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Functional analysis of the circadian clock gene period by RNA interference in nymphal crickets Gryllus bimaculatus
Journal of Insect Physiology 55 (2009) 396–400
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Functional analysis of the circadian clock gene period by RNA interference in nymphal crickets Gryllus bimaculatus Yoshiyuki Moriyama a, Tomoaki Sakamoto a, Akira Matsumoto b, Sumihare Noji c, Kenji Tomioka a,* a
Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan Center for Research and Advancement in Higher Education, Kyushu University, Ropponmatsu, Fukuoka 810-8560, Japan c Department of Life Systems, Institute of Technology and Science, University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima City 770-8506, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 September 2008 Received in revised form 11 November 2008 Accepted 12 November 2008
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. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Circadian rhythm Clock gene Cricket dsRNA Locomotor activity period RNA interference
1. Introduction Most organisms show daily cycles in their lives. In animals the rhythm is robustly observed in their activity (Dunlap et al., 2004). The rhythm is regulated by an endogenous circadian clock that is believed to oscillate in a daily manner using molecular machinery consisting of feedback loops of the so-called circadian clock genes and their product proteins (Hardin, 2006). In Drosophila melanogaster, for example, clock genes such as period (per), timeless (tim), Clock (Clk) and cycle (cyc) and their product proteins PERIOD (PER), TIMELESS (TIM), CLOCK (CLK) and CYCLE (CYC) play a major role. CLK and CYC form heterodimers and promote transcription of per and tim during the late subjective day. As a result, PER and TIM increase during the subjective night, enter the nucleus by forming heterodimers during the late subjective night, and suppress transcriptional activity of the CLK–CYC complex. The reduction of PER and TIM levels through the suppression at the late subjective night initiates the clock’s next cycle. A similar mechanism has been suggested in honey bees (Apis mellifera) and monarch butterflies (Danaus plexippus). But in these two species, mammalian type CRYPTOCHROME (CRY) instead of TIM is assumed to function as a transcriptional repressor (Rubin et al.,
* Corresponding author. Tel.: +81 86 251 8498; fax: +81 86 251 8498. E-mail address: [email protected] (K. Tomioka). 0022-1910/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2008.11.005
2006; Zhu et al., 2008). Vertebrates possess a similar molecular mechanism to the honey bee and the monarch butterfly but the phase of the oscillation is anti-phase: the peak times of per mRNA and its product protein are in the morning and during the day, respectively (Dunlap, 1999; Preitner et al., 2002; Yamaguchi et al., 2003). The clock mechanism synchronizes to the environmental light cycle to oscillate with an exactly 24 h period and to keep an appropriate phase relationship to it (Dunlap et al., 2004). The cricket G. bimaculatus shows a rhythm reversal from diurnal to nocturnal within several days after the imaginal molt (Tomioka and Chiba, 1982). The purposes of the present study are twofold: one is whether the underlying molecular oscillation is the same between nymphs and adults, and the other is to examine the phase angle relationship between the molecular oscillation and the light– dark cycle. In a previous study, we have shown that Gb’per mRNA cycles with a peak during the mid night in the adult optic lobe, the clock locus, and that knock-down of Gb’per mRNA levels by RNA interference disrupts the circadian locomotor rhythm in adults (Moriyama et al., 2008). We thus measured Gb’per mRNA levels with quantitative real-time RT-PCR and investigated the role of Gb’per gene with RNA interference technique. The results show that Gb’per mRNA is expressed cyclically in nymphs with peaks at mid night, as in adults, and its knock-down by RNAi disrupts the circadian rhythm, suggesting that nymphs and adults share a common mechanism involving the clock gene Gb’per.
Y. Moriyama et al. / Journal of Insect Physiology 55 (2009) 396–400
2. Materials and methods 2.1. Experimental animals Third instar nymphal crickets, G. bimaculatus, were used. They were obtained from a laboratory colony maintained under standard environmental conditions: a lighting regimen of 12 h light and 12 h dark (LD 12:12, light: 06:00–18:00; Japanese standard time, JST) and a constant temperature of 25 8C. They were fed laboratory chow and water. 2.2. Measurement of mRNA levels Gb’per mRNA levels were measured with quantitative real-time RT-PCR according to the method described by Moriyama et al. (2008). Total RNA was extracted and purified from seven heads of third instar nymph with ISOGEN (Nipppon gene) and was treated with DNase I to remove contaminating genomic DNA. About 500 ng total RNA of each sample was reverse transcribed with random 6mers using ExscriptTM RT reagent Kit (Takara). Quantitative real-time PCR was performed with Mx3000PTM Real-Time PCR System (Stratagene) using FullVelocityTM SYBR1 Green QPCR Master Mix (Stratagene) including SYBR Green with primers 50 AAGCAAGCAAGCATCCTCAT-30 and 50 -CTGAGAAAGGAGGCCACAAG-30 , which corresponded to a sequence of 2344–2490 bp of Gb’per cDNA. As an internal reference gene we used Gryllus bimaculatus ribosomal protein L18a (rpl18a, GenBank Accession No. DC448653). The primers used for rpl18a were 50 -GCTCCGGATTACATCGTTGC-30 and 50 -GCCAAATGCCGAAGTTCTTG-30 . In both cases, a single expected amplicon was confirmed by melting analysis. The results were analyzed using the software associated with the instrument. The values for Gb’per were normalized with the values for rpl18a at each time point. Results of four independent experiments were pooled to calculate the mean S.E.M.
