**4. Conclusions**

Although mosquito control is a central and crucial component of mosquito-borne disease prevention strategies, insecticide resistance threatens current and future gains in the war against disease vector mosquitoes, and the identification and characterization of new active ingredients and products for mosquito control is critical [1]. The results of this investigation provide further support for the hypothesis that *Irx*.447 kills multiple species of mosquitoes at different life stages (Figures 1–3) ye<sup>t</sup> poses little threat to nontarget species (Table 2). These data, combined with other recent studies [5], sugges<sup>t</sup> that RNAibased yeas<sup>t</sup> pesticides should be further developed as a novel class of insecticides for mosquito control. Characterization of *Irx*.447 yeas<sup>t</sup> demonstrated that it functions as a dual adulticidal and larvicidal IRP with activity in *Aedes, Anopheles,* and *Culex* mosquitoes (Figures 1–3), which possess a conserved *Irx* target site for this insecticide. A loss of *pdm2* transcript expression in the mosquito nervous system, which correlated with silencing of *Irx* (Figure 2D,E, Supplementary Figures S1 and S2), suggests that mortality associated with this insecticide results from disruption of proneural gene expression. Use of *Irx*.447 could facilitate the managemen<sup>t</sup> of insecticide resistance through the addition of an insecticide with a mode of action that differs from that of existing pesticides [1].

The elimination of mosquito-borne diseases will likely require the implementation of new vector control interventions that will complement existing control measures. Thus, in addition to new insecticide classes, new paradigms will be important additions to integrated resistance managemen<sup>t</sup> strategies [1]. To this end, the present investigation provided evidence that *Irx*.447 yeas<sup>t</sup> can be successfully delivered to adult mosquitoes as an ATSB (Figures 2 and 3), a sugar-baited trap, and a new paradigm for vector control [1]. These findings sugges<sup>t</sup> that further development of yeas<sup>t</sup> interfering RNA pesticides, the production of which is likely to be both affordable and scalable [4], should be pursued for use in ATSBs. Confirmation of *Irx*.447 yeas<sup>t</sup> ATSB activity in simulated field trials performed using bait stations in cages (Figures 2 and 3), as well as the analysis of *Irx*.447 yeas<sup>t</sup> activity in outdoor semi-field larvicide trials (Figure 1C), indicates that these new RNAi-based technologies could potentially be useful in the field, a prospect that will be evaluated in future large-scale field trials which will be accompanied by stakeholder engagemen<sup>t</sup> activities and educational campaigns. Such trials will require scaled yeas<sup>t</sup> production in larger fermentation-sized cultures, suggesting that the production of commercial strains that withstand fermentation, as well as the piloting and optimization of scaled yeas<sup>t</sup> IRP production, would be advantageous [4].

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2075-4 450/12/11/986/s1: Table S1. The *Irx*.447 target site conserved in mosquitoes was not found in non-mosquito genomes [47]; Table S2. Mosquito ATSB feeding rates observed in laboratory simulated field trials; Figure S1. *Irx*.447 yeas<sup>t</sup> ATSB induces target gene silencing and reduces *pdm2* expression in *A. aegypti;* Figure S2. *Irx*.447 yeas<sup>t</sup> ATSB induces silencing of the *Irx* target gene and significantly reduces *pdm2* expression in *A. gambiae.*

**Author Contributions:** Conceptualization, M.D.-S.; methodology, J.B.R., K.M., L.K.H., M.D.-S. and N.W.; validation, C.-W.W., J.I., K.M., L.K.H., L.S., M.P.S. and N.D.S.; formal analysis, K.M. and M.D.-S.; investigation, C.-W.W., J.B.R., J.I., L.K.H., L.S., M.P.S., N.D.S. and P.L.; formal analysis, K.M. and M.D.-S.; data curation, K.M. and M.D.-S.; writing—original draft preparation, K.M. and M.D.-S.; writing—review and editing, D.W.S., K.M. and M.D.-S.; supervision, D.W.S., K.M., M.D.-S. and N.W.; project administration, D.W.S., M.D.-S. and N.W.; funding acquisition, M.D.-S., D.W.S. and N.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** Support for this project was provided through the following funding sources: an Indiana University Showalter Scholar award (larvicide screen to M.D.-S.), NIH/NIAID Award 1 R21 AI128116- 01 (yeast strain generation and larvicide assessments to M.D.-S., N.W. and D.W.S.), the Department of the Army, US Army Contracting Command, Aberdeen Proving Ground, Natick Contracting Division, Fort Detrick, MD, USA under Deployed Warfighter Protection (DWFP) Program Grant W911QY-17-1- 0002 (*Aedes aegypti* adulticide to M.D.-S.), DoD Awards W81XWH-17-1-0294 and W81XWH-17-1-0295

(semi-field larvicidal ovitrap trials to M.D.-S. and D.W.S., respectively), W81XWH2120038 (*Culex* adulticides to M.D.-S.), and an Innovative Vector Control Consortium (IVCC) Award (*Anopheles* adulticide to M.D.-S.). These funders played no roles in study design, data collection and analysis, the decision to publish, or preparation of this manuscript.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data are provided within the text and supplementary materials of this manuscript.

**Acknowledgments:** We thank Jacob Realey and Joi Misenti for their technical assistance, Scott Emrich for help with the identification of target site sequences, and IVCC for useful discussions.

**Conflicts of Interest:** M.D.-S., N.W., and D.W.S. are inventors on US Patent Application No.: 62/361,704, European Application No. 17828458.4, but this application did not affect their data interpretation and does not influence their adherence to journal policies on sharing data and materials. All other authors declare no conflict of interest.
