Julie Engell Dahl

Sapho-Lou Marti

Hervé Colinet

Claudia Wiegand

Martin Holmstrup

David Renault

Thermal plasticity and sensitivity to insecticides in populations of an invasive beetle: Cyfluthrin increases vulnerability to extreme temperature

Keywords: Pesticide, thermal variation, tolerance, heat spike, pyrethroid, insect

Climate change increases average temperatures and the occurrence of extreme weather events, in turn accentuating the risk of organism exposure to temperature stress. When thermal conditions become stressful, the sensitivity of insects toward insecticides can be exacerbated. Likewise, exposure of insects to insecticides can subsequently influence their ability to handle stressful temperatures. Here, we investigated the effects of constant temperature and daily heat spikes, in presence/absence of insecticide treatment (cyfluthrin), on the condition (impairment of mobility) and thermal tolerance to cold (-6 °C) and heat (42.5 °C) of the terrestrial beetle Alphitobius diaperinus. The responses of insects from four populations (three farm-collected populations, one laboratory population) to different durations of extreme temperature exposure were compared. The results showed that the laboratory population was generally more sensitive to extreme cold and heat temperatures, with less than 50% of adults recovering after an exposure at -6 or +42.5 °C for 3h. Significant differences in the level of thermal tolerance were also found among insects from poultry farms. Cyfluthrin exposure incurred detrimental effects to insects' condition in all but one population. For two out of the four populations, mobility impairment was increased when adults were exposed to daily heat spikes (6 hours per day at 38 °C) and cyfluthrin simultaneously, compared to cyfluthrin exposure at constant temperatures; yet, no significant interaction between the two stressors was found. Finally, using one farm collected population, effects of pre-exposure to cyfluthrin on extreme temperature tolerance provided another example of the toxicant-induced climate sensitivity in insects.

Introduction

Over the course of their life, all living organisms, including insects, are confronted with a range of environmental conditions which vary spatially and temporally [START_REF] Hirose | Spatial and Diurnal Variation of Precipitation Systems over Asia Observed by the TRMM Precipitation Radar[END_REF][START_REF] Mann | Global-Scale Temperature Patterns and Climate Forcing over the Past Six Centuries[END_REF][START_REF] Soranno | Spatial and Temporal Variation of Ecosystem Properties at Macroscales[END_REF]. In some instances, environmental constraints may be exacerbated by the currently changing climatic conditions. This increases the occurrence of extreme weather events and the probability of organisms' exposure to higher temperatures [START_REF] Hansen | Perception of Climate Change[END_REF]IPCC 2014;[START_REF] Stillman | Heat Waves, the New Normal: Summertime Temperature Extremes Will Impact Animals, Ecosystems, and Human Communities[END_REF], heightening the risk of organism exposure to heat stress. In turn, several life history traits of individual insects can be affected [START_REF] Bozinovic | The Mean and Variance of Environmental Temperature Interact to Determine Physiological Tolerance and Fitness[END_REF][START_REF] Kingsolver | Heat Stress and the Fitness Consequences of Climate Change for Terrestrial Ectotherms[END_REF][START_REF] Renault | Long-Term after-Effects of Cold Exposure in Adult Alphitobius Diaperinus (Tenebrionidae): The Need to Link Survival Ability with Subsequent Reproductive Success[END_REF], with cascading effects on population demography and organism evolution [START_REF] Chown | Adapting to Climate Change: A Perspective from Evolutionary Physiology[END_REF][START_REF] Colinet | Insects in Fluctuating Thermal Environments[END_REF][START_REF] Katz | Extreme Events in a Changing Climate: Variability Is More Important than Averages[END_REF][START_REF] Moe | Combined and Interactive Effects of Global Climate Change and Toxicants on Populations and Communities[END_REF]. When temperature varies, heat spikes are more likely to occur, and they are expected to have greater evolutionary significance -and pose greater threat to species performance -than increase in average temperature [START_REF] Katz | Extreme Events in a Changing Climate: Variability Is More Important than Averages[END_REF][START_REF] Vasseur | Increased Temperature Variation Poses a Greater Risk to Species than Climate Warming[END_REF]).

Yet, whatever the nature of the physiological and biochemical mechanisms triggered by thermal stress, their activation may weaken the ability of organisms to deal with other environmental constraints, or conversely magnify these abilities when the responses elicit cross tolerance phenomena [START_REF] Chen | Multigenerational Analysis of Temperature and Salinity Variability Affects on Metabolic Rate, Generation Time, and Acute Thermal and Salinity Tolerance in Daphnia Pulex[END_REF][START_REF] Noyes | Forecasting the Impacts of Chemical Pollution and Climate Change Interactions on the Health of Wildlife[END_REF][START_REF] Sinclair | Cross-Tolerance and Cross-Talk in the Cold: Relating Low Temperatures to Desiccation and Immune Stress in Insects[END_REF]. In this context, investigating insects' ability to cope with stress under and after exposure to varying temperature regimes provides useful information about their sensitivity to environmental stress, and for future insights into their geographic distribution [START_REF] Bozinovic | Physiological Ecology Meets Climate Change[END_REF][START_REF] Buckley | Can Mechanism Inform Species' Distribution Models?[END_REF][START_REF] Ma | Daily Temperature Extremes Play an Important Role in Predicting Thermal Effects[END_REF].

Besides climatic conditions, adverse environmental effects on insects are also caused by the unintentional spread of man-made contaminants, in particular by the use of various pesticides in agriculture and horticulture [START_REF] Aviles | Effects of DEHP on Post-Embryonic Development, Nuclear Receptor Expression, Metabolite and Ecdysteroid Concentrations of the Moth Spodoptera Littoralis[END_REF][START_REF] J O U R N A L P R E -P R O O F Calow | Physiological Costs of Combating Chemical Toxicants: Ecological Implications[END_REF][START_REF] Köhler | Wildlife Ecotoxicology of Pesticides: Can We Track Effects to the Population Level and Beyond?[END_REF]. The use of pesticides has become widespread in many regions of the world where agricultural intensity has increased [START_REF] Tilman | Agricultural Sustainability and Intensive Production Practices[END_REF], representing a threat to biodiversity in general. While valuable insights of the effects of insecticides on behaviour [START_REF] Dewer | Behavioral and Metabolic Effects of Sublethal Doses of Two Insecticides, Chlorpyrifos and Methomyl, in the Egyptian Cotton Leafworm, Spodoptera Littoralis (Boisduval) (Lepidoptera: Noctuidae)[END_REF], olfaction [START_REF] Lalouette | Unexpected Effects of Sublethal Doses of Insecticide on the Peripheral Olfactory Response and Sexual Behavior in a Pest Insect[END_REF], reproduction and development (Abbes et al. 2015) have been gathered, these investigations were commonly conducted by exposing the organisms to constant and optimal temperatures, as prescribed in standardized tests [START_REF] Camp | Can't Take the Heat: Temperature-Enhanced Toxicity in the Mayfly Isonychia Bicolor Exposed to the Neonicotinoid Insecticide Imidacloprid[END_REF][START_REF] Khan | The Effect of Temperature on the Toxicity of Insecticides against Musca Domestica L.: Implications for the Effective Management of Diarrhea[END_REF]). Yet, the J o u r n a l P r e -p r o o f inclusion of daily thermal fluctuations is needed to better simulate the conditions experienced by free-ranging organisms in natural and anthropogenic environments, and thus increase the quality and realism of the outcomes of these investigations [START_REF] Colinet | Insects in Fluctuating Thermal Environments[END_REF]Verheyen and Stoks 2019).

Moreover, thermal tolerance can vary significantly among insect populations originating from different environments [START_REF] Dahl | Thermal Tolerance Patterns of a Carabid Beetle Sampled along Invasion and Altitudinal Gradients at a Sub-Antarctic Island[END_REF][START_REF] Käfer | Temperature Tolerance and Thermal Environment of European Seed Bugs[END_REF], as is also the case for pesticide exposure (see Hawkins et al. (2019) for a review of the different mechanisms that may confer different level of insecticide resistance among insect populations). When assessing the effects of environmental stressors on insects' ecology, it has thus become crucial to consider multiple populations (of different exposure or adaptation history) in the experimental designs to get a larger coverage of the range of responses that could be expected at the species level.

