Genomic and Proteomic Analyses of Extracellular Products Reveal Major Virulence Factors Likely Accounting for Differences in Pathogenicity to Bivalves between Vibrio mediterranei Strains.

Vibrio mediterranei, a bacterial pathogen of bivalves, has exhibited strain-dependent virulence. The mechanisms behind the variations in bivalve pathogenicity between V. mediterranei strains have remained unclear. However, a preliminary analysis of the extracellular product (ECP) proteomes has revealed differences in protein compositions between low- and high-virulence strains; in addition to 1265 shared proteins, 127 proteins have been identified to be specific to one low-virulence strain and 95 proteins to be specific to two high-virulence strains. We further studied the ECP proteins of the three V. mediterranei strains from functional perspectives using integrated genomics and proteomics approaches. The results showed that lipid metabolism, transporter activity and membrane transporter pathways were more enriched in the ECPs of the two high-virulence strains than in those of the low-virulence strain. Additionally, 73 of the 95 high-virulence strain-specific proteins were found to have coding genes in the genome but were not expressed in the low-virulence strain. Moreover, comparisons with known virulence factors in the Virulence Factor Database (VFDB) and the Pathogen-Host Interactions Database (PHI-base) allowed us to predict more than 10 virulence factors in the categories of antimicrobial activity/competitive advantage, the effector delivery system and immune modulation, and the high-virulence strain-specific ECP proteins consisted of a greater percentage of known virulence factors than the low-virulence strain. Particularly, two virulence factors, MtrC and KatG, were identified in the ECPs of the two high-virulence strains but not in those of the low-virulence strain. Most coding genes of the ECP proteins including known virulence factors were identified on chromosome 1 of V. mediterranei. Our findings indicate that variations in virulence factor composition in the bacterial ECPs may partially account for the differences in the bivalve pathogenicity between V. mediterranei strains.

Acute Salinity Stress Disrupts Gut Microbiota Homeostasis and Reduces Network Connectivity and Cooperation in Razor Clam Sinonovacula constricta.

Accumulating evidence demonstrates that it is of great importance to maintain a stable and functional gut microbial community for host's growth and health. However, gut microenvironment is constantly affected by diverse environmental factors. Salinity can cause stress, including hypersaline or hyposaline stress to aquatic species, thereby affecting their growth conditions. Razor clam (Sinonovacula constricta), an economically important bivalve species, inhabits in intertidal and estuarine zones and constantly experiences salinity stress. Yet little is known about how and to what extent clam gut microbiota is affected by salinity stress, while this knowledge is fundamental for clam aquaculture health management. To address this concern, this study compared the temporal differences of gut bacterial signatures and community assembly of S. constricta under normal salinity (NS), low salinity (LS), and high salinity (HS) conditions. Acute salinity stress affected the compositions, structures, and functional potentials of clam gut microbial community, of which salinity stress, hours post stress, and their interaction respectively constrained 7.6%, 16.4%, and 7.9% of community variation. Phylogenetic bin-based null model result revealed that the gut bacterial assembly of three salinity groups seemed to be largely driven by stochastic processes. Network analysis indicated that gut bacterial interspecies interaction exhibited less connected and lower cooperative activity under the conditions of LS and HS compared with NS. Notably, some pathogenic bacteria, including Vibrio and Pseudoalteromonas, were identified as keystone taxa of gut microbial networks in LS and HS groups. Above findings suggest that the clams under LS and HS conditions might be at a higher risk of developing disease. Our findings enhance the mechanism understanding of gut microbial assembly in S. constricta under abiotic factor challenge, which has important implications for clam health control from a microbial ecological perspective.

Genome-Wide Analysis of Family I84 Protease Inhibitor Genes in Three Bivalves Reveals Important Information About the Protein Family's Evolution.

