Friday, July 20, 2012

What do we do with all this poop?

Whenever we see one of our hyenas doin’ its business, we try to collect a fecal sample. This may strike you as pretty disgusting (and indeed, it sometimes is), but poop can provide us with a wealth of information about the inner workings of hyenas. We can use a small sample to measure a variety of hormone levels and even extract DNA to determine their genetic relatedness to other individuals. Because physiological changes can be a warning sign of impending population declines, as David Green described in a recent post, we are also planning to explore what stress hormone levels in hyena poop can signal about the status of mammals in the Mara ecosystem (for more on this, and for opportunities to help through our sponsorship by Petridish, check out http://www.petridish.org/projects/a-sentinel-for-african-ecosystems). All of this makes us hyena researchers quite excited when we see a study subject dropping us one of these treasured morsels, can locate it in the grass, get it into a ziplock bag in the field, and then stuff it into vials that are frozen in liquid nitrogen back at camp.

But the road from watching a hyena poop to having usable data is a long one. This spring, before heading out to the Mara for my two-year field season, fellow grad student Sarah Jones and I packed up about 550 hyena fecal samples and headed to the University of Nebraska to see what our hyenas’ various bowel movements over the past six years or so could tell us. Sarah was analyzing samples for androgen levels (a class of hormones including testosterone). I analyzed samples for corticosterone – a main mammalian stress hormone.

After our precious cargo made it safely to Omaha (driving around with a back seat full of coolers of valuable poop is a bit unnerving), we learned from endocrine god Dr. Jeff French how to transform our stinky poop samples into PhD gold. Dr. French normally spends his time exploring the connections between hormones and behavior in marmoset monkeys and humans but takes a break every few years to help us process these more exotic poops transported all the way from the wilds of Africa.

Before even doing hormone assays to determine the concentrations of hormones in our samples, we first had to extract the hormone from the feces. This proved the most labor-intensive part of the whole process and amounted to about five days of thawing poop, weighing poop, heating poop, drying poop, grinding poop with a mortar and pestle, picking hairs out of poop, and…weighing it again. The goal is to get a pure sample of dried feces that, when combined in a known mass with a known volume of liquid, will give us a homogenous solution of hormones. Here are some of the steps we went through to turn hyena diarrhea into some pretty science-y looking tubes of extracted hormone…




Weighing out a precise quantity of feces





Drying out a hundred-or-so fecal samples overnight in an incubator 



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DO NOT drop this tray of incubated samples! Aren’t they colorful!? Dr. French and I think they look like spices set out in a Middle Eastern market. 

In the beginning, this process was super cool to me. Five years after some hyena ate a zebra, here I was sitting in Nebraska seeing all those tiny black and white hairs in its poop and picking them out with the attention of a surgeon. But after doing this for more or less 12 hours a day, Sarah and I were starting to think our morning coffee grounds looked like poop (and wow, so perfectly mortared-and-pesteled!) and had the urge to weigh out a perfect .2 grams of that pepper before adding it our pasta. We were ready to reach the end of the extracting phase….






Voila! We do some chemistry and centrifuging magic with those dried samples and here’s that solution of hormones on the left. The rest of the stuff in our sample – all the stuff we don’t want – remains in the tube on the right.













After using some more fancy science tools that us field biologists don’t usually lay our hands on, like this multi-channel pipetter on the left, we are left with our final extracted product on the right. I swear, these little holders filled with just the precise quantity of yellowish sample, perfectly in solution, looked more beautiful than liquid gold. Now, on to the exciting part…finding out the concentration of corticosterone in each of those little tubes. This will help us determine how a hyena’s stress level is shaped by ecological, social, and physiological factors, as well as conservation management decisions.

And for this, us field folk get to feel even more like real, honest-to-god scientists by utilizing the properties of radioactive substances. We add an antibody to our extracted hormone as well as corticosterone tagged with a radioactive Iodine tracer. What this amounts to in the lab is adding various brightly-colored radioactive liquids to our precious tubes of extracted hormone in specific timed succession, giving us pretty test-tube racks like this…



Radioactive poop! 


