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The structures of fish serum immunoglobulin differ among different fish species. In this study, we accidently isolated a rabbit immunoglobulin (Ig) light chain bound to serum immunoglobulin from different marine fish species using phage display. Fish Ig was separated using a protein A column.The phage library was generated from variable regions of rabbit spleen B cells immunized with bluefin tuna Thunnus orientalis Ig. Fish Ig–specific phages were enriched using two rounds of bio-panning with yellowtail Seriola quinqueradiata serum Ig, followed by two rounds of bio-panning with red seabream Pagrus major serum Ig.The enriched phages demonstrated an increase in binding specificity to the tuna, yellowtail, and red seabream Igs compared to the phages listed in the unpanned library.A recombinant protein of a single clonal phage, which encodes the rabbit Ig light chain, was produced, and the binding specificities to fish Igs were analyzed using enzyme-linked immunosorbent assay (ELISA) and western blotting.The recombinant protein exhibited binding properties to fish Igs in the ELISA. However, the recombinant protein that bound to serum protein(s), but not IgM, was detected via western blotting.The recombinant protein may provide a novel information on the common structural feature in the fish immunoglobulins., 公開日: 2023-07-21

Red sea bream iridovirus (RSIV) is the pathogen that causes red sea bream iridoviral disease. It causes a huge loss to the Japanese aquaculture industry. In 2021, outbreaks of red sea bream iridovirus occurred in South Japan. This study analysed nine whole-genome sequences of RSIV isolated in Oita and Ehime Prefectures in 2021 using a short-read next-generation sequencer. Nine isolates had highly uniform sequences, and there was no variant depending on locations or host species. Phylogenetic analyses with other reported megalocytivirus isolates showed that RSIV isolated in 2021 was genetically different from RSIV previously isolated in Oita and Ehime Prefectures in 2017-2019. These results suggest that RSIV isolated in Oita and Ehime Prefectures in 2021 might spread from a common ancestor different from the recent one. Additionally, it was found that RSIV isolated in 2021 had sequence mutations on protein-coding sequences that may be involved in viral pathogenicity and infectivity.

Virus like particles (VLPs) are non-infectious nanoparticles containing repetitive, high density viral epitopes on the surface and can prevent viral infections in aquatic animals. Here, we evaluated the immuno-stimulation effect of infectious hypodermal and hematopoietic necrosis virus like particle (IHHNV-VLP) using a next generation sequencing in Fenneropenaeus merguiensis to identify the important immune-related genes that may prevent viral infection. The in situ target of IHHNV was predominantly found in gill tissue following IHHNV-VLP administration in juvenile shrimp. Comparative transcriptome analysis in the injected gills showed that there were 326 unigenes expressed differently than the mock-injected samples. One of the most differential genes between the two animal groups was the antioxidative gene, peroxiredoxin (FmPrx), that was up-regulated after 6 h post-VLP injection. Phylogenetic tree analysis showed that this gene could be found among many shrimp species and was closely clustered among Prx families. The expression of FmPrx was also detected in all tissues examined, thus suggesting the multi-functional roles of this gene in many tissues. Administration of IHHNV-VLP in vivo led to a significant increase in peroxidase activity in gill tissue-approximately two-fold versus control animals; the WSSV copy number was significantly reduced. These data suggest that IHHNV-VLP exerts an immune-stimulating effect by enhancing the level of immune-related genes including FmPrx and its corresponding peroxidase activity, which are a well-known part of the shrimp innate immune system.

Red sea bream iridovirus (RSIV) belonging to the genus Megalocytivirus of the family Iridoviridae is the cause of serious mass mortality of cultured marine fishes. RSIV-type megalocytiviruses show extremely high nucleotide sequence identities. Thus, epidemiological studies on this virus are limited. This study developed two primer sets amplifying the regions possessing single nucleotide polymorphism (SNP) to determine the relationships and divergence of RSIV-type megalocytiviruses isolated from cultured marine fishes in Japan. The two regions were designed according to the genome sequences of the representative RSIV genotype II of megalocytivirus members in GenBank. The SNP 1 and 2 regions have sequences homologous to hypothetical protein ORF 24 and ORF 31, respectively, of RSIV (accession no. AP017456.1). By sequencing the regions, 53 polymorphic sites were identified. The phylogenetic analysis of 25 RSIV-type megalocytivirus isolates, classified into RSIV cluster, was clustered into eight haplotypes (seven haplotypes from Oita, two haplotypes from Ehime, and one haplotype shared between Oita and Ehime). These findings suggested that SNP in the RSIV genome is a powerful application for the detection and identification of RSIV-type megalocytiviruses.

Streptococcus agalactiae is a causative agent of streptococcosis disease in various fish species, including Nile tilapia (Oreochromis niloticus Linn.). Vaccination is an effective disease prevention and control method, but limitations remain for protecting against catastrophic mortality of fish infected with different strains of streptococci. Immunoproteomics analysis of S. agalactiae was used to identify antigenic proteins and construct a chimeric multiepitope vaccine. Epitopes from five antigenic proteins were shuffled in five helices of a flavodoxin backbone, and in silico analysis predicted a suitable RNA and protein structure for protein expression. 45F2 and 42E2 were identified as the best candidates for a chimeric multiepitope vaccine. Recombinant plasmids were constructed to produce a recombinant protein vaccine and DNA vaccine system. Overexpressed proteins were determined to be 30 kDa and 25 kDa in the E. coli and TK1 systems, respectively. The efficacy of the chimeric multiepitope construct as a recombinant protein vaccine and DNA vaccine was evaluated in Nile tilapia, followed by S. agalactiae challenge at 1 × 107 CFU/mL. Relative percentage survival (RPS) and cumulative mortality were recorded at approximately 57–76% and 17–30%, respectively. These chimeric multiepitope vaccines should be applied in streptococcosis disease control and developed into a multivalent vaccine to control multiple diseases.

The genomes of Streptococcus agalactiae (group B streptococcus; GBS) collected from diseased fish in Thailand and Vietnam over a nine-year period (2008–2016) were sequenced and compared (n = 21). Based on capsular serotype and multilocus sequence typing (MLST), GBS isolates are divided into 2 groups comprised of i) serotype Ia; sequence type (ST)7 and ii) serotype III; ST283. Population structure inferred by core genome (cg)MLST and Bayesian clustering analysis also strongly indicated distribution of two GBS populations in both Thailand and Vietnam. Deep phylogenetic analysis implied by CRISPR array's spacer diversity was able to cluster GBS isolates according to their temporal and geographic origins, though ST7 has varying CRISPR1-spacer profiles when compared to ST283 strains. Based on overall genotypic features, Thai ST283 strains were closely related to the Singaporean ST283 strain causing foodborne illness in humans in 2015, thus, signifying zoonotic potential of this GBS population in the country.

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin-producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA-like (rPirA) and PirB-like (rPirB) toxins. Whole-egg powders having IgY specific to rPirA (anti-PirA-IgY) and rPirB (anti-PirB-IgY) and IgY from non-immunized hen (control-IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3 days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti-PirA-IgY, anti-PirB-IgY and control-IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti-PirA-IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti-PirA-IgY (87%) compared with the control (12%). These results confirm that addition of anti-PirA-IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.

