Comparative physiological, biochemical and transcriptomic analyses to reveal potential regulatory mechanisms in response to starvation stress in Cipangopaludina chinensis.

Cipangopaludina chinensis, as a financially significant species in China, represents a gastropod in nature which frequently encounters starvation stress owing to its limited prey options. However, the underlying response mechanisms to combat starvation have not been investigated in depth. We collected C. chinensis under several times of starvation stress (0, 7, 30, and 60 days) for nutrient, biochemical characteristics and transcriptome analyses. The results showed that prolonged starvation stress (> 30 days) caused obvious fluctuations in the nutrient composition of snails, with dramatic reductions in body weight, survival and digestive enzyme activity (amylase, protease, and lipase), and markedly enhanced the antioxidant enzyme activities of the snails. Comparative transcriptome analyses revealed 3538 differentially expressed genes (DEGs), which were significantly associated with specific starvation stress-responsive pathways, including oxidative phosphorylation and alanine, aspartate, and glutamate metabolism. Then, we identified 40 candidate genes (e.g., HACD2, Cp1, CYP1A2, and GPX1) response to starvation stress through STEM and WGCNA analyses. RT-qPCR verified the accuracy and reliability of the high-throughput sequencing results. This study provides insights into snail overwintering survival and the potential regulatory mechanisms of snail adaptation to starvation stress.

Genetic diversity and population structure of Bellamya purificata in Guangxi.

Bellamya purificata is an important medicinal value and economically farmed species in China. However, because little is known about the genetic characteristics of this species, the utilization of high-quality germplasm resources is hindered. The study examined the genetic differentiation between, and the structure of 12 B. purificata populations in Guangxi using 7 microsatellite DNA markers. High genetic diversity occurred in each population, with mean observed heterozygosity 0.655 and a mean expected heterozygosity 0.832. Analysis of molecular variance reveals genetic diversity to be greater within (95.2%) than among populations (4.8%). Genetic differentiation between populations is weak (Fst = 0.048, P < 0.001), with mixing of genetic clusters prevalent at the level of the individual. Genetic flow exists between populations (Nm = 3.084-11.778), with Longshui and Guilin populations exchanging frequently. A Mantel test reveals a low correlation between geographic and genetic distances (r = 0.2482, P < 0.071), suggesting that dispersal between neighboring populations facilitates population exchange. No significant heterozygosity excess was observed for any population (P > 0.05), indicating a lack of recent genetic bottlenecks. The results provide important genetic information for B. purificata, and data for potential germplasm discovery and aquaculture development.

Characterization of the Complete Mitochondrial Genome of the Elongate Loach and Its Phylogenetic Implications in Cobitidae.

The elongate loach is an endemic fish in China. Previous studies have provided some insights into the mitochondrial genome composition and the phylogenetic relationships of the elongate loach inferred using protein-coding genes (PCGs), yet detailed information about it remains limited. Therefore, in this study we sequenced the complete mitochondrial genome of the elongate loach and analyzed its structural characteristics. The PCGs and mitochondrial genome were used for selective stress analysis and genomic comparative analysis. The complete mitochondrial genome of the elongate loach, together with those of 35 Cyprinidae species, was used to infer the phylogenetic relationships of the Cobitidae family through maximum likelihood (ML) reconstruction. The results showed that the genome sequence has a full length of 16,591 bp, which includes 13 PCGs, 22 transfer RNA genes (tRNA), 2 ribosomal RNA genes (rRNA), and 2 non-coding regions (CR D-loop and light chain sub-chain replication origin OL). Overall, the elongate loach shared the same gene arrangement and composition of the mitochondrial genes with other teleost fishes. The Ka/Ks ratios of all mitochondrial PCGs were less than 1, indicating that all of the PCGs were evolving under purifying selection. Genome comparison analyses showed a significant sequence homology of species of Leptobotia. A significant identity between L. elongata and the other five Leptobotia species was observed in the visualization result, except for L. mantschurica, which lacked the tRNA-Arg gene and had a shorter tRNA-Asp gene. The phylogenetic tree revealed that the Cobitidae species examined here can be grouped into two clades, with the elongate loach forming a sister relationship with L. microphthalma. This study could provide additional inferences for a better understanding of the phylogenetic relationships among Cobitidae species.

