Single generation epigenetic change in captivity and reinforcement in subsequent generations in a delta smelt (Hypomesus transpacificus) conservation hatchery.

A refugial population of the endangered delta smelt (Hypomesus transpacificus) has been maintained at the Fish Conservation and Culture Laboratory (FCCL) at UC Davis since 2008. Despite intense genetic management, fitness differences between wild and cultured fish have been observed at the FCCL. To investigate the molecular underpinnings of hatchery domestication, we used whole-genome bisulfite sequencing to quantify epigenetic differences between wild and hatchery-origin delta smelt. Differentially methylated regions (DMRs) were identified from 104 individuals by comparing the methylation patterns in different generations of hatchery fish (G1, G2, G3) with their wild parents (G0). We discovered a total of 132 significant DMRs (p < .05) between G0 and G1, 132 significant DMRs between G0 and G2, and 201 significant DMRs between G0 and G3. Our results demonstrate substantial differences in methylation patterns emerged between the wild and hatchery-reared fish in the early generations in the hatchery, with a higher proportion of hypermethylated DMRs in hatchery-reared fish. The rearing environment was found to be a stronger predictor of individual clustering based on methylation patterns than family, sex or generation. Our study indicates a reinforcement of the epigenetic status with successive generations in the hatchery environment, as evidenced by an increase in methylation in hypermethylated DMRs and a decrease in methylation in hypomethylated DMRs over time. Lastly, our results demonstrated heterogeneity in inherited methylation pattern in families across generations. These insights highlight the long-term consequences of hatchery practices on the epigenetic landscape, potentially impacting wild fish populations.

RNA sequencing describes both population structure and plasticity-selection dynamics in a non-model fish.

BACKGROUND: Messenger RNA sequencing is becoming more common in studies of non-model species and is most often used for gene expression-based investigations. However, the method holds potential for numerous other applications as well-including analyses of alternative splicing, population structure, and signatures of selection. To maximize the utility of mRNA data sets, distinct analyses may be combined such as by exploring dynamics between gene expression with signatures of selection in the context of population structure. Here, we compare two published data sets describing two populations of a minnow species endemic to the San Francisco Estuary (Sacramento splittail, Pogonichthys macrolepidotus): a microsatellite data set showing population structure, and an mRNA whole transcriptome data set obtained after the two populations were exposed to a salinity challenge. We compared measures of population structure and genetic variation using single nucleotide polymorphisms (SNPs) called from mRNA from the whole transcriptome sequencing study with those patterns determined from microsatellites. For investigating plasticity and evolution, intra- and inter-population transcriptome plasticity was investigated with differential gene expression, differential exon usage, and gene expression variation. Outlier SNP analysis was also performed on the mRNA data set and signatures of selection and phenotypic plasticity were investigated on an individual-gene basis. RESULTS: We found that mRNA sequencing revealed patterns of population structure consistent with those found with microsatellites, but with lower magnitudes of genetic variation and population differentiation consistent with widespread purifying selection expected when using mRNA. In addition, within individual genes, phenotypic plasticity or signatures of selection were found in almost mutual exclusion (except heatr6, nfu1, slc22a6, sya, and mmp13). CONCLUSIONS: These results show that an mRNA sequencing data set may have multiple uses, including describing population structure and for investigating the mechanistic interplay of evolution and plasticity in adaptation. MRNA sequencing thus complements traditional sequencing methods used for population genetics, in addition to its utility for describing phenotypic plasticity.

Migration-related phenotypic divergence is associated with epigenetic modifications in rainbow trout.

