Physiology & Behavior 151 (2015) 463–468 Contents lists available at ScienceDirect Physiology & Behavior journal homepage: www.elsevier.com/locate/phb Amino acid composition of the bushcricket spermatophore and the function of courtship feeding: Variable composition suggests a dynamic role of the nuptial gift Alicia Jarrige ⁎, Mélanie Body 1, David Giron, Michael D. Greenfield, Marlène Goubault Institut de Recherche sur la Biologie de l'Insecte, UMR CNRS 7261, Université François-Rabelais, Parc Grandmont, 37200 Tours, France H I G H L I G H T S • • • • We investigated the amino acid composition of a bushcricket nuptial gift. We examined amino acids composition in regard to the receiving female’s traits. Gift composition varied both in quality and quantity according to the female traits. Nuptial gift composition may represent a form of cryptic male mate choice. a r t i c l e i n f o Article history: Received 7 May 2015 Received in revised form 3 August 2015 Accepted 4 August 2015 Available online 5 August 2015 Keywords: Ephippiger diurnus Free amino acids Male mate choice Protein bound amino acids Strategic allocation a b s t r a c t Nuptial gifts are packages of non-gametic material transferred by males to females at mating. These gifts are common in bushcrickets, where males produce a complex spermatophore consisting in a sperm-containing ampulla and an edible sperm-free spermatophylax. Two non-mutually exclusive hypotheses have been suggested to explain the function of the spermatophylax: the paternal investment hypothesis proposes that it represents a male nutritional investment in offspring; the mating effort hypothesis proposes that the spermatophylax maximizes the male's sperm transfer. Because gift production may represent significant energy expenditure, males are expected to adjust their investment relative to the perceived quality of the female. In this study, we first examined the free amino acid composition and protein-bound amino acid composition of the nuptial gift in the bushcricket, Ephippiger diurnus (Orthoptera: Tettigoniidae). Second, we investigated whether this composition was altered according to female age and body weight. Our study represents the first investigation of both free and proteinbound amino acid fractions of a bushcricket spermatophylax. We found that composition of the nuptial gift varied both qualitatively and quantitatively with respect to traits of the receiving female: older females received larger amounts of protein-bound amino acids (both essential and non-essential), less water and less free glycine. This result suggests that gift composition is highly labile in E. diurnus, and we propose that gift allocation might represent a form of cryptic male mate choice, allowing males to maximize their chances of paternity according to the risk of sperm competition that is associated with mate quality. © 2015 Elsevier Inc. All rights reserved. 1. Introduction Nuptial feeding, i.e. material donations transferred to the opposite sex at mating, is widespread among insects and encompasses a wide variety of forms, including prey, body parts and glandular secretions [15, 23]. One of the most intensively studied examples of courtship feeding can be found in bushcrickets (Orthoptera: Tettigoniidae), where males ⁎ Corresponding author at: Institut de Recherche sur la Biologie de l'Insecte, UMR CNRS 7261, Université François-Rabelais, Parc Grandmont, 37200 Tours, France. E-mail address: alicia.jarrige@gmail.com (A. Jarrige). 