Insecticide tolerance shapes performance responses to multiple stressors in a crop pest

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M. Bueno, Y. H. Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6156635/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Insect pests are remarkably successful in evolving resistance to management tactics while facing multiple sources of stress in modern agroecosystems. One possible explanation for this success is that repeated exposure to insecticides may enable pests to tolerate additional stressors through cross-protection. Using the Colorado potato beetle ( Leptinotarsa decemlineata Say), we tested whether selection for imidacloprid tolerance influences responses to multiple stressors. We compared imidacloprid-selected and unselected beetles exposed to sublethal imidacloprid (LC 10 ), high temperature (40°C), or their combination, measuring effects on mobility, herbivory, development, fecundity, and mortality. Contrary to our expectations, selected beetles showed increased vulnerability to stress treatments, particularly exhibiting reduced mobility and lower survival when exposed to combined stressors. While both beetle groups maintained similar development times and reproductive output, the imidacloprid-selected beetles demonstrated cross-susceptibility rather than cross-protection when facing multiple stressors. These findings suggest that selection for insecticide tolerance may create vulnerabilities to environmental stress, a dynamic that could inform pest management strategies under climate change. insect pests climate change stress insecticide cross-susceptibility synergistic Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Insect pests are extraordinarily successful in modern agroecosystems, despite frequent and long-term exposure to multiple stressors, such as extreme temperatures, drought, insecticides, fungicides, and herbicides (Kaunisto et al., 2016 ; Gutiérrez, 2020 ; Cutler et al., 2022 ; Bueno et al., 2023 ). While one explanation for this success could stem from insect pests independently developing tolerance to individual stressors, another possibility is that selection for insecticide tolerance may enhance their ability to tolerate different stressors. Continuous exposure to insecticides can lead to the development of stress-tolerant phenotypes in pest populations, thereby conferring elevated tolerance to various stressors, including high temperatures (Gould et al., 2018 ; Matzrafi, 2019 ; Pu et al., 2020 ). For example, insects selected for insecticide resistance can tolerate high temperatures better than unselected individuals (Li et al., 2017 ). Other possible outcomes include reduced tolerance when multiple stressors interact in non-additive ways, or when selection for resistance creates fitness costs that compromise responses to additional stressors. With the frequency of elevated temperatures expected to increase, understanding how selection for insecticide resistance influences an insect’s capacity to handle other stressors is highly relevant for sustainable agriculture, as higher temperatures are projected to accelerate the development and metabolism of insect pests, potentially affecting food security (Deutsch et al., 2018 ; Porter et al., 1991 ; Skendžić et al., 2021 ). Whether natural or anthropogenic, interacting stressors can either enhance or inhibit survival under combined exposure, where stressors occur closely one after another (Kaunisto et al., 2016 ). Depending on the nature of each stressor, interactions can be additive or non-additive (Gunderson et al., 2016 ; Todgham & Stillman, 2013 ). When stressors act independently of each other, the combined effect equals the sum of each stressor in isolation resulting in an additive effect. Conversely, non-additive interactions occur where the combined effect deviates from the additive effect, resulting in synergistic or antagonistic interactions that can be either higher or lower than the sum of the individual effects. For instance, exposure to one stressor can hinder tolerance to other stressors through cross-susceptibility (synergistic), leading to negative effects on insect performance and survival (Todgham & Stillman, 2013 ). Alternatively, cross-protective (antagonistic) interactions occur when exposure to one stressor heightens tolerance towards other stressors. This is particularly relevant for understanding interactions between insecticides and temperature stress, which are generally non-additive (Deng et al., 2016 ; Ge et al., 2013 ; Li et al., 2017 ; Patil et al., 1996 ; Perrin et al., 2022 ; Todgham & Stillman, 2013 ; Wang et al., 2021 ; Bueno et al., 2023 ). While moderate temperature increases can enhance insect performance, the combination of insecticides and high temperatures can lead to either cross-protection or cross-susceptibility, depending on the physiological responses activated (González-Tokman et al., 2020). At the same time, continuous exposure to insecticides in agroecosystems creates strong selection pressure for resistance in insect populations. While selection for insecticide resistance increases insect survival following insecticide exposure, it can also lead to physiological trade-offs that affect performance in other contexts. How selection for increased tolerance to one stressor influences responses to multiple stressors remains poorly understood. Previous research suggests that exposure to one stressor can activate physiological protective mechanisms that may provide cross-protection against subsequent stressors (Boivin et al., 2001 ; Kliot and Ghanim, 2012 ). These shared protective responses, such as increased production of heat shock proteins and metabolites, could enhance an organism's ability to cope with additional environmental challenges. On the other hand, cross-susceptibility may arise from resource allocation conflicts where energetic demands for resistance reduce the ability to tolerate additional stress. Despite these theoretical mechanisms, the relationship between selection for insecticide resistance and responses to multiple stressors has not been thoroughly investigated in agricultural pest species. The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), is a globally invasive pest of potato and other solanaceous crops, known for its resilience to stressful conditions (Alyokhin et al., 2015 ). CPB is an ideal model organism for investigating responses to stress as they are notorious for adapting rapidly to insecticides (Chen et al., 2023 ). The propensity for CPB to develop tolerance is so extreme that it has evolved resistance to over 55 different classes of insecticides, causing billions of dollars in damage to the potato industry (Alyokhin et al., 2008 ; Alyokhin et al., 2015 ). The expansion of CPB into Asia and Europe demonstrates the beetle's resiliency to a wide range of environmental conditions (Alyokhin et al., 2015 ; Lehmann et al., 2014 ; Piiroinen et al., 2013 ). The neonicotinoid imidacloprid, which has been commonly used against CPB populations in the U.S., can interact synergistically with other stressors including pathogens, fungicides and high temperatures, thereby reducing beetle survival (Chen et al., 2016 ; Clements et al., 2018 ; Furlong & Groden, 2003 ). Despite such interactions, it remains unclear how long-term exposure to imidacloprid and thereby selection for tolerance affects the beetle’s ability to overcome exposure to multiple stressors. Here, we investigated whether selection for insecticide tolerance affects the combined impact of insecticides and high temperature stress on CPB. We tested if imidacloprid-selected and unselected beetles responded differently when exposed to single and combined stress. Given that prior stress exposure can improve tolerance to subsequent stressors via cross-protective interactions, we hypothesized that beetles selected for imidacloprid tolerance would exhibit higher survival rates and overall performance compared to unselected counterparts. Specifically, we asked: (1) how does a single exposure to imidacloprid or high temperature affect phenotypic responses between imidacloprid-selected and unselected beetles? (2) How does combined stress exposure (insecticide followed by high temperature), affect phenotypic responses among imidacloprid-selected and unselected beetles? By focusing on phenotypic responses such as life history traits and behavior, our study emphasizes the importance of understanding how prior stress exposure influences an insect's ability to cope with additional stressors. Methods and Materials Insect colonies We started an insect colony using ~ 1,500 adult beetles collected from organic potato farms in northern Vermont in the summer of 2018, and by augmenting the colony with additional collections in 2019 and 2020. Since imidacloprid is not permitted under organic standards, beetles collected from organic potato farms would not have been exposed to imidacloprid previously. However, organic growers can use Spinosad, which exhibits moderate cross-resistance with imidacloprid (Mota-Sanchez et al., 2006 ). As a result, we anticipated that field-collected beetles would exhibit low to moderate levels of tolerance to imidacloprid. In the laboratory, we allowed beetles to freely mate for four generations to reduce the potential impact of maternal effects on performance. We then split the colony into an imidacloprid-selected and unselected group (Fig. 1 ). Egg clutches from each group were collected daily to minimize cannibalism and to prevent the overlap of life stages. All beetles were raised in the laboratory at 25°C with a 16-hour light and 8-hour darkness cycle on six-week-old potato plants ( Solanum tuberosum ) grown in 4-inch pots filled with Pro-Mix HP soil mix (Pro-Mix, PA, USA) inside the University of Vermont greenhouse. The plants were subjected to long-day conditions (16:8 L:D hours, 24°C max, 20°C min) and fertilized three times a week using a 15-16-17 (N-P-K) (Jack’s Professional Peat-Lite, PA, USA). Topical imidacloprid bioassays We selected for imidacloprid tolerance using a LC 50 dose (the lethal dose leading to 50% mortality) for nine generations. To estimate the appropriate LC 50 concentration for each generation, we performed topical bioassays by exposing third instar larvae to four doses (5, 10, 25, 50 ppm) of 99.8% technical grade imidacloprid (Chemservice, PA, USA) dissolved in acetone, including an acetone control group. Additionally, we performed topical bioassays on unselected larvae and calculated LC 50 values to evaluate their sensitivity to imidacloprid compared to selected beetles across each generation (Table 1 ). Table 1 Results from selection assays showing LC 50 values at each generation of selection for imidacloprid tolerance. Selection line Generation n LC 50 (ppm) 95% CI Slope ± SE RR Unselected F0 96–97 13 0.05–4.06 1.39 ± 0.14 G1 103–110 15.6 8.51–28.7 2.25 ± 0.20 G2 105–111 11.2 2.01–27.8 2.44 ± 0.21 G3 141–179 21.5 11.8–46.1 3.20 ± 0.20 G4 204–209 16.9 9.99–28.4 3.50 ± 0.19 G5 198–212 20.5 10.5–52.3 2.36 ± 0.15 G6 239–259 17.5 11.4–28.1 2.12 ± 0.13 G7 205–214 17.2 11.5–26.7 1.78 ± 0.13 G8 202–210 22.8 11.8–67 2.08 ± 0.14 G9 57–64 11.2 9.54–13.1 2.95 ± 0.32 Selected G1 100–110 27.4 17-60.3 2.24 ± 0.21 G2 129–133 31.9 20.9–54 2.52 ± 0.22 G3 161–199 27.8 25.8–29.9 3.36 ± 0.23 G4 240–261 33.6 1.83–2760 3.04 ± 0.20 G5 231–233 72.1 56.9–106 2.51 ± 0.20 G6 237–322 66.5 68.1–91 2.31 ± 0.20 G7 202–225 43.5 38.1–50.2 1.56 ± 0.13 G8 196–208 66.1 60.2–73.4 2.70 ± 0.20 G9 203–210 66.5 60.4–74.0 2.65 ± 0.20 5.9 *NA = Probit model was not able to calculate a CI. RR = Resistance Ratio For all bioassays, 50–100 third-instar larvae were treated with 1 µl of each imidacloprid dose, applied directly on to the dorsal abdominal cuticle using a micropipette. We specifically selected third instar larvae to ensure that larvae would not enter pupation during selection assays. To account for potential delayed effects of imidacloprid on mortality, we checked for mortality 48 hours after imidacloprid exposure. We scored larvae as dead if we observed tissue necrosis, indicated by darkening of the larval cuticle and a shrunken body. We performed a probit analysis with fiducial confidence limits to estimate the LC 50 value for each generation using the ecotox package in R studio (Table 1 ; Hlina et al., 2021 ). Imidacloprid Selection Assays Before starting selection assays, we performed an LC 50 bioassay on the source colony (F 0 ) and used that LC 50 imidacloprid dose to generate the first generation of selected beetles (G1). Specifically, we placed ~ 1000 third instar larvae individually into 6-well plates with a 1.8 cm² potato leaf disk. The LC 50 treatment was prepared by diluting a 1000 ppm solution of technical grade imidacloprid dissolved in acetone. We