Christopher I Keeling

• PhD
• Research Scientist in Forest Genomics at Natural Resources Canada

Genome sequencing methods and assembly tools have improved dramatically since the 2013 publication of draft genome assemblies for the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). We conducted proximity ligation library sequencing and scaffolding to improve contiguity, and then used linkage mapping and recent bi...

Significance trans -Verbenol is a critical aggregation pheromone of the female mountain pine beetle. It is derived from a compound of the host defense, the monoterpene α-pinene. We found that beetles accumulate monoterpenyl esters during early life stages. These may serve as a previously unknown reservoir for the sex-specific release of aggregation...

Background Flight can drastically enhance dispersal capacity and is a key trait defining the potential of exotic insect species to spread and invade new habitats. The phytophagous European spongy moths (ESM, Lymantria dispar dispar) and Asian spongy moths (ASM; a multi–species group represented here by L. d. asiatica and L. d. japonica), are global...

Insects have developed various adaptations to survive harsh winter conditions. Among freeze-intolerant species, some produce “antifreeze proteins” (AFPs) that bind to nascent ice crystals and inhibit further ice growth. Such is the case of the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae), a destructive North American conifer...

The highly diverse insect family of true weevils, Curculionidae, includes many agricultural and forest pests. Pissodes strobi, commonly known as the spruce weevil or white pine weevil, is a major pest of spruce and pine forests in North America. P. strobi larvae feed on the apical shoots of young trees, causing stunted growth and can destroy regene...

Spruce Beetle: A Synthesis of Biology, Ecology, and Management in Canada This book presents a synthesis of published information on the biology, ecology, and management of spruce beetle, Dendroctonus rufipennis (Kirby) (Coleoptera: Curculionidae), in Canada. It is intended as a reference for forest practitioners, researchers, and students. The vol...

This chapter presents our current knowledge of pheromone production in bark beetles, emphasizing the biochemical, molecular, functional genomics, and post-genomic studies in Ips and Dendroctonus species. Ipsdienol, ipsenol and cis-verbenol were identified as the first aggregation pheromone components from bark beetles in 1966. Since then, many addi...

Bark beetles (family: Curculionidae; subfamily: Scolytinae) in the Dendroctonus and Ips genera are the most destructive forest pests in the Northern hemisphere. They use cytochromes P450 (P450 s) to detoxify tree-produced terpenes to produce pheromones, in de novo pheromone production and to oxidize odorants on antennae. Many Dendroctonus spp. use...

Background Olfaction and gustation underlie behaviors that are crucial for insect fitness, such as host and mate selection. The detection of semiochemicals is mediated via proteins from large and rapidly evolving chemosensory gene families; however, the links between a species’ ecology and the diversification of these genes remain poorly understood...

Background Olfaction and gustation underlie behaviors that are crucial for insect fitness, such as host and mate selection. The detection of semiochemicals is mediated via proteins from large and rapidly evolving chemosensory gene families; however, the links between a species’ ecology and the diversification of these genes remain poorly understood...

Background: Olfaction and gustation underlie behaviors that are crucial for insect fitness, such as host and mate selection. The detection of semiochemicals is mediated via proteins from large and rapidly evolving chemosensory gene families; however, the links between a species' ecology and the diversification of these genes remain poorly understo...

Background Olfaction and gustation underlie behaviors that are crucial for insect fitness, such as host and mate selection. The detection of semiochemicals is mediated via proteins from large and rapidly evolving chemosensory gene families; however, the links between a species’ ecology and the diversification of these genes remain poorly understood...

The mountain pine beetle (MPB; Dendroctonus ponderosae) is a forest insect pest that attacks several different pine (Pinus) species in its native range of distribution in western North America. MPB are exposed for most of their life cycle to the chemical defenses of their hosts. These defenses are dominated by oleoresin secretions containing mostly...

Denatured CYP6DJ1, CYP6BW1, CYP6BW3 and empty vector control microsomes on a 12% SDS-Page gel. Lane 1: Precision Plus Protein ladder (Bio-rad). Lane 2: empty vector microsomes. Lane 3: CYP6BW1 microsomes, protein band is visible between 75 kDa and 50 kDa. Lane 4: CYP6BW3 microsomes, protein band is visible between 75 kDa and 50 kDa. Lane 5: Precisi...

