
   bov(3#)-P or(3#)  112  105*  95  0.067  6  9  21 2 6 3 3  1.21  35  15 2 0 1 6  3  0.95  11  37  29  4 
G. austeni                     
   Bovine (bov) 104 99 79 0.059 8 13 18 16 24 1.17 48 11 19 16 5 0.97 14 49 25 8 
   Porcine (por) 88 87 66 0.058 9 12 24 11 10 1.08 36 22 15 9 5 1.00 11 49 22 5 
   bov(1#)-por(4#) 93 91 72 0.060 2 6 12 26 26 1.21 50 8 16 8 7 0.95 11 48 23 7 
   bov(4#)-por(1#) 104 103 82 0.061 2 8 18 22 32 1.19 37 16 28 9 6 0.96 26 43 28 3 
   bov (3#)-por(3#) 95 94 68 0.055 4 7 12 21 24 1.13 35 15 19 16 3 0.95 11 37 29 4 
# Days fed on specific host blood
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Chapter 4: Rearing diet for colonised tsetse flies 
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78 
Chapter 4: Rearing diet for colonised tsetse flies 
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4.3.3 Evaluation of phagostimulants to improve production in the tsetse fly 
colonies 
Bovine blood was spiked with phagostimulants to assess potential stimulated feeding 
responses that could increase overall colony productivity. Glossina brevipalpis that fed on 
the bovine (control) diet had a survival rate of 94% which was similar to those that fed on 
blood with ITP (Table 4.3). All G. brevipalpis that fed on a diet with other phagostimulants 
had a lower survival rate. The diet with ITP as well as with GMP improved the survival rate 
(94% and 92% respectively) of G. austeni as compared with flies fed on the bovine (control) 
diet only (72%) (Table 4.3). The G. brevipalpis (76%) as well as the G. austeni (49%) flies 
fed on a diet with ATP had the lowest survival rate which was significantly lower (P < 0.01) 
from the survival obtained for flies fed on the bovine blood (Table 4.3). 
The fecundity of G. brevipalpis females that fed on bovine blood or blood mixed with 
ITP was the highest  0.056 (Table 4.3). Glossina brevipalpis females fed on pure bovine 
blood produced the largest (98% in the C class and above) pupae (Table 4.3), whereas 
flies that were fed blood containing ATP produced the smallest pupae (86% in the C class 
and above). Glossina austeni females fed on blood containing ATP, ITP and GMP produced 
the most as well as the largest pupae (Table 4.3). 
The insemination rate for G. brevipalpis ranged from 0.87 for flies fed on the bovine 
(control) diet to 0.94 for flies fed on the diet with GMP. The spermatecae fill of G. brevipalpis 
females was low for all the diets with the majority of the femalesa spermatecae filling in the 
0.25 or 0.5 class range. The G. austeni females had an insemination rate of > 0.98 for all 
the diets and the majority of the females had a spermatecae fill of between 0.5 and 0.75 
(Table 4.3). 
All the phagostimulant-treated as well as the bovine (control) diets resulted in QF 
values of above one for G. brevipalpis (Table 4.3; Fig. 4.3) and there was no significant 
differences (P = 0.64).For G. austeni, the QF values of the diet spiked with CMP (0.98) was 
below one (Table 4.3; Fig. 4.3). The highest QF value was obtained with the blood mixed 
with ATP (1.18) and this was significant different (P < 0.01) from that of the bovine (control) 
diet (1.06). 
 
4.3.4 The colonisation of tsetse flies collected from north eastern KwaZulu-Natal 
A total of 16 569 G. brevipalpis and 198 G. austeni were collected from 11 August 2014 to 
25 April 2015 with 34 odour baited H traps. For G. brevipalpis the flies were both males 
(7356) and females (9213) of different ages. For G. austeni the majority were female. All 
collected flies were transferred to the insectary and the sexes separated. Flies were fed on 
bovine blood using an artificial membrane. Very few flies fed on this system and large 
numbers of deaths were observed. At the end of the collection period the flies were 
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Chapter 4: Rearing diet for colonised tsetse flies 
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transported to ARC-OVI tsetse fly colony. A 58% mortality rate was observed for 
G. brevipalpis after collection and transport to the ARC-OVI. 
 
 
 
 
Fig. 4.3. Quality Factor (QF) values for blood mixed with different phagostimulants as 
obtained in the standard bioassay for Glossina brevipalpis and Glossina austeni. Each box 
shows the group median separating the 25th and 75th quartiles, capped bars indicate 
maximum and minimum values, boxes denoted by a different letter indicate that the QF 
values were significantly different for each species at the 5% level. 
80 
 
 
Chapter 4: Rearing diet for colonised tsetse flies 
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Table 4.3. Blood mixed with different phagostimulats tested for their potential use as rearing diet for Glossina brevipalpis and Glossina austeni. 
No. of Uterus 
mature Pupal size classes Quality Viable instar Spermatheca fill 
Pupae Recently Empty 
  Females Fecundity factor larvae Insemination 
produced ovulated due to 
Day Day (QF) 
A B C D E egg abortion I II III 0.25 0.5 0.75 1 
18 30 
G.  
brevipalpis                    
    Control 115 113 83 0.056 1 1 20 23 38 1.09 38 35 22 7 11 0.87 39 17 8 3 
    ATP 92 91 57 0.048 2 6 16 21 12 1.02 26 21 9 13 9 0.87 22 14 13 0 
    ITP 116 113 84 0.056 3 3 19 21 38 1.11 47 28 20 10 6 0.89 42 25 6 4 
    CMP 109 107 75 0.054 0 6 20 24 25 1.10 48 21 26 6 5 0.93 41 17 6 3 
    GMP   116  110  65  0.044  1  5 1 0 2 2 2 7  1.10  44  21  21  18  3  0.94  50  17  14  2 
G. austeni                     
    Control 96 86 66 0.057 9 17 20 17 3 1.06 39 10 20 4 7 0.99 11 38 30 6 
    ATP 71 59 56 0.067 3 7 20 23 3 1.18 22 7 14 13 0 0.98 9 22 22 4 
    ITP 103 94 74 0.058 7 8 29 21 9 1.08 42 10 25 6 4 0.99 5 57 21 4 
   CMP 89 75 52 0.049 16 9 19 7 1 0.98 41 5 17 6 6 0.99 8 40 21 4 
   GMP 103 96 72 0.056 6 4 27 20 15 1.14 50 5 18 13 4 1.00 6 60 21 7 
 
 
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Chapter 4: Rearing diet for colonised tsetse flies 
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81 
Chapter 4: Rearing diet for colonised tsetse flies 
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The 9213 field-collected G. brevipalpis females produced 3722 pupae (40.4% 
pupation rate) out of which 1216 flies emerged (32.7% emergence rate); 768 were females 
and 448 males. These F1 flies subsequently produced 891 pupae (73.3%) from which 288 
flies emerged (32.3%), i.e. 167 females and 121 males. The F2 flies produced 162 pupae 
(56.3%), however, only five flies emerged. 
The use of porcine blood to improve survival and pupae production of G. brevipalpis 
was also assessed. Flies were collected from the field and fed a 100% bovine, 100% 
porcine and a 50% bovine 50% porcine blood meals. Twenty females were used in each of 
the five replicates. High initial mortalities were seen for all three diets, i.e., on day seven, 
50% of flies fed on the porcine diet had died, while on day eight, 50% of flies fed on the 
bovine diet died and on day nine, all flies fed on the combination diet were dead. The flies 
fed on the bovine and the combination diets did significantly better (P < 0.01) than the flies 
that fed on the porcine diet. Additionally, flies that fed on the combination diet produced 
more pupae (138) than flies on the bovine diet (111) and lastly flies that fed on porcine 
blood produced significantly (P = 0.03) fewer pupae (29). A higher percentage of flies died 
of starvation in the porcine (77%) and combination (71%) diet trials compared with the 
bovine diet (48%). 
 
4.4 Discussion 
A high quality rearing diet (blood) is essential for growth and sustainability of tsetse fly 
colonies (Feldmann, 1994b). The extent of the influence of chemicals and microbiological 
contaminants as well as genetic, environmental, chemical and physical factors on the 
quality of the diet can differ between colonies and species. 
Sterile, freshly frozen, defibrinated or heparinized blood used with a silicon membrane 
is considered the most efficient way of maintaining tsetse fly colonies (Bauer & Wetzel, 
1976; Gooding et al., 1997). In the present study, defibrinated bovine blood was the most 
suitable diet for both G. brevipalpis and G. austeni colonies. Additionally, it was shown that 
bovine blood collected with the anticoagulants sodium citrate, citric sodium combination, 
CPDA and citric acid were suitable for both G. brevipalpis and G. austeni feeding. While 
ACD did not impacted negatively on the maintenance of G. austeni it was not suitable for 
the rearing of G. brevipalpis. Although the addition of anticoagulants will simplify collection 
and make the process more sterile, it is expensive and will increase costs for large-scale 
operations. As these anticoagulants did not significantly improve the blood quality and 
colony productivity it would be more economical to use defibrinated blood in tsetse fly mass-
rearing facilities. 
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Both G. brevipalps and G. austeni are known to feed on bovine and porcine hosts in 
the wild (Moloo, 1993; Clausen et al., 1998). Porcine blood has been used successfully for 
the in vitro feeding of colonies of G. morsitans and G. austeni (Gee, 1977; Clausen et al., 
1998). Combinations of fresh/frozen bovine blood and reconstituted lyophilized porcine 
blood have been used for a G. austeni colony in Tanzania (Tarimo et al., 1988). 
Combinations of bovine and porcine blood as supplements in the synthetic diets gave 
survival rates between 70% and 90% for Glossina palpalis palpalis (Kabayo & Taher, 1986). 
In the present study feeding on a 50% / 50% combination of defibrinated bovine and porcine 
blood or feeding on either bovine or porcine on alternating days improved the overall 
G. austeni pupae production, and would be useful to accelerate colony growth. This is not 
surprising as DeLoach & Spates (1991) discovered that porcine erythrocytes contain 
phosphatidycholine wheras bovine erythrocytes do not, choline is considered an essential 
dietary requirement for insects. Although bovine blood seemed to be more appropriate for 
a G. brevipalpis colony, feeding a single source on alternating days did improve 
productivity. Using porcine blood for the ARC-OVI colonies to boost production in times of 
low performance would be beneficial. 
Observed differences in the quality of the blood collected from bovines at commercial 
abattoirs and the closed quarantined herd that effect the production of the tsetse flies clearly 
indicates that the quality of the blood used in the colony is very important. 
Adding phagostimulants to the blood diet at the concentrations used in the present 
study did not improve colony productivity as indicated by the obtained QF values. Taste 
receptors of tsetse flies, which assist in ingestion of blood, are stimulated by adenosine 
triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) 
(Galun & Margalit, 1970). ATP at a concentration of 10-3 M has been used to promote fly 
engorgement and thus improve colony production (Mews et al., 1976; Galun, 1988; Galun 
& Kabayo, 1988). ATP is an expensive component to add to the maintenance diet (Galun, 
1988), especially in mass-rearing but it has been shown that G. tachinoides are more 
sensitive to ATP and that even a low dose will enhance feeding (Galun, 1988). The 
simultaneous use of ATP and sodium bicarbonate will synergistically enhance feeding; 
however, the use of a suitable feeding membrane is also important (Galun, 1988). Other 
nucleotides such as AMP, ADP, as well as mono- and tri-phosphates of inosine (IMP, ITP), 
guanosine (GMP, GTP) and cytosine (CMP, CTP), all at a concentration of 10-4 M are also 
effective phagostimulants for G. brevipalpis and G. austeni (De Beer et al., 2012). 
Increasing the concentration of ITP, CMP and GMP to 10-3 M improved production of 
G. brevipalpis more than the addition of ATP alone. However, ATP and ITP at a 10-3 M 
concentration did not improved the production of G. austeni. These phagostimulants remain 
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Chapter 4: Rearing diet for colonised tsetse flies 
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expensive and is recommended as a tool in colonies where flies struggle to adapt to artificial 
feeding i.e. at the onset of colonization of wild flies. 
Most tsetse fly colonisation was initially done on animals (rabbits, goats, guinea pigs) 
before migrated to in vitro feeding (Mews & Ruhm, 1971; Nash et al., 1971; 1972; Mews et 
al., 1977; Wetzel & Luger, 1978; Mutika et al., 2013). Colonising field-collected tsetse flies 
on in vitro feeding is challenging, due to the initial high mortalities, it was, however, 
successful for G. austeni (Opiyo et al., 2000) and G. palpalis gambiensis (Momar Seck, 
ISRA-LNERV, personal communication). An advantage is that an early selection is made 
for field flies that will feed on the artificial system and this will be beneficial for further 
colonization. However, the high initial mortality necessitates starting with large numbers as 
was the case with G. brevipalpis. A different feeding approach seems to be required for 
G. austeni as none fed on the membrane system. Because of the low initial G. austeni 
numbers there should be a system in place that will reduce the mortality in collected flies. 
The failure to colonise G. brevipalpis from KwaZulu-Natal was mainly due to three 
reasons. Firstly, the field flies were reluctant to feed on blood using an in vitro feeding 
system. Although a combination of bovine and porcine blood can be used to increase 
survival and pupae production, this combined blood diet did not increase the feeding 
response and could not reduce the initial mortalities. High mortalities were mainly due to 
flies not feeding and starvation. Phagostimulants mixed with the combined blood meal 
might improve the initial feeding response; however, this still needs to be evaluated. A 
second contributing factor was the high mortality during transport from the field to the ARC-
OVI rearing facility. Transporting pupae rather than adults might solve this problem. The 
third and most significant problem was the very low adult emergence rate. The reasons for 
this very low emergence are not clear, but sub-optimal environmental conditions at which 
the pupa were kept as well as the use of a blood meal of low quality might be contributing 
factors. Further effort is required to improve fly emergence. Nash et al. (1968) developed a 
pupae maintenance system where pupae were collected in dry sand trays suspended over 
wet sand to increase humidity. The benefits of keeping the G. brevipalis (KwaZulu-Natal 
strain) pupae in such a management system need to be investigated. 
The mass-rearing of tsetse flies remains challenging, especially so if more than one 
species is involved. The optimal rearing diet may differ between colonies and tsetse species 
and might need to be customised for each production unit. Decisions on the most suitable 
rearing diet will not only depend on the biological requirements of the flies involved but will 
also be influenced by the availably of a suitable blood source on a continuous and economic 
basis. Quality control and research on factors to optimise the diet needs to be done 
continuously. 
84 
 
Chapter 5: Radiation sensitivity 
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Chapter 5 
 
