A bacterial binary toxin system that kills both insects and aquatic crustaceans: Photorhabdus insect-related toxins A and B.

Photorhabdus insect-related toxins A and B (PirA and PirB) were first recognized as insecticidal toxins from Photorhabdus luminescens. However, subsequent studies showed that their homologs from Vibrio parahaemolyticus also play critical roles in the pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimps. Based on the structural features of the PirA/PirB toxins, it was suggested that they might function in the same way as a Bacillus thuringiensis Cry pore-forming toxin. However, unlike Cry toxins, studies on the PirA/PirB toxins are still scarce, and their cytotoxic mechanism remains to be clarified. In this review, based on our studies of V. parahaemolyticus PirAvp/PirBvp, we summarize the current understanding of the gene locations, expression control, activation, and cytotoxic mechanism of this type of toxin. Given the important role these toxins play in aquatic disease and their potential use in pest control applications, we also suggest further topics for research. We hope the information presented here will be helpful for future PirA/PirB studies.

Shrimp SIRT4 promotes white spot syndrome virus replication.

In WSSV pathogenesis, the molecular mechanisms and the key host factors that regulate the viral replication and morphogenesis remain unclear. However, like most viruses, WSSV is known to induce metabolic reprogramming in several metabolic pathways including the host glutamine metabolism, and several recent reports have suggested that the sirtuins SIRT3, SIRT4, and SIRT5, which belong to a family of NAD+-dependent deacetylases, play an important role in this regulation. Here we focus on characterizing LvSIRT4 from Litopenaeus vannamei and investigate its role in regulating glutamine dehydrogenase (GDH), an important enzyme that promotes glutaminolysis and viral replication. We found that LvSIRT4 silencing led to significant decreases in both WSSV gene expression and the number of viral genome copies. Conversely, overexpression of LvSIRT4 led to significant increases in the expression of WSSV genes and the WSSV genome copy number. Immunostaining in Sf9 insect cells confirmed the presence of LvSIRT4 in the mitochondria and the co-localization of LvSIRT4 and LvGDH in the same cellular locations. In vivo gene silencing of LvSIRT4 significantly reduced the gene expression of LvGDH whereas LvSIRT4 overexpression had no effect. However, neither silencing nor overexpression had any effect on the protein expression levels of LvGDH. Lastly, although GDH activity in uninfected shrimp was unchanged, the GDH enzyme activity in WSSV-infected shrimp was significantly increased after both LvSIRT4 silencing and overexpression. This suggests that although there may be no direct regulation, LvSIRT4 might still be able to indirectly regulate LvGDH via the mediation of one or more WSSV proteins that have yet to be identified.

Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor.

Acute hepatopancreatic necrosis disease (AHPND) caused by PirABVP-producing strain of Vibrio parahaemolyticus, VPAHPND, has seriously impacted the shrimp production. Although the VPAHPND toxin is known as the VPAHPND virulence factor, a receptor that mediates its action has not been identified. An in-house transcriptome of Litopenaeus vannamei hemocytes allows us to identify two proteins from the aminopeptidase N family, LvAPN1 and LvAPN2, the proteins of which in insect are known to be receptors for Cry toxin. The membrane-bound APN, LvAPN1, was characterized to determine if it was a VPAHPND toxin receptor. The increased expression of LvAPN1 was found in hemocytes, stomach, and hepatopancreas after the shrimp were challenged with either VPAHPND or the partially purified VPAHPND toxin. LvAPN1 knockdown reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VPAHPND bacteria in the stomach after VPAHPND toxin challenge. In addition, LvAPN1 silencing prevented the toxin from causing severe damage to the hemocytes and sustained both the total hemocyte count (THC) and the percentage of living hemocytes. We found that the rLvAPN1 directly bound to both rPirAVP and rPirBVP toxins, supporting the notion that silencing of LvAPN1 prevented the VPAHPND toxin from passing through the cell membrane of hemocytes. We concluded that the LvAPN1 was involved in AHPND pathogenesis and acted as a VPAHPND toxin receptor mediating the toxin penetration into hemocytes. Besides, this was the first report on the toxic effect of VPAHPND toxin on hemocytes other than the known target tissues, hepatopancreas and stomach.

