Comparative evaluation for the performance of environmental DNA and RNA analyses targeting mitochondrial and nuclear genes from ayu (Plecoglossus altivelis).

Environmental DNA and RNA (eDNA and eRNA; collectively eNA) analyses have the potential for non-invasive and cost-efficient biomonitoring compared with traditional capture-based surveys. Although various types of eNA particles, including not only mitochondrial eDNA but also nuclear eDNA and their transcripts, are present in the water, performances of eNA detection and quantification have not yet been evaluated sufficiently across multiple mitochondrial and nuclear genes. We conducted a tank experiment with ayu (Plecoglossus altivelis) to compare the detection sensitivity, yields per water sample, and quantification variability between replicates of each type of eNAs. The assay targeting the multi-copy nuclear gene exhibited a higher sensitivity than the assay targeting the mitochondrial gene, and both the target eDNA and eRNA concentrations per water sample were higher for the nuclear gene. On the contrary, variation in eRNA quantifications per sample does not necessarily correspond to that in eDNA, and the intra-sample quantification variability (represented as the CVs between PCR replicates) tended to be larger for eRNA than eDNA. Our results suggested that, even if suitable to the sensitive detection of species occurrence, the use of eRNA particularly derived from multi-copy nuclear gene may not be necessarily appropriate for the reliable assessment of species abundance. The findings in this study would help optimize eNA analyses for making biomonitoring and stock assessment in aquatic environments more efficient and reliable.

Parasite and genetic diversity of big-scale sand smelt (Atherina boyeri Risso, 1810) populations in their natural and expansion ranges in Ukraine.

The big-scale sand smelt (Atherina boyeri) is an Atlanto-Mediterranean amphidromous fish species found within the Black Sea. Here, we assess differences in the parasite fauna of big-scale sand smelt populations from their natural range in the northwestern Black Sea and from their expansion range in the Lower and Middle River Dnipro. In addition, we undertook a microsatellite analysis to assess the genetic similarity of fish from the different locations. We found that the parasite community of fish in their natural range was wider than that from their expansion range. While the Gulf of Odesa was most distant from all other localities by parasite community composition and the Dnipro Reservoir was characterised by an absence of parasites (newest and most distant expansion locality), only fish from the Danube Delta showed a significant genetic difference. Our results suggest that the parasite community of big-scale sand smelt is primarily influenced by environmental factors, such as habitat type, water salinity and/or prey composition. Both microsatellite analysis and parasite community species composition (e.g. the presence of the marine Telosentis exiguus in the Kakhovka Reservoir and freshwater Raphidascaris sp. in the Gulf of Odesa) confirmed that populations in the River Dnipro reservoirs had, at some time, been connected with native marine populations, thus also confirming the species' amphidromous nature.

Linkage between spatiotemporal distribution of environmental DNA and phenological activity in an amphidromous fish, ayu Plecoglossus altivelis altivelis, in a river located in its northernmost distributional area.

Environmental DNA (eDNA) is a promising tool for the continuous monitoring of fish ecology and diversity. However, its potential for describing the phenological activity of fish has rarely been examined. This study aimed to elucidate a linkage between the spatiotemporal distribution of eDNA and the phenology of an amphidromous fish, ayu Plecoglossus altivelis altivelis, in a river in Hokkaido, Japan, which is its northernmost distributional area. A significant positive correlation between eDNA concentration and catch per unit effort of P. a. altivelis in the river confirmed the use of eDNA as a surrogate for the abundance of P. a. altivelis. eDNA of P. a. altivelis was first detected in late April on a sandy beach adjacent to the river mouth. Subsequent to its first detection at the lowest site in the river in early May, eDNA spread throughout the river, indicating the upstream migration of P. a. altivelis. Spawning activity was also represented by a rapid increase in eDNA concentration and its surge at night in the lowest reaches of the river during September and October. These results suggest that upstream migration and spawning primarily commenced when the water temperature reached 10°C and decreased below 20°C, respectively. This observation is consistent with the behavioral responses observed in P. a. altivelis populations from other regions of Japan. Consequently, this study demonstrated that eDNA distribution was closely linked to the phenological activity of P. a. altivelis and that eDNA is a powerful tool for studying the phenology of migratory fishes.

Population structure and demographic history for year cohort dynamics of landlocked ayu Plecoglossus altivelis altivelis in dam reservoir of Japan.

