A chromosome-level reference genome assembly of the Reeve's moray eel (Gymnothorax reevesii).

Due to potentially hostile behaviors and elusive habitats, moray eels (Muraenidae) as one group of apex predators in coral reefs all across the globe have not been well investigated. Here, we constructed a chromosome-level genome assembly for the representative Reeve's moray eel (Gymnothorax reevesii). This haplotype genome assembly is 2.17 Gb in length, and 97.87% of the sequences are anchored into 21 chromosomes. It contains 56.34% repetitive sequences and 23,812 protein-coding genes, of which 96.77% are functionally annotated. This sequenced marine species in Anguilliformes makes a good complement to the genetic resource of eel genomes. It not only provides a genetic resource for in-depth studies of the Reeve's moray eel, but also enables deep-going genomic comparisons among various eels.

The Complete Mitochondrial Genome of Plectorhinchus Chaetodonoides (Perciformes: Haemulidae).

Plectorhinchus chaetodonoides Lacepède, 1801 is a widespread multicolored sweetlips fish found in the Indo-West Pacific Ocean where its appearance and color patterns drastically change during growth. In this study, the whole mitochondrial genome of P. chaetodonoides was sequenced which revealed it is 16,546 bp long and contains 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and one noncoding regulatory region. The GC content of the whole genome was 47.5% and 48.2%, 46.3%, 46.8%, 42.5% in the protein coding genes, tRNAs, rRNAs, and control regions, respectively. Molecular phylogenetic analysis resolved P. chaetodonoides as closely associated with Diagramma pictum and nested within a clade of Haemulidae that is allied with species from the Lutjanidae, Kyphosidae, Teraponidae, and Sciaenidae families. These results provide an essential genomic resource for future evolutionary and conservation studies of P. chaetodonoides as well as the Haemulidae family.

The complete mitochondrial genome of Pomadasys kaakan (Perciformes: Haemulidae).

Pomadasys kaakan (Cuvier 1830) is a fish found in coastal waters that is widely distributed in the Western Indo-Pacific Ocean and plays an important role in commercial fisheries. The complete mitochondrial genome of P. kaakan was determined for the first time in this study. The genome was 16,808 bp in length and consisted of 13 protein coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and one noncoding control region. The overall base composition was estimated to be A: 27.1%; T: 24.7%; C: 31.7%; and G: 16.5%, with an AT bias of 51.8%. Molecular phylogenetic analysis suggested that P. kaakan was clustered with species of genera Plectorhinchus, Diagramma, and Parapristipoma, which also belonged to the Haemulidae family. Furthermore, the Haemulidae family was closely related to the group containing Oplegnathidae, Kyphosidae, Teraponidae, and Lutjanidae. These results may provide molecular information for the species evolution and phylogenetic status of P. kaakan in the suborder Percoidei.

Genome-wide identification and characterization of toll-like receptor 5 (TLR5) in fishes.

Toll-like receptors 5 (TLR5), a member of the toll-like receptors (TLRs) family, is a class of pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs). It responds to vertebrate recognition of bacterial flagellin and participates in innate immune responses. However, genome-wide identification and characterization of TLR5 in fishes have not been investigated. Here, three TLR5M isotypes (TLR5Ma, TLR5Mb1, and TLR5Mb2) and a TLR5S are all extracted from fish genomes on the basis of phylogenetic and synteny analyses. We confirmed that the non-teleost fishes have one TLR5M gene, as well as additional TLR5 genes (TLR5M and TLR5S) in teleost fishes. In addition, some special teleost fishes possess two to three TLR5 genes, which have undergone the fourth whole-genome duplication (WGD). According to our results, we inferred that the diversity of TLR5 genes in fishes seems to be the result of combinations of WGD and gene loss. Furthermore, TLR5 isoforms displayed differences at the flagellin interaction sites and viral binding sites, and showed lineage-specific, which indicated that TLR5 duplicates may generate functional divergence. Bacterial experiments also supported the idea that CiTLR5Ma and CiTLR5Mb are subfunctionalized to sense bacterial flagellin. In summary, our present comparative genomic survey will benefit for further functional investigations of TLR5 genes in fish.

The complete mitochondrial genome of Diagramma pictum (Perciformes: Haemulidae).

