Genetic variation mining of the Chinese mitten crab (Eriocheir sinensis) based on transcriptome data from public databases.

At present, public databases house an extensive repository of transcriptome data, with the volume continuing to grow at an accelerated pace. Utilizing these data effectively is a shared interest within the scientific community. In this study, we introduced a novel strategy that harnesses SNPs and InDels identified from transcriptome data, combined with sample metadata from databases, to effectively screen for molecular markers correlated with traits. We utilized 228 transcriptome datasets of Eriocheir sinensis from the NCBI database and employed the Genome Analysis Toolkit software to identify 96 388 SNPs and 20 645 InDels. Employing the genome-wide association study analysis, in conjunction with the gender information from databases, we identified 3456 sex-biased SNPs and 639 sex-biased InDels. The KOG and KEGG annotations of the sex-biased SNPs and InDels revealed that these genes were primarily involved in the metabolic processes of E. sinensis. Combined with SnpEff annotation and PCR experimental validation, a highly sex-biased SNP located in the Kelch domain containing 4 (Klhdc4) gene, CHR67-6415071, was found to alter the splicing sites of Klhdc4, generating two splice variants, Klhdc4_a and Klhdc4_b. Additionally, Klhdc4 exhibited robust expression across the ovaries, testes, and accessory glands. The sex-biased SNPs and InDels identified in this study are conducive to the development of unisexual cultivation methods for E. sinensis, and the alternative splicing event caused by the sex-biased SNP in Klhdc4 may serve as a potential mechanism for sex regulation in E. sinensis. The analysis strategy employed in this study represents a new direction for the rational exploitation and utilization of transcriptome data in public databases.

Genetic diversity of the common carp black strain population based on mtDNA (D-loop and cytb).

The common strain black carp (Cyprinus carpio var. baisenensis) is a culturally important carp strain that is raised and cultured in Guangxi Province, China. Its color reflects the interactions between the Burau people and their surrounding environment. The population of the common carp black strain was isolated and cultured in a rice-fish integration system. To explore the genetic diversity and protection of germplasm resources, we analyzed mitochondrial DNA (mtDNA) sequences, specifically the displacement loop (D-loop) and cytochrome b (Cytb), using single-nucleotide polymorphisms (SNP). We compared these sequences with those from four other local common carp populations. The study included a total of 136 adult common carps from five strain populations: the common black carp strain (HJ = 31), Jian (F = 30), Heilongjiang (H = 10), Songpu (S = 31), and Saijiang (SJ = 34). The results of the Cytb and D-loop analyses showed that the Heilongjiang carp (H) and Saijiang (SJ) populations had the highest levels of haplotype diversity (0.867 ± 0.034785) and nucleotide diversity (π = 0.0063 ± 0.000137 and 0.0093 ± 0.000411), respectively. On the other hand, the Common carp black strain population (HJ) exhibited the lowest haplotype diversity in both Cytb and D-loop, with haplotype 2 being the most commonly observed among the populations. Private haplotypes dominated the five common carp populations, which were significantly different at P<0.001. Furthermore, analyzing the coefficient of genetic differentiation (Fst), the highest genetic difference was observed between Saijiang (SJ) and Heilongjiang (H) (Fst = 0.963), whereas the lowest was observed between Songpu (S) and the Common carp black strain population (HJ) (Fst = 0.019) for the Cytb gene sequences. For the D-loop, the Common carp black strain population (HJ) and Songpu (S) (Fst = 0.7) had the highest values, and Heilongjiang (H) and Common black carp strain (HJ) had an Fst of 0.125. Additionally, the AMOVA analysis revealed a higher level of variance for the Cytb and D-loop genes, indicating lower genetic diversity within the local carp community. On the other hand, the phylogenetic tree analysis showed that the five carp populations were closely related and formed a distinct cluster. The distinct cluster of populations suggests a common ancestor or recent gene flow, possibly due to geographic proximity or migration history, and unique genetic characteristics, possibly due to adaptations or selective pressures. The results of this study provide valuable insights into the genetic diversity of the common strain black carp, which can have implications for conservation, breeding programs, evolutionary studies, and fisheries management.

Transcriptomic analysis of hydrogen peroxide-induced liver dysfunction in Cyprinus carpio: Insights into protein synthesis and metabolism.

