Lack of robust evidence for a Wolbachia infection in Anopheles gambiae from Burkina Faso.

The endosymbiont Wolbachia can have major effects on the reproductive fitness, and vectorial capacity of host insects and may provide new avenues to control mosquito-borne pathogens. Anopheles gambiae s.l is the major vector of malaria in Africa but the use of Wolbachia in this species has been limited by challenges in establishing stable transinfected lines and uncertainty around native infections. High frequencies of infection of Wolbachia have been previously reported in An. gambiae collected from the Valle du Kou region of Burkina Faso in 2011 and 2014. Here, we re-evaluated the occurrence of Wolbachia in natural samples, collected from Valle du Kou over a 12-year time span, and in addition, expanded sampling to other sites in Burkina Faso. Our results showed that, in contrast to earlier reports, Wolbachia is present at an extremely low prevalence in natural population of An. gambiae. From 5341 samples analysed, only 29 were positive for Wolbachia by nested PCR representing 0.54% of prevalence. No positive samples were found with regular PCR. Phylogenetic analysis of 16S rRNA gene amplicons clustered across supergroup B, with some having similarity to sequences previously found in Anopheles from Burkina Faso. However, we cannot discount the possibility that the amplicon positive samples we detected were due to environmental contamination or were false positives. Regardless, the lack of a prominent native infection in An. gambiae s.l. is encouraging for applications utilizing Wolbachia transinfected mosquitoes for malaria control.

Entomological baseline data collection and power analyses in preparation of a mosquito swarm-killing intervention in south-western Burkina Faso.

BACKGROUND: Insecticides are currently the main tools used to reduce the transmission of malaria; therefore, the development of resistance to insecticides in malaria vectors is of major concern for malaria control. The resistance level to pyrethroids is particularly high in the Western region of Burkina Faso and may affect the efficacy of insecticidal bed nets and indoor residual spraying. Adult mosquito swarming and other nocturnal behaviours exhibit spatial and temporal patterns that suggest potential vulnerability to targeted space spraying with effective insecticides. Indeed, targeted space-spraying against adult mosquito swarms has been used to crash mosquito populations and disrupt malaria transmission. METHODS: Prior to impact assessment of swarm killing, a baseline data collection was conducted from June to November 2016 in 10 villages divided into two areas in western Burkina Faso. The data considered both ecological and demographic characteristics to monitor the key entomological parameters. RESULTS: The mean number of swarms observed was 35 per village, ranging from 25 to 70 swarms according to the village. Female density in both areas varied significantly as a function of the village and the period of collection. The human biting rate was significantly affected by the period of collection and depended upon whether the collection was carried out indoors or outdoors. Averages of parity rate were high in both areas for all periods of collection, ranging from 60 to 90%. These values ranged from 80 to 100% for inseminated females. Sporozoite rates ranged between 1.6 and 7.2% depending upon the village. The molecular identification of resting and swarming mosquitoes showed the presence of the three major malaria vectors in Burkina Faso, but in different proportions for each village. CONCLUSIONS: The distribution of the potential swarm markers and swarms in villages suggested that swarms are clustered across space, making intervention easier. Power simulations showed that the direct sampling of swarms provides the highest statistical power, thereby reducing the number of villages needed for a trial.

Assessment of novel Lehmann's funnel entry trap prototypes performance to control malaria mosquito populations.

