The Molecular Mechanism Underlying The Olfactory Detection Of Methyl Eugenol By Mature Males Of Bactrocera Dorsalis (Hendel)

The Oriental fruit fly,Bactrocera dorsalis(Hendel),is one of the most destructive fruit/vegetable-eating agricultural pests in the world.It is a typical highly polyphagous pest—with larval stage feeding,multiple mating,a long life span,and a great fecundity of adults—capable of causing severe damage to more than 250commercially-valuable tropical and subtropical crops,especially several staple fruits,including mango,guava,orange,carambola,etc.Damage caused by B.dorsalis consists of oviposition stings in fruits by adult females and subsequent larval feeding and decay on the fruit pulp.Therefore,the application of chemical control of the hidden immature stages is difficult and insecticides proved unsuccessful.Moreover,B.dorsalis field populations developed resistance against organophosphates,synthetic pyrethroids,and abamectin insecticides.Considering the mining habits of the larval stage,pest control strategies focus mainly on the adults.The natural phenylpropanoid,methyl eugenol(ME),is a powerful attractant for mature males of B.dorsalis,has been widely used in male annihilation technique(MAT)systems for detecting,luring,and eradicating B.dorsalis population worldwide.ME functions as a precursor for the synthesis of a sex pheromone in B.dorsalis males,which makes those males more attractive to females.Particularly noteworthy is the fact that ME has its risks,however: it is carcinogenic to humans and its attractiveness is limited to mature males.Although diligently developed of B.dorsalis novel attractants,progress toward development of better and more affordable attractants has been slow.In this regard,using a “computational reverse chemical ecology”,designing of attractants to target specific olfactory communication proteins may promote the development of new baits for pest management.However,the molecular mechanism underlying the olfactory detection of ME is hitherto unknown.Therefore,exploring the molecular mechanismunderlying the olfactory perception of ME in B.dorsalis is of great importance for the development of sustainable pest control strategies based on manipulating chemosensory communication.1.OBP2 plays an indispensable role in the perception of methyl eugenol by mature males of Bactrocera dorsalisFirst,we chosed the male flies without taxis to ME by the double-exposure experiment method under laboratory conditions.We found that that the proportion of males with non-taxis to ME could be increased persistently via 6 generations of artificial selection.Then,we analyzed the differential proteomics profiling of the antennae between ME-responsive and ME-non-responsive males by using isobaric tags for relative and absolute quantitation(iTRAQ).In total,4,622 proteins were identified,of which 277 proteins were significant differentially expressed,with 192 up-regulated and 85down-regulated in responsive male antennae.Quantitative real-time PCR(qRT-PCR)analysis confirmed the authenticity and accuracy of the proteomic analysis.Based on the iTRAQ and qRT-PCR results,we screened four odorant binding proteins(OBP2,OBP50 c,OB56D-1 and OB56D-2)as target genes for further study.Mature males(15-day-old)showed significantly greater taxis toward ME than did immature males(3-day-old)and mature females(15-day-old).Furthermore,in contrast to the low expression observed in immature male and mature female flies,OBP2,OBP50 c,OB56D-1 and OB56D-2 was highly expressed in mature males.Moreover,OBP2,OB56D-1 and OB56D-2 were significantly up-regulated by ME in male antennae.Silencing OBP2 reduced mature males’ responsiveness to ME.These results indicate that OBP2 may play an essential role in the molecular mechanism underlying B.dorsalis olfactory perception of ME.2.OR88 a modulates the responsiveness to methyl eugenol in mature males of Bactrocera dorsalisNevertheless,the specific ORs and their molecular functional involvement in the mature male fly’s reception of ME remain a mystery.To gain new insights into the cellular and molecular mechanisms in ME trapped to the mature fly males,we characterized the olfactory receptors targeted by ME.First,we analyzed the differential genes profiling of the antennae between ME and mineral oil(MO)–treated males by RNA-Seq.A total of 4,433 differentially expressed genes(DEGs)were detected in male flies after exposure to ME,of which 3,813 were up-regulated,and 620 were down-regulated.Meanwhile,quantitative real-time PCR(qRT-PCR)verified that DEGs data were reliable for further transcriptionalanalysis.Specifically,based on the RNA-Seq and qRT-PCR results,we found that two odorant receptors(OR63a-1 and OR88a)were abundantly expressed after ME stimulation.Subsequently,we searched for these receptors in heterologous expression system.In contrast to OR63a-1,OR88 a co-expressed with Orco in Xenopus oocytes elicited dose-dependent inward currents upon ME application.Additional,silencing OR63a-1through the injection of dsRNA has no effects on males’ attractive to ME,while the number of attracted males decreased sharply after OR88 a was silenced.Consequently,we conclude that OR88 a is necessary and sufficient for the mature males observed attraction toward ME.3.Hypothesized modal analysis of ME perception and transportation process in the antennae of Bactrocera dorsalis mature male fliesHere,we propose a model describing how chemosensory proteins might be used by a mature male to distinguish the ME odorant from a noisy environment.Once the ME odorant penetrates the pore tubules of the antennae,the protein products of OBP83a-2 and OBP2 are posited to assist in transporting the ME odorant across the aqueous sensillum lymph.After its release from these proteins,the ME odorant is then transferred to the protein encoded by OR88 a.With the ME odorant now bound,the OR88a/Orco complexes are activated to trigger the signals that lead to neural spikes generated in the fly’s brain,evoking its characteristic response behavior to ME.After the activation of the olfactory receptor,the ME odorant is removed and degraded by the ODEs.