Hyperoxia disproportionally benefits the aerobic performance of large fish at elevated temperature.

Increasing evidence shows that larger fish are more vulnerable to acute warming than smaller individuals of the same species. This size-dependency of thermal tolerance has been ascribed to differences in aerobic performance, largely owing to a decline in oxygen supply relative to demand. To shed light on these ideas, we examined metabolic allometry in 130 rainbow trout ranging from 12 to 358 g under control conditions (17°C) and in response to acute heating (to 25°C), with and without supplemental oxygen (100% versus 150% air saturation). Under normoxia, high temperature caused an average 17% reduction in aerobic scope compared with 17°C. Aerobic performance disproportionally deteriorated in bigger fish as the scaling exponent (b) for aerobic scope declined from b=0.87 at 17°C to b=0.74 at 25°C. Hyperoxia increased maximum metabolic rate and aerobic scope at both temperatures and disproportionally benefited larger fish at 25°C as the scaling exponent for aerobic scope was reestablished to the same level as at 17°C (b=0.86). This suggests that hyperoxia may provide metabolic refuge for larger individuals, allowing them to sustain aerobic activities when facing acute warming. Notably, the elevated aerobic capacity afforded by hyperoxia did not appear to improve thermal resilience, as mortality in 25°C hyperoxia (13.8%, n=4) was similar to that in normoxia (12.1%, n=4), although we caution that this topic warrants more targeted research. We highlight the need for mechanistic investigations of the oxygen transport system to determine the consequences of differential metabolic scaling across temperature in a climate warming context.

Inhibitory effects of Oncorhynchus mykiss lipids in LPS-induced RAW264.7 cells via suppression of NF-κB and MAPK pathways.

Oncorhynchus mykiss, a significant aquaculture species, possesses compounds with numerous biological and pharmacological functions, including antioxidant, anticancer, anti-microbial, and anti-obesity effects. However, possible anti-inflammatory effects of lipids extracted from O. mykiss eggs on RAW264.7 cells induced by LPS have not been elucidated yet. The current study identified 13 fatty acids in lipids extracted from O. mykiss eggs that contained high amounts (51.92% of total fatty acids) of polyunsaturated fatty acids (PUFAs), especially DHA (33.66%) and EPA (7.77%). These O. mykiss lipids (100-400 µg/mL) showed significant anti-inflammatory effects by inhibiting NO and iNOS expression in LPS-stimulated RAW264.7 cells. They also inhibited expression of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α, while upregulating anti-inflammatory cytokines IL-10, IL-11, and TGF-ß. These lipids from O. mykiss effectively inhibited LPS-induced expression CD86 as a surface biomarker on RAW264.7 cells. Additionally, O. mykiss lipids suppressed phosphorylation of p38, JNK, and ERK1/2 and the expression of phosphorylated NF-κB subunit p65. These findings indicate that O. mykiss lipids possess anti-inflammatory properties by inhibiting NF-κB and MAPK signaling pathways.

Evaluation of a DNA vaccine with self-designed CpG sequences against J genotype IHNV infection in rainbow trout (Oncorhynchus mykiss).

Rainbow trout suffer from infectious hematopoietic necrosis virus (IHNV) outbreaks, which lead to massive mortality and huge economic loss worldwide. The approved commercial vaccine is used for the prevention of IHN in Canada. Given that Chinese domestic J-genotype isolates are different from North American IHNV isolates, the development of an effective DNA vaccine against Chinese J-genotype isolates is urgent. In this study, we developed a DNA vaccine encoding glycoprotein based on our previously isolated IHNV GS21 strain and self-designed CpG sequences were supplemented as molecular adjuvants. The vaccinated rainbow trout were significantly protected against IHNV with approximately a relative percent survival (RPS) of 94.74% compared to the unvaccinated group. Moreover, the specific antibody of IgM and neutralizing antibody (NAb) was significantly provoked after the vaccination. Particularly, the antiviral immune response was rapidly evoked in the early stage of vaccination including the up-regulation of Mx-1, IFN-Ⅰ, and IFN-γ. The IHNV load in vaccinated fish was apparently lower than that in the unvaccinated group. Furthermore, the integration of exogenous genes into the host chromosome and the spread of antimicrobial-resistant genes were not found. These results suggested that our vaccine enhances robust immune responses and evokes considerable protection against IHNV with limited genetically modified risk, which is an effective and promising vaccine candidate for further prevention of IHNV outbreaks.

