Pacific herring, a forage fish species widely distributed throughout the North Pacific, are known to be highly sensitive to crude oil toxicity during early development. Embryonic exposure produces a well-characterized syndrome initiated by cardiovascular pathology, attributable to uptake of crude-oil derived polycyclic aromatic hydrocarbons (PAHs). While this syndrome is documented in multiple fish species, a direct comparison between herring stocks has not been conducted. The goal of this work was to determine if the nature and severity of oil-induced cardiotoxicity varied between geographically and ecologically distinct herring stocks. We included stocks from Prince William Sound of southcentral Alaska, Sitka Sound of southeast Alaska, and Cherry Point in northwest Washington. Embryos from each stock were exposed to environmentally realistic and sublethal doses of crude oil (0.01−3.5 µg L−1 ∑PAHs) and assessed for tissue PAH concentrations, congener-specific accumulation patterns, xenobiotic gene expression, and cardiac morphology before and at hatch. Prince William Sound and Sitka Sound embryos had similar responses to oil exposure in terms of PAH composition, xenobiotic response, and the pattern and severity of cardiac injury. However, Cherry Point embryos showed a distinct pattern of PAH accumulation, a differential xenobiotic response, and a lesser degree of injury. This distinct pattern is likely due to metabolic differences associated with the unique life history of this stock. While severity varied between Cherry Point and the Alaskan stocks, all three stocks demonstrated a dose-dependent decrease in outgrowth of the posterior ventricle (ballooning), a pathognomonic sign of early life stage PAH exposure. Our findings demonstrate that the severity of oil-induced cardiotoxicity can vary by stock over a very narrow dose range. They further reinforce that ventricular ballooning is a sensitive and biologically relevant biomarker of oil exposure that can detect oil injury in Pacific herring larvae throughout their geographic range.

6PPD-quinone (6PPDQ), derived from the tire-protectant 6PPD reacting with ozone, is an emerging contaminant of concern owing to its role in coho salmon (Oncorhynchus kisutch) deaths via urban runoff mortality syndrome (URMS). Given the impact of 6PPDQ on aquatic life in urban streams, we addressed the acute toxicity of 6PPDQ exposure on coastal cutthroat trout (CCT) (Oncorhynchus clarkii clarkii), a species sympatric with coho salmon in natal watersheds. Using static exposures coupled with analytical chemistry, we determined the 24-h LC50 values for alevin (297.2 ng/L), swim-up fry (39.6 ng/L), 5-month parr (103.3 ng/L), and 13-month juveniles (185.9 ng/L)─values similar to toxicity observed in coho salmon. Additionally, the 96-h LC50 (77.6 ng/L) was 2.4 times more lethal for juvenile CCT. We assessed potential effects of sublethal 6PPDQ exposure on disease resistance to infectious hematopoietic necrosis (IHN), an endemic viral disease of Pacific salmon, and to swimming performance. Sublethal 6PPDQ (53.6 ng/L) did not affect survival of parr exposed to IHN virus compared to virus alone. Conversely, 6PPDQ exposure as low as 72.2 ng/L significantly reduced 15- and 24-month juvenile swimming performance, and 120.5 ng/L 6PPDQ increased blood hematocrit. Overall, CCT are the second most sensitive species tested to date for 6PPDQ sensitivity which further emphasizes the need for identifying alternatives to 6PPD.

Crude oil toxicity to early life stage fish is commonly attributed to polycyclic aromatic hydrocarbons (PAHs). However, it remains unclear how the polar unresolved complex mixture (UCM), which constitutes the bulk of the water-soluble fraction of crude oil, contributes to crude oil toxicity. Additionally, the role of photomodification-induced toxicity in relation to the polar UCM is not well understood. This study addresses these knowledge gaps by assessing the toxicity of two laboratory generated polar UCMs from Cook Inlet crude oil, representing the readily water-soluble fraction of crude oil and photoproduced hydrocarbon oxidation products (HOPs), to Pacific herring (Clupea pallasii) embryos. A small-scale semi-static exposure design was utilized with a range of polar UCM concentrations (0.5–14 mg/L) in nonvolatile dissolved organic carbon (NVDOC) units, quantifying the entire polar UCM. Compositional analyses revealed a photochemical-driven shift toward more complex aromatic compositions, naphthenic acids, and no detectable levels of PAHs (above 0.3 μg/L). Exposure to the dark polar UCM resulted in higher mortality than exposure to the light polar UCM. Both dark and light polar UCMs induced developmental abnormalities commonly attributed to the PAH fraction, including edema, reduced heart rate, body axis defects, and decreased body lengths, with these effects observed at the lowest dose group (0.5 mg/L NVDOC). These responses suggest photomodification-induced toxicity is driven by exposure to increased concentrations of dissolved HOPs rather than photochemical induced compositional changes. Gene expression analyses focusing on xenobiotic metabolism and cardiac morphogenesis yielded results consistent with previous studies examining the biological mechanisms of crude oil toxicity. In summary, these phenotypic and genotypic responses in Pacific herring embryos indicate that the polar UCM is a significant driver of crude oil toxicity. These findings emphasize the importance of considering the polar UCM in future studies, metric reporting, and risk assessments related to crude oil toxicity.