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by a computer. The chambers and the activity sensing system were placed in an incubator (Sanyo, MIR-153), in which the light and temperature were controlled. Lighting conditions were given by a cool white fluorescent lamp connected to an electric timer, and temperature was kept constant at 25 8C. The light intensity in the activity chamber was 600–1000 lux at the animal’s level, varying with proximity to the lamp. The raw data were displayed as conventional double-plotted actograms to judge activity patterns, and judgment of rhythmicity and calculation of free-running periods were made by the chi-square periodogram (Sokolove and Bushell, 1978). If peaks of periodogram appeared above the 0.05 confidence level, the rhythm was designated as statistically significant. 3. Results 3.1. Rhythmic expression of Gb’per mRNA levels in nymphal head To determine whether Gb’per transcripts oscillate in a circadian manner in nymphs, we measured the levels of Gb’per mRNA in the heads of third instar nymphs under LD 12:12 by performing quantitative real-time RT-PCR analysis (Fig. 1A). The results showed that Gb’per mRNA levels in the nymphal heads changed rhythmically, and a peak of oscillation occurred at mid night (ZT18: ZT is the zeitgeber time within a light–dark cycle experiment; ZT0 corresponds to lights-on and ZT12 to the lights-off events). The expression level of Gb’per mRNA at ZT18 was significantly greater than at other time points (P < 0.05, ANOVA followed by Tuckytest). We then examined the levels of Gb’per mRNA under constant darkness to determine whether this oscillation of Gb’per mRNA was endogenous. The rhythmic expression of Gb’per mRNA in the head was clearly reproduced in the third day after transfer to DD
2.3. RNA interference Double stranded RNAs (dsRNA) for Gb’per and DsRed2, derived from a coral species (Discosoma sp.), were synthesized as previously described (Moriyama et al., 2008) using MEGAscript1 High Yield Transcription Kit (Ambion). Sequence (853–1657 bp) of Gb’per; cloned into TOPO-pCR II vector, was amplified with M13 forward and reverse primer. DsRed2 in pDsRed2-N1 (Clontech) was amplified with the forward and reverse primers containing the T7 promoter. With these linearized DNA fragments, RNAs were synthesized with T7 RNA polymerase and SP6 RNA polymerase. The same amounts of sense and antisense RNAs were mixed, denatured for 5 min at 100 8C, and annealed by a gradual cool down to room temperature. After ethanol precipitation, the obtained dsRNA was suspended in Ultra Pure Water (Invitrogen) and the concentration was adjusted to 20 mM. The dsRNA solutions were stored at 80 8C. 207 nl dsRNA solution was injected into the abdomen of third instar nymphs anesthetized on ice with a nanoliter injector (WPI). 2.4. Behavioral analysis To monitor locomotor activity, we used a computerized system including actographs operated photoelectronically. Third instar nymphs were individually housed in an acrylic rectangular chamber (1.8 cm 1.2 cm 10.0 cm). At one end of the chamber laboratory chow was fixed, and at the other end water was supplied from a bottle with a silicon tube filled with damped absorbent cotton. A moving cricket interrupted an infrared beam and the number of interruptions during each 6 min was recorded
Fig. 1. Rhythmic expression of Gb’per mRNA in G. bimaculatus third instar nymph heads under LD 12:12 (A) and DD (B). The abundance of Gb’per mRNA was measured by quantitative real-time RT-PCR. Total RNA was extracted from heads of the third instar nymphs that were kept in LD 12:12, 25 8C or in constant darkness for 2 days starting ZT12. The heads were collected at 4 h interval starting at ZT2 (A) or CT2 on the third day of DD (B). The white and black bars indicate light and dark fractions, respectively. The data collected from three independent experiments were averaged and plotted as mean S.E.M. for each time point. The abundance of rpl18a was used as a reference.
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(P < 0.01, ANOVA), with a peak occurring at the middle of the subjective night (CT18) (Fig. 1B). 3.2. Locomotor rhythm of G. bimaculatus nymphs To examine the role of the Gb’per gene in circadian rhythm generation in nymphs, we chose the locomotor rhythm as a hand of the clock machinery. We first measured activity of 16 intact nymphs. A representative actogram is shown in Fig. 2A. Under LD 12:12, the nymphs showed a diurnal rhythm entrained to light– dark cycles with a peak in the middle of the day, but with some activity during the night as has been reported previously (Tomioka and Chiba, 1982). Under DD conditions, their diurnal locomotor rhythm persisted and free-ran with an average period of 24.2 0.1 (mean S.E.M.) h. The activity level fluctuated with a period of 7–10 days. This was probably associated with molting (Tomioka and Chiba, 1982). For a control, we injected DsRed2 dsRNA (20 mM, 207 nl) into the abdomen of 29 third instar nymphal crickets. This dsRNA was used as a negative control in our previous study (Moriyama et al., 2008). The injected nymphs showed a clear diurnal locomotor rhythm under LD 12:12, which persisted in the ensuing DD (Fig. 2B). The average free-running period of these crickets was 24.3 0.1 (mean S.E.M.) h, and the period was not significantly different from that of the intact crickets. 3.3. Abolishment of the locomotor rhythm by Gb’per dsRNA in G. bimaculatus nymphs We then injected Gb’per dsRNA (20 mM, 207 nl) into the abdomen of 39 third instar nymphs and investigated its effects on the locomotor rhythm. Five showed a diurnal locomotor rhythm under LD 12:12, running free under the ensuing DD with a period longer than 24 h (example Fig. 2C), but the rhythm under DD was apparently weaker than that of the control (Fig. 2A and B). The average free-running period of these five crickets was 24.9 0.3 (mean S.E.M.) h, and this was significantly longer than that of the control and intact crickets (t-test, P < 0.05). In the remaining 34 nymphs, the activity level was too low to judge whether locomotor rhythm existed or not in LD 12:12. The low level of activity was probably associated with molting. Under DD, they were apparently arrhythmic with activity dispersed over the entire day and no significant periodicity was detected by the chi-square periodogram (Fig. 2D). 