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.

A new bacteria-free strategy induced by MaGal2 facilitates pinewood nematode escape immune response from its vector beetle

Symbiotic microbes play a crucial role in regulating parasite–host interactions;however,the role of bacterial associates in parasite–host interactions requires elucidation.In this study,we showed that,instead of introducing numerous symbiotic bacteria,dispersal of 4th-stage juvenile(JIV)pinewood nematodes(PWNs),Bursaphelenchus xylophilus,only introduced few bacteria to its vector beetle,Monochamus alternatus(Ma).JIV showed weak binding ability to five dominant bacteria species isolated from the beetles’pupal chamber.This was especially the case for binding to the opportunistic pathogenic species Serratia marcescens;the nematodes’bacteria binding ability at this critical stage when it infiltrates Ma for dispersal was much weaker compared with Caenorhabditis elegans,Diplogasteroides asiaticus,and propagative-stage PWN.The associated bacterium S.marcescens,which was isolated from the beetles’pupal chambers,was unfavorable to Ma,because it caused a higher mortality rate upon injection into tracheae.In addition,S.marcescens in the tracheae caused more immune effector disorders compared with PWN alone.Ma_Galectin2(MaGal2),a pattern-recognition receptor,was up-regulated following PWN loading.Recombinant MaGal2 protein formed aggregates with five dominant associated bacteria in vitro.Moreover,MaGal2 knockdown beetles had up-regulated prophenoloxidase gene expression,increased phenoloxidase activity,and decreased PWN loading.Our study revealed a previously unknown strategy for immune evasion of this plant pathogen inside its vector,and provides novel insights into the role of bacteria in parasite–host interactions.

Inducible pine rosin defense mediates interactions between an invasive insect-fungal complex and newly acquired sympatric fungal associates

Mutualism between insects and fungi drives insect evolutionary diversification and niche expansion;for invasive insects,however,mechanisms by which they maintain mutualistic relationships with beneficial fungi have not been clearly explored.Here,we report that an invasive herbivorous insect,the red turpentine beetle(RTB),with its co-invasive mutualistic fungus,Leptographium procerum,has newly acquired a set of sympatric fungi during invasion,which could potentially outcompete the RTB mutualistic fungus.Host pine Pinus tabuliformis exhibited more rosin-based responses to the sympatric fungi than to RTB mutualistic fungus and,in return,the rapidly induced rosin suppressed the sympatric fungi more significantly than L.procerum.In addition,from direct fungal pairing competitions,we found that the antagonistic effects of sympatric fungi on L.procerum were drastically reduced under induced rosin defense.Our results together with previous findings imply that pine oleoresin defense(turpentine and rosin)might have been exploited by the invasive mutualistic fungus L.procerum,which helps to explain its invasion success and,by extension,its mutualistic partner RTB in China.

Differential immune responses of Monochamus alternatus against symbiotic and entomopathogenic fungi

Monochamus alternates,the main vector beetles of invasive pinewood nematode,has established a symbiotic relationship with a native ectotrophic fungal symbiont,Sporothrix sp.1,in China.The immune response ofM.alternates to S.sp.1 in the coexistence of beetles and fungi is,however,unknown.Here,we report that immune responses ofM.alternates pupae to infection caused by ectotrophic symbiotic fungus S.sp.1 and entomopathogenic fungus Beauveria bassiana differ significantly.The S.sp.1 did not kill the beetles while B.bassiana killed all upon injection.The transcriptome results showed that the numbers of differentially expressed genes in M.alternates infected with S.sp.1 were 2-fold less than those infected with B.bassiana at 48 hours post infection.It was noticed that Toll and IMD pathways played a leading role in the beetle's immune system when infected by symbiotic fungus,but upon infection by entomopathogenic fungus,only the Toll pathway gets triggered actively.Furthermore,the beetles could tolerate the infection of symbiotic fungi by retracing their Toll and IMD pathways at 48 h.This study provided a comprehensive sequence resource ofM.alternates transcriptome for further study of the immune interactions between host and associated fungi.

Major ascaroside pheromone component asc-C5 influences reproductive plasticity among isolates of the invasive species pinewood nematode

Pheromones are communication chemicals and regulatory signals used by animals and represent unique tools for organisms to mediate behaviors and make“decisions”to maximize their fitness.Phenotypic plasticity refers to the innate capacity of a species to tolerate a greater breadth of environmental conditions across which it adapts to improve its survival,reproduction,and fitness.The pinewood nematode,Bursaphelenchus xylophilus,an invasive nematode species,was accidentally introduced from North America into Japan,China,and Europe;however,few studies have investigated its pheromones and phenotypic plasticity as a natural model.Here,we demonstrated a novel phenomenon,in which nematodes under the condition of pheromone presence triggered increased reproduction in invasive strains(JP1,JP2,CN1,CN2,EU1,and EU2),while it simultaneously decreased reproduction in native strains(US1 and US2).The bidirectional effect on fecundity,mediated by presence/absence of pheromones,is henceforth termed pheromone-regulative reproductive plasticity(PRRP).We further found that synthetic ascaroside asc-C5(ascr#9),the major pheromone component,plays a leading role in PRRP and identified 2 candidate receptor genes,Bxydaf-38 and Bxysrd-10,involved in perceiving asc-C5.These results suggest that plasticity of reproductive responses to pheromones in pinewood nematode may increase its fitness in novel environments following introduction.This opens up a new perspective for invasion biology and presents a novel strategy of invasion,suggesting that pheromones,in addition to their traditional roles in chemical signaling,can influence the reproductive phenotype among native and invasive isolates.In addition,this novel mechanism could broadly explain,through comparative studies of native and invasive populations of animals,a potential underlying factor behind of the success of other biological invasions.