Monitoring Needs for Gene Drive Mosquito Projects: Lessons From Vector Control Field Trials and Invasive Species
Frontiers in Genetics 12
DOI:10.3389/fgene.2021.780327
Gordana Rašić
Queensland Institute of Medical Research
Neil F Lobo
Eileen Jeffrey Gutiérrez
Héctor Manuel Sánchez Castellanos
As gene drive mosquito projects advance from contained laboratory testing to semi-field testing and small-scale field trials, there is a need to assess monitoring requirements to: i) assist with the effective introduction of the gene drive system at field sites, and ii) detect unintended spread of gene drive mosquitoes beyond trial sites, or resistance mechanisms and non-functional effector genes that spread within trial and intervention sites. This is of particular importance for non-localized gene drive projects, as the potential scale of intervention means that monitoring is expected to be more costly than research, development and deployment. Regarding monitoring needs for population replacement systems, lessons may be learned from experiences with Wolbachia-infected mosquitoes, and for population suppression systems, from experiences with releases of genetically sterile male mosquitoes. For population suppression systems, assessing monitoring requirements for tracking population size and detecting rare resistant alleles are priorities, while for population replacement systems, allele frequencies must be tracked, and pressing concerns include detection of gene drive alleles with non-functional effector genes, and resistance of pathogens to functional effector genes. For spread to unintended areas, open questions relate to the optimal density and placement of traps and frequency of sampling in order to detect gene drive alleles, drive-resistant alleles or non-functional effector genes while they can still be effectively managed. Invasive species management programs face similar questions, and lessons may be learned from these experiences. We explore these monitoring needs for gene drive mosquito projects progressing through the phases of pre-release, release and post-release.
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Monitoring Needs for Gene Drive
Mosquito Projects: Lessons From
Vector Control Field Trials and
, Neil F. Lobo
, Eileen H. Jeffrey Gutiérrez
, Héctor M. Sánchez C.
John M. Marshall
Mosquito Genomics, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia,
Department of Biological Sciences,
University of Notre Dame, Notre Dame, IN, United States,
Divisions of Epidemiology and Biostatistics, School of Public Health,
University of California, Berkeley, Berkeley, CA, United States,
Innovative Genomics Institute, University of California, Berkeley,
As gene drive mosquito projects advance from contained laboratory testing to semi-field
testing and small-scale field trials, there is a need to assess monitoring requirements to: i)
assist with the effective introduction of the gene drive system at field sites, and ii) detect
unintended spread of gene drive mosquitoes beyond trial sites, or resistance mechanisms
and non-functional effector genes that spread within trial and intervention sites. This is of
particular importance for non-localized gene drive projects, as the potential scale of
intervention means that monitoring is expected to be more costly than research,
development and deployment. Regarding monitoring needs for population replacement
systems, lessons may be learned from experiences with Wolbachia-infected mosquitoes,
and for population suppression systems, from experiences with releases of genetically
sterile male mosquitoes. For population suppression systems, assessing monitoring
requirements for tracking population size and detecting rare resistant alleles are
priorities, while for population replacement systems, allele frequencies must be
tracked, and pressing concerns include detection of gene drive alleles with non-
functional effector genes, and resistance of pathogens to functional effector genes. For
spread to unintended areas, open questions relate to the optimal density and placement of
traps and frequency of sampling in order to detect gene drive alleles, drive-resistant alleles
or non-functional effector genes while they can still be effectively managed. Invasive
species management programs face similar questions, and lessons may be learned from
these experiences. We explore these monitoring needs for gene drive mosquito projects
progressing through the phases of pre-release, release and post-release.
Keywords: population replacement, population suppression, Wolbachia, RIDL, invasive species, resistant alleles,
gene drive, monitoring
Atashi Sharma,
Virginia Tech, United States
Diego Alonso,
São Paulo State University, Brazil
Daniel Powell,
University of the Sunshine Coast,
*Correspondence:
john.marshall@berkeley.edu
Specialty section:
This article was submitted to
Evolutionary and Population Genetics,
a section of the journal
RašićG, Lobo NF,
Jeffrey Gutiérrez EH, Sánchez C. HM
and Marshall JM (2022) Monitoring
Needs for Gene Drive Mosquito
Projects: Lessons From Vector Control
Field Trials and Invasive Species.
Front. Genet. 12:780327.
doi: 10.3389/fgene.2021.780327
Frontiers in Genetics | www.frontiersin.org January 2022 | Volume 12 | Article 7803271
As the impact of currently-available tools to control malaria stagnates,
gene drive mosquitoes have been described as a promising and
potentially transformative technology. Exciting progress has been
made in Anopheles vector species towards two general classes of
strategies: i) population replacement, whereby inheritance is biased in
favor of an allele that confers refractoriness to pathogen transmission
(Adolfiet al., 2020;Carballar-Lejarazú et al., 2020), and ii) population
suppression, whereby vector populations are suppressed by biassing
inheritance in favor of an allele that induces a severe fitnesscostorsex
bias (Hammond et al., 2016;Kyrou et al., 2018). A key strength and
challenge facing this technology is its potential scale of impact, and as
products advance from contained laboratory testing to semi-field
testing and small-scale field trials, monitoring programs must be
envisioned that align with the scale of intervention (James et al., 2018).
While Anopheles gene drive projects pose challenges,
precedents exist from monitoring of biological and self-
limiting genetic control trials, albeit in Aedes mosquitoes
(Hoffmann et al., 2011;Carvalho et al., 2015;Utarini et al.,
2021). Field trials of Wolbachia-infected Aedes aegypti in
Queensland, Australia tracked the frequency of Wolbachia
infection in the mosquito population over time, including
heterogeneity in space and spread to areas neighboring the
trial site (Hoffmann et al., 2011;Schmidt et al., 2017), and
more recently, a randomized control trial in Yogyakarta,
Indonesia, monitored dengue incidence in the human
population (Utarini et al., 2021). Field trials of releases of Ae.
aegypti carrying a dominant lethal gene (RIDL) intended for
population suppression tracked mosquito population density
over time with a high degree of spatial resolution (Carvalho
et al., 2015). On a wider scale, precedents also exist for invasive
species monitoring programs (Koch et al., 2020).
