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Thermoperiodic regulation of the circadian eclosion rhythm in the flesh fly, Sarcophaga crassipalpis
Journal of Insect Physiology 57 (2011) 1249–1258
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Thermoperiodic regulation of the circadian eclosion rhythm in the flesh fly, Sarcophaga crassipalpis Yosuke Miyazaki a,b,⇑, Shin G. Goto a, Kazuhiro Tanaka c, Osamu Saito b, Yasuhiko Watari b a
Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan Laboratory of Biotechnology, Faculty of Clinical Education, Ashiya University, Hyogo 659-8511, Japan c Ecological Laboratory, General Education, Miyagi Gakuin Women’s University, Miyagi 981-8557, Japan b
a r t i c l e
i n f o
Article history: Received 17 February 2011 Received in revised form 19 May 2011 Accepted 24 May 2011 Available online 15 June 2011 Keywords: Circadian clock Eclosion rhythm Flesh fly Outdoor condition Thermoperiod
a b s t r a c t We recorded the eclosion time of the flesh fly, Sarcophaga crassipalpis, at different depths in the outdoor soil and under temperature cycles with various amplitudes in the laboratory, to examine the timing adjustment of eclosion in response to temperature cycles and their amplitudes in the pupal stage. In the soil, most eclosions occurred in the late morning, which was consistent with the eclosion time under pseudo-sinusoidal temperature cycles in the laboratory. The circadian clock controlling eclosion was reset by temperature cycles and free-ran with a period close to 24 h. This clock likely helps pupae eclose at an optimal time even when the soil temperature does not show clear daily fluctuations. The eclosion phase of the circadian clock progressively advanced as the amplitude of the pseudo-sinusoidal temperature cycle decreased. This response allows pupae located at any depth in the soil to eclose at the appropriate time despite the depth-dependent phase delay of the temperature change. In contrast, the abrupt temperature increase in square-wave temperature cycles reset the phase of the circadian clock to the increasing time, regardless of the temperature amplitude. The rapid temperature increase may act as the late-morning signal for the eclosion clock. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The appropriate timing of eclosion in many insects is controlled by a circadian clock. Both light–dark cycles (photoperiod) and temperature cycles (thermoperiod) serve as a zeitgeber to entrain the circadian rhythm (Saunders, 2002; Myers, 2003). In some flies, pupation occurs underground where light does not penetrate. Therefore, the photoperiod is not available as the zeitgeber for the eclosion rhythm during the underground pupal stage. It has been suggested that the circadian rhythm entrained by the photoperiod during the larval stage above the ground free-runs with a period close to 24 h after larvae burrow into the soil and then the flies emerge as adults at an appropriate time of the day (Winfree, 1974; Saunders, 1976, 1979; Roberts et al., 1983; Smith, 1985; Joplin and Moore, 1999). However, there are different opinions about the effect of temperature cycles in the pupal stage on entrainment. Roberts et al. (1983) described that pupae of the brown blow fly, Calliphora stygia, respond to temperature cycles and pulses in the eclosion timing but considered it unlikely that temperature cycles in the field continue to entrain the eclosion ⇑ Corresponding author. Present address: Laboratory of Biotechnology, Faculty of Clinical Education, Ashiya University, Rokurokuso-cho 13-22, Ashiya, Hyogo 6598511, Japan. Tel.: +81 797 23 0661; fax: +81 797 23 1901. E-mail address: [email protected] (Y. Miyazaki). 0022-1910/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2011.05.006
clock during the underground pupal stage, because environmental cues are absent beneath the substrate. In contrast, Smith (1985) suggested that in the pupae of the sheep blow fly, Lucilia cuprina – which occasionally remain underground up to several months – daily cycles of soil temperature compensate for slight deviations from exactly 24 h in the free-running period and maintain entrainment with natural daily cycles. However, this has not been sufficiently verified experimentally. The onion fly, Delia antiqua, also ecloses in the soil and the circadian eclosion rhythm is entrained to temperature cycles as well as light–dark cycles (Watari, 2002a,b). However, photoperiods applied in the larval stage play no role in generating the eclosion rhythm and the sensitivity to zeitgebers is found in the late pupal stage (Watari, 2005). In the field, therefore, only temperature cycles in the late pupal stage were considered zeitgebers. Tanaka and Watari (2003) reported an interesting property in the eclosion timing of D. antiqua under temperature cycles. Because of the low heat conductivity of the soil, the natural daily fluctuation of temperature is gradually dampened and the phase of the temperature cycle is more delayed with increasing soil depth. Therefore, if the timing of eclosion is associated only with soil temperature changes, eclosion should occur later when pupae are located deeper in the soil. Tanaka and Watari (2003) compared eclosion times of D. antiqua kept at different depths in the outdoor soil or kept under artificial thermoperiods with different amplitudes in the
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laboratory, and found that D. antiqua compensates for the depthdependent phase delay of the temperature change by advancing the eclosion time as the amplitude of the temperature cycle decreases with an increase in depth. This novel property suggests the importance of temperature cycles and their amplitudes for the eclosion timing in insects, the pupae of which are located in the soil. Such a response, however, has not been confirmed in species other than D. antiqua. In the present study, we focused on the eclosion of the flesh fly, Sarcophaga crassipalpis Macquart. In the field, Sarcophaga pupae are buried 4–8 cm underground (Denlinger, 1981). In laboratory experiments, the eclosion peaks of S. crassipalpis occur shortly after light-on under light–dark cycles (Yocum et al., 1994), but the eclosion pattern under natural conditions has not been investigated. This eclosion rhythm is governed by the circadian clock, which is entrained to light–dark cycles experienced during, at least, the wandering larval stage (Joplin and Moore, 1999). The eclosion of S. crassipalpis may be also responsive to temperature cycles during the pupal stage because the responsiveness to temperature pulses is known in pharate adults of another sarcophagid fly, S. argyrostoma (Saunders, 1979). In the present study, we recorded the eclosion of S. crassipalpis at different depths in the outdoor soil and under temperature cycles with various amplitudes in the laboratory to examine whether the pupae of S. crassipalpis adjust eclosion timing in response to temperature cycles and their amplitudes.
prevent invasion of small animals like ants. The apparatus topped with a plastic box was buried, before dusk, at 5- or 20-cm depth in the soil near the laboratory building, where a recording computer was placed. A corrugated PVC sheet was covered above the ground as protection against rain. The soil temperature near the recording apparatus was monitored by using a portable data logger (Ondotori, TR-71S; T&D Co., Matsumoto, Japan) at 10-min intervals. The hourly data of solar radiation were obtained from the meteorological records of NARCH (http://ss.cryo.affrc.go.jp/seisan/meteo/ dailydatae.html). Laboratory studies were conducted to assess the importance of daily temperature cycle as a cue to adjust the timing of adult eclosion. The recording apparatuses were filled with post-diapause pupae under room light and temperature within 2–3 h after DD exposure at 7.5 °C and then were kept from 16:00 to 18:00 h in an incubator (Nippon Medical and Chemical Instruments Co. Ltd., Tokyo, Japan) in which temperature cycles or thermoperiods can be programmed in DD. The programmed constant temperature was kept within a range of ±1.0 °C. As each experimental group of flies emerged over a 2–10-day period in various thermoperiods, the daily data during the whole emergence period were pooled, unless otherwise stated, and the phase of the eclosion peak (/E) was represented by the median time of eclosion. The free-running period (s) of eclosion rhythm was estimated as the mean value of the period between eclosion peaks.
2. Materials and methods 2.3. Rhythmicity 2.1. Insects A colony of S. crassipalpis Macquart (Pape et al., 2010; also known as Parasarcophaga crassipalpis, Hirashima, 1989) originating from adults captured in Sapporo City (43°040 N) in 2004 was kindly provided by Mr. Shigeki Murayama of Hokkaido University. The colony was maintained as described (Denlinger, 1972). Newly emerged adults under 16-h light and 8-h darkness (LD 16:8) at 25 °C were transferred to LD 12:12 at 25 °C within 2 days after eclosion and provisioned with water, sugar, and a piece of beef liver (day 1). The abdomen of females was dissected on day 12 and larvae in the uterus were collected. The larvae were placed on a piece of beef liver and maintained under LD 12:12 at 20 °C. Under these conditions, 96.5% (N = 314) of individuals entered pupal diapause. Diapause pupae were transferred to continuous darkness (DD) at 7.5 °C, maintained for more than 3 months to terminate diapause, and then used in the experiments.
