Controlling vector-borne disease by releasing modified mosquitoes

Abstract

Aedes mosquito-transmitted diseases such as dengue, Zika and chikungunya, are becoming major global health emergencies while old threats such as yellow fever are re-emerging. Traditional control methods, which have focused on reducing mosquito populations through the application of insecticides or preventing breeding through removal of larval habitat, are largely ineffective, as evidenced by the increasing global disease burden. Here, we review novel mosquito population reduction and population modification approaches with a focus on control methods that are based on the release of mosquitoes, including the release of Wolbachia-infected mosquitos and strategies to genetically modify the vector, that are currently under development and have the potential to contribute to a reversal of the current alarming disease trends.

Introduction

Aedes aegypti is exquisitely adapted to tropical and sub-tropical cities as its preferred habitat, living and breeding within people’s dwellings and the waste that accumulates around them. As tropical cities continue to grow, often outstripping the delivery of adequate infrastructure to manage either water delivery or waste removal, this mosquito has flourished. This rapid urban growth together with widespread global air travel, that enables human pathogens to travel as easily as their hosts, creates the perfect conditions for human disease to rapidly spread, as we are currently experiencing globally.

The burden of Aedes-transmitted disease has significantly increased over the past 50 years ^1,2. The incidence of dengue, now the world’s most common mosquito-borne viral disease, grew more than 30-fold during this period ³. Dengue viruses are estimated to infect around 400 million people per year, and over half the world’s population is at risk of the disease (FIG. 1) ⁴.

Figure 1. The global distribution and burden of dengue.

Evidence consensus map showing the complete absence to complete presence of dengue. Green colours indicate evidence consensus towards absence of dengue, and orange and red colours indicate consensus towards presence of dengue. Darker colouring indicates more data supporting a conclusion about the presence or absence of dengue in a country. Figure adapted from ref ⁴.

More recently, chikungunya virus emerged from Africa in the mid-2000s, spreading first across India and Asia, and then into the Americas in 2013 ⁵. Zika virus outbreaks occurred in the South Pacific in 2013 and the Americas in 2015 ⁵. Infection with Zika virus in the Americas coincided with a surge in cases of microcephaly and other congenital abnormalities. Even yellow fever, for which an effective vaccine exists, is re-emerging. Recent outbreaks started in Angola in late 2015 and the virus quickly spread to the Democratic Republic of Congo, Kenya and China ⁶. In late 2016, hundreds of cases of yellow fever have been reported in Brazil ⁷.

This unprecedented global emergence of viruses that are transmitted by arthropod vectors (arboviruses) is thought to be caused by a combination of human population growth, increasing globalization, a rapid rise in population-dense cities in tropical areas and major expansion of the geographic range of A. aegypti ^1,5,8. Existing methods that aim to reduce disease by suppressing mosquito populations through the physical removal of breeding sites or rely on the application of insecticides targeting either larvae or adults are unable to cope in this new global context. To effectively limit or prevent future outbreaks, novel public health interventions are desperately needed ³.

New vector control approaches that involve the release of mosquitoes currently fall into two broad classes: they either aim to reduce the vector population, or modify the vector to make it refractory to pathogen transmission. Reducing mosquito populations through suppression approaches is based on the intuitive assumption that as virus transmission is dependent on a bite from an infectious mosquito, reducing mosquitoes will lower transmission and disease. However, although this is clearly true if the mosquito population can be completely eliminated, the impact on disease if population suppression is only partial is much less clear. Currently, there is little experimental evidence (for example, randomised controlled trials with epidemiological endpoints) that indicates the effectiveness of imperfect mosquito suppression strategies ^9,10.

Novel population reduction approaches involve rearing and releasing large numbers of male mosquitoes that cannot produce viable offspring when they mate with wild females. Over the course of many generations of continual release of these males, the size of the vector population should be substantially reduced, which in turn should reduce disease transmission. These methods include the sterile insect technique (SIT), the incompatible insect technique (IIT) and various genetic modification strategies (FIG 2).

Figure 2. Modification of vectors for population reduction.

