peerj peerj PeerJ PeerJ PeerJ 2167-8359 PeerJ Inc. San Diego, USA 7802 10.7717/peerj.7802 Agricultural Science Biodiversity Ecology Entomology Genetics The cuticle inward barrier in Drosophila melanogaster is shaped by mitochondrial and nuclear genotypes and a sex-specific effect of diet Dong Wei dongwei@sxu.edu.cn12 Dobler Ralph 2 Dowling Damian K. 3 Moussian Bernard bernard.moussian@unice.fr124 Institute of Applied Biology, Shanxi University, Taiyuan, China Applied Zoology, Faculty of Biology, Technische Universität Dresden, Dresden, Germany School of Biological Sciences, Monash University, Clayton, Victoria, Australia Université Côte d’Azur, CNRS—Inserm, iBV, Parc Valrose, Nice, France Gillespie Joseph 4 10 2019 7 e7802 8 6 2019 31 8 2019 ©2019 Dong et al. 2019 Dong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.

An important role of the insect cuticle is to prevent wetting (i.e., permeation of water) and also to prevent penetration of potentially harmful substances. This barrier function mainly depends on the hydrophobic cuticle surface composed of lipids including cuticular hydrocarbons (CHCs). We investigated to what extent the cuticle inward barrier function depends on the genotype, comprising mitochondrial and nuclear genes in the fruit fly Drosophila melanogaster, and investigated the contribution of interactions between mitochondrial and nuclear genotypes (mito-nuclear interactions) on this function. In addition, we assessed the effects of nutrition and sex on the cuticle barrier function. Based on a dye penetration assay, we find that cuticle barrier function varies across three fly lines that were captured from geographically separated regions in three continents. Testing different combinations of mito-nuclear genotypes, we show that the inward barrier efficiency is modulated by the nuclear and mitochondrial genomes independently. We also find an interaction between diet and sex. Our findings provide new insights into the regulation of cuticle inward barrier function in nature.

 Mitonuclear Mito-nuclear Mitochondria Desiccation Xenobiotics mtDNA Insects Sex differences Lipids Diet Nutrition Deutsche Forschungsgemeinschaft-Exzellenzinitiative Zukunftskonzept to Technische Universität Dresden Australian Research Council DP170100165 FT160100022 National Natural Science Foundation of China NSFC31761133021 31402021 Special Talents Projects in Shanxi Province, China 201805D211019 Deutsche Forschungsgemeinschaft DFG1714/9-1 The study was financially supported by the Deutsche Forschungsgemeinschaft- Exzellenzinitiative Zukunftskonzept to Technische Universität Dresden (Ralph Dobler, and Bernard Moussian), by the Australian Research Council: DP170100165 and FT160100022 (to Damian K. Dowling), by National Natural Science Foundation of China (NSFC31761133021,31402021), Special Talents Projects in Shanxi Province, China [201805D211019] (to Wei Dong) and the Deutsche Forschungsgemeinschaft [DFG1714/9-1] (to Bernard Moussian). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction

The insect cuticle plays an important role in maintaining homeostasis by preventing uncontrolled penetration of xenobiotics and water (Hadley, 1978; Lockey, 1976; Wang et al., 2016). This barrier function relies mainly on the outer cuticular region composed of the envelope and surface lipids including cuticular hydrocarbons (CHCs) at the cuticle surface (Blomquist & Bagnères, 2010; Gibbs, 1995; Gibbs, 2002). In general, CHCs have chain lengths from C23 to C50, and may have double bonds and be branched. The CHC pool is species-specific and shows within-species variation with respect to age, sex and diet (Barbosa et al., 2017; Bonelli et al., 2015; Ishii et al., 2002; Moore et al., 2017; Otte, Hilker & Geiselhardt, 2015; Rouault, Capy & Jallon, 2001). Length variation of CHCs was found in different geographical populations of the fruit fly Drosophila melanogaster, where the ratio of two CHC isomers varied with climatic conditions (Ferveur, 1991; Ferveur & Sureau, 1996). More recently, it was reported that the chain length of CHCs correlates with the latitudinal habitats of D. melanogaster strains from north (Maine) to south (Florida) along the US east coast paralleling increasing temperatures and desiccation threat (Rajpurohit et al., 2017). Rajpurohit and colleagues also found an association of genomic SNPs with the production and chemical profile of CHCs (Rajpurohit et al., 2017). Consistently, it has been shown that CHC variation in recombinant inbred lines depends on the nuclear genotype in D. melanogaster (Dembeck et al., 2015). Based on these works, it is conceivable that CHC chain length is associated with the function of the outward barrier and as a result would affect the desiccation resistance (Rouault et al., 2004).

Two findings indicate that also the inward barrier is based on lipids including CHCs. First, penetration of xenobiotics such as the dye Eosin Y is sensitive to lipid solvents (Wang et al., 2016; Wang, Carballo & Moussian, 2017). Second, cuticle impermeability for water and xenobiotics is disrupted by mutations in genes coding for proteins involved in lipid-based barrier formation (Li et al., 2017; Yu et al., 2017; Zuber et al., 2018). The molecular function of these proteins, including the ABCH transporter Snustorr (Snu) and the extracellular protein Snustorr-Snarlik (Snsl), and their relationship to CHC distribution, however, are yet unexplored. Hence, at least to some extent, the inward and outward barriers share the same molecular constitution.

Diet has a significant impact on lipid composition of D. melanogaster in general (Brankatschk et al., 2018; Buczkowski et al., 2005; Carvalho et al., 2012; Liang & Silverman, 2000; Martin, Helanterä & Drijfhout, 2011; Savarit & Ferveur, 2002; Wurdack et al., 2015). Food composition, sex and their interactions significantly affected CHC composition and amounts in strains of D. melanogaster, maintained on different food types for years (Fedina et al., 2012). Likewise, in the mustard leaf beetle Pheadon cochleariae, different food resources (host plants) contribute to variation in CHC profiles, which in turn affects their courtship behaviour (Geiselhardt, Otte & Hilker, 2009). The diversity of CHCs is assumed to depend on different metabolic pathways associated with digestion processes and adaptations to specific diets.

Mitochondria are important sites of lipid metabolism in the cell, thereby contributing to CHC production. Key steps of this process occur in these organelles. For instance, the bulk of acetyl-CoA required in fatty acid synthesis by the acetyl-CoA carboxylase is produced in the mitochondria (Hardwood, 1988; Kennedy, 1962; Wakil, Stoop & Joshi, 1983). Biosynthesis of haem, a co-factor of cytochrome P-450 enzymes, which modify the long carbon chains in the mitochondria or the smooth endoplasmatic reticulum (ER) (Capdevila, Falck & Estabrook, 1992), involves enzymes acting in the mitochondria. While the role of mitochondria in lipid metabolism is well understood (Goldin & Keith, 1968; Mesmin, 2016; Scharwey, Tatsuta & Langer, 2013; Tyurina et al., 2014; Voelker, 2004), it is not known whether mitochondrial genetic variation is associated with variation of lipid or CHC synthesis, in turn influencing cuticle barrier function.

