Testing configurations of attractive toxic sugar bait (ATSB) stations in Mali, West Africa, for improving the control of malaria parasite transmission by vector mosquitoes and minimizing their effect on non-target insects.

BACKGROUND: Application methods of |Attractive Toxic Sugar Baits (ATSB) need to be improved for wide-scale use, and effects on non-target organisms (NTOs) must be assessed. The goals of this study were to determine, at the village level, the effect of different configurations of bait stations to (1) achieve < 25% Anopheles mosquito vector daily feeding rate for both males and females and (2) minimize the effect on non-target organisms. METHODS: Dye was added to Attractive Sugar Bait Stations (without toxin) to mark mosquitoes feeding on the baits, and CDC UV light traps were used to monitor for marked mosquitoes. An array of different traps were used to catch dye marked NTOs, indicating feeding on the ASB. Stations were hung on homes (1, 2, or 3 per home to optimize density) at different heights (1.0 m or 1.8 m above the ground). Eight villages were chosen as for the experiments. RESULTS: The use of one ASB station per house did not mark enough mosquitoes. Use of two and three stations per house gave feeding rates above the 25% goal. There was no statistical difference in the percentage of marked mosquitoes between two and three stations, however, the catches using two and three bait stations were both significantly higher than using one. There was no difference in An. gambiae s.l. feeding when stations were hung at 1.0 and 1.8 m. At 1.8 m stations sustained less accidental damage. ASB stations 1.8 m above ground were fed on by three of seven monitored insect orders. The monitored orders were: Hymenoptera, Lepidoptera, Coleoptera, Diptera, Hemiptera, Neuroptera and Orthoptera. Using one or two stations significantly reduced percentage of bait-fed NTOs compared to three stations which had the highest feeding rates. Percentages were as follows: 6.84 ± 2.03% Brachycera followed by wasps (Hymenoptera: Vespidae) 5.32 ± 2.27%, and Rhopalocera 2.22 ± 1.79%. Hanging the optimal number of stations per house for catching mosquitoes (two) at 1.8 m above ground, limited the groups of non-targets to Brachycera, Chironomidae, Noctuoidea, Rhopalocera, parasitic wasps and wasps (Hymenoptera). Feeding at 1.8 m only occurred when stations were damaged. CONCLUSIONS: The goal of marking quarter of the total Anopheles population per day was obtained using 2 bait stations at 1.8 m height above the ground. This configuration also had minimal effects on non-target insects.

Estimating the potential impact of Attractive Targeted Sugar Baits (ATSBs) as a new vector control tool for Plasmodium falciparum malaria.

BACKGROUND: Attractive targeted sugar baits (ATSBs) are a promising new tool for malaria control as they can target outdoor-feeding mosquito populations, in contrast to current vector control tools which predominantly target indoor-feeding mosquitoes. METHODS: It was sought to estimate the potential impact of these new tools on Plasmodium falciparum malaria prevalence in African settings by combining data from a recent entomological field trial of ATSBs undertaken in Mali with mathematical models of malaria transmission. The key parameter determining impact on the mosquito population is the excess mortality due to ATSBs, which is estimated from the observed reduction in mosquito catch numbers. A mathematical model capturing the life cycle of P. falciparum malaria in mosquitoes and humans and incorporating the excess mortality was used to estimate the potential epidemiological effect of ATSBs. RESULTS: The entomological study showed a significant reduction of ~ 57% (95% CI 33-72%) in mosquito catch numbers, and a larger reduction of ~ 89% (95% CI 75-100%) in the entomological inoculation rate due to the fact that, in the presence of ATSBs, most mosquitoes do not live long enough to transmit malaria. The excess mortality due to ATSBs was estimated to be lower (mean 0.09 per mosquito per day, seasonal range 0.07-0.11 per day) than the bait feeding rate obtained from one-day staining tests (mean 0.34 per mosquito per day, seasonal range 0.28-0.38 per day). CONCLUSIONS: From epidemiological modelling, it was predicted that ATSBs could result in large reductions (> 30% annually) in prevalence and clinical incidence of malaria, even in regions with an existing high malaria burden. These results suggest that this new tool could provide a promising addition to existing vector control tools and result in significant reductions in malaria burden across a range of malaria-endemic settings.

