Integrative Metabolomics, Enzymatic Activity, and Gene Expression Analysis Provide Insights into the Metabolic Profile Differences between the Slow-Twitch Muscle and Fast-Twitch Muscle of Pseudocaranx dentex.

The skeletal muscles of teleost fish encompass heterogeneous muscle types, termed slow-twitch muscle (SM) and fast-twitch muscle (FM), characterized by distinct morphological, anatomical, histological, biochemical, and physiological attributes, driving different swimming behaviors. Despite the central role of metabolism in regulating skeletal muscle types and functions, comprehensive metabolomics investigations focusing on the metabolic differences between these muscle types are lacking. To reveal the differences in metabolic characteristics between the SM and FM of teleost, we conducted an untargeted metabolomics analysis using Pseudocaranx dentex as a representative model and identified 411 differential metabolites (DFMs), of which 345 exhibited higher contents in SM and 66 in FM. KEGG enrichment analysis showed that these DFMs were enriched in the metabolic processes of lipids, amino acids, carbohydrates, purines, and vitamins, suggesting that there were significant differences between the SM and FM in multiple metabolic pathways, especially in the metabolism of energy substances. Furthermore, an integrative analysis of metabolite contents, enzymatic activity assays, and gene expression levels involved in ATP-PCr phosphate, anaerobic glycolysis, and aerobic oxidative energy systems was performed to explore the potential regulatory mechanisms of energy metabolism differences. The results unveiled a set of differential metabolites, enzymes, and genes between the SM and FM, providing compelling molecular evidence of the FM achieving a higher anaerobic energy supply capacity through the ATP-PCr phosphate and glycolysis energy systems, while the SM obtains greater energy supply capacity via aerobic oxidation. These findings significantly advance our understanding of the metabolic profiles and related regulatory mechanisms of skeletal muscles, thereby expanding the knowledge of metabolic physiology and ecological adaptation in teleost fish.

DNA barcoding, identification, and validation of the pufferfish (Order: Tetraodontiformes) in China coastal waters.

The order Tetraodontiformes are one of the most unique groups of teleostean fish, exhibiting highly derived and greatly diversified phenotypes. It is a difficult task for both professionals and nonprofessionals to accurately identify these species only according to morphological characteristics. DNA barcoding can identify species at the molecular level to overcome the limitations of morphological classification. In this study, we collected 616 specimens of pufferfish from the coastal waters of China. According to the morphological characteristics, they were preliminarily identified as 50 species. Further analysis using DNA barcodes identified these specimens as 46 species, belonging to 23 genera, 6 families. According to the species classification results of DNA barcoding, the three species identified by morphology as Takifugu pseudommus, Takifugu chinensis, and Takifugu rubripes should be the same species. Similarly, Lagocephalus wheeleri is the synonym of Lagocephalus spadiceus. Another important discovery of DNA barcoding analysis is that there are closer interspecific genetic distances within the genus Takifugu. If T. rubripes, T. pseudommus, and T. chinensis are taken as one species, the average interspecific to intraspecific genetic distance ratio of Takifugu is only 6.21 times, which does not reach the DNA barcoding threshold of more than 10 times proposed previously. Although the interspecific genetic distance in the genus Takifugu is relatively small, each species can be clustered into independent clades in the NJ tree. In conclusion, this study not only found that there are synonymous phenomena in the order Tetraodontiformes but also provided molecular evidence for the valid species names of Takifugu rubripes and Lagocephalus Spadiceus. The results can provide reliable DNA barcoding information for the identification of pufferfish species, help solve the problem of classification confusion in this order, and provide technical support for the identification of the original components of related commodities on the aquatic product market.

Sarcocheilichthys vittatus, a new species of gudgeon (Teleostei: Cyprinidae) from the Poyang lake basin in Jiangxi Province, South China.

Sarcocheilichthys vittatus, a new species of gudgeon, is described from the Gan-Jiang and Fu-He flowing into the Poyang Lake Basin in Jiangxi Province, South China. It is similar to S. parvus and S. caobangensis in having a longitudinal black band extending from the snout tip to the caudal-fin base, a character separating them from all other congeneric species, but distinct from S. parvus in having more lateral-line scales, no longitudinal yellowish stripe above the lateral black band on the flank and no barbels, and from S. caobangensis in possessing two shorter lateral lobes of the lower lip restricted only to the side of the lower jaw, no black spots along the dorsal-fin base and a stouter caudal peduncle. Its validity was also warranted by a molecular phylogenetic analysis based on the cyt b gene. Critical notes were provided on some currently recognized Chinese congeners.

Testing the utility of DNA barcodes and a preliminary phylogenetic framework for Chinese freshwater mussels (Bivalvia: Unionidae) from the middle and lower Yangtze River.

