Characterization of the complete mitochondrial genome of brown barracuda, Sphyraena pinguis (Perciformes: Sphyraenidae).

The entire mitochondrial genome sequence of Sphyraena pinguis collected from Korean water was determined by the Next Generation Sequencing (NGS) technology. Its total length was 16,620 bps in length, which possessed the canonical 37 genes in the eukaryotes. Unusual start codon was exclusively found in COX1(GTG), while incomplete stop codons (TA-/T-) were identified in ATP6, COX2, ND3, ND4, and Cyt b. A phylogenetic analysis with currently identified full mitogenomes in Perciformes, S. pinguis was most closely related to S. barracuda (76.87%) and S. jello (76.84%). This mitogenome sequence would explain the evolution of genus Sphyraena.

Characterization of the complete mitochondrial genome of Odontobutis platycephala collected from Nakdong River, South Korea.

The complete mitochondrial genome of Odontobutis platycephala collected from a native Korean river was determined by the bioinformatics assembly of the next-generation sequencing (NGS) reads. The circular mitogenome was 17,590 bp length which harbored canonical 13 protein-coding genes, 22 tRNAs, and 2 rRNAs, which was identical to those of family Odontobutidae. Twenty-eight genes were located on H strand, whereas remaining nine genes were on L strand. Except for COX1 gene (GTG), other 12 protein-coding genes were predicted typical start codons (ATG). Among the currently known mitogenome sequences, O. platycephala showed highest identity (96.98%) to Korean haplotype of O. platycephala (NC010199).

Four Members of Heat Shock Protein 70 Family in Korean Rose Bitterling (Rhodeus uyekii).

Heat shock protein (HSP) 70, the highly conserved stress protein families, plays important roles in protecting cells against heat and other stresses in most animal species. In the present study, we identified and characterized four Hsp70 (RuHSP4, RuHSC70, RuHSP12A, RuGRP78) family proteins based on the expressed sequence tag (EST) analysis of the Korean rose bitterling R. uyekii cDNA library. The deduced RuHSP70 family has high amino acid identities of 72-99% with those of other species. Phylogenetic analysis revealed that RuHsp70 family clustered with fish groups (HSP4, HSC70, HSP12A, GRP78) proteins. Quantitative RT-PCR analysis showed the specific expression patterns of RuHsp70 family members in the early developmental stages and several tissues in Korean rose bitterling. The expression of 4 groups of Hsp70 family was detected in all tested tissue. Particularly, Hsp70 family of Korean rose bitterling is highly expressed in hepatopancreas and sexual gonad (testis and ovary). The expression of Hsp70 family was differentially regulated in accordance with early development stage of Rhodeus uyekii.

Effective RNA-silencing strategy of Lv-MSTN/GDF11 gene and its effects on the growth in shrimp, Litopenaeus vannamei.

Myostatin (MSTN), also known as GDF8, is a member of the transforming growth factor-ß (TGF-ß) superfamily and plays an important role in muscle growth, development, and differentiation. Recently, Lv-MSTN/GDF11, the primitive isoform of MSTN and GDF11, was identified from the shrimp Litopenaeus vannamei. The major production site for Lv-MSTN/GDF11 is in the heart, not the tail muscle, which differs from MSTNs in mammals. Among the three injected RNAs, long dsRNA was the most effective for Lv-MSTN/GDF11 knockdown and transcripts of Lv-MSTN/GDF11 decreased in both the heart (88.85%) and skeletal muscles (43.36%) 72h after injection of 10pmol of long dsRNA. We also found that higher doses of dsRNA did not lead to greater decreases in Lv-MSTN/GDF11 transcripts for amounts between 1pmol and 100pmol. Injection of Lv-MSTN/GDF11 dsRNA did not affect the upregulation of the skeletal actin gene (Lv-ACTINSK) in the tail muscle, but the expression of cytoplasmic and cardiac actins were upregulated in both the heart and tail muscle. Over the course of 8weeks of dsRNA injection, considerably higher mortality (~71%) was seen in the dsRNA-injected group compared to the control group (40%). Surviving shrimp in the dsRNA injected group had a lower growth rate due to the adverse effects of Lv-MSTN/GDF11 knockdown. Lv-MSTN/GDF11 appears to be involved in muscular or neuronal development, but not in doubling muscle fibers, as is the case with mammalian MSTN.

Induction of Primary Male in Juvenile Red Spotted Grouper Epinephelus akaara by Immersion of 17α-Methyltestosterone.

We investigated the androgenic effects of 17α- methyltestosterone (MT) on gonadal sex reversal in juvenile red spotted grouper Epinephelus akaara. The fish were immersed in 17α-MT at 1 and 5 mg/L. Treatment method of 17α-MT was once weekly for 4 and 8 weeks. Fish were sampled at 12 months after end of the treatment period in order to histological analysis. At the initiation of an experiment (70 day after hatching), juvenile red spotted grouper have the paired primordial gonads with somatic cells bellow kidney in the posterior portion of the body cavity. Formation of ovarian cavity indicates that the ovarian differentiation beginning at 70 DAH in red spotted grouper. At 12 months after end of the treatment period, control group, 17α-MT 1 mg/L treatment group for 4 and 8 weeks, and 17α-MT 5 mg/L treatment group for 4 weeks were all female. However, sex-changed males without ovarian cavity were observed in the 17α-MT 5 mg/L treatment group for 8 weeks. In grouper, we firstly reported that the red spotted grouper be able to induce the primary males by hormone treatment prior to gonadal sex differentiation.

