Joffrey Moiroux
email: joffrey.moiroux@gmail.com

Quentin Chesnais

Fabien Spicher

Elise Verrier

Arnaud Ameline

Aude Couty

Moiroux Joffrey

Plant virus infection influences bottom-up regulation of a plant-aphid-parasitoid system

Keywords: Insect physiology, Parasitoid behaviour, Plant physiology, Pathogen, Pest control, Tri-trophic system

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INTRODUCTION

Plant viruses can have various negative effects on host physiology, including an accelerated demise of the photosynthetic apparatus [START_REF] Balachandran | Concepts of plant biotic stress. Some insights into the stress physiology of virusinfected plants, from the perspective of photosynthesis[END_REF]) and the accumulation of nitrogen compounds or expanded oxidase activities [START_REF] Culver | Virus-induced disease: altering host physiology one interaction at a time[END_REF]. As a result, plant viruses are a major cause of crop yield losses throughout the world [START_REF] Strange | Plant disease: a threat to global food security[END_REF]. More than 55% of the >700 known phytoviruses [START_REF] Nault | Arthropod transmission of plant viruses: a new synthesis[END_REF]) are transmitted by hemipteran vectors, 30% of which are aphids [START_REF] Brault | Aphids as transport devices for plant viruses[END_REF]. Because these associations generally result from a tight coevolution (Mauck et al. 2014a), viruses can alter plant-vector interactions to increase their own dissemination, through visual and chemical modifications of infected hosts phenotypes (for a review, see [START_REF] Blanc | Manipulation of hosts and vectors by plant viruses and impact of the environment[END_REF]. These effects have received special attention due to the fact that many insect vectors are important pests of agricultural production. For example, [START_REF] Eigenbrode | Volatiles from potato plants infected with potato leafroll virus attract and arrest the virus vector, Myzus persicae (Homoptera: Aphididae)[END_REF] observed changes in volatile chemical emissions of potatoes infected by a persistently transmitted virus, the Potato leafroll virus PLRV (Polerovirus), which resulted in preferential colonisation of the virus-infected plantlets by the principal vector, the aphid Myzus persicae. This virus, as well as the majority of viruses transmitted in a persistent manner (for reviews, see [START_REF] Fereres | Behavioural aspects influencing plant virus transmission by homopteran insects[END_REF][START_REF] Mackauer | Parasites and parasite impact on aphid populations, in Plant virus epidemics: monitoring, modelling and predicting outbreaks[END_REF][START_REF] Blanc | Manipulation of hosts and vectors by plant viruses and impact of the environment[END_REF], are known to increase the quality of host plants for their vectors, conferring increased fecundity and/or shorter development time [START_REF] Castle | Rates of growth and increase of Myzus persicae on virus-infected potatoes according to type of virus-vector relationship[END_REF], increasing their population growth and the virus' dispersal rate. Such effects may also result from interaction between virus and vector phenotype.

Studies on tri-trophic interactions involving plant viruses, aphid vectors and natural enemies have mostly focused on top-down effects. Originally, aphid parasitoids were thought to limit dissemination of viruses by limiting aphid populations [START_REF] Mackauer | Parasites and parasite impact on aphid populations, in Plant virus epidemics: monitoring, modelling and predicting outbreaks[END_REF], but there has been increasing evidence that the presence of a parasitoid in the environment or in host body improves virus dispersal by increasing aphids' movements [START_REF] Weber | Effects of parasitism by Lysiphlebus testaceipes (Hymenoptera: Aphidiidae) on transmission of Beet yellows closterovirus by bean aphid (Homoptera: Aphididae)[END_REF][START_REF] Hodge | Do plant viruses facilitate their aphid vectors by inducing symptoms that alter behavior and performance?[END_REF][START_REF] Hodge | Parasitoids aid dispersal of a nonpersistently transmitted plant virus by disturbing the aphid vector[END_REF].

Studies of the effects of plant viruses on bottom-up regulation have mainly been restricted to plantaphid systems and little attention has been paid to tri-trophic systems including parasitoids. Viruses may change the direct effects of plants on parasitoid behaviour, but differences in parasitoid attraction towards odours from non-infected and virus-infected plants have not been detected in previous studies [START_REF] Mauck | Infection of host plants by Cucumber mosaic virus increases the susceptibility of Myzus persicae aphids to the parasitoid Aphidius colemani[END_REF]. Indirect effects of infected plants mediated by the aphid host on parasitoid life history traits may also occur, but such effects have rarely been investigated, except for emergence rate and development time. [START_REF] Mauck | Infection of host plants by Cucumber mosaic virus increases the susceptibility of Myzus persicae aphids to the parasitoid Aphidius colemani[END_REF] and [START_REF] Christiansen-Weniger | Plant virus and parasitoid interactions in a shared insect vector/host[END_REF] observed, respectively, an increased and a decreased emergence rate of parasitoids developing in viruliferous aphids. De [START_REF] De Oliveira | A negative effect of a pathogen on its vector? A plant pathogen increases the vulnerability of its vector to attack by natural enemies[END_REF], however, did not detect any effects. [START_REF] Christiansen-Weniger | Plant virus and parasitoid interactions in a shared insect vector/host[END_REF] also observed that parasitoid development was delayed when Aphidius ervi developed in Sitobion avenae infected by a luterovirus from the Barley yellow dwarf virus BYDV/ Cereal yellow dwarf virus CYDV pathosystem (BYDV-MAV). Such host-mediated effects on parasitoid fitness may modulate behavioural decisions made by females facing virusinfected versus uninfected aphids. De [START_REF] De Oliveira | A negative effect of a pathogen on its vector? A plant pathogen increases the vulnerability of its vector to attack by natural enemies[END_REF] observed that aphids infected by a polerovirus from the same pathosystem (CYDV-RPD) were more frequently attacked by Aphidius colemani females than uninfected aphids, although they were not emitting odors attractive to parasitoid females. It was proposed that indirect effects of the virus altered aphid physiology and/or behaviour in a way that made them more acceptable or suitable for parasitoids.

In the present study, we investigated the bottom-up effects of a plant virus on a plant-aphidparasitoid tri-trophic system. We measured parameters linked to plant physiology (photosynthesis, stomatal conductance, chlorophyll content index, biomass), aphid performance and fitness (reproductive and demographic parameters, body size and stored energetic resources) and parasitoid fitness-related traits (emergence rate, tibia size, fecundity and stored energetic resources). We also measured the effects of plant and aphid infection by the virus on parasitoid host selection behaviour (long-distance attraction, attack rate). We focused on a persistent, circulative and non-propagative Polerovirus, the Turnip yellows virus (TuYV), also known as the Beet western yellow virus (BWYV). This virus, which has been reported in Europe, USA and Australia [START_REF] Stevens | Turnip yellows virus (syn Beet western yellows virus): an emerging threat to European oilseed rape production[END_REF], can infect plant species from at least 13 families. These include many species of agronomic importance, such as oilseed rape or lettuce [START_REF] Walkey | Studies on resistance to Beet western yellows virus in lettuce (Lactuca sativa) and the occurrence of field sources of the virus[END_REF].

TuYV is probably the most important viral disease of cultivated Brassicaceae in several countries, including United Kingdom [START_REF] Stevens | Turnip yellows virus (syn Beet western yellows virus): an emerging threat to European oilseed rape production[END_REF]). It may be responsible for yield losses of up to 46% in Brassica napus [START_REF] Jones | Yield-limiting potential of Beet western yellows virus in Brassica napus[END_REF]). We focused on Camelina sativa, an historically important oilseed crop in temperate Europe until the nineteenth century. This Brassicaceae has recently been re-introduced because its oil is promising as a biofuel and functional food [START_REF] Faure | Camelina, a Swiss knife for plant lipid biotechnology[END_REF]. Many common aphid species are able to transmit the TuYV from plant to plant. We studied its main vector and a major pest, the generalist green peach aphid, Myzus persicae [START_REF] Stevens | Detection of the luteoviruses, Beet mild yellowing virus and Beet western yellows virus, in aphids caught in sugar-beet and oilseed rape crops, 1990-1993[END_REF], for which experimental transmission rates of over 90% have been reported [START_REF] Schliephake | Investigations on the vector transmission of the Beet mild yellowing virus (BMYV) and the Turnip yellows virus (TuYV)[END_REF]. Of the various natural enemies used to limit Myzus persicae populations, we used Aphidius colemani in this study, because it is a cosmopolitan parasitoid commercially available as a biological control agent [START_REF] Boivin | Aphid parasitoids in biological control[END_REF].

We thus investigated the influence of TuYV on its host Camelina sativa, its vector Myzus persicae and the parasitoid Aphidius colemani. Based on previous studies, we expected to observe in presence of TuYV: (i) a decrease in physiological traits for plants, and (ii) an increase in reproductive and demographic parameters for aphids. Such effect may be paid off by (iii) decreased body size and quantity of energetic resources for vectors, which would result in (iv) decreased body size and fecundity for parasitoids; although no study to date investigated such bottom-up regulation. Contradictory observations on influence of plant virus on parasitoid behaviour [START_REF] De Oliveira | A negative effect of a pathogen on its vector? A plant pathogen increases the vulnerability of its vector to attack by natural enemies[END_REF][START_REF] Mauck | Infection of host plants by Cucumber mosaic virus increases the susceptibility of Myzus persicae aphids to the parasitoid Aphidius colemani[END_REF] did not allow us to make any prediction for this experiment.

To our knowledge, this is the first study of its kind to integrate physiological approaches on all three trophic levels, as well as a behavioural approach for the third trophic level.

II-MATERIALS AND METHODS

1-Plants, insects and virus

Seeds of camelina Camelina sativa cv. "Celine" (Brassicales: Brassicaceae) were provided by an agronomical technical institute (Terres Inovia, Thiverval-Grignon, France). Plants were cultivated in plastic pots (90×90×90mm) containing commercial sterilised potting soil in a growth chamber under 20 ± 1°C, 60 ± 5% relative humidity (RH), and 16L:8D photoperiod at 2.5 klx.

The green peach aphid Myzus persicae Sulzer (Hemiptera: Aphididae) colony was established from one parthenogenetic female collected in 1999 in a potato field in the vicinity of Loos-en-Gohelle (France). Aphid clones were reared on rapeseed Brassica napus cv. "Adriana" (Brassicales: Brassicaceae). Pots (90×90×90mm) containing 3-4 rapeseed plants each were placed in ventilated plastic cages (360x240x110mm) and maintained in a climate chamber at 20 ± 1°C, 60 ± 5% relative humidity (RH), and 16L:8D photoperiod at 2.5 klux. To obtain second instar aphids used in the experiments with parasitoids, we transferred adult aphids from the colony to a Petri dish (Ø90mm) containing a control C. sativa leaf set in 1.5 % agar. Then, their progeny was removed twice per day, transferred and maintained in a Petri dish (Ø90mm) containing a control C. sativa leaf until being placed on control or virus infected C. sativa plantlets in ventilated cages for experiments, which were conducted under similar conditions to those used in colony maintenance.

