Larval fish and aquatic macroinvertebrates are believed to be at greater predation risk by visual riverine predators during periods of higher lunar illumination. Predation rates may also be affected by the taxonomic composition and relative abundance of alternative co-distributed prey. We experimentally tested whether differences in larval lake sturgeon (Acipenser fulvescens) predation levels were attributed to changes in prey selectivity, or to an overall decrease in consumption of co-distributed and dispersing prey consumed by predatory riverine species under different light conditions. Larvae from three common riverine prey taxa observed nocturnally dispersing together (lake sturgeon, white suckers (Catostomus commersonii), and Hetptageniid mayflies) were released concurrently into raceway mesocosms. Raceways contained one of two abundant predacious riverine fishes [either rock bass (Ambloplites rupestris) or hornyhead chub (Nocomis biguttatus)]. Trials were conducted in light conditions simulating a full or new moon. Both predators consumed fewer prey under new moon relative to full moon light conditions. In full moon conditions, rock bass selected for mayflies and against white suckers, but in new moon conditions rock bass selectivity was similar for all prey. Hornyhead chub selected for mayflies under both light conditions. Results indicate rock bass and hornyhead chub rely on visual cues to detect prey. Rock bass also appeared to visually differentiate between prey taxa, whereas hornyhead chub may use alternative senses to select prey. Variation in larval lake sturgeon mortality during the downstream drifting period was a function of the taxonomic composition and size distribution of predators, and composition and relative abundance of alternative co-distributed prey. Nocturnal light levels likely also influence predation rates in natural populations of nocturnally dispersing aquatic larvae. Light levels during larval dispersal should be considered when interpreting intra- and inter-annual variation in recruitment in species of regional conservation concern.

Long-lived iteroparous organisms vary resource expenditures toward migration and reproduction in response to individual physical factors and conspecific interactions, which can affect future reproductive timing and interval. Reproductive actions can lead to trade-offs associated with allocations to current vs. future reproduction, including longer reproductive interval, require additional study. The objective of this study was to evaluate associations between physical stream characteristics, individual behaviors, and breeding demographics and spawning periodicity in lake sturgeon (Acipenser fulvescens). We used Radio Frequency Identification tags to monitor spawning migration by male (N = 1931) and female (N = 683) adults over seven consecutive years (2016 through 2022) in the Black River, Cheboygan Co., MI. We used ordinal regression models to quantify associations. Male spawning periodicity (1.60 ± 0.63 years; mean ± SE) decreased with increasing body size and intra-sex interactions and increased with increasing cumulative temperature, discharge, number of inter-sex interactions, and complete river migrations in a season. Female spawning periodicity (3.19 ± 0.05 years, mean ± SE) decreased with increasing upstream swimming time and inter-sex interactions. Results demonstrated spawning periodicity shortened as male lake sturgeon age, and future breeding opportunities decreased, while female periodicity may be more individualized and is more likely to be affected by resource acquisition.

Measures of genetic diversity among individuals within and between broodstock year classes of Arctic Grayling (Thymallus arcticus) were quantified as part of a large inter-disciplinary effort to repatriate the species into Michigan fluvial habitats. Genetic pedigree analysis of 200 juveniles from each of the 2019 and 2021 year classes indicated that 205 and 218 adults from a natural stream population in central Alaska contributed to offspring genotyped. Because broodstock sampling occurred in the same stream location each year and adults were not marked, 22.4 % of successful adults were estimated to have contributed to offspring genotyped in both years, indicating that the local adult population was philopatric to the spawning site and of moderate size. Measures of genetic diversity including heterozygosity and allelic diversity were high, while inbreeding coefficients (FIS), and estimates of inter-individual relatedness (RXY) of offspring genotyped within and between year classes were low. Collectively, results indicate that crosses conducted among adults to establish broodstock year classes had achieved restoration program goals of creating a genetically diverse domestic broodstock to use for future repatriation efforts.

