Sylvia Warren

A Diet of Worms

March 13, 2019

Archaeometry is an enticing and important bridge between the humanities and sciences, and applying scientific methodologies to archaeological sites has fundamentally transformed archaeology as a discipline. Initially described as ‘revolutionary’, radiocarbon dating has become convention, [1] and its general reliability is widely accepted. Ancient DNA (aDNA), however, is emerging as another tool that can not only be cross-referenced with early written documents to calibrate chronology, but also to investigate aspects of diet, trade, migration, and human history.

From Pääbo’s first extraction of mitochondrial DNA from soft tissues in 1997 [2] to bone and teeth substrates in the mid-2000s, [3] fledgling aDNA explorations were primarily mammalian. The early-day vedettes of these investigations were also pathogens with high mortality rates (the plague, tuberculosis [*]), while those that caused persistent but non-lethal infection subject to more limited molecular research. When I joined Dr (then Herr) Flammer and Dr Smith at Oxford to start looking at parasite aDNA, extant research focused mainly on using sequence analysis for clinical diagnosis rather than for any overarching archaeological purpose. [4]

So, why parasites? Why look past the wondrous artefacts unearthed in graves and cesspits to zoom into soil samples, to obsessively hydrate and spin and gaze at them under the microscope? One answer is practical: nematode eggs have been microscopically detected in archaeological samples since the seventies, and two of our target parasites have protective egg-casing that reduce aDNA degradation over time [5] [6]. Another is that we were simply being difficult. We already had a tame friend with access to the cesspits and the single graves whom we could use to our advantage. Last but not least is that modern Internal Transcribed Spacer 1 (ITS1) Trichuris trichiura (T. Trichiura) sequences showed geographical grouping on sequence similarity, and we thought ancient sequences might also show this signature. Though it was probably a combination of all of these factors, what we really wanted to test is whether we could move beyond diagnosis and into a more subtle evaluation of archaeological change.

A word of warning: this topic cannot be dealt with without jargon. We had samples from nine sites across Europe, from 3355BCE to 1550CE; for which we designed a workflow between two laboratories that minimised the risk of cross-contamination, and only handled samples from a single site and date at any one time. To break the parasite eggs we would beat them with glass beads, add chloroform to wash out soil dyes and take them through two rounds of the polymerase chain reaction (PCR) to amplify the scant aDNA present. Aliquots were made to peer at microscopically, as we targeted Cox1 in Ascaris lumbricoides (A. lumbricoides) and β-tubulin in T. trichiura. PCR exponentially amplifies a particular section of aDNA: it heats and cools the fragments to melt them, bind primers to the split strands, then fills in the corresponding base pairs on to the exposed nucleotides until one has a quantity that is large enough to analyse. Thus, Adenine=Thymine CytosineºGuanine (tea for two is the mnemonic for the number of hydrogen bonds that keep them together). To date the samples, we recurred to a variety of methods. Our samples from Lübeck used stratigraphy; [7] those from Zurich, radiocarbon dating; Viborg used dendrochronology, and London and Bristol turned to artefacts found in the communal pits.

Once we had sufficient aDNA, we had to check whether it had been correctly amplified. The samples were measured via electrophoresis on agarose gel (do not pour sloppily, ease the comb out of the set gel with care, be sure not to mix in too much dye, pipette into those transparent wells and set running–simple, but not necessarily easy) against a standard ladder of DNA marker weights to check for successful amplification. The lighter the fragment, the further it runs down the gel in a given time period, and only if it matched with the weight we were expecting could we assume the sample to have successfully amplified the aDNA sequence we were looking for.

There were many blank gels. When we had some successful samples, we cloned them using the simple and fast TA cloning method, picked the resultant colonies, then prepared them for in-house base-pair sequencing. (The published methodology of this runs to over a thousand words, condensing days of preparation—with nights spent in lab coats swearing at each other, at equipment, at ourselves; the frustration of blank results; weeks of failed experiments and rare jubilation when something went well—into a neat and replicable recipe. To introduce this level of personal recollection when writing about science feels subversive, as it is only too easy to slip into the comforting impersonality of the passive voice.)

