An introduction to lesions and histology of scleractinian corals.

Stony corals (Scleractinia) are in the Phylum Cnidaria (cnidae referring to various types of stinging cells). They may be solitary or colonial, but all secrete an external, supporting aragonite skeleton. Large, colonial members of this phylum are responsible for the accretion of coral reefs in tropical and subtropical waters that form the foundations of the most biodiverse marine ecosystems. Coral reefs worldwide, but particularly in the Caribbean, are experiencing unprecedented levels of disease, resulting in reef degradation. Most coral diseases remain poorly described and lack clear case definitions, while the etiologies and pathogenesis are even more elusive. This introductory guide is focused on reef-building corals and describes basic gross and microscopic lesions in these corals in order to serve as an invitation to other veterinary pathologists to play a critical role in defining and advancing the field of coral pathology.

Gill epithelial cell nuclear virus disease is prevalent and widespread in Mya arenaria clams of Chesapeake Bay, USA.

Several historic investigations have reported intranuclear virus infections of Mya arenaria soft-shell clams from the Atlantic coast of North America, but their descriptive details are limited. Among numerous multi-clam samples of Chesapeake Bay M. arenaria that were analyzed histopathologically during clam population surveys from 2000-2009, virus replication apparently caused extreme hypertrophy among the infected nuclei of gill epithelial cells. Infected cells were often abundant within the gill epithelia of affected clams, where their nuclear abnormalities suggested compromised genetic controls of critical cellular physiological functions. Infection prevalences were generally elevated, reaching 90% in 25% of samples. A grand mean prevalence of 67% resulted for all (69) M. arenaria samples of the decadal investigation, which included 1934 individual clams. Infected nuclei of gill epithelial cells were microscopically conspicuous by their extreme hypertrophic diameters of 10 µm or more and their prominent DNA-inclusion bodies. Cells with abnormal, hypertrophic nuclei were often abundant in the epithelia of M. arenaria gills. Transmission electron microscopy revealed abundant, replicating, icosahedral viral particles of 65-85 nm diameter within such hypertrophic nuclei. Viruses frequently occurred in paracrystalline nuclear arrays, showed granular internal contents, and had radial structures that suggested capsid surface ornamentation. Normal heterochromatin of infected nuclei appeared emarginated by dense central masses of replicating virions. Large, electron-dense DNA inclusion bodies routinely occurred at the internal margins of virus-infected nuclei. These may be virus replication centers based on their ultrastructural features and close proximity to replicated viral particles.

Ecology, histopathology, and microbial ecology of a white-band disease outbreak in the threatened staghorn coral Acropora cervicornis.

This study is a multi-pronged description of a temperature-induced outbreak of white-band disease (WBD) that occurred in Acropora cervicornis off northern Miami Beach, Florida (USA), from July to October 2014. We describe the ecology of the disease and examine diseased corals using both histopathology and next-generation bacterial 16S gene sequencing, making it possible to better understand the effect this disease has on the coral holobiont, and to address some of the seeming contradictions among previous studies of WBD that employed either a purely histological or molecular approach. The outbreak began in July 2014, as sea surface temperatures reached 29°C, and peaked in mid-September, a month after the sea surface temperature maximum. The microscopic anatomy of apparently healthy portions of colonies displaying active disease signs appeared normal except for some tissue atrophy and dissociation of mesenterial filaments deep within the branch. Structural changes were more pronounced in visibly diseased fragments, with atrophy, necrosis, and lysing of surface and basal body wall and polyp structures at the tissue-loss margin. The only bacteria evident microscopically in both diseased and apparently healthy tissues with Giemsa staining was a Rickettsiales-like organism (RLO) occupying mucocytes. Sequencing also identified bacteria belonging to the order Rickettsiales in all fragments. When compared to apparently healthy fragments, diseased fragments had more diverse bacterial communities made up of many previously suggested potential primary pathogens and secondary (opportunistic) colonizers. Interactions between elevated seawater temperatures, the coral host, and pathogenic members of the diseased microbiome all contribute to the coral displaying signs of WBD.

Growth and reproduction of Echeneis naucrates from the eastern Gulf of Mexico.

