Recent results of high-altitude windborne mosquito migration raised questions about the viability of these mosquitoes despite ample evidence that many insect species, including other dipterans, have ...been known to migrate regularly over tens or hundreds of kilometers on high-altitude winds and retain their viability. To address these concerns, we subjected wild Anopheles gambiae s.l. Giles mosquitoes to a high-altitude survival assay, followed by oviposition (egg laying) and blood feeding assays. Despite carrying out the survival assay under exceptionally harsh conditions that probably provide the lowest survival potential following high altitude flight, a high proportion of the mosquitoes survived for 6- and even 11-h assay durations at 120- to 250-m altitudes. Minimal differences in egg laying success were noted between mosquitoes exposed to high altitude survival assay and those kept near the ground. Similarly, minimal differences were found in the female's ability to take an additional bloodmeal after oviposition between these groups. We conclude that similar to other high-altitude migrating insects, mosquitoes are able to withstand extended high-altitude flight and subsequently reproduce and transmit pathogens by blood feeding on new hosts.
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.
Current mark–release–recapture methodologies are limited in their ability to address complex problems in vector biology, such as studying multiple groups overlapping in space and time. Additionally, ...limited mark retention, reduced post‐marking survival and the large effort in marking, collection and recapture all complicate effective insect tracking.
We have developed and evaluated a marking method using a fluorescent dye (SmartWater®) combined with synthetic DNA tags to informatively and efficiently mark adult mosquitoes using an airbrush pump and nebulizer. Using a handheld UV flashlight, the fluorescent marking enabled quick and simple initial detection of recaptures in a field‐ready and non‐destructive approach that when combined with an extraction‐free PCR on individual mosquito legs provides potentially unlimited marking information.
This marking, first tested in the laboratory with Anopheles gambiae s.l. mosquitoes, did not affect survival (median ages 24–28 days, p‐adj > 0.25), oviposition (median eggs/female of 28.8, 32.5, 33.3 for water, green, red dyes, respectively, p‐adj > 0.44) or Plasmodium competence (mean oocysts 5.56–10.6, p‐adj > 0.95). DNA and fluorescence had 100% retention up to 3 weeks (longest time point tested) with high intensity, indicating marks would persist longer.
We describe a novel, simple, no/low‐impact and long‐lasting marking method that allows separation of multiple insect subpopulations by combining unlimited length and sequence variation in the synthetic DNA tags. This method can be readily deployed in the field for marking multiple groups of mosquitoes or other insects.
Résumé
Dans la biologie des vecteurs, Les méthodologies actuelles de marquage, lâcher et recapture sont limitées dans leur habilité à résoudre les problèmes complexes de la biologie des vecteurs tels que l’étude de plusieurs groupes se chevauchant dans l’espace et dans le temps. En plus, la courte durée de vie des marquages, la réduction de la longévité après marquage et les gros efforts que demande le marquage, la collecte et la recapture, compliquent davantage le suivi efficace des insectes.
Nous avons développé et évalué une technique de marquage utilisant un colorant fluorescent (SmartWater®) combiné à des marqueurs d’ADN synthétiques pour marquer de manière informative et efficace des moustiques adultes à l'aide d'une pompe à air munie d'un nébuliseur. À l'aide d'une lampe de poche portable à UV, la marque fluorescente est détectée de façon rapide et simple lors des recaptures sur le terrain. Cette approche non destructive, combinée à une PCR sans extraction d’ADN sur des pattes de moustiques individuelles peut fournir potentiellement des informations illimitées sur le marquage.
Ce type de marquage, testé d'abord au laboratoire avec des moustiques d’Anopheles gambiae s.l., n'a pas affecté leur survie (âge médian 24‐28 jours, p‐adj> 0.25), la ponte (médiane œufs / femelle de 28,8, 32,5, 33,3 pour l'eau, colorants verts et rouges, respectivement, p‐adj> 0.44) ou leur compétence de s’infecter avec Plasmodium (oocystes moyens de 5.56 à 10.6, p‐adj> .95). L'ADN et la fluorescence avaient une rétention de 100% jusqu'à 3 semaines (le plus long moment testé) avec une intensité élevée, indiquant que les marques persisteraient plus longtemps.
Nous décrivons une nouvelle méthode de marquage simple, durable avec sans/ou faible impact et durable qui permet de séparer plusieurs sous‐populations d'insectes en combinant avec une longueur illimitée et la variation de séquences dans les marqueurs d'ADN synthétiques. Cette méthode peut être facilement déployée sur le terrain pour marquer plusieurs groupes de moustiques ou d'autres insectes.
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 4 months using tissue mass fraction 2H measurements, providing opportunities for long‐term mark–capture studies on a large scale.
A laboratory study followed by a field experiment of mosquito larval habitat 2H enrichment was conducted in Mali, to evaluate potential labelling 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.
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 c. 24 days later (to a mean of c. 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.
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.
