Prevalence, intensity and pathology of the nasal parasite Nasicola hogansi in Atlantic bluefin tuna (Thunnus thynnus).

Ectoparasitic flatworms of Nasicola (Monogenoidea: Capsalidae), which infect nasal epithelium of true tunas (Thunnus spp.), are not well studied, nor have their impacts on the host's olfactory organ been evaluated. Infections of Nasicola hogansi on Atlantic bluefin tuna, Thunnus thynnus, were investigated with emphasis on the relationship between infection prevalence, abundance and mean intensity with bluefin tuna size, sex, body condition and capture month, as well as histopathological effects. Commercially caught Atlantic bluefin tuna (n = 161, 185-305 cm curved fork length) from the Gulf of Maine were sampled during June through August 2009 for infections by N. hogansi. A total of 247 specimens of N. hogansi were collected, with a prevalence of 45.3%, mean abundance of 1.57 (CI: 1.21-2.03) and mean intensity of 3.45 (CI: 2.91-4.22). Neither fish sex nor landing month had a significant effect on parasite parameters. Larger and better-conditioned Atlantic bluefin tuna had a higher mean intensity of infection. Pathology associated with infection by N. hogansi included extensive necrosis, sloughing of the nasal epithelium and associated inflammation of underlying connective tissues. Further epidemiological and pathological study of this host-parasite system is warranted since impaired olfaction, if present, could adversely affect spawning and migration of this top ocean predator.

Impact of disease on the survival of three commercially fished species.

Recent increases in emergent infectious diseases have raised concerns about the sustainability of some marine species. The complexity and expense of studying diseases in marine systems often dictate that conservation and management decisions are made without quantitative data on population-level impacts of disease. Mark-recapture is a powerful, underutilized, tool for calculating impacts of disease on population size and structure, even in the absence of etiological information. We applied logistic regression models to mark-recapture data to obtain estimates of disease-associated mortality rates in three commercially important marine species: snow crab (Chionoecetes opilio) in Newfoundland, Canada, that experience sporadic epizootics of bitter crab disease; striped bass (Morone saxatilis) in the Chesapeake Bay, USA, that experience chronic dermal and visceral mycobacteriosis; and American lobster (Homarus americanus) in the Southern New England stock, that experience chronic epizootic shell disease. All three diseases decreased survival of diseased hosts. Survival of diseased adult male crabs was 1% (0.003-0.022, 95% CI) that of uninfected crabs indicating nearly complete mortality of infected crabs in this life stage. Survival of moderately and severely diseased striped bass (which comprised 15% and 11% of the population, respectively) was 84% (70-100%, 95% CI), and 54% (42-68%, 95% CI) that of healthy striped bass. The disease-adjusted yearly natural mortality rate for striped bass was 0.29, nearly double the previously accepted value, which did not include disease. Survival of moderately and severely diseased lobsters was 30% (15-60%, 95% CI) that of healthy lobsters and survival of mildly diseased lobsters was 45% (27-75%, 95% CI) that of healthy lobsters. High disease mortality in ovigerous females may explain the poor recruitment and rapid declines observed in this population. Stock assessments should account for disease-related mortality when resource management options are evaluated.

Temperature, hypoxia, and mycobacteriosis: effects on adult striped bass Morone saxatilis metabolic performance.