1 Present address: Division of Plant Sciences, 312 Christopher S. Bond Life Sciences Center, 1201 Rollins Street, University of Missouri, Columbia, MO 65211, USA. http://dx.doi.org/10.1016/j.physbeh.2015.08.009 0031-9384/© 2015 Elsevier Inc. All rights reserved. transfer the product of their accessory glands as an edible sperm-free spermatophylax attached to a sperm-containing ampulla, together forming the spermatophore [6]. Following copulation, the female consumes the spermatophylax while the sperms migrate to her genital tract [6]. The function of nuptial feeding has been the focus of considerable debate among evolutionary biologists for several decades and two main, non-mutually exclusive, hypotheses have been proposed [15,23, 24]. According to the paternal investment hypothesis, donations would consist of substances, potentially nutritive, that enhance female ‘condition’ (e.g. higher egg load or survival and larger eggs), and ultimately increase the number or quality of the male's offspring. Alternatively, the mating effort hypothesis proposes that donations ‘protect’ the donor's 464 A. Jarrige et al. / Physiology & Behavior 151 (2015) 463–468 sperm by prolonging the female remating interval, hence reducing the risk of sperm competition. Current studies show that spermatophore function probably differs among bushcricket species [7,23,27]. Bushcricket spermatophylaxes mainly consist of water, protein (4– 27% of the wet mass), and a small amount of lipids [10,11]. Female condition is thought to be predominantly determined by the amount of free and protein-bound amino acids obtained via the spermatophylax. In line with the parental effort hypothesis, protein-bound amino acids, especially essential ones, may strongly affect female fecundity, because vitellogenesis is protein-limited [14,27,28,32]. In support of this hypothesis, ingested protein-bound amino acids have been found to be incorporated into female soma and eggs in bushcrickets [18,29,30], and experimental evidence showed that gift consumption positively affects female longevity and reproductive output [6,7,23]. In contrast, free amino acids are phagostimulants in many insects and may instead improve the gift's gustatory appeal and/or texture [2,4,19,21,26]. Besides, the low concentration of free amino acids in the spermatophylax implies that these substances are unlikely to represent a significant contribution to the female's diet. Consequently, this fraction may be more indicative of the male's mating effort than his parental effort. Despite the considerable variation in spermatophylax size between species (from 2 to 40% of male body mass), gift-giving behaviors are costly for males and strongly limit their reproductive rate [6,15]. Consistent with this premise, males from various bushcricket species strategically allocate their resources by selectively mating with particular females, or by adjusting the size of their donation, and thereby protein content, according to i) female quality [13,29], ii) the risk of sperm competition [20] and iii) their own physiological condition [13,31]. In contrast to other types of nuptial gifts transferred prior to mating [23], bushcricket spermatophylaxes are manufactured by male reproductive glands during copulation [6]. This opens the opportunity for cryptic male mate choice through the manipulation of the biochemical composition of the gift according to female quality. However, to our knowledge, no study has yet jointly investigated the amino-acid composition of nuptial gifts and their variation with regard to female traits. In this study we focused on determining the free amino acid composition and protein-bound amino acid composition of spermatophylaxes produced by males in Ephippiger diurnus (Orthoptera: Tettigoniidae) and measuring the extent to which males modify spermatophylax composition with respect to traits (age and body weight) of the receiving female. In this species, males produce an unusually large spermatophore (20–30% of their body mass) that appears to be particularly costly: its transfer results in a 4–5 day refractory period [31]. Moreover, the nitrogen content of the spermatophylax decreases with successive matings [13,31], suggesting that at least part of these resources cannot be replenished via feeding. Consequently, males allocate their scarce resources strategically by modifying the size, and thereby the total protein content, of their donation according to mate quality [13]. The present study provides a detailed analysis of the amino acid content in the E. diurnus spermatophylax. Our results revealed quantitative, as well as qualitative, variations in the gift's amino acid composition according to female body mass and age. Together, these results indicate that gift production represents a plastic process by which males adjust their reproductive expenditure according to the potential fitness returns associated with mate quality. 