Getting Ready to Head Back to the U.S.

Well, my days in Chile are numbered- I'll be flying home on Monday, October 31st (and yes, Chileans do celebrate Halloween, so I'm hoping for some candy on the plane ride home). The last few days have been crazy busy since I've been scrambling to collect last-minute data, steadily writing up my thesis proposal which I'm defending a week after I get back to Tufts, and cleaning/packing/organizing for the trip home. This means I'll have to wait till next week to write about the endocrinology of parental care, so for this post I'll just be uploading a bunch of photos from my Chilean field season, enjoy!

Cueca dancers, the "cueca" is the traditional Chilean dance. 

Me in front of the Moneda (the president's house). Plus a stray dog, which are everywhere in Santiago.

Hiking in the Andes.

Black-billed shrike-tyrant

Parque Nacional La Campana

Pablo Neruda, Chile's famous poet and humanitarian.

Chilean swallow.

Magellanic horned owl

Street art in Valparaiso

Enormous male sea lion in Valparaiso (it smelled bad, too)

More cool street art, Valparaiso.

I've witnessed the sun rise pretty much everyday for the past two months.

And some days it's foggy and cold.

View from my apartment balcony after a student demonstration.

I caught an iguana with a TV antenna and some string a few days ago.

What a cutie!

Degu Sex

Reproductive physiology is amazingly super cool and fascinating. With the exception of stress physiology, I’d say that reproduction is the most interesting topic in biology. So, in honor of reproduction, let me tell you about the endocrinology behind mating, pregnancy, and lactation in the degu.

The mating period for degus is typically around the month of June. Because degus have a three-month gestation period, this means that the baby degus will be born around late August or September. Degus, like many seasonal animals, optimize their time for breeding so their offspring will be able to develop and grow in ideal conditions. The austral spring is a good time for degus to give birth because there’s plentiful food due to the rainy winter. The mattoral (that’s the habitat type I’ve been working in, it’s similar to the chaparral of Southern California) is very dry during the austral summer, so it’s important that the degus time their reproduction so all energetically expensive processes (growth in pups and lactation in mothers) will be completed before the dry season hits.

For male degus, testosterone is a key player during the mating season. Testosterone is an important hormone for spermatogenesis (sperm production) and several different mating behaviors such as gaining access to a mate, wooing a female, and performing the physical act of copulation. For species where mate access is competitive (elephant seals are a classic example), the more dominant and aggressive males are usually the most successful when it comes to reproducing. This aggressive behavior is partially modulated by testosterone, and dominant males oftentimes have higher levels of testosterone compared to subordinate males. Aggressive behavior is enhanced when testosterone binds to brain receptors (either by converting to 5-alpha-dihydrotestosterone and binding to androgen receptors, or by aromatizing to estradiol and binding to estrogen receptors). And while most of us think of aggressive behavior as fighting, aggression can also be displayed through song and other non-physical behaviors (ex: marine iguanas bob their heads up and down to caution other males encroaching on their territory).

Like many other seasonal vertebrates, male degus have high levels of testosterone during the mating season and low levels of testosterone during the rest of the year. Why do we see seasonal differences in testosterone levels, and if testosterone helps increase reproductive fitness through aggression and other behaviors, then why not pump out a ton of testosterone all the time? Well, the answer is that it’s costly; high circulating levels of testosterone create an energetically-expensive lifestyle, and it has been shown that testosterone-implanted animals can have low body mass, a suppressed immune system, and a higher rate of mortality.

The Challenge Hypothesis, posited by Wingfield and colleagues, states that the seasonal changes we see in testosterone levels are due to a tradeoff between aggression and parenthood. In bird species where males provide significant parental care (ex: providing chicks with food), testosterone levels usually fall after the chicks are born. After the chicks fledge, and if the parents are able to breed again, the male will then up-regulate his testosterone levels. For species where males don’t provide much parental care (like degus, who may do a little bit of thermoregulatory huddling, but that’s about it), the drop in testosterone is often longer and slower.

So what’s going on with the females during the mating season? Well, they’re probably checking out the males and judging them, but in addition to that, females are preparing themselves for pregnancy. Compared to male reproduction, the female reproductive cycle is quite complicated, but let’s see if I can break it down into the major steps (note: most of the information I’m providing is typical of guinea pigs, which are close relatives of degus):

Proestrous:

This stage lasts a day or two, and the female is agitated and may mount other con-specifics. During this time period, the ovarian follicles are growing and the endometrium (lining of the uterus) is slowly building up. This follicle development is fueled by follicle stimulating hormone (FSH).

At the same time, the some outer cells in the follicles (specifically, the granulosa cells) are producing estrogens. Estrogen production is due to the work of two hormones: FSH and luteinizing hormone (LH), both of which are secreted from the anterior pituitary. LH stimulates the production of androgens from the theca interna (an inner layer of cells in the follicle), and FSH stimulates the production of an enzyme that takes the androgens and converts them to estrogens. This increase in estrogen levels is very important for the next stage:

Estrous:

First, the vaginal membrane opens, and after a few hours the estrous period begins. Estrous lasts 9-11 hours and this is when the female is sexually receptive (or “in heat”). In guinea pigs and other mammals, a female needs to be in estrous in order to perform “lordosis,” which is when the female arches up her back so the male can mount from behind. Copulation cannot occur without lordosis, so the males can only successfully mate with the females during this short time window. The binding of estradiol to certain neurons in the brain controls lordosis (note: I’m not sure if female degus perform lordosis, but it’s likely since guinea pigs do).

