The impact of primary colonizers on the community composition of river biofilm.

As a strategy for minimizing microbial infections in fish hatcheries, we have investigated how putatively probiotic bacterial populations influence biofilm formation. All surfaces that are exposed to the aquatic milieu develop a microbial community through the selective assembly of microbial populations into a surface-adhering biofilm. In the investigations reported herein, we describe laboratory experiments designed to determine how initial colonization of a surface by nonpathogenic isolates from sturgeon eggs influence the subsequent assembly of populations from a pelagic river community, into the existing biofilm. All eight of the tested strains altered the assembly of river biofilm in a strain-specific manner. Previously formed isolate biofilm was challenged with natural river populations and after 24 hours, two strains and two-isolate combinations proved highly resistant to invasion, comprising at least 80% of the biofilm community, four isolates were intermediate in resistance, accounting for at least 45% of the biofilm community and two isolates were reduced to 4% of the biofilm community. Founding biofilms of Serratia sp, and combinations of Brevundimonas sp.-Hydrogenophaga sp. and Brevundimonas sp.-Acidovorax sp. specifically blocked populations of Aeromonas and Flavobacterium, potential fish pathogens, from colonizing the biofilm. In addition, all isolate biofilms were effective at blocking invading populations of Arcobacter. Several strains, notably Deinococcus sp., recruited specific low-abundance river populations into the top 25 most abundant populations within biofilm. The experiments suggest that relatively simple measures can be used to control the assembly of biofilm on the eggs surface and perhaps offer protection from pathogens. In addition, the methodology provides a relatively rapid way to detect potentially strong ecological interactions between bacterial populations in the formation of biofilms.

Recovery of the gut microbiome following enteric infection and persistence of antimicrobial resistance genes in specific microbial hosts.

Enteric pathogens cause widespread foodborne illness and are increasingly resistant to important antibiotics yet their ecological impact on the gut microbiome and resistome is not fully understood. Herein, shotgun metagenome sequencing was applied to stool DNA from 60 patients (cases) during an enteric bacterial infection and after recovery (follow-ups). Overall, the case samples harbored more antimicrobial resistance genes (ARGs) with greater resistome diversity than the follow-up samples (p < 0.001), while follow-ups had more diverse gut microbiota (p < 0.001). Although cases were primarily defined by genera Escherichia, Salmonella, and Shigella along with ARGs for multi-compound and multidrug resistance, follow-ups had a greater abundance of Bacteroidetes and Firmicutes phyla and resistance genes for tetracyclines, macrolides, lincosamides, and streptogramins, and aminoglycosides. A host-tracking analysis revealed that Escherichia was the primary bacterial host of ARGs in both cases and follow-ups, with a greater abundance occurring during infection. Eleven distinct extended spectrum beta-lactamase (ESBL) genes were identified during infection, with some detectable upon recovery, highlighting the potential for gene transfer within the community. Because of the increasing incidence of disease caused by foodborne pathogens and their role in harboring and transferring resistance determinants, this study enhances our understanding of how enteric infections impact human gut ecology.

Evaluating the utility of effective breeding size estimates for monitoring sea lamprey spawning abundance.

Sea lamprey (Petromyzon marinus) is an invasive species that is a significant source of mortality for populations of valued fish species across the North American Great Lakes. Large annual control programs are needed to reduce the species' impacts; however, the number of successfully spawning adults cannot currently be accurately assessed. In this study, effective breeding size (N b) and the minimum number of spawning adults (N s) were estimated for larval cohorts from 17 tributaries across all five Great Lakes using single nucleotide polymorphisms (SNP) genotyped via RAD-capture sequencing. Reconstructed larval pedigrees showed substantial variability in the size and number of full- and half-sibling groups, N b (<1-367), and N s (5-545) among streams. Generalized linear models examining the effects of stream environmental characteristics and aspects of sampling regimes on N b and N s estimates identified sample size, the number of sampling sites, and drainage area as important factors predicting N b and N s. Correlations between N b, N s, and capture-mark-recapture estimates of adult census size (N c) increased when streams with small sample sizes (n < 50) were removed. Results collectively indicate that parameters estimated from genetic data can provide valuable information on spawning adults in a river system, especially if sampling regimes are standardized and physical stream covariates are included.