Our Aphidius colemani Viereck (Hymenoptera: Aphidiinae) colony was established from mummies purchased from Viridaxis S.A. (Gosselies, Belgium). These mummies were isolated in plastic tubes (L=75mm, Ø10mm) placed in a climate chamber at 20 ± 1°C, 60 ± 5% relative humidity (RH), and 16L:8D photoperiod at 2.5 klux. Upon emergence, parasitoids were sexed and fed ad libitum with a water-honey solution (50% vol/vol) for 48h. After that time, each female was allowed to mate with two males for 4h in a Petri dish (Ø45mm). These mated F0 parasitoid females were then used to produce F1 parasitoid females that allowed us to investigate the plant-mediated and aphid-mediated effects of TuYV on parasitoids' life history traits and behaviour.

The Turnip yellows virus (TuYV, Luteoviridae) [START_REF] Leiser | Agroinfection as an alternative to insects for infecting plants with beet western yellows luteovirus[END_REF]) used in our experiment was provided by Véronique Ziegler-Graff at IBMP-CNRS (Strasbourg, France) and maintained in Montia perfoliata (Caryophylalles: Portulaceae). Camelina sativa plants were inoculated with TuYV by placing five viruliferous aphids on a single 7 day-old camelina plant for a three days' inoculation period. Viruliferous aphids were obtained by placing them for 24 hours on infected M. perfoliata. Sham-inoculated plants (i.e. control plants) were treated in the same manner using control aphids (i.e. non-viruliferous aphids), and were therefore not infected with the virus. For all of bioassays described below, plants were used three weeks after virus inoculation or sham inoculation The TuYV infection status of plants was confirmed visually (smaller plants, reddening/yellowing leaf margins, interveinal discoloration) and using double antibody sandwich enzyme-linked immunosorbent assay using polyclonal antibodies produced by LOEWE [START_REF] Clark | Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses[END_REF].

2-Effects of TuYV on plants

Plant physiology. Physiological traits of ten control and TuYV-infected plants were measured on the third fully expanded leaf from bottom of a same plant, at 21 and 29 days after inoculation to cover the duration of the presence of aphids on the plant. Net assimilation rate of CO2 (An) and stomatal conductance (gs) to water were assessed using the 2 cm² leaf chamber LCF LI6400-40 linked to the portable photosynthesis system LI-6400 (LICOR Biosciences, Lincoln, Nebraska, USA). Before each measurement, leaves were acclimated during 15-20 minutes at 250 µmol m-2 s-1 of photosynthetically active photons flux density (PPFD). Both parameters were measured in the same environmental conditions: 400 µmol mol-1 of CO2, leaf temperature at 25°C and PPFD at saturating light considering previous measurements (data not shown), i.e. 800 µmol m-2 s-1. The chlorophyll content index (CCI) was measured with CCM200 (Opti-Sciences, Tyngsboro, Massachusetts, USA) in growth chamber conditions.

Biomass. Twenty control and TuYV-infected plants were harvested at soil level 21 and 29 days after inoculation, and their fresh above-ground biomass was measured.

3-Plant-mediated effects of TuYV on aphid life history traits

Aphid performance. Pools of synchronised first instar (<24h) aphids were obtained from parthenogenetic adult females placed on Brassica napus leaves set in 1.5 % agar in Petri dishes (Ø90 mm). To measure nymph survival rate, twenty groups of five first instar aphids per treatment were transferred onto control or virus-infected C. sativa plantlets. These groups of aphids were enclosed in clip cages on leaves at mid-height of each plantlet and their survival was recorded every day until they reached the adult stage. Thirty individuals per treatment were placed individually in a clip-cage on control or virus-infected plantlets to measure adult aphid performance. Their survival was checked daily and newly larviposited aphids were counted and removed with a brush. This procedure allowed us to estimate the duration of the pre-reproductive period and to measure the fecundity of each adult aphid, which was assessed daily for a time equivalent to the duration of the pre-reproductive period [START_REF] Hackett | Unpredicted impacts of insect endosymbionts on interactions between soil organisms, plants and aphids[END_REF][START_REF] Pointeau | Characterization of antibiosis and antixenosis to the woolly poplar aphid (Hemiptera: Aphididae) in the bark of different poplar genotypes[END_REF]. The daily fecundity and the intrinsic rate of natural increase (rm) were then calculated using the DEMP 1.5.2 Software (Giordanengo, 2014), which uses Jackknife technique. The intrinsic rate of natural increase (rm) was calculated as , where x is the age, lx the age-specific survival, and mx the mean number of female offspring produced in a unit of time by a female (Birch, 1948).

Aphid body size. In parallel, aphids were reared on control or virus-infected plants and twenty 2day and 8-day old apterae aphids per treatment were sampled, frozen and photographed under a stereomicroscope (LEICA M165C). Their body size was then measured from the tip of the head to the base of the cauda using ImageJ analysis software [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF]. Aphids were measured in the beginning and in the end of their larval development to estimate influence of virus infection on aphid growth and resource quantity available for parasitoids.

Aphid lipid quantity and content. The quantity of lipid stores of thirty apterae aphids per treatment was measured using a chloroform-methanol extraction, as described in [START_REF] Moiroux | Local adaptations of life-history traits of a Drosophila parasitoid, Leptopilina boulardi: does climate drive evolution?[END_REF]. Briefly, thirty 8-days old aphids previously placed at -80°C were dried at 40°C for three days in an air oven and weighed with a Metler Toledo M3 microbalance (Max= 3g; Low=1µg; T=-3G; [dd]=1µg). These individuals were then placed for two weeks in a Chloroform-Methanol solution (2:1) and dried again at 40°C for one day before being weighed. Lipid quantity and lipid content were respectively calculated as the difference between dry mass after and before lipid extraction and as the ratio between lipid quantity and lean dry mass.

Aphid sugar quantity and content. Sugar content was measured on thirty 8-day old apterae aphids per treatment using the colorimetric analysis developed by [START_REF] Giron | The physiology of host feeding in parasitic wasps: implications for survival[END_REF]. Aphids were individually placed in Eppendorf tubes with 40 µL of Ringer solution and crushed with a plastic pestle in 300 µL of methanol. After centrifugation for 15 minutes at 4°C and 1400 rpm, we added 150 µL of chloroform and 60 µL of 2% sodium sulphate solution to the tubes which were vortexed and stored at 4°C for one night. The next day, tubes were centrifuged again for 15 minutes at 1400 rpm. We then transferred 150 µL of the supernatant to new tubes which were heated at 90°C on aluminium block and removed before complete evaporation. We then added 1 ml of anthrone reagent in tubes, heated them for 15 min at 90°C, and cooled them on ice for 5 min. Absorbance was read at 620 nm with a spectrophotometer. We used glucose to establish the calibration curve for sugars. Attack rate. In a no-choice experiment, the attack rate of twenty A. colemani on non-viruliferous or viruliferous aphids was measured by placing a standardised F1 parasitoid female in a Petri dish (Ø90 mm) pierced on its side to allow introduction of a fresh C. sativa leaf on which twenty synchronised second instar control or viruliferous aphids were placed. Parasitoids were continuously observed until they left the leaf for more than one minute or when they did not move for more than five minutes. The number of attacks on aphids and the number of host rejections were recorded using the behavioural coding software CowLog 2.0 [START_REF] Hänninen | CowLog: Open source software for coding behaviors from digital video[END_REF]).

4-Plant

An attack was noted when a parasitoid female bent its abdomen and touched the aphid with its ovipositor. Host rejection was recorded when antennal contact with an aphid was not followed by an attack. After observation, aphids were dissected to detect parasitoid eggs (ensure that an attack resulted in an oviposition), and a mean true oviposition rate was calculated.

The same procedure was used to measure attack and rejection rate of twenty A. colemani in a dual choice experiment. Parasitoid females were offered simultaneously ten non-viruliferous and ten viruliferous aphids placed on a C. sativa leaf. A drop of blue or red painting (Posca®) was deposited on aphids to distinguish between control and viruliferous hosts, and colour was switched every day between treatments.

6-Statistical analyses

Plants. Photosynthetic activity, stomatal conductance and chlorophyll content index measured at 21 and 29 days post-inoculation were compared between control and TuYV-infected plants using linear mixed-effect models with treatment and time as factors and individuals as the random variable. Because of the normal distribution and homoscedasticity of the data, above-ground biomass was compared between treatments using distinct ANOVA for each age since this trait was measured on different individuals.

Aphids. Influence of plant status on aphid survival rate was tested with a Cox proportional hazards model. Body length was compared between treatments using distinct ANOVA for each age since this trait was measured on different individuals. As data were normally distributed and met the assumption of homoscedasticity, other aphid parameters were analysed using ANOVA except for differences in lipid content which were tested using an ANCOVA with lipid quantity as variable and lean dry mass as a covariate.

Parasitoids. Differences between treatments in tibia length and development time were assessed using an ANOVA as data were normally distributed and met the assumption of homoscedasticity.

We tested differences in wing area using an ANCOVA with wing area as variable and tibia length as a covariate. Initial egg load was compared between treatments using a GLM, with treatment as fixed factor and tibia length as a covariate, and a Poisson error distribution. Differences in lipid content between treatments were tested using an ANCOVA with lipid quantity as variable and lean dry mass as a covariate.

Attack rates, true oviposition rates (i.e. attacks which resulted in egg deposition), host rejection rates and emergence rates were compared between treatments using a GLM, with treatment as fixed factor and a binomial error distribution. Preference for control or TuYV-infected plants, without or with aphids, was tested using a chi-square test.

All statistical analyses were carried out using the statistical program 'R' (R 3.2.2 -R Development Core Team 2015; Downloadable from: https://cran.r-project.org/mirrors.html; nlme package loaded for linear mixed-effect models). Statistical significance was considered when p-value was inferior to 0.05.

III-RESULTS

1-Effects of TuYV on plants.

