The Ca^(2+)/CaN/ACC and cAMP/PKA/HK signal pathways are required for PBAN-mediated sex pheromone biosynthesis in Conogethes punctiferalis

Conogethes punctiferalis is a crop and fruit pest that has caused serious economic losses to agricultural production.This pest relies heavily on its sex pheromone to ensure sexual encounters and subsequent mating success.However,the molecular mechanism underlying sex pheromone biosynthesis in this species remains elusive.The present study investigated the detailed mechanism underlying PBAN-regulated sex pheromone biosynthesis in C.punctiferalis by transcriptome sequencing of the C.punctiferalis pheromone glands(PGs)and subsequent functional identification of the target genes.The results showed that female mating started from the first scotophase,and peaked at the second to fifth scotophases in accordance with the release of sex pheromones.PBAN regulated sex pheromone biosynthesis by employing Ca^(2+)and cAMP as secondary messengers,as demonstrated by RNA interference(RNAi),pharmacological inhibitors,and behavioral assays.Further investigation revealed that calcineurin(CaN)and acetyl-CoA carboxylase(ACC)were activated by PBAN/Ca^(2+)signaling,and the RNAimediated knockdown of CaN and ACC transcripts significantly reduced sex pheromone production,ultimately leading to a significantly reduced ability of females to attract males.Importantly,hexokinase(HK)was found to regulate sex pheromone biosynthesis in response to the PBAN/cAMP/PKA signaling pathway,as demonstrated by RNAi,enzyme activity,and pharmacological inhibitor assays.Furthermore,Far2 and Desaturase1 were found to participate in PBAN-regulated sex pheromone biosynthesis.Altogether,our findings revealed that PBAN regulates sex pheromone biosynthesis through the PBANR/Ca^(2+)/CaN/ACC and PBANR/cAMP/PKA/HK pathways in C.punctiferalis,which enriches our comprehension of the details of sex pheromone biosynthesis in moths.

Functional assessment of cadherin as a shared mechanism for cross/dual resistance to Cry1Ac and Cry2Ab in Helicoverpa zea

Helicoverpa zea is a major target pest of pyramided transgenic crops expressing Cry1,Cry2 and/or Vip3Aa proteins from Bacillus thuringiensis(Bt)in the United States.Laboratory-selected Cry1Ac/Cry2Ab cross resistance and fieldevolved practical dual resistance of H.zea to these two toxins have been widely reported.Whether the widespread Cry1Ac/Cy2Ab dual resistance of H.zea has resulted from the selection of one shared or two independent resistance mechanisms by pyramided Bt crops remains unclear.Cadherin is a well-confirmed receptor of Cry1Ac and a suggested receptor of Cry2Ab in at least three Lepidopteran species.To test whether cadherin may serve as one shared mechanism for the cross and dual resistance of H.zea to Cry1Ac and Cry2Ab,we cloned H.zea cadherin(HzCadherin)cDNA and studied its functional roles in the mode of action of Cry1Ac and Cry2Ab by gain-and lossof-function analyses.Heterologous expression of HzCadherin in H.zea midgut,H.zea fat body and Sf9 cells made all three of these cell lines more susceptible to activated Cry1Ac but not activated Cry2Ab,whereas silencing HzCadherin of H.zea midgut and fat body cells significantly reduced the susceptibility to Cry1Ac but not Cry2Ab.Likewise,suppressing HzCadherin with siRNA made H.zea larvae resistant to Cry1Ac.These results clearly demonstrate that HzCadherin is not a receptor for Cry2Ab,and thus it is unlikely to serve as one shared mechanism for the cross and dual resistance of H.zea to Cry1Ac and Cry2Ab.

Cloning and characterization of a bursicon-regulated gene Su（H） in the house fly Musca domestica

Bursicon is a neuropeptide that regulates cuticle sclerotization （hardening and tanning） in insect via a G-protein coupled receptor. However, the signal transduction pathway downstream of the G-protein coupled receptor is currently not well known. In our recent microarray analysis, we identified a panel of genes regulated by bursicon in Drosophila. One of the genes, Suppressor of Hairless, or Su（H）, has drawn our attention because its product acts down-stream of the bursicon receptor. In the present study, we cloned the Drosophila homolog, mdSu（H）, from the house fly Musca domestica using 3＇ and 5＇ rapid amplification of complementary DNA ends. Real-time polymerase chain reaction analysis revealed that the level ofmdSu（H） transcript is up-regulated by ~3-fold 1 h after recombinant bursicon injection, which correlates well with the cuticle sclerotization process observed in the recombinant bursicon-injected flies. We infer that Su（H） is an essential gene involved in the insect cuticle sclerotization process.

Molecular characterization of glutaredoxin 2 from Ostrinia furnacalis

Glutaredoxins(GRXs)play very important roles in maintaining intracellular redox homeostasis.In the present study,the full-length cDNA sequence encoding GRX2,named OfurGRX2(GenBank accession no.GU393246),was obtained from Ostrinia furnacalis,using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends.Sequence analysis revealed that the open reading frame of OfurGRX2 consists of 351 nucleotides encoding 116 amino acid residues with a predicted molecular weight of 12.6 kDa.Homolog research revealed that OfurGRX2 shares a common active site,CPYC/CPFC,with other insect counterparts.Expression profiles revealed that OfurGRX2 is a ubiquitous gene expressed in insect heads,fat bodies,epidermises,mid guts and muscles.The OfurGRX2 transcript peaked in 36-h larvae of 4th instars,and then suddenly declined in the molting stage.Hormone treatment experiments revealed that 20-hydroxyecodyson(20e)significantly induces the expression of the OfurGRX2 transcript,whereas juvenile hormone(JH)counteracts 20e effects.Adverse stress factors(including starvation,ultraviolet light,mechanical injury,Escherichia coli exposure,and high and low temperatures)dramatically induced OfurGRXGRX2 transcript expression,which confirmed for the first time that GRX2 play important roles in insecta during exposure to adverse environments.