Calreticulin functions in antimicrobial immunity of obscure puffer Takifugu obscurus.

Calreticulin (Crt) is a highly conserved and multi-functional protein with lectin-like properties and important immunological activities. In this study, a Crt homolog, namely, ToCrt, was cloned and characterized from the obscure puffer Takifugu obscurus with an open reading frame of 1278 bp encoding a putative protein of 425 amino acids. The deduced amino acid sequence of ToCrt consisted of three conserved structural domains: N-domain, P-domain, and C-terminal domain. In the phylogenetic tree, ToCrt formed a separate cluster with three Crts from other pufferfish species (Takifugu rubripes, Takifugu flavidus, and Takifugu bimaculatus). The mRNA transcript of ToCrt was ubiquitously expressed in all the examined tissues in a decreasing order: liver, spleen, kidney, gills, intestine, and heart. After Vibrio harveyi, Edwardsiella tarda, and Aeromonas hydrophila stimulations, the levels of ToCrt mRNA in the kidney and spleen were significantly upregulated compared with that in the control group. The recombinant calreticulin domain of ToCrt (rToCrt) could bind three Gram-negative bacteria (V. harveyi, E. tarda, and A. hydrophila) and polysaccharides from bacterial cell walls such as lipopolysaccharide and peptidoglycan. Meanwhile, rToCrt could agglutinate different kinds of microorganisms and exhibit antimicrobial activity. These results suggested that T. obscurus ToCrt could serve as an antimicrobial effector in the host immune response against invading microorganisms.

Functional characterization of TNF-α in pufferfish (Takifugu obscurus) in immune response and apoptosis against Aeromonas hydrophila.

Tumour necrosis factor-α (TNF-α) is a multifunctional cytokine involved in immune system homeostasis, antimicrobial defence, regulation of apoptosis, cell proliferation and differentiation. Although the pro-inflammatory property of TNF-α has been made new progress, detailed research on host defence against bacterial infection and inducing apoptosis remains to be revealed in early vertebrates. Here, we reported the TNF-α homologue (ToTNF-α) from pufferfish (Takifugu obscurus). The open reading frame (ORF) of ToTNF-α was 753 bp, encoding a protein of 250 aa contained the TNF family signature and conserved cysteine residues. The mRNA expression of ToTNF-α had a wide range of tested tissues, with the highest expression in the skin. After Aeromonas hydrophila infection, the mRNA expression of ToTNF-α was significantly up-regulated both in vivo and in vitro experiments. After stimulation by recombinant protein of ToTNF-α ((r)ToTNF-α), the relative expressions of endogenous TNF-α, caspase 8, caspase 3, p53, and Bax inhibitor-1 in head kidney leucocytes were all notably up-regulated. These results showed that ToTNF-α might induce apoptosis depend on pro- and anti-apoptotic proteins at mRNA level. Moreover, flow cytometry analysis indicated that the (r)ToTNF-α can induce apoptosis of head kidney leucocytes. Taken together, these characteristics suggest that ToTNF-α can participate in immune response against A. hydrophila and induce apoptosis at mRNA and cellular level, which will help to understand the mechanism of apoptosis and immune response in teleost fish.

In silico characterization and expression analysis of eight C-type lectins in obscure puffer Takifugu obscurus.

C-type lectins (CTLs) are a group of carbohydrate-binding proteins that play crucial roles in innate immune defense against invading pathogens. CTLs have been extensively studied in lower vertebrates, such as fish, for their roles in eliminating pathogens; however, their homologs in pufferfish are not well known. In the present study, eight CTLs from obscure puffer Takifugu obscurus (designated as ToCTL3-10 according to the order they were discovered) were obtained. All predicted ToCTL proteins contained a single carbohydrate recognition domain (CRD). ToCTL7 also contained one calcium-binding epidermal growth factor (EGF)-like domain (EGF_CA) and a transmembrane region. ToCTL9 also contained an SCP domain, an EGF domain, and an EGF-like domain. Bioinformatics analysis revealed that ToCTL3-10 mainly clustered with the corresponding CTL homologs of other pufferfish species. Tissue distribution analysis detected ToCTL3-10 in all tissues examined, including kidneys, liver, gills, spleen, intestines, and heart. Moreover, the expressions of ToCTL3-10 were significantly induced in the kidneys of obscure puffer following challenges with three Gram-negative bacterial pathogens, namely, Vibrio harveyi, Aeromonas hydrophila, and Edwardsiella tarda, and a synthetic analog of double-stranded RNA poly(I:C). The expression patterns of ToCTL3-10 in response to different immune stimulants were different. Our results indicated that the eight ToCTLs obtained herein might be involved in host defense against bacterial and poly(I:C) infections in T. obscurus.

