Since spermatophore compounds are also incorporated into the somatic tissue of females (Bowen et al. 1984; Wedell 1993; Voigt et al. 2006), nuptial gifts could also be important nutritional sources for females (reviewed in Gwynne 2008). For example, in the European bushcricket Isophya kraussi, nuptial gifts may provide sufficient energy to cover 2 or 3 days of a female’s metabolic requirements (Voigt et al. 2005). Fuelling the metabolism of females with spermatophore nutrients may be advantageous for females, since they would then need to feed less on plant matter (Heller 1996; see Boggs (1990) for a model using butterflies) and may therefore be less exposed to predation (Heller 1992). Accordingly, spermatophores may benefit a male’s offspring indirectly by increasing the survival rate of the egg-producing female (Wickler 1994). Therefore, we asked whether female bushcrickets may gain immediate benefits from nuptial gift consumption by routing spermatophore nutrients to metabolism. We enriched male donors of nuptial gifts with different levels of 13C, and predicted that females feeding on nuptial gifts should exhibit stable carbon isotope ratios in their exhaled breath according to the ratio of the male donor, if they combust spermatophore nutrients. Biol. Lett. (2008) 4, 476–478 doi:10.1098/rsbl.2008.0282 Published online 1 July 2008 Animal behaviour Female bushcrickets fuel their metabolism with male nuptial gifts Christian C. Voigt1,*, Antje S. Kretzschmar2, John R. Speakman3 and Gerlind U. C. Lehmann4 1 Evolutionary Ecology Research Group, Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Strasse 17, 10315 Berlin, Germany 2 Department of Biology, Humboldt University of Berlin, Invalidenstrasse 42, 10115 Berlin, Germany 3 Aberdeen Centre for Energy Regulation and Obesity, School of Biological Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK 4 Research Group Evolutionary Biology, Institute of Biology/Zoology, Free University of Berlin, Königin-Luise-Strasse 1-3, 14195 Berlin, Germany *Author for correspondence (voigt@izw-berlin.de). In many arthropods, such as bushcrickets, males donate protein-rich nuptial gifts—so-called spermatophores—to females, which females ingest while the sperm enter the female’s reproductive tract. Previously, it was shown that females route spermatophore nutrients over the course of hours and days to egg production or body synthesis. We investigated whether female bushcrickets fuel their metabolism with spermatophores immediately after consumption. We fed two male groups diets that were either enriched or depleted in 13C, and then tracked the isotopic changes in exhaled breath in female bushcrickets after spermatophore consumption. Within 3 hours, the stable carbon isotope ratio (d13C) of female breath converged on the ratio of the male donor of the nuptial gift. This supports the idea that females quickly routed nutrients to metabolism, receiving immediate benefits from spermatophore feeding. 2. MATERIAL AND METHODS (a) Insect maintenance and diet We collected 21 male and 22 female Poecilimon ornatus as nymphs in Slovenia (45.611 N; 13.936 O; 900 m NN). Females were maintained until the onset of the experiment on a diet of flowering Taraxacum officinale (Asteraceae), Plantago major (Plantaginaceae) and Bellis perennis (Asteraceae). Immediately after capture, the males were assigned to two groups with different feeding regimes. Group 1 (nZ12) was maintained on a diet of Lactuca sativa (Asteraceae) and group 2 (nZ9) on a mixed diet of L. sativa (Asteraceae) and seaweed (Sushi Nori, OKF Corporation, South Korea). We started the experiment approximately three weeks after mature moulting. From each individual, we took an initial breath sample prior to the experiment (see below for a description of breath collection). The females were introduced each to a single male of either group 1 or 2 in small observational cages (70!80!23 cm). Once nuptial gifts were transferred to females, males were removed from the cages. We noted the onset of nuptial feeding in females and collected breath samples from females at the following time intervals: 1, 3, 5, 7, 9, 11 and 24 hours after