Ancient insect DNA, stable isotopes and synanthropic environments


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.
In terms of 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 extensive changes in the distribution of the insect fauna (e.g. Panagiotakopulu 2014). 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 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 (idem).
Historically, biogeographic genetic research has been based primarily on modern DNA studies, and 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 species using stable isotope geochemistry (e.g. Groecke et al. 2010).
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. This database coupled with stable isotope analysis of beetle chitin will provide valuable information with respect to palaeoclimatic/ palaeoenvironmental changes.
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. 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 beetle lived. The candidate will further compare these results with modern 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

Figure 1. Trogoderma granarium (Everts), the khapra beetle from Qasr Ibrim, Egypt. Source: E. 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 pilot 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 2017). DNA will be extracted using a non-destructive silica-based protocol following the implementations introduced in Simpson (2017). DNA will be amplified following a combination of low (PCR and Sanger sequencing) and high throughput (Illumina Libraries and Next Generation Sequencing) approaches. 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 primers for PCR and SNP selection for hybridization capture using modern variability as a proxy. Genetic data analysis will follow establish ancient DNA bioinformatic pipelines (Feldman et al. 2019).
Stable isotopes of mobility will be analysed in insect chitin following Groecke 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 1-6. Sample collection and experimental design. Primer design and design of DNA capture arrays using current genetic variability. Will require a compilation of a worldwide database of DNA sequences and SNPs from the different species using DNA sequence repositories.
Months 7-12. DNA laboratory analysis part 1. DNA extraction, PCR amplification and Sanger Sequencing, Illumina library preparation, quantification and amplification.

Year 2

Months 13-22. DNA laboratory analysis part 2. Shotgun sequencing and DNA capture of specific SNPs in samples with well-preserved endogenous DNA.
Months 13-18. Stable isotope preparation and analysis.
Months 22-24. Genetic data analysis part 1. Data filtering and identification of endogenous DNA.

Year 3

Months 25-32. Genetic data analysis part 2. Comparison of DNA obtained from different sites/locations with modern data from the same geographic areas using specialised software.
Months 25-30. Interpretation of stable isotope values with respect to palaeoclimate, and comparison of that with other independent palaeoclimate data.
Months 33-36. Start thesis write-up and dissemination of results in seminars, workshops and conferences.

Year 3.5

Months 37-42. Finish thesis write up. Preparation of at least 2 high impact publications in international journals.

& Skills

This project will provide the student with valuable transferrable skills in fossil insect recovery and identification, evolutionary and population genetics and stable isotopes. The PhD candidate will be introduced to fossil insect research, identification skills using entomological keys and reference collections and interpretation of fossil insect assemblages. The student will also be trained to work in an ancient DNA laboratory and will learn genetic analysis techniques: DNA extraction from insect chitin, PCR design and reaction preparation, Illumina library preparation, amplification and indexing of Illumina libraries, qPCR, in- solution DNA capture. The student will also be trained in sample preparation for stable isotope analysis, and the use of isotope ratio mass spectrometry. Moreover, the student will be introduced to population genetics data analysis using specialised software.

References & further reading

Feldman, M., Fernández-Domínguez, E., Reynolds, L., Baird, D., Pearson, J., Hershkovitz, I., May, H., Goring-Morris, N., Benz, M., Gresky, J., Bianco, R.A., Fairbairn, A., Mustafaoğlu, G., Stockhammer, P.W., Posth, C., Haak, W., Jeong, C., Krause, J. (2019). Late Pleistocene human genome suggests a local origin for the first farmers of central Anatolia. Nature Communications 10, 1218.
Groecke, 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. (2014). Hitchhiking across the North Atlantic – Insect immigrants, origins, introductions and extinctions. Quaternary International 41:59-68.
Panagiotakopulu, E., Buckland, P.C. (2017). A thousand bites – Insect introductions and late Holocene environments. Quaternary Sci Rev 156:23-35.
Simpson, A. (2017). Ancient DNA preservation and genetic diversity in fossil insects from Qasr Ibrim, an Egyptian Archaeological Site. MSc dissertation. Durham University.

Further Information

Dr. Eva Fernandez-Dominguez, Department of Archaeology, Durham University, Durham DH1 3LE. Tf:+44 (0) 191 33 41141. email:

Dr. Luc Bussiere, Biological and Environmental Sciences, 3A133 Cottrell Building, University of Stirling, Stirling FK9 4LA. Tf:+44 (0)1786467758. email:

Dr. Darren R. Groecke, Department of Earth Sciences, Durham University, Durham DH1 3LE. Tf:+44 (0) 191 33 42282. email:

Dr. Eva Panagiotakopulu, School of GeoSciences, Drummond Street, University of Edinburgh, EH8 9XP. Tf:+44 1316502531. email:

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