Comparative Blood Transcriptome Analysis of Semi-Natural and Controlled Environment Populations of Yangtze Finless Porpoise.

The Yangtze finless porpoises (Neophocaena asiaeorientalis asiaeorientalis) living in different environments display significant differences in behavior and physiology. To compare and analyze gene expression differences between an ex situ population and a controlled environment population of the Yangtze finless porpoise, we sequenced the transcriptome of blood tissues living in a semi-natural reserve and an artificial facility, respectively. We identified 6860 differentially expressed genes (DEGs), of which 6603 were up-regulated and 257 were down-regulated in the controlled environment vs ex situ comparison. GO and KEGG enrichment analysis showed that the up-regulated genes in the controlled environment population were significantly associated with glucose metabolism, amino acid metabolism, and the nervous system, while those up-regulated in the ex situ population were significantly associated with energy supply and biosynthesis. Further analysis showed that metabolic and hearing-related genes were significantly affected by changes in the environment, and key metabolic genes such as HK, PFK, IDH, and GLS and key hearing-related genes such as OTOA, OTOF, SLC38A1, and GABBR2 were identified. These results suggest that the controlled environment population may have enhanced glucose metabolic ability via activation of glycolysis/gluconeogenesis, the TCA cycle, and inositol phosphate metabolism, while the ex situ population may meet higher energy requirements via enhancement of the amino acid metabolism of the liver and muscle and oxidative phosphorylation. Additionally, the acoustic behavior and auditory-related genes of Yangtze finless porpoise may show responsive changes and differential expression under different environment conditions, and thus the auditory sensitivity may also show corresponding adaptive characteristics. This study provides a new perspective for further exploration of the responsive changes of the two populations to various environments and provides a theoretical reference for further improvements in conservation practices for the Yangtze finless porpoise.

Gap-free genome assembly of anadromous Coilia nasus.

The Chinese tapertail anchovy, Coilia nasus, is a socioeconomically important anadromous fish that migrates from near ocean waters to freshwater to spawn every spring. The analysis of genomic architecture and information of C. nasus were hindered by the previously released versions of reference genomes with gaps. Here, we report the assembly of a chromosome-level gap-free genome of C. nasus by incorporating high-coverage and accurate long-read sequence data with multiple assembly strategies. All 24 chromosomes were assembled without gaps, representing the highest completeness and assembly quality. We assembled the genome with a size of 851.67 Mb and used BUSCO to estimate the completeness of the assembly as 92.5%. Using a combination of de novo prediction, protein homology and RNA-seq annotation, 21,900 genes were functionally annotated, representing 99.68% of the total predicted protein-coding genes. The availability of gap-free reference genomes for C. nasus will provide the opportunity for understanding genome structure and function, and will also lay a solid foundation for further management and conservation of this important species.

Gapless genome assembly of East Asian finless porpoise.

In recent years, conservation efforts have increased for rare and endangered aquatic wildlife, especially cetaceans. However, the East Asian finless porpoise (Neophocaena asiaeorientalis sunameri), which has a wide distribution in China, has received far less attention and protection. As an endangered small cetacean, the lack of a chromosomal-level reference for the East Asian finless porpoise limits our understanding of its population genetics and conservation biology. To address this issue, we combined PacBio HiFi long reads and Hi-C sequencing data to generate a gapless genome of the East Asian finless porpoise that is approximately 2.5 Gb in size over its 21 autosomes and two sex chromosomes (X and Y). A total of 22,814 protein-coding genes were predicted where ~97.31% were functionally annotated. This high-quality genome assembly of East Asian finless porpoise will not only provide new resources for the comparative genomics of cetaceans and conservation biology of threatened species, but also lay a foundation for more speciation, ecology, and evolutionary studies. Measurement(s) Neophocaena asiaeorientalis sunameri • Gapless genome assembly • sequence annotation Technology Type(s) MGISEQ. 2000 • PacBio HiFi Sequencing • Hi-C Sample Characteristic - Organism Neophocaena asiaeorientalis sunameri Sample Characteristic - Environment seawater Sample Characteristic - Location Yellow Sea near Lianyungang City, Jiangsu Province, China.

A proteomics approach reveals digestive and nutritional responses to food intake in anadromous Coilia nasus.

