MSX Disease Research Articles

Investigating The Life Cycle ofHaplosporidium Nelsoni(Msx): A Review

ABSTRACT Attempts to decipher the life cycle of Haplosporidium nelsoni began almost immediately after it was identified as the pathogen causing MSX disease in eastern oysters, Crassostrea virginica. But transmission experiments failed and the spore stage, characteristic of haplosporidans, was extremely rare. Researchers concluded that another host was involved: an intermediate host in which part of the life cycle was produced, or—if the oyster was an accidental host—an alternate host that produces infective elements. A later finding that spores were found more often in spat (<1 y old) than in adults revived the idea of direct transmission between oysters. The new findings and the availability of molecular diagnostics led us to revive life cycle investigations. Over several years, oyster spat were examined for spores and searched for H. nelsoni in potential non-oyster hosts using both histological and polymerase chain reaction (PCR) methodologies. Although spores occurred in a high proportion of spat with ...

Anthropogenic impacts on an oyster metapopulation: Pathogen introduction, climate change and responses to natural selection

Abstract Humans rely on marine ecosystems for a variety of services but often impact these ecosystems directly or indirectly limiting their capacity to provide such services. One growing impact is the emergence of marine disease. We present results from a unique case study examining how oysters, a dominant organism in many coastal bays and estuaries that is often harvested for food, have responded to pathogens influenced by human activities, namely the introduction of novel pathogens. Climate change has enabled a northward spread and establishment of Dermo disease in oysters along the eastern seaboard of North America and human activities inadvertently introduced MSX disease along this same coast. Oysters in Delaware Bay have responded differently to each pathogen, and uniquely to MSX disease by developing a highly resistant baywide population not documented in any other bay. Offspring were produced using parents collected from low or high disease (MSX and Dermo) regions of Delaware Bay and exposed in a common garden experiment along with a naïve population from Maine. Results indicated widespread resistance to MSX disease, but not to Dermo disease, across Delaware Bay. One striking result was the demonstration of resilience in the population through its capacity to spread, presumably through larval transport, resistance to MSX disease into portions of the population that have experienced little to no MSX disease pressure themselves. Related studies indicated that larval transport mechanisms allowed widespread dispersal such that the entire metapopulation could acquire a high level of resistance over time if disease resistance is sufficiently heritable. The findings have implications for restoration, management and recovery of diseased populations. Namely, that if left to their own devices, natural selection may find a solution that enables populations to recover from introduced pathogens.

Outcomes of asymmetric selection pressure and larval dispersal on evolution of disease resistance: a metapopulation modeling study with oysters

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 531:221-239 (2015) - DOI: https://doi.org/10.3354/meps11349 Outcomes of asymmetric selection pressure and larval dispersal on evolution of disease resistance: a metapopulation modeling study with oysters Daphne M. Munroe1,*, Eric N. Powell2, Susan E. Ford1, Eileen E. Hofmann3, John M. Klinck3 1Haskin Shellfish Research Laboratory, Rutgers University, 6959 Miller Ave., Port Norris, NJ 08349, USA 2Gulf Coast Research Laboratory, University of Southern Mississippi, 703 East Beach Drive, Ocean Springs, MS 39564, USA 3Center for Coastal Physical Oceanography, Department of Ocean, Earth and Atmospheric Sciences, 4111 Monarch Way, 3rd Floor, Old Dominion University, Norfolk, VA 23529, USA *Corresponding author: dmunroe@hsrl.rutgers.edu ABSTRACT: Marine diseases are a strong selective force that can have important economic and ecological consequences. Larval dispersal patterns, selective mortality and individual growth rates can modulate metapopulation responses to disease pressure. Here, we use a modeling framework that includes distinct populations, connected via larval transport, with varying disease selection pressure and connectivity to examine how these dynamics enhance or inhibit the evolution of disease resistance in metapopulations. Our system, oysters and MSX disease, is one in which disease resistance is highly and demonstrably heritable. Simulations show that under conditions of population isolation (i.e. local retention of larvae) and strong disease selection, populations rapidly evolve genetic disease resistance. Varying the patterns of larval dispersal alone doubles the time for evolution of disease resistance. Spatially varying disease in the absence of larval dispersal leaves some populations unable to respond to the disease, whereas adding larval dispersal slows the response of populations under strong selection and speeds the response in populations under low selection when fitness is based on relatively limited genetic structure (‘juvenile fitness’ in our simulations). Under spatially variable disease pressure, larval dispersal generates a fourfold greater variance in fitness outcomes across the dispersal patterns tested. In a metapopulation, populations experiencing lower selection pressure will tend to slow the development of other, more heavily selected populations. This suggests that conservation efforts aimed at improving overall metapopulation resistance in the face of marine diseases should target those populations under modest or high disease pressure, rather than protecting those experiencing low selective pressure. KEY WORDS: Larval dispersal · Metapopulation dynamics · Connectivity · Disease · Oyster · Resistance · Structured population model · Genetic adaptation · Evolution Full text in pdf format PreviousNextCite this article as: Munroe DM, Powell EN, Ford SE, Hofmann EE, Klinck JM (2015) Outcomes of asymmetric selection pressure and larval dispersal on evolution of disease resistance: a metapopulation modeling study with oysters. Mar Ecol Prog Ser 531:221-239. https://doi.org/10.3354/meps11349 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 531. Online publication date: July 02, 2015 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2015 Inter-Research.