Individual-based analyses reveal effects of behavioral and demographic variables associated with multi-annual reproductive success of male and female lake sturgeon.

Quantifying effects of individual attributes and population demographic characteristics that affect inter- and intrasexual interactions and adult reproductive success, and the spatial and temporal contexts in which they are expressed is important to effective species management. Multi-year individual-based analyses using genetically determined parentage allowed the examination of variables associated with the reproductive success of male and female lake sturgeon (Acipenser fulvescens) in the well-studied population in Black Lake, Michigan, USA. Spawning lake sturgeon (a total of 599 individuals where many were captured more than once based on 1024 total captures) and larvae (N = 3436) were genotyped during each of seven consecutive years (2001-2007). Factors associated with individual reproductive success differed between sexes and varied among spawning groups within a year and among years depending on spawning date (higher reproductive success earlier in the season for females) and spawning locations (higher reproductive success in upstream spawning zones for females). Female reproductive success increased nonlinearly with increasing body size. Male reproductive success increased with increasing residence time in spawning areas and, to a modest degree, with increasing body size in a nonlinear fashion. Fixed effects of repeatability in spawn timing and location across years led to consistently higher or lower reproductive success for females. Results identified factors, including time spent at spawning areas by males and intersexual encounters and mate number, that contributed to higher interindividual variance in reproductive success and affected population levels of recruitment, the degree of subpopulation genetic structure (lack of isolation by time), and effective population size.

Behavioral evidence of olfactory imprinting during embryonic and larval stages in lake sturgeon.

Many migratory fishes are thought to navigate to natal streams using olfactory cues learned during early life stages. However, direct evidence for early-life olfactory imprinting is largely limited to Pacific salmon, and other species suspected to imprint show life history traits and reproductive strategies that raise uncertainty about the generality of the salmonid-based conceptual model of olfactory imprinting in fishes. Here, we studied early-life olfactory imprinting in lake sturgeon (Acipenser fulvescens), which have a life cycle notably different from Pacific salmon, but are nonetheless hypothesized to home via similar mechanisms. We tested one critical prediction of the hypothesis that early-life olfactory imprinting guides natal homing in lake sturgeon: that exposure to odorants during early-life stages results in increased activity when exposed to those odorants later in life. Lake sturgeon were exposed to artificial odorants (phenethyl alcohol and morpholine) during specific developmental windows and durations (limited to the egg, free-embryo, exogenous feeding larvae and juvenile stages), and later tested as juveniles for behavioral responses to the odorants that were demonstrative of olfactory memory. Experiments revealed that lake sturgeon reared in stream water mixed with artificial odorants for as little as 7 days responded to the odorants in behavioral assays over 50 days after the initial exposure, specifically implicating the free-embryo and larval stages as critical imprinting periods. Our study provides evidence for olfactory imprinting in a non-salmonid fish species, and supports further consideration of conservation tactics such as stream-side rearing facilities that are designed to encourage olfactory imprinting to targeted streams during early life stages. Continued research on lake sturgeon can contribute to a model of olfactory imprinting that is more generalizable across diverse fish species and will inform conservation actions for one of the world's most imperiled fish taxonomic groups.

Determination of thyroid hormones in lake sturgeon (Acipenser fulvescens) tissues at different developmental stages using a sensitive liquid chromatography-mass spectrometry method.

Thyroid hormones (TH) are known to play an important role in the growth and development of vertebrates. In fish species, TH regulates the larval-juvenile metamorphosis, and is crucial for development during early life stages. Monitoring the variations in TH levels at different life stages can provide insights into the regulation of metamorphosis and fish development. In this study, we developed an extremely sensitive method for the quantification of thyroxine (T4), triiodothyronine (T3), and reverse-triiodothyronine (rT3), in lake sturgeon (Acipenser fulvescens) tissues from eggs, free embryos, larvae, and juveniles. The target compounds were extracted by an enzymatic digestion method, followed by protein precipitation. Further cleanup was performed by liquid-liquid extraction (LLE) and solid phase extraction (SPE) using SampliQ OPT cartridges. The liquid-chromatography tandem mass spectrometry (LC-MS/MS) method used to quantify TH compounds showed remarkably high sensitivity with the limit of detection (LOD) and the limit of quantification (LOQ) ranging from < 1 pg/mL to 10 pg/mL and linearity in the range of 10-50,000 pg/mL. This method was validated for tissue samples across several early developmental stages and was checked for intra- and inter-day accuracy (78.3-111.2 %) and precision (0.1-4.9 %), matrix effect (75.4-134.1 %), and recovery (41.2-69.0 %). The method was successfully applied for the quantification and comparison of T4, T3 and rT3 in hatchery raised lake sturgeon samples collected at unique time points (i.e., days post fertilization dpf) including fertilized eggs (11 dpf), free embryos (14 dpf), larvae (22 dpf), juveniles (40 dpf) and older juveniles (74 dpf). With modifications, this method could be applied to other species important for agriculture or conservation.

