Effects of temperature on viral load, inclusion body formation, and host response in Pacific Herring with viral erythrocytic necrosis (VEN).

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.

A Long-Standing Hybrid Population Between Pacific and Atlantic Herring in a Subarctic Fjord of Norway.

Atlantic herring (Clupea harengus) and Pacific herring (C. pallasii) are sister species that split from a common ancestor about 2 million years ago. Balsfjord, a subarctic fjord in Northern Norway, harbors an outpost population of Pacific herring within the range of the Atlantic herring. We used whole genome sequencing to show that gene flow from Atlantic herring into the Balsfjord population has generated a stable hybrid population that has persisted for thousands of generations. The Atlantic herring ancestry in Balsfjord was estimated in the range 25-26%. The old age and large proportion of introgressed regions suggest there are no obvious genetic incompatibilities between species. Introgressed regions were widespread in the genome and large, with some in excess of 1 Mb, and they were overrepresented in low-recombination regions. We show that the distribution of introgressed material is non-random; introgressed sequence blocks in different individuals are shared more often than expected by chance. Furthermore, introgressed regions tend to show elevated divergence (FST) between Atlantic and Pacific herring. Together, our results suggest that introgression of genetic material has facilitated adaptation in the Balsfjord population. The Balsfjord population provides a rare example of a stable interspecies hybrid population that has persisted over thousands of years.

Delayed trophic response of a marine predator to ocean condition and prey availability during the past century.

Understanding the response of predators to ecological change at multiple temporal scales can elucidate critical predator-prey dynamics that would otherwise go unrecognized. We performed compound-specific nitrogen stable isotope analysis of amino acids on 153 harbor seal museum skull specimens to determine how trophic position of this marine predator has responded to ecosystem change over the past century. The relationships between harbor seal trophic position, ocean condition, and prey abundance, were analyzed using hierarchical modeling of a multi-amino-acid framework and applying 1, 2, and 3 years temporal lags. We identified delayed responses of harbor seal trophic position to both physical ocean conditions (upwelling, sea surface temperature, freshwater discharge) and prey availability (Pacific hake, Pacific herring, and Chinook salmon). However, the magnitude and direction of the trophic position response to ecological changes depended on the temporal delay. For example, harbor seal trophic position was negatively associated with summer upwelling but had a 1-year delayed response to summer sea surface temperature, indicating that some predator responses to ecosystem change are not immediately observable. These results highlight the importance of considering dynamic responses of predators to their environment as multiple ecological factors are often changing simultaneously and can take years to propagate up the food web.

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.

Genetic structure of marine and lake forms of Pacific herring Clupea pallasii.

The Pacific herring (Clupea pallasii) is one of the most important species in the commercial fisheries distributed in the North Pacific Ocean and the northeastern European seas. This teleost has marine and lake ecological forms a long its distribution in the Holarctic. However, the level of genetic differentiation between these two forms is not well known. In the present study, we used ddRAD-sequencing to genotype 54 specimens from twelve wild Pacific herring populations from the Kara Sea and the Russian part of the northwestern Pacific Ocean for unveiling the genetic structure of Pacific herring. We found that the Kara Sea population is significantly distinct from Pacific Ocean populations. It was demonstrated that lake populations of Pacific herring differ from one another as well as from marine specimens. Our results show that fresh and brackish water Pacific herring, which inhabit lakes, can be distinguished as a separate lake ecological form. Moreover, we demonstrate that each observed lake Pacific herring population has its own and unique genetic legacy.

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.

Long-term shedding from fully convalesced individuals indicates that Pacific herring are a reservoir for viral hemorrhagic septicemia virus.

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.

Functional genetic diversity in an exploited marine species and its relevance to fisheries management.

