Interaction of Two Bacillus thuringiensis d-Endotoxins with the Digestive System of Lygus hesperus

Abstract

The active-toxin form of CrylAc (65 kDa) or Cry2Ab was fed to a non-susceptible insect, Lygus hesperus, in an artificial diet. Biochemical and immunocytochemical methods were used to determine the distribution of ingested toxin. The toxins did not elicit a feeding deterrent response. CrylAc and Cry2Ab were ingested; small amounts were absorbed into the hemolymph as holoproteins, but most was excreted. SDS-PAGE analysis of CrylAc and Cry2Ab incubations with salivary gland homogenate showed a small decrease in the molecular weight of the active toxins. Proteolytic processing of the toxins also occurred in vivo, within the digestive system of L. hesperus. Excreted CrylAc and Cry2Ab retained activity toward lepidopteran larvae. Immunocytochemical in vivo localization studies showed negligible association of CrylAc with L. hesperus tissues. In contrast, strong extracellular association of Cry2Ab was observed with L. hesperus midgut brush border microvilli and basement membrane, as well as with cellular outlines within the hemolymph and fat body.

Acquisition of Cry1Ac Protein by Non-Target Arthropods in Bt Soybean Fields

“…A similar difference between life stages has been reported for Lygus rugulipennis (Hemiptera: Miridae) collected in Cry3Bb1-expressing Bt maize [44]. An artificial diet study with Lygus hesperus (Hemiptera: Miridae) revealed that only a small portion of the ingested Cry1Ac toxin was absorbed into the hemolymph while most was excreted in a still biologically active form [64]. Field investigations revealed that the abundances of Lygus spp., C. viridis , and D. Baccarum were similar in Bt soybean and control soybean (Yu et al, unpublished data).…”

Recommendations for the design of laboratory studies on non-target arthropods for risk assessment of genetically engineered plants

“…Examples are stomach poisons such as the growth regulator teflubenzuron (Stacey et al 2006), potassium arsenate (Duan et al 2006, 2007), or the proteinase inhibitor E-64 (Duan et al 2007). Alternatively, ingestion of test substance could also be confirmed by immuno-assays (e.g., ELISA test) of the treated arthropods (Vojtech et al 2005; Li et al 2008; Chen et al 2009; Meissle and Romeis 2009b; Li and Romeis 2010; Álvarez-Alfageme et al 2010) or their frass (Brandt et al 2004; Christeller et al 2005; Mulligan et al 2010), by incorporation of a dye into the prepared diet and subsequent examination of the diet uptake (Rodrigo-Simón et al 2006), by labeling the protein of interest with a fluorescent compound such as rhodamine and confirm its uptake by the test organisms (Hogervorst et al 2006), or by simply determining the weight of the test arthropods prior to and after exposure to the test substance (Romeis et al 2004; Li et al 2008). …”

Toxins for Transgenic Resistance to Hemipteran Pests

“…Lygus spp. are reported to feed on 117 non-crop plants and over 25 cultivated plants and are primarily known as pests of cotton ( Gossypium hirsutum L.) and seed alfalfa ( Medicago sativa L.) [12]. Nymphs and adults feed on the flowers and fruits of many plants causing abscission and deformation [13].…”

“…Although functional receptors for Cry toxins have not been identified in sap-sucking pests, some studies have been carried out to investigate the association of Cry toxins with specific tissues following Cry toxin ingestion [12,56]. Immunocytochemical analysis of L. hesperus tissues after feeding on trypsin-activated Cry1Ac and Cry2Ab showed differential association of the toxins [12].…”

“…Immunocytochemical analysis of L. hesperus tissues after feeding on trypsin-activated Cry1Ac and Cry2Ab showed differential association of the toxins [12]. Cry1Ac did not associate with any of the L. hesperus tissues indicating the lack of a specific midgut binding receptor, whereas Cry2Ab showed extensive binding to brush border microvilli, the basement membrane of midgut epithelial cells, and to cellular structures within the hemolymph and fat body.…”