Ent. exp. & appl. 28(1980) 204--212. Ned. Entomol. Ver. Amsterdam

T E R M I N A T I O N O F T H E F A C U L T A T I V E D I A P A U S E IN T H E C O D L I N G M O T H , LASPEYRESIA POMONELLA

(LEPIDOPTERA, T O R T R I C I D A E )

13n

ROBERT SIEBER; and GEORG BENZ

Department of Entomology, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092, Zfirich, Switzerland

The abiotic factors regulating the termination of the facultative diapause of the mature larva of the codling moth, Laspeyresia pomonella (L.), are described. The termination of diapause under long-day conditions (LD) is influenced by: (i) the rearing temperature of the larvae during prediapause development, (ii) the duration of the preincubation period, i.e, the time for which the diapausing larvae remain under prediapause rearing conditions, (iii) the reactivating incubation, i.e. the period for which the larvae are chilled, and (iv) the complementary incubation (LD and, except in one experiment, 26~ i.e. the period after the chilling needed for the pupation of the insects. The complementary incubation is distinctly shorter if the prediapause development of the larvae takes place at rearing temperatures below 26 ~ , i.e. 21 ~ or 19 ~ . The latter conditions led to 100% pupae and pupation could be accelerated by prolonging the preincubation period. In larvae reared at 26 ~ the prolongation of the preincubation period raised the rate of pupation to a maximum of only 56% and also caused higher mortality. On the other hand, diapause was terminated under short-day conditions if the temperature was raised to 26 ~ after a rearing temperature of 190 and a preincubation period of 90 days.

Diapause of the mature larvae of the codling moth is either obligate, i.e. independent of ambient factors, or facultative, i.e. primarily induced by a short- day photoperiod (SD) (Dickson, 1949; Russ, 1966; Wildbolz & Riggenbach, 1969). The length of the critical illumination period for the induction of the facultative diapause varies between populations from different geographical latitudes. Besides the photoperiod, the maturity of the host fruit may also influence diapause induction (Ivancich-Gambaro, 1960; Saringer, 1977). On the other hand, diapause in the codling moth may be terminated by the reversal of the photoperiod to long day (LD) alone, or in combination with a reactivating incubation at low temperatures (Wildbolz & Riggenbach, 1969). According to the definitions of Mfiller (1970), this type of dormancy belongs to the oligopauses and specifically may be classified as "diapausing oligopause". It differs from the true "eudiapause" in so far as the latter may be broken by a reactivating period at low temperature only.

If the termination of diapause is achieved by simply applying LD after a

i Present address: I.C.I.P.E., P.O. Box 30772, Nairobi, Kenya

TERMINATION DIAPAUSE IN CODLING MOTH 205

reactivation period, good synchronisation of diapause is rarely obtained, the insects pupating any time over a long period. Since we were interested in the elucidation of the hormonal control of diapause in the codling moth (Sieber & Benz, 1980), for which the synchronisation of the different stages of diapause was a prerequisite, we first analyzed the rearing conditions which lead to the best synchronisation.

Presented here is a detailed investigation of those factors which influence the termination of diapause. Most experiments were first performed with a laboratory strain and then verified with a freshly collected field strain.

M A T E R I A L A N D M E T H O D S

A laboratory strain of the codling moth with the genetic constitution for 100% facultative diapause, which has been reared in our laboratory already for several years at 26 ~ , was used for most experiments. The development of this strain is controlled by the photoperiod. Under LD (= constant light in our experiments) the insects develop without interruption from egg to adult, whereas SD (= 10 hr light: 14 hr dark in our experiments) during the first four larval instars induces facultative diapause in the mature larva. Under both light regimes, mature larvae spin a cocoon. However, whereas LD-reared larvae pupate within a few days after spinning, SD-reared larvae enter diapause.

