Sunday, January 25, 2015

The Copious Little Coprolite: A Tale of Underrated Informants

As you may or may not already know, a great deal of my dissertation work focuses on the analysis of desiccated human feces (i.e. "coprolites"). These remains can come from archaeological sites such as caves and trash middens, or from direct human contexts such as mummified intestines or even burials in some special cases (e.g. Nivelles). Coprolites most often form in arid environments, preserving within them a vast amount of information about their depositors' diets, medicinal plant usage, and pathogens.  By analyzing these tiny cornucopias of data, we are able to learn not only what people were eating and what was eating them, but also to use these data to elucidate patterns of human behavior as our ancestors interacted with the world around them. Today, we are going to pay homage to something that doesn't often get the recognition that it deserves...the copious little coprolite.

A coprolite from Hinds Cave, Texas.
A Brief History of Coprolite Analysis
The word "coprolite" began as a descriptor of mineralized dinosaur feces used for the first time by a paleontologist named William Buckland in 1829. By the 1960s, the term had been applied to other fecal forms preserved via desiccation in addition to mineralization and was being used to describe fecal materials from archaeological contexts  in addition to paleontological contexts. There have been three distinct phases in this history of coprolite analysis stretching from 1829 up to the present.

The first phase (1829-1960) began with the birth of the term "coprolite". The value of human coprolites was not recognized until 1896, when a botanist named John William Harshberger suggested that looking at seeds in coprolites could reveal information about ancient diets. The early 1900s that followed Harshberger's suggestion saw researchers examining coprolites for other plant remains, like leaves and twigs, in addition to faunal remains, such as the tiny bones found in fecal deposits from a wide range of archaeological contexts. By the 1950s, people began looking more at the neat stuff in human coprolites. They started looking at hair and feathers as well as shell fragments and insect remains. Today, the study of "macrofossils" (macroscopic plant and animal tissues found in coprolites) is a crucial element of coprolite analysis.

Eventually, people began seeking smaller sources of evidence that today we call "microfossils" (things like pollen, starch granules, and our beloved parasite eggs). The first evidence of parasitism in the prehistoric New World came from whipworm (Trichuris trichiura) eggs found in an Incan mummy in 1954. This began the transition of coprolite analysis into the second phase (1960-1970). This phase saw the development of specialized techniques for examining microfossils and laid the foundations for later expansion.

The superstar researcher of the second phase was indisputably a man by the name of Eric Callen. Callen is known to many as the first true coprolite specialist. He completed three major analyses of coprolites recovered from New World archaeological sites and was working on a fourth when he died tragically in 1970. Despite being ridiculed by colleagues for his interest in coprolites (which were regarded as useless by researchers of that time), Callen persevered to become a legend in a now much more respected field. He developed methods for rehydration (a crucial first step in analyzing coprolites) and standardized other techniques for evaluating fecal deposits.

Various pollen grains
A wealth of other researchers also worked on coprolites during this second phase. It was during this era that pollen analysis was integrated into coprolite analysis. These early studies yielded incredible insights into the diets, medicinal plant usage, and even seasonal site occupations of prehistoric peoples of the New World. This era also saw the flickering of parasite studies that would ignite the third phase.

The current phase of coprolite analysis began in 1970. This phase has been characterized by both refined methods of analysis and by the expanse of these analyses into more interdisciplinary realms. Coprolite analysis grew to be applied in a broader sense to archaeological questions beyond the direct discoveries of dietary remains and evidence of diseases in antiquity. Techniques for quantifying macrofossils were developed over the course of a decade. Macrofossil identification techniques were also becoming more sophisticated and researchers began comparing coprolite data from various archaeological sites to one another. Pollen analyses also became more refined as methods for quantification and interpretation were tested and standardized. The study of phytoliths, fungal spores, and starch granules also became integrated into coprolite analysis. This allowed for more rigorous and fruitful assessments of nutrition through time and space.
A variety of phytoliths from the Tangue site in China.
{A–c = rice bulliform; d–g = rice double peaked; h, i = phytoliths from broomcorn millet husk;
j = long saddle; k = scutiform-bulliform from reed; l = common bulliform; m = Cyperus type;
n = trapeziform sinuate (tooth type); o = woody phytolith}
Which brings us up to the birth and subsequent growth of the field of archaeoparasitology. With foci in Brazil, Canada, Chile, England, Germany, Peru, and the United States, analyzing coprolites for evidence of parasitism grew exponentially. From the late 1970s right up through the early 1990s, methods for extracting and quantifying parasite eggs were developed. From the refinement of these methods came the ability of researchers to begin examining epidemiology of the past through the lens of parasitism. The following decades would usher in the integration of molecular techniques for finding evidence of parasitism that could not be seen with the naked eye. Studies using PCR and ELISA would revolutionize coprolite analyses to give researchers an even broader perspective of epidemiology in antiquity.

