Effects of simulated gut pH environment on bacterial composition and pheromone production of Dendroctonus valens

Bark beetles are an economically and ecologically important insect group,with aggregation behavior and thus host colonization success depends on pheromone-mediated communication.For some species,such as the major invasive forest pest in China,red turpentine beetle(Dendroctonus valens),gut microbiota participates in pheromone production by converting tree monoterpenes into pheromone products.However,how variation in gut microenvironment,such as pH,affects the gut microbial composition,and consequently pheromone production,is unknown.In this study,we fed wild caught D.valens with 3 different pH media(main host diet with natural pH of 4.7;a mildly acidic diet with pH 6 mimicking the beetle gut pH;and highly acidic diet with pH 4),and measured their effects on the gut pH,bacterial community and production of the main aggregation and anti-aggregation pheromone(verbenone).We further tested the verbenone production capacity of 2 gut bacterial isolates in different pH environments(pH 6 and 4).Compared to natural state or main host diet,feeding on less acidic diet(pH 6)diluted the acidity of the gut,whereas feeding on highly acidic diet(pH 4)enhanced it.Both changes in gut pH reduced the abundance of dominant bacterial genera,resulting in decreased verbenone production.Similarly,the highest pheromone conversion rate of the bacterial isolates was observed in pH mimicking the acidity in beetle gut.Taken together,these results indicate that changes in gut pH can affect gut microbiota composition and pheromone production,and may therefore have the potential to affect host colonization behavior.

The effect of inactivation of aldehyde dehydrogenase on pheromone production by a gut bacterium of an invasive bark beetle,Dendroctonus valens

Semiochemical-based management strategies are important for controlling bark beetles,such as invasive Red Turpentine Beetle(Denroctonus valens),the causal agent for mass mortality of pine trees(Pinus spp.)in China.It has been previously shown that the pheromone verbenone regulates the attack density of this beetle in a dose-dependent manner and that the gut bacteria of D.valens are involved in verbenone production.However,molecular functional verification of the role of gut bacteria in the pheromone production of D.valens is still lacking.To better understand the molecular function of gut bacterial verbenone production,we chose a facultative anaerobic gut bacterium(Enterobacter xiangfangensis)of D.valens based on its strong ability to convert cis-verbenol to verbenone,as shown in our previous study,and investigated its transcriptomics in the presence or absence of cis-verbenol under anaerobic conditions(simulating the anoxic environment in the beetle's gut).Based on this transcriptome analysis,aldehyde dehydrogenase(ALDH1)was identified as a putative key gene responsible for verbenone production and was knocked-down by homologous recombination to obtain a mutant E.xiangfangensis strain.Our results show that these mutants had significantly decreased the ability to convert the monoterpene precursor to verbenone compared with the wild-type bacteria,indicating that ALDH1 is primarily responsible for verbenone conversion for this bacterium species.These findings provide further mechanistic evidence of bacterially mediated pheromone production by D.valens,add new perspective for functional studies of gut bacteria in general,and may aid the development of new gene silencing-based pest management strategies.