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Circadian clocks (oscillators) regulate multiple aspects of insect behaviour and physiology. The circadian system located in the male reproductive tract of Lepidoptera orchestrates rhythmic sperm release from testis and sperm maturation in the upper vas deferens (UVD). Our previous research on the cotton leafworm, Spodoptera littoralis, suggested rhythmic changes in the V-ATPase levels in the UVD epithelium, which correlated with rhythmic pH fluctuations in the UVD lumen. However, it was not known whether UVD cells contain clock mechanism that generates these daily fluctuations. In the current paper, we show circadian rhythm in the expression of clock gene period at the mRNA and protein level in the UVD epithelium. To determine the role of PER in V-ATPase and pH regulation, testes–UVD complexes were treated in vitro with double-stranded fragments of per mRNA (dsRNA). This treatment, which transiently lowered per mRNA and protein in the UVD, altered expression of V-ATPase c subunit. In addition, per RNAi caused a significant delay in the UVD lumen acidification. These data demonstrate that the UVD molecular oscillator involving the period gene plays an essential role in the regulation of rhythmic V-ATPase activity and periodic acidification of the UVD lumen.

Disruption of the circadian clock, which directs rhythmic expression of numerous output genes, accelerates aging. To enquire how the circadian system protects aging organisms, here we compare circadian transcriptomes in heads of young and old Drosophila melanogaster. The core clock and most output genes remained robustly rhythmic in old flies, while others lost rhythmicity with age, resulting in constitutive over- or under-expression. Unexpectedly, we identify a subset of genes that adopted increased or de novo rhythmicity during aging, enriched for stress-response functions. These genes, termed late-life cyclers, were also rhythmically induced in young flies by constant exposure to exogenous oxidative stress, and this upregulation is CLOCK-dependent. We also identify age-onset rhythmicity in several putative primary piRNA transcripts overlapping antisense transposons. Our results suggest that, as organisms age, the circadian system shifts greater regulatory priority to the mitigation of accumulating cellular stress., Disruption of circadian rhythms leads to reduced healthspan, but the mechanisms by which the normal clock protects aging organisms are not known. Here, the authors show that a subset of genes becomes more rhythmically expressed in older flies, and these are enriched for response to oxidative stress.

Circadian clock genes are ubiquitously expressed in the nervous system and peripheral tissues of complex animals. While clock genes in the brain are essential for behavioral rhythms, the physiological roles of these genes in the periphery are not well understood. Constitutive expression of the clock gene period was reported in the ovaries of Drosophila melanogaster; however, its molecular interactions and functional significance remained unknown. This study demonstrates that period( per) and timeless( tim) are involved in a novel noncircadian function in the ovary. PER and TIM are constantly expressed in the follicle cells enveloping young oocytes. Genetic evidence suggests that PER and TIM interact in these cells, yet they do not translocate to the nucleus. The levels of TIM and PER in the ovary are affected neither by light nor by the lack of clock-positive elements Clock( Clk) and cycle( cyc). Taken together, these data suggest that per and tim are regulated differently in follicle cells than in clock cells. Experimental evidence suggests that a novel fitness-related phenotype may be linked to noncircadian expression of clock genes in the ovaries. Mated females lacking either per or tim show nearly a 50% decline in progeny, and virgin females show a similar decline in the production of mature oocytes. Disruption of circadian mechanism by either the depletion of TIM via constant light treatment or continuous expression of PER via GAL4/UAS expression system has no adverse effect on the production of mature oocytes.

Release of sperm bundles from moth testes is controlled by the local circadian oscillator. The mechanism which restricts migration of sperm bundles to a few hours each day is not understood. We demonstrate that a daily cycle of sperm release is initiated by the migration of folded apyrene sperm bundles through a cellular barrier at the testis base. These bundles have conspicuous concentrations of actin filaments at their proximal end. Inhibition of actin polymerization by cytochalasin at a specific time of day inhibited sperm release from the testis. Likewise, application of double-stranded actin RNA specifically inhibited sperm release. This RNA-mediated interference (RNAi) lowered the pool of actin mRNA in tissues involved in sperm release. The decline in mRNA levels resulted in the selective depletion of F-actin from the tip of apyrene sperm bundles, suggesting that this actin may be involved in the initiation of sperm release. Combined results of RNAi experiments at physiological, cellular and molecular levels identified unique cells that are critically involved in the mechanism of sperm release.

