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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 deacetylase (CDA, EC 3.5.1.41), belonging to a family of extracellular chitin-modifying enzymes, can catalyze the deacetylation of chitin. In this study, the full-length cDNA sequence encoding chitin deacetylase 1 ( EcCDA1 ) was obtained from Exopalaemon carinicauda . The complete nucleotide sequence of EcCDA1 contained a 1611 bp open reading frame (ORF) encoding EcCDA1 precursor of 536 amino acids. The domain architecture of the deduced EcCDA1 protein contained a signal peptide, a chitin-binding peritrophin-A domain (ChtBD2), a low-density lipoprotein receptor class A domain (LDLa) and a Polysacc_deac_1 domain. EcCDA1 mRNA was predominantly expressed in the gills. The expression of EcCDA1 in the prawns challenged with Vibrio parahaemolyticus and Aeromonas hydrophila changed in a time-dependent manner. The expression of EcCDA1 in the prawns challenged with V. parahaemolyticus was up-regulated at 12 h ( p  < 0.05), and significantly up-regulated at 24 h and 48 h ( p  < 0.01), and then returned to the control levels at 96 h post-challenge ( p  > 0.05). At the same time, the expression in Aeromonas -challenged group was significantly up-regulated at 12, 24 and 48 h ( p  < 0.01) and returned to the control levels at 120 h post-challenge ( p  > 0.05). Then, EcCDA1 was recombinantly expressed in Pichia pastoris and the purified recombinant EcCDA1 could not inhibit the growth of V. parahaemolyticus or A. hydrophila , which indicated that the CDA1 may play its biological activity in immune defense by deacetylation from chitin. [ABSTRACT FROM AUTHOR]

Chitinase belongs to the glycosyl hydrolases family 18 and plays key role in the development and pathogen resistance of crustaceans. In this study, the enzymatic characterization of chitinase 3C (EcChi3C) of Exopalaemon carinicauda was analyzed. In addition, we analyzed the expression profiles of EcChi3C at different tissues and different molting stages. In the all tested tissues, it was predominantly expressed in hepatopancreas, and then stomach, but poor in other tissues. In all tested molting periods, it was mainly expressed in intermolt and molting stages, but poor in other stages. The results of molting, mortality and the uropod ultrastructure of prawns after being injected with EcChi3C dsRNA were in accordance with those of the control group. In addition, there is no difference for endopodite morphology between the survival and dead individuals in experimental group. After being challenged with bacteria, the expression of EcChi3C was up-regulated significantly at 12 h and followed with a comeback at 96 h. These results suggest that EcChi3C is an important immune related gene but not a necessary gene in the molting process of E. carinicauda . [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]

The recently emerged CRISPR/Cas9 technology is the most flexible means to produce targeted mutations at the genomic loci in a variety of organisms. In Crustaceans, molt-inhibiting hormone (MIH) is an important negative-regulatory factor and plays a key role in suppressing the molting process. However, whether precise disruption of MIH in crustacean can be achieved and successfully used to improve the development and growth has not been proved. In this research, the complementary DNA (cDNA) and genomic DNA, including flanking regions of the MIH gene ( EcMIH ) of ridgetail white prawn Exopalaemon carinicauda, were cloned and sequenced. Sequence analysis revealed that EcMIH was composed of three exons and two introns. Analysis by RT-PCR showed that EcMIH mainly expressed in eyestalks. During different development periods, EcMIH was highest in juvenile stage and extremely low in others but adult prawns eyestalks. In addition, we applied CRISPR/Cas9 technology to generate EcMIH knock-out (KO) prawns and then analyzed the changes in their phenotypes. We efficiently generated 12 EcMIH-KO prawns out of 250 injected one-cell stage embryos and the mutant rate reached 4.8% after embryo injection with one sgRNA targeting the second exon of EcMIH . The EcMIH -KO prawns exhibited increased the body length and shortened the metamorphosis time of larvae from mysis larva to postlarva. Meanwhile, EcMIH -KO did not cause the health problems such as early stage death or deformity. In conclusion, we successfully obtained EcMIH gene and generated EcMIH -KO prawns using CRISPR/Cas9 technology. This study will certainly lead to a wide application prospect of MIH gene in prawns breeding. [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]

Arginine kinase (AK, EC 2.7.3.3) plays an important role in cells with high, fluctuating energy requirements. In invertebrates, AK is the major phosphagen kinase that modulates the energy metabolism. Here, the full-length cDNA sequence encoding arginine kinase (EcAK) was obtained from the Exopalaemon carinicauda. The complete nucleotide sequence of EcAK contained a 1068 bp open reading frame (ORF) encoding EcAK precursor of 355 amino acids. The genomic DNA fragment of EcAK with the corresponding cDNA sequence is composed of 4 exons and 3 introns. The domain architecture of the deduced EcAK protein contained an ATP-gua_PtransN domain and an ATP-gua_Ptrans domain. EcAK mRNA was predominantly expressed in the muscle. The expression of EcAK in the prawns challenged with Vibrio parahaemolyticus and Aeromonas hydrophila changed in a time-dependent manner. Then, EcAK was recombinantly expressed in Pichia pastoris and the purified recombinant EcAK had the same enzymatic characterization as AK from the muscle of Euphausia superba. In conclusion, EcAK may play the same biological activity in E. carinicauda as those from other crustaceans. Image 1 • EcAK was obtained from E. carinicauda and it is composed of 4 exons and 3 introns. • EcAK was predominantly expressed in muscle of E. carinicauda. • EcAK expression changed in a time-dependent manner after prawns were challenged with V. parahaemolyticus or A. hydrophila. • EcAK was recombinantly expressed in Pichia pastoris and the enzymatic characterization of recombinant EcAK was studied. [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]

Abstract The gC1qR is a ubiquitously expressed cell protein that interacts with the globular heads of C1q (gC1q) and many other ligands. In this study, one gC1qR homolog gene was obtained from Exopalaemon carinicauda and named EcgC1qR. The complete nucleotide sequence of EcgC1qR contained a 774 bp open reading frame (ORF) encoding EcgC1qR precursor of 257 amino acids. The deduced amino acid sequence of EcgC1qR revealed a 55-amino-acid-long mitochondrial targeting sequence at the N-terminal and a mitochondrial acidic matrix protein of 33 kDa (MAM33) domain. The genomic organization of EcgC1qR gene showed that EcgC1qR gene contained five exons and four introns. EcgC1qR could express in all of the detected tissues and its expression was much higher in hepatopancreas and hemocytes. The expression of EcgC1qR in the hepatopancreas of prawns challenged with Vibrio parahaemolyticus and Aeromonas hydrophila changed in a time-dependent manner. The expression of EcgC1qR in prawns challenged with V. parahaemolyticus was up-regulated at 6 h (p < 0.05), and significantly up-regulated at 12 h and 24 h (p < 0.01), and then returned to the control levels at 48 h post-challenge (p > 0.05). At the same time, the expression in Aeromonas -challenged group was significantly up-regulated at 6, 12 and 24 h. The recombinant EcgC1qR could inhibit the growth of two tested bacteria. In addition, we successfully deleted EcgC1qR gene through CRISPR/Cas9 technology and it was the first time to obtain the mutant of gC1qR homolog gene in crustacean. It's a great progress to study the biological function of gC1qR in crustacean in future. Graphical abstract Image 1 Highlights • The expression profiles of EcgC1qR was were demonstrated. • EcgC1qR was up-regulated after challenge with V. parahaemolyticus or A. hydrophila. • EcgC1qR was successfully deleted through CRISPR/Cas9. [ABSTRACT FROM AUTHOR]