White perch health relative to urbanization and habitat degradation in Chesapeake Bay tributaries. I. Biliary neoplasms and hepatic lesions.

White perch Morone americana (Gmelin, 1789) from the Chesapeake Bay (USA) watershed have a high incidence of liver disease, including neoplasms of bile duct origin. Fish collected seasonally from spring 2019 to winter 2020 from the urban Severn River and the more rural Choptank River were evaluated for hepatic lesions. Biliary hyperplasia (64.1%), neoplasms (cholangioma and cholangiocarcinoma, 27%), and dysplasia (24.9%) were significantly higher in Severn River fish compared to Choptank River fish (52.9, 16.2, and 15.8%, respectively). Hepatocellular lesions were less common, including foci of hepatocellular alteration (FHA, 13.3%) and hepatocellular neoplasms (1%). There was also a progressive age-related increase in copper-laden granules in hepatocytes, which was a significant risk factor for FHA and could be a source of oxidative stress in the liver. Significant risk factors for biliary neoplasms included age, bile duct fibrosis, and infections by the myxozoan parasite Myxidium murchelanoi, but the prevalence and relative intensity of M. murchelanoi infections did not differ significantly between fish populations. Hepatic disease in this species appears to be chronic and may stem from an age-related accumulation of damage, possibly from parasitic infections and contaminants such as polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and copper. Watershed development and exposures to PCBs and PAHs were generally higher for white perch in the Severn River, but similar suites of chemical contaminants were detected in the Choptank River. A broader survey of white perch within and outside Chesapeake Bay may allow determination of the extent of biliary neoplasia in this species.

White perch health relative to urbanization and habitat degradation in Chesapeake Bay tributaries. II. Hepatic and splenic macrophage aggregates.

Macrophage aggregate (MA) abundance in fish is a useful general biomarker of contaminant exposures and environmental stress. Hepatic and splenic MAs were evaluated in semi-anadromous white perch Morone americana (Gmelin, 1789) from the urbanized Severn River (S) and the more rural Choptank River (C), Chesapeake Bay. Fish were collected from different sites in the annual migratory circuit in each river that corresponded to active spawning in late winter-early spring, summer regenerating, autumn developing, and winter spawning-capable phases. An age-associated progressive increase in the total volume of MAs (MAV) was evident in the liver and spleen. Mean hepatic MAV (range in seasonal means, C: 6.4-23.1 mm3; S: 15.7-48.7 mm3) and mean splenic MAV (C: 7.3-12.6 mm3; S: 16.0-33.0 mm3) differed significantly among seasons and were significantly greater in females and in Severn River fish. Age and river were the most influential factors, suggesting that increased MAV in Severn River fish resulted from chronic exposures to higher concentrations of environmental contaminants. Hepatic MAV was directly related to the relative volume of copper granules in the liver. Less influential factors on splenic MAV included fish condition, trematode infections, and granulomas, indicating possible functional differences in MAs by organ. While organ volumes were strongly linked to gonadosomatic index (GSI) and reproductive phase, the reason for seasonal differences in MAV was less clear. Water temperature, salinity, and dissolved oxygen were not significantly related to MAV, and indicators of reproductive phase (hepatosomatic index and GSI) were significant but less important in explaining variation in MAV.

Seasonal variation of polycyclic aromatic hydrocarbon metabolites in bile of white perch Morone americana from two Chesapeake Bay tributaries.

