Serological affinities of the oyster pathogen Perkinsus marinus (Apicomplexa) with some dinoflagellates (Dinophyceae)

The protozoan oyster pathogen Perkinsus marinus is classified in the phylum Apicomplexa, although molecular-genetic and ultrastructural evidence increasingly concur on its closer phylogenetic relationship with the dinoflagellates. To test for evidence of serological epitopes common to P. marinus and dinoflagellates, we probed 19 free-living and 8 parasitic dinoflagellate, or dinoflagellate-like, species for cross-reactivity with polyclonal antibodies to P. marinus. Three of 19 free-living dinoflagellates (16%), and 7 of 8 parasitic dinoflagellates (88%) were labeled by anti-P. marinus antibodies. In reciprocal immunoassays using polyclonal antibodies to the Hematodinium sp. dinoflagellate parasite of Norway lobsters, Nephrops norvegicus, P. marinus and the same 7 parasitic dinoflagellates labeled by anti-P. marinus antibodies, were again labeled. The dinoflagellate-like parasite of prawns Pandalus platyceros was not labeled by either antibody reagent. These reciprocal results confirm the presence of shared antibody-binding epitopes on cells of P. marinus and several dinoflagellates. The apparent widespread serological affinity between P. marinus and the parasitic dinoflagellates suggests a closer phylogenetic link to the syndinean dinoflagellate lineage. The consistent failure of the dinoflagellate-like prawn parasite to bind either antibody reagent shows that this parasite is serologically distinct from both P. marinus and Hematodinium-species parasitic dinoflagellates.     

Complete genome sequence of Staphylothermus marinus Stetter and Fiala 1986 type strain F1

Staphylothermus marinus Fiala and Stetter 1986 belongs to the order Desulfurococcales within the archaeal phylum Crenarchaeota. S. marinus is a hyperthermophilic, sulfur-dependent, anaerobic heterotroph. Strain F1 was isolated from geothermally heated sediments at Vulcano, Italy, but S. marinus has also been isolated from a hydrothermal vent on the East Pacific Rise. We report the complete genome of S. marinus strain F1, the type strain of the species. This is the fifth reported complete genome sequence from the order Desulfurococcales.     

Natural and cultured populations of the mangrove oyster Saccostrea palmula from Sinaloa, Mexico, infected by Perkinsus marinus

The mangrove oyster Saccostrea palmula coexists with the pleasure oyster Crassostrea corteziensis in coastal lagoons of northwest Mexico. Recent discovery of Perkinsus marinus infecting the pleasure oyster in the region prompted evaluation of S. palmula as an alternative P. marinus host. An analysis to determine the possible presence of P. marinus in natural and cultured populations of S. palmula at four coastal lagoons in Sinaloa, Mexico was carried out during October-November 2010. Tissues from apparently healthy S. palmula were evaluated using Ray's fluid thioglycollate method (RFTM), which revealed a Perkinsus sp. to be present in all four locations at 6.7-20.0% prevalence. Histopathological analysis of these specimens showed tissue alterations and parasite forms consistent with moderate P. marinus infection, which was confirmed by ribosomal non-transcribed spacer (NTS)-based PCR assays on DNA samples from oysters positive by RFTM and histology. DNA sequencing of amplified NTS fragments (307 bp) produced a sequence 98-100% similar to GenBank-deposited sequences of the NTS from P. marinus. Fluorescent in situ hybridization for Perkinsus spp. and P. marinus corroborated the PCR results, showing clear hybridization of P. marinus in host tissues. This is the first record of P. marinus infecting a species from genus Saccostrea and the first record of the parasite from coastal lagoons in Sinaloa, Mexico.     

Comparison of in vitro-cultured and wild-type Perkinsus marinus. III. Fecal elimination and its role in transmission

Perkinsus marinus, a pathogen of the eastern oyster Crassostrea virginica, is transmitted directly among oysters. Previous studies found viable P. marinus parasites in the feces and pseudofeces of oysters within hours of injection with parasites, suggesting that the parasite may be voided from live oysters and subsequently dispersed in the water column. The experiments described here were designed to quantify P. marinus shed in the feces and pseudofeces of experimentally infected oysters. The results indicated that parasites were shed in 2 phases. A 'decreasing' phase occurred within 2 wk of challenge and before net parasite proliferation began in the host. An 'increasing' phase occurred after P. marinus had begun replicating. The quantity of P. marinus recovered in the feces and pseudofeces of exposed oysters was only about 5 % of the dose administered. In vitro-cultured P. marinus were eliminated at a greater rate than wild-type P. marinus and the fraction discharged was not associated with culture phase. Oysters that were continuously dosed with P. marinus in their food gradually lost the ability to discard the parasite in pseudofeces. The quantity of P. marinus shed in feces of infected oysters was correlated with both the P. marinus body burden and subsequent survival time, suggesting that noninvasive fecal counts could predict infection intensity and survival. The results indicate that in an epizootic, shedding of P. marinus via feces is relatively small compared to the potential number released by cadavers of heavily infected oysters, but that fecal discharge may be important in transmission before infections become lethal.     