BACKGROUND: There is a global consensus that new intervention tools are needed for the final steps toward malaria elimination/eradication. In a recent study in Burkina Faso, the Lehmann Funnel Entry Trap (LFET) has shown promising results in the reduction of mosquito densities, even in areas where insecticide resistance is as high as 80%. The LFET requires no chemicals and is self-operated. However, one of the issues with the original LFET is the size of the funnel, which often occupies too much space within users' homes. Here, the performance of three new, smaller-sized LFET prototypes that combine a screening and killing effect on mosquitoes was assessed. METHODS: The study was carried out over three months during the rainy season in low and high malaria vector density sites, Soumousso and Vallée du Kou, respectively. The original LFET (or 'Prototype 1'/'P1') was modified to produce three new prototypes, which were referred to as prototype 2 ('the Medium' or 'P2'), prototype 3 (P3) and prototype 4 (P4). Each of the new prototypes was tested on eight days per month over the three-month period to assess their effectiveness in trapping and killing mosquitoes entering houses through the windows compared to the original LFET. RESULTS: Overall, 78,435 mosquitoes (mainly Anopheles gambiae sensu lato) were collected in the two study sites, both in the traps and in the houses. A total of 56,430 (72%) mosquitoes were collected from the traps. In Vallée du Kou, the original LFET caught a greater number of mosquitoes than the medium (prototype 2), whereas no difference was observed between the other new prototypes (3 and 4) and the medium. In Soumousso, both the original and medium LFETs collected significantly greater numbers of mosquitoes compared to prototypes 3 and 4. CONCLUSION: This study has shown that the new LFET prototypes are effective in trapping mosquitoes in high mosquito density settings. A large-scale study with one of the prototypes will be needed to assess community acceptance of the traps and their ability to control malaria vectors.

Mating competitiveness of sterile male Anopheles coluzzii in large cages.

BACKGROUND: Understanding the factors that account for male mating competitiveness is critical to the development of the sterile insect technique (SIT). Here, the effects of partial sterilization with 90 Gy of radiation on sexual competitiveness of Anopheles coluzzii allowed to mate in different ratios of sterile to untreated males have been assessed. Moreover, competitiveness was compared between males allowed one versus two days of contact with females. METHODS: Sterile and untreated males four to six days of age were released in large cages (~1.75 sq m) with females of similar age at the following ratios of sterile males: untreated males: untreated virgin females: 100:100:100, 300:100:100, 500:100:100 (three replicates of each) and left for two days. Competitiveness was determined by assessing the egg hatch rate and the insemination rate, determined by dissecting recaptured females. An additional experiment was conducted with a ratio of 500:100:100 and a mating period of either one or two days. Two controls of 0:100:100 (untreated control) and 100:0:100 (sterile control) were used in each experiment. RESULTS: When males and females consort for two days with different ratios, a significant difference in insemination rate was observed between ratio treatments. The competitiveness index (C) of sterile males compared to controls was 0.53. The number of days of exposure to mates significantly increased the insemination rate, as did the increased number of males present in the untreated: sterile male ratio treatments, but the number of days of exposure did not have any effect on the hatch rate. DISCUSSION: The comparability of the hatch rates between experiments suggest that An. coluzzii mating competitiveness experiments in large cages could be run for one instead of two days, shortening the required length of the experiment. Sterilized males were half as competitive as untreated males, but an effective release ratio of at least five sterile for one untreated male has the potential to impact the fertility of a wild female population. However, further trials in field conditions with wild males and females should be undertaken to estimate the ratio of sterile males to wild males required to produce an effect on wild populations.

Using artificial odors to optimize attractiveness of host decoy traps to malaria vectors.

Malaria vector surveillance tools often incorporate features of hosts that are attractive to blood-seeking females. The recently developed host decoy trap (HDT) combines visual, thermal, and olfactory stimuli associated with human hosts and has shown great efficacy in terms of collecting malaria vectors. Synthetic odors and yeast-produced carbon dioxide (CO2) could prove useful by mimicking the human odors currently used in HDTs and provide standardized and easy-to-use olfactory attractants. The objective of this study was to test the attractiveness of various olfactory attractant cues in HDTs to capture malaria vectors. We compared 4 different odor treatments in outdoor field settings in southern Benin and western Burkina Faso: the standard HDT using a human, HDT with yeast-produced CO2, HDT with an artificial odor blend, and HDT with yeast-produced CO2 plus artificial odor blend. In both experimental sites, the standard HDT that incorporated a real human produced the greatest catch of Anopheles gambiae s.l (Diptera: Culicidae). The alternatives tested were still effective at collecting target vector species, although the most effective included CO2, either alone (Benin) or in combination with synthetic odor (Burkina Faso). The trap using synthetic human odor alone caught the fewest An. gambiae s.l. compared to the other baited traps. Both Anopheles coluzzii and Anopheles gambiae were caught by each trap, with a predominance of An. coluzzii. Synthetic baits could, therefore, represent a more standardized and easier-to-deploy approach than using real human odor baits for a robust vector monitoring strategy.

