CRISPR/Cas9 Technology for Enhancing Desirable Traits of Fish Species in Aquaculture.

Aquaculture, the world's fastest-growing food production sector, is critical for addressing food security concerns because of its potential to deliver high-quality, nutrient-rich supplies by 2050. This review assesses the effectiveness of CRISPR/Cas9 genome editing technology in enhancing desirable traits in fish species, including growth rates, muscle quality, disease resistance, pigmentation, and more. It also focuses on the potential effectiveness of the technology in allowing precise and targeted modifications of fish DNA to improve desirable characteristics. Many studies have reported successful applications of CRISPR/Cas9, such as knocking out reproductive genes to control reproduction and sex determination, enhancing feed conversion efficiency, and reducing off-target effects. Additionally, this technology has contributed to environmental sustainability by reducing nitrogen-rich waste and improving the nutritional composition of fish. However, the acceptance of CRISPR/Cas9 modified fish by the public and consumers is hindered by concerns regarding public perception, potential ecological impacts, and regulatory frameworks. To gain public approval and consumer confidence, clear communication about the editing process, as well as data on the safety and environmental considerations of genetically modified fish, are essential. This review paper discusses these challenges, provides possible solutions, and recommends future research on the integration of CRISPR/Cas9 into sustainable aquaculture practices, focusing on the responsible management of genetically modified fish to enable the creation of growth and disease-resistant strains. In conclusion, this review highlights the transformative potential of CRISPR/Cas9 technology in improving fish traits, while also considering the challenges and ethical considerations associated with sustainable and responsible practices in aquaculture.

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.

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.

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.

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.

Dynamic changes in microbial community structure in farming pond water and their effect on the intestinal microbial community profile in juvenile common carp (Cyprinus carpio L.).

Water quality parameter dynamics, gut, sediment and water bacteria communities were studied to understand the environmental influence on the gut microbial community of a new strain of Huanghe common carp. A total of 3,384,078 raw tags and 5105 OTUs were obtained for the gut, water and sediment bacteria. The water quality had a stronger influence on the water bacteria community than gut and sediment bacteria communities. The ambient water quality parameters also significantly influenced the water and sediment bacteria communities. Comparing the gut, sediment, and water microbial communities, a relationship was found among them. However, gut bacteria were more closely related to sediment bacterial communities than to water bacteria communities. The results showed that the top three bacterial taxa were identical in gut and sediment samples in the early days of rearing. Interestingly, bacterial communities in the carp gut, water, and sediment had different adaptabilities to variations in environmental factors.

Leptin promotes methionine adenosyltransferase 2A expression in hepatic stellate cells by the downregulation of E2F-4 via the ß-catenin pathway.

Most obese patients develop hyperleptinaemia. Leptin, mainly produced by adipocytes, demonstrates a promotional role in liver fibrosis. Hepatic stellate cell (HSC) activation, a key step in liver fibrogenesis, requires global reprogramming of gene expression. The remodeling of DNA methylation is a mechanism of the epigenetic regulation of gene expression. The biosynthesis of S-adenosylmethionine, a principle biological methyl donor, is catalyzed by methionine adenosyltransferase (MAT) such as MATⅡ which has been shown to promote HSC activation in vitro. This study was mainly aimed to determine the effect of leptin on MAT2A expression (the catalytic subunit of MATⅡ) in HSCs. Results showed that MAT2A knockdown reduced leptin-induced HSC activation and liver fibrosis in the leptin-deficient mouse model. Leptin promoted MAT2A expression in HSCs and increased MAT2A promoter activity. The axis of the ß-catenin pathway/E2F-4 mediated the effect of leptin on MAT2A expression. Leptin-induced ß-catenin signaling reduced E2F-4 expression and thus abated E2F-4 binding to MAT2A promoter at a site around -2779 bp, leading to an increase in the MAT2A promoter activity. These data might shed more light on the mechanisms responsible for liver fibrogenesis in obese patients with hyperleptinaemia.

Molecular insights into information processing and developmental and immune regulation of Eriocheir sinensis megalopa under hyposaline stress.

Eriocheir sinensis is an important euryhaline catadromous crustacean of the Yangtze River and an important commercial species for breeding in China. However, wild E. sinensis have suffered serious damage attributed to overfishing, climate change, etc. The Ministry of Agriculture of China issued a notice banning the commercial fishing of wild E. sinensis. E. sinensis megalopa migrates upriver into fresh water for growth and fattening, which creates optimal conditions to experimentally explore its hyposaline osmoregulation mechanism. We performed comparative transcriptome analyses of E. sinensis megalopae under hyposaline stress. The results suggest that KEGG pathways and genes related to genetic information processing, developmental regulation, immune and anti-stress responses were differentially expressed. The present study reveals the most significantly enriched pathways and functional gene groups, and explores the hyposaline osmoregulation mode of E. sinensis megalopae. This study lays a theoretical foundation for further studies on the osmoregulation and developmental mechanisms of E. sinensis.

Molecular insights into the sex-differential regulation of signal transduction in the cerebral ganglion and metabolism in the hepatopancreas of Eriocheir sinensis during reproduction.