We were still looking at all of our samples–from the Czech Republic, Bristol, Switzerland–when we landed on Lübeck, a seat of the Hanseatic League. Medieval Lübeck was one of the most influential trading cities in Europe and the main port from which colonists left for the Baltic territories. In the thirteenth century, it was designated an Imperial Free City, autonomous under the Holy Roman Emperor. Our inquiry led us to hone into Fischstrasse and Alfstrasse, two parallel streets with individual houses producing samples from the early thirteenth to the late seventeenth centuries. Archival material described the living conditions, socioeconomic class and changing social dynamics of the excavation sites (a beaded purse rotting in a latrine, a growth of floorplans) which we hoped would allow us a more nuanced interpretation of our genetic data.

I imagined this as sinking my fingers into the decomposing soil of the past, bringing up handfuls of history as shit. Men and women ate and drank and did their accounts and worked as merchants and fucked as wives and went to the loo and dropped their purses or lost their rings, possibly when drunk, or tired, or both. A direct link exists between these parasites and their hosts, whom they did not kill but on whom they levied nausea, cramps and diarrhoea. There were people here, as there are today, and we are combing through their excrement to gain insight into their daily lives.

There were thirty-three samples from houses on each of these two streets, and the faecal deposits had a high incidence of both A. lumbricoides and T. trichiura. To determine the presence of parasites, we would hydrate the earth and pipette 40µl on to a microscope slide, then sweep across the square of material, counting eggs. Soil looks strange under a microscope: washed-fragments of plant fibre float like rafts, grains of sand and other minerals glow sepia, organic matter presents as amorphous dark smears. It takes a while to become accustomed to this world, to learn to see what one is looking for.

T. trichuria is easy and elegant, an elongated oval with a lighter cap at either end, thick brown walls encasing dark and bright granules. A. lumbricoides is more difficult, roundish and brown with a scalloped edge. It is tempting to see it in smudges, but the real ones shine out with such clarity and obviousness it seems impossible that one could ever have been on the fence.

Left. Modern fertilised A. lumbricoides egg (CDC dpd.cdc.gov). Right. Micrograph of A. lumbricoides from London, ~1500CE.

 

I counted them. I took my samples, slid the lens across each slide tallying the eggs, then scaled up into eggs per gram in order to compare across sites. Lübeck is vermicular compared to the other cities; over 80% of the latrine samples had A. lumbricoides or T. trichiura. The highest counts showed 4934 T. trichiura eggs per gram of soil sample, which sounds like a staggering quantity. One must however remember that the female T. trichiura can produce between 2000-10,000 single-celled eggs a day, deposited into the soil via human faeces. These eggs embryonate and become infectious, are ingested and then hatch within their new host, larvae burrowing into the villi of the small intestine; to mature into adults and move through the mucosa into the large intestine. They can live for up to a year inside their human homes.

Although the eggs are beautiful—minute time capsules preserved for millennia perchance—they have not been eradicated globally. It is crucial to remember these infections cause pain, bloody stool, and distension of the abdomen. Today, T. trichiura afflicts almost one billion people, chiefly in South-East Asia (with Laos, Thailand, and Malaysia having the highest incidences of infection), coastal countries in Southern Africa and parts of South America, especially Brazil. A. lumbricoides is similarly distributed, with high levels of infection in Central and Southern Africa, Central and South America, and South-East Asia. These parasites may be historical in Europe, but they remain among the largest and most widespread of the soil-transmitted helminth infections, which lead to malnutrition and physical impairment. I may be writing of the past, but one should be minded this is not entirely history.

That said, Trichuris and Ascaris’ European extinction is of consequence for the validity of our project. As with journalists, the horror of all academic scientists is to be ‘scooped’—that is, to have the results of something you are researching announced by another, unrelated, group before one’s work is published. Since parasite aDNA was very much in its infancy at the time of our investigation, Dr Flammer and I would scour publication records, looking at our competition. Oh et al [8] released a paper with Ascaris aDNA sequences from Korean mummies of the Joseon Dynasty (1392-1910) noting their amplified aDNA was different to surface-soil and extraction controls. We had not been scooped; it turned out that the sequences indicated A. suum rather than A. lumbricoides –the pig-based variant– and there was also significant overlap with extant modern Ascaris DNA, which could indicate contamination from modern sources. We ran sequential negative controls and found no trace. (Subsequent work, alongside that of Dong Hoon Shin, has shown fascinating developments in Korean medieval history.) We could be confident our aDNA was truly ancient.

