Wendy Kain

Parallel Evolution of Bacillus thuringiensis Toxin Resistance in Lepidoptera

Despite the prominent and worldwide use of Bacillus thuringiensis (Bt) insecticidal toxins in agriculture, knowledge of the mechanism by which they kill pests remains incomplete. Here we report genetic mapping of a membrane transporter (ABCC2) to a locus controlling Bt Cry1Ac toxin resistance in two lepidopterans, implying that this protein plays a critical role in Bt function.

Parallel Evolution of Bt Toxin Resistance in Lepidoptera

Despite the prominent and worldwide use of Bacillus thuringiensis (Bt) insecticidal toxins in agriculture, knowledge of the mechanism by which they kill pests remains incomplete. Here we report genetic mapping of a membrane transporter (ABCC2) to a locus controlling Bt Cry1Ac toxin resistance in two lepidopterans, implying that this protein plays a critical role in Bt function.

Mechanism of Resistance to Bacillus thuringiensis Toxin Cry1Ac in a Greenhouse Population of the Cabbage Looper, Trichoplusia ni

ABSTRACT The cabbage looper, Trichoplusia ni, is one of only two insect species that have evolved resistance to Bacillus thuringiensis in agricultural situations. The trait of resistance to B. thuringiensis toxin Cry1Ac from a greenhouse-evolved resistant population of T. ni was introgressed into a highly inbred susceptible laboratory strain. The resulting introgression strain, GLEN-Cry1Ac-BCS, and its nearly isogenic susceptible strain were subjected to comparative genetic and biochemical studies to determine the mechanism of resistance. Results showed that midgut proteases, hemolymph melanization activity, and midgut esterase were not altered in the GLEN-Cry1Ac-BCS strain. The pattern of cross-resistance of the GLEN-Cry1Ac-BCS strain to 11 B. thuringiensis Cry toxins showed a correlation of the resistance with the Cry1Ab/Cry1Ac binding site in T. ni. This cross-resistance pattern is different from that found in a previously reported laboratory-selected Cry1Ab-resistant T. ni strain, evidently indicating that the greenhouse-evolved resistance involves a mechanism different from the laboratory-selected resistance. Determination of specific binding of B. thuringiensis toxins Cry1Ab and Cry1Ac to the midgut brush border membranes confirmed the loss of midgut binding to Cry1Ab and Cry1Ac in the resistant larvae. The loss of midgut binding to Cry1Ab/Cry1Ac is inherited as a recessive trait, which is consistent with the recessive inheritance of Cry1Ab/Cry1Ac resistance in this greenhouse-derived T. ni population. Therefore, it is concluded that the mechanism for the greenhouse-evolved Cry1Ac resistance in T. ni is an alteration affecting the binding of Cry1Ab and Cry1Ac to the Cry1Ab/Cry1Ac binding site in the midgut.

Novel Chitinolytic Enzymes with Biological Activity Against Herbivorous Insects

The soil bacteria, Streptomyces albidoflavus, secretes endochitinases and chitobiosidases that are active over a broad range of pH (4–10). Ingestion of this mixture of chitinolytic enzymes significantly reduced the growth and development of Trichoplusia ni and significantly reduced survival of Myzus persicae, Bemisia argentifolii, and Hypothenemus hampei. Perfusion chromatography was used to separate endochitinases from chitobiosidases. The endochitinases had significantly greater biological activity against Bemisia argentifolii than the chitobiosidases. The utility of chitinolytic enzymes as regulators of populations of herbivorous insects is discussed.

Inheritance of Resistance to Bacillus thuringiensis subsp. kurstaki in Trichoplusia ni

ABSTRACT The genetic inheritance of resistance to a commercial formulation of Bacillus thuringiensis subsp. kurstaki was examined in a Trichoplusia ni colony initiated from a resistant population present in a commercial vegetable greenhouse in British Columbia, Canada. Progeny of F1 reciprocal crosses and backcrosses between F1 larvae and resistant (PR) and susceptible (PS) populations were assayed at different B. thuringiensis subsp. kurstaki concentrations. The responses of progeny of reciprocal F1 crosses were identical, indicating that the resistant trait was autosomal. The 50% lethal concentration for the F1 larvae was slightly higher than that for PS, suggesting that resistance is partially recessive. The responses of both backcross progeny (F1 × PR, F1 × PS) did not correspond to predictions from a single-locus model. The inclusion of a nonhomozygous resistant parental line in the monogenic model significantly increased the correspondence between the expected and observed results for the F1 × PR backcross but decreased the correspondence with the F1 × PS backcross results. This finding suggests that resistance to B. thuringiensis subsp. kurstaki in this T. ni population is due to more than one gene.

Partial characterization of chitinolytic enzymes from Streptomyces albidoflavus

Streptomyces albidoflavus NRRL B‐16746 secreted three types of chitinolytic enzymes: N‐acetyl‐glucosaminidase, chitobiosidase and endochitinase. Optimal activity for all three types of enzymes occurred at pH 4–6; however 55–74% of the chitobiosidase and endochitinase activity was detectable at pH 8–10. Chitobiosidase activity originated from two strongly acidic (pI < 3.0) proteins with molecular mass of 27 kDa and 34 kDa, while endochitinase activity originated from five major acidic proteins (pI 5.1, 5.3, 5.75, 5.8–5.9 and 6.4) with molecular mass of 59, 45, 38.5, 27 and 25.5 kDa. Purified chitobiosidases significantly reduced spore germination and germ tube elongation of Botrytis cinerea and Fusarium oxysporum. Chitinolytic enzymes with significant activity at pH 4–10 may be used, transgenically, to reduce the growth and/or development of a broad spectrum of insects and fungi that are major economic pests.