Rainbow trout (Oncorhynchus mykiss, Walbaum 1792) adenocarcinoma investigation with various diagnostic imaging techniques.

Diagnostic imaging techniques provide a new aspect of the ante-mortem and post-mortem diagnostics in fish medicine. Ultrasonography, computed tomography (CT) and magnetic resonance imaging (MRI) can provide more information about the internal organs and pathognomic lesions. The authors used diagnostic imaging techniques to evaluate and describe the neoplastic malformation in a 3-year-old female rainbow trout (Oncorhynchus mykiss). The fish was examined with Siemens Somatom Definition AS + CT scanner and Siemens Biograph mMR scanner. The animal was lethargic and showed anorectic signs and muscular dystrophy. During the post-mortem investigation, histopathology and immunohistochemistry were also performed allowing us to identify the neoplasms. The results showed a large soft tissue mass in the first mid-intestine segment, which proved to be an adenocarcinoma. This subsequently led to digestion problems and absorption disorders. Immunohistochemically, neoplastic cells of carcinoma revealed E-cadherin and pancytokeratin positivity. This is the first study to report the use of MRI and CT for studying gastrointestinal adenocarcinoma in rainbow trout.

Identification of new amoebae strains in rainbow trout (Oncorhynchus mykiss, Walbaum) farms affected by nodular gill disease (NGD) in Northeastern Italy.

Nodular gill disease (NGD) is an emerging condition associated with amoeba trophozoites in freshwater salmonid farms. However, unambiguous identification of the pathogens still must be achieved. This study aimed to identify the amoeba species involved in periodic NGD outbreaks in two rainbow trout (Oncorhynchus mykiss) farms in Northeastern Italy. During four episodes (February-April 2023), 88 fish were euthanized, and their gills were evaluated by macroscopic, microscopic and histopathological examination. The macroscopic and microscopic severity of the lesions and the degree of amoebae infestation were scored and statistically evaluated. One gill arch from each animal was put on non-nutrient agar (NNA) Petri dishes for amoeba isolation, cultivation and subsequent identification with SSU rDNA sequencing. Histopathology confirmed moderate to severe lesions consistent with NGD and mild to moderate amoeba infestation. The presence of amoebae was significantly correlated with lesion severity. Light microscopy of cultured amoebae strains and SSU rDNA analysis revealed the presence of a previously characterized amoeba Naegleria sp. strain GERK and several new strains: two strains from Hartmannelidae, three vannelid amoebae from the genus Ripella and cercozoan amoeba Rosculus. Despite the uncertainty in NGD etiopathogenesis and amoebae pathogenic role, identifying known and new amoebae leans towards a possible multi-aetiological origin.

Transcriptomic and Epigenomic Responses to Cortisol-Mediated Stress in Rainbow Trout (Oncorhynchus mykiss) Skeletal Muscle.

