RNA interference of timeless gene does not disrupt circadian locomotor rhythms in the cricket Gryllus bimaculatus

Molecular studies revealed that autoregulatory negative feedback loops consisting of so-called “clock genes” constitute the circadian clock in Drosophila. However, this hypothesis is not fully supported in other insects and is thus to be examined. In the cricket Gryllus bimaculatus, we have previously shown that period (per) plays an essential role in the rhythm generation. In the present study, we cloned cDNA of the clock gene timeless (tim) and investigated its role in the cricket circadian oscillatory mechanism using RNA interference. Molecular structure of the cricket tim has rather high similarity to those of other insect species. Real-time RT-PCR analysis revealed that tim mRNA showed rhythmic expression in both LD and DD similar to that of per, peaking during the (subjective) night. When injected with tim double-stranded RNA (dstim), tim mRNA levels were significantly reduced and its circadian expression rhythm was eliminated. After the dstim treatment, however, adult crickets showed a clear locomotor rhythm in DD, with a free-running period significantly shorter than that of control crickets injected with Discosoma sp. Red2 (DsRed2) dsRNA. These results suggest that in the cricket, tim plays some role in fine-tuning of the free-running period but may not be essential for oscillation of the circadian clock.     

Functional analysis of the circadian clock gene period by RNA interference in nymphal crickets Gryllus bimaculatus

The circadian clock gene period (Gryllus bimaculatus period, Gb’per) plays a core role in circadian rhythm generation in adults of the cricket Gryllus bimaculatus. We examined the role of Gb’per in nymphal crickets that show a diurnal rhythm rather than the nocturnal rhythm of the adults. As in the adult optic lobes, Gb’per mRNA levels in the head of the third instar nymphs showed daily cycling in light-dark cycles with a peak at mid night, and the rhythm persisted in constant darkness. Injection of Gb’per double-stranded RNA (dsRNA) into the abdomen of third instar nymphs knocked-down the mRNA levels to 25% of that in control animals. Most Gb’per dsRNA injected nymphs lost their circadian locomotor activity rhythm, while those injected with DsRed2 dsRNA as a negative control clearly maintained the rhythm. These results suggest that nymphs and adults share a common endogenous clock mechanism involving the clock gene Gb’per.     

Circadian oscillations outside the optic lobe in the cricket Gryllus bimaculatus

Although circadian rhythms are found in many peripheral tissues in insects, the control mechanism is still to be elucidated. To investigate the central and peripheral relationships in the circadian organization, circadian rhythms outside the optic lobes were examined in the cricket Gryllus bimaculatus by measuring mRNA levels of period (per) and timeless (tim) genes in the brain, terminal abdominal ganglion (TAG), anterior stomach, mid-gut, testis, and Malpighian tubules. Except for Malpighian tubules and testis, the tissues showed a daily rhythmic expression in either both per and tim or tim alone in LD. Under constant darkness, however, the tested tissues exhibited rhythmic expression of per and tim mRNAs, suggesting that they include a circadian oscillator. The amplitude and the levels of the mRNA rhythms varied among those rhythmic tissues. Removal of the optic lobe, the central clock tissue, differentially affected the rhythms: the anterior stomach lost the rhythm of both per and tim; in the mid-gut and TAG, tim expression became arrhythmic but per maintained rhythmic expression; a persistent rhythm with a shifted phase was observed for both per and tim mRNA rhythms in the brain. These data suggest that rhythms outside the optic lobe receive control from the optic lobe to different degrees, and that the oscillatory mechanism may be different from that of Drosophila.     

Secretion of collagen by insects: Autoradiographic study of l-proline 3H-5 incorporation by the firebrat Thermobia domestica

The biosynthesis of collagenous endoskeletal endosterna by fibroblasts was studied by electron microscope autoradiography after tritium-labelled proline administration in the firebrat Thermobia domestica at various time intervals. Autoradiographs were quantitatively analyzed and the relative concentration of label was determined for various cellular compartments. The labelling in the rough endoplasmic reticulum, ground cytoplasm, Golgi apparatus and endosterna was compared during the secretion of radioactive products. The label, initially located in the rough endoplasmic reticulum was found in the Golgi apparatus before it accumulated in the endosternum. The involvement of the rough endoplasmic reticulum and Golgi apparatus in the transport of secreted collagen is discussed, comparing our results with current knowledge of collagenous secretion.     

The circadian clock genes affect reproductive capacity in the desert locust Schistocerca gregaria

The circadian clocks govern many metabolic and behavioral processes in an organism. In insects, these clocks and their molecular machinery have been found to influence reproduction in many different ways. Reproductive behavior including courtship, copulation and egg deposition, is under strong influence of the daily rhythm. At the molecular level, the individual clock components also have their role in normal progress of oogenesis and spermatogenesis. In this study on the desert locust Schistocerca gregaria, three circadian clock genes were identified and their expression profiles were determined. High expression was predominantly found in reproductive tissues. Similar daily expression profiles were found for period (per) and timeless (tim), while the clock (clk) mRNA level is higher 12 h before the first per and tim peak. A knockdown of either per or tim resulted in a significant decrease in the progeny produced by dsRNA treated females confirming the role of clock genes in reproduction and providing evidence that both PER and TIM are needed in the ovaries for egg development. Since the knockdown of clk is lethal for the desert locust, its function remains yet to be elucidated.     

