Does the temperature–size rule apply to idiobiont parasitoids?

In most ectotherms, several life‐history traits, including body size, respond to environmental conditions through the temperature–size rule (TSR). The mechanisms underlying the TSR are still being debated, but studying idiobiont insect parasitoids, which develop with a fixed amount of resources, may shed light on this relationship. In this study, we conducted experiments to determine how the developmental temperature affects various characteristics of male and female Trichogramma euproctidis (Girault) (Hymenoptera: Trichogrammatidae), an idiobiont egg parasitoid of Lepidoptera. The study tested various hypotheses, including the cellular or oxygen diffusion hypotheses and the resource acquisition hypothesis, to understand whether T. euproctidis follows the TSR. The developmental time of both male and female T. euproctidis decreased with increasing temperature. Both males and females displayed a unimodal distribution for size, dry mass, and lipid content, with individuals at lower and higher temperatures being smaller, weighing less, and containing fewer lipids. Female lifetime fecundity increased from 13 to 24 °C and then decreased at 31 °C. Additionally, the number and size of gametes in male and female T. euproctidis displayed a unimodal distribution with increasing temperature. Trichogramma euproctidis deviates from the TSR as it follows a non‐linear reaction norm with an optimal developmental temperature. This result supports the hypothesis that for species following TSR and having unlimited access to food resources, the resource acquisition hypothesis is a significant mechanism explaining the TSR. With climate change affecting temperature, understanding the TSR is crucial, and research on insect parasitoids may help reveal how the interplay between environmental temperature and resource allocation affects the TSR in natural populations.


INTRODUC TION
Life-history traits, being connected to the fitness of an individual, are subject to selection and evolution in response to local environmental conditions, and may display phenotypic plasticity in response to developmental conditions (Bernardo, 1994;Bernardo & Reagan-Wallin, 2002;Pigliucci, 2005;Colinet et al., 2007).Temperature, influenced by elevation or latitude, is recognized as a factor that impacts various life-history traits including body size; Bergmann's rule states that there is a general intraspecific trend towards larger body size in cooler environments (Blackburn et al., 1999, Ashton, 2001;Bernardo & Reagan-Wallin, 2002;Angilletta & Dunham, 2003).
The temperature-size rule (TSR) is a special case of Bergmann's rule that describes reaction norms relating temperature to body size in ectotherms (Angilletta & Dunham, 2003); it represents a form of phenotypic plasticity in which the phenotype (adult size) of an individual is affected by temperature during immature development (Kingsolver & Huey, 2008;Boivin, 2010).According to that rule, ectotherms that develop at higher temperatures will become smaller adults relative to individuals that develop at lower temperatures (Kingsolver & Huey, 2008).However,