Migration is essential for the reproduction and survival of many animals, yet little is understood about its underlying molecular mechanisms. We used the salmonid Oncorhynchus mykiss to gain mechanistic insight into smoltification, which is a morphological, physiological and behavioural transition undertaken by juveniles in preparation for seaward migration. O. mykiss is experimentally tractable and displays intra- and interpopulation variation in migration propensity. Migratory individuals can produce nonmigratory progeny and vice versa, indicating a high degree of phenotypic plasticity. One potential way that phenotypic plasticity might be linked to variation in migration-related life history tactics is through epigenetic regulation of gene expression. To explore this, we quantitatively measured genome-scale DNA methylation in fin tissue using reduced representation bisulphite sequencing of F2 siblings produced from a cross between steelhead (migratory) and rainbow trout (nonmigratory) lines. We identified 57 differentially methylated regions (DMRs) between smolt and resident O. mykiss juveniles. DMRs were high in magnitude, with up to 62% differential methylation between life history types, and over half of the gene-associated DMRs were in transcriptional regulatory regions. Many of the DMRs encode proteins with activity relevant to migration-related transitions (e.g. circadian rhythm pathway, nervous system development, protein kinase activity). This study provides the first evidence of a relationship between epigenetic variation and life history divergence associated with migration-related traits in any species.

Evaluating environmental DNA detection of a rare fish in turbid water using field and experimental approaches.

Detection sensitivity of aquatic species using environmental DNA (eDNA) generally decreases in turbid water but is poorly characterized. In this study, eDNA detection targeted delta smelt (Hypomesus transpacificus), a critically endangered estuarine fish associated with turbid water. eDNA sampling in the field was first paired with a trawl survey. Species-specific detection using a Taqman qPCR assay showed concordance between the methods, but a weak eDNA signal. Informed by the results of field sampling, an experiment was designed to assess how turbidity and filtration methods influence detection of a rare target. Water from non-turbid (5 NTU) and turbid (50 NTU) estuarine sites was spiked with small volumes (0.5 and 1 mL) of water from a delta smelt tank to generate low eDNA concentrations. Samples were filtered using four filter types: cartridge filters (pore size 0.45 µm) and 47 mm filters (glass fiber, pore size 1.6 µm and polycarbonate, pore sizes 5 and 10 µm). Prefiltration was also tested as an addition to the filtration protocol for turbid water samples. eDNA copy numbers were analyzed using a censored data method for qPCR data. The assay limits and lack of PCR inhibition indicated an optimized assay. Glass fiber filters yielded the highest detection rates and eDNA copies in non-turbid and turbid water. Prefiltration improved detection in turbid water only when used with cartridge and polycarbonate filters. Statistical analysis identified turbidity as a significant effect on detection probability and eDNA copies detected; filter type and an interaction between filter type and prefilter were significant effects on eDNA copies detected, suggesting that particulate-filter interactions can affect detection sensitivity. Pilot experiments and transparent criteria for positive detection could improve eDNA surveys of rare species in turbid environments.

Genomics and 20 years of sampling reveal phenotypic differences between subpopulations of outmigrating Central Valley Chinook salmon.

Intraspecific diversity plays a critical role in the resilience of Chinook salmon populations. California's Central Valley (CV) historically hosted one of the most diverse population complexes of Chinook salmon in the world. However, anthropogenic factors have dramatically decreased this diversity, with severe consequences for population resilience. Here we use next generation sequencing and an archive of thousands of tissue samples collected across two decades during the juvenile outmigration to evaluate phenotypic diversity between and within populations of CV Chinook salmon. To account for highly heterogeneous sample qualities in the archive dataset, we develop and test an approach for population and subpopulation assignments of CV Chinook salmon that allows inclusion of relatively low-quality samples while controlling error rates. We find significantly distinct outmigration timing and body size distributions for each population and subpopulation. Within the archive dataset, spring run individuals that assigned to the Mill and Deer Creeks subpopulation exhibited an earlier and broader outmigration distribution as well as larger body sizes than individuals that assigned to the Butte Creek subpopulation. Within the fall run population, individuals that assigned to the late-fall run subpopulation also exhibited an earlier and broader outmigration distribution and larger body sizes than other fall run fish in our dataset. These results highlight the importance of distinct subpopulations for maintaining remaining diversity in CV Chinook salmon, and demonstrates the power of genomics-based population assignments to aid the study and management of intraspecific diversity.

Critical considerations for communicating environmental DNA science.