First Isolation of a Herpesvirus (Family Alloherpesviridae) from Great Lakes Lake Sturgeon (Acipenser fulvescens).

The lake sturgeon (Acipenser fulvescens; LST) is the only native sturgeon species in the Great Lakes (GL), but due to multiple factors, their current populations are estimated to be <1% of historical abundances. Little is known about infectious diseases affecting GL-LST in hatchery and wild settings. Therefore, a two-year disease surveillance study was undertaken, resulting in the detection and first in vitro isolation of a herpesvirus from grossly apparent cutaneous lesions in wild adult LST inhabiting two GL watersheds (Erie and Huron). Histological and ultrastructural examination of lesions revealed proliferative epidermitis associated with herpesvirus-like virions. A virus with identical ultrastructural characteristics was recovered from cells inoculated with lesion tissues. Partial DNA polymerase gene sequencing placed the virus within the Family Alloherpesviridae, with high similarity to a lake sturgeon herpesvirus (LSHV) from Wisconsin, USA. Genomic comparisons revealed ~84% Average Nucleotide Identity between the two isolates, leading to the proposed classification of LSHV-1 (Wisconsin) and LSHV-2 (Michigan) for the two viruses. When naïve juvenile LST were immersion-exposed to LSHV-2, severe disease and ~33% mortality occurred, with virus re-isolated from representative skin lesions, fulfilling Rivers' postulates. Results collectively show LSHV-2 is associated with epithelial changes in wild adult LST, disease and mortality in juvenile LST, and is a potential threat to GL-LST conservation.

A new GTSeq resource to facilitate multijurisdictional research and management of walleye Sander vitreus.

Conservation and management professionals often work across jurisdictional boundaries to identify broad ecological patterns. These collaborations help to protect populations whose distributions span political borders. One common limitation to multijurisdictional collaboration is consistency in data recording and reporting. This limitation can impact genetic research, which relies on data about specific markers in an organism's genome. Incomplete overlap of markers between separate studies can prevent direct comparisons of results. Standardized marker panels can reduce the impact of this issue and provide a common starting place for new research. Genotyping-in-thousands (GTSeq) is one approach used to create standardized marker panels for nonmodel organisms. Here, we describe the development, optimization, and early assessments of a new GTSeq panel for use with walleye (Sander vitreus) from the Great Lakes region of North America. High genome-coverage sequencing conducted using RAD capture provided genotypes for thousands of single nucleotide polymorphisms (SNPs). From these markers, SNP and microhaplotype markers were chosen, which were informative for genetic stock identification (GSI) and kinship analysis. The final GTSeq panel contained 500 markers, including 197 microhaplotypes and 303 SNPs. Leave-one-out GSI simulations indicated that GSI accuracy should be greater than 80% in most jurisdictions. The false-positive rates of parent-offspring and full-sibling kinship identification were found to be low. Finally, genotypes could be consistently scored among separate sequencing runs >94% of the time. Results indicate that the GTSeq panel that we developed should perform well for multijurisdictional walleye research throughout the Great Lakes region.

Aquatic insects differentially affect lake sturgeon larval phenotypes and egg surface microbial communities.

