Genome assembly of redclaw crayfish (Cherax quadricarinatus) provides insights into its immune adaptation and hypoxia tolerance.

BACKGROUND: The introduction of non-native species is a primary driver of biodiversity loss in freshwater ecosystems. The redclaw crayfish (Cherax quadricarinatus) is a freshwater species that exhibits tolerance to hypoxic stresses, fluctuating temperatures, high ammonia concentration. These hardy physiological characteristics make C. quadricarinatus a popular aquaculture species and a potential invasive species that can negatively impact tropical and subtropical ecosystems. Investigating the genomic basis of environmental tolerances and immune adaptation in C. quadricarinatus will facilitate the development of management strategies of this potential invasive species. RESULTS: We constructed a chromosome-level genome of C. quadricarinatus by integrating Nanopore and PacBio techniques. Comparative genomic analysis suggested that transposable elements and tandem repeats drove genome size evolution in decapod crustaceans. The expansion of nine immune-related gene families contributed to the disease resistance of C. quadricarinatus. Three hypoxia-related genes (KDM3A, KDM5A, HMOX2) were identified as being subjected to positive selection in C. quadricarinatus. Additionally, in vivo analysis revealed that upregulating KDM5A was crucial for hypoxic response in C. quadricarinatus. Knockdown of KDM5A impaired hypoxia tolerance in this species. CONCLUSIONS: Our results provide the genomic basis for hypoxic tolerance and immune adaptation in C. quadricarinatus, facilitating the management of this potential invasive species. Additionally, in vivo analysis in C. quadricarinatus suggests that the role of KDM5A in the hypoxic response of animals is complex.

Effects of dietary chitosan oligosaccharide on the growth, intestinal microbiota and immunity of juvenile red claw crayfish (Cherax quadricarinatus).

This study aimed to evaluate the potential benefits of chitosan oligosaccharide (COS) on red claw crayfish (Cherax quadricarinatus) and explore its underlying mechanisms. The crayfish were randomly divided into six groups, and the diets were supplemented with COS at levels of 0 (C0), 0.2 (C1), 0.4 (C2), 0.6 (C3), 0.8 (C4), and 1 (C5) g kg-1. Treatment with COS significantly improved the growth performance of the crayfish with a higher weight gain rate (WGR) and specific growth rate (SGR) in the C2 group compared to the C0 group. Additionally, the content of crude protein in the crayfish muscles in the C1 group was significantly higher than that of the C0 group. Regarding non-specific immunity, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and alkaline phosphatase (AKP), and the levels of expression of the genes related to immunity (SOD; anti-lipopolysaccharide factor [ALF]; thioredoxin1 [Trx1]; C-type lysozyme, [C-LZM]; and GSH-Px) in the hepatopancreas and hemolymph increased significantly (P < 0.05) after supplementation with 0.4 g kg-1 of COS, while the content of malondialdehyde (MDA) decreased (P < 0.05). The survival rate of C. quadricarinatus increased (P < 0.05) in the C2, C3, C4, and C5 groups after the challenge with Aeromonas hydrophila. This study found that COS has the potential to modulate the composition of the intestinal microbiota and significantly reduce the abundance of species of the phylum Proteobacteria and the genera Aeromonas and Vibrio in the gut of C. quadricarinatus, while the abundance of bacteria in the phylum Firmicutes and the genus Candidatus_Hepatoplasma improved significantly. This study suggests that the inclusion of COS in the diet of C. quadricarinatus can enhance growth, boost immunity, and increase resistance to infection with A. hydrophila, especially when supplemented at 0.4-0.8 g kg-1.

Effects of antimicrobial peptides from dietary Hermetia illucens larvae on the growth, immunity, gene expression, intestinal microbiota and resistance to Aeromonas hydrophila of juvenile red claw crayfish (Cherax quadricarinatus).