The painted sweetlips Diagramma pictum (Thunberg 1792) is an important fish for commercial fisheries which is widely distributed in the Indo-West Pacific Ocean. It can change its external coloration and pattern during their lives. The complete mitochondrial genome of D. pictum was determined in this study. The genome was 16,531 bp in length and consisted of 13 protein coding genes, 22 transfer RNA (tRNA), 2 ribosomal RNA (rRNA), and one noncoding control region. The overall base composition was estimated to be A: 27.5%; T: 24.7%; C: 30.9% and G: 16.9% with AT bias of 52.2%. The molecular phylogenetic result revealed that D. pictum did not form an independent branch but was tightly clustered inside the Plectorhinchus groups, closely related to the species Plectorhinchus chaetodonoides, indicating the close relationships between genera Diagramma and Plectorhinchus. These results may provide important genomic information for species evolution and mitogenome based phylogenetic analyses of D. pictum in the family Haemulidae.

Insight into the Regulatory Relationships between the Insulin-Like Androgenic Gland Hormone Gene and the Insulin-Like Androgenic Gland Hormone-binding Protein Gene in Giant Freshwater Prawns (Macrobrachium rosenbergii).

Giant freshwater prawns (Macrobrachium rosenbergii) are commonly found throughout the world. The size of the male giant freshwater prawn is much larger than that of the female. Therefore, understanding the molecular mechanism that underlies the sexual differentiation of M. rosenbergii is of both commercial and scientific importance. Insulin-like androgenic gland hormone (IAG) plays a key role in the differentiation of sex in M. rosenbergii. Although IAG has been investigated, the regulatory relationship between IAG and its binding protein partner, the insulin-like androgenic gland hormone-binding protein (IAGBP), has not been studied in M. rosenbergii. Here, we cloned and characterized the IAGBP from M. rosenbergii (Mr-IAGBP) for the very first time. Transcriptomic analysis showed that Mr-IAGBP mRNA was detected in a wide array of tissues with the highest expression found in the androgenic gland. The importance of IAG in male development was further demonstrated by an increase in IAG transcripts during the development of the androgenic gland and Mr-IAG was only highly transcribed in the androgenic gland of M. rosenbergii. Interestingly, we found that the Mr-IAG gene expression started during the 20th-day larva after hatching stage (LH20), followed (20th-day post-larval stage, PL20) by a gradual elevation of Mr-IAGBP levels. The levels of both genes peaked at the adult stage. The relationship between Mr-IAGBP and Mr-IAG was further analyzed using RNA interference. The injection of Mr-IAGBP double-stranded RNA (dsRNA) significantly reduced the transcription of Mr-IAG, while the amount of Mr-IAGBP mRNA and the translation of IAGBP protein was significantly reduced by the injection of Mr-IAG dsRNA. These results revealed that IAGBP is involved in IAG signaling. Furthermore, our data supports the hypothesis that (IAG and IAGBP)-IAG receptor signaling schemes exist in M. rosenbergii. Our results will provide important information for the further study of determining the sex of M. rosenbergii.

A Comparative Genomic and Transcriptional Survey Providing Novel Insights into Bone Morphogenetic Protein 2 (bmp2) in Fishes.

Intermuscular bones (IBs) are only found in the muscles of fish. Bone morphogenetic protein 2 (bmp2) is considered to be the most active single osteogenesis factor. It promotes cell proliferation and differentiation during bone repair, as well as inducing the formation of bones and cartilages in vivo. However, detailed investigations of this family in fish are incredibly limited. Here, we have used a variety of published and unpublished bmp2 sequences for teleosts and cartilage fish in order to explore and expand our understanding of bmp2 genes in fish. Our results confirmed that teleost genomes contain two or more bmp2 genes, and the diversity of bmp2 genes in vertebrates appears to be as a result of a combination of whole genome duplication (WGD) and gene loss. Differences were also observed in tissue distribution and relative transcription abundance of the bmp2s through a transcriptomic analysis. Our data also indicated that bmp2b may play an important role in the formation of IBs in teleosts. In addition, protein sequence alignments and 3D structural predictions of bmp2a and bmp2b supported their similar roles in fishes. To summarize, our existing work provided novel insights into the bmp2 family genes in fishes through a mixture of comparative genomic and transcriptomic analysis.

Transcriptomic analysis reveals differentially expressed genes and a unique apoptosis pathway in channel catfish ovary cells after infection with the channel catfish virus.