Documentation of how interactions among members of different stream communities [e.g., microbial communities and aquatic insect taxa exhibiting different feeding strategies (FS)] collectively influence the growth, survival, and recruitment of stream fishes is limited. Considerable spatial overlap exists between early life stages of stream fishes, including species of conservation concern like lake sturgeon (Acipenser fulvescens), and aquatic insects and microbial taxa that abundantly occupy substrates on which spawning occurs. Habitat overlap suggests that species interactions across trophic levels may be common, but outcomes of these interactions are poorly understood. We conducted an experiment where lake sturgeon eggs were fertilized and incubated in the presence of individuals from one of four aquatic insect FS taxa including predators, facultative and obligate-scrapers, collector-filterers/facultative predators, and a control (no insects). We quantified and compared the effects of different insect taxa on the taxonomic composition and relative abundance of egg surface bacterial and lower eukaryotic communities, egg size, incubation time to hatch, free embryo body size (total length) at hatch, yolk-sac area, (a measure of resource utilization), and percent survival to hatch. Mean egg size varied significantly among insect treatments. Eggs exposed to predators had a lower mean percent survival to hatch. Eggs exposed to predators had significantly shorter incubation periods. At hatch, free embryos exposed to predators had significantly smaller yolk sacs and total length. Multivariate analyses revealed that egg bacterial and lower eukaryotic surface community composition varied significantly among insect treatments and between time periods (1 vs 4 days post-fertilization). Quantitative PCR documented significant differences in bacterial 16S copy number, and thus abundance on egg surfaces varied across insect treatments. Results indicate that lethal and non-lethal effects associated with interactions between lake sturgeon eggs and free embryos and aquatic insects, particularly predators, contributed to lake sturgeon trait variability that may affect population levels of recruitment.

Compositional Dynamics of Gastrointestinal Tract Microbiomes Associated with Dietary Transition and Feeding Cessation in Lake Sturgeon Larvae.

Compromised nutritional conditions associated with dietary transitions and feeding cessation in the wild and during fish aquaculture operations are common and can impact growth and survival. These effects are especially prevalent during early ontogenetic stages. We quantified phenotypic and GI tract microbial community responses with an emphasis on protease-producing bacteria of lake sturgeon (Acipenser fulvescens) larvae, a species of aquacultural and conservational importance. To quantify responses associated with experimental food transition and feeding cessation, we performed a 36-day feeding experiment using two treatments: control and diet transition. However, larvae in the diet transition treatment failed to undergo transition and ceased feeding. Larvae in the diet transition treatment exhibited lower growth (total length and body weight) and survival than control larvae. Treatment had a greater effect than ontogenetic changes on taxonomic composition and diversity of the GI tract microbial community. Proteobacteria dominated the GI tract microbial community of the diet transition larvae whereas Firmicutes dominated the GI tracts of control larvae. Most of the 98 identified protease-producing isolates in both treatments were from genera Pseudomonas and Aeromonas: taxonomic groups that include known fish pathogens. Overall, failing to transition diets affected responses in growth and GI tract microbiome composition and diversity, with the later dysbiosis being an indicator of morbidity and mortality in larval lake sturgeon. Thus, microbiological interrogations can characterize responses to dietary regimes. The results can inform fish culturalists and microbiologists of the importance of dietary practices consistent with the establishment and maintenance of healthy GI tract microbiota and optimal growth during early ontogeny.

Changes in Lake Sturgeon Gut Microbiomes Relative to Founding Origin and in Response to Chemotherapeutant Treatments.

Antibiotics, drugs, and chemicals (collectively referred to as chemotherapeutants) are widely embraced in fish aquaculture as important tools to control or prevent disease outbreaks. Potential negative effects include changes in microbial community composition and diversity during early life stages, which can reverse the beneficial roles of gut microbiota for the maintenance of host physiological processes and homeostatic regulation. We characterized the gut microbial community composition and diversity of an ecologically and economically important fish species, the lake sturgeon (Acipenser fulvescens), during the early larval period in response to weekly treatments using chemotherapeutants commonly used in aquaculture (chloramine-T, hydrogen peroxide, and NaCl2 followed by hydrogen peroxide) relative to untreated controls. The effects of founding microbial community origin (wild stream vs. hatchery water) were also evaluated. Gut communities were quantified using massively parallel next generation sequencing based on the V4 region of the 16S rRNA gene. Members of the phylum Firmicutes (principally unclassified Clostridiales and Clostridium_sensu_stricto) and Proteobacteria were the dominant taxa in all gut samples regardless of treatment. The egg incubation environment (origin) and its interaction with chemotherapeutant treatment were significantly associated with indices of microbial taxonomic diversity. We observed large variation in the beta diversity of lake sturgeon gut microbiota between larvae from eggs incubated in hatchery and wild (stream) origins based on nonmetric dimensional scaling (NMDS). Permutational ANOVA indicated the effects of chemotherapeutic treatments on gut microbial community composition were dependent on the initial source of the founding microbial community. Influences of microbiota colonization during early ontogenetic stages and the resilience of gut microbiota to topical chemotherapeutic treatments are discussed.

