Background
Although insecticide-treated nets (ITNs) and indoor residual spraying (IRS) have been the backbone for malaria vector control, resistance to insecticides used for these applications is a growing problem. Furthermore, these interventions cannot control mosquitoes that bite or rest outdoors [
1]. As residual transmission of malaria becomes increasingly problematic, there is renewed interest in the use of larval source management (LSM) for reduction of residual transmission. In the first half of the twentieth century, LSM was a large-scale and highly effective method for control of malaria. Although large-scale LSM programmes were disbanded and replaced by IRS with dichlorodiphenyltrichloroethane (DDT) in the latter part of the century, LSM is still a major component of integrated mosquito control programmes in some parts of the world [
2]. Moreover, LSM, unlike IRS and ITNs, can reduce the number of mosquitoes that enter houses and the number of outdoor-biting mosquitoes [
3].
Larviciding, a form of LSM, involves the application of chemical or biological agents to water bodies for targeting of immature aquatic larvae and pupae before they become malaria vector mosquitoes. Integrated control programmes that included larviciding were successful in Tanzania, Sudan, and Mauritius [
2,
3], and a recent study in Kenya demonstrated that long-lasting FourStar™ briquette larvicides significantly reduced mean densities of indoor- and outdoor-biting adult malaria vector mosquitoes [
4]. The World Health Organization (WHO) recommended that larviciding, when used as a supplement to ITNs and IRS in sub-Saharan Africa, is cost-effective for malaria control in urban settings where vector breeding sites are few, fixed, and findable [
2]. However, given the increase of reported insecticide resistance and rising concern for the negative effects of pesticides on non-target organisms, the current pesticide repertoire is faced with great challenges to sustainability [
1]. The identification of new, environmentally safe, cost-effective larvicides is critical if the current levels of larviciding are to be sustained or expanded.
Interfering RNA molecules represent a novel class of larvicides with untapped potential for sustainable mosquito control. Although RNA interference (RNAi) is beginning to attract attention in agricultural biotechnology communities [
5‐
7], RNAi is still a largely unexplored approach for control of disease vector mosquitoes. The RNAi pathway is initiated by Dicer, which cleaves long pieces of double stranded RNA (dsRNA) into small interfering RNAs (siRNAs) which silence genes that are complementary in sequence. Most mosquito researchers use longer (300–400 bp) dsRNA molecules for RNAi. However, the short length (21–25 bp) of custom siRNAs and their short hairpin RNA (shRNA) counterparts facilitates the design of interfering RNA that targets mosquitoes, but not humans or other non-target organisms [
8,
9]. siRNAs have facilitated our functional genetic characterization of mosquito larval development [
10‐
14]. This investigation tested the hypothesis that short interfering RNA molecules can be utilized as larvicidal agents for control of
Anopheles mosquitoes. The authors recently conducted a high throughput screen (MDS, in preparation) that resulted in the identification of > 100 mosquito siRNA larvicides. Here, the characterization of highly toxic interfering RNAs targeting three larval lethal mosquito genes identified in the screen,
suppressor of actin (
Sac1),
leukocyte receptor complex member (
lrc), and
offtrack (
otk), is described. The study then describes the bioengineering and testing of a
Saccharomyces cerevisiae (baker’s yeast) system for delivery of these interfering RNA molecules to
Anopheles gambiae mosquito larvae. The results of this investigation demonstrate that heat-inactivated dry yeast interfering RNA pellets targeting these genes induce severe defects in the mosquito central nervous system and up to 100% larval mortality.
Discussion
Numerous studies have demonstrated that target gene silencing through RNAi can promote insect death [
6]. For example, Baum et al. [
38] engineered transgenic corn plants expressing dsRNA targeting the western corn rootworm
Diabrotica virgifera virgifera. dsRNA insecticidal corn targeting this species was recently registered by the US Environmental Protection Agency [
39], which deemed that this intervention meets the regulatory standards described in the Federal Insecticide, Fungicide, and Rodenticide Act [
40]. Likewise, Whyard et al. [
41] showed that
Tribolium castaneum,
Acyrthosiphon pisum, and
Manduca sexta were killed when fed species-specific dsRNA targeting
vATPase. The sequence specificity of RNAi facilitated their design of dsRNAs that targeted specific chosen insects, but which did not kill non-target insects, including closely related insect species [
41]. Similarly, this study designed yeast interfering RNA larvicides with target sequences in
Anopheles larval lethal genes that are not perfectly conserved in other organisms (Additional file
1) and which failed to kill
A. aegypti (Additional file
2). Of course, the ability of an siRNA to have off-target effects is complicated [
42]. It is difficult to predict, on the basis of sequence information alone, whether these yeast interfering RNA larvicides could impact gene expression in non-target organisms [
42], and it will be important to conduct more detailed toxicity studies on these larvicides. However, in general, interfering RNA pesticides are believed to be much safer than chemical pesticides [
43]. MonSanto [
43] concluded, in an extensive evaluation of RNA molecules prepared for the United States Environmental Protection Agency, that RNA based products are an important technology with great promise for pest mitigation applications while presenting an overwhelmingly desirable safety profile, particularly when compared to conventional pesticides. Moreover, although many had questioned whether interfering RNAs were sufficiently stable for field applications, dsRNA stability has actually been reported to be quite high [
44,
45]. These and other studies suggest that RNAi can be exploited to control insect agricultural pests.
