Modulation of the paracellular pathway in the lepidopteran larval midgut

Control of insect pests is still mainly performed using broad spectrum chemical pesticides even though the limits of this strategy are presently well known: the reduction in pest population is associated with an unfavourable alteration of food quality and safety, with a strong impact on non-target species and the rise of a widespread resistance in target insects. Therefore, significant research efforts are devoted to the development of innovative technologies based on the use of biological control agents, which include small organic molecules and peptide or protein toxins, deriving from plants and insect natural antagonists. For an effective oral delivery of these bioinsecticides, it will be crucial to develop strategies to facilitate their passage through the midgut barriers. They can reach the haemocoel either through the cellular pathway, crossing the two polarized plasma membranes of the epithelial cells, or through the paracellular pathway, along the aqueous channels formed by the junctional complexes, according to the physico-chemical and biological properties of each molecule. The insect midgut paracellular pathway is particularly interesting for the delivery of bioinsecticides of low molecular weight targeting hemocelic receptors. We have previously demonstrated that the permeability of the septate junction (SJ) of lepidopteran larval midgut can be modulated by a precise regulation of cytosolic Ca2+ concentration. We have now studied one of the signaling pathway that may lead to the intracellular release of Ca2+ responsible for the increase of SJ permeability. We used C10, a medium-chain fatty acid, known to modulate mammalian tight junctions by activation of a Ca2+ ions-mediated intracellular signaling pathway. Addition of C10 to the luminal side of lepidopteran larval midguts isolated in conventional Ussing chambers, causes a decrease of the transepithelial shunt resistance, i.e. an increase of the junction ion conductance, and an increase of the paracellular fluxes of proctolin and fluorescein, two organic molecules that cross the epithelium exclusively by the paracellular route. Our data show that C10 triggers an IP3- dependent signaling cascade by activation of phopholipase C. As a result, Ca2+ is released from the intracellular stores and a calmodulin-dependent kinase is activated. This event leads to the phosphorylation of the myosin light chain by myosin light chain kinase. Myosin light chain phosphorylation presumably causes a modification of the cytoskeleton organization connected to the SJ and thereby an increase of the paracellular permeability to ions and small organic molecules.