Ayu Plecoglossus altivelis altivelis is a valuable osmeroid species for inland fishery in Japan. It is classified into two ecological forms of amphidromous migrating between rivers and sea and landlocked migrating between rivers and lakes or dam reservoirs. The number of dams and their reservoirs has remarkably increased in the twenty-first century under climate change, because of their respective roles in hydropower generation with negligible carbon emissions and in flood control. Dam reservoirs therefore become increasingly important as inland nursery grounds of ayu. In this study, we investigated the reproduction status of landlocked ayu migrating in the Haidzuka Dam reservoir and the Tabusa River in western Japan by molecular phylogenetic analysis based on population structure and demographic history for year cohort dynamics. A total of 849 individuals were collected monthly from October 2018 to September 2021 according to an annual life cycle of ayu. Nucleotide sequences of the partial mitochondrial DNA control region yielded 31 haplotypes, consisting of 4 shared haplotypes among the 2019, 2020 and 2021 cohorts and 27 unique haplotypes. The overall haplotype diversity and nucleotide diversity were calculated to be relatively low at 0.3503 ± 0.0206 and 0.0077 ± 0.0045, respectively, suggesting a founder event by dominant haplotypes. Star-shaped radiational haplotypes from dominant shared haplotypes on the median-joining network likely support a founder event. Although pairwise ФST values were determined to be very low among the year cohorts, only the 2019 cohort was found to have a significant difference from the 2020 and 2021 cohorts, for both of which Tajima's D values were also statistically significant. For the overall population, multimodal mismatch distribution and negative Tajima's D and Fu's Fs values in the neutrality test suggested population expansion or population subdivision. The native riverine population in the Tabusa River suffered habitat fragmentation and population bottleneck from dam construction, and therefore severe founder effect remained behind the artificially landlocked population with a low level of genetic diversity in the Haidzuka Dam reservoir.

Ecological variation in invasive brown trout (Salmo trutta) within a remote coastal river catchment in northern Patagonia complicates estimates of invasion impact.

Salmonids were first introduced into the Chilean fresh waters in the 1880s, and c. 140 years later, they are ubiquitous across Chilean rivers, especially in the southern pristine fresh waters. This study examined the brown trout (Salmo trutta) and native taxa ecology in two adjacent but contrasting rivers of Chilean Patagonia. During spring 2016 and spring-fall 2017 we examined the variation in benthic macroinvertebrate and fish community composition and characterized fish size structure, stomach contents, and stable isotopes (δ13 C and δ15 N) to understand population structure, fish diet, and trophic interactions between S. trutta and native taxa. The native Galaxias maculatus (puye) dominated the fish community (74% of abundance). S. trutta was less abundant (16% of survey catch) but dominated the fish community (over 53%) in terms of biomass. S. trutta showed distinct diets (stomach content analysis) in the two rivers, and individuals from the larger river were notably more piscivorous, consuming native fish with a relatively small body size (<100-mm total length). Native fishes were isotopically distinct from S. trutta, which showed a wider isotopic niche in the smaller river, indicating that their trophic role was more variable than in the larger river (piscivorous). This study provides data from the unstudied pristine coastal rivers in Patagonia and reveals that interactions between native and introduced species can vary at very local spatial scales.

Gorges partition diversity within New Zealand flathead Galaxias populations.

Understanding the landscape factors governing population connectivity in riverine ecosystems represents an ongoing challenge for freshwater biologists. We used DNA sequence analysis to test the hypothesis that major geomorphological features underpin freshwater-limited fish diversity in a tectonically dynamic region of New Zealand. Phylogeographic analysis of 101 Galaxias depressiceps cytochrome b sequences, incorporating 55 localities from southern New Zealand, revealed 26 haplotypes, with only one shared among rivers. We detect strong hierarchical genetic differentiation both among and within river systems. Genetic structuring is particularly pronounced across the Taieri River system (63 individuals from 35 sites, 18 haplotypes), with 92% of variation partitioned among locations. Distinctive within-river genetic clusters are invariably associated with major subcatchment units, typically isolated by substantial gorges. The anomalous distribution of a single lineage across a major drainage divide is consistent with local, tectonically driven headwater capture. We conclude that major landscape features such as gorges can strongly partition riverine fish diversity and constrain freshwater biodiversity.

