egg diapause in ephippiger cruciger (orthoptera: tettigoniidae)

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Diapause may supervene at stage I-4, as demonstrated by fixing pricked eggs in. Pampels fluid and staining the primordium with 2% alum carmine (Dean, 1974) ...
'.exp. Biol. (1977), 66, 173-183 ith 9 figures Printed in Great Britain

EGG DIAPAUSE IN EPHIPPIGER CRUCIGER (ORTHOPTERA: TETTIGONIIDAE) I. THE INCIDENCE, VARIABLE DURATION AND ELIMINATION OF THE INITIAL DIAPAUSE BY R. L. DEAN* AND J. C. HARTLEY Department of Zoology, University of Nottingham, England (Received 2 August 1976)

SUMMARY

Eggs of the bush-cricket Ephippiger cruciger may be subject to two periods of diapause during the course of embryogenesis. After oviposition not all eggs develop at temperatures favourable to morphogenesis, some cease activity at the unsegmented embryonic primordium stage. If these eggs are cooled for 10-30 days some develop on subsequent exposure to higher temperatures, but further prolongation of the cooling period does not render more eggs competent to develop. Although cooling for longer than the critical period does not affect the proportion of eggs which subsequently start to develop, it does affect the time within which reactivated eggs begin to develop. The delay in the initiation of development after cooling increases with the duration of the chilling period. Excessively long cooling periods, however, impair the eggs' ability to complete embryogenesis. It is evident that eggs laid in one season will develop and hatch over a number of years.

INTRODUCTION

Individuals of many temperate species belonging to the family Tettigoniidae are unusual in that they may be subject to two periods of diapause during the course of embryogenesis (Hartley & Warne, 1972). Further, in the Ephippigerinae, the arrest of growth which occurs early in development may be very persistent even when eggs are maintained, for alternating periods, at low and high temperatures expected to eliminate the diapause state and favour subsequent embryonic activity. Although not previously reported for the egg-state, developmental arrests of the ' winter diapause' type, which may persist for several years, have been reported in the larval and pupal stages of a number of insect species. In these cases it has been thought probable that either the incubation temperatures applied failed to tally with the thermal requirements of the diapause stage, or that the diapause is so intense that it is eliminated extremely slowly even at suitable temperatures (Lees, 1955). Results gained from work on the laboratory cultures of Ephippiger cruciger suggest that, in this case, neither assumption is correct. • Present address: Department of Zoology, University of Western Ontario, London, Canada. 13 EXB 66