What's in a Coprolite and What Can They Teach Us?
As you likely gathered from the above, coprolites can treasure troves of taxonomic data. Breaking these into discrete components is how researchers conduct analysis, but only by re-combining dataset after discrete analyses are we able to get the full story that coprolites are trying to tell us. Reinhard and Bryant (1992) broke coprolites into their components in the following way:

1) Biological (Bacteria, Viruses, Fungi, Parasites, Insects, Pollen, Phytoliths, Macrobotanicals, and Macrofaunal Remains)

2) Mineral and Chemical (Sand, Grit, and Flakes, Charcot-Leyden Crystals, and Chemical Components)

All of these different elements that can be recovered from coprolites give dimension to the overall analysis. Taking in discrete datasets examining coprolites for a variety of components and synthesizing the information leads to the emergence of the bigger picture. By finding the eggs of fish tapeworms and tiny fish vertebrae in a land-locked population's coprolites, we can begin to understand prehistoric patterns of trade between this population an a coastal population. By finding the minuscule bones of rodents in the coprolites of cave dwellers, we can begin to picture the resource utilizations and ecological displacements that contributed to the origins of now-established zoonotic diseases among human populations today.

It is obvious, though not always intuitive for some, that diet and disease are intrinsically linked. This is true for the modern world as it was true for populations of the past. Understanding the nature of the diet-disease relationship comes to light by combining the data one can gather from coprolites. These little packets of poop are warehouses of information for understanding such relationships. They not only provide direct evidence (e.g. parasite eggs or pollen grains) but also proxy evidence (e.g. neotropical parasites found in pre-clovis coprolites from the pacific northwest point towards coastal human migration patterns into the new world).

Coprolite analysis is a vital aspect of archaeoparasitology, but also reaches far into other disciplines. Such studies are important for dietary reconstructions, understanding the interactions of people and their environments, and inferring aspects of early human behaviors. Advances in the areas of medicine, food technology, and environmental adaptation can be reflected by the composition of macrofossils and microfossils present in coprolites.

Today, coprolite analyses are being used to examine the origins of many diseases that plague modern societies. Diseases as different in their etiologies as Chagas' disease (caused by a parasitic protozoan) and diabetes (a metabolic disorder). Tracing the origins of such diseases is no small or simple task, but can be done through the analysis of the copious little coprolites that await researchers interesting in unveiling their stories.

The Moral of the Story
I could go on with a long, eloquent speech doting on the awesomeness of coprolite analysis and droning on about how excited I am about my own dissertation work with these remains, but I think by now you've probably read enough to wet your appetite for exploring coprolite analyses on your own. (Or, at least I hope I've so piqued your interest.) Instead, I will leave you with a somewhat crude, but appropriate message: Don't let anyone give you sh*t for liking coprolites. Seriously, people are quick to put down great work that originates from looking at things that some deem as "gross". (This is typically due to their own ignorance as to the significance of said work.) As a parasitophile, you are probably no stranger to the disgusted reactions of people who don't understand the value of parasitiology. But we don't do it for them, now do we? We do it for us. We do it to better understand the intricacies of the world around us. We do it because our passion knows no bounds. As a coffee cup that sits in an unnamed parasitology lab states: Don't let the bastards get you down!

Sunday, January 18, 2015

Show Me the Mummy!: A Journey into the World of Mummy Studies

This week I embarked upon my first collaborations with an amazing person that I met my very first semester here. She was an undergraduate back in those days, but she has since grown into working on a graduate degree through a dental program and...oh yeah, she's a Fulbright scholar. ;) She spent a year in Chile examining the hair (which, apparently wasn't always attached to the head) of mummies. I am honored to be joining the effort to get a few papers out from the data that she collected before she made her way back here for dental school. Until these data are published, I can't really say too much about what we are doing, but meeting with her earlier this week got me to thinking that I should do a blogpost on mummy studies.