the female started to feed on the nuptial gift. Keywords: female choice; homeostasis; nutrient routing (b) Breath collection and isotopic analysis For breath collection, bushcrickets were transferred singly into a 65-ml plastic container to which a needle was hermetically fused with the tip facing the outside and the base the inside of the container. After closing the lid of the container (except for the needle as an air outlet), the container air was washed of CO2 by flushing ambient air through NaOH and subsequently through the container via a plastic tube (diameter 3 mm) for 5 min at a flow-through rate of ca 0.5 l minK1. Then, we sealed the plastic container for 1 hour to let the exhaled CO2 accumulate in the container. To extract the exhaled CO2 from the container, we penetrated the Teflon membrane of an evacuated vacutainer (LabcoTM, Buckinghamshire, UK) with the needle tip attached to the plastic container. Thus, approximately 10 ml of air was sucked via the needle into the vacutainer. Afterwards, the experimental animals were transferred back to their cage, where they continued to feed on the nuptial gift. Breath samples were automatically flushed from the vacutainers in a stream of chemically pure helium, after which a gas chromatograph separated the CO2 gas from the other gases before admitting it into the isotope ratio mass spectrometer in a continuous flow. Breath samples, together with internal standards that had been previously characterized relative to an international 13C standard (IAEA-CO-1), were analysed in duplicates. All 13C/12C values were expressed relative to the international standard using the d notation (‰) and the following equation:   Rsample K 1 !1000; d13 C Z Rstandard 1. INTRODUCTION Nuptial feeding—the transfer of a gift from a male to a female in exchange for mating—is a common phenomenon among arthropods (reviewed in Vahed 1998) and represents an interesting evolutionary scenario of cooperation and conflict between the sexes (Parker & Simmons 1989; Simmons & Parker 1989). In bushcrickets, spermatophore production is widespread (Gwynne 2001) and imposes severe costs on males (Vahed 2007a). Female bushcrickets usually prefer larger males as mates, since they provide larger spermatophores (Gwynne 1982; Lehmann & Lehmann 2008). Several studies found positive effects of spermatophylax consumption on female reproductive output, e.g. an increase in fecundity or offspring survival (reviewed in Gwynne 2001). But such positive effects on female fitness have not been detected in other species (Wedell & Arak 1989; Vahed & Gilbert 1997; Vahed 2003), questioning the generality of direct benefits of male nuptial gifts for female fitness (Vahed 2007b). Received 20 May 2008 Accepted 12 June 2008 476 This journal is q 2008 The Royal Society Metabolized nuptial gifts C. C. Voigt et al. 477 13 C breath (‰) –26 males of group 1 –27 –28 –29 males of group 2 0 5 10 15 20 time elapsed since initial nuptial feeding (hours) Figure 1. Mean d13Cbreath (G1 s.e; ‰) of female P. ornatus during a 24 hour time period after they started to feed on nuptial gifts from males of groups 1 and 2, which differed in their stable carbon isotope signature. The mean d13Cbreath of group 1 males is indicated by a long-dashed line and those of group 2 males by a short-dashed line. The d13Cbreath of females feeding on nuptial gifts from males of group 1 (open circles) remained constant, whereas that of females feeding on nuptial gifts from males of group 2 (closed circles) decreased in 13C enrichment over time. where R sample and R standard represent the ratio 13C/12C of the sample and standard, respectively. Precision was better than G0.01 ‰ (1s). All samples were analysed using a blind experimental protocol. (c) Exponential model and statistics We expected that changes in isotopic composition of the breath follow a single-pool exponential model after the females fed on nuptial gifts. Therefore, we calculated equations of the following type for females feeding on nuptial gifts contrasting in stable carbon isotope ratio to their previous diet, d13 CðtÞ Z d13 CðNÞ C ðd13 Cð0ÞKd13 CðNÞÞ !e Kkt : In this equation, d13C(t) is