Résumé
Suivre les moustiques dans la nature à l'aide des techniques actuelles de Marquage‐Lâcher‐Recapture est réservé à des études spatio‐temporelles à petites échelles, faisant ressortir de nombreuses insuffisances dans la compréhension des mouvements à longue distance et pour une survie prolongée. Des approches innovantes sur le suivi des moustiques peuvent fournir de nouvelles informations sur leur biologie enfin d’améliorer les activités d’intervention qui visent à réduire la transmission de la maladie. L'enrichissement en isotopes stables des gîtes larvaires naturels des moustiques permet un marquage à grande échelle des moustiques sauvages sans manipulation manuelle de l’homme. Les moustiques issus des larves qui se développent dans une eau enrichie en Deutérium (2H) sont supposés être identifiables pendant plus de quatre mois à l'aide des mesures du 2H dans la fraction de masse tissulaire, offrant ainsi des opportunités pour des études à grande échelle de Marquage‐Lâcher‐Recapture à long terme.
Une étude de laboratoire suivie d'expérimentations sur le terrain en vue d’enrichir les gîtes larvaires des moustiques en 2H a été menée au Mali afin d'évaluer un marquage potentiel des moustiques sauvages. Douze gîtes larvaires naturels ont été enrichis à l’aide d’oxyde de Deutérium 2H (D2O, 99%). Le niveau d'enrichissement a été maintenu par une compensation après dilution des gîtes par les eaux de pluies. Pour augmenter le niveau de marquage des larves, un régime alimentaire composé de microorganismes (protozoaires, algues et bactéries) collectés localement et élevés dans de l’eau très enrichie en Deutérium leur a été fournis. Les présumés prédateurs naturels ont été retirés des gîtes larvaires et un apport externe supplémentaire de larves de premier stade d’Anopheles gambiae s.l. a été fait tous les deux jours. Des pièges de capture après émergence ont permis la collecte des imagos. Les moustiques adultes collectés ont été maintenus au laboratoire pour des analyses de détection du niveau de marquage en fonction de l’âge.
L'enrichissement en Deutérium des moustiques sauvages au‐dessus du niveau référentiel (maximum = 143,1 ppm) est devenu apparent 5 à 6 jours après exposition, ensuite les valeurs de 2H ont régulièrement augmenté jusqu'à environ 24 jours au plus tard (où une moyenne d'environ 220 ppm est atteinte). Les Anophèles et Culex présentaient des valeurs de 2H significativement différentes (211 et 194,2 ppm respectivement). Les deux genres ont présenté une usure exponentielle des traces de marquage (e(‐x)) s'élevant à 21,6% au 30e jour après émergence, puis le taux d'usure du marquage a continuellement diminué. Les mâles des deux taxons présentaient une valeur moyenne de 2H supérieure à celle des femelles.
Malgré le coût, l'oxyde de Deutérium s'est montré efficace dans le marquage des moustiques dans leurs gîtes larvaires naturels ; cela serait d’une grande importance dans les études concernant les moustiques et autres insectes aquatiques. A partir de nos résultats sur terrain dans cette étude, nous venons de produire un protocole sur le marquage des gîtes larvaires de moustiques à l’oxyde de deutérium.
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.
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.
The identification and dissection of the recurrent laryngeal nerve is essential to guarantee its anatomical and functional integrity. The superior approach of the recurrent nerve is a reliable ...surgical alternative. Various indications are recognized with a reliable landmark. This is the entry point into the larynx under the inferior horn of the thyroid cartilage. The limits of this technique, namely, the extralaryngeal divisions and the hemorrhages encountered at the point of entry of the larynx are a source of morbidity of the recurrent laryngeal nerve. A careful dissection, respect for the surgical steps, and the surgeon's experience are guarantees of a good result. We wanted through a review of the literature and our experience in the superior approach to discuss surgical indications, to identify landmarks at the point of entry of the larynx, to determine the limits of this approach, and to take precautions to mitigate the risk of recurrent laryngeal nerve injury.
Recent results of high-altitude windborne mosquito migration raised questions about the viability of these mosquitoes despite ample evidence that many insect species, including other dipterans, have ...been known to migrate regularly over tens or hundreds of kilometers on high-altitude winds and retain their viability. To address these concerns, we subjected wild Anopheles gambiae s.l. Giles mosquitoes to a high-altitude survival assay, followed by oviposition (egg laying) and blood feeding assays. Despite carrying out the survival assay under exceptionally harsh conditions that probably provide the lowest survival potential following high altitude flight, a high proportion of the mosquitoes survived for 6- and even 11-h assay durations at 120- to 250-m altitudes. Minimal differences in egg laying success were noted between mosquitoes exposed to high altitude survival assay and those kept near the ground. Similarly, minimal differences were found in the female's ability to take an additional bloodmeal after oviposition between these groups. We conclude that similar to other highaltitude migrating insects, mosquitoes are able to withstand extended high-altitude flight and subsequently reproduce and transmit pathogens by blood feeding on new hosts. Key words: altitude, disease-vector, egg-laying, long-range dispersal, wind
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.
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