The interaction between lipid and vitamin D3 impacts lipid metabolism and innate immunity in Chinese mitten crabs Eriocheir sinensis.

As a fat-soluble vitamin, vitamin D3 relies on fat to perform its biological function, affecting lipid metabolism and innate immunity. This study used different percentages of lipid and vitamin D3 diets to evaluate the synergistic effects on the growth, lipid metabolism and immunity of juvenile Eriocheir sinensis (5.83 ± 0.01 g) for 56 days, including low lipid (LL, 1.5%) and normal lipid (NL, 7.5%) and three levels of vitamin D3: low (LVD, 0 IU/kg), medium (MVD, 9000 IU/kg) and high (HVD, 27,000, IU/kg). The synergistic effect of lipid and vitamin D3 was not significant on growth but significant on ash content, total protein, hepatopancreas lipid content, hemolymph 1α,25-hydroxy vitamin D3 [1α,25(OH)2D3] content, hepatopancreas lipolysis and synthesis genes. Crabs fed normal lipid (7.5%) and medium vitamin D3 (9000 IU/kg) had the highest hepatopancreas index, hemolymph 1α,25(OH)2D3 content, antibacterial ability, immune-related genes and hepatopancreatic lipid synthesis genes expression, but down-regulated the lipolysis genes expression. In contrast, crabs fed diets with low lipid percentage (1.5%) had low growth performance, hemolymph 1α,25(OH)2D3, mRNA levels of lipid synthesis genes, antibacterial ability and immune-related gene expression. At the 1.5% lipid level, excessive or insufficient vitamin D3 supplementation led to the obstruction of ash and protein deposition, reduced growth and molting, aggravated the reduction in antioxidant capacity, hindered antimicrobial peptide gene expression and reduced innate immunity, and resulted in abnormal lipid accumulation and the risk of oxidative stress. This study suggests that diets' lipid and vitamin D3 percentage can enhance antioxidant capacity, lipid metabolism and innate immunity in E. sinensis. A low lipid diet can cause growth retardation, reduce antioxidant capacity and innate immunity, and enhance lipid metabolism disorder.

Molecular characterization, spatio-temporal expression patterns of crtc2 gene and its immune roles in yellow catfish (Pelteobagrus fulvidraco).

cAMP response element binding (CREB) protein 2 (CRTC2) is a transcriptional coactivator of CREB and plays an important role in the immune system. Thus far, the physiological roles of Crtc2 in teleost are still poorly understood. In this study, the crtc2 gene was identified and characterized from yellow catfish (Pelteobagrus fulvidraco; therefore, the gene is termed as pfcrtc2), and its evolutionary and molecular characteristics as well as potential immunity-related roles were investigated. Our results showed that the open reading frame of pfcrtc2 was 2346 bp in length, encoding a protein with 781 amino acids. Gene structure analysis revealed its existence of 14 exons and 13 introns. A phylogenetic analysis proved that the tree of crtc2 was clustered into five groups, exhibiting a similar evolutionary topology with species evolution. Multiple protein sequences alignment demonstrated high conservation of the crtc2 in various vertebrates with similar structure. Syntenic and gene structural comparisons further established that crtc2 was highly conserved, implying its similar roles in diverse vertebrates. Tissue distribution pattern detected by quantitative real-time PCR showed that the pfcrtc2 gene was almost expressed in all detected tissues except for eyes, with the highest expression levels in the gonad, indicating that Crtc2 may play important roles in various tissues. In addition, pfcrtc2 was transcribed at all developmental stages in yellow catfish, showing the highest expression levels at 12 h after fertilization. Finally, the transcriptional profiles of crtc2 were significantly increased in yellow catfishes injected with Aeromonas hydrophila or Poly I:C, which shared a consistent change pattern with four immune-related genes including IL-17A, IL-10, MAPKp38, and NF-κBp65, suggesting pfCrtc2 may play critical roles in preventing both exogenous bacteria and virus invasion. In summary, our findings lay a solid foundation for further studies on the functions of pfcrtc2, and provide novel genetic loci for developing new strategies to control disease outbreak in teleost.

Growth and Hepatopancreas Health of Juvenile Chinese Mitten Crab (Eriocheir sinensis) Fed Different Levels of Black Soldier Fly (Hermetia illucens) Larvae Meal for Fish Meal Replacement.

