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Pharmacological and Genetic Evidence for Gap Junctions as Potential New Insecticide Targets in the Yellow Fever Mosquito, Aedes aegypti.

Calkins TL, Piermarini PM - PLoS ONE (2015)

Bottom Line: We show that the injection of pharmacological inhibitors of gap junctions (i.e., carbenoxolone, meclofenamic acid, or mefloquine) into the hemolymph of adult female mosquitoes elicits dose-dependent toxic effects, with mefloquine showing the greatest potency.When added to the rearing water of 1st instar larvae, carbenoxolone and meclofenamic acid both elicit dose-dependent toxic effects, with meclofenamic acid showing the greatest potency.Injecting a double-stranded RNA cocktail against innexins into the hemolymph of adult female mosquitoes knock down whole-animal innexin mRNA expression and decreases survival of the mosquitoes.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, United States of America.

ABSTRACT
The yellow fever mosquito Aedes aegypti is an important vector of viral diseases that impact global health. Insecticides are typically used to manage mosquito populations, but the evolution of insecticide resistance is limiting their effectiveness. Thus, identifying new molecular and physiological targets in mosquitoes is needed to facilitate insecticide discovery and development. Here we test the hypothesis that gap junctions are valid molecular and physiological targets for new insecticides. Gap junctions are intercellular channels that mediate direct communication between neighboring cells and consist of evolutionarily distinct proteins in vertebrate (connexins) and invertebrate (innexins) animals. We show that the injection of pharmacological inhibitors of gap junctions (i.e., carbenoxolone, meclofenamic acid, or mefloquine) into the hemolymph of adult female mosquitoes elicits dose-dependent toxic effects, with mefloquine showing the greatest potency. In contrast, when applied topically to the cuticle, carbenoxolone was the only inhibitor to exhibit full efficacy. In vivo urine excretion assays demonstrate that both carbenoxolone and mefloquine inhibit the diuretic output of adult female mosquitoes, suggesting inhibition of excretory functions as part of their mechanism of action. When added to the rearing water of 1st instar larvae, carbenoxolone and meclofenamic acid both elicit dose-dependent toxic effects, with meclofenamic acid showing the greatest potency. Injecting a double-stranded RNA cocktail against innexins into the hemolymph of adult female mosquitoes knock down whole-animal innexin mRNA expression and decreases survival of the mosquitoes. Taken together these data indicate that gap junctions may provide novel molecular and physiological targets for the development of insecticides.

No MeSH data available.


Related in: MedlinePlus

Effect of eGFP (black squares) and innexin (red circles) dsRNA injection on mosquito survival.Values are means ± SEM. n = 3 replicates of 24 mosquitoes. Asterisks indicate a significant difference in survival between eGFP and innexin dsRNA injected mosquitoes as determined by a two-way ANOVA with a Holm-Sidak’s post-hoc test.
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pone.0137084.g007: Effect of eGFP (black squares) and innexin (red circles) dsRNA injection on mosquito survival.Values are means ± SEM. n = 3 replicates of 24 mosquitoes. Asterisks indicate a significant difference in survival between eGFP and innexin dsRNA injected mosquitoes as determined by a two-way ANOVA with a Holm-Sidak’s post-hoc test.

Mentions: Injection of an innexin dsRNA cocktail containing dsRNAs for each innexin (333 ng per innexin, 2 μg total) resulted in significant knockdown of Inx1 (32 ± 8%), Inx2 (69 ± 3%), Inx3 (51 ± 5%), Inx4 (71 ± 10%) and Inx7 (86 ± 2%) by 3 days after injection compared to expression levels in the eGFP-injected controls (Fig 6). The expression levels of Inx8 were not significantly knocked down, but the mRNA levels were very low to begin (Fig 5). The knockdown of innexin expression in mosquitoes injected with innexin dsRNA persisted until at least day 11 (data not shown). In addition, mosquitoes injected with innexin dsRNA exhibited a significantly lower survival than those injected with eGFP dsRNA that progressed over the next 11 days, and started as early as 1 day after injection (Fig 7).


