Dyadic contests for possession of indivisible resources are of longstanding and ongoing interest as they occur across a wide range of animal taxa and have provided key examples for the game-theoretic approach to understanding behavioural evolution (Maynard Smith and Price, 1973; Hardy and Briffa, 2013). Two major predictors of contest outcomes are the competitive ability of individuals (resource holding potential, RHP) and the value (V) that contestants place on possession of the resource (Maynard Smith and Parker, 1976; Kokko, 2013): each of these may be affected by the nutritional status of the contestants. For example, individuals that have been well fed may be able to persist for longer during contests (RHP) due to their enhanced energy reserves (Poole, 1989; Kemp and Alcock, 2003; Martinez-Lendech et al., 2007; Briffa et al., 2013b) while individuals with depleted reserves may compete more intensely for a contested resource, due to placing a higher value (V) on its exploitation (Riechert, 1998). Further, the availability of macronutrients (protein, fats and carbohydrates) may promote dominance-related behaviours (Davidson, 1997) while other metabolites may physiologically constrain contest behaviour. For instance, in hermit crabs and other crustaceans, metabolites related to energy reserves (glycogen and glucose) and aerobic capacity (lactic acid) as well as hormones (biogenic amines) influence contestant motivation and contest duration (Briffa and Sneddon, 2007; Briffa et al., 2013b).

In this paper we explore relationships between nutritional status and performance using the parasitoid wasp Goniozus legneri Gordh (Hymenoptera: Bethylidae). Female G. legneri parasitize host larvae that tunnel into crop tissues such as pistachio nuts, almonds and apples (Steffan et al., 2001). Attack by the wasp results in host paralysis and the female then remains in a restricted space with the host before oviposition approx. 24 h later. Additionally, G. legneri exhibits aggressive post-oviposition brood guarding behaviour, during which time a female may spend several days with each host both prior and post oviposition (Hardy et al., 2013). Because of the long time spent with a host, guarding females may encounter a series of conspecific intruders, with whom they compete for host possession (Bentley et al., 2009). A number of prior studies have shown that contest outcomes are affected by components of both RHP (contestant body size) and V (host size and nutritional quality, contestant age and reproductive status) (reviewed in Hardy et al., 2013). Effects of adult nutrition on Goniozus contest performance have, however, not been examined.

In common with other parasitoids (Ellers, 1996; Eijs et al., 1998; Casas et al., 2005; Jervis et al., 2008; Visser and Ellers, 2008), G. legneri adults lack de novo lipogenesis (Visser et al., 2010) and adults are thus limited to utilizing fat reserves accumulated throughout larval development. In many species of parasitoids, adults acquire nutrients from feeding on plant nectar, honey dew and host haemolymph (Jervis et al., 1993; Eijs et al., 1998; Giron et al., 2002; Visser and Ellers, 2008) and decline in lipid reserves can be slower when carbohydrate and lipid-rich diets are available (Ellers, 1996; Heimpel et al., 1997; Lee et al., 2004; Wäckers et al., 2006; Winkler et al., 2006; Desouhant et al., 2010; Ellers et al., 2011; Gómez et al., 2012; Harvey et al., 2012). Feeding on carbohydrates can also enhance adult parasitoid longevity (Heimpel et al., 1997; Pexton and Mayhew, 2002; Lee et al., 2004; Wäckers et al., 2006; Kapranas and Luck, 2008; Gómez et al., 2012) and preliminary laboratory observations (CJPS & AK pers. obs.) indicate that G. legneri fed with honey live longer than starved adults, suggesting that G. legneri can use dietary carbohydrates as a maintenance energy supply. Feeding on honey has the potential to counter the effect of the increased value, V, that older contestants place on host possession (Stockermans and Hardy, 2013, see also Humphries et al., 2006); if so, we might expect starved wasps to win contests against similarly aged but fed wasps. Conversely, honey-fed contestants may have enhanced contest abilities, RHP, due to higher energy reserves (Tsai et al., 2014); if so we might expect starved wasps to lose such contests.

