Multiple-Integrated Biomarker Indexes to Assess the Responses of the Flatfish Achirus lineatus during Exposure to Light Crude Oil Water Accommodated Fraction.

In the present study, we evaluated the biological response of Achirus lineatus to water accommodated fraction (WAF) of light crude oil (American Petroleum Institute gravity 35°) during a sub-chronic bioassay (14 and 28 days) at two different concentrations: 5% v/v (1.20 µg∙L- 1 expressed as total polycyclic aromatic hydrocarbons [∑25 PAH]) and 10% v/v (6.61 µg∙L- 1 [∑25 PAH]). The responses were evaluated through the biomarker response index (BRI), the integrated biomarker response (IBRv2) and the bioconcentration factor (BCF). The results showed an increase in biological response in relation to WAF concentration and exposure time, which resulted in a slight and moderate disturbance in the basal condition and bioconcentration level of metals (Pb > Ni > V > Cd) in fish tissue. Results in the present study denote that flatfish such as A. lineatus may be negatively influenced by spilled light crude oil.

THIN AND ULTRATHIN STRUCTURE OF ENVELOPES OF STEPHANOSTOMUM BACCATUM (TREMATODA: ACANTHOCOLPIDAE) METACERCARIAE IN YELLOWFIN SOLE, LIMANDA ASPERA.

The structure of the envelopes (capsule and cyst) surrounding metacercariae of Stephanostomum baccatum (Nicoll, 1907) in the second intermediate host, the yellowfin sole Limanda aspera (Pallas 1814), is examined with the methods of light and transmission electron microscopy. The cyst, presumably formed by secretions of the metacercarial tegument, consists of 2 layers: the outer, very thin layer of an electron-dense, finely granular substance and the inner layer composed of loose material of a moderate electron density that includes dense bodies varying in size, shape, and localization. The capsule, formed by the host's cells, is also organized into 2 distinct layers. The inner layer of the capsule is loose, consisting of evenly spaced debris of degenerated cells and lipid droplets with inclusions of intact macrophages between them. The outer layer of the capsule consists of parallel rows of cells arranged around the parasite, with fibroblasts and macrophages being dominant types and granulocytes and lymphocytes found in smaller numbers. Aggregations of collagen fibers are located in narrow spaces between the cells. The number of lipid droplets in the outer layer is significantly smaller than in the inner layer. The capsules formed around the examined trematodes have several structural features that distinguish them from those of S. baccatum and Stephanostomum sp. metacercariae recovered from other fishes of the family Pleuronectidae. The major morphological features of such capsules are the lack of epithelioid or giant multinucleated cells and the presence of numerous lipid droplets. Investigating the structural details of the envelopes surrounding metacercariae in trematodes, as well as other helminths, contributes to our scientific understanding of parasite biology, which can, in turn, have broader implications for understanding host-parasite interactions and evolutionary biology.

Comparison of the presence of high production volume chemicals in farmed and wild fish highly consumed in catalonia and their risk assessment.

The decline in fish populations and the depletion of marine resources have sparked concerns about sustainable fish production, driving the innovation of new aquaculture methods. While some argue that wild fish are healthier than farmed fish due to less exposure to contaminants and pathogens, wild fish can accumulate contaminants from more contaminated water sources. The slower growth of wild fish and their longer exposure to the environment may contribute to higher pollutant levels in fish tissues. In this study, we focus on 25 contaminants considered as high production volume chemicals (HPVCs), such as organophosphate esters (OPEs), benzothiazoles (BTs), benzosulfonamides (BSAs) and phthalates (PAEs). The compounds were extracted from the edible part of the fish using the QuEChERS method and analysed by gas chromatography-tandem mass spectrometry. A total of 74 samples were analysed from three of the most commonly consumed species in Catalonia, Spain (turbot, sea bass and sea bream). Two samples of each species were collected each month, one form farmed and one from wild origin. In general, the compounds were found in all the samples in a wide concentrations range, although no significant differences were observed between the mean concentration of wild and farmed samples. Although similar mean concentrations for the OPEs, BTs and BSAs were found between farmed and wild origin samples, PAEs were more frequently detected in farmed samples. Di-n-octyl phthalate and diethyl phthalate showed the highest concentrations in all fish samples, with values up to 19505 and 17605 ng g-1 (d.w.), in sea bass and sea bream, respectively. Di-(2-ethylexyl)-adipate proved to be the most relevant carcinogenic compound, with no associated health risk. Despite the detection of the studied HPVCs, no health risk was associated with the consumption of these three fish species.

