Getting Wasted with Old Foes and New Friends: Microbial Ecology of Sea Star Wasting Disease

Sea star wasting disease (SSWD) has affected over 20 species of asteroids on the North American Pacific Coast. The current geographic extent of this disease is from the Kenai Peninsula to Baja California, making this the most geographically extensive marine disease ever observed. The cause of the disease (at the time of grant funding) was not fully resolved, however previous work by the Team Aquatic Virus and colleagues established that SSWD signs could be elicited by taking filtered tissue homogenates and injecting them into asymptomatic animals, and comparisons of the microbiota inhabiting asymptomatic and symptomatic (i.e. healthy and diseased) sea stars across multiple species suggested association between the disease and the Sea Star Associated Densovirus (SSaDV) (Hewson et al., 2014, PNAS). Furthermore, comparing genes expressed by healthy and diseased animals showed that SSWD signs are associated with apoptotic and degradative processes, as well as changes in the way sea star tissues are bound (Gudenkauf and Hewson, 2015, PLoS One).

There remained at the time of grant funding enormous questions about SSWD that were the focus of this 3-year study.

First, was SSaDV actually associated with SSWD and if so how did it cause disease? Densoviruses belong to a unique group of parvoviruses that in some cases cause disease, while in others do not. From what we knew about parvoviruses generally, they do not cause disease in isolation, but rather are associated with other pathogens that elicit symptoms. The project examined progression of the disease, from entirely healthy and uninfected, to SSWD and eventually animal death through monitoring temporal changes in the microbiome and the host gene expression response.

Second, our previous work highlighted great heterogeneity in the association between SSaDV and SSWD signs, with many individuals bearing SSaDV infection, but lacking SSWD signs. While we attribute this to the ‘incubation period’ (i.e. preclinical infection) at the time those sea stars were sampled, we examined whether there is a relationship between SSWD signs and genetic variation of sea stars at the population level.

Finally, we discovered that SSaDV, based on detection of two genes on its genome, has been present on the west coast for at least 72 years. A key question was why the disease has happened now if it was associated with SSaDV? One clue came from parvoviruses generally, which experience random mutation in genes responsible for viral entry that can strongly influence their ability to infect (i.e. their virulence). We went back in time through museum specimen studies to understand what changes, on the nucleic acid level, had occurred in SSaDV, to try and understand whether such mutations corresponded with the current event.

This project was a collaboration between the Team Aquatic Virus at Cornell University, Seattle Aquarium, Vancouver Aquarium, and the Natural History Museum of Los Angeles County.

Significant Results:

Through this project we have generated transformative new knowledge about disease process in aquatic ecosystems, elucidated the wide distribution and diversity of asteroid viruses, and highlighted hitherto unknown impacts of environmental conditions on in vivo viral dynamics.

Sea star wasting is a condition brought on by biological oceanographic processes, and similar phenomena may impact other marine invertebrates: Sea star wasting disease, which describes a condition affecting over 20 species of asteroid, has caused significant mortality of over 20 species of asteroids. The disease was first observed en masse in June 2013 and caused mass mortality between late September 2013 and April 2014, and has continued to affect localized populations of asteroids since. The condition is not new, and shared characteristics with mass mortality affecting asteroids in the Gulf of Maine, Channel Islands, and elsewhere worldwide since at least 1898. The prominence and intensity of sea star wasting in the northeast Pacific Ocean 2013-2014 led to hypotheses that it was a unique and novel event. However, sea star wasting has no pathognomic signs on either gross or histologic levels (Work et al. submitted). Rather, it comprises a syndrome of common stress responses and autocatalyzed tissue degradation.

We had originally hypothesized that sea star wasting disease is accompanied by changes in holobiont composition and host transcriptomic patterns starting with viral infection, followed by host induction of apoptosis, and finally bacterial proliferation.   Work on this project, which was only possible through concerted characterization of microbiome constituents during wasting progression, has elucidated a mechanism for sea star wasting that relates directly to biological oceanography. Indeed, one of the challenges in determining the cause of sea star wasting disease has been concurrent disciplinary (i.e. marine ecology vs biological oceanography vs disease biology)- and subdisciplinary (e.g. pathology vs microbiology)- driven investigations of etiology which each did not find convincing agents of disease. Work on this project strongly suggests that sea star wasting is not a transmissible disease at all (even though it appeared to be based on transmission studies and longshore movement of the disease and into aquarium intakes), but rather a manifestation of elevated heterotrophic bacterial remineralization adjacent to respiratory surfaces (Fig. 1; (Aquino et al. submitted)). Stimulation of bacterial respiration by organic matter produced by phytoplankton (and perhaps macroalgae), derived from terrestrial runoff, and from decaying animal carcasses cause limitation across the diffusive boundary layer overlying animal surfaces. In turn, this causes induction of hypoxia-sensitive processes, including apoptosis and autocatalyzed tissue remodeling, in animal tissues. The process is more pronounced under warmer conditions since oxygen gradients between animals and overlying waters are driven into suboxic conditions. Moreover, the apparent transmissibility of the disease is likely a consequence of rapid remineralization of tissue homogenates during challenge trials, decaying carcasses adjacent to aquarium intake pipes, and local- to regional-scale hikes in primary production. Hence, we rejected our original hypothesis that sea star wasting was the result of a primary viral insult, followed by host apoptosis and then bacterial proliferation and accepted an alternate hypothesis that bacterial proliferation preceded host apoptosis followed by (concomitant with) viral proliferation.

