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Triacylglycerol lipases (TGLs) can catalyze the hydrolysis reaction of triacylglycerol serving multiple functions in most organisms. Based on the genomic and transcriptomic databases of Neocaridina denticulata sinensis, two TGL genes from N. denticulata sinensis designated NdTGL1 and NdTGL2 were identified and characterized. NdTGL1 showed the highest expression in the stomach, followed by the testis and hepatopancreas, and NdTGL2 exhibited the maximum expression in the hepatopancreas, followed by the stomach and heart. Under the stimulation of copper ion, the expression of NdTGL1 peaked at 12 h and the expression of NdTGL2 elevated significantly at 24 h after stimulation (P < 0.05). It is speculated that NdTGLs may play an important role in the stress response of N. denticulata sinensis. Challenged with Vibrio parahaemolyticus, the expression profiles of NdTGL1 and NdTGL2 in the hepatopancreas was different, which indicates that the immune response of the V. parahaemolyticus challenge might lead to changes in triglyceride metabolism. The recombinant NdTGL (recNdTGL1 and recNdTGL2) were obtained and the enzymatic characterization of recNdTGL1 and recNdTGL2 were determined. The common maximum activity and stability of the recNdTGL1 and recNdTGL2 were observed at 45 °C and 10 °C, respectively. Both recNdTGL1 and recNdTGL2 exhibited the highest activity at pH 10.0. Furthermore, the recNdTGL1 and recNdTGL2 displayed the maximum stability at pH 5.0 and pH 8.0, respectively. In presence of different metal ions, the enzyme activity of recNdTGL1 and recNdTGL2 were inhibited by Cu 2+ and Zn 2+ , and decreased by about 25%. Studies on the triacylglycerol lipases of N. denticulata sinensis provide theoretical support for studies related to fat metabolism in crustaceans and studies on response mechanism of digestive enzymes to microbial pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Vibrio parahaemolyticus causes serious economic losses to the shrimp farming industry. There is still a lack of adequate understanding of the changes in the overall response of N. denticulata sinensis caused by V. parahaemolyticus, particularly with respect to gill tissue, which is severely damaged by the pathogen. In this study, a total of 1358 differentially expressed genes were identified between the PBS control and Vibrio stimulation groups using transcriptome sequencing techniques. After further screening and analysis, many immune-related genes were obtained, involving lysosome pathway, metabolic process, chitin-binding protein, and serine protease family members. In addition, we randomly selected six DEGs in the lysosome pathway for qRT-PCR verification, and the results showed that their expression patterns were consistent with the RNA-seq. The results demonstrate the molecular regulation of the gill tissue response to V. parahaemolyticus infection in N. denticulata sinensis, contributing to the understand of the complex and efficient innate immune system and defense mechanisms in crustaceans., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

CRISPR/Cas9 technology is an efficient genome editing tool for producing genetically modified animals. Carotenoids color the world around us and their accumulation in animals could be used to culture colorful new verities in animal breeding. β, β-carotene 9′, 10′-oxygenase (BCO2) is an important enzyme during β-carotene metabolism. In this research, one full-length cDNA sequence encoding BCO2 (named EcBCO2) were obtained from Exopalaemon carinicauda. The genomic structure analysis showed that EcBCO2 gene was composed of 9 exons and 8 introns. Then, the CRISPR/Cas9-mediated deletion of EcBCO2 gene was generated by co-microinjection of Cas9 mRNA and EcBCO2 sgRNA into one-cell stage embryos of E. carinicauda. Subsequently, the phenotype of EcBCO2 -KO prawns was compared with that of wild-type prawns, which showed that EcBCO2 -KO resulted in the color change in the hepatopancreas of prawns. In addition, the EcBCO2 -KO prawns had a higher survival rate than wild-type prawns when the prawns were challenged with Vibrio parahaemolyticus or Aeromonas hydrophila. These results indicate that BCO2 gene could be used as a candidate gene in molecular marker-assistant breeding of prawns. [ABSTRACT FROM AUTHOR]