Lake trout reproduction has increased in Lake Michigan since the 2000 s. Previous genetic studies reported that the strains of stocked adults did not contribute equally to wild recruits. Consequently, reproductive potential of spawning biomass estimated in stock assessments will depend upon strain composition, complicating comparisons across time and space. We integrated data from a stock assessment with genetic data to estimate an effective lake trout spawning biomass that accounts for strain-specific reproductive efficiency. A reproductive power index (RPI) was developed for six strains of hatchery-reared lake trout using genetic data from lakes Michigan and Huron. The RPI is the ratio of the observed to expected genetic contribution of a strain to wild recruits. The Seneca Lake strain had the highest RPI, followed by Lake Manitou, Lewis Lake, Green Lake, Lake Superior, and Lake Huron strains. The RPI in southern Lake Michigan was 2.56 for Seneca Lake, 0.74 for Lake Superior, 0.50 for Lewis Lake, and 0.32 for Green Lake strains. Strain-specific effective spawning biomass in southern Lake Michigan was estimated using numbers stocked, population demographics from a stock assessment, and RPI to develop an annual effective spawning biomass index (ESBI) as a measure of reproductive potential. After 1996, ESBI increased faster than spawning biomass, and continued to increase when spawning biomass leveled off, reflecting the shift toward lake trout strains with higher RPI. The contribution to the ESBI after 2010 was 46 % Seneca Lake, 34 % wild adults, 12 % Lake Superior, and 4 % for the Lewis Lake and Green Lake strains.

Eukaryotic microorganisms are an important, but understudied, component of freshwater aquatic ecosystems, and are significant sources of mortality in early life stages of fishes in natural and aquaculture systems. The eukaryotic microbiome colonizing egg surfaces of the lake sturgeon (Acipenser fulvescens) was characterized from eggs collected in natural stream habitats and a streamside hatchery in the Cheboygan River watershed in MI, USA. The taxonomic diversity of members of the Kingdoms Fungi and Stramenopile associated with infections of lake sturgeon eggs during spawning is contributing to lake sturgeon mortality in the hatchery. Characterization of the microbial communities from deposited eggs demonstrated heavy influence of spawning location on the diversity of Pythium, an Oomycete predominating in the microbiome. The Ascomycota also had a strong and distinguishing presence, with members of the Dothidiales found only on eggs from the streamside hatchery. Aureobasidium pullulans, a ubiquitous pigmented yeast, was present in the greatest numbers of egg samples, and Helotiales were found only on samples from the Black River. Independent isolates were collected from egg surfaces and tested for chemical sensitivity to the oomicides ethaboxam and mefenoxam, which are used for control of Oomycete agricultural pathogens. Ethaboxam inhibited mycelial growth almost completely for all Saprolegnia strains tested, while mefenoxam, at 20 × strength, was largely ineffective. Water prevents the natural inactivation of mefenoxam by light, thus is not advisable in aquatic systems, where it could accumulate. Alternatively, ethaboxam may be a nonpersistent, welcome control option for these fish pathogens.

Conventional dietary assessments are challenging in hematophagous species, particularly in sea lamprey (Petromyzon marinus). However, recent technological developments and molecular approaches have provided an attractive alternative through the use of DNA metabarcoding. While DNA metabarcoding has been used for dietary analyses in numerous species, including lampreys, applications of universal primers that detect a diverse set of prey items can be limited by the amplification of predator DNA. In this study, we designed and tested eight blocking primers designed to suppress the amplification of sea lamprey DNA with vertebrate-universal primers targeting the mitochondrial 12S rRNA gene. This approach allowed for the use of a single marker to amplify a taxonomically diverse suite of host species, in contrast to previous studies that used multiple taxon-specific primer pairs (e.g., Salmonidae, Cyprinidae, and Catostomidae). Candidate blocking primers evaluated in this study differed in base pair length, end sequence modification, and purification method. Samples with different sea lamprey-to-host DNA ratios were subjected to multiple detection methods including gel electrophoresis, quantitative PCR, and DNA metabarcoding to assess the ability of each blocking primer to selectively suppress amplification of the sea lamprey 12S gene region. All blocking primers tested performed well and demonstrated high effectiveness, suppressing sea lamprey reads by > 99.9% in mock communities and improving host DNA sequence recovery across various sample types, including wild-caught lamprey. Results show that the blocking primers evaluated can facilitate molecular diet analysis in sea lamprey, allowing the amplification of a taxonomically diverse range of host fish species with universal primers.