3.4. Gb’per dsRNA suppresses levels of Gb’per transcripts To examine whether Gb’per dsRNA suppressed the Gb’per transcript levels, we measured levels of Gb’per mRNA in the heads of intact third instar nymphs and nymphs with Gb’per dsRNA injected under LD12:12 at 25 8C by quantitative real-time RT-PCR (Fig. 3). For the injected nymphs, heads were collected at ZT18 seven days after injection. In injected crickets, the Gb’per mRNA expression level was reduced to about 25% of that in intact crickets, suggesting that injected Gb’per dsRNA suppressed the expression of Gb’per mRNA through RNA interference. 4. Discussion 4.1. Rhythmic expression of period in G. bimaculatus nymphs The present study revealed that Gb’per is rhythmically expressed in the third instar nymphal head. The rhythmic profile was similar to that of the adult optic lobe, peaking in mid night (Moriyama et al., 2008), suggesting that the phase of the underlying molecular rhythm is similar between nymphs and adults irrespective of the reversed phase of the locomotor rhythm
(Tomioka and Chiba, 1982). The similar phasing of the Gb’per transcripts rhythm between nymphs and adults is consistent with the fact that electrical activity of the optic lobe and the light induced responses of optic lobe interneurons are always nocturnally increasing, both in nymphs and adults (Tomioka and Chiba, 1992; Uemura and Tomioka, 2006). The phase reversal in locomotor activity is probably caused by the switch of the clock’s connection with diurnal to nocturnal locomotor centers, associated with an increase of cerebral 5-HT levels (Tomioka and Chiba, 1982, 1992; Nishinokubi and Tomioka, 2000). The nocturnal increase in per mRNA is commonly observed in insects through diverse genera, including the fruit fly D. melanogaster (Hardin et al., 1990), the honey bees Apis serana japonica and A. mellifera (Rubin et al., 2006; Fuchikawa and Shimizu, 2007), the monarch butterfly D. plexippus (Zhu et al., 2008) and the silkmoth Antheraea pernyi (Sauman and Reppert, 1996). Thus the phase relationship between the core molecular oscillatory machinery and the light cycle may be common throughout the insect phylum and opposite to that of vertebrate species, in which per mRNA levels peak during the early day (Stanewsky, 2003). 4.2. Possible role of per in rhythm generation In the present study, we have shown that Gb’per mRNA levels in the nymphal head were knocked-down to 25% of the level found in intact crickets and that most crickets injected with Gb’per dsRNA lost their circadian locomotor activity. These results clearly indicate the importance of the Gb’per gene in circadian rhythm generation in the nymphal cricket G. bimaculatus. The injection of DsRed2 dsRNA derived from a coral species, Discosoma sp. confirms specificity of the gene knock-down by Gb’per dsRNA: DsRed2 dsRNA had no effect on the rhythmicity of the cricket. The specificity in the knock-down of gene expression agrees well with our previous report using the same dsRNA in adult crickets (Moriyama et al., 2008) as well as other reports on different genes (Meyering-Vos et al., 2006; Meyering-Vos and Muller, 2007). The circadian rhythmic expressions of Gb’per mRNA in nymphal crickets and the disruption of circadian rhythm by Gb’per dsRNA suggest that Gb’per expression may be the core part of the circadian clock like in Drosophila, as we have suggested in our previous paper (Moriyama et al., 2008). The product protein quite likely oscillates to serve as a feedback suppressor of its own transcription. The knock-down of per mRNA by RNAi may result in a reduced level of PERIOD protein that stops the clock machinery as in per0 mutant flies of Drosophila (Hardin et al., 1990). A small fraction of nymphs treated with Gb’per dsRNA showed a weak locomotor rhythm in DD with a period significantly longer than control animals. Considering dosage effects of the per gene on the locomotor rhythm in Drosophila (Smith and Konopka, 1982), these weak rhythms with longer periods are probably due to a weak oscillation that still persisted irrespective of reduced levels of per transcripts. There are some differences in effects of RNAi between nymphs and adults. First, the knock-down was more prominent in nymphs. The level of mRNA was about 25% in nymphs treated with Gb’per dsRNA (Fig. 3), whereas it was about 50% in adults (Moriyama et al., 2008). This might be caused by the different amount of dsRNA injected: 207 nl was injected in nymphs, while 760 nl was used for adults in our previous experiment (Moriyama et al., 2008). Second, the free-running period of the locomotor rhythm of crickets that maintained the rhythm after the treatment with Gb’per dsRNA was significantly shorter in nymphs (24.9 0.29 h) than in adults (26.5 0.72 h) (t-test, P < 0.05). Although the reason for this different effect is unclear, the required level of Gb’per mRNA abundance for maintenance of rhythmicity may be different between nymphs and adults.
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Fig. 2. Double-plotted actograms (left) and chi-square periodograms (right) of four representative intact third instar G. bimaculatus nymphs (A), or nymphs injected with DsRed2 dsRNA (B) or Gb’per dsRNA (C and D). The crickets were first kept in LD12:12, 25 8C for 5 days, then transferred to DD. White and black bars above actograms indicate light and dark phases, respectively. Arrows in the actograms indicate the day of transfer from LD12:12 to DD. An oblique line in the periodogram indicates significant level of P < 0.05 and the peak value above the line was designated as significant.
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during the sensitive stage, we may be able to test this hypothesis directly. Acknowledgments This study was supported in part by grants from JSPS to KT. References
Fig. 3. Relative abundance of Gb’per mRNA in heads of intact third instar nymphs and of Gb’per dsRNA injected third instar nymphs (per dsRNA). Ordinate indicates relative abundance of Gb’per mRNA. Samples were collected at ZT18 in LD 12:12, 25 8C. In Gb’per dsRNA injected third instar nymphs, heads were collected on the seventh day after injection. The abundance of Gb’per mRNA was measured by quantitative real-time RT-PCR with total RNA extracted from heads of the third instar cricket nymphs. The data collected from three independent experiments were averaged and plotted as mean S.E.M. for intact and Gb’per dsRNA injected crickets. The abundance of rpl18a was used as reference.