treated larvae by pipetting a 1 µl droplet of the LC 50 dose (13 ppm) onto the dorsal abdominal cuticle. We assessed mortality 48 hours post-exposure and scored larvae as dead or alive. Surviving larvae were individually placed in petri dishes with potato leaves. At the end of the fourth instar, larvae were transferred to soil-filled plastic bins (86.36 cm x 45.72 cm x 17.78 cm) with ventilation to allow for pupation and adult emergence. Upon emergence, all adult beetles were placed together into a rearing cage with potted potato plants to allow for mating. All subsequent generations (G1 to G9) belonging to imidacloprid-selected beetles were reared in separate cages and selected for imidacloprid tolerance using the same procedure as the parental generation. Specifically, for each generation (G1 to G9), we estimated the LC 50 dose by performing dose-response assays as described above and then used the resulting LC 50 value to perform the selection assays on third instar larvae (see Table 1 for LC 50 estimates). Experimental Design For the initial stress exposure treatment, we chose a sublethal concentration of imidacloprid that resulted in approximately 10% mortality for the imidacloprid-selected and unselected beetles. We used an LC 10 dosage to prevent high mortality rates and to be able to detect any synergistic interactions between stressors. After nine generations of selection, imidacloprid-selected larvae could tolerate a LC 10 of 22 ppm, whereas unselected larvae could tolerate a LC 10 of 8 ppm. We pipetted a 1 µl LC 10 dose of the imidacloprid treatment on the dorsal abdominal cuticle of third instar larvae. For the control treatment, we opted to use water instead of acetone. While not extremely lethal to insects, acetone has been shown to modify insect fecundity and behavior (Critchley & Almeida, 1973 ; Sahota et al., 1998 ; Sanada-Morimura & Matsumura, 2011 ). Therefore, to mitigate any potential effects induced by acetone, we applied a 1 µl droplet of water to the dorsal abdominal cuticle of the control beetles. Since the potential priming effects of initial stress are enhanced with recovery time between stressors, we allowed larvae to recover from imidacloprid stress for 24 hours on fresh potato foliage before exposure to another stressor (Rodgers & Gomez Isaza, 2021 ). Afterward, we exposed imidacloprid-treated and untreated larvae to either 25℃ (ambient control) or 40℃ for 3 hours, which corresponds to the lethal temperature LT 10 (lethal time to 10% mortality, unpublished results). Each treatment was replicated over ten randomly assigned third instar larvae and the experiment was repeated at least five times (n = ~ 50 larvae per treatment). Performance Metrics Mobility To test the single and combined effects of imidacloprid and high temperature treatments on beetle behavior, we measured larval mobility. Larval mobility is a sensitive assay for detecting the sublethal effects of stress (Kjærsgaard et al., 2015 ; Nansen et al., 2016 ). We monitored larval mobility using high-definition cameras (GoPro HERO 5) to record the distance traveled by third instar larvae for ten minutes. We set up two camera stations with each overhead camera mounted on a stand above four petri dishes (9 cm x 1.7 cm) with a direct field of view to prevent any image distortion. To create even lighting and prevent any unwanted reflections, the four petri dishes were placed on top of one of two light pads (31 cm x 23.4 cm) under each camera station. Given the multiple camera setup, we were able to track larval behavior in eight larvae simultaneously. Before recording, larvae were placed at the center of each petri dish and allowed to acclimate for five minutes. After each recording, we wiped down each petri dish with distilled water before introducing the next set of larvae. We processed the videos using the open-source animal tracking software, Toxtrac, which can track and analyze the movement of individuals in multiple arenas (Rodriguez et al., 2018 ). Using default Toxtrac parameters, we analyzed the total distance traveled (mm). Herbivory We measured the impact of stress treatments on larval herbivory, assessing both whether beetles fed and how much they consumed. Immediately after completing the recordings, we transferred individual larvae to a new petri dish (9 cm diameter) containing a single potato leaf disk (Area: 1.8 cm²). We limited the herbivory assays to one hour to allow sufficient time for larvae to consume the leaf disks without compromising the quality of the food. After an hour, we transferred each larva to a separate petri dish to measure the other life-history traits. We calculated herbivory as the percent area consumed for each leaf disk using the iOS application, LeafByte (Getman-Pickering et al., 2020 ). We photographed each leaf disk on top of a light pad and assessed the area using a standardized scale provided by the LeafByte application. Development time and survival to adult stage Following exposure to the stress treatments, we measured developmental time based on the number of days it took for the third instar larvae to reach the pupal and adult stage. We monitored development daily by checking larvae for signs of pupation, such as reduced feeding and burying activity. Once these behaviors were detected, we provided each individual with one tablespoon of sterilized soil to help them burrow inside a petri dish (9 cm diameter). All pupae were maintained at room temperature and monitored daily for signs of adult emergence. At the adult stage, all individuals were sexed by examining the outline of the last abdominal segment under a dissecting microscope (Pelletier, 1993 ). Model testing for Synergistic Effects on Mortality Rates Presuming that stressors are independent, interactions among stressors can be identified using null models that predict the effect of combined exposure (Schäfer & Piggott, 2018 ; Tekin et al., 2020 ). Additive models, such as generalized linear models, can be useful for identifying additive effects among stressors, but they can also produce errors by mistakenly identifying interaction types, including both additive and antagonistic effects (Delnat et al., 2019 ; Schäfer & Piggott, 2018 ). Multiplicative models, on the other hand, are appropriate for testing the combined effect of stressors that differ in mode of action, useful for identifying synergistic interactions (Delnat et al., 2019 ; Schäfer & Piggott, 2018 ). Given that insect pests typically encounter stressors with distinct modes of action, multiplicative models such as the independent action model (IA model), are suitable for evaluating the synergistic effects of combined stress on insect fitness and performance (Delnat et al., 2019 ). Specifically, the IA model predicts that the likelihood of surviving two stressors is dependent on the likelihood of surviving each stressor when applied separately (Schäfer & Piggott, 2018 ). The IA model has been previously applied to identify stressor interactions, demonstrating that it is suitable for assessing the impacts of combined stress on insect survival (Delnat et al., 2019 ; Furlong & Groden, 2003 ). We monitored the survival of all individuals across all treatment groups for 30 days. The proportion of beetles dying each day was calculated by dividing the total number of surviving beetles by the total number of beetles exposed to the same treatment. For the combined stress treatment (imidacloprid followed by high temperature), we used the IA model to determine if the treatment effects acted independently or synergistically using Eq. 1: Equation 1 : E Imid+40C = E Imid + E 40C − (E Imid × E 40C ) Here, the probability of dying from combined exposure to imidacloprid and 40°C ( E Imid+40C ) is equal to the sum of the probability of dying from single exposure to imidacloprid ( E Imid. ) and 40°C ( E 40C ) minus the product of the probabilities for each stressor (as in Meyling et al., 2018 ). If the observed mortality rates are higher or lower than the mortality predicted by the IA model, then the interaction between imidacloprid and heat stress is considered non-additive. If the predicted curve is higher than the observed curve confidence interval (95% CI) the interaction was considered antagonistic. On the other hand, if the predicted curve was lower than the observed curve confidence interval, the interaction was considered synergistic. Female fecundity To test for the effects of stress on female fecundity, each adult female was paired with a virgin male from the same treatment group. To ensure males were unmated, we sexed and isolated males from each group as pupae. We placed the mating pairs into a petri dish with potato leaves, which we monitored daily for eggs. We carefully transferred the egg clutches to a clean petri dish lined with filter paper with a camel-hair brush and counted the number of eggs per clutch. After collecting the first three clutches per mating pair, we checked the eggs for hatchlings. We calculated the percent hatching success by calculating the number of emerged neonates divided by the total number of eggs laid. Statistical Analysis Mobility To test for the effects of selection, insecticide, and temperature on mobility, we performed a three-factor analysis of variance (ANOVA) with experimental trials as a random effect term using the lmerTest package in R studio (Kuznetsova et al., 2017 ). The total distance traveled data was modified with a square root transformation to meet the normality assumptions for parametric tests. All significant effects were followed up with a post-hoc Tukey’s significant difference test to identify significant pairwise differences between levels of each treatment group with a P-value of < 0.05. Herbivory We tested whether selection, followed by additional insecticide and temperature stress influenced herbivory with a generalized linear mixed-effect model (GLMM) using the R package glmmTMB (Brooks et al., 2017 ). This package employs a zero-inflated beta distribution to account for the excess of zeros in the dataset, and it simultaneously fits GLMMs to continuous data. We used this package to examine how treatments affected the likelihood of feeding, using a zero-inflated model, as well as their impact on the proportion of leaf area consumed, using a continuous model. We used the package DHARMa to test for model overdispersion (Hartig, 2015), Due to an unbalanced number of replicates among factor levels, we calculated the significance values ( P < 0.05) using an analysis of deviance Type II Wald Chi-square tests (R package car ; (Fox & Weisberg, 2019 ). If significant main effects were found, we calculated pairwise contrasts between all treatment levels using Tukey’s honest significant difference test via the emmeans package in R (Lenth, 2023 ). Development time and survival to adult stage To test for interactions and main effects among selection, insecticide, and temperature on developmental time, we performed a three-factor analysis of variance (ANOVA) with experimental trials as a random effect term using the lmerTest package in R studio (Kuznetsova et al., 2017 ). If significant effects were found, we followed up with post-hoc Tukey’s honest significant difference tests to identify significant pairwise differences with a p-value of < 0.05. To examine interactions and main effects among selection, insecticide, and temperature on survival to adulthood, we applied a generalized linear mixed model with a binomial error distribution and logit link using the package lme4 in R (Bates et al., 2015 ). We conducted a post-hoc Tukey's honest significant difference test to assess significant pairwise differences between treatment groups. A p-value of < 0.05 was used to determine statistical significance. Female fecundity We tested if selection, insecticide, and temperature, influenced female clutch size (number of eggs per clutch) using a three-factor analysis of variance (ANOVA) using the lmerTest package in R studio (Kuznetsova et al., 2017 ), followed with a post-hoc Tukey’s honest significant difference test. Additionally, we tested if selection, insecticide, and temperature influenced egg hatching success (proportion of eggs hatched per female) using a logistic regression with a generalized linear model assuming a binomial error distribution ( glm in R-base). We tested for the significance of the treatment factors using a Chi-square Wald Test with a P < 0.05. Results Larval mobility Analysis of variance revealed significant effects of high temperature (F 1,371 = 208.51, P < 0.001) and insecticide exposure (F 1,371 = 24.58, P < 0.001) on larval mobility, with significant interactions between selection and both insecticide (F 1,371 = 6.97, P < 0.01) and temperature (F 1,371 = 14.01, P < 0.001) treatments (Tables S1.1 and S2.1). High temperature (40°C) significantly reduced mobility compared to the control group (25°C) in both imidacloprid-selected and unselected beetles. Insecticide exposure significantly reduced mobility in imidacloprid-selected beetles at 25°C. When combined with high temperature, insecticide further reduced mobility in imidacloprid-selected beetles, with selected larvae walking significantly less than unselected beetles (Fig. 2 ). Herbivory Selection group and imidacloprid exposure influenced how likely larvae fed after treatments (Selection: χ 2 = 4.37, P < 0.05, Imidacloprid: χ 2 = 12.4 P < 0.0001; Tables S1.1 and S2.1). In imidacloprid-selected beetles, insecticide exposure significantly reduced feeding at 25°C, and this effect persisted under the combined stress treatment (Fig. 3 ). In contrast, unselected beetles showed no significant changes in feeding behavior across all treatments (Fig. 3 ). Development Time and Survival to Adult Stage Colorado potato beetles demonstrated resilience to stress, as neither insecticide exposure nor high temperature significantly affected development time from third instar to adult emergence (Fig. 4 , Table S1 .1). While statistical analysis indicated a significant effect of beetle group (F 1,224 = 13.24, P < 0.001, Table S2.1), the difference in development time between imidacloprid-selected and unselected beetles was minimal with all beetles taking approximately 20 days to complete development. Neither insecticide exposure nor high temperature alone significantly affected survival to adult stage in either selection group when compared to controls (Fig. 5 , Table S1 .1). However, under combined stress, imidacloprid-selected beetles were less likely to survive compared to unselected beetles (Fig. 5 , z = 2.627, P < 0.001; Table S2.1). Synergistic Effects on Mortality Rates Rather than finding cross-protection, we found evidence of cross-susceptibility. Selection for imidacloprid tolerance elevated larval susceptibility to combined stress. In imidacloprid-selected larvae, mortality rates under combined stress consistently exceeded those predicted by the independent action model (dashed line), ranging from 35 to 85% over the 27-day period following high temperature exposure (Fig. 8 ). In contrast, unselected larvae showed more modest deviation from the predicted mortality curve, with rates only 16 to 31% higher than IA predictions during the first 10 days after heat exposure, before returning to expected levels (Fig. 8 ). Female Fecundity Female fecundity was highly resilient following stress exposure. Neither selection group, insecticide exposure, nor high temperature stress affected mean clutch size, either individually or in combination (Fig. 6 , Tables S1.1 and S2.1). Similarly, egg hatching success remained consistently high across all treatments, with no significant effects of beetle group or stress exposure, either alone or in combination (Fig. 7 , Tables S1.1 and S2.1). Discussion Given that the Colorado potato beetle can overcome exposure to a multitude of insecticidal compounds, we predicted that the beetle may use cross-protective responses to tolerate insecticides and high temperatures. Contrary to our prediction, we found evidence of cross-susceptibility instead of cross-protection. Surprisingly, imidacloprid-selected beetles were more vulnerable to combined imidacloprid and high temperature exposure. Furthermore, we observed that selected beetles suffered from synergistic effects, with higher mortality relative to the rates predicted by the independent action model. Below, we expand on the effects of each stressor treatment, highlighting differences and similarities in responses between unselected and imidacloprid-selected beetles. Effect of Combined Stress Prolonged selection for insecticide tolerance adversely affected CPB tolerance to other stressors. It is possible that the length of time spent inbreeding in the laboratory setting reduced genetic diversity or elevated genetic drift, negatively affecting the responsiveness of selected beetles to stress. Due to the nature of laboratory selection assays, the imidacloprid-selected beetles underwent multiple genetic bottlenecks events for nine generations. Therefore, our results indicate that multiple genetic bottlenecks may impose constraints on stress tolerance in the field-collected population. However, it is possible that selection for imidacloprid tolerance might have induced pleiotropic effects, where genes associated with tolerance influence multiple unrelated traits, impacting overall fitness (Boivin et al., 2001 ; Carrière & Roff, 1995 ; Kliot & Ghanim, 2012 ; Pu et al., 2020 ). Pleiotropic effects associated with insecticide tolerance are typically described as fitness costs (or life-history trade-offs), including reduced fecundity, life span, body mass, and mating ability (ffrench-Constant & Bass, 2017 ; Freeman et al., 2021 ; Kliot & Ghanim, 2012 ). Although we did not detect any fitness costs related to development time and female fecundity in selected beetles, imidacloprid selection could have imposed fitness costs on the ability to withstand combined stress. Previous work has found that fitness costs linked to insecticide tolerance may be exacerbated under additional stress. For instance, Helitohis virescens selected for Bt ( Bacillus thuringiensis ) tolerance developed more poorly than the non-selected strain when subjected to elevated temperatures (Gulzar et al., 2012 ). Likewise, selection of the brown planthopper ( Nilaparvata lugens ) for chlorpyrifos tolerance lowered relative fitness and lengthened recovery times following high temperature treatment compared to the susceptible strain (Yang et al., 2018 ). Thus, the fitness costs associated with imidacloprid tolerance may account for the heightened sensitivity of selected beetles to combined insecticide and high temperature stress compared to unselected beetles. We found that selection reduced mobility and survival to adult stage in response to combined stress resulting in cross-susceptibility which, if the term genotype is interpreted broadly, aligns with previous studies showing genotype-dependent responses (Delnat et al., 2022 ; Liu et al., 2008 ). For instance, Daphnia magna exposed to combined exposure to heat and insecticide showed a genotype-dependent effect on survival and performance (Delnat et al. 2022 ). Insects selected to tolerate insecticides may become more sensitive to environmental stressors due to the depletion of energy reserves resulting from prolonged activation of detoxification pathways, reducing their ability to withstand additional stressors. In general, when insects encounter stressful conditions, protective cellular responses are activated such as the upregulation of heat shock proteins ( Hsp ), which are known to be energetically demanding (González-Tokman et al., 2020). For instance, insecticide-resistant and susceptible diamondback moth strains varied in their production of Hsp70 in response to heat stress, with insecticide-resistant strains producing less Hsp70 than susceptible strains (Liu et al. 2008 ). It is possible that prolonged expression of stress tolerance pathways resulted in energetic trade-offs in imidacloprid-selected beetles, thereby impairing their ability to ramp up protective physiological responses under further stress. Effect of Imidacloprid Exposure to sublethal doses of insecticide can have profound impacts on insects, including reduced feeding and altered behavior (Kenna et al., 2019 ). We found that exposure to imidacloprid immediately caused imidacloprid-selected beetles to reduce their mobility. Sublethal doses of imidacloprid caused similar effects in other studies, in L. decemlineata adults and Drosophila melanogaster larvae (Alyokhin & Miller, 2015 ; Young et al., 2020 ). It is possible that reduced locomotor activity in response to sublethal imidacloprid was only temporary since we only measured mobility 24 hours following exposure. Exposure to imidacloprid resulted in reduced feeding behavior within both beetle groups. It is possible that lower food consumption reduced an individual's total energy budget, impairing growth, development, and survival. Reductions in feeding activity following sublethal insecticide exposure have been reported in other insects, including leaf beetles, green peach aphids, and mayflies (Alexander et al., 2007 ; Cho et al., 2011 ; Wolz et al., 2021 ). Despite reducing larval mobility and herbivory, imidacloprid exposure did not have a negative impact on survival to adult stage, development time, or female fecundity in both beetle groups. Many other insecticide-resistant insects have been observed to experience reductions in fecundity and egg viability, which may be associated with energy reserve trade-offs related to detoxification mechanisms (Alyokhin & Ferro, 1999 ; Argentine et al., 1989 ; Boivin et al., 2001 ; Cao & Han, 2006 ). Effect of High Temperature Stress The high temperature treatment immediately reduced mobility in both unselected and imidacloprid-selected CPB larvae. Exposure to high temperatures has been shown to decrease mobility in other insect species, such as reducing locomotor activity and flight durations in house flies and decreasing jumping distance in crickets (Kjærsgaard et al., 2015 ; Lachenicht et al., 2010 ). We found that exposure to high temperature did not impact herbivory rates among unselected and imidacloprid-selected beetles. Given that insects are poikilothermic, high temperatures can be beneficial to some extent, promoting faster development and other performance traits (Colinet et al., 2015 ; Kiritani, 2013 ). For example, previous studies have reported that high temperatures increase insect consumption rates (Lemoine et al., 2013 , Lemoine et al. 2014 ). In the case of CPB, prior studies have shown that all stages perform optimally between 25°C and 32°C (Ferro et al., 1985 ; Logan et al., 1985 ). Although the high temperature treatment exceeded the optimal temperature range for CPB, the heat exposure did not significantly affect herbivory rates. Therefore, it is plausible that the high temperature treatment was not highly stressful. Following the high temperature treatment, both imidacloprid-selected and unselected larvae showed similar rates of survival to the adult stage, development time, or female fecundity. These results suggests that CPB larvae can tolerate brief periods of exceedingly high temperatures without apparent costs on development or female reproductive success. However, it is important to mention that extended durations above 40°C has been demonstrated to negatively impact CPB survival (Chen et al., 2016 ). Hence, we cannot dismiss the possibility that at longer durations, our 40°C treatment might elevate mortality. Nevertheless, our findings differ from earlier studies that looked at the relationship between exposure to high temperatures and life history traits in insects. For example, exposure to high temperatures reduced egg laying in the cotton bollworm and slowed development time in the brown planthopper (Mironidis & Savopoulou-Soultani, 2010 ; Piyaphongkul et al., 2012 ). Since we did not find any trade-offs in development and female fecundity, it is possible that brief exposure to 40°C was not severe enough to cause major heat injury, allowing larvae to recover quickly after exposure with no negative effects on their overall fitness. However, since we only assessed offspring survival during the hatchling stage, it is unclear if there any potential generational effects on survival and performance. Limitations and Future Directions Due to logistical constraints, our study was limited to comparing the effects of a single selection process, which limits our ability to generalize regarding broader responses of CPB to various selection pressures and stressors. However, the observed patterns may also be due to the genetic architecture, genetic drift, or other stochastic processes that arose during the selection process itself. Using multiple independent selection lines would allow one to assess how comparable interactions between insecticides and high temperatures are and help distinguish between consistent evolutionary responses and population-specific effects. To better understand the mechanisms underlying our findings, several key areas warrant further investigation. First, examining physiological responses to combined stressors, including heat shock protein expression and cellular stress markers, may help identify how insecticide-resistant and insecticide-susceptible insects differ in stress tolerance mechanisms. Second, exploring a wider range of imidacloprid concentrations would provide a more comprehensive understanding of dose-dependent effects on CPB performance. Although we did not assess physiological parameters related to