New vector control approaches that involve the release of mosquitoes aim to reduce the vector population. A) In the sterile insect technique (SIT) approach, male insects are exposed to either irradiation or sterilizing chemicals, causing large-scale random damage to the insect chromosomes or dominant-lethal mutations in the sperm. These males are then released in the wild population, and when they mate with wild females, viable offspring are rarely produced, eventually leading to a substantial decrease in vector population size. B) In the incompatible insect technique approach, a Wolbachia strain is stably introduced into a colony of a mosquito species. Only Wolbachia-infected males are released, which when mated to females that do not harbour the same Wolbachia strain or that do not carry Wolbachia, results in the death of their offspring due to CI. A combination of IIT and SIT could be used to suppress mosquito populations. During this approach, Wolbachia-infected mosquitoes are treated with low level irradiation. As in IIT alone, Wolbachia-males mating with wild females will not produce offspring. In the case of accidental female releases, these irradiated females are sterile and cannot reproduce with wild or Wolbachia-infected males. C) RIDL (Release of Insects carrying a Dominant Lethal) is a suppression strategy, whereby males that carry a transgene that causes late-acting lethality are released in the open field. These males mate with wild-type females, and the resulting offspring die before reaching the pupal stage.

By contrast, population modification approaches involve the release of both male and female mosquitoes that carry a heritable factor that reduces or blocks their ability to transmit viruses such as dengue or Zika viruses. As these mosquitoes mate with wild mosquitoes, the factor will spread through the population, effectively rendering the mosquitoes incapable of transmitting the pathogen without the need for population suppression. These approaches include the deployment of pathogen-blocking endosymbiotic bacteria Wolbachia pipientis (FIG. 3), and gene-drive mechanisms such as the CRISPR-Cas9 system coupled with transmission-blocking gene constructs (FIG. 4).

Figure 3. Wolbachia use for population reduction and modification approaches.

A) Using Wolbachia in the incompatible insect technique (IIT) approach, results in population reduction. In IIT, a Wolbachia strain is stably introduced into a colony of a mosquito species. Only Wolbachia-infected males are released, which when mated to females that do not harbour the same Wolbachia strain or that do not carry Wolbachia, results in the death of their offspring due to CI. Large numbers of males are released to increase the number of incompatible matings that are occurring. Over time, the population of disease-competent mosquitoes will decrease. B) Wolbachia can also be used to modify a mosquito population. Both Wolbachia-infected male and female mosquitoes are generally released over a 12-16 week period. CI provides a reproductive advantage to Wolbachia-infected females resulting in the spread and establishment of Wolbachia in the population. These Wolbachia-infected females are resistant to arboviruses such as dengue, Zika, and chikungunya.

Figure 4. Gene-drive approaches for population modification and reduction.

A) Homing endonuclease genes (HEGs) encode endonuclease genes that recognize a specific DNA sequence and catalyse a break, which is then naturally repaired through homology-directed repair resulting in non-Mendelian inheritance. B) The CRISPR-Cas9 system is analogous to HEGs; however, a guide RNA (gRNA) provides sequence specificity for DNA cleavage by the Cas9 nuclease which is then repaired through homology-directed repair. Examples of CRISPR-Cas9 used in population modification and reduction approaches include C) the addition of vector competence genes with the Cas9-gRNA construct resulting in virus-resistant offspring ⁹⁹, D) creating a gRNA to targeting female fertility genes resulting in sterile females ¹⁰⁰, and creating a gRNA to target X-chromosome-specific sequences resulting in a reduction of female offspring ¹⁰¹. The Cas9-gRNA constructs inherited by any surviving offspring resulting in its continual spread.

Before any new vector control approach can be deployed at scale, it should progress from laboratory-based proof-of-concept experiments to semi-field and then open-field releases ¹¹. To achieve this, researchers must adapt the vector-control methodologies for large-scale releases, satisfy regulatory requirements and commit to investing great effort in community engagement. Strong public support is critical for adoption of any new technology, as these stakeholders have the power to help “pull” a technology to the field – or alternatively prevent its implementation. Finally, if field releases are successful, the task of demonstrating epidemiological impact for a particular technology still remains. Effective epidemiological studies require the support and involvement of the community at the trial site, approval by the government, collaboration with the existing health system and robust financial support.

In this Review, we describe, evaluate and compare novel vector-control methodologies that are based on either the modification of the mosquito population or mosquito population suppression approaches and that require the active release of modified mosquitoes. We highlight knowledge gaps and discuss lessons learned from field releases that may aid in the success of these or other approaches going forward.

Population modification approaches

Wolbachia to target virus transmission

The endosymbiotic bacterium Wolbachia pipientis, referred to as Wolbachia from here on, naturally infects an estimated 40-60% of all insect species ^12,13. It is vertically transmitted via the host egg, and many Wolbachia strains manipulate host reproduction to provide an advantage to infected females – most commonly by inducing cytoplasmic incompatibility (CI). Infected females can mate successfully with both infected and uninfected males, which enables the rapid spread of Wolbachia throughout a population (FIG. 3B). The expression of CI also provides a method to suppress insect populations, by releasing Wolbachia-infected males into a population of naturally uninfected female insects, thus effectively sterilizing those females (see below, also FIG. 3A).