Fc receptors (FcRs) are specific to the Fc portion of immunoglobulin (Ig) molecules. Here, four Fc receptor-like proteins, JF-FcR-like protein 1-4, were identified in Japanese flounder. Their open reading frames encoded 358, 255, 519 and 441 amino acid residues, respectively. JF-FcR-like protein mRNAs were mainly detected in kidney and spleen of healthy fish. Injection of formalin-killed cells (FKCs) of Edwardsiella tarda significantly increased the spleen mRNA levels of JF-FcR-like protein 1 but not the other JF-FcR-like proteins. Injection of FKC of Streptococcus iniae did not significantly affect any of the JF-FcR-like protein mRNAs. These findings suggest that the FcR-like proteins have different involvements in pathogen responses.

We examined lysozyme activities in the serum and the leukocyte extracts of the banded houndshark Triakis scyllium.The serum exhibited lytic activity, but not the leukocyte extracts.The lytic substance in the serum was of approximately 14 kDa and the N-terminal amino acid sequence was YVYSK.cDNA cloning identified a C-type lysozyme (TsLysC) gene and two G-type lysozyme (TsLysG) cDNA clones of different lengths.The TsLysC gene encodes 149 amino acids residues, and the sequence derived from the N-terminal amino acid sequencing was displayed at position 17–21. TsLysG, on the other hand, contains two ORFs that are homologous to the N- and C-terminal regions of G-type lysozyme of other fish species.TsLysC mRNA levels were high in the liver. TsLysG mRNA level was significantly lower than TsLysC mRNA in the liver., 公開日: 2023-06-04

The white spot syndrome virus (WSSV) has been considered a serious threat to shrimp aquaculture. Besides, the activation of cell metabolism as an immune reaction to the virus is now recognized as a piece of the pivotal puzzle of the antiviral responses. Hence, this study explores the relationship between metabolic gene expression and antiviral responses in shrimp using transcriptome analysis. The RNA-seq libraries of Fenneropenaeus merguensis hemocytes after WSSV challenge at early (6 hpi) and late (24 hpi) stages of infection were analyzed to identify differentially expressed genes (DEGs) that the WSSV subverted the expression. One-hundred-thirty-three DEGs that were expressed in response to WSSV infection at both stages were identified. Based on the GO annotation, they were related to innate immunity and metabolic pathway. The expression correlation between "full term" (NGS) and qRT-PCR of 16 representative DEGs is shown. Noticeably, the expression profiles of all the selected metabolic genes involved in glucose metabolism, lipid metabolism, amino acid metabolism, and nucleotide metabolism showed a specific correlation between NGS and qRT-PCR upon WSSV infection. Of these, we further characterized the function related to the WSSV response of glutamine: fructose-6-phosphate aminotransferase (FmGFAT), the rate-limiting enzyme of the hexosamine biosynthesis pathway, which was found to be up-regulated at the late stage of WSSV infection. Suppression of FmGFAT by RNA interference resulted in postponing the death of WSSV-infected shrimp and reduction of viral copy number. These results suggested that the FmGFAT is linked between metabolic change and WSSV responses in shrimp, where the virus-induced metabolic rewiring hijack biological compounds and/or energy sources to benefit the viral replication process.

The kuruma shrimp Marsupenaeus japonicus (order Decapoda, family Penaeidae) is an economically important crustacean that occurs in shallow, warm seas across the Indo-Pacific. Here, using a combination of Illumina and Oxford Nanopore Technologies platforms, we produced a draft genome assembly of M. japonicus (1.70 Gbp; 18,210 scaffolds; scaffold N50 = 234.9 kbp; 34.38% GC, 93.4% BUSCO completeness) and a complete mitochondrial genome sequence (15,969 bp). As with other penaeid shrimp genomes, the M. japonicus genome is extremely rich in simple repeats, which occupies 27.4% of the assembly. A total of 26,381 protein-coding gene models (94.7% BUSCO completeness) were predicted, of which 18,005 genes (68.2%) were assigned functional description by at least one method. We also produced an Illumina-based transcriptome shotgun assembly (40,991 entries; 93.0% BUSCO completeness) and a PacBio Iso-Seq transcriptome assembly (25,415 entries; 67.5% BUSCO completeness). We envision that the M. japonicus genome and transcriptome assemblies will serve as useful resources for the basic research, fisheries management, and breeding programs of M. japonicus.

Antibodies of cartilaginous fish are of scientific interest due to their phylogenetic position. In the present study, we developed antiserum against IgM of the banded houndshark, Triakis scyllium, and characterized binding activity of the IgM against fish pathogenic bacteria.Pentameric and monomeric IgM antibodies were separated by gel filtration chromatography using high performance liquid chromatography and SDS–PAGE. Antisera were developed by immunizing rabbits with unfractionated IgM antibodies separated by SDS–PAGE electrophoresis.Shark serum antibodies were found to have binding affinity for Aeromonas hydrophila, Vibrio anguillarum, Edwardsiella tarda, and Pseudomonas plecoglossicida antigens but not Lactococcus garvieae by enzyme-linked immunosorbent assay.We speculate the binding activities of shark antibodies may confer protection against certain bacterial pathogens., 公開日: 2022-07-08

In mammals, the intestine harbors numerous bacteria that play an important role in health. Intestinal microbiota have also been thought to be an important factor in the health of shrimp. However, the barrier systems of the digestive tracts of shrimp seem to be different from those of mammals. In this study, we analyzed the bacterial composition in the stomach and midgut of two species of shrimp during administration of a probiotic, Bacillus amyloliquefaciens strain TOA5001 by analysis of 16S rRNA genes with Illumina sequencing technology. Whiteleg shrimp Litopenaeus vannamei were observed under laboratory conditions and kuruma shrimp Marsupenaeus japonicus were observed in an aquaculture farm. The diversities of bacteria in the stomachs of both shrimps were significantly higher than those in the midgut. Also, the microbiota changed during probiotic feeding. Feeding whiteleg shrimp the probiotic after being challenged with an acute hepatopancreatic necrosis disease (AHPND)-causing strain of Vibrio parahaemolyticus increased their survival compared to the control group, which suggested that the probiotic prevented AHPND. These results appear to show that a probiotic can affect the microbiota throughout digestive tract of penaeid shrimps and that probiotic can have a role in preventing disease.

In this study, we isolated and characterized natural antibodies found in serum samples from Bester sturgeon (Huso huso × Acipenser ruthenus). Natural antibodies specifically detected hen egg lysozyme (HEL), keyhole limpet hemocyanin (KLH), and several species of pathogenic bacteria. Interestingly, we detected no antibodies with similar specificity in serum samples from rainbow trout (Oncorhynchus mykiss) or from Japanese flounder (Paralichthys olivaceus). Binding capacity of the sturgeon natural serum antibodies increased slightly at 7 months compared to 3 months after hatching. Antigen-specific antibodies against KLH, Aeromonas hydrophila and Streptococcus iniae were affinity-fractionated from naive sera of Bester sturgeon; specific detection of the corresponding antigens was observed. We conclude that Bester sturgeon are capable of generating unique natural antibodies including those that are pathogen-specific.