Alterations in hematological and biochemical parameters, oxidative stress, and immune response in Takifugu rubripes under acute ammonia exposure.

Ammonia is a major pollutant in aquatic environments and poses a considerable threat to the survival of fish. In this study, we investigated the toxic effects of ammonia on the hematological and biochemical parameters, oxidative stress, and immune responses in Takifugu rubripes. Juvenile T. rubripes (average weight 246.17 ± 3.54 g) were exposed to different concentrations of ammonia (0, 5, 50, 100, and 150 mg/L) for 96 h. The results showed that the hematological parameters (hemoglobin, hematocrit, red blood cell, and white blood cell count) were significantly reduced in response to ammonia exposure. Of the plasma components, such as serum total protein, albumin, glucose, glutamic-oxalacetic transaminase, and glutamic-pyruvic transaminase, were significantly altered in response to ammonia exposure. Additionally, the levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GPx) were increased after exposure to low concentration ammonia exposure. However, when fish were exposed to a high concentration of ammonia, these parameters showed the opposite trend, suggesting that an increase in antioxidant enzymes during the early stages of ammonia exposure may contribute to the removal of the induced reactive oxygen species (ROS) and protect the cells from oxidative damage. However, as the ammonia concentration and exposure time increased, the overproduction of ROS accelerated the depletion of antioxidant enzymes. Ammonia exposure significantly increased the expression of heat shock proteins (HSP70 and HSP90). Ammonia poisoning elevated gene expressions of TLR-3, TNF-α, IL-6, IL-12, and IL-1ß in the gills, causing an inflammatory response. Our findings provide new insights into the mechanisms involved in ammonia-induced aquatic toxicology in marine fish, which may aid in their captive management.

Molecular cloning, characterization and expression analysis of major histocompatibility complex class I alpha gene of pufferfish (Takifugu obscurus).

The major histocompatibility complex (MHC) genes play a key role in immune response in vertebrates. In this study, an MHC I alpha homolog gene (PfMHC Ⅰα) from pufferfish (Takifugu obscurus) was identified and its subcellular localization and expression patterns of PfMHC Ⅰα after challenge in vivo and in vitro were analysed. The open reading frame of PfMHC Ⅰα was 1,089 bp in length, encoding 362 aa. The immunofluorescence result revealed that PfMHC Ⅰα was presented on the membrane of lymphocytes. qRT-PCR analysis indicated that PfMHC Ⅰα was expressed in all examined tissues, with the highest expression in skin, followed by the expression in gills and whole blood. After challenge of Aeromonas hydrophila or polyinosinic: polycytidylic acid (Poly I:C) in vitro, the expression levels of PfMHC Ⅰα on pufferfish kidney lymphocytes were significantly up-regulated, with the highest expression level at 48 hr post-challenge. After infection with A. hydrophila or Poly I:C in vivo, the expression levels of PfMHC Ⅰα in the skin, whole blood and kidneys were significantly up-regulated. Taken together, it is speculated that PfMHC Ⅰα associates with resistance to both intracellular and extracellular antigens and plays an important role in the host response against pathogen infection in pufferfish.

Expression profile of kalliklectin, a soluble-type mannose receptor, during embryogenesis and early larval development in fugu (Takifugu rubripes).

