University of Huddersfield Repository Giordani, Giorgia Non-invasive approaches to morphological and molecular identification of insects from museum, archaeo-funerary and forensic contexts Original Citation Giordani, Giorgia (2019) Non-invasive approaches to morphological and molecular identification of insects from museum, archaeo-funerary and forensic contexts. Doctoral thesis, University of Huddersfield. This version is available at http://eprints.hud.ac.uk/id/eprint/34893/ The University Repository is a digital collection of the research output of the University, available on Open Access. Copyright and Moral Rights for the items on this site are retained by the individual author and/or other copyright owners. Users may access full items free of charge; copies of full text items generally can be reproduced, displayed or performed and given to third parties in any format or medium for personal research or study, educational or not-for-profit purposes without prior permission or charge, provided: • The authors, title and full bibliographic details is credited in any copy; • A hyperlink and/or URL is included for the original metadata page; and • The content is not changed in any way. For more information, including our policy and submission procedure, please contact the Repository Team at: [email protected]. http://eprints.hud.ac.uk/ Non-invasive approaches to morphological and molecular identification of insects from museum, archaeo-funerary and forensic contexts Author: Supervisors: Giorgia Giordani Dr. Stefano Vanin Prof. Martin Richards A thesis submitted to the University of Huddersfield in partial fulfilment of the requirements for the degree of Doctor of Philosophy Department of Biological and Geographical Sciences, School of Applied Sciences University of Huddersfield September 2018 I Copyright statement i- The author of this thesis (including any appendices and/or schedules to this thesis) owns any copyright in it (the “Copyright”) and s/he has given The University of Huddersfield the right to use such copyright for any administrative, promotional, educational and/or teaching purposes. ii- Copies of this thesis, either in full or in extracts, may be made only in accordance with the regulations of the University Library. Details of these regulations may be obtained from the Librarian. This page must form part of any such copies made. iii- The ownership of any patents, designs, trademarks and any and all other intellectual property rights except for the Copyright (the “Intellectual Property Rights”) and any reproductions of copyright works, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property Rights and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property Rights and/or Reproductions. II Student Declaration Form I, Giorgia Giordani, hereby declare that - while registered as a candidate for the research degree, I have not been a registered candidate or enrolled student for another award of the University of Huddersfield or other academic or professional institution; - my thesis/dissertation entitled “Non-invasive approaches to morphological and molecular identification of insects from museum, archaeo-funerary and forensic contexts” is the result of my own work. The outcome of work done in collaboration is declared. I am the main author responsible of the writing of the following publications (Appendix B) results of this thesis 1. Giordani G., Grzywacz A. and Vanin S. (2018) Characterization and identification of puparia of Hydrotaea Robineau-Desvoidy, 1830 (Diptera: Muscidae) from forensic and archaeological contexts. Journal of Medical Entomology. https://doi.org/10.1093/jme/tjy142 2. Giordani G., Tuccia F., Zoppis S., Vecchiotti C. and Vanin S. (2018) Record of Leptometopa latipes (Diptera: Milichiidae) from a human cadaver in the Mediterranean area. Forensic Sciences Research. https://doi.org/10.1080/20961790.2018.1490473 3. Giordani G., Tuccia F., Floris I. and Vanin S. (2018) First record of Phormia regina (Meigen, 1826) (Diptera: Calliphoridae) from mummies at the Sant’Antonio Abate Cathedral of Castelsardo, Sardinia, Italy. PeerJ 6:e4176 https://doi.org/10.7717/peerj.4176 The coauthors of the previous publications contributed in the following way: - Dr A. Grzywacz provided the specimens polish specimens of Hydrotaea specimens; - Dr S. Zoppis and Prof. C. Vecchiotti provided the specimens and the general information about the forensic case; - Prof. I. Floris provided part of the entomological material from Sant’Antonio Abate Cathedral; - Tuccia F. provided historical information about the cases or the samplings. - Dr Vanin S. made available his entomological collection and supervised