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.

Species extinctions

Of all the factors harming frogs and other species, the most ubiquitous and devastating is habitat destruction. Humans are radically altering more habitats than ever. Forests are cut down for lumber and turned into pastures or agricultural lands. Grasslands are plowed for agriculture leading to erosion and subsequent siltation of streams and lakes. Reservoirs flood streams and overuse of surface water and groundwater dries vital habitats. Wetlands and ponds are drained or filled for other uses. Urbanization, suburbanization, and exurbanization (domestic development outside of the suburbs) are destroying habitat for many species. Most land suitable for cultivation has been converted to cropland. Aquatic and terrestrial habitats are under assault every place on earth with even moderate human population densities.

These trends of global impact are global, with loss of most of the area of tropical rain forests (one of the top areas with biodiversity) predicted in the next few decades. The habitat destruction is causing extinctions at far greater rates than evolution can replace the species. I argue in my book, Humanity’s Footprint, that the pressures on natural habitats will intensify as population grows and our insatiable appetite for ever greater resource uses increases. Without drastic changes in the way we treat the natural habitats, extinction is inevitable for many species.

One of the strongest results of ecological theory and field research is that the number of species found decreases as the area of the habitat is decreased. Small islands have few species relative to continental regions. We know if we decrease the total habitat by 90%, we will loose around half of the species. Habitat destruction, even if it does not destroy the entire habitat, creates small islands of habitats. Ecological principles tell us that these small islands cannot support as many species as one very large area of natural habitat.

Frogs are but one of the species threatened by habitat loss; habitat destruction is a primary cause of a very rapid ongoing major extinction of plants and animals on Earth. This extinction is one of the 6 mass extinctions in Earth’s 3.5 billion year history. About 65 million years ago an asteroid slammed into the earth causing loss of about half the species in the oceans and many terrestrial groups of organisms, including the dinosaurs. Around 200 million years ago massive volcanic activity caused another mass extinction, and 250 million years ago 95% of Earth’s species were lost (the exact cause has not been determined). About 360 million and 440 million years ago there were two more mass extinctions.

Back to the species area calculation and how this influences extinction. Considering it is likely that we will decrease habitat by 90% in the speciose tropics, and that carbon dioxide increases in our atmosphere will continue to acidify the oceans and ultimately make it difficult for corals to survive in a warmer ocean, humans are causing an extinction that will rival other major catastrophes on earth.

The loss of amphibians has been compared to the loss of the dinosaurs. That was the last time a major taxonomic group was almost completely removed from the entire Earth’s fauna. The amount of terrestrial and aquatic habitats humanity conserves will ultimately determine the number of species that survive into the anthropocene (the current geological era of the Earth’s history where humans dominate the biosphere).

Amphibians arose around 400 million years and made it through four all four major extinction events that occurred since they arose. Modern frogs arose during the period dominated by the dinosaurs and survived the extinction that eliminated the dinosaurs. Now, amphibians, including frogs, are some of the hardest hit species of the current (human-caused) mass extinctions.

The Global Amphibian Assessment, the effort of a large international group of herpetologists, estimates that one third of the 5,918 known species of amphibians are endangered or extinct. In Panama, 55, or over 1/4th of the species, are threatened or extinct. Habitat loss and degradation affect almost 4000 species globally. Pollution is the second most common threat, and unknown causes, fires, and invasive species follow. Most of these causes are controllable or reversible. Disease is not.

No one factor can account for the disappearance or decline of frog species around the world. In some areas that seem pristine, there have still been substantial losses of frogs. In other areas the effect of chytrid fungal disease, UV exposure, pesticides and herbicide pollution, or habitat destruction are obvious. In most cases multiple impacts are occurring. Whatever the cause, frogs are becoming rare or extinct.

Amphibians are excellent indicators of ecological health. They are commonly surveyed by field biologists to indicate if an environment is degraded. Frogs and salamanders are among the first organisms to disappear when environmental damage occurs because most of them need both terrestrial and aquatic habitats, and their eggs develop in such an exposed fashion. Lakes, streams and wetlands where eggs and tadpoles develop integrate all the processes occurring in the stream.

Humanity should be deeply troubled that an entire group of organisms is disappearing from our planet, if nothing else this could indicate that the fundamental ability of earth to support us is being damaged. A broader moral interpretation, that other species have the right to exist, or for some, that humanity was placed on earth in part to take care of creation, provides reasons, other than the purely utilitarian reasons, to be concerned about amphibian declines

Introduced species and disease harming frogs

Not only do humans introduce harmful chemicals into aquatic habitats, but they also add harmful species. We have purposefully or accidentally introduced many predators that harm frogs around the world. These predators include sport fishes and other frogs. Invasive species have been deposited into aquatic habitats everywhere people live or go to recreate.