There are indeed some examples demonstrating the importance of mitochondrial genetic variation for insect ecology, especially in D. melanogaster (Ballard, Pichaud & Fox, 2014; Camus & Dowling, 2018; Dowling, 2014; Wolff et al., 2014). In the case of heat tolerance in D. melanogaster, for example, Camus et al. (2017) recently showed that variation in this phenotype in Australia was in part associated with mitochondrial single nucleotide polymorphisms (SNPs) that did not change the protein sequence.

An issue complicating experimental approaches to assess the contribution by mitochondrial genetic variation on vital body functions is that mitochondrial effects (caused by variation in the mitochondrial genome) on the phenotype can differ across nuclear genomes, across sex and with environmental factors such as diet (Aw et al., 2018; Zhu, Ingelmo & Rand, 2014). It is well known that some organismal dysfunctions associated with mitochondrial mutations are expressed in only some nuclear backgrounds, but not in others (Blumberg, Rice & Kundaje, 2017; Connallon et al., 2018; Dobler et al., 2014; Dobler et al., 2018; Dowling, 2014; Kenney et al., 2014; Latorre-Pellicer et al., 2016; Patel et al., 2016; Reinhardt, Dowling & Morrow, 2013; Wolff et al., 2014). Several of these effects are sex-specific (e.g., Immonen et al., 2016). Addition of long-chain fatty acids to the diet has been shown to influence mitochondrial physiology (Holmbeck & Rand, 2015; Stanley, Khairallah & Dabkowski, 2012), and even offset mitochondrial genetic defects (Senyilmaz et al., 2015). Moreover, such nuclear or environmental influences on mitochondrial effects can differ across mitochondrial genotypes (Ballard and Youngson, 2015; Mossman et al., 2016a; Mossman et al., 2016b).

To detect an effect of genotypic mitochondrial variation on cuticle differentiation requires an advanced experimental protocol in which the effects of mitochondrial variation are isolated from diet, sex and nuclear genome effects. Here, we use a sophisticated experimental design employing Drosophila lines with distinct mito-nuclear genotypes reared on different diets. We coupled this with a new in situ method that generically measures the cuticle inward barrier function (Wang et al., 2016) and thereby examine the effects of mitochondrial and nuclear genomic variation on the cuticle inward barrier function.

This new in situ method is based on the ability of the dye Eosin Y to penetrate the cuticle, thereby reflecting the cuticle barrier function in Drosophila and other insects (Wang et al., 2016; Wang, Carballo & Moussian, 2017). Penetration of Eosin Y is regionalised, with different body parts taking up the dye at distinct temperatures. Eosin Y staining provides a simple and reliable way to detect cuticle inward barrier properties that are possibly mediated by lipids and CHCs. Here, we analyse the Eosin Y penetration pattern in the wing cuticle that allows fast and efficient assessment of differences in the cuticle inward barrier function caused by the mitochondrial genome, the nuclear genome, sex or diet.

 Material and Methods Fly line generation and maintenance

We created nine fly lines with different combinations of mitochondrial and nuclear genomes by specific crossing of males and females from three source populations. The three source populations originated from Coffs Harbour, Australia (A) (Dowling, Williams & García-Gonzaléz, 2014; Williams et al., 2012), Benin (B) (formerly Dahomey), Africa (Clancy, 2008) and Dundas (near Hamilton), Canada (C) (MacLellan, Whitlock & Rundle, 2009). According to the “Climate Data for Cities Worldwide” database (https://en.climate-data.org/), Coffs Harbour lies in a zone with a humid subtropical climate (Cfa according to the climate classification of Köppen, average temperature of 18.8 °C, 1,688 mm of precipitation), Dundas lies in a warm-summer, humid continental climate (Dfb, average temperature of 8.5 °C, 834 mm of precipitation) zone and Benin lies in a zone with tropical monsoon climate (Aw, average temperature of 27.4 °C, 1,320 mm of precipitation). We initially crossed 45 virgin females from the source population with the desired mitochondrial genotype to 45 males from the source population with the desired nuclear genotype to create the first generation of each mito-nuclear line. To avoid skewed effects of mito-nuclear combinations due to non-random sampling from each source population, we created each line three times independently (resulting in a total of 27 lines) and kept them separated from each other since then. To generate the second generation (and all subsequent generations) we backcrossed 45 virgin female offspring from the line (harbouring the desired mitochondrial genotype due to maternal inheritance of the mitochondria Birky, 2001) with 45 males from the source population with the desired nuclear genotype (Fig. 1). With this crossing scheme, we removed 50% of the remaining nuclear genome from the maternal source population in each generation, leading to theoretical 99.99% removal of the maternal nuclear genome after 17 generations. We continued with the described crossing scheme for another 21 generations (until the experiment started) to avoid selective co-adaptation processes between the nuclear and the mitochondrial genome. We labelled the generated mito-nuclear lines as AA, AB, AC, BA, BB, BC, CA, CB and CC, the first letter denoting the origin of the mitochondrial genome and the second letter denoting the origin of the nuclear genome (hence mito-nuclear lines). We further distinguished the three replicates of each line with a suffix (1 to 3). We kept the mito-nuclear lines and the source populations as 14 day non-overlapping generations at 25 °C on a 12:12 h day-night rhythm. We kept these flies on 7 ml standard corn-yeast-sugar medium (corn 90 g/l, yeast 40 g/l, sugar 100 g/l, agar 12 g/l, Nipagin 20 ml/l, propionic acid 3 ml/l) in 25 mm vials.

10.7717/peerj.7802/fig-1 Crossing scheme of fly line generation.

We initially crossed 45 virgin females from population (A) to 15 males from (A, B and C), respectively, to create the first generation. After then, we backcrossed 45 female offspring from the line with 45 males from the desired nuclear genotype. All subsequent generations repeat the back cross scheme. Similarly, we generated mito-nuclear lines as BA, BB, BC, CA, CB and CC by crossing males from the three source populations to females from the populations B and C, respectively.

All lines we used for the experiment were free of Wolbachia. This was confirmed for all lines by diagnostic PCR for Wolbachia- specific primers after infected lines were treated with Tetracycline (0.3 g/l added to the food) for three generations (Clancy & Hoffmann, 2010). We applied the Tetracycline treatment at least four generations before the start of our experiment.