Resilience and probiotic interventions to prevent and recover from shrimp gut dysbiosis.

To counter the recurrent outbreaks of bacterial (acute hepatopancreatic necrosis disease; AHPND) and viral (white spot disease; WSD) shrimp diseases, which still remain a threat to the global industry, shrimp gut microbiota research has been gaining more attention in recent years, and the use of probiotics in aquaculture has had promising results in improving shrimp gut health and immunity. In this review based on our studies on AHPND and WSD, we summarize our current understanding of the shrimp gastrointestinal tract and the role of the microbiota in disease, as well as effects of probiotics. We focus particularly on the concept of microbiota resilience, and consider strategies that can be used to restore shrimp gut health by probiotic intervention at a crucial time during gut microbiota dysbiosis. Based on the available scientific evidence, we argue that the use of probiotics potentially has an important role in controlling disease in shrimp aquaculture.

Multiple Nucleocapsid Structural Forms of Shrimp White Spot Syndrome Virus Suggests a Novel Viral Morphogenetic Pathway.

White spot syndrome virus (WSSV) is a very large dsDNA virus. The accepted shape of the WSSV virion has been as ellipsoidal, with a tail-like extension. However, due to the scarcity of reliable references, the pathogenesis and morphogenesis of WSSV are not well understood. Here, we used transmission electron microscopy (TEM) and cryogenic electron microscopy (Cryo-EM) to address some knowledge gaps. We concluded that mature WSSV virions with a stout oval-like shape do not have tail-like extensions. Furthermore, there were two distinct ends in WSSV nucleocapsids: a portal cap and a closed base. A C14 symmetric structure of the WSSV nucleocapsid was also proposed, according to our Cryo-EM map. Immunoelectron microscopy (IEM) revealed that VP664 proteins, the main components of the 14 assembly units, form a ring-like architecture. Moreover, WSSV nucleocapsids were also observed to undergo unique helical dissociation. Based on these new results, we propose a novel morphogenetic pathway of WSSV.

Litopenaeus vannamei peritrophin interacts with WSSV and AHPND-causing V. parahaemolyticus to regulate disease pathogenesis.

Peritrophins are peritrophic membrane (PM) proteins that can interact with chitin fibers via chitin-binding domains. Peritrophins have essential roles in providing porosity and strength to the PM that lines the shrimp midgut. Acute hepatopancreatic necrosis disease (AHPND), caused by strains of V. parahaemolyticus, is known to initially colonize the shrimp stomach and simultaneously disrupt its structural barriers (e.g., cuticle or epithelial tissues) to reach the hepatopancreas. Although stomach and hepatopancreas were identified as target tissues involved in AHPND pathogenesis, our results indicated that peritrophin in peritrophic membrane has a crucial role in determining not only colonization of AHPND-causing bacteria but also their tissue distribution. As the interaction between LvPeritrophin (LvPT) and WSSV (white spot syndrome virus) is not well understood, we noted that LvPT expression was upregulated in shrimp stomach challenged with either WSSV or AHPND. In an in vitro pathogen binding assay, there was strong binding of recombinant LvPT WSSV and AHPND-causing V. parahaemolyticus, and various bacteria. Furthermore, dsRNA-mediated LvPT silencing inhibited WSSV gene expression and viral genome replication. However, downregulation of LvPT gene expression increased copies of AHPND-causing bacteria in shrimp digestive tract, and facilitated bacterial colonization in stomach. In conclusion, we speculated that LvPT might regulate bacterial colonization during AHPND, whereas in WSSV infection, LvPT silencing favored the host. Although recombinant LvPT had strong binding with WSSV, the precise role of LvPT in WSSV infection needs further investigation. These findings increased our understanding of host-pathogen interactions in AHPND and WSSV infection that can be applied in shrimp aquaculture for developing effective antibacterial and antiviral strategies.

