Frog-killing fungus is a skin-loving hybrid

 Frog killing fungus is a skin loving hybrid

These are not the best of times for amphibians. All around the world, populations of frogs, salamanders and newts are declining. At least 489 species (7.8% of all known amphibians) are nearing extinction. More than a hundred of these endangered species have not been seen in recent years, and have likely gone extinct already.

Who is to blame for this wave of extinction? While climate change, pollution and habitat destruction certainly play a supporting role in this amphibian drama, biologists now agree that a fungus is the major villain. the fungus in question is chytrid fungus, and bears the name Batrachochytrium dendrobatidis, or Bd for short, and causes a disease called chytridiomycosis.

What Bd does to an amphibian’s skin is not pretty. the fungus grows right within the cells of the skin. When it has produced enough spores, they burst out of the cells and re-enter uninfected skin cells. This cycle of growth and infection causes lesions and a premature shedding of the top layer of the skin. Lee Berger, who first identified Bd in sick and dying frogs in 1998, wrote that “these specialized adaptations suggest that B. dendrobatidis has long evolved to live in skin.”

This seems strange. Bd only became a problem somewhere in the second half of the twentieth century. Analyses of museum skins show that Bd was absent from most affected localities prior to the 1970s. it has spread over the world at an alarming rate since then, killing frogs, salamanders and newts wherever it goes. But if Bd has really existed for a long time and kills its hosts with such vigour, shouldn’t it have burned itself out by now?

Biologists have come up with two general explanations for the sudden emergence and spread of Bd in the 20th century. some have suggested that environmental changes make amphibians more susceptible to Bd. others have proposed that Bd is a novel disease to which amphibians have no resistance. these two hypotheses are far from exclusive, and come with many flavours in between.

Feeling at home in skin

When the Bd genome was sequenced in 2006, biologists hoped it would reveal how Bd became a slayer of frogs. But without similar genomes to compare the Bd genome to, it was hard to draw conclusions about what makes Bd special. the chytrids, the branch of fungi that Bd belongs to, turned out to be particularly understudied.

Conventional chytrids are quite harmless. They are microscopic fungi that usually live in water or wet soil where they degrade leaves and other organic material. the closest known relative of Bd is Homolaphlyctis polyrhiza (or Hp). This fungus was isolated from leaf litter in Maine by Joyce Longcore. Erica Bree Rosenblum, evolutionary biologist at the University of Idaho, and her colleagues have now sequenced the DNA of this leaf muncher, to see what makes it different from Bd.

Rosenblum discovered several types of genes that are abundant in the Bd genome, but not in the Hp genome. the proteases were on of them. Proteases are like molecular scissors. these enzymes recognize, cut and cleave other proteins. Rosenblum found that three different protease families have expanded in the Bd lineage. They have between between four to ten times as much members as the same families in the Hp genome.

Fungi that infect human skin or nails are known to carry similarly large and diverse sets of protein slicers. They help the fungus to invade tissues and obtain nutrients by breaking down the proteins and cells of its host. It’s likely that the numerous proteases in the Bd genome also play a role in the colonization of amphibian skin.

Another abnormal group of genes in the Bd genome is the crinkler family. Crinklers have never been found in other fungi. They were originally discovered in oomocytes, which are single-celled organisms that infect plants and cause diseases such as late blight and sudden oak death. True to their name, crinklers cause the leaves of the plants they infect to crinkle. it is unknown what these proteins do to amphibian skin and whether they are important for infection, but their presence in the Bd genome is certainly intriguing.

Another group had described these Bd-crinklers earlier. They proposed that crinkler genes hopped from oomycetes to Bd and that this transfer of genes possibly led to the Bd-epidemic. Sophien Kamoun, who works with oomycetes and discovered crinklers in 2003, thinks that this conclusion is premature. “There are 62 crinklers in the Bd genome and they only resemble oomycete crinklers on a general level. if it is true that oomycetes are the source of the crinklers, they must have been transferred a long time ago.”

Rosenblum says the origins of Bd proteases are similarly ancient. “the Bd protease families expanded recently on an evolutionary time scale. This still means that most expansions are a millions years old. They certainly didn’t happen in the last 50 years.” So while the presence of crinklers and proteases might explain how Bd evolved to live in skin, but not why it became a global menace. these proteins loaded the gun, but they didn’t pull the trigger.