The degree of rhythmicity in eclosion was measured by the parameter R (Winfree, 1970; Saunders, 1976; Smith, 1985). Several days of eclosion data were pooled to calculate the total number of eclosions for each hour of the day. The 8-h period (gate) of the day containing the highest number of eclosions was then determined. The measure R was calculated by dividing the number of eclosions outside this 8-h period by the number of eclosions within it and multiplying by 100. The theoretical range of R is from 0, if all eclosions occur within the gate, to 200, if eclosions are distributed uniformly through the day. R values of about 150 or greater show statistically uniform eclosion (Winfree, 1970). R values of 60 or less represent rhythmic eclosion, those between 60 and 90 are weakly rhythmic, and those greater than 90 are arrhythmic (e.g., Saunders, 1979; Smith, 1985; Watari, 2005). 3. Results
2.2. Recording of eclosion rhythms
3.1. Eclosion under outdoor conditions
The method of recording eclosion under laboratory and outdoor conditions was according to the previous studies of D. antiqua (Watari, 2002a; Tanaka and Watari, 2003), but the recording apparatus was redesigned for the bigger size of S. crassipalpis. The apparatus is made of a plastic box flanked with an infrared-light emitter and a detector (GT2; Takenaka Electronic Industrial, Kyoto, Japan) and is based on the ‘‘falling ball’’ principle (e.g., Truman, 1972; Saunders, 1976; Lankinen and Lumme, 1982). Holes in the plastic plate used to load pupae are about 7.0 mm in diameter. When a stainlesssteel ball is pushed out by an eclosing fly’s head and crosses the infrared beam, a signal is fed to a computer and the number of eclosions is counted. All measurements under outdoor conditions were performed at the experimental farm of the National Agricultural Research Center for Hokkaido Region (NARCH) at Sapporo (43°030 N) in August 2007 and June–July 2008. The recording apparatus filled with post-diapause pupae was completely covered with a net laundry bag to
The adult eclosion of S. crassipalpis and soil temperature were recorded under outdoor conditions at different soil depths (5 and 20 cm; Fig. 1). An investigation in August 2007 showed that the average soil temperatures were 26.6 and 24.6 °C at 5- and 20-cm depths, respectively. The phase and the amplitude of the temperature cycle were distinctly different depending on the depth. The soil temperature began to increase from 08:00 h at 5-cm depth, but from 13:00 h at 20-cm depth. The range of temperature change averaged 3.5 °C at 5-cm depth but was only 0.6 °C at 20-cm depth (Fig. 1A and B, right panels). Daily fluctuation of soil temperature varied considerably from day to day (Fig. 1A and B, left panels), depending on the solar radiation (Fig. 1C). At 5-cm depth on August 12–14, for example, the soil temperature clearly showed cyclic fluctuations, whereas on August 15 the fluctuations attenuated. On August 16, when the solar radiation was minor, the soil temperature continued to decline gradually all through the day. Clearly cyclic fluctuations of the soil temperature were again observed
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Fig. 1. Outdoor soil temperature and distribution of adult eclosion from pupae of Sarcophaga crassipalpis. Field experiments were performed at 5-cm (A) and 20-cm (B) depths in the soil in August 2007 and at 5-cm (D) and 20-cm (E) depths in the soil in June–July 2008. Each left panel shows the soil temperature and the record of adult eclosions for a certain 10-day period of the experiment period (A,B,D, and E), and also the corresponding data of solar radiation in August 2007 (C) and in June–July 2008 (F). Each right panel (A,B,D, and E) shows the data for a 5-day period indicated by a double-headed arrow in the left panel, as the mean temperature at 5- or 20-cm depth in the soil and the pooled record of daily eclosion. Triangles in right panels represent /E (closed) and the time when the temperature cycle began to increase (open). R values show the degree of rhythmicity. Time is Japan Standard Time. The values of /E were 9.2, 11.1, 9.8, and 11.6 h in A, B, D, and E, respectively. Average sunrise times for a 5-day record period were 04:38, 04:39, 03:57, and 03:58 h in A, B, D, and E, respectively. Average sunset times for a 5-day record period were 18:40, 18:38, 19:18, and 19:18 h in A, B, D, and E, respectively.
from August 19 (Fig. 1A, left panel). The timing of adult eclosion was synchronous but differed with soil depth (Fig. 1A and B, right panels). The eclosion occurred earlier at 5-cm depth than at 20-cm depth, but it is noteworthy that the difference between the medians (/E) of the 2 depths was only 1.9 h, compared with the differ-
ence of 5 h in the phase of temperature cycle between the respective soil depths. This investigation was repeated in June–July 2008. The average soil temperatures were 23.5 and 20.1 °C at 5- and 20-cm depths, respectively. The soil temperature began to increase from 07:00 h
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at 5-cm depth, but from 12:00 h at 20-cm depth. The range of temperature change averaged 10.5 °C at 5-cm depth but was only 1.5 °C at 20-cm depth (Fig. 1D and E, right panels). The daily solar radiation was relatively stable (Fig. 1F) as compared to an investigation in August 2007 (Fig. 1C), and the daily fluctuation of soil temperature did not vary considerably from day to day (Fig. 1D and E, left panels). The timing of adult eclosion was similar to an investigation in August 2007 (Fig. 1D and E, right panels). Again, the difference in /E between the 2 depth groups was only 1.8 h in spite of the 5 h difference in the phase of temperature cycle between the respective soil depths. Thus, most flies emerged in the late morning in both investigations and the R values were all less than 5, which indicate highly synchronous eclosion (Fig. 1, right panels). /E at 5-cm depth was about 2–3 h after the lowest phase point of the soil temperature cycle and /E at 20-cm depth occurred at about the lowest phase point of the temperature cycle. This means that the flies can, to some extent, compensate for the depth-dependent phase delay of zeitgeber, and the compensation was achieved by advancing the eclosion timing of pupae located deeper in the soil relative to the temperature fluctuations. In addition, this advance of the eclosion timing may be established by a circadian clock controlling eclosion rather than as a direct response to temperature change, because flies eclosed at the appropriate time on August 16, 2007, despite the continuous decline of soil temperature. 3.2. Eclosion under laboratory conditions To examine whether the timing adjustment of eclosion of S. crassipalpis is a response to the amplitude of temperature cycles, the adult eclosion was recorded under laboratory conditions with various amplitudes (0, 1, 2, 4, 6, or 8 °C) of temperature cycles under DD. First, the adult eclosion was recorded under pseudo-sinusoidal temperature cycles where temperature was changed every 1 h (Fig. 2). When the average temperature was 20 °C, the /E values were 10.8, 10.0, 9.5, 7.7, and 6.1 h in amplitudes of 8, 6, 4, 2, and 1 °C, respectively (Fig. 2A–E). There were statistical differences in the timing of adult eclosion among these regimes, except between regimes with amplitudes of 6 and 4 °C (Steel–Dwass test; P < 0.05). /E occurred a few hours after the temperature began to increase under the temperature cycles whose amplitude was large, whereas /E was at the lowest temperature of the temperature cycle with the amplitude of 1 °C. Such phase relationships between /E and the temperature cycle were coincident with those under outdoor conditions. At a constant 20 °C, the eclosion was also rhythmic (R = 54.0), but the peak was considerably broader than under pseudo-sinusoidal temperature cycles and the /E was 3.5 h (Fig. 2F). These results show that the eclosion rhythm of S. crassipalpis was entrained to the pseudo-sinusoidal temperature cycle, and /E progressively advanced as the amplitude of the temperature cycle decreased. When the average temperature was 25 °C, the pseudosinusoidal temperature cycle also entrained the eclosion rhythm and similarly /E advanced as the temperature amplitude decreased, although the advance response was weaker than when the average temperature was 20 °C (Fig. 2G–L). Thus, we observed that the timing adjustment of eclosion under laboratory conditions was similar to that observed under outdoor conditions. Therefore, we conclude that these advances are caused in response to the amplitude of temperature cycles. Second, the adult eclosion was recorded under a square-wave temperature cycle that consisted of a warm phase and a cool phase; the temperature difference between the 2 phases was varied (0, 1, 2, 4, 6, or 8 °C) with the same average temperature (20 °C; Fig. 3). Under a 12-h warm phase and a 12-h cool phase (WC 12:12), the /E values were close to the time of the temperature increase in the thermoperiod, regardless of the magnitude of temper-
ature change (Fig. 3A–E), although at a constant 20 °C the eclosion peak was unclear because eclosion was observed in only a small number of individuals during the experimental period (Fig. 3F). There was no statistical difference in the timing of eclosion among the regimes with the amplitude of 2 °C or more (Steel–Dwass test; P > 0.05; Fig. 3A–D). In these regimes, most flies emerged within 1 h after the temperature increase. Under the 1 °C amplitude, eclosion was less synchronous but many individuals eclosed within 1 h after the temperature increase (Fig. 3E). Therefore, the shift of /E in response to the amplitude of temperature cycles was not observed under square-wave cycles of WC 12:12, in contrast to under pseudo-sinusoidal temperature cycles. Under WC 6:18, the /E values were also close to the time of temperature increase (Fig. 3G–L). Statistical differences in the timing of eclosion were found among some regimes (Steel–Dwass test; P < 0.05), but there was no correlation with the magnitude of temperature change. Third, to examine whether differences in the amplitude of pseudo-sinusoidal temperature cycles affect a circadian clock controlling eclosion or merely influence more downstream eclosion processes, the adult eclosion was recorded at 20 °C after pupae had experienced pseudo-sinusoidal cycles with high (8 °C) or low (1 °C) amplitude under DD; these measurements were compared to those of pupae continuously exposed to temperature cycles and pupae that had not experienced temperature cycles (Fig. 4). When flies were exposed to high-amplitude temperature change and then were transferred to 20 °C, circadian rhythmicity was shown in adult eclosion. The free-running period (s) was 24.3 h (Fig. 4B). The /E value in the pooled record was 10.9 h, which was close to that (10.6 h) under continuous temperature cycles (Fig. 4A and B). There was no statistical difference in eclosion timing between these 2 regimes (Wilcoxon rank sum test; Z = 0.31, P = 0.754). Flies that did not experience temperature cycles began to emerge several days later than those experiencing temperature cycles. The rhythmicity was weak (R = 63.3) and the /E value in the pooled record was 1.4 h, which is distinctly different from the /E in flies experiencing temperature cycles (Fig. 4C). When flies were exposed to low-amplitude temperature change and then were transferred to 20 °C, circadian rhythmicity was shown and s was 23.2 h (Fig. 4E). The /E value in the pooled record was 6.7 h, which was close to that (7.2 h) under continuous temperature cycles (Fig. 4D and E). There was no statistical difference in eclosion timing between these 2 regimes (Wilcoxon rank sum test; Z = 0.83, P = 0.406). In flies not experiencing temperature cycles, circadian rhythmicity was also observed and s was 24.0 h. However, the /E value in the pooled record was 20.1 h, which is far from the /E in flies experiencing temperature cycles (Fig. 4F). These results indicate that differences in the amplitude of pseudo-sinusoidal temperature cycles affect a circadian clock controlling eclosion because the advance response of the eclosion timing was persistent even in the free-running rhythm after temperature was fixed at 20 °C. In the above exposure to low-amplitude temperature change, at the same time, we also recorded eclosion under antiphase conditions, by shifting the phase of temperature fluctuations by 12 h (Fig. 5). The peak time of eclosion was also shifted by about 12 h, compared to that in Fig. 4D and E. Under continuous temperature cycles, the eclosion peak occurred at the lowest temperature point and the /E value in the pooled record was 18.9 h (Fig. 5A). When flies were transferred to 20 °C, s was 23.4 h and the /E value in the pooled record was 17.8 h (Fig. 5B). These R values were much less than that in flies not experiencing temperature cycles (Fig. 4F). Thus, the circadian eclosion clock was clearly entrained to temperature cycles with the amplitude of 1 °C. Lastly, to examine the effects of the abrupt temperature change in square-wave temperature cycles on eclosion and on the circadian clock controlling it, adult eclosion was recorded at 20 °C after
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Fig. 2. Various pseudo-sinusoidal temperature cycles and distribution of adult eclosion from pupae of Sarcophaga crassipalpis under continuous darkness. The average temperature was 20 °C (A–F) or 25 °C (G–L). The amplitudes of temperature cycles were 8 °C (A and G), 6 °C (B and H), 4 °C (C and I), 2 °C (D and J), 1 °C (E and K), or 0 °C (F and L). R values show the degree of rhythmicity. At the average temperature of 20 °C, the values of /E (triangle) were 10.8, 10.0, 9.5, 7.7, 6.1, and 3.5 h in A, B, C, D, E, and F, respectively. At the average temperature of 25 °C, the values of /E were 9.6, 9.6, 8.1, 8.1, 7.0, and 16.6 h in G, H, I, J, K, and L, respectively. The differences in /E were examined statistically by the Steel–Dwass test for nonparametric multiple comparison in A–E and in G–K, separately (Hochberg and Tamhane, 1987). The /E values with the same letter (a–d in A–E; e, f in G–K) are not significantly different (P > 0.05).