There are two important issues to be addressed in our future study. One is the role of Gb’per gene in the circadian molecular machinery. The oscillation and the subcellular localization of its product protein need to be clarified. The other is the identification of clock neurons within the optic lobe. Although, in crickets, the circadian clock has been localized in the optic lobe, the location of the clock neurons remains to be determined (Tomioka and Abdelsalam, 2004). 4.3. Advantage of nymphal RNA interference In this study we found that Gb’per RNAi effectively disrupts the circadian rhythm of nymphal crickets. Although we did not examine how long the effect of Gb’per dsRNA continues, we confirmed that the arrhythmicity persisted up to 44 days after the injection. The reason for this prolonged effect of Gb’per dsRNA after a single injection remains unknown. In most insects the effect is rather transient and special techniques such as RNAi constructs which endogenously produce dsRNA are usually required to continuously silence target gene expression. G. bimaculatus seems thus exceptionally suitable for RNAi experiments. In addition to the analysis of the functional role of clock genes, nymphal RNAi would be a useful technique for analysis of photoperiodic response in the nymphal stage. Some species of crickets show photoperiodism in their nymphal development, and the sensitive stage for photoperiods is often restricted to certain developmental stages. The circadian clock is hypothesized to be involved in the photoperiodic time measurement, and if the circadian clock were stopped by RNA interference
Dunlap, J.C., 1999. Molecular bases for circadian biological clocks. Cell 96, 271–290. Dunlap, J.C., Loros, J., DeCoursey, P.J., 2004. Chronobiology: Biological Timekeeping. Sinauer, Sunderland, MA. Fuchikawa, T., Shimizu, I., 2007. Effects of temperature on circadian rhythm in the Japanese honeybee, Apis cerana japonica. Journal of Insect Physiology 53, 1179– 1187. Hardin, P.E., 2006. Essential and expendable features of the circadian timekeeping mechanism. Current Opinion in Neurobiology 16, 686–692. Hardin, P.E., Hall, J.C., Rosbash, M., 1990. Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels. Nature 343, 536–540. Meyering-Vos, M., Muller, A., 2007. RNA interference suggests sulfakinins as satiety effectors in the cricket Gryllus bimaculatus. Journal of Insect Physiology 53, 840– 848. Meyering-Vos, M., Merz, S., Sertkol, M., Hoffmann, K.H., 2006. Functional analysis of the allatostatin A gene in the cricket Gryllus bimaculatus and the armyworm Spodoptera frugiperda. Insect Biochemistry and Molecular Biology 36, 492–504. Moriyama, Y., Sakamoto, T., Karpova, S.G., Matsumoto, A., Noji, S., Tomioka, K., 2008. RNA interference of the clock gene period disrupts circadian rhythms in the cricket Gryllus bimaculatus. Journal of Biological Rhythms 23, 308–318. Nishinokubi, I., Tomioka, K., 2000. Analysis of the mechanism underlying the rhythm reversal from diurnal to nocturnal in the cricket Gryllus bimaculatus, with special reference to the role of serotonin. Zoological Science 17, 1075–1080. Preitner, N., Damiola, F., Lopez-Molina, L., Zakany, J., Duboule, D., Albrecht, U., Schibler, U., 2002. The orphan nuclear receptor REV-ERBa controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell 110, 251–260. Rubin, E.B., Shemesh, Y., Cohen, M., Elgavish, S., Robertson, H.M., Bloch, G., 2006. Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee (Apis mellifera) and shed new light on the molecular evolution of the circadian clock. Genome Research 16, 1352–1365. Sauman, I., Reppert, S.M., 1996. Circadian clock neurons in the silkmoth Antheraea pernyi: novel mechanisms of period protein regulation. Neuron 17, 889–900. Smith, R.F., Konopka, R.J., 1982. Effects of dosage alterations at the per locus on the period of the circadian clock of Drosophila. Molecular and General Genetics 185, 30–36. Sokolove, P.G., Bushell, W.N., 1978. The chi square periodogram: its utility for analysis of circadian rhythm. Journal of Theoretical Biology 72, 131–160. Stanewsky, R., 2003. Genetic analysis of the circadian system in Drosophila melanogaster and mammals. Journal of Neurobiology 54, 111–147. Tomioka, K., Chiba, Y., 1982. Post-embryonic development of circadian rhythm in the cricket, Gryllus bimaculatus. Journal of Comparative Physiology A 147, 299– 304. Tomioka, K., Chiba, Y., 1992. Characterization of optic lobe circadian pacemaker by in situ and in vitro recording of neuronal activity in the cricket Gryllus bimaculatus. Journal of Comparative Physiology A 171, 1–7. Tomioka, K., Abdelsalam, S.A., 2004. Circadian organization in hemimetabolous insects. Zoological Science 21, 1153–1162. Uemura, H., Tomioka, K., 2006. Postembryonic changes in circadian photo-responsiveness rhythms of optic lobe interneurons in the cricket Gryllus bimaculatus. Journal of Biological Rhythms 21, 1–11. Yamaguchi, S., Isejima, H., Matsuo, T., Okura, R., Yagita, K., Kobayashi, M., Okamura, H., 2003. Synchronization of cellular clocks in the suprachiasmatic nucleus. Science 302, 1408–1412. Zhu, H., Sauman, I., Yuan, Q., Casselman, A., Emery-Le, M., Emery, P., Reppert, S.M., 2008. Cryptochromes define a novel circadian clock mechanism in monarch butterflies that may underlie sun compass navigation. PLoS Biology 6, 138–155.
