Trunk Kidney Research Articles

Comparison and evaluation of DNA vaccines against Nocardia seriolae infection in largemouth bass (Micropterus salmoides)

Chronic granulomatous diseases caused by Nocardia seriolae have resulted in substantial economic losses for the aquaculture industry. DNA vaccines are considered a promising approach for controlling infectious diseases. To develop effective DNA vaccines against N. seriolae, we selected Ag85L, MmaA4, HspX, Rv3407, and HtpG as candidate antigens. Among these, pcDNA-Ag85L and pcDNA-MmaA4 were the most effective against N. seriolae infection in largemouth bass (Micropterus salmoides), achieving the highest relative percentage survival (RPS) rates of 78.6 % and 92.9 %, respectively. Tissue bacterial loads and histopathological tests revealed a significant reduction in bacterial abundance in the liver, spleen, head kidney, and trunk kidney of the immunised groups compared to that in the control group. Additionally, no distinct pathological changes were observed in the spleen or head kidney tissues. The expression levels of immune-related genes including IL-1β, IL-8, MHCI, MHCII, CD4, and CD8α were significantly upregulated in the pcDNA-Ag85L and pcDNA-MmaA4 immunised groups, thus indicating that the two DNA vaccines mediated immune protection by promoting both humoral and cellular immune responses. Overall, the pcDNA-Ag85L and pcDNA-MmaA4 DNA vaccines demonstrated strong potential for protecting largemouth bass against N. seriolae infection, offering an innovative solution for controlling granulomatous diseases in fish.

Mass mortality associated with carpione rhabdovirus in golden pompano (Trachinotus ovatus) in China: First report

From August to November 2023, a severe epizootic outbreak occurred in an offshore cultured golden pompano (Trachinotus ovatus) population in Zhanjiang City, Guangdong Province, China, resulting in a cumulative mortality rate of 65–82 %. The diseased fish showed clinical signs of lethargy, anorexia, whirling movements, and hemorrhage at the base of the fins and in the upper and lower jaws before mass mortality. A rhabdovirus strain, designated as CARPV2023, was successfully isolated using fathead minnow (FHM) cells. The cytopathic effects of the tissue filtrate from the diseased golden pompano on FHM and epithelioma papulosum cyprini (EPC) cell monolayers were characterized by rounded cells, grape-like cluster formation, detachment, and lysis. Histopathology revealed congestion, hemorrhage, and necrosis in the spleen, liver, trunk kidney, and intestinal tissues of naturally and experimentally infected golden pompanos. Electron microscopy showed that bullet-shaped viral particles, measuring 183.5–217.6 nm in length and 57.3–82.7 nm in width, budded from the cell membrane or aggregated around the infected FHM cells. The virus remained stable for 7 d at 4–33 °C and replicated optimally at 28 °C. Whole-genome sequencing and subsequent phylogenetic analysis further indicated that the carpione rhabdovirus (CARPV)2023 strain is a new carpione rhabdovirus, and is related to novirhabdoviruses, however, is thus far unclassified. A specific nested reverse transcription-polymerase chain reaction assay confirmed the presence of CARPV in the affected golden pompano. Significantly higher mortality was observed in challenged golden pompanos than that in the controls through intraperitoneal injection and immersion infection. Fluorescence in situ hybridization showed showed positive reactions for RNA in multiple tissues (liver, spleen, trunk kidney, brain, heart, gill, and muscle) from infected fish. The highest expression level was confirmed in the spleen through qPCR. This is the first study to report natural CARPV infections in marine fish.

A comparative study on the immune response in the head and trunk kidney of yellow catfish infected with Edwardsiella ictaluri

The teleost kidneys are anatomically divided into head kidney and trunk kidney, each performing distinct physiological functions. Although previous research has elucidated the role of the head kidney in immune responses, there is a paucity of literature on the comparative studies of the head and trunk kidney response to bacterial infection. Therefore, an Edwardsiella ictaluri infection model of yellow catfish was constructed to investigate and compare the immune responses between the two kidney types. The findings indicated that E. ictaluri infection induced significant pathological changes in both the head and trunk kidney. Despite variances in structure, both the head and trunk kidney of yellow catfish exhibit robust immune responses following E. ictaluri infection. Unexpectedly, the up-regulation level of IgM was found to be higher in the trunk kidney compared to the head kidney. Additionally, both the IgM+ and IgD+ B cells were increased after bacterial infection. This research elucidates the parallels and distinctions in immune functions between both the head and trunk kidney in fish, enriching the immune theory of the fish kidney, and also providing a theoretical basis for the immune response of teleost kidney against bacterial infections.