The possible combined effects of climate change and environmental pollution on organisms, and wildlife in general, have been reviewed by [START_REF] Noyes | Forecasting the Impacts of Chemical Pollution and Climate Change Interactions on the Health of Wildlife[END_REF]. These authors reported that chemical exposure may affect organisms' sensitivity to climate change by disrupting physiological responses to diurnal and seasonal thermal fluctuations. In particular, the alteration of endocrine system functioning and energy balance in toxicant-exposed organisms may subsequently alter their thermal performance curves, potentially challenging their ability to survive thermal stress. In several instances, it has been suggested that the simultaneous exposure to pollutants, including endocrine disrupting chemicals, and climate change could result in synergistic actions [START_REF] Cairns | The Effects of Temperature upon the Toxicity of Chemicals to Aquatic Organisms[END_REF][START_REF] Hooper | Interactions between Chemical and Climate Stressors: A Role for Mechanistic Toxicology in Assessing Climate Change Risks[END_REF][START_REF] Laskowski | Interactions between Toxic Chemicals and Natural Environmental Factors -A Meta-Analysis and Case Studies[END_REF][START_REF] Moe | Combined and Interactive Effects of Global Climate Change and Toxicants on Populations and Communities[END_REF]. When sequential exposures occur, climatic stress can influence sensitivity towards pollutants, and the reverse is also true, e.g. previous exposure to a pollutant can influence the tolerance to climatic stress [START_REF] Hooper | Interactions between Chemical and Climate Stressors: A Role for Mechanistic Toxicology in Assessing Climate Change Risks[END_REF].

Nowadays, studies assessing the impacts of multiple stressors, including temperature, are increasingly needed [START_REF] Kaunisto | Can We Predict the Effects of Multiple Stressors on Insects in a Changing Climate?[END_REF][START_REF] Orr | Towards a Unified Study of Multiple Stressors: Divisions and Common Goals across Research Disciplines[END_REF]), as such investigations greatly improve our estimates of the effects of interactions among environmental stressors on insects. [START_REF] Holmstrup | Interactions between Effects of Environmental Chemicals and Natural Stressors: A Review[END_REF] reviewed the available studies that assessed the combined effects of pesticide and temperature exposures, and reported only few works that considered daily thermal fluctuations, or daily heat spikes, in their experimental designs. Since this review has been published, the number of studies that combined temperature and toxicant exposures has started to accumulate, reporting how thermal conditions could modify the effects of pesticides on insects (Op J o u r n a l P r e -p r o o f de Beeck, Verheyen, and Stoks 2018;[START_REF] Camp | Can't Take the Heat: Temperature-Enhanced Toxicity in the Mayfly Isonychia Bicolor Exposed to the Neonicotinoid Insecticide Imidacloprid[END_REF][START_REF] Delnat | Whether Warming Magnifies the Toxicity of a Pesticide Is Strongly Dependent on the Concentration and the Null Model[END_REF][START_REF] Harwood | Temperature as a Toxicity Identification Evaluation Tool for Pyrethroid Insecticides: Toxicokinetic Confirmation[END_REF][START_REF] Verheyen | Increased Daily Temperature Fluctuations Overrule the Ability of Gradual Thermal Evolution to Offset the Increased Pesticide Toxicity under Global Warming[END_REF]Verheyen and Stoks 2019;[START_REF] Willming | Effects of Environmentally Realistic Daily Temperature Variation on Pesticide Toxicity to Aquatic Invertebrates[END_REF]. For example, Verheyen and Stoks (2019) found that exposure of damselfly larvae to the organophosphate chlorpyrifos did not affect the survival of individuals maintained at constant 20 °C.

Conversely, the mortality of the larvae was increased under daily thermal fluctuations of 5 (17.5-22.5 °C) or 10 °C (15-25 °C). [START_REF] Delnat | Whether Warming Magnifies the Toxicity of a Pesticide Is Strongly Dependent on the Concentration and the Null Model[END_REF] reported that daily thermal fluctuations of 7 (16.5-23.5 °C) or 14 °C (13-27 °C) slightly increased the mortality of the larvae of the mosquito Culex pipiens exposed to chlorpyrifos, while having no effect on adults. Working with heat waves (constant heat exposure of damselfly larvae over three consecutive days), Arambourou and Stoks (2015) reported that the subsequent exposure to chlorpyrifos did not change mortality rates of the larvae. However, the higher inhibition of acetylcholine esterase suggested that heat waves may increase the sensitivity of damselflies to this chemical. More recent studies have examined the effects of successive heat spikes and pesticide exposure [START_REF] Meng | The Exposure Order Strongly Modifies How a Heat Spike Increases Pesticide Toxicity[END_REF], and the delayed effects of pesticide exposures on life histories of insects [START_REF] Dinh | Exposure to a Heat Wave under Food Limitation Makes an Agricultural Insecticide Lethal: A Mechanistic Laboratory Experiment[END_REF].

Even though [START_REF] Delnat | Whether Warming Magnifies the Toxicity of a Pesticide Is Strongly Dependent on the Concentration and the Null Model[END_REF] found the effect of warming on pesticide toxicity to be dependent on other factors of their experimental setup, results from the available literature generally underline the importance of incorporating more realism into the experimental designs when investigating the interaction between pesticide and temperature [START_REF] Böttger | Closer to Reality -the Influence of Toxicity Test Modifications on the Sensitivity of Gammarus Roeseli to the Insecticide Imidacloprid[END_REF]. Moreover, all but one of these studies used insect larvae, and all investigations were based on aquatic species, meaning that terrestrial insect species and adult life stages are severely underrepresented from this research. Likewise, some studies have investigated the thermal tolerance of insects after they were exposed to pesticides (Op de Beeck, Verheyen, andStoks 2017, 2018;[START_REF] Delnat | Whether Warming Magnifies the Toxicity of a Pesticide Is Strongly Dependent on the Concentration and the Null Model[END_REF][START_REF] Meng | The Exposure Order Strongly Modifies How a Heat Spike Increases Pesticide Toxicity[END_REF]). Yet again, all of these investigations have focused on aquatic larvae, and used the organophosphate chlorpyrifos. Other chemicals, including pyrethroids, which are also highly toxic to insects [START_REF] Tang | Pyrethroid Pesticide Residues in the Global Environment: An Overview[END_REF], should be considered. Finally, no study examined the effects of sequential exposure to daily thermal fluctuations and pesticide on the subsequent thermal tolerance in terrestrial insects.

In order to contribute to the abovementioned research gaps concerning the interaction of climate and pollutant stress, we aimed at investigating the effects of daily heat spikes and insecticide treatment on the thermal tolerance of adults of the lesser mealworm Alphitobius diaperinus J o u r n a l P r e -p r o o f (Panzer) (Coleoptera: Tenebrionidae), as well as comparing the performance of populations of A. diaperinus of different exposure histories to temperature and insecticide. This insect pest, pullulating in poultry farms from many regions worldwide [START_REF] Geden | Research and Extension Needs for Integrated Pest Management for Arthropods of Veterinary Importance[END_REF][START_REF] Johnson | Insectes Des Bâtiments d'élevages Avicoles Des Fermes de Bingerville, Côte d'Ivoire : Alphitobius Diaperinus (Panzer, 1797) (Coleoptera: Tenebrionidae)[END_REF][START_REF] Rueda | Arthropods in Litter of Poultry (Broiler Chicken and Turkey) Houses[END_REF], represents a fitting model for research combining ecophysiological and ecotoxicological investigations. Insects can be collected in large numbers from geographically distant and unconnected habitats (poultry farms), making it possible to work with different and locally adapted strains. In these anthropogenic environments, the lesser mealworms are exposed to fluctuating temperatures, as broilers optimize poultry production by running a standardized cycle (which has to be closely followed to maintain profitability [START_REF] Donald | Ross Broiler Report Environmental Management in the Broiler House[END_REF])) of weekly decreasing temperatures, i.e. starting from ≈ 31 and reaching ≈ 17 °C over approximately seven weeks [START_REF] Salin | Controlling the Mealworm Alphitobius Diaperinus (Coleoptera: Tenebrionidae) in Broiler and Turkey Houses: Field Trials with a Combined Insecticide Treatment: Insect Growth Regulator and Pyrethroid[END_REF]. This provides an excellent habitat for lesser mealworms, that have been found to grow well, i.e. with a very high energy conversion efficiency at 23 to 31 °C [START_REF] Bjørge | Role of Temperature on Growth and Metabolic Rate in the Tenebrionid Beetles Alphitobius Diaperinus and Tenebrio Molitor[END_REF]. Temperature extremes up to 48 °C have also been measured in the litter where A. diaperinus thrive [START_REF] Salin | Rapport scientifique Bayern AG, Université de Rennes-1 et UMR 6553 Efficiency Tests in Natural Conditions of Baycidal (Triflumuron) and Solfac (Cyfluthrine) against Coleoptera in Poultry Breeding Systems[END_REF], meaning that the insect can be exposed to heat spikes. The poultry houses are disinfected from time to time with chemicals, including the structural type II pyrethroid, cyfluthrin (the use of this insecticide will be banned in France from July 21 st , 2021 (ANSES 2020)). This insecticide is an inhibitor of insect nerve cell sodium channels, leaving them open and causing paralysis, tremors, and eventually death [START_REF] Lushchak | Pesticide Toxicity: A Mechanistic Approach[END_REF]. It is sprayed at the end of each broiler flock for controlling outbreaks of A. diaperinus, and during the disinfection and cleaning of the poultry farm, the heating system is stopped, doors of the building are left opened, and temperature can vary from -5 °C to 31 °C [START_REF] Salin | Controlling the Mealworm Alphitobius Diaperinus (Coleoptera: Tenebrionidae) in Broiler and Turkey Houses: Field Trials with a Combined Insecticide Treatment: Insect Growth Regulator and Pyrethroid[END_REF].

