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Alginate oligosaccharide (AOS) is widely used in agriculture because of its many excellent biological properties. However, the possible beneficial effects of AOS and their underlying mechanisms are currently not well known in grass carp (Ctenopharyngodon idellus). Here, grass carp were fed diets supplemented with 5, 10, or 20 g/kg AOS for six weeks. HE and PAS staining showed that the diets of AOS significantly increased the number of goblet cells in the intestinal. According to transcriptome and quantitative real-time PCR (qRT-PCR) data, AOS-supplemented diets activated the expression of fat metabolism-related pathways and genes. The 16S rRNA sequencing results showed that supplementation with AOS affected the distribution and abundance of the gut bacterial assembly. qRT-PCR and activity assays revealed that the AOS diets significantly increased the antioxidant resistance in gut of grass carp, and down-regulated the expression of inflammatory and up-regulated anti-inflammatory cytokines. Finally, the Aeromonas hydrophila infection assay suggested that the mortality in the groups fed dietary AOS was slightly lower than that in the control. Therefore, supplementing the diet of grass carp with an appropriate amount of AOS can improve fat metabolism and immune responses and alter the intestinal bacterial community, which may help to fight bacterial infection.

Superoxide dismutases (SODs) are the main antioxidant enzymes involved in alleviating oxidative stress. Although mitochondrial manganese SOD (mMnSOD) has been reported to be correlated with the immune response in crustaceans, its biological properties and role in the immune response remain unclear. Here, we cloned the Macrobrachium rosenbergii mMnSOD (MrmMnSOD), analyzed its activity and expression pattern under Staphylococcus aureus and Vibrio parahaemolyticus infection, and further explored its possible mechanism during antibacterial immune response. The results showed that both enzyme activity and the expression of MrmMnSOD were significantly up-regulated by bacterial infection. MrmMnSOD knockdown made the prawn susceptible to Vibrio infection, which increased the mortality rate and the number of bacteria in haemocytes. The bacterial agglutination assay confirmed that MrmMnSOD decreases bacterial abundance via agglutination. Overall, this work identified antibacterial function of MrmMnSOD in the immune response. In addition to contributing to immunological theory, these findings aid disease prevention and control in crustacean aquaculture.

An increasing important area in immunology is the process cell death mechanism, enabling the immune system triggered thru extrinsic or intrinsic signals to effectively remove unwanted or virus infected cells called apoptosis. A recently isolated infectious Snakehead fish vesiculovirus (SHVV), comprising negative strand RNA and encoded viral matrix (M) proteins, is responsible for causing cytopathic effects in infected fish cells. However, the mechanism by which viral M protein mediates apoptosis has not been elucidated. Therefore, in the present experiments, it was investigated the regulatory potential of apoptosis signals during SHVV infection. By employing the model of SHVV infection in SSN-1 cells, the accelerated apoptosis pathway involves an intrinsic pathway requiring the activation of caspase-9 but not caspase-3 or -8. In the groups of infection (SHVV) or treatment (hydrogen peroxide) were induced apoptotic morphological changes and indicated the activation of the main caspases, i.e.; executioner caspase-3, initiators caspase-8 and caspase-9 using colorimetric assays. Turning to the role of viral M protein when it was overexpressed in SSN-1 cells, it was indicated that the viral M gene alone has the ability to induce apoptosis. To elucidate the mechanism of apoptosis in SSN-1 cells, the activation inhibitors of main caspases were used showing that inhibiting of caspase-3 or caspase-8 activation did not seize induction of apoptosis in virus-infected SSN-1 cells. However, the inhibiting of caspase-9 activation reduced significantly the apoptosis initiation process and sharply the expression of viral M gene, suggesting that SHVV plays a major role in the early induction of apoptosis by caspase-9. Interestingly, there were also differences in the mitochondrial membrane potential after the apoptotic induction of caspases, which confirm that caspase-9 is primarily responsible for the cleavage of caspases during apoptosis. Taken together, these findings can therefore be assumed that viral M protein induces apoptosis via the intrinsic apoptotic pathway in SHVV infecting SSN-1 cells.