Pedigree analysis and estimates of effective breeding size characterize sea lamprey reproductive biology.

The sea lamprey (Petromyzon marinus) is an invasive species in the Great Lakes and the focus of a large control and assessment program. Current assessment methods provide information on the census size of spawning adult sea lamprey in a small number of streams, but information characterizing reproductive success of spawning adults is rarely available. We used RAD-capture sequencing to genotype single nucleotide polymorphism (SNP) loci for ~1600 sea lamprey larvae collected from three streams in northern Michigan (Black Mallard, Pigeon, and Ocqueoc Rivers). Larval genotypes were used to reconstruct family pedigrees, which were combined with Gaussian mixture analyses to identify larval age classes for estimation of spawning population size. Two complementary estimates of effective breeding size (N b), as well as the extrapolated minimum number of spawners (N s), were also generated for each cohort. Reconstructed pedigrees highlighted inaccuracies of cohort assignments from traditionally used mixture analyses. However, combining genotype-based pedigree information with length-at-age assignment of cohort membership greatly improved cohort identification accuracy. Population estimates across all three streams sampled in this study indicate a small number of successfully spawning adults when barriers were in operation, implying that barriers limited adult spawning numbers but were not completely effective at blocking access to spawning habitats. Thus, the large numbers of larvae present in sampled systems were a poor indicator of spawning adult abundance. Overall, pedigree-based N b and N s estimates provide a promising and rapid assessment tool for sea lamprey and other species.

A chromosome-anchored genome assembly for Lake Trout (Salvelinus namaycush).

Here, we present an annotated, chromosome-anchored, genome assembly for Lake Trout (Salvelinus namaycush) - a highly diverse salmonid species of notable conservation concern and an excellent model for research on adaptation and speciation. We leveraged Pacific Biosciences long-read sequencing, paired-end Illumina sequencing, proximity ligation (Hi-C) sequencing, and a previously published linkage map to produce a highly contiguous assembly composed of 7378 contigs (contig N50 = 1.8 Mb) assigned to 4120 scaffolds (scaffold N50 = 44.975 Mb). Long read sequencing data were generated using DNA from a female double haploid individual. 84.7% of the genome was assigned to 42 chromosome-sized scaffolds and 93.2% of Benchmarking Universal Single Copy Orthologues were recovered, putting this assembly on par with the best currently available salmonid genomes. Estimates of genome size based on k-mer frequency analysis were highly similar to the total size of the finished genome, suggesting that the entirety of the genome was recovered. A mitochondrial genome assembly was also produced. Self-versus-self synteny analysis allowed us to identify homeologs resulting from the salmonid specific autotetraploid event (Ss4R) as well as regions exhibiting delayed rediploidization. Alignment with three other salmonid genomes and the Northern Pike (Esox lucius) genome also allowed us to identify homologous chromosomes in related taxa. We also generated multiple resources useful for future genomic research on Lake Trout, including a repeat library and a sex-averaged recombination map. A novel RNA sequencing data set for liver tissue was also generated in order to produce a publicly available set of annotations for 49,668 genes and pseudogenes. Potential applications of these resources to population genetics and the conservation of native populations are discussed.

Quantifying nonlinear temporal effects of ethanol preservation on round goby (Neogobius melanostomus) anatomical traits.