 Food treatment

We used two distinct diets to assess the effect of food composition on the function of the cuticle barrier. The diets were developed by Carvalho et al. (2012) and Brankatschk et al. (2018) and differ in lipid composition while being isocaloric. One food type was plant food (PF, 788 kcal/l). Compared to the standard food, extra malt (80 g/l), cold pressed sunflower oil (2 ml/l) and treacle (22g/l) were added, while yeast and glucose were removed. The other food type was yeast food (YF, 809 kcal/l). Compared to the standard food, fresh yeast (80 g/l) and extra yeast extract (20 g/l) were supplied and cornmeal was removed. A detailed description of the recipes for the two food types can be found elsewhere (Brankatschk et al., 2018; Carvalho et al., 2012). In principle, the nutritional value of both types of food is similar. By contrast, their lipid composition differs: the plant food contains longer and more unsaturated lipids that the yeast food. The mito-nuclear lines completed an entire life cycle on either PF food or YF food (i.e., females laid eggs on the designated food type and larvae subsequently developed on this food type) before we collected adult flies for wing staining and measurements (see below). To roughly control larvae density, we used 10 males and 10 females to lay eggs and we standardised egg-laying time to 12 h for each line. Finally, we checked the egg density roughly by eye to make sure it was approximately equal across all lines. We kept all vials at 25 °C on a 12:12 h day-night rhythm. We collected virgin males and females within six hours of eclosion and kept them in vials (10 flies/vial) separated by sex and line for five days, thereafter we started wing staining and measurements.

 Wing staining and measurement

We used 10 flies (20 wings) of each combination (per line/food/sex) for wing staining. We carried out Eosin Y staining according to Wang et al. (2016) with a slight modification. Instead of two days old flies in the original protocol, we used five days old flies. We anaesthetized the flies with CO2, transferred them into a micro-centrifuge tube containing 1 ml of the red dye solution (0,5% Eosin Y (W/V) and 0.1% Triton X-100) and incubated them at 55 °C for 30 min. We washed the Eosin Y-stained flies three times with distilled water, isolated wings using tweezers and mounted them in 50% glycerol on glass slides. We collected images using a Leica DMI8 microscope with a built-in camera and the software LAS X. For quantification of staining, we converted the images to 8-bit format and recorded the mean grey values that reflect the staining intensity using the Fiji software (Schindelin et al., 2012). We compared the mean grey values in the areas expected to take up Eosin Y and in surrounding areas. In a previous work, we discovered that the Eosin Y staining pattern depended on the genetic background of D. melanogaster (Wang et al., 2016). If the mean grey values of potentially Eosin Y-positive areas (in the posterior, lower half), were higher than those of potentially Eosin Y-negative areas (in the anterior, upper half), we scored a “presence of staining”, otherwise we scored an “absence of staining”. We used a semi-quantitative method to classify staining patterns as ‘no staining’, ‘front area staining’, ‘back area staining’ or ‘front and back area staining’.

 Statistical analyses

Prior to data analysis we inspected our data and found that one staining pattern (front patch unstained, back patch stained) occurred in only seven out of 1108 individuals distributed across four of the nine mito-nuclear combinations. We omitted these seven measurements from our analyses and used the remaining 1101 measurements assigned to one of the three staining pattern as ordinate response variable (0, 1, 2 stained spots) for our analysis. We analysed the data with generalised linear mixed models (GLMMs) using the lme4 package (Bates et al., 2013) in R 3.4.2. (R Development Core Team, 2017). We started with a full model including the factors diet, sex, mitochondrial and nuclear genome and all their higher-order interactions. We then reduced the model stepwise by excluding factor combinations to improve the Bayesian information criterion (BIC). We used the BIC for model comparison because of the large number of degrees of freedom in our models. Using BIC reduces the chance to have false positive factors in the final model because a high number of degrees of freedom is more penalised than with the AIC (Dziak et al., 2012). We stopped the model reduction when the removal of factor combinations did not increase the explanatory power of the model (the final model). The four linear factors remained in the final model because they were experimentally manipulated and we were a priori interested in their effects.

The full statistical model included the observed patterns as dependent variable (ordinal data type). Mitochondrial genotype, nuclear genotype, sex and food type, with all their higher-level interactions, were fixed effects of the full model and line was used as random factor to avoid pseudo-replication of data in the analyses. As the response variable was ordinal (see above), we used a Binomial distribution with a log-link error function to analyse the data. We further changed the number of maximal iterations for the model to converge from 1,000 to 500,000 in the glmer Control to assure model convergence. To find the optimal grouping of the fly lines we ran a principal component analysis (PCA) using SNP analyses of the mitochondrial genomes from all our mito-nuclear lines (GenBank accession PRJNA532313) and the origin of the nuclear genome. In brief we ran PCAs on the frequencies of the three observed staining patterns using 34 SNPs and the nuclear background as explanatory variables. The aim of the PCAs was to see whether and how the 27 lines cluster in the plane of the first two principal components. Based on the results from the PCA we grouped the fixed factor mitochondrial genotype to three levels (Fig. S1). We grouped the mitochondrial genotypes AA1, AA2, AB1-3 and AC1-3 (hereafter type ‘A’), the mitochondrial genotypes BA1-3, BB1-3, BC1-3, CA1 and CA3 (hereafter type ‘B’) and the mitochondrial genotypes AA3, CA2, CB1-3 and CC1-3 (hereafter type ‘C’).

 Results

We scored the frequency of occurrence of two Eosin Y staining areas at the posterior half of the wing blade of flies from population lines with putatively co-evolved or newly constituted mitochondrial and nuclear genome combinations in order to test barrier efficiency. It should be noted that as these fly lines were derived from natural populations with assumed nucleotide diversity in the mitochondrial and nuclear genomes, and not from isogenic stocks, we did not expect to observe only one staining pattern in flies from a single population. We found variation in the wing-staining pattern across our D. melanogaster lines (Fig. 2). The frequencies of the wing staining patterns for each of the four fixed factors are visualised in Fig. S2.

10.7717/peerj.7802/fig-2 Eosin Y staining pattern of the fly wing.

As represented by a drawing (A), a proportion of wings does not take up Eosin Y after staining (B). Some wings, by contrast, show staining of a posterior region close to the hinge (C, D). Another set of wings takes up the dye in two posterior regions (E, F).

The final model revealed that nuclear (p < 0.001, Table 1) and mitochondrial (p = 0.002) genotypes, as well as the interaction between sex and food (p < 0.001), best explained the frequency of the wing-staining pattern (Table 1, Figs. 3 and 4). Of note, our data do not provide evidence for a significant mito-nuclear interaction effect on the inward barrier function of the cuticle (p = 0.141).

10.7717/peerj.7802/table-1 ANOVA table for the statistical analysis of the wing pattern frequency.

Analysis of deviance table (type III Wald χ2 test) for the optimised model. The interaction between sex and food was statistically significant, with both of the two linear terms to be found non-significant. The mitochondrial genotype and the nuclear genotype had also a significant effect on the staining pattern.

Factor χ2 df p-value
Intercept 21.337 1 <0.001
Mitochondrial genotype 12.740 2 0.002
Nuclear genotype 134.358 2 <0.001
Sex 0.161 1 0.688
Food 0.646 1 0.422
Sex × food 14.146 1 <0.001
10.7717/peerj.7802/fig-3 Relative frequencies of wing patterns.

The relative occurrence of each wing pattern for each mitochondrial genotype - nuclear genotype -sex-food combination. A, B and C in the first line of the x-axis label are for the mitochondrial genotype. The A, B and C in the second line stand for the nuclear genotype. The third line indicates the sex of the flies m for males and f for females and the forth line label are for plant food (P) or yeast food (Y).

