Integrated Application of Multiomics Strategies Provides Insights Into the Environmental Hypoxia Response in Pelteobagrus vachelli Muscle.

Increasing pressures on aquatic ecosystems because of pollutants, nutrient enrichment, and global warming have severely depleted oxygen concentrations. This sudden and significant lack of oxygen has resulted in persistent increases in fish mortality rates. Revealing the molecular mechanism of fish hypoxia adaptation will help researchers to find markers for hypoxia induced by environmental stress. Here, we used a multiomics approach to identify several hypoxia-associated miRNAs, mRNAs, proteins, and metabolites involved in diverse biological pathways in the muscles of Pelteobagrus vachelli. Our findings revealed significant hypoxia-associated changes in muscles over 4 h of hypoxia exposure and discrete tissue-specific patterns. We have previously reported that P. vachelli livers exhibit increased anaerobic glycolysis, heme synthesis, erythropoiesis, and inhibit apoptosis when exposed to hypoxia for 4 h. However, the opposite was observed in muscles. According to our comprehensive analysis, fishes show an acute response to hypoxia, including activation of catabolic pathways to generate more energy, reduction of biosynthesis to decrease energy consumption, and shifting from aerobic to anaerobic metabolic contributions. Also, we found that hypoxia induced muscle dysfunction by impairing mitochondrial function, activating inflammasomes, and apoptosis. The hypoxia-induced mitochondrial dysfunction enhanced oxidative stress, apoptosis, and further triggered interleukin-1ß production via inflammasome activation. In turn, interleukin-1ß further impaired mitochondrial function or apoptosis by suppressing downstream mitochondrial biosynthesis-related proteins, thus resulting in a vicious cycle of inflammasome activation and mitochondrial dysfunction. Our findings contribute meaningful insights into the molecular mechanisms of hypoxia, and the methods and study design can be utilized across different fish species.

Effects of dietary plant protein sources intake on growth, digestive enzyme activity, edible tissue nutritional status and intestinal health of the omnivorous Redclaw crayfish, Cherax quadricarinatus.

For the omnivorous Cherax quadricarinatus crayfish, plant raw materials can be good alternatives to dietary fish meal (FM). A 56-d feeding trial was conducted in C. quadricarinatus (11·70 (se 0·13) g). Diet with 100 % FM as the protein source was the control. Seven experimental diets were formulated by replacing 75 or 100 % of FM with soyabean meal (SM75, SM100) or cottonseed meal (CM75 and CM100), and a mixture of SM and CM (protein content is 1:1) replacing 50, 75 or 100 % of FM (SC50, SC75 and SC100). Crayfish fed the CM100 and SC100 showed significantly lower weight gain (WG), specific growth rate, trypsin and pepsin activities compared with the control diet. Crayfish in CM100 group showed significantly higher GPx, alanine aminotransferase, aspartate aminotransferase activities and malondialdehyde content than the control. SM100 and CM100 diets can cause slight separation of the peritrophic membrane from the intestinal folds. The pepsin activity of crayfish in SC50 was significantly higher than those in other experimental diets. The highest WG and muscle arginine content were also found in crayfish fed SC50. The relative abundance of Proteobacteria, Unclassified Enterobacteriaceae and Candidatus Bacilloplasma was significantly higher, but Actinobacteriota was significantly lower in SM100, CM100 and SC100 than in control. Microbiota functional prediction indicated that the relative abundance of 'cell motility' pathway in crayfish fed CM100 was significantly decreased compared with the control. In conclusion, only half of the FM can be effectively substituted with a mixture of SM and CM (protein content is 1:1) for C. quadricarinatus.

Changes in the gene expression and gut microbiome to the infection of decapod iridescent virus 1 in Cherax quadricarinatus.