Systematic evaluation of factors controlling Perkinsus marinus transmission dynamics in lower Chesapeake Bay

The transmission of Perkinsus marinus in eastern oysters Crassostrea virginica in relation to water temperature, host oyster mortality, and water-column abundance of anti-P. marinus antibody-labeled cells was systematically examined for 20 mo at a site in the lower York River, Virginia, USA. Uninfected sentinel oysters were naturally exposed to the parasite at 2 wk intervals throughout the course of the study to determine the periodicity and rates of parasite transmission. The timing and magnitude of disease-associated oyster mortalities in a local P. marinus-infected oyster population were estimated by monitoring a captive subset of the local oyster population. Flow cytometric immunodetection methods were employed to estimate the abundance of P. marinus cells in water samples collected 3 times each week. The acquisition of P. marinus infections by na?ve sentinel oysters occurred sporadically at all times of the year; however, the highest incidence of infection occurred during the months of August and September. This window of maximum parasite transmission coincided with the death of infected hosts within the captive local oyster population. Counts of antibody-labeled cells ranged from 10 to 11900 cells l(-1), with the highest abundances in July and August coincident with maximum summer temperatures. A statistically significant relationship between water-column parasite abundance and infection-acquisition rate was not observed; however, highest parasite-transmission rates in both years occurred during periods of elevated water-column abundance of parasite cells. These results support the prevailing model of P. marinus transmission dynamics by which maximum transmission rates are observed during periods of maximum P. marinus-associated host mortality. However, our results also indicate that transmission can occur when host mortality is low or absent, so alternative mortality-independent dissemination mechanisms are likely. The results also suggest that atypically early-summer oyster mortality from Haplosporidium nelsoni infection, at a time when infections of P. marinus are light, has a significant indirect influence on P. marinus transmission dynamics. Elimination of these hosts prior to late-summer P. marinus infection-intensification effectively reduces the overall number of P. marinus cells disseminated.     

Bufo marinus oocytes as a model for ion channel protein expression and functional characterization for electrophysiological studies.

Oocytes from Xenopus laevis are commonly used as an expression system for ion channel proteins. The aim of this study was to determine whether oocytes from the Colombian native toad, Bufo marinus, could be used as an alternative expression system for ion channel protein expression and functional characterization using the two-microelectrode voltage clamp method. B. marinus oocytes and X. laevis were isolated and cultured in similar conditions. The mean resting membrane potential of B. marinus oocytes was similar to that of X. laevis oocytes as well as the whole-cell basal currents. The potassium ion channel Kv1.1 was successfully expressed in B. marinus oocytes and showed a typical outward rectifying current. Potassium channel blockers reduced these currents. The similarities on electrical properties and expression of ion channel proteins show that B. marinus oocytes can be used effectively to express these proteins, making these cells a viable heterologous system for the expression of ion channel proteins and their electrophysiological characterization.     

Migration of Antarctic minke whales to the Arctic

The Antarctic minke whale (Balaenoptera bonaerensis), and the common minke whale found in the North Atlantic (Balaenoptera acutorostrata acutorostrata), undertake synchronized seasonal migrations to feeding areas at their respective poles during spring, and to the tropics in the autumn where they overwinter. Differences in the timing of seasons between hemispheres prevent these species from mixing. Here, based upon analysis of mitochondrial and microsatellite DNA profiles, we report the observation of a single B. bonaerensis in 1996, and a hybrid with maternal contribution from B. bonaerensis in 2007, in the Arctic Northeast Atlantic. Paternal contribution was not conclusively resolved. This is the first documentation of B. bonaerensis north of the tropics, and, the first documentation of hybridization between minke whale species.     

