Responses of Anopheles gambiae complex mosquitoes to the use of untreated bednets in The Gambia

Population dynamics of the Anopheles gambiae complex of malaria vector mosquitoes were studied in four small hamlets in The Gambia. Bednets were used to reduce man/vector contact in two of the hamlets. High densities of An. gambiae, sensu lato, were present for only 3-8 weeks during the rainy season, depending on the position of the hamlet within the study area. The proportions of blood-fed mosquitoes caught indoors (83.0%) and existing from houses (11.6%) were lower in hamlets where bednets were used than in hamlets without (96.5% and 33.1% respectively). Fewer of the blood-fed mosquitoes had fed on man in houses where people slept under bednets (68.2%) than in those without (81.5%). However, the average number of infective bites received by children was still greater than one a year in hamlets where bednets were used. Consequently bednets are considered unlikely to be an effective malaria control measure so long as they are untreated with insecticide.     

A behavioural test of the sensitivity of a nocturnal mosquito, Anopheles gambiae, to dim white, red and infra-red light

Abstract. A behavioural test was used to determine the light sensitivity of the nocturnal mosquito Anopheles gambiae Giles s.s. to low intensities of 'white' light (tungsten filament), 'red' light (white light filtered by a darkroom safelight filter) and 'infra-red' light) of two types (white light filtered by a λ>700 nm filter, and light-emitting diodes with λ>900 nm). Mosquitoes were placed in a 20 cm diameter flight-tunnel and their 'optomotor' response to a pattern of stripes moving across their visual field (at 14.5 cm s^-1) was recorded with infra-red-sensitive video. In free-flight, with ample light, the mosquitoes controlled their flight speed and direction in relation to the stripe movement, so that the stripes always appeared to move across their visual field from front to back. They did this by flying either with the moving stripes fast enough to overtake them (19.5 ± 0.7 cm s^-1), or against them more slowly (10.3 ± 0.7 cm s^-1)- The net ground speed of the mosquitoes was thus c. 4–5 cm s^-1. This response was significant down to 10^-5 W m^-2 in 'white' light, and 10^-3 W m^-2 in 'red' light. At light intensities below threshold and in infra-red light, however, they appeared to fly at random with respect to the stripe movement. The assumption commonly made, that mosquitoes do not 'see' in red light, may thus have to be revised.     

Pyrethroid-treated bednet effects on mosquitoes of the Anopheles gambiae complex in The Gambia

The response of Anopheles gambiae complex mosquitoes to men sleeping under insecticide-impregnated or untreated bednets in six verandah trap huts was studied during the dry season in The Gambia. With this type of hut it was possible to collect live and dead indoor-resting mosquitoes and estimate the number of wild mosquitoes which entered, bloodfed on man, and exited each night. Bednets were treated with emulsions targetted to leave deposits of 25 mg/m2 lambda-cyhalothrin, or 5, 50 or 500 mg/m2 permethrin, diluted from emulsifiable concentrates (EC), or a blank formulation similar to the EC except that the permethrin was omitted; the sixth net was left untreated. Nets and sleepers were rotated between huts on different nights, the design being based on a series of Latin squares and conducted double-blind. Permethrin-impregnated bednets deterred mosquitoes from entering the huts. The degree of deterrency was proportional to the dosage of permethrin. This effect was also caused by the blank formulation and therefore attributed to other components of the formulation, rather than to the permethrin itself. The net impregnated with 500 mg permethrin per square metre gave the best individual protection, reducing mosquito bloodfeeding by 91% compared with untreated nets. However, lambda-cyhalothrin was proportionately more insecticidal than permethrin at doses of equivalent deterrency. At this stage of research, it remains conjectural whether chemical deterrency or killing of malaria vectors is better for community protection.     

