Surveying an insect collection from a 17th-century Northeastern agrarian settlement to determine changes in beneficial insects, pests, and climate

1. Using Ancient DNA to make a species-level identification of a group of rove beetle remains entombed in a 1680s plantation owner’s grave to draw conclusions about pest pressure at the site

The vast collection of insect fragments was first recovered starting in 1992 and identified to the family level by the late interdisciplinary entomologist Dr. Theodore “Ted” Suman (1939 – 2020). Since Dr. Suman completed the family-level identifications, revised identification keys, many based on pictures provide more points of reference. This increases the likelihood of being able to make positive identifications from fragmentary remains based on visual characteristics. Rove beetles will be used as a measurement in changes of beneficial insects that act as “natural enemies” to pest insects in agricultural systems. To do this, the rove beetles from the Historic St. Mary’s City archaeological grave site in Maryland will be compared to the modern rove beetle fauna of the location. A combination of unique characteristics makes rove beetles an adequate model through which to measure this change. Specifically, many species have narrow preferences for environmental conditions and season. The vast number of species within the family of rove beetles means that by analyzing the collection, or assemblage of rove beetles at the site will result in a clearer environmental picture. Of the ~5,000 fragments identified as rove beetles, a subsection of ~15 head capsules will be selected for genetic testing. This number was chosen for economic feasibility and to ensure the bulk of the fragments remain undamaged to be used in case of future advances in molecular techniques. Prior to selection of specimens, head capsules will be separated into groups of potential sub-families and/or genus based on visual morphology of the structure of the head as assessed through the Arnett & Thomas (2000) morphological key for rove beetles of North America. The groups will then be sub-sampled equally until approximately 15 head capsules are selected for genetic testing. All samples used in the analysis will be photographed with high-resolution photography, advanced microcopy techniques (i.e. stacked imaging) and pencil-sketched, with sketches emphasizing the key morphological character(s) used to make the visual identification.

Genetics work will take place in June 2023 at the laboratory at Arizona State University. June was selected so that this analysis can coincide with presentation of our project’s current findings at the 2023 conference of the North American Forensic Entomology Association (NAFEA). The molecular protocol we have designed is based on publications from Sproul & Maddison (2017); Hung et al. (2013); Smith et al. (2021); and Brunke et al. (2021). In the period prior to extraction, ancient DNA specialists from the Swedish SciLifeLab will assist in producing a finalized extraction and library preparation protocol in accordance with current best practices in ancient DNA studies. I will extract DNA from historical insect specimens with a reagent kit designed for small amounts of damaged DNA. To quantify the DNA concentrations extracted, I will perform a serial dilution with a Qubit model Fluorometer then measure the fragment length distributions on a 2100 Bioanalyzer. To magnify the DNA, the qPCR protocol will use the specialized StaphBaits probe set developed by Brunke et al. (2021) built to anneal to specific genetic loci in family Staphylinidae. The amplified products will be compared to public databases such as NCIB to ensure I have extracted verifiable ancient DNA adequate for sequencing.  A DNA repair process for ancient specimens will repair damages in bases prior to library preparation. Genetic library preparation is designed for low-input and will recover ancient DNA taken from the specimens. Library quantification will be performed on a Qubit Flurometer. The DNA extracted from all specimens will be used to perform paired-end shotgun sequencing. The CLC Genomics Workbench software will remove duplicate reads, assemble non-duplicate paired end reads to BLAST the sequences against the genomes of beetle species sequenced in databanks. The computer program will also remove genetic reads from any contamination such as human or fungi DNA. Results from Sproul & Maddison (2017) and Hung et al. (2013) were generally of high quality with 86-99% and 97.5% recovery, respectively.