Evaluation of radiation sensitivity of tsetse males 
 
5.1 Introduction 
The idea of using tsetse flies to control tsetse flies was conceived by F.L. Vanderplank and 
colleagues in the 1930s and 1940s. Several studies (Corson, 1932; Potts, 1944; 
Vanderplank, 1944; 1947; 1948) demonstrated that laboratory crosses between Glossina 
morsitans and Glossina swynnertoni produced offspring with low fertility. These hybrid 
males were sterile and the females partially so (Vanderplank, 1947). Vanderplank (1944) 
suggesting that this induced sterility, by crossing two closely related species, could be used 
to control populations, especially as random matings between these two species had been 
recorded (Jackson, 1945). 
The successful implementation of this control technique (hybrid sterility) was 
demonstrated in an arid area in Tanzania where only G. swynnertoni was found. For a 
period of seven months, from August 1944 to February 1945, 101 000 fertile G. morsitans 
were released in this area from adults that had emerged from field collected pupae. The 
size of the target area was 26 km2 and geographically isolated from other tsetse fly infested 
areas with a barrier of approximately 19 km (Klassen & Curtis, 2005). After the release of 
these G. morsitans adults, the numbers of G. swynnertoni declined drastically and 
continued doing so into 1946 (Vanderplank, 1947). This release of G. morsitans virtually 
eliminated G. swynnertoni from the area. Since the released G. morsitans were not able to 
become established in this arid habitat their numbers also declined. The area was returned 
to the local inhabitants who, then, cleared the bush to eliminate any tsetse fly habitat 
(Klassen & Curtis, 2005). This was one of the first examples showing that sterility, induced 
when different species are hybridized, can be used for tsetse fly control (Robinson, 2005). 
Independent of Vanderplank, E.F. Knipling suggested in the 1930s that sterile males, 
although he was unable to sterilise them, could be used to reduce or eradicate wild 
populations of pest insects (Lindquist, 1955; Klassen & Curtis, 2005). Although it was 
already known before the 1930s that X-rays and ionizing radiation could induce sterility in 
insects (Runner, 1916; Muller 1927), it was only in the 1950s that Knipling became aware 
of the detrimental biological effects of radiation (Muller, 1950) and its potential for insect 
control (Baumhover, 2001; 2002). This led to the most successful area-wide integrated pest 
management (AW-IPM) programme, integrating a Sterile Insect Technique (SIT) 
component, that was implemented over 50 years, i.e. the eradication of the New World 
screwworm Cochliomyia hominivorax (Diptera: Calliphoridae) from the southern USA, 
85 
 
Chapter 5: Radiation sensitivity 
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Mexico and Central America to Panama (Van der Vloedt & Klassen, 1991; Vargas-Terán 
et al., 2005). Presently the SIT is used for the control of a variety of insects of agricultural, 
veterinary and medical importance (Dyck et al., 2005). 
After the successful control of G. swynnertoni in Tanzania (Vanderplank, 1947; 
Klassen & Curtis, 2005), the SIT was used in feasibility studies (chemical or radiation 
sterilization) for the control of Glossina morsitans morsitans in Zimbabwe and Tanzania, 
Glossina tachinoides in Chad and Glossina palpalis gambiensis in Burkina Faso (Dame & 
Schmidt, 1970; Cuisance & Itard, 1973; Dame et al., 1975; 1981; Van der Vloedt et al., 
1980; Williamson et al., 1983a; Klassen & Curtis, 2005). The first eradication campaign that 
integrated the use of radiation-sterilized adults with other suppression methods such as 
insecticide impregnated targets was implemented in Burkina Faso in the 1980’s against 
Glossina morsitans submorsitans, G. p. gambiensis and G. tachinoides (Politzar & 
Cuisance, 1984). Glossina palpalis palpalis was simultaneously targeted in Nigeria and 
populations of all these four species were eradicated from the target areas (Takken et al., 
1986). These control strategies were, however, not following area-wide IPM principles, and 
their pest free status was lost due to reinvasion (Klassen & Curtis, 2005). 
The most successful AW-IPM programme, with a SIT component to eradicate tsetse 
flies, was implemented on Unguja Island, Zanzibar, in the 1990’s (Vreysen et al., 2000). 
Suppression of Glossina austeni by means of insecticide-treated screens and cattle was 
started in 1988 and from August 1994 to December 1997, 8.5 million sterile male flies were 
released on the iland (Vreysen et al., 2000). This was also the first aerial releases of sterile 
tsetse males. The last wild tsetse fly was collected on the island in September 1996 and to 
date Unguja Island is still free of tsetse flies and Trypanosomosis. 
The current eradication campaign of a G. p. gambiensis population in the Niayes, 
Senegal also integrates SIT into an AW-IPM programme (Bouyer et al., 2010; Dicko et al., 
2014). The Niayes area was divided into three zones for control. Suppression with 
insecticide treated targets and cattle started in the first zone in December 2010 and sterile 
males were released as of March 2012, and the last wild female was collected in August 
2012 (Dicko et al., 2014). In the second zone suppression started in November 2012 and 
sterile males were released in March 2014. During 2016 sterile males were still being 
released in zone two with suppression underway in the third zone (Dicko et al., 2014). 
To be successful, the released sterile males must be able to compete with local wild 
males (Vreysen et al., 2011). In this chapter the focus will be on the influence of radiation 
on the productivity of the insect. The effect of gamma radiation on the reproduction and 
competitiveness of several tsetse species has been investigated. Radiation doses ranging 
from 50 Gy for Glossina brevipalpis up to 170 Gy for G. tachinoides has been reported to 
86 
 
Chapter 5: Radiation sensitivity 
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induce acceptable or complete sterility in these two species (Itard, 1968; Vreysen et al., 
1996). A dose of 110 Gy to 120 Gy has been reported sufficient to induce sterility in a 
number of species, i.e. G. austeni (Curtis, 1968), G. tachinoides (Offori & Clock, 1975), 
G. p. palpalis (Van der Vloedt et al., 1978), Glossina pallidipes (Opiyo, 2001) and 
G. p. gambiensis (Sow et al., 2012). 
The G. brevipalpis and G. austeni colonies at the Agricultural Research Council – 
Onderstepoort Veterinary Institute (ARC-OVI) in Pretoria, South Africa have been in culture 
for longer than 30 years (Chapter 4). Very little is known on the radiation sensitivity of 
G. brevipalpis adults and there is no information on irradiation of pupae. The radiation 
sensitivity of G. austeni has been investigated previously, however, the sensitivity of these 
long colonised tsetse flies was re-evaluated. 
 
5.2 Materials and methods 
5.2.1 Colony tsetse flies 
For radiation sensitivity studies of adults and pupae of G. brevipalpis and G. austeni, the 
flies were derived from the laboratory colonies housed at the ARC-OVI. The origin and 
holding conditions (Feldmann, 1994a; FAO/IAEA standard operating procedures 2006) of 
these colonies are described in detail in Chapter 4. 
Adult males of G. brevipalpis and G. austeni were irradiated four days after 
emergence. For pupae irradiation they were collected from the colonies in 24 hour 
increments to synchronise adult emergence. The pupae were irradiated on three specific 
times, i.e., three (group 1), five (group 2) or seven (group 3) days before expected 
emergence. The G. brevipalpis were irradiated on day 41 (group 1), 39 (group 2) or 37 
(group 3) of pupation and the G. austeni on day 36 (group 1), 34 (group 2) or 32 (group 3) 
of pupation. 
 
5.2.2 Radiation evaluation procedures 
Adult males and pupae were given a radiation dose of either 40 Gy, 80 Gy, 100 Gy, 120 Gy 
or 140 Gy using a Caesium Gammacell providing a dose rate of 0.69 Gy/min. To determine 
their reproductive success, six-day-old males from all treatments (pupae and adults) were 
mated with three-day-old virgin females at a 1 : 2 male (N = 15) : female (N = 30) ratio. All 
treatments were repeated three to four times. Males and females were kept together for 
four days in standard holding cages under colony conditions (23-24 °C, 75-80% RH and 
sub-dued/indirect lighting) (Feldmann, 1994a; FAO/IAEA standard operating procedures, 
2006). The experimental flies were fed daily on bovine blood using an artificial membrane. 
87 
 
Chapter 5: Radiation sensitivity 
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The blood was collected with a closed sterile system as described in Chapter 4 (Feldmann, 
1994a; FAO/IAEA standard operating procedures, 2006). 
Male and female survival was monitored daily and female pupae production recorded. 
Fecundity was determined by the number of pupae produced per mature female day (Curtis, 
1968). Mature female days were calculated for each treatment by adding the number of 
flies alive each day, starting on day 18 after emergence until the end of the experiment on 
day 60 (Curtis, 1968). 
All pupae produced were mechanically sorted into five distinct size classes as 
described in Chapter 4. Adult emergence rate was also recorded. Abortions of eggs and 
immature larval stages were monitored daily. After 60 days all surviving females were 
dissected to determine their reproductive status, insemination rate and spermatecae fill 
(Feldmann, 1994a; FAO/IAEA standard operating procedures, 2006). The spermatecae 
were removed and the fill microscopically scored as either, empty (0), quarter full (0.25), 
half (0.5), three quarters (0.75) or full (1) (Nash, 1955). Male mortality was monitored until 
all the males had died. 
 
5.2.3 Statistical analysis 
Data were analysed using the statistical software GraphPadInstat (version 3.00, 2003). 
Proportional differences in adult emergence rates were determined with Chi-square (χ2) 
analysis with the Yate’s continuity correction. Linear and multiple regression analyses were 
carried out on fecundity as well as male survival in relation to radiation dose. All tests were 
done at the 5% significance level. 
 
5.3 Results 
5.3.1 Adult emergence rate 
Glossina brevipalpis females irradiated as pupae three days before expected emergence 
(group 1), started emerging between day 37 and 38 after larviposition, continued for two 
days and peaked on day 39. The males from this group started to emerge between day 40 
and 41, peaked from day 42 to 43 and no emergence was seen after day 44 post 
larviposition. Total adult emergence varied from four days for the non-irradiated pupae and 
pupae irradiated with 40 Gy to seven days for pupae treated with 120 Gy. Although the 
dose did not affect the day emergence started it extend this period markedly. 
This same trend was observed for pupae irradiated five (group 2) and seven days 
(group 3) before emergence. The females from group 2 pupae started to emerge between 
day 38 and 39 and peaked at 39 and 40 post larviposition. The males started to emerge 
between day 41 and 43, peaked on day 43 and no more flies emerged after 49 days. The 
88 
 
Chapter 5: Radiation sensitivity 
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total adult emergence period ranged from seven days for the un-irradiated pupae and those 
irradiated with 40 Gy to 10 days for pupae done with 140 Gy. The females from group 3 
pupae started to emerge between day 39 and 41 and peaked on day 42. The males started 
on day 43, peaked on day 45, and emergence stopped on day 47 post larviposition. The 
total adult emergence period varied from six days for un-irradiated pupae to eight days for 
pupae irradiated with 140 Gy. The differences seen in the emergence period between 
pupae irradiated three, five and seven days before emergence was within the normal 
variation seen in the controls (non-irradiated pupae) of each group. 
Adult emergence from the G. brevipalpis pupae that were treated with the five doses 
was compared with that of the controls for each of the three radiation groups (Table 5.1). 
Adult emergence from the G. brevipalpis pupae (group 1) that had been treated with a dose 
of 100 Gy or higher was significantly lower (100 Gy: P < 0.01, χ2 = 20.14, d.f. = 1; 120 Gy: 
P < 0.01, χ2 = 29.07, d.f. = 1; 140 Gy: P < 0.01, χ2 = 25.62, d.f. = 1) as compared to 
emergence of the control group (95.2%) (Table 5.1). For group 2 pupae, adult emergence 
from pupae irradiated with a dose of 40 Gy (P = 0.03, χ2 = 4.69, d.f. = 1), 80 Gy (P = 0.03, 
χ2 = 4.69, d.f. = 1) and 120 Gy (P < 0.01, χ2 = 13.29, d.f. = 1) was significantly lower from 
that of the controls (77.3%). For group 3 pupae, adult emergence from pupae that had been 
irradiated with a dose of 140 Gy was significantly higher (P = 0.04, χ2 = 4.12, d.f. = 1) from 
that of the control ones (69.6%) (Table 5.1). On average the male to female ratio of 
G. brevipalpis that emerged from all three pupal treatment groups and the control pupae 
was 1 : 1 and did not differ significantly between treatments (Table 5.1). 
 
Table 5.1. Comparison of emergence rates of adult Glossina brevipalpis from pupae 
irradiated with different doses and on different days before expected emergence. 
Radiation dose Males Females Total Male / Female 
No. pupae 
(Gy) (%) (%) (%) ratio 
Pupae irradiated 3 days before emergence (group 1) 
   Control 167 88 (55.4) 71 (44.7) 159 (95.2) 1 : 0.8 
   40 167 88 (54.7) 73 (45.3) 161 (96.4) 1 : 0.8 
   80 167 74 (49.7) 75 (50.3) 149 (89.2) 1 : 1 
   100 167 63 (48.5) 67 (51.5) 130 (77.8)* 1 : 1.1 
   120 167 47 (38.5) 75 (61.5) 122 (73.1)* 1 : 1.6 
   140 167 72 (57.6) 53 (42.4) 125 (74.9)* 1 : 0.7 
Pupae irradiated 5 days before emergence (group 2) 
   Control 132 65 (63.7) 37 (36.3) 102 (77.3) 1 : 0.6 
   40 132 57 (67.1) 28 (32.9) 85 (64.4)* 1 : 0.5 
   80 132 59 (69.4) 26 (30.6) 85 (64.4)* 1 : 0.5 
   100 132 59 (60.8) 38 (39.2) 97 (73.5) 1 : 0.6 
   120 132 32 (43.8) 41 (56.2) 73 (55.3)* 1 : 1.3 
   140 132 50 (54.4) 42 (45.7) 92 (69.7) 1 : 0.8 
Pupae irradiated 7 days before emergence (group 3)   
   Control 112 34 (43.6) 44 (56.4) 78 (69.6) 1 : 1.3 
   40 112 38 (52.8) 34 (47.2) 72 (64.3) 1 : 0.9 
   80 112 44 (55.0) 36 (45.0) 80 (71.4) 1 : 0.8 
   100 112 44 (54.3) 37 (45.7) 81 (72.3) 1 : 0.8 
   120 112 47 (57.3) 35 (42.7) 82 (73.2) 1 : 0.7 
   140 112 55 (59.8) 37 (40.2) 92 (82.1)* 1 : 0.7 
*Emergence rate statistical significant different from the control group at the 5% level. 
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On average adult emergence started five days (± 1.55) earlier in G. austeni than in 
G. brevipalpis. Different to G. brevipalpis dose rate did affect the commencement day for 
the total duration of the emergence period for all of the pupae groups. Female emergence 
from group 1 pupae irradiated three days before expected emergence started between day 
33 and 34 post larviposition and peaked on day 37. Male emergence in this group 
commenced between day 36 and 37, peaked on day 38 and stopped on day 40 post 
larviposition. A similar pattern was seen in the pupae irradiated five (group 2) and seven 
(group 3) days before expected emergence. The females started to emerge between day 
33 and 35 for group 2 pupae and between day 31 and 35 post larviposition for group 3 
pupae. Female emergence from group 3 pupae peaked between day 36 and 38 and for 
group 2 pupae on day 36 post larvipositon. The male flies started to emerge from group 2 
and 3 pupae between day 36 and 38 and between days 36 and 37, respectively. Adult male 
emergence peaked on day 39 for both pupae groups 2 and 3 and no more emergence was 
seen in both groups 41 days post larviposition. 
The emergence rate of the G. austeni pupae irradiated with five doses was also compared 
separately with those of the untreated controls for each treatment group (Table 5.2).  
 