Ocean acidification can affect the immune responses of fish, but effects on pathogen susceptibility remain uncertain. Pacific herring Clupea pallasii were reared from hatch under 3 CO2 partial pressure (pCO2) treatments (ambient, similar to 650 mu atm; intermediate, similar to 1500 mu atm; high, similar to 3000 mu atm) through metamorphosis (98 d) to evaluate the effects of ocean acidification on bioenergetics and susceptibility to an endemic viral disease. Mortality from viral hemorrhagic septicemia (VHS) was comparable between herring reared under ambient and intermediate pCO2 (all vulnerability testing at ambient pCO2). By contrast, fish reared under high pCO2 experienced significantly higher rates of VHS mortality, and the condition factor of survivors was significantly lower than in the other pCO2 treatments. However, the prevalence of infection among survivors was not influenced by pCO2 treatment. Pre-flexion larval development was not affected by elevated pCO2, as growth rate, energy use, and feeding activity were comparable across treatments. Similarly, long-term growth (14 wk) was not affected by chronic exposure to elevated pCO2. Herring reared under both elevated pCO2 treatments showed an average reduction in swimming speed; however, wide intra-treatment variability rendered the effect nonsignificant. This study demonstrates that the VHS susceptibility and bioenergetics of larval and post-metamorphic Pacific herring are not affected by near-future ocean acidification predicted for coastal systems of the North Pacific. However, increased susceptibility to VHS in fish reared under 3000 mu atm pCO2 indicates potential health and fitness consequences from extreme acidification.

We characterized a natural sea louse epizootic of Caligus clemensi and the effects of parasitism on Pacific herring Clupea pallasii in Port Angeles Harbor, WA, USA. Infestation prevalence on newly metamorphosed age 0 Pacific herring reached 100% prevalence by mid-August. At this time, the mean louse intensity was 4.6 lice/fish, and a positive correlation occurred between louse intensity and herring body length. The epizootic then waned, with infestation prevalence decreasing to less than 25% and the mean parasite intensity falling below 1 louse. While skin injuries were not detected, motile lice preferentially aggregated around head and anterior dorsal areas. However, louse tropism became evenly distributed over the body as the parasite intensity increased. Louse-induced mortality in herring was negligible in controlled experiments. These results indicate that Caligus clemensi epizootics reach high prevalence, but also fade from mid-summer to early fall. Due to the predominant presence of motile copepod stages, we suggest that the epizootic fades because lice complete their life cycle and dislodge from the host; however, multiple explanations for epidemic fading are possible.

Causes of population collapse and failed recovery often remain enigmatic in marine forage fish like Pacific herring (Clupea pallasii) that experience dramatic population oscillations. Diseases such as ichthyophoniasis are hypothesized to contribute to these declines, but lack of long-term datasets frequently prevents inference. Analysis of pathogen surveillance and population assessment datasets spanning 2007-2019 indicate that the age-based prevalence estimate of Ichthyophonus infection was, on average, 54% greater among a collapsed population of Pacific herring (Prince William Sound, Alaska, USA) as compared to a nearby population (Sitka Sound, Alaska, USA) that is relatively robust. During the study years, the age-based infection prevalence ranged from 14 to 44% in Prince William Sound and 5 to 33% in Sitka Sound. At both sites, the age-based infection prevalence declined over time, with an average decrease of 7% per year. Statistical analyses indicated that infection prevalence between the two populations was reduced by regional factors affecting both sites, and that these factors were independent of herring density. Infection prevalence in both populations was positively correlated with herring age and negatively correlated with the Pacific Decadal Oscillation. This study demonstrates how synthesis of environmental, stock assessment, and disease assessment data can be leveraged to elucidate epidemiological trends in diseases of wild fish.

