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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., (© 2021 The Authors. Methods in Ecology and Evolution published by John Wiley & Sons Ltd on behalf of British Ecological Society. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

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 2 H-enriched water are expected to be detectable for over four months using tissue mass-fraction 2 H 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 2 H-enrichment was conducted in Mali, to evaluate potential labeling of wild mosquitoes. Twelve natural larval sites were enriched using [ 2 H]-Deuterium-oxide (D 2 O, 99%). Enrichment level was maintained by supplementation following dilution by rains. Availability of 2 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 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 2 H values increased steadily until ~24 days later (to a mean of approx. 220 ppm). Anopheles and Culex mosquitoes showed significantly different 2 H 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 2 H 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.