ABOVE: Artist's rendition of an African lungfish (Protopterus spp.) © ISTOCK.COM, 7ACTIVESTUDIO

African lungfish (Protopterus spp.) live in water for part of each year. During yearly dry seasons, though, they dig into mud, curl up, and secrete staggering amounts of mucus that then hardens to form a cocoon of protection. This process, known as estivation, allows the animals to survive on land for years, protecting them from complete desiccation until water returns, at which point they rehydrate their bodies and go back to swimming. Most scientists believed the cocoon to be inert, but a study published today (November 17) in Science Advances suggests otherwise. The authors found that the mucus cocoon is filled with white blood cells called granulocytes, which create extracellular traps to nab bacteria and protect the animals from sepsis, skin infections, and blood loss. 

“This is an animal that, for this brief moment in time, can—like a force field—extend its body, its actual living body beyond its scope,” says Ashley Poust, a vertebrate paleontologist at the San Diego Natural History Museum who did not participate in the study. “The fact that they have actively dividing cells, and they carry around these granulocytes in their tissues, almost like a storehouse, is really cool,” he continues. It’s a new example of “storing up something that you need for this different part of your life cycle, so that is exciting.”

There have long been hints of a connection between the immune system and lungfish cocoons. A 1931 paper that characterized all the blood cells in lungfish pointed to a possible role for granulocytes in estivation because of how prevalent they are during the animals’ time on land, and other studies found that lungfish have tons of granulocytes. “These animals are like bags of granulocytes,” says evolutionary immunologist Irene Salinas of the University of New Mexico. There are so many granulocytes that when the researchers try to look at other cell types, they have to find ways to circumvent the granulocytes, which autofluoresce.

This is an animal that, for this brief moment in time, can—like a force field—extend its body, its actual living body beyond its scope.

—Ashley Poust, San Diego Natural History Museum

Salinas and her colleagues found further evidence for a granulocyte-cocoon connection in a study published in 2018 in which they analyzed the proteomes of the skin mucus and cocoon during the transition from water to land. “We started to see things that were exciting from an immunological point of view,” Salinas says, including granulocyte markers in the cocoon proteome. They also showed that granulocytes, which are usually found in the kidney and gut wall, migrate into the skin during the process of estivation, which the researchers induce in the lab by gradually removing water from the lungfish tanks.

In the new work, the researchers looked more closely at where the granulocytes that show up in the skin were coming from. First, they found them migrating in the blood, Salinas says, which indicated that “they must be leaving the tissues where they usually reside and moving to the skin, [but] then once they get to the skin, we didn’t know what to expect.”

Using both brightfield and fluorescent imaging, the researchers figured out that the granulocytes were moving outside of the skin and migrating into the cocoon. “All of a sudden, we realized that the cocoon was not just dry mucus. It was like a living structure, and there were loads of cells in there, and they were actually alive,” Salinas explains.

See “Why Immune Cells Extrude Webs of DNA and Protein

With imaging and bacterial species profiling, the researchers found that the live granulocytes in the cocoon make extracellular traps filled with immobilized bacteria—creating a microbiome of sorts that’s different from the skin microbiome of water-dwelling lungfish. When they sprayed cocoons with DNases that eliminated these extracellular traps, the animals experienced skin infections, hemorrhage, and a huge increase in circulating bacteria, consistent with septicemia. By analyzing gene expression, the researchers also found that many proinflammatory genes are activated during estivation, which could help the granulocytes stay alive, they write in the paper.

“This is a new function for an old organ that was considered just to be a physiological barrier to avoid the desiccation or drying of the fish,” says Oriol Sunyer, Salinas’s former postdoc mentor and an immunologist at the University of Pennsylvania School of Veterinary Medicine who was not involved in the work. “We might consider that as a new form of immunity, from the perspective that this is like an extracorporeal immunity,” he explains, along the lines of an “immune vest” that the animals put on as they transition to a terrestrial lifestyle.

From the new study, the team has long lists of genes that change expression levels in the skin during estivation. Trying to make sense of that data is a challenge, because while the lungfish genome was published earlier this year, it’s huge—around 37 gigabases—and only about 60 percent of the genes that Salinas’s team identified have known functions. “Now we’re going one by one, trying to find out what is in that list that is really exciting,” she says.

Another future direction is to look in older cocoons. In this paper, the cocoons were about two weeks old. In 2022, the researchers plan to travel to Tanzania to analyze the cells and microbes present in cocoons in wild lungfish that have been estivating for months.

 “An interesting angle to follow up on would be to what extent they naturally get infections [and whether] there are microorganisms that are favored by the conditions of estivation,” says Warren Burggren, a comparative physiologist at the University of North Texas who did not participate in the study. Regarding the finding that lungfish have a complex cocoon microbiome, he says: “I was not at all surprised to see that the lungfish—like just about every other organism—is actually a community.”