Antimicrobial peptides (AMPs), which are widely present in animals and plants, have a broad distribution, strong broad-spectrum antibacterial activity, low likelihood of developing drug resistance, high thermal stability and antiviral properties. The present study investigated the effects of adding AMPs from Hermetia illucens larvae on the growth performance, muscle composition, antioxidant capacity, immune response, gene expression, antibacterial ability and intestinal microbiota of Cherax quadricarinatus (red claw crayfish). Five experimental diets were prepared by adding 50 (M1), 100 (M2), 150 (M3) and 200 (M4) mg/kg of crude AMP extract from H. illucens larvae to the basal diet feed, which was also used as the control (M0). After an eight-week feeding experiment, it was discovered that the addition of 100-150 mg/kg of H. illucens larvae AMPs to the feed significantly improved the weight gain rate and specific growth rate of C. quadricarinatus. Furthermore, the addition of H. illucens larvae AMPs to the feed had no significant effect on the moisture content, crude protein, crude fat and ash content of the C. quadricarinatus muscle. The addition of 100-150 mg/kg of H. illucens larvae AMPs in the feed also increased the antioxidant capacity, nonspecific immune enzyme activity and related gene expression levels in C. quadricarinatus, thereby enhancing their antioxidant capacity and immune function. The H. illucens larvae AMPs improved the structure and composition of the intestinal microbiota of C. quadricarinatus, increasing the microbial community diversity of the crayfish gut. Finally, the addition of 100-150 mg/kg of H. illucens larvae AMPs in the feed enhanced the resistance of C. quadricarinatus against Aeromonas hydrophila, improving the survival rate of the crayfish. Based on the aforementioned findings, it is recommended that H. illucens larvae AMPs be incorporated into the C. quadricarinatus feed at a concentration of 100-150 mg/kg.

Functional characterization of serine proteinase inhibitor Kazal-Type in the red claw crayfish Cherax quadricarinatus.

Serine protease inhibitors Kazal type (SPINKs) function in physiological and immunological processes across multicellular organisms. In the present study, we identified a SPINK gene, designated as CqSPINK, in the red claw crayfish Cherax quadricarinatus, which is the ortholog of human SPINK5. The deduced CqSPINK contains two Kazal domains consisting of 45 amino acid residues with a typical signature motif C-X3-C-X5-PVCG-X5-Y-X3-C-X6-C-X12-14-C. Each Kazal domain contains six conserved cysteine residues forming three pairs of disulfide bonds, segmenting the structure into three rings. Phylogenetic analysis revealed CqSPINK as a homolog of human SPINK5. CqSPINK expression was detected exclusively in hepatopancreas and epithelium, with rapid up-regulation in hepatopancreas upon Vibrio parahaemolyticus E1 challenge. Recombinant CqSPINK protein (rCqSPINK) was heterologously expressed in Escherichia coli and purified for further study. Proteinase inhibition assays demonstrated that rCqSPINK could potently inhibit proteinase K and subtilisin A, weakly inhibit α-chymotrypsin and elastase, but extremely weak inhibit trypsin. Furthermore, CqSPINK inhibited bacterial secretory proteinase activity from Bacillus subtilis, E. coli, and Staphylococcus aureus, and inhibited B. subtilis growth. These findings suggest CqSPINK's involvement in antibacterial immunity through direct inhibition of bacterial proteases, contributing to resistance against pathogen invasion.

Optimizing the nested PCR method for Decapod iridescent virus 1 (DIV1) targeting ATPase gene by reselecting the inner primers.

DIV1 has the characteristics of fast transmission and a broad host range. Its infection leads to a high mortality rate, posing a serious threat to the global crustacean aquaculture industry. In order to increase the accuracy of DIV1 detection and reduce the difficulty of result interpretation, this study modified the original nested PCR method targeting the DIV1 ATPase gene. The internal primers for the nested PCR were redesigned to produce a 338 bp amplification product in the second step PCR, effectively distinguishing the target band from primer dimers. The newly established nested PCR method exhibits strong specificity and high sensitivity, with a detection limit as low as 1.37 × 101 copies/reaction. The developed nested PCR assay provides new technical support for the accurate detection of DIV1 in global crustacean aquaculture.