pupae had experienced WC 12:12 with high (8 °C) or low (1 °C) amplitude under DD; this was compared to eclosion of pupae continuously exposed to temperature cycles and pupae not experiencing temperature cycles (Fig. 6). When flies were exposed to
high- and low-amplitude temperature change, and not exposed to any temperature change during the experimental period, circadian rhythmicity was shown after transfer to a constant temperature; the values of s were 24.4, 23.7, and 24.5 h, respectively
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Fig. 3. Various square-wave temperature cycles (average temperature, 20 °C) and distribution of adult eclosion from pupae of Sarcophaga crassipalpis under continuous darkness. Thermoperiod was 12 h warm phase and 12 h cool phase (WC 12:12, A–F) or 6 h warm phase and 18 h cool phase (WC 6:18, G–L). The temperature difference between the 2 phases was 8 °C (A and G), 6 °C (B and H), 4 °C (C and I), 2 °C (D and J), 1 °C (E and K), or 0 °C (F and L). R values show the degree of rhythmicity. Under WC 12:12, the values of /E (triangle) were 12.6, 12.5, 12.5, 12.7, and 12.0 h in A, B, C, D, and E, respectively. Under WC 6:18, the values of /E were 12.5, 11.5, 12.4, 12.7, and 12.7 h in G, H, I, J, and K, respectively. The differences in /E were examined statistically by the Steel–Dwass test for nonparametric multiple comparison in A–E and in G–K, separately (Hochberg and Tamhane, 1987). The /E values with the same letter (a, b in A–E; c–e in G–K) are not significantly different (P > 0.05).
(Fig. 6B, D and E). The eclosion records showed that under squarewave temperature cycles, /E was reset close to, or just after, the time at which temperature increased in the thermoperiod, irrespective of the amplitude of the temperature exposure – although WC 12:12, with a 1 °C amplitude, was insufficient to reset the
eclosion phase in all individuals. The advance of eclosion timing that was dependent upon the exposed temperature amplitude was also not observed in the free-running rhythm. In the exposure to high-amplitude change, the /E value in the pooled record after transfer to 20 °C was significantly different from that under WC
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Fig. 4. Effects of pseudo-sinusoidal temperature cycles on the circadian eclosion rhythm of Sarcophaga crassipalpis under continuous darkness. Pupae were exposed to temperature cycles with the temperature amplitude of 8 °C continuously (A); to 20 °C after transfer from temperature cycles with the temperature amplitude of 8 °C (B); to temperature cycles with the temperature amplitude of 1 °C continuously (D); to 20 °C after transfer from temperature cycles with the temperature amplitude of 1 °C (E); or to 20 °C continuously (C and F). Each left panel shows temperature and the record of adult eclosions for a certain 7-day period of the experiment. Each right panel shows the data for the period indicated by a double-headed arrow in the left panel, as the pooled record of daily eclosion. R values show the degree of rhythmicity. The values of /E (triangle) in right panels were 10.6, 10.9, 1.4, 7.2, 6.7, and 20.1 h in A, B, C, D, E, and F, respectively.
12:12 (Wilcoxon rank sum test; Z = 4.77, P < 0.001; Fig. 6A and B), in contrast to that under pseudo-sinusoidal temperature cycles (Fig. 4A and B). This may be attributed to a burst of eclosion
directly invoked by an abrupt temperature increase. This direct response is induced to some extent by even low-amplitude temperature change (Fig. 6C and D).
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Fig. 5. Effects of shifting the phase of the pseudo-sinusoidal temperature cycle by 12 h on the circadian eclosion rhythm of Sarcophaga crassipalpis under continuous darkness. Pupae were exposed to temperature cycles with the temperature amplitude of 1 °C continuously (A); or to 20 °C after transfer from temperature cycles with the temperature amplitude of 1 °C (B). The phase of the temperature cycle in A was shifted by 12 h from that in Fig. 4D. The phase of the temperature cycle in B was shifted by 12 h from that in Fig. 4E. Each left panel shows temperature and the record of adult eclosions for a certain 7-day period of the experiment. Each right panel shows the data for the period indicated by a double-headed arrow in the left panel, as the pooled record of daily eclosion. R values show the degree of rhythmicity. The values of /E (triangle) in right panels were 18.9 and 17.8 h in A and B, respectively.
4. Discussion 4.1. Entrainment to temperature cycles during the pupal stage Although the circadian rhythm of eclosion has been reported in many insects (Saunders, 2002; Myers, 2003), there are few reports associated with the location where eclosion occurs. In the laboratory and when exposed to temperature cycles that simulate soiltemperature fluctuations, D. antiqua ecloses at the same time as in the outdoor soil (Tanaka and Watari, 2003). The present results demonstrated that the eclosion pattern of S. crassipalpis in the soil was consistent with that observed in response to pseudo-sinusoidal temperature cycles in the laboratory. This indicates the importance of temperature cycles for the eclosion timing of S. crassipalpis. Flies are generally thought to eclose around dawn (Pittendrigh, 1954; Roberts et al., 1983; Denlinger and Zˇdárek, 1994; Saunders, 2002). Experiments under light–dark cycles in the laboratory indicate that S. crassipalpis ecloses around dawn (Yocum et al., 1994). The present studies of S. crassipalpis eclosion in the soil, however, showed that this fly ecloses from early morning to mid-afternoon, with a peak in the late morning rather than around dawn. However, similar experiments performed in August 2000 showed that D. antiqua eclosed from mid-night to noon, with a peak just after dawn. Most eclosions at both 5- and 20-cm depths were completed before the temperature increase, in contrast to S. crassipalpis (Tanaka and Watari, 2003). These results indicate that the eclosion time of flies in the field varies depending on the species. For example, the tsetse fly, Glossina morsitans, emerges as an adult from the soil in mid-afternoon. This eclosion rhythm acutely responds to the thermoperiod but not the photoperiod, and the peak occurs during the late temperature increase (Zˇdárek and Denlinger, 1995). The eclosion clock of S. crassipalpis is highly sensitive to light– dark cycles during the wandering larval stages and free-runs with a period close to 24 h in DD (Joplin and Moore, 1999). Joplin and Moore (1999) suggested that these properties of the circadian clock allow S. crassipalpis to determine precisely the eclosion phase with respect to environmental conditions despite a pupal stage
that is spent underground, as suggested by other researchers (Winfree, 1974; Saunders, 1976, 1979; Roberts et al., 1983; Smith, 1985). In the present study, when pupae of S. crassipalpis were transferred to DD at constant temperature within 2–3 h after chilling – i.e., in control experiments without daily temperature cycles – the eclosion patterns were not consistent in terms of the rhythmicity and days of adult emergence (Figs. 2F,L, 3F,L, 4C,F, 6E). We cannot sufficiently explain what factors caused these variable patterns, because there are various possibilities, such as photoperiodic entrainment of the larval clock, resetting of the pupal clock by room light for a few hours and a temperature step-up from 7.5 °C before the experiments, and the difference of responsiveness to external stimuli among pupae. However, we found that temperature cycles applied to the pupal stage did produce eclosion entrainment. Temperature cycles not only initiated high rhythmicity in eclosion (e.g., Fig. 2E and F) but also reset the circadian clock (e.g., Fig. 4E and F), so that the eclosion peak occurred at the optimal time, even under a 1 °C temperature amplitude. We therefore consider that in insects that pupate underground, the eclosion time under natural conditions should be identified based on the responsiveness to thermoperiods rather than to photoperiods. This is likely important for pupae that overwinter for a long time, especially in areas such as Sapporo, where the winter soil temperature does not fluctuate, because of snow cover, but begins to fluctuate again in the spring, after diapause (Watari, 2005). Daily changes in the soil temperature are varied depending on the weather and do not show clear fluctuations when the solar radiation is low. Under outdoor conditions, the soil temperature on August 16, 2007, continued to decline only gradually during the day, but the eclosion peak occurred in the late morning (Fig. 1A and B, left panels). This suggests that the circadian clock was entrained by soil temperature fluctuations that were experienced before August 16, with eclosion at the appropriate time established by the internal periodicity, as occurs in the laboratory after the transfer from temperature cycles to constant temperature under DD. Therefore, a free-running period close to 24 h likely helps pupae emerge as adults at the optimal time when the circadian rhythm persists without temperature cycles, rather than
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Fig. 6. Effects of square-wave cycles of 12 h warm phase and 12 h cool phase on the circadian eclosion rhythm of Sarcophaga crassipalpis under continuous darkness. Pupae were exposed to temperature cycles with the temperature difference of 8 °C continuously (A); to 20 °C after transfer from temperature cycles with the temperature difference of 8 °C (B); to temperature cycles with the temperature difference of 1 °C continuously (C); to 20 °C after transfer from temperature cycles with the temperature difference of 1 °C (D); or to 20 °C continuously (E). Each left panel shows temperature and the record of adult eclosions for a certain 7-day period of the experiment. Each right panel shows the data for the period indicated by a double-headed arrow in the left panel, as the pooled record of daily eclosion. R values show the degree of rhythmicity. The values of /E (triangle) in right panels were 12.5, 13.5, 14.1, 15.8, and 0.2 h in A, B, C, D, and E, respectively.
