Elevated CO2 impairs olfactory‐mediated neural and behavioral responses and gene expression in ocean‐phase coho salmon (Oncorhynchus kisutch)

Elevated concentrations of CO₂ in seawater can disrupt numerous sensory systems in marine fish. This is of particular concern for Pacific salmon because they rely on olfaction during all aspects of their life including during their homing migrations from the ocean back to their natal streams. We investigated the effects of elevated seawater CO₂ on coho salmon (Oncorhynchus kisutch) olfactory‐mediated behavior, neural signaling, and gene expression within the peripheral and central olfactory system. Ocean‐phase coho salmon were exposed to three levels of CO₂, ranging from those currently found in ambient marine water to projected future levels. Juvenile coho salmon exposed to elevated CO₂ levels for 2 weeks no longer avoided a skin extract odor that elicited avoidance responses in coho salmon maintained in ambient CO₂ seawater. Exposure to these elevated CO₂ levels did not alter odor signaling in the olfactory epithelium, but did induce significant changes in signaling within the olfactory bulb. RNA‐Seq analysis of olfactory tissues revealed extensive disruption in expression of genes involved in neuronal signaling within the olfactory bulb of salmon exposed to elevated CO₂, with lesser impacts on gene expression in the olfactory rosettes. The disruption in olfactory bulb gene pathways included genes associated with GABA signaling and maintenance of ion balance within bulbar neurons. Our results indicate that ocean‐phase coho salmon exposed to elevated CO₂ can experience significant behavioral impairments likely driven by alteration in higher‐order neural signal processing within the olfactory bulb. Our study demonstrates that anadromous fish such as salmon may share a sensitivity to rising CO₂ levels with obligate marine species suggesting a more wide‐scale ecological impact of ocean acidification.     

Xenoma formation during microsporidial gill disease of salmonids caused by Loma salmonae is affected by host species (Oncorhynchus tshawytscha,O. kisutch,O. mykiss) but not by salinity

Host species and salinity often affect the development of disease in aquatic species. Eighty chinook salmon Oncorhynchus tshawytscha, 80 coho salmon O. kisutch and 80 rainbow trout O. mykiss were infected with Loma salmonae. Forty of each species were reared in seawater and 40 in freshwater. The mean number of xenomas per gill filament was 8 to 33 times greater in chinook salmon than in rainbow trout (RBT). Coho salmon had a mean xenoma intensity intermediate to that of chinook salmon and RBT. In contrast to the differences between species, salinity had no significant effect on xenoma intensity in any of these host species. The onset of xenoma formation occurred at Week 5 postexposure (PE) for chinook salmon and RBT, and at Week 6 PE for coho salmon. RBT had cleared all visible branchial xenomas by Week 9 PE, whereas xenomas persisted in coho and chinook salmon at Week 9 PE. Histologically, xenomas were visible in the filament arteries of the branchial arch in chinook and coho salmon gills but were absent from RBT gills. Fewer xenomas were seen in the central venous sinusoids of RBT than in chinook and coho salmon. The lower xenoma intensity, shorter duration of infection and pathological characteristics, common to microsporidial gill disease in RBT, suggest a degree of resistance to clinical disease that is not seen in coho and chinook salmon.     

Bioaccumulation of organochlorines by yearling coho salmon (Oncorhynchus kisutch walbaum) fed diets containing Great Lakes' coho salmon, and the pathophysiological responses of the recipients

1. Yearling coho salmon were fed diets containing the flesh of sexually mature adult coho salmon from Lakes Ontario, Erie and Michigan; Pacific Ocean coho salmon and a commercial trout chow were used to prepare control diets. The fish were fed the experimental diets for 27 weeks. Samples were taken of fed fish and fish fed for 27 weeks and then food deprived for 4 weeks. 2. Total accumulated organochlorine in the recipient was directly proportional to dietary total organochlorine levels, although there were considerable interlake differences in the degree of clearance of specific organochlorine compounds. 3. There was evidence of changes in thyroid physiology, hematocrit, liver structure and inter-renal gland activity in recipients fed the Great Lakes' coho salmon, apparently correlated with organochlorine bioaccumulation.     