We evaluated the effects of Freund's adjuvants (FCA/FIA) on protection and immune response of Japanese flounder Paralichthys olivaceus immunized by the formalin-killed cell (FKC) of Edwardsiella tarda. Combination of FKC and FCA/FIA did not confer protection against the challenge, while they significantly induced higher antibody titers than that of FKC only. The suppression of FKC-dependent induction of interferon γ (IFNγ) mRNA levels by FCA/FIA was observed by gene expression profiling. Similarly, interleukin (IL)-12 p35 mRNA levels were not detected after FKC+FCA or +FIA. These results suggest that the mineral oil in Freund's adjuvants might suppress the signaling pathway(s) that induce IFNγ and IL-12 gene expression.

Crustins are well known cysteine-rich cationic antimicrobial peptides (AMPs) in crustaceans that have WFDC [WAP (whey acidic protein) four-disulfide core] domain at the carboxyl terminus. Proteins containing a WFDC domain have been discovered in many invertebrates and vertebrates. Although, there have been many WFDC domain containing nucleotide sequences found in NCBI GenBank database, their distinct sequential characteristics and their role in the innate immune system is not well understood. Here, we identified a new crustin isoform from Marsupenaeus japonicus by transcriptome analysis. The full-length cDNA of this isoform (MjCRS7) consists of 537 bp that include a 489 bp open reading frame (ORF) encoding 162 deduced amino acids (aa). The sequence contains the eight conserved cysteine residues characteristic of the WFDC domain. A phylogenetic analysis showed that MjCRS7 is a type II crustin. We also identified the full-length cDNA of a M. japonicus MjWFDC-like gene. MjWFDC-like has a 543 bp ORF encoding 180 aa. In an RT-PCR analysis, MjCRS7 and MjWFDC-like transcripts were mainly detected in gill tissue. An alignment of MjCRS7 and MjWFDC-like with previously reported M. japonicus crustin isoform 1–5 (MjCRS1–5) showed variation in the WFDC-like domain. Neither of the genes was responsive to Vibrio parahaemolyticus, Vibrio penaeicida or white spot syndrome virus (WSSV) either by immersion or injection challenge test. Although crustins are mainly antimicrobial peptides, the present results suggest that MjCRS7 may have other roles in M. japonicus.

Crustins are diverse group of antimicrobial peptides (AMPs) that have numerous isoforms mainly identified from hemocytes in decapods crustacean. However, little is known about its presence solely in gills tissue. In this study, we found two new crustin isoforms MjCRS8 and MjCRS9 by using transcriptome analysis from gills. Open reading frame of MjCRS8 and MjCRS9 were 593 bp and 459 bp encoding 197aa and 152aa, respectively. Tissue distribution analysis indicated that both MjCRS8 and MjCRS9 are expressed only in gills tissue. Multiple sequence alignment and phylogenetic analysis with previously reported crustin suggested that both MjCRS8 and MjCRS9 belong to type 2 crustin family. Experimental infection was conducted against Vibrio parahaemolyticus and white spot syndrome virus (WSSV) by immersion test. However, no significant upregulation was observed.

A viral responsive protein (MjVRP) was characterized from Marsupenaeus japonicus haemocytes. In amino acid homology and phylogenetic tree analyses, MjVRP clustered in the same group with the viral responsive protein of Penaeus monodon (PmVRP15), showing 34% identity. MjVRP transcripts were mainly expressed in haemocytes and the lymphoid organ. Western blotting likewise showed that MjVRP was strongly expressed in haemocytes and the lymphoid organ. Immunostaining detected MjVRP within the cytosol next to the perinuclear region in some haemocytes. Experimental challenge with white spot syndrome virus (WSSV) significantly up-regulated the mRNA level of MjVRP in the M. japonicus haemocytes at 6 and 48 h. Flow cytometry and indirect immunofluorescence assays revealed that the ratio of MjVRP+ haemocytes significantly increased 24 and 48 h post-WSSV infection. These results suggest that MjVRP+ haemocytes have a supporting role in the pathogenesis of WSSV.

Antibodies are widely considered to be essential tools for detection of immune responses in various fish species. Here we produced the peptide polyclonal antisera (anti-fish IgH-1 and anti-fish IgH-2) to detect IgM of various fish species. The peptides were designed based on the conserved sequence of the fish immunoglobulin heavy chains of seven fish species (Japanese flounder, seabream, yellowtail, carp, rainbow trout, hybrid sturgeon and banded houndshark). By Western blotting, anti-fish IgH-1 antiserum detected the IgMs of all fish species except banded houndshark. Anti-fish IgH-2 antiserum clearly reacted with the IgMs of only three of the fish species (seabream, yellowtail and rainbow trout). Attempts to use the antisera to measure fish antibody titer by ELISA were unsuccessful. These results demonstrate that anti-fish IgH-1 peptide polyclonal antiserum is a potentially applicable tool for detecting immunoglobulins in various fish species by Western blotting.

Flavobacterium columnare is one of the deadliest fish pathogens causing devastating mortality in various freshwater fish species globally. To gain an insight into bacterial genomic contents and structures, comparative genome analyses were performed using the reference and newly sequenced genomes of F. columnare including genomovar I, II and I/II strains isolated from Thailand, Europe and the USA. Bacterial genomes varied in size from 3.09 to 3.39Mb (2714 to 3101 CDSs). The pan-genome analysis revealed open pan-genome nature of F. columnare strains, which possessed at least 4953 genes and tended to increase progressively with the addition of a new genome. Genomic islands (GIs) present in bacterial genomes were diverse, in which 65% (39 out of 60) of possible GIs were strain-specific. A CRISPR/cas investigation indicated at least two different CRISPR systems with varied spacer profiles. On the other hand, putative virulence genes, including those related to gliding motility, type IX secretion system (T9SS), outer membrane proteins (Omp), were equally distributed among F. columnare strains. The MLSA scheme categorized bacterial strains into nine different sequence types (ST 9-17). Phylogenetic analyses based on either 16S rRNA, MLSA and concatenated SNPs of core genome revealed the diversity of F. columnare strains. DNA homology analysis indicated that the estimated digital DNA-DNA hybridization (dDDH) between strains of genomovar I and II can be as low as 42.6%, while the three uniquely tilapia-originated strains from Thailand (1214, NK01 and 1215) were clearly dissimilar to other F. columnare strains as the dDDH values were only 27.7-30.4%. Collectively, this extensive diversity among bacterial strains suggested that species designation of F. columnare would potentially require re-emendation.