photoperiodic cycles. The sensitivity of larvae to photoperiods may be meaningful for the eclosion rhythm only when the pupal period is short and there is no reliable temperature signal during the period, such as in locations deep underground or during long periods with cloudy days. 4.2. Effects of the amplitude of temperature cycles on the eclosion timing In the present study, the eclosion phase of S. crassipalpis progressively advanced as the amplitude of the pseudo-sinusoidal temperature cycle decreased and coincided with the lowest temperature when the amplitude was 1 °C. As reported in D. antiqua
(Tanaka and Watari, 2003), this response allows pupae located at any depth in the soil to eclose at the appropriate time of day, despite the depth-dependent phase delay of the temperature change. Insects other than flies that pupate underground may also have an eclosion timing that is linked to the amplitude of temperature cycles (Tanaka and Watari, 2003). A transfer from pseudo-sinusoidal temperature cycles to constant temperature revealed that the eclosion time is set in response to the amplitude based on the phase of a circadian clock. This tempts us to investigate, at the molecular level, the responsiveness of the circadian clock of flies to the amplitude of temperature change. The eclosion peak of D. antiqua occurs 3–4 h earlier in WC 12:12 with a temperature difference of 1 °C than with a temperature dif-
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ference of 4 °C (Tanaka and Watari, 2003; Watari and Tanaka, 2010). However, the present results did not show an alteration of eclosion timing in response to the amplitude of the square-wave temperature cycle in S. crassipalpis, even after transfer to constant temperature. Although it is unclear why such a difference was observed between S. crassipalpis and D. antiqua, it may be attributed to the optimal eclosion time in the field. Delia antiqua ecloses around dawn, and the peak occurs during the decline of the sinusoidal temperature cycle and during the cool phase of the square-wave temperature cycle (Watari, 2002b; Tanaka and Watari, 2003; Watari and Tanaka, 2010). However, near-surface pupae of S. crassipalpis emerge as adults in the late morning when the soil temperature rapidly begins to increase (see Fig. 1A and D). The rapid temperature increase may be the reliable time signal of late morning for the circadian clock, and therefore, resets the eclosion rhythm to the particular phase. The rapid temperature increase of 2 °C or more directly invoked the extremely narrow eclosion peak (Figs. 3 and 6). This high sensitivity indicates the importance of the temperature environment for the eclosion timing of S. crassipalpis. There was a difference in the eclosion time between the pseudosinusoidal cycle and the square-wave cycle with the 1 °C amplitude. Under the pseudo-sinusoidal cycle, the eclosion peak occurred at the lowest temperature point, whereas under the square-wave cycle, the peak occurred around the temperature increase (Figs. 2E, 3E, 4D, and 6C). This difference indicates our limited ability to simulate natural conditions based on a square-wave environmental cycle. Recent experiments on the locomotor activity of the fruit fly, Drosophila melanogaster, also demonstrated different results between pseudo-sinusoidal and the square-wave temperature cycles (Yoshii et al., 2009). These results suggest the significance of applying sinusoidal temperature cycles to discuss temperature entrainment of circadian rhythms under natural conditions. In summary, the present study of the circadian clock underlying eclosion of S. crassipalpis revealed the following 3 points. First, the clock is entrained to temperature cycles in the pupal stage with high sensitivity under both natural and laboratory conditions. Second, the eclosion phase of a clock is progressively advanced as the amplitude of the sinusoidal temperature cycle decreases, as shown in D. antiqua. Third, the abrupt temperature increase reset the eclosion phase of the circadian clock to the increasing time, regardless of the amplitude of temperature cycles, unlike D. antiqua. These properties of the circadian clock of S. crassipalpis would enhance the likelihood of emerging as adults from the soil at the optimal time of the day (late morning).
Acknowledgements We thank Dr. Toru Shimizu for providing the computer program for eclosion recording. We also thank Drs. Kiyomitu Ito, Junichi Kaneko, and Kazuhiko Konishi for allowing us to use an experimental field of NARCH. This study was supported by a Grant-in-Aid for Scientific Research (C) (19570075) from the Japan Society for the Promotion of Science and, in part, by a special research Grant from Miyagi Gakuin Women’s University (2008).
References Denlinger, D.L., 1972. Induction and termination of pupal diapause in Sarcophaga (Diptera: Sarcophagidae). Biological Bulletin 142, 11–24. Denlinger, D.L., 1981. The physiology of pupal diapause in flesh flies. In: Bhaskaran, G., Friedman, S., Rodriguez, J.G. (Eds.), Current Topics in Insect Endocrinology and Nutrition. Plenum, New York, pp. 131–160. Denlinger, D.L., Zˇdárek, J., 1994. Metamorphosis behavior of flies. Annual Review of Entomology 39, 243–266. Hirashima, Y., 1989. A Check List of Japanese Insects II. Entomological Laboratory, Faculty of Agriculture. Kyushu University and Japan Wild Life Research Center, Fukuoka. Hochberg, Y., Tamhane, A.C., 1987. Multiple Comparison Procedures. Wiley, New York. Joplin, K.H., Moore, D., 1999. Effects of environmental factors on circadian activity in the flesh fly, Sarcophaga crassipalpis. Physiological Entomology 24, 64–71. Lankinen, P., Lumme, J., 1982. An improved apparatus for recording the eclosion rhythm in Drosophila. Drosophila Information Service 58, 161–163. Myers, E.M., 2003. The circadian control of eclosion. Chronobiology International 20, 775–794. Pape, T., Dahlem, G., de Mello Patiu, C.A. Giroux, M., 2010. The world of flesh flies (Diptera: Sarcophagidae). http://www.zmuc.dk/entoweb/sarcoweb/sarcweb/ sarc_web.htm (accessed 22.1.2011). Pittendrigh, C.S., 1954. On temperature independence in the clock system controlling emergence time in Drosophila. Proceedings of the National Academy of Sciences of the United States of America 40, 1018–1029. Roberts, R.M., Northover, J.M., Lewis, R.D., 1983. Circadian clock control of the eclosion rhythm of the brown blowfly, Calliphora stygia (Diptera: Calliphoridae). New Zealand Entomologist 4, 424–431. Saunders, D.S., 1976. The circadian eclosion rhythm in Sarcophaga argyrostoma: some comparisons with the photoperiodic clock. Journal of Comparative Physiology 110, 111–133. Saunders, D.S., 1979. The circadian eclosion rhythm in Sarcophaga argyrostoma: delineation of the responsive period for entrainment. Physiological Entomology 4, 263–274. Saunders, D.S., 2002. Insect Clocks, third ed. Elsevier, Amsterdam. Smith, P.H., 1985. Responsiveness to light of the circadian clock controlling eclosion in the blowfly, Lucilia cuprina. Physiological Entomology 10, 323– 336. Tanaka, K., Watari, Y., 2003. Adult eclosion timing of the onion fly, Delia antiqua, in response to daily cycles of temperature at different soil depths. Naturwissenschaften 90, 76–79. Truman, J.W., 1972. Involvement of a photoreversible process in the circadian clock controlling silkmoth eclosion. Zeitschrift für Vergleichende Physiologie 76, 32– 40. Watari, Y., 2002a. Comparison of the circadian eclosion rhythm between nondiapause and diapause pupae in the onion fly, Delia antiqua. Journal of Insect Physiology 48, 83–89. Watari, Y., 2002b. Comparison of the circadian eclosion rhythm between nondiapause and diapause pupae in the onion fly, Delia antiqua: the effect of thermoperiod. Journal of Insect Physiology 48, 881–886. Watari, Y., 2005. Comparison of the circadian eclosion rhythm between nondiapause and diapause pupae in the onion fly, Delia antiqua: the change of rhythmicity. Journal of Insect Physiology 51, 11–16. Watari, Y., Tanaka, K., 2010. Interacting effect of thermoperiod and photoperiod on the eclosion rhythm in the onion fly, Delia antiqua supports the two-oscillator model. Journal of Insect Physiology 56, 1192–1197. Winfree, A.T., 1970. Integrated view of resetting a circadian clock. Journal of Theoretical Biology 28, 327–374. Winfree, A.T., 1974. Suppressing Drosophila circadian rhythm with dim light. Science 183, 970–972. Yocum, G.D., Zˇdárek, J., Joplin, K.H., Lee, R.E., Smith, D.C., Manter, K.D., Denlinger, D.L., 1994. Alteration of the eclosion rhythm and eclosion behavior in the flesh fly, Sarcophaga crassipalpis, by low and high temperature stress. Journal of Insect Physiology 40, 13–21. Yoshii, T., Vanin, S., Costa, R., Helfrich-Förster, C., 2009. Synergic entrainment of Drosophila’s circadian clock by light and temperature. Journal of Biological Rhythms 24, 452–464. Zˇdárek, J., Denlinger, D.L., 1995. Changes in temperature, not photoperiod, control the pattern of adult eclosion in the tsetse, Glossina morsitans. Physiological Entomology 20, 362–366.
