The protistan parasite Perkinsus marinus is resistant to selected reactive oxygen species

The parasite Perkinsus marinus has devastated natural and farmed oyster populations along the Atlantic and Gulf coasts of North America. When viable P. marinus trophozoites are engulfed by oyster hemocytes, the typical accumulation of reactive oxygen species (ROS) normally associated with phagocyte activity is not observed. One hypothesis to explain this is that the parasite rapidly removes ROS. A manifestation of efficient ROS removal should be a high level of resistance to exogenous ROS. We investigated the in vitro susceptibility of P. marinus to ROS as compared to the estuarine bacterium Vibrio splendidus. We find that P. marinus is markedly less susceptible than V. splendidus to superoxide and hydrogen peroxide (H(2)O(2)), but equally sensitive to hypochlorite. Viable P. marinus trophozoites degrade H(2)O(2) in vitro, but lack detectable catalase activity. However, extracts contain an ascorbate dependent peroxidase activity that may contribute to H(2)O(2) removal in vitro and in vivo.     

Real-time PCR for detection and quantification of the protistan parasite Perkinsus marinus in environmental waters

The protistan parasite Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica along the east coast of the United States. Very few data have been collected, however, on the abundance of the parasite in environmental waters, limiting our understanding of P. marinus transmission dynamics. Real-time PCR assays with SybrGreen I as a label for detection were developed in this study for quantification of P. marinus in environmental waters with P. marinus species-specific primers and of Perkinsus spp. with Perkinsus genus-specific primers. Detection of DNA concentrations as low as the equivalent of 3.3 x 10(-2) cell per 10-microl reaction mixture was obtained by targeting the multicopy internal transcribed spacer region of the genome. To obtain reliable target quantification from environmental water samples, removal of PCR inhibitors and efficient DNA recovery were two major concerns. A DNA extraction kit designed for tissues and another designed for stool samples were tested on environmental and artificial seawater (ASW) samples spiked with P. marinus cultured cells. The stool kit was significantly more efficient than the tissue kit at removing inhibitors from environmental water samples. With the stool kit, no significant difference in the quantified target concentrations was observed between the environmental and ASW samples. However, with the spiked ASW samples, the tissue kit demonstrated more efficient DNA recovery. Finally, by performing three elutions of DNA from the spin columns, which were combined prior to target quantification, variability of DNA recovery from different samples was minimized and more reliable real-time PCR quantification was accomplished.     

Gene organization and expression of the divalent cation transporter Nramp in the protistan parasite Perkinsus marinus

Trophozoites of the protistan parasite Perkinsus marinus reside and proliferate inside phagosomelike structures of hemocytes from the host, the eastern oyster Crassostrea virginica. In a murine model, it has been proposed that the outcome of intracellular parasite-host interactions is determined, at least in part, by the activity of the host's divalent cation transporter natural resistance-associated macrophage protein 1 (Nramp1). Although nucleotide sequences from members of the Nramp family in protozoan parasites have recently become available in public databases, little is known about their molecular, structural, and functional aspects that may relate to the parasite's survival of intracellular killing by the host. The complementary DNA (cDNA) sequence of the Nramp from P. marinus (PmNramp) was obtained by polymerase chain reaction amplification with degenerated primers, followed by rapid amplification of cDNA ends. The 2,082-bp cDNA sequence encoded a predicted protein of 558 amino acids. PmNramp is a single-copy gene composed of 7 exons and 6 short introns (44-61 bp) with the canonical splicing signal (GT/AG). A phylogenetic analysis indicates that P. marinus and apicomplexan Nramp genes derive from a common "archetype" Nramp ancestor. However, the apicomplexan Nramps are highly divergent from the P. marinus sequence and the rest of the archetype Nramp group. Preliminary studies suggest that expression of PmNramp in in vitro-cultured P. marinus trophozoites is modulated by metals and by exogenous oxidative stress.     

