Toxic effects of combined exposure to cadmium and diclofenac on freshwater crayfish (Procambarus clarkii): Insights from antioxidant enzyme activity, histopathology, and gut microbiome.

In recent years, excessive discharge of pollutants has led to increasing concentrations of cadmium (Cd) and diclofenac (DCF) in water; however, the toxicity mechanism of combined exposure of the two pollutants to aquatic animals has not been fully studied. Procambarus clarkii is an economically important aquatic species that is easily affected by Cd and DCF. This study examined the effects of combined exposure to Cd and DCF on the tissue accumulation, physiology, biochemistry, and gut microflora of P. clarkii. The results showed that Cd and DCF accumulated in tissues in the order of hepatopancreas > gill > intestine > muscle. The hepatopancreas and intestines were subjected to severe oxidative stress, with significantly increased antioxidant enzyme activity. Pathological examination revealed lumen expansion and epithelial vacuolisation in the hepatopancreas and damage to the villous capillaries and wall in the intestine. The co-exposure to Cadmium (Cd) and Diclofenac (DCF) disrupts the Firmicutes/Bacteroidetes (F/B) ratio, impairing the regular functioning of intestinal microbiota in carbon (C) and nitrogen (N) cycling. This disturbance consequently hinders the absorption and utilization of energy and nutrients in Procambarus clarkii. This study offers critical insights into the toxicological mechanisms underlying the combined effects of Cd and DCF, and suggests potential approaches to alleviate their adverse impacts on aquatic ecosystems.

Single-cell transcriptome analysis reveals a cellular immune response in freshwater dark sleeper (Odontobutis potamophila) after infection with Aeromonas veronii.

The bacterium Aeromonas veronii is a co-pathogenic species that can negatively impact the health of both humans and aquatic animals. In this study, we used single-cell transcriptome analysis (scRNA-seq) to investigate the effects of infection with A. veronii on head kidney cells and the regulation of gene expression in the dark sleeper (Odontobutis potamophila). scRNA-seq was used to assess the effects of infection with A. veronii in O. potamophila B cells, endothelial cells, macrophages, and granulocytes, and differential enrichment analysis of gene expression in B cells and granulocytes was performed. The analyses revealed a significant increase in neutrophils and decrease in eosinophils in granulocytes infected with A. veronii. Activation of neutrophils enhanced ribosome biogenesis by up-regulating the expression of RPS12 and RPL12 to fight against invading pathogens. Crucial pro-inflammatory mediators IL1B, IGHV1-4, and the major histocompatibility class II genes MHC2A and MHC2DAB, which are involved in virulence processes, were upregulated, suggesting that A. veronii activates an immune response that presents antigens and activates immunoglobulin receptors in B cells. These cellular immune responses triggered by infection with A. veronii enriched the available scRNA-seq data for teleosts, and these results are important for understanding the evolution of cellular immune defense and functional differentiation of head kidney cells.

The early function of cortisol in liver during Aeromonas hydrophila infection: Dynamics of the transcriptome and accessible chromatin landscapes.

In China, channel catfish (Ictalurus punctatus) is an important aquaculture species; however, haemorrhagic disease (Aeromonas hydrophila induced disease) in these fish has caused tremendous economic loss due to high morbidity and mass mortality in the breeding industry. The role of cortisol in bacterial diseases, particularly in the acute phase, remains unclear. In this study, liver transcriptome (RNA-seq) and chromatin accessibility (ATAC-seq) analyses were employed to investigate the early functional role of cortisol in Aeromonas hydrophila-stimulated responses. Our experiments confirmed that A. hydrophila infection can initially significantly increase serum cortisol levels at 1 h after infection. At this time point, the increased serum cortisol levels can significantly regulate A. hydrophila-regulated genes by affecting both transcriptome and chromatin accessibility. Cross-analysis of RNA-seq and ATAC-seq revealed that a certain gene group (92 target_DEGs) was regulated at an early time point by cortisol. KEGG enrichment analysis revealed that the top three pathways according to target_DEGs were cancer, glutathione metabolism, and the Notch signalling pathway. The protein-protein interaction analysis of target_DEGs revealed that they may be primarily involved in cell proliferation, CD8+ T cell function, glutathione synthesis, and activation of the NF-κB signalling pathway. This suggests that after the emergence of immune stress, the early regulation of cortisol is positive against the immune response. It is possible that in this situation, the animal is attempting to avoid dangerous situations and risks and then cope with the imbalance produced by the stressor to ultimately restore homeostasis. Our results will contribute to future research on fish and provide valuable insight regarding the mechanism of immune regulation by cortisol and the study of bacterial haemorrhagic disease in channel catfish.