Hydrogen peroxide (H2O2), a prevalent reactive oxygen species (ROS) found in natural aquatic environments, has garnered significant attention for its potential toxicity in fish. However, the molecular mechanisms underlying this toxicity are not yet comprehensively understood. This study aimed to assess H2O2-induced liver dysfunction in common carp (Cyprinus carpio) and elucidate the underlying molecular mechanisms via biochemical and transcriptomic analyses. Common carp were divided into normal control (NC) and H2O2-treated groups (1 mM H2O2), the latter of which was exposed to H2O2 for 1 h per day over a period of 14 days. Serum biochemical analyses indicated that exposure to H2O2 resulted in moderate liver damage, characterized by elevated alanine aminotransferase (ALT) activity and lowered albumin (Alb) level. Concurrently, H2O2 exposure induced oxidative stress and modified the hepatic metabolic enzyme levels. Transcriptome analysis highlighted that 1358 and 1188 genes were significantly downregulated and upregulated, respectively, in the H2O2-treated group. These differentially expressed genes (DEGs) were significantly enriched in protein synthesis and a variety of metabolic functions such as peptide biosynthetic processes, protein transport, ribonucleoprotein complex biogenesis, oxoacid metabolic processes, and tricarboxylic acid metabolic processes. Dysregulation of protein synthesis is principally associated with the downregulation of three specific pathways: ribosome biogenesis, protein export, and protein processing in the endoplasmic reticulum (ER). Furthermore, metabolic abnormalities were primarily characterized by inhibition of the citrate cycle (TCA) and fatty acid biosynthesis. Significantly, anomalies in both protein synthesis and metabolic function may be linked to aberrant regulation of the insulin signaling pathway. These findings offer innovative insights into the mechanisms underlying H2O2 toxicity in aquatic animals, contributing to the assessment of ecological risks.

Genomics and transcriptomics of the Chinese mitten crabs (Eriocheir sinensis).

To gain a deeper understanding of the genetic factors influencing the growth and development of Eriocheir sinensis, a well-known species of hairy crab found in Yangcheng Lake, this study focused on the de novo genome and full-length transcriptome information of the selected subjects. Specifically, Yangcheng Lake hairy crabs were chosen as the experimental samples. Initially, a genome analysis was performed, resulting in the identification of gene fragments with a combined length of 1266,092,319 bp. Subsequently, a transcriptome analysis was conducted on a mixture of tissues from four different sites, namely muscle, brain, eye, and heart, to further investigate the genetic characteristics at the transcriptome level. The Pacific Biosciences (Pacio) single-molecule real-time sequencing system generated a total of 36.93 G sub-fragments and 175,90041 effective inserts. This research contributes to the indirect comprehension of genetic variations underlying individual traits. Furthermore, a comparison of the obtained data with relevant literature emphasizes the advantages of this study and establishes a basis for further investigations on the Chinese mitten crab.

The Role of Biogeography in Shaping Intestinal Flora and Influence on Fatty Acid Composition in Red Swamp Crayfish (Procambarus clarkii).

Intestinal microbiota plays an important role in promoting digestion, metabolism, and immunity. Intestinal microbiota and fatty acids are important indicators to evaluate the health and nutritional composition of Procambarus clarkii. They have been shown to be strongly influence by environmental and genetic factors. However, it is not clear whether environmental factors have a greater impact on the intestinal microbiota and fatty acid composition of crayfish. The link between the intestinal microbial communities and fatty acid (FA) compositions of red swamp crayfish from different geographical has not yet been studied. Thus, the current paper focuses on the influence of different environments on the fatty acids in muscles of crayfish and the possible existence between gut microbiota and fatty acids. Therefore, in this study, we compared the fatty acid compositions and intestinal microbiota of five crayfish populations from different geographical locations. The results were further analyzed to determine whether there is a relationship between geographical location, fatty acid compositions and intestinal microbiota. The gut microbial communities of the crayfish populations were characterized using 16S rRNA high-throughput gene sequencing. The results showed that there were significant differences in FA compositions of crayfish populations from different geographical locations. A similar trend was observed in the gut microbiome, which also varied significantly according to geographic location. Interestingly, the analysis revealed that there was a relationship between fatty acid compositions and intestinal microbes, revealed by alpha diversity analysis and cluster analysis. However, further studies of the interactions between the P. clarkii gut microbiota and biochemical composition are needed, which will ultimately reveal the complexity of microbial ecosystems with potential applications in aquaculture and species conservation.

Effect of Dietary Selenium on the Growth and Immune Systems of Fish.

