Molecular and in silico species typing of Anopheles cell lines indicates 4a-3A and 4a-3B are primarily An. coluzzii in origin. A The S200 X6.1 assay [49] molecular species diagnostic was run on genomic DNA isolated from cell lines. Lane 1 contains a 100-bp ladder; lanes 2 and 3 show amplified products from known An. gambiae and An. coluzzii samples, respectively. Lane 4 is amplified product from an equal volume mixture of the PCR template used in lanes 2 and 3 and thus represents a species hybrid. Lanes 5–8 show amplified product from 4a-3B, 4a-3A, Ag55 and Sua4.0 genomic DNA, respectively. Expected band sizes are 479 bp for An. coluzzii and 249 bp for An. gambiae. Lane 9 is a no template control, and lane 10 contains GeneRuler 1-kb Plus DNA Ladder. (B) In silico species typing performed following a previously published method [51]. SNP names follow the naming scheme provided by Lee et al. 2014 [51] with the last five digits of the AGAP gene name followed by the position of the SNP in the coding sequence of the gene (e.g. 01706-129 is a SNP at position 129 in AGAP001706). The second column of the table gives the proportion of An. gambiae ancestry ranging from 0 to 1 with 0 indicating complete An. coluzzii ancestry and 1 indicating complete An. gambiae ancestry. Gray shading indicates the degree of An. gambiae ancestry; dark gray 100% An. coluzzii, light gray 100% An. gambiae and medium gray species hybrids. In silico species typing was also done for the AgamP4 genome assembly; however, this may be an over- or underestimate of An. gambiae ancestry because genome assembly lacks heterozygosity