Ovulation:

Right at the end of estrus, ovulation happens. Ovulation occurs when the estrogen levels reach a certain high point and activate a special control center in the hypothalamus. This control center causes a positive feedback cycle to start: the hypothalamus increases secretion of gonadotropin-releasing hormone (GnRH) which goes to the anterior pituitary and causes increased secretion of LH and FSH, which then go to the ovaries and cause the developing follicles to rupture and release the ova. The ova then begin their journey down the fallopian tubes where fertilization must take place (there’s about a 20 hour time window for fertilization to occur). The vaginal membrane may stay open for several more days, and then it will close until mid-pregnancy.

Immediately after ovulation, the ruptured follicles in the ovaries turn into corpus lutea. The corpus lutea produce high levels of estrogens and progesterone, which inhibit FSH and LH production (thus, preventing more follicles from developing). After about three days the corpus lutea start to degrade, and within six more days the corpus lutea are gone and the animal is ready to start the whole cycle again unless fertilization occurs and the ova successfully implant in the uterus lining, which brings us to:

Pregnancy:

The ova reach the uterus about three days after fertilization and take another three to four days to implant in the endometrium. The fertilized ova then begin to develop placenta, which produce and secrete a hormone called chorionic gonadotropin (CG). CG prevents the corpus lutea from degrading, so estrogens and progesterone levels stay elevated and no more follicles develop for the duration of the pregnancy. The estrogens and progesterone help stimulate development of the mammary glands for lactation, but lactation cannot begin until after the animals give birth because of the high levels of progesterone. (Question to think about: Birth control pills prevent ovulation from occurring. What hormone(s) do you predict are in birth control pills and how do these hormones prevent ovulation?)

Parturition and Lactation:

Degus have a gestation period of about 90 days (which can be longer or shorter, depending on the number of developing fetuses) and typically give birth during the night. During parturition (giving birth), each pup takes 10-30 minutes with 1-16 minutes between each pup. When parturition begins, the hormone “relaxin” helps the relax the cartilage holding the pubic bones together, and then oxytocin, released from the posterior pituitary gland, causes cervical distention and smooth muscle contraction in the uterine wall. Twelve to fifteen hours after parturition the mother has a post-partum estrous period of about 3 hours, which means she can get pregnant again right away. In humans, nursing and lactation are an effective means of inhibiting ovulation, but degus and some other small mammals can nurse their current young while carrying another litter. Milk production is stimulated by prolactin and glucocorticoids, while the effects of oxytocin cause milk letdown. During nursing, the stimulation of the nipple causes increased secretion of prolactin and oxytocin from the hypothalamus.

While I don’t have any photos of nursing degus, here are some pictures of degu pups:

The first degu pup capture of the year.

Getting ready to be released.

It will be hard to not take one home.

What I’m Doing When I’m Not Trapping Degus

My advisor suggested that I talk a little bit about the challenges of working at an international field site. While it would be easy for me to list off the difficulties I’ve faced and the obstacles I’ve overcome, I feel that I should also talk about some of the terrific experiences that I only could have gained by traveling to Chile. So in this post, I’ll be writing about both the ups and downs of fieldwork and living in Santiago.

Driving

In Chile (and pretty much the rest of the world minus the U.S.) everyone drives stick shift. Because my parents didn’t want me to burn out the clutch on their old Volvo, I never really learned how to drive manual. Not long before I left for Chile, I was informed that I would have to drive a truck with manual transmission… surprise! So, after two short lessons with some trusting colleagues, I flew to Chile and prayed that I could “wing it.” I mean, where better to learn how to drive stick than in a major, South American city? (To be fair, Santiago drivers are no crazier than Boston drivers.)

Luckily, I didn’t have to start driving right away, so I had some time to practice before driving out to the field myself. Our daily commute takes about 45 minutes (depending on traffic) and involves driving through the heart of Santiago, taking a highway for a few miles, and then navigating a very long, bumpy dirt road. I started practicing on the dirt road and then one sleepy, Sunday morning my patient co-mentor, Dr. Loren Hayes, had me drive around the city until I got comfortable with shifting through all the gears. As time went on my stalls became less frequent and my shifting started working on a subconscious level, and so, thanks to the degus, I now know how to drive manual.

The truck in front of our cabin at Parque Nacional Fray Jorge 

Wildlife

Working at the same field site for five months has given me the chance to see many cool plants and animals. Spending hours upon hours in the same place has allowed me to observe some things that I would have never seen otherwise. Here are some of my favorite wildlife memories:

-While observing degus one day I saw little sprays of dirt flying into the air nearby. After a couple dirt sprays a tiny, shiny black head would pop out of a hole like a Whack-a-Mole. The culprit was a cururo, a type of subterranean rodent that has glossy black fur, tiny little ears, and large yellow teeth. The coruros construct little dirt mounds (kind of like molehills), which threaten to break my ankle every time I have to walk down one particular hill.

-One time while on my way to the boulder field I suddenly saw an enormous hummingbird. It was hovering in front of a tall, red flower, and it made a loud “PEEP!” every time it went in to drink some nectar. After consulting my bird book, I found out that I had seen a “Giant Hummingbird” (appropriately named, in my opinion).

-A few weeks ago my friend Meredith (she’s an American studying for her PhD here in Chile) found a Southern lapwing nest. The four eggs were very well camouflaged, so much so that we sometimes had difficulty re-finding the nest! But after checking on the eggs everyday, we were lucky enough to witness the baby lapwings hatching!

