Bd isolation from French Guiana

Last week was clear out week for the toe clip and tadpole mouthparts that hadn’t produced any Bd zoospores or sporangia or were full of contamination by other fungi and bacteria. That was a lot of samples to throw out – the success rate for Bd isolation is extremely low (about 1/100) but it’s still difficult to throw away so many samples that we all worked so hard to collect!

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82 samples for the bin

BUT two samples, numbers 32 and 184, started producing zoospores and sporangia and we are now in the process of culturing them. With any luck, we’ll be able to grow up these cultures and carry out DNA sequencing to shed some more light on what type of Bd is in French Guiana. The pictures below are from the early stages of the isolation. Bd normally grows well at 18ºC in liquid medium with antibiotics added. These two seem a little different however – at this early stage they appear to be doing better without antibiotics, and growth is fairly slow at 18ºC, so we’re mixing it up a bit and trying some higher temperatures (they’ve come from the tropics, after all!). We aren’t yet home and dry, they’re growing slowly, but now we know for sure that Bd is in French Guiana and that it is possible to isolate it despite the distinctly un-sterile conditions! The first sign that you might have caught Bd is when you spot the motile zoospores – the Chytridiomycota, the phylum which Bd belongs to, is unusual among fungi in that it reproduces via motile zoospores which are released from the large sporangia. In this video you can see the tiny zoospores darting around the larger, fixed sporangia.

1) Isolate 32 – Sporangia in toe clip skin 2) Isolate 184 – Sporangia and zoospores 3) Isolate 184 – Fungal contamination
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Anamoglossus baeobatrachus (L) & Allobates femoralis (R)

Isolate 32 is from an Anamoglossus baeobatrachus, isolate 184 is from an Allobates femoralis. Now that we’ve found them, we need to keep them alive, grow them up and get some DNA sequencing done on them. Getting them into a pure cell culture is likely to be challenging, but even if we stumble at that hurdle, we’ve shown that it is possible to isolate whatever the Bd is in French Guiana, even in (to put it mildly) less than sterile laboratory conditions. Meanwhile over the next few weeks we’ll be extracting the DNA from all the swabs and carrying out qPCR to get an idea of Bd prevalence and intensity in Nouragues.

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The first of many, many DNA extractions

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