Chemical Ecological Mechanism Of Conexistence Among Three Tomicus Species

Bark beetle is one of the important forest pests,which not only seriously affect the growth of pine trees,but also cause a great threat to the ecological environment.Three sympatric bark beetle: Tomicus minor,T.yunnanensis,and T.brevipilosus infested Pinus yunnanensis in southwest part of China,which have caused tremendous loss to forestry production and ecological environment.We studied the intraspecific and interspecific relationships and their chemical and ecological mechanisms of three Tomicus species,which compete and coexist in the same host during the shoot-feeding and trunk-breeding phase.Based on behavior ecology,chemical ecology and molecular ecology,the key genes involved in the regulation of the synthesis of pheromones related to the coexistence and competition of the Tomicus species were analysed in the transcriptome level.Hoping to lay a molecular foundation for the behavioral regulation techniques of three bark beetles.Finally,the highly active pheromone and anti-aggregation pheromone are used in the practice of “Push-Pull” control strategy in order to find a better way to manage the population and reduce the harm of Tomicus spp.The results are showed as follows.1.The population density of T.yunnanensis was significant higher than that of T.minor in heavily infested stand,this is contrary in the lightly infested stand.The population densities of T.minor and T.yunnanensis in the heavily infested stand were significant higher than that in the moderate and lightly infested stands.The results from traditional index methods showed that the spatial distribution patterns of three sympatric Tomicus species were all aggregated in the different degrees of infested stands.The cause for aggregation distribution of T.minor and T.yunnanensis was their aggregation behavior and environment.The results from geostatistical methods showed that all presented the aggregation distribution,in addition to T.brevipilosus in the heavily infested stand.The level of spatial dependence of all population ranged from 4.01 to 7.45 m except the T.minor population in the heavily infested stand.The semivariogram models of T.minor and T.yunnanensis switched between spherical model and gaussian model.2.The time of flight peak in both sexes of T.minor was later than that of T.yunnanensis in the crowns of P.yunnanensis,respectively,but there was no significant flight peak in T.brevipilosus.T.minor distributed at the base of crowns,on the contrary,T.yunnanensis and T.brevipilosus were the upper parts.All stems of P.yunnanensis could been initially infested by T.yunnanensis,which then attacked the middle-upper part of stem along with infestation of T.minor from the base to upper part of the stem.The population density of T.minor was significantly different among different heights of Yunnan pine trees,while that of T.yunnanensis was significant difference during the late of trunk-breeding phase.There was significant differences on the temporal-and spatial-niche among three Tomicus species,as well as that between the sexes.The temporal-niche in T.minor was wider than that T.yunnanensi,on the contrary,the spatial-niche in T.minor was narrower.3.Both species were significantly attracted by their own species and the same sex,and this attraction behavior was inhibited by exposure to additional beetles or to the hindgut extracts of beetles which had shown interaction.Female and male of T.minor and T.yunnanensis hindguts contained 0.19,0.09,0.22,and 0.05 ng/individual averagely of(-)-trans-verbenol,respectively;following interaction with additional beetles,this increased to 16.74 – 292.71 ng/individual in T.minor females.Mean concentration of verbenone detected in the hindguts of female/male individuals of T.minor and T.yunnanensis under natural conditions were 0.16,0.06,0.03,and 0.05 ng/individual,respectively,but these correspondingly increased to 5.90,2.43,0.06,and 0.19 ng/individual after exposure to additional insects.In T.yunnanensis,the amounts of detectable(-)-trans-verbenol and verbenone extracted from hindguts were lower than those from T.minor.The levels of cis-verbenol and(-)-trans-verbenol most attractive to walking T.yunnanensis and T.minor were 0.1 and 1.0 ng/μl,respectively.Verbenone was not attractive at any of the concentrations tested.The addition of verbenone to cis-verbenol or(-)-trans-verbenol reduced the attraction responses.4.When the population density of Tomicus was high,two or more bark beetles infested and bored one shoot of the pine tree.When T.minor and T.yunnanensis population compete and coexist in the same tree,the distance between two borer holes was about 2 cm.Comparing to 5-cm and 10-cm distance between females of Tomicus with the same sex and the same species,the distance of 2 cm significantly reduces the number of attractant of the same species.The absolute amounts of trans-verbenol and verbenone in the hindgut of female T.minor was significantly higher than that of the control group and longer distances when the distance between two T.minor females were 2 cm apart.Based on GC-EAD analysis,both male and female of two Tomcius species reacted strongly against trans-verbenol and verbenone.5.The transcriptomes analysis of the intestines and fat bodies of T.minor and T.yunnanensis within and between intraspecific and interspecific interactions for 24 and 48 h were analyzed using the Illumina sequencing.A total of 204.14 Gb Clean Data were obtained.The Q30 base percentage was more than 92.44%.After assembly by Trinity software,a total of 56,917 unigenes were obtained.Unigene’s N50 was 3,804,and 13,578 SSR markers based on SSR analysis were obtained.Further annotation and functional analysis of unigene were performed using GO,COG and KEGG databases,and 29998,17017 and 25267 genes were annotated,respectively.The differentially expressed genes(DEGs)of two Tomicus species were analyzed.The KEGG database was used to annotate the DEGs in the insect hormone synthesis pathway,and the genes involved in the regulation of pheromone biosynthesis were discovered: CYP306A1,CYP302A1,CYP314A1,CYP18A1,CYP307 A.6.The field bioassay was carried out using the attractant trans-verbenol and the inhibition,verbenone to test the effect of the "Push-Pull" control technique.The results showed that the trapping numbers of T.minor and T.yunnanensis were significantly greater than the blank control and control groups.There were fewer traps for T.brevipilosus,and the difference between treatments was not significant.The number of traps per unit area significantly affected the trap catches of T.minor and T.yunnanensis,with negatively correlation.The suspended position of traps also affect the trap catches of bark beetles,and more bark beetles were traped when traps suspended in the middle and lower parts of the trunk.