Pergamon 0022-1910(94)ooo91-3

J. Insect Physiol. Vol. 41, No. 2, 143-152, 1995 pp. Copyright 0 1995 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 0022-1910/95 $9.50 + 0.00

Postembryonic Development of Zophobas atratus Fab. (Coleoptera: Tenebrionidae) under Crowded or Isolated Conditions and Effects of Juvenile Hormone Analogue Applications ANDRB QUENNEDEY,* NADIA ARIBI,* CLAUDE EVERAERTS,* JEAN-PAUL DELBECQUE*

Received 25 ApriI 1994; revised 25 Jury 1994

Pupation in Zopbobas stratus is dependent on isolation and this provides a model for study of the onset of metamorphosis in beetles. We have analyzed the effects of isolation vs grouping on certain developmental parameters; the number of larval instars; their duration and weight. Sex differences were detected, such as the occurrence of regressive moults in females. Moreover, the development of Z. atrutus is discussed in relation to Tenebrio molitor, the physiological timing of which appeared in part extendable to Z. stratus. Experiments show that 4-6 days of isolation are required for commencement of metamorphosis, as indicated by an immobile crooked posture observed at the end of this period, characterizing the beginning of prepupal development. During this period, return to grouped conditions, as well as applications of a juvenile hormone analogue (ZR 515) prevented metamorphosis and precipitated another larval moult. This suggests that grouped conditions may delay the onset of metamorphosis through the modulation of juvenile hormone titres.

Zophobas Tenebrio Group effect Juvenile hormone Pupal commitment

INTRODUCTION

Zophobas atratus Fab. (= rugipes Kirsch; Tschinkel, 1984) is a large neotropical tenebrionid beetle living in fruit-bat guano and organic litter (Tschinkel and Willson, 1971). It resembles the mealworm Tenebrio molitor, but is several-fold larger. This insect is strictly dependent on isolation for the onset of metamorphosis. Tschinkel and Willson (1971) have shown that, if artifi- cially maintained in crowded conditions, larvae increase in size and in weight, making supernumerary larval instars without pupating, until death. These authors have also shown that metamorphosis was primarily inhibited by the mechanical contacts between larvae and they suggested that the nervous impulses arising from a crowded environment could maintain juvenile hormone (JH) titres in larvae, compelling them to undertake a supernumerary larval moult.

Therefore, Z. atratus serves as a model for studying the relationships between external events and endocrine factors regulating the onset of insect metamorphosis (or pupal commitment, according to Riddiford, 1976, 1978).

*CNRS URA 674, Laboratoire de Zoologie, Facultk des Sciences, Universitt de Bourgogne, Bd. Gabriel, F-21000 Dijon, France.

Several studies have already been published on the biology and development of Z. atratus (Tschinkel and Willson, 1971; Tschinkel and van Belle, 1976; Tschinkel, 1978, 1981, 1993) but questions still arise concerning the number and duration of larval instars under isolated or crowded conditions and the characteristics of super- numerary instars. In this paper, our aim was to answer these questions by giving a detailed chronology of Z. atratus development and examining the effects of a JH analogue application. Moreover, a comparison was made with T. molitor, for which numerous chronological and physiological data are available (Stellwaag-Kittler, 1954; Delbecque et al., 1978), including the effects of isolation vs crowding (Connat et al., 1984, 1991; Weaver and McFarlane, 1989, 1990).

MATERIALS AND METHODS

Insects

For 5 years, Z. atratus mass rearing has been con- ducted in our laboratory, from 100 larvae obtained from the “Jardin des Plantes” in Paris. The exact geographic origin of these animals, which were seized by French customs, remains unknown. Each year, a hun-

143

144 ANDR6 QUENNEDEY et al.

dred pairs of young adult beetles were bred at 25°C in a 60 x 50 x 30 cm glass box, filled with about 5 cm of peat moss (turf). Cardboard egg-packing was used as shelter and breeding-places for adults. Wheat bran and fruit peelings were the insect diet. About 2000 larvae hatched monthly and were reared at 25°C either in the same glass boxes with a larger amount of turf (about 25 cm) and fed bran laid on the surface, or placed into a 50 x 30 x 10 cm plastic box containing only bran (about 5 cm) and fresh fruit peelings. In this case, bran and larvae were sieved weekly to eliminate frass. Although larvae became larger in turf, due to a higher and more regular relative humidity, bran and fruit peelings were also used, as recommended by Tschinkel and Willson (1971), in order to avoid infestations by parasitic arthropods living in turf.