As a new emerging viral pathogen, Decapod iridescent virus 1 (DIV1) seriously threatens crustacean farming in recent years. However, limited research progresses have been made on the immune mechanism between host and viral factors in response to DIV1 infection. In the current study, a natural occurrence of DIV1 infection with obvious clinical signs was found in farmed redclaw crayfish Cherax quadricarinatus, and confirmed by nested PCR detection and histopathological examination. Besides, gene expression profiles were analyzed after being challenged with DIV1, and results showed that 27 immune related genes were upregulated compared with the control group. Moreover, the gut microbiota from healthy and DIV1-infected crayfish were investigated by 16S rDNA high-throughput sequencing. Results showed that significant differences in the microbial composition and function were observed after DIV1 challenge. Furthermore, we discovered that changes in gene expression profiles were correlated with microbiota alterations under DIV1 challenge. Taken together, our findings will provide new insights into the immune response mechanism of DIV1 infection in crustaceans.

Evaluation of the Role of Soybean Lecithin, Egg Yolk Lecithin, and Krill Oil in Promoting Ovarian Development in the Female Redclaw Crayfish Cherax quadricarinatus.

The optimal supplementation of lipid nutrients in the diet showed crucial physiological functions in gonadal development and maturation in adult female aquatic animals. Four isonitrogenous and isolipidic diets with no extra lecithin supplementation (control), 2% soybean lecithin (SL), egg yolk lecithin (EL), or krill oil (KO) supplementation were formulated for Cherax quadricarinatus (72.32 ± 3.58 g). Ovary development and physiological characteristics of crayfish were evaluated after a 10-week feeding trial. The results indicated that SL, EL, or KO supplementation all significantly increased the gonadosomatic index, especially in the KO group. Crayfish fed the diet with SL showed the highest hepatosomatic index compared with those fed the other experimental diets. KO was more efficient than SL and EL in promoting triacylglycerol and cholesterol deposition in the ovary and hepatopancreas but also showed the lowest concentration of low-density lipoprotein cholesterol in the serum. KO significantly increased yolk granule deposition and accelerated oocyte maturation than other experimental groups. Furthermore, dietary phospholipids significantly enhanced the gonad-stimulating hormone concentration in the ovary and reduced the secretion of gonad-inhibiting hormones in the eyestalk. KO supplementation also significantly improved organic antioxidant capacity. From the ovarian lipidomics results, phosphatidylcholine and phosphatidylethanolamine are two main glycerophospholipids that respond to different dietary phospholipids. Polyunsaturated fatty acids (especially C18:2n-6, C18:3n-3, C20:4n-6, C20:5n-3, and C22:6n-3) were pivotal participants during ovarian development of crayfish regardless of lipid type. Combined with the ovarian transcriptome, the best positive function of KO was due to activated steroid hormone biosynthesis, sphingolipid signaling, retinol metabolism, lipolysis, starch and sucrose metabolism, vitamin digestion and absorption, and pancreatic secretion. As a consequence, dietary supplementation with SL, EL, or KO all improved the ovarian development quality of C. quadricarinatus, especially KO, which was the optimum choice for promoting ovary development in adult female C. quadricarinatus.

Genomic structure, expression, and functional characterization of the Fem-1 gene family in the redclaw crayfish, Cherax quadricarinatus.

The Fem-1 (Feminization-1) gene, encoding an intracellular protein with conserved ankyrin repeat motifs, has been proven to play a key role in sex differentiation in Caenorhabditis elegans. In the present study, three members of the Fem-1 gene family (designating Fem-1A, Fem-1B, and Fem-1C, respectively) were cloned and characterized in the redclaw crayfish, Cherax quadricarinatus. Sequence analysis showed that all three Fem-1 genes contained the highly conserved ankyrin repeat motifs with variant repeat numbers, which shared similarity with other reported crustaceans. In addition, a phylogenetic tree revealed that the Fem-1 proteins from C. quadricarinatus were clustered with the crustacean Fem-1 homologs, and had the closest evolutionary relationship with Eriocheir sinensis. Quantitative real-time PCR (qRT-PCR) results demonstrated that Fem-1B exhibited a significant higher expression abundance in the ovary than in other tissues. In addition, a regular mRNA expression pattern of the Fem-1B gene appeared in the reproductive cycle of ovarian development. Furthermore, RNA interference experiments were employed to investigate the role of Fem-1B in ovarian development. Moreover, knockdown of Fem-1B by RNAi decreased the expression of VTG in the ovaries and hepatopancreas. In summary, this study pointed out that Fem-1B was involved in the sex differentiation process through regulating VTG expression in C. quadricarinatus, and provided new insights into the role of Fem-1B in ovary development.