Here, we draw from these programs to explore the monitoring
needs of Anopheles gene drive projects as they move through the
phases of pre-release, release and post-release (Table 1). We
consider distinct requirements for population replacement versus
suppression, focusing on low-threshold approaches.
PRE-RELEASE MONITORING
Pre-release monitoring data serves a range of purposes. First, it
allows us to better understand the temporal dynamics of
mosquito populations, their drivers, and effective sampling
tools. Second, it enables us to design efficient release strategies
TABLE 1 | Monitoring priorities for Anopheles gene drive projects as they move through the phases of pre-release, release and post-release. "R"refers to population
replacement, and "S"refers to population suppression.
Question/endpoint Activity Indicators Priority
Pre-release During
Post-
Target mosquito vector abundance Adult and pupal sampling throughout
the study area and across seasons
Number of adults per trap per period,
number of pupae per breeding site per
High (R, S) Medium (R),
High (S)
Medium (R),
Environmental drivers of mosquito
Measuring environmental data within
and across seasons
Daily rainfall, temperature, humidity etc. High (R, S) High (R, S) Medium
(R, S)
Target mosquito local-scale
Mark-release-recapture experiments Local dispersal kernel, average dispersal
High (R, S) Medium
Low (R, S)
Target mosquito intermediate and
wide-scale movement
Adult and larval sampling over a wide
scale and population genetic analysis
Effective dispersal distance, migration
rates at larger spatial scales
Target mosquito insecticide
Larval sampling, rearing and laboratory
Fraction of knockdown and dead
mosquitoes after exposure to insecticide
High (R),
Medium (S)
Low (S)
Target and non-target vector biting
Adult sampling by human landing
catch, or proxy
Human biting rate (mosquitoes per person
per night)
Target and non-target vector
Larval sampling and laboratory rearing
and testing
Fraction of exposed mosquitoes with
disseminated infection
High (R, S) High (R),
sporozoite rate
Adult sampling, dissection and
Fraction of examined mosquitoes with
sporozoites
Malaria incidence and prevalence Passive case detection, cohort
studies, cross-sectional surveys
Health system case reports, cohort-based
incidence, cross-sectional prevalence
High (R, S) High (R, S) High (R, S)
Prevalence of gene drive allele in
Adult and larval sampling in target and
non-target areas and molecular
Allele frequency throughout the study area
and allele presence elsewhere over time
N/A High (R, S) High (R, S)
Presence of gene drive allele in non-
target mosquito species
Allele presence in non-target species N/A Medium
Phenotypic stability of gene drive
construct in target mosquito
non-target areas and laboratory
Gene drive efficacy, effectiveness of
effector gene (R), sex bias or fitness
cost (S)
Fitness of gene drive mosquitoes Fitting models to data, parameter
Male mating competitiveness, female
fecundity, adult lifespan
Prevalence of resistance to gene
drive in target mosquito species
Adult and larval sampling, molecular
assays, laboratory evaluation
Prevalence of drive-resistance
mechanisms throughout the study area
Rašićet al. Monitoring of Gene Drive Mosquitoes
that enhance the likelihood of intervention success, and to
minimize risks such as escape of transgenic organisms to non-
target populations. And third, baseline data provides an
understanding of intervention-related changes both to the
vector population and to vector-borne disease transmission.
One of the most common forms of pre-release monitoring data
for genetic and biological control systems is the temporal measure of
local mosquito population size, alongside environmental data such
as temperature and rainfall. Population size estimates and seasonal
patterns help to inform the timing and size of releases for both
suppression and replacement gene drive systems, and represent a
fundamental entomological endpoint to assess the efficacy of a
suppression strategy. Highly seasonal mosquito population
dynamics, such as in the Sahel of Africa, are especially important
to monitor, as gene drive modeling studies reveal a large influence of
seasonality on the outcome of a control program (North et al., 2019).
Effective sampling tools to measure relative mosquito population
density depend on the species and location of interest. For the RIDL
trial in Juazeiro, Brazil, and the Wolbachia trial in Queensland, Ae.
aegypti was the main species of interest and a combination of
ovitraps and BG Sentinel traps were used, with human landing
catches (HLCs) also being used for the RIDL trial (Hoffmann et al.,
2011;Carvalho et al., 2015). For Anopheles species, evaluation of
sampling devices that accurately represent local mosquito densities is
needed, and selection of these tools will depend on local regulations
(e.g., HLCs are not always permissible). Most entomological
monitoring is directed at adult female mosquitoes, so details of
the adult male population may need to be inferred from captures of
adult females and juvenile stages.
Monitoring data on mosquito movement patterns, although
difficult to collect, is essential prior to field trials of gene drive
mosquitoes. This can help to inform: i) the spatial resolution
required for releases, and ii) the risk of escape to non-target
populations. Data on autonomous dispersal of Ae. aegypti has
been used to determine the size of buffer zones around treatment
areas for suppression programs, and the size of the release area for the
Wolbachia replacement programs (Schmidt et al., 2017). Similar
Anopheles data could inform gene drive programs. Mark-release-
recapture experiments are an effective way to estimate Anopheles
dispersal patterns on a village or suburban scale (Taylor et al., 2001;
Epopa et al., 2017), and have previously been used to quantify
movement patterns of genetically sterile male Aedes prior to field
trials (Lacroix et al., 2012). Population genetics offers complementary
tools to infer local (Filipovićet al., 2020) and intermediate to large-
scalemovementpatternsthatresultfromhuman-assisteddispersal
(Marsden et al., 2013).Thiscanhelptoidentifypotentialroutesof
escape, with monitoring encompassing mosquito populations close to
the target population or those connected via transport routes.
One form of pre-release monitoring uniquely required by gene
drive mosquito projects is assessment of DNA sequence
polymorphisms at the specific genetic locus targeted by the
drive system. Some of these alternate alleles may confer a
drive-resistant phenotype to the mosquitoes carrying them.
