Content uploaded by John M Marshall
Author content
All content in this area was uploaded by John M Marshall on Jan 06, 2022
Content may be subject to copyright.
Chapter 2
Field Trials of Gene Drive Mosquitoes:
Lessons from Releases of Genetically Sterile
Males and Wolbachia-infected Mosquitoes
John M. Marshall and Váleri N. Vásquez
Abstract The discovery of CRISPR-based gene editing and its application to
homing-based gene drive has been greeted with excitement, for its potential to
control mosquito-borne diseases on a wide scale, and concern, for the invasiveness
and potential irreversibility of a release. At the same time, CRISPR-based gene
editing has enabled a range of self-limiting gene drive systems to be engineered with
much greater ease, including (1) threshold-dependent systems, which tend to spread
only when introduced above a certain threshold population frequency, and (2) tem-
porally self-limiting systems, which display transient drive activity before being
eliminated by virtue of a fitness cost. As these CRISPR-based gene drive systems are
yet to be field-tested, plenty of open questions remain to be addressed, and insights
can be gained from precedents set by field trials of other novel genetics-based and
biological control systems, such as trials of Wolbachia-transfected mosquitoes,
intended for either population replacement or suppression, and trials of genetically
sterile male mosquitoes, either using the RIDL system (release of insects carrying a
dominant lethal gene) or irradiation. We discuss lessons learned from these field
trials and implications for a phased exploration of gene drive technology, including
homing-based gene drive, chromosomal translocations, and split gene drive as a
system potentially suitable for an intermediate release.
Keywords Trial · Gene drive · Mosquitoes · Genetically sterile males · Wolbachia
J. M. Marshall (*)
Divisions of Epidemiology and Biostatistics, School of Public Health, University of California,
Berkeley, CA, USA
Innovative Genomics Institute, University of California, Berkeley, CA, USA
e-mail: john.marshall@berkeley.edu
V. N. Vásquez
Energy and Resources Group, University of California, Berkeley, CA, USA
Berkeley Institute for Data Science, University of California, Berkeley, CA, USA
e-mail: vnvasquez@berkeley.edu
©The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021
B. K. Tyagi (ed.), Genetically Modified and other Innovative Vector Control
Technologies,https://doi.org/10.1007/978-981-16-2964-8_2
21
2.1 Introduction
The discovery of CRISPR and its application as a gene editing tool has enabled gene
drive systems to be engineered with much greater ease (Doudna and Charpentier
2014; Champer et al. 2016). Recent attention has focused on homing-based drive
systems and their potential to control mosquito-borne diseases on a wide scale, either
by spreading disease-refractory genes (Gantz et al. 2015) or by spreading genes that
confer a fitness load or sex bias thereby suppressing mosquito populations
(Hammond et al. 2016; Kyrou et al. 2018). However, there is growing awareness
of the invasiveness of homing-based drive systems (Noble et al. 2018) and interest in
alternatives that could be confined to partially isolated populations and remediated—
properties that are well aligned to the conduct of field trials (Marshall and Akbari
2018).
In addition to homing-based gene drive, the increased ease of gene editing has
advanced the entire field of gene drive, including systems that, by design, limit their
spread in space and time (Marshall and Akbari 2018). Such systems would ideally be
capable of enacting local population control by: (a) effectively spreading into
populations to the extent required to achieve the desired epidemiological or ecolog-
ical effect and (b) being recallable from the environment in the event of unwanted
consequences, public disfavor, or the end of a trial period. Two varieties of these
systems have been recently engineered: (1) threshold-dependent systems that tend to
spread when introduced above a certain population frequency (Akbari et al. 2013;
Buchman et al. 2018) and (2) temporally self-limiting systems that display transient
drive activity before being eliminated by virtue of a fitness cost (Gould et al. 2008;Li
et al. 2020).
In this chapter, we discuss considerations for field trials of gene drive systems,
with a specific focus on confinement and reversibility criteria, and lessons learned
from other genetics-based and biological control systems (Table 2.1). We pay
special attention to reciprocal chromosomal translocations (Buchman et al. 2018),
as an example of a threshold-dependent system that is confineable and reversible,
and then extend our consideration to CRISPR-based homing gene drive systems and
temporally self-limiting systems, such as split gene drive (Li et al. 2020), which
could be used as confineable and reversible intermediate systems in a development
pathway of homing-based systems. While these gene drive systems are yet to be
trialed in the wild, lessons can be learned from trials of several varieties of sterile
male mosquitoes, specifically, those sterilized through radiation (sterile insect tech-
nique, SIT), transfection with Wolbachia (incompatible insect technique, IIT)
(Zheng et al. 2019; Crawford et al. 2020), and release of insects carrying a dominant
lethal (RIDL) gene (Harris et al. 2011; Carvalho et al. 2015), as well as releases of
Wolbachia-infected mosquitoes for population replacement (Hoffmann et al. 2011).
We begin by discussing trials of these systems and discuss threshold-dependent,
self-limiting, and nonlocalized gene drive systems in this context.
22 J. M. Marshall and V. N. Vásquez
2.2 Lessons from Releases of Genetically Sterile Male
Insects
Releases of irradiated sterile male insects as a means of population suppression have
been discussed since the early twentieth century (Klassen and Curtis 2005), and a
transgenic version of this technology was the first transgenic mosquito product to be
trialed in the field (Harris et al. 2011). As the first transgenic mosquito release, this
intervention has come under high levels of scrutiny and serves as an important case
study for potential releases of gene drive mosquitoes. The traditional SIT approach
involves mass-rearing insects and applying a carefully calibrated amount of radiation
such that their genetic material is mutated to render them sterile while still being able
Table 2.1 Genetics-based and biological mosquito control strategies and their potential to be
confineable and reversible
Strategy Variant Mechanism of action Confineable Reversible
Sterile insect
technique (SIT)
Ionizing radiation
or
chemosterilization
Offspring of released
females and males are
unviable
Yes Yes
Wolbachia Incompatible
insect technique
(IIT)
Offspring of released
males are unviable
Yes, if no
females
released
Yes, if no
females
released
Population
replacement
Spreads through popula-
tion due to cytoplasmic
incompatibility
Yes, for
moderate-
to-high fit-
ness costs
Possibly,
for high fit-
ness costs
Release of
insects carrying
a dominant
lethal (RIDL)
gene
Bisex RIDL
(bi-RIDL)
Both female and male off-
spring having the RIDL
allele are unviable
Yes Yes
Female-specific
RIDL (fs-RIDL)
Only female offspring
having the RIDL allele are
unviable
Yes Yes
Chromosomal
translocations
CRISPR or other
endonucleases
Translocation heterozy-
gotes with unbalanced
chromosome sets are
unviable, leading to
bistable dynamics
Yes Yes
CRISPR-based
gene drive
Homing-based
drive systems
Bias inheritance by cleav-
ing a target sequence and
serving as a template for
DNA repair, effectively
turning a heterozygote into
a homozygote
Potentially,
but with
difficulty
Potentially,
but with
difficulty
Split gene drive
systems
Components of drive sys-
tem are split across two
loci, leading to transient
drive when they co-occur
before being eliminated
due to a fitness cost
Yes Yes
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 23
to compete for female mates in the field (Knipling 1955). Upon release, sterile males
(preferably the majority of released insects) seek out wild females, essentially
wasting their reproductive potential as the females produce no or significantly less
viable offspring. Consecutive releases over a sufficiently wide area result in less
productive matings and a progressive reduction in insect population size over
subsequent generations (Hendrichs and Robinson 2009).