The economic and methodological efficiencies of environmental DNA (eDNA) based survey approaches provide an unprecedented opportunity to assess and monitor aquatic environments. However, instances of inadequate communication from the scientific community about confidence levels, knowledge gaps, reliability, and appropriate parameters of eDNA-based methods have hindered their uptake in environmental monitoring programs and, in some cases, has created misperceptions or doubts in the management community. To help remedy this situation, scientists convened a session at the Second National Marine eDNA Workshop to discuss strategies for improving communications with managers. These include articulating the readiness of different eDNA applications, highlighting the strengths and limitations of eDNA tools for various applications or use cases, communicating uncertainties associated with specified uses transparently, and avoiding the exaggeration of exploratory and preliminary findings. Several key messages regarding implementation, limitations, and relationship to existing methods were prioritized. To be inclusive of the diverse managers, practitioners, and researchers, we and the other workshop participants propose the development of communication workflow plans, using RACI (Responsible, Accountable, Consulted, Informed) charts to clarify the roles of all pertinent individuals and parties and to minimize the chance for miscommunications. We also propose developing decision support tools such as Structured Decision-Making (SDM) to help balance the benefits of eDNA sampling with the inherent uncertainty, and developing an eDNA readiness scale to articulate the technological readiness of eDNA approaches for specific applications. These strategies will increase clarity and consistency regarding our understanding of the utility of eDNA-based methods, improve transparency, foster a common vision for confidently applying eDNA approaches, and enhance their benefit to the monitoring and assessment community.

Sall2 is a novel p75NTR-interacting protein that links NGF signalling to cell cycle progression and neurite outgrowth.

By screening a fetal brain two-hybrid library with the death domain of the p75 neurotrophin receptor (NTR), we identified the Sall2 transcription factor as a novel interacting protein. Sall2 is a unique member of the Sall gene family, which is believed to be a tumour suppressor. Here, we show that Sall2 contains a p75NTR interaction domain not found in other Sall proteins and that p75NTR/Sall2 complexes co-immunoprecipitate from brain lysates. NGF dissociates p75NTR/Sall2 complexes and activates TrkA, which has an obligate function in the nuclear translocation of Sall2. NGF also increases Sall2 expression and this is mediated by p75NTR, but may not require TrkA. Depletion of Sall2 from cells decreases the expression and activity of p21(WAF1/CIP1), as well as the ability of NGF to induce growth arrest and the development of neurites. Overexpression of Sall2 activates p21(WAF1/CIP1), induces growth arrest, and promotes neurite outgrowth independently of NGF. These data establish Sall2 as a link between NTRs and transcriptional events that regulate the growth and development of neuronal cells.

Temporal expression patterns of rainbow trout immune-related genes in response to Myxobolus cerebralis exposure.

Infection of salmonids by the myxozoan parasite Myxobolus cerebralis can cause whirling disease, which is responsible for high mortalities in rainbow trout hatcheries and natural populations in the United States. Although considerable research has provided insight into disease pathology, host invasion, and inheritance patterns of resistance, the causal genetic variants and molecular mechanisms underlying host resistance or susceptibility remain elusive. A previous study found that expression changes of specific metallothionein genes following M. cerebralis infection are implicated in whirling disease resistance. The present study examines the dynamic transcriptional response to infection of several upstream regulators of the metallothionein gene family (IL-1ß, KLF2, STAT3, STAT5), along with innate immune response genes (IFN-γ, IRF1 and iNOS). Pathogen loads and gene expression were compared across multiple time points after M. cerebralis exposure to elucidate how resistant and susceptible rainbow trout strains transcriptionally respond to early invasion. IL-1ß, IFN-γ, IRF1, and iNOS all showed increased expression following M. cerebralis exposure for one or both strains across multiple time points. The interferon-related genes IFN-γ and IRF1 had consistently increased expression in the susceptible strain in comparison to the resistant strain, likely due to a less effective initial immune response. STAT3 was the only gene with consistently increased expression in the resistant strain following infection while remaining unchanged in the susceptible strain. Given its pleiotropic effects on immune response, STAT3 is an excellent candidate for future research of whirling disease resistance mechanisms.

Rapid CRISPR-Cas13a genetic identification enables new opportunities for listed Chinook salmon management.

