Hustling for funds to get back to Panama

The frog extinctions were starting to generate professional and public interest. Matt published results from his Panama studies done on the trips before ours in a very high profile, non-technical, ecological journal (the publicity magazine for the Ecological Society of America) and other more specialized papers from the group were coming out in the peer reviewed literature. Meanwhile Karen and Matt were receiving numerous invitations to speak at universities across the country where they would tell the story of the disappearing frogs of Panama and how the effects of these extinctions were reverberating through the ecosystems.

The research group also continued to make presentations at scientific meetings. Such meetings are great places to share scientific ideas and results with hundreds of others who are working on related issues. They provide the one time where scientific specialists are not the oddballs; nerd talk is the norm.

Matt and I attend a meeting of stream scientists every year and room together. Rooming with me may not be the most pleasant experience, but it is one way we could both use our grant money more efficiently. The day starts at 8:00 with scientific presentations till 5:00. Dinner is followed by a boisterous mixer in the meeting hall with hundreds of people discussing and arguing science. As with any large group of talking people, the volume increases to a roar. This makes for a long day, and after this Matt and I would lie in our beds and talk about science, our colleagues, and the state of the world.

Often our conversations would turn to the frogs in Panama. We kept trying to think of ways to strengthen the research. As in all science, every experiment leads to more experiments. In addition, the impending extinction of the frogs was something to worry about, even if we could do nothing to stop it. One thing we learned early on from rooming together at meetings, earplugs are essential.

One of the gaps in our research after our trip to Panama, and an issue Matt and the rest of us worried about for the next proposal, was measuring how much nitrogen is excreted by tadpoles. The tadpoles could be what we refer to as “ecosystem engineers”. Ecosystem engineers are organisms that have a disproportionately large effect on the environment and this effect cascades to the other species. Beavers that fill valleys with their dams, hippopotami grazing in fields and bringing nutrients they excrete back to the water, alligators digging water holes in the Everglades that last through the dry season, and bison eating dead grass and recycling the nutrients locked up in the grass available for the new growth of grass, are all examples of ecosystem engineers.

The tadpoles at El Valle were abundant and active enough that they could be major nutrient contributors to their streams. We were concerned with what happens when these potential ecosystem engineers in streams are lost. Some species of the tadpoles break down the organic material that falls into the stream in the form of leaves, and others clean the algae off the rocks. An interesting feedback is that the leaves become better to eat and the algae grow better when supplied with nitrogen. The tadpoles excrete nitrogen, and it actually stimulates production of their food. This excretion may also stimulate the microbes that serve as the food source for many of the tadpoles, insect larvae, shrimps, and fish in the stream. There were so many tadpoles in the streams that their effect had to be large, but how large was what we wanted to know.

Nitrogen excretion measurements were made while we were in the field with live tadpoles collected directly from the stream. The animals were placed in test tubes and then removed and released back into the stream. The amount of ammonia (the form of nitrogen excreted by the tadpoles) left after the tadpoles were removed was used to estimate the rate of their excretion. We did quite a few of these experiments streamside on the first trip with the samples returned to our rooms in Hotel Campestre for late night analyses. Our initial experiments indicated the rates of excretion were very high over the first few minutes and then decreased over time.

Bob argued that the tadpoles quit eating when they were placed in the experimental tubes and their excretion rates were slowing after a few minutes because of that. Alex and I thought that maybe stress was causing them to excrete more at the beginning of the experiment. Matt, being the diplomatic group leader, and a fine scientist to boot, suggested experiments were needed to settle the argument. This is the scientific processes. Our speculations formed hypotheses and now we needed the experiments to test them.

These discussions led Matt and Alex to run a series of experiments on tadpoles from the Midwestern US that are taxonomically-related to the tadpoles dominant at Rio Maria. These experiments proved that the higher excretion rates at the beginning were from the stress of being handled and put into a tube. Excretion rates from later in the experiment were more applicable to what the tadpoles were doing in the stream. Matt and Alex proved that handling tadpoles and putting them in large test tubes literally scared the piss out of them. These results were written up and submitted for publication in the scientific literature. They also strengthened the next proposal and would guide our experiments if we got funded to go back to Panama.

