The influence of photoperiod and temperature on the induction of diapause in pupae of Heliothis punctigera

1. Insect Physiol., Vol. 24, pp. 595 to 601. (G Pergamon Press Ltd. 1978. Printed in Great Britain.

THE INFLUENCE OF PHOTOPERIOD AND TEMPERATURE ON THE INDUCTION OF DIAPAUSE PUPAE OF HELIOTHIS PUNCTIGERA

IN

J. M. CULLEN* and T. 0. BROWNING

Waite Agricultural Research Institute, (Received 22 September

P.B.

I, Glen Osmond.

1976; revised 6 January

South Australia

1978)

Abstract-Heliothis punctigera exhibits a facultative pupal diapause that is regulated by photoperiod and temperature. The greatest proportion of pupae enter diapause when the larvae are reared at 19” C and 12-12.5 hr of light per day. The proportion diapausing falls steeply at both longer and shorter photoperiods. Diapause induction is also increased with a decrease in photoperiod betwen the egg and larval stages. Diapause is averted in a high proportion of pupae if they are held at 28” C compared with those held at 19” C. The significance of these results is discussed in relation to the life-cycle in South Australia. in particular to its capacity for overwintering.

induction

INTRODUCTION

Heliothis punctigera Wallengren (hepidoptera: Noctoidae) a pest of several crops in South Australia has several generations each year commencing with the appearance of adults in early spring (August) (CULLEN, 1969). Neither adults nor larvae occur in the field after May (late autumn). Pupae however, may be found in the soil during late autumn and winter. A study of the rate of development in relation to temperature showed that temperatures are sufficiently high even in winter, for development to continue after formation of pupae in autumn and for adults to emerge during winter unless diapause intervenes (CULLEN, 1%9). The same study showed that diapause occurred subsequent to larval-pupal ecdysis and prior to any observable development of the pharate adult within the pupal cuticle. The terms pupae and pupal diapause are therefore used throughout this account in referring to this stage. While care must be taken in the interpretation of developmental stages (HINTON, 1976) this usage seems justifiable on the basis of present knowledge and offers the minimum of opportunity for confusion. The number of H. punctigera are usually greatest in spring when there is an abundance of food for the larvae, and they are least in autumn. following the long, dry summer, when very little food for the larvae is available. Thus it’ seemed unlikely that the large numbers of moths flying in spring could be derived solely from the previous autumn generation when diapausing pupae might be formed. The question therefore arises as to whether it is possible for pupae formed at other times of the year, e.g. spring, to enter a diapause strong enough to carry them right through to the subsequent season. In order to understand the ecology of H. punctigera in southern Australia it was therefore necessary to study the factors responsible for the

and termination

of H. punctigera

of diapause

in the species.

There is an extensive literature on the influence of various factors in inducing diapause in insects. well reviewed by LEES (1968). DANILEVSKII (l965), and BECK (1968). This paper is concerned with the influence of daylength and temperature on the induction of diapause. MATERIALS

AND

METHODS

Rearing The method used for rearing H. punctigeru was adapted from CALLAHAN (1962) with modifications to reduce the incidence of disease among the larvae. Adult moths were placed in pairs to mate in large perspex containers which contained several strips of coarse cotton fabric and a feeding wick containing a IO% solution of honey in water. Most eggs were laid on the cotton and could then be removed readily and incubated at controlled temperatures.

When

larvae

hatched

they were

transferred

in

groups of IO-12 to small unwaxed paper cups containing a layer of sawdust about I cm deep and several small pieces (about 2 cm) of fresh French bean. The tops were covered with tightly fitting Petri

dishes. At the third instar the larvae were separated into individual paper cups covered with squares of clear glass. They also were fed with cut pieces of French bean. After larval-pupal ecdysis the pupae were either removed, the cups discarded and the glass washed in sodium hypochlorite solution. ot they were left so as to allow the adults to emerge in situ, where they could be fed individually on 10% honey solution via a feeding wick. All experiments were carried out in constant temperature cabinets fitted with 2 x 6 W fluorescent tubes operated by a time switch. The light intensity within the larval containers was checked with a photocell and always exceeded I Im/fP in all parts of the cabinets. The recognition of diapause

* P.O.

Present address: CSIRO Division of Box 1700, Canberra City, Australia.

The Occurrence of diapause was recognised in two

Entomology,

ways: 595

(a) by observable

non-development

in the

J. M. CULLEN

596

Fig.

