Isotopic evidence that aestivation allows malaria mosquitoes to persist through the dry season in the Sahel.

Data suggest that the malaria vector mosquito Anopheles coluzzii persists during the dry season in the Sahel through a dormancy mechanism known as aestivation; however, the contribution of aestivation compared with alternative strategies such as migration is unknown. Here we marked larval Anopheles mosquitoes in two Sahelian villages in Mali using deuterium (2H) to assess the contribution of aestivation to persistence of mosquitoes through the seven-month dry season. After an initial enrichment period, 33% of An. coluzzii mosquitoes were strongly marked. Seven months following enrichment, multiple analysis methods supported the ongoing presence of marked mosquitoes, compatible with the prediction that the fraction of marked mosquitoes should remain stable throughout the dry season if local aestivation is occurring. The results suggest that aestivation is a major persistence mechanism of An. coluzzii in the Sahel, contributing at least 20% of the adults at the onset of rains. This persistence strategy could influence mosquito control and malaria elimination campaigns.

Diversity, dynamics, direction, and magnitude of high-altitude migrating insects in the Sahel.

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.

Induction of long-lived potential aestivation states in laboratory An. gambiae mosquitoes.

BACKGROUND: How anopheline mosquitoes persist through the long dry season in Africa remains a gap in our understanding of these malaria vectors. To span this period in locations such as the Sahelian zone of Mali, mosquitoes must either migrate to areas of permanent water, recolonize areas as they again become favorable, or survive in harsh conditions including high temperatures, low humidity, and an absence of surface water (required for breeding). Adult mosquitoes surviving through this season must dramatically extend their typical lifespan (averaging 2-3 weeks) to 7 months. Previous work has found evidence that the malaria mosquito An. coluzzii, survives over 200 days in the wild between rainy seasons in a presumed state of aestivation (hibernation), but this state has so far not been replicated in laboratory conditions. The inability to recapitulate aestivation in the lab hinders addressing key questions such as how this state is induced, how it affects malaria vector competence, and its impact on disease transmission. METHODS: In effort to induce aestivation, we held laboratory mosquitoes in climate-controlled incubators with a range of conditions that adjusted humidity (40-85% RH), temperature (18-27 °C), and light conditions (8-12 h of light) and evaluated their survivorship. These conditions were chosen to mimic the late rainy and dry seasons as well as relevant extremes these mosquitoes may experience during aestivation. RESULTS: We found that by priming mosquitoes in conditions simulating the late wet season in Mali, and maintaining mosquitoes in reduced light/temperature, mean mosquito survival increased from 18.34 ± 0.65 to 48.02 ± 2.87 days, median survival increased from 19 (95% CI 17-21) to 50 days (95% CI 40-58), and the maximum longevity increased from 38 to 109 days (P-adj < 0.001). While this increase falls short of the 200 + day survival seen in field mosquitoes, this extension is substantially higher than previously found through environmental or dietary modulation and is hard to reconcile with states other than aestivation. This finding will provide a platform for future characterization of this state, and allow for comparison to field collected samples.

Windborne long-distance migration of malaria mosquitoes in the Sahel.

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 Africa1,2. 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 migration3. 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.

Marking mosquitoes in their natural larval sites using 2H-enriched water: a promising approach for tracking over extended temporal and spatial scales.

BACKGROUND: 1.Tracking mosquitoes using current methods of mark-release-recapture are limited to small spatial and temporal scales exposing major gaps in understanding long-range movements and extended survival. Novel approaches to track mosquitoes may yield fresh insights into their biology which improves intervention activities to reduce disease transmission.Stable isotope enrichment of natural mosquito breeding sites allows large-scale marking of wild mosquitoes absent human handling. Mosquito larvae that develop in 2H-enriched water are expected to be detectable for over four months using tissue mass-fraction 2H measurements, providing opportunities for long-term mark-capture studies on a large scale. APPROACH: 2.A laboratory study followed by a field experiment of mosquito larval habitat 2H-enrichment was conducted in Mali, to evaluate potential labeling of wild mosquitoes. Twelve natural larval sites were enriched using [2H]-Deuterium-oxide (D2O, 99%). Enrichment level was maintained by supplementation following dilution by rains. Availability of 2H to mosquito larvae was enhanced by locally collected and cultured microorganisms (i.e. protozoa, algae and bacteria) reared in deuterated water, and provided as larval diet. Putative natural predators were removed from the larval sites and first instar larvae Anopheles gambiae s.l. larvae were added every other day. Emergence traps enabled collection of eclosing adults. Adult mosquitoes were kept at laboratory conditions for analysis of label attrition with age. RESULTS: 3.Deuterium enrichment of wild mosquitoes above background levels (maximum = 143.1 ppm) became apparent 5-6 days after initial exposure, after which 2H values increased steadily until ~24 days later (to a mean of approx. 220 ppm). Anopheles and Culex mosquitoes showed significantly different 2H values (211 and 194.2 ppm respectively). Both genera exhibited exponential label attrition (e (-x)) amounting to 21.6% by day 30 post emergence, after which attrition rate continuously decreased. Males of both taxa exhibited a higher mean 2H value compared to females. CONCLUSIONS: 4.Deuterium-oxide proved useful in marking mosquitoes in their natural larval sites and although costly, may prove valuable for studies of mosquitoes and other aquatic insects. Based on our field study, we provide a protocol for marking mosquito larval sites using deuterium-oxide.