This study describes growth and reproductive characteristics of a facultative elasmobranch symbiont, Echeneis naucrates. Females grew slower but achieved a larger size than males (growth coefficient, K = 0.25 and 0.38 year-1 , and mean maximum size, L∞ = 603 and 477 mm, respectively). Mean relative batch fecundity was 39.5 (s.d. = 13.1). Gonadosomatic indices peaked in July and August for males and females, respectively, with histology evidence of readiness to spawn or active spawning in August. Host-symbiont length ratios increased linearly with sharksucker length (y = 0.0402 + 0.0003x, adjusted R2 = 0.56).

Immunolocalization of skeletal matrix proteins in tissue and mineral of the coral Stylophora pistillata.

The precipitation and assembly of calcium carbonate skeletons by stony corals is a precisely controlled process regulated by the secretion of an ECM. Recently, it has been reported that the proteome of the skeletal organic matrix (SOM) contains a group of coral acid-rich proteins as well as an assemblage of adhesion and structural proteins, which together, create a framework for the precipitation of aragonite. To date, we are aware of no report that has investigated the localization of individual SOM proteins in the skeleton. In particular, no data are available on the ultrastructural mapping of these proteins in the calcification site or the skeleton. This information is crucial to assessing the role of these proteins in biomineralization. Immunological techniques represent a valuable approach to localize a single component within a calcified skeleton. By using immunogold labeling and immunohistochemical assays, here we show the spatial arrangement of key matrix proteins in tissue and skeleton of the common zooxanthellate coral, Stylophora pistillata. To our knowledge, our results reveal for the first time that, at the nanoscale, skeletal proteins are embedded within the aragonite crystals in a highly ordered arrangement consistent with a diel calcification pattern. In the tissue, these proteins are not restricted to the calcifying epithelium, suggesting that they also play other roles in the coral's metabolic pathways.

Expression and Characterization of a Bright Far-red Fluorescent Protein from the Pink-Pigmented Tissues of Porites lobata.

Members of the anthozoan green fluorescent protein (GFP) family display a diversity of photo-physical properties that can be associated with normal and damaged coral tissues. Poritid coral species often exhibit localized pink pigmentation in diseased or damaged tissues. Our spectral and histological analyses of pink-pigmented Porites lobata lesions show co-localization of bright red fluorescence with putative amoebocytes concentrating in the epidermis, suggesting an activated innate immune response. Here we report the cloning, expression, and characterization of a novel red fluorescent protein (plobRFP) from the pink-pigmented tissues associated with lesions on Porites lobata. In vitro, the recombinant plobRFP exhibits a distinct red emission signal of 614 nm (excitation maximum: 578 nm), making plobRFP the furthest red-shifted natural fluorescent protein isolated from a scleractinian coral. The recombinant protein has a high molar extinction coefficient (84,000 M-1 cm-1) and quantum yield (0.74), conferring a notable brightness to plobRFP. Sequence analysis suggests the distinct brightness and marked red shift may be inherent features of plobRFP's chromophore conformation. While plobRFP displays a tendency to aggregate, its high pH stability, photostability, and spectral properties make it a candidate for cell imaging applications and a potential template for engineering optimized RFPs. The association of plobRFP with a possible immune response furthers its potential use as a visual diagnostic and molecular biomarker for monitoring coral health.

Correction: Phylogenetic, genomic, and biogeographic characterization of a novel and ubiquitous marine invertebrate-associated Rickettsiales parasite, Candidatus Aquarickettsia rohweri, gen. nov., sp. nov.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genotypic variation in disease susceptibility among cultured stocks of elkhorn and staghorn corals.

Disease mortality has been a primary driver of population declines and the threatened status of the foundational Caribbean corals, Acropora palmata and A. cervicornis. There remain few tools to effectively manage coral disease. Substantial investment is flowing into in situ culture and population enhancement efforts, while disease takes a variable but sometimes high toll in restored populations. If genetic resistance to disease can be identified in these corals, it may be leveraged to improve resistance in restored populations and possibly lead to effective diagnostic tests and disease treatments. Using a standardized field protocol based on replicated direct-graft challenge assays, we quantified this important trait in cultured stocks from three field nurseries in the Florida Keys. Field tests of 12 genotypes of A. palmata and 31 genotypes of A. cervicornis revealed significant genotypic variation in disease susceptibility of both species measured both as risk of transmission (percent of exposed fragments that displayed tissue loss) and as the rate of tissue loss (cm2 d-1) in fragments with elicited lesions. These assay results provide a measure of relative disease resistance that can be incorporated, along with consideration of other important traits such as growth and reproductive success, into restoration strategies to yield more resilient populations.

