Partner study description
15 crosses were contributed to Ag1000G phase 3, 11 of which were previously released in Ag1000G phase 2. Crosses were generated using parents from eight different colonies: G3 (MRA-112); Kisumu (MRA-762); Pimperena (canonical representative of An. gambiae species; MRA-861); Ghana (recent colony of An. coluzzii from Okyereko, southern Ghana(1); Mali-NIH (canonical representative of An. coluzzii species; Niono, MRA-860); (P)Akron (Benin, MRA-913); Nagongera (Tororo, Uganda); and Tiassalé (southern Côte d’Ivoire) (1). The cross family labels, e.g. “29-2”, are identifiers used for each of the crosses within the contributor project and have no special meaning.
Anopheles gambiae is a swarm-mater and crosses were therefore undertaken in mixed groups involving 4-10 females from a single colony with 1-4 males from each from a different colony in plastic cups covered with netting with 10% sugar water provided ad libitum. Females were fed on human blood. After 3 days, males were removed and individually preserved in 95% ethanol. Gravid or half-gravid females were then removed and placed in 1.5ml Eppendorf tubes. Females that did not appear gravid were given a second blood meal before placing in Eppendorf tubes for egg laying. Following egg deposition, females were removed and stored in tubes containing ethanol for subsequent DNA extraction. Eggs were floated in clear plastic trays (15x10x5cm) and, following hatching, larvae were raised on finely-ground fish food (Tetramin). Trays were checked daily and pupae were placed individually into small, labelled centrifuge tubes. Offspring were removed on eclosion and stored in individual tubes containing ethanol. DNA was extracted from parents and offspring using the Qiagen DNeasy kit.
A preliminary assessment of the father of each cross was obtained by genotyping seven microsatellite loci in the mother, potential fathers and five or six offspring. Where possible, the colony of origin of each father was established using individual clustering of the mothers and fathers in BAPS version 5.2, with cluster identity mapped to colony of origin via the mothers (for which the colony was known). The four crosses that are novel to phase 3: B5, K2, K4 and K6, required further analysis to ascertain the true father of each cross, given mother and offspring. For each cross for which the father was in doubt, the list of potential parental pairs was determined. For each of these pairs Mendelian error was computed for every sample of the progeny and the median value (among samples) was plotted. In these four crosses (B5, K2, K4 and K6) one pair yielded median Mendelian errors significantly lower for every autosome than all other pairs, identifying the parsimonious parents. Two of the novel crosses, K4 and K6, were found to be fathered by the same male,
1. Constant V. Edi, Luc Djogbénou, Adam M. Jenkins, Kimberly Regna, Marc A. T. Muskavitch, Rodolphe Poupardin, Christopher M. Jones, John Essandoh, Guillaume K. Kétoh, Mark J. I. Paine, Benjamin G. Koudou, Martin J. Donnelly, Hilary Ranson, and David Weetman. Cyp6 p450 enzymes and ace-1 duplication produce extreme and multiple insecticide resistance in the malaria mosquito anopheles gambiae. PLoS Genet., 10:e1004236, March 2014. https://doi.org/10.1371/journal.pgen.1004236
Craig S. Wilding (C.S.Wilding [at] ljmu.ac.uk) School of Biological and Environmental Sciences, Liverpool John Moores University, Liverpool L3 3AF, UK.
David Weetman (david.weetman [at] lstmed.ac.uk) Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.