Accurate taxonomic identification is foundational for effective species monitoring and management. When visual identifications are infeasible or inaccurate, genetic approaches provide a reliable alternative. However, these approaches are sometimes less viable (e.g., need for near real-time results, remote locations, funding concerns, molecular inexperience). In these situations, CRISPR-based genetic tools can fill an unoccupied niche between real-time, inexpensive, but error-prone visual identification and more expensive or time-consuming, but accurate genetic identification for taxonomic units that are difficult or impossible to visually identify. Herein, we use genomic data to develop CRISPR-based SHERLOCK assays capable of rapidly (<1 h), accurately (94%-98% concordance between phenotypic and genotypic assignments), and sensitively (detects 1-10 DNA copies/reaction) distinguishing ESA-listed Chinook salmon runs (winter- and spring-run) from each other and from unlisted runs (fall- and late fall-run) in California's Central Valley. The assays can be field deployable with minimally invasive mucus swabbing negating the need for DNA extraction (decreasing costs and labour), minimal and inexpensive equipment needs, and minimal training to conduct following assay development. This study provides a powerful genetic approach for a species of conservation concern that benefits from near real-time management decision-making but also serves as a precedent for transforming how conservation scientists and managers view genetic identification going forward. Once developed, CRISPR-based tools can provide accurate, sensitive, and rapid results, potentially without the prohibitive need for expensive specialty equipment or extensive molecular training. Further adoption of this technology will have widespread value for the monitoring and protection of our natural resources.

Captive-reared Delta Smelt (Hypomesus transpacificus) exhibit high survival in natural conditions using in situ enclosures.

Conservation of endangered fishes commonly includes captive breeding, applied research, and management. Since 1996, a captive breeding program has existed for the federally threatened and California endangered Delta Smelt Hypomesus transpacificus, an osmerid fish endemic to the upper San Francisco Estuary. Although this program serves as a captive refuge population, with experimental releases being initiated to supplement the wild population, it was uncertain how individuals would survive, feed, and maintain condition outside hatchery conditions. We evaluated this and the effects of three enclosure designs (41% open, 63% open, and 63% open with partial outer mesh wrap) on growth, survival, and feeding efficacy of cultured Delta Smelt at two locations (Sacramento River near Rio Vista, CA and in Sacramento River Deepwater Ship Channel) in the wild. Enclosures exposed fish to semi-natural conditions (ambient environmental fluctuations and wild food resources) but prevented escape and predation. After four weeks, survival was high for all enclosure types (94-100%) at both locations. The change in condition and weight was variable between sites, increasing at the first location but decreasing at the second location. Gut content analysis showed that fish consumed wild zooplankton that came into the enclosures. Cumulatively, results show that captive-reared Delta Smelt can survive and forage successfully when housed in enclosures under semi-natural conditions in the wild. When comparing enclosure types, we observed no significant difference in fish weight changes (p = 0.58-0.81 across sites). The success of housing captive-reared Delta Smelt in enclosures in the wild provides preliminary evidence that these fish may be suitable to supplement the wild population in the San Francisco Estuary. Furthermore, these enclosures are a new tool to test the efficacy of habitat management actions or to acclimate fish to wild conditions as a soft release strategy for recently initiated supplementation efforts.

Non-invasive genetic monitoring for the threatened valley elderberry longhorn beetle.

The valley elderberry longhorn beetle (VELB), Desmocerus californicus dimorphus (Coleoptera: Cerambycidae), is a federally threatened subspecies endemic to the Central Valley of California. The VELB range partially overlaps with that of its morphologically similar sister taxon, the California elderberry longhorn beetle (CELB), Desmocerus californicus californicus (Coleoptera: Cerambycidae). Current surveying methods are limited to visual identification of larval exit holes in the VELB/CELB host plant, elderberry (Sambucus spp.), into which larvae bore and excavate feeding galleries. Unbiased genetic approaches could provide a much-needed complementary approach that has more precision than relying on visual inspection of exit holes. In this study we developed a DNA sequencing-based method for indirect detection of VELB/CELB from frass (insect fecal matter), which can be easily and non-invasively collected from exit holes. Frass samples were collected from 37 locations and the 12S and 16S mitochondrial genes were partially sequenced using nested PCR amplification. Three frass-derived sequences showed 100% sequence identity to VELB/CELB barcode references from museum specimens sequenced for this study. Database queries of frass-derived sequences also revealed high similarity to common occupants of old VELB feeding galleries, including earwigs, flies, and other beetles. Overall, this non-invasive approach is a first step towards a genetic assay that could augment existing VELB monitoring and accurately discriminate between VELB, CELB, and other insects. Furthermore, a phylogenetic analysis of 12S and 16S data from museum specimens revealed evidence for the existence of a previously unrecognized, genetically distinct CELB subpopulation in southern California.

