Nature

12
Mar

On the Trail of the Bengal Tiger—And Its Feces

Walking through the jungles of Nepal with a team of conservation biologists is a bit like walking anywhere with a toddler: you have to stop every few moments to examine something small and seemingly mundane. There are frequent group huddles when someone spots a strange plant or an insect that sucks nutrients from seeds or tiger marks clawed deep into a tree trunk or, in this case, poop.

In fact, it’s the poop that interests Dibesh Karmacharya the most. Tiger scat, like all feces, including human waste, is covered in a layer of cells from the intestinal wall. When the animal defecates, some of the epithelial cells that line the colon are sloughed off along with the waste. By carefully collecting the scat and extracting DNA from the outer layer, biologists like Karmacharya, who founded and runs the Nepal-based Center for Molecular Dynamics, can determine which species left it, the sex of the animal, and even its individual identity.

bengal-tiger
Tigers are difficult to track, so wildlife biologists search for scat samples to reveal insights about the cats' lives.

Earlier tiger surveys that used visual surveys or photo traps have suggested that only about 200 Bengal tigers remain in Nepal, but DNA tell a much deeper story. Tiger genes reveal secrets about the cats’ movements, mating behaviors, eating habits, and even clues about their future: After the scientists have collected a range of tiger samples, they can then compare known key markers to see just how genetically diverse—and potentially how resilient—the tiger population is.

Our expedition is finally underway after idling for a day, having had to wait for government permits to be sorted out. (The required paper copy was eventually delivered by motorcycle.) We are heading into Chitwan National Forest, home to the Bengal tiger, the greater one-horned rhinoceros and the Asian elephant. We ferry across a low river in a dug-out canoe—me, Karmacharya, and two techs from his lab, two local trained trackers, and two Australian biologists.

chitwan-river
The Chitwan National Forest lies on the other side of the East Rapti River.

We’re not even into the forest when we spot the first tiger pugprint pressed deep into the muddy river bank. It had rained a few days ago, and from the print’s crisp edges, wildlife technician Harka Man Lama guesses the big cat was here this morning. He’s not surprised. Lama lives near Chitwan and has gathered much of the scat collected for the Tiger Genome Project. He knows the tigers’ habits and haunts, and this is one of them.

pugprint
One of several tiger pugprints we spotted that day

Lama and the other local wildlife techs have been trained by Karmacharya’s team on how to identify tiger scat and how to carefully scrape up a sample with a stick, keeping the outer layer intact as they slip it into a vial filled with alcohol that will preserve the DNA. Lama says tiger scat appears rough with visible hair in it, and it’s often covered in a smattering of dry leaves the big cat kicked over the scat, similar to how housecats will throw a layer of kitty litter over their mess. Lama has developed a keen eye for these telltale scratches in the dirt.

Australian biologist Marc Jean-Hero fiddles with his GPS unit to point us toward the first plot where we’ll be gathering samples. It’s midday already, and the sun is hot overhead, but the leafy green canopy offers a cool reprieve. It’s not long before someone cries “poop!” Lab tech Priya Joshi snaps on a pair of latex gloves and kneels down to collect the sample. Marc-Hero jots down the GPS coordinates in a log book, and Lama squats down to examine the specimen. “Rhesus monkey,” he says definitively, though they won’t be entirely sure until they run the sample in the lab.

priya-collecting-sample
Priya Joshi, one of the Tiger Genome Project's technicians, collects a sample.

So far, the wildlife technicians have correctly eyeballed tiger scat about 60 percent of the time. Most of mistaken tiger scats turn out to be common leopard.

“Things that weren’t tigers, most others would say it’s simply a ‘sampling error’ and throw it out,” Joshi says. “But we looked at these and found leopards living where tigers are, genetic evidence of civet cats, wolves, and foxes in the park. These findings are happy accidents and equally important for judging the health of the forest.”

Several hours slip by, and we’ve barely made it a third of a mile through the forest. Despite the promising pugprints on the riverbank, we don’t net any tiger scat on this outing. Still, we’ve collected nearly 10 scats which will be run through the DNA sequencer as part of a larger biodiversity project. The excursion has also served to teach the Australian biologists how to collect scat samples, as they’ll be surveying a series of plots over the next few weeks with a group of biology students and gathering new samples for the Karmacharya’s lab.

