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Comparative transcriptomics of mountain pine beetle pheromone-biosynthetic tissues and functional analysis of CYP6DE3

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Comparative transcriptome analysis to tentatively identify MPB genes encoding enzymes active in pheromone-biosynthetic pathways is based on the hypothesis that the genes for each pathway are coordinately regulated. It has proven useful for prior studies [15, 18]. We extended this approach here by comparing expression profiles in pheromone-biosynthetic tissues of fed and unfed female and male mountain pine beetles. Extensive RNA-Seq profiling yielded nearly 425 million paired-end reads, with 74.9% aligning to the reference genome. The clear separation of gender and feeding status shown by PCA underscores the remarkable shifts in genome usage exhibited by these beetles [7, 8]. The close correlation between RNA-Seq and qRT-PCR data (Table 2) supports that the expression values reported here reliably indicate in vivo mRNA levels.

We used a combination of bioinformatics analyses to narrow the pool of candidate pheromone-biosynthetic genes, beginning with four straight-forward comparisons of relative expression levels between physiological conditions. For example, exo-brevicomin-biosynthetic genes would be expected to have elevated expression levels in unfed males compared to both unfed females and fed males, whereas frontalin-biosynthetic genes would be higher in fed males compared to both unfed males and fed females. This produced preliminary pools of only ~200 – 1,200 candidates, depending on the analysis (Table 3; Fig. 3). These pools were enriched for P450 production and activity, consistent with increased metabolic activity upon feeding [7, 8, 18], which complicates identifying enzymes involved primarily in pheromone biosynthesis. Nevertheless, mevalonate pathway enzymes are more predominantly represented in fed males compared to females, consistent with frontalin production (Fig. 5). In parallel, co-expression network analysis by petal using a Spearman Correlation Coefficient and similarity threshold of 0.808 also isolated candidate genes. As these expression data were not normally distributed, the Spearman Correlation Coefficient supplied a robust non-parametric alternative to the standard Pearson Correlation Coefficient. Twenty-two of the final genes selected by the petal analysis appear relevant to exo-brevicomin biosynthesis, while another 71 may be involved in frontalin production.

(–)-trans-Verbenol is produced by a single P450-mediated hydroxylation of (–)-α-pinene, a reaction that may be catalyzed by multiple enzymes as part of a detoxification process [37]. Thus, a “pheromone-biosynthetic” P450 that specifically produces trans-verbenol in females may be an artificial designation. Our current study notes three P450s with relatively high expression levels in fed females compared to unfed females and males (Fig. 6a). A fourth P450, CYP4BD4, showed the highest mRNA levels in females compared to males, though in a pattern that is not consistent with feeding-induced trans-verbenol production.

exo-Brevicomin production from long chain fatty acid precursors in the fat body of unfed males [3] involves steps catalyzed by P450s, a short chain dehydrogenase, and a cyclase (Fig. 1c). High probability candidate genes for exo-brevicomin biosynthesis are likely in the same petal group containing CYP6CR1 and ZnoDH (Fig. 3a). Interestingly, this gene group includes a putative cyclase (YQE_04789) that may catalyze the terminal reaction. The two P450s (CYP6DE3 and CYP4EX1) may be active upstream of ZnoDH to produce and/or hydroxylate 3-nonene. In this respect, the CYP4G56-like P450 (YQE_03851) was not part of the gene group but is of interest given its similar expression profile (Fig. 6b) and identity as a CYP4G. While predicting P450 function from sequences is very difficult, CYP4G family P450s appear to be insect-specific and function as oxidative decarbonylases – yielding hydrocarbons from long chain fatty aldehydes [38, 39]. Thus, YQE_03851 may contribute to 3-nonene production.