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Thermoperiodic regulation of the circadian eclosion rhythm in the flesh fly, Sarcophaga crassipalpis Yosuke Miyazaki a,b,⇑, Shin G. Goto a, Kazuhiro Tanaka c, Osamu Saito b, Yasuhiko Watari b a
Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan Laboratory of Biotechnology, Faculty of Clinical Education, Ashiya University, Hyogo 659-8511, Japan c Ecological Laboratory, General Education, Miyagi Gakuin Women’s University, Miyagi 981-8557, Japan b
a r t i c l e
i n f o
Article history: Received 17 February 2011 Received in revised form 19 May 2011 Accepted 24 May 2011 Available online 15 June 2011 Keywords: Circadian clock Eclosion rhythm Flesh fly Outdoor condition Thermoperiod
a b s t r a c t We recorded the eclosion time of the flesh fly, Sarcophaga crassipalpis, at different depths in the outdoor soil and under temperature cycles with various amplitudes in the laboratory, to examine the timing adjustment of eclosion in response to temperature cycles and their amplitudes in the pupal stage. In the soil, most eclosions occurred in the late morning, which was consistent with the eclosion time under pseudo-sinusoidal temperature cycles in the laboratory. The circadian clock controlling eclosion was reset by temperature cycles and free-ran with a period close to 24 h. This clock likely helps pupae eclose at an optimal time even when the soil temperature does not show clear daily fluctuations. The eclosion phase of the circadian clock progressively advanced as the amplitude of the pseudo-sinusoidal temperature cycle decreased. This response allows pupae located at any depth in the soil to eclose at the appropriate time despite the depth-dependent phase delay of the temperature change. In contrast, the abrupt temperature increase in square-wave temperature cycles reset the phase of the circadian clock to the increasing time, regardless of the temperature amplitude. The rapid temperature increase may act as the late-morning signal for the eclosion clock. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The appropriate timing of eclosion in many insects is controlled by a circadian clock. Both light–dark cycles (photoperiod) and temperature cycles (thermoperiod) serve as a zeitgeber to entrain the circadian rhythm (Saunders, 2002; Myers, 2003). In some flies, pupation occurs underground where light does not penetrate. Therefore, the photoperiod is not available as the zeitgeber for the eclosion rhythm during the underground pupal stage. It has been suggested that the circadian rhythm entrained by the photoperiod during the larval stage above the ground free-runs with a period close to 24 h after larvae burrow into the soil and then the flies emerge as adults at an appropriate time of the day (Winfree, 1974; Saunders, 1976, 1979; Roberts et al., 1983; Smith, 1985; Joplin and Moore, 1999). However, there are different opinions about the effect of temperature cycles in the pupal stage on entrainment. Roberts et al. (1983) described that pupae of the brown blow fly, Calliphora stygia, respond to temperature cycles and pulses in the eclosion timing but considered it unlikely that temperature cycles in the field continue to entrain the eclosion ⇑ Corresponding author. Present address: Laboratory of Biotechnology, Faculty of Clinical Education, Ashiya University, Rokurokuso-cho 13-22, Ashiya, Hyogo 6598511, Japan. Tel.: +81 797 23 0661; fax: +81 797 23 1901. E-mail address: [email protected] (Y. Miyazaki). 0022-1910/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2011.05.006
clock during the underground pupal stage, because environmental cues are absent beneath the substrate. In contrast, Smith (1985) suggested that in the pupae of the sheep blow fly, Lucilia cuprina – which occasionally remain underground up to several months – daily cycles of soil temperature compensate for slight deviations from exactly 24 h in the free-running period and maintain entrainment with natural daily cycles. However, this has not been sufficiently verified experimentally. The onion fly, Delia antiqua, also ecloses in the soil and the circadian eclosion rhythm is entrained to temperature cycles as well as light–dark cycles (Watari, 2002a,b). However, photoperiods applied in the larval stage play no role in generating the eclosion rhythm and the sensitivity to zeitgebers is found in the late pupal stage (Watari, 2005). In the field, therefore, only temperature cycles in the late pupal stage were considered zeitgebers. Tanaka and Watari (2003) reported an interesting property in the eclosion timing of D. antiqua under temperature cycles. Because of the low heat conductivity of the soil, the natural daily fluctuation of temperature is gradually dampened and the phase of the temperature cycle is more delayed with increasing soil depth. Therefore, if the timing of eclosion is associated only with soil temperature changes, eclosion should occur later when pupae are located deeper in the soil. Tanaka and Watari (2003) compared eclosion times of D. antiqua kept at different depths in the outdoor soil or kept under artificial thermoperiods with different amplitudes in the
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laboratory, and found that D. antiqua compensates for the depthdependent phase delay of the temperature change by advancing the eclosion time as the amplitude of the temperature cycle decreases with an increase in depth. This novel property suggests the importance of temperature cycles and their amplitudes for the eclosion timing in insects, the pupae of which are located in the soil. Such a response, however, has not been confirmed in species other than D. antiqua. In the present study, we focused on the eclosion of the flesh fly, Sarcophaga crassipalpis Macquart. In the field, Sarcophaga pupae are buried 4–8 cm underground (Denlinger, 1981). In laboratory experiments, the eclosion peaks of S. crassipalpis occur shortly after light-on under light–dark cycles (Yocum et al., 1994), but the eclosion pattern under natural conditions has not been investigated. This eclosion rhythm is governed by the circadian clock, which is entrained to light–dark cycles experienced during, at least, the wandering larval stage (Joplin and Moore, 1999). The eclosion of S. crassipalpis may be also responsive to temperature cycles during the pupal stage because the responsiveness to temperature pulses is known in pharate adults of another sarcophagid fly, S. argyrostoma (Saunders, 1979). In the present study, we recorded the eclosion of S. crassipalpis at different depths in the outdoor soil and under temperature cycles with various amplitudes in the laboratory to examine whether the pupae of S. crassipalpis adjust eclosion timing in response to temperature cycles and their amplitudes.
prevent invasion of small animals like ants. The apparatus topped with a plastic box was buried, before dusk, at 5- or 20-cm depth in the soil near the laboratory building, where a recording computer was placed. A corrugated PVC sheet was covered above the ground as protection against rain. The soil temperature near the recording apparatus was monitored by using a portable data logger (Ondotori, TR-71S; T&D Co., Matsumoto, Japan) at 10-min intervals. The hourly data of solar radiation were obtained from the meteorological records of NARCH (http://ss.cryo.affrc.go.jp/seisan/meteo/ dailydatae.html). Laboratory studies were conducted to assess the importance of daily temperature cycle as a cue to adjust the timing of adult eclosion. The recording apparatuses were filled with post-diapause pupae under room light and temperature within 2–3 h after DD exposure at 7.5 °C and then were kept from 16:00 to 18:00 h in an incubator (Nippon Medical and Chemical Instruments Co. Ltd., Tokyo, Japan) in which temperature cycles or thermoperiods can be programmed in DD. The programmed constant temperature was kept within a range of ±1.0 °C. As each experimental group of flies emerged over a 2–10-day period in various thermoperiods, the daily data during the whole emergence period were pooled, unless otherwise stated, and the phase of the eclosion peak (/E) was represented by the median time of eclosion. The free-running period (s) of eclosion rhythm was estimated as the mean value of the period between eclosion peaks.
2. Materials and methods 2.3. Rhythmicity 2.1. Insects A colony of S. crassipalpis Macquart (Pape et al., 2010; also known as Parasarcophaga crassipalpis, Hirashima, 1989) originating from adults captured in Sapporo City (43°040 N) in 2004 was kindly provided by Mr. Shigeki Murayama of Hokkaido University. The colony was maintained as described (Denlinger, 1972). Newly emerged adults under 16-h light and 8-h darkness (LD 16:8) at 25 °C were transferred to LD 12:12 at 25 °C within 2 days after eclosion and provisioned with water, sugar, and a piece of beef liver (day 1). The abdomen of females was dissected on day 12 and larvae in the uterus were collected. The larvae were placed on a piece of beef liver and maintained under LD 12:12 at 20 °C. Under these conditions, 96.5% (N = 314) of individuals entered pupal diapause. Diapause pupae were transferred to continuous darkness (DD) at 7.5 °C, maintained for more than 3 months to terminate diapause, and then used in the experiments.