From Nebraska to Nepal

This type of noninvasive wildlife genetic research is fast becoming integral to conservation efforts. The first successful DNA sequencing of fecal matter was with endangered bears in Europe in 1992, but the practice has spread as costs of DNA sequencing have dropped. The work at the Center for Molecular Dynamics Nepal is the brainchild of Karmacharya. Tall and built like a rugby player, with intense black eyes, Karmacharya spent 14 years in the United States studying and working as a microbiologist. But Nepal was always his home.

Karmacharya has long been passionate about wildlife. As a college student in the cornfields of Nebraska, he studied tigers and cats, experimenting with using cat pugprints to determine an individuals’ sex. He graduated in 1996 and worked in various labs in the U.S. and Canada. Then, in 2007, Karmacharya made his way back to Nepal and founded the center, equipping it with PCR machines and other advanced instruments. Initially, the center was largely dedicated to diagnosing and studying human diseases, but Karmacharya kept finding ways to incorporate conservation research. In 2011, he began the Nepal Tiger Genome Project.

With more than $250,000 in funding from USAID, the Nepal Tiger Genome Project has set out to gather tiger DNA samples from across Nepal’s Terai Arc, the jungle zone that stretches the length of the country, paralleling the mighty Himalayas. The project has trained scat collectors and sent them out to over 100 preexisting 25-kilometers-square grids where biologists have surveyed tigers in previous years.

“In the past, we used to look at pugmarks to do a population census,” Karmacharya says. “That is very inaccurate way of doing it.” Camera traps change that, giving scientists photographic evidence they could use to more accurately identify individuals—and their numbers—by their stripe patterns. “But genetics, in the long run, gives you more information not only about the population—because now you can count each individual—but also information about the gene flow and the diversity.”

Getting to the Good Stuff

At the Center for Molecular Dynamics in Kathmandu, Karmacharya shows me around the lab, introducing each gleaming piece of equipment like a proud parent. As is the custom in most homes in Nepal, we had removed our shoes before entering and slipped into a pair of indoor Crocs to scoot around the lab. This, Karmacharya says, is where the vials of scat are transformed into extensive data about each tiger. Ultimately, that information makes its way onto a color-coded map showing each tiger’s travels and how genetically similar it is to its neighbors.

Karmacharya walks me through the process. First, lab techs extract DNA from the scat sample, spinning out 100 microliters of the fecal sample that’s suspended in alcohol. Though alcohol helps preserve the DNA, the genetic material could be in rough shape—it has been exposed to not only the gut’s digestive bile but also the weather. If Karmacharya and his team were just looking at nuclear DNA, which only has two copies, that could pose a problem, but cells also produce hundreds of copies of mitochondrial DNA. Lab techs first extract and compare the mitochondrial DNA against known tiger samples.

nepal-pcr
Preparing a PCR gel at the Center for Molecular Dynamics Nepal

If it’s a tiger, then the techs use nuclear DNA to test for sex. Finally, they look at nine markers, or loci, in the nuclear DNA. These sites are known to be very different between individual tigers, which helps establish the individual’s DNA fingerprint. No other tiger will have this exact same combination of genes. (Though the tiger’s parents, siblings, or cousins will share some genes; that will help sort out the family tree.)

On the Brink

Since the Nepal Tiger Genome project began in 2011, Karmacharya and his team have collected 1,200 putative tiger samples across four national parks in the Terai Arc. They have screened around 800 of those samples and identified 125 tigers total: 72 individuals in Chitwan, 32 in Bardia National Park, and 20 in the other two parks. Their data is the first to use genetic evidence, and they estimate that just over 220 Bengal tigers live in Nepal.

That’s not very many tigers, but potentially more worrisome than their low numbers is their genetic diversity. “The lower the genetic diversity, the more likely the population is to decline because of vulnerability to diseases or genetic disorders or problems with reproduction,” Karmacharya says.

dibesh-sample-collection
Dibesh Karmacharya collects a sample in Chitwan National Forest.

To measure genetic diversity, he traces a tiger’s family heritage by looking at select spots on the genome. Each individual tiger has two copies of DNA—called alleles—one from each parent. The lab analyzes the nine loci for each individual and determines how often the maternal allele is different from the paternal copy, a measure of diversity known as heterozygosity. The more instances where the genes are the same, the lower the heterozygosity.

So far, his data shows the tigers in Chitwan National Park have a diversity level of about 45%. “From a conservation point of view,” Karmacharya says, “a genetic diversity of about 60% or higher is considered to be a healthy, viable population.” 

There is hope: the Chitwan population is measurably genetically different from the tigers in Bardia National Park and the two other parks in the Terai Arc. For now, however, it appears that most tigers aren’t leaving their home parks.