A field strain with facultative diapause was reared from larvae collected in an orchard in the eastern part of Switzerland. They were trapped in strips of corrugated cardboard which had been wound around the trunks of apple trees. The insects were brought into the laboratory and reared under LD at 26 ~ and 70% R.H. The larvae pupating under these conditions within 40 days were considered as facultatively diapausing insects and were used as the parent generation of the field strain.

Both strains were reared on a semisynthetic medium after the method of Huber et al. (1972) with individual rearing of the larvae. The rearing temperatures were kept constant at 19 ~ 21 ~ or 26 ~ with 70% R.H. The light regime depended on the phase of development (see below). For each experiment 80--100 larvae were used.

Manipulation of diapause development Since diapause had to be induced in all experimental insects, the larvae were

always reared under SD up to a more or less extended, but defined time after they had spun their cocoons and entered diapause. In this paper the time for the development of the neonate to the diapausing larvae is called the prediapause period.

In this paper the time during which the diapausing larvae remained under the rearing conditions of the prediapause period is called the preincubation period.

The facultative diapause of the codling moth may be broken by a period of chilling under SD (the reactivating incubation), followed by a complementary incubation at a relatively high temperature (26 ~ in the present experiments) and

206 ROBERT SIEBER AND GEORG BENZ

LD. In this case the reactivation period (4 ~ for 70 days in these experiments) and the complementary incubation are clearly defined. However, the diapause of the codling moth may also be broken by simply reversing the photoperiod to LD and by raising the temperature from 19 ~ to 26 ~ (see Results). Since in these cases the reactivation period cannot be distinguished from the period of complementary incubation, the latter expression is used for the whole period following the preincubation period.

RESULTS

Diapause termination after rearing the larvae at different temperatures Fig. i shows that the termination of diapause under LD and 26 ~ is influenced by

the rearing temperature of the larvae during prediapause development but also by the length of the preincubation period. After a preincubation period of 10 days, the first adults emerged after a complementary incubation of 20--30 days if the larvae had been reared at 19 ~ and 30--40 or 70--80 days if the larvae had been reared at 21 ~ or 26 ~ respectively. A prolongation of the preincubation period from 10 to 30 days increased adult emergence during the 100 days of observation at all temperatures, biat accelerated diapause termination only in larvae which had been reared at 26 ~ Consequently, adult emergence was best synchronized after a prediapause development at 19 ~ followed by a preincubation period of 30 days.

At the end of the observation period, almost all larvae that had not pupated were alive, which indicates that they were still in diapause.

Adult emergence in the field strain was slightly lower than in the laboratory strain, but the differences were statistically not significant.

100

90

80

70

8o

i,o 30

20

/ ~ o o o o o

/ / "

days at 26 ~ LD Fig. 1. Diapause termination (ordinate) of larvae, reared under SD and 19 ~ (circles), 21 ~ (triangles) or 26 ~ (squares), after preincubation periods of 10 (open signs) or 30 days (filled signs), at different times

of complementary incubation at 26 ~ and LD (abscissa). Each curve based on 80 larvae.

T E R M I N A T I O N D I A P A U S E IN C O D L I N G M O T H 207

Diapause termination of larvae reared at 19 o, after different preincubation periods The previous experiments gave the best results with a rearing temperature of

19 ~ and the longer of the two preincubation periods. In order to find out whether or not a further prolongation of the preincubation period would improve the results, larvae reared at 19 ~ were kept under prediapause conditions for 30, 60 or 90 days. Subsequently the insects were transferred to LD and 26 ~ The time of adult emergence was checked daily.

Fig. 2 shows that the longer the preincubation lasted, the earlier and better synchronized was the diapause terminated. After a preincubation period of 90 days, all adults emerged between 16 and 36 days.

2~ 15

15 i2~ 10'

~ s eo

20

B

?.5 30 35 40 45 50 55

15 20 25 30 35 40 45 50

15 20 25 30 35 4 0

days a t 2 6 ~ L D

Fig. 2. Diapause termination of larvae, reared under SD at 19% after preincubation periods of 30 (A), 60 (B), and 90 days (C); 80 larvae for each experiment.