CO Spectra of CYP6DJ1, CYP6BW1, CYP6BW3 and empty vector control microsomes. (PDF)

Mass spectra of peaks 1–8 from the gas chromatograms of extracts of CYP6DJ1 or female beetles treated with (–)-(4S)-limonene along with the standards. Gas chromatograms with peak numbers can be found in Fig 2. (PDF)

Mass spectra of peaks 15–18 from the gas chromatograms of extracts of CYP6BW1 or CYP6BW3 or female beetles treated with isopimaric, palustric, or dehydroabietic acid. Gas chromatograms with peak numbers can be found in Fig 7A–7C. (PDF)

Mass spectra of peaks 3–9 from the gas chromatograms of extracts of CYP6DJ1 or female beetles treated with (+)-(4R)-limonene along with the standards. Gas chromatograms with peak numbers can be found in Fig 1. (PDF)

Mass spectra of peaks 9 and 10 from the gas chromatograms of extracts of CYP6DJ1 or female beetles treated with with (–)-(4S)-limonene along with the standards. Gas chromatograms with peak numbers can be found in Fig 2. (PDF)

Mass spectra of peaks 11–14 from the gas chromatograms of extracts of CYP6DJ1 or female beetles treated with terpinolene. Gas chromatograms with peak numbers can be found in Fig 3. (PDF)

Mass spectra of peaks 19–21 from the gas chromatograms of extracts of CYP6BW1 or CYP6BW3 or female beetles treated with levopimaric, abietic or neoabietic acid. Gas chromatograms with peak numbers can be found in Fig 7D–7F. (PDF)

Activity assay of CYP6DJ1, CYP6BW1 and CYP6BW3 with ten selected monoterpenes and six diterpene resin acid substrates. Red crosses indicate that no product was detected in GC chromatograms of the assay, green checkmarks indicate that one or more products were detected in the assay. (PDF)

The retention index of all limonene and terpinolene products of CYP6DJ1 and from extracts of MPB after treatment. All samples were injected onto a DB-Wax column. See Figs 1–5 and S3–S6 Figs. for the gas chromatograms, structures and mass spectra of these peaks. (PDF)

The retention index of diterpene resin acid products of CYP6BW1 and CYP6BW3 and from extracts of MPB after treatment. All samples were injected onto a HP-5 column. See Fig 7 and S7 and S8 Figs for the gas chromatograms and mass spectra of these peaks. (PDF)

The recent outbreak of the mountain pine beetle (Dendroctonus ponderosae; MPB) has affected over 20 M hectares of pine forests in western North America. During the colonization of host trees, female MPB release the aggregation pheromone (−)-trans-verbenol. (−)-trans-Verbenol is thought to be produced from the pine defense compound (−)-α-pinene by c...

The mountain pine beetle (MPB, Dendroctonus ponderosae) is a forest insect pest endemic to western North America. During dispersal and host colonization, MPB identify suitable host trees by olfaction of monoterpene volatiles, contend with host terpene defenses, and communicate with conspecifics using terpenoid and other pheromones. Cytochromes P450...

The seed coat of fir (Abies), hemlock (Tsuga), and cedar (Thuja) species contain terpenoid resin vesicles. Although information is limited about the morphology and allelopathy of these vesicles, their damage during seed processing can negatively impact germination success. We examined resin vesicle morphology of western redcedar (Thuja plicata Donn...

The mountain pine beetle (Dendroctonus ponderosae; MPB) is an eruptive bark beetle species affecting pine forests of western North America. MPB are exposed to volatile monoterpenes, which are important host defense chemicals. We assessed the toxicity of the ten most abundant monoterpenes of lodgepole pine (Pinus contorta), a major host in the curre...

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...

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...

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...

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...

Bark beetles (Coleoptera: Scolytinae) are pests of many forests around the world. The rapid development of molecular tools and high-throughput sequencing has permitted the examination of the biological processes of pheromone biosynthesis and host col- onization at the genomic and molecular levels. I briefly review the available information on host...

Bark beetles (Coleoptera: Scolytinae) are pests of many forests around the world. The mountain pine beetle (MPB), Dendroctonus ponderosae Hopkins, is a significant pest of western North American pine forests. The MPB is able to overcome the defences of pine trees through pheromone-assisted aggregation that results in a mass attack of host trees. Th...

White spruce (Picea glauca), a gymnosperm tree, has been established as one of the models for conifer genomics. We describe the draft genome assemblies of two white spruce genotypes, PG29 and WS77111, innovative tools for the assembly of very large genomes, and the conifer genomics resources developed in this process. The two white spruce genotypes...

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,...

Insights from sequenced genomes of major land plant lineages have advanced research in almost every aspect of plant biology. Until recently, however, assembled genome sequences of gymnosperms have been missing from this picture. Conifers of the pine family (Pinaceae) are a group of gymnosperms that dominate large parts of the world's forests. Despi...