The estuarine tapertail anchovy, Coilia nasus, is an anadromous fish that undertakes over a 600-km spawning migration along the Yangtze River of China. They generally cease feeding during this process, but we recently documented that a small proportion of them appear to feed. Research on proteomic responses is essential for understanding the phenomenon of C. nasus feeding. In this study, we used an iTRAQ-based proteomics approach to study the changes in protein expression in response to food intake in C. nasus following voluntary fasting. Coilia nasus in the feeding group (CSI) were fed shrimp or small fish, whereas those in the control group (CSN) were starved. We identified 3279 proteins in the gastric tissue/stomach, of which 279 were significantly differentially expressed. In all, 133 differentially expressed proteins (DEPs) were upregulated and 146 proteins were downregulated in CSI compared with those in CSN C. nasus. In addition to gastric acid secretion caused by gastric distention, a functional analysis suggested that a series of DEPs were involved mainly in the regulation of protein digestion (e.g., carboxypeptidase A1 and chymotrypsin A-like), immune response (e.g., lysozyme and alpha 2-macroglobulin), and nutrition metabolism (e.g., glyceraldehyde 3-phosphate dehydrogenase, glycogenin, long-chain acyl-CoA synthetase, and creatine kinase). Real-time PCR confirmed that the mRNA levels of the DEPs were similar those obtained using iTRAQ. These results indicate that the nutrients obtained through food were effectively utilized by C. nasus, thereby providing energy for swimming, gonadal maturation, primary metabolism, and an enhanced immune function to better resist pathogen interference. This research contributes to the elucidation of nutritional regulation mechanisms of C. nasus to better protect the wild population.

The bacteria of Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) are site-specific and distinct from freshwater environment.

Bacteria play an essential role in the health of marine mammals, and the bacteria of marine mammals are widely concerned, but less is known about freshwater mammals. In this study, we investigated the bacteria of various body sites of Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) and analyzed their association with freshwater environmental bacteria. The bacterial community and function of Yangtze finless porpoise showed apparent site-specificity. Various body sites have distinct differences in bacteria and have their dominant bacteria. Romboutsia, Plesiomonas, Actinobacillus, Candidatus Arthromitus dominated in the intestine (fecal and rectal samples). Fusobacterium, Streptococcus, and Acinetobacter dominated in the oral. The dominant genera in the blowhole include Suttonella, Psychrobacter, and two uncultured genera. Psychrobacter, Flavobacterium, and Acinetobacter were dominant in the skin. The alpha diversity of intestinal (fecal and rectal) bacteria was the lowest, while that of skin was the highest. The oral and skin bacteria of Yangtze finless porpoise significantly differed between the natural and semi-natural conditions, but no sex difference was observed. A clear boundary was found between the animal and the freshwater environmental bacteria. Even the skin bacteria, which are more affected by the environment, are significantly different from the environmental bacteria and harbor indigenous bacteria. Our results provide a comprehensive preliminary exploration of the bacteria of Yangtze finless porpoise and its association with bacteria in the freshwater environment.

Integrated analysis of blood mRNAs and microRNAs reveals immune changes with age in the Yangtze finless porpoise (Neophocaena asiaeorientalis).

Populations of Yangtze finless porpoises (YFPs) have rapidly declined in recent decades, raising the specter of extinction. In order to protect YFPs, a greater understanding of their biology is needed, including studying how their immune functioning changes with age. Here, we systematically studied the hematologic and biochemical parameters, as well as mRNAs and miRNAs profiles of old, adult, and young YFPs. The lymphocyte (LYMPH), neutrophils (NEUT) and eosinophils (EOS) counts in old YFPs were lower than those in young or adult YFPs. When comparing old to adult YFPs, the latter showed higher expression of genes associated with the innate and adaptive immune systems, including complement components, major histocompatibility complex, interleukins, TNF receptors, and chemokines/cytokines. When comparing old to young YFPs, the most striking difference was in higher toll-like receptor signaling in the latter. When comparing adult to young YFPs, the former exhibited higher expression of genes related to adaptive immunity and the FoxO signaling pathway, but lower expression of genes associated with the PI3K-Akt signaling pathway. Negative miRNA-mRNA interactions were predicted in comparisons of the old and adult (326), old and young (316), adult and young (211) groups. Overall, these results delineate a progression from early innate immune function dominance to adaptive immune function enhancement (young to adult) and deterioration (adult to old), and the changes in miRNAs profile correlate with the effects of age on immune functions. This study is the first to observe the changes of immune function of Yangtze finless porpoise with age using transcriptome method, and the study's findings are of great significance for protecting this endangered species.

Metabolic mechanisms of Coilia nasus in the natural food intake state during migration.

As a prominent member of freshwater and coastal fish faunas, Coilia nasus migrates annually from the sea up the Yangtze River in China to spawn. It is traditionally believed that C. nasus generally do not feed during their spawning migration. However, we recently documented the occurrence of food intake phenomenon in C. nasus following voluntary fasting. The purpose of the current study is to explore the metabolic mechanisms on C. nasus in response to food intake during migration. A total of 23,159 differentially expressed mRNA molecules and 204 metabolites were identified in transcriptome and metabolome analyses. Our results provide insights into the activation of energy consumption and reinforcement of energy storage during migration, and also identify key genes involved in food intake regulation. Our findings will be useful for future research on population recruitment and energy utilization in wild C. nasus.