Two novel type I interferon genes (JfIFN3 and JfIFN4) have been identified in Japanese flounder Paralichthys olivaceus. Open reading frames of JfIFN3 and JfIFN4 were 555bp and 528bp, encoding 184aa and 175aa, respectively. The genomic structures of JfIFN3 and JfIFN4 are composed of 5 exons and 4 introns. JfIFN4 has 2 conserved cysteine residues, while JfIFN3 has 4. JfIFN3 and JfIFN4 showed the highest amino acid sequence identities to turbot IFN1 (74%) and IFN2 (62%), respectively. Interestingly, JfIFN3 and JfIFN4 were clustered in distinct branches with JfIFN1 and JfIFN2, which have reported so far. The mRNA levels of JfIFN4 were apparently increased in the kidney and spleen at 3 h after ployI:C injection, while JfIFN1-3 were not detected by RT-PCR.

Aims This study aimed to biotype Streptococcus agalactiae isolated from tilapia farms in Thailand based on molecular biotyping methods and to determine the correlation between the serotype and virulence of bacteria. In addition to a biotyping (serotyping) technique based on multiplex PCR of cps genes, in this study, we developed multiplex PCR typing of Group B streptococcus (GBS) virulence genes to examine three clusters of virulence genes and their correlation with the pathogenicity of S. agalactiae. The epidemiology of S. agalactiae in Thailand was analysed to provide bacterial genetic information towards a future rational vaccine strategy for tilapia culture systems. Methods and Results Streptococcus agalactiae were isolated from diseased tilapia from different areas of Thailand. A total of 124 S. agalactiae isolates were identified by phenotypic analysis and confirmed by 16S rRNA PCR. Bacterial genotyping was conducted based on (i) molecular serotyping of the capsular polysaccharide (cps) gene cluster and (ii) virulence gene profiling using multiplex PCR analysis of 14 virulence genes (lmb, scpB, pavA, cspA, spb1, cyl, bca, rib, fbsA, fbsB, cfb, hylB, bac and pbp1A/ponA). Only serotypes Ia and III were found in this study; serotype Ia lacks the lmb, scpB and spb1 genes, whereas serotype III lacks only the bac gene. Virulence tests in juvenile Nile tilapia demonstrated a correlation between the pathogenicity of the bacteria and their virulence gene profile, with serotype III showing higher virulence than serotype Ia. Epidemiological analysis showed an almost equal distribution in all regions of Thailand, except serotype III was found predominantly in the southern areas. Conclusions Only two serotypes of S. agalactiae were isolated from diseased tilapia in Thailand. Serotype Ia showed fewer virulence genes and lower virulence than serotype III. Both serotypes showed a similar distribution throughout Thailand. Significance and Impact of the Study We identified two major serotypes of S. agalactiae isolates associated with the outbreak in tilapia culture in Thailand. We developed multiplex PCR assays for 14 virulence genes, which may be used to predict the pathogenicity of the isolates and track future infections. Multiplex PCR typing of the GBS virulence genes was developed and might be further used to predict the pathogenicity of S. agalactiae.

Neobenedenia melleni is a monogenean parasite that causes significant mortality and economic losses in fish aquaculture. Changes in the antigenic composition of this parasite occur during its developmental stages. In this study, we evaluated humoral parameters in serum and transcriptional immune responses of yellowtail naturally infected with N. melleni. In addition, in vitro assays were performed to study the stimulatory effects of antigens from larvae and adults on spleen leucocytes from non-infected fish at 6 and 24 h post-stimulation. The results showed enhanced total protein, myeloperoxidase and antiprotease activities in N. melleni-infected fish compared with non-infected ones. The induction of Toll-like receptors (TLRs) and pro-inflammatory cytokines in spleen leucocytes during natural infection with N. melleni suggests that these immune-related genes play an important role in the initiation of the immune defence mechanism for controlling parasite infection. Interestingly, the magnitude of in vitro responses of spleen leucocytes was dependent on the parasitic stage. An important stimulation of gene expression by adult antigens on spleen leucocytes was observed. Differential expression patterns of TLRs and target cytokines in yellowtail leucocytes in both in vivo and in vitro studies suggest that the quality of yellowtail immune response is conditioned by N. melleni development.

5-Aminolevulinic acid synthase (ALAS) is the rate-limiting enzyme in the biosynthesis of heme, a prosthetic group that is found in hemoproteins, including those involved in molting. To better understand the roles of ALAS in L. vannamei (LvALAS), we analyzed its sequence and tissue distribution, the effects of age and bacterial infection on its gene expression, and the effects of LvALAS gene silencing. We also examined the expressions of three hemoproteins, the cytochrome oxidase subunit I (COX I) and subunit IV (COX IV) and catalase. Three LvALAS splicing variants were found in the hepatopancreas, with the main splicing variant having an open reading frame that encodes 532 aa. LvALAS transcripts were found in each of the eleven tissues tested in this study, with the highest gene expression in the intestine. The transcript abundances of LvALAS, COX I and COX IV in the hepatopancreas and stomach tended to decrease with age. LvALAS and catalase gene expressions significantly increased in the stomach after V. parahaemolyticus infection. LvALAS gene expression in the hepatopancreas, stomach and intestine (12- and 24-hours post-injection) was relatively lower in dsALAS-injected shrimp than in PBS-injected shrimp. All the PBS-injected shrimp molted after 8-10 days while no molting activity was observed in the dsALAS-injected shrimp group within the 14 days post-injection period. Our results provide evidence that (1) only the housekeeping form of ALAS exists in L. vannamei; LvALAS gene expression (2) decreases with age and (3) increases after bacterial infection; and (4) an ALAS-dependent pathway is necessary for proper molting in L. vannamei.

Chemokines are categorized into five families; one of the families is the CXC chemokines, which are critical in the pro-inflammatory process. CXC chemokines transmit signals and mediate a cell's biological activities by binding to cell surface receptors known as chemokine receptors (CXCRs). In this study, the CXCR2 from Japanese flounder (Paralichthys olivaceus) (JfCXCR2) was identified and characterized at the molecular level. The JfCXCR2 gene has a 1077 bp open reading frame that encodes a protein of 359 amino acid residues with seven transmembrane domains. Phylogenetic analysis of JfCXCR2 revealed that it belonged to the fish CXCR2 subfamily. Furthermore, JfCXCR2 was compared with the previously identified Japanese flounder CXCR1 (JfCXCR1). The expression analysis of uninfected Japanese flounder showed that JfCXCR1 and JfCXCR2 were expressed in all the tissues and organs tested but mainly in immune-related organs, including the kidney and spleen. Infection by Streptococcus iniae significantly increased the level of JfCXCR1 and JfCXCR2 mRNA in the kidney at days 1 and 3 post-infection. On the other hand, VHSV (viral hemorrhagic septicemia virus) and Edwardsiella tarda infection significantly increased JfCXCR2 mRNA levels in the kidney at days 3 and 6 post-infection, respectively. Conversely, JfCXCR1 expression was not significantly changed by either E. tarda or VHSV infection. Additionally, the peripheral blood leukocytes (PBLs) stimulated by recombinant proteins rCXCL8_L1a and rCXCL8_L1b were found to have significantly increased levels of JfCXCR1 and JfCXCR2 mRNA. Interestingly, even higher levels of JfCXCR1 and JfCXCR2 expression were observed in PBLs stimulated with rCXCL8_L1a and rCXCL8_L1b than in PBLs stimulated with either recombinant protein. These data suggest that bacterial infections may activate JfCXCR1. By contrast, JfCXCR2 may be activated by both bacterial and viral infection to mediate the immune response. These data can contribute to further understanding the functions of CXCR1 and CXCR2 in the fish immune system.