As a first part of the study, we compared the basal thermal tolerance of A. diaperinus from four populations, by measuring their ability to recover from exposure to extreme cold or heat temperatures for different durations. We hypothesized that the beetles' recovery from thermal stress would be faster in populations that experienced temperature fluctuations in their original environment, as previously reported on several other insect models (Arias, Poupin, and Lardies 2011;[START_REF] Bozinovic | The Mean and Variance of Environmental Temperature Interact to Determine Physiological Tolerance and Fitness[END_REF][START_REF] Cavieres | Transgenerational and within -Generation Plasticity Shape Thermal Performance Curves[END_REF]. As a second part of the study, we were interested in comparing the effects of exposure to an environmentally relevant dose of the insecticide cyfluthrin on the condition of insects maintained at constant temperatures or subjected to daily heat spikes. We hypothesized that populations which had experienced frequent pesticide exposure J o u r n a l P r e -p r o o f would perform better than laboratory-reared adults [START_REF] Hamm | Resistance to Cyfluthrin and Tetrachlorvinphos in the Lesser Mealworm, Alphitobius Diaperinus, Collected from the Eastern United States[END_REF][START_REF] Lambkin | Baseline Responses of Alphitobius Diaperinus (Coleoptera: Tenebrionidae) to Cyfluthrin and Detection of Strong Resistance in Field J o u r n a l P r e -p r o o f Populations in Eastern Australia[END_REF].

Finally, the population with the highest pesticide tolerance was exposed to different temperature regimes, without pesticide exposure or with different concentrations of cyfluthrin (up to those recommended by commercial formulations). Subsequently, the recovery times of individuals from the different treatments after an exposure to temperature extremes were assessed. Insects preexposed to daily heat spikes were expected to recover faster from heat shock, but slower from cold shock treatments, compared to insects kept at constant temperature. Furthermore, insects exposed to insecticides were hypothesized to be characterized by a lower ability to cope with temperature stress, either due to an increased toxicity of the chemical with daily heat spikes or decreased ability to cope with heat shock after chemical stress (Op de Beeck, [START_REF] Op De Beeck | Integrating Both Interaction Pathways between Warming and Pesticide Exposure on Upper Thermal Tolerance in Highand Low-Latitude Populations of an Aquatic Insect[END_REF][START_REF] Delnat | Whether Warming Magnifies the Toxicity of a Pesticide Is Strongly Dependent on the Concentration and the Null Model[END_REF][START_REF] Meng | The Exposure Order Strongly Modifies How a Heat Spike Increases Pesticide Toxicity[END_REF].

Materials and methods

Rearing of the insects

Adults of A. diaperinus were collected from three poultry farms in Brittany, France, thereafter referred to as FARM1 (sampled at Miniac-sous-Bécherel, 48°17'10'' N, 1°55'51'' W), FARM2 (sampled at Plaine Haute, 48°26'44'' N, 2°51'16'' W), and FARM3 (sampled at Pommerit-le-Vicomte, 48°37'10'' N, 3°05'15'' W). In addition, larvae of A. diaperinus were obtained from Proti-Farm via a pet shop (Envies Animales, Saint-Jacques-de-la-Lande, Brittany, France). Insects from this latter population, hereafter referred to as LAB, were reared over many generations under controlled conditions (25 °C, relative humidity 50%, no light) in a supposedly pollutant free environment.

Because LAB insects are produced and used as a dietary food supply for pets (e.g. lizards), they are in very good physiological condition [START_REF] Bjørge | Role of Temperature on Growth and Metabolic Rate in the Tenebrionid Beetles Alphitobius Diaperinus and Tenebrio Molitor[END_REF].

All adults were kept at 25 °C, in darkness, in plastic containers with sawdust for minimum 4 weeks prior to experimental start. They were supplied with water and food ad libitum, in the form of water in microtubes with cotton and dry dog food pellets. This procedure allowed standardisation of the temperature, humidity, and trophic conditions experienced by the adults before the assays. The LAB larvae were additionally provided with a block of polystyrene to burrow in for pupation, and oat bran for the first four weeks to further encourage their development into adults [START_REF] Rice | A New Culture Method for Lesser Mealworm, Alphitobius Diaperinus[END_REF]. The sex and age of the adults was not considered in our experiments.

J o u r n a l P r e -p r o o f

Experimental design

Characterization of the thermal tolerance of the insects from different populations

In order to compare the thermal tolerance of the insects from the four populations, adults were exposed to two extreme temperatures, -6 or +42.5 °C, in a cryothermostat (VWR Collection, AP15R-30). This procedure is derived from protocols commonly used for assessing the thermal tolerance in insects [START_REF] Hazell | A Method for the Rapid Measurement of Thermal Tolerance Traits in Studies of Small Insects[END_REF][START_REF] Sinclair | An Invitation to Measure Insect Cold Tolerance: Methods, Approaches, and Workflow[END_REF]. At -6 and + 42.5 °C, the lesser mealworms are expected to fall into coma (see Supplementary File 1 for the results of the preliminary tests that allowed us designing the methodology). Each insect was exposed to one of these two extreme temperatures for one of six durations: 15 min, 30 min, 1h, 2h, 3h or 4h, in darkness. This range of stress durations allowed us to describe and compare thermal tolerance among populations. At least four replicates of 10 adults (test tube with rubber cap) were used for each exposure duration and temperature combination. At the end of the thermal treatment (-6 or +42.5 °C), each group of 10 insects was directly transferred into Petri dishes and observed for up to 40 seconds every 5 minutes over 2h at room temperature (19 ± 1 °C) (Supplementary File 2). During each observation period, the recovery of the insects was scored by registering the individuals as either "Active" or "Impaired/immobile"; insects were characterized as "Active" when they exhibited coordinated movements of all limbs when stimulated, all other states were registered as "Impaired/immobile". The temperatures, both in the cryothermostat and in the room where recovery observations took place, were monitored using iButtons® (iButtonLink, LLC., Whitewater, USA).

Effect of daily heat spikes and cyfluthrin exposure

Here, we combined two abiotic stresses by jointly exposing adult A. diaperinus to both moderately stressful heat spikes and insecticide for eight days, before assessing any visible impairment or death of exposed individuals. One temperature cabinet was set to constant 26 °C (control temperature), and another cabinet varied the temperature by holding a 6h period at 38 °C, followed by 18h at 26 °C (Supplementary File 2); the two cabinets were MIR 154 Panasonic programmable incubators. The experiment was run in dark conditions.

