Oyster Host Research Articles

Serine protease inhibitor cvSI-1 potential role in the eastern oyster host defense against the protozoan parasite Perkinsus marinus

The serine protease inhibitor cvSI-1, purified from plasma of eastern oysters, inhibited the proliferation of the protozoan parasite Perkinsus marinus in vitro. In situ hybridization located cvSI-1 gene expression in basophil cells of the digestive tubules and cvSI-1 expression measured by real-time quantitative reverse transcriptase polymerase chain reaction was several hundred folds greater in digestive glands than in other organs examined or circulating hemocytes. cvSI-1 gene expression was also significantly greater in winter than in summer. Finally, cvSI-1 gene expression and plasma protease inhibitory activity in oysters selected for increased resistance to P. marinus were significantly greater than in unselected oysters. These findings support the hypothesis that cvSI-1 plays a role in eastern oyster host defense against P. marinus possibly through inhibition of parasite proliferation.

Determination of the effects of temperature on viability, metabolic activity and proliferation of two Perkinsus species, and its significance to understanding seasonal cycles of perkinsosis

The range of water temperatures in which Perkinsus species can survive and proliferate remains ill-defined, particularly at lower temperatures. The in vitro viability, metabolic activity, and proliferation of 3 isolates each of P. marinus and P. olseni trophozoites at 28 degrees C, and at 15 and 4 degrees C, after transfer from 28 degrees C, were compared. Both species showed declines in metabolic activity and proliferation from 28 degrees C to 15 degrees C. At 4 degrees C, both species had viability after 30 days incubation time (P. marinus 49%, P. olseni 58%), but limited metabolic activity and no proliferation. Perkinsus marinus viability was further compared when transferred directly from 28 degrees C, 18 degrees C and progressively from 18 degrees C (0.5 degrees C/day) to 2, 4 and 6 degrees C and maintained for up to 4 months. Viability was highest under progressive transfer (77% and 54% after 30 and 60 days exposure to test temperatures). The decrease in P. marinus viability at the lower temperatures in vitro only partially explains decreasing parasite infection intensities in eastern oysters in the colder months of the year. Moreover, the significant decrease in parasite infection intensities in late winter and early spring, as temperatures increase, is likely due to an active process of elimination by oyster host defences.

CDNA cloning and tissue expression of plasma lysozyme in the eastern oyster, Crassostrea virginica

The cDNA sequence of a 17,861 Da lysozyme first purified from plasma of eastern oysters ( Crassostrea virginica) was identified and its complete amino acid sequence deduced. The amino acid sequence of the plasma lysozyme, designated cv-lysozyme 1, contained both a unique and a conserved region when compared to the amino acid sequences of other bivalve lysozymes. In situ hybridisation located cv-lysozyme 1 gene expression in mantle and gill cells in standard histological sections. Quantitative real-time RT-PCR detected cv-lysozyme 1 expression in all organs examined and circulating haemocytes. The number of cv-lysozyme 1 mRNA transcripts was particularly high in mantles and labial palps suggesting those organs are the main sites of cv-lysozyme 1 synthesis. Cv-lysozyme 1 enzyme activity measured by lysing Micrococcus lysodeikticus bacteria and expressed in units per gram tissue was highest in mantles, labial palps and gills. Most cv-lysozyme 1 enzyme activity in oysters was found in plasma. Cv-lysozyme 1 main organs of synthesis, its abundance in plasma and its strong antimicrobial properties suggest its main role is in oyster host defences.