The timing of reproduction influences key evolutionary and ecological processes in wild populations. Variation in reproductive timing may be an especially important evolutionary driver in the marine environment, where the high mobility of many species and few physical barriers to migration provide limited opportunities for spatial divergence to arise. Using genomic data collected from spawning aggregations of Pacific herring (Clupea pallasii) across 1600 km of coastline, we show that reproductive timing drives population structure in these pelagic fish. Within a specific spawning season, we observed isolation by distance, indicating that gene flow is also geographically limited over our study area. These results emphasize the importance of considering both seasonal and spatial variation in spawning when delineating management units for herring. On several chromosomes, we detected linkage disequilibrium extending over multiple Mb, suggesting the presence of chromosomal rearrangements. Spawning phenology was highly correlated with polymorphisms in several genes, in particular SYNE2, which influences the development of retinal photoreceptors in vertebrates. SYNE2 is probably within a chromosomal rearrangement in Pacific herring and is also associated with spawn timing in Atlantic herring (Clupea harengus). The observed genetic diversity probably underlies resource waves provided by spawning herring. Given the ecological, economic and cultural significance of herring, our results support that conserving intraspecific genetic diversity is important for maintaining current and future ecosystem processes.

Urban stormwater and crude oil injury pathways converge on the developing heart of a shore-spawning marine forage fish.

Understanding how aquatic organisms respond to complex chemical mixtures remains one of the foremost challenges in modern ecotoxicology. Although oil spills are typically high-profile disasters that release hundreds or thousands of chemicals into the environment, there is growing evidence for a common adverse outcome pathway (AOP) for the vulnerable embryos and larvae of fish species that spawn in oiled habitats. Molecular initiating events involve the disruption of excitation-contraction coupling in individual cardiomyocytes, which then dysregulate the form and function of the embryonic heart. Phenanthrenes and other three-ring (tricyclic) polycyclic aromatic hydrocarbons (PAHs) are key drivers for this developmental cardiotoxicity and are also relatively enriched in land-based urban runoff. Similar to oil spills, stormwater discharged from roadways and other high-traffic impervious surfaces contains myriad contaminants, many of which are uncharacterized in terms of their chemical identity and toxicity to aquatic organisms. Nevertheless, given the exceptional sensitivity of the developing heart to tricyclic PAHs and the ubiquitous presence of these compounds in road runoff, cardiotoxicity may also be a dominant aspect of the stormwater-induced injury phenotype in fish early life stages. Here we assessed the effects of traffic-related runoff on the embryos and early larvae of Pacific herring (Clupea pallasii), a marine forage fish that spawns along the coastline of western North America. We used the well-characterized central features of the oil toxicity AOP for herring embryos as benchmarks for a detailed analysis of embryolarval cardiotoxicity across a dilution gradient ranging from 12 to 50% stormwater diluted in clean seawater. These injury indicators included measures of circulatory function, ventricular area, heart chamber looping, and the contractility of both the atrium and the ventricle. We also determined tissue concentrations of phenanthrenes and other PAHs in herring embryos. We find that tricyclic PAHs are readily bioavailable during cardiogenesis, and that stormwater-induced toxicity is in many respects indistinguishable from canonical crude oil toxicity. Given the chemical complexity of urban runoff, non-tricyclic PAH-mediated mechanisms of developmental toxicity in fish remain likely. However, from the standpoint of managing wild herring populations, our results suggest that stormwater-driven threats to individual survival (both near-term and delayed mortality) can be understood from decades of past research on crude oil toxicity. Moreover, Pacific herring embryos are promising sentinels for water quality monitoring in nearshore marine habitats, as in situand sensitive indicators of both toxic runoff and the effectiveness of pollution reduction efforts such as green stormwater infrastructure.

A geographic hot spot of Ichthyophonus infection in the southern Salish Sea, USA.

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.

Polycyclic aromatic hydrocarbons in Pacific herring (Clupea pallasii) embryos exposed to creosote-treated pilings during a piling-removal project in a nearshore marine habitat of Puget Sound.