The mountain pine beetle (MPB; Dendroctonus ponderosae Hopkins), a major pine forest pest native to western North America, has extended its range north and eastward during an ongoing outbreak. Determining how the MPB has expanded its range to breach putative barriers, whether physical (nonforested prairie and high elevation of the Rocky Mountains)...

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 lar...

Significance A long-standing question in pheromone biosynthesis is the origin of the mevalonate pathway-derived precursor to frontalin, a key pheromone to the successful mass attack of conifer hosts by Dendroctonus bark beetles. Using a combination of genome and transcriptome analysis, functional characterization of expressed proteins, RNA interfer...

Sandalwood oil is one of the world's most highly prized essential oils, appearing in many high-end perfumes and fragrances. Extracted from the mature heartwood of several Santalum species, sandalwood oil is comprised mainly of sesquiterpene olefins and alcohols. Four sesquiterpenols, α-, β-, and epi-β-santalol and α-exo-bergamotol, make up approxim...

White spruce (Picea glauca) is a dominant conifer of the boreal forests of North America, and providing genomics resources for this commercially valuable tree will help improve forest management and conservation efforts. Sequencing and assembling the large and highly repetitive spruce genome though pushes the boundaries of the current technology. H...

Background The mountain pine beetle (MPB, Dendroctonus ponderosae) epidemic has affected lodgepole pine (Pinus contorta) across an area of more than 18 million hectares of pine forests in western Canada, and is a threat to the boreal jack pine (Pinus banksiana) forest. Defence of pines against MPB and associated fungal pathogens, as well as other p...

Background The mountain pine beetle, Dendroctonus ponderosae Hopkins, is the most serious insect pest of western North American pine forests. A recent outbreak has destroyed more than 15 million hectares of pine forests with major environmental impacts on forest health, and economic impacts on the forest industry. The outbreak has in part been driv...

Background The European spruce bark beetle, Ips typographus, and the North American mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae: Scolytinae), are severe pests of coniferous forests. Both bark beetle species utilize aggregation pheromones to coordinate mass-attacks on host trees, while odorants from host and non-host tre...

Accession numbers for genes encoding olfactory proteins that were submitted to GenBank and the Transcriptome Shotgun Assembly sequence database (TSA) at NCBI.

Gene ontology results. Gene ontology analyses as in Figure 1, but here represented as bar diagrams that have a higher resolution. A) Molecular function level 3 in Ips typographus, B) molecular function level 3 in Dendroctonus ponderosae, C) biological process level 2 in I. typographus, and D) biological process level 2 in D. ponderosae.

Presence of chemosensory proteins in various tissues of Dendroctonus ponderosae. Analyses of Sanger-specific data and normalized as well as non-normalized transcriptome assemblies from various body parts of D. ponderosae indicate that 4 of the 11 identified chemosensory proteins were found exclusively in non-antennal tissues. The numbers in the tab...

Shared chemosphere of I. typographusandD. ponderosae. List of 54 semiochemicals that are produced by the two bark beetle species, or present in their host or non-host plants and whether the compounds are active at a physiological and/or behavioral level in each species.

Presence of odorant binding proteins in various tissues of Dendroctonus ponderosae. Analyses of Sanger-specific data and normalized as well as non-normalized transcriptome assemblies from various body parts of D. ponderosae indicate that large sets of odorant binding proteins were found exclusively in non-antennal tissues. The numbers in the tables...

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 mi...

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 Columb...

In order to successfully reproduce, adult mountain pine beetles must attack a host tree and overwhelm its defenses. The female is the first-attacking sex in this species. Arriving females, and the later-arriving males, produce aggregation pheromones as they invade the phloem tissue of host pines. Both sexes inoculate the tree with symbiotic, pathog...

Conifers have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. The large diversity of terpenoid metabolites is determined by the size and composition of the terpene synthase (TPS) gene family, and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3-caren...

The levopimaradiene/abietadiene synthase from Norway spruce (Picea abies; PaLAS) has previously been reported to produce a mixture of four diterpene hydrocarbons when incubated with geranylgeranyl diphosphate as the substrate: levopimaradiene, abietadiene, neoabietadiene, and palustradiene. However, variability in the assay products observed by GC-...

This article documents the addition of 238 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Alytes dickhilleni, Arapaima gigas, Austropotamobius italicus, Blumeria graminis f. sp. tritici, Cobitis lutheri, Dendroctonus ponderosae, Glossina morsitans morsitans, Haplophilus subterr...

The levopimaradiene/abietadiene synthase from Norway spruce (Picea abies) (PaLAS) has previously been reported to produce a mixture of four diterpene hydrocarbons when incubated with geranylgeranyl diphosphate as the substrate: levopimaradiene, abietadiene, neoabietadiene, and palustradiene. However, variability in the assay products observed by GC...