I,

The positions of the “eye-spots

changes.

Five

AND

T. 0.

BROWNING

” in the newly moulted

pupa of H. punctigera

stages (A-E) can be recognised. whose durations WC without diapause are: A-2-5. B-1, C-2-3.

in days during D-l. E-2-3.

Emergence

0; 0

I

10

20 ’

8

’

60

’

’

e

1

1’00

DAYS FROM LARVAL Fig. 2. Duration

of time till adult emergence

lb0

and subsequent development

at

at 28 OC

zbo ’

- PUPAL ECDYSIS

from 300 pupae held at 28” C after larval-pupal

ecdysis.

Induction

pupae and (b) by the time taken for the adult to emerge. SHUMAKOV and YAKHIMOVICH (1955) described the presence of “eye-spots”in the post-genal region of the newly-moulted pupae of H. armigera, and their disappearance during subsequent development. The same phenomenon occurs in H. :ea (PHILLIPS and NEWSOM, 1966) and in H. punctigercl. Immediately following larval-pupal ecdysis the spots are distinct, occurring in a straight line across the area (Fig. I A). During development of the pharate adult their prominence and position both change as shown in Fig. I, and five stages can be recognised, whose durations in uninterrupted development at 28” C are as given on the figure. Diapause always occurs in stage A. Once stage B occurs, development proceeds without interruption until the adult emerges. For any temperature. the duration of the period stage B-eclosion varies little, most of the variability in the time between larval-pupal ecdysis and emergence of the adult being due to the variation in the time spent in stage A. Despite the variation however, the ability to distinguish the “eye-spots ” any longer than the time normally taken to reach stage C was a good sign of diapause. Conversely. the movement of “eye-spots” was the first sign that development had resumed. Development within pupae was considered to have occurred without diapause if the adult emerged within a specified period at a particular temperature. CULLEN (1969) showed that the mean duration of development from larval-pupal ecdysis to emergence of the adult at 28” C was 12.3 days, with arange from IO-20 days. Similarly the mean at 19” C was 32.5 days with a range from 30-50 days. Accordingly. diapause was considered to have occurred in pupae if emergence took longer than 20 days at 28Cand50daysat 19°C. Figure 2 represents the cumulative emergence of adults from 300 pupae, reared under various conditions in the laboratory and held at 28” C. Two hundred and twenty-five adults emerged within 20 days with a mean development time close to the figure of 12.3 days quoted previously. It is reasonable to regard all those adults taking longer than 20 days to complete their development and emerge, as having developed from pupae in diapause. Figure 2 shows that there was great variability in the duration of diapause development among the 75 diapausing pupae, from a few days to approximately 200 days. A diapause of such intensity that 200 days is required before normal development resumes could possibly enable the pupa to survive from one spring to the next, and experiments were designed to investigate the influence of certain environmental factors on the induction of diapause. RESULTS The influence the laborator!

AND DISCUSSION

of temperature

and photoperiod

597

in pupae of Heliofhis punctigera

of diapause

in

The effect on eggs and larvae. Eggs were obtained from a group of laboratory reared females (19” C) and assigned at random among five sets of conditions representing approximations to the climate of southern Australia.

Treatment I. Nine hours at 28” C, 9 hr at 16” C in each day, with 4.5 hr heating and I .5 hr cooling, and 14 hr light and IO hr darkness (l4L: IOD). This treatment was chosen to represent summer conditions, for 28” C is the mean daily maximal air temperature for January and February obtained from meteorological records at the Waite Agricultural Research Institute, and 16” C is the mean daily minimum. l4L: IOD was taken as a standardised long day regime for these experiments. Treatment 2. Nine hours at 22” C, 9 hr at I I ’ C with 5 hr heating and I hr cooling. and 13L: I ID. This regime was chosen to represent the conditions under which the first generation of larvae live in spring. Treatment 3. Constant 22°C. 14L: IOD. Treatment 4. Constant 16.5”C. l3L: I ID. Treatment 5. Constant 28°C. 14L: IOD. 22O C represents the mean summer temperature and 16.5”C the mean for spring and is also very close to the mean minimal summer temperature. “ Lights on ” and “heat on ” and ” lights off ” and “heat off” were as synchronous as possible. The eggs were incubated and the larvae reared under these conditions and the pupae were (ransferred to constant 28” C and assessed for further development. This temperature was chosen to represent the mean soil temperature to which pupae would be exposed in summer whether they were formed in spring or summer. Table I shows that only under the regime representing spring conditions was diapause induced in an appreciable proportion of pupae. This result differs from that of BENSCHOTER (1968) who consistently obtained only decreased induction when H. zea and H. virescens were reared in alternating temperatures. The next experiment was designed to assess the influence of a range of standard photoperiods. to determine whether the temperature at which the pupae were held following larval-pupal ecdysis influenced induction and to gain information about likely events in autumn. The larvae were reared at 19” C, the mean daily air temperature in autumn. A regime of 16” C and l3L: I ID was also included as a control against the results obtained earlier (Table I). The larvae were obtained from a large group of females brought from the field and confined at 28” C l4L: IOD to oviposit. After laying, the eggs were transferred to their respective treatments for hatching and larval development, except for one group. in which the eggs remained at 28” C. l4L: IOD until they hatched and the larvae were Table

