Crustacean female sex hormone: More than a female phenotypes-related hormone in a protandric simultaneous hermaphroditism shrimp.

Crustacean female sex hormone (CFSH) is believed to regulate the development of female-related phenotypes in crustaceans. However, its role in gonadal development has been understudied. This study identified a CFSH gene, Lvit-CFSH1b, in the peppermint shrimp Lysmata vittata, a protandric simultaneous hermaphroditism (PSH) species. Lvit-CFSH1b is only expressed in the eyestalk ganglion. qRT-PCR showed that the expression level of Lvit-CFSH1b significantly increased with the gonad development from stage I to III (male phase) and decreased at stage IV (euhermaphrodite phase). Gene knockdown of Lvit-CFSH1b resulted in retardation of female phenotypes and stimulated the development of male phenotypes. At the same time, ovarian development was inhibited, and spermatogenesis was promoted. In addition, injection of rCFSH1b increased ovarian expression of vitellogenin (Lvit-Vg) and hepatopancreas expression of vitellogenin receptor (Lvit-VgR), while suppressing the expressions of insulin-like androgenic gland hormones (Lvit-IAG1 and Lvit-IAG2) in androgenic glands. The addition of rCFSH1b induced the in vitro expression of Lvit-Vg in ovarian and Lvit-VgR in hepatopancreas explants. In conclusion, this study provides convincing evidence that CFSH expedites the feminization process and impedes masculinization by inhibiting IAG in hermaphroditic crustaceans.

Complete genome sequence of Pseudomonas stutzeri S116 owning bifunctional catalysis provides insights into affecting performance of microbial fuel cells.

BACKGROUND: Pseudomonas stutzeri S116 is a sulfur-oxidizing bacteria isolated from marine sludge. It exhibited excellent electricity generation as bioanode and biocathode applied in microbial fuel cells (MFCs). Complete genome sequencing of P. stutzeri and cyclic voltammetry method were performed to reveal its mechanism in microbial fuel cells system. RESULTS: This study indicated that the MFCs generated a maximum output voltage of 254.2 mV and 226.0 mV, and maximum power density of 765 mW/m2 and 656.6 mW/m2 respectively. Complete genome sequencing of P. stutzeri S116 was performed to indicate that most function genes showed high similarities with P. stutzeri, and its primary annotations were associated with energy production and conversion (6.84%), amino acid transport and metabolism (6.82%) and inorganic ion transport and metabolism (6.77%). Homology of 36 genes involved in oxidative phosphorylation was detected, which suggests the strain S116 possesses an integrated electron transport chain. Additionally, many genes encoding pilus-assembly proteins and redox mediators (riboflavin and phenazine) were detected in the databases. Thiosulfate oxidization and dissimilatory nitrate reduction were annotated in the sulfur metabolism pathway and nitrogen metabolism pathway, respectively. Gene function analysis and cyclic voltammetry indicated that P. stutzeri probably possesses cellular machinery such as cytochrome c and redox mediators and can perform extracellular electron transfer and produce electricity in MFCs. CONCLUSION: The redox mediators secreted by P. stutzeri S116 were probably responsible for performance of MFCs. The critical genes and metabolic pathways involved in thiosulfate oxide and nitrate reduction were detected, which indicated that the strain can treat wastewater containing sulfide and nitrite efficiently.

The complete mitogenome of Lysmata vittata (Crustacea: Decapoda: Hippolytidae) with implication of phylogenomics and population genetics.

In this study, the complete mitogenome of Lysmata vittata (Crustacea: Decapoda: Hippolytidae) has been determined. The genome sequence was 22003 base pairs (bp) and it included thirteen protein-coding genes (PCGs), twenty-two transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and three putative control regions (CRs). The nucleotide composition of AT was 71.50%, with a slightly negative AT skewness (-0.04). Usually the standard start codon of the PCGs was ATN, while cox1, nad4L and cox3 began with TTG, TTG and GTG. The canonical termination codon was TAA, while nad5 and nad4 ended with incomplete stop codon T, and cox1 ended with TAG. The mitochondrial gene arrangement of eight species of the Hippolytidae were compared with the order of genes of Decapoda ancestors, finding that the gene arrangement order of the Lebbeus groenlandicus had not changed, but the gene arrangement order of other species changed to varying degrees. The positions of the two tRNAs genes (trnA and trnR) of the L. vittata had translocations, which also showed that the Hippolytidae species were relatively unconserved in evolution. Phylogenetic analysis of 50 shrimp showed that L. vittata formed a monophyletic clade with Lysmata/Exhippolysmata species. This study should be helpful to better understand the evolutionary status, and population genetic diversity of L. vittata and related species.