The production and release of cortisol during stress responses are key regulators of growth in teleosts. Understanding the molecular responses to cortisol is crucial for the sustainable farming of rainbow trout (Oncorhynchus mykiss) and other salmonid species. While several studies have explored the genomic and non-genomic impacts of cortisol on fish growth and skeletal muscle development, the long-term effects driven by epigenetic mechanisms, such as cortisol-induced DNA methylation, remain unexplored. In this study, we analyzed the transcriptome and genome-wide DNA methylation in the skeletal muscle of rainbow trout seven days after cortisol administration. We identified 550 differentially expressed genes (DEGs) by RNA-seq and 9059 differentially methylated genes (DMGs) via whole-genome bisulfite sequencing (WGBS) analysis. KEGG enrichment analysis showed that cortisol modulates the differential expression of genes associated with nucleotide metabolism, ECM-receptor interaction, and the regulation of actin cytoskeleton pathways. Similarly, cortisol induced the differential methylation of genes associated with focal adhesion, adrenergic signaling in cardiomyocytes, and Wnt signaling. Through integrative analyses, we determined that 126 genes showed a negative correlation between up-regulated expression and down-regulated methylation. KEGG enrichment analysis of these genes indicated participation in ECM-receptor interaction, regulation of actin cytoskeleton, and focal adhesion. Using RT-qPCR, we confirmed the differential expression of lamb3, itga6, limk2, itgb4, capn2, and thbs1. This study revealed for the first time the molecular responses of skeletal muscle to cortisol at the transcriptomic and whole-genome DNA methylation levels in rainbow trout.

Constitutive Neurogenesis and Neuronal Plasticity in the Adult Cerebellum and Brainstem of Rainbow Trout, Oncorhynchus mykiss.

The central nervous system of Pacific salmon retains signs of embryonic structure throughout life and a large number of neuroepithelial neural stem cells (NSCs) in the proliferative areas of the brain, in particular. However, the adult nervous system and neurogenesis studies on rainbow trout, Oncorhynchus mykiss, are limited. Here, we studied the localization of glutamine synthetase (GS), vimentin (Vim), and nestin (Nes), as well as the neurons formed in the postembryonic period, labeled with doublecortin (DC), under conditions of homeostatic growth in adult cerebellum and brainstem of Oncorhynchus mykiss using immunohistochemical methods and Western Immunoblotting. We observed that the distribution of vimentin (Vim), nestin (Nes), and glutamine synthetase (GS), which are found in the aNSPCs of both embryonic types (neuroepithelial cells) and in the adult type (radial glia) in the cerebellum and the brainstem of trout, has certain features. Populations of the adult neural stem/progenitor cells (aNSPCs) expressing GS, Vim, and Nes have different morphologies, localizations, and patterns of cluster formation in the trout cerebellum and brainstem, which indicates the morphological and, obviously, functional heterogeneity of these cells. Immunolabeling of PCNA revealed areas in the cerebellum and brainstem of rainbow trout containing proliferating cells which coincide with areas expressing Vim, Nes, and GS. Double immunolabeling revealed the PCNA/GS PCNA/Vim coexpression patterns in the neuroepithelial-type cells in the PVZ of the brainstem. PCNA/GS coexpression in the RG was detected in the submarginal zone of the brainstem. The results of immunohistochemical study of the DC distribution in the cerebellum and brainstem of trout have showed a high level of expression of this marker in various cell populations. This may indicate: (i) high production of the adult-born neurons in the cerebellum and brainstem of adult trout, (ii) high plasticity of neurons in the cerebellum and brainstem of trout. We assume that the source of new cells in the trout brain, along with PVZ and SMZ, containing proliferating cells, may be local neurogenic niches containing the PCNA-positive and silent (PCNA-negative), but expressing NSC markers, cells. The identification of cells expressing DC, Vim, and Nes in the IX-X cranial nerve nuclei of trout was carried out.

Tire Rubber Based Microplastic Particles Cause Adverse on Quality Parameters of Rainbow Trout Sperm Cells.