So here I am...finally getting back into that series that I wanted to start back in October, when things got crazy. I hope y'all enjoy this journey into the exciting and totally underrated world of mummy studies.

Mummies Across Time and Space
Xin Zhui, a.k.a. "The Diva Mummy"
As soon as you read the word "mummy", I'm sure your first thought was "EGYPT!"...or at least that word was in whatever your first thought was. Yes, there are some awesome mummies in Egypt and those are the mummies that have garnered the post popularity in global media. However, there are LOTS of other places in the world that can boast of their own mummies. There are mummies in Asia...that's right, Japan, Korea, Mongolia, China all have mummies. In fact, one of the most well-preserved mummies ever discovered was the body of a noble woman named Xin Zhui who died in 163 BCE. She lived a lavish lifestyle, but died of a heart attack around the age of 50. The care taken to preserve her body along with all of the artifacts found over 2,000 years after she was entombed have earned her the nickname "The Diva Mummy". Her body was so perfectly preserved that when researchers examined the body, they commented that it was almost like doing an autopsy of a recently deceased person. Her limbs were flexible and her organs were remarkably in-tact. Mummy researchers learned a lot about the health of this person, including that she harbored tapeworms! (Yay parasites!)

Mummies have also been found in other parts of the world like Europe and South America. I've done a little bit of work with European mummies (from Lithuania and from Italy) that were much younger (1700s and forward) than the Egyptian mummies or most of the mummies from Asia. The previously mentioned work with the Chilean mummies will be the first work I'll have ever done with South American mummies. The Chinchorro mummies, found in present day Chile and Peru, are the oldest artificially mummified human remains in the world. You heard that right. The oldest of these mummies predates the oldest Egyptian mummies by about 4,000 years!

Now, I could easily spend hours talking to you about the differences in mummies across the world because they are just so diverse and fascinating, but this is a blog post...despite my propensity to sometimes get a little long-winded. Suffice it to say that mummies can (and are) found in a variety of places on this planet and that they range in age from around 7,000 BC to much more modern mummies who died in say, the early 1900s (AD).

Types of Mummies
There are several ways that would could split up mummies by "type", but I'm just going to break it into two broad categories for today's purposes. First, you could have a "prepared", "artificial", or "anthropogenic" mummy. These terms all refer to bodies that did not undergo natural mummification as a product of the corpse's depositional environment. These mummies were instead created by intentional preparation of the bodies. Most people think of these kinds of mummies when they picture mummies. Long before embalming, mummification was common practice for dealing with the remains of the deceased in certain parts of the world. Most people think of Egypt, with their whole wrapping, organs in jars, and pulling the brain out through the nose things, but the way that various cultures prepared mummies are as unique as the cultures themselves. Often times, the bodies were eviscerated and packed with plant material like straw to help maintain the shape of the now hollowed out body. There were frequently local (or sometimes imported) oils, vinegars, and herbs used on the bodies. Bodies were typically tightly wrapped in linen or other textiles and placed in a well-ventilated area to allow for drying. Some bodies were later placed into coffins, sarcophagi, or even glass viewing cases.

Ötzi the Iceman
However, there's more than one way to make a mummy. Some of the most famous mummies in the world are the bodies of people who were mummified unintentionally as a product of the environment in which they died. These of most frequently referred to as "spontaneous" mummies. Arid environments are particularly good for naturally drying out the body. Thus we have some excellent mummies found in desert regions, like the Chinchorro mummies found in South America's Atacama desert that I mentioned earlier. Mummies can also be found in arid environments that are cold, like the mummy known famously as Ötzi the Iceman. This mummy was discovered by some Germans hiking in the Alps. The hikers thought they had stumbled upon the body of another hiker who had had an accident, but it turns out that the body was 5,000 years old. Political issues arose when Italy and Austria both tried to claim the body, but in the end it was determined to have been on Italian soil.

