Incompatible and sterile insect techniques combined eliminate mosquitoes

Abstract

The radiation-based sterile insect technique (SIT) has successfully suppressed field populations of several insect pest species, but its effect on mosquito vector control has been limited. The related incompatible insect technique (IIT)—which uses sterilization caused by the maternally inherited endosymbiotic bacteria Wolbachia—is a promising alternative, but can be undermined by accidental release of females infected with the same Wolbachia strain as the released males. Here we show that combining incompatible and sterile insect techniques (IIT–SIT) enables near elimination of field populations of the world’s most invasive mosquito species, Aedes albopictus. Millions of factory-reared adult males with an artificial triple-Wolbachia infection were released, with prior pupal irradiation of the released mosquitoes to prevent unintentionally released triply infected females from successfully reproducing in the field. This successful field trial demonstrates the feasibility of area-wide application of combined IIT–SIT for mosquito vector control.

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Fig. 1: Characterization of the triple-Wolbachia-infected A. albopictus HC line.
Fig. 2: Field sites, release schedule and larval suppression by HC male release.
Fig. 3: Adult suppression by HC male release.
Fig. 4: HC male release ratios, mating competitiveness, mass rearing and quality control.
Fig. 5: Community support and reduction in mosquito biting.

Data availability

Source Data for the main and Extended Data figures are provided in the online version of this paper. Any other relevant data are available from the corresponding authors upon reasonable request.

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Acknowledgements

This work was supported by Guangdong Innovative Research Team Program (No. 2011S009), Scientific and Technological Leading Talents of Guangzhou Development District (No. 2013L-P116), Science and Technology Planning Project of Guangdong Province (2016A020251001), a grant from the Foundation for the NIH through the Grand Challenges in Global Health Initiative of the Bill and Melinda Gates Foundation, the joint Food and Agricultural Organization (FAO) of the United Nations and International Atomic Energy Agency (IAEA) Division of Nuclear Techniques in Food and Agriculture and the IAEA Department of Technical Cooperation (RAS5066, RAS5082, D42016 and D44002), the 111 Project (grant no. B12003), Key Project of NNSF of China (11631005), China Postdoctoral Innovation Program (BX20180394), and a grant-in-aid for joint research (2017-AH-04) from the NJAU-MSU Asia-Hub Project. A.A.H. was supported by an NHMRC Fellowship. We thank X. Zhou, S. O’Neill, S. L. Dobson, G. Bian and E. Walker for their support, suggestions and technical assistance.

Author information

Z.X., X.Z., D.Z., Y. Li, C.Y., Y. Wu, A.G.P., J.R.L.G., K.B., Z.W., L.A.B. and A.A.H. developed the concept and methodology; D.Z. performed radiation and male mating-competitiveness assay; Y. Liang and C.Y. performed population suppression and population replacement in laboratory cages; Y. Li and X.Z. performed human-landing assay; C.Y. performed mosquito quality control; Y. Li, Y. Wu, X.L. and X.P. performed vector competence assays; A.G.P designed the X-ray irradiator; D.Z., K.B. and J.R.L.G. performed the population-suppression experiment in semi-field cages; X.Z., Z.Y., Y. Wu and J. Zhuang performed community engagement; X.L., X.P., Q.S., J.-T.G. and M.Z. performed cell culture, virus titration and Wolbachia density quantification; Z.Y., Zhigang Hu, Z.Z., L.L. and Q.L. identified the field sites; B.Z., L.H. M.T. and J.Y. developed the mathematical model and performed spatial analyses; X.W. and J. Zhu performed mosquito mass rearing; Y. Wei and W.Q. performed release and field surveillance; J. Zhu, W.Q., X.-Y.H., Zhiyong Hu and Z.W. performed coordination for the project; W.Q. obtained regulatory approvals for mosquito releases; J.L. performed mosquito crosses and maintenance of mosquito lines; J.B. and Z.X. performed cost-effectiveness analysis; Z.X. provided oversight of the project and contributed to all experimental designs, data analysis and data interpretation; Z.X., L.A.B., X.Z., D.Z., Y.L. and A.A.H. wrote the manuscript. All authors participated in manuscript editing and final approval.

Correspondence to Zhiyong Xi.

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Competing interests

Y. Li, X.W., Y. Wei, J. Zhu, W.Q., J.L. and Z.X. are affiliated with Guangzhou Wolbaki Biotech Co., Ltd. This does not alter our adherence to all Nature policies.

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Peer review information Nature thanks William Sullivan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Illustration of the procedures used to establish A. albopictus HC line by embryonic microinjection and for PCR verification of the Wolbachia strains in HC.

a, The Wolbachia strain wPip from C. pipiens molestus (donor) was transinfected into the wAlbA/wAlbB superinfected HOU line of A. albopictus (recipient) by embryonic microinjection, to generate a mosquito line infected with three Wolbachia strains (HC). A circle containing a cross indicates that the line was discarded, and a tick indicates that the line was maintained. Red indicates wPip-positive, and white indicates wPip-negative individuals. b, Wolbachia infection status was verified by PCR in both male and female HC mosquitoes. Results indicate that HC contains both the native Wolbachia strains (wAlbA and wAlbB) and the new transinfected strain wPip. The experiments were repeated at least three times independently with similar results.

