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Role of the juvenile hormone on the differentiation of specialized integumental areas, “fenestrae”, in Schistocerca gregaria (Acrididae: Orthoptera)
J. Insect Physiol. Vol. 32, No. 11, pp. 941-953,
Printed in Great
0022-1910/86 $3.00+0.00 Pergamon Journals Ltd
1986
Britain
ROLE OF THE JUVENILE HORMONE ON THE DIFFERENTIATION OF SPECIALIZED INTEGUMENTAL AREAS, “FENESTRAE”, IN SCHISTOCERCA GREGARIA (ACRIDIDAE: ORTHOPTERA) SBRENNA and ANNA MICCIARELLI-SBRENNA of Zoology, Via L. Borsari, 46-44100 Ferrara, Italy
GIOVANNI Institute
(Received 19 December
1985; revised 7 March 1986)
Ah&act---“Fenestrae” or “Slifer’s patches” are highly permeable integumentary areas of locusts, segmentally arranged in the adult abdominal terga of males and females. In S. gregaria they arise, after being newly programmed, from epidermal cells, and the developmental pattern proceeds during the last two larval instars. In order to study the mechanism by which the cells that form these areas respond to the hormonal stimuhzs to change commitment, 4th- and Sth-instar larvae were surgically allatectomizcd or treated with precocene. When performed within the first 24 h of the 4th-instar, allatectomy, whether surgical or chemical, leads to the appearance of precocious adults, with the integumentary areas on all terga. If removal of the corpora allata is done after that period, the precocious adults have smaller integumentary areas, and if the removal is delayed for 48 h, differentiation of the areas does not occur at all in the last terga. Topical application of juvenile hormone to the sites of the presumptive areas of the 4th-instar larvae, allatectomized during the first 12 h, reduces the specialized differentiation. The presence or absence of juvenile hormone commits the integumentary-area-forming cells to the deposition of larval or specialized
cuticle, respectively. The hormonal events during the larval instars and an asynchronism of the integumentary cellular cycle may explain the distribution of these integumentary areas. Key Word Index: Allatectomy, Slifer’s patches, specialized integumental pattern, Schisfocerca gregaria, precocious morphogenesis
INTRODUCI’ION The effect of juvenile hormone on epidermal cells has
been called “maintenance of the S~U~US quo” (Williams, 1953). In holometabolous insects levels of ecdysteroids and juvenile hormone in the different phases of development suggest that commitment of cells forming adult epidermally derived structures requires that the cells be exposed to ecdysteroids with no juvenile hormone (Mitsui and Riddiford, 1976; Kiguchi and Riddiford, 1978), and that the different structures require different and characteristic periods of time for changing their commitment (Truman et al., 1974). Some authors (Wigglesworth, 1964; Riddiford, 1980) hypothesize that this scheme could be valid also for the control of the metamorphosis of hemimetabolous insect. On the other hand, in these insects, experiments involving bioassay of haemolymph extracts or corpora allata (Wigglesworth, 1952; Joly, 1972; Johnson and Hill, 1973) lend weight to the suggestion that both the maintenance of larval characteristics and the metamorphosis are controlled in a similar way. In the median dorsal-zone of each abdominal tergum of adults of Schistocerca gregaria there is a pair of specialized integumentary areas that have been variously termed “fenestrae” (Slifer, 1951) and “Slifer’s patches” (Uvarov, 1966). Their cells differ 941
greatly from those of the surrounding integument and synthesize a cuticular layer composed of exocuticle alone, containing many large pore-canals (Sbrenna and Antoneili, 1977). This specialized integumentary area is involved in the active control of water content (Makings, 1968; Loveridge, 1980) and since the earlier terms “fenestra” and “Slifer’s patches” are not descriptive of their activity, we have called these structures as “highly permeable integumentary areas” (abbreviated to integumentary areas). The pattern of these areas in adults is reached gradually during the final-larval stages (Sbrenna and Sbrenna-Micciarelli, 1980a) indicating that the initial processes of this morphogenesis begin before the last-larval instar. This morphogenesis, which is realized in different successive stages, offers an excellent test-system for studying the action of ecdysteroids and juvenile hormone on integumental differentiation in hemimetabolous insects. Some preliminary results have already been published (Sbrenna and Sbrenna-Micciarelli, 1980b). In the present work we report and discuss the effect on morphogenesis of the integumentary areas when juvenile hormone deficiency is created by surgical removal of the corpora allata or by treatment with Precocene 2.