Assessment of Culicidae collection methods for xenomonitoring lymphatic filariasis in malaria co-infection context in Burkina Faso.

BACKGROUND: Entomological surveillance of lymphatic filariasis and malaria infections play an important role in the decision-making of national programs to control, or eliminate these both diseases. In areas where both diseases prevalence is low, a large number of mosquitoes need to be sampled to determine vectors infection rate. To do this, efficient mosquito collection methods must be used. This study is part in this framework, to assess appropriate mosquito collection methods for lymphatic filariasis xenomonitoring in a coexistence context with malaria in Burkina Faso. METHODOLOGY/PRINCIPAL FINDINGS: Mosquito collections were performed between August and September 2018 in four villages (Koulpissi, Seiga, and Péribgan, Saptan), distributed in East and South-West health regions of Burkina Faso. Different collection methods were used: Human Landing Catches (HLC) executed indoor and outdoor, Window Exit-Trap, Double Net Trap (DNT) and Pyrethrum Spray Catches (PSC). Molecular analyses were performed to identify Anopheles gambiae s.l. sibling species and to detect Wuchereria bancrofti and Plasmodium falciparum infection in Anopheles mosquitoes. A total of 3 322 mosquitoes were collected among this, Anopheles gambiae s.l. was the vector caught in largest proportion (63.82%). An. gambiae s.l. sibling species molecular characterization showed that An. gambiae was the dominant specie in all villages. The Human Landing Catches (indoor and outdoor) collected the highest proportion of mosquitoes (between 61.5% and 82.79%). For the sampling vectors infected to W. bancrofti or P. falciparum, PSC, HLC and Window Exit-Trap were found the most effective collection methods. CONCLUSIONS/SIGNIFICANCE: This study revealed that HLC indoor and outdoor remained the most effective collection method. Likewise, the results showed the probability to use Window Exit-Trap and PSC collection methods to sample Anopheles infected.

Anopheles aquatic development kinetic and adults' longevity through different seasons in laboratory and semi-field conditions in Burkina Faso.

ABSTRACT: BACKGROUND: Anopheles mosquitoes are ectothermic and involved in numerous pathogen transmissions. Their life history traits are influenced by several environmental factors such as temperature, relative humidity and photoperiodicity. Despite extensive investigations of these environmental conditions on vector population ecology, their impact on the different life stages of Anopheles at different seasons in the year remains poorly explored. This study reports the potential impact of these abiotic factors on the immature and adult stages of Anopheles gambiae sensu lato during different seasons. METHODS: Environmental conditions were simulated in the laboratory using incubators to mimic the environmental conditions of two important periods of the year in Burkina Faso: the peak of rainy season (August) and the onset of dry season (December). Eggs from wild An. coluzzii and An. gambiae s.l. were reared separately under each environmental condition. For Anopheles coluzzii or An. gambiae s.l., eggs were equally divided into two groups assigned to the two experimental conditions. Four replicates were carried out for this experiment. Then, egg hatching rate, pupation rate, larval development time, larva-to-pupae development time, adult emergence dynamics and longevity of Anopheles were evaluated. Also, pupae-to-adult development time from wild L3 and L4 Anopheles larvae was estimated under semi-field conditions in December. RESULTS: A better egg hatching rate was recorded overall with conditions mimicking the onset of the dry season compared to the peak of the rainy season. Larval development time and longevity of An. gambiae s.l. female were significantly longer at the onset of the dry season compared than at the peak of the rainy season. Adult emergence was spread over 48 and 96 h at the peak of the rainy season and onset of dry season conditions respectively. This 96h duration in the controlled conditions of December was also observed in the semi-field conditions in December. CONCLUSIONS: The impact of temperature and relative humidity on immature stages and longevity of An. gambiae s.l. adult females differed under both conditions. These findings contribute to a better understanding of vector population dynamics throughout different seasons of the year and may facilitate tailoring of control strategies.

