Fitness cost of resistance to Bt cotton linked with increased gossypol content in pink bollworm larvae

Fitness costs of resistance to Bacillus thuringiensis (Bt) crops occur in the absence of Bt toxins, when individuals with resistance alleles are less fit than individuals without resistance alleles. As costs of Bt resistance are common, refuges of non-Bt host plants can delay resistance not only by providing susceptible individuals to mate with resistant individuals, but also by selecting against resistance. Because costs typically vary across host plants, refuges with host plants that magnify costs or make them less recessive could enhance resistance management. Limited understanding of the physiological mechanisms causing fitness costs, however, hampers attempts to increase costs. In several major cotton pests including pink bollworm (Pectinophora gossypiella), resistance to Cry1Ac cotton is associated with mutations altering cadherin proteins that bind this toxin in susceptible larvae. Here we report that the concentration of gossypol, a cotton defensive chemical, was higher in pink bollworm larvae with cadherin resistance alleles than in larvae lacking such alleles. Adding gossypol to the larval diet decreased larval weight and survival, and increased the fitness cost affecting larval growth, but not survival. Across cadherin genotypes, the cost affecting larval growth increased as the gossypol concentration of larvae increased. These results suggest that increased accumulation of plant defensive chemicals may contribute to fitness costs associated with resistance to Bt toxins.     

Non-Recessive Bt Toxin Resistance Conferred by an Intracellular Cadherin Mutation in Field-Selected Populations of Cotton Bollworm

Transgenic crops producing Bacillus thuringiensis (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.     

Glycosphingolipid expression in pig aorta: identification of possible target antigens for human natural antibodies

Total non-acid glycosphingolipids were isolated from the aortas of more than 80 pigs. The glycolipids were separated by HPLC, analysed by thin- layer chromatography, and tested for reactivity with monoclonal anti- blood group antibodies. The fractions were structurally characterized by NMR spectroscopy and mass spectrometry. Reactivity with both anti- blood group A and H antibodies was seen. The major glycosphingolipid constituents were globotri- and globotetraosylceramides and blood group H pentaglycosylceramides based on type 1 and type 2 core saccharide chains. Globopentaosylceramides, blood group H hexaglycosylceramides based on type 4 chain, and blood group A hexaglycosylceramides based on type 1 core chain were also present. Two structures, that may be important targets for human antibodies initiating hyperacute rejection following pig to human xenotransplantation, were present as minor constituents compared to the blood group components. These were Galalpha1,3neolactotetraosylceramide and a Galalpha1, 3Lexstructure. A Leb/Y hexaglycosylceramide was also present.      

Control of Resistant Pink Bollworm (Pectinophora gossypiella) by Transgenic Cotton That Produces Bacillus thuringiensis Toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 μg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 μg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 μg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.     

Next‐generation transgenic cotton: pyramiding RNAi and Bt counters insect resistance

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are extensively cultivated worldwide. To counter rapidly increasing pest resistance to crops that produce single Bt toxins, transgenic plant ‘pyramids’ producing two or more Bt toxins that kill the same pest have been widely adopted. However, cross‐resistance and antagonism between Bt toxins limit the sustainability of this approach. Here we describe development and testing of the first pyramids of cotton combining protection from a Bt toxin and RNA interference (RNAi). We developed two types of transgenic cotton plants producing double‐stranded RNA (dsRNA) from the global lepidopteran pest Helicoverpa armigera designed to interfere with its metabolism of juvenile hormone (JH). We focused on suppression of JH acid methyltransferase (JHAMT), which is crucial for JH synthesis, and JH‐binding protein (JHBP), which transports JH to organs. In 2015 and 2016, we tested larvae from a Bt‐resistant strain and a related susceptible strain of H. armigera on seven types of cotton: two controls, Bt cotton, two types of RNAi cotton (targeting JHAMT or JHBP) and two pyramids (Bt cotton plus each type of RNAi). Both types of RNAi cotton were effective against Bt‐resistant insects. Bt cotton and RNAi acted independently against the susceptible strain. In computer simulations of conditions in northern China, where millions of farmers grow Bt cotton as well as abundant non‐transgenic host plants of H. armigera, pyramided cotton combining a Bt toxin and RNAi substantially delayed resistance relative to using Bt cotton alone.     