LECT2 mediates antibacterial immune response induced by Nocardia seriolae infection in the northern snakehead

Leukocyte-derived chemotaxin-2 (LECT2) is a multifunctional immunoregulator that plays several pivotal roles in the host's defense against pathogens. This study aimed to elucidate the specific functions and mechanisms of LECT2 (CaLECT2) in the northern snakehead (Channa argus) during infections with pathogens such as Nocardia seriolae (N. seriolae). We identified CaLECT2 in the northern snakehead, demonstrating its participation in the immune response to N. seriolae infection. CaLECT2 contains an open reading frame (ORF) of 459 bp, encoding a peptide of 152 amino acids featuring a conserved peptidase M23 domain. The CaLECT2 protein shares 62%–84 % identities with proteins from various other fish species. Transcriptional expression analysis revealed that CaLECT2 was constitutively expressed in all examined tissues, with the highest expression observed in the liver. Following intraperitoneal infection with N. seriolae, CaLECT2 transcription increased in the spleen, trunk kidney, and liver. In vivo challenge experiments showed that injecting recombinant CaLECT2 (rCaLECT2) could protect the snakehead against N. seriolae infection by reducing bacterial load, enhancing serum antibacterial activity and antioxidant capacity, and minimizing tissue damage. Moreover, in vitro analysis indicated that rCaLECT2 significantly enhanced the migration, respiratory burst, and microbicidal activity of the head kidney-derived phagocytes. These findings provide new insights into the role of LECT2 in the antibacterial immunity of fish.

The Effects of Acute Ammonia Nitrogen Stress on Antioxidant Ability, Phosphatases, and Related Gene Expression in the Kidney of Juvenile Yellowfin Tuna (Thunnus albacares)

This study investigated the effects of acute ammonia nitrogen (NH3-N) exposure on kidney antioxidant ability and phosphatases and related gene expression in juvenile yellowfin tuna (Thunnus albacares). The 180 juvenile yellowfin tuna (260.39 ± 55.99 g, 22.33 ± 2.28 cm) were exposed to ammonia for 6, 24, and 36 h using natural seawater (0 mg/L) as a control and NH3-N at 5 and 10 mg/L. The lipid peroxidation byproduct malondialdehyde (MDA) and the levels of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), alkaline phosphatase (AKP), and acid phosphatase (ACP), were measured using the colorimetric method in the trunk kidney to determine changes in antioxidant ability and phosphatase activity of juvenile yellowfin tuna exposed to NH3-N. Results indicated that, at 36 h, MDA, SOD, CAT, and GSH-PX levels rose in the 5 mg/L group versus the control. In the 10 mg/L group, MDA and SOD, CAT, and GSH-PX activities significantly increased after 24 and 36 h exposure compared to the control. Phosphatases play a pivotal role in the immune system. AKP activity significantly increased at 6 h, and ACP activity markedly rose at 36 h in the 5 mg/L group versus the control. Real-time fluorescence quantitative PCR was applied to detect alterations in the antioxidant genes SOD2, CAT, and glutathione peroxidase 1b (GPX1b) and immune cytokines-related genes Interleukin 10 (IL-10) and Interleukin 6 receptor (IL-6r) expression in the head kidney in juvenile tuna. Relative to the control, antioxidant gene expression in the 5 mg/L group significantly rose at 6 and 36 h, and in the 10 mg/L group, SOD2 and GPX1b were significantly elevated at 36 h. Compared to the control group, IL-10 expression in the 5 mg/L group significantly increased at 6 h, whereas IL-6r expression decreased. In the 10 mg/L group, both IL-10 and IL-6r levels were observed to be lower. Low ammonia nitrogen concentrations boost antioxidant defenses, phosphatase activities, and gene expression levels, whereas higher levels may induce suppressive effects. In yellowfin tuna juvenile farming, NH3-N concentration significantly affects the health of the juveniles. When the NH3-N concentration is between 5–10 mg/L, the stress duration should be limited to 24 h; if the concentration is below 5 mg/L, the stress duration can be extended to 36 h.

