Loss of the membrane anchor of the target receptor is a mechanism of bioinsecticide resistance

Abstract

The mosquitocidal activity of Bacillus sphaericus is because of a binary toxin (Bin), which binds to Culex pipiens maltase 1 (Cpm1), an ␣-glucosidase present in the midgut of Culex pipiens larvae. In this work, we studied the molecular basis of the resistance to Bin developed by a strain (GEO) of C. pipiens. Immunohistochemical and in situ hybridization experiments showed that Cpm1 was undetectable in the midgut of GEO larvae, although the gene was correctly transcribed. The sequence of the cpm1GEO cDNA differs from the sequence we previously reported for a susceptible strain (cpm1IP) by seven mutations: six missense mutations and a mutation leading to the premature termination of translation. When produced in insect cells, Cpm1IP was attached to the membrane by a glycosylphosphatidylinositol (GPI). In contrast, the premature termination of translation of Cpm1GEO resulted in the targeting of the protein to the extracellular compartment because of truncation of the GPI-anchoring site. The interaction between Bin and Cpm1GEO and the enzyme activity of the receptor were not affected. Thus, Bin is not toxic to GEO larvae because it cannot interact with the midgut cell membrane, even though its receptor site is unaffected. This mechanism contrasts with other known resistance mechanisms in which point mutations decrease the affinity of binding between the receptor and the toxin. E nvironmentally safe toxins produced by Bacillus thuringiensis and͞or Bacillus sphaericus have been integrated in management programs to control crop pests such as Heliothis virescens and Plutella xylostella, and disease vectors such as the mosquitoes Anopheles gambiae and Culex pipiens (1, 2). However, the potential benefits of these biopesticides may be rapidly lost because of the proliferation of highly resistant insect populations (3-6). Control strategies to delay or prevent the development of resistance have been developed, based on several assumptions. The most important of these assumptions are that the resistance gene is recessive and that the rate of mutation to generate resistance alleles is low. Currently, it is difficult to evaluate the success of these strategies because we lack adequate methods for monitoring resistance alleles because of our very restricted knowledge of the mechanisms of resistance to bioinsecticides. Bacillus sphaericus is toxic to mosquitoes, mainly because it produces a binary toxin (Bin) in crystals during sporulation. Following the ingestion and solubilization of crystals by larvae, the released toxin is activated and interacts with the brushborder membrane of the midgut epithelium. In a previous study, we reported the partial purification of a Bin-binding protein from IP, a susceptible strain of C. pipiens. This receptor displayed sequence similarity to ␣-glucosidases and other maltase-like proteins, and was thus named Cpm1, for Culex pipiens maltase 1 (7). We recently isolated the cDNA encoding Cpm1 from IP larvae (cpm1 IP ) and showed that Cpm1 has ␣-glucosidase activity when produced in bacteria (8). ...

Induction and transmission of Bacillus thuringiensis tolerance in the flour moth Ephestia kuehniella

“…The proposed immune-related tolerance mechanism is different from previously described Bt-resistance mechanisms (3) in which the observed reductions in Bt toxicity are explained almost exclusively in terms of alterations to receptor properties on the gut epithelium (4,5). However, our results in E. kuehniella are consistent with recent observations of malaria-refractory Anopheles gambiae colonies, which have elevated oxidative activities compared with susceptible insects (13,14).…”

“…However, despite the use of Bt endotoxins in transgenic crops covering Ͼ11.4 million hectares (2), the precise details of how endotoxins bind to gut cells to kill insects are poorly understood (3). This limitation impedes our understanding of potential mechanisms of insect resistance to Bt endotoxins other than the loss or modification of receptors (4,5), altered proteolysis of protoxin and͞or toxin (6,7), and repair and͞or replacement of damaged cells (8). Although few insect species have developed resistance in the field (3,9), genetic resistance to Bt-endotoxins has been selected in several species in the laboratory (3,10).…”

Detection of an Allele Conferring Resistance to Bacillus sphaericus Binary Toxin in Culex quinquefasciatus Populations by Molecular Screening

“…Bioassays are further limited in that they demand large sample size and maintenance of F1 or F2 progenies in the laboratory and assays should be performed under specific conditions to provide reliable data. In the context of Culex resistance to B. sphaericus, the improvement in DNA screening is needed to track other resistance alleles already described (10,11), since the PCR used in the study is specific for the cqm1 REC allele. The ␣-glucosidade in-gel assay can also be useful to detect individuals not expressing Cqm1 whose resistance can be associated with alleles other than cqm1 REC , although this assay is able to detect only homozygous resistant larvae.…”

“…Four cpm1/cqm1 resistance alleles were found in Culex populations of different origins. The cpm1 GEO allele, detected in a C. pipiens laboratory-selected colony (GEO) from California, contains a nonsense mutation leading to the synthesis of a truncated protein lacking the glycosylphosphatidylinositol anchor and is therefore not able to localize to the apical membrane of the midgut epithelium (11,38). A 19-nucleotide deletion in the gene cqm1, here named cqm1 REC , was associated with the resistance of a C. quinquefasciatus laboratory colony (CqRL1/ 2362) originating from the city of Recife (Brazil), and this mutation prevents the expression of Cqm1 on the midgut brush border membranes (28,29,30).…”

Recombinant bacteria for mosquito control

“…In addition, Cyt1A can overcome very high levels of resistance to the Bin toxin of B. sphaericus 2362 when combined with this species (Wirth et al, 2000a) and can extend its target spectrum to A. aegypti (Wirth et al, 2000b). Recent studies using fluorescent dyes have shown that the lack of sensitivity in B. sphaericus-resistant C. quinquefasciatus is due to the absence of the Bin toxin receptor in the midgut microvillar membrane (Darboux et al, 2002). Studies in our laboratory have shown that Cyt1A forms lesions in this membrane that enable the Bin toxin to enter these cells and exert toxicity (Fig.·3).…”