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Egg hatching of two locusts, Schistocerca gregaria and Locusta migratoria, in response to light and temperature cycles
Journal of Insect Physiology 76 (2015) 24–29
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Egg hatching of two locusts, Schistocerca gregaria and Locusta migratoria, in response to light and temperature cycles Yudai Nishide a, Seiji Tanaka a,⇑, Shinjiro Saeki a,b a b
Locust Research Laboratory, National Institute of Agro-biological Sciences at Ohwashi, Tsukuba, Japan Graduate School of Agricultural Science, Kobe University, Japan
a r t i c l e
i n f o
Article history: Received 16 December 2014 Received in revised form 4 March 2015 Accepted 6 March 2015 Available online 18 March 2015 Keywords: Egg hatching Locusta migratoria Photoperiod Schistocerca gregaria Thermoperiod
a b s t r a c t The present study showed that the eggs of the desert locust, Schistocerca gregaria, and the migratory locust, Locusta migratoria, responded to photoperiod by hatching when placed on sand in the laboratory. S. gregaria mainly hatched during the dark phase and L. migratoria during the light phase. The importance of light as a hatching cue depended on the magnitude of the temperature change during the thermoperiod; photoperiod played a more important role in the control of hatching time in both species when the magnitude of the temperature change was small. In addition, the eggs of the two species that were covered with sand did not respond to photoperiod and hatched during both the light and dark phases, indicating that light did not penetrate through the sand. Because locust eggs are normally laid as egg pods and a foam plug is deposited between the egg mass and the ground surface, we tested a possibility that naturally deposited eggs perceived light through the foam plug. The eggs that were deposited and left undisturbed in the sand hatched during the light and dark phases at similar frequencies. These results suggest that the eggs of both locust species responded to light and controlled their hatching timing accordingly but would not use light as a hatching cue in the field. The evolutionary significance of the ability of eggs to respond to light in these locusts was discussed. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction In many organisms, egg hatching occurs at the end of embryogenesis, but the time of hatching is not always at the end of embryogenesis. Hatching often occurs at a particular time of the day, e.g., shortly after dawn or dusk (Saunders, 2002). In orthopteran insects, hatching occurs mostly at dawn or during the night and its timing is controlled by temperature and/or light conditions (Arai, 1977; Tomioka et al., 1991; Shimizu and Masaki, 1997). Based on information on field observations, Uvarov (1966, 271p.) suggested that temperature, moisture and light were involved in the control of hatching in locusts. In the desert locust, Schistocerca gregaria, oviposition is observed in the field at all times during both the day and night (Stower et al., 1958; Popov, 1958), but the eggs hatch at a particular time of the day (Ellis and Ashall, 1957). These observations suggest the involvement of a sophisticated mechanism that controls the time of hatching in these insects. Padgham (1981) has reported that hatching in S. gregaria is controlled by thermoperiod. Based on observations under field
⇑ Corresponding author. http://dx.doi.org/10.1016/j.jinsphys.2015.03.010 0022-1910/Ó 2015 Elsevier Ltd. All rights reserved.
conditions, this author reported that neither light nor soil moisture were influential on egg hatching of this locust. In a previous study that compared the hatching time of S. gregaria and Locusta migratoria, we confirmed the observations of Padgham (1981) in that the S. gregaria eggs hatched in response to thermoperiod, and further demonstrated that a thermal difference as small as 1 °C was sufficient for both species to respond by hatching, although hatching occurred during either the cryophase (low-temperature phase) or the thermophase (high-temperature phase) depending on the species (Nishide et al., 2015). In addition, the two species adjusted their hatching time to a new thermal environment that occurred shortly before the expected time of hatching. This result suggests the presence of an endogenous mechanism. These observations were made under continuous illumination, because we assumed that light was not important in the control of hatching time, as Padgham (1981) had suggested for S. gregaria eggs. However, in a preliminary laboratory study (Nishide, Y. and Tanaka, S., unpublished observations), the eggs of both S. gregaria and L. migratoria responded to light or photoperiod by hatching at specific times of the day. Therefore, the present study was undertaken to clarify the following questions: How does photoperiod affect the hatching time and which of the two factors, photoperiod and temperature, is more important in the control of hatching time? How do these
Y. Nishide et al. / Journal of Insect Physiology 76 (2015) 24–29
factors affect the hatching times of the two locust species? What is the ecological and evolutionary significance of photoperiod or light in the control of hatching time in these species? This paper describes the results of experiments to answer these questions.
2. Materials and methods 2.1. Insects and rearing methods The experiments were performed with gregarious (crowdreared) colonies of S. gregaria and L. migratoria. The details of these locusts and the rearing methods were previously described (Nishide et al., 2015). Briefly, groups of approximately 100–200 individuals of each species were reared in large wooden-framed cages (42 24 42 cm) that were covered with nylon screen. Desert locusts were reared at 31 ± 1 °C and a 16L:8D photoperiod and migratory locusts at 30 ± 1 °C and a 12L:12D photoperiod. Both of the locusts were fed leaves of Bromus catharticus, Dactylis glomerata or Sorghum vulgare depending on the season and wheat bran was constantly supplied. Cabbage, Brassica oleracea, was also supplied constantly for the desert locust.
