Mass Spectrometry Imaging of Specialized Metabolites for Predicting Lichen Fitness and Snail Foraging

Abstract

Lichens are slow-growing organisms supposed to synthetize specialized metabolites to protect themselves against diverse grazers. As predicted by the optimal defense theory (ODT), lichens are expected to invest specialized metabolites in higher levels in reproductive tissues compared to thallus. We investigated whether Laser Desorption Ionization coupled to Mass Spectrometry Imaging (LDI-MSI) could be a relevant tool for chemical ecology issues such as ODT. In the present study, this method was applied to cross-sections of thalli and reproductive tissues of the lichen Pseudocyphellaria crocata. Spatial mapping revealed phenolic families of metabolites. A quantification of these metabolites was carried out in addition to spatial imaging. By this method, accumulation of specialized metabolites was observed in both reproductive parts (apothecia and soralia) of P. crocata, but their nature depended on the lichen organs: apothecia concentrated norstictic acid, tenuiorin, and pulvinic acid derivatives, whereas soralia mainly contained tenuiorin and pulvinic acid. Stictic acid, tenuiorin and calycin, tested in no-choices feeding experiments, were deterrent for N. hookeri while entire thalli were consumed by the snail. To improve better knowledge in relationships between grazed and grazing organisms, LDI-MSI appears to be a complementary tool in ecological studies.

Keywords: Chemical Ecology; Lichens; Lobariaceae; Mass Spectrometry Imaging; Notodiscus hookeri; Optimal Defense Theory; Pseudocyphellaria crocata; Specialized Metabolites.

Figure 1

Reproductive parts of the lichen P. crocata. The thalli of the lichen species collected on the fern Blechnum penna-marina (a). Insets correspond to the sexual reproductive parts apothecia (b) and asexual reproductive part soralia (c) which are marginal (c1) and laminal (c2). Blue lines allow visualization of the cross sections for MSI experiments. Scales bars correspond to 1 cm, 500 µm, and 100 µm in panels (a–c), respectively.

Figure 2

P. crocata thallus cross section that shows four distinctive layers and the basal part fixed to the substrate, called tomentum. Scale bar corresponds to 500 µm.

Figure 3

Structures of the phenolic specialized metabolites identified in P. crocata.

Figure 4

Distribution of the specialized metabolites in the lichen P. crocata. Optical image (a) of the cross-sections of an apothecium (upper panel) and a thallus with soralium (lower panel). Mass spectrometry imaging shows (b) norstictic acid at m/z 371, (c) fragment of depsides at m/z 149 and (d) calycin at m/z 305. Image (e) compiled all the previous results. Intensity scales were adjusted to maximize the visualization of each family compound.

Figure 5

Gel consumption, estimated by a feeding score (Mean±s. e.), by the snails (N = 150 × four groups), according to the metabolite tested on starch gel. Starch gel without metabolites was considered as the positive control. Significant differences between positive control and metabolite containing gels (stictic acid, tenuiorin, or calycin) are highlighted by the lowercase superscript letters a, b and c.