For population suppression strategies, these confer a
significant selective advantage over intact drive alleles,
preventing the success of suppression programs. For
population replacement strategies, alternate alleles may have a
mild selective advantage over intact drive alleles; but even if not,
could prevent the drive system from reaching a high frequency in
the population. Modeling studies suggest that pre-existing drive-
resistant alleles with population frequencies less than 
1% are
tolerable for population replacement programs (Lanzaro et al.,
2021). A recent study found an abundance of conserved sites that
could potentially be targeted by gene drive systems by screening
Anopheles specimens from the UC Davis Vector Genetics Lab
archive and An. gambiae 1,000 Genomes Consortium (Schmidt
et al., 2020). Modeling would ideally inform pre-release
monitoring requirements, with samples corresponding to the
location or region of interest.
Pre-release monitoring should focus on the target species, but
include some consideration of: i) other local vector species for the
pathogen of interest, ii) species between which there is some gene
flow, albeit at potentially low levels, and iii) species that may
compete for a similar niche (for population suppression
strategies). Other local vector species are important to quantify
in order to understand the proportion of pathogen transmission
attributed to the target species, and hence the expected impact on
disease transmission. Species between which there is a low level of
gene flow could result in spread of the construct with ecological
consequences. For Anopheles, limited gene flow occurs between
members of the An. gambiae species complex, which includes An.
coluzzii,An. arabiensis, and others (Taylor et al., 2001;Weetman
et al., 2014). The potential for niche replacement is an important
consideration for population suppression strategies where non-
target species have some vectorial capacity.
MONITORING DURING A RELEASE
Monitoring during a release serves three key purposes. First, it
allows us to monitor the progress of the release, as measured by
changes in genotype frequencies and population size. This also
allows us to adapt the release scheme in an iterative fashion.
Second, it allows us to compare changes in the vector population
and, ideally, vector-borne disease incidence, to the pre-release
baseline or control area. And third, it allows us to assess biosafety
features such as confinement to a trial site.
For population replacement systems, there is a need to monitor: i)
how intact drive alleles spread through the population, ii) the extent
to which drive-resistant alleles emerge and spread, and iii)
effectiveness of the effector gene. As a case study in Aedes,initial
trials of Wolbachia-infected Ae. aegypti in Queensland demonstrated
how a network of ovitraps (∼1 per two houses) and BG Sentinel traps
(∼1per30–45 houses) combined with a PCR assay to determine
mosquito species and Wolbachia status successfully documented the
spread of Wolbachia over time (Hoffmann et al., 2011). For
population replacement strategies in Anopheles, additional assays
will be needed to monitor for the intact drive allele and alternative
alleles in target mosquito species, as well as their presence in non-
target species, which can be efficiently achieved through targeted
NGS amplicon sequencing, for instance.
For population suppression systems, there may be no effector
gene to monitor, but there is a need to monitor the stability of
suppression phenotype (e.g., fecundity reduction, lethality of juvenile
stages, or sex ratio bias) and reduction in mosquito density. As a case
study in Aedes, a grid of ovitraps spanning treated and control areas
was used to provide an indirect measure of adult Ae. aegypti
abundancefortheRIDLtrialinJuazeiro(Carvalho et al., 2015).
Larvae were also scored for the transgene based on a red fluorescent
marker phenotype, and non-fluorescent larvae were reared to adults
to check for other vector species. For population suppression gene
drive projects in Anopheles, it is especially important to assay for
drive-resistant alleles which, due to their selective advantage over
intact drive alleles, are expected to rapidly spread through
populations following emergence. These assays can be informed,
in part, by experiments on caged populations that mimic genotype
fixation and can be designed to rapidly select and identify functional
resistance alleles among the detected variants in CRISPR-based
suppression gene drives (Fuchs et al., 2021). For large-scale
interventions, it will be essential to detect these alleles quickly if
their spatial spread is to be curtailed.
Close monitoring of intervention progress is highly valuable as
it enables releases to occur in an adaptive and iterative manner.
For instance, as Wolbachia-infected Ae. aegypti were released and
monitored in Queensland, the fitness cost parameter estimate was
refined and model predictions ensured the release scheme would
result in Wolbachia exceeding its threshold frequency (Hoffmann
et al., 2011). For releases of An. gambiae with low-threshold gene
drive systems, ongoing monitoring will allow us to refine model
parameters and ensure release schemes achieve entomological
and epidemiological targets. Parameters such as mating
competitiveness and adult lifespan are essential to refine, as
lab measurements are not reliable in the field.
In seeking to understand the drivers of pathogen transmission
when a gene drive intervention targets only one of several local
vector species, multiple sampling tools will be needed to capture
entomological indicators of transmission corresponding to each
local vector species. Sampling tools should be selected that take
advantage of vector species behaviors, and that collect data
reflective of the question to be addressed. Other factors should
be monitored that could explain potential epidemiological
outcomes, such as the inclusion of additional interventions as
part of the gene drive release (e.g., insecticide-treated nets), and
the inclusion of insecticide-susceptibility in the released modified
mosquitoes, potentially combined with ongoing spread of
insecticide resistance alleles in the local wild mosquito
population. Epidemiological data is often detected passively
through reported cases of symptomatic disease, and should be
compared leading up to, during and after the release (Utarini
Lastly,monitoringduringareleaseallowsbiosafetyfeaturesofa
gene drive intervention to be assessed - most importantly,
confinement to the target area. As a case study, monitoring of
Wolbachia-infected Ae. aegypti in Queensland included a site
across a major highway from one release site, and another
separated by over 1 km from the second release site (Hoffmann
et al., 2011). Wolbachia was sporadically detected in both non-release
locations, suggesting occasional movement of Ae. aegypti spanning
more than 20 times their average dispersal distance (Muir and Kay,
1998;Russell et al., 2005). Anopheles mosquitoes disperse greater
distances than Aedes (Guerra et al., 2014), suggesting the potential for
low-threshold gene drive systems in Anopheles to spread on a wide-
scale. This highlights the need for rigorous monitoring of non-target
populations during trials and interventions, even more so for
Anopheles, including at sites that could facilitate wide-scale spread,
such as nearby sea and airports.