Aims To investigate the virulence of the Vp_PirAB-like genes in Vibrio parahaemolyticus-AHPND causing strain and factors that associate with the virulence level. Methods and Results The virulence of Vp_PirAB-like were examined using a non-virulent strain FP11 of V. parahaemolyticus transformed with a plasmid harboring Vp_PirAB-like genes and then used it to challenge shrimp Litopenaeus vannamei and Marsupenaeus japonicus. Both species experienced 100% mortality 10 days post-infection. Analysis of a mutant strain (E1M), that was originally identified as virulent strain (E1) but lost its virulence to L. vannamei, revealed that it lacked a part of the Vp_PirA-like gene and all of Vp_PirB-like gene. The copy numbers of Vp_PirA-like and Vp_PirB-like genes varied among virulent strains and were not correlated with their virulence. In Western blotting, Vp_PirA-like and Vp_PirB-like proteins were detected in both cell lysate and culture supernatant. The strongest intensity of detecting band in culture supernatant could be observed in the strain that caused the highest mortality. V. parahaemolyticus AHPND-causing strain, unlike the human tdh-positive strain, did not show any enterotoxicity. Conclusion V. parahaemolyticus AHPND-causing strains secret the Vp_PirA-like and Vp_PirB-like proteins during growing phase. The amount of secreted proteins affects the shrimp mortality. Significance and Impact of the Study The secreted proteins of Vp_PirAB-like are key factors of virulence in V. parahaemolyticus AHPND-causing strain, but not gene copy. This article is protected by copyright. All rights reserved.

Suppressor of cytokine signaling (SOCS) family members are key regulators of the immune system, particularly cytokine action, and have now been discovered in a number of fish species. Here we identified eight SOCS proteins (CISH, SOCS1a, SOCS1b, SOCS3a, SOCS3b, SOCS5, SOCS6 and SOCS9) in the Japanese flounder and analyzed their mRNA expressions after injection of poly (I:C) and formalin-killed cells (FKC) of Edwardsiella tarda. The expressions of all eight SOCS genes were detected in all the tissues examined. Stimulation of Japanese flounder reared at 15 or 25 °C with poly (I:C) affected the gene expressions of CISH, SOCS1a, SOCS1b and SOCS3a. All SOCS genes mRNA levels were significantly changed after FKC injection. Significant up-regulation of SOCS1a, SOCS1b, SOCS3a and SOCS3b genes was detected at 3, 12 and 24 hpi. SOCS5 and SOCS6 genes were significantly down-regulated at 3 hpi. SOCS9 gene was significantly up-regulated at 12 hpi. These results suggest that all eight of the SOCS genes are involved in immune responses, and that the CISH, SOCS1 and SOCS3 genes have functions distinct from those of the other SOCS members.

Serine proteases and their inhibitors play vital roles in biological processes. Serine protease inhibitors, including Kunitz-type protease inhibitors play important roles not only in physiological process (i.e. blood clotting and fibrinolysis) but also in immune responses. In this study, we characterized a Kunitz-type protease inhibitor, designated MjKuPI, from kuruma shrimp Marsupenaeus japonicus. An expression profile showed that MjKuPI was mainly expressed in hemocytes. Immunostaining revealed that some hemocytes expressed MjKuPI (MjKuPI(+) hemocytes) and others did not (MjKuPI(-) hemocytes). Injection of shrimp with Vibrio penaeicida and white spot syndrome virus (WSSV) upregulated the mRNA level of MjKuPI, and a flow cytometry analysis revealed that the proportion of MjKuPI(+) hemocytes increased significantly 24 h after injection. Together, these results suggest that MjKuPI and MjKuPI(+) hemocytes have a role in the innate immune system of kuruma shrimp.

The purpose of this study was to characterize the TLR21 gene from yellowtail (Seriola lalandi) and its functional activity using TLR agonist stimulation and Aeromonas antigens. The TLR21 nucleotide sequence from yellowtail was obtained using the whole-genome shotgun sequencing method and bioinformatics tools. Basal TLR21 gene expression was analyzed in several tissues. Subsequently, the gene expression of TLR21 and cytokines IL-1β and TNF-α was evaluated in TLR agonist (CpG-ODN2006, LPS, and Poly I:C) exposing head kidney leucocytes, which were then subjected to Aeromonas antigen stimulation. The yellowtail full-length cDNA sequence of SlTLR21 was 3615 bp (980 aa) showing a high degree of similarity with the counterparts of other fish species and sharing the common structural architecture of the TLR family, including LRR domains, one C-terminal LRR region, and a TIR domain. Gene expression studies revealed the constitutive expression of TLR21 mRNA in all the analyzed tissues; the highest levels were observed in spleen and head kidney where they play an important role in the fish immune system. Transcripts of TLR21 and the downstream IL-1β and TNF-α cytokine genes were most strongly up-regulated after exposure to the TLR agonists following Aeromonas antigen stimulation, suggesting they are involved in immune response. The results indicated that TLR agonists, in combination with Aeromonas antigens in head kidney leucocytes, synergistically enhance TLR21 and cytokines in yellowtail.

In invertebrates, hemocytes play an important role in immune responses. Recently, a novel form of innate immune mechanism called extracellular traps (ETs) was identified in shrimps, where DNA and antimicrobial peptides form complex structure to entrap the invading microbes. In this study, we detected the formation of ETs from hemocytes of kuruma shrimp in response to various stimulations, including phorbol myristate acetate (PMA), lipopolysaccharide (LPS), peptidoglycan (PGN) and Escherichia coli. E. coli cells were also found to be trapped by ET fibers. Fluorescence imaging revealed that c-type lysozyme proteins were released around the ET complex after E. coli stimulation, suggesting the presence of a coupled antimicrobial immune response involving ET formation and AMP release.

Adipose tissue is a lipid storage organ characterized by the pronounced accumulation of adipocytes. Although adipose tissues are found in various parts of the vertebrate body, it is unclear whether these tissues have a common ancestral origin or have evolved in several phylogenetic lineages by independent adipocyte accumulation events. To gain insight into the evolutionary history of vertebrate adipose tissues, we determined the distribution of adipocytes by oil red O staining in skeletal muscle of 10 teleost species spanning eight orders: Tetraodontiformes, Pleuronectiformes, Spariformes, Salmoniformes, Clupeiformes, Beloniformes, Osmeriformes, and Cypriniformes. Accumulation of adipocytes in the myoseptum was observed in many species, including red seabream, rainbow trout, Pacific herring, Pacific saury, zebrafish and giant danio. We also found some order-, species-, and swimming mode-specific distribution patterns of adipocytes: 1) almost complete absence of intramuscular adipocytes in the order Tetraodontiformes (torafugu and spotted green pufferfish), 2) clear adipocyte accumulation in the inclinator muscles of fin in Japanese flounder, 3) a large intramuscular adipose tissue at the root of the dorsal fin in ayu, and 4) thick lipid layers consisting of subcutaneous adipose tissue and red muscle lipids in pelagic migratory fish (Pacific herring and Pacific saury). Of note, Pacific herring and Pacific saury are phylogenetically distinct species sharing a similar niche and swimming mode, suggesting that their analogous adipocyte/lipid distribution patterns are the consequence of convergent evolution. The potentially heterogeneous origin of adipose tissues has significant implications for the interpretation of their functional diversity.