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

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

MicroRNA transcriptome analysis for elucidating the immune mechanism of the redclaw crayfish Cherax quadricarinatus under Decapod iridescent virus 1 infection.

Redclaw crayfish (Cherax quadricarinatus) is a large, tropical freshwater crustacean species with considerable potential of commercial production. In recent years, infection with DIV1 in redclaw crayfish is being reported in aquaculture industries, causing high mortality and huge economic losses. However, many characteristics of this virus, including pathogenesis, transmission mechanism, and host immunity, remain largely unknown.MicroRNAs are known to play important roles in numerous biological processes, and many microRNAs are reported to be involved in the regulation of immune responses. In this study, nine-small RNA libraries were constructed using hemocytes of redclaw crayfish to characterize the differentially expressed miRNAs (DE-miRNAs) at 24 and 48 h postinfection (hpi). A total of 14 and 22 DE-miRNAs were identified in response to DIV1 infection at 24 and 48 hpi, respectively. Further, functional annotation of the predicted host target genes using GO and KEGG pathway enrichment analyses indicated that relevant biological processes and signal pathways underwent miRNA-mediated regulation after DIV1 infection. Our results enhanced the understanding of the mechanisms of miRNA-mediated regulation of immune responses under DIV1 infection in crustaceans.

The effect of white spot syndrome virus (WSSV) envelope protein VP28 on innate immunity and resistance to white spot syndrome virus in Cherax quadricarinatus.

VP28 is the most abundant membrane protein of WSSV, and the recombinant protein VP28 (VP26 or VP24) was constructed for the immune protection experiment in this study. Crayfish were immunized by intramuscular injection of recombinant protein V28 (VP26 or VP24) at a dose of 2 µg/g. The survival rate of crayfish immunized by VP28 showed a higher value than by VP26 or VP24 after WSSV challenge. Compared with the WSSV-positive control group, the VP28-immunized group could inhibit the replication of WSSV in crayfish, increasing the survival rate of crayfish to 66.67% after WSSV infection. The results of gene expression showed that VP28 treatment could enhance the expression of immune genes, mainly JAK and STAT genes. VP28 treatment also enhanced total hemocyte counts and enzyme activities including PO, SOD, and CAT in crayfish. VP28 treatment reduced the apoptosis of hemocytes in crayfish, as well as after WSSV infection. In conclusion, VP28 treatment can enhance the innate immunity of crayfish and has a significant effect on resistance to WSSV, and can be used as a preventive tool.

New insights into the regulation mechanism of red claw crayfish (Cherax quadricarinatus) hepatopancreas under air exposure using transcriptome analysis.

Red claw crayfish (Cherax quadricarinatus) is an important freshwater shrimp species worldwide with enormous economic value. Waterless transportation is an inherent feature of red claw crayfish transportation. However, the high mortality of red claw crayfish is a severe problem in the aquaculture of crayfish after waterless transportation. In this study, we investigated the responses of the hepatopancreas from the red claw crayfish undergoing air exposure stress and normal conditions on transcriptome levels. We used Illumina-based RNA sequencing (RNA-Seq) to perform a transcriptome analysis from the hepatopancreas of red claw crayfish challenged by air exposure. An average of 57,148,800 clean reads per library was obtained, and 33,567 unigenes could be predicted and classified according to their homology with matches in the National Center for Biotechnology Information (NCBI) non-redundant protein sequences (Nr), Gene Ontology (GO), a manually annotated and reviewed protein sequence database (Swiss-Prot), protein families (Pfam), Clusters of Orthologous Groups (COG) of proteins, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. 690 and 3407 differentially expressed genes (DEGs) were identified between the two stress stages of the red claw crayfish. More DEGs were identified in 12 h, indicating that gene expressions were largely changed at 12 h. Some immune-related pathways and genes were identified according to KEGG and GO enrichment analysis. A total of 12 DEGs involved in immune response and trehalose mechanism were verified by quantitative real-time-polymerase chain reaction (qRT-PCR). The results indicated that the red claw crayfish might counteract the stress of air exposure at the transcriptomic level by increasing expression levels of antioxidant-, immune-, and trehalose metabolism-related genes. These transcriptome results from the hepatopancreas provide significant insights into the influence mechanism of air exposure to the trehalose mechanism and immune response in the red claw crayfish.

