Progress report for GNE22-292
Since the dawn of agrarian society humans have been managing natural resources, climate conditions, and crop damage caused by pest pressure. Our project is taking a novel approach to generating quantifiable data on historical pest pressure in the Northeast through an extensive evaluation of an insect collection from 1680s Maryland. Our analysis is focusing on the historical and modern diversity of rove beetles (Coleoptera, Staphylinidae) as a proxy indicator of climate and the system’s capacity to subdue pests. Completing a DNA analysis of historical rove beetle specimens will build on recent insect genetic technologies while advancing sustainable interdisciplinary agriculture research. The other agriculturally significant specimens are also being analyzed, including, and not limited to thrips (Thysanoptera), true insects (Hemiptera), and mites (Acari). Our project crosses disciplines to utilize techniques from archaeology, forensics, genetics, and geology to better understand historical pest dynamics in the Northeastern USA. My planned outreach and communication plan includes workshops for Master Gardeners’ educators on identification of insect specimen fragments, fact sheets for Extension professionals, and conference presentations in 2023 including the 10th International Congresses of Dipterology (ICDX), the North American Forensic Entomology Association (NAFEA) Conference and the Northeast Organic Farmer’s Association (NOFA) 2023 Summer Conference. This research will also result in an open-source scholarly article.
- Complete a DNA 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.
- Advance the study of a specific group of beneficial insects by using DNA to study their role in controlling pest pressure at a historical agricultural settlement
- Location: Arizona, New Jersey, Sweden
- Complete a three-year survey of the site’s modern insect biodiversity which started in 2021
- Trapping modern insects to provide site-specific references to compare historical specimens to.
- Location: Maryland
- Finalize morphological identifications noting specific identifiable characters using updated morphological keys
- Complete a thirty-year research endeavor first started in 1992
- Use advanced imaging techniques to discern morphologically identifiable characters
- Location: New Jersey, Arizona, Sweden
- Analyze data
- Complete descriptive and inferential statistics with the R Studio open-source coding language.
- Compile statistics on both rove beetle and wider insect biodiversity for both modern and historical groups
- Location: New Jersey
The purpose of this project is to address the need to use novel cross-disciplinary approaches to strengthen sustainable agriculture in the Northeastern USA. We will do this by quantifying historical beneficial insect diversity and pest pressure of an agrarian settlement in 1640 Maryland. The results of an in-depth analysis of this unique collection of insect specimens can be used in creative and diverse ways to promote sustainable agriculture. Pursuing this question is important because understanding the historical diversity of beneficial insects is likely to reveal insect-related information important to sustainable food security. For example, documenting and analyzing the species of insects historically associated with low-impact agriculture in the region will contribute to diagnosing the health of modern agroecosystems.
Functional biodiversity is an ecosystem’s interconnected web of vital or beneficial services performed by the collection organisms in the habitat. These ecological services driven by biodiversity directly benefit our agricultural system. A wide range of beneficial insects are directly responsible for performing these services in our production fields and the surrounding forested or meadowed lands. Evidence is indicating warming climates and off-target chemical pollution put a significant amount of pressure on many insect communities. Our project will use new technological advances in interdisciplinary entomology to address this issue with an in-depth study of the only insect collection recovered from the site of a 17th century Northeastern agricultural settlement. Our methods incorporate techniques from archaeology, genetics, pest management, and other fiends to pursue this investigation. Completing this investigation is likely to indicate shifts in populations within the key natural enemy group of rove beetles (Coleoptera, Staphylinidae).
This study will address future issues in agriculture by expanding the scope of Integrated Pest Management to document changes in beneficial insect biodiversity in agricultural systems over time. This specifically addresses the need to study pest management from an evolutionary perspective by using time-calibrated historical specimens (Madison, 2016). An excavation at the site of a seventeenth-century brick chapel at Historic St. Mary’s City, Maryland in 1992 led to the discovery of three lead coffins interred within the structure. From the 1990s to the early 2000s a recovery and examination of the arthropod (insects and related animals) remains was completed. Over 41,000 arthropod fragments were recovered from within the coffins of the two adults. Consistent with insect remains from archaeological deposits, specimens consist mainly of fragments of arthropod exoskeleton and snail shells. The original analyses asserted the lack of fly (Diptera) indicated the insects assembled in winter. However, the presence of large quantities of primary predators of fly larvae, mainly rove beetles and ants (Hymenoptera, Formicidae) and apex predators (Araneae) suggests the coffins were stored for a period of time long enough to allow a complex microhabitat to form. The number of true insects (Hemiptera) and thrips found within the plantation owner’s coffin is unusual and suggests the agricultural operation may have been under considerable pest pressure. Since summer 2021 I have been conducting an ongoing insect survey at the location the coffins were originally stored before burial.
