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

Guy Boivin

Jacques Brodeur

Ovigeny index increases with temperature in an aphid parasitoid: is early reproduction better when it is hot?

Highlights

 Ovigeny index (OI) has been developed to study timing of reproduction in insects.

 OI = ratio of the mature egg load upon emergence to the lifetime fecundity.

 We investigated the influence of temperature on OI in an aphid parasitoid.

 OI increased with temperature between 16 and 28°C but was the highest at 12°C.

 OI thermal variations were the consequence of physiological and adaptive changes. a

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Abstract. Studying relative investment of resources towards early and delayed reproduction is central to understand life history evolution since these traits are generally negatively correlated and traded-off against several other fitness components. For this purpose, ovigeny index (OI), which is calculated as the fraction of the maximum potential lifetime egg complement that is mature upon female emergence, has been developed in insects. Despite the central role of temperature on life history evolution in ectotherms, its influence on ovigeny index has never been tested. Adaptive models imply that OI should increase with temperature because of changes in body size, but the same influence may be expected considering physiological effects of temperature on egg maturation rate or amount of energy available. We investigated in the aphid parasitoid Aphidius ervi the influence of temperature experienced by the immature and/or the adult (from 12°C to 28°C) on ovigeny index and oviposition behaviour.

As predicted, OI increased between 16 and 28°C, i.e. females were able to reproduce earlier as temperature increased but this was traded off against a lower delayed reproduction. The highest OI was however observed at 12°, probably because this temperature was too low for females to mature eggs. Females that developed at 20°C and were transferred as adult at 24°C and 28°C had the highest ovigeny index and laid more eggs during the early oviposition period while those transferred at 16°C laid more eggs at the end of their life. Our results suggest that ovigeny index is not only influenced by body size -i.e. the adaptive explanation-but also by adult egg maturation rate, lifespan or amount of energy available -i.e. a physiological and adaptive explanation.

Keywords. Aphidius ervi; fecundity; life history; oviposition behaviour; synovigeny; trade-off.

Introduction

Life history theory aims at understanding how natural selection and other evolutionary forces shape organisms in the face of their environment (Roff 1992, Stearns 1992). This theory is based on the study of fitness components, so-called life history traits, such as age at maturity, number and size of offspring, progeny sex ratio or lifespan, that are assumed to be optimised in a given environment (Roff 2002). Among those traits, early reproduction is central since it is generally traded-off against several other fitness components such as future reproduction (e.g. [START_REF] Desouhant | Host and food searching in a parasitic wasp Venturia canescens : a trade-off between current and future reproduction[END_REF][START_REF] Harshman | The cost of reproduction: the devil in the details[END_REF], Stearns 1989), survival [START_REF] Jervis | Egg maturation strategy and survival trade-offs in holometabolous insects: a comparative approach[END_REF][START_REF] Papaj | Ovarian dynamics and host use[END_REF], or mobility [START_REF] Papaj | Ovarian dynamics and host use[END_REF], because of limited resources (de Jong and van Noordwijk 1992). Investigating environmental and intrinsic factors that constrain timing of reproduction is thus crucial to understand how species evolved and to help make prediction on potential evolution in a changing world.

Different approaches have been developed to investigate reproductive strategy. For oviparous insects which are able to mature eggs during their development and/or during adult life, [START_REF] Jervis | Life-history strategies in parasitoid wasps: a comparative analysis of 'ovigeny[END_REF] proposed an ovigeny index which is calculated as "the fraction of the maximum potential lifetime egg complement that is mature upon female emergence into the environment".

An ovigeny index of 1 indicates that all eggs are mature upon emergence, such organisms being called pro-ovigenic, while an index of 0 applies to females that emerge with no mature oocytes.

A continuum exists between these two extremes, and females that produce eggs during their reproductive life are called synovigenic; although this term may sometimes refer only to females with an ovigeny index of 0, females with an index between 0 and 1 being considered as "partly synovigenic". This ovigeny index has been first proposed to investigate the diversity of egg maturation strategies found in insects [START_REF] Jervis | Life-history strategies in parasitoid wasps: a comparative analysis of 'ovigeny[END_REF]. It offers the benefit of linking resource capital (i.e. larval resources carried-over to adult), income (through adult feeding) and expenditure (i.e. egg production and somatic maintenance) [START_REF] Jervis | Body size and the timing of egg production in parasitoid wasps: a comparative analysis[END_REF]. Several studies showed that such an index is an interesting tool to study life history evolution since it is related with other biological characteristics such as lifespan [START_REF] Jervis | Egg maturation strategy and survival trade-offs in holometabolous insects: a comparative approach[END_REF], female mobility or degree of polyandry [START_REF] Jervis | Egg maturation strategy and its associated tradeoffs: a synthesis focusing on Lepidoptera[END_REF]. When considering egg maturation strategy as a keystone in life history evolution, parasitoids -i.e. insects that lay their eggs on or in the bodies of other insects, the larva feeding on its host and eventually killing it [START_REF] Godfray | Parasitoids: behavioral and evolutionary ecology[END_REF]) -are very interesting as they are generally unable to synthesise lipids as adults (Visser et al. 2010).

Resources used for early reproduction will thus never be regained for delayed reproduction or for other traits.

Temperature is considered to play a central role in life history evolution in insects (e.g. Abram et al. 2016[START_REF] Angilletta | The evolution of thermal physiology in ectotherms[END_REF][START_REF] Chown | Physiological diversity in insects : ecological and evolutionary contexts[END_REF], including timing of reproduction. [START_REF] Ismail | What are the possible benefits of small size for energy-constrained ectotherms in cold stress conditions?[END_REF] observed for example that cold storage resulted in a higher early fecundity in the parasitoid Aphidius ervi, but those females were exposed to stressful temperatures which may be quite different from field temperatures. To the best of our knowledge, influence of non-stressful temperatures on ovigeny index has never been tested in insects, although temperature-dependent egg maturation received some attention in Lepidoptera [START_REF] Berger | What limits insect fecundity? Body size-and temperature-dependent egg maturation and oviposition in a butterfly[END_REF]. Non-stressful temperatures may have several influences on parasitoid reproduction strategy. First, higher egg maturation rate may occur because of higher metabolic rate with increasing temperature [START_REF] Berger | What limits insect fecundity? Body size-and temperature-dependent egg maturation and oviposition in a butterfly[END_REF][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].

Second, such influence of temperature on insect physiology implies that more energy is needed to sustain metabolism [START_REF] Brown | Toward a metabolic theory of ecology[END_REF]; the amount of energy available for delayed reproduction would thus decrease and the ovigeny index increase. Third, lifespan is known to commonly decrease as temperature increases because of higher metabolic rate (Brown et al. 2004, Nylin and[START_REF] Nylin | Plasticity in life-history traits[END_REF]. Organisms should thus invest more resources in early reproduction at high temperature to lay eggs before reaching average lifespan [START_REF] Olsen | Maturation trends indicative of rapid evolution preceded the collapse of northern cod[END_REF]). Finally, body size, which generally decreases with increasing developmental temperature in arthropods [START_REF] Atkinson | Temperature and organism size: a biological law for ectotherms?[END_REF], may influence ovigeny index. [START_REF] Ellers | Body size and the timing of egg production in parasitoid wasps[END_REF] proposed theoretical models to link ovigeny index with body size. Although initial egg load and lifetime potential fecundity typically increase with body size, ovigeny index is expected to decrease with increasing body size as a result of a lower relative increase in initial egg load compared to the relative increase in the maximum potential complement of eggs [START_REF] Ellers | Body size and the timing of egg production in parasitoid wasps[END_REF]. The adaptive explanation, proposed by these authors, is that small, short-lived females perceive the environment as more stochastic than large, longe-lived females because they sample fewer host patches. In a stochastic environment, individuals should allocate a larger proportion of resources to initial egg load [START_REF] Ellers | Egg load evolution in parasitoids[END_REF], small females should thus allocate a larger portion of resources to initial egg load than large females. Thorne et al. (2006) confirmed this negative relationship between body size and ovigeny index in the Drosophila parasitoid Aphaereta genevensis, considering intraspecific variability in body size, while [START_REF] Fischbein | Linking reproductive and feeding strategies in the parasitoid Ibalia leucospoides: does feeding always imply profit[END_REF] found a constant relationship between these two traits in the woodwasp parasitoid Ibalia leucospoides.