Transcriptome profiling towards understanding of the morphogenesis in the scale development of blunt snout bream (Megalobrama amblycephala).

Skin appendages in vertebrates have individual morphological differences, but share the same evolutionary origin. In this study, we used Megalobrama amblycephala as a fish model to study the developmental regulation mechanism of a common skin appendage in fish: scales. By combining in-toto live imaging method and transcriptomic analysis during the scale development, we elucidated core features of scale patterning containing three distinct regions and experiencing four stages. Differentially expressed genes in skin tissues at the initial site before and after scale development were analyzed and some key regulatory genes (Wnt3, Wnt6, Fgf8, Fgf10, Fgf16, Fgfr1a, Ihhb and BMP6) which are crucial for scale morphogenesis were selected. This study provides a strong reference for further exploration of the function of genes related to the molecular regulation mechanism of scale development in M. amblycephala, as well as in other fishes.

The Optimum Lipid Level for the Juvenile Redclaw Crayfish Cherax quadricarinatus: Practical Diets with Soybean Oil as the Lipid Source.

As a new species in aquaculture, the lipid nutrition requirement for the juvenile redclaw crayfish Cherax quadricarinatus on a dietary basis on a practical formula needs to be evaluated accurately. In this study, the optimum dietary lipid level was explained by analyzing the growth performance, antioxidant state, lipid metabolism, and gut microbiota of C. quadricarinatus after an eight-week cultivation trial. Six diets with different soybean oil levels (named L0, L2, L4, L6, L8, and L10) were fed to C. quadricarinatus (11.39 ± 0.28 g). The results indicated that the specific growth rate and weight gain of crayfish fed the L4 and L6 diets were significantly higher than those of the other groups (P < 0.05). By the analysis of a second-order polynomial regression model according to growth performance (weight gain rate), the optimum lipid level in a practical diet for juvenile C. quadricarinatus was 9.67%. The survival, condition factor, and hepatosomatic index of crayfish were not significantly affected by dietary oil levels (P > 0.05). As the level of dietary lipids increased, the total antioxidant capacity and glutathione peroxidase activity in serum showed a tendency to rise and then fall and the enzyme activity was highest in crayfish fed the L6 diet. Gut lipase and pepsin activities showed the highest value in crayfish fed the L6 diet. There was no significant difference in acetyl-CoA carboxylase and carnitine palmitoyltransferase-1 contents in crayfish among all the groups (P > 0.05). The relative abundance of Proteobacteria in the phylum and Citrobacter in the genus showed a significant decrease in crayfish of the L10 diet, while the relative abundance of Firmicutes in the phylum markedly increased compared to that of the other groups (P < 0.05). In summary, the results indicated that the 10.39% (L6 diet) dietary lipid level could induce better growth performance, antioxidant ability, and digestive enzyme activity. Most of the fatty acid composition of muscle is not closely related to the fatty acid composition of the diet. Moreover, the composition and diversity of the gut microbiota of C. quadricarinatus were changed by high dietary lipid levels.

Different pituitary action of NK3Ra and NK3Rb in grass carp.