energy reserves, such as lipid content or mitochondrial function, future studies should examine the effects of insecticides on feeding behavior and how changes in food consumption may impact survival. Third, research is necessary to elucidate how reductions in feeding under heat stress affect growth rates and other life history traits that contribute to overall fitness. Future studies should also incorporate longer selection duration and different stressor orders to provide further insight into the interactions between insecticides and high temperature stress. Although complicated to perform, such studies would be particularly valuable for predicting CPB responses under various field conditions where both insecticide exposure and temperature fluctuations are common. Conclusions By exposing Colorado potato beetles to imidacloprid followed by high temperature stress, we found negative synergistic effects on survival, mobility, and herbivory, that were magnified in beetles selected for imidacloprid tolerance. Overall, while beetles performed poorly in the short term, those that recovered showed no long-term impacts on development or reproductive fitness. These findings have important implications for pest management in agricultural systems, particularly as climate change increases the frequency of temperature extremes. The increased vulnerability of imidacloprid-selected beetles to combined stress suggests that resistance management strategies should consider environmental contexts. Specifically, targeted applications of insecticides during periods of predicted high temperatures could potentially improve control efficacy against insecticide resistant populations. As such, cross-susceptibility could be leveraged in integrated pest management programs to delay resistance development or better manage existing insecticide resistance issues. Declarations Conflict of Interest The authors declare no conflict of interest. Authorship Contribution Statement Erika M. Bueno: Conceptualization, Methodology, Data collection, Formal Analysis, Writing – original draft. Yolanda H. Chen: Conceptualization, Funding Acquisition, Methodology, Supervision, Writing- review and editing. 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Journal of Evolutionary Biology, 33 (2), 151–164. https://doi.org/10.1111/jeb.13555 Additional Declarations No competing interests reported. Supplementary Files TableS1Final021225.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6156635","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":424599241,"identity":"dbfa7ac8-c303-4e59-b8b3-8becfa4983f8","order_by":0,"name":"E. M. Bueno","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYDACCTBpA8QJYMTAcIA4LWmkaznMAFVPhBZ+6R7DxwUV5xP725OfSTz4wyDHdyMBvxbJOWeMjWecuZ0448wzM4nENgZjSUJaDG7kmEnztt1O3CCRYGyQ2MCQuIGQFvsbOea/edvOAbWkfzZI+MNQT1CLgUSOGTNv2wGglhzDBwlsDAkGhLRI3DlWLM1zJhnonzeFDxLbJAxnnnmAXwv/7OaNn3kq7GT729M3HPzxx0ae7zgBWzBsJU35KBgFo2AUjALsAACUfUfgAnEmxwAAAABJRU5ErkJggg==","orcid":"","institution":"University of Vermont","correspondingAuthor":true,"prefix":"","firstName":"E.","middleName":"M.","lastName":"Bueno","suffix":""},{"id":424599242,"identity":"541d30c6-eb5f-4c22-a31d-8ad6e26e99a6","order_by":1,"name":"Y. H. Chen","email":"","orcid":"","institution":"University of Vermont","correspondingAuthor":false,"prefix":"","firstName":"Y.","middleName":"H.","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-03-04 18:53:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6156635/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6156635/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77971634,"identity":"14c9c0ce-a019-4eba-aae4-b65973f61fb2","added_by":"auto","created_at":"2025-03-07 11:05:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":260659,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical illustration of the experimental design used to examine the impacts of insecticide and high temperature stress on CPB. Third instar larvae were initially selected for imidacloprid tolerance and then exposed to imidacloprid (LC\u003csub\u003e10\u003c/sub\u003e) or water. After a 24-hour recovery period, the larvae were exposed to either 40°C or 25°C (control) for three hours.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6156635/v1/ed79d2215218338df6b38346.png"},{"id":77972660,"identity":"11e511f4-87e7-48d9-902e-0d8b3b0ab8a9","added_by":"auto","created_at":"2025-03-07 11:13:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":108237,"visible":true,"origin":"","legend":"\u003cp\u003eMean distance traveled (mobility) in response to single and combined exposure to insecticide and high temperature treatments. Grey and black lines with an asterisk indicate significant within-group differences for unselected and selected groups, respectively, at P \u0026lt; 0.05 (Tukey's HSD). The dashed line with an asterisk represents significant between-group differences at P \u0026lt; 0.05 (Tukey's HSD). Lines represent standard error (se) bars (mean ± se).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6156635/v1/b5c159f71d6db7b19c7a9428.png"},{"id":77971674,"identity":"b7cfcc4e-e80e-4371-acda-d05d036987f1","added_by":"auto","created_at":"2025-03-07 11:05:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":111553,"visible":true,"origin":"","legend":"\u003cp\u003eMean proportion of leaf area consumed (herbivory) in response to single and combined exposure to insecticide and high temperature treatments. Black lines with an asterisk indicate significant within-group differences for the selected group at P \u0026lt; 0.05 (Tukey's HSD). Lines represent standard error (se) bars (mean ± se).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6156635/v1/f490319b3aeb8d49c367aab4.png"},{"id":77971641,"identity":"2ab39b03-f156-4a35-acaf-e5966bfd7a4f","added_by":"auto","created_at":"2025-03-07 11:05:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":116466,"visible":true,"origin":"","legend":"\u003cp\u003eMean development time (days to adult emergence from the third instar stage) in response to single and combined exposure to insecticide and high temperature treatments. Lines represent standard error (se) bars (mean ± se).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6156635/v1/0e3165d1cd2e37df67096471.png"},{"id":77972661,"identity":"1f64a41c-5495-4ad4-97e9-24ffe435cd59","added_by":"auto","created_at":"2025-03-07 11:13:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":114539,"visible":true,"origin":"","legend":"\u003cp\u003eThe percentage of beetles surviving to adult stage after exposure to single and combined insecticide and high temperature treatments. Dashed line with an asterisk represents significant between-group differences at P \u0026lt; 0.05 (Tukey's HSD). Lines represent standard error (se) bars (mean ± se).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6156635/v1/f203a1c925eb3e2f79f224ad.png"},{"id":77971638,"identity":"23c7be3d-83e7-46e9-9c42-4c5fcbec09b4","added_by":"auto","created_at":"2025-03-07 11:05:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":103229,"visible":true,"origin":"","legend":"\u003cp\u003eMean clutch size (female fecundity) from females exposed to single and combined insecticide and high temperature treatments. Lines represent standard error (se) bars (mean ± se).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6156635/v1/7635daa21dccae3354d1ae95.png"},{"id":77971647,"identity":"f5a68728-5bc5-4042-ab4c-4a40fc3f1729","added_by":"auto","created_at":"2025-03-07 11:05:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":107569,"visible":true,"origin":"","legend":"\u003cp\u003eMean percent egg hatching (female fecundity) from unselected and selected females exposed to single and combined insecticide and high temperature treatments. Lines represent standard error (se) bars (mean ± se).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6156635/v1/a0137002d49db9bf09854445.png"},{"id":77972663,"identity":"b24af34d-63db-47fd-96b5-123eed5ba4a8","added_by":"auto","created_at":"2025-03-07 11:13:13","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":445210,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of single and combined exposure to insecticide and high temperature stress on mortality over time. Line graphs represent cumulative mortality over time starting from day of insecticide (imidacloprid) exposure. Panels from left to right show mortality over time for control, insecticide, high temperature, and combined stress treatments, respectively. For the combined stress treatment, a two-parameter logistic model (solid black line) was fitted on observed mortality using a 95% confidence interval (blue shading). The predicted IA model (dashed line) was compared to the observed mortality (95% CI) to identify synergistic interactions among stressors.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6156635/v1/29ebc78eabe1fd96c064bfb8.jpeg"},{"id":78478871,"identity":"c7dd5a22-b10c-4260-b2e8-c0efa163e83b","added_by":"auto","created_at":"2025-03-13 18:01:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2344331,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6156635/v1/3e12a75d-6d4c-4e63-8140-bd520ccf7b03.pdf"},{"id":77972668,"identity":"212f29a6-b188-4f8b-9243-b97c0fb20388","added_by":"auto","created_at":"2025-03-07 11:13:13","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19214,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1Final021225.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6156635/v1/d3a58da7b39d7e67d9fa78c2.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Insecticide tolerance shapes performance responses to multiple stressors in a crop pest","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInsect pests are extraordinarily successful in modern agroecosystems, despite frequent and long-term exposure to multiple stressors, such as extreme temperatures, drought, insecticides, fungicides, and herbicides (Kaunisto et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Guti\u0026eacute;rrez, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Cutler et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Bueno et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While one explanation for this success could stem from insect pests independently developing tolerance to individual stressors, another possibility is that selection for insecticide tolerance may enhance their ability to tolerate different stressors. Continuous exposure to insecticides can lead to the development of stress-tolerant phenotypes in pest populations, thereby conferring elevated tolerance to various stressors, including high temperatures (Gould et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Matzrafi, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pu et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For example, insects selected for insecticide resistance can tolerate high temperatures better than unselected individuals (Li et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Other possible outcomes include reduced tolerance when multiple stressors interact in non-additive ways, or when selection for resistance creates fitness costs that compromise responses to additional stressors. With the frequency of elevated temperatures expected to increase, understanding how selection for insecticide resistance influences an insect\u0026rsquo;s capacity to handle other stressors is highly relevant for sustainable agriculture, as higher temperatures are projected to accelerate the development and metabolism of insect pests, potentially affecting food security (Deutsch et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Porter et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Skendžić et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhether natural or anthropogenic, interacting stressors can either enhance or inhibit survival under combined exposure, where stressors occur closely one after another (Kaunisto et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Depending on the nature of each stressor, interactions can be additive or non-additive (Gunderson et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Todgham \u0026amp; Stillman, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). When stressors act independently of each other, the combined effect equals the sum of each stressor in isolation resulting in an additive effect. Conversely, non-additive interactions occur where the combined effect deviates from the additive effect, resulting in synergistic or antagonistic interactions that can be either higher or lower than the sum of the individual effects. For instance, exposure to one stressor can hinder tolerance to other stressors through cross-susceptibility (synergistic), leading to negative effects on insect performance and survival (Todgham \u0026amp; Stillman, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Alternatively, cross-protective (antagonistic) interactions occur when exposure to one stressor heightens tolerance towards other stressors. This is particularly relevant for understanding interactions between insecticides and temperature stress, which are generally non-additive (Deng et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ge et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Patil et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Perrin et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Todgham \u0026amp; Stillman, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bueno et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While moderate temperature increases can enhance insect performance, the combination of insecticides and high temperatures can lead to either cross-protection or cross-susceptibility, depending on the physiological responses activated (Gonz\u0026aacute;lez-Tokman et al., 2020).