Another five months went by and the proposal was rejected again. This was extremely disheartening because once again, the reviews were fabulous. Matt called the program officers at the National Science Foundation to try to figure out how to get the proposal funded. The funding situation was getting dire because Piet’s and several graduate students’ salary depended upon continued funding.

We had agreed to fund the follow-up experiments in Panama out of our own pockets if need be, but we could not expect post-doctoral researchers or graduate students already living on poverty-level salaries to do the same. Luckily the program directors at the National Science Foundation agreed to some stop-gap funding to keep the project going and allow Matt time to apply for funding in the next round. One of the investigators on the grant actually paid Piet’s salary out of her personal funds for awhile. Scientists generally don’t enter the field of ecology to get rich, and many are quite generous with their time and resources, but this was exceptional. There is a long tradition of using personal funds for research; Charles Darwin had to pay his own way as naturalist on the Beagle.

Scientists through the years have had to scrape for external funding and we were no exception. Matt and the rest of us had to strategize on how to make the proposal sexy, compelling, and stress how imperative it was to fund the research immediately. We needed to write the proposal so well that the agency simply could not justify declining to fund it. Matt worked with the group on the best way to sell the research, and the proposal was submitted yet again.

The third time was a charm. I received an email from Matt telling me to plan to travel to Panama the following February. This was very good news and just in the nick of time. Matt had scouted out one site not far from the other side of Panama City where there were still frogs and additional experiments could be conducted as part of this new grant. If the disease spread much further into Panama the work would be finished for good. The remote Darien rainforest between Panama City and Columbia presumably contains the last areas the disease has not reached, but the eastern part of it is inhabited by drug runners and rebels and is not a safe place to work without an entire protective army. Going to the Darien was out of the question; we had a hard enough time getting funding, but getting support for a private protective army was out of the realm of possibility.

The disease, and getting more resources to study it

While all the causes described above are factors in global declines of amphibians, the chytrid fungal disease is primary cause of the current cascade of extinctions in higher altitude regions throughout Central America. Every place that still has a diverse amphibian diversity associated with intact jungle is losing species of frogs.

Dr. Karen Lips has been the main scientist who has described this loss in Central America. By 2007, she had worked west of El Valle, at El Cope, Fortuna, and in Costa Rica for over a decade. She studied frogs intensively at these sites, and collected and identified as many species she could.
Karen was working on her dissertation in Costa Rica when the frogs suddenly disappeared. She was at Fortuna in Panama and the frogs vanished there. There was no obvious cause of mortality. The Fortuna site is old growth jungle, minimally impacted by humans.

Perhaps the most intense experience Dr. Lips had was in the forest at El Cope in 2004 when the disease front swept through the site. In a period of a couple of weeks most of the adult frogs died. Dead frogs were literally falling from the trees. She found species that she had never described before dying on the jungle floor. This was the first time she had seen these species because they spent all or much of their lives up in the trees where they were extremely difficult to collect.

The dying frogs all had the telltale sign of chytrid infections, sloughing skin. It is difficult to imagine how Karen felt in this situation. This was an area she had been studying for years, and she was watching the animals she had dedicated her career to die in front of her. Many people would have simply given up at this point. Karen kept going. She took samples and sent them out to experts to confirm that the chytrid was indeed present and causing the infection.

As time went on, Dr. Lips mapped the spread of the infection. It killed frogs in the high altitude rain forests in a wave of infection spreading generally from north to south in Central America (but from west to east in Panama because of the orientation of the country). The disease apparently can infect, but not kill lowland species. It is not lethal but can spread through these populations in warmer areas. When the disease is passed to the next mountain range where the temperatures are lower, the disease becomes fatal and extinctions begin again as it sweeps through populations of susceptible frogs.

