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.     

Comparison of Anopheles gambiae and Culex pipiens acetycholinesterase 1 biochemical properties

Selection of insensitive acetycholinesterase 1 (AChE1) has occurred in several mosquito species controlled with carbamate (CX) and organophosphate (OP) insecticides. In case of pyrethroid resistance, these insecticides represent an alternative for disease vector control program. Their heavy use in agriculture has selected resistant populations of Anopheles gambiae in West Africa. The evolution of resistance has to be studied to prevent, or at least slow down, the spread of resistant mosquito in wild populations. An. gambiae shares the same resistance mechanism to CX and OP insecticides as Culex pipiens, which was attributed to the G119S substitution in the AChE1 enzyme. By comparing resistant AChE1 from both species, we show here that similar resistance levels are obtained toward 10 insecticides of both classes. Moreover, similar AChE1 activity levels are recorded between either susceptible or resistant mosquitoes of both species. Enzymes belonging to both species seem thus to share identical properties. Consequently, we hypothesize that fitness cost associated with AChE1 insensitivity in C. pipiens mosquitoes should be similar in An. gambiae and thus be used in strategies to control resistant populations where malaria is prevalent.     

A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila

Acetylcholinesterase (AChE) is the target of two major insecticide families, organophosphates (OPs) and carbamates. AChE insensitivity is a frequent resistance mechanism in insects and responsible mutations in the ace gene were identified in two Diptera, Drosophila melanogaster and Musca domestica. However, for other insects, the ace gene cloned by homology with Drosophila does not code for the insensitive AChE in resistant individuals, indicating the existence of a second ace locus. We identified two AChE loci in the genome of Anopheles gambiae, one (ace-1) being a new locus and the other (ace-2) being homologous to the gene previously described in Drosophila. The gene ace-1 has no obvious homologue in the Drosophila genome and was found in 15 mosquito species investigated. In An. gambiae, ace-1 and ace-2 display 53% similarity at the amino acid level and an overall phylogeny indicates that they probably diverged before the differentiation of insects. Thus, both genes are likely to be present in the majority of insects and the absence of ace-1 in Drosophila is probably due to a secondary loss. In one mosquito (Culex pipiens), ace-1 was found to be tightly linked with insecticide resistance and probably encodes the AChE OP target. These results have important implications for the design of new insecticides, as the target AChE is thus encoded by distinct genes in different insect groups, even within the Diptera: ace-2 in at least the Drosophilidae and Muscidae and ace-1 in at least the Culicidae. Evolutionary scenarios leading to such a peculiar situation are discussed.     

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.     

Resistance to carbosulfan in Anopheles gambiae from Ivory Coast,based on reduced sensitivity of acetylcholinesterase

Resistance to carbosulfan, a carbamate insecticide, was detected in field populations of the malaria vector mosquito Anopheles gambiae Giles (Diptera: Culicidae) from two ecologically contrasted localities near Bouaké, Ivory Coast: rural M'bé with predominantly M form of An. gambiae susceptible to pyrethroids; suburban Yaokoffikro with predominantly S form of An. gambiae highly resistant to pyrethroids (96% kdr). The discriminating concentration of 0.4% carbosulfan (i.e. double the LC100) was determined from bioassays with the susceptible An. gambiae Kisumu strain. Following exposure to the diagnostic dosage (0.4% carbosulfan for 1 h), mortality rates of female An. gambiae adults (reared from larvae collected from ricefields) were 62% and 29% of those from M'bé and Yaokoffikro, respectively, 24 h post-exposure. Exposure for 3 min to netting impregnated with the operational dosage of carbosulfan 200 mg/m2 gave mortality rates of 88% of those from M'bé and only 12.2% for Yaokoffikro. In each case the control untreated mortality rate was insignificant. Biochemical assays to detect possible resistance mechanism(s) revealed the presence of insensitive AChE in populations of An. gambiae at both localities, more prevalent in the S form at Yaokoffikro than in M form at M'bé, as expected from bioassays results. Our study demonstrates the need to monitor carbamate resistance among populations of the An. gambiae complex in Africa, to determine its spread and anticipate vector control failure if these insecticides are employed.     