- (Educator and Researcher)
- 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
- 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 1st or 15th 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
- 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
- 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
The assemblage of archaeological insect remains first cataloged during sorting and preliminary identifications are more extensive than originally reported. This has included at least two beetle (Coleoptera) families, remains of several fly (Diptera) species and the remains of what is potentially a bedbug (Siphonaptera). The fly remains are allowing us to compare how soil nutrient recycling catalyzed by fly-driven decomposition may have changed between the 17th century and today by studying the biodiversity of carrion flies at the site. The inclusion of additional beetle specimens beyond what was originally cataloged is allowing us to complete a more nuanced analysis of how pest pressure in coastal Maryland in the late 1600s likely impacted the colony.
Bringing together a robust group of interdisciplinary researchers to evaluate insect remains from agrarian funerary sites has the benefit of leveraging expertise from varying fields. Revisiting previously unidentified specimens with the aid of updated taxonomic keys has allowed us to make identifications of specimens beyond the family level. Deploying multiple trapping schema to catch modern site insects has provided reference material necessary to confirm the identification of the rove beetle Creophilus maxillosus in our archaeological material, and demonstrate that this species has existed at the site since the late 1600s. Capturing C. maxillosus and Ontholestes cingulatus during modern sampling indicates to us that the site vegetation can likely be categorized as "open fields" in both historical and modern contexts.
Education & Outreach Activities and Participation Summary
The likely audience for my project as it is shaping up will be organic and/or low-impact growers seeking to incorporate new techniques to foster functional biodiversity. Additionally, the progression of my project is helping me to meet my goal of addressing the lack of classical entomology and agricultural science perspectives in the field of archaeological entomology. Currently I am mentoring my two of my project’s participants at the Historic St. Mary’s City site who are both classically trained archaeologists. One of these individuals is currently planning to present our new Diptera findings as an invited talk at the 10th International Congress of Dipterology (ICDX). I am assisting her in the construction of the presentation which will span the modern fly taxa we have collected via trapping and will focus on the fly puparia and larval skins we have discovered from the coffin assemblage. I am also mentoring a Swedish graduate student who focuses on archaeoentomology but has no traditional education in modern insect science. Together, he and I are using macroscopic photography of specimens of this project to author a manuscript on standardizing the descriptive language used to write about sub-fossil insect specimens.
Because of the opportunity I have to examine these specimens with one of the few experts in my field, I have decided to postpone hosting an in-person outreach activity. Earlier in the fall term I reached out to the director of my institution’s Master Gardener’s Program with my proposal for hosting insect identification workshops and I hope to connect with him soon about moving these plans forward. My current plan still includes creating and disseminate a factsheet for Master Gardeners identifying fragmentary insect remains. An additional fact sheet will be created for Integrated Pest Management professionals using our results to describe a healthy agricultural insect community for the region based on historical data.
I presented data from this project at an invited lecture to the Umeå University Department of Historical, Religious & Philosophical Studies in November 2022. I will also present current project findings at the World Wildlife Fund of Sweden and the Stockholm Natural History Museum both in February 2023. Currently, our findings are scheduled for presentation at the aforementioned ICDX in July 2023 in Reno, Nevada. Finally I will present this project’s findings through the Northeast Organic Farmer’s Association (NOFA) at their NOFA Summer Conference 2023 in Amherst, Massacushettes and a TBD educational event for the New Jersey chapter of NOFA for the purpose of receiving feedback from local stakeholders on ways in which this project’s findings can help bolster sustainable and food security in the American Northeast.
No growers are directly participating in this research, however we are taking actions to ensure this project does make direct contributions to the agricultural community. The results of the soil analysis from Site 1 was made available to the grower (1) operating the farm at the location of Site 1 as a gesture of gratitude for allowing our study to be completed on their land. Presentation of our project at the NOFA summer conference and hosting a NOFA NJ educational event will be paired with a discussion component where we will outline how we think this study will benefit sustainable agriculture in the American Northeast so stakeholders can provide feedback on how our community can receive the maximum benefits from our research.