The present study aimed to investigate the effects of five chitooligosaccharide monomers of different molecular weights on immunomodulatory activity in macrophage-like RAW264.7 cells. The incubation of various chitooligosaccharide monomers enhanced phagocytosis and pinocytosis activity toward Staphylococcus aureus and Escherichia coli in RAW264.7 cells. The incorporation of chitooligosaccharide monomers significantly boosted the generation of reactive oxygen species and reactive nitrogen species, as well as the release of inflammatory cytokines. To further explore the mechanism of inflammation regulated by chitooligosaccharide, the activation inhibitors of NF-кB (CAPE) and TLR-4 (TAK-242) were utilized, the determination data demonstrated that chitobiose suppressed the expression of inflammatory cytokines and NF-кB p65. In addition, the investigation results revealed that the presence of the mannose receptor inhibitor (mannan) suppressed chitohexaose-induced phagocytic activity and inflammatory cytokines. These results suggested that the five distinct chitooligosaccharide monomers had inconsistent effects, the chitobiose and chitohexaose exhibiting the best biological activity in activating RAW264.7 cells, promoting cell proliferation, and increasing non-specific immunity.

Snakehead vesiculovirus (SHVV) causes enormous economic losses in snakehead fish (Ophicephalus striatus) culture. Understanding replication mechanisms of virus is considerable significance in preventing and treating viral disease. In our previous studies, we have reported that glutamine starvation could significant inhibit the replication of SHVV. Furthermore, we also showed that SHVV infection could cause apoptosis of striped snakehead fish cells (SSN-1). However, the underlying mechanisms remain enigmatic. To decipher the relationships among the viral infection, glutamine starvation and apoptosis, SSN-1 cells transcriptomic profilings of SSN-1 cells infected with or without SHVV under glutamine deprived condition were analyzed. RNA-seq was used to identify differentially expressed genes (DEGs). Our data revealed that 1215 up-regulated and 226 down-regulated genes at 24 h post-infection were involved in MAPK, apoptosis, RIG-1-like and toll-like receptors pathways and glutamine metabolism. Subsequently, DEGs of glutamine metabolism and apoptosis pathways were selected to validate the sequencing data by quantitative real-time PCR (qRT-PCR). The expression patterns of both transcriptomic data and qRT-PCR were consistent. We observed that lack of glutamine alone could cause mild cellular apoptosis. However, lack of glutamine together with SHVV infection could synergistically enhance cellular apoptosis. When the cells were cultured in complete medium with glutamine, overexpression of glutaminase (GLS), an essential enzyme for glutamine metabolism, could significantly enhance the SHVV replication. While, SHVV replication was decreased in cells when GLS was knocked down by specific siRNA, indicating that glutamine metabolism was essential for viral replication. Furthermore, the expression level of caspase-3 and Bax was significantly decreased in SHVV infected cells with GLS overexpression. By contrast, they were significantly increased in SHVV infected cells with GLS silence by SiRNA, indicating that SHVV infection activated the Bax and caspase-3 pathways to induce apoptosis independent of glutamine. Our results reveal that SHVV replication and starvation of glutamine could synergistically promote the cellular apoptosis, which will pave a new way for developing strategies against the vial infection.