Kalliklectin is a unique fish-specific lectin, whose sequence is similar to the heavy chain of mammalian plasma kallikrein and coagulation factor XI. In this study, we aimed to evaluate dynamic expression profiles of the lectin gene, during early developmental stages, in fugu, Takifugu rubripes. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the kalliklectin gene was not expressed until 14 h post-fertilization (hpf), while the mRNA was detected after 30 hpf. In real-time quantitative PCR (qPCR), the gene was first expressed at 10.5 hpf; then, the expression level increased with a peak at 30 hpf and then gradually decreased. On the other hand, western blotting with specific antibody detected the lectin protein at all tested stages, including the unfertilized egg, which suggests that the lectin detected in the early stages was a maternal factor. Immunohistochemistry demonstrated that kalliklectin was localized at the basement membranes of the newly hatched larvae, while the lectin was widely detected in epidermal cells in larva at 5 dph. A 40-kDa lectin was partially purified from unfertilized eggs using mannose-affinity chromatography, and the lectin was determined as kalliklectin by liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS) analysis, which indicated that the lectin is functional in the eggs. The egg lectin can bind to Gram-positive bacterial pathogens of fish, such as Lactococcus garvieae and Streptococcus iniae. We conclude that fugu kalliklectin might be an important immunocomponent, transferred from mother to offspring.

Interleukin-17A/F1 from Japanese pufferfish (Takifugu rubripes) stimulates the immune response in head kidney and intestinal cells.

In mammals, interleukin (IL)-17A and IL-17F, mainly produced by Th17 cells, are hallmark inflammatory cytokines that play important roles in the intestinal mucosal immune response. In contrast, three mammalian IL-17A and IL-17F counterparts (IL-17A/F1-3) have been identified in teleosts, and most of their functions have been described in the lymphoid organs. However, their function in the intestinal mucosal immune response is poorly understood. In this study, a recombinant (r) tiger puffer fish fugu (Takifugu rubripes) IL-17A/F1 was produced and purified using a mammalian expression system, and was used to stimulate cells isolated from fugu head kidney and intestines. The gene expression levels of TNF-α, IL-1ß, IL-6, and ß-defensin-like protein-1 (BD-1) genes were evaluated at 0, 3, 6 and 12 h post-stimulation (hps). Phagocytic activity and superoxide anion production were evaluated at the same time points using an NBT assay. The rIL-17A/F1 protein was shown to induce the expression of pro-inflammatory cytokines and antimicrobial peptides in both head kidney and intestinal cells. Expression levels for IL-1ß, TNF-α, and IL-6 were all up-regulated between 3 and 12 hps. In addition, stimulation with rIL-17A/F1 enhanced phagocytic activity at 24 hps. Superoxide anion production was increased at 48 hps in the head kidney cells and moderately increased at 48 hps in intestinal cells. This study suggests that fugu IL-17A/F1 plays an important role in promoting the innate immune response and may act as a bridge between innate and adaptive immunity in the head kidney and intestine.

Functional characterization of an immunity-related GTPase gene in immune defense from obscure puffer (Takifugu obscurus).

Immunity-related GTPases (IRGs) are a family of large interferon-inducible GTPases that function in effective host defense against invading pathogens. IRGs have been extensively studied in mammals for their roles in the elimination of intracellular pathogens; however, their homologs in lower vertebrates are not well known. In this study, an IRG from obscure puffer (Takifugu obscurus), ToIRG, was identified and further characterized for its functional activity. The ToIRG gene encodes a protein of 396 amino acids containing a typical N-terminal GTPase domain with three conserved motifs. Phylogenetic analysis revealed that it has a closer evolutionary relationship with mammalian GKS IRGs. Gene expression profile analysis revealed that ToIRG was ubiquitously expressed in all tested healthy tissues of obscure puffer and upregulated in response to Aeromonas hydrophila or Edwardsiella tarda challenge. The subcellular localization of ToIRG is characterized as condensed forms around the nucleus. Importantly, an antimicrobial assay in vitro suggested that ToIRG enhanced the ability of host cells to resist both intracellular (E. tarda) and extracellular pathogens (A. hydrophila). Taken together, these results provide the functional characterization of obscure puffer IRGs in immune defense, which is the first study to reveal the function of IRGs in bony fish and will provide important insights into the evolutionary divergence of IRGs.

Serum amyloid P-component/C-reactive proteins in fugu (Takifugu rubripes) egg with binding ability to disease-causing bacteria by carbohydrate-recognition.

Two galactose-binding proteins were purified from the eggs of Takifugu rubripes by affinity chromatography. These proteins were detected at 26 and 23 kDa under reducing and at 40 and 45 kDa under non-reducing conditions at SDS-PAGE. The peptide sequences from both proteins matched to short-type pentraxin. The 26-kDa lectin was glycosylated, while the other one was not, indicating that these could be glycoforms of pentraxin. Messenger RNA of pentraxin was detected in eggs and embryos at 1-cell stage, was undetectable till blastula, and finally detected again after gastrula, suggesting that the mRNAs in eggs and 1-cell embryos were maternal in origin, and autologous transcription of the gene occurred after blastula. Since they bind to pathogenic bacteria, egg pentraxins may have immunological functions during embryogenesis. This is the first study to show the presence of short-type pentraxin in fish eggs and the diversity of fish egg lectins.