Location: Arizona, Arizona State University

2. Complete a three-year survey of the site’s modern insect biodiversity which started in 2021

While prescribed as best practice by Elias (1994), most archaeological entomology studies do not complete or report extensive surveys of a site’s modern insect taxa. Because insects’ natural history and ecological preferences have changed very little in thousands to millions of years, comparing a site’s historical versus modern insect biodiversity can measure the degree of ecological change the site has undergone (Elias, 2010). Several traditional insect collection methods are being used to survey a wide range of modern insect species diversity at the Historic St. Mary’s City, Maryland site. The site of the original building which likely held the coffins prior to burial, is immediately adjacent to a lean-to structure which is now being used to simulate the coffin scenario. This is accomplished by insect sampling from a deceased piglet (Sus scrofa domesticus)

The piglet is placed in a replica lead-encased coffin. Piglets are obtained deceased from the Penn Veterinary School of Medicine and are replaced 1-2 times per season depending on availability. Insects are sampled from the remains 1-2 times per week until they are skeletonized. These methods meet the standards of forensic entomology best practice in research. The land directly adjacent to the lean-to is arable and in yearly production of various crops (i.e. sweet corn, soybeans). Other insect taxa are sampled through a series of 6 pitfall traps. Our pitfall trapping schema is a modified version of Hoffman et al., 2006 which was a study pairing beetle diversity with soil biogeochemical analysis which determined land use could be elucidated from this study design. Pitfall traps are at two sites; the agricultural land directly adjacent to the lean-to where the pig is located (Site 1) and the meadow adjacent to the chapel where the human remains were excavated (Site 2) in the early 1990s. Each site has three pitfall traps orientated in a NW-SE direction where traps were set in a straight line approximately 10m apart from one another. Traps are harvested twice a month on the 1^st or 15^th unless that date falls on a weekend or holiday, in which case they are harvested on the next following business day. At the time when traps were dug, approximately 0.5kg of soil from below the root zone was collected for biogeochemical analysis. The land surrounding the lean-to and production field (Site 1) is heavily forested, increasing the likelihood of diverse insect species while trapping. The area around the chapel (Site 2) is a planned meadow using local plants that were determined to be indigenous to the area and was designed to reflect the likely meadow-land plant communities that existed at the time of the original colony in the late 1600s. Subterranean insects at Site 1 are collected with a PVC tube-trap outfitted with ingress channels and a collection receptacle (mason jar) filled half-way with antifreeze to kill and preserve insects which fall in. The subterranean trap was inserted into the ground on the outer southeast corner of the lean-to.

Location: Maryland, Historic St. Mary’s City

3. Using updated morphological keys to make likely genus or lower-level taxonomic identifications of the other specimens in the assemblage including true insects (Hemiptera) and mites (Acari)

At the original examination of the remains, Dr. Suman separated the invertebrate remains from the coffin debris; which mainly consisted of wood fragments from the coffins, some bone and cloth fragments. The debris were examined under a stereo microscope where all arthropod and snail fragments were removed using a small-pointed brush dampened with alcohol. Specimens were stored for long-term storage in shell vials with 80% ethyl alcohol. Since then, the updated true insect (Hemiptera) and mite (Acari) morphological keys published since the Historic St. Mary’s City collection was first recovered and described provide a fresh opportunity for a deeper analysis of these specimens in lieu of other molecular packages like Brunke et al. (2021)’s StaphBaits. Other arthropod taxa within the assemblage includes the remains of thrips, ants, spiders, centipedes, millipedes, and remains that have not currently been identified. Texts that will be consulted will include but will not be limited to; Triplehorn & Johnson (2015), Balogh & Balogh (2012), Arnett & Thomas (2000) and Ubick & Cushing (2005).

Location: New Jersey, Rutgers University

4. Plan of data analysis:

The final DNA identifications will be compared to and incorporated with the interpretation of the wider group of rove beetle remains from the 1680s coffin. The molecular genetic identifications will be used to verify the morphological identification of the wider collection of rove beetle fragments from one coffin. These will then be compared to the rove beetles collected during the multi-year survey which began in 2021 at the location archaeologists determined was the likely site the coffins were stored before final burial. Descriptive and inferential statistics will be coded in R Studio. Data will be checked for normal distribution. The R Studio ‘VEGAN’ statistical package will be used to measure biodiversity with a Simpson’s Diversity Index, performed to evaluate four different aspects of the historical versus modern insect groups. These four aspects are the 1640 rove beetles, the wider collection of 1680s insects including agricultural pests, the site’s modern insects, and finally a comparison of the modern vs historical site biodiversity. Because no single historical assemblage will account for the breadth of a site’s insect biodiversity, the procedures designed by Kenward (1976) and King et al. (2014) will be used to contextualize the functional biodiversity indicated by the ancient insect assemblage.

Location: New Jersey, Rutgers University