With their moist, breathable skin, frogs and toads are particularly vulnerable to drying out. Their water balance isn't helped by a very leaky circulatory system, which causes them to produce up to five times more lymph for a given amount of tissue than mammals. All the lymph that oozes out is returned to the blood stream by the lymphatic system, where two pairs of dorsal lymph hearts contract rhythmically to squeeze lymph from the lymph sacs and back into the blood stream. Despite the fact that lymph gathering in the sacs builds up a pressure, this pressure is not enough to transport the lymph lying in sacs on the ventral side of the animal vertically up a few centimetres to the beating hearts. Suspecting that other mechanisms were helping the lymph on its way, Robert Drewes, Michael Hedrick and their colleagues Stanley Hillman and Philip Withers investigated further(p. 3931).
Since many of the muscles in the pelvis and connected to the hind legs of frogs and toads run very close to lymph sacs, it's possible that a muscular`pump' could squeeze the sacs to help move lymph. To find out if this was the case, the team operated on anaesthetised cane toads and bullfrogs to insert recording wires into the leg and pelvic muscles and pressure gauges into their lymph sacs. They recorded the muscle activity and lymph sac pressure as the animals rested in a container 12–24 h after surgery. They found that pressure changes in the lymph sacs always correlated with muscle activity, and that the pressures generated were enough to move lymph vertically to the lymph hearts. One muscle called the piriformis moved the urostyle, a thin bone made up of fused vertebrae at the base of the spine. As the urostyle bended and flexed, it acted like a pump handle, changing the volume and pressure of the pubic lymph sac and contributing to vertical lymph movement.
In a second group of cane toads, the team severed the tendons of some of the muscles that attached to the skin beneath the thighs and the pelvic region, finding that 10 days later around 20 times more lymph had collected in one of the sacs. This confirmed that the movements of these muscles are important for helping to move the lymph out of the sacs.
The team suspected, however, that muscle movements weren't the whole story(p. 3940). There are fewer muscles associated with the front legs in frogs and toads, so it was unlikely that muscle movements would help move lymph in the front half of the body. They already knew that lung movements and muscle movements were coordinated, so suspected that lung movements also helped move lymph to the lymph hearts. As before, they inserted pressure gauges into the lymph sacs of anaesthetised frogs and toads. They also inserted a tube into the lungs, so that they could artificially inflate and deflate the lungs, and then measure what happened to the pressure in the lymph sacs. They found that as the lungs expanded and shrank, the pressure in the lymph sacs surrounding the lungs changed too: as the lungs inflated, the pressure in the sacs increased, and when the lungs deflated, the pressure in the sacs went down.
Having shown that filling the lungs with air influenced lymph sac pressure,the team were interested to know what happens in awake animals. The team carried out another operation to insert pressure gauges into a forelimb lymph sac and in a sac overlying the lungs. As the amphibians recovered in their tank, the team monitored pressure in the sacs. They found that when the animals breathed out, the pressure in the sac above the lungs went down,probably because it expanded in size to compensate for the decrease in lung size. At the same time, the pressure in the forelimb sac went down, and lymph was sucked vertically from this sac into the sac overlying the lungs. When the amphibians breathed in, filling their lungs with air, the team suspect that lymph in all the sacs surrounding the lungs was squeezed towards the lymph hearts, which then pumped the lymph back into the blood stream.
This shows that breathing has two important roles in frogs and toads. When oxygen levels in the blood drop and carbon dioxide levels rise, breathing rate will go up to restore the balance of gases in the blood. However the role of the lungs in moving lymph means that breathing rate will also change in response to lymph pooling, helping squeeze lymph to the lymph hearts and on into the blood, maintaining blood volume.