Supernumerary instars produced by chilled wax moth larvae: Endocrine mechanisms

J. Insect Physiol., 1976, Vol. 22, pp. 1641 to 1641. Pergamon Press. Printed in Great Britain.

SUPERNUMERARY INSTARS PRODUCED BY CHILLED WAX MOTH LARVAE: ENDOCRINE MECHANISMS RUDOLPH L. PIPA Division of Entomology and Parasitology, University of California, Berkeley, California 94720, U.S.A. (Received 11 May 1976) Abstract-Young last instar larvae of Galleria mellonella underwent supernumerary ecdyses within 3 to 6 davs after beinrr chilled at 0 to 1°C for 30min. The freauencv diminished from 89 f 9.4X for the sur;ivors of thoie that were chilled < 16 hr after their la& edysis, to 25 + 11.2% fo< those 46 to 88 hr old, and was no longer evident beyond 123 hr. Irrespective of their ages, the larvae never became “superlarvae” unless they had fed after they had been chilled. This was unlike the requirement for metamorphosis, when a feeding period of 40 to 48 hr immediately following ecdysis allowed half the larvae that were subsequently chilled and starved to pupate. The propensity to become superlarvae could be extended by starvation. Chilling signaled the occurrence of the larval moulting program, but its expression was held in abeyance until the larvae had fed. Brains from chilled or unchilled donors were equally effective initiators of supernumerary larval apolyses. The capacity to respond to chilling was abolished following bilateral extirpation of the corpora cardiaca and corpora alla@ but not after the corpus cardiacum and corpus allatum,of one side were removed. This effect of bilateral cardiacectomy and allatectomy could be remedied by applying Altosid, a juvenile hormone analog. Potentiation of the larval-larval apolysis by chilling and by JH may involve separate mechanisms, for the analog was less effective on unchilled larvae than on those that had been chilled. The results are discussed with reference to the hypothesis that the brains of young larvae produce an “allatotropic hormone”.

INTRODUCTION KRISHNAKUMARAN(1972) reported that larvae of the wax moth, Galleria mellonella (L.), would undergo supernumerary apolyses if they were injured shortly after their final larval-larval ecdysis. He also noted that brains from such injured larvae would induce extra apolyses when implanted into larvae that were in the second half of the last instar, but that brains from uninjured larvae would not have this effect. This led him to suggest that injury causes the brain to release both an allatotropic and a prothoracotropic hormone. The effect of injury was critically dependent on the way the young larvae had been anesthetized; those injured while chilled on ice would undergo supernumerary apolyses, those operated upon while anesthetized with COZ or ether would not. My unpublished attempts to confirm the observation that surgical injury will induce extra apolyses by young last instar Galleria larvae have led to a different conclusion. In those experiments the incidence of supernumerary larvae was as great amongst chilled, uninjured controls as it was amongst the test animals. The question whether chilling, by itself, stimulates the brain to release the “neurohumoral allatotropic factor” hypothesized by KRISHNAKUMARAN (1972), GFCANGER and SEHNAL (1974), and SEHNAL and GRANGER (1975) motivated me to investigate the phenomenon more closely. Beyond this, the likelihood that chilling may provide a convenient means

of synchronizing the larval moulting program for endocrinological investigations on this species made it seem worthwhile to identify certain of the conditions that influence the effect. MATERIALS

AND

METHODS

Larvae hatched from eggs laid during 24-hr intervals were reared on a cereal-honey-glycerine diet in total darkness at 33 to 35°C (PIPA, 1963). Newly emerged last instar larvae were obtained by segregating penultimate instar larvae in Petri dishes with food, then periodically removing those that had undergone ecdysis. These were kept under the above rea.ring conditions until needed. Groups of last instar larvae of uniform age were routinely chilled for 30min on a sheet of aluminum foil on the surface of melting ice (0 to 1°C). To retard melting, the ice tray was placed in a refrigerator (6 to 9OC) during the treatment period. The larvae were allowed to recover for 30min at room temperature (22 to 25”C), and two of them were placed into each wire gauze-topped, snap-cap vial (30 x 40mm) containing cu. 5 ml of diet. Except for brief periods of daily inspection, these and all the other experimental insects described below were kept in constant darkness at 33 to 35°C. The methods used to extirpate the corpora cardiaca and corpora allata (CC-CA) or to implant brains were those employed previously (PIPA, 1971). After

