Mass mortalities of echinoderms have led to major shifts in the ecology of coral reefs in the Caribbean and of kelp forests and rocky shores on the north-west Pacific coast. These have had major implications for biodiversity, carbon capture, and economic impacts on coastal communities. Each case involves microbial activities, echinoderms in two different classes, and algae, but the nature of the ecological changes is very different. Recent research is shedding light on the cause of these mortalities and the prospects for restoration.
Urchin death on Caribbean coral reefs
Mass mortalities of the long-spined black sea urchin Diadema antillarum began near Panama in 1983 and spread rapidly to affect 3.5 million km2 of the Caribbean. Mortality was >90% and urchins were completely wiped out in some areas. These urchins play a key role in maintaining coral reefs by keeping the growth of turf algae in check and providing space for coral larvae to settle. Within a short time, loss of this top-down control resulted in a phase shift to an algal-dominated system, resulting in a loss of biodiversity from which the reefs have never fully recovered. Although a host-specific waterborne disease agent was suspected, methods for investigating marine diseases were very rudimentary at that time. Deposition of dust from North Africa, following a severe drought, was implicated as a contributing factor but a causative agent for the 1983 outbreak could not be identified. Since early 2022, a new mass mortality event has occurred,providing researchers with the opportunity to apply a range of modern molecular methods combined with histopathology and cytology to investigate the condition (Hewson et al., 2023; video).
No evidence for bacteria or viruses as the causative agents. To determine differences in gene expression and the presence of microbes associated with infection, transcriptome libraries were prepared from the coelomic fluid and body tissue of urchins showing disease signs and compared with those from normal individuals collected from affected and unaffected sites. When transcriptomes were analysed to assess difference between the three specimen types, they did not reveal any viruses, archaea, or bacteria uniquely associated with abnormal tissues. It is surprising that so few bacterial sequences were found in this study, since many species of other bacteria can be readily cultured from diseased invertebrate tissues or demonstrated with other DNA-based methods. The authors note that filamentous and coccoid bacterial cells were observed in some histopathological sections and coelomic fluid, but this is not discussed further.
A scutociliate is implicated as the causative agent. When eukaryotic 18S and 23S rRNA sequences were examined, two sequences with significantly greater representation in abnormal urchin tissues were matched to a group of unicellular protists known as scutociliates, which have previously been isolated from diseased corals (Sweet & Séré, 2016) and fish (Rossteuscher et al., 2008). The closest matches were to members of the Philaster genus. Also, when coelomic fluid of affected urchins was examined microscopically, cells with similar morphology to known scutociliate taxa were found. A qPCR assay was used to show that the Philaster-like 23S rRNA gene was highly enriched in tissue samples from abnormal specimens, especially in the body wall and spine base. It was also detected in seawater samples collected close to the urchins. Pure cultures of the ciliate were obtained by growth of coelomic fluid from affected urchins in an enrichment medium, followed by a dilution-to-extinction method. To test whether the isolated ciliates could initiate disease, fluid containing low numbers of the cultured ciliates were pipetted close to the surface of urchins in small aquarium tanks. Within 72-168 h, about 60% of the inoculated specimens showed levels of spine loss not seen in the controls. Using qPCR, the DNA of the Philaster-like ciliate was shown to be enriched in the urchins with spine loss, and in the aquarium water of treated specimens. Ciliates were also observed by microscopy in grossly abnormal tissue samples but absent from controls. Hewson et al. (2023) conclude that the experimental challenge fulfils Koch’s postulates and give a name to the mass mortality syndrome—“D. antillarum scuticociliatosis (DaSc).” In a study of the association of diverse ciliates with coral diseases, Sweet & Séré (2016) concluded that many of the ciliates observed are feeding on the bacteria or other protists which are likely colonizing the disease lesion or the necrotic tissue itself. Whilst there were indications that some ciliates—including Philaster spp.—may directly attack host tissues, Sweet & Séré could not determine their role in disease causation in their study. In the case of DaSc, there is clearly a very strong association between the ciliate and disease signs, but a stronger association between the timing of tissue pathology changes and the conditions that lead to invasion and proliferation of the ciliate would support the conclusion that it can be considered as the sole aetiological agent. Also, whether the ciliate was associated with the 1983 outbreak cannot be determined, as there is unfortunately no preserved material from that time.