DNA probes for species identification of mosquitoes in the Anopheles gambiae complex

Abstract. Identification of species within the Anopheles gambiae Giles species complex is essential for the correct evaluation of malaria vector ecology studies and control programmes. The development of DNA probes to distinguish species of the An.gambiae complex is described. Genomic libraries were prepared for four members of the An.gambiae complex. These were screened using radiolabeled DNA from different species of An. gambiae sensu lato and a number of clones selected on the basis of their species specificity. These clones could be divided into two groups, each containing homologous sequences. Sequences homologous to group 1 inserts are highly reiterated in the genomes of Anopheles arabiensis Patton and Anopheles merus Dönitz, present in low copy number in Anopheles melas Theobald, but were not detected in Anopheles gambiae sensu stricto. Studies on the organization of this sequence in the genome of An.arabiensis show that homologous sequences are male specific and interspersed within the chromatin. Sequences homologous to group 2 inserts are highly repeated in the genomes of An.merus and An.melas, but present in low copy number in An.gambiae s.s. and An.arabiensis. Group 2 homologous sequences are not sex-specific in the species tested and appear to be tandemly repeated. When used as hybridization probes, these sequences provide a sensitive means for the identification of species within the Anopheles gambiae complex.     

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.     

Human sweat and 2-oxopentanoic acid elicit a landing response from Anopheles gambiae

A wind tunnel bioassay and video to observe mosquitoes landing on heated glass cylinders were used to test sweat and some derivatives for responses of Anopheles gambiae Giles (Diptera: Culicidae), a highly anthropophilic African species of malaria vector. Filter papers impregnated with human sweat and a diethyl ether extract from the filter papers elicited significantly more landings than a water control (P<0.001). The concentration of lactic acid in the extract was determined by GLC assay, but bioassays of an equivalent dose of lactic acid (from a commercial supplier) did not elicit landings. Chemical analysis of the extract by combined GLC/mass spectrometry indicated the presence of 73 compounds, of which 40 were tentatively identified. The major components of the extract were aliphatic carboxylic acids. An artificial blend of 22 carboxylic acids did not elicit landings. Bioassays of 2-oxopentanoic acid elicited significantly more, landings (P<0.001). The possible importance of oxo-carboxylic acids for host-seeking by anthropophilic mosquitoes is discussed and their use for trapping is suggested.     

Pteridine fluorescence for age determination of Anopheles mosquitoes

The age structure of mosquito populations is of great relevance to understanding the dynamics of disease transmission and in monitoring the success of control operations. Unfortunately, the ovarian dissection methods currently available for determining the age of adult mosquitoes are technically difficult, slow and may be of limited value, because the proportion of diagnostic ovarioles in the ovary declines with age. By means of reversed-phase HPLC this study investigated the malaria vectors Anopheles gambiae and An. stephensi to see if changes in fluorescent pteridine pigments, which have been used in other insects to determine the age of field-caught individuals, may be useful for age determination in mosquitoes. Whole body fluorescence was inversely proportional to age (P < 0.001, r2 > 91%) up to 30 days postemergence, with the regression values: y = 40580-706x for An. gambiae, and y = 52896-681x for An. stephensi. In both species the main pteridines were 6-biopterin, pterin-6-carboxylic acid and an unidentified fluorescent compound. An. gambiae had only 50-70% as much fluorescence as An. stephensi, and fluorescent compounds were relatively more concentrated in the head than in the thorax (ratios 1:0.8 An. gambiae; 1:0.5 An. stephensi). The results of this laboratory study are encouraging. It seems feasible that this simpler and faster technique of fluorescence quantification could yield results of equivalent accuracy to the interpretation of ovarian dissection. A double-blind field trial comparing the accuracy of this technique to marked, released and recaptured mosquitoes is required to test the usefulness of the pteridine method in the field.     

