Evidence for Regular Sporulation by Haplosporidium nelsoni (MSX) (Ascetospora; Haplosporidiidae) in Spat of the American Oyster, Crassostrea virginica

The spore stage of Haplosporidium nelsoni , the ascetosporan parasite causing multinucleated sphere unknown (MSX) disease in oysters, Crassostrea virginica , has been reported so rarely (≥0.01% of infected oysters) that a second host has been postulated. However, recent intensive sampling of young (≥1 year) oysters in Delaware Bay, U.S. suggests that spore formation occurs regularly in this group and that spores are produced in at least 75–85% of all infections reaching the advanced stage. Sporulation was seasonal, occurring over two to three weeks in late June/early July and again in late summer/early fall. Our data indicate that sporulation by H. nelsoni in oysters is more common than previously suspected, occurring in a segment of the host population that may not have been sufficiently sampled in the past, and that a direct life cycle should be reconsidered.     

Infection and mortality patterns in strains of oysters Crassostrea virginica selected for resistance to the parasite Haplosporidium nelsoni (MSX)

Strains of oysters Crassostrea virginica resistant to mortality caused by the parasite Haplosporidium nelsoni (MSX) were developed and tested through 6 generations. In addition, strains in each generation were followed for up to 6 yr of continuous exposure to the parasite in nature. Selected strains responded to challenge by the parasite with gradually improved survival in successive generations. They were slower to develop patent infections than were unselected groups and were able to delay mortality after infections did develop, but under repeated exposure most oysters eventually died with H. nelsoni parasitism. Many selected strains, however, reached market size before significant mortalities occurred. The data suggest that resistance to H. nelsoni mortality is under the influence of many genes. No clear defense mechanism has been described and we hypothesize that resistance to H. nelsoni may, in part, involve a physiological state in which selected oysters temporarily fail to provide a suitable habitat for the parasite. Temporary insusceptibility would, in this view, be followed by an increased ability to tolerate the parasite when conditions for its development are present. Selection would then favor individuals that are able to prolong periods of insusceptibility and/or to carry out basic life processes while parasitized.     

Widespread survey finds no evidence of Haplosporidium nelsoni (MSX) in Gulf of Mexico oysters

The advent of molecular detection assays has provided a set of very sensitive tools for the detection of pathogens in marine organisms, but it has also raised problems of how to interpret positive signals that are not accompanied by visual confirmation. PCR-positive results have recently been reported for Haplosporidium nelsoni (MSX), a pathogen of the oyster Crassostrea virginica in 31 of 40 oysters from 6 sites in the Gulf of Mexico and the Caribbean Sea. Histological confirmation of the PCR results was not undertaken, and no haplosporidian has been reported from the numerous histological studies and surveys of oysters in the region. To further investigate the possibility that H. nelsoni is present in this region, we sampled 210 oysters from 40 sites around the Gulf of Mexico and Puerto Rico using PCR and 180 of these using tissue-section histology also. None of the oysters showed evidence of H. nelsoni by PCR or of any haplosporidian by histology. We cannot, therefore, confirm that H. nelsoni is present and widespread in the Gulf of Mexico and the Caribbean Sea. Our results do not prove that H. nelsoni is absent from the region, but taken together with results from previous histological surveys, they suggest that for the purposes of controlling oyster importation, the region should continue to be considered free of the parasite.     

Detection of Minchinia sp., in rock oysters Saccostrea cuccullata (Born, 1778) using DNA probes

Haplosporidian parasites infect various invertebrate hosts including some commercially important shellfish. Haplosporidium nelsoni (along with Perkinsus marinus) has severely affected Eastern oyster production on the eastern seaboard of the United States and flat oyster production in Europe has been severely impacted by Bonamia ostreae. These parasites are also often present at a very low prevalence and there are a variety of morphologically similar species that can be difficult to differentiate during cytological or histological diagnosis hence the need to develop specific tests. Recently, a Minchinia sp. was described affecting rock oysters (Saccostrea cuccullata) in north Western Australia. In this study, two in situ hybridisation (ISH) assays and a PCR assay have been developed and optimised for use in investigating these parasites. The first ISH assay used a 166bp polynucleotide probe while the second used a 30bp oligonucleotide probe. The specificity of each ISH assay was assessed by applying each probe to a variety of haplosporidian (5), a paramyxian (1) or ciliophora (1) parasites. The polynucleotide probe produced strong hybridisation signals against all of the haplosporidian parasites tested (Minchinia sp., Minchinia teredinis, Bonamia roughleyi, H. nelsoni and Haplosporidium costale) while the oligonucleotide probe recognised only the Minchinia sp. Both probes failed to detect the paramyxian (Marteilia sp.) or the Rhynchodid-like ciliate. The PCR assay amplifies a 220bp region and detected Minchinia sp. DNA from 50ng of genomic DNA extracted from the tissues of infected oysters and 10fg of amplified Minchinia sp. DNA. The assay did not react to oysters infected with H. nelsoni or H. costale. The ability of the PCR and oligonucleotide ISH assay to diagnose Minchinia sp. infected oysters was compared to histological examination from a sample of 56 oysters. The PCR assay revealed 26 infections while histological examination detected 14 infections. The oligonucleotide ISH assay detected 29 infections. The oligonucleotide ISH and PCR assays were found to be significantly more sensitive than histology for detecting the parasite.     

