Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

Navy Entomology Center of Excellence

Lovelace Respiratory Research Institute

Puerto Rico (PR) has a long history of vector-borne disease and insecticide-resistant Aedes aegypti (L.). Defining contributing mechanisms behind phenotypic resistance is critical for effective vector control intervention. However, previous studies from PR have each focused on only one mechanism of pyrethroid resistance. This study examines the contribution of P450-mediated enzymatic detoxification and sodium channel target site changes to the overall resistance phenotype of Ae. aegypti collected from San Juan, PR, in 2012. Screening of a panel of toxicants found broad resistance relative to the lab susceptible Orlando (ORL1952) strain. We identified significant resistance to representative Type I, Type II, and nonester pyrethroids, a sodium channel blocker, and a sodium channel blocking inhibitor, all of which interact with the sodium channel. Testing of fipronil, a chloride channel agonist, also showed low but significant levels of resistance. In contrast, the PR and ORL1952 strains were equally susceptible to chlorfenapyr, which has been suggested as an alternative public health insecticide. Molecular characterization of the strain indicated that two common sodium channel mutations were fixed in the population. Topical bioassay with piperonyl butoxide (PBO) indicated cytochrome P450-mediated detoxification accounts for approximately half of the resistance profile. Transcript expression screening of cytochrome P450s and glutathione-S-transferases identified the presence of overexpressed transcripts. This study of Puerto Rican Ae. aegypti with significant contributions from both genetic changes and enzymatic detoxification highlights the necessity of monitoring for resistance but also defining the multiple resistance mechanisms to inform effective mosquito control.

Resistance Status and Resistance Mechanisms in a Strain of Aedes aegypti (Diptera: Culicidae) From Puerto Rico.

House flies (Musca domestica) are worldwide agricultural pests with estimated control costs at $375 million annually in the U.S. Non-target effects and widespread resistance challenge the efficacy of traditional chemical control. Double stranded RNA (dsRNA) has been suggested as a biopesticide for M. domestica but a phenotypic response due to the induction of the RNAi pathway has not been demonstrated in adults. In this study female house flies were injected with dsRNA targeting actin-5C or ribosomal protein (RP) transcripts RPL26 and RPS6. Ovaries showed highly reduced provisioning and clutch reductions of 94-99% in RP dsRNA treated flies but not in actin-5C or GFP treated flies. Gene expression levels were significantly and specifically reduced in dsRNA injected groups but remained unchanged in the control dsGFP treated group. Furthermore, injections with an Aedes aegypti conspecific dsRNA designed against RPS6 did not impact fecundity, demonstrating species specificity of the RNAi response. Analysis of M. domestica tissues following RPS6 dsRNA injection showed significant reduction of transcript levels in the head, thorax, and abdomen but increased expression in ovarian tissues. This study demonstrates that exogenous dsRNA is specifically effective and has potential efficacy as a highly specific biocontrol intervention in adult house flies. Further work is required to develop effective methods for delivery of dsRNA to adult flies.

Reduction in Musca domestica fecundity by dsRNA-mediated gene knockdown.

In this semi-field study, a new polymer-enhanced deltamethrin formulation, K-Othrine PolyZone, was compared to a standard deltamethrin product for residual activity against a susceptible strain of laboratory-reared Anopheles gambiae using standard WHO cone bioassays.

Evaluation of the long-term efficacy of K-Othrine PolyZone on three surfaces against laboratory reared Anopheles gambiae in semi-field conditions.

Recent outbreaks of locally transmitted dengue and Zika viruses in Florida have placed more emphasis on integrated vector management plans for Aedes aegypti (L.) and Aedes albopictus Skuse. Adulticiding, primarily with pyrethroids, is often employed for the immediate control of potentially arbovirus-infected mosquitoes during outbreak situations. While pyrethroid resistance is common in Ae. aegypti worldwide and testing is recommended by CDC and WHO, resistance to this class of products has not been widely examined or quantified in Florida. To address this information gap, we performed the first study to quantify both pyrethroid resistance and genetic markers of pyrethroid resistance in Ae. aegypti and Ae. albopictus strains in Florida. Using direct topical application to measure intrinsic toxicity, we examined 21 Ae. aegypti strains from 9 counties and found permethrin resistance (resistance ratio (RR) = 6-61-fold) in all strains when compared to the susceptible ORL1952 control strain. Permethrin resistance in five strains of Ae. albopictus was very low (RR<1.6) even when collected from the same containers producing resistant Ae. aegypti. Characterization of two sodium channel kdr alleles associated with pyrethroid-resistance showed widespread distribution in 62 strains of Ae. aegypti. The 1534 phenylalanine to cysteine (F1534C) single nucleotide polymorphism SNP was fixed or nearly fixed in all strains regardless of RR. We observed much more variation in the 1016 valine to isoleucine (V1016I) allele and observed that an increasing frequency of the homozygous V1016I allele correlates strongly with increased RR (Pearson corr = 0.905). In agreement with previous studies, we observed a very low frequency of three kdr genotypes, IIFF, VIFF, and IIFC. In this study, we provide a statewide examination of pyrethroid resistance, and demonstrate that permethrin resistance and the genetic markers for resistance are widely present in FL Ae. aegypti. Resistance testing should be included in an effective management program.

Christy M. Waits

CMAVE Detachment

Navy Entomology Center of Excellence

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Christy M. Waits

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Navy Entomology Center of Excellence

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

CMAVE Detachment