GIOVANNI SBRENNA and ANNA MICCIARELLI-SBRENNA
942 Table
I. Effect of surgical allatectomy on metamorphosis of 4th- and Sth-instar larvae of S. areaaria
Instar 5th
4th
Larval death
Time after ecdysis (hours)
Allatect. larvae
at next ecdysis
&I2 12-24
12 I7
0 0
0 0
I2 I7
6 6
&I2 12-24 2436 3648
27 I2 I9 8
2 I 3 I
25 II I4 4
0 0 2 3
8 6 6 4
*Sham-operated
Normal adults
Sham-operat. larvae*
larvae all moulted to normal adults.
MATERIAL AND METHODS
Experimental animals
We used a strain of S. gregaria raised in our laboratory from eggs provided by the C.O.P.R. of London. The life history and rearing procedures have been described elsewhere (Sbrenna and Antonelli, 1977). We utilized 4th-instar nymphs at O-12, 12-24, 24-36 and 3648 h following ecdysis, and Sth-instar nymphs O-12 and 12-24 h after ecdysis. Extirpation of larval corpora allata
During the operation the larvae were anaesthetized with carbon dioxide; allatectomy was performed using Pener’s technique (persona1 demonstration). For the sham-operated insects, the same procedure was followed, but without removing the glands. Other intact insects of the same age were used as controls. The allatectomized, sham-operated and control insects were marked individually and observed daily and killed after metamorphosis from the 6th to the 20th day of adult life (Table 1). Juvenile hormone applications
Juvenile hormone 1 (Fluka A.G.) was dissolved in olive oil (10 pg/ ~1). Five pg were applied topically, with a Hamilton microlitre syringe, along the dorsai midline of the 7th abdominal tergum of carbon dioxide-anaesthetized 4th-instar larvae. Larvae were allatectomized 24 h prior to hormone application. After the application, the insects were kept in a glass jar with the same temperature and lighting conditions as the controls. Olive oil alone was applied to the controls. Precocene-2
Precocious adults
deposited on the surface of the Petri dishes (14 x 2 cm) at different concentrations (see Table 2) as described by Unnithan et al. (1980). Groups of 8 newly moulted 4th-instar nymphs were exposed to the precocene for 24 h at 35°C. After treatment the insects were transferred to rearing cages. Petri dishes for controls were prepared using acetone alone. The effects of the treatment were recorded daily. The animals were sacrificed and dissected after methamorphosis from the 6th to 20th day of adult life. Histological procedures
The terga from the 1st to the 8th abdominal segment of each insect were excised and then fixed in 2% glutaraldehyde in 0.1 M phosphate buffer at pH 7.0 or in 2% glutaraldehyde in 0.05 M cacodylate buffer at pH 7.2 with 4% sucrose. Some of these terga, with the muscles, tracheae and fat bodies removed, were mounted whole in balsam, after staining using one of the following dyes: Giemsa solution, Nile blue, Azure II or paracarmine (Chevreau et al., 1977). Others, after being fixed in glutaraldehyde, were post-fixed in 1% osmium tetroxide, dehydrated, and embedded in Epon-Araldite. The sections (1 pm thick) were stained with Azure II-methylene blue. Drawings of whole mounts of the integumentary areas, stained with Azure II, were made using a projection microscope (Reichert) and their surface areas were measured with a planimeter. The cell density was determined using three radomly chosen points of the integumentary areas of each insect. Cell counts were made on these areas. RESULTS
Normal development of the integumentary areas
treatments
Precocene 2, purchased from the Aldrich Chemical Co., was dissolved in spectra1 grade acetone and
Figure 1 shows a diagram of the developmental pattern of the specialized abdominal areas of S.