the work. Date: Signature: Print Name: III "I suppose you are an entomologist" "Not quite so ambitious as that, Sir. I should like to put my eyes on the individual entitled to that name. No man can be truly called an entomologist, Sir; the subject is too vast for any single human intelligence to grasp." Oliver Wendell Holmes, The Poet at the Breakfast Table. IV V Abstract Species identification is the starting point for the formulation of any further ipothesis in insect sciences and they are particularly important in forensic entomology and funerary arcaheoentomology. Two main approaches are currently used for identifying insects, the morphological one, preferred by old-school taxonomists and the more recent molecular one, favoured by non-specialists. Both approaches have respectively some limitations: lack of morphological identification keys, especially for the immature stages and the destruction of the specimens during the DNA extraction, as examples. The development of easy-access identification keys and of non-invasive DNA extraction methods permitting the repeatability of the analyses have been the main target of this thesis. To achieve this aims morphological analysis and DNA based methods were integrated and used on samples belonging to different contexts – forensic, museum and archaeological – and different timeframe – 0 to 1000 years old -. The attention was paid to adults and the poor investigate puparia of flies from each context and the different timeframes. For the DNA analysis commercial kits and homemade digestion buffers were tested on modern and old specimens but applying operational procedure to preserve the morphology of the specimens. More then 10 puparia belonging to species in the family Heleopmyzidae, Sphaeroceridae, Piophilidae, Muscidae and Calliphoridae have been described and illustrated. Forty new DNA sequences, mainly from poor investigated taxa, were generated and 37 deposited in GenBank despite some problems arised, mainly due to presence of PCR inibithors in and on the cuticle. The results, reported in this work, will impact in the anlysis of forensic and archaeological material but as well in the study of museum material, that especialy for the species typus is particularly precius and can not be destroid or altered during the identification process. In addition specific question as the separation and identification of Hydrotaea puparia belonging to different species, the identification of poorly investigated species, the systematic of Sarcophaga species in the subgenus Sarcophaga and the evaluation of pre- Columbian funerary cerimonies have been answered. In addition the pictorial material produced will help entomologists working with fragmented material especially from old VI cases or archaeological sites. Several questions are still open and they request further investigations to be done in the future. VII Acknowledgements The PhD thesis is the result of a journey in which you have to put yourself personally at stake. Firstly, I would like to express my sincere gratitude to the F.L.E.A. group. I could not have imagined having a better team for these three years and the credit for this success belongs to a great leader, Dr. Stefano Vanin. Thanks for the incredible support even in the most discouragement moments of the doctorate course, for your patience, motivation, and advices. Fabiola Tuccia, Jennifer Pradelli, your support was essential, in work and in life. We shared knowledge, joy and fun but also moment of difficulty and you were always there to rejoice or comfort me. Prashasti Singh and Subham Mukherjee, we shared lab knowledge but, in particular, we shared the writing-thesis stress. I really felt no one understood me better than you did in this period. Philip Jackson was right when he said “The strength of the team is each individual member. The strength of each member is the team.” even if he was talking about a basketball team. I would like to thank Dr. Gareth Parkes and Dr. Maria Pala (University of Huddersfield), for their insightful comments and encouragement, but also for the hard questions that motivated me to improve my research from various perspectives. I also would like to thank all the Archaeogenetic group for the constructive suggestions during Lab Meetings. My sincere thanks also go to Lorenzo Munari (expert of Sphaeroceridae family, Museum of Natural History, Venice, Italy), Dr. Andrzej J. Woźnica (expert of Heleomyzidae family, Wrocław University, Wrocław, Poland), Dr. Daniel Martín-Vega (expert of Piophilidae family, Universidad de Alcalá, Madrid, Spain), Dr. Daniel Whitmore (expert of Sarcophagidae family, Natural History Museum, London, UK),