Insects were placed in glass Petri dishes (10 cm diameter) with a fitting filter paper covering the bottom. For insecticide application, cyfluthrin (pure molecule standard, CAS-number 68359-37-5, J o u r n a l P r e -p r o o f Sigma-Aldrich) was dissolved in acetone, and a volume of 2 mL of appropriately diluted solution was transferred to the filter paper. Pre-testing the insects' susceptibility to cyfluthrin led to preparation of two distinct insecticide concentrations, so that the applied dose was 20 mg cyfluthrin/m 2 for populations FARM1, 2 and 3, and 0.5 mg cyfluthrin/m 2 for LAB. We had to reduce the cyfluthrin dose significantly for the LAB population, as the 20 mg cyfluthrin / m² concentration killed all adults within a day at our experimental temperatures. These cyfluthrin concentrations induced negligible knockdown (<5%) over prolonged exposures in the works of Desvignes-Labarthe (2018) and Renault and Colinet (Submitted). These concentrations also range in the values that may be encountered by the insects in poultry houses [START_REF] Salin | Controlling the Mealworm Alphitobius Diaperinus (Coleoptera: Tenebrionidae) in Broiler and Turkey Houses: Field Trials with a Combined Insecticide Treatment: Insect Growth Regulator and Pyrethroid[END_REF]. In the other Petri dishes, 2 mL of pure acetone was added to control for any effect of the solvent. All Petri dishes were left to dry under a fume hood for at least 10 min before the tests, which effectively evaporated all acetone.

The exiting literature reveals that cyfluthrin remains efficient over seven days [START_REF] Guillebeau | Influence of Weather on Efficacy of Pyrethroid Insecticides for Boll Weevil (Coleoptera: Curculionidae) and Bollworm (Lepidoptera: Noctuidae) in Cotton[END_REF], with knockdown effects remaining unchanged when the beetle Tribolium castaneaum were assessed eight weeks after the chemical was applied [START_REF] Arthur | Effect of Temperature on Residual Toxicity of Cyfluthrin Wettable Powder[END_REF] Finally, starting from an initial concentration of 17.3 mg / m², [START_REF] Nakagawa | Pyrethroid Concentrations and Persistence Following Indoor Application[END_REF] reported that cyfluthrin concentration decreased by ca. 17% after 56 days. Thus, even if changes in cyfluthrin concentration were not monitored here, it is assumed that it remained close to the nominal concentration.

For all treatments, the insects were supplied with a water source (0.5 mL microtube containing water and cotton) and a food source (approx. 0.8 g dog food pellet). For each population, a set of Petri dishes treated with the insecticide and a set of dishes without pesticide treatment were placed in the temperature cabinets (8 to 30 dishes of each, as specified in Table 1) along with an iButton® (iButtonLink, LLC., Whitewater, USA) measuring both temperature and humidity. Each Petri dish in experiments with poultry house collected populations contained 30 individuals, and for the LAB population, each Petri dish contained 20 individuals, due to limited number of individuals (Table 1).

Previous experiments that examined the sensitivity of A. diaperinus to insecticides (including cyfluthrin) revealed that densities from 10 to 30 adults per Petri dish had no effects on the results (Desvignes-Labarthe 2018; Renault and Colinet Submitted).

J o u r n a l P r e -p r o o f

Table 1 Overview of number of dishes and number of individuals per dish used for assessing the effects of temperature regime (constant temperature or daily heat spikes), with and without the insecticide cyfluthrin, on the locomotory capacities of adult Alphitobius diaperinus.

After eight days, and 17 hours after the last peak of heat, the state of each individual was noted: the insects were considered as either "Active" (no apparent effect of the treatment on the locomotory activities of the insects), "Impaired" (locomotor deficits: difficulties in moving limbs, or laying on their backs while moving) or "Immobile" (no observable movement).

Effect of daily heat spikes and cyfluthrin exposure on recovery from exposure to extreme temperatures

In the third part of the study, we measured the capabilities of adult A. diaperinus to recover from cold or heat shocks after they had been exposed to insecticide and/or daily heat spikes for eight days. As a non-negligible part of the adults from LAB, FARM2 and FARM3 were impaired or immobile after they were exposed to cyfluthrin concentration of 20 mg / m² (see the results presented in Figure 3), we had to focus on adults from FARM1 for this experiment. Lesser mealworms from this population survive exposure to cyfluthrin concentrations encountered in the field (as recommended applications contain 20 mg cyfluthrin/m 2 , C. [START_REF] Salin | Controlling the Mealworm Alphitobius Diaperinus (Coleoptera: Tenebrionidae) in Broiler and Turkey Houses: Field Trials with a Combined Insecticide Treatment: Insect Growth Regulator and Pyrethroid[END_REF], which thus allowed working with this concentration without incurring selection of individuals due to mortality or locomotor deficits during the pre-exposure period.

For this experiment, three temperature conditions were used: (i) constant 26 °C for eight days (control), 18h at 26 °C daily interrupted by exposures of 6h at (ii) 32 °C (low heat spike), or (iii) 38 °C (high heat spike). The exposure ran over eight days in darkness. Glass Petri dishes were prepared with four doses of cyfluthrin (0, 5, 10 or 20 mg cyfluthrin/m 2 ) as described above, including addition of food and water. Then, 30 adults from FARM1 were added to each dish, and dishes of every pesticide dose were placed into each of the three temperature cabinets (MIR 154 Panasonic After eight days, and 17 hours after the last peak of heat, the Petri dishes were removed from the temperature cabinets, and the beetles were directly exposed to either -6 or +42.5 °C for 2h, in darkness (Supplementary File 2). Extreme temperature exposure was performed as described above (section 2.2.1.), including testing the recovery of at least 40 individuals to each combination of treatment (temperature regime, insecticide dose, and temperature regime x insecticide dose), but the temperature of the recovery room was higher (approx. 24 °C instead of 20 °C).

Data treatment

The recovery time of every single individual was used to compute the temporal recovery curves using Kaplan Meier estimates computed in R version 3.5.2, using the Survminer package insecticide dose), and their interactions, were analysed using the analysis of deviance (Anova function in the car package [START_REF] Fox | An R Companion to Applied Regression[END_REF]). We assume that this insecticide and heat peaks have different modes of actions, meaning that their individual-and interaction effects on A.

diaperinus adults are assumed to be multiplicative. Differences among the treatments were finally analysed by Sidak's multiple comparisons using the emmeans and multcompView packages (Graves, Piepho, and Selzer 2019; Lenth 2020), with a significance value of 0.05.

Results

Characterization of the thermal tolerance of the insects from different populations

The recovery probability over time of adult A. diaperinus was dependent on the exposure duration to hot or cold temperature extremes (Figures 1 and2; Supplementary File 3). Adults from LAB J o u r n a l P r e -p r o o f recovered more slowly than adults from the poultry house-collected populations for all durations of exposure at 42.5 °C (P ≤ 0.05), and all but 30 minutes exposure at -6 °C (P ≤ 0.05) (Supplementary File 3). After a 30 min exposure at -6 °C, the recovery from LAB was comparable to that measured from the three other populations (P ≥ 0.05); after 1 hour exposure at 42.5 °C, the recovery curve of LAB was also similar to FARM3 (P = 0.33). When the duration of exposure to heat or cold temperatures was increased, the proportion of insects that fully recovered was decreased and the duration of recovery was prolonged (Figures 1 and2).

The recovery probability of the lesser mealworms from FARM1 and FARM2 after they were exposed to heat (42.5 °C) was similar for all experimental conditions. When exposed to temperature extremes for 0.25, 0.5, 1, 2, or 3h, insects from FARM3 were characterised by a lower recovery probability as compared with the two other poultry house-collected populations (Figure 1).

Effect of daily heat spikes and cyfluthrin exposure

The number of active lesser mealworms was significantly reduced when insects were exposed to cyfluthrin at constant temperature (26 °C) as compared with control insects (P < 0.05 (95% confidence level)) for all populations (Figure 3), except for FARM1 (Figure 3, A; Supplementary File J o u r n a l P r e -p r o o f

3). Sidak's multiple comparisons post-hoc tests following a two-way ANOVA showed that the insecticide cyfluthrin and daily heat spikes (18h at 26 °C followed by 6h period at 38 °C for 8 days) significantly decreased the proportion of active insects as compared with control and heat treatment conditions (P < 0.05) for all populations (Figure 3A-D; Supplementary File 3). Yet, the proportion of insects that were scored as active remained close to 100% for FARM1. The proportion of insects scored as active significantly differed among the "Cyfluthrin" and "Daily heat spikes &

Cyfluthrin" treatments for FARM2 (P < 0.05) and LAB (P < 0.05). There was no significant interaction between daily heat spikes and cyfluthrin for any of the populations (P = 0.28 or higher) (Figure 3, A-D; Supplementary File 3). The recovery probability of adult A. diaperinus from FARM1 decreased with increasing doses of cyfluthrin after the insects were maintained for eight days under different temperature regimes and cyfluthrin concentrations prior to extreme temperature exposure (P < 0.001) (Figure 4; Supplementary File 3). This was particularly evident for beetles exposed to heat (42.5 °C) (Figure 4).