For grouped conditions, a density of 2-10 larvae (according to their size) in 50 cm3 of turf or bran was chosen. These population densities, higher than those used by Tschinkel and Willson (1971), entirely inhibited metamorphosis.

Plastic boxes of various sizes were used for isolation experiments, without any significant difference on the metamorphosis percentage.

The sex of animals was impossible to determine in larvae, but was easily determined for pupae, by the observation of the abdominal end, as described by Konok (1955) for T. molitor.

Hormonal treatments

The juvenile hormone analogue ZR 515 (Zoecon, Sandoz), dissolved in 1 ~1 acetone, was applied topically on abdominal tergites of isolated larvae, at a single dose of 10 pg per animal.

Data

Experimental animals were regularly observed, counted or weighed. Statistical analyses were made using StatView software (Abacus Concepts, Inc., Berkeley) on an Apple Macintosh computer. The Mann-Whitney U-test (Campbell, 1981) was used for small sample sizes. However, most results were analyzed by single-factor analysis of variance (Anova) and classified using Sheffe’s F-test (Parker, 1979). Means given in the results are followed by standard deviations (SD.).

RESULTS

The onset of metamorphosis in larvae

Isolation of large larvae. In the first experiment, a large number of larvae were taken from a l-year-old mass rearing turf box (sample 1). These larvae (443 individ- uals), all weighing more than 900 mg, were isolated with or without food. Within a few days (6.2 days, S.D. = 1.7), 89.1% of animals (395 larvae) took an immobile crooked posture, laying on one side [Fig. l(A)] They all became pupae about 7 days later, i.e. 12.9 days (S.D. = 1.4) after isolation and adult emergence

occurred 28 days (S.D. = 2.2) after isolation, with no effect for the presence or absence of food.

The 10.9% remaining larvae [Fig. l(B)] underwent another larval instar, 6.9 days (S.D. = 2.7) after iso- lation, prior to metamorphosis. Interestingly, none made more than one larval moult.

Such an experiment has been repeated several times on more than 2000 large larvae of various ages. Though a variable number of animals (up to 10%) died without moulting, this percentage was not the consequence of starvation but was related with the old age of some of these larvae (up to 18 months old). In all cases, 8&95% of fed or unfed larvae pupated immediately. The others underwent a larval moult before metamorphosis, except for only two larvae, that made two successive larval moults. Moreover, the metamorphosis percentage was 100% when animals were sampled according to a physiological timing (see below), and by discarding those already engaged in a new larval moult.

Percentage of metamorphosis according to the larval weight. In a second experiment, more than 1500 larvae of various ages and weights, taken at random (sample 2, Table 1) were isolated and the nature of the follow- ing moult(s) was noted. No pupation was observed for larvae weighing less than 300 mg, but isolated larvae weighing from 300 to 600 mg directly pupated in 3&46% of cases. This percentage reached 85-89% for larvae weighing 600-1300 mg, then decreased to 57% for the older larvae, weighing more than 1400 mg.

The whole development in grouped and isolated conditions

The isolation of newly hatched larvae (about 0.5 mg in weight and 3 mm long) greatly protracted their devel- opment by increasing the duration of the first instars. Because of this disturbance and the difficulty of working with such small animals, most of the following studies on isolated conditions were made from larvae that were grouped until the end of the 4th instar.

Mean weight gain during development. Newly ecdysed Sth-instar larvae (8-10 mg in weight and 1&12mm long) were used for the following experiment. For the first condition, 21 larvae (sample 3) were isolated in plastic Petri dishes (5 cm in diameter) containing turf and bran which were regularly renewed to avoid mould development. For the other condition, 170 larvae (sample 4) were grouped in a 25 x 20 x 10 cm plastic box, three quarters filled with wet turf, and fed bran laid on the surface. Figure 2 shows the mean weight of isolated and grouped larvae, for 9 months. The growth curve of isolated larvae was sigmo’idal but lagged far behind the growth curve of grouped larvae. Pupation occurred 132.9 days (S.D. = 4.3) after iso- lation and adult moult 13.5 days (S.D. = 0.75) after pupation. Isolated larvae reached a maximum of 533.6mg (S.D. = 75.3) weight before pupation while 9-month-old grouped larvae reached 1085.9 mg (S.D. = 117.2). Larval moults occurred regularly and no pupation was obtained in grouped conditions during the experiment.

