Yeast interfering RNA larvicides targeting neural genes induce high rates of Anopheles larval mortality.

BACKGROUND: Although larviciding can reduce the number of outdoor biting malaria vector mosquitoes, which may help to prevent residual malaria transmission, the current larvicide repertoire is faced with great challenges to sustainability. The identification of new effective, economical, and biorational larvicides could facilitate maintenance and expansion of the practice of larviciding in integrated malaria vector mosquito control programmes. Interfering RNA molecules represent a novel class of larvicides with untapped potential for sustainable mosquito control. This investigation tested the hypothesis that short interfering RNA molecules can be used as mosquito larvicides. RESULTS: A small interfering RNA (siRNA) screen for larval lethal genes identified siRNAs corresponding to the Anopheles gambiae suppressor of actin (Sac1), leukocyte receptor complex member (lrc), and offtrack (otk) genes. Saccharomyces cerevisiae (baker's yeast) was engineered to produce short hairpin RNAs (shRNAs) for silencing of these genes. Feeding larvae with the engineered yeasts resulted in silenced target gene expression, a severe loss of neural synapses in the larval brain, and high levels of larval mortality. The larvicidal activities of yeast interfering RNA larvicides were retained following heat inactivation and drying of the yeast into user-friendly tablet formulations that induced up to 100% larval mortality in laboratory trials. CONCLUSIONS: Ready-to-use dried inactivated yeast interfering RNA larvicide tablets may someday be an effective and inexpensive addition to malaria mosquito control programmes and a valuable, biorational tool for addressing residual malaria transmission.

Comparison of the Centers for Disease Control and Prevention-backpack and insectazooka aspirators for sampling Aedes polynesiensis in French Polynesia.

The efficiency of the recently developed handheld InsectaZooka (IZ) aspirator was compared to that of the Centers for Disease Control and Prevention-Backpack (CDC-BP) aspirator by conducting human bait collections on 2 islets (locally called motus) of the atoll of Tetiaroa, French Polynesia. Abundance of mosquitoes was compared between the wind-exposed and wind-protected sides of each motu to measure the effect of wind on mosquito distribution. The number of host-seeking Aedes polynesiensis mosquitoes collected on the 2 motus with either sampling device was not significantly different. Collection of male mosquitoes was low irrespective of the type of aspirator used. Wind had an effect on mosquito distribution, as females were more abundant on the protected sides of both motus. The IZ aspirator is a lighter and equally efficient alternative to the CDC-BP aspirator for collecting Ae. polynesiensis.

Population studies of the filarial vector Aedes polynesiensis (Diptera: Culicidae) in two island settings of French Polynesia.

A mark-release-recapture study was conducted to estimate the adult population size, migration, and dispersal patterns of male and female Aedes (Stegomyia) polynesiensis (Marks) in a valley of Moorea, a volcanic island, and a motu (islet) on the atoll of Tetiaroa, two settings typical of the Society Islands. Aedes polynesiensis recapture rate was high for females and low for males. The distribution of Aedes species in the valley was heterogeneous. Marked individuals dispersed to most parts of the motu and over great distances in the valley for some females. The study provides insights into the field dynamics of Ae. polynesiensis populations and confirms that more efficient sampling methods are warranted. There was no evidence of active migration between motus on the atoll, suggesting that Tetiaroa is a suitable site for small-scale initial open releases of Wolbachia incompatible insect technique and other sterile insect technique-like suppression or replacement strategies.

Field evaluation of selected traps and lures for monitoring the filarial and arbovirus vector, Aedes polynesiensis (Diptera: Culicidae), in French Polynesia.

