Population-specific responses in eastern oysters exposed to low salinity in the northern Gulf of Mexico.

Eastern oysters, Crassostrea virginica, are facing rapid environmental changes in the northern Gulf of Mexico and can respond to these changes via plasticity or evolution. Plastic responses can immediately buffer against environmental changes, although this buffering may impact the organism's ability to evolve in subsequent generations. While plasticity and evolution are not mutually exclusive, the relative contribution and interaction between them remains unclear. In this study, we investigated the roles of plastic and evolved responses of C. virginica acclimated to low salinity using a common garden experiment with four populations exposed to two salinities. We used three transcriptomic analyses (edgeR, PERMANOVA and WGCNA) combined with physiology data to identify the effect of genotype (population), environment (salinity) and the genotype-environment interaction on both whole-organism and molecular phenotypes. We demonstrate that variation in gene expression is mainly driven by population, with relatively small changes in response to salinity. In contrast, the morphology and physiology data reveal that salinity has a larger influence on oyster performance than the population of origin. All analyses lacked signatures of the genotype×environment interaction and, in contrast to previous studies, we found no evidence for population-specific responses to low salinity. However, individuals from the highest salinity estuary displayed highly divergent gene expression from that of other populations, which could potentially drive population-specific responses to other stressors. Our findings suggest that C. virginica largely rely on plasticity in physiology to buffer the effects of low salinity, but that these changes in physiology do not rely on large persistent changes in gene expression.

Lack of genotype-by-environment interaction suggests limited potential for evolutionary changes in plasticity in the eastern oyster, Crassostrea virginica.

Eastern oysters in the northern Gulf of Mexico are facing rapid environmental changes and can respond to this change via plasticity or evolution. Plasticity can act as an immediate buffer against environmental change, but this buffering could impact the organism's ability to evolve in subsequent generations. While plasticity and evolution are not mutually exclusive, the relative contribution and interaction between them remains unclear. In this study, we investigate the roles of plastic and evolved responses to environmental variation and Perkinsus marinus infection in Crassostrea virginica by using a common garden experiment with 80 oysters from six families outplanted at two field sites naturally differing in salinity. We use growth data, P. marinus infection intensities, 3' RNA sequencing (TagSeq) and low-coverage whole-genome sequencing to identify the effect of genotype, environment and genotype-by-environment interaction on the oyster's response to site. As one of first studies to characterize the joint effects of genotype and environment on transcriptomic and morphological profiles in a natural setting, we demonstrate that C. virginica has a highly plastic response to environment and that this response is parallel among genotypes. We also find that genes responding to genotype have distinct and opposing profiles compared to genes responding to environment with regard to expression levels, Ka/Ks ratios and nucleotide diversity. Our findings suggest that C. virginica may be able to buffer the immediate impacts of future environmental changes by altering gene expression and physiology, but the lack of genetic variation in plasticity suggests limited capacity for evolved responses.

Transcriptomic signatures of temperature adaptation in the eastern oyster Crassostrea virginica.

The large geographic distribution of the eastern oyster, Crassostrea virginica, makes it an ideal species to test how populations have adapted to latitudinal gradients in temperature. Despite inhabiting distinct thermal regimes, populations of C. virginica near the species' southern and northern geographic range show no population differences in their physiological response to temperature. In this study, we used comparative transcriptomics to understand how oysters from either end of the species' range maintain enantiostasis across three acclimation temperatures (10, 20, and 30°C). With this approach, we identified genes that were differentially expressed in response to temperature between individuals of C. virginica collected from New Brunswick, Canada and Louisiana, USA. We observed a core set of genes whose expression responded to temperature in both populations, but also an even larger set of genes with expression patterns that were unique to each population. Intriguingly, the genes with population-specific responses to temperature had elevated FST and Ka/Ks ratios compared to the genome-wide average. In contrast, genes showing only a response to temperature were found to only have elevated FST values suggesting that divergent FST may be due to selection on linked regulatory regions rather than positive selection on protein coding regions. Taken together, our results suggest that, despite coarse-scale physiological similarities, natural selection has shaped divergent gene expression responses to temperature in geographically separated populations of this broadly eurythermal marine invertebrate.

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.

Energetic budget of diploid and triploid eastern oysters during a summer die-off.