Plasma and tissue transferrin and ferritin, and gene expression of ferritin, transferrin, and transferrin receptors I and II in channel catfish Ictalurus punctatus fed diets with different concentrations of inorganic or organic iron.

Ferritin, transferrin, and transferrin receptors I and II play a vital role in iron metabolism, health, and indication of iron deficiency anaemia in fish. To evaluate the use of high-iron diets to prevent or reverse channel catfish (Ictalurus punctatus) anaemia of unknown causes, we investigated the expression of these iron-regulatory genes and proteins in channel catfish fed plant-based diets. Catfish fingerlings were fed five diets supplemented with 0 (basal), 125, and 250 mg/kg of either inorganic iron or organic iron for 2 weeks. Ferritin, transferrin, and transferrin receptor I and II mRNA and protein expression levels in fish tissues (liver, intestine, trunk kidney, and head kidney) and plasma were determined. Transferrin (iron transporter) and TfR (I and II) genes were generally highly expressed in fish fed the basal diet compared to those fed the iron-supplemented diets. In contrast, ferritin (iron storage) genes were more expressed in the trunk kidney of fish fed the iron-supplemented diets than in those fed the basal diet. Our results demonstrate that supplementing channel catfish plant-based diets with iron from either organic or inorganic iron sources affected the expression of the iron-regulatory genes and increased body iron status in the fish.

Comparative Transcriptomics Analysis Reveals Unique Immune Response to Grass Carp Reovirus Infection in Barbel Chub (Squaliobarbus curriculus).

Grass carp (Ctenopharyngodon idella) and barbel chub (Squaliobarbus curriculus)-both Leuciscinae subfamily species-demonstrate differences in grass carp reovirus (GCRV) infection resistance. We infected barbel chubs with type II GCRV and subjected their liver, spleen, head kidney, and trunk kidney samples to investigate anti-GCRV immune mechanisms via RNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR). We identified 139, 970, 867, and 2374 differentially expressed genes (DEGs) in the liver, spleen, head kidney, and trunk kidney, respectively. Across all four tissues, gene ontology analysis revealed significant immune response-related DEG enrichment, and the Kyoto Encyclopedia of Genes and Genomes analysis revealed pattern recognition receptor (PRR) and cytokine-related pathway enrichment. We noted autophagy pathway enrichment in the spleen, head kidney, and trunk kidney; apoptosis pathway enrichment in the spleen and trunk kidney; and complement- and coagulation-cascade pathway enrichment in only the spleen. Comparative transcriptome analysis between GCRV-infected barbel chubs and uninfected barbel chubs comprehensively revealed that PRR, cytokine-related, complement- and coagulation-cascade, apoptosis, and autophagy pathways are potential key factors influencing barbel chub resistance to GCRV infection. qRT-PCR validation of 11 immune-related DEGs confirmed our RNA-seq data's accuracy. These findings provide a theoretical foundation and empirical evidence for the understanding of GCRV infection resistance in barbel chub and hybrid grass carp-barbel chub breeding.

Identification, expression and molecular polymorphism of T-cell receptors α and β from the glacial relict Hucho bleekeri