2. Methods 2.1. Rearing and maintenance E. diurnus (Dufour, 1841) (Orthoptera: Tettigoniidae) used in experiments were offspring of individuals collected in the field at Col de Mantet (42°29′N, 2°3′E, Pyrénées Orientales, France) in July 2008. Eggs were cultured following the standard methods for this species [8] which consisted of two diapauses of 60 days at 4 ± 2 °C, separated by an interval of 120 days during which eggs were kept at 20 ± 2 °C. Eggs were placed on cotton covered by filter paper in Petri dishes and regularly sprayed with a 1% methyl-4-hydroxybenzoate solution to prevent desiccation and mold development. Nymphs were reared individually in 5 cm diameter × 8 cm height plastic containers and fed ad libitum with cabbage bee pollen, and flaked goldfish food. After the final molt, adults were individually transferred to larger plastic cages (10 cm diameter × 15 cm height) and provided with the same diet as nymphs. Rearing and experiments took place in environmental chambers maintained at 25 ± 2 °C on a L:D 16 h:8 h cycle. 2.2. Mating and spermatophores 2.2.1. Mating To obtain spermatophores, 28 males aged from 25 to 35 days after final ecdysis were randomly paired with virgin females ranging from 16 to 63 days old; 24 females were 24-to-49 days old, 1 female was 16 days old and 3 females were between 56 and 63 days old. E. diurnus, have a long lifespan, and females within this age range are able to reproduce and produce viable eggs. Mating took place between 8 am and 15 pm, the peak period for singing and mating in E. diurnus (Busnel 1955). Males and females were weighed on a microbalance (±1 mg; Mettler-Toledo, Greifensee, Switzerland) prior to mating sessions, and female age was recorded. 2.2.2. Spermatophore collection and analysis Immediately after mating, spermatophores were carefully removed from the females' genitalia. Fresh ampullae and spermatophylaxes (SPFx) were weighed separately on a microbalance (±1 mg; MettlerToledo, Greifensee, Switzerland). We estimated the water content of a spermatophylax by comparing its fresh and dry weights. Desiccation was achieved by freeze-drying (primary drying: 1 h at − 10 °C, 25 mbar, secondary drying: − 76 °C, 0.001 mbar overnight; Bioblock Scientific Alpha1-4LDplus lyophilizator). The dried subsamples were ground to powder with a mortar and stored at −80 °C until subsequent analysis. 2.3. Free and protein-bound amino acid analysis From a subset of 5 mg of powdered spermatophylax, free amino acids were extracted with 1.2 mL acetonitrile 25% in HCl 0.01 N (1:3, v:v). From another subset of 5 mg of powdered spermatophylax, proteins were hydrolyzed into their protein-bound amino acids in a sealed glass tube at 150 °C for 2 h with 500 μL of 4 M methanesulfonic acid after flushing out air with a gentle stream of nitrogen gas. Following hydrolysis, the hydrolysates were partially neutralized with 1 mL sodium carbonate 1 M. Prior to analysis, samples were transferred to a 1.5 mL Eppendorf tube, and pH was checked to confirm that it was between 1.5 and 5.0. Free and hydrolyzed protein-bound amino acids were extracted and derivatized as described in the EZ:faast amino acid analysis kit (Phenomenex Ltd, Aschaffenburg, Germany). Subsequent samples were then concentrated under a stream of nitrogen gas and immediately injected into the GC–MS system composed of an AutoSystem XL gas chromatograph (ZB-AAA column (10 m × 0.25 mm), Phenomenex Ltd) coupled to a TurboMass mass spectrometer (Perkin-Elmer, Courtabœuf, France). Helium served as the carrier