Recently, it was discovered that in addition to inducing CI in insects Wolbachia can protect its natural host Drosophila melanogaster from pathogenic viruses such as Drosophila C virus ^14,15. Since that initial observation, a number of different Wolbachia strains were shown to prevent the transmission of a range of viruses and parasites in laboratory studies ^16–23 by preventing pathogen replication within the insect ^16,24

The properties of CI coupled with the inhibition of virus replication provide the basis for a novel intervention strategy against mosquito-transmitted diseases. By releasing both male and female mosquitoes that are infected with Wolbachia into a wild population it should be possible for Wolbachia to invade that population. Wolbachia-infected females would have a reproductive advantage compared with wild-type females owing to the induction of CI, and Wolbachia would naturally spread throughout the population until nearly all mosquitoes carried it. The Wolbachia-infected females would then have greatly reduced ability to transmit a virus to humans, and disease should decline and potentially be eliminated from communities ^25,26.

In contrast to many insects, including many mosquito species, A. aegypti is not a natural host for Wolbachia, and therefore to use Wolbachia to modify a population, the bacteria must be introduced into the mosquito through microinjection and a stable colony needs to be established ^24,27. Subsequently, it needs to be determined if Wolbachia can reduce the vector competence of the mosquito. To date, eight different Wolbachia strains have been transinfected into A. aegypti: wMel, wMelPop-CLA, wMelCS, wRi, wAu, wAlbA, wAlbB and wPip ^24,27–30. Importantly, it was shown that Wolbachia can limit the transmission of a range of human pathogens by A. aegypti, including dengue, Zika and chikungunya viruses ^16,17,19,24, which suggests that this intervention could simultaneously target multiple diseases.

Using Wolbachia to reduce the ability of the mosquito population to transmit disease has a number of desirable attributes. The method requires the release of far fewer mosquitoes than population reduction methods such as SIT or IIT (Table I). Moreover, once Wolbachia are established in a population, they are expected to be maintained at a high frequency indefinitely ³¹. In Australia, initial releases of Wolbachia infected male and female insects were undertaken for 10 weeks, and Wolbachia infection has persisted in wild mosquito populations at frequencies above 90% ³². Therefore, mosquitoes infected with Wolbachia only need to be deployed once, which is in contrast to population suppression strategies (which requires the repeated deployments of modified mosquitoes as the natural vector population recovers). As a result, Wolbachia-based replacement strategies are cost effective and, moreover, as costs occur only for the initial deployment, donor fatigue’ might be less of a problem for this approach as no ongoing recurrent funding is needed to sustain the intervention. Finally, as this method involves the release of both female and male mosquitoes, there is no need for the laborious and error-prone process of sex sorting before release.

Table 1. Comparison of different vector control technologies that are currently being developed.

Technology	Laboratory proof-of-concept	Field release	Scaled deployment beyond 50 km2	Reapplication required	Approximate release rate (mosquitoes/ha/week)
Population modification
Wolbachia	+	+	+	No	10-100 75,109
CRISPR-Cas9	+	-	-	No	<1
Population suppression
SIT	+	+	-	Yes	1,000* 67
IIT (Wolbachia)	+	+	-	Yes	1,000-10,000* 72,73,114
RIDL	+	+	-	Yes	25,000-50,000* 35,90

^*males/ha/week

Ongoing field trials

Currently, the World Mosquito Program (previously known as the Eliminate Dengue Program) ³³ is undertaking deployments of A. aegypti infected with Wolbachia in five countries with strong community support. These studies have shown that the wMel strain of Wolbachia can quickly spread to near fixation in the wild mosquito population, and in the field sites in Australia, where this approach has been studied the longest, the frequency of the wMel strain in the mosquito population has remained stable since the initial deployment in 2011 at rates of around 90% or greater ^32,34. Large-scale releases are now underway in Brazil (Niteroi and Rio de Janeiro), Colombia (Bello and Medellin) and Indonesia (Yogyakarta) ³⁵ in the form of randomized cluster trials or large non-experimental deployments covering more than 2 million inhabitants each.

Work in Australia has shown that the method can be deployed successfully at low cost across small cities. Moreover, early time series observational data from these sites indicates no observations of local dengue transmission once Wolbachia is established in the local mosquito population ³⁶. A randomised cluster trial of the method is currently in progress in Yogyakarta ³³ (ClinicalTrials.gov, # NCT03055585). This trial, which is estimated to finish in 2019, is expected to provide high quality epidemiological evidence on the degree of disease reduction through the use of Wolbachia-infected mosquitoes.