The T-cell receptor (TCR) is a specific molecule on the surface of all T cells that mediates cellular adaptive immune responses to antigens. Hucho bleekeri is a critically endangered species and is regarded as a glacial relict that has the lowest-latitude distribution compared with any Eurasian salmonid. In the present study, two TCR genes, namely, TCR α and β, were identified and characterized in H. bleekeri. Both TCR α and TCR β have typical TCR structures, including the IgV domain, IgC domain, connecting peptide, transmembrane and cytoplasmic domains. The two TCR genes were constitutionally expressed in various tissues, with the highest expression found in the spleen for TCR α and in the trunk kidney for TCR β. Challenge of H. bleekeri with LPS or poly(I:C) resulted in significant upregulation of both TCR α and β expression in headkidney and spleen primary cells, indicating their potential roles in the immune response. Molecular polymorphism analysis of the whole ORF regions of TCR α and β in different individuals revealed high diversity of IgV domains of these two genes, especially in complementarity-determining region (CDR) 3. The ratio of nonsynonymous substitution occurred at a significantly higher frequency than synonymous substitution in the CDR of TCR α and β, demonstrating the existence of positive selection. The results obtained in the present study enhance our understanding of TCR roles in regulating immune mechanisms and provide new information for the study of TCR lineage diversity in fish.

Potential to grow carp oedema virus (genogroup I) in monolayers of carp-derived primary cells with further implication in cell analysis.

Carp oedema virus (CEV) has distinct molecularly identified genogroups of viral mutations, denoted as I, IIa, and IIb. Failure to propagate CEV invitro limits studies towards understanding its interactions with host cells. Here, virus isolates belonging to genogroup I collected during natural outbreaks in the Czech Republic were employed for routine CEV cultivation in monolayers of carp-derived primary cells, common carp brain (CCB) cells, and epithelioma papulosum cyprinid (EPC) cells. Induction of cytopathic effects (CPEs) was observed and recorded in affected cells. Cell survival rate was evaluated under serial dilutions of the CEV inoculum. Virus cell entry was quantified and visualized by qPCR and transmission electron microscopy, respectively. Study findings indicate primary gills epithelia likely present the most suitable matrix for CEV growth invitro. Cells of the head kidney and spleen facilitate virus entry with microscopically confirmed CPEs and the presence of cytoplasmic pleomorphic virus particles. Cells of the trunk kidney and gonads are unlikely to permit virus cell entry and CPEs development. Although CEV cultivation in cell lines was inconclusive, EPC cells were CEV permissible. Monolayers of carp-derived primary cells show promise for CEV cultivation that could enable elaborate study of mechanisms underlying cellular binding and responses.

Identifying the in vivo-induced antigenic genes is a strategy to develop DNA vaccine against Nocardia seriolae in hybrid snakehead (Channa maculata ♀ × Channa argus ♂)

Nocardia seriolae has been identified as the causative agent of fish nocardiosis, resulting in serious economic losses in aquaculture. With an aim to screen potential candidates for vaccine development against N. seriolae, the in vivo-induced genes of N. seriolae in hybrid snakehead (Channa maculate ♀ × Channa argus ♂) model were profiled via in vivo-induced antigen technology (IVIAT) in the present study, and 6 in vivo-induced genes were identified as follows: IS701 family transposase (is701), membrane protein insertase YidC (yidC), ergothioneine biosynthesis glutamate-cysteine ligase (egtA), molybdopterin respectively-dependent oxidoreductase (mol), phosphoketolase family protein (Ppl), hypothetical protein 6747 (hp6747). Additionally, the yidC was inserted into eukaryotic expression vector pcDNA3.1-myc-his-A to construct a DNA vaccine named as pcDNA-YidC to evaluate immunoprotection in hybrid snakehead after artificial challenge with N. serioale. Results showed that the transcription of yidC was detected in spleen, trunk kidney, muscle and liver in vaccinated fish, suggesting that this antigenic gene can be recombinantly expressed in fish. Meanwhile, indexes of humoral immunity were evaluated in the vaccinated fish through assessing specific-antibody IgM and serum enzyme activities, including lysozyme (LZM), superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). Quantitative real-time PCR analysis indicated that pcDNA-YidC DNA vaccine could notably enhance the expression of immune-related genes (CD4、CD8α、MHCIIα、TNFα、IL-1β and MHCIα) in 4 tissues (spleen, trunk kidney, muscle and liver) of the vaccinated fish. Finally, an immuno-protection with a relative survival rate of 65.71 % was displayed in vaccinated fish in comparison to the control groups. Taken together, these results indicate that pcDNA-YidC DNA vaccine could boost strong immune responses in hybrid snakehead and show preferably protective efficacy against N. seriolae, indicating that IVIAT is a helpful strategy to screen the highly immunogenic antigens for vaccine development against fish nocardiosis.