We used manually spawned, field-deployed embryos of a common marine fish species, Pacific herring (Clupea pallasii), to evaluate accumulation of polycyclic aromatic hydrocarbons (PAHs) associated with an incomplete creosote-treated piling (CTP) removal project. Embryos near undisturbed 100-year-old CTPs (before removal) accumulated higher PAHs and exhibited higher cyp1a gene expression than embryos from reference areas. Embryos incubated close to CTP debris after CTP removal showed PAHs 90 times higher than reference areas up to a year after CTP removal. cyp1a fold-induction correlated with total embryo PAHs in all three years. Patterns of individual PAH chemicals differed slightly between embryos, wood sampled from CTPs, and passive samplers. This study illustrates the importance of using appropriate techniques and procedures to remove CTPs in aquatic environments to prevent release of toxic chemicals. Of particular concern is that incomplete CTP removal could expose sensitive life stages of fishes to chemicals that may reduce their survival.

Preparation and Identification of Antioxidative Peptides from Pacific Herring (Clupea pallasii) Protein.

The aim of this study was to isolate and purify antioxidative peptides from Pacific herring (Clupea pallasii) protein. Five enzymes (pepsin, trypsin, papain, flavourzyme, and neutrase) were used for protein hydrolysis, and Pacific herring protein hydrolysates (PHPH) were separated by ultrafiltration. The fraction with the molecular weight below 3500 Da exhibited the highest in vitro antioxidant activities and cellular antioxidant activity. The PHPH was isolated and purified by consecutive chromatographic methods including gel filtration chromatography and reverse high-performance liquid chromatography (RP-HPLC). The purified antioxidant peptides were identified as Leu-His-Asp-Glu-Leu-Thr (MW = 726.35 Da) and Lys-Glu-Glu-Lys-Phe-Glu (MW = 808.40 Da), and the IC50 values of cellular antioxidant activity were 1.19 ± 0.05 mg/mL and 1.04 ± 0.06 mg/mL. The results demonstrate that is possible to produce natural antioxidative peptides from Pacific herring protein via enzymatic hydrolysis and purification.

Distribution and Phylogeny of Erythrocytic Necrosis Virus (ENV) in Salmon Suggests Marine Origin.

Viral erythrocytic necrosis (VEN) affects over 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. However, the distribution and strain variation of its viral causative agent, erythrocytic necrosis virus (ENV), has not been well characterized within Pacific salmon. Here, metatranscriptomic sequencing of Chinook salmon revealed that ENV infecting salmon was closely related to ENV from Pacific herring, with inferred amino-acid sequences from Chinook salmon being 99% identical to those reported for herring. Sequence analysis also revealed 89 protein-encoding sequences attributed to ENV, greatly expanding the amount of genetic information available for this virus. High-throughput PCR of over 19,000 fish showed that ENV is widely distributed in the NE Pacific Ocean and was detected in 12 of 16 tested species, including in 27% of herring, 38% of anchovy, 17% of pollock, and 13% of sand lance. Despite frequent detection in marine fish, ENV prevalence was significantly lower in fish from freshwater (0.03%), as assessed with a generalized linear mixed effects model (p = 5.5 × 10-8). Thus, marine fish are likely a reservoir for the virus. High genetic similarity between ENV obtained from salmon and herring also suggests that transmission between these hosts is likely.

Intraspecific DNA contamination distorts subtle population structure in a marine fish: Decontamination of herring samples before restriction-site associated sequencing and its effects on population genetic statistics.

Wild specimens are often collected in challenging field conditions, where samples may be contaminated with the DNA of conspecific individuals. This contamination can result in false genotype calls, which are difficult to detect, but may also cause inaccurate estimates of heterozygosity, allele frequencies and genetic differentiation. Marine broadcast spawners are especially problematic, because population genetic differentiation is low and samples are often collected in bulk and sometimes from active spawning aggregations. Here, we used contaminated and clean Pacific herring (Clupea pallasi) samples to test (a) the efficacy of bleach decontamination, (b) the effect of decontamination on RAD genotypes and (c) the consequences of contaminated samples on population genetic analyses. We collected fin tissue samples from actively spawning (and thus contaminated) wild herring and nonspawning (uncontaminated) herring. Samples were soaked for 10 min in bleach or left untreated, and extracted DNA was used to prepare DNA libraries using a restriction site-associated DNA (RAD) approach. Our results demonstrate that intraspecific DNA contamination affects patterns of individual and population variability, causes an excess of heterozygotes and biases estimates of population structure. Bleach decontamination was effective at removing intraspecific DNA contamination and compatible with RAD sequencing, producing high-quality sequences, reproducible genotypes and low levels of missing data. Although sperm contamination may be specific to broadcast spawners, intraspecific contamination of samples may be common and difficult to detect from high-throughput sequencing data and can impact downstream analyses.

