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Incompatible and sterile insect techniques combined eliminate mosquitoes

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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.
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).
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ARTICLE https://doi.org/10.1038/s41586-019-1407-9
Incompatible and sterile insect techniques
combined eliminate mosquitoes
Xiaoying Zheng1,14, Dongjing Zhang1,2,14, Yongjun Li1,3,14, Cui Yang1,3,14, Yu Wu 1,14, Xiao Liang4, Yongkang Liang1,3,
Xiaoling Pan4,5, Linchao Hu1, Qiang Sun1,4, Xiaohua Wang3, Yingyang Wei3, Jian Zhu3, Wei Qian3, Ziqiang Yan6,
Andrew G. Parker2, Jeremie R. L. Gilles2, Kostas Bourtzis2, Jérémy Bouyer2, Moxun Tang7, Bo Zheng8, Jianshe Yu8,
Julian Liu3, Jiajia Zhuang1, Zhigang Hu6, Meichun Zhang1, Jun-Tao Gong9, Xiao-Yue Hong9, Zhoubing Zhang6, Lifeng Lin10,
Qiyong Liu11, Zhiyong Hu12, Zhongdao Wu1, Luke Anthony Baton4, Ary A. Hoffmann13 & Zhiyong Xi1,3,4*
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.
SIT, in which artificially reared radiation-sterilized males are released
into the field to mate with wild females—thereby preventing them
from producing viable offspring—has successfully suppressed
populations of several insect pests of agricultural and veterinary
importance1. However, despite various trials, SIT has not been widely
used against mosquitoes because of the difficulty of irradiating males
without reducing their mating competitiveness and survival2–4. A
promising alternative approach is the related IIT
5
, in which released
males are infected with the maternally inherited endosymbiotic bacteria
Wolbachia, resulting in sterile matings with field females that are not
infected with the same Wolbachia strain, a phenomenon known as cyto-
plasmic incompatibility6,7. An advantage of IIT is that Wolbachia-based
sterilization has little or no effect on male mating competitiveness and
survival
8–10
. Historically, in a small-scale pilot field trial IIT success-
fully eradicated the primary filariasis vector Culex quinquefasciatus
5
although another trial showed limited success11—but the approach
has not been deployed operationally, primarily because theaccidental
release of fertile females risks causing population replacement, whereby
individuals infected with the same Wolbachia strain as released males
replace the wild-type field population, preventing future population
suppression (as matings between released males and field females
are no longer incompatible)
1113
. Consequently, previous studies
1417
haveproposed combining IIT and SIT so that any residual females
that are not removed from the released males are sterilized using low-
dose irradiation without affecting the males’ mating competitiveness
or survival. There has been a resurgence of interest in IIT
1820
in the
past decade, partly because of the development of the ability to artifi-
cially transfer Wolbachia strains between mosquitoes21,22, and the first
small-scale field release of artificially Wolbachia-infected mosquitoes
for IIT was recently reported
18
. Concurrently, the combined IIT–SIT
approach has also been under renewed consideration and develop-
ment2326, but has not yet been deployed.
The globally invasive mosquito A. albopictus is an important vector
of arboviruses—including dengue and Zika viruses—that is particularly
challenging to control using traditional approaches
27,28
. Unlike some
other mosquito vectors, A. albopictus is superinfected with two native
Wolbachia strains (wAlbA and wAlbB), complicating the development
of Wolbachia-based control strategies. Various Wolbachia strains have
previously been artificially introduced into A. albopictus
2932
, including
wPip in mosquitoes cured of their native double wAlbA/wAlbB infec-
tion
33
, but these endosymbiont–host associations are either unsuitable
for IIT—as they are pathogenic or do not inhibit arboviruses—or their
appropriateness has not been fully determined. Here we report the
generation and characterization of an A. albopictus line (termedHC)
with an artificial triple-Wolbachia infection, and demonstrate its use
in an open-release field trial of the combined IIT–SIT approach. We
show thatthe mass release of millions of factory-reared incompatible
adult HC males over a two-year period enabled near-elimination of
wild-type A. albopictus field populations, without their replacement
by released HC mosquitoes.
Generation and characterization of HC
For use in IIT, Wolbachia must induce high levels of cytoplasmic incom-
patibility to effectively sterilize wild females, and have high maternal
transmission to enable efficient mass production of only infected males
for release as well as low fitness costs to ensure that released males can
1Key Laboratory of Tropical Disease Control of the Ministry of Education, Sun Yat-sen University–Michigan State University Joint Center of Vector Control for Tropical Diseases, Zhongshan School
of Medicine, Sun Yat-sen University, Guangzhou, China. 2Insect Pest Control Laboratory, Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture, Vienna International Centre,
Vienna, Austria. 3Guangzhou Wolbaki Biotech Co., Ltd, Guangzhou, China. 4Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA. 5School of
Medicine, Hunan Normal University, Changsha, China. 6Guangzhou Center for Disease Control and Prevention, Guangzhou, China. 7Department of Mathematics, Michigan State University,
East Lansing, MI, USA. 8Center for Applied Mathematics, College of Mathematics and Information Sciences, Guangzhou University, Guangzhou, China. 9Department of Entomology, Nanjing
Agricultural University, Nanjing, China. 10Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, China. 11State Key Laboratory of Infectious Disease Prevention and Control,
Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and
Prevention, Beijing, China. 12Lingnan Statistical Science Research Institute, Guangzhou University, Guangzhou, China. 13Bio21 Institute, School of BioSciences, University of Melbourne, Melbourne,
Victoria, Australia. 14These authors contributed equally: Xiaoying Zheng, Dongjing Zhang, Yongjun Li, Cui Yang, Yu Wu. *e-mail: xizy@msu.edu
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Article
reSeArcH
mate competitively with respect towild males. In addition, as a respon-
sible safety precaution, any released mosquitoes should have a lower
vector competence for human pathogens than the target field popula-
tion
34
. Accordingly, we created an endosymbiont infection appropriate
for IIT by transferring wPip from its native mosquito host Culex
pipiens into the A. albopictus HOU line by embryonic microinjection
21
,
generating the mosquito line HC, which possesses a triple-Wolbachia
infection (the artificially transinfected wPip as well as the original
native wAlbA and wAlbB strains)
9
(Extended Data Fig.1). HC females
were subsequently outcrossed with males of a wild-type mosquito line
(GUA) with the native double wAlbAandwAlbB infection, initially
collected from the area of our field trial, to create comparable nuclear
genetic backgrounds in both mosquito lines, as well as with wild mos-
quitoes in our study region9.
Laboratory reciprocal-cross experiments demonstrated that wPip in
HC causes complete unidirectional cytoplasmic incompatibility with
GUA—of 7,578 eggs resulting from crosses between HC males and
GUA females, none hatched, whereas HC females rescued cytoplasmic
incompatibility when mated with either GUA or HC males (Fig.1a).
The total density of Wolbachia was higher in the ovaries of HC com-
pared to GUA (Fig.1b, c), and was stably maintained by 100% maternal
transmission across subsequent generations. In laboratory cage popula-
tions, wPip also had no apparent effects on the fitness of HC males and
females9,24,25, and only very minor effects on the mating competitiveness
of HC males (Fig.1d). In addition, wPip significantly reduced the vector
competence of HC females for both horizontal and vertical transmission
of dengue and Zika viruses (Extended Data Fig.2 and Supplementary
Information), similar to that of some other mosquito–Wolbachia asso-
ciations
3538
. These results demonstrate that the A. albopictus HC line
has the characteristics required for use in IIT control programs.
Laboratory cage experiments showed that wPip invades wild-type
GUA populations following release of only HC females, and that this
population replacement may be facilitated by simultaneous inundative
release of HC males (Fig.1e and Supplementary Information). These
observations indicate that the accidental release of HC females during
an IIT program could result in the introduction of wPip into the target
field population, and that this risk increases as HC males are released.
Therefore, we tested the effectiveness of low-dose irradiation
24,25
forthe
prevention of unintended population replacement during population
suppressionand/orelimination by simulating, in semi-field cage pop-
ulations of GUA, accidental HC female release during an IIT inter-
vention using the release of HC males (Fig.1f, g and Supplementary
Information). During these experiments, sufficient HC females were
released to mimic a 2.0% contamination rate of the released males,
and the number of released HC males was chosen to result in an initial
5:1 ratio of released HC to GUA males. The level of pupal irradiation
used was previously shown to completely sterilize HC females24 without
affecting male mating competitiveness
24,25
. Successful eradication of
wild-type GUA mosquitoes occurred in the semi-field cages, without
population replacement by the released wPip-infected HC mosquitoes
(Fig.1f, g). Mathematical modelling accurately described and predicted
target population dynamics in the semi-field cage experiments, and
supported the notion that a 5:1 ‘over-flooding’ ratio of HC to wild-type
males is sufficient for effective population suppressionand/or
elimination (Fig.1f, g, Extended Data Fig.3 and Supplementary
Information).