Family I84 serine protease inhibitors are believed to be mollusk specific proteins involved in host defense. The molecular evolution of the family, however, remains to be understood. In this study, the genes of Family I84 protease inhibitors in 3 bivalves, Crassostrea gigas, Crassostrea virginica and Tegillarca granosa, were analyzed at the genomic level. A total of 66 Family I84 genes (22 in C. gigas, 28 in C. virginica and 16 in T. granosa) were identified from the 3 species. They distributed unevenly in the genomes involving 4 chromosomes in C. gigas and 5 chromosomes in C. virginica and T. granosa and some genes were tandemly duplicated. Most genes had 3 exons with 12 genes having 4 exons and 1 gene having 2 exons. All genes but 1 from C. gigas and 1 from T. granosa encoded peptides with a signal sequence at the N-terminus, and the properties of the predicted mature molecules were similar. Four conserved motifs were identified in the 66 amino acid sequences. Collinear analysis revealed higher collinearity between the 2 oyster species in general genes and in Family I84 genes. Phylogenetic analysis of the 66 genes with those previously reported from 3 other bivalves and 1 gastropod showed that Family I84 protease inhibitor genes from the same species tended to be grouped together in terminal branches of the constructed Maximum likelihood tree, but most internal nodes were poorly supported by the bootstrap values. In addition, differences in expression patterns between the genes of a same species were observed in the developmental stages and tissues of C. gigas and T. granosa. Moreover, the co-expression of genes within Family I84 and Family I84 genes with non-Family I84 were also detected in C. gigas and T. granosa. These results suggested that Family I84 protease inhibitor genes evolved by active duplications and structural and functional diversifications after the speciation of related mollusks, and the diversified protease inhibitor family was likely multifunctional.

Characterization of Vibrio mediterranei Isolates as Causative Agents of Vibriosis in Marine Bivalves.

Marine bivalves include species important globally for aquaculture and estuary ecology. However, epizootics of variable etiologies often pose a threat to the marine fishery industry and ecosystem by causing significant mortalities in related species. One of such diseases is larval vibriosis caused by bacteria of the genus Vibrio, which frequently occurs and causes mass mortalities in bivalve larvae and juveniles in hatcheries. During a mass mortality of razor clam, Sinonovacula constricta, juveniles in a shellfish hatchery in 2019, Vibrio mediterranei was identified as a dominant bacterial species in diseased animals and their rearing water. In this study, we selected and characterized 11 V. mediterranei isolates and studied their pathogenicity to the larvae and juveniles of S. constricta and Crossostrea sikamea. We found that V. mediterranei isolates showed various degrees of pathogenicity to the experimental animals by immersion. Injection of the extracellular products (ECPs) of the strains into clam juveniles resulted in similar pathogenicity with strain immersion. Furthermore, the measurements of enzyme activity suggested the existence of virulence factors in the ECPs of disease-causing V. mediterranei strains. Additionally, proteomic analysis revealed that more than 700 differentially expressed proteins were detected in the ECPs among V. mediterranei strains with different levels of virulence, and the higher expressed proteins in the ECPs of highly virulent strains were involved mainly in the virulence-related pathways. This research represented the first characterization of the V. mediterranei strains as causative agents for larval bivalve vibriosis. The mechanisms underlying the pathogenicity and related strain variability are under further study. IMPORTANCE In the marine environment, Vibrio members have a significant impact on aquatic organisms. Larval vibriosis, caused by bacteria of the genus Vibrio, often poses a threat to the marine fishery industry and ecosystem by causing the mortality of bivalves. However, the emerging pathogens of larval vibriosis in bivalves have not been explored fully. Vibrio mediterranei, the dominant bacterium isolated from moribund clam juveniles in a mortality event, may be responsible for the massive mortality of bivalve juveniles and vibriosis occurrence. Thus, it is necessary to study the pathogenic mechanisms of V. mediterranei to bivalve larvae. We found that V. mediterranei was the pathogen of larval bivalve vibriosis, and its extracellular products contributed a critical role for virulence in juveniles. This research is the first report of V. mediterranei as a causative agent for vibriosis in bivalve juveniles. Our results provide valuable information for understanding the pathogenic mechanism of V. mediterranei to bivalve larvae.

Changes in Bacterial Communities of Kumamoto Oyster Larvae During Their Early Development and Following Vibrio Infection Resulting in a Mass Mortality Event.

