Water intake and the termination of diapause in the European corn borer, Ostrinia nubilalis

r. Insect Physic& 1967, Vol. 13, pp. 739 to 750. Pergamon Press Ltd. Printed in Great Britain

WATER INTAKE AND THE TERMINATION OF DIAPAUSE IN THE EUROPEAN CORN BORER, OSTRINIA NUBILALIS* STANLEY Department

of Entomology, (Received

D. BECK

University 5 October

of Wisconsin,

Madison

1966)

Abstract-Partially dehydrated diapausing corn borers were found to be incapable of terminating diapause and completing prepupal development until after they had had access to water. Long-day photoperiods would not terminate diapause in partially dehydrated borers, but such dehydration did not prevent the insects from responding to the photoperiod. Partial dehydration followed by water imbibition did not accelerate development unless the borer’s photoperiodic requirements had been satisfied. After prolonged (30 days’) exposure to dry conditions and long daylengths, diapause termination and pupation were dependent upon the production of proctodone for about 48 hr and upon the production of prothoracotropic hormone for about 72 hr following water imbibition. Diapause termination in this species is postulated to be a two-phase developmental process. The first phase is rate controlled by the photoperiodic synchronization of rhythmic functions, and the second phase includes the physiological action of proctodone and the activation of the neuroendocrine system. Completion of the second phase is dependent upon an appropriate water balance.

INTRODUCTION

OVERWINTEREDlarvae of the European corn borer require contact with water in order to terminate d&pause and to pupate (BABCOCK,1927; MONCHADSKII,1935). The latter worker observed that overwintered larvae would pupate readily if incubated in the presence of free moisture, but not if incubated without contact moisture, even though the relative humidity was maintained near 100 per cent. Under field conditions, the water content of overwintering borers was found to fall from a prediapause value of about 80 per cent to a mid-winter level of approximately 52 per cent (BECK and HANEC, 1960). This marked loss of water is thought to be restored by an active imbibition of water prior to pupation in the spring. MELLAN~Y (1958) observed that such larvae would drink 9 or 10 ~1 of water once or more times during the first 24 hr in which water was made available to them. Water balance has been implicated in the termination of diapause in many insect species that diapause as embryos (SLIFER, 1946), larvae (KOIDSUMI, 1952; CHURCH, 1955), or adults (HODSON, 1937; NAYAR, 1960). There has been some * Approved for publication by the Director of the Wisconsin Agricultural Experiment Station. This study was supported in part by research grants from the National Science Foundation (GB-2366) and the National Institutes of Health (GM-07.557) of the U.S. Public Health Service. 739

740

STANLEY D. BECK

question, however, as to the role of water in diapause. An influx of water, by natural or experimental means, does not necessarily terminate the diapause state. Some authors (CHURCH,1955 ; LEES,1955) have suggested that a lack of water may inhibit the further development of an insect in which diapause has been terminated. In such cases, the inhibition of development has been considered to be a post-diapause phenomenon. Diapause is generally considered to be an arrest, or suppression of growth processes occurring at a specific growth stage and under the control of genetic, and therefore biochemical, mechanisms. The European corn borer enters diapause as a mature, non-feeding larva, properly termed a ‘prepupa’ (FOLSOMand WARDLE, 1934). During the prepupal stage, biochemical and physiological developmental changes occur, apparently in preparation for the pupal moulting cycle (BECKand ALEXANDER, 1964a, b; BECKet al., 1965; CHIPPENDALE and BECK,1966; BECK, 1967). From the beginning of the pupal moult to the occurrence of ecdysis, the insect is considered to be a ‘pharate pupa’ (HINTON,1946). The termination of diapause is, probably, closely related to the completion of prepupal development, but has been termed ‘diapause development’ (ANDREWARTHA, 1952). The relationship of water loss and subsequent water intake to the termination of diapause is the subject of the present report. MATERIALS AND METHODS The European corn borer larvae used in this study were reared at 30°C under a photoperiod of 12 hr of light and 12 hr of darkness per day (12L : 12D). The meridic rearing medium of CHIPPENDALE and BECK(1964) was used. Under these conditions, the larvae matured in about 16 days. Fewer than 5 per cent of the borers pupated, and the rest were considered to have entered diapause and were removed from the culture vials at 20 days of age. The diapausing larvae were transferred to cotton-plugged shell vials (23 x 85 mm), each of which contained a strip of two-ply absorbent paper towelling. Diapausing larvae that were to be incubated under dry conditions were held in vials as described above. Incubation was at 3O”C, about 40 per cent relative humidity, and photoperiods of 12L : 12D (short day), 16L : SD (long day), or continuous darkness. Wet-incubated borers were treated in exactly the same manner, except that the absorbent paper strips were maintained in a moistureladen condition throughout the incubation period. About 50 larvae were used for each experimental treatment. The groups of experimental larvae were inspected daily, with mortality and pupation being recorded. Treatments or series of treatments were begun when the borers were 20 days of age, which is taken as day 0 in all of the results presented below. Different combinations of moisture and photoperiod were used, and for convenience in the discussion of experimental results, the following abbreviated designations will be employed : LD-wet = long-day photoperiod with excess contact moisture; LD-dry = long-day photoperiod without contact moisture;

