تاثیر خاموشی ژن E93 روی بیان ژن‌های دخیل در مسیر سیگنالینگ JH در تخمدان ماده های بالغ B. germanica

The german cockroach, Blattella germanica (L.) (Blattaria: Blattellidae), is one of the most sanitary and economic important pests in Iran. This species causes severe economic damage because of using any agricultural productions in storages and residential areas and transmitting of a variety of diseases. Most control techniques mainly rely on using chemical insecticides. Because of the high economic and environmental costs of insecticides and evolution of insecticides resistant populations, there is an urgent need to develop new pest management strategies that based on IPM. In recent years, RNA interference (RNAi) has shown great potential in controlling insect pests by silencing vital genes. RNAi is a gene silencing mechanism in which the expression of a gene is specifically inhibited by its cognate double-stranded RNA. Juvenile hormone (JH) signaling pathway involves in insects’ metamorphosis and reproduction. E93 (Ecdysteroid- dependent transcription factor Eip 93F) is a transcriptional factor that placed in downstream of JH and Met and Kr-h1 genes and transmits the anti-metamorphic signaling of JH in insects. For clarifying the role of E93 in reproduction, E93 mRNA levels were knocked down by using systemic RNAi. The colony of B. germanica reared on dry dog food (Panlab 125C3) and water in the dark at 30 ± 1°C and 60-70% relative humidity. A dose of 2 µg of dsE93 was injected into the abdomen of freshly emerged adult females and injection was repeated on third day. For control, females equivalently treated with dsMock. The dissection and transcriptome analysis of genes that involve in JH signaling pathway, carried out on fifth day of adult stage. Total RNA extraction from ovary and cDNA synthesizing were carried out. Quantitative real time PCR (qRT-PCR) reactions were carried out in triplicate by using SYBR Green. Levels of mRNA were calculated relative to BgActin-5c expression by using the iQ5 Real-Time PCR Detection System (Bio-Rad Laboratories). All data were analyzed by using of T test student and Rest (2009) and Excel. E93 mRNA levels were examined in the ovary of dsE93 treated females on day 5. The results showed that E93 expression was 38.39% lower in comparison with control significantly (p˂0.05). Beside, depletion of E93 expression didn’t affect the expression of Met and Kr-h1 in ovary in comparison with control significantly. In terms of phenotypic, E93 RNAi reduced significantly the total protein content of ovary, basal oocytes growth (9.7%), size of ovary compares with control. E93 appears to play key role in oogenesis and basal oocyte development by using another signaling pathway that independent on JH signaling. So, E93 can be an appropriate candidate gene as potential target site for controlling of B. germanica by using the RNAi technique in future.