Of insects and men: Biomolecular study of fossil insects to trace human movement, trade and environmental impact in the past

The study of ancient insects can inform about past human activities in a variety of ways, for example, as palaeoclimatic indicators that increase our understanding of climate and environmental change, or as testimonies of human impact on past environments, including detailed information on introductions and extinctions, human environments, health and disease.
Insects can provide a wealth of information about biogeographic change. The last glaciation in northwest Europe was accompanied with insect regional extinctions and faunas dominated by cold-adapted species interspersed with warmer ones. The warming climate at the beginning of the Holocene saw also extensive changes in the distribution of the insect fauna. Human impact resulting from the development of agriculture in turn promoted further biogeographic changes. Human mobility during this time, and the domestication of plants and animals were accompanied by uninvited insect guests (Panagiotakopulu & Buckland 2017). The study of these synanthropic and anthropochorous insect species from archaeological contexts can provide important information on human mobility, the introduction of farming, trade, and the spread of insect borne diseases (Simpson et al. 2020).
In this context, the genetic characterisation of fossil insects from natural and archaeological contexts emerges as a powerful tool to study inter and intraspecific variability patterns to allow a refined mapping of the distribution of these species in the past. This can provide information and a better understanding of biogeographic patterns which includes evidence for survival in refugia, re-immigration, synanthropic introductions, their geographic origins, dispersal patterns of insects associated with disease, etc. Genetic analysis coupled with stable isotope analysis of insect chitin can therefore provide valuable information with respect to palaeoclimatic/ palaeoenvironmental changes.
Historically, biogeographic genetic research on insects has been based primarily on modern DNA studies. While there are few studies of fossil insect DNA; these have until recently provided rather limited results (e.g. Heintzman et al. 2014). Very little research has so far been applied to archaeological insect specimens using stable isotope geochemistry (e.g. Gröcke et al. 2010). Recently, we have successfully extracted DNA from desiccated specimens of Musca domestica L., the house fly, which has probable origins in the Nile Valley and has become virtually cosmopolitan. This technical success has indicated the potential application of the technique and the importance of this research for understanding long-term biogeographic change (Simpson et al 2020).
The proposed PhD project, by using specimens from a variety of contexts and geographic areas, aims to further develop a methodology for the extraction of ancient DNA from fossil insects addressing complications arising from specimen size, differential preservation, contamination, etc. It will then use the genetic information obtained from the specimens to explore patterns of expansion of important synanthropic insects during different periods. Subsequently, stable isotope analysis of the specimens will be used to reconstruct palaeoclimatic conditions at the time the insects lived.
The PhD candidate will further compare these results with a database of modern and ancient DNA data in order to better understand pathways for dispersal, human mobility patterns and the processes behind the current cosmopolitan status of many synanthropic insects.

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Image Captions

ArchaeoDNA lab, Durham University. Image: Jeff Veitch.,Trogoderma granarium, the khapra beetle, from pharaonic Egypt. Image: Eva Panagiotakopulu


The analysis will focus primarily on a range of synanthropic insects, representative of different environments and preservation, including Musca domestica L. and three of the most important storage pest species: Sitophilus granarius (L.), Trogoderma granarium (Everts) and Tribolium castaneum (Hbst). In a previous study, we were able to extract ancient DNA from desiccated house fly puparia from the archaeological site of Qasr Ibrim in Egypt, thus demonstrating the viability of the project in terms of DNA preservation (Simpson et al 2020).
DNA will be extracted using a non-destructive silica-based protocol following the implementations introduced in Simpson et al. 2020. Illumina libraries will be constructed from extracted DNA and informative regions will be isolated by in-solution hybridisation techniques, copied, and sequenced. The analysis will target variable regions of the mitochondrial DNA (COI, COII and 18S) together with barcoding genomic regions variable at intraspecific level.
The project will include an important component of experimental design of SNP selection for hybridization capture using modern variability as a proxy.
Stable isotopes of mobility will be analysed in insect chitin following Gröcke et al. 2010.
Analyses will be performed in the ancient DNA laboratory (Archaeo-DNA lab) and the Stable Isotope Biogeochemistry Laboratory (SIBL) at the Department of Archaeology, Durham University.

Project Timeline

Year 1

Months 1-4. Sample collection and laboratory training. Samples will be collected at museums and collections in the UK and continental Europe. The student will be trained in the identification of insects using entomological keys with our end-user collaborator (Edinburgh), in ancient DNA extraction and preparation of Illumina libraries (Durham , Stirling) and in the preparation of chitin samples for isotope analysis (Durham).
Months 4-8. Experimental design and implementation of laboratory protocols. DNA capture arrays will be designed using the current insect genetic variability as a proxy (Stirling, Durham). Will require a compilation of a worldwide database of DNA sequences and SNPs from the different species using DNA sequence repositories.
Months 9-12. Start of Laboratory analysis. DNA extraction, Illumina library preparation, quantification, amplification, shotgun sequencing and DNA capture of specific SNPs in samples with well-preserved endogenous DNA. Stable isotope preparation and analysis.

Year 2

Months 13-24. Continuation and completion of laboratory analysis.
Months 12-24. Start of Data analysis (in parallel to data production). Data filtering and identification of endogenous DNA. Comparison of DNA obtained from different sites/locations with modern data from the same geographic areas using specialised software. Interpretation of stable isotope values with respect to palaeoclimate, and comparison of that with other independent palaeoclimate data.

Year 3

Months 25-30. Continuation and completion of data analysis.
Months 31-36. Start of thesis write-up and dissemination of results. Publication of at least 2 high impact publications in international journals and participation in relevant seminars and conferences.

Year 3.5

Months 37-42. Continuation and completion of thesis write-up and dissemination of results.

& Skills

This project will provide the student with valuable transferrable skills in fossil insect recovery and identification, evolutionary and population genetics analysis and stable isotope analysis. The PhD candidate will be introduced to fossil insect research, identification skills using entomological keys and reference collections and interpretation of fossil insect assemblages (training by end-user Edinburgh). The student will also be trained to work in an ancient DNA laboratory and will learn genetic analysis techniques: DNA extraction from insect chitin, Illumina library preparation, amplification and indexing of Illumina libraries, qPCR, in- solution DNA capture (Durham, Stirling). The student will also be trained in sample preparation for stable isotope analysis, and the use of isotope ratio mass spectrometry (Durham). Moreover, the student will be introduced to population genetics data analysis using specialised software (Durham, Stirling). The project will also include an initial stage of sample collection in the UK and in continental Europe.

References & further reading

– Gröcke, D. R., van Hardenbroek, M., Sauer, P. E. & Elias, S. A. (2010). Hydrogen Isotopes in Beetle Chitin. In: Chitin: formation and diagenesis. Gupta, Neal S. Dordrecht: Springer Netherlands, pp. 105-116.
– Heintzman, P., Elias, S.A., Moore, K., Pasziewicz, K., Barnes, I. (2014) Characterizing DNA preservation in degraded specimens of Amara alpina (Carabidae: Coleoptera). Molecular Ecology Resources 14:606-615.
– Panagiotakopulu, E., Buckland, P.C. (2017). A thousand bites – Insect introductions and late Holocene environments. Quaternary Sci Rev 156:23-35.
– Simpson, A., Fernandez-Dominguez, E., Panagiotakopulu, E., Clapham A. (2020). Ancient DNA Preservation, Genetic Diversity and Biogeography: A Study of Houseflies from Roman Qasr Ibrim, Lower Nubia, Egypt’. Journal of Archaeological Science 120: 105180.

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