Rainbow smelt (Osmerus mordax) influence on walleye (Sander vitreus) recruitment decline: mtDNA evidence supporting the predation hypothesis.

Rainbow smelt (Osmerus mordax) have been introduced widely but are associated with declines in walleye (Sander vitreus) recruitment. A primary hypothesis for these declines is that O. mordax consume larval S. vitreus. We confirmed overlapping spatial-temporal distributions of larval S. vitreus and O. mordax in our study system and used mtDNA analyses to determine if O. mordax stomach contents contained S. vitreus. Approximately 20% of O. mordax composite stomach samples were considered positive for S. vitreus consumption. These findings support the predation hypothesis and have S. vitreus management/stocking implications.

Expanding non-invasive approaches for fish-health monitoring: A survey of the epidermal mucous metabolomes of phylogenetically diverse freshwater fish species.

There is a pressing need for more-holistic approaches to fisheries assessments along with growing demand to reduce the health impacts of sample collections. Metabolomic tools enable the use of sample matrices that can be collected with minimal impact on the organism (e.g., blood, urine, and mucus) and provide high-throughput, untargeted biochemical information without the requirement of a sequenced genome. These qualities make metabolomics ideal for monitoring a wide range of fish species, particularly those under protected status. In the current study, we surveyed the relative abundances of 120 endogenous metabolites in epidermal mucus across eight freshwater fish species belonging to seven phylogenetic orders. Principal component analysis was used to provide an overview of the data set, revealing strong interspecies relationships in the epidermal mucous metabolome. Normalized relative abundances of individual endogenous metabolites were then used to identify commonalities across multiple species, as well as those metabolites that showed notable species specificity. For example, taurine was measured in high relative abundance in the epidermal mucus of common carp (Cyprinus carpio), northern pike (Esox lucius), golden shiner (Notemigonus crysoleucas), rainbow trout (Oncorhynchus mykiss), and rainbow smelt (Osmerus mordax), whereas γ-amino butyric acid (GABA) exhibited a uniquely high relative abundance in flathead catfish (Pylodictis olivaris). Finally, hierarchical cluster analysis was used to evaluate species relatedness as characterized by both the epidermal mucous metabolome (phenotype) and genetic phylogeny (genotype). This comparison revealed species for which relatedness in the epidermal mucous metabolome composition closely aligns with phylogenetic relatedness (e.g., N. crysoleucas and C. carpio), as well as species for which these two measures are not well aligned (e.g., P. olivaris and Polyodon spathula). These, and other findings reported here, highlight novel areas for future research with fish, including development of epidermal mucous-based markers for non-invasive health monitoring, sex determination, and hypoxia tolerance.

Bifenthrin, a Ubiquitous Contaminant, Impairs the Development and Behavior of the Threatened Longfin Smelt during Early Life Stages.

The Longfin Smelt (Spirinchus thaleichthys) population in the San Franscisco Bay/Sacramento-San Joaquin Delta (Bay-Delta) has declined to ∼1% of its pre-1980s abundance and, as a result, is listed as threatened under the California Endangered Species Act. The reasons for this decline are multiple and complex, including the impacts of contaminants. Because the spawning and rearing seasons of Longfin Smelt coincide with the rainy season, during which concentrations of contaminants increase due to runoff, we hypothesized that early life stages may be particularly affected by those contaminants. Bifenthrin, a pyrethroid insecticide commonly used in agricultural and urban sectors, is of concern. Concentrations measured in the Bay-Delta have been shown to disrupt the behavior, development, and endocrine system of other fish species. The objective of the present work was to assess the impact of bifenthrin on the early developmental stages of Longfin Smelt. For this, embryos were exposed to 2, 10, 100, and 500 ng/L bifenthrin from fertilization to hatch, and larvae were exposed to 2, 10, and 100 ng/L bifenthrin from one day before to 3 days post-hatch. We assessed effects on size at hatch, yolk sac volume, locomotory behavior, and upper thermal susceptibility (via cardiac endpoints). Exposure to these environmentally relevant concentrations of bifenthrin did not significantly affect the cardiac function of larval Longfin Smelt; however, exposures altered their behavior and resulted in smaller hatchlings with reduced yolk sac volumes. This study shows that bifenthrin affects the fitness-determinant traits of Longfin Smelt early life stages and could contribute to the observed population decline.