A quantitative competitive polymerase chain reaction assay for the oyster pathogen Perkinsus marinus

A quantitative competitive polymerase chain reaction (QCPCR) assay was developed for the oyster parasite Perkinsus marinus. PCR primers for the rRNA gene region of P. marinus amplified DNA isolated from P. marinus but not from Perkinsus atlanticus, Crassostrea virginica, or the dinoflagellates Peridinium sp., Gymnodinium sp., or Amphidinium sp. A mutagenic primer was used to create a competitor plasmid molecule identical to the P. marinus target DNA sequence except for a 13-bp deletion. Both P. marinus and competitor DNA amplified with equivalent efficiencies. Each of 25 oysters was processed by 5 P. marinus diagnostic methods--Ray's fluid thioglycollate medium (FTM) tissue assay, FTM hemolymph assay, whole oyster body burden assay, QCPCR of combined gill and mantle (gill/mantle) tissue, and QCPCR of hemolymph. The QCPCR assay enabled detection of 0.01 fg of P. marinus DNA in 1.0 microg of oyster tissue. QCPCR of gill/mantle tissue or hemolymph as well as the body burden assay detected infections in 24 of 25 oysters. Ray's FTM tissue assay detected only 19 infections. The FTM hemolymph assay detected only 22 infections. Regression analysis of QCPCR results and FTM results indicated that the QCPCR assays were effective in quantitating P. marinus infections in oyster tissues.     

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 degrees 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 degrees 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.     

Biochemical characterization of a thermophilic cellobiose 2-epimerase from a thermohalophilic bacterium, Rhodothermus marinus JCM9785

Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of β-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinus DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 °C at pH 6.3, and stable in a range of pH 3.2-10.8 and below 80 °C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.     

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.     

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.     

Impact of the invasive cane toad (Bufo marinus) on an Australian frog (Opisthodon ornatus) depends on minor variation in reproductive timing

Invasive species are widely viewed as unmitigated ecological catastrophes, but the reality is more complex. Theoretically, invasive species could have negligible or even positive effects if they sufficiently reduce the intensity of processes regulating native populations. Understanding such mechanisms is crucial to predicting ultimate ecological impacts. We used a mesocosm experiment to quantify the impact of eggs and larvae of the introduced cane toad (Bufo marinus) on fitness-related traits (number, size and time of emergence of metamorphs) of a native Australian frog species (Opisthodon ornatus). The results depended upon the timing of oviposition of the two taxa, and hence the life-history stages that came into contact. Growth and survival of O. ornatus tadpoles were enhanced when they preceded B. marinus tadpoles into ponds, and reduced when they followed B. marinus tadpoles into ponds, relative to when tadpoles of both species were added to ponds simultaneously. The dominant tadpole-tadpole interaction is competition, and the results are consistent with competitive priority effects. However, these priority effects were reduced or reversed when O. ornatus tadpoles encountered B. marinus eggs. Predation on toxic toad eggs reduced the survival of O. ornatus and B. marinus. The consequent reduction in tadpole densities allowed the remaining O. ornatus tadpoles to grow more rapidly and to metamorphose at larger body sizes (>60% disparity in mean mass). Thus, exposure to B. marinus eggs reduced the number of O. ornatus metamorphs, but increased their body sizes. If the increased size at metamorphosis more than compensates for the reduced survival, the effective reproductive output of native anurans may be increased rather than decreased by the invasive toad. Minor interspecific differences in the seasonal timing of oviposition thus have the potential to massively alter the impact of invasive cane toads on native anurans.     

Generation of targeted deletions in the genome of Rhodothermus marinus

The aim of this work was to develop an approach for chromosomal engineering of the thermophile Rhodothermus marinus. A selection strategy for R. marinus had previously been developed; this strategy was based on complementing a restriction-negative trpB strain with the R. marinus trpB gene. The current work identified an additional selective marker, purA, which encodes adenylosuccinate synthase and confers adenine prototrophy. In a two-step procedure, the available Trp(+) selection was used during the deletion of purA from the R. marinus chromosome. The alternative Ade(+) selection was in turn used while deleting the endogenous trpB gene. Since both deletions are unmarked, the purA and trpB markers may be reused. Through the double deletant SB-62 (ΔtrpB ΔpurA), the difficulties that are associated with spontaneous revertants and unintended chromosomal integration of marker-containing molecules are circumvented. The selection efficiency in R. marinus strain SB-62 (ΔtrpB ΔpurA) was demonstrated by targeting putative carotenoid biosynthesis genes, crtBI, using a linear molecule containing a marked deletion with 717 and 810 bp of 5' and 3' homologous sequences, respectively. The resulting Trp(+) transformants were colorless rather than orange-red. The correct replacement of an internal crtBI fragment with the trpB marker was confirmed by Southern hybridization analysis of the transformants. Thus, it appears that target genes in the R. marinus chromosome can be readily replaced with linear molecules in a single step by double-crossover recombination.     

