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Zoology

In the dry downpour

Caatinga savanna toads physiologically adapt to survive months of drought

Alert sleep: Pleurodema diplolistris in its hiding place during the drought

When there is a downpour in the hinterlands of the state of Rio Grande do Norte, the landscape changes abruptly. From one minute to the next, rivers form, lakes fill up and hundreds of toads jump out of the ground. This is what happens near Angicos, in the middle of the state, where the Pleurodema diplolistris toads spend some 10 to 11 months of drought a year buried in the sand, from which the males emerge singing in unison, like a choir, promptly jumping into the nearest lake. Drawn by the singing, the females choose their partners and release dozens of eggs that, once fertilized, are wrapped in mucus that is somewhat like egg white, whipped up by the males. Within one month, or two at most, when the rains cease and the rivers vanish as if by magic, the little toads must be fully formed and ready to burrow into the sand. Trying to understand how these amphibians survive so long with no water or food has proven to be an enigma, which is now being explored by the physiologists Carlos Navas, from the University of São Paulo (USP), and José Eduardo Carvalho, from the Diadema campus of the Federal University of São Paulo (Unifesp).

Throughout the dry spell, the Pleurodema remain buried and they do not eat while in aestivation (the summer equivalent of winter hibernation). Understanding the physiological processes that enable such a feat is where the projects coordinated by the two researchers meet. One project, led by Navas, combines physiology and conservation within the climate change context. The project led by Carvalho, on the other hand, concerns comparing the physiology of reptiles and amphibians, under the scope of the INCT (National Institute of Science and Technolgy) for Compared Physiology. Both studies have FAPESP financing. “During aestivation, inactivity occurs when the environment is unfavorable,” explains Carvalho; “when the temperature is high, the animals’ metabolism tends to become quicker, not the other way round.” In search of knowledge about various aspects (gene activity, the effect on the muscles and what one can observe in the fossil registry) of different animals (ranging from sponges to mammals), the two researchers edited the book Aestivation: molecular and physiological aspects, written by authors from several countries and published this year by Springer, an international publishing house. “The summary of each chapter may help us outline which mechanisms are common to different groups,” says Navas.

Even if one brings together these works, a consensus defining aestivation in ecological and physiological terms is yet to be reached. Moreover, this might never be attained, as each organism embraces its own set of solutions for the difficulties that the environment dictates. The case of the amphibians, including the results of the São Paulo research, is set out in one of the chapters, written by Carvalho, Navas and Isabel Cristina Pereira, for whose master’s degree Carvalho and Navas acted as advisors. They found that the Pleurodema does not enter a state of torpor as deep as that of the species studied in other countries, as these toads remain buried in the sand with their eyes open and, whenever they are found, promptly jump out. “It is a state of moderate physiological depression,” defines Carvalho. Furthermore, the group led by Carlos Jared, from the Butantan Institute , had already observed that this amphibian from the Caatinga savanna does not form cocoons. In one month of preparation for the drought, the Australian toad Neobatrachus aquilonius, for instance, secretes 45 layers of skin, which form a wrapping around it like millefeuille pastry, while the Scaphiopus couchii from the American deserts takes approximately four hours to emerge from its dormant state when it is disturbed.

Physiology
During the drought, the stomach of the Pleurodema is empty, the intestines are atrophied and masses of fat account for 12% of their weight. The ovary of the females is full, ready to release the ova as soon as it rains. Isabel took some of these animals to the lab to measure their oxygen consumption, which indicates their energy expenditure, and found that during the dry season consumption at rest drops by half. This indicates a restriction of the body?s functions. “It’s like metabolic valves that are closed,” explains Navas. However, when the researcher forced the toads to jump, the oxygen consumption did not oscillate with humidity, making it clear that the animals were able to switch on all their “valves” fast.

To find out which metabolic paths are kept active and which are switched off, the group examined the activity of several enzymes that are essential for the toads’ metabolism. They found a drop in the activity of the metabolic rates that depend on oxygen – not because there was any shortage of the gas, but to save energy. During the drought, the metabolism was indeed reduced in the liver and the muscles of the rear legs. A low concentration of proteins in the heart suggests that this organ also becomes less active during aestivation. The legs, on the other hand, maintain normal protein levels. This is what apparently enables the toads to promptly start jumping about at any time within this inactive spell, which can last for as long as two years, whereas a person who spends one month in bed will suffer muscle atrophy and have to relearn how to use his legs.

ISABEL CRISTINA PEREIRA/USP Hugging a female, a male beats mucus to make a nest for the eggs.ISABEL CRISTINA PEREIRA/USP

Carlos Jared and Marta Antoniazzi, from the Butantan Institute , are also trying to add pieces to the Caatinga savanna jigsaw puzzle, through natural history and morphology studies. By analyzing the skin of the Pleurodema with a scanning electron microscope , they are discovering that the density of the skin’s blood vessels is greater during the dry season. They must complete their analyses to achieve full quantification, but so far they believe that this is a means of maintaining greater efficiency of gas exchanges and water absorption. “The skin of these toads is thicker than in other species,” explains Jared, “which is why greater vascularization becomes necessary, presumably.”