O R I G I N A L A R T I C L E S p e c i a l I s s u e : 7 t h I n t e r n a t i o n a l E n t o m o p h a g o u s I n s e c t s C o n f e r e n c e
Does the temperature-size rule apply to idiobiont parasitoids?Annie-Ève Gagnon 1  | Véronique Martel 2 | Guy Boivin 1,3 the TSR is not universal and approximately 20% of all ectotherm species tested did not follow TSR (Atkinson, 1994), and more than one mechanism seems to be responsible for the TSR (Angilletta & Dunham, 2003).
The mechanisms behind the effect of developmental temperatures on the adult body size of ectotherms are still debated (Atkinson & Sibly, 1997;Kingsolver & Huey, 2008;Walczyńska & Sobczyk, 2017).The three main hypotheses are presented here.(1) The resource acquisition mechanisms postulate that as temperature increases, both resource acquisition and resource utilization rates increase but at different rates (Atkinson & Sibly, 1997).When resource utilization increases faster than resource acquisition under higher temperatures, an organism has access to fewer resources per unit of development time and therefore grows to a smaller size.
(2) The oxygen availability hypothesis states that when temperature increases, oxygen use increases faster than the diffusion of oxygen in cells (Woods, 1999;Verberk et al., 2021).The cells are thus smaller at high temperatures to overcome the relatively slow diffusion rate of oxygen at these temperatures.This hypothesis is the only one that postulates an adaptive value of the TSR (Walczyńska et al., 2015).( 3) The cellular hypothesis states that when temperature increases, the growth rate of cells responds differently to temperature than the differentiation rate.In that case, as temperature increases, the reaction norm of cell differentiation is steeper than that of cell growth, and the size of cells decreases as temperature increases, resulting in a smaller adult body.Globally, it is unclear whether the TSR is adaptive in ectotherms or if it is merely the result of physiological processes and constraints at the cellular or individual behavior level (Forster & Hirst, 2012;Pimentel et al., 2012).
It is difficult to design experiments that could differentiate between these hypotheses because temperature affects not only the size of an individual ectotherm but also the duration of its development (Jarośík et al., 2004).Ectotherms developing at low temperature have access to resources for a longer period of time and therefore could accumulate more resources.Insect parasitoids could provide such an opportunity; they develop in or on another individual, known as their host, and kill it as a result of their development (Eggleton & Gaston, 1990).Depending on their life history, insect parasitoids may be divided into koinobionts and idiobionts (Haeselbarth, 1978;Askew & Shaw, 1986).Koinobiont parasitoids allow their host to continue to feed, grow, and function after parasitism, whereas hosts attacked by idiobiont parasitoids cease development immediately upon being parasitized or shortly thereafter (Brodeur & Boivin, 2004).Immature idiobiont parasitoids must, therefore, complete their development with a fixed amount of food, regardless of the duration of their development.Parasitoids, and especially idiobiont parasitoids, are thus constrained in size by the fact that they must develop with the resources offered by a single host and by their lack of lipogenesis (Visser & Ellers, 2008;Ruther et al., 2021).
The TSR has been observed in several hymenopteran parasitoid species, all of which are koinobionts (Nealis et al., 1984;Bazzocchi et al., 2003;Colinet et al., 2007;Wu et al., 2011;Le Lann et al., 2012).These species have shown a general trend of increasing adult body size as temperature decreases, but it should be noted that the period during which their host remains alive and able to feed also increases with decreasing temperature.Therefore, it cannot be assumed that the quantity of resources gathered by the parasitized host remains constant across temperatures, as this would only be true if the rate of change of both behavioral and physiological components of nutrition respond to temperature in the exact same way as the rate of development, which is an untested assumption.
In this paper, we measured the impact of developmental temperature on several characteristics of males and females of Trichogramma euproctidis (Girault) (Hymenoptera: Trichogrammatidae), a solitary idiobiont egg parasitoid of Lepidoptera.We predicted that if the oxygen or cellular diffusion hypotheses (hypotheses 2 and 3) were true for idiobiont parasitoids, T. euproctidis should follow the TSR.However, if the resource acquisition hypothesis (hypothesis 1) is valid, it should not follow the TSR, as the resources available to the idiobiont parasitoid are fixed regardless of the developmental temperature.

MATERIALS AND METHODS
The T. euproctidis strain used in this study originated from Egypt (GenBank accession number HM116410), and was maintained at 24 ± 1 °C, 50% r.h., and L16:D8 on cold-killed eggs of the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae).
In all experiments, three mated T. euproctidis females of <24 h old were offered 400 cold-killed E. kuehniella eggs for 3 h at 24 ± 1 °C.This number of host eggs was much higher than what the three females could attack in 3 h, in order to minimize superparasitism.After the 3-h period, the eggs were placed in small glass vials and randomly assigned to four temperatures: 13, 17, 24, and 31 ± 1 °C, as no development occurred below 10 ± 1 °C and above 35 ± 1 °C (G Boivin, pers.obs.).The eggs were considered parasitized if they turned black, which indicated that the meconium had been discharged just prior to pupation (Duval et al., 2018).Each parasitized egg was then placed individually in a 300-μL Beem polyethylene capsule until emergence.For each experiment, 30 replicates were performed for each temperature (except when noted), with around 40 parasitized eggs per replicate.The following tests were conducted.

