It’s been a couple of months since last I updated this blog, but this isn’t for lack of things happening… far from it! The team has been extremely busy in the first couple of months of 2022 working to establish new protocols, validate results, and process samples from our field experiment in November 2021. Not to mention the start of teaching (in person), seminars, and for some of our team lots of outside commitments. Here’s a description of what we’ve been up to!
First, the team has been working to process samples collected during organic matter amendment and hypoxia experiments performed in Sitka last November. Coming home with over 1000 samples of various flavors means it takes a bit of time to get through them – not least of which is finding the right samples for each analysis! So far we have extracted nucleic acids from all tissue samples and performed qRT-PCR. These have then been subject to a protocol called loop-mediated isothermal amplification (LAMP for short) to determine which had our target virus, the Apostichopus californicus associated flavivirus (PcaFV). While we attempted this in the field in Sitka, unfortunately we ran into contamination issues which precluded us using those results. Our results showed that ~ 30% of samples were positive for the virus, which is in line with population-level studies that undergraduate researcher Jay is performing as part of his honors thesis. LAMP is performed using a master mix from a different supplier than our normal protocol for detecting viruses by qPCR. We ran into inordinate problems last year trying to amplify both PcaFV and more widely the Tamanavirus family to which PcaFV belongs. Hence, Ian tried using a new master mix for qRT-PCR using the same reverse transcriptase (which converts RNA to DNA)…and it worked. Hence, we’ve used this new master mix to amplify all our RNA samples, both from Sitka as well as population-level surveys on animals collected last year in Ketchikan, Friday Harbor and California. The results not only confirm that LAMP-positive animals have detectable PcaFV by qRT-PCR, but also that the number of virus copies per unit tissue (or ng of RNA) shows some very interesting dynamics over time with organic matter amendment and hypoxia. Stay tuned for super interesting and somewhat unexpected results in future blog posts!
We’re also revisiting our original goal of the project, which is to survey the [micro] diversity of tamanaviruses in natural sea cucumber populations. This work, which is being performed by rotation student Angie, has so far yielded some rather confirmatory results: While there is some minor variation in PcaFV on the nucleotide level, it’s insufficient to call these new variants – so diversity does look very low in natural populations.
In the lab, undergraduate researcher Katie has started to process samples to look at the rate of bacterial activity in our Sitka experiments by first trialling a previously published approach with some samples from our aquarium room. So far we’ve been unable to replicate that protocol, but there are signs that we are able to see some activity using the antibody-based BrDU approach. This approach is entirely new to the Team Aquatic Virus, so there is lots of painstaking validation and optimization, but this approach should allow us to look at microbial remineralization on and near animals, which is critical to understanding boundary layer oxygen limitation.
Also in the lab, undergraduate researcher Ashley has started to process samples for two key indicators of sea cucumber metabolism and health: Lipid and Protein content. Both of these respond on slightly different time scales to changes in the overall condition of animals, and we hope to look at how these vary with different PcaFV loads and with treatments. Again, this is entirely new for the team!
Second, the team has continued to work on seagrass viruses, starting with more viral metagenomics to survey overall RNA viral diversity. Unfortunately some libraries that Ian prepared in December yielded nothing but host transcripts, which was unfortunate and likely due to poor enzyme treatment to remove them. However, Ian has started to use an entirely new, in-house approach to prepare sequence libraries, the first data from which has recently arrived in the lab. Cross your fingers that this time we are able to extract useful information from the somewhat expensive sequencing efforts! Jordan has continued to work on seagrass-associated viruses in our original 3 libraries while awaiting more sequencing data from his microbiome study. So far we have pulled out numerous interesting and unique viruses, some of which bear some resemblance to plant pathogenic viruses. Late last year and early this year we were joined by rotation student Carolina, who attempted to directly amplify some of these viruses from plant tissues; this project is being continued by undergraduate researcher Ricardo who is currently processing the amplicons for Sanger sequencing.
The lab’s also been busy concluding a couple of publications. The first is based on a project led by Caroline Solomon (Gallaudet) which examines RNA virioplankton from the Anacostia River in Washington DC; it reports on putative plant, animal and wastewater viruses and their patterns across an impact landscape in the river. The second (by Elliot Jackson, PhD 2021) is a comprehensive description of RNA viruses in echinoderms, in which he and collaborators surveyed a huge number of published transcriptomes and pulled out a huge number of new viruses which were previously unrecognized in this phyla.
Coming up shortly the BioMI3500 Marine Diseases class will perform group experiments to examine the impact of various stressors on seagrass viruses, cyanobacteria epibionts, and photosynthesis. In groups of 6, students design, propose, execute and analyze a small project on this topic.
Excited to see how things progress!