Identification Of Close Range Sex Pheromones And Analysis Of Sensory Proteins In Bactrocera Dorsalis

Bactrocera dorsalis(Hendel)(Diptera: Tephritidae)has caused extremely serious damage to over 200 varieties of fruits and vegetables in south China.The larvae of B.dorsalis always hide in the fruits,which lead to great difficulties for chemical control.Extensive use of chemical pesticides causes pesticide residues and ecological destruction,which seriously affects human health,ecological balance and sustainable development of agriculture.At present,one of the most important strategies for the controlling of B.dorsalis is trapping them using lures that with methyl eugenol as the main component.However,methyl eugenol reveals attraction to at least a dozen Tephritidae flies.Therefore,the use of methyl eugenol is bound to cause local ecosystem damage.In addition,methyl eugenol also induces multiple malignancies in mammals.Obviously,methyl eugenol has great potential hazards to ecological balance,food safety and human health.At present,it is very important to find new strategies for B.dorsalis control.The courtship and mating of B.dorsalis is a composed of long-distance attraction and close-range perception.Insect cuticular hydrocarbons(CHCs)can act as close or contact sex pheromones to regulate insect courtship and mating behavior.In this study,we screened and obtained active components regulating the courtship and mating behaviors of B.dorsalis,and carried out thorough studies on their sensory proteins.The results of this study will reveal the chemosensory mechanism of courtship and mating behavior of B.dorsalis.It also provides a new strategy and idea for pest controlling based on the theory of behavior regulation.The main research contents and results of this paper are as follows:(1)Comparative study on the differences of CHCs between females and males of Bactrocera dorsalisIn this study,nine compounds with significantly different contents in female than male CHCs were screened by gas chromatography-mass spectrometry(GC-MS),and their electrophysiological activities were determined by gas chromatography-antenna potentiometer(GC-EAD).The results showed that only 6 compounds,Methyl Laurate,Ethyl Laurate,Methyl Myristate,Ethyl Myristate,Palmitoleic Acid Methyl Ester,Ethyl-9-Hexadecenoate,could induce the antennal electrophysiological responses of male Bactrocera dorsalis.At the same time,the behavioral activities of these nine compounds were measured by Y-olfactometer.The results showed that only 4-Allyl-2,6-Dimethoxyphenol had strong attractant activity to male at 50 and 100 equivalents.On this basis,the activity of compounds in the head and foot of Bactrocera dorsalis was measured by antennal potentiometer(EAG),and the results showed that only the foot of Bactrocera dorsalis had electrophysiological response to 4-DMP.(2)Screening of 4-DMP response proteins in males of Bactrocera dorsalisA suitable amount of 4-DMP was used to stimulate male Bactrocera dorsalis during sexual maturity,and transcriptome sequencing was performed on stimulated and unstimulated male Bactrocera dorsalis respectively.The chemosensory genes differentially expressed between the two were obtained by screening the conditions that the difference multiples were more than twice and the Qvalue was less than 0.001.The expression of differential genes was further analyzed by fluorescence quantification to identify target genes that responded to 4-DMP stimulation.Nine odorant binding protein(OBPs)genes were obtained(BdorOBP5 、 BdorOBP2 、 BdorOBP4 、 BdorOBP56 a 、 BdorOBP22 、BdorOBP19d、BdorOBP50e、BdorOBP19a、BdorOBP56h).Among them,4 OBP genes(BdorOBP2、BdorOBP4、BdorOBP19a、BdorOBP56h)were significantly down-regulated after being stimulated by 4-DMP,and the other 5 OBP genes(BdorOBP19d、BdorOBP50e、BdorOBP56a 、 BdorOBP5 、 BdorOBP22)were significantly up-regulated after being stimulated by 4-DMP.(3)Determination of binding capacity of protein to 4-DMP using fluorescence quenching competitive binding assayFour response proteins(BdorOBP2、BdorOBP4、BdorOBP19a、BdorOBP56h)were expressed in prokaryotic cells,and purified proteins were obtained by Ni-NTA affinity purification method.The binding coefficient Ki of 4-DMP to 4 proteins was obtained by fluorescence quenching test.The results showed that BdorOBP2,the protein with the strongest binding capacity to 4-DMP,preliminary analysis speculated that BdorOBP2 may be the target protein to recognize binding to 4-DMP.(4)Validation of BdorOBP2 protein function using RNAi TechnologyIn this study,we synthesized dsRNA to inhibit the normal expression of BdorOBP2,and then conducted a behavioral selection test of Bactrocera dorsalis.The results showed that the transcription level of BdorOBP2 decreased significantly 48 h and 72 h after RNA interference compared with the control,And the attracting activity of 4-DMP to male Bactrocera dorsalis after BdorOBP2 was disturbed decreased significantly.Therefore,this study demonstrated that BdorOBP2 is a key target protein for the recognition of 4-DMP by Bactrocera dorsalis.