The efficacy of the BG-Sentinel (BGS) and the BG-Mosquitito (BGM) mosquito traps for sampling populations of the important filariasis and dengue vector Aedes (Stegomyia) polynesiensis (Marks) was evaluated in French Polynesia against human bait collections (HBC) using a modified Centers for Disease Control and Prevention backpack aspirator. Traps were baited with BG-Lure (a combination of lactic acid, ammonia, and caproic acid) or carbon dioxide plus octenol (1-octen-3-ol) known as attractants to aedine mosquitoes. Mosquito sampling was conducted on two typical islands of French Polynesia: the high, volcanic island of Moorea, and the low, coral island (atoll) of Tetiaroa Sampling efficacy was measured in a randomized Latin Square design. Production of carbon dioxide from yeast-sugar fermentation was used as an alternative source of CO2 because supply via dry ice, gas cylinders, or propane combustion in remote tropical islands is costly and challenging. Although the BGS trap captured the greatest number ofAe. polynesiensis in both island settings, catch rates of BGS or BGM baited with either lure were not significantly different from that of HBC. On Moorea, the number of collected aedes species in the BGS trap baited with either lure was significantly greater than the BGM with BG-lure. On Tetiaroa, BGM trapping was severely hampered by damage from rats, and the traps were removed from the study. Our study confirms the efficiency, comparability, and convenience of the BGS trap, a robust and safe alternative to HBC for sampling Aedes mosquitoes in research and surveillance efforts against filariasis and arboviruses in the South Pacific.

Sugar-Baited Delivery of Small Interfering RNA for Gene Silencing in Adult Mosquitoes.

RNA interference (RNAi), an innate regulatory mechanism that is conserved across many eukaryotic species, has been harnessed for experimental gene silencing in many organisms, including mosquitoes. This protocol describes an optimized method for inducing RNAi in adult Aedes aegypti and Anopheles gambiae mosquitoes that involves feeding them a red-colored sugar bait containing small interfering RNA (siRNA). This oral delivery method is less physically disruptive than delivery by subcutaneous injection, and the use of siRNAs (in contrast to long dsRNAs) for RNAi enables the design of molecules that target conserved sites so that gene function can be studied in multiple species. After feeding, the behavioral and morbidity phenotypes that result from the suppression of target gene expression can then be analyzed.

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

RNA interference (RNAi) has played a key role in the field of insect functional genomics, a discipline that has enhanced the study of developmental, evolutionary, physiological, and molecular biological phenomena in a wide variety of insects, including disease vector mosquitoes. Here we introduce a recently optimized RNAi procedure in which adult mosquitoes are fed with a colored sugar bait containing small interfering RNA (siRNA). This procedure effectively and economically leads to gene silencing, is technically straightforward, and has been successfully used to characterize a number of genes in adult mosquitoes. We also discuss how, in addition to laboratory applications, this oral RNAi procedure might one day be used in the field for controlling insect pests.

Advancing climate change health adaptation through implementation science.

To date, there are few examples of implementation science studies that help guide climate-related health adaptation. Implementation science is the study of methods to promote the adoption and integration of evidence-based tools, interventions, and policies into practice to improve population health. These studies can provide the needed empirical evidence to prioritise and inform implementation of health adaptation efforts. This Personal View discusses five case studies that deployed disease early warning systems around the world. These cases studies illustrate challenges to deploying early warning systems and guide recommendations for implementation science approaches to enhance future research. We propose theory-informed approaches to understand multilevel barriers, design strategies to overcome those barriers, and analyse the ability of those strategies to advance the uptake and scale-up of climate-related health interventions. These findings build upon previous theoretical work by grounding implementation science recommendations and guidance in the context of real-world practice, as detailed in the case studies.

Correction: Mysore et al. A Broad-Based Mosquito Yeast Interfering RNA Pesticide Targeting Rbfox1 Represses Notch Signaling and Kills Both Larvae and Adult Mosquitoes. Pathogens 2021, 10, 1251.

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A functional requirement for sex-determination M/m locus region lncRNA genes in Aedes aegypti female larvae.