Releasing female mosquitoes is not without issue. As female mosquitoes bite, their release can be a source of discomfort for individuals. During the period of active releases, the number of female mosquitoes present in the population will temporarily increase and the community may experience higher biting pressure. The released Wolbachia-carrying female mosquitoes are expected to decrease, rather than increase, the transmission of arboviruses such as dengue, Zika and chikungunya viruses ^16,19,24; however, communities need to accept an approach that superficially seems to counter years of health-promotion messages advising to kill mosquitoes. This issue needs to be addressed with strong community engagement ³⁷.

Publicly available risk analyses provide confidence about the safety of the method ^33,38,39, but some concern has been raised that Wolbachia infection might enhance the transmission of other pathogens. For example, it has been suggested that transient Wolbachia infections (that is temporary infections of Wolbachia injected into the body of the mosquito followed by pathogen challenge) may enhance infection rates, although not dissemination or transmission rates, of West Nile virus in the mosquito Culex tarsalis ⁴⁰. However, in stably-transmitted Wolbachia infections (where the Wolbachia infection is stable, infects germ line tissues and is maternally transmitted by the mosquito), no enhancement of transmission of any virus, including West Nile virus, has been shown ^{16–18,21,24,41–43}. Similarly, transient Wolbachia infections in anopheline mosquitoes have suggested that in certain contexts vector competence for infections with Plasmodium spp. might be enhanced ^44–46, whereas in naturally infected anophelines vector competence is reduced ^47,48. By contrast, natural Wolbachia infections of Culex pipiens mosquitos have been shown to increase susceptibility to infections with Plasmodium relictum ⁴⁹. Clearly, extensive testing is needed before release to ensure that the control measure does not inadvertently exacerbate disease.

Although Wolbachia is a vertically transmitted endosymbiont, comparisons of host and bacterial phylogenies suggest that horizontal transmission can occur ^50,51, leading to the concern that the introduction of Wolbachia to a novel host such as A. aegypti could result in the horizontal transfer of the bacterium to predators or other insects. Laboratory- and field-based experimental testing for horizontal transfer of Wolbachia from A. aegypti have found no evidence of transfer ^52,53. Moreover, 40-60% of all insect species are naturally infected with Wolbachia ^13,54, and it is unlikely that introducing Wolbachia into one more species will increase the frequency of horizontal transmission, especially when closely related, Wolbachia-infected mosquitoes such as A. albopictus and A. notoscriptus already inhabit the same larval habitats as A. aegypti. Furthermore, the fact that A. aegypti is not a natural host for Wolbachia despite shared habitats with the aforementioned species provides further support that horizontal transmission is unlikely to occur.

It has also been suggested that viruses might develop mutations over time that render them less susceptible or resistant to Wolbachia ⁵⁵. The mechanistic basis of Wolbachia-mediated pathogen blocking remains to be fully elucidated, but current data suggest that multiple pathways underlie this effect, which suggest that resistance will not evolve easily ^56–58. Moreover, assessments of field-released mosquitoes suggest that if resistance does develop, it may not happen quickly ³⁴. And even if resistance were to develop in the future, a great reduction in disease burden may have been afforded to communities in the intervening period.

Population reduction approaches

Sterile insect technique

Reducing mosquito populations has long been a focus of disease control programs with the underlying assumption that reducing the number of mosquitoes present in a population will limit the probability of transmission for viruses such as dengue or Zika. Population reduction approaches have been widely used, despite limited experimental evidence supporting the epidemiological effectiveness of these approaches ¹⁰. One such method of population reduction, the SIT, involves irradiating or chemically treating male mosquitos to sterilize them. When these males are released and mate with wild females, no offspring are produced, eventually leading to a substantial decrease in population size (FIG. 2A) ⁵⁹. SIT has been used to reduce mosquito populations with some success, although it has generally been more successful in other agricultural pest species ^60,61.

Work to explore the use of SIT in Aedes species has begun ^62–66, and the results of a pilot field trial have been published. One study, trialing SIT in A. albopictus at four different sites, found that eggs collected in ovitraps from treated areas had induced egg sterility rates of 18-68% compared to eggs from untreated areas, with two sites showing a significant reduction (50-72%) ⁶⁷.

The use of SIT has a number of advantages. As females will not be released, communities should experience no increase in biting rates and therefore the intervention may be more acceptable than population modification approaches described above. It is also aligned with existing health-promotion messaging of reducing mosquito population size. Finally, if the population can be suppressed then reductions in disease transmission are expected with little chance of pathogen resistance developing.