Pharmacological and Genetic Evidence for Gap Junctions as Potential New Insecticide Targets in the Yellow Fever Mosquito, Aedes aegypti.

Calkins TL, Piermarini PM - PLoS ONE (2015)

Effect of eGFP (black squares) and innexin (red circles) dsRNA injection on mosquito survival.Values are means ± SEM. n = 3 replicates of 24 mosquitoes. Asterisks indicate a significant difference in survival between eGFP and innexin dsRNA injected mosquitoes as determined by a two-way ANOVA with a Holm-Sidak’s post-hoc test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556631&req=5

pone.0137084.g007: Effect of eGFP (black squares) and innexin (red circles) dsRNA injection on mosquito survival.Values are means ± SEM. n = 3 replicates of 24 mosquitoes. Asterisks indicate a significant difference in survival between eGFP and innexin dsRNA injected mosquitoes as determined by a two-way ANOVA with a Holm-Sidak’s post-hoc test.
Mentions: Injection of an innexin dsRNA cocktail containing dsRNAs for each innexin (333 ng per innexin, 2 μg total) resulted in significant knockdown of Inx1 (32 ± 8%), Inx2 (69 ± 3%), Inx3 (51 ± 5%), Inx4 (71 ± 10%) and Inx7 (86 ± 2%) by 3 days after injection compared to expression levels in the eGFP-injected controls (Fig 6). The expression levels of Inx8 were not significantly knocked down, but the mRNA levels were very low to begin (Fig 5). The knockdown of innexin expression in mosquitoes injected with innexin dsRNA persisted until at least day 11 (data not shown). In addition, mosquitoes injected with innexin dsRNA exhibited a significantly lower survival than those injected with eGFP dsRNA that progressed over the next 11 days, and started as early as 1 day after injection (Fig 7).

Bottom Line: We show that the injection of pharmacological inhibitors of gap junctions (i.e., carbenoxolone, meclofenamic acid, or mefloquine) into the hemolymph of adult female mosquitoes elicits dose-dependent toxic effects, with mefloquine showing the greatest potency.When added to the rearing water of 1st instar larvae, carbenoxolone and meclofenamic acid both elicit dose-dependent toxic effects, with meclofenamic acid showing the greatest potency.Injecting a double-stranded RNA cocktail against innexins into the hemolymph of adult female mosquitoes knock down whole-animal innexin mRNA expression and decreases survival of the mosquitoes.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, United States of America.

ABSTRACT
The yellow fever mosquito Aedes aegypti is an important vector of viral diseases that impact global health. Insecticides are typically used to manage mosquito populations, but the evolution of insecticide resistance is limiting their effectiveness. Thus, identifying new molecular and physiological targets in mosquitoes is needed to facilitate insecticide discovery and development. Here we test the hypothesis that gap junctions are valid molecular and physiological targets for new insecticides. Gap junctions are intercellular channels that mediate direct communication between neighboring cells and consist of evolutionarily distinct proteins in vertebrate (connexins) and invertebrate (innexins) animals. We show that the injection of pharmacological inhibitors of gap junctions (i.e., carbenoxolone, meclofenamic acid, or mefloquine) into the hemolymph of adult female mosquitoes elicits dose-dependent toxic effects, with mefloquine showing the greatest potency. In contrast, when applied topically to the cuticle, carbenoxolone was the only inhibitor to exhibit full efficacy. In vivo urine excretion assays demonstrate that both carbenoxolone and mefloquine inhibit the diuretic output of adult female mosquitoes, suggesting inhibition of excretory functions as part of their mechanism of action. When added to the rearing water of 1st instar larvae, carbenoxolone and meclofenamic acid both elicit dose-dependent toxic effects, with meclofenamic acid showing the greatest potency. Injecting a double-stranded RNA cocktail against innexins into the hemolymph of adult female mosquitoes knock down whole-animal innexin mRNA expression and decreases survival of the mosquitoes. Taken together these data indicate that gap junctions may provide novel molecular and physiological targets for the development of insecticides.

No MeSH data available.


Related in: MedlinePlus