Based on direct physiological influence of temperature on egg maturation rate or resources available and on indirect influence through changes in lifespan or body size, small-warm developed females living at high temperature should be more pro-ovigenic and reproduce earlier than females that developed and live at lower temperature. We tested this prediction by investigating the influence of developmental and adult temperature on ovigeny index and oviposition behaviour in a synovigenic parasitic wasp, the aphid parasitoid Aphidius ervi (Sequeira and Mackauer 1994), which is known to be unable to synthesize lipids as adult (Visser et al. 2010). We measured the influence of temperature on ovigeny index and oviposition behaviour in A. ervi by rearing females at five temperatures, from 12°C to 28°C, during their entire life. We also distinguished the effects of developmental temperature and adult temperature by transferring females that developed at optimal temperature (20°C) to different temperatures. Such protocol would help to understand if ovigeny index and oviposition behaviour are mainly determined by body size or metabolic rate. If body size alone explains changes in ovigeny index, we should observe no difference between transferred females. Any change in oviposition behaviour and ovigeny index would thus result from a change in egg maturation rate and/or resources available and/or lifespan, which are all dependent on metabolic rate [START_REF] Berger | What limits insect fecundity? Body size-and temperature-dependent egg maturation and oviposition in a butterfly[END_REF][START_REF] Brown | Toward a metabolic theory of ecology[END_REF]). Such study should help to predict how climate change (IPCC 2014) may influence parasitoid and aphid population dynamics, and may be useful to develop better conditions for mass-rearing parasitoids commonly used for biological control [START_REF] Boivin | Aphid parasitoids in biological control[END_REF].

Material & Methods

Insect colonies. Aphidius ervi is a solitary koinobiont parasitoid that attacks several aphid species. Our colony was established with individuals bought from BioBest Canada Company (Leamington, Canada). Parasitoids were reared in cages on the potato aphid Macrosiphum euphorbiae at 20±1°C, 60±10% RH, 16L: 8D photoperiod. The aphid colony was initiated with individuals collected from potato fields near Québec City, Canada, and maintained on potato plants, Solanum tuberum, cultivar "Norland" under the same conditions.

Ovigeny index.

General procedure and preliminary experiment on lifespan. Ovigeny index is defined as the initial egg load divided by potential lifetime fecundity [START_REF] Jervis | Life-history strategies in parasitoid wasps: a comparative analysis of 'ovigeny[END_REF]. Since these two variables cannot be measured on a given individual, it is necessary to pair different females to calculate the index. In our experiment, we paired sisters to reduce genetic variability between individuals used for calculation of ovigeny index. Moreover, only paired sisters that had similar tibia length were used for calculation (Thorne et al. 2006). Initial egg load can be measured by dissecting females at emergence while estimation of potential lifetime fecundity requires an experimental design in which females can mature eggs throughout their life, providing them oviposition opportunities (Vayssade et al. 2012). To estimate the influence of non-stressful temperatures on potential lifetime fecundity, A. ervi females were reared at different temperatures, were offered aphids at one third (early oviposition) and two third (late oviposition) of their average lifespan, and were dissected one day before average lifespan. Since longevity changes with temperature in most ectotherms (e.g. [START_REF] Atkinson | Temperature and organism size: a biological law for ectotherms?[END_REF][START_REF] Colinet | Manipulation of parasitoid size using the temperaturesize rule: fitness consequences[END_REF], we first calculated average lifespan of A. ervi at the five tested temperatures. Parasitoids were randomly collected from our laboratory colony and females were allowed to parasitise 25 second instar aphids in a Petri dish (Ø90 mm) at 20±1°C. Those parasitised aphids were reared on potato plants at 12,16,20,24 or 28±1°C until mummification. This thermal range was chosen as it corresponds to the linear part of Aphidius ervi thermal curve for locomotor activity (Gilchrist 1996) and mainly for development time, although 28°C was a suboptimally high temperature for this parameter (Moiroux et al., unpublished data). Mummies were then individually placed in gelatine capsules until parasitoid emergence, which was observed twice daily (at 8:00 and 18:00). Thirty emerging females per temperature were placed separately in a Petri dish (Ø90 mm) containing moistened cotton and honey with a male. They were reared at their developmental temperature and mortality was checked twice daily (at 8:00 and 18:00).

Females' average lifespan was next used to determine ages of early and late oviposition and age of dissection at the five temperatures (Table 1).

Initial egg load, potential lifetime fecundity and ovigeny index: influence of developmental

and adult temperature. Parasitoids were randomly collected from the colony and fifteen couples were each placed for a day with 60 second instar aphids in a Petri dish (Ø90 mm) at 20±1°C. Parasitised aphids from a cohort were divided into 4 groups of 10 aphids that were reared on potato plants at 12, 16, 20, 24 or 28±1°C, respectively. The remaining 10 aphids were reared at 20±1°C for a second experiment. This experimental design allowed allocating sisters among temperatures in order to avoid lineage effect. Once parasitised aphids turned into mummies, they were placed individually in gelatine capsules until parasitoid emergence, which was observed every two hours from 07:30 to 18:30, aphid parasitoids rarely emerging at night. At emergence, two females per mother (i.e., 30 females per temperature) were frozen and their hind tibia length measured under a stereomicroscope using PixeLINK® µScope Microscopy Software (Ottawa, Canada). To measure initial egg load, these females were next placed in a drop of Ringer's solution on a microscope slide and dissected under a stereomicroscope to count the number of mature eggs, which were distinguished from immature ones by their shell-like chorion and their lemon-shaped size (Völkl and Stadler 1991). To measure potential lifetime fecundity, the remaining females were placed in a Petri dish (Ø90 mm) containing moistened cotton and honey together with a male from the same thermal treatment until they reached one third of their average lifespan. Females were then allowed to parasitise 60 second-instar aphids placed on a potato leaf in a Petri dish for 4 hours at 20°C. Those aphids were then reared at 20±1°C until mummification. The number of mummies was divided by an average mummification rate measured at 20°C (i.e. 0.765, J. Moiroux, unpublished data) to evaluate the number of eggs laid by parasitoids. After this early oviposition period, A. ervi females were once again isolated with a male at their developmental temperature until they reached two third of their average lifespan, and the same oviposition procedure was conducted. Parasitoids were thus offered 120 aphids throughout their lifetime, which is closed to the maximum lifetime fecundity measured by [START_REF] Ismail | What are the possible benefits of small size for energy-constrained ectotherms in cold stress conditions?[END_REF] in this species. After this late oviposition, females were kept at their developmental temperature with a male in a Petri dish containing moistened cotton and honey until they reached one day before average lifespan. Females were then frozen and their tibia and the number of mature eggs in their ovaries were measured as described above.

The number of eggs laid during early and late oviposition was added to the egg load measured in ovaries to calculate the potential lifetime fecundity of each female. An ovigeny index was next calculated by dividing initial egg load by the potential lifetime fecundity of a sister with a similar tibia length.

Potential lifetime fecundity and ovigeny index: influence of adult temperature. To discriminate between the influence of temperature encountered during the entire life vs. adult life, parasitoid females emerging from the 10 remaining aphids reared at 20°C in the previous experiment were placed following emergence at 12, 16, 24 or 28±1°C. The same procedure as the one described above was performed to calculate potential lifetime fecundity and ovigeny index. Ages of early and late oviposition and age at dissection were the same than for individuals tested in the previous experiment at a given temperature.

Statistical analyses. We used the survival package provided by R software to test for differences in longevity between the five thermal treatments, using a Weibull distribution.

The effect of developmental temperature on body size of parasitoid females was analysed with an ANOVA, as data were normally distributed. We compared initial egg load using a Generalised Linear Model (GLM) with temperature as fixed factor and tibia length as a covariate, considering a Poisson distribution of errors. GLMs were also used to compare the number of eggs laid at early and late oviposition periods, the number of eggs counted in ovaries at the end of life, and the lifetime potential fecundity using thermal treatment as fixed factor and tibia length as a covariate. Females tested in the first and second experiment were treated altogether in these analyses, to compare the influence of temperature on reproductive parameters during the whole life vs. adult life. Ovigeny index was compared using an ANCOVA with temperature as fixed factor and average tibia length of paired sisters as a covariate. We used the multcomp package, available in R software, to perform post hoc tests following a GLM. All statistical analyses were carried out using R software version 2.14.1 (R Development Core Team, 2011).

Results

Parasitoid longevity significantly decreased as temperature increased (Survival analysis, χ2 = 41.36, p <0.001; Table 1). Female ages of early and late oviposition and age of dissection deduced from longevity measures are presented in Table 1 for the five temperatures.