Kelp forest ecosystems—the role of keystone predators
On the Pacific northwest coast, the balance of kelp forest ecosystems depends on interactions between herbaceous invertebrates, especially purple sea urchins Strongylocentrotus purpuratus, which graze on the juvenile kelp, and their keystone predators. Historically, sea otters were found all the way from the Aleutian Islands to Baja California and played a critical role in controlling the urchins and allowing vast kelp forests to develop. Two centuries of hunting by fur traders reduced the population of sea otters from an estimated 300,000 individuals to 13 small populations totalling one or two thousand, before an international ban was imposed in 1911. The population in Alaska had recovered to about 100,000 by the 1970s, but has declined since, thought to be due to changes in predation behaviour by orcas and to oil pollution. Populations south of Alaska remain small and patchy in distribution and absent from many parts of the coasts of British Columbia, Washington State, Oregon, and California (apart from an isolated population of ~3000 individuals around the central California coast). Alongside sea otters, grazing by sea stars (asteroids) plays a major role in controlling urchin populations. Several species feed on sea urchins, but the most voracious are the sunflower sea star Pycnopedia helianthoides—which can grow up to a metre across, with 24 arms (rays), and can consume about 5 urchins per week—and the smaller ochre sea star Pisaster ochraceus (five arms, 10-25 cm).
Sea star wasting disease
In the summer of 2013, diseased sea stars began washing up onto the shores of the Olympic Peninsula and British Columbia. The animals showed loss of body turgor, missing limbs, and tissue decay, quickly leading to a disintegrated mush littering the seabed. The condition became known as “sea star wasting disease” (SSWD) and intensive efforts were mobilized to elucidate the cause and investigate the ecological effects of the epizootic. Over 20 species of asteroids have been affected, with the ochre and sunflower stars being particularly hard hit. Within a year, surveys showed that populations were drastically reduced along the entire Pacific coast from Alaska to southern California, with declines of over 75% in all areas and 100% in some areas. (GalLoss of these keystone predators has resulted in trophic cascade, with a catastrophic shift in coastal ecology, with kelp forests replaced by urchin barrens. Restoration programs are in progress, but the recovery of sea star populations and regrowth of kelp beds remains uncertain (Galloway et al., 2023).
Many attempts have been made to identify viruses, bacteria, or fungi that might be responsible for SSWD. A promising lead came from studies by Ian Hewson and colleagues at Cornell University who found that t 0.22 μm-filtered extracts of infected tissue reproduced disease signs in experimental infection of the sunflower sea star, followed by identification of a ssDNA Densovirus as a likely pathogen in the sunflower star (Hewson et al., 2014.) Follow-up studies with other sea star species found that there was no consistent link between the virus and presence of disease (Hewson et al., 2020) and it soon became clear that the syndrome involves a complex repertoire of interactions between geographic locations, host species, pathogenic agents, increased water temperature, and lower levels of dissolved oxygen (Hewson et al., 2018).
Microbiome shifts and altered gene expression. Recent work by a team at the University of Vermont has followed disease progression in both the sunflower and ochre sea stars. In an experimental study of SSWD progression in ochre sea stars, Lloyd & Pespeni (2018) used repeated time sampling to analyse microbial community composition. Animals were collected from an area that had previously experienced an SSWD outbreak, but all were asymptomatic at the start of the experiment. Kept in aquaria, some of the sea stars developed symptoms of varying severity during the two-week experiment, while others remained healthy. Biopsy samples showed no evidence of the densovirus. To characterize the microbial community composition of healthy and sick sea stars, Lloyd & Perspeni amplified the V3-V4 variable region of 16S rRNA genes. Although species richness (the estimated number of microbial taxa) did not differ between healthy and sick individuals, the community composition differed significantly, as measured by Principal Coordinate Analysis. Following analysis of taxonomy-based functional profiling with the KEGG database, 20 pathways were shown to be enriched in healthy individuals and 20 were enriched in sick individuals, The authors suggest that microbes abundant in healthy animals perform functions that inhibit growth of other potentially pathogenic microbes. Comparison of the initial microbial community composition of sea stars that remained healthy versus those that became sick showed that numerous taxa were more abundant in healthy animals, and a small number were higher in animals that eventually became sick. Analysis of the microbiome at different stages of the disease showed distinctive sequential changes, with some bacteria known to be beneficial decreasing in abundance as the disease progressed, while the abundance of some types known to be pathogenic increased.