Rapid and accurate species identification for ecological studies and monitoring using CRISPR-based SHERLOCK.

One of the most fundamental aspects of ecological research and monitoring is accurate species identification, but cryptic speciation and observer error can confound phenotype-based identification. The CRISPR-Cas toolkit has facilitated remarkable advances in many scientific disciplines, but the fields of ecology and conservation biology have yet to fully embrace this powerful technology. The recently developed CRISPR-Cas13a platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) enables highly accurate taxonomic identification and has all the characteristics needed to transition to ecological and environmental disciplines. Here we conducted a series of "proof of principle" experiments to characterize SHERLOCK's ability to accurately, sensitively and rapidly distinguish three fish species of management interest co-occurring in the San Francisco Estuary that are easily misidentified in the field. We improved SHERLOCK's ease of field deployment by combining the previously demonstrated rapid isothermal amplification and CRISPR genetic identification with a minimally invasive and extraction-free DNA collection protocol, as well as the option of instrument-free lateral flow detection. This approach opens the door for redefining how, where and by whom genetic identifications occur in the future.

Inter-population differences in salinity tolerance of adult wild Sacramento splittail: osmoregulatory and metabolic responses to salinity.

The Sacramento splittail (Pogonichthys macrolepidotus) is composed of two genetically distinct populations endemic to the San Francisco Estuary (SFE). The allopatric upstream spawning habitat of the Central Valley (CV) population connects with the sympatric rearing grounds via relatively low salinity waters, whereas the San Pablo (SP) population must pass through the relatively high-salinity Upper SFE to reach its allopatric downstream spawning habitat. We hypothesize that if migration through SFE salinities to SP spawning grounds is more challenging for adult CV than SP splittail, then salinity tolerance, osmoregulatory capacity, and metabolic responses to salinity will differ between populations. Osmoregulatory disturbances, assessed by measuring plasma osmolality and ions, muscle moisture and Na+-K+-ATPase activity after 168 to 336 h at 11‰ salinity, showed evidence for a more robust osmoregulatory capacity in adult SP relative to CV splittail. While both resting and maximum metabolic rates were elevated in SP splittail in response to increased salinity, CV splittail metabolic rates were unaffected by salinity. Further, the calculated difference between resting and maximum metabolic values, aerobic scope, did not differ significantly between populations. Therefore, improved osmoregulation came at a metabolic cost for SP splittail but was not associated with negative impacts on scope for aerobic metabolism. These results suggest that SP splittail may be physiologically adjusted to allow for migration through higher-salinity waters. The trends in interpopulation variation in osmoregulatory and metabolic responses to salinity exposures support our hypothesis of greater salinity-related challenges to adult CV than SP splittail migration and are consistent with our previous findings for juvenile splittail populations, further supporting our recommendation of population-specific management.

Discovery of genes implicated in whirling disease infection and resistance in rainbow trout using genome-wide expression profiling.

