Joffrey Moiroux
email: joffrey.moiroux@univ-avignon.fr

Jacques Brodeur

Guy Boivin

Sex ratio variations with temperature in an egg parasitoid: behavioural adjustment and physiological constraint

Keywords: Sex allocation, climate change, haplodiploidy, egg fertilization

Sex allocation in haplodiploid arthropods is a central fitness-related decision that received much attention in insect parasitoids. The effect of temperature on the reproductive strategy of female parasitoids has rarely been addressed, despite evidence of its influence on other fitness-related traits. We explored mechanisms inducing the higher production of males typically observed at low and high temperature in parasitic wasps, considering that this pattern may result from both behavioural adjustment and physiological constraint. By observing the oviposition behaviour of an egg parasitoid, Trichogramma euproctidis, we were able to distinguish the sex ratio intended by the female from the secondary sex ratio, thereby discriminating between a change in behaviour and a physiological constraint on egg fertilization. More males emerged from eggs laid at low (+45%) or high (+80%) temperature than at medium temperature but the underlying mechanisms were different between the two conditions. We observed a behavioural change in sex allocation at high temperature, suggesting that laying sons may be advantageous at high temperature. At low temperature, the females intended a sex ratio similar to that at medium temperature but physiological constraints prevented egg fertilization during oviposition, resulting in an increased number of males emerging from eggs expected to be females. To our knowledge, this is the first experimental evidence that temperature modulates both sex allocation and physiological constraints in egg fertilization in parasitoids.

Introduction

Sex allocation is a central fitness-related decision that should be selected in response to environmental factors, as predicted by theory and confirmed in many empirical studies [START_REF] Charnov | The theory of sex allocation[END_REF][START_REF] West | Sex Allocation[END_REF]. Despite the role of temperature on life history traits (e.g., [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]) and behaviours (e.g. [START_REF] Traniello | Ant foraging behavior: ambient temperature influences prey selection[END_REF][START_REF] Le Lann | Thermal plasticity of metabolic rates linked to life history traits and foraging behaviour in a parasitic wasp[END_REF], only a few studies proposed theoretical models or empirical approaches to understand how organisms should adjust their sex allocation in response to this environmental factor. Among them, [START_REF] Werren | Facultative sex ratios and population dynamics[END_REF] developed a perturbation and a cyclical model, assuming that parents can adjust the sex ratio of their progeny. In their perturbation model, they predicted that if periods of exceptional mortality during cold or heat waves occur and that mortality of one sex is higher than the other, mothers would gain to bias sex allocation towards the sex with the highest mortality because of the expected reduced competition in the next generation. Alternatively, Roy et al. (2002) suggested that a change in sex allocation might be adaptive under such dramatic perturbations when one sex has better capacities than the other to disperse and survive under harsh conditions. The cyclical model developed by [START_REF] Werren | Facultative sex ratios and population dynamics[END_REF] predicted that adjustment of sex allocation in response to seasonal factors such as temperature should be selected if the relative reproductive success of males and females differs seasonally [START_REF] Werren | Facultative sex ratios and population dynamics[END_REF]. Based on this model, it is also likely that organisms may adjust their sex allocation according to microclimates encountered in their habitat.

Temperature can also physiologically modulate sex ratio. Temperature-dependent sex determination during embryonic development is known in species from the "reptiles" group (e.g., [START_REF] Ewert | Patterns of temperature-dependent sex determination in turtles[END_REF], Lang & Andrews 2005). In some birds [START_REF] Göth | Temperature-dependent sex ratio in a bird[END_REF] and insects [START_REF] Wilkes | Effects of high temperatures during postembryonic development on the sex ratio of an arrhenotokous insect, Dahlbominus fuliginosus (Nees) (Hymenoptera: Eulophidae)[END_REF], developmental temperature is also known to cause differential mortality between males and females, resulting in biased sex ratio. In haplodiploid arthropods, the fertilization of an egg produces a female offspring while no fertilization results in a male [START_REF] Flanders | The mechanisms of sex ratio regulation in the parasitic Hymenoptera[END_REF]. Temperature can thus physiologically influence sex ratio by sterilizing males or incapacitating sperm [START_REF] Wilkes | Effects of high temperatures during postembryonic development on the sex ratio of an arrhenotokous insect, Dahlbominus fuliginosus (Nees) (Hymenoptera: Eulophidae)[END_REF][START_REF] Wilkes | Environmental causes of variation in the sex ratio of an arrhenotokous insect, Dahlbominus fuliginosus (Nees) (Hymenoptera: Eulophidae)[END_REF][START_REF] King | Offspring sex ratios in parasitoid wasps[END_REF][START_REF] Nguyen | Heat stress affects male reproduction in a parasitoid wasp[END_REF]. Such an effect of temperature on egg fertilization implies that the primary sex ratio (i.e., the sex ratio at oviposition) or the secondary sex ratio (i.e., the sex ratio at emergence of the adult) may differ from the intended sex ratio, the sex ratio "decided" by the mother, because a physiological constraint occurs between the decision of the female and the end of the oviposition process. If a physiological constraint prevents egg fertilization, a son may be produced from an egg expected to be a daughter by the mother. Among animal taxa, parasitoid Hymenoptera is one of the most studied groups when investigating sex allocation variations with environment [START_REF] Ode | Parasitoid sex ratios and biological control[END_REF]. This is mainly explained by their reproductive system: mated females store sperm in their spermathecae and are generally able to control the sex of their progeny at oviposition by releasing or retaining sperm [START_REF] Flanders | The mechanisms of sex ratio regulation in the parasitic Hymenoptera[END_REF]). Typically, more males are produced when parents and/or eggs are exposed to low or high temperatures (for reviews see [START_REF] King | Offspring sex ratios in parasitoid wasps[END_REF][START_REF] Colinet | Insect parasitoids cold storage: A comprehensive review of factors of variability and consequences[END_REF], although some species produce a constant sex ratio across a large range of temperatures (e.g., [START_REF] Ichiki | Effects of temperature on immature development of the parasitic fly Bessa parallela (Meigen) (Diptera: Tachinidae)[END_REF][START_REF] Jacas | Thermal requirements of Fidiobia dominica (Hymenoptera: Platygastridae) and Haeckeliania sperata (Hymenoptera: Triсhogrammatidae), two exotiс egg parasitoids of Diaprepes abbreviates (Coleoptera: Curculionidae)[END_REF]). However, the mechanisms underlying sex ratio variations at low and high temperatures have rarely been investigated [START_REF] King | Offspring sex ratios in parasitoid wasps[END_REF]. By comparing intended sex ratio with primary or secondary sex ratio across a temperature gradient, one may discriminate between an ovipositional decision by the female and a physiological constraint on egg fertilization.

