Gene expression analysis of overwintering mountain pine beetle larvae suggests multiple systems involved in overwintering stress, cold hardiness, and preparation for spring development.

Cold-induced mortality has historically been a key aspect of mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), population control, but little is known about the molecular basis for cold tolerance in this insect. We used RNA-seq analysis to monitor gene expression patterns of mountain pine beetle larvae at four time points during their overwintering period-early-autumn, late-autumn, early-spring, and late-spring. Changing transcript profiles over the winter indicates a multipronged physiological response from larvae that is broadly characterized by gene transcripts involved in insect immune responses and detoxification during the autumn. In the spring, although transcripts associated with developmental process are present, there was no particular biological process dominating the transcriptome.

Proteomics indicators of the rapidly shifting physiology from whole mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae), adults during early host colonization.

We developed proteome profiles for host colonizing mountain pine beetle adults, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). Adult insects were fed in pairs on fresh host lodgepole pine, Pinus contorta Dougl. ex Loud, phloem tissue. The proteomes of fed individuals were monitored using iTRAQ and compared to those of starved beetles, revealing 757 and 739 expressed proteins in females and males, respectively, for which quantitative information was obtained. Overall functional category distributions were similar for males and females, with the majority of proteins falling under carbohydrate metabolism (glycolysis, gluconeogenesis, citric acid cycle), structure (cuticle, muscle, cytoskeleton), and protein and amino acid metabolism. Females had 23 proteins with levels that changed significantly with feeding (p<0.05, FDR<0.20), including chaperones and enzymes required for vitellogenesis. In males, levels of 29 proteins changed significantly with feeding (p<0.05, FDR<0.20), including chaperones as well as motor proteins. Only two proteins, both chaperones, exhibited a significant change in both females and males with feeding. Proteins with differential accumulation patterns in females exhibited higher fold changes with feeding than did those in males. This difference may be due to major and rapid physiological changes occurring in females upon finding a host tree during the physiological shift from dispersal to reproduction. The significant accumulation of chaperone proteins, a cytochrome P450, and a glutathione S-transferase, indicate secondary metabolite-induced stress physiology related to chemical detoxification during early host colonization. The females' activation of vitellogenin only after encountering a host indicates deliberate partitioning of resources and a balancing of the needs of dispersal and reproduction.

Comparison of lodgepole and jack pine resin chemistry: implications for range expansion by the mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae).

The mountain pine beetle, Dendroctonus ponderosae, is a significant pest of lodgepole pine in British Columbia (BC), where it has recently reached an unprecedented outbreak level. Although it is native to western North America, the beetle can now be viewed as a native invasive because for the first time in recorded history it has begun to reproduce in native jack pine stands within the North American boreal forest. The ability of jack pine trees to defend themselves against mass attack and their suitability for brood success will play a major role in the success of this insect in a putatively new geographic range and host. Lodgepole and jack pine were sampled along a transect extending from the beetle's historic range (central BC) to the newly invaded area east of the Rocky Mountains in north-central Alberta (AB) in Canada for constitutive phloem resin terpene levels. In addition, two populations of lodgepole pine (BC) and one population of jack pine (AB) were sampled for levels of induced phloem terpenes. Phloem resin terpenes were identified and quantified using gas chromatography. Significant differences were found in constitutive levels of terpenes between the two species of pine. Constitutive α-pinene levels - a precursor in the biosynthesis of components of the aggregation and antiaggregation pheromones of mountain pine beetle - were significantly higher in jack pine. However, lower constitutive levels of compounds known to be toxic to bark beetles, e.g., 3-carene, in jack pine suggests that this species could be poorly defended. Differences in wounding-induced responses for phloem accumulation of five major terpenes were found between the two populations of lodgepole pine and between lodgepole and jack pine. The mountain pine beetle will face a different constitutive and induced phloem resin terpene environment when locating and colonizing jack pine in its new geographic range, and this may play a significant role in the ability of the insect to persist in this new host.

Disentangling detoxification: gene expression analysis of feeding mountain pine beetle illuminates molecular-level host chemical defense detoxification mechanisms.

The mountain pine beetle, Dendroctonus ponderosae, is a native species of bark beetle (Coleoptera: Curculionidae) that caused unprecedented damage to the pine forests of British Columbia and other parts of western North America and is currently expanding its range into the boreal forests of central and eastern Canada and the USA. We conducted a large-scale gene expression analysis (RNA-seq) of mountain pine beetle male and female adults either starved or fed in male-female pairs for 24 hours on lodgepole pine host tree tissues. Our aim was to uncover transcripts involved in coniferophagous mountain pine beetle detoxification systems during early host colonization. Transcripts of members from several gene families significantly increased in insects fed on host tissue including: cytochromes P450, glucosyl transferases and glutathione S-transferases, esterases, and one ABC transporter. Other significantly increasing transcripts with potential roles in detoxification of host defenses included alcohol dehydrogenases and a group of unexpected transcripts whose products may play an, as yet, undiscovered role in host colonization by mountain pine beetle.

Global and comparative proteomic profiling of overwintering and developing mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae), larvae.

BACKGROUND: Mountain pine beetles, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), are native to western North America, but have recently begun to expand their range across the Canadian Rocky Mountains. The requirement for larvae to withstand extremely cold winter temperatures and potentially toxic host secondary metabolites in the midst of their ongoing development makes this a critical period of their lives. RESULTS: We have uncovered global protein profiles for overwintering mountain pine beetle larvae. We have also quantitatively compared the proteomes for overwintering larvae sampled during autumn cooling and spring warming using iTRAQ methods. We identified 1507 unique proteins across all samples. In total, 33 proteins exhibited differential expression (FDR < 0.05) when compared between larvae before and after a cold snap in the autumn; and 473 proteins exhibited differential expression in the spring when measured before and after a steady incline in mean daily temperature. Eighteen proteins showed significant changes in both autumn and spring samples. CONCLUSIONS: These first proteomic data for mountain pine beetle larvae show evidence of the involvement of trehalose, 2-deoxyglucose, and antioxidant enzymes in overwintering physiology; confirm and expand upon previous work implicating glycerol in cold tolerance in this insect; and provide new, detailed information on developmental processes in beetles. These results and associated data will be an invaluable resource for future targeted research on cold tolerance mechanisms in the mountain pine beetle and developmental biology in coleopterans.