Immature developmental time and survival
The duration of immature development, from oviposition to adult emergence, was recorded daily for all experiments.The number of individuals observed per temperature varied from 701 to 2402 females and from 223 to 653 males.
To determine immature survival at different temperatures, egg-larval and pupal survival was estimated separately.Larval survival at 13, 17, and 31 °C was determined individually by comparing the percentage of eggs that turned black at a given temperature to the percentage at 24 °C.For example, this comparison involved calculating the survival rate for each temperature as a ratio in relation to the survival rate at 24 °C.The latter temperature was chosen as a standard rearing condition with minimal mortality (Moiroux et al., 2014).Pupal survival was assessed by determining the percentage of black (and thus parasitized) eggs from which parasitoids emerged.The sex ratio of the emerging individuals was also recorded.

Adult size, mass, and lipid content
Thirty males and 30 females were randomly selected for each temperature, and their size was estimated by measuring the length of both their hind tibiae.The average tibia length of each individual was used.
The dry mass of male and female T. euproctidis that developed at different temperatures was estimated as follows.Freshly emerged individuals were placed in an oven at 60 °C for 3 days.For each temperature, groups of 10 individuals of the same sex were weighted using a Cahn 29 microbalance (n = 20 groups of 10 individuals per sex and temperature).The weights were divided by 10 and reported as individual dry mass.
The lipid content of both males and females was estimated using a colorimetric method (Rivero & West, 2002).Groups of 10 males or 10 females were placed in a glass tube (0.3 mL Reacti-vial) with 100 μL of chloroform (n = 30 per sex and temperature).After 16 h at room temperature (between 20-22 °C), 80 μL of the solution was pipetted, poured into glass tubes (0.3 mL Reacti-vial), and heated at 90 °C until all the chloroform had evaporated.The same procedure was followed for the controls (blank) containing no insects between each extraction measurements.Then, 4.8 μL of sulphuric acid was added to the tubes, which were heated at 90 °C for 2 min.After cooling, 115 μL of vanillin reagent was added (for details of reagent preparation, see Van Handel, 1985), left for 15 min to allow the color to develop, and read in the spectrophotometer (GE, NanoVue Plus) at OD525 against the control.Lipid concentrations were obtained from a standard curve done with commercial vegetable oil (1 mg mL −1 in chloroform; Van Handel, 1985).The lipid content was calculated by dividing the mass of lipids in a group of 10 individuals by the dry mass of the same group of individuals.Lipid mass and lipid contents are reported per individual.

Longevity and lifetime fecundity
Longevity of males from each temperature was obtained by placing freshly emerged virgin males individually in a ventilated glass vial at 24 ± 1 °C.A drop of honey was placed into the vial for nutrition, and it was changed every 2 days until the male died.The individuals were observed daily in order to record the time of death.
To measure longevity and lifetime fecundity of females, mated freshly emerged females were used.To do this, a freshly emerged female was placed in a Beem capsule with a freshly emerged virgin male that had developed at 24 ± 1 °C.The mating was visually confirmed under a stereomicroscope (20× magnification), and then the mated female was placed individually in a ventilated glass vial with a drop of honey and ca. 100 host eggs at 24 ± 1 °C.The honey and host eggs were changed every 2 days, whereas the individuals were monitored daily to assess the timing of their death.The host eggs were incubated at 24 ± 1 °C until emergence of the progeny, which were counted and sexed.

Number and size of gametes
Freshly emerged virgin males from each temperature were dissected, and the sperm present in both seminal vesicles was then expelled onto a microscope slide in a saline solution (NaCl 0.1%).The slide was then dried and fixed with 95% ethanol, followed by treatment with DAPI solution (2 × 10 −6 g mL −1 ) for 15 min.DAPI (4′-6-diamidino-2-phenylindole) forms fluorescent complexes with natural double-stranded DNA, and thus stains only the head of the sperm.The sperms were counted under fluorescent microscopy at 320× magnification (Damiens & Boivin, 2005;Martel et al., 2011), and the length of the sperm tail of five sperms per male was also measured at 640× magnification.
Freshly emerged virgin females from each temperature were dissected, and the mature oocytes (i.e., fully chorionated eggs) present in the ovaries were counted.The lengths and widths of five mature oocytes per female were measured at 320× magnification, and average measurements were used for each female.The oocyte volume was estimated using the formula: 4/3(πab 2 ) (Martel et al., 2011).As female T. euproctidis are synovigenic and continue to produce oocytes during their adult life (Ferracini et al., 2006), the volume of oocytes produced after emergence was obtained by allowing freshly emerged and mated females (as described previously) to oviposit on five host eggs for 48 h.These females were then placed in a vial with water and honey but without hosts for 24 h before being dissected (thus at approximately 3 days old), and the number and size of five mature oocytes per female were measured as described above.