A 56-day feeding trial assessed the effects of black soldier fly larvae meal (BSFLM) on the growth performance and hepatopancreas health of juvenile Eriocheir sinensis. Six isoproteic and isolipidic diets with 0% (FM), 10% (BSFLM10), 20% (BSFLM20), 30% (BSFLM30), 40% (BSFLM40), or 50% (BSFLM50) replacement of fish meal by BSFLM were formulated. Compared to FM, replacing 10%-40% of fish meal with BSFLM did not significantly affect the weight gain rate (WGR) or specific growth rate (SGR), while BSFLM50 significantly decreased the WGR and SGR. Crabs fed BSFLM50 had significantly lower T-AOC activity than those fed other diets, and crabs fed BSFLM30, BSFLM40, or BSFLM50 had significantly lower activities of antioxidant enzymes (SOD and GSH-Px) in the hepatopancreas than those fed FM or BSFLM10. Compared to FM, BSFLM10, BSFLM20, and BSFLM30 did not affect the relative expression of genes related to the nonspecific immunity, while BSFLM40 and BSFLM50 upregulated the relative expression of these genes. Furthermore, histological analysis showed that the hepatopancreas was deformed in the BSFLM50 group, with widened lumens and loss of basal membrane integrity. In summary, BSFLM replacing 50% of fish meal reduced growth and structural damage to the hepatopancreas. An immune response was activated when the replacement level was over 30%. Therefore, the replacement level of dietary fish meal by BSFLM is recommended to be not more than 30% of the juvenile E. sinensis feed.

Comprehensive transcriptional and metabolomic analysis reveals the neuroprotective mechanism of dietary gamma-aminobutyric acid response to hypoxic stress in the Chinese mitten crab (Eriocheir sinensis).

Hypoxia is one of the serious stress challenges that aquatic animals face throughout their life. Our previous study found that hypoxia stress could induce neural excitotoxicity and neuronal apoptosis in Eriocheir sinensis, and observed that gamma-aminobutyric acid (GABA) has a positive neuroprotective effect on juvenile crabs under hypoxia. To reveal the neuroprotective pathway and metabolic regulatory mechanism of GABA in E. sinensis exposed to hypoxia stress, an 8-week feeding trial and acute hypoxia challenge were performed. Subsequently, we performed a comprehensive transcriptomic and metabolomic analysis of the thoracic ganglia of juvenile crabs. Differential genes and differential metabolites were co-annotated to 11 KEGG pathways, and further significant analysis showed that only the sphingolipid signaling pathway and the arachidonic acid metabolism pathway were significantly enriched. In the sphingolipid signaling pathway, GABA treatment significantly increased long-chain ceramide content in thoracic ganglia, which exerted neuroprotective effects by activating downstream signals to inhibit hypoxia-induced apoptosis. Moreover, in the arachidonic acid metabolism pathway, GABA could increase the content of neuroprotective active substances and reduce the content of harmful metabolites by regulating the metabolism of arachidonic acid for inflammatory regulation and neuroprotection. Furthermore, the decrease of glucose and lactate levels in the hemolymph suggests the positive role of GABA in metabolic regulation. This study reveals the neuroprotective pathways and possible mechanisms of GABA in juvenile E. sinensis exposed to hypoxia stress and inspires the discovery of new targets for improving hypoxia tolerance in aquatic animals.

Molecular characterization of a teleost-specific toll-like receptor 22 (tlr22) gene from yellow catfish (Pelteobagrus fulvidraco) and its transcriptional change in response to poly I:C and Aeromonas hydrophila stimuli.

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that can recognize pathogen-associated molecular patterns (PMPs) and play important roles in the innate immune system in vertebrates. In this study, we identified a teleost-specific tlr22 gene from yellow catfish (Pelteobagrus fulvidraco) and its immune roles in response to different pathogens were also determined. The open reading frame (ORF) of the tlr22 was 2892 bp in length, encoding a protein of 963 amino acids. Multiple protein sequences alignment, secondary and three-dimensional structure analyses revealed that TLR22 is highly conserved among different fish species. Phylogenetic analysis showed that the phylogenetic topology was divided into six families of TLR1, TLR3, TLR4, TLR5, TLR7 and TLR11, and TLR22 subfamily was clustered into TLR11 family. Meanwhile, synteny and gene structure comparisons revealed functional and evolutionary conservation of the tlr22 gene in teleosts. Furthermore, tlr22 gene was shown to be widely expressed in detected tissues except barbel and eye, with highest expression level in liver. The transcription of tlr22 was significantly increased in spleen, kidney, liver and gill tissues at different timepoints after Poly I:C infection, suggesting TLR22 plays critical roles in defensing virus invasion. Similarly, the transcription of tlr22 was also dramatically up-regulated in spleen, kidney and gill tissues with different patterns after Aeromonas hydrophila infection, indicating that TLR22 is also involved in resisting bacteria invasion. Our findings will provide a solid basis for the investigation the immune functions of tlr22 gene in teleosts, as well as provide useful information for disease control and treatment for yellow catfish.