In the present study, we aimed to determine the parameters of oxidative stress markers, motility and kinematics of rainbow trout (Oncorhynchus mykiss) sperm cells exposed to different doses (0.001, 0.01, 0.1, 1.0, and 10 mg/L, in vitro 4 h) of tire rubber based microplastic particles (TRMP-Ps) the leachates procedure of rubber pieces. First of all, TRMP-Ps were prepared by abrasion method in accordance with the literature. Structural and morphological features of TRMP-Ps were determined by Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) methods, respectively. Energy dispersive X-ray (EDX) analysis technique was used to characterize the elemental composition of TRMP-Ps. Particle size of microplastic structures was measured hydrodynamically with dynamic light scattering analysis (DLS). After exposure, the effect of TRMP-Ps was defined by the observations of kinematics and antioxidant activities in sperm cells. Our findings showed that the straight line velocity, the curvilinear velocity, the angular path velocity, and the amplitude of lateral displacement of sperm cells decreased. Moreover, while the level of superoxide dismutase decreased dose-dependently against the toxicity of TRMP-Ps, no significant change was observed in the levels of malondialdehyde and total glutathione. The 4-h median effective concentrations (EC50) of TRMP-Ps based on mobility parameters of sperm ranged from 0.31 mg/L for reduced straight line velocity of sperm cells to 0.51 mg/L for reduced amplitude of lateral displacement of the spermatozoa head. Therefore, we concluded that TRMP-Ps can be a risk for the reproduction cycle of fish in aquatic environments.

Whole genome resequencing identifies local adaptation associated with environmental variation for redband trout.

Aquatic ectotherms are predicted to harbour genomic signals of local adaptation resulting from selective pressures driven by the strong influence of climate conditions on body temperature. We investigated local adaptation in redband trout (Oncorhynchus mykiss gairdneri) using genome scans for 547 samples from 11 populations across a wide range of habitats and thermal gradients in the interior Columbia River. We estimated allele frequencies for millions of single nucleotide polymorphism loci (SNPs) across populations using low-coverage whole genome resequencing, and used population structure outlier analyses to identify genomic regions under divergent selection between populations. Twelve genomic regions showed signatures of local adaptation, including two regions associated with genes known to influence migration and developmental timing in salmonids (GREB1L, ROCK1, SIX6). Genotype-environment association analyses indicated that diurnal temperature variation was a strong driver of local adaptation, with signatures of selection driven primarily by divergence of two populations in the northern extreme of the subspecies range. We also found evidence for adaptive differences between high-elevation desert vs. montane habitats at a smaller geographical scale. Finally, we estimated vulnerability of redband trout to future climate change using ecological niche modelling and genetic offset analyses under two climate change scenarios. These analyses predicted substantial habitat loss and strong genetic shifts necessary for adaptation to future habitats, with the greatest vulnerability predicted for high-elevation desert populations. Our results provide new insight into the complexity of local adaptation in salmonids, and important predictions regarding future responses of redband trout to climate change.

Characterization of the activity of 2'-C- methylcytidine against infectious pancreatic necrosis virus replication.

Infectious pancreatic necrosis virus (IPNV) is the pathogen of infectious pancreatic necrosis (IPN), which can cause high mortality in salmonids, harm the healthy development of salmon-trout aquaculture, and lead to huge economic losses. However, in China, there is currently neither a commercially available vaccine to prevent IPNV infection nor antiviral drugs to treat IPNV infection. The genome of IPNV consists of two segments of dsRNA named A and B. Segment B encodes the RNA-dependent RNA-polymerase (RdRp) VP1 which is essential for viral RNA replication and is therefore considered an important target for the development of antiviral drugs. In this study, we investigate whether 2'-C-methylcytidine (2CMC), a nucleoside analog which target viral polymerases, has an inhibitory effect on IPNV both in vitro and in vivo. The results show that 2CMC inhibits IPNV infection by inhibiting viral RNA replication rather than viral internalization or attachment. In vivo experiment results showed that 2CMC could inhibit viral RNA replication and reduce viral load in rainbow trout (Oncorhynchus mykiss). In our study, we have revealed that 2CMC has a potent inhibitory effect against IPNV infection. Our data suggest that 2CMC is an attractive anti-IPNV drug candidate which will be highly valuable for the development of potential therapeutics for IPNV.

Effects of artificial photoperiods on antigen-dependent immune responses in rainbow trout (Oncorhynchus mykiss).