Characterization of type II IFNs and their receptors in a cyprinid fish, the blunt snout bream Megalobrama amblycephala

Type II interferons (IFNs) are a key class of molecules regulating innate and adaptive immunity in vertebrates. In the present study, two members of the type II IFNs, IFN-γ and IFNγ-rel, were identified in the blunt snout bream (Megalobrama amblycephala). The open reading frame (ORF) of IFN-γ and IFNγ-rel was found to have 564 bp and 492 bp, encoding 187 and 163 amino acids, with the first 26 and 24 amino acids being the signal peptide, respectively. IFN-γ and IFNγ-rel genes showed a high degree of similarity to their zebrafish homologues, being 76.9 % and 58.9 %, respectively. In the phylogenetic tree, IFN-γ and IFNγ-rel were clustered with homologous genes in cyprinids. In blunt snout bream, IFN-γ and IFNγ-rel were constitutively expressed in trunk kidney, head kidney, spleen, liver, heart, muscle, gill, intestine and brain and were significantly up-regulated by poly (I:C) induction in head kidney, spleen, liver, gill and intestine. Using recombinant proteins of IFN-γ and IFNγ-rel, the surface plasmon resonance (SPR) results showed that IFN-γ was bound to CRFB6, CRFB13 and CRFB17, but mainly to CRFB6 and CRFB13, whereas IFN-γrel bound mainly to CRFB17 and had no affinity with CRFB6. These results contribute to a better understanding on type II IFNs and their receptor usage in teleost fish.

Characteristics of CD4-1 gene and its immune responses against Aeromonas veronii infection by activating NF-κB signaling in Qihe crucian carp Carassius auratus

CD4-1 found in bony fish contains four extracellular immunoglobulin (Ig)-like domains similar to that of mammalian CD4, which is crucial for the activation of CD4+ helper T-cell. However, there is limited knowledge regarding the molecular markers, immune functions and regulation mechanism of CD4-1 in teleosts due to their vast diversity. In this study, we cloned and characterized two isoforms of Qihe crucian carp CD4-1, designated as CaCD4-1.1 and CaCD4-1.2. We further explored their expression responses upon stimulation with Aeromonas veronii, and the regulation of their immune responses against A. veronii by NF-κB. The ORF of CaCD4-1.1 and CaCD4-1.2 cDNA encoded 477 and 466 amino acids, respectively. Both proteins contained seven conserved cysteine residues in the extracellular domain, and a CCC motif in their cytoplasm, respectively. However, CaCD4-1.1 exhibited a relatively limited similarity with CaCD4-1.2 in the ectodomain. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the mRNA expression of CaCD4-1.1 and CaCD4-1.2 exhibited differential constitutive expression across all examined tissues. Furthermore, the expression level of CD4-1.2 was higher than that of CD4-1.1 in the gills, head kidney, and spleen of Qihe crucian carp subjected to A. veronii challenge, while it was lower in the trunk kidney. Inhibition of NF-κB activity resulted in a decrease in the expression levels of CD4-1.1 and CD4-1.2 mRNA in the gill, while inducing an increase in expression levels in the spleen, in accordance with the observed ultrastructural changes in both organs. Interestingly, the impact of NF-κB on the mRNA expression level of CD4-1.1 appears to be stronger than that of CD4-1.2. Our results suggest that CaCD4-1.1 and CaCD4-1.2 could be expressed on T cells and antigen-sampling cells that exhibit similar characteristics to mammalian M cells, respectively, and differentially regulated by NF-κB in adaptive immune responses against bacterial infection. This research contributes to a better understanding of the crucial role of CD4-1 in the immune response of Qihe crucian carp and provide novel insights for the prevention and treatment of fish diseases in aquaculture.