Leptins regulate the migration, proliferation, apoptosis, and synthesis of sexual steroid in tongue sole (Cynoglossus semilaevis) ovarian cells.

Leptin is an important hormone in mammals, which plays a key role in regulating reproduction and energy metabolism. However, there are few studies on the function of leptin in reproductive regulation in fish, especially on tongue sole (Cynoglossus semilaevis). Thus, in this study, we firstly exploited the basic function of tongue sole leptins, the migration and growth rate of ovarian cells were reduced after knocking down lepA and lepB in ovarian cells, while increasing the apoptosis rate. Then both rlepA and rlepB were proved to be combined with lepR to further exert functions by dual luciferase assay. Transcriptome sequencing showed that differentially expressed genes (DEGs) were mainly enriched in KEGG pathways related to membrane receptors, fatty acid synthesis, growth, etc. when lepA and lepB were knocked down or additionally added in vitro. Additionally, the estradiol (E2) hormone was increased significantly after knocking down lepB. Finally, based on DEGs and the signaling pathways they participated in, we proposed a hypothesis about the signaling pathways in which leptin may be involved in ovarian cells. Taken together, these results provide new insights into the role of leptin in the regulation of physiological functions such as ovarian growth and development.

How contaminated is flatfish living near World Wars' munition dumping sites with energetic compounds?

Seas worldwide are threatened by an emerging source of pollution as millions of tons of warfare materials were dumped after the World Wars. As their metal shells are progressively corroding, energetic compounds (EC) leak out and distribute in the marine environment. EC are taken up by aquatic organisms and pose a threat to both the marine ecosphere and the human seafood consumer because of their toxicity and potential carcinogenicity. Here, sediment samples and fish from different locations in the German North Sea of Lower Saxony were examined to determine whether EC transfer to fish living close to munition dumping areas. EC were found in sediments with a maximum concentration of 1.5 ng/kg. All analyzed fish muscle tissues/fillets and bile samples were positive for EC detection. In bile, the max. EC concentrations ranged between 0.25 and 1.25 ng/mL. Interestingly, while detected TNT metabolites in the muscle tissues were in concentrations of max. 1 ng/g (dry weight), TNT itself was found in concentrations of up to 4 ng/g (dry weight). As we found considerable higher amounts of non-metabolized TNT in the fish muscle, rather than TNT metabolites, we conclude an additional absorption route of EC into fish other than per diet. This is the first study to detect EC in the edible parts of fish caught randomly in the North Sea.

Immune and regulative characterization of complement-related gene cfhl5 in response to Vibrio harveyi challenge in Cynoglossus semilaevis.

Complement factor H-related protein (CFHR) plays an important role in regulating complement activation and defensive responses. The function of CFHR2 (complement factor H related 2), a member of the CFHR family, in fish remains unclear. Here, we report the genetic relationship, expression characteristics and regulatory mechanism of cfhl5 (complement factor H like 5) gene, which encodes CFHR2 in Chinese tongue sole. We observed that the cfhl5 gene was widely expressed in several tissues, such as brain, heart and immune organs, and was most abundantly expressed in liver. After injection with Vibrio harveyi, the expression of cfhl5 was up-regulated significantly in liver, spleen and kidney at 12 or 24 hours post infection (hpi), suggesting an involvement of this gene in the acute immune response. Knockdown of cfhl5 in liver cells significantly up-regulated the expression of the pro-inflammatory cytokines tnf-α (tumor necrosis factor-alpha) and il1ß (interleukin-1beta), the immunomodulatory factor il10 (interleukin-10) and the lectin complement pathway gene masp1 (MBL-associated serine protease 1), and down-regulated the expression of complement components c3 (complement 3) and cfi (complement factor I). In our previous work, we found that cfhl5 gene was significantly higher methylated and lower expressed in the resistant family compared with the susceptible family. Therefore, we used dual-luciferase reporter system to determine the effect of DNA methylation on this gene and found that DNA methylation could inhibit the promoter activity to reduce its expression. These results demonstrated that the expression of cfhl5 is regulated by DNA methylation, and this gene might play an important role in the immune response by regulating the expression of cytokines and complement components genes in Chinese tongue sole.