Effects of triclosan on growth, viability and fatty acid synthesis of the oyster protozoan parasite Perkinsus marinus

Perkinsus marinus, a protozoan parasite of the Eastern oyster Crassostrea virginica, has severely impacted oyster populations from the Mid-Atlantic region to the Gulf of Mexico coast of North America for more than 30 yr. Although a chemotherapeutic treatment to reduce or eliminate P. marinus from infected oysters would be useful for research and hatchery operations, an effective and practical drug treatment does not currently exist. In this study, the antimicrobial drug triclosan 5-chloro-2-(2,4 dichlorophenoxy) phenol, a specific inhibitor of Fab1 (enoyl-acyl-carrier-protein reductase), an enzyme in the Type II class of fatty acid synthetases, was tested for its effects on viability, proliferation and fatty acid synthesis of in vitro-cultured P. marinus meronts. Treatment of P. marinus meront cell cultures with concentrations of > or = 2 microM triclosan at 28 degrees C (a temperature favorable for parasite proliferation) for up to 6 d stopped proliferation of the parasite. Treatment at > or = 5 microM at 28 degrees C greatly reduced the viability and fatty acid synthesis of meront cells. Oyster hemocytes treated with > or = 20 microM triclosan exhibited no significant (p < 0.05) reduction in viability relative to controls for up to 24 h at 13 degrees C. P. marinus meronts exposed to > or = 2 microM triclosan for 24 h at 13 degrees C exhibited significantly (p < 0.05) lower viability relative to controls. Exposure of P. marinus meronts to triclosan concentrations of > or = 20 microM resulted in > 50% mortality of P. marinus cells after 24 h. These results suggest that triclosan may be effective in treating P. marinus-infected oysters.     

A cryptic algal group unveiled: a plastid biosynthesis pathway in the oyster parasite Perkinsus marinus

Plastids are widespread in plant and algal lineages. They are also exploited by some nonphotosynthetic protists, including malarial parasites, to support their diverse modes of life. However, cryptic plastids may exist in other nonphotosynthetic protists, which could be important in studies on the diversity and evolution of plastids. The parasite Perkinsus marinus, which causes mass mortality in oyster farms, is a nonphotosynthetic protist that is phylogenetically related to plastid-bearing dinoflagellates and apicomplexans. In this study, we searched for P. marinus methylerythritol phosphate (MEP) pathway genes, responsible for de novo isoprenoid synthesis in plastids, and determined the full-length gene sequences for 6 of 7 of these genes. Phylogenetic analyses revealed that each P. marinus gene clusters with orthologs from plastid-bearing eukaryotes, which have MEP pathway genes with essentially the same mosaic pattern of evolutionary origin. A new analytical method called sliding-window iteration of TargetP was developed to examine the distribution of targeting preferences. This analysis revealed that the sequenced genes encode bipartite targeting peptides that are characteristic of proteins targeted to secondary plastids originating from endosymbiosis of eukaryotic algae. These results support our idea that Perkinsus is a cryptic algal group containing nonphotosynthetic secondary plastids. In fact, immunofluorescent microscopy indicated that 1 of the MEP pathway enzymes, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, was localized to small compartments near mitochondrion, which are possibly plastids. This tiny organelle seems to contain very low quantities of DNA or may even lack DNA entirely. The MEP pathway genes are a useful tool for investigating plastid evolution in both of the photosynthetic and nonphotosynthetic eukaryotes and led us to propose the hypothesis that ancestral "chromalveolates" harbored plastids before a secondary endosymbiotic event.     

Nucleotide sequence variability in the nontranscribed spacer of the rRNA locus in the oyster parasite Perkinsus marinus.

We examined the sequence variability of the nontranscribed spacer (NTS) and internal-transcribed spacer (ITS1 and ITS2) domains of the rRNA locus of Perkinsus marinus from Maryland, Florida, and Louisiana. The sequence of P. marinus DNA including the 5S rRNA, NTS, small subunit (SSU) rRNA, ITSI, and ITS2 regions confirmed their contiguity in the rRNA locus and revealed differences at 28 positions with the SSU rRNA sequences published earlier. The 307-bp polymerase chain reaction (PCR)-amplified fragments from the NTS domain of the various P. marinus isolates revealed the presence of 2 distinct sequences, designated as types I and II, that differed at 6 defined nucleotide positions. Based on these differences, nested PCR and restriction enzyme digests were used to distinguish between the 2 types. Sequences of the ITS1 and ITS2 domains of samples from either NTS type I (n = 3) or type II (n = 3) showed no variation and were identical to published sequences. Frequencies of the P. marinus NTS sequence types I and II in infected oysters varied with the geographic origin of the samples. All Maryland samples examined (n = 19) corresponded to the NTS type I sequence, the type II was the most frequent in the Florida samples (n = 17), and both types were about equally represented in the Louisiana samples (n = 19), with both sequence types found in individual oyster specimens. Although it has been suggested that P. marinus is diploid, it remains to be determined if both NTS sequence types can be present in a single P. marinus trophozoite.     

