Tea polyphenols, astaxanthin, and melittin can significantly enhance the immune response of juvenile spotted knifejaw (Oplegnathus punctatus).

The frequent occurrence of diseases seriously hampers the sustainable development of the spotted knifejaw (Oplegnathus punctatus) breeding industry. Our previous genome-wide scan and cross-species comparative genomic analysis revealed that the immune gene family (Toll-like receptors, TLR) members of O. punctatus underwent a significant contraction event (tlr1, tlr2, tlr14, tlr5, and tlr23). To address immune genetic contraction may result in reduced immunity, we investigated whether adding different doses (0, 200, 400, 600, and 800 mg/kg) of immune enhancers (tea polyphenols, astaxanthin, and melittin) to the bait after 30 days of continuous feeding could stimulate the immune response of O. punctatus. We found that the expression of tlr1, tlr14, tlr23 genes in immune organs (spleen and head kidney) was stimulated when tea polyphenols were added at 600 mg/kg. The tlr2 (400 mg/kg), tlr14 (200 mg/kg), tlr5 (200 mg/kg), and tlr23 (200 mg/kg) genes expression of intestine were elevated in the tea polyphenol group. When the addition of astaxanthin is 600 mg/kg, it can effectively stimulate the expression of tlr14 gene in immune organs (liver, spleen and head kidney). In the astaxanthin group, the expression of the genes tlr1 (400 mg/kg), tlr14 (600 mg/kg), tlr5 (400 mg/kg) and tlr23 (400 mg/kg) reached their highest expression in the intestine. Besides, the addition of 400 mg/kg of melittin can effectively induce the expression of tlr genes in the liver, spleen and head kidney, except the tlr5 gene. The tlr-related genes expression in the intestine was not significantly elevated in the melittin group. We hypothesize that the immune enhancers could enhance the immunity of O. punctatus by increasing the expression of tlr genes, and thereby leading to increased resistance to diseases. Meanwhile, our findings further demonstrated that significant increases in weight gain rate (WGR), visceral index (VSI), and feed conversion rate (FCR) were observed at 400 mg/kg, 200 mg/kg and 200 mg/kg of tea polyphenols, astaxanthin and melittin in the diet, respectively. Overall, our study provided valuable insights for future immunity enhancement and viral infection prevention in O. punctatus, as well as offered guidance for the healthy development of the O. punctatus breeding industry.

Reconstruction of the Origin of a Neo-Y Sex Chromosome and Its Evolution in the Spotted Knifejaw, Oplegnathus punctatus.

Sex chromosomes are a peculiar constituent of the genome because the evolutionary forces that fix the primary sex-determining gene cause genic degeneration and accumulation of junk DNA in the heterogametic partner. One of the most spectacular phenomena in sex chromosome evolution is the occurrence of neo-Y chromosomes, which lead to X1X2Y sex-determining systems. Such neo-sex chromosomes are critical for understanding the processes of sex chromosome evolution because they rejuvenate their total gene content. We assembled the male and female genomes at the chromosome level of the spotted knifejaw (Oplegnathus punctatus), which has a cytogenetically recognized neo-Y chromosome. The full assembly and annotation of all three sex chromosomes allowed us to reconstruct their evolutionary history. Contrary to other neo-Y chromosomes, the fusion to X2 is quite ancient, estimated at 48 Ma. Despite its old age and being even older in the X1 homologous region which carries a huge inversion that occurred as early as 55-48 Ma, genetic degeneration of the neo-Y appears to be only moderate. Transcriptomic analysis showed that sex chromosomes harbor 87 genes, which may serve important functions in the testis. The accumulation of such male-beneficial genes, a large inversion on the X1 homologous region and fusion to X2 appear to be the main drivers of neo-Y evolution in the spotted knifejaw. The availability of high-quality assemblies of the neo-Y and both X chromosomes make this fish an ideal model for a better understanding of the variability of sex determination mechanisms and of sex chromosome evolution.

Transcriptome comparative analysis of immune tissues from asymptomatic and diseased Epinephelus moara naturally infected with nervous necrosis virus.

Epinephelus moara is an economically important fish in Southeast Asian countries but is suffering from nervous necrosis virus (NNV) infection. A deeper understanding of the host-NNV interaction mechanisms makes sense for disease control, however, at present, the pathogenesis of natural NNV infection and the resistance mechanism in host remains poorly understood. In this study, asymptomatic and diseased E. moara with clinical symptoms of viral nervous necrosis (VNN) from a grouper farm were both detected with a positive RT-PCR signal of NNV, then transcriptome sequencing of their immune tissues (liver, spleen and kidney) were performed for comparation analysis. The de novo assemblies yielded 53,789 unigenes which had a length varied from 201 to 19,675 bp and a N50 length of 2115 bp, and 29,451 unigenes were functionally annotated, with 83, 250 and 5632 unigenes being differentially expressed in liver, spleen and kidney respectively. KEGG pathway enrichment analysis of the DEGs showed many DEGs were enriched in immune related pathways. Although the expression of class I major histocompatibility complex (MHC) was significantly higher in three immune tissues of the diseased grouper, many immune related genes, including humoral immune molecules (such as antibodies), the cellular mediated cytotoxic molecules (such as perforin) and some adhesion related genes were down regulated in the diseased grouper. Our results provided many unigenes that might play important roles in NNV resistance for further research. Furthermore, a total of 8666 unigenes containing 11,623 simple sequence repeats (SSRs) were identified, which provided useful information for screening molecular markers associated with NNV resistance in E. moara.