I The influence of the temperature anh photoperiod experienced by developing eggs and larvae on the rnduction of diapause in pupae Temperature CC) 28/16 22/l I 16.5

22 28

Photoperiod 14L: IOD 13L:llD 13L:llD 14L: IOD 14L: IOD

Diapausing pupae (3 ) -. ‘7 22.0 5.5 2.7 0.0

J. M. CULLEN AND T. 0. BROWNING

598 Table 2. Q of pupae

in diapause at 28 and 19°C following development and larvae at different photoperiods and temperatures

Temperature

I ID llL:l3D IZL: I2D l3L: IID l4L: IOD I2L: IZD 13L:

16

then transferred to 19” C 12L: 12D. The pupae were divided at random into two groups. one group being transferred to 28” C within 24 hr of larval-pupal ecdysis and the other remaining at 19” C. The results are given in Table 2. The value of 6.3% diapause recorded for the regime 16” C, 13L: I 1D and 28” C is very close to the value of 5.5% found for these same conditions in the first experiment, and was significantly different from the proportion in diapause from 19” C, f3L: I ID, whether the pupae were held at 19” C or transferred to 28” C. Of considerable interest was the High level of diapause induction obtained with l2L: f2D and this was significantly different from the lower levels obained with either 1fL: f3D or l3L: I ID. In all cases more pupae entered diapause when they were held at 19” than at 28” C. Thus the insect remains sensitive todiapause inducing stimuli until after larval-pupal ecdysis. Also holding the eggs at 28°C and a long photoperiod until they hatched induced a significantly greater proportion of pupae to enter diapause following a larval regime of 19” C 12L: f2D, than if the eggs were incubated at the lower temperature and shorter photoperiod, and this was irrespective of the temperature at which the pupae were held. It seems that all the juvenile stages including part of the pupal stage following larval-pupal ecdysis are sensitive to temperature and photoperiodic influences. With this in mind and the parental effects demonstrated by PHILLIPS and NEWSOM (1966) and WELLSO and ADKISSON (1966) in H. zea, a further experiment was performed to investigate the nature and extent of this and any further similar effects

Table

Pupae

Eggs and larvae Photoperiod (“Cl

19 I9 I9 I9 28°C l4L: IOD-+ 19°C

of eggs

28°C

19°C

6.3 1.4 25.0 1.9 0.0 80.0

86.7 34.4 85.7 25.0 14.0 100.0

which might be observed in H. punctigera. Particufar attention was paid to the regimes for the adults and parental pupae (plus, in this case, the pharate adult) and to separating the effects of photoperiod and temperature. Adults that had developed as larvae at 19” C and l4L: IOD and that had developed from pupae either in cool conditions (19” C), or warm (28” C), were maintained at one of these fatter temperatures for mating. The eggs were transferred as soon as they were laid to a series of photoperiods ranging from f4L: IOD to IOL: 14D and either 28” C or 19” C. On hatching, the larvae were all maintained at 19” C and the resulting pupae were assessed for diapause. A summary of the conditions under which the generations were kept and the proportion of diapausing pupae is shown in Table 3. It is clear that the

overwhelming influence on pupal diapause was exerted by the conditions under which the eggs and larvae were kept. No influence of the adult regime was apparent under the conditions tested but when overall induction was low, a small increase in the induction was just significant of diapause (P= < 0.05) when parental pupae (and the devefoping pharate adult) had been maintained at 19” C

rather than 28” C. When eggs and larvae were reared at f2L: f2D the level of diapause induction was again very high and significantly more than that produced by either constant 14L: IOD or IOL: 14D. The effect of 14L: IOD for eggs followed by IOL: 14D for larvae was very marked and the high level of diapause induction was significantly different from using either a constant f4L: f0D or IOL: 14D regime. The

3. Proportion of diapausing pupae reared under different conditions and resulting non-diapausing parents reared in different conditions of photopericxf and temperature

Parental history Adults Pupae* (“C) 28

28 14L:IOD

.. .,..

