Sofia G Costa
email: sgcosta@fc.ul.pt

Sara Magalhães

Inês Santos

Flore Zélé

Leonor R Rodrigues

A sex-specific trade-off between pesticide resistance and thermal tolerance in Tetranychus urticae

Keywords: reproductive success, climate warming, crop pests, fertility

Current pest management relies extensively on pesticide application worldwide, despite the frequent rise of pesticide resistance in crop pests. This is particularly worrisome because resistance is often not costly enough to be lost in populations after pesticide application, resulting in increased dependency on pesticide application. As climate warming increases, effort should be put into understanding how thermal tolerance will affect the persistence of pesticide resistance in populations. To address this, we measured thermal tolerance in two populations of the spider mite crop pest Tetranychus urticae that differ in the presence or absence of a target-site mutation conferring resistance to etoxazole pesticide.

We found that developmental time and fertility, but not survival, were negatively affected by increasing temperatures in the susceptible population. Furthermore, we found no difference between resistant and susceptible populations in all life-history traits when both sexes developed at control temperature, nor when females developed at high temperature. Resistant heat-stressed males, in contrast, showed lower fertility than susceptible ones, indicating a sex-specific trade-off between thermal tolerance and pesticide resistance. This suggests that global warming can lead to reduced pesticide resistance in natural populations. However, resistant females, being as affected by high temperature as susceptible individuals, may buffer the toll in resistant male fertility. In addition, the decrease in developmental time of both sexes at high temperature may accelerate adaptation to both temperature and pesticide, which can promote the maintenance of resistant populations under climate warming.

Introduction

Crop pests are responsible for enormous economic losses worldwide [START_REF] Culliney | Crop Losses to Arthropods[END_REF][START_REF] Oliveira | Crop losses and the economic impact of insect pests on Brazilian agriculture[END_REF]. Particularly, insects and mites account for most of the damage to crops, with estimated losses of 10-20% pre-harvest and 5-10% post-harvest [START_REF] Culliney | Crop Losses to Arthropods[END_REF]. Current pest management relies extensively on pesticide application, despite its unambiguous adverse effects (IPBES 2019; Janssen and van Rijn 2021; [START_REF] Jacquet | Pesticide-free agriculture as a new paradigm for research[END_REF]. Indeed, the use of pesticides is one of the main drivers of biodiversity loss, including of biological control agents [START_REF] Geiger | Persistent negative effects of pesticides on biodiversity and biological control potential on European farmland[END_REF][START_REF] Sánchez-Bayo | Worldwide decline of the entomofauna: A review of its drivers[END_REF]IPBES 2019), and it is highly detrimental to the environment and human health [START_REF] Bourguet | The Hidden and External Costs of Pesticide Use[END_REF][START_REF] Kim | Exposure to pesticides and the associated human health effects[END_REF]. The evolution of pesticide resistance in crop pests [START_REF] Bass | The global status of insect resistance to neonicotinoid insecticides[END_REF][START_REF] Hawkins | The evolutionary origins of pesticide resistance[END_REF] along with the loss of biocontrol agents and other ecosystem services, in turn, increases the dependency on pesticide application to ensure pest control, resulting in the emergence of a "pesticide treadmill" (van den Bosch 1989).

Current climate warming is leading to a continuous increase in intensity and frequency of temperature extremes on a global scale [START_REF] Seneviratne | Weather and Climate Extreme Events in a Changing Climate[END_REF]. The response of arthropod crop pests to high temperature can lead to increased or decreased crop losses depending on the pest species and other varying biotic and abiotic factors [START_REF] Skendžić | The Impact of Climate Change on Agricultural Insect Pests[END_REF]. For instance, decreases in crop losses have been attributed to reduced physiological performance at extreme temperatures and to range contraction of insect pests [START_REF] Lehmann | Complex responses of global insect pests to climate warming[END_REF]. In these cases, one could expect the need for pest control measures to be reduced. In turn, many crop pests respond to high temperatures by increasing in number [START_REF] Lehmann | Complex responses of global insect pests to climate warming[END_REF] due, for example, to a rise in metabolic rate and population growth [START_REF] Deutsch | Increase in crop losses to insect pests in a warming climate[END_REF], or to the disruption of biological control as biocontrol agents show lower tolerance to high temperatures [START_REF] Montserrat | Can climate change jeopardize predator control of invasive herbivore species? A case study in avocado agro-ecosystems in Spain[END_REF]. When this increase in crop number occurs, it may be necessary to increase pesticide use [START_REF] Delcour | Literature review: Impact of climate change on pesticide use[END_REF].

Given that pest management greatly depends on pesticide application, effort should be put into understanding the reciprocal interaction between thermal tolerance and pesticide resistance, such as to determine how persistent will pesticide resistance be under climate warming. In fact, even if agricultural practices are moving towards a reduction in the use of pesticides, resistant genotypes can remain in the environment for many generations [START_REF] Kontsedalov | Effects of Insecticides on Immature and Adult Western Flower Thrips (Thysanoptera: Thripidae) in Israel[END_REF][START_REF] Bielza | Stability of spinosad resistance in Frankliniella occidentalis (Pergande) under laboratory conditions[END_REF], especially those that are not costly in pesticide-free environments [START_REF] Bielza | Stability of spinosad resistance in Frankliniella occidentalis (Pergande) under laboratory conditions[END_REF][START_REF] Abbas | Fitness cost and realized heritability of resistance to spinosad in Chrysoperla carnea (Neuroptera: Chrysopidae)[END_REF]. How these genotypes will respond to climate change is thus key to understand the effect of climate change on crop pest populations. Up until now, various studies have shown that resistance to pesticide changes with temperature, often becoming reduced under heat (reviewed in [START_REF] Matzrafi | Climate change exacerbates pest damage through reduced pesticide efficacy[END_REF].

However, evidence on whether thermal tolerance differs between resistant and susceptible individuals in a pesticide-free environment is not abundant (but see [START_REF] Fulton | Fitness costs of pesticide resistance in Hyalella azteca under future climate change scenarios[END_REF][START_REF] Langmüller | Fitness effects for Ace insecticide resistance mutations are determined by ambient temperature[END_REF][START_REF] Li | Temperature-related fitness costs of resistance to spinosad in the diamondback moth, Plutella xylostella (Lepidoptera: Plutelidae)[END_REF][START_REF] Ma | Climate warming promotes pesticide resistance through expanding overwintering range of a global pest[END_REF][START_REF] Yang | Effects of temperature on fitness costs in chlorpyrifosresistant brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)[END_REF][START_REF] Zhang | Trade-off between thermal tolerance and insecticide resistance in Plutella xylostella[END_REF]. This is unfortunate because such knowledge is crucial to predict the dynamics of crop pests under climate warming given that, depending on the outcome, one could expect the maintenance or loss of pesticide resistance in a population and thus the failure or success of pesticide applications, respectively.