Although many putative long non-coding RNA (lncRNA) genes have been identified in insect genomes, few of these genes have been functionally validated. A screen for female-specific larvicides that facilitate Aedes aegypti male sex separation uncovered multiple interfering RNAs with target sites in lncRNA genes located in the M/m locus region, including loci within or tightly linked to the sex determination locus. Larval consumption of a Saccharomyces cerevisiae (yeast) strain engineered to express interfering RNA corresponding to lncRNA transcripts resulted in significant female death, yet had no impact on male survival or fitness. Incorporation of the yeast larvicides into mass culturing protocols facilitated scaled production and separation of fit adult males, indicating that yeast larvicides could benefit mosquito population control strategies that rely on mass releases of male mosquitoes. These studies functionally verified a female-specific developmental requirement for M/m locus region lncRNA genes, suggesting that sexually antagonistic lncRNA genes found within this highly repetitive pericentromeric DNA sequence may be contributing to the evolution of A. aegypti sex chromosomes.

A Broad-Based Mosquito Yeast Interfering RNA Pesticide Targeting Rbfox1 Represses Notch Signaling and Kills Both Larvae and Adult Mosquitoes.

Prevention of mosquito-borne infectious diseases will require new classes of environmentally safe insecticides and novel mosquito control technologies. Saccharomyces cerevisiae was engineered to express short hairpin RNA (shRNA) corresponding to mosquito Rbfox1 genes. The yeast induced target gene silencing, resulting in larval death that was observed in both laboratory and outdoor semi-field trials conducted on Aedes aegypti. High levels of mortality were also observed during simulated field trials in which adult females consumed yeast delivered through a sugar bait. Mortality correlated with defects in the mosquito brain, in which a role for Rbfox1 as a positive regulator of Notch signaling was identified. The larvicidal and adulticidal activities of the yeast were subsequently confirmed in trials conducted on Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus, yet the yeast had no impact on survival of select non-target arthropods. These studies indicate that yeast RNAi pesticides targeting Rbfox1 could be further developed as broad-based mosquito larvicides and adulticides for deployment in integrated biorational mosquito control programs. These findings also suggest that the species-specificity of attractive targeted sugar baits, a new paradigm for vector control, could potentially be enhanced through RNAi technology, and specifically through the use of yeast-based interfering RNA pesticides.

A Yeast RNA-Interference Pesticide Targeting the Irx Gene Functions as a Broad-Based Mosquito Larvicide and Adulticide.

Concerns for widespread insecticide resistance and the unintended impacts of insecticides on nontarget organisms have generated a pressing need for mosquito control innovations. A yeast RNAi-based insecticide that targets a conserved site in mosquito Irx family genes, but which has not yet been identified in the genomes of nontarget organisms, was developed and characterized. Saccharomyces cerevisiae constructed to express short hairpin RNA (shRNA) matching the target site induced significant Aedes aegypti larval death in both lab trials and outdoor semi-field evaluations. The yeast also induced high levels of mortality in adult females, which readily consumed yeast incorporated into an attractive targeted sugar bait (ATSB) during simulated field trials. A conserved requirement for Irx function as a regulator of proneural gene expression was observed in the mosquito brain, suggesting a possible mode of action. The larvicidal and adulticidal properties of the yeast were also verified in Aedes albopictus, Anopheles gambiae, and Culexquinquefasciatus mosquitoes, but the yeast larvicide was not toxic to other nontarget arthropods. These results indicate that further development and evaluation of this technology as an ecofriendly control intervention is warranted, and that ATSBs, an emerging mosquito control paradigm, could potentially be enriched through the use of yeast-based RNAi technology.

Assessment of Trinidad community stakeholder perspectives on the use of yeast interfering RNA-baited ovitraps for biorational control of Aedes mosquitoes.