 10.7717/peerj.7802/fig-4 Nuclear, mitochondrial and interaction of sex and diet on wing pattern frequencies.

The relative occurrence of each wing pattern for each mitochondrial genotype, nuclear genotype and sex- food combination, respectively. The interaction between sex and diet affects the number of wings with no staining and with a single spot staining, but not the number of wings with both spots stained. Females (f) on yeast food (Y) show a much higher proportion of single stained wings than males (m) on yeast food and males and females on plant food (P). Males on yeast food have a higher occurrence of unstained wings than females on yeast food and males and females on plant food. In summary, males and females show similar frequencies for double stained area (m: 71.61%, f: 72.09%). However, the frequencies between non-stained (m: 21.61%, f: 11.46%) and single stained areas (m: 6.79%, f: 16.45%) showed some difference. Staining patterns between plant food and yeast food were quite different (non-stained: P: 11.07%, Y: 22.56%; single stained: P: 3.51%, Y: 20.11%; double stained: P: 85.41%, Y: 57.33%).

For different nuclear genotypes, flies with the Australian nuclear genotype generally showed the highest frequency of non-stained wing area (39.94%) and the lowest frequency of both wing areas being stained (43.30%) among all three nuclear genotypes (Fig. 4 and Fig. S1). Flies with both Benin and Canadian nuclear genotypes showed high frequencies of double stained areas patterns (Benin: 96.26%, Canada: 74.80%) (Fig. 4 and Fig. S1). The frequency of wing staining pattern with only the rear area being stained was about the same in flies with the Australian and the Canadian nuclear genotype (Australia: 16.76%; Canada: 17.07%) (Fig. 4 and Fig. S1). This pattern was almost absent in flies with the Benin nuclear genotype (1.07%) (Fig. 4 and Fig. S1).

For different mitochondrial genotypes, the staining frequency for the rear area was similar in all three mitochondrial genotype groups (A: 10.80%, B: 14.96%, C: 7.60) (Fig. 4), whereas there was substantial variation in whether no or both wing spots were stained (no staining: A: 19.44%, B: 18.08%, C: 11.85%; staining both areas: A: 69.75%, B: 66.96%, C: 80.55%) (Fig. 4).

The significant interaction effect between food and sex is underlined by the striking differences of a male–female difference in wing staining pattern for plant food or yeast food (Fig. 4). Specifically, most wings of flies reared on plant food showed staining at both posterior areas (female: 234, male: 252), and only a few wings of these flies had non-stained wings (female: 29, male: 34). The frequency of wing staining patterns with only the rear area being stained was lowest in males reared on plant food (Table S1, Fig. S2).

 Discussion

The physiological and genetic mechanisms involved in the constitution of the inward barrier function of the insect cuticle are largely unexplored. To contribute to our understanding of these mechanisms, we investigated the effects of nuclear and mitochondrial genetic variation, sex and diet and their interactions on the function of the wing cuticle inward barrier. These factors had an effect on cuticle barrier function at different hierarchical levels.

Interactions between sex and diet

According to our statistical model, if we consider sex or diet alone (as main effects), they do not have any significant effect on the frequency of the wing-staining pattern and, by consequence, on the cuticle inward barrier efficiency. Yet, they are involved in a statistically significant interaction, with the effects of each of these factors is contingent on the other. Simply, this suggests an important role of sex on cuticle barrier function that is modulated by diet. Thus, with respect to the cuticle barrier function, we conclude that the responses to food quality between males and females are different. In other words, food utilization for barrier construction and optimization (that may at least partially rely on CHCs) varies between males and females. Indeed, in recent studies it was shown that the impact of the food source on the CHC pattern differed in males and females (Otte, Hilker & Geiselhardt, 2015). Thus, it seems that the CHC composition is not only sexually dimorphic with respect to communication (Antony & Jallon, 1982), but also with respect to penetration resistance against xenobiotics.

Next, we need to determine sex-specific single nucleotide polymorphisms (SNPs) and identify the CHC composition in males and females of our lines on different food sources to better understand inward barrier function in dependence of diet and sex. These experiments would also allow us to reveal possible differences and similarities between the molecular constitution of the inward and the outward barrier for which rich data is available.

Indeed, a difference of the outward barrier between sexes has been observed in D. melanogaster. Due to specific modifications of CHC composition after induction of desiccation resistance, the survival rate of females is higher than the survival rate of males under prolonged desiccation conditions (Foley & Telonis-Scott, 2011; Stinziano et al., 2015). This effect is independent of starvation. These findings with respect to our results are interesting insofar as they suggest that the outward and inward barriers display independent efficiencies; while, for instance, females are more resistant to water loss than males, resistance to dye penetration is comparable between females and males if we ignore the effect of diet (see below). A simple conclusion is that these two barriers do not rely on CHCs and the respective physical properties alone, but employ distinct and non-analogous factors. Further experiments are needed to unravel the molecular basis of this difference.

Likewise, it has previously been demonstrated that diet does control the efficiency of the outward barrier, probably by influencing the amounts and, importantly, the composition of CHCs (Fedina et al., 2012). In line with this work, a recent study reported that the proportion of desaturated CHCs defines the efficiency of the barrier against desiccation (Ferveur et al., 2018). This may be explained by an influence of food metabolites on fatty acid synthesis and, by consequence, on CHC amounts and composition (Fedina et al., 2012; Ferveur, 2005; Pavković-Lučić et al., 2016). However, the situation is certainly more complex. The effect of food alone on CHC quality is not sufficient to explain the outward barrier function. Indeed, the effects of food source on desiccation resistance have been repeatedly reported to depend on multiple complex factors including, in addition to lipid metabolism, carbohydrate metabolism and body size (Andersen et al., 2010; Kristensen et al., 2016; Mikkelsen et al., 2010). Moreover, diet has been demonstrated in large-scale studies to have a significant and variable effect on genome-wide gene expression in 20 D. melanogaster wild-type strains in various complex traits including puparial adhesion, metamorphosis and central energy metabolic functions (Reed et al., 2010; Reed et al., 2014; Williams et al., 2015). Overall, we therefore conclude that the outward and inward barriers are differentially sensitive to diet.

 The nuclear genotype

We observed significant effects of the nuclear genotype on the dye penetration efficiency. In particular, the nuclear genotype of flies from Coffs Harbour with a humid subtropical climate (Cfa according to the climate classification of Köppen) correlates with a wing cuticle inward barrier that is more efficient than in flies with the Dundas (warm-summer, humid continental climate, Dfb) or with the Benin (tropical monsoon climate, Am) nuclear genotype, which correlates with the lowest cuticle inward barrier efficiency. Rough temperature or humidity profiles of the locations (see Materials & Methods) do not explain these correlations arguing that the cuticle inward barrier may not be directly dependent on these factors. In any case, we conclude that the inward barrier function depends largely on varying fly line-specific expression profiles of nuclear genes, some of which presumably are needed for CHC production and deposition including genes coding for Cyp proteins, fatty acid synthases and elongases. Nuclear genetic variations that comply with differences in CHC composition in inbred or geographically separated lines of D. melanogaster have been reported to be represented by quantitative trait loci (Foley et al., 2007; Foley & Telonis-Scott, 2011) or SNPs (Dembeck et al., 2015; Rajpurohit et al., 2017). The genomic differences between our lines and the associated differences in cuticle permeability are either due to genetic adaptation to the original environmental conditions in Coffs Harbour, Australia, Benin, Africa and Dundas (near Hamilton), Canada, or due to genetic drift within the populations. This remains to be tested.

