effects of skeleton photoperiods on the induction of larval diapause in the indian meal moth ...

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Physiological Entomology (2009) 34, 180–184 DOI: 10.1111/j.1365-3032.2009.00673.x © 2009 The Authors 180 Journal compilation © 2009 The Royal Entomological Society Introduction The Indian meal moth Plodia interpunctella Hübner (Lepidoptera: Pyralidae) may enter diapause as a last (fifth)- instar larva in response to the photoperiod during its larval life (Tsuji, 1963; Bell, 1976). The photoperiodic response curve reported by Kikukawa et al. (2008) is a long-day type at 25 °C. In continuous darkness (DD), the incidence of diapause is 43%. It is low (< 12%) under very short days (LD 2 : 22 h to 4 : 20 h), increases to > 84% under photope- riods between LD 10 : 14 h and 12 : 12 h and then decreases sharply to 13% under an LD 14 : 10 h photoperiod, showing a critical day length of 13 h. Diapause is averted completely Effects of skeleton photoperiods on the induction of larval diapause in the Indian meal moth Plodia interpunctella SHIGERU KIKUKAWA, YOUSUKE ARAKAWA, KANAKO HAYAKAWA, MIHO HAYASHI, KOUICHI KATOU, JUNJI KANESHIGE, MASASHI KIMURA, TOMOMI NAKAMURA, YASUHIKO NAKAMURA and HITOMI WATANABE Biological Institute, Faculty of Science, University of Toyama, Toyama, Japan Abstract. The Indian meal moth Plodia interpunctella Hübner (Lepidoptera: Pyralidae) may measure night length to determine its developmental programme with respect to entering diapause or continuing development in the last-larval instar. Its photoperiodic clock appears to be hourglass-like, in that the response to a symmetrical skeleton photoperiod consists of two 2-h light pulses. If the insects read this as lights-on and lights-off signals, an oscillator should be involved in the clock system. In the present study, the insects are maintained under a normal photoperiod for 1 day at 25 and 20 °C and then transferred to the skeleton photoperiod or constant darkness (DD) for 1, 2 or 3 days. These photoperiodic regimes are repeated during the larval sensitive stage. There is no difference in the percentage diapause between the effects of skeleton photoperiod and DD treatment. At 25 °C, the incidence of diapause is relatively low under very short days (< 8-h main photophase) and long (> 14-h main photophase) days and relatively high under a 12-h main photophase. At 20 °C, the percentage of diapause is generally high under any photoperiodic treatment. Diapause is prevented slightly under LD 14 : 10 h and 16 : 8 h photoperiods. If the diapause clock is an oscillator that damps rap- idly, under DD or constant light (LL) for 5–15 days, it may stop its oscillation but, after exposure to the skeleton photoperiod, the clock system might be reset. There is no dif- ference in the diapause-inducing effect between the skeleton and normal photoperiodic treatments after DD. The incidence of diapause is moderately high under short days (<12 h) but low under long days (>16 h). On the other hand, when the insects are exposed to the skeleton photoperiod after LL, the induction of diapause is low, regard- less of the interval between the two light pulses in skeleton photoperiods. Under normal photoperiodic conditions after LL, a clear photoperiodic response curve is obtained. Thus, P. interpunctella does not show any sign of the oscillatory function in the night- time measurement and is regarded as being ‘hourglass-like’. Key words. diapause clock, Indian meal moth, larval diapause, skeleton photoperiod. Correspondence: Shigeru Kikukawa, Biological Institute, Faculty of Science, University of Toyama, Toyama 930-8555, Japan. Tel.: +76 445 6635; fax: +76 445 6549; e-mail: [email protected]. ac.jp

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Page 1: Effects of skeleton photoperiods on the induction of larval diapause in the Indian meal moth               Plodia interpunctella

Physiological Entomology (2009) 34, 180–184 DOI: 10.1111/j.1365-3032.2009.00673.x

© 2009 The Authors180 Journal compilation © 2009 The Royal Entomological Society

Introduction

The Indian meal moth Plodia interpunctella Hübner (Lepidoptera: Pyralidae) may enter diapause as a last (fifth)-

instar larva in response to the photoperiod during its larval life ( Tsuji, 1963; Bell, 1976 ). The photoperiodic response curve reported by Kikukawa et al. (2008) is a long-day type at 25 °C. In continuous darkness (DD), the incidence of diapause is 43%. It is low (< 12%) under very short days (LD 2 : 22 h to 4 : 20 h), increases to > 84% under photope-riods between LD 10 : 14 h and 12 : 12 h and then decreases sharply to 13% under an LD 14 : 10 h photoperiod, showing a critical day length of 13 h. Diapause is averted completely

Effects of skeleton photoperiods on the induction of larval diapause in the Indian meal moth Plodia interpunctella