Promoting effect of amorphous support on ruthenium-based catalyst for electrochemical hydrogen evolution reaction.

Nano-network Ru with definite lattice defects on amorphous Co nanosheets is obtained for the first time. Amorphous Co support can promote the surface Ru to obtain special morphology and modified electronic structure, thus improving HER activity in alkaline solution. A current density of 10 mA cm-2 can be obtained only with an overpotential of 33.5 mV.

Preparation of Acidic Electrolyzed Water by a RuO2@TiO2 Electrode with High Selectivity for Chlorine Evolution and Its Sterilization Effect.

The food hygiene problems caused by bacterial biofilms in food processing equipment are directly related to human life safety and health. Therefore, it is of great strategic significance to study new food sterilization technology. An acidic electrolyzed water (AEW) disinfectant is an electrochemical sterilization technology which has the characteristics of wide adaptability, high efficiency, and environmental friendliness. However, since the sterilization efficiency of AEW for biofilms is not ideal, it is necessary to increase the available chlorine content (ACC) in AEW. A feasible method to increase the ACC is by increasing the chlorine evolution reaction (CER) selectivity of the electrode for AEW preparation. In this paper, the RuO2@TiO2 electrode was prepared by thermal decomposition combined with high-vacuum magnetron sputtering. Compared with the oxygen evolution reaction (OER) activity of an ordinary RuO2 electrode, the OER activity of the RuO2@TiO2 electrode is significantly reduced. However, the CER activity of the RuO2@TiO2 electrode is close to the OER activity of RuO2. The CER mechanism of the RuO2@TiO2 electrode is the second electron transfer, and the OER mechanism is the formation and transformation of OHads. The potential difference between the CER and OER of the RuO2@TiO2 electrode is 174 mV, which is 65 mV higher than that of the RuO2 electrode, so the selectivity of the CER of the RuO2@TiO2 electrode is remarkably improved. During the preparation of AEW, the ACC obtained with the RuO2@TiO2 electrode is 1.7 times that obtained with the RuO2 electrode. In the sterilization experiments on Escherichia coli and Bacillus subtilis biofilms, the logarithmic killing values of AEW prepared the by RuO2@TiO2 electrode are higher than those of AEW prepared by the RuO2 electrode.

Effects of Acute Exposure to Polystyrene Nanoplastics on the Channel Catfish Larvae: Insights From Energy Metabolism and Transcriptomic Analysis.

Microplastics (nanoplastics) pollution has been a major ecological issue threatening global aquatic ecosystems. However, knowledge of the adverse effects of nanoplastics and the effects on freshwater ecosystems is still limited. To understand the impacts of nanoplastics on freshwater ecosystems, it is essential to reveal the physiological changes caused by nanoplastics in freshwater organisms, especially at their early life-history stages. In the present study, the larval channel catfish Ietalurus punetaus were exposed to gradient concentrations (0, 5, 10, 25, and 50 mg/L) of 75-nm polystyrene nanoplastics (PS-NPs) for 24 h or 48 h, and changes in contents of energy metabolites, metabolic enzyme activities and transcriptome were assessed. The results showed that glucose and triglyceride contents increased after 24 h of exposure to 10 or 25 mg/L of PS-NPs but decreased with increased concentrations or prolonged exposure duration. Activities of most metabolic enzymes analyzed decreased in the larvae after 48 h of exposure, especially in 25 or 50 mg/L of PS-NPs. These suggested that PS-NPs caused huge energy consumption and disturbed the energy metabolism in larval fish. Transcriptomic analysis showed that 48 h of exposure to 50 mg/L PS-NPs affected the expression of genes involved in protein digestion and induced response of proteasomes or heat shock proteins in the larval I. punetaus. The genes involved in peroxisome proliferator-activated receptors (PPAR) pathway and biosynthesis of amino acids were activated after the exposure. PS-NPs also depressed the expression of the genes involved in gonad development or muscle contraction in the larval I. punetaus. Overall, acute exposure to 75-nm PS-NPs disrupted the energy metabolism by consuming the energy reserves, and affected a series of molecular pathways which may further affect the development and survival of fish. This study provided the information about adverse effects of nanoplastics on the fish larvae and revealed the molecular pathways for the potential adverse outcomes.