Geometric morphometrics provides a powerful means of evaluating differences in phenotypic traits among specimens. However, inferences of trait variability can be confounded when measurements are based on preserved samples. We evaluated effects of ethanol preservation on morphology over a 22-week time period for a Laurentian Great Lakes invasive fish, round goby (Neogobius melanostomus, Pallas 1814), using sets of 17 lateral and six dorsal landmarks. We tested whether ethanol preservation affected the magnitude of inter-population variation between individuals collected from lake and river habitats. Generalized least square regression determined that length did not significantly vary through the preservation time series for fish from either population, while mass decreased significantly. Body shape variation was summarized using principal component analysis, which revealed that most preservation-associated changes occurred in the first 14 days. The lateral shape experienced a large magnitude change during the first 24 h in ethanol then only minor changes for the remainder of the study. The dorsal shape began to revert to pre-preservation measurements about 14 days following preservation. Additionally, differences in shape were apparent between the two populations throughout the experiment; however, the magnitude of differences between populations varied depending on whether dorsal or lateral landmarks were considered. Our study demonstrates that tissue responses to ethanol preservation can be more complex than a simple loss of mass, resulting in difficult to predict consequences for geometric morphometric analyses, including variable responses depending on the anatomical region being analyzed.

A recovery network leads to the natural recolonization of an archipelago and a potential trailing edge refuge.

Rapid environmental change is reshaping ecosystems and driving species loss globally. Carnivore populations have declined and retracted rapidly and have been the target of numerous translocation projects. Success, however, is complicated when these efforts occur in novel ecosystems. Identifying refuges, locations that are resistant to environmental change, within a translocation framework should improve population recovery and persistence. American martens (Martes americana) are the most frequently translocated carnivore in North America. As elsewhere, martens were extirpated across much of the Great Lakes region by the 1930s and, despite multiple translocations beginning in the 1950s, martens remain of regional conservation concern. Surprisingly, martens were rediscovered in 2014 on the Apostle Islands of Lake Superior after a putative absence of >40 yr. To identify the source of martens to the islands and understand connectivity of the reintroduction network, we collected genetic data on martens from the archipelago and from all regional reintroduction sites. In total, we genotyped 483 individual martens, 43 of which inhabited the Apostle Islands (densities 0.42-1.46 km-2 ). Coalescent analyses supported the contemporary recolonization of the Apostle Islands with progenitors likely originating from Michigan, which were sourced from Ontario. We also identified movements by a first-order relative between the Apostle Islands and the recovery network. We detected some regional gene flow, but in an unexpected direction: individuals moving from the islands to the mainland. Our findings suggest that the Apostle Islands were naturally recolonized by progeny of translocated individuals and now act as a source back to the reintroduction sites on the mainland. We suggest that the Apostle Islands, given its protection from disturbance, complex forest structure, and reduced carnivore competition, will act as a potential refuge for marten along their trailing range boundary and a central node for regional recovery. Our work reveals that translocations, even those occurring along southern range boundaries, can create recovery networks that function like natural metapopulations. Identifying refuges, locations that are resistant to environmental change, within these recovery networks can further improve species recovery, even within novel environments. Future translocation planning should a priori identify potential refuges and sources to improve short-term recovery and long-term persistence.

Spatial genetic structure and landscape connectivity in black bears: Investigating the significance of using different land cover datasets and classifications in landscape genetics analyses.

Landscape genetic analyses allow detection of fine-scale spatial genetic structure (SGS) and quantification of effects of landscape features on gene flow and connectivity. Typically, analyses require generation of resistance surfaces. These surfaces characteristically take the form of a grid with cells that are coded to represent the degree to which landscape or environmental features promote or inhibit animal movement. How accurately resistance surfaces predict association between the landscape and movement is determined in large part by (a) the landscape features used, (b) the resistance values assigned to features, and (c) how accurately resistance surfaces represent landscape permeability. Our objective was to evaluate the performance of resistance surfaces generated using two publicly available land cover datasets that varied in how accurately they represent the actual landscape. We genotyped 365 individuals from a large black bear population (Ursus americanus) in the Northern Lower Peninsula (NLP) of Michigan, USA at 12 microsatellite loci, and evaluated the relationship between gene flow and landscape features using two different land cover datasets. We investigated the relative importance of land cover classification and accuracy on landscape resistance model performance. We detected local spatial genetic structure in Michigan's NLP black bears and found roads and land cover were significantly correlated with genetic distance. We observed similarities in model performance when different land cover datasets were used despite 21% dissimilarity in classification between the two land cover datasets. However, we did find the performance of land cover models to predict genetic distance was dependent on the way the land cover was defined. Models in which land cover was finely defined (i.e., eight land cover classes) outperformed models where land cover was defined more coarsely (i.e., habitat/non-habitat or forest/non-forest). Our results show that landscape genetic researchers should carefully consider how land cover classification changes inference in landscape genetic studies.

