Complete genome sequences of Edwardsiella ictaluri strains isolated from ayu (Plecoglossus altivelis altivelis) in the Kagami and Shimanto Rivers, Kochi, Japan.

The complete genome sequences of seven Edwardsiella ictaluri strains, isolated from the kidneys of dead ayu (Plecoglossus altivelis altivelis) in Kochi's Kagami and Shimanto Rivers, Japan, were determined. Multilocus sequencing typing revealed that their genotypes were sequence-type ST26.

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.

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.

Characterization of the bacterial community structure in traditional Gifu ayu-narezushi (fermented sweetfish).

In this study, we aimed to elucidate the bacterial biota of ayu-nazushi, which is a fermented salted fish dish made in Gifu City, Japan. In traditional Gifu ayu-nazushi, Lactobacillaceae (mainly Latilactobacillus sakei) was the most dominant family, followed by Enterobacteriaceae. Moreover, fermentation bacteria in ayu-nazushi came from the salted fish, and the bacterial biota in the ayu-nazushi transferred as the fermentation process progressed. In the early stage of fermentation, Leuconostoc mesenteroides was main species, and then in the late stage, L. sakei became predominant. We also observed that when non-salted fish was used for the manufacture of ayu-nazushi, Aeromonas veronii, which is a pathogen for humans, was observed in significant quantities. These results indicate that L. sakei and L. mesenteroides were influential lactic acid bacteria for the fermentation of Gifu ayu-narezushi, and that salting treatment of the fish is an indispensable step in the manufacturing process in order to suppress the growth of Aeromonas species.

Prior exposure to ciprofloxacin disrupts intestinal homeostasis and predisposes ayu ( Plecoglossus altivelis) to subsequent Pseudomonas plecoglossicida-induced infection.

With the rapid development of intensive farming, the aquaculture industry uses a great many antibiotics for the prevention and treatment of bacterial diseases. Despite their therapeutic functions, the overuse and accumulation of antibiotics also pose a threat to aquaculture organisms. In the present study, ayu ( Plecoglossus altivelis) was used as a fish model to study the impacts of ciprofloxacin (CIP) overuse on intestinal homeostasis and immune response during subsequent Pseudomonas plecoglossicida infection. Based on 16S rRNA gene amplification and Illumina sequencing, we found that CIP pre-exposure caused significant variation in intestinal microbiota, including increased species richness, altered microbiota composition and interaction networks, and increased metabolic dysfunction. Furthermore, immunohistochemical analysis indicated that CIP pre-exposure resulted in severe mucosal layer damage, goblet cell reduction, and epithelial cell necrosis of the intestinal barrier in infected ayu. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that disruption of intestinal homeostasis impaired systemic anti-infection immune responses in the intestine, gill, spleen, and head kidney, while inhibiting IL-1ß, TNF-α, and IL-10 expression and promoting TGF-ß expression. Our findings indicated that CIP administration can directly affect intestinal microbiota composition and intestinal integrity in ayu fish. This perturbation of intestinal homeostasis is likely responsible for the lower survival rate of hosts following subsequent infection as the capacity to mount an effective immune response is compromised. This study also provides preliminary clues for understanding the effects of antibiotic overuse on higher vertebrates through trophic transfer.

Isolation of purple nonsulfur bacteria from the digestive tract of ayu (Plecoglossus altivelis).

Purple nonsulfur bacteria (PNSB) reportedly have probiotic effects in fish, but whether they are indigenous in the digestive tract of fish is a question that requires answering. We attempted to isolate PNSB from the digestive tract of ayu (Plecoglossus altivelis) from the Kuma River (Kumamoto, Japan) and successfully isolated 12 PNSB strains. All the isolated PNSB belonged to the genus Rhodopseudomonas. Five Rhodopseudomonas strains were also isolated from the soil samples collected along the Kuma River. The phylogenetic tree based on the partial sequence of pufLM gene indicated that the PNSB from ayu and soil were similar. The effects of NaCl concentration in growth medium on growth were also compared between the PNSB from ayu and soil. The PNSB from ayu showed a better growth performance at a higher NaCl concentration, suggesting that the intestinal tract of ayu, a euryhaline fish, might provide suitable environment for halophilic microorganisms.

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.

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.

Environmental factors affecting Edwardsiella ictaluri-induced mortality of riverine ayu, Plecoglossus altivelis (Temminck & Schlegel).