Synthesized effects of medium-term exposure to seawater acidification and microplastics on the physiology and energy budget of the thick shell mussel Mytilus coruscus.

Ocean acidification (OA) and microplastics (MPs) contamination are two results of human excises. In regions like estuarine areas, OA and MPs exposure are happening at the same time. The current research investigated the synthesized effects of OA and MPs exposure for a medium-term duration on the physiology and energy budget of the thick shell mussel Mytilus coruscus. Mussels were treated by six combinations of three MPs levels (0, 10 and 1000 items L-1) × two pH levels (7.3, 8.1) for 21 d. As a result, under pH 7.3, clearance rate (CR), food absorption efficiency (AE), respiration rate (RR), and scope for growth (SFG) significantly decreased, while the fecal organic dry weight ratio (E) significantly increased. 1000 items L-1 MPs led to decrease of CR, E, SFG and increase of AE under pH 8.1. Interactive effects from combination of pH and MPs were found in terms of CR, AE, E and RR, but not for SFG of M. coruscus.

A novel bifunctional catalyst for overall water electrolysis: nano IrxMn(1-x)Oy hybrids with L12-IrMn3 phase.

A nano IrxMn(1-x)Oy hybrid electrode with a L12-IrMn3 phase was used as a bifunctional catalyst with ultra-low iridium loading for overall water electrolysis in an acid solution for the first time. The HER activity of the IrxMn(1-x)Oy hybrid electrode not only exceeded that of IrO2, but also exceeded that of Pt/C. The OER activity of the IrxMn(1-x)Oy hybrid electrode also exceeded that of IrO2.

Comparative transcriptome analysis of the gills and hepatopancreas from Macrobrachium rosenbergii exposed to the heavy metal Cadmium (Cd2+).

Heavy metal Cadmium (Cd2+) pollution has become a severe environmental problem for aquatic organisms. In crustaceans, gills (Gi) and hepatopancreas (Hp) play a vital role in the toxicology. However, in Macrobrachium rosenbergill, there are few researches about gill and hepatopancreases responding to Cd2+ stress at a molecular level. In this study, transcriptomic analysis was applied to characterize gene expression profiles of gills and hepatopancreas of M. rosenbergill after Cd2+ exposure for 0 h, 3 h and 3 d. Six cDNA libraries (Gi 0 h, Gi 3 h, Gi 3 d, Hp 0 h, Hp 3 h, and Hp 3 d) were constructed and a total of 66,676 transcripts and 48,991 unigenes were annotated. Furthermore, differentially expressed genes (DEGs) were isolated by comparing the Cd2+ treated time-point libraries (3 h and 3 d group) with the control library (0 h group). The results showed that most of the DEGs were down-regulated after Cd2+ exposure and the number of DEGs among gill groups were significantly higher than those among hepatopancreas groups. GO functional and KEGG pathway analysis suggested many key DEGs in response to the Cd2+ stress, such as metallothionein and Hemocyanin. Additionally, a total of six DEGs were randomly selected to further identify their expressional profile by qPCR. The results indicated that these DEGs were involved in the response to Cd2+. This comparative transcriptome provides valuable molecular information on the mechanisms of responding to Cd2+ stress in M. rosenbergii, which lays the foundation for further understanding of heavy metal stress.