2.2. The experimental manipulations Sexually mature females of both species laid eggs into moist sand in transparent plastic cups (diameter, 10 cm; height, 6 cm). The egg pods that were laid in sand could be seen through the wall of the cups. The plastic cups were changed daily, and the eggs that were laid within 24 h (day 0) were used for experiments. The eggs of both of the locusts hatched 15 days after oviposition at 30 °C. As previously reported (Nishide et al., 2015), hatching time of the day was little influenced by the temperature conditions during the first 10 days at 30 °C. Therefore, the eggs were kept at 30 °C for the first 10 days and were then exposed to test conditions in incubators (Bio Incubator LR-600S; Fuji Ika Sangyo, Chiba, Japan). The test temperature and light intensity were monitored with thermo recorders (Ondotori TR-74Ui, T and D Co., Tokyo, Japan) every hour, and the light intensity was approximately 200 lx on the floor of each incubator during the light period. The test temperatures fluctuated within 0.2 °C of the set value. The hatching activity was observed by placing eggs individually in a 24-well plastic dish (CostarÒ, 24 well cell culture cluster). Each well (diameter, 15 mm; depth, 18 mm) was half-filled with moist white sand (Brisbane White Sand, Hario Co. Ltd., Japan) and one egg was placed on the sand. The hatching activity was recorded by counting the hatchlings every one or 12 h. The hatchlings were visually counted in the experiments that observed hatching activity every 12 h. In experiments that recorded the number of hatchlings every hour, the plastic Petri dishes that contained the eggs were photographed with a digital camera every hour during the light phase, and the hatched nymphs in the photographs were counted on a computer. During the dark phase the hatchlings were counted visually under dim red light every hour. Our previous study (Nishide et al., 2015) showed that the hatching timing was influenced by temperature cycles: S. gregaria hatched during the cryophase and L. migratoria hatched during the thermophase when exposed to 24-h thermoperiod cycles. As will be shown later, these eggs also responded to photoperiod: S. gregaria hatched during the dark phase and L. migratoria hatched during the light phase. Therefore, we examined which of the two factors, temperature or light, was the more important in the control of hatching time for these locusts by exposing them to a 12L:12D photoperiod combined with different thermoperiods with an identical mean temperature of 27.5 °C. In this case, the
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cryophase and thermophase occurred during the light and dark periods, respectively. Locusts deposit eggs in the ground where light is unlikely to reach. Therefore, we determined whether locust eggs would respond to photoperiod when buried in the sand. For comparison, we observed the hatching time of eggs buried in a layer of transparent micro glass beads (diameter, 1.0 mm; Toshinriko Co. Ltd, Tokyo, Japan) that allowed the light to penetrate. In both of the treatments, 20 eggs were placed at the bottom of a plastic Petri dish and covered with a layer of either sand or glass beads (17 mm thick). In this case, care was taken not to let the eggs touch one another to avoid possible interaction among them. We confirmed that the light reached the bottom of the bead-filled dishes (approximately 20 lx) in the incubator, whereas it was not detectable at the bottom of the sand-filled dishes. The bottom and outer wall of the dish were wrapped with aluminum foil to prevent light penetration. All of the dishes were covered with a transparent lid and exposed to a 12L:12D photoperiod at 30 ± 0.2 °C. The mean temperatures during the light (30.0 ± 0.2 °C) and dark (30.0 ± 0.2 °C) periods did not differ significantly (t-test; P > 0.05). Locust eggs that are deposited as an egg pod are plugged with the foam material that is produced by the female parent (Uvarov, 1966). This foam plug is approximately 2 cm in length. Although the top of the plug is usually covered with sand by the female parent at the end of oviposition, it is sometimes exposed. We thus tested the possibility that light reached the locust eggs through the egg pod plug. A female was allowed to lay an egg pod into moist sand that was filled to the top of a steel can (diameter, 66 mm; height, 166.7 mm). River sand (Joyful Honda, Ibaraki, Japan) was used for this experiment. The can that contained the egg pod was covered with a transparent plastic bag and was carefully transferred to an incubator with a 12L:12D photoperiod at 30 °C. The can was checked at the beginning and the end of the light phase to record the number of hatchlings. 2.3. Statistical analysis The average time of hatching was determined and analyzed among different treatments with the Scheffe’s test for multiple comparison after ANOVA. The proportions of hatchlings were analyzed with a v2 test. 3. Results 3.1. Responses to photoperiod The eggs of S. gregaria and L. migratoria that were transferred from 30 °C to 25 °C on day 10 began to hatch on day 16 at a 12L:12D photoperiod. Most of the S. gregaria eggs hatched during the dark phase (Fig. 1A), whereas the L. migratoria eggs hatched during the light phase (Fig. 1B). To determine the effects of photoperiod and temperature on the time of egg hatching, eggs were exposed to 35, 30 or 25 °C at a 12L:12D photoperiod. At all of the tested temperatures, almost all of the S. gregaria eggs hatched during the dark phase (Fig. 2A, C and E). The hatching time was influenced by temperature (ANOVA; F = 100.44; df = 2, 353; P < 0.001) and tended to occur later as the temperature decreased (r = 0.49; n = 545; P < 0.001). Some of the eggs that were incubated at the lowest temperature hatched at the beginning of the light phase (Fig. 2E). Similar results were obtained for L. migratoria except that the eggs hatched during the light phase (Fig. 2B, D and F). The hatching time was different among the 3 temperatures (ANOVA; F = 147.46; df = 2, 487; P < 0.001) and tended to occur later as the temperature decreased (r = 0.637; n = 490; P < 0.001).
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Y. Nishide et al. / Journal of Insect Physiology 76 (2015) 24–29
(A) S. gregaria
No. of hatchlings
50
(B) L. migratoria
60
n = 112
n = 87
50
40
40
30
30
20
20
10
10
0
0
12 24 36 48 60 72 84
0
D
L
D
L
D
L
D
96 108 h L
0
12 L
D
24 D
36 L
Photoperiod
48 D
60 L
72 h D
Photoperiod
Fig. 1. The number of individuals that hatched during the dark phase (closed bars) and the light phase (open bars) of 12L:12D cycles at constant 25 °C in S. gregaria (A) and L. migratoria (B). The horizontal axis indicates the time in hours after the beginning of hatching. The photoperiodic schedule is shown below each panel; D, dark phase; L, light phase.
S. gregaria 40
40 Mean = 6.8 n = 127
30
No. of hatchlings
L. migratoria
(A) 35 ºC
30 20
10
10
0
0
(C) 30 ºC
50 Mean = 7.4 n = 279
20
Mean = 14.3 n = 120
0.1
20
30
(B) 35 ºC
0.2
0.2
(D) 30 ºC Mean = 16.5 n = 216
40
0.1
30 20
10
10 0
0 40
(E) 25 ºC
40 Mean = 9.6 n = 139
30
0.1
20
10
10 0
6 Dark
12
18 Light
24
Mean = 18.7 n = 154
30
20
0
(F) 25 ºC
0
0
6 Dark
0.2
12
18 Light
24
The time of the day Fig. 2. The effect of temperature on the hatching time at 12L:12D in S. gregaria (A, C and E) and L. migratoria (B, D and F). A and B, 35 °C; C and D, 30 °C; E and F, 25 °C. The mean hatching time (±SD h) and sample size (n) are shown for each treatment. The horizontal axis indicates the time in hours. The photoperiodic schedule is shown below each panel; D, dark phase; L, light phase.