Reversing insect adaptation to transgenic insecticidal plants.

The refuge-high-dose strategy for delaying insect adaptation to transgenic plants produces non-transgenic plants that enable survival of susceptible individuals. Previous theoretical work has suggested three requirements for success of the refuge-high-dose strategy: a low initial frequency of the resistance allele, extensive mating between resistant and susceptible adults and recessive inheritance of resistance. In order to understand an observed decrease in resistance frequency and improve the potential for managing resistance better, we used analytical and simulation models for exploring the conditions that prevent or reverse the evolution of resistance, even when resistance is not rare initially. Assuming random mating and recessive or nearly recessive inheritance of resistance, the factors favouring reversal of resistance are non-recessive costs of resistance, low initial resistance allele frequency, large refuges, incomplete resistance and density-independent population growth in refuges.     

Large-scale management of insect resistance to transgenic cotton in Arizona: can transgenic insecticidal crops be sustained?

A major challenge for agriculture is management of insect resistance to toxins from Bacillus thuringiensis (Bt) produced by transgenic crops. Here we describe how a large-scale program is being developed in Arizona for management of resistance to Bt cotton in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), and other insect pests of cotton. Financial support from growers makes this program possible. Collaboration between the Arizona Cotton Research and Protection Council, the University of Arizona, and government agencies has led to development of resistance management guidelines, a remedial action plan, and tools for monitoring compliance with the proposed guidelines. Direct participation in development of resistance management policies is a strong incentive for growers to invest in resistance management research. However, more research, regularly updated regulations, and increased collaboration between stakeholders are urgently needed to maintain efficacy of Bt toxins in transgenic crops.     

Cyt1A from Bacillus thuringiensis lacks toxicity to susceptible and resistant larvae of diamondback moth (Plutella xylostella) and pink bollworm (Pectinophora gossypiella)

We tested Cyt1Aa, a cytolytic endotoxin of Bacillus thuringiensis, against susceptible and Cry1A-resistant larvae of two lepidopteran pests, diamondback moth (Plutella xylostella) and pink bollworm (Pectinophora gossypiella). Unlike previous results obtained with mosquito and beetle larvae, Cyt1Aa alone or in combination with Cry toxins was not highly toxic to the lepidopteran larvae that we examined.     

Insect resistance to transgenic Bt crops: lessons from the laboratory and field

Transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) grew on >62 million ha worldwide from 1996 to 2002. Despite expectations that pests would rapidly evolve resistance to such Bt crops, increases in the frequency of resistance caused by exposure to Bt crops in the field have not yet been documented. In laboratory and greenhouse tests, however, at least seven resistant laboratory strains of three pests (Plutella xylostella [L.], Pectinophora gossypiella [Saunders], and Helicoverpa armigera [Hübner]) have completed development on Bt crops. In contrast, several other laboratory strains with 70- to 10,100-fold resistance to Bt toxins in diet did not survive on Bt crops. Monitoring of field populations in regions with high adoption of Bt crops has not yet detected increases in resistance frequency. Resistance monitoring examples include Ostrinia nubilalis (Hübner) in the United States (6 yr), P. gossypiella in Arizona (5 yr), H. armigera in northern China (3 yr), and Helicoverpa zea (Boddie) in North Carolina (2 yr). Key factors delaying resistance to Bt crops are probably refuges of non-Bt host plants that enable survival of susceptible pests, low initial resistance allele frequencies, recessive inheritance of resistance to Bt crops, costs associated with resistance that reduce fitness of resistant individuals relative to susceptible individuals on non-Bt hosts ("fitness costs"), and disadvantages suffered by resistant strains on Bt hosts relative to their performance on non-Bt hosts ("incomplete resistance"). The relative importance of these factors varies among pest-Bt crop systems, and violations of key assumptions of the refuge strategy (low resistance allele frequency and recessive inheritance) may occur in some cases. The success of Bt crops exceeds expectations of many, but does not preclude resistance problems in the future.