POST-EMBRYONIC DEVELOPMENT OF ZOPHOBAS 145

A. pG

Days

B. w= 6.9 g= 17.6 u= 26.4 Lk39.5 16 4 (Sk2.7) (SD=3.6) (SD=P.B) (Sk3.;

t 10

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0 0 5 10 15 20 25 45 Days

FIGURE 1, Effects of isolation on the development of 443 old larvae taken at random (sample 1). A: 395 larvae (90%) directly

undertook metamorphosis. An immobile crooked posture (black charts) was observed in larvae, before pupation (white charts)

and adult emergence (grey charts). B: Another larval instar (striped charts) occurred for the remaining other larvae (48) before

metamorphosis. Mean fj~) and standard deviations (S.D.) are indicated for each event.

Duration of larval instars. As was mentioned above, isolation of newly hatched larvae altered development, increasing the duration of the first larval instar to 1 month instead of about 9 days for grouped larvae. To avoid this disturbance, instar duration was principally analyzed in grouped conditions: 19 larvae (sample 5), hatched from a single batch of eggs from a couple of 6-week-old adults, were weighed daily for about 9

TABLE 1. Occurrence of pupal moults

according to the weight of isolated larvae

taken at random

Pupal Weight moults

(mg) n % (dead)

<300 69 0 - 301400 100 30.0 401~00 179 46.4 -

601~800 90 88.9 (1) 801~1000 648 88.0

1001-1200 354 85.0 0 1201~1300 60 85.0 - 1301-1400 34 73.5 -

> 1400 14 57.1

When it occurred, mortality is indicated in parentheses.

months. White ink spots were applied for recognition of different larvae. These progeny were reared together before being isolated at the beginning of the 15th larval instar, more than 200 days after eclosion. As shown in Fig. 3, the duration of larval instars lasted less than 10 days before the 10th instar and then it increased to more than 20 days at the 1 lth instar and to about 40 days at the 13th. No significant difference was observed between males and females for larval instar duration under crowded conditions.

Number of larval instars. Twenty-three small larvae, at the beginning of their 5th instar, were randomly taken from mass rearing and isolated (sample 6). Each larva was examined daily and the number of larval moults counted until the emergence of adults occurred at 125-140 days after isolation. One female larva pu- pated after a total of 11 larval instars, 14 larvae (8 females and 6 males) after 12 larval instars and 8 larvae (4 females and 4 males) after 13 larval instars [Table 2(A)].

The exact number of potential supernumerary larval instars in grouped conditions varies greatly according to the longevity of animals. Nevertheless, taking into account the maximum duration of the larval life under crowded conditions (up to 18 months), it appears that

146

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ANDRl? QUENNEDEY et al.

~1 GROUPED (sample 4)

I I I I I

0 50 100 150 200 250 300 Days

FIGURE 2. Comparison of mean weight increase between 21 isolated (sample 3, white circles) and 170 grouped (sample 4,

black circles) larvae weighed the 5th larval instar. Except for pupal and adult stages (white squares), measures did not

correspond to any particular stage and the curves only reflect the weight gain of samples. (Vertical bars = standard deviations).

grouped animals can undergo a total of about 18-20 larval instars before dying.

Weight increase within larval instars. The weight curve given in Fig. 4(A-D) corresponds to that of the biggest larva (male) among the 19 grouped animals shown in Fig. 3 (sample 5). No significant weight gain was observed during the first larval instar, but from the 2nd instar, weight rapidly increased, apart from a slight decrease about 2-3 days before every new larval moult corresponding to an arrest of feeding. Larval weight approximately doubles during each of the first 9 instars. Thereafter, it increased about 25550% per instar. For the later larval instars under grouped conditions, the weight curve was more irregular due to irregular feeding [Fig. 4(D)]. This phenomenon was amplified because of the lengthening of the non-feeding period to about 5 days at the end of supernumerary larval instars. The weight losses were particularly striking in female larvae,

for which stationary or regressive moults were frequently observed. The same phenomenon was also possible with male larvae [13th larval instar in Fig. 4(C)], however, they more consistently increased their weight during the feeding periods.

In the last larval instar, the weight curve was different from the previous stages [Fig. 4(D)], because the weight gain, occurring during the feeding period, was followed by an obvious decrease corresponding to the longer non-feeding period of the pharate pupal stage.

A slight and regular weight decrease was also observed during the 13-l 5 days of the pupal stage. A drastic loss of weight (100 mg and more) occurred at the adult emergence [Fig. 4(D)].