1641

RUDOLPH L. PIPA

1642

these operations, the larvae were left at room temperature for cu. 12 hr before placing them in the snapcap vials with food. The period required for apolysis was calculated from the time the larvae were put into the incubator. The juvenile hormone analog Altosid (ZR515), isopropyl 11 -methoxy-3,7,11 -trimethyldodeca-2,4dienoate (HENRICK et al., 1973), was dissolved in acetone (100 pg/pl), and to the dorsum of each larva 1~1 was applied topically with a 10~1 capacity syringe (701) fitted with the Chaney Adaption (Hamilton Co., Inc., Whittier CA). Larvae that served as controls in these experiments received 1~1 topical applications of the diluent. Allatectomized larvae were kept at room temperature overnight, and the analog or diluent was applied the following morning, immediately after the larvae had been chilled. The insects were inspected twice daily for the occurrence of ecdyses. Because larval or pupal ecdysis normally happens within 48 to 72 hr after apolysis, the former event is a convenient indicator of the approximate onset of the moulting cycle. For those cases where ecdysis did not follow apolysis, the time it should have occurred was estimated. The pupae will usually cast the larval exuviae and commence to tan within cu. 18 hr after the pharate “Stage III” of PIPA (1963). They also will tan within that period if ecdysis is postponed. Therefore, the presence of a darkening cuticle can be used to estimate when ecdysis by a pharate pupa ought to have taken place. Similarly, the time of larval ecdysis can be predicted by an event that occurs 12 to 24 hr earlier: the protrusion of the head capsule that follows apolysis. RESULTS Ages-related effectiveness

of chilling

The results obtained with last instar larvae that had been chilled at different times after their ecdysis are

summarized in Table 1. The effectiveness of chilling in stimulating supernumerary apolyses by the larvae was clearly related to their age. An average of 89% of the survivors from those that were chilled when less than 16 hr beyond their previous ecdysis underwent apolysis (i.e. became “superlarvae”) within 2 to 3 days. By 46 to 65 hr this diminished to 24% and by 90 to 120hr only 6% responded that way. Unchilled controls rarely became superlarvae; only 2 of 54 that were 24 to 40 hr old and one of 46 that were 46 to 65 hr old did so. Chilling for 30min did not cause an immediate sharp increase in mortality. More than 80% of the larvae survived to become superlarvae or pupae. The superlarvae occasionally underwent an additional larval-larval apolysis, but such a response was erratic; for all of the age groups it ranged from 0 to 40% and averaged 17%. Mortality amongst all superlarvae was high. On average, only 60% pupated (range = 25 to 75%). The development of those that failed to become superlarvae after being chilled seemed, nevertheless, to be affected by the treatment in an age-dependent manner. Twenty-one of the 58 survivors that were 24 to 40hr old when chilled did not become superlarvae (Table 1). These underwent apolysis and, correspondingly, larval-pupal ecdysis 2 days sooner than the 52 non-chilled controls (t = 3.15; P < 0.01). Quite the opposite effect was obtained with larvae older than 123 hr, when chilling caused the length of the larval instar to be extended 150 to 200% beyond that of the controls. Last instar larvae did not need to be chilled at so low a temperature as 0 to 1°C to stimulate supernumerary apolyses. Somewhat higher temperatures were effective, but the treatment period had to be prolonged. When 24 last instar larvae less than one day beyond ecdysis were chilled at 6 to 9°C for 1 hr, only 3 of them became superlarvae, but 8 of 12 chilled

Table 1. Effects of chilling (0 to 1°C for 30min) on subsequent development of last instar Galleria larvae of increasing age Days to larvalpupal ecdysis No. of Age (hr postecdysis) when chilled

No. of replications

<16 2440 46-65 72-88 90-120 123-148 168-228 (Cocoonspinning)

4 6 4 5 5 3 3

chilled larvae surviving 36 58 45 40 31 20 27

Per cent superlarvae ( f SD.) 89 64 24 25 6

* 9.4 + 3.3 f 11.5 f 10.9 + 10.2 0 0

for those not becoming superlarvae Cr,,,* zk S.D.)