They look much cuter after their feathers dry.

-And sometimes I see things that are slightly cool/slightly disturbing, like the time I saw a tarantula walk in front of me (I’ve been told that they’re pretty rare in Chile), or the time I saw an eagle take out a degu (you just have to tell yourself that it’s the Circle of Life).

Student Protests

Chilean students have been waging a long protest against the government. The students are calling for more educational funding, both for public secondary schools and universities. Chile previously boasted an exceptional public education system, but when General Augusto Pinochet came to power in 1973 (through a military coup which ousted socialist president Salvador Allende) the government immediately cut education funding and encouraged privatization of the education sector. Because of the low number of public schools today, many young Chileans only have the option of attending a private school, which has subsequently saddled these student with lots of debt. The main aim of the protestors is to make education affordable for the middle and lower classes.

While the protests in Chile haven’t really affected my fieldwork, I have still felt a lot of the effects of the protests themselves. Not long after moving into my apartment, the university across the street was taken over by the students. One day I woke up and found that the students had blockaded the gate entrances with tables and chairs and had put up banners on the buildings. The next two weeks were rather annoying because the students put on their own little mini-concerts/rallies while I was trying to sleep. Earlier this month, some students also blocked my street with burning trash, but the police were quick to arrive and dispersed the protestors with water cannons and tear gas. I’ve gotten a few whiffs of tear gas while walking around the city, and let’s just say that it’s not a pleasant experience. 

The students.

And the police.

In addition to school takeovers, strikes, and random street fires, students have also been organizing many protest marches. The government denied a permit for one march in mid-July, so when the students tried to march without a permit the police immediately swooped in and used water cannons and tear gas to break up the protest. The citizens of Santiago sympathized with the students and showed their solidarity by banging pots and pans out of their windows and honking their car horns for several hours. Banging pots and pans was a form of protest during the food-shortages of Allende’s presidency, but was used as a way to anonymously protest during the dictatorship of Pinochet.

Public Transportation

Because of funding availability, I only had access to a field vehicle from June through August. For September and October, I’ve been getting rides to the field with the technicians of our Chilean host, Dr. Luis Ebensperger at the Pontificia Catolica Universidad de Chile. This has ended up working well, but I needed a little more time in the field during early September to collect the rest of my late-pregnancy seasonal samples. So, with the help of my friend Meredith, I learned how to use the public transportation system to travel to my field site.

Getting to my field site involved a 15-minute walk to the subway, a subway ride of approximately 30 minutes (with only one transfer), a bus ride of 30 minutes, and then an hour-long walk through the field. It was pretty exhausting to do the trek both there and back, but most days I had a ride to the field in the morning. I actually enjoyed the commute because I could see lots of cool animals during my walk (which was also good exercise) and I could read on the bus and subway. One of the frustrating things about fieldwork is that there are always limits on time and resources, so it’s always nice when you have backup plans.

Here's a burrowing owl I spotted while walking to the bus.

 Life in the City

Overall, I like living in Santiago. I can buy fresh bread from the corner store everyday, I can walk up a picturesque hill in the middle of the city and watch the sunset over the Andes, and I can even go to the university and practice my Spanish with the ecology department security guard. Some of the downsides of living in Santiago, though, are the air pollution, the earthquakes, and the loud traffic.

When we first arrived to Chile in June we found ourselves in the midst of a soccer-crazed nation; the Copa America was about to start! Whenever Chile played a match the whole city would watch, and we would immediately know when Chile scored a goal because we’d hear people cheering and taxi’s honking their horns. After every game, win or lose, there’d be an impromptu march on the Alameda (the city’s major street), which would be promptly dispersed with police water cannons and tear gas. It was really cool to see a whole city cheer for their country; I can’t really compare it to anything in the U.S. (sadly, Chile lost in the quarter finals, Uruguay went on to win the cup).

I was also lucky enough to be in Santiago for Chile’s independence day, the Dieciocho de Septiembre (September 18^th). There are activities and events before, during, and after the Dieciocho that basically involve eating lots of BBQ and drinking chicha, a type of sweet, fermented wine. My Chilean friend Cecilia took me to a “fonda,” which is sort of like a U.S. fair but a little smaller. I looked at livestock animals, ate some BBQ, and tried some chicha. Rodeos are a big part of the Dieciocho celebration, but unlike a typical U.S. rodeo, Chilean rodeo only involves two cowboys on horseback trying to force a cow up against the arena wall. While they do knock the cow into a padded section of the wall, it does seem pretty hard on the cow so it was tough for me to sit through. I also watched the Chilean bomberos (firemen) do a little skit, but everything they did went wrong: they started a mini-fire so they could hose it down but the fire went out on its own, and then when a bombero holding a Chilean flag tried to zipline down from stage he got stuck. Nevertheless, I had a great time and I hope to be in Chile for another Dieciocho!

The cowboys are just about to knock the cow into the padded wall.

Field Stress Techniques

One of my projects this field season is to determine the seasonal variation of cortisol concentrations in wild degus during the breeding season. As I blogged earlier, I’ll be collecting four blood samples from each degu: baseline, stress-induced, dexamethasone (DEX) challenge, and adrenocorticotropic hormone (ACTH) challenge. Here’s a quick overview of the four samples:

The baseline sample is taken within 3 minutes of capture (remember, CORT starts to increase 3 minutes after a stressor) and represents the typical levels of CORT an animal experiences throughout the day. The stress-induced sample is taken 30 minutes after capture and tells me how much an animal increases its CORT levels after encountering a major stressor. After the stress-induced sample, I then inject the animal with a DEX (a synthetic version of CORT) and wait 90 minutes before taking another blood sample. DEX binds to CORT receptors, thus initiating negative feedback, so the DEX sample gives me a good idea of how well the animal can turn of its stress response. After collecting the DEX sample, I then inject the animal with ACTH, wait 15 minutes, and then take my final blood sample. ACTH stimulates CORT release from the adrenal glands, so this sample will tell me the maximum amount of CORT an animal can release.