Developmental temperature significantly influenced A. ervi female body size (ANOVA, F = 675.5, p < 0.001); tibia length decreased with increasing temperature (Table 2). Body size differed between treatments, except for females that developed at 16°C which were similar in size to females that developed at 12 and 20°C. Initial egg load was significantly influenced by the interaction between developmental temperature and body size (GLM, χ² = 10.27, p = 0.021), body size (GLM, χ² = 21.04, p < 0.001), but not by temperature alone (GLM, χ² = 0.027, p = 0.412). Small warm-developed females (i.e. 24 and 28°C treatments) emerged with more eggsrelatively to their body sizethan larger females, while no significant difference was observed between females that developed at 12, 16 and 20°C (Table 2).

Number of eggs laid during early and late oviposition, egg load measured one day before average lifespan, and lifetime fecundity were significantly influenced by thermal treatments, body size and the interaction between thermal treatment and body size (Table 3). The number of eggs laid during early oviposition increased with temperature experienced during immature and adult phases and with decreasing body size, while the number of eggs laid during the last oviposition period was the lowest at 12°C-developed females, the highest at 16-and 20°Cdeveloped females and intermediary at 24-and 28°C-developed females. Egg load measured at the end of life and lifetime fecundity decreased as immature and adult temperature increased and as body size decreased between 16 and 28°C, but the lowest values were detected at 12°C. Those relationships were observed considering both temperature alone and the interaction between temperature and body size. We did not observe any significant differences between females from experiment 1 which experienced different temperatures during their lifetime, and females from experiment 2 which experienced different temperatures only as adults, except for the number of eggs counted at the end of life which was higher in females that developed at 20 °C and were transferred at 12, 24 and 28°C than females that respectively developed and were reared as adults at 12, 24 and 28°C (Figure 1).

Ovigeny index was significantly influenced by thermal treatments (ANCOVA, F = 18.77, p = 0.003), body size (ANCOVA, F = 36.04, p < 0.001) and the interaction between these two variables (ANCOVA, F = 41.30, p < 0.001). It increased between 16 and 28°C, but was the highest at 12°C (Figure 2). We observed linear negative relationships between body size and ovigeny index for each temperature, except at 12°C. The intercept of the model temperature*body size was significantly higher at 28 and 24°C than at 20 and 16°C. Females that developed at 20°C and next transferred at 12, 24 or 28°C had respectively a lower ovigeny index than females kept at 12, 24 or 28°C for their whole life, while no difference was observed between females kept at 16°C and those transferred from 20 to 16°C (Figure 3).

Discussion

We investigated in an aphid parasitoid the influence of non-stressful temperature experienced by the immature and the adult on ovigeny index and oviposition behaviour. We considered a thermal range which mainly corresponds to the linear part of A. ervi thermal curve. We expected to observe a higher ovigeny index in females reared throughout their life at high temperature than in cold-developed females living at low temperature as adult, because of higher metabolic rate [START_REF] Berger | What limits insect fecundity? Body size-and temperature-dependent egg maturation and oviposition in a butterfly[END_REF][START_REF] Brown | Toward a metabolic theory of ecology[END_REF] or smaller body size [START_REF] Ellers | Body size and the timing of egg production in parasitoid wasps[END_REF]. Our results partly support this prediction since the ovigeny index increased between 16 and 28°C although we also observed the highest value in females reared at the lowest temperature (i.e. 12°C). Females that developed at 20°C and were transferred as adult at 24°C and 28°C had the highest ovigeny index and laid more eggs during the early oviposition period while those transferred at 16°C laid more eggs at the end of their life. These results suggest that ovigeny index is not only influenced by body size but also by adult egg maturation rate, lifespan or amount of energy available.

Adaptive and physiological explanation. The proportion of the maximum potential lifetime egg complement that is mature upon female emergence, i.e. ovigeny index, is expected to have evolved in parasitoids in response to environmental variables such as host spatial distribution [START_REF] Ellers | Why are so few parasitoid wasp species pro-ovigenic?[END_REF]) and life history traits such as egg size [START_REF] Ellers | Why are so few parasitoid wasp species pro-ovigenic?[END_REF] or body size [START_REF] Ellers | Body size and the timing of egg production in parasitoid wasps[END_REF]. The increase in ovigeny index that we observed in A. ervi between 16 and 28°C supports predictions proposed by [START_REF] Ellers | Body size and the timing of egg production in parasitoid wasps[END_REF] who stated that small parasitoids -those which developed at high temperatures in our study-should allocate more of resources to early reproduction, thereby increasing ovigeny index, as they experience a more stochastic environment because they sample fewer patches than the larger females with a longer life span. This prediction has previously been confirmed in a Drosophila parasitoid (Thorne et al. 2006) and has been suggested by Abram et al. (2016 b) who observed that small Telenomus podisi females shifted resource allocation to early reproduction compared to larger females. Such negative relationship between ovigeny index and body size is also partly supported when A. ervi females were kept at a given temperature their entire life; but may be limited to species with a low ovigeny index as proposed by [START_REF] Fischbein | Linking reproductive and feeding strategies in the parasitoid Ibalia leucospoides: does feeding always imply profit[END_REF] who did not observe this relationship in a species with a high index.

Results from our second experiment in which females developed at 20°C and were transferred at a different temperature following adult emergence suggest that others factors than body size influence ovigeny index. Indeed, we observed an increase in ovigeny index with temperature experienced by adult females with similar body size. Moreover, females kept throughout their life at a given temperature had similar ovigeny index than females that developed at 20°C and were transferred at the same given temperature. Body size alone thus cannot explain temperature-dependent variations in ovigeny index in A. ervi.

Variations in ovigeny index are generally considered to be adaptive but temperature-mediated variations may be mainly physiological and result from the increase in metabolic rate with increasing temperature observed in ectotherms [START_REF] Brown | Toward a metabolic theory of ecology[END_REF][START_REF] Clarke | Temperature and the metabolic theory of ecology[END_REF]. A higher metabolic rate implies a higher consumption rate of energy and consequently fewer resources available for other life history traits, including reproduction. Such effect would result in a higher ovigeny index since females exposed to high temperatures would have less resources to mature additional eggs. Moreover, egg maturation rate is expected to increase with metabolic rate and thus with temperature [START_REF] Berger | What limits insect fecundity? Body size-and temperature-dependent egg maturation and oviposition in a butterfly[END_REF]); females will thus mature eggs faster at high temperature and have a higher ovigeny index. Temperature-mediated egg maturation rate may partly explain variations in ovigeny index in A. ervi, as observed in Lepidoptera [START_REF] Berger | What limits insect fecundity? Body size-and temperature-dependent egg maturation and oviposition in a butterfly[END_REF]). This hypothesis is partly supported by our results, especially the highest ovigeny index observed in A. ervi females which developed at 12°C, or in females that developed at 20°C and were transferred at 12°C. It is likely that egg maturation was slowed down at low temperature and few eggs were produced during adult life, resulting in the highest ovigeny index. Similar results were observed by [START_REF] Ismail | What are the possible benefits of small size for energy-constrained ectotherms in cold stress conditions?[END_REF] who exposed A. ervi to cold storage, resulting in an increase in early fecundity.

Physiological and adaptive explanations are not mutually exclusive. Life expectancy decreases with increasing metabolic rate, and thus with temperature, in insects [START_REF] Nylin | Plasticity in life-history traits[END_REF], including A. ervi (Table 1). Females should thus allocate more resources to early reproduction -as indicated by the higher number of eggs laid during the early oviposition period (i.e. at one third of average lifespan) in females transferred to the highest temperatures-in order to maximise reproduction before dying. This higher investment in early reproduction would however be paid off by a lower quantity of resources available for survival and other life history traits [START_REF] Harshman | The cost of reproduction: the devil in the details[END_REF]Zera 2007, Papaj 2000). Such physiological and adaptive hypothesis may partly explain why females which were kept throughout their life at a given temperature had similar ovigeny index than females that developed at 20°C and were transferred at the same given temperature.