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Functional analysis of the circadian clock gene period by RNA interference in nymphal crickets Gryllus bimaculatus Yoshiyuki Moriyama a, Tomoaki Sakamoto a, Akira Matsumoto b, Sumihare Noji c, Kenji Tomioka a,* a
Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan Center for Research and Advancement in Higher Education, Kyushu University, Ropponmatsu, Fukuoka 810-8560, Japan c Department of Life Systems, Institute of Technology and Science, University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima City 770-8506, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 September 2008 Received in revised form 11 November 2008 Accepted 12 November 2008
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. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Circadian rhythm Clock gene Cricket dsRNA Locomotor activity period RNA interference
1. Introduction Most organisms show daily cycles in their lives. In animals the rhythm is robustly observed in their activity (Dunlap et al., 2004). The rhythm is regulated by an endogenous circadian clock that is believed to oscillate in a daily manner using molecular machinery consisting of feedback loops of the so-called circadian clock genes and their product proteins (Hardin, 2006). In Drosophila melanogaster, for example, clock genes such as period (per), timeless (tim), Clock (Clk) and cycle (cyc) and their product proteins PERIOD (PER), TIMELESS (TIM), CLOCK (CLK) and CYCLE (CYC) play a major role. CLK and CYC form heterodimers and promote transcription of per and tim during the late subjective day. As a result, PER and TIM increase during the subjective night, enter the nucleus by forming heterodimers during the late subjective night, and suppress transcriptional activity of the CLK–CYC complex. The reduction of PER and TIM levels through the suppression at the late subjective night initiates the clock’s next cycle. A similar mechanism has been suggested in honey bees (Apis mellifera) and monarch butterflies (Danaus plexippus). But in these two species, mammalian type CRYPTOCHROME (CRY) instead of TIM is assumed to function as a transcriptional repressor (Rubin et al.,
* Corresponding author. Tel.: +81 86 251 8498; fax: +81 86 251 8498. E-mail address: [email protected] (K. Tomioka). 0022-1910/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2008.11.005
2006; Zhu et al., 2008). Vertebrates possess a similar molecular mechanism to the honey bee and the monarch butterfly but the phase of the oscillation is anti-phase: the peak times of per mRNA and its product protein are in the morning and during the day, respectively (Dunlap, 1999; Preitner et al., 2002; Yamaguchi et al., 2003). The clock mechanism synchronizes to the environmental light cycle to oscillate with an exactly 24 h period and to keep an appropriate phase relationship to it (Dunlap et al., 2004). The cricket G. bimaculatus shows a rhythm reversal from diurnal to nocturnal within several days after the imaginal molt (Tomioka and Chiba, 1982). The purposes of the present study are twofold: one is whether the underlying molecular oscillation is the same between nymphs and adults, and the other is to examine the phase angle relationship between the molecular oscillation and the light– dark cycle. In a previous study, we have shown that Gb’per mRNA cycles with a peak during the mid night in the adult optic lobe, the clock locus, and that knock-down of Gb’per mRNA levels by RNA interference disrupts the circadian locomotor rhythm in adults (Moriyama et al., 2008). We thus measured Gb’per mRNA levels with quantitative real-time RT-PCR and investigated the role of Gb’per gene with RNA interference technique. The results show that Gb’per mRNA is expressed cyclically in nymphs with peaks at mid night, as in adults, and its knock-down by RNAi disrupts the circadian rhythm, suggesting that nymphs and adults share a common mechanism involving the clock gene Gb’per.
Y. Moriyama et al. / Journal of Insect Physiology 55 (2009) 396–400
2. Materials and methods 2.1. Experimental animals Third instar nymphal crickets, G. bimaculatus, were used. They were obtained from a laboratory colony maintained under standard environmental conditions: a lighting regimen of 12 h light and 12 h dark (LD 12:12, light: 06:00–18:00; Japanese standard time, JST) and a constant temperature of 25 8C. They were fed laboratory chow and water. 2.2. Measurement of mRNA levels Gb’per mRNA levels were measured with quantitative real-time RT-PCR according to the method described by Moriyama et al. (2008). Total RNA was extracted and purified from seven heads of third instar nymph with ISOGEN (Nipppon gene) and was treated with DNase I to remove contaminating genomic DNA. About 500 ng total RNA of each sample was reverse transcribed with random 6mers using ExscriptTM RT reagent Kit (Takara). Quantitative real-time PCR was performed with Mx3000PTM Real-Time PCR System (Stratagene) using FullVelocityTM SYBR1 Green QPCR Master Mix (Stratagene) including SYBR Green with primers 50 AAGCAAGCAAGCATCCTCAT-30 and 50 -CTGAGAAAGGAGGCCACAAG-30 , which corresponded to a sequence of 2344–2490 bp of Gb’per cDNA. As an internal reference gene we used Gryllus bimaculatus ribosomal protein L18a (rpl18a, GenBank Accession No. DC448653). The primers used for rpl18a were 50 -GCTCCGGATTACATCGTTGC-30 and 50 -GCCAAATGCCGAAGTTCTTG-30 . In both cases, a single expected amplicon was confirmed by melting analysis. The results were analyzed using the software associated with the instrument. The values for Gb’per were normalized with the values for rpl18a at each time point. Results of four independent experiments were pooled to calculate the mean S.E.M.