We analysed the predisposing factors for Edwardsiella ictaluri infection in the riverine ayu Plecoglossus altivelis on the basis of environmental and epidemiological data obtained in a tributary to and the lower reaches of the Tama River, Japan, in July and August 2011-2015. Mortality of ayu due to E. ictaluri infection was observed only in the tributary in August 2012 and 2013; both periods were unusually hot. During these mortality events, daily average water temperatures rose approximately 3-4°C over 4-8 days, reaching the optimum temperature for E. ictaluri infection (>25°C) and approaching the upper tolerable limit for ayu (30°C). Diurnal water temperature ranges (DWTRs) in the tributary during the mortality events exceeded 6°C, which was 1-2°C greater than in the lower reaches. Experimental infection of ayu with E. ictaluri resulted in higher mortality when exposed to 6°C DWTR than to 4°C DWTR. Furthermore, water levels in the tributary were generally low in August 2012 and 2013 because of low rainfall. From these results, we conclude that unusually high-water temperatures combined with high DWTRs and low water levels drove riverine ayu mortality from E. ictaluri infection.

Hypoxia-inducible factor-1α involved in macrophage regulation in ayu (Plecoglossus altivelis) under hypoxia.

Hypoxia-inducible factor-1α (HIF-1α) plays a critical role in immune and inflammatory responses and is important in controlling a variety of processes in monocytes and macrophages. However, the role of HIF-1α in the teleost immune system remains less known. In this study, we cloned the cDNA sequence of HIF-1α from the ayu (Plecoglossus altivelis, PaHIF-1α). Sequence and phylogenetic tree analysis showed that PaHIF-1α clustered within the fish HIF-1α tree and was closely related to that of Northern pike (Esox lucius). PaHIF-1α was expressed in all tested tissues and expression increased in liver, head kidney, and body kidney upon Vibrio anguillarum infection. PaHIF-1α was found to regulate the expression of cytokines in ayu monocytes/macrophages (MO/MФ). PaHIF-1α mediated hypoxia-induced enhancement of MO/MФ phagocytic and bactericidal activities to enhance host defenses. Compared with the control, intermittent hypoxia further increased the expression of PaHIF-1α mRNA, improved the survival rate, and reduced the bacterial load of V. anguillarum-infected ayu. Therefore, PaHIF-1α may play a predominant role in the modulation of ayu MO/MФ function.

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.

Distribution of Carotenoids and Protective Effects of Zeaxanthin on Retina of Ayu Sweetfish (Plecoglossus altivelis).

Ayu sweetfish (Plecoglossus altivelis) is a diurnal freshwater fish that are surface swimmers and active under broad and short wavelength-dominated light. Biochemical analyses have shown that the ayu fish have abundant carotenoids including zeaxanthin in their integuments. Although zeaxanthin plays an important role in the physiological function of the retina, the amount and location of zeaxanthin in the ayu eye have not been accurately determined. In this study, circular dichroism spectral data and chiral high-performance liquid chromatography analysis showed that zeaxanthin was the primary carotenoid in the ayu eye, and the eye had the highest carotenoid content compared to those in the integuments, subcutaneous fat, and digestive tract. Interestingly, zeaxanthin in the ayu eyeball was expressed in the photoreceptor layer and near the retinal pigmented epithelium. In vitro assays showed that zeaxanthin could protect photoreceptors and retinal pigmented epithelial cell lines against the oxidative stress induced by exposure to L-buthionine-(S,R)-sulfoximine/glutamate. These findings indicate that zeaxanthin plays protective roles against oxidative stress in the vision of wild ayu.

[A CASE OF ANAPHYLAXIS CAUSED BY LANDLOCKED AYU FISH, KOAYU FISH, FROM LAKE BIWA].

A 28-year-old man was transferred to our emergency room for dyspnea and wheals on the entire body. He had eaten landlocked ayu fish (Plecoglossus altivelis) the so-called "koayu fish", from Lake Biwa, and had immediately experienced a stomachache. Wheals and dyspnea developed one hour later and were successfully treated with intravenous corticosteroids. The patient was examined for koayu fish and related allergens by skin prick and allergen-specific immunoglobulin E (IgE) (ImmunoCAP®) tests. Positive skin prick results were obtained for Lake Biwa koayu fish (raw and heated) as well as for standard skin test allergens (prepared by Torii pharmaceuticals) including shrimp, crab, and squid. Negative prick test results were observed for regular ayu fish and other fish such as horse mackerel, sardine, salmon, mackerel, codfish, and tuna. Allergen-specific IgE tests (ImmunoCAP ®) showed positivity for shrimp, crab, ticks, moths, and mosquitoes, while ImmunoCAP® tests were negative for the allergen components rGad c 1 (pollackparvalbumin) and rPen a 1 (shrimp tropomyosin). Moreover, enzyme-linked immunosorbent assay (ELISA) tests were negative for mackerel parvalbumin and collagen. We considered this case to be of anaphylaxis caused by koayu fish from Lake Biwa and speculated that a novel koayu-specific antigen might have been the cause of the condition.

Beta-adrenergic receptor stimulation influences the function of monocytes/macrophages in ayu (Plecoglossus altivelis).

