Transcriptomic analysis of resistance and short-term induction response to pyrethroids, in Anopheles coluzzii legs.

BACKGROUND: Insecticide-treated bed nets and indoor residual spraying comprise the major control measures against Anopheles gambiae sl, the dominant vector in sub-Saharan Africa. The primary site of contact with insecticide is through the mosquitoes' legs, which represents the first barrier insecticides have to bypass to reach their neuronal targets. Proteomic changes and leg cuticle modifications have been associated with insecticide resistance that may reduce the rate of penetration of insecticides. Here, we performed a multiple transcriptomic analyses focusing on An. coluzzii legs. RESULTS: Firstly, leg-specific enrichment analysis identified 359 genes including the pyrethroid-binder SAP2 and 2 other chemosensory proteins, along with 4 ABCG transporters previously shown to be leg enriched. Enrichment of gene families included those involved in detecting chemical stimuli, including gustatory and ionotropic receptors and genes implicated in hydrocarbon-synthesis. Subsequently, we compared transcript expression in the legs of a highly resistant strain (VK7-HR) to both a strain with very similar genetic background which has reverted to susceptibility after several generations without insecticide pressure (VK7-LR) and a lab susceptible population (NG). Two hundred thirty-two differentially expressed genes (73 up-regulated and 159 down-regulated) were identified in the resistant strain when compared to the two susceptible counterparts, indicating an over-expression of phase I detoxification enzymes and cuticular proteins, with decrease in hormone-related metabolic processes in legs from the insecticide resistant population. Finally, we analysed the short-term effect of pyrethroid exposure on An. coluzzii legs, comparing legs of 1 h-deltamethrin-exposed An. coluzzii (VK7-IN) to those of unexposed mosquitoes (VK7-HR) and identified 348 up-regulated genes including those encoding for GPCRs, ABC transporters, odorant-binding proteins and members of the divergent salivary gland protein family. CONCLUSIONS: The data on An. coluzzii leg-specific transcriptome provides valuable insights into the first line of defense in pyrethroid resistant and short-term deltamethrin-exposed mosquitoes. Our results suggest that xenobiotic detoxification is likely occurring in legs, while the enrichment of sensory proteins, ABCG transporters and cuticular genes is also evident. Constitutive resistance is primarily associated with elevated levels of detoxification and cuticular genes, while short-term insecticide-induced tolerance is linked with overexpression of transporters, GPCRs and GPCR-related genes, sensory/binding and salivary gland proteins.

Efficient genome editing in the olive fruit fly, Bactrocera oleae.

The olive fruit fly, Bactrocera oleae, causes great damage to the quality and quantity of olive production worldwide. Pest management approaches have proved difficult for a variety of reasons, a fact that has brought about a need for alternative tools and approaches. Here we report for the first time in B. oleae the development of the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene editing tool, using the well-known eye colour marker gene scarlet. Two synthetic guide RNAs targeting the coding region of the scarlet gene were synthesized and shown to work efficiently in vitro. These reagents were then microinjected along with purified Cas9 protein into early-stage embryos. Successful CRISPR-induced mutations of both copies of the scarlet gene showed a striking yellow eye phenotype, indicative of gene disruption. Multiple successful CRISPR events were confirmed by PCR and sequencing. The establishment of an efficient CRISPR-based gene editing tool in B. oleae will enable the study of critical molecular mechanisms in olive fruit fly biology and physiology, including the analysis of insecticide resistance mechanisms and the discovery of novel insecticide targets, as well as facilitate the development of novel biotechnology-based pest control strategies.

The Anopheles gambiae ATP-binding cassette transporter family: phylogenetic analysis and tissue localization provide clues on function and role in insecticide resistance.