Pre-exposure to a daily heat spike for eight days (Figure 4, B-C) accelerated the recovery of the insects after they were exposed at 42.5 °C for 2h, as compared with animals exposed at constant 26 °C (Figure 4, A; Supplementary File 3). Conversely, the recovery curves remained comparable for insects that were exposed to extreme cold (-6 °C for 2h; Figure 4, D-F), even though they were previously subjected to different heat spikes for 8 days. regime used as the reference recovery probability). Different letters indicate differences among the recovery probability curves within each temperature pre-treatment group.

Discussion

Characterization of the thermal tolerance of the insects from different populations

In this study, we were interested in comparing the basal thermal tolerance of adult A. diaperinus from four populations through assessments of their ability to recover from different durations of exposure to cold or heat. Adults from FARM1 and FARM2 were characterized by the greatest ability to recover from thermal stress, in particular to heat exposure, while laboratory-reared adults exhibited the lowest thermal tolerance, in line with our expectations. The thermal plasticity of lesser mealworm populations is also consistent with the wide range of temperatures that can occur during a poultry house production cycle. Acclimation or adaptation to thermally variable habitats may widen the temperature tolerance range, as reported from other insect models (reviewed in [START_REF] Colinet | Insects in Fluctuating Thermal Environments[END_REF][START_REF] Lachenicht | Effects of Acclimation Temperature on Thermal Tolerance, Locomotion Performance and Respiratory Metabolism in Acheta Domesticus L. (Orthoptera: Gryllidae)[END_REF]. For instance, working with Drosophila melanogaster reared at varying temperature regimes, [START_REF] Bozinovic | The Mean and Variance of Environmental Temperature Interact to Determine Physiological Tolerance and Fitness[END_REF] observed a reduction of the number of flies entering into chill or heat coma when transferred to extreme temperatures, as compared with flies reared at constant temperature. [START_REF] Overgaard | Assessing Population and Environmental Effects on Thermal Resistance in Drosophila Melanogaster Using Ecologically Relevant Assays[END_REF] evidenced that acclimation of D. melanogaster to variable temperatures increased tolerance to both low and high thermal stress. In addition, these authors showed that populations originating from temperate areas exhibited a wider thermal tolerance than those sampled from more thermally stable tropical environments. In sum, the lower thermal tolerance of adult A. diaperinus from LAB is consistent with the available literature, and the hypothesis that poultry house-collected insects have higher recovery probabilities than laboratory-bred individuals was confirmed.

In this work, insects from FARM3 exhibited a significantly lower thermal tolerance than their counterparts sampled from the other two poultry farms. However, many other environmental factors experienced by the insects in the poultry farm, such as pH and ammonia concentration in the litter, or the presence and diversity of bacteria and viruses which trigger immune responses that can alter the performance of other physiological traits, may have shaped the thermal tolerance of the insects from poultry farms. Further, we can also expect the populations to be genetically distinct J o u r n a l P r e -p r o o f temperatures. For instance, daily thermal fluctuations as low as 5 °C increased toxicity of chlorpyrifos in damselfly larvae (Verheyen and Stoks 2019). The synergistic effects of thermal variations and insecticides may occur under large thermal variations only, as reported in the vector mosquitoes Culex pipiens [START_REF] Delnat | Whether Warming Magnifies the Toxicity of a Pesticide Is Strongly Dependent on the Concentration and the Null Model[END_REF]. The magnification of the effects of pyrethroids on A.

diaperinus we have found emphasizes the importance of considering daily thermal variations and heat spikes in risk assessments of the sensitivity of biodiversity to pollution under different scenarios of climate change, although a higher constant temperature equivalent of the average temperature experienced by animals exposed to daily heat peaks would allow us to make stronger conclusions on this point. At higher temperatures, pesticide uptake may be increased, as found by [START_REF] Lydy | Effects of Temperature on the Toxicity of M-Parathion, Chlorpyrifos, and Pentachlorobenzene to Chironomus Tentans[END_REF] and [START_REF] Buchwalter | Temperature Influences on Water Permeability and Chlorpyrifos Uptake in Aquatic Insects with Differing Respiratory Strategies[END_REF]. In parallel, elimination rates of the toxicants, for example through temperature-dependent activation of enzymes, should also be enhanced at warmer conditions. In sum, the available literature and our results could indicate a promotion of insecticide resistant genotypes with increasing occurrence of extreme weather events, which is consistent with documented links between climatic variables and evolution of pesticide resistance in arthropod pests [START_REF] Maino | Climate Contributes to the Evolution of Pesticide Resistance[END_REF]. Facilitation of insecticide resistance as a result of climate change could be a result of pleiotropy, shared genetic mechanisms or genetic linkage [START_REF] Pu | Climate Change and the Genetics of Insecticide Resistance[END_REF].

Effect of daily heat spikes and cyfluthrin exposure on recovery from exposure to extreme temperatures

Using adult A. diaperinus from FARM1, we tested how the sensitivity to extreme temperatures was affected by thermal and insecticide pre-treatments. We expected higher proportion of recovery and faster recovery from heat stress in insects exposed to a daily heat spike over eight consecutive days, as extreme temperature tolerance is readily modified by a thermal conditioning processes, even in the short-term [START_REF] Chidawanyika | Rapid Thermal Responses and Thermal Tolerance in Adult Codling Moth Cydia Pomonella (Lepidoptera: Tortricidae)[END_REF][START_REF] Kingsolver | Plasticity of Upper Thermal Limits to Acute and Chronic Temperature Variation in Manduca Sexta Larvae[END_REF][START_REF] Weldon | Time-Course for Attainment and Reversal of Acclimation to Constant Temperature in Two Ceratitis Species[END_REF]. Our results are consistent with this expectation for heat-shocked insects. Daily peaks at high temperatures may have acclimated the insects to heat, which does not necessarily prepare them for cold exposures. Indeed, cold tolerance requires different mechanisms compared to heat tolerance, by for instance increasing membrane fluidity instead of decreasing it [START_REF] Neven | Physiological Responses of Insects to Heat[END_REF][START_REF] Teets | Physiological Mechanisms of Seasonal and Rapid Cold-Hardening in Insects[END_REF]. This can explain why heat pre-exposures did not consistently affect cold tolerance in this experiment. Similarly, it has been shown that acclimation to low temperature J o u r n a l P r e -p r o o f increased cold tolerance, but did not affect heat tolerance in Drosophila melanogaster [START_REF] Overgaard | Metabolomic Profiling of Rapid Cold Hardening and Cold Shock in Drosophila Melanogaster[END_REF].

For insects exposed to extreme heat, insecticide pre-treatment significantly reduced recovery probability, both by generally increasing the recovery time and the number of beetles that did not recover within the recovery period. In fact, beetles pre-exposed to the sublethal dose of 20 mg/m 2 at control temperature never recovered from the applied sublethal heat exposure. We also observed that even a relatively low exposure dose (5 mg cyfluthrin/m²) had a significant effect on the recovery probability of the individuals, thus providing supporting evidence for toxicant-induced climate sensitivity [START_REF] Hooper | Interactions between Chemical and Climate Stressors: A Role for Mechanistic Toxicology in Assessing Climate Change Risks[END_REF][START_REF] Moe | Combined and Interactive Effects of Global Climate Change and Toxicants on Populations and Communities[END_REF]) in adult A. diaperinus. Several studies have

shown that an increase in temperature generally enhances negative effects of contaminants [START_REF] Noyes | The Toxicology of Climate Change: Environmental Contaminants in a Warming World[END_REF][START_REF] Noyes | Forecasting the Impacts of Chemical Pollution and Climate Change Interactions on the Health of Wildlife[END_REF], but the number of studies that have tested the effects of insecticide pre-treatments is rather limited as compared with investigations assessing the effects of simultaneous exposures. Importantly, when working sequentially, i.e. by applying one stress before the other, results can greatly vary depending on the specific chemical investigated [START_REF] Matzrafi | Climate Change Exacerbates Pest Damage through Reduced Pesticide Efficacy[END_REF]).