Dam construction contributes to declines in the distribution and abundance of many fishes. Increasing connectivity through adult transfer can be demographically and genetically beneficial, but assessing the effects resulting from transfer can be difficult if resident fish exist upstream. Genotypes of adult and larval lake sturgeon ( Acipenser fulvescens) were used to quantify contributions to larval recruitment from adults transferred upstream of dams on the Menominee River, USA. We evaluated whether transfer timing, sex, and adult size were associated with the odds of reproduction. Elevator transfer operations in Fall 2019, Fall 2020, and Spring 2021 resulted in 152 male and 81 female lake sturgeon transferred upstream. In 2020 and 2021, 580 and 518 larvae were genotyped. We found that 86% (201/233) of adults reproduced and 62.3% (684/1098) of offspring had transferred parents. In total, we estimated that 392 resident adults contributed to offspring production. Mixed matings accounted for 53% of offspring genotyped, increasing levels of offspring genetic diversity relative to offspring produced from resident-only matings. Transferring adults may be a viable restoration alternative for other iteroparous fish in river systems where connectivity to spawning areas has been impeded.

The population abundance and distribution of many Great Lakes fish species, particularly those that are adapted to cold water, have changed greatly in recent times due in part to changes in physical lake and biotic environments. Burbot (Lota lota) is an understudied cold water benthivore of conservation concern. To characterize levels of genetic diversity and spatial genetic structure associated with historical phylogeographic isolation or contemporary reproductive isolation, microsatellite genotypes and mitochondrial DNA sequences were analyzed for burbot (n = 253) collected from 14 open water locations in Lakes Huron, Michigan, and Superior and the upper St. Clair River. Spatial variation in levels of diversity and degree of genetic distinction among samples in different geographic locations was most evident over broader geographic scales between Lake Superior and Lakes Michigan and Huron. Members of open-water sampling locations within basins did not differ significantly in microsatellite allele or mtDNA haplotype frequency. Data from this study and from previous results in other North American locations indicated that burbot sampled from the upper Great Lakes likely originated from multiple glacial refugia, and that geographic representations were admixed at the lake basin level. Analyses differentiating populations of burbot will inform managers how to appropriately target the genetically differentiated populations to support species management.

ABSTRACT Migratory fishes are renowned for their ability to home to natal streams for spawning. Learned olfactory cues play a critical role in natal homing of Pacific salmon and other fishes, but the underlying chemical signature of streams remains poorly understood after decades of study. The molecules that convey a stream‐specific odour must differ among sites but remain constant over time. Among leading odorant candidates are amino acids; however, little research has assessed the spatial and temporal variability of amino acid profiles in streams. We report a comprehensive chemical study of dissolved amino acids as potential olfactory cues for homing by migratory fish. Specifically, we profiled amino acids in water from 23 streams in the upper Laurentian Great Lakes basin over 2 years. We investigated variation in amino acid profiles (1) among regions and rivers within a year, (2) between years and (3) among sites and across the seasons of migration and early life history within a stream. Liquid‐chromatography tandem mass spectrometry revealed nanomolar concentrations for most of the 20 L‐amino acids measured, above the levels detectable by studied migratory fishes. Moreover, amino acid profiles were temporally stable between 2 years and across an annual season from adult spawning migration through offspring early‐life development within a stream. However, spatial differences in amino acid profiles were evident primarily over large geographic distances (among regions) but not among tributaries within regions or among sites within a stream. Collectively, our results indicate dissolved amino acids may be consistent components of rivers' odorant profiles but suggest additional molecules are likely important for natal homing of migratory fishes to specific spawning sites. We suggest that future studies consider the combined importance of amino acids and molecules from other chemical classes. Understanding the chemical basis of olfactory‐guided natal homing is especially important as human activities could alter the odorant profiles of streams and thereby disrupt fish migrations and negatively impact population recruitment.

Characterizing inter- and intra-population levels of variability in age at sexual maturation for long-lived fishes provides insight into year-class strength and recruitment dynamics, allowing for more effective management practices. Here we analyzed the ontogenetic chronology of pectoral fin ray annuli trace elemental concentration profiles as well as changes in annuli growth in lake sturgeon ( Acipenser fulvescens) to determine onset of sexual maturity (OSM). Elemental concentrations and growth-zone width were used to build a random forest classification model to discriminate year-specific signatures to before or after OSM from 98 individual fin rays from both sexes across multiple populations and watersheds. The model demonstrated an overall accuracy of 98.8%. Ba was the most important variable related to OSM discrimination success followed by zone width, Pb, Mn, Mg, Zn, Cu, and Sr. Fin ray elemental concentrations began to increase at approximately age 24 (±4.7 years) in females and 15 (±3.1 years) in males while zone width decreased. This study implies that new applications for fin ray microchemistry are possible and could benefit future fisheries management strategies specifically by adding nonlethal or less invasive sampling techniques for evaluating OSM in sturgeon.