There is no known cure for the fungal disease in the wild, but infected individuals can be treated with chemicals in the lab if they are not too far gone. Presently, the only way to save susceptible species from extinction is to collect them and grow them in aquaria where they are not exposed to the disease. Tadpoles have been re-released into the wild a few years after the disease has killed all the adult frogs. They die from the disease when they metamorphose and emerge as adults. Apparently, once an area is infected by the chytrid, the deadly fungus remains even if the frogs that it infects are gone.

Karen was a colleague of Matt’s at Southern Illinois University and she got Matt interested in the ecosystem consequences of the loss of these species. She knew the Central American frogs as well as anybody, but did not have the expertise in stream ecology to assess the effects on the other plants, animals and microbes in the streams.

Matt had the expertise in stream ecology, and his lifetime interest in herpetology (he has kept poisonous snakes since college) drew him into the project. Matt takes vacations to the deserts of Arizona and New Mexico to see snakes and lizards; his idea of a good time is turning over rocks to see what is under them. Even after he got bit (tagged) by one of his “pet” rattlesnakes he still kept them as pets. It was not until he had children that he thinned back his considerable collection of venomous snakes.

In one of their discussions, Karen told Matt how the rocks at Fortuna had gotten slippery and greener after the disease hit. At that point a light bulb went on and Matt realized that the loss of the extremely high density of tadpoles that occurred naturally in the streams could have important ecosystem consequences. Karen got Matt to go down to Panama and see her sites, and Matt cooked up the idea of assessing the ecosystem consequences of frog extinctions.

All researchers involved with this phenomenon realize the necessity of rapid work on the frog populations. Researching the Panamanian frog extinctions is essential because there will not be a second chance. Once the tadpoles are gone from a stream, they will not be back. This was a problem Matt could not ignore. Practically, this meant obtaining funding to work on figuring out the consequences of the disease that was killing the frogs. Now, the first round of that funding was almost gone, and more was needed to keep the research going.

Matt’s proposal to continue the work previously funded by the National Science Foundation received excellent scores, but it was rejected. This was not completely unexpected, but was still very disappointing. Less than five percent of the proposals in the competition were funded. Only one out of every twenty proposals written by the top academic researchers in the world was selected for funding by this program. The National Science Foundation funds the vast majority of the basic ecological research done in the United States.

The rejection put Matt in a difficult position. He needed to re-submit the proposal immediately to meet the next deadline (proposals were only evaluated every 6 months) and it was difficult to decide how to revise the proposal because the reviews were so positive. Nonetheless, he strengthened and re-wrote the proposal in only a few weeks. After thorough review by all the official project investigators (Karen Lips, Cathy Pringle, Susan Kilham) and associated participants (Bob, Alex, and me), it was sent off. The clock was ticking, and longer delays would make research impossible.

Science is not only about writing good proposals, but also about self promotion. When funding is so competitive, only the researchers that are able to publish their work in the top science journals in the world are able to continue getting funded.

Peer reviewed publication is the ultimate yardstick of scientific success. The system is set up to increase the probability that only the most robust and exciting science gets published in the journals. A scientist or group of scientists writes a paper, and then submits it to a journal. The editor at that journal reads it and decides if it is worthy for review. If it is, the editor obtains 2-4 reviewers to look at the paper. Each reviewer spends hours looking at the paper and searching for the tiniest flaw. They are also asked to rank the importance of the work (more subjective than answering the question was the work done soundly).

The editor reads the reviews of the papers and decides if it should be rejected, accepted, or to ask the authors for a revision that will be reviewed again. If one of the later two decisions is made, the paper and the anonymous reviews are passed back to the author who is invited to re-write the paper. This is a painstaking process but it does lead to a high degree of certainty that work is done well before it is published.

Human pollution and frog declines

The impending loss of the frogs in Panama led me to delve more deeply into the literature on amphibian extinctions. The decline of amphibians is global pattern that was noticed by individual researchers comparing observations from around the world. The researchers at international meetings realized that one talk after another was telling the same story, from sites around the world. Some of these sites were pristine, others heavily influenced by humans. The picture that has emerged over the last few decades as a result of the herpetologists realizing the global pattern is sobering, but is also a testament to science as an international endeavor and the broad geographic approach that leads to generalizations that could not be reached if research was constrained to one small area.