Dietary selenium (Se) is an essential component that supports fish growth and the immune system. This review attempts to provide insight into the biological impacts of dietary Se, including immunological responses, infection defense, and fish species growth, and it also identifies the routes via which it enters the aquatic environment. Dietary Se is important in fish feed due to its additive, antioxidant, and enzyme properties, which aid in various biological processes. However, excessive intake of it may harm aquatic ecosystems and potentially disrupt the food chain. This review explores the diverse natures of dietary Se, their impact on fish species, and the biological methods for eliminating excesses in aquatic environments. Soil has a potential role in the distribution of Se through erosion from agricultural, industrial, and mine sites. The research on dietary Se's effects on fish immune system and growth can provide knowledge regarding fish health, fish farming strategies, and the health of aquatic ecosystems, promoting the feed industry and sustainable aquaculture. This review provides data and references from various research studies on managing Se levels in aquatic ecosystems, promoting fish conservation, and utilizing Se in farmed fish diets.

Effects of Different Sources of Culture Substrate on the Growth and Immune Performance of the Red Swamp Crayfish (Procambarus clarkii).

The substrate in the aquatic environment plays a crucial role in nutrient deposition and recovery for the growth of aquatic organisms. In order to optimize the culture medium of Procambarus Clarkii, culture media from different sources were selected in this study to explore their effects on the growth and immune performance of red swamp crayfish. The results showed that the weight gain rate (WGR), body length growth rate (BLGR) and specific growth rate (SGR) in group I2 were the highest, followed by group I1 and group I3. The WGR and SGR of crayfish in the I1 and I2 groups were significantly higher than those in the I3 group (p < 0.05). The activities of acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) were the highest in group I2, followed by group I3, and the lowest in group I1. The expression trends in growth-related genes, nuclear hormone receptor (E75), molt-inhibiting hormone (MIH) and chitinase genes were similar, and the expression levels in the I2 group were higher than those in the I1 and I3 groups. It was noted that the expression levels of E75 and MIH genes in the I2 group were significantly higher than those in the I3 group (p < 0.05). α diversity analysis of 16S rRNA data showed that there was no statistically significant difference in the abundance of intestinal flora among the three culture substrate groups. The ß diversity in the Xitangni group, crayfish Tangni group and Shuitangni group was significantly different. These changes in microbiota suggest that using different substrates to culture crayfish leads to differences in gut microbiota diversity. To sum up, the growth in crayfish and immune performance influenced by the culture substrate condition and aquatic breeding sediment substrates, rather than crab pool and paddy field pond sediment substrates, showed a better effect.

Screening of Suitable Reference Genes for Immune Gene Expression Analysis Stimulated by Vibrio anguillarum and Copper Ions in Chinese Mitten Crab (Eriocheir sinensis).

The reference gene expression is not always stable under different experimental conditions, and screening of suitable reference genes is a prerequisite in quantitative real-time polymerase chain reaction (qRT-PCR). In this study, we investigated gene selection, and the most stable reference gene for the Chinese mitten crab (Eriocheir sinensis) was screened under the stimulation of Vibrio anguillarum and copper ions, respectively. Ten candidate reference genes were selected, including arginine kinase (AK), ubiquitin-conjugating enzyme E2b (UBE), glutathione S-transferase (GST), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF-1α), α-tubulin (α-TUB), heat shock protein 90 (HSP90), ß-actin (ß-ACTIN), elongation factor 2 (EF-2) and phosphoglucomutase 2 (PGM2). Expression levels of these reference genes were detected under the stimulation of V. anguillarum at different times (0 h, 6 h, 12 h, 24 h, 48 h and 72 h) and copper ions in different concentrations (11.08 mg/L, 2.77 mg/L, 0.69 mg/L and 0.17 mg/L). Four types of analytical software, namely geNorm, BestKeeper, NormFinder and Ref-Finder, were applied to evaluate the reference gene stability. The results showed that the stability of the 10 candidate reference genes was in the following order: AK > EF-1α > α-TUB > GAPDH > UBE > ß-ACTIN > EF-2 > PGM2 > GST > HSP90 under V. anguillarum stimulation. It was GAPDH > ß-ACTIN > α-TUB > PGM2 > EF-1α > EF-2 > AK > GST > UBE > HSP90 under copper ion stimulation. The expression of E. sinensis Peroxiredoxin4 (EsPrx4) was detected when the most stable and least stable internal reference genes were selected, respectively. The results showed that reference genes with different stability had great influence on the accurate results of the target gene expression. In the Chinese mitten crab (E. sinensis), AK and EF-1α were the most suitable reference genes under the stimulation of V. anguillarum. Under the stimulation of copper ions, GAPDH and ß-ACTIN were the most suitable reference genes. This study provided important information for further research on immune genes in V. anguillarum or copper ion stimulation.