Sonic hedgehog (shh) gene from Pseudopleuronectes yokohamae (Teleostei: Pleuronectidae): Molecular cloning, characterization, and expression profile during early embryonic, juvenile, and adult stages.

The hedgehog signaling pathway plays an important role in early development and growth of most vertebrates. Sonic hedgehog (shh) gene is a critical regulator of embryonic development in many species, including humans. However, it is not clear what roles shh can play in the development of fish. In this paper, shh gene was cloned from Pseudopleuronectes yokohamae. The full-length complementary DNA (cDNA) of P. yokohamae sonic hedgehog gene (Pyshh) comprises 3194 bp, with a 1317-bp open reading frame (ORF) that encodes a polypeptide of 438 amino acids with a typical HH-signal domain and Hint-N domain. The conserved sequences of the protein among species were predicted by using multiple sequence comparison. The phylogenetic tree construction showed that PySHH is clustered in a branch of Pleuronectidae. To explore the expression of Pyshh gene in various tissues of P. yokohamae, we used real-time fluorescence quantitative PCR technology to detect it. The results showed that Pyshh gene is widely distributed in various tissues of P. yokohamae juveniles, different tissues of adult males and females, and is particularly expressed in immune organs. The Pyshh gene expression was higher in the muscle and brain of juvenile fish, and higher in bone, gill, and skin of male fish than that of female fish, suggesting that Pyshh might be involved in the formation of immune organs of P. yokohamae. The expression of Pyshh gene significantly upregulated from the gastrula stage to the hatching stage. Western blotting of the expression levels of PySHH during different embryonic development stages revealed that PySHH levels increased gradually during development stages from oosperm stage to hatching stage. These results indicate that Pyshh is highly conserved among species and plays a critical role in the complex process of embryonic development. Its precise regulation is essential for the proper formation of many organs and tissues in the body, and disruptions in its function may have serious consequences for the formation of immune organs in fish.

CRISPR/dCas9-Mediated DNA Methylation Editing on emx2 in Chinese Tongue Sole (Cynoglossus semilaevis) Testis Cells.

DNA methylation is a key epigenetic mechanism orchestrating gene expression networks in many biological processes. Nonetheless, studying the role of specific gene methylation events in fish faces challenges. In this study, we validate the regulation of DNA methylation on empty spiracles homeobox 2 (emx2) expression with decitabine treatment in Chinese tongue sole testis cells. We used the emx2 gene as the target gene and developed a new DNA methylation editing system by fusing dnmt3a with catalytic dead Cas9 (dCas9) and demonstrated its ability for sequence-specific DNA methylation editing. Results revealed that utilizing dCas9-dnmt3a to target emx2 promoter region led to increased DNA methylation levels and decreased emx2 expression in Chinese tongue sole testis cells. More importantly, the DNA methylation editing significantly suppressed the expression of MYC proto-oncogene, bHLH transcription factor (myc), one target gene of emx2. Furthermore, we assessed the off-target effects of dCas9-dnmt3a and confirmed no significant impact on the predicted off-target gene expression. Taken together, we developed the first DNA methylation editing system in marine species and demonstrated its effective editing ability in Chinese tongue sole cells. This provides a new strategy for both epigenetic research and molecular breeding of marine species.

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.

The extracellular matrix protein EFEMP2 is involved in the response to VHSV infection in the olive flounder Paralichthys olivaceus.

The EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) is involved in connective tissue development, elastic fiber formation, and tumor growth. In this study, we characterized the cDNA of EFEMP2 (PoEFEMP2), a member of the fibulin family of ECM proteins, in the olive flounder Paralichthys olivaceus. The coding region of PoEFEMP2 encodes a protein that contains six calcium-binding EGF-like (EGF-CA) domains and four complement Clr-like EGF-like (cEGF) domains. PoEFEMP2 shows 67.51-96.77 % similarities to orthologs in a variety of fish species. PoEFEMP2 mRNA was detected in all tissues examined; the highest levels of PoEFEMP2 mRNA expression were observed in the heart, testis, ovary and muscle. The PoEFEMP2 mRNA level increases during early development. In addition, the PoEFEMP2 mRNA level increased at 3 h post-infection (hpi) and decreased from 6 to 48 hpi in flounder Hirame natural embryo (HINAE) cells infected with viral hemorrhagic septicemia virus (VHSV). Disruption of PoEFEMP2 using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system resulted in a significant upregulation of VHSV G mRNA levels and immune-related genes expression in knockout cells. These findings implicate PoEFEMP2 in antiviral responses in P. olivaceus.

Manipulated C5aR1 over/down-expression associates with IL-6 expression during bacterial inflammation in half-smooth tongue sole (Cynoglossus semilaevis).

The complement component 5a/complement component 5 receptor 1 (C5a/C5aR1) pathway plays a crucial role in the onset and development of inflammation, but relevant studies in fish are lacking. In this study, we successfully characterized the relationship between half-smooth tongue sole (Cynoglossus semilaevis) C5aR1 (CsC5aR1) and bacterial inflammation. First, we showed that the overexpression of CsC5aR1 significantly increased bacterial pathological damage in the liver and intestine, whereas inhibition attenuated the damage. The in vitro experiments suggested that CsC5aR1 was able to positively regulate the phagocytic activity and respiratory burst of tongue sole macrophages. In terms of both transcriptional and translational levels, overexpression/inhibition of CsC5aR1 was followed by a highly consistent up-regulation/decrease of its downstream canonical inflammatory factor interleukin-6 (CsIL-6). Furthermore, we stimulated macrophages by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and found a broad-spectrum response to bacterial infections by the C5a/C5aR1 complement pathway together with the downstream inflammatory factor CsIL-6. Subsequently, we directly elucidated that CsIL-6 is an indicator of C5a/C5aR1-mediated inflammation at different infection concentrations, different infectious bacteria (Vibrio anguillarum and Mycobacterium marinum), and different detection levels. These results might provide a new inflammation bio-marker for early warning of bacteria-induced hyperinflammation leading to fish mortality and a promising target for the treatment of bacterial inflammation in teleost.

Determination and Characterization of Novel Papillomavirus and Parvovirus Associated with Mass Mortality of Chinese Tongue Sole (Cynoglossus semilaevis) in China.

A massive mortality event concerning farmed Chinese tongue soles occurred in Tianjin, China, and the causative agent remains unknown. Here, a novel Cynoglossus semilaevis papillomavirus (CsPaV) and parvovirus (CsPV) were simultaneously isolated and identified from diseased fish via electron microscopy, virus isolation, genome sequencing, experimental challenges, and fluorescence in situ hybridization (FISH). Electron microscopy showed large numbers of virus particles present in the tissues of diseased fish. Viruses that were isolated and propagated in flounder gill cells (FG) induced typical cytopathic effects (CPE). The cumulative mortality of fish given intraperitoneal injections reached 100% at 7 dpi. The complete genomes of CsPaV and CsPV comprised 5939 bp and 3663 bp, respectively, and the genomes shared no nucleotide sequence similarities with other viruses. Phylogenetic analysis based on the L1 and NS1 protein sequences revealed that CsPaV and CsPV were novel members of the Papillomaviridae and Parvoviridae families. The FISH results showed positive signals in the spleen tissues of infected fish, and both viruses could co-infect single cells. This study represents the first report where novel papillomavirus and parvovirus are identified in farmed marine cultured fish, and it provides a basis for further studies on the prevention and treatment of emerging viral diseases.

EPA and DHA promote cell proliferation and enhance activity of the Akt-TOR-S6K anabolic signaling pathway in primary muscle cells of turbot (Scophthalmus maximus L.).

