RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design

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Circadian rhythm of glycoprotein secretion in the vas deferens of the moth, <it>Spodoptera littoralis</it>

<p>Abstract</p> <p>Background</p> <p>Reproductive systems of male moths contain circadian clocks, which time the release of sperm bundles from the testis to the upper vas deferens (UVD) and their subsequent transfer from the UVD to the seminal vesicles. Sperm bundles are released from the testis in the evening and are retained in the vas deferens lumen overnight before being transferred to the seminal vesicles. The biological significance of periodic sperm retention in the UVD lumen is not understood. In this study we asked whether there are circadian rhythms in the UVD that are correlated with sperm retention.</p> <p>Results</p> <p>We investigated the carbohydrate-rich material present in the UVD wall and lumen during the daily cycle of sperm release using the periodic acid-Shiff reaction (PAS). Males raised in 16:8 light-dark cycles (LD) showed a clear rhythm in the levels of PAS-positive granules in the apical portion of the UVD epithelium. The peak of granule accumulation occurred in the middle of the night and coincided with the maximum presence of sperm bundles in the UVD lumen. These rhythms persisted in constant darkness (DD), indicating that they have circadian nature. They were abolished, however, in constant light (LL) resulting in random patterns of PAS-positive material in the UVD wall. Gel-separation of the UVD homogenates from LD moths followed by detection of carbohydrates on blots revealed daily rhythms in the abundance of specific glycoproteins in the wall and lumen of the UVD.</p> <p>Conclusion</p> <p>Secretory activity of the vas deferens epithelium is regulated by the circadian clock. Daily rhythms in accumulation and secretion of several glycoproteins are co-ordinated with periodic retention of sperm in the vas deferens lumen.</p

Molecular Oscillator Affects Susceptibility of Caterpillars to Insecticides: Studies on the Egyptian Cotton Leaf Worm—<em>Spodoptera littoralis</em> (Lepidoptera: Noctuidae)

The molecular oscillator is the core of the biological clock and is formed by genes and proteins whose cyclic expression is regulated in the transcriptional-translational feedback loops (TTFLs). Proteins of the TTFLs are regulators of both their own and executive genes involved in the control of many processes in insects (e.g., rhythmic metabolism of xenobiotics, including insecticides). We disrupted the clock operation in S. littoralis larvae by injecting the dsRNA of clock genes into their body cavity and culturing the larvae under continuous light. As a result, the daily susceptibility of larvae to insecticides was abolished and the susceptibility itself increased (in most cases). In the fat body, midgut, and Malpighian tubules (the main organs metabolizing xenobiotics) of the larvae treated with injected-dsRNA, the daily activity profiles of enzymes involved in detoxification—cytochrome P450 monooxygenases, Glutathione-S-transferase, and esterase—have changed significantly. The presented results prove the role of the molecular oscillator in the regulation of larvae responses to insecticides and provide grounds for rational use of these compounds (at suitable times of the day), and may indicate clock genes as potential targets of molecular manipulation to produce plant protection compounds based on the RNAi method