Host location by hyperparasitoids: an ecogenomic approach Feng Zhu Thesis committee Promotor Prof. Dr Marcel Dicke Professor of Entomology Wageningen University Co-promoter Dr Erik H. Poelman Assistant professor, Laboratory of Entomology Wageningen University Other members Prof. Dr Niels P. R. Anten, Wageningen University Prof. Dr Monique M. van Oers, Wageningen University Dr T. Martijn Bezemer, Netherlands Institute of Ecology, Wageningen Dr Klaas Vrieling, Leiden University This research was conducted under the auspices of the graduate school Experimental Plant Sciences. Host location by hyperparasitoids: an ecogenomic approach Feng Zhu Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr. A.P.J. Mol in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 2 October 2015 at 1:30 p.m. in the Aula Feng Zhu Host location by hyperparasitoids: an ecogenomic approach, 192 pages. PhD thesis, Wageningen University, Wageningen, NL (2015) With references, with summary in English ISBN 978-94-6257-444-1 To my beloved parents 谨以此书，献给我最亲爱的父母 Abstract It is fascinating that our ecological systems are structured by both direct and indirect spe- cies interactions. In terrestrial ecosystems, plants interact with many species of insects that include both harmful herbivores and beneficial natural enemies of herbivores. During the last 30 years, substantial progress has been made in different plant-insect systems regarding plant trait-mediated species interactions in a tritrophic context. However, plant- based food webs generally consist of more than three trophic levels. For example, hy- perparasitoids are parasitic wasps at the fourth trophic level within the plant-associated insect community. They parasitize larvae or pupae of primary parasitoids that are broadly used in biological pest control programmes. Surprisingly, the cues that hyperparasitoids use for host location have remained largely unknown. The studies presented in this thesis aimed to investigate the cues that are used by hyper- parasitoids in host location using an ecogenomic approach that combines metabolomic, transcriptomic and proteomic tools with behavioural studies and field experiments. In addition, we addressed the role of herbivore-associated organisms in plant-mediated indirect species interactions. A naturally existing study system of the Brassica oleracea plant-based food web, including four trophic levels was used. In this system, the two herbivorous insect species, Pieris brassicae and P. rapae, are specialists on Brassica plants. The plants emit herbivore-induced plant volatiles (HIPVs) in response to Pieris caterpillar feeding damage which results in attraction of natural enemies of the herbi- vores, i.e. Cotesia wasps. These parasitic wasps, in turn, are attacked by hyperpara- sitoids, such as Lysiba nana. The results presented in this thesis show that hyperpara- sitoids also use HIPVs for host searching. Interestingly, they are especially attracted by plant odours induced by parasitized caterpillars. Moreover, hyperparasitoids can also use caterpillar body odours to find their hosts at close distance. These findings indicate that infochemicals are the major cues that mediate host searching behaviour of hyper- parastioids. Similar to other herbivore-associated organisms, parasitoid larvae feeding inside a herbivore host can induce both behavioral and physiological changes in the host. To further investigate how parasitoid larvae indirectly affect plant responses to her- bivory and plant volatile-mediated multitrophic interactions, the role of caterpillar labial salivary glands in plant-hyperparasitoid interactions were investigated. The secretions of labial saliva were eliminated by using an ablation technique. Remarkably, the results show that when the labial salivary glands of the caterpillars were completely removed, plants induced by either unparasitized or Cotesia glomerata-parasitized caterpillars were equally attractive to the hyperparasitoid. Moreover, plants became less attractive to the hyperparasitoid when damaged by ablated caterpillars compared to plants damaged by mock-treated caterpillars and the hyperparasitoids were not able to distinguish between volatiles emitted by herbivore-damaged plants and undamaged control plants when cat- erpillar salivary glands had been removed. These results suggest that parasitism alters the composition of labial saliva of parasitized caterpillar, which thereby alters the plant phenotype and subsequently plant-hyperparasitoid interactions. The outcomes of this thesis contribute to our understanding of the role of infochemicals in foraging decisions of hyperparasitoids. 