Effects of age and size on Anopheles gambiae s.s. male mosquito mating success.

Before the release of genetically-modified or sterile male mosquitoes in an attempt to control local populations of malaria vectors, it is crucial to determine male traits involved in mating success. The effects of male size and age as determinants of male mating success in Anopheles gambiae s.s. were measured in the field and under laboratory conditions in Burkina Faso. First, the body sizes (estimated by wing length) of mating, swarming, and indoor-resting male mosquitoes were compared over a 3-yr period (2006-2009) from July to October in Soumousso and Vallée du Kou, two villages in western Burkina Faso. Second, the age structure of swarming and resting male mosquitoes were characterized based on the number of spermatocysts and the proportion of sperm in the reservoir of wild-caught male testis. Third, male age effects on the insemination rate of female An. gambiae were investigated in the laboratory. The mean size of males collected in copula was significantly larger than the mean for swarming males and indoor-resting males. The optimum male age for successful insemination of females was 4-8 d. These results suggest that male size is an important trait in determining male mating competitiveness in the field. Although age was not found to be a significant factor in mating competitiveness, it was significantly correlated with swarming behaviors in the field and insemination success in the laboratory. The implications of these results in terms of sexual selection in An. gambiae and vector control programs are further discussed.

Mosquito (Diptera: Culicidae) populations in contrasting areas of the western regions of Burkina Faso: species diversity, abundance and their implications for pathogen transmission.

BACKGROUND: Mosquitoes (Diptera: Culicidae) can have a significant negative impact on human health. The urbanization of natural environments and their conversion for agricultural use, as well as human population growth, may affect mosquito populations and increase the risk of emerging or re-emerging mosquito-borne diseases. We report on the variety and number of adult mosquitoes found in four environments with varying degrees of human impact (rural, urban, rice fields, and forest) located in a savannah zone of West Africa. METHODS: Mosquitoes were collected from two regions (Hauts-Bassins and Sud-Ouest) of Burkina Faso during five periods between August 2019 and June 2021. Sampling sites were grouped according to environment. Mosquitoes were collected using BG-Sentinel traps and double net traps, and Prokopack Aspirators. Statistical analyses were performed using R software version 4.1.2. Logistic regression, using generalised mixed linear models, was used to test the effect of environment on mosquito abundance and diversity. Alpha diversity analysis was also performed, using the vegan package. RESULTS: A total of 10,625 adult mosquitoes were collected, belonging to 33 species and five genera: Culex, Aedes, Anopheles, Mansonia, and Ficalbia. The most dominant species were Culex quinquefasciatus, Anopheles gambiae sensu lato and Aedes aegypti. Alpha diversity was similar in the two regions. Habitat had a significant effect on mosquito species richness, the Shannon index and the Simpson index. The rural environment had the highest species richness (n = 28) followed by the forest environment (n = 24). The highest number of mosquitoes (4977/10,625) was collected in the urban environment. CONCLUSIONS: The species composition of the mosquito populations depended on the type of environment, with fewer species in environments with a high human impact such as urban areas and rice fields. Due to the diversity and abundance of the mosquito vectors, the human populations of all of the environments examined are considered to be at potential risk of mosquito-borne diseases.

Perfect association between spatial swarm segregation and the X-chromosome speciation island in hybridizing Anopheles coluzzii and Anopheles gambiae populations.