Now let me tell you in a little more detail of how I collected these samples. When I first came down to Chile in June I was working with another graduate student and three undergraduates (well, they’re really post-grads because all of the them had graduated from college that May). After a week or two of practicing our bleeding and handling skills, we set out 120 tomahawk traps at our first site and got down to business. Here’s how a typical trapping day went:

After arriving at our field site, we would set up a degu-processing station near our trapping area. Then, the five of us would each grab a bag of oats, spread out and start opening and baiting the traps. Once the traps were all open and baited, we would station ourselves around the trapping perimeter and begin monitoring the traps through our binoculars. Observing the degus could be boring and tedious- oftentimes we would go hours without catching a degu. The degus also liked to taunt us in various ways, usually by studiously avoiding the areas with traps. Some degus liked to enter the traps partway, gently rest their paw on the treadle, and then quickly run out of the trap. And occasionally, a few particular degus would sit by the traps for long periods of time, just staring back at us.

Degu processing station

Nevertheless, we did catch quite a few degus. When one of us saw a degu set off a trap, we would yell, “DEGU!” and then the closest graduate student (designated bleeder) and post-grad (designated handler) would run towards the trapped degu. The handler (using a gardening glove because the degus will bite) would get the degu out of the trap and hold it for the bleeder. The bleeder would use an electric razor to shave the degu’s leg and would then prick the saphenous vein (one of the main leg veins) with a needle. We first collected blood in a glass capillary tube for future cortisol analysis, and then we would switch over to an Eppendorf tube to collect blood for leptin and ghrelin analysis (these are two important hormones for energy regulation). After we collected the blood, the handler would hold a piece of cotton or gauze to the degu’s leg if it was still bleeding and then the degu would be taken to the processing center while the three other people continued to watch the traps.

Once at the processing center, the first thing we would do was ear-tag the degu so we could properly identify it (this is important because if the animal escaped during processing, then we could complete our measurements if we ever re-caught the animal). After ear-tagging the animal, we would then weigh the degu so we could figure out how much DEX and ACTH to inject. Then we would take a variety of measurements including ectoparasite levels (I covered this procedure in my last post), reproductive condition, glucose levels (we use the same monitor a diabetic might use), and anogenital distance (this is the distance from the top of the anus to the base of the penis or vaginal opening). By the time we were done taking all of these measurements, it was usually time to take the stress-induced blood sample and inject the degus with DEX. Then, we would stick a paper towel under the degu’s trap to collect any feces, cover the degu with a white sheet to keep off the sun, and head back to the trapping area to continue watching the traps.

After catching a few degus, the day would become a game of coordination between watching degus and taking further blood samples on the degus at the processing station. Because we had five people, we were usually able to keep the traps open the whole day. On the occasions when we caught multiple degus within a short period of time, we would have to close some traps for a while because there was no way we could watch all of the traps while processing the degus. This is one of the challenges of fieldwork; you often have long chunks of time where you’re essentially doing nothing, interrupted by brief periods of intense activity.

Fieldwork could sometimes be very frustrating, like when a degu would get caught in a trap and we didn’t notice. This mostly happened at the beginning of the field season- we eventually got better at preventing unnoticed captures by checking that all the traps were visible to at least one person before opening them, and also by tying a piece of orange flagging tape to the door of trap so we could easily tell from a distance whether the trap was open or closed. We also got better at watching the degus and recognizing the sound of a trap being triggered.

Other frustrations included when a degu would go into the trap, step on the treadle, but the trap would fail to shut. Some of the traps are better than others, and we oftentimes had to fiddle with the older traps to make then more sensitive. Sometimes we’d catch a degu but it’d escape from the trap while we were trying to get it out; it’s something that happens to everyone, no matter how long you’ve been working with the degus. And finally, the most frustrating thing about trapping degus is watching the birds eat all of the bait and set off the traps. Sometimes the birds would be so bad that we’d have to re-bait the traps every hour or so. Over the past few months, we’ve caught (in order of capture frequency) rufous-collared sparrows, common diuca-finches, long-tailed meadowlarks, band-tailed sierra-finches, Chilean mockingbirds, mustached turcas, mourning sierra-finches, white-throated tapaculos, eared doves, and shiny cowbirds. Here are some pictures of a few of these birds:

Rufous-collared sparrow 

My advisor, Dr. Michael Romero, holding a long-tailed meadowlark

Mustached turca

Common diuca-finch

White-throated tapaculo

Mourning sierra-finch

Chilean mockingbird

But even with the difficulties of trap-shy degus, unnoticed trappings, escaped animals, and hungry birds, we managed to get our first seasonal samples within two weeks (full stress series on 8 males and 14 females). Because we had a lot of time before gathering our next seasonal samples and preparing for my other project (examining the effects of poor maternal care on the pup stress response), we decided to do a side project to determine whether the stress response differs by habitat. We moved all of our traps up to a nearby field that had lots of boulders and began trapping up there. The boulder field, unexpectedly, was degu heaven, and we collected full stress series from 12 males and 8 females within just three days! Emboldened by our success, we decided to try trapping in an area with lots of trees and vegetation. This site proved to be more difficult, and it took a week to collect full stress series from 10 females and 5 males, after which we had to remove all of traps because it was time to start trapping at nearby national park. Which brings me to….