White Spot Syndrome Virus Benefits from Endosomal Trafficking, Substantially Facilitated by a Valosin-Containing Protein, To Escape Autophagic Elimination and Propagate in the Crustacean Cherax quadricarinatus.

As the most severely lethal viral pathogen for crustaceans in both brackish water and freshwater, white spot syndrome virus (WSSV) has a mechanism of infection that remains largely unknown, which profoundly limits the control of WSSV disease. By using a hematopoietic tissue (Hpt) stem cell culture from the red claw crayfish Cherax quadricarinatus suitable for WSSV propagation in vitro, the intracellular trafficking of live WSSV, in which the acidic-pH-dependent endosomal environment was a prerequisite for WSSV fusion, was determined for the first time via live-cell imaging. When the acidic pH within the endosome was alkalized by chemicals, the intracellular WSSV virions were detained in dysfunctional endosomes, resulting in appreciable blocking of the viral infection. Furthermore, disrupted valosin-containing protein (C. quadricarinatus VCP [CqVCP]) activity resulted in considerable aggregation of endocytic WSSV virions in the disordered endosomes, which subsequently recruited autophagosomes, likely by binding to CqGABARAP via CqVCP, to eliminate the aggregated virions within the dysfunctional endosomes. Importantly, both autophagic sorting and the degradation of intracellular WSSV virions were clearly enhanced in Hpt cells with increased autophagic activity, demonstrating that autophagy played a defensive role against WSSV infection. Intriguingly, most of the endocytic WSSV virions were directed to the endosomal delivery system facilitated by CqVCP activity so that they avoided autophagy degradation and successfully delivered the viral genome into Hpt cell nuclei, which was followed by the propagation of progeny virions. These findings will benefit anti-WSSV target design against the most severe viral disease currently affecting farmed crustaceans.IMPORTANCE White spot disease is currently the most devastating viral disease in farmed crustaceans, such as shrimp and crayfish, and has resulted in a severe ecological problem for both brackish water and freshwater aquaculture areas worldwide. Efficient antiviral control of WSSV disease is still lacking due to our limited knowledge of its pathogenesis. Importantly, research on the WSSV infection mechanism is also quite meaningful for the elucidation of viral pathogenesis and virus-host coevolution, as WSSV is one of the largest animal viruses, in terms of genome size, that infects only crustaceans. Here, we found that most of the endocytic WSSV virions were directed to the endosomal delivery system, strongly facilitated by CqVCP, so that they avoided autophagic degradation and successfully delivered the viral genome into the Hpt cell nucleus for propagation. Our data point to a virus-sorting model that might also explain the escape of other enveloped DNA viruses.

Bile acid and bile acid transporters are involved in the pathogenesis of acute hepatopancreatic necrosis disease in white shrimp Litopenaeus vannamei.

Acute hepatopancreas necrosis disease is a recently emerged shrimp disease that is caused by virulent strains of Vibrio parahaemolyticus. Although AHPND poses a serious threat to the shrimp industry, particularly in Asia, its underlying pathogenic mechanisms are not well characterized. Since a previous transcriptomic study showed upregulation of the apical sodium bile acid transporter (LvASBT), our objective here was to explore the role of bile acids and bile acid transporters in AHPND infection. We confirmed that mRNA expression of LvASBT was upregulated in the stomach of AHPND-infected shrimps. Bile acid concentrations were also higher in the stomach of AHPND-infected shrimp and correlated with high expression of pVA plasmid and Pir toxins. In vitro assays showed that bile acids enhanced biofilm formation and increased the release of PirABvp toxins in AHPND-causing V. parahaemolyticus, while in vivo inhibition of LvASBT by GSK2330672 reduced the copy numbers of pVA plasmid, Pir toxin and reduced the amounts of bile acids in AHPND-infected shrimp stomach. Transcriptomics data for AHPND-causing V. parahaemolyticus treated with bile acids showed upregulation of various genes involved in membrane transport, RND efflux pumps and a bacterial secretion system. Taken together, our results show that AHPND-causing V. parahaemolyticus virulence is positively regulated by bile acids and that LvASBT and bile acids in shrimp stomach have important roles in AHPND pathogenesis.