Intravascular hemolysis is a fundamental feature of hemorrhagic venereal infection or tissue and releases the endogenous damage-associated molecular pattern hemoglobin (Hb) into the plasma or tissues, which results in systemic inflammation, vasomotor dysfunction, thrombophilia, and proliferative vasculopathy. However, how the cytotoxic Hb affects the tissues of grass carp remains unclear. Here, we established a hemolysis model in grass carp by injecting phenylhydrazine (PHZ). The data revealed that the PHZ-induced hemolysis increased the content of Hb and activated the antioxidant system in plasma. The histopathology analysis data showed that the PHZ-induced hemolysis increased the accumulation of Hb and iron both in the head and middle kidney. The results of quantitative real-time PCR (qRT-PCR) detection suggested that the hemolysis upregulated the expressions of iron metabolism-related genes. In addition, the immunofluorescence and immunohistochemistry data revealed that the hemolysis caused an obvious deposition of collagen fiber, malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE) accumulation and increased the content of oxidative-related enzymes such as β-galactosidase (β-GAL), lipid peroxide (LPO), and MDA in both the head and middle kidney. Furthermore, the PHZ-induced hemolysis significantly increased the production of reactive oxygen species (ROS), which resulted in apoptosis and modulated the expressions of cytokine-related genes. Taken together, excess of Hb released from hemolysis caused tissue oxidative damage, which may be associated with ROS and inflammation generation.

Gonad-inhibiting hormone (GIH) belongs to a family of neuropeptides that are released from the eyestalks of male crustaceans and plays key roles in gonadal maturity, reproduction, and molting. However, the detailed mechanisms underlying the effects of GIH on sexual regulation have yet to be elucidated. In the present study, we aimed to demonstrate how GIH mediate the activity of the androgenic gland (AG) to affect sexual regulation. To do this, we cloned and characterized a GIH sequence from Macrobrachium rosenbergii (MrGIH). The open reading frame (ORF) of MrGIH was 360 bp and codes for a polypeptide of 119 amino acids and a putative protein of 13.56 KDa. Tissue analysis showed that MrGIH is widely expressed in a range of tissues but particularly, the eyestalk, intestine, and nerve cord. Following the dsRNA silencing of MrGIH for 24 h, the expression levels of MrGIH were down-regulated in both the eyestalk and AG when compared with the negative control, but significantly increased the expression of Macrobrachium rosenbergii insulin-like androgenic gland hormone-binding protein (MrIAGBP) in AG, thus suggesting that MrGIH is an inhibitory factor for MrIAGBP. In addition, we found that eyestalk removal on certain days led to increased expression levels of MrIAGBP expression. The expression levels of MrIAGBP peaked at 2 d in the AG after unilateral and bilateral eyestalk ablation, exhibiting a 7.27- and 6.03-fold increase, respectively. Afterward, the expression of GIH protein levels were down-regulated and IAGBP protein levels were up-regulated after GIH silencing using immunohistochemistry method, combined with the increase of IAGBP protein levels after eyestalk ablation, confirming that MrGIH is an inhibitory factor that can moderately regulate AG development and IAGBP expression. Collectively, our findings enriched the mechanisms that control the sexual regulation pathway of male M. rosenbergii, and provided significant information for further explorations of the mechanism underlying sex regulation in other decapod crustaceans.

Red blood cells (RBCs) are widely accepted as their primary function in respiration. Recent studies in mammals have revealed a vital role in immune responses of RBCs; however, little is known about immune function of teleost erythrocytes. Here we demonstrated that RBCs from grass carp (Ctenopharyngodon idella) were capable of binding and aggregating the bacteria with apparent morphological alterations. The phagocytosis by teleost RBCs (erythrophagocytosis) was visualized by confocal, scanning and transmission electron microscopy. Hb-FeII of hemoglobin (Hb) could quickly be auto-oxidated to Hb-FeIII (methemoglobin/metHb) in the presence of oxygen (O2), and release superoxide radical (O2-.) which could be spontaneously dismutated into H2O2 that could further oxidize Hb-FeIII to transient HbFeIV-OH (ferryl-Hb). Furthermore, bacterial extracellular proteases and pathogen-associated molecular patterns (PAMPs) binding to Hb could synergistically activate pseudoperoxidase, subsequently facilitated the generation of reactive oxygen species (ROS) which were toxic to the bacteria. Our results indicated that erythrocyte pertains anti-bacterial activity using unique ROS generation pathway via oxidation of hemoglobin and associated with its phagocytosis.