Effects of physico-chemical treatments on hemagglutination activity of Anopheles gambiae haemolymph and midgut extract

Abstract. Anopheles gambiae midgut extracts and haemolymph possessed agglutinins, titre 1:16 to 1:256, against human red blood cells (RBCs). Subjection of both tissues to protein precipitation reagents, organic chemical and selected protease, neuraminidase and other glycosidic hydrolase treatments revealed the haemagglutinins to be protein, most likely glycoprotein, in nature - not lipoprotein, lipid, glycolipid or nucleic acid. An.gambiae agglutinins were thermo-labile >40^oC, affected by freezing and thawing treatments, and contained disulphide and hydrogen bonds on the basis of sensitivity following exposure to dithio-threitol and urea respectively. Optimum haemagglutination depended generally on slightly acid to neutral pH conditions and agglutinin activity was Ca²⁺ ion, albeit to a lesser extent Mg²⁺ ion, dependent. The midgut extract agglutinin subunit molecule had a relative molecular weight (M_r) of 65kDa whilst that of haemolymph was 40kDa.
This study presents the first report on selected physico-chemical properties, the glycoproteinaceous nature and tentative subunit M_r of mosquito midgut extract and haemolymph anti-RBC agglutinin(s).     

Movement of Anopheles gambiae s.l. malaria vectors between villages in The Gambia

ovement of mosquitoes belonging to the Anopheles gambiae complex (mixed wild populations of An.arabiensis, An.gambiae and An.melas ) between three neighbouring rural villages in The Gambia was investigated by mark-release-recapture. A total of 12,872 mosquitoes were collected in bednets, marked with a magenta fluorescent powder and released over a 15-day period in one of the villages. A further 15,507 mosquitoes were collected in exit traps, marked with a yellow powder and released over the same period. Mosquitoes were captured daily in all three villages using pyrethrum spray catches, as well as bednet and exit trap catches. The catching period extended for 6 days after the last day of release.
Of the mosquitoes released, 372 (1.3%) were recaptured 2–21 days later. Of these recaptures, 272 were caught in the release village, and 98 were caught in other villages situated 1–1.4 km away. The 'movement index' between villages was calculated as 17.2% (12.2–22.4% confidence limits) for mosquitoes released after feeding and 20.1% (14.7–25.3%) for those released unfed.
These results suggest that movement of mosquitoes between neighbouring villages in The Gambia seriously affects the entomological evaluation of pyrethroid-impregnated bednet programmes in areas where treated and untreated villages are interspersed.     

Comparison of carbon dioxide-baited trapping systems for sampling outdoor mosquito populations in Tanzania

For collecting mosquitoes (Diptera: Culicidae) the outdoor catching efficiency of four types of trapping devices baited with carbon dioxide (CO2, 300 ml/ min) was evaluated and compared in two areas of Tanzania. The types of traps employed were: the CDC miniature trap with the incandescent light bulb switched on or off; electric nets (ENT) and a Counterflow Geometry (CFG) trap. In Njage, southeast Tanzania, Anopheles gambiae Giles sensu stricto was the most abundant of the seven mosquito species obtained, comprising of 74.3% of the total number caught (n=2,171). In Muheza, north-east Tanzania, Culex quinquefasciatus Say was the predominant species (90.9%) among 1,080 caught. At both localities the CFG trap was superior to the CDC trap with light-on or light-off for sampling both An. gambiae and Cx. quinquefasciatus. Efficiency of the CFG trap and ENT were similar for sampling these species of mosquitoes (P > 0.05). However, ENT was superior to the CDC trap with light-off for collecting both species. Significantly more (P < 0.05) Cx. quinquefasciatus were obtained by the CDC trap with light-off than with light-on, especially outdoors. It is concluded that both ENT and the CFG are effective tools for sampling populations of An. gambiae and Cx. quinquefasciatus outdoors.     