The most widely celebrated application of SIT involved the use of ionizing
radiation to eradicate the screwworm fly, Cochliomyia hominivorax, from North
America—a program that began in 1957 following successful field trials on the
Island of Curacao—and continues to this day to prevent reinvasion of the continent
(Wyss 2000; Klassen and Curtis 2005). In this intervention, large-scale releases of
sterilized insects led the screwworm fly population in the USA to crash within a
decade. Subsequent releases progressively shifted the eradication zone southward,
eventually covering all of North and Central America by 2001 (Robinson 2002).
The success of the screwworm SIT project motivated the application of SIT to a
range of other insect pest species, including mosquito vectors of disease (Knipling
1968). Both irradiation and chemosterilization were initially explored for applica-
tions to mosquitoes, and in the 1960s and 1970s, large SIT field trials were
conducted using chemosterilized Culex quinquefasciatus in India and Anopheles
albimanus in El Salvador (Klassen and Curtis 2005). The trial in India was halted in
the mid-1970s, following false accusations that the project was being used to collect
data to engage in biological warfare (Nature 1975), highlighting the importance of
effective community and political engagement for international biocontrol programs.
Nevertheless, benefits of chemosterilization were demonstrated for this particular
intervention due to reduced fitness costs as compared to irradiation.
A significant advancement in SIT technology was ushered with specific DNA
changes introduced by the RIDL construct (Thomas et al. 2000; Alphey 2002).
Insects sterilized through mutagens are subject to a myriad of random genetic
mutations, which are invariably associated with significant fitness costs. In theory,
releases of insects carrying (in homozygous form) a dominant lethal gene (RIDL)
have essentially the same population impact as SIT—i.e., offspring of released males
are unviable—although in a more controlled way that has potential for smaller
associated fitness costs. Benedict and Robinson (2003) argued that a transgenic
version of SIT should be the first application of transgenic mosquitoes in the wild
(as it was), both for enhanced efficacy and for biosafety features—i.e., lethality-
inducing transgenes should be quickly eliminated from the environment, causing the
intervention to be reversible within a few generations. Quick elimination of
transgenes also leads to confinement, since released mosquitoes can only travel so
far in a few generations.
Sterile insect approaches based on genetic engineering present more opportunities
than those based on mutagenesis, as genes and their associated traits can be modified
in a more precise way. The original RIDL strain in Aedes aegypti, OX513A, causes
lethality in both female and male offspring (bi-RIDL) (Thomas et al. 2000); how-
ever, an alternative construct was engineered soon after that only causes female
offspring to be inviable (female-specific RIDL, or fs-RIDL). This allows the
24 J. M. Marshall and V. N. Vásquez
population-suppressing trait to persist for a few more generations through the male
line, while continuing to suppress the female population, which is effective since
only female mosquitoes bite and transmit diseases to humans. Furthermore, the
introduced trait is late acting, affecting the development of wing muscles in adult
females (Fu et al. 2010). This has the benefit that viable population reduction is not
seen until the adult stage, delaying the reduction in larval density and hence
maintaining high larval mortality rates for longer due to density-dependent compe-
tition of larvae in breeding sites (Black et al. 2011).
The first field trials of Ae. aegypti mosquitoes having the RIDL construct were
conducted using the OX513A strain in Malaysia and the Cayman Islands. In
Malaysia, Oxitec Ltd. and the Institute for Medical Research, Malaysia, worked
closely with the Malaysian government in conducting a risk assessment. Releases
were carried out in an uninhabited area to assess the mortality and dispersal
characteristics of released RIDL mosquitoes; however, negative reactions were
encountered from nongovernmental organizations and the media, preventing a trial
from being conducted in an inhabited area where the impact on wild Ae. aegypti
populations could be assessed (Enserink 2011).
In the Cayman Islands, Oxitec Ltd. worked with the local Mosquito Research and
Control Unit (MRCU), initially conducting smaller releases over the course of
4 weeks to assess the fitness of genetically modified (GM) sterile males relative to
wild males and subsequently conducting a population suppression field trial over the
course of several months, again using the OX513A Ae. aegypti strain. In a lab cage
study, GM sterile males were found to be more or less of equal competitiveness in
mating with wild females, and the lethality trait was found to be effective in all
crosses between GM sterile males and wild females (Harris et al. 2011). Subsequent
field releases over a 4-week period found that GM males successfully mated with
wild females in the field and fertilized their eggs resulting in unviable offspring;
however, the field competitiveness of the GM males was estimated at ~56% that of
wild males, albeit with a very wide 95% confidence interval of 3.2–197% (Harris
et al. 2011).
The subsequent suppression field trial in the Cayman Islands was carried out
across three contiguous areas on Grand Cayman island (denoted areas A, B, and C)
over a period of 23 weeks (Harris et al. 2012). The initial goal had been to achieve a
10:1 GM-to-wild male ratio by releasing across all three areas (55 ha in total);
however, production limitations led the actual achieved ratio to be significantly
less (~2:1 GM-to-wild males), and a subsequent release in areas A and B still only
achieved a ratio of ~5:1 GM-to-wild males. The third phase of the release was
carried out solely in area A, achieving a release ratio of ~25:1 GM-to-wild males and
demonstrating the benefit of a smaller trial area. Another benefit of the area A release
was that area C served as a control and area B served as a buffer region. Significant
population reduction was seen in this phase, with an 80% reduction in the mean
ovitrap index in area A relative to areas B and C over the last 7 weeks of the release
period (Harris et al. 2012).
Releases of GM sterile males in the Cayman Islands faced some controversy
(Nightingale 2010; Enserink 2010); however, the major criticisms concerned the
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 25
manner in which information about the trials was disseminated, rather than the
conduct of the trials themselves. The releases did abide by national regulations, in
particular, a draft biosafety bill that had yet to become law, the MRCU obtained a
permit from the Cayman Islands Department of Agriculture, and a risk analysis and
environmental impact assessment were carried out. The degree of community
engagement was questioned; however, with several groups complaining, they had
not been given details of the releases in advance (Enserink 2010).
Subsequent releases in Brazil followed a much more transparent approach. From
the outset, a joint project was agreed, the Projeto Aedes Transgênico (PAT), between
the University of São Paulo and Oxitec Ltd. to explore the potential use of GM sterile
male Ae. aegypti as a form of urban mosquito control in terms of its social, technical,
and operational dimensions. The project was launched by Moscamed, a Brazilian
not-for-profit organization dependent on the Brazilian Ministry of Agriculture. The
project enjoyed significant support in its early years as the government and public
were aware of dengue outbreaks caused by this mosquito, and governmental support
showed that they were being proactive in using the latest technology to control these
outbreaks. The PAT worked closely with the Brazilian regulatory system to obtain
required permits for field activities and adopted a vigorous community engagement
campaign including school presentations, public events, interviews on TV and radio,
house visits, and involvement of the community in trap monitoring and surveillance
(de Campos et al. 2017).