Statistical analysis
Developmental times, larval and pupal survival, adult size, dry weight, lipid mass and content, longevity, lifetime fecundity, sex ratio, number and size of sperm, and oocytes were analyzed to examine their relationship with temperature using polynomial regressions using the lm function.
The polynomial regression degree selected was the minimum degree that yielded statistically significant polynomial coefficients (α = 0.05) and the highest R 2 value.ANOVA was also performed for each parameter to compare the impact of each temperature tested, using the aov function.When significant, a multiple comparison of means was performed using a Tukey test.The normality assumption of ANOVA was first tested using the Shapiro test.All statistical analyses were conducted using R software v. 4.2.2 (R Core Team, 2018).

Immature developmental time and survival
The duration of development of both male and female T. euproctidis decreased with increasing temperature (male: F 3,174 = 8286.90;female: F 3,174 = 9836.40,both P < 0.0001; Figure 1).
Larval survival was not affected by temperature (F 3,144 = 1.25, P = 0.30), with survival rates at 13, 17, and 31 °C being approximately 90% of that observed at 24 °C.Pupal survival was affected by temperature (F 2,115 = 72.42,P < 0.001); it displayed a unimodal distribution across temperatures, with the optimum temperature for pupal survival around 20 °C (Figure 2).

Longevity and lifetime fecundity
When provided with access to water, honey, and hosts (in the case of females), adult longevity for males (F 2,117 = 20.66) and females (F 2,117 = 14.04, both P < 0.001) also demonstrated a unimodal distribution, although with high variability (Figure 4A).Lifetime fecundity of females increased from 13 to 24 °C and then decreased at 31 °C (Figure 4B).

Number and size of gametes
The number and size of gametes in T. euproctidis were also influenced by the rearing temperature.In males, the number of sperms (F 2,117 = 58.55)and their size (F 2,117 = 23.65,both P < 0.001) increased to a maximum at 24 °C (Figure 5).In females, the number of oocytes at emergence displayed a unimodal distribution (F 2,117 = 55.03,P < 0.001), with the maximum reached between 24 and 31 °C (Figure 6A).When females were to oviposit for 48 h and then rested for 24 h, the number of oocytes present in the ovaries was lower than at emergence, but it followed a similar distribution over temperature (F 2,117 = 72.22,P < 0.001; Figure 6A).The size of the oocytes present at emergence increased to a maximum at 24 °C and then decreased at 31 °C (F 2,117 = 11.35,P < 0.001; Figure 6B).However, the size of the oocytes present in the ovaries of females aged 72 h after ovipositing for 48 h did not vary with temperature (F 3,115 = 2.42, P = 0.070; Figure 6B).