Insulin-like androgenic gland hormone 1 (IAG1) regulates sexual differentiation in a hermaphrodite shrimp through feedback to neuroendocrine factors.

Insulin-like androgenic gland hormone (IAG) is regarded as a key sexual differentiation regulator in gonochoristic crustaceans. However, until now the knowledge concerning its functions in hermaphroditic crustaceans is scanty. Herein, we investigated the function of IAG (Lvit-IAG1) in peppermint shrimp Lysmata vittata, a species that possesses protandric simultaneous hermaphroditism (PSH) reproductive system, which is rare among crustaceans. Lvit-IAG1 was exclusively expressed in the androgenic gland. The qRT-PCR demonstrated that its mRNA expression level was relatively high at the functional male phase but decreased sharply in the subsequent euhermaphrodite phase. Both the short-term and long-term silencing experiments showed that Lvit-IAG1 negatively regulated both the gonad-inhibiting hormone (Lvit-GIH) and crustacean female sex hormone (Lvit-CFSH) expressions in the eyestalk ganglion. Besides, Lvit-IAG1 gene knockdown induced a retarded development of the appendices masculinae (AM) and male gonopores while suppressing the germ cells at the primary spermatocyte stage. Also, Lvit-IAG1 gene silencing hindered ovarian development. This in turn led to small vitellogenic oocytes and decreased expression of vitellogenin and vitellogenin receptor genes in hepatopancreas and ovarian region, respectively. Generally, this study's findings imply that Lvit-IAG1 modulated the male sexual differentiation in PSH species L. vittata, and exhibited negative feedback on Lvit-GIH and Lvit-CFSH genes expression in the species' eyestalk ganglion.

Comparison of the complete mitochondrial genome of Phyllophorus liuwutiensis (Echinodermata: Holothuroidea: Phyllophoridae) to that of other sea cucumbers.

Sea cucumber species are abundant (>1400 species) and widely distributed globally. mtDNA sequencing is frequently used to identify the phylogenetic and evolutionary relationships among species. However, there are no reports on the mitochondrial genome of Phyllophorus liuwutiensis. Here, we performed mtDNA sequencing of P. liuwutiensis to examine its phylogenetic relationships with other echinoderms. Its mitochondrial genome (15 969 bp) contains 37 coding genes, including 13 protein-coding genes, 22 tRNA genes and 2 rRNA genes. Except for one protein-coding gene (nad6) and five tRNA genes encoded on the negative strand, all other genes were encoded on the positive strand. The mitochondrial bases of P. liuwutiensis were composed of 29.55% T, 22.16% C, 35.64% A and 12.64% G. The putative control region was 703 bp in length. Seven overlapping regions (1-10 bp) were found. The noncoding region between the genes ranged from 1 to 130 bp in length. One putative control region has been found in the P. liuwutiensis mitogenome. All of the tRNA genes were predicted to fold into a cloverleaf structure. In addition, we compared the gene arrangements of six echinoderms, revealing that the gene order of P. liuwutiensis was a new arrangement.

Ontogenetic development of gonads and external sexual characters of the protandric simultaneous hermaphrodite peppermint shrimp, Lysmata vittata (Caridea: Hippolytidae).