Isolation and characterization of 17 polymorphic microsatellite loci with tri- and tetra-nucleotide repeat motifs for Ayu (Plecoglossus altivelis) using next-generation sequencing.

BACKGROUND: Ayu or sweetfish, Plecoglossus altivelis, an amphidromous fish ranging in the northwestern Pacific, is economically important inland fisheries and aquaculture resources. Genetic characterization of wild Ayu and derived culture seeds with competent molecular genetic markers is still insufficient for their sustainable use. Microsatellite DNA markers with larger repeat motifs (e.g. tri- and tetra-nucleotide motifs) are convenient and accurate compared with those having mono- and di-nucleotide motifs, but the latter motifs characterized most Ayu microsatellite markers developed previously. METHODS AND RESULTS: Here, we isolated and characterized 17 polymorphic microsatellite DNA markers with tri- and tetra-nucleotide repeat motif using next-generation sequencing. Alleles per locus varied from 6 to 23. The observed and expected heterozygosities ranged from 0.542 to 1.000 and 0.709 to 0.951, respectively. Polymorphic information content (PIC) of 15 out of the 17 loci were high (≧ 0.700), suggesting them to be highly informative. Twelve of the 17 loci were used for preliminary assignment test among three collections, and successfully allocated the examined fish to the original populations. CONCLUSION: The novel polymorphic microsatellite markers developed herein will be useful to examine the genetic diversity and population structure of wild Ayu and the effect of seed transplantation on native populations, providing a tool for conservation and sustainable adaptive management of this species.

No observed developmental effects in early life stages of capelin (Mallotus villosus) exposed to a water-soluble fraction of crude oil during embryonic development.

The rise in offshore oil and gas operations, maritime shipping, and tourism in northern latitudes enhances the risk of oil spills to sub-Arctic and Arctic coastal environments. Therefore, there is a need to understand the potential adverse effects of petroleum on key species in these areas. Here, we investigated the effects of oil exposure on the early life stages of capelin (Mallotus villosus), an ecologically and commercially important Barents Sea forage fish species that spawns along the coast of Northern Norway. Capelin embryos were exposed to five different concentrations (corresponding to 0.5-19 µg/L total PAHs) of water-soluble fraction (WSF) of crude oil from 6 days post fertilization (dpf) until hatch (25 dpf), and development of larvae in clean seawater was monitored until 52 dpf. None of the investigated endpoints (embryo development, larval length, heart rate, arrhythmia, and larval mortality) showed any effects. Our results suggest that the early life stages of capelin may be more robust to crude oil exposure than similar life stages of other fish species.

Improvement of the ayu (Plecoglossus altivelis) draft genome using Hi-C sequencing.

OBJECTIVE: The ayu or sweetfish Plecoglossus altivelis is ray-finned fish that is widely distributed in East Asia. The genome size of ayu was estimated at approximately 420 Mb. Previously, we reported on ayu draft genome assembly by whole-genome shotgun using Illumina short reads and PacBio long reads; however, the assembly was not to chromosome level. Therefore, to improve the draft genome sequence of ayu to chromosome level, we performed in situ Hi-C sequencing as a source of linkage information. RESULTS: The ayu genome assembly yielded 28 large scaffolds that corresponded to the karyotype of ayu (n = 28). The resulting ayu genome assembly has a N50 scaffold length of 17.0 Mb, improved from 4.3 Mb. The high-quality reference genome will be helpful for phylogenetic research on bony fishes and for breeding programs in ayu aquaculture.

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

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

Decreased resistance to bacterial cold-water disease and excessive inflammatory response in ayu (Plecoglossus altivelis) reared at high water temperature.