S H I G E R U K I K U K A W A , Y O U S U K E A R A K A W A , K A N A K O H A Y A K A W A , M I H O H A Y A S H I , K O U I C H I K A T O U , J U N J I K A N E S H I G E , M A S A S H I K I M U R A , T O M O M I N A K A M U R A , Y A S U H I K O N A K A M U R A and H I T O M I W A T A N A B E Biological Institute, Faculty of Science, University of Toyama, Toyama, Japan

Abstract . The Indian meal moth Plodia interpunctella Hübner (Lepidoptera: Pyralidae) may measure night length to determine its developmental programme with respect to entering diapause or continuing development in the last-larval instar. Its photoperiodic clock appears to be hourglass-like, in that the response to a symmetrical skeleton photoperiod consists of two 2-h light pulses. If the insects read this as lights-on and lights-off signals, an oscillator should be involved in the clock system. In the present study, the insects are maintained under a normal photoperiod for 1 day at 25 and 20 °C and then transferred to the skeleton photoperiod or constant darkness (DD) for 1, 2 or 3 days. These photoperiodic regimes are repeated during the larval sensitive stage. There is no difference in the percentage diapause between the effects of skeleton photoperiod and DD treatment. At 25 °C, the incidence of diapause is relatively low under very short days (< 8-h main photophase) and long (> 14-h main photophase) days and relatively high under a 12-h main photophase. At 20 °C, the percentage of diapause is generally high under any photoperiodic treatment. Diapause is prevented slightly under LD 14 : 10 h and 16 : 8 h photoperiods. If the diapause clock is an oscillator that damps rap-idly, under DD or constant light (LL) for 5 – 15 days, it may stop its oscillation but, after exposure to the skeleton photoperiod, the clock system might be reset. There is no dif-ference in the diapause-inducing effect between the skeleton and normal photoperiodic treatments after DD. The incidence of diapause is moderately high under short days (<12 h) but low under long days (>16 h). On the other hand, when the insects are exposed to the skeleton photoperiod after LL, the induction of diapause is low, regard-less of the interval between the two light pulses in skeleton photoperiods. Under normal photoperiodic conditions after LL, a clear photoperiodic response curve is obtained. Thus, P. interpunctella does not show any sign of the oscillatory function in the night-time measurement and is regarded as being ‘hourglass-like’.

Key words . diapause clock , Indian meal moth , larval diapause , skeleton photoperiod .

Correspondence: Shigeru Kikukawa, Biological Institute, Faculty of Science, University of Toyama, Toyama 930-8555, Japan. Tel.: +76 445 6635; fax: +76 445 6549; e-mail: [email protected]

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in photoperiods longer than 16 h. At 20 °C, the incidence of diapause is generally high (> 87%) under any photoperiod, except for the range between LD 14 : 10 h and 20 : 4 h, under which the percentage diapause is moderate and reaches its lowest level under LD 16 : 8 h and 18 : 6 h photoperiods.

It is proposed that the time-measuring system underlying the seasonal determination of diapause and related phenom-ena can be either an oscillator or hourglass type ( Saunders, 2002 ). Vaz Nunes & Saunders (1999) list 13 types of clock models in different species. For the diapause determination in P. interpunctella , no evidence of an oscillator system is reported to date ( Takeda & Masaki, 1976; Masaki & Kikukawa, 1981; Kikukawa & Masaki, 1984; Kikukawa et al. , 1998 , 2005; Kikukawa & Ohde, 2007 ); accordingly, all experimental results including night interruptions can be explained by an hourglass timer that measures night length. However, Saunders et al. (2004) propose that an oscillator that damps rapidly can act as if it is an hourglass timer. One experimental protocol to detect an operation of the oscillator mechanism(s) comprises a skeleton photoperiod ( Saunders, 2002 ). Subsequently, this technique has been adopted in several insect species ( Saunders, 2002 ). In the present study, the effects of symmetrical skeleton photoperiods on diapause determination in P. interpunctella are examined to determine whether an oscillator component is involved in the night-time measurement.

Materials and methods

A laboratory colony of P. interpunctella was established from larvae collected at Toyama in January 1998. Eggs obtained from adult females ( Tsuji, 1960 ) at 25 °C under an LD 16 : 8 h photoperiod and approximately 75% RH were placed in transparent plastic cups (diameter 15 cm, height 9 cm) in groups of 100. Food (1 g per egg) consisted of commercial rice bran, the moisture content of which was standardized ( Kikukawa & Masaki, 1984 ). Experiments were conducted at 20 and 25 °C (with an approximate sample size of 100 for each treatment). Under these conditions, nondiapauing larvae pupated in the food and emerged as adults by day 60 after oviposition. Diapausing larvae did not pupate by day 130. Thus, the incidence of diapause could be resolved simply by determining the proportion of adults that had emerged by day 60. As a light source, two 10-W daylight fluorescent tubes were used. Photoperiodic cycles were controlled by a 24-h time switch or manually. The appropriate experimental protocols are described along with the results obtained.

