Figure 4
RNA interference (RNAi) of Se-5HTR and suppression of hemocyte behaviors. (a) RNAi of Se-5HTR expression with a gene-specific dsRNA (dsSe-5HTR). About 900 ng of dsSe-5HTR was injected to L5. At 0, 12, 24, and 48 h post-injection (PI), expression levels of Se-5HTR were assessed from whole body. Expression levels of Se-5HTR in different tissue parts were assessed at 24 h PI. As a constitutive expressional control, a ribosomal gene, RL32, was used for expression analysis in RT-PCR and RT-qPCR. As a control RNAi (dsCON), dsRNA specific to CpBV-ORF302 (a viral gene) was used for expression analysis. In RT-qPCR, each treatment was triplicated. (b) Influence of RNAi on up-regulation of total hemocyte count (THC) in response to bacterial challenge. At 24 h PI of dsSe-5HTR, hemolymph was collected from L5 larvae for THC assessment. Each treatment was replicated three times. (c) Influence of RNAi on hemocyte-spreading behavior. For spreading assay, hemocytes from larvae treated with dsRNA were collected at 24 h. Each treatment was independently replicated three times. Spread cells were stained with F-actin and FITC-labeled phalloidin. Nuclei were stained with DAPI. Histogram bars indicate percentages of spread hemocytes and error bars indicate standard deviation. Different letters above standard error bars indicate significant difference among means at type I error = 0.05 (LSD test). Asterisks represent significant difference between control and treatment in each tissue.