The role of ATP-binding cassette (ABC) transporters in conferring insecticide resistance has received much attention recently. Here we identify ABC transporters differentially expressed in insecticide-resistant populations of the malaria vector, Anopheles gambiae. Although we found little evidence that the orthologues of the multidrug resistance proteins described in other species are associated with resistance in An. gambiae we did identify a subset of ABC proteins consistently differentially expressed in pyrethroid-resistant populations from across Africa. We present information on the phylogenetic relationship, primary sites of expression and potential role of ABC transporters in mediating the mosquito's response to insecticides. Furthermore we demonstrate that a paralogous group of eight ABCG transporters, clustered on chromosome 3R, are highly enriched in the legs of An. gambiae mosquitoes, consistent with a proposed role for this ABC subfamily in transport of lipids to the outer surface of the cuticle. Finally, antibodies raised against one of the most highly expressed ABC transporters in adult females, ABCG7 (AGAP009850), localized this transporter to the pericardial cells. These data will help prioritize members of this gene family for further localization and functional validation studies to identify the in vivo function of these transporters in the mosquito and determine whether elevated expression of members of this family contribute to insecticide resistance.

Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by the epiphytic yeasts Rhodotorula glutinis and Rhodotorula rubra.

The possible involvement of the epiphytic yeasts Rhodotorula glutinis and Rhodotorula rubra in the biodegradation of the insecticide chlorpyrifos and its metabolite 3,5,6-trichloro-2-pyridinol (TCP), in pure cultures and in plant surfaces (tomato fruits) was investigated. Higher biodegradation rates were observed as the concentration of chlorpyrifos and the inoculum of the microorganisms were increased, while the yeasts proved to be more active at 25 and 15 °C. The presence of glucose in the mineral nutrient medium, as an extra source of carbon, delayed the biodegradation by Rhodotorula glutinis, while Rhodotorula rubra proved to be more active. The detection and quantification of the parent compound and TCP was successfully achieved using a LC/MS/MS chromatographic system. The in vitro enzymatic assays applied suggested that esterases may be involved in the biodegradation of chlorpyrifos, a fact that was further enhanced after the addition of the synergists triphenyl phosphate, diethyl maleate and piperonyl butoxide in the biodegradation trials. The decrease of chlorpyrifos residues on tomato fruits confirmed the corresponding on pure cultures, resulting in the suggestion that the yeasts R. glutinis and R. rubra can possibly be used successfully for the removal or detoxification of chlorpyrifos residues on tomatoes.

Identification of a point mutation in the ace1 gene of Therioaphis trifolli maculata and detection of insecticide resistance by a diagnostic PCR-RFLP assay.

Aphids are important agricultural pests worldwide. Their control is largely based on chemical insecticides. One species that shows important invasive abilities and host-plant-related differences is Therioaphis trifolii (Monell) (Hemiptera: Aphididae). T. trifolii maculata, also known as spotted alfalfa aphid (SAA), can be very injurious to alfalfa crops in certain regions, such as in Saudi Arabia for effective control it is essential to diagnose and monitor the resistance mechanisms in the SAA populations. In the present study, we analysed acetylcholinesterase (ace) target site insensitivity mechanisms. A 650 bp length DNA containing the putative acetylcholinesterase (ace1) precursor was obtained and compared with other Hemipteran species. The sequences of many individual aphids collected from alfalfa crops in Saudi Arabia were analysed for the presence of resistance mutations: no resistance mutations were found at the resistance mutation loci 302; however, the presence of a serine-phenylalanine substitution (S431F) was identified in one individual. The S431F substitution, has been shown to confer significant levels of both organophosphate and carbamate resistance in other aphid species, and is now found for the first time in T. trifolii. We subsequently developed a simple polymerase chain reaction-restriction fragment length polymorphism assays for the S431F mutation, using a TaqI restriction site destroyed by the S431F mutation. The novel diagnostic assay may support the implementation of Insecticide Resistance Management strategies, for the control of SAA in alfalfa crops in the Kingdom of Saudi Arabia, and other countries worldwide.

