Unexpected regulatory functions of cyprinid Viperin on inflammation and metabolism.

BACKGROUND: Viperin, also known as radical S-adenosyl-methionine domain containing protein 2 (RSAD2), is an interferon-inducible protein that is involved in the innate immune response against a wide array of viruses. In mammals, Viperin exerts its antiviral function through enzymatic conversion of cytidine triphosphate (CTP) into its antiviral analog ddhCTP as well as through interactions with host proteins involved in innate immune signaling and in metabolic pathways exploited by viruses during their life cycle. However, how Viperin modulates the antiviral response in fish remains largely unknown. RESULTS: For this purpose, we developed a fathead minnow (Pimephales promelas) clonal cell line in which the unique viperin gene has been knocked out by CRISPR/Cas9 genome-editing. In order to decipher the contribution of fish Viperin to the antiviral response and its regulatory role beyond the scope of the innate immune response, we performed a comparative RNA-seq analysis of viperin-/- and wildtype cell lines upon stimulation with recombinant fathead minnow type I interferon. CONCLUSIONS: Our results revealed that Viperin does not exert positive feedback on the canonical type I IFN but acts as a negative regulator of the inflammatory response by downregulating specific pro-inflammatory genes and upregulating repressors of the NF-κB pathway. It also appeared to play a role in regulating metabolic processes, including one carbon metabolism, bone formation, extracellular matrix organization and cell adhesion.

Regulatory gene network for coffee-like color morph of TYRP1 mutant of oujiang color common carp.

BACKGROUND: Neither a TYRP1-mediated highly conserved genetic network underlying skin color towards optimum defense nor the pathological tendency of its mutation is well understood. The Oujiang Color Common Carp (Cyprinus carpio var. color) as a model organism, offering valuable insights into genetics, coloration, aquaculture practices, and environmental health. Here, we performed a comparative skin transcriptome analysis on TYRP1 mutant and wild fishes by applying a conservative categorical approach considering different color phenotypes. RESULTS: Our results reveal that an unusual color phenotype may be sensitized with TYRP1 mutation as a result of upregulating several genes related to an anti-inflammatory autoimmune system in response to the COMT-mediated catecholamine neurotransmitters in the skin. Particularly, catecholamines-derived red/brown, red with blue colored membrane attack complex, and brown/grey colored reduced eumelanin are expected to be aggregated in the regenerated cells. CONCLUSIONS: It is, thus, concluded that the regenerated cells with catecholamines, membrane attack complex, and eumelanin altogether may contribute to the formation of the unusual (coffee-like) color phenotype in TYRP1 mutant.

Downregulation of miR-1388 Regulates the Expression of Antiviral Genes via Tumor Necrosis Factor Receptor (TNFR)-Associated Factor 3 Targeting Following poly(I:C) Stimulation in Silver Carp (Hypophthalmichthys molitrix).

MicroRNAs (miRNAs) are highly conserved endogenous single-stranded non-coding RNA molecules that play a crucial role in regulating gene expression to maintain normal physiological functions in fish. Nevertheless, the specific physiological role of miRNAs in lower vertebrates, particularly in comparison to mammals, remains elusive. Additionally, the mechanisms underlying the control of antiviral responses triggered by viral stimulation in fish are still not fully understood. In this study, we investigated the regulatory impact of miR-1388 on the signaling pathway mediated by IFN regulatory factor 3 (IRF3). Our findings revealed that following stimulation with the viral analog poly(I:C), the expression of miR-1388 was significantly upregulated in primary immune tissues and macrophages. Through a dual luciferase reporter assay, we corroborated a direct targeting relationship between miR-1388 and tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3). Furthermore, our study demonstrated a distinct negative post-transcriptional correlation between miR-1388 and TRAF3. We observed a significant negative post-transcriptional regulatory association between miR-1388 and the levels of antiviral genes following poly(I:C) stimulation. Utilizing reporter plasmids, we elucidated the role of miR-1388 in the antiviral signaling pathway activated by TRAF3. By intervening with siRNA-TRAF3, we validated that miR-1388 regulates the expression of antiviral genes and the production of type I interferons (IFN-Is) through its interaction with TRAF3. Collectively, our experiments highlight the regulatory influence of miR-1388 on the IRF3-mediated signaling pathway by targeting TRAF3 post poly(I:C) stimulation. These findings provide compelling evidence for enhancing our understanding of the mechanisms through which fish miRNAs participate in immune responses.