Table 2. Mode of application, time, and effect on metamorphosis of precocene treatment of 4th-instar larvae of S. gregaria Dose of precocene pg/cm’ Petri dishes (fig/larva) 45 pg/cm’ (865 pg/larva) 60 pgjcm’ (I 153 pg/larva)
Time after 3rd/4th instar ecdysis (hours)
Treated larvae
Precocious adult
Normal adult
Death’ during treatment
O-24
32
8(25%)
18(56.2%)
6(18%)
&24
40
16(40%)
14(35%)
10(25%)
Control with acetone
O-24
24
-
22(91.6%)
2(8.3%)
*The death occurred mostly during the 24 h of treatment.
Differentiation of integumental areas
m
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sIsAa33 w
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943
944
GIOVANNI
SBRENNA
and ANNA MICCIARELLI-SBRENNA
P2
dd
1000 -
I
0
Control
q
V
(O-12 h
allat.
IV allat.10.12
ul
IV
Precoc.
h II
800-
;
5
m
m ;
400
2 :
200
0 2nd seg.
3rd seg.
4th
seg
5th
seg.
6th seg.
P2
7th seg.
I
99
1000
0 fzl q
8th seg.
Control V
allat.
(O-12
IV allat.lO-12
h 1 h )
: 800 CT Y m .;
600
g n m ;
400
: iii 200
L
0 d seg.
3rd seg.
4th seg.
5th seg.
6th seg.
7th seg.
8th seg.
Fig. 2. Effects of removal of the corpora allata or precocene-2 treatment on the area of abdominal integumentary areas according to abdominal segment. Each histogram represent the mean of 640 samples (+SD).
gregaria. These areas are not present during the first three larval instars: in the medial zone of each abdominal tergum there is only a heavily pigmented area called “cardiac band” (Nickerson, 1956). Differentiation begins during the 4th instar. During the mitotic period (36th hour) two or three small light-coloured zones appear, in the 2nd, 3rd and 4th terga, in the cardiac bands. In the hours that follow
these zones grow larger and then blend together to form two lighter strips parallel to the dorsal medial line. At about the 72nd hour differentiation ceases, before all the presumptive cells of the integumentary areas in these first terga have actually differentiated, a certain number of them (“peripheral presumptive cells”, Sbrenna and Sbrenna-Micciarelli, 1983) differentiate later in the next instar (5th). This inter-
Figs 3 and 4. Integumentary areas of a male control adult, present on the 3rd and 5th abdominal tergum respectively. Azure II-stained whole mounts (asterisk: tergal middle line). Bar = 0.5 mm. Fig. 5. Section of the highly permeable integument of a male control adult. Below the specialized exocuticle (ex) there is no endocuticle (ss: subcuticular space). Bar = 10 pm. Fig. 6. Whole mount of a female control integument. The integumentary area is outlined by the characteristic cuticular bulge (cb.). Bar = 0.15 mm. Fig. 7. Integumentary areas present on the 5th tergum of an allatectomized adult (deprived of corpora allata at O-12 h of the 5th instar). The edge of the areas are clearly delineated by the cuticular bulge (arrows) as the control (see Fig. 4). Fig. 8. Section of the highly permeable integument of an allatectomized adult (deprived of corpora allata at &I2 h of the 5th instar). The aspects of the specialized cuticle and epidermis are similar to HP integument of the control adult (see Fig. 5). Bar = IOpm.
945
Figs 9 and 10. 3rd and 5th tergum of a precocious adult (allatectomized at &12 h of 4th instar). The integumentary areas have a reduced surface area and an irregular outline (see Figs 4 and 7). Azure IIstained whole mounts (asterisk: tergal middle line). Bar = 0.5 mm. Fig. 11. Section of highly permeable integument of a precocious adult similar to Figs 9 and 10. The cuticular bulge (cb) secreted by normal epidermis (EI) outlines the highly permeable integument of a 5th tergum as in the control adult (see Fig. 6). Bar = 10 pm. Figs 12 and 13. 3rd and 5th tergum of a male precocious adult (allatectomized at 2436 h of 4th instar). The integumentary areas of the 3rd tergum have reduced surface, whereas those of the posterior tergum (5th) are broken up into islets. Fig. 14. Greater
magnification
of the Fig. 13. The islets of integumentary area are consistently by a cuticular bulge (arrows). Bar = 0.25 mm.