Triploid oysters are widely used in off-bottom aquaculture of eastern oysters, Crassostrea virginica. However, farmers of the Gulf of Mexico (GoM) and Atlantic coast estuaries have observed unresolved, late-spring die-offs of triploid oysters, threatening the sustainability of triploid aquaculture. To investigate this, the physiological processes underlying oyster growth (e.g., feeding, respiration) and mortality of one-year-old diploid and triploid oysters were compared in early summer following an uptick in mortality. It was predicted that higher triploid mortality was the result of energetic imbalances (increased metabolic demands and decreased feeding behavior). Oyster clearance rates, percentage of time valves were open, absorption efficiency, oxygen consumption rates (basal and routine), ammonia excretion rate were measured in the laboratory and scope for growth was calculated. In addition, their condition index, gametogenic stage, Perkinsus marinus infection level, and mortality were measured. Mortality of triploids in the laboratory was greater than for diploids, mirroring mortality observed in a related field study. The physiological parameters measured, however, could not explain triploid mortality. Scope for growth, condition index, and clearance rates of triploids were greater than for diploids, suggesting sufficient energy reserves, while all other measurements where similar between the ploidies. It remains to be determined whether mortality could be caused from disruption of energy homeostasis at the cellular level.

Comparison of haemocytic parameters among flat oyster Ostrea edulis stocks with different susceptibility to bonamiosis and the Pacific oyster Crassostrea gigas.

Farming of the flat oyster Ostrea edulis in Europe is severely constrained by the protozoan Bonamia ostreae. The introduction of the resistant species Crassostrea gigas has been a relief for the farmers, while the pilot programmes to select O. edulis strains resistant to bonamiosis performed in various countries can be seen as a promising strategy to minimise the effects of bonamiosis. However, the physiological bases of this differential susceptibility remain unknown. A search for an explanation of the intra and interspecific differences in oyster susceptibility to bonamiosis was accomplished by comparing some immune parameters among various O. edulis stocks and C. gigas. On December 2003, naïve and Bonamia-relatively resistant flat oysters from Ireland, Galician flat oysters and Pacific oysters C. gigas were deployed in a Galician area affected by bonamiosis; haemolymph samples were taken in February and May 2004. A new oyster deployment at the same place was carried out on June 2004 and haemolymph sampling was performed on April 2005. On November 2004, new sets of Irish flat oysters and C. gigas were deployed in Ireland and haemolymph sampling was performed in June 2005. Various haemocytic parameters were measured: total and differential haemocyte count, phagocytic ability, respiratory burst (superoxide anion [O(2)(-)] and hydrogen peroxide [H(2)O(2)]) and nitric oxide [NO] production. The comparison of the parameters was carried out at 3 levels: (1) between O. edulis and C. gigas, (2) among O. edulis stocks with different susceptibility to bonamiosis, and (3) between Bonamia-infected and non infected O. edulis. In addition, haemocyte-B. ostreaein vitro encounters were performed to analyse interspecific differences in the haemocytic respiratory burst, using flow cytometry. Significant differences associated with total and differential haemocyte count, and respiratory burst between O. edulis and C. gigas were detected, which could be linked to differences in susceptibility to bonamiosis between both species. Additionally, significant changes in total and differential haemocyte count, and respiratory burst of O. edulis associated with B. ostreae infection were found. However, no consistent difference in any haemocyte parameter between the O. edulis stocks involved in the study was recorded.

Comparison of antibacterial activity in the hemolymph of marine bivalves from Galicia (NW Spain).

A screening study of in vitro antibacterial activity was conducted in marine bivalves with economical importance and widespread along the coast of Galicia (NW Spain). Hemocyte lysate supernatant (HLS) and plasma of Mytilus galloprovincialis, Ostrea edulis, Crassostrea gigas, Ruditapes decussatus, Ruditapes philippinarum, and Cerastoderma edule were incubated with Vibrio splendidus and Micrococcus sp. HLS and plasma for all the species demonstrated antibacterial activity, and C. edule had the highest activity per unit of protein in these hemolymph fractions. Significant differences were not found between HLS and plasma activities. Furthermore, antibacterial activity against Micrococcus sp. (Gram-positive) was stronger than against V. splendidus (Gram-negative).

