Ent. exp. & appl. 28 (1980) 259-261. Ned. Entomol. Ver. Amsterdam

INDUCTION OF SUPERNUMERARY MOLTING BY STARVATION IN MANDUCA SEXTA LARVAE

BY

DAVY JONES*, GRACE JONES*, AND GOVINDAN BHASKARAN

Institute of Developmental Biology, Texas A & M University, College Station, Texas 77843, U.S.A.

Studies were carried out on the effects of prolonged starvation on the development of fifth- (last-) instar larvae of the tobacco hornworm Manduca sexta. Following ecdysis, larvae were starved for varying lengths of time and subsequently fed normal diet. The percent of starved larvae molting to sixth instars increased, while the percent survival decreased with increasing length of the starvation period. When larvae were provided with agar as a source of water during the starvation period, the percent survival increased, but the percent undergoing supernumerary molting decreased. The optimal condition for maximum survival and supernumerary molting appeared to be 3 days of starvation with 0.5- 1.0 g of agar provided on day 0. The endocrine basis for the supernumerary larval molt induced by starvation is briefly discussed.

In insects, molting and morphogenesis are controlled directly by hormones secreted by the prothoracic glands (PG) and the corpora allata (CA). The activity of these glands is regulated by neurohormonal or neural signals from the brain. These control centers in the brain are in turn influenced by environmental factors either directly or indirectly through altered internal milieu (Wigglesworth, 1954). The number and nature of the molts can frequently be altered by external factors, chiefly temperature and the quality and quantity of food. The morphogenetic responses to these factors vary with the species. Thus, starvation causes Trogoderma larvae to undergo stationary molts and the larvae even decrease in size during this period (Beck, 1971). Shortage of food induces some species of lepidopteran larvae to prolong larval life by increasing the duration of the intermolt period to allow for full growth (Skoblo, 1935). Reduced food supply leads to precocious pupation in some species (Lower, 1961), while in others it causes supernumerary larval molts (Kellog & Bell, 1904; Gaines & Campbell, 1935; Beck, 1950).

Starvation of mid-fourth instar larvae of the tobacco hornworm Munduca sextu (L) produces undersized fifth instars with undersized head capsules and such larvae undergo one or more supernumerary molts (Nijhout, 1975a). Fifth- (final-) instar larvae (l-2 days old) weighing 3.0-4.0 g or less do not undergo extra larval

* Present address: Department of Entomology, University of California, Riverside, California 92521 U.S.A.

260 DAVY JONES. GRACE JONES AND GOVINDAN BHASKARAN

molts when starved but form larval-pupal intermediates. Starvation above those weights does not affect either the timing of prothoracicotropic hormone (PTTH) release or pupation (Nijhout & Williams, 1974a).

The fact that starvation of young fifth instars gives rise to larval-pupal intermediates suggests that during the initiation of the molt a sufficient amount of juvenile hormone (JH) is present to inhibit the normal larval-pupal transformation. Evidentlyqin such larvae the CA are either not turned off (Nijhout & Williams, 1974b) or are reactivated due to starvation.

The possibility of using such starved larvae for studying the regulation of CA activity in the last larval instar led us to a more detailed investigation of the effects of starvation on the timing and nature of the subsequent molt. The present paper describes some physiological parameters of normal fourth- and fifth-instar development in our culture of M. sexra and the conditions of starvation which disrupt normal development by inducing a supernumerary molt. The neuro- endocrine bases for the starvation induced supernumerary molt are reported else- where (Bhaskaran & Jones, 1980).

MATERIALS AND METHODS

M. sexru larvae were reared on artificial diet (Yamamoto, 1969) under a 16 : 8 light-dark cycle (with lights off at midnight, 24: 00) at 27” and 60% RH. Observations on the gating phenomenon (Truman, 1972) in larvae under these conditions were made by collecting third-stage larvae showing head capsule slippage and noting the time of ecdysis to the fourth instar. Thereafter larvae were weighed daily and the time of head capsule slippage and ecdysis to the fifth instar were recorded. Since more larvae ecdysed to the fifth instar between 18 : 00 and 21 : 00 than at any other time interval, larvae collected during this period were used for all starvation experiments. Larvae were maintained without food and water until lO:OO-13:OO of the following day, which is regarded as day 0. They were then either fed on normal diet (controls) or starved with or without agar as noted in the results. Normally-fed fifth-stage larvae were weighed daily and the timing of appearance of the frosted frass (fecal pellets coated with a white substance appear approximately 24 hr prior to purging of the gut), and/or exposure of dorsal vessel (accompanies purging of the gut and marks the onset of the “wandering stage”), were recorded (Nijhout 8c Williams, 1974a). For the starvation experiments larvae were weighed on day 0, as well as prior to refeeding, and randomly selected individuals were weighed either at head capsule slippage in the event of a supernumerary molt or at frosted frass for a pupal molt. In all cases, weights and developmental changes were recorded between lO:OO-13:OO hr.