Lineage-specific molecular probing reveals novel diversity and ecological partitioning of haplosporidians

Haplosporidians are rhizarian parasites of mostly marine invertebrates. They include the causative agents of diseases of commercially important molluscs, including MSX disease in oysters. Despite their importance for food security, their diversity and distributions are poorly known. We used a combination of group-specific PCR primers to probe environmental DNA samples from planktonic and benthic environments in Europe, South Africa and Panama. This revealed several highly distinct novel clades, novel lineages within known clades and seasonal (spring vs autumn) and habitat-related (brackish vs littoral) variation in assemblage composition. High frequencies of haplosporidian lineages in the water column provide the first evidence for life cycles involving planktonic hosts, host-free stages or both. The general absence of haplosporidian lineages from all large online sequence data sets emphasises the importance of lineage-specific approaches for studying these highly divergent and diverse lineages. Combined with host-based field surveys, environmental sampling for pathogens will enhance future detection of known and novel pathogens and the assessment of disease risk.

Diseases of oysters Crassostrea ariakensis and C. virginica reared in ambient waters from the Choptank River, Maryland and the Indian River Lagoon, Florida

To assess potential benefits and liabilities from a proposed introduction of Asian Suminoe oysters, susceptibilities of exotic Crassostrea ariakensis and native C. virginica oysters were compared during exposures to pathogens endemic in temperate, mesohaline waters of Chesapeake Bay and sub-tropical, polyhaline Atlantic waters of southern Florida, USA. Cohorts of diploid, sibling oysters of both species were periodically tested for diseases while reared in mesocosms receiving ambient waters from the Choptank River, Maryland (>3 yr) or the Indian River Lagoon, Florida (10 to 11 mo). Haplosporidium sp. infections (e.g. MSX disease) were not detected in oysters from either site. Perkinsus sp. infections (dermo disease) occurred among members of both oyster species at both sites, but infections were generally of low or moderate intensities. A Bonamia sp. was detected by PCR of DNAs from tissues of both oyster species following exposure to Florida waters, with maximum PCR prevalences of 44 and 15% among C. ariakensis and C. virginica oysters respectively during June 2007. Among C. ariakensis oysters sampled during April to July 2007, a Bonamia sp. was detected in 31% of oysters by PCR (range 11 to 35%) and confirmed histologically in 10% (range 0 to 15%). Among simultaneously sampled C. virginica oysters, a Bonamia sp. was detected in 7% by PCR (range 0 to 15%), but histological lesions were absent. Although this is the first report of a Bonamia sp. from Florida waters, sequences of small subunit (SSU) rDNA and in situ hybridization (ISH) assays both identified the Florida pathogen as Bonamia exitiosa, which also infects oysters in the proximate waters of North Carolina, USA.