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

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

Dietary quercetin protects Cherax quadricarinatus against white spot syndrome virus infection.

Quercetin is a plant flavonoid with a molecular formula C15H10O7. It has antioxidant, anti-inflammatory, antibacterial, and anti-apoptotic effects in animals. We used red claw crayfish (Cherax quadricarinatus) infected with white spot syndrome virus (WSSV) to investigate quercetin's effects on innate immunity of crustaceans. Quercetin supplementation significantly reduced the mortality of crayfish caused by WSSV infection and the number of VP28 copies in WSSV-infected crayfish. Quantitative real-time PCR analysis showed that dietary quercetin supplementation increased the expression of immune-related genes, like JAK, STAT and ALF. Quercetin supplementation affected the activity of six immune-related enzymes and increased the total number of hemocytes in crayfish. It also significantly reduced the rate of hemocyte apoptosis in both WSSV-infected and uninfected crayfish. These results demonstrate the potential for commercial use of quercetin for the prevention of WSSV disease in crustaceans.

Transcriptome Analysis Reveals the Effect of Stocking Density on Energy Metabolism in the Gills of Cherax quadricarinatus under Rice-Crayfish Co-Culture.

Stocking density is a crucial factor affecting productivity in aquaculture, and high stocking density is a stressor for aquatic animals. In this study, we aimed to investigate the effects of stocking densities on oxidative stress and energy metabolism in the gills of Cherax quadricarinatus under rice-crayfish farming. The C. quadricarinatus were reared at low density (LD), medium density (MD), and high density (HD) for 90 days. The results showed that the superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA) levels were higher in the HD group than those in the LD group. Transcriptomic analysis revealed 1944 upregulated and 1157 downregulated genes in the gills of the HD group compared to the LD group. Gene ontology (GO) enrichment analysis indicated that these differentially expressed genes (DEGs) were significantly associated with ATP metabolism. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis also showed that high stocking density resulted in the dysregulation of oxidative phosphorylation. Furthermore, high stocking density upregulated six lipid metabolism-related pathways. Overall, our findings, despite the limited number of samples, suggested that high stocking density led to oxidative stress and dysregulation of energy metabolism in the gills of C. quadricarinatus under rice-crayfish co-culture. Alteration in energy metabolism may be an adaptive response to adverse farming conditions.

Dietary supplementations modulate the physiological parameters, fatty acids profile and the growth of red claw crayfish (Cherax quadricarinatus).

An optimal diet is an important factor for the proper growth and health of crustaceans. However, the regulation of antioxidant activity and non-specific immunity related to the consumption of feed additives has not been studied in RC-crayfish. Triplicate groups of 20 crayfish/tank (36.72 ± 0.70 g) fed with a basal diet and sixteen experimental diets that contained five feed additives with four grade levels (40, 160, 240 and 320 mg/kg vitamin E, 2, 4, 6 and 8 g/kg nucleotides, 2, 4, 6 and 8 g/kg Haematococcus pluvialis, 5, 10, 15 and 20 g/kg arachidonic acid and 2.5, 5, 10 and 15 g/kg yeast extract) on physiological parameters, fatty acids profile and growth of Cherax quadricarinatus for a period of 70 days by using orthogonal array method (L16 45 ). The results showed that the antioxidants activity in the haemolymph and hepatopancreas were both higher in crayfish fed with diets NO. 9 to 12 than others. Also, all the diets except diets NO. 13 to 16 showed lower free radicals contents than the control group. Similarly, significantly higher non-specific immune parameters were observed in the hepatopancreas of crayfish supplementations than those fed a control diet. Biochemical parameters related to protein profile in haemolymph increased in diets NO. 9 to 12 and then decreased in control and diets NO. 13 to 16, while the highest biochemical parameters related to lipid profile except HDL-c contents in haemolymph were observed in crayfish fed the control diet. Fatty acid composition in the hepatopancreas, muscle and ovary of RC-crayfish was significantly influenced by using the combination of Vit E, NT, H. pluvialis and YP compared to the control group. Compared to all treatments, RC-crayfish fed with diets NO. 2 and 12 had significantly stimulated higher growth performance and feed utilisation. Overall, our results suggest that diets supplemented with Vit E level of 240 mg/kg, in combination with 8 g/kg NT, 4 g/kg, H. pluvialis, 5 g/kg ARA and 10 g/kg YP are the promising treatments to increase antioxidants activity, non-specific immune response, fatty acids composition and growth of RC-crayfish. However, high dietary supplementations level can reduce antioxidants activity, immunity and inhibit growth.