3.2. Importance of photoperiod under different thermoperiods When the thermoperiod and photoperiod were in phase, i.e., the high (32.5 °C) and low (22.5 °C) temperature occurred during the light and dark phase, respectively, 93.3% of the S. gregaria eggs hatched during the dark phase and 99.2% of the L. migratoria eggs hatched during the light phase (Fig. 3A and B), as observed at constant temperatures. When the thermoperiod and photoperiod were out of phase, however, the egg hatching of each species was commonly observed during both of the phases (Fig. 3C and D). However, as the magnitude of the temperature difference between the thermo- and cryophases decreased from 10 °C to 7 or 3 °C with the mean temperature kept constant (27.5 °C), photoperiod played a more important role in the control of hatching time in both species. That is, when the temperature difference between the thermo- and cryophases decreased from 10 °C to 7 °C, the proportion of S. gregaria eggs that hatched during the dark phase increased from 70.7% to 85.7% (v2 = 8.85; df = 1; P < 0.01;
Fig. 3C and E). When the temperature difference further decreased to 3 °C (Fig. 3G), 95.1% of the eggs hatched during the dark phase, which was significantly larger than the values in the treatments with a difference of 10 or 7 °C (v2 = 31.30 or 7.92; df = 1; P < 0.01). In L. migratoria, the importance of photoperiod was also more emphasized as the temperature difference between the thermo- and cryophases decreased (Fig. 3D, F and H): the proportion of eggs that hatched during the light phase increased from 73.4% in the eggs that were exposed to a difference of 10 °C to 94.6 and 95.0% in those that were exposed to a difference of 7 or 3 °C (v2 = 24.16 or 21.99; df = 1; P < 0.01). 3.3. Importance of photoperiod for the eggs that were deposited in the sand Almost all of the S. gregaria eggs that were buried in transparent glass beads, which allowed light to penetrate, hatched during the dark phases (Fig. 4A), and most of the L. migratoria eggs that were
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Y. Nishide et al. / Journal of Insect Physiology 76 (2015) 24–29
S. gregaria
No. of hatchlings
80 60 40 20 0
50 40 30 20 10 0
0
12
24
36
48
60
L
D
L
D
L
C
T
C
T
C
T
0
12
36
24
48
C
60
D
L
D
L
D
T
C
T
C
T
C
T
12
24
48
36
60
72
L
D
L
D
L
D
T
C
T
C
T
C
T
(G)
0
n = 162
12
24
36
48
60
72 84
D
L
D
L
C
T
C
T
C
T
96 h
D
L
C
T
n = 139
12
24
48
36
60
72
84
96 h
D
L
D
L
D
L
D
L
T
C
T
C
T
C
T
C
0
n = 147
12
80 60 40 20 0
60 h
48
36
L
(F)
50 40 30 20 10 0
84 h
D
24
12 D
0
n = 147
n = 136
(D)
84 h
L
0
40 30 20 10 0
72
D
(E)
0
D
n = 116
L. migratoria
(B)
80 60 40 20 0
84 h
72
D
(C)
50 40 30 20 10 0
80 60 40 20 0
n = 150
(A)
24
48
36
60
72
84
96 h
D
L
D
L
D
L
D
L
T
C
T
C
T
C
T
C
(H)
0
n = 121
12
24
48
36
60
72
84 h
D
L
D
L
D
L
D
L
D
L
D
L
T
C
T
C
T
C
T
C
T
C
T
C
Fig. 3. The number of hatchlings during the dark phase (closed bars) and the light phase (open bars) in different combinations of photoperiod and thermoperiod in S. gregaria (A, C, E and G) and L. migratoria (B, D, F and H). Thermoperiod in A–D was thermophase:cryophase (T:C) 32.5:22.5 °C and 31:24 °C in E and F, respectively, and T:C 29:26 °C in G and H. D, L, T and C indicate the dark phase, light phase, thermophase and cryophase, respectively. The horizontal axis indicates the time in hours after the beginning of hatching.
S. gregaria No. of hatchlings
140
L. migratoria 120
n = 283
(A)
120
100
100
80
80 60
60
40
40
20
20 0
0
12 D
120
No. of hatchlings
n = 216
(B)
24 L
48
36 D
L
60 D
(C)
72 L
84 h
0
D
12 L
80
n = 204
100
0
24 D
48
36 L
D
60 L
(D)
72 D
84 h L
n = 141
70 60
80
50
60
40
30
40
20 20 0
10 0
12 D
24 L
36 D
48 L
60 h
0
0
D
12 L
24 D
36 L
48 D
60 h L
Photocycles Fig. 4. The number of individuals that hatched during the dark phase (closed bars) and the light phase (open bars) of 12L:12D cycles at a constant 30 °C in S. gregaria and L. migratoria eggs that were buried in transparent glass beads (A and B) or sand (C and D). The photoperiodic schedule is shown below each panel; D, dark phase; L, light phase.
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Y. Nishide et al. / Journal of Insect Physiology 76 (2015) 24–29
(A) S. gregaria
No. of hatchlings (%)
100
(B) L. migratoria n = 1021
100
80
80
60
60
40
40
20
20
0
0
n = 465
Fig. 5. Hatching time of S. gregaria (A) and L. migratoria (B) eggs that were left intact in the egg pods in sand at a 12L:12D photoperiod at constant 30 °C. Closed and open bars indicate the percentages of hatchlings during the dark and light phases, respectively.
similarly treated hatched during the light phases (Fig. 4B). This result suggests that both species apparently responded to the photoperiod. In addition, the S. gregaria eggs that were covered with sand hatched during the both light and dark phases with a unimodal pattern of hatching activity over time. This result suggested that photoperiod did not influence the hatching time (Fig. 4C). Similar results were obtained for the L. migratoria eggs that were covered with sand (Fig. 4D): they hatched in a unimodal pattern independent of the photoperiod. As mentioned above, locust eggs are deposited as an egg pod that consists of eggs and a foam plug. The last experiment was designed to determine whether the eggs that were deposited in sand responded to light above the ground through the foam plug and used the daily light–dark cycles as a cue for hatching. Of the eggs that were kept intact in the egg pod in the sand, hatching was observed at similar frequencies during the light and dark phases in either species of locust, and the ratio did not deviate significantly from unity or from each other (v2-test; P > 0.05 in each species; Fig. 5). 4. Discussion 4.1. Characteristics of the responses to light conditions In various organisms, including insects, photoperiod plays a pivotal role in the control of daily activities and development, but temperature may also exert a significant effect in many organisms (Saunders, 2002). This study demonstrated that the eggs of S. gregaria and L. migratoria were capable of responding to photoperiod by hatching at species-specific times of the day. The timings of the hatching of these species were in sharp contrast to each other. At a 12L:12D photoperiod with a constant temperature, the eggs of S. gregaria primarily hatched during the dark phase, whereas those of L. migratoria mainly hatched during the light phase. In the field, S. gregaria eggs hatch over a short period from shortly before dawn to the first four hours after sunrise (Ellis and Ashall, 1957). Hatching during the dark phase may be an adaptation to the desert where this locust lives, because the temperature in the desert is often over 50 °C and humidity is low during the day (Ellis and Ashall, 1957; Stower et al., 1958). On the other hand, in L. migratoria, a grassland species, little is known about hatching time in the field. The differences in the habitats could affect the hatching strategy and time of hatching (Nishide et al., 2015). The temperature also affected the hatching time in the S. gregaria and L. migratoria eggs that were exposed to light–dark cycles. The eggs of both species tended to hatch later during the day as the temperature decreased (Fig. 2). Because some eggs of these locusts hatched in the ‘unexpected’ phase, i.e., in the light phase for S.