Head of the Tollund Man
Bodies can also be preserved by the environments peat bogs. When a person's body is left in a peat bog, the bones tend to dissolve because of the acidity of the bog itself (remember that bones have lots of calcium phosphate, which is basic in nature). However, the acidity of bogs along with having little to no oxygen, and lower ambient temperatures creates an amazing preservation environment for human skin. The skin preserves extremely well, though it does get crazy dark in color making them appear almost like statues in the photographs that I've seen. These conditions include highly acidic water, low temperature, and a lack of oxygen, and combine to preserve but severely tan their skin. While the skin is well-preserved, the bones are generally not, due to the acid in the peat having dissolved the calcium phosphate of bone. The Tollund Man is one of the most famous bog bodies, belonging to a man who was hanged sometime between 375-210 BCE.

As a fun side note, mummies don't have to be humans. (But you've probably heard of how the Egyptians mummified cats...because you're a smart one!) Egyptians also mummified dogs...and lots of them. I read a neat study a while back that looked at the ectoparasites on Egyptian dog mummies. I did a post about it, and later a presentation at a parasite seminar. (You can read it here, but please keep in mind that I wrote it a few years ago and I've learned a lot more about taphonomy and parasitology since then.) Egyptians also commonly mummified pet monkeys, gazelles, mongooses, and a variety of birds. Aside from pets, Egyptians mummified other animals, including crocodiles, baboons, fish, snakes, and even bulls, for religious purposes. I haven't really heard of any other cultures that mummified animals, but I wouldn't be the least bit surprised if such cultures existed.

Mummy Studies
The field of mummy studies is an ever-growing one. As we become more technologically advanced, we are given the opportunity to really examine mummies to help us answer questions about life in the past. Mummy studies give us insights into the worlds of people who lived long ago. By analyzing mummies, we are able to understand when these people died and often times under what circumstances. We learn about their diets, medical practices, and funerary rituals. We learn about their societies and are able to tell their long-since forgotten stories.

Mummy studies brings together researchers from all kinds of educational backgrounds. The expertise of archaeologists, anthropologists, radiologists, epidemiologists, forensic scientists, palynologists, medical historians, and, of course, archaeoparasitologists, are brought together to put together the stories of these mummies. The patterns of culture, diet, and disease begin to emerge as mummies reveal their secrets to these researchers.

If any of you are interested in mummy studies, I'd like to inform you of a mummy field school that is currently in the making to begin in the summer of 2016. The course will consist of 15 days in Italy studying the mummies of the region. You'll actually get to do hands-on analyses of some of these mummies as class projects after you learn from experts all about how such analyses are conducted! There's even a possibility that yours truly will be there as either faculty or staff...but let's not get too far ahead of ourselves! (Dissertation OP.)

Mummies and Archaeoparasitology
Many of you may have gotten this far asking the question, "So, when do we get to learn more about parasites?!?!" Okay, okay...let's get to the parasites! Like other areas of mummy studies, the recovery of parasite data is largely dependent on the preservation environment and on the available technology of the people studying mummy parasitism. The analysis of mummified remains can (and has) revealed evidence of ectoparasites (as you already know from talking about the ticks and hippoboscids found on the mummies of dogs from Egypt), helminths (i.e. "worms"), and even protozoans. Yes, there's a little something for every kind of parasitophiliac when it comes to mummy studies!

Lice from a pre-columbian, Chilean mummy.
Click here for a link to the paper.
One of the coolest things about ectoparasites is that they tend to preserve well since we are typically referring to arthropods like ticks, fleas, and lice when we use the term "ectoparasite". In terms of human mummies, lice are the paydirt of ectoparasite-related archaeoparasitology. Lice can not only be found in their adult states on mummies, but also exist in the form of nits and nymphs. For those who don't know, "nits" are cases that house developing lice and are cemented onto the shafts of hair in an infested person. These nits, both with and without nymphs inside of them, can be found on the hairs of mummies. Counting the number of these nits on a small section of hair can allow for quantified comparative data across various analyses of head lice and their mummified hosts. Currently, I'm involved in the preparation of a paper or two that will look at the lice of mummies from the Atacama desert of South America. I'll be sure to post all about it when this paper (or papers) is (are) published. Be on the lookout! ;)