Polycyclic aromatic hydrocarbon (PAH) accumulation in the bile is a reliable biomarker of recent exposure to environmental PAH and elevated concentrations have been identified as significant risk factors for hepatic disease in some fish species. Recent surveys (spring 2019) revealed a high incidence of hepatic lesions in white perch Morone americana from Chesapeake Bay, but this initial survey did not detect an association between PAH metabolites and lesions. Seasonal sampling of fish was extended through the winter 2020 to increase the metabolite and hepatic lesion database and better understand annual PAH exposures to white perch. Naphthalenes, phenanthrenes, and benzo[a]pyrenes (equivalents) were analyzed in bile specimens (n = 400) using high-performance liquid chromatography with fluorescence detection. Mean concentrations of metabolites were highest in fish collected from the urbanized Severn River in summer and were lowest in fish from the more rural Choptank River in winter. Variance in metabolite concentrations were primarily due to location and season. Seasonal fluctuations in water temperature, river discharge, or feeding status had little influence on the pattern of metabolite concentrations observed. The results suggest higher exposures of fish to environmental PAH in the more developed Severn River with significant increases during summer, which could correspond to a spike in seasonal recreational motor boat use or other human activities. A better understanding of the factors that contribute to variability in PAH metabolism and the enterohepatic biliary circulation in white perch will improve the usefulness of biliary metabolites as a biomarker of PAH exposure in Chesapeake Bay and elsewhere.

Biological and anthropogenic influences on macrophage aggregates in white perch Morone americana from Chesapeake Bay, USA.

The response of macrophage aggregates in fish to a variety of environmental stressors has been useful as a biomarker of exposure to habitat degradation. Total volume of macrophage aggregates (MAV) was estimated in the liver and spleen of white perch Morone americana from Chesapeake Bay using stereological approaches. Hepatic and splenic MAV were compared between fish populations from the rural Choptank River (n = 122) and the highly urbanized Severn River (n = 131). Hepatic and splenic MAV increased with fish age, were greater in females from the Severn River only, and were significantly greater in fish from the more polluted Severn River (higher concentrations of polycyclic aromatic hydrocarbons, organochlorine pesticides, and brominated diphenyl ethers). Water temperature and dissolved oxygen had a significant effect on organ volumes, but not on MAV. Age and river were most influential on hepatic and splenic MAV, suggesting that increased MAV in Severn River fish resulted from chronic exposures to higher concentrations of environmental contaminants and other stressors. Hemosiderin was abundant in 97% of spleens and was inversely related to fish condition and positively related to fish age and trematode infections. Minor amounts of hemosiderin were detected in 30% of livers and positively related to concentrations of benzo[a]pyrene metabolite equivalents in the bile. This study demonstrated that hepatic and splenic MAV were useful indicators in fish from the 2 tributaries with different land use characteristics and concentrations of environmental contaminants. More data are needed from additional tributaries with a wider gradient of environmental impacts to validate our results in this species.

Synopsis of the species of the genus Zschokkella Auerbach, 1910 (Myxozoa: Bivalvulida: Myxidiidae).

A synopsis of the species of Zschokkella Auerbach, 1910 (Myxozoa: Bivalvulida: Myxidiidae) is presented, including 94 nominal species from piscine hosts and 3 additional nominal species from amphibian or reptilian hosts. The most relevant morphological and morphometric features of both myxospores and trophozoite stages (when available) are presented for each species in tabulated format.

High prevalence of biliary neoplasia in white perch Morone americana: potential roles of bile duct parasites and environmental contaminants.

Recent surveys of white perch Morone americana from Chesapeake Bay, USA, revealed a high prevalence of hepatic and biliary lesions, including neoplasia, and bile duct parasites. Here, we describe lesions in the liver and gallbladder and evaluate for statistical associations among lesions, parasites, and biomarkers of chemical exposure in fish from 2 tributaries of Chesapeake Bay. Fish were collected from an estuarine site in the Choptank River (n = 122, ages 3-11), a tributary with extensive agriculture within the watershed, and the Severn River (n = 131, ages 2-16), a tributary with extensive urban development. Passive integrative samplers were deployed at the fish collection site and an upstream, non-tidal site in each river for 30 d. Intrahepatic biliary lesions observed in fish from both rivers included neoplasia (23.3%), dysplasia (16.2%), hyperplasia (46.6%), cholangitis (24.9%), and dilated ducts containing plasmodia of Myxidium sp. (24.9%). Hepatocellular lesions included foci of hepatocellular alteration (FHA, 15.8%) and neoplasia in 4 Severn River fish (2.3%). Age of fish and Myxidium sp. infections were significant risk factors for proliferative and neoplastic biliary lesions, age alone was a risk factor for FHA, and Goussia bayae infections were associated with cholangitis and cholecystitis. Lesion prevalence was higher in fish from the Severn River, which contained higher concentrations of PAHs, organochlorine pesticides, and brominated diphenyl ethers. Metabolite biomarkers indicated higher PAH exposures in Severn River fish. This study suggests Myxidium sp. as a promoter of bile duct tumors, but more data are needed to evaluate the biological effects of environmental contaminants in this species.

