title: Proteolytic digestive enzymes and peritrophic membranes during the development of Plodia interpunctella (Lepidoptera: Piralidae): targets for the action of soybean trypsin inhibitor (SBTI) and chitin-binding vicilin (EvV).
abstract: The digestive system of P. interpunctella was characterized during its larval development to determine possible targets for the action of proteinaceous enzyme inhibitors and chitin-binding proteins. High proteolytic activities using azocasein at pH 9.5 as substrate were found. These specific enzymatic activities (AU/mg protein) showed an increase in the homogenate of third instar larvae, and when analyzed by individual larvae (AU/gut), the increase was in sixth instar larvae. Zymograms showed two bands corresponding to those enzymatic activities, which were inhibited by TLCK and SBTI, indicating that the larvae mainly used serine proteinases at pH 9.5 in their digestive process. The presence of a peritrophic membrane in the larvae was confirmed by chemical testing and light microscopy. In a bioassay, P. interpunctella was not susceptible to the soybean trypsin inhibitor, which did not affect larval mass and mortality, likely due to the weak association with its target digestive enzyme. EvV (Erythrina velutina vicilin), when added to the diet, affected mortality (LD50 0.23%) and larval mass (ED50 0.27%). This effect was associated with EvV-binding to the peritrophic membrane, as seen by immunolocalization. EvV was susceptible to gut enzymes and after the digestion process, released an immunoreactive fragment that was bound to the peritrophic matrix, which probably was responsible for the action of EvV.
authors.affiliation: Chemistry and Bioactive Protein Function Laboratory, Department of Biochemistry, Bioscience Center, Federal University of Rio Grande do Norte, Natal, Brazil.
authors.forename: Ticiana M L
authors.initials: TM
authors.lastname: Amorim
authors.forename: Leonardo L P
authors.initials: LL
authors.lastname: Macedo
authors.forename: Adriana F
authors.initials: AF
authors.lastname: Uchoa
authors.forename: Adeliana S
authors.initials: AS
authors.lastname: Oliveira
authors.forename: Joelma C M
authors.initials: JC
authors.lastname: Pitanga
authors.forename: Francisco P
authors.initials: FP
authors.lastname: Macedo
authors.forename: Elizeu A
authors.initials: EA
authors.lastname: Santos
authors.forename: Mauricio P
authors.initials: MP
authors.lastname: de Sales
mesh_terms: D000818:Animals; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D041981:Gastrointestinal Tract / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007306:Insecticides; D007814:Larva / Q000201:enzymology / Q000254:growth & development; D007915:Lepidoptera / Q000201:enzymology* / Q000254:growth & development*; D010447:Peptide Hydrolases / Q000378:metabolism*; D010940:Plant Proteins / Q000494:pharmacology*; D055314:Seed Storage Proteins; D013025:Glycine max / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000494:pharmacology*
chemical_list: D005456:Fluorescent Dyes; D007306:Insecticides; D010940:Plant Proteins; D055314:Seed Storage Proteins; D014361:Trypsin Inhibitors; C015575:vicilin protein, plant; D010447:Peptide Hydrolases; D016650:Fluorescein-5-isothiocyanate