Virus-infected plants had a reduced photosynthetic activity (An) (LME, treatment effect: F1,17 = 22.11, p < 0.001) and a reduced stomatal conductance (gs) (LME, treatment effect: F1,17 = 76.51, p < 0.001) compared to control plants (Table 1). These variables both decreased with time (LME, An: F1,17 = 19.79, p < 0.001; gs: F1,17 = 25.51, p < 0.001) but there was no interaction between treatment and time (LME, An: F1,17 = 2.24, p = 0.144; gs: F1,17 = 1.25, p = 0.271). Virus infection had no significant effect on chlorophyll content index (LME, treatment effect: F1,17 = 3.598, p = 0.074), but we observed a significant effect of time (LME, F1,17 = 32.30, p < 0.001) and of the interaction between treatment and time (LME, F1,17 = 40.25, p < 0.001). These effects were explained by the strong decrease in CCI which occurred in virus infected plants, while it remained constant in control plants.

We also observed a smaller above-ground biomass in TuYV-infected plants than in control plants 21 days (ANOVA, F1,38 = 170.70, p < 0.001) and 29 days (ANOVA, F1,38 = 24.06, p < 0.001) after inoculation (Table 1).

2-Plant-mediated effects of TuYV on aphids' life history traits.

Myzus persicae nymphs reared on TuYV-infected plants had a significantly lower survival rate than nymphs reared on control plants (Cox model, chisq = 10.41, p = 0.001). They were also significantly smaller at 2 days (ANOVA, F1,38 = 4.114, p = 0.044) and 8 days (ANOVA, F1,38 = 6.217, p = 0.027), reproduced earlier (ANOVA, F1,58 = 19.84, p < 0.001), but we did not observe any significant difference between treatments for daily fecundity (ANOVA, F1,58 = 2.087, p = 0.154) (Table 2). Aphid populations consequently had a higher intrinsic rate of natural increase (t = 4.125, df = 48.902, p = 0.0001) (Table 2).

Aphids reared on TuYV-infected plants had less lipids (ANOVA, F1,58 = 70.5, p < 0.001) and less carbohydrates (ANOVA, F1,58 = 28.72, p < 0.001) than aphid adults reared on control plants, mainly because of their smaller size. We also observed a lower lipid content in adult aphids reared on

TuYV-infected plants (ANCOVA, F1,57 = 30.37, p < 0.001) (Figure 1A) but a similar sugar concentration between aphids from the two treatments (ANOVA, F1,57 = 1.83, p = 0.181).

4-Plant and aphid-mediated effects of TuYV on parasitoids' life history traits.

Parasitoid females that developed in viruliferous aphids were significantly smaller (ANOVA, F1,48 = 6.171, p = 0.017) (Table 3), and emerged with fewer eggs (GLM, z = -5.08, p < 0.001, Figure 2) and smaller lipid stores (ANCOVA, F1,37 = 28.43, p < 0.001, Figure 1B) compared to females developing in non-viruliferous aphids. We did not observe any significant difference in development time (ANOVA, F1,164 = 1.914, p = 0.651), wing area (ANCOVA, F1,47 = 2.568, p = 0.212) or emergence rate between treatments (GLM, z = -0.954, p = 0.565) (Table 4).

3-Plant-mediated and aphid-mediated effects of TuYV on parasitoids' behaviour.

There was no significant difference in parasitoid preference for control versus TuYV-infected plants without (χ² = 1.460, p = 0.377) or with aphids (χ² = 1.129, p = 0.508) (Figure 3). Moreover, attack rates were similar between non-viruliferous and viruliferous aphids when they were offered separately (GLM, z = 1.474, p = 0.561) or in a dual choice experiment (GLM, z = 1.866, p = 0.427).

The rates of true oviposition and of host rejection were also similar between treatments when nonviruliferous and viruliferous aphids were offered separately (True oviposition: GLM, z = -1.097, p = 0.509; Host rejection: GLM, z = 1.902, p = 0.484) or in a dual choice experiment (True oviposition: GLM, z = 2.544, p = 0.371; Host rejection: GLM, z = -1.142, p = 0.453).

20

DISCUSSION

In this study, the bottom-up regulation of a plant-aphid-parasitoid tri-trophic system by virus infection was investigated. Based on previous studies, we expected to observe in presence of a persistently transmitted virus: (i) a decrease in physiological traits for plants, and (ii) an increase in reproductive and demographic parameters for aphids, which may be paid off by (iii) decreased body size and quantity of energetic resources. Such effect would result in (iv) decreased body size, fecundity and lipid content for parasitoids. Our results mainly supported our predictions. The plant virus TuYV negatively affected Camelina sativa physiology, had positive effects on demographic parameters of Myzus persicae but negative effects on its body size and quantity of energetic resources, and negative effects on parasitoid fitness. Virus infection did not modify parasitoid host selection behaviour.

Plant physiology.

We observed that virus infection resulted in reduced photosynthetic activity in C. sativa, as described for many other plant species infected by viruses (e.g. [START_REF] Chia | Photosynthetic capacity in Oncidium (Orchidaceae) plants after virus eradication[END_REF]Swiech et al. 2001;[START_REF] Sampol | Analysis of the virus-induced inhibition of photosynthesis in malmsey grapevines[END_REF]. Several mechanisms have been proposed to explain the negative effect of virus infection on photosynthesis rate (for a review, see Balachadran et al. 1997), such as stomatal closure [START_REF] Guo | Photosynthetic rate and chlorophyll fluorescence in leaves of stem mustard (Brassica juncea var. tsatsai) after turnip mosaic virus infection[END_REF] or reduced chlorophyll content [START_REF] Funayama-Noguchi | Ecophysiology of virus-infected plants: a case study of Eupatorium makinoi infected by geminivirus[END_REF]. In our study, we observed a decrease in stomatal conductance that may partly explain the decreased photosynthetic activity in TuYV infected plants, as proposed by [START_REF] Guo | Photosynthetic rate and chlorophyll fluorescence in leaves of stem mustard (Brassica juncea var. tsatsai) after turnip mosaic virus infection[END_REF], who observed similar results in leaves of stem mustard infected by the Turnip mosaic virus. However, chlorophyll content does not explain differences in photosynthesis rate since this variable was similar between treatments 21 days after virus infection. Thus it is likely that other mechanisms were involved in the reduced photosynthesis rate associated with virus infection, such as a decrease in mesophyll conductance to CO2 and RUBISCO activity [START_REF] Sampol | Analysis of the virus-induced inhibition of photosynthesis in malmsey grapevines[END_REF] or a reduction in photosynthetic electron transport in photosystem II [START_REF] Rahoutei | Inhibition of photosynthesis by viral infection: effect on PSII structure and function[END_REF].

The influence of the virus on photosynthetic rate likely explains the lower above-ground biomass measured in virus-infected plants, as proposed by [START_REF] Funayama | Effects of virus infection and growth irradiance on fitness components and photosynthetic properties of Eupatorium makinoi (Compositae)[END_REF], who stated that the reduction in photosynthetic production they observed in virus infected Eupatorium makinoi was a major factor explaining lower performance in those plants.

Aphid host performance and physiology. Virus infection improved some aphid fitness-related traits despite the alteration of the plant traits. Aphids reared on infected plants had a higher nymphal survival rate and reproduced earlier than control aphids, however their daily fecundity was not affected. This influence resulted into a higher intrinsic rate of natural increase (rm). These results are consistent with several studies showing a positive effect of plant infection by persistently transmitted viruses, especially Luteoviruses, on aphid fitness (for reviews, see [START_REF] Fereres | Behavioural aspects influencing plant virus transmission by homopteran insects[END_REF][START_REF] Mackauer | Parasites and parasite impact on aphid populations, in Plant virus epidemics: monitoring, modelling and predicting outbreaks[END_REF][START_REF] Blanc | Manipulation of hosts and vectors by plant viruses and impact of the environment[END_REF]. Such effects are thought to be adaptive for circulative viruses that generally require a long feeding period to be acquired and inoculated [START_REF] Hogenhout | Insect vector interactions with persistently transmitted viruses[END_REF]. By changing plant physiology and chemistry, virus infection influences plant suitability for aphids, and may lead to enhanced vector populations and amplified virus propagation. This positive impact could be achieved indirectly through changes in plant physiology, such as down-regulation of defence pathways or altered amino acid content of the sap (Mauck et al. 2014b;[START_REF] Casteel | Disruption of ethylene responses by Turnip mosaic virus mediates suppression of plant defense against the aphid vector, Myzus persicae[END_REF][START_REF] Su | Tomato yellow leaf curl virus differentially influences plant defence responses to a vector and a non-vector herbivore[END_REF]), but mechanisms are still poorly understood. A direct effect of the virus on aphids after acquisition is also possible, as circulative viruses realise a complex circuit in its insect vector, thus potentially affecting its physiology or behaviour [START_REF] Chuche | Use the insiders: could insect facultative symbionts control vector-borne plant diseases?[END_REF].

We also observed that the reduction of infected plant performance between 21 and 29 days after virus inoculation negatively affected some aphid traits important for parasitoid success, such as body size and body mass. A trade-off between body size and developmental rate is common in insects [START_REF] Roff | The evolution of life histories: theory and analysis[END_REF]Nylin and Gothard 1998) and is likely to explain the smaller body size observed in the fast-growing viruliferous aphids. Viruses would benefit from increasing growth rate of their vectors, which would also result in a smaller aphid nymphal and adult body sizeparameters which are less important for virus spread.

The smaller size we observed was associated with lower lipid content in M. persicae. Viruses are known to affect plant nutritional quality, for example by disrupting ratios of carbohydrates to free amino acids in the phloem [START_REF] Mauck | Infection of host plants by Cucumber mosaic virus increases the susceptibility of Myzus persicae aphids to the parasitoid Aphidius colemani[END_REF] where M. persicae feeds. To our knowledge, the influence of plant quality on lipid content has not been tested in aphids but results on Lepidoptera confirm that better plant quality generally results in higher lipid levels [START_REF] Liu | Effects of larval host plants on over-wintering preparedness and survival of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)[END_REF]). In aphids, lipid content is not correlated to fecundity [START_REF] Gwynn | Resistance is costly: trade-offs between immunity, fecundity and survival in the pea aphid[END_REF]; such differences between treatments should thus have limited influence on aphid intrinsic rate of natural increase. This impact of virus on fat content may however have important consequences on virus and vector dispersal since lipids are the main resource used for long-range flight in alate aphids [START_REF] Liquido | Longevity, fecundity, change in degree of gravidity and lipid content with adult age, and lipid utilisation during tethered flight of alates of the corn leaf aphid, Rhopalosiphum maidis[END_REF]Irwin 1986, Yao and[START_REF] Yao | Comparing wing loading, flight muscle and lipid content in ant-attended and non-attended Tuberculatus aphid species[END_REF], but our results on apterae vectors should first be confirmed on winged aphids before conclusions are drawn.