Mycobacteriosis, a chronic bacterial disease of fishes, is prevalent in adult striped bass from Chesapeake Bay (USA). Although environmental factors may play a role in disease expression, the interaction between the disease and environmental stress remains unexplored. We therefore examined the individual and interactive effects of elevated temperature, hypoxia, and mycobacteriosis on the metabolism of wild-caught adult striped bass from Chesapeake Bay using respirometry. Because the spleen is the primary target organ of mycobacteriosis in striped bass, we hypothesized that the disease interferes with the ability of fish to increase their hematocrit in the face of increasing oxygen demands. We determined standard metabolic rate (SMR), maximum metabolic rate under normoxia (MMRN), critical oxygen saturation (S(crit)), and MMR under hypoxia (3 mg O(2) l-1: MMR(H)) for healthy and visibly diseased fish (i.e. exhibiting skin lesions indicative of mycobacteriosis). Measurements were taken at a temperature within the preferred thermal range (20°C) and at an elevated temperature (28°C) considered stressful to striped bass. In addition, we calculated aerobic scope (AS(N) = MMR(N) - SMR, AS(H) = MMR(H) - SMR) and factorial scope (FS(N) = MMR(N) SMR-1, FS(H) = MMR(H) SMR-1). SMR increased with increasing temperature, and hypoxia reduced MMR, AS, and FS. Mycobacteriosis alone did not affect either MMR(N) or MMR(H). However, elevated temperature affected the ability of diseased striped bass to tolerate hypoxia (S(crit)). Overall, our data indicate that striped bass performance under hypoxia is impaired, and that elevated water temperatures, hypoxia, and severe mycobacteriosis together reduce aerobic scope more than any of these stressors acting alone. We conclude that the scope for activity of diseased striped bass in warm hypoxic waters is significantly compromised.

Dermal mycobacteriosis and warming sea surface temperatures are associated with elevated mortality of striped bass in Chesapeake Bay.

Temperature is hypothesized to alter disease dynamics, particularly when species are living at or near their thermal limits. When disease occurs in marine systems, this can go undetected, particularly if the disease is chronic and progresses slowly. As a result, population-level impacts of diseases can be grossly underestimated. Complex migratory patterns, stochasticity in recruitment, and data and knowledge gaps can hinder collection and analysis of data on marine diseases. New tools enabling quantification of disease impacts in marine environments include coupled biogeochemical hydrodynamic models (to hindcast key environmental data), and multievent, multistate mark-recapture (MMSMR) (to quantify the effects of environmental conditions on disease processes and assess population-level impacts). We used MMSMR to quantify disease processes and population impacts in an estuarine population of striped bass (Morone saxatilis) in Chesapeake Bay from 2005 to 2013. Our results supported the hypothesis that mycobacteriosis is chronic, progressive, and, frequently, lethal. Yearly disease incidence in fish age three and above was 89%, suggesting that this disease impacts nearly every adult striped bass. Mortality of diseased fish was high, particularly in severe cases, where it approached 80% in typical years. Severely diseased fish also had a 10-fold higher catchability than healthy fish, which could bias estimates of disease prevalence. For both healthy and diseased fish, mortality increased with the modeled average summer sea surface temperature (SST) at the mouth of the Rappahannock River; in warmer summers (average SST ≥ 29°C), a cohort is predicted to experience >90% mortality in 1 year. Regression of disease signs in mildly and moderately diseased fish was <2%. These results suggest that these fish are living at their maximum thermal tolerance and that this is driving increased disease and mortality. Management of this fishery should account for the effects of temperature and disease on impacted populations.

Monoclonal antibody analysis of Perkinsus marinus extracellular products.

The protozoan oyster parasite Perkinsus marinus releases a complex set of extracellular products (ECP) during in vitro culture. These products have been previously implicated in parasite virulence, and their expression can be altered by medium supplementation with oyster tissue homogenate. Little is known regarding ECP function, regulation, or mechanism of storage and release. Perkinsus marinus ECP were purified from a protein-free medium and used to produce a panel of five monoclonal antibodies. Several of the antibodies recognised series of proteins implying that the ECP may originate from comparatively few parental molecules. The ECP are secreted by several pathways, including the release of one product from an external cell layer, and two other products from two morphologically distinct intracellular compartments. Antibodies against separate epitopes on one protein provided information about possible protein structure. A sandwich ELISA format allowed sensitive quantification of that protein and showed significantly reduced protein expression in oyster tissue homogenate supplemented cultures. Immunopurification allowed tandem mass spectroscopic amino acid sequencing of that protein. Another antibody was used to characterise the P. marinus cell wall. This antibody specifically bound to trophozoite and tomont walls, and was used to investigate the morphological and antigenic changes in these walls during Ray's fluid thioglycollate medium-induced formation of hypnospores. It was also used to confirm that oyster tissue homogenate supplementation could induce formation of hypnospores. This antibody labeled P. marinus cells in fixed oyster tissue in a species-specific manner.