White Spot Syndrome Virus (WSSV) remains the most widespread and devastating infectious agent that hit the shrimp aquaculture industry worldwide. To date, there are no known effective strategies yet to combat WSSV infection. Hence, functional studies on genes critical for viral infection is essential in elucidating shrimp-virus interaction. Here we report the function of a gene from WSSV coding for a non-structural protein, VP9, utilizing RNA interference. Silencing of VP9 gene also effectively suppressed other gene region in the WSSV genome (wsv168 gene) as early as day 1 post infection (dpi). Three set-ups using Macrobrachium rosenbergii shrimp were prepared for treatment using VP9-dsRNA, GFP-dsRNA, and PBS. Each shrimp was challenge with WSSV, and survival rate was recorded. VP9- and GFP-dsRNA injected shrimps showed a significant survival rate of 80% and 70%, respectively, in contrast to 0% of the PBS injected shrimps at 25dpi. Re-infection of shrimp survivors using a higher viral titer concentration, concurrent with the infection of new shrimp samples for the PBS control group, resulted in a significant 67% survival rate for VP9-dsRNA compared to 0% with that of GFP-dsRNA and PBS group. Challenge test on two more species, Penaeus monodon and Marsupenaeus japonicus, also significantly increased survival after VP9-dsRNA treatment. Our results provided evidence that VP9 gene plays an essential role in WSSV replication and it can be a potent target gene in the development of RNAi therapeutics for shrimps.

Lectins are found in most living organisms, providing immune surveillance by binding to carbohydrate ligands. In fishes, C-type lectins were isolated from mucus of respiratory organs (skin and gills), where they aid the mucosal immune response in regulating microbiota and suppressing pathogens. In shrimp, however, no mucosal immunity or any form of gill-specific immune defense has been reported, and most identified C-type lectins are associated with hemocyte cellular and humoral responses. Interestingly, our microarray analysis revealed the localization of highly expressed novel biodefense genes in gills, among which is

White tail disease caused by Macrobrachium rosenbergii nodavirus (MrNV) infection takes place only in nauplii, not adults, of M. rosenbergii prawn. Hemocyte homeostasis and immune-related functions derived from the hematopoietic tissue (Hpt) in adult prawn are presumed to play roles in resisting viral infection. To elucidate the role of the Hpt cell response to MrNV, a comparative transcriptome analysis was performed with MrNV-infected prawn at various time intervals. The results showed that there were 462 unigenes that were differentially expressed between mock and infected samples. BlastX sequence analysis revealed that two proteins, crustacean hematopoietic factor (CHF) and cell growth-regulating zinc finger protein (Lyar), are involved in hemocyte hematopoiesis and are up-regulated during MrNV infection. In fact, genes involved in cell growth regulation and immunity were highly expressed at 6 h and decreased within 24 h post-infection. Localization studies in the Hpt tissue revealed the presence of anti-lipopolysaccharide factor (ALF) and CHF mRNAs in Hpt cells. Considering these findings, we concluded that resistance to MrNV infection in adult prawn is due to an increase in humoral immune factors and the acceleration of hemocyte homeostasis by the dual roles of the Hpt organ in M. rosenbergii.

In mammals, M cells can take up antigens through mucosal surfaces of the gut and the respiratory tract. Since M cells are deficient of lysosomes and phagosomes, the antigens are directly delivered to the mucosa-associated lymphoid tissue (MALT) without degradation. In teleost fish, the entire body surface (gills, skin, and intestinal system) is covered by mucus; however, specific antigen-sampling cells have not yet been identified in their mucosal tissues. Here, we show that two phenotypes of antigen-sampling cells take up antigens through epithelial surfaces of the rainbow trout gill. One phenotype of antigen-sampling cells has features of monocyte/macrophage/dendritic cell-type cells; they have large vacuoles in the cytoplasm and express PTPRC (CD45), CD83, IL-1β, and IL-12p40b. The second phenotype exhibits similar characteristics to mammalian M cells; the corresponding cells bind the lectin UEA-1 but not WGA and show expression of M cell marker gene Anxa5. In contrast to mammalian M cells, teleost M-type cells were found to exhibit small vacuoles in their cytoplasm and to express almost all genes related to the "phagosome", "lysosome," and "antigen processing and presentation" pathways. Furthermore, MHC class II was constitutively expressed on a fraction of M-type cells, and this expression was significantly increased after antigen uptake, suggesting that the MHC class II is inducible by antigen stimulation. Here, we suggest that teleost M-type cells play a role in the phylogenetically primitive teleost immune system, similar to bona-fide M cells. In addition, the presence of MHC class II expression suggests an additional role in antigen presentation in the gills, which are an organ with high T cell abundance, especially in interbranchial lymphoid tissue. The present results suggest an unconventional antigen presentation mechanism in the primitive mucosal immune system of teleosts, which generally lack highly organized lymphoid tissues. Moreover, the results of this work may be valuable for the development of mucosal vaccines that specifically target M-type cells; mucosal vaccines significantly reduce working costs and the stress that is usually induced by vaccination via injection of individual fish.

Acute hepatopancreatic necrosis disease (AHPND) is a shrimp farming disease, caused by the pathogenic Vibrio parahaemolyticus carrying a plasmid encoding Vp_PirAB-like toxins. Formalin-killed cells of V. parahaemolyticus AHPND-causing strain D6 (FKC-VpD6) were used to select Vp_PirAB-like toxin-resistant Litopenaeus vannamei by oral administration. Stomach and hepatopancreas tissues of shrimps that survived for one week were subjected to RNA sequencing. Differentially expressed genes (DEGs) between surviving shrimp, AHPND-infected shrimp, and normal shrimp were identified. The expressions of 10 DEGs were validated by qPCR. Only one gene (a gene homologous to L. vannamei anti-lipopolysaccharide factor AV-R isoform (LvALF AV-R)) was expressed significantly more strongly in the hepatopancreas of surviving shrimp than in the other groups. Significantly higher expression of LvALF AV-R was also observed in shrimp that survived two other trials of FKC-VpD6 selection. Recombinant ALF AV-R bound to LPS, PGN, Gram-negative bacteria, and some Gram-positive bacteria in ELISAs. ALF AV-R recombinant protein did not interact with native Vp_PirAB-like toxin in an ELISA or a Far-Western blot. For L. vannamei orally fed ALF AV-R protein for 3 days, the survival rate following challenge with VpD6-immersion was not significantly different from that of shrimp fed two control diets. These results suggest that LvALF AV-R expression was induced in the hepatopancreas of shrimp in response to the presence of Vp_PirAB-like toxin, although other factors might also be involved in the resistance mechanism.

