Evidence of a hydraulically challenging reach serving as a barrier for the upstream migration of infection-burdened adult steelhead.

Anadromous fishes such as steelhead trout, Oncorhynchus mykiss, are exposed to a suite of infectious agents and migratory challenges during their freshwater migrations. We assessed infectious agent load and richness and immune system gene expression in gill tissue of Bulkley River (British Columbia, CA) steelhead captured at and upstream of a migratory barrier to evaluate whether infectious burdens impacted migration success. We further considered the potential influences of water temperature, sex and fish size on host infectious agents and transcription profiles. There were eight infectious agents detected in steelhead gill tissue, with high prevalence of the bacteria Candidatus Branchiomonas cysticola (80%) and Flavobacterium psychrophilum (95%) and the microparasite Sphaerothecum destruens (53%). Fish sampled at the falls had significantly greater relative loads of Ca. B. cysticola and F. psychrophilum, higher infectious agent richness and differential gene expression compared to fish captured upstream. Flavobacterium psychrophilum was only associated with immune gene expression (particularly humoral immunity) of fish sampled at the falls, while water temperature was positively correlated with genes involved in the complement system, metabolic stress and oxidative stress for fish captured upstream. This work highlights interesting differences in agent-host interactions across fisheries and suggests that hydraulic barriers may reduce the passage of fish with the heaviest infectious agent burdens, emphasizing the selective role of areas of difficult passage. Further, this work expands our knowledge of infectious agent prevalence in wild salmonids and provides insight into the relationships between infectious agents and host physiology.

Olfactory gene expression in migrating adult sockeye salmon Oncorhynchus nerka.

Expression of 12 olfactory genes was analysed in adult sockeye salmon Oncorhynchus nerka nearing spawning grounds and O. nerka that had strayed from their natal migration route. Variation was found in six of these genes, all of which were olfc olfactory receptors and had lower expression levels in salmon nearing spawning grounds. The results may reflect decreased sensitivity to natal water olfactory cues as these fish are no longer seeking the correct migratory route. The expression of olfactory genes during the olfactory-mediated spawning migration of Pacific salmon Oncorhynchus spp. is largely unexplored and these findings demonstrate a link between migratory behaviours and olfactory plasticity that provides a basis for future molecular research on salmon homing.

Facing warm temperatures during migration: cardiac mRNA responses of two adult Oncorhynchus nerka populations to warming and swimming challenges.

The main findings of the current study were that exposing adult sockeye salmon Onchorhynchus nerka to a warm temperature that they regularly encounter during their river migration induced a heat shock response at an mRNA level, and this response was exacerbated with forced swimming. Similar to the heat shock response, increased immune defence-related responses were also observed after warm temperature treatment and with a swimming challenge in two different populations (Chilko and Nechako), but with some important differences. Microarray analyses revealed that 347 genes were differentially expressed between the cold (12-13° C) and warm (18-19° C) treated fish, with stress response (GO:0006950) and response to fungus (GO:0009620) elevated with warm treatment, while expression for genes involved in oxidative phosphorylation (GO:0006119) and electron transport chain (GO:0022900) elevated for cold-treated fish. Analysis of single genes with real-time quantitative PCR revealed that temperature had the most significant effect on mRNA expression levels, with swimming and population having secondary influences. Warm temperature treatment for the Chilko population induced expression of heat shock protein (hsp) 90α, hsp90ß and hsp30 as well as interferon-inducible protein. The Nechako population, which is known to have a narrower thermal tolerance window than the Chilko population, showed even more pronounced stress responses to the warm treatment and there was significant interaction between population and temperature treatment for hsp90ß expression. Moreover, significant interactions were noted between temperature treatment and swimming challenge for hsp90α and hsp30, and while swimming challenge alone increased expression of these hsps, the expression levels were significantly elevated in warm-treated fish swum to exhaustion. In conclusion, it seems that adult O. nerka currently encounter conditions that induce several cellular defence mechanisms during their once-in-the-lifetime migration. As river temperatures continue to increase, it remains to be seen whether or not these cellular defences provide sufficient protection for all O. nerka populations.

Major histocompatibility complex and kin discrimination in Atlantic salmon and brook trout.

Many species of salmonids can discriminate kin from unrelated conspecifics using olfactory cues. In this study, we determined the role of the major histocompatibility complex (MHC) in kin discrimination by juvenile Atlantic salmon (Salmo salar) and brook trout (Salvelinus fontinalis). Genetic variation at the highly polymorphic exon coding for peptide-binding region of an MHC class II gene was studied using polymerase chain reaction and denaturing gradient gel electrophoresis. Experiments compared discrimination ability based on MHC haplotypes both within and among kin and non-kin groups. Juveniles chose kin sharing both alleles over kin sharing no alleles. Juveniles also preferred non-kin sharing both alleles to non-kin sharing no alleles. These data suggest that the MHC class II gene influence kin discrimination in juvenile Atlantic salmon and brook trout. The influence of additional genes was also apparent in trials where juveniles were able to recognize kin sharing no alleles over non-kin sharing no alleles. However, the inability of juveniles to discriminate between kin sharing no alleles and non-kin sharing either one or both alleles indicates that MHC is as potent as the rest of the genome in producing distinguishable odours.

