Distribution of genetic diversity in relation to chromosomal inversions in the malaria mosquito Anopheles gambiae

The epidemiology of malaria in Africa is complicated by the fact that its principal vector, the mosquito Anopheles gambiae, constitutes a complex of six sibling species. Each species is characterized by a unique array of paracentric inversions, as deduced by karyotypic analysis. In addition, most of the species carry a number of polymorphic inversions. In order to develop an understanding of the evolutionary histories of different parts of the genome, we compared the genetic variation of areas inside and outside inversions in two distinct inversion karyotypes of A. gambiae. Thirty-five cDNA clones were mapped on the five arms of the A. gambiae chromosomes with divisional probes. Sixteen of these clones, localized both inside and outside inversions of chromosome 2, were used as probes in order to determine the nucleotide diversity of different parts of the genome in the two inversion karyotypes. We observed that the sequence diversity inside the inversion is more than threefold lower than in areas outside the inversion and that the degree of divergence increases gradually at loci at increasing distance from the inversion. To interpret the data we present a selectionist and a stochastic model, both of which point to a relatively recent origin of the studied inversion and may suggest differences between the evolutionary history of inversions in Anopheles and Drosophila species.Correspondence to: K.D. Mathiopoulos     

Towards a species-selective acetylcholinesterase inhibitor to control the mosquito vector of malaria, Anopheles gambiae

Anopheles gambiae is the major mosquito vector of malaria in sub-Saharan Africa. At present, insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides are widely used in malaria-endemic regions to reduce infection; however the emergence of pyrethroid-resistant mosquitoes has significantly reduced the effectiveness of the pyrethroid ITNs. An acetylcholinesterase (AChE) inhibitor that is potent for An. gambiae but weakly potent for the human enzyme could potentially be safely deployed on a new class of ITNs. In this paper we provide a preliminary pharmacological characterization of An. gambiae AChE, discuss structural features of An. gambiae and human AChE that could lead to selective inhibition, and describe compounds with 130-fold selectivity for inhibition of An. gambiae AChE relative to human AChE.     

The defensin peptide of the malaria vector mosquito Anopheles gambiae: antimicrobial activities and expression in adult mosquitoes

A recombinant Anopheles gambiae defensin peptide was used to define the antimicrobial activity spectrum against bacteria, filamentous fungi and yeast. Results showed that most of the Gram-positive bacterial species tested were sensitive to the recombinant peptide in a range of concentrations from 0.1 to 0.75 microM. No activity was detected against Gram-negative bacteria, with the exception of some E. coli strains. Growth inhibitory activity was detected against some species of filamentous fungi. Defensin was not active against yeast. The kinetics of bactericidal and fungicidal effects were determined for Micrococcus luteus and Neurospora crassa, respectively. Differential mass spectrometry analysis was used to demonstrate induction of defensin in the hemolymph of bacteria-infected adult female mosquitoes. Native peptide levels were quantitated in both hemolymph and midgut tissues. The polytene chromosome position of the defensin locus was mapped by in situ hybridization.     

The Anopheles gambiae genome: next steps for malaria vector control

Malaria remains a major public health problem that is made worse by poor implementation of control measures, and by the spread of drug- and insecticide-resistant parasites and vectors, respectively. Availability of the Anopheles gambiae genome sequence will accelerate identification and exploitation of new target genes in this insect vector. This provides unique opportunities to improve on existing vector control tools and to generate new tools within a global partnership. However, significant capacity needs to be built for investigators in disease-endemic countries to exploit the genome data. When integrated with existing strategies, the new tools will form an effective package for selective vector control in an effort to prevent mortality and morbidity due to malaria.     

Identification of electrophysiologically-active compounds for the malaria mosquito, Anopheles gambiae, in human sweat extracts