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.     

Perkinsus marinus, a protozoan parasite of the eastern oyster, has a requirement for dietary sterols

Perkinsus marinus, a protozoan parasite of the eastern oyster, Crassostrea virginica, causes high mortality in its host along the Atlantic and Gulf coasts of North America. P. marinus meronts cultured in vitro in medium containing complete lipid supplement (cod liver oil, cholesterol and alpha tocopherol acetate in detergent) are able to synthesize a wide variety of lipids, yet cultures cannot be maintained in lipid-free medium. To determine P. marinus lipid requirements meronts were inoculated into media containing different combinations of lipid components in detergent. Treatments included complete lipid supplement (positive control), detergent only (negative control), cholesterol in detergent, alpha tocopherol acetate in detergent and cholesterol+alpha tocopherol acetate in detergent. Meronts proliferated in the positive control medium and media containing cholesterol or cholesterol+alpha tocopherol acetate, but failed to proliferate in the negative control medium and the medium containing just alpha tocopherol acetate. Gas chromatography analysis of P. marinus meronts grown in medium with added (13)C sodium acetate (0.5 mg mL(-1)) revealed the presence of fatty acids containing (13)C, but the only sterol present was cholesterol containing no (13)C. These results suggest that P. marinus cannot synthesize sterols and must sequester them from its host.     

Supplementation of Perkinsus marinus cultures with host plasma or tissue homogenate enhances their infectivity

The protozoan oyster parasite Perkinsus marinus can be cultured in vitro in a variety of media; however, this has been associated with a rapid attenuation of infectivity. Supplementation of defined media with products of P. marinus-susceptible (Crassostrea virginica) and -tolerant (Crassostrea gigas, Crassostrea ariakensis) oysters alters proliferation and protease expression profiles and induces differentiation into morphological forms typically seen in vivo. It was not known if attenuation could be reversed by host extract supplementation. To investigate correlations among these changes as well as their association with infectivity, the effects of medium supplementation with tissue homogenates from both susceptible and tolerant oyster species were examined. The supplements markedly altered both cell size and proliferation, regardless of species; however, upregulation of low-molecular-weight protease expression was most prominent with susceptible oysters extracts. Increased infectivity occurred with the use of oyster product-supplemented media, but it was not consistently associated with changes in cell size, cell morphology, or protease secretion and was not related to the susceptibility of the oyster species used as the supplement source.     

Superoxide dismutases from the oyster parasite Perkinsus marinus: purification,biochemical characterization,and development of a plate microassay for activity

We have isolated and biochemically characterized superoxide dismutase (SOD) activity in cell extracts of clonally cultured Perkinsus marinus, a facultative intracellular parasite of the Eastern oyster, Crassostrea virginica. In order to assess the SOD activity throughout the purification, we developed and optimized a 96-well-plate microassay based on the inhibition of pyrogallol oxidation. The assay was also adapted to identify SOD activity type (Cu/Zn-, Mn-, or FeSOD), even in mixtures of more than one type of SOD. All SOD activity detected in the cell extracts was of the FeSOD type. Most of the SOD activity in P. marinus trophozoites resides in a major component of subunit molecular weight 24 kDa. The protein was purified by affinity chromatography on an anti-SOD antibody-Sepharose column. Amino-terminal peptide sequence of the affinity-purified protein corresponds to the predicted product of the PmSOD1 gene and indicates that amino-terminal processing has taken place. The results are discussed in the context of processing of mitochondrially targeted SODs.     

