Incompatible Aedes aegypti male releases as an intervention to reduce mosquito population-A field trial in Puerto Rico

Abstract

Mosquito-transmitted viruses such as dengue are a global and growing public health challenge. Without widely available vaccines, mosquito control is the primary tool for fighting the spread of these viruses. New mosquito control technologies are needed to complement existing methods, given current challenges with scalability, acceptability, and effectiveness. A field trial was conducted in collaboration with the Communities Organized to Prevent Arboviruses project in Ponce, Puerto Rico, to measure entomological and epidemiological effects of reducing Aedes aegypti populations using Wolbachia incompatible insect technique. We packed and shipped Wolbachia-males from California and released them into 19 treatment clusters from September 2020 to December 2020. Preliminary evaluation revealed sub-optimal Wolbachia-male densities and impact on the wild-type population. In 2021, we shifted to a phased release strategy starting in four clusters, reducing the mosquito population by 49% (CI 29-63%). We describe the investigation into male quality and other factors that may have limited the impact of Wolbachia-male releases. Laboratory assays showed a small but significant impact of packing and shipping on male fitness. However, mark-release-recapture assessments suggest that male daily survival rates in the field may have been significantly impacted. We compared induced-sterility levels and suppression of the wild population and found patterns consistent with mosquito population compensation in response to our intervention. Analysis of epidemiological impact was not possible due to very low viral transmission rates during the intervention period. Our entomological impact data provide evidence that Wolbachia incompatible-male releases reduced Ae. aegypti populations, although efficacy will be maximized when releases are part of an integrated control program. With improvement of shipping vessels and shipped male fitness, packing and shipping male mosquitoes could provide a key solution for expanding access to this technology. Our project underscores the challenges involved in large and complex field effectiveness assessments of novel vector control methods.

Fig 1. Location and project assignment of COPA clusters, Ponce, Puerto Rico, September 2020–December 2021.

A) 38 polygons showing clusters defined for the COPA study. Phase I releases were conducted in 19 treatment clusters (blue) in 2020. B) Phase II treatment clusters are highlighted in purple and control clusters highlighted in blue. Remaining clusters received no extra activity, but monitoring traps remained active during Phase II. Map source: USDA Farm Service Agency, National Agriculture Imagery Program. Map created using ArcGIS Pro 2.6.0. USDA:NRCS:Geospatial Data Gateway:Home:Direct DownLoad.

Fig 2. Male mosquitoes with Wolbachia released in COPA clusters, September 2020–December 2021, Ponce, Puerto Rico.

A) Weekly male shipment totals (in Millions) shown on y-axis, with shipment date on the x-axis. Each bar is one week, summarizing five shipments per week. The orange bars show the total number of males that arrived on-time and were successfully released. The green bars show the number of males that arrived between 24–48 hrs that were released. Black bars show the number of males from shipments delayed beyond ~48 hrs were not released. B) The mean number of Ae. aegypti males collected in AGO traps in 2021 calculated across 4 treatment clusters (yellow) and all control clusters (grey). Shaded area shows 95% bootstrap confidence intervals. The vertical dotted lines show when releases began and ended in 2021. C) Overflooding ratio shown on y-axis from BG Sentinel collections according to dates on x-axis, with the cluster name shown for each sub-panel. Each dot is one BG trap collection. The middle horizontal line shows the mean for a weekly collection, with the 95% confidence interval shown with the vertical bar.

Fig 3

A) Induced sterility calculated as the scaled difference between treatment clusters aggregated hatch rate vs. all control clusters aggregated scaled by the control clusters hatch rate. Shaded area shows the 95% bootstrap confidence interval. Dotted lines show when releases started on left and ended on right. B) Mean number of female Aedes aegypti per trap per week in treatment clusters versus control clusters. December 2020–May 2022. Ponce, Puerto Rico. Dotted lines are the same as panel A. C) Cumulative rain over 21-day windows plotted on the same weekly schedule as in B for three weather stations in Ponce. D) Ratio of female Aedes aegypti mosquitoes in treatment clusters (n = 4) to control clusters (n = 4), Ponce, Puerto Rico, January–December 2021. Shaded area shows point-wise 95% confidence region. Dotted lines same as panel A.

Fig 4

Hotspot areas in cluster ST02. A) Heatmap showing Ae. aegypti female counts by trap in rows and collection week in columns with colors according to key above. Trap labels to the right of heatmap. Actual counts are labeled in each cell. Specific traps discussed in text shown with yellow and green boxes. Some traps were added after the releases began. Yellow and green bars below indicate weeks used for means shown in panels B (green) and C (yellow). B) Four-week mean number of females in April 2021 (green bar under heatmap) in traps in ST02 according to legend colors showing outlier collection numbers in trap 14–6. Weekly collections for this trap shown in panel A. C) Same as B but for June 2021 (yellow bar under heatmap) showing high female means in the center of the cluster corresponding to an apartment complex. Map source: OpenStreetMap.org (CC BY-SA 2.0). Open Street maps were labeled in R using the Rgooglemaps package. https://www.openstreetmap.org/#map=14/18.01318/-66.60985.

Fig 5. Male quality assessments.

A) Mating competitiveness cage assays. Each dot indicates the proportion of females mated with a Wolbachia-male in competition with wild-type males. Violin plots show median of distribution with white dot and quartiles. B) Reaction norm plot comparing mean lifespan for samples of males from the same batch assayed in San Francisco (held back) or in Ponce (shipped), either on-time or delayed according to legend. Violin plots summarize distribution of held back and shipped samples separately. C) Proportion recaptured (y-axis) in mark-release recapture (MRR) study as a function of distance from release point (x-axis) for two replicates according to legend. Each dot shows the total collected for a trap, and the lines show corrected collection rate (see methods). D) Proportion recaptured (y-axis) in MRR study over days since release (x-axis). Solid lines show empirical data and dotted lines show best-fit model for each replicate. E) MRR summary statistics. Estimated recapture rate and estimated proportion daily survival rate refer to values inferred from model fitting with 95% confidence intervals calculated from simulations (see Methods). Mean distance traveled in meters after correcting for variable trap density (see Methods).

Fig 6. Evidence for ecological compensation and overcompensation.

Each dot represents the mean for a four-week window for each Phase II treatment cluster according to the legend. A) Change in suppression calculated using before-after-control-impact comparing windows separated by three weeks. Analysis of fitted Generalized Mixed Model shown in black. Ovals show regions where values under Additive, Compensation, and Over-compensation models would be expected. B) Impact on suppression as a function of mean female densities at the time of application. C) Impact on suppression as a function of mean egg densities at the time of application. D) Induced sterility as a function of female densities in the same window. Dotted lines show best-fit line and results of Pearson’s correlation test shown in bottom right.