D-mannose-specific Immunoglobulin M in Grass Puffer (Takifugu niphobles), a Nonhost Fish of a Monogenean Ectoparasite Heterobothrium okamotoi, Can Act as a Trigger for its Parasitism.

Heterobothrium okamotoi, a monogenean gill parasite, exhibits high host specificity for the tiger puffer, Takifugu rubripes, and it has been experimentally verified that the parasite cannot colonize either closely related species such as the grass puffer Takifugu niphobles or distantly related fish such as the red seabream Pagrus major. Previously, we demonstrated in T. rubripes that immunoglobulin M (IgM) with d-mannose affinity induced deciliation of the oncomiracidia, the first step of parasitism, indicating that the parasite utilizes the molecule as a receptor for infection. In the present study, we purified mannose-specific IgM from 2 nonhost species, T. niphobles and P. major, by affinity and gel-filtration chromatography techniques and compared their deciliation-inducing activity against H. okamotoi oncomiracidia. The IgM of the former showed activity, whereas the latter had no effect, suggesting that in addition to d-mannose-binding ability, the crystallizable fragment domain of IgM, which is not part of the antigen-binding domain, plays an important role in host recognition by the oncomiracidia, such as direct binding to the parasites. It also suggests that the host specificity of H. okamotoi is relatively low upon initial recognition, and the specificity is established by exclusion in nonhosts during a later stage.

Characterization and Functional Analysis of Two Transmembrane C-Type Lectins in Obscure Puffer (Takifugu obscurus).

C-type lectins (CTLs) have received widespread attention in animal immune responses. In the present study, two CTLs (ToCTL1 and ToCTL2) were identified from obscure puffer Takifugu obscurus. The open reading frames of ToCTL1 and ToCTL2 were 687 and 1,380 bp, respectively. The predicted ToCTL1 and ToCTL2 proteins contained a single transmembrane region and one typical carbohydrate recognition domain (CRD). Quantitative real-time polymerase chain reaction detected ToCTL1 and ToCTL2 transcripts in all examined tissues, with high levels in the intestine and kidney, and their expression levels were remarkably altered upon Vibrio harveyi and Aeromonas hydrophila infection. The recombinant proteins ToCTL1-CRD and ToCTL2-CRD agglutinated the Gram-negative and Gram-positive bacteria in a Ca2+-dependent manner. rToCTL1-CRD and rToCTL2-CRD exhibited evident binding activities against seven kinds of bacteria and polysaccharides (lipopolysaccharide and peptidoglycan) in a Ca2+-independent manner. Moreover, rToCTL1-CRD and rToCTL2-CRD could inhibit the growth of four types of bacteria in vitro. These findings collectively demonstrated that ToCTL1 and ToCTL2 could be involved in host defense against bacterial infection in T. obscurus.

Toll-like receptors (TLRs) respond to tributyltin chloride (TBT-Cl) exposure in the river pufferfish (Takifugu obscurus): Evidences for its toxic injury function.

Tributyltin chloride (TBT-Cl) residual in water body had become a noticeable ecological problem for aquatic ecosystems. Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors that play key roles in detecting nonself antigens and immune system activation. In this study, we explored the effect of TBT-Cl exposure on four TLRs expression in river pufferfish, Takifugu obscurus. The four T. obscurus Toll-like receptors (To-TLRs) contained different types of domains such as leucine-rich repeats (LRRs), leucine-rich repeats, typical subfamily (LRR_TYP) and other special domains. The To-TLRs mRNA transcripts expressed in all tissues, also To-TLR2 was investigated with higher level in kidney, as well as To-TLR3 in kidney, while To-TLR18 in liver and To-TLR22 in intestine. After the acute and chronic exposure of TBT-Cl, To-TLR2 and To-TLR3 mRNA transcripts were significantly down-regulated in gill. However, To-TLR18 and To-TLR22 were significantly up-regulated in gill and liver. Moreover, the histology and immunohistochemistry (IHC) results showed the different injury degrees of TBT-Cl in liver and gill and implied the cytoplasm reorganization after TBT-Cl stress and the function of immunoregulation for To-TLRs to TBT-Cl exposure. All the results indicated that To-TLRs might involve in sensing and mediating innate immune responses caused by TBT-Cl for keeping detoxification homeostasis.

