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.

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.

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.

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.

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.

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.

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.

Molecular and functional characterization of a ladderlectin-like molecule from ayu (Plecoglossus altivelis).

Ladderlectin is a member of C-type lectins (CTLs) in teleost fish and involved in innate immune defense. In this study, ayu (Plecoglossus altivelis) ladderlecin-like (PaLL-like) sequence was cloned, which encodes a polypeptide of 172 amino acids that includes a signal peptide and characteristic C-type lectin-like domains (CTLDs). Phylogenetically, PaLL-like was most closely related to its teleost counterpart from shishamo smelt (Spirinchus lanceolatus). Expression analysis revealed a ubiquitous expression profile, with highest expression detected in liver and its expression was up-regulated following Vibiro anguillarum infection. Similar to canonical CTLs, PaLL-like exhibited carbohydrate-binidng capacities to a wide range of well-defined mono-/di-saccharides and likely confer PaLL-like the ability to agglutinate all tested bacterial, including three Gram-positive species (i.e., Listeria monocytogenes, Staphylococcus aureus and Streptococcus iniae) and eight Gram-negative species (i.e., Edwardsiella tarda, Aeromonas (A.) hydrophila, Escherichia coli, Vibrio (V.) harveyi, V. anguillarum, V. parahemolyticus, A. versoni and V. vulnificus), in a calcium-dependent manner. Further functional studies revealed that PaLL-like displayed immunomodulatory activities leading to enhanced bactericidal activity of serum, pathogen opsonization and macrophage activation with increased expression of pro-inflammatory cytokines (i.e., PaIL-1ß and PaTNF-α). Collectively, these immunomodulatory activities of PaLL-like suppressed proliferations of V. anguillarum in targeted tissued in vivo and likely contributed to the increased survival rate of infected-fish. Overall, our results demonstrated PaLL-like is a critical component of innate immunity and provides protective effects against bacterial infection.

Environmental DNA detects Spawning Habitat of an ephemeral migrant fish (Anadromous Rainbow Smelt: Osmerus mordax).

BACKGROUND: Anadromous rainbow smelt (Osmerus mordax) have experienced a large range reduction in recent decades and the status of remnant spawning populations is poorly known in Maine, where these fish have significant ecological, cultural, and commercial relevance. Defining the remnant range of anadromous smelt is more difficult than for many declining fish species because adults are only ephemerally present while spawning in small coastal streams at night during spring runoff periods when traditional assessments can be unreliable or even hazardous. We hypothesized that eDNA might facilitate improved survey efforts to define smelt spawning habitat, but that detection could also face challenges from adult eDNA quickly flushing out of these small stream systems. We combined daytime eDNA sampling with nighttime fyke netting to ascertain a potential window of eDNA detection before conducting eDNA surveys in four streams of varying abundance. Hierarchical occupancy modeling was in turn employed to estimate eDNA encounter probabilities relative to numbers of sampling events (date), samples within events, and qPCR replicates within samples. RESULTS: Results from the combined eDNA and fyke net study indicated eDNA was detectable over an extended period, culminating approximately 8-13 days following peak spawning, suggesting developing smelt larvae might be the primary source of eDNA. Subsequently, smelt eDNA was readily detected in eDNA surveys of four streams, particularly following remediation of PCR inhibitors. Hierarchical occupancy modeling confirmed our surveys had high empirical detection for most sites, and that future surveys employing at least three sampling events, three samples per event, and six qPCR replicates can afford greater than 90% combined detection capability in low abundance systems. CONCLUSIONS: These results demonstrate that relatively modest eDNA sampling effort has high capacity to detect this ephemerally present species of concern at low to moderate abundances. As such, smelt eDNA detection could improve range mapping by providing longer survey windows, safer sampling conditions, and lower field effort in low density systems, than afforded by existing visual and netting approaches.

Polygenic discrimination of migratory phenotypes in an estuarine forage fish.