Here, we can hypothesize that coma, which is related to nerve dysfunction, through decreased membrane potential and reduced excitability of the neuromuscular system, occurs more quickly and severely during heat treatment after cyfluthrin exposure, as cyfluthrin is an inhibitor of nerve cell sodium channels. Meanwhile, further research is required to reveal the mechanistic processes explaining the recovery patterns we observed here. For the insects exposed to extreme cold, the effect was only observable after exposure to a dose that is close to the one recommended for use of the insecticide in commercial products (20 mg cyfluthrin/ m²). A previous study did not find an effect of pesticide exposure on freeze tolerance in an earthworm [START_REF] Bindesbøl | Impacts of Heavy Metals, Polyaromatic Hydrocarbons, and Pesticides on Freeze Tolerance of the Earthworm Dendrobaena Octaedra[END_REF], which indicates that cold tolerance is not as sensitive to insecticide exposure as heat exposure.

Our results also fit well with effects of pesticide exposure on temperature tolerance found in other studies. For example, Op de Beeck, Verheyen, andStoks (2017, 2018) found that damselfly larvae that were sensitive to the pesticide chlorpyrifos also showed a reduction in heat tolerance after exposure to the pesticide. The authors also showed that the reduction in heat tolerance was smaller when the animals had been pre exposed to higher temperatures, a finding that is in line with our study. In addition, [START_REF] Delnat | Whether Warming Magnifies the Toxicity of a Pesticide Is Strongly Dependent on the Concentration and the Null Model[END_REF] found that pesticide exposure decreased temperature tolerance in mosquito larvae and males, and [START_REF] Meng | The Exposure Order Strongly Modifies How a Heat Spike Increases Pesticide Toxicity[END_REF] showed that pesticide J o u r n a l P r e -p r o o f exposure prior to experiencing a heat spike reduced heat tolerance of mosquito larvae. This reduction in temperature tolerance following pesticide exposure is also true for other taxa, as critical thermal maximum was also reduced in fishes after exposure to endosulfan and chlorpyrifos [START_REF] Heath | Heat and Cold Tolerance of the Fathead Minnow, Pimephales Promelas , Exposed to the Synthetic Pyrethroid Cyfluthrin[END_REF][START_REF] Patra | The Effects of Three Organic Chemicals on the Upper Thermal Tolerances of Four Freshwater Fishes[END_REF]).

The effect of fluctuating temperature regime was also observable in animals who had been exposed

to insecticide prior to extreme temperature treatment. It is however difficult to determine whether the observed increase in thermal tolerance after varying temperature regimes was caused by increased tolerance to extreme heat, to insecticide exposure or a combination of the two (Oliver and Brooke 2017). Further research into pesticide uptake and elimination in the animals in the course of this experimental setup would be of interest to further elucidate the processes behind the observed effect.

Conclusion

The present work reports that populations of the lesser mealworms have distinct abilities to recover from thermal and insecticide stress, and their interactions. This finding emphasises the importance of designing experiments including several populations when making assessments of the sensitivity of a given species to environmental factors, and more particularly when these data nourish models aiming at forecasting the future of biodiversity. No synergistic interaction was found when the lesser mealworms were jointly subjected to daily heat spikes and cyfluthrin; yet, this type of experimental procedure, which simulates the conditions that could be experienced by free-ranging organisms in natural and anthropogenic environments, increase the ecological realism of the study. This is even truer in a global warming context, as daily heat spikes are expected to become more frequent in several habitats. Here, we also found the importance of working with field-collected insects.

  resulting in a total of four different pesticide exposures at three different temperature regimes.

(

  [START_REF] Kassambara | Survminer: Drawing Survival Curves Using 'Ggplot2[END_REF][START_REF] Core | R: A Language and Environment for Statistical Computing[END_REF]. The probability of recovery at any given time point therefore represents the proportion of individuals that have recovered by that time. Pairwise comparisons using Log-Rank test with adjusted P-values[START_REF] Benjamini | Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing[END_REF] were performed post-hoc, with a significance value of 0.05. Generalized linear models (GLMs) with logit link function for proportions outcome (i.e. number of active versus impaired or immobile individuals per dish) were run on data reporting the state of animals after eight days of exposure to the different temperature regimes and pesticide doses. The effect of each variable (temperature, and

Figure 1 .

 1 Figure 1. Recovery probability of individuals from four different populations of A. diaperinus exposed at 42.5 °C for six different durations: A) 0.25 hour (overall P < 0.001); B) 0.5 hour (overall P < 0.001); C) 1 hour (overall P < 0.001); D) 2 hours (overall P < 0.001); E) 3 hours (overall P < 0.001); F)

Figure 2 .

 2 Figure 2. Recovery probability of individuals from four different populations of A. diaperinus exposed at -6 °C for six different durations: A) 0.25 hour (overall P < 0.001); B) 0.5 hour (P = 0.074); C) 1 hour (overall P < 0.01); D) 2 hours (overall P < 0.001); E) 3 hours (overall P < 0.001); F) 4 hours (overall P < 0.001). The red lines represent insects from FARM1, the black lines represent the insects from FARM2, the green lines represent the insects from FARM3, and the blue lines represent the insects from LAB. Different letters indicate differences among the recovery probability curves within each exposure duration.

Figure 3 .

 3 Figure 3. Status of adult A. diaperinus from populations A) FARM1, B) FARM2, C) FARM3 after they were exposed for eight days to control conditions (constant 26 °C), daily heat spikes (26 °C for 18h, 38 °C for 6h), cyfluthrin (20 mg/m 2 ) or daily heat spikes and cyfluthrin and D) LAB after eight days of same exposure treatments, but with a cyfluthrin concentration of 0.5 mg/m 2 . Different letters indicate differences among the number of active insects within each population.

Figure 4 .

 4 Figure 4. Recovery probability of A. diaperinus from FARM1 observed every 5 minutes after a 2-hour exposure at 42.5 °C (A, B and C) or -6 °C (D, E and F). Before the test, the insects were exposed to different temperature regimes: (A) and (D) constant 26 °C; (B) and (E) 26 °C daily interrupted by 6h at 32 °C; (C) and (F) 26 °C daily interrupted by 6h at 38 °C. For each temperature regime, the insects were exposed to four different concentrations of the insecticide cyfluthrin: 0 (control), 5 (black lines), 10 (red lines) or 20 (blue lines) mg cyfluthrin/m 2 . Stars (*) represent significant differences between the marked curve and the curve of the same colour drawn in Fig. 4A (constant temperature

  The thermal tolerance and sensitivity to insecticide of the laboratory population was very different than that of the field specimens, meaning that different conclusions would be drawn if working with the former population only. Finally, and in line with earlier investigations, our study pointed out the importance of considering the effects of multiple stressors, and the lasting effects they can have on other traits of the insects.Appendix table 2: Detailed statistical results of experiment 2 (corresponding to results presented in Figure3in the main text of the article). Overall ANOVA tests (type II) to the left, Sidak adjusted post hoc test to the right. All data made using 95% confidence level, significant P-values are presented in Appendix table 3: Detailed statistical result of experiment 3 (corresponding to results presented in Figure4in the main text of the article). Pairwise comparisons using Log-Rank test were performed using the Survdiff function in R. Significant p values are presented in bold.