The sibling species An. coluzzii and An. gambiae s.s. are major malaria vectors thought to be undergoing sympatric speciation with gene flow. In the absence of intrinsic post-zygotic isolation between the two taxa, speciation is thought possible through the association of assortative mating and genomic regions protected from gene flow by recombination suppression. Such genomic islands of speciation have been described in pericentromeric regions of the X, 2L and 3L chromosomes. Spatial swarm segregation plays a major role in assortative mating between sympatric populations of the two species and, given their importance for speciation, genes responsible for such pre-mating reproductive barriers are expected to be protected within divergence islands. In this study 2063 male and 266 female An. coluzzii and An. gambiae s.s. individuals from natural swarms in Burkina Faso, West Africa were sampled. These were genotyped at 16 speciation island SNPs, and characterized as non-hybrid individuals, F1 hybrids or recombinant F1+n backcrossed individuals. Their genotypes at each speciation island were associated with their participation in An. coluzzii and An. gambiae-like swarms. Despite extensive introgression between the two species, the X-island genotype of non-hybrid individuals (37.6%), F1 hybrids (0.1%) and F1+n recombinants (62.3%) of either sex perfectly associated to each swarm type. Associations between swarm type and the 3L and 2L speciation islands were weakened or broken down by introgression. The functional demonstration of a close association between spatial segregation behaviour and the X speciation island lends further support to sympatric speciation models facilitated by pericentric recombination suppression in this important species complex.

Comparison of entomological impacts of two methods of intervention designed to control Anopheles gambiae s.l. via swarm killing in Western Burkina Faso.

Outdoor biting constitutes a major limitation of current vector control based primarily on long-lasting insecticidal nets and indoor residual spraying, both of which are indoor interventions. Consequently, malaria elimination will not be achieved unless additional tools are found to deal with the residual malaria transmission and the associated vector dynamics. In this study we tested a new vector control approach for rapidly crashing mosquito populations and disrupting malaria transmission in Africa. This method targets the previously neglected swarming and outdoor nocturnal behaviors of both male and female Anopheles mosquitoes. It involved accurate identification and targeted spraying of mosquito swarms to suppress adult malaria vector populations and their vectorial capacities. The impact of targeted spraying was compared to broadcast spraying and evaluated simultaneously. The effects of the two interventions were very similar, no significant differences between targeted spraying and broadcast spraying were found for effects on density, insemination or parity rate. However, targeted spraying was found to be significantly more effective than broadcast spraying at reducing the number of bites per person. As expected, each intervention had a highly significant impact upon all parameters measured, but the targeted swarm spraying required less insecticide.

Cuticular hydrocarbons are associated with mating success and insecticide resistance in malaria vectors.

Anopheles coluzzii females, important malaria vectors in Africa, mate only once in their lifetime. Mating occurs in aerial swarms with a high male-to-female ratio, where traits underlying male mating success are largely unknown. Here, we investigated whether cuticular hydrocarbons (CHCs) influence mating success in natural mating swarms in Burkina Faso. As insecticides are widely used in this area for malaria control, we also determined whether CHCs affect insecticide resistance levels. We find that mated males have higher CHC abundance than unmated controls, suggesting CHCs could be determinants of mating success. Additionally, mated males have higher insecticide resistance under pyrethroid challenge, and we show a link between resistance intensity and CHC abundance. Taken together, our results suggest that CHC abundance may be subject to sexual selection in addition to selection by insecticide pressure. This has implications for insecticide resistance management, as these traits may be sustained in the population due to their benefits in mating even in the absence of insecticides.

The Anopheles coluzzii microbiome and its interaction with the intracellular parasite Wolbachia.