POST-RELEASE MONITORING
Post-release monitoring is needed at multiple scales. At the scale
of the release and its immediate vicinity, it is needed to monitor
the continued persistence and effectiveness of the intervention,
and on a wider scale, it is needed to assess the extent to which a
low-threshold gene drive system has spread spatially, as well as
the extent to which alternative alleles are spreading.
Local-scalemonitoringneedsdependonthegenedrivesystem
being implemented. For population replacement systems, it is
important to monitor: i) persistence of intact drive alleles, ii)
prevalence of drive-resistant alleles, and iii) continued effectiveness
of the effector gene. A key concern for these systems is that drive-
resistant alleles may emerge that are less costly than intact drive
alleles, as when there are fewer cleavable wild-type alleles left in the
population, these resistant alleles may replace the drive alleles
(Eckhoff et al., 2017). Monitoring for this scenario will be essential
in the aftermath of a successful release. Loss of effector gene function
is also essential to monitor post-release, as this could conceivably
happen in the months and years following a release, whether through
loss-of-function mutations in the effector gene, evolution of effector
gene-resistant pathogen strains, or some combination thereof
(Marshall et al., 2019).
For population suppression systems, it is important to monitor:
i) target species population size, ii) presence and abundance of
non-target vector species, iii) presence of drive-resistant alleles, and
iv) persistence of intact drive alleles. Modeling of population
suppression drive systems in spatially-structured populations
warns against the expectation of local target species elimination.
Although this is a possibility for an ideally-functioning drive
system, a more likely scenario is a fluctuating level of
suppression, as populations are repeatedly eliminated and
recolonized by wild and drive-carrying organisms (Eckhoff
et al., 2017;North et al., 2019). This means that populations
must continue to be monitored even following an initially
successful release. In addition to monitoring the target species,
local non-target vector species should be closely monitored, as
suppression programs may leave vacant or partially vacant
ecological niches that other species may inhabit. Along with
adult trapping and "dipping"for larvae, presence and relative
abundance of target and non-target species could be monitored
through a molecular technique such as eDNA that detects trace
DNA shed by mosquito larvae and pupae in water and sediment
samples (Boerlijst et al., 2019;Krol et al., 2019). As previously
mentioned, it is essential to rigorously monitor for drive-resistant
alleles, as early detection is key to preventing their spatial spread,
and the more time that passes, the more likely they are to emerge.
For both replacement and suppression strategies, epidemiological
data shouldcontinue to bemonitored post-release, and may help to
signal program failures.
Gene drive-modified mosquito projects are unique in the field of
vector control in that they involve transgenes that could potentially
spread on a wide scale. This means that, in certain cases, monitoring
will be required on a wide scale. When a gene drive system is only
intended to spread locally, for instance during a trial, it will be
essential to monitor areas of unwanted spread for the intact drive
allele, and when the gene drive system is intended to spread on a
wide scale, it will be of interest to monitor on this scale for alternative
alleles (e.g., homing-resistance alleles and non-functional effector
genes). The scale, cost and projected effectiveness of wide-scale
monitoringprogramsisimportanttoconsiderasthisisexpectedto
be a major cost driver of gene drive mosquito projects as they
advance from lab to field.
Wide-scale monitoring programs to detect intact or alternative
gene drive alleles stand to learn from experiences with invasive
species, in which early invasions may be halted through effective
monitoring programs (Koch et al., 2020). These programs often take
into account the life history of the species in question, its predicted
geographic distribution, expected pattern of spread, and the costs of
monitoring activities. Multiple scenarios are then modeled to
determine the most cost-effective option. A key result is that
early detection is critical to minimizing the impact of an invasion
and to preserving the possibility of local elimination (Holden et al.,
2016). Future modeling analyses should explore the optimal density
and placement of traps and frequency of sampling in order to detect
gene drive alleles, drive-resistant alleles or non-functional effector
genes while they can still be effectively managed (Figure 1).
Given the expected expense of these monitoring programs, cost-
efficiency will be a priority. Difficult questions must be addressed
regarding what can feasibly be achieved by wide-scale monitoring
FIGURE 1 | Optimal density and placement of traps to detect gene drive alleles, drive-resistant alleles and non-functional effector genes. (A–B) Transects may be
used to optimize monitoring efforts to detect gene drive and alternate alleles in the vicinity of a release site during and post-release. (A) In the pictured scenario, during
release, basic molecular tests would be conducted on adults to establish presence/a bsence of the construct and percent allele prevalence. Tested sites include primary
sites (red triangles) adjacent to the release site (crimson circle), and secondary sites (green triangles) adjacent to primary sites, in order to capture early spread. In the
pictured scenario, the lower-right arm of the sampling transect extends along a natural corridor of increased dispersal (e.g., prevailing wind direction). Traps along this
section are placed at shorter intervals, extending further beyond the release site. (B) Post-release, molecular testing becomes less frequent, but more specific, once a site
has reached high frequency or fixation for the gene drive construct. Once a large fraction of mosquitoes at a site has the construct, sampled mosquitoes having the
construct will go on to have the construct and surrounding insertion site sequenced to verify construct integrity and functionality. Criteria should also be discussed
regarding when, how and the frequency at which resistance in the malaria pathogen should be tested. (C–F) A mosquito metapopulation is denoted by a set of circles,
each circle corresponding to a partially-isolated population connected to others by migration. Populations without the gene drive system are open circles with blue
outlines, those with the gene drive are purple circles, those with traps are magenta circles, and the circle of first detection is plum, also denoted by an arrow. (C) In this
simulation, with only 1 trap per 128 populations, the gene drive allele invades 101 populations before first detection. (D–F) As the number of traps is increased, the
number of populations invaded at the time of detection declines: in this simulation, there are 56, 46 and 9 invaded populations for the cases of 5, 9 and 15 traps,
respectively. Questions arise as to the density of traps required to detect a gene drive or alternate allele in time for it to be effectively managed, and how much investment
would be required to achieve this.
programs. Thus far, monitoring of the invasion of An. stephensi in
the Horn of Africa (Seyfarth et al., 2019), and of Ae. aegypti in
California (Lee et al., 2019), has resulted in documentation and
genetic reconstruction of the invasions, rather than control or
elimination. Hence, for Anopheles gene drive mosquito projects,
how much investment is required to detect intact or alternate gene
drive alleles in time for them to be effectively managed?