Relationships between sex and weight

Crowded larvae. Small larvae, weighing S-lOmg, were reared together for 1 year and then isolated (sample

Experimental isolation

, 2 3 4 5 6 7 8 9 10 11 12 13 14 15 P

Larval lnstars

FIGURE 3. Mean duration of larval instars depending on the sex (sample 5). The longest durations were obtained for supernumerary larval instars (13, 14). Experimental isolation (arrow) strongly reduced the duration of the last larval instar

(n = 19; vertical bars = confidence limits).

POSTEMBRYONIC DEVELOPMENT OF ZOPHOBAS 147

TABLE 2. Weight comparison between sex for last instar larvae resulting: A from 23 isolated small larvae and undertaking

metamorphosis at the end of the 1 lth to 13th larval instars, and B from 19 grouped little larvae hatched from the same egg laying,

and isolated at the beginning of the 15th larval instar

Number of larval instars 11 A: Isolated larvae

12 13

B. Grouped larvae

(until 15)

Sex (n) Mean SD. (n) Mean SD. (n) Mean S.D. (n)

Male 621.5 51 714.0 61 4 978.7 35 6 a (1) b NS c ***

Female 478 1 599.3 53 8 49 4 71 13 a a c

(’ & ? Mean comparisons both between for each of larvae isolation, and between instars of

isolation for each sex. (I): case letters to between instars of isolation F-test). with the same are not

significantly different at level P = 0.05. ? Bold uppercase symbols refer to comparisons (Anova) between sexes for each instar of isolation. NS: test not significant at

level P = 0.05; ***: test significantly different at level P = 0.001.

Among 211 174 (82.4%), weighing between the weight becoming from to 1550 directly underwent pupation few male female (male mean = 1267 days later. sex of prepupae and was evaluated S.D. 105, n 97; female mean = 1055 [Fig. 5(A, B)]. observed S.D. 89.3, n 77; P 0.05, Anova): The

mg mg

0 10 20 30 40

mg

30 40 50 40 50 60 70 80 90 100 110 120 130 140 150 160 170 160 Days

Prepupal Pupal Adult Ecdysis Ecdysis Ecdysis

I : 660 a I

I I :: I

b 800 14 -15 -1

750 J I I I I ._.. 190 200 210 220 230 240 250 260 Days

FIGURE 4. Example of daily weight increase of a male larva (from sample 5), reared in grouped conditions, from hatch to the 13th larval instar (A, B, C). (D) Detailed curve of weight increase during the following supernumerary larval instar (14)

and after isolation (open arrow), which induced metamorphosis with prepupal (15), pupal stages and adult emergence. The occurrence of the crooked posture of the prepupa is also given (black arrow). A: feeding period; B: pharate period;

T: transitory period (behavioral change).

148 ANDRE QUENNEDEY et al.

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1 20.

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600 700 800 900 -1000 1100 1200 1300 1400 1500 1600

Prepupal weights (mg)

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600 700 800 900 1000 1100 1200 1300

Pupal weights (mg)

1600

FIGURE 5. Weight difference occurring between sexes of 174 last

instar larvae (sample 7) taken at their maximum weight (A) and just

after pupation (B). General decrease observed for pupae of both sexes

resulted from the loss of weight during pharate pupal B period and

pupal ecdysis. In both stages, because of supernumerary larval instars,

males were bigger than females.

pupae are generally male, although a few small male pupae were also obtained. The mean average time for pupation was 15.5 days (S.D. = 1.7) for males and 14.5 days (S.D. = 1.5) for females.

In sample 5, 13 females and 6 males were obtained after isolation, as indicated above. No significant differ- ence was observed in the weight increase between larvae of both sexes during the first 11 larval instars (Mann- Whitney U-test). From the 12th larval instar however, a difference (P = 0.05) was observed between the two sexes, which appeared more and more evident (P = 0.01) as the number of supernumerary larval instars increased [Fig. 6, Table 2(B)]. No difference was found for the last larval weight between this sample and sample 7.

Isolated larvae. No difference was observed between males and females for prepupal, pupal and adult weight (22 individuals out of the 23 from sample 6) obtained after 12 and 13 larval instars in isolated conditions

[Table 2(A) and Fig. 61. However, a slight difference may be found depending on the number of larval instars occurring before pupation.