Adrenergic receptors (ARs) are members of the G-protein-coupled receptor superfamily that can be categorized into αARs and ßARs. The specific function of ARs in teleost monocytes/macrophages (MO/MФ) remains unknown. We determined the cDNA sequence of ARs from ayu (Plecoglossus altivelis; PaαAR and PaßAR). Sequence comparisons showed that PaαAR was most closely related to the αAR of the Japanese flounder and Nile tilapia, while PaßAR was most closely related to the ßAR of Atlantic salmon. The AR transcripts were mainly expressed in the spleen, and their expression was altered in various tissues upon infection with Vibrio anguillarum. PaαAR and PaßAR proteins were upregulated in MO/MФ after infection, and PaßAR knockdown resulted in a pro-inflammatory status in ayu MO/MФ upon V. anguillarum infection and lowered the phagocytic activity of MO/MФ. Our results indicate that PaßAR plays the role of an anti-inflammatory mediator in the immune response of ayu against bacterial infection.

The host defense peptide ß-defensin confers protection against Vibrio anguillarum in ayu, Plecoglossus altivelis.

ß-defensin is a cationic host defense peptide actively participating in host innate immune response against pathogens. In teleost fish, ß-defensin exhibits a diversity in genotypes and functions. Herein, a ß-defensin homolog (PaBD) was identified from ayu, Plecoglossus altivelis, showing multiple tissues' upregulation against Vibrio anguillarum challenge. In vivo experiments revealed that intraperitoneal injection of chemically synthesized mature PaBD (mPaBD) increased the survival rate of V. anguillarum-infected ayu, accompanied by reduced bacterial load and decreased tissue mRNA levels of tumor necrosis factor α (PaTNF-α) and interleukin 1ß (PaIL-1ß). However, in vitro, mPaBD showed weak bactericidal activity against V. anguillarum. Interestingly, mPaBD enhanced phagocytosis, intracellular bacterial killing, and respiratory burst of ayu monocytes/macrophages (MO/MΦ). Moreover, it inhibited mRNA levels of PaIL-1ß and PaTNF-α in MO/MФ upon V. anguillarum infection. In conclusion, PaBD protects ayu against V. anguillarum challenge not only through its direct antibacterial ability, but also through its immunomodulation in MO/MΦ.

The interleukin-6 regulates the function of monocytes/macrophages (MO/MФ) via the interleukin-6 receptor ß in ayu (Plecoglossus altivelis).

Interleukin-6 (IL-6) is one of the most pleiotropic cytokines because of its wide range of effects on cells of the immune and non-immune systems in the body. However, the role of IL-6 in fish monocytes/macrophages (MO/MФ) is poorly understood. In this study, we cloned the cDNA sequence of the IL-6 gene from ayu (Plecoglossus altivelis) and demonstrated using a tissue distribution assay that ayu interleukin-6 (PaIL-6) mRNA is expressed in all tested tissues. Changes in expression were observed in immune tissues as well as in MO/MФ after a Vibrio anguillarum infection; subsequently, PaIL-6 was expressed and purified to prepare anti-PaIL-6 antibodies. Recombinant PaIL-6 protein (rPaIL-6) treatment enhanced pro-inflammatory cytokine expression. Ayu interleukin-6 receptor ß (PaIL-6Rß) knockdown resulted in decreased pro-inflammatory cytokine expression in MO/MФ treated with rPaIL-6, whereas no significant changes were observed after ayu interleukin-6 receptor α (PaIL-6Rα) knockdown in MO/MФ. PaIL-6 and PaIL-6Rß knockdown in MO/MФ inhibited the phosphorylation of signal transducer and activator of transcription 1. Moreover, PaIL-6Rß knockdown inhibited the phagocytic and bactericidal ability of ayu MO/MФ treated with rPaIL-6. These data indicate that PaIL-6 may be able to regulate the function of ayu MO/MФ.

Glucocorticoid receptor in ayu (Plecoglossus altivelis): Genomic and non-genomic effects on monocytes/macrophages function.

The glucocorticoid receptor (GR) is an important feedback regulator of the hypothalamic-pituitary-interrenal (HPI) axis. However, there are a limited number of studies focused on host-pathogen interactions in which an association between GR and immune response has been evaluated in monocytes/macrophages (MO/MФ) after being challenged with highly pathogenic bacteria. Here, we cloned the cDNA sequence of the glucocorticoid receptor (PaGR) gene from ayu fish. The PaGR transcript was expressed in all tissues, and changes in expression were observed in immune tissues and MO/MФ after live Vibrio anguillarum infection. Subsequently, PaGR was expressed and purified to prepare anti-PaGR antibodies. We analyzed the subcellular localization of PaGR. PaGR was expressed not only in the intracellular space but also in the plasma membrane. PaGR activation decreased the expression of pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokines. However, PaGR activation suppressed the phagocytosis activity of V. anguillarum-infected ayu MO/MФ via a non-genomic pathway. Interestingly, PaGR activation could enhance MO/MФ bacterial killing capability and apoptosis. Therefore, PaGR may modulate the immune response in ayu MO/MФ by genomic and non-genomic pathways.