The main challenge when studying sex allocation is to discriminate between sex allocation and progeny sex ratio. Several methods have been proposed over the last decades, including genetic analyses (e.g., [START_REF] Ratnieks | Queen control of egg fertilization in the honey bee[END_REF][START_REF] Khidr | Primary and secondary sex ratios in a gregarious parasitoid with local mate competition[END_REF] or the observation of female behaviour during oviposition in species where the oviposition behaviour differs with the sex of the progeny deposited (e.g., [START_REF] Cole | A visible sign of a fertilization act during oviposition by an ichneumonid wasp, Itoplectis maculator[END_REF][START_REF] Suzuki | Sex allocation and effects of superparasitism on secondary sex ratios in the gregarious parasitoid, Trichogramma chilonis (Hymenoptera: Trichogrammatidae)[END_REF], van Dijken & Waage 1987). In the genus Trichogramma (Hymenoptera: Trichogrammatidae), when a female offspring is deposited, a pause in abdominal movements during oviposition, which corresponds to the fertilization of the egg by a spermatozoid, is observed. When a male progeny is deposited, no such pause is observed and the abdominal movements are continuous [START_REF] Suzuki | Sex allocation and effects of superparasitism on secondary sex ratios in the gregarious parasitoid, Trichogramma chilonis (Hymenoptera: Trichogrammatidae)[END_REF][START_REF] Martel | Unequal distribution of local mating opportunities in an egg parasitoid[END_REF]. It is thus possible in these species to calculate an intended sex ratio by observing the oviposition sequence of a female and subsequently to compare it to the primary or secondary sex ratio.

We investigated the effect of temperature during female oviposition on offspring sex ratio in the egg parasitoid Trichogramma euproctidis Girault. We compared the intended sex ratio to the secondary sex ratio of progeny laid at three temperatures to explore the behavioural and physiological mechanisms involved.

Material & Methods

Trichogrammatidae are minute endoparasitoids that usually parasitize eggs of a wide range of Lepidoptera species. The Trichogramma euproctidis (Girault) strain used in this study originates from Egypt and was maintained at 24 ±1˚C, 50% RH, and LD 16:8 h on coldkilled eggs of the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Using cold-killed eggs is common in studies on Trichogramma and does not affect oviposition behaviour of females nor quality of their progeny [START_REF] Daumal | Trophic quality of eggs of Ephestia kuehniella Zell. after freezing treatment for the rearing of Trichogramma species[END_REF][START_REF] Smith | Biological Control with Trichogramma: Advances, successes, and potential of their use[END_REF]). In Ephestia kuehniella eggs, T. euproctidis only lays one egg per oviposition sequence. Superparasitism may occur but it requires a second oviposition sequence.

Sex ratio experiment. We measured intended and secondary sex ratio of T. euproctidis at three temperatures during oviposition: 14, 24 or 34˚C. We considered that a 10-degree variation above and below the average temperature (24°C in our colony) is likely to occur during a cold or a heat wave, as well as across a day or across microhabitats. Such a variation did not induce any short-term lethal effects on parasitoids and females remained active despite changes in walking speed (unpublished data). Parasitized eggs were withdrawn from the colony and isolated in 300μL Beem ® polyethylene capsules with a drop of honey. The next morning, freshly emerged females were mated once with a virgin male, stored at 24˚C during one to five hours and transferred to a climate room at 14, 24 or 34˚C one hour before observation. After this acclimation period, 20 cold-killed E. kuehniella eggs, placed side by side in 4 lines of 5 eggs on a filter paper disc were offered to each female. The experimental arena was delimited by the plastic end piece of a camera (Ø20 mm) covered with Fluon ® to prevent females from climbing. Ovipositions 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). Oviposition sequences were divided into three components: host drilling, egg deposition (indicated by abdominal movements) and, if any, pause in abdominal movements during egg deposition (i.e., when an egg was fertilized). During these pauses, the whole body typically stopped moving for a few seconds (Video 1). Sex of each egg deposited was determined, based on the absence or presence of a pause in abdominal movements, and the intended sex ratio was calculated. This protocol was successfully used in several Trichogramma species (e.g. [START_REF] Suzuki | Sex allocation and effects of superparasitism on secondary sex ratios in the gregarious parasitoid, Trichogramma chilonis (Hymenoptera: Trichogrammatidae)[END_REF]), including T. euproctidis (e.g. Martel & Boivin 2007 -previously named T. turkestanica in this study-, [START_REF] Martel | Partial local mate competition in the wasp Trichogramma euproctidis: the role of emergence sex ratio on female mating behaviour[END_REF]. Duration of pauses in the oviposition sequence was also recorded to make sure that they were long enough, especially at 34˚C, to be accurately observed.

Superparasitism was quantified when a second oviposition sequence was observed on a parasitized host. Observation stopped when a female walked off the patch for more than 3 minutes. Wasps were thus exposed to 14, 24 or 34˚C for one hour before observation plus duration of patch exploitation, which increased as temperature decreased. At the end of an observation, the wasp was removed; eggs were isolated in 300μL Beem ® polyethylene capsules and incubated at 24 ±1˚C until emergence to determine the sex of the emerging adult. Emerging sex was then compared to intended sex and secondary sex ratio was compared to intended sex ratio for each mother. Superparasitized hosts were not included in the calculation of intended and secondary sex ratios as we were not able to directly compare intended and emerging sex of a given egg. Hatching success was measured for both sexes to examine if temperature during oviposition had a differential effect on males and females egg survival, which may explain differences in secondary sex-ratio.

Survival experiment.

A behavioural change in sex allocation in response to temperature may have been selected for by sex-dependent mortality during a cold or a heat wave [START_REF] Werren | Facultative sex ratios and population dynamics[END_REF]. To test this hypothesis, we measured survival to emergence of female and male parasitoids developing at 14, 24 or 32 ˚C (T. euproctidis failed to emerge at 34˚C).

Using only mated females would have limited the number of sons produced, since sex ratios were close to 0.13 at 24˚C. We thus used virgin females to produce sons. Sixty mated and virgin females were allowed to parasitize twenty eggs each at 24˚C. The number of ovipositions was noted for virgin females, since only sons could be produced, while the predicted number of sons and daughters was noted for mated females using the method described above. Eggs were then incubated at 14, 24 or 32˚C (twenty replicates per temperature), and the number of emerging sons and daughters was compared to the number of ovipositions. Hatching success was calculated for each sex at each temperature.

Statistical analyses.

We performed generalized linear models (GLMs) with temperature included as a factor to test for differences in the number of ovipositions, the number of intended and emerging males, and intended sex ratio and secondary sex ratio. We considered a Poisson error structure for the number of ovipositions and the number of expected and emerging males, and a binomial error structure for intended and secondary sex ratios. The "multcomp" package, available in R software, was used to perform post-hoc tests after a GLM. The numbers of intended and emerging males were compared with a Wilcoxon signed-rank test for each temperature, as well as the intended and secondary sex ratios. Mean durations of the pause in abdominal movements (indicating fertilization) was compared using an ANOVA with temperature as factor, with a Tukey test used as post-hoc test. The influence of sex, temperature and the interaction between these factors on hatching success of eggs laid at the three temperatures (reared at 24˚C) was analyzed using a GLM considering a binomial error structure. The number of superparasitized hosts was compared using a GLM with temperature as factor, considering a Poisson error structure. The influence of sex, temperature and the interaction between these two factors on hatching success of parasitoids laid at 24˚C and reared at the three temperatures was analyzed using a GLM, considering a binomial error structure.