Field trial of combined IIT–SIT using HC
The preceding experimental and theoretical observations indicated
that combined IIT–SIT using HC has the potential to eradicate
a c
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Hatch rate (%)
HC × HC
GUA × GUA
HC × GUA
GUA × HC
Ƃ × ƃ
c
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abGUAHCGT
HCGUA
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copies per rps6
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NC
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GUAƂ:GUAƃ:HCƃ
1:1:01:1:1 1:1:51:1:10
Observed
Expected
Fig. 1 | Characterization of the triple-Wolbachia-infected A. albopictus
HC line. a, Reciprocal crosses between HC and GUA lines. Letters above
columns indicate significant differences between groups (mean±s.e.m.;
n=5 for each cross, ANOVA and Tukey’s multiple comparisons test, F(3, 16)
=513.5, P<0.0001). b, Fluorescence insitu hybridization (FISH),
showing Wolbachia distribution and density in ovaries. Scale bar, 100µm.
c, Real-time quantitative PCR (RT-qPCR) analysis of the relative number
of Wolbachia wsp gene copies (mean±s.e.m.; n=7 pools of two ovary
pairs for each group, two-sided Mann–Whitney test, P=0.006). d, Egg
hatch rate in laboratory cage populations with different GUA female:GUA
male:HC male ratios. Two-sided binomial test: n=3,681, P=0.0002
(1:1:1); n=4,083, P<0.0001 (1:1:5); n=2,392, P=0.0009 (1:1:10).
e,Invasion of wPip in laboratory GUA populations after a single release
of different numbers of HC females at generation 0. For release of 6% and
12% HC females, a single simultaneous inundative release of HC males at a
4:1 ratio with GUA males was also used, to mimic accidental female release
during IIT. NC, negative control. f, g, Combined IIT–SIT in semi-field
cages: egg hatch rate (f) and adult female population sizes (g). Target GUA
populations were established in six replicate cages for 12 weeks before the
release (indicated by the dashed red lines) of HC males, with HC females
to mimic female contamination. Black triangles represent mathematical
model outputs (mean goodness of fit: egg hatch rate R20.9325; number
of females captured R20.8417).
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Article reSeArcH
A. albopictus field populations, as well as prevent unintended
population replacement caused by accidental release of HC females.
Therefore, we optimized mass rearing of HC for large-scale produc-
tion39,40 and, with approval from the Chinese Ministry of Agriculture,
undertook an open-release field trial in residential areas of two
isolated riverine islands in Guangzhou, the city with the highest
dengue transmission rate in China, and where A. albopictus is the
only vector (Fig.2a, b).
In the year before HC release, baseline data were collected weekly in
both release and control sites using ovitraps during the local mosquito
breeding season between March and November (site 1 in 2014 and site
2 in 2015) (Fig.2c). Overall, A. albopictus were highly abundant during
this period, with no significant differences and strong temporal correla-
tions in egg numbers and hatch rates between control and release sites
(Fig.2d, e and Extended Data Fig.4a–f ), validating the appropriateness
of the control sites selected.
During the intervention period, adult male HC mosquitoes were
released at multiple locations within each site, three times per week
during the mosquito breeding season, for either three (site 1) or two
(site 2) consecutive years (Fig.2c), and A. albopictus populations were
monitored weekly with ovitraps and adult-collecting BG-Sentinel traps
(Extended Data Fig.5).
In 2015, at site 1 only, at the beginning of the field trial, a limited
number of HC mosquitoes were released because only manual checks
were carried out for contaminant females (that is, IIT only, without
irradiation). As cytoplasmic incompatibility causes embryonic death
when HC males mate with wild-type females, the number of eggs
hatching in each ovitrap was recorded. Initially, HC male release
resulted in 55% population suppression, based on the number of eggs
hatching per ovitrap (from 12 March 2015 to 21 May 2015), but this
effect diminished as the mosquito season peaked (late May to early
June, Fig.2d), consistent with a low ratio of HC to wild-type males
(see below). Consequently, the site 1 area was reduced from 16 June
to increase the density of released males, after which the population-
suppression effect increased. Overall, there was a yearly mosquito
population reduction in release sites compared to control sites of 62%
and 65% as measured at the larval and adult female stages, respec-
tively (Figs.2d, 3a). These observations demonstrate that IIT alone
can suppress field populations, but only if sufficient numbers of male
mosquitoes are released.
In 2016 and 2017, pupal irradiation to sterilize contaminant females
(that is, combined IIT–SIT) replaced manual checks, enabling the
production and release of larger numbers of mosquitoes (Fig.4c) and
allowing high-density releases throughout sites 1 and 2. In both release
a
c
d
e
b
Release site 1:
Pre-release 1st release: IIT 2nd release: IIT–SIT 3rd release: IIT–SIT
6/5/2014 3/12/2015 11/14/2015 3/22/2016 11/29/2016 3/23/2017 11/30/2017
Release site 2:
Pre-release 1st release: IIT–SIT 2nd release: IIT–SIT
5/5/2015 4/6/2016 11/22/2016 3/23/2017 11/30/2017
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Per cent suppression
Release site 1 Control site 1 Suppression efciency
Release site 2 Control site 2 Suppression efciency
2014 2015
2015 2016 2017
2016 2017
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Per cent suppression
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Release site 1
Control site 1
Control site 2a
Control site 2b
Release site 2
200 m 200 m
North North
Fig. 2 | Field sites, release schedule and larval suppression by HC male
release. a, b, Satellite images of control and release sites 1 (a) and 2 (b) in
Guangzhou city (map data: Google, DigitalGlobe). c, Release schedule.
d,e, Effect of HC male release on A. albopictus larval stages in release sites
1 (d) and 2 (e). Vertical green dashed lines indicate onset of HC release.
Red dashed line in d indicates that only IIT was used in release site 1 in
2015. Red solid lines in d and e indicate period of combined IIT–SIT
in both release sites in 2016 and 2017. Two-sided Mann–Whitney test.
Pre-release period: site 1 2014, n=22, P=0.164; site 2 2015, n=26,
P=0.0805. Release period: IIT only: site 1 2015, n=34, P<0.0001;
12 March–21 May, n=11, P=0.0032. Release period: combined IIT–SIT:
site 1 (2016, n=37, P<0.0001; 2017, n=34, P<0.0001); site 2 (2016,
n=32, P<0.0001; 2017, n=35, P<0.0001).
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sites, the numbers of eggs and adults collected markedly declined
(Fig.2d, e, 3a, b, Extended Data Fig.6a–f). In 2016 and 2017, we
observed a yearly reduction of more than 94% in the average number
of hatched eggs per ovitrap in release sites compared to control sites, and
no viable eggs for up to 13 weeks (Fig.2d, e). Similarly, there were yearly
reductions of 83% to 94% in the average number of wild-type adult
females caught per trap, with none detected for up to 6 weeks (Fig.3a, b).
Furthermore, declines in egg hatching coincided with declines in num-
bers of collected eggs and adults, consistent with cytoplasmic incompati-
bility rather than other factors driving the loss of wild-type A. albopictus.
The spatial dynamics of population suppression were analysed
across different zones within the release sites (Fig.3c, d). Consistently,
the highest levels of population suppression were observed in more-
isolated areas surrounded by vegetation and with limited transport links
(zones 12–19 in site 1 and zones 2 and 3 in site 2), whereas less isolated
zones nearer transportation routes with frequent traffic were relatively
resistant to population suppression (in site 1, zones 10 and 11 had
an ongoing bridge construction and zones 20–22 were adjacent to a
shipping harbour, and there was considerable motor traffic in zones
7 and 8 of site 2),which suggests that human activities facilitate mos-
quito immigration into release sitesand compromise the efficiency of
A. albopictus elimination.
As the ratio of released HC to wild-type males is critical for popula-
tion suppression, we measured this by detecting wPip in field-collected
A. albopictus males (Fig.4a). As indicated by our mathematical mod-
elling and semi-field cage studies, we set a 5:1 ratio of HC to wild-type
males as the target over-flooding ratio. As expected, in 2015, when
fewer HC males were released and population suppression was rela-
tively weak, the average male release ratio was 4.4:1 HC to wild-type
males. However, in 2016 and 2017, when there was an increase in HC
male release and population suppression was strong, yearly averaged
ratios varied between 8.7 and 15.8.
The relative mating performance of HC to wild-type males in the
field was also inferred from egg hatch rates (Fig.4b). In 2015, when the
released HC mosquitoes were not irradiated, expected and observed
egg hatch rates were not significantly different, indicating that non-
irradiated HC and wild-type males had similar mating competitive-
ness (as found in laboratory studies
25
)(Fig.1d). However, in 2016 and
2017, when the released HC mosquitoes were irradiated the observed
egg hatch was between 1.5 and 2.1-fold higher than expected, which
suggests that the relative competitive mating ability of irradiated HC
to wild-type males was 0.5 to 0.7—consistent with other laboratory-
based cage experiments (Extended Data Table1). Nevertheless, the
reduced mating competitiveness of HC males as a result of irradiation
was apparently offset by the increased number of mosquitoes released.
As accidental HC female release could lead to unintended popula-
tion replacement, thereby preventing further population suppression
(particularly as population suppression proceeds and the ratio of
wPip-positive females relative to wild-type field females increases),the
number of adult female wPip-positive mosquitoes was carefully
monitored both before and after their release (Fig.4c, d). A mean of
0.24%±0.03% (s.e.m.) contaminant HC females were released in
2016 and 2017 (Fig.4d), which is below the 2% level simulated in the
semi-field cage experiments that successfully prevented population
replacement (Fig.1f, g). Relative to the number of HC males, the pro-
portion of wPip-positive females in release sites was generally higher
than the pre-release contamination rate (Fig.4d), possibly reflect-
ing sex-specific differences in mortality and/or trap collection18,41.