Dengue, Zika, chikungunya and yellow fever viruses continue to be a major public health burden. Aedes mosquitoes, the primary vectors responsible for transmitting these viral pathogens, continue to flourish due to local challenges in vector control management. Yeast interfering RNA-baited larval lethal ovitraps are being developed as a novel biorational control tool for Aedes mosquitoes. This intervention circumvents increasing issues with insecticide resistance and poses no known threat to non-target organisms. In an effort to create public awareness of this alternative vector control strategy, gain stakeholder feedback regarding product design and acceptance of the new intervention, and build capacity for its potential integration into existing mosquito control programs, this investigation pursued community stakeholder engagement activities, which were undertaken in Trinidad and Tobago. Three forms of assessment, including paper surveys, community forums, and household interviews, were used with the goal of evaluating local community stakeholders' knowledge of mosquitoes, vector control practices, and perceptions of the new technology. These activities facilitated evaluation of the hypothesis that the ovitraps would be broadly accepted by community stakeholders as a means of biorational control for Aedes mosquitoes. A comparison of the types of stakeholder input communicated through use of the three assessment tools highlighted the utility and merit of using each tool for assessing new global health interventions. Most study participants reported a general willingness to purchase an ovitrap on condition that it would be affordable and safe for human health and the environment. Stakeholders provided valuable input on product design, distribution, and operation. A need for educational campaigns that provide a mechanism for educating stakeholders about vector ecology and management was highlighted. The results of the investigation, which are likely applicable to many other Caribbean nations and other countries with heavy arboviral disease burdens, were supportive of supplementation of existing vector control strategies through the use of the yeast RNAi-based ovitraps.

Community acceptance of yeast interfering RNA larvicide technology for control of Aedes mosquitoes in Trinidad.

RNA interference (RNAi), a technique used to investigate gene function in insects and other organisms, is attracting attention as a potential new technology for mosquito control. Saccharomyces cerevisiae (baker's yeast) was recently engineered to produce interfering RNA molecules that silence genes required for mosquito survival, but which do not correspond to genes in humans or other non-target organisms. The resulting yeast pesticides, which facilitate cost-effective production and delivery of interfering RNA to mosquito larvae that eat the yeast, effectively kill mosquitoes in laboratory and semi-field trials. In preparation for field evaluation of larvicides in Trinidad, a Caribbean island with endemic diseases resulting from pathogens transmitted by Aedes mosquitoes, adult residents living in the prospective trial site communities of Curepe, St. Augustine, and Tamana were engaged. Open community forums and paper surveys were used to assess the potential acceptability, societal desirability, and sustainability of yeast interfering RNA larvicides. These assessments revealed that Trinidadians have good working knowledge of mosquitoes and mosquito-borne illnesses. A majority of the respondents practiced some method of larval mosquito control and agreed that they would use a new larvicide if it were proven to be safe and effective. During the community engagement forums, participants were educated about mosquito biology, mosquito-borne diseases, and the new yeast larvicides. When invited to provide feedback, engagement forum attendees were strongly supportive of the new technology, raised few concerns, and provided helpful advice regarding optimal larvicide formulations, insecticide application, operational approaches for using the larvicides, and pricing. The results of these studies suggest that the participants are supportive of the potential use of yeast interfering RNA larvicides in Trinidad and that the communities assessed in this investigation represent viable field sites.

Characterization of a dual-action adulticidal and larvicidal interfering RNA pesticide targeting the Shaker gene of multiple disease vector mosquitoes.

The existing mosquito pesticide repertoire faces great challenges to sustainability, and new classes of pesticides are vitally needed to address established and emerging mosquito-borne infectious diseases. RNA interference- (RNAi-) based pesticides are emerging as a promising new biorational mosquito control strategy. In this investigation, we describe characterization of an interfering RNA pesticide (IRP) corresponding to the mosquito Shaker (Sh) gene, which encodes an evolutionarily conserved voltage-gated potassium channel subunit. Delivery of the IRP to Aedes aegypti adult mosquitoes in the form of siRNA that was injected or provided as an attractive toxic sugar bait (ATSB) led to Sh gene silencing that resulted in severe neural and behavioral defects and high levels of adult mortality. Likewise, when provided to A. aegypti larvae in the form of short hairpin RNA (shRNA) expressed in Saccharomyces cerevisiae (baker's yeast) that had been formulated into a dried inactivated yeast tablet, the yeast IRP induced neural defects and larval death. Although the Sh IRP lacks a known target site in humans or other non-target organisms, conservation of the target site in the Sh genes of multiple mosquito species suggested that it may function as a biorational broad-range mosquito insecticide. In support of this, the Sh IRP induced both adult and larval mortality in treated Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus mosquitoes, but was not toxic to non-target arthropods. These studies indicated that IRPs targeting Sh could one day be used in integrated biorational mosquito control programs for the prevention of multiple mosquito-borne illnesses. The results of this investigation also suggest that the species-specificity of ATSB technology, a new paradigm for vector control, could be enhanced through the use of RNAi-based pesticides.