Increase in the efficacy of pesticides under global warming would be expected when pesticide resistance and thermal tolerance trade-off, given the expected decline in the number of resistant individuals in such a scenario. This has been shown in a few studies [START_REF] Li | Temperature-related fitness costs of resistance to spinosad in the diamondback moth, Plutella xylostella (Lepidoptera: Plutelidae)[END_REF][START_REF] Zhang | Trade-off between thermal tolerance and insecticide resistance in Plutella xylostella[END_REF][START_REF] Yang | Effects of temperature on fitness costs in chlorpyrifosresistant brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)[END_REF]. For example, in the diamondback moth Plutella xylostella, chlorpyrifos-resistant individuals have significantly lower survival, adult emergence rates and higher damages of wing veins under heat stress than susceptible ones [START_REF] Zhang | Trade-off between thermal tolerance and insecticide resistance in Plutella xylostella[END_REF]. In turn, the efficacy of pesticides would be expected to decrease whenever resistant individuals are also more tolerant to heat stress than susceptible ones, meaning they will spread quicker in the population under climate warming. For example, in Drosophila simulans, differences in viability between haplotypes susceptible and resistant to carbamate and organophosphate are reduced at high temperatures, meaning the resistant haplotypes have lost their disadvantage compared to susceptible ones under these conditions [START_REF] Langmüller | Fitness effects for Ace insecticide resistance mutations are determined by ambient temperature[END_REF].

The majority of studies focusing on the costs of pesticide resistance disregard the possibility of sex-specific responses. This is unfortunate, as the meagre evidence of such a phenomenon call attention to its important implications. Indeed, in Drosophila melanogaster, resistance to DDT is costly in males but increases female reproductive output in the absence of pesticide [START_REF] Smith | DDT resistance, epistasis and male fitness in flies[END_REF], which helps explaining why the resistance allele was present in populations at low frequency prior to the widespread use of DDT [START_REF] Rostant | Sexual conflict maintains variation at an insecticide resistance locus[END_REF]. Likewise, extensive work has demonstrated that understanding the effect of temperature in each sex is a crucial step to accurately predict the impact of climate change on organisms. For instance, evidence of sex-specific thermal sensitivity in different taxa accumulate [START_REF] Vasudeva | Developmental temperature affects the expression of ejaculatory traits and the outcome of sperm competition in Callosobruchus maculatus[END_REF][START_REF] Iossa | Sex-Specific Differences in Thermal Fertility Limits[END_REF], with reproductive traits being affected at lower temperatures in males than in females (e.g., [START_REF] David | Male sterility at extreme temperatures: A significant but neglected phenomenon for understanding Drosophila climatic adaptations[END_REF][START_REF] Iossa | Sex-Specific Differences in Thermal Fertility Limits[END_REF]Rodrigues et al. 2022b;[START_REF] Sales | Experimental heatwaves compromise sperm function and cause transgenerational damage in a model insect[END_REF][START_REF] Van Heerwaarden | Male fertility thermal limits predict vulnerability to climate warming[END_REF]. As such, male, not female, thermal fertility limits have been identified as the best predictors of the response of populations to climate change [START_REF] Parratt | Temperatures that sterilize males better match global species distributions than lethal temperatures[END_REF][START_REF] Van Heerwaarden | Male fertility thermal limits predict vulnerability to climate warming[END_REF]. In haplodiploid species, heat-induced sterility can also lead to variation in sex ratio and productivity, depending on the sex that is being affected [START_REF] Costa | Multiple mating rescues offspring sex ratio but not productivity in a haplodiploid exposed to developmental heat stress[END_REF]. Indeed, because fertilized eggs only generate daughters, whereas sons develop from unfertilized eggs, male sterility should lead to a more male-biased sex ratio and female sterility to fewer offspring. Such sex-specific responses are bound to affect plastic and/or evolutionary responses of populations [START_REF] Costa | Multiple mating rescues offspring sex ratio but not productivity in a haplodiploid exposed to developmental heat stress[END_REF]. Therefore, disregarding sex-specific responses compromises our ability to accurately predict the impact of climate warming on the persistence of pesticide-resistance in populations. Yet, to date no study has investigated the existence of such sexspecific responses.

Here, we tested the effect of high temperature and its interaction with pesticide resistance on three life-history traits (survival, developmental time and fertility) in Tetranychus urticae. This spider mite is a haplodiploid polyphagous agricultural pest that occurs on a wide variety of plant species across the world, some of which of great economic significance [START_REF] Migeon | Spider Mites Web: A comprehensive database for the Tetranychidae[END_REF]. Pesticides are commonly used to control the spread of mite population in crops, but the success of this control strategy if often compromised by the spider mites' exceptional ability to rapidly evolve resistance [START_REF] Van Leeuwen | Acaricide resistance mechanisms in the twospotted spider mite Tetranychus urticae and other important Acari: A review[END_REF]. We have created two populations with the same genetic background but a different resistance status (susceptible vs resistant) to etoxazole, an acaricide commonly used on populations of this species worldwide [START_REF] Kim | Effect of temperature on development and life table parameters of Tetranychus urticae koch (Acari: Tetranychide) reared on eggplants[END_REF]. This allows us to attribute any observed differences between populations to the resistance allele or to evolutionary changes in the genome due to its presence. Etoxazole resistance has been thoroughly characterized in T. urticae [START_REF] Van Leeuwen | Mechanisms of Acaricide Resistance in the Two-Spotted Spider Mite Tetranychus urticae[END_REF], 2012): this pesticide exhibits high efficacy as a chitin biosynthesis inhibitor in T. urticae during the developmental stages, being lethal to eggs and juveniles, but not adults [START_REF] Nauen | Mode of action of etoxazole[END_REF][START_REF] Van Leeuwen | Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods[END_REF] and resistance is recessive and conferred by a single nonsynonymous mutation in the chitin synthase [START_REF] Van Leeuwen | Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods[END_REF]. Despite this wealth of information, no study has yet tested if the resistance status of each sex elicits specific responses to temperature. Here, we set out to fill this gap.

