Oral RNAi for Gene Silencing in Mosquitoes: From the Bench to the Field

Abstract

RNA interference (RNAi) has played a key role in the field of insect functional genomics, a discipline which has revolutionized the study of developmental, evolutionary, physiological, and molecular biological phenomena in a wide variety of insects, including disease vector mosquitoes. Recently optimized oral RNAi procedures have facilitated the technically straightforward, high-throughput, effective and economical generation of reproducible gene silencing data, which has been used for the successful characterization of a number of genes in adult mosquitoes. In addition to laboratory applications, oral RNAi could one day be utilized in the field for control of insect pests.

TOPIC INTRODUCTION

Oral RNAi for Gene Silencing in Mosquitoes

Vector mosquitoes transmit a variety of medically important human disease-causing pathogens (Centers for Disease Control 2021). Vector control is the primary method for preventing mosquito-borne illnesses in humans. However, as a result of the increasing global incidence of insecticide resistance and concerns for potential negative impacts of chemical pesticides on non-target organisms, current mosquito control methods are reaching the limits of sustainability, necessitating the development and introduction of innovative vector control strategies (Airs and Bartholomay 2017, Centers for Disease Control 2021). Mosquito genome projects (Holt et al. 2002, Nene et al. 2007) facilitated research in new aspects of mosquito biology, including functional genetic studies in medically important Aedes (dengue, Zika, chikungunya, and yellow fever vector) and Anopheles (malaria vector) human disease vector mosquitoes (Centers for Disease Control 2021). These advancements have precipitated the development of novel gene-centered vector control strategies, resulting in research studies focused on the identification of potential gene targets for vector control, as well as methods for manipulating mosquito gene function both in the laboratory as well as in the field. RNAi, which facilitates functional characterization of mosquito genes in the lab, could potentially be applied as a new method of vector control (Wiltshire and Duman-Scheel 2020).

RNAi, an innate genetic regulatory mechanism which is conserved in many eukaryotes, prevents expression of mRNA transcripts that are silenced by interfering RNA species, including 100–300 bp double-stranded RNAs (dsRNAs), 21–25 bp small interfering RNAs (siRNAs), or short hairpin RNAs (shRNAs), in a sequence-specific manner (Fire 1998, Timmons, 2001). In the laboratory, RNAi has facilitated characterization of gene function in a wide variety of insects, including mosquitoes (Airs and Bartholomay, 2017). Despite the successful use of this technique by many laboratory research programs, the efficiency of RNAi is historically inconsistent and seemingly dependent on a number of variables (Wiltshire and Duman-Scheel, 2020), including the interfering RNA species administered (dsRNA, siRNA or shRNA), the stability of the interfering RNA and the mRNA target, the interfering RNA delivery system utilized (Whitten, 2019), the arthropod species targeted and the life stage at which RNAi is introduced, and the cellular physiology of the tissue(s) targeted (Airs and Bartholomay 2017, Kunte 2019). Such variables have often challenged RNAi studies in the lab and hamper development of RNAi as an insect pest control technology. Recent advances in the oral administration of interfering RNA to both larvae (Singh et al. 2013, Whyard et al. 2015, Hapairai 2017) and adult mosquitoes (Hapairai 2020, Mysore 2020) may facilitate translation of this technology to the field (Wiltshire and Duman-Scheel, 2020). As a result, orally administered RNAi pesticides may represent a new class of biorational insecticides that could combat the increased global incidence of insecticide resistance and be successfully deployed as a new component of integrated mosquito control programs.

Procedures for analysis of gene function in which adult mosquitoes are fed with a colored sugar bait containing small interfering RNA (siRNA) were recently optimized (Hapairai et al. 2020, Mysore et al. 2020). This methodology, which is an adaptation of a previously published technique for feeding long dsRNAs to A. aegypti (Coy et al. 2012), can be used for delivery of siRNAs to adult A. aegypti and A. gambiae mosquitoes. The technique, which is less technically challenging and physically disruptive than subcutaneous injection of adult mosquitoes, facilitates analysis of behavioral and morbidity phenotypes resulting from suppression of target gene expression. The revised method includes procedural modifications that were designed to enhance sugar feeding and yield reproducible data. Moreover, the use of siRNAs facilitates the design of interfering RNA with conserved target sites that can be used to study gene function in multiple species, but which have less potential for off-site targeting of other genes or in non-target organisms (Hapairai et al. 2020, Mysore et al. 2020). This methodology could likely be adapted for the study of other laboratory insects that consume sugar bait and could potentially be applied for control of mosquitoes or other insect pests in the field.

