The Regulation Mechanisms Of Sex Pheromone Recognition And Postmating Behavior In Helicoverpa Armigera

Mating is an essential innate behavior to ensure the insect population renewal.Olfactory serves as a crucial role in insect sensory system and mediates many behavioral contexts,such as finding sexual partners.For most moth species,the male selects a suitable mate by recognizing the specific sex pheromones emitted by its conspecific female.Sensory Neuron Membrane Proteins(SNMPs),a transmembrane protein,which have been reported to be required to sense cVA in Drosophila melanogaster.However,there is little evidence about its specific in vivo function in moth.Except for olfactory elements which are necessary for detecting pheromones,many studies had explored that host plant volatiles also could mediate pheromone detection in insects.In this study,we analyzed the mechanisms of regulating sex pheromone recognition and postmating behavior in an important agricultural pest,Helicoverpa armigera by using bioinformatics,CRISPR/Cas9,insect behavioral and electrophysiological techniques.This study would provide new strategies for pest control.The main results are as follows:1.HarmSNMP1 has an important role in detection of long-chain sex pheromones,but is not essential for detecting shorter chain sex pheromone.In this work,we found that SNMP1 was expressed in antennae,labial palps and legs,with high levels of expression in the antennae of both sexes.To investigate the function of SNMP 1,we developed a SNMP1^-/-homozygous mutant line of H.armigera using CRISPR/Cas9.Wind-tunnel behavioral experiments showed that HarmSNMP1^-/-males could not be attracted by sex pheromones(Z11-16:Ald/Z9-16:Ald=97/3),while mating behavior observations revealed that the SNMP1 mutant males responsed more slowly to calling females in contrast to wild type males(3.73±2.38 h;6.75±1.63 h)and the rate of copulation was significantly decreased(55.55%±3.85%;11.11%±3.85%).The EAG results indicated that HarmSNMP1 contributes to the detection of(Z)-11-hexadecenal(Z11-16:Ald)and(Z)-9-hexadecenal(Z9-16:Ald),but not to(Z)-9-tetradecenal(Z9-14:Ald)and host plant volatiles.The SSR results indicated that HarmSNMP1 contributes to the detection of 16-carbon liner sex pheromones Z11-16:Ald,Z9-16:Ald and(Z)-11-hexadecanol(Z11-16:OH)and 16-carbon acetate(Z)-11-hexadecenyl acetate(Z11-16:OAc),but is not required for detecting the 14-carbon sex pheromone component Z9-14:Ald and an analogue of Z11-16:Ald,(Z)-9-tetradecen-1-yl formate(Z9-14:OFor),which can activate the Z11-16:Ald-responsive neuron.2.SNMP2 is not involved in the process of detecting the sex pheromones,but may be involved in the metamorphosis development process.HarmS NMP2 was expressed in all tissues during adult stage,but it showed higher expression in antennae and the expression of SNMP2 in male antennae was significantly higher than that of female.In this study,we mainly focused on the function of HarmSNMP2 in detecting sex pheromones.We designed the sgRNA target site at exon 6 and knocked out HarmS NMP2 by using the CRISPR/Cas9 technique.The SSR results indicated that the SNMP2 mutants could sense the sex pheromones Z11-16:Ald,Z9-16:Ald,Z9-14:Ald and Z11-16:OH normally.We also found the SNMP2 is not involved in the process of detecting the host plant volatiles by using EAG.In pupal stage,the pupation deformity rate of SNMP2 mutants(63.8%)were significantly higher than wild type(23.3%),the results implied that the SNMP2 may be involved in the metamorphosis development process.3.Plant volatile linalool synergizes the sex pheromones of the H.armigera.To date,numerous studies have reported on interactions between insect pheromones and host plant volatiles.There is mass of leaf-emitted volatile,linalool,in the shelter of H.armigera.Wind-tunnel assay indicated that the behaviors of males induced by sex pheromone alone(Z11-16:Ald/Z9-16:Ald=97/3): flighting,upwind,closing the source,landing are 62.4%,62.4%,and 35.3%,respectively and the induced behaviors by linalool alone are 32%,32% and 0,however,when the mixture of pheromone and linalool were used to attract the males,the induced behaviors: flighting(?