marked
Figs 15 and 16. 3rd and 5th tergum of a male precocious adult (allatectomized at 3648 h of 4th instar). The anterior integumentary areas are very small and irregular, whereas the posterior ones do not differentiate. Respective bars = 0.5 mm and 0.2 mm. 946
Figs 17 and 18 Effects of topical application of juvenile hormone to 4th-instar larvae (allatectomized during the first 12 h after ecdysis). Islets of integumentary area are evident on the female 7th tergum (Fig. 17). The areas on the male 7th tergum are missing (Fig. 18). Respective bars = 0.5 mm and 0.2 mm. Figs 19 and 20. Whole mounts of 3rd and 5th tergum of male precocious adult after precocene-2 treatment during the 4th instar. The integumentary-area pattern is similar to that of a control and to a precocious adult surgically allatectomized during the first CL12h of the 4th instar (see Figs 3, 4, 9 and IO).
947
Differentiation of integumental areas ruption of the differentiation means that during the 4th instar, no lighter strips appear in the remaining terga (Sth-8th), but the cardiac bands are evident. After ecdysis to the 5th instar, pairs of lighter strips (called “larval integumentary areas”) appear in the 2nd, 3rd and 4th terga (Fig. 1). No differences in the larval form of the areas are present between males and females. Differentiation recommences during the 5th instar (the final larval instar) on about the 3rd day after ecdysis. From this moment both the peripheral presumptive cells, located around the lighter strips, and the cells forming the areas of the last four terga begin the differentiation process. After the next (imaginal) ecdysis, all the abdominal terga in both males and females have assumed the definitive pattern of integumentary areas (Figs 1, 3 and 4), and the normal epidexmal cells synthesize the endocuticular bulge (Sbrenna and Antonelli, 1977) between the normal integument and the integumentary areas (Fig. 6). The bulge, secreted during the first days of adult life, is missing in the “larval integumentary areas” of the 5th instar. Allatectomy during the 5th~larval instar Allatectomized Sth-instar larvae undergo methamorphosis on day 12-13. The removal of the corpora allata thus lengthens the final-larval instar by about 3 days. Morphologically the allatectomized insects resemble those described by Pener (1967). The adults do not take on the characteristic yellow colour which is observed in normal males reaching sexual maturation, and sexual behaviour and locomotor activity are less intense than in controls and shamoperated insects. Removal of the glands during the first hours of the instar (O-12 and 12-24 h) resulted in a small but statistically significant reduction in the sizes of the integumentary areas (Fig. 2) yet allatectomy did not modify the differentiation pattern of the areas and they were present on all the abdominal terga in their characteristic shape and appearance (Fig. 7). The integument is these areas presented a cuticle composed of exocuticle alone crossed by numerous straight, wide pore canals. Between the exocuticle and the specialized cells there was a wide subcuticular zone (Sbrenna and Antonelli, 1977), resembling that of control adults (Figs 5 and 8). Allatectomy during the 4th-larval instar O-I2-h old larvae In the controls and sham-operated insects the 4th-5th ecdysis occurs on about day 6 of the instar, and a last-instar larva emerges from the exuviae. When 4th-instar larvae were allatectomized within 12 h of ecdysis, the instar was lengthened by 34 days. Then they moulted to precocious adults (Pener et al., 1978) [Table 11. Removal of the corpora allata during the first O-12 h resulted in a reduction in the sizes of the integumentary areas (Fig. 2). The dimensions of these areas of the precocious adults was however much less than that of the control adults, especially in females, where the areas were roughly half the size of those in normal insects (Fig. 2). The cell density of the areas remains however unchanged: the 3rd tergum has
949
4.040cells/mm2 compared to 4.070 cells in control adults; and the 5th tergum has 5.560 cells/mm2 compared to of 5.670 observed in the controls. The normal integument of the precocious adults was found to be cytologically different from that of control adults, due to the presence of pigment granules in. the epithelial cells. Also these precocious adults had quiescent integumentary glands as previously described by Cassier and Delorme-Joulie (1976). On the contrary in all the abdominal terga of both male and female precocious adults there were integumentary areas resembling those in the control adults in shape and appearance (Figs 9 and 10). The cuticle and also the specialized cells resembled those of the specialized areas of normal adults and were separated from the epidermal cells with a very clearcut endocuticular boundary (Fig. 1I). 