A genome-wide survey of photoreceptor and circadian genes in the coral, Acropora digitifera

Corals exhibit circadian behaviors, but little is known about the molecular mechanisms underlying the regulation of these behaviors. We surveyed the recently decoded genome of the coral, Acropora digitifera, for photoreceptor and circadian genes, using molecular phylogenetic analyses. Our search for photoreceptor genes yielded seven opsin and three cryptochrome genes. Two genes from each family likely underwent tandem duplication in the coral lineage. We also found the following A. digitifera orthologs to Drosophila and mammalian circadian clock genes: four clock, one bmal/cycle, three pdp1-like, one creb/atf, one sgg/zw3, two ck2alpha, one dco (csnk1d/cnsk1e), one slim/BTRC, and one grinl. No vrille, rev-ervα/nr1d1, bhlh2, vpac2, adcyap1, or adcyaplr1 orthologs were found. Intriguingly, in spite of an extensive survey, we also failed to find homologs of period and timeless, although we did find one timeout gene. In addition, the coral genes were compared to orthologous genes in the sea anemone, Nematostella vectensis. Thus, the coral and sea anemone genomes share a similar repertoire of circadian clock genes, although A. digitifera contains more clock genes and fewer photoreceptor genes than N. vectensis. This suggests that the circadian clock system was established in a common ancestor of corals and sea anemones, and was diversified by tandem gene duplications and the loss of paralogous genes in each lineage. It will be interesting to determine how the coral circadian clock functions without period.     

Circadian rhythms in the morphology of neurons in <Emphasis Type="Italic">Drosophila</Emphasis>

Neurons have an enormous capacity to adapt to changing conditions through the regulation of gene expression, morphology, and physiology. In the fruit fly Drosophila melanogaster, this plasticity includes recurrent changes taking place within intervals of a few hours during the day. The rhythmic alterations in the morphology of neurons described so far include changes in axonal diameter, branching complexity, synapse numbers, and the number of synaptic vesicles. The cycles of these changes have larger amplitude when the fly is exposed to light, but they persist in constant darkness and require the expression of the clock genes period and timeless, leading to the concept of circadian plasticity. The molecular mechanisms driving these cycles appear to require the expression of these genes either inside the neurons themselves or in other peripheral pacemaker cells. Loss-of-function mutations in period and timeless not only abolish the morphological rhythms, but also often cause abnormal axonal branching suggesting that circadian plasticity is relevant for the maintenance of normal morphology. Research into whether (1) circadian plasticity is a common feature of neurons in all animals and (2) our own neurons change shape between day and night will be of interest.     

Rhythmic clock gene expression in heart, kidney and some brain nuclei involved in blood pressure control in hypertensive TGR(mREN-2)27 rats

Hypertensive TGR(mREN-2)27 rats exerting inverted blood pressure (BP) profile were used to study clock gene expression in structures responsible for BP control. TGR and control Sprague Dawley male rats were synchronized to the light:dark cycle 12:12 with food and water ad libitum. Daily rhythm in per2, bmal1, clock and dbp expression in the suprachiasmatic nucleus (SCN), rostral ventrolateral medulla (RVLM), nucleus of the solitary tract (NTS), heart and kidney was determined in both groups. Sampling occurred in regular 4 h intervals when rats of both strains were 11-weeks-old. Blood pressure and relative heart weight were significantly elevated in TGR rats in comparison with control. Expression of bmal1 and clock was up regulated in SCN of TGR rats but daily rhythm in per2 and dbp expression was similar in both groups. Mesor of per2 expression in RVLM was significantly higher in TGR than in control rats. In NTS of TGR rats expression of per2 was phase delayed by 3.5 h in comparison with control and bmal1 did not exert rhythmic pattern. Our study provided the first evidence about modified function of central and peripheral circadian oscillators in TGR rats at the level of clock gene expression. Expression of clock genes exerted up regulation in SCN and RVLM and down regulation in NTS. Circadian oscillators in selected brain structures were influenced more than oscillators in the heart and kidney by additional renin gene. Interactions of RAS and circadian system probably contribute to the development of inverted BP profile in TGR rats.     

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.     