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by a computer. The chambers and the activity sensing system were placed in an incubator (Sanyo, MIR-153), in which the light and temperature were controlled. Lighting conditions were given by a cool white fluorescent lamp connected to an electric timer, and temperature was kept constant at 25 8C. The light intensity in the activity chamber was 600–1000 lux at the animal’s level, varying with proximity to the lamp. The raw data were displayed as conventional double-plotted actograms to judge activity patterns, and judgment of rhythmicity and calculation of free-running periods were made by the chi-square periodogram (Sokolove and Bushell, 1978). If peaks of periodogram appeared above the 0.05 confidence level, the rhythm was designated as statistically significant. 3. Results 3.1. Rhythmic expression of Gb’per mRNA levels in nymphal head To determine whether Gb’per transcripts oscillate in a circadian manner in nymphs, we measured the levels of Gb’per mRNA in the heads of third instar nymphs under LD 12:12 by performing quantitative real-time RT-PCR analysis (Fig. 1A). The results showed that Gb’per mRNA levels in the nymphal heads changed rhythmically, and a peak of oscillation occurred at mid night (ZT18: ZT is the zeitgeber time within a light–dark cycle experiment; ZT0 corresponds to lights-on and ZT12 to the lights-off events). The expression level of Gb’per mRNA at ZT18 was significantly greater than at other time points (P < 0.05, ANOVA followed by Tuckytest). We then examined the levels of Gb’per mRNA under constant darkness to determine whether this oscillation of Gb’per mRNA was endogenous. The rhythmic expression of Gb’per mRNA in the head was clearly reproduced in the third day after transfer to DD
2.3. RNA interference Double stranded RNAs (dsRNA) for Gb’per and DsRed2, derived from a coral species (Discosoma sp.), were synthesized as previously described (Moriyama et al., 2008) using MEGAscript1 High Yield Transcription Kit (Ambion). Sequence (853–1657 bp) of Gb’per; cloned into TOPO-pCR II vector, was amplified with M13 forward and reverse primer. DsRed2 in pDsRed2-N1 (Clontech) was amplified with the forward and reverse primers containing the T7 promoter. With these linearized DNA fragments, RNAs were synthesized with T7 RNA polymerase and SP6 RNA polymerase. The same amounts of sense and antisense RNAs were mixed, denatured for 5 min at 100 8C, and annealed by a gradual cool down to room temperature. After ethanol precipitation, the obtained dsRNA was suspended in Ultra Pure Water (Invitrogen) and the concentration was adjusted to 20 mM. The dsRNA solutions were stored at 80 8C. 207 nl dsRNA solution was injected into the abdomen of third instar nymphs anesthetized on ice with a nanoliter injector (WPI). 2.4. Behavioral analysis To monitor locomotor activity, we used a computerized system including actographs operated photoelectronically. Third instar nymphs were individually housed in an acrylic rectangular chamber (1.8 cm 1.2 cm 10.0 cm). At one end of the chamber laboratory chow was fixed, and at the other end water was supplied from a bottle with a silicon tube filled with damped absorbent cotton. A moving cricket interrupted an infrared beam and the number of interruptions during each 6 min was recorded
Fig. 1. Rhythmic expression of Gb’per mRNA in G. bimaculatus third instar nymph heads under LD 12:12 (A) and DD (B). The abundance of Gb’per mRNA was measured by quantitative real-time RT-PCR. Total RNA was extracted from heads of the third instar nymphs that were kept in LD 12:12, 25 8C or in constant darkness for 2 days starting ZT12. The heads were collected at 4 h interval starting at ZT2 (A) or CT2 on the third day of DD (B). The white and black bars indicate light and dark fractions, respectively. The data collected from three independent experiments were averaged and plotted as mean S.E.M. for each time point. The abundance of rpl18a was used as a reference.
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(P < 0.01, ANOVA), with a peak occurring at the middle of the subjective night (CT18) (Fig. 1B). 3.2. Locomotor rhythm of G. bimaculatus nymphs To examine the role of the Gb’per gene in circadian rhythm generation in nymphs, we chose the locomotor rhythm as a hand of the clock machinery. We first measured activity of 16 intact nymphs. A representative actogram is shown in Fig. 2A. Under LD 12:12, the nymphs showed a diurnal rhythm entrained to light– dark cycles with a peak in the middle of the day, but with some activity during the night as has been reported previously (Tomioka and Chiba, 1982). Under DD conditions, their diurnal locomotor rhythm persisted and free-ran with an average period of 24.2 0.1 (mean S.E.M.) h. The activity level fluctuated with a period of 7–10 days. This was probably associated with molting (Tomioka and Chiba, 1982). For a control, we injected DsRed2 dsRNA (20 mM, 207 nl) into the abdomen of 29 third instar nymphal crickets. This dsRNA was used as a negative control in our previous study (Moriyama et al., 2008). The injected nymphs showed a clear diurnal locomotor rhythm under LD 12:12, which persisted in the ensuing DD (Fig. 2B). The average free-running period of these crickets was 24.3 0.1 (mean S.E.M.) h, and the period was not significantly different from that of the intact crickets. 3.3. Abolishment of the locomotor rhythm by Gb’per dsRNA in G. bimaculatus nymphs We then injected Gb’per dsRNA (20 mM, 207 nl) into the abdomen of 39 third instar nymphs and investigated its effects on the locomotor rhythm. Five showed a diurnal locomotor rhythm under LD 12:12, running free under the ensuing DD with a period longer than 24 h (example Fig. 2C), but the rhythm under DD was apparently weaker than that of the control (Fig. 2A and B). The average free-running period of these five crickets was 24.9 0.3 (mean S.E.M.) h, and this was significantly longer than that of the control and intact crickets (t-test, P < 0.05). In the remaining 34 nymphs, the activity level was too low to judge whether locomotor rhythm existed or not in LD 12:12. The low level of activity was probably associated with molting. Under DD, they were apparently arrhythmic with activity dispersed over the entire day and no significant periodicity was detected by the chi-square periodogram (Fig. 2D). 