In this study, we investigated the effects of the artificial photoperiods that mimic summer (16L:8D; 16 h Light: 8 h Dark) and winter (8L:16D) solstices, equinoxes (12L:12D), and the artificial 24-h light regimen (24L:0D) on the leukocyte populations and the T helper and regulatory type responses on rainbow trout (Oncorhynchus mykiss). Using flow cytometry analysis, we found that photoperiod induces changes in head kidney leukocyte subsets. The lymphoid subset increased in the 16L:8D summer solstice regime. The analysis using antibodies against B and T cells showed the increase of CD4-1+ T lymphocytes and other unidentified lymphoid cells, with no changes in the B cells. To investigate the modulatory influence of the photoperiod on the fish T cell response, we quantified in the head kidney the transcript levels of genes involved in the Th1 type response (t-bet, ifn-Æ´, il-12p35, il-12p40c), Th2 type response (gata3, il-4/13a), Th17 response (ror-Æ´t, il-17a/f), T regulatory response (foxp3α, il-10a, tgf-ß1), and the T cell growth factor il-2. The results showed that the seasonal photoperiod alone has a limited influence on the expression of these genes, as the only difference was observed in il-14/13a and il-10a transcripts of fish kept on the 16L:8D regimen. In addition, the 24L:0D treatment used in aquaculture produces a reduction of il-14/13a and il-17a/f. We also evaluated the effect of photoperiod in the presence of an antigenic stimulus. Thus, in fish immunized with the recombinant viral protein 1 (rVP1) of infectious pancreatic necrosis virus (IPNV), the photoperiod had a striking influence on the type of adaptive immune response. Each photoperiod fosters a unique immune signature of antigenic response. A classical type 1 response is observed in fish subjected to the 16L:8D photoperiod. In contrast, fish in the 12L:12D photoperiod showed only the upregulation of il-12p40c. Furthermore, none of the cytokines were increased in fish maintained on the artificial 24L:0D regimen, and a decrease in the master transcription factors (t-bet, ror-Æ´t, and foxp3α) was observed. Thus, fish on the 12L:12D and 24L:0D photoperiod appear hyporesponsive regarding the T cell response. Altogether, this study showed that photoperiods modify the magnitude and quality of the T-helper response in rainbow trout and thus impact essential mechanisms for the generation of immune memory and protection against microorganisms.

Application of UV-C irradiation prevented a severe outbreak of proliferative kidney disease in rainbow trout aquaculture.

There is an urgent need to establish protocols on how to protect salmonids in aquaculture from outbreaks of proliferative kidney disease (PKD). For this purpose, systems for a continuous application of peracetic acid (PAA, 0.1 mg l-1) and of ultraviolet C light (UV-C, 323.5-158.6 mW s cm-2) were installed in the inlet of raceway-channels within a sub-unit of a commercial rainbow trout Oncorhynchus mykiss farm. After 127 d of rearing, a fish health examination was conducted. Fish in the control and PAA treatment groups showed signs of PKD. In contrast, fish in the UV-C treatment group showed almost no signs of disease based on clinical examinations and necropsy. This observation indicates that UV-C irradiation could be a promising tool to protect fish from PKD in the future.

Amoeba species colonizing the gills of rainbow trout (Oncorhynchus mykiss) in Swiss aquaculture.

