DNA profiling from the crop content of sarcophagidae spp. larvae - Barnard, Adri Marlene (University of the Free State, 2017)
Morphological analysis of insect evidence plays a significant role in crime scene investigation. With the influence of DNA analysis in forensic cases, which now also plays a key role in forensic entomology, more emphasis has been placed on a dual preservation goal when collecting insect evidence. Previous studies indicated that it might be possible to identify the last meal of sarcophagid maggots using gut content combined with DNA profiling. For gut content analysis it is imperative to be familiar with the internal morphology as well as maggot gastric emptying. However, insufficient information is available on the morphology of Sarcophaga cruentata maggot alimentary canals as well as the rate of maggot gastric emptying. Also, considering the use of insects for both PMI and DNA analysis, the current preservation methods are not necessarily suitable for maggot preservation for DNA analysis of the crop content. Various preservation methods were examined for optimal preservation of both morphology and gut content. In order to understand gastric emptying, the internal gut structures and movement of food through the gut was examined. Fully fed maggots were removed from the food source and hot water killed at 3 hour intervals for up to 30 hours to investigate gastric emptying. Crop content DNA analyses were performed to attempt identification and DNA profiling of the maggots’ last meal. Due to the inhibiting effect of formalin on DNA analysis and the extensive dehydration of prolonged ethanol storage, -80°C was investigated for sample preservation which generally provided good results. Inconsistent results were obtained using the various preservation and DNA analysis combinations tested. Sarcophagidae gut morphology analysis indicated gross anatomical similarity to Chrysomya megacephala. External tracking of food movement proved difficult. After digestive tract dissection it was found that the mid- and hindgut coiled around each other. Due to the coiled structure of the gut, the exact location of the food bolus could therefore not be determined without dissection. The expected gastric emptying was not consistently observed with pronounced variability in results. Nevertheless, it was observed that the maggot crops were completely empty by 30 hours post removal from food source. DNA profiling of the crop content supported previous findings, although only partial STR profiles were obtained. It is unlikely that full profiles will be obtained when analyzing gut content due to the degraded nature of the food source, as well as the effect of digestive enzymes present in the maggot saliva regurgitated onto the food source. Various recommendations can be made based on the results. At crime scenes maggots should be killed in warm water (± 60°C for 30 seconds), dried on paper towel and stored in 80% ethanol while transported to the laboratory. Samples should be removed from the ethanol within 24 hours after collection, dried and stored individually in microcentrifuge tubes at -80°C (or similar low temperatures) until analysis is performed. During analysis samples should be handled on ice, ensuring the integrity of the sample, as it was found that samples defrosted rapidly after removal from the -80°C storage. It is further recommended to use commercially available kits when analyzing maggot gut samples due to the additional clean-up steps present in the kits. These clean-up steps aid in limiting the addition of fats and lipids from the maggot internal structures that could inhibit downstream DNA analysis of samples. Overall this study reinforced the possibility of using maggot crop content for providing STR profiles of the victim and/or perpetrator. Although only partial STR profiles were obtained, it indicated that, with further investigation and optimization, this is an interesting avenue for future research with many unexplored avenues for aiding in crime scene investigation.

