Identification of serine proteases in Gracillariid leaf miners and role of foliar protease inhibitors in Millettia pinnata (L.) Panigrahi, a biofuel crop

A well-known coevolutionary mechanism in plant-insect interactions involves insect digestive proteases and plant protease inhibitors. Digestion of dietary protein by gut serine proteases is a complex process and its disruption can affect growth and development in lepidopteran larvae. This study investigates recurrent infestations of an economically important tree legume by two microlepidoptera (Genbank accessions # MH020177 and MN149606). A protocol was devised to maintain hydrated twigs under lab conditions and monitor insect growth and development. In vitro spectrophotometric assays were used to detect trypsin and chymotrypsin activities in fourth instars, as well as determine their inhibition by diagnostic protease inhibitors. Gelatinolytic zymograms were used to visualize protease activities for the two leaf miners, feeding on the same plant. Preliminary studies with whole leaf extracts of M. pinnata indicated phenological changes in putative trypsin inhibitory activities in foliage used as diet by the insects. During peak insect infestations, PTI levels were significantly low (p≤0.05) in mature leaves, suggesting an altered defense responses in these trees upon herbivory. Effort is underway to identify serine protease genes and trypsin inhibitor genes in Millettia species to study adaptations in digestive physiology of these insects while feeding on host plant tissues with varying levels of PTI. Improved understanding of unique interactions between tropical leaf miners and their host trees can help in developing effective management strategies.