Detection and Monitoring of Insecticide Resistance Mutations in Anopheles gambiae: Individual vs Pooled Specimens.

Bioassays and molecular diagnostics are routinely used for the monitoring of malaria vector populations to support insecticide resistance management (IRM), guiding operational decisions on which insecticides ought to be used for effective vector control. Previously developed TaqMan assays were optimised to distinguish the wild-type L1014 from the knockdown resistance (kdr) point mutations 1014F and 1014S (triplex reaction), and the N1575 wild-type from the point mutation 1575Y (duplex reaction). Subsequently, artificial pools of Anopheles gambiae (An. gambiae) specimens with known genotypes of L1014F, L1014S, and N1575Y were created, nucleic acids were extracted, and kdr mutations were detected. These data were then used to define a linear regression model that predicts the allelic frequency within a pool of mosquitoes as a function of the measured ΔCt values (Ct mutant - Ct wild type probe). Polynomial regression models showed r2 values of >0.99 (p < 0.05). The method was validated with populations of variable allelic frequencies, and found to be precise (1.66⁻2.99%), accurate (3.3⁻5.9%), and able to detect a single heterozygous mosquito mixed with 9 wild type individuals in a pool of 10. Its pilot application in field-caught samples showed minimal differences from individual genotyping (0.36⁻4.0%). It allowed the first detection of the super-kdr mutation N1575Y in An. gambiae from Mali. Using pools instead of individuals allows for more efficient resistance allele screening, facilitating IRM.

A novel window entry/exit trap for the study of endophilic behavior of mosquitoes.

To overcome the limitations of currently used window entry/exit traps, we developed an efficient new glue net entry/exit trap (GNT) that is economical, easily transported and assembled, and can be utilized for a variety of studies which aim to investigate the dynamics of mosquito movements between indoor and outdoor habitats. Cage experiments were conducted to determine what percentage of mosquitoes trying to pass through the netting are actually being caught. The GNT caught 97% of female and 98% of male Anopheles gambiae s.s., as well as 97.5% of female and 98% of male Culex pipiens attempting to cross into a bait chamber adjoining the release cage. During a six day field study, the bedroom windows of 12 homes in Mali were fitted with entry/exit GNTs. Traps without glue were fitted over the inside and outside bedroom windows of an additional 12 homes as a control. A total of 446 An. gambiae s.l. were caught attempting to exit dwellings while 773 An. gambiae s.l. were caught attempting to enter. The number of males and females attempting to exit dwellings were roughly similar (215 and 231 respectively) while there was a slight difference in the number of males and females trying to enter (382 and 430 respectively). Pyrethrum spray catches (PSC's) conducted inside the dwellings on the last day of the experiment yielded only six females and a single male.

First record of Aedes albopictus in inland Africa along the River Niger in Bamako and Mopti, Mali.

The distribution of Aedes albopictus in Africa has thus far been known to be restricted to coastal Sub-Saharan countries. This report describes the first record of the tiger mosquito in habitats located in Mali, at a significant distance from the coastal areas of the continent. Aedes albopictus was observed over several years in increasing frequency in Mopti in Central Mali and later in the capital city Bamako, both adjacent to the Niger River. These findings suggest further dissemination of Ae. albopictus could be facilitated by river transport of goods and commodities which harbor larvae and eggs of this species. If correct, the distribution of Ae. albopictus is expected to extend to areas located upstream of the Niger River and its tributaries.

Indoor use of attractive toxic sugar bait (ATSB) to effectively control malaria vectors in Mali, West Africa.