This result has considerable implications for other benthic invertebrates, especially those that do not actively ventilate using lungs or gills. For example, sponges, which have experienced disease in the Salish Sea in 2016, may have been affected by a similar process limiting oxygen near animal surfaces because of organic matter uptake. Similarly, and perhaps more strikingly, this same etiology has been described in scleractinian corals and various described coral diseases, which are fuelled by oxygen deficit generated by algal exudates (Dinsdale and Rohwer 2011; Haas et al. 2011; Roach et al. 2017). Moreover, based on the results of this project, it would be imperative to exclude this phenomenon before or in concert with searching for novel pathogenic agents in other marine invertebrate diseases.

Densoviruses are widespread and diverse in asteroids but unlikely to generate significant pathology in hosts: Based on early observations of SSaDV in museum asteroid specimens dating back to 1959, we initially hypothesized that SSaDV antagonism in contemporary asteroids is accompanied by changes in genome sequence compared to historical SSaDV which was not associated with SSWD. Subsequent genomics efforts on museum specimens revealed that qPCR primers initially used to determine SSaDV presence in tissues also picked up on previously unknown densoviruses within those tissues. This led to a further effort to characterize densoviral diversity in historical and contemporary samples.

Through this project we have identified numerous novel densoviruses through viral surveillance (Jackson et al. 2020; Jackson et al. submitted) and through efforts comparing wasting and asymptomatic individuals (Hewson et al. 2018; Jackson et al. submitted). Moreover, we have learned a great deal about how the diversity of such closely related viral groups can cause spurious results, as was the case in 2014, which may lead to inaccurate hypotheses about disease association
(Hewson 2019; Hewson et al. 2018). However, these studies have also revealed important and novel information about densoviruses as a viral group, including their widespread association with echinoderms and persistent infection. The association of specific densoviral clades with echinoderms, and difference between these and endogenized viral elements suggest that they coevolved over time to form stable symbioses. Furthermore, these results suggest that densoviruses likely do not generate considerable pathology of their hosts, but rather may form a stable part of the sea star microbiome. Hence, we rejected our initial hypothesis and accepted the null that numerous densoviruses are associated with asteroids but do not yield pathogenic infections.

Metazoan viruses are responsive to environmental conditions: Our observation of viral proliferation during animal death, and the link between disease process and suboxic conditions, suggest that inter-and intra-cellular RNA viruses may respond to extracellular physico-chemical conditions. The rapid replication of RNA viruses under stress may be coupled with cytopathic effects, which may in turn exacerbate those stressors. The relative load of these viruses may therefore dictate severity of ex vivo stressors. Hence, there is great need to further understand the interplay between presumably asymptomatic viral infections and their dynamics during stressful environmental conditions.

Wasting susceptibility in context of host parameters: We originally hypothesized that asteroid larvae and juvenile sea stars do not experience the same antagonistic effects of SSaDV as adult asteroids, and larvae may be a source of viral dispersal. This hypothesis was based on field observations in 2013-2014 that larger stars appeared to be more susceptible to wasting than smaller specimens. Unfortunately, we were never able to survey wasting or microbial associations in larval asteroids, save for the presence of SSaDV in gametes released from stressed asteroids. Furthermore, study of SSaDV tropism suggested that gonads are not a large reservoir of SSaDV (Jackson et al. submitted). However, we examined the size-wasting susceptibility relationship in context of theoretical respiration and surface area: volume ratios.

