Seasonal shifts in accumulation of glycerol biosynthetic gene transcripts in mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), larvae.

Winter mortality is a major factor regulating population size of the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). Glycerol is the major cryoprotectant in this freeze intolerant insect. We report findings from a gene expression study on an overwintering mountain pine beetle population over the course of 35 weeks. mRNA transcript levels suggest glycerol production in the mountain pine beetle occurs through glycogenolytic, gluconeogenic and potentially glyceroneogenic pathways, but not from metabolism of lipids. A two-week lag period between fall glycogen phosphorylase transcript and phosphoenolpyruvate carboxykinase transcript up-regulation suggests that gluconeogenesis serves as a secondary glycerol-production process, subsequent to exhaustion of the primary glycogenolytic source. These results provide a first look at the details of seasonal gene expression related to the production of glycerol in the mountain pine beetle.

Overexpression of PPARγ specifically in pancreatic ß-cells exacerbates obesity-induced glucose intolerance, reduces ß-cell mass, and alters islet lipid metabolism in male mice.

The contribution of peroxisomal proliferator-activated receptor (PPAR)-γ agonism in pancreatic ß-cells to the antidiabetic actions of thiazolidinediones has not been clearly elucidated. Genetic models of pancreatic ß-cell PPARγ ablation have revealed a potential role for PPARγ in ß-cell expansion in obesity but a limited role in normal ß-cell physiology. Here we overexpressed PPARγ1 or PPARγ2 specifically in pancreatic ß-cells of mice subjected to high-fat feeding using an associated adenovirus (ß-PPARγ1-HFD and ß-PPARγ2-HFD mice). We show ß-cell-specific PPARγ1 or PPARγ2 overexpression in diet-induced obese mice exacerbated obesity-induced glucose intolerance with decreased ß-cell mass, increased islet cell apoptosis, and decreased plasma insulin compared with obese control mice (ß-eGFP-HFD mice). Analysis of islet lipid composition in ß-PPARγ2-HFD mice revealed no significant changes in islet triglyceride content and an increase in only one of eight ceramide species measured. Interestingly ß-PPARγ2-HFD islets had significantly lower levels of lysophosphatidylcholines, lipid species shown to enhance insulin secretion in ß-cells. Gene expression profiling revealed increased expression of uncoupling protein 2 and genes involved in fatty acid transport and ß-oxidation. In summary, transgenic overexpression of PPARγ in ß-cells in diet-induced obesity negatively impacts whole-animal carbohydrate metabolism associated with altered islet lipid content, increased expression of ß-oxidative genes, and reduced ß-cell mass.

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.

Transcriptome and full-length cDNA resources for the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major insect pest of pine forests.

Bark beetles (Coleoptera: Curculionidae: Scolytinae) are major insect pests of many woody plants around the world. The mountain pine beetle (MPB), Dendroctonus ponderosae Hopkins, is a significant historical pest of western North American pine forests. It is currently devastating pine forests in western North America--particularly in British Columbia, Canada--and is beginning to expand its host range eastward into the Canadian boreal forest, which extends to the Atlantic coast of North America. Limited genomic resources are available for this and other bark beetle pests, restricting the use of genomics-based information to help monitor, predict, and manage the spread of these insects. To overcome these limitations, we generated comprehensive transcriptome resources from fourteen full-length enriched cDNA libraries through paired-end Sanger sequencing of 100,000 cDNA clones, and single-end Roche 454 pyrosequencing of three of these cDNA libraries. Hybrid de novo assembly of the 3.4 million sequences resulted in 20,571 isotigs in 14,410 isogroups and 246,848 singletons. In addition, over 2300 non-redundant full-length cDNA clones putatively containing complete open reading frames, including 47 cytochrome P450s, were sequenced fully to high quality. This first large-scale genomics resource for bark beetles provides the relevant sequence information for gene discovery; functional and population genomics; comparative analyses; and for future efforts to annotate the MPB genome. These resources permit the study of this beetle at the molecular level and will inform research in other Dendroctonus spp. and more generally in the Curculionidae and other Coleoptera.