BACKGROUND: Whirling disease, caused by the pathogen Myxobolus cerebralis, afflicts several salmonid species. Rainbow trout are particularly susceptible and may suffer high mortality rates. The disease is persistent and spreading in hatcheries and natural waters of several countries, including the U.S.A., and the economic losses attributed to whirling disease are substantial. In this study, genome-wide expression profiling using cDNA microarrays was conducted for resistant Hofer and susceptible Trout Lodge rainbow trout strains following pathogen exposure with the primary objective of identifying specific genes implicated in whirling disease resistance. RESULTS: Several genes were significantly up-regulated in skin following pathogen exposure for both the resistant and susceptible rainbow trout strains. For both strains, response to infection appears to be linked with the interferon system. Expression profiles for three genes identified with microarrays were confirmed with qRT-PCR. Ubiquitin-like protein 1 was up-regulated over 100 fold and interferon regulating factor 1 was up-regulated over 15 fold following pathogen exposure for both strains. Expression of metallothionein B, which has known roles in inflammation and immune response, was up-regulated over 5 fold in the resistant Hofer strain but was unchanged in the susceptible Trout Lodge strain following pathogen exposure. CONCLUSION: The present study has provided an initial view into the genetic basis underlying immune response and resistance of rainbow trout to the whirling disease parasite. The identified genes have allowed us to gain insight into the molecular mechanisms implicated in salmonid immune response and resistance to whirling disease infection.

Inter-population differences in salinity tolerance and osmoregulation of juvenile wild and hatchery-born Sacramento splittail.

The Sacramento splittail (Pogonichthys macrolepidotus) is a minnow endemic to the highly modified San Francisco Estuary of California, USA and its associated rivers and tributaries. This species is composed of two genetically distinct populations, which, according to field observations and otolith strontium signatures, show largely allopatric distribution patterns as recently hatched juveniles. Juvenile Central Valley splittail are found primarily in the nearly fresh waters of the Sacramento and San Joaquin rivers and their tributaries, whereas San Pablo juveniles are found in the typically higher-salinity waters (i.e. up to 10‰) of the Napa and Petaluma Rivers. As the large salinity differences between young-of-year habitats may indicate population-specific differences in salinity tolerance, we hypothesized that juvenile San Pablo and Central Valley splittail populations differ in their response to salinity. In hatchery-born and wild-caught juvenile San Pablo splittail, we found upper salinity tolerances, where mortalities occurred within 336 h of exposure to 16‰ or higher, which was higher than the upper salinity tolerance of 14‰ for wild-caught juvenile Central Valley splittail. This, in conjunction with slower recovery of plasma osmolality, but not ion levels, muscle moisture or gill Na(+),K(+)-ATPase activity, in Central Valley relative to San Pablo splittail during osmoregulatory disturbance provides some support for our hypothesis of inter-population variation in salinity tolerance and osmoregulation. The modestly improved salinity tolerance of San Pablo splittail is consistent with its use of higher-salinity habitats. Although confirmation of the putative adaptive difference through further studies is recommended, this may highlight the need for population-specific management considerations.

Sequencing improves our ability to study threatened migratory species: Genetic population assignment in California's Central Valley Chinook salmon.

Effective conservation and management of migratory species requires accurate identification of unique populations, even as they mix along their migratory corridors. While telemetry has historically been used to study migratory animal movement and habitat use patterns, genomic tools are emerging as a superior alternative in many ways, allowing large-scale application at reduced costs. Here, we demonstrate the usefulness of genomic resources for identifying single-nucleotide polymorphisms (SNPs) that allow fast and accurate identification of the imperiled Chinook salmon in the Great Central Valley of California. We show that 80 well-chosen loci, drawn from a pool of over 11,500 SNPs developed from restriction site-associated DNA sequencing, can accurately identify Chinook salmon runs and select populations within run. No other SNP panel for Central Valley Chinook salmon has been able to achieve the high accuracy of assignment we show here. This panel will greatly improve our ability to study and manage this ecologically, economically, and socially important species and demonstrates the great utility of using genomics to study migratory species.

Ten real-time PCR assays for detection of fish predation at the community level in the San Francisco Estuary-Delta.

The effect of predation on native fish by introduced species in the San Francisco Estuary-Delta (SFE) has not been thoroughly studied despite its potential to impact species abundances. Species-specific quantitative PCR (qPCR) is an accurate method for identifying species from exogenous DNA samples. Quantitative PCR assays can be used for detecting prey in gut contents or faeces, discriminating between cryptic species, or detecting rare aquatic species. We designed ten TaqMan qPCR assays for fish species from the SFE watershed most likely to be affected by non-native piscivores. The assays designed are highly specific, producing no signal from co-occurring or related species, and sensitive, with a limit of detection between 3.2 and 0.013 pg/µL of target DNA. These assays will be used in conjunction with a high-throughput qPCR platform to compare predation rates between native and non-native piscivores and assess the impacts of predation in the system.