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

Results

Sex ratio experiment. After removing superpasitized hosts, the number of hosts considered for analyses was respectively 549, 558 and 554 at 14, 24 and 34 ˚C. Observation of a pause appears to be a reliable method to predict sex of the progeny in T. euproctidis since 98% of intended females resulted in the emergence of a female at 24˚C while 96% of intended males resulted in the emergence of a male.

Temperature did not influence the number of ovipositions by T. euproctidis (Table 1), but females laid more sons at 34˚C than at 14˚C (p < 0.001) and 24˚C (p < 0.001), the last two treatments being similar (p = 0.541) (Figure 1). The intended sex ratio was thus higher at the highest temperature (GLM, df = 2, 89, z = 4.12, p < 0.001). Secondary sex ratio was also influenced by temperature (GLM, df = 2, 89, z = 3.74, p<0.001) but the pattern was different. It was significantly higher at 34˚C than at 14˚C (p = 0.005) and 24˚C (p < 0.001), intermediary at 14˚C and the lowest at 24˚C (Figure 1). The number of intended and emerging males were similar at 34˚C (V =114, p = 0.737) and 24˚C (V = 69, p = 0.315) while more males than expected emerged at 14˚C (V = 9, p < 0.001) (Figure 2), resulting in a secondary sex ratio higher than the intended sex ratio (V = 11, p < 0.001).

The pause duration in abdominal movements associated with egg fertilization significantly decreased when temperature increased (ANOVA, F2,89, = 178.5, p < 0.001) (Figure 3). The pause lasted a minimum of 2.4 seconds at 34°C, a duration long enough to be accurately quantified.

We only observed 14 cases of superparasitism out of the 1675 parasitized eggs and temperature did not influence superparasitism frequency (GLM, df = 2, 89, z = -0.53, p = 0.644). Every host on which two oviposition sequences were observed resulted in the emergence of two parasitoids while only one parasitoid emerged from host attacked only one time.

Hatching success was very high and was not affected by temperature, sex (Table 1) or the interaction between these two factors (Table 1).

Survival experiment.

Hatching success was significantly influenced by developmental temperature (p = 0.036), but we did not observe a significant effect of sex (p = 0.60) or interaction between both factors (p = 0.78). Hatching success was significantly lower at 32˚C (Mean [95% CI], ♂: 43% [33-52%], ♀: 51% [46-59%]) than at 14˚C (♂: 81% [75-88%], ♀: 86% [80-91%]) and 24˚C (♂: 90% [87-94%], ♀: 93% [90-96%]).

Discussion

This study explored the mechanisms inducing the higher production of males typically observed at low and high temperature in parasitoids, which can vary from a slight increase to the production of only males according to the species and the protocol used [START_REF] King | Offspring sex ratios in parasitoid wasps[END_REF][START_REF] Colinet | Insect parasitoids cold storage: A comprehensive review of factors of variability and consequences[END_REF]. Our main results are that (1) female T. euproctidis laid more sons at high temperature and (2) the secondary sex ratio was higher than the intended sex ratio at low temperature. These results indicate that temperature influences the sex allocation by the ovipositing female and physiologically constrains the secondary sex ratio.

Intended sex ratio. Variation in temperature can be a reliable indicator of environmental quality for arthropods and trigger the production of individuals with different capacities to develop, reproduce or disperse [START_REF] Wellington | Weather and insects[END_REF]. The increase in the intended proportion of T. euproctidis males at high temperature is likely an evolutionary response to these environmental conditions. In the field, parasitoid females may encounter not only temporal variations in temperature, during a heat wave or along seasonal variations, but also spatial variations, when foraging in habitats with different microclimates.

The influence of extreme variations in temperature on sex allocation has mainly been investigated in the perturbation model developed by [START_REF] Werren | Facultative sex ratios and population dynamics[END_REF], who predicted that if mortality is male-biased during a cold or a heat wave, parasitoid females would gain by laying more sons than under optimal weather conditions as competition would be reduced between sons. Hatching success indeed decreased when T. euproctidis was reared at high temperature, but mortality was not biased towards one sex under this climatic condition. The perturbation model thus does not explain the higher production of males at high temperature. Alternatively, [START_REF] Roy | Temperature and sex allocation in a spider mite[END_REF] proposed that a change in sex allocation may be an adaptive response to such perturbations if one sex has better capacities to disperse than the other. However, females are generally more prone to initiate flight and disperse than males [START_REF] Jones | Relative movement patterns of a tephritid fly and its parasitoid wasps[END_REF] in parasitic wasps, including a closely related species, Trichogramma minutum [START_REF] Forsse | Flight initiation in the egg parasitoid Trichogramma minutum: Effects of ambient temperature, mates, food, and host eggs[END_REF]). The cyclical model on seasonal variations developed by [START_REF] Werren | Facultative sex ratios and population dynamics[END_REF] is also unlikely to explain the variations in sex allocation observed in our study as it implies that both sexes should live long enough to mate with a partner that developed during a different season, while Trichogramma only lives for a few days. Moreover, temperature is not a reliable cue of seasonality in comparison to photoperiod because it fluctuates widely from day to day [START_REF] Danilevskii | Photoperiodism and seasonal development in insects[END_REF][START_REF] Danks | The elements of seasonal adaptations in insects[END_REF][START_REF] Bradshaw | Genetic response to rapid climate change: it's seasonal timing that matters[END_REF]. If seasonal variations in sex allocation occur, parasitoids should thus rely on photoperiod and not temperature.

It is thus likely that thermal variation in sex allocation has been selected in response to spatial thermal variations. Trichogramma are polyphagous species that parasitize eggs of a wide range of Lepidoptera species that are known to select their oviposition site according to microclimate, species laying their eggs in either shady habitats or sunny habitats [START_REF] Rausher | Larval habitat suitability and oviposition preference in three related butterflies[END_REF]. Trichogramma females are thus likely to exploit different microclimates at a small spatial scale, for example within a crop or along a hedgerow, and may vary offspring sex ratios accordingly if the relative reproductive success of males and females differs. This hypothesis would hold if Trichogramma females forage during a short period of time per day as observed in Drosophila parasitoids [START_REF] Fleury | Adaptive significance of a circadian clock: temporal segregation of activities reduces intrinsic competitive inferiority in Drosophila parasitoids[END_REF] -otherwise the hourly variations in temperature would alter the reliability of microclimate as an estimate of the average temperature for progeny development-or if they can integrate both temperature and time of the day. In insects, a strong correlation exists between body size and several life history traits, especially fecundity [START_REF] Honěk | Intraspecific variation in body size and fecundity in insects: A general relationship[END_REF], even though this relationship is not always true in the field (e.g. [START_REF] Kazmer | Field tests of the size-fitness hypothesis in the egg parasitoid Trichogramma pretiosum[END_REF]. Moreover female fecundity is more closely correlated with body size than the components of male reproductive success [START_REF] Honěk | Intraspecific variation in body size and fecundity in insects: A general relationship[END_REF]Jarošik and Honěk 2007). As body size generally decreases when temperature increases in ectotherms [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], it is likely that female fitness would be more affected by high temperature than male fitness. Laying more males may thus be adaptive under hot microclimate.

