DNA in Strange Places: Hippo Poop, Zoo Air, and Cave Dirt

Many years ago, a dear friend took me to the Detroit zoo to see the Hippoquarium. Much to my delight, the resident hippo positioned her rear to the glass of the enclosure and let loose, her whirring tail distributing the intestinal contents like blowing on an open milkweed pod.

A few years later I saw the same demonstration at the Tampa zoo, a hippo’s whirligig-of-a-tail in action.

Hippo Microbiomes

Recently, researchers from the US and Kenya described in Scientific Reports their investigation of the ejection of hippo feces into the pools of the Serengeti’s Mara River. A more natural environs than a zoo, the river is home to more than 4,000 hippos wallowing in some 170 hippo pools in the Kenyan part of the territory.

By night the hippos graze on land. By day they laze in their pools, spewing organic matter in their fecal flush. The effluence includes the residents of their gut microbiomes, the microbes’ jettisoned genetic material instantly becoming environmental or “eDNA.” Meanwhile, hippo urine adds phosphorus as the solid stuff sinks, depleting oxygen. And that’s good for the microbes.

“As hippo pools become anoxic, the pool environment becomes more similar to the hippo gut, increasing the likelihood that some of the enteric microbes will survive and even function outside the host gut,” the researchers write. Flatulence might churn things up anew.

“We used natural field gradients and experimental approaches to examine fecal and pool water microbial communities and aquatic biogeochemistry across a range of hippo inputs,” the report states.

The investigators tracked the microbial excrement residents with an old technique. They compared the RNA sequences of part of the ribosomes, where protein synthesis takes place in a cell. And they discovered, perhaps not surprisingly, that poolmates tended over time to harbor identical gut microbiomes, forming “a meta-gut system,” like toddlers with leaky diapers in a swimming pool.

In fact, the hippo description echoes one of the very first published microbiome studies. The researcher began with his newborn’s diapers and compared the microbiome within to those of other babies. The experiment revealed that after their first year, the microbial poop communities in babies’ soiled diapers came to be very much alike. I can’t find a link because of the vast literature that has grown since then analyzing diaper contents and what they may mean.

The new hippo findings are intriguing.

Hippo feces tend to harbor a high viral load, but only tiny populations of protozoa.

Hippo pool residents swallow emissaries of each other’s microbiomes. And as is true for all gut microbiomes, those of hippos supplement the mammals’ digestive repertoire, expanding the nutrients they enjoy and helping to digest carbs.

Other species may tap into the feast of hippo doo too. Aquatic insects and fish regularly dine on it, then spread the material in their own emissions. And hippo crap is essential for the well being of tilapia in lakes in the Democratic Republic of Congo, where efforts are underway to boost the hippo population to up tilapia numbers. Bandits selling hippo meat during the decade-long civil war in the DRC depleted the population; human residents are anxious to get back to fresh fish after eating the frozen stuff for years.

Other recent studies track DNA in the air and in sediments.

Zoo DNA

Two papers in Current Biology, from the University of Copenhagen and from Queen Mary University in Canada, explore the ability to identify zoo residents by DNA wafting in the air. Anyone who has ever inhaled in a zoo can attest that this is indeed possible. In fact, identifying zoo DNA may prove easier than using cameras, human observers, or tracking hoofprints or feces to check attendance.

Team leader Kristine Bohmann from the University of Copenhagen describes the inspiration.

“Earlier in my career, I went to Madagascar hoping to see lots of lemurs. But in reality, I rarely saw them. Instead, I mostly just heard them jumping away through the canopy. For many species it can be a lot of work to detect them by direct observation, especially if they are elusive and live in very closed or inaccessible habitats.”

Monitoring zoo air works, even though the researchers were initially skeptical because the molecules are so diluted. They used a fan attached to a filter to collect DNA from fur, excrement, saliva, and even the breath of approachable beasts. Then they PCRed (amplified) the DNA enough to identify it, like a COVID test that increases viral RNA to a quantifiable level.