Behavioural variations. In this study, variations in timing of reproduction were both physiological and behavioural since warm-developed females laid more eggs early in life (i.e. one third of average lifespan) while females which developed and lived at 16 or 20°C laid more eggs at later age (i.e. two third of average lifespan). Females that developed at 20°C and were transferred at 24 or 28°C as adults also shifted their egg laying early in life, proportionally to their lifespan. Several non-exclusive mechanisms may explain such behavioral changes with temperature, as proposed by Abram et al. (2016 a) who recently developed a conceptual framework to unify thermal physiology and behavioural plasticity. Parasitoids may first change their behaviour according to changes in metabolic rate, which may inform females about their lifespan, or egg maturation rate, a direct physiological effect of temperature [START_REF] Berger | What limits insect fecundity? Body size-and temperature-dependent egg maturation and oviposition in a butterfly[END_REF].

Such mechanism is referred as a medium-term kinetic effect (Abram et al. 2016 a), i.e. insects change their behaviour according to temperature-dependent physiological changes. Parasitoids may also integrate temperature as an environmental cue for decision-making. Females may perceive hot environment as a risky one and thus lay their eggs early in life. Amat el al. (2006) observed for example that parasitoid females laid more eggs following a sudden change in temperature, and proposed that such integrated mechanism may explain their results.

Conclusion and significance to biological control.

To our knowledge, this study is the first to explore influence of non-stressful temperatures on ovigeny index in parasitoids. In A. ervi, ovigeny index fluctuated from 0.24 at 24°C to 0.53 at 12°C, i.e. an estimated range of 0.29. Such large temperature-mediated range should favour the selection of egg maturation strategy adapted to different climates, as suggested by [START_REF] Moiroux | Local adaptations of life-history traits of a Drosophila parasitoid, Leptopilina boulardi: does climate drive evolution[END_REF] who observed pro-ovigenic and synovigenic populations originating from contrasting climates in a Drosophila parasitoid, Leptopilina boulardi. Predicted global warming (IPCC 2014) may therefore rapidly select for a higher ovigeny index in the next decades since our results suggest that early reproduction is better when temperature increases. However, it will remain difficult to disentangle the physiological effects of temperature via metabolic rate on ovigeny index from adaptive allocation of resources when studying thermal response in ectotherms.

This plasticity in the ovigeny index could be used in biological control programmes. Different strains of parasitoids could be produced by varying immature developmental temperature according to the need of growers: a first strain developing at high temperature and characterised by a high ovigeny index that would result in a rapid impact at reducing pest populations, tradedoff against a lower lifetime fecundity and lower longevity, and a second strain developing at low temperature and characterised by a low ovigeny index that would have a more long-term impact on pest populations. Such possibility should however be investigated further since a higher immature temperature may decrease ability of Aphidius ervi to successfully parasitise aphids [START_REF] Moiroux | Temperature influences host-instar selection in an aphid parasitoid: support for the relative fitness rule[END_REF]. Moreover, photoperiod should be considered in such studies since this environmental factor is known to influence reproduction in parasitoids (e.g. Sagarra et al. Vayssade, C., Martel, V., Moiroux, J., Fauvergue, X., van Alphen, J.J.M., van Baaren, J. 2012.

The response of life-history traits to a new species in the community: a story with Drosophila parasitoids from the Rhône and Saone valleys. Biological Journal of the Linnaean Society, 107, 125-165 Visser, B., Le Lann, C., den Blanken, F.J., Harvey, J.A., van Alphen, J.J.M., Ellers, J., 2010. Loss of lipid synthesis as an evolutionary consequence of a parasitic lifestyle. Proceedings of the National Academy of Sciences of the United States of America, 107, 8677-8682. Völkl, W., Stadler, B., 1991. Interspecific

  ., Mackauer, M., 1994. Variation in selected life-history parameters of the parasitoid wasp, Aphidius ervi: influence of host developmental stage. Entomologia Experimentalis et Applicata, 71, 15-22. Stearns, S.C., 1989. Trade-offs in life-history evolution. Functional Ecology, 3, 259-268. Stearns, S.C., 1992. The evolution of life histories Oxford University Press, New York. Thorne, A.D., Pexton, J.J., Dytham, C., Mayhew, P.J., 2006. Small body size in an insect shifts development, prior to adult eclosion, towards early reproduction. Proceedings of the Royal Society B: Biological Sciences, 273, 1099-1103.

Figure 1 .

 1 Figure 1. Number of eggs laid during early (dark grey) and late oviposition (light grey) period, and egg load measured in ovaries one day before average lifespan (white) for Aphidius ervi females reared at five temperatures throughout their life (left panel) or reared at 20°C and next transferred at a different temperature upon emergence (listed as 20-x, right panel). Different letters within each panel mean significant differences thermal treatments for the potential lifetime fecundity, i.e. the sum of the three reproductive parameters cited above. Mean ± s.e.

Figure 2 .

 2 Figure 2. Relationship between ovigeny index and body size in Aphidius ervi females reared at five temperatures: 12°C (open diamonds, n=28), 16°C (filled diamonds, n=29), 20°C (grey squares, n=29), 24°C (open triangles, n=27) and 28°C (filled triangles, n=23). Intercept was higher for females reared at 24 and 28 °C than females reared at 16 or 20°C. No significant relationship at 12°C.

Figure 3 .

 3 Figure 3. Ovigeny index in Aphidius ervi reared at 20°C and next transferred at 12 (n=27), 16 (n=28), 20 (n=28), 24 (n=26) or 28°C (n=25) upon emergence. Different letters mean significant differences. Mean ± C.I.

Table 1 .

 1 larval competition between Lysiphlebus testaceipes and Aphidius colemani (Hym., Aphidiidae). Journal of Applied Entomology, 111, 63-71. Average lifespan (Mean ± s.e.), age of early and late oviposition, and age of dissection used for measurements of potential lifetime fecundity when Aphidius ervi females were reared at five temperatures. Different letters mean significant differences between thermal treatments.

	1
	2
	3
	4	Temperature (°C)	Average lifespan (days)	Age of early/late oviposition	Age of dissection
	5	12	15.6 ± 1.5 a	5/10	14
		16	14.9 ± 1.2 a	5/10	14
	6	20	11.4 ± 1.2 b	4/8	10
		24	9.1 ± 1.0 c	3/6	8
		28	6.9 ± 0.7 d	2/4	6
	7
	8

Table 2 .

 2 Tibia length (1) and initial egg load of Aphidius ervi females reared at five temperatures, tibia length (2) of individuals used for estimating potential lifetime fecundity, and tibia length (3) of individuals transferred from 20°C to other temperatures upon emergence. Different letters mean significant differences between thermal treatments, considering body size as covariate for initial egg load. Mean ± s.e.

Acknowledgements. We are grateful to Cécile Gerardin 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.

Table 3. Results of generalized linear models testing the influence of body size, temperature and interaction between these two factors on reproductive parameters measured on Aphidius ervi females reared at five different temperatures.


Joffrey Moiroux

Cécile Le Lann

Majeed A Seyahooei

Philippe Vernon

Jean- Sébastien Pierre

Joan Van Baaren

Jacques J M Van Alphen

Local adaptations of life history traits of a Drosophila parasitoid, Leptopilina boulardi: does climate drive evolution?

Keywords: Life-history theory, reproductive strategies, trade-offs, temperature, precipitation, resource distribution, parasitoids

1. Climate is an important source of selection on life histories, and local adaptations to climate have been described in several cline studies. Temperature is the main climatic factor that has been considered as an agent of selection, whereas other factors may vary with it, such as precipitation.

2. We compared life-history traits of five populations of Leptopilina boulardi, a Drosophila parasitoid, originating from contrasting climates. Referring to cline studies, we hypothesised shorter lifespan, earlier reproduction, and lower lipid content in populations from the hottest and driest areas if life histories have been selected in response to temperature and/or humidity.

3. Our results are opposite to these predictions. Females from humid and mild climates invested more in early reproduction and lived for fewer days than females from dry and hot areas, which were synovigenic (i.e. they matured additional eggs during adult life) and able to synthesise lipids during adult life.

4. We suggest that life histories are more adapted to host distribution than to climatic factors.

Drosophila patches are more abundant in the humid area, allowing the parasitoids to spend less energy and time finding hosts. This may result in selection for early reproduction tradedoff against longevity. In the hot and dry climate, females have to fly large distances to find host patches. Synovigeny, a long lifespan, lipogenesis, and high dispersal ability may be adaptive there. This is the first time that between-population differences in the ability to synthesise lipids have been described in parasitoids.