 The mitochondrial genotype

We found a significant effect of the mitochondrial genotype on the wing-staining frequency patterns. Cuticle inward barrier efficiency was similarly reduced in flies with a Dundas mitochondrial genotype. Rough climate profiles of the original locations of our fly populations are insufficient to explain the correlation between the mitochondrial genotype and cuticle inward barrier efficiency. More detailed assessment of the local climate situation and the use of more geographically disjunct fly populations are thus necessary to allow relating cuticle inward barrier efficiency to any climatic factor. A relationship between cuticle inward barrier efficiency, mitochondrial genotype and a climatic factor would, in turn, allow the testing of whether this relationship is in agreement with the mitochondrial climatic adaptation hypothesis (Camus et al., 2017). This hypothesis proposes that latitudinal climatic differences shape patterns of standing variation in mitochondrial genotypes across a species distribution, and that these genotypes play a role in determining temperature sensitivity of individuals.

In a recent work, no significant effect of the mitochondrial genotype on lipid content was found in D. melanogaster lines engineered from laboratory and natural populations (Aw et al., 2017). That study thus indicated that lipid (including CHC) homeostasis is to a large extent independent from the mitochondrial genotype in D. melanogaster. Together, we conclude that the function of the inward barrier is sensitive to variation in the mitochondrial genomes of the lines tested. We also conclude that the mitochondrial-dependent inward barrier function is possibly not mediated by CHCs.

Interestingly, Aw and colleagues (2017, 2018) did find a significant interaction of the mitochondrial genotype with diet and/or sex on a number of physiological traits including survival and fecundity. According to our data, however, these genotype-by-environment interactions do not seem to play any role with respect to the cuticle inward barrier function. Likewise, we do not detect any interaction between the mitochondrial and the nuclear genomes that would influence dye penetration in our assays. The seeming independence of the inward barrier function from mito-nuclear interaction possibly represents a particular case. Indeed, mito-nuclear interactions have been demonstrated to affect a wide range of biological processes such as developmental time, sex-specific transcription, hypoxia and longevity (Dowling, Meerupati & Arnqvist, 2010; Mossman et al., 2016a; Mossman et al., 2016b; Mossman et al., 2017; Rand, Fry & Sheldahl, 2006; Rand et al., 2018). We will need to expand the number of different geographical populations of D. melanogaster or refine the quantification of our dye-penetration assay in our analyses to uncover whether any subtle mito-nuclear effects on cuticle barrier function exist.

 Conclusions

In summary, in this study we find that Eosin Y penetration through the wing cuticle of mass bred populations from three global populations is variable and that the pattern of variability differs between the lines. Our data indicate that along with CHCs, other cuticle components are sensitive to genetic variation within the nuclear and mitochondrial genomes as well as to sex-diet interactions. Candidates are factors acting in the Snu-Snsl pathway that contribute to the construction of the outermost cuticle layer termed envelope that serves as a physical barrier against penetration and desiccation (Zuber et al., 2018). Nuclear and mitochondrial SNP analyses will help to shed light on this complex trait that is crucial for insect survival. Mitochondrial genes comprising 13 polypeptides of the electron transfer-chain (ETC), 2 rRNAs and 22 tRNAs, are, however, probably not directly involved in cuticle barrier formation (Ballard & Rand, 2005; Burton, Pereira & Barreto, 2013; Piomboni et al., 2012; St John, Jokhi & Barratt, 2005).

 Supplemental Information 10.7717/peerj.7802/supp-1 Mitochondrial genotype grouping

The mitochondrial genotypes were grouped based on the result by a PCA. We distinguish three groups. The mitochondrial genotypes AA1, AA2, AB1-3 and AC1-3 were grouped and named type ‘A’, the mitochondrial genotypes BA1-3, BB1-3, BC1-3, CA1 and CA3 were grouped and named type ‘B’ ,the mitochondrial genotypes AA3, CA2, CB1-3 and CC1-3 were grouped and named type ‘C’.

 10.7717/peerj.7802/supp-2 Sex and diet linear effects on wing pattern frequencies

Relative frequencies by sex are: male no staining: 21.61%, rear area stained: 6.79%, both areas stained: 71.61%; female no staining: 11.46%, rear area stained: 16.45%, both stained: 72.09%. Staining frequencies by food: plant food no staining: 11.07%, rear area stained: 3.51%, both areas stained: 85.41%; yeast food no staining: 22.56%, rear area stained: 20.11%, both areas stained: 57.33%.

 10.7717/peerj.7802/supp-3 Wing pattern frequencies

∘ ∘ indicates that none of the two areas was stained, ∘ • indicates that the rear wing area was stained and • • indicates that both areas on the wing were stained. Empty cells indicate that the specific combination of all four factors did not occur in our experiment after recording of the mitochondrial genotypes to five levels based on the sequence data.

 10.7717/peerj.7802/supp-4 Raw data

We thank Klaus Reinhardt and three anonymous referees for helpful comments on previous versions of the manuscript.

 Additional Information and Declarations Competing Interests

The authors declare there are no competing interests.

 Author Contributions

Wei Dong and Ralph Dobler conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

Damian K Dowling analyzed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft.

Bernard Moussian conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

 Data Availability

The following information was supplied regarding data availability:

The raw statistics are available in the Supplemental Files. The SNP data is available at GenBank: PRJNA532313.