With the emergence of several infectious diseases in shrimp aquaculture, there is a growing interest in the use of feed additives to enhance shrimp immunity. Recently, the use of 5-aminolevulinic acid (5-ALA), a non-protein amino acid that plays a rate-limiting role in heme biosynthesis, has received attention for its positive effect on immunity in livestock animals. To evaluate the effect of 5-ALA in the Pacific white shrimp, Litopenaeus vannamei, we conducted microarray analysis, a Vibrio parahaemolyticus immersion challenge test, an ATP level assay, and gene expression analysis of some hemoproteins and genes associated with heme synthesis and degradation. Out of 15,745 L. vannamei putative genes on the microarray, 101 genes were differentially expressed by more than fourfold (p

A novel Gram-stain-negative, rod-shaped (0.3 × 4–6 μm), non-flagellated, aerobic strain with gliding motility, designated JBKA-6T, was isolated in 1991 from a yellowtail fish, Seriola quinqueradiata, showing symptoms of bacterial haemolytic jaundice. 16S rRNA gene sequence analysis showed that strain JBKA-6T was related most closely to members of the family Flavobacteriaceae in the phylum ‘Bacteroidetes’. Furthermore, based on gyrB gene sequence analysis, JBKA-6T was classified into a single clade within the order Flavobacteriales, which was distinct from the known clades of the families Flavobacteriaceae, Blattabacteriaceae and Cryomorphaceae. The predominant isoprenoid quinone was identified as MK-6 (97.9 %), and the major cellular fatty acids (>10 %) were C14 : 0 and iso-C15 : 0. The main polar lipids were phosphatidylethanolamine, three unidentified phospholipids, two unidentified aminophospholipids and two unidentified polar lipids. The DNA G+C content of JBKA-6T, as derived from its whole genome, was 33.4 mol%. The distinct phylogenetic position and phenotypic traits of strain JBKA-6T distinguish it from all other described species of the phylum ‘Bacteroidetes’, and therefore it was concluded that strain JBKA-6T represents a new member of the phylum ‘Bacteroidetes’, and the name Ichthyobacterium seriolicida gen. nov., sp. nov. is proposed. The type strain of Ichthyobacterium seriolicida is JBKA-6T ( = ATCC BAA-2465T = JCM 18228T). We also propose that Icthyobacterium gen. nov. is the type genus of a novel family, Ichthyobacteriaceae fam. nov.

Streptococcus agalactiae, or Group B streptococcus (GBS), is a highly virulent pathogen in aquatic animals, causing huge mortalities worldwide. In Thailand, the serotype Ia, β-hemolytic GBS, belonging to sequence type (ST) 7 of clonal complex (CC) 7, was found to be the major cause of streptococcosis outbreaks in fish farms. In this study, we performed an in silico genomic comparison, aiming to investigate the phylogenetic relationship between the pathogenic fish strains of Thai ST7 and other ST7 from different hosts and geographical origins. In general, the genomes of Thai ST7 strains are closely related to other fish ST7s, as the core genome is shared by 92-95% of any individual fish ST7 genome. Among the fish ST7 genomes, we observed only small dissimilarities, based on the analysis of clustered regularly interspaced short palindromic repeats (CRISPRs), surface protein markers, insertions sequence (IS) elements and putative virulence genes. The phylogenetic tree based on single nucleotide polymorphisms (SNPs) of the core genome sequences clearly categorized the ST7 strains according to their geographical and host origins, with the human ST7 being genetically distant from other fish ST7 strains. A pan-genome analysis of ST7 strains detected a 48-kb gene island specifically in the Thai ST7 isolates. The orientations and predicted amino acid sequences of the genes in the island closely matched those of Tn5252, a streptococcal conjugative transposon, in GBS 2603V/R serotype V, Streptococcus pneumoniae and Streptococcus suis. Thus, it was presumed that Thai ST7 acquired this Tn5252 homologue from related streptococci. The close phylogenetic relationship between the fish ST7 strains suggests that these strains were derived from a common ancestor and have diverged in different geographical regions and in different hosts.

Galectins belong to the family of galactoside-binding proteins and play a major role in the immune and inflammatory responses of vertebrates and invertebrates. The galectin family is divided into three subtypes based on molecular structure; prototypes, chimera types, and tandem-repeated types. We isolated and characterized the cDNA of galectin-8 (OnGal-8) in Nile tilapia (Oreochromis niloticus). OnGal-8 consisted of a 966 bp open reading frame (ORF) that encoded a 321 amino acid protein (43.47 kDa). Homology and phylogenetic tree analysis suggested the protein was clustered with galectin-8s from other animal species and shared at least 56.8% identity with salmon galectin-8. Structurally, the amino acid sequence included two distinct N- and C- terminus carbohydrate recognition domains (CRDs) of 135 and 133 amino acids, respectively, that were connected by a 39 amino acid polypeptide linker. The N- and C-CRDs contained two conserved WG-E-I and WG-E-T motifs, suggesting they have an important role in mediating the specific interactions between OnGal-8 and saccharide moieties such as β-galactoside. The structure of OnGal-8 was characterized by a two-fold symmetric pattern of 10-and 12-stranded antiparallel ß-sheets of both N- and C-CRDs, and the peptide linker presumably formed a random coil similar to the characteristic tandem-repeat type galectin. The expression of OnGal-8 in healthy fish was highest in the skin, intestine, and brain. Experimental challenge of Nile tilapia with S. agalactiae resulted in significant up-regulation of OnGal-8in the spleen after 5 d. Our results suggest that OnGal-8 is involved in the immune response to bacterial infection.

Gene expression profiling of poly (I:C)-treated Japanese flounder, Paralichthys olivaceus, under different temperatures was investigated using microarray analysis. The response was analyzed in spleen tissue at 3 and 24 h post injection (hpi) at 15 °C and 25 °C. A large number of genes in fish treated with poly (I:C) at 25 °C were expressed at 3 hpi, whereas the expression profiles at 24 hpi appeared to be similar to those of the controls. Cluster analysis of the different expression profiles showed three distinct groups of up-regulated genes in fish reared at 15 °C. These were early (3 hpi), early-to-late (3 and 24 hpi), and late (24 hpi) up-regulated genes. These genes included type I IFN-related genes and inflammatory genes. Among the up-regulated genes, most of the type I IFN-related genes played early-to-late- and late-responding genes at 15 °C but early-responding genes at 25 °C. Thus, several up-regulated genes in these groups from the microarray result were further verified by qPCR. These results indicate that the type I IFN gene expressions of P. olivaceus treated with poly (I:C) can be regulated in a temperature-dependent manner.

Penaeidins are a unique family of antimicrobial peptides specific to penaeid shrimp and have been reported mainly function as anti-bacterial and anti-fungal. In order to investigate whether penaeidins could also respond to virus or not, we examined the effect of WSSV on MjPen-II (penaeidin in kuruma shrimp, Marsupenaeus japonicus) expression. In the control group, MjPen-II transcript level can be detected in almost all test tissues but was expressed most strongly in hemocytes. After WSSV infection, MjPen-II transcript level was significantly downregulated in hemocytes. Moreover, the proportion of MjPen-II+ hemocytes was not significantly different between non-infected and WSSV-infected shrimp, but the number of MjPen-II+ highly expressing hemocytes decreased after infection. In addition, MjPen-II was observed in the cytoplasm of granule-containing hemocytes. These results suggest that WSSV suppresses MjPen-II expression in hemocytes.

