A comparison of sample preparation techniques for quantifying iridoid glycosides sequestered by lepidopteran larvae

This study compared different methods of tissue preparation for extraction of iridoid glycosides sequestered by three species of lepidopteran larvae. Junonia coenia is a specialist on plant species that produce iridoid glycosides, while the arctiids Estigmene acrea and Spilosoma congrua are both polyphagous and will eat plants that produce iridoid glycosides. Larvae of all three species were reared on leaves of Plantago lanceolata, which produces two primary iridoid glycosides, aucubin and catalpol. Three methods of preparing the specimens before extraction in methanol were compared in all three species: 1) larvae were flash-frozen in liquid nitrogen, 2) larvae were macerated fresh in boiling methanol, or 3) larvae were macerated fresh in room temperature methanol. A set of J. coenia larvae was oven-dried before maceration as an additional treatment for this species only. Junonia coenia sequestered the most iridoid glycosides, while E. acrea sequestered the least, and S. congrua was intermediate. Estigmene acrea was poor at sequestering catalpol. Tissue preparation method only significantly influenced iridoid glycoside recovery from S. congrua, with maceration in room-temperature methanol being the most effective of the three methods. This study shows that treatment of insects prior to iridoid glycoside extraction can influence recovery of the compounds, and that the effects of treatment may vary among different species.