The most well-documented trial of GM sterile male Ae. aegypti in Brazil was
carried out in the Itaberaba suburb of the city of Juazeiro in Bahia, Brazil. This site
had generally low socioeconomic indicators and relied on stored water to a large
extent, providing breeding sites for mosquitoes and leading to relatively high dengue
transmission. Similar to the Cayman Islands, the study area was divided into
treatment areas A and B and a control area, with treatment eventually being restricted
to area A in order to maintain sufficiently high release ratios. A Moscamed mass-
rearing facility was built specifically for the project, producing millions of GM
sterile males over the course of the study. Releases began with a “range finder”
phase lasting a little over a month, which allowed the release requirements to be
calibrated and estimates of parameters such as male mating competitiveness to be
refined. GM male mating competitiveness was estimated to be ~3.1% that of wild
males (95% CI: 2.5–3.6%), suggesting that releases for the “suppression”phase
would need to be increased ninefold in order to achieve the target of 50% of mating
events involving a GM sterile male (Carvalho et al. 2015).
The GM sterile male field trial in Brazil was successful, achieving a ~95%
reduction in mosquito density at the release site, albeit with large release require-
ments of ~140,000 mosquitoes per week over a 5.5 ha control site for ~3 months
(Carvalho et al. 2015). Enthusiasm for the GM sterile male approach was initially
raised when the Zika outbreak began in 2015; however, an unexpected complication
arose as untrue claims began to circulate in social media linking the Zika outbreak to
past releases of the GM mosquitoes (de Campos et al. 2017). This draws attention to
the importance of an enduring community engagement effort as well as political
engagement and stakeholder messaging.
26 J. M. Marshall and V. N. Vásquez
While this is not an invasive technology, these releases of sterile male mosquitoes
do provide lessons from which potential field trials of gene drive mosquitoes may
learn. Releases both of chemosterilized Cx. quinquefasciatus in India and of GM
sterile Ae. aegypti in Brazil highlight the crucial importance of an effective and
sustained community engagement effort. This especially applies to technologies
developed in the Global North and applied in the Global South, which provide
much potential for community mistrust. Furthermore, releases of GM sterile Ae.
aegypti in both the Cayman Islands and Brazil highlight the importance of choosing
a study site in which the required release sizes can be achieved and in conducting a
range finder release phase to refine release requirements. For threshold-dependent
gene drive systems, this will be important to determine release sizes that exceed the
threshold, while for nonlocalized gene drive systems, this will be important to
determine release sizes that are expected to demonstrate population control within
the timeframe of the trial.
2.3 Lessons from the Wolbachia-based Incompatible Insect
Technique
A promising alternative to SIT and GM sterile male releases is IIT, in which male
mosquitoes are released that are infected with a Wolbachia strain absent from the
wild population, resulting in sterile matings with wild females that lack the
Wolbachia strain due to a phenomenon referred to as cytoplasmic incompatibility
(CI) (LePage et al. 2017) (Fig. 2.1). This strategy has proceeded with much less
resistance than GM approaches in recent years and serves as a case study for
potential releases of novel biological control technologies, particularly regarding
the use of factory rearing facilities (Zheng et al. 2019; Crawford et al. 2020).
The first field trial of IIT was conducted in Burma (now Myanmar) in 1967. The
technique was seen as an alternative to insecticide-based strategies given the grow-
ing insecticide resistance among target species, Cx. pipiens fatigans, a vector of
lymphatic filariasis (LF) which had proliferated in Southeast Asia at the time (Laven
1967). Despite successful elimination of the vector species from that trial site, the
approach has not been deployed operationally until recently due to concern that
accidental releases of Wolbachia-infected fertile females could result in the
Wolbachia strain spreading into the population, preventing further suppression
efforts. This is because Wolbachia is maternally inherited, and in most cases, the
only incompatible crosses are between infected males and uninfected females. In
2009 and 2010, however, subsequent trials were carried out in French Polynesia to
suppress populations of Aedes polynesiensis, a primary vector of LF in the South
Pacific(O’Connor et al. 2012). Results from those field experiments showed that
(1) Wolbachia-transfected Ae. polynesiensis males successfully competed for mates
following release and (2) the trial did not result in population replacement
eventuating.
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 27
In the last few years, two factory-scale IIT projects have moved forward to
achieve community-scale mosquito population suppression: (1) an IIT program
supplemented with sterilizing irradiation (also termed IIT-SIT) in Guangzhou,
China (Zheng et al. 2019), and (2) an IIT program supplemented with factory-
scale automation of production and sex sorting in Fresno, California (Crawford
et al. 2020). The two projects represent different approaches to prevent population
replacement: (1) through greatly reducing the fertility of any Wolbachia-infected
females that may be accidentally released and (2) through using automation and
machine learning to reduce the number of accidentally released Wolbachia-infected
females effectively to zero.
In the IIT-SIT program in Guangzhou, Aedes albopictus, the main vector of
dengue and other arboviruses in Guangzhou, were generated having an artificial
triple Wolbachia infection (termed HC), through the addition of the wPip Wolbachia
strain to the native double infection of the wAlbA and wAlbB strains of Wolbachia.
High levels of CI were confirmed such that matings of HC males with wild females
produced no viable offspring and maternal transmission of the triple Wolbachia
infection was confirmed, allowing efficient mass production of HC males. HC males
were exposed to low-dose irradiation at the pupal stage to reduce the fecundity of
any accidentally released HC females, and semi-field cage studies confirmed that the
irradiated HC males effectively competed for mates leading to population suppres-
sion, without population replacement occurring due to released HC females. Fur-
thermore, as an additional safety precaution, HC females were shown to be less
competent at disease transmission than their wild counterparts (Zheng et al. 2019).
Fig. 2.1 (a) The use of Wolbachia as a means for both population suppression (incompatible insect
technique, IIT) and population replacement hinges on the inheritance pattern in which crosses
between Wolbachia-infected males and uninfected females produce unviable offspring due to
cytoplasmic incompatibility (CI), while crosses involving Wolbachia-infected females produce
Wolbachia-infected offspring due to Wolbachia being maternally inherited. (b) In IIT,
Wolbachia-infected males are released into a wild population lacking that strain of Wolbachia.
This leads to population suppression as mating events involving Wolbachia-infected males produce
no viable offspring. (c)InWolbachia-based population replacement, Wolbachia-infected females
are included in the release. This leads to population replacement as CI biases inheritance in favor of
Wolbachia when Wolbachia-infected females are present
28 J. M. Marshall and V. N. Vásquez
A trial carried out by the Wolbaki Biotech Company in 2016–2017 demonstrated
the high degree of population suppression possible when factory rearing of mosqui-
toes is involved. Irradiated HC males were released on a weekly basis on two
riverine islands within the jurisdiction of Guangzhou, with the ratio of released
HC to wild males varying between 8.7:1 and 15.8:1 over the 38-week intervention
period. Population suppression was highly successful, achieving a >94% reduction
in the number of hatched eggs per ovitrap, as compared to control sites, and an
83–94% reduction in the number of wild adult females caught per trap. The success
of the program also led to a significant increase in community support, with
interviews suggesting 13% of residents were supportive prior to the intervention
(notably, with 76% being neutral) and 54% were supportive following the interven-
tion (Zheng et al. 2019).
