## 3 Discussion  
The phylogenetic analyses of MVA pathway enzymes in _D. rhizophagus_ produced well supported topologies (bootstrap values >63%), suggesting the monophyly of these enzymes in _Dendroctonus_ species. Phylogenies also showed a general topology congruent with the integrity of taxonomical groups (Figure 1). Despite the low number of sequences of these enzymes available in different databases, the integration of consistent monophyletic groups in each MVA pathway enzyme of _Dendroctonus_ spp. suggests that their functions and biochemical characteristics are conserved. In fact, many mutational changes observed in the sequences of genes occurred in the third position of the codons, as suggested by the high frequency of synonym mutations in amino acids located outside the catalytic region. The in silico analysis suggested subcellular localizations for all MVA pathway enzymes in _D. rhizophagus_, which partially agrees with those known for other insect or mammal species (Table 2). For example, the mitochondrial prediction of the AACT enzyme in _D. rhizophagus_ has also been reported as mitochondrial in human [50], but as cytosolic in _Ostrinia scapulalis_[51]. The HMGS enzyme, inferred as cytoplasmic in _D. rhizophagus_, has been recorded as mitochondrial and cytosolic in humans [52] and rats [53], and as cytosolic in German cockroach, _Blattella germanica_[54] and the Pacific beetle cockroach, _Diplottera punctata_[55]. The HMGR, predicted as an enzyme of the endoplasmic reticulum membrane in _D. rhizophagus_, has also been reported in the same site in mammals [56; 57] and insects [58]. The prediction for MK, PMK and MDPC enzymes in _D. rhizophagus_ agree with the cytosolic location [59; 60] or in peroxisomes [61; 62] reported in insects and humans. Lastly, the mitochondrial and cytoplasmic predictions for GPPS/FPPS and GGPPS enzymes in this bark beetle were in concordance with the findings for these enzymes in mammals [63; 64] and _Drosophila melanogaster_[65]. The IPPI enzyme of _D. rhizophagus_ was the only protein whose predicted location (cytoplasmic) disagrees with those reported in mammals (peroxisomes) [66]. The expression analysis showed that phloem feeding and JH III topical application differentially induced several MVA pathway genes in _D. rhizophagus_ (Figure 2). Most genes reached higher expression levels in fed rather than stimulated insects, with solitary and paired males showing the highest levels in almost all genes. These findings agree with previous reports on the expression of these genes in bark beetles, which demonstrated that MVA pathway genes presented higher upregulation levels in males than females in the majority of analyzed genes. In addition, male genes are continuously reported as upregulated, whereas female gene expression can be upregulated, downregulated or remain at basal levels (Table 4). With respect to MVA pathway genes in JH III stimulated insects, an irregular \begin{table}
\begin{tabular}{c c c} \hline \hline
**Compound** & **Female** & **Male** \\ \hline \multirow{2}{*}{fenchyl alcohol} & 0.01–0.041  & - & - \\  & 2/12 2  & 0/12 \\ \hline \multirow{2}{*}{cis-verbenol} & - & - \\  & 0/12 & 0/12 \\ \hline \multirow{2}{*}{trans-verbenol} & - & - \\  & 0/12 & 0/12 \\ \hline \multirow{2}{*}{myrtenal} & - & 0.007 \\  & 0/12 & 1/12 \\ \hline \multirow{2}{*}{myrtenol} & - & 0.03–0.1 \\  & 0/12 & 3/12 \\ \hline \multirow{2}{*}{verbenone} & 0.002–0.003 & 0.002–0.05 \\  & 2/12 & 3/12 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: The range of oxygenated monoterepenes quantities (ng/beelele) extracted from the gut of pre-emerged unfred _D. rhizophagus_. expression pattern was displayed, and there was no tendency for a specific sex or time (Figure 2). However, other studies in bark beetles have reported that JH III induced some of these genes, showing high expression levels mainly in males, or at least higher compared to females [25; 27; 28; 48; 67; 68]. Upregulation was observed mainly in the _AACT_, _HMGS_, _HMGR_, and _PMK_ genes in fed males of _D.