Coral persistence despite marginal conditions in the Port of Miami.

Coral cover has declined worldwide due to anthropogenic stressors that manifest on both global and local scales. Coral communities that exist in extreme conditions can provide information on how these stressors influence ecosystem structure, with implications for their persistence under future conditions. The Port of Miami is located within an urbanized environment, with active coastal development, as well as commercial shipping and recreational boating activity. Monitoring of sites throughout the Port since 2018 has revealed periodic extremes in temperature, seawater pH, and salinity, far in excess of what have been measured in most coral reef environments. Despite conditions that would kill many reef species, we have documented diverse coral communities growing on artificial substrates at these sites-reflecting remarkable tolerance to environmental stressors. Furthermore, many of the more prevalent species within these communities are now conspicuously absent or in low abundance on nearby reefs, owing to their susceptibility and exposure to stony coral tissue loss disease. Natural reef frameworks, however, are largely absent at the urban sites and while diverse fish communities are documented, it is unlikely that these communities provide the same goods and services as natural reef habitats. Regardless, the existence of these communities indicates unlikely persistence and highlights the potential for coexistence of threatened species in anthropogenic environments, provided that suitable stewardship strategies are in place.

Transmission of stony coral tissue loss disease (SCTLD) in simulated ballast water confirms the potential for ship-born spread.

Stony coral tissue loss disease (SCTLD) remains an unprecedented epizootic disease, representing a substantial threat to the persistence and health of coral reef ecosystems in the Tropical Western Atlantic since its first observation near Miami, Florida in 2014. In addition to transport between adjacent reefs indicative of waterborne pathogen(s) dispersing on ocean currents, it has spread throughout the Caribbean to geographically- and oceanographically-isolated reefs, in a manner suggestive of ship and ballast water transmission. Here we evaluate the potential for waterborne transmission of SCTLD including via simulated ballast water, and test the efficacy of commonly-used UV radiation treatment of ballast water. Two species of reef-building corals (Orbicella faveolata and Pseudodiploria strigosa) were subjected to (1) disease-exposed or UV-treated disease-exposed water, and (2) a ballast hold time series of disease-exposed water in two carefully-controlled experiments to evaluate transmission. Our experiments demonstrated transmission of SCTLD through water, rather than direct contact between diseased and healthy corals. While UV treatment of disease-exposed water led to a 50% reduction in the number of corals exhibiting disease signs in both species, the statistical risk of transmission and volume of water needed to elicit SCTLD lesions remained similar to untreated disease-exposed water. The ballast hold time (24 h vs. 120 h) did not have a significant effect on the onset of visible disease signs for either species, though there appeared to be some evidence of a concentration effect for P. strigosa as lesions were only observed after the 120 h ballast hold time. Results from both experiments suggest that the SCTLD pathogens can persist in both untreated and UV-treated ballast water and remain pathogenic. Ballast water may indeed pose a threat to the continued spread and persistence of SCTLD, warranting further investigation of additional ballast water treatments and pathogen detection methods.

Erection of a New Genus and Species for the Pathogen of Hard Clams 'Quahog Parasite Unknown' (QPX): Mucochytrium quahogii gen. nov., sp. nov.

Quahog Parasite Unknown (QPX) is a facultative parasite of the hard clam, Mercenaria mercenaria. Although it has been observed in clams since the 1960's and cultivated since the 1990's, conflicting reports on important aspects of its biology have prevented its formal description. 18S rRNA gene sequences identify QPX as a thraustochytrid, but its production of copious mucus is atypical for this group. There are also conflicting reports about whether QPX shares common features of thraustochytrids, such as the production of an ectoplasmic net and biflagellate zoospores. This study reaffirms the previous descriptions of zoospore production by QPX in culture, in multiple strains from several geographic locations, and provides detail on how to maintain QPX cultures under conditions that promote the production of zoospores. Furthermore, we describe new aspects of the life cycle not previously observed. Finally, we erect Mucochytrium quahogii gen. nov., sp. nov. to accommodate this unusual thraustochytrid.