Since the establishment of the invasive sea lamprey (Petromyzon marinus) in the Great Lakes, extensive management efforts have attempted to reduce negative impacts on native fishes. Despite a significant reduction in sea lamprey population abundance following application of several control methods, uncertainties remain concerning damage caused by sea lamprey predation on Great Lakes fish populations. While conventional dietary assessments are hindered by the hematophagous nature of sea lamprey, DNA metabarcoding offers a promising alternative by identifying prey species DNA from sea lamprey digestive tract samples. DNA metabarcoding has been used for dietary analyses in numerous species, including lampreys; however, initial assessments using polymerase chain reaction (PCR) primers designed to amplify the 12S rRNA gene in vertebrate taxa indicated a high presence of sea lamprey amplification product per sample. To minimize sea lamprey DNA co-amplification, we designed and tested eight blocking primers that suppress the amplification of sea lamprey 12S sequences while allowing amplification of host species DNA. This approach allowed for the use of a single marker to amplify a taxonomically diverse suite of host species, in contrast to previous studies that used multiple taxon-specific primer pairs (e.g. Salmonidae, Cyprinidae, and Catostomidae). Candidate blocking primers evaluated in this study differed in base pair length, end sequence modification, and purification method. Samples with different sea lamprey-to-host DNA ratios were subjected to multiple detection methods including gel electrophoresis, quantitative PCR, and DNA metabarcoding to assess the ability of each blocking primer to selectively suppress amplification of the sea lamprey 12S gene region. All blocking primers tested performed well and demonstrated high effectiveness. Results show that the blocking primers evaluated can facilitate molecular diet analysis in sea lamprey, allowing the amplification of a taxonomically diverse range of host fish species with universal primers.

ABSTRACTMany species of reptiles and amphibians (herpetofauna) rely on wetlands that are being degraded and lost at a high rate. Characterization of herpetofauna diversity in different wetland types may help guide conservation strategies. However, traditional survey methods often involve sampling within small temporal windows, and the gear deployed may be taxonomically biased, thus, they may fail to accurately characterize species presence/absence and diversity. In contrast, environmental (e)DNA metabarcoding has been shown to effectively survey entire aquatic communities and can provide a useful complement to traditional surveys. The objective of this study was to design and optimize eDNA sampling and laboratory protocols for wetland herpetofauna. Protocols evaluated included different water sampling approaches (point versus transect sampling), seasonality of sampling, and choice of metabarcoding marker (mitochondrial 12S versus 16S rDNA). Samples collected from 10 sites across southern Michigan detected 17 amphibian and five reptile species, including four species of conservation concern (Ambystoma texanum, Clemmys guttata, Rana palustris, and Sternotherus odoratus). We observed no difference in the number of species detected between point and transect samples (p = 0.70), but point sampling required less time (p = 0.03) and allowed significantly larger volumes of water to be filtered (p = 1.13e‐5). No difference in species richness was observed between the 12S and 16S mitochondrial DNA markers (p = 0.96). However, a greater number of taxa were identifiable at the species level when using the 16S locus. There was also a significant difference in the number of species detected between early and late summer sampling periods (more species detected in the earlier period; p = 6.31e‐6), and some species were only found in the early or late sampling period. Sampling during multiple periods to fully characterize species composition, the use of point sampling, and the 16S mtDNA marker for herpetofauna eDNA metabarcoding studies may increase efficiency and reliability of results.