In the 1990’s, Andrew Blaustein, at Oregon State University, began to see declines in the species of frogs he was studying in the mountains of the Pacific Northwest. He understood that ultraviolet radiation (UVR) was increasing world-wide because of global ozone depletion in the upper atmosphere. Chlorofluorocarbons used in aerosol cans, now being phased out from use in air conditioners, refrigerators and freezers, enter the air at ground level and work their way into the upper atmosphere. Here, ozone that forms naturally absorbs the ultraviolet rays from the sun. The chlorofluorocarbons destroy the ozone that protects life on the Earth’s surface.

Blaustein hypothesized that increased UVR might be harming amphibians. This hypothesis was reasonable because UVR is known to cause damage to organisms and most amphibians lay their eggs in shallow water where they are exposed to high levels of sunlight and UVR. At higher elevations, UVR is more intense, plus it is possible that amphibians were already close to their upper tolerance levels before UVR started to increase. While these animals did evolve under such exposure, it was not certain if increases in the levels of UVR were responsible for harming the frogs. A series of experiments confirmed that the UVR increases similar to those caused by global ozone depletion lowered survival rates of eggs. One piece of the puzzle was starting to come together.

So this begs the question, will UVR increase or decrease in future years? The use of CFC’s has decreased since the Montreal Protocol was signed in 1991. The decline in stratospheric ozone has led to a halt in the loss of ozone, but still, almost 20 years later, the upper atmosphere has not healed itself. Now we know part of the reason why, nitrous oxide produced as a byproduct of agricultural nitrogen fertilization is also damaging stratospheric ozone. As humanity struggles to feed more and more people, we have been and continue to use ever more fertilizers. Some of this fertilizer is converted by bacteria into nitrous oxide, and this gas works its way into the upper atmosphere, destroying ozone and allowing more UVR to pass to the surface of the earth. Thus, UVR will continue to be a problem for some species of amphibians.

Subsequent research by Carlos Davidson and colleagues from the University of California at Davis established the role of pesticides in amphibian declines. This research examined the declining red-legged frog in California. Careful analyses of data on red-legged frog populations from 237 sites revealed that one of the most important factors in population declines was distance downwind from agricultural fields. Other research over last decades has demonstrated that pesticides and other volatile chemical contaminants can be transported substantial distances and in some cases concentrated by atmospheric processes. High mountain areas as well as arctic habitats are areas where volatile pesticide pollutants are concentrating. These are habitats that seem pristine.

Amphibians are particularly sensitive to these contaminants because their eggs require direct exposure to water, and water carries the contaminants from where they are deposited on land and water upstream, to the eggs. The potential for concentration of these contaminants in the food chain is well recognized; almost a half century ago Rachel Carson wrote “Silent Spring” and illuminated the propensity of these toxins to bioconcentrate and cause environmental harm. Before Silent Spring, most people did not realize that very low levels of these chemicals added to the environment could have adverse biological effects because they were concentrated by natural processes occurring in all ecosystems. Now, the effect could be harming species of frogs and contributing to their declines. Furthermore, a new and unanticipated threat of the pesticides has been documented.

Dr. Tyrone Hayes of University of California Berkeley, was conducting experiments on a common weed killer, atrazine. Seventy-seven million pounds of this herbicide are applied each year to fields in the US. The experiments were designed to detect minimum levels of atrazine which would cause negative effects on aquatic animals. In these experiments, frog eggs are exposed to a range of concentrations of the atrizine to extremely low concentrations that nobody would think had any effect on any organism.

