Insect Origins

Lodgepole pine bolts infested with D. ponderosae were harvested from an area near to Penticton, British Columbia, with outbreak population levels of insects in May of 2010 (UTM: 5478504 northing, 321764 easting, zone 11 U). Bolts were provided by Doug Batemen Logging from the company harvest for that area; no further permissions were required. The collection of bolts did not involve endangered or protected species, and ethics approval is not required by an Animal Care and Use Committee for research on insects. Bolt ends were waxed with paraffin to prevent drying and to ensure optimal beetle development. During late-instar larval development, bolts were contained in vented plastic storage bins at ambient outdoor spring and summer temperatures and were misted with water every few days. During beetle emergence (July through August 2010), cages were checked daily to collect emerged mountain pine beetle adults. The insects were separated by sex according to [13] and were stored at 4°C in petri dishes containing lightly moistened Kimwipes. Beetles were stored for a maximum of 10 days prior to use in experiments and were checked to ensure a lack of storage-related damage prior to use.

Feeding Treatments

As we were interested in changes in transcript levels for adult female and male insects feeding on host plant tissue, we used two treatments: insects starved for 24 hours (control), and insects fed in male-female pairs on host tissues for 24 hours (treatment).

The control beetles were kept in the dark, at room temperature, for 24 hours and those insects were maintained individually in 1.5 mL microcentrifuge tubes with a small hole in the lid. After 24 hours, the insects were flash frozen in liquid nitrogen and stored at −80°C until RNA extraction.

Simultaneously, the treatment insects were placed into the phloem tissue of freshly cut (less than 24 hours before the experiment began) and waxed bolts of lodgepole pine. After drilling a small entrance hole (approximately 3 mm in diameter), insects were placed under the bark in randomly chosen pairs of females and males. Females were placed under the bark first, followed by the males. Insects were held in the holes by wire mesh stapled to the outside of the bark. The treatment insects were allowed to feed under the bark for 24 hours. We removed adults from galleries showing excavation of frass (an indication of feeding) and once again separated the insects into males and females. Beetles were then flash frozen in liquid nitrogen and stored at −80°C for subsequent RNA extraction.

RNA Extraction

RNA extraction was performed with individual whole beetles using the MagMAX™-96 Total RNA Isolation Kit (Ambion). For each beetle, RNA quality was determined using an Experion Automated Electrophoresis Station (BioRad) without heating the extracted RNA to 70°C degrees because of the tendency for the ribosomal RNA 28S subunit band to break in some insects [14], including mountain pine beetle [15]. RNA quantity was determined using a Qubit 2.0 fluorometer (Invitrogen).

A minimum number of four high quality extractions [RNA integrity numbers (RIN) >7] were pooled in order to achieve the 10 µg total RNA required for library construction and RNA-seq analysis at Canada’s Michael Smith Genome Sciences Centre.

Data Analysis

Sequence read information was mapped to the gene models of the male mountain pine beetle genome sequence [16]. Sequence information was converted to fastq format using bam2fastq software (http://www.hudsonalpha.org/gsl/information/software/bam2fastq). The paired-end fastq files were mapped to the genome via CLC Genomics Workbench (http://www.clcbio.com/; CLC bio) using the parameters listed in Table S1. The raw RNA-seq data is available at the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) database (accession numbers SRS421461-64) under the TRIA umbrella BioProject (PRJNA169907) that identifies aggregated research project data generated from the TRIA research collaborations on mountain pine beetle systems genomics.

Statistics

The RNA-Seq data were analysed using DESeq [17] package available from The R Project for Statistical Computing (www.r-project.org). The DESeq package calculates differential expression using a negative binomial distribution and a shrinkage estimator for the distribution’s variance. The program calculates fold changes for each gene as well as p-values adjusted (padj) for multiple comparisons using a Benjamini-Hochberg correction [18]. We used a 1% false discovery rate (padj <0.01) to identify transcript accumulation that was significantly different between treatments. The DESeq output is publicly available via figshare.

Cytochromes P450

Insect cytochrome P450 enzymes have previously been implicated in the detoxification of exogenous compounds. Brattsten et al. [19] showed the induction of mixed function oxidase activity after southern armyworm (Spodoptera eridania) larvae exposure to conifer secondary metabolites. These enzymes are ubiquitous in nature; they are found in bacteria, plants, fungi, and animals. Cytochrome P450 enzymes have diverse functions, but in metazoan they are often involved in the oxygenation of xenobiotics thereby reducing toxicity or facilitate excretion by increasing hydrophilicity. In insects, cytochromes P450 perform a large array of detoxification reactions [20]. Bark beetles express functional cytochromes P450, and the expression and amount of some of these enzymes or their transcripts varies with feeding on host tissues [21]–[23] and with developmental stage [24]; or with treatment juvenile hormone levels [25] suggesting a potential role in metabolite detoxification. Cytochromes P450s are also involved in key physiological processes in bark beetles such as pheromone biosynthesis [26]–[28].