Materials and Methods

Spider mite populations

Two populations of the spider mite Tetranychus urticae red form (a.k.a. T. cinnabarinus; [START_REF] Crawley | Wolbachia and host intrinsic reproductive barriers contribute additively to postmating isolation in spider mites[END_REF] that share the same genetic background, except for the presence or absence of an allele conferring resistance to the pesticide etoxazole (i.e., a single nucleotide mutation on the chitin synthase 1 gene, CHS1; [START_REF] Van Leeuwen | Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods[END_REF], were used in the experiments. The procedure used to create these populations is depicted in Fig. 1 and fully described in the sections below.

Creation of the Wi.SS populations

Five Wolbachia-infected homozygous etoxazole-susceptible ("Wi.SS") population replicates were initially created in 2015, each by merging six compatible Wolbachia-infected populations collected in the region of Lisbon, Portugal, in 2013 (Zélé et al. 2018a(Zélé et al. , 2020b)). Each population replicate was initially created with a census population size of 200 mated females and subsequently maintained with discrete generations at this same population size (cf. detailed procedure for the creation and maintenance of these populations, called "Infected controls; iC", in Rodrigues et al. 2022a). Full homozygosity for the etoxazole susceptible allele was subsequently confirmed by PCR-RFLP (as described in [START_REF] Van Leeuwen | Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods[END_REF] for each of the field-derived populations and each of the Wi.SS replicates.

Creation of the Wi.RR populations

Five Wolbachia-infected homozygous etoxazole-resistant ("Wi.RR") population replicates were created in 2018 by introgression of an etoxazole-resistance allele into each of the Wi.SS population replicates (Fig. 1 and detailed procedure in Suppl. Fig. 1). The etoxazole-resistance allele originated from the homozygous etoxazole-resistant (RR) red lab strain SB9, collected in Crete, Greece, in 2006[START_REF] Van Leeuwen | Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods[END_REF]). As coinfection with Wolbachia and Rickettsia was detected in this strain (using the multiplex PCR procedure described in Zélé et al. 2018b), and because these endosymbionts may cause reproductive incompatibilities with the Wolbachia strain that is present in the Wi.SS populations, SB9 was treated with a rifampicin solution at 0.05%, w/v for one generation as described in Zélé et al. (2020a) to create the symbiont-free donor parent strain SB9.rif. We then used a backcross procedure ensuring the complete introgression of the etoxazole-resistance allele from the SB9.rif donor strain into each of the Wi.SS recurrent parent populations, with an estimated retention of 99.6% of their nuclear background, and complete retention of their mitochondrial background and Wolbachia infection.

At the onset of the backcross procedure, SB9.rif males were crossed with females from each of the Wi.SS population replicates to obtain a 100% heterozygous F1 female progeny (hence all "Wi.RS":

Wolbachia-infected with a resistant and a susceptible allele). Given haplodiploidy in spider mites and because the allele conferring resistance to etoxazole is recessive, these females were isolated as virgin to produce "Wi.S" and "Wi.R" F2 haploid males (note that this step replaces the usual self-cross step for the introgression of recessive alleles in diploid organisms). Resistant F2 males (Wi.R) were then selected by being placed on etoxazole-soaked cotton (diluted in water at 0.5g/L) during the development of the male offspring, and subsequently backcrossed with females from the recurrent parent population Wi.SS to produce a new generation of heterozygous females (i.e., the first generation of backcrossed females; BC 1 ). Heterozygous BC 1 females were, as their grandmothers, isolated as virgin to produce a new generation of males (i.e., the progeny of "non crossed" females; NC 1 ), which were again selected for etoxazole resistance during their development. This procedure was repeated for six additional backcrosses (BC 2 NC 2 to BC 7 NC 7 ). At the end of the procedure, a subset of the progeny resulting from the last generation of backcross (Wi.RS females and Wi.S males; BC 7 ) was selfcrossed (SC 1 ) to obtain Wi.RS and Wi.SS females, which were crossed with NC 7 selected cousins.

Their offspring (SC 2 ) was then exposed to etoxazole to select the homozygous resistant Wi.RR females and Wi.R males that founded the replicated populations "Wi.RR". A census population size of at least 200 females and 100 males was used at each step of the procedure (cf. detailed procedure in Suppl.

Fig. 1). The concentration of etoxazole used is higher than the LC100 seen in [START_REF] Van Leeuwen | Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods[END_REF], having the same effect on our populations as it led them to be 100% homozygous resistant.

Creation and merge of the Wu.SS and Wu.RR populations

The five Wi.SS and the five Wi.RR population replicates (each with a population size of 200 females)

were subsequently treated for one generation with rifampicin (0.05% w/v), following the procedure described in Zélé et al. (2020a). Each newly created Wu.SS and Wu.RR population replicates was subsequently maintained in discrete generations (population size of 200 females) in absence of antibiotics (i.e., under the same conditions as the untreated populations). Three generations later, the presence/absence of Wolbachia and that of the etoxazole-resistance allele were confirmed in a pool of 100 females from each Wi.SS, Wi.RR, Wu.SS and Wu.RR population replicate by multiplex PCR (see Zélé et al. 2018b) and PCR-RFLP (see [START_REF] Van Leeuwen | Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods[END_REF], respectively. All replicates from either the Wu.SS or the Wu.RR populations, were then merged in September 2020 to create the susceptible and resistant base populations used in this study, respectively.

Rearing conditions

Since their creation and merging, both Wu.SS and Wu.RR were maintained in large numbers (> 2000) under continuous generations on entire plants. These populations were kept on bean leaves (Phaseolus vulgaris, variety Contender, provided by Germisem, Portugal) under controlled conditions (25°C; photoperiod of 16L:8D). Bean plants were germinated and grown in an isolated and herbivore-free room for 14 days under controlled conditions (photoperiod of 16L at 25°C: 8D at 20°C) before being infested with spider mites.

Experimental setup

Effect of high developmental temperatures on life-history traits

First, we characterized the impact of four temperatures above the control temperature in the laboratory (control temperature: 25°C, high temperatures: 33, 35, 36 and 37°C) during development on the developmental time, survival and fertility of pesticide-susceptible spider mites (from Wu.SS).

To assess the effect of temperature on developmental time and juvenile survival, mite cohorts were created by placing on a bean leaf patch (ca. 75 cm 2 ) 100 mated females for 2 days at 25°C, so they could oviposit synchronously. Once adult, the offspring of those females was transferred to one of the tested temperatures (25,33,35,36 or 37°C) for three days, after which a maximum of 32 offspring females were individually placed on bean leaf discs (2.55 cm 2 ) to oviposit for 24 hours at the same temperature. Egg development was followed daily, and the number of days elapsed until the first adult male and female offspring emerged (i.e., developmental time), as well as the number of eggs that reached adulthood (i.e., number of surviving juveniles) were recorded.