Diapause termination of larvae reared at 26 ~ after different preincubation periods To show whether the termination of diapause could also be accelerated and

synchronized in larvae reared at 26 ~ and SD, diapausing larvae were preincubated under the same conditions for 20--130 days (Fig. 3). Subsequently they were

208 ROBERT $1EBER AND GEORG BENZ

60 '

40"

20"

(15)

2'o s'o 7'0 ~o 1~o 15o

preincubation period in days

Fig. 3. Diapause termination of larvae, reared under SD at 26 ~ after different preincubation periods (abscissa) and complementary incubation at 26 ~ and LD for 70 days (�9 and 140 days (t) . Values in

brackets = mortality in %. Hundred larvae per point .

transferred to LD at the same temperature. The adults were counted 70 days later and after another 70 days the adults, the diapausing larvae and the dead larvae or pupae were counted. Fig. 3 shows that diapause termination within 1413 days increased from 7% after a preincubation period of 20 days to 56% after a preincubation period of 110 days. The shape of the curve is not regular and may be interpreted as a plateau for the preincubation periods of 50--90 days and a maximum at i 10 days, as suggested by the curve with a complementary incubation of 70 days. In either case, however, the number of adults decreased after a preincubation period of 130 days because of a high mortality rate of 45%.

Influence of chilling The rate of the adult emergence of insects reared at 26 ~ could be further

100"

80" O ~ �9

~ 40"

20" /

2'0 7b 1�89 preincubation period in days

Fig. 4. Diapause termination of larvae, reared under SD at 26 ~ after different preincubation periods (abscissa), a reactivation period of 70 days at 4 ~ and complementary incubation at 26 ~ and LD. Each

preincubation period tested with 84 larvae.

TERMINATION DIAPAUSE IN CODLING MOTH 209

increased, if the preincubation period was followed by a reactivating period with chilling for 70 days. The subsequent evaluation during a complementary incubation of another 70 days under LD showed that almost all larvae (98.8%) had terminated diapause if the preincubation period was 120 days, but only 71.2% after a preincubation period of 70 days (Fig. 4). Emphasis is placed on the fact that in this experiment almost all animals survived after the long preincubation period of 120 days. No difference between the field and the laboratory strain was found.

Diapause termination by changes in temperature or photoperiod To investigate whether the termination of diapause is induced either by the

change of the photoperiod to LD alone or by a rise in temperature, larvae which had been reared at 19 ~ and SD, were kept under the same rearing conditions for another 90 days and then subdivided into two groups. One group remained at 19 ~ but the photoperiod was prolonged to LD and one group remained under SD, but the temperature was raised to 26 ~ As shown in Fig. 5, the termination of diapause could be induced both by raising the temperature alone (Fig. 5A) and by changing the photoperiod to LD alone (Fig. 5B).

If diapause was terminated by increasing only the temperature, adult emergence was more or less the same as in the experiment where diapause was terminated by a combination of LD and 26 ~ (Fig. 2). However, at the end of the observation period, i.e. after 100 days at 26 ~ 6.2% of the larvae were still in diapause.

Due to the low temperature of 19 ~ the first adults had a delayed emergence if diapause was terminated by LD only (Fig. 5B). Nevertheless, adult emergence was well synchronized and all larvae terminated diapause.

15 20 25 30 35 40 45

d a y s at 2 6 ~

15 20 25 30 35 40 45 50 55

d a y s at 19 ~ LO

Fig. 5. Diapause termination by either a rise in temperature from 19 ~ to 26 ~ (A) or by changing the photoperiod from SD to LD (B). Rearing conditions = 19 ~ and SD; preincubation period = 90 days;

complementary incubation at 26 ~ and SD (A) or 19 ~ and LD (B); each experiment with 80 larvae.