..

1:

ib

11

*1

ii

11

3, 3.

,,

,.,.

* Includes

. .

I.

+,

pharate

,.

IOL1’14D adult.

from

Present regime Larvae

Eggs (“C) 28 I9 19 19 I9 I9 I9 I9 I9

l4L: IOD l2L: l2D IOL: l4D 14L: 10D 14L: IOD 1OL:l4D l4L: 10D IOL: l4D IOL: l4D

19 19 19 I9 19 19 19 19 I9

14L:lOD 12L:l2D IOL:l4D 12L:12D lOL:14D lOL:14D lOL:l4D IOL:l4D lOL:l4D

Diapause (70)

31 90 8 93 87 IS 91 30 25

Induction of diapause in pupae

of Heliothis

punctigera

effect of a temperature

Table 4. Proportion of pupae

period,

different

decrease at constant photoas has been shown for H. zea (ROACH and ADKISSON, 1970). was not clear. The figure of 31% diapause obtained in a l4L: IOD regime with a 9” C decrease from eggs to larvae was higher, than the 14% obtained previously with no decrease (Table 2). but the two results are not directly comparable. A final experiment was performed to define more closely the unique response to a constant 12 hr photoperiod compared with higher and lower values. Eggs were laid, larvae reared and pupae held at 19” C. The photoperiodic regimes (constant for all immature stages) and results are given in Table 4 which shows the significantly greater effect of I2 and 12.5 hr of light. All the resultsusingconstantphotoperiodsat 19” C have been graphed in Fig. 3 but with a distinction made according to whether the pupae of the parents were held for subsequent development in warm (38” C or summer) or cool (19” C or spring/autumn) conditions. The extremely narrow range of effective photoperiod is very apparent.

constant photoperiod

s99

in diapause at 19°C following regimes for eggs and larvae

Treatment Temperature (“C) 19 19

Photoperiod

Diapause f”;)

llL:13D

37.7

I1.5L: 12.5D I’L:l2D 12.5L: 1I .5D

19 19

36.8 92.0 84.ti

The induction of diapause in H. punctigera depends strongly on the temperature and photoperiod experienced by the developing eggs and larvae and the temperature experienced by the newly moulted pupa. The marked difference in the proportion of diapausing pupae when they were transferred to different temperatures at larval-pupal ecdysis (Table 2) could have been due either to the induction of diapause not being complete by this stage, or to a

. Pupae of parents 19O 0

of parents So

Pupae

I

I

9

I

I

II

10 HOURS

I

I2

OF LIGHT PER DAY

/

13

I

1.4

c

15

(EGG 8 LARVA)

Fig. 3. The influence of the photoperiodic regime experienced by the eggs and larvae on the incidence of diapause in the pupae of H. punctigera.