wATBR

INTAKE AND

DIAPAUSE INTHEEUROPEAN

741

CORNBORER

SD-wet = short-day photoperiod with excess contact moisture; SD-dry = short-day photoperiod without contact moisture; DD-wet = continuous darkness with excess contact moisture; DD-dry = continuous darkness without contact moisture. Experiments that involved the injection of water into the haemocoel were carried out by the use of an automatic calibrated microinjector using a 30-gauge hypodermic needle. Larval weights were determined by means of a semimicro analytical balance. EXPERIMENTAL

RESULTS

When 20-day-old diapausing borer larvae were transferred to dry vials and incubated indefinitely in a long-day photoperiod (LD-dry), none pupated. Similar borers that were incubated under conditions of both contact moisture and a longday (LD-wet) showed a normal curve of pupation incidence, with 50 per cent having pupated by about the thirtieth day of incubation. These results demonstrated that both diapause termination and pupation will occur in the presence of water and a long-day photoperiod. Under dry conditions, pupation does not take place, but the demonstration does not indicate the cause of the developmental failure. Large groups of diapausing borers were incubated LD-dry for either 20 or 30 days. At the end of these times, groups of 50 were moistened and held under conditions of long day, short day, or continuous darkness. The number of days required for 50 per cent of each group to pupate, and the total time (dry period+ wet period) required to attain the 50 per cent level of pupation was recorded (Table 1). Borers that had not been exposed to LD-dry incubation served as controls (Table 1, series A), and under LD-wet conditions, the control groups TABLED-EFFECTS

OFPHOTOPERIODSAND CONTACTWATERONTHRTERMINATIONOFDIAPAUSE IN THEEUROPEAN CORNBORER

Wet days to 50% pupation Experimental series A B C

LD-dry (days) 0 20 30

Total days to 50% pupation

LD

SD

DD

LD

SD

DD

32 10 10

>90 16 7

>90 11 11

32 30 40

P-90 36 37

>90 31 41

reached 50 per cent pupation in 32 days. Neither the short-day nor the continuous darkness (SD-wet and DD-wet) control groups pupated at a significant rate. Twenty days’ exposure to LD-dry conditions (Table 1, series B) did not appear to delay subsequent pupation, and upon receiving water the larvae pupated at a high rate under LD, SD, or DD conditions. Fifty per cent of each treatment group had pupated within 10 to 16 days, and the total numbers of days required to terminate diapause were not significantly different under any of the wet conditions employed. 48

STANLEYD. BECK

742

When groups of diapausing borers had been deprived of water for 30 days under long-day conditions before being given contact moisture, subsequent pupation was also quite rapid (Table 1, series C). In these cases, however, the total numbers of days from the beginning of LD-dry to the time of 50 per cent pupation were higher than in the previous series, but the delay was approximately equal to the additional 10 days that the larvae had been held in dry vials. Pupation curves were obtained by plotting the cumulative incidence of pupation against time in days (Fig. 1). These pupation curves showed that 20 days of

0

15

20

25

30

35

40

45

50

DAYS FIG. 1. Effect of moisture and photoperiod on rates of diapause development and pupation among diapausing European corn borers.

