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Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum
Published on: October 13, 2014
R-loop Scarlet-RL suppresses scarlet transcription to mediate attraction to plant volatile in migratory locusts
Zongyuan Ma1, Yajing Sun1, Jing Qu1
1College of Life Sciences, University of Chinese Academy of Sciences, No.1 Yanqihu East Rd, Huairou District, Beijing 101408, China.
Introduction:
The ABC transporter Scarlet is known for its role in pigment deposition within insect eye pigment granules. However, its extraocular functions remain largely unexplored.
Objectives:
To investigate the role of Scarlet(LOCMI17149) in olfactory modulation and its regulatory mechanisms in the brains of the migratory locusts.
Methods:
The expression patterns and functions of scarlet(LOCMI17149) were analyzed by quantitative PCR (qPCR), fluorescence in situ hybridization and RNAi knockdown. Enzyme linked immunosorbent assay (ELISA) was employed to measure the xanthurenic acid (XA) titer. Pharmacological interventions were performed to validate the role of XA and scarlet(LOCMI17149) in multi-unit electrophysiology. DNA-RNA immunoprecipitation (DRIP), chromatin immunoprecipitation (ChIP) and qPCR were conducted to measure the levels of R-loop and histone modification.
Results:
Among the four Scarlet orthologs, one scarlet(LOCMI17149) is predominantly expressed in the antennae and brain. This expression pattern implicates its roles in olfactory processing. RNAi knockdown of scarlet(LOCMI17149) impaired olfactory preference for plant volatiles and common odorants (indole and β-ionone). This behavioral deficit caused by scarlet(LOCMI17149) RNAi knockdown is linked to a metabolic shift, characterized by decreased 3-hydroxykynurenine (3-HK) and elevated levels of its metabolite XA in the brain. Intracerebral injection of XA recapitulates the olfactory impairment and implicates it as a key behavioral effector. Local field potential (LFP) and electroantennogram (EAG) analysis support that XA suppresses the neurophysiological activity in the antennae and brains of locusts. Furthermore, we discover that an R-loop structure (Scarlet-RL) forms at scarlet(LOCMI17149) locus. Knockdown of Scarlet-RL with antisense oligonucleotides (ASO) increases scarlet(LOCMI17149) mRNA expression, indicating that Scarlet-RL acts as a transcriptional repressor in scarlet(LOCMI17149) transcription. Scarlet-RL mediates the synthesis of nascent mRNAs through histone modification. This study provides the first evidence that Scarlet-RL directly controls the transcription of this chemosensory regulator, revealing a novel epigenetic mechanism that orchestrates insect olfactory preference.

