Space-time Dynamics of Dendroctonus valens Population in China and Spatial Sampling Technique based on Its Spatial Distribution Pattern

Red turpentine beetle （RTB）, Dendroctongs valens LeConte, is a destructive forest invasive species in China, it mainly attacks Pings tabuliformis and P. bungeana. So far it has spread rapidly to the provinces of Shanxi, Hebei, Henan, Shanxi and Beijing since its first outbreak in Shanxi Province in 1998, and has caused extensive tree mortality. Space-time dynamics of D. valens population and spatial sampling technique based on its spatial distribution pattern were ana- lyzed using geostatistical methods in the pure P. tabuliforis forests and mixedwood stands which were at different damage levels. According to the spatial distribu- tion of D. valeas population, the specific spatial sampling technique was also studied, and then was compared with traditional sampling technique. The spatial sam- piing technique combined with sampling theory and the biological characteristics of D. valens population, which not only could calcnlate the error of the sampling, but also could discuss the optimal sampling number and the optimum size of plot according to different damage levels and different stand types. This helps to explain population expansion and colonization mechanism of D. valens, and to provide a good reference for adopting snitable control measures.

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.

Evaluation of Beauveria bassiana （Hyphomycetes） isolates as potential agents for control of Dendroctonus valens

The red turpentine beetle （RTB）, Dendroctonus valens LeConte, as a destructive invasive pest, has become one of the most economically important forest pest in China. Effective control measures are desperately needed. Entomopathogenic fungi, such as Beauveria bassiana, have shown great potential for the management of some bark beetle species. In this study, 12 isolates of B. bassiana from bark beetle were examined for biological characteristics and virulence, to assess their potential as biocontrol agents for RTB. There were significant differences （at P = 0.05） in colony growth rate, conidial yield, conidial germination, tolerance to UV light and extracellular proteases activity among the tested B. bassiana isolates. Isolates, including Bbl801, Bb1906, Bb789 and Bb773, exhibited the best characteristics, because they have faster hyphal growth rate, higher spore production and faster spore germination, higher UV tolerance and protease （Prl） production. The results of a pathogenicity test ofB. bassiana on RTB larvae showed that most isolates of B. bassiana have demonstrated high efficacy and the highest virulent isolate was Bb 1801, which killed 100% of the treated insects and had a median lethal time （LT50） of 4.60 days at a concentration of 1 ×107 conidia/mL. Therefore, isolate Bb1801 has a great potential for sustainable control of RTB in the forest. The correlation between biological characteristics and virulence of the fungal isolates is discussed and the possibility of combination of entomopathogenic fungi with semiochemicals, as one of the promising strategy for RTB control, is considered.

Colonization patterns of the red turpentine beetle, Dendroctonus valans （Coleoptera： Curculionidae）, in the Luliang Mountains, China

The alien red turpentine beetle （RTB）, Dendroctonus valens LeConte, is one of the most economically destructive forest pests in China, having killed more than 6 million pines in recent years. There is a need to understand the basic biology and ecology of the beetle in order to develop an effective monitoring and management strategy. In this study, the effects of hillside exposure （south- and north-facing）, host-tree locations according to relief （valley, mid-slope, and ridge-top） and tree diameters on RTB colonization were investigated in one valley （3 sites）. The results showed that （i） RTB clearly preferred colonizing pines growing on south-facing hillsides, especially in the valley; （ii） RTB preferred to colonize the pines growing at the valley rather than pines growing at mid-slope or on ridge-top; （iii） RTB preferred to colonize trees with large diameter over small and medium-sized pines; （iv） the attack density of RTBs （measured by pitch tubes/pine） was obviously higher on larger trees standing in the valley than other trees standing at other places. We conclude from RTB colonization patterns, that RTB prefers to attack large trees in the valley, which may be useful in developing a pest-management strategy.

Invasive bark beetle-associated microbes degrade a host defensive monoterpene

Conifers respond to herbivore attack with defensive chemicals, which are toxic to both insects and their associated microorganisms. Microorganisms associated with insects have been widely reported to metabolize toxic chemicals, which may help both microorganisms and host insects overcome host conifer defense. Dendroctonus valens LeConte, an introduced exotic pest from North America to China, has killed millions of healthy pines. Alpha-pinene is the most abundant defensive monoterpene in Chinese Pinus tabuliformis. Although microorganisms associated with D. valens have already been investigated, little is known about their bioactivities when encountering host defensive monoterpenes. In this study, we evaluated the influences of different concentrations ofα- pinene to D. valens and the three most frequently isolated yeasts and bacteria ofD. valens, and further assayed microorganisms＇ capabilities to degrade α-pinene. Results showed that the gallery lengths and body weight changes of bark beetles were significantly affected by 6 mg/mL and 12 mg/mL of α-pinene applied in media compared to controls. The tolerance of experimental microorganisms to α-pinene varied depending on the microbial species. Two out of three yeast strains and all three bacterial strains degraded 20%-50% of α-pinene compared to controls in 24 h in vitro. The microorganisms capable of α-pinene degradation in vitro and their tolerance to high levels ofα-pinene suggested that D. valens- associated microorganisms may help both microorganisms and the bark beetle overcome host α-pinene defense.

Physical contact, volatiles, and acoustic signals contribute to monogamy in an invasive aggregating bark beetle

The behavioral strategies and mechanisms by which some insects maintain monogamous mating systems are not well understood.We investigated the mating system of the bark beetle Dendroctonus valens,and identified several contributing mechanisms.Field and laboratory observations suggest the adults commonly form permanent bonds during host colonization.Moreover,it showed mated females that remained paired with males produced more offspring than mated females that were alone in galleries.In bioassays,a second female commonly entered a gallery constructed by a prior female.Videos show she commonly reached the location of the first female,but they did not engage in actual fighting.Rather,the second female typically departs to form her own gallery.Acoustic signaling likewise does not appear to influence female-female encounters,based on controlled muting experiments.Instead the intruder appears to perceive the residents presence by physical contact.Both acoustic signals and volatiles released by females during gallery constructing were shown to attract males.After a male joined a female in a gallery,the male-produced aggressive sounds,which were shown by playback to deter other males from entering the gallery.Unlike female-female interactions,resident males use their head and rear to push intruders out of galleries.Additionally,volatiles released by males during feeding repelled intruding males,discouraging them from entering the gallery.Males also construct plugs that block the entrance,which may prevent subsequent males and predators from entering the gallery.Thus,D.valens has evolved multifaceted mechanisms contributing to single pairings that confer benefits to both sexes.