Environmental factors and the infectious disease caused by the protozoan parasite, Perkinsus marinus, in eastern oysters (Crassostrea virginica)

Temperature and salinity are two important factors limiting the distribution and abundance of Perkinsus marinus, a protozoan parasite of Eastern oysters (Crassostrea virginica). Results of laboratory studies ale consistent with field observations and clearly demonstrate that P. marinus susceptibility and disease advancement are positively correlated with temperature, salinity and in situ number of infective cells. Laboratory findings also suggest that environmental degradation may enhance the epizootic, although disease caused by P. marinus in oysters is known to be predominantly exacerbated by elevated temperature and salinity. Oysters cellular defence mechanisms appear ineffective in defence against P. marinus. Also, pollutant esposure caused no significant effects on defence-related activities measured in oysters

Disease processes of the parasite Perkinsus marinus in eastern oyster Crassostrea virginica: Minimum dose for infection initiation, and interaction of temperature, salinity and infective cell dose

Experiments were conducted to: (1) test the response of oysters to different doses of the oyster parasite Perkinsus marinus and to 2 stages, meronts or prezoosporangia; and (2) investigate the synergistic effects of temperature, salinity and infective cell concentration on P. marinus infection in oysters. A dose-dependent response of P. marinus infection was found in oysters inoculated with 0, 10, 10(2), 10(4), and 10(5) meronts or prezoosporangia per oyster and maintained at 22 to 25 degrees C and 14 to 21 ppt for 8 to 12 wk. The minimum dose required to infect oysters was 10(2) meronts or prezoosporangia per oyster through shell cavity inoculation. Interactive effects between temperature, salinity, and infective cell dose on P. marinus prevalence was insignificant in the experiment, in which oysters were challenged by 0, 2.5 x 10(3) or 2.5 x 10(4) meronts per oyster and held at 9 temperature-salinity regimes (10, 15 and 25 degrees C at 3, 10 and 20 ppt). However, there was a significant positive interaction relevant to infection intensity between temperature and salinity, and between temperature and meront dose. Temperature was the most important factor followed, respectively, by the infective cell dose and salinity in determining the susceptibility to P. marinus in oysters. Reduced condition index was observed in moderately to heavily infected oysters and in oysters at 25 degrees C

Seasonal Variation Of Heat Shock Protein 70 In Eastern Oysters (Crassostrea Virginica) Infected With Perkinsus Marinus (Dermo)

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 marinas (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 relationships between hsp70 and these variables. Hsp70 was analyzed at 4 seasonal samplings (fall, winter, spring, and summer months), while all other variables were measured bimonthly. Patterns and amounts of hsp70 expression varied significantly across different seasons, but did not correspond with seasonal temperature. Total amounts of hsp70 were significantly highest in the fall. Seasonal variation in specific isoforms of hsp70 (69 kDa and 72 kDa) was observed. Highest amounts of each were expressed in the spring and fall, respectively, and they were inversely proportional to each other. Differential expression was observed during the winter and spring, with several individuals expressing only hsp72 in the winter and only hsp69 in the spring. Although hsp72 changed concurrently with seasonal changes in infection, both hsp72 and hsp69 did not vary significantly between stocks or with levels of P. marinus infection. This study reveals that measuring total levels of hsp70 do not sufficiently describe the effect of seasonal temperatures on hsp70 expression. Stock mortalities were consistent with the patterns of disease resistance exhibited by their stock parentage, implying existence of a strong genetic component to resistance to Dermo disease. Differences in shell heights, condition index, and P. marinus infection differences showed significant associations among stock, site, and time. Variation in hsp70 did not reflect differences in infection among oyster stocks, indicating that hsp70 may not be a useful indicator to distinguish the effects of pathogenic stress between resistant and susceptible oyster stocks. Differences in expression between hsp69 and hsp72 suggest that seasonal patterns of specific hsp70 isoforms must be understood to determine the role of hsp70 proteins in stress and disease resistance in oysters

An unidentified haplosporidian parasite of bay scallop Argopecten irradians cultured in the Shandong and Liaoning provinces of China