The pre-release contamination rate correlated with the wPip-positive
field rate in site 2 but not in site 1 (Fig.4d), suggesting that—at least
in the former—the wPip-positive adult females that were caught were
those that were released and did not originate from reproduction in
the field. If a viable breeding wPip-infected field population had estab-
lished, the numbers of A. albopictus would be expected to increase after
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27 Apr
11 May
25 May
8 Jun
22 Jun
6 Jul
20 Jul
3 Aug
17 Aug
31 Aug
14 Sep
28 Sep
12 Oct
26 Oct
9 Nov
23 Nov
7 Dec
16 Mar
30 Mar
13 Apr
27 Apr
11 May
25 May
8 Jun
22 Jun
6 Jul
20 Jul
3 Aug
17 Aug
31 Aug
14 Sep
28 Sep
12 Oct
26 Oct
9 Nov
23 Nov
Per cent suppression
2016 2017
2015 2016 2017
11
10
98
7654
2
3
1
12
13
14
15
1617
18
19 21 22
20
60–70%
70–80%
80–90%
90–100%
100%
17 Aug 21 Sep 29 Oct 23 Nov Suppression levels
17 Aug 21 Sep 29 Oct 23 Nov
c
d
a
b
876542
31
Fig. 3 | Adult suppression by HC male
release. a, b, Relative density of adult females
collected weekly in control and release sites 1
(a) and 2 (b). The red dashed line in a indicates
period of IIT only in 2015. Red solid lines in a
and b indicate the period of combined IIT–SIT
in 2016 and 2017. Two-sided Mann–Whitney
test. Site 1 2015, n=34, P<0.0001; site 1
2016, n=37, P<0.0001; site 1 2017, n=37,
P<0.0001; site 2 2016, n=37, P<0.0001;
site 2 2017, n=38, P<0.0001. c, d, Spatial
dynamics of adult suppression at release sites 1
(c) and 2 (d) during the dengue transmission
season in Guangzhou in 2017.
NATURE| www.nature.com/nature
Article reSeArcH
an initial period of population suppression, with a concomitant decline
in the observed level of cytoplasmic incompatibility (as compatible mat-
ings with wPip-infected females would have increased). However, there
was no evidence of an increase over time in either absolute or relative
numbers of eggs and adult females collected, regardless of whether all
or only wPip-positive mosquitoes were considered (Fig.3a, b, Extended
Data Figs.6b, e, 7). Notably, there was also no evidence of an increase in
the proportion of eggs hatching (Fig.2d, e), which would be expected if
compatible matings were becoming more frequent. Additionally, larvae
hatched from collected eggs were also wPip-negative in nearly all ovit-
raps (Extended Data Fig.8a–c). Overall, 16 ovitraps with wPip-positive
larvae were detected on 14 separate spatially and/or temporally isolated
occasions among 1,844 ovitrap weeks (Extended Data Fig.8a–c), indi-
cating that very few accidentally released HC females had offspring
in the field. Whether this might drive population replacement in the
long run is uncertain, as the viability of the wPip-positive larvae is not
known. Nevertheless, irradiation provides protection against accidental
female release, especially compared to manual checking, as wPip-
positive larvae did not increase despite a more-than-tenfold increase in
the number of mosquitoes being released. In addition, irradiated HC
males also induced HC female sterility (Extended Data Fig.9), further
reducing the risk of population replacement.
Successful population suppression resulted in a significant increase
in community support for the field trials. Interviews carried out before
the mosquito releases indicated that 13.0% of residents were supportive,
with 76.4% and 10.6% being neutral and negative, respectively (Fig.5a).
However, after successful population suppression, there was a marked
shift in attitudes, with a majority of the residents interviewed being
supportive (54.3%)—probably owing to reduced mosquito nuisance
biting27,42. Human landing catches verified the efficiency of A. albopictus
population suppression, indicating its epidemiological importance
for vector-borne disease transmission. The mosquito-biting rate by
wild-type A. albopictus significantly decreased by 96.6% and 88.7%,
respectively, in release sites 1 and 2, compared to their respective con-
trol sites (Fig.5b).
In conclusion, combined IIT–SIT nearly eliminated two field pop-
ulations of A. albopictus over a two-year period. The few mosquitoes
remaining were probably migrants from outside the study area, as
indicated by population genetic analyses43 and their presence in areas
with good transport links, whereas isolated areas were mosquito-free.
The possibility of population replacement emphasizes the importance
of releasing mosquitoes that cannot increase pathogen transmission. As
shown here, wPip markedly reduces arbovirus transmission by wild-type
A. albopictus with their native double wAlbAandwAlbB infection, so
unintended population replacement could even be beneficial in the
short- and long-term, by initially collapsing vector populations and
No. released (ha
–1
week
–1
)
Male ratio (release vs wild)
Egg hatch rate (%)
Male production per week
(×10
6
)
Number released Male ratio
Observed
Expected
180,000
160,000
140,000
120,000
100,000
80,000
60,000
40,000
20,000
0
0
10
20
30
40
50
60
70
9 Mar
13 Apr
18 May
22 Jun
22 Jun
27 Jul
31 Aug
5 Oct
21-Mar
25 Apr
30 May
4 Jun
9 Aug
12 Sep
17 Oct
12 Nov
27 Mar
1 May
5 Jun
10 Jul
14 Aug
18 Sep
23 Oct
27 Nov
Female contamination rate (%)
13 Nov
27 Nov
30 Oct
15 Apr
29 Apr
14 May
28 May
11 Jun
25 Jun
31 Oct
27-Mar
17 Apr
8 May
26 Jul
8 Aug
23 Aug
7 Sep
21 Sep
4 Oct
18 Oct
8 Apr
18 Apr
2 May
16 May
30 May
13 Jun
27 Jun
11 Jul
25 Jul
8 Aug
22 Aug
5 Sep
19 Sep
3 Oct
17 Oct 31 Oct
14 Nov
8 Apr
18 Apr
2 May
16 May
30 May
13 Jun
27 Jun
11 Jul
25 Jul
8 Aug
22 Aug
5 Sep
19 Sep
3 Oct
17 Oct
12 Jul
15 Apr
29 Apr
14 May
28 May
11 Jun
25 Jun
26 Jul
8 Aug
23 Aug
7 Sep
21 Sep
4 Oct
18 Oct
12 Jul
23 May
5 Jun
19 Jun
11 Jul
24 Jul
7 Aug
21 Aug
4 Sep
18 Sep
2 Oct
pct 16
6 Nov
20 Nov
11 Apr
2 May
17 May
31 May
14 Jun
18 Jul
8 Jul
31 Jul
14 Aug
29 Aug
11 Sep
29 Sep
9 Oct
23 Oct
2015 2016 2017
2015
2015 2015 2016 20172016 2017
2016 2017
0
20
40
60
80
100
Positive female rate (%)
0
1
2
3
4
5
0
1
2
3
4
5
6
Males production Female residues
Release site 1Release site 2Lab quality co ntrol
a
b
cd
0.0
0.2
0.4
0.6
1
2
3
4
5
Aug
Sep
Oct
Apr
May
Jun
Jul
Aug
Sep
Oct
May
Jun
Jul
Aug
Sep
Oct
Nov
Mar
Apr
Fig. 4 | HC male release ratios, mating
competitiveness, mass rearing and quality
control. a, Number of HC males released weekly
and observed ratios of HC to wild-type males
in the field at release site 1. Blue dashed line
indicates target overflooding ratio of 5:1.
b, Comparison of observed and expected weekly
egg hatch rates at release site 1. Two-sided
paired t-test after arcsine transformation. 2015,
n=27, P=0.6522; 2016, n=31, P<0.0001;
2017, n=33, P<0.0001. c, Total number
of HC males produced weekly and female
contamination rate at adult stage in mass
rearing facility. d, Comparison of monthly
positive female rate detected in HC males
in mass rearing facility (laboratory quality
control) and that observed in adults collected
from the field. Two-sided paired t-test after
arcsine transformation: Laboratory (n=19)
versus site 1 (n=18), P<0.0001; versus site 2
(n=16), P=0.0012. Pearson correlation: site
1, r=0.110, n=18, P=0.664; site 2, r=0.839,
n=16, P<0.0001.
ControlRelease
Site 1 Site 2
0
2
4
6
8
Before release
SupportNeutralReject
After release
Mosquito biting rate
** *
ab
Fig. 5 | Community support and reduction in mosquito biting. a, Pie
chart showing community support for the field trial in release site 1 before
(13.0%, n=123 interviews) and after (54.3%, n=431) mosquito releases
(χ2=71.29, P<0.0001). b, Mosquito human landing catches in release
and control sites 1 and 2, July to November 2017. Mean±s.e.m.; n=4
independent biological replicates for both control and release sites; two-
sided paired t-test. Site 1, t=6.988, 3 degrees of freedom, P=0.006; site 2,
t=3.566, 3 degrees of freedom, P=0.0376.
NATURE| www.nature.com/nature
ArticlereSeArcH
rendering any newly established populations incompetent for pathogen
transmission. However, the aim of population suppression is preferable
and has greater public acceptance, as it enables reduction of nuisance
biting and pathogen transmission, and long-term mosquito eradica-
tion in the absence of immigration. The combined IIT–SIT approach
is environmentally friendly and cost-effective (see Supplementary
Information), enabling vector control in complex and inaccessible
urban habitats in which implementation of standard vector control is
difficult
27,28
, as released males actively seek wild females, and allows
release of much higher numbers of male mosquitoes in comparison to
IIT alone, while simultaneously protecting against accidental female
release. Area-wide application of this approach will necessitate the
development of novel technologies, especially with regard to scaling-up
production capacity and enabling efficient mass release of mosquitoes.
Online content
Any methods, additional references, Nature Research reporting summaries,
source data, extended data, supplementary information, acknowledgements, peer
review information; details of author contributions and competing interests; and
statements of data and code availability are available at https://doi.org/10.1038/
s41586-019-1407-9.
Received: 5 November 2018; Accepted: 19 June 2019;
Published online xx xx xxxx.
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Article reSeArcH
METHODS
No statistical methods were used to predetermine sample size. The experiments
were not randomized. The investigators were not blinded to allocation during
experiments and outcome assessment.