LD-dry led to the termination of diapause in most, but not all, of the borers. Although the 50 per cent level of pupation was reached on about the thirty-sixth day among borers that had been incubated LD-dry for 20 days and then SD-wet, the pupation curve was truncated; about 40 per cent of the larvae failed to pupate within the 50 days of observation (Fig. 1, curve A). Experimental groups that were incubated LD-dry for 30 days followed by SD-wet showed a rapid and untruncated curve (Fig. 1, curve B). Twenty days of LD-dry, followed by ED-wet incubation, also permitted an uninterrupted pupation record (Fig. 1, curve C). These results were interpreted as showing that a substantial proportion of the borers were unable to complete diapause development during a 20 day LD-dry incubation; their growth and differentiation processes remained susceptible to a reinduction of the low developmental rates characteristic of diapause, as demonstrated by MCLEOD and BECK (1963) and BECK and ALEXANDER(1964a). After 30 days of LD-dry incubation, however, the insects’ developm.ental rates could not be suppressed by short-day photoperiods, and diapause development was considered to have been completed during that incubation.

WATER

INTAKEANDDIAPAUSE

INTHEEUROPEAN

CORNBORER

743

The borer’s requirement for water during diapause and prepupal development was further investigated by exposing groups of diapausing larvae to LD-wet incubation for different lengths of time, followed by LD-dry treatment. The pupation data (Table 2) clearly show that the availability of contact moisture during TABLE

~-EFFECT

OF LIMITED ZXPOSURE TO CONTACT MOISTURE (LD-WET) PUPATION(LD-DRY)OFDIAPAUSINGEUROPEAN CORNBORERS

Days LD-wet (No.)

Pupation in LD-dry (%)

1 4 7 11 15 17 40 (controls)

0.0 0.0 6.1 9-l 11.1 33.3 86.3

ON SUBSEQUENT

Forty day observation period.

the early phases of diapause development had little effect on subsequent pupation, Contact moisture was required during the later prepupal stages, but did not appear to be essential to the early phases of diapause development. Larvae were not incubated under LD-wet conditions for longer than 17 days (except for the control group), because pupation begins to occur at about 18 days under LD-wet conditions. Quantitative aspects of the relationship between water intake and the termination of diapause were studied by means of two types of experiments : (1) administration of measured volumes of water by injection, and (2) determination of water uptake and pupation rates of partially dehydrated diapausing corn borers. In experiments of the first type, different amounts of water were injected into the haemocoels of larvae that had been held LD-dry for 30 days. Following administration of the water, the borers were again incubated LD-dry and observed for pupation. The maximum injected volume that could be tolerated by the partially dehydrated larvae was 30 ~1. Dosages higher than 30 ~1 were administered as two treatments 2 days apart. The results (Table 3) show that the larvae required an astoundingly large volume in order to complete their prepupal development. MELLANBY (1958) reported that overwintered borers imbibed relatively small volumes of water (9-10 ~1) once or twice during the first 24 hr that they were exposed to contact moisture. In the present experiments, little or no pupation occurred when the water treatment was in an amount less than about 30 ~1. Experiments were also run to determine whether or not the borers could absorb water via the integument, and the results showed that no significant absorption occurred (also see Table 7). The possibility that sensory stimulation might play a part in the

STANLEYD. BECK

744

TABLE ~-EFFECT OF DIFFERENTVOLUMESOF WATER ON PUPATION OF LD-DRY INCUBATED DIAPAUSINGEUROPEANCORNBORERS Water dosage (ELI) 0 12 17 22 30 45* 60T Ad lib&m

Larvae treated (No.)

Pupae

No.

y.

30 26 20 23 28 26 30 27

0 2 1 4 9 16 28 27

0.0 7.7 5.0 17.4 32.2 61.5 93.6 100

Thirty day post-treatment observation period. * Administered as 30 p1 followed by 15 ~12 days later. t Administered as two 30 ~12 days apart. insect’s injecting

response

to water was also tested.

30 ~1 of water into the haemocoel

Moistening

the mouth-parts

while

did not increase the incidence of pupa-

tion, as compared to that observed after injection alone. Volumes

of water injected

into the gut tract via either mouth or anus by means of fine capillary tubing were also effective, but were no more effective haemocoel injection. Some.of

the relationships

diapause were determined.

than similar volumes

administered

between natural water intake and the termination Borer larvae that had been incubated

days were given access to water for limited

times (1, 5, or 24 hr).

LD-dry After

by of

for 30 access to

TABLE 4.--WATER CONSUMPTIONAND PUPATION INCIDENCEAMONG PARTIALLY DEHYDRATED (30 DAYSLD-DRY) DIAPAUSINGEUROPEANCORNBORERS Pupation Hydration time @r)

Days

Initial (mg + S.E.)