Introduction: Temporal elevation of water temperature positively affects immune activity and disease resistance in poikilothermic teleost fish. The ayu, Plecoglossus altivelis, an important fish species for Japanese freshwater fisheries, is usually produced under higher water temperatures than the natural conditions to facilitate rapid growth. However, it has been reported that rearing fish at higher water temperatures inhibits the development of the thymus, suggesting that resistance to infectious diseases is reduced in ayu reared at higher water temperatures. Here, we show that decreased resistance to bacterial cold-water disease and excessive inflammatory responses occurred in ayu reared at 22°C compared with those reared at lower temperatures. Methods: Ayu larvae were reared at 12°C, 15°C and 22°C for 77 days and fed 3% of their body weight. Thymus index and condition factor was calculated after the fish rearing. Then, ayu reared at the different temperatures were challenged with Flavobacterium psychrophilum and the fish were sampled for histopathology and gene expression analyses. Further, the fish were vaccinated with formalin-killed F. psychrophilum and continuously reared at the three different water temperatures. Serum antibody titer was determined by ELISA and cumulative mortality in each group was recorded after the bacterial challenge. Results: Ayu reared at 22°C showed a significantly lower thymus index and higher condition factor than those reared at lower temperatures. Infiltrated leukocytes and many melanin pigments were frequently observed in the adipose tissues and spleens of ayu reared at 22°C, respectively, but not in those reared at 12°C. The gene expression levels of inflammatory cytokines such as IL-1ß, IL-8 and TNFα in the spleen were significantly higher in the 22°C group than in the 12°C group. The cumulative survival rate after challenge with Flavobacterium psychrophilum was 51.7%, 40.0% and 13.3% in the 12°C, 15°C and 22°C groups, respectively. The relative percent survival values of vaccinated fish reared at 15°C and 22°C groups were lower than those reared at 12°C. Moreover, the specific antibody titer of the vaccinated fish was the lowest in the 22°C group and the highest in the 12°C group. Discussion: These results suggest that rearing the fish under high water temperature causes excessive inflammatory responses similar to metabolic inflammation in human obesity, resulting in a decrease of disease resistance. In addition, thymic involution induced by higher water temperature probably leads the poor response to vaccination. The present study provides insights into the physiological and immunological changes of fish under global warming.

Transposon mutagenesis and genome sequencing identify two novel, tandem genes involved in the colony spreading of Flavobacterium collinsii, isolated from an ayu fish, Plecoglossus altivelis.

Bacteria of the family Flavobacteriaceae (flavobacteria) primarily comprise nonpathogenic bacteria that inhabit soil and water (both marine and freshwater). However, some bacterial species in the family, including Flavobacterium psychrophilum and Flavobacterium columnare, are known to be pathogenic to fish. Flavobacteria, including the abovementioned pathogenic bacteria, belong to the phylum Bacteroidota and possess two phylum-specific features, gliding motility and a protein secretion system, which are energized by a common motor complex. Herein, we focused on Flavobacterium collinsii (GiFuPREF103) isolated from a diseased fish (Plecoglossus altivelis). Genomic analysis of F. collinsii GiFuPREF103 revealed the presence of a type IX secretion system and additional genes associated with gliding motility and spreading. Using transposon mutagenesis, we isolated two mutants with altered colony morphology and colony spreading ability; these mutants had transposon insertions in pep25 and lbp26. The glycosylation material profiles revealed that these mutants lacked the high-molecular-weight glycosylated materials present in the wild-type strain. In addition, the wild-type strains exhibited fast cell population movement at the edge of the spreading colony, whereas reduced cell population behavior was observed in the pep25- and lbp26-mutant strains. In the aqueous environment, the surface layers of these mutant strains were more hydrophobic, and they formed biofilms with enhanced microcolony growth compared to those with the wild-type. In Flavobacterium johnsoniae, the Fjoh_0352 and Fjoh_0353 mutant strains were generated, which were based on the ortholog genes of pep25 and lbp26. In these F. johnsoniae mutants, as in F. collinsii GiFuPREF103, colonies with diminished spreading capacity were formed. Furthermore, cell population migration was observed at the edge of the colony in wild-type F. johnsoniae, whereas individual cells, and not cell populations, migrated in these mutant strains. The findings of the present study indicate that pep25 and lbp26 contribute to the colony spreading of F. collinsii.

No difference between critical and sprint swimming speeds for two galaxiid species.

