# The Infestation of Olive Fruits by _Bactrocera oleae_ ([PERSON])
## 2 Results and Discussion
### Effect of Bactrocera oleae Infestation on the Phenolic Profile of VOO
Picual fruits showed a high oxidative degradation of the secoiridoid orthodiphenyls 3,4-DHPEA-EDA, and 3,4-DHPEA-EA, but practically no alteration of the secoiridoid monophenols [19]. The changes induced by _B. oleae_ infestation in the relative expression levels of the selected LOX, \(\beta\)-glucosidase, and PPO olive genes were analyzed by RT-QPCR (Figure 5). The two selected LOX genes have 13-LOX activity and therefore produce 13-hydroperoxides that are later transformed by 13-HPL into C6 aldehydes and subsequently into alcohols and volatile esters that determine the green aroma notes of VOO [14]. The data we have on the regulation of their expression suggest that _Oep2 LOX2_ is mainly responsible for the biosynthesis of the VOO aroma, while _Oep1 LOX2_ is more related to the response to wounding, and probably focused on the synthesis of jasmonic acid. No significant differences were found in the expression level of either of the two _LOX_ genes in olive fruits infested by _B. oleae_ (Figure 5). These results apparently do not agree with the highest concentration of volatile compounds derived from LOX pathway found in oils obtained from infested fruits, especially in terms of (_E_)-hex-2-enal content. However, we have previously observed that, although the expression of _Oep2 LOX2_ is not affected, the expression of _Oep1 LOX2_ is rapidly but transiently induced as a consequence of mechanical damage in the olive fruit, reaching its maximum level 90 min after wounding [14]. This brief induction of gene expression caused an increase in LOX activity, sustained over time, which could explain the increase in the biosynthesis of C6 compounds during oil extraction from infested fruits despite not detecting a higher expression levels of the _Oep1 LOX2_ gene. Regarding the metabolism of phenolic compounds, the two \(\beta\)-glucosidase genes directly involved in the formation of the secoiridoid derivatives present in VOO did not show a significant modification of their expression that could explain the lower content of phenols found in the oils obtained from _B. oleae_-infested fruits (Figure 5). The expression of _OeBGLUIA_, whose product has a powerful hydrolytic activity on oleupopein and is mainly responsible for the hydrolysis of phenolic glucosides during the oil extraction process [42], experimented a slight but not statistically significant increase, which seems to support previous findings on the active role of \(\beta\)-glucosidase in the olive defense system [42]. The parallel decrease of _OeBGLUIB_, whose molecular characteristics suggest that it could also be involved in the synthesis of VOO secoiridoids, although with a very modest participation according to its very low expression level [43], does not justify the drastic decrease observed in the content of phenolic compounds in the oils from infested fruits. Unlike \(\beta\)-glucosidase, the study of PPO gene expression levels did show very significant differences between control fruits and fruits infested by _B. oleae_. The expression of _OepPO1_ showed a negligible level in both groups of fruits, control and infested (Figure 5), in line with the level that we had previously found in different olive cultivars, including _cv._ Picul [44]. However, a strong induction of _OepPO2_ expression, which has a significantly higher mean expression level in the olive transcriptome [44], was observed as a consequence of _B. oleae_ infestation, 30 times higher in infested fruits than in the control (Figure 5). The effects of PPO-mediated oxidative defenses against different herbivore pathogens have been reported in plants such as tomato, tea, or strawberry [21]; however, the specific mechanisms are largely unknown. [PERSON] et al. [33] already proposed that the increase in endogenous PPO activity in olive fruits, together with the greater exposure to oxygen due to cell breakage and damage associated to the wound caused by the fly, could explain the notable decrease that they observed in the phenolic content of oils obtained from _B.