Low incidence and limited effect of the oyster pathogen dermo (Perkinsus marinus) on an artificial reef in Delaware's Inland Bays

Delaware's Inland Bays comprise a large estuarine system with a restricted access to the Atlantic Ocean (Indian River Inlet). As part of a local oyster stock enhancement and restoration effort, we conducted a survey for the protozoan pathogen Perkinsus marinus (Dermo) in oysters from a newly established reef. Using standardized methods for the polymerase chain reaction (PCR) amplification of the non-transcribed spacer (NTS) region, we were surprised to find no detectable titers of this pathogen in the 30 oysters sampled in the first year of the project. The detection threshold of the PCR coupled with chemiluminescent detection was 30 fg P. marinus NTS DNA. We were able to detect a trace presence of this pathogen in a few hard clams (Mercenaria mercenaria) from the same locale, indicating that a Perkinsus sp. was present in the Inland Bay system. Subsequent monitoring of the reef system using a fluid thioglycollate assay over 3 yr revealed no epizootic outbreaks of this pathogen within the planted oyster population. Two large mortality episodes that did appear in the oyster population were attributable to abiotic conditions and not pathogen exposure. This study emphasizes that all potential sources of mortality in the environment are important to consider when designing oyster seeding projects. In the Delaware Inland Bays, P. marinus does not appear to have a large enough oyster host population to become a significant disease threat at present. Because of the low parasite incidence levels in the Inland Bay system in 2000, the James Farm oyster reef restoration project presents an ideal model system to follow the population dynamics between an oyster-host population and a latent or reservoir pathogen population.

A novel slow-tight binding serine protease inhibitor from eastern oyster ( Crassostrea virginica) plasma inhibits perkinsin, the major extracellular protease of the oyster protozoan parasite Perkinsus marinus

A serine protease inhibitor was purified from plasma of the eastern oyster, Crassostrea virginica. The inhibitor is a 7609.6 Da protein consisting of 71 amino acids with 12 cysteine residues that are postulated to form 6 intra-chain disulfide bridges. Sequencing of the cloned cDNA identified an open reading frame encoding a polypeptide of 90 amino acids, with the 19 N-terminal amino acids forming a signal peptide. No sequence similarity with known proteins was found in sequence databases. The protein inhibited the serine proteases subtilisin A, trypsin and perkinsin, the major extracellular protease of the oyster protozoan parasite, Perkinsus marinus, in a slow binding manner. The mechanism of inhibition involves a rapid binding of inhibitor to the enzyme to form a weak enzyme-inhibitor complex followed by a slow isomerization to form a very tight binding enzyme-inhibitor complex. The overall dissociation constants K i⁎ with subtilisin A, perkinsin and trypsin were 0.29 nM, 13.7 nM and 17.7 nM, respectively. No inhibition of representatives of the other protease classes was detected. This is the first protein inhibitor of proteases identified from a bivalve mollusk and it represents a new protease inhibitor family. Its tight binding to subtilisin and perkinsin suggests it plays a role in the oyster host defense against P. marinus.

EFFECT OF HOMOGENATE FROM DIFFERENT OYSTER SPECIES ON PERKINSUS MARINUS PROLIFERATION AND SUBTILISIN GENE TRANSCRIPTION

Abstract The modulation of Perkinsus marinus proliferation and subtilisin gene transcription by host (oyster) tissue was examined. Perkinsus marinus cells were cultured for 4 weeks in media supplemented with extract from either one of four different Crassostrea virginica stocks or with extract from one of two other Crassostrea species, C. ariakensis and C. gigas. After 4 weeks in culture, we determined cell counts and relative subtilisin gene transcription levels using quantitative real-time polymerase chain reaction (qRTPCR). Cell proliferation and subtilisin gene transcription were significantly lower when P. marinus cells were grown in the presence of homogenate from any of the three oyster species than in unsupplemented media. Perkinsus marinus subtilisin gene transcription was also significantly lower in cells cultured in media supplemented with homogenate from either C. ariakensis or C. gigas, than in media containing extract from the native oyster host, C. virginica. Gene transcription levels among...

SEASONAL VARIATION OF HEAT SHOCK PROTEIN 70 IN EASTERN OYSTERS (PERKINSUS MARINUS (DERMO)

Abstract Eastern oysters (Crassostrea virginica) inhabit highly variable environments and are exposed to large seasonal shifts in temperature. Prevalence and intensity of oyster diseases, particularly Perkinsus marinus (Dermo), increase during thermally stressful periods, thus posing additional stress on the oyster host. Heat shock proteins (hsps) are important in protecting organisms from thermal and overall environmental stress. Additionally, hsps may play protective roles for both the host and parasite during infection. The interactive effects of temperature and disease on heat shock protein expression in oysters, however, are unknown. In this study, using slot and western blotting assays, seasonal and intraspecific variation in heat shock protein 70 (hsp70) expression was compared among stocks of C. virginica known to be resistant or susceptible to Dermo at two sites in the Chesapeake Bay. Mortalities, shell heights, condition, and P. marinus infections were also compared among stocks to examine relat...