Changes in the fecal microbiome of the Yangtze finless porpoise during a short-term therapeutic treatment.

The fecal microbiome is an integral part of aquatic mammals, like an inner organ. But we know very little about this inner organ of the threatened aquatic species, Yangtze finless porpoise (YFP). Four YFPs were placed into a purse seine for skin ulceration treatment, and this opportunity was taken to nurse the animals closer. In particular, we collected the feces of the YFPs before and after the paired healing and therapeutic treatment, along with samples of their fish diet and water habitat, to explore the changes in their fecal microbiome. Firmicutes (20.9-96.1%), Proteobacteria (3.8-78.7%), Actinobacteria (0.1-35.0%) and Tenericutes (0.8-17.1%) were the most dominant phyla present in the feces. The proportion of Proteobacteria and Actinobacteria increased after the treatment. Firmicutes showed a significant decrease, and most potential pathogens were absent, which reflected the administration of ciprofloxacin hydrochloride. Moreover, environmental shifts can also contribute to changes in the fecal microbiome. These results indicate that certain microbial interactions can be affected by environmental shifts, dietary changes and health-care treatments, which can also help maintain the internal environment of YFPs. These findings will inform the future enhanced protection and management of endangered YFPs and other vulnerable aquatic animals.

The intestinal microbiota of lake anchovy varies according to sex, body size, and local habitat in Taihu Lake, China.

Lake anchovy (Coilia ectenes taihuensis) is a sedentary, dominant fish species that forms an unmanaged fishery in Taihu Lake, eastern China. The environment and developmental stage of lake anchovy are likely important drivers of their gut microbiome, which is linked to host health and development. To investigate the relationship between the gut microbiome and three defined factors (fish sex, fish body size, and the local habitat), high-throughput sequencing of the 16S ribosomal RNA gene was used to study the microorganisms of 184 fish samples and four water samples collected in Taihu Lake. Four dominant bacterial phyla (Proteobacteria, Firmicutes, Planctomycetes, and Cyanobacteria) were present in all fish samples. We compared the microbial communities of males and females and found that the relative abundance of Corynebacteriaceae was significantly higher in males than in females, while the opposite trend was detected for Sphingomonadaceae. We also discovered that the relative abundance of Firmicutes was positively correlated with fish body size and that the proportions of Proteobacteria and Tenericutes were lower in larger fish than in fish of other sizes. Finally, we found that the difference in microbial richness between eastern and northern Taihu Lake was the most marked. Lake anchovy was rich in Lactobacillus and Clostridium in the eastern site, while those in the northern site had the highest abundance of Sphingomonas and Methylobacterium, suggesting that the local habitat may also influence the intestinal microbiome. These findings will not only help researchers understand the community composition of the intestinal microflora of lake anchovy but also contribute to the protection of fish resources in Lake Taihu and the sustainable use of lake anchovy.

Digestive enzyme activity of the Japanese grenadier anchovy Coilia nasus during spawning migration: influence of the migration distance and the water temperature.

In this study, we investigated the activity levels of two major digestive enzymes (pepsin and lipase) in the commercially important Japanese grenadier anchovy Coilia nasus during its upstream migration to analyse the digestive physiological responses to starvation and to analyse the influence of the water temperature on enzyme activity. Water temperature had a significant effect on pepsin activity, while long-term starvation resulted in a significant decrease in pepsin activity. As starvation continued, however, a slight increase in pepsin activity between the Wuhu (440 river km) and Anqing (620 river km) regions may indicate that C. nasus had refeeding behaviour due to its large expenditure of energy reserves. In contrast, lipase activity was not significantly affected by the water temperature but the effect of fasting increased as much as 13% of lipase activity from the Chongming region (20 river km) to Anqing region, suggesting that the stored lipids of grenadier anchovy were mobilised to meet energy requirements of upstream migration activity and gonad development. Lipid mobilisation activated lipoprotein lipase (LPL; proteins with lipase activity) to hydrolyse triacylglycerides (TAG), which is the first step of lipid assimilation and obtained energy from fatty acids under fasting conditions. Therefore, the increased lipase activity is attributed mainly to the lipase that is involved in endogenous lipid hydrolysis. Grenadier anchovy appears to adapt to long-term starvation during migration and the increased lipase activity may indicate a crucial effect on lipid metabolism. This study demonstrated that distinct alterations occur in pepsin and lipase activities during the spawning migration of grenadier anchovy due to exogenous nutrition and endogenous metabolism. Furthermore, it provides a basis for further research on the digestive physiology and energy metabolism in this species.

