Biological Responses of Pacific Herring Embryos to Crude Oil Are Quantifiable at Exposure Levels Below Conventional Limits of Quantitation for PAHs in Water and Tissues.

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.

A phylogeny based on cytochrome-c oxidase gene sequences identifies sympatric Ichthyophonus genotypes in the NE Pacific Ocean.

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.

Annual Recurrences of Viral Hemorrhagic Septicemia Epizootics in Age 0 Pacific Herring Clupea pallasii Valenciennes, 1847.

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.

Differential susceptibility of Yukon River and Salish Sea stocks of Chinook salmon Oncorhynchus tshawytscha to ichthyophoniasis.

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 Sea-origin 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.

Ichthyophonus sp. Infection in Opaleye (Girella nigricans).

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.

Ichthyophonus in sport-caught groundfishes from southcentral Alaska.

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.

High-Prevalence and Low-Intensity Ichthyophonus Infections in Pacific Halibut.

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, Alaska (58-77%). Additionally, intra-annual differences occurred at Albatross-Portlock, Alaska (71% versus 32% within 2012), and interannual differences occurred along coastal Oregon (50% in 2012 versus 12% in 2015). The infection prevalence was influenced by host age, increasing from 3% or less 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.

Ichthyophonus parasite phylogeny based on ITS rDNA structure prediction and alignment identifies six clades, with a single dominant marine type.

Despite their widespread, global impact in both wild and cultured fishes, little is known of the diversity, transmission patterns, and phylogeography of parasites generally identified as Ichthyophonus. This study constructed a phylogeny based on the structural alignment of internal transcribed spacer (ITS) rDNA sequences to compare Ichthyophonus isolates from fish hosts in the Atlantic and Pacific oceans, and several rivers and aquaculture sites in North America, Europe, and Japan. Structure of the Ichthyophonus ITS1-5.8S-ITS2 transcript exhibited several homologies with other eukaryotes, and 6 distinct clades were identified within Ichthyophonus. A single clade contained a majority (71 of 98) of parasite isolations. This ubiquitous Ichthyophonus type occurred in 13 marine and anadromous hosts and was associated with epizootics in Atlantic herring, Chinook salmon, and American shad. A second clade contained all isolates from aquaculture, despite great geographic separation of the freshwater hosts. Each of the 4 remaining clades contained isolates from single host species. This study is the first to evaluate the genetic relationships among Ichthyophonus species across a significant portion of their host and geographic range. Additionally, parasite infection prevalence is reported in 16 fish species.

Displaced phylogeographic signals from Gyrodactylus arcuatus, a parasite of the three-spined stickleback Gasterosteus aculeatus, suggest freshwater glacial refugia in Europe.

We examined the global mitochondrial phylogeography of Gyrodactylus arcuatus, a flatworm ectoparasite of three-spined stickleback Gasterosteus aculeatus. In accordance with the suggested high divergence rate of 13%/million years, the genetic variation of the parasite was high: haplotype diversity h=0.985 and nucleotide diversity π=0.0161. The differentiation among the parasite populations was substantial (Φst=0.759), with two main allopatric clades (here termed Euro and North) accounting for 54% of the total genetic variation. The diversity center of the Euro clade was in the Baltic Sea, while the North clade was spread across the Barents and White Seas. A single haplotype within the North clade was found in the western and eastern Pacific Ocean. Divergence of main clades was estimated to be circa 200 thousand years ago. Each main clade was further divided into six distinct subclades, estimated to have diverged in isolation since 135 thousand years ago. This second division corresponds approximately to the Eemian interglacial predating the last glacial maximum. A demographic expansion of the subclades is associated with colonisation of northern Europe since the last glacial maximum, circa 15-40 thousand years ago. The parasite phylogeny is most likely explained by sequential isolated bottlenecks and expansions in numerous allopatric refugia. The postglacial intermingling and high variation in the marine parasite populations, separately in the Baltic and Barents Seas, suggest low competition of divergent parasite matrilines, coupled with a large population size and high rate of dispersal of hosts. The genetic contribution of the assumed refugial fish populations maintaining the parasite during the last glacial maximum was not detected among the marine sticklebacks, which perhaps were infected after range expansion.

Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.

Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

Molecular identification of erythrocytic necrosis virus (ENV) from the blood of Pacific herring (Clupea pallasii).

Viral erythrocytic necrosis (VEN) is a condition affecting the red blood cells of more than 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. Among populations of Pacific herring (Clupea pallasii) on the west coast of North America the disease causes anemia and elevated mortality in periodic epizootics. Presently, VEN is diagnosed by observation of typical cytoplasmic inclusion bodies in stained blood smears from infected fish. The causative agent, erythrocytic necrosis virus (ENV), is unculturable and a presumed iridovirus by electron microscopy. In vivo amplification of the virus in pathogen-free laboratory stocks of Pacific herring with subsequent virus concentration, purification, DNA extraction, and high-throughput sequencing were used to obtain genomic ENV sequences. Fragments with the highest sequence identity to the family Iridoviridae were used to design four sets of ENV-specific polymerase chain reaction (PCR) primers. Testing of blood and tissue samples from experimentally and wild infected Pacific herring as well as DNA extracted from other amphibian and piscine iridoviruses verified the assays were specific to ENV with a limit of detection of 0.0003 ng. Preliminary phylogenetic analyses of a 1448 bp fragment of the putative DNA polymerase gene supported inclusion of ENV in a proposed sixth genus of the family Iridoviridae that contains other erythrocytic viruses from ectothermic hosts. This study provides the first molecular evidence of ENV's inclusion within the Iridoviridae family and offers conventional PCR assays as a means of rapidly surveying the ENV-status of wild and propagated Pacific herring stocks.