However, the temporary nature of vector population suppression has some disadvantages. Complete elimination of a vector population in an area would require large numbers of males to be released over a long time period (Table I) – especially considering the biology of A. aegypti (for example, mosquito eggs can withstand drying for many months) - and unless the population is completely eliminated it is expected to recover quickly if no further control measures are in place. Similarly, migration of mosquitoes from regions other than the treatment area would spark a population resurgence. This means that SIT releases need to be repeated regularly to maintain community protection from disease. It also requires that mosquitoes are sorted by sex before release, which is not straightforward, and any females that escape sorting may be a competent disease vector. Another difficulty in using SIT is the generation of sterile males that have high fitness and are reproductively competitive with wild-type males ^65,68. Finally, as for all suppression methods, a likely scenario is that the mosquito population is only reduced and not eliminated. Unfortunately, there are no experimental studies available that robustly measure the impact of incomplete suppression on epidemiological endpoints, and therefore the effects on disease are currently unclear.

Incompatible insect technique

A modified version of SIT, termed the IIT, can overcome the fitness costs that are associated with irradiation or chemical treatment of males by using Wolbachia to effectively sterilize males ⁶⁹. To implement IIT, a Wolbachia strain is stably introduced into a colony of a mosquito species. In contrast to population modification approaches, only male mosquitoes carrying Wolbachia are released into a wild population to mate with wild-type females; owing to the CI induced by Wolbachia, no offspring can be produced. If males are released in high enough numbers, more incompatible matings will occur, and ultimately, the population collapses (FIG 3A).

IIT has a long history of field trials. In 1967, Wolbachia was first used as a population reduction strategy to control Culex quinquefasciatus in Burma ⁷⁰. Subsequently, semi-field and pilot field studies of IIT have been performed for A. albopictus and A. polynesiensis ^71–73. Wolbachia-infected males of the lymphatic filiariasis vector, A. polynesiensis, were released over a thirty-week span in French Polynesia, leading to a significant decrease (17%) in egg brood-hatch success in the treated area relative to an untreated area ⁷². In Kentucky (USA), A. albopictus males transinfected with the wPip strain of Wolbachia were released over a 17 week period, causing a significant decrease in the mean number of females collected, as well as a reduction in egg hatch in treated compared to untreated areas ⁷³.

IIT has many advantages as a method of vector population reduction. Using Wolbachia to ‘sterilize’ males is not associated with the fitness costs that can reduce male mating competitiveness in SIT approaches ⁶⁸. Depending on the strain of Wolbachia used, females that escape sorting and are released may have greatly reduced ability to transmit pathogens. Finally, as Wolbachia is naturally occurring and already ubiquitous, the public may accept this technique more easily than genetic modification or irradiation.

By contrast, the IIT still shares many of the limitations of SIT. It requires the continual release of large numbers of males to suppress the population (Table I), and migration of mosquitos from surrounding (untreated) areas will limit long-term effectiveness of this method. As only males are introduced into the environment, an effective sex-sorting system is still required. If non-negligible numbers of females are also released, Wolbachia could spread through a population as in a replacement approach rather than suppress it – although the probability of this is dependent on the overall fitness effects that the Wolbachia strain has on the vector ^31,74,75. Although the IIT approach was tested on a small scale in pilot studies more than 50 years ago, it has yet to be shown that this approach can be scaled-up sufficiently to be an effective operational tool for disease control.

Using a combination of IIT and SIT could further reduce the need to carefully sort females from males before release. In this method, Wolbachia-infected mosquitoes are treated with low level irradiation, which sterilizes females whereas males are unaffected. Females that escape sex sorting and are released into the wild cannot produce offspring and therefore would not interfere with the induced CI in the population (FIG. 2B) ^76,77. The low dose of irradiation has minimal effects on male fitness in a laboratory setting, which suggests that this combined method could be effective in the field ^76–79.

Genetically modified mosquitoes

Although a number of transgenic systems have been developed to suppress mosquito populations, few have progressed to field releases ^80–83. Oxitec has developed a number of transgenic approaches that are based on their Release of Insects carrying a Dominant Lethal (RIDL) methodology ^80,84. The OX513A mosquito strain has been used most successfully to date. This mosquito strain has a tetracycline-repressible transcriptional activator (tTAV) under the control of its own binding site (TetO) creating a positive-feedback loop in which the expression of tTAV results in late-larval lethality. ⁸⁴. When the mosquitoes are reared on a diet supplemented with tetracycline, it binds tTAV preventing its binding to TetO, decreasing the production of tTAV allowing the mosquitoes to thrive. When OX513A males are released into the wild and mate with wild-type females, they pass on the transgene to their offspring; owing to the lack tetracycline in their diet, the transgene is expressed leading to late-larval death (FIG. 2C) ^84–86.