Wolbachia, an endosymbiotic alpha-proteobacterium commonly found in insects, can inhibit the transmission of human pathogens by mosquitoes. Biocontrol programs are underway using Aedes aegypti mosquitoes trans-infected with a non-natural Wolbachia strain to reduce dengue virus transmission. Less is known about the impact of Wolbachia on the biology and vectorial capacity of Anopheles mosquitoes, the vectors of malaria parasites. A naturally occurring strain of Wolbachia, wAnga, infects populations of the major malaria vectors Anopheles gambiae and Anopheles coluzzii in Burkina Faso. Previous studies found wAnga infection was negatively correlated with Plasmodium infection in the mosquito and wAnga influenced mosquito egg-laying behavior. Here, we investigate wAnga in natural populations of An. coluzzii and its interactions with other resident microbiota using targeted 16S sequencing. Though we find no major differences in microbiota composition associated with wAnga infection, we do find several taxa that correlate with the presence or absence of wAnga in female mosquitoes following oviposition, with the caveat that we could not rule out batch effects due to the unanticipated impact of wAnga on oviposition timing. These data suggest wAnga may influence or interact with the Anopheles microbiota, which may contribute to the impact of wAnga on Anopheles biology and vectorial capacity.

Semi-field and indoor setups to study malaria mosquito swarming behavior.

BACKGROUND: The recent resurgence of interest in sterile insect techniques to control vector mosquitoes has renewed interest in novel methods for observing mating behavior. Malarial vectors of the Anopheles gambiae complex are known to mate in swarms at specific locations at dawn and dusk. Most knowledge of mosquito swarming behavior is derived from field observations and a few experimental studies designed to assess critical parameters that affect mosquito swarming. However, such studies are difficult to implement in the field because of uncontrollable environmental factors and mosquito conditions. Here, we present two experimental setups specifically designed to analyze mosquito swarming behavior and provide evidence that swarming behavior of mosquitoes can be generated and accurately assessed under both semi-field and laboratory conditions. METHODS: The Mosquito Ecology Research Facility setup is a semi-field enclosure made of 12 compartments (10.0 × 6.0 × 4.5 m L × W × H each) exposed to ambient meteorological and lighting conditions. The laboratory setup consists of a windowless room (5.1 × 4.7 × 3.0 m) in which both environmental and mosquito conditions can be controlled. In the two setups, 300 3-6-days-old An. coluzzii virgin males were released and some swarm characteristics were recorded such as the time at which the swarm started, the number of mosquitoes in the swarm and the height. Climatic conditions in the semi-field setup were also recorded. RESULTS: In both setups, An. coluzzii males displayed stereotyped and consistent swarming behavior day after day; males gradually gather into a swarm over a ground marker at sunset, flying in loops in relation to specific visual features on the ground. Although semi-field climatic conditions were slightly different from outdoors conditions, they did not impede swarming behavior and swarm characteristics were similar to those observed in the field. CONCLUSIONS: Swarm characteristics and their consistency across days provide evidences that these facilities can be used confidently to study swarming behavior. These facilities come to complement existing semi-field setups and pave the way for new experimental studies which will enhance our understanding of mating behavior but also mosquito ecology and evolution, a prerequisite for application of genetic approaches to malaria control.

Exploiting Anopheles responses to thermal, odour and visual stimuli to improve surveillance and control of malaria.

Mosquito surveillance and control are at the heart of efforts to eliminate malaria, however, there remain significant gaps in our understanding of mosquito behaviour that impede innovation. We hypothesised that a combination of human-associated stimuli could be used to attract and kill malaria vectors more successfully than individual stimuli, and at least as well as a real human. To test this in the field, we quantified Anopheles responses to olfactory, visual and thermal stimuli in Burkina Faso using a simple adhesive trap. Traps baited with human odour plus high contrast visual stimuli caught more Anopheles than traps with odour alone, showing that despite their nocturnal habit, malaria vectors make use of visual cues in host-seeking. The best performing traps, however, combined odour and visual stimuli with a thermal signature in the range equivalent to human body temperature. When tested against a human landing catch during peak mosquito abundance, this "host decoy" trap caught nearly ten times the number of Anopheles mosquitoes caught by a human collector. Exploiting the behavioural responses of mosquitoes to the entire suite of host stimuli promises to revolutionise vector surveillance and provide new paradigms in disease control.