Then, the hormone in our sample and this radioactive antigen compete for binding sights on the antibody. This is one of the highlights of the process because as the magic of “competitive binding” occurs within each of these tubes, you get to take a 2-hr break from pipetting and go grab some lunch. When you return, chemistry magic has happened… and I have never been this excited about basic chemistry principles in my life. We can then add another brightly colored liquid to our tubes to separate the radioactive-labeled hormone that has bound to the antibody from the hormone that has not. After adding this precipitant and centrifuging our tubes, we’ve successfully completed this separation.



Tubes of radioactive poop about to be centrifuged


And finally comes the most nerve-wracking step of the entire procedure…decanting. Imagine, after spending days extracting the hormones, and then many hours carefully pipetting small amounts of somewhat dangerous substances in and out of small containers, you manage to get each of these precious tubes safely in and out of the massive centrifuge. You’ve also managed to not spill anything and are trying not to think about just how much each of these little tubes actually costs if you were to sum up the price of all the different liquids you’ve added. Now comes the time when you put these tubes in a special holder, turn them upside down, and literally pour all the fancy chemicals you’ve added down the drain. For me, this was the most stressful step in the process. I always had a mental flash of all those valuable tubes crashing into the sink and having to start the whole thing over again!


Pouring it all down the drain...

...but not everything goes down the drain.  What we are left with is a small pellet in each tube that can be read in a Gamma Counter to measure the amount of radioactivity. The higher the radioactivity, the more the radioactive hormone won out in our competitive binding battle and the less corticosterone there actually is in our fecal sample. By comparing our samples with various solutions of known standard concentration, we can determine precisely the amount of stress hormone in each of our samples.

In the coming months, we will be using these new data to address several questions in addition to the hypothesis that spotted hyenas can serve as an "indicator species" for the Mara ecosystem.  For example, I will be asking whether hyenas living in areas within the Mara with different management strategies show different stress profiles. I will also be looking at the behavioral, physiological, and demographic consequences of maternal stress for mother-offspring relations and cub development.  As these questions form the heart of my PhD research, you'll hear more about them in future blog posts...

My first foray into endocrine lab work would not have been possible without the help of the French Lab. Thanks to Dr. French for opening his doors to smelly hyena poop samples and all his guidance. Also, thanks to undergrad Benjamin Hochfelder in the French lab. Can you believe Ben came in on a weekend to help us grind and weigh poop!? Finally, Sarah and I want to give a huge thank you to Drew Bernie. Drew’s title is “lab tech” but his endless help and trouble-shooting abilities during our stay in Omaha quickly earned him the nickname “Superman.” Drew turned me from a pipette-na├»ve hyena watcher into an assay-machine and seemed to be able to put out any fire that Sarah and I started. Thanks, Drew!














Left: undergrad-extraordinaire Ben Hochfelder
Right: Drew didn’t want a commemorative photograph taken, but this picture pretty much sums him up.

Wednesday, July 11, 2012

Tall Grass be Gone!

Here in the Mara Triangle, the lack of grazing and extended long rains in April has made for some really tall grass.

Like REALLY tall. Upwards of six feet in some places.

Luckily, The Mara Conservancy has cut the grass on tracks in preparation for the high season and flood of tourists. Unfortunately in a small car like the Maruti, our car wanted to bring some back from the bush as a souvenir for camp!

Wednesday, July 4, 2012

A Sentinel for African Ecosystems and an Opportunity to Help


An up and coming website called Petridish is giving us the opportunity to raise funds for research, not from big granting agencies, but from ordinary people like you. Below is a general synopsis of the project, but for more information and ways to donate go to http://www.petridish.org/projects/a-sentinel-for-african-ecosystems.

Monitoring an entire ecosystem is an incredibly daunting task. There are many species of sympatric herbivores and carnivores, important ecological processes taking place, and interactions between all three that maintain and sustain ecosystem functioning. If disturbed, the ecosystem (and species within it) may become threatened, endangered, and risk extinction.

Although sentinel and indicator species have been monitored to prevent damage to ecosystems and specific species, they have historically been used on a presence/absence basis.  In other words, if the indicator species (i.e. spotted owl) is present in an ecosystem, then the ecosystem is deemed to be in “good” condition, or recovering from previous disturbances.