Monitoring Aedes populations for arboviruses, Wolbachia, insecticide resistance and its mechanisms in various agroecosystems in Benin.

Aedes mosquitoes are the main vectors of arboviruses in Benin. Cases of dengue have been reported in Benin with all four serotypes of the virus actively circulating in this region. Some agricultural settings are known to harbor Aedes vectors responsible for the transmission of arboviruses. The massive use of certain insecticides in agricultural settings has probably contributed to insecticide resistance in these vectors. In Benin, the susceptibility of arbovirus vectors to insecticides is poorly studied. In addition, the distribution of Wolbachia spp., which is used against some arboviruses is unknown. Moreover, there is limited information regarding the vectors responsible for the transmission of arboviruses in Benin. This present study monitored the species composition, arboviruses, and Wolbachia symbiont status, as well as the phenotypic and molecular insecticide resistance profile of Aedes populations from three agroecosystems in Benin. Aedes species identification was performed morphologically and confirmed using qPCR. (RT)-qPCR assay was applied for monitoring the presence of DENV, CHIKV, ZIKV, and WNV pathogens as well as for naturally occurring Wolbachia symbionts. Insecticide resistance was assessed phenotypically, by permethrin (0.75%) exposure of Adults (F0) using World Health Organization (WHO) bioassay protocols, and at the molecular level, using TaqMan (RT)-qPCR assays for assessing knock-down resistance (kdr) mutations (F1534C, V1016G/I, and S989P) and the expression levels of eight detoxification genes (P450s from the CYP9 and CYP6 families, carboxylesterases and glutathione-S-transferases). Aedes aegypti (Ae. aegypti) mosquitoes were the most abundant (93.9%) in the three agroecosystems studied, followed by Aedes albopictus (Ae. albopictus) mosquitoes (6.1%). No arboviruses were detected in the study's mosquito populations. Naturally occurring Wolbachia symbionts were present in 7 pools out of 15 pools tested. This could influence the effectiveness of vector control strategies based on exogenously introduced Wolbachia, all present in the three agroecosystems. Full susceptibility to permethrin was observed in all tested populations of Ae. albopictus. On the contrary, Ae. aegypti were found to be resistant in all three agroecosystem sites except for banana plantation sites, where full susceptibility was observed. Molecular analysis revealed that individual target site resistance kdr mutations F1534C and V1016G/I were detected in most Ae. aegypti populations. Additionally, double mutant (F1534C + V1016G/I) mosquitoes were found in some populations, and in one case, triple mutant (F1534C + V1016G/I + S989P) mosquitoes were detected. Metabolic resistance, as reflected by overexpression of three P450 genes (CYP6BB2, CYP9J26, and CYP9J32), was also detected in Ae. aegypti mosquitoes. Our study provides information that could be used to strategize future vector control strategies and highlights the importance of continuing vector surveillance. Future studies should assess the effect of piperonyl butoxide (PBO) on metabolic resistance and identify the different strains of Wolbachia spp., to choose the best vector control strategies in Benin.

Transcription profiling of eleven cytochrome P450s potentially involved in xenobiotic metabolism in the mosquito Aedes aegypti.

Transcription profiles of 11 Aedes aegypti P450 genes from CYP6 and CYP9 subfamilies potentially involved in xenobiotic metabolism were investigated. Many genes were preferentially transcribed in tissues classically involved in xenobiotic metabolism including midgut and Malpighian tubules. Life-stage transcription profiling revealed important variations amongst larvae, pupae, and adult males and females. Exposure of mosquito larvae to sub-lethal doses of three xenobiotics induced the transcription of several genes with an induction peak after 48 to 72 h exposure. Several CYP genes were also induced by oxidative stress and one gene strongly responded to 20-hydroxyecdysone. Overall, this study revealed that these P450s show different transcription profiles according to xenobiotic exposures, life stages or sex. Their putative chemoprotective functions are discussed.