Nodular gill disease (NGD) is an infectious condition characterized by proliferative gill lesions leading to respiratory problems, oxygen deficiency and mortality in fish. Globally, NGD primarily impacts freshwater salmonids in intensive aquaculture systems. In recent years, numerous outbreaks of severe gill disease have affected more than half of the larger rainbow trout (Oncorhynchus mykiss) farms in Switzerland, mainly during spring and early summer. Mortality has reached up to 50% in cases where no treatment was administered. Freshwater amoeba are the presumed aetiologic agent of NGD. The gross gill score (GS) categorising severity of gill pathology is a valuable first-line diagnostic tool aiding fish farmers in identifying and quantifying amoebic gill disease (AGD) in farmed marine salmonids. In this study, the GS was adapted to the NGD outbreak in farmed trout in Switzerland. In addition to scoring disease severity, gill swabs from NGD-affected rainbow trout were sampled and amoeba were cultured from these swabs. Morphologic and molecular methods identified six amoeba strains: Cochliopodium sp., Naegleria sp., Vannella sp., Ripella sp., Saccamoeba sp. and Mycamoeba sp. However, the importance of the different amoeba species for the onset and progression of NGD still has to be evaluated. This paper presents the first description of NGD with associated amoeba infection in farmed rainbow trout in Switzerland.

Bioenergetic consequences of repeated catch-and-release fisheries interactions on adult steelhead across a range of ecologically relevant water temperatures.

The biological consequences of catch-and-release angling have been studied for decades, yet little is known about the compounding effects of repeated recreational fisheries recaptures on the physiology and behaviour of angled fish. Using heart rate biologgers and behavioural assays, this study investigated the physiological and behavioural consequences of multiple simulated angling events (i.e., repeated stressors) on female steelhead (Oncorhynchus mykiss), under current (6 °C) and future (11 °C) water temperature scenarios. While steelhead in the warmer water temperature scenario demonstrated alterations in cardiac function (e.g., increases in maximum heart rate and scope of heart rate) and evidence of behavioural impairments (e.g., decreases in chase activity and landing time) over the course of two simulated angling events, cold water treated fish had negligible change. Fish subjected to two simulated angling events under warm water temperature conditions tended to demonstrate an increase in recovery time and scope for heart rate, and a decrease in resting heart rate. A second experiment was conducted to test for sex-specific differences in the heart rate response of steelhead subjected to an increase in water temperature. Females demonstrated a higher scope for heart rate when compared to males during the event and during recovery. More work is needed to better understand the interaction between multiple angling events and recovery from these events at various water temperatures, and the biological basis for sex-specific differences in cardiac function and response to challenges. This study contributes to a growing body of evidence on the effects of repeated stressors on wild fish.

Comparing dominance relationships and movement of native marble trout (Salmo marmoratus) and introduced rainbow trout (Oncorhynchus mykiss).

Behaviour observations of the endangered native marble trout (Salmo marmoratus, Cuvier, 1829) and introduced rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) in the laboratory and in a tributary to the Idrijca River in Slovenia were combined to study the movements and dominance relationships between individuals of the two species in an open field test. Under laboratory conditions, no difference between the species was detected for neither time spent actively or distance moved. In species paired tests, rainbow trout initiated more aggressive behaviours towards marble trout than vice versa, and rainbow trout were clearly the dominant individuals. After simultaneous release in the river, marble trout immediately left the release area and spent twice as long time as rainbow trout until they settled in an area of the river; therefore, the release site was immediately occupied exclusively by rainbow trout. Thus, the dominant and aggressive behaviour of rainbow trout seen in the laboratory before release might have influenced marble trout's subsequent behaviour in the river, by marble trout leaving the areas occupied by rainbow trout and moving to locations further away from the release site. In the field, the marble trout occupied sites individually, whereas rainbow aggregated at a few locations. Rainbow trout showed higher movement activity in the morning compared to marble trout. There was a positive correlation between swimming speed in the laboratory and movement in the field for marble trout but not for rainbow trout. In conclusion, the results in this study support the need to end stocking of rainbow trout in rivers with native marble trout. To better understand the interaction between the species, and to develop efficient management plans to protect the native marble trout, reference behaviours should first be understood, and future research in sites where the two species do not co-exist is needed. This is especially important for marble trout for which behavioural research and data are lacking.

Teleost Eye Is the Portal of IHNV Entry and Contributes to a Robust Mucosal Immune Response.