The IIT program in Fresno, CA, showcased the role that large-scale, automated
rearing and sex sorting of mosquitoes can play in increasing the scale of an IIT
intervention. In this case, Ae. aegypti, the main arboviral vector through much of the
Americas, was transfected with the wAlbB strain of Wolbachia, and sterility of
crosses between infected males and wild females was confirmed. An automated
larval rearing system was designed that, at maximum capacity, was able to produce
almost 3 million pupae per week. A multistep sex-separation process was then
designed that removed 95% of females at the pupal stage and the remainder at the
adult stage based on a machine learning algorithm informed by photographic images
as emerging adults walked down a narrow path. Estimates from the operation of this
system suggested that a single Wolbachia-infected female mosquito would be
released for every 900 million males, making the sex-sorting system near perfect
(Crawford et al. 2020).
A trial carried out through a partnership between the Debug Project of Verily Life
Sciences, MosquitoMate, and the Consolidated Mosquito Abatement District of
Fresno County in 2018 demonstrated dramatic population suppression over an
area nine times larger than that of the Guangzhou study. A total of more than
14 million Wolbachia-infected males were released as part of the study
(an average of more than 78,000 per day), which led to a 96% reduction in the
wild adult mosquito population; however, despite the large size of the releases,
elimination was not achieved, likely due to inward migration of wild mosquitoes
from neighboring untreated areas (Crawford et al. 2020). A public information
campaign was conducted around the trial; however, formal documentation of this
campaign is not yet available. A similar project is currently underway in Singapore
through a partnership between Verily Life Sciences and the National Environment
Agency of Singapore.
While neither a transgenic nor invasive technology, these IIT releases do provide
lessons regarding the scale of releases that can be achieved when investment is made
into automated rearing and sex-sorting facilities. Release requirements for
low-threshold gene drive mosquitoes will be orders of magnitude lower than those
for sterile male releases, and hence a facility capable of producing tens of millions of
mosquitoes, such as the one designed by Verily Life Sciences, would be capable of
achieving control over a much greater spatial scale than for IIT. The technological
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 29
capacity for sex sorting is also encouraging given that male mosquitoes don’t bite or
transmit diseases to humans and hence may also be preferable for gene drive
mosquito releases. The IIT releases enjoyed much less resistance from communities
and regulatory agencies than GM sterile male releases, despite acting through a
similar mechanism, highlighting issues that trials of gene drive mosquitoes will
likely also face and must invest in.
2.4 Lessons from Wolbachia-based Population Replacement
A second approach to the use of Wolbachia to control mosquito-borne disease
transmission is to intentionally include Wolbachia-infected females in a release. In
IIT, care is taken to only release Wolbachia-infected males, as CI causes matings
between Wolbachia-infected males and wild females to be sterile; however,
CI-induced sterility, combined with the fact that Wolbachia is maternally inherited,
provides an inheritance bias in favor of Wolbachia when Wolbachia-infected
females are also present (Turelli and Hoffmann 1991) (Fig. 2.1). For Wolbachia
strains that also block pathogen transmission, this can be used to drive the pathogen-
blocking trait into the mosquito population (Moreira et al. 2009). This strategy has
advanced significantly over the last decade (Hoffmann et al. 2011) and, like IIT, has
faced much less resistance than GM strategies. It serves as an interesting case study
for potential releases of transgenic population replacement technologies, as it has
faced many of the non-GM issues that future gene drive programs will face.
The first Wolbachia population replacement program was carried out by the
Eliminate Dengue project (now known as the World Mosquito Program) in the
communities of Yorkeys Knob and Gordonvale in Queensland, Australia (Hoffmann
et al. 2011). In this program, Ae. aegypti, the main vector of dengue and other
arboviruses in Queensland, was transfected with the wMel strain of Wolbachia from
Drosophila melanogaster, a strain that has been shown to (1) block dengue trans-
mission, (2) have a small associated fitness cost, and (3) be capable of driving into a
small field cage (Walker et al. 2011). Wolbachia displays threshold properties in the
presence of a fitness cost such that releases above a certain population frequency
tend to spread, while releases below that frequency tend to be eliminated. The exact
value of the threshold is determined by the point at which the inheritance bias
induced by CI outweighs the fitness cost associated with the infection and has
been estimated at ~20–30% for the Wolbachia strain used in this release (Hoffmann
et al. 2011; Hancock et al. 2019).
The releases in Yorkeys Knob and Gordonvale were a clear success—after
10 weekly releases of 11,000–22,000 Wolbachia-infected Ae. aegypti per week,
the Wolbachia infection reached near fixation in both populations within 3 months,
despite a tropical storm postponing one of the releases in Gordonvale (Hoffmann
et al. 2011) (Fig. 2.2). The finer details of this program provide an excellent example
of how gene drive systems may be successfully trialed in the future. To begin, they
highlight the importance of a detailed monitoring effort and adaptive release
30 J. M. Marshall and V. N. Vásquez
protocol. The releases in Yorkeys Knob and Gordonvale were accompanied by a
network of 29 Biogents Sentinel mosquito traps that monitored Wolbachia infection
frequency at the block level. Heterogeneity in Wolbachia infection frequency was
–16.805
–16.810
–16.815
–16.820
145.695
1.0
0.5
0
1.0
0.5
≥9
8
7
6
≤5
00 250 500
0 50 100
Time (days) Time (days)
Wolbachia frequency
Drive allele frequency
Number of releases
Field observations
Model predictions
150
145.700 145.705
Trinity
Park
a
bc
Yorkeys
Knob
Longitude
Latitude
N
~0.533 m
145.710 145.715 145.720 145.725
Fig. 2.2 (a) Landscape of Yorkeys Knob and Trinity Park in Queensland, Australia, where field
trials of Wolbachia-based population replacement for Aedes aegypti were carried out and where
trials of reciprocal chromosomal translocations were simulated. (b) Blue lines depict data for
Wolbachia frequency over time from the Wolbachia population replacement field trial conducted
in Yorkeys Knob in 2011 (Hoffmann et al. 2011), with line thickness representing 95% binomial
confidence intervals around observed proportions. Red lines depict simulated data for an analogous
release scheme consisting of 20 Wolbachia-infected mosquitoes per household at a coverage of
30% over 10 weeks, demonstrating good agreement with field data (Sánchez et al. 2020). (c)
Translocation frequency over time for a given number of weekly releases of 20 adult male Ae.
aegypti mosquitoes homozygous for the translocation per household with the intent of population
replacement in Yorkeys Knob. Results are depicted for a coverage of 50%, at which seven or more
releases result in the translocation being driven into the population (Sánchez et al. 2020). Due to the
50% threshold property of translocations, the same release scheme for wild types can be used to
remediate translocations from the population
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 31
observed, and releases were supplemented in areas where Wolbachia frequency was
low.
Monitoring for unintended spread outside the study area was also conducted, and
this did indeed reveal limited long-distance spread into a neighboring suburb from
Yorkeys Knob and across a freeway from Gordonvale (Hoffmann et al. 2011).
Although these migrants were expected to be lost due to being present at subthresh-
old levels, continued monitoring was important to confirm this. Continued monitor-
ing was also conducted at the trials sites to confirm enduring intervention efficacy,
and while the Wolbachia infection remained at near fixation for several years
following the release, a low frequency of uninfected mosquitoes has also persisted,
likely due to immigration (Hoffmann et al. 2014).
The releases in Yorkeys Knob and Gordonvale also highlight the importance of
preparing for unexpected events. In addition to the tropical storm that affected both
release sites and postponed one of the releases in Gordonvale, releases in a portion of
Yorkeys Knob ceased two-thirds of the way into the intervention following a
reported dengue case (Hoffmann et al. 2011). Although this dengue case likely
originated elsewhere, a reactive insecticide intervention was carried out in surround-
ing households in agreement with local disease control protocols. Trials of mosqui-
toes with gene drive systems should make allowances for events such as these.