“After DNA sequencing, we processed the millions of sequences and ultimately compared them to a DNA reference database to identify the animal species,” explained Christina Lynggaard from the University of Copenhagen. Both teams found DNA from animals within the zoo and outside too, such as a rare Eurasian hedgehog’s DNA found just outside Hamerton Zoo in London, and red squirrel and water vole DNA outside the Copenhagen Zoo.

“In just 40 samples, we detected 49 species spanning mammal, bird, amphibian, reptile, and fish. In the Rainforest House we even detected the guppies in the pond, the two-toed sloth, and the boa. When sampling air in just one outdoor site, we detected many of the animals with access to an outdoor enclosure in that part of the zoo, for example kea, ostrich, and rhino,” said Bohmann.

Elizabeth Clare’s group from Queen Mary University of London identified DNA from 25 animal species, including “tigers, lemurs and dingoes, 17 of which were zoo species. We were even able to collect eDNA from animals that were hundreds of meters away from where we were testing, and even from outside sealed buildings. The animals were inside, but their DNA was escaping,” she reported.

The Sands of Time – DNA in Ancient Sediments

E-DNA has been crucial in reconstructing long-gone ecosystems, even when fossils have deteriorated into nothingness. For many years studies were restricted to the snippets of DNA found in mitochondria, which resist damage. But more recently, researchers have been able to reconstitute sequences of DNA from cell nuclei, fleshing out the pictures of the past.

Recent “dirt DNA” studies have come from:

• Estatuas Cave in Spain, whose Neanderthal residents, both male and female, lived there from 80,000 to 113,000 years ago.
• Satsurblia Cave in Georgia, where a female from an unknown group of Neanderthals left her DNA some 25,000 years ago, plus DNA from a wolf and a bison.
• Chiquihuite Cave in Mexico, which yielded DNA from 12,000-year-old black bears, the forebears of Alaskan bears kept south by glaciers.

Most intriguing is Denisova Cave. The famed domicile in the Altai Mountains in south central Siberia has served as a hotel of sorts for ancient humanity, housing Neanderthals, Denisovans, and others yet to be named as they mated over the millennia. Researchers from the Max Planck Institute for Evolutionary Anthropology in Leipzig report in Proceedings of the National Academy of Sciences their analysis of “cave dirt” to detail life in the famous cave.

A little background. The Denisovan Cave was discovered in 2010 with the initial description of a pinky bone from a girl whom researchers named Denise. Intense research followed. Here is how I describe the goings on in my human genetics textbook:

“We do not yet have all of the puzzle pieces—the Denisovan genome clearly indicates admixture with at least one other, yet unknown, type of archaic human. Genes also entered Denisovan and Neanderthal genomes from our very distant ancestors. Because we have such scant evidence, we cannot really know what happened as humanity sorted itself out. Several small populations of archaic humans probably coexisted and eventually mixed
for at least 100,000 years before we modern humans, retaining some archaic genes, emerged and persisted. A few ancient gene variants remain in some of us.”

I’m happy to report that my genome includes the maximal input from Neanderthals.

The Max Planck researchers recovered samples of human DNA from the Denisovan cave in tiny bits of what were once bodies, rather than just the molecules scattered about, as is the case for the hippo pools and the airborne zoo DNA. Geoarchaeologists collect the pieces and embed them in plastic resin. But until the recent work, it wasn’t known whether the samples would yield much information on past residents. But it can!

When lead researcher Diyendo Massilani was able to extract substantial Neanderthal DNA from only a tiny nubbin of Denisovan cave sediment, he could identify the sex of the individuals (from the SRY gene only in males) and show that they were related to other Neanderthals whose genomes had been sequenced. An ancient version of getting one’s 23andMe ancestry results, perhaps.

And we’ll learn more.

“The Neanderthal DNA in these small samples of plastic-embedded sediment was far more concentrated than what we typically find in loose material. With this approach it will become possible in the future to analyze the DNA of many different ancient human individuals from just a small cube of solidified sediment. It is amusing to think that this is presumably so because they used the cave as a toilet tens of thousands of years ago,” Massilani said.

These sorts of studies make me wonder what we humans will leave behind of our genomes for future Earth residents to discover and attempt to interpret.