The ocular mucosa (OM) is an important and unique part of the vertebrate mucosal immune system. The OM plays an important role in maintaining visual function and defending against foreign antigens or microorganisms, while maintaining a balance between the two through complex regulatory mechanisms. However, the function of ocular mucosal defense against foreign pathogens and mucosal immune response in bony fish are still less studied. To acquire deeper understanding into the mucosal immunity of the OM in teleost fish, we established a study of the immune response of rainbow trout (Oncorhynchus mykiss) infected with the infectious hematopoietic necrosis virus (IHNV). Our findings revealed that IHNV could successfully infiltrate the trout's OM, indicating that the OM could be an important portal for the IHNV. Furthermore, qPCR and RNA-Seq analysis results showed that a large number of immune-related genes were significantly upregulated in the OM of trout with IHNV infection. Critically, the results of our RNA-Seq analysis demonstrated that viral infection triggered a robust immune response, as evidenced by the substantial induction of antiviral, innate, and adaptive immune-related genes in the OM of infected fish, which underscored the essential role of the OM in viral infection. Overall, our findings revealed a previously unknown function of teleost OM in antiviral defense, and provided a theoretical basis for the study of the mucosal immunity of fish.

Effects of salinity acclimation on histological characteristics and miRNA expression profiles of scales in juvenile rainbow trout (Oncorhynchus mykiss).

BACKGROUND: The scales serve as an ideal model for studying the regulatory mechanism of bone homeostasis in fish. To explore the effect of salinity acclimation on bone metabolism of juvenile rainbow trout (Oncorhynchus mykiss), three sampling time points during salinity acclimation (7D, 14D and 21D) were selected to detect variations in histological characteristics. In the histological analysis, osteoblast marker enzymes alkaline phosphatase (ALP), osteoclast marker tartrate-resistant acid phosphatase (TRAcP) and calcium salt deposit areas (Von Kossa's) were detected. Changes in calcium (Ca), phosphorus (P) and the molar mass ratio of calcium to phosphorus (Ca/P) in the scales were also detected by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). In addition, the global MicroRNA (miRNA) expression profiles during salinity acclimation were examined using Illumina sequencing platform because of their important regulatory roles in teleost biological processes. RESULTS: Twelve independent miRNA libraries were constructed, a total of 664 known and 92 putative novel miRNAs were identified. A total of 290 differentially expressed (DE) miRNAs were found in clusters with significant trends in the cluster analysis, and five types of clustering patterns were obtained; 22,374 DE predicted target genes of the aforementioned 290 DE miRNAs were obtained, 5957 of which clustered in six types of clustering patterns with a significant trend. To better understand the functions of the DE miRNAs, GO and KEGG analysis was performed on the 5957 target genes, as a result, they were significantly enriched in bone metabolism related signaling pathways such as MAPK signaling pathway, Calcium signaling pathway, Wnt signaling pathway, Mineral absorption and NF-kappa B signaling pathway. Six DE miRNAs were randomly selected and their expression were verified by quantitative real-time PCR (qRT-PCR), the expression trends were consistent with the results of transcriptome sequencing. CONCLUSIONS: The DE miRNAs and DE target genes identified in this study might play an important role in regulation of bone metabolism during salinity acclimation, relative genes or pathways could serve as key candidates for further studies to elucidate molecular mechanism of teleost bone metabolism, and help performing salinity acclimation and developing marine culture of salmonid species.

Deefgea salmonis sp. nov., isolated from gills of rainbow trout (Oncorhynchus mykiss).