BACKGROUND: Attractive toxic sugar bait (ATSB) solutions containing any gut toxins can be either sprayed on plants or used in simple bait stations to attract and kill sugar-feeding female and male mosquitoes. This field study in Mali demonstrates the effect of ATSB bait stations inside houses as a vector control method that targets and kills endophilic African malaria vectors. METHODS: The studies were conducted in five villages located near the River Niger, Mali. Baseline village-wide assessments of densities for female and male Anopheles gambiae sensu lato were performed by pyrethrum spray collections (PSC) in ten houses in each of five villages. To determine the rate of mosquito feeding on bait stations, one bait station per house containing attractive sugar bait (ASB) (without toxin) plus a food dye marker, was set up in ten houses in each of the five villages. PSC collections were conducted on the following day and the percentage of female and male mosquitoes that had fed was determined by visual inspection for the dye marker. Then, a 50-day field trial was done. In an experimental village, one bait station containing ATSB (1% boric acid active ingredient) was placed per bedroom (58 bedrooms), and indoor densities of female and male An. gambiae s.l. were subsequently determined by PSC, and female mosquitoes were age graded. RESULTS: In the five villages, the percentages of An. gambiae s.l. feeding inside houses on the non-toxic bait stations ranged from 28.3 to 53.1% for females and 36.9 to 78.3% for males. Following ATSB indoor bait station presentation, there was a significant reduction, 90% in female and 93% in male populations, of An. gambiae s.l. at the experimental village. A 3.8-fold decrease in the proportion of females that had undergone four or more gonotrophic cycles was recorded at the experimental village, compared to a 1.2-fold increase at the control village. CONCLUSION: The field trial demonstrates that An. gambiae s.l. feed readily from ATSB bait stations situated indoors, leading to a substantial reduction in the proportion of older female mosquitoes. This study demonstrates that ATSB inside houses can achieve impressive malaria vector control in Africa.

Field experiments of Anopheles gambiae attraction to local fruits/seedpods and flowering plants in Mali to optimize strategies for malaria vector control in Africa using attractive toxic sugar bait methods.

BACKGROUND: Based on recent studies in Israel demonstrating that attractive toxic sugar bait (ATSB) methods can be used to decimate local anopheline and culicine mosquito populations, an important consideration is whether the same methods can be adapted and improved to attract and kill malaria vectors in Africa. The ATSB approach uses fruit or flower scent as an attractant, sugar solution as a feeding stimulant, and an oral toxin. The ATSB solutions are either sprayed on vegetation or suspended in simple bait stations, and the mosquitoes ingesting the toxic solutions are killed. As such, this approach targets sugar-feeding female and male mosquitoes. This study examines the attractiveness of African malaria vectors to local fruits/seedpods and flowering plants, key biological elements of the ATSB approach for mosquito control. METHODS: Three field experiments were conducted at sites in Mali. The attraction of Anopheles gambiae s.l. to 26 different local fruits and seedpods was determined at a site in the semi-arid Bandiagara District of Mali. Wire mesh glue traps with fruits/seedpods suspended on skewers inside were set along a seasonal lagoon. Seven replicates of each fruit/seedpod species were tested, with a water-soaked sponge and a sugar-soaked sponge as controls. The attraction of An. gambiae s.l. to 26 different types of flowering plants was determined at a site near Mopti in Mali. The flowering plants held in a water-filled buried container were tested using the same glue traps, with controls including water only and sugar solution. Six replicates of each selected plant type were tested on transects between rice paddies. Additional studies using CDC light traps were done to determine the relative densities and periodicity of An. gambiae s.l. attraction to branches of the most highly attractive flowering plant, branches without flowers, human odor, and candescent light. RESULTS: Of the 26 fruits and seedpods tested, 6 were attractive to An. gambiae s.l. females and males, respectively. Guava (Psidium guajava) and honey melon (Cucumis melo) were the two most attractive fruits for both females and males. Of the 26 flowering plants tested, 9 were significantly attractive for females, and 8 were attractive for males. Acacia macrostachya was the most attractive flowering plant. Periodicity studies using this plant showed peaks of An. gambiae s.l. attraction between 1930 and 2200 h and 0400-0500 h, which differed considerably from the response to human odors, which expectedly peaked at around midnight. CONCLUSION: These field experiments in Mali highlight that female and male An. gambiae s.l. have pronounced differences in attraction for diverse types of indigenous fruits/seedpods and flowering plants. The identification of attractive fruits and seedpods shows that a variety of indigenous and locally abundant natural products could potentially be used as juices to make ATSB solution for mosquito control. As well, the simple methods used to identify the most attractive flowering plants provide valuable insights into the natural history of sugar feeding for An. gambiae s.l. These observations can be used to guide future strategies for employing ATSB methods for malaria vector control in Africa. They also provide a basis for subsequent chemical analysis and development of attractive baits for mosquito control.

