Evaluation of synthetic repellents on mosquito nets in experimental huts against insecticide-resistant Anopheles gambiae and Culex quinquefasciatus mosquitoes

Owing to the development of pyrethroid resistance in Anopheles gambiae, there is a need to develop chemical alternatives for use on mosquito nets. Synthetic insect repellents are widely used for personal protection as skin or clothing applications. The efficacy of repellent-treated nets (RTN) was evaluated in experimental huts in C?te d'Ivoire against pyrethroid-resistant populations of An. gambiae and Culex quinquefasciatus. The repellents tested were DEET (N,N-diethyl-3-methylbenzamide) at 7.9 g/m2 and two formulations of ethyl butylacetylaminopropionate (IR3535) at 7.6 g/m2 and 7.3 g/m2. Over 45 nights there was a 74-82% reduction in the number of An. gambiae entering the huts containing RTNs but no significant reduction in entry of C. quinquefasciatus. There was a 63-64% reduction in the proportion of An. gambiae blood feeding but no reduction in the proportion of C. quinquefasciatus blood feeding in huts with RTNs. An unexpected result was the 69-76% mortality of An. gambiae and 51-61% mortality of C. quinquefasciatus in huts containing RTNs. Treated filter paper bioassays in WHO test kits confirmed that confined contact with DEET induces mortality. The DEET-based product provided better and longer protection; tunnel test bioassays confirmed that residual activity lasted for up to 6 weeks. Application of repellents to nets warrants further investigation and development.     

Assessment of the kill of Anopheles gambiae by the fumigant insecticide dichlorvos in experimental huts

As part of the World Health Organization''s programme for the evaluation and testing of new insecticides, different types of dichlorvos dispenser were installed in experimental huts in Tanganyika for study of their effectiveness in killing Anopheles gambiae. It was found that satisfactorily high mosquito mortalities of 75% or more were maintained for one to two months after installation of a dispenser. Mortalities were about 10% higher in huts with grass roofs than in those with mud-lined roofs. The vapour toxicity was similar in the two types of hut, but the results of bio-assay tests on different types of surface showed that there was a residual contact effect of the insecticide as well as the fumigant effect; the lower mortalities in the mud-roofed huts may be due in part to removal or decomposition of the dichlorvos by the mud surface.     

A preliminary test of the protective efficacy of permethrin-treated bed nets in an area of Anopheles gambiae metabolic resistance to pyrethroids in north Cameroon

A trial of permethrin-treated nets (PTNs) versus untreated nets (UTNs) was conducted in Pitoa (north Cameroon), where the main malaria vectors, Anopheles gambiae s.l. and Anopheles arabiensis, show metabolic-based permethrin resistance. The deterrent effect of permethrin greatly reduced A. gambiae biting rate inside rooms where PTNs were installed. After 3 months of net use, malaria reinfection rate was significantly lower in children sleeping under a PTN, but no such effect was observed after 6 months. Parasitaemia was not significantly different between the two arms. These findings suggest good, although transitory, personal protection against malaria conferred by PTNs in an area of metabolic-based permethrin resistance.     

Ecophysiology of Anopheles gambiae s.l.: Persistence in the Sahel

The dry-season biology of malaria vectors is poorly understood, especially in arid environments when no surface waters are available for several months, such as during the dry season in the Sahel. Here we reappraise results on the dry-season physiology of members of the Anopheles gambiae s.l. complex in the broad context of dormancy in insects and especially in mosquitoes. We examine evidence on seasonal changes in reproduction, metabolism, stress tolerance, nutrition, molecular regulation, and environmental conditions and determine if the current results are compatible with dry-season diapause (aestivation) as the primary strategy for persistence throughout the dry season in the Sahel. In the process, we point out critical gaps in our knowledge that future studies can fill. We find compelling evidence that members of the An. gambiae s.l. complex undergo a form of aestivation during the Sahelian dry season by shifting energetic resources away from reproduction and towards increased longevity. Considering the differences between winter at temperate latitudes, which entails immobility of the insect and hence reliance on physiological solutions, as opposed to the Sahelian dry season, which restricts reproduction exclusively, we propose that behavioral changes play an important role in complementing physiological changes in this strategy.     

Pyrethroid cross resistance spectrum among populations of Anopheles gambiae s.s. from C?te d'Ivoire.

