| Eriocheir sinensis,commonly known as river crab and hairy crab,is an economical crab species widely cultivated in China in recent years.The mitten crab is rich in long-chain polyunsaturated fatty acids(LC-PUFAs)such as eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA),and was well received by consumers.At present,limited knowledge is known about the biosynthesis ability of PUFAs in mitten crab.The demands of PUFAs in mitten crab are mainly satisfied by fish oil in the diets.However,the price of fish oil has been increasing year by year,which has seriously affected the development of the mitten crab industry,there is an urgent need for new sources to substitute fish oil.Therefore,transcriptomic and proteomic analysis were used in the present study to investigate the effects of the substitution of fish oil by vegetable oils on mitten crab.In view of the fact that the main difference between different oil sources is the composition of fatty acids,we also analyzed the effects of different fatty acid diets on the hepatopancreas transcriptome and proteome of mitten crab.The biosynthesis ability of PUFAs in mitten crab has been a research hotspot in recent years.In this study,both in vitro and in vivo experiments were performed to investigate the effects of fish oil substitution on the genes expression associated with the biosynthesis of PUFA.And from the transcriptomic analysis,we find a pancreatic lipase,which is a key enzyme in the utilization of dietary lipid,the full length of pancreatic lipase is then cloned,and the expression pattern of pancreatic lipase in different tissue and different development larvae stage are analyzed,as well as the expression pattern in the crabs fed with different lipid diets.Experiment 1To illustrate the effects of dietary lipids on mitten crab and to improve the use of vegetable oils in the diet of the crabs,60 male juvenile Chinese mitten crabs were fed one of five diets for 116 days: fish oil(FO),soybean oil(SO),linseed oil(LO),FO + SO(1:1,FSO),and FO + LO(1:1,FLO).Changes in the crab hepatopancreas transcriptome were analyzed using RNA sequencing.There were a total 55,167 unigenes obtained from the transcriptome,of which the expression of 3030 was significantly altered in the FLO vs.FO groups,but the expression of only 412 unigenes was altered in the FSO vs.FO groups.The diets significantly altered the expression of many enzymes involved in lipid metabolism,such as pancreatic lipase,long-chain acyl-CoA synthetases,carnitine palmitoyltransferase I,acetyl-CoA carboxylase,fatty acid synthase,and fatty acyl ?9-desaturase.The dietary lipids also affected the Toll-like receptor and Janus activated kinase-signal transducers and activators of transcription signaling pathways.Our results indicate that in transcript level,the substituting fish oil with vegetable oils in the diet of mitten crabs might decrease the digestion and absorption of dietary lipids,fatty acids biosynthesis,and immunologic viral defense,and increase ?-oxidation by altering the expression of the relevant genes.Our results lay the foundation for further understanding of lipid nutrition in mitten crab.Experiment 2Since the transcriptomic analysis is mainly based on the mRNA,while the function of a gene is performed by the proteins translated according to the gene.Based on the experiment 1,the present study analyzed the effects of the substitute of fish oil by soybean oil and linseed oil on the hepatopancreas proteome in mitten crab.From the results,70 and 108 proteins were differentially expressed in SO and LO compared with FO.Many proteins relative to immunity,including hemocyanin subunit 6,cryptocyanin,and C-type lectin were significantly down-regulated in SO than FO.Expression of fatty acid synthase,long-chain fatty acid transport protein 4,acyl-CoA delta-9 desaturase,and fatty acid-binding protein 1,was higher in crabs fed FO than LO.In summary,at the protein level,the substitute of FO by SO can significantly reduce the abundance of the proteins related to immune response of mitten crabs.And comparing with LO,dietary FO can significantly increase the abundance of proteins related to fatty acid transport,thus providing a reason of the high content of EPA and DHA in crabs fed with FO.Experiment 3Through experiment 1 and experiment 2,we have preliminarily understood the effects of substitute of fish oil by vegetable oil on mitten crab.The main difference between different oil sources is the composition of fatty acids.In order to further optimize the study of the substitute of fish oil in mitten crab,it is necessary to further understand the different values of different fatty acid components on mitten crab,thus laying a foundation of the optimal ratio of different fatty acids in the diets of mitten crab.In the present study,two experiment diets were formulated using linoleic acid(n-6,LA)and ?