Increasing the in vitro proliferation rate of Perkinsus mediterraneus, a parasite of the European flat oyster Ostrea edulis.

Perkinsus mediterraneus is an alveolate parasite first described in Ostrea edulis from the Balearic Islands (Mediterranean Sea, Spain), and little is known about its biology or the disease it causes. Continuous in vitro cultures of P. mediterraneus have recently been established in the protein-deficient culture medium JL-ODRP-2F to facilitate its study. Parasite proliferation rate in vitro however was low, with densities increasing 2- to 6-fold between subcultures at 6-week intervals. To increase the proliferation rate of P. mediterraneus cultures to rates similar to other Perkinsus species, various culture conditions (temperature, osmolality, pH, O(2), and CO(2) concentrations), culture procedures (seeding density and frequency of medium changes), concentrations of medium components, and addition of medium supplements (oyster tissue lysate, oyster plasma, animal sera, growth factors, and hormones) were tested. All treatments were evaluated by measuring parasite densities after 2 weeks of culture. The greatest increase in parasite densities, a 35-fold increase over the cell seeding density and 18 times that of the control (cells without supplementation), occurred in medium supplemented with 1,000 µg/mL of O. edulis tissue lysate. P. mediterraneus proliferation was also significantly increased by oyster tissue lysate concentration as low as 125 µg/mL.

Divergence in salinity tolerance of northern Gulf of Mexico eastern oysters under field and laboratory exposure.

The eastern oyster, Crassostrea virginica, is a foundation species within US Gulf of Mexico (GoM) estuaries that has experienced substantial population declines. As changes from management and climate are expected to continue to impact estuarine salinity, understanding how local oyster populations might respond and identifying populations with adaptations to more extreme changes in salinity could inform resource management, including restoration and aquaculture programs. Wild oysters were collected from four estuarine sites from Texas [Packery Channel (PC): 35.5, annual mean salinity, Aransas Bay (AB): 23.0] and Louisiana [Calcasieu Lake (CL): 16.2, Vermilion Bay (VB): 7.4] and spawned. The progeny were compared in field and laboratory studies under different salinity regimes. For the field study, F1 oysters were deployed at low (6.4) and intermediate (16.5) salinity sites in Alabama. Growth and mortality were measured monthly. Condition index and Perkinsus marinus infection intensity were measured quarterly. For the laboratory studies, mortality was recorded in F1 oysters that were exposed to salinities of 2.0, 4.0, 20.0/22.0, 38.0 and 44.0 with and without acclimation. The results of the field study and laboratory study with acclimation indicated that PC oysters are adapted to high-salinity conditions and do not tolerate very low salinities. The AB stock had the highest plasticity as it performed as well as the PC stock at high salinities and as well as Louisiana stocks at the lowest salinity. Louisiana stocks did not perform as well as the Texas stocks at high salinities. Results from the laboratory studies without salinity acclimation showed that all F1 stocks experiencing rapid mortality at low salinities when 3-month oysters collected at a salinity of 24 were used and at both low and high salinities when 7-month oysters collected at a salinity of 14.5 were used.

The combined influence of sub-optimal temperature and salinity on the in vitro viability of Perkinsus marinus, a protistan parasite of the eastern oyster Crassostrea virginica.

Perkinsus marinus is a major cause of mortality in eastern oysters along the Gulf of Mexico and Atlantic coasts. It is also well documented that temperature and salinity are the primary environmental factors affecting P. marinus viability and proliferation. However, little is known about the effects of combined sub-optimal temperatures and salinities on P. marinus viability. This in vitro study examined those effects by acclimating P. marinus at three salinities (7, 15, 25 ppt) to 10 degrees C to represent the lowest temperatures generally reached in the Gulf of Mexico, and to 2 degrees C to represent the lowest temperatures reached along the mid-Atlantic coasts and by measuring changes in cell viability and density on days 1, 30, 60 and 90 following acclimation. Cell viability and density were also measured in 7 ppt cultures acclimated to each temperature and then transferred to 3.5 ppt. The largest decreases in cell viability occurred only with combined low temperature and salinity, indicating that there is clearly a synergistic effect. The largest decreases in cell viability occurred only with both low temperature and salinity after 30 days (3.5 ppt, 2 degrees C: 0% viability), 60 days (3.5 ppt, 10 degrees C: 0% viability) and 90 days (7 ppt, 2 degrees C: 0.6+/-0.7%; 7 ppt, 10 degrees C: 0.2+/-0.2%).