DISCUSSION
Trichogramma euproctidis did not follow the temperaturesize rule (TSR).If they had followed the TSR, most parameters such as size, body mass, lipid mass, lipid content, fecundity, and size and number of gametes would have gradually declined with increasing temperature.Instead, in T. euproctidis, these parameters followed a non-linear convex reaction norm typical of a species with an optimal developmental temperature.
In this study, both male and female T. euproctidis, an idiobiont parasitoid, exhibited an optimal unimodal reaction norm on all size-related parameters, including tibia length dry mass, which were to be highest at a temperature range of 20-24 °C.This reaction to developmental temperature is different from what has been observed in other parasitoids, which were koinobionts.For example, the braconids Cotesia glomeratus (L.) and Cotesia rubecula Marshall showed a reduction in pupal weight with increasing rearing temperature (Nealis et al., 1984), whereas the eulophid wasp Diglyphus isaea (Walker) showed a reduction in female hind tibia length as rearing temperature increased (Bazzocchi et al., 2003).However, in all of these examples, the availability of food resources was not restricted, given that these parasitoids do not kill or paralyze their host immediately and allow their host to continue developing.
Nutrition availability can explain the mechanism for the TSR because it influences an insect's growth rate and ability to allocate resources to growth, which ultimately determines its final size (Atkinson & Sibly, 1997).The results obtained in this study with T. euproctidis demonstrate that all parameters related to the size and fitness of the species increase with temperature, up to an optimum, before decreasing.This result supports the resource acquisition hypothesis, which states that if an organism's resource utilization increases faster than its resource acquisition under higher temperatures, it will have less access to resources per unit of development time, resulting in reduced growth in size.As idiobiont parasitoids have always access to the same amount of resource, the host egg, they must complete their development with a fixed amount of food, explaining why these organisms did not follow the TSR.Therefore, although T. euproctidis undergoes faster development at higher temperatures, with a decrease from 50 days at 13 °C to only 7 days at 31 °C, the quantity and quality of food available remains unchanged.Other studies have revealed evidence that some organisms, such as diatoms, are exception to the TSR with no consistent relationship between size and temperature at either the population or the community level (Adams et al., 2013).Svensson et al. (2014) investigated the relationship between diatom size and three environmental factors: temperature, salinity, and nutrient supply, and found that nutrient concentrations and nutrient limitation played key roles in all changes in the diatom cell size spectra.Furthermore, Lee et al. (2015) demonstrated how the nutritional quality affects the response of body size to temperature in the caterpillar Spodoptera litura (Fabricius).When the protein:carbohydrate ratio was low, more food was ingested at lower temperatures, but this effect was less pronounced under balanced diet conditions and with a high protein:carbohydrate ratio.In this case, the TSR was explained by an increase in the quantity of ingested nutrients (carbohydrates) and their efficiency in being converted to lipids (Lee et al., 2015).Thus, typical TSR response species could be explained the variability in the amount of ingested nutrients as a function of temperature, whereas the absence of TSR response in T. euproctidis would be explained by the fixed availability of nutrients.
The oxygen limitation hypothesis represents another non-exclusive hypothesis to explain the mechanism of TSR.Several recent studies have attempted to confirm the role of oxygen limitation in determining the size and fitness of ectothermic organisms, mostly in aquatic organisms.Einum et al. (2021) attempted to model the expected strength of the TSR in the aquatic ectotherm Daphnia magna Straus by considering the impact of temperature on both oxygen supply and demand, while accounting for phenotypic plasticity.However, their predictions did not align well with observed temperature-size responses, suggesting that their results do not offer quantitative evidence for the hypothesis that oxygen limitation drives temperature-size clines in aquatic ectotherms.In a study comparing the size and fertility of two aquatic insects, Neocloeon triangulifer (McDunnough) and Cloeon dipterum (L.), under a range of temperatures and oxygen concentrations, Funk et al. (2021) concluded that a failure to meet tissue oxygen demands is not a viable hypothesis for explaining TSR pattern in these species.Verberk et al. (2021) suggested that oxygen limitation acts as a selective pressure for decreasing body size under warm conditions, rather than being a proximate factor.This could be due to the elimination of genotypes that are more susceptible to oxygen limitation, resulting in evolved responses that ensure adequate oxygen provisioning in warmer conditions, reflecting the balance between oxygen supply and demands experienced by ancestors, also known as the 'ghost of oxygen-limitation past.'Despite recent studies exploring hypotheses that can explain the mechanism behind the TSR, it remains uncertain whether the TSR is an adaptive physiological or ecological response, or a response to cellular-level constraints (Audzijonyte et al., 2019).These studies support the notion that the TSR may not always be explained by oxygen limitation alone.Instead, accessibility to food resources may more commonly underlie the mechanism of TSR in natural environments where resource variability is higher than in laboratory settings.
By influencing aspects ranging from the fecundity of an organism to the functioning of communities and ecosystems, body size holds a central position in ecology (Berger et al., 2008).Larger individuals potentially produce more offspring, live longer, be superior competitors, and be better at avoiding predators (Brown & Sibly, 2006).Among parasitoids, various measures of fitness, such as longevity and fecundity, have been demonstrated to have a positive correlation with size (Visser, 1994).However, Colinet et al. (2007) examined the effects of rearing temperature on various fitness components of the koinobiont parasitoid Aphidius colemani (Dalman) and found that the relationship between size and fitness was not always linear due to plasticity in energy reserves.Insufficient reserves of lipids may lead to decreased productivity in wasps that developed at high and low temperatures.This result is in accordance with our study where female fecundity measurements were inferior under suboptimal temperatures (i.e., high and low temperatures).This trend, in the case of our study, could be explained by the lipid content, which is known to be directly involved in egg production (Ellers & van Alphen, 1997;Zhao & Zera, 2002).The low lipid content at both low and high temperatures would reduce egg production in T. euproctidis.Some studies have emphasized the trade-off between reproduction and longevity in parasitoid females (Ellers et al., 2000;Colinet et al., 2007;Jervis et al., 2008).In the case of A. colemani, females exhibiting high fertility had a lower longevity at low temperatures (Colinet et al., 2007).In the case of T. euproctidis, females reared at low temperatures also exhibited low fecundity, with fewer and smaller oocytes at emergence, but their longevity was also reduced.The low allocation of lipid resources at low temperatures should have a significant impact on the development of T. euproctidis, preventing any trade-off regarding resource allocation.Vayssade et al. (2012) also showed that in Leptopilina heterotoma (Thompson) there was no trade-off between fecundity and longevity, and they explained it by a change in metabolic rate.
From an applied perspective, it is relevant to determine the appropriate developmental temperature for producing parasitoids that will be released as part of biological control strategies.For example, A. colemani reared at higher temperatures exhibited the highest heat coma threshold, which could be helpful in designing release strategies for regions experiencing heat waves or higher temperature (Jerbi-Elayed et al., 2021).To ensure the efficacy of T. euproctidis in a biological control context, mass rearing facilities should maintain a temperature of 24 °C as our study found that this temperature resulted in the best performance of the parasitoid.Furthermore, the introduction of parasitoids in the field under arid climatic conditions could impair the establishment of the population as both reproductive success and adult longevity would be reduced.These findings have significant implications in the context of climate change, as increasing temperatures and more frequent extreme weather events may threaten the sustainability of insect populations and alter their distribution pattern (Boggs, 2016;Skendžić et al., 2021;Harvey et al., 2023).The impact of temperature on behaviors can also provide a more comprehensive explanation for ecological shifts related to climate change compared to its influence on metabolism or physiology alone.For example, numerous ectothermic organisms possess the ability to adapt their behavior in order to modify or endure their thermal environment (Abram et al., 2017).Furthermore, natural enemies, such as parasitoids, play a vital role in regulating populations of pest insects in agricultural and forestry sectors (Schmidt et al., 2003;Bianchi et al., 2006), and loss in biodiversity or their population size could have a severe impact on ecosystem equilibrium (Letourneau et al., 2009).It is worth mentioning that understanding the TSR on biological control agents, such as parasitoids or other ectothermic organisms, has become crucial due to the impact of climate change on temperatures.

