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Changing responses to temperature and moisture of diapausing and developing eggs of Allonemobius fasciatus (Orthoptera: Gryllidae)
J. Insect Physiol. Vol. 33, No. 9, pp. 635441, Printed in Great Britain. All rights reserved
1987 Copyright
0
0022-1910/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
CHANGING RESPONSES TO TEMPERATURE AND MOISTURE OF DIAPAUSING AND DEVELOPING EGGS OF ALLONEMOBIUS FASCIATUS (ORTHOPTERA: GRYLLIDAE) SEIJI TANAKA Department of Entomology, Oregon State University, Corvallis, Oregon, U.S.A. (Received 10 October 1986; revised 5 January 1987) Abstract-A portion of the Allonemobius fasciatus eggs laid in September averated embryonic diapause when transferred from 20 to 27°C in the first 12 days, but this proportion decreased if the transfer was delayed until day 16 or 20. This response may be explained by the interacting effects of low and high temperatures on the two types of diapause: Incubation at 20°C decreases the incidence of summer diapause during early embryonic stages but increases the incidence of winter diapause at a later stage. The intensity of winter diapause at 20°C was greatly increased by a 5-day exposure to 27°C occurring around the diapause stage. Exposure of eggs in winter diapause to different temperatures indicates that the thermal optimum for diapause development is relatively high (16-1l’C) at the beginning and decreases to 6°C later. In the field, diapause development occurred rapidly in autumn but slowly during the winter. A short exposure of eggs to dry conditions (4-6 days) increased the incidence of summer diapause at 30°C but a long exposure ( > 15 days) terminated summer diapause. In this cricket, the effects of different temperature and moisture conditions on diapause regulation thus change during the course of morphogenetic and diapause development. Key Word Index: Allonemobius fasciatus, Gryllidae, embryonic development, diapause, temperature, moisture
INTRODUCllON The striped ground cricket, Allonemobius farciatus, is a univoltine species which overwinters as an egg
(Sarai, 1967; Howard, 1983). In Oregon, eggs laid in summer enter summer diapause in early embryonic stages at high temperatures (Tanaka, 1984). If such eggs are transferred to a low temperature of 2O”C, they resume development but enter a second, winter diapause at the end of anatrepsis. Eggs laid in autumn tend to avert diapause at high temperatures, although they enter winter diapause if constantly incubated at 20°C. In this case, however, the intensity of diapause is much less than that in eggs laid earlier in the season. This seasonal variation in diapause character is mainly due to the environmental conditions, particularly temperature, to which their parents are exposed (Tanaka, 1984, 1986a). Photoperiod, which influences the duration of the nymphal stage and wing form in this species, has no effect on diapause regulation (Tanaka and Brookes, 1983; Tanaka, 1986a). In other species, diapause characteristics are influenced by environmental conditions impinging on the eggs. For example, in the field cricket, Teleogryllus commodus, the incidence of diapause is greatly reduced when the eggs are first incubated at a low temperature and then transferred to 26°C or Present Address: Department of Entomology, The Ohio State University, 1735 Neil Avenue Columbus, OH 43210, U.S.A.
higher during the pre-diapause period (Browning, 1982; Hogan, 1960a). On the other hand, eggs of T. emma increase the intensity of diapause in response to high incubation temperature (Masaki, 1962). Moisture is another environment factor that commonly influences embryonic diapause (for reviews, see Andrewartha, 1952; Lees, 1955; Uvarov, 1966; Tauber et al., 1986). Most attention has focussed on the role of water in termination of diapause. Wardhaugh (1980) pointed out that whereas in some species uptake of water is associated with completion of diapause, in others such as the Australian plague locust, Chortoicetes terminifera, uptake of water is actively involved in diapause induction. In the present study, I examined the effects of different combinations of temperature and moisture conditions on induction, maintenance, and termination of embryonic diapause and development in A. fascuatus. The seasonal progression of diapause development in the field was also monitored by transferring eggs from the field to the laboratory at different intervals. MATRRULS AND METHODS
Eggs of A. fasciatus were obtained from > 40 female adults collected in Corvallis, Oregon, in early August of mid-September in 1980 and 1981. To examine the thermal responses, groups of eggs were collected and placed on moist tissue paper in Petri dishes, as pre.viously described (Tanaka, 1984), and exposed to various temperature-regimes. The hatched 635
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nymphs were checked daily and removed. Temperatures of 0, 3, 6, 11, 16, 20 and 27°C were controlled within a range of f 1°C but the 16°C conditions fluctuated from 14.5 to 17.5”C. To observe the progression of diapause development under natural conditions, eggs laid in September were kept on moist paper for a month at room temperature and then exposed to field conditions on October 15, 1980. Using the method previously reported (Tanaka and Brookes, 1983), bags made of nylon gauze (2 x 3 cm) each containing about 50 eggs were deposited about 1 cm deep in soil in habitats of this species that were open and exposed to the sun during the day; the ground was covered with short forbs. The bags were taken back to the laboratory on different dates from November 16 to May 16, and hatches were recorded daily for 90 days at 20°C. To examine the effects of low moisture on embryonic development, eggs laid in mid September were placed in dry soil (10% moisture content) in a plastic container (4 x 4 x 3 cm) with an air tight lid. The soil (clay loam) had been collected from 1-3 cm below the ground surface covered with green forbs in habitats of this species in August, 1981, and stored in a plastic bag until used. Moisture content was determined based on the difference in weight before and after the soil (about 50 g) was dried at 120°C for 24 h. Because this species can be heard only in those fields where some forbs are kept green throughout the summer, it is likely that eggs are laid in such sites. During the summer, soil moisture at 1-3 cm in depth is between 10 and 15% where the ground is covered with green forbs, while it is about 5% where the ground is bare or covered with wilted forbs (Tanaka, unpublished data). After rain falls in September, soil moisture increases above 30%. After different periods of incubation at 3O”C, eggs were removed from the soil and dissected to determine the embryonic stages according to the method and criterion previously described (Tanaka, 1984): embryonic development before katatrepsis is divided into 7 stages; stage I, embryo dumb-bell shaped; stage II, embryo elongating; stage III to VII, embryo developing appendages. The last stage (VIII) includes all embryos after stage VII. Results for eggs placed on moist paper at 30°C for various periods (Tanaka, 1984) will be cited here for comparison since the observations were made concurrently. In the same way, other groups of eggs laid in September were used to examine the effects of dry conditions on diapause. Such eggs were first kept in soil with 10% moisture content for various periods at 30°C and then placed on moist paper in Petri dishes at 30°C and examined every day for hatching. RESULTS
Eflects of temperature
on diapause regulation
When freshly laid eggs were incubated at 20°C for various lengths of time and then transferred to 27°C a bimodal pattern of hatching was recognized by a probit analysis. Usually, a proportion of the eggs hatched within 3 weeks after transfer to 27°C (identified as ‘early hatching’) and the remainder hatched sporadically over a period of months.
