Odor exposure during imprinting periods increases odorant-specific sensitivity and receptor gene expression in coho salmon (Oncorhynchus kisutch).

Pacific salmon are well known for their homing migrations; juvenile salmon learn odors associated with their natal streams prior to seaward migration, and then use these retained odor memories to guide them back from oceanic feeding grounds to their river of origin to spawn several years later. This memory formation, termed olfactory imprinting, involves at least in part, sensitization of the peripheral olfactory epithelium to specific odorants. We hypothesized that this change in peripheral sensitivity is due to exposure-dependent increases in the expression of odorant receptor (OR) proteins that are activated by specific odorants experienced during imprinting. To test this hypothesis, we exposed juvenile coho salmon, Oncorhynchus kisutch Walbaum, to the basic amino acid odorant L-arginine during the parr-smolt transformation (PST), when imprinting occurs, and assessed sensitivity of the olfactory epithelium to this and other odorants. We then identified the coho salmon orthologue of a basic amino acid odorant receptor (BAAR) and determined the mRNA expression levels of this receptor and other transcripts representing different classes of OR families. Exposure to L-arginine during the PST resulted in increased sensitivity to that odorant and a specific increase in BAAR mRNA expression in the olfactory epithelium relative to other ORs. These results suggest that specific increases in ORs activated during imprinting may be an important component of home stream memory formation and this phenomenon may ultimately be useful as a marker of successful imprinting to assess management strategies and hatchery practices that may influence straying in salmon.

Dietary Exposure to Environmentally Relevant Levels of Chemical Contaminants Reduces Growth and Survival in Juvenile Chinook Salmon.

Chemical pollution can degrade aquatic ecosystems. Chinook salmon in contaminated habitats are vulnerable to health impacts from toxic exposures. Few studies have been conducted on adverse health outcomes associated with current levels and mixtures of contaminants. Fewer still address effects specific to the juvenile life-stage of salmonids. The present study evaluated contaminant-related effects from dietary exposure to environmentally relevant concentrations and mixture profiles in juvenile Chinook salmon from industrialized waterways in the U.S. Pacific Northwest using two end points: growth assessment and disease susceptibility. The dose and chemical proportions were reconstituted based on environmental sampling and analysis using the stomach contents of juvenile Chinook salmon recently collected from contaminated, industrialized waterways. Groups of fish were fed a mixture with fixed proportions of 10 polychlorinated biphenyls (PCBs), 3 dichlorodiphenyltrichloroethanes (DDTs), and 13 polycyclic aromatic hydrocarbons (PAHs) at five concentrations for 35 days. These contaminant compounds were selected because of elevated concentrations and the widespread presence in sediments throughout industrialized waterways. Fork length and otolith microstructural growth indicators were significantly reduced in fish fed environmentally relevant concentrations of these contaminants. In addition, contaminant-exposed Chinook salmon were more susceptible to disease during controlled challenges with the pathogen Aeromonas salmonicida. Our results indicate that dietary exposure to contaminants impairs growth and immune function in juvenile Chinook salmon, thereby highlighting that current environmental exposure to chemicals of potential management concern threatens the viability of exposed salmon.

Crude oil exposure of early life stages of Atlantic haddock suggests threshold levels for developmental toxicity as low as 0.1 µg total polyaromatic hydrocarbon (TPAH)/L.

Atlantic haddock (Melanogrammus aeglefinus) embryos bind dispersed crude oil droplets to the eggshell and are consequently highly susceptible to toxicity from spilled oil. We established thresholds for developmental toxicity and identified any potential long-term or latent adverse effects that could impair the growth and survival of individuals. Embryos were exposed to oil for eight days (10, 80 and 300 µg oil/L, equivalent to 0.1, 0.8 and 3.0 µg TPAH/L). Acute and delayed mortality were observed at embryonic, larval, and juvenile stages with IC50 = 2.2, 0.39, and 0.27 µg TPAH/L, respectively. Exposure to 0.1 µg TPAH/L had no negative effect on growth or survival. However, yolk sac larvae showed significant reduction in the outgrowth (ballooning) of the cardiac ventricle in the absence of other extracardiac morphological defects. Due to this propensity for latent sublethal developmental toxicity, we recommend an effect threshold of 0.1 µg TPAH/L for risk assessment models.

Sublethal neurotoxicity of organophosphate insecticides to juvenile coho salmon.