While swimming through these rusty worlds, I began to notice new eggs in some of the samples. They couldn’t be anything else. One looked a little like a larger Ascaris; the other like a bloated oval with a single, darker cap. Dr Flammer and Dr Smith looked at them–Diphyllobothrium spp. and Taenia spp, they said. These were only in the Lübeck samples, in a couple from London and a few from Bristol, and so we didn’t have enough of them to extrapolate anything concrete.

Now, Diphyllobothrium and Taenia are not quite like Ascaris and Trichuris as they are not spread via faeces. The first spreads through fish, the other through pork or beef, depending on the species. Dr Flammer and I sat at one of the white Formica tables in the zoological café, crappy Sunday vending-machine coffee in thin-ridged plastic cups at hand, when he drew The Graph. We had sample dates along the x-axis and Diphyllobothrium and Taenia quantities on two y-axes, messing around with our numbers, trying to see what new conclusions could be drawn from these new counts. The picture was almost too orderly.

The (very) rough graph of Diphyllobothrium spp. vs Taenia spp.

 

There was a fairly clear temporal pattern for both Diphyllobothrium and Taenia, with the former primarily present in the pre-1300CE samples, and the Taenia increasing in prevalence post-1300CE. Human Taenia infection was likely from undercooked beef: we obtained sequences from a fragment of the mitochondrial 16S gene and sequenced them, finding more sequences that matched the cow-borne T. saginata, as opposed to the pork tapeworm, T. solium. Diphyllobothrium sequence analysis, on the other hand, revealed a range of fish species that could act as intermediate hosts. So, why was there a decrease in fish consumption with a corresponding rise in meat?

In 1188 and 1226, imperial charters granted Lübeck formal control of fishing rights over its surrounding rivers and lakes, [9][10] which could explain an easy and reliable source of fish as protein pre-1300CE. But this does not explain why people would stop eating a plentiful resource. Status? Some other event? A general reduction in fish stock, such as overfishing? Digging deeper into historical documents (and I have to thank Dr Flammer here for his skill as a native speaker of Swiss-German and Hochdeutsch), we uncovered a few possibilities. Lübeck expanded around 1300CE, and the river to the west -the Wakenitz- became polluted with the overrun of meat and leather production. [11] This could have killed intermediate fish hosts, or simply made the produce from the river less appealing for human consumption. In the meantime, a Benedictine monastery was converted into a nunnery, losing its fishing rights close to the city. The years between 1245 and 1247CE saw the Black Death happen, and one can easily imagine that the surplus of able-bodied fishermen would drop.

This is where we came upon a danger. We were using primary sources–written at the time–to try to match with primary sources of an altogether different kind–the genetics of intestinal parasites–. And we wanted this to work.

A side note on fish, palatability, and The Hanse. The Hanseatic League was not a country, but it would often behave as if it were one with its own  laws, economic rules, even a navy. Though it operated under a vaguely Northern Germanic cultural base, it reached into what is now Novgorod in the Kievan Rus. Stockfish: unsalted dried fish (often cod, but also many other white fish) dried in the wind under wooden racks. The byproduct has been compared to oil today as relates to its importance as a commodity. Dr Flammer and I looked up recipes. You need to soak the fish for 20 hours in water, remove the bones. A common method advised that one put the rehydrated fish on a warmer and melt a square of butter or puddle of oil to moisten the flesh. Judgements of palatability may not be easily gauged through our eyes.

Raw or undercooked beef suddenly looks like a better prospect. (We had some evidence that a diplomatic squabble stopped supply of stockfish to the Hanse, but unfortunately could not substantiate this well enough to be more confident in drawing the connection at the appropriate dates.) Part of the increase could due to status: oysters, after all, had once been paupers’ food in London. Another was, again, the Black Death: a reduction in manpower for arable cultivation led to an increase in beef-eating, which required less human intervention. The Rinderpest virus was introduced to Europe in the early 1300CE, perhaps increasing the proportion of infective beef.