600

J. M.

CIJLLEN

AND

stimulating effect of high temperature such that diapause was terminated and normal development proceeded almost immediately. When a group of 30 diapausing pupae, that had been reared at 12L: 12D and 19°C for 4 weeks without observable development, were transferred to 28”C, 9 of them commenced development within a few days. The higher temperature could thus be considered to have had astimulatingeffect on development. However, from Table 2 it is clear that more the group would have been expected to have commenced development if stimulation by 28” C was the sole difference between the different proportions formed in diapause at 28” C and 19” C. Development of the pharate adult without prior diapause occurred in only 15% of the pupae at 19” C , whereas at 28” C it occurred in 75%. The transfer of pupae from 19-28” C induced development in only an additional 30%. i.e. total of 45% cf. 75%. Thus it seems that the influence of high temperature on pupae that had experienced a diapauseinducing photoperiodic regime as eggs and larvae both stimulated pupae already in diapause, and also acted against the effect of the diapause-inducing conditions already experienced. The latter effect could possibly be considered as an extension past the larval-pupal ecdysis. of the accepted influence of high temperature in supressing diapause. GIBBS (1975) has recently shown a similar effect in pupariating larvae of Sarcophaga argyrostoma prior to pupal diapause. There are few reports of a stimulatory effect of high temperature though VINOGRADOVA (1974) working with diapausing larvae of Calfiphora vicina obtained rapid development, i.e. pupation, on transfer from 12.5-25” C. It is possible that this result was also influenced by a more rapid termination of diapause at high temperatures, a further, separate effect also found in H. punctigeru as well as other insects, e.g. Leptinotarsa decemlineata (DE WILDE, 1953). In H. punctigera, diapausing pupae at 28” C were found to terminate diapause more rapidly than at 19” C and at an intermediate rate at 25” C (CULLEN, 1969). The influence of constant photoperiodic regimes on the induction of diapause in H. punctigercl is reminiscent of the effect found by DICKSON (1949) in Laspeyresia molesfa and by DANILEVSKII and GEYSPITZ (1948) (in DANILEVSKII (1965)) in Pieris brussicae in that there is an optimal dark/light regime that induces a high incidence of diapause and all other regimes are less effective. These cases form type III in Beck’s classification (BECK, 1968). The difference here lies in the extremely narrow range of photoperiodic conditions that are effective (Fig. 3). H. punctigera also shows a pronounced increase in diapause induction as temperatures are decreased (Table 2) similar to the response observed by GORYSHIN (1958) in H. arm&era and by BENS~‘HOTER (1968) in H. virescens. There was no evidence of any subsequent decrease in the response as observed by KOMAROVA (1959) in H. armigera at lower temperatures and by BENSCHOTER (1968) in H. zea. Of considerable significance is the greatly increased induction following a decrease in photo-

T. 0. BROWNING period between the regime for the egg and that for the larvae. In H. :ea a similar effect first reported by WELLSO and ADKISSON (1966) was investigated further by ADKISSON and ROACH (1971). These latter authors proposed a model incorporating two phases of response the first possessed by the adults and eggs and the second possessed by the larvae. Only at a very narrow range of photoperiods where the two response ranges overlap would diapause induction be possible at constant photoperiods. In fact, in H. zea it would seem the ranges are well separated and high levels of diapause induction are very difficult to obtain at constant photoperiods (WELLSO and ADKISSON, 1966). In H. punctigerathe limits of response have not been defined but the effect of decreasing photoperiods would fit a similar model and the restricted response at 12-12.5 hr photoperiods would imply a narrow but very definite area of overlap. ADKISSON and ROACH (1971) further suggest that this is the mechanism whereby H. zea distinguishes between spring and autumn conditions, ensuring that no diapausing pupae are formed in spring. In H. pun&gem however it is not clear that this necessarily follows. An effect of decreasing photoperiod has been demonstrated, but only at 19” C. the mean autumn air temperature. The increased incidence of diapause at 16.5” C, the further increase when fluctuating temperatures were used at spring temperatures and the low soil temperatures experienced by the pupae would suggest that the incidence of diapause might be higher in spring or at least that ranges of sensitivity established experimentally at 19” C might not apply at lower temperatures. The incidence of diapause in the field

Table 5 shows the percentage of pupae in diapause developing from samples of last instar larvae collected in the field and allowed to form pupae in outdoor cages. At no other time during the summer were more than 5% of pupae found to be in diapause. From March onwards larvae became extremely scarce but in the samples obtained on April 17 and 25.95 and 85% respectively of the pupae formed entered diapause. In autumn the natural photoperiod does not reach 12.5 hr until late March and even with the effects of a decrease, it seems that induction of diapause before this time would be unlikely, but highly probable subsequently. The available field data, while inadequate to confirm this conclusion. tend to support it. In spring, the mechanism is undoubtedly complex. If, as seems likely. successive responses to differing photoperiod and temperature ranges exist. Table 5. Proportion of pupae formed in diapause from field collected larvae. Dates correspond to mean dates of larval-pupal ecdysis for each sample Date

%

13 October 20 October 25 October 31 October

16 17 28 93

3 5 9 I7

Date

Q

November November November November

50 45 22 ?