Two field studies have compared the fitness of OX513A mosquitoes to that of wild-type mosquitoes for male competitiveness ⁸⁷ as well as dispersal and longevity ⁸⁸. The life expectancy and maximal dispersal distance of OX513A is similar to that of wild-type mosquitoes, but the mean distance travelled is significantly lower ⁸⁸.

Field releases of these mosquitoes have been performed in the Cayman Islands and Bahia, Brazil. The release of OX513A in the Cayman Islands allowed researchers to perform real-time comparisons of the effective numbers of males required to achieve a significant decrease in mosquito populations. Under their highest release ratios, they found an 80% relative reduction in treated versus untreated areas over a 23-week period ⁸⁹. In Brazil, mosquitoes were released over the course of one year. A 95% reduction in the local population of A. aegypti was observed based on adult trapping data, and an 81% reduction based on egg trapping data ⁹⁰. Currently, Oxitec is performing releases in the Cayman Islands, Panama and Brazil, with plans for substantial expansion in their Brazil release sites ³⁵.

Oxitec’s methodology has several advantages over traditional suppression methods. The radiation used in SIT generates dominant lethals in a non-specific manner – which can also lead to strong fitness effects and lowered mating competitiveness of males. The RIDL method specifically engineers a dominant lethal, thereby limiting off-target effects. Additionally, engineering allows for control of when the lethality is induced (that is, the presence of absence of tetracycline) ⁸⁴. In contrast to the SIT, which induces lethality generally at the embryonic stage, lethality of OX513A is induced at late-larval stages ⁵⁹, suggesting that although OX513A larvae ultimately die before adulthood, they still compete with wild-type larvae for food, possibly enhancing population suppression ⁸⁴. Although the public may have concerns about the release of genetically modified insects, their concerns may be alleviated by the fact that the OX513A-based approach is a self-limiting technology. As the transgenic mosquitoes require tetracycline in their diet for survival, mosquitoes that carry the transgene cannot survive more than one generation in the field.

One of the biggest limitations of a RIDL technology such as the OX513A-based approach is that it requires large numbers of males to be released for successful suppression (Table I), and this can be technologically and financially difficult. For the release of OX513A in both the Cayman Island and Brazil, the planned field site sizes had to be decreased owing to rearing limitations and a requirement to maintain a mating fraction of 50% for genetically modified males ^89,90. This suggests that large-scale releases could be difficult to maintain. As with SIT and IIT, accurate sex sorting is required for this RIDL method. Although sex-sorting methods have become more efficient, rates of accidental release of females were previously reported to be between 0.02-0.33% ^88–90. For large scale releases, such as those planned in Brazil for which Oxitec estimates releases of 30-60 million males per week ³⁵, this would result in the unintended daily release of thousands of females. In addition, a considerable community engagement effort to build sufficient trust for widespread deployment of genetically modified mosquitoes is required ⁹¹.

Emerging technologies

A number of developing technologies exist that have not yet progressed to field trials. Numerous laboratory-based studies have shown that the use of transgenes can be effective in limiting pathogen transmission through the expression of genes that target the pathogen, or that are effective in suppressing mosquito populations by targeting genes involved in reproduction or by using sex distortion systems. Although these systems show promise, the difficulty lies in how to spread the transgenes to all mosquitoes in a population. One of the most promising methods to solve this problem is the use of transgenes to generate a gene-drive system, a strategy that was first proposed nearly 15 years ago ⁹². Gene-drive systems alter normal Mendelian inheritance to greatly increase the odds that the drive system will be passed on to offspring. An effective gene-drive system could be used to establish disease inhibitors or population repressors into a population. Homing endonuclease genes (HEGs) were the initial inspiration for a gene drive system. HEGs encode proteins that recognize and cleave a 15-30 bp DNA sequence. By placing HEGs within their target sequences, the chromosome on which it was located would be resistant to cleavage. Cleavage of chromosomes that only contain the recognition site would occur, and owing to homology-directed repair (HDR) a heterozygote would be converted into a homozygote (FIG. 4A). HEGs have been developed in Anopheles and Aedes mosquitoes in proof-of-concept experiments ^93–95.