gregaria and in the dark phase for L. migratoria, their hatching may not be a direct response to the presence or absence of light. The underlying mechanism that controls the hatching time in these locusts remains unknown. A population of the marine midge, Clunio marinus, that was kept in light–dark cycles shows a peak of adult emergence during the light phase. When transferred from the light–dark cycles to continuous illumination, the population exhibits one further emergence peak and then arrhythmicity (Pflüger and Neumann, 1971). This phenomenon was explained by an hour-glass mechanism in which a non-repetitive (i.e., non-oscillatory) timer was initiated by a light-on stimulus and then ran its allotted time (Saunders, 1976). The present results with the locusts might be explained by this mechanism: the hatching time could be set by a light-on or light-off stimulus and the process leading to hatching proceeds as a function of temperature. Another possibility is the presence of an endogenous oscillator that is entrained by an environmental cue. We previously explored this possibility for these locusts by transferring the eggs from a thermoperiod to a constant temperature (Nishide et al., 2015). The results indicate that peak hatching activity was observed over 2 consecutive days after the eggs were transferred to a constant temperature. Therefore, it is possible that an endogenous oscillator is involved in the control of hatching in these locusts, although further experiments using other thermoperiods or photoperiods are necessary to determine whether or not the hatching activity of these locusts is under the control of a circadian rhythm. The eggs of S. gregaria and L. migratoria adjusted the hatching time by responding to the photoperiod that occurred during the last 5 days before hatching. In a previous study (Nishide et al., 2015), the eggs of the two locust species adjusted the hatching time to a new thermo-environment that occurred within 24 h before hatching. A similar prompt adjustment might be achieved by these locusts in response to photoperiod, but appropriate experiments to evaluate this possibility have not yet been performed. 4.2. The responses to photoperiod under various thermoperiods The responses to photoperiod to control hatching time were greatly influenced by the magnitude of temperature differences of thermoperiod in S. gregaria and L. migratoria. Under a thermoperiod with a difference of 10 °C, hatching occurred commonly during both the light and dark phases (Fig. 3C and D). This pattern was likely caused by the interacting effects of the two factors. As the temperature difference of thermoperiod decreased while the mean daily temperature was kept constant, the hatching time tended to become more dependent on the photoperiod than on the thermoperiod. In a previous study (Nishide et al., 2015), S. gregaria and L. migratoria eggs that were exposed to various thermoperiods with different magnitudes of temperature differences under continuous illumination responded to even a difference of 1 °C by hatching at the expected time of the day. In the present study, however, most of the eggs that were exposed to a thermoperiod with a difference of 3 °C at a 12L:12D photoperiod hatched as if they entirely depended on the photoperiod. There is a possibility that, as observed with temperature, variation in light intensity produces different effects on the control of hatching time in these locusts, especially when interacting with the thermoperiod. 4.3. The influence of photoperiod on the hatching time Photoperiod or light affects the hatching time of various orthopteran species that lay eggs in soil (e.g., Arai, 2012; Shimizu and Masaki, 1997; Tomioka et al., 1991). However, whether or not the eggs of these insects respond to photoperiod in the field has not been confirmed. As described above, the eggs of S. gregaria
Y. Nishide et al. / Journal of Insect Physiology 76 (2015) 24–29
and L. migratoria responded to photoperiod by hatching at the specific times of the day when they were removed from the egg pod and placed on sand, but photoperiod did not affect their hatching time if the eggs were covered with sand. Locusts deposit eggs as an egg pod, which is 5.3–7.2 cm long in a Japanese strain of L. migratoria (n = 11; Tanaka, S. and Nishide, Y., unpublished observations). The upper portion of an egg pod normally consists of a foam plug that is 2.2 cm long on average (range, 1.6–3.1 cm; n = 11). In the present study, we tested whether the light reached the eggs in the egg pod through the foam plug. Because the locust eggs that were left intact in the egg pod in the sand hatched at similar frequencies during the light and dark phases at a constant temperature, it was concluded that the light did not penetrate the foam plug. Similar results were observed for S. gregaria when the egg pods were laid in the sand that was brought from a desert habitat in Mauritania (Nishide, Y. and Tanaka, S., unpublished observations). If the light does not reach the locust eggs that were deposited in the soil, it would then follow that the light sensitivity and the light-driven mechanism that controls the hatching time have no ecological significance, which raises an intriguing question: Why do locusts have these functions? One possibility is that an embryo that is ready to hatch has already developed all of the sensory organs and functions that are necessary for nymphal life so that it can perceive light if exposed to it. However, this hypothesis does not explain how the locust egg can use light to control hatching time. Another possibility is that an unidentified organ or molecules that are responsible for the perception of temperature and the control of hatching time also perceive light so that a photoperiod can act like a thermoperiod for the locust eggs. A possible candidate for such molecules might be rhodopsin, which is a photoreceptor that is also involved in circadian rhythms in the animal kingdom including insects (White, 1978; Shichida and Matsuyama, 2009). In Drosophila melanogaster, rhodopsin functions not only as a photoreceptor but also as a thermosensor (Shen et al., 2011). This hypothesis is highly speculative but might explain why the eggs of the locusts and possibly other insects that are buried under ground are often capable of responding to light to control their behavior and activity. Acknowledgements We thank Ms. H. Ikeda, Ms. N. Totsuka and Ms. M. Higuchi for maintaining the locust colonies. Thanks are also due to Dr.
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Takashi Suzuki (University of Western Ontario) for his stimulating discussion and information on the light perception mechanisms. We are indebted to Dr. Sinzo Masaki (Professor emeritus of Hirosaki University) for his invaluable comments on the manuscript and correcting our English. This study was founded in part by a Kakenhi Grant of Japan (No. 23380038) to S.T. The grass used was raised by the Field Management Section of NIASO and we would like to thank Mr. Kameo Tsukada for growing the grass. Two anonymous reviewers greatly improved the manuscript. References Arai, T., 1977. Effects of the daily cycles of light and temperature on hatchability and hatching time in Metrioptera hime Furukawa (Orthoptera: Tettigoniidae). Kontyu 45, 107–120. Arai, T., 2012. Egg hatching rhythm in insects. Academic Archives of Yamaguchi Prefectural University (Bulletins and Records of Research Activities) 5, 1–42. (In Japanese with an English abstract). Ellis, P.E., Ashall, C., 1957. Field studies on diurnal behaviour, movement and aggregation in the desert locust (Schistocerca gregaria Forskål). Anti-locust Bull. 25, 1–94. Nishide, Y., Tanaka, S., Saeki, S., 2015. Adaptive difference in daily timing of hatch in two locust species. Schistocerca gregaria and Locusta migratoria: the effects of thermocycles and phase polyphenism. J. Insect Physiol. 72, 79–87. Padgham, D.E., 1981. Hatching rhythms in the desert locust, Schistocerca gregaria. Physiol. Entomol. 6, 191–198. Pflüger, W., Neumann, D., 1971. Die Steuerung einer gezeitenparallelen Schlüpfrhythmik nach dem Sanduhr-Prinzip. Oecologia, Berl. 7, 262–266. Popov, G.B., 1958. Ecological Studies on oviposition be swarms of the desert locust (Schistocerca gregaria Forskål) in Eastern Africa. Anti-Locust Bull. 31, 1–70. Saunders, D.S., 1976. The circadian eclosion rhythm in Sarcophaga argyrostoma: some comparisons with the photoperiodic ‘‘clock’’. J. Comp. Physiol. A. 110, 111–133. Saunders, D.S., 2002. Insect Clocks, third ed. Elsevier, Amsterdam. Shen, W.L., Kwon, Y., Adegbola, A.A., Luo, J., Chess, A., Montell, C., 2011. Function of rhodopsin in temperature discrimination in Drosophila. Science 331, 1333– 1336. Shichida, Y., Matsuyama, T., 2009. Evolution of opsins and phototransduction. Philos. Trans. R. Soc. B 364, 2881–2895. Shimizu, T., Masaki, S., 1997. Daily time of hatching in Nemobiine crickets. Jpn. J. Entomol. 65, 335–342. Stower, W.J., Popov, G.B., Greathead, D.J., 1958. Oviposition behaviour and egg mortality of the desert locust (Schistocerca gregaria Forskål) on the coast of Eritrea. Anti-Locust Bull. 30, 1–33. Tomioka, K., Wakatsuki, T., Shimono, K., Chiba, Y., 1991. Circadian control of hatching in the cricket, Gryllus bimaculatus. J. Insect Physiol. 37, 365–371. Uvarov, B., 1966. Grasshoppers and Locusts, vol. 1. Cambridge University Press, Cambridge. White, R.H., 1978. Insect visual pigments. Adv. Insect Physiol. 13, 35–67.