For decades, organophosphate (OP) insecticides have been used as chemical control agents in watersheds that support at-risk populations of Pacific salmon throughout western North America. Spray drift, runoff, and other processes transport OPs to critical surface water habitats for migratory salmonids. While most OPs share a common mechanism of action (i.e., inhibition of neuronal acetylcholinesterase, or AChE), they typically vary in toxic potency. Moreover, dose-response relationships for exposure and sublethal neurotoxicity (e.g., brain AChE inhibition) in salmonids have not been defined for many OPs. Here we exposed juvenile coho salmon (Oncorhynchus kisutch) to five common anticholinesterase insecticides (dimethoate, ethoprop, naled, phorate and phosmet) that are widely used on agricultural, commercial, residential, and public lands. Each of the five pesticides produced a concentration-dependent inhibition of AChE enzyme activity. The effective concentration for 50 % AChE inhibition (96-hr EC50) indicated the highest toxicity for phorate (EC50 = 0.57 µg/L) followed by phosmet (3.3 µg/L), naled (7.8 µg/L), ethoprop (90.6 µg/L) and dimethoate (273 µg/L). These findings can inform 1) relative hazard analyses for OP use near sensitive aquatic habitats, 2) predictions of sublethal OP mixture toxicity, and 3) ecological risk assessments for threatened or endangered species of Pacific salmon.

Crude oil cardiotoxicity to red drum embryos is independent of oil dispersion energy.

The potential bioavailability of toxic chemicals from oil spills to water column organisms such as fish embryos may be influenced by physical dispersion along an energy gradient. For example, a surface slick with minimal wave action (low energy) could potentially produce different toxic effects from high energy situations such as pressurized discharge from a blown wellhead. Here we directly compared the toxicity of water accommodated fractions (WAFs) of oil prepared with low and high mixing energy (LEWAFs and HEWAFs, respectively) using surface oil samples collected during the 2010 Deepwater Horizon spill, and embryos of a representative nearshore species, red drum (Sciaenops ocellatus). Biological effects of each WAF type was quantified with several functional and morphological indices of developmental cardiotoxicity, providing additional insight into species-specific responses to oil exposure. Although the two WAF preparations yielded different profiles of polycyclic aromatic hydrocarbons (PAHs), cardiotoxic phenotypes were essentially identical. Based on benchmark thresholds for both morphological and functional cardiotoxicity, in general LEWAFs had lower thresholds for these phenotypes than HEWAFs based on total PAH measures. However, HEWAF and LEWAF toxicity thresholds were more similar when calculated based on estimates of dissolved PAHs only. Differences in thresholds were attributable to the weathering state of the oil samples.

Roads to ruin: conservation threats to a sentinel species across an urban gradient.

Urbanization poses a global challenge to species conservation. This is primarily understood in terms of physical habitat loss, as agricultural and forested lands are replaced with urban infrastructure. However, aquatic habitats are also chemically degraded by urban development, often in the form of toxic stormwater runoff. Here we assess threats of urbanization to coho salmon throughout developed areas of the Puget Sound Basin in Washington, USA. Puget Sound coho are a sentinel species for freshwater communities and also a species of concern under the U.S. Endangered Species Act. Previous studies have demonstrated that stormwater runoff is unusually lethal to adult coho that return to spawn each year in urban watersheds. To further explore the relationship between land use and recurrent coho die-offs, we measured mortality rates in field surveys of 51 spawning sites across an urban gradient. We then used spatial analyses to measure landscape attributes (land use and land cover, human population density, roadways, traffic intensity, etc.) and climatic variables (annual summer and fall precipitation) associated with each site. Structural equation modeling revealed a latent urbanization gradient that was associated with road density and traffic intensity, among other variables, and positively related to coho mortality. Across years within sites, mortality increased with summer and fall precipitation, but the effect of rainfall was strongest in the least developed areas and was essentially neutral in the most urbanized streams. We used the best-supported structural equation model to generate a predictive mortality risk map for the entire Puget Sound Basin. This map indicates an ongoing and widespread loss of spawners across much of the Puget Sound population segment, particularly within the major regional north-south corridor for transportation and development. Our findings identify current and future urbanization-related threats to wild coho, and show where green infrastructure and similar clean water strategies could prove most useful for promoting species conservation and recovery.

Coho salmon spawner mortality in western US urban watersheds: bioinfiltration prevents lethal storm water impacts.

