Kecheng Zhu

Characterization of complete mitochondrial genome of fives tripe wrasse (Thalassoma quinquevittatum, Lay & Bennett, 1839) and phylogenetic analysis

To further supplement the genome-level features in related species, T. quinquevittatum complete mtDNA was firstly sequenced and de novo assembled by next-generation sequencing. The full-length mtDNA of T. quinquevittatum was a 16,896bp fragment, which was atypical of Labridae, with 2 ribosomal RNA (rRNA) genes, 13 protein-coding genes (PCGs), 23 transfer RNA (tRNA) genes, and a major non-coding control region (D-loop region). Additionally, the mtDNA of T. quinquevittatum exhibited characteristics of A (27.1%), T (29.3%), G (17.8%), and C (25.8%) with a high A+T content (56.4%). Furthermore, the analysis of the average Ka/Ks in the 13 PCGs of three Labridae species indicated a strong purifying selection within this group. Additionally, the phylogenetic analysis based on 13 concatenated PCGs nucleotide and amino acid datasets, showed high value support for the following sister clade among the four genera (T. quinquevittatum, Halichoeres trimaculatus, Halichoeres melanurus, Parajulis poecilepterus). The complete mtDNA of the T. quinquevittatum provided important information for the study in population genetics and evolutionary theory.

Shotgun assembly of the mitochondrial genome from Fenneropenaeus penicillatus with phylogenetic consideration.

The complete mitochondrial genome is of great importance for better understanding of the genome-level characteristics and phylogenetic relationships among related species. In this study, Fenneropenaeus penicillatus mitochondrial genome sequence was determined by next-generation sequencing. The complete genome DNA was 16,040 bp in length and consisted of a typical set of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and a putative control region (CR). The gene arrangement is identical to the pancrustacean pattern. The overall base composition of its mitochondrial genome is estimated to be 34.1% for A, 34.1% for T, 12.5% for G and 19.3% for C with a high A+T content (68.2%). The analysis of the average Ka/Ks in the 13 mitochondrial protein-coding genes of penaeid shrimps indicated a strong purifying selection within this group. The phylogenetic analysis based on mitochondrial sequences and 13 concatenated protein-coding genes showed strong statistic support for the following relationship among the five genera ((Penaeus s.s+Fenneropenaeus)+(Litopenaeus+Farfantepenaeus))+Marsupenaeus. The sequence data of F. penicillatus can provide useful information for the studies on molecular systematics, population structure, stock evaluation and conservation genetics.

Feed type regulates the fatty acid profiles of golden pompano Trachinotus ovatus (Linnaeus 1758)

ABSTRACT The aim of the present study was to evaluate the effect of different feed types on the fatty acid profiles of golden pompano (Trachinotus ovatus). Three feed types (pelleted feed, frozen squid and frozen fish) were assigned to triplicate groups of fish (146.22 ± 3.26 g) in a total of 9 floating cages (50 fish per cage). Analysis of the fatty acid profiles of the three feed types revealed that frozen squid had the highest levels of long-chain polyunsaturated fatty acids (LC-PUFA), while the pelleted feed and frozen fish had lower levels of LC-PUFA, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). After three months of being fed a specific feed type, the fatty acid profiles of golden pompano muscle tissues were analysed. The highest levels of LC-PUFA were detected in fish fed with frozen squid. Golden pompano fed with frozen fish had intermediate levels of LC-PUFA content. The results suggest that the fatty acid composition of prey items can significantly affect the muscle fatty acid composition of the consumer. Muscle fatty acid composition could also be used as an indicator of diet type for golden pompano in the aquaculture industry.

Functional characteristic and differential expression of myostatin in Chlamys nobilis

ABSTRACT Myostatin (MSTN) was a conserved negative regulatory protein of muscle growth and development in numerous species. In this study, the MSTN gene was cloned and characterized from a noble scallop, Chlamys nobilis, and it was named CN-MSTN. To investigate the molecular characterization of MSTN and its gene expression profile of spatio-temporal, we isolated the MSTN cDNA sequence in C. nobilis and analysed expression patterns using quantitative real-time PCR. CN-MSTN cDNA contained a 1,374 bp open reading frame that encoded a 458 amino acids. Furthermore, the result of 3D model indicated that CN-MSTN mature peptide was similar to that of Chlamys farreri. Additionally, based on both the nucleotide and amino acid datasets, phylogenetic trees indicated that C. nobilis has the highest similarity with homologues of C. farreri. The expression patterns of different stages showed that MSTN expression was markedly higher in the gastrulae period. Additionally, remarkable expression of MSTN was observed in adductor muscle than with other tissues. Our data would provide expression and phylogenetic information of this economical important sea food, noble scallop.