Maintenance of mosquito lines. The four A. albopictus lines (HOU, HC, GUA
and GT) and Culex pipiens molestus used in this study were maintained on 10%
sugar solution at 27±1 °C and 80±10% relative humidity (RH), with a 12:12h
light:dark photoperiod, according to standard rearing procedures. For routine
colony maintenance and experimental studies, including viral infection assays,
female mosquitoes were blood-fed on mice according to protocols approved by the
Michigan State University Institutional Animal Care and Use C ommittees (03/14-
036-00), and the Ethics Committee on Laboratory Animal Care of the Zhongshan
School of Medicine, Sun Yat-sen University (No. 2014-003 and No. 2017-041).
Transinfection and the generation of the HC and GT lines. The natively super-
infected HOU line of A. albopictus, and C. pipiens molestus natively infected with
wPip were used as a recipient and donor, respectively, for transinfection accord-
ing to thepreviously described approach21. Cytoplasm was withdrawn from
donor embryos through embryonic microinjection, and immediately injected
into the posterior of 60–90-min-old recipient embryos using an IM300 micro-
injector (Narishige Scientific). After injection, embryos were incubated at 80%
RH and 27 °C for approximately 1 h and transferred to wet filter paper. Embryos
were then allowed to develop for 5–7 days before being hatched. Adult females
(G0) that survived embryo microinjection were isolated as pupae and mated
with HOU males. Following blood-feeding and oviposition, G0 females were
assayed for wPip infection using PCR primers specific for the WO phage orf7
gene of wPip44. G1 females from the infected G0 female were then sib-mated,
blood-fed, isolated and allowed to oviposit, followed by PCR assay for the wPip
infection. From the wPip-infected G1 females, one line (designated HC) with
a stable association was chosen for further studies, including confirmation of
infection with wAlbA and wAlbB using strain-specific primers for PCR diag-
nosis. The primers for wAlbA have previously been reported45. The primers
for wAlbB were: wsp, forward 5-ACGTTGGTGGTGCAACATTTG-3; reverse
5-TAACGAGCACCAGCATAAAGC-3. Males of a wild-type mosquito line GUA,
with the native double wAlbA and wAlbB infection(initially collected from the
field in Guangzhou, China), were subsequently outcrossed with HC females for
seven generations to create comparable nuclear genetic backgrounds in both mos-
quito lines for subsequent experiments and the field release of HC
9
. To generate the
aposymbiotic line GT, the HC line was fed with 10% sucrose containing 1 mg/ml
tetracycline solution for five consecutive generations. Removal of Wolbachia f rom
the mosquito was confirmed by PCR in the subsequent generations.
Experimental crosses to determine cytoplasmic incompatibility. Cytoplasmic
incompatibility assays were conducted as previously described29. In brief, ten virgin
females were mated with ten virgin males, with five replicates for each cross. Mated
females were blood-fed weekly using mice. Oviposition sites were constantly avail-
able to females, and oviposition paper was changed weekly for three weeks. After
egg maturation for 5–7 days on wet f ilter paper, eggs were immersed in water. Two
days later, hatched eggs were counted to determine the hatch rate.
Wolb a c hi a visualization in ovaries. Wolbachia were visualized in the ovaries by
FISH, as previously described with slight modifications21. Dissected ovaries from
females (about10 days old) were fixed in 4% formaldehyde for 15 min. The ova-
ries were washed consecutively in methanol, acetone and finally PBST, and then
incubated overnight at 37 °C in hybridization solution (Dig Easy Hyb Granules,
Roche) containing 200 ng of Wolbachia-specific 5-FITC-labelled 16S rDNA
oligonucleotide probes W1 and W2 (Bioneer)
46
. Following hybridization, samples
were washed with PBST and stained with DAPI (Roche) for 5 min. Samples were
then mounted on a glass slide with neutral resin and a cover slip, before viewing
with an Olympus IX70 fluorescence microscope.
Virus culture and titration. Zika virus (ZIKV) strain Z16006 was isolated from a
patient in February 2016 by Guangdong Provincial Center for Disease Control and
Prevention (CDC). Both ZIKV and DENV-2 (New Guinea C strain) were cultured
in C6/36 cells before infecting mosquitoes according to standard procedures
35
.
ZIKV was passaged for only three generations, after initially seeding cultures at a
multiplicity of infection of ~1 virus particle per cell. Infected cells were grown in
DMEM supplemented with 10% FBS, and incubated for 6 days at 35 °C and 5%
CO
2
. Cells were subsequently collected, thawed and frozen to facilitate release of
the virus particles. ZIKV used for mosquito oral infection was titrated on BHK
cells in 96-well plates at half-maximal tissue culture infection dose, and DENV
was titrated using the plaque assay method as previously reported47. C6/36 and
BHK cell lines were purchased from the ATCC. None of these cell lines was found
in the database of commonly misidentified cell lines maintained by ICLAC and
NCBI Biosample. All these cell lines were authent icated by ATCC and did not have
mycoplasma contamination.
Nucleic acid extraction and RNA reverse transcription. Total RNA was extracted
from whole mosquitoes, their tissues or saliva samples, as well as virus cell
culture supernatant, using RNAiso (Takara) according to the manufacturer’s
protocol. Extracted RNA was dissolved in RNase-free water, DNase-treated and
then immediately reverse-transcribed using HiScript Q RT SuperMix for qPCR
(Vazyme). cDNA was stored at 20 °C for subsequent RT-qPCR analyses. To
measure Wolbachia genome copy number, ovaries were dissected from female
mosquitoes, and total DNA was ext racted from p ools of two ovary pairs using the
phenol–chloroform method, then dissolved in ddH2O, and stored at 20 °C for
subsequent PCR analysis.
Virus and Wolbachia quantific ation. The genome copy numbers of ZIKV and
Wolbachia were measured using RT-qPCR as previously described37,48. Plasmids
containing target gene fragments of ZIKV NS1, wsp or rps6 were cloned, quanti-
fied using a NanoDrop 2000 (Thermo), and then used for serial dilutions (from
10
1
to 10
7
) to construct the standard curve
49
. RT-qPCR was performed using
SYBR Premix Ex Taq (Takara) on a Roche 480 instrument using the following
conditions: 95 °C for 30 s, then 40 cycles of 90 °C for 5 s, and finally 60 °C for 20 s,
followed by melting curve analysis. ZIKV and Wolbachia copies in mosquito
tissues were normalized using the mosquito rps6 gene. The genome copy numbers
of Wolbachia were measured usingpreviously reported
50
primers (440F, 691R),
and the primers used to measure ZIKV in RT-qPCR were newly designed and had
the following sequences: NS1: forward 5-GAGACGAGATGCGGTACAGG-3,
and reverse 5-GGGGGAGTCAGGATGGTACT-3; rps6: forward 5-CGT
CGTCAGGAACGTATTCG-3, and reverse 5-TCTTGGCAGCCTTGACAGC-3.
Vector competence assay using oral infection. Mosquitoes were infected with
ZIKV or DENV-2 through bloo d-feeding as previously described47. In brief, freshly
propagated ZIKV or DENV-2 supernatant was mixed 1:1 with human blood, and
then the mixture was added into glass feeders covered wit h pig intestine as a mem-
brane. Glass feeders were connected to a water bath circulating system (Fisher)
to keep the blood at 37 °C. Mosquitoes were allowed to feed on the mixture for
30–45 min. Only engorged mosquitoes were collected and maintained in standard
rearing conditions, and were kept in a double cage system to pre vent escape. ZIKV
replication was determined by viral genome copy numbers in mosquito abdomens
at 7 days post infection (dpi), and dissemination was measured by ZIKV infection
status in one mosquito hind leg at 14 dpi. Total RNA of the dissected tissues was
extracted, reverse-transcribed and quantified by RT-qPCR. To study ZIKV and
DENV-2 horizontal transmission potential, at 14 dpi, saliva of each mosquito was
collected by the forced salivation technique with modifications
51
. In brief, mos-
quitoes were anaesthetized with CO2, and their legs and wings were removed.
The mosquito proboscis was inserted into a 10-µl pipette tip containing 6 µl FBS
for 30 min at room temperature. A plaque assay was used to determine DENV-2
infection level in saliva. To quantify ZIKV genome copies in the saliva, RNA of 16
saliva samples from each mosquito line were extracted, reverse-transcribed and
quantified by RT-qPCR. To determine the infectivity of the viruses from orally
infected mosquitoes, 24 saliva samples from each mosquito line (in total 48 saliva
samples) were immediately separately injected into 4–5-day-old adult female GUA
mosquitoes. Each mosquito was injected with ~1µl supernatant, and each individ-
ual saliva sample was injected into 4–6 mosquitoes. After seven days incubation
under standard rearing conditions, t he injected mosquitoes for each saliva sample
were killed and their bodies were pooled, homogenized, and tested by RT-qPCR
for ZIKV detection. Samples with positive results indicated that infectious ZIKV
particles were present in the saliva that was originally used to inject the mosquitoes,
suggesting that the corresponding mosquitoes from whom the saliva was originally
taken had the potential to transmit ZIKV37.
Mosquito transmission assay using ZIKV-infected suckling mice. After propa-
gation in C6/36 cells, ZIKV supernatant, with 10
7.4
viral genome copies per ml, was
used to inject female GUA mosquitoes via thorax inoculation using a Nanoject II
microinjector (Drummond). Ten days post-infection, 10 mosquitoes were allowed
to bite 4 1-day-old suckling Kunming mice (KM) with each mouse receiving 3–4
bites. Mice were sex-matched and randomized for the experiment. During the
48–72 h post-biting during which the viraemia developed, each suckling mouse
was used to feed and infect 6–8 day-old GUA and HC mosquitoes. For each mouse,
HC was allowed to feed for 1 h and then removed, immediately followed by GUA
for the same period of time. In total 4–6 engorged mosquito es were collec te d from
each suckling mouse. The engorged mosquito es were kept under standard rearing
conditions for 7 days, as described above. Then, tot al RNA was extracted from each
mosquito whole body, followed by PCR with reverse transcription (RT–PCR) to
check their ZIKV infection status.