Extended Data Fig. 2 Inhibition of both horizontal and vertical transmission of Zika and dengue viruses in the A. albopictus HC line.

a, b, ZIKV (a) and DENV-2 (b) were significantly decreased in the saliva of HC compared to wild type, GUA and HOU, respectively. Fourteen days post-infection, mosquito saliva samples were collected, ZIKV copies were quantified by RT-qPCR, and the titre of DENV-2 was measured by plaque assay. Horizontal lines indicate the median value (two-sided Mann–Whitney test: ZIKV, n = 16 for both HC and GUA, P = 0.0049; DENV-2, n = 39 for HC and n = 36 for HOU, P < 0.0001). c, Experimental design to measure the horizontal transmission of ZIKV. d, Viral positive rate in mosquitoes at day 7 after feeding on Zika-infected suckling mice (two-sided Fisher’s exact test, n = 19 for GUA and 20 for HC, P = 0.047). e, Experimental design to measure the vertical transmission of ZIKV in mosquitoes. f, The minimum ZIKV vertical transmission rate in HC and GUA lines (two-sided Fisher’s exact test, n = 35 biologically independent samples, P = 0.004). g, h, ZIKV replication and dissemination in HC were both significantly decreased. Mosquitoes were infected with ZIKV by oral feeding. ZIKV replication was determined by viral genome copy numbers in mosquito abdomens at 7 dpi (n = 20), and dissemination was measured by ZIKV infection status in one mosquito hind leg at 14 dpi (n = 20). The observations showed that ZIKV replication (g) and dissemination (h) were both significantly inhibited in HC. The infection prevalence is shown as a percentage. Horizontal lines indicate the median number of viral copies (two-sided Mann–Whitney test: abdomen, P = 0.018; hind legs, P = 0.002).

Extended Data Fig. 3 Sensitivity analysis of the robustness of the 5:1 HC:GUA male release ratio to induce population suppression.

ac, The mathematical model of the semi-field cage experiments shown in Fig. 1e, f and described in the Supplementary Information provides an accurate approximation to the semi-field data when r = 5 and one of three parameters listed in (5) in the Supplementary Information are varied across a wide range of values. a, R2 [0.9259, 0.9355] for ξ0 [0.6, 0.9]. b, R2 [0.9301, 0.9329] for μ [0.75, 0.95]. c, R2 [0.9325, 0.9573] for λ [0.5, 1]. d, The effect of mosquito migration and the efficiency of population suppression as measured by egg hatch rate. We fixed λ = 0.6, μ = 0.85, ξ0 = 0.80 and b0 = 75. The 5:1 ratio is sufficient to offset 20% migration with a suppression efficiency 92.20% as compared to 98.71% suppression efficiency without migration.

Extended Data Fig. 4 The proportion of egg-positive ovitraps, the average number of eggs per ovitrap, and the average percentage of eggs hatching per ovitrap in release and control sites before release of HC males.

ac, Site 1: the proportion of egg-positive ovitraps (a), average number of eggs per ovitrap (b) and the average percentage egg hatch per ovitrap (c) in 2014, compared to the control site. df, Site 2: the proportion of egg-positive ovitraps (d), average number of eggs per ovitrap (e) and the average percentage egg hatch per ovitrap (f) in 2015, compared to the control site. The proportion of egg-positive ovitraps was calculated from the number of ovitraps with eggs divided by the total number of ovitraps used. The average number of eggs per ovitrap was calculated as the total number of eggs collected divided by the number of ovitraps used. The average percentage of eggs hatching per ovitrap was calculated as the mean of the percentage of hatched eggs per individual ovitrap for all the ovitraps that collected eggs. Data were collected weekly. The proportion of egg-positive ovitraps (two-sided Mann–Whitney test: site 1, n = 26, P = 0.591; site 2, n = 32, P = 0.3239), the average number of eggs per ovitrap (two-sided Mann–Whitney test: site 1, n = 26, P = 0.4516; site 2, n = 32, P = 0.6940), and the average percentage of eggs hatching per ovitrap (two-sided Mann–Whitney test: site 1, n = 26, P = 0.3186; site 2, n = 32, P = 0.8232) did not differ significantly between the control and release sites. In addition, there were significant and strong correlations across time between the release and their respective control sites for these three parameters, demonstrating similar temporal fluctuations in them: the proportion of egg-positive ovitraps (Pearson correlation: site 1, r = 0.88, n = 26, P < 0.0001; site 2, r = 0.85, n = 32, P < 0.0001), the average number of eggs per ovitrap (Pearson correlation: site 1, r = 0.77, n = 26, P < 0.0001; site 2, r = 0.96, n = 32, P < 0.0001), and the average percentage of eggs hatching per ovitrap (Pearson correlation: site 1, r = 0.67, n = 26, P = 0.0002; site 2, r = 0.70, n = 32, P < 0.0001).