Conquering the world

I’ve painted a grim picture of Bd so far, but the truth is that not every strain of Bd is a global killer. in a recent PNAS paper, scientists describe two lineages of Bd from Switzerland and South Africa that are genetically distinct from the globally occurring lineage that is killing amphibians worldwide. They’re also not nearly as lethal. the researchers infected tadpoles with the South African strain and found that more than 7 out of 10 of them survived. Tadpoles that were exposed to global varieties of Bd were much worse off. in the most severe cases, less than 20% survived infection.

Isolated pockets of Bd such as those in South Africa and Switzerland have likely existed for a long time. Geneticists have uncovered some clues as to how a global killer fungus could emerge from such local varieties. To see what they found, we first have to take a short trip into basic Bd genetics. Bd is a diploid fungus, which means that it has two copies of each chromosome, just like humans have. Diploid organisms can thus carry two different versions of any given genetic variant, a situation which is known heterozygosity. When the same genetic variation is present on both chromosomes, this is called homozygosity.

Genomes of sexual organisms are a mixed bag of homozygous and heterozygous variants. But not that of Bd. its genome is way more heterozygous than is normal. “the simplest explanation for this pattern is that the hypervirulent lineage of Bd is the product of two undiscovered parents”, says Matthew Fisher a geneticist from Imperial College London and co-author of the PNAS paper. in other words, the killer lineage of Bd is a hybrid fungus. it has received two different sets of chromosomes from its parents, which explains the high degree of heterozygosity in its genome. “Sex is rare for this species. But when it happens, a new strain with new properties might emerge”, says Fisher. “We think this is how hypervirulent Bd originated, somewhere in the 20th century.”

Fisher thinks the international trade in amphibians is directly responsible for the emergence of Bd. “From the 1950s onwards the African clawed frog (Xenopus laevis) was shipped all over the world, first as a pregnancy test and later as a laboratory animal. This global trade in amphibian increased the possibility that two divergent lineages of Bd come into contact with each other. I’m pretty sure that hypervirulent Bd wouldn’t have evolved without the amphibian trade. We can clearly see the ongoing effects of this trade as it spreads the killer lineage ever more widely.”

Fisher’s team used the genetic relationships between the different lineages to date the emergence of Bd to 35 to 257 years ago, depending on which genome segment they analysed. Rosenblum points out that this analysis depends on a number of assumptions, each of which could affect the outcome. “my suspicion is that our next wave of analyses will suggest these genetic transitions are more ancient than that”, she says. Fisher admits that it is hard to argue what the exact emergence date is. “But since it underwent its spread in the 20th century, we think it is likely that the recombination event took place in recent history.”

A firmer answer to the questions where and when Bd evolved will have to await the sequencing of more genomes, from other lineages of Bd and from additional chytrids. Each genome will provide another piece of the puzzle. There are still corners of this world where Bd hasn’t penetrated yet, such as Madagascar. the sooner we understand this amphibian scourge, the better we can prevent its spread. the frogs will thank us for it.

Images:Dead Limosa Harlequin Frog by Brian Gratwicke.Petri dishes with flaked skin from second reference.North American bullfrog by Carl Howe.

References:Carey, C., Bruzgul, J., Livo, L., Walling, M., Kuehl, K., Dixon, B., Pessier, A., Alford, R., & Rogers, K. (2006). Experimental Exposures of Boreal Toads (Bufo boreas) to a Pathogenic Chytrid Fungus (Batrachochytrium dendrobatidis) EcoHealth, 3 (1), 5-21 DOI: 10.1007/s10393-005-0006-4Joneson S, Stajich JE, Shiu SH, & Rosenblum EB (2011). Genomic transition to pathogenicity in chytrid fungi. PLoS pathogens, 7 (11) PMID: 22072962Farrer RA, Weinert LA, Bielby J, Garner TW, Balloux F, Clare F, Bosch J, Cunningham AA, Weldon C, du Preez LH, Anderson L, Pond SL, Shahar-Golan R, Henk DA, & Fisher MC (2011). Multiple emergences of genetically diverse amphibian-infecting chytrids include a globalized hypervirulent recombinant lineage. Proceedings of the National Academy of Sciences of the United States of America, 108 (46), 18732-6 PMID: 22065772

Frog-killing fungus is a skin-loving hybrid

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