Stole this one from my major professor's Facebook page.
It's an adult louse from a South American mummy!
Most of the studies published with regard to archaeoparasitology of mummies focus on the discovery of parasitic helminths. In fact, the first archaeoparasitological study ever published (Ruffer in 1910) described the discovery of calcified Schistosoma sp. eggs in the kidneys of two 12th dynasty Egyptian mummies. Since those days, mummies from around the world have  revealed evidence of infections with roundworms (Ascaris lumbricoides, Strongyloides stercoralis, Trichostrongylus sp., Trichuris trichiura, etc.), tapeworms (most often Taenia sp.), and flukes (Clonorchis sinensisDicrocoelium dendriticum, Gymnophalloides seoi, Metagonimus yokogawai, Schistosoma sp., etc.).

Paragonimus westermani eggs from
the liver of a female, Korean mummy.
Click here for a link to the paper.
My personal experience with mummies is limited, but growing with every passing semester. I've seen Ascaris lumbricoides and Trichuris trichiura eggs in a mummy from Lithuania and Clonorchis sinensis eggs from a Korean mummy. I've also analyzed mummies from other places and not found any parasite eggs. I'm hoping to expand this in the future as I become more involved with mummy studies.

Another aspect of studying mummy parasites is to look for things that can't actually be seen with our human eyes. I'm talking of course about protozoan parasites (one of my favorite groups of parasitic organisms!). Because these are delicate, single-celled organisms, they don't preserve in the way that helminth eggs preserve. Instead of leaving behind a physical form that can be found with the aid of a microscope, these parasites leave behind molecular traces that can be detected with the use of serological test kits, such as enzyme-linked immunosorbent assays (ELISA), or through the use of DNA detection techniques, such as polymerase chain reaction (PCR). These techniques have been most frequently utilized to identify parasites in archaeological materials such as coprolites and latrine sediments, but they have also been applied to mummy studies. For example, researchers have revealed that ancient peoples were infected with malaria (caused by Plasmodium sp.) by analyzing bone, muscles, and skin.

The conclusion of this section brings us back to the infinite awesomeness that will be the mummy field school mentioned earlier. Students will be working on independent research projects with the mummies. Some of those students will be looking specifically at the parasites that infected these individuals in life. It will be fascinating to see what new information will come from the systematic examination of these individuals over time.

The Moral of the Story
The world of mummy studies is a complex, interdisciplinary area with lots of discoveries just waiting to emerge from hard work of enthusiastic researchers. What we can learn from the past through archaeological material is always a puzzle, but mummies give us the unique ability to equate data with a particular individual rather than guessing at how many people are represented by a group of coprolites or a gram of latrine sediments. Understanding the diets, medical advancements, seasonality of death, and of course the diseases of mummies allows us to paint an epidemiological picture of past societies one person at a time. As I grow to be a more competent archaeoparasitologist, I can only hope that my path will cross with more and more of these astoundingly interesting individuals and the parasites that they hold on or within them.

Sunday, January 11, 2015

The Nivelles Story: My Second Peer-Reviewed Paper Makes JAS

I get it. I kind of dropped the ball for, oh, three months. I can't even guarantee that I'm officially picking it back up again today. Last semester was insanely busy and went by before I could blink. I'm happy to report that other than being crazy it went really well for me. I published a book, got a paper out (for which I was lead author), passed my comprehensive exams, and lined up a ton (technical term) of new projects for the Spring. This semester doesn't look like it will be any slower (which isn't a bad thing), so I will try my best to keep up with posts, but please forgive me if I again drop the ball. (I promise to pick it up again when I get the chance!)

Today, I'm going to talk to you a little bit about my latest publication, which was published this month. This paper was quite a doozy! ("Doozy" is defined by Merriam-Webster as: "something that is unusually good, bad, big, severe, etc."... this paper was unusually good, bad, big, and a whole host of other words.)

My involvement with this paper began long after the analyses were complete. The story begins back in 2011, long before I made my way up to Nebraska. Dr. Karl Reinhard's Archaeoparasitology class began analyzing coproites excavated from skeletonized remains resting in Nivelles, Belgium. The coprolites were many, but hailed from only three individuals. These individuals had lived in this part of Europe during the Medieval period, a time notoriously ridden with filth and disease. As one would expect, these individuals were found to have been hosts to parasitic worms, namely Trichuris trichiura (the human whipworm) and Ascaris lumbricoides (the human mawworm or "giant intestinal roundworm"). European archaeoparasitology is known to yield evidence of such worms in great abundance, especially if you go back in time when our understanding of disease was dominated by miasmatism (the belief that diseases were caused by smells and "bad air" rather that pathogens as we know them to be caused by today).