In conifers, terpene synthases (TPSs) of the gymnosperm-specific TPS-d subfamily form a diverse array of mono-, sesqui-, and diterpenoid compounds, which are components of the oleoresin secretions and volatile emissions. These compounds contribute to defence against herbivores and pathogens and perhaps also protect against abiotic stress. The avail...

Conifers are extremely long-lived plants that have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. In these species, terpenoid diversity is determined by the size and composition of the terpene synthase (TPS) gene family and the single- and multi-product profiles of these enzymes...

Table S1. Excel file containing the supplementary table with all unigene locations, their coordinates on the G. clavigera genome, the ESTs that mapped to each UL, relevant annotations, and expression analysis results.

Conifers produce terpenoid-rich oleoresin in specialized resin ducts as a main line of defence against pests and pathogens. In spruce species (Picea spp.), axial resin ducts are either present constitutively in the cortex tissue (cortical resin ducts, CRDs) or are formed de novo as traumatic resin ducts (TRDs) in the cambial zone upon attack by ins...

Grosmannia clavigera is a bark beetle-vectored fungal pathogen of pines that causes wood discoloration and may kill trees by disrupting nutrient and water transport. Trees respond to attacks from beetles and associated fungi by releasing terpenoid and phenolic defense compounds. It is unclear which genes are important for G. clavigera's ability to...

Table S3: Information for genes shown in Figure 5. Names, GenBank accession numbers, and tentative BLSTX identifications of genes (Features) in clusters incorporated into Figure 5.

Table S1: Summary of qRT-PCR and microarray comparisons. Relative expression values for nine selected genes (Features) in 10 different biological states, each compared to unfed females (F24u). The ratios were determined by qRT-PCR and from microarray data as described in the text. Values in this table were used to create Figure 4.

Table S2: Cleansed, normalized and clustered microarray expression values. Microarray expression values are provided for those genes (Features) with at least 0.85 average pairwise correlation across the 11 biological states to create clusters. GO identifiers are also provided.

The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene s...

The mountain pine beetle (Dendroctonus ponderosae) is a significant coniferous forest pest in western North America. It relies on aggregation pheromones to colonize hosts. Its three major pheromone components, trans-verbenol, exo-brevicomin, and frontalin, are thought to arise via different metabolic pathways, but the enzymes involved have not been...

Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs...

Figure S1 - Alignment of nucleic acid sequences of four closely related 3CAR gDNA fragments from white spruce (Picea glauca, Pg_3CAR1-4) and Sitka spruce (Picea sitchensis) (+)-3-carene synthase (Ps_Q09). The numbering above the alignment corresponds to the nucleotide position of the complete 3CAR gene of PGB02. Underlined sequences correspond to p...

Figure S2 - Size and read allocation of the PHRAP assembled contigs of PGB02 (A) and PGB04 (B). The upper panel in each of A and B shows the number of reads in all contigs with the relative percentage of total reads given on top of the bars. The lower panel in A and B shows the length of all contigs given in bp with the relative percent of the leng...

Figure S3 - Effect of sequencing depth on assembly quality. The sequence reads from five plates were used in all possible permutations to build assemblies corresponding to one, two, three and four combined plates. (A) The number of contigs and the number of nucleotides represented in the contigs. (B) Coverage relative to the manually curated sequen...

Table S1. Sequencing summary of plasmid libraries for PGB02 and PGB04.

Table S2. Impact of sequencing depth on assembly quality of PGB04.

Sandalwood, Santalum album (Santalaceae) is a small hemi-parasitic tropical tree of great economic value. Sandalwood timber contains resins and essential oils, particularly the santalols, santalenes and dozens of other minor sesquiterpenoids. These sesquiterpenoids provide the unique sandalwood fragrance. The research described in this paper set ou...

Terpenoids (isoprenoids) encompass more than 40 000 structures and form the largest class of all known plant metabolites. Some terpenoids have well-characterized physiological functions that are common to most plant species. In addition, many of the structurally diverse plant terpenoids may function in taxonomically more discrete, specialized inter...

Terpenoids are the largest class of all known natural products. Plants produce a variety of terpenoid compounds that number in the thousands. Some terpenoids are involved in plant growth and development directly (i.e., in primary metabolism), but most plant terpenoids are thought to function in interactions of plants with their biotic and abiotic e...

We isolated a cDNA of unknown function from a juvenile hormone III (JH III)-treated male midgut cDNA library prepared from the pine engraver beetle, Ips pini, and examined its genomic structure. The gene, tentatively named "Ipi10G08", encoded a 410 amino acid translation product that shared 26-37% identity with unannotated matches from several inse...