Field samples of Anopheles gambiae s.s. from C?te d'Ivoire were tested with 5 pyrethroids (cyfluthrin, lambda-cyhalothrin, alpha-cypermethrin, deltamethrin, permethrin), 1 pseudo-pyrethroid (etofenprox), and an organochlorine (DDT). With the use of World Health Organization diagnostic tests, 5 out of 6 samples were found cross-resistant to these insecticides. A strong decrease in knockdown effect and mortality was also observed when testing deltamethrin-impregnated nettings. With a polymerase chain reaction amplification of specific alleles diagnostic test, resistance was found associated with the presence of a kdr mutation. The strong correlation between kdr allelic frequency and resistance to DDT or etofenprox indicated that kdr was the main resistance factor for these 2 insecticides. On the contrary, a lower correlation was observed between kdr frequency and resistance to 4 of the 5 pyrethroids tested, suggesting that another mechanism was also involved, likely a metabolic detoxification. These results point out the necessity to monitor pyrethroid resistance and the presence of kdr before implementation of any impregnated bed-net programs for malaria control.     

Traditional use of mosquito-repellent plants in western Kenya and their evaluation in semi-field experimental huts against Anopheles gambiae: ethnobotanical studies and application by thermal expulsion and direct burning

Ethnobotanical survey in 2 communities in western Kenya revealed that the most commonly known repellent plants were Ocimum americanum L. (64.1%), Lantana camara L. (17.9%), Tagetes minuta L. (11.3%) and Azadirachta indica A. Juss (8.7%) on Rusinga Island, and Hyptis suaveolens Poit. (49.2%), L. camara (30.9%) and O. basilicum L. (30.4%) in Rambira. Direct burning of plants is the most common method of application for O. americanum (68.8%), L. camara (100%) and O. basilicum (58.8%). Placing branches or whole plants inside houses is most common for H. suaveolens (33.3 and 57.8% for the respective locations), A. indica (66.7 and 100%), and T. minuta (54.8 and 56.0%). The repellency of plants suggested by the ethnobotanical survey and other empirical information was evaluated against the malaria vector Anopheles gambiae s.s. Giles in experimental huts within a screenwalled greenhouse. Thermal expulsion and direct burning were tested as alternative application methods for the selected plants O. americanum, O. kilimandscharicum Guerke, O. suave Willd., L. camara, A. indica, H. suaveolens, Lippia uckambensis Spreng and Corymbia citriodora Hook. When thermally expelled, only H. suaveolens failed to repel mosquitoes, whereas the leaves of C. citriodora (74.5%, P < 0.0001), leaves and seeds of O. suave (53.1%, P < 0.0001) and O. kilimandscharicum (52.0%, P < 0.0001) were the most effective. Leaves of C. citriodora also exhibited the highest repellency (51.3%, P < 0.0001) by direct burning, followed by leaves of L. uckambensis (33.4%, P = 0.0004) and leaves and seeds of O. suave (28.0%, P = 0.0255). The combination of O. kilimandscharicum with L. uckambensis repelled 54.8% of mosquitoes (P < 0.0001) by thermal expulsion. No combination of plants increased repellency by either method. The semi-field system described appears a promising alternative to full-field trials for screening large numbers of candidate repellents without risk of malaria exposure.     

No evidence for positive selection at two potential targets for malaria transmission-blocking vaccines in Anopheles gambiae s.s

Human malaria causes nearly a million deaths in sub-Saharan Africa each year. The evolution of drug-resistance in the parasite and insecticide resistance in the mosquito vector has complicated control measures and made the need for new control strategies more urgent. Anopheles gambiae s.s. is one of the primary vectors of human malaria in Africa, and parasite-transmission-blocking vaccines targeting Anopheles proteins have been proposed as a possible strategy to control the spread of the disease. However, the success of these hypothetical technologies would depend on the successful ability to broadly target mosquito populations that may be genetically heterogeneous. Understanding the evolutionary pressures shaping genetic variation among candidate target molecules offers a first step towards evaluating the prospects of successfully deploying such technologies. We studied the population genetics of genes encoding two candidate target proteins, the salivary gland protein saglin and the basal lamina structural protein laminin, in wild populations of the M and S molecular forms of A. gambiae in Mali. Through analysis of intraspecific genetic variation and interspecific comparisons, we found no evidence of positive natural selection at the genes encoding these proteins. On the contrary, we found evidence for particularly strong purifying selection at the laminin gene. These results provide insight into the patterns of genetic diversity of saglin and laminin, and we discuss these findings in relation to the potential development of these molecules as vaccine targets.     