During the immune defense reaction of invertebrate, a plenty of reactive oxygen species (ROS) could be induced to product. Though ROS can kill foreign invaders, the accumulation of these reactive molecules in animals will cause serious cell damage. Carotenoids could function as scavengers of oxygen radicals. In this research, cDNA and genomic DNA of one carotenoid isomerooxygenase gene (named EcNinaB-X1) were cloned from Exopalaemon carinicauda. EcNinaB-X1 gene was composed of 12 exons and 11 introns. EcNinaB-X1 knock-out (KO) prawns were produced via CRISPR/Cas9 technology and the change of their phenotypes were analyzed. Of the 400 injected one-cell stage embryos with cas9 mRNA and one sgRNA targeting the first exon of EcNinaB-X1 gene, 26 EcNinaB-X1- KO prawns were generated and the mutant rate reached 6.5% after embryo injection. The EcNinaB-X1- KO prawns had significant lower mortality than those in wild-type group when the prawns were challenged with Vibrio parahaemolyticus or Aeromonas hydrophila. In conclusion, we first demonstrate the function of the carotenoid isomerooxygenase gene in immune defense of E. carinicauda by performing directed, heritable gene mutagenesis. Image 1 • EcNinaB-X1 gene was cloned from E. carinicauda. • The expression profiles of EcNinaB-X1 were demonstrated. • CRISPR-Cas9 system efficiently generated indels in EcNinaB-X1 loci. • EcNinaB-X1 -KO prawns had lower mortality than wild-type after bacterial challenge. [ABSTRACT FROM AUTHOR]

Chitin degradation is catalyzed by a two-component chitinolytic enzyme system, chitinase and β-N-acetylglucosaminidase (NAGase). In this paper, the full-length cDNA sequence encoding NAGase ( EcNAG ) was obtained from Exopalaemon carinicauda . The deduced amino acid sequence of EcNAG open reading frame (ORF) contained one Glycohydro_20b2 domain and one Glyco_hydro_20 domain. Based on the cDNA sequence, the genomic structure of EcNAG was characterized and it was composed of six exons and five introns. EcNAG mRNA majorly expressed in the hepatopancreas and epidermis. During the molting stages, EcNAG mRNA expression was well-regulated and its expression reached the highest level at the molting stage E. In addition, EcNAG was recombinant expressed in Pichia pastoris and the partial enzymatic characterization of recombinant EcNAG was confirmed. After being challenged with Vibrio parahaemolyticus and Aeromonas hydrophila , the expression of EcNAG was up-regulated significantly at 6 h and reached the peak at 12 h. And then, the expression began to down-regulated and came to the normal level at 72 h. It is helpful to research the relationship between the molt-related hormones and chitinlytic enzymes. [ABSTRACT FROM AUTHOR]

Chitinase, belonging to family 18 glycosyl hydrolase, is a multi-gene family and it has many functions. Generation of loss-of-function mutant targeting an interesting gene is a common way to clarify its function based on reverse genetics. In this study, we first reported the immune defense of a chitinase gene ( EcChi4 ) in Exopalaemon carinicauda using its EcChi4 -deletion mutant. EcChi4 was predominantly expressed in hepatopancreas and was upregulated after challenge with Vibrio parahaemolyticus or Aeromonas hydrophila . After knockout EcChi4 gene using CRISPR/Cas9 tool, the prawns in EcChi4 -deletion group had significant higher mortality than those in wild-type group when the prawns were challenged with V. parahaemolyticus or A. hydrophila . In conclusion, we first demonstrate the function of a chitinase gene in immune defense of E. carinicauda by performing directed, heritable gene mutagenesis. In the future, CRISPR/Cas9 should be widely applicable as a feasible means for gene editing in E. carinicauda for the study of important biological questions that cannot be easily addressed in other decapods. [ABSTRACT FROM AUTHOR]

Peroxiredoxin (Prx) is an antioxidant protein that widely exists in various organisms. To further investigate the role of Prx in the antioxidant and immune responses of Neocaridina denticulata sinensis , the full-length cDNA sequence of a Prx gene (Nd-Prx) from N. denticulata sinensis was obtained. The open reading frame (ORF) of Nd-Prx is 597 bp and encodes 198 amino acids. Amino acid similarity alignment showed that Nd-Prx contained a conserved sequence region "FYPLDFTFVCPTEI". qRT-PCR assay showed that Nd-Prx was expressed in all tested tissues and its expression was highest in the ovary. Nd-Prx was most highly expressed at 36 h after copper stimulation. Nd-Prx expression levels in hepatopancreas were significantly upregulated after Vibrio parahaemolyticus challenge (P < 0.05). In addition, the recombinant Nd-Prx was prepared and its enzyme activity was most stable at 70 °C with pH of 6.0. The antioxidant activity and DNA protection of recombinant Nd-Prx was also demonstrated. In summary, this study investigated the role of Prx in antioxidant and immune responses of N. denticulata sinensis , which might provide a foundation for further exploring Prx in immune system of crustaceans and for the application in disease control. [Display omitted] • Nd-Prx gene was cloned and its recombinant protein was prepared to analyze its enzymatic activity. • Nd-Prx was expressed in all tested tissues. • Expression of Nd-Prx was elevated under copper exposure or V. parahaemolyticus challenge. • The recombinant Nd-Prx exhibited high reducibility. [ABSTRACT FROM AUTHOR]