Encouragingly, the Wolbachia infection continued to spread through the Yorkeys
Knob Ae. aegypti population despite this, and no secondary dengue cases were
documented following the reported case.
The Yorkeys Knob and Gordonvale releases provide an example of a successful
community and regulatory engagement process. Community engagement was car-
ried out over 2 years leading up to the releases and consisted of informal interviews,
semi-structured in-depth interviews, qualitative and quantitative surveys, focus
groups, historical research, and face-to-face presentations at community meetings
(Hoffmann et al. 2011; McNaughton 2012). Issues explored through these activities
included the sociopolitical context, lay knowledge of dengue fever and biological
control programs, and acceptability of the project. Community members did raise
concerns about a previous local biological control program—the introduction of the
cane toad near Gordonvale in the 1930s. Largely seen as a failed biological control
program, this was raised as a cautionary tale indicating the limits of scientific
knowledge and the unpredictability of ecological interventions (McNaughton 2012).
The Queensland releases enjoyed substantial community support, with 85% of
respondents viewing Wolbachia as an acceptable dengue prevention strategy in a
March 2010 telephone survey (ahead of insecticides, at 66% acceptance) and 84% of
respondents stating they would support a release that they were informed and
updated about, that had regulatory oversight, and that was shown to be safe for
people and the environment by a risk assessment carried out by Australia’s Com-
monwealth Scientific and Industrial Research Organisation (CSIRO) (McNaughton
2012). The releases were ultimately approved by the Australian Pesticides and
Veterinary Medicines Authority (APVMA) following risk assessments by CSIRO
(Murphy et al. 2010) and the APVMA with support from the Federal Common-
wealth Government’s Department of the Environment, Water, Heritage and the Arts
32 J. M. Marshall and V. N. Vásquez
(Marshall 2011). The World Mosquito Program is now exploring application of their
technology beyond Australia, with active collaborations throughout Latin America,
Asia, and Oceania.
In summary, key lessons from the Wolbachia-based population replacement
strategy include the importance of (1) a detailed monitoring protocol to assess
heterogeneity of spread at the field site, (2) an adaptive release scheme to supplement
releases in areas of low Wolbachia frequency, (3) additional monitoring to assess
levels of unintended spread to neighboring areas, and (4) preparing for unexpected
events. The fact that Wolbachia infection has the potential to persist in the mosquito
population for extended periods, and perhaps indefinitely, also emphasizes the need
for a long-term, comprehensive, and multifaceted community engagement program.
2.5 Considerations for Trials with Reciprocal
Chromosomal Translocations
Lessons from field trials of Wolbachia-based population replacement systems apply
most closely to threshold-dependent gene drive systems, which are also expected to
spread if released above a certain threshold frequency and to be eliminated if present
below that frequency. One of the first of these systems to be proposed (Serebrovskii
1940; Curtis 1968), and perhaps currently one of the most promising (Sánchez et al.
2020), is reciprocal chromosomal translocations. These result from a mutual
exchange between terminal segments of two nonhomologous chromosomes and
produce a heterozygote reproductive disadvantage because, when translocation
heterozygotes mate, several crosses result in unbalanced genotypes and hence
unviable offspring. This produces a threshold frequency of 50%, which increases
in the presence of a fitness cost (Curtis 1968). Early attempts to generate trans-
locations through radiation-induced mutagenesis were abandoned due to high asso-
ciated fitness costs (Laven et al. 1972; Lorimer et al. 1972); however, interest has
been reignited as site-specific translocations have recently been generated using
CRISPR (Lekomtsev et al. 2016; Jiang et al. 2016), and translocations generated
in D. melanogaster using endonucleases were recently shown to drive in laboratory
experiments with a threshold frequency of ~50% (Buchman et al. 2018).
A recent modeling study suggests that translocations represent one of the best
systems to implement in field trials due to their symmetrical threshold properties and
strong confinement potential. A key advantage of translocations is that releases
required to introduce them into a population are of a similar magnitude to wild-
type releases required to eliminate them once they have been introduced (Sánchez
et al. 2020). Population replacement and reversion were modeled at the household
level in the suburb of Yorkeys Knob, the site of the Wolbachia population replace-
ment study, with low levels of migration modeled to the neighboring suburb of
Trinity Park in Queensland, Australia (Fig. 2.2). Population replacement could be
achieved in simulations with seven or more weekly releases of 20 Ae. aegypti males
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 33
homozygous for the translocations per household per week (a similar magnitude to
that used in the Wolbachia population replacement trial at the same site) and for a
coverage of 50% of the households in the community. Elimination could be achieved
for the same release scheme using wild Ae. aegypti mosquitoes.
One benefit of translocations, and other underdominant systems that have a
threshold in the absence of a fitness cost, is that their release threshold is more
robust than that for Wolbachia, which only arises in the presence of a fitness cost.
This property leads to translocations being more robustly confineable to a field site
than a Wolbachia infection, since they are unlikely to exceed the release threshold in
a neighboring population purely through migration, even if they spread to near
fixation at the trial site. In the translocation modeling study in Yorkeys Knob and
Trinity Park (Sánchez et al. 2020), it was considered unlikely that Ae. aegypti
mosquitoes would travel from one suburb to another by their own flight, especially
in numbers sufficient to exceed the release threshold there, and so “batch migration”
was instead considered, in which several mosquitoes are carried, perhaps by a
vehicle, from one suburb to another at once. Batch migration events were modeled
as occurring between randomly chosen neighborhoods, and the number of daily
migration events and effective number of adults carried per event were varied.
Results from this modeling study made a strong case for the potential to confine
translocations to the release site, as the number of daily migration events required for
the translocation to exceed the threshold in the neighboring suburb exceeded those
inferred from field data. Specifically, 3–4 daily migration events consisting of
batches of ten adults were required for translocations to spillover to the neighboring
suburb in simulations (Sánchez et al. 2020), while field data suggested 1–2 daily
migration events consisting of batches of less than five adult females (Hoffmann
et al. 2011).
Collectively, these modeling results for translocations are encouraging for the
potential to conduct field trials of Ae. aegypti mosquitoes with translocations
because (1) translocations could be introduced on a suburban scale and remediated
through releases of non-disease-transmitting male mosquitoes with release sizes on
the scale of what has been previously implemented and (2) spillover of transloca-
tions into neighboring suburbs is unlikely. Lessons for the conduct of field trials with
translocations may be drawn from the field trials previously described in this
chapter—most importantly, for Wolbachia-based population replacement. These
lessons highlight the importance of a detailed monitoring effort, including outside
the study area, and of an adaptive release protocol that can respond to heterogeneities
in spread at the trial site. They also highlight the importance of preparing for
unexpected events and for conducting a long-term and comprehensive community
engagement program, given that translocations have the potential to persist in the
environment long term. A comparison of the RIDL and IIT releases suggests that
community engagement and regulatory requirements for translocations may be
stricter than for those for Wolbachia due to the fact that mosquitoes with trans-
locations, generated using CRISPR or other endonucleases, will be considered GM
organisms. Finally, regarding the release protocol, including a range finder release
34 J. M. Marshall and V. N. Vásquez
phase may help to refine fitness cost estimates and release requirements for trans-
locations, as per a lesson from the RIDL field trial in Brazil.