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Effects of temperature shift from 20 to 27°C on (A) the proportion of early hatching eggs of Allonemobius fasciutw laid in August (0) or September (0) and (B) the time spent at 27°C by the early hatching eggs. Eggs that hatched soon after transfer to 27°C were identified as early hatching and distinguished from the remainder which hatched sporadically over a period of months. Vertical lines indicates SD. Each point in (A) is based on 50 eggs.
Among eggs laid in August, no early hatching occurred when eggs were transferred from 20 to 27°C before day 40 (Fig. IA). The proportion of early hatching eggs increased as the time of transfer from 20 to 27°C was further delayed. About 13 days was required for hatching at 27°C (Fig. 1B). Eggs laid in September showed a different response. The proportion of early hatching eggs increased as the transfer to 27°C was delayed until day 12 (Fig. 1A). The proportion then declined and reached a minimum when the eggs were transferred from 20 to 27°C on day 25. Thereafter, a shift in temperature again increased the proportion of early hatching individuals and all eggs hatched shortly after transfer on day 50. Eggs incubated at 20°C reached the stage where winter diapause intervenes in 16 or 20 days (Fig. 1B). Eggs kept at 20°C for longer (20-50 days) developed no further as demonstrated by the fact that they hatched after 12 days when subsequently transferred to 27°C. Eggs kept at 20°C for less than 16 days before transfer to 27°C hatched in 13-18 days. A 4- or 8-day exposure of August eggs to 27°C slightly increased the mean time for 50% hatch compared with that in those constantly kept at 20°C (Fig. 2). After day 8, the duration of the egg stage increased as the transfer to 20°C occurred later (r = 0.81; P < 0.05). However, the time spent at 20°C was almost constant and there was no significant correlation between the time of transfer and the time spent at 20°C (r = 0.24; P > 0.05). This suggests that development was suppressed at a certain stage at 27°C as previously reported (Tanaka, 1984) and a
Embryonic diapause in Allonemobius
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Fig. 2. The time for 50% hatch in eggs of Allonemobius fbsciarus kept at 21°C for various periods before transfer to 20°C. Each point indicates the mean of 3 replicates, each consisting of 25 eggs laid in August. Vertical lines indicate SD.
long exposure to this temperature did not influence subsequent development at 20°C. Figure 3 shows the results of transfer from 20 to 27°C as obtained in the experiment described in Fig. 1. When eggs laid in August were transferred to 27°C within 12 days after laying, they required a much longer time to hatch than did those transferred later. The former probably entered summer diapause in response to high temperature. When transferred on day 16 to 20, the time required for 50% hatch was reduced by about 50 days. It was reduced further until the 50th day, and then increased. These results indicate that the duration of the egg stage was increased by early shifts to 27°C and decreased by later shifts. The duration of the egg stage was increased by a May exposure to high temperature (Fig. 4). The sensitivity to this exposure increased almost linearly as the eggs developed, and reached its maximum on
Days
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20
kept
30 at
40
60
2D’C
60
before
70
150
transfer
to 27’C
Fig. 3. The time for 50% hatch in eggs of Aflonemobius f0sciaru.s kent at 20°C for various neriods before transfer to 27°C. E&h point indicates the-mean of 3 replicates, each consisting of 25 eggs laid in August. Vertical lines
indicate SD.
50 exposure
60
a? 27’C
Fig. 4. Effects of 5-day exposure to 27°C on the time required for 50% hatch in eggs of Ailonemobius fasciatus otherwise kept at 20°C. Eggs kept at 20°C for various periods were exposed to 27°C for 5 days and returned to 20°C. Each point indicates the mean of 3 replicates, each consisting of 25 eggs laid in August. Vertical lines indicate SD.
day 20. This effect was less pronounced but still detected after day 25. Because summer diapause is neither induced nor maintained at 20°C (Tanaka, 1984), a short exposure to 27°C is likely to have intensified the winter diapause. When exposure to 27°C occurred after day 30, the time for 50% hatch was shorter than when eggs were maintained at 20°C throughout the egg stage. When exposure to 27°C was delayed until day 60, more than 50% of eggs hatched soon after return to 2o”C, suggesting that the high temperature stimulated termination of winter diapause. To examine the effects of low temperature on termination of winter diapause, eggs laid in September were incubated at 20°C for various periods, exposed to a second temperature of 16, 11, 6, or 0°C for 45 or 90 days, and returned to 20°C for hatching. Upon transfer to the last temperature, a group of eggs hatched within 40 days which could be distinguished from the remaining eggs which hatched later in a sporadic fashion. The former were considered as early hatching eggs and the proportion of such eggs determined. When 25-day old eggs were exposed to different second temperatures for 90 days, the proportion of early eggs hatching increased in proportion to the temperature increase (Fig. 5). A 90 day exposure of 40-, 50-, or 75-day old eggs to different second temperatures caused most eggs to hatch soon after transfer to 20°C. If, however, eggs were exposed to the second temperature for only 45 days, the proportion of early hatching eggs was highest at a second temperature of 6°C and lower at other temperatures. Development
0
to 5 day
in the field
Diapause development proceeded relatively rapidly in autumn. When eggs were kept at room temperature for a month and then directly incubated at 20°C on October 15, none hatched within 40 days while 53.6% hatched if they were kept in the field for 31 days before incubation at 20°C (Fig. 6). During the winter, development was slow but hatching occurred more synchronously when transferred from the field to 20°C on February 16 than on December
SEIJITANAKA
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Second trmperoturs PC 1 Fig. 5. Effects of low temperatures on the proportion of early hatching eggs of Allonemobiusjizrciat. Eggs were first incubated at 20°C for 25, 40, 50, or 75 days, exposed to different second temperatures for 45 (0) or 90 (0) days, and returned to 20°C for hatching. Each point is based on 90 eggs. Numbers on the right indicate the times of transfer to second temperatures. Percentages between different set-
ond temperatures followed by different letter significant at 5% (X2-test). 16. Eggs kept in the field until March 17 took longer to hatch upon transfer to 20°C than those kept at 3°C until March 17 and transferred to 20°C (t = 10.8;
,
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15 zz after laying
Fig. 7. Effects of moisture on embryonic development at 30°C in eggs of Allonemobius fasciatus laid in September. Eggs were placed in dry soil with 10% moisture content (0) or on moist paper (0) for various periods and dissected to determine the embryonic stage. For the embryonic stages, see Tanaka (1984). Each point indicates one individual.