Among the sequences annotated as cytochromes P450 in the male mountain pine beetle genome [16], we identified six transcripts (Table 2) that significantly increased (four in females, one in males, and one in both) and five whose transcript levels significantly decreased following feeding in host phloem (three in females, one in males, and one in both) (Table 3). The cytochrome P450 transcripts that increased significantly with feeding in our experiments were also identified in EST libraries [29] generated from whole adults treated with terpenes and juvenile hormone, whereas those that decreased significantly were predominantly found in libraries originating from the head or antennal region (Table 3). Those that increased in the midgut, fatbody, and the whole adult insects and larvae are more likely to be involved in detoxification of ingested host plant secondary metabolites. One of the cytochrome P450 transcripts that decreased with feeding (CYP345E2) was found only in EST libraries derived from antennal-specific or head-specific tissue. This suggests that this cytochrome P450 is potentially involved in olfaction, a process that is more important during prior host colonization and mate selection prior to feeding.

Although function is almost impossible to predict from the sequence information of a cytochrome P450 gene alone [30], comparison of phylogenies in addition to information of expression in different tissues or under different treatment conditions, may suggest reasonable hypotheses for functional characterization efforts. The annotated cytochrome P450 transcripts identified here align to full-length cDNAs of mountain pine beetle cytochromes P450 identified in [29] and [16]. The cytochromes P450 from our study fall into several CYP families including CYP4, CYP6, and CYP9 (Figure 2). Of the cytochrome P450 transcripts that increased significantly with feeding, four showed a female-specific change (CYP6DH1, CYP345F1, CYP6DJ1, and CYP6DJ2), one showed a male-specific change (CYP6DE4), and one increased significantly in both sexes (CYP6DK1). Of the cytochrome P450 transcripts that decreased significantly with feeding, three of the five showed female-specific changes (CYP6CR2, CYP4BQ1, CYP9AP1), one showed a male-specific shift (CYP6BW2), and one decreased significantly in both sexes (CYP345E2) (Table 3).

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Figure 2. Identified Mountain Pine Beetle Cytochrome P450 Gene Transcripts.

Phylogeny of all identified full-length cytochromes P450 identified in the mountain pine beetle genome as compared to cytochrome P450 sequences from the genomes of Apis melifera, Bombyx mori, and Tribolium castaeum (figure modified from 29). Significantly changing cytochrome P450 transcripts are highlighted, and the direction of change is designated by arrows; female transcripts are in pink, male transcripts are in blue.

https://doi.org/10.1371/journal.pone.0077777.g002

Host tree defense compounds are primary substrate candidates for the functional characterization of up-regulated cytochromes P450. These include terpenoid compounds and phenolics. Sex specific up-regulation of cytochromes P450 suggests a detoxification mechanism that is linked to other sex-specific physiological processes, for example in the production of hormones or pheromones for excretion. Recently, changes in transcripts annotated as cytochrome P450s in the closely related Dendroctonus spp. midguts, fatbodies, and antennae were shown to increase in response to common conifer host monoterpenes [31]–[32]. Our data showed a large significant female-specific increase in CYP6DH1. As female beetles are the pioneers (females initiate attacks, excavate the initial galleries, then are joined by the male beetles), our data suggest a possible increased importance of female detoxification of components of oleoresin in the first 24 hours after colonization. As the males show the largest increase in CYP6DE4 shortly after colonization, functional characterization of this enzyme would shed further light on the host colonization roles and physiological activities of males in the first 24 hours after attack.