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。     

Anopheles arabiensis and An. gambiae chromosomal inversion polymorphism, feeding and resting behaviour in relation to insecticide house-spraying in Tanzania

Abstract. Differential responses of the mosquitoes Anopheles arabiensis and An. gambiae sensu stricto to house-spraying with DDT or lambda-cyhalothrin were evaluated in relation to chromosomal inversion polymorphism, feeding and resting behaviour of these malaria vectors in Tanzania. Blood-fed mosquitoes from pit traps outdoors, exit traps on windows and indoor-resting catches were identified cytogenetically and the chromosomal inversion frequencies compared between samples and species. Their outdoor-resting behaviour was assessed by a mark–release–recapture experiment and by determining the proportion of freshly blood-fed individuals in exit traps. The source of bloodmeals was analysed by an ELISA method. Endophagic females of An. arabiensis were more likely than those of An. gambiae to exit from a house on the night of blood-feeding. Only in one out of three villages was there evidence that chromosomally distinct individuals within a species had different preferences for resting sites. There were indications, but not conclusive evidence, that mosquitoes caught indoors or outdoors had a tendency to return to the same type of resting site. In villages sprayed with either insecticide, the mean age of the vector populations was greatly reduced, compared with those in the unsprayed villages. An. arabiensis females exited from DDT sprayed houses after blood-feeding, whereas with lambda-cyhalothrin those exiting were mostly unfed and there was a decline in the human blood index. The excitorepellency of DDT was perceived as a disadvantage, whereas lambda-cyhalothrin apparently had more impact on malaria transmission by An. arabiensis.     

Long-term use of permethrin-impregnated nets does not increase Anopheles gambiae permethrin tolerance

Abstract. Previous use of permethrin-impregnated bednets (mosquito nets) and curtains in four Kenyan villages for one year, 1990-91, raised the permethrin LT₅₀ of Anopheles gambiae to 2.4-fold above its baseline value, designated permethrin tolerance (PT), as measured by exposure to 0.25% permethrin-impregnated papers in W.H.O. test-kits. During 1992-93, with ongoing use of permethrin-impregnated nets and curtains, PT regressed slightly compared with the contemporary susceptibility level of An. gambiae from non-intervention villages, to 1.8-fold in 1992 and only 1.6-fold in 1993. Thus the selection pressure of impregnated nets for PT in Angambiae appears to be minimal in our study villages, although the impact of permethrin was demonstrated by a significantly lower parous-rate of Angambiae females in the intervention (63–66%) than in non-intervention (79%) villages, and by reduced malaria transmission (reported elsewhere).
In a selected stock of An. gambiae from the study area, PT did not affect the susceptibility to deltamethrin, fenitrothion, propoxur or DDT. Bioassays described herein provide easy procedures for field-monitoring of mosquito susceptibility/tolerance/resistance to insecticides used for net impregnation in operational programmes.     

Behaviour and fitness of γHCH/dieldrin resistant and susceptible female Anopheles gambiae and An.stephensi mosquitoes in the absence of insecticide

The effects of gamma HCH/dieldrin resistance genes on various fitness components of mosquito larvae and adult females in the absence of insecticide were investigated in backcrossed strains of Anopheles gambiae Giles and An.stephensi Liston. Among larvae, heterozygotes (RS) developed slightly but significantly faster than homozygotes for resistance (RR) or susceptibility (SS). The lifetime fecundity of RR females in population cages was only half to two-thirds that of SS and RS females despite similar longevities; several reasons were identified: RR gravid females were less responsive to oviposition-site stimuli, their spontaneous activity--as measured in an acoustic actograph--was only half that of SS or RS females, and RR females produced fewer eggs per unit bloodmeal. When inseminated females were recorded in LD 12:12, RR were again less active than SS or RS. When the lighting was switched to a regime simulating full-moonlight, the activity pattern of SS and RS changed and they flew for longer periods. In contrast, the activity of RR females was the same in LD 12:12 as in 'moonlight'. In a test simulation of potential predation, RR mosquitoes took to flight least readily. All component tests on adult females therefore point to RR as being the least fit of the three genotypes. The behavioural tests suggest that resistance has raised the response threshold of RR females to diverse stimuli. A possible physiological mechanism underlying RR behaviour is that a change in the cyclodiene receptor on the chloride channels has increased their permeability to chloride ions, causing hyper-inhibition of the nervous system.     