P < 0.05), although all eggs hatched within 40 days at 20°C. This suggests that eggs which hatched within 40 days at 20°C contained not only those free from diapause but also those still in diapause at the time of transfer to 20°C. The time required for hatching was further reduced when the time of transfer was delayed, but some eggs in April were still in diapause. Effects of moisture on diapause regulation
Eggs laid in September and placed on moist paper at 30°C developed rapidly and a half of the embryos were beyond stage VII by day 7 (Fig. 7). Embryos beyond this.- stage _ are._^no longer __ capable of entering . 7. HATCHING dtapausc (l‘anaka, lYg4). However, some eggs remained at stage II or III and they probably entered summer diapause (see Tanaka, 1984). Embryos which appeared to enter diapause at these stages were also found among eggs deposited in the dry soil with 10% moisture content. Some embryos reached stage IV or V by day 10 but further development did not take place. Instead, their body and appendages became smaller. It was not possible to distinguish precisely those which remained at stage II or III from those which probably developed beyond stage III and became smaller after prolonged incubation in the dry soil. All eggs became slightly smaller than the initial size but few were completely collapsed even after 30 days of dry incubation. When eggs laid in September were kept in soil with 10% moisture content for different periods and then moistened at 30°C some began to hatch in 2 weeks, Days to hatch ot 2O*C and hatching continued for several days. Eggs which Fig. 6. Effects of outdoor temperature on egg hatching ot hatched during this early period were separated from Allonemobiw fasciatus. Eggs laid in September were kept at the others by probit analysis of the hatching curves room temperature for a month before they were transferred (indicated by the d&continuities of the lines)[Fig. 81. to the field on October 15, 1980. They were taken back to When maintained in the soil for 2 days, the proporthe laboratory (20°C) for hatching on each of 8 occasions tion of eggs in the first hatching group was slightly from November 15 to May 26. For comparison, two groups decreased and it became significantly lower when of eggs were incubated at 20°C on October 15 without being kept in the soil for 4 (x2 = 6.52; P < 0.05) or 6 days transferred to the field and on March 17 after a 5-month (x2 = 5.11; P < 0.05) than when incubated under exposure to 3°C. Numbers on the right indicate the propormoist conditions throughout. This may indicate that tions of eggs hatching within 40 days at 20°C. Arrows indicate the times for 50% hatch. the induction of summer diapause is more common
Embryonic diapause in Allonemobiu
Fig. 8. Effects of moisture on cumulative percentage of hatch in eggs of Alfonepobiw fasciotus at 30°C. Eggs laid in Sptember were first incubated in dry soil with 10% moisture content and transferred to moist conditions. Numbers in the figure indicate the times of transfer. By a probit
analysis, the first hatching group was distinguished from the rest hatching later, as indicated by the discontinuities of the lines. N = 90 for each treatment. under dry conditions than under wet conditions.
Eggs developing without diapause appeared to enter a state of quiescence when water deficiency was encountered (Fig. 7). This would explain why their hatching started later as the time of transfer to moist conditions was delayed (Fig. 8). Embryonic development stopped after 4 days under dry conditions as indicated by the observation that the first hatching group kept under dry conditions for 4, 6 or 8 days hatched 10 days after transfer to moist conditions. Those transferred to moist conditions on day 0 or 2 took longer (12-14 days) for hatching. When the period of exposure to dry conditions was prolonged 15 days or more, the proportion of eggs in the first hatching group gradually increased (Fig. 8). In those transferred to moist conditions on day 20, it was 84.4% which was significantly higher than the percent hatching attained in the control during the same period of incubation at 30°C (x2 = 8.56; P < 0.05). This, together with the fact that the embryonic stages of summer diapause can be reached in dry soil, may suggest that the termination rather than prevention of summer diapause occurred under dry conditions or upon transfer to moist conditions. When exposed to dry conditions for 30 days, all surviving eggs except one appeared in the first hatching group. The time spent by the first hatching group after transfer to moist conditions gradually increased from 10 to 12 days as the time of transfer was delayed from day 8 to day 30. This was because (1) eggs exposed longer to dry conditions probably took longer to gain sufficient water to resume embryogenesis and (2) those which had been in summer diapause at early stages joined the first hatching group. Mortality at the end of the observations was 2.2% or less except for eggs exposed to dry conditions for 30 days (7.8%). DISCUSSION
The present data suggest physiological processes responsible for the determination of diapause charac-
639
teristics in the eggs of A. farciutus. These processes are highly sensitive to temperature and moisture and their sensitivity greatly changes during the course of embryonic development and diapause. If eggs were transferred from 20 to 27°C before the stage of winter diapause was reached, many hatched without diapause, although few or none did so when constantly kept at 27 or 20°C (Fig. 1). A similar response has been reported in other orthopterans (Browning, 1952; Rakshpal, 1962; Dean and Hartley, 1977; Deura and Hartley, 1982; Ingrisch, 1984) including the Edmonton strain of A. farciatus (Sarai, 1967). Browning (1952) attributed this phenomenon in T. commodus to the completion of diapause development during the low temperature in the prediapause stage. Hogan (196Oa,b), working with the same species, found that an exposure to low temperature during the pre-diapause period itself did not have a diapause-eliminating effect, although the tendency to enter diapause at such a low temperature is so weakened that it can be easily averted upon transfer to high temperature. In the Oregon strain of A. fasciatus, the observed pattern of response to temperature shifts may be explained in terms of the interacting effects of low and high temperatures on the two types of diapause. As already reported (Tanaka, 1984), summer diapause is induced at early embryonic stages (II-IV) at a high temperature (e.g. 27°C) while no development suppression occurs at a lower temperature (e.g. 20°C) until the end of anatrepsis (stage VII) at which winter diapause occurs. Thus, the later the time of transfer from 20 to 27°C the greater would be the proportion of eggs that fail to enter summer diapause and hatch upon transfer to 27°C (Fig. IA). However, as embryos become closer to stage VII at 20°C more eggs would enter winter diapause, and the