gas and its flow was held constant at 1.1 mL/min. The oven temperature program was a 30 °C/min ramp from 110 °C to 320 °C, with the temperature of the injection port maintained at 250 °C. The MS ion source (electronic impact) and inlet line temperatures were 200 °C and 310 °C, respectively. The scan range was 3.5 scans/s and atomic masses between 45 and 450 Da were detected. Under these conditions, a 2 μL sample was injected in splitless mode during 30 s. We used Norvaline at 200 nmol·mL−1 as an internal standard. Calibration curves for each of the standard physiological amino acids were produced using an original concentration of A. Jarrige et al. / Physiology & Behavior 151 (2015) 463–468 Table 1 Composition (mean ± SEM) of E. diurnus spermatophylaxes in free and protein-bound amino acids. Ala Gly Val* Leu* Ile* Thr* + Ser Pro Asn Arg + Asp Met* Glu Phe* + Cys Gln Lys* His* Tyr Trp* Essential Non-essential Total Free amino acids (mg) Protein-bound amino acids (mg) Free amino acid (% total) Protein-bound amino acids (% total) 0.04 ± 0.004 2.20 ± 0.14 0.09 ± 0.01 0.09 ± 0.09 0.01 ± 0.002 0.05 ± 0.01 0.07 ± 0.01 0.04 ± 0.01 0.03 ± 0.004 0.003 ± 0.001 0.53 ± 0.02 0.010 ± 0.02 0.01 ± 0.001 0.004 ± 0.001 0.25 ± 0.054 0.06 ± 0.012 0.011 ± 0.003 0.62 ± 0.07 2.50 ± 0.14 3.12 ± 0.17 0.82 ± 0.09 3.96 ± 0.30 5.70 ± 0.57 15.13 ± 1.72 3.20 ± 0.41 0.72 ± 0.07 3.18 ± 0.24 0.14 ± 0.02 1.024 ± 0.20 0.42 ± 0.08 4.19 ± 0.60 6.71 ± 0.87 0.06 ± 0.02 13.95 ± 1.92 8.71 ± 1.21 4.59 ± 0.61 0.20 ± 0.04 54.74 ± 5.89 17.96 ± 1.62 72.70 ± 7.11 1.51 ± 0.22 70.46 ± 2.5 3.09 ± 0.27 2.82 ± 0.21 0.42 ± 0.07 1.74 ± 0.19 2.52 ± 0.23 1.27 ± 0.21 1.08 ± 0.30 0.10 ± 0.01 1.60 ± 0.41 3.36 ± 0.53 0.15 ± 0.03 0.14 ± 0.02 7.59 ± 1.40 1.79 ± 0.31 0.35 ± 0.07 0.97 ± 0.15 3.82 ± 0.26 4.78 ± 0.34 1.17 ± 0.05 5.85 ± 0.34 8.08 ± 0.37 20.94 ± 1.06 4.33 ± 0.26 1.10 ± 0.10 4.72 ± 0.29 0.19 ± 0.02 1.43 ± 0.21 0.67 ± 0.12 6.21 ± 0.72 9.07 ± 0.65 0.10 ± 0.04 18.06 ± 1.09 11.54 ± 1.17 6.28 ± 0.51 0.27 ± 0.05 70.55 ± 1.29 24.67 ± 1.15 95.22 ± 0.34 N = 28. * indicates essential amino acids [33]. 465 weighed on average 823.00 ± 24.33 (Md: 791.98, Q1: 637.39, Q3: 892.13) mg and contained a spermatophylax of 678.44 ± 20.24 (Md: 659.56, Q1: 612.65, Q3: 733.36) mg. Spermatophylax and ampulla weights were positively correlated (rs = 0.49, P = 0.007, N = 28). Water represented 85.12 ± 0.31 (Md: 85.34, Q1: 80.83, Q3: 86.05) % of the spermatophylax fresh mass. In total, spermatophylaxes contained 75.81 ± 7.16 (Md: 55.68, Q1: 35.04, Q3: 95.64) mg of amino acids, representing 11.15 ± 1.03% of their fresh mass and 74.20 ± 6.46% of their dry mass. Free amino acids represented only 4.78 ± 0.34% of this amount, while protein-bound amino acids represented the remaining 95.22 ± 0.34%. Amino acid composition is detailed in Table 1. Two pairs of amino acids, serine and threonine, and cysteine and phenylalanine, were co-eluted, and our data therefore show the combined amounts of each pair (Table 1). 3.1.1. Free amino acids In total, the spermatophylaxes contained on average 3.12 ± 0.17 (Md: 3.26, Q1: 0.97, Q3: 3.66) mg of free amino acids. Most amino acids (2.50 ± 0.14, Md: 2.40, Q1: 6.31, Q3: 2.81 mg) were nonessential, with glycine predominating (70.46 ± 2.5% of the total free amino acid content). Essential free amino acids, including serine and cysteine, represented only 0.62 ± 0.07 (Md: 0.52, Q1: 0.15, Q3: 0.73) mg (Table 1). 200 nmol·mL− 1. Chromatogram analyses were completed using TurboMass™ Software (version 5.4.2; Perkin-Elmer, Courtabœuf, France). The total free amino-acid content and protein bound aminoacid content of spermatophylaxes were calculated by multiplying their content in 1 mg of dry spermatophylax by the spermatophylax total dry weight. 3.1.2. Protein-bound amino acids Spermatophylaxes contained a total of 72.70 ± 7.11 (Md: 52.76, Q1: 32.49, Q3: 92.15) mg of protein-bound amino acids. In this fraction, leucine, lysine and histidine, all essential amino acids, dominated, accounting for respectively 20.94 ± 1.06%, 18.06 ± 1.09% and 11.54 ± 1.17% of the total. Essential amino acids formed the major part of this fraction with 54.74 ± 5.89 (Md: 40.31, Q1: 23.75, Q3: 68.73; 70.56 ± 1.29%) mg (Table 1). 