Protease activity in the plasma of American oysters, Crassostrea virginica, experimentally infected with the protozoan parasite Perkinsus marinus

Perkinsus marinus is responsible for disease and mortality of the American oyster, Crassostrea virginica. To investigate the interactions between P. marinus and oyster hemocytes, protease activity was measured in plasma of oysters collected 4 hr, 24 hr, 4 days, and 2 mo after experimental infection with P. marinus. A significant increase in protease activity was observed in oyster plasma 4 hr after injection with P. marinus, followed by a sharp decrease within 24 hr. Gelatin-impregnated gel electrophoresis showed the presence of 2 major bands (60 and 112 kDa) and 3 less prevalent bands (35, 92, and 200 kDa) with metalloproteinaselike activity in the plasma of noninfected oysters. Additional bands in the 40- to 60-kDa range, corresponding to P. marinus serine proteases, were observed in oyster plasma at early time points after infection. A transient, but significant, decrease in the activity of oyster metalloproteinases was observed at early time points after infection. Coincubation of oyster plasma with P. marinus extracellular products resulted in a decrease in oyster metalloproteinases and several P. marinus proteases. This study provides insights into the role of proteases in the pathogenesis of Dermo disease.     

Ability of predatory native Australian fishes to learn to avoid toxic larvae of the introduced toad Bufo marinus

Of two native Australian fishes naïve to the introduced toad Bufo marinus most barramundi Lates calcarifer rapidly learned to avoid B. marinus tadpoles, while sooty grunter Hephaestus fuliginosus exhibited considerable intraspecific variation in their learning ability. Some sooty grunter learned to avoid tadpoles after only a few attacks, while other individuals continued to attack and reject tadpoles throughout the entire laboratory trials. Individuals of both species recognized and avoided tadpoles 1 day after their previous encounter. None of the fishes died during the trials. The observed variation in behavioural responses of fishes to B. marinus may be due to differences in (1) learning ability, (2) fish hunger levels, and or (3) tadpole palatability and toxicity. The results demonstrate that most barramundi and sooty grunter learn to avoid B. marinus tadpoles with minimal trauma. Consequently, it is anticipated that the toads are unlikely to have a significant negative impact on wild populations of these fishes through direct toxic effects.     

The sequence of the single 16S rRNA gene of the thermophilic eubacterium Rhodothermus marinus reveals a distant relationship to the group containing Flexibacter, Bacteroides, and Cytophaga species.

Rhodothermus marinus, a gram-negative heterotrophic marine thermophile, has been the subject of several recent studies. Isolation, sequencing, and analyses of a 16S rRNA gene have shown that R. marinus diverges sharply from major bacterial phyla and is most closely allied to the Flexibacter-Cytophaga-Bacteroides group. Further analyses revealed that the R. marinus chromosome contains a single rRNA operon with a 16S-23S intergenic region coding for tRNA(Ile) and tRNA(Ala).     

Phylogeography of Bufo marinus from its natural and introduced ranges.

The marine toad, Bufo marinus, has a broad natural distribution extending from the south-west of the USA to southern Peru and the central Amazon. It was introduced to several localities in the Caribbean and Pacific Oceans to control sugar cane pests. We sequenced 468 bp of mitochondrial DNA (mtDNA) containing the ND3 gene, and flanking tRNA genes from toads spanning the broad natural and introduced ranges. Consistent with the known history of introductions and expected effects of serial bottlenecks, mtDNA within introduced populations in Hawaii and Australia was uniform and most closely related to samples from eastern Venezuela and French Guiana. However, mtDNA nucleotide diversity in the geographic region spanning the source areas is also relative low (0.18-0.46%) and the absence of variation in the introduced populations precludes quantitative assessment of the reduction in genetic diversity. Unexpectedly, there was a large phylogeographic break (5.4% sequence divergence) within the natural range separating populations east and west of the Venezuelan Andes. We hypothesize that the two major lineages of B. marinus were isolated by the uplift of the eastern Andean cordillera which was completed approximately 2.7 Ma. Another species of the marinus group, B. paracnemis, had mtDNA paraphyletic, with marinus, being nested within the eastern lineage. Thus, at least one speciation event within the marinus group postdates the split within marinus. These findings suggest that the taxonomy of B. marinus should be re-evaluated and that the search for pathogens to control Australian populations should be conducted in populations from both lineages in the natural range.     

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.