Shrimp, as invertebrates, have an open vasculature that allows circulating hemocytes to infiltrate the tissues, where they are referred to as sessile hemocytes. Sessile hemocytes are known to express immune-related genes, but it is not known whether their functions differ from those of circulating hemocytes. To answer this question, we enriched them from suspensions of different tissues using discontinuous density gradient centrifugation and analyzed their transcripts by RNA-seq. The results suggest that circulating hemocytes and sessile hemocytes of the gills are in a state that could react quickly to pathogens, immune-related genes expression of sessile hemocytes differ from circulating hemocytes, and the gills, heart and lymphoid organs have cells that express immune-related genes that are different from hemocytes.

cDNA of a newly recognized white spot syndrome virus (WSSV)-induced gene (MjVIG1) was characterized from Marsupenaeus japonicus hemocytes; this gene encodes a protein that lack similarity to any known characterized protein. To identify this novel gene, we mainly conducted transcript level analysis, immunostaining and flow cytometry after WSSV infection. MjV1G1 transcript levels were also measured after Yellow head virus (YHV) and Vibrio parahaemolyticus infection tests. In non-infected and WSSV-infected shrimp, MjVIG1 was observed in granule-containing hemocytes. In addition, the MjVIG1 transcript level and ratio of MjVIG1-positive hemocytes both significantly increased, and number of MjVIG1-positive hemocytes slightly increased after WSSV infection. In contrast, MjVIG1 transcript level did not change after YHV and V. parahaemolyticus infection. These results indicated that MjVIG1 might be a WSSV-specific induced gene in M. japonicus hemocytes.

Phagocytosis is main cellular immunity, however, it is still unknown or debated upon which types of hemocyte contributes phagocytosis in penaeid shrimps. The hemocyte characterization in kuruma shrimp have been mainly performed based on its morphology by microscopic observation. Therefore, establishment of molecular markers to distinguish phagocytic hemocytes is required. In this study, using magnetic fluorescent beads, we enriched phagocytic hemocytes and conducted RNA-seq analysis between total and enriched phagocytic hemocytes. The data demonstrated functional difference between total and phagocytic hemocytes. In addition, a transcript homologous to integrin-alpha was highly expressed in phagocytic hemocytes, and named Mj-Intgα. Using anti-serum against Mj-Intgα revealed that around 60% of total hemocytes and more than 90% of phagocytic hemocytes showed positive for Mj-Intgα. This study presents Mj-Intgα as a candidate molecular marker for future functional characterization of hemocytes.

Aims Genomic characterization of Harveyi clade vibrio strain Y6 causing 'Scale drop and Muscle Necrosis syndrome' (SDMN) isolated from barramundi (Lates calcarifer) in Vietnam. Methods and results A bacterial genome was sequenced using Illumina MiSeq platform. Multilocus sequence analysis confirmed that the bacterium belongs to Vibrio harveyi species. Further phylogenetic analysis inferred from core genome SNPs revealed a close relationship between our bacterium and the V. harveyi isolated from groupers in Taiwan and China. blastp results indicated that V. harveyi piscine strains carried numerous adhesin, secretion system, siderophore and toxin-related genes. Genome comparison between Y6 and 32 strains of V. harveyi from different origins showed that at least 17 potential virulence genes were present exclusively in the strain Y6. Many of these (six of 17 genes) were homologous to pyoverdine siderophore, a secreted high-affinity iron chelator, clusters originally found in Pseudomonas aeruginosa. Genome of V. harveyi Y6 was incorporated by a bacteriophage VHY6φ and replication protein of the phage was most similar to CTXφ described previously in Vibrio cholerae and Vibrio fischeri. However, the cholera toxin-encoding genes, namely ctxA and ctxB, were absent from VHY6φ, while the CTXφ-enterotoxin gene (zonula occludens toxin; zot) remained intact. Conclusions Several putative virulence genes and a phage carrying toxin gene were identified in the genomes of SDMN-associated V. harveyi Y6. Significance and impact of the study This study confers genomic information of the piscine pathogenic V. harveyi which recently caused widespread mortality. Such information is of importance to gain insight into bacterial molecular pathogenesis.

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins known to assist the innate immune system as pattern recognition receptors (PRRs). The binding specificity of CTLs lies in the motif of their carbohydrate recognition domain (CRD), the tripeptide motifs EPN and QPD bind to mannose and galactose, respectively. However, variants of these motifs were discovered including a QAP sequence reported in shrimp believed to have the same carbohydrate specificity as QPD. Here, we characterized a novel C-type lectin (MjGCTL) possessing a CRD with a QAP motif. The recombinant MjGCTL has a calcium-dependent agglutinating capability against both Gram-negative and Gram-positive bacteria, and its sugar specificity did not involve either mannose or galactose. In an encapsulation assay, agarose beads coated with rMjGCTL were immediately encapsulated from 0 h followed by melanization at 4 h post-incubation with hemocytes. These results confirm that MjGCTL functions as a classical CTL. The structure of QAP motif and carbohydrate-specificity of rMjGCTL was found to be different to both EPN and QPD, suggesting that QAP is a new motif. Furthermore, MjGCTL acts as a PRR binding to hemocytes to activate their adherent state and initiate encapsulation.

Influence of temperature on the susceptibility of fish against virus infection has been studied for a decade. Recent reports have been shown the effects of rearing temperatures on the fish immune system against virus infection. However, the roles of temperature in regulation of type I interferon (IFN) system has not yet been investigated. Thus, the effects of temperature on type I IFN response were investigated in this study using poly (I:C) injection in sevenband grouper and Mx gene was used as a marker for type I IFN expression. Quantitative real-time PCR (qPCR) result showed that Mx expression profiles were moderately different between temperatures. The highly up-regulated Mx transcripts at 3 h post injection (hpi) were observed in high temperatures (25 °C and 30 °C) but not in low temperatures (15 °C and 20 °C). Meanwhile, low temperatures (15 °C and 20 °C) could detect the highly up-regulated Mx transcripts at 24 hpi. Expression of Mx transcripts was also observed at 72 hpi at 15 °C. Poly (I:C)-injected fish were challenged with RGNNV after 72 and 168 hpi. At 72 hpi, 100% of fish survived at all temperatures, whereas 95% survival rate was observed at 168 hpi at 25 °C during 14 days of observation. To further verify the duration period of an antiviral state at different temperatures, qPCR and endpoint dilution assay were used to quantify the number of virus in fish challenged with RGNNV. The reduction of viral copy numbers and viral titers could be observed at 72 and 168 hpi. However, high viral copy numbers and viral titers could be detected at 168 hpi at 30 °C. These results demonstrate that temperatures influenced on the Mx expression profiles and the duration period of an antiviral state efficiently interfered with virus replication at different temperatures.