To explore whether feeding on carbohydrates, such as honey, during the adult stage affects the subsequent energy reserves, longevity and contest performance of female G. legneri, we: (1) Employ a metabolomics approach (Snart et al., 2015; Kapranas et al., 2016) to assess how feeding on a carbohydrate-rich honey diet affects the concentrations of lipids, carbohydrates and other metabolites in individual parasitoids; (2) Compare the lifespans of starved wasps with those of wasps provided with carbohydrate-rich honey diet; (3) Stage contests for host possession between pairs of females, either both young or both old, in which one contestant was fed on honey and the other one was left unfed.

While several previous studies have considered the metabolomic effects of dietary intake in a range of organisms (German et al., 2002, 2003; Davis and Milner, 2004; Zeisel et al., 2005; Wang et al., 2009; Ametaj et al., 2010; Primrose et al., 2011), ours is the first to consider dietary effects on the metabolome in the context of animal resource competition. Results from our dyadic contest setup indicate that dietary intake does not influence the outcome of contests between G. legneri females, while we also find that feeding enhances female longevity considerably and that a large number of polar and non-polar metabolites associated with energy storage and metabolism were elevated in older fed wasps. There are a number of possible implications of these findings for the understanding of parasitoid behaviour and life-histories and we discuss these in turn along with suggestions for further investigations.

The energetic costs of animal contests have previously been examined in red deer (Clutton-Brock et al., 1979), cichlid fish (Neat et al., 1998a,b), hermit crabs (Briffa and Elwood, 2000, 2001, 2004, 2007), shore crabs (Sneddon et al., 1999), swimming crabs (Thorpe et al., 1994), and spiders (Prestwich, 1983a,b, 1988; DeCarvalho et al., 2004; Elwood and Prenter, 2013). These studies have predominantly focused on the depletion of immediately available energy reserves, such as glycogen, and the accumulation of muscle waste products, such as lactic acid (Briffa and Sneddon, 2007). Although fed wasps contained higher levels of polar-metabolites associated with the rapid mobilization of energy (e.g., glucose, trehalose, glutamate, glycerol) than did starved wasps, this does not appear to have enhanced their resource holding potentials (RHP); an affect that might be expected if contest interactions are energetically demanding. In a study on dyadic contests between male parasitoid wasps, Tasi et al. (2014) also found no effect of honey feeding on outcome when wasps were young (1 day old) but they did find that feeding by 4-day old males enhanced their contest success (although it was not possible to conclude whether this likely effect on RHP was operating via increased body mass after ingestion or improved energetic reserves). It may be that the Goniozus contest interactions we observed were too brief (typically < 60 s of agonistic interactions, including just a few seconds of fully escalated fighting, Petersen and Hardy, 1996) for energetic constraints on RHP to become apparent. Experiments that force G. legneri females into more sustained interactions, or that use bethylid species in which contests appear to be of naturally longer durations (Batchelor et al., 2005), may prove useful to explore this possibility further.

Our study also assessed the levels of non-polar metabolites, typically associated with long-term energy storage. As for the carbohydrates, the higher levels of glycerolipids (storage lipids) and diacylglycerides (the major form of fat mobilisation in insect haemolymph (Arrese and Soulages, 2010) and one of the primary energy sources for insect flight (Vogt et al., 2000; Harrison and Fewell, 2002) of 7-day old honey-fed wasps suggest that these contestants could have greater available energy and thus higher RHPs than their starved opponents. However, starved wasps could potentially place a greater value (V) on winning possession of the contested host than would honey-fed wasps, due to their lower life-expectancy (Humphries et al., 2006; Stockermans and Hardy, 2013) and/or the opportunity to host-feed, whereas fed wasps may perceive a host as having lower value due to their more abundant reserves. Thus, it remains possible that diet influences G. legneri contests between starved and fed wasps, but via opposing and coincidentally equal effects.

Further investigations could assess individual contest behaviours, rather than contest outcome. Older G. legneri females behave more aggressively during contests (Stockermans and Hardy, 2013), due to RV increasing as females age; thus increased aggression in contests between two starved wasps compared to two honey-fed wasps may similarly indicate that starved wasps value hosts more greatly, while higher aggression among fed wasps may indicate that diet affects RHP, and both influences may interact with female age. Preliminary observations on 3-day old female G. legneri, however, suggest that feeding does not affect behavioural interactions during contests (A.K. unpublished data). In other words, starved wasps could be on one hand weaker, due to lack of energetic reserves (lower RHP), but may on the other hand be bolder due to their limited future reproductive opportunities, and as a result fight more fiercely than honey fed wasps (higher RV). Similarly, Tasi et al. (2014) found no effect of honey feeding on the degree of agonism during male-male contests, despite feeding by older males affecting the outcome of contests.

