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.
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 (
H) 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.
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.
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
H-enriched water are expected to be detectable for over four months using tissue mass-fraction
H measurements, providing opportunities for long-term mark-capture studies on a large scale.
2.A laboratory study followed by a field experiment of mosquito larval habitat
H-enrichment was conducted in Mali, to evaluate potential labeling of wild mosquitoes. Twelve natural larval sites were enriched using
H-Deuterium-oxide (D
O, 99%). Enrichment level was maintained by supplementation following dilution by rains. Availability of
H 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
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.
3.Deuterium enrichment of wild mosquitoes above background levels (maximum = 143.1 ppm) became apparent 5-6 days after initial exposure, after which
H values increased steadily until ~24 days later (to a mean of approx. 220 ppm).
and
mosquitoes showed significantly different
H values (211 and 194.2 ppm respectively). Both genera exhibited exponential label attrition (
) amounting to 21.6% by day 30 post emergence, after which attrition rate continuously decreased. Males of both taxa exhibited a higher mean
H value compared to females.
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.
Recent studies have reported
mosquitoes captured at high-altitude (40-290 m above ground) in the Sahel. Here, we describe this migration modality across genera and species of African Culicidae and ...examine its implications for disease transmission and control. As well as
, six other genera-
, and
comprised 90% of the 2,340 mosquitoes captured at altitude. Of the 50 molecularly confirmed species (
= 2,107), 33 species represented by multiple specimens were conservatively considered high-altitude windborne migrants, suggesting it is a common migration modality in mosquitoes (31-47% of the known species in Mali), and especially in
(45-59%). Overall species abundance varied between 2 and 710 specimens/species (in
and
, respectively). At altitude, females outnumbered males 6:1, and 93% of the females have taken at least one blood meal on a vertebrate host prior to their departure. Most taxa were more common at higher sampling altitudes, indicating that total abundance and diversity are underestimated. High-altitude flight activity was concentrated between June and November coinciding with availability of surface waters and peak disease transmission by mosquitoes. These hallmarks of windborne mosquito migration bolster their role as carriers of mosquito-borne pathogens (MBPs). Screening 921 mosquitoes using pan-
assays revealed that thoracic infection rate in these high-altitude migrants was 2.4%, providing a proof of concept that vertebrate pathogens are transported by windborne mosquitoes at altitude. Fourteen of the 33 windborne mosquito species had been reported as vectors to 25 MBPs in West Africa, which represent 32% of the MBPs known in that region and include those that inflict the heaviest burden on human and animal health, such as malaria, yellow fever, dengue, and Rift Valley fever. We highlight five arboviruses that are most likely affected by windborne mosquitoes in West Africa: Rift Valley fever, O'nyong'nyong, Ngari, Pangola, and Ndumu. We conclude that the study of windborne spread of diseases by migrating insects and the development of surveillance to map the sources, routes, and destinations of vectors and pathogens is key to understand, predict, and mitigate existing and new threats of public health.
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