RESULTS

Normal development of larvae Ecdysis to fourth andfifth instars. Since in mass cultures ecdysis to fourth and fifth

MOLTING BY STARVATION IN MANDIJCA 261

TABLE I

Time of ecdysis to fourth orfifth instars of Manduca sexta larvae

Number Larval stage conditmn % ecdysing condmon’ and % % ecdysmg durmg mterval

at 15.00 ‘5’CKL-‘8:oo ““ma;‘;;~$’ 18.00--21 00 21~00-24 00 after24.00

96 HCSb 3rd mixture of 14.6 39.6 BM 36 5 21 I.0 GM, BM’ 458GM 2. I 9.4 34.3

98 HCS 4th GM 0.0 lOO.OGM, BM 0.0 12.2 87.8

87 HCS 4th BM 27 6 72.4 BM 43.7 24. I 4.6

a Mandibles of the next instar green (GM) or black (BM) beneath slipping head capsule. b HCS - head capsule slippage.

instars was seldom observed during the first half of the photophase, observations on selected groups of animals were made only during the second half of the photophase.

Third-stage larvae showing head capsule slippage were collected at 15.00 hr and examined at 18.00 hr. In 40% of these larvae the fourth-instar mandibles beneath the old (third instar) head capsule were black while in 46% the mandibles were still green (GM). Almost all of the black mandible larvae ecdysed during the following 3 hr(18.00--2l.OO)(TableI).

Fourth-stage larvae with head capsule slippage were separated at 15.00 hr into black and green mandible types. Nearly all of the black mandible larvae ecdysed to the fifth instar by 24.00 hr. A majority of the black mandible larvae ecdysed between 18.00 and 21.00 hr with lesser but equal numbers between 15.00-18.00 and 21.00-24.00 hr periods (Table I). It is thus evident that ecdysis of black mandible pharate fourth and fifth instars occurs predominantly during the late photophase. Most green mandible larvae ecdysed during the following scotophase.

Gating and weight gain. Fifty-five third-stage larvae showing head capsule slippage were used for observations on gating (Truman, 1972) and weight gain, and 47 of these survived to the prepupal stage. Gate 1 fourth instars gained weight faster than gate 2 larvae (Fig. 1, A, B) and ecdysed to the fifth instar during the late photophase of day 2 (Table I). Gate 2 larvae ecdysed one day later and were heavier than gate 1 larvae.

Gate 1 and 2 larvae after ecdysis to the fifth instar again differed in the rate of growth. About 36% of them exhibited frosted frass on day 2 at which time they weighed close to 6.0 g. These larvae exposed the dorsal vessel on day 3 and weighed almost 8.0 g by then. A majority of the remaining larvae weighed less than 5.0 g on day 2 and waited until day 3 to excrete frosted frass. Similar variations were also observed among gate 2 larvae after ecdysis to the fifth stage (Fig. 1) that is, gate 2 fourths could give rise to either Gate 1 or gate 2 fifths.

A group of 42 fifth instar larvae which ecdysed between 18.00 and 21.00 hr was starved until 10.00 hr of the following day, that is, day 0. A few exhibited frosted frass on day 2 (19%) while most (83%) did so on day 3 and exposed the dorsal

262 DAVY JONES, GRACEJONES AND GO~INDANB~ASKARAN

0 2 3fOl 1 2 3 4

IV lnstaf (day1 V lnstar ldayf

AGE

IV lnster (day) V lnstar (day)

AGE

Fig. 1. Growth of fourth- and fiith-instar M. Sextu larvae reared at 27O and 16 : 8 L : D cycle, A Gate 1 larvae, B. Gate 2 larvae. Timing of ecdysis to fifth instar (arrow) and appearance of frosted

frass also indicated.

MOLTING BY STARVATION IN MANDUCA 263

vessel on day 4. Thus their maturation was much more synchronized than that of the above groups. Their weights on day C&4 averaged 1.1 f 0.2, 2.3 f 0.4, 4.7 f 0.7, 7.2 f 1.0 and 5.3 + 0.5 g respectively. Because of the degree of synchronization achieved by this procedure it was adoptkd for all subsequent work on dietary (D. Jones, G. Jones & G. Bhaskaran, in lift.) and neuroendocrine factors (Bhaskaran & Jones, 1980) regulating the starvation-induced super- numerary molt.