Impact of nitrite exposure on plasma biochemical parameters and immune-related responses in Takifugu rubripes.

Nitrite is a major environmental pollutant in aquatic environments that negatively affects aquatic species. In this study, we investigated the impact of nitrite exposure on plasma biochemical parameters and immune responses in Takifugu rubripes. Fish were exposed to various concentrations of nitrite (0, 0.5, 1, 3, and 6 mM) for 96 h. After 0, 12, 24, 48, and 96 h of exposure, fish blood samples were collected to assay the levels of total protein (TP), albumin (Alb), glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (ALT), complement C3 (C3), complement C4 (C4), immunoglobulin (IgM), and lysozyme activity (LZM). The gills were sampled to analyze the mRNA levels of heat shock protein 70 (hsp70), heat shock protein 90 (hsp90), tumor necrosis factor α (tnf-α), B-cell activating factor (baff), interleukin-6 (il-6), and interleukin-12 (il-12). Levels of GOT, ALT, C3, and C4 were significantly enhanced in the high nitrite concentration group (3 and 6 mM), whereas those of TP, Alb, LZM, and IgM decreased significantly with the same treatments. Nitrite significantly upregulated hsp70, hsp90, tnf-α, il-6, il-12, and baff mRNA levels after 96 h of exposure. These results indicated that nitrite exposure altered the blood physiological status and immune system response, resulting in dysfunction and immunotoxicity in T. rubripes. Furthermore, our results reveal the possible mechanism of aquatic-nitrite-induced toxicity in fish.

Effects of dietary monocalcium phosphate supplementation on the anti-oxidative capacity, anti-bacteria function in immune organs of obscure puffer (Takifugu obscurus) after infection with Aeromonas hydrophila.

The purpose of the present study was to explore the impaired anti-bacteria ability in immune organs and immune systems of obscure puffer induced by chronic dietary phosphorus (P) deficiency. Fish were fed diets supplemented with 6 g/kg P (P6) and 0 g/kg P (P0) respectively for 15 weeks, and lower final body weight, feed intake, weight gain, whole body P content and bone P content were observed in fish fed P0 diet (P < 0.05). Then the fish were continued to feed for 3 weeks and intraperitoneal injection with PBS (P6+PBS) and Aeromonas hydrophila (A.hydrophila) (P6 + A.hydrophila and P0 + A.hydrophila), and sampled at 3, 6, 12 and 24 h. The results showed that dietary P deficiency lowered survival rate, total hemocyte count, whereas enhanced ROS production and apoptosis rate of obscure puffer compared to the 6 g/kg P supplemented group after infection. Moreover, compared to the P sufficient group, puffer fish fed P deficient diet decreased the expressions of antioxidant genes catalase (cat) and glutathione reductase (gr), immune-related genes toll-like receptor 2 (tlr-2) and anti-inflammatory factors transforming growth factor ß1 (tgf-ß1) and interleukin 11 (il-11) while increased pro-inflammatory cytokines tumor necrosis factor α (tnf-α), interleukin 1ß (il-1ß) and interleukin 8 (il-8) in head kidney post-infection. In addition, dietary P deficiency decreased the hepatic gene expressions of anti-apoptotic factor B-cell lymphoma 2 (bcl-2) and bax-inhibitor 1 (bi-1), accompanied by increasing the mRNA expressions of pro-apoptotic factor caspase 3, caspase 8 and caspase 9 compared to the P sufficient group after A.hydrophila infection. In conclusion, dietary P deficiency impaired the anti-bacteria function of the immune system as well as immune organs by increasing oxidative stress and aggravating the inflammatory response and apoptosis in obscure puffer under the A.hydrophila challenge.

Fugu, Takifugu ruberipes, mucus keratins act as defense molecules against fungi.

