Expression of clock genes period and timeless in the central nervous system of the Mediterranean flour moth, Ephestia kuehniella.

Homologous circadian genes are found in all insect clocks, but their contribution to species-specific circadian timing systems differs. The aim of this study was to extend research within Lepidoptera to gain a better understanding of the molecular mechanism underlying circadian clock plasticity and evolution. The Mediterranean flour moth, Ephestia kuehniella (Pyralidae), represents a phylogenetically ancestral lepidopteran species. We have identified circadian rhythms in egg hatching, adult emergence, and adult locomotor activity. Cloning full-length complementary DNAs and further characterization confirmed one copy of period and timeless genes in both sexes. Both per and tim transcripts oscillate in their abundance in E. kuehniella heads under light-dark conditions. PER-like immunoreactivity (PER-lir) was observed in nuclei and cytoplasm of most neurons in the central brain, the ventral part of subesophageal complex, the neurohemal organs, the optic lobes, and eyes. PER-lir in photoreceptor nuclei oscillated during the day with maximal intensity in the light phase of the photoperiodic regime and lack of a signal in the middle of the dark phase. Expression patterns of per and tim messenger RNAs (mRNAs) were revealed in the identical location as the PER-lir was detected. In the photoreceptors, a daily rhythm in the intensity of expression of both per mRNA and tim mRNA was found. These findings suggest E. kuehniella as a potential lepidopteran model for circadian studies.

Continuous activity and no cycling of clock genes in the Antarctic midge during the polar summer.

The extreme seasonal shifts of day length in polar regions, ranging from constant light in the summer to constant darkness in the winter, pose an intriguing environment for probing activity rhythms and the functioning of circadian clocks. Here, we monitor locomotor activity during the summer on the Antarctic Peninsula and under laboratory conditions, as well as the accompanying patterns of clock gene expression in the Antarctic midge, the only insect endemic to Antarctica. Larvae and adults are most active during the warmest portion of the day, but at a constant temperature they remain continuously active regardless of the photoregime, and activity also persists in constant darkness. The canonical clock genes period, timeless, Clock, and vrille are expressed in the head but we detected no cycling of expression in either the field or under diverse photoregimes in the laboratory. The timekeeping function of the clock has possibly been lost, enabling the midge to opportunistically exploit the unpredictable availability of permissive thermal conditions for growth, development, and reproduction during the short summer in Antarctica.

Functional molecular analysis of a circadian clock gene timeless promoter from the Drosophilid fly Chymomyza costata.

The circadian transcription of the tim gene is tightly regulated by the protein complex dCLK/CYC, which directly interacts with a series of closely spaced E-box and E-box-like elements in the Drosophila timeless promoter. The tim promoter from D. melanogaster has been studied in detail both in tissue cultures and in living flies yet has never been investigated in other species. This article presents a detailed functional analysis of the tim promoter from the drosophilid fly, Chymomyza costata, in Drosophila tissue cultures. A comparison of tim promoters from wt and npd-mutants confirmed that the 1855 bp deletion in the latter removes crucial regulatory cis-elements as well as the minimal promoter, being subsequently responsible for the lack of tim mRNA expression. Deletion and substitution mutations of the wt tim promoter showed that the region containing the canonical E-box, TER-box, and 2 incomplete E-box sequences is essential for CLK/CYC-mediated expression, while the PERR element appears to be a repressor in S2 cells. Furthermore, the expression of the circadian genes timeless, period , vrille, and doubletime was quantified in C. costata adults. Striking differences were found in expression profiles for tim, per, and vri between wild-type and npd-mutant individuals.

Cell cycle arrest as a hallmark of insect diapause: changes in gene transcription during diapause induction in the drosophilid fly, Chymomyza costata.

The division cycle of CNS cells was arrested in G0/G1 (86.6%) and G2 (12.8%) phases in diapausing larvae of Chymomyza costata. A two-step response was observed when the diapause was induced by transferring the 3rd instar larvae from long-day to short-day conditions: first, the proportion of G2-arrested cells increased rapidly within a single day after transfer; and second, the increase of G0/G1-arrested cells started with a delay of 5 days after transfer. The changes of relative mRNA levels of seven different genes, which code for important cell cycle regulatory factors [Cyclins D and E, kinases Wee1 and Myt1, phosphatase Cdc25 (String), Dacapo (p27), and PCNA] were followed using qRT-PCR technique. Two reference genes (Rp49 and ss-tubulin) served as a background. Significant transcriptional responses to photoperiodic transfer were observed for two genes: while the relative levels of dacapo mRNA increased during the rapid entry into the G2 arrest, the pcna expression was significantly downregulated during the delayed onset of G0/G1 arrest. In addition, moderate transcriptional upregulations of the genes coding for two inhibitory kinases, wee1 and myt1 accompanied the entry into diapause. The other genes were expressed equally in all photoperiodic conditions.