Fish growth and health are predominantly governed by dietary nutrient supply. Although the beneficial effects of omega-3 polyunsaturated fatty acids supplementation have been shown in a number of fish species, the underlying mechanisms are still mostly unknown. In this study, we conducted an investigation into the effects of EPA and DHA on cell proliferation, nutrient sensing signaling, and branched-chain amino acids (BCAA) transporting in primary turbot muscle cells. The findings revealed that EPA and DHA could stimulate cell proliferation, promote protein synthesis and inhibit protein degradation through activation of target of rapamycin (TOR) signaling pathway, a pivotal nutrient-sensing signaling cascade. While downregulating the expression of myogenin and myostatin, EPA and DHA increased the level of myogenic regulatory factors, such as myoD and follistatin. Furthermore, we observed a significant increase in the concentrations of intracellular BCAAs following treatment with EPA or DHA, accompanied by an upregulation of the associated amino acid transporters. Our study providing valuable insights into the mechanisms underlying the growth-promoting effects of omega-3 fatty acids in fish.

Differentially expressed proteins and microbial communities of the skin regulate disease resistance to Chinese tongue sole (Cynoglossus semilaevis).

Vibriosis, caused by Vibrio, seriously affects the health of fish, shellfish, and shrimps, causing large economic losses. Teleosts are represent the first bony vertebrates with both innate and adaptive immune responses against pathogens. Aquatic animals encounter hydraulic pressure and more pathogens, compared to terrestrial animals. The skin is the first line of defense in fish, constituting the skin-associated lymphoid tissue (SALT), which belongs to the main mucosa-associated lymphoid tissues (MALT). However, little is known about the function of immunity related proteins in fish. Therefore, this study used iTRAQ (isobaric tags for relative and absolute quantitation) to compare the skin proteome between the resistant and susceptible families of Cynoglossus semilaevis. The protein integrin beta-2, the alpha-enolase isoform X1, subunit B of V-type proton ATPase, eukaryotic translation initiation factor 6, and ubiquitin-like protein ISG15, were highly expressed in the resistant family. The 16S sequencing of the skin tissues of the resistant and susceptible families showed significant differences in the microbial communities of the two families. The protein-microbial interaction identified ten proteins associated with skin microbes, including immunoglobulin heavy chain gene (IGH), B-cell lymphoma/leukemia 10 (BCL10) and pre-B-cell leukemia transcription factor 1 isoform X2 (PBX2). This study highlights the interaction between skin proteins and the microbial compositions of C. semilaevis and provides new insights into understanding aquaculture breeding research.

Effect of tea polyphenol-trehalose complex coating solutions on physiological stress and flesh quality of marine-cultured Turbot Scophthalmus maximus during waterless transport.

OBJECTIVE: The waterless transport of live fish has changed the present situation of live-fish transport. However, the waterless transport environment may cause stress in fish. This research evaluated the effect of tea polyphenol-trehalose (TPT) coating solutions on Turbot Scophthalmus maximus during waterless transport. METHODS: After cold acclimation, Turbot were coated and subsequently transported in a waterless environment for 18 h. Physiological and biochemical parameters were measured, including lysozyme (LZM) and immunoglobulin M (IgM) activities, serum creatinine (Cr) and uric acid (UA) concentrations, and nutritional flavor. RESULT: The results showed that the nonspecific immunity of Turbot was inhibited during the waterless transport; the LZM activity first increased and then decreased, and the serum Cr and UA concentrations significantly increased. In addition, the waterless transport promoted the breakdown of Turbot flesh proteins, leading to changes in nucleotides and free amino acids (FAAs). After waterless transport, the LZM and IgM activities in the TPT-treated Turbot were higher than those in the control group (CK), and the changes in FAA content and nucleotides were smaller than those observed in the CK group. CONCLUSION: This study shows that the use of TPT coating solution can reduce the impact of waterless transportation stress on the immune and metabolic functions of Turbot and can maintain the meat quality and flavor of Turbot.

Water-in-oil adjuvant challenges in fish vaccination: An experimental inactivated adjuvanted vaccine against betanodavirus infection in Senegalese sole.