Uptake and interconversion of fluorescent lipid analogs in the protozoan parasite, Perkinsus marinus, of the oyster, Crassostrea virginica

Uptake, distribution, and interconversion of fluorescent lipid analogs (phosphatidylcholine, PC; cholesteryl ester, CHE; phosphatidylethanolamine, PE; palmitic acid, C16; sphingomyelin, SM) by the two life stages, meront and prezoosporangium, of the oyster protozoan parasite, Perkinsus marinus, were investigated. Class composition of these two life stages and lipid contents in meront cells were also examined. Both meronts and prezoosporangia incorporated and modified fluorescent lipids from the medium, but their metabolic modes differ to some extent. Results revealed that among the tested analogs, neutral lipid components (CHE and C16) were incorporated to a greater degree than the phospholipids (PC, PE, and SM). HPLC analysis of meront lipids showed that while the majority of the incorporated PC, CHE, and PE remained as parent compounds, most of the incorporated C16 was in triacylglycerol (TAG) and SM was in ceramide and free fatty acids. The cellular distribution of fluorescent labels varied with lipid analogs and the extent of their metabolism by the parasite. Fluorescence distribution was primarily in cytoplasmic lipid droplets of both life stages after 24 h incubation with PC. After 24 h incubation with SM, fluorescence appeared in the membrane and cytosol. Total lipid contents in meront cultures increased during proliferation and TAG accounted for most of the increased total lipids. Since total lipid content per meront cell did not increase until the day of culture termination, the lipid increase in the meront culture was mainly a result of increased cell numbers. Both life stages contain relatively high levels of phospholipids, 53.8% in 8-day-old meronts and 39.4% in prezoosporangia. PC was the predominant phospholipid.     

Microsatellite marker development in the protozoan parasite Perkinsus olseni

The analysis of an enriched partial genomic library and of public expressed sequence tag (EST) resources allowed the characterization of the first microsatellite loci in the protozoan parasite Perkinsus olseni. Clonal cultures from laboratory isolates derived from infected clams Ruditapes decussatus (from Spain), R. philippinarum (from Spain and Japan), and Austrovenus stutchburyi (from New Zealand) were used for the characterization of 12 microsatellites. Low variation was detected at most loci, with the number of alleles at polymorphic loci ranging from 2 to 7 (average 3.20 +/- 0.51) and gene diversity from 0.11 to 0.79 (average 0.40 +/- 0.07). Preliminary results show that (1) isolates of P. olseni are diploid cells, and (2) multiple infections can occur within a single host. Eight of the loci analyzed successfully cross-amplified in the congeneric species P. mediterraneus. These microsatellite markers will be useful to analyze in detail the population genetic structure of P. olseni, crucial for the efficient management of this parasitic disease.     

Salinity effects on viability, metabolic activity and proliferation of three Perkinsus species

Little is known regarding the range of conditions in which many Perkinsus species may proliferate, making it difficult to predict conditions favorable for their expansion, to identify conditions inducing mortality, or to identify instances of potential cross-infectivity among sympatric host species. In this study, the effects of salinity on viability, metabolic activity and proliferation of P. marinus, P. olseni and P. chesapeaki were determined. Specifically, this research examined the effects of 5 salinities (7, 11, 15, 25, 35 per thousand), (1) without acclimation, on the viability and metabolic activity of 2 isolates of each Perkinsus species, and (2) with acclimation, on the viability, metabolic activity, size and number of 1 isolate of each species. P. chesapeaki showed the widest range of salinity tolerance of the 3 species, with high viability and cell proliferation at all salinities tested. Although P. chesapeaki originated from low salinity areas (i.e. <15 per thousand), several measures (i.e. cell number and metabolic activity) indicated that higher salinities (15, 25 per thousand) were more favorable for its growth. P. olseni, originating from high salinity areas, had better viability and proliferation at the higher salinities (15, 25, 35 per thousand). Distinct differences in acute salinity response of the 2 P. olseni isolates at lower salinities (7, 11 per thousand), however, suggest the need for a more expansive comparison of isolates to better define the lower salinity tolerance. Lastly, P. marinus was more tolerant of the lower salinities (7 and 11 per thousand) than P. olseni, but exhibited reduced viability at 7 per thousand, even after acclimation.     

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.     