“If the suggestion is that tigers are not moving across the landscape, then from a conservation and management perspective you can evaluate what is necessary to provide that geographic linkage,” says Lisette Waits, a professor of conservation genetics at the University of Idaho. Waits has studied the genetics of endangered wolves, grizzlies, pygmy rabbits, and pronghorns in the United States, and she has visited the Center for Molecular Dynamics Nepal twice to train staff biologists how to extract DNA from feces to evaluate genetic diversity.

Waits says the genetic information gleaned from this kind of work is invaluable for making conservation decisions. When there are pockets of genetically similar species, land managers could work to restore habitat between the pockets, creating corridors for individuals to travel and mate with a more heterozygous partner. In some cases, scientists might even conduct a “genetic rescue,” moving an individual female to another patch where her peers are more genetically distinct. If all goes well, she will mate and her offspring will diversify the population.

Putting DNA to Work

It’s too soon to make those kinds of decisions in Nepal. But as Karmacharya pulls up the Tiger Genome Project’s map, it’s markedly clear that related individuals are clustered together. There’s a pocket of red dots in Bardia, then a cluster of blue ones in Chitwan, and green in the other two parks. Each marks an individual tiger, and the color represents its genetic similarity to the other individuals.

The map also tells a broader story, Karmacharya says. By superimposing the tiger maps on those with human settlements, “we can tell whether they are moving into areas where tigers are, and it gives us more information to design conservation plans and to mitigate these problem areas.”

The data could even help scientists catch poachers. Karmcharya is working with Interpol to establish a system where the police could send the lab tissue samples from any tiger parts they confiscate. The lab could then determine if the animal is from Nepal, and if so, if there is a particular region that’s being targeted by poachers. Officials could then take steps to ramp up enforcement in that area.

sample-vials
Karmacharya's lab at the Center for Molecular Dynamics stores hundreds of scat samples, including several dozen from tigers.

Karmacharya continues, guiding me through the map and pointing out the family histories it reveals. “These two reds here somehow came from there,” he says, pointing to two lone red tigers mingling in amidst the blue Chitwan tigers. The two dots are promising because they suggest that tigers have braved the territory between, crossing villages, roads, and farm fields before finding their way into the park. Karmacharya says ultimately he and his fellow conservationists would like to connect the four parks in the Terai Arc with forested corridors, creating one large habitat that will not only benefit these charismatic cats, but numerous other species, too.

“Many creatures are not as cute or sexy, but we still want to know about their health,” says Joshi, one of the lab techs. She and her colleagues have already collected samples from and studied many of the so-called charismatic megafauna, including the endangered greater one-horned rhino, the Asian elephant, musk deer, and snow leopards living in the Himalayas. So beginning in 2013, the lab started gathering and analyzing DNA samples from all kinds of species living in the Terai Arc—plants, birds, amphibians, mammals, and reptiles.

“We don’t have baseline information for anything” in Nepal, Karmacharya says. “Right now we are going to Chitwan and collecting samples from existing grids,” or plots which the Australian biologists have been studying. “This will be first time genetics will be incorporated.”

rhino-in-chitwan
A great one-horned rhino in Chitwan National Forest

Previously much of the research on wildlife in Nepal was done by recording the size and proportions of an animal. It’s an age-old method that relies on being able to catch the animal. Identifying the exact species is done using physical appearance alone, which isn’t always the most reliable technique. But with rapid advances in genetic sequencing, scientists can now peer into an animal’s identity in ways they never could before. Sometimes they stumble upon a cryptic species, which looks like one species but is, in fact, another. For example, Karmacharya says they’ve analyzed musk deer scat and were surprised to find that DNA evidence suggests there are two separate species, even though they all look the same.

Karmacharya says sequencing technology is advancing so quickly they may soon have affordable, handheld devices that trackers could take into the field to evaluate sample DNA on the spot. That could speed a broad genetic survey of Nepal’s teeming lowlands, potentially uncovering new species or revealing unheard of evolutionary histories. It could even lay bare previously cryptic or unknown animal behaviors.

Nepal is a country rich in biodiversity, yet so many creatures remain unknown to scientists. But the work Karmacharya is doing—and championing—could help Nepal become not just a biodiversity hotspot, but a hotbed of ecological research. To think, it all started with poop.

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masterson

Kathleen Masterson

Kathleen Masterson is a science and environment reporter. She has written about food production, climate change, ecology, and land use, including topics ranging from antibiotic use in raising meat animals to the rise of mining frack sand.