Patterns of intra- and inter-population genetic diversity in Alaskan coho salmon: Implications for conservation

Little is known about the genetic diversityof coho salmon in Alaska, although this arearepresents half of the species' North Americanrange. In this study, nine microsatellite lociwere used to genotype 32 putative coho salmonpopulations from seven regions of Alaska. Theprimary objectives were to estimate andevaluate the degree and spatial distribution ofneutral genetic diversity within and amongpopulations of Alaskan coho salmon. Geneticanalysis yielded four results that provideinsight into forces influencing geneticdiversity in Alaskan coho salmon and haveimportant conservation implications: 1)significant population differentiation wasfound within each region; 2) the degree ofdifferentiation (F _ST = 0.099) amongpopulations was as large or larger than thatreported for other Pacific salmon species inAlaska; 3) phenetic clustering of populationsshowed weak geographic concordance; 4) stronggenetic isolation by distance was only apparentat the finest geographic scale (within adrainage). These results suggest that cohosalmon populations are small relative topopulations of other Pacific salmon, and thegenetic diversity within and among coho salmonpopulations is influenced primarily by geneticdrift, and not gene flow. Resource managementand conservation actions affecting coho salmonin Alaska must recognize that the populationsare generally small, isolated, and probablyexhibit local adaptation to different spawningand freshwater rearing habitats. These factorsjustify managing and conserving Alaskan cohosalmon at a fine geographic scale.     

刀鲚嗅觉受体基因MOR-51I2克隆、序列分析及组织表达

为研究刀鲚的嗅觉受体（Olfactory receptor,OR）是否参与其生殖洄游过程,利用基因组步移技术,从洄游型刀鲚（Coilia nasus）中克隆出嗅觉受体基因MOR-51I2的基因序列全长。该基因为单外显子结构,编码区长为999 bp。在3'UTR区域具有一段微卫星序列,以（AC）n为重复单位,并夹有若干T或G碱基,且在不同生态型间具有明显长度差异。MOR-51I2基因所编码的蛋白具有7次疏水性α-螺旋的跨膜结构,为G-蛋白偶联受体。MOR-51I2基因与已报道的其他鱼类的OR基因所编码的氨基酸序列的同源性在51%以上,其中与大西洋鲱（Clupea harengus）的OR51I2-like基因的同源性高达83%。经qRT-PCR分析显示,在定居型刀鲚中,MOR-51I2基因主要在嗅囊和性腺中表达,在肝脏、鳃、肌肉微弱表达,在心脏和眼睛中几乎不表达。其中,雌性嗅囊中的表达量约是雄性嗅囊中的2倍,是精巢和卵巢中的80-100倍。在洄游型刀鲚中,该基因在雄性嗅囊的表达量约是雌性嗅囊中的6倍。MOR-51I2基因在洄游型刀鲚的雌性嗅囊中的表达量约是定居型刀鲚的雌性嗅囊中的1/5,而在洄游型刀鲚的雄性嗅囊中的表达量却是定居型刀鲚的雄性嗅囊中的3倍。这些结果表明,MOR-51I2基因不但参与刀鲚的嗅觉功能,而且可能参与了刀鲚的性腺发育及生殖洄游过程,同时也可能与其生态型的分化相关。     

Discovery and characterization of single nucleotide polymorphisms in coho salmon,Oncorhynchus kisutch

Molecular population genetic analyses have become an integral part of ecological investigation and population monitoring for conservation and management. Microsatellites have been the molecular marker of choice for such applications over the last several decades, but single nucleotide polymorphism (SNP) markers are rapidly expanding beyond model organisms. Coho salmon (Oncorhynchus kisutch) is native to the north Pacific Ocean and its tributaries, where it is the focus of intensive fishery and conservation activities. As it is an anadromous species, coho salmon typically migrate across multiple jurisdictional boundaries, complicating management and requiring shared data collection methods. Here, we describe the discovery and validation of a suite of novel SNPs and associated genotyping assays which can be used in the genetic analyses of this species. These assays include 91 that are polymorphic in the species and one that discriminates it from a sister species, Chinook salmon. We demonstrate the utility of these SNPs for population assignment and phylogeographic analyses, and map them against the draft trout genome. The markers constitute a large majority of all SNP markers described for coho salmon and will enable both population‐ and pedigree‐based analyses across the southern part of the species native range.     

