The range of the mosquito Aedes aegypti continues to expand, putting more than two billion people at risk of arboviral infection. The sterile insect technique (SIT) has been used to successfully combat agricultural pests at large scale, but not mosquitoes, mainly because of challenges with consistent production and distribution of high-quality male mosquitoes. We describe automated processes to rear and release millions of competitive, sterile male Wolbachia-infected mosquitoes, and use of these males in a large-scale suppression trial in Fresno County, California. In 2018, we released 14.4 million males across three replicate neighborhoods encompassing 293 hectares. At peak mosquito season, the number of female mosquitoes was 95.5% lower (95% CI, 93.6–96.9) in release areas compared to non-release areas, with the most geographically isolated neighborhood reaching a 99% reduction. This work demonstrates the high efficacy of mosquito SIT in an area ninefold larger than in previous similar trials, supporting the potential of this approach in public health and nuisance-mosquito eradication programs.
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Adult count data from field traps analyzed in this study are included as supplementary tables. Per-site male release numbers are also included as supplementary tables. Genome sequencing data can be found under Bioproject PRJNA600991 at NCBI. Training image data and the trained neural-net model for male–female classification can be accessed by visiting https://github.com/verilylifesciences/classifaedes.
Scripts for analysis of trap data are available upon request. Scripts for organizing machine learning training data and conducting model training can be found at https://github.com/verilylifesciences/classifaedes.
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We are grateful to the residents of Fresno County for their support and participation in the study. We are also thankful to the many staff members at Verily and Consolidated Mosquito Abatement District for their contributions.
J.E.C., D.W.C, V.C., M.D., K.G., K.C.H., P. H., J.S.H., J.L., C.B., R.B., W.C., C.E., D.G., Y.H., B.H., E.K., J.K., A.K., E.L., T.L., J.L., M.L., W.M., M.M., S.N.M., D.M., J.R.O., K.P., A.P., C.R., M.S., R.S., P.S., J.S., J.S., B.W. A.M.W., M.W., J.W., A.Y., W.C.C., N.S., L.U., T.Z., P.M. and B.J.W. were paid employees of Verily Life Sciences, a for-profit company developing products for mosquito control, at the time they performed research for this study. K.L.D., J.W.M. and S.L.D. were paid employees of Mosquito Mate, a for-profit company developing products for mosquito control, at the time they performed research for this study. S.L.D. and The University of Kentucky Research Foundation hold a patent (US7868222B1) on the use of Wolbachia for mosquito control.
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Integrated supplementary information
Supplementary Figure 1 Spatial distribution of released males under housing-density model and after releases.
Top row: satellite maps with treatment areas outlined in white and treatment buffers indicated with B in T2. Middle row: Target density of Wolbachia-males based on local housing density per street segment. Bottom row: Density of Wolbachia-males, as measured by the onboard mosquito counter, averaged over six months of releases assuming a 100 m dispersal kernel.
Mean number of eggs per trap from twice-weekly egg paper collection in each treatment area on left (red) and each control area (yellow) on right. Mean was calculated from all papers for a given collection day. Shaded areas indicate 95% bootstrap confidence intervals calculated by bootstrap sampling of egg papers, such that confidence interval size corresponds to variability among papers and number of papers intervals (nT1 = 61, nT2 = 35, nT3 = 41, nC1 = 30, nC2 = 32, nC3 = 15 independent trap samples per collection day). Dotted lines indicate first and last days of 2018 releases.
Hatch rates from twice-weekly egg paper collection in each treatment area on left (red) and each control area (yellow) on right. Hatch rate was calculated by combining all egg-positive papers for a given collection day. Shaded areas indicate 95% bootstrap confidence intervals calculated by bootstrap sampling of egg-positive papers, such that confidence interval size corresponds to variability among papers and number of papers (nT1 = 61, nT2 = 35, nT3 = 41, nC1 = 30, nC2 = 32, nC3 = 15 independent trap samples per collection day). Dotted lines indicate first and last days of 2018 releases.
Proportion of Fresno-Clovis ancestry in backcross colonies WB1-CL4 and WB1-CL5 estimated using whole-genome sequencing (~5x) of backcrossed individuals at generations 4 (BC4), 5 (BC5), and 6 (BC6) compared to ‘parental’ WB1-Waco and G1 generations of Fresno-Clovis colonies (CL4 and CL5) . Each dot is an individual mosquito and black horizontal lines indicate the mean across all samples within a generation and colony.