Crustins are a kind of antibacterial peptides (AMP) existing in crustaceans, and their antibacterial abilities are considered to be related to the conserved WAP domain. In this study, a novel type I Crustin gene was identified in Exopalaemon carinicauda , named EcCru. The deduced amino acid sequence revealed that the conserved cysteine at position 7 in the WAP domain was replaced by aspartic acid. The gene is 405 bp in length, encoding 134 amino acids, and is mainly distributed in gills and hepatopancreas. After Vibrio parahaemolyticus and Aeromonas hydrophila stimulation, the expression of EcCru was significantly up-regulated within 12 h, and then returned to normal levels. The recombinant protein was obtained using the Pichia pastoris expression system, and the recombinant protein had neither antibacterial activity against gram-positive or gram-negative bacteria. But the antibacterial ability emerged when Asp101 was mutated to Cys. Notably, we also obtained a mutant that had a deletion at the 6 th conserved Cys in the WAP domain, and this mutant had antibacterial ability against gram-positive bacteria Bacillus subtilis and B. cereus. This indicates that the conserved cysteine with different positions in WAP domain can have different effects on the antibacterial ability of Crustins. [Display omitted] • A novel type I crustin gene was obtained from Exopalaemon carinicauda (EcCru). • EcCru expression profiles were determined after prawns were challenged with pathogens. • Two mutations of conserved Cys in the WAP domain of EcCru lead to increase its antibacterial ability. [ABSTRACT FROM AUTHOR]

As a common aquatic pathogen, Vibrio parahaemolyticus can cause a variety of diseases of shrimp, especially acute hepatopancreatic necrosis disease (AHPND), which leads to great losses to the aquaculture industry around the world. However, the molecular mechanism of V. parahaemolyticus infection is still unclear. Neocaridina denticulate sinensis is a kind of small ornamental shrimp that is popular in aquarium trade, and due to its tenacious vitality, rapid growth, high reproductive capacity, it is very suitable to be developed as an animal model for basic research on decapod crustaceans. Thus, in this paper, transcriptomes of N. denticulate sinensis hepatopancreas with or without V. parahaemolyticus injection were explored. The results showed that a total of 23,624 genes with the N50 of 2705 bp were obtained. Comparative transcriptomic analysis revealed 21,464 differentially expressed genes between the V. parahaemolyticus infected and non-infected group, of which, 11,127 genes were up-regulated and 10,337 genes were down-regulated. Functional enrichment analysis suggested that many DEGs enriched in immune related pathways, including MAPK signaling pathway, Phosphatidylinositol signaling system, Chemokine signaling pathway, Phagosome and Jak-STAT signaling pathway and so on. Eight genes were selected randomly for qRT-PCR to verify the transcriptome sequencing results and the results showed the expression of these genes were consistent with the transcriptome results. Our work provides a unique and important dataset that contributes to the understanding of the molecular mechanisms of the immune response to V. parahaemolyticus infection and may further provide the basis for the prevention and resolution of bacterial diseases. • Transcriptomic analysis of hepatopancreas from Neocaridina denticulate sinensis challenged with Vibrio parahemolyticus. • A total of 21,464 DEGs were identified, including 11127 up-regulated genes and 10337 down-regulated genes. • Many DEGs were enriched in immune-related pathways, and expression of some PRRs and immune related factors were also altered. • Expression tendencies of 8 randomly selected genes were consistent with the result from transcriptomic analysis. [ABSTRACT FROM AUTHOR]

Trehalose, a nonreducing disaccharide, is present in a wide variety of organisms and plays a key role in many organisms under different stress conditions. In the study, the full-length cDNA sequence encoding trehalose-6-phosphate synthase (EcTPS) was obtained from Exopalaemon carinicauda. The complete nucleotide sequence of EcTPS contained a 2532 bp open reading frame (ORF) encoding a putative protein of 843 amino acids. The domain architecture of the deduced EcTPS contained a glycol_transf_20 domain and a trehalose_PPase domain. EcTPS mRNA was predominantly expressed in the hepatopancreas. The expression of EcTPS in the prawns challenged with Vibrio parahaemolyticus and Aeromonas hydrophila changed in a time-dependent manner. The function of EcTPS was also studied by double-strand RNA interference. The results showed that the knock-down of EcTPS increased the mortality of the Vibrio -challenged group and Aeromonas -challenged group compared with the control group. The present study provides some new insight into the immune function of the trehalose-6-phosphate synthase in prawns. • EcTPS was identified in E. carinicauda. • EcTPS was predominantly expressed in the hepatopancreas of E. carinicauda. • EcTPS was upregulated after the challenge with V. parahaemolyticus or A. hydrophila. • Knock-down of EcTPS increased the mortality of bacteria-challenged prawns. [ABSTRACT FROM AUTHOR]