Since 1988 growers of bay scallop Argopecten irradians in China have been experiencing mortality in their cultured stocks. Although poorly documented, mortality apparently began near Qingdao and has since spread to other areas of Shandong and Liaoning provinces. Samples of cultured scallops were collected from several growing areas in these provinces and analyzed by histological methods for pathogens. An unidentified haplosporidian parasite was observed in a high proportion of scallops from two of the stocks examined. Most infections were of low intensity, but one heavy infection was also observed. Only plasmodia stages were observed; they occurred intercellularly in connective tissues throughout the scallops. Plasmodia were spherical to oval, varied from 4.0 to 17.0 mu m in diameter and contained from 2 to 18 nuclei. Absence of spores prevented generic assignment of the parasite. The source and pathogenicity of the haplosporidian could not be assessed without additional research. No other microbial parasites (i.e. rickettsia-like, chlamydia-like or kidney coccidia) were observed in any of the scallops examined

Heat-Shock Proteins Of The Oyster Parasite Perkinsus-marinus

The susceptibility of the eastern oyster Crassostrea virginica to infection by the protozoan parasite Perkinsus marinus is influenced by temperature. Because of the crucial roles of heat shock proteins in cellular thermal tolerance and in host-parasite adaptations in other species, we compared the in vitro heat shock responses of cultured P. marin us and of oyster hemocytes. The parasite and host heat shock proteins were different in size and in immunochemical specificity. In addition, the thermal threshold for inducing the response was higher for P. marinus acclimated to the same temperature as the oysters. The results suggest that EI marinus is likely to employ heat shock proteins as part of its adaptive survival repertoire and that it may be able to function normally under conditions of hyperthermia that evoke an emergency physiological response from the oyster defense cells. Furthermore, they indicate that it is feasible to investigate the host\u27s and parasite\u27s adaptive responses to each other, since their individual responses are readily distinguishable

Immunomodulation by Different Types of N-Oxides in the Hemocytes of the Marine Bivalve Mytilus galloprovincialis

The potential toxicity of engineered nanoparticles (NPs) for humans and the environment represents an emerging issue. Since the aquatic environment represents the ultimate sink for NP deposition, the development of suitable assays is needed to evaluate the potential impact of NPs on aquatic biota. The immune system is a sensitive target for NPs, and conservation of innate immunity represents an useful basis for studying common biological responses to NPs. Suspension-feeding invertebrates, such as bivalves, are particularly at risk to NP exposure, since they have extremely developed systems for uptake of nano and microscale particles integral to intracellular digestion and cellular immunity. Evaluation of the effects of NPs on functional parameters of bivalve immunocytes, the hemocytes, may help understanding the major toxic mechanisms and modes of actions that could be relevant for different NP types in aquatic organisms.In this work, a battery of assays was applied to the hemocytes of the marine bivalve Mytilus galloprovincialis to compare the in vitro effects of different n-oxides (n-TiO2, n-SiO2, n-ZnO, n-CeO2) chosen on the basis of their commercial and environmental relevance. Physico-chemical characterization of both primary particles and NP suspensions in artificial sea water-ASW was performed. Hemocyte lysosomal and mitochondrial parameters, oxyradical and nitric oxide production, phagocytic activity, as well as NP uptake, were evaluated. The results show that different n-oxides rapidly elicited differential responses hemocytes in relation to their chemical properties, concentration, behavior in sea water, and interactions with subcellular compartments. These represent the most extensive data so far available on the effects of NPs in the cells of aquatic organisms. The results indicate that Mytilus hemocytes can be utilized as a suitable model for screening the potential effects of NPs in the cells of aquatic invertebrates, and may provide a basis for future experimental work for designing environmentally safer nanomaterials

Viability, infectivity and fatty acid synthetic activity of Perkinsus marinus meront cells incubated in estuarine and artificial seawater