Vibrio and Ostreid herpesvirus 1 are responsible for mass mortalities of oyster larvae in hatcheries. Relevant works have focused on their relationships with the disease when larval mortality occurs. On the contrary, little is known about how the resident microbiota in oyster larvae responds to Vibrio-infected disease causing mortality as the disease progressed, whereas this knowledge is fundamental to unveil the etiology of the disease. Here, we analyzed the temporal succession of the microbiome of Kumamoto oyster (Crassostrea sikamea) larvae during their early development, accompanied by a Vibrio-caused mortality event that occurred at the post D-stage of larval development in a shellfish hatchery in Ningbo, China, on June 2020. The main causative agent of larval mortality was attributable to Vibrio infection, which was confirmed by linearly increased Vibrio abundance over disease progression. Larval bacterial communities dramatically changed over host development and disease progression, as highlighted by reduced α-diversity and less diverse core taxa when the disease occurred. Null model and phylogenetic-based mean nearest taxon distance analyses showed that the relative importance of deterministic processes governing larval bacterial assembly initially increased over host development, whereas this dominance was depleted over disease progression. Furthermore, we screened the disease-discriminatory taxa with a significant change in their relative abundances, which could be indicative of disease progression. In addition, network analysis revealed that disease occurrence remodeled the co-occurrence patterns and niche characteristics of larval microbiota. Our findings demonstrate that the dysbiosis of resident bacterial communities and the shift of microecological mechanisms in the larval microbiome may contribute to mortality during oyster early development.

Transcriptomic identification of key genes in Pacific oysters Crassostrea gigas responding to major abiotic and biotic stressors.

Oysters are commercially important intertidal filter-feeding species. Mass mortality events of oysters often occur due to environmental stresses, such as exposure to fluctuating temperatures, salinity, and air, as well as to metal pollution and pathogen infection. Here, RNA-seq data were used to identify shared and specific responsive genes by differential gene expression analysis and weighted gene co-expression network analysis. A total of 18 up-regulated and 10 down-regulated shared responsive genes were identified corresponding to five different stressors. Total 27 stressor-specific genes for temperature, 11 for salinity, 80 for air exposure, 51 for metal pollution, and 636 for Vibrio mediterranei pathogen stress were identified in oysters. Elongin-ß was identified as a crucial gene for thermal stress response. Some HSP70s were determined to be shared responsive genes while others were specific to thermal tolerance. The proteins encoded by these stress-related genes should be further investigated to characterize their physiological functions. In addition, the uncharacterized proteins and ncRNAs that were identified may be involved in species-specific stress-response and regulatory mechanisms. This study identified specific genes related to stressors relevant to oyster cultivation. These findings provide useful information for new selective breeding strategies using a data driven method.

Bacterial Community Dynamics in Kumamoto Oyster Crassostrea sikamea Hatchery During Larval Development.

Increasing evidence indicates that microbes colonized in early life stages have a long-term effect on animal wellbeing in later life stages. Related research is still limited in aquatic animals, particularly in bivalve mollusks. In this study, we analyzed the dynamics of the bacterial composition of the pelagic larval stages (fertilized egg, trochophore, D-stage, veliger, and pediveliger) and the sessile postlarval stage (spat) of Kumamoto oyster (Crassostrea sikamea) and their relationships with the rearing water bacterioplankton in a hatchery by using Illumina sequencing of bacterial 16S rRNA gene. Both bacterioplankton and larval bacterial communities changed greatly over larval development, and the two communities remarkably differed (r = 0.956, P < 0.001), as highlighted by the differences in the dominant taxa and bacterial diversity. Ecological processes of larval bacterial communities were measured by abundance-unweighted and abundance-weighted standardized effect sizes of the mean nearest taxon distance (ses.MNTD). The unweighted ses.MNTD analysis revealed that the deterministic process constrained the larval bacterial assembly, whereas the weighted ses.MNTD analysis showed that larval bacterial composition was initially governed by stochasticity and then gradually by determinism in the later stages. SourceTracker analysis revealed that the larval bacteria were primarily derived from an internal source, mainly from larvae at the present stage. Additionally, the abundances of larval bacterial-mediated functional pathways that were involved in the amino acid, energy, lipid and carbohydrate metabolisms significantly altered with the larval development. These findings suggest that bacteria assemble into distinct communities in larvae and rearing water in the hatchery system, and the dynamics of bacterial community composition in larvae is likely associated with larval developmental stages.

Genome Wide Identification and Expression Profiling Indicate Expansion of Family I84 Protease Inhibitor via Gene Tandem Duplication and Divergence in Razor Clam Sinonovacula constricta.