2.4. Statistical analysis 3.2. Male body mass Relationships between amino acid content and female body mass, female age, and male body mass were investigated using Spearman correlation tests, as most of the data were non-normally distributed. To account for the number of comparisons being performed and avoid false positives, the alpha value was adjusted following the Benjamini– Hochberg procedure with a false discovery rate of 0.10 [1,16]. All data were analyzed using SigmaStat 3.5 Software. The amount of non-essential amino acids of both types, free and protein-bound, was negatively correlated with male body mass (Table 2). In contrast, spermatophylax water content increased with male body mass (Table 2). 3. Results 3.1. Spermatophore composition Data are presented as mean ± SEM in Table 1, and mean ± SEM (median, Q1, Q3) in the text, except for percentages. Spermatophores 3.3. Female body mass and age No significant relationship was found between female body mass and the general features or amino acid content of the spermatophylax that they received (i.e. mass, water content and ampulla weight), although larger females tended to receive more essential free amino acids than smaller females (Table 2). In contrast, older females received larger spermatophylaxes (Table 2 & Fig. 1a), containing more proteinbound amino acids, and less water and free glycine (Table 2 & Fig. 1b, Table 2 Relationship between male and female general features and amino acid content of spermatophylaxes. Spermatophylax features Mass (mg) Water content (% of fresh mass) Ampulla weight (mg) Free amino acids (mg) Essential Non-essential (without glycine) Glycine Total Protein-bound amino acids (mg) Essential Non-essential Total Male body mass (mg) Female body mass (mg) Female age (days) rs = 0.01, P = 0.95, α = 0.1 rs = 0.57, P = 0.002, α = 0.01 rs = −0.15, P = 0.44, α = 0.07 rs = −0.14, P = 0.48, α = 0.06 rs = −0.16, P = 0.42, α = 0.02 rs = −0.14, P = 0.48, α = 0.07 rs = 0.49, P = 0.008, α = 0.04 rs = −0.59, P b 0.0001, α = 0.01 rs = 0.60, P b 0.0001, α = 0.02 rs = −0.27, P = 0.17, α = 0.05 rs = −0.39, P = 0.04, α = 0.04 rs = −0.13, P = 0.52, α = 0.08 rs = −0.40, P = 0.04, α = 0.03 rs = 0.43, P = 0.02, α = 0.01 rs = 0.05, P = 0.78, α = 0.09 rs = −0.04, P = 0.84, α = 0.1 rs = 0.16, P = 0.43, α = 0.04 rs = 0.15, P = 0.46, α = 0.1 rs = 0.30, P = 0.12, α = 0.08 rs = −0.39, P = 0.04, α = 0.07 rs = 0.23, P = 0.23, α = 0.09 rs = −0.12, P = 0.54, α = 0.09 rs = −0.48, P = 0.01, α = 0.02 rs = −0.24, P = 0.22, α = 0.06 rs = 0.16, P = 0.42, α = 0.03 rs = 0.10, P = 0.61, α = 0.08 rs = 0.15, P = 0.43, α = 0.05 rs = 0.51, P = 0.005, α = 0.03 rs = 0.41, P = 0.03, α = 0.06 rs = 0.50, P = 0.008, α = 0.05 Spearman rank correlation, α-values are given following Benjamini–Hochberg procedure and for a false discovery rate of 0.10. N = 28. Significant results are in bold. 466 A. Jarrige et al. / Physiology & Behavior 151 (2015) 463–468 d, e, f). They also received a heavier ampulla (Table 2 & Fig. 1c). No significant relationship between female age and the total amount of free amino acids (without glycine) contained in the spermatophylax was found (Table 2). Female age and body weight were not correlated (rs = 0.09, P = 0.65, N = 28). 