Parasitoid fitness. We observed strong effects of infected plants mediated by the aphid host on some life history traits of the parasitoid Aphidius colemani. Females that developed in aphids reared on virus-infected plants suffered from reduced body size, initial fecundity and lipid content compared to females that developed in control aphids and plants. To our knowledge, this is the first study showing negative effects of plant virus infection on natural enemy traits important for pest management. These effects are likely the result of bottom-up effects whereby the body size of the vectors and the quantity of energetic resources (lipid and carbohydrates quantity) available for the parasitoid are reduced because of the lower photosynthetic activity of the plant. Lipid resources are particularly important for parasitoids belonging to the genus Aphidius that lack adult lipogenesis and thus rely on lipids accumulated during larval development for activities during adult life [START_REF] Visser | Loss of lipid synthesis as an evolutionary consequence of a parasitic lifestyle[END_REF]. Thus, the reduction in host body size and resource quantity probably explain the observed smaller body size and initial fecundity, which are commonly observed in parasitoids developing in small hosts [START_REF] Godfray | Parasitoids: behavioral and evolutionary ecology[END_REF]. Moreover, lipid content is generally positively correlated with several life history traits, such as egg production during adult life [START_REF] Casas | Lifetime nutrient dynamics reveal simultaneous capital and income breeding in a parasitoid[END_REF] longevity [START_REF] Ellers | Fat and eggs: An alternative method to measure the trade-off between survival and reproduction in insect parasitoids[END_REF], and dispersal of parasitic wasps [START_REF] Ellers | A field study of size-fitness relationship in the parasitoid Asobara tabida[END_REF]. The reduction in lipid content may thus have important consequences on parasitoid population dynamics, and biological control through a reduction in their abundance and between-field movement. However, while negative effects of virus infection on parasitoid fitness are likely the result of bottom-up effects they may also be due to direct interference or resource competition between the pathogen and the parasitoid larva, both organisms co-occurring in the same host for a long period of time [START_REF] Hodge | Do plant viruses facilitate their aphid vectors by inducing symptoms that alter behavior and performance?[END_REF][START_REF] De Oliveira | A negative effect of a pathogen on its vector? A plant pathogen increases the vulnerability of its vector to attack by natural enemies[END_REF].

We observed similar emergence rates and development times between parasitoids developing in control aphids and aphids infected by the circulative virus TuYV. These parameters received particular intention in studies focusing on plant-aphid-parasitoid systems. Our results are consistent with observations of de [START_REF] De Oliveira | A negative effect of a pathogen on its vector? A plant pathogen increases the vulnerability of its vector to attack by natural enemies[END_REF] who did not observe any influence of infection by a persistent virus on parasitoid emergence rate. However, there are contradictory results in the literature. Development of parasitoid in aphid carrying circulative viruses can also result in delayed development and decreased emergence rate [START_REF] Christiansen-Weniger | Plant virus and parasitoid interactions in a shared insect vector/host[END_REF]). On the contrary, [START_REF] Mauck | Infection of host plants by Cucumber mosaic virus increases the susceptibility of Myzus persicae aphids to the parasitoid Aphidius colemani[END_REF] observed an increased emergence rate of parasitoids developing in aphid reared on plants infected by the non-circulative Cucumber mosaic virus (CMV -Cucumovirus). These authors proposed that the contrast between their results and those of Christiansen-Weniger et al.

(1998) could be explained by differences in virus modes of transmission. Indeed, close interactions between circulative viruses and their insect vector may offer greater opportunity for direct effects on insect physiology, and consequently alter parasitoid success [START_REF] Mauck | Infection of host plants by Cucumber mosaic virus increases the susceptibility of Myzus persicae aphids to the parasitoid Aphidius colemani[END_REF]. However, in light of the contrast between our results and previous studies [START_REF] Christiansen-Weniger | Plant virus and parasitoid interactions in a shared insect vector/host[END_REF]), it appears that the effect of virus on parasitoid emergence rate does not depend on virus mode of transmission and such effects may be system-specific.

Parasitoid behaviour. Aphid parasitoids are natural enemies that rely on both visual and chemical cues released by plants to detect their hosts (e.g. [START_REF] Du | Identification of semiochemicals released during aphid feeding that attract parasitoid Aphidius ervi[END_REF][START_REF] Wajnberg | Behavioral Ecology of Insect Parasitoids: From theoretical approaches to field application[END_REF] Asterisks indicate significant differences between treatments (p < 0.001). Mean ± s.e.

  -mediated and aphid-mediated effects of TuYV on parasitoid life history traits Emergence rate. Twenty standardised F0 parasitoid females were placed separately in a Petri dish (Ø90 mm) pierced on its side to allow introduction of a fresh C. sativa leaf, and allowed to parasitise each twenty synchronised second instar aphids. Parasitoids were continuously observed until they left the leaf for more than one minute or when they did not move for more than five minutes, and the number of attacks on aphid hosts was recorded. An attack was noted when a parasitoid female bent its abdomen and touched the aphid with its ovipositor. Parasitised aphids were then reared on control plants or on virus-infected plants until parasitoid emergence. We then calculated an emergence rate corresponding to the number of emerging parasitoids divided by the number of aphids attacked by a female.Body size, fecundity and lipid content. Standardised F0 females were placed separately in a Petri dish (Ø90 mm) and were offered 25 synchronised second instar control aphids placed on a control C. sativa leaf set in 1.5 % agar for one hour. Parasitised aphids were then transferred in equal subsets onto control or virus-infected C. sativa plants until mummification. Mummies were isolated in gelatine capsules and emergence was checked twice daily (9:00 am and 7:00 pm) to calculate oviposition to emergence development time. Upon emergence, fifty virgin females (<1hour-old) per treatment were killed by placing them at -80°C. The left hind tibia and left wing of twenty-five of those individuals were photographed and measured using ImageJ analysis software. These females were then dissected in a drop of PBS solution under a binocular (x40, Leica M165C). Both ovaries were removed and the number of mature eggs was counted. The lipid quantity and content of twenty of the remaining parasitoid females per treatment were measured using the same procedure as for aphids.5-Plant-mediated and aphid-mediated effects of TuYV on parasitoid behaviourPlant attraction. Preference of F1 A. colemani females for control or virus-infected plants, with or without aphids, was assessed using dual choice tests. To obtain these F1 females, standardized F0 parasitoid females were offered second instar M. persicae placed on a C. sativa leaf set in 1.5 % agar in a Petri dish (Ø45mm) for one hour. Parasitised aphids were transferred in ventilated plastic cages containing C. sativa plantlets under standard rearing conditions until emergence of F1 females, which were fed ad libitum with a water-honey solution (50% vol/vol) for 48h and allowed to mate with two males for 4h in a Petri dish before experiments. In a first test, parasitoid preference was assessed using only plants, while in a second test, twenty second instar aphids reared on control or virus-infected plants were placed on control or virus-infected plants. Using a paintbrush, female parasitoids were gently placed on a take-off platform in the centre of a ventilated plastic chamber (360x240x110 mm) inside which control and virus-infected plants were placed 30 cm apart on opposite sides. In order to ensure that parasitoids were exposed to similar amounts of VOCs emitted by each side, we standardised plants biomass by using different numbers of infected and control plants[START_REF] Chesnais | Aphid Parasitoid Mothers Don't Always Know Best through the Whole Host Selection Process[END_REF]: the first pot contained one control plant while the second pot contained three virus-infected plants. Parasitoids were continuously observed until they landed on a plant, and their choice was noted. Parasitoids that did not leave the platform or did not land on a plant after 30 minutes were discarded from analyses. Four plastic chambers were observed at the same time in a climate-controlled room (20±1°C, 60±5% RH), and control and virus-infected plants were switched for every replicate (n=20 for each combination control vs. virus-infected plant, control plant and aphids vs. virus infected plant and viruliferous aphids).

Figures captions Figure 1 .

 captions1 Figures captions

Figure 2 .

 2 Figure 2. Initial egg load of twenty-five newly emerged Aphidius colemani females developing in

Figure 3 .

 3 Figure 3. Percentage of Aphidius colemani females (n=20 for each test) orienting towards one

  Figure 1.

  Figure 2.

Figure 3 .

 3 Figure 3.

Table 1 .

 1 Moreover, chemical cues emitted by infected camelina did not influence parasitoid female attraction. This result was consistent with other studies testing parasitoid choice in Y-tube olfactometers, in which no preference for the odors of control and viruliferous aphids[START_REF] De Oliveira | A negative effect of a pathogen on its vector? A plant pathogen increases the vulnerability of its vector to attack by natural enemies[END_REF] or for the odors of aphid-infested control and virus-infected plants[START_REF] Mauck | Infection of host plants by Cucumber mosaic virus increases the susceptibility of Myzus persicae aphids to the parasitoid Aphidius colemani[END_REF] was observed.[START_REF] Mauck | Infection of host plants by Cucumber mosaic virus increases the susceptibility of Myzus persicae aphids to the parasitoid Aphidius colemani[END_REF] proposed that the increased emissions of volatiles observed in Cucurbita pepo infected by CMV may be offset by the smaller size of the infected plants, leading to similar chemical profiles between those one and healthy plants. However, in our experiment, we standardised biomass of infected and control plants to avoid this effect, and A. colemani still did not orientate preferentially towards one type of plant. This result is thus likely due to the similarity between emission profiles of plants, at least qualitatively[START_REF] Mauck | Infection of host plants by Cucumber mosaic virus increases the susceptibility of Myzus persicae aphids to the parasitoid Aphidius colemani[END_REF], or to the inability of A. colemani to discriminate between odors of healthy and infected plants.After plant host and aphid host location, viruses may influence parasitoid host acceptance behaviour. This may be especially true if viruliferous aphids are of different quality for the developing parasitoid offspring (i.e. different fitness 'payoff' for the parasitoid). In our experiment, we did not observe any difference in oviposition rate between aphids reared on infected (i.e. Infection of Camelina sativa by the Turnip yellows virus may have agronomical consequences, as this virus alters bottom-up regulation of the major pest Myzus persicae by improving host-plant quality for the aphid vector and decreasing aphid host quality for its parasitoid. Damages on Camelina may increase because of (i) the virus itself, (ii) an increase in the number of aphid pests which feed on plants and transmit the virus, and/or (iii) a decrease in biological control of M. persicae by one of its main natural enemies caused by lower parasitoid Mean (± s.e.) of parameters measured on sham-inoculated (=control) or TuYV-infected Camelina sativa. Different letters indicate significant differences between treatments and time.