Coumarin protects Cherax quadricarinatus (red claw crayfish) against white spot syndrome virus infection.

Coumarin is a natural compound from plants with the molecular formula C9H6O2. Cherax quadricarinatus (red claw crayfish) is an aquaculture species exhibiting high economic efficiency and quality that is mainly distributed and cultivated in the southeast provinces in China. In order to identify an effective herbal immunopotentiator against white spot syndrome virus (WSSV) infection, this study examined the effect of coumarin as a feed additive in protecting C. quadricarinatus against WSSV infection. The expression of immune-related genes and WSSV copies were analyzed by Q-PCR. Challenge experiments were conducted to analyze the survival rate and determine the optimal concentration of coumarin. The Phenoloxidase activity (PO), Acid phosphatase (ACP) and superoxide dismutase activity (SOD) activity and lysozyme activity were also analyzed. Total hemocyte count (THC) and apoptosis rate were determined by flow cytometry. The WSSV challenge results showed that 40 mg/kg coumarin reduced the mortality of C. quadricarinatus and delayed the WSSV infection process. Further investigation showed that coumarin treatment had a positive effect on the important immunity-related parameters THC, ACP activity, SOD activity, LZM and PO activity. Coumarin up-regulated the expression of proPO, JAK, STAT, ALF, Hsp70 and down-regulated the expression of caspase at the mRNA level. After WSSV infection, the hemocyte apoptosis rate was lower in the 40 mg/kg coumarin + WSSV group compared with the WSSV only group. These data illustrate that coumarin enhances innate immunity in C. quadricarinatus and exhibits a protective effect against WSSV infection by reducing the number of WSSV copies and slowing the process of infection, which provides a potential theoretical basis for studies of coumarin as a new aquatic feed additive in crustacean aquaculture.

Redclaw crayfish (Cherax quadricarinatus) responds to Vibrio parahaemolyticus infection by activating toll and immune deficiency signaling pathways and transcription of associated immune response genes.