The establishment and spread of invasive species are directly related to intersexual interactions as dispersal and reproductive success are related to distribution, effective population size, and population growth. Accordingly, populations established by r-selected species are particularly difficult to suppress or eradicate. One such species, the red swamp crayfish (Procambarus clarkii) is established globally at considerable ecological and financial costs to natural and human communities. Here, we develop a single nucleotide polymorphism (SNP) loci panel for P. clarkii using restriction-associated DNA-sequencing data. We use the SNP panel to successfully genotype 1800 individuals at 930 SNPs in southeastern Michigan, USA. Genotypic data were used to reconstruct pedigrees, which enabled the characterization of P. clarkii's mating system and statistical tests for associations among environmental, demographic, and phenotypic predictors and adult reproductive success estimates. We identified juvenile cohorts using genotype-based pedigrees, body size, and sampling timing, which elucidated the breeding phenology of multiple introduced populations. We report a high prevalence of multiple paternity in each surveyed waterbody, indicating polyandry in this species. We highlight the use of newly developed rapid genomic assessment tools for monitoring population reproductive responses, effective population sizes, and dispersal during ongoing control efforts.

Species distributions are often indicative of historical biogeographical events and contemporary spatial biodiversity patterns. The Harlequin Duck Histrionicus histrionicus is a sea duck of conservation concern that has a disjunct distribution, with discrete portions of its range associated with northern Pacific and Atlantic Ocean basins. Movement data indicate migratory connectivity within regions of each ocean basin but not cross‐continent dispersal, suggesting that genetic structuring could exist at multiple spatial scales. Little is known regarding the impacts of past vicariance events on the species phylogeographical structure and historical demography, or rates of gene flow at different spatial scales. We used data from microsatellite loci and mitochondrial DNA (mtDNA) sequences to quantify levels of genetic diversity within, and the extent of spatial genetic differentiation among locations sampled at multiple spatial scales across the species range. Samples were collected at nonbreeding locations, which represent groupings appropriate for characterizing genetically differentiated subgroups at regional and continental scales. Collectively, genetic data and coalescence modelling suggested that individuals colonized regions currently occupied within both ocean basins in the Holocene from a single refuge in the Atlantic. Further, it seems likely there was secondary contact with lineages derived from populations in Asia, based on the shallow species‐wide mtDNA phylogeny and high incidence of recently derived private mtDNA haplotypes. Estimates of inter‐location variance in microsatellite allele and mtDNA haplotype frequency were moderate and significant between western (Pacific – North America) and eastern (Atlantic – North America, Greenland and Iceland) ocean basins and among sampling groups within each ocean basin. Genetic differentiation among sampling groups was particularly evident at the species distributional margins in the Atlantic (Iceland) and the Pacific (Shemya Island) Ocean basins. Coalescent modelling results suggest that contemporary spatial genetic patterns in the species arose through the combined influences of secondary contact, shared ancestry and gene flow after the last glacial maxima.

Artificial propagation and wild release may influence the genetic integrity of wild populations. This practice has been prevalent in fisheries for centuries and is often termed 'stocking'. In the Laurentian Great Lakes (Great Lakes here-on), walleye populations faced declines from the 1950s to the 1970s, prompting extensive stocking efforts for restoration. By the mid-2010s, walleye populations showed signs of recovery, but the genetic legacy of stocking on population structure at the genomic level remains unclear. Using a dataset of 45,600 genome-aligned SNP loci genotyped in 1075 walleye individuals, we investigated the genetic impacts of over 50 years of stocking across the Great Lakes. Population structure was associated with both natural geographic barriers and stocking from non-native sources. Admixture between Lake Erie walleye and walleye from the re-populated Tittabawassee River indicate that stocking may have re-distributed putatively adaptive alleles around the Great Lakes. Genome scans identified FST outliers and evidence of selective sweeps, indicating local adaptation of spawning populations is likely. Notably, one genomic region showed strong differentiation between Muskegon River and walleye from the Tittabawassee River, which was re-populated by Muskegon strain walleye, suggesting admixture and selection both impact the observed genetic diversity. Overall, our study underscores how artificial propagation and translocations can significantly alter the evolutionary trajectory of populations. The findings highlight the complex interplay between stocking practices and population genetic diversity, emphasising the need for careful management strategies to preserve the genetic integrity of wild populations amidst conservation efforts.