	LAB	0.0004 20 < 2.0E-16 < 2.0E-16 5.9E-07 3.20E-14 < 2.00E-16	LAB	0.0720 20 5.2E-16	0.1430 1.2E-05 0.00038 0.2050
		FARM3		Chisq Df P-value	FARM3		LCL	UCL Group
	Duration Daily heat spikes Insecticide conc.	1 hour exposure 1.52 1 Pre-treatment peaks: 38 °C	0.218	Duration Daily heat spikes & Cyfluthrin 0.329 0.469 1 hour exposure Insecticide conc. Pre-treatment peaks: 38 °C	a
	Population FARM 3 FARM 2 Cyfluthrin treatment mg cyfluthrin/ m2 0	FARM 1 603.76 1 2.00E-16 5 10	Population FARM 3 Cyfluthrin treatment mg cyfluthrin/ m2 0	FARM 2 0.377 0.519 FARM 1 a 5 10
	FARM 2 Daily heat spikes & Cyfluthrin 0.0001 -5 0.17	-0.02 1 -	-	0.8857	FARM 2 Control	5	0.5049 -0.961 0.25 -	1	-	-	b
	FARM 1	4.90E-07 10 2.7E-05	0.9446 -1.3E-05		-		FARM 1 Daily heat spikes	10	0.8946 0.7	0.5049 -0.961 1 0.65	-	b
	LAB	0.3298 20 1.4E-14	2.00E-08 1.9E-15	2.70E-12 7.7E-05	LAB	0.0039 20 5.0E-07	0.0449 8.3E-08 1.3E-07 0.0080
		LAB			Chisq Df P-value	LAB		LCL	UCL Group
	Duration Daily heat spikes	2 hours exposure 9.53 1 0.002018	Duration Daily heat spikes & Cyfluthrin 0.084 0.193 2 hours exposure	a
	Population FARM 3 FARM 2 Cyfluthrin treatment	FARM 1 631.06 1 2.20E-16	Population FARM 3 Cyfluthrin treatment	FARM 2 0.177 0.315 FARM 1 b
	FARM 2 Daily heat spikes & Cyfluthrin 2.80E-06 -	-0.27 1 0.604759	FARM 2 Control		0.3069 -0.929 0.999 -	c
	FARM 1 LAB Duration Population FARM 3 FARM 2 2.30E-05 0.2720 -0.0190 1.00E-07 3 hours exposure FARM 1 1.70E-08 FARM 2 1.70E-08 --FARM 1 3.40E-10 0.6024 -LAB 0.0002 5.30E-14 1.50E-14 Duration 4 hours exposure Population FARM 3 FARM 2 FARM 1 FARM 2 0.0591 --FARM 1 5.00E-06 0.0591 -LAB 0.0121 0.0002 1.10E-08 J o u r n a l P r e -p r o o f FARM 1 1.80E-07 LAB < 2.00E-16 Duration 3 hours exposure 0.0042 -2.20E-11 Population FARM 3 FARM 2 FARM 1 1.60E-10 FARM 2 0.4633 --FARM 1 0.0019 0.0080 -LAB 2.00E-13 < 2.00E-16 < 2.00E-16 Duration 4 hours exposure Population FARM 3 FARM 2 FARM 1 FARM 2 0.4142 --FARM 1 0.0002 0.0137 -LAB < 2.00E-16 < 2.00E-16 < 2.00E-16 Daily heat spikes 0.929 0.999 c J o u r n a l P r e -p r o o f
	bold.
	FARM1 Extreme heat (42.5 °C) tolerance data Chisq Df P-value Daily heat spikes 0.6414 1 0.4232 Insecticide conc. Pre-treatment peaks: non Cyfluthrin treatment 16.6845 1 4.41E-05 mg cyfluthrin/ m2 0 5 10 Daily heat spikes & Cyfluthrin 1.1667 1 0.2801 5 1.60E-06 --10 < 2.00E-16 3.40E-09 -20 < 2.00E-16 < 2.00E-16 9.20E-14	FARM1 Extreme cold (-6 °C) tolerance data LCL UCL Group Daily heat spikes & Cyfluthrin 0.931 0.973 Insecticide conc. Pre-treatment peaks: non a Cyfluthrin treatment mg cyfluthrin/ m2 0 5 10 0.947 0.983 ab Control 0.968 0.994 5 0.4598 --b Daily heat spikes 0.973 0.996 10 0.0627 0.1072 -b 20 0.0064 0.0341 0.7462
	FARM2 Daily heat spikes Insecticide conc. Pre-treatment peaks: 32 °C Chisq Df P-value 13.7 1 0.000214 Cyfluthrin treatment 973.44 1 2.20E-16 mg cyfluthrin/ m2 0 5 10 Daily heat spikes & Cyfluthrin 0.08 1 0.775383 5 2.2E-06 --10 2.0E-14 9.4E-09 -	FARM2 Daily heat spikes & Cyfluthrin 0.484 0.567 LCL UCL Group Insecticide conc. Pre-treatment peaks: 32 °C a Cyfluthrin treatment 0.569 0.651 mg cyfluthrin/ m2 0 5 10 b Control 0.981 0.999 5 1.0E-05 --c Daily heat spikes 0 1 abc 10 3.6E-07 0.25036 -
												27

Acknowledgements

J o u r n a l P r e -p r o o f

We would like to thank Guillaume Weecxsteen, Héloïse Moullec and Baptiste Vasseur for their contribution in collecting data for this study. We also thank Maxime Dahirel, Mathias Holmstrup and Stine Slotsbo for interesting discussions on this work. This study was supported by France-Agri-Mer (Research grant 'Teneblimit'), and by the International Research Project (IRP) 'Phenomic responses of invertebrates to changing environments and multiple stress (PRICES) funded by InEE-CNRS.

from each other. As a result, variance in thermal tolerance among the insects from the three farm populations is not surprising [START_REF] Miller | Genetic Variability in Susceptibility and Response to Toxicants[END_REF].

Effect of daily heat spikes and cyfluthrin exposure

The previous experiment reported differences in the thermal tolerance among populations of A. diaperinus. In particular, insects from FARM1 and FARM2 exhibited slightly higher capacity to recover from heat exposure, suggesting that they would be better able to cope with daily heat spikes, while insects from the LAB population should have had the lowest ability to deal with peaks of heat. Yet, none of the populations were visibly affected by the daily heat spikes on their own. Insects from FARM1 were capable of coping with the investigated dose of cyfluthrin at constant temperature, while this condition was detrimental for the three other populations. As expected, adults of A. diaperinus from LAB were more sensitive to the insecticide than those of the three FARM populations. Differences in cyfluthrin resistance have already been reported in A. diaperinus populations from Australia [START_REF] Lambkin | Baseline Responses of Alphitobius Diaperinus (Coleoptera: Tenebrionidae) to Cyfluthrin and Detection of Strong Resistance in Field J o u r n a l P r e -p r o o f Populations in Eastern Australia[END_REF], where resistance was generally related to the extent and frequency of cyfluthrin applications experienced by the insects. Similarly, [START_REF] Chernaki-Leffer | Susceptibility of Alphitobius Diaperinus (Panzer) (Coleoptera, Tenebrionidae) to Cypermethrin, Dichlorvos and Triflumuron in Southern Brazil[END_REF] found strains of A. diaperinus of different pesticide (cypermethrin, dichlorvos and triflumuron) susceptibility in Brazil, and [START_REF] Hamm | Resistance to Cyfluthrin and Tetrachlorvinphos in the Lesser Mealworm, Alphitobius Diaperinus, Collected from the Eastern United States[END_REF] found strains of A. diaperinus with different pesticide (cyfluthrin and tetrachlorvinphos) resistance in the United States. The latter authors suggested that resistance alleles are present in the resistant populations of the lesser mealworm, as may have been the case with our poultry house collected insects.

While daily heat spikes alone did not affect the recovery of the insects of the four tested populations, the combination of insecticide and daily heat spikes significantly reduced the number of active individuals in two (FARM2 and LAB) out of the four assayed populations. Warm temperatures have been found to decrease the toxicity of the pyrethroids cypermethrin and deltamethrin in the fly Musca domestica [START_REF] Khan | The Effect of Temperature on the Toxicity of Insecticides against Musca Domestica L.: Implications for the Effective Management of Diarrhea[END_REF], and in the Spotted bollworms Earias vittella exposed to pyrethroids at higher temperature regimes [START_REF] Satpute | Temperature-Dependent Variation in Toxicity of Insecticides against Earias Vitella (Lepidoptera: Noctuidae)[END_REF]). Yet, these authors used constant temperatures in their experiments. The increased negative effects of the insecticide for adult A. diaperinus experiencing a daily heat spike of 6 hours is consistent with the findings of several other studies [START_REF] Delnat | Whether Warming Magnifies the Toxicity of a Pesticide Is Strongly Dependent on the Concentration and the Null Model[END_REF][START_REF] Willming | Effects of Environmentally Realistic Daily Temperature Variation on Pesticide Toxicity to Aquatic Invertebrates[END_REF][START_REF] Zhu | Toxicity of Biocide GCSC-BtA on Arthropod Pests under Different Temperature Conditions[END_REF], who found increasing pesticide toxicity in insects subjected to daily bouts of high

J o u r n a l P r e -p r o o f

Supplementary File 1

To begin this study, we had to test the effects of different temperatures on the recovery capacity of adult Alphitobius diaperinus, that is, the effects of exposure to extreme temperatures (cold, heat) on the recovery ability and duration of the individuals.

Insects from the population FARM1 were used to describe the general temperature tolerance of the species, and to find temperature exposures that would cause the adults to enter chill coma, yet keeping the ability to recover (capacity to regain full movement) afterwards. Four to eight replicates of 10 individuals in each container (test tube with rubber cap) were created for each exposure temperature, starting at -4 °C for cold exposure, and at +42 °C for heat. At the end of the thermal treatment, the insects were directly transferred to Petri dishes and observed every 5 minutes for 2h.