Neurobehavioral disorders induced by environmental zinc in female zebrafish (Danio rerio): Insights from brain and intestine transcriptional and metabolic signatures.

Human activities can cause zinc (Zn) contamination of aquatic environments. Zn is an essential trace metal, but effects of environmentally relevant Zn exposure on the brain-intestine axis in fish are poorly understood. Here, six-month-old female zebrafish (Danio rerio) were exposed to environmentally relevant Zn concentrations for six weeks. Zn significantly accumulated in the brain and intestine, causing anxiety-like behaviors and altered social behaviors. Zn accumulation altered levels of neurotransmitters, including serotonin, glutamate, and γ-aminobutyric acid, in the brain and intestine, and these changes were directly associated with changes in behavior. Zn caused oxidative damage and mitochondrial dysfunction, and impaired NADH dehydrogenase, thereby dysregulating the energy supply in brain. Zn exposure resulted in nucleotide imbalance and dysregulation of DNA replication and the cell cycle, potentially impairing the self-renewal of intestinal cells. Zn also disturbed carbohydrate and peptide metabolism in the intestine. These results indicate that chronic exposure to Zn at environmentally relevant concentrations dysregulates the bidirectional interaction of the brain-intestine axis with respect to neurotransmitters, nutrients, and nucleotide metabolites, thereby causing neurological disorder-like behaviors. Our study highlights the necessity to evaluate the negative impacts of chronic environmentally relevant Zn exposure on the health of humans and aquatic animals.

Effects of simulated winter short photoperiods on the microbiome and intestinal metabolism in Huanghe carp (Cyprinus carpio haematopterus).

Objective: As one of the most important environmental signals, photoperiod plays a crucial role in regulating the growth, metabolism, and survival of organisms. The photoperiod shifts with the transition of the seasons. The difference in photoperiod between summer and winter is the greatest under natural conditions. However, the effect of photoperiod on Huanghe carp (Cyprinus carpio haematopterus) was paid little attention. We investigated the impact of artificial manipulation of seasonal photoperiod on Huanghe carp by integrating growth performance, intestinal flora, and intestinal metabolome. Method: We conducted an 8-week culture experiment with summer photoperiod (14 h light:10 h dark, n = 60) as the control group and winter photoperiod (10 h light:14 h dark, n = 60) based on the natural laws. Results: Winter photoperiod provokes significant weight increases in Huanghe carp. The altered photoperiod contributed to a significant increase in triglyceride and low-density lipoprotein cholesterol levels and the gene expressions of lipid metabolism in the intestine of Huanghe carp. 16s rDNA sequencing revealed that winter photoperiod diminished intestinal flora diversity and altered the abundance. Specifically, the relative abundances of Fusobacteria and Acidobacteriota phyla were higher but Proteobacteria, Firmicutes, and Bacteroidetes phyla were reduced. Analogously, photoperiodic changes induced a significant reduction in the Pseudomonas, Vibrio, Ralstonia, Acinetobacter, and Pseudoalteromonas at the genus level. Additionally, metabolomics analysis showed more than 50% of differential metabolites were associated with phospholipids and inflammation. Microbiome and metabolome correlation analyses revealed that intestinal microbe mediated lipid metabolism alteration. Conclusion: The winter photoperiod induced intestinal flora imbalance and lipid metabolism modification, ultimately affecting the growth of Huanghe carp. This study provides new insights into the effects of seasonal photoperiodic alteration on the well-being of fish.

Changes in aggression and locomotor behaviors in response to zinc is accompanied by brain cell heterogeneity and metabolic and circadian dysregulation of the brain-liver axis.

