Increased Host Investment in Extrafloral Nectar (EFN) Improves the Efficiency of a Mutualistic Defensive Service Marcia Gonza´ lez-Teuber 1 *, Juan Carlos Silva Bueno 2 , Martin Heil 2 , Wilhelm Boland 1 1 Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Beutenberg Campus, Jena, Germany, 2 Departamento de Ingenierı ´a Gene´tica, CINVESTAV-Irapuato, Km. 9.6 Libramiento Norte, Guanajuato, Me´xico Abstract Extrafloral nectar (EFN) plays an important role as plant indirect defence through the attraction of defending ants. Like all rewards produced in the context of a mutualism, however, EFN is in danger of being exploited by non-ant consumers that do not defend the plant against herbivores. Here we asked whether plants, by investing more in EFN, can improve their indirect defence, or rather increase the risk of losing this investment to EFN thieves. We used the obligate plant-ant Acacia- Pseudomyrmex system and examined experimentally in the field during the dry and the rainy seasons how variations in EFN secretion are related to (i) ant activity, to (ii) the ant-mediated defence against herbivores and (iii) the exploitation of EFN by non-ant consumers. Extrafloral investment enhanced ant recruitment and was positively related to the ant mediated defence against herbivores. The ant-mediated protection from exploiters also increased in proportion to the nectar sugar concentration. Although the daily peak of EFN production coincided with the highest activity of EFN thieves, Pseudomyrmex ferrugineus ants protected this resource effectively from exploiters. Nevertheless, the defensive effects by ants differed among seasons. During the dry season, plants grew slower and secreted more EFN than in the rainy season, and thus, experienced a higher level of ant-mediated indirect defence. Our results show that an increased plant investment in an indirect defence trait can improve the resulting defensive service against both herbivores and exploiters. EFN secretion by obligate ant-plants represents a defensive trait for which the level of investment correlates positively with the beneficial effects obtained. Citation: Gonza´lez-Teuber M, Silva Bueno JC, Heil M, Boland W (2012) Increased Host Investment in Extrafloral Nectar (EFN) Improves the Efficiency of a Mutualistic Defensive Service. PLoS ONE 7(10): e46598. doi:10.1371/journal.pone.0046598 Editor: Minna-Maarit Kyto¨ viita, Jyva¨skyla¨ University, Finland Received June 26, 2012; Accepted September 3, 2012; Published October 4, 2012 Copyright: ß 2012 Gonza´ lez-Teuber et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Financial support by the DFG (grant He 3169/4-2), CONACyT (Consejo Nacional de Ciencia y Tecnologı ´a) and Max-Planck-Society is gratefully acknowledged. MGT is supported by a postdoctoral fellowship from the Alexander von Humboldt Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The co-author Martin Heil is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: mgonzalez-teuber@ice.mpg.de Introduction Plants have evolved multiple strategies to defend themselves against herbivores. They can either directly reduce herbivore attack through the production of chemical and physical defences [1,2] or indirectly by producing rewards that attract the natural enemies of herbivores [3]. Most commonly reported in the latter context are herbivore-induced volatile organic compounds (VOCs) and extrafloral nectar (EFN) [4]. Even though the role of direct and indirect defences has been widely demonstrated, it remains an open question for most defensive traits how variations in the investment into a given trait translate into variations in its defensive effect [5]. A higher concentration of glucosinolates and a greater density of trichomes in genetic lines of Arabidopsis thaliana reduced the herbivory by two specialist flea beettles [6]. Similar quantitative effects have also been reported for the cyanogenic glyosides of lima bean (Phaseolus lunatus) [7]. Nevertheless, other studies have not found an enhanced resistance to specialist and generalist herbivores due to a higher concentration of secondary compounds [8–11]. Thus, it remains open for most defensive secondary compounds whether an increased herbivore pressure can exert a positive selective pressure on their quantitative levels. This problem might be even more pertinent for indirect defences, which are achieved by interactions of plants with members of the third trophic level. These plant-carnivore interactions are mainly maintained by the provisioning of rewards to the defending carnivores [12]. However, such mutualistic interactions are also prone to exploitation [13]. Does, under these circumstances, a higher investment into the reward cause a better protection by ants? Or, in the context of plant-ant mutualisms: can ants defend their food rewards against exploiters and at the same time efficiently fulfil their mutualistic role in the indirect defence? In the present study we used an obligate plant-ant system to investigate whether an increased rate of EFN secretion improves the defensive effects of EFN or rather its exploitation by non- defending ‘EFN thieves’. EFN is rich in carbohydrates and amino acids that usually function in the attraction of ants and contains PR-proteins as protection from microbial infections [14,15]. Ants that are attracted to EFN can effectively reduce the rates of PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e46598