The channel catfish virus (CCV) can cause lethal hemorrhagic infection in juvenile channel catfish, thereby resulting in a huge economic loss to the fish industry. The genome of the CCV has been fully sequenced, and its prevalence is well documented. However, less is known about the molecular mechanisms and pathogenesis of the CCV. Herein, the channel catfish ovary cells (CCO) were infected with CCV and their transcriptomic sketches were analyzed using an RNA sequencing technique. In total, 72,686,438 clean reads were obtained from 73,231,128 sequence reads, which were further grouped into 747,168 contigs. These contigs were assembled into 49,119 unigenes, of which 20,912 and 18,333 unigenes were found in Nr and SwissProt databases and matched 15,911 and 14,625 distinctive proteins, respectively. From these, 3641 differentially expressed genes (DEGs), comprising 260 up-regulated and 3381 down-regulated genes, were found compared with the control (non-infected) cells. For verification, 16 DEGs were analyzed using qRT-PCR. The analysis of the DEGs and their related cellular signaling pathways revealed a substantial number of DEGs that were involved in the apoptosis pathway induced by CCV infection. The apoptosis pathways were further elucidated using standard apoptosis assays. The results showed that CCV could induce extrinsic apoptosis pathway (instead of a mitochondrial intrinsic apoptosis pathway) in CCO cells. This study helps our understanding of the pathogenesis of CCV and contributes to the prevention of CCV infection in channel catfish.

Pathogenicity of snakehead vesiculovirus in rice field eels (Monopterus albus).

Snakehead vesiculovirus (SHVV) has caused mass mortality to cultured snakehead fish in China, resulting in enormous economic losses in snakehead fish culture. In this report, the whole genome of SHVV was sequenced. Interestingly, it shared more than 94% nucleotide sequence identity with Monopterus albus rhabdovirus (MoARV), which has caused great economic loss to cultured rice field eel (Monopterus albus). Therefore, the concern of cross-species infection of these viruses prompted us to investigate the susceptibility of rice field eel to SHVV infection. The results showed that rice field eel was susceptible to SHVV in both intracoelomical injection and immersion routes. Severe hemorrhage was observed on the skin and visceral organs of SHVV-infected rice field eels. Histopathological examination showed vacuoles in the tissues of infected liver, kidney and heart. Viral RNA or protein was detected in the tissues of infected fish by reverse transcription polymerization chain reaction (RT-PCR), in situ hybridization (ISH), or immunohistochemistry assay (IHC). Investigation of the epidemic of vesiculovirus in rice field eel as well as other co-cultured fish is invaluable for the prevention of vesiculovirus infection.

Characterization of microRNAs in orange-spotted grouper (Epinephelus coioides) fin cells upon red-spotted grouper nervous necrosis virus infection.

Nervous necrosis virus (NNV), one of the most prevalent fish pathogens, has caused fatal disease of viral nervous necrosis (VNN) in many marine and freshwater fishes, and resulted in heavy economic losses in aquaculture industry worldwide. However, the molecular mechanisms underlying the pathogenicity of NNV remain elusive. In this study, the expression profiles of microRNA (miRNA) were investigated in grouper fin (GF-1) cells infected with red-spotted grouper nervous necrosis virus (RGNNV) via deep sequencing technique. The results showed that a total of 220 miRNAs were identified by aligning the small RNA sequences with the miRNA database of zebrafish, and 18 novel miRNAs were predicted using miRDeep2 software. Compared with the non-infected groups, 51 and 16 differentially expressed miRNAs (DE-miRNAs) were identified in the samples infected with RGNNV at 3 and 24 h, respectively. Six DE-miRNAs were randomly selected to validate their expressions using quantitative reverse transcription polymerase chain reaction (qRT-PCR), the results showed that their expression profiles were consistent with those obtained by deep sequencing. The target genes of the DE-miRNAs covered a wide range of functions, such as regulation of transcription, oxidation-reduction process, proteolysis, regulation of apoptotic process, and immune response. In addition, the effects of four DE-miRNAs including miR-1, miR-30b, miR-150, and miR-184 on RGNNV replication were evaluated, and the results showed that over-expression of each of the four miRNAs promoted the replication of RGNNV. These data provide insight into the molecular mechanism of RGNNV infection, and will benefit for the development of effective strategies to control RGNNV infection.

Molecular phylogenetic relationships of some common sweetlips (Haemulidae: Plectorhynchinae) and the synonyms controversy of two Plectorhinchus species.