Table 5.2. Comparison of emergence rates of Glossina austeni pupae irradiated with 
different doses and on different days before expected emergence. 
Radiation Males Females Total Female / Male 
No. pupae   
dose (Gy) (%) (%) (%) Ratio 
Pupae irradiated 3 days before emergence (group 1) 
   Control 106 43 (49.4) 44 (50.6) 87 (82.1) 1 : 1 
   40 103 47 (52.8) 42 (47.2) 89 (86.4) 1 : 0.9 
   80 103 51 (58.0) 37 (42.1) 88 (85.4) 1 : 0.7 
   100 103 53 (52.5) 48 (47.5) 101 (98.1)* 1 : 1.1 
   120 103 55 (55.6) 44 (44.4) 99 (96.1)* 1 : 0.8 
   140 103 47 (51.1) 45 (48.9) 92 (89.3) 1 : 1 
      
Pupae irradiated 5 days before emergence (group 2) 
   Control 112 49 (62.0) 30 (38.0) 79 (70.5) 1 : 0.6 
   40 110 60 (53.5) 50 (46.5) 110 (100.0)* 1 : 0.8 
   80 110 60 (58.0) 47 (42.1) 107 (85.4)* 1 : 0.8 
   100 110 55 (52.5) 47 (47.5) 102 (98.1)* 1 : 0.9 
   120 110 59 (55.6) 46 (44.4) 105 (96.1)* 1 : 0.8 
   140 110 45 (51.1) 57 (48.9) 102 (89.3)* 1 : 1.3 
      
Pupae irradiated 7 days before emergence (group 3)   
   Control 152 75 (54.4) 63 (45.7) 138 (90.8) 1 : 0.8 
   40 149 61 (49.2) 63 (50.8) 124 (83.2) 1 : 1 
   80 149 68 (52.7) 61 (47.3) 129 (86.6) 1 : 0.9 
   100 149 82 (60.3) 54 (39.7) 136 (91.3) 1 : 0.7 
   120 149 67 (51.9) 62 (48.1) 129 (86.6) 1 : 0.9 
   140 149 78 (56.5) 60 (43.5) 138 (92.6) 1 : 0.8 
* Emergence rate statistical significant different from the control group at the 5% level. 
 
Adult emergence from group 1 pupae (irradiated three days before emergence) that 
had been treated with a dose of 100 Gy (P < 0.01, χ2 = 13.05, d.f. = 1) and 120 Gy (P < 0.01, 
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χ2 = 9.13, d.f. = 1) was significantly greater as compared with that of the controls (82.1%) 
(Table 5.2). Adult emergence from group 2 pupae (irradiated five days before emergence) 
was significantly greater than that of the controls (70.5%) (40 Gy: P < 0.01, χ2 = 35.78, 
d.f. = 1; 80 Gy: P < 0.01, χ2 = 27.27, d.f. = 1; 100 Gy: P < 0.01, χ2 = 16.71, d.f. = 1; 120 Gy: 
P < 0.01, χ2 = 22.57, d.f. = 1; 140 Gy: P < 0.01, χ2 = 16.701, d.f. = 1) (Table 5.2). Adult 
emergence from group 3 pupae was similar for all doses as compared with the untreated 
control group.  In all treatment groups G. austeni emerged in equal female to male ratios, 
with the exception of the pupae irradiated with 140 Gy in group 2 (P = 0.02, χ2 = 5.02, 
d.f. = 1) (Table 5.2). 
 
5.3.2 Reproduction in females mated with males irradiated as adults or pupae 
Of the 2820 G. brevipalpis females (30 for each treatment replicate) at the onset of the 
experiments, 2668 survived to day 18 post emergence. The survival of mature 
G. brevipalpis females in all individual experiments exceeded 95.9% on day 18, except in 
the group 3 pupae (irradiated seven days before emergence) where mature female survival 
was lower (88.8%). The survival rate decrease at the end of the experiment (day 60) for all 
treatmnets. 
Of the 2730 G. austeni females (30 for each treatment replicate) at the onset of the 
experiments, 2449 survived to day 18 post emergence. Although the survival for the mature 
females was lower than that of G. brevipalpis on day 18, it still exceeded 88.6%. The 
survival rate for G. austeni females also decreases at the end of the experiment (day 60) 
for all treatments. 
As the radiation dose increased, fecundity (number of pupae produced per mature 
female) for both species decreased (Table 5.3 & 5.4). A negative linear regression was 
found between fecundity of the untreated female G. brevipalpis that had mated with adult 
males treated at the five radiation levels (r2 = 0.616, P < 0.01), or that had been irradiated 
as pupae in all the pupae groups (r2 = 0.57, P = 0.01). A similar negative linear regression 
was found between fecundity of untreated female G. austeni and radiation dose 
administered to their male mates as adults (r2 = 0.81, P < 0.01) and as group 1 (r2 = 0.72, 
P < 0.01), group 2 (r2 = 0.73, P < 0.01) and group3 (r2 = 0.71, P < 0.01), pupae. This showed 
fecundity to be dose dependent. The number of pupae produced by untreated female 
G. brevipalpis that had mated with males treated as adults with 40 Gy (number of pupae = 
16) and 80 Gy (N = 2) was 6.7% and 0.8%, respectively as compared with the untreated 
controls (N = 240). The pupal production (Table 5.3) relative to the control group for the 
females mated with G. brevipalpis males irradiated with 40 Gy as pupae was 1.4% for group 
1, 1.9% for group 2 and 2.9% for group 3 pupae. A radiation dose of 40 Gy and 80 Gy was 
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thus sufficient to induce 93% and 99% sterility, respectively in G. brevipalpis females when 
the males had been treated as adults. Induced sterility was 97% or higher when the males 
had been irradiated with 40 Gy for the different groups of pupae. 
A dose of 40 Gy induced only 61.5% sterility in female G. austeni that had mated with 
males irradiated as adults. In females mated with males emerging from pupae irradiated 
three and five days before emergence the sterility was 61.6%, whereas it was 77.3% when 
the pupae were treated seven days before emergence. Higher doses of 80 Gy and 100 Gy 
induced 97% to 99% sterility in females that mated with males treated as adults or pupae. 
The pupal production (Table 5.4) relative to the controls for females mated with males 
irradiated as adults with a dose of 80 Gy was 2.6% and for males treated as pupae it was 
2.2% in group 1, 3.1% in group 2 and 1.8% in group 3 pupae. Using a dose of 100 Gy the 
pupal production (Table 5.4) relative to the controls was 1.3%, 2.5% and 2.2% for females 
mated with males irradiated as pupae of group 1, 2 and 3, respectively. No pupae were 
produced by females that mated with males treated as adults with a dose of 100 Gy. 
The number of eggs aborted during the 60 days experimental period was lower for 
both species in females that mated with non-irradiated males than in females mated with 
any of the males in the experimental groups (Table 5.3 & 5.4). For both species and all 
treatment groups the majority of the pupae produced were in or above the pupal size class 
C (Table 5.3 & 5.4). The male to female ratio that emerged from pupae produced by females 
mated with irradiated males were similar for both species and equally distributed (1 : 1 ratio) 
(Table 5.3 & 5.4). 
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Table 5.3. Production of Glossina brevipalpis females mated with males irradiated with different doses at different developmental stages. 
      Pupal size classes (mg)   
No. of A (%) B (%) C (%) D (%) E (%) 
No. of No. 
 Radiation dose mature % Emergence Replications aborted pupae Fecundity# 
(Gy) females <56 56-<68 68-<76 76-<84 >84 / % females 
eggs produced 
(day 18) 
Irradiated Male Adults 
   Control 4 119 36 240 7 (2.9)  18 (7.5) 60 (25.0) 85 (35.4) 79 (29.2) 0.046 92.9 / 49.3 
   40 4 116 341 16 2 (12.5) 1 (6.3) 6 (37.5) 3 (18.8) 4 (25.0) 0.003 93.8 / 46.7 
   80 4 114 326 2 0 1 (50.0) 0 1 (50.0) 0 0 2 / 2 / 1 / 2 
   100 4 119 402 0 0 0 0 0 0 0  
   120 4 112 287 0 0 0 0 0 0 0  
   140 4 119 307 0 0 0 0 0 0 0  
 
Pupae irradiated 3 days before emergence (group 1) 
   Control 4 114 20 294 15 (6.2) 45 (18.7) 87 (36.1) 68 (28.2) 26 (10.8) 0.060 67.2 / 61.4 
   40 4 116 248 4 0 0 2 (100) 0 0 0.001 3 /4 /2 / 4 
   80 4 116 218 0 0 0 0 0 0 0  
   100 4 109 187 1 1 (100) 0 0 0 0 <0.001 0 / 1 / 0 / 1 
   120 4 119 240 1 0 0 0 1 (100) 0 <0.001 0 / 1 / 0 / 1 
   140 3 88 152 0 0 0 0 0 0 0  
 
Pupae irradiated 5 days before emergence (group 2) 
   Control 4 115 13 269 25 (9.3) 59 (21.9) 92 (34.1) 83 (30.7) 11 (4.1) 0.057 86.3 / 58.2 
   40 4 115 230 5 1 (20) 2 (40) 1 (20) 1 (20) 0 0.001 80.0 / 25.0 
   80 4 118 252 0 0 0 0 0 0 0  
   100 4 113 232 3 3 (100) 0 0 0 0 0.001 66.7 / 50.0 
   120 4 116 223 1 0 1 (100) 0 0 0 <0.001 0 / 1 / 0 / 1 
   140 4 117 221 1 0 0 0 0 0 <0.001 0 / 1 / 0 / 1 
 
Pupae irradiated 7 days before emergence (group 3) 
   Control 4 116 10 343 24 (7.8) 64 (20.7) 109 (35.3) 90 (29.1) 22 (71.2) 0.058 91.8 / 52.9 
   40 4 106 181 10 1 (11.1) 3 (33.3) 6 (55.6) 0 0 0.023 30.0 / 66.7 
   80 4 111 176 0 0 0 0 0 0 <0.001  
   100 4 109 174 0 0 0 0 0 0 0  
   120 4 96 150 2 0 1 (50) 1 (50) 0 0 0.001 0 / 0 / 0 / 0 
   140 3 75 118 2 0 0 2 (100) 0 0 <0.001 0 / 0 / 0 / 0 
# Number of pupa produced per the mature female day 
 
 
 
 
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Table 5.4. Production of Glossina austeni females mated with males irradiated with different doses at different developmental stages. 
      Pupal size classes (mg)   
No. of A (%) B (%) C (%) D (%) E (%) 
No. of 
Radiation mature No. pupae % Emergence / 
Replications aborted Fecundity# 
dose (Gy) females produced <16 16-<19 19-<21 21-<23 >23 % females 
eggs 
(day 18) 
Irradiated Male Adults 
   Control 4 109 40 234 18 (7.7) 17 (7.3) 30 (12.8) 51 (21.8) 118 (50.4) 0.063 93.2 / 55.1 
   40 4 106 201 90 7 (7.78) 10 (11.1) 11 (12.2) 15 (16.7) 47 (52.2) 0.022 92.2 / 53.0 
   80 4 107 209 6 2 (33.33) 0 1 (16.67) 1 (16.67) 2 (33.33) 0.001 83.3 / 60.0 
   100 4 102 210 0 0 0 0 0 0 0  
   120 4 109 224 1 0 0 1 (100) 0 0 <0.001 1 / 1 / 0 / 1 
   140 4 105 225 1 0 0 1 (100) 0 0 <0.001 0 / 1 
 
Pupae irradiated 3 days before emergence (group 1) 
   Control 4 97 45 226 4 (1.8) 9 (4.0) 30 (13.3) 82 (23.0) 132 (58.0) 0.064 89.4 / 59.4 
   40 4 110 261 70 1 (1.4) 2 (2.9) 7 (10.0) 10 (14.3) 50 (71.4) 0.015 87.1 / 54.4 
   80 4 114 268 5 0 0 0 2 (40.0) 3 (60.0) 0.001 4 / 5 / 1 / 5 
   100 4 110 280 3 0 0 0 1 (33.33) 2 (66.66) 0.001 2 / 3 / 1 / 3 
   120 4 111 241 2 0 0 0 0 2 (100) 0.001 2 / 2 / 2 / 2 
   140 4 107 288 4 0 2 (50.0) 1 (25.0) 0 1 (25.0) 0.001 4 / 4 / 1 / 4 
 
Pupae irradiated 5 days before emergence (group 2) 
   Control 3 75 40 159 16 (10.1) 10 (6.3) 40 (28.9) 29 (18.2) 58 (36.5) 0.062 57.9 / 45.7 
   40 3 78 121 61 2 (3.3) 3 (4.9) 21 (34.4) 18 (29.5) 17 (27.9) 0.021 80.3 / 63.3 
   80 3 78 156 5 0 0 1 (20.0) 3 (60.0) 1 (20.0) 0.001 3 / 5 / 2 / 5 
   100 3 87 151 4 1 (25.0) 0 1 (50.0) 0 1 (25.0) 0.001 2 / 4 / 1 / 4 
   120 3 88 167 0 0 0 0 0 0 0  
   140 4 112 178 0 0 0 0 0 0 0  
 
Pupae irradiated 7 days before emergence (group 3) 
   Control 4 99 44 277 11 (40.9) 21 (78.1) 36 (13.4) 62 (23.1) 139 (51.7) 0.070 53.4 / 65.5 
   40 4 111 168 63 5 (79.4) 7 (11.1) 13 (20.6) 13 (20.6) 25 (39.7) 0.014 69.8 / 65.9 
   80 4 106 239 5 0 0 1 (20.0) 2 (40.0) 2 (40.0) 0.002 2 / 5 / 1 / 2 
   100 4 97 200 6 0 0 4 (60.0) 2 (40.0) 0 0.004 3 / 6 / 2 / 3 
   120 4 118 230 0 0 0 0 0 0 0  
   140 4 113 223 2 0 0 0 0 2 (100) <0.001 2 / 2 / 1 / 2 
# Number of pupa produced per the mature female day 
 
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Chapter 5: Radiation sensitivity 
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Table 5.5. Reproductive status of Glossina brevipalpis females mated with irradiated males at different developmental stages and radiation levels 
and dissected after an experimental period of 60 days. 
       Uterus  
   Spermatecae fill   Viable instar larvae 
Empty due 
Recently to abortion 
Radiation dose (Gy) Insemination %  I II III 
ovulated egg (abortion 
0.25 (%) 0.5 (%) 0.75 (%) 1 (%) rate) 
Irradiated Male Adults         
   Control 92.2 16 (13.8) 27 (23.3) 60 (51.7) 4 (3.5) 50 17 (0.15) 16 14 16 
   40 100 10 (8.9) 25 (22.1) 62 (54.9) 16 (14.2) 15 98 (0.87) 0 0 0 
   80 91.9 23 (20.7) 29 (26.1) 37 (33.3) 13 (11.7) 8 103 (0.93) 0 0 0 
   100 99.1 5 (4.3) 33 (28.5) 56 (48.3) 21 (18.1) 18 98 (0.84) 0 0 0 
   120 100 15 (14.0) 36 (33.6) 40 (37.4) 16 (15.0) 19 88 (0.82) 0 0 0 
   140 100 0 53 (48.2) 46 (41.8) 11 (10.0) 31 79 (0.72) 0 0 0 
       