Physiological timing during postembryonic development

. Larvae. The eyestage (ES) timing, established by Stellwaag-Kittler (1954) using the ocular migration of T. molitor larvae, is partly usable for 2. atratus. ES l-3 constitute the first part of the instar, which is conven- tionally named A period (Connat et al., 1991) and which approximately encompasses the feeding period. ES 4-6 constitute the end period (B period), approximately corresponding to the pharate larval stage, i.e. a non- feeding period [see Fig. 4(D)]. As in T. molitor (Connat et al., 1991), the physiological significance of the three first ES remains questionable, since some larvae gradu- ally undertook the three ES, while others remained at the same ES during a great part of the A period. In T. molitor, the ES method is thus very useful in the B period only, but in Z. atratus, the darkness of the head capsule prevented following the shifting of ommatidia after ES 4. Nevertheless in Z. atratus, the ES method remains easy to check to determine if individuals are in the A or B period.

The duration of the A and B periods was recorded on 100 Z. atratus larvae of various stages (sample 8), except for the three first larval instars, because of their size and fragility. No significant sex difference was observed for A and B period length, so the results of both sexes were pooled in Table 3. A gradual lengthening in duration was observed for the two periods which was more obvious for the A period (630 days in average between 4th and 12th larval instars) than for the B period (2-5 days only). Similar data were obtained after the 12th larval instar and, in particular, during supernumerary larval ones (14 and more).

As the larval B period approximately corresponds to the pharate larval stage, it was interesting that larvae, isolated from the mass rearing during this period, always underwent a larval and never a pupal moult. Conversely, isolation of mature larvae during their A period greatly increased the pupal moult percentages, reaching nearly 100%.

Prepupae. In the last larval stage of T. molitor, the B period, corresponding approximately to the prepupa (pharate pupa), is marked by a more complex ocular migration, which allows the discrimination of a total of 14 ES (ES l-6 being similar with larval ES and ES 7-14 typical of prepupae). In T. molitor, a transitory period (T period) was observed between A and B periods in last instar larvae, characterizing the onset of metamor- phosis and probably similar to the wandering stage of other holometabolous insects (Connat et al., 1984). Such behaviour also occurred in Z. atratus at the beginning of the B period. If pieces of dry-rotted wood were given to isolated prepupae at this moment, they dug a hole within it to pupate. Under natural conditions, the animals do display a digging behaviour after dispersal from other larvae by wandering (Tschinkel and van Belle, 1976).

POSTEMBRYONIC DEVELOPMENT OF ZOPHOBAS 149

Isolation

1000

900

800

Grouped Larvae (19)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

: .iJp

.+

.:* Pp

‘t,

P

..bp P A

: A

Pb A

isolated Larvae (23)

Last larval instar 11 12 13

Female 0 0 A

Male W A

Larval lnstars

FIGURE 6. Weight evolution for 19 male and female larvae (sample 5, black and open circles) grouped from hatching to the beginning of the 15th larval instar. The difference between male and female weights is significant at level P = 0.05 (*) for the

12th instar, at level P = 0.01 (**) for the 13th and 14th instars and at level P = 0.005 (***) for the following stages (Anova).

Right insert refers to the evolution of prepupal (Pp), pupal (P) and adult (A) weights for 23 isolated larvae (sample 6, metamorphosis occurring after 1 l-13 larval instars). No significant difference was observed between males and females which

underwent the same number of larval instars.

Within wood or in the open, the crooked posture [Fig. 4(D)] corresponds to the beginning of ES 7 of T. molitor.

If isolation of large larvae occurred just after ecdysis, the durations of A and B periods were respectively 12.4 days (S.D. = 3.5) and 9 days (SD. = 0.8, IZ = 41). The immobile crooked posture was observed 3.6 days (SD. = 0.5) after the beginning of the B period. If isolation of prepupae occurred later during the A period (about l&30 days after larval ecdysis), the duration of the B period was 9.6 days (S.D. = 0.8, II = 33) and the crooked posture happened 3.7 days (S.D. = 0.7) after isolation. For larvae taken randomly [Fig. l(A)], a few were isolated at the end of the A period, so the crooked posture appeared more precociously (l-2 days later).

Pupae. The duration of Z. atratus pupal stage reached 13-15 days at 25X, depending on the pupal weight and the small pupae had shorter development. It was also reduced with higher temperatures (11 days at 29°C). The physiological timing of T. molitor pupae, using eye development and cuticular sclerotization (Wigglesworth, 1948; Delbecque et al., 1978), can also be applied to Z. atratus pupae. Comparison with T. molitor and correlations between pupal stages and ages, expressed in days, are given in Fig. 7.