The increased male-biased sex ratio at high temperature might also be not adaptive but rather reflects a constraint. High temperature may alter the neural system of parasitoid females and interfere with oviposition decision. However, the high temperature (34°C) used in our experiment did not result in alteration of locomotor activity or foraging behaviours, it is thus likely that sex allocation variation was adaptive.

Secondary sex ratio. The low temperature treatment did not induce a strong thermal stress on parasitoid females since they remained active and parasitized as many hosts as did females at higher temperatures. However more males were obtained than what the female intended to deposit at low temperature whereas no difference between intended and secondary sex ratio was observed at other temperatures. Low temperature thus acted as a physiological sex ratio distorter during oviposition, as mating and rearing temperatures were kept constant at 24°C. This pattern does not result from a difference in mortality between sexes as hatching success was similar and very high (>95%) for males and females laid at low temperature and reared at 24°C. Moreover, this pattern cannot arise from misleading behavioural observations when assessing egg fertilization because the pause in abdominal movements lasted longer (> 15 sec) at low temperature and was easily noticeable. A physiological constraint on egg fertilization likely occurred during oviposition at low temperature. [START_REF] Richards | The rate of sperm locomotion in the cockroach as a function of temperature[END_REF] observed that sperm velocity in the American cockroach Periplaneta americana decreased with temperature. At low temperature, T. euproctidis spermatozoids probably did not move fast enough from the spermatheca to the egg despite the long pause in abdominal movements occurring during oviposition. A reduction in spermatozoid speed may thus affect fertilization, and consequently distort intended sex ratio. In insects, fertilization of eggs is induced by muscular contractions of the spermathecal sac that are controlled by a neural loop [START_REF] Clarke | Evidence of a neural loop involved in controlling spermathecal contractions in Locusta migratoria[END_REF]. It may also be that this neural loop was affected at low temperature or that the frequency or amplitude of the muscular contractions of the spermatheca and associated ducts decreased to the point that spermatozoid transfer was reduced.

Conclusion.

Models of climate change predict an increase in mean global temperature and in its variance in the next decades (IPCC 2007). Our study underlines the influence of such variations in temperature on insect behaviour, especially the allocation of sex. These climatic variations have significant demographic impacts, as a drop in temperature resulted in a 50% increase in secondary sex ratio while a rise in temperature resulted in a 65% increase in intended sex ratio, in comparison to females foraging under constant thermal conditions. In the next decades, sex ratio may thus be distorted under extreme climatic events either through a physiological constraint on egg fertilization or a behavioural adjustment in response to temperature. Our results suggest, together with other studies (e.g., [START_REF] Petchey | Predicting the effects of temperature on food web connectance[END_REF][START_REF] Rall | Temperature, predator-prey interaction strength and population stability[END_REF][START_REF] Sentis | Parsing handling time into its components: implications for responses to a temperature gradient[END_REF], that models predicting the evolution of insect populations and communities in response to climate change should integrate the effect of temperature on behaviour and physiology, and that more empirical studies are needed to understand how organisms will cope with changing environment.

Fig 1 .

 1 Fig 1. Box plots of intended sex ratio (white) and secondary sex ratio (grey) measured at

Fig 2 .Fig. 3 .

 23 Fig 2. Box plots of the number of males that emerged from hosts in which the female T.

Figure 1 Figure 2 Figure 3

 123 Figure 1

Table 1 .

 1 Number of ovipositions and hatching success, measured on T. euproctidis females allowed to parasitize 20 cold-killed E. kuehniella eggs at three temperatures. Mean ± s.e.

	Temperature (˚C)	Oviposition	♂ hatching success	♀ hatching success
	14	18.60 ± 0.23	0.96	0.95
	24	18.63 ± 0.33	0.96	0.96
	34	18.57 ± 0.33	0.96	0.95
	z value	0.13		0.48
	p-value	0.913		0.810


Joffrey Moiroux
email: joffrey.moiroux@gmail.com

Emilie Delava

Frederic Fleury²

Joan Van Baaren

Local adaptation of a Drosophila parasitoid: habitat-specific differences in thermal reaction norms

Keywords: climate, drosophila parasitoid, environmental canalization, genetic differentiation, Leptopilina boulardi, life history traits, phenotypic plasticity, temperature

Local climate is an important source of selection on thermal reaction norms that has been well investigated in cline studies, where populations sampled along altitudinal or latitudinal gradients are compared. Several biotic factors vary with climate but are rarely integrated as alternative agents of selection to climatic factors. We tested the hypothesis that habitat may select for thermal reaction norms and magnitude of phenotypic plasticity in a drosophila parasitoid, independently of the climate of origin.

We sampled populations of Leptopilina boulardi, a Drosophila parasitoid in two different habitats, orchards and forests. Orchards offer laying opportunities over small distances for parasitoids, with a low variability in the number of hosts per patch, while forests offer more dispersed and more variable patches. The sampling was realised in a temperate and a Mediterranean climate. We measured egg load, volume of eggs, longevity and lipid content for parasitoids reared at two temperatures.

Reaction norms were opposite for populations from forests and orchards for investment in reproduction, independently of the climate of origin. The maximal investment of resources in reproduction occurred at the lower temperature in orchards and the higher temperature in forests. Host distribution differences between habitats may explain these opposite reaction norms. We also observed a flatter reaction norm for egg load in forests than in orchards. This relative canalization may have been selected in response to the higher variability in laying opportunities observed in forests. Our results demonstrate the potential role of resource distribution in evolution of thermal plasticity.

Introduction

According to the theory of evolution, natural selection should drive organisms to an optimal combination of life history traits in a given environment [START_REF] Roff | The Evolution of Life Histories: Theory and Analysis[END_REF]. In ectotherms, the evolution of life history traits in response to temperature has been well investigated as this factor directly affects their fitness. To study the effect of temperature, authors have developed several approaches such as selection in the lab at different temperatures over several generations (e.g. [START_REF] Huey | Thermal Sensitivity of Drosophila melanogaster Responds Rapidly to Laboratory Natural Selection[END_REF][START_REF] Partridge | Rapid laboratory evolution of adult life-history traits in Drosophila melanogaster in response to temperature[END_REF], or clinal studies. By comparing populations or species originating from different climates and reared in a common garden (i.e. common temperatures), authors have observed clinal pattern for the great majority of traits in various taxa such as an increase in cold resistance in insects and gastropods (Addo-Bediako et al. 2000[START_REF] Hilbish | Latitudinal variation in freezing tolerance of Melampus bidentatus (Say) (Gastropoda:Pulmonata)[END_REF]) and growth rate in amphibians and fishes (Laugen et al. 2003[START_REF] Yamahira | Intra-vs interspecific latitudinal variation in growth: adaptation to temperature or seasonality?[END_REF] in populations/species from cold climates, or an increase in heat resistance in insects from hot areas [START_REF] Hoffmann | Opposing clines for high and low temperature resistance in Drosophila melanogaster[END_REF], Sørensen et al. 2005). [START_REF] Schmidt | Geographic variation in diapause incidence, life-history traits, and climatic adaptation in Drosophila melanogaster[END_REF], [START_REF] Schmidt | Reproductive diapause and life-history clines in north American populations of Drosophila melanogaster[END_REF] and [START_REF] Mitrovski | Postponed reproduction as an adaptation to winter conditions in Drosophila melanogaster: evidence for clinal variation under semi-natural conditions[END_REF] have observed that reproduction of Drosophila melanogaster females from colder environments was delayed compared to females from hotter areas while they lived longer. These studies have shown that in a common garden, organisms from different climates exhibit locally adapted life history traits and these differences are genetically determined.