Factors influencing the sporulation and cyst formation of Aphanomyces invadans, etiological agent of ulcerative mycosis in Atlantic menhaden, Brevoortia tyrannus.

Oomycete infections caused by Aphanomyces invadans occur in freshwater and estuarine fishes around the world. Along the east coast of the USA, skin ulcers caused by A. invadans are prevalent in Atlantic menhaden, Brevoortia tyrannus. From laboratory observations low salinities appear crucial to transmission of the pathogen. To better understand aspects of transmission, we characterized sporulation and cyst formation of secondary zoospores of two isolates of A. invadans at different salinities and temperatures. Sporulation occurred only at low salinities. At room temperature (ca. 20-22 C), using "pond water" augmented with artificial sea salts, the endemic strain WIC and the Thailand strain PA7 of A. invadans produced free-swimming secondary zoospores at salinities of 0, 1 and 2 psu (practical salinity unit = per thousand), but not at 4 psu or higher. Secondary zoospores of another species, ATCC-62427 (Aphanomyces sp.), were observed at 1, 2, 4 and 8 psu but not at 0 and 12 psu. Secondary zoospores of all three isolates, especially WIC, were abundant and motile 1-2 d postsporulation. Sporulation was temperature dependent and occurred over a relatively narrow range. No sporulation occurred at 4, 30 or 35 C for either WIC or PA7. For both strains zoospore production within 1-3 d after the initiation of sporulation was more prolific at 25 C than at 20 and 15 C. At 15 C production of zoospores was sustained over 11 d for WIC and 5 d for PA7. At room temperature single WIC secondary zoospores remained motile 12-18 h. Salinities exceeding 4 psu or vigorous shaking caused immediate cyst formation of WIC secondary zoospores. Exposure to menhaden tissue, but not tissues of other fishes to secondary zoospores (WIC), caused rapid (2 h) cyst formation. Cysts were capable of excysting when transferred to 1 psu water within 2-3 h of cyst formation. Cysts that had remained encysted in 6.5 psu for 24 h did not excyst when transferred to 1 psu water. Salinity and temperature requirements for sporulation indicate that juvenile menhaden must acquire infections during rain or in low salinity oligohaline waters.

Morphology and ontogeny of multiple lateral-line canals in the rock prickleback, Xiphister mucosus (Cottiformes: Zoarcoidei: Stichaeidae).

The structure and ontogeny of lateral-line canals in the Rock Prickleback, Xiphister mucosus, were studied using cleared-and-stained specimens, and the distribution and morphology of neuromasts within lateral-line canals were examined using histology. X. mucosus has seven cephalic canals in a pattern that, aside from four branches of the infraorbital canals, is similar to that of most teleostean fishes. Unlike most other teleosts, however, X. mucosus features multiple trunk lateral-line canals. These include a short median posterior extension of the supratemporal canal and three paired, branching canals located on the dorsolateral, mediolateral, and ventrolateral surfaces. The ventrolateral canal (VLC) includes a loop across the ventral surface of the abdomen. All trunk canals, as well as the branches of the infraorbitals, are supported by small, dermal, ring-like ossifications that develop independently from scales. Trunk canals develop asynchronously with the mediodorsal and dorsolateral canals (DLC) developing earliest, followed by the VLC, and, finally, by the mediolateral canal (MLC). Only the mediodorsal and DLC connect to the cephalic sensory canals. Fractal analysis shows that the complexity of the trunk lateral-line canals stabilizes when all trunk canals develop and begin to branch. Histological sections show that neuromasts are present in all cephalic canals and in the DLC and MLC of the trunk. However, no neuromasts were identified in the VLC or its abdominal loop. The VLC cannot, therefore, directly function as a part of the mechanosensory system in X. mucosus. The evolution and functional role of multiple lateral-line canals are discussed.