Inhibition of metamorphosis by contacts between individ- uals or a JH analogue

Re -crowding. 120 larvae weighing 800-900 mg (sample 9) were isolated from the mass rearing during their A period. Four days after isolation, crooked prepu- pae appeared. Then they were put again in crowded

TABLE 3. Respective durations of the A and B periods for each conditions (respectively 4, 6, 8 and 10 days after iso-

Zophobas larval instars from the 4th instar (length, mean and standard lation) and divided in groups of 30 prepupae into

deviation) 18 x 11 x 6 cm plastic boxes containing bran (1 cm) and

A Period B Period B/(A + B) 50 active larvae in the 7th larval instar (5MO mg weight

Instar n Mean S.D. Mean S.D. %

4 3 6.00 - 2.00 25.0

5 8 6.00 1.77 2.13 0.35 26.2

6 17 6.18 1.13 2.06 0.24 25.0 7 22 6.82 1.68 2.36 0.49 25.7 8 28 9.36 2.08 2.71 0.46 22.5 9 23 12.00 3.83 3.48 0.51 22.5

10 11 15.64 2.20 3.91 0.54 20.0 11 6 20.50 5.47 4.17 0.41 16.9 12 9 30.44 6.97 4.78 0.44 13.6

14-15 25 33.44 6.51 4.88 0.33 12.7

12 3 4 5678 9 Zophobas i i j i i j j i ::: I

i :

. ; i

4 56789 i ; i : Tenebrio : : : : : : : : : :

j j j i :, :2:3:4:5:6:7:8:9:,o:,,:,2:,3: DAYS

FIGURE 7. Comparison of pupal stage duration (at 25°C) between Z. stratus and T. molifor. Physiological pupal ages (l-9) are the same

for both species.

150 ANDRE? QUENNEDEY et al.

80 -

20 -

o- 1 I I I 1 I I 0 1 2 3 4 5 6 Days

FIGURE 8. Percentages of larval moults induced by ZR 515 applied

at various times after artificial isolation. 140 large larvae, weighing more than 800 mg, were isolated during their A period (sample 10).

From 0 to 6 days after isolation, about 20 animals each day were

topically applied with 10 pg ZR 515, dissolved in 1 ~1 acetone, and 20

control larvae were treated with 1~1 pure acetone. A few days later,

all the control larvae underwent the pupal moult (0% larval score), whereas the JH analogue-treated larvae underwent either larval or

pupal moults, after a variable delay: the larval moult percentages are

represented with their standard errors (vertical bars).

and about 25 mm long). The crowding disturbance induced, in 25% of animals isolated during 4 days (i.e. 7 prepupae), the reversal of the crooked posture and the recovery of larval activity a few days later. The other crooked larvae pupated normally, so re-crowding had no inhibitory effect on metamorphosis after 6 days of isolation.

JH analogue application. 120 larvae weighing more than 800 mg (sample 10) were isolated during their A period and ZR 515 was topically applied on the tergites from 0 to 6 days after isolation. All the control larvae (20) treated with or without 1 ~1 acetone on isolation day (day 0), underwent metamorphosis a few days later. Treatment by a JH analogue clearly inhibited pupation when applied during the 4 first days of isolation, fre- quently in association with a renewal of activity and a delay of ecdysis, even for several individuals showing characteristic crooked posture. The inhibition of meta- morphosis, measured by the larval moult score (Fig. 8), was close to 100% at day 0, then it decreased progress- ively and significantly at days 1, 4 and 5 respectively. Finally, it is noteworthy that ZR 515 was no more capable of inhibiting pupal moult from the 6th day after isolation, as already observed for re-crowding. A few pupal malformations were observable after treatment at day 4 or later, and consisted of a mildly pronounced prognathous position of the head.

DISCUSSION

Developmental features of Zophobas and comparison with other beetles

Our study corroborates the previous data of Tschinkel and Willson (1971) showing the inhibition of pupation by crowding in Zophobas atratus because the larvae artificially maintained in grouped conditions undertake supernumerary larval instars until death. This inhibition of pupation by crowding has been also described in another tenebrionid species, Tribolium jiieemani

(Nakakita, 1982; Kotaki et al., 1993). In fact, crowding probably induces a pupation delay in numerous other species of tenebrionid beetles, as observed by Tschinkel and Willson (1971). In Tenebrio molitor, several authors have observed the occurrence of supernumerary larval instars when larvae were placed in crowded conditions (Tschinkel and Willson, 1971; Weaver and McFarlane, 1990; Connat et al., 1991; Quennedey and Quennedey, 1993). However, unlike Z. atratus or T. freemani, the metamorphosis of T. molitor is not totally inhibited in mass rearing and mealworms manage to pupate after a variable number of larval instars, even without isolation.