The peppermint shrimp Lysmata vittata (Caridea: Hippolytidae) is a marine caridean shrimp popular in marine aquarium trade. The species is known to display the sexual system of protandric simultaneous hermaphrodite. In this study, based on captive bred specimens, the complete ontogenetic gonad development of L. vittata was studied both morphologically and histologically, from newly settled juveniles until they reached euhermaphrodite phase. It was found that in all specimens examined (carapace length: 1.8-8.5 mm), including the newly settled juveniles, possessed ovotestes, which comprised of an anterior ovarian and a posterior testicular part. Based on both morphological (e.g., size, color and shape) and histological features (e.g., oogenesis and spermatogenesis), four gonadal development stages were defined and described for L. vittata. From Stage I to III, the testicular part of the gonad became gradually mature but the ovarian part was still immature, which is defined as the male phase. At the male phase, cincinulli (5-8 hooks) presented at the tips of the appendix interna on the first pair of pleopods while appendices masculinae (AM), in a form of a stick structure with spines, presented at the inner edge of the appendix interna (AI) on the second pair of pleopods. At Stage IV, both the testicular part and the ovarian part were mature and hence is defined as euhermaphrodite phase. At the euhermaphrodite phase, most individuals lacked cincinulli and appendices masculinae on the first and second pair of pleopods respectively. This is the first time that complete ontogenetic gonadal and external sexual character development have been described and staged for a species from the genus Lysmata from newly settled juveniles to euhermaphrodite phase.

Complete mitochondrial genome of Holothuria leucospilata (Holothuroidea, Holothuriidae) and phylogenetic analysis.

The complete Holothuria leucospilata mitochondrial genome was determined and analyzed in this work. It had a circular mapping molecular with a total length of 15,904 bp and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 putative control region. Phylogenetic analysis showed that H. leucospilata clustered together with Holothuria scabra and Holothuria forskali. The complete mitochondrial genome provided in this work would be used for elucidation of Holothuroidea conservation genetics and evolutionary relationships.

Immunity to nervous necrosis virus infections of orange-spotted grouper (Epinephelus coioides) by vaccination with virus-like particles.

Nervous necrosis virus (NNV) is a kind of the betanodaviruses, which can cause viral nervous necrosis (VNN) and massive mortality in larval and juvenile stages of orange-spotted grouper (Epinephelus coioides). Due to the lack of viral genomes, virus-like particles (VLPs) are considered as one of the most promising candidates in vaccine study to control this disease. In this study, a type of VLPs, which was engineered on the basis of orange-spotted grouper nervous necrosis virus (OGNNV), was produced from prokaryotes. They possessed the similar structure and size to the native NNV. In addition, synthetic oligodeoxynucleotide (ODN) containing CpG motif was added in vaccines, and the expression patterns of several genes were analyzed after injecting with VLP and VLP with adjuvant (VA) to assess the regulation effect of vaccine for inducing immune responses. RT-PCR assays showed that six related genes in healthy tissues were ubiquitously expressed in all nine tested tissues. The vaccine alone was able to enhance the expression of genes, including MHCIa, MyD88, TLR3, TLR9 and TLR22 after vaccination, indicating that the vaccine was able to induce immune response in grouper. In liver, spleen and kidney, the gene expressions of VA group were all significantly higher than that of VLP group at 72 h post-stimulation, showing that the fish of VA challenge group obtained the longer-lasting protective immunity and resistance to pathogen challenge than that of VLP group. The data indicated that the efficacy of vaccine could be further enhanced by CpG ODN after vaccination and provided the reference for the development of future viral vaccine in grouper.

Characterization of 40 full-length MHC class IIA functional alleles in miiuy croaker: Polymorphism and positive selection.

The major histocompatibility complex is a highly polymorphic gene superfamily in vertebrates that plays an important role in adaptive immune response. In the present study, we identified 40 full-length miiuy croaker MHC class IIA (Mimi-DAA) functional alleles from 26 miiuy croaker individuals and found that the alleles encode 30 amino acid sequences. A high level of polymorphism in Mimi-DAA was detected in miiuy croaker. The rate of non-synonymous substitutions (d(N)) occurred at a significantly higher frequency than that of synonymous substitutions (d(S)) in the peptide-binding region (PBR) and non-PBR. This result suggests that balancing selection maintains polymorphisms at the Mimi-DAA locus. Phylogenetic analysis based on the full-length sequences showed that the Mimi-DAA alleles clustered into three groups. However, the phylogenetic tree constructed using the exon 2 sequences indicated that the Mimi-DAA alleles clustered into two groups. A total of 22 positively selected sites were identified on the Mimi-DAA alleles after testing for positive selection, and five sites were predicted to be associated with the binding of peptide antigen, suggesting that a few selected residues may play a significant role in immune function.