Comparisons of Stereological and Other Approaches for Quantifying Macrophage Aggregates in Piscine Spleens.

Macrophage aggregates (MAs) are focal accumulations of pigmented macrophages in the spleen and other tissues of fish. A central role of MAs is the clearance and destruction of degenerating cells and recycling of some cellular components. Macrophage aggregates also respond to chemical contaminants and infectious agents and may play a role in the adaptive immune response. Tissue damage or physiological stress can result in increased MA accumulation. As a result, MAs may be sensitive biomarkers of environmental stress in fish. Abundance of MAs in tissues has been reported in a variety of ways-most commonly as density, mean size, and relative area-but the utility of these estimates has not been compared. In this study, four different types of splenic MA abundance estimates (abundance score, density, relative area, and total volume) were compared in two fish populations (Striped Bass Morone saxatilis and White Perch M. americana) with a wide range in ages. Stereological estimates of total volume indicated an increase in MA abundance with spleen volume, which generally corresponded to fish age, and with splenic infections (mycobacteria or trematode parasites). Abundance scores were generally limited in the ability to detect changes in MA abundance by these factors, whereas density estimates were greatly influenced by changes in spleen volume. In some instances, densities declined while the total volume of MAs and spleen volume increased. Experimentally induced acute stress resulted in a decrease in spleen volume and an increase in MA density, although the total volume of MAs remained unchanged. Relative area estimates accounted for the size and number of MAs but not for changes in organ volume. Total volume is an absolute measure of MA abundance irrespective of changes in organ volume or patterns of accumulation and may provide an improved means of quantifying MAs in the spleens of fish.

Seasonal development of the coccidian parasite Goussia bayae and hepatobiliary histopathology in white perch Morone americana from Chesapeake Bay.

The coccidium Goussia bayae infects the gallbladder and bile ducts of white perch Morone americana from Chesapeake Bay, USA. Seasonal changes in coccidian infections were analyzed from bile specimens of 1588 fish from the Choptank River during 2016-2018 using wet mount preparations with a Sedgwick-Rafter counting chamber. Histopathology of the gallbladder and liver was analyzed from a subset (n = 480) of these fish. Maximum parasite prevalence (100%) and intensities in the gallbladder occurred during the fish spawning season in March and April. Asynchronous coccidian development and prevalence of infections in fish increased gradually during autumn and winter, but coccidian intensity increased sharply 2-4 wk prior to the onset of fish spawning activity and decreased after spawning activity concluded. Sporulation was internal, and the gallbladder was the primary reservoir for oocysts. Two previously undescribed species of coccidia were observed in the intestine. Lesions in the gallbladder were rare and included cholecystitis and epithelial necrosis. Intrahepatic bile duct lesions were more common and included distension, cholangitis, epithelial erosion and necrosis, cholestasis, hyperplasia, and neoplasia. Cholangitis and necrosis of intrahepatic bile ducts were significantly associated with coccidial infections, while plasmodia of a myxosporean (spore morphology consistent with the genera Myxidium and Zschokella) were significantly associated with bile duct hyperplasia. Biliary neoplasia included cholangiomas (5% prevalence) and cholangiocarcinomas (1% prevalence). No association was detected between G. bayae and biliary neoplasms, but an association may exist between these lesions and the myxosporean plasmodia.

