Effects Of Diets Supplemented With Cholesterol On The Development Of Apis Mellifera Ligustica

Lipids play an important role in the growth and development,production performance and life span of Honeybee.Cholesterol is one of the sterols in Lipids,it is not only the component of bio-membrane but also the precursor of ecdysone synthesis,which plays an important role in the growth and development of Honeybee.If the bees lack cholesterol in the body,their development will be delayed and their life expectancy will be shortened.Insects can’t make cholesterol by themselves,they can only get it from food.At present,there are few studies on the effect of Dietary Cholesterol supplementation on the growth and development of Honeybee.In this experiment,Apis mellifera ligustica(Am)was used as the research object.The larvae were fed diets with different levels of cholesterol,and the techniques of histomorphology and molecular biology were used,the purpose of this study was to investigate the effects of cholesterol on the development of Am by detecting the growth and development,the morphological development,ovarian development and expression of related genes.We collected 1 440 1-day-old larvae of Am and divided them into 6 groups random Ly,each group had 10 replications,each replications had 24 larvae.One group was fed basal diet with 0.00% cholesterol,the other five groups were fed basal diet with 0.05%,0.10%,0.25%,0.50% and 1.00% cholesterol,respectively.The larvae of Am were reared in a constant temperature and humidity incubator(temperature 34.5 °C,relative humidity 90%)until emergence.The larval pupation weight,pupation rate,newborn weight,emergence rate,developmental duration and morphological index of Larvae were measured.Samples of 3,4and 5 day Old Larvae,white-pupae(Pw),pink-pupae(Pp)and new emergely bee(NEW)were collected from each group,detection of gene expression and other molecular biological indicators.The results are as follows:Effects of dietary cholesterol supplementation on the growth and development of Am: 1)the pupation rate and eclosion rate of each experimental group increased firstly and then decreased with the increase of dietary cholesterol level,there was no significant difference compared with the Control Group(P > 0.05),but the pupation rate increased significantly compared with Tc1.00% group(P < 0.05),2)compared with the control group,when the dietary cholesterol level was 0.25% and 0.50%,the larval weight and newborn weight of Am were significantly increased(P < 0.05),3)compared with the control group,the development period of the experimental groups with cholesterol was significantly shorter(P < 0.05),4)compared with the control group,the width of forewing increased significantly when the level of dietary cholesterol was 0.05%(P < 0.05).0.25%-1.00% cholesterol level significantly increased the length of femoral joint(P < 0.05).The length of Tibiae,Basal Tarsus and Basal tarsus width were significantly shortened(P < 0.05),the length of body,the length of forewing and the number of cubitus veins of forewing were not affected significantly(P >0.05),5)dietary cholesterol significantly increased the number of ovarian tubules and ovarian development.Compared with the control group,the number of ovarian tubules increased significantly when dietary cholesterol level was 0.25%-1.00%(P < 0.05),suggesting that the addition of cholesterol in the Diet could promote the development of honeybee toward the Queen.RNA transcriptome sequencing results show that,the differential genes produced by adding cholesterol to bee diets were enriched in biosynthesis of insect hormones pathways,and the CYP302A1,CYP314A1 and CYP315A1 genes were up-regulated in the biosynthetic pathway of insect hormones,promote the synthesis of ecdysone.Effects of diets supplemented with cholesterol on the gene expression of Am: 1)cholesterol has a significant effect on the expression of CYP450 family genes,which are key regulators of ecdysone synthesis(P < 0.05),When the content of cholesterol in bee diet is0.50%,the expression of CYP302A1,CYP314A1 and CYP315A1 in Larva and pupae of Honeybee increased significantly(P < 0.05),which suggested that cholesterol had a strong effect on the ecdysone synthesis in Larva and pupae of Am,2)the ecdysone Response Gene(E74)is the downstream gene of ecdysone which regulates the growth and development of honeybee.Compared with the control group,the level of added cholesterol in diet was 0.5%of the experimental group significantly increased the expression(P < 0.05),it is suggested that ecdysone regulates E74 and affects grade differentiation,3)Vitellogenin gene(Vg)is closely related to egg laying performance of honeybee.Cholesterol can significantly increase the expression of Vg(P < 0.05).The dietary cholesterol level was 0.50% increased the expression of Vg significantly(P < 0.05),which suggested that adding cholesterol in diet could enhance the oviposition performance of honeybee.The results showed that the high expression of CYP302A1,CYP314A1,CYP315A1,which are key genes of ecdysone synthesis pathway,was affected by dietary cholesterol supplementation,then the downstream gene E74 and other related genes Vg were regulated to influence the growth and development of Honeybee Larvae and the morphology of new bee.By comparison,it was found that 0.50% cholesterol could promote the pupation rate,eclosion rate,body weight,number of ovarian tubes and wing width of Am,which was beneficial to the growth and development of Am.