Aquariums, Gels, and Fieldwork!

Summer is almost here, but classes are already done for the semester, and the lab has been hard at work on a couple of our new projects- with some promising results to boot! After a [somewhat frustrating] time validating PCR-based protocols for detecting aquatic flaviviruses in sea cucumbers from January – April, we finally had a solid pipeline for detecting (by qRT-PCR) these aiFVs, which are then targeted with general PCR using degenerate primers targeting a conserved (NS5) region. So far so good. After validating these, optimizing PCR conditions, and and generating new cDNA from our bank of RNA extracts from sea cucumbers (from 2016 – 2017), we applied the new approach and came up with our first estimate of aiFV prevalence at a specific site (Ketchikan) – which is around 20%. Hooray! These viruses are not so incredibly rare that one needs to sample hundreds to get a solid estimate of their abundance, nor do we have to perform innumerable replicates in experiments to ensure that we have aiFVs included amongst our specimens.

The first field trip of the season was to our -80 freezer to retrieve samples of Apostichopus californicus from the Salish Sea that we collected on board the R/V Barnes in January 2016. We have a total of 18 intact, small specimens which have been sitting at -80 since collection. These will provide an important backdrop for our current work.

The second field trip fo the season so far was to the supermarket, where we sought samples of A. californicus. Unfortunately we could only obtain samples of Apostichopus japonicus which were farmed in Taiwan, but we’ve included these since they may provide interesting comparisons to A. californicus. However, turning to online ordering, we obtained 30 A. californicus from last year’s sea cucumber fishery in Southeast Alaska. Jay extracted RNA from these, converted this to cDNA and ran it through our pipeline – drum roll… about 5 specimens (4 Alaskan, 1 from Taiwan) produced a qRT-PCR signal which now awaits confirmation through general PCR and sequencing. But it’s exciting to see even if these are from an undisclosed site!

Next week, we’ll be assessing extraction efficiency of this virus by first creating amplified RNA of the NS5 gene (via in vitro transcription) and then spiking this in samples before RNA extraction. We’ll also be testing a brand new approach which will lead to a field-deployable, isothermal test for aiFV infection (RT-LOOP). This is the first time the lab has used this approach for any purpose, and we’re excited to test this, and crude extraction methods to determine distribution of aiFVs with fishery, tribal, and secondary school partners in SE Alaska.

Jordan’s also been on fieldwork in Cape Cod looking at seagrass microbiomes in concert with Katie Haviland (graduate student, Cornell Natural Resources). He’s collecting rhizome and blade samples for a time series survey of microbiome constituents – and in collaboration with Katie and Audrey Vinton (Cornell ’22) will be looking at seagrass viruses and their covariation with the putative seagrass pathogen Labyrinthula zosterae. As part of this work, Jordan’s going to be taking back cores of seagrasses to the lab in Ithaca, so Ian and Chris have been busy preparing our aquarium room for use.

The aquarium facility in Wing Hall has been shut down since August 2019 because our work on sea stars (we’d housed 5 – 20 sea stars in there between 2015 and 2019) has concluded. After a thorough clean, refit (with lights!) and warming up (sea stars were housed at 8oC, while these seagrasses are at around 17oC) it’s like a whole new facility!

Finally, Ian learned that he’d been promoted to full professor starting July 1st. It’s a great honor and many thanks to many people for their support, including administrative staff for helping to prepare his dossier, reviewers for their review, and all those who wrote letters in support.

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