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Egg hatching of two locusts, Schistocerca gregaria and Locusta migratoria, in response to light and temperature cycles Yudai Nishide a, Seiji Tanaka a,⇑, Shinjiro Saeki a,b a b
Locust Research Laboratory, National Institute of Agro-biological Sciences at Ohwashi, Tsukuba, Japan Graduate School of Agricultural Science, Kobe University, Japan
a r t i c l e
i n f o
Article history: Received 16 December 2014 Received in revised form 4 March 2015 Accepted 6 March 2015 Available online 18 March 2015 Keywords: Egg hatching Locusta migratoria Photoperiod Schistocerca gregaria Thermoperiod
a b s t r a c t The present study showed that the eggs of the desert locust, Schistocerca gregaria, and the migratory locust, Locusta migratoria, responded to photoperiod by hatching when placed on sand in the laboratory. S. gregaria mainly hatched during the dark phase and L. migratoria during the light phase. The importance of light as a hatching cue depended on the magnitude of the temperature change during the thermoperiod; photoperiod played a more important role in the control of hatching time in both species when the magnitude of the temperature change was small. In addition, the eggs of the two species that were covered with sand did not respond to photoperiod and hatched during both the light and dark phases, indicating that light did not penetrate through the sand. Because locust eggs are normally laid as egg pods and a foam plug is deposited between the egg mass and the ground surface, we tested a possibility that naturally deposited eggs perceived light through the foam plug. The eggs that were deposited and left undisturbed in the sand hatched during the light and dark phases at similar frequencies. These results suggest that the eggs of both locust species responded to light and controlled their hatching timing accordingly but would not use light as a hatching cue in the field. The evolutionary significance of the ability of eggs to respond to light in these locusts was discussed. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction In many organisms, egg hatching occurs at the end of embryogenesis, but the time of hatching is not always at the end of embryogenesis. Hatching often occurs at a particular time of the day, e.g., shortly after dawn or dusk (Saunders, 2002). In orthopteran insects, hatching occurs mostly at dawn or during the night and its timing is controlled by temperature and/or light conditions (Arai, 1977; Tomioka et al., 1991; Shimizu and Masaki, 1997). Based on information on field observations, Uvarov (1966, 271p.) suggested that temperature, moisture and light were involved in the control of hatching in locusts. In the desert locust, Schistocerca gregaria, oviposition is observed in the field at all times during both the day and night (Stower et al., 1958; Popov, 1958), but the eggs hatch at a particular time of the day (Ellis and Ashall, 1957). These observations suggest the involvement of a sophisticated mechanism that controls the time of hatching in these insects. Padgham (1981) has reported that hatching in S. gregaria is controlled by thermoperiod. Based on observations under field
⇑ Corresponding author. http://dx.doi.org/10.1016/j.jinsphys.2015.03.010 0022-1910/Ó 2015 Elsevier Ltd. All rights reserved.
conditions, this author reported that neither light nor soil moisture were influential on egg hatching of this locust. In a previous study that compared the hatching time of S. gregaria and Locusta migratoria, we confirmed the observations of Padgham (1981) in that the S. gregaria eggs hatched in response to thermoperiod, and further demonstrated that a thermal difference as small as 1 °C was sufficient for both species to respond by hatching, although hatching occurred during either the cryophase (low-temperature phase) or the thermophase (high-temperature phase) depending on the species (Nishide et al., 2015). In addition, the two species adjusted their hatching time to a new thermal environment that occurred shortly before the expected time of hatching. This result suggests the presence of an endogenous mechanism. These observations were made under continuous illumination, because we assumed that light was not important in the control of hatching time, as Padgham (1981) had suggested for S. gregaria eggs. However, in a preliminary laboratory study (Nishide, Y. and Tanaka, S., unpublished observations), the eggs of both S. gregaria and L. migratoria responded to light or photoperiod by hatching at specific times of the day. Therefore, the present study was undertaken to clarify the following questions: How does photoperiod affect the hatching time and which of the two factors, photoperiod and temperature, is more important in the control of hatching time? How do these
Y. Nishide et al. / Journal of Insect Physiology 76 (2015) 24–29
factors affect the hatching times of the two locust species? What is the ecological and evolutionary significance of photoperiod or light in the control of hatching time in these species? This paper describes the results of experiments to answer these questions.
2. Materials and methods 2.1. Insects and rearing methods The experiments were performed with gregarious (crowdreared) colonies of S. gregaria and L. migratoria. The details of these locusts and the rearing methods were previously described (Nishide et al., 2015). Briefly, groups of approximately 100–200 individuals of each species were reared in large wooden-framed cages (42 24 42 cm) that were covered with nylon screen. Desert locusts were reared at 31 ± 1 °C and a 16L:8D photoperiod and migratory locusts at 30 ± 1 °C and a 12L:12D photoperiod. Both of the locusts were fed leaves of Bromus catharticus, Dactylis glomerata or Sorghum vulgare depending on the season and wheat bran was constantly supplied. Cabbage, Brassica oleracea, was also supplied constantly for the desert locust.