Infectivity and pathogenicity of the oomycete Aphanomyces invadans in Atlantic menhaden Brevoortia tyrannus.

Atlantic menhaden Brevoortia tyrannus develop characteristic skin ulcers in response to infection by the oomycete Aphanomyces invadans. To investigate pathogenicity, we conducted a dose response study. Juvenile menhaden were inoculated subcutaneously with 0, 1, 5, 10, 100, and 500 secondary zoospores per fish and monitored for 37 d post-injection (p.i.). Survival rates declined with increasing zoospore dose, with significantly different survivorship curves for the different doses. Moribund and dead fish exhibited characteristic ulcerous lesions at the injection site starting at 13 d p.i. None of the sham-injected control fish (0 zoospore treatment) died. The LD50 (lethal dose killing 50% of exposed menhaden) for inoculated fish was estimated at 9.7 zoospores; however, some fish receiving an estimated single zoospore developed infections that resulted in death. Menhaden were also challenged by aqueous exposure and confirmed that A. invadans was highly pathogenic by this more environmentally realistic route. Fish that were acclimated to culture conditions for 30 d, and presumably free of skin damage, then aqueously exposed to 100 zoospores ml(-1), exhibited 14% lesion prevalence with 11% mortality. Net-handled fish that were similarly infected had a significantly higher lesion prevalence (64%) and mortality (64%). Control fish developed no lesions and did not die. Scanning electron microscopy of fish skin indicated that zoospores adhered to intact epidermis, germinated and penetrated the epithelium with a germ tube. Our results indicate that A. invadans is a primary pathogen of menhaden and is able to cause disease at very low zoospore concentrations.

Genetic structure and mtDNA diversity of Fundulus heteroclitus populations from polycyclic aromatic hydrocarbon-contaminated sites.

Genetic structure and diversity of mummichog (Fundulus heteroclitus) populations were investigated using mitochondrial DNA (mtDNA) sequences. Forty-six haplotypes were identified among 208 mummichog from the Elizabeth and York Rivers in Virginia, USA. No evidence of decreased gene or nucleotide diversity for mummichog from polycyclic aromatic hydrocarbon (PAH)-contaminated sites was observed. However, based on mtDNA data from 17 sites, a significant correlation (Mantel analysis, p = 0.035) was noted between genetic distance (F(ST)) and PAH concentration but not between genetic distance and geographic distance. Mummichog from the most heavily PAH-contaminated site, Atlantic Wood (AW), were genetically distinct from those of other Elizabeth River sites. At AW, high frequencies of several divergent haplotypes were observed that were more closely allied to the northern mummichog than to the more abundant southern form in the Chesapeake Bay. These data suggested that a locally stable population existed at the AW site. This conclusion is consistent with the observation that mummichog from the AW site display enhanced tolerance to PAH contamination relative to mummichog from noncontaminated sites. Conclusions about gene diversity and the correlation between genetic distance with site differences in PAH concentrations were also consistent with those from tandem genetic analyses based on allozymes.

Fish (Fundulus heteroclitus) populations with different exposure histories differ in tolerance of creosote-contaminated sediments.