Researchers have used laboratory experiments to examine how fish might be affected by anthropogenic alterations and conclude how best to adjust fish passage and culvert remediation designs in response. A common way to document swimming performance for this purpose is measuring fish critical swimming speed (Ucrit ). Nonetheless, the Ucrit protocol as defined by Brett [(1964) Journal of the Fisheries Research Board of Canada, 21, 1183-1226] may be inappropriate for studying swimming performance and determining how it relates to upstream migration in benthic fish, as they may not actively swim throughout the entire Ucrit test. An alternative method to estimate swimming performance is sprint swimming speed (Usprint ), which is suggested to be a measure of the burst speed of fish rather than maximum sustained swimming speed. The authors conducted comparative swimming performance experiments to evaluate whether Usprint can be used to compare swimming performance of benthic species to that of pelagic, actively swimming species. They measured individual swimming speeds of inanga (Galaxias maculatus), an actively swimming pelagic species, and banded kokopu (Galaxias fasciatus), a fish that exhibits benthic station-holding behaviour, using both the Usprint and Ucrit test. Experiments revealed that no significant statistical difference between swimming speeds was estimated using the Ucrit and Usprint test protocols for both G. maculatus and G. fasciatus. The result of this study suggests that fish swimming speeds obtained using these two methods are comparable for the species used in this study. By using Usprint for benthic-associated fish and Ucrit for pelagic fish, we may be able to compare a broader range of species' swimming abilities for use in a fish passage context.

Genome-wide DNA methylation predicts environmentally driven life history variation in a marine fish.

Epigenetic modifications are thought to be one of the molecular mechanisms involved in plastic adaptive responses to environmental variation. However, studies reporting associations between genome-wide epigenetic changes and habitat-specific variations in life history traits (e.g., lifespan, reproduction) are still scarce, likely due to the recent application of methylome resequencing methods to non-model species. In this study, we examined associations between whole genome DNA methylation and environmentally driven life history variation in 2 lineages of a marine fish, the capelin (Mallotus villosus), from North America and Europe. In both lineages, capelin harbor 2 contrasting life history tactics (demersal vs. beach-spawning). Performing whole genome and methylome sequencing, we showed that life history tactics are associated with epigenetic changes in both lineages, though the effect was stronger in European capelin. Genetic differentiation between the capelin harboring different life history tactics was negligible, but we found genome-wide methylation changes in both lineages. We identified 9,125 European and 199 North American differentially methylated regions (DMRs) due to life history. Gene ontology (GO) enrichment analysis for both lineages revealed an excess of terms related to neural function. Our results suggest that environmental variation causes important epigenetic changes that are associated with contrasting life history tactics in lineages with divergent genetic backgrounds, with variable importance of genetic variation in driving epigenetic variation. Our study emphasizes the potential role of genome-wide epigenetic variation in adaptation to environmental variation.

Does the loss of diadromy imply the loss of salinity tolerance? A gene expression study with replicate nondiadromous populations of Galaxias maculatus.

The recurrent colonization of freshwater habitats and subsequent loss of diadromy is a major ecological transition that has been reported in many ancestrally diadromous fishes. Such residency is often accompanied by a loss of tolerance to seawater. The amphidromous Galaxias maculatus has repeatedly colonized freshwater streams with evidence that freshwater-resident populations exhibit stark differences in their tolerance to higher salinities. Here, we used transcriptomics to gain insight into the mechanisms contributing to reduced tolerance to higher salinities in freshwater resident populations. We conducted an acute salinity challenge (0 ppt to 23-25 ppt) and measured osmoregulatory ability (muscle water content) over 48 h in three populations: diadromous, saltwater intolerant resident (Toltén), and saltwater tolerant resident (Valdivia). RNA sequencing of the gills identified genes that were differentially expressed in association with the salinity change and associated with the loss of saltwater tolerance in the Toltén population. Key genes associated with saltwater acclimation were characterized in diadromous G. maculatus individuals, some of which were also expressed in the saltwater tolerant resident population (Valdivia). We found that some of these "saltwater acclimation" genes, including the cystic fibrosis transmembrane conductance regulator gene (CFTR), were not significantly upregulated in the saltwater intolerant resident population (Toltén), suggesting a potential mechanism for the loss of tolerance to higher salinities. As the suite of differentially expressed genes in the diadromous-resident comparison differed between freshwater populations, we hypothesize that diadromy loss results in unique evolutionary trajectories due to drift, so the loss of diadromy does not necessarily lead to a loss in upper salinity tolerance.

