Improved reference genome of Aedes aegypti informs arbovirus vector control methods
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Flow cytometry
Genome size was estimated by flow cytometry as described, except that the propidium iodide was added at a concentration of 25 µl mg -1, not 50 µl mg -1, and samples were stained in the cold and dark for 24 h to allow the stain to fully saturate the sample. In brief, nuclei were isolated by placing a single frozen head of an adult sample along with a single frozen head of an adult Drosophila virilis female standard from a strain with 1C = 328 Mb into 1 ml of Galbraith buffer (4.26 g MgCl 2, 8.84 g sodium citrate, 4.2 g 3-[ N -morpholino] propane sulfonic acid (MOPS), 1 ml Triton X-100 and 1 mg boiled RNase A in 1 l of ddH 2 O, adjusted to pH 7.2 with HCl and filtered through a 0.22-µm filter) and grinding with 15 strokes of the A pestle at a rate of 3 strokes per 2 s. The resultant ground mixture was filtered through a 60-µm nylon filter (Spectrum La...
Pacific Biosciences library construction, sequencing and assembly
HMW DNA extraction for Pacific Biosciences sequencing was performed using the Qiagen MagAttract Kit (67563) following the manufacturer's protocol with approximately 80 male sibling pupae in batches of 25 mg.
Bionano optical mapping
HMW DNA extraction was performed using the Bionano Animal Tissue DNA Isolation Kit (RE-013-10), with a few protocol modifications. A single-cell suspension was made as follows. First, 47 mg of frozen male pupae was fixed in 2% v/v formaldehyde in Homogenization Buffer from the kit (Bionano 20278), for 2 min on ice. Then, the pupae were roughly homogenized by blending for 2 s, using a rotor-stator tissue homogenizer (TissueRuptor, Qiagen 9001271). After another 2 min fixation, the tissue was finely homogenized by running the rotor-stator for 10 s. Subsequently, the homogenate was filtered with a 100-µm nylon filter, fixed with ethanol for 30 min on ice, spun down, and washed with more Homogenization Buffer (to remove residual formaldehyde). The final pellet was resuspended in Homogenization Buffer. A single agarose plug was made using the resuspended cells, using the C...
ATAC-seq
The previously described ATAC-seq protocol was adapted for Ae. aegypti brains. Individual brains from LVP_MR4 non-blood-fed females (Extended Data Fig. ) or females 48 h or 96 h after taking a human blood meal (data not shown) were dissected in 1 × PBS, immediately placed in 100 µl ice-cold ATAC lysis buffer (10 mM Tris-Hcl, pH 7.4, 10 mM NaCl, 3 mM MgCl 2, 0.1% IGEPAL CA-630), and homogenized in a 1.5-ml Eppendorf tube using 50 strokes of a Wheaton 1-ml PTFE-tapered tissue grinder. Animals at 96 h after the blood meal were deprived of access to a water oviposition site and were considered gravid at the time of dissection. Lysed brains were centrifuged at 400 g for 20 min at 4 °C and the supernatant was discarded. Nuclei were resuspended in 52.5 µl 1 × Tagmentation buffer (provided in the Illumina Nextera DNA Library Prep Kit) and 5 µl were removed t...
QTL mapping of DENV vector competence
Ultimately, AaegL5 has a markedly improved QTL mapping resolution over AaegL3. For instance, we mapped the same QTL underlying systemic DENV dissemination at the extremity of chromosome 2 with both AaegL3 and AaegL5. The 1.5 LOD support interval was much larger for the AaegL3-guided linkage map (0-50 cM, 74% of the linkage group) than for the AaegL5-guided linkage map (0-17 cM, 9% of the linkage group). We present this analysis in Extended Data Fig..
