Identification And Functional Analysis Of The Odorant Receptor Coreceptor Orco Gene In Agasicles Hygrophila

Agasicles hygrophila is a monophagous natural enemy insect of the worldwide notorious invasive Alternanthera philoxeroides,which mainly relies on its olfaction to recognize special ratios of plant volatiles(E)-4,8-dimethyl-1,3,7-nonatriene(DMNT)and(Z)-3-hexenol to locate its host plant.Odorant receptor(OR)plays an important role in its olfactory signal transduction.Study on the function of odorant receptor coreceptor(Orco)in A.hygrophila can provide preliminary basis for elucidating the olfactory recognition mechanism underlying its host specificity.In this study,Orco gene was cloned and identified by PCR and its sequence was analyzed by biological information analyses.Then the expression level of Orco in different developmental stages and body parts of A.hygrophila was detected by real-time quantitative PCR(q PCR).Finally,the function of Orco was identified by RNAi and behavioral analysis.The main results are as follows:The full length of Orco sequence was 1771 bp,encoding 479 amino acids,with molecular size of53943.64 Da.Its isoelectronic point was 8.36,belonging to alkaline protein.This protein had seven transmembrane structure domain,an inverted Nin-Cout topology,localized in cell membrane,had no signal peptide indicating non-secretory protein.It had five potential phosphorylation sites,a conserved domain of Pfam 7tm-6 at amino acid 70-469.The secondary structure was consisted of alpha helix(Hh),extended strand(Ee),beta turn(Bt)and random coil(Cc).The tertiary structure of Orco was predicted as a homotetramer,with four subunits encircling a central pore.One subunit had potential ligand binding site residues Trp(W108),Ser(S111),Gln(Q231),Arg(R337)and His(H340).Multiple alignment and phylogenetic analysis showed that A.hygrophila Orco was conserved and had the closest relationship with Diabrotica virgifera virgifer,another Coleoptera insect,and their similarity was90.23%.The relative expression level of Orco gene in different developmental stages(eggs,2nd instar larvae,3rd instar larvae,pupae and adults)and different adult body parts(antennae,heads,thoraxes,abdomen,legs and wings)was detected by q PCR.The results showed that Orco gene was highly expressed at adults.In other stages,the expression levels were extremely low.This gene was highly expressed in female and male antennae,with no significant difference between them.Orco had lower expression level in heads(without antennaes)but hardly expressed in other body parts(thoraxes,abdomen,legs and wings).The expression of Orco gene was decreased by RNAi technology,and the interference efficiency of A.hygrophila Orco gene was detected by q PCR.The results showed that the expression of A.hygrophila Orco gene was significantly decreased after ds RNA injection for 4 days and 5 days,and the interference efficiency was the highest,about 50%.Adults in the interference group(injection of ds Orco)were thiner and weighed less than the control group(injection of ds GFP),indicating that RNA interference affects A.hygrophila feeding.Behavioral choice experiments showed that A.hygrophila adults injected with ds EGFP were more active and could accurately locate host plants.However,it is difficult for the adults injected with ds Orco to find their host plants,and the choice to host plants is blind due to their insensitivity to odor molecules.Analysis on the choice behavior of male and female adults showed that female adults were more sensitive to host plants than the male adults.After interference,female adults had no choice or negative choice to the host plants,while male adults had low preference for host plants,interference had little effect on their host choice.In conclusion,our study indicates that Orco gene plays a vital role in the host-plant recognition of A.hygrophila,and disrupting expression of Orco leads to serious olfactory defects of A.hygrophila and significantly reduces their preference to host plants.This research will lay the foundation for further explore the characterization of odorant receptor genes recognizing key plant volatiles and illuminating the olfactory mechanism underlying host specificity of A.hygrophila.