The CRISPR-Cas9 system has been used in genome-editing for a number of years in diverse organisms ^96,97. A study showed that placing the genes that encode Cas9 and a guide RNA (gRNA) into the template used for HDR generated a mutagenic ‘chain reaction’ capable of gene drive (FIG. 4B) ⁹⁸. Subsequent work showed that in laboratory settings, CRISPR-Cas9 could be used to spread anti-Plasmodium falciparum effector genes into an Anopheles stephensi population ⁹⁹, to target genes required for female fertility in Anopheles gambiae ¹⁰⁰, and to create a sex distortion system that targets female A. gambiae ¹⁰¹, suggesting that that this system can be used for both population suppression and population modification (FIG. 4C, D). However, optimization of this methodology is still required before commencing field trials. The first two studies discussed above ^99,100 used the regulatory regions of the germline-specific gene vasa to induce the expression of Cas9 in the germline thus causing only heritable mutations. However, one study found that the expression of Cas9 was not completely restricted to the germline resulting in somatic mutations ¹⁰⁰. A second study found that maternal deposition of the Cas9 protein from the mother into the developing egg caused double-stranded DNA breaks during early embryonic development before a homologous chromosome was present as a repair template, resulting in an increase in non-homologous end joining repair (NHEJ) rather than HDR ⁹⁹. Repair via NHEJ often results in mutations, or insertions or deletions of sequences which destroy the Cas9-recognition site and thus the generation of resistant alleles ^99,100.

The CRISPR-Cas9 gene-drive method can potentially be extremely powerful. Only small numbers of the modified mosquitoes might need to be released (Table I) as the modification should drive itself throughout a population, ⁹⁸, and nearly any sequence of interest can be targeted. However, similarly to HEGs, the CRISPR-Cas9 system is susceptible to developing resistance owing to mutations that can occur in the recognition site. As described above, multiple, laboratory-based studies using CRISPR-Cas9 for gene drive have reported the accumulation of mutations that led to CRISPR-resistant alleles ^98–100, which halt the spread of any modifications throughout a population. Furthermore, based on theoretical modeling, evolution of resistance against the CRISPR-Cas9 system is inevitable ^102,103. The emergence of resistance might be avoided, or at least prolonged, by targeting multiple sequences, by targeting conserved sequences that cannot tolerate disruption or by being more mindful of when releases occur in relation to seasonality of the vector population ¹⁰². Whereas the OX513A strain is self-limiting, the CRISPR-Cas9-drive is self-promoting. The potential for uncontrolled spread of genetic modifications has caused concern among the scientific community ¹⁰⁴, resulting in the publication of guidelines not only pertaining to field releases of such modified organisms but also preventing the accidental release of the modified organisms from laboratories ^104–107.

Lessons learned

Most of the different technologies described above are still at early developmental stages, with limited examples of field releases, and only Wolbachia-based population modification approaches, as undertaken by the World Mosquito Program (WMP) ³³, being done at operational scales in medium-sized cities. A number of lessons are being learned that generally apply to all of the approaches.

Importance of field-cage studies

Advocates of phased testing approaches have stressed the importance of preliminary testing of technologies in semi-field cages prior to open field release ¹¹. The construction of these facilities is expensive and time consuming, and evidence suggests that they may not actually provide data that are more useful than data collected from small laboratory cages in regards to evaluating an approach. Even very elaborate field cages ¹⁰⁸ do not mimic the true field situation. For example, semi-field cage experiments demonstrated successful establishment of the wMelPop strain of Wolbachia in a mosquito population, but it was later shown that this Wolbachia strain could not be established following open field releases ^24,109. Those findings together with the expense of such preliminary testing strategies indicate that field-cage studies should be carefully considered and relevant to the question being addressed and not automatically recommended.

When the WMP first started to undertake field releases there was some concern that Wolbachia might spread in an uncontrolled manner. There was good evidence documenting regional ^110,111 and even global sweeps ¹¹² of Wolbachia infections in naturally infected hosts, raising the prospect that once Wolbachia was released it might spread to locations that might not have approved its release. Initial field testing was done very carefully in Australia in geographically isolated areas to evaluate the ability of Wolbachia to spread ⁷⁵. Over time it was realised that in A. aegypti, the spreading rates of the wMel strain of Wolbachia were very slow ¹¹³, and the initial concern was unfounded. The current controversy around the potential uncontrolled spread of CRISPR-Cas9 gene-drive technology is injecting an even greater sense of caution into this area ^104,105,107. The theoretical ability of gene-drive systems to spread from very small numbers of released individuals and to alter an entire wild population is of concern as we do not fully understand possible adverse consequences of such a release and may not be able to assess it prior to release. Current gene-drive methodologies do not have reversibility built into the system, so if negative consequences are observed, it would be difficult to stop the intervention from spreading. However, the emerging issues of resistance with this technology suggest that similar to Wolbachia-based approaches, the power of the gene drive systems that are being developed might be similarly overstated. Although there is merit in a cautious framework to evaluate and test these methods, it must be balanced against the public health need of new technologies to protect people from ongoing disease outbreaks. Testing and regulatory frameworks need to be sufficiently flexible to be able to adapt to less stringent and time consuming testing procedures if empirical evidence shows that risks are likely to be overstated. Otherwise technology that is urgently needed may be unnecessarily impeded in its adoption and use.

