Factors influencing the developmental capacity of Galleria larval epidermis

3. Insect Phytid., 1975, Vol. 21, pp. 705 to 712. Pergumon Press. Printed in Great Britain

FACTORS INFLUENCING THE DEVELOPMENTAL CAPACITY OF GALL.Z3RlTA LARVAL EPIDERMIS WILLIAM

H. KASTERN”

and A. KRISHNAKUMARAN-I_

Department of Biology, Marquette University, Milwaukee, Wisconsin 53233, U.S.A. (Received

10 September

1974)

Abstract-The nature of the cuticle secreted by integument from a day-l penultimate instar larval Galleria when cultured in uivo in the abdomen of a last instar larva varied with the age of the host. When placed in a day-5 last instar larva, the implanted integument secreted a pupal cuticle at the time the host metamorphosed and became a pupa. However, when placed in a day-7 last instar larva the implant, from the same stage donor, secreted a larval cuticle at the time the host pupated. Experimental studies involving implantation of the integument for a 24 hr period, into various developmental stages of normal and ligated last instar larvae, pupae, and pharate adults, prior to placing it in a day-7 last instar larva suggest that a non-hormonal factor present in day-4 and -5 last instar larvae is important to initiate pupal syntheses. INTRODUCTION

DURINGpost-embryonic development of endopterygote insects, the epidermal cells after secreting a succession of larval cuticles deposit a pupal cuticle and then an adult cuticle. This successive morphogenetic expression of the epidermal cell genome has been shown to be under the control of two insect hormones, ecdysone and juvenile hormone. It is assumed that in the presence of high titres of both ecdysone and juvenile hormone (JH) the larval epidermal cells secrete another larval cuticle, whereas in the presence of a reduced titre of JH the cells secrete a pupal cuticle and in the absence of JH (but in the presence of ecdysone) they secrete an adult cuticle (PIEPHO, 1952; WIGGLESWORTH,1964). Although normally a larval insect maintains its larval morphology through a definite number of instars, in Lepidoptera and some other insects, premature metamorphosis can be achieved by surgical removal of the corpora allata, the source of JH, before completion of the characteristic number of larval instars (BQUNHIOL, 1939; FUKUDA, 1944). In addition, even a first or second instar Gall&a larval integument will skip the other larval stages and will deposit a pupal cuticle if implanted into a last larval instar (PIEPHO, 1938). In the course of a study of the relationship between reprogramming of Gnlleria larval epidermal cells (to secrete a pupal cuticle) and DNA synthesis we observed * Present address: Department of Biology, Wesleyan University, Middletown, Connecticut 01457. -I_To whom reprint requests should be sent. 705

706

WILLIAMH. KASTEFCN ANDA. KRISHNAKUMARAN

that the nature of the cuticle deposited by the epidermis from a penultimate instar larva on implantation into last instar larvae depended on the age of the host as well as the age of the donor. This afforded us an opportunity to examine the factors involved in the regulation of the type of cuticle secreted by the penultimate larval epidermis. The data to be presented in this communication demonstate that day-l penultimate instar larval epidermis secretes a larval cuticle if placed in a day-7 last instar larva and a pupal cuticle if placed in day-5 last instar larvae. Further, the observations suggest that the time available from implantation to stimulation of cuticular synthesis and a factor present in the 3 to 5 day last instar larvae influence the metamorphosis. The significance of these observations in the understanding of the time of action of JH is discussed.

