RNA Interference of the Period Gene Affects the Rhythm of Sperm Release in Moths

Abstract

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.

Diurnal rhythm in expression and release of yolk protein in the testis of Spodoptera littoralis (Lepidoptera: Noctuidae)

“…Reduced per mRNA levels affected the egg hatching rhythm in the silk moth Bombyx mori (Sandrelli et al, 2007) and inhibited the locomotor activity rhythm and rhythmic electrical activity in the optic lobes in crickets Gryllus bimaculatus (Moriyama et al, 2008). Furthermore, we have recently shown that per RNAi inhibited sperm release rhythm in the moth S. littoralis (Kotwica et al, 2009a).…”

“…Another difference is that PER and TIM, which translocate to cell nuclei in the fruit fly, are predominantly cytoplasmic in neurons of other insects. However, PER was detected in cell nuclei in peripheral organs, such as larval midgut of Antheraea pernyi (Sauman and Reppert, 1998), epithelial cells in the antenna of Manduca sexta (Schuckel et al, 2007) and vas deferens epithelium in the Cydia pomonella (Gvakharia et al, 2000) and Spodoptera littoralis (Kotwica et al, 2009a).…”

Male reproduction is affected by RNA interference of period and timeless in the desert locust Schistocerca gregaria

“…Giebultowicz, 2004;Howlader and Sharma, 2006;Sellix and Menaker, 2010;Yoshikawa et al, 2009). In the fruit fly and moths, the circadian clock genes per and tim have been found to be directly connected with male reproductive capacity (Beaver et al, 2002;Kotwica et al, 2009). Previously, the effect of a knockdown of these genes in the desert locust has been reported for female S. gregaria which was comparable to the effect in female per and tim loss-of-function mutant fruit flies (Beaver et al, 2003).…”

“…RNAi experiments in Spodoptera littoralis indicated that per has a role in the regulation of sperm release in the male reproductive system (Kotwica et al, 2009). Moreover, sperm that is released from the testes undergoes further maturation in the male reproductive tract and accessory glands.…”

“…Earlier research had already demonstrated that when moths are kept in constant light, both the size of the testes and the number of sperm bundles in the testes decrease significantly (Bebas and Cymborowski, 1999). Sperm release throughout the male reproductive system is regulated in a circadian manner in moths (Giebultowicz et al, 1989) and it is under direct control of the circadian per gene as per RNAi causes a delay in sperm release (Kotwica et al, 2009).…”