Studies on multiple feeding by Anopheles gambiae s.l. in a Sudan savanna area of north Nigeria

The detection of haptoglobins in Anopheles gambiae s.l. has been used to obtain an estimate of the incidence of multiple feeding for the village of Barmawa, Garki District, Kano State, Nigeria. The results indicated that the incidence of multiple feeding was approximately 10% but problems were encountered by the high incidence of ahaptoglobinaemia in the population. In four villages in Garki District the incidence of ahaptoglobinaemia varied between 65 and 76% while in young children and personnel under constant malaria chemoprophylaxis it was less than 30%. A strong correlation between ahaptoglobinaemia and malaria infections was seen. The results show evidence of selection of hosts by mosquitoes at Barmawa although this does not necessarily imply a preference per se. The results provide evidence of movement of blood-fed mosquitoes, between houses and from houses to resting sites.     

Laboratory and field efficacy of Bacillus thuringiensis var. Israelensis and Bacillus sphaericus against Anopheles gambiae s.l. and Culex quinquefasciatus in Ouagadougou, Burkina Faso

Two wettable powders (Bactimos and Vectobac) and one flowable concentrate (Teknar) of Bacillus thuringiensis var. israelensis (B.t.i.) and primary powders of Bacillus sphaericus isolates 1593 and 2362 were evaluated (laboratory) against field-collected larvae of Anopheles gambiae s.l. and Culex quinquefasciatus in Ouagadougou, Burkina Faso. Bactimos, Vectobac and a Corn-cob B.t.i. formulation (ABG-6138G) were field tested against Cx. quinquefasciatus and An. gambiae s.l. The isolates of B. sphaericus were also tested against An. gambiae s.l. in artificial ponds. Both wettable powders of B.t.i. showed superior activity than the flowable concentrate formulation against An. gambiae s.l. in the laboratory. Culex quinquefasciatus was more susceptible (3-4X) to B.t.i. (Bactimos) than An. gambiae s.l. The isolates of B. sphaericus were more effective (2-3X) against both mosquito species than Bactimos. In a ditch and two channels, Bactimos, Vectobac and ABG-6138G at 0.65, 1.5 and 5.6 kg/ha, respectively, gave 91-100% control of Cx. quinquefasciatus within 3 days of treatment. The same formulations at rates ranging from 0.25 to 5.6 kg/ha, produced 82-97% control of An. gambiae s.l. in rainwater pools 24 h after treatment. Isolates 1593 and 2362 at 0.12 and 0.24 kg/ha gave excellent control of An. gambiae s.l. in artificial ponds.     

Efficacy of Bacillus sphaericus against the malaria vector Anopheles gambiae and other mosquitoes in swamps and rice fields in Zaire.

The microbial control of Anopheles gambiae and other mosquitoes with a granular formulation of Bacillus sphaericus (Vectolex) was evaluated in rice fields and swamps, located around the suburban region of Kingabwa-village in Kinshasa, Zaíre. Ten treatment cycles with 15-day intervals were carried out with the same application rate, 10 kg/ha, during the dry season (May to September 1991). The treatments reduced larval populations of An. gambiae by 98% after 48 h, but repetitive applications were required every 15 days to maintain control. The persistence of B. sphaericus spores was more apparent in rice fields than in swamps. A significant reduction in nuisance biting by Culex quinquefasciatus and Mansonia uniformis was observed. For An. gambiae, a decrease of 13.6% in human biting was noted during the post-treatment period. The entomological inoculation rate was reduced from 0.238 to 0.143. The efficacy of B. sphaericus does not appear to offer outstanding potential for control of An. gambiae in rice fields and swamps and seems to be limited due to different factors tied to ecology and natural conditions in the fields.     

Chromosomal differentiation and adaptation to human environments in the Anopheles gambiae complex.

Speciation in the Anopheles gambiae complex is reviewed and discussed with emphasis on the patterns of chromosomal differentiation, particularly at the intraspecific level. The significance of inversion polymorphism in gambiae and arabiensis (the two species of greatest medical importance) is evaluated with reference to recent field investigations carried out in Nigeria. In both sibling species some of the inversions show clinical geographical changes in frequencies, with evident correlations with climatic conditions and vegetation zones. Microgeographical variations in species distribution and in intraspecific inversion frequencies are also present, which appear mostly related to man-made environmental contrasts. Parallel indoor-/outdoor collections of samples from polymorphic populations of arabiensis and gambiae show that adult mosquitoes carrying certain inversion karyotypes do not distribute at random in relation to the human environment, being significantly more frequent in outdoor than in indoor samples, or vice-versa. Optimal habitat choice appears to be involved in such variations of indoor resting behaviour, since the chromosomal types carried by less endophilic individuals are those more adapted to humid climates, i.e. those which tend to avoid the higher nocturnal saturation deficit of the indoor environment. This phenomenon, producing non-uniform exposure of the vector population to residual insecticides sprayed in houses, might explain the mediocrity of the results of malaria control projects based on house-spraying against endophilic vectors in the African savannas.     