Pupae irradiated 3 days before emergence (group 1)       
   Control 94.6 13 (11.7) 35 (31.5) 57 (51.4) 0 65 23 (0.21) 7 9 7 
   40 92.7 18 (16.4) 36 (32.7) 48 (43.6) 0 31 76 (0.71) 0 0 0 
   80 87.6 16 (14.2) 46 (40.7) 33 (29.2) 4 (3.5) 18 95 (0.84) 0 0 0 
   100 95.3 28 (26.2) 32 (29.9) 41 (38.3) 1 (0.9) 21 86 (0.80) 0 0 0 
   120 97.4 27 (23.3) 35 (30.2) 51 (44.0) 0 38 78 (0.67) 0 0 0 
   140 89.9 18 (20.2) 35 (28.1) 35 (39.3) 2 (2.3) 10 76 (0.88) 0 0 0 
       
Pupae irradiated 5 days before emergence (group 2)       
   Control 96.9 5 (5.2) 22 (22.9) 63 (65.6) 3 (3.1) 29 12 (0.13) 6 15 31 
   40 95.9 14 (14.4) 33 (34.0) 45 (46.4) 1 (1.0) 26 75 (0.74) 0 0 0 
   80 94.3 12 (11.4) 41 (39.1) 45 (42.9) 1 (1.0) 23 81 (0.78) 0 0 0 
   100 94.3 18 (17.1) 18 (17.1) 60 (57.1) 3 (2.9) 20 86 (0.81) 0 0 0 
   120 97.2 12 (11.1) 34 (31.5) 58 (53.7) 1 (0.9) 28 79 (0.73) 1 0 0 
   140 96.4 16 (14.6) 44 (40.0) 46 (41.8) 0 22 88 (0.80) 0 0 0 
       
Pupae irradiated 7 days before emergence (group 3)       
   Control 97.1 11 (10.5) 26 (24.8) 64 (61.0) 1 (1.0) 49 24 (0.23) 1 14 16 
   40 100 7 (7.9) 26 (29.2) 55 (61.8) 1 (1.1) 27 61 (0.69) 1 0 0 
   80 97.98 14 (14.1) 26 (26.3) 56 (56.6) 1 (1.0) 28 71 (0.72) 0 0 0 
   100 97.67 11 (12.8) 23 (26.7) 50 (58.1) 0 15 69 (0.82) 0 0 0 
   120 93.98 12 (14.5) 28 (33.7) 38 (45.8) 0 17 66 (0.80) 0 0 0 
   140 96.88 2 (3.1) 28 (43.8) 32 (50.0) 0 16 48 (0.75) 0 0 0 
 
 
 
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Table 5.6. Reproductive status of Glossina austeni females mated with irradiated males at different developmental stages and radiation levels 
and dissected after an experimental period of 60 days. 
      Uterus  
  Spermatecae fill   Viable instar larvae 
Empty due 
Radiation dose Recently to abortion 
Insemination %  I II III 
(Gy) ovulated egg (abortion 
0.25 (%) 0.5 (%) 0.75 (%) 1 (%) rate) 
Irradiated Male Adults         
   Control 93.9 12 (18.2) 19 (28.8) 29 (43.9) 2 (3.0) 33 16 (0.26) 2 3 8 
   40 100 13 (15.7) 28 (33.7) 38 (45.8) 4 (4.8) 0 71 (0.84) 0 2 12 
   80 93.3 12 (14.0) 19 (33.7) 24 (27.9) 15 (17.4) 1 74 (0.99) 0 0 0 
   100 97.0 13 (19.4) 21 (31.3) 30 (44.8) 1 (1.5) 1 66 (0.99) 0 0 0 
   120 100 11 (12.4) 38 (42.7) 35 (39.3) 5 (5.6) 7 80 (0.92) 0 0 0 
   140 100 9 (10.5) 39 (45.4) 31 (36.1) 7 (8.1) 2 84 (0.98) 0 0 0 
       
Pupae irradiated 3 days before emergence (group 1)       
   Control 100 12 (18.5) 18 (27.7) 35 (53.9) 0 32 3  (0.05) 11 8 6 
   40 100 10 (10.8) 40 (43.0) 42 (45.2) 1 (1.1) 37 45 (0.48) 2 4 5 
   80 99.0 4 (4.0) 33 (32.7) 62 (61.4) 1 (1.0) 17 84 (0.83) 0 0 0 
   100 100 9 (10.0) 38 (42.2) 43 (47.8) 0 23 67 (0.74) 0 0 0 
   120 100 16 (12.0) 36 (43.4) 37 (44.6) 0 21 62 (0.75) 0 0 0 
   140 100 13 (14.0) 47 (49.0) 36 (37.5) 0 14 82 (0.85) 0 0 0 
       
Pupae irradiated 5 days before emergence (group 2)       
   Control 100 3 (7.0) 11 (25.6) 27 (62.8) 2 (4.7) 17 12 (0.28) 1 4 9 
   40 100 0 7 (11.3) 52 (83.9) 3 (4.8) 10 45 (0.73) 0 3 4 
   80 100 4 (6.0) 8 (11.9) 49 (73.1) 6 (9.0) 12 54 (0.81) 1 0 0 
   100 100 5 (8.3) 10 (16.7) 42 (70.0) 3 (5.0) 9 51 (0.85) 0 0 0 
   120 100 2 (2.6) 10 (19.2) 60 (75.7) 2 (2.6) 10 68 (0.87) 0 0 0 
   140 100 7 (7.0) 26 (26.0) 67 (67.0) 0 7 93 (0.93) 0 0 0 
       
Pupae irradiated 7 days before emergence (group 3)       
   Control 98.8 7 (8.4) 28 (33.7) 44 (53.0) 3 (36.1) 42 15 (0.19) 4 8 12 
   40 97.9 6 (6.5) 29 (31.2) 53 (60.0) 3 (32.3) 17 64 (0.71) 1 5 3 
   80 100 6 (6.3) 25 (26.3) 62 (65.3) 2 (21.1) 15 81 (0.84) 0 0 0 
   100 100 4 (4.5) 22 (25.3) 59 (67.8) 2 (23.0) 11 76 (0.87) 0 0 0 
   120 99.1 5 (4.6) 27 (25.0) 72 (66.7) 3 (27.8) 14 83 (0.85) 1 0 0 
   140 99.1 9 (8.6) 24 (22.9) 67 (63.8) 4 (38.1) 18 87 (0.83) 0 0 0 
 
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Chapter 5: Radiation sensitivity 
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5.3.3 Reproductive status of females inseminated by males irradiated as adult or 
pupae 
Average insemination of female G. brevipalpis (Table 5.5) and G. austeni (Table 5.6) flies, 
that were mated with males irradiated as adults was 98.0% ± 3.55 and 98.1% ± 2.96, 
respectively, as shown by dissection at the end of the experimental period of 60 days. 
Average insemination of untreated controls was 92.2% for G. brevipalpis and 93.9% for 
G. austeni which clearly showed that treatment of adult males did not affect their ability to 
transfer sperm to the females. Similar high insemination rates, independent of dose, were 
seen for both species irradiated as pupae (Table 5.5 & 5.6). 
The reproductive status of the females of both species dissected at the end of the 
experiment at 60 days was markedly different between the females that had mated with 
non-irradiated males compared with treated males (Table 5.5 & 5.6). For both species the 
uterus of females mated with males irradiated as adults were, either empty due to abortions 
or contained a recently ovulated egg. Except G. austeni females of the 40 Gy-group, almost 
none of these females had any viable larvae in utero (Table 5.6). The same trend was found 
for all the pupal groups of both species, irrespective of dose (Table 5.5 & 5.6). In contrast, 
for both species the uterus of females that had mated with non-irradiated males as well as 
G. austeni females mated with males of the 40 Gy dose contained either a recently ovulated 
egg or a viable instar larva, and fewer females were found with an empty uterus due to an 
abortion (Table 5.5 & 5.6). All the females of both species at the end of 60 days appeared 
to be in good health, however, a build-up of fat bodies was observed in the females mated 
with males irradiated as adults or pupae. 
 
5.3.4 Male survival 
The average lifespan of G. brevipalpis males was radiation dose dependent with survival 
decreasing with increasing doses. A relatively strong negative linear regression was found 
between lifespan and dose rate in males treated as adults (r2 = 0.67, P < 0.01), males 
irradiated as group 1 (r2 = 0.83, P < 0.01), group 2 (r2 = 0.79, P < 0.01) and group 3 
(r2 = 0.82, P < 0.01) pupae (Fig. 5.1). The age of the G. brevipalpis pupae at the time of 
irradiation affected the lifespan of the adult males. The males irradiated as adults lived the 
longest followed by males from the group 1 pupae, then group 2, and finally group 3 pupae 
(Fig. 5.1). 
In G. austeni this negative linear correlation was only found for the males irradiated 
as adults (r2 = 0.72, P < 0.01) and for males treated as pupae seven days before emergence 
(r2 = 0.23, P = 0.02) (Fig. 5.2). The age of the pupae at the time of treatment did not affect 
the lifespan of the adult males as observed with G. brevipalpis males. 
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Fig. 5.1. Lifespan of Glossina brevipalpis males irradiated either as adults or as pupae 
three- (group 1), five- (group 2) or seven- (group 3) days before expected emergence. 
 
 
Fig. 5.2. Lifespan of Glossina austeni males irradiated either as adults or as pupae three- 
(group 1), five- (group 2) or seven- (group 3) days before expected emergence. 
 
 
 
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5.4 Discussion 
Selecting an appropriate radiation dose to sterilise insects destined for release in a 
programme that includes a SIT component is very important, as high radiation doses can 
have a negative effect on the quality of these insects (Bakri et al., 2005; Calkins & Parker, 
2005; Lance & McInnis, 2005). A dose below the optimal will result in insects that are not 
sufficiently sterile and a too high dose may result in insects that are not competitive with 
wild flies (Bakri et al., 2005). Factors such as the developmental stage of the insect, its age 
and the atmosphere used during irradiation can influence dose and the level of sterility 
achieved (Bakri et al., 2005; Calkins & Parker, 2005). 
Similar to other studies (Curtis, 1968; Vreysen et al., 1996), the rate of induced sterility 
or the proportions of dominant lethal mutations induced in the sperm of G. brevipalpis and 
G. austeni when exposed to radiation increased with increasing dose. In the present study 
a dose of 40 Gy and 80 Gy induced 93% and 99% sterility respectively in G. brevipalpis 
females mated with males irradiated as adults, and a 97% or higher sterility when irradiated 
as pupae with a dose of 40 Gy. This is comparable with data presented by Vreysen et al. 
(1996), which indicated that a dose of 50 Gy administered to four- to six-day-old males in 
air induced about 95% sterility. This relatively low dose inducing more than 93% sterility 
indicates that the sperm of G. brevipalis are more sensitive to radiation than the sperm of 
others species such as G. fuscipes fuscipes, G. tachinoides (Vreysen et al., 1996), 
G. pallidipes (Opiyo, 2001), G. morsitans (Curtis & Langley, 1972) and G. austeni (Curtis, 
1969). Although Vreysen et al. (1996) suggested that this higher susceptibility might be 
chromosome related, the main reasons for this difference remain unclear. 
Compared with G. brevipalpis, higher doses of 80 Gy and 100 Gy were needed to 
induce more than 97% sterility in G. austeni females that mated with males treated either 
as adults or pupae. The dose rate of 100 Gy or even 80 Gy is lower than that of 120 Gy 
proposed by Curtis (1970). The same dose (120 Gy) was also used in the successful 
eradication of G. austeni from Unguja Island (Vreysen et al., 2000). Curtis (1969) used 
doses of 50 Gy, 60 Gy and 70 Gy to produce partially sterile G. austeni males and showed 
that these rates induced dominant lethality in 70-90% of their sperm, which is comparable 
to our data. 
The somewhat longer emergence periods and high variation in emergence rate of 
irradiated pupae obtained in the present study may be an artefact of the basic rearing 
conditions in the colony. Due to logistical constrains these two species are kept under the 
same rearing conditions which may not be the most optimal for both species. Although the 
longer emergence periods were still within the natural variation observed for non-irradiated 
pupae these potentially extended pupal periods will have to be taken into consideration in 
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the implementation of an AW-IPM programme with an SIT component, as this can have 
time management and operational coast implications. Furthermore, due to the variation in 
emergence rates obtained for the different irradiation doses and time of irradiation for both 
species no clear effect of these variables could be established.Our data indicates a 
relatively higher irradiation susceptibility of G. brevipalpis and G. austeni pupae as 
compared to adults, and this was also observed by Van der Vloed & Taher (1978) for 
G. p. palpalis. These authors found that the production rate relative to the control for 
G. p. palpalis irradiated with 80 Gy was 7.1 for the adults and 4.08 for the pupae (Van der 
Vloedt et al., 1978). 
Dissection results revealed a clear abortion pattern for almost all of the G. brevipalpis 
females that mated with males, irrespective of treatment dose and life stage when 
irradiated. The same pattern was seen for G. austeni females that mated with males treated 
with 80 Gy or higher. The uterus of females that mated with irradiated males contained 
either an egg or was empty as a result of abortion of the embryo or the immature larva. Van 
der Vloedt & Barnor (1984) suggested that this observation could be used to monitor the 
impact of sterile male releases on a natural tsetse fly population. The imbalance between 
uterus content and the follicle next in ovulation sequence (Vreysen et al., 1996), was indeed 
used successfully to monitor induced sterility in the wild female G. austeni population in the 
eradication campaign on Unguja Island (Vreysen et al., 2000) and against G. p. gambiensis 
in Niayes Senegal (Bouyer et al., 2010). The natural abortion rate of the population needs 
to be determined first for comparison with abortion rates during the sterile male release 
programme. It is clear from the dissection results that this method of assessing reproductive 
abnormalities as a result of mating of wild females with irradiated males, and hence, as a 
tool to monitor induced sterility in the targeted population, can, as in G. austeni and 
G. p. gambiensis, also be applied to G. brevipalpis. In addition, radiation did not affect 
G. brevipalpis and G. austeni males’ insemination ability whether irradiated as adults or as 
pupae. 
The reduction in average longevity of irradiated males as compared with untreated 
males is a manifestation of the somatic damage caused by irradiation (Vreysen et al., 2000). 
This relationship was clearly observed for G. brevipalpis and it was furthermore shown that 
irradiation as pupae reduced adult longevity even more than treatment as adults. This 
negative linear regression between dose and longevity for G. austeni was, however, only 
observed when adults were irradiated. In contrast, an apparent increase in average 
longevity was seen when G. austeni pupae were treated three days before emergence. 
This radiation induced increase in average lifespan of males was also documented for 
G. morsitans pupae irradiated in air (Dean & Wortham, 1969) as well as in nitrogen (Curtis 
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& Langley, 1972). Many reasons for this increase in average lifespan have been suggested 
and it is most likely that a range of radiation induced repair mechanisms are involved 
(Calabrese, 2013). 
The results of the present study clearly indicate that G. brevipalpis adults as well as 
pupae are highly sensitive to irradiation. It also confirms that the 30-year old colonised 
G. austeni is still susceptible to radiation. The results indicate that G. brevipalpis can be 
treated ether as adult or late pupa (up to seven days before emergence) with a dose of 
80 Gy and G. austeni as late pupa (also seven days before emergence) with a dose of 
100 Gy for use in a tsetse fly control programme with a SIT component. In addition to the 
small differences in the quality of sterile males when irradiated as adults or pupae, other 
logistical requirements, e.g. distance of mass-rearing facility and radiation source from 
release site, may also need to be taken into consideration when selecting the most efficient 
irradiation protocol. Treatment of pupae has its advantages, i.e., large numbers of pupae 
can be irradiated at a time, the handling and transport of pupae is less cumbersome, and 
pupae are less fragile than adult flies. The competitiveness of the males irradiated as adults 
or pupae as compared with untreated males needs to be assessed and taken into account 
in any proposed radiation protocol. 
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Chapter 6 
 