Abstract. Human sweat samples were chemically fractionated into acid and non-acid components. The most abundant volatile compounds present in the fractions were identified by linked gas chromatography mass spectrometry. The acid fractions were found to be composed of a range of twenty aliphatic and three aromatic carboxylic acids ranging, on average, from 0.02 to 20 ig per ml of sweat sampled. Non-acid fractions were found to contain: 6-methyl-5-hepten-2-one, l-octen-3-ol, decanal, benzyl alcohol, dimethylsulphone, phenylethanol, phenol and 4-mefhylphenol, collectively amounting to 0.1 and 3 |ig per ml of sweat. The major component of sweat was found to be L-lactic acid which constituted from 1 to 5 mg/ml.
Using the intact antennae of the anthropophilic malaria vector mosquito Anopheles gambiae Giles, the peripheral olfactory activities of compounds identified in the sweat fractions were investigated by electroantennography (EAG). Short-chain saturated carboxylic acids, methanoic, ethanoic, propanoic, butanoic, pentanoic and hexanoic acids were found to elicit significantly larger EAG responses than longer chain saturated carboxylic acids from female An.gambiae. For a given dose the largest amplitude EAG response was elicited by methanoic acid. Pentanoic acid elicited larger EAG responses than either butanoic or hexanoic acids. Two non-acidic compounds, l-octen-3-ol and 4-methylphenol, were found to elicit significant dose-dependent EAG responses from female An.gambiae. 1 -Octen-3-ol elicited larger EAG responses than 4-methylphenol for a given dose, but both compounds elicited smaller EAG responses than the same dose of C_]-C₆straight-chain aliphatic carboxylic acids. The possible behavioural significance of the EAG-active compounds identified in human sweat samples is discussed.     

Mosaic: a position-effect variegation eye-color mutant in the mosquito Anopheles gambiae

The Mosaic (Mos) mutation, isolated in the F1 of 60Co-irradiated mosquitoes, confers variegated eye color to third and fourth instar larvae, pupae, and adults of the mosquito Anopheles gambiae. Mos is recessive in wild pink eye (p+) individuals, but is dominant and confers areas of wild-type pigment in mutant pink eye backgrounds. Mos is located 14.4 cM from pink eye on the X chromosome and is associated with a duplication of division 2B euchromatin that has been inserted into division 6 heterochromatin. Various combinations of Mos, pink eye alleles, and the autosomal mutation red eye were produced. In all cases, the darker pigmented regions of the eye in Mos individuals show the phenotypic interactions expected if the phenotype of those regions is due to expression of a p+ allele. Expression of Mos is suppressed by rearing larvae at 32 degrees C relative to 22 degrees C. All of these characteristics are consistent with Mos being a duplicated wild copy of the pink eye gene undergoing position-effect variegation.     

Impact of permethrin-treated bednets on malaria transmission by the Anopheles gambiae complex in The Gambia

Malaria vector mosquitoes belonging to the Anopheles gambiae complex were studied in four hamlets in The Gambia. All inhabitants were given bednets treated either with a placebo (milk) in two hamlets or with the pyrethroid insecticide permethrin (500 mg/m2) in two other hamlets. Malaria transmission occurred mainly during a few weeks of the rainy season, in September and October 1987. The indoor resting densities of mosquitoes in permethrin-treated hamlets were reduced, and we estimated over 90% reduction in biting on man by An. gambiae Giles sensu stricto in these hamlets. No mosquitoes were found under permethrin-treated bednets compared with eighty-one recovered from placebo-treated bednets. Mosquitoes exited more readily from rooms where permethrin-treated bednets were used than from rooms with placebo-treated nets. The annual mean probability that a child would receive an infective bite was estimated to be 0.09 in hamlets with insecticide-treated bednets, compared with 1.9 where placebo-treated bednets were used. Permethrin-treated bednets are therefore recommended as a means of effectively reducing the risk of exposure to malaria transmission, particularly in areas of low seasonal transmission.     

Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito,Anopheles gambiae

Abstract Chitin synthase (CHS) is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control. However, our understanding of biochemical properties of insect CHSs has been very limited. We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito, Anopheles gambiae. Our study, which represents the first time to use a nonradioactive method to assay CHS activity in an insect species, determined the optimal conditions for measuring the enzyme activity, including pH, temperature, and concentrations of the substrate uridine diphosphate N‐acetyl‐d ‐glucosamine (UDP‐GlcNAc) and Mg⁺⁺. The optimal pH was about 6.5–7.0, and the highest activity was detected at temperatures between 37°C and 44°C. Dithithreitol is required to prevent melanization of the enzyme extract. CHS activity was enhanced at low concentration of GlcNAc, but inhibited at high concentrations. Proteolytic activation of the activity is significant both in the 500 ×g supernatant and the 40 000 ×g pellet. Our study revealed only slight in vitro inhibition of A. gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration (2.5 μmol/L) examined. There was no in vitro inhibition by polyoxin D at any concentration examined. Furthermore, we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined. Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A. gambiae.     