Induction

of diapause

in pupae of

as suggested by ADKISSON and ROACH (1971) for H. ;eu.then (a) the modification of such ranges at lower temperatures, (b) the exposure of all larvae to photoperiods in the 13-12.5 hr range at some stage and (c) lower soil temperatures ensure that over a limited period a considerable proportion of pupae formed enter diapause. Finally it is apparent that whereas pupae entering diapause in autumn would remain in diapause over winter to yield adults in the spring, those entering diapause in spring would encounter high soil temperatures which would promote termination of diapause. The accelerated termination of diapause at 28°C in the laboratory has already been mentioned and in fact all pupae formed in spring entering diapause under field conditions, have been observed to produce adults in summer with no pupae persisting through the autumn. The problem therefore remains that at the only time of the season when pupae can be formed which will overwinter. larval numbers are extremely low and seem unable to account for subsequent spring flights of adults. It will undoubtedly be necessary to examine very carefully the distribution and density of larvae and the movements and numbers of adults over very large areas, to resolve this problem satisfactorily. Ackno~,lrd~ernrnts-The authors wish to thank Drs. B. M. DOUBE. A. D. LEES, K. G. WARDHAUGH and J. A. L.. WATSON for critical reading of the manuscript.

REFERENCES ADKISSO~ P. L. and ROACH S.H. (1971) A mechanism for seasonal discrimination in the photoperiodic induction of pupal diapause in the bollworm Hekothis zea (Boddie). In S~~mposium on Biochrmistr.v(Ed. by MENAKER M .I no. . . 271-280. Nat. Acad. Sci.. Biol. and Agric. Div.. Washington. BECK S. D. ( 1968) Insrct Photoperiodism. Academic Press. New York. BENSCHOrER C. A. (1968) Diapause and development of Heliothis ;ea and H. virescens in controlled environments. Ann. em. Sot,. Am. 61, 953-956.

Heliothis

punctigera

601

CALLAHAN P. S. (1962) Techniques for rearing the corn earworm Heliothis :ea. J. econ. Ent. 55. 4.53--157. CULLEN J. M. (1969) The reproduction and survival of Heliothis puncfigera Wallengren in South Australia. Ph.D. thesis. University of Adelaide. DANILEVSK~~ A. S. (1965) Photoperiodism and Seusonul Development of Insects. Oliver & Boyd, London. DICKSON R. C. (1949) Factors governing the induction of diapause in the oriental fruit moth. Ann. enf. Sot,. Am. 42, s I I-537. GIBBS D. ( 1975) Reversal of pupal diapause in Surcophugu argvrostoma by temperature shifts after puparium formaiion. J. Insect. Phvsiol. 21, 1179-l 186. GORYSHIN N. I. (1958) The ecological analysis of lhe seasonal cycle of development of the cotton boHworm (Chlorideu obsoleta F.) in the northern areas of its range. Lichen. Zap. Leningr. pas Univ. 240. 3-N. HINTON H. E. (1976) Notes on neglected phases in metamorphosis. and a reply to J. M. Whitten. .4nn. enl. SK Am. 69, 560-O-(66. KOMAROVA 0. S. (1959) On the conditions determimng diapause of hibernating pupae of Chloridea obsoleta F. I Lepidoptera: Noctuidae). Ent. Re\,. Wush. .I& 3 18-325. LEES A. D. (1968) Photoperiodism in insects. Pholophysiology 4, 47-137. PHILLIPS J. R. and NEWSOM L.. D. (1966) Diapause m Heliothis :eu and Heliothis \tirescens (Lepidoptera: Noctuidae). Ann. ent. Sot. Am. 59, 154-159. ROACH S. H. and ADKISSON P. L. (1970) Rale of photoperiod and temperature in the induction of pupal diapause in the bollworm Heliothi.7 ;eu. J. Insecf Plry.sio/. 16, 1591-1597. SHUMAKOV E. M. and YAKHIMOVICH L. A. (1955) Morphological and histological peculiarities of the metamorphosis of the cotton bollworm Chloridea obsoletu F. in connection with the phenomenon of diapause. Rev. appl. Ent. 44, I l-12. VINOGRADOVA E. B. (1974). The pattern of reactivation of diapausing larvae in the blowfly. Culliphom \,icinu. J. Insect Physiol. 20. 3487-2496. WELLSO S. G. and ADKISSON P. L. (1966) A long-day short-day effect in the photoperiodic control of the pupal diapause of the bollworm Heliofhis :ea (Boddie). J. Insect Physiol. 12, 145%1465. WILDE J. DE (1953) Provisional analysis of the imaginal diapause in an insect I Leptinotursu decemlineuru Sah ). .4ctu PhvsioI. Phurm. NeCrl 3, ?33-249.