In mammals, NK3R is the specific receptor for NKB, which played an important role in reproduction. Recently, two NK3R isoforms, namely NK3Ra and NK3Rb, have been identified in fish. However, little is known about the pituitary actions of the two NK3R isoforms in fish. In this study, both NK3Ra and NK3Rb were isolated from grass carp pituitary. Although their sequence similarity was only 61.6%, the two NK3R isoforms displayed similar ligand selectivity and binding affinity to TAC3 gene products (NKBa, NKBRPa and NKBRPb). In addition, both NK3Ra and NK3Rb displayed similar signaling pathways, including PKA, PKC, MAPK and Ca2+ cascades. Tissue distribution indicated that both NK3Ra and NK3Rb were highly detected in grass carp pituitary. Further study found that NK3Ra was mainly located in pituitary LHß cells, while NK3Rb was only detected in pituitary SLα cells. Furthermore, NK3Ra and NK3Rb activation could induce LHß and SLα promoter activity, respectively. These results suggested that the two NK3R isoforms displayed different pituitary actions in fish. Using grass carp pituitary cells as model, we found that PACAP could significantly reduce NK3Ra, but induce NK3Rb mRNA expression coupled with cAMP/PKA and PLC/PKC pathways. Interestingly, PACAP could also significantly inhibit LHß, but stimulate SLα mRNA expression in grass carp pituitary cells. Furthermore, NK3R antagonist could not only inhibit LHß mRNA expression, but also block PACAP-induced SLα mRNA expression in grass carp pituitary cells. These results suggested that NK3Ra and NK3Rb could mediate PACAP-reduced LHß and -induced SLα mRNA expression in grass carp pituitary, respectively.

Effect of single and combined immunostimulants on growth, anti-oxidation activity, non-specific immunity and resistance to Aeromonas hydrophila in Chinese mitten crab (Eriocheir sinensis).

This study evaluates the effect of dietary supplementation of immunostimulants on the Chinese mitten crab (Eriocheir sinensis) with a single administration of mannan oligosaccharide (MOS), or its combination with either ß-glucan or with inulin for 8 weeks. Four diets included an untreated control diet (C), MOS alone (3 g kg-1, M), MOS with ß-glucan (3 g kg -1 MOS + 1.5 g kg -1 ß-glucan, MB), and MOS with inulin (3 g kg -1 MOS + 10 g kg -1 inulin, MI). The weight gain and specific growth rate of the crabs fed M, MB, and MI diets were improved by lowing feed conversion ratio. The growth and feed utilization of the crabs fed the MB diet were improved compared with the other three groups. The crabs fed the M, MB and MI diets showed a higher intestinal trypsin activity than that in the M and control groups. The highest trypsin activity in the hepatopancreas was observed in the MB group. Crabs fed M, MB and MI diets increased antioxidant system-related enzyme activities, but reduced malondialdehyde. The highest activities of alkaline phosphatase, acid phosphatase, lysozyme and phenol oxidase in the gut and the respiratory burst of the crabs were found in the MB group. The MB diet promoted the mRNA expression of E. sinensis immune genes (ES-PT, ES-Relish, ES-LITAF, p38MAPK and Crustin) compared with the control. After 3 days of infection with Aeromonas hydrophila, the highest survival of crabs was also found in the MB group. This study indicates that the combination of MOS with ß-glucan or with inulin can improve growth, antioxidant capacity, non-specific immunity and disease resistance in E. sinensis.

Arginine supplementation improves growth, antioxidant capacity, immunity and disease resistance of juvenile Chinese mitten crab, Eriocheir sinensis.