He was surprised to find that vanishingly small concentrations of atrazine feminized leopard frogs while they were developing in the laboratory. Then, Dr. Hayes and his graduate students found similar results in field experiments. Further investigation showed that African clawed frogs exposed to water with as little as one part per billion of atrazine developed improperly, with inhibited larynx growth. A larynx is important to the males of many species of frogs because singing is how they attract mates. The question was how could these compounds be active at such low concentrations? This was a particularly vexing question because negative effects were expected at higher concentrations, and the effects diminished as concentrations decreased. Then, suddenly at very low concentrations, negative effects on the animals popped up again.

The answer is that the chemicals such as atrazine mimic hormones that all animals use in cell-to-cell communication. These compounds signal cells in developing animals to take one or another developmental pathway. The compounds are only biologically active at very low concentrations; animals only produce very low concentrations of hormones to signal developing cells. Many compounds made by humans and released into the environment mimic estrogen, a hormone with strong influence on sex characteristics of both males and females as embryos develop.
Ecoestrogens have been implicated in a number of cases of feminization of wildlife. It is not currently known how these compounds influence development of human embryos, and potential effects of ecoestrogens on humans is an area at the forefront of environmental concern. In this case, amphibians serve as the canaries in the mine; they are quite sensitive to pollutants in the water because they develop fully exposed to the water.

The increased used of fertilizers in agriculture was already mentioned, but more intensive agriculture also requires more use of pesticides. The use of pesticides has skyrocketed in the decades since their widespread introduction in the mid 1900’s, and we have fed the world in part because we have limited some pests (insects and weeds) by using such compounds. The chances that use of these compounds and their release into the environment will decrease in future years are slim.

Other aquatic pollutants could be harming frogs as well. Mercury concentrations are so high that many fish from waters in the US and Europe should not be consumed. Even fishes from the mighty and mostly remote Amazon can have high enough burdens of mercury that regular consumption is not advised (gold mining upstream causes mercury contamination). Mercury can harm all animals, and less attention is paid to frogs than fish with respect to contamination. This is because fewer people consume frogs than fish.

Back to the fertilizers, further work by Dr. Blaustein demonstrated that nitrite, a common byproduct of runoff from fertilized agricultural lands, also harms development of frog eggs. Nitrate contamination downstream of fertilized areas is common with very high concentrations in the rivers and streams of many developed lands, particularly Western Europe and the Midwestern United States. To make things worse, combustion of fossil fuels leads to substantial amounts of nitrogen deposition and causes nitrogen contamination of many aquatic habitats. The effect is particularly strong near industrial or urban areas. More pieces of the puzzle of global amphibian declines were falling into place.

A motley crew

The immensity of the loss of an entire group of animals was abstract to me, until I took my first trip to Panama and saw a jungle stream teeming with so many types of frogs I had never seen before in my life; so the story begins with the trip to get there. Most of our research group met in February 2006 in the Atlanta airport to fly to Panama. This was an eclectic group of individuals who are science nerds (Ph.D.s), globe trotters, adventurers, and generally workaholics. Our common bond is a drive to understand the natural world. Everyone in the group has research experience in aquatic ecology around the world; studying streams is the thread that flows through all of our lives. Matt Whiles, Bob Hall, Alex Huryn and I were the lead researchers that traveled together on this trip.

Our connection in Atlanta was tight, and a bit of running was required to make it from the domestic terminal to the international departure gates. Bob Hall is the kind of person who is amusing to watch run for an airplane. Although I am told he is an excellent skier, he is tall, gangly and awkward looking when he runs, like a camel or a stork. He has the look and the accent of an Ivy League aristocratic intellectual. Bob looked a bit worse for the wear because he had recently returned from a research trip to streams in Venezuela and had not yet decompressed from that experience. He had a touch of the Chavez’s revenge as well.

Currently, Bob is an Associate Professor at the University of Wyoming. We have been friends since we met years ago in the North Carolina Adirondacks. We were attending a workshop to explore the use of stable chemical compounds to trace movement of elements (nitrogen) in streams. This new technique opened up an extremely productive area of research. We could use it to dissect out the basic processes that nourish all the plants and animals living in the streams. The method ultimately allowed comparison of streams from the tropics to the tundra, and resulted in a large project and ultimately a publication in the prestigious journal Science. Many investigators never are able to publish in this journal, as it is one of the top scientific outlets in the world and extremely competitive to get papers into.