Phylogenetic, genomic, and biogeographic characterization of a novel and ubiquitous marine invertebrate-associated Rickettsiales parasite, Candidatus Aquarickettsia rohweri, gen. nov., sp. nov.

Bacterial symbionts are integral to the health and homeostasis of invertebrate hosts. Notably, members of the Rickettsiales genus Wolbachia influence several aspects of the fitness and evolution of their terrestrial hosts, but few analogous partnerships have been found in marine systems. We report here the genome, phylogenetics, and biogeography of a ubiquitous and novel Rickettsiales species that primarily associates with marine organisms. We previously showed that this bacterium was found in scleractinian corals, responds to nutrient exposure, and is associated with reduced host growth and increased mortality. This bacterium, like other Rickettsiales, has a reduced genome indicative of a parasitic lifestyle. Phylogenetic analysis places this Rickettsiales within a new genus we define as "Candidatus Aquarickettsia." Using data from the Earth Microbiome Project and SRA databases, we also demonstrate that members of "Ca. Aquarickettsia" are found globally in dozens of invertebrate lineages. The coral-associated "Candidatus A. rohweri" is the first finished genome in this new clade. "Ca. A. rohweri" lacks genes to synthesize most sugars and amino acids but possesses several genes linked to pathogenicity including Tlc, an antiporter that exchanges host ATP for ADP, and a complete Type IV secretion system. Despite its inability to metabolize nitrogen, "Ca. A. rohweri" possesses the NtrY-NtrX two-component system involved in sensing and responding to extracellular nitrogen. Given these data, along with visualization of the parasite in host tissues, we hypothesize that "Ca. A. rohweri" reduces coral health by consuming host nutrients and energy, thus weakening and eventually killing host cells. Last, we hypothesize that nutrient enrichment, which is increasingly common on coral reefs, encourages unrestricted growth of "Ca. A. rohweri" in its host by providing abundant N-rich metabolites to be scavenged.

Successful cryopreservation of coral larvae using vitrification and laser warming.

Climate change has increased the incidence of coral bleaching events, resulting in the loss of ecosystem function and biodiversity on reefs around the world. As reef degradation accelerates, the need for innovative restoration tools has become acute. Despite past successes with ultra-low temperature storage of coral sperm to conserve genetic diversity, cryopreservation of larvae has remained elusive due to their large volume, membrane complexity, and sensitivity to chilling injury. Here we show for the first time that coral larvae can survive cryopreservation and resume swimming after warming. Vitrification in a 3.5 M cryoprotectant solution (10% v/v propylene glycol, 5% v/v dimethyl sulfoxide, and 1 M trehalose in phosphate buffered saline) followed by warming at a rate of approximately 4,500,000 °C/min with an infrared laser resulted in up to 43% survival of Fungia scutaria larvae on day 2 post-fertilization. Surviving larvae swam and continued to develop for at least 12 hours after laser-warming. This technology will enable biobanking of coral larvae to secure biodiversity, and, if managed in a high-throughput manner where millions of larvae in a species are frozen at one time, could become an invaluable research and conservation tool to help restore and diversify wild reef habitats.

Disease dynamics and potential mitigation among restored and wild staghorn coral, Acropora cervicornis.

The threatened status (both ecologically and legally) of Caribbean staghorn coral, Acropora cervicornis, has prompted rapidly expanding efforts in culture and restocking, although tissue loss diseases continue to affect populations. In this study, disease surveillance and histopathological characterization were used to compare disease dynamics and conditions in both restored and extant wild populations. Disease had devastating effects on both wild and restored populations, but dynamics were highly variable and appeared to be site-specific with no significant differences in disease prevalence between wild versus restored sites. A subset of 20 haphazardly selected colonies at each site observed over a four-month period revealed widely varying disease incidence, although not between restored and wild sites, and a case fatality rate of 8%. A tropical storm was the only discernable environmental trigger associated with a consistent spike in incidence across all sites. Lastly, two field mitigation techniques, (1) excision of apparently healthy branch tips from a diseased colony, and (2) placement of a band of epoxy fully enclosing the diseased margin, gave equivocal results with no significant benefit detected for either treatment compared to controls. Tissue condition of associated samples was fair to very poor; unsuccessful mitigation treatment samples had severe degeneration of mesenterial filament cnidoglandular bands. Polyp mucocytes in all samples were infected with suspect rickettsia-like organisms; however, no bacterial aggregates were found. No histological differences were found between disease lesions with gross signs fitting literature descriptions of white-band disease (WBD) and rapid tissue loss (RTL). Overall, our results do not support differing disease quality, quantity, dynamics, nor health management strategies between restored and wild colonies of A. cervicornis in the Florida Keys.