7 Table of contents Abstract 7 Chapter 1 General introduction 11 Chapter 2 Insect herbivore-associated organisms affect plant 21 responses to herbivory Chapter 3 Hyperparasitoids use herbivore-induced plant volatiles in 35 host location under field conditions Chapter 4 Parasitism overrides herbivore identity allowing 47 hyperparasitoids to locate their parasitoid host using herbivore-induced plant volatiles Chapter 5 Labial saliva of parasitized caterpillars affects plant- 85 mediated indirect species interactions Chapter 6 Body odours of parasitized caterpillars give away 119 the presence of parasitoid larvae to their primary hyperparasitoid enemies Chapter 7 Intrinsic competition between primary hyperparasitoids 137 of the solitary endoparasitoid Cotesia rubecula Chapter 8 General discussion 153 References 165 Summary 179 Acknowledgements 183 Curriculum vitae 187 Publication list 188 Education Statement 189 Chapter 1 General introduction Feng Zhu Chapter 1 Chapter 1 General introduction Ecological studies have extensively demonstrated the complexity of species interactions in communities that range from direct trophic relationships to intricate indirect interaction networks (Polis & Strong 1996). One of the most famous examples of food chains and their indirect interactions was reported in The Origin of Species by Darwin (1859), involving bumble bees that pollinate red clover; though some bees may be eaten by field mice, in turn, the mice may be attacked by domestic cats. Thereafter, Darwin made a remarkable speculation that if the cats were removed 1 from this food chain, the red clover plants would eventually remain unpollinated, because the mice would eliminate the bees. The major components of a food chain in terrestrial ecosystem are primary producers (such as plants), consumers (herbivores), intermediate-level predators and top predators. The relationships and interactions between organisms at the same or different trophic levels significantly affect the structure of food webs, as well as population dynamics. Thus far, people have extended their observations and predictions on species interactions in food webs in different types of ecosystems, attempting to explain possible similarities and differences among them (Chase 2000). In terrestrial ecosystems, plants, besides struggling for survival under various abiotic stresses, are constantly challenged by herbivorous organisms because they are the primary sources of energy for this second trophic level. One of the important groups of herbivores on plants are herbivorous insects. In order to defend themselves against attack by herbivorous insects, plants have evolved a suite of constitutive and induced defence mechanisms (Mithofer & Boland 2012). On the one hand, constitutive defences are generally “static” plant traits and act as physical barrier (wax layer or lignification in plant tissue), or stored plant toxins that act as feeding or oviposition deterrents or can intoxicate feeding herbivores (Gatehouse 2002; Wittstock & Gershenzon 2002). On the other hand, induced defence mechanisms become “active” upon tissue damage by attackers; for example the production of defensive compounds can be initiated in response to herbivory (Gatehouse 2002). Price et al. (1980) pointed out that plant-herbivore interactions cannot be studied realistically without consideration of natural enemies of herbivores at the third trophic level, because it is essential to understand the role of natural enemies in plant- herbivore interactions, as well as the role of plants in predator-prey relationships. Soon after, the importance of plant infochemicals (Dicke & Sabelis 1988b) in plant- herbivore-carnivore interactions had been acknowledged and further developed into plant indirect defence theory (Vet & Dicke 1992; Heil 2008). In response to herbivory, plants actively produce so-called herbivore-induced plant volatiles (HIPVs) that have been demonstrated to be used by natural enemies of herbivores for host location (Dicke & Sabelis 1988a; Godfray 1994; Agelopoulos et al. 1995; Turlings et al. 2012). Thus, recruiting natural enemies of herbivores by HIPVs may benefit plants by top- 13 Chapter 1 down control of their herbivorous attackers. Besides natural enemies of herbivores, however, increasing evidence indicates that a wide range of other members of the plant-associated community (including antagonists) use HIPVs in their foraging decisions (De Moraes et al. 2001; Runyon et al. 2006; Dicke & Baldwin 2010; Karban et al. 2014). Thereby, plant volatiles become “public” cues and make a plant apparent to all other community members (Dicke & Baldwin 2010; Heil & Karban 2010). 1 It has been long recognized that natural food webs generally contain more than three trophic levels