Comparative assessment of the mating performance of tsetse males under field cage 
conditions3 
 
6.1 Introduction 
The successful implementation of an area-wide integrated pest management (AW-IPM) 
programme with a sterile insect technique (SIT) component depends on a number of 
prerequisites (Vreysen et al., 2007) of which the biological quality and sexual 
competitiveness of the sterile males are amongst the more important ones (Vreysen, 2005). 
The mass-reared, released sterile males must be able to compete with wild males for 
mating opportunities with the local virgin females (Vreysen et al., 2011). Releasing low 
quality sterile males will necessitate higher release rates, requiring more funding that might 
prolong the duration of the programme and potential failure (Vreysen, 2005). 
The age and radiation dose discussed in Chapter 5 can influence the mating success 
of released sterile males (Liedo et al., 2002; Flores et al., 2014). The competitiveness of 
Glossina fuscipes fuscipes, Glossina palpalis palpalis (Mutika et al., 2001; Abila et al., 
2003), Glossina palpalis gambiensis (Van der Vloedt & Barnor, 1984), and Glossina 
pallidipes (Olet et al., 2002) was significantly influenced by the age of the sterilised males. 
Mutika et al. (2001) indicated that a sterilizing radiation dose of 120 Gy did not affect the 
ability of G. pallidipes males to compete with untreated males. He also found that irradiated 
males that emerged from pupae kept at a low temperature of 15 °C for 24-72 h 
demonstrated increased competitiveness (Mutika et al., 2001). Determining the optimal 
mating age of colonised tsetse flies under field conditions as well as their competitiveness 
after irradiation will be challenging and costly, while the results might also be influenced by 
several environmental, climatic and ecological parameters which cannot be controlled. 
Since 1999, large walk-in field cages have been utilised successfully as a surrogate 
for open field studies to conduct mating compatibility, mating competitiveness and other 
behavioural studies on fruit flies, tsetse flies and Lepidoptera (Cayol et al., 1999; Mutika et 
al., 2001; Vera et al., 2003; Taret et al., 2010). Field cages have been used successfully to 
determine the optimal mating age for G. f. fuscipes, G. p. palpalis and G. p. gambiensis 
(Abila et al., 2003). 
                                                          
3 Partially published as: 
De Beer, C.J., Venter, G.J. & Vreysen, M.J.B. (2015) Determination of the optimal mating age of colonised 
Glossina brevipalpis and Glossina austeni using walk-in field cages in South Africa. Parasites & Vectors, 8, 
467. 
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The SIT has never been used or evaluated for Glossina brevipalpis and no data are 
available on the optimal mating age and competitiveness of irradiated sterilised flies. In the 
successful eradication campaign of G. austeni on Unguja Island, Zanzibar (1993-1997), 
sterile males that were mass-reared at the Vector & Vector-Borne Diseases Research 
Institute, Tanga, United Republic of Tanzania were released when three to five days old 
(Vreysen et al., 2000). Notwithstanding the success on Unguja Island, no studies were 
carried out to assess the optimal mating age of G. austeni or the competitiveness of the 
sterile males. 
In Chapter 5 G. brevipalpis adults as well as its pupal stages were shown to be highly 
sensitive to radiation. It was furthermore confirmed that the 30-year-old colonised 
G. austeni at the Agricultural Research Council–Onderstepoort Veterinary Institute (ARC-
OVI) are still susceptible to the same level of radiation as previously reported. In the present 
study, the optimal mating age and mating performance of colonised G. brevipalpis and 
G. austeni males irradiated at different levels were determined using walk-in field cages. 
 
6.2 Materials and methods 
6.2.1 Colony tsetse flies 
The mating performance of colonised G. brevipalpis and G. austeni housed at the ARC-
OVI was determined. The origin and holding conditions (Feldmann, 1994a; FAO/IAEA 
standard operating procedures, 2006) of these colonies are given in Chapter 4. 
 
6.2.2 Walk-in field cage and environmental conditions 
Comparative assessment of the mating performance of G. brevipalpis and G. austeni was 
conducted separately using walk-in field cages under “near-natural” conditions (Calkins & 
Webb, 1983; Mutika et al., 2001). The cylindrical field cages (Ø 2.9 m x 2.0 m) were made 
of cream polyester netting with a flat floor and ceiling (Fig. 6.1 A). Black nylon strips, 
connecting the panels of polyester netting, encircles the top and bottom of the cage where 
the ceiling and floor meet the sides of the cage (Fig. 6.1 B). A 1.5 m potted weeping boer-
bean tree (Schotia brachypetala) was placed in the middle of the cage. A zip, also in a black 
nylon strip, from top to bottom sealed the entrance of the cage. The field cages were 
deployed in a irrigated small forest, at the ARC-OVI in Pretoria South Africa, of 
approximately 15 m x 70 m, consisting of a lane of century old Chir pines (Pinus roxburghii) 
on one side and water berry trees (Syzygium cordatum) on the other. The forest also 
contained two large karee trees (Searsia lancea) that reduced the natural light intensity and 
a plentiful undergrowth (below 3 m) of a variety of tree species; Hyphaene coriacea, 
Strelitzia nicolai, Ziziphus mucronata, Cussonia spicata, Syringa persica, Ligustrum 
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Chapter 6: Male mating performance 
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lucidum, Melia azedarach, Dracena aletriformis and Jacaranda species. The shrub and 
herb foliage layer, below 0.5 m, consisted of Cyperus rotundus, Asparagus densiflorus, 
Tradescantia albiflora, Alpinia species as well as Hedera helix growing on the pine trees. 
The forest floor had a thick carpet of leaf litter and pine needles. Compared to the 
surroundings, the forest was a cool, humid area with low natural light intensity. 
 
 
Fig. 6.1. The outside (A) and inside (B) of a cylindrical walk-in field cage, made of panels 
of polyester netting joined with black nylon strips, deployed in a small forest at the ARC-
OVI, Pretoria, South Africa. 
 
Throughout the experiment, temperature and relative humidity were recorded every 
10 minutes using a DS1923-F5# Hygrochron iButton data logger. Light intensity at the top 
and the bottom of the cage and at tree level was recorded every 15 minutes using a Major 
Tech MT940 light meter. 
To determine the time of peak mating activity, optimal mating age and sterile male 
mating performance, 30 (three-day-old) females of either G. brevipalpis or G. austeni were 
released in the middle of the cage 5 minutes before 90 males of the corresponding species 
were to be released, giving a male to female ratio of 3 : 1. The observer remained inside 
the cage for the 3-hour duration of the experiment and movements were kept to a minimum. 
The time of mating was recorded to determine mating latency. The mating pairs were 
collected individually into small vials, and duration of the mating recorded. Although no 
direct adverse effect on mating behaviour was seen when the pairs were collected, its 
potential influence on mating behaviour cannot be ruled out. To minimise this effect mating 
pairs were similarly collected in all experiments. They were not replaced. 
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Except for the sterile versus fertile flies experiment, where the females were kept for 
60 days, the mated females were immobilised at -5 °C and dissected the following day to 
determine insemination rate and spermatecae fill (Feldmann, 1994a; FAO/IAEA standard 
operating procedures 2006). The spermatecae were removed and their fill microscopically 
scored as either, empty (0), quarter full (0.25), half (0.5), three quarters (0.75) or full (1) 
(Nash, 1955). Females that did not mate were dissected to confirm virginity. All male flies 
remaining in the cages at the end of the experiments were collected and returned to the 
colony. Glossina brevipalpis and G. austeni were evaluated separately. 
 
6.2.3 Time of peak mating activity 
In an initial set of experiments, the time of day at which the flies showed a peak in mating 
performance was recorded. The performance of nine-day-old males with three-day-old 
virgin females at a male : female ratio of 3 : 1 was assessed in the morning (9:00 h to 
12:00 h) and the afternoon (13:00 h to 16:00 h). The experiment was replicated five times 
for each species for two weeks in March 2012. 
 
6.2.4 Optimal mating age 
The optimal mating age of males was assessed using walk-in field cages. Three-, six- and 
nine-day-old males (30 of each age) competed for 30 three-day-old virgin females of the 
same species, giving a sex ratio of 3 : 1 (90 males : 30 females). To distinguish different 
male age groups, they were marked with a dot of different coloured polymer paint on the 
notum (Mutika et al., 2001). The males were marked 24 hours before being released. The 
experiments with G. brevipalpis were conducted in the afternoon from 12:00 h to 15:00 h in 
March 2012 and those with G. austeni in the afternoon from 12:00 h to 15:00 h in March 
2013. 
 
6.2.5 Sterile versus fertile males 
The mating performance of males sterilised with different radiation doses was assessed 
using walk-in field cages. Based on the results obtained in Chapter 5, G. brevipalpis adult 
males were irradiated four days after emergence with a dose of 40 Gy or 80 Gy and G. 
austeni adult males with 80 Gy or 100 Gy. Two groups of sterile males of each species, 
irradiated with different doses, and one group of fertile males (30 nine-day-old males in 
each group) competed for 30 three-day-old virgin females of the same species, giving a sex 
ratio of 3 : 1 (90 males : 30 females). To differentiate the various male groups, they were 
marked with a dot of different coloured polymer paint on the notum 24 hours before being 
released (Mutika et al., 2001). The experiments with G. brevipalpis and G. austeni were 
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Chapter 6: Male mating performance 
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conducted in the afternoon from 12:00 h to 15:00 h in February 2013 and September 2014, 
respectively. 
The mated females and males were transferred to individual holding cages and kept 
for 60 days. They were, fed daily on abattoir collected defibrinated bovine blood using an 
artificial in vitro membrane feeding system (Feldmann, 1994a; FAO/IAEA standard 
operating procedures, 2006). 
As a control for the field cage experiments, 15 irradiated males (nine-day-old) were 
mated with 30 three-day-old virgin females under controlled laboratory conditions. For these 
control experiments, males and females were kept together for one day in colony cages 
under standard colony conditions (23-24 °C, 75-80% RH and subdued/indirect lighting) 
(Feldmann, 1994a; FAO/IAEA standard operating procedures 2006). These experimental 
flies were fed on bovine blood collected with the closed sterile system using an artificial 
membrane as described in Chapter 4. Male and female survival, female fecundity and 
evaluation of pupa produced were determined as described in Chapter 5. 
 
6.2.6 Mating performance indicators 
The propensity of mating (PM), relative mating index (RMI) and relative mating performance 
(RMP) were the mating indices used to compare the mating performance of the males of 
the various treatments. Propensity of mating (PM) was defined as the overall proportion of 
released females that had mated. Relative mating index (RMI) was defined as the number 
of pairs of one treatment group as a proportion of the total number of matings (Mutika et 
al., 2001). Relative mating performance (RMP) was defined as the difference between the 
number of matings of two treatments of males as a proportion of the total number of matings 
(Mutika et al., 2001). In addition, the mating latency time, mating duration, insemination rate 
and the spermatecae fill of each mated female was determined. 
 
6.2.7 Statistical analysis 
Data were analysed using the statistical software GraphPadInstat (version 3.00, 2003). 
Differences in the overall proportions of peak mating activity were analysed with Chi-square 
(χ2) analysis with the Yate’s continuity correction. The P value was two-sided and a relative 
risk, p1-p2 was determined. Additionally, an unpaired test was used to differentiate between 
the average mating latency, mating duration and spermatecae fill. Where the data passed 
the normality test, standard (parametric) methods were used with Welch correction. If the 
data was not normally distributed a nonparametric method (Mann-Whitney test) was used. 
For the optimal mating age and sterile versus fertile comparisons a one-way analysis 
of variance (ANOVA) was used to differentiate between the relative mating index, average 
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Chapter 6: Male mating performance 
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mating latency, mating duration and spermatecae fill. Where the data passed the normality 
test, standard (parametric) methods were used and the Tukey’s test was applied. If the data 
was not normally distributed the nonparametric Kruskal-Wallis test was used. All tests were 
done at the 5% significance level. 
 
6.3 Results 
6.3.1 Environmental conditions 
All field cage experiments were conducted in summer, March 2012, February to March 
2013 and September 2014, outdoors in a small forest at the ARC-OVI. 
During the ten replicates (five for each species) conducted in the mornings in March 
2012 the mean temperature gradually increased from 21.4 ± 1.4 °C at the onset (9:00 h) to 
25.0 ± 2.8 °C at the end (12:00 h) (Fig. 6.2 A). The mean temperature in the field cages 
during these ten replicates was 24.4 ± 2.4 °C. The increase in temperature was 
accompanied by a decrease in relative humidity (Fig. 6.2 B) which dropped from an average 
of 68.0 ± 7.3% to 52.3 ± 13.0%, the mean being 58.6 ± 11.0%. 
During the ten replicates (five for each species) in the afternoons the temperature and 
relative humidity were more stable (Fig. 6.2). The temperature ranged from 27.6 ± 1.3 °C 
to 29.0 ± 2.0 °C, the mean being 28.6 ± 2.0 °C. The relative humidity ranged from 
37.9 ± 7.8% to 46.5 ± 6.8% with a mean of 41.1 ± 4.3%. 
The light intensity at the top and bottom of the cage and also at the potted plant was 
usually higher in the afternoon (433.0 ± 271.6 Lx) than the morning (301.9 ± 94.5 Lx). During 
the morning the intensity was the highest at the top (351.0 ± 102.7 Lx) of the cage. In the 
afternoons the difference in light intensity was less pronounced with the top 
(432.3 ± 155.8 Lx) and bottom (510.2 ± 387.1 Lx). 
Comparisons of optimal age and mating performance between sterile and fertile 
males were conducted in the afternoons from 12:00 h to 15:00 h in March 2012, February 
to March 2013 and September 2014. The variation in the temperature and relative humidity 
for these periods is reflected in Fig. 6.3. The lowest mean temperature of 27.0 ± 2.5 °C was 
recorded in March 2012 (Fig. 6.3). In February and March 2013, the mean temperatures of 
29.0 ± 3.0 °C and 28.2 ± 1.8 °C respectively, were on average higher than in March 2012 
(Fig. 6.3). The average relative humidity in March 2012 (44.2 ± 13.3%) was similar to that 
of February 2013 (46.2 ± 12.9%) and March 2013 (43.4 ± 8.7%) (Fig. 6.3). The highest 
mean temperature of 29.4 ± 2.2 °C was recorded in September 2014 which was also the 
period with the lowest relative humidity of 21.0 ± 7.1% (Fig. 6.3). September is considered 
to be the last month of the cold dry season in Pretoria. 
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Chapter 6: Male mating performance 
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The light intensity, ranging from 910.25 ± 351.87 Lx at the top to 653.47 ± 363.67 Lx 
at the bottom of the cages was the highest in September. This was due to the seasonal 
change in leaf cover of the trees, being less in the cold dry season. The light intensity 
ranged from an average of 657.41 ± 280.20 Lx and 604.17 ± 266.54 Lx measured at the 
tree inside the cage in February and March 2013 to 517.22 ± 390.77 Lx and 
440.32 ± 258.21 Lx at the bottom. In March 2012 the bottom (582.29 ± 468.56 Lx) of the 
cage had on average a higher light intensity than the top (404.96 ± 137.22 Lx) and the 
lowest taken at the tree (366.07 ± 221.89 Lx), was also the lowest light intensity recorded. 
 