I believe developing the concepts behind my work in collaboration with Integrated Pest Management specialists would lead to direct outreach and increased services to growers. If our project is successful, it would provide the basis to find and analyze other historical insect collections within the Northeast region. Eventually this would result in enough data through which novel Integrated Pest Management algorithms can be generated to assess the “health” of an agroecosystem based on what its insect community should be.
We recovered an additional 5 distinct species of rove beetles from unsorted project specimens. Along with the unsorted specimens were Coleopterans from the weevil family Curculionidae (Fig. 1) and Scolytidae, both of which have the potential for significant pest pressure depending on species, determination of which is ongoing. From the previously unsorted beetle remains, we have determined that Creophilus maxillosus was present at the site in the 1680s. Creophilus maxillosus (Coleoptera, Staphylinidae) has also been trapped over several consecutive warm-weather months from piglet carcasses. Additionally, the gold-and-brown rove beetle Ontholestes cingulatus (Coleoptera, Staphylinidae) was also trapped at the Site 1 pig carcass. Both species are large rove beetles that are commonly associated with open meadowland and fields and are voracious predators of blow fly (Diptera, Calliphoridae) larvae as well as various other insect taxa. Our preliminary analysis of these findings indicates that insect prey at Site 1 has been adequate for robust populations of these competing beetle species and is likely indicative that the system has been continually capable of suppressing agricultural pests to some degree. Identifying the same beetle species in the archaeological assemblage and modern site trapping is a finding suggesting that the site may be classified as the same ecological type (i.e. open field, meadow) in both the late 1680s through today.
Two rare finds we recovered from the unsorted sample were several Dipteran puparia and larval skins/body (Fig 2) and remains which have been tentatively identified as a bedbug (Siphonaptera, Cimex spp.) (Fig. 3). These two groups of insects preserve poorly in contexts similar to the coffin they were recovered from, mainly that within the open space of the coffin an aerobic environment is created and because the specimens were likely subjected to repeated freeze-thaw cycles. Our preliminary determinations that Cuculionidae, Siphonaptera and Scolytidae were present in 1680s indicates to us that the original colony was likely under considerable pest pressure on several fronts where plant production, stored products, and human health were all vulnerable. The unexpected presence of both weevils and the bedbug are leading us to consider reallocating some of the DNA testing resources currently slated for rove beetle testing for the purpose of making definitive identifications of these specimens. This is because, depending on the species present, they may represent the oldest known, and/or potentially first arrival, of these pests in Maryland.
From 6 pitfall traps, a total of 220 insect specimens have been collected. Within this data, currently 22 genera have been identified. Of this, a subset of 19 individuals are rove beetles, our target taxa. However all insect taxa will be incorporated into our final analysis. Within our pitfall traps, ants (Hymenoptera, Formicidae) are also strongly represented with 22 individuals from 6 genera. Identifications of specimens from pitfall traps are ongoing. The beetle family Nitidulidae is also been trapped frequently with 18 individuals which we determine to be likely significant for our analysis as this beetle family contains many genera and species that are highly ecologically specialized meaning that we will be able to collate our Staphylinidae and Nitidulidae analysis for a more nuanced determination of ecological health and functional biodiversity across both trapping sites.
From collections at the pig carcass, three families of flies (Diptera) have been collected, which are: flesh flies (Sarcophagidae), coffin flies (Phoridae), and blow flies (Calliphoridae). Identifications of Phoridae and Sarcophagidae are ongoing, while most Calliphoridae specimens collected are the species Phormia regina (Meigen) and Lucilia sericata (Meigen). Notably, a TBD Lucilia sp. was trapped in November which spent approximately two months in the post-feeding “wandering” stage before pupation occurred. Currently, 1 individual from this group has eclosed into its adult stage. We believe this to be a notable observation because this behavior is not typical of most blow fly species, especially from temperate regions and may represent a novel observation and potentially a previously undescribed species or a species not frequently found at this location. The number of blow flies and other carrion colonizing flies recovered from within the lead coffin housing the pig carcass. This bolsters our preliminary analysis that the fly remains extracted from the archaeological assemblage are likely carrion flies. Further, this is also indicative that the location’s fly biodiversity is capable of overcoming significant barriers to colonizing decomposing remains which catalyzes both the decomposition process and soil biogeochemical nutrient cycling.