To investigate the effects of arginine (Arg) on the growth, antioxidant capacity, immunity and disease resistance of juvenile Chinese mitten crab, three diets containing Arg levels at 1.72% (control), 2.73% and 3.72% were formulated and fed to Chinese mitten crab (0.22 ±â€¯0.03 g) for eight weeks. The weight gain, ecdysterone and growth hormone in the serum, relative expression of insulin-like growth factor 2 in the hepatopancreas significantly increased in crabs fed the 2.73% and 3.72% Arg diets. The protein and lipid contents significantly increased in crabs fed the 3.72% Arg diet. The feed conversion ratios in crabs fed the diets with Arg additions were lower than in the control. Arg supplementation also enhanced the antioxidative capacity by increasing the activities of superoxide dismutase, catalase and the relative expression of Kelch-like ECH-associated protein 1 gene in the hepatopancreas, which subsequently decreased malondialdehyde content in the hepatopancreas. Besides, Arg also decreased nitric oxide content in the serum and the activity of nitric oxide synthetase in the hepatopancreas. The relative mRNA levels of crustin, relish, lysozyme and cryptocyanin genes were significantly upregulated by Arg supplementation. The activities of acid phosphatase and alkaline phosphatase in the serum significantly increased in crabs fed the 3.72% Arg diet than those in the control. Similarly, the relative mRNA levels of crustin, cryptocyanin and proPO genes were significantly upregulated in crabs fed the 2.73% Arg diet after lipopolysaccharide challenge, and in crabs fed the 3.72% Arg diet after the Poly (I:C) challenge. The crabs fed the 2.73% and 3.72% Arg diets had higher survival rate after bacterial infection than those fed the control diet. This study indicates that the addition of Arg to the diet at 2.7-3.7% can improve the growth, survival, antioxidant capacity, immunity and disease resistance in juvenile Chinese mitten crab.

Evaluation of salinomycin isolated from Streptomyces albus JSY-2 against the ciliate, Ichthyophthirius multifiliis.

The present study was undertaken to investigate the antiparasitic activity of extracellular products of Streptomyces albus. Bioactivity-guided isolation of chloroform extracts affording a compound showing potent activity. The structure of the compound was elucidated as salinomycin (SAL) by EI-MS, 1H NMR and 13C NMR. In vitro test showed that SAL has potent anti-parasitic efficacy against theronts of Ichthyophthirius multifiliis with 10 min, 1, 2, 3 and 4 h (effective concentration) EC50 (95% confidence intervals) of 2.12 (2.22-2.02), 1.93 (1.98-1.88), 1.42 (1.47-1.37), 1.35 (1.41-1.31) and 1.11 (1.21-1.01) mg L-1. In vitro antiparasitic assays revealed that SAL could be 100% effective against I. multifiliis encysted tomonts at a concentration of 8.0 mg L-1. In vivo test demonstrated that the number of I. multifiliis trophonts on Erythroculter ilishaeformis treated with SAL was markedly lower than that of control group at 10 days after exposed to theronts (P < 0.05). In the control group, 80% mortality was observed owing to heavy I. multifiliis infection at 10 days. On the other hand, only 30.0% mortality was recorded in the group treated with 8.0 mg L-1 SAL. The median lethal dose (LD50) of SAL for E. ilishaeformis was 32.9 mg L-1.

Molecular identification of dmrt1 and its promoter CpG methylation in correlation with gene expression during gonad development in Culter alburnus.

Dmrt1, a member of the Dmrt family, is an important transcription regulator of gender determination. To study the biological function of dmrt1 in sexual differentiation and its potential implication in breeding technology, we obtained the full-length cDNA and proximal promoter sequence of dmrt1 in Culter alburnus, and analyzed the impact of promoter CpG methylation on the gene expression pattern of dmrt1 during gonad development. Dmrt1 was 922 bp in length and consisted a 150 bp 5'-UTR, a 28 bp 3'-UTR, and a 744 bp open reading frame (ORF). Based on the coding sequence of the dmrt1 gene, the deduced amino acid sequence was detected, and the protein structure of this gene was predicted in C. alburnus. The results indicate that the structure and function of dmrt1 were highly conservative compared to other vertebrates. The expression level of dmrt1 mRNA in different tissues was explored by qRT-PCR, which was only highly expressed in the testes and almost undetectable in other tissues. The CpG methylation pattern of the dmrt1 promoter was studied using DNA sequencing of sodium bisulfite in adult testes and ovaries, and it was found that dmrt1 promoter CpGs were not methylated in the testes, whereas hypermethylated in the ovaries. These findings demonstrate that DNA methylation can regulate sexual dimorphic expression of dmrt1, and therefore epigenetic modifications may play a critical role in the gonad differentiation of C. alburnus.