Analytical and diagnostic performance of a qPCR assay for Ichthyophonus spp. compared to the tissue culture 'gold standard'.

Parasites of the genus Ichthyophonus infect many fish species and have a non-uniform distribution within host tissues. Due in part to this uneven distribution, the comparative sensitivity and accuracy of using molecular-based detection methods versus culture to estimate parasite prevalence is under debate. We evaluated the analytical and diagnostic performance of an existing qPCR assay in comparison to the 'gold standard' culture method using Pacific herring Clupea pallasii with known exposure history. We determined that the assay is suitable for use in this host, and diagnostic specificity was consistently high (>98%) in both heart and liver tissues. Diagnostic sensitivity could not be fully assessed due to low infection rates, but our results suggest that qPCR is not as sensitive as culture under all circumstances. Diagnostic sensitivity of qPCR relative to culture is likely affected by the amount of sample processed. The prevalence values estimated by the 2 methods were not significantly different when sample amounts were equal (heart tissue), but when the assayed sample amounts were unequal (liver tissue), the culture method detected a significantly higher prevalence of the parasite than qPCR. Further, culture of liver also detected significantly more Ichthyophonus infections than culture of heart, suggesting that the density and distribution of parasites in tissues also plays a role in assay sensitivity. This sensitivity issue would be most problematic for fish with light infections. Although qPCR does not detect the presence of a live organism, DNA-based pathogen detection methods provide the opportunity for alternate testing strategies when culture is not possible.

How relative size and abundance structures the relationship between size and individual growth in an ontogenetically piscivorous fish.

While individual growth ultimately reflects the quality and quantity of food resources, intra and interspecific interactions for these resources, as well as individual size, may have dramatic impacts on growth opportunity. Out-migrating anadromous salmonids make rapid transitions between habitat types resulting in large pulses of individuals into a given location over a short period, which may have significant impact on demand for local resources. We evaluated the spatial and temporal variation in IGF-1 concentrations (a proxy for growth rate) and the relationship between size and concentration for juvenile Chinook salmon in Puget Sound, WA, USA, as a function of the relative size and abundance of both Chinook salmon and Pacific herring, a species which commonly co-occurs with salmonids in nearshore marine habitats. The abundance of Chinook salmon and Pacific herring varied substantially among the sub-basins as function of outmigration timing and spawn timing, respectively, while size varied systematically and consistently for both species. Mean IGF-1 concentrations were different among sub-basins, although patterns were not consistent through time. In general, size was positively correlated with IGF-1 concentration, although the slope of the relationship was considerably higher where Pacific herring were more abundant than Chinook salmon; specifically where smaller individual herring, relative to Chinook salmon, were more abundant. Where Pacific herring were less abundant than Chinook salmon, IGF-1 concentrations among small and large Chinook salmon were more variable and showed no consistent increase for larger individuals. The noticeable positive effect of relative Pacific herring abundance on the relationship between size and individual growth rates likely represents a shift to predation based on increased IGF-1 concentrations for individual Chinook salmon that are large enough to incorporate fish into their diet and co-occur with the highest abundances of Pacific herring.

The parasite Ichthyophonus sp. in Pacific herring from the coastal NE Pacific.