Effects of Environmental Hypoxia on Serum Hematological and Biochemical Parameters, Hypoxia-Inducible Factor (hif) Gene Expression and HIF Pathway in Hybrid Sturgeon (Acipenser schrenckii ♂ × Acipenser baerii ♀).

Hypoxia is a globally pressing environmental problem in aquatic ecosystems. In the present study, a comprehensive analysis was performed to evaluate the effects of hypoxia on physiological responses (hematology, cortisol, biochemistry, hif gene expression and the HIF pathway) of hybrid sturgeons (Acipenser schrenckii ♂ × Acipenser baerii ♀). A total of 180 hybrid sturgeon adults were exposed to dissolved oxygen (DO) levels of 7.00 ± 0.2 mg/L (control, N), 3.5 ± 0.2 mg/L (moderate hypoxia, MH) or 1.00 ± 0.1 mg/L (severe hypoxia, SH) and were sampled at 1 h, 6 h and 24 h after hypoxia. The results showed that the red blood cell (RBC) counts and the hemoglobin (HGB) concentration were significantly increased 6 h and 24 h after hypoxia in the SH group. The serum cortisol concentrations gradually increased with the decrease in the DO levels. Moreover, several serum biochemical parameters (AST, AKP, HBDB, LDH, GLU, TP and T-Bil) were significantly altered at 24 h in the SH group. The HIFs are transcription activators that function as master regulators in hypoxia. In this study, a complete set of six hif genes were identified and characterized in hybrid sturgeon for the first time. After hypoxia, five out of six sturgeon hif genes were significantly differentially expressed in gills, especially hif-1α and hif-3α, with more than 20-fold changes, suggesting their important roles in adaptation to hypoxia in hybrid sturgeon. A meta-analysis indicated that the HIF pathway, a major pathway for adaptation to hypoxic environments, was activated in the liver of the hybrid sturgeon 24 h after the hypoxia challenge. Our study demonstrated that hypoxia, particularly severe hypoxia (1.00 ± 0.1 mg/L), could cause considerable stress for the hybrid sturgeon. These results shed light on their adaptive mechanisms and potential biomarkers for hypoxia tolerance, aiding in aquaculture and conservation efforts.

Discovery and Characterization of the ddx41 Gene in Atlantic Salmon: Evolutionary Implications, Structural Functions, and Innate Immune Responses to Piscirickettsia salmonis and Renibacterium salmoninarum Infections.

The innate immune response in Salmo salar, mediated by pattern recognition receptors (PRRs), is crucial for defending against pathogens. This study examined DDX41 protein functions as a cytosolic/nuclear sensor for cyclic dinucleotides, RNA, and DNA from invasive intracellular bacteria. The investigation determined the existence, conservation, and functional expression of the ddx41 gene in S. salar. In silico predictions and experimental validations identified a single ddx41 gene on chromosome 5 in S. salar, showing 83.92% homology with its human counterpart. Transcriptomic analysis in salmon head kidney confirmed gene transcriptional integrity. Proteomic identification through mass spectrometry characterized three unique peptides with 99.99% statistical confidence. Phylogenetic analysis demonstrated significant evolutionary conservation across species. Functional gene expression analysis in SHK-1 cells infected by Piscirickettsia salmonis and Renibacterium salmoninarum indicated significant upregulation of DDX41, correlated with increased proinflammatory cytokine levels and activation of irf3 and interferon signaling pathways. In vivo studies corroborated DDX41 activation in immune responses, particularly when S. salar was challenged with P. salmonis, underscoring its potential in enhancing disease resistance. This is the first study to identify the DDX41 pathway as a key component in S. salar innate immune response to invading pathogens, establishing a basis for future research in salmonid disease resistance.

