The poorly understood mechanisms of dry season persistence of Anopheles spp. mosquitoes through the dry season in Africa remain a critical gap in our knowledge of Plasmodium disease transmission. ...While it is thought that adult mosquitoes remain in a dormant state throughout this seven-month dry season, the nature of this state remains unknown and has largely not been recapitulated in laboratory settings. To elucidate possible connections of this state with microbial composition, the whole body microbiomes of adult mosquitoes in the dry and wet seasons in two locations of Mali with varying water availability were compared by sequencing the 16S ribosomal RNA gene. These locations were a village near the Niger River with year-round water sources (N'Gabakoro, "riparian"), and a typical Sahelian area with highly seasonal breeding sites (Thierola Area, "Sahelian"). The 16S bacterial data consisted of 2057 sequence variants in 426 genera across 184 families. From these data, we found several compositional differences that were seasonally and spatially linked. Counter to our initial hypothesis, there were more pronounced seasonal differences in the bacterial microbiome of riparian, rather than Sahelian areas. These seasonal shifts were primarily in Ralstonia, Sphingorhabdus, and Duganella spp. bacteria that are usually soil and water-associated, indicating these changes may be from bacteria acquired in the larval environment, rather than adulthood. In Sahelian dry season mosquitoes, there was a unique intracellular bacteria, Anaplasma, which likely was acquired through non-human blood feeding. Cytochrome B analysis of blood meals showed greater heterogeneity in host choice of An. coluzzii independent of season in the Thierola area compared to N'Gabakoro (77.5% vs. 94.6% human-origin blood meal, respectively), indicating a relaxation of anthropophily. Overall, this exploratory study provides valuable indications of spatial and seasonal differences in bacterial composition which help refine this difficult to study state.
Anopheles gambiae mates in flight at particular mating sites over specific landmarks known as swarm markers. The swarms are composed of males; females typically approach a swarm, and leave in copula. ...This mating aggregation looks like a lek, but appears to lack the component of female choice. To investigate the possible mechanisms promoting the evolution of swarming in this mosquito species, we looked at the variation in mating success between swarms and discussed the factors that structure it in light of the three major lekking models, known as the female preference model, the hotspot model, and the hotshot model.
We found substantial variation in swarm size and in mating success between swarms. A strong correlation between swarm size and mating success was observed, and consistent with the hotspot model of lek formation, the per capita mating success of individual males did not increase with swarm size. For the spatial distribution of swarms, our results revealed that some display sites were more attractive to both males and females and that females were more attracted to large swarms. While the swarm markers we recognize help us in localizing swarms, they did not account for the variation in swarm size or in the swarm mating success, suggesting that mosquitoes probably are attracted to these markers, but also perceive and respond to other aspects of the swarming site.
Characterizing the mating system of a species helps understand how this species has evolved and how selective pressures operate on male and female traits. The current study looked at male mating success of An. gambiae and discussed possible factors that account for its variation. We found that swarms of An. gambiae conform to the hotspot model of lek formation. But because swarms may lack the female choice component, we propose that the An. gambiae mating system is a lek-like system that incorporates characteristics pertaining to other mating systems such as scramble mating competition.
Long-distance migration of insects impacts food security, public health, and conservation-issues that are especially significant in Africa. Windborne migration is a key strategy enabling exploitation ...of ephemeral havens such as the Sahel, however, its knowledge remains sparse. In this first cross-season investigation (3 years) of the aerial fauna over Africa, we sampled insects flying 40-290 m above ground in Mali, using nets mounted on tethered helium-filled balloons. Nearly half a million insects were caught, representing at least 100 families from thirteen orders. Control nets confirmed that the insects were captured at altitude. Thirteen ecologically and phylogenetically diverse species were studied in detail. Migration of all species peaked during the wet season every year across localities, suggesting regular migrations. Species differed in flight altitude, seasonality, and associated weather conditions. All taxa exhibited frequent flights on southerly winds, accounting for the recolonization of the Sahel from southern source populations. "Return" southward movement occurred in most taxa. Estimates of the seasonal number of migrants per species crossing Mali at latitude 14°N were in the trillions, and the nightly distances traversed reached hundreds of kilometers. The magnitude and diversity of windborne insect migration highlight its importance and impacts on Sahelian and neighboring ecosystems.