2.2. The experimental manipulations Sexually mature females of both species laid eggs into moist sand in transparent plastic cups (diameter, 10 cm; height, 6 cm). The egg pods that were laid in sand could be seen through the wall of the cups. The plastic cups were changed daily, and the eggs that were laid within 24 h (day 0) were used for experiments. The eggs of both of the locusts hatched 15 days after oviposition at 30 °C. As previously reported (Nishide et al., 2015), hatching time of the day was little influenced by the temperature conditions during the first 10 days at 30 °C. Therefore, the eggs were kept at 30 °C for the first 10 days and were then exposed to test conditions in incubators (Bio Incubator LR-600S; Fuji Ika Sangyo, Chiba, Japan). The test temperature and light intensity were monitored with thermo recorders (Ondotori TR-74Ui, T and D Co., Tokyo, Japan) every hour, and the light intensity was approximately 200 lx on the floor of each incubator during the light period. The test temperatures fluctuated within 0.2 °C of the set value. The hatching activity was observed by placing eggs individually in a 24-well plastic dish (CostarÒ, 24 well cell culture cluster). Each well (diameter, 15 mm; depth, 18 mm) was half-filled with moist white sand (Brisbane White Sand, Hario Co. Ltd., Japan) and one egg was placed on the sand. The hatching activity was recorded by counting the hatchlings every one or 12 h. The hatchlings were visually counted in the experiments that observed hatching activity every 12 h. In experiments that recorded the number of hatchlings every hour, the plastic Petri dishes that contained the eggs were photographed with a digital camera every hour during the light phase, and the hatched nymphs in the photographs were counted on a computer. During the dark phase the hatchlings were counted visually under dim red light every hour. Our previous study (Nishide et al., 2015) showed that the hatching timing was influenced by temperature cycles: S. gregaria hatched during the cryophase and L. migratoria hatched during the thermophase when exposed to 24-h thermoperiod cycles. As will be shown later, these eggs also responded to photoperiod: S. gregaria hatched during the dark phase and L. migratoria hatched during the light phase. Therefore, we examined which of the two factors, temperature or light, was the more important in the control of hatching time for these locusts by exposing them to a 12L:12D photoperiod combined with different thermoperiods with an identical mean temperature of 27.5 °C. In this case, the
25
cryophase and thermophase occurred during the light and dark periods, respectively. Locusts deposit eggs in the ground where light is unlikely to reach. Therefore, we determined whether locust eggs would respond to photoperiod when buried in the sand. For comparison, we observed the hatching time of eggs buried in a layer of transparent micro glass beads (diameter, 1.0 mm; Toshinriko Co. Ltd, Tokyo, Japan) that allowed the light to penetrate. In both of the treatments, 20 eggs were placed at the bottom of a plastic Petri dish and covered with a layer of either sand or glass beads (17 mm thick). In this case, care was taken not to let the eggs touch one another to avoid possible interaction among them. We confirmed that the light reached the bottom of the bead-filled dishes (approximately 20 lx) in the incubator, whereas it was not detectable at the bottom of the sand-filled dishes. The bottom and outer wall of the dish were wrapped with aluminum foil to prevent light penetration. All of the dishes were covered with a transparent lid and exposed to a 12L:12D photoperiod at 30 ± 0.2 °C. The mean temperatures during the light (30.0 ± 0.2 °C) and dark (30.0 ± 0.2 °C) periods did not differ significantly (t-test; P > 0.05). Locust eggs that are deposited as an egg pod are plugged with the foam material that is produced by the female parent (Uvarov, 1966). This foam plug is approximately 2 cm in length. Although the top of the plug is usually covered with sand by the female parent at the end of oviposition, it is sometimes exposed. We thus tested the possibility that light reached the locust eggs through the egg pod plug. A female was allowed to lay an egg pod into moist sand that was filled to the top of a steel can (diameter, 66 mm; height, 166.7 mm). River sand (Joyful Honda, Ibaraki, Japan) was used for this experiment. The can that contained the egg pod was covered with a transparent plastic bag and was carefully transferred to an incubator with a 12L:12D photoperiod at 30 °C. The can was checked at the beginning and the end of the light phase to record the number of hatchlings. 2.3. Statistical analysis The average time of hatching was determined and analyzed among different treatments with the Scheffe’s test for multiple comparison after ANOVA. The proportions of hatchlings were analyzed with a v2 test. 3. Results 3.1. Responses to photoperiod The eggs of S. gregaria and L. migratoria that were transferred from 30 °C to 25 °C on day 10 began to hatch on day 16 at a 12L:12D photoperiod. Most of the S. gregaria eggs hatched during the dark phase (Fig. 1A), whereas the L. migratoria eggs hatched during the light phase (Fig. 1B). To determine the effects of photoperiod and temperature on the time of egg hatching, eggs were exposed to 35, 30 or 25 °C at a 12L:12D photoperiod. At all of the tested temperatures, almost all of the S. gregaria eggs hatched during the dark phase (Fig. 2A, C and E). The hatching time was influenced by temperature (ANOVA; F = 100.44; df = 2, 353; P < 0.001) and tended to occur later as the temperature decreased (r = 0.49; n = 545; P < 0.001). Some of the eggs that were incubated at the lowest temperature hatched at the beginning of the light phase (Fig. 2E). Similar results were obtained for L. migratoria except that the eggs hatched during the light phase (Fig. 2B, D and F). The hatching time was different among the 3 temperatures (ANOVA; F = 147.46; df = 2, 487; P < 0.001) and tended to occur later as the temperature decreased (r = 0.637; n = 490; P < 0.001).
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(A) S. gregaria
No. of hatchlings
50
(B) L. migratoria
60
n = 112
n = 87
50
40
40
30
30
20
20
10
10
0
0
12 24 36 48 60 72 84
0
D
L
D
L
D
L
D
96 108 h L
0
12 L
D
24 D
36 L
Photoperiod
48 D
60 L
72 h D
Photoperiod
Fig. 1. The number of individuals that hatched during the dark phase (closed bars) and the light phase (open bars) of 12L:12D cycles at constant 25 °C in S. gregaria (A) and L. migratoria (B). The horizontal axis indicates the time in hours after the beginning of hatching. The photoperiodic schedule is shown below each panel; D, dark phase; L, light phase.
S. gregaria 40
40 Mean = 6.8 n = 127
30
No. of hatchlings
L. migratoria
(A) 35 ºC
30 20
10
10
0
0
(C) 30 ºC
50 Mean = 7.4 n = 279
20
Mean = 14.3 n = 120
0.1
20
30
(B) 35 ºC
0.2
0.2
(D) 30 ºC Mean = 16.5 n = 216
40
0.1
30 20
10
10 0
0 40
(E) 25 ºC
40 Mean = 9.6 n = 139
30
0.1
20
10
10 0
6 Dark
12
18 Light
24
Mean = 18.7 n = 154
30
20
0
(F) 25 ºC
0
0
6 Dark
0.2
12
18 Light
24
The time of the day Fig. 2. The effect of temperature on the hatching time at 12L:12D in S. gregaria (A, C and E) and L. migratoria (B, D and F). A and B, 35 °C; C and D, 30 °C; E and F, 25 °C. The mean hatching time (±SD h) and sample size (n) are shown for each treatment. The horizontal axis indicates the time in hours. The photoperiodic schedule is shown below each panel; D, dark phase; L, light phase.