2.6 Considerations for Trials with CRISPR-based Gene
Drive Systems
Finally, lessons from the field trials discussed here have implications for the spec-
trum of CRISPR-based gene drives, from those that are nonlocalized to those that are
self-limiting. Recent attention has focused on CRISPR-based homing gene drives,
for their ability to spread widely and their potential to control vector-borne diseases
on a wide scale (Gantz et al. 2015; Kyrou et al. 2018); however, there are also
threshold-dependent gene drive systems that can now be engineered using CRISPR,
such as chromosomal translocations (Buchman et al. 2018) and various forms of
underdominance (Akbari et al. 2013), as well as temporally self-limiting gene drive
systems, such as split drive (Li et al. 2020), which display transient drive activity
before being eliminated by virtue of a fitness cost. The CRISPR revolution has also
enabled gene drive countermeasures to be engineered, such as homing-based drive
remediation systems, ERACR (element for the reversal of the autocatalytic chain
reaction) and e-CHACR (erasing construct hitchhiking on the autocatalytic chain
reaction) (Gantz and Bier 2016; Xu et al. 2020).
CRISPR-based homing gene drive systems bias inheritance in their favor by
cleaving a highly specific target sequence in the host genome and copying them-
selves to the cut chromosome through a mechanism known as homology-directed
repair (Gantz and Bier 2015; Champer et al. 2016). For high homing efficiencies and
low-to-moderate fitness costs, these systems are capable of driving into populations
from arbitrarily low initial frequencies. This property allows them to spread widely,
and hence they are considered “nonlocalized.”For these gene drive systems, while
we may learn from field trials of Wolbachia-based population replacement systems,
the scale of their potential spread and impact leads to additional and unique chal-
lenges that we must carefully consider.
One way to manage the risks associated with the potential wide-scale spread of
homing-based gene drive systems is for testing to proceed iteratively through
multiple phases, with each phase involving a larger spatial scale and a higher degree
of human or environmental exposure (James et al. 2018) (Fig. 2.3). In this phased
release pathway, initial studies are to be conducted in contained laboratories and
Fig. 2.3 Phased release pathway for CRISPR-based homing gene drive systems
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 35
insectaries, where product efficacy and safety are studied. Entering field-testing is a
big decision, given the anticipated difficulty of remediating a homing-based gene
drive system that is capable of spreading widely. Large outdoor cages present one
option for moving beyond the laboratory; however, this is not considered essential
since some mosquito behaviors, such as mating, and parameters, such as fitness, can
only be meaningfully studied in the field. Furthermore, studies in outdoor cages must
anticipate the possibility of escape occurring, and hence similar safety and efficacy
criteria must be met before either outdoor cage studies or small-scale isolated
releases are performed. Initial outdoor testing should be conducted at field sites
within which the gene drive system is expected to be contained, for instance, on
oceanic islands, following which open releases would be conducted on iteratively
larger spatial scales (James et al. 2018).
Another consideration for trials of nonlocalized gene drive systems is that
regulators are likely to require that a remediation plan be in place prior to field-
testing (James et al. 2018). The chosen remediation strategy will depend on a
number of factors, including the mode of action of the drive system and the scale
and geography of the field site. A default remediation plan would be a large-scale
insecticide-based campaign to eliminate the vector population at the field site. This
would require an assessment of insecticide resistance in the local vector population
prior to the gene drive trial. Failing this, releases of non-disease-transmitting male
mosquitoes carrying a drive-resistant allele that restores the function of the gene
targeted by the drive system are an attractive option, especially if the drive-resistant
allele is sourced from a wild population.
Gene drive countermeasures such as ERACR and e-CHACR are another option
for remediation. The ERACR system consists of a second homing system with a
target site corresponding to the original drive system, essentially removing the
original system as it homes into it, while utilizing the Cas9 of the original system
and thus removing that as well (Gantz and Bier 2016; Xu et al. 2020). The e-CHACR
system uses the Cas9 from the original homing system to home into a second site in
the genome in addition to the site of the original drive system, thus driving itself into
the population while removing the original system and its Cas9 in the process (Gantz
and Bier 2016, Xu et al. 2020). Both of these systems hold promise; but they may not
be the first choice for remediation efforts as they introduce additional transgenes into
populations from which transgenes are trying to be removed.
Another potential phased release pathway is to precede the release of a
nonlocalized gene drive system with a self-limiting one. Ideally, such a release
would provide insights into the expected behavior of the nonlocalized system, and
hence there should be strong resemblance between the two systems, to the extent
possible. For a CRISPR-based homing gene drive system, one possibility is to begin
with a trial of a split drive system, in which the Cas9 and guide RNA components are
separated at different loci (Li et al. 2020). In the split drive system, transient drive
activity occurs at the guide RNA locus when the Cas9 and guide RNA alleles
co-occur in an organism; however, the Cas9 allele is gradually eliminated from the
population due to its fitness cost, followed by the guide RNA if it also has a fitness
cost. This transient drive activity also leads to spatial confinement, since a gene can
36 J. M. Marshall and V. N. Vásquez
only disperse so far in a limited number of generations. Intermediate technologies
also exist for other systems. For instance, a driving Y chromosome that spreads by
cleaving the X chromosome at multiple sites during spermatogenesis is expected to
spread on a wide scale (Galizi et al. 2014); however, if linked to an autosome, it is
self-limiting, providing an opportunity for intermediate study in the field.
For self-limiting CRISPR-based gene drive systems that could be used as an
intermediate system in a field trial, similar field trial considerations apply as for
chromosomal translocations. Namely, the ability to confine the release to the trial
site, and to remediate transgenes from the environment as needed, is a great strength.
Furthermore, it is important to combine a detailed monitoring effort, both in and
outside the trial site, with an adaptive release protocol to respond to heterogeneities
in spread, and to make allowances for unexpected events. A range finder release
phase may help to refine fitness cost estimates and release requirements.
For nonlocalized CRISPR-based gene drive systems, the potentially wide scale of
spread and difficulty of remediation emphasize the need to monitor for the gene drive
system both in and outside the field trial area. Additionally, a range finder release
phase may help to predict release schemes capable of achieving population control
within the desired timeframe. Finally, as the spatial scale of the release grows,
lessons may be learned from the experience of the Fresno IIT trial regarding
automated rearing and sex sorting of mosquitoes. Knowledge of the potential scale
of mosquito production will allow us to set expectations for wide-scale vector-borne
disease control.
As for all of the systems discussed in this chapter, effective community and
regulatory engagement is essential prior to field trials of mosquitoes engineered with
CRISPR-based gene drive systems; however, this is especially important for trials of
nonlocalized gene drive systems. Mosquitoes engineered with these systems are GM
organisms capable of spreading widely, potentially across international borders, and
are often developed in the Global North for application in the Global South. Their
potential to spread across international borders highlights the desirability of a
multicountry or regional agreement on their release, especially when a country that
shares a border with another is being considered for field trials. Indeed, such
agreements may be required by the Cartagena Protocol on Biosafety, which governs
the safe transfer, handling, and use of GM organisms (referred to as “living modified
organisms”in the protocol), including their transboundary spread (Secretariat of the
Convention on Biological Diversity 2000; Marshall 2010).
