Antibacterial activity study of a novel piscidin 5-like type 4 from Larimichthys crocea.

As one of the key components of innate immune system, piscidins are likely to play pivotal role in the first defense line in fish. Piscidins own multiple resistance activity. A novel piscidin 5-like type 4 was excavated from Larimichthys crocea (termed Lc-P5L4) liver transcriptome immuned by Cryptocaryon irritans, and upregulated at 7 days post infection when secondary bacterial infection occurred. In the study, we characterized the antibacterial activity of Lc-P5L4. The liquid growth inhibition assay detected the recombinant Lc-P5L4 (rLc-P5L) had potent antibacterial activity to Photobacterium damselae. Scanning electron microscope (SEM) observed the cell surface of P. damselae collapsed to form pit, and membrane of some bacteria ruptured after co-incubation with rLc-P5L. Further, transmission electron microscope (TEM) was also employed to observe the intracellular microstructural damage, rLc-P5L4 caused cytoplasm contraction, pores formation and contents leakage. After knowing about its antibacterial effects, the preliminary antibacterial mechanism was also explored, western blot analysis showed rLc-P5L4 could bind to P. damselae through targeting to LPS. Agarose gel eletrophoresis analysis further showed rLc-P5L4 could also penetrate into cells and brought about genome DNA degradation. Therefore, rLc-P5L4 was of potential being a candidate to explore new antimicrobial drug or additive agent, especially to P. damselae.

Dual RNA-seq provides novel insight into the roles of dksA from Pseudomonas plecoglossicida in pathogen-host interactions with large yellow croakers ( Larimichthys crocea).

Pseudomonas plecoglossicida is a rod-shaped, gram-negative bacterium with flagella. It causes visceral white spot disease and high mortality in Larimichthys crocea during culture, resulting in serious economic loss. Analysis of transcriptome and quantitative real-time polymerase chain reaction (PCR) data showed that dksA gene expression was significantly up-regulated after 48 h of infection with Epinephelus coioides (log 2FC=3.12, P<0.001). RNAi of five shRNAs significantly reduced the expression of dksA in P. plecoglossicida, and the optimal silencing efficiency was 96.23%. Compared with wild-type strains, the symptoms of visceral white spot disease in L. crocea infected with RNAi strains were reduced, with time of death delayed by 48 h and mortality reduced by 25%. The dksA silencing led to a substantial down-regulation in cellular component-, flagellum-, and ribosome assembly-related genes in P. plecoglossicida, and the significant up-regulation of fliC may be a way in which virulence is maintained in P. plecoglossicida. The GO and KEGG results showed that RNAi strain infection in L. crocea led to the down-regulation of inflammatory factor genes in immune-related pathways, which were associated with multiple immune response processes. Results also showed that dksA was a virulence gene in P. plecoglossicida. Compared with the wild-type strains, RNAi strain infection induced a weaker immune response in L. crocea.

Novel insights into host-pathogen interactions of large yellow croakers ( Larimichthys crocea) and pathogenic bacterium Pseudomonas plecoglossicida using time-resolved dual RNA-seq of infected spleens.

Host-pathogen interactions are highly complex, involving large dynamic changes in gene expression during infection. These interactions are fundamental to understanding anti-infection immunity of hosts, as well as the pathogenesis of pathogens. For bacterial pathogens interacting with animal hosts, time-resolved dual RNA-seq of infected tissue is difficult to perform due to low pathogen load in infected tissue. In this study, an acute infection model of Larimichthys crocea infected by Pseudomonas plecoglossicida was established. The spleens of infected fish exhibited typical symptoms, with a maximum bacterial load at two days post-injection (dpi). Time-resolved dual RNA-seq of infected spleens was successfully applied to study host-pathogen interactions between L. crocea and P. plecoglossicida. The spleens of infected L. crocea were subjected to dual RNA-seq, and transcriptome data were compared with those of noninfected spleens or in vitro cultured bacteria. Results showed that pathogen-host interactions were highly dynamically regulated, with corresponding fluctuations in host and pathogen transcriptomes during infection. The expression levels of many immunogenes involved in cytokine-cytokine receptor, Toll-like receptor signaling, and other immune-related pathways were significantly up-regulated during the infection period. Furthermore, metabolic processes and the use of oxygen in L. crocea were strongly affected by P. plecoglossicida infection. The WGCNA results showed that the metabolic process was strongly related to the entire immune process. For P. plecoglossicida, the expression levels of motility-related genes and flagellum assembly-related genes were significantly up-regulated. The results of this study may help to elucidate the interactions between L. crocea and P. plecoglossicida.