In the West African Sahel, mosquito reproduction is halted during the 5-7 month-long dry season, due to the absence of surface waters required for larval development. However, recent studies have ...suggested that both Anopheles gambiae sensu stricto (s.s.) and Anopheles arabiensis repopulate this region via migration from distant locations where larval sites are perennial. Anopheles coluzzii engages in more regional migration, presumably within the Sahel, following shifting resources correlating with the ever-changing patterns of Sahelian rainfall. Understanding mosquito migration is key to controlling malaria-a disease that continues to claim more than 400,000 lives annually, especially those of African children. Using tethered flight data of wild mosquitoes, the distribution of flight parameters were evaluated as indicators of long-range migrants versus appetitive flyers, and the species specific seasonal differences and gonotrophic states compared between two flight activity modalities. Morphometrical differences were evaluated in the wings of mosquitoes exhibiting high flight activity (HFA) vs. low flight activity (LFA).
A novel tethered-flight assay was used to characterize flight in the three primary malaria vectors- An. arabiensis, An. coluzzii and An. gambiae s.s. The flights of tethered wild mosquitoes were audio-recorded from 21:00 h to 05:00 h in the following morning and three flight aptitude indices were examined: total flight duration, longest flight bout, and the number of flight bouts during the assay.
The distributions of all flight indices were strongly skewed to the right, indicating that the population consisted of a majority of low-flight activity (LFA) mosquitoes and a minority of high-flight activity (HFA) mosquitoes. The median total flight was 586 s and the maximum value was 16,110 s (~ 4.5 h). In accordance with recent results, flight aptitude peaked in the wet season, and was higher in gravid females than in non-blood-fed females. Flight aptitude was also found to be higher in An. coluzzii compared to An. arabiensis, with intermediate values in An. gambiae s.s., but displaying no statistical difference. Evaluating differences in wing size and shape between LFA individuals and HFA ones, the wing size of HFA An. coluzzii was larger than that of LFAs during the wet season-its length was wider than predicted by allometry alone, indicating a change in wing shape. No statistically significant differences were found in the wing size/shape of An. gambiae s.s. or An. arabiensis.
The partial agreement between the tethered flight results and recent results based on aerial sampling of these species suggest a degree of discrimination between appetitive flyers and long-distance migrants although identifying HFAs as long-distance migrants is not recommended without further investigation.
Anopheles gambiae, the major malaria vector in Africa, can be divided into two subgroups based on genetic and ecological criteria. These two subgroups, termed the M and S molecular forms, are ...believed to be incipient species. Although they display differences in the ecological niches they occupy in the field, they are often sympatric and readily hybridize in the laboratory to produce viable and fertile offspring. Evidence for assortative mating in the field was recently reported, but the underlying mechanisms awaited discovery. We studied swarming behaviour of the molecular forms and investigated the role of swarm segregation in mediating assortative mating. Molecular identification of 1145 males collected from 68 swarms in Donéguébougou, Mali, over 2 years revealed a strict pattern of spatial segregation, resulting in almost exclusively monotypic swarms with respect to molecular form. We found evidence of clustering of swarms composed of individuals of a single molecular form within the village. Tethered M and S females were introduced into natural swarms of the M form to verify the existence of possible mate recognition operating within-swarm. Both M and S females were inseminated regardless of their form under these conditions, suggesting no within-mate recognition. We argue that our results provide evidence that swarm spatial segregation strongly contributes to reproductive isolation between the molecular forms in Mali. However this does not exclude the possibility of additional mate recognition operating across the range distribution of the forms. We discuss the importance of spatial segregation in the context of possible geographic variation in mechanisms of reproductive isolation.
