title: A viral chitinase enhances oral activity of TMOF.
abstract: In this study we investigate the combined effect on Heliothis virescens (Lepidoptera, Noctuidae) larvae of Aedes aegypti-Trypsin Modulating Oostatic Factor (Aea-TMOF), a peptide that inhibits trypsin synthesis by the gut, impairing insect digestive function, and Autographa californica nucleopolyhedrovirus Chitinase A (AcMNPV ChiA), an enzyme that is able to alter the permeability of the peritrophic membrane (PM). Aea-TMOF and AcMNPV ChiA were provided to the larvae by administering transgenic tobacco plants, co-expressing both molecules. Experimental larvae feeding on these plants, compared to those alimented on plants expressing only one of the two molecules considered, showed significantly stronger negative effects on growth rate, developmental time and mortality. The impact of AcMNPV ChiA on the PM of H. virescens larvae, measured as increased permeability to molecules, was evident after five days of feeding on transgenic plants expressing ChiA. This result was confirmed by in vitro treatment of PM with recombinant ChiA, extracted from the transgenic plants used for the feeding experiments. Collectively, these data indicate the occurrence of a positive interaction between the two transgenes concurrently expressed in the same plant. The hydrolytic activity of ChiA on the PM of tobacco budworm larvae enhances the permeation of TMOF molecules to the ectoperitrophic space, and its subsequent absorption. The permeation through the paracellular route of Aea-TMOF resulted in a spotted accumulation on the basolateral domain of enterocytes, which suggests the occurrence of a receptor on the gut side facing the haemocoel. The binding of the peptide, permeating at increased rates due to the ChiA activity, is considered responsible for the enhanced insecticide activity of the transgenic plants expressing both molecules. These data corroborate the idea that ChiA can be effectively used as gut permeation enhancer in oral delivery strategies of bioinsecticides targeting haemocoelic receptors.
authors.affiliation: Dipartimento di Biologia, Università di Milano, Milano, Italy.
authors.forename: Luisa
authors.initials: L
authors.lastname: Fiandra
authors.forename: Irma
authors.initials: I
authors.lastname: Terracciano
authors.forename: Paolo
authors.initials: P
authors.lastname: Fanti
authors.forename: Antonio
authors.initials: A
authors.lastname: Garonna
authors.forename: Lia
authors.initials: L
authors.lastname: Ferracane
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Fogliano
authors.forename: Morena
authors.initials: M
authors.lastname: Casartelli
authors.forename: Barbara
authors.initials: B
authors.lastname: Giordana
authors.forename: Rosa
authors.initials: R
authors.lastname: Rao
authors.forename: Francesco
authors.initials: F
authors.lastname: Pennacchio
mesh_terms: D000330:Aedes / Q000235:genetics; D000818:Animals; D002688:Chitinases / Q000235:genetics / Q000494:pharmacology*; D005247:Feeding Behavior; D006458:Hemolymph / Q000378:metabolism; D007814:Larva / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D009036:Moths / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000201:enzymology / Q000235:genetics; D009842:Oligopeptides / Q000235:genetics / Q000494:pharmacology*; D010572:Pest Control, Biological; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000494:pharmacology*
chemical_list: D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C065612:Peptide oostatic hormone; D002688:Chitinases