8.3 8.8 7.6 7.6 7.7 9.3

+ f + + f +

0.94 0.83 0.80 1.02 0.41 1.25

No. of nonchilled larvae (controls) pupating? 34 52 45 47 26 16 26

* Average time required for half the population to undergo ecdysis. t The three non-chilled controls that underwent another larval-larval ecdysis are excluded.

Days to larval-pupal ecdysis for controls (TX,,* k S.D.) 11.5 10.3 8.8 8.6 7.8 5.0 4.3

f 1.82 + 1.25 f 0.87 f 1.02 +_ 1.10 + 0.08 f 0.89

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Supernumerary instars produced by chilled wax moth larvae Table 2. &e&s

of feeding periOd on incidence of supernumerary apolysis and metamorphosis Galleriu larvae

by chikd

last instar

hr. fed (from ecdysis) before chilling <24 24-30 2531

No. of larvae and treatment after chilling Fed Starved 18

13 0 0 80 0 0 63 0 47 0

18 23 19 20 22

3 t-39 40-4s

8

49-54

15

Per cent superlarvae

27 16

Per cent pupae (N = normal T = malformed*) N T 17 x 20 0 5 25 19 53 69

0 0 a, 0 10 18 0 31 0 25

Per cent dying without apolysing 10 100 96 0 90 77 12 44 0 6

* These were uharate and incompletely tanned. Their antenna& wing, and leg cases were only partly extended, but no larval feat&s were evident. at this temperature

for 2 hr and 5 of 8 chilled for

5 hr did so. Regulation of chill-induced trolled ,feeding

moulting cycles

by con-

Last instar larvae that were chilled had to feed subsequently, or they would not become superlarvae. This was so irrespective of their age or, correspondingly, the length of time they had access to food before being chilled (Table 2). Not unexpe~t~ly, the fate of those individuals that did not become superlarvae after they had been chilled and starved depended on the period they had been held with food beforehand. Nearly all of those that had been fed throughout the first 3Ohr after ecdysis before being chilled and starved died without pupating, but only about 45% did so when fed for 40 to 48 hr before being tested (Table 2). Most of those that had been fed during the entire 49 to 54 hr after ecdysis pupated after they had been chilled and starved. These results reflect the necessity for an obligatory feeding period if last instar Galleria larvae Table 3. Su~rnumerary

Age (hi- postecdysis) when chihed

apolysis and pupation by last instar G~Z~e~ialarvae that were starved before or after being chilled Hr starved (B = before chill A = after chill) B A

<21 24-36 unc~l~ 24-36 48-53

are to pupate (BOUNHIOL,1938; WOOLEVER and PIPA, 1970). The discovery that supem~erary apolyses by the chilled larvae could be prevented simply by depriving them of food provided a means of studying some of the temporal characteristics of the relationship. Last instar larvae younger than 21 hr that had been chilled, starved for 48 hr, and then fed responded like those that had been chilled at the same age, but not starved (Table 3); 82% of them became superlarvae. Similarly, the incidence of supernumerary apolyses amongst last instar larvae that had been chilled when 24 to 36hr old, starved 48 to 68 hr and then fed (67x, Table 3) was like that amongst larvae chilled when 24 to 40 hr old, but not starved (Table 1). Because none of the unchilled controls in this experiment became superlarvae after they had been starved 48 to 68 hr and then fed, it is clear that chilling, not the subsequent starvation, signalled their moulting program. The incidence of supernumerary apolyses depended on the age of the larvae at the time they were chilled and the av~Iab~ity of food afterward. The observation that the percent of superlarvae obtained was