Differential Gene Expression in Five Isolates of the Clam Pathogen, Quahog Parasite Unknown (QPX).

Quahog parasite unknown (QPX) is a thraustochytrid protist that infects the hard clam, Mercenaria mercenaria, causing significant economic losses along the northeastern coast of North America. Previous investigations noted differences in growth dynamics and virulence in QPX cells from different geographic locations. In order to probe the molecular determinants for these variations, we investigated the transcriptomic profiles of five geographically distinct QPX isolates using custom 15k 60-mer oligonucleotide arrays. A total of 1,263 transcripts were differentially expressed (DE) among the five QPX isolates. The hierarchical clustering of gene expression profiles showed that the QPX isolates from Raritan Bay (RB, NY) and from Provincetown Harbor (MA) were more similar to each other and diverged from QPX isolates from Peconic Bay (PB, NY) and Old Plantation Creek (VA), which had more similar gene expression profiles. The most prominent difference was based on 78 transcripts coding for heat shock proteins DE between the five QPX isolates. The study generated contrasting transcriptomic profiles for QPX isolated from northern (MA) and deeper (RB, NY) locations as compared to southern (VA) and shallower (PB, NY) areas, suggesting the adaptation of the parasite to local environmental, in particular temperature, conditions.

Identification and characterization of peptidases secreted by quahog parasite unknown (QPX), the protistan parasite of hard clams.

Quahog parasite unknown (QPX) is a protistan parasite capable of causing deadly infections in the hard clam Mercenaria mercenaria, one of the most valuable shellfish species in the USA. QPX is an extracellular parasite found mostly in the connective tissue of clam mantle and, in more severe cases of infection, other clam organs. Histopathologic examinations revealed that QPX cells within clam tissues are typically surrounded by hollow areas that have been hypothesized to be, at least in part, a result of extracellular digestion of clam proteins by the parasite. We investigated peptidase activity in QPX extracellular secretions using sodium dodecyl sulfate-polyacrylamide gels containing gelatin as a co-polymerized substrate. Multiple peptidase activity bands of molecular weights ranging from 20 to 100 kDa were detected in QPX secretions derived from a variety of culture media. One major band of approximately 35 kDa was composed of subtilisin-like peptidases that were released by QPX cells in all studied media, suggesting that these are the most common peptidases used by QPX for nutrient acquisition. PCR quantification of mRNA encoding QPX subtilisins revealed that their expression changes with the protein substrate used in the culture media. A fast protein liquid chromatography (FPLC) was used to fractionate QPX extracellular secretions. An FPLC-fraction containing a subtilisin-type serine peptidase was able to digest clam plasma proteins, suggesting that this peptidase might be involved in the disease process, and making it a good candidate for further investigation as a possible virulence factor of the parasite.

Characterisation of the secretome of the clam parasite, QPX.

Secreted and cell surface-associated molecules play a major role in disease development processes and host-pathogen interactions, and usually determine the virulence of invading organisms. In this study, we investigated proteins secreted by quahog parasite unknown, a thraustochytrid protist that infects the hard clam, Mercenaria mercenaria. In silico analysis of quahog parasite unknown transcripts predicted over 1200 proteins to possess an amino-terminal signal peptide which directs proteins into the classical eukaryotic secretory pathway. Proteomic analysis using LC/MS technology identified 56 proteins present in the extracellular secretion of quahog parasite unknown cells grown in vitro, including six mucin-like molecules, four glycosyl hydrolases and eight peptidases. Transcription levels of 19 quahog parasite unknown extracellular proteins were investigated in clam tissue lesions (in vivo) using quantitative PCR. The overexpression of six of these extracellular proteins in clam tissues compared with in vitro cultures suggests that they are involved in interaction with the clam host.

Characterization of the transcriptome and temperature-induced differential gene expression in QPX, the thraustochytrid parasite of hard clams.