Adult coho salmon Oncorhynchus kisutch return each autumn to freshwater spawning habitats throughout western North America. The migration coincides with increasing seasonal rainfall, which in turn increases storm water run-off, particularly in urban watersheds with extensive impervious land cover. Previous field assessments in urban stream networks have shown that adult coho are dying prematurely at high rates (>50%). Despite significant management concerns for the long-term conservation of threatened wild coho populations, a causal role for toxic run-off in the mortality syndrome has not been demonstrated.We exposed otherwise healthy coho spawners to: (i) artificial storm water containing mixtures of metals and petroleum hydrocarbons, at or above concentrations previously measured in urban run-off; (ii) undiluted storm water collected from a high traffic volume urban arterial road (i.e. highway run-off); and (iii) highway run-off that was first pre-treated via bioinfiltration through experimental soil columns to remove pollutants.We find that mixtures of metals and petroleum hydrocarbons - conventional toxic constituents in urban storm water - are not sufficient to cause the spawner mortality syndrome. By contrast, untreated highway run-off collected during nine distinct storm events was universally lethal to adult coho relative to unexposed controls. Lastly, the mortality syndrome was prevented when highway run-off was pretreated by soil infiltration, a conventional green storm water infrastructure technology.Our results are the first direct evidence that: (i) toxic run-off is killing adult coho in urban watersheds, and (ii) inexpensive mitigation measures can improve water quality and promote salmon survival. Synthesis and applications. Coho salmon, an iconic species with exceptional economic and cultural significance, are an ecological sentinel for the harmful effects of untreated urban run-off. Wild coho populations cannot withstand the high rates of mortality that are now regularly occurring in urban spawning habitats. Green storm water infrastructure or similar pollution prevention methods should be incorporated to the maximal extent practicable, at the watershed scale, for all future development and redevelopment projects, particularly those involving transportation infrastructure.

Corresponding morphological and molecular indicators of crude oil toxicity to the developing hearts of mahi mahi.

Crude oils from distinct geological sources worldwide are toxic to developing fish hearts. When oil spills occur in fish spawning habitats, natural resource injury assessments often rely on conventional morphometric analyses of heart form and function. The extent to which visible indicators correspond to molecular markers for cardiovascular stress is unknown for pelagic predators from the Gulf of Mexico. Here we exposed mahi (Coryphaena hippurus) embryos to field-collected crude oil samples from the 2010 Deepwater Horizon disaster. We compared visible heart defects (edema, abnormal looping, reduced contractility) to changes in expression of cardiac-specific genes that are diagnostic of heart failure in humans or associated with loss-of-function zebrafish cardiac mutants. Mahi exposed to crude oil during embryogenesis displayed typical symptoms of cardiogenic syndrome as larvae. Contractility, looping, and circulatory defects were evident, but larval mahi did not exhibit downstream craniofacial and body axis abnormalities. A gradation of oil exposures yielded concentration-responsive changes in morphometric and molecular responses, with relative sensitivity being influenced by age. Our findings suggest that 1) morphometric analyses of cardiac function are more sensitive to proximal effects of crude oil-derived chemicals on the developing heart, and 2) molecular indicators reveal a longer-term adverse shift in cardiogenesis trajectory.

Very low embryonic crude oil exposures cause lasting cardiac defects in salmon and herring.

The 1989 Exxon Valdez disaster exposed embryos of pink salmon and Pacific herring to crude oil in shoreline spawning habitats throughout Prince William Sound, Alaska. The herring fishery collapsed four years later. The role of the spill, if any, in this decline remains one of the most controversial unanswered questions in modern natural resource injury assessment. Crude oil disrupts excitation-contraction coupling in fish heart muscle cells, and we show here that salmon and herring exposed as embryos to trace levels of crude oil grow into juveniles with abnormal hearts and reduced cardiorespiratory function, the latter a key determinant of individual survival and population recruitment. Oil exposure during cardiogenesis led to specific defects in the outflow tract and compact myocardium, and a hypertrophic response in spongy myocardium, evident in juveniles 7 to 9 months after exposure. The thresholds for developmental cardiotoxicity were remarkably low, suggesting the scale of the Exxon Valdez impact in shoreline spawning habitats was much greater than previously appreciated. Moreover, an irreversible loss of cardiac fitness and consequent increases in delayed mortality in oil-exposed cohorts may have been important contributors to the delayed decline of pink salmon and herring stocks in Prince William Sound.

Toward enhanced MIQE compliance: reference residual normalization of qPCR gene expression data.

Normalization of fluorescence-based quantitative real-time PCR (qPCR) data varies across quantitative gene expression studies, despite its integral role in accurate data quantification and interpretation. Identification of suitable reference genes plays an essential role in accurate qPCR normalization, as it ensures that uncorrected gene expression data reflect normalized data. The reference residual normalization (RRN) method presented here is a modified approach to conventional 2(-ΔΔCt)qPCR normalization that increases mathematical transparency and incorporates statistical assessment of reference gene stability. RRN improves mathematical transparency through the use of sample-specific reference residuals (RR i ) that are generated from the mean Ct of one or more reference gene(s) that are unaffected by treatment. To determine stability of putative reference genes, RRN uses ANOVA to assess the effect of treatment on expression and subsequent equivalence-threshold testing to establish the minimum permitted resolution. Step-by-step instructions and comprehensive examples that demonstrate the influence of reference gene stability on target gene normalization and interpretation are provided. Through mathematical transparency and statistical rigor, RRN promotes compliance with Minimum Information for Quantitative Experiments and, in so doing, provides increased confidence in qPCR data analysis and interpretation.