Geographic heterogeneity in natural selection on an MHC locus in sockeye salmon.

Balancing selection maintains high levels of polymorphism and heterozygosity in genes of the MHC (major histocompatibility complex) of vertebrate organisms, and promotes long evolutionary persistence of individual alleles and strongly differentiated allelic lineages. In this study, genetic variation at the MHC class II DAB-beta1 locus was examined in 31 populations of sockeye salmon (Oncorhynchus nerka) inhabiting the Fraser River drainage of British Columbia, Canada. Twenty-five percent of variation at the locus was partitioned among sockeye populations, as compared with 5% at neutral genetic markers. Geographic heterogeneity of balancing selection was detected among four regions in the Fraser River drainage and among lake systems within regions. High levels of beta1 allelic diversity and heterozygosity, as well as distributions of alleles and allelic lineages that were more even than expected for a neutral locus, indicated the presence of balancing selection in populations throughout much of the interior Fraser drainage. However, proximate populations in the upper Fraser region, and four of six populations from the lower Fraser drainage, exhibited much lower levels of genetic diversity and had beta1 allele frequency distributions in conformance with those expected for a neutral locus, or a locus under directional selection. Pair-wise FST values for beta1 averaged 0.19 and tended to exceed the corresponding values estimated for neutral loci at all levels of population structure, although they were lower among populations experiencing balancing selection than among other populations. The apparent heterogeneity in selection resulted in strong genetic differentiation between geographically proximate populations with and without detectable levels of balancing selection, in stark contrast to observations at neutral loci. The strong partitioning and complex structure of beta1 diversity within and among sockeye populations on a small geographic scale illustrates the value of incorporating adaptive variation into conservation planning for the species.

The isolation of a novel metallothionein-related cDNA expressed in somatic and zygotic embryos of Douglas-fir: regulation by ABA, osmoticum, and metal ions.

To isolate genes which are expressed preferentially during embryogenesis, a Douglas-fir embryogenesis cDNA library was constructed and differentially screened with cDNA probes made with mRNA from developing and mature embryos, respectively. The cDNA clone PM 2.1 was isolated based on its abundance in developing seeds and absence in mature seeds, and its predicted amino acid sequence was shown to have structural features characteristic of plant MT-like proteins. Alignment of the PM 2.1 predicted amino acid sequence with other plant MT-like protein sequences revealed a general paucity of Cys and Cys-Xaa-Cys sequences and the presence of novel serine residues within the conserved Cys-Xaa-Cys motifs in the C-terminal domain. The consensus sequence following the Cys-poor spacer in type 2 MT-like proteins, CXCXXXCXCXXCXCX, was modified in PM 2.1 to CXSXXXSXYXX-XCX. Phylogenetic analysis supported PM 2.1 was distinct from other MT and grouped with MT-like proteins from Arabidopsis (OEST), rice (AEST) and kiwifruit (AD1), which do not belong to type 1 or 2. The PM 2.1 gene was expressed in somatic and zygotic embryos, in haploid maternal tissue, as well as in hormone- and metal-treated seeds and seedlings. The PM 2.1 transcripts were detected in the needles of 14-week-old seedlings, but not the root tissue or mature pollen. The expression of the PM 2.1 gene in embryos was dependent upon ABA and osmoticum and in seedlings was differentially modulated by metals, suggesting a role of the PM 2.1 gene product in the control of microelement availability during Douglas-fir seed development and germination. The novel structural features, and the developmental, hormonal and metal modulation of PM 2.1 expression, are evidence for a new type of MT-related protein in plants.

Post-termination-induced and hormonally dependent expression of low-molecular-weight heat shock protein genes in Douglas fir.

We have isolated and sequenced two cDNA clones (PM 18.2A; PM 18.2B) from Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) which encode for the low-molecular-weight heat shock proteins (LMW HSPs) of 18.2 kDa. The predicted amino acid sequences of the two Douglas fir proteins are 97.5% identical. A phylogenetic tree of class I LMW HSPs showed that the PM LMW HSPs are found within a subgroup consisting exclusively of dicot species indicating that class I LMW HSPs evolved from a common ancestor predating the divergence of gymnosperms and angiosperms. Northern blots of RNA from dry, imbibed, stratified and germinated seeds revealed a notable induction of LMW HSP transcripts during post-germination and early seedling growth. Unlike previous reports, the expression of these HSPs appears to be primarily restricted to seedlings as mRNA transcripts were detected at very low levels during seed development and desiccation. Maximum induction of LMW HSPs in seedlings occurred during heat shock treatment at 38-40 degrees C, whereas cold shock or wounding failed to induce HSP transcripts. The transcription of HSP genes is up regulated by GA, MeJA and auxin and is down regulated by ABA. Methyl jasmonate treatment induced expression of these genes in dormant seeds of Douglas fir. The expression of class I cytoplasmic LMW HSPs in seedlings and their regulation by plant growth regulators suggests specific roles in plant development other than desiccation tolerance.