Ummm… what’s been going on?

…pretty much nothing and everything’s been happening. The last year (technically 15 months…) has been a busy time for the project(s… more about this later). In the last episode, Ian had just returned from the Aquatic Virus Workshop in Plymouth, UK, and we were expecting sea star wasting to kick in sometime in September – October. The only problem was… there we very few reports of wasting at all. In contrast, we had reports in October 2016 that sea cucumbers in southeast Alaska were experiencing some kind of disease. Given sea cucumbers and sea stars are both echinoderms and the diseases had similar signs – the team jumped into action!

The sea cucumber fishery of southeast Alaska is open from late October through December. Individuals are caught by hand at somewhat secretive locations, the animals are retrieved onto a fishing vessel, where they are ‘poked’ – which basically punctures their coelomic cavity so they take up less room in the ship’s hold – and then placed into a cooler. In late 2016, there were reports from several vessels that recovered and ‘poked’ individuals were rapidly falling apart – in a similar fashion to sea stars. The lab received 10 individuals from fisheries in the region – 5 asymptomatic and 5 ‘sick’ – and put them through the same viral metagenomic workup that we had applied to sea star wasting in 2013-2014. Work on comparing these libraries is still underway, however as a preliminary finding we haven’t really seen anything that was different between healthy and diseased individuals- and SSaDV (or other densoviruses) seemed to be absent from any specimen. Learning from our past experiences, we sought to determine the composition of viral communities in completely asymptomatic populations, so we mounted a sampling expedition to the region in February 2017, where Elliot and Mitch sampled asymptomatic sea cucumbers – and also took the opportunity to sample asymptomatic sea stars, in collaboration with Mike Donnellan (Alaska Department of Fish & Game). The idea here was to then compare the microbial ecology of asymptomatic cucumbers with wasting cucumbers when it appeared in October 2017. Fast forward to the present (Dec 2017) and… no reports of additional cucumber wasting in 2017, sadly…

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Mitch exploring the shoreline of Ketchikan

 

Over the last couple of years we have been trying to induce sea star wasting in the lab – starting with animals collected from Unalaska (see post about shipping disaster!), then attempting to work with animals collected from Whidbey Island. In October – November 2016, we tried once more (for the 5th time) to transmit SSaDV and/or induce wasting using virus-sized homogenates. But after 2 weeks of monitoring the Pisaster ochraceus, Pisaster brevispinus and Evasterias troscheli were totally fine. The definition of insanity is doing the same thing over and over again expecting a different result… so after 7 attempts, this was becoming insane! We’d tried everything, from working with virus-sized material from sea stars in 2013, to whole tissue homogenates from sick stars in 2016, and nothing seems to be transmissible. We even tried transplanting whole tissues from one animal into another animal, to no avail. The sea stars we have in our tanks at Cornell are incredibly robust!

The world’s hardiest sea stars… numerous experiments later, and we still can’t induce disease…

Getting a bit disappointed by the negative results, we turned to other potential stressors to induce SSWD. Temperature has been put forward as a key correlate of disease in wild populations and experiments where aquarium temperature is either raised or lowered seem to suggest that SSWD speeds up or slows down with water temperature increase and decrease, respectively (see Eisenlord et al., 2016 and Kohl et al., 2016). Hence, we performed a couple of experiments in which temperature was really elevated (6 degrees difference between controls and treatments) on Pisaster ochraceus and Pisaster brevispinus – and nothing happened to the animals after 14d. Animals were fine, very active, no SSWD, and behaving normally. Its hard to make definite conclusions based on a single experiment, but these animals are very hardy!!

In April 2017, Ian’s BioMI3500 class performed their annual group projects. This year, all three groups proposed to work with sea stars examining different stressors and their impacts on SSWD. The first group examined differences in salinity (from 25 – 42) on SSWD in Evasterias troscheli; the second examined the impact of viruses on animal activity (righting time) in the east coast species Asterias forbesii, and the third examined the impact of a bacterial culture (a Vibrio strain cultivated from sick stars) on development of SSWD in Pisaster ochraceus. Net result of these experiments? Neither salinity, viral, or bacterial challenge elicited SSWD in the lab. Seeing a pattern?