Effects ofprolonged starvation on pupation Limited feeding followed by starvation and refeeding. When larvae were reared in

large numbers we occasionally found sixth instars with deformed pupal mouth parts. These abnormal larvae were found in cultures which had gone unattended for several days after ecdysis to the fifth instar and were then provided with a normal amount of food. This finding led us to vary the feeding regime of newly ecdysed fifth instar larvae in an attempt to produce viable sixth instars.

Newly ecdysed fifth-instars were fed on 1 .O g of food and then totally starved for 4, 5 or 6 days. This amount of food was consumed in about 12 hr and larvae increased their body weight by about 25%-40%. Table II shows that all larvae starved for 4 days eventually pupated after refeeding; 50% of larvae starved for 5 days and 98% of those starved for 6 days underwent a supernumerary molt. When the initial quantity of food provided was increased to 2.0 g (consumed in about 24 hr and larvae almost doubled their weight) even 6 days of starvation caused only

TABLE II

Nature of molt following various degrees of starvation during the last-larval instar of M. sexta

amount days

of food (g)’ starvation

1 4

1 5 1 6

2 5

2 6

larvae

23

31 46

13

13

surviving

(%I

22( 96)

28( 90) 42( 91)

13(100)

13( 10)

% 6th % pupae

0 100

50 50 98 2

0 100

15 85

* Newly ecdysed animals were fed on a limited amount of food, starved for a variable length of time

and then refed normal diet.

15% of the larvae to undergo a supernumerary molt (Table II). The sixth instars were completely larval in external morphology except for a small change in mouth parts to the pupal type. This prevented the larvae from feeding and all died within a few days.

Prolonged initial starvation followed by refeeding. In an attempt to find a starvation regime of shorter duration which would still produce sixth instars, starvation was

264 DAVY JON@S. GRACE JONES AND GOVINDAN BHASKARAN

TABLE Ill

Summary of the effects of various starvation treatments on the development offith-instar M. sexta larvae

Treatment ammals

Srarw 2 days No Agar I 8 Agar Day 0 I g Agar Day I

Starw 3 days

No Agar I g Agar Day 0 I g Agar Day I I g Agar Day 2

Smrve4dqvs No Agar 1 g Agar Day 0 I g Agar Day 1 I 8 Agar Day 2 I g Agar Day 3

28 0.82+0.13 93 640 2.63+050 4.3f0.7 36.0 4 752091 5 o+o.o 28 1.16kO.19 86 39.3 305*105 4.3io 8 60.7 5 62+ I.20 4 I to.8 29 l.ll~O.13 73 42 8 2.57tO.39 45+05 57 I 446kO83 52+ I.0

26 0.74+0.12 31 loo.0 2 14+046 5.2+0 5 00 14 1.10+0.17 80 70.3 2.62+0 57 4.4?09 29.7 4 55 0.0 6.0 00 19 1.01 +o I7 68 loo.0 2.20?0 25 4.2tO8 00

30 0.93io I7 53 loo.0 2.38?0 39 4.9t 0.8 0.0

30 0.83iO.15 20 lao.0 1.48fO 12 5.7fO.8 00 20 1.0410.12 30 800 2.80-tO.28 48f05 20.0 4 21 to.0 5.0+0,0 19 0.92+0.16 26 1000 1.97?0 82 46f0.5 00 30 089+0 I7 65 loo.0 2 1250.34 4.6f09 0.0 30 0.91~0.14 41 91 6 1.85?0 35 4lkO.9 83 5.37+0.0 6.0+00

Xweight % % 6th X-werght daysb to % X-wetght days? when refed SUWlV*“g mstars at HCS’ HCS pupatron at FF* to FF

a HCS = Head Capsule Slippage; includes rare larval-pupal intermediates; FF = Frosted Frass. b From the day of refeeding on regular diet.

begun at ecdysis to the fifth instar (Table III). The percentage of starved larvae molting to sixth instars increased, while the percentage survival decreased with increasing length of the starvation period. Provisioning with agar on day 0 decreased the percentage of larvae molting to sixths but increased survival of larvae starved for 3 days. Nijhout (1975a) also prolonged survival of starving larvae with agar. It was noticed that larvae provided agar late in starvation, such as on day 3 of the 4-day regime, often were weak and did not eat all of it.

An effort was made to correlate various factors with percentage of supernumerary larvae. No correlation of high predictive value was found between percentage of supernumerary larvae and weight on day 0, weight at refeeding, percentage or weight loss during starvation, days after refeeding until head capsule slippage or weight at head capsule slippage. However, the weight of starved and refed larvae which were destined to pupate (see weight at frosted frass) was much higher than the weight of larvae undergoing a supernumerary molt. Perhaps further study will relate rate of weight gain early in the refeeding period to percentage supernumerary molting and percentage pupation.