Inorganic pyrophosphatase: a new polymorphic allozyme locus in Pacific salmon

Genetic polymorphism of inorganic pyrophosphatase was investigated in 2799 individuals in four species of Pacific salmon: chinook salmon (Oncorhynchus tshawytscha), coho salmon (O. kisutch), kokanee (O. nerka), and steelhead (O. mykiss), using horizontal starch gel electrophoresis. This enzyme system appears to be an isolocus system with electrophoretically indistinguishable allozymes encoded by two loci (PP-1,2*) expressed in retinal tissue. Mendelian inheritance was observed with a breeding study in three family crosses. Population variability in four species was characterized in 44 populations from the U.S. Pacific coast. Three alleles were found in chinook salmon; two alleles each were found in coho salmon, kokanee, and steelhead. Chinook salmon and kokanee populations differed enough with respect to PP-1,2* frequencies that this isolocus is useful for genetic stock identification in these species.     

Effects of species, culture history, size and residency on relative competitive ability of salmonids

The relative competitive ability of juvenile farm and wild salmonids was investigated to provide insight into the potential effects of introduction of cultured salmon on wild Pacific salmonid ( Oncorhynchus ) species. Aquarium experiments involving equal contests ( i.e. size matched, simultaneously introduced individuals) indicated that two wild coho salmon Oncorhynchus kisutch populations were competitively equal to a farm coho salmon population. In equal contests between farm Atlantic salmon Salmo salar (Mowi strain) and these wild coho salmon populations or coastal cutthroat trout Oncorhynchus clarki clarki , Atlantic salmon were subordinate in all cases. When Atlantic salmon were given a residence advantage, however, they were competitively equal to both wild coho salmon populations, but remained subordinate to coastal cutthroat trout. When Atlantic salmon were given a 10–30% length advantage, they were competitively equal to one wild coho salmon population but remained subordinate to the other. In equal contests in semi-natural stream channels, both wild coho and farm Atlantic salmon grew significantly more in the presence of the other species than when alone. It appears that coho salmon obtain additional food ration by out competing Atlantic salmon, whereas Atlantic salmon were stimulated to feed more in the presence of coho salmon competitors. These results suggest that wild coho salmon and cutthroat trout should out compete farm Atlantic salmon of a similar size in nature. As the relative competitive ability of Atlantic salmon improves when they have a size and residence advantage, should feral populations become established, they may exist on a more equal competitive footing owing to the long freshwater residence of Atlantic salmon.     

Physiological and Behavioral Effects of Zinc and Temperature on Coho Salmon (Oncorhynchus kisutch)

Pacific salmon species including the U.S. federally endangered coho salmon (Oncorhynchus kisutch) and the U.S. federally threatened steelhead trout (Oncorhynchus mykiss) have declined at an alarming rate in the last 40 years. Two of the main causes for the decline in coastal coho populations include increases in temperature and contaminant loads in coastal watersheds. Zinc, in particular, is one of the most common contaminants in aquatic systems. Using an experimental mesocosm design, we examined physiological, biochemical, and behavioral responses of coho salmon to excess dietary zinc and increased temperatures, with the ultimate goal of relating results to wild populations of coho salmon and steelhead in the Navarro River, California. Fish were obtained from a hatchery and divided into four treatments: low water temperature-no dietary zinc, high temperature-no zinc, low temperature-zinc, and high temperature-zinc. Each treatment had four replicate tanks. Zinc concentrations in liver increased during exposure to a high zinc diet. Iron concentrations in liver increased during simultaneous exposure to high zinc diet and increased temperature, and growth was reduced in this experimental treatment. Expression of hsp-70 was not significantly different between treatments, but showed decreasing trends with high dietary zinc and high temperature. Feeding rate increased with exposure to a high zinc diet. Comparison with steelhead trout samples from the Navarro River, California, showed levels of zinc, iron, and hsp-70 greater than those found in the experimental Coho salmon. All comparisons between the hatchery coho salmon and wild steelhead should be viewed with caution due to the differences between species, the laboratory and natural environment, and the genetic differences between wild and hatchery fish.     