The Molecular Clockwork of the Fire Ant Solenopsis invicta

The circadian clock is a core molecular mechanism that allows organisms to anticipate daily environmental changes and adapt the timing of behaviors to maximize efficiency. In social insects, the ability to maintain the appropriate temporal order is thought to improve colony efficiency and fitness. We used the newly sequenced fire ant (Solenopsis invicta) genome to characterize the first ant circadian clock. Our results reveal that the fire ant clock is similar to the clock of the honeybee, a social insect with an independent evolutionary origin of sociality. Gene trees for the eight core clock genes, period, cycle, clock, cryptochrome-m, timeout, vrille, par domain protein 1 & clockwork orange, show ant species grouping closely with honeybees and Nasonia wasps as an outgroup to the social Hymenoptera. Expression patterns for these genes suggest that the ant clock functions similar to the honeybee clock, with period and cry-m mRNA levels increasing during the night and cycle and clockwork orange mRNAs cycling approximately anti-phase to period. Gene models for five of these genes also parallel honeybee models. In particular, the single ant cryptochrome is an ortholog of the mammalian-type (cry-m), rather than Drosophila-like protein (cry-d). Additionally, we find a conserved VPIFAL C-tail region in clockwork orange shared by insects but absent in vertebrates. Overall, our characterization of the ant clock demonstrates that two social insect lineages, ants and bees, share a similar, mammalian-like circadian clock. This study represents the first characterization of clock genes in an ant and is a key step towards understanding socially-regulated plasticity in circadian rhythms by facilitating comparative studies on the organization of circadian clockwork.     

Clock genes period and timeless are rhythmically expressed in brains of newly hatched, photosensitive larvae of the fly, Sarcophaga crassipalpis

While roles of the clock genes period (per) and timeless (tim) are relatively well understood in relation to circadian clocks, their potential roles in insect photoperiodism remain enigmatic. In this study, the expression of per and tim genes under two contrasting photoperiods is described in the central nervous system of photoperiodically sensitive, newly hatched first instar larvae of the flesh fly, Sarcophaga crassipalpis. Using qPCR, diel oscillations were observed in the mRNA levels of both genes under long-day (15 h light:9 h dark, promotes direct development) and short-day conditions (11 h light:13 h dark, induces pupal diapause). Peak per and tim mRNA oscillations were closely associated with the light/dark transition. The conspicuous difference between the two photoperiodic conditions was that the sharp increase in per and tim mRNA abundance occurred during the light phase under long days but during the dark phase under short days. The diel oscillations were, at least in part, driven by an endogenous component, as demonstrated by transferring larvae to continuous darkness. The cells displaying Tim- and Per-like immunoreactivities (Tim- and Per-LIRs) were localized using anti-Drosophila-Per and anti-Chymomyza-Tim antibodies. Per-LIR and Tim-LIR co-localized in three groups of cells in each brain hemisphere. Two other groups, one in the brain hemispheres and the other in the fused ventral nerve ganglion, expressed only the Per-LIR.     

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.     

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.     

Changes in the Daily Rhythm of Lipid Metabolism in the Diabetic Retina

Disruption of circadian regulation was recently shown to cause diabetes and metabolic disease. We have previously demonstrated that retinal lipid metabolism contributed to the development of diabetic retinopathy. The goal of this study was to determine the effect of diabetes on circadian regulation of clock genes and lipid metabolism genes in the retina and retinal endothelial cells (REC). Diabetes had a pronounced inhibitory effect on the negative clock arm with lower amplitude of the period (per) 1 in the retina; lower amplitude and a phase shift of per2 in the liver; and a loss of cryptochrome (cry) 2 rhythmic pattern in suprachiasmatic nucleus (SCN). The positive clock arm was increased by diabetes with higher amplitude of circadian locomotor output cycles kaput (CLOCK) and brain and muscle aryl-hydrocarbon receptor nuclear translocator-like 1 (bmal1) and phase shift in bmal1 rhythmic oscillations in the retina; and higher bmal1 amplitude in the SCN. Peroxisome proliferator-activated receptor (PPAR) α exhibited rhythmic oscillation in retina and liver; PPARγ had lower amplitude in diabetic liver; sterol regulatory element-binding protein (srebp) 1c had higher amplitude in the retina but lower in the liver in STZ- induced diabetic animals. Both of Elongase (Elovl) 2 and Elovl4 had a rhythmic oscillation pattern in the control retina. Diabetic retinas lost Elovl4 rhythmic oscillation and had lower amplitude of Elovl2 oscillations. In line with the in vivo data, circadian expression levels of CLOCK, bmal1 and srebp1c had higher amplitude in rat REC (rREC) isolated from diabetic rats compared with control rats, while PPARγ and Elovl2 had lower amplitude in diabetic rREC. In conclusion, diabetes causes dysregulation of circadian expression of clock genes and the genes controlling lipid metabolism in the retina with potential implications for the development of diabetic retinopathy.     