A common feature of all of these new vector control tools is that they rely on the release of mosquitoes into the environment to control the diseases they transmit. This requires communities to have high levels of trust to willingly participate given that the health promotion messages of decades have been based on the dangerous nature of mosquitoes and the need to kill them to reduce disease risk. Even the most robust and elegant technology will fail to be implemented if communities will not accept it. Recently, this was exemplified by the difficulties Oxitec has faced in applying the RIDL methodology in open releases in Florida where deep issues of mistrust toward GM technology, government and industry have led to open protests and stalling of testing plans of a potentially robust and useful technology ⁹¹. Serious attention and resourcing is required for effective community engagement programs associated with these technologies so that trust and acceptance can be built with the communities that will be the end recipient of the technology ³⁷. This engagement is costly and time consuming and needs to start early, even before a given technology is fully developed. Unfortunately, many of the scientists involved in the development of new technologies are laboratory focused specialists with little experience in field application or the principles of effective community engagement.

Conclusion

Existing vector control methods are clearly unable to cope with the unprecedented emergence and reemergence of arboviral diseases. A number of novel methods under development show promise in curbing the ability of A. aegypti mosquitoes to transmit pathogens. Within the next few years we expect that the evidence for the effectiveness of these new interventions will accumulate. Critical to wide scale adoption of any of these approaches will be rigorous epidemiological evidence showing the impact on disease, not just entomological indicators. Many of these technologies are being developed by scientists that are not located in disease endemic countries. Ultimately, collaborations between scientists and governments of affected countries are needed to test and apply the technology. This requires open and authentic partnerships to be developed very early with these collaborators so that they are active participants in the development and implementation of the technology in their countries. Without their full support and ownership there is no pathway to adoption. Just as important will be the sustainability and cost effectiveness of the different approaches for disease endemic countries with limited resources for control programs. Hopefully, at least some of these technologies will prove to be cost saving for health ministries in which case adoption pathways will be more straight forward. With solid epidemiological evidence and community support, their widespread implementation might reverse the current, alarming global disease trend.

Glossary.

Aedes aegypti

Aedes aegypti is the primary mosquito vector of epidemic transmission for viruses, such as dengue, zika and chikungunya. A. aegypti occurs primarily in tropical and sub-tropical regions of the world and is particularly adapted to urban habitats.

Wolbachia pipientis

A naturally occurring bacterial endosymbiont that is estimated to be present in 40-60% of all insect species. Commonly referred to as just Wolbachia.

Sterile insect technique

The radiation or chemical treatment of male mosquitos, which renders them sterile. When they are released in the field and they mate with wild-type females they cannot produce offspring.

incompatible insect technique

The release of Wolbachia-infected males, which when mated with wildtype females who contain no Wolbachia or a different, incompatible strain of Wolbachia, produce no offspring due to cytoplasmic incompatibility.

CRISPR-Cas9

A genome editing tool that was developed from adaptive immune systems found in bacteria and archaea. The system is composed of a nuclease, Cas9, and a guide RNA which targets the nuclease to a specific DNA sequence for cleavage.

Cytoplasmic incompatibility

When Wolbachia-infected male mosquitos mate with uninfected females, the resulting progeny die during early embryogenesis. If the female is also infected with the same Wolbachia strain, that infection can rescue the embryonic lethality resulting in viable progeny.

Vector competence

A measure of ability of arthropod vectors to acquire and transmit viruses in their saliva.

Ovitraps

Traps designed for the collection of mosquito eggs.

Homing endonuclease genes

Selfish genetic elements encoding endonucleases that recognize a specific DNA sequence and catalyse a break, which is then naturally repaired through homologous repair.

Homology-directed repair

A repair mechanisms of a DNA double-strand break, whereby the homologous chromosome is used as a template for repair.

Non-homologous end joining repair

A repair mechanism for DNA double-strand breaks, whereby the two DNA ends are ligated without the need for a homologous template, often resulting in small indels or the introduction of mutations.