Zinc is an essential nutrient for life, but over-accumulation can result in toxicity. Anthropogenic activities can increase zinc concentrations in aquatic environments (e.g., to ∼0.46-1.00 mg/L), which are above the safe level of 0.1 mg/L. We investigated the behavior and physiology of zebrafish (Danio rerio) in response to environment-related exposure to zinc chloride at 0.0 (Ctrl), 1.0 (ZnCl2-low) and 1.5 (ZnCl2-high) mg/L for 6 weeks (the zinc conversion ratio of zinc chloride is ∼0.48 and the nominal (measured) values were: Ctrl, 0 (∼0.01); ZnCl2-low, 0.48 (∼0.51); ZnCl2-high, 0.72 (∼0.69) mg/L). Low-zinc exposure resulted in significantly increased locomotion and fast moving behaviors, while high-zinc exposure resulted in significantly increased aggression and freezing frequency. Single cell RNA-seq of neurons, astrocytes, and oligodendrocytes of the brain revealed expression of genes related to ion transport, neuron generation, and immunomodulation that were heterogeneously regulated by zinc exposure. Astrocyte-induced central nervous system inflammation potentially integrated neurotoxicity and behavior. Integrated analyses of brain and hepatic transcriptional signatures showed that genes (and pathways) dysregulated by zinc were associated with sensory functions, circadian rhythm, glucose and lipid metabolism, and amyloid ß-protein clearance. Our results showed that environment-related zinc contamination can be heterogeneously toxic to brain cells and can disturb coordination of brain-liver physiology. This may disrupt neurobehavior and cause a neurodegeneration-like syndrome in adult zebrafish.

Exploration of the regulatory mechanisms of regeneration, anti-oxidation, anti-aging and the immune response at the post-molt stage of Eriocheir sinensis.

Eriocheir sinensis is widely appreciated by the surrounding population due to its culinary delicacy and rich nutrients. The E. sinensis breeding industry is very prosperous and molting is one of the important growth characteristics. Research on the regulation of molting in E. sinensis is still in the initial stages. There is currently no relevant information on the regulatory mechanisms of heart development following molting. Comparative transcriptome analysis was used to study developmental regulation mechanisms in the heart of E. sinensis at the post-molt and inter-molt stages. The results indicated that many regulatory pathways and genes involved in regeneration, anti-oxidation, anti-aging and the immune response were significantly upregulated after molting in E. sinensis. Aside from cardiac development, the differentially expressed genes (DEGs) were relevant to myocardial movement and neuronal signal transduction. DEGs were also related to the regulation of glutathione homeostasis and biological rhythms in regard to anti-oxidation and anti-aging, and to the regulation of immune cell development and the immune response. This study provides a theoretical framework for understanding the regulation of molting in E. sinensis and in other economically important crustaceans.

Effects of Copper Exposure on Oxidative Stress, Apoptosis, Endoplasmic Reticulum Stress, Autophagy and Immune Response in Different Tissues of Chinese Mitten Crab (Eriocheir sinensis).

High concentrations of copper (Cu2+) pose a great threat to aquatic animals. However, the mechanisms underlying the response of crustaceans to Cu2+ exposure have not been well studied. Therefore, we investigated the alterations of physiological and molecular parameters in Chinese mitten crab (Eriocheir sinensis) after Cu2+ exposure. The crabs were exposed to 0 (control), 0.04, 0.18, and 0.70 mg/L of Cu2+ for 5 days, and the hemolymph, hepatopancreas, gills, and muscle were sampled. The results showed that Cu2+ exposure decreased the antioxidative capacity and promoted lipid peroxidation in different tissues. Apoptosis was induced by Cu2+ exposure, and this activation was associated with the mitochondrial and ERK pathways in the hepatopancreas. ER stress-related genes were upregulated in the hepatopancreas but downregulated in the gills at higher doses of Cu2+. Autophagy was considerably influenced by Cu2+ exposure, as evidenced by the upregulation of autophagy-related genes in the hepatopancreas and gills. Cu2+ exposure also caused an immune response in different tissues, especially the hepatopancreas, where the TLR2-MyD88-NF-κB pathway was initiated to mediate the inflammatory response. Overall, our results suggest that Cu2+ exposure induces oxidative stress, ER stress, apoptosis, autophagy, and immune response in E. sinensis, and the toxicity may be implicated following the activation of the ERK, AMPK, and TLR2-MyD88-NF-κB pathways.

Comparative Transcriptome Analysis on the Regulatory Mechanism of Thoracic Ganglia in Eriocheir sinensis at Post-Molt and Inter-Molt Stages.