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Newly laid eggs were obtained from stocks of adults maintained in a greenhouse as described elsewhere (Hartley & Dean, 1974). The eggs, abstracted at monthly intervals, were cleaned and randomized by washing in water, then placed on filter paper on moist cotton wool in Petri dishes. The eggs were subjected to different incubation programmes consisting of various periods spent in a sequence of temperatures. The temperatures used were selected from those in six cooled incubators maintained at 4, 8, 12, 16, 20 and 25 °C ( ± i ° C ) and in an oven at 30 °C (±i°C). The state of development of eggs was determined by visual inspection at low magnification. When the water in the shell is allowed to evaporate, the shell can be rendered transparent by immersion in xylene (Hartley, 1971). This procedure does not harm the egg or embryo at any stage, even when immersed for several hours (Warne, 1972), neither does it terminate diapause as is apparently the case in Melanoplus eggs (Slifer, 1946). The stage of development attained by the embryos was identified by reference to published illustrations of the embryonic development of Pholidoptera griseoaptera, which is very similar to that of E. cruciger (Warne, 1972). The nomenclature of the stages of embryonic development follows the system proposed by Warne. The principal stages mentioned in the following work are I-4, where the blastoderm has condensed to form a roughly circular embryonic primordium; III-12, a stage in mesentrepses (between anatrepsis and catatrepsis) where the appendages become directed into the mid-line; and VT-23, where the completed embryo fills the shell but the posterior femurs have not attained their full length. Diapause may supervene at stage I-4, as demonstrated by fixing pricked eggs in Pampels fluid and staining the primordium with 2 % alum carmine (Dean, 1974), and at stage VI-23. All newly laid eggs develop to stage I-4 within the temperature range of 16-30 °C before any arrest of growth occurs. Only development beyond this stage is referred to in this account and the terms ' initial development' and ' initiation of development' refer to development beyond stage I-4. The first readily identifiable stage of development in living eggs is III-12 and, for convenience, it has been used to denote the beginning of development. The proportion of newly laid eggs which develop directly at suitable high temperatures is variable. Preliminary results showed that hardly any eggs began to develop beyond stage I-4 at 20 °C, whereas between 2-11 % started to develop within 3 months at 25 °C and 23-75 % started to develop within 3 months at 30 °C. In any egg batch the percentage of eggs which begin to develop is not greatly increased by extending the duration of the period at 25 or 30 °C. In fact, most eggs which ultimately develop begin to do so within the first 2 months. Ephippiger eggs which do not develop in the initial incubation period have been described as' diapausing' (Hartley & Warne, 1972; Hartley & Dean, 1974). Unlike the diapausing egg of Bombyx mori, which is readily identified by its pigment-laden serosa (Tirelli, 1941), those of E. cruciger give no visual indication of their diapause or non-diapause potential. In the absence of any obvious criterion for deciding which eggs would not develop without a period of cooling the ' standard diapausing eggs' chosen for experimental purposes were those which had failed to initiate development

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within 3 months at 30 °C. Such eggs had resisted initial development in conditions which strongly favoured it. (a) The influence of the cooling temperature on subsequent development at suitable temperatures in initially non-developing eggs To compare the effect of different low temperatures on the elimination of diapause, 15 replicates of 100 diapausing eggs were cooled for 90 days at each of 4, 8 and 12 °C. Five replicates from each low-temperature treatment were then incubated at 20, 25 and 30 °C. The replicate groups were inspected at 10-day intervals and the numbers of developing, non-developing and deteriorating eggs recorded. (b) The influence of the duration of the cooling period on the subsequent development of initially non-developing eggs Ten groups, each containing 10 replicates of 100 eggs, were cooled at 8 °C for 1, 5, 10, 20, 30, 60, 90, 120, 150 and 180 days. After cooling, five groups from each treatment were incubated at 20 °C and the remaining five groups at 25 °C for a further 200 days. Eggs were inspected at 10-day intervals as before. Since the eggs used were not all available at one time it was not possible to randomize them before treatment. Thus, results obtained from eggs cooled for longer than 30 days are not strictly comparable, but are used to illustrate a trend demonstrated more critically in section (c). (c) The influence of the duration of a second cooling period on the subsequent development of eggs which remained undeveloped after two periods at high temperature with an intervening cooling period Eggs used were the undeveloped, but apparently healthy, residue from a large stock batch subjected to the following incubation programme in a preliminary study: 60 days at 25 °C; 30 days at 30 °C; 150 days at 8 °C; 150 days at 25 °C. The remaining n o eggs were randomized and divided into replicates of 100. Two replicates were cooled at 8 °C for 30 days and the remaining eggs were cooled for 60, 90 and 120 days, three replicates to each treatment. After cooling the groups were incubated at 25 °C. Every 30 days the eggs were inspected and the stage of development attained by them recorded as before. Observation ceased when all eggs able to reach stage VI-23 had done so. RESULTS

(a) The cooling temperature As no development occurred in the egg groups cooled at 4, 8 or 12 °C even after 200 days of incubation at 20 °C, it was assumed that cooling did not lower the developmental threshold temperature. Figures 1 and 2 show that, although more eggs began to develop at 30 °C than at 25 °C, the effect of the cooling temperature was similar in both cases. Eggs cooled at 4 °C showed a lower percentage of development, although many of them started to develop sooner than eggs from other cooling treatments. The greatest number of eggs began to develop after exposure to 8 °C. Although the number of eggs starting to develop after cooling at 12 °C was similar to that of eggs cooled at 8 °C, relatively few reached stage VI-23, ^ shown in Fig. 3. Many of the eggs ceased development at