Genetic variation in phenotypic plasticity of life history traits also exists along thermal clines. Climate may select for the thermal range and the slope of the reaction norm (i.e. a function with the value of the environmental variable as argument and the genotypic value of the trait as function value, [START_REF] De | Quantitative genetics of reaction norms[END_REF], which gives information on the level of phenotypic plasticity. There are two main assumptions. The first one is a shift of the reaction norm toward higher temperatures in warm-adapted populations or toward lower temperatures in cold-adapted populations, such as found in D. melanogaster populations for wing and thorax length [START_REF] Morin | Divergence of reaction norms of size characters between tropical and temperate populations of Drosophila melanogaster and D. simulans[END_REF] or offspring production (Trotta et al. 2006).

The second theoretical assumption, which has been rarely investigated, is a higher level of phenotypic plasticity in morphological and physiological traits [START_REF] Bradshaw | Evolutionary significance of phenotypic plasticity in plants[END_REF][START_REF] De Jong | Phenotypic plasticity as a product of selection in a variable environment[END_REF] and canalization (i.e. an absence of plasticity or at least a lower level of plasticity) in fitness-related traits in variable climates, as these traits should be buffered against environmental variation (Stearns andKawecki 1994, Wagner et al. 1997). [START_REF] Liefting | Plasticity versus environmental canalization: population differences in thermal responses along a latitudinal gradient in Drosophila serrata[END_REF] have observed that developmental rate, a fitness trait, of Drosophila serrata was more canalized in populations from temperate climate (i.e. a variable climate) than tropical climate (i.e. a stable climate) in response to different temperatures, whereas morphological traits were more plastic in temperate climate. [START_REF] Liefting | Habitat-specific differences in thermal plasticity in natural populations of a soil arthropod[END_REF] also observed habitat-specific differences in growth rate thermal plasticity by comparing springtail populations inhabiting close forests and heathlands. The authors found flatter thermal reaction norms in heath populations compared to forests populations. It is likely that microclimate explained these differences, as temperature and other climatic parameters are more variable in heathland. Most studies only consider climate as a selective force on thermal reaction norms in cline studies, but the environment experienced by an organism is the combination of many interacting biotic and abiotic factors. Other parameters than temperature vary with climate, such as interacting species [START_REF] Kraaijeveld | Geographic Patterns in the Evolution of Resistance and Virulence in Drosophila and Its Parasitoids[END_REF][START_REF] Fleury | Ecological and genetic interactions in Drosophila-parasitoids communities: a case study with D. melanogaster, D. simulans and their common Leptopilina parasitoids in southeastern France[END_REF] or nature and distribution of available resources [START_REF] Ellers | Life history evolution in Asobara tabida: plasticity in allocation of fat reserves to survival and reproduction[END_REF], and may potentially affect evolution of thermal plasticity. In parasitoids, fitness is directly dependent on the number of hosts parasitized.

Host distribution, varying with climate, may thus be a major force in the evolution of life histories of parasitic wasps, even stronger than climate, as suggested by [START_REF] Moiroux | Local adaptations of life history traits of a Drosophila parasitoid, Leptopilina boulardi: does climate drive evolution?[END_REF]. They found strong differences in life history traits between populations of parasitoids originating from contrasting climates, but temperature of origin did not explain these differences. They suggested that life histories have been selected mainly in response to the spatial and temporal distribution of hosts and not climatic factors, but the absence of data on host distribution did not allow the authors to provide evidence for this hypothesis.

In this study, we tested the hypothesis that temperature-induced plasticity in fitness traits may have differently evolved between habitats mainly characterized by different host distribution. We compared life histories of females of a Drosophila parasitoid, Leptopilina boulardi, from two different habitats with particular host distribution: orchards and forests.

During the fruiting season, orchards offer nearby laying opportunities with low variability in the number of hosts per patch while the number of hosts per patch and the distance between them are more variable in forests, where Drosophila lay their eggs on scarce fruits or sapfluxes on wounded trees. These habitat differences were investigated in a temperate and a Mediterranean area with two contrasting climates. We measured fecundity and longevity thermal reaction norms as they are major components of fitness and a trade-off generally occurs between these two traits in parasitoids (e.g. [START_REF] De Jong | Acquisition and allocation of resources-genetic (co)variances, selection, and life histories[END_REF][START_REF] Ellers | Fat and eggs: An alternative method to measure the trade-off between survival and reproduction in insect parasitoids[END_REF]. Lipid content thermal reaction norm was also studied as these nutrients represent the main and limited energetic resources allocated to survival and/or reproduction in parasitoids [START_REF] Rivero | Incorporating physiology into parasitoid behavioral ecology: the allocation of nutritional resources[END_REF][START_REF] Ellers | Life history evolution in Asobara tabida: plasticity in allocation of fat reserves to survival and reproduction[END_REF]. Moreover we tested for genetic differentiation between orchards and forests using AFLP to ensure that populations evolved separately in different habitats and may thus be locally adapted. If populations were not genetically distinct, two genotypes with different habitat preferences may occur in a same population, as found in Drosophila (Taylor & Powell 1976). We expected to find similar reaction norms between similar habitats sampled in both climate and different reaction norms between habitats from a similar climate.

Material and Methods

Leptopilina boulardi (Hymenoptera: Figitidae) is a solitary endoparasitoid that mainly attacks Drosophila melanogaster and D. simulans larvae (Diptera: Drosophilidae). This species occurs in the Mediterranean Basin, in tropical Africa, in North and South America and in the West Indies [START_REF] Fleury | Ecology and life-history evolution of frugivorous Drosophila parasitoids[END_REF].

Sampling and Field data

We sampled four populations of Leptopilina boulardi near the France-Spain border in orchards and forests in two contrasting climates. Populations were sampled in 2007 in Casteil (Pyrénées-Orientales, South of France; 42°31'55''N, 2°23'39''E) and Calonge (Cataluña, North of Spain; 41°51'45''N, 3°04'35"E), which have different climates (table 1). Casteil is located in Pyrenean Mountains where winters are cold and snowy and summers are sunny. There, we installed traps in apple trees in four close orchards in the village and in a forest of broadleaf trees, at four to five kilometres of the village.

Calonge is a coastal city located 100 km north-east of Barcelona, with a Mediterranean climate (mild winters, very hot summers, little precipitation). Traps were installed in four orchards (apple and peach trees) in the city, and in a Mediterranean forest (mainly cork oaks) at eight to ten kilometres from the orchards, in direction of the land.