Genomic structure, expression pattern and polymorphisms of GILT in golden pompano Trachinotus ovatus (Linnaeus 1758)

The interferon-g-inducible lysosomal thiol reductase (GILT) plays a significant character in the processing and presentation of MHC class II restricted antigen (Ag) by catalyzing disulfide bond reduction in mammals. To explore the function of GILT in the immune system of fish, we cloned a GILT gene homologue from Trachinotus ovatus, the full-length cDNA of GILT, which consisted of 2, 747 bp with a 771 bp open reading frame, encoding a protein of 256 amino acids. Moreover, similar to other species GILT gene, 7 exons and 6 introns were identified in T. ovatus, the deduced protein also possessed a representative characteristic of known GILT proteins. The result of real-time quantitative PCR showed that GILT mRNA was dramatically expressed in immune-associated tissues, such as spleen (p < 0.01) and kidney (p < 0.05). Bacterial challenge revealed that GILT mRNA level remarkably up-regulation in liver, spleen, kidney and intestine after induction with Photobacterium damsela. Furthermore, based on cloned sequences and genome BLAST, only one SNP site (ToGILT-S1-g.148C>G) was identified, and the allele C was significantly associated with high-susceptibility (HS) group, nevertheless, the allele G was dramatically associated with high-resistance (HR) group, indicating potential application for disease resistant breeding selection in T. ovatus.

The Transcriptional Factor PPARαb Positively Regulates Elovl5 Elongase in Golden Pompano Trachinotus ovatus (Linnaeus 1758)

The nuclear peroxisome proliferator-activated receptors (PPARs) regulate the transcription of elongases of very long-chain fatty acids (Elovl), which are involved in polyunsaturated fatty acid (PUFA) biosynthesis in mammals. In the present study, we first characterized the function of Elovl5 elongase in Trachinotus ovatus. The functional study showed that ToElovl5 displayed high elongation activity toward C18 and C20 PUFA. To investigate whether PPARαb was a regulator of Elovl5, we also reported the sequence of T. ovatus PPARαb (ToPPARαb). The open reading frame (ORF) sequence encoded 469 amino acids possessing four typical characteristic domains, including an N-terminal hypervariable region, a DNA-binding domain (DBD), a flexible hinge domain and a ligand-binding domain (LBD). Thirdly, promoter activity experiments showed that the region from PGL3-basic-Elovl5-5 (-146 bp to +459 bp) was defined as the core promoter by progressive deletion mutation of Elovl5. Moreover, PPARαb overexpression led to a clear time-dependent enhancement of ToElovl5 promoter expression in HEK 293T cells. Fourth, the agonist of PPARαb prominently increased PPARαb and Elovl5 expression, while PPARαb depletion by RNAi or an inhibitor was correlated with a significant reduction of Elovl5 transcription in T. ovatus caudal fin cells (TOCF). In conclusion, the present study provides the first evidence of the positive regulation of Elovl5 transcription by PPARαb and contributes to a better understanding of the transcriptional mechanism of PPARαb in fish.

Genomic structure and molecular characterization of Toll-like receptors 1 and 2 from golden pompano Trachinotus ovatus (Linnaeus, 1758) and their expression response to three types of pathogen-associated molecular patterns

Abstract Toll‐like receptors (TLRs) play an essential role in the immune response. Here two Toll‐like receptors from golden pompano (Trachinotus ovatus), ToTLR1 and ToTLR2, were characterized, the full‐length cDNAs were 3126 bp and 7430 bp, and the deduced proteins consisted of 801 and 825 amino acids, respectively. ToTLR1 and ToTLR2 both contained the typical TLR domain architecture including signal peptide, leucine rich repeat (LRR), C‐terminal LRR domain at the extracellular region and Toll/interleukin (IL)‐1 receptor (TIR) domain in the cytoplasmic region. ToTLR1 only had one intron and two exons, but ToTLR2 consisted of twelve introns and thirteen exons. The promoters of ToTLR1 and ToTLR2 contained several putative transcription factor binding sites. Phylogenetic analysis showed that ToTLR1 and ToTLR2 were clustered into the clade of TLR1 and TLR2, respectively. Tissues distribution analysis indicated that both genes were ubiquitously expressed in all examined tissues, with higher expression levels observed in blood, head‐kidney and spleen. After injection with poly inosinic:cytidylic [poly(I:C)], flagellin and lipopolysaccharides (LPS), ToTLR1 and ToTLR2 mRNAs were significantly up‐regulated in the immune related tissues, indicating the possible the role of ToTLR1 and ToTLR2 in defense against pathogenic microbes. Further research should be carried out to identify ligands of fish TLR1 and TLR2 in order to understand the function of these receptors. HighlightsThe full‐length TLR1 and TLR2 cDNA and genomic sequences were identified and characterized from golden pompano.ToTLR1 and ToTLR2 were clustered into an independent clade of TLR1 family.ToTLR1 and ToTLR2 might play the important roles in defense against bacterial and parasite infection.