The Role of Z Chromosome Localization Gene psmd9 in Spermatogenesis of Cynoglossus semilaevis.

Proteasome 26S Subunit, Non-ATPase 9 (psmd9) plays an important role in the balance of protamine and the stability of the nucleolar structure during spermatogenesis. In this study, we cloned the psmd9 of Cynoglossus semilaevis and analyzed its expression pattern. psmd9 was identified on the Z chromosome of C. semilaevis, which is considered an interesting candidate gene for spermatogenesis. qRT-PCR and FISH experiments showed that the psmd9 gene was significantly highly expressed in the testes. It is worth noting that the expression level of psmd9 in male fish testes is significantly higher than that in pseudomales. In order to further explore the role of psmd9 in spermatogenesis, a male testicular cell line was used as the experimental material. The results of the psmd9-RNAi and overexpression experiments showed that psmd9 had a synergistic effect with spermatogenesis-related genes dnd1, cfap69, dnah3 and dnajb13, but had an antagonistic effect with ccne2. Our findings offer a scientific foundation for comprehending the role of psmd9 in the spermatogenesis regulatory network of C. semilaevis.

The anti-inflammatory role of zDHHC23 through the promotion of macrophage M2 polarization and macrophage necroptosis in large yellow croaker (Larimichthys crocea).

Zinc finger Asp-His-His-Cys motif-containing (zDHHC) proteins, known for their palmitoyltransferase (PAT) activity, play crucial roles in diverse cellular processes, including immune regulation. However, their non-palmitoyltransferase immunomodulatory functions and involvement in teleost immune responses remain underexplored. In this study, we systematically characterized the zDHHC family in the large yellow croaker (Larimichthys crocea), identifying 22 members. Phylogenetic analysis unveiled that each of the 22 LczDHHCs formed distinct clusters with their orthologues from other teleost species. Furthermore, all LczDHHCs exhibited a highly conserved DHHC domain, as confirmed by tertiary structure prediction. Notably, LczDHHC23 exhibited the most pronounced upregulation following Pseudomonas plecoglossicida (P. plecoglossicida) infection of macrophage/monocyte cells (MO/MΦ). Silencing LczDHHC23 led to heightened pro-inflammatory cytokine expression and diminished anti-inflammatory cytokine levels in MO/MΦ during infection, indicating its anti-inflammatory role. Functionally, LczDHHC23 facilitated M2-type macrophage polarization, as evidenced by a significant skewing of MO/MΦ towards the pro-inflammatory M1 phenotype upon LczDHHC23 knockdown, along with the inhibition of MO/MΦ necroptosis induced by P. plecoglossicida infection. These findings highlight the non-PAT immunomodulatory function of LczDHHC23 in teleost immune regulation, broadening our understanding of zDHHC proteins in host-pathogen interactions, suggesting LczDHHC23 as a potential therapeutic target for immune modulation in aquatic species.

Teleost Hox code defines regional identities competent for the formation of dorsal and anal fins.