The protistan parasite Ichthyophonus occurred in populations of Pacific herring Clupea pallasii Valenciennes throughout coastal areas of the NE Pacific, ranging from Puget Sound, WA north to the Gulf of Alaska, AK. Infection prevalence in local Pacific herring stocks varied seasonally and annually, and a general pattern of increasing prevalence with host size and/or age persisted throughout the NE Pacific. An exception to this zoographic pattern occurred among a group of juvenile, age 1+ year Pacific herring from Cordova Harbor, AK in June 2010, which demonstrated an unusually high infection prevalence of 35%. Reasons for this anomaly were hypothesized to involve anthropogenic influences that resulted in locally elevated infection pressures. Interannual declines in infection prevalence from some populations (e.g. Lower Cook Inlet, AK; from 20-32% in 2007 to 0-3% during 2009-13) or from the largest size cohorts of other populations (e.g. Sitka Sound, AK; from 62.5% in 2007 to 19.6% in 2013) were likely a reflection of selective mortality among the infected cohorts. All available information for Ichthyophonus in the NE Pacific, including broad geographic range, low host specificity and presence in archived Pacific herring tissue samples dating to the 1980s, indicate a long-standing host-pathogen relationship.

Persistence of external signs in Pacific herring Clupea pallasii Valenciennes with ichthyophoniasis.

The progression of external signs of Ichthyophonus infection in Pacific herring Clupea pallasii Valenciennes was highly variable and asynchronous after intraperitoneal injection with pure parasite preparations; however, external signs generally persisted through the end of the study (429 days post-exposure). Observed signs included papules, erosions and ulcers. The prevalence of external signs plateaued 35 days post-exposure and persisted in 73-79% of exposed individuals through the end of the first experiment (147 days post-exposure). Among a second group of infected herring, external signs completely resolved in only 10% of the fish after 429 days. The onset of mortality preceded the appearance of external signs. Histological examination of infected skin and skeletal muscle tissues indicated an apparent affinity of the parasite for host red muscle. Host responses consisted primarily of granulomatous inflammation, fibrosis and necrosis in the skeletal muscle and other tissues. The persistence and asynchrony of external signs and host response indicated that they were neither a precursor to host mortality nor did they provide reliable metrics for hindcasting on the date of exposure. However, the long-term persistence of clinical signs in Pacific herring may be useful in ascertaining the population-level impacts of ichthyophoniasis in regularly observed populations.

Evaluating hypoxia-inducible factor-1α mRNA expression in a pelagic fish, Pacific herring Clupea pallasii, as a biomarker for hypoxia exposure.

Hypoxia [dissolved oxygen (DO)<2 mg L(-1)] is a major environmental perturbation for many aquatic ecosystems, particularly highly productive estuaries. Most research attention and understanding about the impacts of hypoxia on estuarine species has focused on the benthos, where hypoxia is most common. Although the pelagic zone is also susceptible to the effects of hypoxia, the biological interactions and consequences are not as well understood in marine environments because documenting exposure or avoidance of hypoxia is often difficult. Physiological biomarkers may provide a way to gain more detailed spatiotemporal information regarding species' exposure to hypoxia. Here, we identified and tested a hypoxia-specific responsive gene, hypoxia-inducible factor-1α (hif-1α), to evaluate its potential as a biomarker for hypoxia exposure in Pacific herring (Clupea pallasii). We conducted controlled laboratory experiments to establish the level of hepatic hif-1α elevated gene expression (>1 sd normoxic mean), exposure amplification (≥2 hours), reduction rate (ca. 24 hours), and some evidence of a lethal hypoxic limit (ca. 2 mg L(-1), ≥4 hours). We then used these findings to evaluate the spatiotemporal patterns of hif-1α for Pacific herring in a seasonally hypoxia estuary, Hood Canal, Washington, USA. Although expression did not parallel the local hypoxic conditions in the estuary, herring from the more severe hypoxic year (2013) had a higher probability of having elevated mRNA levels. These patterns indicate that hepatic hif-1α levels may not be directly indicative of local DO levels for pelagic marine fish, but rather provide insight into hypoxia exposure over broader scales.