ZIKV vertical-transmission assay. Thirty 3–4-day-old female GUA and HC mos-
quitoes were infected with ZIKV by intrathoracic inoculation using ZIKV culture
supernatant, with 10
7.2
viral genome copies per ml. At 7, 14 and 21 dpi, mosquitoes
were blood-fed, and eggs were collected 3 days after each bloodmeal. After egg
hatch, fourth-instar larvae from each gonotrophic cycle were collected, and five
larvae were pooled for RNA extraction. ZIKV infection status was evaluated by
RT–PCR. For the f irst and se cond gonotrophic cycles, 10 pools were collected; for
the third gonotrophic cycle, 15 pools were collected.
NATURE| www.nature.com/nature
ArticlereSeArcH
Laboratory cage male mating competitiveness assays. Four adult cages were
prepared with fifty GUA males and fifty females. Varying numbers of HC males
(0, 50, 250 or 500) were released into the cages, so that the ratio of GUA females:GUA
males:HC males was 1:1:0, 1:1:1, 1:1:5 or 1:1:10. Mosquitoes were allowed to mate
for two days. The mosquitoes were then blood-fed for approximately 20 min. Two
days after blood-feeding, egg cups were inserted into the cages for collecting eggs.
Eggs were collected for two nights, and the egg hatch rate was then determined
as described in the cytoplasmic incompatibility cross experiment. The egg hatch
rate was compared to the expected hatch rate assuming: (i) random mating and
equal mating competitiveness between HC and GUA males, and (ii) complete
unidirectional cytoplasmic incompatibility between HC males and GUA females.
Population replacement in laboratory cages. The population cage experimental
design was as previously described
22,52
. Each population cage started with 50 GUA
females and 50 GUA male adults. Three days after cage establishment, cages were
provided with mice for blood-feeding, followed by the release of blood-fed HC
females into the cages. The number (3, 7, 13 and 33) of HC females introduced
into each cage was varied to produce an initial female infection frequency of 6%
12%, 20% and 40%, respectively. No additional HC females were released. To pro-
mote population replacement in the release cages with 6% and 12% initial female
infection frequency, a single release of 200 HC males was used at the start of the
experiment to induce cytoplasmic incompatibility and suppress viable progeny
production by GUA females. An uninfected control GUA population cage was
set up without any additional int ro duc tions of mosquitoes. Oviposition sites were
provided in population cages two days post-blood meal. Eggs were collected for
two consecutive nights, matured for an additional 5–7 days, and then hatched. All
hatched larvae were reared to adults, and 50 females and 50 males were randomly
selected to establish the next generation. After eggs were collected at each gen-
eration, approximately 10–20 females were randomly selected in each cage and
examined for wPip infection by PCR to determine female infection frequency.
Semi-field cage population suppression experiments. Each of the six semi-field
cages (1.75×1.75×1.75 m, 5.36 m
3
, Live Monarch) were s et up w ith: (i) 1 plastic
container filled with 300 ml deionized water and lined with filter paper for oviposition;
(ii) 1 plastic cylinder filled with 200 ml deionized water for holding larvae, which
was covered with a plastic board to prevent oviposition (the plastic board was
removed every day to release newly emerged adults and then replaced); and
(iii) 2 plastic cups each with a piece of filter paper and filled with 80 ml sugar
solution (10%) (the sugar feeders were changed twice a week). The environmental
conditions were 25.0±0.5 °C, and 36.0±6.0% RH, measured by means of 3 data
loggers (HoBo) located on the top of 3 randomly selected cages.
To establish GUA populations in the treatment and control cages, 200 third-
instar GUA larvae were transferred to the plastic cylinder containers in each cage
weekly, from week 0 to week 4. From week 3, females were fed on a sausage filled
with pig blood (60–70 ml per sausage) placed on the top of the cages 3 times a week.
From weeks 5 to 12, the GUA populations were maintained by returning 150 third-
instar larval offspring into each cage every week. Eggs laid in each cage were
collected twice a week, counted, dried, stored in a plastic bag in the climate-
controlled room (25±1.0 °C, 60±10% RH), and then hatched after being left to
mature for 7–12 days. The hatch rates of eggs from each cage were recorded each
week. To monitor the population dynamics inside the experimental cages, adults
were randomly collected using aspirators placed in the centre of each cage for 10 min
each week. After immobilization at 4 °C, the captured adults were counted and
sexed, and then returned to their respective cages.
Starting from week 12, 375 HC male adults were released weekly into the treat-
ment cages, representing a 5:1 (375 HC males: 75 GUA males) initial release ratio
of HC to GUA males based on the assumption that 75 fertile GUA males would
eclose from the 150 larvae introduced weekly into the target populations. To mimic
a 2% female contamination rate, which could happen during mass rearing owing
to lack of perfect sex-separation, eight HC female adults were released together,
each time, with the HC males.
Before release, both HC male and female pupae were irradiated at 28 Gy, which is
a dose known to effectively sterilize females but tonot negatively affect male mating
performance24. HC pupae were collected and placed in the centre of a plastic plate,
which was placed in the middle of the irradiation cylinder. Irradiation was per
-
formed with 4.2 s transit time at the dose rate 2.144 Gy/s by a Gammacell irradi ator
220 (Atomic Energy of Canada). Irradiated pupae were placed into a plastic cage
in the climate-controlled room for emergence. The release frequency was twice a
week with a 48-h interval. HC males were 3–4 or 5–6 days old during the first and
second releases, respectively. E ach time, either 188 or 187 HC males were released
into each treatment cage, resulting in a total of 375 HC males per week per cage.
Starting from week 13 (week 1 post-release), the number of GUA larvae returned
to each treatment cage was adjusted to ref lect the effect of HC male releases on the
mosquito population. To maintain a stable population, 150 larvae were returned to
each control cage every week. The number of larvae returned to each HC treatment
cage was calculated to reflect the level of population suppression as determined by
egg hatch rate in the treatment cage in relation to the control cage in the previous
week. For example, if the egg hatch rate in week 15 was 80% and 50% in a control
and treatment cages, respectively, then 94 larvae (150×0.5/0.8) were returned to
the treatment cage in week 17.
To assess whether combined IIT–SIT can prevent population replacement
caused by HC female contamination, from week 19 (week 7 post-release), the
larvae in excess of those that had been returned to the experimental cages were
randomly sampled each week to examine wPip infection. Each time, up to 300
larvae were tested for wPip infection (all larvae were examined if there were <300
larvae). Larvae were tested in groups of 10 or 20 pooled larvae if numbers fell
between 30 and 300, and larvae were tested individually if there were less than 30.
Mass-production and irradiation of HC males. Mass-production of HC males
included five steps: adult rearing, larvae rearing, sex separation, X-ray irradiation
and packaging according to a proto col des cribed pre viously with slight modifica-
tions
39,40,53
. Approximately 3,000 female pupae and 1,000 male pupae (3:1 ratio of
female to male) were placed into an adult cage (stainless steel, 30×30×30 cm)
for eclosion
40
. Adults were continuously provided with 10% sugar solution. Sheep
blood, provided weekly by a local abattoir, was mixed with ATP (500 mg ATP in
100 ml blood) and then used to feed females at 5–6 and 9–10 days old. Two days
after blood-feeding, mosquitoes were provided oviposition sites to collect eggs
for two days. After their eggs were collected twice, mosquitoes were euthanized
by putting adult cages in a freezer at 20 °C overnight. Eggs were matured for one
week before hatching in water. After hatching, 6,600 larvae were added to each tray
(length×width×height=58 cm×38 cm×4 cm) filled with water at a depth
of 1.5 cm39. Larvae were fed daily with food containing 60% liver powder, 30%
shrimp powder and 10% yeast for six days. No lar val fo od was added when larvae
started to develop into pupae at day 7. At day 8, pupae mixed with a few larvae
were collected and then went through a Fay–Morlan sorter to separate male pupae
from female pupae and larvae
54
. Before 2016, approximately 1,000 male pupae
were transferred to each plastic ‘release’ bucket (17-cm diameter×17-cm height),
which contained water (1-cm depth) at the bottom and had a lid with a large hole
covered on top by mesh gauze to allow for air exchange and prevent the escape of
adult mosquitoes as they eclosed. Cotton soaked in 10% sugar solution was placed
on top of the gauze, when pupae started to eclose into adults 24 h after transfer
into buckets. After one day, water was removed through the gauze by turning the
bucket upside down. After the mosquitoes were immobilized at 5 °C, a manual
visual check was used to individually remove any residual females mixed in with
the adult males. The quality-controlled males were then transported to the field for
release. Starting from 2016, the male pupae collected through mechanical sorters
were exposed to irradiation at 45 Gy for 1,000 s to sterilize any residual females
using an X-ray irradiator (Wolbaki) developed specifically to treat mosquito pupae.
Approximately 65,000 to 75,000 pupae were placed together in a canister (diameter
7.5 cm×height 7.5 cm), with 2 canisters being simultaneously irradiated. After
treatment, pupae were packaged into buckets for release, as described above, but
without the manual check of adults for contaminant females.