Due to logistic constraints, three assays were performed, always including the control temperature (25°C): i) 25°C, 33°C and 37°C; ii) 25°C and 35°C; iii) 25°C and 36°C. In all cases, replicates with damaged (i.e., hurt during manipulation) mothers were excluded from the analyses.

Additionally, in the analysis of all independent variables, patches with females that did not lay eggs were excluded. Overall, we analysed 67, 65 and 82 replicates (i.e., patches) of female developmental time, male developmental time and number of surviving juveniles, respectively (see Table S1 for more details). Due to the low number of adult females surviving and ovipositing at 37°C, this temperature was not included in the statistical analyses of developmental time and number of surviving juveniles (see Table S2).

To determine the impact of heat on fertility, we focused only on female fertility, as we assumed that male fertility would also be hindered at the temperature females are affected, since males generally suffer from a reduction in fertility at lower high temperatures than females in several arthropod species [START_REF] David | Male sterility at extreme temperatures: A significant but neglected phenomenon for understanding Drosophila climatic adaptations[END_REF][START_REF] Sales | Experimental heatwaves compromise sperm function and cause transgenerational damage in a model insect[END_REF][START_REF] Iossa | Sex-Specific Differences in Thermal Fertility Limits[END_REF][START_REF] Zwoinska | Phenotypic Responses to and Genetic Architecture of Sterility Following Exposure to Sub-Lethal Temperature During Development[END_REF][START_REF] Van Heerwaarden | Male fertility thermal limits predict vulnerability to climate warming[END_REF]. Cohorts were created with mated females as before, but they were directly maintained at one of the tested temperatures (25, 33, 35, 36 or 37°C). Before the offspring reached adulthood, deutonymph quiescent males and females were isolated separately to ensure virginity. Once adults (ca. 24 hours later), a maximum of 40 pairs of virgin females and males were isolated on bean leaf discs (2.55 cm 2 ) to mate and oviposit. Five treatments were established: pairs where both individuals developed at 25°C (control; ♀25 x ♂25) and pairs where only the female developed at one of the high temperatures (♀33 x ♂25, ♀35 x ♂25, ♀36 x ♂25 and ♀37 x ♂25). Pairs were maintained during 24 hours, after which males were discarded. Females were left to lay eggs for two more days, after which they were also discarded, and the eggs were counted. Leaf discs were kept at the female developmental temperature from pairing until the female was removed, being placed at 25°C afterwards. The survival status of both male and female was recorded until removal. Once reaching adulthood, offspring was counted, sexed and removed 12 and 14 days after the onset of oviposition. Daily fecundity and offspring number were thus used as proxies for female fertility.

As in the previous experiment, temperature treatments were distributed in three assays: (i) 25°C, 33°C and 37°C; (ii) 25°C and 35°C; (iii) 25°C and 36°C. For the first assay, we performed two blocks, which occurred in two consecutive days. The other two assays were done in a single block. In all cases, replicates with individuals forming the mating pairs that were damaged during manipulation were excluded from the analyses. In addition, patches with females that did not lay eggs were excluded from the analysis of offspring number. Overall, we analysed 140 and 117 replicates (i.e., patches) of daily fecundity and offspring number, respectively (see Table S1 for more details).

Interaction between pesticide resistance and response to high developmental temperature

To test if the effect of high developmental temperature on developmental time, survival and fertility differed depending on the resistance status of individuals, we exposed both resistant (Wu.RR) and susceptible (Wu.SS) populations to either 25 or 36°C and repeated the experiments described above. In this case, when testing fertility, we measured both sexes, with offspring number being used to access female fertility and sex ratio to access male fertility, as only daughters contain the genetic material of the father. Here, females were given four days to oviposit instead of three, and four treatments were established to separately test the response of males and females to high developmental temperature: pairs where both individuals developed at 25°C (control; ♀25 x ♂25), and pairs where each or both sexes developed at 36°C (♀36 x ♂25, ♀25 x ♂36 and ♀36 x ♂36, respectively). Pairs were maintained at 25°C when both individuals developed at that temperature. Otherwise, they were kept at 36°C until the sex that developed at 36°C was removed, at which time they were moved to 25°C.

When both sexes developed at 36°C, the transfer to 25°C occurred when the last individual of the pair was removed. All other experimental details are as above.

Replicates were excluded according to the same criteria as described above. Overall, we analysed the developmental time of females and males from 97 and 95 patches, respectively, the number of surviving juveniles from 114 patches and the number and sex of offspring from 233 patches (see Table S1 for more details).

Statistical analyses

All statistical analyses were performed using the software R (version 4.0.3; R Core Team 2020).

Depending on the data and error structure, we performed linear models (LM), linear mixed-effects models (LMM), generalized linear models (GLM) and generalized mixed-effects models (GLMM) implemented in lme4 (version 1.1.26; [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF] and glmmTMB (version 1.0.2.1; [START_REF] Brooks | glmmTMB Balances Speed and Flexibility Among Packages for Zero-inflated Generalized Linear Mixed Modeling[END_REF]; see Table S3 andS4). Maximal models were simplified by removing non-significant terms (p > 0.05) from the highest-to the lowest-order interaction [START_REF] Crawley | Wolbachia and host intrinsic reproductive barriers contribute additively to postmating isolation in spider mites[END_REF]. The significance of each explanatory variable was determined by chi-squared tests for discrete distributions, and Wald F tests for continuous distributions [START_REF] Bolker | Generalized linear mixed models: a practical guide for ecology and evolution[END_REF]. Graphic representations of the data were produced with the software package ggplot2 (version 3.3.3;[START_REF] Wickham | ggplot2: Elegant Graphics for Data Analysis[END_REF].

Effect of high developmental temperatures on life-history traits

The effect of developmental high temperatures on juvenile traits was assessed by analysing two traits: the number of days from egg until adulthood (i.e., number of days the first male or female took to reach adulthood in a patch), used as a proxy for developmental time of each sex; and the number of surviving juveniles (i.e., number of juveniles surviving until the adult stage; Table S3). The effect of developmental heat stress on female fertility, was determined by analysing daily fecundity and offspring number (Table S3).

As temperatures were not tested simultaneously, we analysed the differences of each high temperature to the control temperature. For that, within each assay, the mean trait value measured at the control temperature was subtracted to the individual trait values measured at the high temperature tested (trait value at the high temperature -x trait value at 25°C). When the assay was done in more than one block, the mean of the control temperature was calculated within each block, to account for that variability. The new variable was used as dependent variable in the subsequent models.