Transcriptome and metabolome analyses of Coilia nasus in response to Anisakidae parasite infection.

Parasites from the family Anisakidae are capable of infecting a range of marine fish species worldwide. Coilia nasus, which usually feeds and overwinters in coastal waters and spawns in freshwater, is highly susceptible to infection by Anisakidae. In this study, we used scanning electron microscopes to show that C. nasus infected by Anisakidae exhibited damage and fibrosis of the liver tissue. To better understand host immune reaction and metabolic changes to Anisakidae infection, we used a combination of transcriptomic and metabolomic method to characterize the key genes and metabolites, and the signaling pathway regulation of C. nasus infected by Anisakidae. We generated 62,604 unigenes from liver tissue and identified 391 compounds from serum. Of these, Anisakidae infection resulted in significant up-regulation of 545 genes and 28 metabolites, and significant down-regulation of 416 genes and 37 metabolites. Seventy-four of the 961 differentially expressed genes were linked to immune response, and 1, 2-Diacylglycerol, an important immune-related metabolite, was significantly up-regulated after infection. Our results show activation of antigen processing and presentation, initiation of the T cell receptor signaling pathway, disruption of the TCA cycle, and changes to the amino acid and Glycerolipid metabolisms, which indicate perturbations to the host immune system and metabolism following infection. This is the first study describing the immune responses and metabolic changes in C. nasus to Anisakidae infection, and thus improves our understanding of the interaction mechanisms between C. nasus and Anisakidae. Our findings will be useful for future research on the population ecology of C. nasus.

Identification and characterization of a TAB1 gene involved in innate immunity of amphioxus (Branchiostoma belcheri).

Transforming growth factor-ß activated kinase-1 (TAK1) is an essential regulator in toll-like receptor (TLR), tumor necrosis factor (TNF) and interleukin-1 (IL-1) signaling pathways, and plays very important roles in animal innate immunity. TAK1-binding protein, TAB1, can specifically regulate the activation of TAK1. However, the TAB1 gene in amphioxus has not yet been identified to date. In this study, we identified and characterized a TAB1 gene from Branchiostoma belcheri (designed as AmphiTAB1). Our results showed that the full-length cDNA of AmphiTAB1 is 2281bp long with an open reading frame (ORF) of 1659bp that encodes a predicted protein of 553 amino acids containing a typical PP2Cc domain. Phylogenetic analysis indicated that the AmphiTAB1 gene was located between invertebrates and vertebrates, suggesting that the AmphiTAB1 gene is a member of the TAB1 gene family. Real-time PCR analysis indicated that the AmphiTAB1 was ubiquitously and differentially expressed in six investigated tissues (gills, hepatic cecum, intestine, muscles, notochord and gonad). After lipopolysaccharide stimulation, the expression of AmphiTAB1 was significantly up-regulated at 6h, which shows that AmphiTAB1 may be involved in the host immune response. In addition, the recombinant TAB1 expressed in vitro shows a molecular mass of 62kDa and Western blot confirmed it, which proved it is an encoding isoform. Taken together, our findings provide an insight into innate immune response of amphioxus and evolution of the TAB1 gene family.

Identification and characterization of complement factor H in Branchiostoma belcheri.

Complement factor H (CFH) is an essential regulator of the complement system and plays very important roles in animal innate immunity. Although the complement system of amphioxus has been extensively studied, the expression in amphioxus and evolution of CFH gene remain unknown. In this study, we identified and characterized an amphioxus (Branchiostoma belcheri) CFH gene (designated as AmphiCFH). Our results showed that the full-length cDNA of AmphiCFH gene consists of 1295 bp nucleotides containing an 855 bp open reading frame (ORF) that was predicted to encode a 284 amino acid protein. The putative AmphiCFH protein possessed the characteristic of the CFH protein family, including typical CCP (complement control protein) domain. Real-time PCR analysis showed that the AmphiCFH was ubiquitously and differentially expressed in five investigated tissues (intestine, gills, notochord, muscles, and hepatic cecum). The expression level of the AmphiCFH gene was induced upon lipopolysaccharide stimulation, indicating that the AmphiCFH gene might be involved in innate immunity. In addition, phylogenetic analysis showed that the AmphiCFH gene was located between that of invertebrates and vertebrates, suggesting that the AmphiCFH gene is a member of the CFH gene family. In conclusion, our findings provided an insight into animal innate immunity and evolution of the CFH gene family.