 

BioMI3500 Class Experiments 2017 – Cultures of Vibrios on TCBS media, Asterias forbesii and 16S rRNA sequences…

In June 2017, Ian started to analyze climate data from 2013 – 2017 (well, technically he’s been tracking it continuously since 2013… but needed at least 4 years to make sense of repeating patterns…). His analysis made is clear that the onset of mass mortality of SSWD in 2013 and 2014 (at different geographic locations) was associated with significant drought conditions on land – and an analysis of precipitation data revealed that SSWD was almost always associated with below-average rainfall for 1 – 3 months prior to the outbreak. Remembering a talk at ASM in 2015 in which the presenter described the presence of freshwater cyanobacterial toxins (microcystins) in sea otters, Ian measured the abundance of microcystins in a subset of sea stars collected in 2013, representing asymptomatic and symptomatic individuals. He also performed an experiment in which Pisaster brevispinus was challenged with high concentrations of microcystin. After 14 days, the brevispinus challenged with microcystin were actually healthier than the controls (with no challenge)…

So if viruses can’t make these ‘hardy’ sea stars sick with viruses, elevated temperature repeatedly didn’t elicit disease, and they don’t respond to common cyanobacterial toxins, then what might cause SSWD? Retrospective analysis of patterns of SSaDV occurrence and load with SSWD signs, especially using a new detection protocol that is more specific to a group of viruses that is associated with wasting sea stars from China, New England and the Northeast Pacific, suggests that the epidemiological association between SSWD and viruses is significant only for one species: Pycnopodia helianthoides. Our prior work in 2014 on sea star wasting included viral-size fraction challenge of only Pycnopodia helianthoides (in which we could induce SSWD). Looking at data on sea star population declines in the Salish Sea (Mondecino-LaTorre et al., 2016), it is clear that SSWD has affected some species more heavily than others – and Pycnopodia helianthoides is by far the worst hit. In 2015 Ian and Jason performed a survey for SSaDV in Kodiak, Alaska and found none (and SSWD hasn’t yet appeared…); in August 2016, there were reports of SSWD at Kodiak, and samples of affected Pycnopodia helianthoides from Kodiak that were sent to the lab were screaming with SSaDV. Although far from being a definitive result, current thought around the lab is that SSaDV (or indeed other closely related densoviruses) are associated with any kind of disease in only Pycnopodia helianthoides. What about other species that also went down in 2013 and beyond? Well, probably different things happened in different places, there may have been spillover of opportunistic infections, or some disease may have been pathogenic and some not pathogenic. It’s a complex tango of various different things happening simultaneously. But our best candidate pathogen-host ineraction is for sunflower stars, which are not functionally extinct in certain areas (i.e. wiped out).

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Ian posing with a Pycnopodia helianthoides in the Salish Sea, Jan 2016. Though they’re still there, they’re rare.

As any good scientist knows, correlation does not equal causation, even if the only thing present in a diseased animal doesn’t happen to be complementary healthy animals. So what are we doing now? In June 2017 we were awarded another grant from the National Science Foundation (co-PIs are Mike Dawson, John Wares and Pete Raimondi, and we’ll be looking to Lauren Schiebelhut to lead!) to examine the impacts of SSWD on population structure of Pisaster ochraceus on the west coast. The idea is that we will perform challenge experiments to test various sea stars of various populations to determine their resistance and determine which genes are under selection (through transcriptomics) during challenge. You might be asking: How will we do this given our failure to induce disease with viral challenge over the last few years? This is where cool science and new approaches come in.

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Sick Pisaster ochraceus from Olympic National Park in 2013 (Photo courtest of Steve Fradkin, NPS). Same thing affecting these as affecting P. ochraceus elsewhere? Or between species? We don’t know.

Rather than simply repeat the same experiment again and again (=insanity?) expecting a different result, Elliot Jackson (who recently passed his A-exam) is currently generating an infectious clone of the densovirus which we can use in controlled (and quarantined) experiments. He’s also employing some new techniques to histopathologically localize SSaDV in animal tissues (i.e. work out which tissues are infected… something we still don’t know… and allow us to ‘see’ the virus) – and having some success in east coast animals. These approaches are still more than a year out from being ready for deployment to experimental settings, but in the meantime, we’re going to throw a new stressor into the experiments (related to observations of climate) and keep trying. We’re also going to revisit a group of microorganisms (and potential pathogens) that we haven’t looked at much… eukaryotic microorganisms. And we also have a nascent collaboration with folks at UBC looking at an enigmatic group of bacteria that inhabits sea star guts, but isn’t likely related to disease (Tenericutes… also known as Mycoplasmas). There’s still much to be learned!

So while we’ve mostly been doing everything and finding mostly nothing over the last 15 months, and wasting continues to wane, we continue to ramp up investigations of the microbial ecology of wasting disease.

Stay tuned.


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