The spread of SARS-CoV-2 cannot be well monitored and understood in areas without capacity for effective disease surveillance. Countries with a young population will have disproportionately large ...numbers of asymptomatic or pauci-symptomatic infections, further hindering detection of infection. Sero-surveillance on a country-wide scale by trained medical professionals may be limited in a resource-limited setting such as Mali. Novel ways of broadly sampling the human population in a non-invasive method would allow for large-scale surveillance at a reduced cost.
Here we evaluate the collection of naturally blood-fed mosquitoes to test for human anti-SARS-CoV-2 antibodies in the laboratory and at five field locations in Mali.
Immunoglobulin-G antibodies to multiple SARS-CoV-2 antigens were readily detected in mosquito bloodmeals by bead-based immunoassay through at least 10 h after feeding mean sensitivity of 0.92 (95% CI 0.78-1) and mean specificity of 0.98 (95% CI 0.88-1), indicating that most blood-fed mosquitoes collected indoors during early morning hours (and likely to have fed the previous night) are viable samples for analysis. We found that reactivity to four SARS-CoV-2 antigens rose during the pandemic from pre-pandemic levels. The crude seropositivity of blood sampled via mosquitoes was 6.3% in October and November 2020 across all sites, and increased to 25.1% overall by February 2021, with the most urban site reaching 46.7%, consistent with independent venous blood-based sero-surveillance estimates.
We have demonstrated that using mosquito bloodmeals, country-wide sero-surveillance of human diseases (both vector-borne and non-vector-borne) is possible in areas where human-biting mosquitoes are common, offering an informative, cost-effective, and non-invasive sampling option.
Persistence of African anophelines throughout the long dry season (4-8 months) when no surface waters are available remains one of the enduring mysteries of medical entomology. Recent studies ...demonstrated that aestivation (summer diapause) is one mechanism that allows the African malaria mosquito, Anopheles gambiae, to persist in the Sahel. However, migration from distant localities - where reproduction continues year-round - might also be involved.
To assess the contribution of aestivating adults to the buildup of populations in the subsequent wet season, two villages subjected to weekly pyrethrum sprays throughout the dry season were compared with two nearby villages, which were only monitored. If aestivating adults are the main source of the subsequent wet-season population, then the subsequent wet-season density in the treated villages will be lower than in the control villages. Moreover, since virtually only M-form An. gambiae are found during the dry season, the reduction should be specific to the M form, whereas no such difference is predicted for S-form An. gambiae or Anopheles arabiensis. On the other hand, if migrants arriving with the first rain are the main source, no differences between treated and control villages are expected across all members of the An. gambiae complex.
The wet-season density of the M form in treated villages was 30% lower than that in the control (P < 10-4, permutation test), whereas no significant differences were detected in the S form or An. arabiensis.
These results support the hypothesis that the M form persist in the arid Sahel primarily by aestivation, whereas the S form and An. arabiensis rely on migration from distant locations. Implications for malaria control are discussed.
Malaria parasites undergo complex developmental transitions within the mosquito vector. A commonly used laboratory model for studies of mosquito-malaria interaction is the rodent parasite, P. ...berghei. Anopheles funestus is a major malaria vector in sub-Saharan Africa but has received less attention than the sympatric species, Anopheles gambiae. The imminent completion of the A. funestus genome sequence will provide currently lacking molecular tools to describe malaria parasite interactions in this mosquito, but previous reports suggested that A. funestus is not permissive for P. berghei development.
An A. funestus population was generated in the laboratory by capturing female wild mosquitoes in Mali, allowing them to oviposit, and rearing the eggs to adults. These F1 progeny of wild mosquitoes were allowed to feed on mice infected with a fluorescent P. berghei strain. Fluorescence microscopy was used to track parasite development inside the mosquito, salivary gland sporozoites were tested for infectivity to mice, and parasite development in A. funestus was compared to A. gambiae.