Wolbachia infections in natural Anopheles populations affect egg laying and negatively correlate with Plasmodium development.

The maternally inherited alpha-proteobacterium Wolbachia has been proposed as a tool to block transmission of devastating mosquito-borne infectious diseases like dengue and malaria. Here we study the reproductive manipulations induced by a recently identified Wolbachia strain that stably infects natural mosquito populations of a major malaria vector, Anopheles coluzzii, in Burkina Faso. We determine that these infections significantly accelerate egg laying but do not induce cytoplasmic incompatibility or sex-ratio distortion, two parasitic reproductive phenotypes that facilitate the spread of other Wolbachia strains within insect hosts. Analysis of 221 blood-fed A. coluzzii females collected from houses shows a negative correlation between the presence of Plasmodium parasites and Wolbachia infection. A mathematical model incorporating these results predicts that infection with these endosymbionts may reduce malaria prevalence in human populations. These data suggest that Wolbachia may be an important player in malaria transmission dynamics in Sub-Saharan Africa.

Does extreme asymmetric dominance promote hybridization between Anopheles coluzzii and Anopheles gambiae s.s. in seasonal malaria mosquito communities of West Africa?

BACKGROUND: Anopheles gambiae s.s. and An. coluzzii are two of the most important malaria vector species in sub-Saharan Africa. These recently-diverged sibling species do not exhibit intrinsic post-zygotic barriers to reproduction and are thought to be separated by strong assortative mating combined with selection against hybrids. At present, little is known about the ecological conditions that determine hybridization and introgression between these cryptic taxa. METHODS: Swarm segregation and assortative mating were studied in Western Burkina Faso in the villages of Vallée du Kou (VK7) and Soumousso which differed in terms of which sibling species was much rarer than the other, and in Bana where both occurred in similar proportions. Swarms and pairs in copula were collected and genotyped, the proportion of intra and interspecific mating determined, and interspecific sperm transfer checked genetically. Females were collected through larval and adult indoor collections and genotyped or sexed-and-genotyped via a novel multiplex PCR. RESULTS: A total of 3,687 males and 220 females were collected and genotyped from 109 swarms. Only 3 swarms were composed of males from both species, and these were from the village of VK7 where An. gambiae s.s. was comparatively rare. Mixed-species pairs captured in copula were only detected in that area and made for 3.62 % and 100 % of mating pairs involving An. coluzzii and An. gambiae s.s. individuals, respectively. The high An. gambiae s.s. cross-mating rate was mirrored by high rates of hybridizations estimated from female larvae and adults indoor collections. This contrasted with Soumousso, where despite being much less common than An. gambiae s.s., An. coluzzii males did not form mixed swarms, females were not found in interspecific swarms or copula and hybridization rates were low in both sibling species. CONCLUSIONS: These data suggest that ecological conditions leading to rare An. gambiae s.s. in populations dominated by An. coluzzii may promote a breakdown of spatial swarm segregation and assortative mating between the two species. The lower overall hybridization rates observed at the larval and adult indoor stages compared to cross-mating rates support the idea that post-mating selection processes acting against hybrids may occur mostly prior to and/or at the early larval stages.

Role of nutritional reserves and body size in Anopheles gambiae males mating success.