Morfologiese analise van insek bewyse speel ‘n noemenswaardige rol in misdaadtoneel ondersoeke. Met die invloed van deoksiribonukleïensuur (DNS) analise in forensiese sake, wat nou ook ‘n sleutelrol speel in forensiese entomologie, word meer klem geplaas op ‘n dubbele bergingsdoel met versameling van insek bewyse. Vorige studies dui aan dat dit moontlik kan wees om die laaste maaltyd van sarcophagid larwes te identifiseer deur gebruik te maak van dermkanaalinhoud gekombineer met DNS profielbepaling. Vir dermkanaalinhoudsbepaling is dit noodsaaklik om vertroud te wees met die larwale interne morfologie as ook larwale gastriese lediging. Onvoldoende inligting is egter beskikbaar oor die morfologie van Sarcophaga cruentata spysverteringskanale asook die tempo van larwale gatriese lediging. Verder, met die gebruik van insekte vir beide PMI en DNS analise, is die huidige bergingsmetodes nie noodwendig geskik vir larwale berging vir DNS analises van die dermkanaal inhoud nie. Verskeie bergingsmetodes vir optimale storing vir beide morfologie en dermkanaalinhoud analise is ondersoek. Om die gastriese-lediging te verstaan is die interne spysverteringskanaal struktuur asook die beweging van voedsel deur die dermkanaal ondersoek. Ten volle gevoede larwes is vanaf die voedselbron verwyder en met warm water doodgemaak teen 3-uur intervalle vir 30 ure na verwydering vanaf die voedselbron. Kropinhoud analise was uitgevoer in ‘n poging tot identifikasie en DNS profielbepaling van die larwe se laaste maaltyd. Weens die inhiberende effek van formalien op DNS analise asook die ekstensiewe dehidrasie effek van langdurige etanol berging, is -80°C berging ondersoek wat oor die algemeen goeie resultate verskaf het. Strydige resultate is verkry met gebruik van die verskillende bergings en DNS analise kombinasies getoets. Morfologiese analise van Sarcophagidae dermkanale het aangedui dat dit ooreenstem met die van Chrysomya megacephala. Beweging van voedsel kon nie ekstern maklik gevolg word nie. Disseksie van die spysverteringskanaal het aangetoon dat die middel en agterderm om mekaar gedraai is. Die presiese posisie van die voedselbolus kon weens hierdie gedraaide struktuur van die dermkanaal, nie bepaal word sonder disseksie nie. Die verwagte gastriese-lediging is nie konsekwent waargeneem nie, met merkbare variasie in resultate. Nietemin is dit waargeneem dat larwale kroppe teen 30 uur na verwydering van die voedselbron, heeltemal leeg was. DNS profielbepaling het vorige bevindings ondersteun al is slegs gedeeltelike profiele verkry. Dit is egter onwaarskynlik dat vol profiele verkry sou word weens die gedegradeerde natuur van die voedselbron, asook die effek van verteringsensieme teenwoordig in die larwale spoeg, wat help met gedeeltelike vertering van die voedselbron. Verskeie voorstelle kan gemaak word vanaf die resultate. By misdaad tonele moet larwes doodgemaak word in warm water (± 60°C vir 30 sekondes), drooggemaak word op papierdoek en gestoor word in 80% etanol tydens vervoer na die laboratorium. Monsters moet binne 24 uur verwyder word uit die etanol, drooggemaak en individueel gestoor word in buise teen -80°C (of soortgelyke temperature) tot analise uitgevoer word. Tydens analise moet eksemplare op ys hanteer word aangesien daar waargeneem is dat larwes baie vinnig ontdooi na verwydering uit -80°C berging. Daar word verder voorgestel dat kommersieel beskikbare stelle gebruik word om larwale dermkanaal inhoud te analiseer aangsien dit addisionele skoonmaakstappe vereis. Hierdie skoonmaakstappe beperk die invloed van vette en lipiede vanaf die larwale interne strukture wat DNS analise van monsters kan inhibeer. Oor die algemeen versterk hierdie studie die moontlikheid dat larwale kropinhoud gebruik kan word om STR profiele van die slagoffer en/of oortreder op te lewer. Al was slegs gedeeltelike STR profiele verkry dui dit steeds daarop dat, met verdere navorsing en optimisering, hierdie ‘n interesante veld vir toekomstige navorsing met verskeie onverkende moontlikhede is wat hulp kan verskaf in die ondersoek van misdaadtonele.
Dissertation (M.Sc. (Genetics and Entomology))--University of the Free State, 2017
Keywords: Alimentary canal; Dissection; DNA; Ethanol; Formalin; Gut content; Maggot; Preservation; Sarcophagidae; STR; Larvae