SMRT Sequencing Reveals Candidate Genes and Pathways With Medicinal Value in Cipangopaludina chinensis.

Cipangopaludina chinensis is an economically important aquatic snail with high medicinal value. However, molecular biology research on C. chinensis is limited by the lack of a reference genome, so the analysis of its transcripts is an important step to study the regulatory genes of various substances in C. chinensis. Herein, we conducted the first full-length transcriptome analysis of C. chinensis using PacBio single-molecule real-time (SMRT) sequencing technology. We identified a total of 26,312 unigenes with an average length of 2,572 bp, of which the largest number of zf-c2h2 transcription factor families (120,18.24%) were found, and also observed that the majority of the 8,058 SSRs contained 4-7 repeat units, which provided data for subsequent work on snail genetics Subsequently, 91.86% (24,169) of the genes were successfully annotated to the four major databases, while the highest homology was observed with Pomacea canaliculata. Functional annotation revealed that the majority of transcripts were enriched in metabolism, signal transduction and Immune-related pathways, and several candidate genes involved in drug metabolism and immune response were identified (e.g., CYP1A1, CYP2J, CYP2U1, GST, ,PIK3, PDE3A, PRKAG). This study lays a foundation for future molecular biology research and provides a reference for studying genes associated with the medicinal value of C. chinensis.

Comparison of the composition and function of gut microbes between adult and juvenile Cipangopaludina chinensis in the rice snail system.

Cipangopaludina chinensis is an important economic value snail species with high medicinal value. The gut microbes of aquatic animals plays a vital role in food digestion and nutrient absorption. Herein, we aimed at high-throughput sequencing of 16S rRNA to further investigate whether there were differences in the composition and function of gut microbes of adult and juvenile C. chinensis snails, as well as sediments. This study found that the microbial diversity of the sediment was significantly higher than that of the snails gut (P < 0.001), but there was no significant difference between the gut flora of adult and juvenile snails (P > 0.05). A total of 47 phyla and 644 genera were identified from all samples. Proteobacteria and Verrucomicrobia were the two dominant phyla in all samples, and overall relative abundances was 48.2% and 14.2%, respectively. Moreover, the relative abundances of Aeromonas and Luteolibacter in the gut of juvenile snails (30.8%, 11.8%) were higher than those of adults (27.7%, 10.6%) at the genus level (P > 0.05). Then, four indicator genera were found, namely Flavobacterium, Silanimonas, Geobacter and Zavarzinella, and they abundance in the gut of juvenile snails was significantly higher than that of adults (P < 0.05). This results imply the potential development of Silanimonas as a bait for juvenile snail openings. We observed that Aeromonas was the primary biomarker of the snail gut and sediments (P < 0.001), and it may be a cellulose-degrading bacteria. Function prediction revealed significantly better biochemical function in the snail gut than sediments (P < 0.001), but no significant differences in adult and juvenile snail (P > 0.05). In conclusion, studies show that the snail gut and sediment microbial composition differ, but the two were very similar. The microbial composition of the snail gut was relatively stable and has similar biological functions. These findings provide valuable information for in-depth understanding of the relationship between snails and environmental microorganisms.

Liver and spleen transcriptome reveals that Oreochromis aureus under long-term salinity stress may cause excessive energy consumption and immune response.

To investigate the physiological responses of Oreochromis aureus to salinity fluctuations at the molecular level. We used RNA-seq to explore the differentially expressed genes (DEGs) in the liver and spleen of O. aureus at 0, 3, 7 and 11 ppt (parts per thousand) salinity levels. Herein, De novo assembly generated 71,009 O. aureus unigenes, of which 34,607 were successfully mapped to the four major databases. A total of 120 shared DEGs were identified in liver and spleen transcripts, of which 83 were up-regulated and 37 were down-regulated. GO and KEGG analysis found a total of 26 significant pathways, mainly including energy metabolism, immune response, ion transporters and signal transduction. The trend module category of DEGs showed that the genes (e.g., FASN, ODC1, CD22, MRC, TRAV and SLC7 family) involved in the change-stable-change (1) and the constant-change categories (2) were highly sensitive to salinity fluctuations, which were of great value for further study. Based on these results, it would help provide basic data for fish salinity acclimation, and provide new insights into evolutionary response of fish to various aquatic environments in the long-term stress adaptation mechanism.