12-24-h old larvae The precocious adults formed after allatectomy of 12-24-h old, 4th-instar larvae had specialized areas on all the abdominal terga. This delayed removal of the corpora allata did not influence the differentiation of the presumptive cells. The integumentary areas of the anterior terga were like those present in precocious adults obtained from 4th-instar larvae which were deprived of their glands on the 12th hour of life. By contrast the integumentary areas of the last terga had a very irregular outline and, instead of lying within the areas as they normally do, the trichoid sensilla remained outside them. 24-36-h old larvae After, allatectomy of 24-36-h old, 4th-instar larvae, most formed precocious adults. The specialized areas of the first three terga of these precocious adults differentiated normally, but those of the last terga were much smaller or broken up into tiny islets (Figs 12 and 13). The cells of these islets however resembled those of the controls, with regularly spaced nuclei, clear-cut cell boundaries and the characteristic stellate shape. These small areas could be distinguished because their borders were consistently marked by an endocuticular thickening (Fig. 14). In males the integumentary areas of the 4th tergum were also smaller, and sometimes one of the two areas is broken up into small round areas. 3648-h old larvae When 36-48-h old, 4th-instar larvae were allatectomized, only half formed precocious adults. The remainder formed larvae (Table 1). The specialized areas of the first terga of the precocious adults differentiated normally but were very small and irregular. They resembled the lighter strips (larval integumentary areas) of 5th~instar controls but these areas were bounded by endocuticular border (Fig. 15). On the contrary the areas of the four posterior terga did not differentiate. At this level of the abdomen all the cells appeared identical to those of normally developing epidermal cells before the onset of differentiation (Fig. 16). EJect of juvenile hormone on 4th -I’nstarallatectomized larvae We have investigated the juvenilizing activities of the juvenile hormone using juvenile hormone I.
950
GIOVANNI SBRENNAand ANNA MICCIARELLI-SBRENNA
Hormones 1 and 11 are more active than hormone III in many bioassays (Roussel, 1976; Lanzrein, 1979). Fourth-instar larvae, allatectomized O-12 h after ecdysis and treated 24 h after the operation with juvenile hormone, moulted into precocious adults on the 10th day following ecdysis. The morphogenetic response of the integumentary areas to topical treatment was different in the two sexes, the females being less sensitive to the hormone than the males. In females, the specialized areas appeared on all the abdominal terga, although the areas on the 7th tergum (i.e. where the hormone was applied) and on the 8th were smaller or irregularly shaped and lobed. These areas however were always clearly identifiable, due to the endocuticular thickening marking their boundaries (Fig. 17). In the males the specialized areas on the 7th (juvenile hormone-treated) and 8th terga were missing (Fig. 18) or present in the form of small islets which were often difficult to distinguish. Effect of precocene-2
treatment in the 4th instar
Precocene-2 treatment of 4th-instar larvae of S. gregaria induces some insects to moult to precocious adults whereas others moult to normal Sth-instar larvae and then to morphogenetically normal adults (Table 2). These effects are in accord with the results of Unnithan et al. (1980) and Chenevert et al. (1981). The larvae moulting to precocious adults moult 4-5 days later than the controls and have the specialized areas on all the abdominal terga. In females the integumentary areas of the anterior terga have the same shape as those of the controls, whereas in the posterior terga the outline, marked by the endocuticular border, is irregular. In males the integumentary areas of the 4th tergum were often composed of small islets, and those of the terga behind of the 4th, though having the same basic inverted-L shape as in the controls, often had irregular outlines (Fig. 20). The surface area of these regions in the males treated with precocene was about half that of normal adult males and smaller than that of precocious adults obtained by surgical allatectomy (Fig. 2). After treatment with precocene, the cell density in the integumentary areas resembled that of both allatectomized and normal adults (3.910 cells/mm2 for the 3rd tergum and 5.270 cells/mm’ for the 5th tergum). DISCUSSION
Allatectomy and morphogenetic pattern of the integumentary areas
The presence of specialized areas on all the terga of the precocious adults of S. gregaria signifies that, in the 9-10 days which elapse between the moment of allatectomy and the successive ecdysis, there is differentiation not only of the presumptive integumentary area cells of the first terga (2nd, 3rd and 4th) as in the controls (Fig. I), but also of the integumentary area cells present in the last terga (5th, 6th, 7th and 8th). Thus the morphogenetic pattern of the areas which during normal development extends over two instars may occur more rapidly, within a single instar.