Understanding the physiology and morphology of these animals calls for cutting-edge science, but it is relatively useless if the more basic work of observing how these animals live is not carried out. Isabel, for instance, observed that at the end of each night of dating the toads try to eat and then burrow into the sand again. “I would follow each one into the night to see where they went,” she tells us. However, she could hardly even blink, as the mottled skin of the Pleurodema makes them almost invisible in the sand. Moreover, they vanish from the surface in just about 30 seconds. The next day, they emerge from the ground, until the wet spell reaches its end.

We are yet to find out where most of the roughly 40 species of amphibians that live in the Caatinga savanna aestivate. However, they launch themselves by the hundreds into unbridled singing and reproduction in the newly-formed lakes. Jared and Marta have played a major role in reducing this lack of knowledge: every year they explore savannas in various northeastern states to observe the animals and to try to figure out where they hide. It was Jared who discovered where the Pleurodema spend the summer and who opened the way for other studies. “It took me seven years to discover this, as of 1987, when I first started going to the Caatinga every year,” he recalls. “Without his contribution we’d still be looking for the toads,” comments Navas, who, for this very reason, dedicated his chapter in the book Aestivation to Jared.

Strategies
Year after year, the researcher from Butantan observed that as the drought progressed and the soil became dryer, the Pleurodema burrowed further and further down, by as much as 1.8 meters, always near a bit of moisture. Isabel measured these depths several times during the year and agrees that the toads really do travel vertically in their quest for less arid areas. She measured the moisture at different depths and found that at 40 centimeters from the surface water is lost quickly, which, however, is not the case when one digs down 80 centimers. She also found that the temperature remains fairly stable and that the sand’s oxygen content drops only little – from 21% at the surface to 20.7% at a depth of 1.5 meters. “It is like two people sleeping in a closed room,” Navas compares; “this change doesn’t even tickle the amphibians.”

“In the Caatinga, each amphibian embraces a strategy to face the environmental conditions,” Jared tells us. As a member of the Toxinology INCT, financed by FAPESP, he is looking for the relation between the amphibians’ toxins and the Caatinga savanna environment. The Proceratophrys cristiceps toads are at least twice the size of the Pleurodema and their skin is four times thinner, which should make absorbing water easier. Another species, Rhinella granulosa, is active during the day and tolerates temperatures as high as 44 degrees Celsius, according to an article written jointly by the Navas and the Jared groups and published in 2007 in Comparative Biochemistry and Physiology, Part A. It was found that among young toads, which are active during daylight hours, the citrate synthase enzyme, which important for the aerobic metabolism, remains stable even at far higher temperatures than those tolerated by the nocturnal adults. According to Jared, this species has a calcified layer of skin that keeps water from evaporating. Furthermore, changes in the skin channel the dew to an area in the groin that is specialized in absorption. Along with the Rhinella jimi, a cururu (toad of the Bufo genus), this amphibian is the only one to disregard the Caatinga drought.

The lack of more specialized resources, such as a cocoon, may reflect the history of this environment, which is believed to be some 10 thousand years old. This is very young in evolutionary terms. Previously, the Northeastern hinterlands held a mosaic of different woodlands, far more humid than the almost desert-like, thorny ecosystem we see today. “Perhaps the Caatinga amphibians didn?t have time to specialize,” speculates Navas.

It is in the capacity to adapt to environmental changes that the knowledge of aestivation physiology fits into the project that the USP researcher is coordinating under the FAPESP Program of Research into Global Climate Change. He tells us that “extreme events will become increasingly greater; we need to evaluate the fauna’s capacity to deal with the physiological challenges that the changes dictate.” Thus, he aims to put physiology at the service of conservation within the context of climate change. “How will things turn out if the drought becomes longer, if rainfall becomes more concentrated? Will the amphibians have enough time to reproduce?”

The projects
1.
Effects of global climate change on the Brazilian fauna: a conservation physiology approach (nº 2008/57687-0); Modality Thematic Project – FAPESP Program of Research into Global Climate Change; Coordinator Carlos Arturo Navas Iannini – IB/USP; Investment R$ 1,007,071.66
2. National Institute of Research into Compared Physiology (nº 2008/57712-4); Modality Thematic Project; Coordinator Augusto Shinya Abe – Unesp-Rio Claro; Investment R$ 200,000.00

Scientific article
CARVALHO, J. E. et al. Energy and water in aestivating amphibians. In: Aestivation. Carvalho, J. E. and Navas, C. A., eds. p. 141-169. 2010.

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