3.4. Gb’per dsRNA suppresses levels of Gb’per transcripts To examine whether Gb’per dsRNA suppressed the Gb’per transcript levels, we measured levels of Gb’per mRNA in the heads of intact third instar nymphs and nymphs with Gb’per dsRNA injected under LD12:12 at 25 8C by quantitative real-time RT-PCR (Fig. 3). For the injected nymphs, heads were collected at ZT18 seven days after injection. In injected crickets, the Gb’per mRNA expression level was reduced to about 25% of that in intact crickets, suggesting that injected Gb’per dsRNA suppressed the expression of Gb’per mRNA through RNA interference. 4. Discussion 4.1. Rhythmic expression of period in G. bimaculatus nymphs The present study revealed that Gb’per is rhythmically expressed in the third instar nymphal head. The rhythmic profile was similar to that of the adult optic lobe, peaking in mid night (Moriyama et al., 2008), suggesting that the phase of the underlying molecular rhythm is similar between nymphs and adults irrespective of the reversed phase of the locomotor rhythm
(Tomioka and Chiba, 1982). The similar phasing of the Gb’per transcripts rhythm between nymphs and adults is consistent with the fact that electrical activity of the optic lobe and the light induced responses of optic lobe interneurons are always nocturnally increasing, both in nymphs and adults (Tomioka and Chiba, 1992; Uemura and Tomioka, 2006). The phase reversal in locomotor activity is probably caused by the switch of the clock’s connection with diurnal to nocturnal locomotor centers, associated with an increase of cerebral 5-HT levels (Tomioka and Chiba, 1982, 1992; Nishinokubi and Tomioka, 2000). The nocturnal increase in per mRNA is commonly observed in insects through diverse genera, including the fruit fly D. melanogaster (Hardin et al., 1990), the honey bees Apis serana japonica and A. mellifera (Rubin et al., 2006; Fuchikawa and Shimizu, 2007), the monarch butterfly D. plexippus (Zhu et al., 2008) and the silkmoth Antheraea pernyi (Sauman and Reppert, 1996). Thus the phase relationship between the core molecular oscillatory machinery and the light cycle may be common throughout the insect phylum and opposite to that of vertebrate species, in which per mRNA levels peak during the early day (Stanewsky, 2003). 4.2. Possible role of per in rhythm generation In the present study, we have shown that Gb’per mRNA levels in the nymphal head were knocked-down to 25% of the level found in intact crickets and that most crickets injected with Gb’per dsRNA lost their circadian locomotor activity. These results clearly indicate the importance of the Gb’per gene in circadian rhythm generation in the nymphal cricket G. bimaculatus. The injection of DsRed2 dsRNA derived from a coral species, Discosoma sp. confirms specificity of the gene knock-down by Gb’per dsRNA: DsRed2 dsRNA had no effect on the rhythmicity of the cricket. The specificity in the knock-down of gene expression agrees well with our previous report using the same dsRNA in adult crickets (Moriyama et al., 2008) as well as other reports on different genes (Meyering-Vos et al., 2006; Meyering-Vos and Muller, 2007). The circadian rhythmic expressions of Gb’per mRNA in nymphal crickets and the disruption of circadian rhythm by Gb’per dsRNA suggest that Gb’per expression may be the core part of the circadian clock like in Drosophila, as we have suggested in our previous paper (Moriyama et al., 2008). The product protein quite likely oscillates to serve as a feedback suppressor of its own transcription. The knock-down of per mRNA by RNAi may result in a reduced level of PERIOD protein that stops the clock machinery as in per0 mutant flies of Drosophila (Hardin et al., 1990). A small fraction of nymphs treated with Gb’per dsRNA showed a weak locomotor rhythm in DD with a period significantly longer than control animals. Considering dosage effects of the per gene on the locomotor rhythm in Drosophila (Smith and Konopka, 1982), these weak rhythms with longer periods are probably due to a weak oscillation that still persisted irrespective of reduced levels of per transcripts. There are some differences in effects of RNAi between nymphs and adults. First, the knock-down was more prominent in nymphs. The level of mRNA was about 25% in nymphs treated with Gb’per dsRNA (Fig. 3), whereas it was about 50% in adults (Moriyama et al., 2008). This might be caused by the different amount of dsRNA injected: 207 nl was injected in nymphs, while 760 nl was used for adults in our previous experiment (Moriyama et al., 2008). Second, the free-running period of the locomotor rhythm of crickets that maintained the rhythm after the treatment with Gb’per dsRNA was significantly shorter in nymphs (24.9 0.29 h) than in adults (26.5 0.72 h) (t-test, P < 0.05). Although the reason for this different effect is unclear, the required level of Gb’per mRNA abundance for maintenance of rhythmicity may be different between nymphs and adults.
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Fig. 2. Double-plotted actograms (left) and chi-square periodograms (right) of four representative intact third instar G. bimaculatus nymphs (A), or nymphs injected with DsRed2 dsRNA (B) or Gb’per dsRNA (C and D). The crickets were first kept in LD12:12, 25 8C for 5 days, then transferred to DD. White and black bars above actograms indicate light and dark phases, respectively. Arrows in the actograms indicate the day of transfer from LD12:12 to DD. An oblique line in the periodogram indicates significant level of P < 0.05 and the peak value above the line was designated as significant.
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during the sensitive stage, we may be able to test this hypothesis directly. Acknowledgments This study was supported in part by grants from JSPS to KT. References
Fig. 3. Relative abundance of Gb’per mRNA in heads of intact third instar nymphs and of Gb’per dsRNA injected third instar nymphs (per dsRNA). Ordinate indicates relative abundance of Gb’per mRNA. Samples were collected at ZT18 in LD 12:12, 25 8C. In Gb’per dsRNA injected third instar nymphs, heads were collected on the seventh day after injection. The abundance of Gb’per mRNA was measured by quantitative real-time RT-PCR with total RNA extracted from heads of the third instar cricket nymphs. The data collected from three independent experiments were averaged and plotted as mean S.E.M. for intact and Gb’per dsRNA injected crickets. The abundance of rpl18a was used as reference.