 
Fig. 6.2. Mean temperature (A) and relative humidity (B) recorded in field cages in the 
mornings and the afternoons during March 2012. Each box shows the group median 
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Chapter 6: Male mating performance 
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separating the 25th and 75th quartiles, capped bars indicate maximum and minimum values, 
black dots indicating the outliers. 
 
Fig. 6.3. Temperature and relative humidity recorded in the field cage during March 2012, 
February to March 2013 and September 2014. Each box shows the group median 
separating the 25th and 75th quartiles, capped bars indicate maximum and minimum values, 
circles indicating the outliers. 
 
6.3.2 Activity in field cage 
After release, males and females of both species dispersed immediately with most of the 
G. brevipalpis (males and females) settling in the top half of the cage and finding a resting 
site on the black band (Fig. 6.1) that connects the top and the vertical netted panels of the 
cage. In contrast, male and female G. austeni settled mostly in the bottom half of the cage, 
once again favouring the black band that connects the bottom and the vertical netted 
panels. No other notable behavioural differences were observed in G. austeni and 
G. brevipalpis towards the field cage environment. 
For both species, most of the flies, males and females, settled in the more shaded 
areas and only a few on the tree. Some flies remained immobile after being released until 
recaptured and did not mate. After male release, there were immediate matings, the overall 
minimum mating latency time was 2 minutes. Occasionally more than one male tried to 
mate with the same female. Some attempted matings were met with clear rejection from 
the female. 
 
6.3.3 Time of peak mating activity 
The propensity of mating (PM) in the morning was 0.70 and 0.49 for G. brevipalpis and 
G. austeni, respectively. This was not significantly higher than that of 0.66 for G. brevipalpis 
(P = 0.62) and 0.59 for G. austeni (P = 0.15) in the afternoon (Table 6.1). The average 
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Chapter 6: Male mating performance 
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mating latency was significantly longer in the mornings than in the afternoons for 
G. brevipalpis (P = 0.01) and G. austeni (P < 0.01) (Table 6.1). Fig. 6.4 indicates that for 
both species, more flies mated in the first hour of the afternoon as compared with the 
morning. 
The G. brevipalpis couples mated on average for 174.21 ± 0.06 minutes in the 
morning and 165.13 ± 0.06 minutes in the afternoon which was not significantly different 
(P = 0.56). For G. austeni the average mating duration of 204.50 ± 0.06 minutes in the 
morning was significantly (P < 0.01) longer than the mating duration in the afternoon 
(138.63 ± 0.04 minutes). The mean spermatecae fill value for G. brevipalpis in the morning 
(0.75 ± 0.20) was slightly lower than in the afternoon (0.86 ± 0.10) (P < 0.01), the overall 
insemination rate was above 99% (Table 6.1). The insemination rate for G. austeni was 
above 94%. The mean spermatecae fill value of G. austeni was significantly higher 
(P < 0.01) in the morning (0.80 ± 0.20) than in the afternoon (0.68 ± 0.30) (Table 6.1). 
 
 
Fig. 6.4. Cumulative mating for Glossina austeni and Glossina brevipalpis in the morning 
and in the afternoon. 
 
 
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Chapter 6: Male mating performance 
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6.3.4 Optimal mating age  
For the optimal mating age assessment, the overall proportions of released females that 
mated (propensity of mating) was 0.46 for G. brevipalpis and 0.43 for G. austeni (Table 
6.1). The relative mating performance (RMP) for G. brevipalpis and G. austeni was 0.84 
and 0.54, both being in favour of nine-day-old males. The mean relative mating index (Table 
6.1) for nine-day-old males (0.68 ± 0.23 for G. brevipalpis and 0.54 ± 0.12 for G. austeni) 
was significantly higher than that of the six-day-old (0.25 ± 0.20, P < 0.01 for G. brevipalpis 
and 0.30 ± 0.16, P < 0.01 for G. austeni) and the three-day-old (0.06 ± 0.06, P < 0.01 for 
G. brevipalpis and 0.17 ± 0.14, P < 0.01 for G. austeni) males for both species (Fig. 6.4). 
The relative mating index (RMI) was not significantly different (P > 0.050) between six-day- 
and three-day-old males for both species (Fig. 6.5). 
For G. brevipalpis the mean mating latency, ranging from 40.3 ± 0.05 minutes for 
three-day-old to 56.4 ± 0.03 minutes for six-day-old males was not significantly different 
(P = 0.74) between the age groups (Table 6.1). Similarly mean mating duration ranging 
from 152.2 ± 0.04 minutes for six-day-old to 193.5 ± 0.04 minutes for nine-day-old males 
was also not significantly different (P = 0.21) between the age groups (Table 6.1). There 
were, however, significant differences in the mean spermatecae fill between age groups 
(P = 0.01). The mean fill of 0.25 ± 0.30 in three-day-old males was significantly different 
from that of 0.79 ± 0.30 in six-day- (P < 0.01) and 0.74 ± 0.30 in the nine-day-old (P < 0.05) 
males. 
Similarly, for G. austeni the mean mating latency (ranging from 84.1 ± 0.04 minutes 
for six-day- to 103.6 ± 0.05 minutes for three-day-old males) and mean duration of mating 
(ranging from 126.8 ± 0.04 minutes for three-day- to 144.8 ± 0.06 min for nine-day-old 
males) was not significantly different (P = 0.44, P = 0.74) between the age groups (Table 
6.1). In contrast to G. brevipalpis no significant differences (P = 0.37) in the mean 
spermathecal fill were observed between the different age groups of G. austeni (Table 6.1). 
For G. brevipalpis, the age of the male did affect the insemination rate. The rate for 
females mated with three-day-old males was only 0.33 compared to 0.96 for six-day-old 
and 0.94 for nine-day-old males (Table 6.1). For G. austeni the insemination rate ranged 
from 0.80 for three-day-old to 0.96 for six-day-old males (Table 6.1). 
 
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Chapter 6: Male mating performance 
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Fig. 6.5. Number of males of different age groups that mated with Glossina brevipalpis and 
Glossina austeni females in the field cage. Each box shows the group median separating 
the 25th and 75th quartiles, capped bars indicate maximum and minimum values, circles 
indicating the outliers. Boxes with different letters indicate that the numbers were 
significantly different at the 5% level. 
 
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Chapter 6: Male mating performance 
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Table 6.1. Mating parameters for Glossina brevipalpis and Glossina austeni in the field cages for assessing the time of peak mating activity, 
optimal mating age and the effect of radiation on male competiveness. 
Mean 
Relative Mating Mating spermatecae 
mating index latency time duration (min fill value (± 
  Possible pairs Actual mated Overall proportion(PM) (RMI ±SD) (min ± SD) ± SD) SD) Insemination rate 
G. brevipalpis         
     Fly activity morning 90 63 0.7 - 73.2 ± 0.04 174.2 ± 0.06 0.75 ± 0.20 1.00 
     Fly activity afternoon 120 79 0.66 - 47.2 ± 0.03 165.1 ± 0.06 0.86 ± 0.12 0.99 
     Male age (days) 210 97 0.46 - 54.4 ± 0.03 173.2 ± 0.03 0.72 ± 0.31 0.94 
          9 - 67 - 0.68 ± 0.23 55.0 ± 0.03 193.5 ± 0.04 0.74 ± 0.28 0.94 
          6 - 24 - 0.25 ± 0.20 56.4 ± 0.03 152.2 ± 0.04 0.79 ± 0.28 0.96 
          3 - 6 - 0.06 ± 0.06 40.3 ± 0.05 176.0 ± 0.04 0.25 ± 0.35 0.33 
  Male sexual status 360 204 0.57 - 66.7 ± 0.04 220.3 ± 0.05 0.59 ± 0.17 0.99 
Fertile - 53 - 0.41 ± 0.13 61.1 ± 0.04  225.1 ± 0.05 0.60 ± 0.17 1.00 
Sterile (40 Gy) - 70 - 0.33 ± 0.19 71.8 ± 0.51 215.8 ± 0.05 0.55 ± 0.15 1.00 
Sterile (80 Gy) - 81 - 0.27 ± 0.12 68.6 ± 0.04 218.6 ± 0.05 0.60 ± 0.18 0.98 
         
G. austeni         
     Fly activity morning 120 59 0.49 - 94.3 ± 0.04 204.5 ± 0.06 0.80 ± 0.20 0.98 
     Fly activity afternoon 150 88 0.59 - 58.4 ± 0.04 138.6 ± 0.04 0.68 ± 0.25 0.94 
     Male age (days) 360 153 0.43 - 94.3 ± 0.05 137.4 ± 0.05 0.57 ± 0.30 0.93 
          9 - 83 - 0.54 ± 0.12 97.1 ± 0.05 144.8 ± 0.06 0.61 ± 0.30 0.95 
          6 - 45 - 0.30 ± 0.16 84.1 ± 0.04 139.2 ± 0.04 0.54 ± 0.25 0.96 
          3 - 25 - 0.17 ± 0.14 103.6 ± 0.05 126.8 ± 0.04 0.51 ± 0.34 0.80 
Male sexual status 360 225 0.63 - 76.8 ± 0.04 149.2 ± 0.04 0.67 ± 0.15 0.99 
Fertile - 72 - 0.33 ± 0.13 81.0 ± 0.04 155.1 ± 0.04 0.64 ± 0.19 0.96 
Sterile (80 Gy) - 77 - 0.35 ± 0.16 79.0 ± 0.49 147.5 ± 0.05 0.72 ± 0.12 1.00 
Sterile (100 Gy) - 76 - 0.32 ± 0.12 69.9 ± 0.04 144.5 ± 0.05 0.65 ± 0.14 1.00 
 
 
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Chapter 6: Male mating performance 
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6.3.5 Sterile versus fertile males 
The propensity of mating (PM) was 0.57 and 0.63 for G. brevipalpis and G. austeni, 
respectively (Table 6.1). A RMI of 0.27 ± 0.12, 0.33 ± 0.19 and 0.41 ± 0.13 was obtained 
for 80 Gy-, 40 Gy-treated and untreated control G. brevipalpis males, respectively and these 
values were not significantly different (P = 0.08) (Fig. 6.6). Similarly, the RMI of untreated 
male G. austeni (0.33 ± 0.13) and of those irradiated with100 Gy (0.32 ± 0.12), and 80 Gy 
(0.35 ± 0.16) was also not significantly different (P = 0.87) (Fig. 6.6). 
The RMP of irradiated (40 Gy) to untreated G. brevipalpis males was -0.07, which 
indicates that the mating performance of the 40 Gy-treated and untreated males was almost 
equal. Similar observations were made for males irradiated with 80 Gy versus untreated 
males, i.e. a RMP of -0.21. The RMP of 80 Gy and 100 Gy-treated G. austeni males versus 
untreated ones was 0.01 (in favour of treated) and -0.03, (in favour of untreated males). 
Untreated G. brevipalpis males formed mating pairs sooner (on average 
61.1 ± 0.04 minutes) than the males irradiated with 40 Gy (71.8 ± 0.51 minutes) and 80 Gy 
(68.6 ± 0.04 minutes) and they also mated longer (225.1 ± 0.05 minutes) (Table 6.1). The 
opposite was observed for G. austeni: where the irradiated males formed mating pairs 
sooner (80 Gy: 79.0 ± 0.49 minutes and 100 Gy: 69.9 ± 0.04 minutes) than the untreated 
males (81.0 ± 0.04 minutes), however, the untreated males mated longer (155.1 ± 0.04 
minutes) (Table 6.1). 
Mated females and males were collected and placed in individual fly holding cages 
and kept for 60 days, female survival and pupae production were monitored and surviving 
females were dissected after 60 days. Ninety percent of the G. brevipalpis females that 
mated with untreated or 80 Gy-irradiated males and 81% of females that mated with 40 Gy-
treated males survived to day 60 (Table 6.2). The survival on day 60 of G. austeni was 81% 
and 73% for females that mated respectively with 80 Gy- and 100 Gy- irradiated males and 
59% of the females mated with untreated males (Table 6.2). 
Females of both species that mated with untreated males produced more pupae 
(Table 6.2) than those that mated with irradiated males. In parallel, the females of both 
species that mated with untreated fertile males aborted a lower number of eggs compared 
to those that had mated with irradiated males (Table 6.2). In both G. brevipalpis and 
G. austeni pupae production was dose dependent and fecundity decreased as the radiation 
dose increased (Table 6.2). A dose of 40 Gy and 80 Gy was sufficient to induce 93% and 
98% sterility respectively in G. brevipalpis. With G. austeni 80 Gy and 100 Gy was sufficient 
to induce 79% and 89% sterility, respectively. 
 
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Chapter 6: Male mating performance 
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Fig. 6.6. Number of irradiated male (40, 80 or 100 Gy) and untreated male 
Glossina brevipalpis and Glossina austeni that mated with untreated females in a field cage. 
Each box shows the group median separating the 25th and 75th quartiles, capped bars 
indicate maximum and minimum values, circles indicating the outliers. 
 
The reproductive status of females of both species on the day of dissection indicated 
a strong difference between those that mated with fertile males compared to irradiated 
males. In the majority of females of both species that mated with the irradiated males the 
uterus was either empty due to abortions or contained a recently ovulated egg. Only a few 
females of both species that had mated with fertile males showed an empty uterus due to 
abortions and most had either recently ovulated eggs or viable instar larvae in the uterus 
(Table 6.2). The insemination rate was above 0.98 for G. brevipalpis and 0.96 for G. austeni, 
both for females mated with untreated and with irradiated males, which indicated that 
radiation did not influence the males’ ability to transfer sperm. 
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Chapter 6: Male mating performance 
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Table 6.2. Production of Glossina brevipalpis and Glossina austeni females mated with sterile and fertile males in field cages. 
Uterus Spermatecae fill % 
% No. of 
No. of mature No. pupae Recently Empty Viable instar 
Fecundity Emergence / aborted Insemination   
   females produced ovulated due to larvae 
% females eggs 
egg abortion I II III 0.25 0.5 0.75 1 
Day 18 Day 60               
G. brevipalpis                  
 Fertile 74 67 81 0.058 91.5 / 44.7 14 100 27 17 5 6 14 10.1 37.7 52.2 0 
 Sterile (40 Gy) 65 57 6 0.010 100.0 / 25.0 52 100 19 35 0 0 2 10.7 57.1 32.1 0 
 Sterile (80 Gy) 53 48 2 0.006 75.0 / 25.0 37 98 8 40 0 0 0 6.3 39.6 52.1 0 
                   
G. austeni                  
 Fertile 76 45 141 0.026 86.4 / 46.2 7 96 8 14 9 9 5 4.4 26.7 68.9 0 
 Sterile (80 Gy) 77 63 29 0.002 70.8 / 70.8 23 100 16 39 0 0 3 0 19 75.9 5.2 
  Sterile (100 Gy) 70 53 15 0.001 73.7 / 42.1 24 100 13 39 0 0 0 3.9 29.4 66.7 0 
 