Response of gut health and microbiota to sulfide exposure in Pacific white shrimp Litopenaeus vannamei.

Sulfide is a natural and widely distributed toxicant. It can be commonly found on the interface between water and sediment in the aquatic environment. The Pacific white shrimp Litopenaeus vannamei starts life in the benthic zone soon after the mysis stage, an early stage of post larvae. Therefore, L. vannamei is inevitably affected by exposure to sulfide released from pond sediment. This study explored the toxicant effect of different concentrations of sulfide on the intestinal health and microbiota of Pacific white shrimp by monitoring the change of expression of inflammatory, immune related cytokines, and the structure of the intestinal microbiota. The gut histology, expressions of inflammatory and immune related cytokines (tumor necrosis factor-alpha, C-type lectin 3, myostatin and heat shock transcription factor 1), and the microbiota were determined in L. vannamei after exposure to 0 (control), 425.5 (1/10 LC 50-96 h), and 851 µg/L (1/5 LC 50-96 h) of sulfide for 21 days. With the increase of sulfide concentration, intestinal injury was aggravated and the inflammatory and immune related cytokines generated a range of reactions. The expression of myostatin (MSTN) was significantly down-regulated by the concentration of sulfide exposure. No difference in the expression of heat shock transcription factor 1 (HSF1) was found between the control and shrimp exposed to 425.5 µg/L, but significantly higher HSF1 expression was found in shrimp exposed to 851 µg/L of sulfide. Significantly higher values of tumor necrosis factor-alpha (TNF-α) and C-type lectin 3 (CTL3) were found in the shrimp exposed to 425.5 µg/L of sulfide compared to the control, but a lower value was found in the shrimp exposed to 851 µg/L (P < 0.05). Sulfide also changed the intestinal microbial communities. The abundance of pathogenic bacteria, such as Cyanobacteria, Vibrio and Photobacterium, increased significantly with exposure to the increasing concentration of sulfide. The abundance of some anti-stress bacteria, such as Chlorobi and Fusobacterium, increased. Nitrospirae which can alleviate nitrite toxicity decreased. Microbacterium, Parachlamydia, and Shewanella were all commonly found and down-regulated in both sulfide groups, which is associated with an adaptation to sulfide stimulation. This study indicates that chronic exposure to sub-lethal levels of sulfide could lead to damage of the gut structure, stimulate the response of the inflammatory and immune systems, and shape the structure of the gut microbiota in L. vannamei.

Analysis of the Differences in Muscle Nutrition among Individuals of Different Sexes in Redclaw Crayfish, Cherax quadricarinatus.

Redclaw crayfish (Cherax quadricarinatus) was introduced to China many years ago. In recent years, a breeding boom for C. quadricarinatus has been set off in China due to a breakthrough in key technology of seedling breeding. The size and growth rate of C. quadricarinatus vary greatly between female and male individuals, usually the size and growth rate of male individuals are bigger than that of female individuals. There is usually a certain linkage relationship between the sex traits of crustaceans and their own nutrition. In order to explore the linkage relationship between the sex traits of C. quadricarinatus and its nutritional components, this study measured and analyzed the muscle nutritional components of female and male individuals. The results showed that the meat yield rate of male individuals was significantly higher than that of females (p < 0.05), and the crude fat content was significantly lower than that for females (p < 0.05). The ratios of essential amino acids to total amino acids for females and males were 39.61% and 38.49%, respectively. The ratios of essential amino acids to non-essential amino acids were 79.69% and 75.66%, respectively, which far exceed FAO/WHO standards and both belong to high-quality protein. The total amount of flavor amino acids of male individuals was significantly higher than that of female individuals (p < 0.05). The total amount of polyunsaturated fatty acids and the polyunsaturated fatty acid eicosapentaenoic acid of males are both significantly higher than that of females (p < 0.05). Studies have shown that there are certain differences in nutrition between male and female individuals. Compared with female individuals, the meat yield rate, crude protein content, and edible value of the muscles of male individuals is higher.