Molecular characterization of the B lymphocyte-induced maturation protein-1 (blimp1) gene of common carp (Cyprinus carpio) and its transcription repression involves recruitment of histone deacetylase HDAC3

Blimp1 is the master regulator of B cell terminal differentiation in mammals, it inhibits expression of many transcription factors including bcl6, which provides the basis for promoting further development of activated B lymphocytes into plasma cells. Blimp-1 is thought to act as a sequence-specific recruitment factor for chromatin-modifying enzymes including histone deacetylases (HDAC) and methyltransferases to repress target genes. The cDNA of Ccblimp1a (Cyprinus carpio) open reading frame is 2337 bp encoding a protein of 777 amino acids. CcBlimp1a contains a SET domain, two Proline Rich domains, and five ZnF_C2H2 domains. Blimp1 are conserved in vertebrate species. Ccblimp1a transcripts were detected in common carp larvae from 1 dpf (day post fertilization)to 31 dpf. Ccblimp1a expression was up-regulated in peripheral blood leukocytes (PBL) and spleen leukocytes (SPL) of common carp stimulated by intraperitoneal lipopolysaccharide (LPS) injection. Ccblimp1a expression in PBL and SPL of common carp was induced by TNP-LPS and TNP-KLH. The results indicated TNP-LPS induced a rapid response in PBL and TNP-KLH induced much stronger response in SPL and PBL. IHC results showed that CcBlimp1 positive cells were distributed in the head kidney, trunk kidney, liver, and gut. Immunofluorescence stain results showed that CcBlimp1 was expressed in IgM + lymphocytes. The subcellular localization of CcBlimp1 in the nuclei indicated CcBlimp1 may be involved in the differentiation of IgM + lymphocytes. Further study focusing on the function of CcBlimp1 transcriptional repression was performed using dual luciferase assay. The results showed that the transcription repression of CcBlimp1 on bcl6aa promoter was affected by the histone deacetylation inhibitor and was synergized with histone deacetylase 3 (HDAC3). The results of Co-IP in HEK293T and immunoprecipitation in SPL indicated that CcBlimp1 recruited HDAC3 and might be involved in the formation of complexes. These results suggest that CcBlimp1 is an important transcription factor in common carp lymphocytes. Histone deacetylation modification mediated by HDAC3 may have important roles in CcBlimp1 transcriptional repression during the differentiation of lymphocytes.

Molecular Identification and Expression Analysis of an Intelectin Gene in the Yellow Catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae)

Intelectins (ITLNs) are a family of calcium-dependent lectins with carbohydrate-binding capacity, are distributed across various vertebrates, and play an important role in the innate immune response against pathogen infection. The yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae) is an economically important fish in China. The aim of this study was to quantify the gene expression of ITLN in response to pathogen-associated molecular patterns (PAMPs) stimulation. Here, the ITLN gene of P. fulvidraco was characterized and named PfITLN. The full-length cDNA of PfITLN was 1132 bp, including a 5’-untranslated region (UTR) of 140 bp, a 3’-UTR of 110 bp, and an open reading frame (ORF) of 882 bp encoding a polypeptide of 293 amino acids, which contains a signal peptide and two fibrinogen-related domains (FReDs). PfITLN had a molecular weight of 32.39 kDa with a theoretical pI of 5.03. The deduced PfITLN amino acid sequence had 81%, 64%, and 55% homology with Ictalurus furcatus, Danio rerio, and Homo sapiens, respectively. Moreover, the predicted tertiary protein structure of PfITLN was highly similar to that of other animals, and phylogenetic analysis showed that the PfITLN protein was close to those of other Teleostei. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed PfITLN expression in all examined tissues, with the highest abundance seen in the liver, followed by the head kidney, spleen, trunk kidney, and muscle. After PAMP infection with lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid (poly I:C), the expression levels of PfITLN were significantly upregulated at different time points. These results suggested that PfITLN might be involved in innate immunity.

