title: Using Stable Isotopes of Carbon and Nitrogen to Mark Wild Populations of Anopheles and Aedes Mosquitoes in South-Eastern Tanzania.
abstract: 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 (15N) and carbon (13C), and describe potential field applications.
  
  
  METHODS
  We created man-made aquatic mosquito habitats and added either 15N-labelled potassium nitrate or 13C-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).
  
  
  RESULTS
  Anopheles gambiae s.l and Aedes spp. from enriched 13C and enriched 15N larval habitats had significantly higher isotopic levels than controls (P = 0.005), and both isotopes produced sufficient distinction between marked and unmarked mosquitoes. Mean δ15N for enriched females and males were 275.6±65.1 and 248.0±54.6, while mean δ15N in controls were 2.1±0.1 and 3.9±1.7 respectively. Similarly, mean δ13C 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 δ15N and δ13C 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Environmental Health and Ecological Sciences Thematic Group, Ifakara Health Institute, Ifakara, Tanzania.|Vector Biology Department, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.
authors.forename: Mercy A
authors.initials: MA
authors.lastname: Opiyo
authors.affiliation: Department of Entomology, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Gabriel L
authors.initials: GL
authors.lastname: Hamer
authors.affiliation: Environmental Health and Ecological Sciences Thematic Group, Ifakara Health Institute, Ifakara, Tanzania.
authors.forename: Dickson W
authors.initials: DW
authors.lastname: Lwetoijera
authors.affiliation: Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Lisa D
authors.initials: LD
authors.lastname: Auckland
authors.affiliation: Environmental Health and Ecological Sciences Thematic Group, Ifakara Health Institute, Ifakara, Tanzania.|Vector Biology Department, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.|Innovative Vector Control Consortium, Liverpool, United Kingdom.
authors.forename: Silas
authors.initials: S
authors.lastname: Majambere
authors.affiliation: Environmental Health and Ecological Sciences Thematic Group, Ifakara Health Institute, Ifakara, Tanzania.|School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, Parktown, Republic of South Africa.
authors.forename: Fredros O
authors.initials: FO
authors.lastname: Okumu
mesh_terms: D000330:Aedes / Q000737:chemistry / Q000502:physiology*; D000823:Animal Identification Systems / Q000379:methods*; D000818:Animals; D000852:Anopheles / Q000737:chemistry / Q000502:physiology*; D001947:Breeding; D002247:Carbon Isotopes / Q000032:analysis*; D017753:Ecosystem; D005260:Female; D008297:Male; D009587:Nitrogen Isotopes / Q000032:analysis*; D013636:Tanzania
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes