Suppression of Argonaute 2 Transcript Levels in Du182A Cells

RNA interference (RNAi) uses double-stranded RNA (dsRNA) molecules to degrade and suppress the transcript level of a complementary mRNA target1. The RNAi pathway is complex and includes many different proteins, like argonautes, in the core machinery. Argonautes are dsRNA binding proteins which help recognize and cleave target mRNA molecules. In our experiments, we attempted to suppress the transcript level of argonaute 2 (Ago2) in a Diabrotica undecimpunctata cell line (Du182A) using dsRNA, with the idea of disrupting the RNAi pathway using an RNAi of RNAi technique. Ago2 transcript levels were suppressed following treatment with dsRNA. Future experiments can now use this technique, with some modification to better understand the RNAi pathway

Aspirin-Induced Mortality in AedesaegyptiMosquitoes

The Aedes aegypti mosquito is a vector for many dangerous diseases including yellow fever, Zika, Chikungunya, and Dengue viruses! Currently, the best method for suppressing infection with these viruses is by controlling their arthropod vectors, however the continual use of conventional insecticides to control Ae. aegypti mosquitoes has led to the development of significant levels of resistance to these chemicals. Accordingly, new methods are needed. Anecdotal evidence has suggested that mosquitoes may be sensitive to treatment with aspirin or its metabolites in a blood meal. Previous studies with aspirin in insects have mainly focused on the ability of aspirin to enhance longevity and lifespan in Drosophila melanogaster and crickets2.4, but one study showed that aspirin in water where larvae are developing causes significant mortality and signs of neurotoxicity. While treating larvae in this manner is not practical for vector control, this result, in addition to the anecdotal evidence, prompted the question of whether aspirin consumed in a blood meal can cause mortality in adult mosquitoes. Accordingly, we fed adult female Ae. aegypti mosquitoes differing concentrations of aspirin in human blood and measured mortality for four days following the blood meal. The concentrations chosen correspond to plasma concentrations of aspirin in humans who have consumed 100 mg (a baby aspirin), 1 g (normal adult dose), or 10 g (overdose), respectively. Our results indicate that these concentrations of aspirin are insufficient to cause significant mortality in the mosquitos. Although unsuccessful, modification of our methods, use of a different mosquito species, or use of aspirin metabolites in our bioassays may yet show a significant impact of aspirin consumption on mosquito survival

Effects Of Acclimation Position On Detachment Of Tethered Culex Quinquefasciatus

Electropenetrography (EPG) allows researchers to observe, record,and quantify the feeding behaviors of arthropods. [2] EPG has beentypically used to study the relationship between plants and plant-eating arthropods. EPG involves wiring insects into electrical circuits to visualizeelectrical signals associated with each insect feeding behavior. (A) By using EPG, researchers can understand how various factors affectarthropod feeding patterns. [1] Culex quinquefasciatus (Cx. quinqs.) are known vectors of virusessuch as Rift Valley fever and Japenese encephalitis, which cansignificantly affect veterinary and human health. To safely study how viruses affect mosquito feeding behavior,detachment from the EPG wire must be zero. Since July 2021, thedetachment rate in our studies has been 0.47%. Different wiring protocols have the mosquitoes either hanging orstanding following the attachment of EPG wires, but it is not clear ifusing one method or the other results in more insect detachments

Evaluation of Several Genes as Targets for RNAi in German Cockroach

Citation: Banister, H. (2017). Evaluation of Several Genes as Targets for RNAi in German Cockroach . 1st Annual Undergraduate Research Experience in Entomology Symposium, November 16, 2016. Manhattam, KS.RNA interference (RNAi) was first discovered in nematodes when exogenous double-stranded RNA (dsRNA) complementary to a specific gene suppressed expression of that gene [1]. Subsequently, much effort has been devoted to developing RNAi as a highly specific tool for therapeutic interventions and control of insect pests [2]. However, there are still many challenges associated with using RNAi to control insects, including efficient delivery and selection of appropriate targets. In this study, we evaluated three genes as potential targets for causing mortality via RNAi in German cockroach, Blatella germanica. German cockroaches are ubiquitous structural pests that can serve as reservoirs for species of pathogenic bacteria, viruses, or fungi in humans [3,4] and is an excellent organism for exploring insect control using RNAi. Injection of dsRNA complementary to either tubulin (Tub, a cytoskeletal structural protein), VATPase subunit 1 (Vha, an integral membrane protein), or Snf7 (an ESCRT III protein) caused decreased survival with Snf7 causing the greatest and fastest mortality (LT50 = 8.2 days). Our results demonstrate that when suppressed with RNAi, these genes could be effective targets for cockroach control. Furthermore, knowing these genes can be effectively used for RNAi, we can now attempt to understand why methods other than injection for RNAi delivery are less efficient in an effort to improve the utility of RNAi in insect control