Cellular responses in sea fan corals: granular amoebocytes react to pathogen and climate stressors.

BACKGROUND: Climate warming is causing environmental change making both marine and terrestrial organisms, and even humans, more susceptible to emerging diseases. Coral reefs are among the most impacted ecosystems by climate stress, and immunity of corals, the most ancient of metazoans, is poorly known. Although coral mortality due to infectious diseases and temperature-related stress is on the rise, the immune effector mechanisms that contribute to the resistance of corals to such events remain elusive. In the Caribbean sea fan corals (Anthozoa, Alcyonacea: Gorgoniidae), the cell-based immune defenses are granular acidophilic amoebocytes, which are known to be involved in wound repair and histocompatibility. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate for the first time in corals that these cells are involved in the organismal response to pathogenic and temperature stress. In sea fans with both naturally occurring infections and experimental inoculations with the fungal pathogen Aspergillus sydowii, an inflammatory response, characterized by a massive increase of amoebocytes, was evident near infections. Melanosomes were detected in amoebocytes adjacent to protective melanin bands in infected sea fans; neither was present in uninfected fans. In naturally infected sea fans a concurrent increase in prophenoloxidase activity was detected in infected tissues with dense amoebocytes. Sea fans sampled in the field during the 2005 Caribbean Bleaching Event (a once-in-hundred-year climate event) responded to heat stress with a systemic increase in amoebocytes and amoebocyte densities were also increased by elevated temperature stress in lab experiments. CONCLUSIONS/SIGNIFICANCE: The observed amoebocyte responses indicate that sea fan corals use cellular defenses to combat fungal infection and temperature stress. The ability to mount an inflammatory response may be a contributing factor that allowed the survival of even infected sea fan corals during a stressful climate event.

Cellular reactions to sedimentation and temperature stress in the Caribbean coral Montastraea cavernosa.

We present evidence of cellular responses to increased sedimentation and temperature in Montastraea cavernosa collected off Broward County, Florida. We sampled corals from six different sites approximately, 500-1000 m off shore, 10-15m depth. Six samples were collected from four sites adjacent to areas of underwater marine dredging (project sites), while the remaining two samples were obtained far away from the influence of the marine dredging (control sites). SSTs around collection time ranged 0.6-0.9 degrees C over the 40-year monthly mean. All specimens collected at project sites exhibited histopathological evidence of mild to moderate sedimentation stress including changes in size and number of mucocytes in epidermis and gastrodermis, attenuation of the epidermal and gastrodermal tissues, presence of cellular debris, and changes in number of zooxanthellae. These findings corroborate results of laboratory-based, sand-application experiments. In addition to the above-noted changes, one specimen exhibited multiple lesions consisting of unusual gastrodermal detachment with infiltration of amoebocytes into the adjacent mesoglea. Tissues surrounding detachment injuries exhibited marked to severe cellular changes. Accumulations of amoebocytes at lesion sites are seldom observed in wild corals. This response may be part of an organized reaction to injury and infection, as has been documented in sea anemones and gorgonians; however, further research is needed on the nature and role(s) of the scleractinian amoebocytes.

The etiology of white pox, a lethal disease of the Caribbean elkhorn coral, Acropora palmata.

Populations of the shallow-water Caribbean elkhorn coral, Acropora palmata, are being decimated by white pox disease, with losses of living cover in the Florida Keys typically in excess of 70%. The rate of tissue loss is rapid, averaging 2.5 cm2 x day(-1), and is greatest during periods of seasonally elevated temperature. In Florida, the spread of white pox fits the contagion model, with nearest neighbors most susceptible to infection. In this report, we identify a common fecal enterobacterium, Serratia marcescens, as the causal agent of white pox. This is the first time, to our knowledge, that a bacterial species associated with the human gut has been shown to be a marine invertebrate pathogen.