Prophenoloxidase (proPO) is the zymogen form of phenoloxidase (PO), a key enzyme in melanization cascade that has been co-opted in invertebrate immune reactions. There have been reported that proPO plays many essential roles in the crustacean immune system. However, little is known about the function of proPO from red swamp crayfish (Procambarus clarkii) which is an important cultured species worldwide. Here, we cloned and expressed proPO gene from red swamp crayfish (PcproPO). Subsequently, specific antibody against PcproPO was generated. The immune function of PcproPO was further characterized in vitro and in vivo. The results showed that the expression of PcproPO mRNA could be significantly up-regulated during the challenge of Gram-positive-negative (Vibrio parahaemolyticus) and Gram-positive-positive bacterial (Staphylococcus aureus). Furthermore, the purified recombinant PcproPO protein had a strong affinity binding to both bacteria and polysaccharides. In vivo knockdown of PcproPO could significantly reduce the crayfish bacterial clearance ability, resulting in the higher mortality of the crayfish during V. parahaemolyticus infection. In addition, in vitro knockdown of PcproPO in the hemocytes significantly reduced the phenoloxidase (PO) activity and the bacterial clearance ability, indicating that PcproPO might involve in hemocyte-mediated melanization. Our results will shed a new light on the immune function of PcproPO in the crayfish.

The freshwater prawn, Macrobrachium rosenbergii naturally lives in the freshwater, though it migrates to the brackish water environment during spawning that claimed to be resistant on a broad range of saline fluxes. However, little is known about the osmoregulatory patterns and the effect of an enzyme glutamine synthetase (GS) in M. rosenbergii under stress. Here, we described the identification and functional characterization of GS from M. rosenbergii (Mr‐GS) at molecular and protein levels. The identified Mr‐GS was comprised of 361 amino acids that phylogenetically shared the highest identity with other crustaceans and predicted to contain Gln‐synt_C and Gln‐synt_N domains at the respective terminal regions. Tissue distribution analysis in M. rosenbergii revealed that the Mr‐GS was highly expressed in muscle, and commonly existed in other examined tissues in the following order gills > heart > stomach > brain > haemolymph. Whereas, the mRNA of Mr‐GS was significantly up‐regulated in the muscle and gill tissues following challenges with either hyper (0 → 13‰), or hypo (13 → 0‰) osmotic stress at 3, 6 and 12 hr. Furthermore, the level of Glutamine concentration was positively correlated with the GS mRNA and protein expression patterns in hyper‐osmotic stress, whereas in hypo‐osmotic stress a slight decrease in the gills and maintained a level in the muscle tissues at 3, 6 and 12 hr post‐treatments. Our findings suggest that Mr‐GS potentially exhibited the osmoregulation responses in the gill and muscle tissues of M. rosenbergii throughout the time of osmotic stress, which will benefit for future study on osmoregulation.

Cyclophilin A (CypA) is a ubiquitously expressed cellular protein and involves in diverse pathological conditions, including infection and inflammation. CypA acts as a key factor in the replication of several viruses. However, little is known about the role of CypA in the replication of the red-spotted grouper nervous necrosis virus (RGNNV). In the present report, grouper CypA (GF-CypA) was cloned from the grouper fin cell line (GF-1) derived from orange-spotted grouper (Epinephelus coioides). Sequence analysis found that GF-CypA open reading frame (ORF) of 495 bp encodes a polypeptide of 164 amino acids residues with a molecular weight of 17.4 kDa. The deduced amino acid sequence shared highly conserved regions with CypA of other animal species, showing that GF-CypA is a new member of Cyclophilin A family. We observed that GF-CypA was up-regulated in the GF-1 cells infected with RGNNV. Additionally, overexpression of CypA could significantly inhibit the replication of RGNNV in GF-1 cells. By contrast, when the GF-CypA was knock-downed by siRNA in GF-1 cells, the replication of RGNNV was enhanced. Furthermore, the expressions of pro-inflammatory factors, such as TNF-2, TNF-α, IL-1b, and ISG-15, were increased in GF-CypA transfected GF-1 cells challenged with RGNNV, indicating that GF-CypA might be involved in the regulation of the host pro-inflammatory factors. Altogether, we conclude that GF-CypA plays a vital role in the inhibitory effect of RGNNV replication that might be modulating the cytokines secretion in GF-1 cells during RGNNV infection. These results will shed new light on the function of CypA in the replication of RGNNV and will pave a new way for the prevention of the infection of RGNNV in fish., Highlights • Cloned and analyzed GF-CypA from GF-1 (grouper fish, Epinephelus coioides) cell line. • Overexpression or knockdown of GF-CypA significantly regulated RGNNV replication. • Up-regulation of TNF-2, TNF-α, IL-1b, and ISG-15 cytokines during RGNNV infection in GF-1 cells were observed.