\u003c/p\u003e \u003cp\u003eAt the same time, continuous exposure to insecticides in agroecosystems creates strong selection pressure for resistance in insect populations. While selection for insecticide resistance increases insect survival following insecticide exposure, it can also lead to physiological trade-offs that affect performance in other contexts. How selection for increased tolerance to one stressor influences responses to multiple stressors remains poorly understood. Previous research suggests that exposure to one stressor can activate physiological protective mechanisms that may provide cross-protection against subsequent stressors (Boivin et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Kliot and Ghanim, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These shared protective responses, such as increased production of heat shock proteins and metabolites, could enhance an organism's ability to cope with additional environmental challenges. On the other hand, cross-susceptibility may arise from resource allocation conflicts where energetic demands for resistance reduce the ability to tolerate additional stress. Despite these theoretical mechanisms, the relationship between selection for insecticide resistance and responses to multiple stressors has not been thoroughly investigated in agricultural pest species.\u003c/p\u003e \u003cp\u003eThe Colorado potato beetle (CPB), \u003cem\u003eLeptinotarsa decemlineata\u003c/em\u003e Say (Coleoptera: Chrysomelidae), is a globally invasive pest of potato and other solanaceous crops, known for its resilience to stressful conditions (Alyokhin et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). CPB is an ideal model organism for investigating responses to stress as they are notorious for adapting rapidly to insecticides (Chen et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The propensity for CPB to develop tolerance is so extreme that it has evolved resistance to over 55 different classes of insecticides, causing billions of dollars in damage to the potato industry (Alyokhin et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Alyokhin et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The expansion of CPB into Asia and Europe demonstrates the beetle's resiliency to a wide range of environmental conditions (Alyokhin et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lehmann et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Piiroinen et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The neonicotinoid imidacloprid, which has been commonly used against CPB populations in the U.S., can interact synergistically with other stressors including pathogens, fungicides and high temperatures, thereby reducing beetle survival (Chen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Clements et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Furlong \u0026amp; Groden, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Despite such interactions, it remains unclear how long-term exposure to imidacloprid and thereby selection for tolerance affects the beetle\u0026rsquo;s ability to overcome exposure to multiple stressors.\u003c/p\u003e \u003cp\u003eHere, we investigated whether selection for insecticide tolerance affects the combined impact of insecticides and high temperature stress on CPB. We tested if imidacloprid-selected and unselected beetles responded differently when exposed to single and combined stress. Given that prior stress exposure can improve tolerance to subsequent stressors via cross-protective interactions, we hypothesized that beetles selected for imidacloprid tolerance would exhibit higher survival rates and overall performance compared to unselected counterparts. Specifically, we asked: (1) how does a single exposure to imidacloprid or high temperature affect phenotypic responses between imidacloprid-selected and unselected beetles? (2) How does combined stress exposure (insecticide followed by high temperature), affect phenotypic responses among imidacloprid-selected and unselected beetles? By focusing on phenotypic responses such as life history traits and behavior, our study emphasizes the importance of understanding how prior stress exposure influences an insect's ability to cope with additional stressors.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInsect colonies\u003c/h2\u003e \u003cp\u003eWe started an insect colony using\u0026thinsp;~\u0026thinsp;1,500 adult beetles collected from organic potato farms in northern Vermont in the summer of 2018, and by augmenting the colony with additional collections in 2019 and 2020. Since imidacloprid is not permitted under organic standards, beetles collected from organic potato farms would not have been exposed to imidacloprid previously. However, organic growers can use Spinosad, which exhibits moderate cross-resistance with imidacloprid (Mota-Sanchez et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). As a result, we anticipated that field-collected beetles would exhibit low to moderate levels of tolerance to imidacloprid.\u003c/p\u003e \u003cp\u003eIn the laboratory, we allowed beetles to freely mate for four generations to reduce the potential impact of maternal effects on performance. We then split the colony into an imidacloprid-selected and unselected group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Egg clutches from each group were collected daily to minimize cannibalism and to prevent the overlap of life stages. All beetles were raised in the laboratory at 25\u0026deg;C with a 16-hour light and 8-hour darkness cycle on six-week-old potato plants (\u003cem\u003eSolanum tuberosum\u003c/em\u003e) grown in 4-inch pots filled with Pro-Mix HP soil mix (Pro-Mix, PA, USA) inside the University of Vermont greenhouse. The plants were subjected to long-day conditions (16:8 L:D hours, 24\u0026deg;C max, 20\u0026deg;C min) and fertilized three times a week using a 15-16-17 (N-P-K) (Jack\u0026rsquo;s Professional Peat-Lite, PA, USA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTopical imidacloprid bioassays\u003c/h3\u003e\n\u003cp\u003eWe selected for imidacloprid tolerance using a LC\u003csub\u003e50\u003c/sub\u003e dose (the lethal dose leading to 50% mortality) for nine generations. To estimate the appropriate LC\u003csub\u003e50\u003c/sub\u003e concentration for each generation, we performed topical bioassays by exposing third instar larvae to four doses (5, 10, 25, 50 ppm) of 99.8% technical grade imidacloprid (Chemservice, PA, USA) dissolved in acetone, including an acetone control group. Additionally, we performed topical bioassays on unselected larvae and calculated LC\u003csub\u003e50\u003c/sub\u003e values to evaluate their sensitivity to imidacloprid compared to selected beetles across each generation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults from selection assays showing LC\u003csub\u003e50\u003c/sub\u003e values at each generation of selection for imidacloprid tolerance.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSelection line\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eGeneration\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003en\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eLC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e(ppm)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e95% CI\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSlope\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eRR\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUnselected\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96\u0026ndash;97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.05\u0026ndash;4.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e103\u0026ndash;110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.51\u0026ndash;28.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e105\u0026ndash;111\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.01\u0026ndash;27.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e141\u0026ndash;179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.8\u0026ndash;46.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e204\u0026ndash;209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.99\u0026ndash;28.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e198\u0026ndash;212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.5\u0026ndash;52.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e239\u0026ndash;259\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.4\u0026ndash;28.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e205\u0026ndash;214\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.5\u0026ndash;26.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e202\u0026ndash;210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.8\u0026ndash;67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57\u0026ndash;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.54\u0026ndash;13.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSelected\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u0026ndash;110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17-60.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e129\u0026ndash;133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.9\u0026ndash;54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e161\u0026ndash;199\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.8\u0026ndash;29.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e240\u0026ndash;261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.83\u0026ndash;2760\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e231\u0026ndash;233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56.9\u0026ndash;106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e237\u0026ndash;322\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e68.1\u0026ndash;91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e202\u0026ndash;225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.1\u0026ndash;50.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e196\u0026ndash;208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60.2\u0026ndash;73.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e203\u0026ndash;210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60.4\u0026ndash;74.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e5.9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e*NA\u0026thinsp;=\u0026thinsp;Probit model was not able to calculate a CI. RR\u0026thinsp;=\u0026thinsp;Resistance Ratio\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFor all bioassays, 50\u0026ndash;100 third-instar larvae were treated with 1 \u0026micro;l of each imidacloprid dose, applied directly on to the dorsal abdominal cuticle using a micropipette. We specifically selected third instar larvae to ensure that larvae would not enter pupation during selection assays. To account for potential delayed effects of imidacloprid on mortality, we checked for mortality 48 hours after imidacloprid exposure. We scored larvae as dead if we observed tissue necrosis, indicated by darkening of the larval cuticle and a shrunken body. We performed a probit analysis with fiducial confidence limits to estimate the LC\u003csub\u003e50\u003c/sub\u003e value for each generation using the \u003cem\u003eecotox\u003c/em\u003e package in R studio (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Hlina et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eImidacloprid Selection Assays\u003c/h3\u003e\n\u003cp\u003eBefore starting selection assays, we performed an LC\u003csub\u003e50\u003c/sub\u003e bioassay on the source colony (F\u003csub\u003e0\u003c/sub\u003e) and used that LC\u003csub\u003e50\u003c/sub\u003e imidacloprid dose to generate the first generation of selected beetles (G1). Specifically, we placed\u0026thinsp;~\u0026thinsp;1000 third instar larvae individually into 6-well plates with a 1.8 cm\u0026sup2; potato leaf disk. The LC\u003csub\u003e50\u003c/sub\u003e treatment was prepared by diluting a 1000 ppm solution of technical grade imidacloprid dissolved in acetone. We treated larvae by pipetting a 1 \u0026micro;l droplet of the LC\u003csub\u003e50\u003c/sub\u003e dose (13 ppm) onto the dorsal abdominal cuticle. We assessed mortality 48 hours post-exposure and scored larvae as dead or alive. Surviving larvae were individually placed in petri dishes with potato leaves. At the end of the fourth instar, larvae were transferred to soil-filled plastic bins (86.36 cm x 45.72 cm x 17.78 cm) with ventilation to allow for pupation and adult emergence. Upon emergence, all adult beetles were placed together into a rearing cage with potted potato plants to allow for mating.