Family I84 protease inhibitors represent a novel family in the MEROPS peptidase database and are likely unique for molluscan host defense. Two Family I84 members, scSI-1 and scSI-2, were reported from the razor clam Sinonovacula constricta in a previous research. In the present study, 12 additional genes, named scSI-3 to scSI-14, were identified via genome wide sequence analyses. Among them, 10 genes were predicted to have a signal sequence, but one (scSI-7) was not. Besides, one sequence (scSI-14) was likely to encode a prematurely terminated peptide. The predicted mature peptides shared characteristics including 12 conserved cysteine residues, isoelectric points of 4.98 to 6.11, and molecular weights of 7.1 to 9.3 kDa with previously reported family members. Four motifs were characterized in 13 predicted mature peptides (with exception of scSI-14), which shared two to four conserved cysteine residues, are possibly to form two functional domain comprised 6 cysteine residues, respectively. At genomic level, all the 14 razor clam Family I84 genes were organized into 3 exons and 2 introns; 13 of them clustered in 3 regions of 100 kb on 3 separate chromosomes, suggesting tandem duplications of related genes. The promoter region of all the 14 genes was predicted to share some transcription factor binding sites, in particular those responsive to pathological and physiological stimuli, but no shared motifs were identified. Analyses also revealed differences in expression patterns among the genes. One gene in a tandem duplicated gene pairs usually showed a higher expression level than the other whereas non-tandem duplicated genes exhibited a higher degree of correlation in expression level. In addition, 8 of the 14 genes demonstrated higher level of expression in Vibrio tolerant clams than in non-tolerant clams following challenges with Vibrio parahaemolyticus. These results generated important information about the evolution of Family I84 protease inhibitors in S. constricta.

Genome-wide identification and characterization of superoxide dismutases in four oyster species reveals functional differentiation in response to biotic and abiotic stress.

BACKGROUND: Oysters inhabit in the intertidal zone and may be suffered from environmental stresses, which can increase the production of reactive oxygen species (ROS), resulting in mass mortality. Superoxide dismutases (SODs) protect oysters from ROS damage through different mechanisms compared with vertebrates. However, the molecular and functional differentiation in oyster SODs were rarely analyzed. RESULT: In this study, a total of 13, 13, 10, and 8 candidate SODs were identified in the genome of Crassostrea gigas, Crassostrea virginica, Crassostrea hongkongensis, and Saccostrea glomerata respectively. The domain composition, gene structure, subcellular locations, conserved ligands, and cis-elements elucidated the SODs into five groups (Mn-SODs, Cu-only-SODs, Cu/Zn ion ligand Cu/Zn-SOD with enzyme activity, Zn-only-SODs, and no ligand metal ions Cu/Zn-SODs). For single domain Cu/Zn-SODs, only one cytosolic Cu/Zn-SOD (cg_XM_034479061.1) may conserve enzymatic activity while most extracellular Cu/Zn-SOD proteins appeared to lose SOD enzyme activity according to conserved ligand amino acid analysis and expression pattern under biotic and abiotic stress in C. gigas. Further, multi-domain-SODs were identified and some of them were expressed in response to biotic and abiotic stressors in C. gigas. Moreover, the expression patterns of these genes varied in response to different stressors, which may be due to the cis-elements in the gene promoter. CONCLUSION: These findings revealed the most extracellular Cu/Zn-SOD proteins appeared to lose SOD enzyme activity in oysters. Further, our study revealed that only one cytosolic Cu/Zn-SOD (cg_XM_034479061.1) may conserve enzymatic activity of SOD. Moreover, the expression patterns of these genes varied in response to different stressors, which may be due to the cis-elements in the promoter. This study provides important insights into the mechanisms through which oysters adapt to harsh intertidal conditions, as well as potential biomarkers of stress response in related species.

Molecular characterization of two CuZn-SOD family proteins in the Pacific oyster Crassostrea gigas.

