Host plant shifts affect a major defense enzyme inChrysomela lapponica

Abstract

Chrysomelid leaf beetles use chemical defenses to overcome predatory attack and microbial infestation. Larvae of Chrysomela lapponica that feed on willow sequester plant-derived salicin and other leaf alcohol glucosides, which are modified in their defensive glands to bioactive compounds. Salicin is converted into salicylaldehyde by a consecutive action of a β-glucosidase and salicyl alcohol oxidase (SAO). The other leaf alcohol glucosides are not oxidized, but are deglucosylated and esterified with isobutyricand 2-methylbutyric acid. Like some other closely related Chrysomela species, certain populations of C. lapponica shift host plants from willow to salicin-free birch. The only striking difference between willow feeders and birch feeders in terms of chemical defense is the lack of salicylaldehyde formation. To clarify the impact of host plant shifts on SAO activity, we identified and compared this enzyme by cloning, expression, and functional testing in a willow-feeding and birch-feeding population of C. lapponica. Although the birch feeders still demonstrated defensive glandspecific expression, their SAO mRNA levels were 1,000-fold lower, and the SAO enzyme was nonfunctional. Obviously, the loss of catalytic function of the SAO of birch-adapted larvae is fixed at the transcriptional, translational, and enzyme levels, thus avoiding costly expression of a highly abundant protein that is not required in the birch feeders.host plant adaptation | glucose-methanol-choline oxidoreductase M ost phytophagous insects are specialized to a restricted set of host plant species (1-4). Host affiliation/specialization has been shown to be influenced by geographical, genetic, biophysical, and ecological enforcements (3, 5). But the most important barriers are toxic metabolites of the host plant (6-8), which all phytophagous insects must overcome by developing appropriate detoxification mechanisms. Adapting to plant-specific chemicals provides insects with a niche that allows them to survive, but narrows the range within which host plant shifts can occur, including only plants with similar metabolite patterns.Chrysomelina leaf beetles are an excellent taxon for investigating host plant adaptation and relevant factors associated with host plant shifts. Most leaf beetle species are highly specialized on a single plant genus, where they spend their whole life cycle. Their well-defended larvae exhibit different degrees of dependence on the host plant's secondary metabolites (9-13).

Independently recruited oxidases from the glucose-methanol-choline oxidoreductase family enabled chemical defences in leaf beetle larvae (subtribe Chrysomelina) to evolve

“…However, among the predicted enzymes in the Chrysomelina secretions, only the functionally characterized salicyl alcohol oxidases (SAOs) from salicin-sequestering species have been analysed with respect to their ancestry [16,20,21]. These SAOs belong to the glucose-methanol-choline (GMC) oxidoreductase multi-gene family [22,23] and convert salicyl alcohol into salicylaldehyde [20].…”

Precise RNAi-mediated silencing of metabolically active proteins in the defence secretions of juvenile leaf beetles

“…Targeting the defensive glands of juvenile poplar leaf beetles by RNA interference Recently, a 1872-bp CpopSAO cDNA (Genbank/ HQ245154.1) encoding a 69 kDa protein for conversion of 2 into 3 was identified from the larval defensive glands of C. populi [13,14] (figure 2a). It belongs to the glucose -methanol -choline (GMC) family of oxidoreductases [38].…”

“…This shows a distinct function of this enzyme in vivo. Before, Kirsch et al [13] showed activity only in in vitro assays. Evidently, RNAi is a valuable technique for identifying in vivo relevance for unknown proteins in defensive glands.…”

“…The hydrophobic aldehyde forms an organic layer, accounting for 15 per cent of the total discharge volume, while the aqueous phase constitutes 85 per cent [12]. The latter contains the precursor salicyl alcohol (2) and a flavine-dependent salicyl alcohol oxidase (SAO); the SAO uses molecular oxygen as an electron acceptor for alcohol oxidation, yielding the aldehyde and hydrogen peroxide [12][13][14] (figure 1). Salicyl aldehyde is considered as a potent repellent against generalist predators [11,15] and as an antimicrobial agent [16].…”

“…Despite the many current genome-and transcriptomesequencing projects, up to now it has only been shown for SAO sequences to be entangled in allomone production in the defensive secretions of the leaf beetle species Chrysomela tremulae, Chrysomela populi, Chrysomela lapponica and Phratora vitellinae [13,14,26]. To demonstrate the in vivo relevance of a target sequence, gene silencing by RNA interference (RNAi) is a suitable method.…”