Functional Analysis Of The Halloween Family Genes In Agasicles Hygrophila,a Natural Enemy Insect Of Alligatorweed

Alternanthera philoxeroides,a worldwide malignant invasive weed,was introduced into China in the late 1930s,and subsequently spread widely to southern China.It has caused a great threat to agriculture,forestry,animal husbandry and fisheries in China.Agasicles hygrophila,as an effective host-specific biological control agent for A.philoxeroides,mainly feeds on A.philoxeroides by larvae and adults with significant control effects.The population dynamics of A.hygrophila has a dircet impact on the control of A.philoxeroides.The Halloween gene family is involved in the synthesis of ecdysone,which affects the growth and reproduction of insects,and then has certain effect on their population dynamics.In this study,we focused on the function of Halloween gene family in A.hygrophila,aiming to find out the factors that affect the population dynamics of this insect,and then provide a new idea for the propagation and utilization of this biological control agent.1.The ovarian transcriptome data were screened with the combination of bioinformatics analysis and six Halloween family genes in A.hygrophila were cloned,which were named AhCYP302A1,AhCYP306A1,AhCYP307A1,AhCYP307A2,AhCYP314A1 and AhCYP315A1,respectively.The analysis indicated that they belonged to the P450 superfamily and had certain conservatism.Among them,AhCYP306A1,AhCYP307A1,AhCYP307A2 were grouped into CYP2 group and the other three genes belonged to mitochondrial group.Meanwhile,all of AhCYP306A1,AhCYP314A1 and AhCYP315A1 had one transmembrane region at their 5'end.2.Transcript levels of the six Halloween family genes at different developmental stages?egg,larva,pupa,female?and in various tissues?Head,Thorax,Midgut,Ovary,Fatbody?of A.hygrophila were analyzed by qRT-PCR.We found that the expression levels of the six Halloween genes differed at different developmental stages and in different tissues of A.hygrophila.The expression level of AhCYP302A1 was the highest on the 6^th to 7^th day of pupal stage,but it was lower at the adult stage.The expression level of AhCYP306A1 fluctuated throughout the life cycle of A.hygrophila and it was the highest on the 10^th day of larval stage.Higher expressions of AhCYP307A2 were detected at the late larval and early pupal stages of A.hygrophila.AhCYP307A1 was highly expressed during the egg stage,and its expression level fluctuated and declined along with the developmental stages.The expression levels of AhCYP314A1 and AhCYP315A1 were higher in adults and eggs than those in larvae and pupae.Among the tested tissues of head,thorax,midgut,ovary and fatbody in A.hygrophila,most of the six Halloween genes were highly expressed in the ovary except AhCYP307A1.The expression level of AhCYP307A1 was significantly higher in the head of A.hygrophila female adult than in other tissues.3.The function of two key Halloween genes of A.hygrophila was verified by utilizing RNA interference?RNAi?.RNAi results showed that after the interference with AhCYP307A2 or AhCYP314A1 gene,the larvae exhibited abnormal molting and a significantly reduced rate of emergence,and the female adults showed delayed ovary development and incapability of oviposition.These phenomena indicate that Halloween gene family plays important roles in the larval development and adult reproduction of A.hygrophila by affecting its molting and ovary development.