Heat shock protein 70 levels and post-harvest survival of eastern oysters following sublethal heat shock in the laboratory or conditioning in the field.

A major problem of storing and shipping eastern oysters (Crassostrea virginica) from the Northern Gulf of Mexico in summer and early fall is their elevated mortality. A study was therefore conducted to determine whether heat shocking the oysters or conditioning them to aerial exposure prior to harvest could reduce their mortality during cold storage. Increasing the levels of stress proteins in bivalves has been shown to reduce their mortality when exposed to additional stressors. In this study, the levels of heat shock protein 70 (HSP70) proteins and cumulative mortality during cold storage, out of water, of market-sized oysters were measured, in summer, following (1) sublethal heat shocks (41 °C, 1 h) in the laboratory or (2) 3 weeks to 6 weeks of daily exposures to air (0 h, ~ 10 h, or ~ 18 h) in the field. In total, four heat shock and two aerial exposure studies were done. Consistently, heat shocks or 6 weeks of daily aerial exposures increased HSP70 levels in oysters but did not reduce their mortality during cold storage. Three weeks of daily aerial exposure did not increase HSP70 levels and only marginally reduced mortality; a significant reduction in cumulative mortality occurred in one of the aerial exposure studies after 7 days of cold storage (0 h [26%], ~ 18 h [8%]). In conclusion, upregulation of HSP70 proteins or aerial exposure during grow-out was not an effective tool in reducing the mortality of oysters harvested in summer and held in cold storage.

In vitro effects of growth factors and hormones on three Perkinsus species and increased proliferation of P. marinus during cloning.

Clonal cultures are essential for the genotypic and phenotypic characterization of Perkinsus species but their cloning, especially of P. marinus, can be tedious. The use of a growth factor and hormone supplement to facilitate cloning was, therefore, investigated. Many of the 16 supplements tested significantly increased P. marinus and P. olseni proliferation but only two significantly increased P. chesapeaki proliferation. The concentration of the most effective supplement for all three Perkinsus species (i.e., endothelial cell growth supplement, ECGS) and medium dilution were then optimized for P. marinus cultured at low densities. Finally, the advantage of using conditioned culture medium, a feeder layer, and ECGS alone and in different combinations to improve cloning of P. marinus were compared. Using conditioned culture medium, a feeder layer and ECGS in combination, each cell (N=7) seeded singly yielded clonal cultures with 253+/-167 cells after 21 days. In contrast, only 4 out of 7 cells seeded singly in culture medium yielded clonal cultures with 5+/-4 cells after 21 days.

Study of perkinsosis in the carpet shell clam Tapes decussatus in Galicia (NW Spain). II. Temporal pattern of disease dynamics and association with clam mortality.

Temporal dynamics of the infection by Perkinsus olseni in a clam (Tapes decussatus) bed was studied over 5 yr (March 1996 to December 2000). Diagnostic techniques were compared to assess their suitability for epizootiological purposes. A technique based on incubation of 2 gill lamellae in Ray's fluid thioglycollate medium (RFTM) was more sensitive, quicker and cheaper than examination of histological sections. Incubation of the whole-clam soft tissues in RFTM allowed detection of very light infections that were not detected with incubation of only 2 gill lamellae. Nevertheless, the correlation between the infection intensity estimated by both RFTM incubations was high. Infection intensity was significantly and positively correlated with clam size/age. No infected clam smaller than 20 mm was found. There was an annual pattern of infection involving lower mean infection intensity and prevalence in winter and higher values for both variables from spring to autumn, with 2 main annual peaks in spring and late summer-early autumn. This temporal pattern was significantly associated with the seawater temperature. The annual spring peak of infection intensity occurred when seawater temperature was around 15 degrees C. Monthly mortality in the clam bed peaked in spring and summer--after peaks of P. olseni infection intensity and concurrently with high seawater temperature. A comparison of percentage mortality between clams from 2 sources (a perkinsosis-affected and a non-affected area) placed in the same clam bed revealed significantly higher mortality in the clams originating from the perkinsosis-affected area.