Circadian clock of Drosophila montana is adapted to high variation in summer day lengths and temperatures prevailing at high latitudes

Photoperiodic regulation of the circadian rhythms in insect locomotor activity has been studied in several species, but seasonal entrainment of these rhythms is still poorly understood. We have traced the entrainment of activity rhythm of northern Drosophila montana flies in a climate chamber mimicking the photoperiods and day and night temperatures that the flies encounter in northern Finland during the summer. The experiment was started by transferring freshly emerged females into the chamber in early and late summer conditions to obtain both non-diapausing and diapausing females for the studies. The locomotor activity of the females and daily changes in the expression levels of two core circadian clock genes, timeless and period, in their heads were measured at different times of summer. The study revealed several features in fly rhythmicity that are likely to help the flies to cope with high variation in the day length and temperature typical to northern summers. First, both the non-diapausing and the diapausing females showed evening activity, which decreased towards the short day length as observed in the autumn in nature. Second, timeless and period genes showed concordant daily oscillations and seasonal shifts in their expression level in both types of females. Contrary to Drosophila melanogaster, oscillation profiles of these genes were similar to each other in all conditions, including the extremely long days in early summer and the cool temperatures in late summer, and their peak expression levels were not locked to lights-off transition in any photoperiod. Third, the diapausing females were less active than the non-diapausing ones, in spite of their younger age. Overall, the study showed that D. montana clock functions well under long day conditions, and that both the photoperiod and the daily temperature cycles are important zeitgebers for seasonal changes in the circadian rhythm of this species.     

Circadian control of permethrin-resistance in the mosquito Aedes aegypti

Daily fluctuation of permethrin-resistance was found in adult mosquito Aedes aegypti, the major vector of dengue viruses in Taiwan. We hypothesized there is a relationship between resistance and the circadian clock. To test our hypothesis we correlated changes in the knock-down time (KT₅₀) response to permethrin with the expression of the pyrethroid-resistant gene CYP9M9 and the clock gene period (per) during a 12:12 h photoperiodic cycle. Rhythmic expression of per peaked at early scotophase of the light-dark cycle and at early subjective night in constant darkness. The values of KT₅₀ and the expression of CYP9M9 also exhibited circadian rhythms in both susceptible and permethrin-resistant mosquito strains, from which we inferred a link to the circadian clock. The KT₅₀ was significantly longer in the light than in the dark phase, and the level of CYP9M9 mRNA was maximal in early scotophase, dropped to a minimum in the midnight and then slowly increased through the photophase. Existence of a clock control over mosquito sensitivity to permethrin was further indicated by reduced expression of CYP9M9 and reduced mosquito resistance to permethrin after temporal silencing of the per gene. These data provide the first evidence on the circadian control of insect resistance to permethrin.     

Drosophilarhythms: from brain to behavior

Theperiod(per) gene and thetimeless(tim) gene are essential components of the circadian clock inDrosophila melanogaster. Both gene products interact in interdependent feedback loops, producing a self-sustained cellular rhythmin situ. Several oscillating cells are combined to discrete pacemaker centers that control rhythmic behavior. This paper reviews the work on localizing the circadian pacemaker neurons controlling activity and eclosion, leading to questions about how these pacemaker cells are synchronized to the external light–dark cycle, and how they impose periodicity on behavior. The circadian system ofDrosophilais also compared with that of other arthropods.     

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.