The Localization Mechanism Of Quadrastichus Mendeli Mediated By Eucalypt Volatiles

Quadrastichus mendeli is an effective obligate biological control agent,preferred for targeting the invasive gall-forming insect Leptocybe invasa larvae,and has demonstrated a significant controlling effect.However,the mechanism by which Q.mendeli accurately locates its host,L.invasa remains unclear.The author hypothesizes that Q.mendeli can precisely locate L.invasa through the mediation of plant volatiles.To test this hypothesis,the study delves into the localization mechanism of Q.mendeli mediated by volatile.Observations of the parasitic behavior of female Q.mendeli were conducted in a small container.Additionally,the ultrastructures of the antennae and ovipositors of female Q.mendeli,as well as volatiles from undamaged,mechanically damaged,and gall-induced plants,were examined.The olfactory response of female Q.mendeli to the active components of volatiles was investigated,along with the identifcation of major olfactory-related genes in female Q.mendeli.The study also involved the selection of reference genes in different adult tissues of female Q.mendeli and explored the expression characteristics of 9 QmenOBPs(Olfactory Binding Proteins)using all-weather detection technology,scanning electron microscopy(SEM),transmission electron microscope(TEM),gas chromatography-mass spectrometry(GC-MS),gas chromatography(GC),gas chromatographic-electroantennographic detection(GC-EAD),Y-tube olfactometer tests,high throughput sequencing,and real-time quantitative polymerase chain reaction(qRT-PCR).Furthermore,the ligand binding characteristics of QmenOBP2 was analyzed through molecular cloning,prokaryotic expression,fluorescence competitive binding,and molecular docking.The results clarified the localization mechanism of Q.mendeli mediated by plant volatile and provide a theoretical basis for developing attractants for Q.mendeli.The main findings are as follows:(1)The parasitic behavior of female Q.mendeli has been defined.The primary processes of parasitic behaviour of Q.mendeli include walking,resting or grooming,antennating,probing,and stinging.Among these,antennating is the most time-consuming and frequent behaviour.Olfactory and vibrational stimuli could be used for Q.mendeli to locate their host.(2)The types of sensilla on the antennae and ovipositor of Q.mendeli have been ascertained.The antenna of Q.mendeli is geniculate,and six distinct types of sensilla were observed,including SBCP,Bohm,SCh I and II,SCa,SPⅠ and Ⅱ,ST.The ovipositors of Q.mendeli consist of the first,second,and third valvulae,and two distinct types of sensilla and four microtrichia were observed,including SCh Ⅰ and Ⅱ,SCa,and MT I,Ⅱ,Ⅲ and Ⅳ.(3)Q.mendeli could be attracted by eucalypt volatiles.Q.mendeli was significantly attracted to volatiles from gall-induced plants.GC-EAD results demonstrated that 3-carene,decanal,d-limonene,ethanone,1-(4-ethylphenyl)-,p-cymene,and benzene,1-methyl-4-(1-methylpropyl)-elicited significant antennal responses in Q.mendeli.Q.mendeli was repelled by decanal and d-limonene and attracted to 3-carene,benzene,1-methyl-4-(1-methylpropyl)-,ethanone,1-(4-ethylphenyl),and p-cymene.Quaternary blends containing 3-carene,p-cymene,benzene,1-methyl-4-(1-methylpropyl)-,and ethanone,1-(4-ethylphenyl)-at a ratio of 1:1:1:1 were found to be attractive to Q.mendeli.(4)Olfactory genes in the antennae and abdomen of Q.mendeli have been explored.A total of 215 olfactory genes were identified from the transcriptomes of the antennae and abdomen of Q.mendeli,including 7 chemosensory proteins(CSPs),24 gustatory receptors(GRs),68 ionotropic receptors(IRs),58 odorant binding proteins(OBPs),34 odorant receptors(ORs),and 24 sensory neuron membrane proteins(SNMPs).By comparing the transcriptomes of the antennae and abdomen of Q.mendeli,10314 DEGs were identified,of which 99 DEGs were olfactory genes.(5)The distribution of QmenOBPs in different tissues of Q.mendeli was investigated.Based on the obtained transcriptome data,4 QmenOBPs were successfully chosen and cloned,namely QmenOBP1,QmenOBP2,QmenOBP3,and QmenOBP4.RPS3 and SDHA were recommended as reference genes for gene quantification in different adult tissues(antennae,head,thorax,and abdomen)in Q.mendeli.qRT-PCR analysis revealed that 9 QmenOBPs were distributed in all tissues(antennae,head,thorax,and abdomen),with the highest abundance detected in the antennae.Among them,QmenOBP1,QmenOBP6,and QmenOBP8 in the thoraxes,QmenOBP2 in the heads,QmenOBP3,QmenOBP4,QmenOBP5,and QmenOBP7 in the antennae,QmenOBP9 in the abdomens were highly expressed.(6)The binding ability of QmenOBP2 with eucalypt volatiles has been ascertained.QmenOBP2 was successfully expressed using a bacterial system.The recombinant protein was purified by passing it through a Ni-ion affinity chromatography column,and the His-tag was cleaved off with recombinant bovine enterokinase to obtain purified recombinant QmenOBP2.The fluorescence competitive binding experiment between QmenOBP2 and 16 plant volatiles showed that QmenOBP2 had a binding ability with 13 plant volatiles,including alcohols,ketones,aldehydes,alenes and benzenes.Notably,QmenOBP2 exhibited a strong binding ability with eucalyptol and trans-beta-ocimene.(7)The key binding sites of QmenOBP2 with eucalypt volatiles have been identified.QmenOBP2 exhibits six α-helices and three disulphide bridges,as indicated by the predicted 3D structure.Evaluation results from the Ramachandran plot energy analysis demonstrate that the model conformation is reasonable.Molecular docking results reveal that QmenOBP2 exhibits binding affinity with 13 plant volatiles.Notably,trans-beta-ocimene demonstrates the lowest binding energy(-6.806)with QmenOBP2.The primary driving forces in the docking interaction between QmenOBP2 and p-cymene include Van der Waals force,hydrophobic interactions,and hydrogen bonding.In conclusion,our study elucidated the parasitic behavior of female Q.mendeli and highlighted the curcial role of the antennae in the process of host location and acceptance.Importantly,our findings confirm that the antennae of Q.mendeli play a vital role in recognizing eucalyptus plant volatiles in the environment,providing clarity on its molecular mechanism of chemosensory.