Characterization and expression of the CXCR1 and CXCR4 in miiuy croaker and evolutionary analysis shows the strong positive selection pressures imposed in mammal CXCR1.

The innate immune system can recognize non-self, danger signals, and pathogen associated molecular patterns and provides a first line of antimicrobial host defense. Therefore, it plays an instructive role and is pretty important in vertebrates. In innate immune responses, CXCRs act as the main receptors of CXC chemokines and play a vital role in host defense and inflammation. In present study, we cloned two cDNA molecules of CXCR1 and CXCR4 in Miichthys miiuy (miiuy croaker). In these two genes, we found the most highly conserved DRY motif in the second intracellular loop adjacent to the third transmembrane domain. The expressions of CXCR1 and CXCR4 showed that they were ubiquitously expressed in ten normal tissues. After infection with Vibrio anguillarum and Vibrio harveyi, the expressions of CXCRs in the immune tissues were significantly regulated in most of tissues except that of CXCR1 in the kidney after V. harveyi injection. Evolutionary analysis showed that only the ancestral lineages of CXCR4 in amphibians underwent positive selection, indicating that the ancestors of amphibians boarded the land and had to further evolve to adapt to terrestrial environments. Multiple ML methods were implemented to detect the robust positively selected candidates for sites. In total, we detected 12 and 3 positively selected sites in the subsets of current mammal and fish CXCR1 genes, and only one site under positive selection was found in mammalian CXCR4 subsets. These positively selected sites were mainly located in the extracellular domains of CXCRs. The sliding window analysis and evolution test tended to favor positive selection acting on the N-terminal domain of CXCR1, which was the critical region for ligand/receptor signaling for neutrophils and receptor-ligand interaction, indicating that the N-terminal of CXCR1 in mammals underwent more positive selection than that of fish.

Characterization of the CCR3 and CCR9 genes in miiuy croaker and different selection pressures imposed on different domains between mammals and teleosts.

The innate immune system can recognize non-self through pattern recognition receptors and provides a first line of antimicrobial host defense. Thus innate immunity plays a very important role in resistance against major bacterial disease in vertebrates. In the innate immune responses, the chemokine receptors act as the main receptors of chemokines which are released at the sites of infection, inflammation and injury. In this study, the Miichthys miiuy CCR3 and CCR9 genes were cloned and characterized, showing that MIMI-CCR3 possesses a highly conserved DRYLA motif similar to that of other fishes. MIMI-CCR3 and CCR9 were ubiquitously expressed in all tested tissues and the expressions were significantly up-regulated after infection with Vibrio anguillarum except that of CCR9 in spleen. Evolutionary analysis showed that all the ancestral lineages of CCR3 and CCR9 in mammals and teleosts underwent positive selection, indicating that the ancestor of terrestrial animals further evolved to adapt to terrestrial environments and the continuous intrusion of microbes stimulated the evolution of CCR genes in the ancestor of teleost. Multiple ML methods were used to detect the robust candidates for sites under positive selection. In total, 11 and 8 positively selected sites were found in the subsets of current mammal and teleost CCR3 genes, and 3 and 2 sites were detected in the subsets of current mammals and teleosts in CCR9. Interestingly, for mammal CCR3 and CCR9 genes, the robust candidates of positively selected sites were mainly located in the extracellular domains which were the ligand binding and pathogen interaction regions. For teleost CCR3 and CCR9 genes, the positively selected sites were not only located in the extracellular domains but also in the C-terminal and intracellular domains, indicating mammals and teleosts experienced different selection pressures upon their N-terminus, C-terminus and intracellular loops of CCRs.

Evolutionary analysis of TLR9 genes reveals the positive selection of extant teleosts in Perciformes.