Some people think fish from one area should be transported to other areas so the fisheries can be “improved” with more desirable species. Many people prefer fish they had in the area where they grew up to the native species where they live. Thus, we have common carp in the US. This was a regular food and sport fish in Europe, and people thought it would be good idea to introduce it here. Now, it is rarely caught as a sport or food fish in the US, but has spread across the country. Carp stir up aquatic habitats, destroys habitat for other fish, and eat most anything they can fit in their mouths. Likewise, people have introduced bass into many habitats, and trout wherever they can.

We have introduced relatively large predators into virtually all major drainages of the world. Many of these introduced predators will eat frogs or tadpoles. Predator introduction is one more factor contributing to amphibian declines.
A very important example of an introduced species, with respect to amphibian declines, is the cane toad was a native to Central and South America. It has been introduced in many places in the world for pest control. It was introduced into Australia in an effort to control beetles that are pests on sugarcane. Unfortunately, the toad is ineffective against the pests it was introduced to control; the toad lives on the ground, and the beetles are higher up in the sugarcane.
Cane toads are very toxic, native snakes die when they eat the adults, and fish die when they eat the cane toad tadpoles. Having no evolutionary experience with the introduced toad, Australian predators continue to try to eat both the adults and the tadpoles. In addition, the large toad will consume any smaller frogs or other amphibians it comes into contact with as well as any other prey that can be gulped and squeezed past its jaws. The cane toad is one amphibian that, unfortunately, is not in decline.

Bullfrogs have spread from Central and Eastern US all the way to California. Because its eggs and tadpoles are not palatable, this is one of the few species with tadpoles that can avoid being preyed upon by fishes. Being the largest North American frog, bullfrogs can ingest mice, other frogs, toads, worms, salamanders, and many types of insects. These frogs may be hastening the disappearance of some rarer species of frogs, particularly those found in California. The bullfrog is resistant to the chytrid fungal disease to which many other frog species are susceptible. It may actually serve as a reservoir to move the fungus around (known as a vector to epidemiologists). Introduced species not only include animals, but also include diseases.

The chytrid fungus, Batrachochytrium dendrobatidis, causes a lethal infection to many species of frogs. The disease could have initially been spread by African clawed frogs, although multiple introductions have probably occurred. These frogs were transported around the world to be used for pregnancy tests and as research species (such as in the work of Dr. Hayes mentioned previously). The theory is that some frogs escaped and then passed the disease on to local amphibians. The African frog and many frogs from warmer habitats are resistant to the disease, but many other species of frogs are not. The resistant frogs can carry the disease from one habitat to the next.

The fungus infects the frog’s skin and causes a catastrophic loss of ability to control water balance across their skin; frogs need healthy skin to survive and are particularly sensitive to factors that alter the exchange of moisture and oxygen across their skin. The fungus causes the frog’s skin to thicken to the point where they suffocate. Diseased frogs look terrible with their skin sloughing and their beautiful colors hidden behind a thick layer of infected skin. The fungal disease seems to be hitting particularly hard in Central America and Australia, though these are the areas where the main study on the effects of the disease has occurred, and it is possible that other areas have also been hit that are not as well documented.

Global warming might interact with the chytrid fungal disease. Even a slight warming of climate represents a significant change in temperature in the tropics, where temperature extremes are not so wide. Warming paradoxically leads to cooler days at higher altitudes in Central America by pumping more moisture from the oceans and increasing cloud cover. Cloud cover also causes warmer evenings. Dr. J. Alan Pounds, of the Monteverde Preserve in Costa Rica (the same preserve that used to be home to the Golden Toad), found that these climate changes are creating the perfect conditions for spread of the chytrid disease. Over 100 species of frogs have probably gone extinct in Central America from the combination of local effects of global climate change and the disease. Frogs have experienced periods of warming and cooling over their evolutionary history, but never in concert with the chytrid fungus.

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.

Saying goodbye to the frogs

As we continued working at Rio Maria, it became more important to me to take in the experience of watching the tadpoles swarming in the stream and the constant movement of small frogs scattering as I walked down the jungle trails. This place was the “zen” of frogs and it was easy to sit and meditate on them. Our discussions more frequently turned to the impending disappearance of the frogs and the prospects for saving other parts of the jungle.

While the road to the research site from El Valle was tremendously difficult, it connected with a road that came up the other side of the volcano from the coast highway. This area up high on the slopes of the volcano had seen years of agricultural use, mainly cleared jungle for pasture. This development was patchy, but now it was being converted into high priced houses and ranchettes for wealthy Panamanians. The site also attracted retirees from the US and Europe who realized their money could buy more in Panama than it could at home. The site also attracted retired “snow birds” who migrated down to avoid the cold winter, and then closed up their homes in Panama and returned to the States in the summer. The mountain sites were attractive because of the fabulous scenery and the pleasant year-round temperatures. Not as hot as the lowlands, yet never actually very cold. The land of eternal green and spring was very attractive indeed.