Cytogenetics of the Anopheles gambiae complex in Sudan, with special reference to An. arabiensis: relationships with East and West African populations

The species composition of malaria vector mosquitoes belonging to the Anopheles gambiae complex (Diptera: Culicidae) from >40 localities in Sudan, representing most ecological situations, was determined by analysis of ovarian polytene chromosomes. Of 2162 females, 93% were identified as An. arabiensis Patton and 7% were An. gambiae Giles sensu stricto. No hybrids were found between the two species. Anopheles arabiensis occurred in all but two sites, whereas An. gambiae s.s. was effectively limited to the southernmost, more humid localities. For chromosomal paracentric inversions, the degree of polymorphism was low in An. gambiae s.s. (inversions 2La, 2Rb and 2Rd), higher in An. arabiensis (inversions Xe, 2Ra, b, bc, d1, s; 3Ra, d). Anopheles gambiae samples from Sudan were all apparently panmictic, i.e. they did not show restricted gene flow such as observed among West African populations (interpreted as incipient speciation). Chromosomal inversion patterns of An. gambiae in southern Sudan showed characteristics of intergrading Savanna/Forest populations similar to those observed in comparable eco-climatic situations of West Africa. Anopheles arabiensis was polymorphic for inversion systems recorded in West Africa (2Ra, 2Rb, 2Rdl, 3Ra) and for a novel 2Rs polymorphism, overlapping with inversion systems 2Rb and 2Rd1. Samples carrying the 2Rs inversion were mostly from Khashm-el-Girba area in central-eastern Sudan. In the great majority of the samples all polymorphic inversions were found to be in Hardy-Weinberg equilibrium. Sudan populations of An. arabiensis should therefore be considered as generally panmictic. Anopheles arabiensis shows more inversion polymorphism in west than in east African populations. Sudan populations have more evident similarities with those from westwards than those from eastwards of the Great Rift Valley. The possible influence of the Rift on evolution of An. arabiensis is discussed.     

Can Anopheles gambiae be infected with Wolbachia pipientis? Insights from an in vitro system

Wolbachia pipientis are maternally inherited endosymbionts associated with cytoplasmic incompatibility, a potential mechanism to drive transgenic traits into Anopheles populations for malaria control. W. pipientis infections are common in many mosquito genera but have never been observed in any Anopheles species, leading to the hypothesis that Anopheles mosquitoes are incapable of harboring infection. We used an in vitro system to evaluate the ability of Anopheles gambiae cells to harbor diverse W. pipientis infections. We successfully established W. pipientis infections (strains wRi and wAlbB) in the immunocompetent Anopheles gambiae cell line Sua5B. Infection was confirmed by PCR, antibiotic curing, DNA sequencing, and direct observation using fluorescence in situ hybridization. The infections were maintained at high passage rates for >30 passages. Our results indicate that there is no intrinsic genetic block to W. pipientis infection in A. gambiae cells, suggesting that establishment of in vivo W. pipientis infections in Anopheles mosquitoes may be feasible.     

Contribution of Ldace1 gene to acetylcholinesterase activity in Colorado potato beetle

The Colorado potato beetle (CPB), Leptinotarsa decemlineata is an important economic pest of potato worldwide. Resistance to organophosphates and carbamates in CPB has been associated in some cases to point mutations in the acetylcholinesterase (AChE) gene Ldace2, an orthologue of Drosophila melanogaster Dmace2. In this paper we report cloning and sequencing of Ldace1, an orthologue of Anopheles gambiae Agace1 that was previously unknown in CPB. The Ldace1 coding enzyme contains all residues conserved in a functionally active AChE. Ldace1 is expressed at higher levels (between 2- and 11-fold) than Ldace2 in embryos, in the four larval instars and in adults. Specific interference of Ldace1 by means of dsRNA injection resulted in a reduction of AChE activity to an approximate 50% compared to control, whilst interference of Ldace2 reduced AChE activity to an approximate 85%. Analysis of zymograms of AChE activity after interference indicates that LdAChE1 is the enzyme predominantly responsible for the activity visualised. Interference of Ldace1 in CPB adults caused a significant increase in mortality (43%) as early as three days post-injection (p.i.), suggesting the essential role of Ldace1. Interference of Ldace2 also caused a significant increase in mortality (29%) compared to control, although at seven days p.i. The effect of the interference of Ldace1 on susceptibility to the organophosphate chlorpyrifos points out that LdAChE1 could be a main target for this insecticide. In the light of our results, studies associating resistance in CPB to mutations in Ldace2 should be reviewed, taking into consideration analysis of the Ldace1 gene.     