 References Andersen LHKTN Loeschcke V Toft S Mayntz D 2010 Protein and carbohydrate composition of larval food affects tolerance to thermal stress and desiccation in adult Drosophila melanogaster Journal of Insect Physiology 56 336 340 10.1016/j.jinsphys.2009.11.006 Antony C Jallon JM 1982 The chemical basis for sex recognition in Drosophila melanogaster Journal of Insect Physiology 28 873 880 10.1016/0022-1910(82)90101-9 Aw WC Garvin MR Melvin RG Ballard WO 2017 Sex-specific influences of mtDNAmitotype and diet on mitochondrial functions and physiological traits in Drosophila melanogaster PLOS ONE 12 11 e0187554 10.1371/journal.pone.0187554 Aw WC Towarnicki SG Melvin RG Youngson NA Garvin MR Hu Y Nielsen S Thomas T Pickford R Bustamante S Vila-Sanjurjo A Smyth GK Ballard JWO 2018 Genotype to phenotype: diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness PLOS Genetics 14 11 e1007735 10.1371/journal.pgen.1007735 Ballard JWO Pichaud N Fox C 2014 Mitochondrial DNA: more than an evolutionary bystander Functional Ecology 28 1 218 231 10.1111/1365-2435.12177 Ballard JWO Rand DM 2005 The population biology of mitochondrial DNA and its phylogenetic implications Annual Review of Ecology, Evolution, and Systematics 36 621 642 10.1146/annurev.ecolsys.36.091704.175513 Ballard JWO Youngson NA 2015 Review: can diet influence the selective advantage of mitochondrial DNA haplotypes? Bioscience Reports 35 6 e00277 Barbosa RR Braga MV Blomquist GJ Queiroz MMDC 2017 Cuticular hydrocarbon profiles as a chemotaxonomic tool for three blowfly species (Diptera: Calliphoridae) of forensic interest Journal of Natural History 51 1491 1498 10.1080/00222933.2017.1337944 Bates D Maechler M Bolker B Walker D 2013 lme4: linear mixed-effects models using Eigen and S4 http://CRAN.R-project.org/package=lme4 R package version 1.0-5 Birky CW 2001 The inheritance of genes in mitochondria and chloroplasts: laws, mechanisms, and models Annual Review of Genetics 35 125 148 10.1146/annurev.genet.35.102401.090231 Blomquist GJ Bagnères A-G 2010 Insect hydrocarbons: biology biochemistry and chemical ecology Cambridge University Press Cambridge Blumberg A Rice EJ Kundaje A 2017 Initiation of mtDNA transcription is followed by pausing, and diverges across human cell types and during evolution Genome Research 27 362 373 10.1101/gr.209924.116 Bonelli M Lorenzi MC Christidès J-P Dupont S Bagnères A-G 2015 Population diversity in cuticular hydrocarbons and mtDNA in a mountain social wasp Journal of Chemical Ecology 41 22 31 10.1007/s10886-014-0531-0 Brankatschk M Gutmann T Knittelfelder O Palladini A Grzybek M Brankatschk B Coskun U Eaton S 2018 A temperature-dependent switch in feeding preference improves drosophila development and survival in the cold Developmental Cell 46 781 793 10.1016/j.devcel.2018.05.028 Buczkowski G Kumar R Suib SL Silverman J 2005 Diet-related modification of cuticular hydrocarbon profiles of the argentine ant, Linepithemahumile, diminishes intercolony aggression Journal of Chemical Ecology 31 4 829 843 10.1007/s10886-005-3547-7 Burton RS Pereira RJ Barreto FS 2013 Cytonuclear genomic interactions and hybrid breakdown Annual Review of Ecology, Evolution, and Systematics 44 281 302 10.1146/annurev-ecolsys-110512-135758 Camus MF Dowling DK 2018 Mitochondrial genetic effects on reproductive success: signatures of positive intrasexual, but negative intersexual pleiotropy Proceedings of the Royal Society B: Biological Sciences 285 20180187 10.1098/rspb.2018.0187 Camus MF Wolff JN Sgrò CM Dowling DK 2017 Experimental support that natural selection has shaped the latitudinal distribution of mitochondrial haplotypes in Australian Drosophila melanogaster Molecular Biology and Evolution 34 2600 2612 10.1093/molbev/msx184 Capdevila JH Falck JR Estabrook RW 1992 Cytochrome P450 and the arachidonate cascade FASEB Journal 6 731 736 10.1096/fasebj.6.2.1537463 Carvalho M Sampaio JL Palm W Brankatschk M Eaton S Shevchenko A 2012 Effects of diet and development on the Drosophila lipidome Molecular Systems Biology 8 600 10.1038/msb.2012.29 Clancy DJ 2008 Variation in mitochondrial genotype has substantial lifespan effects which may be modulated by nuclear background Aging Cell 7 795 804 10.1111/j.1474-9726.2008.00428.x Clancy DJ Hoffmann AA 2010 Environmental effects on cytoplasmic incompatibility and bacterial load in Wolbachia-infected Drosophil asimulans Entomologia Experimentalis Et Applicata 86 1 13 24 10.1046/j.1570-7458.1998.00261.x Connallon T Camus MF Morrow EH Dowling DK 2018 Coadaptation of mitochondrial and nuclear genes, and the cost of mothers curse Proceedings of the Royal Society B: Biological Sciences 285 1871 20172257 10.1098/rspb.2017.2257 Dembeck LM Böröczky K Huang W Schal C Anholt RR Mackay TF 2015 Genetic architecture of natural variation in cuticular hydrocarbon composition in Drosophila melanogaster elife 4 121 127 10.7554/eLife.09861 Dobler R Dowling DK Morrow EH Reinhardt K 2018 A systematic review and meta-analysis reveals pervasive effects of germline mitochondrial replacement on components of health Human Reproduction Update 24 519 534 10.1093/humupd/dmy018 Dobler R Rogell B Budar F Dowling DK 2014 A meta-analysis of the strength and nature of cytoplasmic genetic effects Journal of Evolutionary Biology 27 2021 2034 10.1111/jeb.12468 Dowling DK 2014 Evolutionary perspectives on the links between mitochondrial genotype and disease phenotype Biochimicaet Biophysica Acta (BBA)—General Subjects 1840 1393 1403 10.1016/j.bbagen.2013.11.013 Dowling DK Meerupati T Arnqvist G 2010 Cytonuclear interactions and the economics of mating in seed beetles The American Naturalist 176 131 140 10.1086/653671 Dowling DK Williams BR García-Gonzaléz F 2014 Maternal sexual interactions affect offspring survival and ageing Journal of Evolutionary Biology 27 88 97 10.1111/jeb.12276 Dziak JJ Coffman DL Lanza ST Li R 2012 Sensitivity and specificity of information criteria Methodology Center Technical Report 12-119 Fedina TY Kuo TH Dreisewerd K Dierick HA Yew JY Pletcher SD 2012 Dietary effects on cuticular hydrocarbons and sexual attractiveness in Drosophila PLOS ONE 7 49799 10.1371/journal.pone.0049799 Ferveur JF 1991 Genetic control of pheromones in Drosophila simulans. I. Ngbo, a locus on the second chromosome Genetics 128 293 301 Ferveur JF 2005 Cuticular hydrocarbons: their evolution and rolesin Drosophila pheromonal