The degree of rhythmicity in eclosion was measured by the parameter R (Winfree, 1970; Saunders, 1976; Smith, 1985). Several days of eclosion data were pooled to calculate the total number of eclosions for each hour of the day. The 8-h period (gate) of the day containing the highest number of eclosions was then determined. The measure R was calculated by dividing the number of eclosions outside this 8-h period by the number of eclosions within it and multiplying by 100. The theoretical range of R is from 0, if all eclosions occur within the gate, to 200, if eclosions are distributed uniformly through the day. R values of about 150 or greater show statistically uniform eclosion (Winfree, 1970). R values of 60 or less represent rhythmic eclosion, those between 60 and 90 are weakly rhythmic, and those greater than 90 are arrhythmic (e.g., Saunders, 1979; Smith, 1985; Watari, 2005). 3. Results
2.2. Recording of eclosion rhythms
3.1. Eclosion under outdoor conditions
The method of recording eclosion under laboratory and outdoor conditions was according to the previous studies of D. antiqua (Watari, 2002a; Tanaka and Watari, 2003), but the recording apparatus was redesigned for the bigger size of S. crassipalpis. The apparatus is made of a plastic box flanked with an infrared-light emitter and a detector (GT2; Takenaka Electronic Industrial, Kyoto, Japan) and is based on the ‘‘falling ball’’ principle (e.g., Truman, 1972; Saunders, 1976; Lankinen and Lumme, 1982). Holes in the plastic plate used to load pupae are about 7.0 mm in diameter. When a stainlesssteel ball is pushed out by an eclosing fly’s head and crosses the infrared beam, a signal is fed to a computer and the number of eclosions is counted. All measurements under outdoor conditions were performed at the experimental farm of the National Agricultural Research Center for Hokkaido Region (NARCH) at Sapporo (43°030 N) in August 2007 and June–July 2008. The recording apparatus filled with post-diapause pupae was completely covered with a net laundry bag to
The adult eclosion of S. crassipalpis and soil temperature were recorded under outdoor conditions at different soil depths (5 and 20 cm; Fig. 1). An investigation in August 2007 showed that the average soil temperatures were 26.6 and 24.6 °C at 5- and 20-cm depths, respectively. The phase and the amplitude of the temperature cycle were distinctly different depending on the depth. The soil temperature began to increase from 08:00 h at 5-cm depth, but from 13:00 h at 20-cm depth. The range of temperature change averaged 3.5 °C at 5-cm depth but was only 0.6 °C at 20-cm depth (Fig. 1A and B, right panels). Daily fluctuation of soil temperature varied considerably from day to day (Fig. 1A and B, left panels), depending on the solar radiation (Fig. 1C). At 5-cm depth on August 12–14, for example, the soil temperature clearly showed cyclic fluctuations, whereas on August 15 the fluctuations attenuated. On August 16, when the solar radiation was minor, the soil temperature continued to decline gradually all through the day. Clearly cyclic fluctuations of the soil temperature were again observed
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Fig. 1. Outdoor soil temperature and distribution of adult eclosion from pupae of Sarcophaga crassipalpis. Field experiments were performed at 5-cm (A) and 20-cm (B) depths in the soil in August 2007 and at 5-cm (D) and 20-cm (E) depths in the soil in June–July 2008. Each left panel shows the soil temperature and the record of adult eclosions for a certain 10-day period of the experiment period (A,B,D, and E), and also the corresponding data of solar radiation in August 2007 (C) and in June–July 2008 (F). Each right panel (A,B,D, and E) shows the data for a 5-day period indicated by a double-headed arrow in the left panel, as the mean temperature at 5- or 20-cm depth in the soil and the pooled record of daily eclosion. Triangles in right panels represent /E (closed) and the time when the temperature cycle began to increase (open). R values show the degree of rhythmicity. Time is Japan Standard Time. The values of /E were 9.2, 11.1, 9.8, and 11.6 h in A, B, D, and E, respectively. Average sunrise times for a 5-day record period were 04:38, 04:39, 03:57, and 03:58 h in A, B, D, and E, respectively. Average sunset times for a 5-day record period were 18:40, 18:38, 19:18, and 19:18 h in A, B, D, and E, respectively.
from August 19 (Fig. 1A, left panel). The timing of adult eclosion was synchronous but differed with soil depth (Fig. 1A and B, right panels). The eclosion occurred earlier at 5-cm depth than at 20-cm depth, but it is noteworthy that the difference between the medians (/E) of the 2 depths was only 1.9 h, compared with the differ-
ence of 5 h in the phase of temperature cycle between the respective soil depths. This investigation was repeated in June–July 2008. The average soil temperatures were 23.5 and 20.1 °C at 5- and 20-cm depths, respectively. The soil temperature began to increase from 07:00 h
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at 5-cm depth, but from 12:00 h at 20-cm depth. The range of temperature change averaged 10.5 °C at 5-cm depth but was only 1.5 °C at 20-cm depth (Fig. 1D and E, right panels). The daily solar radiation was relatively stable (Fig. 1F) as compared to an investigation in August 2007 (Fig. 1C), and the daily fluctuation of soil temperature did not vary considerably from day to day (Fig. 1D and E, left panels). The timing of adult eclosion was similar to an investigation in August 2007 (Fig. 1D and E, right panels). Again, the difference in /E between the 2 depth groups was only 1.8 h in spite of the 5 h difference in the phase of temperature cycle between the respective soil depths. Thus, most flies emerged in the late morning in both investigations and the R values were all less than 5, which indicate highly synchronous eclosion (Fig. 1, right panels). /E at 5-cm depth was about 2–3 h after the lowest phase point of the soil temperature cycle and /E at 20-cm depth occurred at about the lowest phase point of the temperature cycle. This means that the flies can, to some extent, compensate for the depth-dependent phase delay of zeitgeber, and the compensation was achieved by advancing the eclosion timing of pupae located deeper in the soil relative to the temperature fluctuations. In addition, this advance of the eclosion timing may be established by a circadian clock controlling eclosion rather than as a direct response to temperature change, because flies eclosed at the appropriate time on August 16, 2007, despite the continuous decline of soil temperature. 3.2. Eclosion under laboratory conditions To examine whether the timing adjustment of eclosion of S. crassipalpis is a response to the amplitude of temperature cycles, the adult eclosion was recorded under laboratory conditions with various amplitudes (0, 1, 2, 4, 6, or 8 °C) of temperature cycles under DD. First, the adult eclosion was recorded under pseudo-sinusoidal temperature cycles where temperature was changed every 1 h (Fig. 2). When the average temperature was 20 °C, the /E values were 10.8, 10.0, 9.5, 7.7, and 6.1 h in amplitudes of 8, 6, 4, 2, and 1 °C, respectively (Fig. 2A–E). There were statistical differences in the timing of adult eclosion among these regimes, except between regimes with amplitudes of 6 and 4 °C (Steel–Dwass test; P < 0.05). /E occurred a few hours after the temperature began to increase under the temperature cycles whose amplitude was large, whereas /E was at the lowest temperature of the temperature cycle with the amplitude of 1 °C. Such phase relationships between /E and the temperature cycle were coincident with those under outdoor conditions. At a constant 20 °C, the eclosion was also rhythmic (R = 54.0), but the peak was considerably broader than under pseudo-sinusoidal temperature cycles and the /E was 3.5 h (Fig. 2F). These results show that the eclosion rhythm of S. crassipalpis was entrained to the pseudo-sinusoidal temperature cycle, and /E progressively advanced as the amplitude of the temperature cycle decreased. When the average temperature was 25 °C, the pseudosinusoidal temperature cycle also entrained the eclosion rhythm and similarly /E advanced as the temperature amplitude decreased, although the advance response was weaker than when the average temperature was 20 °C (Fig. 2G–L). Thus, we observed that the timing adjustment of eclosion under laboratory conditions was similar to that observed under outdoor conditions. Therefore, we conclude that these advances are caused in response to the amplitude of temperature cycles. Second, the adult eclosion was recorded under a square-wave temperature cycle that consisted of a warm phase and a cool phase; the temperature difference between the 2 phases was varied (0, 1, 2, 4, 6, or 8 °C) with the same average temperature (20 °C; Fig. 3). Under a 12-h warm phase and a 12-h cool phase (WC 12:12), the /E values were close to the time of the temperature increase in the thermoperiod, regardless of the magnitude of temper-
ature change (Fig. 3A–E), although at a constant 20 °C the eclosion peak was unclear because eclosion was observed in only a small number of individuals during the experimental period (Fig. 3F). There was no statistical difference in the timing of eclosion among the regimes with the amplitude of 2 °C or more (Steel–Dwass test; P > 0.05; Fig. 3A–D). In these regimes, most flies emerged within 1 h after the temperature increase. Under the 1 °C amplitude, eclosion was less synchronous but many individuals eclosed within 1 h after the temperature increase (Fig. 3E). Therefore, the shift of /E in response to the amplitude of temperature cycles was not observed under square-wave cycles of WC 12:12, in contrast to under pseudo-sinusoidal temperature cycles. Under WC 6:18, the /E values were also close to the time of temperature increase (Fig. 3G–L). Statistical differences in the timing of eclosion were found among some regimes (Steel–Dwass test; P < 0.05), but there was no correlation with the magnitude of temperature change. Third, to examine whether differences in the amplitude of pseudo-sinusoidal temperature cycles affect a circadian clock controlling eclosion or merely influence more downstream eclosion processes, the adult eclosion was recorded at 20 °C after pupae had experienced pseudo-sinusoidal cycles with high (8 °C) or low (1 °C) amplitude under DD; these measurements were compared to those of pupae continuously exposed to temperature cycles and pupae that had not experienced temperature cycles (Fig. 4). When flies were exposed to high-amplitude temperature change and then were transferred to 20 °C, circadian rhythmicity was shown in adult eclosion. The free-running period (s) was 24.3 h (Fig. 4B). The /E value in the pooled record was 10.9 h, which was close to that (10.6 h) under continuous temperature cycles (Fig. 4A and B). There was no statistical difference in eclosion timing between these 2 regimes (Wilcoxon rank sum test; Z = 0.31, P = 0.754). Flies that did not experience temperature cycles began to emerge several days later than those experiencing temperature cycles. The rhythmicity was weak (R = 63.3) and the /E value in the pooled record was 1.4 h, which is distinctly different from the /E in flies experiencing temperature cycles (Fig. 4C). When flies were exposed to low-amplitude temperature change and then were transferred to 20 °C, circadian rhythmicity was shown and s was 23.2 h (Fig. 4E). The /E value in the pooled record was 6.7 h, which was close to that (7.2 h) under continuous temperature cycles (Fig. 4D and E). There was no statistical difference in eclosion timing between these 2 regimes (Wilcoxon rank sum test; Z = 0.83, P = 0.406). In flies not experiencing temperature cycles, circadian rhythmicity was also observed and s was 24.0 h. However, the /E value in the pooled record was 20.1 h, which is far from the /E in flies experiencing temperature cycles (Fig. 4F). These results indicate that differences in the amplitude of pseudo-sinusoidal temperature cycles affect a circadian clock controlling eclosion because the advance response of the eclosion timing was persistent even in the free-running rhythm after temperature was fixed at 20 °C. In the above exposure to low-amplitude temperature change, at the same time, we also recorded eclosion under antiphase conditions, by shifting the phase of temperature fluctuations by 12 h (Fig. 5). The peak time of eclosion was also shifted by about 12 h, compared to that in Fig. 4D and E. Under continuous temperature cycles, the eclosion peak occurred at the lowest temperature point and the /E value in the pooled record was 18.9 h (Fig. 5A). When flies were transferred to 20 °C, s was 23.4 h and the /E value in the pooled record was 17.8 h (Fig. 5B). These R values were much less than that in flies not experiencing temperature cycles (Fig. 4F). Thus, the circadian eclosion clock was clearly entrained to temperature cycles with the amplitude of 1 °C. Lastly, to examine the effects of the abrupt temperature change in square-wave temperature cycles on eclosion and on the circadian clock controlling it, adult eclosion was recorded at 20 °C after
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Fig. 2. Various pseudo-sinusoidal temperature cycles and distribution of adult eclosion from pupae of Sarcophaga crassipalpis under continuous darkness. The average temperature was 20 °C (A–F) or 25 °C (G–L). The amplitudes of temperature cycles were 8 °C (A and G), 6 °C (B and H), 4 °C (C and I), 2 °C (D and J), 1 °C (E and K), or 0 °C (F and L). R values show the degree of rhythmicity. At the average temperature of 20 °C, the values of /E (triangle) were 10.8, 10.0, 9.5, 7.7, 6.1, and 3.5 h in A, B, C, D, E, and F, respectively. At the average temperature of 25 °C, the values of /E were 9.6, 9.6, 8.1, 8.1, 7.0, and 16.6 h in G, H, I, J, K, and L, respectively. The differences in /E were examined statistically by the Steel–Dwass test for nonparametric multiple comparison in A–E and in G–K, separately (Hochberg and Tamhane, 1987). The /E values with the same letter (a–d in A–E; e, f in G–K) are not significantly different (P > 0.05).