Unfortunately, by thinking of an indicator species only from a presence/absence basis, we may be ignoring subtle cues emitted by good sentinels to help prevent damage to ecosystems and other sensitive species. By using a species that is known to emit subtle cues, we may be able to curb disturbances before it becomes too late. And this is where the spotted hyenas come in!

Previous research in our lab has shown spotted hyenas to emit early warning signals about how they themselves are negatively influenced by human encroachment on their habitat. Changes in space use behavior, often one of the first ways that organisms adjust to changing environments, pre-date demographic changes in hyenas by 3 years.  Changes in stress physiology, another signal that a population could be in trouble, pre-date demographic changes in hyenas by 5 years. What we want to understand with this new research program is if these same metrics can be used to predict population changes in more sensitive species in African ecosystems, like lions, cheetahs, and threatened herbivores.

To evaluate the hyena’s capability to act as an indicator species, we will use GPS collar technology to look at how hyenas utilize space, monitor stress physiology through non-invasive collection of feces, and then relate these measures to the spatial patterns and population trends of other carnivores and herbivores that live in our hyenas’ territories. This may sound easy, but it will require deploying new collars to many of our study animals, and months of driving around our territories to effectively monitor multiple animals on the landscape. However, the payoff will be extraordinary if we are able to use changes in our hyenas to aid in the conservation of biodiversity!

To learn more about this exciting research, and to help support and donate to its completion, please see more at http://www.petridish.org/projects/a-sentinel-for-african-ecosystems.

Petridish.org is a great site that allows readers and hyena enthusiasts (like yourself!) to get involved with research taking place around the globe. Please go to the website and learn more about using spotted hyenas as a sentinel for African ecosystems, and think about donating. Every little bit helps! 

Tuesday, July 3, 2012

Pan ate Target!


While in Talek, I have been doing experiments to test an individual hyena’s boldness. We have this foam hyena that we call Target that I have been showing to hyenas to see how they respond.

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Alfredo and Target

Most hyenas who have seen Target will notice it, try to get downwind of it to smell it, maybe head bob to it, and leave. There are a few variations to this pattern, but we think that the hyenas eventually realize that Target is not a hyena as they get closer.

A couple days ago, I saw the two most extreme responses yet.

In the morning, we showed Target to Blue, an adult female. She is relatively high ranking. Blue was very upset by Target. We had placed Target in the road she was going down, and once she saw Target, she stopped dead. Slowly, Blue got a little closer to Target. By the time she was barely 30m away from Target, she turned around, walked over 100m away, walked around Target putting our car between her and Target, then continued down the road. Blue didn’t really even bother to try to sniff out what it was; she just avoided it.

In the evening, we were lucky enough to find another test subject. It was Morpheus, a higher ranking female than Blue. Morpheus saw Target, stopped briefly to stare at it, then walked straight up to it. She was the first hyena I have seen to touch Target. If someone goes close, they’re still usually about 5m away. Morpheus was walking circles around Target, sniffing and licking it. All the sudden, she stopped abruptly and walked off, and we realized that her younger sister, Pan, was coming straight at Target with another older sister, Adonis, hanging back about 80m. For the second time ever, we watched a hyena walk straight up to Target and start sniffing and licking her. Next thing we knew, Pan had grabbed Target’s tail, dragged her a couple meters, and knocked her off her stand. Adonis started to approach from where she was hanging back with her daughter, Grape Escape, at this time, and Pan started to drag Target into the grass. This series of events all happened very quickly, and once Pan started taking Target into the grass, we stopped the session immediately to go save Target.

Unfortunately, we were a bit too late. Within the 30 seconds it took to drive to where they were, Pan had bitten a few bites out of Target’s rump, detached its head (which is meant to slide off), and bitten off about half of her nose. Poor Target. One of the weird things is that the parts that were bitten are the exact places hyenas would attack an intruder. So maybe they think Target is a hyena, or at least enough hyena-like, longer than we had thought. Or this could just be the case for these high ranking and bold sisters.

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Target’s injuries, with Jenna looking shocked and worried in the background

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Ian (one of the IRES students) and me with Target in the beautiful Hilux

Michigan State University | College of Natural Science