Study of perkinsosis in the carpet shell clam Tapes decussatus in Galicia (NW Spain). I. Identification of the aetiological agent and in vitro modulation of zoosporulation by temperature and salinity.

Morphological characters of zoosporulation stages and DNA sequence of the internal transcribed spacer (ITS) region and the small subunit ribosomal RNA (SSU rRNA) gene confirmed that the aetiological agent of perkinsosis in the clam Tapes decussatus from Galicia (NW Spain) was Perkinsus atlanticus Azevedo, 1989. In vitro modulation by temperature and salinity of the zoosporulation of the parasite was studied. The optimum temperature range for zoosporulation was 19 to 28 degrees C. The temperature range allowing zoosporulation in vitro was 15 to 32 degrees C, which is broader than previously reported (24 to 28 degrees C) for P. atlanticus, and strongly suggests that zoospores can be produced in Galician Rias, where temperature ranges from 10 to 22 degrees C. Prezoosporangia held at 10 degrees C for 2 mo (similar to winter conditions in Galician waters) gave rise to viable zoospores after they were transferred to higher temperatures. This suggests that prezoosporangia could overwinter and zoosporulate in the next spring. Zoospores could survive for up to 22 and 14 d at 28 and 10 degrees C, respectively. The optimum salinity range for zooporulation was 25 to 35 per thousand. Zoospore production was abruptly reduced as salinity decreased. The lowest salinity at which zoosporulation was observed was 10 per thousand. The effectiveness of different chlorine concentrations and exposure lengths to kill prezoosporangia and zoospores was tested. No survival of free zoospores, free prezoosporangia and prezoosporangia included in gill tissue was observed after incubation for 1 h with 50, 200 and 3,000 ppm of chlorine, respectively.

Perkinsus mediterraneus n. sp., a protistan parasite of the European flat oyster Ostrea edulis from the Balearic Islands, Mediterranean Sea.

A new species, Perkinsus mediterraneus, a protistan parasite of the European oyster Ostrea edulis (L.), farmed along the coast of the Balearic Islands, Mediterranean Sea, is described. Morphological examinations with light and transmission electron microscopy, DNA sequence-analysis and enlargement in Ray's fluid thioglycollate medium (RFTM) confirmed that this parasite belongs to the genus Perkinsus. Specific morphological and genetic characteristics indicated that it should be considered a new species in the genus. Sequencing of the small subunit ribosomal (ssu rRNA) gene confirmed that the parasite belongs to the genus Perkinsus, and sequences of the internal transcribed spacer (ITS) were distinct from any Perkinsus ITS sequences previously published and/or deposited in the GenBank. Phylogenetic analysis revealed that the ITS sequences of the new species formed a monophyletic group comprising a sister clade to the P. atlanticus/olseni group. In addition, morphological differences were observed between the new species and the other described Perkinsus spp.. After incubation in RFTM for 1 wk, the prezoosporangium had reached an extremely large size (97.4 +/- 1.99 microm) (mean +/- SE), and after 2 wk incubation had again almost doubled in size (167.1 +/- 8.09 microm). The discharge-tube length was one sixth the diameter of the zoosporangium, i.e. a ratio of 17.36:97.38, the lowest ratio observed for any Perkinsus species. At the ultrastructural level, zoosporangia and zoospores exhibited some differences compared to other Perkinsus species.

Continuous in vitro culture of the carpet shell clam Tapes decussatus protozoan parasite Perkinsus atlanticus.

Continuous in vitro cultures of the clam Tapes decussatus parasite Perkinsus atlanticus were established from infected gill fragments, infected haemolymph and parasite hypnospores isolated from infected gill fragments following incubation in Ray's fluid thioglycollate medium (RFTM). No continuous cultures could be initiated from P. atlanticus zoospores. Cultures initiated from hypnospores yielded the highest percentage of continuous cultures (100%, 6/6), followed by cultures initiated from gill fragments (93%, 43/46) and from haemolymph (30%, 3/10). Failures to establish continuous cultures were due to microbial contamination. The source of parasite influenced the success rate, the time taken to establish cultures and the size of cultured cells. In vitro proliferation of parasite cells was mainly by vegetative multiplication. Zoosporulation, yielding motile biflagellated zoospores, was observed at a low frequency (< 1% of dividing cells) in every culture. Morphology of cultured cells examined with light and transmission electron microscopy corresponded to that of P. atlanticus found in clam tissues. Cultured cells enlarged in RFTM and stained blue-black with Lugol's solution, which are characteristics of the Perkinsus species cells. DNA sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex matched those of P. atlanticus. All cultures were established in a medium designated JL-ODRP-2A that was similar in composition to the culture medium JL-ODRP-1 originally used to propagate Perkinsus marinus in vitro. Proliferation of P. atlanticus in vitro could be supported by the commercial culture medium (1:2 v/v) DME:Ham's F-12 with fetuin.