communication Behavior Genetics 35 279 295 10.1007/s10519-005-3220-5 Ferveur JF Cortot J Rihani K Cobb M Everaerts C 2018 Desiccation resistance: effect of cuticular hydrocarbons and water content in Drosophila melanogaster adults Peer J 6 4318 10.7717/peerj.4318 Ferveur JF Sureau G 1996 Simultaneous influence on male courtship of stimulatory and inhibitory pheromones produced by live sex-mosaic Drosophila melanogaster Proceedings of the Royal Society of London Series B: Biological Sciences 263 967 973 10.1098/rspb.1996.0143 Foley B Chenoweth SF Nuzhdin SV Blows MW 2007 Natural genetic variation in cuticular hydrocarbon expression in male and female Drosophila melanogaster Genetics 175 1465 1477 10.1534/genetics.106.065771 Foley BR Telonis-Scott M 2011 Quantitative genetic analysis suggests causal association between cuticular hydrocarbon composition and desiccation survival in Drosophila melanogaster Heredity 106 68 77 10.1038/hdy.2010.40 Geiselhardt S Otte T Hilker M 2009 The role of cuticular hydrocarbons in male mating behavior of the mustard leaf beetle, Phaedon cochleariae (F.) Journal of Chemical Ecology 35 1162 1171 10.1007/s10886-009-9704-7 Gibbs A 1995 Physical properties of insect cuticular hydrocarbons: model mixtures and lipid interactions Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 112 667 672 10.1016/0305-0491(95)00119-0 Gibbs AG 2002 Lipid melting and cuticular permeability: new insights into an old problem Journal of Insect Physiology 48 391 400 10.1016/S0022-1910(02)00059-8 Goldin HH Keith AD 1968 Fatty acid biosynthesis by isolated mitochondria from Drosophila melanogaster Journal of Insect Physiology 14 887 899 10.1016/0022-1910(68)90001-2 Hadley NF 1978 Cuticular permeability of desert tenebrionid beetles: correlations with epicuticular hydrocarbon composition Insect Biochemistry 8 17 22 10.1016/0020-1790(78)90005-7 Hardwood JL 1988 Fatty acid metabolism Annual Review of Plant Physiology and Plant Molecular Biology 39 101 138 10.1146/annurev.pp.39.060188.000533 Holmbeck MA Rand DM 2015 Dietary fatty acids and temperature modulate mitochondrial function and longevity in Drosophila The Journals of Gerontology: Series A 70 1343 1354 10.1093/gerona/glv044 Immonen E Collet M Goenaga J Arnqvist G 2016 Direct and indirect genetic effects of sex-specific mitonuclear epistasis on reproductive ageing Heredity 116 338 339 10.1038/hdy.2015.112 Ishii K Hirai Y Katagiri C Kimura MT 2002 Mate discrimination and cuticular hydrocarbons in Drosophila elegans and D. gunungcola Zoological Science 19 1191 1196 10.2108/zsj.19.1191 Kennedy EP 1962 The metabolism and function of complex lipids Harvey Lectures 57 143 171 Kenney MC Chwa M Atilano SR Falatoonzadeh P Ramirez C Malik D Tarek M Del Carpio JC Nesburn AB Boyer DS Kuppermann BD Vawter MP Jazwinski M Miceli MV Wallace DC Udar N 2014 Molecular and bioenergetic differences between cells with African versus European inherited mitochondrial DNA haplogroups: implications for population susceptibility to diseases Biochimicaet Biophysica Acta (BBA) - Molecular Basis of Disease 1842 208 219 10.1016/j.bbadis.2013.10.016 Kristensen TN Henningsen AK Aastrup C Bech-Hansen M Bjerre LBH Carlsen B Hagstrup M Jensen SG Karlsen P Kristensen L Lundsgaard C Møller T Nielsen LD Starcke C Sørensen CR Schou MF 2016 Fitness components of Drosophila melanogaster developed on a standard laboratory diet or a typical natural food source Insect Science 23 771 779 10.1111/1744-7917.12239 Latorre-Pellicer A Moreno-Loshuertos R Lechuga-Vieco AV Sánchez-Cabo F Torroja C Acín-Pérez R Calvo E Aix E González-Guerra A Logan A Bernad-Miana ML Romanos E Cruz R Cogliati S Sobrino B Carracedo Á Pérez-Martos A Fernández-Silva P Ruíz-Cabello J Murphy MP Flores I Vázquez J Enríquez JA 2016 Mitochondrial and nuclear DNA matching shapes metabolism and healthy ageing Nature 535 561 565 10.1038/nature18618 Li K Zhang X Zuo Y Liu W Zhang J Moussian B 2017 Timed Knickkopf function is essential for wing cuticle formation in Drosophila melanogaster Insect Biochemistry and Molecular Biology 89 1 10 10.1016/j.ibmb.2017.08.003 Liang D Silverman J 2000 You are what you eat: diet modifies cuticular hydrocarbons and nestmate recognition in the Argentine ant, Linepithema humile Naturwissenschaften 87 412 416 10.1007/s001140050752 Lockey KH 1976 Cuticular hydrocarbons of Locusta, Schistocerca, and Periplaneta, and their role in waterproofing Insect Biochemistry 6 457 472 10.1016/0020-1790(76)90068-8 MacLellan K Whitlock MC Rundle HD 2009 Sexual selection against deleterious mutations via variable male search success Biology Letters 5 795 797 10.1098/rsbl.2009.0475 Martin SJ Helanterä H Drijfhout FP 2011 Is parasite pressure a driver of chemical cue diversity in ants? Proceedings Biological Sciences 278 496 503 10.1098/rspb.2010.1047 Mesmin B 2016 Mitochondrial lipid transport and biosynthesis: a complex balance Journal of Cell Biology 214 9 11 10.1083/jcb.201606069 Moore HE Butcher JB Day CR Drijfhout FP 2017 Adult fly age estimations using cuticular hydrocarbons and Artificial Neural Networks in forensically important Calliphoridae species Forensic Science International 280 233 244 10.1016/j.forsciint.2017.10.001 Mossman JA Biancani LM Zhu CT Rand DM 2016a Mitonuclear epistasis for development time and its modification by diet in Drosophila Genetics 203 463 484 10.1534/genetics.116.187286 Mossman JA Tross JG Jourjine NA Li N Wu Z Rand DM 2017 Transcriptional responses to hypoxia in Drosophila Molecular Biology and Evolution 34 447 466 Mossman JA Tross JG Li N Wu Z Rand DM 2016b Mitochondrial-nuclear interactions mediate sex-specific transcriptional profiles in Drosophila Genetics 204 613 630 10.1534/genetics.116.192328 Otte T Hilker M Geiselhardt S 2015 The effect of dietary fatty acids on the cuticular hydrocarbon phenotype of an herbivorous insect and consequences for mate recognition Journal of Chemical Ecology 41 32 43 10.1007/s10886-014-0535-9 Patel MR Miriyala GK Littleton AJ Yang H Trinh K Young JM Kennedy SR Yamashita YM Pallanck LJ Malik HS 2016 A mitochondrial DNA hypomorph of cytochrome oxidase specifically impairs male fertility in Drosophila melanogaster eLife 5 e16923 10.7554/eLife.16923 Pavković-Lučić S Todosijević M Savić T Vajs V Trajković J Anđelković B Lučić L Krstić G Makarov S Tomić V Miličić D Vujisić L 2016 ‘Does my diet affect my perfume?’ Identification and quantification of cuticular compounds in five Drosophila melanogaster strains maintained over 300 generations on different diets Chemistry and Biodiversity 13 224 232 10.1002/cbdv.201500064 Piomboni P Focarelli R Stendardi A Ferramosca A Zara V 2012 The role of mitochondria in energy production for human sperm motility International Journal of Andrology 35 109 124 10.1111/j.1365-2605.2011.01218.x R Development Core Team 2017 R: a language and environment for statistical computing R Foundation for Statistical Computing Vienna http://www.R-project.org/ Rajpurohit S Hanus R Vrkoslav V Behrman EL Bergland AO Petrov D Cvačka J Schmidt PS 2017 Adaptive dynamics of cuticular hydrocarbons in Drosophila Journal of Evolutionary Biology 30 66 80 10.1111/jeb.12988 Rand DM Fry A Sheldahl L 2006 Nuclear–mitochondrial epistasis and drosophila aging: introgression of Drosophila simulans mtDNA modifies longevity in D. melanogaster nuclear backgrounds Genetics 172 329 341 10.1534/genetics.105.046698 Rand DM Mossman JA Zhu L Biancani LM Ge JY 2018 Mitonuclear epistasis, genotype by environment interactions, and personalized genomics of complex traits in Drosophila IUBMB Life 70 1275 1288 10.1002/iub.1954 Reed LK Lee K Zhang Z Rashid L Poe A Hsieh B Deighton N Glassbrook N Bodmer R Gibson G 2014 Systems genomics of metabolic phenotypes in wild-type Drosophila melanogaster Genetics 197 781 793 10.1534/genetics.114.163857 Reed LK Williams S Springston M Brown J Freeman K DesRoches CE Sokolowski MB Gibson G 2010 Genotype-by-diet interactions drive metabolic phenotype variation in Drosophila melanogaster Genetics 185 1009 1019 10.1534/genetics.109.113571 Reinhardt K Dowling DK Morrow EH 2013 Medicine. Mitochondrial replacement, evolution, and the clinic Science 341 1345 1346 10.1126/science.1237146 Rouault J Capy P Jallon JM 2001 Variations of male cuticular hydrocarbons with geoclimatic variables: an adaptative mechanism in Drosophila melanogaster? Genetica 110 117 130 Rouault J-D Marican C Wicker-Thomas C Jallon J-M 2004 Relations between cuticular hydrocarbon (HC) polymorphism, resistance against desiccation and breeding temperature; a model for HC evolution in D. melanogaster and D. Simulans Genetica 120 195 212 10.1023/B:GENE.0000017641.75820.49 Savarit F Ferveur JF 2002 Temperature affects the ontogeny of sexually dimorphic cuticular hydrocarbons in Drosophila melanogaster Journal of Experimental Biology 205 3241 3249 Scharwey M Tatsuta T Langer T 2013 Mitochondrial lipid transport at a glance Journal of Cell Science 126 5317 5323 10.1242/jcs.134130 Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S Schmid B Tinevez JY White DJ Hartenstein V Eliceiri K Tomancak P Cardona A A 2012 Fiji: an open-source platform for biological-image analysis Nature Methods 9 676 682 10.1038/nmeth.2019 Senyilmaz D Virtue S Xu X Tan CY Griffin JL Miller AK Vidal-Puig A Teleman AA 2015 Regulation of mitochondrial morphology and function by stearoylation of TFR1 Nature 525 124 128 10.1038/nature14601 St John JC Jokhi RP Barratt CL 2005 The impact of mitochondrial genetics on male infertility International Journal of Andrology 28 65 73 10.1111/j.1365-2605.2005.00515.x Stanley WC Khairallah RJ Dabkowski ER 2012 Update on lipids and mitochondrial function: impact of dietary n-3 polyunsaturated fatty acids Current opinion in clinical nutrition and metabolic care 15 122 126 10.1097/MCO.0b013e32834fdaf7 Stinziano JR Sové RJ Rundle HD Sinclair BJ 2015 Rapid desiccation hardening changes the cuticular hydrocarbon profile of Drosophila melanogaster Comparative Biochemistry and Physiology Part A: Molecular Integrative Physiology 180 38 42 10.1016/j.cbpa.2014.11.004 Tyurina YY Poloyac SM Tyurin VA Kapralov AA Jiang J Anthonymuthu TS Kapralova VI Vikulina AS Jung MY Epperly MW Mohammadyani D Klein-Seetharaman J Jackson TC Kochanek PM Pitt BR Greenberger JS Vladimirov YA Bayır H Kagan VE 2014 A mitochondrial pathway for biosynthesis of lipid mediators Nature Chemistry 6 542 552 10.1038/nchem.1924 Voelker DR 2004 Lipid synthesis and transport in mitochondrial biogenesis Mitochondrial function and biogenesis Springer Press Berlin Wakil SJ Stoop JK Joshi VC 1983 Fatty acid synthesis and its regulation Annual Review of Biochemistry 52 537 579 10.1146/annurev.bi.52.070183.002541 Wang Y Carballo RG Moussian B 2017 Double cuticle barrier in two global pests, the whitefly Trialeurodes vaporariorum and the bedbug Cimex lectularius Journal of Experimental Biology 220 1396 1399 Wang Y Yu Z Zhang J Moussian B 2016 Regionalization of surface lipids in insects Proceedings of the Royal Society B: Biological Sciences 283 20152994 Williams BR Van Heerwaarden B Dowling DK Sgrò CM 2012 A multivariate test of evolutionary constraints for thermal tolerance in Drosophila melanogaster Journal of Evolutionary Biology 25 1415 1426 10.1111/j.1420-9101.2012.02536.x Williams S Dew-Budd K Davis K Anderson J Bishop R Freeman K Davis D Bray K Perkins L Hubickey J Reed LK 2015 Metabolomic and gene expression profiles exhibit modular genetic and dietary structure linking metabolic syndrome phenotypes indrosophila Genes Genomes Genetics 5 2817 2829 Wolff JN Ladoukakis ED Enríquez JA Dowling DK 2014 Mitonuclear interactions: evolutionary consequences over multiple biological scales Philosophical Transactions of the Royal Society of London 369 20130443 10.1098/rstb.2013.0443 Wurdack M Herbertz S Dowling D Kroiss J Strohm E Baur H Niehuis O Schmitt T 2015 Striking cuticular hydrocarbon dimorphism in the mason wasp odynerusspinipes and its possible evolutionary cause (hymenoptera: chrysididae, vespidae) Proceedings Biological Sciences 282 20151777 10.1098/rspb.2015.1777 Yu Z Wang Y Zhao X Liu X Ma E Moussian B Zhang J 2017 The ABC transporter ABCH-9C is needed for cuticle barrier construction in Locusta migratoria Insect Biochemistry and Molecular Biology 87 90 99 10.1016/j.ibmb.2017.06.005 Zhu CT Ingelmo P Rand DM 2014 GxGxE for lifespan in Drosophila: mitochondrial, nuclear, and dietary interactions that modify longevity PLOS Genetics 10 1004354 10.1371/journal.pgen.1004354 Zuber R Norum M Wang Y Oehl K Gehring N Accardi D Bartozsewski S Berger J Flotenmeyer M Moussian B 2018 The ABC transporter Snu and the extracellular protein Snsl cooperate in the formation of the lipid-based inward and outward barrier in the skin of Drosophila European Journal of Cell Biology 97 90 101 10.1016/j.ejcb.2017.12.003