Genetic mapping of genes conferring permethrin resistance in the malaria vector, Anopheles gambiae.

Resistance to permethrin in an East African population of the major malaria vector, Anopheles gambiae is multifactorial. A mutated sodium channel allele and enhanced insecticide metabolism contribute to the resistance phenotype. We used microsatellite markers to scan the genome for quantitative trait loci (QTL) associated with permethrin resistance. Two major and one minor QTL were identified. The first QTL, rtp1, colocalizes with the sodium channel gene on chromosome 2L thus further supporting the importance of mutations in this gene in conferring permethrin resistance. The second two loci are located on the third chromosome and one of these, rtp2, flanks a large cluster of cytochrome P450 genes. Further detailed mapping of these regions will help elucidate the molecular mechanisms of metabolic resistance to insecticides.

Quantitative analysis of gene amplification in insecticide-resistant Culex mosquitoes.

The amplification of carboxylesterase structural genes followed by their overexpression is the most common mechanism of resistance to organophosphorus insecticides in Culex mosquitoes. Most resistant Culex quinquefasciatus mosquitoes have co-amplified estalpha2(1) and estbeta2(1) genes. Recently, Southern, DNA dot-blot analysis and phosphorimaging technology were used to quantify the est gene copy number in aphids and mosquitoes. Although more accurate than autoradiography, this method relies on probe hybridization, which can be variable. We have directly measured gene and mRNA copy number by using real-time quantitative PCRs in mosquitoes. The acquisition of fluorescence from incorporation of the double-strand-specific dye SYBR GreenI into a PCR product once per cycle is used to provide an absolute quantification of the initial template copy number. Thus it has been possible to show that estalpha2(1) and estbeta2(1) are co-amplified approx. 80-fold in the genome of the resistant PelRR strain of C. quinquefasciatus. The two genes, although co-amplified in a 1:1 ratio, are differentially transcribed: the estbeta2(1) gene from this amplicon has greater transcription than estalpha2(1) in all individual mosquito larvae tested, with an average ratio of 10:1. Purified esterases from mosquito homogenates were found in a ratio of 3:1, which, combined with the quantitative mRNA data, suggests the operation of both transcriptional and translational control mechanisms to regulate the expression of the amplified genes in C. quinquefasciatus insecticide-resistant mosquitoes.

Amplification of a serine esterase gene is involved in insecticide resistance in Sri Lankan Culex tritaeniorhynchus.

Culex tritaeniorhynchus, the major vector of Japanese encephalitis in Sri Lanka, is resistant to organophosphorus insecticides, with a 10-fold resistance ratio at the LC50 for chlorpyrifos, and a high heterogenelty factor in the insect field population. The major mechanism of resistance in this species, as in the mosquito C. quinquefasciatus, is elevation of esterase activity. Basic biochemical, immunological and molecular analysis suggests that the C. tritaeniorhynchus CtrEstbeta1 gene is orthologous to the C. quinquefasciatus amplified Estbetas. The Estbeta2(1) antiserum cross-reacts strongly with CtrEstbeta1(1). Its corresponding cDNA, over the 545 base pairs sequenced, has approximately 84% identity with the various C. quinquefasciatus Estbetas. The gene is amplified in C. tritaeniorhynchus. Amplification of the same esterase in two independent species, along with multiple amplification events involving this esterase gene in C. quinquefasciatus suggests that the location of this gene within the genome predisposes it to amplification.

Expression of the Plasmodium berghei ookinete protein Pbs21 in a baculovirus-insect cell system produces an efficient transmission blocking immunogen.

A surface protein of Plasmodium berghei ookinetes, Pbs21, was expressed in a baculovirus-insect cell system in cell culture and in Heliothis virescens larvae. Groups of BALB/c mice received two intraperitoneal inoculations of either i) Tris-buffer or homogenized H. virescens larvae infected with wild-type baculovirus; ii) enriched, homogenized ookinetes, or iii) homogenized H. virescens larvae expressing recombinant Pbs21 (rPbs21). All animals immunized with ookinetes or with rPbs21 had high titres of antibodies (IgG isotype) that bound to native Pbs21. The large majority of antibodies in immune sera of both groups recognized the antigen under non-reducing but not under reducing conditions. The predominant IgG-subclasses in mice immunized with ookinetes was IgG1 and in mice immunized with rPbs21, the subclasses were IgG1 and IgG2a. Immunization with rPbs21 reduced the infectivity of P.berghei to mosquitoes by 91% compared to a 99% reduction following immunization with ookinetes. This preliminary data indicate that rPbs21 expressed in this eukaryotic system induces a transmission-blocking immunity, which is more effective than that achieved using rPbs21 expressed in Escherichia coli (Matsuoka et al. 1994).

Studies on the immunogenicity of a recombinant ookinete surface antigen Pbs21 from Plasmodium berghei expressed in Escherichia coli.

Plasmodium berghei ookinete surface antigen (Pbs21), was produced as a fusion product with maltose binding protein (MBP) in Escherichia coli and used to induce transmission-blocking immunity in mice. Specificity of induced antibody was confirmed by Western blotting with native ookinete Pbs21, and by the indirect immunofluorescent antibody test on ookinete bloodfilms. Immunized mice were infected with P. berghei and transmission to Anopheles stephensi mosquitoes determined by both the intensity and prevalence of oocyst infections. Compared with a control group immunized with MBP alone the maximum blockade of oocyst intensity was 66% in the mice immunized with recombinant MBP-Pbs21. Over nine experiments blockade averaged only 33%. By comparison with native Pbs21 protein, which usually induces > or = 90% blockade, our data suggests the recombinant protein produced in this bacterial system is a less effective immunogen despite expressing epitopes recognized by known transmission-blocking monoclonal antibodies.

Structure and expression of a post-transcriptionally regulated malaria gene encoding a surface protein from the sexual stages of Plasmodium berghei.

The sexual stage-specific protein Pbs21 of the rodent malaria parasite Plasmodium berghei, expressed on the surface of zygotes and ookinetes, has been shown to induce an effective and long-lasting transmission blocking immunity. The gene encoding Pbs21 was cloned by screening a cDNA library prepared from enriched zygotes and ookinetes using the monoclonal antibody 13.1.15, which is capable of blocking subsequent parasite sexual development in the mosquito vector. The Pbs21 gene encoded a protein of 213 amino acids which contained a putative amino-terminal signal sequence and a putative carboxy-terminal hydrophobic membrane anchor. The amino-acid sequence was characterised by a large number of cysteine residues which were organized into 4 epidermal growth factor-like domains. The spacing of the cysteine residues was highly conserved when compared to the 25-kDa ookinete proteins of Plasmodium falciparum (Pfs25), Plasmodium reichenowi (Prs25) and Plasmodium gallinaceum (Pgs25) which were approximately 45%, 45% and 40% homologous to Pbs21 respectively. The gene is located on chromosome 5 and cross-hybridizes to a similarly defined gene unit in the other rodent malaria species Plasmodium chabaudi, Plasmodium vinckei and Plasmodium yoelii. The gene is internally disposed and not in the subtelomeric region of chromosome 5. The gene is transcribed in a stage-specific manner giving rise to an abundant 1.5-kb transcript. This mRNA is synthesised in the precursor cells to female gametes (gametocytes) however the protein is observed only after activation of the gametes, suggesting that translation of the mRNA is controlled by a post-transcriptional process. The Pbs21 gene and the P. berghei parasite system provide an excellent vehicle for the study of stage-specific transcriptional and post-transcriptional control in malaria.