The dorsal and anal fins can vary widely in position and length along the anterior-posterior axis in teleost fishes. However, the molecular mechanisms underlying the diversification of these fins remain unknown. Here, we used genetic approaches in zebrafish and medaka, in which the relative positions of the dorsal and anal fins are opposite, to demonstrate the crucial role of hox genes in the patterning of the teleost posterior body, including the dorsal and anal fins. By the CRISPR-Cas9-induced frameshift mutations and positional cloning of spontaneous dorsalfinless medaka, we show that various hox mutants exhibit the absence of dorsal or anal fins, or a stepwise posterior extension of these fins, with vertebral abnormalities. Our results indicate that multiple hox genes, primarily from hoxc-related clusters, encompass the regions responsible for the dorsal and anal fin formation along the anterior-posterior axis. These results further suggest that shifts in the anterior boundaries of hox expression which vary among fish species, lead to diversification in the position and size of the dorsal and anal fins, similar to how modulations in Hox expression can alter the number of anatomically distinct vertebrae in tetrapods. Furthermore, we show that hox genes responsible for dorsal fin formation are different between zebrafish and medaka. Our results suggest that a novel mechanism has occurred during teleost evolution, in which the gene network responsible for fin formation might have switched to the regulation downstream of other hox genes, leading to the remarkable diversity in the dorsal fin position.

Characteristics of the Vasa Gene in Silurus asotus and Its Expression Response to Letrozole Treatment.

The identification and expression of germ cells are important for studying sex-related mechanisms in fish. The vasa gene, encoding an ATP-dependent RNA helicase, is recognized as a molecular marker of germ cells and plays a crucial role in germ cell development. Silurus asotus, an important freshwater economic fish species in China, shows significant sex dimorphism with the female growing faster than the male. However, the molecular mechanisms underlying these sex differences especially involving in the vasa gene in this fish remain poorly understood. In this work, the vasa gene sequence of S. asotus (named as Savasa) was obtained through RT-PCR and rapid amplification of cDNA end (RACE), and its expression in embryos and tissues was analyzed using qRT-PCR and an in situ hybridization method. Letrozole (LT) treatment on the larvae fish was also conducted to investigate its influence on the gene. The results revealed that the open reading frame (ORF) of Savasa was 1989 bp, encoding 662 amino acids. The SaVasa protein contains 10 conserved domains unique to the DEAD-box protein family, showing the highest sequence identity of 95.92% with that of Silurus meridionalis. In embryos, Savasa is highly expressed from the two-cell stage to the blastula stage in early embryos, with a gradually decreasing trend from the gastrula stage to the heart-beating stage. Furthermore, Savasa was initially detected at the end of the cleavage furrow during the two-cell stage, later condensing into four symmetrical cell clusters with embryonic development. At the gastrula stage, Savasa-positive cells increased and began to migrate towards the dorsal side of the embryo. In tissues, Savasa is predominantly expressed in the ovaries, with almost no or lower expression in other detected tissues. Moreover, Savasa was expressed in phase I-V oocytes in the ovaries, as well as in spermatogonia and spermatocytes in the testis, implying a specific expression pattern of germ cells. In addition, LT significantly upregulated the expression of Savasa in a concentration-dependent manner during the key gonadal differentiation period of the fish. Notably, at 120 dph after LT treatment, Savasa expression was the lowest in the testis and ovary of the high concentration group. Collectively, findings from gene structure, protein sequence, phylogenetic analysis, RNA expression patterns, and response to LT suggest that Savasa is maternally inherited with conserved features, serving as a potential marker gene for germ cells in S.asotus, and might participate in LT-induced early embryonic development and gonadal development processes of the fish. This would provide a basis for further research on the application of germ cell markers and the molecular mechanisms of sex differences in S. asotus.

Sex-specific overdominance at the maturation vgll3 gene for reproductive fitness in wild Atlantic salmon.

