Fig 1.
Overview of the sequencing assay used to characterize blood meal composition of individual mosquitoes.
(i) A first PCR amplification is performed on DNA extracted from each mosquito targeting ~140 bp of the mammalian mt 16S rRNA (gray) using primers modified with a 5’-end tail complementary to the Illumina sequencing primers (red). (ii) A second PCR amplification incorporates the Illumina adaptors and a 6-nucleotide barcode unique to each mosquito at the ends of the individual blood meal PCR products. (iii) After pooling amplification products from 96 samples together, the PCR products are simultaneously sequenced on an Illumina MiSeq to (iv) generate paired-end reads (in grey) and barcode sequences (grey box). (v) Paired-end reads are then merged to provide error-corrected consensus sequence reads. The dotted black line indicates that the 6-nucleotide barcode corresponding to each read is known but is sequenced independently.
Table 1.
Summary of the amplification range and discriminatory power predicted for the mammalian 16S rRNA primers.
The table indicates, for each mammalian order, the number of species deposited in NCBI for the 16S rRNA genes, the number of species predicted to be amplified by the universal primers as well as the percentage of genera and species that would carry a unique sequence for this locus (enabling their rigorous identification).
Table 2.
Summary of the hosts identified in the mosquito blood meals.
For each host, the number of Anopheles mosquitoes carrying a corresponding DNA sequencing is indicated as well as the highest percent identity between the read generated and the host DNA sequence in NCBI and the average number of reads per sample carrying each DNA sequence.
Fig 2.
Composition of the blood meals for mosquitoes collected in Mirap (A), Kokofine (B), Wasab (C), Dimer (D) and Matukar (E). Each vertical bar shows the composition of the blood meal for one mosquito: each color represents a different host species and the height of each stacked bar corresponds to the proportion of reads matching this host DNA sequence. Gray corresponds to human DNA, turquoise to pig, blue to dog, white to mouse, red to bat and orange to cuscus.
Fig 3.
Neighbor-joining tree showing the relationships among the human mtDNA haplotypes amplified from mosquitoes.
Each symbol represents one DNA sequence amplified from one mosquito. Different shapes represent different Anopheles species (squares-An. punctulatus s.s., triangles-An. farauti 4) and is colored according to the collection site (green-Dimer, blue-Wasab, purple-Kokofine). Mixed blood meals are highlighted by boxes of the same color: for example, the two red boxes show two human mtDNA haplotypes amplified from a single An. farauti 4 mosquito collected in Kokofine.