The authors examined patterns of spatial and temporal expression of Drosophila per gene homologue in the codling moth, Cydia pomonella. Since sperm release in moths is regulated in a circadian manner by an autonomous clock that is independent from the brain, the authors investigated per expression in male reproductive system along with its expression in moth heads. per mRNA is rhythmically expressed with the same phase and amplitude in both tissues under light-dark (LD) conditions. The levels of per mRNA are low during the day, start to increase before lights-off, reach the peak in dark, and decrease after lights-on. In constant darkness (DD), cycling of per mRNA continued in heads with severely blunted amplitude. No cycling of per mRNA was detected in testis in DD. In situ hybridization and immunocytochemistry revealed distinct spatial patterns of per expression in the moth reproductive system. There is no expression of per in cells forming the wall of testes or in sperm bundles. However, permRNA and protein are rhythmically expressed in the epithelial cells forming the wall of the upper vas deferens (UVD) and in the cells of the terminal epithelium, which are involved in the circadian gating of sperm release. Increase in permRNA in the UVD coincides with sperm accumulation in this part of the insect reproductive system.

The period (per) and timeless (tim) genes encode key components of the circadian oscillator in Drosophila melanogaster. The per gene is thought to encode three transcripts via differential splicing (types A, B, and C) that give rise to three proteins. Since the three per mRNA types were based on the analysis of cDNA clones, we tested whether these mRNA types were present in vivo by RNase protection assays and reverse transcriptasemediated PCR. The results show that per generates two transcript types that differ only by the presence (type A) or absence (type B*) of an alternative intron in the 3* untranslated region. Transgenic flies containing transgenes that produce only type B* transcripts (per B* ), type A transcripts (per A ), or both transcripts (per G ) rescue locomotor activity rhythms with average periods of 24.7, 25.4, and 24.4 h, respectively. Although no appreciable differences in type A and type B* mRNA cycling were observed, a slower accumulation of PER in flies making only type A transcripts suggests that the intron affects the translation of per mRNA. The period (per) and timeless (tim) genes encode key components of the circadian oscillator in Drosophila melanogaster. The expression of these genes is required for circadian clock function, and an important aspect of their expression is circadian fluctuations in their mRNA and protein levels (17). These rhythms in per and tim gene products are controlled by a circadian feedback loop in which PER and TIM proteins control the expression of their own mRNAs (17, 25, 26). This feedback is mediated predominantly at the transcriptional level, though posttranscriptional regulation is also involved (5, 16, 29, 31, 32). The role of PER in this process is unknown, but its lack of a known DNA binding domain and inability to bind DNA indicate that it does not regulate transcription directly (17, 25). Analysis of per cDNA clones uncovered three splice variants that encode three different PER isoforms (3). The most abundant of these transcripts, type A, defined both the structure of the per gene and the prototypical 1,218-amino-acid PER protein. Type B transcripts differ from type A by having two additional introns; one removes 288 nucleotides (nt) from exon 5 of type A transcripts, and the other excises 89 nt from the 39 untranslated region (39UTR) of exon 8. After excising the intron from exon 5, type B transcripts produce a protein that is 96 amino acids shorter. The least abundant transcript (only one partial cDNA clone was isolated), type C, differs from type A by retaining introns 5, 6, and 7, thereby producing a transcript whose exon 5 spans exons 5 to 8 in type A transcripts. Due to the inclusion of these additional introns, the last 107 amino acids of the putative type C protein sequence are entirely different from the last 149 amino acids of type A protein sequence. All three per cDNAs are capable of rescuing behavioral rhythms in per 01 flies, though the type C construct may mediate behavioral rescue by generating both type A and type B transcripts (3, 4). Given the critical role that PER plays in controlling the circadian feedback loop in Drosophila, it is important to determine which isoforms contribute to the feedback loop mechanism and what impact this contribution may have on behavioral rhythms. Since the initial characterization of per mRNA splice variants was based on the structure and abundance of partial cDNA clones, we tested whether these per transcripts exist in vivo and function equally to rescue locomotor activity rhythms. Our studies failed to detect per splice variants that generate different PER isoforms. However, two per transcripts that differ by an alternatively spliced intron within their 39UTRs were found; type A contains the 89-bp intron, and type B9 lacks this intron. Transgenes that produce type A mRNA, type B9 mRNA, or both mRNA types each rescue robust locomotor activity rhythms, but the period of these rhythms tends to be longer in the transgene that produces only type A transcripts. Type A and type B9 transcripts are indistinguishable with respect to circadian cycling, but the levels of PER derived from the transgene expressing only type A transcripts rise with a later phase than PER derived from transgenes expressing only type B9 or both type A and type B9 transcripts. These results suggest that the alternatively spliced intron alters the translation of per mRNA.

Background 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.