In all panels, dashed lines mark the start and end of releases, while the solid line indicates the switch from the backcross colony (WB1-CL1-BC5) to the laboratory colony (WB1-Waco). (a) Number of Wolbachia-males released per day into T3 in 2017. We released a total of 7,316,993 males, of which 1,278,064 were WB1-CL1-BC5 males and 6,038,929 were WB1-Waco. (b) Mean number of males caught per CO2-baited trap in the 2017 treatment (red) and control site (yellow). More backcross males were caught per trap night despite lower release levels. (nT3 = 45, nC2 = 38 independent trap samples per collection day) (c) Mean number of females per CO2-baited trap night in 2017 treatment and control sites (sample sizes same as panel b). After Wolbachia-males were released, female abundance declined in the treatment relative to the control site. (d) Estimated ratio of Wolbachia-males to wild males in treatment site (n = 40 independent trap samples per collection week). (e) Hatch rate of eggs collected in treatment and control sites (nT3 = 81, nC2 = 64 independent trap samples per collection day). (f) Mean number of larvae per twice weekly collection in treatment and control site (sample sizes same as panel e). (g) Cumulative average number of larvae collected over the 2017 field season in treatment and control site (sample sizes same as panel e). (h) Percent suppression in the treatment site relative to the control site based on two-week rolling averages of females per trap (sample sizes same as panel b). For panels b-h, 95% bootstrap confidence intervals are shown as shaded area or bars.
(a) Wolbachia titers (ratio of Wolbachia genome copies to mosquito genome copies) in whole female mosquitoes from the WB1-CL1-BC5 backcross (yellow) and WB1-Waco (black) at different points in 2017. Each dot is an individual mosquito. A jump in Wolbachia titer occurred in July coincident with the colony collapse. (b) Same as (a), except Wolbachia titers measured in whole male mosquitoes. (c) Wolbachia titers in whole adult female mosquitoes across the 2018 season for the two release colonies. Each dot is the mean of ~96 independent, individual female mosquito titer ratios with the interquartile range indicated by the line. (d) Same as (c), except Wolbachia titers measured in whole male adult mosquitoes.
A receiver operating characteristic (ROC) plot for the machine learning model used to find female mosquitoes that were accepted by the industrial vision system. False positive rate (that is probability of female being misclassified as a male) shown on x-axis and true positive rate (that is probably that a male is improperly classified as female or sensitivity) shown on the y-axis.
(a) BG-Sentinel fan speed values plotted as a function of remaining trap battery voltage. Measurements taken from 20 test BG traps once per day until the battery died. Scraps of paper were added to traps represented by grey dots in order to mimic the effect of captured mosquitoes and other insects and debris on air flow. Pearson’s r = 0.95 (one-sided correlation test: P = 2.2 x 10−16). (b) Number of female mosquitoes captured in a BG trap night at control sites plotted against the trap fan speed as measured immediately before mosquitoes were removed from the trap. Analysis limited to collections after June 01 and before October 01. Pearson’s r = 0.13 (one-sided correlation test: P = 6.7 x 10−5).
(a) Each collection without dry ice (CO2) lasted six days and was conducted in between weekly dry ice collections. Mean number of females collected per six-day collection across the 2018 field season in control and treatment sites (nT1 = 44, nT2 = 24, nT3 = 35, nC1 = 17, nC2 = 28, nC3 = 15 independent trap samples per collection day) with dashed lines denoting the start and stop of releases. (b) Mean number of females in aggregated treatment and control sites per six-day collection (nTRT = 103, nCTRL = 60 independent trap samples per collection week). The results mirror the dry ice trapping results. 95% bootstrap confidence intervals are shown as shaded areas.
(a) Probability curves indicating likelihood of sampling given numbers of wild males on the x-axis under various assay sample sizes indicated by the legend. The subpanels display different true overflooding ratios including 10:1, 20:1, 30:1, and 40:1 indicated by subpanel titles. (b) Probabilities of overflooding ratio estimate bias under a true overflooding ratio of 5:1. Bias was estimated by comparing simulated subsampled collections to the ratio of the full sample before subsampling. Positive bias estimates indicate the strategy will overestimate the overflooding ratio. Each curve corresponds to a different sampling strategy (see Supplementary Text for details). Blue and purple curves are difficult to distinguish because these strategies had nearly identical bias distributions and mask each other (same for c-e). (c) Probability curves under true overflooding ratio of 10:1. (d) Probability curves under true overflooding ratio of 30:1. (e) Probability curves under true overflooding ratio of 50:1. Note the different x and y axis ranges in panels b-e.