Preliminary analysis of soil biogeochemistry indicate a no statistically significant difference in all parameters between Site 1 and Site 2 when using a T-Test with the α value set to P = 0.05. For example, meadowland in Site 2 was higher in manganese compared to Site 1 but was not significant with a P value of 0.153. Further, in all productive areas, soil was rated strongly-moderately acidic with the only the area around the piglet carcass being rated alkaline. A more nuanced statistical approach will be used to elucidate differences in site soil chemistry for comparison with the completed insect biodiversity survey of both sites. Results from soil chemistry analysis can be found in the associated MS Excel file linked below.
Soil analysis data can be found in the following MS Excel file:
We believe that pairing extensive modern site insect biodiversity surveying through trapping of live insects has greatly benefitted our project and that this design should be more regularly incorporated into investigations of archaeoentomology. This is because modern site specimens are providing vital reference material from which comparisons to archaeological insect remains can be compared. Identifying the same beetle species (detailed below) in both the archaeological assemblage and through modern insect sampling has allowed us to draw preliminary conclusions about the degree of change experienced by the site, which appears to likely be a stable and productive environment. We have also found that by taking a cooperative approach, where scientists from multiple fields come together to analyze archaeological insect remains, allows us to leverage varying expertise which has allowed us to identify specimens that were previously overlooked in the primary sorting of our insect material. For example, the original preliminary analysis of this assemblage stated that there were no fly (Diptera) remains within the coffin material which led the archaeologists to determine that the remains were likely deposited in cold weather periods, however advances in microscopy paired with our project's interdisciplinary group of researchers have identified remains of larval dipterans (detailed below, Figure 2) and the head of an adult fly. These findings allow us to demonstrate to our archaeological partners that their timeline of deposition and burial may be revisited. This also indicates to us that our original assertion that advances in morphological identification (i.e. publication of new identification keys) will allow us to make a more nuanced analysis is a sound approach.
Being conferred a Fulbright Sweden Open Research Award has allowed me to examine these remains in the laboratory of the Environmental Archaeology Lab (MAL) at Umeå University under its Director who is one of the few experts in the world in the identification of insect fragmentary remains from archaeological sites. Through this action we have been able to recover and begin genera/species level identifications of beetles (detailed below) that were not reported in the original sorting/cataloging of the material. Funds awarded through this grant cover the cost of travel to and from MAL as well as time spent in-residence at MAL. We have also applied for the Smithsonian Institution's S.W. Williston Diptera Research Fund for the purpose of communicating these results at the 10th International Congress of Dipterology (IDCX) in Reno, Nevada in July 2023. Historical insect morphological analysis is being supervised by Dr. Philip Buckland who is the director of the Environmental Archaeology Lab (MAL) at Umeå University and is one of the few experts in the identification of archaeological insect remains in the world. Morphological identification of extant insects trapped at the site are being overseen by Dr. Lauren M. Weidner, who is an assistant professor at Arizona State University and the only board-certified forensic entomologist in the state of Arizona. This analysis is also incorporating Dr. Weidner’s laboratory manager and entomology master’s student Mr. Andrew Meeds. The two additional early-career researchers assisting in this project are both from the field of archaeology; Ms. Erin Crawford is a master’s student in archaeology at St. Mary’s College and will be presenting the Diptera-focused findings of our project at the 10th International Congress of Dipterology (ICDX) as an invited speaker and Ms. Abigail Kuene a post-baccalaureate researcher at Historic St. Mary’s City who has been integral in trapping modern insect samples. We are also collaborating with the NOFA regional board and NOFA-NJ to disseminate our findings to our regional stakeholders through a conference presentation and educational outreach event. These additions will not impact the budgetary allocation originally outlined for this project.
The addition of using a trapping regimen based on Hoffman et al. (2006), which recommends pairing insect trapping with soil biogeochemical analysis will likely allow us to draw more nuanced conclusions about long term soil health and productive capacity at the site, especially in light of the unexpected finding of several fly (Diptera) species within the material which were not previously cataloged. Incorporating early career researchers from the field of archaeology has allowed us to broaden participation in entomological research and studies of sustainable agriculture.