Chemical defenses of freshwater macrophytes against crayfish herbivory

We measured feeding preferences of the crayfish Procambarus clarkii for fresh tissue from four species of freshwater macrophytes (Habenaria repens, Saururus cernuus, Ceratophyllum demersum and Typha angustifolia). We then determined the role of plant chemical defenses in generating these preferences by incorporating crude aqueous and organic extracts from each species into palatable foods and comparing feeding on these foods to feeding on control foods lacking these extracts. Tissue toughness, dry mass and ash-free mass per volume, and percentages of carbon, nitrogen, and phenolics were also measured for each of the four macrophytes. Although it had a low nutritional value, Ceratophyllum was the preferred food when it was offered as fresh tissue; it did not produce a chemically deterrent extract. The lipophilic crude extract from Typha significantly deterred crayfish feeding, but this highly nutritious plant was preferred when offered in an agar-based diet lacking structural defenses. Habenaria and Saururus were low preference foods that did not appear to be structurally defended; each species contained both lipophilic and water-soluble extracts that significantly deterred feeding. Fractionation of the lipophilic crude extract from Saururus indicated the presence of at least three deterrent compounds. From the orchid Habenaria, we isolated and identified a novel bis-p-hydroxybenzyl-2-alkyl-2-hydroxysuccinoate metabolite, habenariol, that appeared to explain most of the feeding deterrent activity present in the lipophilic extract of this species. The concentration of the metabolite in frozen collections of this plant doubled if we allowed the material to thaw before placing it in extraction solvents.