All variables were analysed using LM models with a gaussian error distribution, except for the number of surviving juveniles that was analysed using a LMM model (Table S3). The data were box cox transformed (female developmental time: λ = 1.94; male developmental time: λ = 0.73; number of surviving juveniles: λ = 0.91; MASS package, boxcox function; version 7. 3.53;[START_REF] Venables | Modern Applied Statistics with S, 4th edn[END_REF] to improve the fit of the model in each variable, except for daily fecundity and offspring number. To analyse the impact of temperature on developmental time and number of surviving juveniles, the developmental temperature (25, 33, 35 or 36°C) was included in the models as fixed factor (Table S3). Fecundity was added as random factor in the model testing the impact of temperature on the number of surviving juveniles, to account for the variation in the number of eggs laid (Table S3). In the analyses of daily fecundity and offspring number, the type of cross between males and females from different developmental temperatures (♀25 x ♂25, ♀33 x ♂25, ♀35 x ♂25, ♀36 x ♂25 or ♀37 x ♂25) was included in the model as fixed factor (Table S3).

To determine if the difference of each temperature to the control was significant, we used the function summary and took the value that reports the difference of the first temperature listed on the dataset to an intercept of zero (which in this case corresponds to the mean trait value at control temperature). This was repeated for all temperatures by reordering this variable on the dataset. A posteriori contrasts with Bonferroni corrections of significant explanatory variables were made to test differences among the different high temperatures using the emmeans package (version 1.5.3; Length 2020; Table 2).

Interaction between pesticide resistance and response to high developmental temperature

To test whether developmental heat stress differentially affected individuals with different resistance status in a sex-specific manner, we analysed four traits: developmental time, the number of surviving juveniles, the offspring number and the offspring sex ratio (Table S4).

Developmental time and the number of surviving juveniles were analysed using GLM and GLMM models, respectively, with a Poisson error distribution (Table S4). The developmental temperature of the individuals (25°C or 36°C), their resistance status (susceptible or resistant) and the interaction between these two factors were included in all models as fixed factors. Fecundity was included as a random factor in the analysis of the number of surviving juveniles.

The offspring number was analysed using a GLMM model with a quasi-Poisson error distribution (Table S4). Offspring sex ratio, analysed as the proportion of daughters, was computed using the function cbind with the number of daughters and sons as arguments (Table S4). This variable was analysed with a GLMM model with a beta-binomial error distribution and a parameter to account for zero inflation (ziformula ~1; package glmmTMB). In the analysis of both variables, the developmental temperature of the female and of the male (25°C or 36°C, in both variables), the resistance status of the pair (susceptible or resistant), as well as all possible interactions, were included as fixed factors. We paired males and females from two different cohorts (i.e., males and females could be 9 or 10 day-old when developed at 25°C, or 7 or 8 day-old when developed at 36°C), so the age of each sex was included as a random factor (Table S4). A posteriori contrasts with Bonferroni corrections were made using the emmeans package (Length 2020; Table 4).

Results

Effect of high developmental temperatures on life-history traits

The developmental time of males and females was affected by the temperature they developed at (Table 1). Indeed, the development of both sexes was faster at any of the high temperatures than at the control temperature, and the individuals developed significantly faster at 35°C than at 36°C (Table 2; Fig. 2a andb). In contrast, the number of surviving juveniles, accounting for the total number of eggs produced, was not affected by the developmental temperature of individuals (Table 1; Fig. 2c).

Female fertility was affected by developmental temperature (Table 1), with both daily fecundity and the offspring number being higher at 33°C and lower at 35, 36 and 37°C, compared to the control temperature (Table 2; Fig. 2d ande). In both cases, 36 and 37°C showed the lowest values compared to the other temperatures (Table 2; Fig. 2d ande).

In sum, fertility was most affected at 36 and 37°C. However, at 37°C adult survival was drastically hindered (ca. 10% survival after 3 days of exposure; Table S2). Therefore, we used 36°C as the sublethal temperature in subsequent tests.

Interaction between pesticide resistance and response to high developmental temperature

The developmental time and number of surviving juveniles were not affected by the interaction between developmental temperature and resistance status (female developmental time: χ 2 1 = 0.118, p = 0.731; male developmental time: χ 2 1 = 0.359, p = 0.549; number of surviving juveniles: χ 2 1 = 0.072, p = 0.789). However, these traits were equally affected by high developmental temperature (36°C) in individuals from susceptible and resistant populations (Table 3; Fig. 3a, b andc; Fig. S2).

Fertility was affected by the interaction between the developmental temperature of the male and the resistance status of the pair (Table 3). Indeed, resistant males produced fewer offspring and a lower proportion of daughters than susceptible ones, but only when they developed at 36°C. (Table 4;

Fig. 3d ande). The female developmental temperature affected both traits similarly in the two populations, as all pairs with females developed at 36°C produced fewer offspring and a lower proportion of daughters than all pairs with females developed at 25°C (Table 3; Fig. 3d ande).

Discussion

Our results show that all life-history traits measured in the individuals from the susceptible population, but the number of surviving juveniles, responded to high developmental temperatures in a non-linear manner. At 36°C, fertility was affected the most, while survival was as yet not compromised. We thus selected this temperature to test for sex-specific effects of temperature on individuals with different resistant status. Developmental time and number of surviving juveniles were affected by temperature but not by resistance status. In contrast, reproductive traits of resistant and susceptible individuals were differentially affected by high developmental temperatures in a sex-specific manner. Indeed, matings involving resistant males developed at high temperature resulted in fewer offspring and more sons than matings involving susceptible males.