The bulldozers were rolling. It was obvious that development was as much of a threat to Rio Maria as the fungus that was creeping toward the frogs, but the developer claimed that the Rio Maria valley would not be developed and the valley and its wildlife would be preserved. This was good news because habitat destruction would take the majority of the plants and animals with it, leaving only the few weedy species that could coexist with humans. Diversity would be far less than in the current jungle.

Cathy Pringle needed to get back to teaching so she took a shuttle bus to the airport. After she was gone we found a pair of her dirty shoes and socks in the back of one of the pickups. It made sense, who would want to carry wet socks on a day long travel home? We now had proof of the rumors about her always leaving behind socks and we all got a good laugh out of it. Someone in El Valle would certainly put them to good use after a good washing.

We spent a lot of time training Piet, Heidi, and Edgardo who would remain behind to finish up the work and continue monitoring the stream. They needed to learn how to collect all the samples and what to do if things did not work out quite right. It was as important to teach them the concepts behind what we were doing as it was for them to learn specific technique. What if sampling is put off for a day, is that ok? What if the pump breaks and samples cannot be filtered right away? Understanding the research allows such questions to be answered. Fortunately, we could maintain contact with them via e-mail and cell phones from El Valle.

Before we left, we also tried to process as many samples as possible to take back to the US. The routine of field work associated with this type of experiment is the same regardless of the setting. Samples need to be dried and weighed, some of them ground up. Water needs to be filtered and analyzed. Voucher specimens need to be preserved for formal identification in the laboratory. Who was bringing what sample back to the states was important and it was all recorded.

The last day at Rio Maria, we turned off the tracer drip. Then we had to wait for the lines to get cleared out, so Alex and I took a walk to the stream above. It was nearing dusk and the frogs were becoming more active. On a rock in the middle of the stream we saw a smaller male golden frog perched on the back of a larger female. This behavior is called anaplexus, and is how the frogs mate. The males are reputed to ride around on the backs of the females for up to two months before an appropriate breeding site is found. I suspect that this is just legend, but you have to admire the tenacity of the male. All the while, the male strokes the female’s chest with nuptial pads on the fingers of their forelegs. This has to be the longest foreplay in the animal kingdom. Eventually the male fertilizes the eggs and the female lays them. This extraordinary courtship continued in spite of the impending demise of these frogs.

We felt like this was the last we, or anybody else, would witness this behavior of this species in the wild. The last chance to see can be profound, and Alex and I were both lost in our thoughts about what the image meant to us.

The trip back to the US from El Valle was uneventful, with a good shopping trip to an artesian market and a night at a very good seafood restaurant in Panama City. We got up very early the next morning to give us plenty of time to make our 8:00 am flight.

The flight back was ordinary; it was amusing to watch Bob talk his way through US customs with more than his allotment of liquor to avoid paying import tax. The customs guy told him not to ever do it again, but let him through. If he knew how many more bottles Bob had in his checked luggage, he might not have been so nice about it. The best strategy in customs is, never lie and never give more information than asked for. Considering we were bringing samples back into the country, even though it was completely legal to bring the types of biological samples we had back, an overzealous customs or agricultural products inspector could delay us long enough to cause us to miss the next flight out.

Before I knew it, we were back in the US and into the world of strip malls and fast food. Culture shock, even though we were only gone for a couple of weeks, still make a mark. The main objectives of the project now fell to Matt and Piet. Matt needed to obtain more funding to repeat the trip after the disease swept through Rio Maria and the El Valle region, and Piet needed to work through most of the samples we brought back. I had my expensive bottles of rum, a batch of digital photos, and memories of Panama, El Valle, and the frogs of Rio Maria.

Our first task on the project after returning was to obtain funding to get back to Panama and complete our experiment. We agreed that we would find a way to bootleg the work out of other projects if we needed to, but could not do as good of a job without substantial research funding. This job fell to Matt because he was the leader and wrote the first grant. He was a bit spoiled because the first grant had been funded the first time it was submitted, and it was also the first grant he had submitted to the National Science Foundation as lead investigator. Such success is rare, but we were optimistic about requesting the next round of funding. The additional funding seemed highly deserved because of the urgent nature of the research, the unique and powerful angle of using the nitrogen tracer experiments to test how the effects of tadpole extinctions in the streams cascade through the rest of the ecosystem, Matt’s skill at grant writing, and the assistance of the rest of the group.