Cold stress after swimming fatigue decreases immunity-related gene expression in the spleen of the Chinese sucker.

For migratory fish, passing through the cold, fast flowing water of a dam causes stress, leading to disease and even death. To determine the immune response to cold stress in a dam-lake after swimming fatigue in Chinese sucker (Myxocyprinus asiaticus), the spleen mRNA expression profiles in response to cold stress (CS) after fatigue stress (FS) were compared with that of the control (SS). We identified 40,952 differentially expressed genes (DEGs) in the spleen for at least one comparison among 211,397 unigenes. We identified 11,869 DEGs (4,968 upregulated and 6,901 downregulated), 17,803 DEGs (10,610 upregulated and 7,193 downregulated), and 30,579 DEGs (20,652 upregulated and 9,927 downregulated) in the SS vs. FS, SS vs. CS, and FS vs. CS comparisons, respectively. Enrichment analysis indicated the involvement of the immune system and infectious diseases, including the toll-like receptor pathway, the complement and coagulation cascade, and the natural killer cell-mediated cytotoxicity pathway. There were 2,991 DEGs (271 upregulated and 2,720 downregulated), and 2,878 DEGs (873 upregulated and 2,005 downregulated) associated with these pathways in the SS vs. FS and SS vs. CS comparisons, respectively. In the cold stress after fatigue group, the expression levels of heat shock protein (HSP) 70 and HSP90 genes were significantly upregulated; however, more immune response genes showed significantly downregulated expression in SS vs. CS compared with that in SS vs. FS, including those encoding tumor necrosis factor, C-C motif chemokines (3, 8, and 13), complement components (C3, C4, C6, and C7), immunoglobulin, and cathepsins. Overall, cold stress combined with swimming fatigue from passing the dam resulted in the downregulation of many immune genes, suggesting that the Chinese sucker might have experienced serious immune suppression.

Dietary gamma-aminobutyric acid (GABA) improves non-specific immunity and alleviates lipopolysaccharide (LPS)-induced immune overresponse in juvenile Chinese mitten crab (Eriocheir sinensis).

Inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is an immunomodulator to inhibit immune-mediated pro-inflammatory response and has been used to treat various immune-related diseases in mammals. However, the immunoregulatory effect of GABA in crustaceans has not been reported. This study evaluates the regulatory effect of dietary GABA supplementation on the innate immune status and immunoregulatory potential in lipopolysaccharide (LPS)-induced immune response in juvenile Eriocheir sinensis. Juvenile crabs were fed with six diets supplemented with graded GABA levels (0, 40, 80, 160, 320 and 640 mg/kg dry matter) for 8 weeks and then 24 h LPS challenge test was carried out. The results showed that dietary GABA supplementation significantly decreased mortality at 4 and 8 weeks. Moreover, the hemocyanin content, acid phosphatase, and alkaline phosphatase activities significantly increased in the crabs fed GABA supplementation compared with the control. On the contrary, the alanine aminotransferase and alanine aminotransferase activities in serum decreased significantly in the GABA supplementation groups compared with the control. Similarly, superoxide dismutase activity, glutathione content, and the transcriptional expression of the antioxidant-related genes and immune-related genes were significantly higher in the GABA supplementation groups than in the control. In addition, the mRNA expressions of anti-lipopolysaccharide factors (ALF 1, ALF 2, ALF 3) and inflammatory signaling pathways related genes (TLR, Myd88, Relish, LITAF, P38-MAPK, ADAM17) were significantly up-regulated in LPS stimulation groups compared with PBS treatment. Meanwhile, pro-apoptosis-related genes' mRNA expressions were significantly up-regulated, and anti-apoptosis-related genes were significantly down-regulated under LPS stimulation compared with PBS treatment. However, GABA pretreatment effectively alleviated LPS-induced immune overresponse and apoptosis. Therefore, this study demonstrates that dietary GABA supplementation could be used as an immunomodulator to improve the non-specific immunity and antioxidant capacity and alleviate the immune-mediated immune overresponse of juvenile E. sinensis.