Toxic effects of waterborne benzylparaben on the growth, antioxidant capacity and lipid metabolism of Nile tilapia (Oreochromis niloticus).

Benzylparaben (BzP) is a potential endocrine disruptor; however, its antioxidant defense, lipotoxicity and underlying mechanism of BzP in aquatic organisms are unknown. This study investigated the impacts of waterborne low-, environmental-related and high-level benzylparaben on the growth, antioxidant capacity, lipid metabolism and lipidomic response of Nile tilapia (Oreochromis niloticus). Juvenile tilapia (0.60 ± 0.11 g) were exposed to 0, 5, 50, 500 and 5000 ng/L benzylparaben for 8 weeks in quadruplicate for each group. Benzylparaben increased the body crude fat content but decreased brain acetylcholinesterase activity in O. niloticus. Benzylparaben caused oxidative stress, leading to hepatic morphology damage and lipid metabolism disorders in fish. Lipidomic analysis identified 13 lipid classes in fish liver. Benzylparaben exposure induced metabolic disorders of glycerol phospholipids, glycerolipids and sphingomyelins in fish liver. These findings indicate that environmentally related benzylparaben levels (5 to 50 ng/L) could induce an antioxidant response, result in triglyceride accumulation, and increase adipocyte formation and fatty acid intake in tilapia. However, high benzylparaben concentrations inhibit lipid deposition, presumably due to the effects of the antioxidant system, and induce tissue inflammation. Therefore, this study provides new insights into the toxic effects and potential mechanism of benzylparaben in fish, especially from the aspect of lipid metabolism.

Sexual dimorphism in Odontobutis sinensis brain-pituitary-gonad axis and liver highlighted by histological and transcriptomic approach.

In this study, sexual dimorphism in Chinese dark sleeper (Odontobutis sinensis) brain-pituitary-gonad axis and liver was highlighted by histological and transcriptomic approach. The results showed that there were two significant differences between males and females. Firstly, males grew larger and faster than females. Transcriptomic analysis and qPCR validation indicated that two key growth genes, insulin-like growth factor (igf) and 25-hydroxyvitamin D-1 alpha hydroxylase (cyp27b), were more highly detected in male liver than that in female liver. Secondly, histological analysis displayed that the liver in males showed an obvious ivory fatty phenomenon with more fat vacuoles and lipid droplet aggregation compared to that in females. Transcriptomic analysis indicated that the transcript level of vitellogenin (vtg) in male liver were significantly lower than that in female liver. After 17ß-estradiol (E2) treatment of primary cultured hepatocytes, the vtg mRNA expression was induced significantly, while dihydrotestosterone (DHT) treatment had little effect on it. Generally, this study will provide some ideas for further exploring the mechanism of sexual dimorphism in Odontobutis sinensis.

A Chromosome-Level Genome Assembly of the Dark Sleeper Odontobutis potamophila.

The dark sleeper, Odontobutis potamophila, is a commercially valuable fish that widely distributed in China and Southeast Asia countries. The phenomenon of sexual dimorphism in growth is conspicuous, which the males grow substantially larger and faster than the females. However, the high-quality genome resources for gaining insight into sex-determining mechanisms to develop sex-control breeding are still lacking. Here, a chromosomal-level genome assembly of O. potamophila was generated from a combination of Illumina reads, 10× Genomics sequencing, and Hi-C chromatin interaction sequencing. The assembled genome was 1,134.62 Mb with a contig N50 of 22.25 Mb and a scaffold N50 of 24.85 Mb, representing 94.4% completeness (Benchmarking Universal Single-Copy Orthologs). Using Hi-C data, 96.49% of the total contig bases were anchored to the 22 chromosomes, with a contig N50 of 22.25 Mb and a scaffold N50 of 47.68 Mb. Approximately 54.18% of the genome were identified as repetitive elements, and 23,923 protein-coding genes were annotated in the genome. The assembled genome can be used as a valuable resource for molecular breeding and functional studies of O. potamophila in the future.

