Finally, a return to field research! After sitting out 2020 because of risk to our team and communities we would visit from the SARS-CoV-2 pandemic, and thanks to vaccination and better awareness of disease prevention, the team travelled to Sitka, Alaska (Sheet’ká) to work with colleagues at the University of Alaska Southeast. In 2019, Ian had visited Sitka to collect whole specimens for an imaging study of asteroid rugosity that informed the boundary layer oxygen depletion hypothesis which was published earlier this year. The work carried out in mid-November this year follows on from this idea as part of our NSF-funded project examining the impacts of microbial remineralization and productivity on holothurian (sea cucumber) flavivirus replication. This work was performed on the unceded territories of the Sheet’ká Kwáan, on what is currently known as Sitka, Alaska. The team acknowledges that Lingít (Tlingit) Peoples have been stewards of this land since time immemorial and we are grateful for that stewardship, care, and knowledge.

After a very quick turnaround between overseas travel (Ian was in Australia visiting Family for the first time in 19 months), a successful PhD defense (congratulations, Elliot!) and frantic packing, PI Ian and senior research support specialist Chris, flew to Southeast Alaska to conduct an experiment testing how hypoxia and organic matter enrichment influence animal surface microbial productivity, composition (i.e. microbiome), viral abundance and replication, and animal health. The hypothesis guiding this experiment is that organic matter enrichment and hypoxia drive viral replication. Since the vast majority of viruses we have detected in the past, including the sea cucumber flavivirus (PcaFV), we do not have any expectation that enhanced replication will lead to disease. However, we took samples to look at histopathology, cytology, and fluid chemistry in case we saw lesions compatible with sea cucumber (and sea star) wasting observed in the past.

Our first day was spent setting up, which included preparation of our mesocosms. While we’d hoped to perform the experiments in rigid cattle feed troughs, unfortunately they were unavailable in time to be shipped out to Alaska. So, we turned to an old style of container which Ian had used extensively in the past: inflatable kiddie pools. While this may seem inappropriate, in fact, these are used in a variety of marine research topics since they hold water, and fish and other animals avoid bumping into the sides. The pools were filled with seawater from the nearby Sitka Sound, which we replaced on a 50% water change basis (i.e. 50% of the water was replaced daily). After filling the pools to soak (to remove contaminants from manifacture), we waited while collaborators at UAS retrieved sea cucumbers.

After a couple of days of hunting, our collaborators retrieved a total of 48 sea cucumbers, which we split into 7 treatments. These were: 1) dinitrogen gas sparking, which reduced ambient oxygen by 90% compared to controls; 2) glucose, which is usable by a good proportion of marine bacteria; 3) peptone, which is a common constituent of marine culture media; 4) N-acetylglucosamine, which is the greatest contributor to organic matter excreted by phytoplankton in seawater; 5) a combination of fucose and rhamnose, which are subunits of macroalgal-derived organic matter; and 6) controls, lots and lots of controls. Each day after removing 50% of the pool volume and replacing it with fresh seawater, the ponds were amended with the substrates – we will check to see whether our anticipated/calculated enrichment levels were reached by NMR spectroscopy later.

Each day, we weighed, photographed, swabbed (for microbiomes), and collected tube feet (for viral analyses) each specimen. We also collected pool water for chemistry, coelomic fluid, and coelomocytes for analysis of cytology. We also measured bacterial production in ponds and on the surface of animals. More invasive sampling (epidermal biopsy punches) were collected every couple of days for tissue transcriptomics and histopathology.

An early result was that hypoxia induces a distinct shift in animal gross morphology – from ‘football’ (when stimulated due to retraction of longitudinal muscles) to ‘sausage’. In fact, sampling these specimens, which were still alive on the last day of the experiment, was very tricky since their tissues became akin of jello! Another early result based on gross observation: specimens in our control tanks appeared to fare less well than those in glucose or monomer treatments.

After 7 days of doing exactly the same thing each day, often under adverse weather conditions, we wrapped up the experiment and shipped off our samples back to the lab in Ithaca. After some minor headaches with shipping, the samples arrived about 6 days later, still viable!

We look forward to analyzing these specimens in coming months!