Superoxide dismutases (SOD) are multifamily antioxidant enzymes, playing an important role in the defense against oxidative stress in all organisms. Genomic information indicated the presence of genetic diversification of the copper and zinc SOD (CuZn-SOD) family in oysters. In the present research, we characterized two CuZn-SOD family proteins, Cg-CuZn-SOD and Cg-dominin3, in the Pacific oyster Crassostrea gigas using comprehensive sequence analyses, recombinant proteins and site-directed mutagenesis, and observations of gene expression in larval and adult oysters. We found that Cg-CuZn-SOD possessed sequence and structural elements conserved in a CuZn-SOD molecule and the recombinant protein was confirmed empirically to have the SOD enzyme activity. In contrast, Cg-dominin3 lacked five of the seven residues essential for the conformation of SOD active center and the recombinant protein did not have the enzyme activity. However, recombinant Cg-dominin3 showed strong binding activities toward zinc and copper ions. Substitutions of five conserved His residues in the active center demolished the SOD activity but enhanced the metal binding capacity in Cg-CuZn-SOD. On the other hand, reinstallation of the five His residues that were assumed to be activity essential and lost in evolution did not restore the SOD enzyme activity in Cg-dominin3. Additionally, the coding genes of the two proteins exhibited different patterns of expression during larval development and in adult oyster in response to zinc challenges. These results have led to the discovery of the first cytoplasmic CuZn-SOD molecule and the confirmation of molecular diversification of extracellular CuZn-SOD homologs in oysters.

The complete mitochondrial genome of Crassostrea hongkongensis from East China Sea indicates species' range may extend northward.

BACKGROUND: Crassostrea hongkongensis is an important mariculture shellfish with a relatively narrow distribution range. Recently, larger wild oysters were identified as C. hongkongensis from Sanmen bay in East China Sea. No natural distribution had been reported for this species here, and its origin remains unknown. METHODS AND RESULTS: We assembled the complete 18,617 bp circular mitochondrial genome of C. hongkongensis from Sanmen bay by next generation sequencing. It included 12 protein-coding genes, 23 tRNAs, and two rRNAs. The A/T content of the mitogenome was higher than its G/C content. Similar values and features were previously found for five other specimens of C. hongkongensis, and were comparable to those of other congeneric species. A phylogenetic analysis based on the 12 protein-coding genes and complete mitochondrial sequence indicated that the six specimens of C. hongkongensis formed a monophyletic group and shared a sister group relationship with C. ariakensis, C. nippona, C. sikamea, C. angulata, C. gigas, and C. iredalei, whereas specimens from the Sanmen bay area clustered later with the five other C. hongkongensis individuals, sharing a sub-clade. The newly sequenced mitogenome had more singleton sites than previously published C. hongkongensis mitogenomes. CONCLUSIONS: Crassostrea hongkongensis may be a native species, and the species' range extends further to the north than previously known. Our data may therefore contribute to a better understanding of the species diversity and conservation of Crassostrea oysters.

Molecular Basis of Taste and Micronutrient Content in Kumamoto Oysters (Crassostrea Sikamea) and Portuguese Oysters (Crassostrea Angulata) From Xiangshan Bay.

Oysters are the most extensively cultivated bivalves globally. Kumamoto oysters, which are sympatric with Portuguese oysters in Xiangshan bay, China, are regarded as particularly tasty. However, the molecular basis of their characteristic taste has not been identified yet. In the present study, the taste and micronutrient content of the two oyster species were compared. Portuguese oysters were larger and had a greater proportion of proteins (48.2 ± 1.6%), but Kumamoto oysters contained significantly more glycogen (21.8 ± 2.1%; p < 0.05). Moisture and lipid content did not differ significantly between the two species (p > 0.05). Kumamoto oysters contained more Ca, Cu, and Zn (p < 0.05); whereas Mg and Fe levels were comparable (p > 0.05). Similarly, there was no significant difference between the two species with respect to total amount of free amino acids, umami and bitterness amino acids, succinic acid (SA), and most flavoring nucleotides (p > 0.05). In contrast, sweetness amino acids were significantly more abundant in Portuguese oysters. Volatile organic compounds profiles of the two species revealed a higher proportion of most aldehydes including (2E,4E)-hepta-2,4-dienal in Kumamoto oysters. Overall, Kumamoto oysters contain abundant glycogen, Ca, Zn, and Cu, as well as volatile organic compounds, especially aldehydes, which may contribute to their special taste. However, free amino acid and flavor nucleotides may not the source of special taste of Kumamoto oyster. These results provide the molecular basis for understanding the characteristic taste of Kumamoto oysters and for utilizing local oyster germplasm resources.

Family I84 protease inhibitors likely constitute a Mollusca-specific protein family functioning in host defense.