Introduction

Life histories evolve in response to environmental conditions [START_REF] Stearns | The Evolution of Life Histories[END_REF]. In the last decade, climate as a selective factor has been well investigated as it can affect physiology and/or behaviour of organisms, particularly that of ectotherms. Genetic differences due to climate-dependent selection onlife-history traits have been shown in several studies of geographic variation, where populations originating from cold and hot climates have been compared in common garden experiments (e.g. [START_REF] Liefting | Plasticity versus environmental canalization: population differences in thermal responses along a latitudinal gradient in Drosophila serrata[END_REF]. Comparative studies of populations of insect species, particularly Drosophilidae, at a continental scale or along an altitudinal cline have documented between-population variation of several but isolated lifehistory and morphological traits. For example, increase in thorax length [START_REF] Dahlgaard | Behavioral differentiation in oviposition activity in Drosophila buzzatii from highland and lowland populations in Argentina: Plasticity or thermal adaptation[END_REF] and decrease in knock-down resistance [START_REF] Sørensen | Altitudinal variation for stress resistance traits and thermal adaptation in adult Drosophila buzzatii from the New World[END_REF] with an increase in altitude have been recorded in Drosophila buzzati, while starvation resistance increases with increasing latitude in Drosophila burchii [START_REF] Griffiths | Clinal variation and laboratory adaptation in the rainforest species Drosophila birchii for stress resistance, wing size, wing shape and development time[END_REF].

Environmental conditions do not only affect one or more traits in isolation but can affect the balance of allocation of resources to a large range of life-history traits. For example, development time generally decreases with increasing temperature but adult body size also decreases in a majority of insects studied [START_REF] Sibly | How rearing temperature affects optimal adult size in ectotherms[END_REF]. Fecundity also increases with temperature whereas longevity decreases, as a trade-off occurs between these two traits [START_REF] Nunney | The effect of temperature on body size and fecundity in female Drosophila melanogaster: Evidence for adaptive plasticity[END_REF]. Thus reproductive allocation strategies and trade-offs can show clinal variation, e.g. an increase in early reproduction with altitude traded off against a shorter lifespan [START_REF] Norry | Altitudinal patterns for longevity, fecundity and senescence in Drosophila buzzatii[END_REF]. Correlations between traits involve studying a large range of traits when interested in climate-dependent evolution of life histories and that consideration has to be taken into account in comparative studies.

Most studies considered only a temperature gradient to explain evolution of life-history traits but humidity and precipitation gradients that occur on a cline should also be explored.

Desiccation resistance is the only life-history trait that has been considered as a result of differences in humidity [START_REF] Dahiya | Desiccation and starvation tolerance of adult Drosophila: Opposite latitudinal clines in natural populations of three different species[END_REF][START_REF] Gilchrist | Clinal patterns of desiccation and starvation resistance in ancestral and invading populations of Drosophila subosbcura[END_REF] but other traits may be under selection of humidity and precipitation, such as timing of egg-laying. Here we propose a study on local adaptations of a large range of life-history traits in a Drosophila parasitoid, Leptopilina boulardi, to understand how resource allocation strategies have evolved in response to climate. We compared populations originating from a mild and humid area with populations from a very dry and hot desert area and a population from a cool environment but intermediate in precipitation. We considered temperature and humidity gradients as climatic factors that may result in adaptation of life histories.

As no formal theory exists about the impact of temperature on life histories, we used data from empirical studies to formulate hypotheses. The few cline studies that have been published can be used to make predictions about the impact of temperature on selection in several life-history traits in insects. In altitudinal and latitudinal clines studies, lifespan [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] of Drosophila melanogaster females, the main host of L. boulardi, from colder environments exceeds that of females from hotter areas while their reproduction is delayed [START_REF] Mitrovski | Postponed reproduction as an adaptation to winter conditions in Drosophila melanogaster: evidence for clinal variation under semi-natural conditions[END_REF][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]. Moreover, a low wing loading [i.e. the ratio of fresh mass to wing surface that corresponds to the pressure exerted by the wings on the surrounding air [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]] is thought to be selected under cool conditions to compensate the lower wing beat frequency at lower temperatures in insects [START_REF] Unwin | Wingbeat frequency, temperature and body size in bees and flies[END_REF]. Compared with cold or mild environments, we can expect that lifespan of the parasitoid wasps from the hottest environment may be reduced. This shorter lifespan may have resulted in the selection of an early reproduction traded-off against lower lipid content at emergence as a trade-off generally occurs between these two traits in parasitic wasps [START_REF] Ellers | Life history evolution in Asobara tabida: plasticity in allocation of fat reserves to survival and reproduction[END_REF]. Lipids have to be considered when investigating parasitoids life histories as they represent the main energetic resources allocated to survival and reproduction in parasitoids.

Importance of humidity in selection of life histories has been poorly investigated and no cline studies can be used to make predictions because only temperature has been considered as an explanative factor. However similar results of selection for the driest areas as in the hottest areas may be expected as lifespan may be reduced because of a high risk of desiccation and early reproduction may be adaptive in very dry environments where favourable reproductive periods are relatively short in comparison to humid environments.

To resume, compared with cold and dry or mild and humid environments, we may observe shorter lifespan, earlier reproduction, lower lipid content and higher wing loading in populations from the hottest and driest areas. We discuss our results in the light of the abovementioned predictions for the importance of temperature and humidity in the selection of life histories.

Material & Methods

Leptopilina boulardi (Hymenoptera: Figitidae) is a solitary endoparasitoid that mainly attacks D. Melanogaster and D. simulans larvae (Diptera: Drosophilidae) living in fermenting fruits. This species occurs principally in regions with a Mediterranean climate and in tropical Africa.

Leptopilina boulardi females have been described as proovigenic [START_REF] Kopelman | Aspects of the reproductive biology of Leptopilina boulardi (Hymenoptera: Eucoilidae)[END_REF], i.e. they mature all their eggs before the start of their adult life.

Source of laboratory samples

Five populations of L. boulardi from contrasting climates (Table 1) were collected in Iran (Fig. 1) in July 2006 using twelve banana bait traps per site. Each open trap (i.e. a plastic container with a 3 cm diameter hole covered with a mesh with 2 mm openings) was colonised by five to twenty females and several hundreds of offspring were produced in each of them.

Leptopilina boulardi and L. heterotoma were the only parasitoids and Drosophila melanogaster and D. simulans were the only fruit flies we found in our traps in all locations.

From the offspring thirty females per population were taken to set up lab cultures. Two populations -Chalus and Seyakhaloriginated from a humid and mild environment, in the coastal plain of the Caspian Sea where orchards, the main habitat for fruit flies, and fruit trees are abundant during a long season. Moreover, there are a lot of forests where Drosophila can be found in the area. Dorcheh and Zamankhan strains originated from a hot and very dry area, although the location of our traps was in the valley of a river fed by melting snow from the mountains that keeps also water throughout summer and allows for agriculture under irrigation in a narrow zone bordering the river. In this area, fruits are produced in few and distant orchards. These few orchards represent the only habitat where fruit flies are present as the remaining area is desert. The last strain -Sorkhabad -originated from the Alborz Mountains, with the coolest climate but intermediate in humidity and number of rainy days between Chalus/Seyakhal and Dorcheh/Zamankhan. These five localities have been chosen within a relatively small area in comparison to continental-scales studies to compare populations with recent isolation (less than 100 km between Seyakhal, Chalus and Sorkhabad and between Zamankhan and Dorcheh, 400 km between Dorcheh/Zamankhan and Chalus/Seyakhal/Sorkhabad).

We carried out between-populations crossings to ensure that all populations indeed belonged to the same species, L. boulardi. For each population, five crossings were performed with males of the other strains. We obtained fertile females in all crossings confirming the conspecificity of the populations, as only inseminated mothers can produce females in haplodiploid species.

Cultures

Drosophila melanogaster used as hosts in the cultures originated from strains collected in the Netherlands in 1960. Ten L. boulardi females oviposited in separate jars for 48h in 100 to 120 second instar D. melanogaster larvae laid in a two hours period (similar size for every host) in a baker's yeast suspension. The parasitized larvae were reared at 25°C, 50% RH, 12L: 12D in glass jars on an Agar-Nipagine substrate. This temperature was chosen as it is the one at which a maximum of offspring is produced for all populations. Progeny of a same mother was separated in three groups. The first one was used for measurements of morphometric traits, number and volume of eggs and lipid content, the second one for the measurement of longevity and the last one to measure activity. Adults were fed with diluted acacia honey distributed ad libitum.