Glochidiosis of salmonid fishes. II. Comparison of tissue response of coho and chinook salmon to experimental infection with Margaritifera margaritifera (L.) (Pelecypoda: Margaritanidae)

Coho salmon (Oncorhynchus kisutch) are more resistant to experimental infection with the glochidia of the freshwater mussel (Margaritifera margaritifera) than are chinook salmon (Oncorhynchus tshawytscha). Histological sections made at intervals during the infection showed that coho salmon sloughed the parasites from their gills by 4.5 days postinfection, but the parasites remained encysted in the gills of chinook salmon for 12 weeks, when metamorphosis to juvenile mussels was complete. Coho salmon sloughed the parasites by a well-developed hyperplasia. No such pronounced hyperplastic reaction was seen in the gills of chinook salmon.     

Effects of anaesthesia on the chemosensory behaviour of Pacific salmon

As Lewis et al . reported in J. Fish Biol. 26 , 355–358 (1985) that the anaesthetic tricaine methanosulphate (MS 222) damaged the olfactory epithelium of channel catfish, two assays were used to determine whether MS 222 or 2-phenoxyethanol (an alternative anaesthetic) influenced subsequent olfaction-mediated behaviour. Tests revealed that the homing of adult chinook salmon, Oncorhynchus tshawytscha , and the l-serine avoidance response of juvenile coho salmon, O. kisutch , were not altered by anaesthesia.     

Gene expression of neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex components in the olfactory organ and brain during seaward and homeward migration by pink salmon (Oncorhynchus gorbuscha)

The expression of synaptic vesicle exocytosis-regulator SNARE complex component genes (snap25, stx1 and vamp2) was examined in the olfactory nervous system during seaward and homeward migration by pink salmon (Oncorhynchus gorbuscha). The expression levels of snares in the olfactory organ were higher in seaward fry than in feeding and homeward adults, reflecting the development of the olfactory nervous system. The expression of snap25a, b and stx1a was upregulated or stable in the adult olfactory bulb and telencephalon. This upregulated expression suggested alterations in olfactory neuronal plasticity that may be related to the discrimination of natal rivers. The expression of stx1b was downregulated in the adult olfactory bulb, but remained stable in the adult telencephalon. The expression of vamp2 was initially strong in seaward fry, but was downregulated in adults in both the olfactory bulb and telencephalon. Pink salmon has the lowest diversity of maturation age, the largest population, and the most evolutional position in Pacific salmon (genus Oncorhynchus). The expression of snares in the olfactory center of pink salmon reflected the timing of sexual maturation and homeward migration. The present results and our previous studies indicate that snares show distinct expression patterns between two salmon species that depend on physiological and ecological features of migration.     

Chemical toxicity testing in vitro using cytochrome P450-expressing cell lines, such as human CYP1B1

This protocol describes how to use cytochrome P450-dependent monooxygenase (CYP)-expressing cell lines in toxicity testing of chemicals in vitro. Selected cells amenable to permanently grow in culture are genetically manipulated to stably express single CYP enzymes originating from any species of interest. This expression can be characterized by, for example, determining CYP mRNA content, CYP protein level (western blotting or in situ immunofluorescence) and CYP-mediated enzyme activity (substrate conversion assays). These cells can be used to determine substrate specificities and species differences, e.g., in the bioactivation of drugs. Once constructed, CYP-expressing cells can serve as a straightforward and reliable tool in toxicity testing and the corresponding assays could be adapted for high-throughput analysis. Using these cells, enzyme assays can be performed in a matter of hours. This protocol is exemplified with V79 fibroblasts from Chinese hamster (Cricetulus griseus), modified to express human cytochrome P450 1B1 (CYP1B1). These cells are characterized for their CYP1B1-linked properties by in situ immunofluorescence and their activity in the 7-ethoxyresorufin-O-deethylase enzyme assay. This is followed by an assay showing metabolic activation of the polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene by CYP1B1, along with the toxicological endpoints of cytotoxicity and micronucleus formation.     