pupae had experienced WC 12:12 with high (8 °C) or low (1 °C) amplitude under DD; this was compared to eclosion of pupae continuously exposed to temperature cycles and pupae not experiencing temperature cycles (Fig. 6). When flies were exposed to
high- and low-amplitude temperature change, and not exposed to any temperature change during the experimental period, circadian rhythmicity was shown after transfer to a constant temperature; the values of s were 24.4, 23.7, and 24.5 h, respectively
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Fig. 3. Various square-wave temperature cycles (average temperature, 20 °C) and distribution of adult eclosion from pupae of Sarcophaga crassipalpis under continuous darkness. Thermoperiod was 12 h warm phase and 12 h cool phase (WC 12:12, A–F) or 6 h warm phase and 18 h cool phase (WC 6:18, G–L). The temperature difference between the 2 phases was 8 °C (A and G), 6 °C (B and H), 4 °C (C and I), 2 °C (D and J), 1 °C (E and K), or 0 °C (F and L). R values show the degree of rhythmicity. Under WC 12:12, the values of /E (triangle) were 12.6, 12.5, 12.5, 12.7, and 12.0 h in A, B, C, D, and E, respectively. Under WC 6:18, the values of /E were 12.5, 11.5, 12.4, 12.7, and 12.7 h in G, H, I, J, and K, respectively. The differences in /E were examined statistically by the Steel–Dwass test for nonparametric multiple comparison in A–E and in G–K, separately (Hochberg and Tamhane, 1987). The /E values with the same letter (a, b in A–E; c–e in G–K) are not significantly different (P > 0.05).
(Fig. 6B, D and E). The eclosion records showed that under squarewave temperature cycles, /E was reset close to, or just after, the time at which temperature increased in the thermoperiod, irrespective of the amplitude of the temperature exposure – although WC 12:12, with a 1 °C amplitude, was insufficient to reset the
eclosion phase in all individuals. The advance of eclosion timing that was dependent upon the exposed temperature amplitude was also not observed in the free-running rhythm. In the exposure to high-amplitude change, the /E value in the pooled record after transfer to 20 °C was significantly different from that under WC
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Fig. 4. Effects of pseudo-sinusoidal temperature cycles on the circadian eclosion rhythm of Sarcophaga crassipalpis under continuous darkness. Pupae were exposed to temperature cycles with the temperature amplitude of 8 °C continuously (A); to 20 °C after transfer from temperature cycles with the temperature amplitude of 8 °C (B); to temperature cycles with the temperature amplitude of 1 °C continuously (D); to 20 °C after transfer from temperature cycles with the temperature amplitude of 1 °C (E); or to 20 °C continuously (C and F). Each left panel shows temperature and the record of adult eclosions for a certain 7-day period of the experiment. Each right panel shows the data for the period indicated by a double-headed arrow in the left panel, as the pooled record of daily eclosion. R values show the degree of rhythmicity. The values of /E (triangle) in right panels were 10.6, 10.9, 1.4, 7.2, 6.7, and 20.1 h in A, B, C, D, E, and F, respectively.
12:12 (Wilcoxon rank sum test; Z = 4.77, P < 0.001; Fig. 6A and B), in contrast to that under pseudo-sinusoidal temperature cycles (Fig. 4A and B). This may be attributed to a burst of eclosion
directly invoked by an abrupt temperature increase. This direct response is induced to some extent by even low-amplitude temperature change (Fig. 6C and D).
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Fig. 5. Effects of shifting the phase of the pseudo-sinusoidal temperature cycle by 12 h on the circadian eclosion rhythm of Sarcophaga crassipalpis under continuous darkness. Pupae were exposed to temperature cycles with the temperature amplitude of 1 °C continuously (A); or to 20 °C after transfer from temperature cycles with the temperature amplitude of 1 °C (B). The phase of the temperature cycle in A was shifted by 12 h from that in Fig. 4D. The phase of the temperature cycle in B was shifted by 12 h from that in Fig. 4E. Each left panel shows temperature and the record of adult eclosions for a certain 7-day period of the experiment. Each right panel shows the data for the period indicated by a double-headed arrow in the left panel, as the pooled record of daily eclosion. R values show the degree of rhythmicity. The values of /E (triangle) in right panels were 18.9 and 17.8 h in A and B, respectively.
4. Discussion 4.1. Entrainment to temperature cycles during the pupal stage Although the circadian rhythm of eclosion has been reported in many insects (Saunders, 2002; Myers, 2003), there are few reports associated with the location where eclosion occurs. In the laboratory and when exposed to temperature cycles that simulate soiltemperature fluctuations, D. antiqua ecloses at the same time as in the outdoor soil (Tanaka and Watari, 2003). The present results demonstrated that the eclosion pattern of S. crassipalpis in the soil was consistent with that observed in response to pseudo-sinusoidal temperature cycles in the laboratory. This indicates the importance of temperature cycles for the eclosion timing of S. crassipalpis. Flies are generally thought to eclose around dawn (Pittendrigh, 1954; Roberts et al., 1983; Denlinger and Zˇdárek, 1994; Saunders, 2002). Experiments under light–dark cycles in the laboratory indicate that S. crassipalpis ecloses around dawn (Yocum et al., 1994). The present studies of S. crassipalpis eclosion in the soil, however, showed that this fly ecloses from early morning to mid-afternoon, with a peak in the late morning rather than around dawn. However, similar experiments performed in August 2000 showed that D. antiqua eclosed from mid-night to noon, with a peak just after dawn. Most eclosions at both 5- and 20-cm depths were completed before the temperature increase, in contrast to S. crassipalpis (Tanaka and Watari, 2003). These results indicate that the eclosion time of flies in the field varies depending on the species. For example, the tsetse fly, Glossina morsitans, emerges as an adult from the soil in mid-afternoon. This eclosion rhythm acutely responds to the thermoperiod but not the photoperiod, and the peak occurs during the late temperature increase (Zˇdárek and Denlinger, 1995). The eclosion clock of S. crassipalpis is highly sensitive to light– dark cycles during the wandering larval stages and free-runs with a period close to 24 h in DD (Joplin and Moore, 1999). Joplin and Moore (1999) suggested that these properties of the circadian clock allow S. crassipalpis to determine precisely the eclosion phase with respect to environmental conditions despite a pupal stage
that is spent underground, as suggested by other researchers (Winfree, 1974; Saunders, 1976, 1979; Roberts et al., 1983; Smith, 1985). In the present study, when pupae of S. crassipalpis were transferred to DD at constant temperature within 2–3 h after chilling – i.e., in control experiments without daily temperature cycles – the eclosion patterns were not consistent in terms of the rhythmicity and days of adult emergence (Figs. 2F,L, 3F,L, 4C,F, 6E). We cannot sufficiently explain what factors caused these variable patterns, because there are various possibilities, such as photoperiodic entrainment of the larval clock, resetting of the pupal clock by room light for a few hours and a temperature step-up from 7.5 °C before the experiments, and the difference of responsiveness to external stimuli among pupae. However, we found that temperature cycles applied to the pupal stage did produce eclosion entrainment. Temperature cycles not only initiated high rhythmicity in eclosion (e.g., Fig. 2E and F) but also reset the circadian clock (e.g., Fig. 4E and F), so that the eclosion peak occurred at the optimal time, even under a 1 °C temperature amplitude. We therefore consider that in insects that pupate underground, the eclosion time under natural conditions should be identified based on the responsiveness to thermoperiods rather than to photoperiods. This is likely important for pupae that overwinter for a long time, especially in areas such as Sapporo, where the winter soil temperature does not fluctuate, because of snow cover, but begins to fluctuate again in the spring, after diapause (Watari, 2005). Daily changes in the soil temperature are varied depending on the weather and do not show clear fluctuations when the solar radiation is low. Under outdoor conditions, the soil temperature on August 16, 2007, continued to decline only gradually during the day, but the eclosion peak occurred in the late morning (Fig. 1A and B, left panels). This suggests that the circadian clock was entrained by soil temperature fluctuations that were experienced before August 16, with eclosion at the appropriate time established by the internal periodicity, as occurs in the laboratory after the transfer from temperature cycles to constant temperature under DD. Therefore, a free-running period close to 24 h likely helps pupae emerge as adults at the optimal time when the circadian rhythm persists without temperature cycles, rather than
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Fig. 6. Effects of square-wave cycles of 12 h warm phase and 12 h cool phase on the circadian eclosion rhythm of Sarcophaga crassipalpis under continuous darkness. Pupae were exposed to temperature cycles with the temperature difference of 8 °C continuously (A); to 20 °C after transfer from temperature cycles with the temperature difference of 8 °C (B); to temperature cycles with the temperature difference of 1 °C continuously (C); to 20 °C after transfer from temperature cycles with the temperature difference of 1 °C (D); or to 20 °C continuously (E). Each left panel shows temperature and the record of adult eclosions for a certain 7-day period of the experiment. Each right panel shows the data for the period indicated by a double-headed arrow in the left panel, as the pooled record of daily eclosion. R values show the degree of rhythmicity. The values of /E (triangle) in right panels were 12.5, 13.5, 14.1, 15.8, and 0.2 h in A, B, C, D, and E, respectively.
