Glands of leaf beetle larvae – protective structures against attacking predators and pathogens1 Plate 34 Jürgen Gross2 and Henrike Schmidtberg3 Abstract. Th e exocrine glandular secretions of leaf beetle larvae of the taxon Chryso- melinae are well-known defensive devices used against generalistic predators. Re- cently it was shown that larvae of Phratora vitellinae produce fl uid and volatile exocrine secretions containing antimicrobial compounds, which were used to dis- infect their microenvironment. Th ey are emitted by specialized openings above the larval glandular reservoirs and inhibit the growth of bacteria as well as the germina- tion of fungi. In the present study we investigated in four further species from dif- ferent genera of the taxon Chrysomelinae the morphological structures underlying this novel principle of fumigation in antimicrobial defense. We found in all genera furrow-shaped openings above the glandular reservoirs, allowing the larvae the per- manent emission of volatile components, which build clouds of antimicrobial fumi- gants. Further, we could identify the antimicrobial active iridoid (epi)chrysomelidial as main component of the emitted fumigant of Phaedon cochleariae. Keywords. Phaedon cochleariae, Chrysomela vigintipunctata, Plagiodera versicolora, Phratora vitellinae, Gastrophysa viridula, antimicrobial defense, fumigants, anti- microbial activity, glandular secretion, ( epi) chrysomelidial. 1. Introduction Larvae of the tribus Chrysomelini, which includes both the subtribes Chryso- melina and Phyllodectina (Daccordi 1994, Reid 1995, Seeno and Wilcox 1982), possess nine pairs of dorsal exocrine glands, which are inserted into their body surface and contain reservoirs fi lled with glandular secretions. In case of molesta- 1 Paper presented to the 7th International Symposium on the Chrysomelidae, held at Durban (South Africa), July 9, 2008. 2 Julius Kuehn Institute – Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Fruit Crops and Viticulture, Dossenheim, Germany; Juergen.Gross@JKI. bund.de 3 Institute of General and Special Zoology, Justus Liebig University of Giessen, Germany Research on Chrysomelidae, Volume 2, pp. 177–189, © Koninklijke Brill, Leiden, 2009 178 Jürgen Gross and Henrike Schmidtberg tion the larvae can evert their reservoirs in order to contaminate attacking assault- ers with fl uid secretion, which vary both in chemical structure and biosynthetic origin. Th us, some generalistic predators like ants and ladybird beetles can be re- pelled. Th e major components secreted by leaf beetle larvae belonging to the genera Phaedon, Gastrophysa, Linaeidea, and most species of the genus Phratora are iri- doid monoterpenes. Th ey are produced either de novo via the acetate-mevalonate pathway or acquired by sequestration of secondary metabolites from host plants (Oldham et al. 1996, Feld et al. 2001, Burse et al. 2007). E.g., the leaf beetle species Gastrophysa cyanea, Phaedon cochleariae and Plagiodera versicolora synthesize a mixture of two diastereomers, 3S,8S-chrysomelidial and 3S,8R-chrysomelidial, the latter called epichysomelidial (Blum et al. 1978, Meinwald et al. 1977, Meinwald & Jones 1978). In contrast, larvae of Chrysomela spp. and Phratora vitellinae emit salicylaldehyde as major component (Wain 1943, Lorenz et al. 1993, Pasteels et al. 1982, Pasteels et al. 1988). Th ese species feed exclusively upon willows and poplars (Salicaceae), and sequester phenolic glycosides (e.g. salicin) from their host plants as precursors to produce this toxin (Wallace & Blum 1969, Pasteels et al. 1988, Gross & Hilker 1995). While salicylaldehyde has repellent eff ects against some generalistic predators (Blum et al. 1972, Hilker & Schulz 1994, Pasteels et al. 1986, Palokangas & Neuvonen 1992, Rank et al. 1996, Gross et al. 2004), it has also detrimental eff ects for the larvae by attracting specialized predators and parasitoids (Köpf et al. 1997, Gross et al. 2004, Zvereva & Rank 2004). Besides mitigation against predator attacks, further biological functions have been reported for larval glandular secretions from leaf beetles. Some prevent in- tra- or interspecifi c competition (Raupp et al. 1986, Gross & Hilker 1995, Gross et al. 2007), while others show antimicrobial activities (Gross et al. 1998, Gross et al. 2002). Recently it was shown that larvae of the brassy willow leaf beetle Phratora vitellinae constitutively release volatile salicylaldehyde through specialized cuticu- lar structures to combat fungal and bacterial pathogens in their microenvironment (Gross et al. 2008). Hence, in the present study we addressed the question whether this newly iden- tifi ed defense mechanism is more common in further species belonging to the taxon Chrysomelinae. Th is was conducted by looking for morphologically similar cuticular structures as well as other antimicrobial toxins in the headspace of lar- vae. Th us, we examined the glandular surface of larvae from four other genera by scanning electron microscopy. Further, we collected the headspace of undisturbed larvae of the mustard leaf beetle Phaedon cochleariae and analyzed it by GC-MS. 2. Material and methods 2.1. Leaf beetles Larvae of Phaedon cochleariae (Fabricius, 1792) were obtained from our laboratory rearing culture. Th ey were fed with leaves of Chinese cabbage (Brassica pekinensis). .