Gill Morphometry in Growth Hormone Transgenic Pacific Coho Salmon,Onchorhynchus kisutch,Differs Markedly from that in GH Transgenic Atlantic Salmon

In a previous study we showed that many of the morphological features of the respiratory system of GH (growth hormone) transgenic Atlantic salmon are greater than similarly sized control salmon. Here we show that the manifestation of GH transgene is similar in two different lines of GH transgenic Pacific coho salmon, but that it is very different from that in the GH transgenic Atlantic salmon. The GH transgenic Pacific coho salmon do not have a larger gill surface area than similarly sized control fish.     

Survey of parasites in threatened stocks of coho salmon (Oncorhynchus kisutch) in Oregon by examination of wet tissues and histology

We are conducting studies on the impacts of parasites on Oregon coastal coho salmon (Oncorhynchus kistuch). An essential first step is documenting the geographic distribution of infections, which may be accomplished by using different methods for parasite detection. Thus, the objectives of the current study were to (1) identify parasite species infecting these stocks of coho salmon and document their prevalence, density, and geographic distribution; (2) assess the pathology of these infections; and (3) for the first time, determine the sensitivity and specificity of histology for detecting parasites compared with examining wet preparations for muscle and gill infections. We examined 576 fry, parr, and smolt coho salmon in total by histology. The muscle and gills of 219 of these fish also were examined by wet preparation. Fish were collected from 10 different locations in 2006-2007. We identified 21 different species of parasites in these fish. Some parasites, such as Nanophyetus salmincola and Myxobolus insidiosus, were common across all fish life stages from most basins. Other parasites, such as Apophallus sp., were more common in underyearling fish than smolts and had a more restricted geographic distribution. Additional parasites commonly observed were as follows: Sanguinicola sp., Trichodina truttae , Epistylis sp., Capriniana piscium, and unidentified metacercariae in gills; Myxobolus sp. in brain; Myxidium salvelini and Chloromyxum majori in kidney; Pseudocapillaria salvelini and adult digenean spp. in the intestine. Only a few parasites, such as the unidentified gill metacercariae, elicted overt pathologic changes. Histology had generally poor sensitivity for detecting parasites; however, it had relatively good specificity. We recommend using both methods for studies or monitoring programs requiring a comprehensive assessment of parasite identification, enumeration, and parasite-related pathology.     

Steroid 11-beta-hydroxylase deficiency caused by compound heterozygosity for a novel mutation, p.G314R, in one CYP11B1 allele, and a chimeric CYP11B2/CYP11B1 in the other allele

AIMS: Steroid 11beta-hydroxylase deficiency (11beta-OHD) is the second most common (5-8%) cause of congenital adrenal hyperplasia (CAH), and results from homozygous or compound heterozygous mutations or deletions of the responsible gene CYP11B1. In order to better understand the molecular basis causing 11beta-OHD, we performed detailed studies of CYP11B1 in a newly described patient diagnosed with the classical signs of 11beta-OHD. METHODS:CYP11B1 of the patient was investigated by polymerase chain reaction (PCR), sequencing, restriction fragment length polymorphism (RFLP) analysis, Southern blotting, and transient cell expression. RESULTS: We identified two new mutated alleles in CYP11B1. In one allele CYP11B1 has a g.940G-->C (p.G314R) missense mutation. On the other allele we found a chimeric gene that consists of part of the aldosterone synthase gene (CYP11B2) at exons 1-3 and part of the 11beta-hydroxylase gene (CYP11B1) at exons 4-9. Inin vitro studies, the g.940G-->C (p.G314R) mutation abolished all hydroxylase activity in comparison with the wild-type 11beta-hydroxylase. The chimeric CYP11B2/CYP11B1 protein retained 11beta-hydroxylase enzymatic activity in vitro. CONCLUSION: This case is caused by compound heterozygosity for a nonfunctional missense mutation and a chimeric CYP11B2/CYP11B1 gene with hydroxylase activity that is controlled by the CYP11B2 promoter. The most likely explanation is that the CYP11B2 promoter does not function in the zona fasciculata/reticularis where cortisol is exclusively synthesized.