Low-threshold gene drive systems, whether intended for
population replacement or suppression, pose significant
demands on monitoring programs, both in terms of their
persistence and potential to spread. Fortunately, lessons can be
learned from examples of monitoring programs for genetic and
biological control systems in Ae. aegypti, including analogs of
high-threshold replacement via Wolbachia, and suppression via
RIDL. Many of the monitoring priorities before, during and after
a release also hold for low-threshold gene drive systems,
especially at the stage of confined field trials. However, it
should be noted that these case study systems have all been
engineered in Ae. aegypti, while the low-threshold systems that
are furthest along in development are all in Anopheles species.
Hence, while many of the high-level considerations apply,
consideration must also be given to their biological differences -
greater dispersal distances, separation of breeding and blood-
feeding sites, and trapping methods for Anopheles species. In
parallel, methodology used for planning monitoring programs
for invasive species appears to apply well to the detection of
intact or alternative gene drive alleles; however, open questions
remain as to what can be achieved with available resources. This
will be an interesting area of research as this potentially
transformative technology advances from lab to field.
The original contributions presented in the study are included in
the article, further inquiries can be directed to the corresponding
author.
All authors listed have made a substantial, direct, and intellectual
contribution to the work and approved it for publication.
This work was supported by funds from the Bill & Melinda Gates
Foundation (INV-017683), the UC Irvine Malaria Initiative, and
a DARPA Safe Genes Program Grant (HR0011-17-2-0047)
awarded to JMM. The funders had no role in the study
design, decision to publish, or preparation of the manuscript.
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A perspective on the expansion of the genetic technologies to support the control of neglected vector-borne diseases and conservation
Robyn Raban
William A.C. Gendron
Omar Akbari
Genetic-based technologies are emerging as promising tools to support vector population control. Vectors of human malaria and dengue have been the main focus of these development efforts, but in recent years these technologies have become more flexible and adaptable and may therefore have more wide-ranging applications. Culex quinquefasciatus , for example, is the primary vector of avian malaria in Hawaii and other tropical islands. Avian malaria has led to the extinction of numerous native bird species and many native bird species continue to be threatened as climate change is expanding the range of this mosquito. Genetic-based technologies would be ideal to support avian malaria control as they would offer alternatives to interventions that are difficult to implement in natural areas, such as larval source reduction, and limit the need for chemical insecticides, which can harm beneficial species in these natural areas. This mosquito is also an important vector of human diseases, such as West Nile and Saint Louis encephalitis viruses, so genetic-based control efforts for this species could also have a direct impact on human health. This commentary will discuss the current state of development and future needs for genetic-based technologies in lesser studied, but important disease vectors, such as C. quinquefasciatus , and make comparisons to technologies available in more studied vectors. While most current genetic control focuses on human disease, we will address the impact that these technologies could have on both disease and conservation focused vector control efforts and what is needed to prepare these technologies for evaluation in the field. The versatility of genetic-based technologies may result in the development of many important tools to control a variety of vectors that impact human, animal, and ecosystem health.
Selective targeting of biting females to control mosquito-borne infectious diseases
Trends Parasitol
Bianca Kojin
Austin Compton
Zach N. Adelman
Zhijian Tu
Mosquitoes are vectors for a number of infectious diseases. Only females feed on blood to provision for their embryos and, in doing so, transmit pathogens to the associated vertebrate hosts. Therefore, sex is an important phenotype in the context of genetic control programs, both for sex separation in the rearing facilities to avoid releasing biting females and for ways to distort the sex ratio towards nonbiting males. We review recent progress in the fundamental knowledge of sex determination and sex chromosomes in mosquitoes and discuss new methods to achieve sex separation and sex ratio distortion to help control mosquito-borne infectious diseases. We conclude by suggesting a few critical areas for future research.
Target Product Profiles for Mosquito Gene Drives: Incorporating Insights From Mathematical Models
Agastya Mondal
Valeri Vasquez
John M Marshall
Mosquito-borne diseases such as malaria continue to pose a major global health burden, and the impact of currently-available interventions is stagnating. Consequently, there is interest in novel tools to control these diseases, including gene drive-modified mosquitoes. As these tools continue to be refined, decisions on whether to implement them in the field depend on their alignment with target product profiles (TPPs) that define product characteristics required to achieve desired entomological and epidemiological outcomes. TPPs are increasingly being used for malaria and vector control interventions, such as attractive targeted sugar baits and long-acting injectable drugs, as they progress through the development pipeline. For mosquito gene drive products, reliable predictions from mathematical models are an essential part of these analyses, as field releases could potentially be irreversible. Here, we review the prior use of mathematical models in developing TPPs for malaria and vector control tools and discuss lessons from these analyses that may apply to mosquito gene drives. We recommend that, as gene drive technology gets closer to field release, discussions regarding target outcomes engage a wide range of stakeholders and account for settings of interest and vector species present. Given the relatively large number of parameters that describe gene drive products, machine learning approaches may be useful to explore parameter space, and an emphasis on conservative fitness estimates is advisable, given the difficulty of accurately measuring these parameters prior to field studies. Modeling may also help to inform the risk, remediation and cost dimensions of mosquito gene drive TPPs.
Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation
PLOS GENET
Silke Fuchs
William T. Garrood
Anna Beber
Tony Nolan
CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying ('homing') therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel adult lethal gene drive (ALGD) in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations.
