The neonate larvae were also fed different concentrations of dsIAP and the higher concentration showed a higher mortality compared to mortality observed at the lower concentrations Fig.
Previous work in Bactrocera dorsalis Diptera: Tephritidae showed that the concentration of dsRNA used influenced the level of gene suppression Although we analyzed the EAB phenotype effect instead of the gene expression level after feeding different concentrations of dsRNAs, a possible explanation for the increase in mortality observed may be due to higher suppression of the IAP gene at higher concentrations of dsRNA. Also, oral delivery of dsRNA has some limitations, such as natural barriers found in saliva and the gut environment in some insects 15 , 29 , 30 , including some coleopterans 31 , Also, oral delivery makes it difficult to determine precise doses of dsRNA ingested by test insects These additional factors could help explain the need for high concentrations of dsRNA in the feeding bioassays in comparison to injection method In conclusion, our findings confirm that A.
We demonstrate that the droplet-feeding assay is an efficient method to orally deliver dsRNA to neonate larvae and quickly screen potential target genes by evaluating EAB neonate mortality. However, while our results raise hopes for the development of a new and highly specific method to control this devastating insect pest with greater efficiency and less off-target effects 34 , 35 , they also showed the need for future research. In addition, human health and environmental risk assessment to confirm the specificity of the product, its safety for off-target organisms and the fate of those molecules in the environment are also need to be performed.
Laboratory-reared A. The labeled dsRNA was injected into larvae and 72 hr later total RNA was isolated and analyzed by Urea-polyacrylamide gel electrophoresis On day four live neonates from each dsRNA treatment were transferred to new parafilm and fed on dsRNA for the next six days.
Experiments were repeated 2—6 times under the same conditions, using 15 neonate larvae per replicate per treatment.
The two genes that showed higher mortality compared to control were selected for further experiments to evaluate RNAi responses in EAB. The larvae were fed on each treatment for four days. On day four, they were transferred to new parafilm and new sucrose solution without dsRNA and held for 24 hr. On the fifth day, live larvae were collected for RNA extraction and gene expression analysis as described below.
A one-way analysis of variance ANOVA was used for statistical analysis, and Student-Newman-Keuls method was used to assess differences among treatments. The experiment was performed three independent times. Candidate genes were selected based on the previous publications reporting mortality in insects exposed to those genes The dsRNA was recovered adding 0. The ethanol was removed and the dsRNA diluted in ultrapure distilled water. At the end of each RT-qPCR reaction, a melting curve was generated to confirm a single peak and rule out the possibility of primer-dimer and non-specific product formation.
A one-tailed t-test was used for statistical analysis to compare the mean of a single variable.
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