Control of female contamination in released HC males. As a key quality con-
trol in the laboratory during mass rearing, the female contamination rate (FCR)
was monitored at both pupal and adult stages. Each batch of sex-sorted pupae
was checked by randomly selecting 4,000 of the pupae, and manually sexing
each individual by microscopic examination of their terminalia55. The batch of
pupae sampled qualified for release if the FCR was below 1%. If the FCR was over
1%, mechanical sex separation and manual screening of the batch of pupae was
repeated until the FCR was less than 1%. This resulted in an average rate of <0.5%
contaminant females present in the pupae, which—before 2016—was further
reduced by manually removing females after they eclosed into adults (as described
above). In 2016 and onwards, following irradiation and packaging, we randomly
selected 10% of the release buckets for checking adults to record the FCR, which
was less than 0.3% in both 2016 and 2017. To further monitor the risk of female
contamination in inducing population replacement in the field, female adults were
also collected weekly from the release sites and assayed for wPip infe ction by PCR .
The female-positive rates from the laboratory and field were compared to test for
a correlation between these variables. In addition, larvae hatched from ovitraps
collected weekly from the release sites were used to assay wPip infection, to monitor
whether contaminant females had produced offspring in the field.
Description of study areas. With an approximately 3.3 km
2
area, Shazai island
(22°5131.99 N,113°3240.51E) is located in the Nansha District, Guangzhou
City, China. There is a human population of 1,865 individuals across 505 houses
in its only residential area (25 ha), which was selected as release site 1 (Fig.2a). An
area with similar size and ecological conditions in Xiaohu island (22°5049.07
N,113°3137.54E), separated from Shazai by a bridge, was selected as its control
site. Located in the Panyu District, Guangzhou, Dadaosha island (22°5456.39′′
N,113°2543.84′′E) is approximately 10.9 km2 in area. One of its residential
areas (7.5 ha) with 350 people across 158 houses was selected as release site 2, and
2 nearby control sites were located either on the same island or Guanlong island
Article reSeArcH
(22°5453.03′′N,113°2648.63′′E) (Fig.2b). S eparated by rivers, these study areas
are relatively isolated, with evidence of passive mosquito dispersal along human
transportation networks, either through terrestrial or marine vehicles. The local
dengue transmission in Guangzhou occurs from the middle of August to the end
of November with A. albopictus as the only vector. As a typical subtropical area,
Guangzhou has a mosquito season from March to November, with a peak from
June to September and almost no mosquitoes detected from December to February.
Pre-release monitoring of release and control sites. Prior to the release of HC
males, A. albopictus populations were monitored weekly using approximately 100
ovitraps (Tianpai) for release site 1 and its associated control site between June and
November 2014, and 24 or 25 ovitraps for release site 2 and one of its control sites
(site 2a) between May and November 2015. With the control site 2b added in 2016,
data from control sites 2a and 2b were combined for their comparison to release
site 2. The ovitraps cont ained a piece of filter paper (10×6 cm) for collecting eggs,
and 50 ml water previously infused with bamboo leaves for attracting females to
oviposit. Every week, ovitraps were placed in the f ield for 7 days, and then collected
and placed in an incubator (Yiheng) for 6 days at 27±1 °C, 80±10% RH, and a
photoperiod of 12:12 h (light: dark). The number of ovitraps that contained eggs
was recorded, and the number of hatched eggs was determined by visual examina-
tion using a stereomicroscope (Olympus). The proportion of positive ovitraps was
calculated as the number of ovitraps containing eggs divided by the total number
of ovitraps used, and the egg hatch rate per trap was calculated as the number of
hatched eggs divided by the total number of eggs collected.
Field release of HC mosquitoes. Mosquitoes were transported from the mass-rear-
ing factory to the release sites by a van three times per week (Tuesday, Thursday and
Saturday), and on the next day released in the morning between 07:00 and 10:00.
The mass-rearing factory was approximately 1h driving time from the release
sites. Cotton soaked with 10% sugar solution was continuously supplied to the
adults before release. Mosquitoes were released every 50 m with approximately
100 and 40 fixed release spots in release sites 1 and 2, respectively, preferably near
vegetation (for example, t rees or underbrush). During release, buckets were opened
by removing the mesh and all mosquitoes immediately flew away. The number
of buckets for release in each spot was adjusted empirically based on the recently
determined HC-to-wild-type male ratio in each zone, with the goal of reaching the
5:1 target ratio. On average, between 1.5 and 2.6 million HC males were released
weekly, making for a total of 52.7 and 92.6 million males released overall at site 1
in 2016 and 2017, respectively. In release site 2, on average between 600,000 and
890,000 males were released weekly, such that a total of 19.7 and 32.1 million males
released overall in 2016 and 2017, respectively.
Monitoring population suppression. A. albopictus populations in all control and
release sites were monitored weekly, throughout the period of HC male release,
using ovitraps and BG-Sentinel traps (Biogents). Release sites 1 and 2 were divided
into 22 and 8 zones, respectively, to precisely monitor mosquito density and
dynamics (Fig.3c, d). Mosquito releases occurred in all the zones, except that a
rolling-carpet approach was used, owing to initial restrictions in male production,
to limit releases to 6 zones in the north end of release site 1 on 16 June 2015, and
then gradually expanded to the neighbouring zones until all zones were covered
from 11 July 2015 to 20 October 2015. To monitor the mosquito population, on
average 5 ovitraps and 2 BG traps were used per zone, with a total of 110 ovitraps
and 44 BG traps distributed in release site 1, and 40 ovitraps and 16 BG traps in
release site 2 (the locations are shown on t he map in Extended Data Fig.5). There
were 100 ovitraps and 30 BG traps distributed in control site 1, and 30 ovitraps
and 12 BG traps distributed in each of the two control sites for release site 2. All
the ovitraps and BG traps were labelled with specific numbers, corresponding to
their locations, to enable sample locations of collected mosquitoes to be tracked.
Seven days after being left in the field, ovitraps were collected and brought back
to the laboratory. The total number of ovitraps collected, those with eggs present
and the number of eggs in each positive ovitrap were recorded. The positive ovitraps
were incubated for six days in an incubator as described above. The hatched eggs
were counted and recorde d under a diss ecting microscope. The average number of
hatched eggs per ovitrap, in b oth release and control sites, was determined each week,
and used to measure population suppression at immature (egg and larval) stages.
BG traps were continuously run for 24 h every Monday, 48 h after the last
release, in both release and control sites. The captured adults were sent to the labo-
ratory, and were put into a freezer for at least 30 min at 20 °C before further char-
acterization. Then, A. albopictus were identified, sexed and the number of males
and females in each trap was counted and recorded. All collected females were
assayed for wPip infection by PCR. After wPip-positive females were removed, the
average number of females in both release and control sites per BG trap was deter-
mined each week, and used to measure population suppression at the adult stage.
To determine t he ratio of released HC to wild-type males, collected males were
tested for wPip infection using PCR. The following criteria were used to determine
the size of the tested samples: (i) all the collected A. albopictus males were individually
assayed for wPip infection if the total number of males collected in a release site
was less than 300, or the number of males in an individual trap was less than 20;
(ii) 50% of collected males were assayed when the total number of collected males
was between 300 to 1000; (iii) 33.3% of collected males were assayed when the
total number of collected males was more than 1,000. For each week, the ratio of
released HC to wild-type males was then calculated by dividing the number of
wPip-positive males by the number of wPip-negative males.
Although the proportion of egg-positive ovitraps and total number of eggs per
trap are useful and important measures of overall relative population size, they do
not distinguish between the dead and viable eggs present in the ovitrap, and thus
are incapable of indicating the direc t re duct ion in t he number of viable eggs caused
by cytoplasmic incompatibility matings. Consequently, the number of hatched
eggs was the main parameter used to compare the level of population suppression
at the larval stage between release and control sites. However, it should be noted
that hatched eggs could be produced by wild females that migrate into the area
after mating outside it, as well as by resident females emerging within the site that
mate with wild males.
Population suppression was also estimated by changes in the number of adult
females collected in BG traps. This will produce an overestimate of the effective
population size contributing to future generations because many of t he fema les will
have been mated by released HC males and therefore contribute no offspring to the
next generation. Adult counts includedonly wPip-negative females.
We expressed population suppression at release sites relative to control sites
(that is, in terms of the per cent reduction in numbers of hatched eggs and adult
females collected at the release sites relative to the numbers observed at control
sites) (Fig.2d, e, 3a, b, Extended Data Figs. 4, 6). We used the averaged observations
from its two control sites for release site 2 and used only one site as a control at site 1.
Mating competitiveness and expected hatch rate. To estimate mating competi-
tiveness of the released HC males in the field, the expected egg hatch rate (H
e
) in
the release site was determined based on the weekly ratio (N) of HC to wild-type
males, and the egg hatch rate (Hc) in the control site in the same week, assuming
complete unidirectional cytoplasmic incomp atibility between HC males and wild-
type females, equal mating competitiveness between HC and wild-type males,
and random mating. As a result, the following equation was used to calculate the
expected egg hatch rate, He=Hc×[1/(N +1)]. The observed egg hatch rate was
then compared to the expected hatch rate in the same week, with the difference
between these rates taken to reflect relative mating competitiveness.
PCR assay of wPip infection. Each individual adult or pool of larvae from an ovit-
rap were homogenized in DNA extract ion buf fer (Daan Gene). After a brief centrif-
ugation, samples were incubate d at 99 °C for 10 min, and the supernatant was then
used as template for PCR assay. A 20 µl RT-qPCR reaction consisted of 2 µl DNA
template, 15 µl A buffer (containing primers, fluorescent probe and dNTPs) and
3 µl B buffer (containing DNA polymerase). In brief, RT-qPCR reaction was per-
formed according to the procedures of Wolbachia wPip Detection Kit (Wolbaki).