We found that the response of several traits to temperature was non-linear, with daily fecundity and offspring number peaking at 33°C and the developmental time of both sexes reaching its lowest at 35°C. Evidence of non-linear responses in this and other arthropods towards increasing temperatures (Umoetok [START_REF] Akpassam | Response of Callosobruchus maculatus (F.) to varying temperature and relative humidity under laboratory conditions[END_REF][START_REF] Zou | Effect of temperature on development and reproduction of the carmine spider mite, Tetranychus cinnabarinus (Acari: Tetranychiae), fed on cassava leaves[END_REF][START_REF] Sales | Experimental heatwaves compromise sperm function and cause transgenerational damage in a model insect[END_REF][START_REF] Zwoinska | Phenotypic Responses to and Genetic Architecture of Sterility Following Exposure to Sub-Lethal Temperature During Development[END_REF]) and other environmental variables [START_REF] Godinho | Effect of Cadmium Accumulation on the Performance of Plants and of Herbivores That Cope Differently With Organic Defenses[END_REF][START_REF] Godinho | Intraspecific variability in herbivore response to elemental defences is caused by the metal itself[END_REF][START_REF] Guedes | Pesticide-induced hormesis in arthropods: Towards biological systems[END_REF]) are not uncommon. In Tetranychus, some studies show a decrease in developmental time with temperature [START_REF] Kasap | Effect of Apple Cultivar and of Temperature on the Biology and Life Table Parameters of the Twospotted Spider Mite Tetranychus urticae[END_REF][START_REF] Praslička | Influence of Temperature and Host Plants on the Development and Fecundity of the Spider Mite Tetranychus urticae (Acarina: Tetranychidae)[END_REF][START_REF] Bayu | Impact of constant versus fluctuating temperatures on the development and life history parameters of Tetranychus urticae (Acari: Tetranychidae)[END_REF][START_REF] Zou | Effect of temperature on development and reproduction of the carmine spider mite, Tetranychus cinnabarinus (Acari: Tetranychiae), fed on cassava leaves[END_REF], while other studies notice a decrease followed by an increase in developmental time with rising temperatures [START_REF] Riahi | Acari: Tetranychidae)[END_REF][START_REF] Hasanvand | Effect of temperature on development and reproduction of Tetranychus kanzawai (Tetranychidae) fed on apple leaves[END_REF][START_REF] Farazmand | Effect of the temperature on development of Tetranychus urticae (Acari: Tetranychidae) feeding on cucumber leaves[END_REF]. A peak in daily fecundity has also been recorded in previous work, but the temperatures leading to such peak differ between studies [START_REF] Kasap | Effect of Apple Cultivar and of Temperature on the Biology and Life Table Parameters of the Twospotted Spider Mite Tetranychus urticae[END_REF][START_REF] Riahi | Acari: Tetranychidae)[END_REF][START_REF] Zou | Effect of temperature on development and reproduction of the carmine spider mite, Tetranychus cinnabarinus (Acari: Tetranychiae), fed on cassava leaves[END_REF]). These differences may be related to the origin of the populations tested, given that populations from different climates are adapted to specific sets of temperatures [START_REF] Deutsch | Impacts of climate warming on terrestrial ectotherms across latitude[END_REF][START_REF] García-Robledo | Limited tolerance by insects to high temperatures across tropical elevational gradients and the implications of global warming for extinction[END_REF]. Clearly, more studies are needed to understand the origin and consequences of non-linear responses to temperature.

Several studies show that resistance to pesticide can trade off with life-history traits in pesticide-free environments (e.g., [START_REF] Carriere | Life-history costs associated with the evolution of insecticide resistance[END_REF][START_REF] Okuma | Inheritance and fitness costs of Spodoptera frugiperda (Lepidoptera: Noctuidae) resistance to spinosad in Brazil[END_REF][START_REF] Yan | Flubendiamide resistance and Bi-PASA detection of ryanodine receptor G4946E mutation in the diamondback moth (Plutella xylostella L.)[END_REF], including in T. urticae [START_REF] Stumpf | Biochemical Markers Linked to Abamectin Resistance in Tetranychus urticae (Acari: Tetranychidae)[END_REF][START_REF] Sato | Abamectin Resistance in Tetranychus urticae Koch (Acari: Tetranychidae): Selection, Cross-Resistance and Stability of Resistance[END_REF][START_REF] Nicastro | Milbemectin resistance in Tetranychus urticae (Acari: Tetranychidae): selection, stability and cross-resistance to abamectin[END_REF][START_REF] Nicastro | Fitness costs associated with milbemectin resistance in the two-spotted spider mite Tetranychus urticae[END_REF], and specifically when resistant to etoxazole [START_REF] Stocco | Stability and fitness costs associated with etoxazole resistance in Tetranychus urticae (Acari: Tetranychidae)[END_REF][START_REF] Bajda | Fitness costs of key point mutations that underlie acaricide target-site resistance in the two-spotted spider mite Tetranychus urticae[END_REF]. However, in our study we found no cost of etoxazole resistance at control temperature for any of the traits measured, which should result in the maintenance of pesticide resistance in these populations long after pesticide use. One possible explanation for the discrepancy between studies could be the existence of different genetic bases of resistance to the same pesticide (e.g., [START_REF] Van Leeuwen | Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods[END_REF][START_REF] Ilias | Global distribution and origin of target site insecticide resistance mutations in Tetranychus urticae[END_REF][START_REF] Adesanya | Mechanisms of resistance to three mite growth inhibitors of Tetranychus urticae in hops[END_REF], some entailing costs and others not. Another possibility could be that the observed costs are associated with, but not due to, resistance. First, there could be non-genetic differences between resistant and susceptible populations that are responsible for the costs observed. For instance, pesticide resistant mosquitoes (Culex pipiens) are often more heavily infected with Wolbachia than susceptible ones, with the costs associated with the resistance phenotype being due in part to the infection and not to resistance [START_REF] Duron | HIGH WOLBACHIA DENSITY CORRELATES WITH COST OF INFECTION FOR INSECTICIDE RESISTANT CULEX PIPIENS MOSQUITOES[END_REF]. In turn, the differences between resistant and susceptible populations could be genetic, the costs being due to differences in the genetic background. This was observed in Culex quinquefasciatus, in which the fitness costs associated with resistance to organophosphate identified when comparing strains from different locations were strongly reduced after backcrossing the resistant gene into the genetic background of the susceptible strain [START_REF] Amin | Relative fitness of organophosphate-resistant and susceptible strains of Culex quinquefasciatus Say (Diptera: Culicidae)[END_REF]. Here, we have eliminated the possibility of non-genetic differences due to symbionts by treating the populations with antibiotics. Conversely to some previous studies (e.g., [START_REF] Okuma | Inheritance and fitness costs of Spodoptera frugiperda (Lepidoptera: Noctuidae) resistance to spinosad in Brazil[END_REF][START_REF] Stocco | Stability and fitness costs associated with etoxazole resistance in Tetranychus urticae (Acari: Tetranychidae)[END_REF][START_REF] Yan | Flubendiamide resistance and Bi-PASA detection of ryanodine receptor G4946E mutation in the diamondback moth (Plutella xylostella L.)[END_REF], we have also minimized genetic differences among resistant and susceptible populations, by following an introgression procedure. Still, because we are studying genetically variable populations, the resistant population may have evolved after introgression to compensate for an existing cost of resistance. Such hypothesis would explain the discrepancy between our study and that of [START_REF] Bajda | Fitness costs of key point mutations that underlie acaricide target-site resistance in the two-spotted spider mite Tetranychus urticae[END_REF], who found a cost of the same resistance gene in T. urticae after performing a similar introgression procedure but on isogenic lines, thereby preventing evolution. All in all, this suggests that the genetic background in which resistance to etoxazole is inserted plays a role in the expression of costs, but more studies should be performed to confirm this possibility.

