Research On The Host Selection Behavior Of Asian Citrus Psyllid(diaphorina Citri Kuwayama)and Its Mechanism

The Asian citrus psyllid(ACP),Diaphorina citri Kuwayama(Hemiptera: Psyllidae),is the most important pest of citrus worldwide because it serves as a vector of “CandidatusLiberibacter asiaticus” that cause citrus huanglongbing(HLB).HLB is also known as citrus greening disease,is presently the most devastating citrus crop disease with no cure,almost all known citrus varieties including those from their closely related genera can be infected by the bacteria and highly infectious.The increase in HLB and its regional spatial spread is strongly related to ACP,therefore,achieving control of ACP is important in controlling HLB.Pest behaviormanipulation is a kind of new ecological control technology which is highlighted by general pest ecological control theory at the beginning of the 21 st century.The host selection behavior of the herbivorous insects to their host plants is a core issue in the relationship between insects and plants,and the pest behavior manipulationtechnology is mainly based on it.Like other insects,ACP often use its olfactory and visual sensory system to detect and preliminary response to its host plants,and then complete their host selection behavior under the support of their moving system and taste system.However,little is known about the influencing factors of ACP's host selection behavior,and the mechanism of chemical receptors ormolecular components of ACP olfaction wasalso hasn't been described,too.In this study,the influencing factors of ACP's host selection behavior,and the mechanism ofACP's host selection behavior wereanalyzed.The main results were as follows:(1)Analysis of the influencing factors of ACP's host selection behavior.Firstly,the wind tunnel assayis used to measure the host selection behavior of ACPunder different light intensity.The results showed that the host selection behavior ofACPwas significantly enhanced with the increase of light intensity.In the 1000 lx light intensity conditions,the highest percentage of male and femaleACPwhich landing on the host plants was 28.89 ± 5.88% and 35.56 ± 4.44% within 4 h,while there were only 11.11±3.85% and 15.56±3.85% at 1 lx light intensity,respectively.Secondly,the wind tunnel assayis also used to measure the host selection behavior of ACPunder different movingability.The results showed that the moving ability of metathoracic legs wasplayed a decisive role in the process of host selection behavior of male and female ACP.There was none of male and female ACP could landing on the tested host plant after removed its metathoracic legs,it was just only 20.00 ± 3.85% and 17.78 ± 5.88%(about 1/4 and 1/5 comparison to control groups)of male and female ACP could arrived half area of the wind tunnel within 4 h,respectively.Thirdly,by comparing the determinationresults of the Y-tube olfactory,we found that the volatiles of the host plants at different growth stages could significantly effect on the host selection behavior of male and female ACP,too.The results showedthat both male and female ACP were didn't displayed significantly response on the volatiles of 10-day-oldseedlings of Murraya paniculata,while the male ACP wasalso didn't displayed significantly response on the volatiles of 0.5-year-oldseedlings of Citrus reticulate.In addition,by further comparing the effects of host plant covered in filter paper or used a simulation plant modelas host plant on its host selection behavior.We also found that male and female ACP not only could land on a host plant orientated by the volatiles of host plant,but also could land on a host plant orientated by the visual or morphology characterization of the simulation plant modelwithin 1.5 m range.Although there was none of male and female ACP landed on the plant model at the end of determination,but there were 4.44 ± 2.22% and 6.67 ± 3.85% of male and female ACP have been tentatively accepted the plant model and landed on it,respectively.Basing on this results,we were preliminary presumed thatthe visual or morphology characterization of host plant might also playa key role in the process of host selection behavior of male and female ACP.