| Both queen and worker bees develop from diploid fertilized eggs,but the food and developmental space they receive during development make them very different not only in appearance and morphology,but also in reproductive capacity,longevity,and behavior,which is the phenomenon of caste differentiation in honey bees.It has been shown that the level of DNA methylation during the larval stage of the honey bee can regulate whether the bee eventually grows into a queen or worker bee.Folic acid is a water-soluble B vitamin that has been associated with DNA methylation due to its involvement in one-carbon metabolism.However,the effect of folic acid on honey bee caste differentiation has not been reported.In this study,we used Apis mellifera ligustica as an object of study.The larvae of honey bees were fed with different doses of folic acid and cultured indoors until they emerged from the colony.The relevant physiological and biochemical indices at different periods,as well as the final developmental and morphological indices were measured to explore the effects of folic acid on honey bee caste differentiation and its molecular mechanisms,with a view to enriching the theoretical basis of honey bee caste differentiation and providing a reference for queen breeding in beekeeping production.In this experiment,2,160 female 2-day-old honey bee larvae were selected from sister colonies and divided equally into six groups of five replicates each,with 72 larvae per replicate.One group was the control group,fed a basal diet without additional folic acid,and the remaining five groups were the experimental groups,with folic acid levels of 0.02%,0.04%,0.06%,0.08%,and 0.10%,respectively.The larvae of each group were reared at a constant temperature of 34.5 °C ± 0.5 °C and relative humidity of 90% ± 5% in an indoor incubator,where they were reared through the larval and pupal stages until they emerged as new bees.Samples of 3-day-old(3 d),4-day-old(4 d)and 5-day-old(5 d),white-eyed pupae(Pw),pink-eyed pupae(Pp)and newly emerged bees(NE)were randomly selected from each group and stored frozen in an ultra-low temperature refrigerator at-80 °C for the determination of folate metabolism indexes;DNA methylation-related gene expression and enzyme activity indexes.To determine the weight of pre-pupae,weight of new bees,pupation rate,eclosion rate and development time of each group,and to determine the morphological index and number of ovarian tubules of newly emerged bees in each group.After statistical analysis,the following results were obtained:Effect of folic acid addition to the diet on folate metabolism in honey bees: 1)the level of folic acid(FA)added to the larvae of the test group at 0.04% was significantly higher than that of the control group at 3,4 and 5 days of age(P < 0.05),suggesting that the appropriate amount of folic acid added to the basal diet promotes the deposition of folic acid in the bee;2)5-Methyltetrahydrofolate(5-MTHF)is a product of FA catabolism.The content of 5-MTHF in the 0.04% test group was significantly higher than that in the control group when the larvae were reared to 3,4 and 5 days old(P < 0.05),suggesting that FA metabolism was more active in the larvae of the 0.04% test group;3)tetrahydrofolate(THF)is also a downstream product of FA metabolism,and the addition of folic acid to the basal diet had no significant effect on the THF content of larvae in each test group(3,4 and 5 days old)(P > 0.05).4)DHFR,TS and MTHFR are key enzymes of folate metabolism,and the 0.04% test group significantly increased the expression of DHFR,TS and MTHFR genes in the larval and pupal stages compared with the control group(P < 0.05),and significantly increased the MTHFR enzyme activity in the larval stage(P < 0.05)and DHFR enzyme activity in the pupal stage(P < 0.05),suggesting that the addition of 0.04% folic acid could promote FA metabolism in honey bees at different times.The effect of folic acid addition to the diet on DNA methylation in honey bees: 1)DNMT1 and DNMT3 are key enzymes in the DNA methylation process of honey bees.0.04%of the test group significantly increased the expression of Dnmt1 a gene and DNMT1 enzyme activity in 3-day-old larvae compared to the control group(P < 0.05),and significantly increased DNMT1 enzyme activity in 4-day-old larvae(P < 0.05).2)The 0.04% test group significantly reduced the enzyme activity of DNMT3 in 3-day-old larvae compared to the control group(P < 0.05),and significantly reduced the expression of Dnmt3 gene and DNMT3 enzyme activity in 4-day-old larvae(P < 0.05).It is suggested that folic acid positively regulates DNMT1 enzyme activity and negatively regulates DNMT3 enzyme activity at 3 and 4 days of age,the critical periods of caste differentiation.The effects of folic acid supplementation in the diet on larval development and adult bee morphology: 1)The folic acid supplementation at 0.04% significantly shortened the developmental length(P < 0.05),significantly increased the pre-pupal weight(P < 0.05)and significantly increased the new bee weight(P < 0.05)compared to the control group.2)The folic acid supplementation at 0.04% in the diet significantly increased the body length(P <0.05),significantly increased the wing length and wing width(P < 0.05)compared to the control group.3)Regarding the number of ovarian tubules,the number of ovarian tubules in the 0.04% test group was significantly higher than that in the control group(P < 0.05),reaching more than 100,suggesting that the larvae in the 0.04% test group developed toward the queen.This study showed that the addition of folic acid to honey bee larval diets can regulate the metabolic process of folic acid during the larval,pupal and newly emerged bees stages of honey bees,which in turn affected the DNA methylation process of honey bees and ultimately led to phenotypic changes in newly emerged bees.This experiment showed that folic acid can have an effect on the caste differentiation of honey bees.Addition of 0.04% folic acid to the diet helped female honey bee larvae to develop towards the queen. |
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