Effects Of Dietary Restriction On Metabolism Of Hemolymph And Individual Longevity Of Bombyx Mori

Limiting calorie by reducing food has been shown to be effective in delaying aging and prolonging life span in many animal models.Scientists have done a lot of research on the mechanism of dietary restriction to prolong life span,including put forward various hypotheses and found out some related genes and regulatory pathways.But there are still many unknown links to be further studied.Bombyx mori as an experimental animal with easy breeding,multiple offspring,clear genetic background and abundant mutation resources,has its own unique advantages in delaying aging and prolonging life span.The larvae of the bivoltine strain "Qiufeng" of silkworm have limited markings,the females are common markings,and the males are vegetarian silkworms,so it is easy to distinguish males from females.Based on the model of silkworm variety Qiufeng,the effects of dietary restriction on hemolymph metabolism physiology and expression level of life-related genes in silkworm larvae were studied by using qRT-PCR and metabolome analysis techniques.The relationship between dietary restriction and life-related genes was explored,and the physiological and biochemical mechanism of dietary restriction for delaying senility was explored.The main results are as follows.(1)Dietary restriction significantly reduced the individual weight of silkworm and prolonged the life span of silkworm.Taking Qiufeng,a bivoltine silkworm strain,as an object,through preliminary preparatory test,free-feeding group(AL group)and restricted-feeding group(DR group)were set up.The AL group was given mulberry twice a day for 24 hours,while the DR group was given mulberry once a day for 16 hours,and the free-feeding time was maintained for 8 hours.The results showed that the feed intake of DR group was 61.03% of that of AL group;compared with AL group,the individual weight of5 th instar larvae of DR group was 36.41%,the average life span of males and females was107 hours(15.46%)longer,reaching a significant level(p < 0.05),and the average life time of males and females was 24 hours(18.18%)longer.There was no significant change in the number of oviposition of females in the two groups(p > 0.05),and the mating of males was normal.(2)Dietary restriction affects the expression of three life-span-related genes in silkworm,Bombyx mori.The homologous genes Bmchico,Bmrpd 3 and BmAkh2 of threelife-related genes in Drosophila melanogaster were selected and primers were designed to extract total RNA from different tissues of fifth instar day-3 larvae in DR group and AL group respectively.The expression level of these genes was analyzed by qRT-PCR.The results showed that compared with AL group,Bmchico in DR group decreased significantly in female silkgland,fat body and male silkworm epidermis,while Bmchico in other tissues was not significantly down-regulated or even up-regulated in some tissues;Bmrpd3expression was different in different tissues of two groups of silkworm;BmAkh2 was up-regulated in male silkworm epidermis of DR group,but in other tissues.Different degrees of down-regulation indicated that dietary restriction could induce or inhibit the expression of life-related genes.(3)Effect of dietary restriction on physiological and biochemical level of silkworm.The haemolymph of the 5th instar larvae of DR group and AL group was extracted for 3days.The activities of superoxide dismutase and catalase,the contents of malondialdehyde,soluble protein and trehalose were measured.The results showed that compared with AL group,the activities of superoxide dismutase and catalase in hemolymph of DR group were higher,the content of malondialdehyde decreased,the content of soluble protein decreased,and the overall level of trehalose decreased.It suggests that dietary restriction may delay aging by improving the body's antioxidant capacity and reducing the level of energy metabolism.(4)Effects of dietary restriction on hemolymph metabolites of silkworm.LC-MS metabonomics analysis was carried out on the fifth instar day-3 larvae haemolymph of larvae from DR and AL groups.The results showed that 74 kinds of differential metabolites(26 up-regulated and 48 down-regulated in male DR group)and 61 kinds of differential metabolites(22 up-regulated and 39 down-regulated in female DR group)were screened in the comparative analysis of the same sex,among which the main differential metabolites were amino acids,organic acids and amines.In DR group,the levels of 17 kinds of amino acids such as histidine changed significantly,and the rearrangement of amino acids accounted for;the contents of tricarboxylic acid cycle intermediates such as citric acid,malic acid,alpha-ketoglutarate increased;the levels of spermine,putrescine and spermidine decreased.Studies have shown that dietary restriction can improve the antioxidant level of silkworm,change the blood sugar level,make significant changes in the hemolymph metabolites of silkworm,thereby prolonging the life of silkworm.The results provide a good animal model for the study of human diet restriction,provide experimental basis forthe study of moderate diet restriction contributing to anti-aging,and lay a foundation for the follow-up study of the theoretical basis of dietary restriction and anti-aging mechanism.