photoperiodic cycles. The sensitivity of larvae to photoperiods may be meaningful for the eclosion rhythm only when the pupal period is short and there is no reliable temperature signal during the period, such as in locations deep underground or during long periods with cloudy days. 4.2. Effects of the amplitude of temperature cycles on the eclosion timing In the present study, the eclosion phase of S. crassipalpis progressively advanced as the amplitude of the pseudo-sinusoidal temperature cycle decreased and coincided with the lowest temperature when the amplitude was 1 °C. As reported in D. antiqua
(Tanaka and Watari, 2003), this response allows pupae located at any depth in the soil to eclose at the appropriate time of day, despite the depth-dependent phase delay of the temperature change. Insects other than flies that pupate underground may also have an eclosion timing that is linked to the amplitude of temperature cycles (Tanaka and Watari, 2003). A transfer from pseudo-sinusoidal temperature cycles to constant temperature revealed that the eclosion time is set in response to the amplitude based on the phase of a circadian clock. This tempts us to investigate, at the molecular level, the responsiveness of the circadian clock of flies to the amplitude of temperature change. The eclosion peak of D. antiqua occurs 3–4 h earlier in WC 12:12 with a temperature difference of 1 °C than with a temperature dif-
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ference of 4 °C (Tanaka and Watari, 2003; Watari and Tanaka, 2010). However, the present results did not show an alteration of eclosion timing in response to the amplitude of the square-wave temperature cycle in S. crassipalpis, even after transfer to constant temperature. Although it is unclear why such a difference was observed between S. crassipalpis and D. antiqua, it may be attributed to the optimal eclosion time in the field. Delia antiqua ecloses around dawn, and the peak occurs during the decline of the sinusoidal temperature cycle and during the cool phase of the square-wave temperature cycle (Watari, 2002b; Tanaka and Watari, 2003; Watari and Tanaka, 2010). However, near-surface pupae of S. crassipalpis emerge as adults in the late morning when the soil temperature rapidly begins to increase (see Fig. 1A and D). The rapid temperature increase may be the reliable time signal of late morning for the circadian clock, and therefore, resets the eclosion rhythm to the particular phase. The rapid temperature increase of 2 °C or more directly invoked the extremely narrow eclosion peak (Figs. 3 and 6). This high sensitivity indicates the importance of the temperature environment for the eclosion timing of S. crassipalpis. There was a difference in the eclosion time between the pseudosinusoidal cycle and the square-wave cycle with the 1 °C amplitude. Under the pseudo-sinusoidal cycle, the eclosion peak occurred at the lowest temperature point, whereas under the square-wave cycle, the peak occurred around the temperature increase (Figs. 2E, 3E, 4D, and 6C). This difference indicates our limited ability to simulate natural conditions based on a square-wave environmental cycle. Recent experiments on the locomotor activity of the fruit fly, Drosophila melanogaster, also demonstrated different results between pseudo-sinusoidal and the square-wave temperature cycles (Yoshii et al., 2009). These results suggest the significance of applying sinusoidal temperature cycles to discuss temperature entrainment of circadian rhythms under natural conditions. In summary, the present study of the circadian clock underlying eclosion of S. crassipalpis revealed the following 3 points. First, the clock is entrained to temperature cycles in the pupal stage with high sensitivity under both natural and laboratory conditions. Second, the eclosion phase of a clock is progressively advanced as the amplitude of the sinusoidal temperature cycle decreases, as shown in D. antiqua. Third, the abrupt temperature increase reset the eclosion phase of the circadian clock to the increasing time, regardless of the amplitude of temperature cycles, unlike D. antiqua. These properties of the circadian clock of S. crassipalpis would enhance the likelihood of emerging as adults from the soil at the optimal time of the day (late morning).
Acknowledgements We thank Dr. Toru Shimizu for providing the computer program for eclosion recording. We also thank Drs. Kiyomitu Ito, Junichi Kaneko, and Kazuhiko Konishi for allowing us to use an experimental field of NARCH. This study was supported by a Grant-in-Aid for Scientific Research (C) (19570075) from the Japan Society for the Promotion of Science and, in part, by a special research Grant from Miyagi Gakuin Women’s University (2008).
References Denlinger, D.L., 1972. Induction and termination of pupal diapause in Sarcophaga (Diptera: Sarcophagidae). Biological Bulletin 142, 11–24. Denlinger, D.L., 1981. The physiology of pupal diapause in flesh flies. In: Bhaskaran, G., Friedman, S., Rodriguez, J.G. (Eds.), Current Topics in Insect Endocrinology and Nutrition. Plenum, New York, pp. 131–160. Denlinger, D.L., Zˇdárek, J., 1994. Metamorphosis behavior of flies. Annual Review of Entomology 39, 243–266. Hirashima, Y., 1989. A Check List of Japanese Insects II. Entomological Laboratory, Faculty of Agriculture. Kyushu University and Japan Wild Life Research Center, Fukuoka. Hochberg, Y., Tamhane, A.C., 1987. Multiple Comparison Procedures. Wiley, New York. Joplin, K.H., Moore, D., 1999. Effects of environmental factors on circadian activity in the flesh fly, Sarcophaga crassipalpis. Physiological Entomology 24, 64–71. Lankinen, P., Lumme, J., 1982. An improved apparatus for recording the eclosion rhythm in Drosophila. Drosophila Information Service 58, 161–163. Myers, E.M., 2003. The circadian control of eclosion. Chronobiology International 20, 775–794. Pape, T., Dahlem, G., de Mello Patiu, C.A. Giroux, M., 2010. The world of flesh flies (Diptera: Sarcophagidae). http://www.zmuc.dk/entoweb/sarcoweb/sarcweb/ sarc_web.htm (accessed 22.1.2011). Pittendrigh, C.S., 1954. On temperature independence in the clock system controlling emergence time in Drosophila. Proceedings of the National Academy of Sciences of the United States of America 40, 1018–1029. Roberts, R.M., Northover, J.M., Lewis, R.D., 1983. Circadian clock control of the eclosion rhythm of the brown blowfly, Calliphora stygia (Diptera: Calliphoridae). New Zealand Entomologist 4, 424–431. Saunders, D.S., 1976. The circadian eclosion rhythm in Sarcophaga argyrostoma: some comparisons with the photoperiodic clock. Journal of Comparative Physiology 110, 111–133. Saunders, D.S., 1979. The circadian eclosion rhythm in Sarcophaga argyrostoma: delineation of the responsive period for entrainment. Physiological Entomology 4, 263–274. Saunders, D.S., 2002. Insect Clocks, third ed. Elsevier, Amsterdam. Smith, P.H., 1985. Responsiveness to light of the circadian clock controlling eclosion in the blowfly, Lucilia cuprina. Physiological Entomology 10, 323– 336. Tanaka, K., Watari, Y., 2003. Adult eclosion timing of the onion fly, Delia antiqua, in response to daily cycles of temperature at different soil depths. Naturwissenschaften 90, 76–79. Truman, J.W., 1972. Involvement of a photoreversible process in the circadian clock controlling silkmoth eclosion. Zeitschrift für Vergleichende Physiologie 76, 32– 40. Watari, Y., 2002a. Comparison of the circadian eclosion rhythm between nondiapause and diapause pupae in the onion fly, Delia antiqua. Journal of Insect Physiology 48, 83–89. Watari, Y., 2002b. Comparison of the circadian eclosion rhythm between nondiapause and diapause pupae in the onion fly, Delia antiqua: the effect of thermoperiod. Journal of Insect Physiology 48, 881–886. Watari, Y., 2005. Comparison of the circadian eclosion rhythm between nondiapause and diapause pupae in the onion fly, Delia antiqua: the change of rhythmicity. Journal of Insect Physiology 51, 11–16. Watari, Y., Tanaka, K., 2010. Interacting effect of thermoperiod and photoperiod on the eclosion rhythm in the onion fly, Delia antiqua supports the two-oscillator model. Journal of Insect Physiology 56, 1192–1197. Winfree, A.T., 1970. Integrated view of resetting a circadian clock. Journal of Theoretical Biology 28, 327–374. Winfree, A.T., 1974. Suppressing Drosophila circadian rhythm with dim light. Science 183, 970–972. Yocum, G.D., Zˇdárek, J., Joplin, K.H., Lee, R.E., Smith, D.C., Manter, K.D., Denlinger, D.L., 1994. Alteration of the eclosion rhythm and eclosion behavior in the flesh fly, Sarcophaga crassipalpis, by low and high temperature stress. Journal of Insect Physiology 40, 13–21. Yoshii, T., Vanin, S., Costa, R., Helfrich-Förster, C., 2009. Synergic entrainment of Drosophila’s circadian clock by light and temperature. Journal of Biological Rhythms 24, 452–464. Zˇdárek, J., Denlinger, D.L., 1995. Changes in temperature, not photoperiod, control the pattern of adult eclosion in the tsetse, Glossina morsitans. Physiological Entomology 20, 362–366.