Diurnal rhythm expression of transcription factor NF-κB subunit p65 in Pelteobagrus vachellii.

The pleiotropic transcription factor nuclear factor-kappa B (NF-κB) has important functions in viral resistance. In the present study, we isolated a p65 subunit of NF-κB cDNA from Pelteobagrus vachellii (termed Pvp65) and characterized it. The full-length p65 cDNA comprised 3651 bp, including a 148-bp 5'-untranslated region (UTR), a 106-bp 3'-UTR, and an open reading frame encoding a 1067-amino acid putative protein. The protein sequence comprised a DNA binding motif, a Rel-homology domain, a Rel protein signature, a putative transcription activation domain, a nuclear localization signal, and a transcription initiation factor IIA domain. The expression of Pvp65 displayed a daily rhythm, with an acrophase at approximately at 15:32 h in the liver, 11:34 h in the spleen, and 16:45 h in the head kidney. In addition, infection with Aeromonas hydrophila caused Pvp65 expression to increase significantly (P < 0.05), and peaking at 12 h post infection in the spleen, at 24 in the head kidney, and at 12 h in the liver. Thus, NF-κB expression might be under light/dark cycle control in P. vachellii, and may be involved in the immune response to A. hydrophila.

Effects of dietary T-2 toxin on gut health and gut microbiota composition of the juvenile Chinese mitten crab (Eriocheir sinensis).

The current study aims to investigate the effects of dietary T-2 toxin on the intestinal health and microflora in the juvenile Chinese mitten crab (Eriocheir sinensis) with an initial weight 2.00 ± 0.05 g. Juvenile crabs were fed with experimental diets supplemented with T-2 toxin at 0 (control), 0.6 (T1 group), 2.5 (T2 group) and 5.0 (T3 group) mg/kg diet for 8 weeks. Dietary T-2 toxin increased the malondialdehyde (MDA) content and the expression of Kelch-like ECH-associated protein 1 (keap1) gene while the expression of cap 'n' collar isoform C (CncC) decreased in the intestine. The activities of glutathione peroxidase (GSH-Px) and total anti-oxidation capacity (T-AOC) in the intestine increased only in the lower dose of dietary T-2. Dietary T-2 toxin significantly increased the mRNA expression of caspase-3, caspase-8, Bax and mitogen-activated protein kinase (MAPK) genes and the ratio of Bax to Bcl-2 accompanied with a reduction of Bcl-2 expression. Furthermore, T-2 toxin decreased the mRNA levels of antimicrobial peptides (AMPs), peritrophic membrane (PM1 and PM2) and immune regulated nuclear transcription factors (Toll-like receptor: TLR, myeloid differentiation primary response gene 88: Myd88, relish and lipopolysaccharide-induced TNF-α factor: LITAF). The richness and diversity of the gut microbiota were also affected by dietary T-2 toxin in T3 group. The similar dominant phyla in the intestine of the Chinese mitten crab in the control and T3 groups were found including Bacteroidetes, Firmicutes, Tenericutes and Proteobacteria. Moreover, the inclusion of dietary T-2 toxin of 4.6 mg/kg significantly decreased the richness of Bacteroidetes and increased the richness of Firmicutes, Tenericutes and Proteobacteria in the intestine. At the genus level, Dysgonomonas and Romboutsia were more abundant in T3 group than those in the control. However, the abundances of Candidatus Bacilloplasma, Chryseobacterium and Streptococcus in T3 group were lower than those in the control. This study indicates that T-2 toxin could cause oxidative damage and immunosuppression, increase apoptosis and disturb composition of microbiota in the intestine of Chinese mitten crab.

Diurnal rhythm and pathogens induced expression of toll-like receptor 9 (TLR9) in Pelteobagrus vachellii.