There are two important issues to be addressed in our future study. One is the role of Gb’per gene in the circadian molecular machinery. The oscillation and the subcellular localization of its product protein need to be clarified. The other is the identification of clock neurons within the optic lobe. Although, in crickets, the circadian clock has been localized in the optic lobe, the location of the clock neurons remains to be determined (Tomioka and Abdelsalam, 2004). 4.3. Advantage of nymphal RNA interference In this study we found that Gb’per RNAi effectively disrupts the circadian rhythm of nymphal crickets. Although we did not examine how long the effect of Gb’per dsRNA continues, we confirmed that the arrhythmicity persisted up to 44 days after the injection. The reason for this prolonged effect of Gb’per dsRNA after a single injection remains unknown. In most insects the effect is rather transient and special techniques such as RNAi constructs which endogenously produce dsRNA are usually required to continuously silence target gene expression. G. bimaculatus seems thus exceptionally suitable for RNAi experiments. In addition to the analysis of the functional role of clock genes, nymphal RNAi would be a useful technique for analysis of photoperiodic response in the nymphal stage. Some species of crickets show photoperiodism in their nymphal development, and the sensitive stage for photoperiods is often restricted to certain developmental stages. The circadian clock is hypothesized to be involved in the photoperiodic time measurement, and if the circadian clock were stopped by RNA interference
Dunlap, J.C., 1999. Molecular bases for circadian biological clocks. Cell 96, 271–290. Dunlap, J.C., Loros, J., DeCoursey, P.J., 2004. Chronobiology: Biological Timekeeping. Sinauer, Sunderland, MA. Fuchikawa, T., Shimizu, I., 2007. Effects of temperature on circadian rhythm in the Japanese honeybee, Apis cerana japonica. Journal of Insect Physiology 53, 1179– 1187. Hardin, P.E., 2006. Essential and expendable features of the circadian timekeeping mechanism. Current Opinion in Neurobiology 16, 686–692. Hardin, P.E., Hall, J.C., Rosbash, M., 1990. Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels. Nature 343, 536–540. Meyering-Vos, M., Muller, A., 2007. RNA interference suggests sulfakinins as satiety effectors in the cricket Gryllus bimaculatus. Journal of Insect Physiology 53, 840– 848. Meyering-Vos, M., Merz, S., Sertkol, M., Hoffmann, K.H., 2006. Functional analysis of the allatostatin A gene in the cricket Gryllus bimaculatus and the armyworm Spodoptera frugiperda. Insect Biochemistry and Molecular Biology 36, 492–504. Moriyama, Y., Sakamoto, T., Karpova, S.G., Matsumoto, A., Noji, S., Tomioka, K., 2008. RNA interference of the clock gene period disrupts circadian rhythms in the cricket Gryllus bimaculatus. Journal of Biological Rhythms 23, 308–318. Nishinokubi, I., Tomioka, K., 2000. Analysis of the mechanism underlying the rhythm reversal from diurnal to nocturnal in the cricket Gryllus bimaculatus, with special reference to the role of serotonin. Zoological Science 17, 1075–1080. Preitner, N., Damiola, F., Lopez-Molina, L., Zakany, J., Duboule, D., Albrecht, U., Schibler, U., 2002. The orphan nuclear receptor REV-ERBa controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell 110, 251–260. Rubin, E.B., Shemesh, Y., Cohen, M., Elgavish, S., Robertson, H.M., Bloch, G., 2006. Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee (Apis mellifera) and shed new light on the molecular evolution of the circadian clock. Genome Research 16, 1352–1365. Sauman, I., Reppert, S.M., 1996. Circadian clock neurons in the silkmoth Antheraea pernyi: novel mechanisms of period protein regulation. Neuron 17, 889–900. Smith, R.F., Konopka, R.J., 1982. Effects of dosage alterations at the per locus on the period of the circadian clock of Drosophila. Molecular and General Genetics 185, 30–36. Sokolove, P.G., Bushell, W.N., 1978. The chi square periodogram: its utility for analysis of circadian rhythm. Journal of Theoretical Biology 72, 131–160. Stanewsky, R., 2003. Genetic analysis of the circadian system in Drosophila melanogaster and mammals. Journal of Neurobiology 54, 111–147. Tomioka, K., Chiba, Y., 1982. Post-embryonic development of circadian rhythm in the cricket, Gryllus bimaculatus. Journal of Comparative Physiology A 147, 299– 304. Tomioka, K., Chiba, Y., 1992. Characterization of optic lobe circadian pacemaker by in situ and in vitro recording of neuronal activity in the cricket Gryllus bimaculatus. Journal of Comparative Physiology A 171, 1–7. Tomioka, K., Abdelsalam, S.A., 2004. Circadian organization in hemimetabolous insects. Zoological Science 21, 1153–1162. Uemura, H., Tomioka, K., 2006. Postembryonic changes in circadian photo-responsiveness rhythms of optic lobe interneurons in the cricket Gryllus bimaculatus. Journal of Biological Rhythms 21, 1–11. Yamaguchi, S., Isejima, H., Matsuo, T., Okura, R., Yagita, K., Kobayashi, M., Okamura, H., 2003. Synchronization of cellular clocks in the suprachiasmatic nucleus. Science 302, 1408–1412. Zhu, H., Sauman, I., Yuan, Q., Casselman, A., Emery-Le, M., Emery, P., Reppert, S.M., 2008. Cryptochromes define a novel circadian clock mechanism in monarch butterflies that may underlie sun compass navigation. PLoS Biology 6, 138–155.