Identifying factors inducing trophozoite differentiation into hypnospores in Perkinsus species.

Trophozoites of species of Perkinsus in host tissues readily differentiate into hypnospores when incubated in Ray's fluid thioglycollate medium (RFTM). In contrast, hypnospores have rarely been observed in vivo, and when reported they have been associated with dying hosts. The objective of this study was to determine what altered environmental conditions trigger the differentiation of Perkinsus trophozoites into hypnospores. In the first part of the study, cultured P. chesapeaki trophozoites were exposed to lowered oxygen, acidic pH, increased nutrient levels, heat shock, or osmotic shock conditions, and hypnospore density was measured. Acidic pH, lowered oxygen, or increased nutrient levels significantly increased P. chesapeaki hypnospore formation. In the second part of the study, P. olseni and P. marinus trophozoites were exposed to acidic pH, lowered oxygen, or increased nutrient levels resulting in hypnospore formation in P. olseni but not P. marinus. This study demonstrated that changes in environmental conditions consistent with changes expected in decaying tissues or with RFTM incubation induce trophozoite differentiation. The response of the cultured trophozoites varied between species and between isolates of the same species.

Continuous culture of Perkinsus mediterraneus, a parasite of the European flat oyster Ostrea edulis, and characterization of its morphology, propagation, and extracellular proteins in vitro.

Continuous in vitro cultures of Perkinsus mediterraneus were established from tissues of infected European flat oysters, Ostrea edulis. The parasite proliferated in protein-free medium and divided by schizogony in vitro. Cell morphology was similar to that observed for P. mediterraneus in tissues of naturally infected O. edulis and for other Perkinsus spp. cultured in vitro. Parasite cells enlarged approximately 8-fold when placed in alternative Ray's fluid thioglycollate medium, and stained black with Lugol's iodine solution, a response characteristic of Perkinsus spp. DNA sequences matched those determined previously for P. mediterraneus, and phylogenetic analyses on three different data sets indicated that this was a Perkinsus species with a close relationship to another recently described species, Perkinsus honshuensis. Parasite viability was high (>90%) in vitro, but the proliferation rate was low, with densities generally increasing 2-to-6-fold between subcultures at 6-wk intervals. Enzyme analysis of cell-free culture supernatants revealed protease-, esterase-, glycosidase-, lipase-, and phosphatase-like activities. Incubation with class-specific protease inhibitors showed that P. mediterraneus produced serine proteases, and eight proteolytic bands with molecular weights ranging from 34 to 79 kDa were detected in the supernatants by gelatin sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Differential diagnosis of Perkinsus species by polymerase chain reaction-restriction fragment length polymorphism assay.

Perkinsosis is an infection of marine molluscs caused by the protistan parasites of the genus Perkinsus, which has been classified by the OIE as a disease that warrants notification. In the present study, we have applied a molecular genetic approach to develop an optional method for the specific identification of Perkinsus species. A species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay of the rRNA ITS region was developed to identify and distinguish among Perkinsus species. A taxonomic key was established that allows successful identification of Perkinsus species using a single restriction enzyme (Rsa I) to discriminate P. chesapeaki and P. marinus or by a combination of two endonucleases (Rsa I plus Hinf I) to discriminate P. olseni and P. mediterraneus. In order to validate the RFLP assay, the PCR products were cloned and sequenced, and its phylogenetic affinity was determined. Phylogenetic analysis confirmed the specific identification carried out by RFLPs. Herein is the first report of P. olseni in Manila clams from the NW Adriatic Sea (Italy), which we identified by employing this method. The PCR-RFLP assay herein described may be useful to provide accurate, rapid and inexpensive identification of Perkinsus species, and may aid in ongoing epizooetiological studies and diseases control programmes.