Identification of pyrethroid resistance associated mutations in the para sodium channel of the two-spotted spider mite Tetranychus urticae (Acari: Tetranychidae).

We investigated pyrethroid resistance mechanisms in Tetranychus urticae strains from Greece. Combined bioassay, biochemical and synergistic data indicated that although P450 mono-oxygenase activities were associated with the trait, target site insensitivity was the major resistance component. A 3.3 kb cDNA fragment of the T. urticae para sodium channel gene encompassing segment 4 of domain II to segment 6 of domain IV was obtained by a degenerate PCR strategy. The T. urticae sequence showed highest identity (56%) to the scabies mite, Sarcoptes scabiei, and was phylogenetically classified within the divergent group of Arachnida. Comparison of resistant and susceptible strains identified the point mutation F1538I in segment 6 of domain III, which is known to confer strong resistance to pyrethroids, along with a second mutation (A1215D) in the intracellular linker connecting domains II and III with an unknown role. Three transcripts were identified corresponding to the k and l alternative exons. The mode of inheritance of resistance was confirmed as incompletely recessive, which is consistent with a target site mechanism for pyrethroids.

Focal distribution of diflubenzuron resistance mutations in Culex pipiens mosquitoes from Northern Italy.

Insecticide resistance is a major threat for vector control and prevention of mosquito borne diseases. In the Culex pipiens mosquitoes, resistance against diflubenzuron (DFB) was firstly detected in Ravenna (Emilia-Romagna region, Northern Italy), in 2015. The resistant phenotypes were associated with two mutations, I1043 M and I1043 L, at the amino acid 1043 of the chitin synthase gene. In this study, we monitored the presence, frequency and geographical distribution of the DFB resistant mutations in Cx. pipiens populations from Northern Italy, and in populations from Greece and France. In the Emilia-Romagna region, the resistant mutations were detected in 20 out of the 30 populations analysed, reaching allelic frequencies over 70%. The presence and distribution of the resistance mutations was highly focal, with a clear pattern of increasing resistant allelic frequencies moving from the Western towards the Eastern provinces of Emilia-Romagna. Contrary to Italy, DFB resistant alleles were not detected in the Cx. pipiens mosquitoes sampled from Greece and France. Following statistical, literature and bibliographical database analyses on the history of DFB insecticide use in the study areas, we suggest that the selection pressures from the intense agricultural DFB applications occurring throughout the' 80-'90 s against orchard pests, followed, from 2000s onwards by mosquito control DFB applications, may account for the high mutation frequencies observed in the Cx. pipiens populations of the Eastern provinces of Emilia-Romagna. The findings are of major concern for public health in Italy and Europe, as DFB remains a very important insecticide used for controlling arbovirus mosquito vectors, where alternative larvicides are extremely limited.

Characterization of inhibitors and substrates of Anopheles gambiae CYP6Z2.

Three CYP6Z genes are linked to a major pyrethroid resistance locus in the mosquito Anopheles gambiae. We have expressed CYP6Z2 in Escherichia coli and produced a structural model in order to examine its role in detoxification. E. coli membranes co-expressing CYP6Z2 and An. gambiae P450 reductase (AgCPR) catalysed the dealkylation of benzyloxyresorufin with kinetic parameters K(m) = 0.13 microM; K(cat) = 1.5 min(-1). The IC(50) values of a wide range of compounds were measured. Pyrethroids cypermethrin and permethrin produced low IC(50) values, but were not metabolized. Plant flavanoids were the most potent inhibitors. Several compounds were shown to be substrates, suggesting that CYP6Z2 has broad substrate specificity and plays an important chemo-protective role during the herbivorous phase of the life-cycle.

Functional characterization of the Tetranychus urticae CYP392A11, a cytochrome P450 that hydroxylates the METI acaricides cyenopyrafen and fenpyroximate.