Effects of an MCPA-based herbicide formulation on the common carp Cyprinus carpio Linnaeus, 1758 – haematological, biochemical and histological evaluation

Herbicides (weed control agents) are used in crops on a massive scale. MCPA (2-methyl-4-chlorophen- oxyacetic acid) is a herbicide used to control weeds in cereals and other crops. The aim of this study was to investigate the toxic effects of an MCPA-based herbicide formulation (Chwastox Extra® 300 SL) in the common carp (Cyprinus carpio). The fish were exposed for 10 days, to a concentration which corresponded to 1 mg/l or 5 mg/l of MCPA. Our analysis showed fluctuations of the haematological parameters during the treatment. Plasma biochemical changes that were statistically significant, i.e.a decrease of the total protein concentration and alanine aminotransferase activity, were observed after 1 day of exposure. No histopathological lesions in the gills, trunk kidney and liver were identified. The results of the present study indicate that Chwastox Extra® 300 SL has a relatively low toxicity for the common carp. It was also observed that the blood indices were more sensitive to the tested herbicide formulation than the microstructure of the selected organs. Further research aimed at studying the effects of Chwastox on water invertebrates and fish of other taxa is recommended.

High starch diets attenuate the immune function of Micropterus salmoides immune organs by modulating Keap1/Nrf2 and MAPK signaling pathways

Based on their good physiological functions and physical properties, carbohydrates are widely used in fish feed. However, excessive use of carbohydrates such as starch in fish feed may reduce the immunity of the fish and cause a series of health problems. In order to more clearly clarify the effects of different starch levels in feed on the immune organs of Micropterus salmoides, this study took the immune organs as the entry point and explored it from several perspectives, including differences in enzyme activity in plasma, changes in gene expression in immune organs, and resistance to pathogenic bacteria. The results showed that (1) high starch feed activates inflammatory responses in the spleen and head kidney through the MAPK signaling pathway. This leads to a decrease in the number of lymphocytes and weakens the resistance to pathogens; (2) high starch diet affects the antioxidant capacity of the trunk kidney by regulating the Keap1/Nrf2 pathway; (3) There was a strong correlation between gene expression patterns in the head kidney and lysozyme content in plasma. This implies that the high starch diet may regulate lysozyme production by affecting gene expression in the head kidney and further affect immune function. This study helps to reveal the interaction between starch and the immune system and provide scientific basis for the development of reasonable dietary recommendations and disease prevention.

TLR pathway signaling molecules in burbot (Lota lota): molecular characterization, basal expression, and their response to Poly(I:C).

Burbot (Lota lota), a fish species of economic and ecological significance found across northern hemisphere freshwater ecosystems, was the focus of this study. We characterized 19 Toll-like receptor (TLR) genes in burbot, tracing their expression patterns following pathogen exposure. TLR genes, crucial to the innate immune system, including TLR13-1/2/3, TLR2/2-2/2-3/2-4/2-5, and TLR22a/22b/22c/22d, were discovered to be tandemly repeated, signifying an evolution in the fish's immune system. Notably, different TLR subfamilies displayed tissue-specific expressions, with TLR1 primarily in spleen and head kidney, TLR13 in head kidney, trunk kidney, and heart, TLR22 in trunk kidney and liver, and TLR3 and TLR9 predominantly in spleen and head kidney, but also in trunk kidney. Further, we investigated the response of TLR genes in burbot to pathogen exposure using qRT-PCR. This involved measuring mRNA expressions of identified TLR genes in spleen and liver tissues after injecting Poly(I:C) to simulate a double-stranded RNA viral infection. The results revealed a time and tissue-specific expression pattern. Specifically, LoTLR3 reached peak expression in the spleen 12h post-injection, declining thereafter, while TLR2 subfamily members only began expressing after 24h. In the liver, activation of the TLR3-IRF7 and TLR3-IRF3 signaling pathways was noted. Integrating these results with transcriptomic data illuminated the pivotal role of TLR genes in the burbot's immune response. Such findings are vital in shaping future disease prevention and treatment strategies.

Nanopeptide C-I20 as a novel feed additive effectively alleviates detrimental impacts of soybean meal on mandarin fish by improving the intestinal mucosal barrier.