In both climatic regions, the sampling was realised in September, respectively a few days before and after the harvest in France and Spain, when many apples are decaying on the ground and drosophila are thus very abundant in orchards. Twenty banana bait traps (i.

e. a plastic container with a 3 cm diameter hole covered with a mesh with 3 mm openings) were used per site. In the field, we counted the number of fruit flies captured per trapused as a proxy of hosts available for parasitoids-and we measured the host inter-patch distance (i.e. the distance from the trap to the closest patch with active drosophila) to estimate host availability. In the lab, fruit flies and parasitic wasps emerging from traps were also counted. Thirty females per population were randomly taken from the offspring to set up lab cultures. Leptopilina boulardi was the only parasitoid found at this time in our traps in all locations.

Preliminay tests

L. boulardi was considered as a proovigenic parasitoid (i.e. females mature all their eggs before the start of their adult life, [START_REF] Kopelman | Aspects of the reproductive biology of Leptopilina boulardi (Hymenoptera: Eucoilidae)[END_REF] unable to synthesize lipids during adult life, but [START_REF] Moiroux | Local adaptations of life history traits of a Drosophila parasitoid, Leptopilina boulardi: does climate drive evolution?[END_REF] found synovigeny (i.e. additional eggs are matured during adult life) and lipogenesis ability in some Iranian populations. We checked for these characters in our four populations but there was no evidence that synovigeny or lipogenesis ability occur in Pyrenean populations (appendix 1). Thus, egg load at emergence corresponds to the total fecundity. Moreover, we checked for the presence of Lbfv viral particles described by [START_REF] Varaldi | Infectious behavior in a parasitoid[END_REF] in some L. boulardi populations and responsible for an increased tendency to superparasitize and changes in several life history traits in infected females, such as fecundity or locomotor activity [START_REF] Varaldi | Cost induced by viral particles manipulating superparasitism behaviour in the parasitoid Leptopilina boulardi[END_REF]. We did not detect any evidence for viral infection in any population. It is thus unlikely that this virus explains differences in life histories (appendix 2).

AFLP analysis

As populations from orchards and forests were collected only a few kilometres apart, we studied if orchard and forest populations did belong to genetically distinct populations, using amplified fragment length polymorphism technique (AFLP) as neutral characters.

From the wasps emerging in the lab from banana bait traps, thirty females and males per population were isolated in Eppendorf tubes, frozen in liquid nitrogen and conserved at -80°C for genetic analyses. The AFLP procedure was performed according to the standard protocol described by [START_REF] Vos | AFLP: a new technique for DNA fingerprinting[END_REF] with slight modifications (Appendix 3). We used eight combinations of primers for the selective PCR.

Life history traits

Rearing. Drosophila melanogaster used as hosts in the cultures originated from strains collected in the Netherlands in 1960. Drosophila larvae used for parasitoid cultures were laid at 25°C during a two-hour period in a baker's yeast suspension. That short time allowed us to limit variation in host size that would be induced by a too long period between first and last oviposition. For our experiment, ten females L. boulardi oviposited in separate jars at 25°C for 24h in 100 second instar D. melanogaster larvae in a baker's yeast suspension. The parasitized larvae were then separated in two groups and placed at 20 °C or 25°C. At emergence, three females from each jar were placed in Eppendorf tubes, frozen in liquid nitrogen to stop instantaneously any metabolic activity and conserved at -80°C.

These females (i.e. 3 females per mother for each temperature) were then used for measurements of egg load, volume of eggs, lipid content and dry mass (i.e. 30 females tested per population per temperature). The remaining females emerging from the jars were equally separated at 20 or 25°C and used for measurement of longevity (per temperature:

France forest, n=37, France orchards, n=51, Spain forest, n = 43, Spain orchards, n=25).

Egg load and volume of eggs. Females were placed in a drop of Ringer's solution on a microscope slide and dissected under a binocular microscope (×40, Olympus SZX9). We removed both ovaries and counted eggs while the rest of the female's body was conserved for later lipid extraction. After counting, eggs were photographed (Olympus Camedia C3040) under a microscope (×4, Olympus BH2). Length (L) and width (w) of 30 eggs per female were measured with the numeric image analysis software AnalySIS to calculate egg volume (taken as a prolate spheroid volume: V=4/3πLw²).

Lipid content. Lipid quantity of the females was measured after removing eggs, using the protocol proposed and successfully used by [START_REF] Vernon | Developmental patterns of supercooling capacity in a subantarctic wingless fly[END_REF] and Terblanche et al. (2004). Wasps were dried at 40 °C during four days in an air oven and weighed to measure dry mass (Sartorius M4 ±0.001 mg). They were then left during two weeks in an Eppendorf tube containing 1 ml of chloroform/methanol solution (2:1) to extract lipids.

Females were then placed at 40°C during 24 hours after extraction and weighed again to deduce lipid quantity and lipid content (=lipid quantity/lean dry mass).

Longevity. Adult parasitoids were reared at the developmental temperature (20°C or 25°C), 50% RH, 12L: 12D in glass jars on an Agar-Nipagine substrate and were fed ad libitum with diluted acacia honey. Substrate was renewed every week. Dead individuals were counted and removed twice a day, each morning and evening.

Statistical analysis

AFLP analysis. The general matrix of presence/absence of DNA fragments was analyzed with the free software AFLP-Surv 1.0. That software estimates genetic diversity and population genetic structure using the [START_REF] Lynch | Analysis of population genetic structure with RAPD markers[END_REF] approach for diploid organisms, assuming an Hardy-Weinberg equilibrium. 1000 permutations were performed to calculate Hj (Nei's gene diversity), Fst and test for genetic differentiation between populations.

Life history traits. We performed generalized linear models with habitat as a paired fixed factor, temperature and climate region as fixed factors, and dry mass as a covariate to test for differences in egg load, volume of eggs and lipid content reaction norms. We considered a Poisson distribution for egg load while egg volume and lipid quantity were normally distributed. Dry mass, measured before lipid extraction, was integrated as a covariate to correct life history traits for the body size.

We used a Cox model with habitat as a paired fixed factor, temperature and climate region as fixed factors, and dry mass as a covariate to test for differences test for differences in longevity between populations.

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

Results

Field data:

In the field, more Drosophila of the melanogaster-group (D. melanogaster and D. simulans) were captured in orchards than forests (χ² = 47.59, p < 0.001), and the inter-patch distance was smaller in the first habitat (χ² = 13.31, p < 0.001). Fruit trees and Drosophila distribution were more homogeneous in orchards (i.e. a small variance in distance between trees and in number of Drosophila captured per trap) with rich patches (180 ± 30 apples per tree in France, 165 ± 35 in Spain), whereas host distribution was more stochastic (i.e. a high variance in number of Drosophila captured per trap and in distance between laying opportunities) in forests and patches were on average poorer (table 1). However we found in both French and Spanish forests some wild apple trees that represent high-quality patches (40 ± 15 apples per tree in France and Spain) for this habitat. Moreover, the ratio of Drosophila found per trap relative to the number of wasps, and hence interspecific competition, was similar in forests and orchards (Table 1).