Effects of Hatchery Rearing Density, Handling, and Nutrition on Renibacterium salmoninarum Infection Prevalence in Juvenile Chinook Salmon.

Bacterial kidney disease, caused by Renibacterium salmoninarum (RS), is a chronic and often fatal disease of salmonid species, and can be particularly harmful to hatchery-reared Chinook Salmon Oncorhynchus tshawytscha. A considerable amount of research has focused on the prevention of vertical and horizontal transmission; however, a comparatively little amount has investigated factors that increase the prevalence of RS infection in captive environments. We evaluated the effects of three common hatchery conditions (handling, nutrition level, and rearing density) on RS infection prevalence. Fish were sampled at 30-d and 60-d postexposure to RS. Of 577 juveniles examined, 65 (11.27%) had anterior kidneys infected with RS. Using a logistic mixed model analysis, we found effects of nutrition level (P = 0.018), handling (P = 0.010), and sampling period (P = 0.003) on the prevalence of RS. The interactions of nutrition and handling (P = 0.008) and nutrition and time (P < 0.001) were also significant. When fed a standard-nutrition diet, proportionately fewer fish were infected with RS when not handled (7.16% versus 0.04%; P = 0.003). Fish in the standard-nutrition group also had a lower prevalence of RS during the second sampling period (4.08% versus 0.08%, respectively; P < 0.001). When not handled, rearing with standard nutrition (11.50% versus 0.04%; P = 0.004) resulted in a reduction in prevalence of RS infection. Additionally, nonhandled fish had a much lower prevalence of RS infection during the second sampling period (2.66% versus 0.21%; P = 0.009). While density did not affect the prevalence of RS infection (P = 0.145), fish reared at a higher density had lower RS infection when not handled (16.48% versus 0.84%, P = 0.004). For fish at a higher density, the RS prevalence was lower during the second sampling period (10.57% versus 1.40%; P = 0.002). Our results suggest that hatchery managers can reduce RS infection prevalence by maintaining an adequate nutritional regime as recommended by the manufacturer. Additionally, the prevalence of RS may be reduced if managers decrease handling of hatchery-reared Chinook Salmon if exposed to RS.

Mapping of Adaptive Traits Enabled by a High-Density Linkage Map for Lake Trout.

Understanding the genomic basis of adaptative intraspecific phenotypic variation is a central goal in conservation genetics and evolutionary biology. Lake trout (Salvelinus namaycush) are an excellent species for addressing the genetic basis for adaptive variation because they express a striking degree of ecophenotypic variation across their range; however, necessary genomic resources are lacking. Here we utilize recently-developed analytical methods and sequencing technologies to (1) construct a high-density linkage and centromere map for lake trout, (2) identify loci underlying variation in traits that differentiate lake trout ecophenotypes and populations, (3) determine the location of the lake trout sex determination locus, and (4) identify chromosomal homologies between lake trout and other salmonids of varying divergence. The resulting linkage map contains 15,740 single nucleotide polymorphisms (SNPs) mapped to 42 linkage groups, likely representing the 42 lake trout chromosomes. Female and male linkage group lengths ranged from 43.07 to 134.64 centimorgans, and 1.97 to 92.87 centimorgans, respectively. We improved the map by determining coordinates for 41 of 42 centromeres, resulting in a map with 8 metacentric chromosomes and 34 acrocentric or telocentric chromosomes. We use the map to localize the sex determination locus and multiple quantitative trait loci (QTL) associated with intraspecific phenotypic divergence including traits related to growth and body condition, patterns of skin pigmentation, and two composite geomorphometric variables quantifying body shape. Two QTL for the presence of vermiculations and spots mapped with high certainty to an arm of linkage group Sna3, growth related traits mapped to two QTL on linkage groups Sna1 and Sna12, and putative body shape QTL were detected on six separate linkage groups. The sex determination locus was mapped to Sna4 with high confidence. Synteny analysis revealed that lake trout and congener Arctic char (Salvelinus alpinus) are likely differentiated by three or four chromosomal fissions, possibly one chromosomal fusion, and 6 or more large inversions. Combining centromere mapping information with putative inversion coordinates revealed that the majority of detected inversions differentiating lake trout from other salmonids are pericentric and located on acrocentric and telocentric linkage groups. Our results suggest that speciation and adaptive divergence within the genus Salvelinus may have been associated with multiple pericentric inversions occurring primarily on acrocentric and telocentric chromosomes. The linkage map presented here will be a critical resource for advancing conservation oriented genomic research on lake trout and exploring chromosomal evolution within and between salmonid species.