P. berghei oocysts were detectable on A. funestus midguts by 7 days post-infection. By 18-20 days post-infection, sporozoites had invaded the median and distal lateral lobes of the salivary glands, and hemocoel sporozoites were observed in the hemolymph. Mosquitoes were capable of infecting mice via bite, demonstrating that A. funestus supports the complete life cycle of P. berghei. In a random sample of wild mosquito genotypes, A. funestus prevalence of infection and the characteristics of parasite development were similar to that observed in A. gambiae-P. berghei infections.
The data presented in this study establish an experimental laboratory model for Plasmodium infection of A. funestus, an important vector of human malaria. Studying A. funestus-Plasmodium interactions is now feasible in a laboratory setting. This information lays the groundwork for exploitation of the awaited genome sequence of A. funestus.
Background: Most malaria-endemic countries use artemisinin-based combination therapy (ACT) as their first-line treatment. ACTs are known to be highly effective on asexual stages of the malaria ...parasite. Malaria transmission and the spread of resistant parasites depend on the infectivity of gametocytes. The effect of the current ACT regimens on gametocyte infectivity is unclear.Objectives: This study aimed to determine the infectivity of gametocytes to Anopheles gambiae following ACT treatment in the field.Methods: During a randomised controlled trial in Bougoula-Hameau, Mali, conducted from July 2005 to July 2007, volunteers with uncomplicated malaria were randomised to receive artemether-lumefantrine, artesunate-amodiaquine, or artesunate-sulfadoxine/ pyrimethamine. Volunteers were followed for 28 days, and gametocyte carriage was assessed. Direct skin feeding assays were performed on gametocyte carriers before and after ACT administration.Results: Following artemether-lumefantrine treatment, gametocyte carriage decreased steadily from Day 0 to Day 21 post-treatment initiation. In contrast, for the artesunateamodiaquine and artesunate-sulfadoxine/pyrimethamine arms, gametocyte carriage increased on Day 3 and remained constant until Day 7 before decreasing afterward. Mosquito feeding assays showed that artemether-lumefantrine and artesunate-amodiaquine significantly increased gametocyte infectivity to Anopheles gambiae sensu lato (s.l.) (p Conclusion: Different ACT regimens could lead to gametocyte populations with different capacity to infect the Anopheles vector. Frequent assessment of the effect of antimalarials on gametocytogenesis and gametocyte infectivity may be required for the full assessment of treatment efficacy, the potential for spread of drug resistance and malaria transmission in the field.
Over the past two decades efforts to control malaria have halved the number of cases globally, yet burdens remain high in much of Africa and the elimination of malaria has not been achieved even in ...areas where extreme reductions have been sustained, such as South Africa
. Studies seeking to understand the paradoxical persistence of malaria in areas in which surface water is absent for 3-8 months of the year have suggested that some species of Anopheles mosquito use long-distance migration
. Here we confirm this hypothesis through aerial sampling of mosquitoes at 40-290 m above ground level and provide-to our knowledge-the first evidence of windborne migration of African malaria vectors, and consequently of the pathogens that they transmit. Ten species, including the primary malaria vector Anopheles coluzzii, were identified among 235 anopheline mosquitoes that were captured during 617 nocturnal aerial collections in the Sahel of Mali. Notably, females accounted for more than 80% of all of the mosquitoes that we collected. Of these, 90% had taken a blood meal before their migration, which implies that pathogens are probably transported over long distances by migrating females. The likelihood of capturing Anopheles species increased with altitude (the height of the sampling panel above ground level) and during the wet seasons, but variation between years and localities was minimal. Simulated trajectories of mosquito flights indicated that there would be mean nightly displacements of up to 300 km for 9-h flight durations. Annually, the estimated numbers of mosquitoes at altitude that cross a 100-km line perpendicular to the prevailing wind direction included 81,000 Anopheles gambiae sensu stricto, 6 million A. coluzzii and 44 million Anopheles squamosus. These results provide compelling evidence that millions of malaria vectors that have previously fed on blood frequently migrate over hundreds of kilometres, and thus almost certainly spread malaria over these distances. The successful elimination of malaria may therefore depend on whether the sources of migrant vectors can be identified and controlled.
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