(2)Preliminary study on chemical receptors mechanism of ACP's host selection behavior.The analysis of the scanning electron microscope pictures indicatesthat there are a large numbers of chemical receptors have distributed on the surface of antennae of ACP,or on the surface of tarsus and protarsus of ACP'slegs,but the species of chemical receptors which have distributed on the surface of antennae of ACP(5 kinds)was significantly more than on its tarsus and protarsus(1 or 2 kinds).When we treated the chemical receptors which have distributed on the surface of antennae of ACP,or on the surface of tarsus and protarsus of ACP'sprothoracic legs,mesothoracic legs andmetathoracic legs with 5 mol/L HClat 10-15 s,it were significantly less ACP could landing on the tested host plant within 4 h after 5 mol/L HCl treatedapart from the mesothoracic legs treatment groups.It suggests that the use of 5 mol/L HCl treated chemical receptors which have distributed on the surface of antennae of ACP,or on the surface of tarsus and protarsus of ACP's legscould also significantly effect on the ACP's host selection behavior.And it was also found that there were most of ACP have been deprived of the ability of keep moving after used 5 mol/L HCl treated chemical receptors which have distributed on the surface of tarsus and protarsus of ACP's metathoracic legs.It suggests that we might be able to completely break off the process of host selection behavior of ACP through applied some specific chemicals in field,thereby further reducing its transmission of HLB.In addition,the comparative study showed all of the treated ACP groups have been tentatively accepted the plant model and landed on it.However,all of the treated ACP would immediately flew away from the plant model after tentatively landing exceptedthe antennae of ACP was treated with 5 mol/L HCl.Basing on this results,we were preliminary presumed thatthe antennae of ACPcould not only perform long-range olfactory orientation of the host plants,but might also play a key role in determining the choice of host plants.(3)Cloning and expression analysis of OBPs and CSPs gene of ACP.We cloned and sequenced 1 cDNA fregmentof OBPs and 6 cDNAfregmentof CSPs,and further obtained two full-length cDNA ofACPwhich named DcitOBP1(GenBank accession number: KY475564)and DcitCSP1(GenBank accession number: KY475563).The predicted molecular weight of DcitOBP1 is 15.8 kDa,and theoretical pI is 9.12,and encodes 148 amino acids,predict half-life theoretical value was 30 h and unstable parameters was 34.98,aliphatic index was 84.46 and GRAVY was-0.050.The predicted molecular weight of DcitOBP1 is 14.9 kDa,and theoretical pI is 8.2,and encodes 128 amino acids,predict half-life theoretical value was 30 h and unstable parameters was 50.68,aliphatic index was 86.80 and GRAVY was-0.507.Relative quantification analysis was performed using the comparative relative standard method.DcitOBP1 demonstrated the highest expression in the antennae of male and female ACP.It suggested that the DcitOBP1 might be mainly plays an important role in the process of ACP's olfaction.In contrast,gene DcitCSP1 not only demonstrated the highest expression in ACP's legs and other body parts,but also demonstrated the lower expression in the antennae of male and female ACP.It suggested that the DcitCSP1 might be performed a variety of different physiological functions including olfaction.(4)Prokaryotic expression of the recombinant proteins of DcitOBP1 and DcitCSP1,and analysis of its ligand binding ability.In order to clarify the function and binding characteristics of OBPs and CSPs of ACP,we successfully expressed gene DcitOBP1 and DcitCSP1 in prokaryotic expression system.About 4 h after induction at 30?,0.06 mM IPTG,thesoluble proteins with the recombinant proteins DcitOBP1 and DcitCSP1 of ACP were expressed at supernatant,and identified by SDS-PAGE,meanwhile,western-blot also checked it.After purified,then the fluorescence competitive binding assays was used to measure the binding capacity of the DcitOBP1/DcitCSP1 recombinant proteins with each of twelve candidate compounds.Then,using molecular docking(in silico)verified the results of fluorescence competitive binding assays.The results showed the higher affinity of DcitOBP1 than DcitCSP1,DcitOBP1 showed the highest affinity to?-Caryophyllene and ?-Caryophyllene with the dissociation constant Ki.The binding ffinity of decreased followed the sequence of ?-Caryophyllene,?-Caryophyllene,Copaene,?-Cubebene,?-Elemene,?-Phellandrene,(1R)-(+)-?-pinene,(1S)-(-)-?-Pinene,?-Phellandrene,Limonene,Linalool,3-carene,?-Ocimene,Myrcene,Methyl salicylate.Combined the displacement binding data and molecular docking data,we conclude that DcitOBP1 could be a crucial OBPs fordetection of attractive semiochemicals.