A better knowledge of the different parameters that account for male mating success in the wild is critical to the development of genetic control strategies. In this study, we measured energy budgets (total sugar and glycogen) as the daily energetic investment in swarming males of An. gambiae s.s. M and S molecular forms from two different field locations, VK7 and Soumousso. We also looked at the difference between energetic reserves in mated males compared to unmated ones, and assessed wing length in both molecular forms to explore whether this phenotypic trait was involved in swarming behavior or mating success. The current study showed that the energetic cost of 25 min of swarming was around 50% of the male's sugar (M form: 48.5%, S form: 56.2%) and glycogen (M form: 53.1%, S form: 59%) reserves. However, no difference in carbohydrate content was observed between mated and unmated males. Mated males were found to be bigger than unmated ones, while intermediate size of males is advantageous in mating system, both in M and S molecular forms and when collected in two different locations. Regardless of the collection location, no difference in wing size was observed in swarming males collected early or late during a particular swarm. The results are discussed in the context sexual selection in different ecological locations.

Differences in timing of mating swarms in sympatric populations of Anopheles coluzzii and Anopheles gambiae s.s. (formerly An. gambiae M and S molecular forms) in Burkina Faso, West Africa.

BACKGROUND: The M and S molecular forms of Anopheles gambiae s.s. Giles appear to have speciated in West Africa and the M form is now formally named An. coluzzii Coetzee & Wilkerson sp.n. and the S form retains the nominotypical name (abbreviated here to An. gambiae). Reproductive isolation is thought to be the main barrier to hybridisation; even though both species are found in the same mating swarms, hybrid fertilisations in copulae have not been found in the study area. The aim of the study, therefore, was to determine whether differences in circadian and/or environmental control over the timing of swarming in the two species contribute to reproductive isolation. METHODS: The timing of male swarming in these species was recorded four nights per month over four years at five swarming sites in each of two villages. The timing of the start and end of swarming, and the concurrent environmental parameters, temperature, humidity and light intensity, were recorded for n = 20 swarms/month/species. The timing of 'spontaneous' activity at dusk of individual An. coluzzii and An. gambiae males was video-recorded in an actograph outdoors for 21 nights. RESULTS: Of the environmental parameters considered, swarming was most strongly correlated with sunset (r2 > 0.946). Anopheles gambiae started and stopped swarming earlier than An. coluzzii (3:35 ± 0:68 min:sec and 4:51 ± 1:21, respectively), and the mean duration of swarming was 23:37 ± 0:33 for An. gambiae and 21:39 ± 0:33 for An. coluzzii. Accordingly, in principle, whenever both species swarm over the same marker, a mean of 15.3 ± 3.1% of An. gambiae swarming would occur before An. coluzzii males arrived, and 19.5 ± 4.55% of An. coluzzii swarming would occurred after An. gambiae males had stopped swarming. These results are consistent with the finding that An. gambiae males became active in the actograph 09:35 ± 00:22 min:sec earlier than An. coluzzii males. CONCLUSIONS: The timing of swarming and spontaneous activity at dusk are primarily under circadian control, with the phase linked closely to sunset throughout the year. The mating activity of these two species is temporally segregated for 15-20% of the swarming period, which may contribute to the observed reproductive isolation of these species in local sympatric populations.

First report of the L1014S kdr mutation in wild populations of Anopheles gambiae M and S molecular forms in Burkina Faso (West Africa).

We investigated the occurrence of the L1014F and L1014S kdr mutations in malaria vector populations in Burkina Faso (West Africa). A cross-sectional survey was conducted at 10 sites all located in cotton cultivation areas which are assumed to be the major insecticide resistance selection foci in Burkina Faso. The hot ligation method was used to detect the two kdr mutations in field collected Anopheles gambiae s.l. samples. For the first time in Burkina Faso the L1014S mutation was identified in both M and S forms of An. gambiae s.s. populations collected from the site of Koupela in the central-eastern region at low frequency. Furthermore, the L1014S mutation was also found in one specimen of An. arabiensis collected from the Dano site. The data generated in this study provides additional evidence of the spread of the L1014S mutation into An. gambiae s.l. populations in West Africa. It is now important to evaluate the role of the L1014S mutation in the pyrethroid resistance phenotype and assess its potential impact on the efficacy of pyrethroid-based control measures in West Africa where several resistance mutations now coexist.