The antibacterial activity and mechanism analysis of piscidin 5 like from Larimichthys crocea.

Chemical drugs, such as antibiotics, were still important materials to prevent and cure diseases of aquatic organisms. However, antibiotics abuse do not only make the effects little, but also cause other bad problems, such as bacterial resistance and drug residues. Therefore, seeking the effective substitutes of antibiotics was an approach needed to be explored. Antibacterial peptides (AMPs) attracted more and more attention in the recent years. The parasitism and secondary bacterial invasion caused by ectroparasite Cryptocaryon irritans was a disaster to almost all host fish, including Larimichthys crocea. Reports indicated many AMPs played a key role in the whole parasitic infection cycle. Piscidin 5 like was a member of piscidin family. In the study, the antibacterial activity and mechanisms of piscidin 5 like from L.coreca (Lc-P5L) were detected. Liquid growth inhibition results showed recombinant Lc-P5L (rLc-P5L) had broad antibacterial spectrum and strong bactericidal activity. The bactericidal activity functioned in dose- and time-dependent manners. SEM (scanning electron microscope) observed the relatively detailed bactericidal process, rLc-P5L treatment resulted in a mass of bacteria piling together, appearing plenty of strange filaments and covering on the bacteria. Besides, S.aureus overgrowed plenty of granules, formed holes on the membrane of a few cells, and contents poured out from the holes. At the same time, antibacterial mechanisms were explored. After direct incubation with bacteria, western blot detected the apparently positive signal of rLc-P5L on bacteria; secondly, the incubation first with LPS (lipopolysaccharide) or LTA (lipoteichoic acid) significantly affect the binding of rLc-P5L to bacteria again, which indicated rLc-P5L could bind to bacteria through interaction with some PAMPs (pathogen-associated molecular patterns). In addition, rLc-P5L could interact with bacterial genome DNA by dose- and time-dependent means. In summary, rLc-P5L binded to bacteria surface through targeting to some PAMPs to damage membrane, and entered into cells to interact with genome DNA to disturb normal metabolism when it reached to some certain time and concentration thresholds, which were likely to be its pathway to exert antibacterial activity.

Antimicrobial peptide Pc-pis: A new cancer cell killer.

The antimicrobial peptide (AMP) Pc-pis, a member of Piscidin family from fish with cationic amphipathic structure, has potent, broad-spectrum antimicrobial activity against bacteria, fungi and parasite, and lower hemolytic activity. Here, we reported that Pc-pis had antitumor activity. Pc-pis killed tumor cells including HeLa cells. Previously, it is reported that AMPs bind to the membrane of bacteria to generate the pores to lyse the target cells, and similarly, the cancer cell incorporate phosphatidyl-serine on the outer leaflet of plasma membrane so that amphipathic AMPs can bind to the membrane to kill it. Our data supported that notion because suitable size osmo-protectant PEG4000 prevented HeLa cells from death induced by Pc-pis. Additionally, Fusion protein GFP-Pc-pis accumulated mainly at the nuclear membranes of HeLa cells and positive net charge in modified Pc-pis intensified but negative net charges eliminated this effect. Thus, positively charged residues were important for its affinity to the membrane. Our work will lay the groundwork of the development of Pc-pis antitumor activity.

Excavating differentially expressed antimicrobial peptides from transcriptome of Larimichthys crocea liver in response to Cryptocaryon irritans.