While resistance was not costly at control temperature, with the individuals from resistant and susceptible populations showing similar values for all life-history traits, fertility was differently affected at 36°C depending on the resistance status of the individuals. Indeed, the reduction in the number of offspring and in the proportion of daughters felt after exposure to the developmental high temperature was steeper in pairs involving resistant individuals than in those involving susceptible individuals. These results are in accordance with a few other studies done on other crop pests such as Plutella xylostella and Nilaparvata lugens [START_REF] Li | Temperature-related fitness costs of resistance to spinosad in the diamondback moth, Plutella xylostella (Lepidoptera: Plutelidae)[END_REF][START_REF] Zhang | Trade-off between thermal tolerance and insecticide resistance in Plutella xylostella[END_REF][START_REF] Yang | Effects of temperature on fitness costs in chlorpyrifosresistant brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)[END_REF],

showing that pesticide resistance trades-off with thermal tolerance. Such a cost of pesticide resistance on fertility at high temperatures should reduce the selective advantage of resistant individuals, which in turn should increase the efficacy of crop management by pesticide application. However, to the best of our knowledge, none of these previous studies has investigated possible sex-specific responses, thus risking overestimating the cost of resistance.

Here we find that the trade-off between pesticide resistance and thermal tolerance can be observed in males but not in females. Several studies have shown that male thermal fertility limits are the best predictors of extinction in several species [START_REF] Van Heerwaarden | Male fertility thermal limits predict vulnerability to climate warming[END_REF]. This suggests that the fertility toll suffered by resistant spider mite males can foster the exclusion of resistance from the population. On the other hand, the observed sex-specific response to temperature may buffer the costs of pesticide resistance, assuming resistant females have the chance to mate with migrant males from cooler places or mate multiply to compensate sterility. This last strategy has been observed in several arthropod species, including spider mites [START_REF] Berger | HIGH TEMPERATURES REVEAL CRYPTIC GENETIC VARIATION IN A POLYMORPHIC FEMALE SPERM STORAGE ORGAN[END_REF][START_REF] Sutter | Flexible polyandry in female flies is an adaptive response to infertile males[END_REF][START_REF] Vasudeva | Facultative polyandry protects females from compromised male fertility caused by heatwave conditions[END_REF][START_REF] Baur | The mating system affects the temperature sensitivity of male and female fertility[END_REF][START_REF] Costa | Multiple mating rescues offspring sex ratio but not productivity in a haplodiploid exposed to developmental heat stress[END_REF].

Given sex-specific responses, the sex-determination system of the species will also influence the outcome of resistance in the population. First, because of haplodiploidy in spider mites, high temperature affected not only the number, but also the sex ratio of the offspring. Each of these traits entails different consequences for population dynamics. For instance, while both fecundity and sex ratio influence population growth rate, they may do so in different ways, with the effects of sex ratio depending on population structure but those of fecundity not being influenced by this factor [START_REF] Metz | How should we define fitness in structured metapopulation models? Including an application to the calculation of evolutionarily stable dispersal strategies[END_REF]. Second, resistance to etoxazole in spider mites is recessive [START_REF] Van Leeuwen | Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods[END_REF]. Therefore, haploid males will express the resistance cost as soon as they carry one resistance allele, promoting a quick elimination of resistance [START_REF] Carrière | Haplodiploidy, Sex, and the Evolution of Pesticide Resistance[END_REF], which would not be the case in diploid organisms, including diploid females from haplodiploid species.

Overall, we show that climate change can delay the growth of spider mite populations as temperatures rise, negatively affecting several life-history traits, with pesticide resistant males being more affected by temperature than susceptible males. While this is good news from a pest management standpoint, the complex expectations stemming from its sex-determination system and the possible buffering effect of females, coupled with the decrease in developmental time of both sexes at high temperature may counter such forces. Indeed, decreased developmental time should result in an increased number of generations per crop period, which in turn should accelerate adaptation to both high temperature and pesticide, and lead to a faster expansion of populations. Effort should be put into studying the evolution of populations exposed to both stressors, temperature and pesticide, to better understand and ultimately predict the dynamics of crop pests in a changing world.

  However, its interaction with the other two factors did not affect the offspring number (♀ temperature * ♂ temperature: χ 2 1 = 3.170, p = 0.075; ♀ temperature * Resistance status: χ 2 1 = 1.471, p = 0.225; ♀ temperature * ♂ temperature * Resistance status: χ 2 1 = 0.207, p = 0.649) or sex ratio (

Fig. 1 Fig. 2

 12 Fig. 1 Backcross procedure used to introgress the etoxazole-resistant allele into the susceptible

Table 1 Statistical results of the effect of high developmental temperatures on life-history traits.

 1 "Df": degrees of freedom. "F": the sum of squares obtained from the F-test. "Developmental time (♀)" and "Developmental time (♂)": number of days the first female or male, respectively, took to reach

	adulthood in a patch; "Number of surviving juveniles": number of juveniles surviving until the adult
	stage; "Daily fecundity": mean number of eggs laid per day, representing female fertility; "Offspring
	number": number of eggs that reached adulthood laid by females exposed to different temperatures
	(proxy for female fertility). "Developmental temperature": temperature at which eggs were exposed
	until adulthood; "Temperature of pair": developmental temperature of both females and males paired
	to mate and oviposit (offspring developed at 25°C). Statistically significant terms (p ≤ 0.05) are
	represented in bold.
	Var. of interest	Explanatory var.	Df (Df residuals)	F	P-value
	Developmental time (♀)	Developmental temperature	2 (10.651)	4.08	<0.001
	Developmental time (♂)	Developmental temperature	2 (6.153)	4.491	0.004
	Number of surviving juveniles	Developmental temperature	2 (31.880)	2.2084	0.126
	Daily fecundity	Temperature of pair	3 (51.478)	2074.07	<0.001
	Offspring number	Temperature of pair	3 (70.384)	14176.4	<0.001

Table 2 A posteriori contrasts of significant explanatory variables for the effect of high developmental temperatures on life-history traits