La colonización recurrente de hábitats de agua dulce y la subsecuente pérdida de diadromía es una transición ecológica importante que ha sido reportada en varias especies de peces con ancestros diádromos. Esta residencia está acompañada frecuentemente por la pérdida de tolerancia a ambientes de agua salada. Galaxias maculatus, especie anfídroma, ha colonizado ríos repetidamente y existe evidencia que las poblaciones residentes presentan diferencias respecto a la tolerancia al agua salada. En este estudio, usamos transcriptómica para dilucidar los mecanismos que contribuyen a la reducida tolerancia a altas salinidades en las poblaciones residentes de agua dulce. Realizamos un desafío agudo de salinidad (0 ppt a 23-2 ppt) y medimos la habilidad osmoreguladora (contenido de agua en músculo) por 48 horas en individuos de tres poblaciones: una diádroma, una intolerante a agua salada (Toltén) y una tolerante a agua salada (Valdivia). Con el secuenciamiento de ARN de las branquias identificamos los genes expresados diferencialmente al cambio de salinidad y cuales están asociados a la pérdida de tolerancia a agua salada en la población de Toltén. Genes claves asociados a la aclimatación al agua salada fueron caracterizados en individuos diádromos, algunos de ellos también se expresaron en la población residente tolerante al agua salada (Valdivia). Sin embargo, algunos genes involucrados en la aclimatación al agua salada, incluyendo el gen regulador de la conductancia transmembrana de la fibrosis quística (CFTR), no se diferenciaron significativamente en la población residente intolerante al agua salada (Toltén), sugiriendo un mecanismo potencial de la pérdida de tolerancia a ambientes con salinidad elevada. Como el conjunto de genes expresados difiere entre las dos poblaciones residentes al compararse con la población diádroma, hipotetizamos que la pérdida de diadromía resulta en trayectorias evolutivas únicas debido a deriva génica, por lo que la pérdida de la diadromía no necesariamente conlleva a la pérdida de la tolerancia a aguas saladas.

Evaluate effects of the dilution medium and holding time on various motility parameters of delta smelt semen.

Sperm preservation is an efficient technique used for the recovery, conservation, and management of some endangered fish species. The present study was conducted to explore how preservation time would affect sperm and spawning performance in the endangered delta smelt (Hypomesus transpacificus). Sperm were preserved with the modified Hanks balanced salt solution at 14.7-16.9 °C. The Kruskal-Wallis test of sperm parameters using OpenCASA plugin in ImageJ software showed that sperm (n = 33♂) had significantly higher velocity and motility within the first 5 s after activation than that of other time points, while sperm had the lowest velocity and motility after 3 min post activation (P < 0.001). The findings (n = 30♂) also showed fresh sperm had higher velocity and motility than preserved sperm, while the sperm preserved for over 24 h showed a significantly low performance (P < 0.001). The nonlinear mixed effects models of fertilization results (n = 14♂ × 70♀) indicated the fresh sperm and sperm preserved for 1 h had higher fertilization rates than other preservation times (P < 0.001). The hatching rate (n = 14♂ × 70♀) also showed the fresh sperm and sperm preserved for 3 min and 1 h had higher hatching rates than other preservation times (P < 0.001). Overall, the study showed the best sperm performance in delta smelt was found within the first 5 s post activation, and the best fertilization and hatching rates were found when the sperm were fresh and preserved for 1 h. The findings of this study provide information for the first time about how long the delta smelt's sperm are motile for quality analysis, and how the preservation time can affect sperm quality, fertility, and hatching of this species for future applications.

Machine learning based assessment of small-bodied fish tracking to evaluate spoiler baffle fish passage design.

Fish passage research is important to mitigate the adverse effects of fragmented river habitats caused by waterway structures. The scale at which this research is undertaken varies from small-scale laboratory prototype studies to in-situ observations at various fish passage structures and bottlenecks. Using DeepLabCut, we introduce and evaluate a machine learning based workflow to track small-bodied fish in order to facilitate improved fish passage management. We specifically studied the behaviour and kinematics of Galaxias maculatus, a widespread diadromous Southern Hemisphere fish species. Upstream fish passage was studied in the presence of three different patches of spoiler baffles at an average water velocity of 0.4 m/s. In semi-supervised mode, the fish locations were extracted, and fish behaviour, such as swimming pathways and resting locations, was analysed based on extracted positions and recorded kinematic parameters. Individual fish behaviour and kinematic parameters were then used to assess the suitability of the three different spoiler baffle designs for enhancing fish passage. Using this technique, we were able to demonstrate where different spoiler baffle configurations resulted in significant differences in fish passage success and behaviour. For example, medium-spaced smaller baffles provided more accessible and uniform resting locations, which were required for efficient upstream passage. Results are discussed in relation to fish passage management at small instream structures.