Finding these individuals to be infected with worms was interesting and important, but it was the infections of a particular individual (from Burial 122) that made this study phenomenal. That individual was an elderly woman. Bone pathology reports tell us that she had no teeth and was suffering from arthritis. The coprolites taken from her remains were abnormally large in size and were much more numerous than would typically be found in such contexts. The coprolites were also backed up beyond her sacral region and into her lumbar region. This indicated that she likely suffered from a bowel obstruction or some other issue that led to constipation. This poor woman probably suffered greatly in her final days.

Examination of her coprolites revealed an abnormal amount of wheat glume in her feces. The fiber rife within her could have contributed to the obstruction, but why would she have been eating it in the first place? And why so much of it? As it turns out, this was a way that Medieval doctors sometimes treated people who were burdened by intestinal worms...and this lady had a major problem with worms.

Figure. 9. Average number of eggs per gram as represented by all three burial sites. 
Gray bars represent Trichuris trichiura while black bars represent Ascaris lumbricoides.
[From Racz et al., 2015]

As you can see from the histogram above, this woman suffered from two different types of worms AND suffered an EXTREMELY heavy infection with whipworms. Keep in mind that the above represents average eggs per gram of feces. That's a LOT of freaking whipworm eggs to find in one person. Let's not forget she also had mawworm eggs alongside those whipworm eggs. Here's a table from the paper that will (or at least should) blow your mind.

[From Racz et al., 2015]

Having so many worms in any one of these coprolite is staggering, but remember that all 8 of these coprolites were inside ONE person! If you add up the "eggs per coprolite" values, you get over 200,000 mawworm eggs and over 1.5 million whipworm eggs...again, all inside of the same person. (Seriously, just let that sink in for a moment.) A whipworm infection this bad has never been reported in anyone. This discovery brings our understanding of Medieval filth and disease to a whole new level. 

Additionally, a female whipworm can only lay about 30K-60K eggs a day (depending on your information source). Though we don't know how long these eggs were sitting inside of this severely constipated woman, we do know that she had to have more than a single worm laying eggs. Having a heavy load of whipworms can cause people to have problems like prolapsed rectum and compromised peristaltic activity of the intestines. (Which translates to: your rectum falls out of your butt and your intestines can't push poo out of you anymore because the muscles aren't working right.) The paper concludes that the extreme parasitism was likely affiliated with the cause of death for this woman.

The paper also discussed the importance of looking at parasite egg taphonomy (how things deteriorate or decay over time) and the co-infection of two worm species as it relates to other aspects of European archaeoparasitology.

I officially joined the Nivelles team as a co-author in my second year, though I had heard all about it in my first semester. You see, the manuscript was pieced together from the term projects of four students and introductory material from colleagues in Europe. The lead author had been working on trying to bring everyone's work together, but it still didn't read like it was written by one person. At the suggestion of my major professor, I worked with the lead author to cut, re-write, and otherwise clean up the manuscript so that it was publishable. This took up much more of my time than I had anticipated, but it was worth it once we finally got it submission-ready. In February, 2014, I took the position of corresponding author and pulled the manuscript through the submission process. We submitted to the Journal of Archaeological Science, and we received excellent reviews. After lots and lots of editing, we finally sent back the final proofs in October, 2014. Our paper was set to be published online in November and in print the following January. That's right, from submitting the first time to print publication was almost a full year. (This doesn't even include the time spent editing prior to submission.)

The Moral of the Story
Despite all the headaches, this was such an amazing project to be a part of, even in the later stages. The author line can boast of researchers spanning four countries on three different continents. It involved an undergraduate, two master's students, two PhD students, and senior researchers from multiple universities. It was a really cool thing to be involved in, and I'm very thankful to have been brought on to help. 

We even got some media attention for the work! Check out the article about it here!

Finally, as might become tradition for parasite papers that I publish, here is a title shot for your viewing pleasure. :)