Derived esterase activity inDrosophila sechelliacontributes to evolved octanoic acid resistance

Abstract

The dietary specialist fruit fly Drosophila sechellia has evolved resistance to the secondary defence compounds produced by the fruit of its host plant, Morinda citrifolia. The primary chemicals that contribute to lethality of M. citrifolia are the medium‐chain fatty acids octanoic acid (OA) and hexanoic acid. At least five genomic regions contribute to this adaptation in D. sechellia and whereas the fine‐mapped major effect locus for OA resistance on chromosome 3R has been thoroughly analysed, the remaining four genomic regions that contribute to toxin resistance remain uncharacterized. To begin to identify the genetic basis of toxin resistance in this species, we removed the function of well‐known detoxification gene families to determine whether they contribute to toxin resistance. Previous work found that evolution of cytochrome P450 enzymatic activity or expression is not responsible for the OA resistance in D. sechellia. Here, we tested the role of the two other major detoxification gene families in resistance to Morinda fruit toxins – glutathione‐S‐transferases and esterases – through the use of the pesticide synergists diethyl maleate and tribufos that inhibit the function of these gene families. This work suggests that one or more esterase(s) contribute to evolved OA resistance in D. sechellia.

Genomics Analysis of L-DOPA Exposure inDrosophila sechellia

“…These experiments were performed in a heterologous host ( D. melanogaster ) based on availability of genetic tools, which is a common approach for tests of gene function, however, confirmation of Est6 function in OA resistance requires functional tests in D. sechellia in future studies. The combination of the OA resistance data and gene expression data collected in this study in conjunction with the prior data showing one or more members of the esterase gene family are involved in OA resistance (Lanno and Coolon 2019), and QTL mapping data showing Est6 is consistent with regions of the genome contributing to OA resistance all together strongly suggest that Est6 plays an important role in toxin resistance in D. sechellia . Altogether, identification of a derived amino acid substitution fixed in D. sechellia that alters a residue that resides in the enzymes active site and was shown to influence Est6 substrate-specificity and kinetics suggest that both protein coding as well as gene expression changes may contribute to evolutionary changes at this locus.…”

“…For example, we found that D. sechellia response to L-DOPA includes marked increase in the expression of Esterase 6 ( Est6 , Supplemental Table 4). In our recent study, we found that one or more esterase genes are involved in D. sechellia derived resistance to OA through experiments with the chemical synergist tribufos (S,S,S-Tributyltrithiophosphate) that inhibits all the esterase genes simultaneously (Lanno and Coolon 2019). To determine if Est6 plays a role in evolved OA resistance we used RNAi in D. melanogaster to reduce the expression of Est6 and examined the effect on OA resistance.…”

“…In response to L-DOPA exposure, we found that Est6 had a significant and marked increase in expression level. This observation was intriguing because our recent study using the chemical synergist tribufos to inactivate all the esterase enzymes simultaneously (Plapp et al 1963; Snoeck et al 2017) found that one or more esterase genes are involved in D. sechellia derived resistance to OA (Lanno and Coolon 2019). We therefore knocked down the expression of Est6 ubiquitously in D. melanogaster adults with RNAi and performed OA resistance assays.…”

“…The mortality assays used in this work were performed according to methods described in prior studies (Andrade López et al 2017; Peyser et al 2017; Lanno & Coolon 2019; Lanno et al 2019). Briefly, control and L-DOPA exposed flies of all three species were transferred into vials (10 per vial) containing 0.75g Drosophila medium supplemented with 1.2% octanoic acid (OA, Sigma) (Andrade López et al 2017; Peyser et al 2017; Lanno & Coolon 2019; Lanno et al 2019). Flies used for mortality assays were all 1-4 day old females for each species and each sample type was collected in replicate six-eight times with 10 flies per replicate (N = 60 to 80 per sample type depending on the experiment, see below).…”

“…A Cox proportional hazards statistical model was used to test the effect of L-DOPA exposure on OA associated mortality using the coxph command in the survival package in R (Cox 1972; Fox 2008; Therneau 2015; Andrade López et al 2017; Peyser et al 2017; R Core Development Team 2017; Lanno & Coolon 2019; Lanno et al 2019). We report relative survival as the regression coefficient (-β) for each treatment group compared to its species-specific control group (with vs. without L-DOPA).…”