We investigated the viability and fatty acid synthetic activity of in vitro cultured Perkinsus marinus (Dermo) in lipid-free medium and estuarine water, and the infectivity of P. marinus maintained in artificial seawater (ASW). Viability and fatty acid synthetic activity in 7 d old R marinus meronts maintained in lipid-free medium and estuarine water were tested. The infectivity of meronts incubated in ASW was examined by first incubating P. marinus meronts in ASW for 2, 3 or 7 d, and then inoculating viable ASW-incubated meronts into the shell cavity of individual oysters Crassostrea virginica. P. marinus infection prevalence and intensity in oysters were determined 9 wk post-inoculation. Heavy mortality occurred in meronts maintained in estuarine water, a drop from an initial value of 100% viable to 7.8 and 6.1% after 3 and 14 d incubation, respectively. Viability was 85 and 67% in meronts maintained in lipid-free medium for 3 and 24 d, respectively. Meronts kept in lipid-free medium for 14 d retained their ability to synthesize fatty acids. Viable meronts incubated in ASW remained infective for up to 7 d. The infection prevalences were 85, 48 and 100%, in the treatments inoculated with viable meronts that were incubated in ASW for 2, 3 and 7 d, respectively. Infection prevalence in the group inoculated with viable meronts immediately after they were transferred to ASW ranged from 61 to 85%. Our results suggest that in nature meronts can survive for at least 14 d outside the host. Viable meronts are not only infective, but are also able to replicate and retain their fatty acid synthetic ability for 7 d

Heat shock protein (hsp70) expression and thermal tolerance in sublethally heat-shocked eastern oysters Crassostrea virginica infected with the parasite Perkinsus marinus

To investigate whether sublethal heat shock protects Perkinsus marinus (Dermo)infected oysters Crassostrea virginica from lethal heat stress, and the effects of P. marinus infection on sublethal heat shock response, oysters were first experimentally challenged with P. marinus. Then, when infections in oysters progressed to moderate levels (parasite burden = 10(4) to 10(5) cells g(-1) wet tissue weight), oysters were treated with a sublethal heat shock at 40 C for 1 h (heat shock + Dermo challenge). Other treatment groups included heat-shocked, unchallenged (non-P. marinus challenged) oysters and non-heat-shocked, P. marinus-challenged and -unchallenged oysters. Thermal tolerance was compared among these treatments by administering a lethal heat treatment at 44 C for 1 h, 7 d after sublethal heat shock. Sublethal heat shock enhanced survival to lethal heat treatment in both P. marinus-challenged and -unchallenged oysters. Although levels of hsp70 isoforms (hsp69 and hsp72) did not vary significantly by heat shock or infection with P. marinus, responses due to these treatments were apparent when comparing hsp70 levels within infected and uninfected oysters. Infection enhanced expression of hsp69, regardless of whether oysters were heat shocked or not. In uninfected oysters, hsp72 increased due to heat shock 2 and 7 d post heat shock. Overall, this study demonstrates that heat shock can improve survival in oysters, even in oysters infected with P, marinus. Expression of hsp70 varied among isoforms after sublethal and lethal heat shocks and in infected and uninfected oysters. The heat shock response was not negatively affected by P. marinus infection

Species-specific differences in long-chain n-3 essential fatty acid, sterol, and steroidal ketone production in six heterotrophic protist species

We investigated the capability and species-specific differences in long-chain n-3 essential fatty acid (LCn-3EFA), sterol, and steroidal ketone production of 6 heterotrophic protists: 3 thecate dinoflagellates (Cryptoperidiniopsis brodyi, Pfiesteria piscicida, and Luciella masanensis), 1 athecate dinoflagellate (Amphidinium longum), 1 herbivorous ciliate (Strombidinopsis sp.), and 1 bacterivorous ciliate (Uronema sp.) by feeding them algae (Rhodomonas salina or Dunaliella tertiolecta) or bacteria. The 3 thecate species did not convert algal sterols to other usual and common sterols. Instead, they produced sterols and steroidal ketones, such as dinosterol, dinostanol, dinosterone, and dinostanone, usually found in autotrophic dinoflagellates when fed R. salina or D. tertiolecta, both of which do not contain them, The A. longum, Strombidinopsis sp., and Uronema sp. did not bioconvert dietary sterols to other sterols or produce sterols and steroidal ketones. Pfiesteria piscicida and L. masanensis grown on the LCn-3EFA-deficient alga D. tertiolecta and Uronema sp. were capable of producing the long-chain n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are essential for organisms at higher trophic levels. The bacterial prey of Uronema sp. lacked EPA, DHA, and LCn-3EFA precursors. Although the nutritional values of the sterols and steroidal ketones produced by the 3 thecate dinoflagellates are not known, the contribution of EPA and DHA by 2 of them and the bacterivorous ciliate are noteworthy. To further understand the intermediate roles of heterotrophic protists and their essential nutrient contribution in planktonic food webs, it is necessary to examine more species, particularly those newly discovered and isolated