proportion of diapause-free eggs decreases as observed when the time of transfer to 27°C was delayed until day 16 or 20. None of the eggs laid in August responded to the same temperature shifts by hatching rapidly until day 40. This absence of response to early temperature shifts could be related to their high potential to enter both types of diapause at high temperatures as well as the high intensity of diapause compared with eggs laid in autumn (Tanaka, 1984). Sarai (1967) examined the effects of incubation temperature on diapause in the Edmonton strain of A. fasciatus. A direct comparison with the present results is not appropriate because this and the Oregon strains differ from each other in the rate of embryonic development and possibly in the stage of diapause (Tanaka, 1986b). A high temperature intensified diapause in A. fasciatus. When eggs laid in August were kept at 20°C and exposed to 27°C for 5 days, the duration of the egg stage was markedly increased by the exposure to 27°C occurring shortly before or after the induction of winter diapause (Fig. 4). A quite similar result has been reported in T. emma (Masaki, 1962) in which the diapause-intensifying action of high temperature also extends to the beginning of the diapause stage, although these two species enter diapause at different embryonic stages. When transferred from 20 to 27°C but not returned
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SEUI TANAKA
to 2o”C, eggs of A. fascia&s appeared to enter summer diapause if the transfer occurred early enough (Fig. 3). Among eggs transferred later, the longer the duration of incubation at 20°C the shorter the time to hatch upon transfer to 27°C. This may suggest that the induced winter diapause was less intense at 20°C and/or that diapause development proceeded more rapidly at 20°C than at 27°C. Lees (1955) proposed that the thermal optimum for diapause development is not constant but varies in the course of diapause. This view has been supported by many examples which were recently summarized by Tauber ef al. (1986). In A. fusciutus, the thermal optimum for winter diapause development is relatively high (16-l 1°C) at the beginning and then decreases to 6°C (Fig. 5). Until diapause development reaches a certain stage, higher temperatures may be less effective because a transfer from 20 to 27°C terminated diapause more rapidly when it occurred on day 50 than on day 30 (Fig. 3). However, the duration of the egg stage became longer again as the transfer from 20 to 27°C occurred later than day 50, suggesting that the final phase of diapause development proceeds more rapidly at a higher temperature, as demonstrated for T. commodus (Masaki ef al., 1979). In the field diapause development appears to proceed relatively rapidly in autumn, but slowly during winter and some individuals are still in diapause in March and April (Fig. 6). When the thermal influence on diapause development is studied, diapausing insects are often exposed to different low temperatures and then transferred to a high temperature for postdiapause development (Andrewartha, 1952). However, information obtained from such studies may be of limited value, because (1) the intensity of diapause could be influenced by the temperature before or at the time of onset of diapause, (2) the thermal optimum for diapause development could change during the course of diapause, as observed in A. fasciatus and other species (Muroga, 1951; Way, 1959; Ando, 1972, 1978, 1983; Deura and Hartley, 1982; Sim and Shapiro, 1983; Tanaka, 1983; Hunter and Gregg, 1984), and (3) there may be interacting effects of the ‘chilling’ and ‘incubation’ temperatures as demonstrated in this paper. Moisture also influenced embryonic diapause and development in A. fusciatus. For the completion of embryonic development, water absorption is necessary in some species while it is not in others (Browning, 1967; Hinton, 1980). Among the former, eggs which enter diapause absorb water either before or after they enter diapause, although in some species noticeable amounts of water are absorbed during the period of diapause (Lees, 1955; Moriarty, 1969; Ando, 1972; Wardhaugh, 1980; Tanaka, 1986b). In the locust, C. terminifera, eggs kept in dry soil at 32S”C can not absorb water and become quiescent before they reach the embryonic stage at which diapause occurs (Wardhaugh, 1980). If such eggs are allowed to absorb water, more eggs fail to enter diapause than do those constantly kept under moist conditions at the same temperature. A similar phenomenon was observed in A. fasciutus. Unlike in C. terminifera, however, embryos of A. fasciatus can reach the stages at which summer diapause occurs
without absorbing water (Fig. 7). Thus, as observed in the luceme flea, Sminthurus viridis (Wallace, 1968) a long exposure to dry conditions probably influenced some physiological process after summer diapause was imposed, although some eggs apparently entered quiescence rather than diapause. On the other hand, when eggs were exposed to dry conditions only for the first 4 or 6 days, the incidence of summer diapause at 30°C was significantly increased. Therefore, these results indicate different effects of dry conditions on the induction and maintenance of summer diapause. Separation of a direct effect of moisture on the termination of diapause from that on postdiapause development is sometimes very difficult (Beck, 1980; Tauber et al., 1986). In A. farciutus, eggs at a high temperature absorb water even when embryogenesis is suppressed (Tanaka, 1986b). Thus, termination of summer diapause is independent of water absorption in this species. Acknowledgement-I
thank Drs Victor J. Brookes (Oregon State Univ.), Sinzo Masaki (Hirosaki Univ.), David L. Denlinger (Ohio State Univ.), and anonymous referees for correcting my English and providing suggestions on the manuscript and Dr T. D. Schowalter (Oregon State Univ.) for a financial support and encouragement. Many thanks are also due to Dr M. T. AliNiazee (Oregon State Univ.) who kindly let me use his environmental chambers. I acknowledged use of the library and computer facilities of Barre Colorado Island, Panama, where the manuscript was prepared.
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on embryonic diapause in Nemobius fasciatus (De Geer) (Orthoptera; Gryllidae). &zest. Em. 3, 107-135. Sims S. R. and Shapiro A. M. (1983) Pupal diapause in Barrus philenor (Lepidoptera; Papilionidae). Ann. ent. Sot. Am. 76, 407412.
Tanaka S. (1983) Seasonal control of nymphal diapause in the spring ground cricket, Pteronemobius nitidus. In Diapause and Life Cycle Strategies in Insects (Ed. by Brown V. K. and Hodec I.). DD.35-53. Dr W. Junk Publ.. The Hague. Tanaka S. (1984) Seasonal variation in embryonic diapause of the striped ground cricket, Allonemobius fasciatus. Physiol.
Ent. 9, 97-105.
Tanaka S. (1986a) Developmental characteristics of two closely related species of Allonemobius and their hybrids. Oecologia (Berlin),
69, 388-394.