Development of resistance to an introduced marine pathogen by a native host

In 1957–1959, the introduced protistan parasite, Haplosporidium nelsoni, killed 90–95% of the oysters (Crassostrea virginica) in lower Delaware Bay and about half of those in the upper bay. Shortly thereafter, H. nelsoni-caused mortality in the wild population of the lower bay declined, approximating that of first-generation selectively bred oysters. For nearly three decades thereafter no further change in survival of the wild population was evident, although steady improvement was achieved by continued selective breeding. Survival of the wild population is thought to have plateaued because the great majority of oysters inhabited the upper bay where they were protected from H. nelsoni infection and selective mortality by low salinity. Consequently, they contributed most of the offspring to the bay population. From 1957 through 1987, H. nelsoni prevalence was cyclic, but overall high (annual maxima of 60 to 85%) in the lower bay. Since 1988, however, prevalence in wild oysters has rarely exceeded 30% anywhere in the bay, even though unselected oysters continue to become heavily infected when exposed, and molecular evidence indicates that the parasite remains present throughout the bay. This apparent “second step” in the development of resistance in the wild oysters occurred after a drought-associated incursion of H. nelsoni into the upper bay in the mid-1980s. Mortalities were widespread, heavy and more extreme than during the 1957–59 epizootic. Resistant survivors of the second epizootic have apparently repopulated the bay. When compared to unselected stocks, common-garden exposure to H. nelsoni of oysters from both upbay and downbay sites indicates that a high degree of resistance to the development of MSX disease has become widespread in the wild oyster population of Delaware Bay after two major selection events separated by nearly 30 years.

The Potential for Oysters,Crassostrea virginica, to Develop Resistance to Dermo Disease in the Field: Evaluation using a Gene-Based Population Dynamics Model

Today, populations of eastern oysters, Crassostrea virginica, are commonly limited by disease mortality. Resistance to MSX disease has developed in a number of cases, but the development of resistance to Dermo disease would appear to be limited, despite the high mortality rates and frequency of epizootics. Can aspects of the host's genetics or population dynamics limit the response to the disease despite the apparent opportunity afforded by alleles conferring disease resistance or tolerance? To answer this question, we use a gene-based population dynamics model, configured for C. virginica, to simulate the development of disease resistance using mortality as the agent of selection. Simulated populations were exposed to 4 levels of mortality covering the range in mortality observed in Delaware Bay in the 1990s. In each case, disease resistance increased in the simulated population over time, normally proportional to the increase in mortality rate imposed by the disease. However, simulations show that the population responds even at its most rapid rate on multidecadal to half-century timescales. As the mortality rate declines with increasing disease resistance, the rate of further improvement in disease resistance likewise declines. Thus, disease resistance develops over decadal timescales at a 40%-per-year mortality rate, but, as mortality rate falls to 25% per year, the rate of further development of disease resistance extends to half-century timescales. The discouraging profundity is that a mortality rate of 25% per year, yielding a rate of selection profoundly slow, is still very high. In northern climes, significant decrements in oyster abundance will occur. Evidence from fisheries retrospectives suggests that oysters cannot withstand a constant removal at this scale without compromising population integrity noticeably. So, a mortality rate that grievously limits the development of disease resistance still sorely strains the species' ability to maintain a vibrant population necessary to its long-term survival.

Using bivalves as particle collectors with PCR detection to investigate the environmental distribution of Haplosporidium nelsoni