210 ROBERT SIEBER AND GEORG BENZ

D I S C U S S I O N

Insect diapause is terminated in most cases either by an exposure to low temperature for a minimum period or by a change in the photoperiod (Lees, 1955; Danilevskii, 1965; Beck, 1968; MiJller, 1970). According to the classification of the last-mentioned author the first way of breaking diapause is typical for the eudiapause, whereas the second, i.e. termination of dormancy by the reversal of the dormancy-inducing factor(s) is found in oligopauses. Russ (1966), and Wildbolz & Riggenbach (1969) have shown that the diapause of L. pomonella is induced by SD and can be terminated by a period of chilling in combination with a complementary incubation at higher temperatures and LD, or LD alone, indicating that the dormancy of the codling moth belongs to the oligopauses.

Our results show that only few larvae reared at 26 ~ terminate diapause during a complementary incubation of 100 days at LD and the same temperature beginning 10 days after the onset of diapause (i.e. a preincubation period of 10 days). On the other hand, more than 80% of larvae reared at 19 ~ terminated diapause if the change in the photoperiod was combined with a rise in temperature to 26 ~ A prolongation of the preincubation period at 26 ~ not only accelerated diapause termination under LD and increased the percentage of pupation but also increased the percentage of larval mortality under LD. However, after a preincubation period of 110 days the pupation rate after 140 days under LD was not higher than 56%.

The duration of the preincubation period was much more effective, if the diapausing larvae were additionally chilled for 70 days. Although this reactivation period resulted in only 12.8% diapause termination after a preincubation period of 2 days, the percentage of diapause termination increased continuously to 98.8% after a preincubation period of 120 days.

Besides the duration of the preincubation period and a reactivation period, the temperature at which the larvae were reared was found to be very important for diapause termination. After rearing temperatures of 19 ~ and 21 ~ larvae terminated diapause at 26 ~ and LD earlier and with less scattering, i.e. within a distinctly shorter period, than after a rearing temperature of 26 ~ Here again the complementary incubation period necessary for the diapause termination was shortened by prolonging the preincubation period.

Moreover, all larvae terminated diapause if, after a rearing temperature of 19 ~ and a preincubation period of 90 days, the same temperature was maintained under LD, the complementary incubation period varying from 29 to 57 days. This result is in contrast to the corresponding experiment at 26 ~ where only 34% of the larvae terminated diapause within 140 days under LD. These findings could indicate that diapause induced at 19 ~ is not of the same intensity as diapause induced at 26 ~ A temperature-dependent diapause induction was also found in Diatraea grandiosella (Chippendale & Reddy, 1973). Unlike the codling moth, all larvae reared at the low temperature of 23 ~ entered diapause, whereas 90% of the larvae reared at the temperature of 27 ~ continued development.

TERMINATION DIAPAUSE IN CODLING MOTH 211

Control larvae never pupated under SD if they remained at the rearing temperature. In contrast to this, larvae terminated diapause at 26 ~ under SD after a rearing temperature of 19 ~ This indicates that diapause in the codling moth can be terminated not only by the photoperiod but also by a rise in temperature, as also reported for Plodia interpunctella (B ell, 1976).

The fact that it was not possible to find a significant difference in diapause termination between a field strain and our laboratory strain indicates that diapause behaviour was not changed by rearing under laboratory conditions for several years.

Our results show that the time of diapause termination in a codling moth larva is not only influenced by the photoperiod and chilling, but also by other factors influencing the larvae during growth and diapause. This seems to confirm our hypothesis that the intensity of the diapause of codling moth larvae may vary according to the conditions of diapause induction. These findings allow the programming of the pupation time of diapausing larvae by using special rearing conditions.

KEY WORDS: Laspeyresia pomonella (L.), Tortricidae, diapause, photoperiod, temperature.