Cyenopyrafen is a Mitochondrial Electron Transport Inhibitor (METI) acaricide with a novel mode of action at complex II, which has been recently developed for the control of the spider mite Tetranychus urticae, a pest of eminent importance globally. However, some populations of T. urticae are cross-resistant to this molecule, and cyenopyrafen resistance can be readily selected in the lab. The cytochrome P450s genes CYP392A11 and CYP392A12 have been strongly associated with the phenotype. We expressed the CYP392A11 and the CYP392A12 genes with T. urticae cytochrome P450 reductase (CPR) in Escherichia coli. CYP392A12 was expressed predominately as an inactive form, witnessed by a peak at P420, despite optimization efforts on expression conditions. However, expression of CYP392A11 produced a functional enzyme, with high activity and preference for the substrates Luciferin-ME EGE and ethoxycoumarin. CYP392A11 catalyses the conversion of cyenopyrafen to a hydroxylated analogue (kcat = 2.37 pmol/min/pmol P450), as well as the hydroxylation of fenpyroximate (kcat = 1.85 pmol/min/pmol P450). In addition, transgenic expression of CYP392A11 in Drosophila melanogaster, in conjunction with TuCPR, confers significant levels of fenpyroximate resistance. The overexpression of CYP392A11 in multi-resistant T. urticae strains, not previously exposed to cyenopyrafen, which had been indicated by microarray studies, was confirmed by qPCR, and it was correlated with significant levels of cyenopyrafen and fenpyroximate cross-resistance. The implications of our findings for insecticide resistance management strategies are discussed.

Global distribution and origin of target site insecticide resistance mutations in Tetranychus urticae.

The control of Tetranychus urticae, a worldwide agricultural pest, is largely dependent on pesticides. However, their efficacy is often compromised by the development of resistance. Recent molecular studies identified a number of target site resistance mutations, such as G119S, A201S, T280A, G328A, F331W in the acetylcholinesterase gene, L1024V, A1215D, F1538I in the voltage-gated sodium channel gene, G314D and G326E in glutamate-gated chloride channel genes, G126S, I136T, S141F, D161G, P262T in the cytochrome b and the I1017F in the chitin synthase 1 gene. We examined their distribution, by sequencing the relevant gene fragments in a large number of T. urticae collections from a wide geographic range. Our study revealed that most of the resistance mutations are spread worldwide, with remarkably variable frequencies. Furthermore, we analyzed the variability of the ace locus, which has been subjected to longer periods of selection pressure historically, to investigate the evolutionary origin of ace resistant alleles and determine whether they resulted from single or multiple mutation events. By sequencing a 1540 bp ace fragment, encompassing the resistance mutations and downstream introns in 139 T. urticae individuals from 27 countries, we identified 6 susceptible and 31 resistant alleles which have arisen from at least three independent mutation events. The frequency and distribution of these ace haplotypes varied geographically, suggesting an interplay between different mutational events, gene flow and local selection.

Abamectin is metabolized by CYP392A16, a cytochrome P450 associated with high levels of acaricide resistance in Tetranychus urticae.

Abamectin is one of the most important insecticides worldwide. It is used against major agricultural pests and insects of public health importance, as well as against endoparasites in animal health. Abamectin has been used successfully for the control of the spider mite Tetranychus urticae, a major agricultural pest with global distribution, an extremely diverse host range, and a remarkable ability to develop resistance against insecticides including abamectin. Target site resistance mutations may explain a large part of resistance, although genetic evidence and transcriptomic data indicated that additional mechanisms may also be implicated in the abamectin resistant phenotype. To investigate a functional link between cytochrome P450-mediated metabolism and abamectin resistance, we recombinantly expressed three cytochrome P450s (CYP392A16, CYP392D8 and CYP392D10) that have been associated with high levels of abamectin resistance in a resistant T. urticae strain isolated from Greece. CYP392A16 was expressed predominately in its P450 form however, both CYP392D8 and CYP392D10 were expressed predominately as P420, despite optimization efforts on expression conditions. CYP392A16 catalyses the hydroxylation of abamectin (Kcat=0.54 pmol/min/pmol P450; Km=45.9 µM), resulting in a substantially less toxic compound as confirmed by bioassays with the partially purified metabolite. However, CYP392A16 did not metabolize hexythiazox, clofentezine and bifenthrin, active ingredients that also showed reduced toxicity in the abamectin resistant strain. Among a number of fluorescent and luminescent substrates screened, Luciferin-ME EGE was preferentially metabolized by CYP392A16, and it may be a potential diagnostic probe for metabolic resistance detection and monitoring.