Population modification strategies for malaria vector control are uniquely resilient to observed levels of gene drive resistance alleles
Gregory C Lanzaro
Travis C. Collier
Anthony A. James
Cas9/guide RNA (gRNA)-based gene drive systems are expected to play a transformative role in malaria elimination efforts., whether through population modification, in which the drive system contains parasite-refractory genes, or population suppression, in which the drive system induces a severe fitness load resulting in population decline or extinction. DNA sequence polymorphisms representing alternate alleles at gRNA target sites may confer a drive-resistant phenotype in individuals carrying them. Modeling predicts that, for observed levels of SGV at potential target sites and observed rates of de novo DRA formation, population modification strategies are uniquely resilient to DRAs. We conclude that gene drives can succeed when fitness costs incurred by drive-carrying mosquitoes are low enough to prevent strong positive selection for DRAs produced de novo or as part of the SGV and that population modification strategies are less prone to failure due to drive resistance.
Efficacy of Wolbachia-Infected Mosquito Deployments for the Control of Dengue
Adi Utarini
Citra Indriani
Riris Andono Ahmad
Cameron Simmons
Background Aedes aegypti mosquitoes infected with the wMel strain of Wolbachia pipientis are less susceptible than wild-type A. aegypti to dengue virus infection. Methods We conducted a cluster-randomized trial involving releases of wMel-infected A. aegypti mosquitoes for the control of dengue in Yogyakarta, Indonesia. We randomly assigned 12 geographic clusters to receive deployments of wMel-infected A. aegypti (intervention clusters) and 12 clusters to receive no deployments (control clusters). All clusters practiced local mosquito-control measures as usual. A test-negative design was used to assess the efficacy of the intervention. Patients with acute undifferentiated fever who presented to local primary care clinics and were 3 to 45 years of age were recruited. Laboratory testing was used to identify participants who had virologically confirmed dengue (VCD) and those who were test-negative controls. The primary end point was symptomatic VCD of any severity caused by any dengue virus serotype. Results After successful introgression of wMel into the intervention clusters, 8144 participants were enrolled; 3721 lived in intervention clusters, and 4423 lived in control clusters. In the intention-to-treat analysis, VCD occurred in 67 of 2905 participants (2.3%) in the intervention clusters and in 318 of 3401 (9.4%) in the control clusters (aggregate odds ratio for VCD, 0.23; 95% confidence interval [CI], 0.15 to 0.35; P=0.004). The protective efficacy of the intervention was 77.1% (95% CI, 65.3 to 84.9) and was similar against the four dengue virus serotypes. The incidence of hospitalization for VCD was lower among participants who lived in intervention clusters (13 of 2905 participants [0.4%]) than among those who lived in control clusters (102 of 3401 [3.0%]) (protective efficacy, 86.2%; 95% CI, 66.2 to 94.3). Conclusions Introgression of wMel into A. aegypti populations was effective in reducing the incidence of symptomatic dengue and resulted in fewer hospitalizations for dengue among the participants. (Funded by the Tahija Foundation and others; AWED ClinicalTrials.gov number, NCT03055585; Indonesia Registry number, INA-A7OB6TW.)
Optimal invasive species surveillance in the real world: Practical advances from research
Frank H Koch
Denys Yemshanov
Robert G Haight
Krista Lee Ryall
When alien species make incursions into novel environments, early detection through surveillance is critical to minimizing their impacts and preserving the possibility of timely eradication. However, incipient populations can be difficult to detect, and usually, there are limited resources for surveillance or other response activities. Modern optimization techniques enable surveillance planning that accounts for the biology and expected behavior of an invasive species while exploring multiple scenarios to identify the most cost-effective options. Nevertheless, most optimization models omit some real-world limitations faced by practitioners during multi-day surveillance campaigns, such as daily working time constraints, the time and cost to access survey sites and personnel work schedules. Consequently, surveillance managers must rely on their own judgments to handle these logistical details, and default to their experience during implementation. This is sensible, but their decisions may fail to address all relevant factors and may not be cost-effective. A better planning strategy is to determine optimal routing to survey sites while accounting for common daily logistical constraints. Adding site access and other logistical constraints imposes restrictions on the scope and extent of the surveillance effort, yielding costlier but more realistic expectations of the surveillance outcomes than in a theoretical planning case.
Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi
Adriana Adolfi
Valentino M Gantz
Nijole Jasinskiene
Cas9/gRNA-mediated gene-drive systems have advanced development of genetic technologies for controlling vector-borne pathogen transmission. These technologies include population suppression approaches, genetic analogs of insecticidal techniques that reduce the number of insect vectors, and population modification (replacement/alteration) approaches, which interfere with competence to transmit pathogens. Here, we develop a recoded gene-drive rescue system for population modification of the malaria vector, Anopheles stephensi, that relieves the load in females caused by integration of the drive into the kynurenine hydroxylase gene by rescuing its function. Non-functional resistant alleles are eliminated via a dominantly-acting maternal effect combined with slower-acting standard negative selection, and rare functional resistant alleles do not prevent drive invasion. Small cage trials show that single releases of gene-drive males robustly result in efficient population modification with ≥95% of mosquitoes carrying the drive within 5-11 generations over a range of initial release ratios.
Next-generation gene drive for population modification of the malaria vector mosquito, Anopheles gambiae
Rebeca Carballar-Lejarazú
Christian Ogaugwu
Taylor Tushar
Significance Genetic systems for controlling transmission of vector-borne diseases are moving from discovery-stage demonstrations of proofs-of-principle to the next phases of development. A successful transition requires meeting safety and efficacy criteria defined in target product profiles. We show here that the Cas9/guide RNA-based gene-drive components of a genetically-engineered malaria mosquito vector, Anopheles gambiae , achieve key target product profile requirements for efficacy and performance. This system is designed to achieve mosquito population modification when coupled with genes encoding antiparasite effector molecules and result in stable and sustainable blocking of malaria parasite transmission.
