Epinephelus bilobatus, Epinephelus maculatus and Epinephelus longispinis are three groupers that share common morphological characteristics and coloration patterns and have been morphologically confused and misidentified with each other for a long time. Complete mitochondrial genomes of the three groupers were determined and analyzed in this study. Mitogenomes of E. bilobatus, E. maculatus and E. longispinis were 17, 354 bp, 17, 066 bp and 17, 221 bp in size respectively and consisted of 13 protein-coding genes, two ribosomal RNA genes and one control region. However, different from most teleosts, which contain canonical 22 tRNAs, more numbers of tRNAs were identified in the three groupers with 27 tRNAs in E. bilobatus and E. longispinis and 25 tRNAs in E. maculatus. The increased number of tRNAs was due to the presence of tandemly duplicated tRNA-Asp genes that were located between tRNA-Ser and COII genes (six duplications in E. bilobatus and E. longispinis, four duplications in E. maculatus). Intact gene tandem duplication was an uncommon feature that was found in the typical teleost mitogenomes. The phylogenetic trees of the 32 groupers (genus Epinephelus) that were constructed based on 12 protein-coding genes revealed that Epinephelus species with tandemly duplicated tRNA-Asp genes were clustered into one monophyletic group, distinct from other Epinephelus species without any duplication features, which indicated that tandemly duplicated tRNA-Asp genes may be the particular linage-specific characteristics that evolve from a common ancestor and have the ability to distinguish them from other Epinephelus species. The results of the mitogenomes comparative analyses of the three groupers revealed the genetic distance of mitogenomes between each two species to be 0.062 (E. bilobatus vs E. maculatus), 0.091 (E. bilobatus vs E. longispinis) and 0.087 (E. maculatus vs E. longispinis). All values were far greater than the minimum value of 0.020 for species identification, which shows that they were three independent species at molecular level. Regarding the relationships between the three groupers, E. bilobatus was found to be more closely related to E. maculatus in comparison to E. longispinis. The results provide valuable molecular data for the species identification and phylogenetic analyses on E. bilobatus, E. maculatus and E. longispinis, and also provided a new insight into the tandem gene duplication features of Epinephelus mitogenomes.

Introduction: Toll-like receptors (TLRs) are pattern recognition receptors essential for immune defense against pathogens, activating the host’s immune response by recognizing conserved pathogen structures. The Chinese spiny frog (Quasipaa spinosa), an amphibian native to southern China and northern Vietnam, has been severely impacted by recent infectious disease outbreaks caused by bacterial, viral, and parasitic infections, which threaten the sustainable development of the Q. spinosa farming industry. However, the roles of Q. spinosaTLRs (QsTLRs) in combating these exogenous pathogens have not yet been explored.Methods: In the study, using the whole genome data of Q. spinosa, bioinformatics tools were employed to identify and analyze the TLR gene family. The bacteria Elizabethkingia miricola, a common pathogen, which causes the cataract disease and can lead to serious death of the frog. Here, we selected the bacteria to conduct the challenge experiment in order to characterize the immune responses of the TLR genes of Q. spinosa against bacterial infection.Results: The analysis identified 17 members of the TLR gene family in Q. spinosa. Phylogenetic analysis revealed that QsTLRs can be classified into seven subfamilies: TLR1, TLR3, TLR4, TLR5, TLR7, TLR11, and TLR13. Conserved synteny analysis indicated that Q. spinosa is more closely related to Rana temporaria than to Xenopus laevis. Protein structure prediction and motif analysis demonstrated that all QsTLRs are relatively conserved in both structure and function. mRNA expression levels of QsTLRs in spleen tissues were measured following stimulation with Elizabethkingia miricola, which revealing that 15 QsTLR genes exhibited up-regulation at various time points post-stimulation.Discussion: These findings provide a comprehensive understanding of the QsTLR gene family and lay the groundwork for future studies exploring the functional evolution of the amphibian TLR gene family.

Extrachromosomal circular DNAs (eccDNAs) play a significant role in regulating various biological processes, including abnormal muscle development. The molecular functions and impact of eccDNAs in the muscle development of fish are poorly understood. To investigate the potential roles of eccDNAs in the muscle development of fish, we analyzed and compared the expression profile of muscle eccDNAs of crisp grass carp, fed a faba bean meal‐based diet, and ordinary grass carp, fed a practical diet. Using the Circle‐seq strategy, we found the eccDNA abundance in crisp grass carp (211,920 eccDNAs) was significantly higher than that in ordinary grass carp (25,857 eccDNAs), suggesting that the faba bean diet likely independently influences eccDNA production. Compared to ordinary grass carp, crisp grass carp exhibited 10,565 upregulated and 129 downregulated eccDNAs, indicating eccDNAs were possibly associated with the muscle development of grass carp. GO and KEGG enrichment analyses indicated that the upregulated eccDNAs were related to muscle fiber development, cellular structure, and cell junctions. Based on our results, we speculated that the overexpression of genes involved in muscle fiber, calcium metabolism, and collagen driven by eccDNAs likely contributes to the observed increase in muscle fiber density, calcium levels, and collagen content in crisp grass carp, thereby enhancing muscle hardness. Notably, eccDNAs were identified as potential innate immunostimulants capable of eliciting immune responses in fish. In summary, our findings demonstrate that eccDNAs are aberrantly expressed in the muscles of fish fed a faba bean diet, offering novel insights into the molecular mechanisms underlying muscle hardening in fish.

This study reports the first record of Gymnothorax emmae Prokofiev, 2010 in China’s coastal regions. Initially discovered in Vietnam in 2010, this species has not been previously reported in any other global regions. Here the morphological characteristics of G. emmae were described in detail. Additionally, the entire mitochondrial genome of this species was sequenced and assembled, providing molecular evidence for species identification and phylogenetic analysis. Key distinguishing features of G. emmae include: a yellow or yellow-brown body with a lighter head; several rows of brown spots, either rounded or irregular, behind the eyes and in the branchial region; numerous tortuous transverse or oblique brown bands on the trunk and fins, some of which are interconnected; uniserial teeth; and a vertebral count of 3–4/45–47/121–125. The complete mitochondrial genome transfer RNAs of G. emmae was sequenced to be 16 577 bp, encompassing 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNA genes, and a non-coding region. The genome organization aligned with that of other Muraenidae species. Phylogenetic relationships were reconstructed to clarify the taxonomic position of G. emmae. Phylogenetic trees were built for 23 Muraenidae species using 12 protein-coding genes, and for 72 Gymnothorax species using the cytochrome c oxidase subunit I (COI) gene. The tree based on 12 protein-coding genes strongly supported that G. emmae was sister to a species complex consisting of Gymnothorax kidako and Gymnothorax niphostigmus. Similarly, the COI-based tree placed G. emmae within a clade containing 7 species (G. kidako, G. niphostigmus, G. mucifer, among others). These species share distinct body coloration and patterns, some of which could be easily confused with G. emmae. Genetic distances, calculated from the COI gene, ranged from 0.072 to 0.262 between G. emmae and other Gymnothorax species, all exceeding the minimum species-identification threshold of 0.020 (2%), confirming that G. emmae is a distinct molecular species.