Protease inhibitors are proteins or small polypeptides functioning in numerous biological processes in all organisms. The I84 family of protease inhibitors in the MEROPS database represents a novel protease inhibitor family that has been reported in 2 bivalves, Crassostrea virginica and Sinonovacula constricta, and is believed to play a role in host defense. In the present study, 7 new members of Family I84 were identified in 2 bivalves, Meretrix meretrix and Mytilus galloprovincialis, and 1 gastropod, Haliotis discus hannai, at the mRNA level via cDNA cloning. The expression patterns of the newly identified genes varied in response to salinity stresses and pathogen-associated molecular pattern stimulations, suggesting their involvement in the host defense of related species. Additionally, analyses of sequence data in public databases did not reveal any Family I84 protease inhibitor molecules in non-molluscan animals. The results indicated that Family I84 protease inhibitors are likely mollusk specific, constituting a unique host defense mechanism in molluscan species.

Genomic Insights into the Origin and Evolution of Molluscan Red-Bloodedness in the Blood Clam Tegillarca granosa.

Blood clams differ from their molluscan kins by exhibiting a unique red-blood (RB) phenotype; however, the genetic basis and biochemical machinery subserving this evolutionary innovation remain unclear. As a fundamental step toward resolving this mystery, we presented the first chromosome-level genome and comprehensive transcriptomes of the blood clam Tegillarca granosa for an integrated genomic, evolutionary, and functional analyses of clam RB phenotype. We identified blood clam-specific and expanded gene families, as well as gene pathways that are of RB relevant. Clam-specific RB-related hemoglobins (Hbs) showed close phylogenetic relationships with myoglobins (Mbs) of blood clam and other molluscs without the RB phenotype, indicating that clam-specific Hbs were likely evolutionarily derived from the Mb lineage. Strikingly, similar to vertebrate Hbs, blood clam Hbs were present in a form of gene cluster. Despite the convergent evolution of Hb clusters in blood clam and vertebrates, their Hb clusters may have originated from a single ancestral Mb-like gene as evidenced by gene phylogeny and synteny analysis. A full suite of enzyme-encoding genes for heme synthesis was identified in blood clam, with prominent expression in hemolymph and resembling those in vertebrates, suggesting a convergence of both RB-related Hb and heme functions in vertebrates and blood clam. RNA interference experiments confirmed the functional roles of Hbs and key enzyme of heme synthesis in the maintenance of clam RB phenotype. The high-quality genome assembly and comprehensive transcriptomes presented herein serve new genomic resources for the super-diverse phylum Mollusca, and provide deep insights into the origin and evolution of invertebrate RB.

Pathogen proteases and host protease inhibitors in molluscan infectious diseases.

The development of infectious diseases represents an outcome of dynamic interactions between the disease-producing agent's pathogenicity and the host's self-defense mechanism. Proteases secreted by pathogenic microorganisms and protease inhibitors produced by host species play an important role in the process. This review aimed at summarizing major findings in research on pathogen proteases and host protease inhibitors that had been proposed to be related to the development of mollusk diseases. Metalloproteases and serine proteases respectively belonging to Family M4 and Family S8 of the MEROPS system are among the most studied proteases that may function as virulence factors in mollusk pathogens. On the other hand, a mollusk-specific family (Family I84) of novel serine protease inhibitors and homologues of the tissue inhibitor of metalloprotease have been studied for their potential in the molluscan host defense. In addition, research at the genomic and transcriptomic levels showed that more proteases of pathogens and protease inhibitor of hosts are likely involved in mollusk disease processes. Therefore, the pathological significance of interactions between pathogen proteases and host protease inhibitors in the development of molluscan infectious diseases deserves more research efforts.

Dominin and Segon Form Multiprotein Particles in the Plasma of Eastern Oysters (Crassostrea virginica) and Are Likely Involved in Shell Formation.