Presence of Lbfv virus. [START_REF] Varaldi | Infectious behavior in a parasitoid[END_REF] described the presence of Lbfv viral particles in some L. boulardi populations and showed that this virus causes an increased tendency to superparasitize in infected females. These viral particles are also involved in changes in other life history traits such as egg load. We first checked the presence or absence of superparasitism to be sure that the virus could not explain variations in other life history traits between populations. We used a protocol similar to the one described by [START_REF] Varaldi | Infectious behavior in a parasitoid[END_REF].

All the experiments were conducted at 25°C. Twenty females (24 to 48h old) originating from each population were isolated for 16 hours (from 6 pm to 10 am) with ten Drosophila melanogaster larvae (48h old) foraging in an arena consisting of a thin yeast spot spread over an agar layer poured into a 5 cm diameter Petri dish. After two days of incubation, Drosophila larvae were dissected and parasitoid eggs were counted to estimate superparasitism.

No self-superparasitism occurred in our experiments. We never found more than a single egg per parasitized larva in experiments with females from all five populations. There is thus no evidence for the presence of the virus Lbfv in these populations. Hence, Lbfv can not explain between-populations differences in egg load and other life history traits.

Life history traits

Morphometric traits, number and volume of eggs and lipid content were measured on twenty females from each population at 24 hours, five days and ten days after emergence except for Sorkhabad strain which had been lost before measurements at five and ten days of life.

Females were placed in Eppendorf tubes, frozen in liquid nitrogen to stop all metabolic activity instantaneously and conserved at -80°C. These females constituted the first group of individuals.

Morphometric traits. After defrosting, fresh mass (FM) was determined with a microbalance (Sartorius M4 0.001mg) while left wing length was measured (0.01mm) with the numeric image analysis software Pegasus Pro V4 under a binocular linked to a camera video (JVC KY-F). Wing loading was then calculated as the ratio of fresh mass to squared wing length.

Wing loading corresponds to the pressure exerted by the wings on the surrounding air [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]. Thus the cost of transport is influenced in an important way by the wing area that supports the body mass [START_REF] Starmer | Causes of variation in wing loading among Drosophila species[END_REF]. The lower the wing loading, the less costly the flight is.

Investment in early and lifetime reproduction. Females used for morphometric traits were placed in a drop of Ringer's solution on a microscope slide and dissected under a binocular (×40, Olympus SZX9). Eggs were removed from both ovaries and counted, while the rest of the female's body was conserved for later lipids extraction. After counting, eggs were placed under a microscope (×4, Olympus BH2) and photographed (Olympus Camedia C3040).

Length (L) and width (w) of 30 eggs of each female were measured with the numeric image analysis software AnalySIS to calculate eggs volume (taken as a prolate spheroid volume: V=4/3πLw²). Investment in reproduction was calculated as the result of number of eggs*volume of eggs. This parameter is a measure of energy invested into reproduction.

Lipid content. After eggs had been removed from all the females measured of different ages, we measured lipid quantity of the females using the protocol proposed by [START_REF] Vernon | Developmental patterns of supercooling capacity in a subantarctic wingless fly[END_REF] and [START_REF] Terblanche | Metabolic rate variation in Glossina pallidipes (Diptera: Glossinidae): gender, ageing and repeatability[END_REF]. Wasps were dried at 40 °C during four days in an air oven and weighed to measure dry mass. Then parasitoids were left during two weeks in an Eppendorf tube containing 1 ml of chloroform/methanol solution (2:1) to extract lipids. Then females were again placed at 40°C during 24 hours and weighed again to measure lipid quantity and lipid content (=lipid quantity/lean dry mass). This lean dry mass (without eggs) was used as an indication of body size in our statistical analyses.

Longevity. This parameter was measured in the second group of wasps (n=20 per population). Females from each population were placed at 25°C on an Agar-Nipagine substrate and were fed with honey. The substrate was renewed every two weeks. Dead individuals were counted and removed twice a day, each morning and evening.

Activity. This parameter was measured in the third group of individuals (n=20 per population for Chalus, Seyakhal and Zamankhan strains). The behavioural data acquisition and analysis software Ethovision (Noldus Information Technology 1997) allowed us to record and analyze locomotive activity of parasitoids. Females were fed with honey during the first 24 hours of their adult life. Then they were isolated in Petri dishes ( 5 cm) thirty minutes before the start of the recording. For each run, four one-day old females belonging to the three populations were filmed simultaneously (camera Panasonic CCTV) in a windowless room during 24 hours: 12 hours with artificial light and 12 hours with red light only, simulating night.. The contrast between individuals and a white background was used to detect females and record their position twelve times per minute. The total distance covered and the average velocities were automatically calculated from the track records.

Statistical analysis

Variations in life history traits between populations.

Correlations between body size parameters (i.e. fresh mass, lean dry mass and wing length) and (1) reproductive parameters and lipid reserves for the first group and (2) longevity for the second group and (3) activity for the third group of individuals were examined for each population and for the complete data set.

ANCOVA and ANOVA were performed to analyze the effect of population and age on the different traits mentioned above. Post hoc tests were done to compare individuals of a same age from different populations and individuals of different ages within a population. Analyses of covariance (ANCOVA) were used when a correlation occurred between the trait tested and body size to remove any effect of size from the measurement and ANOVA tests were used when no correlation was detected.

The number of eggs (r² = 0.6, P < 0.0001) and lipid quantity (r² = 0.64, P < 0.0001) increased with lean dry mass but the volume of eggs and activity were neither correlated with lean dry mass (P > 0.05) nor fresh mass (P > 0.5) respectively. No correlation was found between longevity and dry mass in any population (P > 0.05).

Thus we used ANCOVA to examine differences in number of eggs, in investment in reproduction and in lipid quantity by incorporating lean dry mass as a covariate. ANCOVA was also used to test differences in squared wing length using fresh mass as a covariate. This test was performed to compare wing loading. Differences in the volume of eggs and in activity (i.e. total covered distance and velocity) were analysed with ANOVA. Differences in activity between day and night for a population were tested with Student's t tests.

The survival package provided by R software was used to test differences in longevity between populations, using a Weibull distribution.

Climatic factors in selection of life-histories.

We made Generalized Linear Models using the global dataset of populations to test for correlations between all life history traits and climatic factors: air temperature, thermal amplitude, relative humidity, number of rainy days and precipitation. Climatic factors were included in the base model and removed one by one to obtain the minimum adequate model.

The selection was done on the basis of the AIC criterion (Akaike Criterion, Akaike 1974). Pvalues reported are those of the last model that contains the variables.

All analyses were carried out using R software version 2.5.0 (R Development Core Team, 2007).

Results

Variations in life history traits between populations.

Wing loading (Fig. 2). Females from Chalus, Seyakhal and Sorkhabad had a similar wing loading. Dorcheh and Zamankhan females had a similar wing loading but lower than the other populations (ANCOVA, F= 48.98, df = 5, P < 0.0001). Wing loading did not differ between the different ages for Chalus, Seyakhal and Sorkhabad populations but it increased in Zamankhan (ANCOVA, F = 48.25, P < 0.001) and Dorcheh (ANCOVA, F = 29.4, P < 0.001) populations between five days and ten days of adult life as their fresh mass increased. No change in number of eggs was observed in Chalus females (ANOVA, F = 0.008, df = 2, P = 0.96) or Seyakhal (ANOVA, F = 0.005, df = 2, P = 0.99) between 24 hours and ten days of adult life. Dorcheh females increased the number of eggs in their ovaries (ANOVA, F = 12.67, df = 2, P < 0.001) during the first five days of adult life (P = 0.012) but no change was observed between five and ten days (P = 0.99). Zamankhan females increased the number of eggs in their ovaries during the first ten days of adult life (ANOVA, F = 6.7, df = 2, P < 0.001) (Fig. 3A).

Investment in early and lifetime reproduction (

The Chalus wasps had smaller eggs than the other populations (ANOVA, F = 2.69, P = 0.03) which did not differ between each other. We did not observe any difference in the volume of eggs in any of the populations between the three ages (ANOVA, P > 0.6). We found a negative correlation between the number and the volume of eggs in Chalus females (r² = 0.53, P <0.001). This correlation was not found in any other population (P > 0.05).

Chalus and Seyakhal females invested more in early reproduction (i.e. number of eggs*volume of eggs, corrected by lean dry mass) than Dorcheh and Zamankhan females while Sorkhabad individuals had an intermediate investment in early reproduction (ANCOVA, F = 4.078, P < 0.0001). (Fig. 3B).