The smallest supernumerary larva at head capsule slippage weighed 1.12 g and the largest was 5.22 g. It was also noted that two larvae which exhibited frosted frass showed head capsule slippage on the next day, and they molted to sixth instars. Though frosted frass has generally been taken as an indication of pupation (Nijhout & Williams, 1974a), the type of molt to be undertaken is apparently not irreversibly programmed at this point.

Preliminary experiments during December gave different results than those reported here, done in March and April. For example, 3 days of starvation, with

MOLTING BY STARVATION IN MANDUCA 265

1.0 g agar provided on day 0, induced 52.2% supernumerary larvae in Dec. (N = 67) and 70% during the spring.

Many of the supernumerary larvae were semi-flaccid, had difficulty in shedding the old cuticle, withdrawing old trachea and had malformed though not pupal maxillary palpi. Most died within several days of no or minimal feeding activity. A few larvae fed and attained weights between 8.2-12.1 g by the 6th day at which time they wandered. Only a few of these pupated and even fewer emerged as adults.

DISCUSSION

Truman (1972) observed that at 12 : 12 L : D cycle and 25’, ecdysis from the fourth to the fifth instar usually occurred during the scotophase. However, larvae reared at 28” usually ecdysed during the later part of the photophase. This is similar to the distribution of larvae reared at 27” and 16 : 8 L : D cycle in our laboratory with highest percentage of ecdysis occurring during 18.00-21.00 hr. These observations enabled us to collect large numbers of newly ecdysed larvae of almost identical age for many of the experiments reported here.

Reduced feeding and or less nutritious food has been reported to produce supernumerary molts in several lepidopteran larvae (Kellog & Bell, 1904; Decker, 1931; Gaines & Campbell, 1935). Nijhout & Williams (1974b) observed that starvation of M. sexfu larvae at weights of less than 4.0 g led to formation of larval- pupal intermediates, whereas starvation at 5.0 g or above had no effect on the nature of the molt. The present study demonstrates that starvation of newly ecdysed fifth instars for varying lengths of time prior to feeding induced an extra larval molt. The optimal period of starvation for obtaining good survival and supernumerary larvae appears to be 3 days.

The occurrence of an additional larval molt suggests that starvation prevents the normal decrease in hemolymph JH titer during the final instar (Nijhout & Williams, 1974b; Nijhout, 1975b) and that the program of metamorphosis initiated at the time of ecdysis to the fifth instar (Nijhout, 1975a) can be altered by starvation. This may result from the failure of the starved larvae to reach the second critical threshold size of 5.0 g and consequent failure to inactive the CA (Nijhout & Williams, 1974b; Williams, 1976). Allatectomy prevented the retention of larval characters and such larvae pupated normally. Recent data indicate that the CA are required for the induction of the supernumerary molt but that the activation of the CA is mediated via the median neurosecretory cells of the brain which secrete an allatotropic factor (Bhaskaran & Jones, 1980). Presumably the brain senses a change in some critical hemolymph factor(s), the most conspicious of which is the drastic decline in hemolymph trehalose concentration. Hemolymph protein concentration, on the other hand, remains relatively unaffected during this period (Dahlman, 1973; Jones et al., in lit?.). Consistent with these observations, we find that providing larvae with sugar, but not protein, during starvation suppressed supernumerary molting (Jones et al., in lift.). These

266 DAVYJONES, GRACEJONES AND GOVINDANBHASKARAN

data suggest that sub-threshold trehaiose titers may activate the allatotropic cen- ter.

We thank Drs. H. Riiller, J. Willis and G. Weirich for comments and Vicki Riddle for technical assistance. Supported by grants from NSF (PCM 77-25417) and from Organized Research, Texas A & M University.

ZUSAMMBNFASSUNG

INDVKT~~N ZVS/%TZLICHER H~VTUN~EN DVR~H ~UN~ERN

3Ei MANDUCA SEXTA LARVEN

Es wurden Studien durchgeftihrt iiber die Effekte llngeren Hungerns auf die Entwicklung des fiinften (letzten) Larvenstadiums bei Manducu sexfa. Nach der Hiiutung wurden die Larven wiihrend unterschiedlicher Zeit ohne Nahrung gelassen und dann mit normalem Futter versehen. Der Prozentsatz Htiutungen fiir ein sechstes Larvenstadium nahm mit der Dauer des Hungerns tu, wiihrend die Ueberlebensrate abnahm. Wenn Agar als Quelle fur Wasser gereicht wurde, wurden beide Effekte vermindert. Die optimalen Verhiiltnisse fur maximales Uberleben und zusatzliches Hluten scheinen bei 3 Tagen Hunger mit 0.5-1.0 Gramm Agar am Tage 0 zu sein. Die endokrinologische Basis fur die zusatztiche Larvenhlutung wird kurz diskutiert.

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Accepted: June 26, 1980