Significantly the total surface area of the integumentary areas of precocious adults was always less than that of the controls. The reduction of these areas seems to be isometric in both sexes since the irregular pattern seen in both anterior and posterior terga is the same as that of the controls (Fig. 2). Thus, like the wings of allatectomized individuals of Locusta (Joly, 1960) the integumentary areas are smaller, as a portion of the development preceding metamorphosis has been omitted. The marked size reduction of the integumentary areas in females seems to be the logical consequence of the fact that in normal development the females of S. gregaria locust are always larger than the males (surface area 1.582 mm* for females and 1.219 mm* for males, see in Slifer, 1953). Surgical allatectomy performed approx 36 h into the penultimate instar clearly showed that cell reprogramming in this system occurs over a period of time, as has been found in other hormonally controlled developmental systems (Riddiford, 1978; Lawrence et al., 1978). The outward appearance and the difference in surface areas of the islets in precocious adults shows that the differentiation process starts with 2 or 3 small centres within the presumptive areas and then gradually involves the surrounding cells (Fig. 14). In addition, the competence of the cells to differentiate into integumentary-area cells decreases along the anterior-posterior axis of each tergum in a manner similar to that observed in Manduca by Truman et al. (1974). In both 4th instars allatectomized at 36 h and those allatectomized at O-12 h and given juvenile hormone 24 h later, the integumentary areas or islets with larger surface areas are always located anteriorly. Juvenile hormone and dtyerentiation control
Differentiation of presumptive cells of the integumentary areas is sensitive to changes in the humoral environment and responds to them in predictable ways. When the corpora allata have been removed or inactivated at the beginning of the next to the last larval instar, early differentiation of the integumentary areas can be observed. The presence of corpora allata in situ starting from 3648 h of the 4th instar interferes with the morphogenesis (Fig. 21, upper part). Likewise the topical administration of juvenile hormone on well-defined presumptive areas after surgical allatectomy alters the morphogenesis of the corresponding integumentary areas. Our results can thus be summarized as follows: (a) with juvenile hormone present (corpora allata active), the presumptive cells located in the cardiac bands of the terga are not capable of modifying their fate, (b) when the hormone is lacking (corpora allata surgically removed or inactivated chemically), the presumptive cells lose their capacity to synthesize larval cuticle, become committed to forming integumentary areas with a specialized cuticle which appear in the next moult. This scheme,is compatible with what is known in other insects regarding the change of commitment by other true epidermal cells (Richard, 1981; Riddiford, 1981; Laufer and Borst, 1983) when ecdysteroids are increasing in absence of juvenile hormone.
Differentiation
951
of integumental areas
+davs
Ial is
II iis
1
2
3
4
5
6
7
0
;;34.-
5
davs
Al adult cell normal integum
larval cell normal integum.
cs ,1;5!
anterior morphogenesis pulse c**********
LARVAL -
H.P.l.As
B) H.Rl. areas - forming cells of 2nd. 3rd and 4th tergum
NO morphogenesis
anterior peripher morphogenesis *+*++*********a
c> 9 lmllllm
NO morphogenesis
posterior morphogenesis
*****c+*****c* H.P.1. areas -forming cells of 5th,6th,7th and 6th tergum
specialized H.PI.A. cells of 2nd. 3rd,end 4th tergum
speclellzed H.P.I.A. cells of 5th,6th, ‘Ith,snd 8th tergum
Fig. 21. Summary of the chronological sequence of hormone-dependent events in differentiation of integumentary areas. (A) The epidermal cells of the normal abdominal integument, during 5th instar, undergo adult differentiation in concomitance with the ecdysteroid pulse and with absence of juvenile hormone (adult reprogrammation). (B) Some cells that form integumentary areas of the anterior terga (bl), at the beginning of the 4th instar (perhaps in concomitance with an ecdysteroid pulse and during the gradual increase in juvenile hormone undergo their differentiation in specialized cells (morphogenesis pulse). The remainders (b2) differentiate during the 5th instar (periferal morphogenesis). (C) The cells that form integumentary areas of posterior terga differentiate only in concomitance with the hormonal variations present in the 5th instar (posterior morphogenesis). Ekdysteroids (---) and juvenile hormone (. . .) patterns from Morgan and Poole (1976), Injeyan and Tobe, 1981.
