As part of our new work on aquatic flaviviruses in sea cucumbers, we have begun to explore the microbiome of sea cucumber body wall tissues, which we expect will vary under organic matter enrichment and ultimately we hope to test whether this impacts flavivirus replication. We haven’t yet been in the lab this year aside from a few PCRs to test new primer sets, so our work has focused on samples we have already processed as part of prior work. We’d hoped to incorporate sequence libraries from other researchers that have been published. But, it turns out, there have been as far as we can tell no prior studies (aside from those we have performed in the past) of body wall microbiome composition.

So what comprises the holothurian body wall microbiome? Lots of different types of bacteria and eukaryotes. First, looking at bacteria, there appears to be a large proportion of actinobacteria, alphaproteobacteria, and acidobacteria, with fewer verrucomicrobia and plactomycetes. This isn’t really surprising; in our experience these occur on aquatic surfaces, and thrive in nutrient-rich conditions which are typical of animal surfaces. Within the alphaproteobacteria, there are a very healthy representation of Rhodospiralles and Rhizobiales, both typical of nutrient-(organic matter)-rich habitats. There’s also a good smattering of deltaproteobacteria which are typically anaerobes. What is also interesting is that the phylum- through order-level taxonomy of microbiome doesn’t vary that much between species – on the graph below those labeled “Holothuria” through “Synaptula” are from tropical/sub-tropical habitats in Australia, while Parastichopus (which has been renamed Apostichopus) is from the Pacific Northwest. So essentially this is collection of bacteria which probably thrive in nutrient-rich habitats and on surfaces.

We’ve also explored the protistan microbiome through a technique we call Phylogenetically-assigned ribosomal transcripts (PhaRTs for short); these are ribosome-bound rRNAs that we pull out of metaviromes and are depleted with subtractive hybridization of holothurian 18S rRNAs. On the figure below (which were all prepared from Apostichopus californicus), the communities are dominated by green algae (Chloroplastida) and flagellates (Excavates), with fewer Cryptophytes and Amoebas. If you’re wondering what the various labels mean, extracted ribosomes (MC1, MC2 and MC3) were treated with and without subtractive hybridization (SH) and in the presence and absence of tissue (tissue). We’re still wrapping our heads around this one, but it is interesting to note so many algal-related 18S rRNAs associated with body all tissues.

So we keep looking and investigating. This week, Jay is working to summarize published descriptions of the digestive tract (gut) microbiomes, while Chris is attempting the aiFV primer sets again. Slow going with COVID, but we’ll get there!