-

No. of larvae

Per cent superlarvae

Per cent pupae

48 0 4868

26 27 33

85 82 67

4 11 12

11 7 21

-

29 22 19

0 91 58

79 5 26

21 4 16

Starved 48-68 48 0

Per cent dying without apolysing

1644

RUDOLPHL. PIPA

positively correlated with the age of the larvae at the time they were chilled and not with their age when fed shows that their propensity to undergo an extra larval apolysis was prolonged by starvation. The disposition of last-instar larvae to undergo supernumerary apolyses also will be extended if they are starved before being chilled. When last instar larvae < 5 hr old were starved for 48 hr and then chilled (48 to 53, Table 3), 91% became superlarvae. Only 58% of the controls that had been fed throughout the 48 hr test period before being chilled responded that way. Supernumerary moulting cycles induced by implanting chilled and unchilled brains Brains removed from last instar Galleria larvae and implanted into others less than 48 hr beyond their last ecdysis will cause a significant proportion of the recipients to undergo extra larval apolyses (PIPA, 1971; GRANGER and SEHNAL, 1974). This observation, and the evidence that low temperatures will stimulate the brains of certain diapausing lepidopteran pupae to secrete prothoracotropic hormone (WILLIAMS, 1946; HIGHNAM, 1958), suggested a mechanism for the action of chilling in Galleria larvae. It was to test the possibility that chilling enhances the ability of brains to induce supernumerary larval apolyses that the relative effectiveness of implanted chilled and unchilled brains was determined. If the incidence of superlarvae is increased by implanting chilled brains, this might provide evidence for the involvement of an allatotropic brain hormone. Brains were removed from last instar larvae that were 72 to 90 hr old. The test donors had been chilled 72 hr previously (i.e. when less than 18 hr beyond ecdysis) and, like the unchilled control donors, were kept with food in total darkness at 33 to 35°C until needed. Two brains were routinely implanted into each unchilled host, a last-instar larva that was 21 to 45 hr beyond its ecdysis. To ascertain the specificity of the response, in several of the trials two thoracic ganglia were implanted instead of brains. In none of those cases did supernumerary apolyses occur, confirming previous results (PEA, 1971; SEHNAL and GRANGER, 1975). In six test replications, 24 of 33 (73 k 21.3%) recipients of chilled brains became superlarvae, while 13 of 28 (46 + 26.7%) of the controls that had received unchilled brains did so. Because these values do not differ from each other significantly (t = 0.1863), the experiment failed to substantiate the hypothesis that chilled brains are more effective initiators of supernumerary larval apolyses than are those that are not chilled. Two trials in which brains from larvae 48 hr beyond chilling were tested also failed to reveal a difference. The onset of larval-pupal apolysis by those recipients of chilled and unchilled brains that did not become superlarvae occurred 5 to 9 days after the operation. Because this value was identical for both

groups, there was no evidence for an increased titer of JH that may have prolonged the larval instar of those that received chilled brains. Necessity for the presence of corpora cardiacs-corpora allata if chilling is to promote supernumerary apolyses SEHNAL and GRANGER (1975) reported that early last instar Galleria larvae that received brain implants would undergo extra apolyses if one corpus cardiacum-corpus allatum (CC-CA) had been removed, but not if both had been removed. This, and the observation that implanted CC-CA prolonged the larval instar and could produce superlarvae without implanting brains led them to hypothesize that the brain secretes an allatotropic hormone. According to their interpretation, this hormone stimulates the corpora allata to release juvenile hormone (JH) in amounts sufficient to induce larval development. These considerations motivated me to determine whether intact CC-CA also were required for supernumerary apolyses by chilled larvae. In three replications of one experiment the CC-CA were extirpated from 20 last instar larvae that were less than 18 hr beyond ecdysis, and the larvae were chilled immediately afterward. The CC-CA of 21 control larvae were removed from one side only. All of the 14 surviving test animals pupated. By contrast, nine of the 16 surviving control insects underwent an additional larval apolysis, and only the remaining seven pupated. These trials clearly demonstrated that at least one CC-CA must be present if chilled larvae are to undergo supernumerary apolyses, but it is uncertain which of the two glands is involved. Because the CC and CA are small, contiguous organs in Galleria they cannot be extirpated or transplanted separately, so a different approach to the problem was explored. EfSects of Altosid on supernumerary apolyses by chilled larvae If removal of the CC-CA prevents supernumerary apolysis because of a concomitant lowering of JH titer, it may be possible to alter the effect by applying the growth regular Altosid, a compound that resembles JH both structurally and by its effect on morphogenesis. Two experiments in which last instar larvae 5 to 23 and 30 to 46 hr beyond ecdysis were allatectomized, chilled, and, immediately thereafter, administered 1OOpg of Altosid substantiated that notion (Table 4A). Within 4 to 10 days the compound promoted supernumerary larval apolysis in 88 to 100% of the test insects, while all of the controls transformed into normal pupae. Did chilling contribute to the effectiveness of Altosid in those trials, or would the compound have been equally potent on unchilled, allatectomized larvae The data obtained by testing larvae ~24 hr beyond ecdysis are summarized in Table 4A and support the former interpretation. Superlarvae were produced