the stable carbon isotope ratio of exhaled CO2 at time t, d13C(N) the asymptotic stable carbon isotope ratio of exhaled CO2 when animals are equilibrated to the stable carbon isotope signature of their diet, d13C(0) the stable isotope ratio of exhaled CO2 at time 0 of the experiment, and k the fractional incorporation rate of ingested carbon atoms into the pool of metabolized substrate. Estimation of k was performed on an iterative basis using SIGMAPLOT (SPSS v. 8.0). For each feeding group, we averaged regression coefficients for all individual values. We calculated the time at which 50% of carbon isotopes are exchanged in the animals’ breath (t50; hours) according to the following equation: t50ZKln(0.5)/k, with ln representing the natural logarithm and 0.5 the exchange of 50% isotopes in exhaled CO2. All values are given as meanG1 s.e. and all statistical tests were performed two-tailed at a significance level of 5%. We performed Mann–Whitney (MW) U-tests to test for differences in median d13Cbreath(N) of females and d13Cbreath of males and d13C of the plant diet. 3. RESULTS Shortly before mating, the exhaled breath of the two male groups differed by ca 2.2 ‰ in 13C enrichment (MW U-test: n 1Z9, n 2Z12, UZ17, U 0 Z91, pZ0.0073). The stable carbon isotope ratio (d13Cbreath) averaged K27.11G0.33 ‰ in males of group 1 and K29.34G0.53 ‰ in males of group 2. The d13Cbreath of females averaged K26.72G0.32 ‰, which did not deviate significantly from the isotopic signature of their diet (MW U-test: n1Z19, n 2Z4, UZ29, U 0 Z47, pZ0.51). We then allowed females to copulate with males and monitored the change in d13Cbreath of females for 24 hours following the Biol. Lett. (2008) onset of nuptial feeding. Once females had started to feed on nuptial gifts, their d13Cbreath converged quickly on the d13Cbreath of the donor of the nuptial gift (figure 1). On average, females exchanged 50% of carbon atoms in exhaled breath with those from ingested nuptial gifts within 2.5G0.6 hours. After 24 hours, females that had fed on nuptial gifts of males from groups 1 and 2 differed in d13Cbreath by, on average, 1.9 ‰ (MW U-test: n 1Z9, n 2Z8, UZ8, U 0 Z64, pZ0.0055), which was almost identical to the isotopic difference in exhaled CO2 between the two groups of males. Exhaled CO2 of females feeding on nuptial gifts of group 2 males was depleted in 13C by 2.3 ‰ in relation to the stable carbon isotope signature of the plant food (MW U-test: n 1Z9, n 2Z4, UZ0, U 0 Z36, pZ0.0028), which was available for all females throughout. 4. DISCUSSION In our experiment, d13Cbreath of females that had fed on nuptial gifts levelled off at values close to the d13Cbreath of male donors. Since the d13Cbreath of orthopteran insects closely matches the dietary d13C (DeNiro & Epstein 1978; this study), we suggest that female P. ornatus routed some of the spermatophore nutrients to metabolism. The nutritional benefit of ingesting and combusting a large nuptial gift may be substantial for reproducing female bushcrickets, since males donate up to 30% of their body mass to females (Wedell 1993; Vahed & Gilbert 1996) and dry matter of nuptial gifts mostly consists of energy-rich glycoproteins (Heller et al. 1998). Measurements of field metabolic rate in free-ranging bushcrickets indicated that females could sustain their metabolism for 2 or 3 days with the digestible energy included in a single nuptial gift (Voigt et al. 2005). This time period often elapses between two subsequent matings in similar-sized female bushcrickets (Heller & von Helversen 1991; 478 C. C. Voigt et al. Metabolized nuptial gifts Vahed & Gilbert 1996; Vahed 2006). Females may then benefit from spermatophore consumption and combustion by not having to search for additional food (see Boggs 1990), and by being less likely to fall prey to predators (Heller 1992). Possibly, female bushcrickets follow a similar selective nutrient-routing strategy like egg-producing butterflies (O’Brien et al. 2002), allocating some essential amino acids from nuptial gifts selectively to egg production and some non-essential material to metabolism. 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