Larimichthys crocea, the special marine economy fish, owns the largest annual yield for a single species in China. One of the most significant factors affecting large yellow croaker culture is the diseases, especially the threat of marine white spot disease which caused by a protozoan Cryptocaryon irritans. Antimicrobial peptides (AMPs) have been demonstrated to be active against bacterium, fungi and parasites, showing their potential usefulness in aquaculture as substitutes for antibiotics. Many researches have been carried out about the AMPs concentrating on the activity resist on C. irritans, and piscidin-like of L. crocea owning widely antibacterial spectrum and strong activity against C. irritans was screened in our team. In the paper, taking advantage of the large yellow croaker hepatic comparison transcriptome in response to C. irritans at 3d post infection, seven kinds of AMPs have been excavated from the differently expressed genes, including LEAP2 like, LEAP-2A, hepcidin, hepcidin-like, piscidin-5-like, piscidin-5-like type 4 and bactericidal permeability increasing protein (BPI). Hepcidin, hepcidin-like, piscidin-5-like, piscidin-5-like type4 and BPI were up-regulated to protect large yellow croaker from being damaged by C. irritans infection; while LEAP2 like and LEAP-2A were down-regulated, they might be as a negative-feedback regulation factor or some other regulatory mechanisms to adjust the immune response in the process of C. irritans infection. The differential expression changes were verified with quantitative real-time PCR (qRT-PCR) to illustrate the reliability of the sequenced data. Hearteningly, piscidin-5-like type 4 was a novel type which was high similar to other piscidin-5-like types. Interestingly, the infection may well cause alternative splicing of LEAP-2A mRNA, which was a surprised phenomenon and finding after C. irritans infection, but more further study was needed to be conducted. Therefore, the data showed that these AMPs were involved in the immune response to the C. irritans infection. In all, these results implied that the immune response of AMPs to C. irritans infection was a complex and sophisticated regulatory process.

A novel stimulator of interferon gene (STING) from Larimichthys crocea and their involvement in immune response to ectoparasite Cryptocaryon irritans infection.

The marine white spot disease caused by protozoan ectoparasite Cryptocaryon irritans is a severe problem to the large yellow croaker farming industry. To understand the molecular immune mechanisms and improve its immune capacity are particular important. STING, one of the important second messengers in innate immune response process, plays pivotal roles in defensing against different pathogenic microorganisms. Many reports have pointed that STING could not only combine the uncovered dsDNA, ssDNA directly in the cytoplasm from the pathogens or biology itself, but it also could recognize cyclic diguanylate monophosphate (c-di-GMP), cyclic diadenylate monophosphate (c-di-AMP). Based on the STING sequence, a variant of the L. crocea STING (termed LcSTING2) was found by accident. RACE was used to clone the full-length cDNA of LcSTING2 which contained a 5'- UTR of 154 bp, a 3'-UTR of 592 bp and an ORF of 1227 bp encoding 408 amino acids. The predicted protein molecular weight (Mw) was 45.83 KDa and the estimated theoretical isoelectric point (pI) was 6.24. The deduced protein of LcSTING2 contains no signal peptide. One transmembrane motif (TM) in the N-terminal region, a TMEM173 domain and five putative motifs (RXR) found in resident endoplasmic reticulum proteins were also conserved. According to the partial genomic sequence, the unknown sequences were amplified, it contained 6 exons and 5 introns, and all the exon-intron boundaries were in accordance with classical GT-AG rule (GT/intron/AG). The similarity shared with fishes was higher than other high vertebrates. qRT-PCR results showed that LcSTING (two variants) distributed in all examined tissues, and it was the most abundant in gill. After challenged by C. irritans, LcSTING mRNA only appeared instantaneous up-regulation during the infective stage of theronts in the head kidney and was also up-regulated during the whole infectious cycle of C. irritans in the liver, which implied LcSTING gene was likely to be involved in the defensing against C. irritans infection, it is the first time to explore the STING taking part in the immune response to ectoparasite rather than bacterium or viruses.