The Chinese mitten crab (Eriocheir sinensis), an economically valuable crustacean that is popular for its flavor, exhibits catadromous spawning migration. Overfishing and environmental pollution have inflicted serious damage on wild E. sinensis populations, and the Chinese government has banned the commercial fishing of this species in the Yangtze River. Studies have examined the sexual dimorphism in the body size and morphology of crabs, but there are few reports on the molecular regulatory mechanisms that occur during the reproduction of E. sinensis. In this study, we performed the first comparative transcriptome analyses of the cerebral ganglion and hepatopancreas of E. sinensis during reproduction. The results indicate that E. sinensis has significant sexual dimorphism in signal transduction, metabolism, substance transportation, and cellular protection. This study aims to provide information that can be used as a basis for further research on the molecular mechanisms that underlie sexual dimorphism in E. sinensis during reproduction. Furthermore, the results can be used to support the development of the E. sinensis breeding industry and the restoration of wild E. sinensis.

Mechanistic insights into the effects of SREBP1c on hepatic stellate cell and liver fibrosis.

Sterol regulatory element-binding protein 1c (SREBP1c) plays key roles in maintenance of hepatic stellate cell (HSC) quiescence. The present researches investigated the mechanisms underlying the effects of SREBP1c on HSCs and liver fibrogenesis by HSC-targeted overexpression of the active SREBP1c using adenovirus in vitro and in vivo. Results demonstrated that SREBP1c exerted inhibitory effects on TAA-induced liver fibrosis. SREBP1c down-regulated TGFß1 level in liver, reduced the receptors for TGFß1 and PDGFß, and interrupted the signalling pathways of Smad3 and Akt1/2/3 but not ERK1/2 in HSCs. SREBP1c also led to the decreases in the protein levels of the bromodomain-containing chromatin-modifying factor bromodomain protein 4, methionine adenosyltransferase 2B (MAT2B) and TIMP1 in HSCs. In vivo activated HSCs did not express cyclin D1 and cyclin E1 but SREBP1c down-regulated both cyclins in vitro. SREBP1c elevated PPARγ and MMP1 protein levels in the model of liver fibrosis. The effect of SREBP1c on MAT2B expression was associated with its binding to MAT2B1 promoter. Taken together, the mechanisms underlying the effects of SREBP1c on HSC activation and liver fibrosis were involved in its influences on TGFß1 level, the receptors for TGFß1 and PDGFß and their downstream signalling, and the molecules for epigenetic regulation of genes.

Leptin up-regulates microRNA-27a/b-3p level in hepatic stellate cells.

Obese patients, often accompanied by hyperleptinemia, are prone to liver fibrogenesis. Leptin is an adipocyte-derived hormone and plays a promotion role in liver fibrosis. Sterol regulatory element binding protein-1c (SREBP1c) exerts a crucial role in inhibiting hepatic stellate cell (HSC) activation, a key step in liver fibrogenesis. Our previous studies indicated that leptin inhibited SREBP1c expression, contributing to leptin-induced HSC activation and liver fibrosis. microRNAs (miR) have emerged as important layers of regulatory control and regulate gene expression, and are implicated in numerous diseases. The present study revealed leptin up-regulation of miR-27a/b-3p levels in HSCs in vitro and in vivo. Three signaling pathways were required for leptin regulation of miR-27a/b-3p levels. miR-27a/b-3p could reduce SREBP1c and liver x receptor α (LXRα) levels, increased α-smooth muscle actin (α-SMA, a marker for HSC activation) and α1(I)collagen levels in cultured HSCs. miR-27a/b-3p regulation of SREBP1c and LXRα were independent of 3'-untranslated region of SREBP1c and LXRα mRNA. In vivo experiments further demonstrated the miR-27a/b-3p involved in leptin-associated decrease in SREBP1 level in HSCs, HSC activation, and liver fibrosis. These data might have potential implications for our understanding of molecular mechanisms underlying leptin roles in liver fibrogenesis of obese patients with hyperleptinaemia.

Comparative expression analysis identifies the respiratory transition-related miRNAs and their target genes in tissues of metamorphosing Chinese giant salamander (Andrias davidianus).

BACKGROUND: Chinese giant salamander (Andrias davidianus) undergoes a metamorphosis from aquatic larvae to terrestrial adults, with concomitant transfer of respiration from gills to lungs prior to metamorphosis. These two tissues, as well as skin, were sampled to identify the differentially expressed miRNAs. RESULTS: High-coverage reference transcriptome was generated from combined gill, lung and skin tissues of metamorphosing juveniles, and lung tissue of adults: 86,282 unigenes with total length of approximately 77,275,634 bp and N50 of 1732 bp were obtained. Among these, 13,246 unigenes were assigned to 288 pathways. To determine the possible involvement of miRNAs in the respiratory transition, small RNA libraries were sequenced; 282 miRNAs were identified, 65 among which were known and 217 novel. Based on the hierarchical clustering analysis, the twelve studied samples were classified into three major clusters using differentially expressed miRNAs. We have validated ten differentially expressed miRNAs and some of their related target genes using qPCR. These results largely corroborated the results of transcriptomic and miRNA analyses. Finally, an miRNA-gene-network was constructed. Among them, two miRNAs with target genes related to oxygen sensing were differentially expressed between gill and lung tissues. Three miRNAs were differentially expressed between the lungs of larvae and lungs of adults. CONCLUSIONS: This study provides the first large-scale miRNA expression profile overview during the respiration transition from gills to lungs in Chinese giant salamander. Five differentially expressed miRNAs and their target genes were identified among skin, gill and lung tissues. These results suggest that miRNA profiles in respiratory tissues play an important role in the regulation of respiratory transition.

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.

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.

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.

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.

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.

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.

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.