The application of oral RNAi for functional characterization of mosquito genes

The optimized oral RNAi strategy was successfully used to characterize the function of the Shaker (Sh) and dopamine-1 receptor (Dop1) genes. Detailed findings from these studies have been published (Hapairai et al. 2020, Mysore et al. 2020) and are summarized here to demonstrate the efficacy and utility of the sugar-baited siRNA delivery technique. In these studies, adult female mosquitoes were permitted to feed for four hours on sugar bait alone, sugar bait with control siRNA that lacks a target in mosquitoes, sugar bait with Sh.463 siRNA (which silences Sh), or sugar bait with dop1.462 siRNA (which silences dop1) in replicate trials conducted using 25 adult females per treatment. Although negligible morbidity was observed in mosquitoes fed with sugar bait alone (10 ±2%) or control siRNA sugar bait (10 ±2%), high levels of morbidity were observed in mosquitoes that consumed Sh.463 (89 ±6%, P<0.001 compared to sugar bait or to control siRNA sugar bait) or dop1.462 siRNA (88±4%, P<0.001 compared to sugar bait or to control siRNA sugar bait). Sh.463 and dop1.462-treated adults died over the course of six days, with the time of death spread fairly evenly over the six day trial period (Hapairai et al. 2020, Mysore et al. 2020).

Interesting behavioral phenotypes were observed in Sh.463- and dop1.462-treated mosquitoes (Hapairai et al. 2020, Mysore et al. 2020), both of which failed to fly, displayed uncoordinated walking behavior, often fell, and did not venture beyond the bottom of the vial in which they were housed. Use of the sugar-baited delivery system facilitated analysis of both the behavioral and morbidity phenotypes, as mosquitoes were not subjected to the trauma of siRNA microinjection, ensuring confidence that the resulting behavioral phenotypes and morbidity observed were not secondary to injection trauma. The Sh.462 and dop1.462 target sites are well-conserved in mosquitoes (Hapairai et al. 2020, Mysore et al. 2020), and it would be interesting to use oral sugar-baited RNAi to target these genes or other genes of interest in additional Aedes, Anopheles, or potentially Culex species. It is also likely that the sugar-baited siRNA delivery system could be used to study gene function in many other insects that consume sugar solutions.

Advantages of using siRNA-mediated gene silencing

Although RNAi is sometimes associated with concerns for off-site targeting, which can complicate analysis of gene function, such concerns can be alleviated through the use of multiple different short siRNAs corresponding to the gene of interest (rather than long dsRNA) to confirm phenotypes. Moreover, though oral RNAi does not generate heritable germline mutations, the technique offers several useful advantages (Wiltshire and Duman-Scheel 2020). RNAi, a conditional method of gene silencing, requires no long-term maintenance of genetically modified mosquito strains. The conditional nature of RNAi-mediated gene silencing also allows researchers to control the stage at which gene silencing initiates, circumventing challenges such as developmental lethality, which has been observed in conjunction with Sh and dop1 gene silencing in mosquito larvae (Hapairai et al. 2020, Mysore et al. 2020), and which can hinder the production and maintenance of strains bearing heritable mutations. Finally, despite the significant advancements associated with the advent of CRISPR-Cas9 technology, genetic engineering in non-model insects is labor-intensive, requires skill, and is still relatively expensive. For these reasons, RNAi remains to be a useful strategy for studying gene function in insects (Wiltshire and Duman-Scheel, 2020).

Potential deployment of RNAi pesticides in integrated mosquito control programs

It is interesting to consider whether RNAi-based pesticides, such as Sh.463 and dop1.462, might represent a new class of biorational pesticides that could be utilized in integrated human disease vector mosquito control programs (Hapairai et al. 2020, Mysore et al. 2020). The expression and delivery of these pesticides in yeast expression systems shows great promise as a larvicide intervention (Duman-Scheel 2019), and yeast larvicides corresponding to these siRNAs effectively kill multiple species of mosquito larvae (Hapairai et al. 2020, Mysore et al. 2020). With respect to adults, attractive targeted sugar baits (ATSBs), a new paradigm for vector control (Fiorenzano et al 2017), take advantage of the sugar feeding behavior of adult male and female mosquitoes that are lured to feed on a sugar bait laced with an insecticide. ATSBs, which can be supplied through bait stations or in sprays, can be used either indoors or outdoors (Müller and Galili 2016). Although ATSB technology facilitates targeted delivery of a variety of pesticides, insecticide resistance is nevertheless an ongoing concern (Faraji et al. 2016), and the use of RNAi insecticides, a new class of pesticides, is therefore of interest. The species-specificity associated with RNAi pesticides, which target genes in a sequence-specific manner, would also be advantageous, as the insecticides that are typically used in many ATSBs are not specific to mosquitoes (Faraji et al. 2016). Moreover, despite efforts to limit ATSB applications to non-flowering vegetation as well as the addition of protective bait stations that are more selective to mosquitoes (Müller and Galili 2016), it is still difficult to completely eliminate risks to pollinators and other non-target organisms. RNAi-based pesticides, which appear to have a highly desirable safety profile (Environmental Protection Agency 2014), particularly with respect to conventional chemical pesticides, therefore have the potential to enhance existing ATSB technology. Optimized laboratory sugar feeding protocols, which should be further adjusted for field deployment in the future, will facilitate further analysis of the utility of this promising new technology.