Biosynthetic studies of ladybird defensive alkaloids

When molested or disturbed, ladybirds fall into thanatosis and exude a yellowish fluid from their tibio-femoral articulations, the so-called “reflex bleeding”. It is now well established that may coccinellids owe their protection, at least in part, to the presence of repellent and, in some cases, toxic alkaloids in this fluid. Over 50 alkaloids have been isolated and characterized from ladybird up to now, including perhydroazaphenalene (e.g. coccinelline), azamacrolide (e.g. epilachnene) or homotropane (e.g. adaline).
Although these alkaloids have been the subject of many biological and synthetic studies, only a few investigations have focused on the elucidation of the pathway through which they are biosynthesized. In Coccinella septempunctata, coccinelline was shown to be labelled after the beetle had been fed with [1-14C] and [2-14C] acetate. Results of degradation experiments on the labelled coccinelline were in agreement with a polyacetate origin for that compound. More recently, radioactive adaline was obtained after feeding the same labelled precursors to Adalia bipunctata. Chemical degradation of this sample led also to the conclusion that adaline must derive from the condensation of seven acetate units.
However, it was not established if coccinelline and adaline are biosynthesized through a fatty acid or a polyketide pathway. Moreover, no informations were available concerning the origin of the nitrogen atom and the anatomical localization of alkaloid biosynthesis in the beetles.
We will present the results of in vitro incubation assays using ladybird tissues, which have enabled us to demonstrate that these two alkaloids are most likely biosynthesized through a fatty acid rather than a polyketide pathway, that glutamine is the preferred source of the nitrogen atom, and that the alkaloid biosynthesis takes place in the insect fat body.