Melinda R. Baerwald

A major effect quantitative trait locus for whirling disease resistance identified in rainbow trout (Oncorhynchus mykiss)

Whirling disease, caused by the pathogen Myxobolus cerebralis, leads to skeletal deformation, neurological impairment and under certain conditions, mortality of juvenile salmonid fishes. The disease has impacted the propagation and survival of many salmonid species over six continents, with particularly negative consequences for rainbow trout. To assess the genetic basis of whirling disease resistance in rainbow trout, genome-wide mapping was initiated using a large outbred F2 rainbow trout family (n=480) and results were confirmed in three additional outbred F2 families (n=96 per family). A single quantitative trait locus (QTL) region on chromosome Omy9 was identified in the large mapping family and confirmed in all additional families. This region explains 50–86% of the phenotypic variance across families. Therefore, these data establish that a single QTL region is capable of explaining a large percentage of the phenotypic variance contributing to whirling disease resistance. This is the first genetic region discovered that contributes directly to the whirling disease phenotype and the finding moves the field closer to a mechanistic understanding of resistance to this important disease of salmonid fish.

Otolith Microchemistry Provides Information Complementary to Microsatellite DNA for a Migratory Fish

Abstract We investigated the ability of otolith microchemistry to discriminate natal habitats of the splittail Pogonichthys macrolepidotus, a migratory cyprinid endemic to the San Francisco Estuary, California. Splittails are broadly distributed in the brackish and freshwater portions of the lower estuary and make long-distance upstream migrations during winter to rivers and floodplains for spawning. We found that the ratios of Sr: Ca and 87Sr: 86Sr in the otoliths (ascertained by laser ablation inductively coupled mass spectrometry) of age-0 fish collected from natal habitats significantly varied among four primary spawning rivers. Based on these two constituents, quadratic discriminant function analysis correctly classified 71% of the fish to their natal rivers. Recent work with microsatellite DNA indicates that splittails from these same rivers represent two genetically distinct populations. Thus, integrating data obtained from otolith microchemistry and microsatellite DNA can provide complementary inf...

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

BackgroundWhirling 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.ResultsSeveral 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.ConclusionThe 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.

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.

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.

Genetic analysis reveals two distinct Sacramento splittail (Pogonichthys macrolepidotus) populations

The Sacramento splittail is an endemic cyprinid fish of the San Francisco estuary and its tributaries, which is a highly manipulated, constantly changing ecosystem. Splittail is the only extant member of its genus and is listed as a federal and California Species of Special Concern due to uncertainties regarding long-term abundance trends. Determining population structure for splittail is important because unique populations may contain different adaptive genetic variation, which can allow one population to persist through future environmental or demographic stochasticity while others become extirpated. To assess splittail population structure, 13 microsatellite markers were used to genotype 489 young-of-year splittail from five major rivers draining into the estuary: Cosumnes, Napa, Petaluma, Sacramento, and San Joaquin Rivers. Two genetically distinct populations were found to exist within our study region; one largely comprised of splittail collected from the Petaluma and Napa Rivers and the second comprised of splittail collected from tributaries in Californiaȁ9s Central Valley: Cosumnes, Sacramento, and San Joaquin Rivers. These results were replicated in two consecutive years with both distance and model-based algorithms. The genetic distinction between these two populations appears correlated with salinity differences between migratory regions and spawning grounds. Splittail from the Petaluma River exhibited a significantly higher degree of differentiation from the Central Valley population than did Napa River splittail. Our results suggest on-going monitoring programs are probably highly biased towards sampling splittail from the Central Valley population. Understanding population dynamics of splittail could be improved if monitoring programs were expanded to include all splittail populations.

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.

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-1β, 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-1β, 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.

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.

Characterization of 24 Microsatellite Loci in Delta Smelt, Hypomesus transpacificus, and Their Cross-Species Amplification in Two Other Smelt Species of the Osmeridae Family

We characterized 24 polymorphic tetranucleotide microsatellite loci for delta smelt (Hypomesus transpacificus) endemic to the San Francisco Bay Estuary, CA, USA. Screening of samples (n = 30) yielded two to 26 alleles per locus with observed levels of heterozygosity ranging from 0.17 to 1.0. Only one locus deviated from Hardy–Weinberg equilibrium, suggesting these individuals originate from a single panmictic population. Linkage disequilibrium was found in two pairs of loci after excluding the locus out of Hardy–Weinberg equilibrium. Twenty‐two primer pairs cross‐amplified in wakasagi smelt (Hypomesus nipponensis), and 15 primer pairs cross‐amplified in longfin smelt (Spirinchus thaleichthys).