MATERIALS

AND METHODS

Larvae of the greater wax moth, Galhia mellonella, were reared in the laboratory according to procedures described earlier (KRISHNAKUMARAN,1972). Penultimate and last instar larvae, recognizable by their pigmentation, weight, and the size of the head capsule (BECK, 1960), were selected from the main culture dish and were maintained in plastic Petri dishes. The day of ecdysis into a instar is designated as day 1 and after the subsequent 24 hr intervals day 2, etc. The penultimate larval instar lasts about 4 days whereas the period from ecdysis into the last larval instar to pupal ecdysis is about 9 days. Apolysis preparatory to pupal ecdysis occurs on day 8 and animals after this stage are recognized as the pharate pupae. The terms penultimate larvae and last larvae will be used in the text to denote the penultimate and the last instar larvae, respectively. A 1 mm square piece of integument was excised with iridectomy scissors from larvae anaesthetized on ice, and was immediately implanted into the abdominal cavity of an appropriately aged last larva. The implant was always obtained from the fourth abdominal segment so as to avoid any variation resulting from anteroposterior gradients in the schedule of metamorphic changes. After placing in the wound a mixture of phenyl thiourea, penicillin, and streptomycin (2 : 1 : 1) the wound was sealed with molten wax. The larvae were allowed to remain on ice for an additional hour before they were transferred to a Petri dish containing food. To prepare ligated larval abdomens, anaesthetized larvae were ligated with No. 6 Deknatel silk thread behind the prothoracic segment following which the head and prothoracic segments were removed and the wound sealed with molten wax. Injured larvae, used in this study, were prepared by keeping the larvae on crushed ice for 4 hr and the injury inflicted in the course of implantation of integument together with chilling induced an additional larval ecdysis in 4 days. The last larvae that received the implant pupated either on schedule or after a delay depending on their age at the time of implantation. Within 12 to 18 hr after pupation the implant was retrieved and the nature of the secreted cuticle was determined by microscopic examination. A larval cuticle was recognized by

707

MBTAMORPHOSIS OF LARVALEPIDERMIS

the surface pattern, the plaques, the presence of larval bristles, and the absence of pigment whereas the pupal cuticle had characteristic tanned amber coloration and rough surface ridges (see KASTEBN and KBISHNAKUMABAN, 1974). The implanted integument rounded up and became a closed vesicle with the epidermis on the outer surface and the cuticle facing the interior of the vesicle whenever the implant secreted a pupal cuticle. However, when an implant secreted a larval cuticle a closed vesicle was not formed. Routinely the vesicle was carefully dissected and examined to determine whether the implant secreted more than one cuticle. RESULTS

Nature of the cuticle secreted by the implant in relation to the age of the donor and the age of the host The results reported in Table 1 show that the day-l penultimate larval integument implanted into day-5 last larvae deposited a pupal cuticle more frequently (88*2 per cent) than a larval cuticle (11.8 per cent), whereas the same tissue on implantation into a day-6 or -7 last larva deposited more frequently a larval cuticle (60 and 100 per cent respectively) thereby suggesting that the day-l TABLE l-EPPECT OF THE AGE OF THE DONORAND OF THE HOST ON THE NATUREOF THE CUTICLEDEPOSITED BY A PENULTIMATE LARVALINTEGUMENT IMPLANTEDINTOA LASTINSTAB LARVAIN Galleria

Age of the host* (last instar larva)

Age of the donor* (penultimate instar larva)

No. of successful implants

Type of cuticle secreted by the implant Pupal

Larval

Day 3

Day 1

6

Day 5

Day 1 Day 2 Day 3

34 17 7

4 (11.8%) 10 (58.6%) 6 (85.7%)

30 (88.2%) 7 (41.4%) 1 (14.3%)

Day 6

Day l Day 2 Day 3

50 11 8

30 (60%) 11 (100%) 8 (100%)

20 (40%) 0 0

Day 7

Day 1

16

16 (100%)

0

Day-l injured larvae-t

Day 1

9

9 (100%)

0

0

6 (100%)

* The day of ecdysis into an instar is designated as day 1. t Day-l last instar larvae stimulated to ecdyse into supernumerary larvae by injury were used as hosts. A piece of integument (1 mm) was excised from a penultimate larva and was implanted into a last instar larva. Three to five days later when the host pupated the implant was retrieved and examined to establish the nature of the cuticle.

708

WILLIAM H. KASTERN AND A. KRISHNMCUMARAN

penultimate larval integument is developmentally uncommitted and can make either a larval or pupal cuticle depending on the hormonal milieu. In day-3 last larval hosts the implants make only a pupal cuticle, while in day-8 hosts, which are pharate pupae, they do not make any new cuticle. In contrast to the day-l penultimate larval integument the day-2 integument deposited a larval cuticle more frequently under similar hormonal conditions (58 per cent in day-5 and 100 per cent in day-6 last larval hosts), and the day-3 penultimate larval integument deposited a larval cuticle 100 per cent in day-6 hosts and 85 per cent in day-5 hosts, thereby suggesting that with increasing age, the penultimate larval integument is progressively committed to make a larval integument. In addition, the data show that the implant deposited a larval cuticle when placed in day-l last instar larva that is caused to moult by injury and ecdyse into an additional larva. Analysis of the factors integument