BACKGROUND: The hard clam or northern quahog, Mercenaria mercenaria, is one of the most valuable seafood products in the United States representing the first marine resource in some Northeastern states. Severe episodes of hard clam mortality have been consistently associated with infections caused by a thraustochytrid parasite called Quahog Parasite Unknown (QPX). QPX is considered as a cold/temperate water organism since the disease occurs only in the coastal waters of the northwestern Atlantic Ocean from Maritime Canada to Virginia. High disease development at cold temperatures was also confirmed in laboratory studies and is thought to be caused predominantly by immunosuppression of the clam host even though the effect of temperature on QPX virulence has not been fully investigated. In this study, the QPX transcriptome was sequenced using Roche 454 technology to better characterize this microbe and initiate research on the molecular basis of QPX virulence towards hard clams. RESULTS: Close to 18,000 transcriptomic sequences were generated and functionally annotated. Results revealed a wide array of QPX putative virulence factors including a variety of peptidases, antioxidant enzymes, and proteins involved in extracellular mucus production and other secretory proteins potentially involved in interactions with the clam host. Furthermore, a 15 K oligonucleotide array was constructed and used to investigate the effect of temperature on QPX fitness and virulence factors. Results identified a set of QPX molecular chaperones that could explain its adaptation to cold temperatures. Finally, several virulence-related factors were up-regulated at low temperature providing molecular targets for further investigations of increased QPX pathogenicity in cold water conditions. CONCLUSIONS: This is one of the first studies to characterize the transcriptome of a parasitic labyrinthulid, offering new insights into the molecular bases of the pathogenicity of members of this group. Results from the oligoarray study demonstrated the ability of QPX to cope with a wide range of environmental temperatures, including those considered to be suboptimal for clam immunity (low temperature) providing a mechanistic scenario for disease distribution in the field and for high disease prevalence and intensity at low temperature. These results will serve as basis for studies aimed at a better characterization of specific putative virulence factors.

Reconstruction of family-level phylogenetic relationships within Demospongiae (Porifera) using nuclear encoded housekeeping genes.

BACKGROUND: Demosponges are challenging for phylogenetic systematics because of their plastic and relatively simple morphologies and many deep divergences between major clades. To improve understanding of the phylogenetic relationships within Demospongiae, we sequenced and analyzed seven nuclear housekeeping genes involved in a variety of cellular functions from a diverse group of sponges. METHODOLOGY/PRINCIPAL FINDINGS: We generated data from each of the four sponge classes (i.e., Calcarea, Demospongiae, Hexactinellida, and Homoscleromorpha), but focused on family-level relationships within demosponges. With data for 21 newly sampled families, our Maximum Likelihood and Bayesian-based approaches recovered previously phylogenetically defined taxa: Keratosa(p), Myxospongiae(p), Spongillida(p), Haploscleromorpha(p) (the marine haplosclerids) and Democlavia(p). We found conflicting results concerning the relationships of Keratosa(p) and Myxospongiae(p) to the remaining demosponges, but our results strongly supported a clade of Haploscleromorpha(p)+Spongillida(p)+Democlavia(p). In contrast to hypotheses based on mitochondrial genome and ribosomal data, nuclear housekeeping gene data suggested that freshwater sponges (Spongillida(p)) are sister to Haploscleromorpha(p) rather than part of Democlavia(p). Within Keratosa(p), we found equivocal results as to the monophyly of Dictyoceratida. Within Myxospongiae(p), Chondrosida and Verongida were monophyletic. A well-supported clade within Democlavia(p), Tetractinellida(p), composed of all sampled members of Astrophorina and Spirophorina (including the only lithistid in our analysis), was consistently revealed as the sister group to all other members of Democlavia(p). Within Tetractinellida(p), we did not recover monophyletic Astrophorina or Spirophorina. Our results also reaffirmed the monophyly of order Poecilosclerida (excluding Desmacellidae and Raspailiidae), and polyphyly of Hadromerida and Halichondrida. CONCLUSIONS/SIGNIFICANCE: These results, using an independent nuclear gene set, confirmed many hypotheses based on ribosomal and/or mitochondrial genes, and they also identified clades with low statistical support or clades that conflicted with traditional morphological classification. Our results will serve as a basis for future exploration of these outstanding questions using more taxon- and gene-rich datasets.