The next deadline for grant applications was 5 months after our return and Matt wrote the proposal and circulated it around to the rest of the group for their input via email. Writing a grant proposal such as this is not a trivial amount of work. The body of the proposal was 15 single-spaced pages, and including all the necessary information to satisfy the dozen or so reviewers in this length of document is quite a trick. Completion of many pages of forms is also required and every detail of the proposal must have references from the scientific literature to verify statements and show that adequate procedures will be used. Multiple investigators from multiple institutions requires numerous budgets each with their own requirements and bureaucracies that need to be navigated for many signatures. After a long process, the proposal was polished up and submitted. Then we had to wait for the 5-6 months it takes for grant proposals to be processed.

The jungle stream at night

The next few days were filled with technical details of working in the field and doing lab work in the evening. We got time to call our families at home and eat some nice meals. A highlight was when Heidi and Scott Connelly took Piet and me on a night herping (looking for amphibians and lizards) expedition. I had been taken on a night-time herping expedition in Costa Rica many years before.

The expedition leader on the Costa Rica trip was a professor with lizard-like eyes; they bulged out and he rarely blinked. He carried his cigarettes in one plastic bag tucked under his belt. He had another large bag stuck in the other side of his belt to hold the snakes and frogs he caught on the trip. His plaid polyester pants tucked into the top of his rubber boots and shirt button down polyester shirt, as well as his umbrella, seemed odd at first but his enthusiasm for the animals was contagious. We had a fantastic night tromping through the jungle swamp. He would shine his flashlight and catch the flash of reflective eyes and crash into the swamp after it. He would come back beaming with a poison snake in his hand, or a frog or lizard. First thing the next morning we looked at all our prizes. I jumped at the opportunity to go night herping again.

We secured our headlamps, grabbed our cameras and drove to a local stream as the sun went down. As we walked to the stream Scott and Heidi talked about the incredible diversity of frogs in the region.

In addition to the golden frog are several species of glass frogs. These frogs are clear because they have little pigmentation. You can see their hearts beat by placing a light behind them. They lay their eggs on leaves overhanging the stream where the eggs are less susceptible to egg predators. When the eggs hatch, the tadpoles drop into the stream.

We talked about the tadpoles that live deep in the leaves at the bottom of the streams. Accumulations of leaves on stream bottoms are very low oxygen habitats and the tadpoles have tremendous amounts of hemoglobin (the protein that caries oxygen in our blood and that of other invertebrates). They are bright red when exposed to oxygen. This is a somewhat common adaptation among animals. In temperate lakes, midge larvae known as blood worms inhabit the low-oxygen sediments. They are mostly colorless in their native habitat, but when they are exposed to air at the surface or highly oxygenated water, they turn bright red as their abundant hemoglobin binds all the oxygen it can.

Panama has 195 documented species of amphibians. This is over half the number found in the US in a country slightly smaller than the state of South Carolina. Thirty four of these species are found nowhere else (are endemic) and most are restricted to Central America. Panama is an amphibian-lovers paradise and El Valle has historically been a Mecca for people wanting to see Panamanian frogs and other amphibians. Frog societies plan meetings and collecting trips commonly with El Valle as their base of operations.

During the day, brightly colored toxic frogs such as poison dart frogs are common. These frogs practically glow in the low light of the tropical forest. Their bright colors are a signal to predators that says “I am poison, eat me and you die”. Other species, also brightly colored, look like poisonous species, sending the same signal to predators, but are not toxic. This mimicry allows the frogs to avoid predation without having to synthesize costly toxic chemicals. The two types of frogs are balanced in population numbers, because if there are too many of the non-toxic species, predators will start eating and frog with that coloration. So, the non toxic frogs take advantage of the toxic species that they mimic. Evolution has lead to some amazing things, and “cheaters” are a fact of life!

At night the more cryptically colored frogs come out to forage. Their calls filled the stream bottom as we worked our way up the rocky stream channel in the dark. Our flashlights were reflected by the eyes of freshwater shrimps as long as your hand. These shrimp are very skittish, and they scuttled out of sight under rocks by the time we were within 50 feet. Freshwater shrimp are common in tropical streams. Some species have been taken into captivity for aquaculture, particularly in south-east asia. Some of these shrimp play the part that crayfish (crawdads) fill in more temperate streams (omnivores), but others specialize on fine or coarse particles, or algae, or other food types. The shrimp are most common in smaller streams where large predatory fish will not eat them.

Moths bigger than my hand were hanging over the stream. We saw an occasional snake foraging through the underbrush near the edge of the stream; preying on the abundant frogs. The jungle shows a whole different face at night, animals scurry off through the darkness, bats dart around over the stream, and there are spiders the size of a salad plate with eyes that glow in the darkness when your flashlight happens to strike them. Scott and Heidi discussed how the frogs probably would not be around the next time I came back. We left the stream to get back to our hotel muddy, sweaty from our walk, and thoughtful.