Pacific herring (Clupea pallasii), a cornerstone of marine food webs, generally spawn on marine macroalgae in shallow nearshore areas that are disproportionately at risk from oil spills. Herring embryos are also highly susceptible to toxicity from chemicals leaching from oil stranded in intertidal and subtidal zones. The water-soluble components of crude oil trigger an adverse outcome pathway that involves disruption of the physiological functions of cardiomyocytes in the embryonic herring heart. In previous studies, impaired ionoregulation (calcium and potassium cycling) in response to specific polycyclic aromatic hydrocarbons (PAHs) corresponds to lethal embryolarval heart failure or subtle chamber malformations at the high and low ends of the PAH exposure range, respectively. Sublethal cardiotoxicity, which involves an abnormal outgrowth (ballooning) of the cardiac ventricular chamber soon after hatching, subsequently compromises juvenile heart structure and function, leading to pathological hypertrophy of the ventricle and reduced individual fitness, measured as cardiorespiratory performance. Previous studies have not established a threshold for these sublethal and delayed-in-time effects, even with total (∑)PAH exposures as low as 29 ng/g of wet weight (tissue dose). Here, we extend these earlier findings showing that (1) cyp1a gene expression provides an oil exposure metric that is more sensitive than typical quantitation of PAHs via GC–MS and (2) heart morphometrics in herring embryos provide a similarly sensitive measure of toxic response. Early life stage injury to herring (impaired heart development) thus occurs below the quantitation limits for PAHs in both water and embryonic tissues as a conventional basis for assessing oil-induced losses to coastal marine ecosystems.

OBJECTIVE:The primary objective of this study was to determine the effects of temperature on viral erythrocytic necrosis (VEN) progression under controlled conditions. Secondarily, this study was intended to evaluate the combined effects of temperature and VEN on the Pacific Herring Clupea palasii transcriptome. METHODS:The effects of temperature on VEN progression were assessed by waterborne exposure of laboratory-reared, specific-pathogen-free Pacific Herring to tissues homogenates containing erythrocytic necrosis virus (ENV) at 6.9, 9.0, or 13.5°C. RESULT:Exposure of Pacific Herring to ENV resulted in the establishment of infections characterized by high infection prevalence (89%; 40/45) and mean viral loads (5.5 log10 [gene copies/μg genomic DNA]) in kidney tissues at 44 days postexposure. Mean viral loads were significantly higher in fish from the ambient (mean = 9.0°C) and warm (mean = 13.5°C) treatments (6.1-6.2 log10 [gene copies/total genomic DNA]) than in fish from the cool (mean = 6.9°C) treatment (4.3 log10 [gene copies/μg genomic DNA]). Similarly, the peak proportion of diseased fish was directly related to temperature, with cytoplasmic inclusion bodies detected in 21% of fish from the cool treatment, 52% of fish from the ambient treatment, and 60% of fish from the warm treatment. The mean VEN load in each fish (enumerated as the percentage of erythrocytes with cytoplasmic inclusions) at 44 days postexposure increased with temperature from 15% in the cool treatment to 36% in the ambient treatment and 32% in the warm treatment. Transcriptional analysis indicated that the number of differentially expressed genes among ENV-exposed Pacific Herring increased with temperature, time postexposure, and viral load. Correlation network analysis of transcriptomic data showed robust activation of interferon and viral immune responses in the hepatic tissue of infected individuals independent of other experimental variables. CONCLUSION:Results from this controlled laboratory study, combined with previous observations of natural epizootics in wild populations, support the conclusion that temperature is an important disease cofactor for VEN in Pacific Herring.

In recent decades, evidence has accumulated to suggest that the widespread and highly variable parasite Ichthyophonus hoferi is actually a species complex. Highly plastic morphology and a general lack of defining structures has contributed to the likely underestimate of biodiversity within this group. Molecular methods are a logical next step in the description of these parasites, but markers used to date have been too conserved to resolve species boundaries. Here we use mitochondrial encoded cytochrome-c oxidase (MTCO1) gene sequences and phylogenic analysis to compare Ichthyophonus spp. isolates from several marine and anadromous fish hosts. The resulting phylogeny displays lineage separation among isolates and possible host/niche segregation not previously described. The parasite type that infects Pacific herring Clupea pallasii, Atlantic herring C. harengus, Atlantic salmon Salmo salar, and Pacific staghorn sculpin Oligocottus maculosus (Clade A) is different from that which infects Chinook salmon Oncorhynchus tshawytscha, walleye pollock Gadus chalcogrammus, Greenland halibut Reinhardtius hippoglossoides, and Pacific halibut Hippoglossus stenolepsis (Clade B). MTCO1 sequences confirmed the presence of a more divergent Ichthyophonus sp. isolated from American shad Alosa sapidissima in rivers of eastern North America (Clade C), while American shad introduced to the Pacific Ocean are infected with the same parasite that infects Pacific herring (Clade A). Currently there are no consensus criteria for delimiting species within Ichthyophonidae, but MTCO1 sequences hold promise as a potential species identifying marker and useful epizootiological tool.