Transcriptional changes revealed water acidification leads to the immune response and ovary maturation delay in the Chinese mitten crab Eriocheir sinensis.

Nowadays, due to increasing carbon dioxide released, water acidification poses a series of serious impacts on aquatic organisms. To evaluate the effects of water acidification on crustaceans, we focused on the Chinese mitten crab Eriocheir sinensis, which is a spawning migration and farmed species in China. Based on histological and oocyte transparent liquid observation, we found that the acidified environment significantly delayed the ovarian maturation of E. sinensis. Moreover, RNA-seq was applied to obtain gene expression profile from the crab's gills and ovaries in response to acidified environment. Compared with control groups, a total of 5471 differentially expressed genes (DEGs) were identified in acidified gills and 485 DEGs were identified in acidified ovaries. Enrichment analysis indicated that some pathways also responded to the acidified environment, such as PI3K-Akt signaling pathway, Chemokine signaling pathway, apoptosis, and toll-like receptor signaling pathway. Subsequently, some DEGs involved in immune response (ALF, Cathepsin A, HSP70, HSP90, and catalase) and ovarian maturation (Cyclin B, Fem-1a, Fem-1b, and Fem-1c) were selected to further validate the influence of water acidification on gene expression by qRT-PCR. The results showed that the expression level of immune-related genes was significantly increased to response to the water acidification, while the ovarian maturation-related genes were significantly decreased. Overall, our data suggested that E. sinensis was sensitive to the reduced pH. This comparative transcriptome also provides valuable molecular information on the mechanisms of the crustaceans responding to acidified environment.

Effects of methyl farnesoate on Krüppel homolog 1 (Kr-h1) during vitellogenesis in the Chinese mitten crab (Eriocheir sinensis).

Methyl farnesoate (MF), a de-epoxidized form of juvenile hormone (JH) Ⅲ in insects, may regulate developmental processes such as reproduction and ovarian maturation in crustaceans. Krüppel homolog 1 (Kr-h1) is a target response gene for the methoprene-tolerant (Met) protein that is a component of the JH signaling pathway in insects. In the present study, Es-Kr-h1 was cloned from E. sinensis and characterized to ascertain whether JH/MF signaling in insects is conserved in crustaceans. The findings with molecular structure analysis indicated Es-Kr-h1 contains seven zinc finger motifs (Zn2-Zn8) commonly conserved in other crustaceans, but the Zn1 motif was not detected to be present. The PCR results indicated that relative abundance of Es-Kr-h1 mRNA transcript in the hepatopancreas was greatest in the Stage Ⅱ, followed by the Stage Ⅳ ovarian developmental categories. The relative abundance of Es-Kr-h1 mRNA transcript in vitro was greater after MF addition to the hepatopancreas, however, not the ovarian tissues. The results from in vivo and eyestalk ablation experiments indicated the relative abundance of Es-Kr-h1 mRNA transcript was greater after MF treatment and bilateral eyestalk removal in the hepatopancreas, however, not ovarian tissues. Notably, there were effects of MF on relative abundance of Es-Kr-h1 mRNA transcript pattern. The Es-Kr-h1 protein, therefore, may be involved in MF-mediated vitellogenesis resulting from the response to Es-Met in E. sinensis, and the JH/MF signaling pathway is potentially conserved in crustaceans.

Potential role of Methoprene-tolerant (Met) in methyl farnesoate-mediated vitellogenesis in the Chinese mitten crab (Eriocheir sinensis).