that stimulate

pupal

syntheses in day-l

penultimate

larval

Since the day-l penultimate larval integument secreted a pupal cuticle in day-5 last larval hosts and a larval cuticle in day-7 hosts we examined the factors that influence this reprogramming of the penultimate larval integument. Day-5 last larvae pupate about 5 days after implantation while day-7 last larvae pupate within 3 days. Therefore, we determined the effect of prolonging the duration from excision to exposure to a hormonal milieu on the nature of cuticle deposited. For this purpose day-l penultimate larval integument was first implanted into day-l pupae or day-l, -2, -3, or -4 pharate adults and 24 hr later was retrieved and reimplanted into day-7 last larvae. When these larval hosts pupated the implant was removed and the nature of the cuticle was determined. The results recorded in Table 2(A) sh ow that prolongation of the period by implanting in 2-, 3-, or 4-day-old pharate adults had no effect and all implants secreted a larval cuticle. However, if the primary host was day-l pupa or day-l pharate adult, 20 per cent of the implants secreted a pupal cuticle thereby suggesting that these pupae may have a factor necessary for stimulation of pupal synthesis. As pupae immediately following ecdysis and day-l pharate adults contain ecdysone and brain hormone we examined the effect of using last instar larvae of diverse ages as primary hosts on the syntheses by the implant. The results recorded in Table 2(B) suggest that day-l last larvae lack the factor that stimulates pupal syntheses and that the day-4 and -5 last larvae stimulated pupal syntheses in 85.7 and 88.2 per cent of the implants. Day-2, -3, and -6 last larvae also stimulated pupal syntheses but were not as effective as day-4 and -5 larvae. Because the day-l last larvae did not influence the developmental programme of the implant we next examined the time of appearance and the nature of the factor that was present in older larvae. In one series of experiments we ligated the larvae behind the prothoracic segment, a procedure that removed all known neuroendocrine centres. Twenty-four hours later the ligated abdomen was used as a primary host for the implant. A day later the implant was removed and reimplanted into day-7 last larva where it remained until the host underwent

709

OF LARVALEPIDERMIS

MET.~MOR~HOSIS

TABLEZ-EFFECT OF PRIOR in viva CULTURE ON THE NATURE OF THE CUTICLE SECRETED BY PENULTIMATE LARVAL INTEGUMENT ON IMPLANTATION INTO DAY-7 LAST INSTARLARVAB Type of cuticle secreted by the implant

Primary host Developmental A.

B.

C.

stage

Pupa Pharate adult

Last instar larva

Ligated larvae*

Age (days)

Larval

1 1 2 3 4

PUDd

2 (22%) 2 (20%) 0 0 0

1 2 3 4 5 6

11 11 9 4 4 30

3; 19 24 30 20

(76%) (68%) (85.7%) (88.2%) (40%)

1 2 3

6 7 10 2 5

3 15 I5 12 12

(33%) (68%) (60%) (80%) (70.6%)

tt

* Age at the time of ligation. Larvae were used as primary hosts 24 hr after ligation. t Day 1 last instar larvae that were caused to undergo an extra-larval moult were used as secondary hosts. Integument from a day-l penultimate instar Galleria larva was excised and implanted first into the primary host. Twenty-four hours later the implant was retrieved and reimplanted into a day-7 last instar GaZZerialarva. When the host pupated the implant was retrieved and the nature of the cuticle was determined.

pupal ecdysis. The nature of the cuticle deposited by the implant was determined and is recorded in Table Z(C). The data show that even in the absence of the neuroendocrine centres the ligated larvae had the ability to stimulate pupal syntheses in several implants. Even ligated day-l last larvae stimulated 33 per cent of the implants to deposit pupal cuticle. In another series the fact that the day-l last instar can be caused to moult into supernumerary larvae by injury (KRISHNAKUMARAN, 1972) was exploited. On implantation of an integument from a day-l penultimate larva into an injured day-l last larva, the implant deposited a larval cuticle while the host ecdysed into a supernumerary larva (see Table 1). However, if the same integument was first implanted into a day-5 ligated larval abdomen and 24 hr later implanted into

WILLIAM H, KASTERNANY A. KRISHNAKUMARAN

710

an injured day-l larva, it deposited a pupal cuticle (Table ZC), thereby demonstrating the presence of some factor in these ligated larvae that caused reprogramming of the epidermal cells.