Hydrated (mg + S.E.)

Average water uptake Q&lc S.E.)

Average larval weights

%

1 1 1

5-9 21-7 IO-14 11.7 Non-pupaters

86.4 + 25.3 75.9 + 31.6 84.0 + 14.5

112.6 + 33.0 99.7 * 41.4 105.1 + 18.0

25.9 f 24.5 23.82 9.1 21.1 + 3.8

: 5

5-9 26.7 IO-14 16.8 Non-pupaters

70.5 F 18.7 83.1 + 27.9 73.8 + 14.3

107.1 f. 26.6 116.8 F 39.2 105.8 f 20.4

30.4+ 8.0 33.7 + II.3 34*0* 6.3

24 24 24

5-9 43.3 10-14 13.4 Non-pupaters

87.8 + 23.3 83.8 rf:29.5 72.1 k 15.1

118.3 f 31.1 121.9 Z!I46.9 106.0 + 22.1

30.5 + 8.1 38.1 f 14.5 33.8 -1: 7.3

WATER INTAKE AND DIAPAUSE IN THE EUROPEAN CORN BORER

745

water for the prescribed time, the borers were returned to LD-dry conditions and observed for pupation. The water intake of each larva was determined by measurement of body weight before and after access to water. Average weights, water intake, and pupation data are presented in Table 4. Based on pupation records, each treatment group was divided into three subgroups, depending on whether the borers had pupated in from 5 to 9 days, 10 to 14 days, or not within 14 days after water imbibition. On the fifteenth day after hydration, the ‘non-pupaters’ subgroup from the 1 hr and 5 hr treatment groups were again given water, this time for 24 hr. Water intake was again determined, and the larvae were returned to Of the 67 larvae that were given water on the fifteenth day, LD-dry incubation. 37 pupated prior to the twenty-first day after the original water-access treatment. The water consumption of those that pupated after the second hydration is compared to that of those that failed to pupate prior to the twenty-first day (Table 5). TABLE 5-EFFECT OF REPEATEDWATER INTAKE ON PUPATION OF DIAPAUSING EUROPEAN CORN BORERS

Status on 21st day

Av. 1st weight (mg C S.E.)

Av. 1st water intake (~1 rt S.E.)

Av. 2nd weight (mg f SE.)

Av. 2nd water intake (pl+ S.E.)

Av. total water intake &I F S.E.)

Pupated Not pupated

85.5 ri:19.0 81.6 f 23.2

Group I (1 hr access group, Table 4) 20.7 -1:4-8 83.7 t 18.6 32-l k 7.2 21.7 + 6.4 83.3 f 23.8 28.8 +8-l

52-S+_ 7.3 50.5 * 10.4

Pupated Not pupated

74.9 + 22.3 72-9 I 19.2

Group II (5 hr access group, Table 4) 31.8 f 5.6 76.8 i_ 23.0 21.4 + 6.7 32.3 + 8.5 62.4 + 21-7 22.1+ 5.9

54.2 + 8.7 545 t 10.4

The data of Table 4 show a number of points of interest. The larvae that were given access to water for 1 hr imbibed an average of about 23 ~1. About one-third (21*7+ 11.7 = 33.4 per cent) of these larvae pupated within 14 days, but there was no significant difference in water uptake among the three subgroups. Borers that were given access to water for 5 hr imbibed larger volumes than did the 1 hr group, and they showed a higher incidence of pupation (26*7+ 16-8 = 43.5 per cent) by the fourteenth day. The average weights and water uptake of the larvae that failed to pupate within 14 days were not significantly different from the values observed in the pupating groups, however. Of the partially dehydrated larvae that were given water for 24 hr, 56.7 per cent (43.3 + 13.4) pupated within 14 days. Their average weights and water uptake volumes were not significantly different from those of the previous group. These borers may have excreted some moisture and imbibed additional quantities during the 24 hr access to water. Their water intake was, therefore, somewhat uncertain. Nevertheless, the apparent water consumption of those borers that failed to pupate prior to the fourteenth day was not significantly different from that of the borers that pupated within that observation period.