Glutathione S-transferases (GST) and Glucosyl/Glucuronosyl Transferases

GSTs and glucosyl or glucuronosyl transferases are a large group of enzymes that transfer a glutathione (or glucosyl, or glucuronsyl) moiety to a variety of substrates. GSTs, in particular, have been implicated in the detoxification of insecticides containing organophosphates [33], organochlorines [34]–[35], and pyrethroids [36]–[37]. GSTs conjugate xenobiotic compounds with a glutathione moiety (GSH) and often work in tandem with cytochromes P450 or other enzymes [38] that aid in detoxification, sequestration or excretion of toxic compounds. GSTs have been previously cloned from spruce budworm, Choristoneura fumiferana, an insect that also feeds on conifer tissue [39], and the mountain pine beetle GSTs have been annotated in EST libraries [29] and in the mountain pine beetle genome [16]. We identified three significantly upregulated transcripts annotated as glucosyl/glucuronosyl transferases in the feeding females, and two are significantly downregulated (Table 4). In males, five transcripts annotated as glucosyl/glucuronosyl transferases increased significantly with feeding, and none were found to significantly decrease (Table 4). Three of the up-regulated transferase transcripts increased in both males and females with very similar fold changes. One of these transcripts annotated as a GST (snap_masked-Seq_1102275-abinit-gene-0.28, DpGSTs2) matched EST database sequences extracted from the midgut and fatbody of fed adult beetles. This putative GST is a good candidate for further study on mountain pine beetle detoxification mechanisms.

Esterases

Esterases are a large class of enzymes that are also implicated in insect resistance to insecticides and other xenobiotics. The up-regulation and mutation of esterase genes has been associated with insecticide resistance in insect orders such as Hymenoptera (wasps), Lepidoptera (moths), and Diptera (flies) (reviewed in [40]). We identified two esterase gene models (genemark-Seq_1102891-abinit-gene-0.13 and maker-Seq_1102774-snap-gene-21.56) that were significantly up-regulated in fed females and that may have roles in host chemical detoxification. We also observed two esterase gene transcripts (maker-Seq_1102308-snap-gene-6.52 and maker-Seq_1103039-snap-gene-0.57) that decreased significantly in female beetles allowed to feed on host tissues. Transcripts that decrease shortly after host colonization could be associated with physiological processes such as odorant degradation that are more important prior to entry into the host tree.

ABC Transporters

ABC transporters – a large class of proteins best known for multi-drug resistance in humans and Drosophila melanogaster [41] and Anopheles gambiae [42] – have been shown to aid in the sequestration and elimination of toxic xenobiotic compounds in Lepidopteran insects [43], and may perform a similar function in the mountain pine beetle. Although we identified 12 transcripts that may be important for signaling and transport mechanisms in females allowed to feed on host tissue (Figure 2), transcript levels of only one ABC transporter (maker-Seq_1102955-augustus-gene-1.33) significantly increased in females beetles feeding on host tissue (Table 6). Evidence for this transcript could only be found in the EST libraries (library DPO11) originating from combined midgut/fatbody tissue of beetles feeding on host tissue [29]. We hypothesize that this gene is involved in digestion and detoxification of the ingested, resin-saturated phloem that is present in the tree after the initial attack [12].

Alcohol Dehydrogenases

The transcripts of three putative alcohol dehydrogenases increased significantly in females feeding on host tissue and transcripts for two alcohol dehydrogenases increased significantly in males. In total, four transcripts increased following feeding on host tissue in the two sexes with one (maker-Seq_1093767-snap-gene-0.4) that increased in both males and females. None of the transcripts that decreased significantly in males or females were annotated as alcohol dehydrogenases. Alcohol dehydrogenases may play a role in metabolizing terpenoid alcohols that are a component of lodgepole pine oleoresin [44], and bark beetles are able to sense and respond to terpene alcohols [45]. For example, 3-caren-10-ol altered the sex specificity of attraction to pheromone bait, and the terpene composition of the host tree determined some of the pheromone production during host colonization [46]. Terpene alcohols also impact other coniferophagous beetles, for example the volatile monoterpene, linalool, that is produced de novo by Sitka spruce in response to attack by white pine weevil [47]. Conifers contain a large number of metabolites with alcohol functional groups including ethanol and phenolic compounds that are produced after mountain pine beetle attack [48]. Finally, alcohol dehydrogenases have been functionally characterized in essential bark beetle metabolic processes. For example, ipsdienol dehydrogenase (IDOLDH) acts on hydroxylated myrcene to produce ipsdienol, an important aggregation pheromone in Ips pini to produce ipsdienone [49]. As transcripts for alcohol dehydrogenases showed general increases, and none decreased in our study, this group of enzymes warrants further study for its role in the detoxification of host specialized metabolites in MPB.

Oxidative Stress, Damage Control, and Immune Response

A number of transcripts were identified by their annotations to have a likely role in stress physiology, damage control, and an immune response after host colonization.

Reproduction, Pheromone Flux, and Digestion

Other metabolic processes evident from shifts in transcript levels following feeding include the production of vitellogenin precursors in preparation for a metabolic switch to reproduction in females, the production of the peritrophic matrix involved in digestion, the down-regulation of gene transcripts annotated as key enzymes in the citric acid cycle, and a reduction in the enzymes required for fatty acid synthesis.