		21 Days post-inoculation	29 Days post-inoculation
	Parameter	Control	TuYV-infected	Control	TuYV-infected
	Assimilation rate (µmol.m⁻².s⁻¹)	5.179 ± 0.441 a	3.923 ± 0.541 b	4.027 ± 0.182 b	1.611 ± 0.267 c
	Stomatal conductance (µmol.m⁻².s⁻¹)	0.101 ± 0.007 a	0.055 ± 0.004 c	0.072 ± 0.004 b	0.037 ± 0.004 d
	Chlorophyll content index (mg. m⁻²FW)	5.67 ± 0.05 a	6.57 ± 0.23 a	5.83 ± 0.06 a	3.66 ± 0.11 b
	Above-ground biomass (g)	0.534 ± 0.018 b	0.231 ± 0.014 d	0.702 ± 0.051 a	0.403 ± 0.034 c

. In our experiment, Aphidius colemani females were exposed to both types of cues but they did not show any preference for control or virus-infected plants, infested by aphids or not. Parasitic wasps are known to respond more strongly to yellow than green traps

[START_REF] Rodriguez-Saona | Effect of trap color and height on captures of blunt-nosed and sharp-nosed leafhoppers (Hemiptera: Cicadellidae) and non-target arthropods in cranberry bogs[END_REF]

. We may thus expect that they would be more attracted by yellow leaves, i.e. virus-infected leaves, than by green leaves, i.e. healthy leaves, but this did not appear to be the case for A. colemani. viruliferous aphids) or control plants, despite strong deleterious consequences on parasitoid lifehistory traits. This result contrasts with Christiansen-Weniger et al. (1998) who observed that Aphidius ervi deposited fewer eggs in Sitobion avenae carrying BYDV than in control aphids, likely because of a greater mortality and delayed development in viruliferous hosts. On the contrary, de Oliveira et al. (

2014

), observed a higher rate of ovipositor insertion following antennation into Rhopalosiphum padi infected by CYDV than control aphids. Aphid performance, especially body size and survival rate, were unaffected by acquisition of the virus. The authors hypothesised that parasitoids may preferentially parasitise viruliferous aphids because of a compromised immune response. Our own results suggest that other parameters may be involved in host selection, such as the energetic resources available. However, this did not appear to influence A. colemani behaviour.

Agronomical impact.

Table 2 .

 2 Mean values (± s.e.) of parameters measured on Myzus persicae reared on shaminoculated or TuYV-infected Camelina sativa. p<0.05, ** p<0.01, *** p<0.001, NS = Non significant

		Control	TuYV
	Parameter
	Body length at 2 days (mm)	0.36 ± 0.03	0.30 ± 0.04	*
	Body length at 8 days (mm)	1.50 ± 0.04	1.41 ± 0.05	*
	Body mass at 8 days (mg)	0.152 ± 0.009	0.099 ± 0.005 ***
	Nymph survival rate	0.91 ± 2.53	1.00 ± 0.00	***
	Pre-reproductive period (days)	8.97 ± 0.17	8.00 ± 0.14	***
	Daily fecundity	4.17 ± 0.12	4.54 ± 0.24	NS
	rm	0.270 ± 0.004	0.301 ± 0.006 ***

*

Table 3 .

 3 Mean (± s.e.) life history trait values and emergence rates measured on Aphidius colemani developing in Myzus persicae aphids reared on control or TuYV-infected Camelina sativa. p<0.05, ** p<0.01, *** p<0.001, NS = Non significant

		Control	Viruliferous	Significance
	Parameter
	Tibia length (mm)	0.64 ± 0.04	0.59 ± 0.03	*
	Development time (days)	15.4 ± 0.9	15.0 ± 1.0	NS
	Wing area (mm²)	1.44 ± 0.09	1.36 ± 0.08	NS
	Emergence rate	57.5 ± 5.1	51.9 ± 6.7	NS

*

Table 4 .

 4 Mean (± 95%CI) oviposition and host rejection rates measured on Aphidius colemani attacking Myzus persicae reared on control or infected-plants (i.e. viruliferous) in no-choice or in dual-choice experiments.

		Control	Viruliferous	Significance
	Parameter
	No-choice experiment
	Oviposition rate	0.74 ± 0.09	0.68 ± 0.07 GLM, z = -1.097, p = 0.509
	Host rejection rate	0.19 ± 0.04	0.24 ± 0.06 GLM, z = 1.902, p = 0.484
	Dual-choice experiment
	Oviposition rate	0.69 ± 0.08	0.75 ± 0.07 GLM, z = 2.544, p = 0.371
	Host rejection rate	0.23 ± 0.05	0.20 ± 0.03 GLM, z = -1.142, p = 0.453

Acknowledgements. We thank Paul Abram, Suzi Claflin and Thomas Seth Davis for helpful comments on earlier drafts of this manuscript. This work was performed in partnership with the SAS PIVERT within the framework of the French Institute for the Energy Transition (Institut pour la Transition Energétique (ITE) P.I.V.E.R.T. (www.institut-pivert.com)), selected as an Investment for the Future ("Investissements d'Avenir"). This work was supported, as part of the Investments for the Future, by the French Government under the reference ANR-001-01.

Authors' Contributions.

JM, QC, FS, AA and AC conceived and designed research. JM, QC, FS, and EV conducted experiments. JM, QC and FS analysed data. JM, QC, FS, AA and AC wrote the manuscript. All authors read and approved the manuscript.

Conflict of Interest:

The authors declare that they have no conflict of interest.

abundance, in presence of the TuYV. This last prediction contrasts with the study of [START_REF] De Oliveira | A negative effect of a pathogen on its vector? A plant pathogen increases the vulnerability of its vector to attack by natural enemies[END_REF] who observed that biological control by Aphidius colemani may be more effective in fields infected by CYDV because of parasitoid preference for viruliferous aphids; however those authors did not consider the influence of development in viruliferous aphids on traits related to parasitoid population dynamics such as fecundity or lipid content. Our study underlines the complexity of interactions between plant viruses and plant-vector-natural enemies and their implications for agricultural ecosystems.

Conflict of Interest:

The authors declare that they have no conflict of interest.


Joffrey Moiroux
email: joffrey.moiroux@gmail.com.

Philippe Louâpre

Paul K Abram

Maryse Barrette

Jacques Brodeur

Guy Boivin

Influence of temperature on patch residence time in parasitoids: Physiological and behavioral mechanisms

Keywords: patch time allocation, host-parasitoid interaction, climate, discrimination ability, sex allocation, anti-predator behaviors

1. Patch time allocation has received much attention in the context of optimal foraging theory, including the effect of environmental variables. We investigated the direct role of temperature on patch time allocation by parasitoids through physiological and behavioral mechanisms and its indirect role via behavioral defences of the hosts.

2. We compared the influence of foraging temperature on patch residence time between an egg parasitoid, Trichogramma euproctidis, and an aphid parasitoid, Aphidius ervi that attacks hosts able to actively defend themselves.

3. Patch residence time decreased with an increase in temperature in both species. The increased activity levels with warming, as evidenced by the increase in walking speed, partly explained these variations but other mechanisms were involved. In T. euproctidis, the ability to externally discriminate parasitized hosts decreased at low temperature, resulting in a longer patch residence time. For A. ervi, we observed that aphids frequently escaped at intermediate temperature and defended themselves aggressively at high temperature but displayed few defence mechanisms at low temperature. These defensive behaviors resulted in a decreased patch residence time for the parasitoid, and partly explained the fact that A. ervi remained for a shorter time at the intermediate and high temperatures than at the lowest temperature.

INTRODUCTION

Resources such as food, mate or hosts are usually heterogeneously distributed in the environment and their exploitation requires time. One of the main decisions an organism faces when foraging is how to manage residence time between visited patches in order to maximize fitness yield [START_REF] Wajnberg | Time allocation strategies in insect parasitoids: from ultimate predictions to proximate behavioral mechanisms[END_REF]. Patch time optimization received much attention in the context of optimal foraging, and was first addressed by [START_REF] Charnov | Optimal foraging: the marginal value theorem[END_REF] in his Marginal Value Theorem (MVT). The main assumption of the MVT is that a forager should leave a depleted patch when the current rate of gain falls below the average rate it would come across in the environment, thus maximizing the long term rate of fitness gain. This model is one of the most influential in behavioral ecology but it has been subjected to criticism as it assumes that foragers are omniscient, and does not take into account proximate behavioral mechanisms involved in patch time allocation [START_REF] Van Alphen | Information acquisition, information processing, and patch time allocation in insect parasitoids[END_REF].

Insect parasitoids have been widely used as model organisms to study patch time allocation as their hosts are often patchily distributed and their foraging behavior directly influences progeny production. Over the years, many authors investigated behavioral mechanisms involved in patch time allocation (e.g., [START_REF] Waage | Foraging for patchily-distributed hosts by the parasitoid, Nemeritis canescens[END_REF][START_REF] Driessen | A count-down mechanism for host search in the parasitoid Venturia canescens[END_REF] and have shown that parasitoids can adapt patch residence time to a wide variety of environmental variables (for a review, see [START_REF] Wajnberg | Time allocation strategies in insect parasitoids: from ultimate predictions to proximate behavioral mechanisms[END_REF], among them biotic factors such as presence of competitors [START_REF] Wajnberg | Genetic variation in the mechanisms of direct mutual interference in a parasitic wasp: consequences in terms of patch time allocation[END_REF][START_REF] Goubault | Patch exploitation strategies of parasitic wasps under intraspecific competition[END_REF][START_REF] Le Lann | First in, last out: asymmetric competition influences patch exploitation of a parasitoid[END_REF] and host kairomones [START_REF] Li | Effects of contact kairomone and experience on initial giving-up time[END_REF][START_REF] Louâpre | Carbon dioxide narcosis modifies the patch leaving decision of foraging parasitoids[END_REF] or abiotic factors such as photoperiod during development [START_REF] Roitberg | Seasonal dynamic shifts in patch exploitation by parasitic wasps[END_REF] and barometric pressure [START_REF] Roitberg | Life expectancy and reproduction[END_REF].