Phenoloxidase activity in three commercial bivalve species. Changes due to natural infestation with Perkinsus atlanticus

The phenoloxidase (PO) activity of the haemolymph and haemocytes from three clam species of commercial interest (Ruditapes philippinarum, Chamelea gallina and Tapes decussatus) has been compared. The activity was assayed spectrophotometrically by recording the formation of dopachrome from L-DOPA using sodium dodecyl sulphate, laminarin, trypsin or lipopolysaccharide as elicitors. Fewer PO units were observed in the haemolymph from T. decussatus than in the haemolymph from R. philippinarum, while the highest values were found in C. gallina. In all cases the activity was only significantly increased when sodium dodecyl sulphate was used as elicitor. PO activity in the haemocytes of all three clam species showed a very similar pattern to that found in the haemolymph from the same species. Furthermore, T. decussatus naturally parasitized by Perkinsus atlanticus (Protozoa, Apicomplexa) was used to study the influence of such infestation on PO activity, which was found to increase significantly in both haemolymph and haemocytes compared with non-infected (control) samples. PO activity in the haemocytes and in the haemolymph was higher when the level of parasitization was low or medium, respectively, and SDS was used as elicitor. No statistically significant differences were observed when the parasitization level was high. The present work constitutes the first report on the influence of this parasite on PO activity in haemolymph and haemocytes from T. decussatus.     

Factors associated with the prevalence of Perkinsus marinus in Crassostrea virginica from the southern Gulf of Mexico

The protozoan Perkinsus marinus is considered the most important pathogen of the eastern oyster Crassostrea virginica, causing high mortality in natural and farmed oysters on the Atlantic coast of the US. In Mexico, no serious P. marinus epizootic has been reported. This study describes the current state of P. marinus prevalence in Terminos Lagoon (Mexico) associated with environmental factors including salinity, temperature, ammonium, nitrate, nitrite, silica, and phosphorus. In addition, the association of physiological (hemocyte density, protein concentration) and immunological (lysozyme, agglutination) parameters with the infection were studied. The prevalence was significantly different among seasons with mean values of 70, 23, and 7% in the dry (February to May), rainy (June to September) and north-wind (October to January) seasons, respectively. Only light infection intensity (Mackin scale value < 1) was observed. Prevalence of P. marinus was associated with seasonal salinity, phosphorus, and silica variations. Comparisons of oyster health demonstrates that the rainy and north-wind seasons are stressful periods. Redundancy analysis showed that only 34% of the variation in seasonal P. marinus prevalence was explained by protein concentration (21%), lysozyme (12%), and agglutination (1%). Overall, the data suggest that freshwater input associated with high nutrient concentrations during the rainy and north-wind seasons has a strong negative effect on P. marinus prevalence and also influences the oysters' physiology. It is probable that this seasonal stress was responsible for the absence of an epizootic event in Terminos Lagoon.     

Certain biochemical characteristics of an insulin-like substance from the bivalve mollusk Anodonta cygnea

An insulin-like substance (ILS) was isolated from the visceral organs of the bivalve mollusc Anodonta cygnea by chromatography on a sulfocationite CU-23 and purified by reverse phase liquid chromatography. ILS was shown to be made up to several fractions with Mr ranging from 9 to 20 kDa which have identical amino acid composition but different hydrophobicity and N-terminal amino acids. It was supposed that the heterogeneity of ILS fractions is due to its genetical or posttranslational polymorphism. ILS has a low (0.02%) affinity for the mammalian insulin receptor and a low immune affinity for mammalian insulin and possesses a mitogenic activity which is commensurate with that of the epidermal growth factor. The data obtained suggest that Anodonta cygnea ILS represents a separate branch of a relatively ancient family of insulin-like hormones and growth factors responsible for metabolism and proliferation of invertebrate tissues.     

Isolation and characterization of serine protease gene(s) from Perkinsus marinus

This study reports the first serine protease gene(s) isolated from Perkinsus marinus. Using universal primers, a 518 bp subtilisin-like serine protease gene fragment was amplified from P. marinus genomic DNA and used as a probe to screen a lambda-phage P. marinus genomic library; 2 different lambda-phage clones hybridized to the digoxigenin(DIG)-labeled subtilisin-like gene fragment. Following subcloning and sequencing of the larger DNA fragment, a 1254 bp open reading frame was identified and later confirmed, by 5' and 3' random amplification of cDNA ends (RACE) and northern blot analysis, to contain the entire coding-region sequence. Sequence analysis of the 3' RACE results from 2 isolate cultures, VA-2 (P-1) and LA 10-1, revealed multiple polymorphic sites within and among isolates. We identified 2 different types of cDNA clones with 95.53% nucleotide sequence similarity, suggesting the possibility of 2 closely related genes within the P. marinus genome. Southern blot analysis of genomic DNA from 12 genetically distinct P. marinus isolate cultures revealed 2 different banding patterns among isolates.