Hibernators, or animals that ‘sleep’ through the winter to conserve energy, have always fascinated scientists. To date, researchers have studied the adaptations that allow these animals to be inactive for so long and their ability to come back from their slumber at the beginning of spring. However, no hibernator has been more captivating than the wood frog, Rana sylvatica. That is because this little critter is able to freeze during the winter and then resurrect as if nothing has happened. We know a great deal about how this animal protects itself from the ice crystals that form in its body as it freezes but very little about the role that the symbiotic bacteria in its gut play during hibernation and emergence from hibernation.
One strategy that wood frogs use to preserve water and salts during hibernation is to produce urea when they break down nitrogen-rich proteins. Urea is less toxic than other forms of nitrogen waste, such as ammonia, which has to be diluted and leads to the loss of water and salts. However, the amphibians lack the ability to recuperate the valuable nitrogen from urea. Might the frog enlist some ‘helpers’ – such as their intestinal flora – to retain the nitrogen? A team of researchers from Miami and Pittsburgh Universities, led by James Wiebler, examined the frog's gut to look for bacteria that can produce the enzyme urease, which breaks down urea to produce nitrogen.
First, the researchers isolated the gut from hibernating adult male frogs and separated out three segments: the foregut, midgut and hindgut. In each of these segments, the group measured the size (length and mass) of the segment, the bacterial load and type, and urease activity. They found that, even though the hindgut was the smallest segment, it contained the largest number of bacteria and also had the largest urease activity out of the three. Next, the authors injected active frogs with urea and allowed them to recover for 10 days, to determine whether high levels of urea in the body – which are typical in frogs nearing hibernation to maintain water and ion balance during their slumber – increases urease activity in the gut bacteria. The group determined that high urea levels in the blood did not influence the number of bacteria present in the hindgut but did increase the activity of the urease enzyme by 2.7 times.
The researchers then collected guts from active frogs during the summer for comparison with guts from the hibernating animals. Even though the digestive tracts of the hibernators weighed 61% less and were 25% shorter than those of the active frogs, there was no difference between the size of the hindgut portion in the two groups. In fact, Wiebler and his colleagues reported that the hindguts of the hibernators had 33% fewer bacteria than those of the active frogs, but the amount of the urease enzyme and activity was 2–3 times higher in the hibernators. In addition, the authors reported that the types of bacteria present in the hibernators were different from those in the active frogs, suggesting that gut bacteria composition changes in these animals as they begin their hibernation.
Frogs have a relatively low-energy lifestyle, which allows them to tolerate low-nutrient environments, so retaining as many nutrients as possible is key to survival, particularly during periods of fasting, such as hibernation. Through this work, Wiebler and his group have provided the first evidence of nitrogen recycling from urea in wood frogs and have highlighted the important role that friendly gut bacteria may play in all amphibians. Talk about waste recycling at its best!