Using spatial genetics to quantify mosquito dispersal for control programs
BMC BIOL
Igor Filipović
Chanditha Hapuarachchi
Wei-Ping Tien
Background: Hundreds of millions of people get a mosquito-borne disease every year and nearly one million die. Transmission of these infections is primarily tackled through the control of mosquito vectors. The accurate quantification of mosquito dispersal is critical for the design and optimization of vector control programs, yet the measurement of dispersal using traditional mark-release-recapture (MRR) methods is logistically challenging and often unrepresentative of an insect's true behavior. Using Aedes aegypti (a major arboviral vector) as a model and two study sites in Singapore, we show how mosquito dispersal can be characterized by the spatial analyses of genetic relatedness among individuals sampled over a short time span without interruption of their natural behaviors. Results: Using simple oviposition traps, we captured adult female Ae. aegypti across high-rise apartment blocks and genotyped them using genome-wide SNP markers. We developed a methodology that produces a dispersal kernel for distance which results from one generation of successful breeding (effective dispersal), using the distance separating full siblings and 2nd- and 3rd-degree relatives (close kin). The estimated dispersal distance kernel was exponential (Laplacian), with a mean dispersal distance (and dispersal kernel spread σ) of 45.2 m (95% CI 39.7-51.3 m), and 10% probability of a dispersal > 100 m (95% CI 92-117 m). Our genetically derived estimates matched the parametrized dispersal kernels from previous MRR experiments. If few close kin are captured, a conventional genetic isolation-by-distance analysis can be used, as it can produce σ estimates congruent with the close-kin method if effective population density is accurately estimated. Genetic patch size, estimated by spatial autocorrelation analysis, reflects the spatial extent of the dispersal kernel "tail" that influences, for example, the critical radii of release zones and the speed of Wolbachia spread in mosquito replacement programs. Conclusions: We demonstrate that spatial genetics can provide a robust characterization of mosquito dispersal. With the decreasing cost of next-generation sequencing, the production of spatial genetic data is increasingly accessible. Given the challenges of conventional MRR methods, and the importance of quantified dispersal in operational vector control decisions, we recommend genetic-based dispersal characterization as the more desirable means of parameterization.
How Does eDNA Compare to Traditional Trapping? Detecting Mosquito Communities in South-African Freshwater Ponds
Louie Krol
Berry B. van der Hoorn
Erin Gorsich
Maarten Schrama
Improved biomonitoring of mosquitoes requires an in-depth understanding on occurrences of both vector and non-vector species, in larval, and adult stages. Accurate descriptions of the ecological context in which mosquitoes thrive remain limited, particularly for larval stages. The aim of this study was to develop a mixed-amplicon eDNA approach to assess (i) whether mosquito larval communities of stagnant fresh-water bodies can be detected using a Culicidae-specific primer and (ii) how these results compare to traditional trapping of adult mosquitoes. Results from 32 ponds inside and outside Kruger National Park, South Africa show that our primer detected mosquito eDNA. However, it yielded only a subset of the species found using adult trapping methods. Particularly the less frequent and container-breeding species were not found. Our approach provides the first steps toward an eDNA-based method to assess the entire community of larval-stage mosquitoes. It may thereby overcome current taxonomic hurdles presented by morphological identification of larvae. As such, it holds great promise for biomonitoring and ecological studies of mosquitoes.
Abundance of conserved CRISPR-Cas9 target sites within the highly polymorphic genomes of Anopheles and Aedes mosquitoes
Hanno Schmidt
Mark Hanemaaijer
A number of recent papers report that standing genetic variation in natural populations includes ubiquitous polymorphisms within target sites for Cas9-based gene drive (CGD) and that these "drive resistant alleles" (DRA) preclude the successful application of CGD for managing these populations. Here we report the results of a survey of 1280 genomes of the mosquitoes Anopheles gambiae, An. coluzzii, and Aedes aegypti in which we determine that ~90% of all protein-encoding CGD target genes in natural populations include at least one target site with no DRAs at a frequency of ≥1.0%. We conclude that the abundance of conserved target sites in mosquito genomes and the inherent flexibility in CGD design obviates the concern that DRAs present in the standing genetic variation of mosquito populations will be detrimental to the deployment of this technology for population modification strategies.
Winning the Tug-of-War Between Effector Gene Design and Pathogen Evolution in Vector Population Replacement Strategies
Robyn R. Raban
Nikolay P Kandul
While efforts to control malaria with available tools have stagnated, and arbovirus outbreaks persist around the globe, the advent of clustered regularly interspaced short palindromic repeat (CRISPR)-based gene editing has provided exciting new opportunities for genetics-based strategies to control these diseases. In one such strategy, called "population replacement", mosquitoes, and other disease vectors are engineered with effector genes that render them unable to transmit pathogens. These effector genes can be linked to "gene drive" systems that can bias inheritance in their favor, providing novel opportunities to replace disease-susceptible vector populations with disease-refractory ones over the course of several generations. While promising for the control of vector-borne diseases on a wide scale, this sets up an evolutionary tug-of-war between the introduced effector genes and the pathogen. Here, we review the disease-refractory genes designed to date to target Plasmodium falciparum malaria transmitted by Anopheles gambiae, and arboviruses transmitted by Aedes aegypti, including dengue serotypes 2 and 3, chikungunya, and Zika viruses. We discuss resistance concerns for these effector genes, and genetic approaches to prevent parasite and viral escape variants. One general approach is to increase the evolutionary hurdle required for the pathogen to evolve resistance by attacking it at multiple sites in its genome and/or multiple stages of development. Another is to reduce the size of the pathogen population by other means, such as with vector control and antimalarial drugs. We discuss lessons learned from the evolution of resistance to antimalarial and antiviral drugs and implications for the management of resistance after its emergence. Finally, we discuss the target product profile for population replacement strategies for vector-borne disease control. This differs between early phase field trials and wide-scale disease control. In the latter case, the demands on effector gene efficacy are great; however, with new possibilities ushered in by CRISPR-based gene editing, and when combined with surveillance, monitoring, and rapid management of pathogen resistance, the odds are increasingly favoring effector genes in the upcoming evolutionary tug-of-war.