4. Discussion This study is the first to simultaneously investigate the free and protein-bound fractions of bushcricket nuptial gifts. Consistent with previous findings in E. diurnus [13,28], spermatophylaxes consisted of approximately 85% of water and 11% of amino acids. More detailed measurements revealed that the amino acid fraction consisted of 5% of free amino acids and 95% of protein-bound amino acids. Among free amino acids, glycine was largely predominant. However, glycine was not abundant in the protein-bound fraction, most of which were essential amino acids (Table 1). Larger males produced spermatophylaxes containing more water and a lower amount of non-essential amino acids (both free and protein-bound) (Table 2). These adjustments could reflect a trade-off between somatic maintenance and gift Fig. 1. Relationship between female age and (a) spermatophylax mass, (b) spermatophylax water content, (c) ampulla mass, (d) essential protein-bound amino acids, (e) non-essential protein-bound amino acids and (f) free glycine. A. Jarrige et al. / Physiology & Behavior 151 (2015) 463–468 production, as somatic costs may increase with body mass. However, our results revealed that the most significant variations in gift composition were related to female traits. Indeed, males transferred a larger amount of protein-bound amino acids (both essential and nonessential), less water and less free glycine to older mates (Table 2). These relationships suggest that E. diurnus males adjust the aminoacid composition of their nuptial gifts, and that these adjustments may represent an adaptation for maximizing reproductive success when mating with females of variable ‘quality’. In nature, sexually receptive mates can be rare and of variable quality (Jarrige et al., 2015, in prep.). Rejecting partners would not be advantageous. In contrast, by adjusting the composition of nuptial gifts to female quality, males could invest the adequate resources, thereby moderating their loss in reproductive potential [25] without dismissing any mating opportunity. Because physical contact during pre-mating interactions, as observed in E. diurnus (Busnel 1955), could provide males with reliable cues about female quality [3], we propose that the observed modifications in amino acid composition reflect a form of cryptic male mate choice. In E. diurnus, recent field observations suggested that female body mass is a poor predictor of fecundity, compared to age (Jarrige et al., 2015, in prep.). Indeed, for a similar mass, young females carry few mature eggs but plenty of body fat, while older females carry more mature eggs but less fat (Jarrige et al., 2015, in prep.). Due to their age, older females are also less likely to mate again. These factors, combined with the last male sperm precedence effect observed in E. diurnus [12], reduce the risk of sperm competition in such females. It would therefore be advantageous for males to provide older females with more nutritious donations in order to extend their survival, fecundity, and ultimately the number of offspring that they would sire. Conversely, males mating with younger females would benefit from increasing an investment that prolongs their mate's refractory period. Thus, males would lower the high risk of sperm competition expected in this situation. Our detailed analysis of gift composition in amino acids allows us to infer possible functions of the spermatophore in E. diurnus. In insects, protein content of a nuptial gift is often considered as a measure of its nutritive quality because female vitellogenesis is protein-limited [14, 32]. Hence, females might use protein-bound amino acids in the spermatophylax to sustain their metabolic and/or reproductive activities. In contrast, free amino acids would only marginally contribute to a female's diet, because they are present at extremely low amounts in the spermatophylax (Table 1). Their presence, however, might strongly affect the male fertilizing success by acting as a phagostimulant: it would lengthen the duration of female gift consumption [2,4,26], hence potentially favoring sperm transfer into the female genital tract. High levels of free glycine may enhance this effect by increasing the gummy consistency of the spermatophylax, which prolongs its handling time by females [10,26]. This small amino acid has also been found to increase a female's refractory period [2,5,9,26], thus reducing her remating rate. Finally, because water is not limited in the natural habitat of E. diurnus, its presence in