Spore ornamentation of Minchinia occulta n. sp. (Haplosporidia) in rock oysters Saccostrea cuccullata (Born, 1778)

A Minchinia sp. (Haplosporidia: Haplosporidiidae) parasite was identified infecting rock oysters and morphologically described by Hine and Thorne (2002) using light microscopy and transmission electron microscopy (TEM). The parasite was associated with up to 80% mortality in the host species and it is suspected that the parasite would be a major impediment to the development of a tropical rock oyster aquaculture industry in northern Western Australia. However, attempts to identify the parasite following the development of a specific probe for Haplosporidium nelsoni were unsuccessful. The SSU region of the parasite's rRNA gene was later characterized in our laboratory and an in situ hybridization assay for the parasite was developed. This study names the parasite as Minchinia occulta n sp. and morphologically describes the parasite using histology, scanning electron microscopy and transmission electron microscopy. The non-spore stages were unusual in that they consisted primarily of uninucleate stages reminiscent of Bonamia spp. The parasite's spores were ovoid to circular shaped and measured 4.5 microm-5.0 microm x 3.5-4.1 microm in size. The nucleus of the sporoplasm measured 1.5-2.3 microm and was centrally located. The spores were covered in a branching network of microtubule-like structures that may degrade as the spore matures.     

Application of a multiplex PCR for the detection of protozoan pathogens of the eastern oyster Crassostrea virginica in field samples

Populations of eastern oysters Crassostrea virginica along the east coast of North America have repeatedly experienced epizootic mass mortality due to infections by protozoan parasites, and molecular diagnostic methodologies are fast becoming more widely available for the diagnosis of protozoan diseases of oysters. In this study we applied a modified version of an existing multiplex polymerase chain reaction (PCR) for detection of the eastern oyster parasites Haplosporidium nelsoni, H. costale and Perkinsus marinus from field-collected samples. We incorporated primers for DNA quality control based on the large subunit ribosomal RNA (LSU rRNA) gene of C. virginica. The multiplex PCR (MPCR) simultaneously amplified genomic DNA of C. virginica, and cloned DNA of H. nelsoni, P. marinus and H. costale. In field trial applications, we compared the performance of the MPCR to that of the conventional diagnostic techniques of histopathological tissue examination and the Ray/Mackin fluid thioglycollate medium (RMFT) assay. A total of 530 oysters were sampled from 18 sites at 12 locations along the east coast of the United States from the Gulf of Mexico to southern New England. The modified MPCR detected 21% oysters with H. nelsoni, 2% oysters with H. costale, and 40% oysters with P. marinus infections. In comparison, histopathological examination detected H. nelsoni and H. costale infections in 6 and 0.8% oysters, respectively, and the RMFT assay detected P. marinus infection in 31% oysters. The MPCR is a more sensitive diagnostic assay for detection of H. nelsoni, H. costale, and P. marinus, and incorporation of an oyster quality control product limits false negative results.     

Prevalence of Perkinsus marinus (dermo), Haplosporidium nelsoni (MSX), and QPX in bivalves of Delaware's inland bays and quantitative, high-throughput diagnosis of dermo by QPCR

Restoration of oyster reef habitat in the Inland Bays of Delaware was accompanied by an effort to detect and determine relative abundance of the bivalve pathogens Perkinsus marinus, Haplosporidium nelsoni, and QPX. Both the oyster Crassostrea virginica and the clam Mercenaria mercenaria were sampled from the bays. In addition, oysters were deployed at eight sites around the bays as sentinels for the three parasites. Perkinsus marinus prevalence was measured with a real-time, quantitative polymerase chain reaction (PCR) methodology that enabled high-throughput detection of as few as 31 copies of the ribosomal non-transcribed spacer region in 500 ng oyster DNA. The other pathogens were assayed using PCR with species-specific primers. Perkinsus marinus was identified in Indian River Bay at moderate prevalence ( approximately 40%) in both an artificial reef and a wild oyster population whereas sentinel oysters were PCR-negative after 3-months exposure during summer and early fall. Haplosporidium nelsoni was restricted to one oyster deployed in Little Assawoman Bay. QPX and P. marinus were not detected among wild clams. While oysters in these bays have historically been under the greatest threat by MSX, it is apparent that P. marinus currently poses a greater threat to recovery of oyster aquaculture in Delaware's Inland Bays.     