In invertebrates, several genes controlled by the Toll and immunodeficiency (IMD) signaling cascade are altered during microbial infection. However, little is known about the expression patterns of innate immune response genes in red-clawed crayfish (Cherax quadricarinatus). In the present study, the transcription of five genes was assessed in C. quadricarinatus challenged with Vibrio parahaemolyticus (V. parahaemolyticus). The expression of Relish, Toll-like receptor (TLR), tumor necrosis factor receptor-related factor 6 (TRAF6), Akirin, and IMD in different tissues and at different time points after infection were assessed. In addition, the Relish gene was amplified, the protein conformation of the Relish gene was predicted, and gene expression changes associated with antimicrobial peptide production in C. quadricarinatus were analyzed using RNA interference (RNAi). During V. parahaemolyticus infection, the transcripts of the above five genes were significantly increased in the hepatopancreas of C. quadricarinatus (P < 0.05). In contrast, TLR was significantly downregulated in muscle tissue at the initial stage of infection (P < 0.05); TRAF6 and IMD were significantly down-regulated throughout infection (P < 0.05); Akirin transcripts had the lowest abundance at 24 h post-infection; Relish, IMD and Akirin genes were significantly up-regulated in gill tissue at the early stage of infection (P < 0.05); only TRAF6 was significantly up-regulated at 6, 24 and 48 h after infection. The Relish gene of C. quadricarinatus is closely related to the Exopalaemon carinicauda. When the Relish gene was knocked down by RNAi, the V. parahaemolyticus challenge showed that the mortality rate of the RANi group was significantly higher than that of the NC group; pathological sections showed that the hepatopancreatic tissue damage was the most severe 12 h after the interference; and the interference significantly inhibited IRF4, NF-κB, ALF, laccase, SOD1, and lectin genes. Therefore, it can be hypothesized that the Toll and IMD pathways are activated in C. quadricarinatus in response to bacterial infection and that genes associated with these pathways are differentially transcribed in different tissues. This study provides insights into the Toll and IMD signaling pathways and the spatiotemporal expression of key genes regulating bacterial infection resistance in C. quadricarinatus.

Dietary glycerol monolaurate protects Cherax quadricarinatus against white spot syndrome virus infection.

Glycerol monolaurate (GML), one of the medium-chain fatty acid esters, is often used as an emulsifier or preservative. Its biological functions include antibacterial and antiviral activities. In this study, we examined the effects of dietary GML on the resistance of the red claw crayfish to WSSV infection. Crayfish fed with 4 g/kg GML showed higher survival rate and lower WSSV copy numbers than the control after WSSV infection. A RT-qPCR analysis showed that GML supplementation enhanced the expression of immune-related genes, especially JAK and caspase. Our data indicate that GML affects the immune parameters of crayfish, including the total hemocyte counts and phenoloxidase, acid phosphatase, superoxide dismutase, lysozyme, and peroxidase activities. After treatment with GML, the apoptosis of hemocytes increased significantly in both WSSV-infected and uninfected crayfish. In summary, GML reduced the mortality of WSSV-infected crayfish, perhaps by modulating the innate immunity of the crayfish. Our study shows that GML can be used to induce the innate immunity and enhance the immune protection of the red claw crayfish against WSSV infection, either therapeutically or as a preventive measure.

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

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

Effects of nanoplastic exposure on the immunity and metabolism of red crayfish (Cherax quadricarinatus) based on high-throughput sequencing.

Previous studies have shown that nanoplastics (NPs) are harmful pollutants that threaten aquatic organisms and ecosystems, however, less research has been conducted on the hazards of NPs for aquaculture animals. In this study, Cherax quadricarinatus was used as an experimental model to evaluate the possible effects of three concentrations (25, 250 and 2500 µg/L) of NPs on red crayfish. The toxicological effects of NPs on this species were investigated based on transcriptomics and microbiome. A total of 67,668 genes were obtained from the transcriptome. The annotation rate of the four major libraries (Nr, KEGG, KOG, Swissprot) was 40.17 %, and the functions of differential genes were mainly related to antioxidant activity, metabolism and immune processes. During the experiment, the activities of superoxide dismutase (SOD) and catalase (CAT) in the high concentration group were significantly decreased, while the concentration of malondialdehyde (MDA) increased after nanoplastics (NPs) exposure, and SOD1, Jafrac1 were significantly reduced at high concentrations. expression is inhibited. The immune genes LYZ and PPO2 were highly expressed at low concentrations and suppressed at high concentrations. After 14 days of exposure to NPs, significant changes in gut microbiota were observed, such as decreased abundances of Actinobacteria, Bacteroidetes, and Firmicutes. NPs compromise host health by inducing changes in microbial communities and the production of beneficial bacterial metabolites. Overall, these results suggest that NPs affect immune-related gene expression and antioxidant enzyme activity in red crayfish and cause redox imbalance in the body, altering the composition and diversity of the gut microbiota.