Preliminary evidence suggests that Chinook salmon Oncorhynchus tshawytscha from the Yukon River may be more susceptible to Ichthyophonus sp. infections than Chinook from stocks further south. To investigate this hypothesis in a controlled environment, we experimentally challenged juvenile Chinook from the Yukon River and from the Salish Sea with Ichthyophonus sp. and evaluated mortality, infection prevalence and infection load over time. We found that juvenile Chinook salmon from a Yukon River stock were more susceptible to ichthyophoniasis than were those from a Salish Sea stock. After feeding with tissues from infected Pacific herring Clupea pallasii, Chinook salmon from both stocks became infected. The infection was persistent and progressive in Yukon River stock fish, where infections sometimes progressed to mortality, and histological examinations revealed parasite dissemination and proliferation throughout the host tissues. In Salish Seaorigin fish, however, infections were largely transient; host mortalities were rare, and parasite stages were largely cleared from most tissues after 3-4 wk. Susceptibility differences were evidenced by greater cumulative mortality, infection prevalence, parasite density, proportion of fish demonstrating a cellular response, and intensity of the cellular response among fish from the Yukon River stock. These observed differences between Chinook salmon stocks were consistent when parasite exposures occurred in both freshwater and seawater. These results support the hypothesis that a longer-standing host-pathogen relationship, resulting in decreased disease susceptibility, exists among Salish Sea Chinook salmon than among Yukon River conspecifics.

Throughout a 20 year biosurveillance period, viral hemorrhagic septicemia virus was isolated in low titers from only 6/7355 opportunistically sampled adult Pacific herring, reflecting the typical endemic phase of the disease when the virus persists covertly. However, more focused surveillance efforts identified the presence of disease hot spots occurring among juvenile life history stages from certain nearshore habitats. These outbreaks sometimes recurred annually in the same temporal and spatial patterns and were characterized by infection prevalence as high as 96%. Longitudinal sampling indicated that some epizootics were relatively transient, represented by positive samples on a single sampling date, and others were more protracted, with positive samples occurring throughout the first 10 weeks of the juvenile life history phase. These results indicate that viral hemorrhagic septicemia (VHS) epizootics in free-ranging Pacific herring C. pallasii are more common than previously appreciated; however, they are easily overlooked if biosurveillance efforts are not designed around times and locations with high disease potential.

Processes that allow viral hemorrhagic septicemia (VHS) virus to persist in the marine environment remain enigmatic, owing largely to the presence of covert and cryptic infections in marine fishes during typical sub-epizootic periods. As such, marine host reservoirs for VHS virus have not been fully demonstrated, nor have the mechanism(s) by which infected hosts contribute to virus perpetuation and transmission. Here, we demonstrate that after surviving VHS, convalesced Pacific herring continue to shed virus at a low rate for extended periods. Further, exposure of previously naïve conspecific sentinels to this shed virus can result in infections for at least 6 mo after cessation of overt disease. This transmission mechanism was not necessarily dependent on the magnitude of the disease outbreak, as prolonged transmission occurred from 2 groups of donor herring that experienced cumulative mortalities of 4 and 29%. The results further suggest that the virus persists in association with the gills of fully recovered individuals, and long-term viral shedding or shedding relapses are related to cooler or decreasing water temperatures. These results provide support for a new VHS virus perpetuation paradigm in the marine environment, whereby the virus can be maintained in convalesced survivors and trafficked from these carriers to sympatric susceptible individuals.

Over a 3-year-period, 17 wild-caught opaleye (Girella nigricans) housed in a public display aquarium were found dead without premonitory signs. Grossly, 4 animals had pinpoint brown or black foci on coelomic adipose tissue. Histologically, liver, spleen, heart, and posterior kidney had mesomycetozoan granulomas in all cases; other organs were less commonly infected. Four opaleye had goiter; additional substantial lesions were not identified. Granulomas surrounded melanized debris, leukocytes, and mesomycetozoa represented by folded membranes (collapsed schizont walls), intact schizonts (50- to >200 µm in diameter with a multilaminate membrane), plasmodia (budding from schizonts or free in tissue), or rarely germinal tubes (budding from schizonts). Ichthyophonus was grown from fresh tissues in tissue explant broth cultures of the heart, liver, and/or spleen. Polymerase chain reaction using 18S ribosomal DNA primers amplified a 1730-bp region, and the DNA sequence was most similar to Ichthyophonus hoferi, which is often associated with freshwater aquaculture fish.