Despite the considerable influence of temperature on life history, physiological and behavioral traits of insects [START_REF] Huey | Evolution of thermal sensitivity of ectotherms performance[END_REF][START_REF] Nylin | Plasticity in life-history traits[END_REF][START_REF] Angilletta | Temperature, growth rate, and body size in ectotherms: fitting pieces of a life-history puzzle[END_REF], its influence on patch time allocation has rarely been addressed. Temperature may directly affect patch residence time through physiological or behavioral mechanisms. Firstly, walking speed, search rate, handling time and related behaviors are known to change with temperature in ectotherms because of its influence on metabolic rate (see the Metabolic Theory of Ecology, [START_REF] Brown | Toward a metabolic theory of ecology[END_REF][START_REF] Moiroux | Evolution of metabolic rate in a parasitic wasp: The role of limitation in intrinsic resources[END_REF]. Usually, walking speed and search rate follow a hump-shaped relationship with temperature in insects [START_REF] Sentis | Using functional response modeling to investigate the effect of temperature on predator feeding rate and energetic efficiency[END_REF]. It is thus likely that patch residence time would vary with temperature as a direct consequence of its influence on the speed of movements and behaviors. Secondly, behavioral mechanisms may have been selected in response to temperature. To our knowledge, only [START_REF] Amat | The influence of temperature and host availability on the host exploitation strategies of sexual and asexual parasitic wasps of the same species[END_REF] have convincingly addressed this issue in a parasitoid. They observed that a drop in temperature before foraging increased patch residence time and that this environmental cue was more important than foraging temperature itself. The authors proposed that thermal variations may be perceived as a signal of unfavourable weather conditions and that searching for a new patch would increase mortality risk or physiological costs. Finally, multi-objective behavioral mechanisms related to patch time allocation may be adopted by animals in response to temperature. [START_REF] Wajnberg | Multi-objective behavioural mechanisms are adopted by foraging animals to achieve several optimality goals simultaneously[END_REF] clearly showed that sex allocation influenced patch time allocation in the egg parasitoid Trichogramma chilonis as laying a son did not change its patch residence time while laying a daughter decreased its patch time allocation. Considering that haplodiploid parasitoids can adapt sex allocation to their foraging temperature [START_REF] Moiroux | Sex ratio variations with temperature in an egg parasitoid: behavioural adjustment and physiological constraint[END_REF], we may thus expect that females would also change their patch time allocation according to sex allocation.

Temperature may also indirectly influence patch time allocation via its effect on the nature and frequency of host defences (e.g., [START_REF] Miyatake | Negative relationship between ambient temperature and death-feigning intensity in adult Callosobruchus maculatus and Callosobruchus chinensis[END_REF][START_REF] Ma | Climate warming may increase aphids' dropping probabilities in response to high temperatures[END_REF]. For example, when exposed to alarm pheromone, pea aphids Acyrthosiphon pisum are less likely to drop from the feeding site to the ground when the environment is hot and dry than when it is more benign [START_REF] Dill | The economics of escape behaviour in the pea aphid, Acyrthosiphon pisum[END_REF]. Temperature may thus affect patch time allocation through its influence on host defences since parasitoids can change patch time allocation as a response to host defence behaviors. This was for example observed for the ichneumonid Diadegma semiclausum, which remains longer on a patch when attacked hosts temporally leave [START_REF] Wang | Patch Time Allocation by the Parasitoid Diadegma semiclausum (Hymenoptera: Ichneumonidae). I. Effect of interpatch distance[END_REF].

The aim of this study was to investigate both the direct and indirect effects of temperature on patch time allocation in parasitoids. We compared the influence of temperature on patch residence time of two different parasitoid species exploiting hosts that exhibit or not defensive behaviors: (i) the parasitoid Trichogramma euproctidis Girault (Hymenoptera: Trichogrammatidae), which attacks eggs of the lepidopteran species Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) and for which we were able to distinguish male egg deposition from female egg deposition by observation [START_REF] Moiroux | Sex ratio variations with temperature in an egg parasitoid: behavioural adjustment and physiological constraint[END_REF] to study influence of sex allocation, and (ii) the parasitoid Aphidius ervi Haliday (Hymenoptera: Aphidiinae) which attacks potato aphids, Macrosiphum euphorbiae Thomas (Hemiptera: Aphididae), that are able to actively defend themselves. For this parasitoid species, we were unable to measure sex allocation. Both parasitoid species were maintained at their optimal temperature and placed at low and high temperatures close to their thermal limits before foraging. Their patch residence time and foraging behaviors were then recorded. We also measured thermal variations in walking speed for T. euproctidis as a proxy of physiological changes, while data were already available from [START_REF] Gilchrist | A quantitative genetic analysis of thermal sensitivity in the locomotor performance curve of Aphidus ervi[END_REF] for A. ervi. The nature and frequency of defensive behaviors displayed by aphids attacked by A. ervi was also measured. We expected that patch time allocation should be directly influenced by temperature for both species as a consequence of changes in walking speed, and indirectly via its effect on sex allocation in T. euproctidis [START_REF] Moiroux | Sex ratio variations with temperature in an egg parasitoid: behavioural adjustment and physiological constraint[END_REF]) and via its effects on behavioral defences of hosts in A. ervi.

MATERIALS & METHODS

Trichogramma euproctidis

Trichogrammatidae wasps are small endoparasitoids of eggs of several lepidopteran species.

The T. euproctidis strain used in this study originates from individuals sampled in Egypt and was maintained at 24 ±1˚C, 50% RH, and LD 16:8 h on cold-killed eggs of Ephestia kuehniella.

Patch residence time. Parasitized eggs were taken from the T. euproctidis colony and isolated in 300μL Beem ® polyethylene capsules with a drop of honey. The next morning, newly emerged females were mated once with a virgin male, stored at 24˚C, and transferred to a climate room at either 14, 24 or 34˚C one hour before observation. The first and last temperatures are close to the species thermal limits but do not cause any deleterious effects on walking activity (see results on walking activity). After this acclimatization period, females were offered, at the same temperature, 20 cold-killed E. kuehniella eggs, placed side by side in 4 lines of 5 eggs on a filter paper disc. The experimental arena was delimited by the plastic end piece of a camera (Ø20 mm) covered with fluon to prevent females from climbing up the side. The number and duration of the following behaviors were recorded for 30 females per temperature with a camera (Dino-lite Pro AM413ZT, x 60) and analyzed using The Observer XT software (Noldus, Wageningen, The Netherlands): entering the patch, leaving the patch, walking, host feeding, drumming the host with antennae, drilling, egg male deposition, egg female deposition, host rejection, and resting. Egg deposition was distinguished from drilling by positioning of the abdomen, which was perpendicular to the host when drilling and parallel during egg deposition. Egg female deposition was distinguished from egg male deposition by the presence of a pause during oviposition sequence [START_REF] Suzuki | Sex allocation and effects of superparasitism on secondary sex ratios in the gregarious parasitoid, Trichogramma chilonis (Hymenoptera: Trichogrammatidae)[END_REF]. Host rejection consisted of drilling not followed by egg deposition. Walking was recorded when a female was moving and did not mark a pause while drumming hosts with antennae was noted when a female was on an egg and touched it repeatedly with its antennae. We did not consider host rejection with antennae, as it was sometimes very fast, especially at high temperature, and difficult to distinguish from walking on eggs. Patch residence time was measured from the time the female entered the patch until she remained inactive for more than 30 minutes or walked off the patch for more than 3 minutes at 34˚C, 4min45s at 24˚C and 7min30s at 14˚C. These durations are proportional to speed variation due to temperature measured in the experiment on walking speed described below. Additional analyses were performed considering a similar threshold, i.e. females walked off the patch for more than 3 minutes for the three temperatures, and the results led to the same conclusion in both cases.

Walking speed. Newly emerged (<24 h), unfed females were placed individually in an empty

Petri dish (Ø92 mm) on a light table at 10, 15, 20, 25, 30 or 35±1˚C. The movement of 15 females per temperature was video-recorded (Camera Panasonic WV-BD400) for 300 seconds and then analyzed with a program developed by [START_REF] Vigneault | Automated system to quantify the behavior of small insects in a four-pointed star olfactometer[END_REF]. The contrast between individuals and the light table was used to track females and the program calculated the mean walking speed (mm/sec), deduced from the distance traveled in 300 seconds minus duration of stops.

Aphidius ervi

Insect colonies. Aphidius ervi is a solitary koinobiont parasitoid of several aphid species. A few weeks prior to the tests, an A. ervi colony was established with individuals bought from BioBest Canada (Leamington, Canada). Parasitoids were reared in cages on the potato aphid M. euphorbiae at 20±1°C, 60±10% RH, 16L: 8D photoperiod. The aphid colony was initiated with individuals collected from potato fields in the vicinity of Québec City, Canada. This colony was maintained on potato plants, Solanum tuberum Linnaeus (Solanales: Solanacae), cultivar "Norland" under the same conditions.

Patch residence time.

Parasitized aphids were reared at 20±1°C on potato plants and isolated in gelatine capsules once they mummified. After emergence, each female parasitoid was kept with a male at 20±1°C for 24 hours in a Petri dish containing moistened cotton and honey. This period allowed females to mature eggs [START_REF] Le Lann | First in, last out: asymmetric competition influences patch exploitation of a parasitoid[END_REF]. The next day, their foraging behavior was observed in climate chambers at 12, 20 or 28°C. The first and last temperatures are close to A. ervi thermal extremes but do not induce any lethal effects or deleterious effects on walking activity (according to [START_REF] Gilchrist | A quantitative genetic analysis of thermal sensitivity in the locomotor performance curve of Aphidus ervi[END_REF]. One hour before the observation, a potato leaf (ø 4cm approximately) was taped on a piece of paper and 60 apterous aphids were placed on it, 15 from each larval instar (L1 to L4). A range of different instars were exposed to A. ervi females because their defensive behaviors change with age [START_REF] Barrette | Travel time affects optimal diets in depleting patches[END_REF]. Prior to the test, parasitoid females were acclimatized to the experimental temperature for one hour.

Females were then introduced in the experimental arena (ø 26cm, h 8.5 cm) and the following behaviors were recorded with a camera (Panasonic WV-BD400) on 25 females per temperature and analyzed with The Observer XT software (Noldus, Wageningen, The Netherlands): entering the patch, leaving the patch, walking, successful oviposition, failed oviposition, host rejection and resting. A successful oviposition was recorded when the parasitoid abdomen came into contact with the aphid while a failed oviposition was recorded when a parasitoid initiated an attack but failed to touch the aphid with its abdomen because of aphid behavioral defences. Host rejection was recorded when a female parasitoid came into contact with a motionless aphid and did not attack it. Both the number and duration of these behaviors were recorded, taking into account host instar which was evaluated visually by aphid body size. Patch residence time was measured from the time the female entered the patch until she left it for more than one minute at 28°C, 2 minutes at 20°C and 6 minutes at 12°C or stopped any activity for more than 15 minutes. These durations are proportional to speed variations with temperature measured by [START_REF] Gilchrist | A quantitative genetic analysis of thermal sensitivity in the locomotor performance curve of Aphidus ervi[END_REF]. Additional analyses were performed considering a similar threshold, i.e. females left the patch for more than 1 minute for the three temperatures, and the results led to the same conclusion in both cases.

