David Pauron

Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s. s.

Pyrethroid‐impregnated bednets are playing an increasing role for combating malaria, especially in stable malaria areas. More than 90% of the current annual malaria incidence (c. 500 million clinical cases with up to 2 million deaths) is in Africa where the major vector is Anopheles gambiae s.s. As pyrethroid resistance has been reported in this mosquito, reliable and simple techniques are urgently needed to characterize and monitor this resistance in the field. In insects, an important mechanism of pyrethroid resistance is due to a modification of the voltage‐gated sodium channel protein recently shown to be associated with mutations of the para‐type sodium channel gene. We demonstrate here that one of these mutations is present in certain strains of pyrethroid resistant A. gambiae s.s. and describe a PCR‐based diagnostic test allowing its detection in the genome of single mosquitoes. Using this test, we found this mutation in six out of seven field samples from West Africa, its frequency being closely correlated with survival to pyrethroid exposure. This diagnostic test should bring major improvement for field monitoring of pyrethroid resistance, within the framework of malaria control programmes.

Voltage‐dependent Na+ channels in pyrethroid‐resistant Culex pipiens L mosquitoes

In some insect species, knockdown resistance (kdr) to pyrethroids and DDT is linked to point mutations in the sequence of the para-type voltage-dependent sodium channel gene. The effects of pyrethroids were assayed on six Culex pipiens strains: two were susceptible to pyrethroids and the four others displayed various levels of resistance, but, in each case, a kdr-type mechanism was strongly suggested. Degenerate primers were designed on the basis of the corresponding sequences of the para orthologous gene reported from several orders of insects. These primers were used to amplify the region of the sodium channel gene which includes the positions where the kdr and super-kdr mutations have been found in Musca domestica. As expected, the amplified fragment was highly homologous to the para sequences. The super-kdr-like mutation (methionine to threonine at position 918 of the M domestica para sequence) was never detected in any strain. In contrast, the same kdr mutation (leucine to phenylalanine at position 1014) was present in some Culex pyrethroid-resistant samples. An alternative substitution of the same leucine to a serine was detected in one strain slightly resistant to pyrethroids but highly resistant to DDT. These data have allowed us to design a PCR-based diagnostic test on genomic DNA to determine the presence or the absence of the kdr allele in single C pipiens collected in several countries. The validity of this test was checked by comparing the frequency of the resistance allele and the toxicological data.

The receptor of Bacillus sphaericus binary toxin in Culex pipiens (Diptera: Culicidae) midgut: molecular cloning and expression ☆

Culex pipiens larval midgut is the primary target of the binary toxin (Bin) present in parasporal inclusions of Bacillus sphaericus. Cpm1, a 60-kDa protein purified from brush border membranes, has been proposed as the receptor of the Bin toxin in the midgut epithelial cells of mosquitoes. We have cloned and characterized the corresponding cDNA from midgut of Culex pipiens larvae. The open reading frame predicted a 580 amino-acid protein with a putative signal peptide at the N-terminus and a putative GPI-anchoring signal at the C-terminus. The amino acid sequence of the cloned Cpm1 exhibited 39-43% identities with insect maltases (alpha-glucosidases and alpha-amylases). Recombinant Cpm1 expressed in E. coli specifically bound to the Bin toxin and had a significant alpha-glucosidase activity but no alpha-amylase activity. These results support the view that Cpm1 is an alpha-glucosidase expressed in Culex midgut where it constitutes the receptor for the Bin toxin. To date, this is the first component involved in the mosquitocidal activity of the Bacillus sphaericus Bin toxin to be characterized. Its identification provides a key step to elucidate the mode of action of the Bin toxin and the mechanisms of resistance developed against it by some mosquito strains.

Pyrosequencing of the midgut transcriptome of the poplar leaf beetle Chrysomela tremulae reveals new gene families in Coleoptera

The insect midgut is the primary target site for Bt-derived insecticides and Bt alternatives. However, despite extensive recent study, the precise role and nature of different Bt receptors remains a subject of considerable debate. This problem is fuelled by a lack of understanding of the genes expressed in the insect midgut and their physiological roles. The poplar leaf beetle, Chrysomela tremulae, is an important model for understanding the mode of action of, and resistance to, coleopteran-specific Bt toxins and currently shows the only known naturally occurring case of resistance to Cry3A toxins. Moreover it belongs to the same family as the corn rootworm, Diabrotica virgifera, an economically important beetle pest and target of hybrid corn expressing Cry3 toxins. Pyrosequencing is a fast and efficient way of defining the transcriptome of specific insect tissues such as the larval midgut. Here we use 454 based pyrosequencing to sample the larval midgut transcriptome of C. tremulae. We identify candidate genes of putative Bt receptors including transcripts encoding cadherin-like proteins, aminopeptidase N and alkaline phosphatase. We also describe a wealth of new transcripts predicting rapidly evolving gene families involved in plant tissue digestion, which have no homologs in the genome of the stored product pest the Red Flour beetle, Tribolium castaneum.