Toll-like receptor 9 (TLR9) is activated by bacterial DNA and induces the production of inflammatory cytokines. In this study, the darkbarbel catfish Pelteobagrus vachellii TLR9 cDNA was cloned and sequenced. The daily expression pattern of TLR9 mRNA was investigated in various tissues. Furthermore, its expression was analyzed following exposure to the pathogen Aeromonas hydrophila. The 4249 bp cDNA includes a 3201 bp open reading frame (ORF) encoding 1067 amino acids. The predicted amino acid sequence comprises a leucine-rich domain (LRD), a toll/interleukin-1 receptor (TIR), and a transmembrane domain. P. vachellii TLR9 showed 42-87% amino acid sequence identity with TLR9 sequences of Ictalurus punctatus, Rhincodon typus, and Miichthys miiuy. The P. vachellii TLR9 mRNA was highly expressed in intestines, head kidney, and spleen in an apparently healthy fish. Following pathogen challenge, TLR9 expression increased significantly (P < 0.05) and peaked at 48 h post-exposure in the liver, at 24 in the head kidney, and at 12 h in the spleen. In addition, the pattern of TLR9 expression over a 24-h period showed a circadian rhythm in the head kidney, spleen, and intestine, with the acrophase at 20:34, 18:45, and 3:50, respectively. This result provided the basis for further study of the rhythm of innate immunity against bacteria in catfish.

Molecular characterization and expression of toll-like receptor 5 genes from Pelteobagrus vachellii.

Toll-like receptor 5 (TLR5) is an important pathogen recognition receptor (PRR) that recognizes the flagellin protein of pathogenic bacteria and plays a fundamental role in activating the innate immune response. In this study, full-length pvTLR5m (membrane) and pvTLR5s (soluble) genes were cloned from darkbarbel catfish Pelteobagrus vachellii, and their expression and that of downstream genes were analyzed following exposure to the Aeromonas hydrophila pathogen. The 3009 bp pvTLR5m cDNA includes a 2652 bp open reading frame (ORF) encoding 884 amino acids. The 2422 bp pvTLR5s cDNA includes a 1944 bp ORF encoding a predicted protein of 648 amino acids. The genes are most closely related to TLR5m (75%) and TLR5s (69%) from Ictalurus punctatus, respectively, and both have a typical TLR structure. Both genes were constitutively expressed in all examined tissues, and most abundantly in the head kidney and spleen. Following pathogen challenge, pvTLR5m and pvTLR5s expression was increased significantly (P <0.05) and peaked at 24 and 12 h post-exposure in the liver, 24 and 12 h in the head kidney, and 48 and 24 h in the spleen, respectively. The downstream genes interleukin-1ß (IL-1ß), IL-12 and tumor necrosis factor-alpha (TNF-α) were significantly up-regulated following pathogen exposure in spleen, and the NF-kB inhibitor (IκB) was down-regulated. These findings indicated that pvTLR5 may play an important role in the immune responses to A. hydrophila. These results provide new insight to elucidate the immune signalling pathways of fish TLR.

Molecular cloning, tissue distribution, and effect of fasting and refeeding on the expression of neuropeptide Y in Channa argus.

Neuropeptide Y (NPY) is a 36 amino-acid amidated peptide of the pancreatic polypeptide (PP) family, which plays an important role in appetite regulation and energy expenditure in mammals. Although several teleost NPY have been identified, its roles remain unclear in fish. We herein reported on the molecular cloning, tissue distribution and the effect of fasting on the expression of NPY in Channa argus, and designated as CaNPY. It consisted of a 300 bp open reading frame predicted to encode a prepro-NPY of 99 amino acids. Sequence analysis revealed that CaNPY was highly conserved (>60%) with other vertebrate NPY. Phylogenetic analysis highly supported CaNPY was closely related to piscine NPY. In addition, except for muscle and spleen tissues, CaNPY was found to extensively expressed in all other detected tissues, with the highest level in brain. Futhermore, the CaNPY transcript was found to significantly increase after short-term and long-term food deprivation, and dramatically decrease following refeeding. These findings suggested that CaNPY might be involved in food intake regulation and it could be as a potential target locus to improve commercial production of this kind of fish.

Uncoupling protein 1 in snakehead (Channa argus): Cloning, tissue distribution, and its expression in response to fasting and refeeding.

In mammals, uncoupling protein 1 (UCP1) is well known for its thermogenic role in brown adipose tissue (BAT). However, the UCP1 physiological roles are still unclear in fish, although several teleost ucp1 genes have been identified. The aim of this study is to investigate the potential roles of fish UCP1 involved in food intake regulation and energy homeostasis. We herein report on the molecular cloning, tissue distribution and the effect of fasting and refeeding on the expression of ucp1 in Channa argus. UCP1 consisted of a 921 bp open reading frame predicted to encode 306 amino acids. Sequence analysis revealed that snakehead UCP1 was highly conserved (>80%) with teleost UCP1, but shared a lower identity (60-72%) with mammals. Phylogenetic analysis supported that snakehead UCP1 was closely related to piscine UCP1. In addition, ucp1 was found to extensively expressed in all detected tissues, with the highest level in liver. Futhermore, the hepatic ucp1 was found to significantly increased after short-term and long-term food deprivation, and dramatically increased following refeeding. These findings suggested that snakehead UCP1 might play important roles in food intake regulation and fatty acid metabolism in snakehead fish, and it could be as a potential target locus to improve commercial production of this kind of fish.