Acoustic detection of DNA conformation in genetic assays combined with PCR.

Application of PCR to multiplexing assays is not trivial; it requires multiple fluorescent labels for amplicon detection and sophisticated software for data interpretation. Alternative PCR-free methods exploiting new concepts in nanotechnology exhibit high sensitivities but require multiple labeling and/or amplification steps. Here, we propose to simplify the problem of simultaneous analysis of multiple targets in genetic assays by detecting directly the conformation, rather than mass, of target amplicons produced in the same PCR reaction. The new methodology exploits acoustic wave devices which are shown to be able to characterize in a fully quantitative manner multiple double stranded DNAs of various lengths. The generic nature of the combined acoustic/PCR platform is shown using real samples and, specifically, during the detection of SNP genotyping in Anopheles gambiae and gene expression quantification in treated mice. The method possesses significant advantages to TaqMan assay and real-time PCR regarding multiplexing capability, speed, simplicity and cost.

A horizontally transferred cyanase gene in the spider mite Tetranychus urticae is involved in cyanate metabolism and is differentially expressed upon host plant change.

The genome of the phytophagous two-spotted spider mite Tetranychus urticae was recently sequenced, representing the first complete chelicerate genome, but also the first genome of a highly polyphagous agricultural pest. Genome analysis revealed the presence of an unexpected high number of cases of putative horizontal gene transfers, including a gene that encodes a cyanase or cyanate lyase. In this study we show by recombinant expression that the T. urticae cyanase remained functionally active after horizontal gene transfer and has a high affinity for cyanate. Cyanases were also detected in other plant parasitic spider mites species such as Tetranychus evansi and Panonychus citri, suggesting that an ancient gene transfer occurred before the diversification within the Tetranychidae family. To investigate the potential role of cyanase in the evolution of plant parasitic spider mites, we studied cyanase expression patterns in T. urticae in relation to host plant range and cyanogenesis, a common plant defense mechanism. Spider mites can alter cyanase expression levels after transfer to several new host plants, including the cyanogenic Phaseolus lunatus. However, the role of cyanase is probably not restricted to cyanide response, but likely to the plant nutritional quality as a whole. We finally discuss potential interactions between cyanase activity and pyrimidine and amino acid synthesis.

The cys-loop ligand-gated ion channel gene family of Tetranychus urticae: implications for acaricide toxicology and a novel mutation associated with abamectin resistance.

The cys-loop ligand-gated ion channel (cysLGIC) super family of Tetranychus urticae, the two-spotted spider mite, represents the largest arthropod cysLGIC super family described to date and the first characterised one within the group of chelicerates. Genome annotation, phylogenetic analysis and comparison of the cysLGIC subunits with their counterparts in insects reveals that the T. urticae genome encodes for a high number of glutamate- and histamine-gated chloride channel genes (GluCl and HisCl) compared to insects. Three orthologues of the insect γ-aminobutyric acid (GABA)-gated chloride channel gene Rdl were detected. Other cysLGIC groups, such as the nAChR subunits, are more conserved and have clear insect orthologues. Members of cysLGIC family mediate endogenous chemical neurotransmission and they are prime targets of insecticides. Implications for toxicology associated with the identity and specific features of T. urticae family members are discussed. We further reveal the accumulation of known and novel mutations in different GluCl channel subunits (Tu_GluCl1 and Tu_GluCl3) associated with abamectin resistance in T. urticae, and provide genetic evidence for their causality. Our study provides useful toxicological insights for the exploration of the T. urticae cysLGIC subunits as putative molecular targets for current and future chemical control strategies.