Characterization of an adulticidal and larvicidal interfering RNA pesticide that targets a conserved sequence in mosquito G protein-coupled dopamine 1 receptor genes.

G protein-coupled receptors (GPCRs), key regulators of a variety of critical biological processes, are attractive targets for insecticide development. Given the importance of these receptors in many organisms, including humans, it is critical that novel pesticides directed against GPCRs are designed to be species-specific. Here, we present characterization of an interfering RNA pesticide (IRP) targeting the mosquito GPCR-encoding dopamine 1 receptor (dop1) genes. A small interfering RNA corresponding to dop1 was identified in a screen for IRPs that kill Aedes aegypti during both the adult and larval stages. The 25 bp sequence targeted by this IRP is conserved in the dop1 genes of multiple mosquito species, but not in non-target organisms, indicating that it could function as a biorational mosquito insecticide. Aedes aegypti adults treated through microinjection or attractive toxic sugar bait delivery of small interfering RNA corresponding to the target site exhibited severe neural and behavioral defects and high levels of adult mortality. Likewise, A. aegypti larval consumption of dried inactivated yeast tablets prepared from a Saccharomyces cerevisiae strain engineered to express short hairpin RNA corresponding to the dop1 target site resulted in severe neural defects and larval mortality. Aedes albopictus and Anopheles gambiae adult and larval mortality was also observed following treatment with dop1 IRPs, which were not toxic to non-target arthropods. The results of this investigation indicate that dop1 IRPs can be used for species-specific targeting of dop1 GPCRs and may represent a new biorational strategy for control of both adult and larval mosquitoes.

Preparation and Use of a Yeast shRNA Delivery System for Gene Silencing in Mosquito Larvae.

The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the dengue and Zika virus vector Aedes aegypti and the primary African malaria vector Anopheles gambiae. RNA interference (RNAi) has facilitated gene silencing experiments in both of these disease vector mosquito species and could one day be applied as a new method of vector control. Here, we describe a procedure for the genetic engineering of Saccharomyces cerevisiae (baker's yeast) that express short hairpin RNA (shRNA) corresponding to mosquito target genes of interest. Following cultivation, which facilitates inexpensive propagation of shRNA, the yeast is inactivated and prepared in a ready-to-use dry tablet formulation that is fed to mosquito larvae. Ingestion of the yeast tablets results in effective larval target gene silencing. This technically straightforward and affordable technique may be applicable to a wide variety of mosquito species and potentially to other arthropods that feed on yeast.

Characterization of a yeast interfering RNA larvicide with a target site conserved in the synaptotagmin gene of multiple disease vector mosquitoes.

New mosquito control strategies are vitally needed to address established and emerging arthropod-borne infectious diseases. Here we describe the characterization of a yeast interfering RNA larvicide that was developed through the genetic engineering of Saccharomyces cerevisiae (baker's yeast) to express a short hairpin RNA targeting the Aedes aegypti synaptotagmin (Aae syt) gene. The larvicide effectively silences the Aae syt gene, causes defects at the larval neural synapse, and induces high rates of A. aegypti larval mortality in laboratory, simulated-field, and semi-field trials. Conservation of the interfering RNA target site in multiple mosquito species, but not in humans or other non-target species, suggested that it may function as a broad-range mosquito larvicide. In support of this, consumption of the yeast interfering RNA larvicide was also found to induce high rates of larval mortality in Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus mosquito larvae. The results of these studies suggest that this biorational yeast interfering RNA larvicide may represent a new intervention that can be used to combat multiple mosquito vectors of human diseases.