Eriocheir sinensis is an aquatic species found distributed worldwide. It is found in the Yangtze River of China, where the commercial fishing of this valuable catadromous aquatic species has been banned. As an important member of the phylum Arthropoda, E. sinensis grows by molting over its whole lifespan. The central nervous system of Eriocheir sinensis plays an important regulatory role in molting growth. Nevertheless, there are no reports on the regulatory mechanisms of the nervous system in E. sinensis during the molting cycle. In this study, a comparative transcriptome analysis of E. sinensis thoracic ganglia at post-molt and inter-molt stages was carried out for the first time to reveal the key regulatory pathways and functional genes operating at the post-molt stage. The results indicate that pathways and regulatory genes related to carapace development, tissue regeneration, glycolysis and lipolysis and immune and anti-stress responses were significantly differentially expressed at the post-molt stage. The results of this study lay a theoretical foundation for research on the regulatory network of the E. sinensis nervous system during the post-molt developmental period. Detailed knowledge of the regulatory network involved in E. sinensis molting can be used as a basis for breeding improved E. sinensis species, recovery of the wild E. sinensis population and prosperous development of the E. sinensis artificial breeding industry.

Transcriptome Analysis of Hepatopancreas Provides Insights into Differential Metabolic Mechanisms of Eriocheir sinensis Feeding on Trash Fish and Formulated Feed.

BACKGROUND: The Chinese mitten crab, Eriocheir sinensis (E. sinensis), is a popular crab species in both domestic and foreign markets. Trash fish are essential for E. sinensis breeding, but have caused serious water pollution. The municipal party committee for the main production areas of E. sinensis implemented a ban on feeding on trash fish since 2020. METHODS: In this study, we performed a culture experiment of E. sinensis feeding on trash fish and formulated feed, with comparative transcriptome analysis on hepatopancreas of E. sinensis. RESULTS: The results indicate that formulated feed causes no significant difference in growth, survival rate or content of amino acids in the muscles of adult E. sinensis. Formulated feed caused a slight downregulation of pathways involved in amino acid metabolism, development, energy metabolism and homeostasis maintenance. CONCLUSIONS: On the whole, formulated feed can serve as an undifferentiated substitution for trash fish. This study provides a theoretical foundation for optimizing research on E. sinensis feed.

Betaine Promotes Fat Accumulation and Reduces Injury in Landes Goose Hepatocytes by Regulating Multiple Lipid Metabolism Pathways.

Betaine is a well-established supplement used in livestock feeding. In our previous study, betaine was shown to result in the redistribution of body fat, a healthier steatosis phenotype, and an increased liver weight and triglyceride storage of the Landes goose liver, which is used for foie-gras production. However, these effects are not found in other species and strains, and the underlying mechanism is unclear. Here, we studied the underpinning molecular mechanisms by developing an in vitro fatty liver cell model using primary Landes goose hepatocytes and a high-glucose culture medium. Oil red-O staining, a mitochondrial membrane potential assay, and a qRT-PCR were used to quantify lipid droplet characteristics, mitochondrial ß-oxidation, and fatty acid metabolism-related gene expression, respectively. Our in vitro model successfully simulated steatosis caused by overfeeding. Betaine supplementation resulted in small, well-distributed lipid droplets, consistent with previous experiments in vivo. In addition, mitochondrial membrane potential was restored, and gene expression of fatty acid synthesis genes (e.g., sterol regulatory-element binding protein, diacylglycerol acyltransferase 1 and 2) was lower after betaine supplementation. By contrast, the expression of lipid hydrolysis transfer genes (mitochondrial transfer protein and lipoprotein lipase) was higher. Overall, the results provide a scientific basis and theoretical support for the use of betaine in animal production.

Development of an in vitro culture system for immature oocytes of red swamp crayfish (Procambarus clarkii).

The present study assessed general strategies for the isolation and in vitro culture of immature ovary-derived cells (oocytes) in red swamp crayfish (Procambarus clarkii). In phase 1 of this study, the effects of different digestive enzymes on cell retrieval from developing ovarian tissues were examined to facilitate efficient oocyte isolation. Subsequently, the ovary-derived cells/oocytes were cultured under various conditions to evaluate the effects of basal media and supplement concentrations. Collagenase I treatment at 26 ℃ for 25 min was more effective (approx. 95 %) in oocytes isolation from ovarian tissues than other treatments. Furthermore, DMEM media with HEPES (DMEM-H) were more effective than L15 for oocyte culture (72.4 % survival rate), while oocyte survival improved to 90.6 % using a 10 % fetal bovine serum supplement. In phase 2, the top-performing media were used in immature oocyte culture to assess 17α-Hydroxyprogesterone (17-OHP) induction in vitro. An up-regulated expression of maturity-related genes, i.e., cyclin B, cdc2, ef1α, after 12 h of incubation was evident in qPCR results, demonstrating that the exogenous hormone 17-OHP could induce P. clarkii oocyte maturation in vitro. These results may provide a basis for developing an in vitro system for P. clarkii germline cell culture, which may ultimately lead to the genetic improvement of this species.