Analysis of liver and gill miRNAs of Larimichthys crocea against Cryptocryon irritans challenge.

The white-spot disease caused by marine ciliate Cryptocryon irritans hindered the sustainable development of large yellow croaker Larimichthys crocea industry. Better understandings about the parasite-host interactions in the molecular level will facilitate the prevention of mass mortality of the L. crocea caused by white-spot disease. MicroRNAs (miRNAs) are a class of small RNA molecules about 20-22 nucleotides which post-transcriptionally regulated many protein-coding genes and involved in many biological processes, especially in host-pathogen responses. In this study, we identified known and novel miRNAs in the gill and liver of L. crocea challenged by C. irritans by high throughput sequencing using Solexa technology. The data were further studied to screen differentially expressed miRNAs, and predict their target genes. The differential expression (p < 0.05) between libraries was observed in 103 miRNAs in liver tissue, among which 65 and 38 were conserved and novel miRNAs, 67 and 36 were up- and down-regulated miRNAs. While in gill tissue, 122 significant differentially expressed miRNAs were identified, among which 83 and 39 were conserved and novel miRNAs, 79 and 43 were up- and down-regulated miRNAs. In addition, these differentially expressed miRNAs target a series of genes which involved in many important biological processes including immune response. Here via deep sequencing, we for the first time characterize L. crocea miRNAs in response to C. irritans challenge, the results should help for better understandings about the immune response of the L. crocea under C. irritans challenge.

Identification, expression and antibacterial activities of an antimicrobial peptide NK-lysin from a marine fish Larimichthys crocea.

As fundamental immunologic mechanism, the innate immunity system is more important than the specific immunity system in teleost fishes during pathogens infection. Antimicrobial peptides are integral parts of the innate immune system, and play significant roles against pathogens infection. NK-lysin, the compounds of the natural killer cells and cytotoxic T cells, are potent and effective antimicrobial peptides widely distributed in animals. In this study, we reported the sequence characteristics, expression profiles and antibacterial activities of a NK-lysin gene (Lc-NK-lysin) from a commercially important marine fish, the large yellow croaker (Larimichthys crocea). The open reading frame of Lc-NK-lysin cDNA sequence was 447 bp in length, coding 148 amino acids. The genomic DNA of Lc-NK-lysin has the common features of NK-lysin family, consisting of five exons and four introns, and in its deduced mature peptide, there are six well-conserved cysteine residues and a Saposin B domain. Lc-NK-lysin was expressed in all tested tissues (skin, muscle, gill, brain, head kidney, heart, liver, spleen, stomach and intestine) with different expression patterns. In pathogens infection the expression profiles of Lc-NK-lysin varied significantly in gill, head kidney, spleen and liver, indicating its role in immune response. Two peptides (Lc-NK-lysin-1 and Lc-NK-lysin-2) divided from the core region of the Lc-NK-lysin mature polypeptide were chemically synthesized and their antibacterial activities were examined; the potential function on the inhibition of bacteria propagation was revealed. Our results suggested that Lc-NK-lysin is a typical member of the NK-lysin family and as an immune-related gene it involves in the immune response when pathogens invasion.

Identification and expression analysis of a new invertebrate lysozyme in Kuruma shrimp (Marsupenaeus japonicus).