Parque Nacional Fray Jorge!

Fray Jorge is approximately 350km northwest of Santiago and is an interesting place because while the majority of the park is dry, dusty and covered with cactuses, there are also fragments of rain forest on top of some of the hills. The incoming fog from the ocean sustains these cloud forest fragments, which are more typically found in the southern part of Chile. Alas, we trapped the degus in the drier, scrubbier areas, here’s a good representative picture of our trapping habitat: 

Fray Jorge was a difficult place to trap because the degus rarely ventured out into the open, so we had to place most of the traps in and around the bushes and trap by ear instead of by sight. This usually worked fine, but on really windy days it was hard to hear the traps closing, so we ended up missing a few captures. The cactuses were also a real pain; by the end of the day we’d have tons of cactus spines embedded in the soles of our boots. But we persevered, and by the end of our time at Fray Jorge (about a week and a half) we managed to collect full stress series on 5 males and 13 females.

After we returned from Fray Jorge, we started prepping for my project examining the effects of poor maternal care on the pup stress response. We did some widespread trapping and radio collaring to help determine social groups (I went over these techniques in my last post), and then we started implanting the females with cortisol or placebo pellets after they gave birth.

I will start trapping and bleeding pups in a few days to determine if communal care helps buffer degu pups from negligent parenting. If this hypothesis is supported, then I expect to see high baseline and stress-induced CORT, plus poor negative feedback, from the pups belonging to social groups with cortisol-implanted mothers. You can read my first blog post for a fuller explanation of my experiment.

Buena suerte!

Ecological Field Techniques

As you can tell from my previous posts, my primary interest in Chile is to collect a lot of blood samples so I can determine cortisol concentrations. But my time down here isn’t just spent bleeding degus; in order to get some of my desired blood samples, I have to employ several ecological field techniques. And in addition to collecting these blood samples, I am also gathering other types of data to complement my primary research. Let me describe some of the techniques that I have been using this field season:

Widespread trapping:

For my primary project investigating the effects of poor maternal care on the pup stress response, I have to find and determine several social groups. To do this, my collaborators and I set out 300 live Tomahawk traps over a large area during early August. We tried to place the traps in areas where we found active burrow systems; a good way to determine whether a burrow system is active is to look for fresh feces and dust-bathing sites. If a fecal pellet is fresh, then it should be soft and green when you break it open. Dust-bathing sites are near burrow openings and are little sandy areas that are free of rocks and vegetation. Watching degus dust-bathe is rather cute; they quickly flip onto their side, roll in the dust, and jump back into an upright position all within a second.

An open Tomahawk trap

Anyway, after setting out our traps, we trapped for several hours a day for about 3 weeks. We would typically open and bait the traps in the morning (we use rolled oats for bait, the degus absolutely love it) and then check and re-bait the traps every hour or so. If we caught a degu, we would take it back to the truck, put an ear tag on each ear, weigh the animal, and then check their reproductive condition if they were a female. We would then return the degus to their respective burrows at the end of our trapping period. We weren’t getting many degus at first, so we spent a few days perched on the hillside, watching degus through our binoculars so we could figure out how to better place our traps.

Ear-tagged degu

Male degu

Female degu

Getting weighed

Nighttime Radio-telemetry:

To determine which degus belong to which social groups, it is necessary to supplement trapping numbers with nighttime radio-telemetry data. Figuring out social groups is tricky because degus use multiple burrows, and some degus move around more than others. The accepted rule for determining social group membership is to establish whether a group of animals spend 80% or more of their nights in the same burrow together. So, in order to figure out my social groups, I had to radio collar and track my females for two weeks. Earlier this month I re-trapped my degus and removed the collars so it wouldn’t be an extra burden during their last few weeks of pregnancy. After the degus give birth (about two-thirds of my degus have already given birth) we then re-collar them and do more tracking because social group membership can frequently change composition.

In order to radio-collar an animal, the first thing I had to do was to make sure that the collar was still functioning properly. After checking that the collar had a strong, clear signal, I then took a piece of special wire and flamed some heat-shrink tubing onto it. I then threaded the wire through the transmitter, threaded one end of the wire through a piece of hard, plastic tubing that protects the transmitter’s antenna, and then pulled the two wire ends through a crimp. With one person holding the degu, I then flipped the collar onto the degu’s neck and used two pairs of pliers to tighten the collar. The degus necks are always smaller than they appear because of their thick fur, so I had to rotate the collar to get the fur out of the way and continually test whether I could push the collar over the degu’s head. It’s important to make sure the collar’s tight enough so the degu can’t get it’s front paws stuck in it, but it’s also important to make sure that the collar isn’t too tight because it can irritate the degu’s neck and cause an infection. Once the collar was the right tightness, I then crimped it, cut the wire ends, and returned the degu to its burrow. Every subsequent time that I caught the degu I checked to make sure that the collar wasn’t irritating the skin.

Radiocollar

And a radiocollared degu

Actually tracking the radio-collared degus wasn’t too difficult. By using a receiver attached to a tracking antenna, I would type in the frequency of the degu’s collar (each radio collar has its own, unique frequency) and then wander around listening to the loudness of the beeping. The receiver and collars are usually sensitive enough to pinpoint the degu’s location within a meter or so, but large obstacles like rocks and trees can sometimes bounce the signal.