Successful field trial of attractive toxic sugar bait (ATSB) plant-spraying methods against malaria vectors in the Anopheles gambiae complex in Mali, West Africa.

BACKGROUND: Based on highly successful demonstrations in Israel that attractive toxic sugar bait (ATSB) methods can decimate local populations of mosquitoes, this study determined the effectiveness of ATSB methods for malaria vector control in the semi-arid Bandiagara District of Mali, West Africa. METHODS: Control and treatment sites, selected along a road that connects villages, contained man-made ponds that were the primary larval habitats of Anopheles gambiae and Anopheles arabiensis. Guava and honey melons, two local fruits shown to be attractive to An. gambiae s.l., were used to prepare solutions of Attractive Sugar Bait (ASB) and ATSB that additionally contained boric acid as an oral insecticide. Both included a color dye marker to facilitate determination of mosquitoes feeding on the solutions. The trial was conducted over a 38-day period, using CDC light traps to monitor mosquito populations. On day 8, ASB solution in the control site and ATSB solution in the treatment site were sprayed using a hand-pump on patches of vegetation. Samples of female mosquitoes were age-graded to determine the impact of ATSB treatment on vector longevity. RESULTS: Immediately after spraying ATSB in the treatment site, the relative abundance of female and male An. gambiae s.l. declined about 90% from pre-treatment levels and remained low. In the treatment site, most females remaining after ATSB treatment had not completed a single gonotrophic cycle, and only 6% had completed three or more gonotrophic cycles compared with 37% pre-treatment. In the control site sprayed with ASB (without toxin), the proportion of females completing three or more gonotrophic cycles increased from 28.5% pre-treatment to 47.5% post-treatment. In the control site, detection of dye marker in over half of the females and males provided direct evidence that the mosquitoes were feeding on the sprayed solutions. CONCLUSION: This study in Mali shows that even a single application of ATSB can substantially decrease malaria vector population densities and longevity. It is likely that ATSB methods can be used as a new powerful tool for the control of malaria vectors, particularly since this approach is highly effective for mosquito control, technologically simple, inexpensive, and environmentally safe.

Perspectives of people in Mali toward genetically-modified mosquitoes for malaria control.

BACKGROUND: Genetically-modified (GM) mosquitoes have been proposed as part of an integrated vector control strategy for malaria control. Public acceptance is essential prior to field trials, particularly since mosquitoes are a vector of human disease and genetically modified organisms (GMOs) face strong scepticism in developed and developing nations. Despite this, in sub-Saharan Africa, where the GM mosquito effort is primarily directed, very little data is available on perspectives to GMOs. Here, results are presented of a qualitative survey of public attitudes to GM mosquitoes for malaria control in rural and urban areas of Mali, West Africa between the months of October 2008 and June 2009. METHODS: The sample consisted of 80 individuals - 30 living in rural communities, 30 living in urban suburbs of Bamako, and 20 Western-trained and traditional health professionals working in Bamako and Bandiagara. Questions were asked about the cause of malaria, heredity and selective breeding. This led to questions about genetic alterations, and acceptable conditions for a release of pest-resistant GM corn and malaria-refractory GM mosquitoes. Finally, participants were asked about the decision-making process in their community. Interviews were transcribed and responses were categorized according to general themes. RESULTS: Most participants cited mosquitoes as one of several causes of malaria. The concept of the gene was not widely understood; however selective breeding was understood, allowing limited communication of the concept of genetic modification. Participants were open to a release of pest-resistant GM corn, often wanting to conduct a trial themselves. The concept of a trial was reapplied to GM mosquitoes, although less frequently. Participants wanted to see evidence that GM mosquitoes can reduce malaria prevalence without negative consequences for human health and the environment. For several participants, a mosquito control programme was preferred; however a transgenic release that satisfied certain requirements was usually acceptable. CONCLUSIONS: Although there were some dissenters, the majority of participants were pragmatic towards a release of GM mosquitoes. An array of social and cultural issues associated with malaria, mosquitoes and genetic engineering became apparent. If these can be successfully addressed, then social acceptance among the populations surveyed seems promising.