Phylogenetic analysis of eight species of Anomopoda based on transcriptomic and mitochondrial DNA sequences.

Anomopoda is the widespread planktonic microcrustacean, which plays a crucial role in aquatic ecosystem. There are few studies about the evolutionary relationships among various Anomopoda basing on molecular data. In the present study, phylogenetic analysis of eight Anomopoda was carried out. Firstly, the culture system was developed to breed cladocerans. By using this system, eight species (Daphnia magna, D. pulex, D. sinensis, Ceriodaphnia reticulata, Moina micrura, Scapholeberis kingi, Simocephalus vetulus and Eurycercus lamellatus) were purified and cultured stably in the laboratory. Then, transcriptomic sequences and partial mitochondrial DNA sequences were both used to reconstruct the phylogenetic tree among 8 species. Transcriptomic sequences were sequenced on Illumina Hiseq 2500 platform. After assembly and annotation, transcriptomic sequences were spliced together and aligned for phylogenetic analysis. Basing on the orthologous genes derived from transcriptomic sequences, the phylogenetic analysis showed that 4 genera of Daphniidae were clustered into one group, and among the 4 genera, Ceriodaphnia was closer to Daphnia than Simocephalus, while Scapholeberis was farthest from other species. In addition, Eurycercidae was closer to Daphniidae than Moinidae. The phylogenetic trees based on both 12S rRNA and 16S rRNA sequences were similar with that based on transcriptomic sequences. Meanwhile, the phylogenetic tree based on 16S rRNA sequences was more suitable than that based on 12S rRNA sequences. These results suggested that the phylogenetic analysis basing on the transcriptomic sequences was available in cladocerans, which will help us to effectively understand the phylogenetic relationships among various cladocerans.

Molecular Characterization and Expression Profiling of Three Transformer-2 Splice Isoforms in the Redclaw Crayfish, Cherax quadricarinatus.

Sex determination/sex differentiation is determined by genetics, environmental factors, or the interactions of the two. The Transformer-2 (Tra-2) gene plays an important role in the sex determination cascade signal pathway in insects. In this study, the Tra-2 gene was isolated and characterized from the cDNA library of gonad tissues in the redclaw crayfish, Cherax quadricarinatus. Three splice variants were identified, designated as CqTra-2A, CqTra-2B, and CqTra-2C, and sequence analysis showed that they had a highly conserved RRM domain. Phylogenetic analysis was performed by the NJ method, and the results revealed that the Tra-2 protein of the redclaw crayfish was very closely related to those of Macrobrachium rosenbergii, Fenneropenaeus chinensis, and Macrobrachium nipponense. Real-time PCR analysis showed that the three isoforms were predominantly expressed in the ovary and gradually increased with embryonic development. Additionally, the expression pattern of CqTra-2 at different developmental stages was analyzed by qPCR and revealed that the phase of having a body length of 3 cm may be the key period for the sex differentiation of C. quadricarinatus. RNAi-targeting gene silencing further confirmed the function of CqTra-2 in sexual differentiation in redclaw crayfish. Our experimental data will contribute to understanding the mechanism of sex determination in crustaceans.

Molecular characterization and expression profiles of transcription factor Sox gene family in Culter alburnus.

Sox protein family is characterized by the presence of the conserved high-mobility group (HMG) box. Sox transcription factors are involved in diverse developmental process in animals, including sex-determination, organogenesis, embryogenesis, neurogenesis, and cell fate decision. In this study, 23 Sox genes were identified based on the Culter alburnus whole-genome sequence and categorized into six subfamilies according to the conserved HMG-box domain. The duplicates of four members revealed that Sox genes in the teleost fishes underwent significant expansion. Moreover, their expression pattern in gonad tissues were analyzed by RNA-seq and qRT-PCR, and Sox9b was determined as a key gene that was essential for testis development. This current study will provide new insight into the role of Sox gene family in fish sex determination and differentiation.