Day-3 penultimate larval epidermis usually secreted a larval cuticle when implanted into day-5 or -6 last larvae (Table 1). In order to examine whether this commitment can be altered, day-3 penultimate larvae were ligated behind the prothoracic segment and 48 hr later a piece of integument was excised and implanted into day-5 last larvae. The results, recorded in Table 3, show that such TABISZ~-DE~ELOPMENTAL

Age of donor

CAPACITY OF PENULTIMATE AFTER LIGATION

Treatment

INSTAB

GaZk&u LARVAE

No. that deposited larval cuticle

No. that deposited pupal cuticle

10

7 (41.4%)

2

None

2

Ligated 48 hr before use of the integument

3

11 (785%)

3

None

6

1 (14.3%)

3

Ligated 48 hr before use of the integument

4

10 (71%)

Penultimate instar larvae were ligated on day 2 or 3 after ecdysis into the stadium. Forty-eight hours later a piece of the integument was implanted into day-5 last instar larvae. Following pupation of the host the nature of the cuticle deposited by the implant was examined.

implants deposited pupal cuticle in 10 out of 14 cases, thereby suggesting that the commitment to secrete a larval cuticle is not irreversible. Day-2 penultimate larval epidermis that normally deposits a larval (58 per cent) cuticle when excised and implanted into day-5 last larval hosts, 48 hr after ligation, deposited a pupal cuticle more frequently (78.5 per cent). Similar results were earlier reported by other workers who exposed ligated penultimate Galleria (SEHNAL, 1972) and Bombyx larvae (KIMURA, 1974) to ecdysone. DISCTJSSION

The results reported above clearly show that the developmental programming of the penultimate larval epidermis is influenced by the age of the host and through its hormonal milieu. When exposed to a hormonal environment that stimulates prompt synthesis of cuticle (e.g. day-7 last larvae and injured day-l larvae) the day-l penultimate larval implant secreted a larval cuticle. It is known that ecdysone level in ~0~~ larvae is high just before pupation (SHAAYA and KARLSON, 1965) and

METAMORPHOSIS OF LARVAL EPIDERMIS

711

presumably a similar high ecdysone titre in Galkria caused the expression of the native developmental state of the implant. However, the same implant deposited a pupal cuticle when implanted into day-3 or -5 last larval hosts. Expression of the larval character when exposed to ecdysone titres that stimulate prompt cuticle synthesis and a metamorphic cuticle when given more time is probably similar to the juvenilizing effects of ecdysone first described in Bombyx (KOBAYASHI et al., 1967) and later confirmed in Cynthia (WILLIAMS, 1968) and Tenebrio (SCHNEIDERMAN et al., 1969). However, the physiological basis for the difference between the day-5 and -7 last larval hosts is not clear. It is not merely a question of time available from explantation to stimulation of cuticle syntheses as is obvious from the experiments with the pharate adults as primary hosts (Table 2A). The facts that the use of day-l pupae to some extent and the days 2 to 5 last larvae as the primary hosts more effectively led to reprogramming of the implant suggest that some factor(s) present in these stages influence the developmental state of the implant. The factor(s) present in the mid last instar larvae could be a hormonal agent such as ecdysone. However, the fact that larvae ligated 24 hr prior to implantation also stimulated pupal synthesis suggests that the hormones may not be involved. This is supported by the fact that even ligated day-l last larvae, lacking all the known sources of the insect hormones, stimulated pupal synthesis, if we assume that the endogenous hormones, if any, are likely to be metabolized in the 24 hr period following ligation. Hence we suggest that the factor(s) is a non-hormonal agent probably involved in JH inactivation. Carboxyl esterase reported in the mid-last instar larvae of lManduca (WEIRICN et al., 1973) could be such an agent. In addition the fact that integument from day-3 penultimate larvae ligated 48 hr prior to explantation deposited a pupal cuticle unlike normal day-3 penultimate larval integument supports this view. Since inactivation of JH may be about the only change that may occur after ligation of these larvae, the alteration of the developmental commitment from a larval to a pupal synthesis suggests the change in the developmental programme results from JH inactivation. Earlier, WILLIAMS (1961) suggested that metamorphosis of the Cecropia larval epidermis requires the activation of the pupal genome by ecdysone in the absence of JH. The present observations that the stimulation of pupal synthesis in the day-l and -3 penultimate larval integument occurs apparently after inactivation of JH support this view. Furthermore, the fact that even the day-3 penultimate larval integument (24-36 hr prior to normal larval ecdysis) can be reprogrammed to secrete a pupal cuticle suggests that JH does not evoke any irreversible covert effects, and that JH to inhibit metamorphosis must be present at the time of ecdysone stimulation of cuticle synthesis. Acknowledgements-The research was supported by funds from Marquette University. Thanks are due to Dr. W. F. MILLINGTONfor his helpful comments on the manuscript. REFERENCES