Results

Symmetrical skeleton photoperiod at 25 °C

To determine the effect of symmetrical skeleton photoperi-ods on the diapause induction in P. interpunctella , insects were maintained under normal photoperiods for 1 day, then

transferred to skeleton photoperiods and maintained for 1, 2 or 3 days ( Fig. 1a – c , respectively). The skeleton photoperiod consisted of two 2-h light pulses. For example, larvae placed under an LD 6 : 18 h photoperiod for the first day were trans-ferred to an LDLD 2 : 2 : 2 : 18 h for the next 1, 2 or 3 days. The insects may read LDLD 2 : 2 : 2 : 18 h as being LD 6 : 18 h if their clock system is an oscillator, and thus entrained to the previous normal photoperiod, and they measure day or night length as the time between lights-on and lights-off signals in the subsequent skeleton regime: in this case, the main photophase might be 6 h of light. If the skeleton photoperiod thus mimics the normal photoperiod, the photoperiodic

Fig. 1. Responses to skeleton photoperiods in the determination of the larval diapause of Plodia interpunctella at 25 °C. Groups of insects were placed under a normal photoperiod for 1 day and then transferred to skeleton photoperiods ( � ) consisting of two 2-h light pulses or constant darkness ( ⦁ ) for (a) 1, (b) 2 or (c) 3 days. This photoperiodic treatment was repeated throughout larval development.

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response curve obtained should be the same as that under the stationary photoperiod ( Kikukawa et al. , 2008 ). As a control group, larvae were maintained under the normal photoperiod for 1 day and then under constant darkness (DD) for 1, 2 or 3 days, respectively. These treatments were repeated throughout larval development.

Figure 1 shows that there was no difference in the percent-age diapause between skeleton photoperiod and DD. The incidence of diapause was relatively low under very short days (< 8-h main photophase) and long days (> 14-h main photophase). It was relatively high under a 12-h main pho-tophase. This tendency, as demonstrated in Figure 1(a) , appeared to be less pronounced when the insects experienced a 3-day skeleton or DD ( Fig. 1c ). Therefore, the time-measuring system for the diapause determination did not recognize the skeleton photoperiods as giving lights-on and lights-off signals.

Symmetrical skeleton photoperiod at 20 °C

Because the photoperiodic response for the induction of diapause in P. interpunctella was highly sensitive to tempera-ture ( Kikukawa et al. , 2008 ), exposure to symmetrical skele-ton photoperiods was also tested at 20 °C ( Fig. 2 ). The experimental protocol was exactly the same as that employed at 25 °C. Larvae were maintained under normal photoperiods for 1 day, transferred to symmetrical skeleton photoperiods or DD and again maintained for 1, 2 or 3 days ( Fig. 2a – c , respectively). These sequences of treatment were repeated during the larval development. In Figure 2 , the percentage diapause was generally greater than 50% under any photope-riodic treatment. Diapause was prevented slightly under LD 14 : 10 h and 16 : 8 h photoperiods. There were substantial differences between the treatments that were followed by the skeleton photoperiod and DD. The skeleton photoperiods at 20 °C had no particular effect on diapause determination as at 25 °C.

Skeleton photoperiod after DD treatment at 25 °C

If the diapause clock of P. interpunctella is an oscillator that damps rather rapidly, it might stop its oscillation under DD for 5 – 15 days. Exposure to the skeleton photoperiod after DD might reset the clock system and invoke it to work. Thus, the insects were exposed to DD for 5, 10 or 15 days ( Fig. 3a – c , respectively) after oviposition and then to skele-ton photoperiods consisting of two 2-h light pulses or normal photoperiods. The incidence of diapause appeared to be mod-erately high under short days (< 12 h) but low under long days (> 16 h). If the diapause clock restarts its oscillation as a result of the skeleton photoperiod, the shape of photoperi-odic response curve should be the same as that of the control groups (i.e. the normal photoperiods after DD). There was no difference between the skeleton photoperiod and the normal photoperiod to which the insects were exposed after DD. Five to 15 days of DD suppressed the diapause-

Fig. 2. Experiments were conducted at 20 °C. A 1-day normal photoperiod was followed by the skeleton photoperiod ( � ) or con-stant darkness ( ⦁ ) for (a) 1, (b) 2 or (c) 3 days.

inducing short-day effects and the diapause-averting long-day effects.