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o. o

•S 30

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Days of incubation at 25 °C Fig. t. Mean cumulative percentages of eggs reaching stage III-iz at 25 °C after 90 days at 4, 8, and 12 °C as indicated by prefix number. 8 * 30

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Days of incubation at 30 °C Fig. 2. Mean cumulative percentage of eggs reaching stage III-12 at 30 °C after 90 days at 4, 8, and 12 °C as indicated by prefix number.

intermediate stages of embryogenesis and then began to deteriorate. Cooling at 12 °C has some unsatisfactory side effects which were not found at the other temperatures. Therefore 8 °C was considered to be the best cooling temperature and was used in all the ensuing studies.

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3 30 8, a 20 DO

a 10

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Fig. 3. Mean cumulative percentage of eggs reaching stage VI-23 at as °C after 90 days at 4, 8, and 12 CC, as indicated by prefix number. SOr

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Fig. 4. Mean cumulative percentage of eggs reaching stage III-12 at as °C after periods of 1 to 30 days at 8 °C as indicated by the middle subscript numbers.

Although the highest proportion of initial development can be induced at 30 °C, it is too high for the continued growth of the embryo (Hartley & Dean, 1974). Therefore 25 °C, which was only proportionately less successful, was used as the standard incubation temperature. (b) The influence of the duration of the initial cooling period on subsequent development

Figure 4 shows that fewer eggs started to develop in groups cooled for 1 and 5 days than in those which spent 10, 20 or 30 days at 8 °C. A control, without cooling, was not included as it is known that a few' non-developing' eggs begin to develop spontaneously when maintained at 25 °C for prolonged periods (Dean, 1974). The 9 % of eggs which developed in the groups cooled for only 1 day may not, therefore, have been influenced by the low temperature. The mean percentage of eggs starting to develop after 5 days of cooling was, however, significantly higher than in those cooled for 1 day (P = < o-oi) and the difference between those cooled for 5 and 10 days was highly significant (P = < o-ooi). The eggs cooled for 10, 20 and 30 days showed remarkable homogeneity with

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8 150 25

v^-8^25 •^ -' • 8«o 25

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3 40 '5. I 30 a

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Days of incubation at 25 °C Fig. 5- Mean cumulative percentage of eggs reaching stage Ill-ia at 25 °C after periods fromj6o to 180 days at 8 °C as indicated by middle subscript numbers. 30

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JJ83O25

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Days of incubation at 25 Fig. 6. Mean cumulative percentage of eggs reaching stage VI-23 at 25 °C after periods from 1 to 30 days at 8 °C as indicated by middle subscript numbers.

respect to the mean percentage starting to develop within the 25 °C incubation period. Figure 5 shows considerable similarity in the mean percentages of eggs developing after cooling for 60, 90, 120 and 180 days. The eggs cooled for 150 days conformed less well to this general pattern in that a greater number began to develop more promptly than in the other groups. However, as previously pointed out, these groups are not strictly comparable as they were derived from different sources. In general there was a clear relationship between the proportions of eggs developing and the cooling period within the range 1 to 10 days at 8 °C, but further increments in the cooling period made little difference to the proportions of eggs starting to develop in 200 days at 25 °C. Figures 6 and 7 illustrate the mean percentage of eggs, in the same groups, which developed to stage VI-23. The trends shown in the proportions reaching stage VI-23 are similar to those illustrated with respect to the proportions of eggs starting to develop. The comparatively high rate at which eggs began to develop at 25 °C after 150 days of cooling was not reflected in the proportion completing this phase of embryogenesis. An unusually large number of eggs which had reached stage III-12 in