Lipid content (Fig. 4).

Chalus, Seyakhal and Sorkhabad populations had similar lipid contents and females of these populations emerged with a higher lipid content than Dorcheh and Zamankhan females (ANCOVA, F = 48.98, P < 0.0001) (Fig. 4A).

No change in lipid content was observed for Chalus (ANCOVA, F = 0.009, P = 0.97) and Seyakhal females (ANCOVA, F = 0.007, P = 0.99) during adult life. In contrast, females of the Dorcheh (ANCOVA, F = 9.27, P < 0.001) and Zamankhan populations (ANCOVA, F = 11.28, P < 0.001) increased their lipid content considerably during the first ten days of adult life (Fig. 4B).

Within populations, negative correlations were found for all populations between number of eggs and lipid content (n = 20, r² = (0.34-0.63), P < 0.05).

Longevity. Seyakhal females had the shortest lifespan (longevity = 11.5 ± 1.47 days, mean ± SE) while Zamankhan females lived significantly longer (longevity = 21.7 ± 0.82 days) than females of any other population (Survival analysis, Chisq = 65.46, P < 0.005). Dorcheh females (longevity = 20.2 ± 0.7days) lived longer than Sorkhabad (longevity = 18.7±0.91days) and Chalus individuals (longevity = 17 ± 1.7 days) that had a similar longevity.

Activity (Fig. 5). Seyakhal and Chalus females covered significantly shorter distances than Zamankhan females during the experiments (ANOVA, F = 8.497, df = 2, P < 0.05). The same pattern was found for velocity (ANOVA, F = 4.522, df = 2, P < 0.05). Moreover, Chalus (Student's test, t = 2.18, df = 19, p = 0.032) and Seyakhal (Student's test, t = 2.79, df = 19, p = 0.015) females were more active during day while Zamankhan individuals were more active during night (Student's test, t = -4.95, df = 19, P < 0.005).

Climatic factors in selection of life-histories (Fig. 6).

Climatic factors explained patterns for wing loading (t = 2.625, P < 0.0001) and investment in early reproduction (t = 7.73, P < 0.0001), lipid content (t = 4.8, P < 0.0001), and longevity (t = 10.95, P < 0.001). For these life history traits, the minimum adequate generalized linear models excluded air temperature and thermal amplitude as explanatory factors but included relative humidity, number of rainy days and amount of precipitation.

Investment in early reproduction (Fig. 6A), lipid content at emergence, and wing loading increased with these factors but longevity decreased (Fig. 6B).

Discussion

Compared [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][START_REF] Mitrovski | Postponed reproduction as an adaptation to winter conditions in Drosophila melanogaster: evidence for clinal variation under semi-natural conditions[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].

Our parasitic wasps were collected in a relatively small area in comparison to the above cited latitudinal cline studies on Drosophilidae. The mean temperatures of our coolest and hottest climate were respectively 24.4°C and 31.6°C. Thus, our temperature range was quite smaller but on average warmer than in the other cline studies. This could possibly explain why we did not obtain similar results. Moreover, only Drosophila species were studied until now and parasitoids may respond differently to selection by temperature. Yet, one would expect that temperature should have similar effects in all ectotherms as it directly affects their physiology.

We therefore think that it is more likely that differences in life histories between our populations result from selection by another environmental factor than temperature. We considered precipitation and humidity in our study as possible agents of selection and found a strong link between number of rainy days/ precipitation amount and life history traits (i.e. investment in early reproduction, longevity, wing loading and lipid reserves). This result suggests that precipitation may be more important than temperature in selection of lifehistories.

To our knowledge, no evidence of direct effect of humidity on selection of life history traits has been shown except for desiccation resistance [START_REF] Gilchrist | Clinal patterns of desiccation and starvation resistance in ancestral and invading populations of Drosophila subosbcura[END_REF][START_REF] Dahiya | Desiccation and starvation tolerance of adult Drosophila: Opposite latitudinal clines in natural populations of three different species[END_REF], but patterns for this trait are not really clear. Other studies on a humidity-dependent selection are needed to understand if this climatic factor may directly affect other life history traits than desiccation resistance.

A difference in climate involves more than only the abiotic environment as it may also affect the biotic environment e.g. interspecific interactions in the community or distribution of resources. We did not collect any other parasitoid species than L. boulardi and L. heterotoma in all locations so there is no evidence that differences in interspecific competition may explain differences in life history traits. Moreover, it is unlikely that variation in host species may explain differences between populations as we only found D. melanogaster and D.

simulans in our traps. Our sampling areas differed in climate but also in landscape and biotic environment. Water availability determines fruit distribution -the main source of food for Drosophila melanogaster-and therefore laying opportunities for parasitoids. In the mild and humid area (Chalus and Seyakhal), there is a lot of precipitation during the whole season so fruits, and therefore Drosophila are common throughout the season. In the desert area (Dorcheh and Zamankhan), the lack of precipitation during summer restricts the production of fruits to few and distant areas under irrigation. Here, subsequent populations of parasitoids have to move between crops of different fruits, because when they emerge as adults, the season for the fruit in which they developed is over. This necessitates long flights in search of host habitats. Thus we will mainly explain our results on life history traits in the light of a selection in response to host distribution as it is affected by humidity.

Reproductive strategies and longevity.

We found a high investment in early reproduction of females from mild and wet environments (Chalus and Seyakhal), correlated with a shorter lifespan. These results suggest that females from these populations are pro-ovigenic i.e. they do not mature eggs during adult life but females were not allowed to lay eggs. According to models developed by [START_REF] Ellers | Egg Load Evolution in Parasitoids[END_REF] and [START_REF] Ellers | Why are so few parasitoid wasp species pro-ovigenic?[END_REF], host distribution is the most important factor resulting in the selection of pro-ovigeny and may explain our results: in environments with small distances between host patches and a high encounter rate with hosts, selection results in the allocation of energy to early reproduction (i.e. pro-ovigeny) at the cost of a shorter lifespan.

Sorkhabad females originate from the coldest area where rainy days and the precipitation amount are intermediate between Chalus/Seyakhal and Zamankhan/Dorcheh. Investment in early reproduction and longevity were both intermediate in Sorkhabad females. The results for this population supports the fact that precipitation and not temperature is important in selection on reproductive strategies.

Females from the desert area (Dorcheh and Zamankhan) invested less in early reproduction but lived longer than other populations. Our results also provide evidence that Leptopilina boulardi is not a completely pro-ovigenic species as suggested by [START_REF] Kopelman | Aspects of the reproductive biology of Leptopilina boulardi (Hymenoptera: Eucoilidae)[END_REF]: we observed an increase of more than 50 eggs during the first ten days of adult life in Dorcheh and Zamankhan females. Females from these populations were synovigenic (i.e. they still mature eggs during adult life) and lived longer than those of other populations.

Synovigeny and increased lifespan allow a plastic response in an environment where females have to move over large distances to find hosts and are time-limited [START_REF] Ellers | Life history evolution in Asobara tabida: plasticity in allocation of fat reserves to survival and reproduction[END_REF]. They can adjust egg maturation to the laying opportunities and trade current for future reproduction. This could explain the synovigeny in the Dorcheh and Zamankhan populations.

Our results suggest that pro-ovigenic and synovigenic populations may occur within the same parasitoid species. Such variation in reproductive strategies between populations has never been described in parasitoids. Therefore, we have to confirm the pro-ovigeny of Seyakhal and Chalus females by providing them hosts and checking if laying opportunities lead to the maturation of additional eggs during adult life (i.e. synovigeny) or not (strict pro-ovigeny) in these populations.

Variations in life history traits between populations suggested that a trade-off occurs between longevity and investment in early reproduction. However, this trade-off was not found for females after five days of adult life because Zamankhan females, that lived longer, matured additional eggs while females from mild and humid area did not. Females were not limited by carbohydrate resources in our fecundity experiments, and thus could allocate more of their lipid reserves to reproduction. In nature, female parasitoids from the desert area are food and time limited because they have to travel long distances to find patches of hosts and sources of food. Thus a trade-off may occur between longevity and realised fecundity that we did not detect here because females were not allowed to lay eggs and were fed ad libitum.

Lipid content. In parasitoids, a longer lifespan is often associated with higher lipid reserves at emergence [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 | The physiological costs of being small in a parasitic wasp[END_REF] when initial egg load is traded-off against fat reserves [START_REF] Ellers | Life history evolution in Asobara tabida: plasticity in allocation of fat reserves to survival and reproduction[END_REF]. Indeed we observed a negative correlation between initial number of eggs and lipid content for the five populations studied. A trade-off between fecundity and lipid reserves exists during larval development where resources are limited by the amount available from the host.