While we have found no overall relationships between metabolome of wasps and contest outcomes, we have found relationships between wasp age and metabolomic state. At 3 days, the lipid profiles of honey-fed and starved wasps were similar but differed clearly among 7-day old wasps. This indicates that effects of dietary provision only become important as individuals approach the life-expectancy of unfed wasps. The greatly increased longevity of honey-fed wasps compared to starved wasps suggests that fed wasps are capable of preserving long-term lipid stores by using dietary carbohydrates as a maintenance source for energy metabolism. However, the presence of higher levels of diacylglycerides in 7-day old honey-fed females than in starved females suggests that carbohydrate-rich dietary feeding did not entirely supress lipid reserve mobilisation throughout the experimental time-frame. This result suggests that lipid store deterioration is merely delayed, rather than prevented, in honey-fed wasps.

Parasitoid fecundity has not been assessed in this study, but it is likely that the use of dietary carbohydrates to maintain vital body processes would allow greater allocation of lipid reserves toward egg production and maturation, as demonstrated by a number of studies on other parasitoid species (Jervis and Kidd, 1986; Heimpel et al., 1997; Winkler et al., 2006; Kapranas and Luck, 2008; Harvey et al., 2012). Indeed, it has already been shown for the congener Goniozus nephantidis that, as adult wasps aged, the egg loads of honey-fed females rose higher and declined later than those of starved females (Stokkebo and Hardy, 2000). Provision of such a diet could also conceivably aid parasitoid dispersion, as two of the three main circulating metabolites used during flight (trehalose and lipids) were found to be elevated in wasps that had consumed a honey diet (Beenakkers et al., 1984; Vogt et al., 2000; Harrison and Fewell, 2002). As such it is plausible that a parasitoid with access to a carbohydrate rich diet could exploit a larger field range in search of potential host larvae, and additionally achieve greater exploitation through decreased egg resorption.

Although it has antecedents in contest studies that focussed on a small sub-set of metabolites (e.g., Briffa and Elwood, 2007) and in studies of the effect of feeding on parasitoid contest behaviour without the associated metabolic analysis (Tsai et al., 2014), ours is the first study to explicitly couple the untargeted metabolomics approach (Snart et al., 2015) with the study of dyadic animal contests (Hardy and Briffa, 2013). Despite the small size of the organisms involved, this methodology is capable of reliably producing identifiable NMR spectra associated with different diet treatments (Kapranas et al., 2016) and as such has potential for application to a wide range of studies of the behaviour and ecology of small insects and other organisms. The approach can identify experimental outliers, in this instance individuals that had not ingested the provided diet were detected by absence of the highly conserved honey signal in the NMR spectra of fed wasps. However, as the whole insect is sampled, the method cannot distinguish between resonances generated from insect haemolymph from those generated from diet within an insect's digestive tract (similarly, Tsai et al., 2014 were unable to determine whether mass gained by honey-fed wasps indicated metabolism or simply ingestion). Hence, further experimentation, such as our longevity study, is required to confirm that parasitoids are capable of utilising a particular dietary resource other than merely ingesting it.

This is the first study to explicitly couple the untargeted metabolomics approach with the study of dyadic animal contests. Provision of a carbohydrate-rich diet affected the metabolomic state of G. legneri and enhanced its longevity but did not affect its performance in dyadic contests. While honey-fed wasps had higher levels of energy rich metabolites, these may not have been sufficient to enhance RHP substantially. Alternatively, an effective increase in RHP among fed females may have been countered by an increase in the host's perceived value, V, by starved competitors.

The research reported here using insects is in full compliance with the principles of Replacement, Reduction and Refinement as set by ARRIVE (Animal Research: Reporting of in vivo Experiments) guidelines and enforced by the University of Nottingham Animal Welfare and Ethical Review Body (AWERB).

CS, AK, IH, and DB: Conceived and designed the experiments; CS, AK, IH and HW: Performed the experiments; CS, AK, IH, and HW: Analysed the data; IH, DB, and HW: Provided resources; CS, AK, DB, and IH: Wrote the paper; All authors contributed to final draft approval.