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Supernumerary instars produced by chilled wax moth larvae Table 4. Effects of Altosid on supernumerary

Age (hr postecdysis)

No. of replications

Fraction surviving to next apolysis Test Control

apolysis by chilled last instar G&via

larvae

Percent apolysing and character of next cuticle (L = superlarvae; M = intermediate forms; P = pupae Chilled Chilled Unchilled + + + solvent Altosid Altosid L M L P L M

P

A. Allatectomized 5-23 30-46 <24 B. Unoperated 26-39 74-83 72-83

1 1 4

s/12 5112 24151

9/11 6110 21141

88 100 75

25

2 3 3

15/15 30130 27127

15115 28128 26126

100 67 74

33 26

from 75 + 18.3% of the chilled insects that received the JH-analog, while only 9.5 f 15.6% of the unchilled controls responded that way. Altosid-treated larvae that did not become superlarvae failed to pupate on schedule. The average time from treatment to apolysis was 28 + 4.6 days. All of the resulting individuals were pharate intermediate forms, mostly with pupal exoskeletons, but having larval features expressed to a variable extent. Larval antennae, claws at the tips of the pupal leg cases, and prolegs on the tenth abdominal segment were commonly present. Also, unlike normal pupae, they possessed spiracular peritremes and open spiracles on the eighth abdominal segment. This potentiating effect of Altosid on supernumerary apolyses by chilled last instar larvae also was evident amongst those that had not been allatectomized (Table 4B). When larvae 74 to 83 hr beyond ecdysis were chilled and administered 100,ug of the compound, 67 + 8% became superlarvae. Only 14 + 2% of the chilled controls that received 1~1 of acetone responded that way. Again, it was clear that the effects of chilling and Altosid application were additive, for 74 f 18% of the chilled larvae that had received the JH-analog became superlarvae, while only 15 + 12% of the unchilled controls did so. The chilled and unchilled Altosid-treated larvae apolysed to intermediate forms after a delay of 8 to 18 days, compared to the 3 to 7 days required for the controls to pupate. DISCUSSION The effects of chilling on the development of last instar Galleria larvae were diverse and closely correlated with age. Superlarvae occurred amongst 7.5 to 100% of the survivors of those that were less than 16 hr beyond ecdysis when chilled, but the incidence declined to about 25% for 46 to 88-hr-old larvae and was not evident beyond 120 hr. Those that did not transform into superlarvae after being chilled at 24