Linking reproductive fitness with adaptive traits at the genomic level can shed light on the mechanisms that produce and maintain sex-specific selection. Here, we construct a multigenerational pedigree to investigate sex-specific selection on a maturation gene, vgll3, in a wild Atlantic salmon population. The vgll3 locus is responsible for ~40% of the variation in maturation (sea age at first reproduction). Genetic parentage analysis was conducted on 18,265 juveniles (parr) and 685 adults collected at the same spawning ground over eight consecutive years. A high proportion of females (26%) were iteroparous and reproduced two to four times in their lifetime. A smaller proportion of males (9%) spawned at least twice in their lifetime. Sex-specific patterns of reproductive fitness were related to vgll3 genotype. Females showed a pattern of overdominance where vgll3*EL genotypes had three-fold more total offspring than homozygous females. In contrast, males demonstrated that late-maturing vgll3*LL individuals had two-fold more offspring than either vgll3*EE or vgll3*EL males. Taken together, these data suggest that balancing selection in females contributes to the maintenance of variation at this locus via increased fitness of iteroparous vgll3*EL females. This study demonstrates the utility of multigenerational pedigrees for uncovering complex patterns of reproduction, sex-specific selection and the maintenance of genetic variation.

DNA Methylation Mediates Sperm Quality via piwil1 and piwil2 Regulation in Japanese Flounder (Paralichthys olivaceus).

DNA methylation is an important way to regulate gene expression in eukaryotes. In order to reveal the role of DNA methylation in the regulation of germ cell-specific piwi gene expression during spermatogenesis of Japanese flounder (Paralichthys olivaceus), the expression profiles of piwil1 (piwi-like 1) and piwil2 (piwi-like 2) genes in the gonads of female, male, and sex-reversed pseudo-male P. olivaceus were analyzed, and the dynamic of DNA methylation was investigated. As a result, piwil1 and piwil2 genes were highly expressed in the testis of both male and pseudo-male P. olivaceus, with significant variation among male individuals. The DNA methylation levels in the promoter regions of both piwil1 and piwil2 were negatively correlated with their expression levels, which may contribute to the transcriptional regulation of piwi genes during spermatogenesis. There was also sperm quality variation among male P. olivaceus, and the sperm curvilinear velocity was positively correlated with the expression of both piwil1 and piwil2 genes. These results indicated that the DNA methylation in piwil1 and piwil2 promoter regions may affect the initiation of piwi gene transcription, thereby regulating gene expression and further affecting the spermatogenesis process and gamete quality in P. olivaceus.

Systematic selection of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Lutjanus erythropterus.

Quantitative real-time PCR (qRT-PCR) has been widely employed for the study of gene expression in fish, and accurate normalization is crucial. In this study, we aimed to identify the most stably expressed genes in various tissues, different developmental stages, and within astaxanthin treatment groups in Lutjanus erythropterus. Twelve candidate genes (EEF1A, CYB5R3, DLD, IDH3A, MRPL17, MRPL43, NDUFS7, PABPC1, PAGR1, PFDN2, PSMC3, and RAB10) were examined via qRT-PCR. We employed geNorm and NormFinder to assess their stability. The results revealed that RAB10 and PFDN2 exhibited relatively stable expression patterns across different tissue and astaxanthin treatment groups, while NDUFS7 and MRPL17 proved to be the most reliable reference gene combinations across various developmental stages. The stability of these selected genes was further validated by assessing the expression of two target genes, CRADD and CAPNS1, across developmental stages, reinforcing the reliability of NDUFS7 as it closely aligned with transcriptome-wide expression patterns at these stages. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in L. erythropterus.

In vitro analysis of the expression of inflammasome, antiviral, and immune genes in an Oreochromis niloticus liver cell line following stimulation with bacterial ligands and infection with tilapia lake virus.