STANLEYD. BECK

746

At 1.5 days aftei hydration, unpupated members of the 1 and 5 hr hydrationtime groups were given additional water (Table 5). Comparison of the average original dehydrated weights with the second dehydrated weights showed that the water consumed on the first day had been entirely lost by the fifteenth. Although the total intake of water averaged more than 50 ~1 in each Subgroup, there were no significant differences between those that pupated and those that failed to pupate by the twenty-first day of the experiment. The experimental results discussed above suggest that the amount of water imbibed does not, in itself, determine whether or how soon the borers will pupate. Although some minimum water level appears to be required for the completion of prepupal development (see Table 3), the insect’s requirement for water probably comes into play only after certain previous physiological requirements have been met. The borers that failed to pupate following an intake of water (see Tables 4 and 5) had apparently not yet reached that developmental point at which the lack of appropriate water balance constituted a growth-limiting factor. The results might be interpreted as supporting the hypothesis that the borer’s water intake requirement is a post-diapause phenomenon; those larvae that did not pupate after receiving water had not yet completed diapause development. Some preliminary observations suggested the possibility that a massive water intake following partial dehydration might have an accelerating effect on diapause development, even under the influence of a short-day photoperiod. This hypothesis was tested experimentally by subjecting 20-day-old diapausing borers to a relatively rapid dehydration. The borers were held under either long-day or shortday photoperiods in dry vials over anhydrous calcium chloride. The larvae were weighed at frequent intervals and the desiccating incubation was terminated when the average weight loss was from 27 to 31 per cent of the average initial body weight. The borers were then watered and incubated under either LD-wet or SD-wet conditions. The borers proved to be quite resistant to water loss, and 10 days of desiccating incubation were required in order to realize the required loss of body weight. The results of the experiment (Table 6) clearly showed that the rate of desiccation did not influence the effect of photoperiod on the termination of diapause. Those borers that had been desiccated under short-day conditions TABLE~-EFFECT OFRAPIDDESICCATION ANDPHOTOPERIOD ON DIAPAUSE TERMINATION AND PUPATION IN THEEUROPEAN CORNBORER

Desiccation photoperiod

Av. wt. loss (%I

Wet

Time to 50%

incubation photoperiod

pupation (days)

Long-day

26.6

Long-day Short-day

32 >60

Short-day

31.5

Long-day Short-day

41 >60

WATERINTAKE AND DIAPAUSEIN

TH.E

EUROPEANCORNBORER

747

followed by LD-wet incubation attained the 50 per cent level of pupation about 10 days later than did their long-day counterparts. The delay corresponded almost exactly to the 10 additional days spent under the influence of the short-day photoperiod. The SD-wet groups failed to reach 50 per cent pupation within the 60 days of the experiment, indicating no accelerating effect from the relatively rapid desiccation. The failure of the SD-wet groups to pupate also indicated that partial desiccation did not reduce the larvae’s sensitivity to photoperiod. In this experiment, dehydration occurred during the early part of the diapause period. It is possible that dehydration and water intake might stimulate growth after the photoperiodic, or temporal, requirements of diapause development have been met. Endocrine function at the end of a 30 day LD-dry incubation and at different times after the borers had been given water was studied. Brain removal either by extirpation or by ligation of the head (ligature placed between head and prothorax) prevented the larvae from imbibing water (Table 7). Such treatment effectively TABLE ~--EFFECTS OF LIGATION ON WATERINTARB AND SUBSEQUENT PUPATION IN LD-DRY INCUBATEDDIAPAUSINGEUROPEANCORNBORERS Av. body weights

Position of ligature

Initial (mg)

24 hr hydration (mg)

Head-thorax Sixth abdominal segment Ninth abdominal segment None (controls)

63.5 62.8 60.8 -

66.5 94.1 101.2 -

48 hr hydration (mg) 66.2 97.4 102.2 -

Av. water intake I&l)

Pupation (%)

2.7 34.6 41.4 -

0.0 17.7 51.5 82.9

prevented pupation, but without yielding any information as to the endocrine state of the insects. Abdominal ligations did not prevent water uptake. Ligation at the level of the sixth abdominal segment prevented pupation, however, presumably because the ligature prevented the movement of proctodone from its site of production in the seventh and eighth abdominal segments (BECK and ALEXANDER, 1964b). Ligatures placed at the ninth abdominal segment did not prevent either water uptake or pupation, and over 50 per cent of the larvae so treated pupated within the 60 days (30 days LD-dry, 30 days LD-wet) of the experiment. These results suggested that the dry-incubated borers were still proctodone-dependent, even though they had been exposed to long-day photoperiods for 30 days. After a 30 day LD-dry incubation, borers were given access to water for periods of 24,48, or 72 hr. At the end of these times, subgroups of larvae were subjected to ligation between the head and prothorax (anterior ligation), ligation at the sixth abdominal segment (posterior ligation), or to no treatment (controls). All of the experimental groups were then returned to LD-dry conditions for observation. The use of LD-dry post-treatment conditions was necessary, because in the