Lysozyme is an important component of the innate immunity system against invading pathogens. An invertebrate (i-type) lysozyme from the hepatopancreas of Kuruma shrimp Marsupenaeus japonicus (Mj-ilys) was identified. The full-length cDNA of Mj-ilys was 580bp with a 429 bp open reading frame encoding a 142 amino acid polypeptide. The encoded polypeptide was predicted to have a 17 amino acid signal peptide, and a 125 amino acid mature protein with a theoretical mass of 14.099 kDa and an isoelectric point (pI) of 4.18. A Destabilase conserved domain was predicted in Mj-ilys amino acid sequences which may be stable by 10 cysteine residues forming 5 disulfide bonds. Mj-ilys may loss the muramidase and isopeptidase activities due to the lack of the key catalytic residues. Mj-ilys had high homologous of 80-82% with i-type lysozymes of penaeid shrimps. It was first grouped with other i-type lysozyme of shrimps and crabs in a phylogenetic tree predicted by the Neighbor-Joining method. Mj-ilys mRNA was expressed mainly in hepatopancreas and almost undetectable in other tissues. The mRNA expression of Mj-ilys were all found from fertilized eggs to post-larvae of 17 days (PL17), and its expression exhibited significant differences among each developmental stage. After white spot syndrome virus (WSSV) challenge (3.6 × 10(8) virions/µl), the time-dependent expression pattern of Mj-ilys in hepatopancreas and gills showed significantly different. These results indicated that Mj-ilys is potentially involved in the ontogenesis and immune defense in Kuruma shrimp.

Transcriptome analysis of the Larimichthys crocea liver in response to Cryptocaryon irritans.

The large yellow croaker (Larimichthys crocea) is an economically important marine fish cultured in China and East Asian countries and is facing a serious threat from Cryptocaryon irritans, which is a protozoan ectoparasite that infects most reared marine fish species. To understand the molecular immune mechanisms underlying the response to C. irritans, we first performed a comparative gene transcription analysis using livers from C. irritans-immunized L. croceas and from a control group through RNA-Seq technology. After the removal of low-quality sequences and assembly, 51360 contigs were obtained, with an average length of 1066.93 bp. Further, a blast analysis indicates that 30747 contigs can be annotated based on homology with matches in the NT, NR, gene, and string databases. A gene ontology analysis was used to classify 21598 genes according to three major functional categories: molecular function, cellular component, and biological process. Moreover, 14470 genes were found in 303 KEGG pathways. We used RSEM and EdgeR to determine that 3841 genes were significantly differentially expressed (FDR < 0.001), including 2129 up-regulated genes and 1712 down-regulated genes. A significant enrichment analysis of these differentially expressed genes and isogenes revealed major immune-related pathways, including the toll-like receptor, complement and coagulation cascades, and chemokine signaling pathways. In addition, 28748 potential simple sequence repeats (SSRs) were detected from 12776 transcripts, and 62992 candidate single nucleotide polymorphisms (SNPs) were identified in the L. croceas liver transcriptome. This study characterized a gene expression pattern for normal and C. irritans-immunized L. croceas for the first time and not only sheds new light on the molecular mechanisms underlying the host-C. irritans interaction but also facilitates future studies on L. croceas gene expression and functional genomics.

Identification and expression analysis of a novel stylicin antimicrobial peptide from Kuruma shrimp (Marsupenaeus japonicus).

Antimicrobial peptides (AMPs) are important components of the innate immune system and function as the first line of defense against invading pathogens. In current study we identified, cloned and characterized a novel stylicin AMP from Kuruma shrimp Marsupenaeus japonicus (Mj-sty). The full-length cDNA of Mj-sty was 428 bp with an open reading frame of 315 bp that encoded 104 amino acids. The theoretical molecular mass of mature Mj-sty was 8.693 kDa with an isoelectric point (pI) of 4.79. A proline-rich N-terminal region and a C-terminal region contained 13 cysteine residues were identified. Genomic sequence analysis with respect to its cDNA showed that Mj-sty was organized into two exons interrupted by one intron. Tissue-specific expression revealed that Mj-sty was mainly transcribed in gills and hemocytes. Expression of Mj-sty in early developmental stages demonstrated that Mj-sty mRNA were present from fertilized eggs to post-larvae of 17 days (PL17), and the expression levels showed a significant variation in different developmental stages. After challenge of white spot syndrome virus (WSSV), the time-dependent expression pattern of Mj-sty in both gills and hepatopancrease showed down-regulation at the early hours of infection, subsequently up-regulation and down-regulation, and then up-regulation at the end hours to almost the half of the controls. The results indicate that Mj-sty is potentially involved in the ontogenesis and immune responses against WSSV.

