Preliminary Study On The Growth Regulation Mechanism Of Marsupenaeus Japonicus Based On Multi-omics Analysis

As one of the largest groups of invertebrates,crustaceans have great economic value.Growth traits are one of the most important traits of economical crustaceans,which directly impact the yield and economic benefits of cultured species.However,the biological mechanism related to the growth traits of crustaceans has not been fully studied,especially in decapod animals such as Marsupenaeus japonicus.In this study,zero-exchange water aquaculture model of M.japonicus was constructed.The growth characteristics of M.japonicus under this aquaculture model was studied.The physiological,intestinal flora,gene level,protein level and metabolite level of M.japonicus with significant differences in body weight at different growth stages were studied,aiming to analyze the growth regulation mechanism of M.japonicus.It is expected to provide theoretical basis for perfecting the crustacean growth traits.The main results were as follows:In this study,the full-sib family of M.japonicus were used as the experimental material,and M.japonicus was cultured under zero-exchange water aquaculture model.M.japonicus could survive for more than 90 days under this model.During the aquaculture experiment,the physical and chemical factors of the water was stable,pH was(8.13±0.07)?(8.61±0.01),NH₄⁺-N was 0?(0.769±0.124) mg/L,NO₂^--N was 0?(0.167±0.034) mg/L,and NO₃^--N was 0?(1.267±0.120) mg/L.The growth of M.japonicus was roughly divided into two growth stages:30?50 days of age was the fast growth stage,and the specific growth rate and relative growth rate were the largest,4.28?9.51 %/day and 53.46%?158.83%,respectively;50?100 days of age was in the steady growth stage,the specific growth rate was 2.15?2.37 %/day,and the relative growth rate was 24.00%?26.71%.This study confirmed the feasibility of the zero-exchange water aquaculture model of M.japonicus for the first time and obtained the growth characteristics of M.japonicus,which provided a theoretical basis for the production practice of M.japonicus.In this study,the physiological indicators and intestinal flora of M.japonicus with significant differences in body weight at different growth stages were analyzed.The?-Amylase (AMS) activity of the FG group was higher than that of the SG group at 40 and 100 days of age.The Lipase (LPS) activity of the FG group was higher than that of the SG group at each growth stage,and the Trypsin (TRS) activity was the opposite of the LPS.The Lysozyme (LZM) activity of the FG group was higher than that of the SG group at each growth stage.The Catalase (CAT) and Total Superoxide Dismutase (T-SOD) activities of the FG group were higher than those of the SG group at 40 days of age,whereas opposite was at 100 days of age.Analysis 12 samples of intestinal flora,Q20 and Clean Reads were all above 73.63% and 90.29%,respectively.The OTUs in the FG group was higher than that in the SG group,and 1,484 OTUs were shared by the two groups.There was no significant difference in Alpha Diversity index between FG and SG groups(p>0.05).The abundance of Firmicutes in the FG group was higher than that in the SG group;the abundance of Vibrio in the FG group was lower than that in the SG group.The function of carbohydrate metabolism in the FG group was higher than that in the SG group(p<0.05).According to the results of analysis of body composition of whole shrimp,the water content of FG group was lower than that of SG group (p<0.05),and the crude protein content was higher than that of SG group (p>0.05).The above results indicate that the FG group of M.japonicus may synthesize more protein and achieve growth advantages by enhancing the absorption and utilization of carbohydrates and fat,as well as reducing some unnecessary energy consumption.In this study,a transcriptomics analysis of M.japonicus from the same family in different growth stages with significant differences in body weight was carried out.A total of 111.64 Gb Clean Data and 10,194 genes were obtained.Compared with the next-generation sequencing,the full-length transcriptome had a longer average length of genes and a higher annotation rate.The fast growth stage with high specific growth rate identified more DEGs than the steady growth stage with low specific growth rate.215 DEGs were shared by the two growth stages,106 DEGs showed the same trend in the two growth stages,and 109 were the opposite.The DEGs with consistent expression trend in the two growth stages were verified by qPCR,and the results were consistent with the transcriptome,confirming that the transcriptome data was reliable.The functions of DEGs obtained in the two growth stages were conservative,and 12 significant enrichment pathways including glycolysis were shared by the two growth stages.This study reports for the first time that the Hippo signaling pathway was directly involved in the growth of crustaceans,and the key gene Mj14-3-3-like in this pathway was cloned.Its ORF was 741 bp,encoding 246 AA.Mj14-3-3-like was down-regulated in the FG group,and the expression level was the lowest in the intermolt stage,suggesting that Mj14-3-3-like may regulate the growth of M.japonicus.In this study,a proteomic analysis of M.japonicus from the same family in different growth stages with significant differences in body weight was carried out.A total of 1,720 proteins were identified.52 and 70 DEPs were identified in the fast growth stage and steady growth stage of M.japonicus,respectively.10 DEPs were shared by the two growth stages and each DEP had the same expression trend in the two growth stages,suggesting that they may play an important role in the growth of M.japonicus.According to the results of integrated analysis of proteome and transcriptome,ten and seven DEPs/DEGs were screened in the fast growth stage and steady growth stage,respectively.Most of them had the same expression trend.Proteome,transcriptome and qPCR all confirmed that the expression of MHC-la and MHC-1b were down-regulated in the two growth stages of the FG group,suggesting that they may negative regulate the growth of M.japonicus.The ORFs of Mj14-3-3-epsilon-like,MjGP1 and MjGPD1 were 822,1,671,and 1,098 bp,respectively,encoding 273,556,and 365 AA.Mj14-3-3-epsilon-like was down-regulated in the FG group,and was at a lower level in the intermolt stage;Mj GPI and Mj GPD1 were up-regulated in the FG group,and had a higher level in the intermolt stage;these results suggest that they may regulate the growth of M.japonicus.In this study,a metabonomics analysis of M.japonicus from the same family in different growth stages with significant differences in body weight was carried out.A total of 2,342 and 1,811 metabolites in positive and negative modes were identified,respectively.In positive mode,229 and 275 differential metabolites were identified in the fast growth stage and steady growth stage,respectively.In negative mode,149 and 85 differential metabolites were identified in the fast growth stage and steady growth stage,respectively.In positive and negative modes,there were 75 and 30 differential metabolites shared by the two growth stages,respectively,and the expression trend of each differential metabolite in the two growth stages were consistent,suggesting that they may play an important role in the growth of M.japonicus.The differential metabolites,pyruvate,etc.,were detected by ELISA kit,and the results were consistent with the metabolome,confirming that the metabolome data was credible.In addition,the content of pyruvate in the FG group was also lower than that in the SG group in other growth stages.These results suggest that pyruvate was a potential growth biomarker of M.japonicus.In this study,the phenomenon of relatively slow growth and blue body color of M.japonicus cultured in plastic membrane ponds was analyzed based on transcriptomics.A transcriptomics analysis was performed on M.japonicus from the traditional culture model and the plastic membrane pond culture model.A total of 60,780 genes were obtained.1,005 DEGs were identified in gills,of which 427 were up-regulated and 578 were down-regulated;115 DEGs were identified in hepatopancreas,of which 41 were up-regulated and 74 were down-regulated.These results suggested that the gills were more sensitive to the external environment changes than the hepatopancreas.Many essential genes including actin and amylase,were inhibited,which may be the reason for the slow growth of the M.japonicus cultured in plastic membrane ponds.The higher expression of Crustacyanin subunit A and C in plastic membrane ponds may be the reason for the blue body color.This study provided a theoretical basis for perfecting plastic membrane pond aquaculture model of M.japonicus.