-linolenic acid(n-3,LNA)as the main lipid sources.After feeding with the two diets for 107 d,we performed transcriptome analysis and proteomic analysis using label-free quantification of the hepatopancreas of mitten crabs fed with LA or LNA diet.A total of 186 proteins were differentially expressed with fold change ?1.5 or ?0.666.Among the 186 proteins,116 were upregulated and 70 were downregulated in the LA and LNA groups.A total of 18 proteins were identified to be related to lipid,carbohydrate,and protein metabolism,which illustrated the different role of LA and LNA in mitten crab,and an optimal ratio of LA and LNA should be found to maximize the role of the two fatty acid.Experiment 4Compared with LA and LNA,EPA and DHA are also important components in the diets of mitten crab.In order to find an optimal ratio of EPA and DHA in the diet of mitten crab,the different role of EPA and DHA is investigated according to the proteomic analysis of the hepatopancreas in the crabs fed with EPA and DHA diets.Similar to experiment 3,two diets were formulated in the present study using EPA and DHA as the two main lipid sources.The male crabs of mitten crabs were fed with two diets for 107 days.At the end of the experiment,the protein of hepatopancreas in each group of crabs was analyzed by proteomic analysis.A total of 198 proteins were identified,of which 46 were differential proteins.Compared with the EPA,there were 19 proteins up-regulated in the DHA group and 27 proteins down-regulated.Several proteins related to immunity,stress response and membrane,including lipopolysaccharide and beta-1,3-glucan binding protein,heat shock protein,tetraspanins-like protein CD63,V-type proton ATPase,sarco/endoplasmic reticulum calcium-ATPase,and sodium-potassium ATPase,were significantly differentially expressed between EPA and DHA.The results of the present study clarified the different functions of EPA and DHA in immunity,stress response and maintenance of cell membrane permeability in mitten crabs,and provided new insight in the study of the optimal ratio of EPA/DHA in the mitten crab diet.Experiment 5The biosynthesis ability of PUFAs in mitten crab has been a research hotspot in recent years.And several enzymes related to the biosynthesis of PUFAs have been obtained from mitten crab.To investigate the effects of the substitute of fish oil by vegetable oils on the biosynthesis of PUFAs,the expression pattern of genes related to the biosynthesis of PUFAs in the hepatopancreas tissues cultured with different fatty acids were analyzed using quantitative real-time PCR.The results of the present study showed that oleinic acid(18:1n-9,OA)could significantly increase the gene expression of ELOVL6,FAD6 and FAD9 in the hepatopancreas cultured in-vitro.The effects of different fatty acids on the gene expression of FAD6 and FAD9 were similar.Compared with EPA and DHA,LNA and OA significantly increased the expression of FAD6 and FAD9 genes.In conclusion,our results confirmed that EPA and DHA could inhibit the gene expression related to the synthesis of PUFA.Experiment 6Pancreatic lipase(PL)is an enzyme which mainly involve in the process of catalyzing the conversion of triglyceride into glycerol monoacylglycerol and free fatty acid,and is the first step in the utilization of lipid in the diet of mitten crab.The rapid amplification of cDNA ends technique(RACE)was used to obtain the full-length cDNA of PL in mitten crab(EsPL).And the expression pattern of pancreatic lipase in different tissue and different development larvae stage are analyzed,as well as the expression pattern in the crabs fed with different lipid diets in experiment 1.The full-length PL cDNA was 1 970 bp contained a 5'-untranslated region(UTR)and 3'-UTR of 372 bp and 224 bp,respectively.The sequence encoded a protein of 457 amino acids with a predicted isoelectric point of 4.65 and a predicted molecular weight of 51.066 kD.Three typical characteristics were found including catalytic triad,active site flap and nucleophilic elbow.Multiple sequence alignment showed higher similarity between EsPL and other species.A phylogenetic analysis revealed that the EsPL was in the same branch as most crustacean.Analysis of the tissue expression pattern of EsPL indicated that the EsPL was expressed in all tissues tested,but highest level was found in hepatopancreas,following by the intestines and thoracic ganglia.The expression of PL was gradually increased in zoea larvae stage I-III,while sharply decreased in IV,and was increased again in V.The expression of PL in megalopa was lower than zoea larvae.After feeding with different dietary lipid,the expression of PL showed that comparing with fish oil,soybean oil and linseed oil could increase the transcript levels of EsPL,and the highest transcript level was found in crabs fed with FLO.These results above showed obvious correlation between EsPL and dietary lipid,and provide important information on the utilization of different dietary lipid in mitten crab. |
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