Vector competence
Four low-passage DENV isolates were used to orally challenge the F 2 females as previously described. In brief, four random groups of females from the F 2 progeny were experimentally exposed to two virus isolates belonging to DENV serotype 1 (KDH0026A and KDH0030A) and two virus isolates belonging to DENV serotype 3 (KDH0010A and KDH0014A), respectively. All four virus isolates were derived from human serum specimens collected in 2010 from clinically ill patients who were infected with DENV at the Kamphaeng Phet Provincial Hospital. Because the viruses were isolated in the laboratory cell culture, informed consent of the patients was not necessary for the present study. Complete viral genome sequences were previously deposited into GenBank (accession numbers HG316481, HG316582, HG316483, and HG316484 ). Phylogenetic analysis assigned the viruses to known viral lineages that were...
Mapping insecticide resistance and VGSC
The mosquito population Viva Caucel from Yucatán State in Southern Mexico (longitude -89.71827, latitude 20.99827), was collected in 2011 through the Universidad Autónoma de Yucatán. We identified up to 25 larval breeding sites from 3-4 city blocks and collected around 1,000 larvae. Larvae were allowed to eclose, and twice a day we aspirated the adults from the cartons, discarding anything other than Ae. aegypti. Then, 300-400 Ae. aegypti were released into a 2-foot (61-cm) cubic cage where they were allowed to mate for up to five days with ad libitum access to sucrose, after which they were blood fed to collect eggs for the next generation. Then, 390 adult mosquitoes were phenotyped for deltamethrin resistance. We exposed groups of 50 mosquitoes (3-4 days old) to 3 µg of deltamethrin-coated bottles for 1 h. After this time, active mosquito...
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Evidence quotes (7)
Genome size was estimated by flow cytometry as described, except that the propidium iodide was added at a concentration of 25 µl mg -1, not 50 µl mg -1, and samples were stained in the cold and dark for 24 h to allow the stain to fully saturate the sample. In brief, nuclei were isolated by placing a single frozen head of an adult sample along with a single frozen head of an adult Drosophila virilis female standard from a strain with 1C = 328 Mb into 1 ml of Galbraith buffer (4.26 g MgCl 2, 8.84 g sodium citrate, 4.2 g 3-[ N -morpholino] propane sulfonic acid (MOPS), 1 ml Triton X-100 and 1 mg boiled RNase A in 1 l of ddH 2 O, adjusted to pH 7.2 with HCl and filtered through a 0.22-µm filter) and grinding with 15 strokes of the A pestle at a rate of 3 strokes per 2 s. The resultant ground mixture was filtered through a 60-µm nylon filter (Spectrum Labs). Samples were stained with 25 µg of propidium iodide and held in the cold (4 °C) and dark for 24 h at which time the relative red fluorescence of the 2C nuclei of the standard and sample were determined using a Beckman Coulter CytoFlex flow cytometer with excitation at 488 nm. At least...
HMW DNA extraction for Pacific Biosciences sequencing was performed using the Qiagen MagAttract Kit (67563) following the manufacturer's protocol with approximately 80 male sibling pupae in batches of 25 mg.
HMW DNA extraction was performed using the Bionano Animal Tissue DNA Isolation Kit (RE-013-10), with a few protocol modifications. A single-cell suspension was made as follows. First, 47 mg of frozen male pupae was fixed in 2% v/v formaldehyde in Homogenization Buffer from the kit (Bionano 20278), for 2 min on ice. Then, the pupae were roughly homogenized by blending for 2 s, using a rotor-stator tissue homogenizer (TissueRuptor, Qiagen 9001271). After another 2 min fixation, the tissue was finely homogenized by running the rotor-stator for 10 s. Subsequently, the homogenate was filtered with a 100-µm nylon filter, fixed with ethanol for 30 min on ice, spun down, and washed with more Homogenization Buffer (to remove residual formaldehyde). The final pellet was resuspended in Homogenization Buffer. A single agarose plug was made using the resuspended cells, using the CHEF Mammalian Genomic DNA Plug Kit (BioRad 170-3591), following the manufacturer's instructions. The plug was incubated with Lysis Buffer (Bionano 20270) and Puregene Proteinase K (Qiagen 1588920) overnight at 50 °C, then again the following morning for 2 h (using new buffer and Proteinas...