Molecular characterization and expression analysis of interferon-gamma in the large yellow croaker Larimichthys crocea.

The large yellow croaker Larimichthys crocea is an important mariculture fish species in China, and the bacterium Vibrio harveyi (V. harveyi) and the ciliate protozoan Cryptocaryon irritans (C. irritans) are the two major pathogens in its aquaculture sector. Interferon-gamma (IFN-γ) plays important roles in regulating both innate and cell mediated immune responses as an inflammatory cytokine. In this study, we obtained the nucleotide sequence of IFN-γ from the large yellow croaker (LcIFN-γ). The phylogenetic relationship tree of 18 available IFN-γ genes was constructed based on their sequences. Expression analyses in 10 various tissues were conducted after the croaker challenged with V. harveyi and C. irritans, respectively. Real time PCR analysis showed that the expression of LcIFN-γ was observed broadly in health individuals. After injected with V. harveyi, the 10 tissues had a higher expression of IFN-γ at the first day (1 d); only spleen, muscle, intestine, heart and skin had higher expressions after infected with C. irritans at 1 d. Major immune tissues (skin, gill, head kidney and spleen) and detoxification tissue (liver) were sampled at 0 h, 6 h, 1 d, 2 d, 3 d, 4 d, 5 d and 7 d to understand the expression trends of LcIFN-γ after challenged with C. irritans. The expressions of LcIFN-γ in skin and gill (the primary immune organs) showed a clear correlative relationship with the life cycle of C. irritans.

Identification and analysis of a Marsupenaeus japonicus ferritin that is regulated at the transcriptional level by WSSV infection.

Marsupenaeus japonicus is a shrimp species of great value in the Chinese aquaculture industry. Given the susceptibility to viral diseases, research efforts have focused on the molecular characteristics of the shrimp's immune mechanisms. Ferritin is well known for its iron storage function, but studies have also addressed its immune function in response to pathogens. In this study, an M. japonicus ferritin cDNA was identified by homology cloning and rapid amplification of cDNA ends-PCR. The full-length cDNA is 1244 bp long and contains an open reading frame (513 bp) that encodes a highly conserved protein of 170 amino acids. Quantitative real-time PCR detection of ferritin revealed high expression in eight tested tissues, with the highest levels in hemocytes-consistent with the iron storage capacity of ferritin. We infected M. japonicus with white spot syndrome virus and validated the model by viral copy analysis and histopathology, which demonstrated an increase in viral copies along with acute degeneration of tissues. Transcripts of ferritin increased by 3.1-fold, 2.1-fold, and 1.5-fold in the hepatopancreas, gill, and midgut at 24h post-injection, suggesting that ferritin played an important role in the immune response of M. japonicus.

Characterization of a novel piscidin-like antimicrobial peptide from Pseudosciaena crocea and its immune response to Cryptocaryon irritans.

Piscidins, important components of the innate (nonspecific) immunity system in fish, have potent, broad-spectrum antimicrobial and antiparasitic activities. In this study, we reported a novel antimicrobial cationic peptide from Pseudosciaena crocea. Although this peptide exhibited a genomic (3 exons and 2 introns) and propeptide (signal peptide, mature peptide and prodomain) organization, conserved signal peptide (22 amino acids) and consensus motif I-X5-H-X4-I-H identical to the reported fish piscidins, Pc-pis showed a relatively low overall conservation with other known piscidins, which was obviously embodied in the amino acid composition of the peptide. Pc-pis is strikingly rich in glycine residues (27.3%), which disrupted the amphipathic structure of the peptide. Relative quantitative real-time PCR revealed that Pc-pis is a typically gill-expressed peptide. The sequence analysis, structural features and tissue distribution suggested that Pc-pis was genetically related to the piscidins family and might be a novel piscidin-like antimicrobial peptide. Quantitative PCR analysis revealed that the expression of Pc-pis in the spleen, head-kidney, liver, intestine, skin and gill could be regulated during Cryptocaryon irritans infection and post C. irritans falling off, implicating a role for Pc-pis in immune defense against C. irritans and secondary bacterial infections. Synthetic Pc-pis exhibited broad-spectrum activity against bacteria, fungi and C. irritans in parasitic stages. These results provided the first evidence of piscidins antiparasitic activity against marine fish ectoparasites C. irritants trophonts and further indicated that Pc-pis might be an important component of the P. crocea innate immune system against C. irritans and secondary bacterial infections. Thus, these data provided new insights into P. crocea innate immunity against external protozoan parasite and microbial infections and facilitate the evaluation of Pc-pis as a therapeutic agent against pathogen invasion.