Recently, PCR technology has been applied to search for marine microorganisms in environmental samples. Such sampling, however, has drawbacks, including the need to collect and filter large volumes of water, and the presence of substances in environmental samples that may destroy DNA or interfere with DNA isolation and amplification. We explored the possibility of using suspension-feeding bivalves in conjunction with PCR to investigate the environmental distribution of microparasites using the oyster pathogen Haplosporidium nelsoni (the MSX disease agent) with oysters and mussels in Delaware Bay, USA, as a model system. Delaware Bay oysters Crassostrea virginica have become very resistant to H. nelsoni infection development and rarely exhibit histologically detectable lesions. The ribbed mussel Geukensia demissa is also resistant. Infections were detected in only 6% of the histologically examined oysters in the present study, although PCR-positive signals for H. nelsoni were found in feces, gill or heart samples of up to 100% of oysters. Positive signals were found in the feces and gill (but not heart) samples of up to 50% of mussels. The temporal evolution of PCR signals suggested a wave-like pattern of putative infective particles, which mimicked a pattern documented in early mortality studies. The present study demonstrated that H. nelsoni persists throughout Delaware Bay, although it is rarely detected using standard histology. We propose that the use of suspension-feeding bivalves as particle collectors in combination with PCR could be a method for detecting the presence of marine microparasites and might help answer questions about factors that prevent outbreaks of epizootics in certain regions.

GROWTH AND MORTALITY OF DERMO-DISEASE-FREE JUVENILE OYSTERS (CRASSOSTREA VIRGINICA) AT THREE SALINITY REGIMES IN AN ENZOOTIC AREA OF CHESAPEAKE BAY

Abstract Specific-pathogen-free (SPF) oysters (Crassostrea virginica) were set on shell and deployed on three oyster bars along a salinity gradient in the Patuxent River, MD, to determine growth, time to initial infection by Perkinsus marinus (causative agent for dermo disease), and subsequent mortalities. Initial deployment was in September 2000, during the second year of a 4-y drought. The salinity gradient experienced by these oysters during the drought was 4‰ to 6‰ above normal and provided ideal conditions for proliferation and dissemination of P. marinus. Oysters grew well during the first year, but mortalities rose rapidly during the second year, and reached 97% to 98% at all sites by the end of the second year. Although mean P. marinus body burdens reached levels of 2.3 × 107 cells·g−1 oyster tissue, MSX disease was also detected at both the lower and midriver sites in 2002, and was probably responsible for some mortalities at those sites. Due to extensive mortalities of the first deployment, we b...

A review of recent information on the Haplosporidia, with special reference toHaplosporidium nelsoni(MSX disease)

The current status of the Haplosporidia is reviewed as well as recent information on Haplosporidium nel- soni, the causative agent of MSX disease in oysters. Recent molecular phylogenetic analyses with greatly increased taxon sampling support monophyly of the Haplosporidia and hypothesize placement of the group as sister taxon to the phylum Cercozoa. Oyster pathogens in the genus Bonamia should be considered haplosporidians based on molecular sequence data. Thus, the group contains 4 genera: Uropsoridium, Haplosporidium, Bonamia and Minchinia. Molecular phylogenetic analyses support monophyly of Urosporidium, Bonamia and Minchinia ,b utHaplosporidium forms a pa- raphyletic clade. Reports of haplosporidia worldwide are reviewed. Molecular detection assays have greatly increased our ability to rapidly and specifically diagnose important pathogens in the phylum and have also improved our under- standing of the distribution and biology of H. nelsoni and H. costale. Much of the data available for H. nelsoni has been integrated into a mathematical model of host/parasite/environment interactions. Model simulations support hypotheses that recent H. nelsoni outbreaks in the NE United States are related to increased winter temperatures, and that a host other than oysters is involved in the life cycle. Evidence is presented that natural resistance to H. nelsoni has developed in oysters in Delaware Bay, USA. However, in Chesapeake Bay, USA H. nelsoni has intensified in historically low salinity areas where salinities have increased because of recent drought conditions. Efforts to mitigate the impact of H. nelsoni involve selective breeding programs for disease resistance and the evaluation of disease resistant non-native oysters.