STANLEY D. BECK

748

presence of water, only the anterior ligation groups would be unable to imbibe additional moisture. The percentage pupation data (Table 8) indicated that the TABLE&-EFFECT OFANTERIOR AND POSTERIOR LIGATURES ON PUPATION OF LD-DRY INCUBATED DIAPAUSING EUROPEAN CORNBORERS THATHADBEENGIVENACCESS TO WATERFOR 24, 48, OR72 hr PRIORTO LIGATION Pupae Treatment

Survivors (No.)

No.

24 24 24

Anterior ligation Posterior ligation None (controls)

28 26 27

2 6 17

7.1 23.0 62.9

48 48 48

Anterior ligation Posteriorligation None (controls)

25 16 28

1 7 18

4.0 43.8 64.2

72 72 72

Anterior ligation Posteriorligation None (controls)

23 21 28

4 13 21

17.3 62.0 75-o

LD-wet (hi-)

--

(%)

borers continued to be dependent on brain function (probably secretion of prothoracotropic hormone) for a period greater than 72 hr after water was made available. The physiological processes that are under the control of the abdominal system (probably secretion of proctodone) appeared to be dependent upon that system for nearly at least 48 hr following imbibition of water. DISCUSSION

Diapause in the European corn borer is a stage in which the insect’s developmental rate has been strongly suppressed, but not totally arrested. Long-day photoperiods lead to an acceleration of development, whereas short-day photoperiods tend to maintain the developmental rate at the low level characteristic of the diapause state. In previous work on diapause development (MCLEOD and BECK, 1963 ; BECK and ALEXANDER, 1964a), we considered diapause development to have been completed when the growth rate could no longer be reduced to the diapause level by means of exposure to short-day photoperiods. The completion of diapause development was also considered to be the point at which the neuroendocrine system of the brain had been fully activated, so that prothoracotropic hormone production was independent of photoperiod and the brain system was no longer influenced by the hormone proctodone. The time required for proctodone activation of the brain system depended on the photoperiod, in that the rhythmic functions of the two endocrine organs (ileal epithelium and brain) were synchronized by a long-day photoperiod, but desynchronized (or dissociated) by short-day photoperiods (BECK, 1964). Injections of massive doses of ammonium acetate were

WATRRINTAxIANDDIAPAUSE IN THEEUROPEAN CORNBORER

749

thought to accelerate diapause development through a high degree of stimulation of proctodone production (BECKand ALEXANDER,1964b). The insect’s requirement for contact moisture for the completion of prepupal development may control the developmental process only after the photoperiodic requirement has been met. By this general view of diapause development, the need for water imbibition might be considered to be a post-diapause requirement. The above concept of diapause development in the European corn borer is not fully consistent with all of the experimental results presented in this paper. In view of the finding that the borer requires proctodone for at least 2 days after rehydration and following the hypothetical rhythmic synchronization by long-day photoperiods (Tables 7 and S), the water requirement cannot be considered to be post-diapause if the proctodone requirement is held to be involved in diapause development. Since both the water influx and the action of proctodone necessarily precede the secretion of prothoracotropic hormone (Table 8), neither can rightly be regarded as post-diapause processes, if the ‘reactivation’ of the prothoracotropic hormoneproducing system is taken as marking the end of the diapause state. And, finally, there is reason to question the concept that the neuroendocrine system’s lack of secretory activity constitutes the growth-limiting factor during diapause of the European corn borer. Implantation of brains into diapausing borers may or may not induce pupation of the recipients, and the response obtained does not appear to depend entirely upon whether or not the implanted brain was that of a diapause or non-diapause donor. Diapausing borers will pupate in response to the implantation of either a diapausing or non-diapausing borer brain (CLOUTIERet al,, 1962). But the non-diapausing brain of a wax moth larva (Galleria mellonella) will induce pupation only if the recipient diapausing borer is held under the influence of longday photoperiods (BECK, 1967). It would appear most logical to postulate that the termination of diapause in corn borer requires the completion of a two-step developmental process. The first phase of diapause development is controlled by photoperiod, in which long daylengths accelerate completion of the phase and short-day photoperiods suppress the rate of completion; this phase may involve rhythmic synchronization, as previously suggested (BECK, 1964). The second phase of diapause development involves the secretion of proctodone and the activation of the neuroendocrine system, and very probably some other growth-controlling systems that have yet to be identified. Phase two is inhibited by a lack of tissue water, or the unfavourable water balance resulting from partial dehydration. Completion of phase two is accelerated by ammonium ions, however, although the mode of action of ammonium ions is still unknown. Once the neuroendocrine system has been fully activated, prothoracotropic hormone and ecdyson production is initiated, and the insect enters the moulting cycle that leads to pupation. The characteristic rise in metabolic rate and the cytological changes that mark the beginning of the moulting cycle also mark the termination of diapause. By this interpretation, the insect’s requirement for contact water is not a post-diapause effect, but is a characteristic of the second phase of a two-phase diapause development.