The extensive growth of intensive fish farming has led to a massive spread of infectious diseases. Nervous necrosis virus (NNV) is the causative agent of the viral encephalo- and retinopathy disease which has become a major threat for fish farming all over the globe. The devastating mortality rates recorded in disease outbreaks, especially when infected specimens are at early stages of development, have a high economic impact on the sector. Currently, vaccines are the most cost-effective preventing tool in the fight against viruses. Inactivated vaccines have the advantage of simplicity in their development at the same time as present the antigen in a similar manner than the natural infection in the host. Nevertheless, they usually trigger weaker immune responses needing adjuvants to boost their effectiveness. In this work, we have intraperitoneally vaccinated Senegalese sole juveniles (Solea senegalensis) with a previously designed inactivated vaccine against NNV based on binary ethylenimine (BEI), mixed or not with an oil-adjuvant. Our results demonstrated the potential activation of different immune pathways when the vaccine was administered alone compared to the oil-adjuvanted vaccine, both resulting in an equivalent partial improvement in survival following a NNV challenge. However, whilst the vaccine alone led to a significant increase in specific antibodies, in the adjuvanted version those antibodies were kept basal although with a slight improvement in their neutralization capacity. At transcriptional level, neither vaccine (adjuvanted or not) triggered the immune system activation during the vaccination period. However, after NNV infection, the BEI-inactivated vaccines alone and oil-adjuvanted both elicited the stimulation of antiviral responsive genes (rtp3, herc4), antigen presentation molecules (mhcii) and T-cell markers (cd8a) in the head-kidney. Additionally, the oil-adjuvanted vaccine appears to stimulate mediator cytokines (il6) and B-cell markers (ight and ighm). Surprisingly, when the adjuvant was administered alone, fish showed the highest survival rates concomitantly with a lack of NNV-IgM production, pointing to the possible induction of different immune pathways than the B-cell responses via antibodies by the adjuvant. Since this combined vaccine did not succeed in the full extension of protection against the pathogen, further studies should be performed focusing on unravelling the molecular mechanisms through which adjuvants trigger the immune response, both independently and when added to a vaccine antigen.

Scoliosis and kyphosis in blue-spotted and marbled flathead fish associated with a Myxobolus acanthogobii-like parasite.

Spinal deformities in finfish have the potential to impact aquaculture industries and wild populations by increasing morbidity, mortality, and reducing growth rates. Myxobolus acanthogobii has been implicated in causing scoliosis and lordosis in various aquatic species in Japan. We investigated 4 cases of spinal deformity in 2 flathead (Platycephalus) species that were submitted to the Elizabeth Macarthur Agricultural Institute (EMAI) in New South Wales (NSW), Australia, between 2015 and 2021. Flathead are commercially significant species that are popular among Australian consumers, and are also sought-after species targeted by recreational fishers. Gross deformities are concerning to the community and may impact the quality and quantity of specimens available for consumption. Three blue-spotted flathead (P. caeruleopunctatus) and one marbled flathead (P. marmoratus) were submitted, all with marked scoliosis and kyphosis; 1-2-mm cysts were present on the dorsum of the brain, most often over the optic lobe or cerebellum. Cytology and differential interference microscopy of cyst material revealed numerous oval spores, x̄ 14 ± SD 0.75 µm × x̄ 11.5 ± SD 0.70 µm, with 2 pyriform polar capsules, the morphology of which is consistent with a Myxobolus sp. PCR assay and 18S rDNA sequencing of the cyst material identified a Myxobolus sp. with 96% identity to M. acanthogobii. The identification of this Myxobolus sp. confirms the presence of parasites with the potential to cause spinal deformity in significant aquatic species in NSW waterways.

Optimal dietary lipid levels alleviated adverse effects of high temperature on growth, lipid metabolism, antioxidant and immune responses in juvenile turbot (Scophthalmus maximus L.).