What began as a diversion became something Dr Flammer and I became increasingly obsessed with solving. Is a parasitic genetic validation of disparate sources valuable, or does it let both archaeologists and biologists choose what they want to construe as an authorised interpretation because it has base pairs and science to back it? I would argue against this: as I said at the beginning, science and archaeology are not merely inevitable, but potentially fruitful bedfellows, with the opportunity to set each other’s foibles straight.

Our foremost aim had also been achieved: aDNA sequencing did not have to be just a diagnostic tool. We had found a change in the dietary habits of a population through looking at aDNA sequences and cross-referenced this with primary sources to find plausible explanations. We knew that there was more to find. To return to T. trichiura, the most diverse sequences were traced to Lübeck and Bristol: port sites, hubs of trade-entwined communication. Single graves scattered around disparate sites provided little similarity. It is those that travelled between ports and countries separated to create divergent paths of evolutionary base pair changes. They sailed off into the night with(in) their hosts, leaving their trace across sea routes for us to discover.


[1] Wood, Rachel. “From revolution to convention: the past, present and future of radiocarbon dating.” Journal of Archaeological Science 56 (2015): 61-72.
[2] Pääbo, Svante. “Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification.” Proceedings of the National Academy of Sciences of the United States of America 86 (1989, 6): 1939-1943.
[3] Rohland, Nadin and Michael Hofreiter. “Ancient DNA extraction from bones and teeth.” Nature Protocols 2 (2007, 7): 1756-1762.
[4] Loreille, Odile et al. “Ancient DNA from Ascaris: extraction amplification and sequences from eggs collected in coprolites.” International Journal of Parasitology 31 (2001, 10): 1101-1106.
[5] Aspock, Horst, Heinz Flamm and Otto Picher. “Intestinal parasites in human excrements from prehistoric salt-mines of the Hallstatt period (800-350 B.C.).” Zentralbl Bakteriol Orig A 223 (1973, 4): 9.
[6] Bouchet, Françoise, Stephanie Harter and Matthieu Le Bailly. “The state of the art of paleoparasitological research in the Old World.” Memórias do Instituto Oswaldo Cruz 98 (2003, Suppl 1): 95-101.
[7] Rieger, Dirk. “Zeitliche Tendenzen und Konstruktionskontinuitäten Aktuelle. Befunde zur Holzarchitektur der Großgrabung im Lübecker Gründungsviertel.” Mitteilungsblatt der Deutschen Gesellschaft für Archäologie des Mittelalters und der Neuzeit, issue 24, 2012.
[8] Oh, C. S. et al (2010) “Paleoparasitological report on Ascaris aDNA from an ancient East Asian sample.” Memórias do Instituto Oswaldo Cruz 105 (2010, 2): 255-228.
[9] Zimmerling, Dieter. Die Hanse: Handelsmacht im Zeichen d. Kogge. Düsseldorf/Wien: Econ, 1976.
[10] “Codex diplomaticus Lubecensis. Lübeckisches Urkundenbuch. Urkundenbuch der Stadt Lübeck. (Verein für Lübeck. Gesch.)” Lübeck: 1843.
[11] Berndt, Helmut and Werner Neugebauer. “Lübeck – einemedizinhistorische Studie”. In Archaeologica ludensia III (ed. B Bengtsson). Lund: University of Lund, 1968, pp. 78-92.
 
[*] A minor detour into the modern research trends for disease could be made here, with the Neglected Tropical Diseases being, well, neglected, despite affecting more than a billion people per year. Though a slightly different topic, it highlights some inherent problems with how funding is reserved for ‘newsworthy’ conclusions.


Sylvia Warren read biological sciences at the University of Oxford. She currently works in academic publishing, and lives in Oxford.

For the full, original research behind this piece, see Flammer, Patrik G. et al. “Molecular archaeoparasitology identifies cultural changes in the Medieval Hanseatic trading center of Lübeck.” Proceedings of the Royal Society B. 03 OCT 2018 @ royalsocietypublishing.org/doi/full/10.1098/rspb.2018.0991