Protected by Fumigants: Beetle Perfumes in Antimicrobial Defense Jürgen Gross & Kerstin Schumacher & Henrike Schmidtberg & Andreas Vilcinskas Received: 16 August 2007 / Revised: 30 November 2007 / Accepted: 11 December 2007 / Published online: 31 January 2008 # Springer Science + Business Media, LLC 2007 Abstract Beetles share with other eukaryotes an innate immune system that mediates endogenous defense against pathogens. In addition, larvae of some taxa produce fluid exocrine secretions that contain antimicrobial compounds. In this paper, we provide evidence that larvae of the brassy willow leaf beetle Phratora vitellinae constitutively release volatile glandular secretions that combat pathogens in their microenvironment. We identified salicylaldehyde as the major component of their enveloping perfume cloud, which is emitted by furrow-shaped openings of larval glandular reservoirs and which inhibits in vitro the growth of the bacterial entomopathogen Bacillus thuringiensis. The sug- gested role of salicylaldehyde as a fumigant in exogenous antimicrobial defense was confirmed in vivo by its removal from glandular reservoirs. This resulted in an enhanced susceptibility of the larvae to infection with the fungal entomopathogens Beauveria bassiana and Metarhizium anisopliae. Consequently, we established the hypothesis that antimicrobial defense in beetles can be expanded beyond innate immunity to include external disinfection of their microenvironment, and we report for the first time the contribution of fumigants to antimicrobial defense in animals. Keywords Phratora vitellinae . Beauveria bassiana . Metarhizium anisopliae . Bacillus thuringiensis . Fumigants . Antimicrobial activity . Glandular secretion . Salicylaldehyde Introduction Larvae of the leaf beetle taxon Chrysomelina possess nine pairs of dorsal exocrine glands, which are inserted in the body surface and contain reservoirs of glandular secretions. In case of predator attack, the larvae evert their glandular reservoirs to present the fluid secretions toward the exterior. The secreted toxins vary both in structure and biosynthetic origin. The major components secreted by leaf beetle larvae belonging to the taxa Phaedon, Gastrophysa, Linaeidea, and most Phratora species are iridoid monoterpenes, which are produced de novo via the acetate-mevalonate pathway. In contrast, larvae of Chrysomela spp. and Phratora vitellinae emit secretions in which salicylaldehyde is the major component (Wain 1943; Oldham et al. 1996; Pasteels et al. 1982, 1988). When feeding upon willows (Salicaceae), larvae of the latter species sequester phenolic glycosides (e.g., salicin) from their host plants as precursors to produce salicylaldehyde (Wallace and Blum 1969; Pasteels et al. 1988; Gross and Hilker 1995). Different biological functions have been reported for larval glandular secretions from beetles: Some prevent intraspecific competition (Gross and Hilker 1995), whereas others show insecticidal activities (Dettner et al. 1992) or act as allomones in defense against predators. Although salicylaldehyde has repellent activity against some generalist predators (Blum et al. 1972; Hilker and Schulz 1994; Pasteels et al. 1986; Gross et al. 2004), it also has detrimental effects for the larvae by attracting specialized predators and parasitoids (Köpf et al. 1997; J Chem Ecol (2008) 34:179–188 DOI 10.1007/s10886-007-9416-9 J. Gross (*) : H. Schmidtberg : A. Vilcinskas Institute of Phytopathology and Applied Zoology, Justus-Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany e-mail: juergen.gross@agrar.uni-giessen.de K. Schumacher Institute for Integrated Plant Protection, Federal Biological Research Centre for Agriculture and Forestry, Stahnsdorfer Damm 81, 14532 Kleinmachnow, Germany