An ultrastructural study of the sporogonic development of Plasmodium falciparum in Anopheles gambiae.

This paper describes the fine structure of the sporogonic development of Plasmodium falciparum in its natural vector Anopheles gambiae (Species A) as seen by scanning and transmission electron microscopy. The parasite was derived from naturally infected volunteers and the vector maintained under natural conditions at the MRC Laboratories, Fajara, The Gambia. Sporogonic development of P. falciparum is similar to that described for other Plasmodium spp. There are however greater similarities between P. falciparum and the avian malaria parasites, than those mammalian (primarily rodent) species described to date--particularly with respect to mitochondrial development, crystalloid morphology and nucleolar organization. Nuclear development is similar to that of the murine malaria parasites, but reconstruction of complete mitotic spindles from serial sections suggest the haploid genome of P. falciparum contains 14 chromosomes compared to eight to ten in the murine plasmodia. Sporoblast formation involves a unique process of cleft formation based on the expansion of the cisternal space of the endoplasmic reticulum. Sporozoite budding is almost exclusively confined to these inner membrane surfaces and results in a characteristic sporozoite distribution in the oocyst. High resolution scanning electron microscopy of free sporozoites provides the first surface view of the micropore of Plasmodium.     

Bioefficacy of cyfluthrin (SOLFAC EW050) impregnated bed-nets against Anopheles gambiae in southern Cameroon

The bioefficacy of cyfluthrin-impregnated bed-nets was evaluated in the agro-industrial town of Mbandjock (southern Cameroon). The objectives were to assess the knockdown and mortality rates, the protection against bloodfeeding mosquitoes, and the irritant effect of cyfluthrin (SOLFAC EW050)-impregnated bed-nets against a susceptible strain of Anopheles gambiae. Polyester bed-nets were impregnated and distributed to inhabitants of Mbandjock for use, then nets were retreated after 6 months. The uptake of active ingredient on nets ranged from 30.8 mg/m2 to 75.1 mg/m2 at the initial treatment and from 131.0 mg/m2 to 225.0 mg/m2 at retreatment. The susceptible Kisumu reference strain of Anopheles gambiae reared in our laboratory was used for bioassays. The knockdown rate on freshly treated nets (2 wk after treatment) ranged from 93 to 96% and the mortality rate ranged from 52 to 70%. During the 12-month trial, knockdown and mortality rates showed 2 peaks, respectively, in the 4th month (94-97% knockdown and 61-96% mortality) and 7th month (i.e., soon after retreatment; 89-98% knockdown and 86-100% mortality), separated by breakdowns on the 5th through 6th and 12th months (46-77% knockdown and 8-69% mortality). Knockdown and mortality rates decreased faster on the lower part of nets than on the top, suggesting that some external factors such as dirt and daily hand manipulation could impede the efficacy of treated nets. Exceedingly dirty nets were less effective than clean nets. The protective rate against bloodfeeding mosquitoes ranged from 60 to 100% during the 1st 4 months and decreased at 40-70% during the 5th and 6th months after the initial treatment. After retreatment, the protective rate ranged from 50 to 90% for the 1st 5 months and from 35 to 64% at the 6th month. More than 70% of mosquitoes that attempt to feed through treated or retreated nettings died within 24 h after contact. Cyfluthrin was found to be mildly irritant during the whole evaluation. This trial reveals that cyfluthrin EW050-treated nets were effective against a susceptible strain of An. gambiae.     

Evaluation of the protective effects of alpha-tocopherol and retinol against ochratoxin A cytotoxicity

Ochratoxin A (OTA), a mycotoxin frequently present in food and feedstuffs, produces a wide range of toxic effects, including cell death via lipid peroxidation. In one human and four animal cell lines we determined the half lethal concentration (LC50) of OTA, its effect on reactive oxygen species (ROS) production, and its ability to induce cytochrome p450 activity. We also examined the protective effect of alpha-tocopherol and all-trans-retinol in the most sensitive cell lines (i.e. bovine mammary epithelia, for which LC50 was 0.8 microg/ml (24 h), and Madin Darby canine kidney, for which LC50 was 4.3 microg/ml (48 h)). Pre-incubation for 3 h with either antioxidant significantly (P<0.05) ameliorated the OTA-induced reduction in cell viability and significantly decreased (P<0.05) ROS production. These findings indicate that oxidative stress is an important factor in OTA cytotoxicity. Supplementation with antioxidant molecules may counteract the short-term toxicity of this mycotoxin.