Modeling the radiotelemetry equipment

Vegetation Sampling:

Some of the degus that I’m working with are part of my Chilean collaborator’s long-term study, and one of the measurements that he has always taken is relative food abundance around the different burrow systems. To do this, we take vegetation samples from two areas per burrow system. To determine which areas to measure, we choose a direction at random (N, S, E, or W) and then mark a spot 3 and 9 meters from the center of the burrow system. We then lay down a 20cmx20cm grid and harvest all of the plants within it. This sounds pretty easy, but we also have to take off the roots from the plants and then separate them into monocots and dicots. The samples are then weighed after being dried in a drying oven for 72 hours.

Hematocrit, Soil Hardness, and Fecal Water Content:

One of the other students (a recent Tufts graduate) is doing a side-project to examine the correlation between hematocrit and cortisol levels. Hematocrit is the proportion of red blood cells in the blood and has been linked to hydration, stress, altitude, etc. Measuring hematocrit is fairly easy; when I bring a blood sample back to the lab I have to spin it in the centrifuge to separate the plasma from the red blood cells (I collect the plasma because that’s where the hormones are). The blood samples are in little, glass tubes and after spinning them each tube separates into a bottom layer of red blood cells and a top layer of clear, yellow-ish plasma. I then use some calipers to measure the lengths of the two layers and, presto! There’s the hematocrit measurement.

Because hematocrit is linked to hydration, we’re also measuring rainfall (as determined from local weather stations), soil hardness, and fecal water content. Soil hardness is quick and simple; we just take a penetrometer (it looks like a long, metal tube with a spike sticking out the bottom), place it on our soil site, and push it all the way to the ground. If the spike digs in far, then the soil is soft and we’ll have a low reading. If the spike doesn’t sink into the soil much, then the soil is hard and we’ll have a high reading. To measure fecal water content, we first collect the feces from the degus (you just put a paper towel under the trap and let the degus work their magic, it doesn’t take very long) and then weigh the feces, dry them in a drying oven for 8 hours, and then weigh them again.

Collecting feces

Ectoparasites:

Degus, like many rodents, are often infested with fleas and other ectoparasites. Ectoparasite level is a good indicator of general health and may even be correlated to immune function. To measure ectoparasite levels, we have one person hold a degu over a yellow bucket while another person brushes the degu with a flea comb. We first comb the degu’s back in the direction of the fur 15 times and against the fur 5 times. Then we comb each side with the fur 5 times and against the fur 5 times, and finally we comb the head against the fur 5 times. Before we comb the degu we spray some alcohol into the bottom of the bucket to instantly kill the parasites, which we then collect and store in alcohol.

            

Seasonality of the stress response

The natural world is dynamic, ever-changing environment. As the seasons progress, organisms experience fairly predictable changes in temperature, precipitation, and photoperiod (length of daylight). Just as the environment is not a static world, neither is an animal’s ability to cope with environmental challenges.

Research has shown that many animals display seasonal patterns in both baseline and stress-induced glucocorticoid concentrations. For the white-crowned sparrow (WCS), the highest CORT (in this case, corticosterone) levels occur during the breeding season while the lowest CORT levels are seen during molt. Previous research on degus has shown that the highest concentrations of CORT (cortisol) occur during lactation.

There are many different hypotheses for why we see these seasonal differences in the stress response, but here are the main three:

•  Energy mobilization hypothesis: Because CORT’s metabolic effects help increase the amount of available energy, this hypothesis predicts that high levels of CORT will occur during energetically costly periods. This is the case for both WCS and degus, as breeding (for WCS) and lactation (for degus) are very energetically costly life history stages.

• The behavior hypothesis: CORT also has many functions on behavior- this hypothesis states that animals modulate their CORT levels depending on the type of behavior that the season requires. This is probably best explained by an example: If a bad storm comes through when a WCS has just laid eggs (this means it’s the beginning of the breeding season), then it makes sense for the WCS to abandon its nest and flee to safety since their reproductive effort is, at the time, rather low, and the WCS can start another clutch later. But, if a bad storm occurs when a WCS has several-day old chicks (this would be during the late breeding season), then it is probably in the best interest of the WCS to try to ride out the storm since their reproductive investment is rather high. According to this hypothesis, we would expect to see high levels of CORT during the early breeding season (since high levels of CORT are correlated with movement) and low levels of CORT during the late breeding season. As it turns out, this is exactly what we see in wild WCS. But, what is unexplained is why levels during the late breeding season are still higher than any of the non-breeding life history stages.

• The preparative hypothesis: In my previous blog post I mentioned that CORT helps prime the fight-or-flight response, another essential component of the stress response. In addition to priming the fight-or-flight response, elevated baseline levels of CORT also have permissive functions on the immune, metabolic, and reproductive systems in order to help an animal get ready for a stressful period.  The preparative hypothesis predicts that animals increase baseline levels of CORT during life history stages when there’s a predictably greater risk of encountering severe stressors. This hypothesis hasn’t been as well studied as the other two because determining the risk of experiencing adverse conditions is difficult to define and measure.

While the seasonality of the stress response has been relatively well studied in birds, there are fewer field studies examining the seasonal stress hormone profiles of mammals. Of these mammalian studies, few actually measure baseline glucocorticoid levels. Measuring baseline CORT is difficult because CORT starts to increase after the onset of a stressor, so most animals must be bled within 3 minutes of capture (the 3-minute rule is a pretty universal rule in field endocrinology). Trapping mammals is difficult because you often need many, many traps spread across a large area, so most researchers collect blood samples after trapping for a period of a few hours.