Prior studies suggest that field-collected fish (Fundulus heteroclitus) from a creosote-contaminated Superfund site (Atlantic Wood Industries site, Elizabeth River, VA, USA) have enhanced tolerance to local, contaminated sediments. This study was designed to test whether other populations in the Elizabeth River at less contaminated sites also show similar tolerance and whether this tolerance is heritable. To test this, F. heteroclitus populations were sampled from four sites within the Elizabeth River with varying sediment polycyclic aromatic hydrocarbon (PAH) concentrations (3.9-264 ng PAH/g dry wt 10(3)) and one reference site in a nearby, uncontaminated estuary (York River, VA, USA; 0.27 ng PAH/g dry wt x 10(3)). Embryo assays were performed to quantify population differences in teratogenic effects during contaminated sediment exposure. Atlantic Wood sediment was mixed with reference sediment to achieve a range of sediment concentrations. Minimal differences were observed in teratogenic effects among fish taken from sites within the Elizabeth River; however, embryos of fish collected from a nearby, uncontaminated York River site and exposed to contaminated sediments had a significantly higher proportion of embryos with cardiac abnormalities than those from the Elizabeth River sites. Embryos from wild-caught and laboratory-reared Elizabeth River F. heteroclitus were simultaneously exposed to contaminated sediments, and no significant tolerance differences were found between embryos from fish taken directly from the field and those reared for a generation in the lab. Differences between fish populations from the two estuaries were larger than differences within the Elizabeth River, and these differences in tolerance were heritable.

First report of Streptococcus parauberis in wild finfish from North America.

Streptococcosis is a common cause of pathology and mortality in fishes resulting in significant economic losses for the aquaculture industry. One etiologic agent of the disease, Streptococcus parauberis, has been associated with fish mortalities in Spain and Korea. Here we report the first identification of S. parauberis in wild finfish in Chesapeake Bay, USA. Gram-positive cocci were isolated from the spleens of striped bass, Morone saxatilis, and identified via species-specific primers and 16S rRNA gene sequencing. Biochemical characterization and antibiotic susceptibility tests were used to compare local isolates to isolates infecting aquacultured fishes and dairy cattle. This is also the first report of a plasmid in S. parauberis from any host.

Multistressor interactions in the zebrafish (Danio rerio): Concurrent phenanthrene exposure and Mycobacterium marinum infection.

The simultaneous exposure of organisms to toxicants and disease causing agents poses a serious risk to important stocks. Worldwide, aquatic animal disease outbreaks have been increasing in both frequency and severity, and many have been associated with anthropogenic environmental change. Little is known about the complex interactions of the immune system and biotransformational pathways of vertebrates; however, urbanization and coastal development create a scenario in which a wide range of species are exposed to chemical pollutants in conjunction with a wide spectrum of ubiquitous, opportunistic pathogens. These interactions can severely compromise organismal health. Potential effects include decreased fitness, increased predation, decreased fecundity, reduced metabolic activity, suppressed immune function and mortality. Recent attention has been paid to immunomodulation in toxicant exposed fishes. In our current study we investigated the effects of the common polycyclic aromatic hydrocarbon phenanthrene in conjunction with Mycobacterium marinum infection in the zebrafish, Danio rerio. The goal of our study was to elucidate the interactions between stressors in the host organism. Fish were exposed to either a high or low dose of phenanthrene, infected with M. marinum or received a combination exposure of toxicant and bacteria. Results of our study were evaluated using survivorship analysis, toxicant body burden, and histology. Our data show an interaction between M. marinum infection and exposure to a high dose of phenanthrene in the zebrafish. Survivorship was significantly reduced for animals only exposed to the high dose of phenanthrene as compared to all other experimental groups. The increased survivorship for fish exposed to both Mycobacterium and a high dose of phenanthrene suggests an antagonistic interaction between stressors. Body burden data, which show significant differences in the ratio of phenanthrene:metabolites between experimental groups, suggests a disruption of the biotransformational pathway. We postulate that the inflammatory response, initiated by bacterial infection, is impeding the ability of the zebrafish to completely metabolize phenanthrene. In addition, the correlation between reduced metabolite production and increased survival indicates that phenanthrene metabolites are more toxic than the parent compound. Our study underscores the importance of investigating multiple stressor interactions as a way to better understand complex environmental interactions.

GENETIC VARIATION AMONG STRAINS OF PSEUDOPFIESTERIA SHUMWAYAE AND PFIESTERIA PISCICIDA (DINOPHYCEAE)(1).