New selenoproteins identified in silico from the genome of Anopheles gambiae

Selenoprotein is biosynthesized by the incorporation of selenocysteine into proteins, where the TGA codon in the open reading frame does not act as a stop signal but is translated into selenocysteine. The dual functions of TGA result in mis-annotation or lack of selenoproteins in the sequenced genomes of many species. Available computational tools fail to correctly predict selenoproteins. Thus, we developed a new method to identify selenoproteins from the genome of Anopheles gambiae computationally.Based on released genomic information, several programs were edited with PERL language to identify selenocysteine insertion sequence (SECIS) element, the coding potential of TGA codons, and cysteine-containing homologs of selenoprotein genes. Our results showed that 11365 genes were terminated with TGA codons, 918 of which contained SECIS elements. Similarity search revealed that 58genes contained Sec/Cys pairs and similar flanking regions around in-frame TGA codons. Finally, 7genes were found to fully meet requirements for selenoproteins, although they have not been annotated as selenoproteins in NCBI databases. Deduced from their basic properties, the newly found selenoproteins in the genome of Anopheles gambiae are possibly related to in vivo oxidation tolerance and protein regulation in order to interfere with anopheles' vectorial capacity of Plasmodium. This study may also provide theoretical bases for the prevention of malaria from anopheles transmission.     

Comparative fecundity and associated factors for two sibling species of the Anopheles gambiaecomplex occurring sympatrically in The Gambia

Abstract. For two sibling species of mosquitoes belonging to the Anopheles gambiae complex of malaria vectors, the effects of body size (wing length) and bloodmeal size (haematin excretion) on fecundity of wild females were investigated in The Gambia, West Africa. Freshly blood-fed individuals from sympatric populations of An.arabiensis and An.gambiae sensu stricto were sampled by collection at 07.00–09.00 hours from within bednets during July/August 1993, at the beginning of the rainy season. The possible confounding effect of infection with Plasmodium parasites was removed by eliminating infected mosquitoes from the study samples. An.arabiensis females comprised 75% of the An.gambiae sensu law population and were significantly larger (greater mean wing length) than those of An.gambiae s.s. mosquitoes. Mean egg production per female (for the subsequent gonotrophic cycle, excluding pre-gravids) for the two species was not significantly different, though the relationship between wing length and egg production showed An.gambiae s.s. to be more fecund than the An.arabiensis of the same size. Pre-gravid An.gambiae s.s. had consumed significandy smaller bloodmeals than gravid females but the mean wing length of these two gonotrophic categories was not significantly different. In contrast, An.arabiensis pre-gravids were smaller and had consumed smaller bloodmeals than the gravids.     

Structure of host-odour plumes influences catch of Anopheles gambiae s.s. and Aedes aegypti in a dual-choice olfactometer

The effects of structure, concentration and composition of host‐odour plumes on catch of female Anopheles gambiae Giles sensu stricto and Aedes aegypti (L.) (Diptera: Culicidae) were investigated in a dual‐choice olfactometer. We demonstrate that the fine‐scale structure of host‐odour plumes modulates capture of An. gambiae and Ae. aegypti. In both species homogeneous skin‐odour plumes result in trap entry, whereas homogeneous CO₂ plumes reduce trap catch. Reduced trap catch also result from combining skin odour with a homogeneous CO₂ plume. Trap capture rates in homogeneous CO₂ plumes were concentration‐dependent and differed between the two species. Electric nets placed in front of the trap entrances intercepted mosquitoes before they could enter the traps. This showed that An. gambiae flew along CO₂ plumes, but did not enter the traps. Survivorship analysis of the trap‐entry times of Ae. aegypti indicated interactions between the time until capture and treatment. The assay's duration therefore can alter the distribution in a dual‐choice olfactometer.