the spermatophore is unlikely to represent a critical contribution to female diet. In summary, older females received significantly more essential and non-essential protein-bound amino acids, less water and free glycine and, although not reaching significance after correction for multiple analyses, larger females tended to receive a larger amount of essential free amino acids. This pattern suggests that E. diurnus males provide older females with more nutritious gifts containing less manipulative or deceiving substances, while larger females tend to receive more substances likely to exploit their sensory responses. Thus, the spermatophylax may function primarily as mating effort in younger females in which sperm competition can be high, whereas it would increase paternal investment in older, potentially more fecund females, in which sperm competition is reduced but survival limited [12,13]. Moreover, the positive relationship between spermatophylax and 467 ampulla size suggests that the gift may simultaneously serve as a sperm protection device (mating effort) (Table 2). The notion that nuptial feeding can concomitantly function as parental and mating effort within a species is not novel, and has been documented in numerous taxa [7,15,17,23,25]. However, our study provides evidence that males are not only able to adjust the size of their donation, but also to finely tailor its amino acid content in regard to female traits. Such a sophisticated manipulation of proteins has already been observed in the ejaculate of Drosophila melanogaster [22]. Together with the present work, it suggests that strategic allocation of amino acids and proteins might be widespread, especially in species where mating involves a costly, complex product of male metabolism. Importantly, the potentiality that males might strategically tailor spermatophylax composition according to mate quality put into question the classical attempt to sort the nuptial gift into the dichotomous mating or parental effort function, because the confounding effect of cryptic male mate choice would render gift function highly labile within the successive mating episodes of the same individual. More theoretical and empirical studies are therefore required to investigate the extent to which these variations affect the fitness of males and females and their evolutionary consequences on both sexes. Acknowledgments We thank Jean-Philippe Christidès for laboratory help. We thank two anonymous reviewers who provided helpful comments on earlier drafts of the manuscript. Financial support was provided by the Région Centre Project n°2014 00094521 to D. Giron and the Université François Rabelais de Tours. References [1] Y. Benjamini, Y. Hochberg, Controlling the false discovery rate: a practical and powerful approach to multiple testing, Journal of the Royal Statistical Society B 57 (1995) 289–300. [2] P.A. Calatayud, M.A. Polanía, J. Guillaud, D.F. Múnera, J.C. Hamon, A.C. Bellotti, Role of single amino acids in phagostimulation, growth, and development of the cassava mealybug Phenacoccus herreni, Entomologia Experimentalis et Applicata 104 (2002) 363–367. [3] D.A. Edward, T. Chapman, The evolution and significance of male mate choice, Trends Ecol. Evol. 26 (2011) 647–654. [4] S.N. Gershman, C. Mitchell, S.K. Sakaluk, J. Hunt, Biting off more than you can chew: sexual selection on the free amino acid composition of the spermatophylax in decorated crickets, Proc. R. Soc. B 279 (2012) 2531–2538. [5] D.G. Gordon, S.N. Gershman, S.K. Sakaluk, Glycine in nuptial food gifts of decorated crickets decreases female sexual receptivity when ingested, but not when injected, Anim. Behav. 83 (2012) 369–375. [6] D.T. Gwynne, Katydids and Bush-crickets: Reproductive Behavior and Evolution of the Tettigoniidae, Cornell University Press, 2001. [7] D.T. Gwynne, Sexual conflict over nuptial gifts in insects, Annu. Rev. Entomol. 