It was our expertise in this tracer method that prompted Matt to involve us in the Panama project. In North Carolina, Bob and I hit it off immediately when we discovered our shared interest in home brewing. He had brought several small kegs of some of the best and more exotic beer I had ever tasted, a fact guaranteed to make me sit up, sip up, and take interest. Bob has since published some of the seminal papers on how animals and other organisms mediate movement of nitrogen through stream environments.

Alex Huryn was at the gate when I arrived. Alex is tall and slender, with a matching ambling bearing, thoughtful personality, and a preoccupied air. Many ecological researchers of my generation never left the 1970’s with regard to their hair, and between Alex’s brown hair over his collar and a substantial mustache, it was unlikely that he would be mistaken for a businessman outside of the silicon valley. He has studied stream invertebrates across North America, including the tundra of Alaska. Alex did stints in New Zealand and Maine, among other places, before he landed at the University of Alabama. Like so many ecologists with a pedigree from the University of Georgia (Matt and Bob included) he has a reputation for top level ecological research and partying hard, presumably to balance the tremendous intellectual and work load required to answer the questions that they doggedly pursue (or maybe that is just the excuse). They don’t call them the Georgia bulldogs for nothing.

I had seen Alex out-on-the-town at conferences. The last time I had seen him, he had crawled across a dance floor in New Orleans and bit me on the calf. The bulldog connotation again comes to mind. Before being bit on the calf, I had admired his research papers, but did not know him well. I was hoping he was easy to work with, but was not sure given our prior interaction.

Matt Whiles had a delayed flight from St. Louis and came to the gate at the last minute. He has been a close friend of mine since the 1990’s when we worked at Kansas State University together. Now he is a professor at Southern Illinois University in Carbondale. He is a very gregarious person; people are compelled to tell him their secrets for some reason, probably his friendly face. He has hair about the same as Alex, another child of the 70’s. Matt is gifted in the art of diplomacy and a perfect leader for a group such as ours with strong personalities and the professors’ common trait of always being certain of being right and ready to argue the point exhaustively.

An inkling of a problem

This is a travelogue, a research log, and most of all an extinction log. It describes two research trips to gauge the effects of loss of frogs caused by a fungal disease that is sweeping through Central America. The story is one of extinction, and how people who study these animals respond to the loss. The story is a wake for lost diversity; we will never see these many species of frogs again in the wild. The blog also places the loss in the cultural context of Panama and the ecological context of the rain forest

In 1977, I visited the Costa Rican cloud forest preserve, Monteverde. The small park headquarters had an aquarium that contained a Golden Toad. I did not know at the time that this species was doomed to extinction. The animal was a beautiful, brilliant neon orange frog (Bufo periglenes), about 2 inches long. A staff person at the headquarters mentioned that this species had only been found in this one small area of this cool, wet, and high-altitude forest. He said that very few had been seen recently and we would be very lucky if we saw some in the wild. I had this in the back of my mind as we left to explore the cloud forest, but our main objective was to see the extravagantly colored Resplendent Quetazal, a bird with a two foot long bright green tail. I saw screaming monkeys, I was chased by a giant cat weasel (Jaguarondi). I never saw the frog in the wild.

In 1989 the last Golden Toad was seen, none have been reported since then. The news gradually filtered out through the scientific community that the toad was extinct, but the exact cause was not understood. Monteverde is a pristine cloud forest at the top of a mountain range; it was not developed, and should not have had much human influence. Yet, this extinction was to become emblematic; loss of this species was for many the beginning of general scientific awareness of a global trend of amphibian extinction. The extinction of the Golden Toad bewildered and saddened me, and came to mind when reading the articles that started surfacing in the 1990’s about amphibian extinctions elsewhere in the world. Little did I know that my experience with frog extinction would become very real in future years.