The putatively toxic dinoflagellates Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester and Pfiesteria piscicida Steid. et J. M. Burkh. have been implicated in massive fish kills and of having negative impacts on human health along the mid-Atlantic seaboard of the USA. Considerable debate still remains as to the mechanisms responsible for fish mortality (toxicity vs. micropredation) caused by these dinoflagellates. Genetic differences among these cultures have not been adequately investigated and may account for or correlate with phenotypic variability among strains within each species. Genetic variation among strains of Ps. shumwayae and P. piscicida was examined by PCR-RFLP analysis using cultures obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP), as well as those from our own and other colleagues' collection efforts. Examination of restriction digest banding profiles for 22 strains of Ps. shumwayae revealed the presence of 10 polymorphic restriction endonuclease sites within the first and second internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the rDNA complex, and the cytochrome oxidase subunit I (COI) gene. Three compound genotypes were represented within the 22 Ps. shumwayae strains. Conversely, PCR-RFLP examination of 14 strains of P. piscicida at the same ITS1, 5.8S, and ITS2 regions revealed only one variable restriction endonuclease site, located in the ITS1 region. In addition, a dinoflagellate culture listed as P. piscicida (CCMP 1928) and analyzed as part of this study was identified as closely related to Luciella masanensis P. L. Mason, H. J. Jeong, Litaker, Reece et Steid.

Fundulus as the premier teleost model in environmental biology: opportunities for new insights using genomics.

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.

Morphometry of hepatic neoplasms and altered foci in the mummichog, Fundulus heteroclitus.

The goal of this study was to intensively sample a small number of livers from a population of mummichog exposed to PAH-contaminated sediments and evaluate them for lesion pathology, distribution, shape, and volume, and the number of histological sections needed to adequately describe the extent of various lesions. Volumetric data for each lesion type from each step section was derived from digitized section images. The total number of hepatic alterations ranged from 10-125 per fish. Alterations included: eosinophilic, basophilic, and clear cell foci; hepatocellular carcinomas; hemangiopericytomas; and cholangiomas. Lesion volumes ranged from 0.00012-64 mm3 and represented 0.21%-67% of total liver volume. There was a tendency for the lesions to be more dorsal-ventrally compressed than spherical or ropelike when observed from longitudinal sections. Periodic subsampling of the data indicated that. on average, 6 evenly spaced, longitudinal histological sections were required to accurately estimate lesion volume and extent in our model population. These data provide a formulation for histological sampling techniques and methodological support for piscine and other cancer study models that observe lesion volume changes over time. Further, this study fosters the development of early quantitative endpoints. rather than using a large number of animals and waiting for tumor progression or death to occur.

Pfiesteria shumwayae kills fish by micropredation not exotoxin secretion.

Pfiesteria piscicida and P. shumwayae reportedly secrete potent exotoxins thought to cause fish lesion events, acute fish kills and human disease in mid-Atlantic USA estuaries. However, Pfiesteria toxins have never been isolated or characterized. We investigated mechanisms by which P. shumwayae kills fish using three different approaches. Here we show that larval fish bioassays conducted in tissue culture plates fitted with polycarbonate membrane inserts exhibited mortality (100%) only in treatments where fish and dinospores were in physical contact. No mortalities occurred in treatments where the membrane prevented contact between dinospores and fish. Using differential centrifugation and filtration of water from a fish-killing culture, we produced 'dinoflagellate', 'bacteria' and 'cell-free' fractions. Larval fish bioassays of these fractions resulted in mortalities (60-100% in less than 24 h) only in fractions containing live dinospores ('whole water', 'dinoflagellate'), with no mortalities in 'cell-free' or 'bacteria'-enriched fractions. Videomicrography and electron microscopy show dinospores swarming toward and attaching to skin, actively feeding, and rapidly denuding fish of epidermis. We show here that our cultures of actively fish-killing P. shumwayae do not secrete potent exotoxins; rather, fish mortality results from micropredatory feeding.