| To ensure the environmental stability and food safety,land-based culturing has become a trend in the future development for filter-feeding shellfish.In the development of this farming model,the primary limiting factor is the lack of artificial compound feeds that can be mass-produced and are source-stable and nutritious.However,limited information is available regarding the nutrient requirements and metabolic mechanisms of filter-feeding shellfish.Therefore,based on the current diet formula from our research team,this study selected the optium protein source and carbohydrate/protein levels of formulated diet in pearl oyster Pinctada fucata martensii.Gas chromatography–mass spectrometry(GC-MS)-based metabolomics was performed and combined with multivariate analyses to explore the relevant metabolic differences in P.f.martensii and analyze the relevant metabolic mechanisms.Then,the optium vitamin D3 level of formulated diet in P.f.martensii was selected according to the trait of the pearl,and a control group(CG)was cultured in natural sea.Five diets were formulated,namely,D5-1,D5-2,D5-3,D5-4,and D5-5,with Chlorella sp.powder,Spirulina platensis powder,yeast powder,soybean meal,and corn gluten,respectively,as major protein sources.A feeding experiment was designed to evaluate the effects of formulated diets on the growth performance,immunity,and antioxidant and biomineralization capacity of juvenile P.f.martensii.In the experiments,the five groups were separately fed with D5-1,D5-2,D5-3,D5-4,and D5-5 diets.After 45 days of feeding,the pearl oysters fed on D5-1,D5-2,D5-3,and D5-4 diets showed significantly higher AGRs and protease and amylase activities than those fed on D5-5 diet(P < 0.05).The pearl oysters fed on D5-1,D5-2,D5-3,and D5-4 diets exhibited significantly higher activities of alkaline phosphatase(AKP),superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GPx)(P < 0.05).Significantly higher expression levels of SOD,GPx,CAT,heat shock protein(HSP)70,HSP90,nacrein,pif177,and pearlin mRNA were observed in pearl oysters fed on D5-1,D5-2,D5-3,and D5-4 diets relative to those fed on D5-5(P < 0.05).Results suggested the suitability of Chlorella sp.powder,S.platensis powder,yeast powder,and soybean meal as protein sources for development of formulated diets for P.f.martensii.Two experimental groups were separately fed with D5-3 and D5-5 diets indoors with those of oysters cultured with natural diet outdoors(CG).After 45 days of feeding,GC–TOF/MS-based metabolomics of the hepatopancreas was performed to assess the effect of different formulated diets on P.f.martensii combined with growth performance.D5-3 displayed significantly faster AGRs and higher survival rate than D5-5.In the metabolomic assay,125 metabolites were identified by mass spectrum matching with a spectral similarity value(SV)> 700,and 35 were considered significantly different metabolites(SDMs)(VIP > 1;P < 0.05).Pathway analysis indicated that the SDMs were involved in 34 pathways.Further integrated key metabolic pathway analysis showed that D5-5 exhibited lower capability for inositol phosphate metabolism,ascorbate and aldarate metabolism,phenylalanine metabolism,and tyrosine metabolism.This study suggests that yeast powder is a better protein source of formulated diet for pearl oysters than corn gluten.The survival rate of D5-3 was significantly higher than that of CG.The AGR of the total weight of D5-3 showed no significant difference from that of CG,but the AGRs of the shell length,shell height,and shell width of CG were significantly higher than those of D5-3.D5-3 showed significantly higher amylase activities than CG,and hexokinase and glucose-6-phosphate isomerase concentrations of the former were significantly lower than those of the latter.Metabolomics revealed 125 metabolites via mass spectrum matching with a spectral SV > 700 in the hepatopancreas,and 48 metabolites were considered significantly different between groups(VIP > 1 and P < 0.05).Pathway analysis results indicated that these significantly different metabolites were involved in 34 pathways.Further integrated key metabolic pathway analysis showed that compared with CG,D5-3 presented lower capabilities for cysteine and methionine metabolism,sulfur metabolism,and starch and sucrose metabolism.This study demonstrated that the formulated diet could be an excellent substitute for natural diet.However,the included nutrients levels are insufficient.Effective strategies should be developed to enhance the utilization of formulated diets.To achieve the optimum balance of dietary carbohydrates and proteins and explore the mechanisms behind the phenomenon,five isoenergetic and isolipidic diets(D6-1,D6-2,D6-3,D6-4,and D6-5)with different levels of carbohydrates and proteins were formulated.There were five experimental groups and two control groups(CG1 was fed with mixed powders of yeast and Chlorella sp.,and CG2 was cultured in natural sea).After 60-day feeding,the highest rates of survival and absolute growth appeared in D6-4.D6-4 exhibited significantly higher activities of amylase,protease,AKP,ACP,SOD,CAT,GPx,and phenoloxidase and significantly lower MDA content than D6-1,D6-2,D6-3,D6-5,and CG1.No significant differences were observed between D6-4 and CG2.Furthermore,the T-AOC of the pearl oysters in D6-4 was significantly higher than that in D6-1,D6-2,D6-3,and D6-5.On the basis of these results,the optimal balance of proteins and carbohydrates for pearl oysters was the D6-4 diet.Metabolomics-based profiling of the pearl oysters fed with D6-4 and D6-1 revealed 80 significantly different metabolites(VIP > 1 and P < 0.05).Furthermore,integrated key metabolic pathway analysis showed that changes in starch and sucrose metabolism,alanine,aspartate and glutamate metabolism,and glycine,serine and threonine metabolism resulting in different growth performance between group D6-4 and D6-1.Based on the optimum diet protein source and carbohydrate/protein levels of the previously optimized,different levels(0,500,1000,3000,and 10000 IU/kg)of Vitamin D3 were added to prepare five diets(D7-1,D7-2,D7-3,D7-4,and D7-5).Five experimental groups were land-based cultured,and CG was cultured in natural sea.After 30-days culturing,the survival rate of group D7-3 was the highest and was significantly higher than that of group D7-5 and CG(P < 0.05).Group D7-3 exhibited the highest AKP,ACP,CAT,SOD,and LYS activity,significantly higher AKP and CAT activities than those of group D7-4,D7-5,and CG(P < 0.05),significantly higher ACP activities than group D7-4 and CG(P < 0.05);significantly higher activities of SOD and LYS than the other groups(P < 0.05).After 137-days culturing,group D7-3 presented the highest retention rates,significantly higher than those of group D7-2,D7-4,D7-5,and CG(P < 0.05).Group D7-3 also exhibited the highest quality pearl rate,and significantly higher than those of group D7-1 and D7-5(P < 0.05).The nacre thickness and pearl deposition rate of pearls in group D7-3 and CG were significantly higher than those in other groups(P < 0.05),whereas no significant difference was observed between group D7-3 and CG(P > 0.05). |
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