Affordable assays for genotyping single nucleotide polymorphisms in insects.

Insect genome projects and DNA sequence databases are providing unprecedented amounts of information about variation at specific nucleotides in protein- and RNA-coding genes. Single nucleotide polymorphisms (SNPs) are abundant in all insect species so far examined and are proving useful in population genetics, linkage mapping and marker-assisted selection. A number of studies has already identified SNPs associated with insecticide resistance, especially mutations conferring reduced target site sensitivity. Unfortunately, most modern, high-throughput, automated SNP detection technologies are expensive or require the use of expensive equipment and are therefore not accessible to laboratories on a limited budget or to our colleagues in developing countries. In this review, we provide a chronological and comprehensive list of all SNP methods. We emphasize and explain those techniques in which genotypes can be identified by eye or that only require agarose gel electrophoresis. We provide examples where these techniques have or are currently being applied to insects.

Transcriptional analysis of insecticide resistance in Anopheles stephensi using cross-species microarray hybridization.

A large scale microarray (20k MMC1) from the African malaria vector Anopheles gambiae was used to monitor gene expression in insecticide resistant and susceptible strains of the Asian mosquito Anopheles stephensi. Heterologous hybridization at slightly reduced stringency yielded approximately 7000 significant signals. Thirty-six putative genes were differentially transcribed between the pyrethroid-resistant (DUB-R) and the susceptible (BEECH) strains. The expression profiles of selected transcripts were verified by real-time PCR. A gene putatively involved in the thickening of the adult cuticle showed the most striking up-regulation in DUB-R. A more specialized microarray containing 231 An. gambiae genes putatively involved in insecticide detoxification was used to further analyse classical insecticide resistance genes. Three glutathione S-transferase (GST) transcripts, one esterase and a cytochrome P450 were up-regulated in the resistant strain, while two peroxidases were down-regulated.

The annexin gene family in the malaria mosquito Anopheles gambiae.

Annexins belong to a class of proteins that are known to bind to, and hold together structures such as membranes. Interestingly, Anopheles gambiae (and Drosophila melanogaster) annexins bind Plasmodium ookinetes in vitro. In the malaria mosquito three genes in two cytogenetic loci on chromosome arm 2R encode annexin homologues; their expression, monitored by quantitative real-time PCR during mosquito development, as well as in various tissues, revealed little fluctuation in patterns of expression during all life stages. A different mode of transcription was observed for the three genes in the midgut in relation to the uptake of a blood meal. Immunohistochemical staining of midguts and ovaries with polyclonal anti-annexin sera reveals that the Anopheles polypeptides are present in the epithelial cells of both tissues and associated with the plasma membrane.

Gene expression in insecticide resistant and susceptible Anopheles gambiae strains constitutively or after insecticide exposure.

A microarray containing approximately 20 000 expressed sequence tags (ESTs; 11 760 unique EST clusters) from the malaria vector, Anopheles gambiae, was used to monitor differences in global gene expression in two insecticide resistant and one susceptible strains. Statistical analysis identified 77 ESTs that were differentially transcribed among the three strains. These include the cytochrome P450 CYP314A1, over-transcribed in the DDT resistant ZAN/U strain, and many genes that belong to families not usually associated with insecticide resistance, such as peptidases, sodium/calcium exchangers and genes implicated in lipid and carbohydrate metabolism. Short-term (6 and 10 h) effects of exposure of the pyrethroid resistant RSP strain to permethrin were also detected. Several genes belonging to enzyme families already implicated in insecticide or xenobiotic detoxification were induced, including the carboxylesterase COEAE2F gene and members of the UDP-glucuronosyl transferase and nitrilase families.