Gene silencing through RNA interference (RNAi) has revolutionized the study of gene 98 function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) 99 RNAi has many times proven to be difficult to achieve. Most of the negative results have been 100 anecdotal and the positive experiments have not been collected in such a way that they are 101 possible to analyze. In this review, we have collected detailed data from more than 150 102 experiments including all to date published and many unpublished experiments. Despite a 103 large variation in the data, trends that are found are that RNAi is particularly successful in the 104 family Saturniidae and in genes involved in immunity. On the contrary, gene expression in 105 epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding 106 dsRNA requires high concentrations for success. Possible causes for the variability of success 107 in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further 108 investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the 109 innate immune response. Our general understanding of RNAi in Lepidoptera will be further 110 aided in the future as our public database at http://insectacentral.org/RNAi will continue to 111 gather information on RNAi experiments.

The period (per) gene is 1 of the core elements of the circadian clock mechanism in animals from insects to mammals. In clock cells of Drosophila melanogaster, per mRNA and PER protein oscillate in daily cycles. Consistent with the molecular clock model, PER moves to cell nuclei and acts as a repressor of positive clock elements. Homologs of per are known in many insects; however, specific roles of per in generating output rhythms are not known for most species. The aim of this article was to determine whether per is functionally involved in the circadian rhythm of sperm release in the moth, Spodoptera littoralis. In this species, as in other moths, rhythmic release of sperm bundles from the testis into the upper vas deferens occurs only in the evening, and this rhythm continues in the isolated reproductive system. S. littoralis was used to investigate the expression of per mRNA and protein in the 2 types of cells involved in sperm release: the cyst cells surrounding sperm bundles in the testes, and the barrier cells separating testicular follicles from the vas deferens. In cyst cells, PER showed a nuclear rhythm in light/dark (LD) cycles but was constitutively cytoplasmic in constant darkness (DD). In barrier cells, nuclear cycling of PER was observed in both LD and DD. To determine the role of PER in rhythmic sperm release in moths, testes-sperm duct complexes were treated in vitro with double-stranded fragments of per mRNA (dsRNA). This treatment significantly lowered per mRNA and protein in cyst cells and barrier cells and caused a delay of sperm release. These data demonstrate that a molecular oscillator involving the period gene plays an essential role in the regulation of rhythmic sperm release in this species.

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