Molecular Characterization Of The Juvenile Hormone Methoprene-tolerant Genes And Their Functions In Plutella Xylostella Reproduction

The diamondback moth Plutella xylostella(L.)(Lepidoptera:Plutellidae)is the most destructive pest of economically important Brassicea crops.Currently,optimal strategies and tactics are still required in order to effectively control this pest insect with high fecundity.Although juvenile hormone(JH)has been proven to play a crucial role in stimulating the reproduction of some insects,we know rather little about its molecular mechanisms,especially in non-model insects.Methoprene-tolerant(Met)is a key receptor that plays an important role in reproductive regulation of JH and has obvious species specificity.To better understand the effects of reproductive regulation on reproductive potential of P.xylostella,we proposed studies of the JH functional receptor Met with the reproduction hormone as a core.Three experiments were conducted.First,the molecular characteristics of PxMet and its phylogenetic relationship with other insects were analysed.Subsequently,the expression pattern of PxMet in different development stages and different tissues were measured.Finally,the roles of PxMet in ovary development and fecundity were studied.The main results were as follows:PxMet-1 and PxMet-2 contained ORFs of 1,575 bp encoding 524 aa and 2,100 bp encoding 699 aa,respectively.The predicted molecular mass of PxMet-1 and PxMet-2 were 60.5 kDa and 70.7 kDa,and the proposed isoelectric points were 6.73 and 5.50,respectively.Sequence alignment indicated that PxMet-1 and PxMet-2 contained four conserved domains,including a basic Helix-Loop-Helix(bHLH)domain,two PAS motifs and one PAC motif.Homologous analysis also showed a high similarity of PxMet-1 and PxMet-2 with other Lepidoptera species such as Operophtera brumata(72%),Danaus plexippus(71%),and Bombyx mori(58%).The results of phylogenetic tree analysis showed that PxMet-1 and PxMet-2 were clustered into different branches,indicating that PxMet-1 and PxMet-2 might have different biological functions and evolutionary patterns.However,both molecules were associated with Lepidoptera,and together with Bombyx mori Met-1 and Met-2,respectively,may play the same role as the Lepidoptera Mets.The expression profile showed that PxMet-1 and PxMet-2 were both expressed at the pupal stage(1-3 days)and female adult stage(0-72 h)and peaked at 72 h after eclosion.The expression level of PxMet-1 show no obvious difference in pupal stage,but was significantly higher than that in adult female(0-48 h);In the adult female stage(0-48 h),the mRNA level of PxMet-2 firstly started to increase,with a peak on 12 h,and then decline,moreover,the expression level of PxMet-2(0-36 h)was significantly higher than that in pupa;However,there was a higher expression of PxMet-1 and PxMet-2 occurring at 72 h after eclosion,guessing that it is related to the trade-off between reproduction and life span of P.xylostella.Furthermore,tissue expression pattern of head,fat body,midgut,ovary and residue of P.xylostella revealed that the expression levels of PxMet-1 and PxMet-2 in fat body were significantly higher than those in other tissues.Injection of PxMet-1 and PxMet-2 dsRNA into two day-old female pupa caused severe suppression of PxMet-1 and PxMet-2 mRNA expression within 24 h,but the expression of PxMet-1 and PxMet-2 was rescued after 48 and 72 h,and there was no significant difference between the treatment and control group.PxMet-1 and PxMet-2 silencing had a significant negative effect on fecundity of P.xylostella.The results showed that comparing with the control treatment,the ovary development and yolk deposition of P.xylostella was significantly inhibited,which lead to the number of mature eggs decreased,in addition,the number of eggs laid by females within 5 days was significantly decreased.Our study has illustrated the molecular characteristics,expression pattern and gene function of PxMet-1 and PxMet-2.The results can be used to establish the theoretical foundations of reproductive mechanisms under JH regulation in P.xylostella and then screen potential targets used for genetic manipulation of the P.xylostella populations subject to varying agroecosystems.