Five hepatopancreatic and one epidermal chitinases from a pandalid shrimp (Pandalopsis japonica): cloning and effects of eyestalk ablation on gene expression.

Six cDNAs encoding chitinase proteins in Pandalopsis japonica were isolated by using polymerase chain reaction (PCR) cloning methods and bioinformatic analysis of expressed sequence tags (ESTs). The cDNAs, designated Pj-Cht1, 2, 3A, 3B, 3C, and 4, encoded proteins ranging from 388 to 607 amino acid residues in length (43.61-67.62kDa) and displayed a common structural organization: an N-terminal catalytic domain, a Thr/Pro-rich linker region, and either 0 (Pj-Cht2, 3A), 1 (Pj-Cht1, 3B, and 3C), or 2 (Pj-Cht4) C-terminal chitin-binding domain(s) (CBD). Pj-Cht1 and 2 lacked the 5' end of the open reading frame (ORF); the other Pj-Chts contained the complete ORF. All known decapod crustacean chitinases were segregated into at least four groups based on phylogenetic analysis and domain organization. Group 1 chitinases, represented by Pj-Cht1, were most closely related to insect group I chitinases and may function in the digestion of the peritrophic membrane. Group 2 chitinases including Pj-Cht2 show different domain organizations and pI value from other chitinases and appear to function in degradation of the old exoskeleton during the premolt period. Group 3 chitinases, represented by Pj-Cht3A, 3B, and 3C, may function in digestion of chitin-containing food and defense against pathogens. Group 4 chitinases, represented by Pj-Cht4, have two CBDs and their functions are unknown. Five Pj-Chts (Pj-Cht1, 3A, 3B, 3C, and 4) are expressed in the hepatopancreas and intestine, whereas Pj-Cht2 is expressed in epidermis and SG/XO complex suggesting crustacean chitinases can be classified into two groups (hepatopancreatic and epidermal) based on the expression profile. Eyestalk ablation (ESA) down-regulated the hepatopancreatic chitinase expression (Pj-Cht1, 3A, and 3C); Pj-Cht3B expression was not significantly affected by ESA. By contrast, mRNA levels of Pj-Cht2 were significantly upregulated in 7days post-ESA. Pj-Cht4 mRNA levels were too low for measurement with quantitative polymerase chain reaction. ESA had no significant effect on chitinase expression in the intestine. These data indicate that Pj-Cht1, 3A, 3B, 3C, and 4 are hepatopancreatic chitinases that may function in the digestion of ingested chitin and the modification of peritrophic membrane in the intestine. By contrast, epidermal chitinase, Pj-Cht2 may play a role in chitin metabolism during molt cycle as shown in other crustacean group 2 chitinases.

Characterization of two vitellogenin cDNAs from a Pandalus shrimp (Pandalopsis japonica): expression in hepatopancreas is down-regulated by endosulfan exposure.

Endosulfan is a neurotoxic organochlorine insecticide of the cyclodiene family of pesticides that inhibits molting and reproduction in aquatic crustaceans. In order to determine the molecular mechanism of endosulfan as an endocrine disrupting chemical (EDC), differential display RT-PCR (DDRT-PCR) was used to isolate genes in the shrimp, Pandalopsis japonica, affected by endosulfan exposure. PCR screening of cDNA from the hepatopancreas from control and endosulfan-exposed animals, using 120 sets of random primers, yielded partial cDNAs encoding two vitellogenin-like proteins (Pj-Vg1 and -Vg2). Complete sequences were obtained using a combination of RT-PCR and RACE-PCR. Pj-Vg1 (7883bp) encoded a protein composed of 2533 amino acid residues (283.27 kDa estimated mass), whereas Pj-Vg2 (7792 bp) encoded a protein composed of 2537 amino acids residues (284.87 kDa estimated mass). Alignment of the Pj-Vgs with those of other vitellogenins identified a conserved subtilisin cleavage site (RQKR) and the lipoprotein N-terminal (vitellin), DUF1081, and von Willebrand factor type D domains, indicating both genes encoded functional proteins. Phylogenetic analysis showed that Pj-Vg1 and -Vg2 were most similar to Pandalus hypsinotus Vg. Both Pj-Vg1 and -Vg2 were expressed primarily in the hepatopancreas, although the Pj-Vg2 transcript was also detected in the ovary. The effects of the 3-day endosulfan exposure (2.5 microg/L and 25 microg/L) on Vg expression in the hepatopancreas were determined by quantitative RT-PCR. Expression of both transcripts was significantly inhibited at 25 microg/L suggesting that Pj-Vgs can be used as indicator for endosulfan exposure.