Behavioral Regulation Mechanism Of Monoterpenoid Semiochemical On Important Ips Spp.

Monoterpenoid semiochemicals are important communication signals in Ips bark beetles,include monoterpenes from host plants and monoterpenoid signals produced by the bark beetles themselves.The species of Ips bark beetles are complex in the spruce forest ecosystem in China.In which,Ips typographus as the most important pest of spruce forests in Eurasia.Although chemical ecology of I.typographus has been studied deeply,there are many unknown about the interaction between host monoterpenes and I.typographus at multiple levels.There are several important aspects will be study in this paper,including 1)the toxicity of host monoterpenes to I.typographus,behavioral regulation,the rule of monoterpene metabolism by I.typographus,2)the function of related metabolites for I.typographus,I.subelongatus and the predator Thanasimus substriatus,and 3)the response the gut microbes of I.typographus to host monoterpenes.In addition,the basic data and preliminary studies are still lacking in the study of chemical ecology of Ips hauseri,so the monoterpenoid chemical signals of I.hauseri will be identified and prove the function of these semiochemicals.The research findings are summarized as follows:（1）Insect toxicology,chemical analysis,and behavioral testing were used to investigate the interaction between three host monoterpenes and I.typographus at the tritrophic level.Host monoterpenes including S-（–）-α-pinene,R-（+）-α-pinene,and myrcene showed fumigation and contact toxicity to I.typographus.The results of funigation experiment showed that there was no significant difference in the LC₅₀value of the three monoterpenes against I.tyographus,and the LC₅₀values ranging from 3.3-4.6μl/m L.The LD₅₀value measured by topical application showed that myrcene had the strongest contact toxicity to I.typographus,and the LD₅₀values ranging from 22-32μg/mg of three monoterpenes.Regardless of which method was used,there was no difference in virulence of the three monoterpenes against male and female adults.When topically treated with S-（–）-α-pinene or R-（+）-α-pinene,the amount of volatile metabolites[4S-（–）-cis-verbenol,4S-（+）-/4R-（–）-trans-verbenol,and R-（+）-/S-（–）-verbenone]in the hindgut extract of I.typographus varied significantly between the sexes,and their quality varied significantly depending on the chirality of theα-pinene.More importantly,S-（–）-α-pinene induced male adults to produce large amounts of 4S-（–）-cis-verbenol and S-（–）-verbenone.Topically treatment with myrcene failed to detect a product similar to the chemical structure of myrcene in the hindgut.In field trapping tests,S-（–）-α-pinene and R-（+）-α-pinene increased trap catches of I.typographus and Thanasimus substriatus compared to the pheromone source,while myrcene showed a strong repellent effect on bark beetle but not on its predator.（2）The release ratios of S-（–）-α-pinene and R-（+）-α-pinene in Picea koraiensis and P.jezoensis were 16:1 and 3:1,respectively.Field experiments revealed that bothα-pinenes in natural ratios were highly attractive to I typographus and T.substriatus,and the effect of S-（–）-α-pinene was stronger than that of R-（+）-α-pinene.Using isotope-labelled precursors and tracing by gas chromatography–mass spectrometry,found D³-S-（–）-α-pinene could induce the male produce D³-4S-（–）-cis-verbenol,D³-4R-（–）-trans-verbenol,D³-S-（–）-verbenone,and D³-R-（+）-α-pinene could induce the male produce D³-4S-（+）-trans-verbenol,D³-R-（+）-verbenone.The juvenile hormone III has significant synergistic effect（2-5 times）in the metabolism ofα-pinene by I.typographus,showing a male-specific dominance pattern.The field trap tests show the 4S-（–）-cis-verbenol is irreplaceable by other metabolites.The S-（–）-verbenone and R-（+）-verbenone show inhibiting effect on I.typographus.We also found the4S-（–）-cis-verbenol and 4R-（–）-trans-verbenol show inhibiting effect on I.subelongatus,but R-（+）-verbenone show synergistic effect on I.subelongatus,and S-（–）-ipsenol and S-（+）-ipsdienol show inhibiting effect on I.typographus.And we found the aggregation pheromone of the two species was mainly used as a prey signal by T.substriatus.（3）In this study,sequencing of the 16s RNA was used to determine the composition of the insestinal bacterial community at successive life stages of I.typographus.At the genus level,Erwinia was most dominant in larvae（83.20%relative abundance）,followed by pupae（44.61%）,female adults（36.06%）,and male adults（19.03%）.The genus Pseudoxanthomonas was found in all four developmental stages and was most abundant in pupae（18.83%）,followed by males（4.08%）,larvae（3.18%）,and females（1.23%）.The genus Serratia was dominant in pupae（16.59%）and males（28.09%）,but less than 1.5%in larvae or females.Large differences in the bacterial gut community of male adults were observed when beetles were treated with S-（–）-α-pinene and R-（+）-α-pinene;the relative abundances of Lactococcus increased dramatically when treated with R-（+）-α-pinene,while that of Lelliottia increased significantly when treated with S-（–）-α-pinene.Predictions of major metabolic pathways by KEEG analyses of the gut bacteria of I.typographus adults afterα-pinene treatment showed that the gut bacteria can participate in many metabolic pathways,including carbohydrate,amino acid,lipid,and terpenoid metabolism.R-（+）-α-pinene increased the relative abundances of all major metabolic pathways of intestinal bacteria more than S-（–）-α-pinene treated.（4）By chemical analysis,the quality and quantity of volatile metabolites in hindgut extracts of Ips hauseri among three groups:beetles that were newly emerged,those with a topical application of the JHIII,and those that had been feeding for 24 hr.4S-（–）-Ipsenol,4S-（+）-ipsdienol,4R-（–）-ipsdienol,4S-（–）-cis-verbenol,4R-（–）-trans-verbenol,S-（–）-verbenone are detected in the hindgut of I.hauseri after 24 hr of feeding.The JHIII could induce much S-（–）-verbenone.There are traces of these volatile metabolites in the hindgut of newly emerged beetles.Olfactory electrophysiology showed that 4S-（–）-ipsenol and4S-（–）-cis-verbenol can evoke antennal response of males and females.Field experiments demonstrated that the binary compositions of 4S-（–）-ipsenol and 4S-（–）-cis-verbenol can be used as aggregation pheromone of I.hauseri,which can be applied to monitor the population aggregation behavior of I.hauseri.The aggregation pheromones of the other two Ips species（Ips shangrila and Ips typographus）had no biological activity against I.hauseri,which proved that the aggregation pheromone blends had strong interspecific specificity.This series of studies will deepen our understanding of semiochemically mediated species interactions,and improve the population management effect of these Ips bark beetles.