Molecular Mechanism Of The Homeostasis Of Hemolymph Microbiota Medicated By Spp38 In Scylla Paramamosain

Mud crab（Scylla paramamosain）is one of the major economic crab species cultured in China,especially in Guangdong Province.The previous studies showed that the hemolymph of mud crab is harbored by a certain number of microorganisms that may be important in the physiological homeostasis in mud crabs.The aims of this study were to study the molecular mechanism of the Spp38 gene in controlling the homeostasis of hemolymph microorganisms and to elucidate the relationship between the production of ROS regulated by Spp38 and the homeostasis of hemolymph microorganisms.The results of this study were as follows:1.The changes of ROS content upon different environmental stressors（NH₄Cl,WSSV,p H,salinity,temperature,and light）were investigated and the results showed that these stressors significantly affect the contents of ROS in the hemolymph of mud crab.Moreover,the changes of hemolymph microbial structure under the increase（WSSV infection and p H 10.5）or reduction(high temperature of 35℃and high ammonia nitrogen of 80 mg·L^-1of NH₄Cl)of ROS were conducted.Based on the 16S r DNA full-length sequencing,the phyla Proteobacteria,Firmicutes,Actinobacteria,and Bacteroidetes are found to be dominant in the hemolymph of mud crab.The hemolymph microbial composition was significantly affected by different environmental factors,and the microbial diversity was negatively correlated with changes in the content of ROS.Totally,52 core microial genera were found to be unaffected by changes in environmental factors in hemolymph the mud crab.At the species level,Bacillus pseudofirmus was respected to the changes of NH₄Cl,while Staphylococcus sp.was to temperature,Trichococcus pasteurii was to WSSV,and Alishewanella sp.was to p H.Additionally,three species,Vibrio parahaemolyticus,Exiguobacterium profundum,and Acinetobacter johnsonii,were isolated from the hemolymph of mud crab using the culture-dependent approach.2.The cloned c DNA sequence of the Spp38 gene is 2691 bp in length,including the conserved Thr-Gly-Tyr（TGY）double phosphate structural motif and Ala-Thr-Arg-Trp（ATRW）substrate-binding site.Spp38 has the highest similarity with the p38 from the Chinese mitten crab and has high conservation with those from other crustaceans.Spp38 is expressed in various tissues,with the highest level in the muscle and hepatopancreas.Under the stimulation of V.parahaemolyticus（Vp）and lipopolysaccharide（LPS）,Spp38 was significantly up-regulated in the hemocytes and hepatopancreas of mud crab.The dual luciferase-reporter gene showed that Spp38 can activate the expression of anti-lipopolysaccharide factors-ALFs（SpALF1-6）.After knocking down the expression of Spp38 by RNAi,the SpALFs and dioxidase（SpDuoxs）and the content of ROS were significantly decreased in the hemolymph,while the number of microorganisms was significantly increased（P<0.05）.Furthermore,the bacteria clearance ability was significantly reduced in the hemolymph of Spp38-knocked down mud crab after challenge with Vp.3.Recombinant expression and purification of GST-Spp38 protein and screening by GST pull-down experiment to obtain leucine zipper protein 2,transcription factor ENY2,mitogen-activated protein kinase 14B,dual-specificity mitogen-activated protein kinase 6,mitosis Pro-activated protein kinase 14C and other potential interacting proteins with Spp38.After the knockdown of Spp38,the transcriptome sequencing analysis revealed that the transcription factors ATF-b,Myb/SANT-like,and oxidase-related genes may interact with Spp38 protein NADPH.Furthermore,the microbiota colonizing the hemolymph of S.paramamosain was significantly changed,specifically the members of Saprospiraceae,Marivivens,Vibrio,Hydrogenophilus,and Aeromonas,indicating that the hemolymph microbiota of mud crab is modulated by Spp38.4.The promoter sequences of SpDuox1 and SpDuox2 are 1884 bp and 2823 bp in length,respectively.The predicted binding sites of transcription factors such as FOXO3a,FOXO1,STAT4,c-Jun,c-fos,c-Myb,c-Myc,p53,ATF3 and ATF2.Then the DNA pull-down combined with LC-MS/MS was used to identify the related proteins,such as serine/threonine-protein kinase BRSK1,transcription factor AP-2,transcription mediator 1.The SpATF2 c DNA sequence is 2747 bp in length and encodes 711 aa,which consists of a typical Cys2 His2 zinc finger domain at the N-terminus,and a more conservative leucine zipper（b ZIP）domain responsible for the formation of dimers at the C-terminus.SpATF2 is highly expressed in the muscle and hepatopancreas.Upon Vp or WSSV challenge,SpATF2 was significantly up-regulated in the hemocytes and hepatopancreas of mud crab.The results of Bio-Layer Interferometry analysis showed that SpATF2 protein can interact with Spp38 protein.The dual-luciferase reporter gene showed that SpATF2 can activate the expression of SpDuox1 in mud crab.After knocking down the expression of SpATF2,the expression of SpDuox1 and the production of reactive oxygen species（H₂O₂）were reduced,whereas the number of microorganisms in the hemolymph was significantly increased.Furthermore,the bacteria removal ability was significantly reduced in the hemolymph of SpATF2-knocked down mud crab after challenge with Vp.The results of this study indicated that the Spp38-SpATF2-SpDuox1 signal cascade is involved in the regulation of microbial homeostasis and the process of antibacterial immunity in the hemolymph of mud crab.In order to theoretically gain a new understanding of the regulation mechanism of the hemolymph microbial homeostasis of mud crab,enrich and perfect the theory of invertebrate immune system.