R01 NIH - Understanding mosquito movement and its relevance to control through genetic analysis
Mosquito-borne diseases such as malaria, dengue and Zika have proven to be very difficult to control with currently-available tools. Genetics-based tools could be transformative in controlling thes e diseases, but their safety and efficacy is dependent on how mosquitoes move around, their mating behavior, and their numbers. We will track mosquito movement by determining how far apart closely-related mosquitoes are found, using genome sequences to determine relatedness. In previous studies to measure movement, mosquitoes have been marked with fluorescent dust, but this marking process itself affects the movement. We will also use genomics data to infer other demographic parameters, such as mosquito population size and mating behavior. ... [more]
Improving Estimates of Temperature and Crowding Impacts on Vectorial Capacity in Aedes aegypti Populations.
Goggy Davidowitz
By using age-dependent gene expression for creating sample-specific estimates of temperature conditions during larval and pupal development, and comparing those estimates to body size measurements, we can study temperature impacts on Ae aegypti fitness in the field. ... [more]
Measurements of Body Size as a Proxy for Fitness and Vectorial Capacity of Aedes aegypti mosquitoes.
This study's aim was to characterize the parameters of temperature and resource-availability during larval development that result in body-size -longevity trade-offs. Size can be a reliable proxy f or nutrient- and temperature-dependent, hormonal-metabolic regulation of life-history strategies (Davidowitz, 2016). ... [more]
Size and Fitness in Aedes aegypti Populations at a Rapidly Warming Edge of Their Geographic Range: Observations and Implications for Mosquito Surveillance in Communities at Risk of Emerging Arboviral Transmission.
This study's aim was to use wing length as a proxy for body size, to characterize the temperature and resource-availability during development. Size can be a reliable proxy for nutrient- and temper ature-dependent, hormonal-metabolic regulation of life-history strategies (Davidowitz, 2016). In insect-disease systems, this effectively means that surveillance including size measurements might enable predictions about a population's response to environmental change, with regards to vectorial capacity (Araújo et al. 2012). ... [more]
Population modification strategies for malaria vector control are uniquely resilient to observed lev...
June 2021 · BioEssays
Cas9/guide RNA (gRNA)-based gene drive systems are expected to play a transformative role in malaria elimination efforts., whether through population modification, in which the drive system contains parasite-refractory genes, or population suppression, in which the drive system induces a severe fitness load resulting in population decline or extinction. DNA sequence polymorphisms representing ... [Show full abstract] alternate alleles at gRNA target sites may confer a drive-resistant phenotype in individuals carrying them. Modeling predicts that, for observed levels of SGV at potential target sites and observed rates of de novo DRA formation, population modification strategies are uniquely resilient to DRAs. We conclude that gene drives can succeed when fitness costs incurred by drive-carrying mosquitoes are low enough to prevent strong positive selection for DRAs produced de novo or as part of the SGV and that population modification strategies are less prone to failure due to drive resistance.
Field Trials of Gene Drive Mosquitoes: Lessons from Releases of Genetically Sterile Males and Wolbac...
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 ... [Show full abstract] greater ease, including (1) threshold-dependent systems, which tend to spread only when introduced above a certain threshold population frequency, and (2) temporally 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.
Confinement and reversibility of threshold-dependent gene drive systems in spatially-explicit Aedes...
Jared Bennett
Sean L. Wu
The discovery of CRISPR-based gene editing and its application to homing-based gene drive systems 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. Gene drive systems that display threshold-dependent behavior could potentially be used during the trial phase of this ... [Show full abstract] technology, or when localized control is otherwise desired, as simple models predict them to: a) effectively spread into partially isolated populations in a confineable manner, and b) be reversible through releases of wild-type organisms. Here, we model hypothetical releases of two recently-engineered threshold-dependent gene drive systems - reciprocal chromosomal translocations and a form of toxin-antidote-based underdominance known as UDMEL - to explore their ability to be confined and remediated. We simulate releases of Aedes aegypti, the mosquito vector of dengue, Zika and other arboviruses, in Yorkeys Knob, a suburb of Cairns, Australia, where previous biological control interventions have been undertaken on this species. We monitor spread to the neighboring suburb of Trinity Park, to assess confinement. Our results suggest that 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. UDMEL requires fewer releases to introduce, but more releases to remediate, including of females capable of disease transmission. Both systems are expected to be confineable to the release site; however, spillover of translocations into neighboring populations is less likely. https://youtu.be/lNcIHcJYI_0
Modeling confinement and reversibility of threshold-dependent gene drive systems in spatially-explic...
May 2020 · BMC Biology
The discovery of CRISPR-based gene editing and its application to homing-based gene drive systems 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. Gene drive systems that display threshold-dependent behavior could potentially be used during the trial phase of this ... [Show full abstract] technology, or when localized control is otherwise desired, as simple models predict them to spread into partially isolated populations in a confineable manner, and to be reversible through releases of wild-type organisms. Here, we model hypothetical releases of two recently engineered threshold-dependent gene drive systems-reciprocal chromosomal translocations and a form of toxin-antidote-based underdominance known as UD MEL-to explore their ability to be confined and remediated. We simulate releases of Aedes aegypti, the mosquito vector of dengue, Zika, and other arboviruses, in Yorkeys Knob, a suburb of Cairns, Australia, where previous biological control interventions have been undertaken on this species. We monitor spread to the neighboring suburb of Trinity Park to assess confinement. Results suggest that 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. UD MEL requires fewer releases to introduce, but more releases to remediate, including of females capable of disease transmission. Both systems are expected to be confineable to the release site; however, spillover of translocations into neighboring populations is less likely. Our analysis supports the use of translocations as a threshold-dependent drive system capable of spreading disease-refractory genes into Ae. aegypti populations in a confineable and reversible manner. It also highlights increased release requirements when incorporating life history and population structure into models. As the technology nears implementation, further ecological work will be essential to enhance model predictions in preparation for field trials.