Identification and characterization of the receptor for the Bacillus sphaericus binary toxin in the malaria vector mosquito, Anopheles gambiae

The binary toxin (Bin) from Bacillus sphaericus exhibits a highly insecticidal activity against Culex and Anopheles mosquitoes. The cytotoxicity of Bin requires an interaction with a specific receptor present on the membrane of midgut epithelial cells in larvae. A direct correlation exists between binding affinity and toxicity. The toxin binds with high affinity to its receptor in its primary target, Culex pipiens, and displays a lower affinity to the receptor in Anopheles gambiae, which is less sensitive to Bin. Although the Bin receptor has previously been identified and named Cpm1 in C. pipiens, its structure in Anopheles remains unknown. In this study, we hypothesize that the Anopheles Bin receptor is an ortholog of Cpm1. By screening the Anopheles genomic database, we identified a candidate gene (Agm3) which is expressed primarily on the surface of midgut cells in larvae and which functions as a receptor for Bin. A Cpm1-like gene is also present in the Bin-refractory species Aedes aegypti. Overall, our results indicate that the three mosquito genes examined share a very similar organization and are strongly conserved at the amino acid level, in particular in the NH(2)-terminus, a region believed to contain the ligand binding site, suggesting that relatively few amino acids residues are critical for high affinity binding of the toxin.     

Resistance management strategies in malaria vector mosquito control. Baseline data for a large-scale field trial against Anopheles albimanus in Mexico

Abstract. A high level of DDT resistance and low levels of resistance to organophosphorus, carbamate and pyrethroid insecticides were detected by discriminating dose assays in field populations of Anopheles albimanus in Chiapas, southern Mexico, prior to a large-scale resistance management project described by Hemingway et al. (1997) . Biochemical assays showed that the DDT resistance was caused by elevated levels of glutathione S-transferase (GST) activity leading to increased rates of metabolism of DDT to DDE. The numbers of individuals with elevated GST and DDT resistance were well correlated, suggesting that this is the only major DDT resistance mechanism in this population. The carbamate resistance in this population is conferred by an altered acetylcholinesterase (AChE) -based resistance mechanism. The level of resistance observed in the bioassays correlates with the frequency of individuals homozygous for the altered AChE allele. This suggests that the level of resistance conferred by this mechanism in its heterozygous state is below the level of detection by the WHO carbamate discriminating dosage bioassay. The low levels of organophosphate (OP) and pyrethroid resistance could be conferred by either the elevated esterase or monooxygenase enzymes. The esterases were elevated only with the substrate pNPA, and are unlikely to be causing broad spectrum OP resistance. The altered AChE mechanism may also be contributing to the OP but not the pyrethroid resistance. Significant differences in resistance gene frequencies were obtained from the F₁ mosquitoes resulting from adults obtained by different collection methods. This may be caused by different insecticide selection pressures on the insects immediately prior to collection, or may be an indication that the indoor- and outdoor-resting A. albimanus collections are not from a randomly mating single population. The underlying genetic variability of the populations is currently being investigated by molecular methods.     

The crystal structure of D7r4, a salivary biogenic amine-binding protein from the malaria mosquito Anopheles gambiae

The D7-related (D7r) proteins of the malaria vector Anopheles gambiae have been shown to bind the biogenic amines serotonin, norepinephrine, and histamine with high affinity. One member of the group (D7r1 or hamadarin) has also been shown to have an anticoagulant/antikinin activity. To understand the mechanistic details of its antihemostatic/anti-inflammatory effects, we have determined the crystal structure of one member of this group, D7r4, along with the structures of ligand complexes with serotonin, tryptamine, histamine, and norepinephrine. The D7 fold consists of an arrangement of eight alpha-helices stabilized by three disulfide bonds. The structure is similar to those of the arthropod odorant-binding proteins, a relationship that had been predicted based on sequence comparisons. Although odorant-binding proteins commonly have six alpha-helices, D7r4 has eight, resulting in significantly different positioning and structure of the ligand binding pocket. The pocket itself is lined by hydrophobic side chains along with polar and charged groups oriented to form hydrogen bonds with the aliphatic amino group and with groups on the aromatic portions of the ligands. These structures, along with accompanying mutagenesis studies, have allowed us to identify critical residues for biogenic amine binding and to predict which members of the large D7 protein family found in blood-feeding nematocerous Diptera will function as biogenic amine-binding proteins.