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I G U R E 1 Duration of development (days) of (A) male and (B) female Trichogramma euproctidis reared at 13-31 °C.The equations are based on polynomial regression.F I G U R E 2 Survival (%) of pupae of Trichogramma euproctidis reared at 13-31 °C.Shading around the polynomial regression line shows 95% confidence intervals.

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I G U R E 3 (A) Tibia length (μm), (B) dry mass (μg), (C) lipid mass (μg), and (D) lipid content (proportion of dry mass) of male and female Trichogramma euproctidis reared at 13-31 °C.Shading around the polynomial regression lines indicates 95% confidence intervals.F I G U R E 4 (A) Longevity (days) of male and female Trichogramma euproctidis, and (B) lifetime fecundity (no.offspring) of females reared at 13-31 °C.Shading around the polynomial regression lines indicates 95% confidence intervals.

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I G U R E 6 (A) Number and (B) size (mm 3 ) of mature oocytes present at emergence or after 72 h in the ovaries of female Trichogramma euproctidis reared at 13-31 °C.Shading around the polynomial regression lines indicates 95% confidence intervals.F I G U R E 5 (A) Number and (B) size (μm) of sperm cells present at emergence in both seminal vesicles of male Trichogramma euproctidis reared at 13-31 °C.Shading around the polynomial regression lines indicates 95% confidence intervals.