Tanaka S. (1986b) Uptake and loss of water in diapause and non-diapause eggs of crickets. Physiol. Em. 11, 343-351. Tanaka S. and Brookes V. J. (1983) Altitudinal adaptation of the life cycle in Allonemobius fasciatus De Geer (Orthoptera; Gryllidae). Can. J. 2001. 61, 19861990. Tauber M. J., Tauber C. A. and Masaki S. (1986) Seasonal Adaptations of Insects. Oxford Univ. Press, New York. Wallace M. M. H. (1968) The ecology of Sminthurus viridis (Collembola). II Diapause in the aestivating egg. Aust. J. 2001. 16, 871-883. Wardhaugh K. G. (1980) The effects of temperature and moisture on the inception of diapause in eggs of the Australian plague locust, Chortoicetes terminifera Walker (Orthoptera; Acrididae). Aust. J. Ecol. 5, 187-191. Way M. J. (1959) The effect of temperature, particularly during diapause, on the development of the egg of Leptohylemyia coarctata. Trans. R. ent. Sot. Lond. 111, 351-364. Uvarov B. P. (1966) Grasshoppers and Locusts Vol. 1, Cambridge Univ. Press.
1987 Copyright
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0022-1910/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
CHANGING RESPONSES TO TEMPERATURE AND MOISTURE OF DIAPAUSING AND DEVELOPING EGGS OF ALLONEMOBIUS FASCIATUS (ORTHOPTERA: GRYLLIDAE) SEIJI TANAKA Department of Entomology, Oregon State University, Corvallis, Oregon, U.S.A. (Received 10 October 1986; revised 5 January 1987) Abstract-A portion of the Allonemobius fasciatus eggs laid in September averated embryonic diapause when transferred from 20 to 27°C in the first 12 days, but this proportion decreased if the transfer was delayed until day 16 or 20. This response may be explained by the interacting effects of low and high temperatures on the two types of diapause: Incubation at 20°C decreases the incidence of summer diapause during early embryonic stages but increases the incidence of winter diapause at a later stage. The intensity of winter diapause at 20°C was greatly increased by a 5-day exposure to 27°C occurring around the diapause stage. Exposure of eggs in winter diapause to different temperatures indicates that the thermal optimum for diapause development is relatively high (16-1l’C) at the beginning and decreases to 6°C later. In the field, diapause development occurred rapidly in autumn but slowly during the winter. A short exposure of eggs to dry conditions (4-6 days) increased the incidence of summer diapause at 30°C but a long exposure ( > 15 days) terminated summer diapause. In this cricket, the effects of different temperature and moisture conditions on diapause regulation thus change during the course of morphogenetic and diapause development. Key Word Index: Allonemobius fasciatus, Gryllidae, embryonic development, diapause, temperature, moisture
INTRODUCllON The striped ground cricket, Allonemobius farciatus, is a univoltine species which overwinters as an egg
(Sarai, 1967; Howard, 1983). In Oregon, eggs laid in summer enter summer diapause in early embryonic stages at high temperatures (Tanaka, 1984). If such eggs are transferred to a low temperature of 2O”C, they resume development but enter a second, winter diapause at the end of anatrepsis. Eggs laid in autumn tend to avert diapause at high temperatures, although they enter winter diapause if constantly incubated at 20°C. In this case, however, the intensity of diapause is much less than that in eggs laid earlier in the season. This seasonal variation in diapause character is mainly due to the environmental conditions, particularly temperature, to which their parents are exposed (Tanaka, 1984, 1986a). Photoperiod, which influences the duration of the nymphal stage and wing form in this species, has no effect on diapause regulation (Tanaka and Brookes, 1983; Tanaka, 1986a). In other species, diapause characteristics are influenced by environmental conditions impinging on the eggs. For example, in the field cricket, Teleogryllus commodus, the incidence of diapause is greatly reduced when the eggs are first incubated at a low temperature and then transferred to 26°C or Present Address: Department of Entomology, The Ohio State University, 1735 Neil Avenue Columbus, OH 43210, U.S.A.
higher during the pre-diapause period (Browning, 1982; Hogan, 1960a). On the other hand, eggs of T. emma increase the intensity of diapause in response to high incubation temperature (Masaki, 1962). Moisture is another environment factor that commonly influences embryonic diapause (for reviews, see Andrewartha, 1952; Lees, 1955; Uvarov, 1966; Tauber et al., 1986). Most attention has focussed on the role of water in termination of diapause. Wardhaugh (1980) pointed out that whereas in some species uptake of water is associated with completion of diapause, in others such as the Australian plague locust, Chortoicetes terminifera, uptake of water is actively involved in diapause induction. In the present study, I examined the effects of different combinations of temperature and moisture conditions on induction, maintenance, and termination of embryonic diapause and development in A. fascuatus. The seasonal progression of diapause development in the field was also monitored by transferring eggs from the field to the laboratory at different intervals. MATRRULS AND METHODS
Eggs of A. fasciatus were obtained from > 40 female adults collected in Corvallis, Oregon, in early August of mid-September in 1980 and 1981. To examine the thermal responses, groups of eggs were collected and placed on moist tissue paper in Petri dishes, as pre.viously described (Tanaka, 1984), and exposed to various temperature-regimes. The hatched 635
SEW TANAKA
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nymphs were checked daily and removed. Temperatures of 0, 3, 6, 11, 16, 20 and 27°C were controlled within a range of f 1°C but the 16°C conditions fluctuated from 14.5 to 17.5”C. To observe the progression of diapause development under natural conditions, eggs laid in September were kept on moist paper for a month at room temperature and then exposed to field conditions on October 15, 1980. Using the method previously reported (Tanaka and Brookes, 1983), bags made of nylon gauze (2 x 3 cm) each containing about 50 eggs were deposited about 1 cm deep in soil in habitats of this species that were open and exposed to the sun during the day; the ground was covered with short forbs. The bags were taken back to the laboratory on different dates from November 16 to May 16, and hatches were recorded daily for 90 days at 20°C. To examine the effects of low moisture on embryonic development, eggs laid in mid September were placed in dry soil (10% moisture content) in a plastic container (4 x 4 x 3 cm) with an air tight lid. The soil (clay loam) had been collected from 1-3 cm below the ground surface covered with green forbs in habitats of this species in August, 1981, and stored in a plastic bag until used. Moisture content was determined based on the difference in weight before and after the soil (about 50 g) was dried at 120°C for 24 h. Because this species can be heard only in those fields where some forbs are kept green throughout the summer, it is likely that eggs are laid in such sites. During the summer, soil moisture at 1-3 cm in depth is between 10 and 15% where the ground is covered with green forbs, while it is about 5% where the ground is bare or covered with wilted forbs (Tanaka, unpublished data). After rain falls in September, soil moisture increases above 30%. After different periods of incubation at 3O”C, eggs were removed from the soil and dissected to determine the embryonic stages according to the method and criterion previously described (Tanaka, 1984): embryonic development before katatrepsis is divided into 7 stages; stage I, embryo dumb-bell shaped; stage II, embryo elongating; stage III to VII, embryo developing appendages. The last stage (VIII) includes all embryos after stage VII. Results for eggs placed on moist paper at 30°C for various periods (Tanaka, 1984) will be cited here for comparison since the observations were made concurrently. In the same way, other groups of eggs laid in September were used to examine the effects of dry conditions on diapause. Such eggs were first kept in soil with 10% moisture content for various periods at 30°C and then placed on moist paper in Petri dishes at 30°C and examined every day for hatching. RESULTS
Eflects of temperature
on diapause regulation
When freshly laid eggs were incubated at 20°C for various lengths of time and then transferred to 27°C a bimodal pattern of hatching was recognized by a probit analysis. Usually, a proportion of the eggs hatched within 3 weeks after transfer to 27°C (identified as ‘early hatching’) and the remainder hatched sporadically over a period of months.