Evaluation of Several Genes as Targets for RNAi in German Cockroach

Citation: Wilkins, R. (2017). Evaluation of Several Genes as Targets for RNAi in German Cockroach . 1st Annual Undergraduate Research Experience in Entomology Symposium, November 16, 2016. Manhattam, KS.RNA interference (RNAi) was first discovered in nematodes when exogenous double-stranded RNA (dsRNA) complementary to a specific gene suppressed expression of that gene [1]. Subsequently, much effort has been devoted to developing RNAi as a highly specific tool for therapeutic interventions and control of insect pests [2]. However, there are still many challenges associated with using RNAi to control insects, including efficient delivery and selection of appropriate targets. In this study, we evaluated three genes as potential targets for causing mortality via RNAi in German cockroach, Blatella germanica. German cockroaches are ubiquitous structural pests that can serve as reservoirs for species of pathogenic bacteria, viruses, or fungi in humans [3,4] and is an excellent organism for exploring insect control using RNAi. Injection of dsRNA complementary to either tubulin (Tub, a cytoskeletal structural protein), VATPase subunit 1 (Vha, an integral membrane protein), or Snf7 (an ESCRT III protein) caused decreased survival with Snf7 causing the greatest and fastest mortality (LT50 = 8.2 days). Our results demonstrate that when suppressed with RNAi, these genes could be effective targets for cockroach control. Furthermore, knowing these genes can be effectively used for RNAi, we can now attempt to understand why methods other than injection for RNAi delivery are less efficient in an effort to improve the utility of RNAi in insect control

Effects of Nanoparticles on Double-Stranded RNA Stability in Moth Hemolymph

RNA interference (RNAi) is an immune response in which double-stranded RNA (dsRNA) suppresses a target gene. By designing dsRNA to target genes that are necessary for life, dsRNA can potentially be used as an insecticide. RNAi-based insecticides are badly needed because they are more specific than conventional pesticides and because many insects have developed resistance to pesticides. Unfortunately, some insects produce enzymes that degrade dsRNA and prevent the RNAi response (Cooperet al., 2018). Therefore, RNAi-based insecticides currently cannot be used to control all insects. Here we investigate dsRNA stability when incubated in hemolymph ex vivoto determine if degradation of dsRNA is contributing to the inadequate RNAi response exhibited by lepidopterans, such as the European corn borer (ECB, Ostrinia nubilalis). Our findings indicate that dsRNA is significantly degraded in ECB hemolymph, but encapsulation of dsRNA in chitosan-based nanoparticles (CB-NPs) enhances stability. These findings provide insight into RNAi efficiency limitations in insects, and may provide a method to enhance RNAi efficiency in lepidopterans and other RNAi-refractory pests

Effects of Nanoparticles on Double-Stranded RNA Stability in Corn Soil

Double-stranded RNA (dsRNA) can potentially be used as a pesticide because these molecules trigger an immune response called RNA interference (RNAi). If the expression of essential genes matching the dsRNA sequence are silenced, then the pest dies. New classes of pesticides, including RNAi-based pesticides, are needed to overcome pesticide resistance and reduce the environmental impacts of pesticides. Unfortunately, dsRNA is easily degraded by enzymes in the environment, particularly those produced by microbes in the soil (Dubelmanet al., 2014),severely limiting delivery of dsRNA to cryptic (soil dwelling) species unless transgenic plants are used. Here we investigate dsRNA stability when incubated in corn soil supernatant ex situ to determine if encapsulation of dsRNA in chitosan-basednanoparticles (CB-NPs) enhances stability in corn soil. Interestingly, dsRNA stability was not affected by soil supernatant, possibly due to the time of year when sampling was performed (Icozet al., 2008). Nonetheless, these findings provide insight into dsRNA stability in soil, and in the future may lead to a method for protecting dsRNA from environmental degradation using CB-NPs

