Transcriptome analysis of Neocaridina denticulate sinensis under copper exposure.

Neocaridina denticulate sinensis is a small freshwater economic shrimp, as well as excellent laboratory model for their short life cycle and easy availability. However, the response of N. denticulate sinensis to pervasive copper pollution in aquatic environments has not been deeply investigated yet. Herein, we preformed Illumina sequencing technology to mine the alterations of cephalothorax transcriptome under 2.5 µmol/L of Cu2+ after 48 h. 122,512 unigenes were assembled and 219 unigenes were identified as significantly differentially expressed genes (DEGs) between control and Cu2+ treatment groups. Functional enrichment analysis revealed that DEGs were mostly associated with immune responses and molting, such as endocytosis, Fc gamma R-mediated phagocytosis and chitin metabolic process. Seven genes were chosen for qPCR verification, and the results showed that the transcriptome sequencing data were consistent with the qPCR results. This is the first report of transcriptome information about N. denticulate sinensis. These results provided a direction for the future research of resistance to Cu2+ in this shrimp, and simultaneously enriched gene information of N. denticulate sinensis.

CRISPR/Cas9-mediated deletion of ß, ß-carotene 9', 10'-oxygenase gene (EcBCO2) from Exopalaemon carinicauda.

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. ß, ß-carotene 9', 10'-oxygenase (BCO2) is an important enzyme during ß-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.

Cloning of a trehalose-6-phosphate synthase gene from Exopalaemon carinicauda and its expression response to bacteria challenge.

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.

Biological function of a gC1qR homolog (EcgC1qR) of Exopalaemon carinicauda in defending bacteria challenge.

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.