Table 6.3. Production of Glossina brevipalpis and Glossina austeni females mated with sterile and fertile males under laboratory conditions. 
Uterus Spermatecae fill % 
Viable instar 
No. of mature   % No. of larvae 
No. pupae 
females Recently Empty    Fecundity Emergence aborted Insemination 
produced ovulated due to 
/ % females eggs 
egg abortion I II III 0.25 0.5 0.75 1 
Day 18 Day 60 
G. brevipalpis                  
 Fertile 112 99 116 0.026 100.0 / 62.9 31 91 34 47 1 5 5 17.9 30.5 42.1 0 
 Sterile (40 Gy) 113 104 7 0.002 71.4 / 0.0 97 83 8 94 0 1 1 25 27.9 27.9 1.9 
 Sterile (80 Gy) 108 101 2 <0.001 66.7 / 100.0 80 88 9 62 0 0 0 28 28 32 0 
                   
G. austeni                  
 Fertile 124 83 269 0.065 69.9 / 63.8 52 100 28 30 5 13 4 6.4 42.3 50 1.3 
 Sterile (80 Gy) 112 87 13 0.003 30.8 / 75.0 182 100 17 67 0 0 1 3.5 40 56.5 0 
  Sterile (100 Gy) 119 81 5 0.001 60.0 /33.3 210 100 13 68 0 0 0 6.2 39.5 54.3 0 
 
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Chapter 6: Male mating performance 
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Chapter 6: Male mating performance 
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The same linear relationship as seen in Chapter 5 for male life span was also 
observed as the average life span (119.86 ± 4 7.51 days) of fertile G. brevipalpis males was 
longer than that of irradiated ones, i.e. 101.64 ± 36.98 days for males irradiated with 40 Gy 
and 94.15 ± 28.58 days for the males done with 80 Gy. The average life span of untreated 
G. austeni (72.35 ± 62.63 days) was longer compared to irradiated males (80 Gy: 
65.13 ± 34.30 days; 100 Gy: 66.08 ± 34.15 days). 
The sterility of irradiated males used in the field cages was assessed by selecting a 
subsample (N = 15) of males, that had mated with 30 three-day-old virgin females and been 
maintained under controlled laboratory conditions. Pupae production of females mated with 
untreated control males is indicated in Table 6.3. In both species irradiation did not affect 
the males’ ability to transfer sperm. G. brevipalpis males that had been irradiated with a 
dose of 40 Gy and 80 Gy induced 94% and 98% sterility in untreated females, respectively. 
This is similar to sterility observed in flies used in the field cages for dose 40 Gy and 
somewhat higher than the 80 Gy. Some discrepancies arose when comparing the induced 
sterility for G. austeni females of the field cage experiments with those used in the 
laboratory. In general, the males of the laboratory experiments induced a higher sterility in 
untreated females as compared with males used in the field cage experiments. It might, 
however, have been due to an experimental error in tracking the production of each 
individual fly. The smaller sample size could also have played a role. 
 
6.4 Discussion 
The success of AW-IPM programmes that include a SIT component depends on the 
capability of the released sterile males to compete with their native counterparts (Calkins & 
Parker, 2005; Vreysen, 2005). Assessment of the mating competitiveness of the produced, 
released insects will therefore be a prerequisite before any operational SIT programme can 
be initiated (Vreysen et al., 2007). Biological attributes such as rate of development, 
temperature adaptation, circadian rhythm, flight capability, optimal mating age, weight and 
strain used may affect the biological quality of the produced and released insects (Van der 
Vloedt & Barnor, 1984; Mutika et al., 2001; Liedo et al., 2002; Olet et al., 2002; Abila et al., 
2003). Operational attributes that will contribute to this include collection techniques, 
handling, radiation, and release methods (UI Haq et al., 2010; Teal et al., 2013; Flores et 
al., 2014; UI Haq et al., 2014). Quantification of the impact of each of these attributes on 
the released insects’ competitiveness is paramount to enable the development of 
procedures to mitigate any potential negative effects. 
In the present study, there was no significant difference in male activity and mating 
performance in the mornings and afternoons for both species, indicating that field cage 
experiments could be conducted in either of these two time slots at the ARC-OVI. 
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Chapter 6: Male mating performance 
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Environmental conditions were, however, somewhat more variable in the mornings with a 
lower average temperature and higher average relative humidity in contrast to the 
afternoons when conditions were more stable but with on average, higher temperatures 
and relatively lower humidity. The on average shorter mating latentcy observed in the 
afternoons might also have been a result of the higher environmentsl temperatures. The 
afternoon time of 12:00 h to 15:00 h was selected for all other field cage experiments. 
The equal mating performance for both G. brevipalpis and G. austeni in the afternoon 
and morning seems to conform to the diurnal activity patterns observed for G. brevipalpis 
in South Africa but not for G. austeni. Previous studies have indicated a bimodal activity 
pattern for G. brevipalpis with flies being active early in the morning from dawn until after 
sunrise and then late afternoon (Kappmeier, 2000). Glossina austeni on the other hand 
showed a more pronounced unimodal activity pattern and flies were active from early 
morning until late afternoon (Kappmeier, 2000). The G. austeni data from South Africa was 
in contrast with that of Owaga et al. (1993), who observed two activity peaks, one at 9.00 h 
to 10.00 h and a second between 14.00 h and 17.00 h, for G. austeni in Kenya. 
Like most tsetse species, G. austeni and G. brevipalpis are markedly diurnal and 
show pronounced periodicity in their activity. Tsetse fly activity patterns are known to be 
under the control of an endogenous clock but in nature, these rhythms are also influenced 
by environmental stimuli such as temperature and light (Brady & Crump, 1978). Circadian 
rhythm of tsetse flies can influence the activity of sterile males in the field, and also their 
competitiveness. Whereas the differences in activity patterns of G. austeni seen in the field 
might be related to different environmental conditions and stimuli, the differences observed 
in the circadian rhythm in the laboratory are more difficult to explain. Crump & Brady (1979) 
reported only one afternoon peak of spontaneous activity of G. austeni in the absence of 
any odours or other stimuli. Owaga et al. (1993), however, states that the U-shaped activity 
pattern observed in the field persisted in the laboratory when flies were maintained under 
a 12 h light /12 h dark cycle and stable temperature and humidity conditions. The authors 
therefore concluded that the activity pattern of G. austeni was mainly driven by endogenous 
factors (Owaga et al.,1993). 
The present study indicated that the age of both G. austeni and G. brevipalpis males 
was significantly correlated to their mating performance as shown by the RMI. Nine-day-
old males were significantly more successful in securing a female for mating than six- or 
three-day-old males. These results are in agreement with data obtained for G. f. fuscipes 
and G. p. palpalis (Abila et al., 2003). Although older G. brevipalpis and G. austeni males 
were more competitive in securing a female in field cages, the age of the males did not 
influence mating duration or insemination ability. This confirms the data of Malele and 
Parker (1999) who observed that G. austeni males that had mated on the day after 
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Chapter 6: Male mating performance 
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emergence could successfully inseminate females of the same age in small laboratory 
cages. Our data on optimal mating ages indicate that the propensity of mating for both 
G. austeni and G. brevipalpis can potentially be improved by releasing older sterile males. 
This, however, would necessitate keeping the males longer in the rearing facility, which 
would increase maintenance and production costs. This protocol would require additional 
blood meals for the sterile males, more labour to absorb the increased handling needs and 
larger facilities to stockpile the flies before release. 
In the majority of previous control programmes with a SIT component, the sterile 
males were released at a relatively young age. The sterile male G. austeni were 4 to 7 days 
old when released on Unguja Island, Zanzibar (Vreysen et al., 1999), sterile male 
G. p. palpalis were 3 to 5 days old when released in the Lafia area of Nigeria (Oladunmade 
et al., 1990), and sterile male Glossina tachinoides were 2 to 10 days old when released in 
a pilot trial in Chad (Cuisance & Itard, 1973). Using younger males will be cost effective in 
terms of space and labour. These release protocols were driven by mating observations in 
small laboratory cages that showed mating and insemination was possible in males 
younger than 5 days (Malele & Parker, 1999). Other researchers used males that were 
between 5 to 8 days old for various experiments (Van der Vloedt et al., 1978; Van der Vloedt 
& Barnor, 1984; Vreysen & Van der Vloedt, 1990). In these operational programmes, sterile 
males were offered at least two blood meals that contained a trypanocidal drug Samorin 
(12.5 mg/L blood) before release in a programme on Unguja (Vreysen et al., 1999) that 
significantly reduced the risk of transmitting trypanosomes. 
An entirely different sterile male release strategy was used in the programme against 
Glossina morsitans morsitans in the Tanga area, Tanzania, in the 1970’s. Here sterile males 
were released as pupae from fixed release stations and emerging males were consequently 
teneral and had to find a blood meal right away to build up energy reserves (Williamson et 
al., 1983a). A drawback of this method was that males were exposed to potential predation 
before reaching sexual maturity and could also potentially transmit the Trypanosomosis 
disease. Despite this, the programme was successful and releasing male pupae at a 
density of 135 pupae/km2 resulted in a sterile male wild male overflooding ratio of 1.2 : 1 
which, despite being low, maintained the indigenous wild fly population at the 80-95% 
reduction level obtained after the initial insecticide application (Williamson et al., 1983b). 
Exposure to radiation may affect the biological quality of the produced released 
insects (Simmons et al., 2010). This study, however, shows that radiation of up to 80 Gy 
for G. brevipalpis and 100 Gy for G. austeni did not affect the ability of sterilised males to 
compete with fertile males. This is in accordance with field cage evaluations of G. morsitans 
and G. pallidipes that also showed that the competitiveness of irradiated males did not differ 
from that of untreated males (Dean et al., 1968; Mutika et al., 2001). Dean et al. (1968) 
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Chapter 6: Male mating performance 
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based their findings of competitiveness on pupal production, however, in this study as well 
as that of Mutika et al. (2001) observing individual flies provided more accurate information 
on male competitiveness. Untreated fertile G. brevipalpis males did form mating pairs 
sooner and mated for longer than the irradiated males. Although this was not statistically 
significant, any delay in initial mating by the irradiated males can lead to a potential 
reduction of their competitiveness and need to be reduced. With G. austeni the opposite 
was seen, irradiated males formed mating pairs sooner and mated for a shorter period than 
the fertile males. Females that mated with untreated fertile G. brevipalpis males did have a 
larger spermatecae fill than those that mated with irradiated males. This was not confirmed 
for G. austeni. 
This study has furthermore shown that field cages can be used to assess the mating 
performance of G. brevipalpis and G. austeni with an average propensity of mating above 
56%. Although the propensity of mating was lower than that in similar field cages with 
G. f. fuscipes and G. p. palpalis (Abila et al., 2003), the values obtained indicate adequate 
environmental conditions for the evaluations. This relatively high propensity of mating 
obtained indicated that the potential interference on the mating behaviour of the flies 
because of the observer presence was minimal. It needs to be pointed out that the field 
cage experiments were conducted in Pretoria which has a different climate to the tsetse fly 
infested area in north eastern KwaZulu-Natal. The different environmental conditions might 
influence the circadian rhythm, the activity patterns of the flies and the propensity of mating. 
The results of our mating performance studies using irradiated G. brevipalpis males 
indicate that G. brevipalpis will be well suited for use in programmes that have a SIT 
component. The data obtained for G. austeni strengthens the findings of previous studies 
that indicate that this species can also be used successfully in programmes with a SIT 
component (Vreysen et al., 1999). 
 
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Chapter 7 
 
Concluding remarks and recommendations 
 
After an outbreak of nagana in 1990 in north eastern KwaZulu-Natal an extensive survey 
of tsetse fly distribution and abundance was conducted with odour baited XT sticky traps 
during 1993 to 1999. This data was incorporated in an area-wide integrated pest 
management (AW-IPM) elimination strategy with a sterile insect technique (SIT) component 
to establish a tsetse fly-free South Africa (Kappmeier Green et al., 2007). A prediction model 
(Hendrickx, 2002), based on the XT sticky trap data, indicated a wider distribution range for 
both Glossina brevipalpis and Glosinna austeni in South Africa. Since this survey 
Kappmeier & Nevill (1999a) and Kappmeier (2000) have developped a more effective trap, 
the odour baited H trap, for the collection and monitoring of G. brevipalpis and G. austeni 
and the 1993 - 1999 data set may have become outdated. One of the outcomes (Chapter 
2) of this study was an updated tsetse fly abundance (Fig. 2.4) and distribution (Fig. 2.5) 
maps as well as a Trypanosomosis infection rate map (Fig. 2.8). 
In the AW-IPM tsetse fly elimination strategy proposed by Kappmeier Green et al. 
(2007), the tsetse fly infested area was divided into four zones from south to north. It was 
proposed to implement an operational phased approach successively, starting in zone I in 
the south following a rolling carpet principle (Hendrichs et al., 2005; Kappmeier Green et 
al., 2007). In each zone, depending on species presence and abundance, initial tsetse 
population reduction would first be achieved with spraying of non-residual insecticides 
based on a sequential aerosol technique (SAT) followed by the release of sterile males 
(Kappmeier Green et al., 2007). The initial tsetse population reduction with SAT was 
suggested because the available insecticide-impregnated targets were found to be rather 
inefficient for G. brevipalpis. 
The most conspicuous change in the tsetse fly distribution as determined in Chapter 
2 to that of Kappmeier Green et al. (2007) is that G. brevipalpis was proven to be present 
in Zone III and G. austeni in Zone I. While the odour baited XT sticky trap data indicated 
G. brevipalpis to be restricted to two distinct bands in the north and south of the area 
(Kappmeier Green, 2002) (Figs. 2.2 & 2.5) the present data indicate a more continuous 
distribution for this species. Glossina brevipalpis was also collected further south (10 km 
from the border of Hluhluwe-iMfolozi Park) than previously. The most southerly limit of 
G. brevipalpis therefore remains undefined and similarly the most westerly extent of the 
tsetse fly distribution still needs to be verified. 
This wider tsetse fly distribution as indicated on the updated maps will necessitate 
modifications of the strategy and zone selection as proposed by Kappmeier Green et al. 
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Chapter 7: Concluding remarks and recommendations 
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(2007). The present distribution data indicates that the southern limits of zone I and II have 
to be expanded to approximately 20 km south of the Hluhluwe-iMfolozi border. The 
southernmost distribution of G. brevipalpis still needs to be confirmed or defined and this 
will ultimately define the final southern limits of their distribution. Based on the fact that 
G. brevipalpis was present in the Phinda and Mkhuze game reserves it is recommended 
that the previously defined Zone III must be split into two. 
After the eradication of G. pallidipes in 1952 the two remaining species in the area, 
G. brevipalpis and G. austeni, were considered of lesser importance in the transmission of 
nagana (Du Toit, 1954). From the Trypanosomosis prevalence data it is clear that nagana 
is abundant and widespread in north eastern KwaZulu-Natal. The apparent absence of a 
significant linear correlation between trypanosome prevalence and the relative abundance 
of the tsetse flies in the present study can partly be attributed to the co-existence of the two 
species, each with a different vectorial capacity and/or competence. Both these species 
have been shown to readily feed on cattle (Moloo, 1993; Clausen et al., 1998). Despite a 
perceived lower vector competence for G. brevipalpis (Motloang et al., 2012) our data has 
indicated that both species can play a role in the transmission of Trypanosomosis in 
KwaZulu-Natal. 
AW-IPM approaches require that the control effort is directed against an entire insect 
population. It is therefore of paramount importance to assess the degree of isolation of the 
targeted pest population. Knowledge on gene flow between the target population and 
adjacent populations can provide the necessary guidance in the decision making process. 
It was known that the South African tsetse fly populations of G. brevipalpis and G. austeni 
extend into southern Mozambique and Swaziland (G. austeni). In the present study 
morphometrical analyses showed that gene flow exists between the three neighbouring 
countries and that there are no significant barriers (Chapter 3). The proposed AW-IPM 
tsetse fly eradication strategy should thus be expanded to include southern Mozambique 
and Swaziland. Zone IV as proposed by Kappmeier Green et al. (2007) needs to be 
extended to include the southern Mozambique tsetse populations. An additional zone, 
zone V will be needed for Swaziland. It still needs to be determined whether the tsetse 
populations in southern Mozambique are genetically isolated from the tsetse populations 
north of Maputo (Fig. 1.2 A). 
Any AW-IPM programme that includes on a SIT component will only be successful if 
the target insect can be mass-reared in adequate numbers, and the sterile males destined 
for release, are competitive with their native counterparts (Calkins & Parker, 2005; Vreysen, 
2005). Assessment of the competitiveness of the produced and released insects is 
therefore a prerequisite before any operational SIT programme can be launched (Vreysen 
et al., 2007). Various attributes both biological (e.g. rate of development, temperature 
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Chapter 7: Concluding remarks and recommendations 
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adaptation, circadian rhythm, flight capability, optimal mating age, weight) and operational 
(e.g. insect collection techniques, handling, radiation, release technologies) may impact on 
the quality of the produced and released insects (Simmons et al., 2010). Quantification of 
the impact of each of these on the released insects’ competitiveness is paramount to 
mitigate potential negative effects. 
In Chapter 4, the impact of anticoagulants and phagostimulants that can be added to 
the blood meal and the effect of source of the blood (bovine, porcine) on tsetse fly 
production were determined. The colonies of G. brevipalpis and G. austeni have been 
maintained at the Agricultural Research Council–Onderstepoort Veterinary Institute (ARC-
OVI) on defibrinated irradiated bovine blood that is obtained from a commercial abattoir. 
The current blood diet was proven to be suitable for the maintenance of these two species. 
These colonies would require considerable upscaling to provide sufficient numbers of 
sterile males for a future SIT component. Since we have two species, with potentially 
different diet requirements, this can be challenging. Therefore, the diet requirements of the 
two species were reassessed to provide better protocols that could be used for the 
upscaling of these colonies. It was found that combinations of bovine and porcine blood or 
feeding on these blood sources on alternative days improved fecundity in both species, this 
adjustment in feeding protocols, can therefore be used to boost production in G. brevipalpis 
and G. austeni colonies in times of low performance as well as during the upscaling phase. 
Any alternation or addition to the rearing diet requirements will need to be reassessed 
in terms of operational efficiency e.g., although anticoagulants will simplify blood collection 
and make the process more sterile, the chemicals are expensive and will increase costs in 
large scale operations. 
Furthermore, the current colonies at the ARC-OVI are not from the same strain as the 
tsetse flies in the proposed control area and it is recommended that mating compatibility of 
the two strains be determined. For this purpose, colonies of the KwaZulu-Natal strains need 
to be established. High mortalities and low productivity in the field collected tsetse flies 
showed that the establishment of colonies will be difficult. The collection of sufficient 
numbers of field flies in a good physical condition will be essential for colony establishment. 
In Chapter 3, flies collected at sites with less pronounced fluctuations in environmental 
conditions together with those at the end of a more favourable season e.g. a hot wet 
summer, were shown to be in a better physical condition and will therefore be more suitable 
for colonization. Transportation, feeding and pupae maintenance protocols need to be 
evaluated and adjusted e.g. transportation of pupa rather than adults, phagostimulants to 
improve feeding and maintaining pupae on wet sand. 
In Chapter 5, the radiation sensitivity of G. austeni and G. brevipalpis when treated 
as pupae or adults was assessed. Treating late stage G. brevipalpis pupae with 40 Gy 
123 
 
