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This work is protected by copyright and other intellectual property rights and duplication or sale of all or part is not permitted, except that material may be duplicated by you for research, private study, criticism/review or educational purposes. Electronic or print copies are for your own personal, non- commercial use and shall not be passed to any other individual. No quotation may be published without proper acknowledgement. For any other use, or to quote extensively from the work, permission must be obtained from the copyright holder/s. Chemical and morphological investigations of the Ontogenetic effects of Novel Psychoactive Substances on forensically important blowfly species (Diptera: Calliphoridae). A thesis submitted by Emma Rachael Garrett to Keele University For the degree of Doctor of Philosophy June 2018 Abstract Post Mortem Interval (PMI) estimation is a key concern of forensic entomology research. Numerous factors are recognized to affect these calculations, and have shown to potentially introduce error, leading to an incorrect time of death estimation. One such acknowledged factor is the presence of drugs and toxins. A range of Novel Psychoactive Substances (NPS), common adulterants and one illegal drug were tested on two blowfly species, of forensic importance, Calliphora vicina and Lucilia sericata (Diptera: Calliphoridae). An artificial diet was used to enable effective delivery of the substances; the effect on development was studied by observing larval length, weight, instar and time taken to reach pupariation. As a potential alternative for accurate aging, the cuticular hydrocarbon profile was analysed for changes in response to drug presence. Preliminary investigations were also carried out to detect the presence of NPS within larvae, by extraction and derivatisation. The NPS had a profound effect on the development of immature larval samples; rates were mostly accelerated, shown by larval length, weight and an increase in time taken to reach pupariation. A potential PMI overestimation of 48 hours was presented for a number of substances. Paracetamol was the only drug shown to produce developmental delay, up to 48 hours for a higher dose. The effects of drug dosage and potential to use data from chemically similar drugs for PMI estimations are presented; MDA is compared with NPS, 6APB. Noteworthy, the cuticular hydrocarbon profile showed no major changes in response to the drugs, some minor differences were observed but this was less pronounced than the development data and has lesser effect on PMI estimation. Results encourage the use of cuticular hydrocarbon analysis for accurately aging blowfly, despite showing developmental changes triggered by drug ingestion, which may otherwise cause incorrect PMI estimations. i | P a g e Acknowledgements I would firstly like to thank Keele University for funding my research, partially through the ACORN scheme. Recognition must also go to the following: Professor Nigel Cassidy for initially offering me my PhD studentship, for the endless reassurance and never doubting my ability to be successful. Dr Falko Drijfhout, my supervisor for making this possible, proofreading my monster of a thesis and for your guidance over the years I have been at Keele. Dr Vladimir Zholobenko, your confidence in my ability has helped more than you know. Mark Wilkes, for understanding how difficult it is to juggle full time work with writing a PhD thesis and having faith that I will do everything to the best of my ability. The European Association for Forensic Entomology (EAFE), for providing a nurturing and inviting environment to discuss ideas and expand my knowledge. Thanks also to Dr Katherine Brown, Dr Kate Barnes, Dr Martin Hall and Dr Stefano Vanin for your advice and for helping to top up my colonies! To all my friends and family, without you all I would not be me. Never underestimate the impact of the littlest gesture. I cannot wait to have more time to spend with you all. Special mention to Emma Byrne, Mike Pyle, Krista Jones, Gillian Smith, Mia Wright, Kristopher Wisniewski, Jamie Garrett and Phil Thomson, you always keep a smile on my face. ii | P a g e To my parents Peter and Karen, all I ever wanted was to make you proud and show you what I am able to achieve with all the support and love you have given me. Thank you for always believing in me and teaching me that the hardest times will lead to the greatest moments and it would all be worth it in the end. My grandma Betsy, thank you for always telling me it is time to go home, checking I have eaten and being so proud of me. Our conversations remind me of what is important. I feel so lucky to be surrounded by all of you. My PhD experience has provided some major challenges but if you do not challenge yourself, you will never realise what you can become. This thesis is presented to the reader as a result of impassioned research, throughout my time at Keele. iii | P a g e Contents 1.1 Forensic entomology....................................................................................................................... 1 1.1.1 Post Mortem Interval ............................................................................................................... 2 1.1.1.1 Minimum Post Mortem Interval (PMImin) estimated by insect development ................ 4 1.1.1.2 Minimum Post Mortem Interval (PMImin) estimated by insect succession .................... 4 1.1.2 Stages of decomposition .......................................................................................................... 6 1.1.3 Isomorphen and Isomegalen diagrams .................................................................................... 7 1.1.4 Thermal Summation models .................................................................................................... 9 1.1.5 Lifecycle .................................................................................................................................... 9 1.1.5.1 Feeding to pupariation .................................................................................................... 10 1.1.6 Factors affecting the lifecycle leading to incorrect PMI estimation ...................................... 13 1.1.6.1 Weather, temperature and photo-period ...................................................................... 13 1.1.6.2 Genetic and geographic differences ............................................................................... 14 1.1.6.3 Precocious egg development .......................................................................................... 14 1.1.6.4 Myiasis ............................................................................................................................ 15 1.1.6.5 Delays in oviposition and nocturnal oviposition ............................................................. 15 1.1.6.6 Overcrowding and competition ...................................................................................... 16 1.1.6.7 Other factors to consider, relating to decomposition rate ............................................. 17 1.1.6.8 Body Tissue ..................................................................................................................... 18 1.1.6.9 Drugs ............................................................................................................................... 19 1.1.6.10 Dispersion time ............................................................................................................. 19 1.1.6.11 Pupae as contaminants ................................................................................................. 19 1.1.6.12 Collecting, Killing and preserving .................................................................................. 20 1.2 Entomotoxicology ................................................................................................................... 21 1.2.1 Forensic Entomotoxicology ................................................................................................ 21 1.2.2 Insect use as alternative toxicological samples ................................................................. 21 1.2.3 Effect on blowfly developmental data ............................................................................... 28 1.2.4 Limitations of Entomotoxicology ....................................................................................... 29 1.3 Novel Psychoactive Substances .............................................................................................. 34 1.4 Potential new methods and Cuticular Hydrocarbons as a tool to study development .......... 42 iv | P a g e 1.5 Rationale and Aims ................................................................................................................. 45 2.1 Chromatography ........................................................................................................................... 47 2.1.1 Adsorption Chromatography ................................................................................................. 48 2.1.2 Partition Chromatography ..................................................................................................... 48 2.1.3 Ion-exchange Chromatography ............................................................................................. 49 2.1.4 Molecular exclusion Chromatography ................................................................................... 49 2.1.5 Affinity Chromatography ......................................................................................................
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