Surface bacteria thriving on organic matter leading to oxygen depletion. Another study of SSWD in the ochre sea star by the Cornell team followed shifts in microbial abundance and composition at the animal-water interface (Aquino et al., 2021). They concluded that increased organic matter, enhanced by higher water temperatures, led to proliferation of copiotrophic bacteria, resulting in anaerobic conditions that impaired host defence functions. This is supported by a recent study by the Vermont team. McCracken et al. (2023) studied progression of SSWD in wild populations of the sunflower sea star collected off the coast of Southeast Alaska during emergence of an outbreak in 2016. Apparently healthy sea stars were collected from sites where no SSWD was observed (naïve) and sites where SSWD was occurring (exposed). These were compared with sea stars showing clear signs of disease (wasting). Amplification and sequencing of 16SrRNA genes was used to assess microbial community composition. The relative proportions of bacterial taxa differed considerably in samples from the three groups. The microbiome of sea stars that had been exposed to SSWD but showed no signs of disease had an increase in bacteria associated with facultatively anaerobic processes compared to naïve animals; the biggest change was a 1233-fold increase in the level of vibrios. When exposed and wasting individuals were compared, there was a further increase in taxa abundance and a shift to include obligate anaerobes. Transcriptome analysis showed that many functional pathways were enhanced following exposure to the disease, whilst metabolic functions diminished as signs of wasting appeared.
Dysbiosis compromises the sea star immune system and loss of control of tissue integrity. In the most recent study from the Vermont group, Pespeni & Lloyd (2023) collected ochre sea stars from a site which had been heavily impacted two years previously but was showing low signs of SSWD at the time of sampling. They were transferred to aquaria and transcriptome analysis was performed on biopsy samples in individuals which either remained asymptomatic or progressed to show signs of SSWD. Sea stars that remained asymptomatic had a higher expression of genes connected with immune response and tissue integrity compared to those that were developing SSWD signs. Transcriptome analysis of wasting animals reveals changes in RNA processing that indicates loss of neurological control of the maintenance of integrity of their collagenous tissue system and the death of epidermal cells because of inability to overcome hypoxia. Network analysis which integrates changes in the transcriptome and the microbiome revealed that increased abundance of microbes through disease progression was linked to decreased expression of host genes, and the disintegration and decay of host tissue.
There may be no “smoking gun”. The legacy of the great founders of pathogenic microbiology in the 1880s, such as Robert Koch and Louis Pasteur, have unwittingly conditioned us to always seek a single microorganism as the causative agent of a specific disease. For example, many studies have shown increased abundance of Vibrio species in other diseases of marine animals, especially fish and corals (Munn, 2015) and, in some cases, particular species have been identified as causative agents. However, it is often difficult to disentangle causation (Mera & Bourne, 2018). The reality is that a “smoking gun” can often not be identified; many infectious diseases are multifactorial and depend on shifts in the normal microbiome (dysbiosis) and complex changes in the host, influenced by multiple environmental factors. Our view of the nature of disease is changing— propelled by advances in high-throughput sequencing and bioinformatic analysis, our growing recognition of multicellular organisms and their associated microbes as a holobiont, and knowledge of the importance of the complex interactions with environmental factors. In human medicine, we now have strong evidence that many complex diseases are due to this imbalance in microbial communities and a huge number of variables shape its composition. This is why we find it so difficult to assign causes of disease in marine organisms.
References
Aquino, C. A. et al. (2021). Evidence that microorganisms at the animal-water interface drive sea star wasting disease. Front Microbiol 11, 610009.
Galloway, A. W. E. et al. (2023). Sunflower sea star predation on urchins can facilitate kelp forest recovery. Proc. Roy Soc. B 290.
Hewson, I. et al. (2014). Densovirus associated with sea-star wasting disease and mass mortality. Proc Natl Acad Sci U S A 111, 17278–17283.
Hewson, I. et al. (2018). Investigating the complex association between viral ecology, environment, and northeast Pacific Sea Star Wasting. Front Mar Sci 5, 335838.
Hewson, I. et al. (2023). A scuticociliate causes mass mortality of Diadema antillarum in the Caribbean Sea. Sci Adv9.
Hewson, I. et al. 2020. Variation during sea star wasting disease progression in Pisaster ochraceus (Asteroidea, Echinodermata). Viruses 12, 1332.
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