Antibacterial peptide has been widely developed in cultivation industry as feed additives. However, its functions in reducing the detrimental impacts of soybean meal (SM) remain unknown. In this study, we prepared nano antibacterial peptide CMCS-gcIFN-20H (C-I20) with excellent sustained-release and anti-enzymolysis, and fed mandarin fish (Siniperca chuatsi) with a SM diet supplemented with different levels of C-I20 (320, 160, 80, 40, 0 mg/Kg) for 10 weeks. 160 mg/Kg C-I20 treatment significantly improved the final body weight, weight gain rate and crude protein content of mandarin fish and reduced feed conversion ratio. 160 mg/Kg C-I20-fed fish maintained appropriate goblet cells number and mucin thickness, as well as improved villus length, intestinal cross-sectional area. Based on these advantageous physiological changes, 160 mg/Kg C-I20 treatment effectively reduced multi-type tissue (liver, trunk kidney, head kidney and spleen) injury. The addition of C-I20 did not change the muscle composition and muscle amino acids composition. Interestingly, dietary 160 mg/Kg C-I20 supplementation prevented the reduction in myofiber diameter and change in muscle texture, and effectively increased polyunsaturated fatty acids (especially DHA + EPA) in muscle. In conclusion, dietary C-I20 in a reasonable concentration supplementation effectively alleviates the negative effects of SM by improving the intestinal mucosal barrier. The application of nanopeptide C-I20 is a prospectively novel strategy for promoting aquaculture development.

Identification and characterization of two novel antimicrobial peptides from Japanese sea bass (Lateolabrax japonicus) with antimicrobial activity and MO/MФ activation capability

Piscidins participate in the innate immune response of fish, which aims to eliminate recognized foreign microbes and restore the homeostasis of immune system. We characterized two piscidin-like antimicrobial peptides (LjPL-3 and LjPL-2) isolated from Japanese sea bass (Lateolabrax japonicus). LjPL-3 and LjPL-2 showed different expression patterns in tissues. After Vibrio harveyi infection, the mRNA expression of LjPL-3 and LjPL-2 was upregulated in the liver, spleen, head kidney, and trunk kidney. The synthetic mature peptides LjPL-3 and LjPL-2 exhibited different antimicrobial spectra. Furthermore, LjPL-3 and LjPL-2 treatments decreased inflammatory cytokine production while promoting chemotaxis and phagocytosis in monocytes/macrophages (MO/MФ). LjPL-2, but not LjPL-3, displayed bacterial killing capability in MO/MФ. LjPL-3 and LjPL-2 administration increased Japanese sea bass survival after V. harveyi challenge, which was accompanied by a decline in bacterial burden. These data suggested that LjPL-3 and LjPL-2 participate in immune response through direct bacterial killing and MO/MФ activation.

Bioinformatics characteristics and expression analysis of TLR3 and its adaptor protein TRIF in largemouth bass (Micropterus salmoides) upon Flavobacterium columnare infection

TLR3 and TRIF (adaptor protein for TLR3) are vital to the MyD88-independent pathway mediated by Toll-like receptors (TLRs). In order to identify the role of TLR3 and TRIF in Micropterus salmoides, the Ms_TLR3 and Ms_TRIF (Ms: abbreviation for M. salmoides) were cloned and characterized in this study. The open reading frames (ORFs) of Ms_TLR3 and Ms_TRIF genes were 2736 bp and 1791 bp in length, encoding 911 and 596 amino acids, respectively. The protein structure of Ms_TLR3 includes a signal peptide, 18 LRR-related domains, a low complexity region, a transmembrane region, and a TIR domain. However, only a TIR domain and a coiled coil domain were found in Ms_TRIF. Both Ms_TLR3 and Ms_TRIF showed the highest homology to that of M. dolomieu. Ms_TLR3 and Ms_TRIF showed similar expression patterns in various tissues, with the highest expression level in the head kidney. After stimulation of Flavobacterium columnare, the mRNA expressions of Ms_TLR3 and Ms_TRIF were significantly up-regulated at 1 dpi in the gill, spleen and head kidney, and at 6 hpi in the trunk kidney. Furthermore, morphological changes in the gills of largemouth bass challenged with F. columnare suggested that F. columnare infection can destroy the gill filament. Taken together, Ms_TLR3 and Ms_TRIF are indeed involved in F. columnare infection and the subsequent immune response in largemouth bass. Moreover, Ms_TLR3 and Ms_TRIF might respectively play their potential roles in mucosal (mainly in the gill) and systemic (mainly in the head kidney) immune response to bacterial infection.