The middle and lower portions of the Yangtze River basin is the most species-rich region for freshwater mussels in Asia. The management and conservation of the taxa in this region has been greatly hampered by the lack of a well-developed phylogeny and species-level taxonomic framework. In this study, we tested the utility of two mitochondrial genes commonly used as DNA barcodes: the first subunit of the cytochrome oxidase c gene (COI) and the first subunit of the NADH dehydrogenase gene (ND1) for 34 putative species representing 15 genera, and also generated phylogenetic hypotheses for Chinese unionids based on the combined dataset of the two mitochondrial genes. The results showed that both loci performed well as barcodes for species identification, but the ND1 sequences provided better resolution when compared to COI. Based on the two-locus dataset, Bayesian Inference (BI) and Maximum Likelihood (ML) phylogenetic analyses indicated 3 of the 15 genera of Chinese freshwater mussels examined were polyphyletic. Additionally, the analyses placed the 15 genera into 3 subfamilies: Unioninae (Aculamprotula, Cuneopsis, Nodularia and Schistodesmus), Gonideninae (Lamprotula, Solenaia and Ptychorhychus) and Anodontinae (Cristaria, Arconaia, Acuticosta, Lanceolaria, Anemina and Sinoanodonta). Our results contradict previous taxonomic classification that placed the genera Arconaia, Acuticosta and Lanceolaria in the Unioninae. This study represents one of the first attempts to develop a molecular phylogenetic framework for the Chinese members of the Unionidae and will provide a basis for future research on the evolution, ecology, and conservation of Chinese freshwater mussels.

Reclassification of Lamprotula rochechouartii as Margaritifera rochechouartiicomb. nov. (Bivalvia: Margaritiferidae) revealed by time-calibrated multi-locus phylogenetic analyses and mitochondrial phylogenomics of Unionoida.

The family Margaritiferidae encompasses 12 valid species, which are distributed widely but disjunctively in the Northern Hemisphere. A lack of a well resolved and temporally calibrated phylogenetic framework of Margaritiferidae has made it difficult to discuss the evolutionary pattern and process. Phylogenetic relationships between five major clades, which were revealed in earlier studies, remain elusive and unresolved. Lamprotula rochechouartii has long been classified within the family Unionidae based on shell morphology, but our preliminary molecular study on this species made us hypothesize that it has an affinity with margaritiferids. Hence, five loci (COI, 16S, 18S, 28S and histone H3) were used to investigate the phylogenetic position of L. rochechouartii and intra-familial relationships within Margaritiferidae using various partitioning strategies. Moreover, two mitochondrial genomes were newly obtained to further resolve and validate the five-clade relationships within Margaritiferidae in a broad view of Unionoida evolution. Both five-gene and mitogenome datasets strongly advocated treating Lamprotula rochechouartii as Margaritifera rochechouartiicomb. nov. Maximum likelihood and Bayesian inference analyses using partitioned five-gene dataset resulted in various topologies, but five well-supported clades were obtained. The most probable cladistic relationships generated by five-gene dataset analyses were identical to subsequent whole mitogenome analyses except the position of M. monodonta. M. rochechouartii and M. laosensis had a well-supported sister relationship and formed a basal clade splitting from the rest of the family. Based on six reliable fossils, crown age of the extant Margaritiferidae was estimated during the Late Cretaceous at 88.3 Ma (95% HPD = 66.2-117.4). But we hypothesized a much earlier origin of this family due to the Permian stem age (mean = 257 Ma, 95% HPD = 230.0-296.0) and a high extinction rate in the whole order. Biogeographic scenarios supported a Laurasian origin of extant Margaritiferidae during the Late Cretaceous, and suggested that Asian margaritiferids may have had two origins, having either Asia (M. rochechouartii, M. laosensis) or North America (M. dahurica, M. laevis, and M. middendorffi) as ancestral. The newly added Margaritiferidae species M. rochechouartii expands our recognized distribution range of modern margaritiferids. Our results indicate that whole mitogenome sequences can be used to reconstruct robust phylogenetic relationships for freshwater mussels, especially with the help of adding M-type mitogenomes.

Complete maternal mitochondrial genome of freshwater mussel Aculamprotula tientsinensis (Bivalvia: Unionidae: Unioninae).

Aculamprotula tientsinensis is a rare and endemic species of freshwater mussel in China. This study firstly determined the complete F-type mitochondrial genome of A. tientsinensis. The circle genome (15 695 bp) comprises 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, 1 FORF gene. Except for cob, nad5 and nad6, the remaining protein-coding genes initiate with the orthodox start codon (ATG, ATA, ATT). There are 26 non-coding regions in the mitogenome of A. tientsinensis, ranging in size from 1 to 229 bp. The base composition of the genome is A (37.83%), G (12.69%), T (25.43%) and C (24.06%). Gene order is identical to other female species of Unionidae but for Gonideinae. The phylogenetic analyses of Unionidae indicate that A. tientsinensis is closely related to A. tortuosa and A. coreana, which belong to Unioninae. The complete mitogenome can deepen comparative and evolutionary genomics of Unionidae and be more comprehensive to parse the genetic relationship between the species and the ownership beyond species.

The complete F-type mitochondrial genome of Chinese Anodonta arcaeformis (Bivalvia: Unionidae: Anodontinae).

Anodonta arcaeformis is a Chinese common species. The complete F-type mitochondrial genome was first determined. The complete genome is 15,672 bp in length, with AT content 64.59%. All the 37 typical animal mitochondrial genes were identified, including 13 protein coding genes, 22 tRNA genes and 2 rRNA genes. And a novel FORF (277 bp, 92aa) was found between tRNA(Glu) and ND2, which was considered to be involved in sex determination. The putative control region (270 bp) is located between ND5 and tRNAGln, with an A + T content of 70.07%. The gene order is identical to other species of Unionidae female mitochondrial except Gonideinae.