ZUSAMMENFASSUNG

BEENDIGUNG DER FA KUL TA TI VEN DIA PA USE DES A PFEL WICKLERS, LASPEYRESIA POMONELLA (LEPIDOPTERA, TORTRICIDAE)

Es wurde untersucht, welche abiotischen Faktoren die Beendigung der fakultativen Diapause ausgewachsener Apfelwicklerlarven (Laspeyresia pomonella) steuern. Die Beendigung der Diapause unter Langtagbedingungen (LT) wird beeinflusst durch: 1. die Zuchttemperatur der Larven w~hrend der Prfidiapauseentwicklung; 2. die Dauer der Pr~iinkubation, d.h. die Zeit, wS.hrend der diapausierende Larven unter Pr~idiapause-Zuchtbedingungen bleiben; 3. die Reaktivierungs- inkubation, d.h. eine Periode von 70 d, wfihrend der die Larven auf 4 ~ gekfihlt werden und 4. die Komplement/irinkubation (LT und, ausser in einem Experiment, 26~ d.h. die nach der K/Jhlung bzw. dem Wechsel in der Photoperiode ben6tigte Zeitdauer bis zur Verpuppung. Die Komplement/irinkubation ist deutlich k/irzer, wenn die Pr~idiapauseentwicklung der Larven bei Temperaturen unter 26 ~ stattfindet. Bei 19 ~ verpuppten sich 100% der Larven, wobei der Zeitpunkt der Verpuppung durch die Verlfingerung der Pr/iinkubationszeit beschleunigt wurde. Bei Larven, die bei 26 ~ gezfichtet wurden, erreichte die Verpuppungsrate lediglich 56% bei einer relativ langen Pr/iinkubationsdauer, wobei deren Verlfingerung auch die Larvenmortalit~t erh6hte. Nach einer Zuchttemperatur von 19 ~ und einer Pr~.inkubationsdauer yon 90 Tagen konnte die Diapause unter Kurztagbedingungen beendet werden, wenn die Temperatur auf 26 ~ erh6ht wurde.

REFERENCES

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moth, Plodia interpunctella. Physiol. Ent. 1 : 93--101. CHIPPENDALE, G. M. & REDDV, A. S. (1973). Temperature and photoperiodic regulation of diapause of

the southwestern borer, Diatraea grandiosella. J. Insect Physiol. 19 : 1397--1408.

212 ROBERT SIEBER AND GEORG BENZ

DANILEVSKII, A. S. (1965). Photoperiodism and Seasonal Development of Insects. London: Oliver & Boyd, 282 pp.

DICKSON, R. C. (1949). Factors governing the induction of diapause in the oriental fruit moth. Ann. entomol. Soc. Am. 42 : 511--537.

HUBER, J., BENZ, G. & SCHMIO, K. (1972). Zuchtmethode und semisynthetische N~ihrmedien fiir Apfelwickler. Experientia 28: 1260--1261.

IVANCICH-GAMB^RO, P. (1960). L'alimentazione e it fotoperiodismo nella determinazione della diapausa di Carpocapsapomonella L. (Lepidopt., Tortr.). CI. Sci. Matem. Nat. 72 : 1--3.

M~LLER, H. J. (1970). Formen der Dormanz bei Insekten. Nova Acta Leop. 3S : 1--27. LEES, A. D. (1955). The Physiology of Diapause in Arthropods. London: Cambridge Univ. Press. 151 pp. Russ, K. (1966). Der Einfluss der Photoperiodizitiit auf die Biologie des Apfelwicklers (Carpocapsa

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Populationen, Laspeyresiapomonella L. (Lepid., Tortricidae). Z. ang. Ent. 83: 62--79. SIEBER, R. & BENZ, G. (1980). The hormonal regulation of the larval diapause in the codling moth,

Laspeyresia pomonella (Lep. Tortricidae). J. Insect Physiol. 26 : 213--218. WILDBOLZ, T. & RIGGENSACH, W. (1969). Untersuchungen fiber die Induktion und die Beendigung der

Diapause bei Apfelwicklern aus der Zentral- und Ostschweiz. Mitt. Schweiz. Ent. Ges. 42 : 58--78.

Accepted: May 20, 1980