AFLP analysis

770 loci were scored in this study and 641 of them were polymorphic. Our results indicate that we had similar genetic diversity in the four populations and that genetic differentiation occurs with the global data set (Fst = 0.08, p = 0). When testing separately the two regions, we observed genetic differentiation between orchards and forests in France (Fst = 0.062, p = 0.034) and Spain (Fst = 0.077, p = 0.027) despite distances less than 10 km.

Life history traits

We found significant main effects of habitat and rearing temperature on egg load, lipid content and longevity, with dry mass as a covariate (Table 2). The climatic area of origin (temperate vs Mediterranean) generally did not explain differences between populations. Females from forests invested more in reproduction, had a lower lipid content and lived shorter than females from orchards at 25˚C, while no difference occurred between populations at 20˚C except for longevity, forests females living slightly longer than orchards females.

The reaction norm slopes were opposite for investment in reproduction (i.e. egg load corrected for body size) between females from different habitats, independently of the climate of origin (significant temperature x habitat interaction in Table 2): females from orchards had significantly more eggs at 20°C than 25°C for a given size, whereas females from forests had more eggs at 25°C than 20°C (figure 1). The reaction norm of egg load was flatter in forests than in orchards (figure 3a).

The slopes were also different for lipid content as it decreased between 20 and 25°C in forests, whereas no difference was detected between temperatures in orchards (figure 3b).

Longevity decreased as temperature increased in all populations but reaction norms were flatter in females from orchards.

We did no detect any effect of habitat, temperature or dry mass on the volume of eggs.

Discussion

In this study, we tested the hypothesis that temperature-induced plasticity in fitness-related traits may have differently evolved between habitats characterized by different host distribution in a parasitic wasp, as this environmental factor is expected to be crucial in the evolution of parasitoid life histories. Our main results are that (1) genetic differentiation occurs between habitats over small geographical distances; (2) habitat of origin -that significantly differed in host abundance, variance and distribution-explained differences in thermal reaction norms of several life history traits and in the level of phenotypic plasticity; and (3) the maximal investment in reproduction occurred at the lower temperature in orchards and the higher temperature in forests, independently of the climate of origin.

Genetic differentiation

We first observed that populations from orchards and forests, less than 10 km from each other, were genetically distinct in both climates. Such differentiation between populations suggests that they have evolved separately in different habitats and may thus be locally adapted. This result differs from a study on Drosophila persimilis (Taylor & Powell 1976) in which authors concluded that habitat differences in allozyme frequencies over very short distances (less than 200m) were partly due to behavioral differences (i.e. habitat preferences) among genotypes.

Genetic differentiation over small distances has been rarely investigated in parasitoids but our result is consistent with the one observed between populations of the parasitic wasp Diaeretiella rapae less than 1km from each other in agricultural systems [START_REF] Vaughn | Population genetics of an opportunistic parasitoid in an agricultural landscape[END_REF]. Authors explained their results by the use of different hosts found in cultivated cereal or crucifer vegetable crops that were compared. To our knowledge, in Europe, Leptopilina boulardi only parasitizes Drosophila melanogaster and D. simulans which were both found in orchards and forests, D. melanogaster being largely dominant.

It is thus unlikely that genetic differentiation in L. boulardi can be explained by differences in host species, but several factors can favour it. First, haplodiploidy, as found in L. boulardi, is known to result in a more pronounced genetic structure of populations in comparison to diploidy [START_REF] Anton | Population structure of a large blue butterfly and its specialist parasitoid in a fragmented landscape[END_REF][START_REF] Packer | Population genetic aspects of pollinator decline[END_REF]. Secondly, this population subdivision may be favoured by fidelity to the habitat implying limited movements between forests and orchards [START_REF] Anton | Population structure of a large blue butterfly and its specialist parasitoid in a fragmented landscape[END_REF]. This is consistent with the dispersal ability observed in the field for other parasitoid species such as Asobara tabida [START_REF] Ellers | A field study of size-fitness relationship in the parasitoid Asobara tabida[END_REF], another Drosophila parasitoid, that generally fly less than one kilometre. Our result suggests that the expansion of Leptopilina boulardi from the south to the north of France observed these last years [START_REF] Fleury | Ecological and genetic interactions in Drosophila-parasitoids communities: a case study with D. melanogaster, D. simulans and their common Leptopilina parasitoids in southeastern France[END_REF]) may be more passive than active, for example thanks to the commercial fruits transports produced in the south, as described in Iran (Seyahooei et al. 2011).

Reaction norm patterns

We observed strong differences in reaction norms between females from forests and orchards under both climates. Females from orchards invested more in reproduction (i.e. had more eggs for a given body size, figure 1) at 20°C than 25°C while the opposite pattern was found for females from forests. It is unlikely that this difference in reaction norm is the result of evolution in response to microclimate because temperature is higher in orchards. A shift of the reaction norm toward higher temperatures should thus be observed there if plasticity evolved in response to this factor. At lower temperature, there is a physiological increase in lifespan in ectotherms as metabolic rate decreases and individuals use resources more slowly than at lower temperature [START_REF] Brown | Toward a metabolic theory of ecology[END_REF]). An explanation for the observed pattern may thus be that this increase in longevity may lead to an inversion in the source of limitation of female wasps in orchards. Females from orchards may be time-limited at 25°C because of a short lifespan and should thus invest some resources in lifespan to lay all their eggs. At 20°C, females have a long lifespan because of the low temperature and they could find many laying opportunities over very short distances in orchards. They may thus be egg-limited at this temperature i.e. females have more laying opportunities than eggs, and a shift of resources to egg load may be adaptative in this habitat to prevent egg-limitation.

In insects, flight activity is known to increase with temperature until an optimum (e.g. [START_REF] Cox | The effects of temperature on flight initiation in a range of moths, beetles and parasitoids associated with stored products[END_REF][START_REF] Williams | A new approach to the problem of the optimum temperature for insect activity[END_REF]). [START_REF] Forsse | Flight initiation in the egg parasitoid Trichogramma minutum: Effects of ambient temperature, mates, food, and host eggs[END_REF] have for example observed that, in the egg parasitoid Trichogramma minutum, between 70-80% of the wasps flew at 25 and 30 °C while less than 4% flew at 20 °C. In forests, females have to fly over long distances to find host patches, much farther than orchards parasitoids that can walk to find drosophila larvae. As temperature may increase their probabilities to find hosts, an increase in investment of resources into reproduction may thus be adaptive at higher temperatures.

That would explain that forests females invested more in reproduction, had a lower lipid content, and lived shorter than orchards females at the higher temperature, as a trade-of generally occurs between fecundity and the latter two traits [START_REF] De Jong | Acquisition and allocation of resources-genetic (co)variances, selection, and life histories[END_REF][START_REF] Ellers | Fat and eggs: An alternative method to measure the trade-off between survival and reproduction in insect parasitoids[END_REF][START_REF] Rivero | Incorporating physiology into parasitoid behavioral ecology: the allocation of nutritional resources[END_REF][START_REF] Ellers | Life history evolution in Asobara tabida: plasticity in allocation of fat reserves to survival and reproduction[END_REF].