Newly Described Coccidia Goussia bayae from White Perch Morone americana: Morphology and Phylogenetics Support Emerging Taxonomy of Goussia within Piscine Hosts.

In March and April 2016, 150 white perch ( Morone americana) were collected from various localities in Chesapeake Bay and examined for coccidia. A previously undescribed species of coccidia was observed in the hepatic bile ducts and gallbladder of all white perch (100%) examined. We describe this species using morphological characteristics, histology, and gene sequences of the small-subunit ribosomal DNA ( rDNA), large-subunit rDNA, and mitochondrial genes cytochrome oxidase 1 ( COI), cytochrome oxidase b ( Cytb), and cytochrome oxidase 3 ( COIII). Oocysts of Goussia bayae n. sp. were subspherical with a single-layered smooth wall and measured (length [L] × width [W]) 26.2 × 21.8 µm, with a L/W ratio of 1.2. A micropyle was present but a micropyle cap, polar granules, and oocyst residuum were absent. Each oocyst contained 4 sporocysts that were ellipsoidal and measured (L × W) 12.6 × 7.8 µm, with a L/W ratio of 1.6. A pair of sporozoites was present, but sporocysts lacked a Stieda body and residuua. Meronts and gamonts were epicellular in biliary epithelial cells and oocysts were coelozoic in hepatic and common bile ducts and gallbladder. This is the first report of Goussia spp. from white perch and the first mitochondrial DNA sequence reported from a Goussia species. Phylogenetic analysis indicates basal placement of G. bayae to Eimeriidae, Choleoeimeria, and Sarcocystidae.

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.

A comparison of hematology, plasma chemistry, and injuries in Hickory shad (Alosa mediocris) captured by electrofishing or angling during a spawning run.

BACKGROUND: Declines in Hickory shad (Alosa mediocris) populations in Chesapeake Bay have prompted efforts at captive propagation of wild broodfish for stock enhancement and research. OBJECTIVE: The objectives of this study were to evaluate injuries sustained, and immediate and delayed (24 hours) effects on blood variables related to 2 fish capturing methods (electrofishing [EF] and angling). METHODS: Blood specimens were collected from fish immediately following capture by EF and angling (n = 40 per sex and capture method) from the Susquehanna River (MD, USA). Additional fish (n = 25 per sex and capture method) were collected on the same day, placed in holding tanks and bled 24 hours following capture. Blood data that were non-Gaussian in distribution were transformed (Box-Cox), and effects of sex, method of capture, and holding time were tested using ANOVA with general linear models. Fish were evaluated for injuries by necropsy and radiography. RESULTS: Sex-specific differences were observed for RBC, HGB, PCV, MCH, MCHC, total proteins (TP), globulins, glucose, calcium, AST, CK, and lactate, while RBC, HGB, PCV, MCV, MCH, MCHC, TP, albumin, globulins, glucose, potassium, sodium, AST, CK, and lactate differed significantly by fish capturing method. Electrofishing may have induced greater disruption in blood variables, but mortality (4%) was not significantly different compared to angling. CONCLUSIONS: Electrofishing for Hickory shad using a constant DC voltage resulted in numerous hematologic and biochemical changes, with no additional injuries or deaths compared to angling. Capture method must be considered when evaluating fish condition, and blood variables should be partitioned by sex during spawning season.

Nonlethal laparoscopic detection of intersex (testicular oocytes) in largemouth bass (Micropterus salmoides) and smallmouth bass (Micropterus dolomieu).