In isolation conditions, a minimum of 11-13 larval instars was observed in this study before pupation of Z. atratus. Cotton and St George (1929) recorded a minimum number of 9 larval instars for T. molitor and 12 larval instars for Tenebrio obscurus, while the greatest number reached respectively 20 and 22 larval instars in crowded conditions, due to supernumerary moults. In grouped Z. atratus, the maximum number of moults varies greatly among larvae and seems to be limited by death only (12-18 months length of life). For the

oldest larvae, a maximum of 18-20 larval instars appears likely, because of the duration of supernumerary larval instars.

A noteworthy fact is the influence of crowding on the speed development of very small larvae. Isolation greatly increases the first instar duration (1 month instead of 9 days). As in Tenebrio (Weaver and McFarlane, 1990), a gregarious factor is probably involved, since newly hatched larvae from an egg clutch remain close together during the whole first larval instar and wander more freely at the second larval moult only. Another surpris- ing result is the percentage of metamorphosis induced by isolation which is reduced for old larvae weighing more than 1400 mg. Our results corroborate Tschinkel’s observations (1993) showing that as larvae grew, death at metamorphosis became more frequent. The repeated inhibitions of metamorphosis, resulting from the forced artificial crowding, probably induce endocrinological disorders in some insects that leads to an inability to pupate.

Several studies have shown that larval weight or body size influence the control of metamorphosis (review in Sehnal, 1985). In T. molitor, larvae weighing less than 80 mg generally do not undergo metamorphosis (Connat et al., 1991). In Z. atratus, the threshold larval weight

POSTEMBRYONIC DEVELOPMENT OF ZOPHOBAS 151

is about 400 mg, but great variability was observed for the weight of prepupae (350-700 mg) according to the percentage of humidity of the substrate and the quality of food. The influence of these two factors has been previously observed for T. molitor (Stellwaag-Kittler, 1954; Murray, 1960; Urs and Hopkins, 1973). The same variability also occurred in Z. stratus for maximal weight in supernumerary larvae: in bran with added fruit peelings, the larval weight did not exceed 1300 mg, while a maximum of 1800 mg was obtained for a 18-month-old male larva reared in wet turf and bran.

The empirical Przibram’s rule-a weight doubling at each instar-(Wigglesworth, 1950) can be observed for the 9 first larval instars of Z. stratus, during which larvae feed intensively (A period). In the following instars however, food intake is interrupted with more and more frequent arrests, as the larval instar number increases. So, the percentage of weight gain declined with age. However, weight increase was also correlated with the sex in the supernumerary larval stages obtained in grouped conditions: the bigger larvae, which were male, fed more often during the A period. Such a sexual weight difference, also reported by Tschinkel (1993) and inter- preted as an adaptation for competition between males, was not observed in T. molitor (Weaver and McFarlane, 1990).

In another beetle, Trogodermu glubrum (Beck, 1972; Beck and Bharadwaj, 1972; Klein and Beck, 1980) retromoults have been described as a consequence of starvation while in Z. utrutus (particularly in female larvae), regressive supernumerary moults resulted from crowding only.

As in Tenebrio molitor (Stellwaag-Kittler, 1954; Connat et al., 1991) ES timing easily gives account of the feeding and pharate periods within Z. utrutus larval instars. The maximal weight is generally reached at the end of the A period (ES l-3), which thus corresponds to the feeding period, before decreasing during the B period (ES 4-6) which corresponds to the pharate stage. However, a difference should be noted for the respective durations of A and B periods of both species: the length of the B period is indeed proportion- ally much shorter in Z. utrutus (about 15% of the total larval instar duration vs 25530% for T. molitor). The greater duration of the A period in Zophobus is related to the high percentage of direct metamorphosis obtained after isolation of large larvae taken at random. Our observations have indicated that when larvae are isolated during the A period, the pupation percentage can reach lOO%, whereas larvae isolated during the B period, i.e. pharate larvae, are unable to commence metamorphosis.