\u003c/p\u003e \u003cp\u003eAll subsequent generations (G1 to G9) belonging to imidacloprid-selected beetles were reared in separate cages and selected for imidacloprid tolerance using the same procedure as the parental generation. Specifically, for each generation (G1 to G9), we estimated the LC\u003csub\u003e50\u003c/sub\u003e dose by performing dose-response assays as described above and then used the resulting LC\u003csub\u003e50\u003c/sub\u003e value to perform the selection assays on third instar larvae (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for LC\u003csub\u003e50\u003c/sub\u003e estimates).\u003c/p\u003e\n\u003ch3\u003eExperimental Design\u003c/h3\u003e\n\u003cp\u003eFor the initial stress exposure treatment, we chose a sublethal concentration of imidacloprid that resulted in approximately 10% mortality for the imidacloprid-selected and unselected beetles. We used an LC\u003csub\u003e10\u003c/sub\u003e dosage to prevent high mortality rates and to be able to detect any synergistic interactions between stressors. After nine generations of selection, imidacloprid-selected larvae could tolerate a LC\u003csub\u003e10\u003c/sub\u003e of 22 ppm, whereas unselected larvae could tolerate a LC\u003csub\u003e10\u003c/sub\u003e of 8 ppm. We pipetted a 1 \u0026micro;l LC\u003csub\u003e10\u003c/sub\u003e dose of the imidacloprid treatment on the dorsal abdominal cuticle of third instar larvae. For the control treatment, we opted to use water instead of acetone. While not extremely lethal to insects, acetone has been shown to modify insect fecundity and behavior (Critchley \u0026amp; Almeida, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Sahota et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Sanada-Morimura \u0026amp; Matsumura, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Therefore, to mitigate any potential effects induced by acetone, we applied a 1 \u0026micro;l droplet of water to the dorsal abdominal cuticle of the control beetles.\u003c/p\u003e \u003cp\u003eSince the potential priming effects of initial stress are enhanced with recovery time between stressors, we allowed larvae to recover from imidacloprid stress for 24 hours on fresh potato foliage before exposure to another stressor (Rodgers \u0026amp; Gomez Isaza, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Afterward, we exposed imidacloprid-treated and untreated larvae to either 25℃ (ambient control) or 40℃ for 3 hours, which corresponds to the lethal temperature LT\u003csub\u003e10\u003c/sub\u003e (lethal time to 10% mortality, unpublished results). Each treatment was replicated over ten randomly assigned third instar larvae and the experiment was repeated at least five times (n\u0026thinsp;=\u0026thinsp;~\u0026thinsp;50 larvae per treatment).\u003c/p\u003e\n\u003ch3\u003ePerformance Metrics\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMobility\u003c/h2\u003e \u003cp\u003eTo test the single and combined effects of imidacloprid and high temperature treatments on beetle behavior, we measured larval mobility. Larval mobility is a sensitive assay for detecting the sublethal effects of stress (Kj\u0026aelig;rsgaard et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nansen et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). We monitored larval mobility using high-definition cameras (GoPro HERO 5) to record the distance traveled by third instar larvae for ten minutes. We set up two camera stations with each overhead camera mounted on a stand above four petri dishes (9 cm x 1.7 cm) with a direct field of view to prevent any image distortion. To create even lighting and prevent any unwanted reflections, the four petri dishes were placed on top of one of two light pads (31 cm x 23.4 cm) under each camera station. Given the multiple camera setup, we were able to track larval behavior in eight larvae simultaneously. Before recording, larvae were placed at the center of each petri dish and allowed to acclimate for five minutes. After each recording, we wiped down each petri dish with distilled water before introducing the next set of larvae. We processed the videos using the open-source animal tracking software, Toxtrac, which can track and analyze the movement of individuals in multiple arenas (Rodriguez et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Using default Toxtrac parameters, we analyzed the total distance traveled (mm).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHerbivory\u003c/h3\u003e\n\u003cp\u003eWe measured the impact of stress treatments on larval herbivory, assessing both whether beetles fed and how much they consumed. Immediately after completing the recordings, we transferred individual larvae to a new petri dish (9 cm diameter) containing a single potato leaf disk (Area: 1.8 cm\u0026sup2;). We limited the herbivory assays to one hour to allow sufficient time for larvae to consume the leaf disks without compromising the quality of the food. After an hour, we transferred each larva to a separate petri dish to measure the other life-history traits. We calculated herbivory as the percent area consumed for each leaf disk using the iOS application, LeafByte (Getman-Pickering et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). We photographed each leaf disk on top of a light pad and assessed the area using a standardized scale provided by the LeafByte application.\u003c/p\u003e\n\u003ch3\u003eDevelopment time and survival to adult stage\u003c/h3\u003e\n\u003cp\u003eFollowing exposure to the stress treatments, we measured developmental time based on the number of days it took for the third instar larvae to reach the pupal and adult stage. We monitored development daily by checking larvae for signs of pupation, such as reduced feeding and burying activity. Once these behaviors were detected, we provided each individual with one tablespoon of sterilized soil to help them burrow inside a petri dish (9 cm diameter). All pupae were maintained at room temperature and monitored daily for signs of adult emergence. At the adult stage, all individuals were sexed by examining the outline of the last abdominal segment under a dissecting microscope (Pelletier, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1993\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eModel testing for Synergistic Effects on Mortality Rates\u003c/h2\u003e \u003cp\u003ePresuming that stressors are independent, interactions among stressors can be identified using null models that predict the effect of combined exposure (Sch\u0026auml;fer \u0026amp; Piggott, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tekin et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additive models, such as generalized linear models, can be useful for identifying additive effects among stressors, but they can also produce errors by mistakenly identifying interaction types, including both additive and antagonistic effects (Delnat et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sch\u0026auml;fer \u0026amp; Piggott, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Multiplicative models, on the other hand, are appropriate for testing the combined effect of stressors that differ in mode of action, useful for identifying synergistic interactions (Delnat et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sch\u0026auml;fer \u0026amp; Piggott, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Given that insect pests typically encounter stressors with distinct modes of action, multiplicative models such as the independent action model (IA model), are suitable for evaluating the synergistic effects of combined stress on insect fitness and performance (Delnat et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Specifically, the IA model predicts that the likelihood of surviving two stressors is dependent on the likelihood of surviving each stressor when applied separately (Sch\u0026auml;fer \u0026amp; Piggott, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The IA model has been previously applied to identify stressor interactions, demonstrating that it is suitable for assessing the impacts of combined stress on insect survival (Delnat et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Furlong \u0026amp; Groden, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe monitored the survival of all individuals across all treatment groups for 30 days. The proportion of beetles dying each day was calculated by dividing the total number of surviving beetles by the total number of beetles exposed to the same treatment. For the combined stress treatment (imidacloprid followed by high temperature), we used the IA model to determine if the treatment effects acted independently or synergistically using Eq.\u0026nbsp;1:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003eEquation 1 : E\u003c/em\u003e\u003csub\u003e\u003cem\u003eImid+40C\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e= E\u003c/em\u003e\u003csub\u003e\u003cem\u003eImid\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e+ E\u003c/em\u003e\u003csub\u003e\u003cem\u003e40C\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026minus; (E\u003c/em\u003e\u003csub\u003e\u003cem\u003eImid\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e\u0026times; E\u003c/em\u003e\u003csub\u003e\u003cem\u003e40C\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e)\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eHere, the probability of dying from combined exposure to imidacloprid and 40\u0026deg;C (\u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003eImid+40C\u003c/em\u003e\u003c/sub\u003e) is equal to the sum of the probability of dying from single exposure to imidacloprid (\u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003eImid.\u003c/em\u003e\u003c/sub\u003e) and 40\u0026deg;C (\u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003e40C\u003c/em\u003e\u003c/sub\u003e) minus the product of the probabilities for each stressor (as in Meyling et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). If the observed mortality rates are higher or lower than the mortality predicted by the IA model, then the interaction between imidacloprid and heat stress is considered non-additive. If the predicted curve is higher than the observed curve confidence interval (95% CI) the interaction was considered antagonistic. On the other hand, if the predicted curve was lower than the observed curve confidence interval, the interaction was considered synergistic.