Shrimp SIRT1 activates of the WSSV IE1 promoter independently of the NF-κB binding site.

Since the mechanisms by which cellular factors modulate replication of the shrimp viral pathogen white spot syndrome virus (WSSV) are still largely unknown, here we consider the sirtuins, a family of NAD+-dependent protein deacetylases that are known to function as regulatory factors that activate or suppress viral transcription and replication in mammals. In particular, we focus on SIRT1 by isolating and characterizing LvSIRT1 from white shrimp (Litopenaeus vannamei) and investigating its involvement in WSSV infection. DsRNA-mediated gene silencing led to the expression of WSSV genes and the replication of genomic DNAs being significantly decreased in LvSIRT1-silenced shrimp. The deacetylase activity of LvSIRT1 was significantly induced at the early stage (2 hpi) and the genome replication stage (12 hpi) of WSSV replication, but decreased at the late stage of WSSV replication (24 hpi). Treatment with the SIRT1 activator resveratrol further suggested that LvSIRT1 activation increased the expression of several WSSV genes (IE1, VP28 and ICP11). Lastly, we used transfection and dual luciferase assays in Sf9 insect cells to show that while the overexpression of LvSIRT1 facilitates the promoter activity of WSSV IE1, this enhancement of WSSV IE1 expression is achieved by a transactivation pathway that is NF-κB-independent.

ASC-deficiency impairs host defense against Aeromonas hydrophila infection in Japanese medaka, Oryzias latipes.

Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) is a component of inflammasome, which plays crucial roles in the inflammatory response. In mammals, ASC regulates caspase-1 activation, thereby inducing pyroptosis and producing activated inflammatory cytokines. In addition, ASC also interacts with receptor-interacting protein kinase 2 (RIPK2) and induces nuclear factor-κB (NF-κB) activation. However, the role of ASC remains poorly understood in fish. In this study, we focused on elucidating the role of ASC in fish that were infected with Aeromonas hydrophila using Japanese medaka (Oryzias latipes) as fish model, and ASC-knockout (KO) medaka was established using CRISPR-Cas9 system. ASC-KO and wild type (WT) medakas were infected with A. hydrophila, and mortality was observed. ASC-KO medaka demonstrated higher mortality than WT. Moreover, the expression of immune-related genes in the kidney and intestine of the ASC-KO and WT medakas challenged with A. hydrophila were analyzed. Following A. hydrophila infection, the kidney of ASC-KO medaka exhibited significantly lower expression of NF-κB regulated genes (e.g., IL-1ß, IL-6, IL-8 and TNF-α) and RIPK2 gene than in WT kidney. Moreover, to investigate the immune response against A. hydrophila via ASC in the medaka, bacterial burden, superoxide anion production, and lactate dehydrogenase release in the kidney cells of ASC-KO medaka were measured. After infection, these responses in ASC-KO medaka were significantly decreased compared to those in WT. These results suggest that the medaka ASC plays a critical role against A. hydrophila infection by inducing inflammatory responses and cell death for bacterial clearance.

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.

ICTV Virus Taxonomy Profile: Nimaviridae.

The family Nimaviridae includes the single species White spot syndrome virus, isolates of which infect a wide range of aquatic crustaceans and cause substantial economic losses. Virions are ellipsoid to bacilliform with a terminal thread-like extension. The circular dsDNA genome is 280-307 kbp with several homologous repeat regions. More than 80 structural and functional proteins have been characterized from 531 ORFs. White spot syndrome is a highly lethal, contagious disease associated with white spot syndrome virus infection of shrimps. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Nimaviridae, which is available at www.ictv.global/report/nimaviridae.

The Rho signalling pathway mediates the pathogenicity of AHPND-causing V. parahaemolyticus in shrimp.