A Gram-stain-negative, milky white, aerobic, rod-shaped bacterium named strain H3-26T was isolated from gills of Oncorhynchus mykiss in Lhasa, Tibet Autonomous Region, PR China. Strain H3-26T grew at 4-30 °C and pH 5.0-11.0 (optimum, 25 °C and pH 7.0) with 0-1% (w/v) NaCl (optimum, 0%). The 16S rRNA gene sequence of strain H3-26T showed the highest similarity to Deefgea rivuli WB 3.4-79T (98.42%), followed by Deefgea chitinilytica Nsw-4T (96.91%). Phylogenetic analysis based on 16S rRNA genes indicated that strain H3-26T was a new member of the genus Deefgea. The digital DNA-DNA hybridization and average nucleotide identity values between the genome sequence of strain H3-26T and Deefgea spp. were 21.2-21.9% and 76.3-77.4%, respectively. The genomic DNA G+C content of strain H3-26T was 48.74%. The predominant fatty acids were C12:0, C14:0, C16:0 and C16:1 ω7c. Based on phenotypic, phylogenetic, and genotypic data, strain H3-26T is considered to represent a novel species of the genus Deefgea, for which the name Deefgea salmonis sp. nov. is proposed. The type strain is H3-26T (= JCM 35050T = CICC 25103T).

Molecular characterization and expression analysis of claudin-4-like in rainbow trout involved in Flavobacterium psychrophilum infection.

The claudin family of proteins are pivotal components of tight junction (TJ) participating in the epithelial barrier function in fish. Our previous studies indicated that one of the claudins, claudin-4-like (OmCLDN4L) was differentially expressed in rainbow trout (Oncorhynchus mykiss) spleen post infection of Flavobacterium psychrophilum, which is the causative pathogen of bacterial coldwater disease (BCWD). However, little is known about the function of OmCLDN4L in rainbow trout against bacterial infection. In the present study, the OmCLDN4L was identified and functionally characterized from rainbow trout. The OmCLDN4L has an open reading frame (ORF) of 668 bp, encoding a 22.86 kDa four-transmembrane protein with function of bicellular tight junction and apical tight junction. OmCLDN4L has the highest similarity with CLDN28a, CLDN28b and CLDN30 in amino acid sequence. Phylogenetic analysis showed that all of CLDN4 and CLDN4-like from fish clustered together but diverged from their counterparts in mammals, with main differences lying in their N-terminus. RT-qPCR results indicated that OmCLDN4L was constitutively expressed in all tissues investigated under healthy conditions, primarily in mucus, liver, skin and intestine. The expression of OmCLDN4L in rainbow trout intestine was slightly down-regulated at day 1 while up-regulated at day 3 and day 7 post F. psychrophilum infection, with the similar profiling of CLDN30 and CLDN10e. The expression level of inflammatory cytokines TNF-α, IL4/13A, IL-6 and pattern recognition receptor TLR-2 showed the same trend with OmCLDN4L in the intestine at day 3 and day 7 post F. psychrophilum infection. Collectively, these findings demonstrate that OmCLDN4L participates in the immune response to bacterial infection, offering new insights into the molecular mechanism of intestinal barrier in rainbow trout against F. psychrophilum infection.

Allee effect in a manipulative parasite within poikilothermic host under temperature change.

Temperature and intraspecific competition are important factors influencing the growth of all organisms, including parasites. The temperature increase is suggested to stimulate the development of parasites within poikilothermic hosts. However, at high parasite densities, this effect could be diminished, due to stronger intraspecific competition. Our study, for the first time, addressed the joint effects of warming and parasite abundances on parasite growth in poikilothermic hosts. The growth of the common fish parasite larvae (trematode Diplostomum pseudospathaceum) within the rainbow trout at different infection intensities and temperatures (15°C and 18°C) was experimentally investigated. The results showed that temperature was positively correlated with both parasite infection success and growth rates. The growth rates increased much more compared to those in many free-living poikilothermic animals. Atypically for a majority of parasites, D. pseudospathaceum larvae grow faster when abundant (Allee effect). The possible causes for this phenomenon (manipulation cost sharing, etc.) are discussed in this study. Importantly, limited evidence of the interaction between temperature and population density was found. It is likely that temperature did not change the magnitude of the Allee effect but affected its timing. The impact of these effects is supposed to become more pronounced in freshwater ecosystems under current climate changes.