Red Swamp Crayfish Procambarus clarkii (Girard, 1852) (RSC) are a highly invasive species established on five continents. Recent detections of RSC populations in several locations in Michigan, USA spurred action to develop trap-based early detection and response plans based on the best available science, however, few recommendations are available concerning important sampling logistics such as timing and density of deployed traps. We surveyed 231 waterbodies (mainly lentic systems with relatively few small lotic systems) for RSC in two invasion zones in southern Michigan. We used Bayesian hierarchical models to evaluate baited traps for detection of RSC invasions, and qualitatively evaluated feasibility of eDNA metabarcoding for enhancing trap-based detection strategies. While overall occupancy was low, predicted detection probabilities with baited traps ranged from 27 to 98% and depended on season, density of traps, and relative crayfish abundance. There was high agreement between trapping and eDNA detections (94%). However, an improved eDNA sampling design that increases the number of samples is needed. We conclude that trap-based detections of RSC in Michigan are most likely to occur mid-summer, trapping densities should be increased in the spring and fall to offset lower detection probabilities, and eDNA could feasibly supplement trapping in spring and fall.

Infectious diseases are a leading cause of losses in the aquaculture industry and conservation programs globally. Simultaneously, infectious diseases pose a substantial risk to fish being hatchery-reared and released into natural habitats for conservation purposes, including the Great Lakes lake sturgeon (Acipenser fulvescens, i.e., GL-LST). Recently, an alloherpesvirus (lake sturgeon herpesvirus 2, i.e., LSHV-2) capable of inducing disease and/or mortality in adult and juvenile GL-LSTs was detected in two adult GL-LST populations. To begin developing disease prevention and/or control methods, in vitro experiments were designed to determine the susceptibility of LSHV-2 to disinfectants commonly used in hatchery and aquaculture facilities (Virkon®-Aquatic: potassium peroxymonosulfate; Ovadine®: polyvinylpyrrolidone iodine complex; and Perox-Aid®: hydrogen peroxide). Cultured LSHV-2 was exposed to each disinfectant at two concentrations (Virkon®-Aquatic: 0.5% and 1%; Ovadine®: 50 and 100 ppm; and Perox-Aid®: 500 and 1000 ppm) in duplicate for durations of 1, 10, and 30 min. Following exposure, the disinfectant was neutralized, and after a 14-day incubation period on a white sturgeon × lake sturgeon hybrid cell line (WSxLS), percent reduction was calculated by comparing the 50% tissue culture infectious doses (TCID50/mL) of the virus with and without disinfectant exposure. When exposed to Perox-Aid®, LSHV-2 percent reduction ranged from 58.7% to 99.5%. When exposed to Ovadine®, the percent reduction ranged from 99.4% to 100%. Lastly, the percent reduction when exposed to Virkon®-Aquatic was 100% for both concentrations and all timepoints. The results herein provide evidence that both Virkon®-Aquatic and Ovadine® are virucidal to LSHV-2 and may represent a means to reduce virus transmission risk under field settings.

Grass carp (Ctenopharyngodon idella), an invasive cyprinid within the Laurentian Great Lakes, is naturally reproducing in several Lake Erie tributaries, which has raised concerns of the species' spread throughout Lake Erie and the other Great Lakes.Knowledge of the recent invasion extent outside of the western basin of Lake Erie, particularly in eastern tributaries and nearshore waters, is limited.Understanding the invasion extent would improve the efficacy of ongoing coordinated multi-agency control efforts.Molecular tools, such as environmental DNA (eDNA), have shown promise for early detection of aquatic invasive species.In this study, water samples (N = 476) were collected for grass carp eDNA monthly between May and November in 2018 and 2019, at three sites in the Michigan waters of Lake Erie and the Detroit River.We fit Bayesian multi-scale occupancy models to determine differences in eDNA capture and detection probability among grass carp qPCR assays, sampling sites, and across time.To determine whether grass carp were physically present, and to validate eDNA samples, we quantified recent grass carp presence in sampled areas using an existing acoustic telemetry and field sampling framework.Our results indicate that grass carp eDNA capture probability differed among sites, but there was no difference among months.Positive grass carp eDNA detections were observed across multiple months at each site, with 69% of sitespecific sampling events testing positive for grass carp eDNA on at least one assay and replicate.The majority (65%) of weeks where positive eDNA sampling detections occurred also concurrently had one or more grass carp detected via acoustic telemetry 1-6 days prior.Our results highlight the potential utility of using eDNA to monitor the invasion extent of grass carp within the nearshore waters of Lake Erie.However, further evaluation of the factors that influence grass carp eDNA characteristics among sites within Lake Erie are needed to determine its efficacy for surveillance protocols by natural resource management agencies.

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.

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.