Molecular characterization of Na+/K+/2Cl− cotransporter 1 alpha from Trachinotus ovatus (Linnaeus, 1758) and its expression responses to acute salinity stress

Trachinotus ovatus is widely cultured in the ponds and gulf on the southeast coast of China. The dramatic salinity decrease caused by heavy rainfall could cause mass mortality of T. ovatus in aquaculture. It is very important to understand the osmoregulatory mechanism of T. ovatus. Na+/K+/2Cl- cotransporter 1a (NKCC1a) is involved in the osmoregulation of fish and plays a crucial role in cell volume homeostasis and maintenance of the electrolyte content. In this study, we characterized nkcc1a (designed as Tonkcc1a) from T. ovatus and investigated its expression responses to acute salinity changes. Tonkcc1a is approximately 70 kb in length and contains 26 exons and 25 introns. The phylogenetic analysis confirmed that ToNKCC1a belonged to the NKCC1a subclade. Quantitative real-time (qRT-PCR) analysis indicated that Tonkcc1a was ubiquitously expressed in all examined tissues, with the highest mRNA levels observed in gills, and the lowest level in liver. When T. ovatus were transferred from seawater (30‰) into fresh water, the expression levels of Tonkcc1a mRNA were significantly downregulated in gills and kidney, whereas its expression level was markedly upregulated in intestine. When transferred from seawater (30‰) to 10‰ sea water, the expression levels of Tonkcc1a mRNA were clearly increased in gills and kidney. When transferred from seawater (30‰) to 20‰ sea water, the expression of Tonkcc1a mRNA increased to some extent in gills, kidney, and intestine. When transferred from seawater (30‰) to 40‰ sea water, the expression levels of Tonkcc1a mRNA were dramatically upregulated in gills and intestine compared to that in the control. These results suggested that Tonkcc1a was involved in the response to acute salinity changes.

Comprehensive assessment of the genetic diversity and population structure of cultured populations of golden pompano, Trachinotus ovatus (Linnaeus, 1758), by microsatellites

Golden pompano, Trachinotus ovatus, belongs to the family Carangidae. Within one decade, this species has rapidly become one of the most important cultured marine fish species in South China. However, the lack of a comprehensive genetic diversity assessment hinders the conservation of natural resources and management of cultured populations. Thus, we sampled one wild population and six cultured populations of golden pompano, which represented the whole cultured population, to assess the genetic diversity and population structure. The level of genetic diversity was low compared with those of other cultured fish, as the values of allelic richness, number of effective alleles and expected heterozygosity in the combined whole sample were 3.709, 2.592, and 0.591, respectively. These populations were little differentiated (Fst = 0.02091, p value = 0.00), and the whole sample did not show an explicit population structure at the individual level. The effective population size in each cultured population was small and in the whole sample was acceptable for a closed selection system. The pedigree reconstruction showed no evidence of artificial disturbance in mating. This general survey of cultured golden pompano populations showed the common characteristics of domestication with wild inputs. However, the low level of and potential decrease in genetic diversity should receive close attention in future breeding programs. In addition, we recommend wide surveys of natural resources and the establishment of closed breeding systems to increase genetic gain.

Sequencing and characterization of the complete mitochondrial genome of Japanese Swellshark ( Cephalloscyllium umbratile )

To further comprehend the genome features of Cephalloscyllium umbratile (Carcharhiniformes), an endangered species, the complete mitochondrial DNA (mtDNA) was firstly sequenced and annotated. The full-length mtDNA of C. umbratile was 16,697 bp and contained ribosomal RNA (rRNA) genes, 13 protein-coding genes (PCGs), 23 transfer RNA (tRNA) genes, and a major non-coding control region. Each PCG was initiated by an authoritative ATN codon, except for COX1 initiated by a GTG codon. Seven of 13 PCGs had a typical TAA termination codon, while others terminated with a single T or TA. Moreover, the relative synonymous codon usage of the 13 PCGs was consistent with that of other published Carcharhiniformes. All tRNA genes had typical clover-leaf secondary structures, except for tRNA-Ser (GCT), which lacked the dihydrouridine ‘DHU’ arm. Furthermore, the analysis of the average Ka/Ks in the 13 PCGs of three Carcharhiniformes species indicated a strong purifying selection within this group. In addition, phylogenetic analysis revealed that C. umbratile was closely related to Glyphis glyphis and Glyphis garricki. Our data supply a useful resource for further studies on genetic diversity and population structure of C. umbratile.