²=12.968,P<0.001),upwind(?²=10.390,P=0.01),and closing the source(?²=5.318,P=0.021)were significantly increased in contrast to the sex pheromones alone.Analyzing the time of attracted behaviors,we found that linalool did not affect the time needed to complete the behaviors.By using SSR,we found that neither 1 ?g?10 ?g or 100 ?g linalool could activate the pheromone sensory neurons,the response value of 10 ?g Z11-16:Ald excitation sensory neurons was 65.9±20.7,the response value of was 97.6±21.4 when pheromone sensory neurons were activated by mixture of 10 ?g Z11-16:Ald and 100 ?g linalool,the response value of 10 ?g Z9-16:Ald excitation sensory neurons was 26.4±10.3,the response value of was 23.3±13.1 when pheromone sensory neurons were activated by mixture of 10 ?g Z9-16:Ald and 100 ?g linalool.All the results indicated that stimulation with binary mixtures of a threshold dosage of Z11-16:Ald and increasing dosage linalool significantly synergized the pheromone-specific neuron's firing rates,however,the linalool could not synergize the Z9-16:Ald-specific neuron's firing rates.Application of the mixture of linalool and pheromone enhanced the pheromonal responses of projection neurons innervating the macroglomerular complex in the antennal lobe by using intracellular recording.4.HarmS PR mediates the long-term receptivity,reproductive behavior and longevity in H.armigera female.In the present study,we employed the CRISPR/Cas9 genome editing system to knock out SPR in H.armigera.When getting HarmSPR^-/-homozygous mutant,we measure the female re-mating behavior to determine the function of SPR in female sexual receptivity.After mating,WT females became unreceptive immediately and would not recover in 24-48 h.Comparing to WT females,HarmSPR^-/-mutants became unreceptive in the first 2h,but recovered in 24-48 h.Interestingly,mutant females also became unreceptive in 2h after mating.In addition,we had also analyzed the duration of mating and re-mating,the results indicated that there was no significantly different between the mutants and WT groups.We reasoned that the high re-mating rates in mutants probably due to the rapid recovery of sex pheromone production.However,Z11-16:Ald in female sex pheromone glands were significantly decreased in both WT and mutant females.Although the pheromone productions in mutant females were low,it might be enough to promote the re-mating behavior if the calling behavior is recovered.Therefore,we observed the calling behavior in both WT and mutant females.Both of the WT and mutant females stop calling after in 2h after mating.In addition,mutant females displayed robust calling behaviors in 24 h and 48 h after mating which consistent with the re-mating test.Next,we analyzed the reproductive behaviors and the results showed that virgin females usually laid few eggs(24h,21.3±5.7;48h,61.3±23.8),whereas this increased significantly after mating(24h,278.1±29.8;48h,478.6±29.7).Comparing to WT mated females,the ovipostion of mated HarmSPR^-/-females was significantly decreased by ⁷0%(24h,73%,75±31.4,p<0.0001;48h,69.3%,147.1±51.9,p<0.0001).Those results indicated that SPR is critical for the female oviposition.In order to further verified that whether the decreased oviposition results from the failure of egg development or the diminished oviposition behaviors,we dissect the ovary in mated females to check the number of eggs under stereomicroscope.The eggs were well-developed in the mutants and most of them were staying in its ovary(HarmSPR^-/-,462.1±40.2;WT,288.3±34;p=0.007).Therefore,SPR could affect the fecundity of females by reduce its oviposition behavior but not the eggs development.We also recorded the longevity of different treated females,the results indicated that the mated HarmSPR^-/-females showed longer longevity than mated WT females.In summary,HarmSPR mediates the egg-laying behavior,longevity and the long-term(24 h,48 h)sexual receptivity switch,but not affect the short-term(2h)receptivity and duration of mating and re-mating.