Identification And Functional Analysis Of The Alarm Pheromone Binding Proteins In The Green Peach Aphid Myzus Persicae

The green peach aphid Myzus persicae is a major destructive polyphagous pest.Other than its direct feeding damages to plants,M.persicae can also transmit more than 100 plant viruses,including both persistent viruses such as potato leaf roll virus(PLRV),and non-persistent viruses such as cucumber mosaic virus(CMV).At present,management of this aphid mainly depends on use of broad-spectrum insecticides,including carbonates,organophosphates,pyrethroids and the neonicotinoids.But overuse of these insecticides may cause environmental pollution and evolution of pest resistance to inscticides.Development of environmentally-friendly approaches to control aphids is urgently needed.Insects use their sensitive and selective olfactory organs,mainly the antennae,to perceive the surrounding world.Aphids,like other insects,heavily rely on the chemical signals including plant volatiles and species-specific pheromones to locate correct hosts,find mates,and avoid predators or parasitoids.It has been well known that aphids can release an alarm pheromone to warn other individuals of danger to promote defense or escape,even to attack the predators,when attacked by predators or parasitoids,odorant binding proteins(OBPs)and chemosensory proteins(CSPs)play essential roles in insect chemosensory recognition.Therefore,we can disrupt their chemical communication and control aphids through identification of their olfactory genes and elucidation of their olfactory mechanisms.To achieve the goal of understanding the olfactory mechanism of M.persicae,an artificial diet was developed that allows us to successfully rear M.persicae in the laboratory for over 10 generations and thus produce uniform standard insects for subsequent experiments.Then,we identified 9 OBPs and 9 CSPs from the published transcriptome and genome of M.persicae.Further qRT-PCR analysis showed that three OBP genes(MperOBP6,MperOBP7 and MperOBP10)were specifically expressed in antennae,and five OBP genes(MperOBP2,MperOBP4,MperOBP5,MperOBP8 and MperOBP9)were mainly expressed in antenna.Only,the expression of MperOBP3 in antennae was lower than that in the whole body.Phylogenetic analysis were performed using 237 OBP sequences from 23 Hemiptera insects and 110 CSP sequences from 11 Hemiptera insects.The result showed that the OBPs and CSPs of aphids were clearly diverged from those of plant bugs and plant hoppers and belonged to different subfamilies.The paralogous OBPs and CSPs of the same subfamily were highly concerved among different aphid species,indicating they were diverged from a common ancestral gene.Genomic structure analysis of the 9 OBPs and 9 CSPs in M.persicae showed that the numbers and lengths of the CSP genes are significantly fewer and shorter than those of the OBP genes.MperOBP3/7/8,MperCSP1/4/6,MperCSP2/9 and MperCSP5/8 are tandemly arrayed on the sane scaffold.Similarly arrayed OBPs and CSPs clusters including OBP3/7/8,MperCSP1/4/6,MperCSP2/9 and MperCSP5/8 also exist in the genomes of Acyrthosiphon pisum and Aphis glycines.The structural conservation of OBPs and CSPs among different species of aphids indicates that these OBPs and CSPs genes originate from gene replication events,may be achieved through chromosome recombination.A total of 45 OBPs and 41 CSPs from five aphid species(M.persicae,Aphis gossypii,A.pisum,A.glycines and Sitobion avenae)were analyzed for the presence of motifs and their distribution patterns.Eight conserved motifs were found for both OBPs and CSPs.The 8 OBP motifs yielded 13 distribution patterns in the 45 aphid OBPs,whereas the 8 CSP motifs only formed 5 distribution patterns in the 41 aphid CSPs.We also analyzed the motif distribution patterns of 199 OBPs and 103 CSPs from 18 Hemiptera species.There were 51 different motif distribution patterns in the 199 Hemiptera OBPs,of which 102 OBPs(51.3%)had the most common 6 motif distribution patterns.By contrast,there were 17 different motif distribution patterns in the 103 Hemiptera CSPs,of which 75 CSPs(72.8%)had the most common 4 motif distribution patterns.These results demonstrate that insect CSPs are more conversed than insect OBPs.Eight OBPs(MperOBP2/3/4/5/6/7/8/9)in M.persicae were cloned,expressed and purified.Competitive fluorescence binding assay of MperOBPs with aphid alarm pheromone,sex pheromone and 14 plant volatiles were measured using 1-NPN as the fluorescence probe.The result showed MperOBP3/7/9 exhibited high binding abilities to EBF,with a dissociation constant of 2.47 μmol/L,1.07 μmol/L,5.02 μmol/L,respectively(Ki<6μM).The plant volatiles,hexyl hexanoate had the strongest binding affinity to OBP3 and OBP9,with a dissociation constant of 5.54 μmol/L and 1.99 μmol/L,respectively(Ki<6μM).OBP7 exhibited a medium binding ability to with hexyl hexanoate and nerolidol,with a dissociation constant of 9.33 μmol/L and 6.74 μmol/L(6μM < Ki<12μM).None of these OBPs could specifically bind to sex pheromone.Therefore,MperOBP3/7/9 can be used as important target genes for biological control of aphids.