Testing for more temperatures may be useful to clearly understand patterns of thermal reaction norms, as we measured these traits on females reared at only two temperatures.

However, the thermal range of L. boulardi is really narrow in comparison to other insects, diapause occurring under 20°C and no development being observed above 28°C in our populations. More populations from both habitats should also be sampled in temperate, Mediterranean and other climatic areas to confirm out results on the effect of host distribution on the evolution of thermal reaction norm in parasitoids.

Level of phenotypic plasticity

Populations exhibited different levels of sensitivity to temperature for egg load, lipid content and longevity, depending on the habitat of origin. These results suggest relative canalization i.e. a decrease in the magnitude of plasticity for these three traits. Theoretical models predict that canalization in fitness traits should occur in individuals from variable environments (Stearns & Kawecki 1994;[START_REF] Wagner | A population genetic theory of canalization[END_REF]. However, even if any environmental factor may vary geographically and select for canalization, there are only evidences for thermal canalization of traits in response to climate of origin (Stearns et al. 1995[START_REF] Liefting | Plasticity versus environmental canalization: population differences in thermal responses along a latitudinal gradient in Drosophila serrata[END_REF][START_REF] Gilchrist | Plastic and Genetic Variation in Wing Loading as a Function of Temperature Within and Among Parallel Clines in Drosophila subobscura[END_REF]. This is quite new to observe variations in the level of thermal plasticity according to the habitat structure of origin.

We observed that the slope of egg load thermal reaction norm, the main component of fitness in parasitoids, was flatter for populations from forests, whereas the slope of longevity and lipid content was flatter for populations from orchards. Temperature is expected to be more variable in orchards than in forests; it is thus unlikely that differences in microclimate explain the decrease in the level of plasticity occurring in forests for egg load. Our field data suggest that forests represent a variable environment for laying opportunities; therefore, according to theoretical predictions, canalization should evolve for egg load in this habitat, as observed in our study. Egg load would thus be buffered against environmental variation and maximised in every condition. The opposite pattern for lifespan and lipid content suggests another environmental factor -variable in orchards and stable in forests -to be responsible for these traits. Temperature is more variable in orchards and may have selected for canalization in this habitat, but we should probably also observe differences between populations from the temperate and Mediterranean areas if this environmental factor was involved, as thermal amplitude is highly different between the two climates. Another explanation may be that lipid content and longevity are fitness traits but also physiological traits. Increased plasticity is known to be selected under variable environments for morphological and physiological traits [START_REF] Bradshaw | Evolutionary significance of phenotypic plasticity in plants[END_REF][START_REF] De Jong | Phenotypic plasticity as a product of selection in a variable environment[END_REF]. Thus, increased plasticity for longevity and lifespan as physiological traits may have been selected in forests in response to the variability of host distribution. A third hypothesis is that relative canalization in one trait, egg load in this study, may be traded-off with the strength of plasticity in other traits such as longevity or lipid content.

Conclusion

Parasitic wasps from forests strongly differed from orchards parasitoids for thermal reaction norms and level of phenotypic plasticity. It is likely that parasitoid populations are locally adapted to the host distribution, and not climate, though a larger sample size would be needed to confirm this result. This result is different from studies on Drosophila, in which thermal reaction norms are expected to have evolved in response to climate. This difference between a parasitoid and its host is probably the consequence of the direct link that exists in parasitoids between the number of hosts encountered and their number of

  progeny. Our study suggests that biotic factors varying with climate should be integrated in clinal studies comparing populations originating from different climates. Leptopilina boulardi populations from different climatic zones of Iran. BMC Ecol. 11: 4 (online). Sørensen, J.G., Norry, F.M., Scannapieco, A.C. & Loeschcke, V. (2005). Altitudinal variation for stress resistance traits and adaptation in adult Drosophila buzzatii from the New World. J. Evolution. Biol. 18: 829-837. Stearns, S.C., Kaiser, M. & Kawecki, T.J. 1995. The differential genetic and environmental canalization of fitness components in Drosophila melanogaster. J. Evolution. Biol. 8: 539-557. Stearns, S.C. & Kawecki, T.J. 1994. Fitness sensitivity and the canalization of life-history traits. Evolution. 48: 1438-1450. Taylor, C.E. & Powell, J.R. 1976. Microgeographical differentiation of chromosomal and enzyme polymorphisms in Drosophila persimilis. Genetics. 85: 681-695. Terblanche, J.S., Klok, C.J. & Chown, C.L. 2004. Metabolic rate variation in Glossina pallidipes (Diptera: Glossinidae): gender, ageing and repeatability. J. Insect Physiol. 50: 419-428.

Figure 1 .

 1 Figure 1. Plot of egg load at emergence according to the dry mass for the four populations reared at 20°C (grey

Figure 3 .

 3 Figure 3. Mean ± s.e number of eggs (A), lipid content (B) and longevity (C) reaction norms across two

Table 1 .

 1 France Forest France Orchard Spain Forest Spain Orchard Climatic data recorded for Vernet-les-Bains, close to Casteil (France) and La Bisbal d'Empordà, close to Calonge (Spain), from 2003 to 2007 (Source: MeteoFrance & Gencat). We considered average of each parameter measured every day from April to October, which is the period of activity of Leptopilina boulardi. Thermal amplitude was calculated as the mean of the differences between maximum and minimum air temperature per month. Field data recorded in orchards and forests sampled in France and Spain in 2007 are also provided. Number of fruit flies/trap and distance to the closest patch were measured in the field and Drosophila and parasitoid emerging were counted in the lab. Error bars: ± s.e.

	Mean temperature	17.1		21.0
	Thermal amplitude	20.1		13.1
	Nb of fruit flies found/trap	39 ± 39	268 ± 21	66 ± 49	190 ± 25
	Inter-patch distance (m)	3.95 ± 2.3	1.95 ± 0.2	4 ± 2.4	2.2 ± 0.2
	Nb of fruit flies emerging/trap	9 ± 12	74 ± 24	13 ± 12	68 ± 25
	Nb of parasitoids emerging/trap	17 ± 11	123 ± 21	23 ± 13	116 ± 17

Table 2 .

 2 Results of the GLMs with the effects of habitat, climate of origin and rearing temperature on egg load (Poisson distribution), egg volume and lipid content (normal distribution) with dry mass as a covariate, and the corresponding Cox model for longevity.

ACKNOWLEDGEMENTS

We are grateful to Gladys Mialdea for the technical assistance in genetic analysis and to the owners of orchards who accepted our sampling during the harvest. This research was supported by the Ministère de l'Enseignement Supérieur et de la Recherche (grant to Joffrey Moiroux) and is part of the Marie Curie Excellence Chair COMPAREVOL research program (http://comparevol.univ-rennes1.fr/), ECOCLIM program founded by Region Bretagne and CLIMEVOL program founded by the Agence Nationale de la Recherche.