An emerging bacterial disease, acute hepatopancreatic necrosis disease (AHPND), is caused by strains of Vibrio parahaemolyticus with an additional AHPND-associated plasmid pVA1 encoding a virulent toxin (Pirvp ) that damages the shrimp's hepatopancreas. Like other species of Vibrio, these virulent strains initially colonise the shrimp's stomach, but it is not yet understood how the bacteria or toxins are subsequently able to cross the epithelial barrier and reach the hepatopancreas. Here, by using transcriptomics and system biology methods, we investigate AHPND-induced changes in the stomach of AHPND-causing V. parahaemolyticus (5HP)-infected shrimp and identify host molecular mechanisms that might explain how the integrity of the stomach barrier is compromised. We found that the expression of 376 unique genes was differentially regulated by AHPND infection. Gene ontology, protein interaction, and gene-to-gene correlation expression interaction analyses indicated that in addition to the immune system, a number of these genes were involved in cytoskeleton regulation by Rho GTPase. The involvement of Rho pathway regulation during AHPND pathogenesis was further supported by experiments showing that while Rho inhibitor pretreatment delayed the infection, pretreatment with Rho activator enhanced the pathogenicity of 5HP, and both the bacteria and toxin were detected sooner in the hepatopancreas. Further, disruption of the stomach epithelial structure was found in both Rho preactivated shrimp and in 5HP-infected shrimp. Taken together, we interpret our results to mean that Rho signalling helps to mediate AHPND pathogenesis in shrimp.

LvRas and LvRap are both important for WSSV replication in Litopenaeus vannamei.

The white Spot Syndrome Virus (WSSV) is a pathogen that causes huge economic losses in the shrimp-farming industry globally. At the WSSV genome replication stage (12 hpi) in WSSV-infected shrimp hemocytes, activation of the PI3K-Akt-mTOR pathway triggers metabolic changes that resemble the Warburg effect. In shrimp, the upstream regulators of this pathway are still unknown, and in the present study, we isolate, characterize and investigate two candidate factors, i.e. the shrimp Ras GTPase isoforms LvRas and LvRap, both of which are upregulated after WSSV infection. dsRNA silencing experiments show that virus replication is significantly reduced when expression of either of these genes is suppressed. Pretreatment with the Ras inhibitor Salirasib further suggests that LvRas, which is a homolog to a commonly overexpressed human oncoprotein, may be involved in regulating the WSSV-induced Warburg effect. We also show that while both the PI3K-Akt-mTOR and Raf-MEK-ERK pathways are activated by WSSV infection, LvRas appears to be involved only in the regulation of the mTOR pathway.

Selective expression of a "correct cloud" of Dscam in crayfish survivors after second exposure to the same pathogen.

Arthropod hypervariable Dscam (Down syndrome cell adhesion molecule) may be involved in adaptive-like immune characteristics, namely immune priming, enabling the host to "learn" and "remember" pathogens previously encountered in arthropods. However, expression of Dscam in immune-primed arthropods after a second challenge has apparently not been confirmed. Herein, working with Dscam of Australian freshwater crayfish (Cherax quadricarinatus, i.e. CqDscam), we further investigated whether immune priming is mediated by "clouds" of appropriate (or "correct") CqDscam isoforms. In crayfish that survived a first WSSV challenge (immune priming), long-lasting CqDscam expression remained higher after a second WSSV challenge. Selective CqDscam isoforms were also induced after both challenges. Based on pathogen binding assays, these WSSV-induced CqDscam isoforms had a higher WSSV binding ability, perhaps mainly mediated by Ig3-spliced variants. We therefore hypothesized that in these crayfish survivors, an unknown selection process was generating a "correct cloud" of CqDscam against a previously encountered pathogen.

The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.

Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirA(vp) and PirB(vp)) proteins and found that the overall structural topology of PirA(vp)/PirB(vp) is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirAB(vp) heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirAB(vp) may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.

An invertebrate Warburg effect: a shrimp virus achieves successful replication by altering the host metabolome via the PI3K-Akt-mTOR pathway.