Frontalin-biosynthetic steps through the mevalonate pathway to geranylgeranyl diphosphate are well established in fed and JH treated MPB males [40]. Our analysis also identified mRNAs for mevalonate pathway enzymes, including HMGR and GGPPS, to be elevated in fed males compared to other treatment groups. Later steps are likely catalyzed by P450s, a dioxygenase, and a cyclase that should group together with HMGR and GGPPS in the petal analysis. Two P450 genes, CYP6DE4 (YQE_01868) and CYP6BW3 (YQE_02884), did group with HMGR and GGPS (light blue VN in Fig. 3b) while four other P450 genes, CYP345F1 (YQE_06277), CYP6DK1 (YQE_01078), CYP6DH2 (YQE_01329) and a CYP6DK1-like P450 (YQE_01079), grouped into one different VN (orange VN in Fig. 3b). The two VNs are connected directly by two links, and both gene groups portray increased expression in fed males (Fig. 3c), a pattern consistent with frontalin biosynthesis. Interestingly, a putative dioxygenase was not identified, which may suggest alternative activities on a GGPP precursor, perhaps catalyzed by a cytochrome P450. It is also noteworthy that the cyclase identified in the “exo-brevicomin cluster” (YQE_04789) also shows elevated mRNA levels in fed males (Fig. 6b). Given the structural similarities of the epoxide precursors for both exo-brevicomin and frontalin, it is possible that a single cyclase could serve the terminal steps in both pathways.

While comparative transcriptomics is invaluable to preliminarily identify putative pheromone-biosynthetic genes, a more accurate assessment requires additional information [10]. For MPB, our transcriptomic analyses return more candidate genes than there are reactions to catalyze. We hypothesized that those with elevated expression upon exposure to monoterpenes are more likely to contribute to resin detoxification than pheromone component production (except for the case of trans-verbenol, as noted above). We therefore measured relative mRNA levels for CYP6DE3, which we had tagged as a potential exo-brevicomin biosynthetic enzyme, in beetles that had been exposed to atmospheres saturated with various monoterpenes. The clear elevation observed for all cases (Fig. 7) suggests that CYP6DE3 is induced by monoterpene exposure, particularly in females, implying a resin-detoxifying role. The absence of this induction in fed insects further implicates that CYP6DE3 regulation is complex. The monoterpene-dependent difference in response in males and females is curious, but has been exhibited in another study reporting similar sex-specific transcriptional responses of various D. armandi P450 genes in response to monoterpenes [41]. A detoxification role for CYP6DE3 is supported by functional assays of the recombinant enzyme which showed that it oxidized a variety of monoterpenes, but did not appear to accept exo-brevicomin precursors as substrates (Fig. 8 and data not shown). Interestingly, the products at 15.65 min for (+)-α-pinene and 14.78 min for 3-carene have a m/z peak at 168 suggesting these substrates were oxidized twice (Fig. 8a and b).

De novo pheromone component biosynthesis in pine bark beetles is affected by sex, feeding status, environment, and JH III [

10

], with JH III treatment sometimes being sufficient to elevate mRNA levels of pheromone-biosynthetic genes even in insects that otherwise require feeding to trigger pheromone production [

17

]. Indeed, JH III stimulates both frontalin [

5

,

11

] and

trans-

verbenol biosynthesis, but not

exo

-brevicomin biosynthesis [

11

] in MPB. Our study complements those of Robert et al. [

8

], who compared fed and JH III-treated whole insects and concentrated on a survey of detoxification mechanisms, and Keeling et al. [

11

], who compared starved and JH III-treated midguts and fat bodies. Our study differs in that we focused on midgut and fat body tissues of fed and unfed insects rather than JH III-treated insects because of the evident complexity in regulating production of these three main pheromone components. Several putative pheromone-biosynthetic genes identified in our study agree with those reported by Keeling et al. [

11

] (Table 

4

), and the increased confidence resulting from this concurrence makes the common genes high priorities for functional assays. It is also noteworthy that CYP6DE4 does not accept pheromone precursors despite being induced by JH III [

11

]. The discrepancies in the list of candidate enzymes are likely due to a combination of factors, including differences in experimental design and data analysis. Given that the populations used by Keeling et al. [

11

] and us appear to be geographically and genetically isolated [

42

], it is also possible that their responses to different conditions also differ ([

2

], unpublished data).

Table 4

Candidate genes for MPB pheromone biosynthesis identified by RNA-seq using feeding status or JH treatment

Pheromone biosynthetic pathway

trans-Verbenol

CYP349B2

 

CYP6DJ1

   

CYP6DJ2

exo-Brevicomin

CYP6DE3

CYP6DE4

CYP4EX1

CYP6BW4

CYP18A1

CYP6DF1

CYP4CV2

CYP4BQ1

CYP6CR1

P450 YQE_03851

 

ZnoDH

Cyclase YQE_04789

   

Frontalin

CYP6DE4

CYP6BW1

HMGR YQE_02503

CYP6BW3

 

GGPPS YQE_09494

CYP345F1

 

CYP6DK1

Cyclase YQE_04789

   

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