Dominin and segon are two proteins purified and characterized from the plasma of eastern oysters Crassostrea virginica, making up about 70% of the total plasma proteins. Their proposed functions are in host defense based on their pathogen binding properties and in metal metabolism based on their metal binding abilities. In the present study, the two proteins were further studied for their native states in circulation and extrapallial fluid and their possible involvement in shell formation. Two-dimensional electrophoresis confirmed that the oyster plasma was dominated by a few major proteins and size exclusion chromatography indicated that these proteins were present in circulation in a morphologically homogenous form. Density gradient ultracentrifugation in Cesium Chloride isolated morphologically homogenous particles of about 25 nm in diameter from the plasma and extrapallial fluids. Polyacrylamide gel electrophoresis identified dominin, segon and an unidentified protein as the principal components of the particles and the three proteins likely formed a multiprotein complex that associated to form the particle. Additionally, three major proteins extracted from shell organic matrix were identified based on the apparent molecular weight in SDS-PAGE to correspond to the three major proteins of plasma and protein particles. Moreover, the hemocyte expression of dominin and segon genes measured by real-time RT-PCR increased significantly upon the initiation of shell repair and were significantly greater in younger oysters. These findings suggest that dominin and segon form protein particles by association with each other and perhaps some other major plasma proteins and play a significant role in oyster shell formation.

Testing plasma subtilisin inhibitory activity as a selective marker for dermo resistance in eastern oysters.

Recent findings have suggested that eastern oyster plasma possesses inhibitors of the protease subtilisin, which play a role in the host defense against Perkinsus marinus, a protist parasite causing dermo. A study was conducted to determine whether plasma subtilisin inhibitory activity (PSIA) could be used as a selective marker in breeding programs for dermo resistance. Eastern oysters Crassostrea virginica from 2 wild Louisiana populations shown to differ in dermo resistance were collected and their PSIA was measured. Three groups of oysters were established to spawn from each population. One group was composed of randomly sampled oysters (i.e. unselected) and the other 2 groups were composed of oysters with the highest or lowest PSIA. After spawning, progenies were deployed in October 2014 in a dermo endemic area and sampled quarterly for 2 yr to measure their mortality, growth, P. marinus infection intensity, condition index, PSIA, and the gene expression of 3 subtilisin inhibitors (cvSI-1, cvSI-2, and cvSI-3). Oyster cumulative mortalities of the progenies of all groups increased both years from April to October, concomitant with increasing P. marinus infection intensities. Mortalities and P. marinus infection intensities differed markedly between the 2 populations, but differences between the unselected and selected groups of each population were limited. Measurements of PSIA and cvSI-1, cvSI-2, and cvSI-3 gene expressions between the progenies of all groups showed few differences. CvSI-1 gene expression in surviving oysters of the most susceptible population was increased at the end of the study, adding additional support to the potential role of cvSI-1 defense against P. marinus.

Two I84 family protease inhibitors from Chinese razor clams Sinonovacula constricta expressed in response to environmental challenges.

Protease inhibitors play critical roles in numerous biological processes including host defense in all multicellular organisms. Eighty three evolutionary families of protease inhibitors are currently accommodated in the MEROPS database and the I84 family currently consists of 3 novel serine protease inhibitors from the eastern oyster Crassostrea virginica. In this study, we identified 2 new I84 family members from the Chinese razor clam Sinonovacula constricta, scSI-1 and scSI-2, using cDNA cloning and sequencing. The scSI-1 cDNA consisted of 494 bp with a 282 bp ORF encoding a 93-amino acid polypeptide that was predicted to have a 19-amino acid signal peptide and a 74-residue mature protein with a calculated molecular mass of 8248.5 Da. The scSI-2 cDNA was 490 bp long with a 273 bp ORF encoding a 90-amino acid polypeptide that was predicted to have an 18-amino acid signal peptide and a 72-residue nature protein with a calculated molecular mass of 7528.4 Da. ScSI-1 and scSI-2 shared high sequence similarity with the 3 known members of I84 family and both expressed primarily in the clam digestive glands. Protease inhibitory activity in the clam plasma also exhibited the signature kinetic characteristics of the I84 members from the oyster. In addition, levels of scSI-1 and scSI-2 gene expression in digestive glands and the protease inhibitory activity in plasma elevated significantly in clams challenged by bacterial injections and Vibrio harveyi was more effective than Staphylococcus epidermidis in inducing the gene expression and plasma protease inhibitory activity. Moreover, drastic changes of salinity and temperature also caused significant changes in the gene expression and plasma activity. These results indicated that scSI-1 and scSI-2 represented 2 new members of the I84 family and they likely play a role in clam host defense against infections and in reactions against physiochemical stressors.