The data that support the findings of this study are openly available in Science Base at https://doi.org/10.5066/P9Q6GQVK.

Supplemental Material, Ichthyophonus_Suppl_Table_1 for Ichthyophonus sp. Infection in Opaleye (Girella nigricans) by Elise E. B. LaDouceur, Judy St Leger, Alexandria Mena, Ashley Mackenzie, Jacob Gregg, Maureen Purcell, William Batts and Paul Hershberger in Veterinary Pathology

An analysis of daily water samples collected from an index site on Big Soos Creek, Washington indicated intra-annual differences in the concentrations of waterborne Nanophyetus salmincola. Waterborne concentrations, quantified as gene copies/L, peaked during the fall (October-November 2016), decreased to very low concentrations over the winter (January-March 2017), and then increased in the spring and throughout the summer. High waterborne concentrations of N. salmincola DNA (2 × 106 gene copies/L) corresponded with live N. salmincola cercariae (mean = 3 cercariae/L) that were detected in companion water samples. Spikes in waterborne N. salmincola concentrations in October and November typically coincided with increases in streamflow; this combination resulted in elevated infection pressures during high water events in the fall. The peak in waterborne N. salmincola concentrations corresponded with an accompanying peak in tissue parasite density (metacercariae/posterior kidney) in Coho Salmon Oncorhynchus kisutch that were reared in the untreated water.

The prevalence of Ichthyophonus infection in Pacific herring Clupea pallasii was spatially heterogeneous in the southern Salish Sea, Washington State, USA. Over the course of 13 mo, 2232 Pacific herring were sampled from 38 midwater trawls throughout the region. Fork length was positively correlated with Ichthyophonus infection at all sites. After controlling for the positive relationship between host size and Ichthyophonus infection, the probability of infection was approximately 6-fold higher in North Hood Canal than in Puget Sound and the northern Straits (12 vs. 2% predicted probability for a 100 mm fish and 30 vs. 7% predicted probability for a 180 mm fish). Temporal changes in Ichthyophonus infection probability were explained by seasonal differences in fish length, owing to Pacific herring life history and movement patterns. Reasons for the spatial heterogeneity remain uncertain but may be associated with density-dependent factors inherent to the boom-bust cycles that commonly occur in clupeid populations.

Ichthyophonus occurred at high prevalence but low intensity in Pacific Halibut (Hippoglossus stenolepis) throughout the west coast of North America, ranging from coastal Oregon to the Bering Sea. Infection prevalence in adults was variable on spatial and temporal scales, with the lowest prevalence typically occurring on the edges of the geographic range and highest prevalence consistently occurring inside Prince William Sound, AK (58-77%). Additionally, intra-annual differences occurred at Albatross - Portlock, AK (71% versus 32% within 2012) and inter-annual differences occurred along coastal Oregon (50% versus 12% from 2012 to 2015). The infection prevalence was influenced by host age, increasing from ≤ 3% among the youngest cohorts (≤age 6) to 39-54% among age 9-17 cohorts, then decreasing to 27% among the oldest (age 18+) cohorts. There was little indication of significant disease impacts to Pacific Halibut, as the intensity of infection was uniformly low and length-at-age was similar between infected and uninfected cohorts. These results suggest that Ichthyophonus in Pacific Halibut currently represents a stable parasite-host paradigm in the North Pacific. Received 04 May 2017 accepted 09 Oct 2017 revised 21 Sep 2017.

This report of Ichthyophonus in common sport-caught fishes throughout the marine waters of southcentral Alaska represents the first documentation of natural Ichthyophonus infections in lingcod Ophiodon elongates and yelloweye rockfish Sebastes ruberrimus. In addition, the known geographic range of Ichthyophonus in black rockfish S. melanops has been expanded northward to include southcentral Alaska. Among all species surveyed, the infection prevalence was highest (35%, n = 334) in Pacific halibut Hippoglossus stenolepis. There were no gross indications of high-level infections or clinically diseased individuals. These results support the hypothesis that under typical conditions Ichthyophonus can occur at high infection prevalence accompanied with low-level infection among a variety of fishes throughout the eastern North Pacific Ocean, including southcentral Alaska.