Transcriptional Response to Acute Thermal Exposure in Juvenile Chinook Salmon Determined by RNAseq.

Thermal exposure is a serious and growing challenge facing fish species worldwide. Chinook salmon (Oncorhynchus tshawytscha) living in the southern portion of their native range are particularly likely to encounter warmer water due to a confluence of factors. River alterations have increased the likelihood that juveniles will be exposed to warm water temperatures during their freshwater life stage, which can negatively impact survival, growth, and development and pose a threat to dwindling salmon populations. To better understand how acute thermal exposure affects the biology of salmon, we performed a transcriptional analysis of gill tissue from Chinook salmon juveniles reared at 12° and exposed acutely to water temperatures ranging from ideal to potentially lethal (12° to 25°). Reverse-transcribed RNA libraries were sequenced on the Illumina HiSeq2000 platform and a de novo reference transcriptome was created. Differentially expressed transcripts were annotated using Blast2GO and relevant gene clusters were identified. In addition to a high degree of downregulation of a wide range of genes, we found upregulation of genes involved in protein folding/rescue, protein degradation, cell death, oxidative stress, metabolism, inflammation/immunity, transcription/translation, ion transport, cell cycle/growth, cell signaling, cellular trafficking, and structure/cytoskeleton. These results demonstrate the complex multi-modal cellular response to thermal stress in juvenile salmon.

Survival and reproduction of Myxobolus cerebralis-resistant rainbow trout introduced to the Colorado river and increased resistance of age-0 progeny.

Myxobolus cerebralis caused severe declines in rainbow trout populations across Colorado following its introduction in the 1980s. One promising approach for the recovery of Colorado's rainbow trout populations has been the production of rainbow trout that are genetically resistant to the parasite. We introduced one of these resistant crosses, known as the GR×CRR (cross between the German Rainbow [GR] and Colorado River Rainbow [CRR] trout strains), to the upper Colorado River. The abundance, survival, and growth of the stocked GR×CRR population was examined to determine if GR×CRRs had contributed offspring to the age-0 population, and determine whether these offspring displayed increased resistance and survival characteristics compared to their wild CRR counterparts. Apparent survival of the introduced GR×CRR over the entire study period was estimated to be 0.007 (±0.001). Despite low survival of the GR×CRRs, age-0 progeny of the GR×CRR were encountered in years 2008 through 2011. Genetic assignments revealed a shift in the genetic composition of the rainbow trout fry population over time, with CRR fish comprising the entirety of the fry population in 2007, and GR-cross fish comprising nearly 80% of the fry population in 2011. A decrease in average infection severity (myxospores fish-1) was observed concurrent with the shift in the genetic composition of the rainbow trout fry population, decreasing from an average of 47,708 (±8,950) myxospores fish-1 in 2009 to 2,672 (±4,379) myxospores fish-1 in 2011. Results from this experiment suggest that the GR×CRR can survive and reproduce in rivers with a high prevalence of M. cerebralis. In addition, reduced myxospore burdens in age-0 fish indicated that stocking this cross may ultimately lead to an overall reduction in infection prevalence and severity in the salmonid populations of the upper Colorado River.

TaqMan assays for the genetic identification of delta smelt (Hypomesus transpacificus) and wakasagi smelt (Hypomesus nipponensis).

We have developed species-specific TaqMan assays for two California fish species, the threatened delta smelt (Hypomesus transpacificus) and the introduced wakasagi smelt (Hypomesus nipponensis). The assays are capable of correctly identifying each species with 100% accuracy, with no cross-species amplification. We anticipate these assays will prove useful for future scientific studies requiring genetic species identification (e.g. predation of smelt) or monitoring (e.g. detection of delta smelt near water diversions).