3.2. Importance of photoperiod under different thermoperiods When the thermoperiod and photoperiod were in phase, i.e., the high (32.5 °C) and low (22.5 °C) temperature occurred during the light and dark phase, respectively, 93.3% of the S. gregaria eggs hatched during the dark phase and 99.2% of the L. migratoria eggs hatched during the light phase (Fig. 3A and B), as observed at constant temperatures. When the thermoperiod and photoperiod were out of phase, however, the egg hatching of each species was commonly observed during both of the phases (Fig. 3C and D). However, as the magnitude of the temperature difference between the thermo- and cryophases decreased from 10 °C to 7 or 3 °C with the mean temperature kept constant (27.5 °C), photoperiod played a more important role in the control of hatching time in both species. That is, when the temperature difference between the thermo- and cryophases decreased from 10 °C to 7 °C, the proportion of S. gregaria eggs that hatched during the dark phase increased from 70.7% to 85.7% (v2 = 8.85; df = 1; P < 0.01;
Fig. 3C and E). When the temperature difference further decreased to 3 °C (Fig. 3G), 95.1% of the eggs hatched during the dark phase, which was significantly larger than the values in the treatments with a difference of 10 or 7 °C (v2 = 31.30 or 7.92; df = 1; P < 0.01). In L. migratoria, the importance of photoperiod was also more emphasized as the temperature difference between the thermo- and cryophases decreased (Fig. 3D, F and H): the proportion of eggs that hatched during the light phase increased from 73.4% in the eggs that were exposed to a difference of 10 °C to 94.6 and 95.0% in those that were exposed to a difference of 7 or 3 °C (v2 = 24.16 or 21.99; df = 1; P < 0.01). 3.3. Importance of photoperiod for the eggs that were deposited in the sand Almost all of the S. gregaria eggs that were buried in transparent glass beads, which allowed light to penetrate, hatched during the dark phases (Fig. 4A), and most of the L. migratoria eggs that were
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Y. Nishide et al. / Journal of Insect Physiology 76 (2015) 24–29
S. gregaria
No. of hatchlings
80 60 40 20 0
50 40 30 20 10 0
0
12
24
36
48
60
L
D
L
D
L
C
T
C
T
C
T
0
12
36
24
48
C
60
D
L
D
L
D
T
C
T
C
T
C
T
12
24
48
36
60
72
L
D
L
D
L
D
T
C
T
C
T
C
T
(G)
0
n = 162
12
24
36
48
60
72 84
D
L
D
L
C
T
C
T
C
T
96 h
D
L
C
T
n = 139
12
24
48
36
60
72
84
96 h
D
L
D
L
D
L
D
L
T
C
T
C
T
C
T
C
0
n = 147
12
80 60 40 20 0
60 h
48
36
L
(F)
50 40 30 20 10 0
84 h
D
24
12 D
0
n = 147
n = 136
(D)
84 h
L
0
40 30 20 10 0
72
D
(E)
0
D
n = 116
L. migratoria
(B)
80 60 40 20 0
84 h
72
D
(C)
50 40 30 20 10 0
80 60 40 20 0
n = 150
(A)
24
48
36
60
72
84
96 h
D
L
D
L
D
L
D
L
T
C
T
C
T
C
T
C
(H)
0
n = 121
12
24
48
36
60
72
84 h
D
L
D
L
D
L
D
L
D
L
D
L
T
C
T
C
T
C
T
C
T
C
T
C
Fig. 3. The number of hatchlings during the dark phase (closed bars) and the light phase (open bars) in different combinations of photoperiod and thermoperiod in S. gregaria (A, C, E and G) and L. migratoria (B, D, F and H). Thermoperiod in A–D was thermophase:cryophase (T:C) 32.5:22.5 °C and 31:24 °C in E and F, respectively, and T:C 29:26 °C in G and H. D, L, T and C indicate the dark phase, light phase, thermophase and cryophase, respectively. The horizontal axis indicates the time in hours after the beginning of hatching.
S. gregaria No. of hatchlings
140
L. migratoria 120
n = 283
(A)
120
100
100
80
80 60
60
40
40
20
20 0
0
12 D
120
No. of hatchlings
n = 216
(B)
24 L
48
36 D
L
60 D
(C)
72 L
84 h
0
D
12 L
80
n = 204
100
0
24 D
48
36 L
D
60 L
(D)
72 D
84 h L
n = 141
70 60
80
50
60
40
30
40
20 20 0
10 0
12 D
24 L
36 D
48 L
60 h
0
0
D
12 L
24 D
36 L
48 D
60 h L
Photocycles Fig. 4. The number of individuals that hatched during the dark phase (closed bars) and the light phase (open bars) of 12L:12D cycles at a constant 30 °C in S. gregaria and L. migratoria eggs that were buried in transparent glass beads (A and B) or sand (C and D). The photoperiodic schedule is shown below each panel; D, dark phase; L, light phase.
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Y. Nishide et al. / Journal of Insect Physiology 76 (2015) 24–29
(A) S. gregaria
No. of hatchlings (%)
100
(B) L. migratoria n = 1021
100
80
80
60
60
40
40
20
20
0
0
n = 465
Fig. 5. Hatching time of S. gregaria (A) and L. migratoria (B) eggs that were left intact in the egg pods in sand at a 12L:12D photoperiod at constant 30 °C. Closed and open bars indicate the percentages of hatchlings during the dark and light phases, respectively.
similarly treated hatched during the light phases (Fig. 4B). This result suggests that both species apparently responded to the photoperiod. In addition, the S. gregaria eggs that were covered with sand hatched during the both light and dark phases with a unimodal pattern of hatching activity over time. This result suggested that photoperiod did not influence the hatching time (Fig. 4C). Similar results were obtained for the L. migratoria eggs that were covered with sand (Fig. 4D): they hatched in a unimodal pattern independent of the photoperiod. As mentioned above, locust eggs are deposited as an egg pod that consists of eggs and a foam plug. The last experiment was designed to determine whether the eggs that were deposited in sand responded to light above the ground through the foam plug and used the daily light–dark cycles as a cue for hatching. Of the eggs that were kept intact in the egg pod in the sand, hatching was observed at similar frequencies during the light and dark phases in either species of locust, and the ratio did not deviate significantly from unity or from each other (v2-test; P > 0.05 in each species; Fig. 5). 4. Discussion 4.1. Characteristics of the responses to light conditions In various organisms, including insects, photoperiod plays a pivotal role in the control of daily activities and development, but temperature may also exert a significant effect in many organisms (Saunders, 2002). This study demonstrated that the eggs of S. gregaria and L. migratoria were capable of responding to photoperiod by hatching at species-specific times of the day. The timings of the hatching of these species were in sharp contrast to each other. At a 12L:12D photoperiod with a constant temperature, the eggs of S. gregaria primarily hatched during the dark phase, whereas those of L. migratoria mainly hatched during the light phase. In the field, S. gregaria eggs hatch over a short period from shortly before dawn to the first four hours after sunrise (Ellis and Ashall, 1957). Hatching during the dark phase may be an adaptation to the desert where this locust lives, because the temperature in the desert is often over 50 °C and humidity is low during the day (Ellis and Ashall, 1957; Stower et al., 1958). On the other hand, in L. migratoria, a grassland species, little is known about hatching time in the field. The differences in the habitats could affect the hatching strategy and time of hatching (Nishide et al., 2015). The temperature also affected the hatching time in the S. gregaria and L. migratoria eggs that were exposed to light–dark cycles. The eggs of both species tended to hatch later during the day as the temperature decreased (Fig. 2). Because some eggs of these locusts hatched in the ‘unexpected’ phase, i.e., in the light phase for S.