Molecular characterization of two glutathione peroxidase genes of Panax ginseng and their expression analysis against environmental stresses

Glutathione peroxidases (GPXs) are a group of enzymes that protect cells against oxidative damage generated by reactive oxygen species (ROS). GPX catalyzes the reduction of hydrogen peroxide (H₂O₂) or organic hydroperoxides to water or alcohols by reduced glutathione. The presence of GPXs in plants has been reported by several groups, but the roles of individual members of this family in a single plant species have not been studied. Two GPX cDNAs were isolated and characterized from the embryogenic callus of Panax ginseng. The two cDNAs had an open reading frame (ORF) of 723 and 681 bp with a deduced amino acid sequence of 240 and 226 residues, respectively. The calculated molecular mass of the matured proteins are approximately 26.4 kDa or 25.7 kDa with a predicated isoelectric point of 9.16 or 6.11, respectively. The two PgGPXs were elevated strongly by salt stress and chilling stress in a ginseng seedling. In addition, the two PgGPXs showed different responses against biotic stress. The positive responses of PgGPX to the environmental stimuli suggested that ginseng GPX may help to protect against environmental stresses.     

Circadian expression of clock genes in human peripheral leukocytes

In mammals, behavioral and physiological processes display 24-h rhythms that are regulated by a circadian system. In the present study, we investigated the possibility that the expression of clock genes in peripheral leukocytes can be used to assess the circadian clock system. We found that Per1 and Per2 exhibit circadian oscillations in mRNA expression in mouse peripheral leukocytes. Furthermore, the rhythms of Per1 and Per2 mRNA expression in peripheral leukocytes are severely blunted in homozygous Cry1/2 double-deficient mice that are known to have an abolished biological clock. We have examined the circadian expression of clock genes in human leukocytes and found that Per1 mRNA exhibits a robust circadian expression while Per2 and Bmal1 mRNA showed weak rhythm. These observations suggest that monitoring Per1 mRNA expression in human leukocytes may be useful for investigating the function of the circadian system in physiological and pathophysiological states.     

Molecular characterization and distribution of CYCLE protein from Athalia rosae

cDNA encoding CYCLE (CYC) from the coleseed sawfly, Athalia rosae (Hymenoptera, Symphyta), was amplified by PCR. This is a first determination of hymenopteran CYC structure. ArCYC had an overall identity of 66% with CYC of Anopheles gambiae and ca. 60% of Drosophila melanogaster. Structural investigation revealed that ArCYC contained characteristic motifs of: bHLH, PAS A, PAS B, PAC and BCTR. Detailed analysis indicated high conservation of these regions among insects. Northern blot analysis showed that the mRNA of ca. 3 kb was transcribed both in the head and in the rest of the body. Southern blot analysis suggested the presence of a single copy of the gene in the genome. Western blot indicates that the quantity of CYC protein does not fluctuate under LD 12:12 in either the head or the rest of the body. Immunocytochemical examination revealed CYC-like antigen in the pars intercerebralis, dorsolateral protocerebrum, dorsal optic tract, tritocerebrum of the brain and the subesophageal ganglion.     

Inhibition of reproduction and embryogenesis in the firebrat, Thermobia domestica, by juvenile hormone analogues

Application of compounds with juvenile hormone (JH) activity to reproducing females may result in shortening of the ovipositor and occasionally in some other changes in appearance. Severe defects occur in the ovaries: the differentiation of oögonia and prefollicular cells seems to be hindered and the mature oöcytes are resorbed. The affected ovarioles diminish and in extreme cases tear into the germarium, which remains attached to the terminal filament, and the rest of the ovariole, which shrinks into a small rudiment adjacent to the oviduct. The development of eggs deposited by treated females is often lethally affected. Disorders in embryogenesis also occur in the eggs contaminated with active substances at any time between deposition and 2 days before hatching. The substances interrupt the normal course of embryogenesis but the embryos survive for some time and may develop into strange creatures; in one case, two embryos were found within a single egg shell. One out of 27 tested substances was active in amounts lower than 1 ng/egg.     

Circadian expression of clock and screening of clock-controlled genes in peripheral lymphocytes of rat

In this paper, the circadian pattern of Clock and genes mediated by the Clock was investigated in peripheral lymphocytes of rats. Circadian rhythms of Clock are found under the regimes of constant darkness (DD) and 12-h light-12-h dark (LD12:12h), with the peak phase at CT7 and ZT21, respectively. Ten differential cDNA fragments were identified to be mediated by the Clock, including three known genes (catalase, myelin proteolipid protein, and histone acetylase), four known expressed sequence tags (ESTs), and three novel ESTs. Experiment of the RNA interference revealed that these ESTs were down-regulated by the Clock gene and three of them were identified as clock-controlled genes. Understanding of clock-mediated genes may lead to a new direction in drug design for control of circadian rhythms.     