Intersex in wild fish populations has received considerable attention in the scientific literature and public media. Conventional detection of testicular oocytes, the presence of immature oocytes within testis of male fish, commonly employs transverse sectioning of excised testis and is lethal. The present study used a nonlethal laparoscopic technique to collect biopsies of testis from black bass, entering the body cavity via the genital pore. Detection of testicular oocytes was compared between biopsy and conventional methods using 79 smallmouth bass (Micropterus dolomieu) from 8 sites and 68 largemouth bass (Micropterus salmoides) from 4 sites. The 2 methods performed similarly at sites where testicular oocyte severity was moderate or high (6 of 8 smallmouth bass sites), whereas transverse sectioning resulted in superior testicular oocyte detection at sites where severity was low (2 of 8 smallmouth bass sites and all 4 largemouth bass sites). In smallmouth bass, testicular oocyte prevalence by transverse and biopsy methods was strongly correlated across sites (r2 = 0.81) and severity reported by enumeration of testicular oocytes was moderately correlated across sites (r2 = 0.59). Survival of a subset of largemouth bass (n = 20) to 28 d after laparoscopic surgery was 90%. The present research indicates that laparoscopy may be useful for monitoring the prevalence and severity of testicular oocytes in Micropterus species, particularly when lethal sampling is precluded. Environ Toxicol Chem 2017;36:1924-1933. © 2016 SETAC.

Preserving whole blood in formalin extends the specimen stability period for manual cell counts for fish.

BACKGROUND: Total blood cell counts for fish are challenging, especially due to cell fragility. Thrombocyte aggregation, cell distortion, and lysis can occur within hours of collection; therefore, hemocytometer counts may not be possible for large sample numbers. Preserving whole blood in formalin offers a simple way to extend the specimen stability period for cell counts. OBJECTIVES: The objectives of this study were to characterize and compare the analytical performance of the Natt-Herrick method for fish blood cell counts using freshly collected and formalin-fixed specimens. METHODS: Specimens from 11 elasmobranch species (n = 36) were compared for WBC counts. WBC, RBC, and thrombocyte counts from 50 striped bass, Morone saxatilis, were compared including 2 time points for the formalin-preserved cells (appr. 1-week and 1-month storage). Coefficient of variation (CV), bias, and total error (TEcalc ) were calculated. TEcalc was compared with allowable total error (TEa ) defined by the Clinical Laboratory Improvements Amendment (CLIA) for human blood cell counts. RESULTS: The CV and TEcalc for fresh and fixed WBC and thrombocyte counts met CLIA limits. In both the fresh and preserved cells, the RBC CV and TEcalc were nearly 3 times higher than the TEa . CONCLUSIONS: Preserving freshly collected blood in 10% formalin is a reliable method to maintain cell morphology for manual counts for up to 1 month post collection. This is especially useful for field studies, where laboratory access is limited. Further evaluation is needed to determine the clinical usefulness of the manual RBC count in fish.

Determination of hematology and plasma chemistry reference intervals for 3 populations of captive Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus).

BACKGROUND: The imperiled status of Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus), a large, long-lived, anadromous fish found along the Atlantic coast of North America, has prompted efforts at captive propagation for research and stock enhancement. OBJECTIVE: The purpose of this study was to establish hematology and plasma chemistry reference intervals of captive Atlantic sturgeon maintained under different culture conditions. METHODS: Blood specimens were collected from a total of 119 fish at 3 hatcheries: Lamar, PA (n = 36, ages 10-14 years); Chalk Point, MD (n = 40, siblings of Lamar); and Horn Point, Cambridge, MD (n = 43, mixed population from Chesapeake Bay). Reference intervals (using robust techniques), median, mean, and standard deviations were determined for WBC, RBC, thrombocytes, PCV, HGB, MCV, MCH, MCHC, and absolute counts for lymphocytes (L), neutrophils (N), monocytes, and eosinophils. Chemistry analytes included concentrations of total proteins, albumin, glucose, urea, calcium, phosphate, sodium, potassium, chloride, and globulins, AST, CK, and LDH activities, and osmolality. RESULTS: Mean concentrations of total proteins, albumin, and glucose were at or below the analytic range. Statistical comparisons showed significant differences among hatcheries for each remaining plasma chemistry analyte and for PCV, RBC, MCHC, MCH, eosinophil and monocyte counts, and N:L ratio throughout all 3 groups. Therefore, reference intervals were calculated separately for each population. CONCLUSIONS: Reference intervals for fish maintained under differing conditions should be established per population.