Kinetics of viral load and erythrocytic inclusion body formation in Pacific herring artificially infected with erythrocytic necrosis virus.

Viral erythrocytic necrosis (VEN) is a condition that affects marine and anadromous fish species, including herrings and salmonids, in the Atlantic and Pacific oceans. Infection is frequently associated with severe anemia and causes episodic mortality among wild and hatchery fish when accompanied by additional stressors; VEN can be presumptively diagnosed by (1) light microscopic identification of a single characteristic-a round, magenta-colored, 0.8-µm-diameter inclusion body (IB) within the cytoplasm of erythrocytes and their precursors on Giemsa-stained blood films; or (2) observation (via transmission electron microscopy [TEM]) of the causative iridovirus, erythrocytic necrosis virus (ENV), within erythrocytes or their precursors. To better understand the kinetics of VEN, specific-pathogen-free Pacific herring Clupea pallasii were infected with ENV by intraperitoneal injection. At 1, 4, 7, 10, 14, 21, and 28 d postexposure, samples of blood, spleen, and kidney were collected and assessed (1) via light microscopy for the number of intracytoplasmic IBs in blood smears and (2) via TEM for the number of virions within erythrocytes. The mean prevalence of intracytoplasmic IBs in the blood cells increased from 0% at 0-4 d postexposure to 94% at 28 d postexposure. Viral load within circulating red blood cells peaked at 7 d postexposure, fell slightly, and then reached a plateau. However, blood cells observed within the kidney and spleen tissues demonstrated high levels of ENV between 14 and 28 d postexposure. The results indicate that the viral load within erythrocytes does not correlate well with IB prevalence and that the virus can persist in infected fish for more than 28 d.

Induction of anti-viral genes during acute infection with Viral hemorrhagic septicemia virus (VHSV) genogroup IVa in Pacific herring (Clupea pallasii).

Infection with the aquatic rhabdovirus Viral hemorrhagic septicemia virus (VHSV) genogroup IVa results in high mortality in Pacific herring (Clupea pallasii) and is hypothesized to be a potential limiting factor for herring recovery. To investigate anti-viral immunity in the Pacific herring, four immune response genes were identified: the myxovirus resistance (Clpa-Mx), a major histocompatibility complex IB (named Clpa-UAA.001), the inducible immunoproteosome subunit 9 (Clpa-PSMB9) and the neutrophil chemotactic factor (Clpa-LECT2). Reverse transcriptase quantitative PCR (RT-qPCR) assays were developed based on these gene sequences to investigate the host immune response to acute VHSV infection following both injection and immersion challenge. Virus levels were measured by both plaque assay and RT-qPCR and peaked at day 6 during the 10-day exposure period for both groups of fish. The interferon stimulated genes (Clpa-Mx, -UAA.001, and -PSMB9) were significantly up-regulated in response to VHSV infection at both 6 and 10 days post-infection in both spleen and fin. Results from this study indicate that Pacific herring mount a robust, early antiviral response in both fin and spleen tissues. The immunological tools developed in this study will be useful for future studies to investigate antiviral immunity in Pacific herring.

Larval and juvenile Pacific herring Clupea pallasii are not susceptible to infectious hematopoietic necrosis under laboratory conditions.

Infectious hematopoietic necrosis (IHN) leads to periodic epidemics among certain wild and farmed fish species of the Northeast (NE) Pacific. The source of the IHN virus (IHNV) that initiates these outbreaks remains unknown; however, a leading hypothesis involves viral persistence in marine host species such as Pacific herring Clupea pallasii. Under laboratory conditions we exposed specific pathogen-free (SPF) larval and juvenile Pacific herring to 10(3) to 10(4) plaque-forming units (pfu) of IHNV ml(-1) by waterborne immersion. Cumulative mortalities among exposed groups were not significantly different from those of negative control groups. After waterborne exposure, IHNV was transiently recovered from the tissues of larvae but absent in tissues of juveniles. Additionally, no evidence of viral shedding was detected in the tank water containing exposed juveniles. After intraperitoneal (IP) injection of IHNV in juvenile herring with 10(3) pfu, IHNV was recovered from the tissues of sub-sampled individuals for only the first 5 d post-exposure. The lack of susceptibility to overt disease and transient levels of IHNV in the tissues of exposed fish indicate that Pacific herring do not likely serve a major epizootiological role in perpetuation of IHNV among free-ranging sockeye salmon Oncorhynchus nerka and farmed Atlantic salmon Salmo salar in the NE Pacific.

Release of infectious cells from epidermal ulcers in Ichthyophonus sp.-infected Pacific herring (Clupea pallasii): evidence for multiple mechanisms of transmission.

A common clinical sign of ichthyophoniasis in herring and trout is "sandpaper" skin, a roughening of the epidermis characterized by the appearance of small papules, followed by ulceration and sloughing of the epithelium; early investigators hypothesized that these ulcers might be a means of transmitting the parasite, Ichthyophonus sp., without the necessity of ingesting an infected host. We examined the cells associated with the epidermal lesions and confirmed that they were viable Ichthyophonus sp. cells that were readily released from the skin into the mucous layer and ultimately into the aquatic environment. The released cells were infectious when injected into the body cavity of specific-pathogen-free herring. Our hypothesis is that different mechanisms of transmission occur in carnivorous and planktivorous hosts: Planktonic feeders become infected by ingestion of ulcer-derived cells, while carnivores become infected by ingestion of whole infected fish.