Reduction in \u3ci\u3eMusca domestica\u3c/i\u3e fecundity by dsRNA-mediated gene knockdown

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 \u3ci\u3eMusca domestica\u3c/i\u3e fecundity by dsRNA-mediated gene knockdown

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

Geographic Partitioning of Dengue Virus Transmission Risk in Florida

Dengue viruses (DENVs) cause the greatest public health burden globally among the arthropod-borne viruses. DENV transmission risk has also expanded from tropical to subtropical regions due to the increasing range of its principal mosquito vector, Aedes aegypti. Focal outbreaks of dengue fever (dengue) in the state of Florida (FL) in the USA have increased since 2009. However, little is known about the competence of Ae. aegypti populations across different regions of FL to transmit DENVs. To understand the effects of DENV genotype and serotype variations on vector susceptibility and transmission potential in FL, we orally infected a colony of Ae. aegypti (Orlando/ORL) with low passage or laboratory DENV-1 through -4. Low passage DENVs were more infectious to and had higher transmission potential by ORL mosquitoes. We used these same DENVs to examine natural Ae. aegypti populations to determine whether spatial distributions correlated with differential vector competence. Vector competence across all DENV serotypes was greater for mosquitoes from areas with the highest dengue incidence in south FL compared to north FL. Vector competence for low passage DENVs was significantly higher, revealing that transmission risk is influenced by virus/vector combinations. These data support a targeted mosquito-plus-pathogen screening approach to more accurately estimate DENV transmission risk

Geographic Partitioning of Dengue Virus Transmission Risk in Florida

Dengue viruses (DENVs) cause the greatest public health burden globally among the arthropod-borne viruses. DENV transmission risk has also expanded from tropical to subtropical regions due to the increasing range of its principal mosquito vector, Aedes aegypti. Focal outbreaks of dengue fever (dengue) in the state of Florida (FL) in the USA have increased since 2009. However, little is known about the competence of Ae. aegypti populations across different regions of FL to transmit DENVs. To understand the effects of DENV genotype and serotype variations on vector susceptibility and transmission potential in FL, we orally infected a colony of Ae. aegypti (Orlando/ORL) with low passage or laboratory DENV-1 through -4. Low passage DENVs were more infectious to and had higher transmission potential by ORL mosquitoes. We used these same DENVs to examine natural Ae. aegypti populations to determine whether spatial distributions correlated with differential vector competence. Vector competence across all DENV serotypes was greater for mosquitoes from areas with the highest dengue incidence in south FL compared to north FL. Vector competence for low passage DENVs was significantly higher, revealing that transmission risk is influenced by virus/vector combinations. These data support a targeted mosquito-plus-pathogen screening approach to more accurately estimate DENV transmission risk

Quantification of permethrin resistance and kdr alleles in Florida strains of Aedes aegypti (L.) and Aedes albopictus (Skuse).

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

Average relative expression (RE) of RPS6 in tissues from female <i>M</i>. <i>domestica</i> injected with 5 μg of dsGFP or dsRPS6.

<p>All values are normalized to RE levels from flies injected with dsGFP. Significant reduction (P<0.05) in RPS6 expression compared to the dsGFP control group was observed in the head, thorax, and abdomen for the dsRPS6 treatment. A significant increase in RPS6 expression was observed in ovarian tissues from dsRPS6 treatments when compared to dsGFP injected samples. Error bars represent mean±SE and * represents significance at P<0.05. Tissues samples were pooled from 5 individual flies and injections were replicated 3 times.</p

Average relative expression (RE) of three transcripts in individual whole female <i>M</i>. <i>domestica</i> injected with 5 μg of dsGFP, dsActin-5C, dsRPL26 and dsRPS6.

<p>Individual expression levels were all normalized to a single dsGFP sample for each replicate. RE for actin-5C, RPL26 and RPS6 were all significantly lower in their respective dsRNA treatment groups when compared to RE in dsGFP treated house flies. Non-target transcripts remained at normal expression levels in all treatment groups indicating a clear RNAi response to exogenous dsRNA. Data represents n = 15 flies total from 3 replicate experiments except dsRPS6 (n = 14).</p

Reduction in <i>Musca domestica</i> fecundity by dsRNA-mediated gene knockdown - Fig 3

<p><b>Ovarian development of untreated <i>M</i>. <i>domestica</i> (a) as well as individuals injected with a non-target control (dsGFP—b) or dsRNAs targeting house fly actin-5C (c), RPL26 (d), and RPS6 (e).</b> Dissections were performed after feeding on a protein source for 4 days (6 days post-injection). Untreated, dsGFP and dsActin-5C treated ovaries are all fully gravid and show eggs with high levels of yolk deposition. Ovaries from females injected with dsRPL26 and dsRPS6 show poorly developed eggs which resulted in significantly reduced fecundity. Micron bars are ~1 mm.</p

Reduction in <i>Musca domestica</i> fecundity by dsRNA-mediated gene knockdown

<div><p>House flies (<i>Musca domestica</i>) 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 <i>M</i>. <i>domestica</i> 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 <i>Aedes aegypti</i> conspecific dsRNA designed against RPS6 did not impact fecundity, demonstrating species specificity of the RNAi response. Analysis of <i>M</i>. <i>domestica</i> 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.</p></div