Migration is a complex phenotypic trait with some species containing migratory and nonmigratory individuals. Such life history variation may be attributed in part to plasticity, epigenetics, or genetics. Although considered semianadromous, recent studies using otolith geochemistry have revealed life history variation within the critically endangered Delta Smelt. Broadly categorizable as migratory or freshwater residents, we examined Restriction site Associated DNA sequencing data to test for a relationship between genetic variation and migratory behaviors. As previously shown, we found no evidence for neutral population genetic structure within Delta Smelt; however, we found significant evidence for associations between genetic variants and life history phenotypes. Furthermore, discriminant analysis of principal components, hierarchical clustering, and machine learning resulted in accurate assignment of fish into the freshwater resident or migratory classes based on their genotypes. These results suggest the presence of adaptive genetic variants relating to life history variation within a panmictic population. Mechanisms that may lead to this observation are genotype dependent habitat choice and spatially variable selection, both of which could operate each generation and are not exclusive. Given that the population of cultured Delta Smelt are being used as a refugial population for conservation, as a supply for wild population supplementation, and currently represent the majority of all living individuals of this species, we recommend that the hatchery management strategy consider the frequencies of life history-associated alleles and how to maintain this important aspect of Delta Smelt biological variation while under captive propagation.

Two ACTH analogs exert differential effects on monocytes/macrophages function regulation in ayu (Plecoglossus altivelis).

Adrenocorticotropic hormone (ACTH), a bioactive peptide of the family of melanocortins, is generated from pro-opiomelanocortin (POMC). So far, the research on the specific functions of ACTH in the immune system of teleosts is limited. We determined two complementary DNA (cDNA) sequences of POMC in ayu (Plecoglossus altivelis), termed PaPOMC-A and PaPOMC-B. PaPOMCs transcripts occurred in all examined tissues, and their expression in immune tissues changed following experimental infection with Vibrio anguillarum. PaACTH-B, but not PaACTH-A, suppressed the phagocytosis of monocytes/macrophages (MO/MФ). Two isoforms of PaACTH increased the bactericidal capacity of MO/MФ. PaACTH-A increased anti-inflammatory cytokine expression, while PaACTH-B decreased pro-inflammatory cytokine expression in MO/MФ. Compared with PaACTH-B treatment, the PaACTH-A treatment improved survival rate and reduced the bacterial load in V. anguillarum-infected ayu through interleukin (IL)-10. Our results indicate that the two PaACTH isoforms exert different effects in the host defense against bacterial infection.

A Y-linked anti-Müllerian hormone type-II receptor is the sex-determining gene in ayu, Plecoglossus altivelis.

Whole-genome duplication and genome compaction are thought to have played important roles in teleost fish evolution. Ayu (or sweetfish), Plecoglossus altivelis, belongs to the superorder Stomiati, order Osmeriformes. Stomiati is phylogenetically classified as sister taxa of Neoteleostei. Thus, ayu holds an important position in the fish tree of life. Although ayu is economically important for the food industry and recreational fishing in Japan, few genomic resources are available for this species. To address this problem, we produced a draft genome sequence of ayu by whole-genome shotgun sequencing and constructed linkage maps using a genotyping-by-sequencing approach. Syntenic analyses of ayu and other teleost fish provided information about chromosomal rearrangements during the divergence of Stomiati, Protacanthopterygii and Neoteleostei. The size of the ayu genome indicates that genome compaction occurred after the divergence of the family Osmeridae. Ayu has an XX/XY sex-determination system for which we identified sex-associated loci by a genome-wide association study by genotyping-by-sequencing and whole-genome resequencing using wild populations. Genome-wide association mapping using wild ayu populations revealed three sex-linked scaffolds (total, 2.03 Mb). Comparison of whole-genome resequencing mapping coverage between males and females identified male-specific regions in sex-linked scaffolds. A duplicate copy of the anti-Müllerian hormone type-II receptor gene (amhr2bY) was found within these male-specific regions, distinct from the autosomal copy of amhr2. Expression of the Y-linked amhr2 gene was male-specific in sox9b-positive somatic cells surrounding germ cells in undifferentiated gonads, whereas autosomal amhr2 transcripts were detected in somatic cells in sexually undifferentiated gonads of both genetic males and females. Loss-of-function mutation for amhr2bY induced male to female sex reversal. Taken together with the known role of Amh and Amhr2 in sex differentiation, these results indicate that the paralog of amhr2 on the ayu Y chromosome determines genetic sex, and the male-specific amh-amhr2 pathway is critical for testicular differentiation in ayu.