Apoptosis in the pathogenesis of infectious diseases of the eastern oyster Crassostrea virginica

Apoptosis, or programmed cell death, has been reported as being pivotal in infectious diseases of different organisms. The effects of apoptosis on the progression and transmission of the protistan parasites Perkinsus marinus and Haplosporidium nelsoni in the eastern oyster Crassostrea virginica were studied. Oysters were diagnosed for their respective infections by standard methods, and apoptosis was detected using in situ hybridization to detect DNA fragments by end labeling on paraffin sections. A digoxigenin nucleotide probe was used to label the 200 bp fragment produced by apoptosis and detected immunohistochemically using an antidigoxigenin peroxidase conjugate. The probe/DNA fragment complex was stained with a peroxidase substrate and tissues were counterstained with methyl green. Uninfected oysters had large numbers of apoptotic hemocytes present in the connective tissue underlying the stomach, gill, and mantle epithelia, whereas oysters infected with P. marinus had a reduced number of apoptotic hemocytes. The parasite may prevent hemocyte apoptosis in order to yield a greater number of hemocytes in which to house itself. Large numbers of P. marinus cells in some infected oysters were eliminated via apoptosis in the stomach epithelia, disabling the spread of infectious particles through seawater. The oysters infected with H. nelsoni also had reduced numbers of apoptotic hemocytes, while part of the vesicular connective tissue cells were apoptotic. H. nelsoni plasmodia were eliminated via apoptosis in some oysters. Apoptosis may enhance progression and prevent transmission of infectious oyster diseases.     

Pollen and anther development in Nelumbo (Nelumbonaceae)

The Nelumbonaceae are a small family of aquatic angiosperms comprising Nelumbo nucifera and Nelumbo lutea. Historically, the genus has been considered to be closely related to Nymphaeales, however new systematic work has allied Nelumbo with lower eudicots, particularly Platanus. In recent years, studies of pollen development have contributed greatly to the understanding of phylogenetic relationships, but little has been known about these events in Nelumbo. In this paper, pollen and anther development are morphologically described for the first time in N. lutea. A comprehensive ontogenetic sequence is documented, including the sporogenous tissue, microspore mother cell, tetrad, free spore, and mature pollen grain stages. The deposition of a microspore mother cell coat and callose wall, the co-occurrence of both tetrahedral and tetragonal tetrads, the formation of a primexine in tetrads, and primexine persistence into the late free spore stage are shown. The majority of exine development occurs during the free spore stage with the deposition of a tectate-columellate ectexine, a lamellate endexine, and an unusual granular layer below and intermixed with the endexine lamellae. A two-layered intine forms rapidly during the earliest mature pollen stage. Major events of anther development documented include the degradation of a secretory-type tapetum during the free spore stage and the rapid formation of U-shaped endothecial thickenings in the mature pollen grain stage. The majority of mature pollen grains are tricolpate, however less common monosulcate and diaperturate grains also develop. Co-occurring aperture types in Nelumbo have been suggested to be an important transition in angiosperm aperture number. However, aperture variability in Nelumbo may be correlated with the lateness of aperture ontogeny in the genus, which occurs in the early free spore stage. This character, as well as other details of pollen and anther ontogeny in Nelumbo, are compared to those of Nymphaeales and Platanus in an effort to provide additional insight into systematic and phylogenetic relationships. Although Nelumbo is similar to both groups in several characters, the ontogenetic sequence of the genus is different in many ways.     