BECK S. D. (1960) Growth and development of the greater wax moth, Galhia (L.).

Trans. Wis. Acad. Sci. Arts Lett. 49, 137-148.

mellonella

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WILLIAM H. KASTERNAND A. KRISHNAKUMARAN

BOUNNIOLJ. J. (1938) Recherches experimentales sur le determinisme de la metamorphose chez les lepidoptbres. Bull. Biol. Fr. Belg. (Suppl.) 24, 1-199. FUKUDAS. (1944) The hormonal mechanism of larval moulting and metamorphosis in the silkworm. r. Fat. Sci. Tokyo Univ. (Sect. 4) 6, 477-532. JKASTERNW. H. and KRISHNAKUMARAN A. (1975) Reprogramming in the absence of DNA synthesis in Galleria larval epidermis. In preparation. KIMURA S. (1974) Relationship between hormone titres and RNA and protein syntheses when the change to the pupal programme occurs in the silkworm, Bombyx mori. J. Insect Physiol. 20, 887-895. KOBAYA~HIM., TAKEMOTOT., QGAWAS., and NISHIMOTON. (1967) The moulting hormone activity of ecdysterone and inokosterone isolated from Achyranthes radix. J. Insect Physiol. 13, 1395-1399. KRISHNAKUMARAN A. (1972) Injury induced molting in Galleria mellonella larvae. Biol. Bull., Woods Hole 142, 281-292. PIEPHO H. (1938) aber die experimentielle Ausliisbarkeit iiberzlihliger Hgutungen und vorzeitiger Verpuppung am Hautstucken bei Kleinschmetterlingen. Naturwiss. 26, 841-842. PIEPHO H. (1951) uber die Lenkung der Insektenmetamorphose durch hormone. Verb. dtsch. Zool. Ges (Wilhelmshaven) 62-75. SCHNEIDERMAN H. A., KRISHNAKUMARAN A., BRYANTP. J., and SEHNALF. (1969) Endocrinological and genetic strategies in insect control. Proc. Symp. ala Potentials in Crop Protection. N.Y. St. Agri. Exp. Sta., Geneva, New York, pp. 14-25. SEHNALF. (1972) Action of ecdysone on ligated larvae of Galleria mellonella L. : induction of development. Acta ent. bohemoslov 69, 143-155. SHAA~AE. and KARLSONP. (1965) Der Ecdysontiter WBhrend der InsektenentwicklungIV. Die entwicklung der Lepidopteren, Bombyx mori und Cerura vinula L. Devel. Biol. 11, 424-432. WEIRICH G., WREN J., and SIDDALLJ. B. (1973) Developmental changes of the juvenile hormone esterase activity in haemolymph of the tobacco hornworm, Manduca sexta. Insect Biochem. 3, 397-407. WIGGLESWORTHV. B. (1964) The hormonal regulation of growth and reproduction in insects. Adv. Insect Physiol. 2, 248-335. WILLIAMS C. M. (1961) The juvenile hormone--II. Its role in the endocrine control of molting, pupation and adult development in the Cecropia silkworm. Biol. Bull., Woods Hole 121, 572-585. WILLIAMS C. M. (1968) Ecdysone and ecdysone-analogues: their assay and action on diapausing pupae of the Cynthia silkworm. Biol. Bull., Woods Hole 134, 344-355.