2.7 Conclusion
The limitations of traditional insecticide-based strategies to control mosquito
populations, and, in particular, the widespread emergence of insecticide resistance,
have spurred interest in a variety of novel biological and genetics-based vector
control strategies, including SIT, IIT, RIDL, Wolbachia-based population replace-
ment, and CRISPR-based gene drive (Benelli et al. 2016). Trials of RIDL, IIT, and
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 37
Wolbachia over the last decade provide a series of case studies from which we may
learn in preparing for field trials of CRISPR-based gene drive systems (Table 2.2).
There are challenges associated with gene drive technologies—notably, the
controversies surrounding GM organisms and the potential for spread across inter-
national borders. However, these challenges are also a reason for promise as half of
the world’s population is at risk of vector-borne diseases, and genetic engineering
provides new opportunities to interfere with pathogen transmission. In learning from
recent field trials, we seek to move these technologies forward carefully and respon-
sibly toward the eventual goal of global vector-borne disease control.
Table 2.2 Significant field trials of novel biological and genetics-based mosquito control strategies
Method Species Location Year Outcome Reference
SIT Anopheles
quadrimaculatus
Florida,
USA
1962 Poor mating
competitiveness
Weidhaas
et al. (1962)
IIT Culex pipiens
fatigans
Burma (now
Myanmar)
1967 Successful
suppression
Laven
(1967)
SIT Culex
quinquefasciatus
India 1971–
1975
Modest
suppression
Singh et al.
(1975)
SIT Anopheles
albimanus
El Salvador 1971–
1979
Significant
suppression
Lowe et al.
(1980)
RIDL Aedes aegypti Cayman
Islands
2009 Small-scale
suppression
Harris et al.
(2011,
2012)
IIT Aedes
polynesiensis
French
Polynesia
2009–
2010
Demonstration
of efficacy
O’Connor
et al. (2012)
Wolbachia popu-
lation
replacement
Ae. aegypti Queensland,
Australia
2011 Successful popu-
lation
replacement
Hoffmann
et al. (2011)
RIDL Ae. aegypti Juazeiro,
Brazil
2012–
2013
Community-
scale
suppression
Carvalho
et al. (2015)
RIDL Ae. aegypti Jacobina,
Brazil
2013 Suppression and
resurgence
Garziera
et al. (2017)
IIT Aedes albopictus Kentucky,
USA
2014 Significant
suppression
Mains et al.
(2016)
IIT-SIT Ae. albopictus Guangzhou,
China
2016–
2018
Community-
scale
suppression
Zheng et al.
(2019)
IIT Ae. aegypti California,
USA
2018–
2019
Community-
scale
suppression
Crawford
et al. (2020)
38 J. M. Marshall and V. N. Vásquez
References
Akbari OS, Matzen KD, Marshall JM, Huang H, Ward CM et al (2013) A synthetic gene drive
system for local, reversible modification and suppression of insect populations. Curr Biol
23:671–677
Alphey L (2002) Re-engineering the sterile insect technique. Insect Biochem Mol Biol
32:1243–1247
Benedict MQ, Robinson AS (2003) The first releases of transgenic mosquitoes: an argument for the
sterile insect technique. Trends Parasitol 19:349–355
Benelli G, Jeffries CL, Walker T (2016) Biological control of mosquito vectors: past, present and
future. Insects 7:52
Black WC, Alphey L, James AA (2011) Why RIDL is not SIT. Trends Parasitol 27:362–370
Buchman A, Ivy T, Marshall JM, Akbari OS, Hay BA (2018) Engineered reciprocal chromosome
translocations drive high threshold, reversible population replacement in Drosophila. ACS
Synth Biol 7:1359–1370
Carvalho DO, McKemey AR, Garziera L, Lacroix R, Donnelly CA et al (2015) Suppression of a
field population of Aedes aegypti in Brazil by sustained release of transgenic male mosquitoes.
PLoS Negl Trop Dis 9:e0003864
Champer J, Buchman A, Akbari OS (2016) Cheating evolution: engineering gene drives to
manipulate the fate of wild populations. Nat Rev Genet 17:146–159
Crawford JE, Clarke DW, Criswell V, Desnoyer M, Cornel D et al (2020) Efficient production of
male Wolbachia-infected Aedes aegypti mosquitoes enables large-scale suppression of wild
mosquitoes. Nat Biotechnol 38:482–492
Curtis CF (1968) Possible use of translocations to fix desirable genes in insect pest populations.
Nature 218:368–369
de Campos A, Hartley S, de Koning C, Lezaun J, Velho L (2017) Responsible innovation and
political accountability: genetically modified mosquitoes in Brazil. J Respons Innov 4:5–23
Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with
CRISPR-Cas9. Science 346:1258096
Enserink M (2010) GM mosquito trial alarms opponents, strains ties in Gates-funded project.
Science 330:1030–1031
Enserink M (2011) GM mosquito release in Malaysia surprises opponents and scientists—again.
Science Insider. Available online at: https://www.sciencemag.org/news/2011/01/gm-mosquito-
release-malaysia-surprises-opponents-and-scientists-again
Fu G, Lees RS, Nimmo D, Aw D, Jin L et al (2010) Female-specificflightless phenotype for
mosquito control. Proc Natl Acad Sci U S A 107:4550–4554
Galizi R, Doyle LA, Menichelli M, Bernardini F, Deredec A et al (2014) A synthetic sex ratio
distortion system for the control of the human malaria mosquito. Nat Commun 5:3977
Gantz VM, Bier E (2015) The mutagenic chain reaction: a method for converting heterozygous to
homozygous mutations. Science 348:442–444
Gantz VM, Bier E (2016) The dawn of active genetics. BioEssays 38:50–63
Gantz VM, Jasinskiene N, Tatarenkova O, Fazekas A, Macias VM et al (2015) Highly efficient
Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles
stephensi. Proc Natl Acad Sci U S A 112:E6736–E6743
Garziera L, Pedrosa MC, de Souza FA, Gómez M, Moreira MB et al (2017) Effect of interruption of
over-flooding releases of transgenic mosquitoes over wild populations of Aedes aegypti: two
case studies in Brazil. Entomol Exp Appl 164:327–339
Gould F, Huang Y, Legros M, Lloyd AL (2008) A killer-rescue system for self-limiting gene drive
of anti-pathogen constructs. Proc Biol Sci 275:2823–2829
Hammond A, Galizi R, Kyrou K, Simoni A, Siniscalchi C et al (2016) A CRISPR-Cas9 gene drive
system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat
Biotechnol 34:78–83
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 39
Hancock PA, Ritchie SA, Koenraadt CJM, Scott TW, Hoffmann AA et al (2019) Predicting the
spatial dynamics of Wolbachia infections in Aedes aegypti arbovirus vector populations in
heterogeneous landscapes. J Appl Ecol 56:1674–1686
Harris AF, Nimmo DD, McKemey AR, Kelly N, Scaife S et al (2011) Field performance of
engineered male mosquitoes. Nat Biotechnol 29:1034–1037
Harris AF, McKemey AR, Nimmo DD, Curtis Z, Black I et al (2012) Successful suppression of a
field mosquito population by sustained release of engineered male mosquitoes. Nat Biotechnol
30:828–830
Hendrichs J, Robinson A (2009) Sterile insect technique. In: Resh VH, Carde RT (eds) Encyclo-
pedia of insects, 2nd edn. Academic Press, London
Hoffmann AA, Montgomery BL, Popovici J, Iturbe-Ormaetxe I, Johnson PH et al (2011) Success-
ful establishment of Wolbachia in Aedes populations to suppress dengue transmission. Nature
476:454–457
Hoffmann AA et al (2014) Stability of the wMel Wolbachia infection following invasion into Aedes
aegypti populations. PLoS Negl Trop Dis 8:e3115
James S, Collins FH, Welkhoff PA, Emerson C, Godfray HCJ et al (2018) Pathway to deployment
of gene drive mosquitoes as a potential biocontrol tool for elimination of malaria in sub-Saharan
Africa: recommendations of a scientific working group. Am J Trop Med Hyg 98:1–49
Jiang J, Zhang L, Zhou X, Chen X, Huang G et al (2016) Induction of site-specific chromosomal
translocations in embryonic stem cells by CRISPR/Cas9. Sci Rep 6:21918
Klassen W, Curtis CF (2005) History of the sterile insect technique. In: Dyck VA, Hendrichs J (eds)
Sterile insect technique: principles and practice in area-wide integrated pest management.