RNAi has been used extensively for functional analysis of mosquito genes [
10‐
14]. However, RNAi has not yet emerged as a method for mosquito control. The results of this investigation, in which a number of larval lethal genes to be targeted and an effective delivery system for interfering RNAs targeting these genes were identified, suggest that the addition of RNAi pesticides to integrated mosquito control programmes may be feasible and beneficial. Through the identification of multiple larval lethal genes and target sites in these genes, including those described here and additional RNAi targets in the
An. gambiae genome that have been discovered and tested, the authors hope to build an arsenal of interfering RNAs that can be used to combat resistance to traditional chemical pesticides. This arsenal can also be used to combat future resistance that might develop to any single interfering RNA pesticide.
The broad application of RNAi for pest control is dependent upon production of dsRNA in an economically feasible, scalable, and sustainable fashion. dsRNA manufacture has traditionally relied on expensive, carbon-intensive chemical synthesis, resulting in high costs [
5,
6] and a perception that use of RNAi for mosquito control would be prohibitively expensive. Additionally, a perceived high operational cost and the complexity of
Anopheles larviciding in general has deterred some mosquito control programmes from adopting LSM strategies for malaria vector control [
3]. The use of ready-to-use yeast interfering RNA larvicide tablets, which are a user-friendly economically feasible biorational alternative to conventional larvicides may help to address these concerns. While
Pichia pastoris has been used for expression of recombinant DNA and dsRNA to target
Aedes [
46,
47], in this investigation, we opted to use
S. cerevisiae, for which many mutant strains and plasmid constructs exist. Interfering RNA was affordably propagated through cultivation of the yeast. The first three
Anopheles interfering RNA larvicides generated in
S. cerevisiae, Sac1.1, lrc.51, and otk.16 (Figs.
2,
3), effectively killed mosquitoes, with lrc.51 inducing 100% larval mortality. As with bacteria [
23], the larvicides retained full activity after the yeast was heat-killed (Fig.
2), which circumvents concerns for the introduction of live genetically modified organisms for mosquito control.
Saccharomyces cerevisiae has been cultivated worldwide for thousands of years, and this technology can be adapted to resource-limited countries with constrained infrastructures. Moreover, dried yeast can be packaged and shipped in both active (live) or inactive (dead) forms, which will facilitate regional distribution. Yeast production is readily scaled to industry-sized cultures, and so commercialization of this intervention is feasible. S. cerevisiae, which is non-toxic to humans, is already used globally in food and alcoholic beverage preparation. Dried inactive yeast is sold commercially as a dietary supplement and is available in tablet or flake formulations. It is anticipated that heat-inactivated yeast interfering RNA, which demonstrated high larvicidal activities in this investigation, could be prepared in bulk and distributed in these ready-to-use dried formulations.
Given the many benefits of the yeast system, the encouraging results reported in this study, as well as a recent study in the agricultural pest
Drosophila suzukii [
48], support the pursuit of proof-of-concept semi-field evaluation of yeast interfering RNA larvicides. To this end, semi-field trials with heat inactivated yeast interfering RNA larvicides are planned. This critical next phase of the project will facilitate evaluation of the efficacy, feasibility, and acceptance of introducing biorational yeast interfering RNA larvicides into integrated vector mosquito control programmes. Given that yeast, a strong odorant attractant for larvae, can act as a larval bait [
10], it is likely that yeast interfering RNA pellets could be used to treat volumes of water that are much larger than those used in the present investigation, and this will need to be assessed in the field. It would also be interesting to test additional yeast formulations in the field. For example, it is possible that some species of
Anopheles larvae may more readily ingest yeast interfering RNA flakes that float at the water surface, and this could become critical in the field, where competing food sources are available to larvae. Finally, in addition to assessing the impacts of yeast interfering RNA larvicides on the densities of juvenile and adult mosquitoes in the field, it will be important to examine the best settings for the use of these larvicides, if they can be used for control of other malaria vector mosquitoes, and ultimately to demonstrate that the larvicides can effectively reduce the number of malaria cases.
Authors’ contributions
KM acquired, analysed, and interpreted the yeast larvicide data and assisted with manuscript preparation. LH assisted with study design, acquired, and analysed the yeast larvicide data. LS performed and helped analyse the microinjection screen and assisted with mosquito rearing. EIH and JSR performed and helped analyse the soaking screen and assisted with mosquito rearing. NDS performed and analysed the Aedes aegypti studies. YC prepared the yeast used in these studies and assisted with data analysis. KE, DWS and NW assisted with study design, data analysis, and interpretation. MDS prepared the manuscript, conceived of the study, designed experiments, and assisted with cloning, data analysis and interpretation. All authors read and approved the final manuscript.