Characterization of a broad-based mosquito yeast interfering RNA larvicide with a conserved target site in mosquito semaphorin-1a genes.

BACKGROUND: RNA interference (RNAi), which has facilitated functional characterization of mosquito neural development genes such as the axon guidance regulator semaphorin-1a (sema1a), could one day be applied as a new means of vector control. Saccharomyces cerevisiae (baker's yeast) may represent an effective interfering RNA expression system that could be used directly for delivery of RNA pesticides to mosquito larvae. Here we describe characterization of a yeast larvicide developed through bioengineering of S. cerevisiae to express a short hairpin RNA (shRNA) targeting a conserved site in mosquito sema1a genes. RESULTS: Experiments conducted on Aedes aegypti larvae demonstrated that the yeast larvicide effectively silences sema1a expression, generates severe neural defects, and induces high levels of larval mortality in laboratory, simulated-field, and semi-field experiments. The larvicide was also found to induce high levels of Aedes albopictus, Anopheles gambiae and Culex quinquefasciatus mortality. CONCLUSIONS: The results of these studies indicate that use of yeast interfering RNA larvicides targeting mosquito sema1a genes may represent a new biorational tool for mosquito control.

Mosquito control practices and perceptions: An analysis of economic stakeholders during the Zika epidemic in Belize, Central America.

The tourist-based economy of Belize, a tropical hub for eco-tourism, is at high risk to be disproportionately impacted by established and emerging mosquito-borne diseases such as Zika. An online survey was used to probe economic stakeholders working in the Belize tourism industry about their mosquito control practices and perceptions. Responses demonstrated that the respondents have good working knowledge of mosquitoes and mosquito-borne illnesses. Most businesses surveyed engage in some means of mosquito control, either through larval source reduction or use of insecticides on the premises. Larvicide use was significantly correlated with a general willingness to use insecticides, as well as belief that treatment of water will reduce mosquito densities and disease transmission. A majority of the respondents agreed that they would be interested in buying a new larvicide to be used on the business premises if it were shown to be safe and effective. The safety of mosquito control products for humans, animals, plants, and the environment in general, followed by product effectiveness, are the most critical determinants of mosquito control purchasing decisions. A majority of respondents agreed that control of mosquitoes and mosquito-borne illnesses is central to the success of their tourist-based industry. Respondents expressed significant concern that the Zika epidemic was over-sensationalized by the media, and that this negatively impacted their livelihoods. The respondents, many of whom are associated with eco/sustainable businesses, also voiced concerns that chemical pesticides could have a negative impact on human health and the environment and expressed a desire for balance between effective mosquito control and preservation of the rich biodiversity of Belize. This study provided a framework for further engagement activities in Belize and other Caribbean nations, uncovered both concerns and support for emerging mosquito control technologies, and revealed opportunities for further debate and educational outreach efforts.

Lure-and-Kill Yeast Interfering RNA Larvicides Targeting Neural Genes in the Human Disease Vector Mosquito Aedes aegypti.

New mosquito control strategies are vitally needed to address established arthropod-borne infectious diseases such as dengue and yellow fever and emerging diseases such as Zika and chikungunya, all of which are transmitted by the disease vector mosquito Aedes aegypti. In this investigation, Saccharomyces cerevisiae (baker's yeast) was engineered to produce short hairpin RNAs (shRNAs) corresponding to the Aedes aegypti orthologs of fasciculation and elongation protein zeta 2 (fez2) and leukocyte receptor cluster (lrc) member, two genes identified in a recent screen for A. aegypti larval lethal genes. Feeding A. aegypti with the engineered yeasts resulted in silenced target gene expression, disrupted neural development, and highly significant larval mortality. Larvicidal activities were retained following heat inactivation and drying of the yeast into tabular formulations that induced >95% mortality and were found to attract adult females to oviposit. These ready-to-use inactivated yeast interfering RNA tablets may one day facilitate the seamless integration of this new class of lure-and-kill species-specific biorational mosquito larvicides into integrated mosquito control programs.