按蚊防御素基因家族转录模式和启动子的生物信息学分析

运用生物信息学方法分析冈比亚按（Anopheles gambiae）中防御素基因家族的表达模式和启动子保守区。在ENSEMBL数据库中搜索防御素基因家族序列,然后将基因编号输入angaGEDUCI数据库中进行分析。结果在冈比亚按蚊中存在有4个防御素基因的异构体分子,其中DEF1基因表达模式与其它3个基因存在明显差异。4个防御素基因启动子序列都存在AP-1、GATA-1、GCN4、GR、HNF-3B、TFIID6个转录因子的识别结合位点,表明这6个转录因子是冈比亚按蚊防御素基因家族表达调控所必需的。值得注意的是,DEF1基因启动子序列中存在与性别决定翦关的SOXproteins转录因子的结合位点,暗示DEF1基因的转录调控可能与性别分化有关;而在DEF2基因启动子序列中存在有热激因子（HSTF）的识别结合位点,表明热刺激会调控DEF2基因的表达。     

Evidence for late Pleistocene population expansion of the malarial mosquitoes, Anopheles arabiensis and Anopheles gambiae in Nigeria

Anopheles gambiae Giles s.s. and Anopheles arabiensis Patton (Diptera: Culicidae) are major vectors of malaria in Nigeria. We used 1115 bp of the mitochondrial COI gene to assess their population genetic structures based on samples from across Nigeria (n = 199). The mtDNA neighbour-joining tree, based on F(ST) estimates, separated An. gambiae M and S forms, except that samples of An. gambiae M from Calabar clustered with all the An. gambiae S form. Anopheles arabiensis and An. gambiae could be combined into a single star-shaped, parsimonious haplotype network, and shared three haplotypes. Haplotype diversity values were high in An. arabiensis and An. gambiae S, and intermediate in An. gambiae M; all nucleotide diversities were relatively low. Taken together, patterns of haplotype diversity, the star-like genealogy of haplotypes, five of seven significant neutrality tests, and the violation of the isolation-by-distance model indicate population expansion in An. arabiensis and An. gambiae S, but the signal was weak in An. gambiae M. Selection is supported as an important factor shaping genetic structure in An. gambiae in Nigeria. There were two geographical subdivisions in An. arabiensis: one included all southern localities and all but two central localities; the other included all northern and two central localities. Re-analysing an earlier microsatellite dataset of An. arabiensis using a Bayesian method determined that there were two distinctive clusters, northern and southern, that were fairly congruent with the mtDNA subdivisions. There was a trend towards decreasing genetic diversity in An. arabiensis from the northern savannah to the southern rainforest that corroborated previous data from microsatellites and polytene chromosomes.     

P elements are found in the genomes of nematoceran insects of the genus Anopheles

We report the identification of genomic sequences in various anopheline mosquitoes (family Culicidae: suborder Nematocera: order Diptera) showing homology to the class II, short inverted-terminal-repeat (ITR) transposable element P from Drosophila melanogaster (family Drosophilidae; suborder Brachycera: order Diptera). Anopheles gambiae appears to have at least six distinct P elements. Other anopheline species, including four additional members of the An. gambiae species complex (An. arabiensis, An. merus, An. melas and An. quadriannulatus), Anopheles stephensi (all subgenus Cellia), An. quadrimaculatus (subgenus Anopheles) and Anopheles albimanus (subgenus Nyssorhynchus) also possess P elements similar to those found in An. gambiae. Ten distinct P element types were identified in the genus Anopheles. At least two of the An. gambiae elements appears to be intact and potentially functional. Phylogenetic analysis of the anopheline P elements reveals them to belong to a distinctly different clade from the brachyceran P elements.     

Species of the Anopheles gambiae complex and chromosomal polymorphism in a rice-growing area of the Rusizi Valley (Republic of Burundi)

Chromosome studies on samples of the Anopheles gambiae complex were carried out in a village of the rice-growing area of the Rusizi Valley (Burundi). A large predominance of An. arabiensis was observed throughout the year. Anopheles gambiae was also present but represented about 4% only of the indoor resting An. gambiae s.l. The finding of human blood-fed specimens in animal shelters suggests partial exophily. Abdominal appearance observations indicated a gonotrophic dissociation during the dry season. Both An. arabiensis and An. gambiae showed a low degree of inversion polymorphism, in agreement with the general picture shown in East Africa. The most frequent karyotypes are typical of wet savannas.     

Modulation of Anopheles gambiae Epsilon glutathione transferase activity by plant natural products in vitro

Elevated glutathione transferase (GST) E2 activity is associated with DDT resistance in the mosquito Anopheles gambiae. The search for chemomodulators that inhibit the function of AgGSTE2 would enhance the insecticidal activity of DDT. Therefore, we examined the interaction of novel natural plant products with heterologously expressed An. gambiae GSTE 2 in vitro. Five of the ten compounds, epiphyllocoumarin (Tral-1), knipholone anthrone, isofuranonaphthoquinones (Mr 13/2, Mr13/4) and the polyprenylated benzophenone (GG1) were shown to be potent inhibitors of AgGSTE2 with IC(50) values of 1.5 microM, 3.5 microM, 4 microM, 4.3 microM and 4.8 microM respectively. Non-competitive inhibition was obtained for Tral 1 and GG1 with regards to GSH (K(i) of 0.24 microM and 0.14 microM respectively). Competitive inhibition for Tral1 was obtained with CDNB (K(i) = 0.4 microM) whilst GG1 produced mixed type of inhibition. The K(i) and K(i)' for GSH for Tral-1 and GG1 were 0.2 microM and 0.1 microM respectively. These results suggest that the novel natural plant products, particularly Tral-1, represent potent AgGSTE2 in vitro inhibitors.     