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFemale fecundity\u003c/h2\u003e \u003cp\u003eTo test for the effects of stress on female fecundity, each adult female was paired with a virgin male from the same treatment group. To ensure males were unmated, we sexed and isolated males from each group as pupae. We placed the mating pairs into a petri dish with potato leaves, which we monitored daily for eggs. We carefully transferred the egg clutches to a clean petri dish lined with filter paper with a camel-hair brush and counted the number of eggs per clutch. After collecting the first three clutches per mating pair, we checked the eggs for hatchlings. We calculated the percent hatching success by calculating the number of emerged neonates divided by the total number of eggs laid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003eMobility\u003c/h2\u003e \u003cp\u003eTo test for the effects of selection, insecticide, and temperature on mobility, we performed a three-factor analysis of variance (ANOVA) with experimental trials as a random effect term using the \u003cem\u003elmerTest\u003c/em\u003e package in R studio (Kuznetsova et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The total distance traveled data was modified with a square root transformation to meet the normality assumptions for parametric tests. All significant effects were followed up with a post-hoc Tukey\u0026rsquo;s significant difference test to identify significant pairwise differences between levels of each treatment group with a P-value of \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHerbivory\u003c/h2\u003e \u003cp\u003eWe tested whether selection, followed by additional insecticide and temperature stress influenced herbivory with a generalized linear mixed-effect model (GLMM) using the R package \u003cem\u003eglmmTMB\u003c/em\u003e (Brooks et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This package employs a zero-inflated beta distribution to account for the excess of zeros in the dataset, and it simultaneously fits GLMMs to continuous data. We used this package to examine how treatments affected the likelihood of feeding, using a zero-inflated model, as well as their impact on the proportion of leaf area consumed, using a continuous model. We used the package \u003cem\u003eDHARMa\u003c/em\u003e to test for model overdispersion (Hartig, 2015), Due to an unbalanced number of replicates among factor levels, we calculated the significance values (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) using an analysis of deviance Type II Wald Chi-square tests (R package \u003cem\u003ecar\u003c/em\u003e; (Fox \u0026amp; Weisberg, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). If significant main effects were found, we calculated pairwise contrasts between all treatment levels using Tukey\u0026rsquo;s honest significant difference test via the \u003cem\u003eemmeans\u003c/em\u003e package in R (Lenth, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment time and survival to adult stage\u003c/h2\u003e \u003cp\u003eTo test for interactions and main effects among selection, insecticide, and temperature on developmental time, we performed a three-factor analysis of variance (ANOVA) with experimental trials as a random effect term using the \u003cem\u003elmerTest\u003c/em\u003e package in R studio (Kuznetsova et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). If significant effects were found, we followed up with post-hoc Tukey\u0026rsquo;s honest significant difference tests to identify significant pairwise differences with a p-value of \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eTo examine interactions and main effects among selection, insecticide, and temperature on survival to adulthood, we applied a generalized linear mixed model with a binomial error distribution and logit link using the package \u003cem\u003elme4\u003c/em\u003e in R (Bates et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). We conducted a post-hoc Tukey's honest significant difference test to assess significant pairwise differences between treatment groups. A p-value of \u0026lt;\u0026thinsp;0.05 was used to determine statistical significance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFemale fecundity\u003c/h2\u003e \u003cp\u003eWe tested if selection, insecticide, and temperature, influenced female clutch size (number of eggs per clutch) using a three-factor analysis of variance (ANOVA) using the \u003cem\u003elmerTest\u003c/em\u003e package in R studio (Kuznetsova et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), followed with a post-hoc Tukey\u0026rsquo;s honest significant difference test. Additionally, we tested if selection, insecticide, and temperature influenced egg hatching success (proportion of eggs hatched per female) using a logistic regression with a generalized linear model assuming a binomial error distribution (\u003cem\u003eglm\u003c/em\u003e in R-base). We tested for the significance of the treatment factors using a Chi-square Wald Test with a \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eLarval mobility\u003c/h2\u003e \u003cp\u003eAnalysis of variance revealed significant effects of high temperature (F\u003csub\u003e1,371\u003c/sub\u003e = 208.51, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and insecticide exposure (F\u003csub\u003e1,371\u003c/sub\u003e = 24.58, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) on larval mobility, with significant interactions between selection and both insecticide (F\u003csub\u003e1,371\u003c/sub\u003e = 6.97, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and temperature (F\u003csub\u003e1,371\u003c/sub\u003e = 14.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) treatments (Tables S1.1 and S2.1). High temperature (40\u0026deg;C) significantly reduced mobility compared to the control group (25\u0026deg;C) in both imidacloprid-selected and unselected beetles. Insecticide exposure significantly reduced mobility in imidacloprid-selected beetles at 25\u0026deg;C. When combined with high temperature, insecticide further reduced mobility in imidacloprid-selected beetles, with selected larvae walking significantly less than unselected beetles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eHerbivory\u003c/h2\u003e \u003cp\u003eSelection group and imidacloprid exposure influenced how likely larvae fed after treatments (Selection: χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;4.37, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Imidacloprid: χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;12.4 P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Tables S1.1 and S2.1). In imidacloprid-selected beetles, insecticide exposure significantly reduced feeding at 25\u0026deg;C, and this effect persisted under the combined stress treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, unselected beetles showed no significant changes in feeding behavior across all treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment Time and Survival to Adult Stage\u003c/h2\u003e \u003cp\u003eColorado potato beetles demonstrated resilience to stress, as neither insecticide exposure nor high temperature significantly affected development time from third instar to adult emergence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.1). While statistical analysis indicated a significant effect of beetle group (F\u003csub\u003e1,224\u003c/sub\u003e = 13.24, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Table S2.1), the difference in development time between imidacloprid-selected and unselected beetles was minimal with all beetles taking approximately 20 days to complete development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNeither insecticide exposure nor high temperature alone significantly affected survival to adult stage in either selection group when compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.1). However, under combined stress, imidacloprid-selected beetles were less likely to survive compared to unselected beetles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, z\u0026thinsp;=\u0026thinsp;2.627, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Table S2.1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eSynergistic Effects on Mortality Rates\u003c/h2\u003e \u003cp\u003eRather than finding cross-protection, we found evidence of cross-susceptibility. Selection for imidacloprid tolerance elevated larval susceptibility to combined stress. In imidacloprid-selected larvae, mortality rates under combined stress consistently exceeded those predicted by the independent action model (dashed line), ranging from 35 to 85% over the 27-day period following high temperature exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003e). In contrast, unselected larvae showed more modest deviation from the predicted mortality curve, with rates only 16 to 31% higher than IA predictions during the first 10 days after heat exposure, before returning to expected levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eFemale Fecundity\u003c/h2\u003e \u003cp\u003eFemale fecundity was highly resilient following stress exposure. Neither selection group, insecticide exposure, nor high temperature stress affected mean clutch size, either individually or in combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Tables S1.1 and S2.1). Similarly, egg hatching success remained consistently high across all treatments, with no significant effects of beetle group or stress exposure, either alone or in combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Tables S1.1 and S2.1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eGiven that the Colorado potato beetle can overcome exposure to a multitude of insecticidal compounds, we predicted that the beetle may use cross-protective responses to tolerate insecticides and high temperatures. Contrary to our prediction, we found evidence of cross-susceptibility instead of cross-protection. Surprisingly, imidacloprid-selected beetles were more vulnerable to combined imidacloprid and high temperature exposure. Furthermore, we observed that selected beetles suffered from synergistic effects, with higher mortality relative to the rates predicted by the independent action model. Below, we expand on the effects of each stressor treatment, highlighting differences and similarities in responses between unselected and imidacloprid-selected beetles.\u003c/p\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Combined Stress\u003c/h2\u003e \u003cp\u003eProlonged selection for insecticide tolerance adversely affected CPB tolerance to other stressors. It is possible that the length of time spent inbreeding in the laboratory setting reduced genetic diversity or elevated genetic drift, negatively affecting the responsiveness of selected beetles to stress. Due to the nature of laboratory selection assays, the imidacloprid-selected beetles underwent multiple genetic bottlenecks events for nine generations. Therefore, our results indicate that multiple genetic bottlenecks may impose constraints on stress tolerance in the field-collected population. However, it is possible that selection for imidacloprid tolerance might have induced pleiotropic effects, where genes associated with tolerance influence multiple unrelated traits, impacting overall fitness (Boivin et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Carri\u0026egrave;re \u0026amp; Roff, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Kliot \u0026amp; Ghanim, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Pu et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Pleiotropic effects associated with insecticide tolerance are typically described as fitness costs (or life-history trade-offs), including reduced fecundity, life span, body mass, and mating ability (ffrench-Constant \u0026amp; Bass, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Freeman et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kliot \u0026amp; Ghanim, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Although we did not detect any fitness costs related to development time and female fecundity in selected beetles, imidacloprid selection could have imposed fitness costs on the ability to withstand combined stress. Previous work has found that fitness costs linked to insecticide tolerance may be exacerbated under additional stress. For instance, \u003cem\u003eHelitohis virescens\u003c/em\u003e selected for Bt (\u003cem\u003eBacillus thuringiensis\u003c/em\u003e) tolerance developed more poorly than the non-selected strain when subjected to elevated temperatures (Gulzar et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Likewise, selection of the brown planthopper (\u003cem\u003eNilaparvata lugens\u003c/em\u003e) for chlorpyrifos tolerance lowered relative fitness and lengthened recovery times following high temperature treatment compared to the susceptible strain (Yang et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Thus, the fitness costs associated with imidacloprid tolerance may account for the heightened sensitivity of selected beetles to combined insecticide and high temperature stress compared to unselected beetles.