Behavioral Responses Of Agrilus Mali To Hostplant Volatiles And Function Of Related Olfactory Genes

The apple buprestid beetle,Agrilus mali Masumura,is one of the most destructive trunk borers for various economic forest species in Rosaceae(including Malus sp.).It has caused severe damage to apple trees in Xinjiang since 1990s,especially for Xinjiang wild fruit forests where Malus sieversii is the dominant species.Agrilus mali can inflict serious damage to fruit trees by tunneling under bark during its long larval period.Adults can select suitable locations for oviposition after maturation feeding in host plants during its relatively short adult period,thus this period is most critical for control of this pest.Host plant volatiles might play an important role in the process of plant selection.Adults of A.mali can distinguish host plants by using their sensitive olfactory system to detect the pivotal host plant volatile components.However,there are only very few studies regarding chemical ecology of this pest.Clarifying the mechanism of A.mali host plant selection will help us to understand the interactions between insects and plants,which can provide a theoretical and technical basis for the development of effective prevention tools using attractant and repellent lures.In this study,we first identified leaf volatile components emitted from host and non-host plants of A.mali and obtained its olfactory genes by sequencing its antennal transcriptome.Secondly,we measured the binding ability of A.mali's odorant binding proteins and chemosensory proteins with its host plant leaf volatiles.Finally,we identified several chemical components which were attractive to A.mali adults in electroantennography tests,dual-choice behavioral assays and trapping field experiments.The main results are as follows:Different host selection behaviors and feeding preferences of adult A.mail were found on the four test plants(i.e.,Malus halliana,Malus domestica,Pyrus betulifolia and Amygdalus persica).Female adults preferred to feed on leaves of M.halliana and M.domestica compared to P.betulifolia or A.persica.Compared to those of M.domestica,leaves of M.halliana consumed by A.mali adults had higher area or mass.No feeding behaviors on P.betulifolia or A.persica leaves were observed.The results indicated that both M.halliana and M.domestica were suitable for A.mali adults'maturation feeding,whereas P.betulifolia or A.persica was not suitable in this respect.A total of 71 volatile components were identified via GC-MS analyses,including five alkanes,two ethers,11 alcohols,two ketones,eight aldehydes,23 esters,four alkenes,15 terpenoids and one nitrile,which indicated significant differences in leaf volatile composition and contents among the four test plants.Three composite factors were extracted in the factor analysis,which together accounted for 93.62%of the total variance.Factor 1,2 and 3 explained 41.98%,31.82%and 19.83%of the total variance,respectively.In addition,M.domestica,A.persica and M.halliana contributed the most to the first three factors,respectively.The contents of volatile components were significantly different between M.domestica and A.persica,but exhibited no significant differences between M.halliana and P.betulifolia.We identifed 11 odorant-binding proteins(OBPs),eight chemosensory proteins(CSPs),17 odorant receptors,nine gustatory receptors,17 ionotropic receptors,and one sensory neuron membrane proteins from male and female antennal transcriptome using the Illumina HiSeq2500 platform.Phylogenetic analyses showed that the odorant binding proteins and chemosensory proteins in A.mali,together with those from other coleopteran species,clustered into many different clades,suggesting their divergence in evolution.However,co-odorant receptors(i.e.,ORco,IR8a and IR25a)and SNMP1 in A.mali were found to cluster into the same clade,respectively,indicating their highly conserved status during evolution.We identified 11 full length OBP sequences that encoded 132-149 amino acid residues.The signal peptides,which included 16-20 amino acid residues in N-terminal regions,were predicted.Among these,five OBPs(i.e.,AmalOBP1,AmalOBP2,Amal OBP3,AmalOBP4 and AmalOBP5)were grouped into the“Classic OBP”sub-family that contained six conserved cysteine residues.Five(i.e.,AmalOBP7,AmalOBP8,AmalOBP9,Amal OBP10 and AmalOBP11)were grouped into the“Minus-C OBP”sub-family that was characteristic of the existence of four conserved cysteine residues,and a loss of the second and fifth cysteine residue compared to“Classic OBP”.Moreover,AmalOBP6 fell into the“Plus-C OBP”sub-family that consisted of eight conserved cysteine residues,and had a proline following the sixth conserved cysteine residue.We also identified eight chemosensory genes in the antennal transcriptome.Seven of them had complete opening reading frames(ORFs)encoding 119-137 amino acid residues except that AmalCSP7 was incomplete with a loss of several nucleotides in C-terminal regions.The signal peptides were also predicted to consist of 16-22 amino acid residues for the seven CSPs.Quantitative real-time PCR(q RT-PCR)results showed that six OBP