gregaria and in the dark phase for L. migratoria, their hatching may not be a direct response to the presence or absence of light. The underlying mechanism that controls the hatching time in these locusts remains unknown. A population of the marine midge, Clunio marinus, that was kept in light–dark cycles shows a peak of adult emergence during the light phase. When transferred from the light–dark cycles to continuous illumination, the population exhibits one further emergence peak and then arrhythmicity (Pflüger and Neumann, 1971). This phenomenon was explained by an hour-glass mechanism in which a non-repetitive (i.e., non-oscillatory) timer was initiated by a light-on stimulus and then ran its allotted time (Saunders, 1976). The present results with the locusts might be explained by this mechanism: the hatching time could be set by a light-on or light-off stimulus and the process leading to hatching proceeds as a function of temperature. Another possibility is the presence of an endogenous oscillator that is entrained by an environmental cue. We previously explored this possibility for these locusts by transferring the eggs from a thermoperiod to a constant temperature (Nishide et al., 2015). The results indicate that peak hatching activity was observed over 2 consecutive days after the eggs were transferred to a constant temperature. Therefore, it is possible that an endogenous oscillator is involved in the control of hatching in these locusts, although further experiments using other thermoperiods or photoperiods are necessary to determine whether or not the hatching activity of these locusts is under the control of a circadian rhythm. The eggs of S. gregaria and L. migratoria adjusted the hatching time by responding to the photoperiod that occurred during the last 5 days before hatching. In a previous study (Nishide et al., 2015), the eggs of the two locust species adjusted the hatching time to a new thermo-environment that occurred within 24 h before hatching. A similar prompt adjustment might be achieved by these locusts in response to photoperiod, but appropriate experiments to evaluate this possibility have not yet been performed. 4.2. The responses to photoperiod under various thermoperiods The responses to photoperiod to control hatching time were greatly influenced by the magnitude of temperature differences of thermoperiod in S. gregaria and L. migratoria. Under a thermoperiod with a difference of 10 °C, hatching occurred commonly during both the light and dark phases (Fig. 3C and D). This pattern was likely caused by the interacting effects of the two factors. As the temperature difference of thermoperiod decreased while the mean daily temperature was kept constant, the hatching time tended to become more dependent on the photoperiod than on the thermoperiod. In a previous study (Nishide et al., 2015), S. gregaria and L. migratoria eggs that were exposed to various thermoperiods with different magnitudes of temperature differences under continuous illumination responded to even a difference of 1 °C by hatching at the expected time of the day. In the present study, however, most of the eggs that were exposed to a thermoperiod with a difference of 3 °C at a 12L:12D photoperiod hatched as if they entirely depended on the photoperiod. There is a possibility that, as observed with temperature, variation in light intensity produces different effects on the control of hatching time in these locusts, especially when interacting with the thermoperiod. 4.3. The influence of photoperiod on the hatching time Photoperiod or light affects the hatching time of various orthopteran species that lay eggs in soil (e.g., Arai, 2012; Shimizu and Masaki, 1997; Tomioka et al., 1991). However, whether or not the eggs of these insects respond to photoperiod in the field has not been confirmed. As described above, the eggs of S. gregaria
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and L. migratoria responded to photoperiod by hatching at the specific times of the day when they were removed from the egg pod and placed on sand, but photoperiod did not affect their hatching time if the eggs were covered with sand. Locusts deposit eggs as an egg pod, which is 5.3–7.2 cm long in a Japanese strain of L. migratoria (n = 11; Tanaka, S. and Nishide, Y., unpublished observations). The upper portion of an egg pod normally consists of a foam plug that is 2.2 cm long on average (range, 1.6–3.1 cm; n = 11). In the present study, we tested whether the light reached the eggs in the egg pod through the foam plug. Because the locust eggs that were left intact in the egg pod in the sand hatched at similar frequencies during the light and dark phases at a constant temperature, it was concluded that the light did not penetrate the foam plug. Similar results were observed for S. gregaria when the egg pods were laid in the sand that was brought from a desert habitat in Mauritania (Nishide, Y. and Tanaka, S., unpublished observations). If the light does not reach the locust eggs that were deposited in the soil, it would then follow that the light sensitivity and the light-driven mechanism that controls the hatching time have no ecological significance, which raises an intriguing question: Why do locusts have these functions? One possibility is that an embryo that is ready to hatch has already developed all of the sensory organs and functions that are necessary for nymphal life so that it can perceive light if exposed to it. However, this hypothesis does not explain how the locust egg can use light to control hatching time. Another possibility is that an unidentified organ or molecules that are responsible for the perception of temperature and the control of hatching time also perceive light so that a photoperiod can act like a thermoperiod for the locust eggs. A possible candidate for such molecules might be rhodopsin, which is a photoreceptor that is also involved in circadian rhythms in the animal kingdom including insects (White, 1978; Shichida and Matsuyama, 2009). In Drosophila melanogaster, rhodopsin functions not only as a photoreceptor but also as a thermosensor (Shen et al., 2011). This hypothesis is highly speculative but might explain why the eggs of the locusts and possibly other insects that are buried under ground are often capable of responding to light to control their behavior and activity. Acknowledgements We thank Ms. H. Ikeda, Ms. N. Totsuka and Ms. M. Higuchi for maintaining the locust colonies. Thanks are also due to Dr.
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Takashi Suzuki (University of Western Ontario) for his stimulating discussion and information on the light perception mechanisms. We are indebted to Dr. Sinzo Masaki (Professor emeritus of Hirosaki University) for his invaluable comments on the manuscript and correcting our English. This study was founded in part by a Kakenhi Grant of Japan (No. 23380038) to S.T. The grass used was raised by the Field Management Section of NIASO and we would like to thank Mr. Kameo Tsukada for growing the grass. Two anonymous reviewers greatly improved the manuscript. References Arai, T., 1977. Effects of the daily cycles of light and temperature on hatchability and hatching time in Metrioptera hime Furukawa (Orthoptera: Tettigoniidae). Kontyu 45, 107–120. Arai, T., 2012. Egg hatching rhythm in insects. Academic Archives of Yamaguchi Prefectural University (Bulletins and Records of Research Activities) 5, 1–42. (In Japanese with an English abstract). Ellis, P.E., Ashall, C., 1957. Field studies on diurnal behaviour, movement and aggregation in the desert locust (Schistocerca gregaria Forskål). Anti-locust Bull. 25, 1–94. Nishide, Y., Tanaka, S., Saeki, S., 2015. Adaptive difference in daily timing of hatch in two locust species. Schistocerca gregaria and Locusta migratoria: the effects of thermocycles and phase polyphenism. J. Insect Physiol. 72, 79–87. Padgham, D.E., 1981. Hatching rhythms in the desert locust, Schistocerca gregaria. Physiol. Entomol. 6, 191–198. Pflüger, W., Neumann, D., 1971. Die Steuerung einer gezeitenparallelen Schlüpfrhythmik nach dem Sanduhr-Prinzip. Oecologia, Berl. 7, 262–266. Popov, G.B., 1958. Ecological Studies on oviposition be swarms of the desert locust (Schistocerca gregaria Forskål) in Eastern Africa. Anti-Locust Bull. 31, 1–70. Saunders, D.S., 1976. The circadian eclosion rhythm in Sarcophaga argyrostoma: some comparisons with the photoperiodic ‘‘clock’’. J. Comp. Physiol. A. 110, 111–133. Saunders, D.S., 2002. Insect Clocks, third ed. Elsevier, Amsterdam. Shen, W.L., Kwon, Y., Adegbola, A.A., Luo, J., Chess, A., Montell, C., 2011. Function of rhodopsin in temperature discrimination in Drosophila. Science 331, 1333– 1336. Shichida, Y., Matsuyama, T., 2009. Evolution of opsins and phototransduction. Philos. Trans. R. Soc. B 364, 2881–2895. Shimizu, T., Masaki, S., 1997. Daily time of hatching in Nemobiine crickets. Jpn. J. Entomol. 65, 335–342. Stower, W.J., Popov, G.B., Greathead, D.J., 1958. Oviposition behaviour and egg mortality of the desert locust (Schistocerca gregaria Forskål) on the coast of Eritrea. Anti-Locust Bull. 30, 1–33. Tomioka, K., Wakatsuki, T., Shimono, K., Chiba, Y., 1991. Circadian control of hatching in the cricket, Gryllus bimaculatus. J. Insect Physiol. 37, 365–371. Uvarov, B., 1966. Grasshoppers and Locusts, vol. 1. Cambridge University Press, Cambridge. White, R.H., 1978. Insect visual pigments. Adv. Insect Physiol. 13, 35–67.