Transcriptome analysis of the spleen of the darkbarbel catfish Pelteobagrus vachellii in response to Aeromonas hydrophila infection.

Intensive aquaculture has increased the susceptibility of fish to Aeromonas hydrophila, and this has led to severe economic damage. There has been little study of the host defense mechanism against A. hydrophila infection in scaleless fish. Therefore, in the present study, the transcriptome profiles of the spleen of Pelteobagrus vachellii were examined after infection with A. hydrophila. In total, 37,730 unigenes from 322 KEGG pathways were identified. Following A. hydrophila infection, 27,803 differentially expressed genes were identified, including 13,934 upregulated and 13,869 downregulated genes. Significant enrichment analysis of these differentially expressed unigenes showed that the major immune pathways were involved, including toll-like receptor pathways, B-cell receptor signaling pathways, Fcγ receptor-mediated phagocytosis, complement and coagulation cascades, and natural killer cell-mediated cytotoxicity pathways. From these pathways, 59 key immune-related differentially expressed genes were selected: 53 genes that were upregulated, including those coding for complement components, interferons, and interleukins, and six DEGs that were downregulated, including inhibitor of nuclear factor kappa-B kinase. Finally, nine DEGs, which were randomly selected, were confirmed by qRT-PCR to be differentially expressed. The results indicated that complement components, interferons and Fcγ receptor-mediated phagocytosis played key role in the response to A. hydrophila infection in the spleen of P. vachellii, which may prove useful in the future for the development of therapeutic regimens.

Effect of ammonia-N and pathogen challenge on complement component 8α and 8ß expression in the darkbarbel catfish Pelteobagrus vachellii.

The complement components C8α and C8ß mediate the formation of the membrane attack complex (MAC) to resist pathogenic bacteria and play important roles in innate immunity. Full-length complement C8α (Pv-C8α) and C8ß (Pv-C8ß) cDNA were identified in the darkbarbel catfish Pelteobagrus vachellii, and their mRNA expression levels were analyzed after ammonia-N and pathogen treatment. The Pv-C8α gene contained 1983 bp, including a 1794-bp open reading frame (ORF) encoding 598 amino acids. The Pv-C8ß gene contained 1952 bp, including a 1761-bp ORF encoding 587 amino acids. Pv-C8α and Pv-C8ß had the highest amino acid identity with rainbow trout Oncorhynchus mykiss C8α (62%) and Japanese flounder Paralichthys olivaceus C8ß (83%), respectively. Sequence analysis indicated that both Pv-C8α and Pv-C8ß contained a thrombospondin type-1 (TSP1) domain, a low-density lipoprotein receptor class A (LDLR-A) domain, a membrane attack complex/perforin (MACPF) domain and an epidermal growth factor-like (EGF-like) domain. In addition, Pv-C8α and Pv-C8ß were mainly distributed in the liver, head kidney, spleen, and eggs. Under ammonia-N stress, the Pv-C8α and Pv-C8ß mRNA levels significantly decreased (P < 0.05), with minimum levels, respectively, attained at 24 and 48 h in the liver, 48 and 24 h in the head kidney, and 24 and 24 h in the spleen. After Aeromonas hydrophila challenge, the Pv-C8α and Pv-C8ß mRNA levels significantly increased (P < 0.05), with maximum levels, respectively, attained at 48 and 24 h in the liver, 24 and 48 h in the head kidney, and 48 and 48 h in the spleen. The present study indicated that Pv-C8α and Pv-C8ß exhibited important immune responses to infection and that ammonia-N in water decreased the immune responses of Pv-C8α and Pv-C8ß.

The differential impact of iron on ferroptosis, oxidative stress, and inflammatory reaction in head-kidney macrophages of yellow catfish (Pelteobagrus fulvidraco) with and without ammonia stress.