Localized breeding of the Anopheles gambiae complex (Diptera: Culicidae) along the River Gambia,West Africa

A study was undertaken to identify the major larval habitats of the Anopheles gambiae (Giles) complex in rural Gambia. Mosquito larvae and pupae were sampled along transects and in specific habitats in the central region of the country during the rainy seasons of 1996 and 1997. The sampling showed that the major breeding sites were located on the flooded alluvial soils bordering the river. The largest numbers of larvae were found during September, one month after the peak rains. Polymerase chain reaction analysis of specimens showed that Anopheles melas (Theobald) was the dominant species in the flooded areas (81.5%), followed by A. gambiae sensu stricto (Giles) (18.0%) and A. arabiensis (Patton) (0.5%). By sampling in specific habitats it was evident that A. arabiensis was mainly breeding in rain-fed rice fields along the edge of the alluvial soils. Anopheles melas and A. gambiae s.s. often coexisted but whereas A. melas were found in water with a salinity of up to 72% sea water (25.2 g NaCl l(-1)), A. gambiae s.s. only occurred in water with up to 30% sea water (10.5 g NaCl l(-1)). Anopheles melas larvae were found in association with plant communities dominated by sedges and grasses (Eleocharis sp., Paspalum sp., Sporobolus sp.) and sea-purslane Sesuvium portulacastrum (L.) and the presence of cattle hoof prints, whereas A. gambiae s.s. larvae mainly occurred in association with Paspalum sp. and Eleocharis sp. The study showed that even during the peak rainy season, breeding of the A. gambiae complex is almost entirely restricted to the extensive alluvial areas along the river.     

hAT element population genetics in Anopheles gambiae s.l. in Mozambique

Herves is a functional Class II transposable element in Anopheles gambiae belonging to the hAT superfamily of elements. Class II transposable elements are used as gene vectors in this species and are also being considered as genetic drive agents for spreading desirable genes through natural populations as part of an effort to control malaria transmission. In this study, Herves was investigated in populations of Anopheles gambiae s.s., Anopheles arabiensis and Anopheles merus in Mozambique over a period of 2 years. The copy number of Herves within these three species was approximately 5 copies per diploid genome and did not differ among species or between years. Based on the insertion-site occupancy-frequency distribution and existing models of transposable element dynamics, Herves appears to be transpositionally active currently or, at least recently, in all species tested. Ninety-five percent of the individuals within the populations of the three species tested contained intact elements with complete Herves transposase genes and this is consistent with the idea that these elements are currently active.     

Differential behaviour of Anopheles gambiae sensu stricto (Diptera: Culicidae) to human and cow odours in the laboratory

A dual port olfactometer was used to study the response of Anopheles gambiae Giles sensu stricto to odours of human and animal origin. Human odour consisted of human skin emanations collected on a nylon stocking, which was worn for 24 h. This was tested alone or together with 4.5% carbon dioxide, the concentration in human and cattle breath. Cattle odours consisted of cow skin emanations and/or carbon dioxide. Cow skin emanations were collected by tying a nylon stocking ('cow sock') around the hind leg of a cow for 12 h. Anopheles gambiae s.s. was consistently highly attracted by human odour, which is consistent with the high degree of anthropophily in this mosquito. Anopheles gambiae s.s. was not attracted by human or cattle equivalent volumes of carbon dioxide and this gas did not enhance the effect of human skin residues. Furthermore, A. gambiae s.s. showed a high degree of aversion to cow odour. When human odour and cow odour were tested together in the same port, mosquitoes were still highly attracted, indicating that whilst cattle odour may deter A. gambiae s.s., these mosquitoes can detect human odour in the presence of cattle odour. It was concluded that carbon dioxide plays a minor role in the host seeking behaviour of A. gambiae s.s., whilst host specific cues such as human skin residues play a major role and very effectively demonstrated anthropophilic behaviour in the laboratory.     