During this test, we also recorded aphid anti-predator behaviors, i.e. kicking, body jerking, or escaping from the patch. Kicking was recorded when an aphid extended one of its leg, usually a hind leg, and shook it up and down. Body jerking consisted of spasmodic horizontal movements of the abdomen. Escaping the patch was recorded when an aphid walked away after an attack and left the potato leaf. These behaviors were only recorded when aphids were attacked by the parasitoid.

For this parasitoid species, we were not able to study the influence of sex allocation on patch residence time. In Aphidius parasitoids, sex allocation can not be determined by observation.

In his paper on multi-objective behavioural mechanisms, [START_REF] Wajnberg | Multi-objective behavioural mechanisms are adopted by foraging animals to achieve several optimality goals simultaneously[END_REF] observed the sex of emerging parasitoids to assess sex allocation during oviposition. However the temperature is known to affect sex ratio via physiological constraints on egg fertilization in haplodiploid parasitoids [START_REF] Moiroux | Sex ratio variations with temperature in an egg parasitoid: behavioural adjustment and physiological constraint[END_REF], resulting in differences between sex allocation and realized sex ratio. Observation of the sex of emerging parasitoids was thus not reliable to evaluate sex allocation in our experiment.

Statistical analyses.

Patch residence time was analyzed using a Cox proportional hazard model [START_REF] Cox | Regression models and life tables[END_REF][START_REF] Tenhumberg | Using Cox's proportional hazard models to implement optimal strategies: An example from behavioral ecology[END_REF]. Such model is classically used to analyse censored data such as patch duration [START_REF] Wajnberg | Genetic variation in patch time allocation in a parasitic wasp[END_REF]. It determines a patch-leaving tendency for a foraging parasitoid, i.e. the probability per unit of time that a female leaves the patch, providing that she is still on it. It is formulated in terms of hazard ratio; a hazard ratio above one indicates an increase in the female's patch-leaving tendency and a hazard ratio below one indicates a decrease in the patch-leaving tendency. For T. euproctidis, temperature was considered as fixed factor, while the number of male ovipositions, number of female ovipositions, number of host rejections, number of host feeding, resting periods and temporary exit from the patch were included as time-dependant covariates. The interaction between temperature and the number of male and female ovipositions or with host rejections were also considered. For A.

ervi, temperature, considered as a fixed factor, total number of ovipositions, number of ovipositions per host larval instar, number of failed ovipositions, number of host rejections, number of aggressive behaviors (i.e., kicking + jerking) and of escaping aphids, number of temporary exits from the patch were included in the model, as were interactions between temperature and the number of successful or failed ovipositions. In both Cox models, the adequacy of the model was assessed by plotting deviance residuals [START_REF] Therneau | Martingale-based residuals for survival models[END_REF].

The assumption that hazard rates were proportional for different values of covariates was also checked following [START_REF] Collett | Modelling survival data in medical research[END_REF] and [START_REF] Wajnberg | Genetic variation in patch time allocation in a parasitic wasp[END_REF].

In addition, the influence of temperature on the total number of ovipositions, host rejections, host feeding, resting and grooming periods for T. euproctidis were analysed using Generalized Linear Models (GLM) with a Poisson error distribution. Data on sex allocation have been previously published in [START_REF] Moiroux | Sex ratio variations with temperature in an egg parasitoid: behavioural adjustment and physiological constraint[END_REF]. The influence of temperature on the proportion of time devoted to each of these behaviors and time spent walking and drumming hosts with antennae was analyzed with a GLM with a Binomial error distribution. For A. ervi, the influence of temperature on the number and duration of these behaviors is described in [START_REF] Moiroux | Temperature influences host instar selection in an aphid parasitoid: support for the relative fitness rule[END_REF]. The number of kicking, jerking and escaping aphids was compared using a GLM with a Poisson error distribution.

Finally, we fitted an exponential curve to the data to investigate the effect of temperature on T. euproctidis walking speed.

All statistical analyses were carried out using R software version 2.14.1 (R Development Core Team, 2011).

RESULTS

Trichogramma euproctidis

Patch residence time. Temperature, the number of male and female ovipositions, and the interaction between temperature and the number of hosts rejection significantly influenced the patch residence time of T. euproctidis (Table 1) while host rejection, host feeding, the number of temporary exits and resting periods did not. The tendency to leave the patch increased with temperature (Figure 1) and following a male and female oviposition (i.e. hazard ratio > 1, Table 1). It should be noted that laying a son or of daughter increased patch leaving tendency by very close factors (respectively 0.558 ± 0.166 vs 0.576 ± 0.077). Host rejection increased the tendency to leave the patch at intermediate and high temperature but it decreased this tendency at low temperature (Figure 2). We did not observe any significant influence of the interaction between temperature and the number of male (Temperature( 24 Temperature did not influence the total number of ovipositions while the number of host rejections was significantly higher at 14˚C than at 24 or 34˚C, and females host-fed more at 34˚C (Table 2). Parasitoid females laid more sons at high temperature than at other temperatures while no difference in sex allocation was observed between low and intermediate temperatures [START_REF] Moiroux | Sex ratio variations with temperature in an egg parasitoid: behavioural adjustment and physiological constraint[END_REF]. Females foraging at 14°C spent proportionally less time laying eggs (df = , p < 0.001), walking (df = , p = 0.021) and feeding on hosts (df = , p = 0.014) than females tested at 24 and 34˚C, but spent significantly more time drumming host eggs with their antennae (df = , p = 0.003) and rejecting hosts (df = , p < 0.001) (Figure 3). Walking speed. Walking speed of T. euproctidis increased significantly with increasing temperature (F = 118.7, df = 1, 87, p < 0.001; Figure 4).

Aphidius ervi

Patch residence time. Patch residence time was significantly influenced by temperature (Figure 1B), the number of total successful ovipositions, the number of escaping aphids and the number of aggressive aphids, but not by other time-dependent covariates or the interactions between temperature and any of the time-dependent covariates (Table 3). Females remained longer in the patch after an oviposition and left earlier with increasing temperature and the number of aggressive or escaping aphids.

Aphids. The number of kicks performed by M. euphorbiae (t = 7.718, df = 73, p < 0.001) and the number of aphids jerking (t = 3.146, p = 0.002) were significantly influenced by the foraging temperature. Aggressive defensive behaviors were more frequent at 28°C and less frequent at 12°C (Figure 5). Foraging temperature significantly influenced the number of aphids escaping from the patch when attacked (t = 3.914, p < 0.001). Escape behavior was the most frequent in aphids attacked at 20°C (Figure 5).

DISCUSSION

We investigated the influence of temperature on patch time allocation in two parasitoid species, considering its direct effects through physiological or behavioral mechanisms and its indirect effects via behavioral defences of hosts. Our main results are that: (1) patch residence time decreased as temperature increased, together with an increase in walking speed for both T. euproctidis and A. ervi, (2) patch time allocation was influenced by an interaction between temperature and the number of host rejections in T. euproctidis, and (3) the nature and number of aphid behavioral defences changed with temperature and influenced patch residence time in A. ervi.

Patch time allocation.

Trichogramma euproctidis females used a decremental mechanism when exploiting host patches as each oviposition increased the patch leaving tendency. This decremental mechanism is consistent with the results of a comparative analysis conducted on 17 species of Trichogramma by [START_REF] Wajnberg | A comparative analysis of patch-leaving decision rules in a parasitoid family[END_REF]. An increased tendency to leave a patch after oviposition is expected when resources are uniformly distributed over patches [START_REF] Iwasa | Prey distribution as a factor determining the choice of optimal foraging strategy[END_REF]. However, this hypothesis cannot be confirmed as no reliable distribution data are available for hosts of Trichogramma parasitoids [START_REF] Wajnberg | A comparative analysis of patch-leaving decision rules in a parasitoid family[END_REF].

On the contrary, patch time allocation increased due to an oviposition in Aphidius ervi. This incremental mechanism [START_REF] Waage | Foraging for patchily-distributed hosts by the parasitoid, Nemeritis canescens[END_REF] has been observed in many parasitoid species (for a review, see [START_REF] Wajnberg | Time allocation strategies in insect parasitoids: from ultimate predictions to proximate behavioral mechanisms[END_REF]) including a close and sympatric species, Aphidius rhopalosiphi [START_REF] Outreman | Effects of within-and amongpatch experiences on the patch-leaving decision rules in an insect parasitoid[END_REF]. Such behavior is expected when organisms face a large variance in patch quality [START_REF] Iwasa | Prey distribution as a factor determining the choice of optimal foraging strategy[END_REF], as observed for aphid colonies in the field (e.g., [START_REF] Ward | Cereal aphid populations and the relation between mean density and spatial variance[END_REF].

Patch time allocation and temperature. In both parasitoid species, patch residence time decreased sharply when temperature increased. This result contrasts with a study by van [START_REF] Van Roermund | Influence of intrapatch experiences and temperature on the time allocation of the whitefly parasitoid Encarsia formosa (Hymenoptera: Aphelinidae)[END_REF] who did not observe any influence of temperature on patch time allocation in the whitefly parasitoid Encarsia formosa. It should be noted that in this last paper, parasitic wasps were tested on patches without hosts. These unnatural conditions likely explain the absence of parasitoid response to temperature.

An increase in female parasitoid activity with warming, as evidenced by the increase in walking speed in T. euproctidis (this study) and A. ervi [START_REF] Gilchrist | A quantitative genetic analysis of thermal sensitivity in the locomotor performance curve of Aphidus ervi[END_REF], would likely modulate patch residence time. The higher the temperature, the faster females perform the component behaviors that make up foraging up to an optimum, as predicted in the context of the Metabolic Theory of Ecology [START_REF] Brown | Toward a metabolic theory of ecology[END_REF]). In T. euproctidis and A. ervi, walking speed increased on average 1.7 times between 24 and 34°C and 20 and 28°C, respectively, while patch residence time and duration of the different behaviors recorded (antennation, drilling, egg deposition) decreased 1.7 times. This result suggests a simple physiological change associated with temperature. However, while the walking speed increased 1.6 times from 14 ˚C to 24 ˚C and 2.5 times from 14 ˚C to 34 ˚C in T. euproctidis, patch residence time decreased 2.6 and 8.7 times, respectively. In A. ervi, walking speed increased 4.1 times from 12 to 20°C and 6.9 times from 14 to 34°C while patch residence time decreased 5 and 8.6 times, respectively. These differences suggest that in both species walking speed alone does not explain the increase in patch time allocation at the lowest temperature. From our results, different mechanisms may be involved for each species: a decrease in external discrimination ability in T. euproctidis and variations in nature and frequency of host defensive behaviors in A. ervi. These mechanisms were observed in parasitoids and hosts taken from the lab but field populations living under changing environment may respond differently than these strains.