Surprisingly, females from the dry and hot area lived longer than females from the other areas but emerged with smaller lipids reserves. Parasitoids were thought to be unable to synthesize and accumulate lipids during adult life and to depend only on lipids accumulated during larval development [START_REF] Giron | Lipogenesis in an adult parasitic wasp[END_REF][START_REF] Visser | Lack of lipogenesis in parasitoids: A review of physiological mechanisms and evolutionary implications[END_REF]. However [START_REF] Visser | Loss of lipid synthesis as an evolutionary consequence of a parasitic lifestyle[END_REF] recently described lipogenesis in some parasitoid species and suggest that "the wide range of host species in which generalists are able to develop may impede effective host manipulation and could have resulted in regaining of lipogenic ability in generalist parasitoids". In opposition to the authors who did not find any lipogenesis in L. boulardi, we found a strong increase in lipid quantity in this species for females from desert areas during the first ten days of adult life, evidence that adult lipogenesis occurs in these populations. This is the first evidence that between-populations differences in the ability to synthesize lipids occur in a parasitoid species. Other factors than hosts range may result in existence of lipogenesis in parasitoids, such as host distribution. Lipogenesis may be adaptive in desert areas where females need lipids to fly over large distances and mature eggs once they have found a lot of laying opportunities. Moreover, higher lipid content may limit water loss in this dry climate [START_REF] Hadley | Water Relations of Terrestrial Arthropods[END_REF]. The observed adult lipogenesis could explain the longer lifespan and increased egg maturation observed in these females.

Dispersal ability and locomotor activity. Wing loading was lower in females from hot and dry areas than in females from other environments. This reduced wing loading may facilitate flight [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]) in an environment where females have to move over large distances to find patches of hosts. Greater dispersal ability during the first days of life may compensate for the low number of hosts encounters and be selected because females can search for patches of hosts at lower cost in desert areas.

Flying over large distances to find hosts is an energy demanding activity [START_REF] Ellers | A field study of size fitness relationships in the parasitoid Asobara tabida[END_REF].

In a humid environment like that in Chalus or Seyakhal, females do not have to move over such large distances as in a desert environment. In the latter environment, flying, which was not allowed in our experiment, to find laying opportunities may result in a decreased egg maturation and/or longevity.

Zamankhan females were also more active than females from mild and wet environment. This high activity may have been selected in an environment where females have to move a lot to find hosts in distant patches. Moreover Zamankhan females were more active during the night while females from Chalus and Seyakhal were more active during the day. Avoidance of extreme temperatures in Zamankhan during the day is likely to be adaptive in this desert area.

The nocturnal activity of Zamankhan females is the only trait that supports a direct effect of temperature on the evolution of life history traits.

Conclusion.

Females from the desert area differed considerably from females originating from the mild coastal region, but these populations were also geographically distant and this separation by distance has allowed local adaptation and caused differences in life histories between populations as a result of selection by the environment.

Differences in temperature and thermal amplitude did not explain our results, but precipitation did. The influence of precipitation on fruit availability, the main nutritional resource for hosts, could explain why this climatic factor is more linked to life history traits than others. Our results, especially synovigeny and lipogenesis, strongly support the hypothesis of selection based on distribution of resources and suggests that host distribution, depending on precipitation, is more important in selection of life history traits of parasitoids than temperature. Relative humidity, number of rainy days and precipitation amount had to be included as explanatory factors to obtain the minimum adequate model for this life history trait. Here we represent the plot of a model on longevity with the more significant factor in our model, the number of rainy days.

  Fig. 3). For the same lean dry mass, females originating from Chalus and Seyakhal emerged with more eggs than Dorcheh and Zamankhan females (ANCOVA, F = 83.36, df = 5, P < 0.0001) (Fig 3A, <24 hours). Sorkhabad individuals emerged with an intermediate number of eggs.

Fig. 1 .

 1 Fig. 1. Map of Iran; the arrows indicate the five Leptopilina boulardi sampling sites.

Fig. 2 .

 2 Fig. 2. Wing loading measured at emergence for Chalus, Seyakhal, Sorkhabad, Dorcheh and Zamankhan females. Different letters indicate significant differences of wing loading between populations (n = 20 per population, P < 0.05). Error bars: ± SE.

Fig. 3 .

 3 Fig. 3. (a). Plot of residuals of models comparing mean number of eggs at 24 hours, 5 days and 10 days of adult life with lean dry mass as covariate. A model was established for each population. The higher the residuals, the more females of a same lean dry mass have eggs in their ovaries (n = 20 per age per population). Error bars: ± SE. (b) Plot of residuals of a model comparing investment in early reproduction (=number of eggs x volume of eggs) between populations with lean dry mass as covariate. The model was established with the global data set. The highest the residuals, the more females of a same lean dry mass invested in reproduction. Different letters indicate significant differences of investment in reproduction between populations (n = 20 per population, P < 0.05). Error bars: ± SE.

Fig. 4 .

 4 Fig. 4. (a) Plot of residuals of a model comparing lipid quantity between populations with lean dry mass as covariate at emergence. The model was established with the global data set. The highest the residuals, the more females of a same lean dry mass have lipid reserves. Different letters indicate significant differences of lipid content between populations (n = 20 per population, P < 0.05). Error bars: ± SE. (b) Plot of residuals of models comparing lipid quantity at 24 hours, five days and ten days of adult life with lean dry mass as covariate. A model was established for each population. The higher the residuals, the more females of a same lean dry mass have lipid reserves. Error bars: ± SE. (n = 20 per age per population).

Fig. 5 .

 5 Fig. 5. (a) Mean distance walked during twelve-hours day and night recordings, measured with Ethovision software. Different letters indicate significant differences of activity between populations. Different numbers mean significant differences of activity between day and night recordings for a single population (n=20 per treatment perpopulation, P < 0.05). Error bars : ± SE. (b) Mean velocity during twelve-hours day and night recordings, measured with Ethovision software. Different letters indicate significant differences of velocity between populations.Different numbers indicate significant differences of velocity between day and night recordings for a single population (n=20 per treatment per population, P < 0.05). Error bars : ± SE.

Fig. 6

 6 Fig. 6 (a) Plot of a generalized additive model realized on investment in early reproduction with lean dry mass as covariate and number of rainy days as an explanatory factor. Relative humidity, number of rainy days and precipitation amount had to be included as explanatory factors to obtain the minimum adequate model for this life history trait. Here we represent the plot of a model on investment in early reproduction with the more significant factor in our model, the number of rainy days. (b) Plot of a generalized additive model realized on longevity with number of rainy days as an explanatory factor.

  to females from mild and wet environment (Chalus and Seyakhal) and females from cool environment and intermediate in precipitations (Sorkhabad), females from hot and very dry environments (Dorcheh and Zamankhan) invested less in early reproduction and had a longer lifespan, lower lipid contents and wing loading during the first twenty-four hours of adult life. Females from the coolest climate but intermediate in precipitations and relative humidity (Sorkhabad) invested an intermediate amount of energy into early reproduction and longevity. Our results are not consistent with other latitudinal cline studies on adaptations of life history traits to temperature (e.g.

Table 1 .

 1 Climatic data recorded for the four sampledareas, from 1977 to 2005 for Chalus, 1955 to 2005 for Seyakhal and Zamankhan, 1993 to 2003 for Sorkhabad, 1951 to 2005 for Dorcheh (Source: Islamic Republic Of Iran Meteorological Organization). We considered the average of each parameter measured every day from April to September, 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.

	Sampling	Temperature	Thermal	Relative	Number of	Precipitation
	areas	(°C)	amplitude (°C)	humidity (%)	rainy days	amount (mm)
	Chalus	26.3	7.6	80	43.3	248
	Seyakhal	26.4	9.8	79	56.7	433
	Sorkhabad	24.4	16.7	44	28.6	105
	Dorcheh	31.6	15.6	29.7	14.2	30.9
	Zamankhan	28.6	19.2	36	13.8	55

ACKNOWLEDGEMENTS

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 (http://comparevol.univ-rennes1.fr/), ECOCLIM program founded by Region Bretagne and CLIMEVOL program founded by the Agence Nationale de la Recherche. We are grateful to Jacintha Ellers for helpful comments on a previous version of this manuscript.