 2 . A posteriori contrasts with Bonferroni corrections were done to interpret the significant effect of the fixed factors. "T ratio": the T-test value

	obtained in each comparison. Comparisons were made between offspring developed at 25, 33, 35 or
	36°C ("25°C", "33°C", "35°C" and "36°C"), and between pairs with males developed at 25°C and
	females developed at 25, 33, 35, 36 or 37°C ("♀25°C", "♀33°C", "♀35°C", "♀36°C" and "♀37°C").
	Statistically significant terms (p ≤ 0.05) are represented in bold. *, represents marginally significant
	values.
	Var. of interest	Comparison	T ratio	P-value
		25°C -33°C	-40.712	<0.001
		25°C -35°C	-46.158	<0.001
		25°C -36°C	-39.64	<0.001
	Developmental time (♀)	33°C -35°C	2.454	0.051*
		33°C -36°C	-2.050	0.133
		35°C -36°C	-4.610	<0.001
		25°C -33°C	-27.097	<0.001
		25°C -35°C	-31.599	<0.001
		25°C -36°C	-25.998	<0.001
	Developmental time (♂)	33°C -35°C	1.733	0.264
		33°C -36°C	-1.671	0.299
		35°C -36°C	-3.507	0.003
		♀25°C -♀33°C	7.067	<0.001
		♀25°C -♀35°C	-3.250	0.001
		♀25°C -♀36°C	-6.114	<0.001
		♀25°C -♀37°C	-9.871	<0.001
		♀33°C -♀35°C	7.315	<0.001
	Daily fecundity	♀33°C -♀36°C	9.325	<0.001
		♀33°C -♀37°C	11.883	<0.001
		♀35°C -♀36°C	2.025	0.269
		♀35°C -♀37°C	4.361	<0.001
		♀36°C -♀37°C	2.268	0.150
		♀25°C -♀33°C	6.156	<0.001
	Offspring number	♀25°C -♀35°C	-3.631	<0.001
		♀25°C -♀36°C	-12.327	<0.001

Table 3 Significant statistical results of the interaction between pesticide resistance and response to high developmental temperature

 3 . "Df": degrees of freedom. "χ 2 ": Chi-square value obtained in each analysis. "Developmental time (♀)" and "Developmental time (♂)": number of days the first female or male, respectively, took to reach adulthood in a patch; "Number of surviving juveniles": number of juveniles surviving until the adult stage; "Offspring number": number of eggs that reached adulthood coming from crosses with females and males developed at high or control temperatures (proxy for female fertility); "Offspring sex ratio": proportion of adult daughters in the offspring (proxy for male fertility). "Developmental temperature": temperature at which eggs were exposed until adulthood; "♀ temperature": developmental temperature of the females paired; "♂ temperature": developmental temperature of the males paired; "Resistance status": resistance or susceptibility to etoxazole; "Fecundity": number of eggs laid for 4 days. Statistically significant terms (p ≤ 0.05) are represented in bold.

	Var. of interest	Explanatory var.	Df	χ 2	P-value
		Developmental temperature	1	23.578	<0.001
	Developmental time (♀)
		Resistance status	1	0.001	0.977
		Developmental temperature	1	22.175	<0.001
	Developmental time (♂)
		Resistance status	1	0.032	0.857
	Number of surviving	Developmental temperature	1	62.199	<0.001
	juveniles	Resistance status	1	0.047	0.829
		♀ temperature	1	243.924	<0.001
	Offspring number	♂ temperature	1	2.492	0.114
		Resistance status	1	0.291	0.590
		♂ temperature * Resistance status	1	5.227	0.022
		♀ temperature	1	10.957	<0.001
	Offspring sex ratio	♂ temperature	1	66.215	<0.001
		Resistance status	1	0.181	0.670
		♂ temperature * Resistance status	1	4.895	0.027

Table 4 A posteriori contrasts of significant explanatory variables for the interaction between pesticide resistance and response to high developmental temperature

 4 . A posteriori contrasts with Bonferroni corrections were done to interpret the significant effect of the fixed factors. "T ratio": the T-test value obtained in each comparison. Comparisons were made between males developed at 25°C or 36°C ("♂25" and "♂36") and that were susceptible or resistant to etoxazole ("Susceptible" and "Resistant"). Statistically significant terms (p ≤ 0.05) are represented in bold.

	Var. of interest	Comparison	T ratio	P-value
		Susceptible ♂25°C -Susceptible ♂36°C	-1.659	0.394
		Resistant ♂25°C -Resistant ♂36°C	1.579	0.463
	Offspring number	Resistant ♂25°C -Susceptible ♂25°C	0.540	1.000
		Resistant ♂36°C -Susceptible ♂36°C	-2.705	0.029
		Susceptible ♂25°C -Susceptible ♂36°C	8.560	<0.001
		Resistant ♂25°C -Resistant ♂36°C	8.137	<0.001
	Offspring sex ratio	Resistant ♂25°C -Susceptible ♂25°C	-0.426	1.000
		Resistant ♂36°C -Susceptible ♂36°C	-2.641	0.035

Acknowledgments

We thank all Mite Squad members for unconditional support in the laboratory and elsewhere, particularly Cátia Eira, Juan Lopez and Lucie Santos for valuable help in the maintenance of the populations and rearing of the plants used in these experiments. We also thank Élio Sucena for useful discussions on the backcross procedure, and Thomas Van Leeuwen for providing the SB9 strain. This work was funded by an European Research Council (ERC) consolidating grant (COMPCON GA 725419) to SM, and a Fundação para Ciência e Tecnologia (FCT) project (EXPL/BIAEVL/0131/2021) to LRR. This is publication ISEM-2023-XXX of the Institut des Sciences de l'Évolution.

Data Availability

The data that support the findings of this study will be openly available at Figshare. For reviewing purposes, we provide the corresponding private link: https://figshare.com/s/6ea0871d134c948bd250.

entire procedure, from the creation of the Wi.SS populations to the obtention of all base populations, was performed in five independent replicates. Wu.RR and Wu.SS replicates were then merged before being used in the current study. Antibiotic treatments are represented in blue and selection for etoxazole resistance is in red. Inner and outer circles within male/female symbols represent, respectively, the proportion of nuclear and mitochondrial genomes from the donor (orange) and recurrent (white) parent populations. Dotted-filled outer circles represent Wolbachia-infected cytoplasm. BC: back-cross progeny; NC: no-cross progeny; SC: self-cross progeny.

(red bars) to the pesticide etoxazole. Developmental time was measured by the day the first female (a) and male (b) in a patch reached adulthood, the number of surviving juveniles by the number of eggs that reached adulthood (c), female fertility by the offspring number (d), and male fertility by the offspring sex ratio (proportion of daughters; e). Boxplots display a median line, interquartile range (IQR) boxes, 1.5*IQR whiskers and data points. Black dots represent individual replicates, white diamonds represent the mean values per conditions tested.