Female-specific genomic regions and molecular sex identification of the clearhead icefish (Protosalanx hyalocranius).

BACKGROUND: The clearhead icefish, Protosalanx hyalocranius, is an economically important fishery species in China. Since 1980s, P. hyalocranius was widely introduced into lakes and reservoirs of northern China for aquaculture. However, the lack of a rapid and cost-effective sex identification method based on sex specific genetic markers has hindered study on sex determination mechanisms and breeding applications. RESULTS: Female-specific genomic regions were discovered by comparing whole genome re-sequencing data of both males and females. Two female-specific genomic regions larger than 50 bp were identified, and one (598 bp) contained a putative FOXI gene, which was paralogous to another FOXI gene with sex-associated SNPs. The two FOXI sequences displayed significant length difference with nine deletions of total length of 230 bp. This deletion-type structural variation could be easily and efficiently detected by traditional PCR and agarose gel electrophoresis with one 569 bp band for males and two bands (569 and 339 bp) for females, which were validated in 50 females and 40 males with known phenotypic sexes. CONCLUSIONS: The results provided structural genomic evidence for the ZZ/ZW sex determination system in P. hyalocranius discovered in our previous study with association analysis of SNPs. Moreover, the female-specific markers and rapid and cost-effective PCR-based genetic sex identification method should have applications in further studies of sex determination mechanism for this species.

Draft genome of the Korean smelt Hypomesus nipponensis and its transcriptomic responses to heat stress in the liver and muscle.

Pond smelt (Hypomesus nipponensis) is a cold-freshwater fish species and a winter economic aquaculture resource in South Korea. Because of its high susceptibility to abnormal water temperature from global warming, a large number of smelt die in hot summers. Here, we present the first draft genome of H. nipponensis and transcriptomic changes in molecular mechanisms or intracellular responses under heat stress. We combined Illumina and PacBio sequencing technologies to generate the draft genome of H. nipponensis. Based on the reference genome, we conducted transcriptome analysis of liver and muscle tissues under normal (NT, 5°C) vs. warm (HT, 23°C) conditions to identify heat stress-induced genes and gene categories. We observed a total of 1987 contigs with N50 of 0.46 Mbp, with the largest contig (3.03 Mbp) in the assembled genome. A total of 20,644 protein-coding genes were predicted, and 19,224 genes were functionally annotated: 15,955 genes for Gene Ontology terms and 11,560 genes for KEGG Orthology. We conducted the lost and gained genes analysis compared with three species that: human, zebrafish, and salmon. In the lost genes analysis, we detected that smelt lost 4461 (22.16%), 2825 (10.62%), and 1499 (3.09%) genes compare with above three species, respectively. In the gained genes analysis, we observed that smelt gained 1133 (5.49%), 1670 (8.09%), and 229 (1.11%) genes compared with the above species, respectively. From transcriptome analysis, a total of 297 and 331 differentially expressed genes (DEGs) with a false discovery rate <0.05 were identified in the liver and muscle tissues, respectively. Gene enrichment analysis of DEGs indicates that upregulated genes were significantly enriched for lipid biosynthetic process (GO:0008610, P < 0.001) and regulation of apoptotic process (GO:0042981, P < 0.01), and genes were downregulated by immune responses such as myeloid cell differentiation (GO:0030099, P < 0.001) in the liver under heat stress. In muscle tissue, upregulated genes were enriched for hypoxia (GO:0001666, P < 0.05), transcription regulator activity (GO:0140110, P < 0.001), and calcium-release channel activity (GO:0015278, P < 0.01), and genes were downregulated for a nicotinamide nucleotide biosynthetic process (GO:0019359, P < 0.01). The results of KEGG pathway analysis were similar to that of gene enrichment analysis. The draft genome and transcriptomic of H. nipponensis will be a useful genetic resource for functional and evolutionary studies. Our findings will improve understanding of molecular mechanisms and heat responses and be useful for predicting survival of the smelt and its closely related species under global warming.

Genetic characteristics of the amphidromous fish Ayu Plecoglossus altivelis altivelis (Osmeriformes: Plecoglossidae) on Yaku-shima Island in Japan, the southernmost population of the subspecies.