Expression pattern and cellular localization of pepsinogen in early development and induced by different diets in the spotted knifejaw (Oplegnathus punctatus).

To solve the high mortality rate of early-stage larval feed conversion during aquaculture in Oplegnathus punctatus, the investigation of the structural and functional characteristics of the gastric tissue was conducted. Histological results showed that the gastric gland rudiment appeared at 17 dph. The basic structure of the stomach was fully developed between 26 and 35 dph. Two pepsinogen genes, named OpPGA1 and OpPGA2, were identified in the spotted knifejaw genome. qPCR results of developmental period showed that the two genes were low in expression during early development (5 and 15 dph). At 20 dph, the two genes started to show trace expression, and at 30 dph the mRNA expression levels of OpPGA1 and OpPGA2 reached the highest levels. Results of pepsin activity detection during the development period showed lower activity was detected 22 dph, followed by a peak at 30 dph. Under different feeding inductions, OpPGA1 showed the highest expression in the basic diet group and hard-shell group, while the expression level in the phytophagous group remained consistently low. The mRNA expression level of OpPGA2 in the phytophagous group was significantly higher than in other groups. Enzyme activity determination under different feeding inductions showed slightly higher enzyme activity in the basic diet group and crustacean group. The results of in situ hybridization showed that the mRNA of both OpPGA1 and OpPGA2 genes was both expressed in gastric gland cells. These information can contribute to the development of practical feeding methods in terms of digestive physiology for the development of larvae.

Isolation of a Male-Specific Molecular Marker and Development of a Genetic Sex Identification Technique in Spotted Knifejaw (Oplegnathus punctatus).

Sex-specific DNA markers are very helpful for identifying genetic sex and studying sex determination mechanisms in fish. To identify the sex-specific markers of spotted knifejaw (Oplegnathus punctatus), we performed a comparative analysis of the female and male genomes. In this study, an 18 bp insertion was identified in the male genome after verification by sequencing depth and PCR. An effective and rapid method based on PCR was then developed to identify the genetic sex. A male-female-shared primer pair and a male-specific primer were designed for PCR amplification to avoid false-negative phenomena. To examine the primers in practice, we utilized hundreds of spotted knifejaw fish from different groups to identify their genetic sex, and the results were consistent with their phenotypic sex. The male-specific DNA marker would be helpful for artificial breeding, Y chromosome assembly and further study of the sex determination mechanism. This study is the first to identify an effective sex-specific marker in spotted knifejaw.

The complete mitochondrial genome of the hybrid offspring Epinephelus awoara♀ × Epinephelus tukula♂.

The complete mitochondrial genome of hybrid grouper from Epinephelus awoara (♀) ×E. tukula (♂) was obtained by PCR amplification. The circular genome was 16,801 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region (D-loop region). The overall base composition was as follows: A: 28.46%, T: 27.27%, C: 27.27%, G: 16.49%. The new results may provide valuable data for the genetic and taxonomic research on artificial hybrid grouper.

The complete mitochondrial genome of the hybrid offspring Epinephelus fuscoguttatus ♀ × Epinephelus tukula ♂.

The hybrid offspring Epinephelus fuscoguttatus ♀ × E. tukula ♂ showed heterosis in terms of growth and disease resistance. The mitochondrial genome is 16,629 bp in length and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a control region. The difference of total length is mainly caused by the difference of length in non-coding region. The nucleotide base composition is A = 29.15%, G = 15.62%, T = 26.91%, C = 28.32%, A + T = 56.06%, and C + G = 43.94%. The phylogenetic analysis using neighbour-joining (NJ) method showed that the hybrid offspring has a closer relationship to E. fuscoguttatus × E. lanceolatus.

Characterization of the complete mitochondrial genome of the hybrid grouper Epinephelus akaara ♀ × Epinephelus tukula ♂, with its phylogenetic analysis.

Epinephelus akaara ♀ × Epinephelus tukula ♂ is an economically important fish. The mitochondrial genome of the hybrid grouper had a double-stranded DNA molecule with the length of 16,928 bp and consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region. The gene composition of the hybrid grouper mitochondrial genome was similar to that of most other vertebrates. Furthermore, phylogenetic analysis by maximum-likelihood (ML) method, based on the nucleotide sequences of 13 protein-coding genes, showed that the hybrid grouper has the closer relationship to Epinephelus akaara and confirmed that the mitochondrial genome is maternally inherited.