Each observation period lasted for up to 40 seconds, and individuals were registered as either "Immobile, Impaired" or "Recovered". Animals were characterized as "Recovered" when they had regained the ability to move normally. The temperature both in the cryothermostat and the room where recovery observations took place was monitored using iButtons® (iButtonLink, LLC., Whitewater, USA).

J o u r n a l P r e -p r o o f

Since thermal performance curves are often steep at high temperature extremes, and change less quickly at cold temperature extremes [START_REF] J O U R N A L P R E -P R O O F Huey | Intergrating Thermal Physiology and Ecology of Ecotherms: A Discussion of Approaches[END_REF]Sinclair et al. 2016), we used small increments of temperature in the heat exposure experiments (0.5 °C) and larger increments (2 °C) for cold exposure. When having run three temperatures for a duration of 2 hours, we were able to see the temperatures that would best describe the effects of different durations of exposure on the thermal tolerance of adult A. diaperinus from different populations. These experimental conditions were also used for comparing the effects of different daily heat spikes and insecticide exposures prior to extreme temperature exposure.

In Appendix Figure 1, we see that the insects recovered very quickly from the least extreme investigated temperatures; exposure to +42 °C did not even elicit heat coma from which the animals could recover. Furthermore, the animals showed a low ability to recover from the most extreme temperatures used: after an exposure to +43 °C, many insects were not able to recover within the recovery period (Appendix Figure 1 From these results, it can be concluded that adults of A. diaperinus have an extreme temperature tolerance that is within the range that commonly characterizes insects (Addo-Bediako, Chown, and

J o u r n a l P r e -p r o o f

Gaston 2000). We also see that the changes in recovery time with changing temperature follows a pattern typical from thermal performance curves [START_REF] Colinet | Insects in Fluctuating Thermal Environments[END_REF][START_REF] J O U R N A L P R E -P R O O F Huey | Intergrating Thermal Physiology and Ecology of Ecotherms: A Discussion of Approaches[END_REF].

Specifically, great changes in thermal tolerance happens across a narrower temperature range at extreme heat (here between 42 and 43 °C) compared to extreme cold (here -4 to -8 °C). We also note that adults of A. diaperinus were stressed (went into chill coma), but all individuals recovered from sudden exposure at 42.5 °C and -6 °C. Therefore, these conditions represent suitable temperatures for comparing differences in recovery time among populations or treatment groups.

Supplementary File 2

Appendix figure 1 J o u r n a l P r e -p r o o f

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:


In memoriam, Professor Patrice FRANCOUR (April 13, 1960-October 13, 2019) It is with shock and great sadness that we heard of the sudden passing of our distinguished colleague and friend, Professor Patrice Francour, at the age of 59, from pancreatic cancer against which he courageously fought till the end. During his whole life, he worked to protect the seas and the oceans, especially the Mediterranean Sea, which was so dear to him. As a specialist in fish communities in rocky areas and seagrass beds (Posidonia oceanica), his work mainly took place in marine protected areas (especially Port-Cros National Park, Scandola Marine Reserve, Cap Roux-Saint Raphael Fishery Reserve). His research in fundamental marine ecology was chiefly aimed at understanding the structure and dynamics of fish assemblages, the role of biodiversity in ecosystem functioning and the importance of habitat structure. He also contributed to numerous innovative approaches in applied marine ecology research. He significantly contributed to improving the available knowledge on the effects of protection measures on fish communities, the role of management in Mediterranean marine protected areas, the sustainable exploitation of coastal marine resources by small-scale fisheries and the use of artificial reefs for the restoration of local fishing resources.

Born in

On the strength of his expertise and competences, he was involved in the scientific committees of several marine parks. As a participant and executive board member of the GEM grouper research association (Groupe d'Etude du Mérou), Patrice Francour had a passionate interest in the grouper, these large peaceful fishes, curious and attracted by divers, but severely impacted by humans, quite often locally extirpated because of the impact of recreational and professional fishers. With other scientists, he was committed to the preservation of the emblematic and overexploited dusky grouper Epinephelus marginatus along the French coasts. As a fish expert, he regularly published new or updated records of fish species reported from France or the Mediterranean basin (Gobiidae, Kyphosidae, Siganidae, Fistulariidae, Oplegnathidae). One part of this activity was devoted to the spread of non-indigenous species of fish in the North-Western Mediterranean. He made a major contribution to the development of expertise to assist marine protected area managers and staff members in assessing the effects of multiple human activities (fishing, anchoring, etc.). Recently, he was also involved in citizen science to encourage members of the public to participate in field monitoring of fish populations.

During his scientific career, Patrice Francour authored or co-authored more than 90 publications and 9 book chapters on echinoderms, fishes, seagrasses, artificial reefs and fish invasions in the Mediterranean Sea. He supervised more than fifteen PhD students and a large number of master's degree students. In addition to his teaching activities in biology and ecology, he also undertook a wide range of scientific expert reports for various universities and scientific agencies in France and abroad. In particular, he was since 2006 an active and highly appreciated member of the IUCN Grouper and Wrasse Specialist Group, then too since 2012 of the Snapper, Seabream and Grunt Specialist Group. He was working recently with IUCN colleagues on the preparation of fish species assessments for the IUCN Red List of threatened marine fishes, on both grouper and wrasse and certain other specific fish family groups, and on Mediterranean and Eastern Atlantic marine fishes in general.

From New Zealand to the Mediterranean Sea, Patrice especially enjoyed field work, diving with colleagues, observing fishes in nature, studying fish communities, tracking non-indigenous species…while passing pleasant moments in good company. He especially liked challenges.

One event in particular in Patrice's life deserves to be highlighted. In 1988, Patrice Francour narrowly escaped death during a field-trip devoted to the conservation of the monk seal in

  Paris in 1960, Patrice Francour studied marine biology and ecology at Paris VI University. For his master degree internship (DEA), he went to Marseille in 1984 to study the fauna of the Posidonia oceanica meadows with Prof. C.F. Boudouresque's team. A few years later (1990), he completed his PhD at the Banyuls-sur-Mer Marine Station, under the dual auspices of Paris VI University and Aix-Marseille II University. His PhD was centered on the study of the dynamics of the Posidonia oceanica ecosystem in the Port-Cros National Park. He focused his research on quantifying the biomass variations of the different compartments of this ecosystem in order to develop the modelling of its trophic functioning. Subsequently (1994-1995), he worked as a postdoctoral researcher at the Portobello Marine Laboratory, in New Zealand, collaborating with Prof. E. Harvey's Lab. Patrice Francour worked as an active and much appreciated member at the GIS Posidonie in Marseille, until he was first recruited as an Assistant Professor at the University of Nice-Sophia Antipolis in 1998 in the Coastal Marine Environment Laboratory, headed by Prof. A. Meinesz. In addition to teaching ecology at the University, he was also at that time the director of the Natural Sciences Department, Nice IUFM, as he was deeply committed to the training of natural sciences secondary school teachers. After his HDR (habilitation to supervise research) in 1999, he was nominated Full Professor at the University of Nice-Sophia Antipolis in 2002. From 2008 to 2015, he was the Director of the ECOMERS Laboratory (a lab specialized in Marine Ecology and Ecotoxicology), then Deputy Director since 2016. He was one of the contributors to the development of this laboratory, now renamed as UMR ECOSEAS (Ecology and COnservation Science for sustainable sEAS), specialized in Marine Socio-Ecology.

Figure 1 .Figure 3 .

 13 Figure 1. Patrice Francour. AG GEM 2010. © J.G. Harmelin

great courage in recovering from his injuries, but remained profoundly affected by this tragedy.

If in that tragic event death let him escape, this has unfortunately not been the case with his recent illness. The void he has left to family, friends and colleagues will remain unbridgeable.

But above all, we wish to remember him as he was. Patrice Francour was a researcher motivated by a deeply passionate interest in marine life, he was an excellent underwater diver and photographer, sometimes tempted to dive a little too deep for his diving buddies, attracted by some small cryptic goby, one of his last passions! He dived underwater intensively throughout his life, and again plunged below the surface of the sea for the last time in July 2019, just before the worsening of his illness and his last journey.

Colleagues and friends, researchers and divers, students and collaborators will greatly miss him, but we know for certain that anyone who knew him will keep alive the memory of the good times spent together. Our deepest thoughts and sympathy are with his family.

Vale, ciao, salut Patrice and rest in peace in the divers' paradise with your beloved groupers… Mireille Harmelin-Vivien, Paolo Guidetti and Virginie Raybaud