loor
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20
(B)
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,
,
,
,
,
,
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before transfer to 27*C
Effects of temperature shift from 20 to 27°C on (A) the proportion of early hatching eggs of Allonemobius fasciutw laid in August (0) or September (0) and (B) the time spent at 27°C by the early hatching eggs. Eggs that hatched soon after transfer to 27°C were identified as early hatching and distinguished from the remainder which hatched sporadically over a period of months. Vertical lines indicates SD. Each point in (A) is based on 50 eggs.
Among eggs laid in August, no early hatching occurred when eggs were transferred from 20 to 27°C before day 40 (Fig. IA). The proportion of early hatching eggs increased as the time of transfer from 20 to 27°C was further delayed. About 13 days was required for hatching at 27°C (Fig. 1B). Eggs laid in September showed a different response. The proportion of early hatching eggs increased as the transfer to 27°C was delayed until day 12 (Fig. 1A). The proportion then declined and reached a minimum when the eggs were transferred from 20 to 27°C on day 25. Thereafter, a shift in temperature again increased the proportion of early hatching individuals and all eggs hatched shortly after transfer on day 50. Eggs incubated at 20°C reached the stage where winter diapause intervenes in 16 or 20 days (Fig. 1B). Eggs kept at 20°C for longer (20-50 days) developed no further as demonstrated by the fact that they hatched after 12 days when subsequently transferred to 27°C. Eggs kept at 20°C for less than 16 days before transfer to 27°C hatched in 13-18 days. A 4- or 8-day exposure of August eggs to 27°C slightly increased the mean time for 50% hatch compared with that in those constantly kept at 20°C (Fig. 2). After day 8, the duration of the egg stage increased as the transfer to 20°C occurred later (r = 0.81; P < 0.05). However, the time spent at 20°C was almost constant and there was no significant correlation between the time of transfer and the time spent at 20°C (r = 0.24; P > 0.05). This suggests that development was suppressed at a certain stage at 27°C as previously reported (Tanaka, 1984) and a
Embryonic diapause in Allonemobius
b
637
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loo-
0 0 c
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Fig. 2. The time for 50% hatch in eggs of Allonemobius fbsciarus kept at 21°C for various periods before transfer to 20°C. Each point indicates the mean of 3 replicates, each consisting of 25 eggs laid in August. Vertical lines indicate SD.
long exposure to this temperature did not influence subsequent development at 20°C. Figure 3 shows the results of transfer from 20 to 27°C as obtained in the experiment described in Fig. 1. When eggs laid in August were transferred to 27°C within 12 days after laying, they required a much longer time to hatch than did those transferred later. The former probably entered summer diapause in response to high temperature. When transferred on day 16 to 20, the time required for 50% hatch was reduced by about 50 days. It was reduced further until the 50th day, and then increased. These results indicate that the duration of the egg stage was increased by early shifts to 27°C and decreased by later shifts. The duration of the egg stage was increased by a May exposure to high temperature (Fig. 4). The sensitivity to this exposure increased almost linearly as the eggs developed, and reached its maximum on
Days
10
20
kept
30 at
40
60
2D’C
60
before
70
150
transfer
to 27’C
Fig. 3. The time for 50% hatch in eggs of Aflonemobius f0sciaru.s kent at 20°C for various neriods before transfer to 27°C. E&h point indicates the-mean of 3 replicates, each consisting of 25 eggs laid in August. Vertical lines
indicate SD.
50 exposure
60
a? 27’C
Fig. 4. Effects of 5-day exposure to 27°C on the time required for 50% hatch in eggs of Ailonemobius fasciatus otherwise kept at 20°C. Eggs kept at 20°C for various periods were exposed to 27°C for 5 days and returned to 20°C. Each point indicates the mean of 3 replicates, each consisting of 25 eggs laid in August. Vertical lines indicate SD.
day 20. This effect was less pronounced but still detected after day 25. Because summer diapause is neither induced nor maintained at 20°C (Tanaka, 1984), a short exposure to 27°C is likely to have intensified the winter diapause. When exposure to 27°C occurred after day 30, the time for 50% hatch was shorter than when eggs were maintained at 20°C throughout the egg stage. When exposure to 27°C was delayed until day 60, more than 50% of eggs hatched soon after return to 2o”C, suggesting that the high temperature stimulated termination of winter diapause. To examine the effects of low temperature on termination of winter diapause, eggs laid in September were incubated at 20°C for various periods, exposed to a second temperature of 16, 11, 6, or 0°C for 45 or 90 days, and returned to 20°C for hatching. Upon transfer to the last temperature, a group of eggs hatched within 40 days which could be distinguished from the remaining eggs which hatched later in a sporadic fashion. The former were considered as early hatching eggs and the proportion of such eggs determined. When 25-day old eggs were exposed to different second temperatures for 90 days, the proportion of early eggs hatching increased in proportion to the temperature increase (Fig. 5). A 90 day exposure of 40-, 50-, or 75-day old eggs to different second temperatures caused most eggs to hatch soon after transfer to 20°C. If, however, eggs were exposed to the second temperature for only 45 days, the proportion of early hatching eggs was highest at a second temperature of 6°C and lower at other temperatures. Development
0
to 5 day
in the field
Diapause development proceeded relatively rapidly in autumn. When eggs were kept at room temperature for a month and then directly incubated at 20°C on October 15, none hatched within 40 days while 53.6% hatched if they were kept in the field for 31 days before incubation at 20°C (Fig. 6). During the winter, development was slow but hatching occurred more synchronously when transferred from the field to 20°C on February 16 than on December
SEIJITANAKA
638
VIII
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Second trmperoturs PC 1 Fig. 5. Effects of low temperatures on the proportion of early hatching eggs of Allonemobiusjizrciat. Eggs were first incubated at 20°C for 25, 40, 50, or 75 days, exposed to different second temperatures for 45 (0) or 90 (0) days, and returned to 20°C for hatching. Each point is based on 90 eggs. Numbers on the right indicate the times of transfer to second temperatures. Percentages between different set-
ond temperatures followed by different letter significant at 5% (X2-test). 16. Eggs kept in the field until March 17 took longer to hatch upon transfer to 20°C than those kept at 3°C until March 17 and transferred to 20°C (t = 10.8;
,
.