Methoprene-tolerant (Met) belongs to the basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family of nuclear transcriptional regulators and is a leading candidate receptor for juvenile hormone (JH III) in insects. Methyl farnesoate (MF) is a de-epoxide form of JH III that regulates many developmental processes in crustaceans, including reproduction, molting, and morphogenesis, much like JH III in insects. In this study, the full-length cDNA for Met was cloned from the Chinese mitten crab (Eriocheir sinensis) (EsMet). The amino acid sequence of EsMet contains three conserved domains (bHLH, PAS-A, and PASB) characteristic of the bHLH-PAS family, having six conserved amino acid residues specifically responsible for JH or MF binding. Tissue distribution analysis revealed that EsMet mRNA is highly expressed in the hepatopancreas. In addition, EsMet and EsVg expression in the hepatopancreas were found to be significantly increased in early endogenous vitellogenic oocytes (stage II) during ovarian development, and the hemolymph MF titer was significantly increased in late exogenous vitellogenic oocytes (stage III), indicating that EsMet is involved in vitellogenesis regulation. In vitro, MF addition markedly upregulated EsMet and EsVg expression in hepatopancreatic tissue, but only EsVg was induced in ovarian tissue. In vivo, EsMet and EsVg expression in the hepatopancreas were both significantly and synchronously increased after MF injection, but not in the ovaries. In addition, EsMet and EsVg expression were upregulated in the hepatopancreas after eyestalk ablation, while only EsVg expression was induced in the ovaries. Thus, our results indicate that Met may act as a receptor for MF in MF-mediated vitellogenesis in crustaceans.

Liver transcriptome analysis and cortisol immune-response modulation in lipopolysaccharide-stimulated in channel catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) is an important aquaculture species in China. In channel catfish, diseases such as haemorrhagic, sepsis and tail-rot disease are all caused by bacteria as general in China. Most of the pathogenic bacteria are Gram-negative bacteria. Liver transcriptome analysis of the co-injection of cortisol and lipopolysaccharide (LPS) was performed in this study. Preliminary evidence from the results suggest that after the emergence of immune stress, cortisol will up-regulate the complement cascade pathway, down-regulate the coagulation cascade pathway, down-regulate the platelet activation pathway, down-regulate antigen presentation pathway, and show complex regulation relationship to inflammatory factors. At 12 h, the number of differential genes regulated by cortisol was about half less than the number of differential genes regulated by LPS. At 24 h, there was no significant difference between the number of differential genes regulated by cortisol and LPS, but the types of differential genes vary widely. KEGG enrichment analysis found that cortisol regulated LPS-stimulated immune responses mainly focus on cytokines, complement and coagulation cascades pathways, antigen presentation pathways, haematopoiesis, and inflammation. It is suggested that there may be some strategic choice in the regulation of immune response by cortisol. These results will help understand the pathogenesis and host defence system in bacterial disease caused by Gram-negative bacteria.

Construction of a High-Density Linkage Map and QTL Fine Mapping for Growth- and Sex-Related Traits in Channel Catfish (Ictalurus punctatus).

A high-density genetic linkage map is of particular importance in the fine mapping for important economic traits and whole genome assembly in aquaculture species. The channel catfish (Ictalurus punctatus), a species native to North America, is one of the most important commercial freshwater fish in the world. Outside of the United States, China has become the major producer and consumer of channel catfish after experiencing rapid development in the past three decades. In this study, based on restriction site associated DNA sequencing (RAD-seq), a high-density genetic linkage map of channel catfish was constructed by using single nucleotide polymorphisms (SNPs) in a F1 family composed of 156 offspring and their two parental individuals. A total of 4,768 SNPs were assigned to 29 linkage groups (LGs), and the length of the linkage map reached 2,480.25 centiMorgans (cM) with an average distance of 0.55 cM between loci. Based on this genetic linkage map, 223 genomic scaffolds were anchored to the 29 LGs of channel catfish, and a total length of 704.66 Mb was assembled. Quantitative trait locus (QTL) mapping and genome-wide association analysis identified 10 QTLs of sex-related and six QTLs of growth-related traits at LG17 and LG28, respectively. Candidate genes associated with sex dimorphism, including spata2, spata5, sf3, zbtb38, and fox, were identified within QTL intervals on the LG17. A sex-linked marker with simple sequence repeats (SSR) in zbtb38 gene of the LG17 was validated for practical verification of sex in the channel catfish. Thus, the LG17 was considered as a sex-related LG. Potential growth-related genes were also identified, including important regulators such as megf9, npffr1, and gas1. In a word, we constructed the high-density genetic linkage map and developed the sex-linked marker in channel catfish, which are important genetic resources for future marker-assisted selection (MAS) of this economically important teleost.