Molecular phylogenetic topologies from 40 individuals of 17 sweetlips were constructed based on the mitochondrial cytochrome b (cyt b) and cytochrome c oxidase subunit I (COI) genes. All phylogenetic results strongly revealed the division of the sweetlips into three morphological distinct groups. Group I: sweetlips with colorful patterns, Group II and Group III: species with uniformly dark patterns. The relationships of those morphologically confused Plectorhinchus species were well-resolved in the phylogenetic results. We also confirmed that the genus Diagramma was placed inside the colorful Plectorhinchus groups, suggesting close relationship of Diagramma within Plectorhinchus. Additionally, we found that two species, P. orientalis and P. vittatus, which were traditionally considered as synonyms for oriental sweetlips, which were suggested to be two distinct species. Sequence divergence also revealed a great genetic variation between P. orientalis and P. vittatus (6.0% in Cyt b and 7.4% in COI), which was largely greater than the species diagnosis divergence value (2%) suggested by Hebert et al. This new finding suggested P. orientalis and P. vittatus might be two distinct species and should not be placed as synonyms.

Complete mitochondrial genome of the yellow-banded sweetlips Plectorhinchus lineatus (Perciformes: Haemulidae).

The complete mitochondrial genome of yellow-banded sweetlips Plectorhinchus lineatus was determined in this study. The genome was 16,548 bp in length and consisted of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and one non-coding control region. The overall base composition was estimated to be A, 28.2%; T, 24.8%; C, 30.7% and G, 16.2% with AT bias of 53.0%. All protein-coding genes used ATG as a start codon except for COI, which started with GTG. Six protein-coding genes ended with termination codon TAA, one ended with AGG, the remaining genes had incomplete stop codons TA or T. The genomic composition, organization, and gene order of P. lineatus was similar to most of vertebrates.

The complete mitochondrial genome of the sea carp, Cyprinus acutidorsalis (Cypriniformes: Cyprinidae).

The complete mitochondrial genome of sea carp Cyprinus acutidorsalis was determined in this study. The genome was 16,579 bp in length. As in other vertebrates, it consisted of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and 1 non-coding control region. The overall base composition was estimated to be A, 31.9%; T, 24.8%; C, 27.6% and G, 15.7% with AT bias of 56.7%. The genomic composition, organization, and gene order of C. acutidorsalis was similar to most of vertebrates. This result may provide the basis for the study of genetic structure as well as resource conservation and protection of C. acutidorsalis.

The complete mitochondrial genome of black grunt Hapalogenys nigripinnis.

The complete sequence of the mitochondrial genome of Hapalogenys nigripinnis was determined in this study. The genome was 16,478 bp in size with a base composition of 28.6% A, 15.8% G, 27.4% T, and 28.2% C, containing a typical structure of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 noncoding control region. The genomic composition, organization, and gene order of H. nigripinnis was similar to that obtained in most vertebrates. These results may provide molecular information on the future phylogenetic relationships of H. nigripinnis and its position within the suborder Percoidei.

Molecular phylogenetic relationships of family Haemulidae (Perciformes: Percoidei) and the related species based on mitochondrial and nuclear genes.

Haemulidae species are morphologically diversified fishes with wondrous and changeable coloration. These species are prevalent in global tropical, subtropical, and temperate inshore reef areas. Previous morphological classification within Haemulidae and some related families was problematic, and no comprehensive molecular evaluation was conducted on these groups. In this study, we revealed the first molecular phylogenetic analysis involving representative species within Haemulidae and the relevant families (Nemipteridae and Teraponidae). The analysis was performed using both mitochondrial DNA (16S rRNA) and nuclear DNA (TMO-4c4) genes to construct maximum parsimony, maximum likelihood, and Bayesian analysis tree topologies. The molecular phylogeny recovered well-resolved relationships within Haemulidae species and the problematic taxa. In the trees, the Haemulidae species (except for genus Hapalogenys) were divided into two distinct sister lineages, including grunts (Haemulinae) and sweetlips (Plectorhynchinae). Hapalogenys was positioned outside the major Haemulidae tribe and formed an independent group, which challenged the traditional taxonomy that Hapalogenys was classified into Haemulidae. The results did agree with most current studies that Hapalogenys was only distantly related to Haemulidae and could potentially be removed from this family. Additionally, the genus Diagramma was observed to be tightly grouped inside the clade Plectorhinchus, indicating its highly close affinity within Plectorhinchus. Regarding interfamily relationships, the phylogenetic constructions suggested distant relationships within two pairs: between Scolopsis and Haemulidae and between Teraponidae and Haemulidae. Scolopsis was revealed to be highly close with Nemipteridae, and Teraponidae was clustered in an independent group, which was in accordance with most current taxonomic studies.