Recognition of Gonadal Development in Eriocheir sinensis Based on the Impulse of Love at First Sight.

Given the difficulty in identifying individuals with different degrees of ovarian development, we developed a new device utilizing the hypothesis of mutual attraction behavior between male and female crabs with mature gonads by releasing the sexual pheromone so they could be examined. From a total of 40 female crabs, 10 were isolated within half an hour. Histological analysis showed that the ovaries of crabs in the isolated group were in stage IV, while those of the control groups were in stage III. In addition, progesterone (PROG) in experimental groups was significantly reduced compared with the control group (p < 0.05), but no significant difference was detected in estradiol (E2). In response to the different developmental stages, hemolymph biochemical indices and the determination of gonadal fatty acids profiles were explored. The results indicated only C18:4 showed a significant difference between these two groups. A transcriptome was generated to determine the genes involved in the mutual attraction process; differentially expressed genes (DEGs) were significantly related to gonadal development. Therefore, the device can be used to isolate Chinese mitten crabs with stage IV ovarian development.

Microcystin-LR induces apoptosis in Juvenile Eriocheir sinensis via the mitochondrial pathway.

Microcystin-LR (MC-LR), the toxic substance of cyanobacteria secondary metabolism, widely exists in water environments and poses great risks to living organisms. Some toxicological assessments of MC-LR have performed at physiological and biochemical levels. However, plenty of blanks about the potential mechanism in aquatic crustacean remains. In this study, we firstly assessed the exposure toxicity of MC-LR to juvenile E. sinensis and clarified that the 96 h LD50 of MC-LR was 73.23 µg/kg. Then, hepatopancreas transcriptome profiles of MC-LR stressed crabs were constructed at 6 h post-injection and 37 differential expressed genes (DEGs) were identified. These DEGs were enriched in cytoskeleton, peroxisome and apoptosis pathways. To further reveal the toxicity of MC-LR, oxidative stress parameters (SOD, CAT, GSH-px and MDA), apoptosis genes (caspase 3, bcl-2 and bax) and apoptotic cells were detected. Significant accumulated MDA and rise-fall enzyme activities verified the oxidative stress caused by MC-LR. It is noteworthy that quantitative real-time PCR and TUNEL assay indicated that MC-LR stress-induced apoptosis via the mitochondrial pathway. Interestingly, activator protein-1 may play a crucial role in mediating the hepatotoxicity of MC-LR by regulating apoptosis and oxidative stress. Taken together, our study investigated the toxic effects and the potential molecular mechanisms of MC-LR on juvenile E. sinensis. It provided useful data for exploring the toxicity of MC-LR to aquatic crustaceans at molecular levels.

Zinc alters behavioral phenotypes, neurotransmitter signatures, and immune homeostasis in male zebrafish (Danio rerio).

Anthropogenic activities discharge zinc into aquatic ecosystems, and the effects of long-term and low-concentration zinc exposure on fish behavior are unclear. We evaluated the behavior and physiology of male zebrafish (Danio rerio) after a 6-week exposure to 1.0 or 1.5 ppm (mg/L) zinc chloride. The exposure caused anxiety-like behaviors and altered the social preferences in both exposure groups. Analysis of transcriptional changes suggested that in the brain, zinc exerted heterogenetic effects on immune and neurotransmitter functions. Exposure to 1.0 ppm zinc chloride resulted in constitutive immune dyshomeostasis, while exposure to 1.5 ppm zinc chloride impaired the neurotransmitter glutamate. In the intestine, zinc dysregulated self-renewal of intestinal cells, a potential loss of defense function. Moreover, exposure to 1.5 ppm zinc chloride suppressed intestinal immune functions and dysregulated tyrosine metabolism. These behavioral alterations suggested that the underlying mechanisms were distinct and concentration-specific. Overall, environmental levels of zinc can alter male zebrafish behaviors by dysregulating neurotransmitter and immunomodulation signatures.