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

The mosquitocidal activity of Bacillus sphaericus is because of a binary toxin (Bin), which binds to Culex pipiens maltase 1 (Cpm1), an α-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.

Transposon-mediated resistance to Bacillus sphaericus in a field-evolved population of Culex pipiens (Diptera: Culicidae)

The binary toxin is the major active component of Bacillus sphaericus, a microbial larvicide used for controlling some vector mosquito‐borne diseases. B. sphaericus resistance has been reported in many part of the world, leading to a growing concern for the usefulness of this environmental friendly insecticide. Here we characterize a novel mechanism of resistance to the binary toxin in a natural population of the West Nile virus vector, Culex pipiens. We show that the insertion of a transposable element‐like DNA into the coding sequence of the midgut toxin receptor induces a new mRNA splicing event, unmasking cryptic donor and acceptor sites located in the host gene. The creation of the new intron causes the expression of an altered membrane protein, which is incapable of interacting with the toxin, thus providing the host mosquito with an advantageous phenotype. As a large portion of insect genomes is composed of transposable elements or transposable elements‐related sequences, this new mechanism may be of general importance to appreciate their significance as potent agents for insect resistance to the microbial insecticides.

Characterization of microsomal oxidative activities in a wild-type and in a DDT resistant strain of Drosophila melanogaster

Abstract Resistance of a laboratory selected DDT strain of Drosophila melanogaster (RalDDT R ) has been found to be monofactorial and correlated to an increased level of activity of the cytochrome P450-dependent mixed function oxidase (MFO). Both strains metabolize DDT and deltamethrin via MFO activity. However, the resistant strain does it more rapidly. The amount of DDT metabolites, including kelthane, bis-4-chlorophenyl acid, bis-4-chlorophenyl-ethanol, and 1,1-bis ( p -chlorophenyl)2,2-dichloroethane, is approximately 9-fold greater with RalDDT R microsomes than with the wild-type strain Raleigh (Ral). Production of deltamethrin metabolites is 2.7-fold higher within the resistant strain. As compared to insecticides, lauric acid and the two steroids used as substrates in this study present many more sites for MFO metabolic action. Lauric acid is hydroxylated on positions 11 and 12 by both strains, but the amount of metabolites formed is 10-fold higher with RalDDT R microsomes. The 2,22-dideoxyecdysone is converted to two polar metabolites when incubated with RalDDT R microsomal preparations. These unidentified metabolites are neither 2-deoxyecdysone nor ecdysone. Also reported for the first time is the metabolization of testosterone by insect microsomes, which gives 13 oxiderivatives formed at different rates, depending on the strains.

Bacillus sphaericus Binary Toxin Elicits Host Cell Autophagy as a Response to Intoxication

Bacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cells response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.

Effects of a mosquitocidal toxin on a mammalian epithelial cell line expressing its target receptor

The spread of diseases transmitted by Anopheles and Culex mosquitoes, such as malaria and West Nile fever, is a growing concern for human health. Bacillus sphaericus binary toxin (Bin) is one of the few available bioinsecticides able to control populations of these mosquitoes efficiently. We previously showed that Bin binds to Cpm1, an α‐glucosidase located on the apical side of Culex larval midgut epithelium. We analysed the effects of Bin by expressing a construct encoding Cpm1 in the mammalian epithelial MDCK cell line. Cpm1 is targeted to the apical side of polarized MDCK, where it is anchored by glycosylphosphatidylinositol (GPI) and displays α‐glucosidase activity. Bin bound to transfected cells and induced a non‐specific current presumably related to the opening of pores. The formation of these pores may be related to the location of the toxin/receptor complex in lipid raft microdomains. Finally, Bin promoted the time‐dependent appearance of intracytoplasmic vacuoles but did not drive cell lysis. Thus, the dual functionality (enzyme/toxin receptor) of Cpm1 is fully conserved in MDCK cells and Cpm1 is an essential target protein for Bin cytotoxicity in Culex mosquitoes.

Target modification as a molecular mechanism of pyrethroid resistance in Drosophila melanogaster

Abstract “Knock down” resistance (kdr) to the pyrethroid deltamethrin was investigated using susceptible (Tubingen) and resistant (TubingenDDT) strains of Drosophila melanogaster. Toxicological and pharmacological data show that resistance involves a modification of the affinity of the insecticide for its receptor site on the voltage-dependent sodium channel. Genetic studies indicate that the kdr factor is linked to the second chromosome where one sodium channel gene, sch, is located. Cloning and sequencing the alleles of this gene from both strains revealed a single substitution that may be responsible for the loss of toxicity of deltamethrin in the resistant strain.