15 zz after laying
Fig. 7. Effects of moisture on embryonic development at 30°C in eggs of Allonemobius fasciatus laid in September. Eggs were placed in dry soil with 10% moisture content (0) or on moist paper (0) for various periods and dissected to determine the embryonic stage. For the embryonic stages, see Tanaka (1984). Each point indicates one individual.
P < 0.05), although all eggs hatched within 40 days at 20°C. This suggests that eggs which hatched within 40 days at 20°C contained not only those free from diapause but also those still in diapause at the time of transfer to 20°C. The time required for hatching was further reduced when the time of transfer was delayed, but some eggs in April were still in diapause. Effects of moisture on diapause regulation
Eggs laid in September and placed on moist paper at 30°C developed rapidly and a half of the embryos were beyond stage VII by day 7 (Fig. 7). Embryos beyond this.- stage _ are._^no longer __ capable of entering . 7. HATCHING dtapausc (l‘anaka, lYg4). However, some eggs remained at stage II or III and they probably entered summer diapause (see Tanaka, 1984). Embryos which appeared to enter diapause at these stages were also found among eggs deposited in the dry soil with 10% moisture content. Some embryos reached stage IV or V by day 10 but further development did not take place. Instead, their body and appendages became smaller. It was not possible to distinguish precisely those which remained at stage II or III from those which probably developed beyond stage III and became smaller after prolonged incubation in the dry soil. All eggs became slightly smaller than the initial size but few were completely collapsed even after 30 days of dry incubation. When eggs laid in September were kept in soil with 10% moisture content for different periods and then moistened at 30°C some began to hatch in 2 weeks, Days to hatch ot 2O*C and hatching continued for several days. Eggs which Fig. 6. Effects of outdoor temperature on egg hatching ot hatched during this early period were separated from Allonemobiw fasciatus. Eggs laid in September were kept at the others by probit analysis of the hatching curves room temperature for a month before they were transferred (indicated by the d&continuities of the lines)[Fig. 81. to the field on October 15, 1980. They were taken back to When maintained in the soil for 2 days, the proporthe laboratory (20°C) for hatching on each of 8 occasions tion of eggs in the first hatching group was slightly from November 15 to May 26. For comparison, two groups decreased and it became significantly lower when of eggs were incubated at 20°C on October 15 without being kept in the soil for 4 (x2 = 6.52; P < 0.05) or 6 days transferred to the field and on March 17 after a 5-month (x2 = 5.11; P < 0.05) than when incubated under exposure to 3°C. Numbers on the right indicate the propormoist conditions throughout. This may indicate that tions of eggs hatching within 40 days at 20°C. Arrows indicate the times for 50% hatch. the induction of summer diapause is more common
Embryonic diapause in Allonemobiu
Fig. 8. Effects of moisture on cumulative percentage of hatch in eggs of Alfonepobiw fasciotus at 30°C. Eggs laid in Sptember were first incubated in dry soil with 10% moisture content and transferred to moist conditions. Numbers in the figure indicate the times of transfer. By a probit
analysis, the first hatching group was distinguished from the rest hatching later, as indicated by the discontinuities of the lines. N = 90 for each treatment. under dry conditions than under wet conditions.
Eggs developing without diapause appeared to enter a state of quiescence when water deficiency was encountered (Fig. 7). This would explain why their hatching started later as the time of transfer to moist conditions was delayed (Fig. 8). Embryonic development stopped after 4 days under dry conditions as indicated by the observation that the first hatching group kept under dry conditions for 4, 6 or 8 days hatched 10 days after transfer to moist conditions. Those transferred to moist conditions on day 0 or 2 took longer (12-14 days) for hatching. When the period of exposure to dry conditions was prolonged 15 days or more, the proportion of eggs in the first hatching group gradually increased (Fig. 8). In those transferred to moist conditions on day 20, it was 84.4% which was significantly higher than the percent hatching attained in the control during the same period of incubation at 30°C (x2 = 8.56; P < 0.05). This, together with the fact that the embryonic stages of summer diapause can be reached in dry soil, may suggest that the termination rather than prevention of summer diapause occurred under dry conditions or upon transfer to moist conditions. When exposed to dry conditions for 30 days, all surviving eggs except one appeared in the first hatching group. The time spent by the first hatching group after transfer to moist conditions gradually increased from 10 to 12 days as the time of transfer was delayed from day 8 to day 30. This was because (1) eggs exposed longer to dry conditions probably took longer to gain sufficient water to resume embryogenesis and (2) those which had been in summer diapause at early stages joined the first hatching group. Mortality at the end of the observations was 2.2% or less except for eggs exposed to dry conditions for 30 days (7.8%). DISCUSSION
The present data suggest physiological processes responsible for the determination of diapause charac-
639
teristics in the eggs of A. farciutus. These processes are highly sensitive to temperature and moisture and their sensitivity greatly changes during the course of embryonic development and diapause. If eggs were transferred from 20 to 27°C before the stage of winter diapause was reached, many hatched without diapause, although few or none did so when constantly kept at 27 or 20°C (Fig. 1). A similar response has been reported in other orthopterans (Browning, 1952; Rakshpal, 1962; Dean and Hartley, 1977; Deura and Hartley, 1982; Ingrisch, 1984) including the Edmonton strain of A. farciatus (Sarai, 1967). Browning (1952) attributed this phenomenon in T. commodus to the completion of diapause development during the low temperature in the prediapause stage. Hogan (196Oa,b), working with the same species, found that an exposure to low temperature during the pre-diapause period itself did not have a diapause-eliminating effect, although the tendency to enter diapause at such a low temperature is so weakened that it can be easily averted upon transfer to high temperature. In the Oregon strain of A. fasciatus, the observed pattern of response to temperature shifts may be explained in terms of the interacting effects of low and high temperatures on the