Autophagy is a degradation cellular process which also plays an important role in virus infection. Glutamine is an essential substrate for the synthesis of glutathione which is the most abundant thiol-containing compound within the cells and plays a key role in the antioxidant defense and intracellular signaling. There is an endogenous cellular glutathione pool which consists of two forms of glutathione, i.e. the reduced form (GSH) and the oxidized form (GSSG). GSH serves as an intracellular antioxidant to maintain cellular redox homeostasis by scavenging free radicals and other reactive oxygen species (ROS) which can lead to autophagy. Under physiological conditions, the concentration of GSSG is only about 1% of total glutathione, while stress condition can result in a transient increase of GSSG. In our previous report, we showed that the replication of snakehead fish vesiculovirus (SHVV) was significant inhibited in SSN-1 cells cultured in the glutamine-starvation medium, however the underlying mechanism remains enigmatic. Here, we revealed that the addition of L-Buthionine-sulfoximine (BSO), a specific inhibitor of the GSH synthesis, could decrease the γ-glutamate-cysteine ligase (GCL) activity and GSH levels, resulting in autophagy and significantly inhibition of the replication of SHVV in SSN-1 cells cultured in the complete medium. On the other hand, the replication of SHVV was rescued and the autophagy was inhibited in the SSN-1 cells cultured in the glutamine-starvation medium supplemented with additional GSH. Furthermore, the inhibition of the synthesis of GSH had not significantly affected the generation of reactive oxygen species (ROS). However, it significantly decreased level of GSH and enhanced the level of GSSG, resulting in the decrease of the value of GSH/GSSG, indicating that it promoted the cellular oxidative stress. Overall, the present study demonstrated that glutamine starvation impaired the replication of SHVV in SSN-1 cells via inducing autophagy associated with the disturbance of the endogenous glutathione pool.

It is widely known that red blood cells (RBCs) are responsible for respiration and the transport of gas. However, recent reports have also described the immune properties of RBCs, therefore creating new understanding for the functionality of RBCs. However, little is known about the immunological role of RBCs in bony fish. In this study, we used RBCs from Clarias fuscus as a model and demonstrate that these cells exhibited phagocytic ability with both latex beads and bacteria. Scanning electron microscopy and transmission electron microscopy provided visual confirmation of the phagocytotic process in RBCs. In addition, we used flow cytometry and fluorescence microscopy to analyse the rate of phagocytosis in RBCs. We found that RBCs exhibited stable phagocytotic ability with latex beads ranging from 0.5 to 1.0 μm in size. In response to bacterial stimulation, RBCs produced reactive oxygen species (ROS) and nitric oxide synthase (NOS), which are harmful to bacteria. RBCs also have an antioxidant system. Under conditions of oxidative stress, the expression levels of antioxidant enzymes, and particularly those of superoxide dismutase（SOD） increased significantly. Our results show that the erythrocytes of bony fish are phagocytic and also produce ROS which are toxic to bacteria. In addition, RBCs have an antioxidant system that removes excess ROS production to protect cells from oxidative damage.