Prior to this project, much basic information about the biology and physics of asteroids was poorly resolved. Through our work, we have defined allometric relationships between animal volume and ray length, and relative rugosity between species with varying body plans (Asterinidae vs Asteriidae etc). We furthermore defined the basal respiration rate of asteroids and established a model of theoretical respiration rate against animal size. We found that highly susceptible stars were not only more rugose than those species that were less affected by wasting (which argues for more extensive diffusive boundary layers), but they also had a basal respiration rate that was just below, or at , their theoretical oxygen uptake (calculated based on total surface area). We also found that wasting susceptibility related directly to overall surface area of specimens. Hence, we accepted the hypothesis that smaller, juvenile stars would be more resilient to wasting because they respired less as a fraction of their theoretical respiratory rate, they had a much higher surface area:volume than adult stars, and they were typically less rugose than adult specimens, suggesting greater diffusive boundary layer extent.

Finally, there was very scant information available about the asteroid-associated microbiome leading into our work, and no estimates of bacterial cell abundance on animal surfaces. Through our work we identified the core microbiome of starfish surfaces as comprising a relatively low diversity community dominated by copiotrophic gammaproteobacteria (notably alteromonads), spirochaetes, mycoplasmas, and planctomycetes (Jackson et al. 2018). We also established their moderately high cell density (2 – 10 X higher than surrounding water. In turn, these observations directed standardized approaches for comparing amplicon libraries and metagenomes in studies comparing grossly normal to disease-affected individuals (Hewson 2019).

Work completed in this project:

See blog posts at: https://seastarwastingdisease.wordpress.com

Alaska Sea Star Expedition 2015

Historical Sampling Expedition 2015

Australia Echinoderm Expedition 2015

Salish Sea Research Cruise 2016

East Coast Sea Star Expedition Jun 2016

Puget Sound Sea Star Retrieval and Experiments 2016

China Sea Star Expedition 2018

Southeast Alaska Echinoderm Virus Survey 2017

Puget Sound Sea Star Retrieval 2017

New Zealand Sabbatical Spring 2018

Santa Cruz Summer 2018

Bodega Bay Marine Lab Summer 2019

Sitka and Santa Cruz Imaging Study 2019

Publications on this project to date:

Aquino CA, Besemer RM, DeRito CM, Kocian J, Porter IR, Raimondi P, Rede JE, Schiebelhut LM, Sparks JP, Wares JP, Hewson I (2021) Evidence that microorganisms at the animal-water interface drive sea star wasting diseaseFrontiers in Microbiology. DOI: https://www.frontiersin.org/articles/10.3389/fmicb.2020.610009/

Hewson I, Aquino CA, DeRito CM (2020) Virome variation during sea star wasting disease progression in Pisaster ochraceus (Asteroidea, Echinodermata) Viruses 12: 1332

Hewson I, Johnson MR, Tibbetts I (2020)An unconventional flavivirus and other RNA viruses in the sea cucumber (Holothuroidea; Echinodermata) virome Viruses 12: 1057

Jackson EW, Wilhelm RC, Johnson MR, Lutz HL, Danforth I, Gaydos JK, Hart MW, Hewson I (2020) Diversity of sea star-associated densoviruses and transcribed endogenized viral elements of densovirus originJournal of Virology DOI: 10.1128/JVI.01594-20

Mordecai GJ, Hewson I (2020) “Coronaviruses in the sea” Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2020.01795

Jackson EW, Pepe-Ranney C, Johnson MR, Distel D, Hewson I (2020) A highly prevalent and pervasive densovirus discovered among sea stars from the North American Atlantic Coast Applied and Environmental Microbiology

Hewson I (2019) Technical pitfalls that bias comparative microbial community analyses of aquatic diseasesDiseases of Aquatic Organisms. 137: 109-124

Hewson I, Sullivan B, Jackson EW, Xu Q, Long H, Lin C, Quijano Cardé EM, Seymour J, Siboni N, Jones MRL, Sewell MA (2019) Perspective: Something old, something new? Review of wasting and other mortality in Asteroidea (Echinodermata) Frontiers in Marine Science https://doi.org/10.3389/fmars.2019.00406

Jackson EW, Pepe-Ranney C, Debenport SJ, Buckley DH, Hewson I (2018) “The microbial landscape of sea star and anatomical and interspecies variability of their microbiome” Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2018.01829

Hewson, I, Bistolas, KSI, Quijano Cardé, EM, Button JB, Foster PJ, Flanzenbaum JM, Kocian J, Lewis CK (2018) Investigating the complex association between viral ecology, environment and North Pacific sea star wasting Frontiers in Marine Science 5:77

Jackson EW, Bistolas KSI, Button JBB, Hewson I (2016) “Novel circular single-stranded DNA viruses among an asteroid, echinoid and holothurian (Phylum: Echinodermata)”. PLoS One. DOI:10.1371/journal.pone.0166093

Gudenkauf BM, Hewson I (2016) Comparative metagenomics of viral assemblages inhabiting four phyla of marine invertebratesFrontiers in Marine Science https://doi.org/10.3389/fmars.2016.00023

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