IAEA, Geneva, pp 3–36
Knipling EF (1955) Possibilities of insect control or eradication through the use of sexually sterile
males. J Econ Entomol 48:459–462
Knipling EF (1968) The potential role of sterility for pest control. In: LeBrecque GC, Smith CN
(eds) Principles of insect chemosterilization. Appleton-Century-Crofts, New York, pp 7–40
Kyrou K, Hammond AM, Galizi R, Kranjc N, Burt A et al (2018) A CRISPR-Cas9 gene drive
targeting doublesex causes complete population suppression in caged Anopheles gambiae
mosquitoes. Nat Biotechnol 36:1062–1066
Laven H (1967) Eradication of Culex pipiens fatigans through cytoplasmic incompatibility. Nature
216:383–384
Laven H, Cousserans J, Guille G (1972) Eradicating mosquitoes using translocations: a first field
experiment. Nature 236:456–457
Lekomtsev S, Aligianni S, Lapao A, Bürckstümmer T (2016) Efficient generation and reversion of
chromosomal translocations using CRISPR/Cas technology. BMC Genomics 17:739
LePage DP, Metcalf JA, Bordenstein SR, On J, Perlmutter JI et al (2017) Prophage WO genes
recapitulate and enhance Wolbachia-induced cytoplasmic incompatibility. Nature 543:243–247
Li M, Yang T, Kandul NP, Bui M, Gamez S, Raban R, Bennett JB, Sánchez HM, Lanzaro GC,
Schmidt H, Lee Y, Marshall JM, Akbari OS (2020) Development of a confinable gene-drive
system in the human disease vector, Aedes aegypti. eLife 9:e51701
Lorimer N, Hallinan E, Rai KS (1972) Translocation homozygotes in the yellow fever mosquito,
Aedes aegypti. J Hered 63:159–166
Lowe RE, Bailey DL, Dame DA, Savage KE, Kaiser PE (1980) Efficiency of techniques for the
mass release of sterile male Anopheles albimanus. Am J Trop Med Hyg 29:695–703
Mains JW, Brelsfoard CL, Rose RI, Dobson SL (2016) Female adult Aedes albopictus suppression
by Wolbachia-infected male mosquitoes. Sci Rep 6:33846
Marshall JM (2010) The Cartagena protocol and genetically modified mosquitoes. Nat Biotechnol
28:896–897
Marshall JM (2011) The Cartagena protocol in the context of recent releases of transgenic and
Wolbachia-infected mosquitoes. Asia-Pacific J Mol Biol Biotechnol 19:93–100
Marshall JM, Akbari OS (2018) Can CRISPR-based gene drive be confined in the wild? A question
for molecular and population biology. ACS Chem Biol 13:424–430
40 J. M. Marshall and V. N. Vásquez
McNaughton D (2012) The importance of long-term social research in enabling participation and
developing engagement strategies for new dengue control technologies. PLoS Negl Trop Dis 8:
e1785
Moreira LA, Iturbe-Ormaetxe I, Jeffery JA, Lu G, Pyke AT et al (2009) A Wolbachia symbiont in
Aedes aegypti limits infection with dengue, chikungunya, and plasmodium. Cell
139:1268–1278
Murphy B, Jansen C, Murray J, De Barro P (2010) Risk analysis on the Australian release of Aedes
aegypti (L.) (Diptera: Culicidae) containing Wolbachia. CSIRO, Canberra
Nature (1975) Oh New Delhi, Oh Geneva (editorial). Nature 256:355–357
Nightingale K (2010) GM mosquito wild release takes campaigners by surprise. SciDev.Net.
Available online at: https://www.scidev.net/global/policy/news/gm-mosquito-wild-release-
takes-campaigners-by-surprise.html
Noble C, Adlam B, Church GM, Esvelt KM, Nowak MA (2018) Current CRISPR gene drive
systems are likely to be highly invasive in wild populations. eLife. https://doi.org/10.7554/
eLife.33423
O’Connor L, Pilchart C, Sang AC, Brelsfoard CL, Bossin HC, Dobson SL (2012) Open release of
male mosquitoes infected with a Wolbachia biopesticide: field performance and infection
containment. PLoS Negl Trop Dis 6:e1797
Robinson AS (2002) Mutations and their use in insect control. Rev Mutat Res 511:113–132
Sánchez HM, Bennett JB, Wu SL, RašićG, Akbari OS et al (2020) Confinement and reversibility of
threshold-dependent gene drive systems in spatially-explicit Aedes aegypti populations. BMC
Biol 18:50
Secretariat of the Convention on Biological Diversity (2000) Cartagena protocol on biosafety to the
convention on biological diversity. World Trade Center, Montreal
Serebrovskii AS (1940) On the possibility of a new method for the control of insect pests. Zool
Zhurnal 19:618–630
Singh KRP, Patterson RS, LaBrecque GC, Razdan RK (1975) Mass rearing of Culex fatigans.J
Commun Dis 7:31–53
Thomas DD, Donnelly CA, Wood RJ, Alphey LS (2000) Insect population control using a
dominant, repressible, lethal genetic system. Science 287:2474–2476
Turelli M, Hoffmann AA (1991) Rapid spread of an inherited incompatibility factor in California
Drosophila. Nature 353:440–442
Walker T, Johnson PH, Moreira LA, Iturbe-Ormaetxe I, Frentiu FD et al (2011) The wMel
Wolbachia strain blocks dengue and invades caged Aedes aegypti populations. Nature
476:450–453
Weidhaas DE, Schmidt CH, Seabrook EL (1962) Field studies on the release of sterile males for the
control of Anopheles quadrimaculatus. Mosq News 22:283–291
Wyss JH (2000) Screw-worm eradication in the Americas. In: Tan KH (ed) Area-wide control of
fruit flies and other insect pests. Penerbit Universiti Sains Malaysia, Penang, pp 79–86
Xu XS, Bulger EA, Gantz VM, Klanseck C, Heimler SR et al (2020) Active genetic neutralizing
elements for halting or deleting gene drives. Mol Cell 80:246–262
Zheng X, Zhang D, Li Y, Yang C, Wu Y et al (2019) Incompatible and sterile insect techniques
combined eliminate mosquitoes. Nature 572:56–61
2 Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically... 41