Annual variation of hematology and plasma chemistry in shortnose sturgeon, Acipenser brevirostrum, during a dam-impeded spawning run.

Shortnose sturgeon (Acipenser brevirostrum) spawning migrations on the Cooper River are impeded by Pinopolis Dam, Lake Moultrie, South Carolina. Sturgeon and other species aggregate below the dam in late winter/early spring and are subjected to a variety of stressors stemming from crowding, poor habitat quality, and injuries that appear to be caused by boat propeller or turbine strikes. Spawning has been documented in the tailrace, but reproductive success has not been verified as no juveniles have been captured. Fish within the dam tailrace were captured by gill net during winter, 2005 and 2007-2011, and physiological condition was assessed using a panel of hematologic and biochemical indices. Plasma phosphorus and calcium were significantly higher in females, while PCV and aspartate aminotransferase were significantly higher in males, indicating sex-specific physiological changes triggered during maturity. A marked leucopenia, accompanied by lymphopenia and neutrophilia, was evident in both sexes and was consistent across years, indicating that these fish were under chronic stress. Testosterone and estradiol levels and hematologic and biochemical reference intervals are provided for comparative purposes.

Low prevalence of splenic mycobacteriosis in migratory striped bass Morone saxatilis from North Carolina and Chesapeake Bay, U.S.A.

Mycobacteriosis is a chronic bacterial disease causing an ongoing epizootic in striped bass Morone saxatilis in Chesapeake Bay, U.S.A. Prevalence of disease is high in pre-migratory fish, and multiple species of Mycobacterium spp. have been isolated. However, prevalence of mycobacteriosis in the coastal migratory population is unknown and is of concern to multiple coastal states, as disease-related mortality may impact the long-term health of the population. Histological examinations of spleens collected from fish caught by recreational anglers during the winter fishery in coastal North Carolina (2005-2006, n=249) and during the spring fishery in Chesapeake Bay (2006, n=120) indicated a low prevalence of mycobacteriosis (6.8% of all fish examined) in comparison to smaller, pre-migratory Chesapeake Bay fish. Genus-level PCR and subsequent sequencing of the 16-23S intergenic transcribed spacer (ITS) region revealed that all bacteria were phylogenetically related, but species is unknown. Location of survey, gender of fish, and total length of fish had no significant effect on prevalence of mycobacteriosis, parasitic granulomas, or the density of splenic granulomas (p > 0.05). These results may indicate that either granulomas resolve after Chesapeake Bay fish enter the coastal migratory population, or that there is disease-related mortality among pre-migratory Chesapeake Bay fish.

Observations and treatment of Nitzschia sturionis on Atlantic sturgeon from Chesapeake Bay.

From 2004 to 2008, 10% of Atlantic sturgeon Acipenser oxyrinchus oxyrinchus examined during a bycatch study of Maryland's Chesapeake Bay were infested with the trematode Nitzschia sturionis on the skin and gills. The parasite intensity increased on 75% of infested fish 4-24 months after being placed in captivity. Mean parasite intensity increased to a greater extent on the skin (up to 100-fold) than on the gills (up to 16-fold). Atlantic sturgeon that were infested with N. sturionis gained weight at a reduced rate or lost weight compared with uninfested fish. Skin lesions associated with N. sturionis progressed from a few reddened foci to hemorrhagic ulcers and depigmentation, while gill lesions showed a less-dramatic progression. Host tissues identified in the intestinal ceca of N. sturionis consisted primarily of malpighian cells, but small amounts of blood were also found in worms attached to lesions in the skin and gills. An Atlantic sturgeon infested with approximately 500 worms was successfully treated with a 3-h bath of praziquantel at 10 mg/L. This first report of N. sturionis from Chesapeake Bay extends the parasite's reported southern range in North America.