Comparison of orthologous cytochrome P450 genes relative expression patterns in the bark beetles Dendroctonus rhizophagus and Dendroctonus valens (Curculionidae: Scolytinae) during host colonization

Abstract

Bark beetles of the genus Dendroctonus are important components of coniferous forests. During host colonization, they must overcome the chemical defences of their host trees, which are metabolized by cytochrome P450 (CYP or P450) enzymes to compounds that are readily excreted. In this study, we report the relative expression (quantitative real-time PCR) of four orthologous cytochrome P450 genes (CYP6BW5, CYP6DG1, CYP6DJ2 and CYP9Z20) in Dendroctonus rhizophagus and Dendroctonus valens forced to attack host trees at 8 and 24 h following forced attack and in four stages during natural colonization [solitary females boring the bark (T1); both male and female members of couples before oviposition (T2); both male and female members of couples during oviposition (T3), and solitary females inside the gallery containing eggs (T4)]. For both species gene expression was different compared with that observed in insects exposed to single monoterpenes in the laboratory, and the expression patterns were significantly different amongst species, sex, gut region and exposure time or natural colonization stage. The induction of genes (CYP6BW5v1, CYP6DJ2v1 and CYP9Z20v1 from D. rhizophagus, as well as CYP6DG1v3 from D. valens) correlated with colonization stage as well as with the increase in oxygenated monoterpenes in the gut of both species throughout the colonization of the host. Our results point to different functions of these orthologous genes in both species.

Time-Course of CYP450 Genes Expression From Dendroctonus rhizophagus (Curculionidae: Scolytinae) During Early Hours of Drilling Bark and Settling Into the Host Tree

“…In particular, CYP6DJ2 transcript levels were higher in solitary and paired females than males throughout the different times assayed, suggesting that this gene could be involved in other sex-specific roles in the endogenous metabolism. This CYP pattern is consistent with that reported by Obregón-Molina et al (2015), who registered an elevated number of transcripts in females at 8 and 24 h, as well as through different colonization stages in the field. In addition, expression patterns in which the induction of a gene is paused in one sex while in the other continues, have been reported in other bark beetles (Sandstrom et al 2006, Huber et al 2007), suggesting different functions in each sex or a change of the metabolic pathway in which they are involved (Pitt et al 2014).…”

“…Previous studies have also documented differential expression of some CYP genes of families 4, 6, and 9 in stimulated or fed bark beetles; however, no one has evaluated these genes before 8 h in Dendroctonus species (Huber et al 2007, Cano-Ramírez et al 2013, López et al 2013, Dai et al 2015, Obregón-Molina et al 2015). Nevertheless, our results and those previously reported for CYP6BW5, CYP6DG1, CYP6DJ2 , CYP9Z18 , and CYP9Z20 genes in stimulated and fed D. rhizophagus insects beyond 8 h (Cano-Ramírez et al 2013, Obregón-Molina et al 2015), jointly suggest that the expression of these CYP s is a dynamic process with up- and down-regulations in response to the colonization process and probably to mating and oviposition (Obregón-Molina et al 2015).…”

“…A high expression of these genes has also been observed in the gut and antennae of female and male of both D. rhizophagus (Cano-Ramírez et al 2013) and D. valens (López et al 2013), after exposing them to single host monoterpenes. The expression patterns of these genes are not sex-specific, suggesting that they have a similar function in males and females (Feyereisen 1999, 2011; Huber et al 2007; Schuler 2011; Cano-Ramírez et al 2013; López et al 2013; Robert et al 2013; Dai et al 2015; Obregón-Molina et al 2015).…”

“…For bark beetles, in particular, some CYP genes have demonstrated a capacity to metabolize different toxic monoterpenes, such as α-pinene, and myrcene, through hydroxylation reactions; these genes may also participate in the de novo synthesis of pheromones (Sandstrom et al 2006, 2008; Song et al 2013; Nadeau et al 2017). Several studies have analyzed the expression of specific CYPs under diverse experimental conditions (e.g., insects fed and unfed; solitary and paired; different exposure times; single monoterpenes, racemic mixtures, and combinations; with assays of individuals in both laboratory and field) and documented and evaluated their expression through transcriptome analysis following exposure to stimuli (Huber et al 2007, Keeling et al 2012, Cano-Ramírez et al 2013, López et al 2013, Dai et al 2015, Obregón-Molina et al 2015, Nadeau et al 2017). Some CYP genes from families 4, 6, and 9 in Dendroctonus rhizophagus Thomas and Bright and their respective orthologues in Dendroctonus valens have been characterized molecularly and their expression evaluated in vitro in pre-emerged insects exposed to different monoterpene vapors at 8 and 24 h (Cano-Ramírez et al 2013, López et al 2013), as well as adults at different colonization stages in the field: solitary females, pairs before oviposition, pairs after oviposition, and solitary females once the male has left the gallery (Obregón-Molina et al 2015).…”

The Differential Expression of Mevalonate Pathway Genes in the Gut of the Bark Beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) Is Unrelated to the de Novo Synthesis of Terpenoid Pheromones

“…All amplicons were visualized in 1% agarose gel electrophoresis to evaluate the specificity. Relative expression values were calculated according to the 2 −ΔΔ C t method [104], using CYP4G55v1 as the reference gene for normalizing expression levels [90,91,105,106,107].…”