Use of particle filtration and UV irradiation to prevent infection by Haplosporidium nelsoni (MSX) and Perkinsus marinus (Dermo) in hatchery-reared larval and juvenile oysters

Means to control infection by pathogenic organisms are needed to help ensure that aquaculture is not a source for the spread of infectious diseases in wild and cultured stocks. Questions often arise as to whether larval or juvenile stages become infected in the hatchery or nursery phase of production, and if so, how they might be protected. To help answer these questions, we utilized both traditional and molecular diagnostic methods to detect two eastern oyster, Crassostrea virginica, pathogens ( Haplosporidium nelsoni, cause of MSX disease and Perkinsus marinus, cause of Dermo disease) in larval and juvenile oysters reared at a hatchery/on-shore nursery receiving water in which both parasites are enzootic. Our study indicated that filtration of water to 1 μm using a cartridge filter followed by exposure to 30,000 μW s −1 cm −2 ultraviolet (UV) irradiation was an effective means of preventing infections of the larvae and post-set. Once the juveniles were moved from the highly treated hatchery water to an upweller nursery receiving only roughly (150 μm) bag-filtered water, however, they became infected by both parasites.

Use of Competitive PCR to Detect and Quantify Haplosporidium nelsoni Infection (MSX disease) in the Eastern Oyster (Crassostrea virginica).

This study was undertaken to develop a quantitative polymerase chain reaction assay that would improve the utility of PCR for detecting Haplosporidium nelsoni (MSX), a serious parasite of the eastern oyster Crassostrea virginica. A competitive PCR sequence was generated from the H. nelsoni small subunit ribosomal DNA fragment, originally described by Stokes and colleagues, that was amplified by the same PCR primers and had similar amplification performance. Assays performed using competitor dilutions ranging from 0.05 to 500 pg/µl DNA were used to test oyster samples designated using histological techniques as having "light" or "heavy" MSX infections. Visual diagnoses were confirmed equally well with three methods: densitometry of ethidium-bromide-stained agarose, densitometry of SYBRGreen-stained polyacrylamide gels, and analysis by GeneScan 3.0 of fluorescent products detected in ultrathin gels. Oysters diagnosed as negative for MSX tested as negative or light by PCR. Oysters with light MSX infections generally had less than 5 pg/µl infectious DNA. Oysters with heavy infections generally corresponded to 5 pg/µl or greater competitor dilutions.

A PCR-ELISA Method for Direct Detection of the Oyster Pathogen Haplosporidium nelsoni.

: A rapid method, utilizing both polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), was developed for detection of oyster MSX disease. The technique included using Haplosporidium nelsoni pathogen-specific PCR primers (based on ribosomal RNA genes), a Chelex resin (for rapid DNA extraction from oyster mantle tissues), and cloned H. nelsoni rRNA plasmid DNA (for use as a capture probe). Digoxigenin was incorporated into the pathogen-specific PCR products, which were captured by the coated probe in a fast hybridization reaction and then detected by ELISA. The sensitivity of PCR amplification on cloned plasmid DNA was 10 fg for detection by stained agarose gel, and increased to 0.01 fg for ELISA. Positive signals were observed in infected oysters using the PCR-ELISA technique. This method may be applicable to early detection of infection.

Altered Response of Oyster Hemocytes toHaplosporidium nelsoni(MSX) Plasmodia Treated with Enzymes or Metabolic Inhibitors

To avoid phagocytosis, parasites may mask themselves with host-like molecules that prevent recognition as nonself or they may produce substances that interfere with host cellular defenses. The protozoan parasiteHaplosporidium nelsoni,which causes MSX disease in the eastern oysterCrassostrea virginica,is not ingested by host hemocytes. To assess potential avoidance mechanisms, oyster hemocytes were incubated with plasmodial stages of the parasite that had been pretreated with one of a variety of enzymes (proteases and carbohydrases) to alter surface molecules or with metabolic inhibitors to prevent the synthesis or active uptake of “masking” molecules, as well as the production and discharge of inhibitory substances. The maximum increase in phagocytosis resulting from treatment with carbohydrases was 12.5% (β-galactosidase) and with proteases was 18% (Proteinase K). Inhibitors of aerobic metabolism resulted in a similar level of enhancement. In contrast, treatment of parasites with the glycolysis inhibitor iodoacetate enhanced phagocytosis by up to 66%. Thus, the process that obstructs phagocytosis involves aerobic and, especially, anaerobic pathways. The greater effect of a metabolic inhibitor compared to enzymes suggests that the mechanism involves more than just surface modification and may include the production of interference molecules.