The Ayu (Plecoglossus altivelis altivelis) population on Yaku-shima Island, Kagoshima Prefecture, Japan represents the southernmost population of the subspecies and is considered to be facing extinction. We investigated the genetic characteristics of Ayu in the Miyanoura River (MYU) in the northeast of the island and the Kurio River (KRO) in the southwest of the island, using partial sequences of mitochondrial (mt) control region and polymorphic microsatellite (simple sequence repeat: SSR) loci. The mtDNA analysis revealed that the Yaku-shima Island population was significantly differentiated from other Ayu populations; additionally, the MYU and KRO populations were significantly different from each other in terms of mtDNA (pairwise ΦST = 0.5826, P < 0.01) and SSR (pairwise ΦST = 0.1598, P < 0.01) analyses, indicating very little or no gene flow between them. The mtDNA haplotype diversity values were minimal for KRO and somewhat lower for MYU (h = 0.8176) than for each population from the mainland of Japan (Honshu-Kyushu) and the Korean Peninsula (h = 0.9905-1.0000). The mean values of expected heterozygosity (He) of SSRs were also lower in KRO (mean He = 0.555) than in MYU (mean He = 0.649). A considerably small effective population size (Ne = 100.1 for MYU, 151.2 for KRO) and a bottleneck effect for Yaku-shima Island population were suggested by SSR analysis. These findings underscore the importance of monitoring the genetic diversity of Ayu on Yaku-shima Island and the necessity of designing conservation policies for each river's population.

Insights into the Gut and Skin Microbiome of Freshwater Fish, Smelt (Hypomesus nipponensis).

Freshwater smelt (Hypomesus nipponensis) is a planktivorous fish found in the river of South Korea, Japan, China, and Russia. Because of its specific characteristics living in the cold temperature, this species is economically valuable in the various countries that held winter festival. The body size of the smelt is too small, so people consumed raw smelt during the winter festival sometimes. However, the microbial studies of smelt are nonexistent. Here, we characterized and compared the bacterial communities in the gut and skin of freshwater smelts. We amplified, sequenced, and analyzed the V4 regions of bacterial 16S rRNA genes from freshwater smelts. The microbial diversity in the skin (375 OTUs) was much greater than that in the gut (250 OTUs). At the phylum level, Proteobacteria (gut: 51.5%; skin: 52.9%), Firmicutes (gut: 30.6%; skin: 25.4%), Bacteroidetes (gut: 7.7%; skin: 14.7%), and Actinobacteria (gut: 5.2%; skin: 3.8%) were predominant in both organs. At the genus level, Sphingomonas (gut: 24.9%; skin: 4.4%, P < 0.01) was more abundant in the gut, whereas Acinetobacter (gut: 0.8%; skin: 11.8%, P = 0.02) and Pseudomonas (gut: 0.3%; skin: 2.1%, P = 0.01) were more abundant in the skin. Both beneficial (Lactobacillus) and harmful (Staphylococcus and Streptococcus) bacteria were detected in both organs, even under freshwater conditions. These results revealed that smelts have their own unique microbial communities in the gut and skin. Our findings broaden the understanding of planktivorous freshwater fish microbiomes and provide new insights into fish microbiomes for ensuring food safety.

Two transcription factors PU.1a and PU.1b have different functions in the immune system of teleost ayu.

Transcription factor PU.1 is a regulator of macrophage function, however, the specific function of PU.1 in teleost monocytes/macrophages (MO/MФ) remains unknown. We determined the cDNA sequence of two PU.1 genes from ayu (Plecoglossus altivelis; PaPU.1a and PaPU.1b). Sequence comparisons showed that PaPU.1 were most closely related to the PU.1 of rainbow smelt (Osmerus mordax). The PU.1 transcripts were mainly expressed in the spleen, and their expression was altered in various tissues upon infection with Vibrio anguillarum. PaPU.1a and PaPU.1b proteins were upregulated in MO/MФ, after infection. RNA interference was employed to knockdown PaPU.1a and PaPU.1b to investigate their function in MO/MФ. The expression of inflammatory cytokines was regulated by PaPU.1a, but not PaPU.1b, in ayu MO/MФ upon V. anguillarum infection. Both PaPU.1a and PaPU.1b knockdown lowered the phagocytic activity of MO/MФ. Furthermore, PaPU.1b knockdown attenuated MO/MФ bacterial killing capability. Our results indicate that two PaPU.1 genes differentially modulate the immune response in ayu MO/MФ against bacterial infection.