In this study, we used a systems biology approach to investigate changes in the proteome and metabolome of shrimp hemocytes infected by the invertebrate virus WSSV (white spot syndrome virus) at the viral genome replication stage (12 hpi) and the late stage (24 hpi). At 12 hpi, but not at 24 hpi, there was significant up-regulation of the markers of several metabolic pathways associated with the vertebrate Warburg effect (or aerobic glycolysis), including glycolysis, the pentose phosphate pathway, nucleotide biosynthesis, glutaminolysis and amino acid biosynthesis. We show that the PI3K-Akt-mTOR pathway was of central importance in triggering this WSSV-induced Warburg effect. Although dsRNA silencing of the mTORC1 activator Rheb had only a relatively minor impact on WSSV replication, in vivo chemical inhibition of Akt, mTORC1 and mTORC2 suppressed the WSSV-induced Warburg effect and reduced both WSSV gene expression and viral genome replication. When the Warburg effect was suppressed by pretreatment with the mTOR inhibitor Torin 1, even the subsequent up-regulation of the TCA cycle was insufficient to satisfy the virus's requirements for energy and macromolecular precursors. The WSSV-induced Warburg effect therefore appears to be essential for successful viral replication.

Expression and biological activity of two types of interferon genes in medaka (Oryzias latipes).

Type I interferon (IFN) is one of most important cytokines for antiviral responses in fish innate immunity, after the induction pathway following pattern recognition. In this study, 2 types of type I IFN mRNA from a medaka (Japanese rice fish; Oryzias latipes) were identified and classified (phylogenetic analysis) into subgroup-a and -d by (designated olIFNa and olIFNd, respectively). Both olIFNa and olIFNd (encoding 197 and 187 amino acid residues, respectively) contained 2 cysteines. Gene expression pattern of olIFNa, olIFNd and IFN-stimulated genes (ISGs) was assessed (quantitative real-time reverse transcriptase PCR, qRT-PCR) in various organs (i.e., whole kidney, liver and spleen) of medaka stimulated by polyI:C or infected with nervous necrosis virus (NNV). Expression of olIFNa, olIFNd and ISGs, especially the ISG15 gene, were significantly upregulated after NNV-infection. Furthermore, olIFNa, olIFNd and ISGs mRNAs were sufficiently induced in DIT cells (i.e., medaka hepatoma cell line) transfected with polyI:C or infected with NNV. In addition, in vitro biological activities of recombinant olIFNa and olIFNd (rolIFNa and rolIFNd) produced by mammalian cell line HEK293T were also characterized. Expression of GIG1a and ISG15 genes in kidney cells of adult medaka were induced by rolIFNa or rolIFNd. The olIFNs-overexpressing DIT cells had reduced viral titers following NNV infection. Therefore, we inferred that 2 type I IFNs were involved in innate immunity (antiviral response) in medaka fish.

TALENs-mediated gene disruption of myostatin produces a larger phenotype of medaka with an apparently compromised immune system.

Although myostatin, a suppressor of skeletal muscle development and growth, has been well studied in mammals, its function in fish remains unclear. In this study, we used a popular genome editing tool with high efficiency and target specificity (TALENs; transcription activator-like effector nucleases) to mutate the genome sequence of myostatin (MSTN) in medaka (Oryzias latipes). After the TALEN pair targeting OlMyostatin was injected into fertilized medaka eggs, mutant G0 fish carrying different TALENs-induced frameshifts in the OlMSTN coding sequence were mated together in order to transmit the mutant sequences to the F1 generation. Two F1 mutants with frameshifted myostatin alleles were then mated to produce the F2 generation, and these F2 OlMSTN null (MSTN(-/-)) medaka were evaluated for growth performance. The F2 fish showed significantly increased body length and weight compared to the wild type fish at the juvenile and post-juvenile stages. At the post-juvenile stage, the average body weight of the MSTN(-/-) medaka was ∼25% greater than the wild type. However, we also found that when the F3 generation were challenged with red spotted grouper nervous necrosis virus (RGNNV), the expression levels of the interferon-stimulated genes were lower than in the wild type, and the virus copy number was maintained at a high level. We therefore conclude that although the MSTN(-/-) medaka had a larger phenotype, their immune system appeared to be at least partially suppressed or undeveloped.