In vitro Interactions between Bivalve Hemocytes and the Oyster Pathogen Haplosporidium nelsoni (MSX)

Resistance to disease caused by the oyster pathogen Haplosporidium nelsoni (MSX) has developed in nature and through selective breeding; however, examination of tissue slides shows no evidence that phagocytic cells are involved. To investigate this phenomenon further, we quantified in vitro phagocytosis of plasmodial stages of H. nelsoni by hemocytes from 2 oyster and 1 mussel species. Our results show that hemocytes from oysters Crassostrea virginica, whether selected or unselected for resistance to MSX disease, did not ingest plasmodia unless the parasites were damaged or killed. Time-lapse photography suggested that granular hemocytes retreated rapidly after initial contact with living plasmodia and thatnearby agranular hemocytes showed little movement

Cellular Responses of Oysters Infected with Haplosporidium nelsoni: Changes in Circulating and Tissue-Infiltrating Hemocytes

The protozoan parasite Haplosporidium nelsoni (MSX) elicits an inflammatory-type response in oysters, Crassostrea virginica. We assayed circulating hemocytes of oysters exposed to H. nelsoni to quantify the effects of parasitism, selection for resistance, and season on total and differential counts. All factors had a significant (P < 0.02) effect on total counts, but explained relatively little of the overall variance [season (12%) > selection (4%) > infection (2%)]. Circulating hemocyte densities increased with infection intensity in resistant animals, but were depressed in susceptible oysters with light infections. Counts in both groups were lowest in August and highest in May, at which times densities in susceptible oysters were reduced (P < 0.02) compared to those in resistant animals. No differences existed in November. Susceptible oysters may be more debilitated by infections than resistant animals, resulting in impaired circulation and depressed cell counts. The number of tissue-infiltrating hemocytes increased with infection intensity (P < 0.0001), but showed no statistically significant (P > 0.05) association with numbers of circulating hemocytes in individual oysters. Size-frequency analysis with a Coulter counter indicated that the proportion of large cells (presumed to be granular hemocytes) was lower (P < 0.0001) in susceptible oysters, which were also heavily infected, compared to resistant oysters, which had very few infections. The loss of granular hemocytes may stem from degranulation associated with tissue damage and inflammation. Present evidence suggests that the principal role of the hemocyte response in MSX disease is to plug lesions, remove debris, and repair tissues and that these functions may help oysters survive infection.

Maintenance of Heterozygosity during Selective Breeding of Oysters for Resistance to MSX Disease

Maintenance of Heterozygosity during Selective Breeding of Oysters for Resistance to MSX Disease

The Decline of Private Sector Oyster Culture in Virginia: Causes and Remedial Policies

Oyster production from private grounds in Virginia has declined significantly since 1960. Restoring industry production requires a better understanding of the reasons for this decline. Currently, state fishery managers and the popular press attribute the decline to the oyster disease Haplosporidium nelsoni (MSX). In fact, private planters themselves consider MSX disease as the primary constraint on the future profitability of private oyster culture. This study analyzes potential causes of the decline using a simulation model of oyster production with data from the 1960s, 1970s, and 1980s. The analysis suggests that rising seed prices driven by economic and productivity changes are more important than MSX in reducing the economic incentives to private planting. Therefore, reducing oyster seed prices is an effective policy strategy. However, education of planters will also be needed to assure a better understanding of the profit potential in oyster planting.

Oyster-MSX interactions: Alterations in hemolymph enzyme activities in Crassostrea virginica during the course of Minchinia nelsoni disease development

Hemolymph enzyme activities were investigated during the course of Minchinia nelsoni (MSX) disease development in Crassostrea virginica. Aspartate aminotransferase, alanine aminotransferase, and phosphohexose isomerase activities increase significantly during the gill lesion stage of MSX disease. Enzyme activities are not significantly elevated during the general infection stage of MSX disease. The alteration of hemolymph enzyme activity is discussed with respect to host metabolism and possible humoral defense mechanisms.