Effects of dietary trehalose on growth, trehalose content, non-specific immunity, gene expression and desiccation resistance of juvenile red claw crayfish (Cherax quadricarinatus).

This study was performed to investigate the effects of dietary trehalose on growth, muscle composition, non-specific immune responses, gene expression and desiccation resistance of juvenile red claw crayfish (Cherax quadricarinatus). A total of 540 (body weight of 0.41 ± 0.05) crayfish were randomly divided into six groups for a feeding experiment. Six diets with trehalose levels at 0 (Diet 1), 1 (Diet 2), 2 (Diet 3), 5 (Diet 4), 10 (Diet 5) and 15 (Diet 6) g kg-1 were prepared to feed juvenile red claw crayfish for 8 weeks. The results showed that the weight gain rate (WGR) and specific growth rate (SGR) of crayfish in Diet 4, Diet 5 and Diet 6 groups were significantly improved compared with the control group (Diet 1). Muscle crude protein contents of crayfish fed Diet 4, Diet 5 and Diet 6 were significantly higher than those of the control group. The activities of superoxide dismutase (SOD) and alkaline phosphatase (AKP) in hepatopancreas and hemolymph of crayfish for Diet 4, Diet 5, and Diet 6 groups were significantly increased while malondialdehyde (MDA) content was significantly reduced when compared with the control. The total antioxidant capacity (T-AOC), catalase (CAT) and glutathione peroxidase (GPx) activities in the hepatopancreas and hemolymph of crayfish fed Diet 5 and Diet 6 were significantly higher than those in the control group. However, acid phosphatase (ACP) activity was not significantly different among all experimental groups. The hepatopancreas and intestine trehalose contents of crayfish showed an upward trend with the increase of dietary trehalose levels. Compared with the control group, supplementation of 5-15 g kg-1 trehalose in the feed up-regulated the expression levels of GPx, C-type lysozyme (C-LZM), antilipolysacchride factor (ALF), facilitated trehalose transporter homolog isoform X2 (Tret1-2) and facilitated trehalose transporter isoform X4 (Tret1-4) mRNA. In addition, supplementation of 5-15 g kg-1 trehalose in the feed could improve the survival rate of red claw crayfish under desiccation stress. These results suggested that supplementation of 5-15 g kg-1 trehalose in feed could significantly improve the growth performance, muscle protein, non-specific immunity and desiccation resistance of juvenile red claw crayfish.

Crayfish (Cherax quadricarinatus) susceptibility to acute hepatopancreatic necrosis disease (AHPND).

Acute hepatopancreatic necrosis disease (AHPND) is an OIE-listed enteric disease that has continued to plague the shrimp aquaculture industry since its first discovery in 2009. AHPND is one of the biggest disease threats to the shrimp aquaculture industry along with white spot disease (WSD) which has severely impacted both crayfish and shrimp aquaculture. AHPND is caused by specific marine Vibrio spp. which carry plasmid-borne binary toxins PirAVp and PirBVp. This research investigated if crayfish are susceptible to AHPND-causing Vibrio parahaemolyticus (VpAHPND) to discern the potential risk that AHPND may pose to the crayfish aquaculture industry. Susceptibility was investigated by challenging Cherax quadricarinatus (Australian red claw crayfish) and Penaeus vannamei (Pacific white shrimp) with VpAHPND in a cohabitation immersion bioassay. Upon termination of the bioassay, crayfish survival was significantly higher than shrimp survival (87% vs. 33%). Hepatopancreas dissected from experimentally challenged animals were screened for the binary toxin genes pirAVp and pirBVp by real-time and duplex conventional PCR assays, and also were examined by H&E histology for the detection of characteristic AHPND pathology. Although AHPND toxin genes pirAVp and pirBVp were detected in a subset of crayfish samples, histopathology did not reveal any pathognomonic lesions that are characteristic of AHPND in any crayfish samples examined. These findings suggest that crayfish are likely resistant to AHPND.