Infectious bacterial and viral diseases that cause hemolysis are considered life-threatening to grass carp (Ctenopharyngodon idellus), which is a species used in aquaculture worldwide. After heme and hemeproteins (Hb) are released as a result of hemolysis, the effect of excess Hb and heme on tissues remains to be characterized. To decipher the mechanisms, after incubation with Hb, we showed that lipopolysaccharide (LPS), Hb, and heme increased the cytotoxicity and secretion of inflammatory cytokines such as interleukin (IL)-6, chemokine (C-C motif) ligand 1 (CCL1), tumor necrosis factor (TNF)-α, IL-6, and IL-1β in vitro, which was due to stimulation of the expression of innate immune receptors, such as nucleotide-binding oligomerization domain (NOD2), toll-like receptor 2 (TLR2), TLR 4, and TLR3. The formation of reactive oxygen species (ROS) and the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB were important for increasing the cytokine production to induce heme, Hb, and LPS. Moreover, we confirmed that after LPS, Hb, and heme challenge, superoxide dismutase (SOD) and glutathione (GSH) synthetase (GSS) also caused remarkable destruction. However, catalase (CAT) and heme oxygenase-1 (HO-1) were strongly activated. In summary, our research findings present a framework through which heme and Hb concentrations amplify the secretions of inflammatory cytokines, which are induced by pattern recognition receptor (PRR) activation and present possible paths for immune intervention during infection with viral diseases and hemolytic bacterial.

The BCL2-associated agonist of cell death protein is a key participant in apoptosis dependent on mitochondria and in disease progression that involves the regulation of cell death, such as tumorigenesis, diabetes, sepsis shock, and epilepsy. Nevertheless, the mechanisms underlying the immune responses to teleost BAD bacterial infection and mitochondrial-dependent apoptosis remains unclear. In order to elucidate the mechanisms involved, in this study, a Ctenopharyngodon idella (grass carp) BAD gene named GcBAD1 was firstly cloned and characterized. The results indicated that the ORF (open reading frame) of GcBAD1 was 438 bp in length, encoding a 145-amino acid putative protein of 16.66 kDa. This deduced amino acid sequence has a better identity than another teleost species according to a phylogenetic analysis, and contains a Bcl2-BAD-1 domain. In healthy grass carp fish, the mRNA transcripts of GcBAD1 were widely present in the studied tissues, which could be ranked as follows; spleen > brain > middle-kidney > head-kidney > liver > gills > intestines > heart and muscle. In addition, during infection by Aeromonas hydrophila and Staphylococcus aureus, the mRNA transcription and protein levels expression of GcBAD1 in the head-kidney, spleen, and liver tissues of the fish were significantly up-regulated. Moreover, when the C. idellus kidney cell line (CIK) cells were incubated with Lipopolysaccharide (LPS) and lipoteichoic acid (LTA), the GcBAD1 expression transcripts were also significantly up-regulated. Additionally, overexpression of GcBAD1 in CIK cells was able to activate apoptosis-related genes, including those encoding p53, Cytochrome C (CytoC), caspase-3, and caspase-9. Besides, in the TUNEL assays, when pEGFP-BAD1 was over-expressed, the number of red signals associated with apoptosis were significantly increased in the CIK cells infected with LPS or LTA at 12 h. This study demonstrates that GcBAD1 has a significant role in the mitochondrial apoptosis pathway of grass carp's innate immunity. Our findings provide new insight into the potential mechanisms of teleost antibacterial immunity.