12

100 100

73 14

10

90

15

81

27 86 4

to 40 hr appeared, nevertheless, to be affected by the treatment. They pupated 2 days sooner than the unchilled controls. This behavior contrasted sharply with that by last instar larvae chilled when older than 123 hr. Metamorphosis was delayed in that group, and they remained larvae 3 to 5 days longer than the unchilled controls. This last observation confirms and extends those by BURKETT (1962) and BECK (1970). During a study independent from mine, &anger (personal communication) also observed that chilling stimulated young, last instar larvae to undergo supernumerary apolyses, but the response was not so pronounced. Only 35 to 40% of the larvae less than 8 hr beyond ecdysis became superlarvae after being chilled on melting ice 30 to 45 min. This points to the possibility that various strains of Galleria may react differently. While my report was in manuscript form, B. Cymborowski and M. J. Bogus kindly provided me with a copy of their paper (1976), at that time in galley proof, that documents further the efficacy of chilling as a promoter of supernumerary apolyses by young, last instar Galleria larvae. Their strain however, seems to be much less sensitive to chilling than mine, for they had to cool l-day-old larvae 2 to 3hr at 0 to 1°C in order to elicit the response in 65 to 100% of their test population. Although the relationship needs to be defined quantitatively before its significance with respect to nutrition can be ascertained, there was evidence that food availability influenced the responsiveness of Galleria larvae to chilling. It was essential that the larvae had fed after being chilled if they were to undergo extra apolyses. Those that did not, failed to become superlarvae irrespective of the length of time they were allowed to feed before they were chilled. This contrasted with the requirement for metamorphosis, when a feeding period of 49 to 54 hr immediately following ecdysis enabled most of the larvae to pupate when subsequently chilled and starved.

1646

RUDOLPHL. PIPA

The available data (Pipa, unpublished) indicate that most young (~4 hr) last instar larvae, if they are to become superlarvae, require 35 to 45 mg of diet after they have been chilled. A small fraction, nevertheless, responded after ingesting as little as 25 mg. By comparison, most of those that failed to become superlarvae required 65 to 75 mg of diet before they could pupate normally, and none pupated without ingesting at least 35 mg. The period during which chilling would induce supernumerary apolyses by young last instar larvae could be lengthened simply by depriving the larvae of food beforehand. Similarly, early last instar larvae that had been chilled, starved 48 hr, and then fed demonstrated a frequency of supernumerary apolyses like the controls that had been chilled but not starved. The predisposition to become superlarvae, though correlated with age, could be extended by starvation. Chilling signaled the occurrence of the larval moulting program, but its expression was delayed until after the larvae had fed. The finding that early last instar larvae can be made to undergo supernumerary apolyses simply by chilling them casts considerable doubt on the interpretation by KRISHNAKUMARAN(1972), that in his experiments with cold-anesthetized larvae surgical injury caused the response. This effect of chilling also explains why he was able to obtain superlarvae from young last instar larvae that had been injured while immobilized on melting ice, but by very few of those anesthetized with ether or COZ. Brains removed from normal larvae can initiate supernumerary apolyses by young, unchilled, last instar larvae (PIPA, 1971; SEHNAL and GRANGER, 1975), so it is clear that the donor need not be injured prior to the time of brain extirpation to stimulate production of the hypothetical “neurohumoral allatotropic factor”. Nevertheless, KRISHNAKUMARAN (1972) reported that superlarvae could be produced by implanting brains from 2 to 3-day-old injured larvae into 4-day-old intact larvae. In that experiment none of the control group that received brains from intact donors became superlarvae, though, like the experimental group, they had been anesthetized by chilling. These data do not seem to be explainable in terms of a differential sensitivity of the larvae to chilling. They may support the “allatotropic hormone” hypothesis, or have a different interpretation. The brain implantation experiments performed during the present study failed to substantiate the notion that chilling induces supernumerary larval apolyses by stimulating secretion of an allatotropic brain hormone. When the data were evaluated statistically, the capacity of chilled and unchilled brains to initiate extra moults was found to be equal. Also, in contrast to what might have been expected had the implants stimulated JH production, the pupation of those individuals that failed to become superlarvae was not delayed. If the chilled larvae did not need to feed in order

