Identification and characterization of defense related enzymes in Chrysomelina larvae (Coleoptera : Chrysomelidae) : Contributions to understand the evolutionary and molecular dynamics of chemical defense in leaf beetles
Glandular chemical defense of Chrysomelina larvae is either, not (iridoids), in part, or completely dependent (salicylaldehyde) on host derived glucosides. After uptake of glucoside precursors into the glandular reservoir, deglucosylation and further modification to defensive compounds falls into line. In case of iridoid and salicylaldehyde formation deglucosylation is followed by a common oxidation step, which led to assume a single origin of the oxidases in those species. Salicylaldehyde producers Chrysomela spp. and P. vitellinae are adapted to sequester the phytogenic precursor salicin and therefore are restricted to feed on salicaceous hosts. Whereas this host plant specialization may have favored salicin-based defense and stabilized salicyl alcohol oxidase (SAO) activity in Chrysomelina, shift to Betulaceae within the genus Chrysomela negatively affected the fate of salicylaldehyde formation. SAO transcripts and genes have been identified in C. lapponica and P. vitellinae. Additionally, SAO related sequences were found in iridoid producing species. But the corresponding protein in the defensive secretion and its specific SAO activity verified after heterologous expression followed by in vitro enzyme assays are only present in salicylaldehyde producers. A common origin of all SAOs and related sequences in a specific GMC oxidoreductase subfamily was shown by phylogenetic analyses. The presence of SAO-like genes but lack of their encoded proteins in the defensive secretion of iridoid producers indicate both an independent evolution of SAO from glandular oxidases in iridoid producers and the recruitment of SAO related proteins for functions other than in the defensive system of iridoid protected species. Comparing isolated Salicaceae- and Betulaceae- adapted C. lapponica populations elucidated a loss of SAO activity caused by mutations, alternative splicing, massive transcript down-regulation and N-terminal protein truncation in the birch-feeder.