The role of phenoloxidase suppression in QX disease outbreaks among Sydney rock oysters (Saccostrea glomerata)

Abstract The paramyxean protozoan, Marteilia sydneyi , is the etiological agent of QX disease in Sydney rock oysters ( Saccostrea glomerata ). QX disease affects the farming of oysters in Queensland and on five rivers in New South Wales (NSW), Australia. Disease outbreaks occur during summer months (January to April) and are associated with high mortality rates (up to 98%), which limit the growing out period for oyster production. This study investigated the relationship between oyster host defense systems and QX disease. Oysters from the same brood stock were harvested from QX prone and QX free growing areas over the course of the 2000–2001 outbreak. A variety of parameters—infection intensity in the digestive diverticulum, total hemolymph protein content, condition index and phenoloxidase (PO) activity—were measured in these oysters. Phenoloxidase activity, which is often associated with host defense, was significantly suppressed in oysters from the QX prone area when compared to those from the QX free river. There was also a strict negative correlation between phenoloxidase activity and the intensity of parasitic infection ( p =0.0038). In contrast, neither total protein content nor condition indexes differed between oysters from the QX prone and QX free rivers. These data suggest that inhibition of prophenoloxidase cascade may facilitate lethal infection by M. sydneyi .

Detection of the initial infective stages of the protozoan parasite Marteilia sydneyi in Saccostrea glomerata and their development through to sporogenesis

DNA probes were used in in situ hybridisation on histological sections of oysters exposed for defined intervals to Marteilia sydneyi infection to reveal the early development of the parasite in the oyster host, Saccostrea glomerata. The initial infective stages enter through the palps and gills whereupon extrasporogonic proliferation results in the liberation of cells into surrounding connective tissue and haemolymph spaces. Following systemic dissemination, the parasite infiltrates the digestive gland and becomes established as a nurse cell beneath the epithelial cells in a digestive tubule. Here, cell-within-cell proliferation results in the eventual liberation of daughter cells from the nurse cell into spaces between adjacent epithelial cells. None of these stages had previously been described. Proliferation is associated with host responses, including haemocytic infiltration of the connective tissue and diapedesis across tubule epithelia. The responses cease as sporogenesis begins.

Effects of Physicochemical Factors and Bacterial Colony Morphotype on Association of Vibrio vulnificus with Hemocytes of Crassostrea virginica

Vibrio vulnificus is a naturally occurring marine bacterium that causes invasive disease of immunocompromised humans following the consumption of raw oysters. It is a component of the natural microbiota of Gulf Coast estuaries and has been found to inhabit tissues of oysters, Crassostrea virginica (Gmelin 1791). The interaction of V. vulnificus with oyster host defenses has not been reported in detail. We examined the interaction of V. vulnificus with phagocytic oyster hemocytes as a function of time, temperature, bacterial concentration, pretreatment with hemolymph, and V. vulnificus translucent and opaque colony morphotypes. Within these experimental parameters, the results showed that the association of V. vulnificus with hemocytes increased with time, temperature, and initial V. vulnificus/hemocyte ratio. Pretreatment of V. vulnificus with serum or an increased serum concentration did not enhance V. vulnificus-hemocyte associations, a result suggesting the absence of opsonic activity. More than 50% of hemocytes bound the translucent, avirulent morphotype, whereas 10 to 20% were associated with the opaque, virulent form, a result indicating that the degree of encapsulation was related to resistance to phagocytosis, as previously described for mammalian phagocytes. Understanding these cellular interactions may, in part, explain the persistence of V. vulnificus in oyster tissues and the ecology of V. vulnificus in estuarine environments.