Functional Identification Of PBP1 And Comparative Analysis Of Sex Pheromone Communication Systems In Different Geographic Populations Of Helicoverpa Armigera (Lepidoptera Noctuidae)

In moth species,sex pheromone communication system displays high sensitivity and species specificity.Therefore,in addition to the use of artificial sex pheromone components in pest monitoring and direct control,the proteins(genes)related to perception of the sex pheromone are also potential targets for development of new pest control strategy.Pheromone binding proteins(PBPs)are small water soluble proteins abundantly existed in lymph of olfactory sensillae on antennae and other olfactory organs.Based on tissue expression pattern and in vitro functional study,it is thought the PBPs mainly function as solubilizer and transporter for sex pheromone molecules,carrying pheromones across the hydrophobic sensillar lymph to the olfactory receptor(OR)on the dendrite membrane of the sensory neurons,and thus play important role in the sex pheromone perception.However,due to the lack of the suitable technique,this proposed function has rarely been verified by in vivo study.Fortunately,the newly developed genome editing technique CRISPR/Cas9 system,which has been successfully used in gene functional study in several insect species,makes it possible for us to verify the in vivo function of PBP genes.Besides,as a green pest control technique,sex pheromones have been used in control of the cotton bollworm,Helicoverpa armigera,but the efficacy is not very stable between geographical regions and years.This may due to various biological(such as populations and generations of the pest,host plants)and environmental factors,and among these factors the differences in sex pheromone communication system of geographic population deserves to be studied with priority.Focused on the two aspects mentioned above,we in this study,firstly conducted the in vivo functional verification of PBP1 gene in H.armigera by using the CRISPR/Cas9 system.The males with PBP1 gene knocked out were obtained and these males showed severe reduction in electrophysiological responses to all three sex pheromone components,providing the first in vivo evidence for the important role of PBP1 in female sex pheromone perception.For the comparative study of sex pheromone communication system among different geographic populations,contents and ratios of the two major sex pheromone components in females and responses of males to lures loaded with the two pheromone components in different proportions were measured.The results showed that sex pheromone communication system is generally stable among different populations,but relatively,males in Changji population displayed a different behavioral response pattern with males in other two populations,regarding the variation in proportions between the two pheromone components.To further explore the possible molecular mechanisms underlying this difference in male behavioral response,the nucleotide diversity and genetic differentiation in two PBP genes(PBP1 and PBP2)were analyzed.The AMOVA,phylogenetic tree and other analyses showed no obvious genetic differentiation occurred among populations,however,genetic distance based PCoA analysis showed that Changji population from Xinjiang was obviously isolated from other tested populations,indicating a differentiation between these populations.The major results were as follows.1.Functional characterization of PBP1 in H.armigera gene by using the CRISPR/Cas9 systemSimilar as other noctuid,H.armigera has three different PBP genes,and tissue expression pattern and in vitro binding assay suggest that PBP1 play more important role in sex pheromone perception.Therefore,PBP1 was selected to do the functional assay.The target sequence of gRNA was designed,and accordingly the sgRNA and Cas9 mRNA were synthesized.Microinjection of sgRNA/Cas9 mRNA mixture into the newly laid(within 4h)eggs were performed,and 24h later,20 eggs were randomly selected and subjected to the RED assay(including gDNA extraction,PCR amplification of the target sequence,digestion of the PCR product by an restriction endonuclease,and finally a get detection),which showed high rate mutagenesis of 36.9%(86.7%by sequencing of individual insects).The TA cloning and sequencing revealed various insertion and/or deletion(indel)mutations at the target site of PBP1 gene,in which a mutation(two base insertion)resulted in a stop codon near the target site,leading to a highly truncated protein with only 10 amino acids(143 amino acids for the intact PBP1).This mutation was selected by an inbred strategy,and homozygous mutant insects were obtained in the third generation.To determine the role of the gene in sex pheromone perception,electroantennogram(EAG)recordings were conducted.Results showed that the mutant male adults displayed severely impaired EAG responses by 40%to all three sex pheromone components(Z11-16:Ald,Z9-16:Ald and Z9-14:Ald).Our study provides the first in vivo evidence that HarmPBP1 plays important role in perception of female sex pheromones,and also an effective methodology for using CRISPR/Cas9 system in functional genetic study in H.armigera and other insects.2.Comparative analysis of sex pheromone communication system of different geographic populations of H.armigeraTo explore the reasons for the efficacy instability of sex pheromone lures in different regions,9 geographical populations from Anyang,Xiajin,Dafeng,Huimin,Wenshang,Jingzhou,Nanpi,Gaoyang,and Shawan were collected and comparatively analyzed for contents and ratios of the two sex pheromone components(Z11-16:Ald and Z9-16:Ald)in 2013-14.The pheromones were extracted by the hexane as the solvent,and quantified by GC analyses.Results showed that the proportion between the two components(Z11/Z9)was generally stable among populations,although the contents of each component displayed significant difference among some populations.Based on the range of the proportion(Z11/Z9),4 lures with different Z11/Z9 proportions(99:1,97:3,95:5 and 90:10)were tested for the male attractiveness in Anyang,Huimin and Changji.As a result,no difference in male catches was found among lures of different Z11/Z9 proportions in field tests at Huimin and Anyang,while significant difference in male catches was found between 99:1 and 90:10 proportions in test at Changji.In addition,the male catches were proportionally related with the Z11/Z9 proportion in Changji test,and was not in Anyang and Huimin tests.Taken together,sex pheromone communication systems in different field populations(especially those in Yellow River and Yangtze River areas)was generally the same,and thus was not likely to be a major reason for the efficacy instability of the sex pheromones used in pest control.3.Polymorphism and genetic differentiation PBP1 and PBP2 genes in different geographic populations of H.armigeraFocused on two PBP gens that play important role in sex pheromone perception,polymorphism and genetic differentiation in nucleic acid sequence was analyzed in 6 field populations and one laboratory populations.The haplotype network genetic relationship analysis showed that few haplotypes were existed in two populations,while most haplotypes were existed in only one population;haplotypes from one population were not grouped together but scattered at different branches,indicating no obvious clustering of certain field population.The AMOVA analysis showed tihat variations within populations were larger than those among populations.For PBPI,percentage variation within population was 89.87%,and that among populations was 10.13%;for PBP2,percentage variations within and among population were 81.23%and 18.77%,respectively.The phylogenic analysis with nucleotide sequences also showed that individuals from same filed population were not grouped together,but scattered at different branches.This indicating the there no obvious differentiation among different field populations.However,genetic distance based PCoA analysis showed that PBP1 was obviously divided into three groups,with laboratory population as one group,Shawan population from Xinjiang as one group,and other 5 field populations as another group.This grouping suggests the genetic differentiation in PBP1 between Shawan and other field populations,which is consistent with geographic barrier between Xinjiang and the eastern part where the other populations were collected from.