Effects of Nanoparticles on Double-Stranded RNA Stability in Corn Soil

Double-stranded RNA (dsRNA) can potentially be used as a pesticide because these molecules trigger an immune response called RNA interference (RNAi). If the expression of essential genes matching the dsRNA sequence are silenced, then the pest dies. New classes of pesticides, including RNAi-based pesticides, are needed to overcome pesticide resistance and reduce the environmental impacts of pesticides. Unfortunately, dsRNA is easily degraded by enzymes in the environment, particularly those produced by microbes in the soil (Dubelmanet al., 2014),severely limiting delivery of dsRNA to cryptic (soil dwelling) species unless transgenic plants are used. Here we investigate dsRNA stability when incubated in corn soil supernatant ex situ to determine if encapsulation of dsRNA in chitosan-basednanoparticles (CB-NPs) enhances stability in corn soil. Interestingly, dsRNA stability was not affected by soil supernatant, possibly due to the time of year when sampling was performed (Icozet al., 2008). Nonetheless, these findings provide insight into dsRNA stability in soil, and in the future may lead to a method for protecting dsRNA from environmental degradation using CB-NPs

Effects of EDTA feeding on survival and development of European corn borer larvae

RNA interference (RNAi) is a process that uses double-stranded RNA (dsRNA) to target and suppress the expression of specific genes (Zhang et al., 2010). It is a powerful tool that has been used to understand the function of many genes and has great potential to control insect pests. However, differences in sensitivity to RNAi among insects have limited the use of RNAi (Kim et al., 2015). In some insects, such as European corn borer (ECB), RNAi efficiency is limited by degradation of dsRNA in insect gut. Strategies to overcome this limitation are necessary. Some studies have suggested that high temperatures or treatment with EDTA can inhibit nuclease activity (Garbutt et al., 2014), however the effects of treatment of insects with EDTA are unknown. The objective of this study was to determine if feeding different concentrations of EDTA to ECB larvae will have any effect on larval survival, weight, or development. Our results showed that neither 6 nor 10 mM EDTA had any significant effect on larval survival or development from 2nd instar to 3rd instar larvae. In contrast, average larval weight was suppressed by treatment with 10 mM EDTA. These data show that larvae can be safely treated with up to 6 mM EDTA without adverse effects on development or larval survival, suggesting that 6 mM EDTA can be used in future experiments to test the ability of EDTA to inhibit nuclease activity in ECB gut and possibly improve RNAi efficiency

The Tribolium castaneum cell line TcA: a new tool kit for cell biology

The red flour beetle, Tribolium castaneum, is an agriculturally important insect pest that has been widely used as a model organism. Recently, an adherent cell line (BCIRL-TcA-CLG1 or TcA) was developed from late pupae of the red flour beetle. Next generation transcriptome sequencing of TcA cells demonstrated expression of a wide variety of genes associated with specialized functions in chitin metabolism, immune responses and cellular and systemic RNAi pathways. Accordingly, we evaluated the sensitivity of TcA cells to dsRNA to initiate an RNAi response. TcA cells were highly sensitive to minute amounts of dsRNA, with a minimum effective dose of 100 pg/mL resulting in significant suppression of gene expression. We have also developed a plasmid containing two TcA-specific promoters, the promoter from the 40S ribosomal protein subunit (TC006550) and a bi-directional heat shock promoter (TcHS70) from the intergenic space between heat shock proteins 68a and b. These promoters have been employed to provide high levels of either constitutive (TC006550) or inducible (TcHS70) gene expression of the reporter proteins. Our results show that the TcA cell line, with its sensitivity to RNAi and functional TcA-specific promoters, is an invaluable resource for studying basic molecular and physiological questions