Extended Data Fig. 5 Map of the ovitraps and BG traps distributed in the two release sites.

a, b, There were 110 ovitraps (grey circles) and 44 BG traps (blue circles) in release site 1 (a), and 40 ovitraps and 16 BG traps in release site 2 (b). Release site 1 was divided into 22 zones and release site 2 contained 8 zones. On average, there were five ovitraps and two BG traps in each zone, and collections from all traps were carried out weekly.

Extended Data Fig. 6 The proportion of egg-positive ovitraps, the average number of eggs per ovitrap, and the average percentage of eggs hatching per ovitrap in release and control sites after release of HC males.

ac, Site 1: the proportion of egg-positive ovitraps (a), average number of eggs per ovitrap (b) and the average percentage egg hatch per ovitrap (c) in 2016 and 2017, compared to the control site. df, Site 2: the proportion of egg-positive ovitraps (d), average number of eggs per ovitrap (e) and the average percentage egg hatch per ovitrap (f) in 2016 and 2017, compared to the control site. Significant declines were observed for all three parameters in the two release sites compared to their control sites: the proportion of egg-positive ovitraps (two-sided Mann–Whitney test: site 1 2016, n = 36, P < 0.0001; site 2017, n = 35, P < 0.0001; site 2 2016, n = 32, P < 0.0001; site 2 2017, n = 35, P < 0.0001), the average number of eggs per ovitrap (two-sided Mann–Whitney test: site 1 2016, n = 36, P < 0.0001; site 1 2017, n = 35, P < 0.0001; site 2 2016, n = 32, P < 0.0001; site 2 2017, n = 35, P < 0.0001), and the average percentage of eggs hatching per ovitrap (two-sided Mann–Whitney test: site 1 2016, n = 36, P < 0.0001; site 1 2017, n = 35, P < 0.0001; site 2 2016, n = 32, P < 0.0001; site 2 2017, n = 35, P < 0.0001).

Extended Data Fig. 7 The total number of wPip-positive adult females collected monthly in release sites 1 and 2.

Females were collected weekly using BG traps and tested for wPip infection by PCR. The wPip-positive females were recorded monthly in site 1 and site 2 during the release period. No significant difference was observed in the number of wPip-positive females between 2015 (n = 3), 2016 (n = 7) and 2017 (n = 9) in site 1 (Kruskal–Wallis test, P = 0.6536), or between 2016 (n = 7) and 2017 (n = 9) in site 2 (two-sided Mann–Whitney test, P = 0.1164). No evidence of an increase in the number of wPip-positive females with time was apparent, but would have been expected if population replacement had started in the field.

Extended Data Fig. 8 Temporal and spatial distribution of wPip-positive ovitraps in the two release sites between 2015 and 2017.

ab, Among 110 ovitraps in site 1 (a) and 40 ovitraps in site 2 (b), those from which wPip-positive larvae were detected are shown as red circles. The specific time points at which wPip-positive larvae were detected are also indicated (year.month). Overall, a total of 15 ovitraps with wPip-positive larvae were detected on 13 separate, spatially and/or temporally isolated, occasions in release site 1, whereas only one ovitrap with wPip-positive larvae was detected on a single occasion in release site 2. The first six of the ovitraps with wPip-positive larvae were detected in 2015, before the use of irradiation, while in 2017 only two were found in site 1 and none in site 2. c, Overall, wPip-positive rates of 0.9% (15/1,678 pooled larval samples taken weekly from individual ovitraps, referred to as ‘ovitrap weeks’) and 0.6% (1/166) were found during the release period in the 3 or 2 years of HC releases in sites 1 and 2, respectively. No evidence of an increase in the number of wPip-positive ovitraps with time was apparent, but would have been expected if population replacement had started in the field.

Extended Data Fig. 9 Induction of sterility in HC females after mating with irradiated HC males.

a, b, The effect of irradiating HC males on the egg hatch rate (a) and the level of induced sterility (b) in mated females. For each cross shown in the figure (x axis), a single treatment cage (30 × 30 × 30 cm) was set up containing males and females, at a 1:1 ratio, of the mosquito line with irradiation status indicated. The two control crosses (HC:HC and wild-type:wild-type) were set up with 100 individuals of each sex. All other treatment crosses used 300 individuals of each sex. IHC45Gy are HC males irradiated at the pupal stage with an X-ray dose of 45 Gy, as described in the Methods. None of the other mosquitoes used were irradiated (HC and GUA). Induced sterility was calculated as follows56: 100 − [(egg hatch rate of treatment cages)/(egg hatch rate of control cages) × 100]. Complete sterility (100%) was induced when wild-type females mated with either non-irradiated or irradiated HC males, whereas high levels of partial sterility (86.4%) were induced when HC females mated with IHC45Gy males, showing that irradiation causes sterility between the otherwise-compatible HC males and females. During HC release in the field, there is a high probability that HC females would mate with irradiated HC males owing to their high abundance relative to wild-type males.

Extended Data Table 1 Male mating competitiveness index (C) and fertility of HC (non-irradiated) and IHC (irradiated) males

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