One of the aims of my research is to determine the seasonal stress profile of the degu. I am hoping to improve upon a previous study that measured CORT after a trapping period of 2 hours. Here are the four blood samples that I take from each animal:

Baseline: This sample is taken within 3 minutes of capture. This sample is the most important of the four because it allows me to determine what kind of CORT levels an animal is typically experiencing on a day-to-day basis.

Stress-induced: I take this blood sample 30 minutes after an animal is trapped. Capture is a major stressor (it’s basically a simulated predation event from the animal’s point-of-view), so this sample will help me determine how high an animal’s CORT levels can get after encountering a significant stressor.

DEX: After I take the stress-induced sample, I then give the animal an injection of dexamethasone (DEX). DEX is a synthetic glucocorticoid and binds to CORT receptors, thus initiating negative feedback. After waiting for 90 minutes, I then take another blood sample, which will tell me how well the animal can turn of its stress response. The ability to turn off the stress response is really important because high, continuous levels of circulating glucocorticoids can start to cause problems (this is termed chronic stress, and some of the pathologies include reproductive suppression, immunosuppression, and muscle-wasting). In fact, one of the hallmarks of a chronically stressed animal is poor negative feedback.

ACTH: After taking the DEX blood sample, I then inject that animal with adrenocorticotropic hormone (ACTH). ACTH is released from the anterior pituitary, travels through the bloodstream, and binds to receptors in the adrenal glands to cause an increase in CORT production and release. 15 minutes after injection, I take another blood sample. This sample will tell me the maximum amount of CORT an animal can produce. It will be interesting to compare this sample to the stress-induced sample to see whether animals are reaching their full adrenal output 30 minutes after capture.

The blood samples that I’m collecting are not the full story of the stress profile, though. Changes in levels of the carrier protein, corticosterone binding globulin (CBG), could affect the availability of biologically active CORT. The role of CBG is unclear, though; many biologists claim that because CBG-bound CORT is unable to bind to receptors, then high levels of CBG decrease the amount of “free CORT.” On the other hand, CORT is a steroid hormone (it’s lipid soluble, not water soluble), so in order to circulate through the bloodstream, it may be necessary to have a protein carrier in order to reach all of the target tissues.

Density and distribution of CORT receptors may also play a large role in the seasonal regulation of the vertebrate stress response. CORT can only exert its physiological effects by binding to receptors, so if changes in CORT receptors mirror that of changes in CORT concentrations, then an animal’s stress response may not be changing over the seasons. Measuring CORT receptors takes a lot of time because the receptor protocol needs to be optimized for each tissue type. I won’t be attempting this, but my lab-mate is currently doing some really cool, cutting-edge research on the seasonal regulation of CORT receptors in the house sparrow.

So far, I have collected blood samples from male and female degus during the breeding/early pregnancy and late pregnancy time points. I probably will not have time to take full stress series on the lactating females this year, but I hope to return to Chile next September to get these samples. 

Field site in winter (June)

Field site in spring (September)

I think you can guess which degu is the pregnant female and which degu is the male.

What is stress and how does it help us?

Let’s talk about stress.

When we think of stress, we typically think of events that challenge our comfort or happiness- things like big exams, arguments with a significant other, or financial problems. When stuff like this happens to us, we often tell people that we’re “stressed out.” But where did the word “stress” come from? Prior to the early 1900s, “stress” was a term that belonged to the lingo of engineers and physicists (think of bridges and buildings). Our current connotation of stress can be attributed to an Austro-Hungarian endocrinologist named Hans Selye.  Selye was injecting rats with various organ extracts when he noticed that regardless of what type of organ extract he used, all of his rats developed ulcers, atrophied thymus glands, and enlarged adrenal glands. It turns out that Selye was a very clumsy scientist and often dropped his rats and had to chase them around the room while he was trying to inject them. Selye realized that the constant handling, chasing, and actual injections made the rats…well… stressed. And so that’s how “stress” came to be.

But what is stress? A good, basic definition is that stress is the physiological and behavioral response to unpredictable or noxious stimuli (meaning, stress is what helps you run away from a charging llama). There are two kinds of stress: acute stress and chronic stress. Acute stress may be running away from a territorial llama, but then getting to safety and revising your walking route so you never have to pass by it again. Chronic stress may be when you have to pass by an aggressive llama two times a day, everyday for a month, in order to get to your field site and there’s no alternative route because of an electric fence. This kind of stress is more long term, and the stress of dealing with the llama bypasses the actual encounter because you spend a good part of your day just thinking and worrying about the llama (note: this is just an example, there are no aggressive large animals at my field site). I’ll talk more about chronic stress in future posts, but for now, let me discuss how the stress response works:

There are two waves to the stress response. The first wave, termed the fight-or-flight response, happens within seconds of perceiving a stressor and helps the animal take immediate action to avoid or deal with the stressor. The second wave of the stress response is slower and is mediated by glucocorticoids. In my research, I mostly focus on the physiological responses caused by the glucocorticoids, but the fight-or-flight response is important and is frequently glossed over, so let me tell you a little more about it.