two types of diapause. As already reported (Tanaka, 1984), summer diapause is induced at early embryonic stages (II-IV) at a high temperature (e.g. 27°C) while no development suppression occurs at a lower temperature (e.g. 20°C) until the end of anatrepsis (stage VII) at which winter diapause occurs. Thus, the later the time of transfer from 20 to 27°C the greater would be the proportion of eggs that fail to enter summer diapause and hatch upon transfer to 27°C (Fig. IA). However, as embryos become closer to stage VII at 20°C more eggs would enter winter diapause, and the proportion of diapause-free eggs decreases as observed when the time of transfer to 27°C was delayed until day 16 or 20. None of the eggs laid in August responded to the same temperature shifts by hatching rapidly until day 40. This absence of response to early temperature shifts could be related to their high potential to enter both types of diapause at high temperatures as well as the high intensity of diapause compared with eggs laid in autumn (Tanaka, 1984). Sarai (1967) examined the effects of incubation temperature on diapause in the Edmonton strain of A. fasciatus. A direct comparison with the present results is not appropriate because this and the Oregon strains differ from each other in the rate of embryonic development and possibly in the stage of diapause (Tanaka, 1986b). A high temperature intensified diapause in A. fasciatus. When eggs laid in August were kept at 20°C and exposed to 27°C for 5 days, the duration of the egg stage was markedly increased by the exposure to 27°C occurring shortly before or after the induction of winter diapause (Fig. 4). A quite similar result has been reported in T. emma (Masaki, 1962) in which the diapause-intensifying action of high temperature also extends to the beginning of the diapause stage, although these two species enter diapause at different embryonic stages. When transferred from 20 to 27°C but not returned
640
SEUI TANAKA
to 2o”C, eggs of A. fascia&s appeared to enter summer diapause if the transfer occurred early enough (Fig. 3). Among eggs transferred later, the longer the duration of incubation at 20°C the shorter the time to hatch upon transfer to 27°C. This may suggest that the induced winter diapause was less intense at 20°C and/or that diapause development proceeded more rapidly at 20°C than at 27°C. Lees (1955) proposed that the thermal optimum for diapause development is not constant but varies in the course of diapause. This view has been supported by many examples which were recently summarized by Tauber ef al. (1986). In A. fusciutus, the thermal optimum for winter diapause development is relatively high (16-l 1°C) at the beginning and then decreases to 6°C (Fig. 5). Until diapause development reaches a certain stage, higher temperatures may be less effective because a transfer from 20 to 27°C terminated diapause more rapidly when it occurred on day 50 than on day 30 (Fig. 3). However, the duration of the egg stage became longer again as the transfer from 20 to 27°C occurred later than day 50, suggesting that the final phase of diapause development proceeds more rapidly at a higher temperature, as demonstrated for T. commodus (Masaki ef al., 1979). In the field diapause development appears to proceed relatively rapidly in autumn, but slowly during winter and some individuals are still in diapause in March and April (Fig. 6). When the thermal influence on diapause development is studied, diapausing insects are often exposed to different low temperatures and then transferred to a high temperature for postdiapause development (Andrewartha, 1952). However, information obtained from such studies may be of limited value, because (1) the intensity of diapause could be influenced by the temperature before or at the time of onset of diapause, (2) the thermal optimum for diapause development could change during the course of diapause, as observed in A. fasciatus and other species (Muroga, 1951; Way, 1959; Ando, 1972, 1978, 1983; Deura and Hartley, 1982; Sim and Shapiro, 1983; Tanaka, 1983; Hunter and Gregg, 1984), and (3) there may be interacting effects of the ‘chilling’ and ‘incubation’ temperatures as demonstrated in this paper. Moisture also influenced embryonic diapause and development in A. fusciatus. For the completion of embryonic development, water absorption is necessary in some species while it is not in others (Browning, 1967; Hinton, 1980). Among the former, eggs which enter diapause absorb water either before or after they enter diapause, although in some species noticeable amounts of water are absorbed during the period of diapause (Lees, 1955; Moriarty, 1969; Ando, 1972; Wardhaugh, 1980; Tanaka, 1986b). In the locust, C. terminifera, eggs kept in dry soil at 32S”C can not absorb water and become quiescent before they reach the embryonic stage at which diapause occurs (Wardhaugh, 1980). If such eggs are allowed to absorb water, more eggs fail to enter diapause than do those constantly kept under moist conditions at the same temperature. A similar phenomenon was observed in A. fasciutus. Unlike in C. terminifera, however, embryos of A. fasciatus can reach the stages at which summer diapause occurs
without absorbing water (Fig. 7). Thus, as observed in the luceme flea, Sminthurus viridis (Wallace, 1968) a long exposure to dry conditions probably influenced some physiological process after summer diapause was imposed, although some eggs apparently entered quiescence rather than diapause. On the other hand, when eggs were exposed to dry conditions only for the first 4 or 6 days, the incidence of summer diapause at 30°C was significantly increased. Therefore, these results indicate different effects of dry conditions on the induction and maintenance of summer diapause. Separation of a direct effect of moisture on the termination of diapause from that on postdiapause development is sometimes very difficult (Beck, 1980; Tauber et al., 1986). In A. farciutus, eggs at a high temperature absorb water even when embryogenesis is suppressed (Tanaka, 1986b). Thus, termination of summer diapause is independent of water absorption in this species. Acknowledgement-I
thank Drs Victor J. Brookes (Oregon State Univ.), Sinzo Masaki (Hirosaki Univ.), David L. Denlinger (Ohio State Univ.), and anonymous referees for correcting my English and providing suggestions on the manuscript and Dr T. D. Schowalter (Oregon State Univ.) for a financial support and encouragement. Many thanks are also due to Dr M. T. AliNiazee (Oregon State Univ.) who kindly let me use his environmental chambers. I acknowledged use of the library and computer facilities of Barre Colorado Island, Panama, where the manuscript was prepared.
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