The specific-primers used for the assay were designed for phage WO of wPip and
consisted of: orf7F: 5-GTTTGTGCAGCTAATAG-3; and orf7R: 5-GTCTGCA
AGGCCTATTTCTACTG-3; and the sequence for the fluorescent probe was
5-CTTTCAATTGAAAAGATTCGATCAAC-3. The RT-qPCR conditions com-
prised of 15 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 45 s at 55 °C, 30 s at
72 °C, and finally 30 s in 1 °C steps from 60 to 95 °C to generate the melting curve
for confirmation that the fluorescence detected was for the specific PCR product.
If wPip-positive larvae were observed, the sample was further screened by
standard PCR using primers specific to the ribosomal protein S6 (rps6) gene of
C. quinquefasciatus, the only possible mosquito species with wPip co-occurring in
the field sites, to exclude any false positives resulting from the collection of Culex
larvae in the ovitraps. The specific-primers used for the assay were designed for
rps6 gene and consisted of: rps6F: 5-TGCCGCGTCGTCTTGAATC-3; and rps6R:
5-GTATTGACCTCGTCGCGCTT-3. The 20 µl PCR reaction consisted of 2 µl
DNA template, 10 µl PCR Master Mix (Dongsheng), 1 µl of each primer (10 µM) and
6 µl ddH
2
O. The PCR conditions comprised of 5 min at 98 °C, followed by 40 cycles
of 30 s at 98 °C, 5 s at 55 °C, 30 s at 72 °C, and then 10 min at 72 °C for the final exten-
sion. PCR products were electrophoresed on a 1.5% agarose gel, which contained
1 µg/ml ethidium bromide. If a product size of approximately 350 bp was obtained,
the sample was considered to contain wPip derived from Culex mosquitoes.
Community engagement. Before the open field releases of HC commenced, meet-
ings and seminars involving various st akeholders (for example, public he alt h of fi-
cials, scientific experts and the general public) were held to introduce the principle,
efficacy and biosafety of A. albopictus population suppression through release of
HC males to induce incompatible matings. We then received a permit authorizing
field trials from the Ministry of Agriculture of the People's Republic of China and
declarations of support from the different administrative levels of local government
(that is, district, town and village). A series of community engagement activities
were launched, including organizing meetings with village representatives, visiting
households and distributing basic information on mosquitoes and mosquito-borne
diseases, as well as the aims and methods of our project. During the course of these
ArticlereSeArcH
activities, we answered questions or concerns raised by the residents. A question-
naire survey led by village officials was also undertaken in each household. Signed
informed consent was obtained from residents who had agreed to HC release,
granting us permission to perform necessary activities around their residences,
including releasing mosquitoes and placing monitoring tools (that is, traps) near
their houses. Among 506 households in Shazai, 455 households were contacted
and informed consent, to release HC mosquitoes in or around their property,
was obtained from 453 (99.6%) households. In Dadaosha, informed consent was
obtained from all the 141 contacted households. No mosquitoes were released at
households (or around their neighbouring residences) that did not consent to the
release of HC mosquitoes.
During the release period, we maintained communication with the different
administrative levels of local government, updating them on the status of the
project, and informing them of preliminary and ongoing results as they became
available. In the release sites, we maintained a close relationship with the resi-
dents through performing house-to-house surveys twice per week, in which their
feedback was sought regarding the mosquito releases. Regular information on
the level of mosquito population suppression was provided to those households if
concerns were raised. In addition, we kept the public informed and updated with
the progress of the project through posters, newsletters, radio broadcasts, print and
TV news media, and the mobile phone app WeChat. All these activities ensured
that the residents understood, were satisfied with and supported the continued
release of mosquitoes.
Community surveys were conducted in release site 1 to investigate if residents
shifted their opinion about the field trial before and after release. Residents were
randomly selected and interviewed to determine whether they either supported,
rejected, or were neutral about the release of HC males. Specific reasons for their
views were also sought.
Mosquito human-landing assay. Human-landing catches were performed at both
release sites and their associated control sites, according to a protocol approved by
the Ethics Committee on Medical Research of the Zhongshan School of Medicine,
Sun Yat-sen University. Sixteen localities were selected in each of Shazai and
Xiaohu, and ten localities were selected in each of the Dadaosha release and con-
trol sites. All the selected localities were close to houses, in shaded and sheltered
areas (that is, locations where A. albopictus is most likely tobe found), and near
the release locations. The experiments were conducted between 09:00 and 11:00
or between 16:00 and 18:00. Researchers worked in pairs, by standing at localities
for 15 min, and collecting mosquitoes from the other person. The same pairs
of researchers monitored mosquitoes in both release and control sites to reduce
variation in the attractiveness of different individuals to mosquitoes. During the
collection periods, mosquitoes that landed or flew around the volunteers were
manually captured by mosquito aspirators. All captured mosquitoes were marked
with time, date and location of collection, and sent back to t he lab oratory for spe-
cies and sex identification, and further investigation. The procedure was performed
four times, in both Shazai and Dadaosha and their associated control sites, from
July to November 2017. The mosquito biting index was calculated as the average
number of A. albopictus females caught per person in 15 min.
Statistical analysis. Statistical analyses were performed using GraphPad Prism
software (v.5.00). ANOVA and Tukey’s multiple comparisons test were used to
compare egg hatching in cytoplasmic incompatibility cross exper iment. Differences
in mosquito infective rates were analysed using Fisher’s exact test. Pearson’s corre-
lations were used to test for an association in mosquito numbers between release
and control sites before suppression. Mann–Whitney tests were undertaken to
compare Wolbachia density in ovaries, the number of wPip-positive females within
different release years, and mosquito density between release and control sites,
including the proportion of egg-positive ovitraps, the average total number of
eggs per ovitrap, the number and proportion of eggs hatching per ovitrap, as well
as total number of female adults per trap per 24 h. To compare within each year
these measures between respective control and release sites, we f irst c alculated their
average values for all traps per week, and then compared separately for each year
these weekly averages between the control and release sites using Mann–Whitney
tests. Binomial test was used to compare the expected and observed egg hatch in
mating competitiveness assay in laboratory cage populations, and paired t-tests
after arcsine transformation were used to compare observed and expected egg
hatch rates in the field, and female contamination rate in the laboratory and field.
χ2 test was computed to compare community support and paired t-test was used
to compare mosquito biting before and after the release period.
Reporting Summary. Further information on research design is available in
theNature Research Reporting Summary linked to this paper.
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 contributions 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.
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.
Additional information
Supplementary information is available for this paper at https://doi.org/
10.1038/s41586-019-1407-9.
Correspondence and requests for materials should be addressed to Z.X.
Peer review information Nature thanks William Sullivan and the other,
anonymous, reviewer(s) for their contribution to the peer review of this work.
Reprints and permissions information is available at http://www.nature.com/
reprints.
Article reSeArcH
Extended Data Fig. 1 | Illustration of the procedures used to establish
A. albopictus HC line by embryonic microinjection and for PCR
verification of the Wolb a c hi a 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.
ArticlereSeArcH
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).
Article reSeArcH
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.
ArticlereSeArcH
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).
Article reSeArcH
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.
ArticlereSeArcH
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).
Article reSeArcH
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.
ArticlereSeArcH
Extended Data Fig. 8 | Temporal and spatial distribution of wPip-
positive ovitraps in the two release sites between 2015 and 2017.
a,b,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.
Article reSeArcH
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.
ArticlereSeArcH
Extended Data Table 1 | Male mating competitiveness index (C) and fertility of HC (non-irradiated) and IHC (irradiated) males
The competitive mating experiments (in bold and labelled Ho) were performed in large cages (290-cm diameter by 200-cm height) containing 100 HC males and 100 wild-type males for copulation,
with 100 virgin wild-type females. The fertile or sterile control cages (30×30×30cm) contained either 50 incompatible/sterile (HC/IHC) or fertile (wild) males, and 50 virgin wild females (normal
font and labelled Hn or Hs). Hn, mean egg hatch rate of fertile control cages; Hs, mean egg hatch rate of sterile control cages; Ho, mean egg hatch rate of treatment cages. C, male mating competi-
tiveness index, calculated as: C=[(Hn–Ho)/(Ho–Hs)]×( N/S), in which N and S are the numbers of fertile and sterile males56. Two-tailed one-sample t-tests were used to compare the C values with
the theoretical value of 1 (males equally competitive). No signicant dierence was observed for C of HC and IHC40Gy males when compared to the theoretical value 1, but C showed a 32% reduction
for IHC50Gy males, indicating that a higher irradiation dose aects male competitiveness. The dose of radiation used (45 Gy) for sterilizing HC males for eld release was therefore within the range at
which male mating competitiveness starts to decrease. This suggests that the dose of radiation should be carefully controlled, so that it is high enough to induce complete female sterility, but as low as
possible to minimize any negative eect on male mating performance.
1
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... Additionally, biological control methods may also include the alteration of the genetic materials of vectors, thereby inhibiting them from transmitting the dengue virus [16]. This subclass includes sterile insect techniques (SIT) [94,95], genetically modified mosquito (GMM) methods [96,97], such as the release of insects carrying a dominant lethal (RIDL) gene [96,98] and Wolbachia bacterium introduction (WI) [15,22,23,29,99]. ...
... Prior to formulating models to biologically control vectors fuelling the transmission of dengue, it is worth mentioning, in general, some factors to be considered in governing model interests and initiation. These factors include describing the biological agents (vectors) to be used [16,36,49,53,86,88], understanding ecological patterns between the vector and dengue virus [15,83,100], and identifying the methods of control [15,29,67,95,96]. Different model structures account for the biological vectors used by modelling vector-only transmission dynamics involving the dengue virus [29,40,64]. ...