Design,synthesis and pharmacological evaluation of chalcone derivatives as acetylcholinesterase inhibitors

A novel series of chalcone derivatives (4a–8d) were designed, synthesized, and evaluated for the inhibition activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The log P values of the compounds were shown to range from 1.49 to 2.19, which suggested that they were possible to pass blood brain barriers in vivo. The most promising compound 4a (IC₅₀: 4.68 μmol/L) was 2-fold more potent than Rivastigmine against AChE (IC₅₀: 10.54 μmol/L) and showed a high selectivity for AChE over BuChE (ratio: 4.35). Enzyme kinetic study suggested that the inhibition mechanism of compound 4a was a mixed-type inhibition. Meanwhile, the result of molecular docking showed its potent inhibition of AChE and high selectivity for AChE over BuChE.     

Unexpected anthropophagic behaviour in Anopheles quadriannulatus

The strongly anthropophilic behaviour of Anopheles gambiae Giles sensu stricto (Diptera: Culicidae), the most important malaria vector in Africa, has been demonstrated by field and laboratory studies. Other members of the An. gambiae complex express varied degrees of anthropophily. Anopheles quadriannulatus (Theobald) species A and B are more zoophilic members of the complex and hence are considered to be of no medical importance. Olfactometer experiments with An. quadriannulatus species A have demonstrated attraction to both human and cow odour. To extend these olfactometer observations a choice experiment was conducted in an outdoor cage with a human and a calf as baits, using laboratory-reared mosquitoes. Anopheles gambiae s.s. (from Liberia) and two strains of An. quadriannulatus species A (SKUQUA from South Africa, SANGQUA from Zimbabwe), marked with different coloured fluorescent powders for identification purposes, were released simultaneously and given an equal opportunity to feed on either host. The experiment was repeated six times. Bloodmeals were identified using the precipitin technique. Anopheles gambiae s.s. showed highly anthropophagic behaviour, taking 88% of bloodmeals from the human host. In contrast, both strains of An. quadriannulatus fed with equal frequency on the human or the calf; the response to either host was not significantly different. These results confirm the olfactometer findings and demonstrate anthropophagic behaviour not previously recorded in this species. This finding has implications for prospective manipulation of host preference for genetic control purposes.     

Male accessory gland proteins induce female monogamy in anopheline mosquitoes

The role of male accessory gland (MAG) secretions in inducing refractoriness to further mating in mosquitoes (Diptera: Culicidae) was established in the late 1960s. In a set of simple experiments, MAG extract was injected intra-thoraxically into the hemocoel of virgin Aedes aegypti (L.), Culex pipiens pipiens (L.) and Anopheles quadrimaculatus Say females. This subsequently caused most females to remain unmated when exposed to males. For anophelines these findings were later challenged by a study involving intra-abdominal injections of MAG extracts into Anopheles gambiae Giles s.l. and Anopheles albimanus Wiedmann females, which failed to induce refractoriness to further mating. These findings led to controversy about the respective role of sperm and accessory gland peptides in inducing female monogamy in Anopheles and are at odds with our current understanding of the mating process in Drosophila spp. (Diptera: Drosophillidae) and other dipterans. Here we confirm the function of MAG secretions in anophelines experimentally by showing that intra-thoracic injections in Anopheles stephensi Liston and in the M and S molecular forms of An. gambiae s.s. result in the expected female monogamy. Cross-injections of MAG extracts between the M and S molecular forms of An. gambiae , two cryptic taxa within An. gambiae s.s. which are thought to be undergoing incipient speciation, also elicited effective refractoriness, suggesting that the two sub-taxa have not diverged with regard to sex peptides responsible for female monogamy. Importantly, this also suggests that the rare cases of re-mating following cross-mating observed in this species may not be a form of reproductive barrier between molecular forms.     

尼日利亚南部阿贝奥库塔地区冈比亚按蚊复合体的形态特征研究（双翅目：蚊科）

对采自尼日利亚南部城市阿贝奥库塔冈比亚按蚊复合体Anopheles gambiae complex的形态特征进行了研究。依据2005年8月至2006年7月灯诱捕获的364个冈比亚按蚊复合体标本,分别对它们的触角、翅、喙、前足、中足和后足6个部位的长度进行了测量,对月平均值进行回归分析,同时利用差异系数（co-efficient of difference,CD）进行近缘分析。分析显示,各特征的长度平均值雨季大于旱季,但是回归分析表明长度变化与季节不显著相关（P>0.05）。差异系数分析结果表明,仅触角长度和翅长显示此复合体为两个不同的种群(CD>1.28),而其他特征值表明为同一种群。因此,该研究结果提示触角长度及翅长对冈比亚按蚊复合体近缘种的区分有重要参考价值。