Circadian Clock genes Per2 and clock regulate steroid production, cell proliferation, and luteinizing hormone receptor transcription in ovarian granulosa cells

Circadian Clock genes are associated with the estrous cycle in female animals. Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression in follicle-stimulating hormone FSH-treated granulosa cells. Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom, whereas Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. Similarly, expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. Our data provide a new insight that Per2 and Clock have different action on ovarian granulosa cell functions.     

Molecular cloning and functional analyses of glutathione peroxidase homologous genes from Chlorella sp. NJ-18

The extreme variability of the mitochondrial (mito) genomes of bivalves makes it difficult to understand their evolutionary dynamics, given that species from different families do not share comparable features. We compared the mitogenomes from four Paphia clams (three of them were firstly sequenced) and found that mitogenome reorganization among the four congeneric species is not random but follows phylogenetic trends. Start/stop codon variations are species-correlated rather than gene-correlated, and bear useful phylogenetic information. Unique start/stop codon usage in P. euglypta and A+T content in P. amabilis indicates that these mitogenome-level characters, usually considered to be conservative features in other lineages, may not be phylogenetically evolved, but may have evolved via species-specific mitogenomic maintenance mechanisms. Variable divergence of two trnM genes in different lineages may demonstrate differences in mechanisms by which paralogous trnM genes are maintained. Sequence alignment analysis indicates that the VNTRs in the four mitogenomes have a common origin. The rationale of the subgenus Neotapes Kuroda and Habe, 1971 was supported by evidence from morphological characters, mitogenomic features, as well as phylogenetic analyses using cox1 and rrnS genes. The data suggest that the taxonomic basis of the subgenus should be “smooth surface” but not “undulated lines,” and P. textile should be classified to the Neotapes subgenus.     

Molecular cloning,regulation, and functional analysis of two GHS-R genes in zebrafish

Mammalian ghrelin is derived from stomach and regulates growth hormone release and appetite by modulating GHS-R (Growth hormone secretagogue receptor) activity. Zebrafish has been developed as a forward genetic screening model system and previous screening identified a number of genes involved in multiple signaling pathways. In this system, ghrelin has been identified and its function and regulation have been shown to be highly conserved to that of mammals.     

Short-day and long-day expression patterns of genes involved in the flesh fly clock mechanism: period,timeless, cycle and cryptochrome

Though our knowledge of the molecular details of the circadian clock has advanced rapidly, the functional elements of the photoperiodic clock remain largely unknown. As a first step to approach this issue, we report here the sequences and expression patterns of period (per), timeless (tim), cycle (cyc) and cryptochrome (cry) mRNAs in the flesh fly Sarcophaga crassipalpis. Nucleotide and deduced amino acid sequences of the genes in S. crassipalpis show high similarity to homologous genes in other insects that have been investigated. S. crassipalpis TIM has a unique C-terminus that contains a poly Q region. A diel rhythmicity of per and tim mRNA abundance was detected in the adult heads (peak during scotophase), while cry and cyc mRNA abundance remained fairly constant throughout. The abundance of cyc mRNA was quite low when compared to per, tim and cry mRNA. Rearing temperature affected the amount of per and tim mRNAs: abundance of per mRNA increased at 20 °C when compared to 25 °C, but that of tim mRNA decreased. Photoperiod influenced the expression patterns of per and tim mRNA: the peak of per mRNA expression shifted in concert with onset of the scotophase, while a shift in tim mRNA expression was less pronounced. The amplitude of tim mRNA was severely dampened under long daylength, but that of per mRNA was not affected. These distinct patterns of expression suggest that this information could be used to determine photoperiodic responses such as diapause.     

Antigonadotropic and antiallatal effects of precocene II in the firebrat, Thermobia domestica (Thysanura,Lepismatidae)

The sensitivity of an apterygote insect to precocene II was investigated for the first time in females of Thermobia domestica. Topical applications of this compound, which has been said to possess anti-juvenile hormone activity, were carried out at various times in the postecdysial period of a reproductive cycle, during intense vitellogenesis in the terminal oöcytes. The effects of treatment were observed on mortality, fecundity, insemination and also on the ultrastructure of corpora allata; they depend on the dose used and on the day of treatment. A single application of 10 μg/insect at the beginning of the postecdysial period induces antigonadotropic effects in females reared both with and without males. The effects of precocene add to those of non-insemination, which also elicits inhibition of oöcyte maturation. In addition, the cytotoxic action of precocene, leading to degeneration of the corpora allata, which is most probably irreversible, was demonstrated.