Keratin is a cytoskeletal protein that constitutes the intermediate filament. Its distribution is restricted to epithelial tissues in mammals, but is wider in fish. An interesting feature of fish keratin is that it is abundant in the cutaneous mucus. However, the biological function of keratin in the mucus has not been explored. In the present study, we hypothesized that mucus keratins of fugu Takifugu rubripes function as antimicrobial molecules. To verify this hypothesis, we first identified all of the keratins expressed in the epidermis and present in mucus. Five of 15 keratins including Tr-K4 expressed in the epidermis were identified in the mucus. Subsequently, we examined the interaction of keratin molecules present in fugu mucus with yeast. Affinity chromatography using yeast as a carrier and subsequent LC-MS/MS analysis revealed that three types of keratin were bound to the yeast. Furthermore, yeast incubated with fugu mucus was agglutinated, and this was inhibited by anti-recombinant Tr-K4 (rTr-K4) antibody. Immunohistochemical analysis also revealed that keratin was attached to the surface of agglutinated yeasts. These findings indicate that mucus keratin agglutinates yeast. Furthermore, we found insoluble clumps in fugu mucus, which were mainly comprised of keratin. After incubation of yeast with soluble mucus fraction, insoluble clumps incorporating yeast were formed. This observation suggests that fugu mucus keratin sequesters microbes into insoluble clumps, which are eventually eliminated from the mucus. Here, we present our finding of the novel function of keratin as a defense molecule in fish mucus.

Biochemical and molecular identification of a novel hepcidin type 2-like antimicrobial peptide in the skin mucus of the pufferfish Takifugu pardalis.

Fish skin mucus is considered to act as the first line of defense against waterborne pathogens and to be potential source of novel antimicrobial components. Here we report the purification and characterization of a novel hepcidin type 2-like antimicrobial peptide (TpHAMP2) from the skin mucus of the pufferfish Takifugu pardalis. The purified TpHAMP2 comprised of 23 amino acids (AAs) with eight Cys residues that form four intramolecular disulfide bonds. The TpHAMP2 gene shared overall structural characteristics with all known hepcidins, which have a tripartite exon-intron gene organization and three structural signatures in the precursor protein. Phylogenetically, TpHAMP2 was classified as HAMP2 class in acanthopterygian fish. Interestingly, the AA sequence of TpHAMP2 did not contain a proprotein cleavage site (RXXR motif) that conserved in most hepcidins and showed a highly positive charged (RKR-) short N-terminus and Val18 and Gly22 residues, which are distinctive structures compared to other known active hepcidins. Recombinant TpHAMP2 identical to the native form exhibited a broad spectrum and potent antimicrobial activity against tested gram-positive and -negative bacteria. Expression of TpHAMP2 mRNA was predominant in the liver and was upregulated in the liver, the spleen, the intestine, and the skin of T. pardalis post immune challenge. Thus, our findings suggests that TpHAMP2 might be of importance in the framework of discovering the fish hepcidins, especially type 2s, and provide noteworthy insight into its gene structure and expression and in the innate immunity as well as the mucosal immunity in regard to hepcidins' evolutionary history in fish species.

Alternations in oxidative stress, apoptosis, and innate-immune gene expression at mRNA levels in subadult tiger puffer (Takifugu rubripes) under two different rearing systems.

Tiger puffer (Takifugu rubripes) is one of the major aquaculture fish species in China due to its high economic value. In this study, the transcriptions of hepatic antioxidant enzyme, stress, apoptosis, and immune-related genes of sub-adult tiger puffers (Takifugu rubripes) were evaluated under two different rearing systems [offshore sea cage aquaculture system (OSCS) and recirculating aquaculture system (RAS)]. Results showed that the mRNA expression levels of the antioxidant enzyme (mn-sod, cu/zn-sod, gpx, and gr) and stress-related (hsp70 and hsp90) genes of male tiger puffers reared in the OSCS were significantly higher than female fish reared in the OSCS and fish reared in the RAS. The anti-apoptotic gene bcl2 exhibited the similar results. By contrast, the mRNAs of the pro-apoptotic genes (p53, caspase8, caspase9, and caspase3) of male tiger puffers reared in the OSCS were significantly lower than female fish reared in the OSCS and fish reared in the RAS. Male tiger puffers reared in the OSCS displayed significantly higher complement components (c3) and inflammatory cytokine (il-6) mRNAs, whereas B-cell activating factor (baf) and tumor necrosis factor α (tnf-α) mRNAs remained unchanged. Meanwhile, the mRNA levels of pro-apoptotic (bax, caspase8) and immunity-related (c3, il-6 and il-7) genes of female tiger puffers reared in the OSCS were significantly lower and higher than female fish reared in the RAS, respectively. In conclusion, the hepatic antioxidant, anti-apoptosis, and innate immunity of tiger puffers reared in the OSCS were better than fish in the RAS, male tiger puffer obtained the best values. These results expand the knowledge on the combined RAS and OSCS alternative aquaculture model for tiger puffers and aid in their management in captive.