Skeleton photoperiod after LL at 25 °C

The incidence of diapause in this strain of P. interpunctella is 43% diapause under DD but 0% under LL at 25 °C ( Kikukawa et al. , 2008 ). When the insects were placed under LL after oviposition and then transferred to the skeleton pho-toperiods at days 5, 10 and 15 ( Fig. 4a – c , respectively), the induction of diapause was suppressed, regardless of the simulated photophase length of the skeleton regime. Under

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DD, the incidence of diapause was 43% ( Kikukawa et al. , 2008 ). Thus, the transfer from LL to skeleton photoperiods was found to be diapause-preventive. Under normal photope-riodic conditions after 5, 10 and 15 days of LL, a clear photoperiodic response curve was obtained ( Fig. 4a,b ). Apparently the skeleton photoperiods consisting of two 2-h light pulses could not be accepted by the larvae as compris-ing effective photoperiods. After LL for 15 days ( Fig. 4c ), responses to the normal photoperiods were somewhat decreased; the incidence of diapause was 72% under an LD 12 : 12 h photoperiod and 28% under an LD 16 : 8 h photope-riod. Although LL induced no diapause ( Kikukawa et al. , 2008 ), transfer to long days (> 14 h) from LL at day 15 induced diapause moderately (12 – 28%). If an oscillator is

involved in the diapause clock, its phase should be entrained to the skeleton photoperiod, such that the response to skeleton photoperiods would be similar to that for normal photoperiods.

Discussion

Previous studies of the diapause clock in P. interpunctella ( Takeda & Masaki, 1976; Masaki & Kikukawa, 1981; Kikukawa & Masaki, 1984; Kikukawa et al. , 1998 , 2005; Kikukawa & Ohde, 2007 ) show no evidence for the involvment of an oscillator; all the experimental results can be explained

Fig. 3. The insects were maintained under constant darkness for (a) 5, (b) 10 or (c) 15 days after oviposition and transferred to the skeleton photoperiod ( � ) or normal photoperiod ( ⦁ ) at 25 °C.

Fig. 4. The insects were placed at constant light for (a) 5, (b) 10 or (c) 15 days after oviposition and transferred to the skeleton photoperiod ( � ) or normal photoperiod ( ⦁ ) at 25 °C.

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by an hourglass-like clock measuring night-time. Although various clock models for insect photoperiodism are listed by Vaz Nunes & Saunders (1999), Saunders et al. (2004) suggest that the hourglass-like clock is a rapidly damping circadian system. Damping coefficients of the circadian system may vary among different species. If the diapause clock of P. interpunctella is a circadian oscillator, it might be detected by entrainment to skeleton photoperiods ( Saunders, 2002 ).

The present study examines this possibility, namely whether the components of the diapause clock of P. interpunctella comprise a circadian oscillator system or not. If the light pulses (2 h each) comprising the symmetrical skeleton pho-toperiods provide time cues such as lights-on and/or lights-off, the insects should diapause under short photoperiods (8 – 12 h light at 25 °C and < 14 h at 20 °C) ( Kikukawa et al. , 2008 ). However, as shown in Figures 1 and 2 , there is no difference in the incidence of diapause between the responses of the insects exposed to a skeleton photoperiod and DD subsequent to normal photoperiods. Apparently, the diapause clock fails to be entrained by the skeleton photoperiods.

If the diapause clock is an oscillator that damps quickly, 5 – 15 days of exposure to DD or LL might stop the time measurement, and subsequent skeleton photoperiods may restart it. The incidence of diapause is moderately high under short days after DD and relatively low under long days after DD, and there is no difference in this tendency between the subsequent skeleton and normal photoperiods. Transfer from DD for 5 – 15 days to normal photoperiods does not result in clear photoperiodic response curves. Transfer from LL to the skeleton or normal photoperiods exerts differing responses. Under the skeleton photoperiods following LL, the incidence of diapause is generally low (< 31%), regardless of the skeleton regimes. When insects maintained under LL for 5 – 15 days are transferred to normal photoperiods, a clear photoperiodic response curve ( Kikukawa et al. , 2008 ) is obtained. Very short photoperiods (< 8 h) and long photoperiods (> 14 h) inhibit the induction of diapause, whereas short days (10 – 12 h light) induce diapause. Kikukawa et al. (2008) show that the transfer from an LD 16 : 8 h photoperiod to various photoperiods at day 5 after oviposition enhances the induction of diapause under short days and that at day 15 inhibits diapause completely under any subsequent photoperiod. Although both an LD 16 : 8 h photoperiod and LL have diapause-averting effects, the photoperiodic change from LL to a normal photoperiod does not appear to inhibit diapause expression, and normal photoperiodic response curves are obtained. Apparently, the preceding LL is ignored by the insects under this particular experimental regime.

Acknowledgements

We thank Emeritus Professor Sinzo Masaki (Hirosaki University) for constructive comments on the manuscript.

References

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Accepted 29 January 2009 First published online 23 March 2009