Chapter 7: Concluding remarks and recommendations 
_________________________________________ 
 
induced 97% sterility when mated with untreated females and 99% sterility when irradiated 
with 80 Gy as adults. Glossina austeni required higher doses, as 80 Gy and 100 Gy were 
needed to induce more than 97% sterility in untreated females that mated with males 
treated either as adults or late stage pupae. Since there were no significant differences in 
the quality of males irradiated as adults or pupae both stages can be selected for irradiation. 
There are positive and negative considerations when choosing to irradiate either flies or 
pupae. As was observed in G. brevipalpis, the negative effect of radiation on tsetse fly 
longevity was more pronounced when pupa, instead of adults, were irradiated. This 
relationship was less clear in G. austeni. Pupae are easier to transport and handle and 
more pupae can be irradiated at a time (Pagabeleguem et al., 2015). Radiation, however, 
can delay adult emergence which will negatively impact pupal maintenance time and 
operational costs. Available sex separation procedures are based on differential 
development time between males and females and although efficient are not 100% 
accurate. As a result, there will be some females that are irradiated and released. This is a 
drawback as the females might live longer and have a higher risk then males for becoming 
infected with trypanosomes, however, this risk is reduced for both sexes as all flies are fed 
with trypanocidal drugs before release. The mating competitiveness of males irradiated 
either as adults or pupae needs to be considered in deciding which stage to irradiate. The 
use of field cages to assess mating performance and competitiveness of pest insects has 
gained importance during the last decade. These cages were originally mainly used for 
testing the mating behaviour of several species of fruit flies, and their use has recently been 
expanded to include insect groups such as tsetse flies (Mutika et al., 2013) and Lepidoptera 
(Taret et al., 2010). Walk-in field cages have proved to be good substitutes for field studies, 
which will be less controlled, more complex, and expensive. Although the data obtained 
from field cages are good indicators of the behaviour of reared insects, this still need field 
verification, where the released insects are competing with wild insects and are exposed to 
a number of varying stimuli. 
In Chapter 6, it is indicated that mating performance can be significantly improved in 
G. austeni and G. brevipalpis males by using older males. In an operational programme, 
this would, however, require keeping the males for longer in the rearing facility, with an 
increase of the maintenance and production costs. This would require more blood meals 
for the sterile males, more labour to absorb increased handling needs and larger facilities 
to stockpile the flies before release. In some male thephritid fruit flies, the time needed to 
reach sexual maturity is significantly reduced if they are exposed to juvenile hormone 
mimics (Teal et al., 2013). Similarly, the addition of certain supplements (e.g. protein) to the 
diet of the melon fly Bactrocera cucurbitae (Ul Haq et al., 2014) or exposure of species such 
as Bactrocera carambolae to methyl eugenol aroma can increase the mating performance 
124 
 
Chapter 7: Concluding remarks and recommendations 
_________________________________________ 
 
of the males (Ul Haq et al., 2014). Identifying similar factors that can shorten the period 
before the optimal mating age for G. austeni and G. brevipalpis is reached will be 
advantageous. 
Colonised nine-day-old males irradiated as adults with 80 Gy for G. brevipalpis and 
100 Gy for G. austeni successfully competed with colonised nine-day-old fertile males for 
three-day-old colony females. This data indicates that, under the experimental field cage 
conditions, the irradiated colony flies at the ARC-OVI are suitable to be used for the SIT. 
However, the mating performance of irradiated colonised G. brevipalpis and G. austeni 
males compared to the fertile wild type males still needs to be assessed, preferably under 
field conditions, in the target area. 
 
The main conclusions from this study can be summarised as follows: 
o The updated tsetse fly distribution and Trypanosomosis prevalence maps needs to 
be taken into consideration in the proposed AW-IPM tsetse fly elimination strategy. 
Tsetse fly abundance can, however, be dynamic and will rapidly react to changes in 
environmental conditions. 
o Comparisons of the abundance of G. brevipalpis and G. austeni with trypanosome 
infection rates in the area indicate that both species can play a role in the 
epidemiology of this disease. 
o Gene flow does occur between the populations of tsetse flies found in South Africa, 
southern Mozambique and Swaziland. 
o The current diet (i.e difebrinated bovine blood) and feeding protocols are sufficient for 
the maintenance of the G. brevipalpis and G. austeni colonies at the ARC-OVI. 
o Glossina brevipalpis and G. austeni can be irradiated with a dose of 80 Gy and 100 Gy 
respectively, and can be irradiated either as adults or late stage pupae. 
o Irradiation did not affect the mating performance of colonised G. brevipalpis and 
G. austeni males. 
o Initial results indicate that G. brevipalpis to be a good candidate for the use in SIT. 
 
The continuous improvement of tsetse control strategies will be beneficial in a 
changing environment of the African continent, and the following can be prioritised as future 
research aspects: 
o The southerly and westerly distribution limits of G. brevipalpis and G. austeni in South 
Africa need to be defined before the implementation of an AW-IPM programme. Due 
to its dynamic nature, tsetse fly abundance should be constantly monitored at certain 
key points in the area. 
125 
 
Chapter 7: Concluding remarks and recommendations 
_________________________________________ 
 
o To determine the extent of gene flow between the tsetse fly populations in the 
northern and southern belts of Mozambique. 
o The efficiency of the current monitoring system is low and need to be improved, 
especially for G. austeni. 
o The vector competence and capacity of G. brevipalpis for various trypanosome 
species need to be determined. 
o The role of mechanical transmission by other biting flies needs to be determined. 
o The role of vegatation, game parks and protected areas in maintaining tsetse fly 
populations need to be better characterised. The extent to which these species will 
disperse from and breed outside the parks need to be determined. 
o Assessing suppression methods for the two target species. 
o The G. brevipalpis and G. austeni colonies at the ARC-OVI will need to be enlarged 
in a mass-rearing facility of approximately 500 times of its current size before it to be 
used in an AW-IPM programme with a SIT component. This will necessitate the 
reassessment of the rearing and operational requirements of the two species 
involved. 
o The mating compatibility and competiveness between the colonised G. brevipalpis 
and G. austeni and the wild type need to be assessed. The degree of compatibility 
between these strains will determine the need for the establishment of colonies of the 
KwaZulu-Natal strains of these two species. 
o The mating competitiveness of males irradiated as adults compared to late stage 
pupae needs to be assessed. 
o Factors that can shorten the time to reach sexual maturity in G. brevipalpis and 
G. austeni need to be identified. 
o The mating performance of irradiated colonised G. brevipalpis and G. austeni males 
compared to that of fertile wild type males needs to be studied. 
 
The debilitating effect of nagana can still be observed in it severity at the outer fringes 
of the tsetse distribution belt as in southern Africa. The sustainable control of nagana in the 
north eastern parts of KwaZulu-Natal will contribute to animal health and will stimulate 
economic development in this largely underdeveloped area. The findings of this thesis will 
be used to improve the proposed AW-IPM tsetse fly elimination strategy for southern Africa 
and can be used in the decision making of control in other regions in Africa where these 
two species are encountered.
126 
 
References 
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Appendix 
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APPENDIX 
 
Appendix 1. List of conference presentations and publications: 
 
2009: 
De Beer, C.J., Venter, G.J. & Latif, A.A. (2009) Colonisation of tsetse flies Glossina austeni 
and Glossina brevipalpis for research and development to support an area-wide 
approach for integrated pest management in northern KwaZulu Natal Province, South 
Africa. In: Journal of the South African Veterinary Association. Proceedings of the 
37th Annual Congress of the Parasitological Society of Southern Africa. 1-3 October 
2008. Pretoria, South Africa. 
De Beer, C.J., Venter, G.J. & Potgieter, F. (2009) Developing quality assurance procedures 
for the sustainable supply of high quality blood for mass rearing of tsetse fly colonies. 
In: Journal of the South African Veterinary Association. Proceedings of the 37th 
Annual Congress of the Parasitological Society of Southern Africa. 1-3 October 2008. 
Pretoria, South Africa. 
2010: 
De Beer, C.J. & Venter, G.J. (2010) Colonization of tsetse flies as a prerequisite for the use 
of sterile Insect technique in South Africa. Proceedings of 18th WiN Global Annual 
Conference, 9-14 May 2010, Paradise Hotel Busan, Korea. 
2012: 
De Beer, C.J. & Venter, G.J. (2012) Evaluation of radiation sensitivity of Glossina 
brevipalpis and Glossina austeni (Dipter, Glossinidae). Proceedings of the 41st 
Annual Congress of the Parasitological Society of Southern Africa. 1-3 October 2012. 
Bloemfontein, South Africa. 
De Beer, C.J. & Venter, G.J. (2012) Tsetse flies in the conservation areas of North-Eastern 
parts of KwaZulu-Natal. Proceedings of the 41st Annual Congress of the 
Parasitological Society of Southern Africa. 1-3 October 2012. Bloemfontein, South 
Africa. 
2014: 
De Beer, C.J. & Venter, G.J. (2014) Assessment the factor of age on the mating 
competitiveness of Glossina brevipalpis males under field cage conditions. 
Proceedings of the 19th E-SOVE conference, 13-17 October 2014, Thessaloniki, 
Greece. 
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Appendix 
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Ntantiso, L., De Beer, C., Marcotty, T. & Latif, A.A. (2014) Bovine trypanosomosis 
prevalence at the edge of Hluhluwe-iMfolozi Park, KwaZulu-Natal, South Africa. 
Onderstepoort Journal of Veterinary Research, 81(1), Art. #762, 8. 
2015: 
De Beer, C.J., Venter, G.J., Motloang, M.A. & Latif, A.A. (2015) Tsetse abundance and 
nagana prevalence in north eastern Kwa-Zulu Natal. Proceedings of the joint ESSA 
and ZSSA Congress. 12-17 July 2015, Grahamstown, South Africa. 
De Beer, C.J., Venter, G.J. & Vreysen, M.J.B. (2015) Determination of the optimal mating 
age of colonised Glossina brevipalpis and Glossina austeni using walk-in field cages 
in South Africa. Parasites & Vectors, 8, 467. 
De Beer, C.J., Venter, G.J. & Vreysen, M.J.B. (2015) The competitiveness of sterile male 
Glossina brevipalpis for use in the sterile insect technique (SIT). Proceedings of the 
joint ESSA and ZSSA Congress. 12-17 July 2015, Grahamstown, South Africa. 
2016: 
De Beer, C.J., Venter, G.J., Kappmeier Green, K., Esterhuizen, J., De Klerk, D.G., 
Ntshangase, J., Vreysen, M.J.B., Pienaar, R., Motloang, M., Ntantiso, L. & Latif, A.A. 
(2016) An update of the tsetse fly (Diptera: Glossinidae) distribution and African 
animal trypanosomosis prevalence in North Eastern KwaZulu-Natal, South Africa. 
Onderstepoort Journal of Veterinary Research, 83 (1), a1172. 
De Beer, C., Venter, G. & Vreysen, M.J.B. (2016) Comparison of geometric morphometric 
markers between South Africa, southern Mozambique and Swaziland tsetse 
populations. Abstracts of the XXV International Congress of Entomology, 25-30 
September 2016, Orlando, United States of America. 
Renda, S., De Beer, C.J., Venter, G.J. & Thekisoe, O.M.M. (2016) Evaluation of 
larviposition site selection of Glossina brevipalpis. Veterinary Parasitology, 215, 92-
95. 
Accepted: 
De Beer, C.J., Venter, G.J. & Vreysen, M.J.B. (accepted) Improving the diet for the rearing 
of Glossina brevipalpis Newstead and Glossina austeni Newstead: blood source and 
collection – processing – feeding procedures. PLoS ONE. 
 
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