Characterization of ayu (Plecoglossus altivelis) urocortin: The function of an endocrine factor in monocyte/macrophage regulation.

Urocortin (UCN) is a hormone in the hypothalamic-pituitary-adrenal axis that is expressed in various immune cells. However, the function of teleost UCN in the immune system remains unclear. In this study, we cloned the cDNA sequence of UCN from ayu Plecoglossus altivelis (PaUCN). Sequence and phylogenetic tree analyses showed that PaUCN clustered within the fish UCN 1 group and was most related to the rainbow trout (Oncorhynchus mykiss) UCN. PaUCN was expressed in all tested tissues and its expression increased in the liver, spleen, head kidney, and gill upon Vibrio anguillarum infection. Mature PaUCN protein (mPaUCN) treatment affected the phagocytosis and bacterial killing of monocytes/macrophages (MO/MФ). mPaUCN reduced pro-inflammatory cytokine expression in MO/MФ, which was partially mediated via interaction with ayu interleukin-6. mPaUCN reduced bacterial load and increased the survival of V. anguillarum-infected ayu. Overall, UCN as an endocrine factor regulates the immune response of ayu after infection by activating MO/MФ, thus contributing to enhance fish survival.

Understanding seasonal migration of Shishamo smelt in coastal regions using environmental DNA.

Migratory organisms have their own life histories that efficiently link multiple ecosystems. Therefore, comprehensive understanding of migration ecologies of these organisms is essential for both species conservation and ecosystem management. However, monitoring migration at fine spatiotemporal scales, especially in open marine systems, often requires huge costs and effort. Recently, environmental DNA (eDNA) techniques that utilize DNA released from living organisms into their environment became available for monitoring wild animals without direct handling. In this study, we conducted an eDNA survey for understanding marine migration of an endemic fish species, Shishamo smelt (Spirinchus lanceolatus). We examined 1) seasonal habitat changes in coastal regions and 2) environmental factors potentially driving the migration of this species. The eDNA concentrations along a 100 km-long coastline exhibited spatiotemporal variation, suggesting that this species shifts their habitat away from nearshore areas between spring and summer. We also found a significantly negative association between the eDNA concentration and sea surface temperature. That finding suggests that the offshore migration of this species is associated with increased sea surface temperature. This study reveals new aspects of S. lanceolatus life history in coastal regions. Together with our previous eDNA study on the freshwater migration of S. lanceolatus, this study illustrates the potential of eDNA techniques for understanding the whole life history of this migratory species.

Genomic basis of the loss of diadromy in Galaxias maculatus: Insights from reciprocal transplant experiments.

Diadromy is known for having major effects on the distribution and richness of aquatic species, and so does its loss. The loss of diadromy has led to the diversification of many species, yet research focusing on understanding its molecular basis and consequences are limited. This is particularly true for amphidromous species despite being the most abundant group of diadromous species. Galaxias maculatus, an amphidromous species and one of the most widely distributed fishes in the Southern Hemisphere, exhibits many instances of nonmigratory or resident populations. The existence of naturally replicated resident populations in Patagonia can serve as an ideal system for the study of the mechanisms that lead to the loss of the diadromy and its ecological and evolutionary consequences. Here, we studied two adjacent river systems in which resident populations are genetically differentiated yet derived from the same diadromous population. By combining a reciprocal transplant experiment with genomic data, we showed that the two resident populations followed different evolutionary pathways by exhibiting a differential response in their capacity to survive in salt water. While one resident population was able to survive salt water, the other was not. Genomic analyses provided insights into the genes that distinguished (a) migratory from nonmigratory populations; (b) populations that can vs those that cannot survive a saltwater environment; and (c) between these resident populations. This study demonstrates that the loss of diadromy can be achieved by different pathways and that environmental (selection) and random (genetic drift) forces shape this dynamic evolutionary process.