OK, so say a llama charges you, do you flee or do you fight? This instantaneous response is mediated by a group of hormones called catecholamines. The two main catecholamines responsible for the fight-or-flight response are norepinephrine and epinephrine (also called noradrenaline and adrenaline). When your brain perceives something as dangerous, it activates your sympathetic nervous system (SNS). The SNS activates preganglionic sympathetic nerves that innervate the adrenal medulla (the adrenal medulla is the inner part of the adrenal gland, you have two adrenal glands that sit on top of each of your kidneys). These nerves form synapses with cells that produce norepinephrine and epinephrine (these are called chromaffin cells, each individual cell can produce only norepinephrine or epinephrine, never both).  Activated preganglionic sympathetic nerves release acetylcholine into the synapse, which causes chromaffin cells to increase their membrane conductance for Ca²⁺, which then causes intracellular Ca²⁺ to rise, which then results in exocytosis of norepinephrine and epinephrine into the bloodstream. Norepinephrine and epinephrine then circulate through the bloodstream and bind to adrenergic receptors in many different tissues (question to think about: if catecholamines are protein hormones, where do you think the adrenergic receptors are located in the target cells?). Catecholamines cause a variety of physiological effects including vasoconstriction, increase in heart rate, bronchodilation, inhibition of insulin secretion, stimulation of glycolysis in muscle cells, and increase of lipolysis in fat cells (question to think about: If a person with bee allergies gets stung, how does their EpiPen help them survive?). The net result of the fight-or-flight response is to help an animal activate their muscles (and other tissues) and to mobilize as much energy as possible so they can confront or flee their stressor.

So while your fight-or-flight response is kicking into high gear to help you flee the charging llama (let’s be real here, most of us would probably run away from a charging llama rather than try to fight it), the second wave of the stress response is getting ready to help you deal with some of the more long-term effects of the stressor. The second-wave of the stress response starts when your brain perceives a stressor and then causes increased secretion of corticotropin-releasing hormone (CRH) from the hypothalamus. CRH travels down to the anterior pituitary where it causes increased release of adrenocorticotropic hormone (ACTH). ACTH then travels through the bloodstream to the adrenal glands where it stimulates increased secretion of glucocorticoids (in humans and degus, the main glucocorticoid is cortisol, in most other animals the main glucocorticoid is corticosterone. From now on I’ll refer to cortisol and corticosterone as CORT). CORT then circulates through the bloodstream and binds to receptors in a variety of different tissues. Because CORT is a steroid hormone, most of its receptors are intracellular (although there is evidence that CORT also has some extracellular receptors). Since the majority of CORT’s actions are mediated through intracellular binding, most of the physiological actions of CORT take a long time to take effect (we’re talking at least 30 minutes, here, but many physiological changes are only seen after several hours). Increased concentration of CORT causes an amazing variety of physiological effects to take place:

•  CORT has a permissive function (this means that they help other physiological mediators take full effect) on the action of catecholamines in the cardiovascular system. This means that CORT helps prime your flight-or-fight response for the next stressor that comes around.
• During hemorrhage, CORT has a suppressive function on secretion of arginine vasopressin (AVP) and renin. Without CORT, your body would produce way too much AVP and renin in response to hemorrhage and you would probably die from too much vasoconstriction in the liver and heart.
• The effects of CORT on the immune systems are very complicated. But, to generalize, CORT has mostly suppressive effects on the immune system and helps prevent over-activation of immune function (question to think about: what do you use cortisone cream for?).
• CORT helps mobilize energy stores for quick consumption by inducing lipolysis, increasing gluconeogenesis (synthesizing glucose from amino acids and fats), making cells more resistant to insulin, and stimulating appetite.
• High CORT concentrations can suppress the reproductive system by inhibiting release of gonadotropin-releasing hormone (GnRH) and decreasing the effects of luteinizing hormone (LH) on the gonads.

To put it all together, the second wave of the stress response helps animals recover from stressors and get prepared for new stressors. In terms of our llama escape, we can think about it this way:

After we get away from the llama, our high levels of CORT help us recover from the stressor by helping us not overdo our hemorrhaging (because there’s a chance that we may have been wounded in the stressful encounter) and stimulating our appetite so we can replenish our energy stores. Our increased CORT levels are also helping us prepare for the next llama interaction (since the llama may be searching for us as we recover) by priming our fight-or-flight response, suppressing our inflammatory response so our limbs remain movable, increasing the amount of available glucose, and getting our mind off of reproduction. Basically, the body is diverting energy from unnecessary functions to preparatory functions- after the stressor is gone your body can start to worry about storing glucose, fixing a wound, and having sex.

This was my attempt to give you a basic overview of the purpose of the stress response. Having a basic understanding of the stress response is important for understanding the aims of my research, so continue to bear with me and I’ll eventually tell you more about the degus. If you’re interested in learning more about stress, here are some suggested readings:

-For an unintimidating, highly entertaining introduction to stress and its effects on human health, you must read: “Why Zebras Don’t Get Ulcers” by Robert M. Sapolsky.

-If you’re still wondering “What is stress and how do we define it?” check out this review paper: Romero, L.M., Dickens, M.J., Cyr, N.E. (2009) The reactive scope model- a new model integrating homeostatsis, allostasis, and stress. Hormones and Behavior 55, 375-389.

-For a more in-depth overview of the fight-or-flight response, try reading pgs. 333-339 in “Animal Physiology: Mechanisms and Adaptations” by Randall, Burggren, and French.

-Want to know more about glucocorticoids? This comprehensive review has it all: Sapolsky, R.M., Romero, L.M., Munck, A.U. (2000) How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocrine Reviews 21, 55-89.

And here is a picture of a one-day old degu:

Baby degu!

Chau!