Background: Fish blood contains RBCs (Red blood cells) as the major cells. Unlike the absence of nucleus in mammalian RBC, fish RBC contains a nucleus. Previous researches have indicated that fish RBCs have a significant function in immune responses. However, the mechanism underlying the immune responses against bacterial infection of teleost RBCs remains enigmatic. To decipher the mechanisms, after the infection of A. hydrophila, transcriptomic profiling of grass carp RBCs was analyzed. Results: Outcomes demonstrated that there were 2144 altogether DEGs (Differently Expressed Genes) between the A. hydrophila non-disease groups, which includes 817 up-regulated and 1327 down-regulated DEGs. Differently Expressed Genes were allocated to 45 GO terms, including 20 natural procedure terms, 14 cell related element, and 11 terms related to molecular functions. Likewise, the expression levels of cytokines by Quantitative Reverse Transcription Polymerase Chain Reaction showed that they (e.g. CCL4, CCL11, CCL20, IL-4, IL-12, and IFNα) were significantly increased after A. hydrophila infection, which was identical to the expression patterns of transcriptomic data. The infection could cause the apoptosis of RBCs which was confirmed by annexin V/PI assay. To further elucidate the apoptosis pathway, the expression levels of genes by mRNA included in cellular apoptosis were monitored. The outcomes indicated that the expressions of mRNA of p53, Fas, Leucine-rich repeats, death domain-containing (LRDD) and caspase 8 were all significantly increased, respectively. Conclusions: Our results support the notion that A. hydrophila infection could activate Fas-related death receptor apoptosis pathway in grass carp RBCs, which will reveal another insight on the mechanisms underlying the antibacterial immunity of teleost fish RBCs.

Heme oxygenase (HO)-1, a rate-limiting enzyme in heme catabolism, results in the formation of equivalent amounts of biliverdin (BV), carbon monoxide (CO) and ferrous iron (Fe2+). Previous studies have revealed that HO-1 plays an important role in immune responses. However, the mechanism underlying the immune responses against bacterial infection of teleost HO-1 remains enigmatic. To decipher the mechanisms, we have cloned and characterized the HO-1 gene of grass carp (designated as GcHO-1) in this research. The results showed that the open reading frame (ORF) of GcHO-1 was 819 bp, which encoded a putative protein of 272 amino acids. The deduced amino acid sequence phylogenetically shared the highest identity with other teleosts, and contained two domains of heme-oxygenase and a single-pass transmembrane domain. The mRNA expressions of GcHO-1 in healthy grass carp have widely existed in examined tissues in the following order of spleen > head-kidney > middle head-kidney > intestines > liver > gills > heart > muscle > brain. Besides, the mRNA and protein transcription of GcHO-1 were both significantly up-regulated in the liver and head-kidney tissues after Staphylococcus aureus and Aeromonas hydrophila infection. In addition, overexpression of GcHO-1 in kidney cell line (CIK) cells of grass carp could reduce the expression of inflammatory cytokines (IL-1β, IL-8, TNFα, CCL1 and IL-6). Herein, we demonstrate that GcHO-1 plays an anti-inflammatory role in innate immunity. Our results shed new light on the mechanisms underlying the antibacterial immunity of teleost.

Giant freshwater prawns (Macrobrachium rosenbergii) are commonly found throughout the world. The size of the male giant freshwater prawn is much larger than that of the female. Therefore, understanding the molecular mechanism that underlies the sexual differentiation of M. rosenbergii is of both commercial and scientific importance. Insulin-like androgenic gland hormone (IAG) plays a key role in the differentiation of sex in M. rosenbergii. Although IAG has been investigated, the regulatory relationship between IAG and its binding protein partner, the insulin-like androgenic gland hormone-binding protein (IAGBP), has not been studied in M. rosenbergii. Here, we cloned and characterized the IAGBP from M. rosenbergii (Mr-IAGBP) for the very first time. Transcriptomic analysis showed that Mr-IAGBP mRNA was detected in a wide array of tissues with the highest expression found in the androgenic gland. The importance of IAG in male development was further demonstrated by an increase in IAG transcripts during the development of the androgenic gland and Mr-IAG was only highly transcribed in the androgenic gland of M. rosenbergii. Interestingly, we found that the Mr-IAG gene expression started during the 20th-day larva after hatching stage (LH20), followed (20th-day post-larval stage, PL20) by a gradual elevation of Mr-IAGBP levels. The levels of both genes peaked at the adult stage. The relationship between Mr-IAGBP and Mr-IAG was further analyzed using RNA interference. The injection of Mr-IAGBP double-stranded RNA (dsRNA) significantly reduced the transcription of Mr-IAG, while the amount of Mr-IAGBP mRNA and the translation of IAGBP protein was significantly reduced by the injection of Mr-IAG dsRNA. These results revealed that IAGBP is involved in IAG signaling. Furthermore, our data supports the hypothesis that (IAG and IAGBP)-IAG receptor signaling schemes exist in M. rosenbergii. Our results will provide important information for the further study of determining the sex of M. rosenbergii.