The inflammasome is a multimeric protein complex that plays a vital role in the defence against pathogens and is therefore considered an essential component of the innate immune system. In this study, the expression patterns of inflammasome genes (NLRC3, ASC, and CAS-1), antiviral genes (IFNγ and MX), and immune genes (IL-1ß and IL-18) were analysed in Oreochromis niloticus liver (ONIL) cells following stimulation with the bacterial ligands peptidoglycan (PGN) and lipopolysaccharide (LPS) and infection with TiLV. The cells were stimulated with PGN and LPS at concentrations of 10, 25, and 50 µg/ml. For viral infection, 106 TCID50 of TiLV per ml was used. After LPS stimulation, all seven genes were found to be expressed at specific time points at each of the three doses tested. However, at even higher doses of LPS, NLRC3 levels decreased. Following TiLV infection, all of the genes showed significant upregulation, especially at early time points. However, the gene expression pattern was found to be unique in PGN-treated cells. For instance, NLRC3 and ASC did not show any response to PGN stimulation, and the expression of IFNγ was downregulated at 25 and 50 µg of PGN per ml. CAS-1 and IL-18 expression was downregulated at 25 µg of PGN per ml. At a higher dose (50 µg/ml), IL-1ß showed downregulation. Overall, our results indicate that these genes are involved in the immune response to viral and bacterial infection and that the degree of response is ligand- and dose-dependent.

The Expansion of Sirtuin Gene Family in Gilthead Sea Bream (Sparus aurata)-Phylogenetic, Syntenic, and Functional Insights across the Vertebrate/Fish Lineage.

The Sirtuin (SIRT1-7) family comprises seven evolutionary-conserved enzymes that couple cellular NAD availability with health, nutrition and welfare status in vertebrates. This study re-annotated the sirt3/5 branch in the gilthead sea bream, revealing three paralogues of sirt3 (sirt3.1a/sirt3.1b/sirt3.2) and two of sirt5 (sirt5a/sirt5b) in this Perciform fish. The phylogeny and synteny analyses unveiled that the Sirt3.1/Sirt3.2 dichotomy was retained in teleosts and aquatic-living Sarcopterygian after early vertebrate 2R whole genome duplication (WGD). Additionally, only certain percomorphaceae and gilthead sea bream showed a conserved tandem-duplicated synteny block involving the mammalian-clustered sirt3.1 gene (psmd13-sirt3.1a/b-drd4-cdhr5-ctsd). Conversely, the expansion of the Sirt5 branch was shaped by the teleost-specific 3R WGD. As extensively reviewed in the literature, human-orthologues (sirt3.1/sirt5a) showed a high, conserved expression in skeletal muscle that increased as development advanced. However, recent sirt3.2 and sirt5b suffered an overall muscle transcriptional silencing across life, as well as an enhanced expression on immune-relevant tissues and gills. These findings fill gaps in the ontogeny and differentiation of Sirt genes in the environmentally adaptable gilthead sea bream, becoming a good starting point to advance towards a full understanding of its neo-functionalization. The mechanisms originating from these new paralogs also open new perspectives in the study of cellular energy sensing processes in vertebrates.

Poly (I:C)-Induced microRNA-30b-5p Negatively Regulates the JAK/STAT Signaling Pathway to Mediate the Antiviral Immune Response in Silver Carp (Hypophthalmichthys molitrix) via Targeting CRFB5.

In aquaculture, viral diseases pose a significant threat and can lead to substantial economic losses. The primary defense against viral invasion is the innate immune system, with interferons (IFNs) playing a crucial role in mediating the immune response. With advancements in molecular biology, the role of non-coding RNA (ncRNA), particularly microRNAs (miRNAs), in gene expression has gained increasing attention. While the function of miRNAs in regulating the host immune response has been extensively studied, research on their immunomodulatory effects in teleost fish, including silver carp (Hyphthalmichthys molitrix), is limited. Therefore, this research aimed to investigate the immunomodulatory role of microRNA-30b-5p (miR-30b-5p) in the antiviral immune response of silver carp (Hypophthalmichthys molitrix) by targeting cytokine receptor family B5 (CRFB5) via the JAK/STAT signaling pathway. In this study, silver carp were stimulated with polyinosinic-polycytidylic acid (poly (I:C)), resulting in the identification of an up-regulated miRNA (miR-30b-5p). Through a dual luciferase assay, it was demonstrated that CRFB5, a receptor shared by fish type I interferon, is a novel target of miR-30b-5p. Furthermore, it was found that miR-30b-5p can suppress post-transcriptional CRFB5 expression. Importantly, this study revealed for the first time that miR-30b-5p negatively regulates the JAK/STAT signaling pathway, thereby mediating the antiviral immune response in silver carp by targeting CRFB5 and maintaining immune system stability. These findings not only contribute to the understanding of how miRNAs act as negative feedback regulators in teleost fish antiviral immunity but also suggest their potential therapeutic measures to prevent an excessive immune response.