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40 (N

30 &

to OO

.2 20

S"? 10 a

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Fig. 7. Mean cumulative percentage of eggs reaching stage VI-23 a t 2 5 °C after periods from 60 to 180 days at 8 °C as indicated by middle subscript numbers.

this group subsequently showed signs of deterioration at intermediate stages. This phenomenon also characterized the eggs cooled for 180 days. (c) The influence of the duration of a second cooling period on subsequent development

Figure 8 illustrates the mean cumulative percentages (± 2 x S.E.) of eggs, which had not developed in two incubation cycles, reaching stage III-12 at 25 °C after 30, 60, 90 and 120 days of a second cooling period at 8 °C. The variation between replicates cooled for the same periods was very small. Figure 9 shows the mean cumulative percentages ( + 2XS.E.) of the same eggs which reached stage VI-23. Here again cooling beyond 30 days made no significant difference to the numbers that subsequently developed. Of the eggs cooled for 30 days, 65 % started to develop within 30 days at 25 °C. In the eggs cooled for 60, 90 and 120 days, the proportions starting to develop within 30 days at 25 °C decreased from 8-3% to 3-0%, to 0-03 % respectively. After only 2 months at 25 °C almost all eggs which ultimately started to develop in the groups previously cooled for 30 days did so, but within the same period only a little more than half of the eggs cooled for 60 days, which eventually developed, had shown signs of activity. The groups cooled for 90 and 120 days produced only 13% and 15% of developing eggs. After 30 and 60 days at 25 °C the differences between the mean percentages of developing eggs in the four cooling-treatment groups were all significant. Thus, an increasing proportion of eggs showed a delay, or latent period, before initiation of development after progessively longer periods of cooling. The latent period increased as the cooling time was lengthened over the period of 30 to 120 days. The difference between the latent periods of groups cooled for 90 to 120 days was less than that between groups cooled for 30 and 60 days, suggesting that further periods of cooling would not greatly extend the delay. After 120 days at 25 °C little further initiation of development occurred in any of the groups. The differences between total percentages of eggs eventually starting to develop in the four groups were not significant. This illustrates that extension of cooling from 30 and 120 days does not influence the numbers of initially non-developing eggs which subsequently start to develop. The duration of cooling does, however, affect the time at which eggs start to develop.

Fig. 8

Fig. 9

Fig. 8. Mean cumulative percentage of eggs which had failed to develop in two previous incubation cycles, reaching stage 111-12 after cooling for a second time at 8 "C for variable periods. 0 , after 30 days; M, 60 days; A , 90 days; 0, 120 days at 8 O C . Fig. 9. Mean cumulative percentage of eggs reaching stage VI-23 after a second cooling period as in Fig. 8.

Days of incubation at 25 "C

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The cumulative percentages of eggs reaching stage VT-23 in the four groups show the same trend, although, of course, more time is needed to develop to this stage (Fig. 9). The differences between the mean percentages reaching stage VI-23 in these groups are all significant up to the end of 120 days at 25 °C. The mean percentages which eventually reached this stage are not significantly different. Thus, extending the cooling period to 120 days did not affect the subsequent viability of the embryos. Neither did it appear to affect the rate at which the embryos developed between stages III-12 and VI-23 ^ t n e curves in Figs. 8 and 9 show very similar spatial relationships. The small discrepancies between the final total percentages of eggs reaching stage III-12 and that of those reaching stage VI-23 m ^ groups resulted from the few eggs which failed at intermediate stages of embryogenesis. At the end of the observation period there remained an average of about 13% of undeveloped but apparently healthy eggs.