\u003c/p\u003e \u003cp\u003eWe found that selection reduced mobility and survival to adult stage in response to combined stress resulting in cross-susceptibility which, if the term genotype is interpreted broadly, aligns with previous studies showing genotype-dependent responses (Delnat et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). For instance, \u003cem\u003eDaphnia magna\u003c/em\u003e exposed to combined exposure to heat and insecticide showed a genotype-dependent effect on survival and performance (Delnat et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInsects selected to tolerate insecticides may become more sensitive to environmental stressors due to the depletion of energy reserves resulting from prolonged activation of detoxification pathways, reducing their ability to withstand additional stressors. In general, when insects encounter stressful conditions, protective cellular responses are activated such as the upregulation of heat shock proteins (\u003cem\u003eHsp\u003c/em\u003e), which are known to be energetically demanding (Gonz\u0026aacute;lez-Tokman et al., 2020). For instance, insecticide-resistant and susceptible diamondback moth strains varied in their production of \u003cem\u003eHsp70\u003c/em\u003e in response to heat stress, with insecticide-resistant strains producing less \u003cem\u003eHsp70\u003c/em\u003e than susceptible strains (Liu et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). It is possible that prolonged expression of stress tolerance pathways resulted in energetic trade-offs in imidacloprid-selected beetles, thereby impairing their ability to ramp up protective physiological responses under further stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Imidacloprid\u003c/h2\u003e \u003cp\u003eExposure to sublethal doses of insecticide can have profound impacts on insects, including reduced feeding and altered behavior (Kenna et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We found that exposure to imidacloprid immediately caused imidacloprid-selected beetles to reduce their mobility. Sublethal doses of imidacloprid caused similar effects in other studies, in \u003cem\u003eL. decemlineata\u003c/em\u003e adults and \u003cem\u003eDrosophila melanogaster\u003c/em\u003e larvae (Alyokhin \u0026amp; Miller, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Young et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is possible that reduced locomotor activity in response to sublethal imidacloprid was only temporary since we only measured mobility 24 hours following exposure.\u003c/p\u003e \u003cp\u003eExposure to imidacloprid resulted in reduced feeding behavior within both beetle groups. It is possible that lower food consumption reduced an individual's total energy budget, impairing growth, development, and survival. Reductions in feeding activity following sublethal insecticide exposure have been reported in other insects, including leaf beetles, green peach aphids, and mayflies (Alexander et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Cho et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wolz et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite reducing larval mobility and herbivory, imidacloprid exposure did not have a negative impact on survival to adult stage, development time, or female fecundity in both beetle groups. Many other insecticide-resistant insects have been observed to experience reductions in fecundity and egg viability, which may be associated with energy reserve trade-offs related to detoxification mechanisms (Alyokhin \u0026amp; Ferro, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Argentine et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Boivin et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Cao \u0026amp; Han, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eEffect of High Temperature Stress\u003c/h2\u003e \u003cp\u003eThe high temperature treatment immediately reduced mobility in both unselected and imidacloprid-selected CPB larvae. Exposure to high temperatures has been shown to decrease mobility in other insect species, such as reducing locomotor activity and flight durations in house flies and decreasing jumping distance in crickets (Kj\u0026aelig;rsgaard et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lachenicht et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). We found that exposure to high temperature did not impact herbivory rates among unselected and imidacloprid-selected beetles. Given that insects are poikilothermic, high temperatures can be beneficial to some extent, promoting faster development and other performance traits (Colinet et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kiritani, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). For example, previous studies have reported that high temperatures increase insect consumption rates (Lemoine et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Lemoine et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In the case of CPB, prior studies have shown that all stages perform optimally between 25\u0026deg;C and 32\u0026deg;C (Ferro et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Logan et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Although the high temperature treatment exceeded the optimal temperature range for CPB, the heat exposure did not significantly affect herbivory rates. Therefore, it is plausible that the high temperature treatment was not highly stressful.\u003c/p\u003e \u003cp\u003eFollowing the high temperature treatment, both imidacloprid-selected and unselected larvae showed similar rates of survival to the adult stage, development time, or female fecundity. These results suggests that CPB larvae can tolerate brief periods of exceedingly high temperatures without apparent costs on development or female reproductive success. However, it is important to mention that extended durations above 40\u0026deg;C has been demonstrated to negatively impact CPB survival (Chen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Hence, we cannot dismiss the possibility that at longer durations, our 40\u0026deg;C treatment might elevate mortality.\u003c/p\u003e \u003cp\u003eNevertheless, our findings differ from earlier studies that looked at the relationship between exposure to high temperatures and life history traits in insects. For example, exposure to high temperatures reduced egg laying in the cotton bollworm and slowed development time in the brown planthopper (Mironidis \u0026amp; Savopoulou-Soultani, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Piyaphongkul et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Since we did not find any trade-offs in development and female fecundity, it is possible that brief exposure to 40\u0026deg;C was not severe enough to cause major heat injury, allowing larvae to recover quickly after exposure with no negative effects on their overall fitness. However, since we only assessed offspring survival during the hatchling stage, it is unclear if there any potential generational effects on survival and performance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eLimitations and Future Directions\u003c/h2\u003e \u003cp\u003eDue to logistical constraints, our study was limited to comparing the effects of a single selection process, which limits our ability to generalize regarding broader responses of CPB to various selection pressures and stressors. However, the observed patterns may also be due to the genetic architecture, genetic drift, or other stochastic processes that arose during the selection process itself. Using multiple independent selection lines would allow one to assess how comparable interactions between insecticides and high temperatures are and help distinguish between consistent evolutionary responses and population-specific effects.\u003c/p\u003e \u003cp\u003eTo better understand the mechanisms underlying our findings, several key areas warrant further investigation. First, examining physiological responses to combined stressors, including heat shock protein expression and cellular stress markers, may help identify how insecticide-resistant and insecticide-susceptible insects differ in stress tolerance mechanisms. Second, exploring a wider range of imidacloprid concentrations would provide a more comprehensive understanding of dose-dependent effects on CPB performance. Although we did not assess physiological parameters related to energy reserves, such as lipid content or mitochondrial function, future studies should examine the effects of insecticides on feeding behavior and how changes in food consumption may impact survival. Third, research is necessary to elucidate how reductions in feeding under heat stress affect growth rates and other life history traits that contribute to overall fitness. Future studies should also incorporate longer selection duration and different stressor orders to provide further insight into the interactions between insecticides and high temperature stress. Although complicated to perform, such studies would be particularly valuable for predicting CPB responses under various field conditions where both insecticide exposure and temperature fluctuations are common.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eBy exposing Colorado potato beetles to imidacloprid followed by high temperature stress, we found negative synergistic effects on survival, mobility, and herbivory, that were magnified in beetles selected for imidacloprid tolerance. Overall, while beetles performed poorly in the short term, those that recovered showed no long-term impacts on development or reproductive fitness. These findings have important implications for pest management in agricultural systems, particularly as climate change increases the frequency of temperature extremes. The increased vulnerability of imidacloprid-selected beetles to combined stress suggests that resistance management strategies should consider environmental contexts. Specifically, targeted applications of insecticides during periods of predicted high temperatures could potentially improve control efficacy against insecticide resistant populations. As such, cross-susceptibility could be leveraged in integrated pest management programs to delay resistance development or better manage existing insecticide resistance issues.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eErika M. Bueno:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eConceptualization, Methodology, Data collection, Formal Analysis, Writing \u0026ndash; original draft. Yolanda H. Chen: Conceptualization, Funding Acquisition, Methodology, Supervision, Writing- review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to Joseph Gunn and Edward Marques for providing valuable feedback on earlier versions of the manuscript and Jorge-Ruiz Arocho for illustrating part of Figure 1. We would also like to thank Casey McIlhenny, Ethan Dean, Indira Palmer, Leni Warlick, and Lauren Henzy for their contributions to the study. We acknowledge the generous support of our funding sources: the Vermont Agricultural Experiment Station Funds awarded to Y. H. Chen, as well as an HHMI Gilliam Fellowship and NSF Quantitative and Evolutionary STEM Traineeship (NSF NRT-1735316; PI: M.H. Pespeni) supporting E. M. 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Journal of Evolutionary Biology, \u003cem\u003e33\u003c/em\u003e(2), 151\u0026ndash;164. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jeb.13555\u003c/span\u003e\u003cspan address=\"10.1111/jeb.13555\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"insect pests, climate change, stress, insecticide, cross-susceptibility, synergistic","lastPublishedDoi":"10.21203/rs.3.rs-6156635/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6156635/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInsect pests are remarkably successful in evolving resistance to management tactics while facing multiple sources of stress in modern agroecosystems. One possible explanation for this success is that repeated exposure to insecticides may enable pests to tolerate additional stressors through cross-protection. Using the Colorado potato beetle (\u003cem\u003eLeptinotarsa decemlineata\u003c/em\u003e Say), we tested whether selection for imidacloprid tolerance influences responses to multiple stressors. We compared imidacloprid-selected and unselected beetles exposed to sublethal imidacloprid (LC\u003csub\u003e10\u003c/sub\u003e), high temperature (40\u0026deg;C), or their combination, measuring effects on mobility, herbivory, development, fecundity, and mortality. Contrary to our expectations, selected beetles showed increased vulnerability to stress treatments, particularly exhibiting reduced mobility and lower survival when exposed to combined stressors. While both beetle groups maintained similar development times and reproductive output, the imidacloprid-selected beetles demonstrated cross-susceptibility rather than cross-protection when facing multiple stressors. These findings suggest that selection for insecticide tolerance may create vulnerabilities to environmental stress, a dynamic that could inform pest management strategies under climate change.\u003c/p\u003e","manuscriptTitle":"Insecticide tolerance shapes performance responses to multiple stressors in a crop pest","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-07 11:05:05","doi":"10.21203/rs.3.rs-6156635/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f77b2a2a-d5f6-4964-b8d9-89a90577a5c7","owner":[],"postedDate":"March 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-13T17:53:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-07 11:05:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6156635","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6156635","identity":"rs-6156635","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}