Ammonia toxicity in fish is closely related to ferroptosis, oxidative stress, and inflammatory responses. Iron is an essential trace element that plays a key role in many biological processes for cells and organisms, including ferroptosis, oxidative stress response, and inflammation. This study aimed to investigate the effect of iron on indicators of fish exposed to ammonia, specifically on the three aspects mentioned above. The head kidney macrophages of yellow catfish were randomly assigned to one of four groups: CON (normal control), AM (0.046 mg L-1 total ammonia nitrogen), Fe (20 µg mL-1 FeSO4), and Fe + AM (20 µg mL-1 FeSO4, 0.046 mg L-1 total ammonia nitrogen). The cells were pretreated with FeSO4 for 6 h followed by ammonia for 24 h. The study found that iron supplementation led to an excessive accumulation of iron and ROS in macrophages, but it did not strongly induce ferroptosis, oxidative stress, or inflammatory responses. This was supported by a decrease in T-AOC, and the downregulation of SOD, as well as an increase in GSH levels and the upregulation of TFR1, CAT and Nrf2. Furthermore, the mRNA expression of HIF-1, p53 and the anti-inflammatory M2 macrophage marker Arg-1 were upregulated. The results also showed that iron supplementation increased the progression of some macrophages from early apoptosis to late apoptotic cells. However, the combined treatment of iron and ammonia resulted in a stronger intracellular ferroptosis, oxidative stress, and inflammatory reaction compared to either treatment alone. Additionally, there was a noticeable increase in necrotic cells in the Fe + AM and AM groups. These findings indicate that the biological functions of iron in macrophages of fish may vary inconsistently in the presence or absence of ammonia stress.

Effects of pathogenic bacterial challenge after acute sublethal ammonia-N exposure on heat shock protein 70 expression in Botia reevesae.

The objective of this study was to investigate the effects of pathogenic bacterial challenge after acute sublethal ammonia-N exposure on heat shock protein 70 expression in Botia reevesae. After ammonia-N exposure at a constant concentration of 7.21 ± 0.10 mg L(-1) for 96 h, B. reevesae was challenged with Aeromonas hydrophila. Quantitative PCR analysis showed predominant and significant expression of HSP70 in liver, gill, skin, spleen and kidney (P < 0.05), with significantly upregulated expression of the mRNA transcript in these tissues after sublethal ammonia-N exposure and A. hydrophila challenge. Furthermore, following A. hydrophila challenge after ammonia-N exposure, HSP70 mRNA expression was significantly upregulated in kidney and gill tissues, although its expression levels were significantly lower than those detected following A. hydrophila challenge or ammonia-N exposure individually. These results indicate that B. reevesae HSP70 is involved in resistance to pathogenic bacteria. It is hypothesized that ammonia-N results in the downregulation of HSP70 mRNA in immune organs after an A. hydrophila challenge, thus lowering their resistance to pathogenic stress.

Transcriptome analysis of Chinese sucker (Myxocyprinus asiaticus) head kidney and discovery of key immune-related genes to cold stress after swimming fatigue.

For Chinese sucker (Myxocyprinus asiaticus), passing through a dam with fast flow and cold water are always unavoidable, and this process can cause stress, disease or even death. In this study, comparative transcriptome analysis was conducted to investigate the potential immune mechanism in head kidney of M. asiaticus with swimming fatigue stress and cold stress after fatigue. In general, a total of 181,781 unigenes were generated, and 38,545 differentially expressed genes (DEGs) were identified. In these DEGs, 22,593, 7286 and 8666 DEGs were identified among groups of fatigue vs. cold, control vs. cold, and control vs. fatigue, respectively. Enrichment analysis revealed these DEGs were involved in coagulation cascades and complement, natural killer cell mediated cytotoxicity, antigen processing and presentation, Toll-like receptor signaling pathways, and chemokine signaling pathway. Notably, immune genes including heat shock protein 4a (HSP4a), HSP70 and HSP90α genes were significantly up-regulated in fishes with cold stress after fatigue. Differently, more immune genes in control vs. cold compared with that in control vs. fatigue were significantly down-regulated expression, such as claudin-15-like, Toll-like receptor 13, antimicrobial peptide (hepcidin), immunoglobulin, CXCR4 chemokine receptor, T-cell receptor, complement factor B/C2-A3, and interleukin 8. In this study, the number of DEGs in the head kidney was less than that our previous study in the spleen, which we speculated was more sensitive to changes in water temperature than the head kidney. In summary, lots of immune-related genes in the head kidney were down-regulated under cold stress after fatigue, suggesting that M. asiaticus might have experienced severe immunosuppression in the process of passing through the dam.