Fish physiological health is often negatively impacted by high-temperature environments and there are few studies on how dietary lipids affect fish growth and physiology when exposed to heat stress. The main objective of this research was to examine the impact of dietary lipid levels on growth and physiological status of juvenile turbot (Scophthalmus maximus L.) and determine if dietary lipid concentration could alleviate the possible adverse effects of heat stress. Five diets containing 6.81%, 9.35%, 12.03%, 14.74%, and 17.08% lipid, respectively, were formulated and fed to turbot (initial weight 5.13 ± 0.02 g) under high-temperature conditions (24.0-25.0 °C). Meanwhile, the diet with 12.03% lipid (considered by prior work to be an optimal dietary lipid level) was fed to turbot of the same size at normal temperature. Results suggested that, among the different dietary lipid levels under high-temperature conditions, fish fed the optimal lipid (12.03%) exhibited better growth compared to non-optimal lipid groups, as evidenced by higher weight gain and specific growth rate. Simultaneously, the optimal lipid diet may better maintain lipid homeostasis, as attested by lower liver and serum lipid, along with higher liver mRNA levels of lipolysis-related genes (pgc1α, lipin1, pparα, lpl and hl) and lower levels of synthesis-related genes (lxr, fas, scd1, pparγ, dgat1 and dgat2). Also, the optimal lipid diet might mitigate oxidative damage by improving antioxidant enzyme activity, decreasing malondialdehyde levels, and up-regulating oxidation-related genes (sod1, sod2, cat, gpx and ho-1). Furthermore, the optimal lipid may enhance fish immunity, as suggested by the decrease in serum glutamic-oxalacetic/pyruvic transaminase activities, down-regulation of pro-inflammatory genes and up-regulation of anti-inflammation genes. Correspondingly, the optimal lipid level suppressed MAPK signaling pathway via decreased phosphorylation levels of p38, JNK and ERK proteins in liver. In summary, the optimal dietary lipid level facilitated better growth and physiological status in turbot under thermal stress.

CsIL-20, a tongue sole interleukin-20, negatively mediates leucocyte activity and antibacterial defense.

Interleukin-20 (IL-20), as an essential member of IL-10 family, plays vital roles in mammalian immunological response such as antimicrobial, inflammation, hematopoiesis, and immune diseases. In teleost, the study about immune antimicrobial function of IL-20 is largely scarce. In this article, we revealed the expression profiles and the immunological functions of the IL-20 (CsIL-20) in tongue sole Cynoglossus semilaevis. CsIL-20 is composed of 183 amino acid residues, with seven cysteine residues and a typical IL-10 domain which comprises six α-helices and two ß-sheets, and shares 34.4-71.2 % identities with other teleost IL-20. CsIL-20 was constitutively expressed in a variety of tissues and regulated by bacterial invasion, and the recombinant CsIL-20 (rCsIL-20) could bind to different bacteria. In vitro rCsIL-20 could interact with the membrane of peripheral blood leukocytes (PBLs), leading to the attenuation of reactive oxygen species (ROS) production and acid phosphatase activity in PBLs. In line with In vitro results, In vivo rCsIL-20 could obviously suppressed the host immune against bacterial infection. Furthermore, knockdown of CsIL-20 in vivo could markedly enhance the host antibacterial immunity. Collectively, these observations offer new insights into the negative effect of CsIL-20 on antibacterial immunity.

Butyrate induces STAT3/HIF-1α/IL-22 signaling via GPCR and HDAC3 inhibition to activate autophagy in head kidney macrophages from turbot (Scophthalmus maximus L.).

As one of short-chain fatty acids, butyrate is an important metabolite of dietary fiber by the fermentation of gut commensals. Our recent study uncovered that butyrate promoted IL-22 production in fish macrophages to augment the host defense. In the current study, we further explored the underlying signaling pathways in butyrate-induced IL-22 production in fish macrophages. Our results showed that butyrate augmented the IL-22 expression in head kidney macrophages (HKMs) of turbot through binding to G-protein receptor 41 (GPR41) and GPR43. Moreover, histone deacetylase 3 (HDAC3) inhibition apparently up-regulated the butyrate-enhanced IL-22 generation, indicating HDACs were engaged in butyrate-regulated IL-22 secretion. In addition, butyrate triggered the STAT3/HIF-1α signaling to elevate the IL-22 expression in HKMs. Importantly, the evidence in vitro and in vivo was provided that butyrate activated autophagy in fish macrophages via IL-22 signaling, which contributing to the elimination of invading bacteria. In conclusion, we clarified in the current study that butyrate induced STAT3/HIF-1α/IL-22 signaling pathway via GPCR binding and HDAC3 inhibition in fish macrophages to activate autophagy that was involved in pathogen clearance in fish macrophages.