genes(i.e.,AmalOBP1,AmalOBP2,AmalOBP3,AmalOBP4,AmalOBP6 and AmalOBP7)were all expressed exclusively in male and female antennae,whereas they showed negligible abundance in other adult tissues,such as heads,thoraxes,abdomens,legs and wings,indicating that they might be involved in the olfactory process.Three OBP genes(i.e.,AmalOBP5,AmalOBP8 and AmalOBP9)were not only highly expressed in male and female antennae,but also had relatively high expression levels in abdomens and wings,suggesting their multiple roles in olfactory responses and other physiological processes.AmalOBP10 and AmalOBP11 were specifically expressed in male and female abdomens,respectively,indicating that they could participate in odor molecular perception and pheromone release for adults.Interestingly,AmalCSP2 and AmalCSP3 were all expressed exclusively in antennae of both sexes,suggesting that these two genes might also be involved in the olfactory process.Four OBPs(i.e.,AmalOBP1,AmalOBP3,AmalOBP7 and AmalOBP8)and two CSPs(i.e.,AmalCSP2 and AmalCSP3)were successfully expressed with the Escherichia coli system and they were all detected in inclusion bodies.We obtained high-quality purified recombinant proteins through attenuation and renaturation of refolded proteins following purification with Ni-His Tag Resin column.Western blot analyses showed single protein bands with a molecular weight of approximately 14-17 KDa,and this verified that those protein bands were our target genes.Using the Bicin-choninic Acid(BCA)protein assay kit,we measured the mass concentration of the enriched proteins,which was 1.28,1.53,0.61,0.58,0.90 and 1.12 mg/mL in the abovementioned order.We analyzed the binding affinity of recombinant proteins to the fluorescent probe 1-NPN using nonlinear regression analysis with the GraphPad Prism 5.0 software.The dissociation constants(K_d)as calculated by Scatchard plots for AmalOBP1,AmalOBP3,AmalOBP7,AmalOBP8,AmalCSP2 and Amal CSP3 were 1.17,8.63,4.52,4.69,8.87 and 8.10?M,respectively.Based on the results of fluorescent competitive binding assays,AmalOBP1 and AmalOBP7 showed selective binding profiles:AmalOBP1 bound farnesol(K_i=23.27±0.06?M),(2E,4Z)-ethyl decandienoate(K_i=35.54±0.22?M),(Z)-3-hexenyl hexanoate(K_i=36.67±0.21?M)and camphene(K_i=35.36±0.06?M);AmalOBP7 bound dodecanol(K_i=14.94±0.36?M),farnesol(K_i=21.60±0.35?M)and dodecanal(K_i=14.46±0.21?M),as well as?-ocimene(K_i=32.28±0.26?M).Both AmalOBP3 and AmalOBP8 showed a broad binding profile with15 and 22 compounds(such as alcohols,aldehydes,esters and terpenoids),respectively.But they did not bind well to alkenes and alkanes.Specially,AmalOBP3 and AmalOBP8 showed strong binding affinities to alcohols of C12 to C15 such as dodecanol,tetradecanol,nerolidol and farnesol(K_i?6?M).Moreover,they also exhibited high binding affinities to esters of C10to C12 such as octyl acetate,geranyl formate,?-undecanolactone,(2E,4Z)-ethyl decandienoate and(Z)-3-hexenyl hexanoate(K_i=7.09-31.90?M).In addition,both AmalOBP3 and AmalOBP8 also displayed high binding affinities to two terpenoids,2,6-dmethyl-2,4,6-octatriene and?-ocimene(K_i=7.72-15.71?M).AmalCSP2 and AmalCSP3 displayed a distinct and selective binding profile to nine and seven volatile compounds respectively(K_i<40?M).Particularly,AmalCSP3 showed very strong binding affinities to tetradecanol(K_i=1.52±0.01?M).All of these OBP and CSP genes could play significant roles in recognizing and binding host plant volatiles,suggesting that they were involved in the olfactory process of A.mali.We measured the electro-antennogram responses(EAG)for male and female adults to 23host plant volatiles that showed relatively high binding affinities for AmalOBPs and AmalCSPs.The results indicated that decanal,geranyl formate,(2E,4Z)-ethyl decandienoate,(Z)-3-hexenyl hexanoate and 2,6-dimethyl-2,4,6-octatriene elicited strong eletrophysiological responses(relative EAG values over 1.0)of female antennae,while farnesol,decanal,(Z)-3-hexenyl hexanoate evoked strong eletrophysiological responses of male antennae.Dual-choice behavioral assay results indicated that 2,6-dimethyl-2,4,6-octatriene,geranyl formate,(Z)-3-hexenyl hexanoate,decanal and tetradecanol showed strong attraction to female adults,and only(Z)-3-hexenyl hexanoate had strong attraction to male adults,suggesting that these odorants could play important roles in the process of host-plant selection,feeding and oviposition of A.mali.The results of field trapping experiments showed that male and female adults were most attracted to yellow sticky traps(the mean number of every trap for ten days:female,14.33±1.09;males,5.67±1.52;total adults,20.00±2.57),followed by green trap boards(the mean number of every trap for ten days:female,11.67±0.88;males,5.33±0.72;total adults,17.00±1.16).This suggests that yellow and green traps can be effective in monitoring and manging A.mali in the field.Besides,decanal was found to be attractive to male and female adults(the mean number of every trap for ten days:female,14.00±1.53;male,7.00±1.53).Thus,decanal could be used as a trapping lure component with green and yellow traps for prevention and control of A.mali.