Its hard to believe that it’s already been a year since we were funded by NSF to work on sea cucumber flaviviruses! This time last year, the lab was completely remote and we headed into a new calendar year with the hope that we would be back to lab and field work, somewhat, like in pre-pandemic times. For the first 5 months of this year (and this project) we focused on assessing the diversity of aquatic invertebrate flaviviruses (aiFVs) in sea cucumber specimens we already had from various places in the northeast Pacific, Australia, and New Zealand. This rather frustrating time – which involved many trials and failures with new PCR primer sets – eventually led to the discovery that flaviviruses are not diverse at all within sea cucumber populations. Rather, the virus we recovered in 2016, while still present in 2021, did not have substantial strain-level genome variability. In some ways this was a great result – whatever detection protocol we settled on (see below) was sure to pick up only this one virus, and not closely related strains (which is what we found in our last project, looking at sea star associated viruses). On the other hand, it means that there isn’t an exciting, as-yet-undiscovered pool of flaviviruses in sea cucumbers which we could explore. But this is the nature of science – you will only see what you’re looking for with the approaches we have (PCR), and this result is certainly consistent with the fact that we never see enveloped, ssRNA viruses in metavirome studies.
So into summer, we started to explore the prevalence of the Apostichopus californicus flavivirus (PcaFV) in specimens from the west coast from 2021. While we were unable to travel much due to COVID, several very generous collaborators shared specimens from Ketchikan, Alaska, and Friday Harbor, WA. Jay Crandell, who is an undergraduate researcher, worked diligently over the summer to extract RNA from tissue samples, then apply RT-LAMP (a new approach which does not rely on PCR) to survey its prevalence within the communities for which we had specimens. After much trouble-shooting and ground-truthing, we confirmed that PcaFV is present in around 20% of specimens sampled, and that it is most commonly found in the respiratory tree and ventral body wall tissues. Jay also worked up a citizen science protocol for looking at PcaFV prevalence using RT-LAMP, which we hope to deploy in time for next year’s sea cucumber fishery in October 2022.
Following on from these initial investigations, Ian and Chris traveled to Sitka, Alaska, to work with Joel Markis (University of Alaska Southeast) on an experiment to examine how hypoxia and model organic substrates affect microbiome composition, PcaFV prevalence and load, and animal health. We’re in the early stages of analyses of these samples, but some initial results suggest that PcaFV was present in nearly every specimen collected at the time (November 2021), and that the copy number of viruses was consistently low but detectable in tube feet samples. Work in the new year will focus on processing microbiome samples of surface tissues and swabs; quantitative reverse transcriptase PCR of PcaFV to examine viral load, and histopathology (with Thierry Work, USGS) to look at cytological and histopathologic changes in response to these perturbations. We will return in summer 2022 to perform additional experiments simulating phytoplankton blooms to examine their impacts on microbiome and viral prevalence.
Meanwhile on the seagrass front, Jordan sampled plants in the West Falmouth Harbor (Mass.) over a time course to examine how microbiomes inhabiting leaves and roots change in response to variable productivity. Just before the holiday break, he processed samples for 16S rRNA amplicon sequencing, which are currently in the sequencing queue. Also on the seagrass front, Jordan prepared and sequenced metavirome libraries from seagrass samples collected by collaborators in Japan (Keizo Nagasaki) and New Zealand (Carolyn Lundquist), and discovered novel and somewhat unexpected viruses in those populations. More recently, the lab received additional samples from Sweden (Karin Holmfeldt), Australia, and Sitka, which have been processed and await sequencing by rotation graduate student Carolina Menchaca. Stay tuned for interesting seagrass viruses!
In November, Elliot successfully defended his PhD on sea star densoviruses!
So this year has seen a return to wet lab work and field work, which was all but absent in 2020 because of the SARS-CoV-2 pandemic. What 2022 holds for us is unknown but we expect to make progress on both project foci! In the meantime the team will be taking a well-earned rest over the holidays, but very much look forward to getting back to work in January!
Wishing everyone a safe and healthy holiday period!