Genome-Wide Identification, Molecular Characterization, and Expression Analysis of the HSP70 and HSP90 Gene Families in Thamnaconus septentrionalis.

Heat shock proteins (HSPs) are a class of highly conserved proteins that play an important role in biological responses to various environmental stresses. The mariculture of Thamnaconus septentrionalis, a burgeoning aquaculture species in China, frequently encounters stressors such as extreme temperatures, salinity variations, and elevated ammonia levels. However, systematic identification and analysis of the HSP70 and HSP90 gene families in T. septentrionalis remain unexplored. This study conducted the first genome-wide identification of 12 HSP70 and 4 HSP90 genes in T. septentrionalis, followed by a comprehensive analysis including phylogenetics, gene structure, conserved domains, chromosomal localization, and expression profiling. Expression analysis from RNA-seq data across various tissues and developmental stages revealed predominant expression in muscle, spleen, and liver, with the highest expression found during the tailbud stage, followed by the gastrula, neurula, and juvenile stages. Under abiotic stress, most HSP70 and HSP90 genes were upregulated in response to high temperature, high salinity, and low salinity, notably hspa5 during thermal stress, hspa14 in high salinity, and hsp90ab1 under low salinity conditions. Ammonia stress led to a predominance of downregulated HSP genes in the liver, particularly hspa2, while upregulation was observed in the gills, especially for hsp90b1. Quantitative real-time PCR analysis corroborated the expression levels under environmental stresses, validating their involvement in stress responses. This investigation provides insights into the molecular mechanisms of HSP70 and HSP90 in T. septentrionalis under stress, offering valuable information for future functional studies of HSPs in teleost evolution, optimizing aquaculture techniques, and developing stress-resistant strains.

Magnetic orientation in juvenile Atlantic herring (Clupea harengus) could involve cryptochrome 4 as a potential magnetoreceptor.

The Earth's magnetic field can provide reliable directional information, allowing migrating animals to orient themselves using a magnetic compass or estimate their position relative to a target using map-based orientation. Here we show for the first time that young, inexperienced herring (Clupea harengus, Ch) have a magnetic compass when they migrate hundreds of kilometres to their feeding grounds. In birds, such as the European robin (Erithacus rubecula), radical pair-based magnetoreception involving cryptochrome 4 (ErCRY4) was demonstrated; the molecular basis of magnetoreception in fish is still elusive. We show that cry4 expression in the eye of herring is upregulated during the migratory season, but not before, indicating a possible use for migration. The amino acid structure of herring ChCRY4 shows four tryptophans and a flavin adenine dinucleotide-binding site, a prerequisite for a magnetic receptor. Using homology modelling, we successfully reconstructed ChCRY4 of herring, DrCRY4 of zebrafish (Danio rerio) and StCRY4 of brown trout (Salmo trutta) and showed that ChCRY4, DrCRY4 and ErCRY4a, but not StCRY4, exhibit very comparable dynamic behaviour. The electron transfer could take place in ChCRY4 in a similar way to ErCRY4a. The combined behavioural, transcriptomic and simulation experiments provide evidence that CRY4 could act as a magnetoreceptor in Atlantic herring.

Molecular characterization, expression and immune functional analysis of cystatin 10 in turbot.