Resistance of Trichoplusia ni to Bacillus thuringiensis Toxin Cry1Ac Is Independent of Alteration of the Cadherin-Like Receptor for Cry Toxins

Alteration of binding sites for Bacillus thuringiensis (Bt) toxins in insect midgut is the major mechanism of high-level resistance to Bt toxins in insects. The midgut cadherin is known to be a major binding protein for Bt Cry1A toxins and linkage of Bt-resistance to cadherin gene mutations has been identified in lepidopterans. The resistance to Bt toxin Cry1Ac evolved in greenhouse populations of Trichoplusia ni has been identified to be associated with the down-regulation of an aminopeptidase N (APN1) gene by a trans-regulatory mechanism and the resistance gene has been mapped to the locus of an ABC transporter (ABCC2) gene. However, whether cadherin is also involved with Cry1Ac-resistance in T. ni requires to be understood. Here we report that the Cry1Ac-resistance in T. ni is independent of alteration of the cadherin. The T. ni cadherin cDNA was cloned and the cadherin sequence showed characteristic features known to cadherins from Lepidoptera. Various T. ni cadherin gene alleles were identified and genetic linkage analysis of the cadherin alleles with Cry1Ac-resistance showed no association of the cadherin gene with the Cry1Ac-resistance in T. ni. Analysis of cadherin transcripts showed no quantitative difference between the susceptible and Cry1Ac-resistant T. ni larvae. Quantitative proteomic analysis of midgut BBMV proteins by iTRAQ-2D-LC-MS/MS determined that there was no quantitative difference in cadherin content between the susceptible and the resistant larvae and the cadherin only accounted for 0.0014% (mol%) of the midgut BBMV proteins, which is 1/300 of APN1 in molar ratio. The cadherin from both the susceptible and resistant larvae showed as a 200-kDa Cry1Ac-binding protein by toxin overlay binding analysis, and nano-LC-MS/MS analysis of the 200-kDa cadherin determined that there is no quantitative difference between the susceptible and resistant larvae. Results from this study indicate that the Cry1Ac-resistance in T. ni is independent of cadherin alteration.

Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism

ABSTRACT The resistance to the Bacillus thuringiensis (Bt) toxin Cry2Ab in a greenhouse-originated Trichoplusia ni strain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in the T. ni strain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab in T. ni. From the dual-toxin-resistant T. ni strain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptible T. ni strain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptible T. ni larvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratory T. ni strains. Genetic linkage analysis showed that the Cry2Ab resistance in T. ni was not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter gene ABCC2. Therefore, the Cry2Ab resistance in T. ni is conferred by a novel but unknown genetic mechanism.

Occurrence of the New Invasive Insect Contarinia nasturtii (Diptera: Cecidomyiidae) on Cruciferous Weeds

ABSTRACT Contarinia nasturtii (Kieffer) (Diptera: Cecidomyiidae), a common insect pest in Europe and a new invasive pest in North America, causes severe damage to cruciferous crops. In the United States, C. nasturtii was first reported in western New York in 2004. From 2005 to 2007, field surveys were conducted in western New York to investigate the occurrence of C. nasturtii in weeds that might serve as a reservoir for this pest. The results indicate that 12 cruciferous weed species were found in and around commercial vegetable crucifer plantings, and C. nasturtii emergence was detected from most of them. The number of C. nasturtii that emerged from the weeds was low and varied by species, year, and the timing of sampling. Peak emergence from weeds in fallow fields occurred in June. Nonchoice tests in the laboratory showed that significantly fewer larvae were found on cruciferous weeds than on cauliflower plants, although C. nasturtii could lay eggs on the weeds. When weeds and cauliflower plants were simultaneously exposed to C. nasturtii adults for egg laying (choice tests), 97.3% of the C. nasturtii larvae were found on the cauliflower plants 8 d after oviposition, 2.7% on Sinapis arvensis L., and none on the other five weed species tested. Our results suggest that cruciferous weeds can serve as alternative host plants of C. nasturtii but are less suitable than cauliflower. A method of detecting C. nasturtii on weeds and control of C. nasturtii through weed management are discussed.

Resistance of Trichoplusia ni Populations Selected by Bacillus thuringiensis Sprays to Cotton Plants Expressing Pyramided Bacillus thuringiensis Toxins Cry1Ac and Cry2Ab

ABSTRACT Two populations of Trichoplusia ni that had developed resistance to Bacillus thuringiensis sprays (Bt sprays) in commercial greenhouse vegetable production were tested for resistance to Bt cotton (BollGard II) plants expressing pyramided Cry1Ac and Cry2Ab. The T. ni colonies resistant to Bacillus thuringiensis serovar kurstaki formulations were not only resistant to the Bt toxin Cry1Ac, as previously reported, but also had a high frequency of Cry2Ab-resistant alleles, exhibiting ca. 20% survival on BollGard II foliage. BollGard II-resistant T. ni strains were established by selection with BollGard II foliage to further remove Cry2Ab-sensitive alleles in the T. ni populations. The BollGard II-resistant strains showed incomplete resistance to BollGard II, with adjusted survival values of 0.50 to 0.78 after 7 days. The resistance to the dual-toxin cotton plants was conferred by two genetically independent resistance mechanisms: one to Cry1Ac and one to Cry2Ab. The 50% lethal concentration of Cry2Ab for the resistant strain was at least 1,467-fold that for the susceptible T. ni strain. The resistance to Cry2Ab in resistant T. ni was an autosomally inherited, incompletely recessive monogenic trait. Results from this study indicate that insect populations under selection by Bt sprays in agriculture can be resistant to multiple Bt toxins and may potentially confer resistance to multitoxin Bt crops.