750

STANLEY D. BECK REFERENCES

ANDREWARTIXA I-1. G. (1952) Diapause in relation to the ecology of insects. Riol. Rev. 27, 50-I 07. BABCOCKK. W. (1927) The European corn borer, Pyruustu nubilulis Hiibn.-I. A discussion of its dormant period. Ecology 8, 45-59. BECK S. D. (1964) Time-measurement in insect photoperiodism. Am. Nut. 98, 329-2346. BECK S. D. (1967) Environmental Photoperiod and the Programming of Development. Centennial Program, Peabody Museum of Natural History, Yale University Press. In press. BECK S. D. and ALEXANDERN. (1964a) Chemically and photoperiodically induced diapause development in the European corn borer, Oshinia nubilalis. BioL Bull., Woods Hole 126, 175-184. BECK S. D. and ALEXANUERN. (1964b) Proctodone, an insect developmental hormone. BioZ. Bull. Woods Hole 126, 185-198. BECK, S. D. and HANECW. (1960) Diapause in the European corn borer, Pyraustu nubilalis (Hiibn.). r. Insect Physiol. 4, 304-318. BECK, S. D., SHANEJ. L., and COLVIN I. B. (1965) Proctodone production in the European corn borer, Ostrinia nubilalis. J. Insect Physiol. 11, 297-303. CHIPPENDALEG. M. and BECK S. D. (1964) Nutrition of the European corn borer, Ost&ia nubilalis (Hiibn.)-V. Ascorbic acid as the corn leaf factor. Entomologia exp. appl. 7, 241-248. CHIPPENDALEG. M. and BECK S. D. (1966) Haemolymph proteins of Ostriniu nubilalis during diapause and prepupal differentiation. g. Insect Physiol. 12, 1629-1638. CHURCHN. S. (1955) Moisture and diapause in the wheat stem sawfly, Cephus cinctus Nort. Can. Ent. 87,85-97. CLOUTIER E. J., BECK S. D., MCLEOD D. G. R., and SILHACEKD. L. (1962) Neural transplants and insect diapause. Nature, Lond. 195, 1222-1224. FOLSOM J. W. and WARDLE R. A. (1934) Entomology with Special Reference to its Ecological Aspects. Blakison, Philadelphia. HINTON H. E. (1946) Concealed phases in the metamorphosis of insects. Nature, Lond. 157, 552-553. HODSONA. C. (1937) Some aspects of the r81e of water in insect hibernation. Ecol. Monogr. 7, 271-315. KOIDSUMI K. (1952) Water content and the hormone center for the pupation of hibernating larvae of Chile simplex Butler. Annotnes zool. jap. 25, 156-167. LEES A. D. (1955) The Physiology of Diapause in Arthropods. Cambridge University Press, London. MCLEOD D. G. R. and BECK S. D. (1963) Photoperiodic termination of diapause in an insect. Biol. Bull., Woods Hole 124, 84-96. MELLANBY K. (1958) Water drinking by the larva of the European corn borer. J. econ. Ent. 51,744-745. MONCHADSKIIA. S. (1935) On the rBle of contact moisture after the winter dormant period (diapause) in the corn borer larvae. Plant Protection, 39-50. NAYAR K. K. (1960) Studies on the neurosecretory system of Iphitu Zimbata StHl. 2. ZelEforsch. 51, 320-324. SLIFER E. H. (1946) The effects of xylol and other solvents on diapause in the grasshopper ;;;;:;ether with a possible explanation for the action of these agents. J. exp. Zool. 102,