BACKGROUND: Cystatin is a protease inhibitor that also regulates genes expression linked to inflammation and plays a role in defense and regulation. METHODS AND RESULTS: Cystatin 10 (Smcys10) was cloned from Scophthalmus maximus and encodes a 145 amino acid polypeptide. The results of qRT-PCR showed that Smcys10 exhibited tissue-specific expression patterns, and its expression was significantly higher in the skin than in other tissues. The expression level of Smcys10 was significantly different in the skin, gill, head kidney, spleen and macrophages after Vibrio anguillarum infection, indicating that Smcys10 may play an important role in resistance to V. anguillarum infection. The recombinant Smcys10 protein showed binding and agglutinating activity in a Ca2+-dependent manner against bacteria. rSmcys10 treatment upregulated the expression of IL-10, TNF-α and TGF-ß in macrophages of turbot and hindered the release of lactate dehydrogenase (LDH) from macrophages after V. anguillarum infection, which confirmed that rSmcys10 reduced the damage to macrophages by V. anguillarum. The NF-κB pathway was suppressed by Smcys10, as demonstrated by dual-luciferase analysis. CONCLUSIONS: These results indicated that Smcys10 is involved in the host antibacterial immune response.

MAVS disruption impairs downstream signaling and results in higher virus replication levels of salmonid alphavirus subtype 3 but not infectious pancreatic necrosis virus in vitro.

The mitochondrial anti-viral signaling (MAVS) protein is an intermediary adaptor protein of retinoic acid-inducible gene-1 (RIG-I) like receptor (RLR) signaling, which activates the transcription factor interferon (IFN) regulatory factor 3 (IRF3) and NF-kB to produce type I IFNs. MAVS expression has been reported in different fish species, but few studies have shown its functional role in anti-viral responses to fish viruses. In this study, we used the transcription activator-like effector nuclease (TALEN) as a gene editing tool to disrupt the function of MAVS in Chinook salmon (Oncorhynchus tshawytscha) embryonic cells (CHSE) to understand its role in induction of interferon I responses to infections with the (+) RNA virus salmonid alphavirus subtype 3 (SAV-3), and the dsRNA virus infectious pancreatic necrosis virus (IPNV) infection. A MAVS-disrupted CHSE clone with a 7-aa polypeptide (GVFVSRV) deletion mutation at the N-terminal of the CARD domain infected with SAV-3 resulted in significantly lower expression of IRF3, IFNa, and ISGs and increased viral titer (1.5 log10) compared to wild-type. In contrast, the IPNV titer in MAVS-disrupted cells was not different from the wild-type. Furthermore, overexpression of salmon MAVS in MAVS-disrupted CHSE cells rescued the impaired type I IFN-mediated anti-viral effect against SAV-3.

Gsdf is not indispensable for male differentiation in the medaka species Oryzias hubbsi.

Sex determination mechanisms differ widely among vertebrates, particularly in fish species, where diverse sex chromosomes and sex-determining genes have evolved. However, the sex-differentiation pathways activated by these sex-determining genes appear to be conserved. Gonadal soma-derived growth factor (Gsdf) is one of the genes conserved across teleost fish, especially in medaka fishes of the genus Oryzias, and is implicated in testis differentiation and germ cell proliferation. However, its role in sex differentiation remains unclear. In this study, we investigated Gsdf function in Oryzias hubbsi, a species with a ZW sex-determination system. We confirmed its male-dominant expression, as in other species. However, histological analyses revealed no male-to-female sex reversal in Gsdf-knockout fish, contrary to findings in other medaka species. Genetic sex determination remained intact without Gsdf function, indicating a Gsdf-independent sex-differentiation pathway in O. hubbsi. Instead, Gsdf loss led to germ cell overproliferation in both sexes and accelerated onset of meiosis in testes, suggesting a role in germ cell proliferation. Notably, the feminizing effect of germ cells observed in O. latipes was absent, suggesting diverse germ cell-somatic cell relationships in Oryzias gonad development. Our study highlights species-specific variations in the molecular pathways governing sex determination and differentiation, emphasizing the need for further exploration to elucidate the complexities of sexual development.