In the last larval instar of Z. utrutus, as in T. molitor, the B period is longer than in previous instars because of the beginning of metamorphosis. In natural con- ditions, isolation is preceded by a wandering period of about l-day-duration, during which larvae look for an appropriate pupation place (Tschinkel, 1981, 1993). This behaviour appears to be homologous with the

wandering stage of numerous other holometabolous insects. Then in Z. utrutus, a delay of about 4 days takes place before the occurrence of the immobile crooked posture, characterizing the initiation of metamorphosis. Such a delay, also observed after artificial isolation of large larvae, is in agreement with previous observations of Tschinkel and Willson (197 1). Interestingly, the whole duration of final instar B period appears remarkably constant (9-10 days). Thus, dating by days may easily replace ES dating during the B period.

Endocrinologicul considerations

The peculiarity of metamorphosis induction in Z. utrutus undoubtedly affords an interesting model for further studies on beetle endocrinology. In this species, interactions between animals are necessary for a rapid growth during the first larval instars, but they also maintain feeding and larval growth during the whole larval life. The removal of such a group effect can commit the animals to undertake metamorphosis (Tschinkel and Willson, 1971). It thus appears very likely that mechanical stimulations are capable of maintaining, after nervous integration, the hormonal conditions which are favorable to growth and regular larval moult- ing. As classically observed in numerous other species (Sehnal, 1976) the application of JH analogues in Z. utrutus was found to not only increase the percentage of larval moult, but also to provoke a renewal of larval activity and feeding, in association with a delay of ecdysis. Such features have also been observed in larvae which have been re-grouped after a variable isolation time. These observations also clearly correspond to the characteristics of supernumerary larvae obtained in mass rearings and are also in agreement with previous observations on other beetles: for example in Dermestes muculutus, JH analogues were found to delay pupation but also to increase activity and body metabolism (Slama and Kryspin-Sorensen, 1979); also in T. mofitor, the effects of JH analogues are clearly similar to those of crowding, increasing both the total number of larval stages and the feeding period in each instar (Connat et al., 1984, 1991). Moreover, in T. freemani, precocene, an inhibitor of JH biosynthesis, is able to induce pupa- tion in crowded animals, this effect being counter- balanced by the application of JH analogues (Nakakita, 1990).

Our experiments have evidenced that a precise period of JH analogues sensitivity becomes apparent and pro- gressively declines during the days following artificial isolation. It is important to stress that JH analogues are capable of reversing the induction of metamorphosis in Z. utrutus up to a relatively late stage, 4-6 days after isolation, marked by an immobile crooked posture, which is characteristic of the prepupa. Interestingly, a similar reversal of metamorphosis was also observed, with a similar 6 day limit, in animals which have been re-grouped after a variable isolation time. Both kinds of experiments thus suggest that the decisive period for the nature of the moult, corresponding to a period of

152 ANDRE QUENNEDEY et al.

JH-sensitivity, takes place during the 6 days following the artificial isolation.

A positive signal may also be involved for the initiation of metamorphosis, during the 6 day decisive period. Such a positive control has been evidenced in vitro by Riddiford (1978) on the epidermis of Munduca sexta, consisting of a small 20-hydroxyecdysone pulse, in the absence of JH. This signal has been shown to occur in uivo in the haemolymph of the last larval stages of numerous holometabolous insects (Smith, 1985), including the related species, T. molitor (Delbecque et al., 1978; Connat et al., 1991). In 2. atratus, the ecdysteroid titres also appear to make a small peak 5 days after isolation, which is in agreement with a role of this hormone during the very last steps of the decisive period (Aribi et al., in preparation).

Complementary information. After preparation of this manuscript, Tschinkel offered us his unpublished pre- liminary data on Zophobus endocrinology. Tschinkel found that although JH analogues inhibited metamor- phosis, as also reported in this paper, the volumes of the corpora allata (CA) of both crowded and isolated larvae indicated that the CA were not actively secreting. This was confirmed by transplantation of the CA into Tenebrio pupae. Furthermore, neither allatectomy, nor precocene treatment caused crowded larvae to pupate, and high doses of JH-III injected into isolated larvae did not prevent their pupation. Tschinkel suggested that the inhibition of metamorphosis in Zophobas may not proceed by the “normal” JH mechanism. At this time, an alternate mechanism cannot be eliminated.

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Acknowledgements-We are indebted to Professor Ch. Noirot and to Dr J. Delachambre for the critical reading of the manuscript. We are also particularly grateful to Dr W. R. Tschinkel for the communication

of unpublished data, helpful suggestions and correction of the English manuscript. We also acknowledge Mrs F. Lethelier (Jardin des Plantes, Paris) for the gift of larvae permitting us to set up Zophobas rearing

in our laboratory.