DEVELOPMENT OF JANCZEWSKIA MORIMOTOI (CERAMIALES) ON ITS HOST LAURENCIA NIPPONICA (CERAMIALES,RHODOPHYCEAE)1

Janczewskia morimotoi Tokida was successfully cultured from spore to reproductive maturity on its host Laurencia nipponica Yamada. The spore penetrates the host without requirement for wound or abrasion sites, growing between host cortical cells and developing a superficial and an endophytic system simultaneously. During the juvenile period, when the parasite is nonpigmented, it differentiates a cortex and the proliferating endophytic filaments enlarge causing a displacement of layers of host cells into the parasitic tissue. Host cells contacted by cells of the parasite exhibit increased wall thickness, cytoplasmic density and vesicle formation. Pit connections between host and parasite cells were rarely observed whereas penetration of host cell walls was seen commonly. As the parasite increases in size, its cells become pigmented evenly throughout the cortex and host cells show less obvious reactions to the parasite. At this same time, the parasite develops branches and reproductive structures. Host plant segments less than 3 cm long failed to grow when infected with spores of the parasite whereas longer segments were not significantly affected by the parasite. In the absence of the host, the parasite cannot complete its development. Although J. morimotoi is well pigmented at maturity, the absence of pigmentation in the juvenile stage, penetration of host cells, and effect on host growth in culture strongly suggest that it is parasitic during at least its early development.     

Transmission of the haplosporidian parasite MSX Haplosporidium nelsoni to the eastern oyster Crassostrea virginica in an upweller system

The haplosporidian oyster parasite MSX (Multinucleated Sphere X) Haplosporidium nelsoni was transmitted to eastern oysters Crassostrea virginica. Hatchery-raised, MSX-free juvenile oysters were placed in upweller tanks. Water to the tanks was filtered through a screen with 1 mm2 openings and originated from the water column overlaying naturally infected oysters beds (MSX prevalence 17 to 57%). MSX was diagnosed by histopathological analysis. MSX-disease (57% prevalence) with increased mortality (19%) was observed 11 wk after the beginning of the exposure and mortality of 80% after 16 wk. The study demonstrates transmission of MSX via water-borne infectious agents capable of passing through a 1 mm filter.     

A persistent, productive, and seasonally dynamic vibriophage population within Pacific oysters (Crassostrea gigas)

In an effort to understand the relationship between Vibrio and vibriophage populations, abundances of Vibrio spp. and viruses infecting Vibrio parahaemolyticus (VpVs) were monitored for a year in Pacific oysters and water collected from Ladysmith Harbor, British Columbia, Canada. Bacterial abundances were highly seasonal, whereas high titers of VpVs (0.5 x 10(4) to 11 x 10(4) viruses cm(-3)) occurred year round in oysters, even when V. parahaemolyticus was undetectable (< 3 cells cm(-3)). Viruses were not detected (<10 ml(-1)) in the water column. Host-range studies demonstrated that 13 VpV strains could infect 62% of the V. parahaemolyticus strains from oysters (91 pairings) and 74% of the strains from sediments (65 pairings) but only 30% of the water-column strains (91 pairings). Ten viruses also infected more than one species among V. alginolyticus, V. natriegens, and V. vulnificus. As winter approached and potential hosts disappeared, the proportion of host strains that the viruses could infect decreased by approximately 50% and, in the middle of winter, only 14% of the VpV community could be plated on summer host strains. Estimates of virus-induced mortality on V. parahaemolyticus indicated that other host species were required to sustain viral production during winter when the putative host species was undetectable. The present study shows that oysters are likely one of the major sources of viruses infecting V. parahaemolyticus in oysters and in the water column. Furthermore, seasonal shifts in patterns of host range provide strong evidence that the composition of the virus community changes during winter.     

A Proposed Life Cycle of Minchinia nelsoni (Haplosporida, Haplosporidiidae) in the American Oyster Crassostrea virginica

SYNOPSIS. A developmental sequence is proposed for the haplosporidan Minchinia nelsoni Haskin, Stauber and Mackin, 1966, based on study of oyster infections over the past 5 years in Chesapeake Bay. Uninucleate stages develop by nuclear division into multinucleate plasmodia which proliferate in the tissues by plasmotomy. Relatively small plasmodia containing what are considered to be gametic nuclei originate by unequal plasmotomy of large plasmodia. These have been interpreted to aggregate and fuse to form large plasmodia which contain prozygotes. Pairing and fusion of nuclei occur within each plasmodium to produce zygote nuclei (synkaryons) which undergo division, possibly meiotic, to form sporonts. Sporoblasts differentiate into spores with the development of spore walls and opercula. Cystoid plasmodia develop during times of unfavorable conditions. An anomalous but common sequence involving sexuality and mitosis is described, and the occurrence of various life cycle stages within the host thruout the year is discussed.     