Comparative transcriptomic analysis reveals the gene expression profiles in the liver and spleen of Japanese pufferfish (Takifugu rubripes) in response to Vibrio harveyi infection.

Japanese pufferfish (Takifugu rubripes) is one of the main marine aquatic fish species cultured in Asia due to its high nutritional value. In recent years, disease caused by Vibrio harveyi infections have led to serious mortality in Japanese pufferfish industry. To understand the complex molecular mechanisms between V. harveyi and Japanese pufferfish, we performed a transcriptome analysis of liver and spleen samples from Japanese pufferfish at 1 and 2 day post-infection. Between-group comparisons revealed 922 genes that were significantly differentially expressed. The altered genes emphasized the function in several immune related pathways including MAPK signaling pathway, JAK-STAT signaling pathway, toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and lysosomal pathway. The data generated in this study provided insight into the responses of Japanese pufferfish against V. harveyi at the transcriptome level, promoting our comprehensive understanding of immune responses for aquatic animal against V. harveyi.

The key differentially expressed genes and proteins related to immune response in the spleen of pufferfish (Takifugu obscurus) infected by Aeromonas hydrophila.

The immune mechanism elicited in pufferfish (Takifugu obscurus) against the invasion of Aeromonas hydrophila is still poorly understood. We examined the spleen of pufferfish at the transcriptome and proteome levels by using Illumina-seq and TMT coupled mass spectrometry after 12 h infection by A. hydrophila, respectively. A total of 2,339 genes (1,512 up-regulated and 827 down-regulated) and 537 (237 up-regulated and 300 down-regulated) proteins were identified. GO and KEGG analyses revealed that the responses to stimulus were the main biological processes, intestinal immune network for IgT production and calcium signaling pathway. Fourteen genes (8 up-regulated and 6 down-regulated) and proteins (5 up-regulated and 9 down-regulated) involved immune responses or signal transduction were validated by qRT-PCR and parallel reaction monitoring to confirm the reliability of the transcriptomic and proteomic analyses, respectively. Moreover, qRT-PCR and flow cytometry were used to detect dynamics of the genes in calcium signaling pathway and changes of concentration of cytoplasm Ca2+ in spleen cells within a 72 h challenge. This study provides the findings regarding immune response, especially intestinal immune network for IgT production pathway and calcium signaling pathway at the molecular, protein and cellular in pufferfish after infection by A. hydrophila. These results would provide a new insight and molecular targets into the response to pathogenic infection in pufferfish.

Individual and combined effects of salinity and lipopolysaccharides on the immune response of juvenile Takifugu fasciatus.

Lipopolysaccharides (LPS) and salinity are important variables in aquatic environments. High concentration of LPS and large changes in salinity seriously threat the survival of a variety of organisms, including fish. To reveal the effects of salinity and LPS on a fish immune response, we measured the immune-related parameters (total leukocyte count, total serum protein, albumin and globulin concentrations, complement C3 concentration, and lysozyme activity) and genes (the expressions of TNF-α, IL-1ß, and SOCS1-3 at the mRNA and protein levels) of juvenile Takifugu fasciatus exposed to phosphate buffered saline (PBS) or LPS (25 µg mL-1) under different salinities (0, 15, and 30 ppt) for 24 h. Changes in key immunological indicators suggested that the LPS challenge induced considerable damage to T. fasciatus, whereas an increase in salinity mitigated the harmful effects. Moreover, although the immune responses in blood and other selected tissues (gill and kidney) were suppressed with an increase in salinity, the increased response in liver in saltwater enabled T. fasciatus to conquer large salinity variation during migration. The appropriate addition of salts appeared to be a sensible strategy to mitigate LPS-induced toxicity in the aquaculture of T. fasciatus.