Adverse effects of thiamethoxam on the behavior, biochemical responses, hepatopancreas health, transcriptome and intestinal flora of juvenile Chinese mitten crab (Eriocheir sinensis).

Frequent detection of thiamethoxam in global surface waters has provoked great concern in environmental safety, as thiamethoxam exhibits high toxicity to aquatic arthropods. However, little systematic investigation has been conducted on the chronic toxicity of thiamethoxam to crustaceans. This study exposed Eriocheir sinensis to thiamethoxam (0, 0.5, 5 and 50 µg/L) in water for 28 days. No significant difference in mortality was observed among all groups. A high concentration of thiamethoxam (50 µg/L) impaired the righting ability of E. sinensis. Thiamethoxam significantly increased antioxidant enzyme activities (superoxide dismutase, total antioxidant capacity and glutathione peroxidase) and malondialdehyde levels. Simultaneously, detoxification enzyme activities (aminopyrine N-demethylase, erythromycin N-demethylase and glutathione-S-transferase) increased under chronic thiamethoxam stress. In addition, thiamethoxam caused immune and hepatopancreas damage. Moreover, thiamethoxam induced intestinal flora dysbiosis by altering the microbiome structure. The reduced complexity of the gut microbiota further illustrated that thiamethoxam could disrupt the stability of the microbiota ecological network. The transcriptomic results revealed that the number of downregulated DEGs increased in a dose-dependent manner, and most downregulated DEGs were enriched in energy metabolism-related pathways. These results indicate that thiamethoxam can adversely affect the crab behavior, biochemistry, intestinal microflora and transcriptomic responses.

Transcriptome analysis provides insights into the molecular mechanism of liver inflammation and apoptosis in juvenile largemouth bass Micropterus salmoides fed low protein high starch diets.

The present study was conducted to investigate the regulatory mechanism of liver injury in largemouth bass Micropterus salmoides (LMB) fed low protein high starch diets. Two isolipidic and isoenergetic diets were formulated with different protein and starch ratios, being named as diets P49S9 (48.8 % protein and 9.06 % starch) and P42S18 (42.4 % protein and 18.2 % starch). Each diet was fed to triplicate replicates of LMB (initial body weight, 4.65 ± 0.01 g) juveniles. Fish were fed to visual satiation for 8 weeks. The results indicated that though the P42S18 fish up-regulated the feeding ratio to meet their protein requirements, feeding efficiency ratio and growth performance were impaired in treatment P42S18 as compared to treatment P49S9. Periodic acid-Schiff (PAS) staining showed glycogen accumulated in the liver of LMB fed low protein high starch diets, and the reason should be attributed to down-regulated expression of the glycogenolytic glycogen debranching enzyme. Lower liver lipid level was associated with feeding low protein high starch diets in LMB, which should be resulted from the changes in hepatic glycerolipid metabolism regulated by lipoprotein lipase (representative of triglyceride synthesis, up-regulated) and diacylglycerol acyltransferase (representative of triglyceride breakdown, down-regulated). Though fasting plasma glucose level was comparable, treatment P42S18 performed inferior glucose tolerance to treatment P49S9. Hematoxylin-eosin (HE) and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining suggested that feeding low protein high starch diets induced disruption of structural integrity, inflammation and apoptosis in the hepatocytes of LMB. As expected, KEGG pathways analysis indicated that many of the up-regulated differentially expressed genes were enriched in AGE (advanced glycation end product)/RAGE (receptor for AGE), Toll-like receptor and apoptosis signaling pathways. Our transcriptome data revealed that feeding low protein high starch diets might promote the accumulation of AGEs in LMB, which bound to RAGE and subsequently induced PI3K/Akt signal pathway. The activation of Akt induced NF-κB translocation into the nucleus thus releasing proinflammatory factors including tumor necrosis factor-α (TNF-α) and interleukin-8. The release of these inflammatory factors concomitantly induced T cell stimulation and natural killer cells chemotactic effects through Toll-like receptor signaling pathway. Besides mediating inflammation and immune response, TNF-α signal transduction participated in mediating apoptosis through the receptor of TNF (TNF-R1) pathway by up-regulating the expression of caspase 8 and cytochrome c. In conclusion, our results demonstrated that feeding low protein and high starch diets induced hepatocytes inflammation and apoptosis in LMB through the PI3K/Akt/NF-κB signaling pathway.