53 (2008) 83–101. [8] J.C. Hartley, R.L. Dean, Ephippiger crucigeras a laboratory insect (Orthoptera, Tettigoniidae), J. Nat. Hist. 8 (1974) 349–354. [9] R. Heinrich, K. Rozwòd, N. Elsner, Neuropharmacological evidence for inhibitory cephalic control mechanisms of stridulatory behaviour in grasshoppers, J. Comp. Physiol. A. 183 (1998) 389–399. [10] K.G. Heller, S. Faltin, P. Fleischmann, O.V. Helversen, The chemical composition of the spermatophore in some species of phaneropterid bushcrickets (Orthoptera: Tettigonioidea), J. Insect Physiol. 44 (1998) 1001–1008. [11] K.G. Heller, P. Fleischmann, A. Lutz-Rider, Carotenoids in the spermatophores of bushcrickets (Orthoptera: Ephippigerinae), Proc. R. Soc. Lond. B 267 (2000) 1905–1908. [12] L. Hockham, J. Graves, M. Ritchie, Sperm competition and the level of polyandry in a bushcricket with large nuptial gifts, Behav. Ecol. Sociobiol. 57 (2004) 149–154. [13] A. Jarrige, M.D. Greenfield, M. Goubault, Male song as a predictor of the nuptial gift in bushcrickets: on the confounding influence of male choice, Anim. Behav. 85 (2013) 1427–1434. [14] M.J. Klowden, Physiological Systems in Insects, Elsevier Science, 2013. [15] S. Lewis, A. South, The evolution of animal nuptial gifts, Adv. Study Behav. 44 (2012) 53–97. [16] A. Reiner, D. Yekutieli, Y. Benjamini, Identifying differentially expressed genes using false discovery rate controlling procedures, Bioinformatics 19 (2003) 368–375. [17] K. Reinhold, K.G. Heller, The ultimate function of nuptial feeding in the bushcricket Poecilimon veluchianus (Orthoptera: Tettigoniidae: Phaneropterinae), Behav. Ecol. Sociobiol. 32 (1993) 55–60. 468 A. Jarrige et al. / Physiology & Behavior 151 (2015) 463–468 [18] J. Rooney, S. Lewis, Differential allocation of male-derived nutrients in two lampyrid beetles with contrasting life-history characteristics, Behav. Ecol. 10 (1999) 97–104. [19] S.K. Sakaluk, Sensory exploitation as an evolutionary origin to nuptial food gifts in insects, Proc. R. Soc. Lond. B 267 (2000) 339–343. [20] L.W. Simmons, C. Kvarnemo, Ejaculate expenditure by male bushcrickets decreases with sperm competition intensity, Proc. R. Soc. Lond. B 264 (1997) 1203–1208. [21] S.J. Simpson, C.L. Simpson, Mechanisms controlling modulation by haemolymph amino acids of gustatory responsiveness in the locust, J. Exp. Biol. 168 (1992) 269–287. [22] L.K. Sirot, M.F. Wolfner, S. Wigby, Protein-specific manipulation of ejaculate composition in response to female mating status in Drosophila melanogaster, Proc. Natl. Acad. Sci. 108 (2011) 9922–9926. [23] K. Vahed, The function of nuptial feeding in insects: a review of empirical studies, Biol. Rev. 73 (1998) 43–78. [24] K. Vahed, All that glisters is not gold: sensory bias, sexual conflict and nuptial feeding in insects and spiders, Ethology 113 (2007) 105–127. [25] K. Vahed, Comparative evidence for a cost to males of manipulating females in bushcrickets, Behav. Ecol. 18 (2007) 499–506. [26] S. Warwick, K. Vahed, D. Raubenheimer, S.J. Simpson, Free amino acids as phagostimulants in cricket nuptial gifts: support for the ‘Candymaker’ hypothesis, Biol. Lett. 5 (2009) 194–196. [27] N. Wedell, Dual function of the bushcricket spermatophore, Proc. R. Soc. Lond. B 258 (1994) 181–185. [28] N. Wedell, Variation in nuptial gift quality in bush crickets (Orthoptera: Tettigoniidae), Behav. Ecol. 5 (1994) 418–425. [29] N. Wedell, Mate quality affects reproductive effort in a paternally investing species, Am. Nat. 148 (1996) 1075–1088. [30] N. Wedell, B. Karlsson, Paternal investment directly affects female reproductive effort in an insect, Proc. R. Soc. Lond. B 270 (2003) 2065–2071. [31] N. Wedell, M.G. Ritchie, Male age, mating status and nuptial gift quality in a bushcricket, Anim. Behav. 67 (2004) 1059–1065. [32] D. Wheeler, The role of nourishment in oogenesis, Annu. Rev. Entomol. 41 (1996) 407–431. [33] R.H. Dadd, Nutrition: organisms. In Comprehensive insect physiology, biochemistry and pharmacology, in: G.A. Kekut, L.I. Gilbert (Eds.), vol.4, Pergamon Press, Oxford, UK 1985, pp. 313–390.