Ecological and Ontogenetic Components of Larval Lake Sturgeon Gut Microbiota Assembly, Successional Dynamics, and Ecological Evaluation of Neutral Community Processes.

Gastrointestinal (GI) or gut microbiotas play essential roles in host development and physiology. These roles are influenced partly by the microbial community composition. During early developmental stages, the ecological processes underlying the assembly and successional changes in host GI community composition are influenced by numerous factors, including dispersal from the surrounding environment, age-dependent changes in the gut environment, and changes in dietary regimes. However, the relative importance of these factors to the gut microbiota is not well understood. We examined the effects of environmental (diet and water sources) and host early ontogenetic development on the diversity of and the compositional changes in the gut microbiota of a primitive teleost fish, the lake sturgeon (Acipenser fulvescens), based on massively parallel sequencing of the 16S rRNA gene. Fish larvae were raised in environments that differed in water source (stream versus filtered groundwater) and diet (supplemented versus nonsupplemented Artemia fish). We quantified the gut microbial community structure at three stages (prefeeding and 1 and 2 weeks after exogenous feeding began). The diversity declined and the community composition differed significantly among stages; however, only modest differences associated with dietary or water source treatments were documented. Many taxa present in the gut were over- or underrepresented relative to neutral expectations in each sampling period. The findings indicate dynamic relationships between the gut microbiota composition and host gastrointestinal physiology, with comparatively smaller influences being associated with the rearing environments. Neutral models of community assembly could not be rejected, but selectivity associated with microbe-host GI tract interactions through early ontogenetic stages was evident. The results have implications for sturgeon conservation and aquaculture production specifically and applications of microbe-based management in teleost fish generally.IMPORTANCE We quantified the effects of environment (diet and water sources) and host early ontogenetic development on the diversity of and compositional changes in gut microbial communities based on massively parallel sequencing of the 16S rRNA genes from the GI tracts of larval lake sturgeon (Acipenser fulvescens). The gut microbial community diversity declined and the community composition differed significantly among ontogenetic stages; however, only modest differences associated with dietary or water source treatments were documented. Selectivity associated with microbe-host GI tract interactions through early ontogenetic stages was evident. The results have implications for lake sturgeon and early larval ecology and survival in their natural habitat and for conservation and aquaculture production specifically, as well as applications of microbe-based management in teleost fish generally.

RAPTURE (RAD capture) panel facilitates analyses characterizing sea lamprey reproductive ecology and movement dynamics.

Genomic tools are lacking for invasive and native populations of sea lamprey (Petromyzon marinus). Our objective was to discover single nucleotide polymorphism (SNP) loci to conduct pedigree analyses to quantify reproductive contributions of adult sea lampreys and dispersion of sibling larval sea lampreys of different ages in Great Lakes tributaries. Additional applications of data were explored using additional geographically expansive samples. We used restriction site-associated DNA sequencing (RAD-Seq) to discover genetic variation in Duffins Creek (DC), Ontario, Canada, and the St. Clair River (SCR), Michigan, USA. We subsequently developed RAD capture baits to genotype 3,446 RAD loci that contained 11,970 SNPs. Based on RAD capture assays, estimates of variance in SNP allele frequency among five Great Lakes tributary populations (mean F ST 0.008; range 0.00-0.018) were concordant with previous microsatellite-based studies; however, outlier loci were identified that contributed substantially to spatial population genetic structure. At finer scales within streams, simulations indicated that accuracy in genetic pedigree reconstruction was high when 200 or 500 independent loci were used, even in situations of high spawner abundance (e.g., 1,000 adults). Based on empirical collections of larval sea lamprey genotypes, we found that age-1 and age-2 families of full and half-siblings were widely but nonrandomly distributed within stream reaches sampled. Using the genomic scale set of SNP loci developed in this study, biologists can rapidly genotype sea lamprey in non-native and native ranges to investigate questions pertaining to population structuring and reproductive ecology at previously unattainable scales.