... Of the three factors, the ecological patterns between the vector and the virus can be modelled using human-vector transmission models [52,57,101], which capture the interaction between the viral-infected mosquitoes and uninfected humans and vice versa. These control methods are considered by incorporating a control type, such as Wolbachia-based control, that may consider complex features, such as CI and IMT effects in vectors [35,95,98,99]. These are some of the complexities in model structures used for different forms of biological controls. ...
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... Nevertheless, one of the reasons why SIT is not widely used in mosquito control is that it is difficult to irradiate males without reducing male mating competitiveness and survival [35]. Rodriguez et al. found that ethanol, trimethylglycine, and beer could be used as radiation protection agents to extend the survival time of irradiated males [36]. ...
... Rodriguez et al. found that ethanol, trimethylglycine, and beer could be used as radiation protection agents to extend the survival time of irradiated males [36]. In recent studies, a combination of incompatible and sterile insect technology produced large-scale male sterility with a low irradiation dose without affecting male mating competitiveness and survival [35,37]. In addition, Becker et al. proved that a combination of Bacillus thuringiensis israelensis and SIT is effective for Ae. ...
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The mosquito Aedes albopictus can transmit various arboviral diseases, posing a severe threat to human health. As an environmentally friendly method, sterile insect technology (SIT) is considered an alternative to traditional methods such as chemical pesticides to control Ae. albopictus. In SIT, the sterility of male mosquitoes can be achieved by γ-ray or X-ray radiation. Compared with γ-rays, X-rays are easier to obtain, cheaper, and less harmful. However, there is a lack of comparative assessment of these two types of radiation for SIT under the same controlled conditions. Here, we compared the effects of X-ray and γ-ray radiation on the sterility of Ae. albopictus males under laboratory-controlled conditions. Neither type of radiation affected the number of eggs but significantly reduced the survival time and hatch rate. The same dose of γ-rays caused a higher sterility effect on males than X-rays but had a more significant impact on survival. However, X-rays could achieve the same sterility effect as γ-rays by increasing the radiation dose. For example, X-rays of 60 Gy induced 99% sterility, similar to γ-rays of 40 Gy. In the test of male mating competitiveness, the induced sterility and the male mating competitiveness index were also identical at the same release ratio (sterile males/fertile males). At a release ratio of 7:1, nearly 80% of eggs failed to hatch. Sterile males produced by X-ray and γ-ray radiation had similar male competitiveness in competition with field males. In conclusion, a higher dose of X-rays is required to achieve the same sterility effect, compared to γ-rays. When γ-rays are not readily available, high-dose X-rays can be used instead. This study provides data supporting the selection of more suitable radiation for the field release of sterile male mosquitoes.
... Thanks to these deployments we begin to have promising evidence of its ability to reduce dengue cases for real [69,122]. As for the IIT we find also successful recent deployments in the literature, whether it is used alone (Australia, [21]) or in combination with the SIT (Thailand [74], Mexico [84] and China [152]). With results ranging from a 50% population reduction, to almost complete suppression. ...
Thesis
With vector-borne diseases rising globally and mosquitoes expanding their habitats due to climate change,mosquito control is undoubtedly one of the main challenges for human health in the years to come. This thesis isdevoted to the modeling, analysis and simulation of mosquito and mosquito-borne diseases optimal control strategies using modified vector releases. We first investigate optimal population replacement strategies. These consist in replacing optimally the wild population by a population carrying the endosymbiotic bacterium Wolbachia, since it has been shown that mosquitoes carrying this bacterium are less likely to transmit some arboviruses. By considering a high fecundity limit we reduce the study of the mosquito population to a single equation on the proportion of Wolbachia-infected mosquitoes. First, we study strategies optimizing a convex combination of both the cost of the releases and the performance of the technique. We fully analyse this problem, proving a time monotonicity property on the proportion of Wolbachia-infected mosquitoes and using a reformulation of the problem based on a suitable change of variable. Next, we consider the spatial optimization of the releases, optimizing a single instantaneous release at the initial time maximising the final proportion of Wolbachia-infected mosquitoes throughout the domain at a given time horizon. We fully characterize the solutions under some hypothesis in the non-diffusive case. Moreover, simulations are carried for the case with diffusion. Finally, we extend the focus of the study to humans. We consider an epidemiological model in which both populations are taken into account as well as the dynamics of a vector-borne disease with exclusively human-mosquito and mosquito-human transmission like dengue. In this setting, we minimise the amount of human infections during an outbreak using instantaneous releases of modified vectors, represented by linear combinations of Dirac measures with positive coefficients determining their intensity. Optimal strategies for both population replacement and the sterile insect technique are studied numerically using ad-hoc algorithms, based on writing first-order optimality conditions characterizing the best combination of Dirac measures.
... These control tools are effective, but are currently threatened by the development of wide-spread insecticide resistance [4]. Alternative a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 strategies involving mass-releases of laboratory reared mosquitoes are rapidly becoming a key tool in the management of Aedes populations with releases of mass-reared Aedes in the Cayman Islands [5,6], Brazil [7], Cuba [8], Malaysia [9], China [10] and Singapore [11]. ...
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... When the intervention stops, mosquito populations may re-emerge. Moreover, the strategy can be hard to deploy in practice due to the accidental release of infected females, which may produce infected offspring and undermine the process 5,6 . ...
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Wolbachia infection in Anopheles albimanus mosquitoes can render mosquitoes less capable of spreading malaria. We develop and analyze an ordinary differential equation model to evaluate the effectiveness of Wolbachia-based vector control strategies among wild Anopheles mosquitoes in Haiti. The model tracks the mosquito life stages, including egg, larva, and adult (male and female). It also accounts for critical biological effects, such as the maternal transmission of Wolbachia through infected females and cytoplasmic incompatibility, which effectively sterilizes uninfected females when they mate with infected males. We derived and interpreted dimensionless numbers, including the basic reproductive number and next-generation numbers. The proposed system presents backward bifurcation, which indicates a threshold infection that needs to be exceeded to establish a stable Wolbachia infection. The sensitivity analysis ranks the relative importance of the epidemiological parameters at the baseline. We simulate different intervention scenarios, including pre-release mitigation using larviciding and thermal fogging before the release, multiple releases of infected populations, and different release timing. Our simulations show that the most efficient approach to establishing Wolbachia is to release all the infected mosquitoes immediately after the pre-release mitigation process. Also, the model predicts that it is more efficient to release during the dry season than the wet season.
... Sterile insect technique (SIT) is a target-specific, nondisruptive pest control method among the biologically-based approaches (Orankanok et al. 2007), and SIT is widely used against dipteranpests such as Aedes aegypti (Thomé et al. 2009), Ceratitis capitata (Walther et al. 2015), Cydia pomonella (Thistlewood and Judd 2019), and Aedes albopictus (Zheng et al. 2019b). Conventionally, SIT usually requires releasing a large number of sterile male flies to mate with wildtype females (Zheng et al. 2019a). ...
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The oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is an invasive and polyphagous pest of horticultural crops, and it can cause huge economic losses in agricultural production. The rapid development of CRISPR/Cas9 gene editing technology has provided new opportunities for the scientific control of agricultural pests. Here, we explore the applicability of the B. dorsalis sex peptide receptor (Bdspr) as a target gene for the CRISPR/Cas9-based sterile insect technique (SIT) in B. dorsalis. We screened two high-efficient single guide RNAs (sgRNAs) for gene editing. The results showed that both mutation efficiency and germline transmission rate were 100% in the surviving G0 females (8/8) from injected embryos, and that 75% of mosaically mutated G0 females (6/8) were sterile. The 50% of heterozygous G1 females (4/8) could not lay eggs; 100% of eggs laid by them could not survive; and 62.5% of individual females (5/8) had abnormal ovaries. These results indicate that Bdspr plays an important role in regulating fertility, egg viability, and ovary development in female B. dorsalis, suggesting that the spr gene can be used for CRISPR/Cas9-based SIT in B. dorsalis.
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Aedes aegypti mosquitoes carrying self-spreading, virus-blocking Wolbachia bacteria are being deployed to suppress dengue transmission. However, there are challenges in applying this technology in extreme environments. We introduced two Wolbachia strains into Ae. aegypti from Saudi Arabia for a release program in the hot coastal city of Jeddah. Wolbachia reduced infection and dissemination of dengue virus (DENV2) in Saudi Arabian mosquitoes and showed complete maternal transmission and cytoplasmic incompatibility. Wolbachia reduced egg hatch under a range of environmental conditions, with the Wolbachia strains showing differential thermal stability. Wolbachia effects were similar across mosquito genetic backgrounds but we found evidence of local adaptation, with Saudi Arabian mosquitoes having lower egg viability but higher adult desiccation tolerance than Australian mosquitoes. Genetic background effects will influence Wolbachia invasion dynamics, reinforcing the need to use local genotypes for mosquito release programs, particularly in extreme environments like Jeddah. Our comprehensive characterization of Wolbachia strains provides a foundation for Wolbachia-based disease interventions in harsh climates.
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In the control of wild mosquito populations, releasing Wolbachia-infected males has shown to be a useful technique. We propose to adopt an oscillatory release rate in a simple mathematical model for this procedure in the form of a quasi-periodic and almost periodic release rate. This choice has conceptual advantages since we do not restrict time to be strictly periodic. There is also a practical advantage, since we may consider several non-synchronized fluctuating environmental factors such as annual proliferation, seasonal and daily temperature changes. We give criteria for global stability. In our examination two main phenomena occur: either the wild population is replaced by the infected population or the wild population persists in oscillating. We provide analytical