Molecular cloning and expression analysis of major histocompatibility complex class IIB gene of the Whitespotted bambooshark (Chiloscyllium plagiosum).

Major histocompatibility complex (MHC) plays an important role in the immune response to antigenic peptides in vertebrates. In this study, the full length of MHC IIB cDNA was isolated from the Whitespotted bambooshark (Chiloscyllium plagiosum) by homology cloning, and the rapid amplification of cDNA ends polymerase chain reaction. As a result, the MHC IIB cDNA is 1,407 bp, which contains an open reading frame (ORF) of 831 bp encoding a protein of 276 amino acids. Furthermore, seven alleles of the complete MHC IIB ORF were detected and the variable sites were mainly located in the immunoglobulin-like (ß2) region. Tissue distribution analysis showed that MHC IIB can be detected in all the ten tissues examined, with the highest expression in the spleen and gill. Challenge of C. plagiosum with the pathogenic bacteria, Vibrio harveyi, resulted in significant changes in the expression of MHC IIB mRNA in the three immune-related tissues (gill, liver and spleen). These results show that the MHC IIB plays an important role in response to bacterial infection in elasmobranches.

Molecular cloning, expression analysis of insulin-like growth factor I (IGF-I) gene and IGF-I serum concentration in female and male Tongue sole (Cynoglossus semilaevis).

Insulin-like growth factor I (IGF-I) is a polypeptide hormone that regulates growth during all stages of development in vertebrates. To examine the mechanisms of the sexual growth dimorphism in the Tongue sole (Cynoglossus semilaevis), molecular cloning, expression analysis of IGF-I gene and IGF-I serum concentration analysis were performed. As a result, the IGF-I cDNA sequence is 911 bp, which contains an open reading frame (ORF) of 564 bp encoding a protein of 187 amino acids. The sex-specific tissue expression was analyzed by using 14 tissues from females, normal males and extra-large male adults. The IGF-I mRNA was predominantly expressed in liver, and the IGF-I expression levels in females and extra-large males were 1.9 and 10.2 times as much as those in normal males, respectively. Sex differences in IGF-I mRNA expressions at early life stages were also examined by using a full-sib family of C. semilaevis, and the IGF-I mRNA was detected at all of the 27 sampling points from 10 to 410 days old. An increase in IGF-I mRNA was detected after 190 day old fish. The significantly higher levels of IGF-I mRNA in females were observed after 190 days old in comparison with males (P<0.01). The IGF-I concentrations in serum of mature individuals were detected by ELISA. The IGF-I level in the serum of females was approximately two times as much as that of males. Consequently, IGF-I may play an important role in the endocrine regulation of the sexually dimorphic growth of C. semilaevis.

Characterization of eight polymorphic microsatellite loci for the giant grouper (Epinephelus lanceolatus Bloch).

Eight polymorphic microsatellite loci were isolated and characterized using a small insert genomic DNA library for the giant grouper (Epinephelus lanceolatus Bloch, 1790), a commercially valuable marine fish in tropical waters. They showed polymorphism information content ranging from 0.177 to 0.775, allele numbers ranging from two to 10, effective allele numbers ranging from 1.227 to 5.012, and observed and expected heterozygosities from 0.2 to 0.733 and from 0.185 to 0.801, respectively, which we anticipate will be useful for population genetic studies of the giant grouper.