The innate immune system can recognize non-self through pattern recognition receptors. Toll-like receptors were the best-known members of these receptors, and they could sense, recognize, and bind pathogen-associated molecular patterns. TLRs played an important role in innate immune system and were conserved in both invertebrate and vertebrate lineages. Thereinto, TLR9 could detect unmethylated CpG motifs in dsDNA and was expected to undergo coevolution with its microbial ligands. It was known that aquatic and terrestrial organisms dwelled in different environments which contained different pathogens, and they had to adapt to their local environmental conditions. Therefore, we collected TLR9 genes from invertebrate to vertebrate to further explore whether the huge differences between aquatic and terrestrial environments affected the TLR9s evolution between aquatic and terrestrial organisms. Molecular evolution analysis detected positively selected sites in the ancestral lineages of vertebrates, teleosts, and Perciformes but not in the ancestral lineage of mammals. In PAML, site model revealed that extant mammalian TLR9 genes underwent positive selection. However, the positive selection of extant teleosts appeared primarily in Perciformes in which there were 14 positively selected sites. Among these sites, two of them were located on the amino acid insertions of the leucine-rich repeats which could create DNA binding sites, three were found on the convex surface which might possibly affect the flexibility of the TLR solenoids, and six were located on the ß-face of concave surface which contained the ligand-binding sites of the TLR solenoids. In other ML methods, we also found three sites under selection that coincided with the codons identified by M8 and these sites were all located in LRRs. The diverse aquatic and terrestrial environments might possess different pathogens to make the living organisms adapt to their local environmental conditions. The positive selection on LRRs in TLR9s of Perciformes might be associated with the adaptation to the rapidly evolving pathogens in the water.

Characterization and comprehensive analysis of the miiuy croaker TLR2 reveals a direct evidence for intron insert and loss.

Toll-like receptor 2 (TLR2) is a member of an ancient pattern recognition receptor family, conserved from insects to mammals and it is best known as a receptor for recognizing conserved components of Gram-positive bacteria. In present study, the genomic structure of TLR2 gene from miiuy croaker was identified and characterized. It comprises twelve exons and eleven introns. The lengths of exons 3 to 10 of miiuy croaker TLR2 and exons 2 to 9 of fugu and pufferfish TLR2 are exactly the same, but most importantly, both of fugu and pufferfish have only eleven exons and ten introns. An intron insert event probably happened on exon 1 of miiuy croaker TLR2 after its divergence from ancestor of zebrafish, and an intron loss event probably happened on those of Tetraodontiformes TLR2 after the divergence with ancestor of miiuy croaker. Our study showed the direct evidence and strongly supported the intron insert and loss on fish TLR2. The pathogen injection experiments indicated that TLR2 might not be an important responder to Gram-negative bacteria in miiuy croaker. Molecular evolutionary analyses indicated TLR2 genes were under strong purifying selection pressure, showing a quite strong functional constraint in both of fish and mammals, despite of their distinct living environment conditions.

Miiuy croaker transferrin gene and evidence for positive selection events reveal different evolutionary patterns.

Transferrin (TF) is a protein that plays a central role in iron metabolism. This protein is associated with the innate immune system, which is responsible for disease defense responses after bacterial infection. The clear link between TF and the immune defense mechanism has led researchers to consider TF as a candidate gene for disease resistance. In this study, the Miichthys miiuy (miiuy croaker) TF gene (MIMI-TF) was cloned and characterized. The gene structure consisted of a coding region of 2070 nucleotides divided into 17 exons, as well as a non-coding region that included 16 introns and spans 6757 nucleotides. The deduced MIMI-TF protein consisted of 689 amino acids that comprised a signal peptide and two lobes (N- and C-lobes). MIMI-TF expression was significantly up-regulated after infection with Vibrio anguillarum. A series of model tests implemented in the CODEML program showed that TF underwent a complex evolutionary process. Branch-site models revealed that vertebrate TF was vastly different from that of invertebrates, and that the TF of the ancestors of aquatic and terrestrial organisms underwent different selection pressures. The site models detected 10 positively selected sites in extant TF genes. One site was located in the cleft between the N1 and N2 domains and was expected to affect the capability of TF to bind to or release iron indirectly. In addition, eight sites were found near the TF exterior. Two of these sites, which could have evolved from the competition for iron between pathogenic bacteria and TF, were located in potential pathogen-binding domains. Our results could be used to further investigate the function of TF and the selective mechanisms involved.