How poison arrow frogs come by their microbiomes

How do frogs come by their microbiomes? We know that a human baby picks up its microbiome as it makes its entry into the world, usually in the form - as one microbiologist put it - of a mouthful of muck. Amphibians, though, develop in spawn as a tadpole. So what’s their story? This is an important nut to crack because of the threat posed to these creatures by a rampant “chytrid” fungus, first documented as a threat about 20 years ago. Maisie Fischer is at the University of Vienna…

Maisie - We're currently witnessing the greatest loss of biodiversity that can be attributed to a pathogen, and that pathogen is a fungus and it targets various species of amphibians. This fungus infects and destroys their skin, which would usually kill them. However, we know that skin microbiome can protect amphibians from these infections. And so I set out to explore how and when skin microbiota of tadpoles are acquired. And in this regard, I was particularly interested to find out about the role of parental care in this process.

Chris - Which amphibians were you looking at specifically?

Maisie - So because I was particularly interested in the parental care aspect, I chose amphibians that show like a high diversity of parental care strategies. So I was working with poison frogs, and one particular, which is the variable poison frog, very colourful, day active, which makes the life of us as scientists much easier. And they are also dedicated parents. So as many poison frogs, they deposit their clutch terrestrial because they live in the neotropics, where the high humidity allows them to do so. However, when the tadpoles develop far enough, they still need to be transported to water to complete their development. And so these parents check regularly on their eggs. And then once the tadpoles are ready, they help them hatch. And then the dad actually does the parental care, and he takes these tadpoles onto his back, piggyback. He then transports them to tiny pools of water that form in plants. And so I was interested in whether we have microbiota transferred from the dad to the offspring in the course of this transport period.

Chris - And are the tadpoles, before they're a tadpole, when they're developing in their egg stage, are they sterile at that stage? So there's no microbes on them at that stage, and then they pick up microbes from their environment and their dad, potentially, after hatching. Is that sort of where you were coming from?

Maisie - Yes. So that was the very first question that I wanted to answer. People had not thought about the tadpoles being sterile at that point. However, other vertebrate eggs, for example, chicken eggs, the embryo is surrounded by a membrane that is called the vitilin envelope. And this envelope serves as microbial barrier. And so just because chicken are much more interesting to human beings because of their importance for human nutrition, this is very well investigated, and nobody has ever looked at that in frogs. And so this was the very first question of my whole project that I wanted to answer. When can microbes actually access the embryo?

Chris - And is that the case, that the tadpoles, before they escape from the egg, are sterile, and as soon as they're out into the environment, that's when they pick up bugs?

Maisie - So we found that the content of microbes in the surrounding jelly, so the egg surrounding the embryo, is much higher than in the embryo itself. So this is a good hint that actually the vitilin envelope also in tadpoles protects the embryo from major microbial colonisation. So this colonisation will just occur after these tadpoles are old enough to hatch from the vitilin envelope, which in our study species happens right before they are transported.

Chris - And then when dad picks them up, is there evidence that there is a colonisation of the tadpole with the parental microbiome at that stage?

Maisie - Yes. I see this as one of the major achievements of this research, that we could actually provide the very first evidence that this is indeed happening during tadpole transport. It doesn't seem to happen in the egg because the embryo is still protected. However, during tadpole transport, there is a direct skin-to-skin contact between the dad and the tadpole. And so we used an experiment where we could actually trace the microbes found in a tadpole after they had been transported. To do that, we used the fact that skin microbiota are influenced by the species. That means that different species of poison frog have very distinct skin microbiota. And so what we did was that we worked with sibling tadpoles and we had one tadpole transported by the biological dad and fostered a second tadpole onto the back of a very different poison frog species. And so that helped us show that whatever species transported that tadpole, this microbiota can then be found on the tadpole skin.

Chris - And it also nails the question, well, they didn't just pick up their particular unique clutch of microbes from the environment. They must have got it from the dad because otherwise both of those experiments would have ended up ultimately with the same microbiome, which they didn't.

Maisie - Exactly. So the other question was whether probably tadpoles can also spend a lot of time in the jelly already acquiring some microbiota there. And that was like one of the major reasons to choose this species, because in our species that dad actually helps the tadpoles hatch. And so if this is the case, really like microbiota of the transporting frogs are like among the very first microbiota that these tadpoles encounter. And they are definitely transferred from the transporting frog to the tadpole in the course of this transport.