At the lowest temperature, the number of host rejections (i.e. a drilling not followed by egg deposition) by T. euproctidis females was higher than at other temperatures. Parasitized eggs were thus mainly rejected after perforation of the chorion by the ovipositor but females were unable to reject them while drumming with their antennae, unlike at 24 or 34˚C. These results indicate that a low temperature during oviposition affected the external discrimination ability of T. euproctidis females, which probably contributed to the increased patch residence time recorded at 14°C. Overall, the same number of eggs was parasitized by females across tested temperatures, but it took more time to exploit the patch at low temperature. Several non-exclusive hypotheses can explain the decrease in external discrimination ability. First, the amount or composition of the marking pheromone produced by a female and left on the host after oviposition [START_REF] Roitberg | On the evolutionary ecology of marking pheromones[END_REF] may be changed at low temperature, as observed for the marking pheromone produced by beewolf males, Philanthus triangulum, to mark their territories [START_REF] Roeser-Mueller | Larval rearing temperature influences amount and composition of the marking pheromone of the male beewolf, Philanthus triangulum[END_REF]. Secondly, the receptivity of antennae to chemical signals or their integration in the olfactory lobe may also be affected, as suggested for European corn borer males, Ostrinia nubilalis, exposed to female sex pheromone at different temperatures [START_REF] Hilker | Chemical and behavioral ecology in insect parasitoids: how to behave optimally in a complex odorous environment[END_REF]). An effect of low temperature on the nervous system, especially on learning ability, was also proposed by [START_REF] Van Baaren | Effect of low temperature exposure on oviposition behaviour and patch exploitation strategy in parasitic wasps[END_REF] to account for the absence of external discrimination ability in cold-stored females of the mymarid Anaphes victus.

This negative effect of low temperature on external discrimination ability probably explains that we observed a significant influence of the interaction between temperature and host rejection on patch residence time in T. euproctidis. At intermediate and high temperature, Trichogramma females left earlier the patch after host rejection. This decremental mechanism is assumed to be adaptive and has been commonly reported in parasitoids [START_REF] Wajnberg | Time allocation strategies in insect parasitoids: from ultimate predictions to proximate behavioral mechanisms[END_REF] including species from the Trichogramma genus [START_REF] Wajnberg | A comparative analysis of patch-leaving decision rules in a parasitoid family[END_REF]. At low temperature, we observed an incremental mechanism associated with host rejection. This behavioral mechanism has only been reported in one parasitoid species (Nelson & Roitberg, 1995) and is probably the consequence of deleterious effect of temperature on sensory structures in our experiment. A. ervi did not suffer as much as T. euproctidis from low temperature. It is likely that interspecific variations or local adaptations of reproductive behaviors at low temperature exist since oviposition does not seem to be negatively affected by temperatures close to 0°C in some parasitoids [START_REF] Meiners | Oviposition at low temperatures -late season negatively affects the leaf beetle Galeruca tanaceti (Coleoptera: Galerucinae) but not its specialised egg parasitoid Oomyzus galerucivorus (Hymenoptera: Eulophidae)[END_REF]. Size differences between the species might for example explain these differences. Due to their small size and thermoregulatory constrains, egg parasitoids might be physiologically more affected by changes in temperature than the larger aphid parasitoids.

We hypothesised that temperature may influence patch time allocation via its effect on sex allocation [START_REF] Wajnberg | Multi-objective behavioural mechanisms are adopted by foraging animals to achieve several optimality goals simultaneously[END_REF]) since in T. euproctidis, [START_REF] Moiroux | Sex ratio variations with temperature in an egg parasitoid: behavioural adjustment and physiological constraint[END_REF] observed that the number of sons laid by a female was greater at high temperature than at low and intermediate temperatures. Laying a son or of daughter increased patch leaving tendency by very close factors. This result contrasts with the paper of [START_REF] Wajnberg | Multi-objective behavioural mechanisms are adopted by foraging animals to achieve several optimality goals simultaneously[END_REF] who observed in T. chilonis that laying a son did not influence patch residence time while it decreased with oviposition of a daughter. Our result, as well as the absence of interaction between male or female egg deposition and temperature, imply that differences in patch residence time with temperature could not be explained by differences in sex allocation.

In A. ervi, the nature and frequency of defensive behaviors displayed by aphids changed with temperature. They frequently defended themselves by kicking and jerking at the highest temperature while they tended to escape at the intermediate temperature. This observation is consistent with results on pea aphids, Acyrthosiphon pisum, which are less likely to drop from their feeding site when the environment is hot and dry than when it is more benign [START_REF] Dill | The economics of escape behaviour in the pea aphid, Acyrthosiphon pisum[END_REF]. Aphids may be less prone to leave a leaf when temperature is high as they need more resources to compensate for the increased metabolic rate [START_REF] Brown | Toward a metabolic theory of ecology[END_REF] and should actively defend against parasitoids instead of escaping. Aphid aggressive behaviors resulted in an increase in patch leaving tendency for the parasitoid A.

ervi. This effect may explain why the patch residence time of aphid parasitoids was shorter at 28°C than expected from the change in walking speed when compared to the lowest temperature, at which aphids rarely defended. Since aggressive host behavioral defences increases injury risk for parasitoids, it is likely that the optimal behavior would be to search for another patch with less aggressive hosts. To our knowledge, this is the first experimental evidence that such aggressive behavioral defences influence patch time allocation in insect natural enemies. We also observed that patch residence time decreased with the number of aphids leaving the patch when exposed to a parasitoid. This last result contrasts with observation on the ichneumonid parasitoid Diadegma semiclausum that remained longer on a patch when hosts temporally left after attack [START_REF] Wang | Patch Time Allocation by the Parasitoid Diadegma semiclausum (Hymenoptera: Ichneumonidae). I. Effect of interpatch distance[END_REF], waiting for the discovered host to climb back to the leaf [START_REF] Wang | A comparison of the host-searching efficiency of two larval parasitoids of Plutella xylostella[END_REF]. Aphidius ervi instead left the patch earlier, probably because females perceived a decrease in patch quality (e.g., [START_REF] Boivin | Optimal patch residence time in egg parasitoids: innate versus learned estimate of patch quality[END_REF][START_REF] Outreman | Can imperfect host discrimination explain partial patch exploitation in parasitoids?[END_REF]. This result may explain why A. ervi remained shorter than expected at the intermediate temperature compared to the lowest temperature, since aphids escaped more frequently at intermediate temperature.

Conclusion.

Patch time allocation is influenced by several environmental variables, including temperature. We present evidence that foraging temperatures directly influences patch time allocation in parasitoids, mainly through physiological mechanisms, and indirectly through changes in the nature and frequency of host behavioral defences. In the next decades, temperature variance and the frequency of extreme climatic events are expected to increase [START_REF] Ipcc | Climate change 2007: The physical science basis. Contribution of working group I to the fourth assessment report of the intergovernmental panel on climate change[END_REF]. From our results, we could predict that some species will be more affected than others, as a change in temperature modified the influence of an host rejection on patch leaving tendency in T. euproctidis but not in A. ervi. In parasitoid species attacking hosts that actively defend themselves, the influence of the predicted climate change on patch time allocation might be more closely related to the induced changes in host behavioral defences than the direct effect of temperature on the parasitoid itself. This result suggests that both parasitoids and hosts should be considered when investigating the role of climate on patch residence time. euproctidis females allowed to parasitize 20 E. kuehniella eggs at three temperatures.

  ): β = -0.243 ± 0.263, p = 0.666; Temperature(34): β = -0.864 ± 0.473, p = 0.167) or female ovipositions (Temperature(24): β = -0.047 ± 0.217, p = 0.827; Temperature(34): β = -0.058 ± 0.220, p = 0.791) on patch leaving tendency.

FIGURE CAPTIONS Figure 1 .

 CAPTIONS1 FIGURE CAPTIONS

Figure 2 .Figure 3 .

 23 Figure 2. Graphical representation of the interaction between temperature and the effect of

Figure 4 .Figure 5 .

 45 Figure 4. Effect of temperature on the walking speed of Trichogramma euproctidis females

  Figure 1

Table 1 .

 1 Estimated regression coefficients (β) and their standard errors, and hazard ratios (exp (β)) obtained for covariates of a Cox proportional hazard model relating patch residence time and behaviors observed in T. euproctidis.

		β ± s.e.	exp (β)	z	p-value
	Temperature(24)	2.189 ± 0.765	8.933	2.862	0.004
	Temperature(34)	2.654 ± 0.949	14.208	2.797	0.005
	Male oviposition	0.558 ± 0.166	1.747	3.496	< 0.001
	Female oviposition	0.576 ± 0.077	1.779	7.407	< 0.001
	Host rejection	-0.059 ± 0.035	0.942	-1.686	0.091
	Temperature(24) : rejection a	0.136 ± 0.062	1.146	2.201	0.027
	Temperature(34) : rejection a	0.754 ± 0.184	2.126	4.088	< .001
	a Interaction

Table 2 .

 2 Total number of ovipositions, host rejections, host feeding and resting periods measured on T. euproctidis females allowed to parasitize 20 cold-killed E. kuehniella eggs at three temperatures. Host rejection refers to a drilling not followed by egg deposition. Mean ± SE.

	616	.
	617

Table 3 .

 3 Estimated regression coefficients (β) and their standard errors, and hazard ratios (exp (β)) for significant covariates of a Cox proportional hazard model relating patch residence time, temperature and behaviors of A. ervi females allowed to attack four stages of M. euphorbiae.

		β ± s.e.	exp (β)	z	p-value
	Temperature(20)	1,817 ± 0.528	6,153	2,823	0.024
	Temperature(28)	2,344 ± 0.712	10,425	3,046	0.009
	Oviposition	-0,348 ± 0.104	0,706	-2,370	0.035
	Aggressive aphids	0,333 ± 0.092	1,395	2,972	0.016
	Escaping aphids	0,289 ± 0.084	1,335	2,838	0.031

Acknowledgements. We are grateful to Josiane Vaillancourt and Josée Doyon for help during this study. This research was part of the project "Impact of climate change on synchronism between pests and their natural enemies" supported by the Consortium on Regional Climatology and Adaptation to Climate Change, OURANOS (Québec, Canada), and the Fonds vert of the Québec Ministry of Agriculture, Fisheries and Food.