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Background  Marking wild mosquitoes is important for understanding their ecology, behaviours and role in disease transmission. Traditional insect marking techniques include using fluorescent dyes, protein labels, radioactive labels and tags, but such techniques have various limitations; notably low marker retention and inability to mark wild mosquitoes at source. Stable isotopes are gaining wide spread use for non-invasive marking of arthropods, permitting greater understanding of mosquito dispersal and responses to interventions. We describe here a simple technique for marking naturally-breeding malaria and dengue vectors using stable isotopes of nitrogen ( 15 N) and carbon ( 13 C), and describe potential field applications.    Methods  We created man-made aquatic mosquito habitats and added either  15 N-labelled potassium nitrate or  13 C-labelled glucose, leaving non-adulterated habitats as controls. We then allowed wild mosquitoes to lay eggs in these habitats and monitored their development  in situ . Pupae were collected promptly as they appeared and kept in netting cages. Emergent adults (in pools of ~4 mosquitoes/pool) and individually stored pupae were desiccated and analysed using Isotope Ratio Mass Spectrometry (IRMS).    Findings   Anopheles gambiae  s.l and  Aedes  spp. from enriched  13 C and enriched  15 N larval habitats had significantly higher isotopic levels than controls (P = 0.005), and both isotopes produced sufficient distinction between marked and unmarked mosquitoes. Mean δ 15 N for enriched females and males were 275.6±65.1 and 248.0±54.6, while mean δ 15 N in controls were 2.1±0.1 and 3.9±1.7 respectively. Similarly, mean δ 13 C for enriched females and males were 36.08±5.28 and 38.5±6.86, compared to -4.3±0.2 and -7.9±3.6 in controls respectively. Mean δ 15 N and δ 13 C was significantly higher in any pool containing at least one enriched mosquito compared to pools with all unenriched mosquitoes, P<0.001. In all cases, there were variations in standardized isotopic ratios between mosquito species.    Conclusion  Enrichment of semi-natural mosquito larval habitats with stable isotopes of nitrogen and carbon resulted in effective marking of  Anopheles  and  Aedes  mosquitoes colonizing these habitats. This approach can significantly enhance studies on mosquito eco-physiology, dispersal, pathogen transmission and responses to control measures.

Using Stable Isotopes of Carbon and Nitrogen to Mark Wild Populations of Anopheles and Aedes Mosquitoes in South-Eastern Tanzania