Homeostatic Regulation Of Intestinal Microbiota In The Kuruma Shrimp Marsupenaeus Japonicus

The shrimp culture industry is one of important mainstay industries at the coastal region in China. Shrimp culture was inflicted heavy losses because of shrimp diseases since 1990’s. To solve the diseases trouble of shrimp culture, scientists work hard on host (shrimp immune defense), pathogens (including pathogen detection, pathogenesis and prevention) and environment (culture environment control) and achieved great progress. However, the old trouble of diseases has not been solved, and novel diseases would appear constantly in the shrimp aquaculture. If we study the integrity of host-pathogens-environment, the novel strategy for disease control would be discovered. Research for the function and homeostasis regulation of bacterial community in shrimp intestine is the bridge for relationship of host-pathogens-environment, because bacteria in shrimp intestine are related with both host immunity and environment. Therefore, the homeostatic regulation of intestinal bacterial community was studied in kuruma shrimp Marsupenaeus japonicus.1. Dual oxidases participate in the regulation of intestinal microbiotic homeostasis in the kuruma shrimp M. japonicusIntestinal innate immune response is an important defense mechanism of animals and humans against external pathogens. In the study of Drosophila, reactive oxygen species (ROS) and antimicrobial peptides have been reported to play important roles in eliminating pathogenic bacteria. However, how to maintain the ROS and microbiota homeostasis in crustacean intestine needs to be elucidated. A novel dual oxidase (named MjDUOXI) was cloned and identified in the kuruma shrimp M. japonicus. MjDUOX2 is a transmembrane protein with an N-signal peptide region (19 aa) and a peroxidase homology domain (PHD,554 aa) in the extracellular region; seven transmembrane regions; and three EF (calcium-binding region) domains (110 aa), a FAD-binding domain (104 aa), and a NAD-binding domain (156 aa) in the intracellular region. The novel MjDUOX2 exhibits a relatively low similarity (26.84% identity) to a previously reported DUOX in the shrimp (designated as MjDUOX1).Two kinds of MjDUOX mRNA were widely distributed in the hemocytes, heart, hepatopancreas, gills, stomach and intestine. Oral infection of the shrimp with Vibrio anguillarum (V. anguillarum) upregulated the mRNA expression of MjDUOXs and increased the ROS level in the intestine. Knockdown of MjDUOXs by RNA interference (RNAi) decreased the ROS level, increased the bacterial count in the intestine, and decreased the survival rate of the MjDUOX-RNAi shrimp. These results suggest that MjDUOXs can generate ROS and play an important role in intestinal microbiota homeostasis.2. Catalase eliminates reactive oxygen species and participates in regulation the homeostasis of intestinal microbiota of shrimpReactive oxygen species include peroxide, superoxide anion and hydroxyl radical. As one of strong oxidants, ROS have broad bactericidal spectrum, including bacteria, virus and fungi. Recent studies show that ROS are involved in regulation for cellular proliferation, differentiation and apoptosis and considered as a new second messenger. Meanwhile, excessive ROS can lead to oxidative damages of biomacromolecule and biological tissue. Our above study finds that DUOX could produce ROS after the intestinal bacteria stimulation. However, it is not clear how the excessive ROS can be eliminated. In this study, we cloned a novel catalase (named MjCAT) in kuruma shrimp, M.japonicus. The tissue distribution of MjCAT was detected using semi-quantitative PCR method detection. The result showed that MjCAT mRNA was widely distributed in hemocytes, heart, hepatopancreas, gills, stomach and intestine. After shrimp was challenged with pathogene-V. anguillarum via oral infection, the expression level of MjCAT was upregulated, and the enzyme activity was increased in the intestine. ROS level was also increased in the intestine at early time after oral infection and recovered rapidly. When MjCAT was knocked down by RNAi, high ROS level maintained longer time, and the number of bacteria number was declined in the shrimp intestinal lumen than those in the control group, but the survival rate of the MjCAT-RNAi shrimp was declined. Further study demonstrated that the intestinal villi protruded from epithelial lining of the intestinal wall were damaged by the high ROS level in MjCAT-knockdown shrimp. These results suggested that MjCAT participated in the intestinal host-microbe homeostasis by regulating ROS level.3. Molecular mechanism of intestinal bacteria flora recognition and homeostasisA DUOX plays an important role in homeostasis of intestinal bacterial community in Drosophila. However, it is not clear how host recognizes intestinal bacteria and how activates downstream signaling pathway induced by intestinal bacteria. In this research, we screened 5 Guanosine-binding protein coupled receptors (GPCRs) gained by transcriptome sequencing and found one GPCR related with MjDUOXs expression. This GPCR was named by MjMeGPCR according to the structural domain. When MjMeGPCR expression was interfered by RNAi, MjDUOXs expression and ROS level decreased. On the contrary, the expression of MjDUOXs and ROS level increased after .M/MeGPCR over-expression. Additionally, MjMeGPCR could bind to several bacteria. We also studied the signal pathway downstream moleculars activating transcription factor-2 (ATF2) and designed as MjATF2 and mitogen-activated protein kinase (MAPK) p38 (Mjp38). After inhibiting the cytoplasm Ca2+ reflux to the endoplasmic reticulum, ROS level increased. The transcription of MjDUOXs was also declined through inhibiting the activation of Mjp38 or MjATF2-RNAi. Meanwhile, ROS generation was hindered. These results showed that intestinal bacteria could be recognized by MjMeGPCR, and regulated MjDUOXs expression by p38 and ATF2. Additionally, enzymatic activity of MjDUOXs was regulated by Ca2+ in intracellular. Therefore, the intestine generated sufficient ROS and regulated intestinal bacteria homeostasis.