Supplementary Figure 11 Alternative relatedness statistics and impact of total site counts on HETHET/IBS0 relatedness estimates.
(a) Histogram showing the distribution of HETHET/IBS0 relatedness scores for pairwise comparisons within control area egg collections. Values of 10 or above indicate full sibling relationships, while values of approximately two indicate the pair are not closely related (Ann. Hum. Genet. 67, 618-619, 2003; Am. J. Hum. Genet. 81, 559-575, 2007). (b) Proportion of sites that are IBS2 plotted as a function of the proportion of sites that are IBS0 for each pairwise comparison within control area egg collections. Full siblings are expected to cluster with high IBS2 values and low IBS0 values while unrelated individuals are expected to plot low on the IBS2 axis and middle to high on the IBS0 axis (Ann. Hum. Genet. 70, 841,847, 2006). (c) For each pairwise comparison within control area egg collections, the fraction of sites that are informative is plotted as a function of IBS2*_ratio (PLoS Genet.7, e1002287, 2011). Full siblings are expected to exhibit relatively high informative site counts and high IBS2*_ratio scores while unrelated individuals are expected to have low informative sites counts and intermediate IBS2*_ratio values (PLoS Genet.7, e1002287, 2011). (d) HETHET/IBS0 values plotted as a function of total number of sites with data as a proxy for read depth. Colors indicate the site class of individuals for each comparison. The distribution indicates that comparisons represented by fewer sites with data are not biased towards low or high HETHET/IBS0 scores suggesting that read depth is not biasing relatedness estimates.
Heatmap showing relatedness estimates calculated between all pairs of individuals. Trap collections are separated by black lines and each tile indicates a pairwise comparison among individuals with the color corresponding to the IBS2/IBS0 relatedness estimate (Ann. Hum. Genet. 67, 618-619, 2003; Am. J. Hum. Genet. 81, 559-575, 2007) according to colors in the legend. Values of 10 or above indicate full sibling relationships, while values of approximately two indicate the pair are not closely related. Within-trap-collection comparisons are outlined in orange for emphasis. See Supplementary Text for further details.
Supplementary Figures 1–12, Supplementary Note 1, and Supplementary Tables 1 and 3–8
Larval Rearing System (LRS) and container unload. The first clip in the video shows an example of robotic arm inside incubated LRS retrieving a tray carrying two larval containers in preparation for container unload and draining at the end of larval development. The second clip shows the larval containers being transferred out of the unload station on the LRS to the draining system where the containers are opened with a blade and contents allowed to drain towards the pupal sieving station.
Adult inspection during visual sex-sorting The video shows an example of adult male mosquitoes walking singularly down the path of a real-time visual sex-sorter. Adults are inspected by the camera-based vision system while walking along the path and either moved into the release tube or into the discard channel based on a decision made by the vision-system algorithm. In addition, multiple photos of each adult are stored for further cloud-based evaluation.
Number of Wolbachia males released in each site in 2018. Excel spreadsheet (.xlsx) with table indicating the number of males from each colony released each day for each treatment area in 2018.
2018 Adult BG-Sentinel data from treatment and control sites. Excel spreadsheet (.xlsx) with legend sheet and raw data sheet containing raw adult trap counts for treatment and control areas in 2018.
2017 Adult BG-Sentinel data from treatment and control sites. Excel spreadsheet (.xlsx) with legend sheet and raw data sheet containing raw adult trap counts for treatment and control areas in 2017.
2017 Egg trap data from treatment and control sites. Excel spreadsheet (.xlsx) with legend sheet and raw data sheet containing raw egg trap counts and larval hatch counts for treatment and control areas in 2017.
Number of Wolbachia males released in each site in 2017. Excel spreadsheet (.xlsx) with table indicating the number of males from each colony released each day for each treatment area in 2017.
2018 Egg trap data from treatment and control sites. Excel spreadsheet (.xlsx) with legend sheet and raw data sheet containing raw egg trap counts and larval hatch counts for treatment and control areas in 2018.
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Crawford, J.E., Clarke, D.W., Criswell, V. et al. Efficient production of male Wolbachia-infected Aedes aegypti mosquitoes enables large-scale suppression of wild populations. Nat Biotechnol 38, 482–492 (2020). https://doi.org/10.1038/s41587-020-0471-x
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