to undergo supernumerary apolyses, it might have been possible to determine by ligation or extirpation experiments whether the effect of chilling requires the presence of the brain. Unfortunately that was not the case, and the likelihood that chilling activates the larval moulting program by a non-neuroendocrine mechanism remains to be considered. Allatectomy affected the sensitivity of young, last instar larvae to chilling like it did their responsiveness to brain implants (SEHNAL and GRANGER, 1975): supernumerary apolysis was abolished when both corpora cardiaca and corpora allata were extirpated, but not when the corpus cardiacum and corpus allaturn of one side were removed. For young larvae that had been allatectomized bilaterally and then chilled, the capacity to become superlarvae was restored by applying the JH-analog Altosid. This compound appeared to enhance, not replace, the effect of chilling, for it was much less active when administered to unchilled, allatectomized larvae than when it was applied to those that had been chilled. This suggests that although supernumerary larval apolyses will not occur in the absence of JH, the effects of chilling and JH are additive, and may involve separate mechanisms. A similar difference in activity related to chilling was observed after the compound was applied to intact larvae. The reason why larvae without corpora allata would not undergo extra apolyses unless Altosid was applied is not clear. The assumption that is most consistent with the “classical scheme” (WIGGLESWORTH, 1964; DOANE, 1973) is that allatectomy reduced the JH-titer below the level that would permit expression of the larval genetic program at the next apolysis. Another possibility is that this compound promoted ecdysone production, an effect that the juvenile hormones seem to have on certain lepidopteran pupae (GILBERT and SCHNEIDERMAN,1959; OBE~LANDERand SCHNEIDERMAN,1966). Acknowledgements-I thank Dr. G. B. STAAL,Zoecon Corporation, Palo Alto, CA, for the gift of Altosid, and Dr. NOELLEGRANGER,University of California, Irvine, for permission to include some of her unpublished observations.

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Supernumerary instars produced by chilled wax moth larvae DOANEW. W. (1973) Role of hormones in insect development. In Developmental Systems: Insects (Ed. by COLJNCE S. J. and WADDINGTONC. H.) pp. 291497. Academic Press, London. GILBERTL. I. and SCHNEIDERMAN H. A. (1959) Prothoracic gland stimulation by juvenile hormone extracts of insects. Nature, Lond. 184, 171-173. GRANGERN. A. and SEHNALF. (1974) Regulation of larval corpora allata in Galleria mellonella. Nature, Lond. 251, 415417. HENRICKC. A., STAALG. B., and SLDDALLJ. B. (1973) Alkyl 3,7,1l-trimethyl-2,4_dodecadienoates, a new class of potent insect growth regulators with juvenile hormone activity. J. Agric. Fd. Chem. 21, 354-359. HIGHNAMK. C. (1958) Activity of the brain/corpora cardiaca system during pupal diapause “break” in Mimas tiliae (Lepidoptera). Quart. J. micr. Sci. 99, 73-88. KRISHNAKUMARAN A. (1972) Injury induced molting in Galleria mellonella larvae. Biol. Bull., Woods Hole 142, 281-292. OBWLANDERH. and SCHNEIDERMAN H. A. (1966) Juvenile

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hormone and RNA synthesis in pupal tissues of saturniid moths. J. Insect Physiol. 12, 37-41. PIPA R. L. (1963) Studies on the hexapod nervous system-VI. Ventral nerve cord shortening; a metamorphic process in Galleria mellonella (L.) (Lepidoptera, Pyrallidae). Biol. Bull., Woods Hole 124, 293-302. PIPA R. L. (1971) Neuroendocrine involvement in the delayed pupation of space-deprived Galleria mellonella (Lepidoptera). J. Insect Physiol. 17, 2441-2450. SEHNALF. and GRANGEXN. A. (1975) Control of corpora allata function in larvae of Galleria mellonella. Biol. Bull., Woods Hole 148, 106-116. WIGGLESWORTH V. B. (1964) The hormonal regulation of growth and reproduction in insects. Adv. Insect Physiol. 2, 247-336.

WILLIAMSC. M. (1946) Physiology of insect diapause: the role of the brain in the production and termination of pupal dormancy in the giant silkworm, Platysamia cecropia. Biol. Bull., Woods Hole 90, 234-243.

WCJCILEVER P. and PIPA R. L. (1970) Spatial and feeding requirements for pupation of last instar larval Galleria mellonella (Lepidoptera). J. Insect PhysioJ. 16, 251-262.