DISCUSSION

Unlike the eggs of Bombyx mori, which are determined with respect to diapause or non-diapause development well in advance of oviposition, the incidence of initial diapause in E. cruciger eggs depends, at least in part, on the prevailing temperature at the time at which stage I-4 is reached. Other factors which control whether an egg will develop directly or diapause in this species are not known. If the criterion for successful completion of diapause development is the percentage of eggs in which embryogenesis starts on subsequent incubation then, of the temperatures used, 4 °C is less effective than 8 or 12 CC. However, it is essential that embryogenesis is completed, and after treatment at 12 °C there was a high failure rate. Therefore the optimum temperature for diapause development appears to be around 8 °C. This is in general agreement with many other insects from temperate climates, with moderately severe winters, which complete diapause development most rapidly within the general range of o to 12 °C (Lees, 1955). The early diapause in the egg of E. cruciger conforms well to this generalization with an optimum temperature close to 8 °C. Danilyevsky (1965) has pointed out that reactivation in unsuitable conditions impedes further development and causes decreased viability. The irregular development of eggs cooled at 12 °C probably indicates that this temperature lies near the upper end of the effective diapause development range. The lower temperature limit has not been determined but one reason for the high failure rate in another study of egg diapause in E. cruciger (Dumortier, 1967) may have resulted from the exclusive use of a cooling temperature of 2 °C. In this species the optimum temperature for diapause development lies well below the threshold necessary for active embryogenesis. The subsequent growth promoted by temperatures that are too low to permit the growth of nondiapause stages is among the most striking features of the diapause condition (Lees, 1955)Although prolonged cooling does not increase the proportion of eggs which develop subsequently, it does have an effect on the rate at which they start to develop. In contrast to eggs which develop in the first period at high temperature, there was a marked delay in the initiation of development after cooling. This increasing latent period was unexpected and seems to have no equal, indeed the usual result of increasing

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the cooling period is to increase the response (Lees, 1955). Danilyevsky (1965J pointed out that, in diapausing species in temperate climates, the whole wintering stock is potentially capable of resuming development by early spring, and that this begins uniformly as soon as the temperature rises to the necessary level. After cooling, the Ephippiger embryos do not resume development at suitable temperatures until after the lapse of a considerable period of time. In Chloridea obsoleta, the resumption of development in spring may be greatly delayed by a raised threshold temperature for the first stages of post-diapause development (Goryshin, 1958). This, however, is not the case in Ephippiger where a delay in the resumption of development occurs even at 30 °C (Fig. 2). Howe (1967) noted that after cooling the fully developed embryo of Leptohylemyia coarctata, a delay in hatching of many days could occur even though the post-diapause development was apparently entirely physiological. He suggested that such a delay might occur when diapause supervenes earlier in egg development. Evidence for this has been found in the egg of E. cruciger. It is not known whether the latent period has any adaptive significance or whether it is a non-adaptive consequence of some aspect of the physiology of the initial diapause. A second effect of prolonged cooling was similar to the effect of exposing eggs to the unsatisfactory cooling temperature of 12 °C. Although large numbers of eggs started to develop at 25 °C after 150 days of cooling at 8 °C (Fig. 5), many of them were unable to complete development as far as stage VI-23 (Fig. 7). The same failure was seen in many of the eggs after 180 days of cooling. Since 8 °C is below the threshold temperature for morphogenesis any reactivated eggs would remain quiescent until after the eggs are exposed to a higher temperature. The quiescent embryos evidently remain healthy after exposure to low temperatures for periods considerably in excess of that required for the completion of diapause development. Eventually, however, the viability of some of the embryos is reduced. The following summary suggests that the arrest of growth or initial diapause may persist, in natural circumstances, for several years. (i) Between 2 and 122% of eggs began to develop within 6 months during an initial incubation period at 25 °C (Dean, 1974). (ii) 23 % of eggs, which had failed to develop within 3 months at 25 °C in the initial incubation period, began to develop within 200 days at 25 °C after 20 days of cooling at 8 °C (Dean, 1974). (iii) 75-5 % of eggs, which had failed to develop within 150 days during a second incubation period at 25 °C, began to develop within 90 days at 25 °C after 30 days of cooling at 8 °C (Fig. 8). In this group no further development occurred, so that even after 180 days at 25 °C there remained 18 % of undeveloped but apparently healthy eggs, some of which it is assumed would develop in a fourth incubation period at 25 °C after a further period of cooling. This residue of undeveloped eggs had been laid over 600 days previously and would have been at stage I-4 for most of that period. There is an apparent tendency for an increasing proportion of the residual undeveloped eggs to develop as the number of warming and cooling cycles increases. Thus, the assumption that the persistent initial diapause in E. cruciger is so intense that it is eliminated extremely slowly, even at the thermal optimum, is not tenable. In fact diapause was eliminated rapidly in the eggs which were reactivated in the first and second cooling periods. Longer cooling periods did not significantly affect the number