The previously described ATAC-seq protocol was adapted for Ae. aegypti brains. Individual brains from LVP_MR4 non-blood-fed females (Extended Data Fig. ) or females 48 h or 96 h after taking a human blood meal (data not shown) were dissected in 1 × PBS, immediately placed in 100 µl ice-cold ATAC lysis buffer (10 mM Tris-Hcl, pH 7.4, 10 mM NaCl, 3 mM MgCl 2, 0.1% IGEPAL CA-630), and homogenized in a 1.5-ml Eppendorf tube using 50 strokes of a Wheaton 1-ml PTFE-tapered tissue grinder. Animals at 96 h after the blood meal were deprived of access to a water oviposition site and were considered gravid at the time of dissection. Lysed brains were centrifuged at 400 g for 20 min at 4 °C and the supernatant was discarded. Nuclei were resuspended in 52.5 µl 1 × Tagmentation buffer (provided in the Illumina Nextera DNA Library Prep Kit) and 5 µl were removed to count nuclei on a haemocytometer. In total, 50,000 nuclei were used for each transposition reaction. The concentration of nuclei in Tagmentation buffer was adjusted to 50,000 nuclei in 47.5 µl Tagmentation buffer and 2.5 µl Tn5 enzyme was added (provided in the Illumina Nextera DNA Libra...
Ultimately, AaegL5 has a markedly improved QTL mapping resolution over AaegL3. For instance, we mapped the same QTL underlying systemic DENV dissemination at the extremity of chromosome 2 with both AaegL3 and AaegL5. The 1.5 LOD support interval was much larger for the AaegL3-guided linkage map (0-50 cM, 74% of the linkage group) than for the AaegL5-guided linkage map (0-17 cM, 9% of the linkage group). We present this analysis in Extended Data Fig..
Four low-passage DENV isolates were used to orally challenge the F 2 females as previously described. In brief, four random groups of females from the F 2 progeny were experimentally exposed to two virus isolates belonging to DENV serotype 1 (KDH0026A and KDH0030A) and two virus isolates belonging to DENV serotype 3 (KDH0010A and KDH0014A), respectively. All four virus isolates were derived from human serum specimens collected in 2010 from clinically ill patients who were infected with DENV at the Kamphaeng Phet Provincial Hospital. Because the viruses were isolated in the laboratory cell culture, informed consent of the patients was not necessary for the present study. Complete viral genome sequences were previously deposited into GenBank (accession numbers HG316481, HG316582, HG316483, and HG316484 ). Phylogenetic analysis assigned the viruses to known viral lineages that were circulating in southeast Asia in the previous years. Each isolate was amplified twice in C6/36 ( Aedes albopictus ) cell lines (maintained at AFRIMS in Bangkok, Thailand; used only to grow virus, not explicitly authenticated or checked for mycoplasma contamination) before vector competence assays....
The mosquito population Viva Caucel from Yucatán State in Southern Mexico (longitude -89.71827, latitude 20.99827), was collected in 2011 through the Universidad Autónoma de Yucatán. We identified up to 25 larval breeding sites from 3-4 city blocks and collected around 1,000 larvae. Larvae were allowed to eclose, and twice a day we aspirated the adults from the cartons, discarding anything other than Ae. aegypti. Then, 300-400 Ae. aegypti were released into a 2-foot (61-cm) cubic cage where they were allowed to mate for up to five days with ad libitum access to sucrose, after which they were blood fed to collect eggs for the next generation. Then, 390 adult mosquitoes were phenotyped for deltamethrin resistance. We exposed groups of 50 mosquitoes (3-4 days old) to 3 µg of deltamethrin-coated bottles for 1 h. After this time, active mosquitoes were transferred to cardboard cups and placed into an incubator (28 °C and 70% humidity) for 4 h; these mosquitoes were referred to as the resistant group. Immobile mosquitoes were transferred to a second cardboard cup. After 4 h, newly recovered mosquitoes were aspirated, frozen and labelle...
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