New ruminant hosts and wider geographic range identified for Babesia odocoilei (Emerson and Wright 1970)

Babesia odocoilei was found to infect two previously unknown host species, desert bighorn sheep (Ovis canadensis nelsoni) and musk oxen (Ovibos moschatus), both of which are members of the family Bovidae. Previously, B. odocoilei has been reported in only Cervidae hosts. New geographic regions where B. odocoilei infections have not been reported previously include Pennsylvania and New York, where fatal babesiosis has occurred in reindeer (Rangifer tarandus tarandus); New Hampshire, where elk (Cervus elaphus canadensis) have been affected; and California, home of the infected desert bighorn sheep. Infection with B. odocoilei in these hosts was confirmed by parasite small subunit ribosomal RNA gene sequence analysis. A serosurvey for B. odocoilei antibody activity in New Hampshire showed prevalence rates of 100% at two elk farms and 12% at another farm. Control of potential vector ticks, Ixodes scapularis, especially when translocating livestock, is imperative to prevent outbreaks of babesiosis in managed herds of potential host species.     

Stage-specific inductive signals in the Drosophila neuroectoderm control the temporal sequence of neuroblast specification

One of the initial steps of neurogenesis in the Drosophila embryo is the delamination of a stereotype set of neural progenitor cells (neuroblasts) from the neuroectoderm. The time window of neuroblast segregation has been divided into five successive waves (S1-S5) in which subsets of neuroblasts with specific identities are formed. To test when identity specification of the various neuroblasts takes place and whether extrinsic signals are involved, we have performed heterochronic transplantation experiments. Single neuroectodermal cells from stage 10 donor embryos (after S2) were transplanted into the neuroectoderm of host embryos at stage 7 (before S1) and vice versa. The fate of these cells was uncovered by their lineages at stage 16/17. Transplanted cells adjusted their fate to the new temporal situation. Late neuroectodermal cells were able to take over the fate of early (S1/S2) neuroblasts. The early neuroectodermal cells preferentially generated late (S4/S5) neuroblasts, despite their reduced time of exposure to the neuroectoderm. Furthermore, neuroblast fates are independent from divisions of neuroectodermal progenitor cells. We conclude from these experiments that neuroblast specification occurs sequentially under the control of non-cell-autonomous and stage-specific inductive signals that act in the neuroectoderm.     

Enzyme-linked immunosorbent assay (ELISA) used in quantification of reproductive output in the pacific oyster,Crassostrea gigas,in Korea

Polyclonal antibody developed from egg proteins of the Pacific oysters, Crassostrea gigas, was used in quantification of the eggs. Quantity of the egg protein was measured using ELISA and a weight-normalized gonadosomatic index (GSI) was then established. Histology indicated that the oysters collected from Goseong Bay, Korea, in 2000 initiated gametogenesis as early as late February and spawned mostly in late June to early July when water temperature reached 23–25 °C. A second spawning peak was also observed in late August and the spawning activity continued to late October. Oyster egg proteins could be detected by ELISA in all months except March and November; a few oysters collected during December and January contained a measurable amount of eggs. The highest monthly mean GSI, 0.423, was observed in mid-June when the oysters were ready to spawn. The maximum GSI recorded in this study was 0.667. A positive correlation was found between oyster size and fecundity; the number of eggs increased as oyster biomass increased (r²=0.7497). Our data suggest that oysters in Goseong Bay produce 3–196 million eggs during the spawning season. The immunological method developed in this study was sensitive enough to measure a small quantity of eggs and considered to be method of choice for studying bivalve reproduction.     

Removal of Toxoplasma gondii Oocysts from Sea Water by Eastern Oysters (Crassostrea virginica)

SUMMARY. Toxoplasma gondii infections have been reported in a number of marine mammals. Presently it is not known how these animals acquire T. gondii from their aquatic environment. The eastern oyster, Crassostrea virginica , has been shown to remove Cryptosporidium oocysts from seawater and a similar phenomenon may be occurring with T. gondii oocysts and marine invertebrates. The present study was done to determine if eastern oysters could remove and retain T. gondii oocysts from seawater. Oocysts of the VEG strain of T. gondii (1 × 10⁶ oocysts) were placed in seawater (32 ppt NaCl) containing live eastern oysters. The infected seawater was removed one day postinoculation (PI) and replaced with fresh seawater. Selected oysters were removed at 1, 3 and 6 days PL Hemolymph, gill washes, and oyster tissue were collected separately at each observation time. The oyster tissue was homogenized and all 3 samples fed separately to mice. Toxoplasma gondii positive mice were observed at each time period. The results indicate that T. gondii oocysts can be removed from seawater by eastern oysters and retain their infectivity. Contaminated raw oysters may serve as a source of T. gondii infection for marine mammals and humans.