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which developed. The results presented here explain why Ephippiger eggs laid in a single year and maintained in an outdoor terrarium hatched over a period of three years (Dumortier, 1967). It could be postulated that a group of eggs in initial diapause included sub-groups with sharply delineated degrees of diapause intensity. This seems improbable as the variation in diapause intensity within a population would be expected to be continuous, even if the limits were extreme. Alternatively, it appears that eggs which are not reactivated in short time at low temperature need to be' primed* by exposure to a higher temperature before they are able to complete diapause development on subsequent exposure to low temperature. Such a mechanism is not dependent on time, but rather on the number of high- or low-temperature events. A mechanism for ' counting the instars' has been invoked to explain the constancy of the number of larval instara which precede metamorphosis in Rhodnius. This function was assumed to reside in the nervous system (Wigglesworth, 1948). Ephippiger eggs experience the initial diapause prior to the differentiation of the embryonic nervous system. We wish to thank Professor B. C. Clarke and Dr D. T. Parkin for their advice on statistical problems. The work was supported by a postgraduate studentship from the Science Research Council of Great Britain. REFERENCES DANILYBVSKY, A. S. (1965). Photoperiodism and Seasonal Development of Insects (English edition). Edinburgh and London: Oliver and Boyd. DEAN, R. L. (1974). Embryonic diapause and development in Ephippiger cruciger (Fieber). Thesis submitted to the University of Nottingham for the degree of Doctor of Philosphy. DUMORTIER, B. (1967). Essais in vitro pour la rupture de la diapause embryonnaire chez quelques Tettigonioides (Insectes: Orthopteres). Annls Epiphyt. 18, 367^400. GORYBHIN, H. I. (1958). The ecological analysis of the seasonal cycle of development of the Cotton-boll worm (Chloridea obsoleta F.) in the northern areas of its range. Sclent. Mem. of Leningrad State University 240, 3-20. HARTLEY, J. C. (1971). The respiratory system of the egg-shell of Homorocoryphus nitidulus vidnus (Orthoptera; Tettigoniidae). J. exp. Biol. 55, 165-76. HARTLEY, J. C. & DEAN, R. L. (1974). Ephippiger cruciger as a laboratory insect (Orthoptera; Tettigoniidae). J. nat. Hist. 8, 349-54. HARTLEY, J. C. &WARNE, A. 0.(1972). The developmental biology of the egg stage of Western European Tettigoniidae (Orthoptera). J. Z00L, Lond. 168, 267-98. HOWE, R. W. (1967). Temperature effects on embryonic development in insects. A. Rev. Ent. la, 15-42. LEES, A. D. (1955). The Physiology of Diapause in Arthropods. London: Cambridge University Press. SLIFER, E. H. (1946). The effects of xylol and other solvents on diapause in the grasshopper egg; together with a possible explanation for the action of these agents. J. exp. Zool. 103, 333-56. TIRELLI, M. (1941). The karyoplasmic ratio in the embryos of monovoltine and bivoltine races of silkworm Bombyx mori L. Physiol. Zool. 14, 70-7. WARNE, A. C. (1972). Embryonic development and the systematics of the Tettigoniidae (Orthoptera: Saltatoria). Int. J. Insect Morphol. and Embryol. I, 267-87. WIGGLESWORTH, V. B. (1948). The functions of the corpus allatum in Rhodnius prolixus (Hemiptera). J. exp. Biol. 25, 1-14.