Permeability of lepidopteran larval midgut to the potential insecticidal peptides proctolin and TMOF

Abstract

Great efforts have been recently devoted to the identification of innovative gene products to be developed as biopesticides. A large number of studies constantly detect new naturally derived peptides and proteins with haemocoelic receptors that could be used as potential insecticides. However, it is still to be defined their efficacy by oral delivery, because very limited information is available on how the epithelial barrier of the insect gut handles these kind of molecules. We have considered two small peptides, both active as internal regulative factors in insects, that have lately been considered potential bioinsecticides, the pentapeptide proctolin and the decapeptide Trypsin Modulating Oostatic Factor (TMOF). We have performed a functional study of proctolin and TMOF permeability across the two barriers of Bombyx mori larval midgut, the peritrophic membrane (PM) and the epithelium, separately isolated and perfused in vitro in Ussing chambers. We observed that both peptides crossed easily the peritrophic membrane, but their permeability coefficient was highly affected by the molecular weight and steric conformation. Once crossed the PM, peptides present in vivo in the ectoperitrophic space have to pass through the second and more selective barrier represented by the intestinal epithelial cell monolayer. While proteins can cross B. mori epithelium from the lumen by transcytosis, no data are available for small peptides: here we tested the hypothesis that peptides with few amino acid residues could reach the insect haemocoel through the paracellular pathway, lined by the smooth septate junctions. First, we characterised the selectivity properties of midgut paracellular pathway by measuring the lumen to haemolymph transepithelial fluxes of a number of organic molecules with different size and electric charge. The results obtained indicated that septate junction is highly selective to both the dimension and the charge of the permeant compounds. Then, we determined the route followed by proctolin and TMOF. The confocal images of whole-mount midguts incubated for two hours with rhodamine(rh)-proctolin or FITC-TMOF revealed that rh-proctolin was localized exclusively in the intercellular spaces, while labelled TMOF was present only inside the enterocytes\u2019 cytoplasm. Therefore, proctolin does not enter midgut cells but crosses the epithelium through the paracellular pathway, whereas TMOF follows the transcellular route by a mechanisms to be clarified