The red blood cells (RBCs) of fish make up around 95% of the total peripheral blood cells, and the long-held paradigm is that RBCs are mainly responsible for transporting oxygen. Previous studies have showed that the RBCs can be involved in the immune response against bacterial infection; however, this mechanism remains enigmatic. Here, we explored the structure of grass carp RBCs (GcRBCs). The results showed that the GcRBCs released a pseudopodia-like structure when grown in a 24-well plate, and the transmission electron microscopy (TEM) result showed that GcRBCs contained some organelle-like structures. To further verify the organelle-like structures might be the mitochondria and lysosome which similar to other immune cells, a fluorescent labeling assay was used to verify it. To decipher the antibacterial immunity of GcRBCs, transcriptomic profiling of grass carp RBCs after the incubation with E. coli was analyzed. The results showed that there were 4099 differently expressed genes (DEGs) of GcRBCs upon E. coli incubation, including 2041 up-regulated and 2058 down-regulated genes. In addition, to validate our transcriptomic data, we checked the expression of several cytokines, such as CCL4, CCL20, IL4, IL12 and IFN-α, and the results showed that all the selected gens were significantly up-regulated after E. coli incubation. Furthermore, E. coli incubation induced hemoglobin oxidation and increased the heme in GcRBCs, which further activated the expression of heme oxygenase 1 (HO-1), autophagy related genes 5 (ATG5), and ferritin. In contrast, E. coli incubation inhibited the expression of Ferroportin-1 (FPN1), which increased intracellular iron levels, induced Fenton reaction to release reactive oxygen species (ROS), and activated the ferroptosis signaling pathway in GcRBCs. Herein, we demonstrate that E. coli can induce teleost RBCs cell death through an iron-mediated ferroptosis pathway, which sheds new light on the interaction between bacteria and teleost RBCs.

Functional ingredients such as Bacillus subtilis are used in aquaculture to improve fish condition, modulate microbiota and promote a healthy intestinal system. However, the underlying mechanisms of grass carp treated with B. subtilis are not fully characterized. This study investigated the gut microbes of grass carp after treated with B. subtilis H2 (106 CFU/mL) and Aeromonas hydrophila (106 CFU/mL). The intestinal flora was found that the dominant bacterial phyla identified in all samples were Proteobacteria, Actinobacteria, Fusobacteria, Bacteroidetes and Acidobacteria. Compared with the control group, the relative abundance of Proteobacteria and Bacteroidetes in B. subtilis group were significantly increased. In addition, the relative abundances of Aeromonas and Shewanella in A. hydrophila group were more than the control group. For the intestinal transcriptomic profiling of the grass carp treated with B. subtilis H2, 824 different expressed genes (DEGs) between the B. subtilis H2 treated and non-treated groups were detected, including 365 up-regulated and 459 down-regulated genes. Six DEGs were randomly selected for further validation by quantitative real-time RT-PCR (qRT-PCR) and the results were consistent with the RNA-seq data. Additionally, eight immunomodulatory genes (IL-4, IL-11, IFN-α, CSF, FOSB, MAPK12b, IGHV3-11 and IGHV3-21) were significantly up-regulated after treated with B. subtilis H2. Furthermore, almost all the lipid metabolism-associated genes were significantly up-regulated after treated with B. subtilis H2 according to the lipid metabolism pathways. Eleven lipid metabolism-associated genes were selected by qRT-PCR, which showed that the expressions of almost all the selected genes were increased, especially Apob-48, ABCG8 and DGAT. Taken together, our results support that B. subtilis could modulate the immune response, fat metabolism and bacterial assembly in the gut of grass carp.