Genome-wide identification, phylogeny and expressional profile of the Sox gene family in channel catfish (Ictalurus punctatus).

The Sox gene family has been systematically characterized in some fish species but not in catfish Ictalurus punctatus. In this study, 25 Sox genes were identified in the channel catfish genome and classified into seven families based on their conserved domains as follows: eight genes in SoxB group (six in SoxB1 subgroup and two in SoxB2 subgroup); five genes in SoxC group; three genes in SoxD and SoxF groups; four genes in SoxE group; and one gene in SoxH and SoxK groups. The mammalian Sox groups SoxA, G, I, and J were not present in catfish. The number of introns in channel catfish Sox genes varied from zero to 13. Sox genes were distributed unevenly across 17 chromosomes. Five members of the ancestral vertebrate Sox genes (Sox1, Sox4, Sox9, Sox11 and Sox19) experienced teleost-specific whole genome duplication during evolution, and now have two copies on different chromosomes. Expression profiles analyses indicated that the accumulation of Sox genes was associated with different tissues, and the expression pattern also differed among each Sox gene group and duplicated gene. This study constitutes a comprehensive overview of the Sox gene family in channel catfish and provides new insights into the evolution of this gene family.

Identification and Characterization of Reference Genes for Normalizing Expression Data from Red Swamp Crawfish Procambarus clarkii.

qRT-PCR is a widely used technique for rapid and accurate quantification of gene expression data. The use of reference genes for normalization of the expression levels is crucial for accuracy. Several studies have shown that there is no perfect reference gene that is appropriate for use in all experimental conditions, and research on suitable reference genes in red swamp crawfish (Procambarus clarkii) is particularly scarce. In this study, eight commonly used crustacean reference genes were chosen from P. clarkii transcriptome data and investigated as potential candidates for normalization of qRT-PCR data. Expression of these genes under different experimental conditions was examined by qRT-PCR, and the stability of their expression was evaluated using three commonly used statistical algorithms, geNorm, NormFinder and BestKeeper. A final comprehensive ranking determined that EIF and 18S were the optimal reference genes for expression data from different tissues, while TBP and EIF were optimal for expression data from different ovarian developmental stages. To our knowledge, this is the first systematic analysis of reference genes for normalization of qRT-PCR data in P. clarkii. These results will facilitate more accurate and reliable expression studies of this and other crustacean species.

Transcriptome analysis of red swamp crawfish Procambarus clarkii reveals genes involved in gonadal development.

BACKGROUND: The red swamp crawfish, Procambarus clarkii, has become one of the most economically important cultured species in China. Currently, little is known about the gonadal development of this species. Isolation and characterization of genes are an initial step towards understanding gonadal development of P. clarkii. RESULTS: Using the 454 pyrosequencing technology, we obtained a total of 1,134,993 high quality sequence reads from the crawfish testis and ovary libraries. We aimed to identify different genes with a potential role in gonad development. The assembly formed into 22,652 isotigs, distributed by GO analysis across 55 categories in the three ontologies, 'molecular function', 'cellular component', and 'biological processes'. Comparative transcript analysis showed that 1,720 isotigs in the ovary were up-regulated and 2138 isotigs were down-regulated. Several gonad development related genes, such as vitellogenin, cyclin B, cyclin-dependent kinases 2, Dmc1 and ubiquitin were identified. Quantitative real-time PCR verified the expression profiles of 14 differentially expressed genes, and confirmed the reliability of the 454 pyrosequencing. CONCLUSIONS: Our findings provide an archive for future research on gonadal development at a molecular level in P. clarkii and other crustacean. This data will be helpful to develop new ideas for artificial regulation of the reproductive process in crawfish aquaculture.