The scheme also agrees with Wigglesworth’s gestion (1952) according to which the
sug-
“premetamorphic” imaginal differentiation occurs only during brief periods of juvenile hormone absence that supposedly exist in larval instars. Unfortunately accurate measurements of change in juvenile hormone synthesis in hemimetabolous insects during larval life are not numerous (Szibbo et al., 1982; Tobe er al., 1985; Lanzrein et al., 1985) and some of the reported identifications should be treated with caution (see e.g. Bergot et al., 1981; Schooley et al., 1984 for comments on this matter). However in support of our scheme (a) the gradual increase of juvenile hormone
synthesis during the first days of the 4th instar both of Diploptera (Szibbo et al., 1982) and S. gregaria (Injeyan and Tobe, 1981) and (b) also the ecdysteroid pulse present at the beginning of the penultimate instar of Nauphoeta (Lanzrein et al., 1985) are to be held in consideration. Cellular competence and imaginal dlferentiation
It is generally agreed that imaginal differentiation commences when the presumptive cells have acquired competence (Laufer and Borst, 1983). During the final (4th/5th) larval moult the abdominal epidermis and the presumptive cells of the integumentary areas
952
GIOVANNI SBRENNA and
in the 5th-8th segments continue to secrete larval cuticle whereas the presumptive cells of the anterior integumentary areas (abdominal segments 2nd4th) undergo their specialized differentiation (Fig. 1). One explanation for these differing responses might be that the cells of the integumetary areas differ in their sensitivity to juvenile hormone, as found in Nuuphoeta (Lanzrein, 1979). Although we have no experimental evidence to exclude this, we suggest, instead, that some cells (“anterior peripheral presumptive cells”, Sbrenna and Sbrenna-Micciarelli, 1983) and cells forming the integumentary areas of Sth-8th segments during the 4th instar are not committed to differentiation to integumentary areas. In various other morphogenetic systems regulated by ecdysteroids (the eye of S. cynthiu, see Willis, 1969; gin-trap region of Manduca see Riddiford, 1978; the intrasegmental pocks see Roseland and Riddiford, 1980; integument of Locusta see Brehelin and Aubry, 1981) the response of the cells has also been observed to be asynchronous. In larvae of S. gregaria asynchronism in the cellular cycle (Sbrenna, unpublished observations) may come into play to regulate the reprogramming of epiderrnal cells. Thus, not all of the presumptive cell population present at the beginning of the 4th instar is able to start differentiation. Only a certain number of cells, depending on their abdominal tergal position, can acquire competence to form integumentary areas, then lose the possibility of larval cuticle synthesis at about 36-48 h, differentiating themselves into larval integumentaryarea cells. The other presumptive cells, both in these anterior terga and in the posterior ones become competent to differentiation during the pre-apolysis period of the 5th instar. In this way, after the imaginal ecdysis the specialized areas reach their final pattern. Figure 21 shows a schematic summary of progress of developmental programme of the integumentary areas in two last-instar larvae of Schistocerca.
Some results of this report support this hypothesis although further investigations are necessary for the solution of these problems. Hormone applications and in vitro cultures are now underway to explore the conditions and hormonal requirements of these different tergal segments. authors wish to thank Professor Lynn M. Riddiford for her critical reading of the manuscript and Professor G. Colombo for his continuous encouragement during the course of this work. Acknowledgements-The
ANNA MICCIARELLI-SBRENNA
Morphogenetic effect of precocene I and II on Schistocerca gregaria (Forsk.). Experientia 37, 32-33. Chevreau J., Bellot J. and Cabanier M. J. (1977) Formuiaire de Techniques Histologiques. Maloine S. A., Paris, Injeyan H. S. and Tobe S. S. (1981) Phase polymorphism in Schistocerca gregaria: assessment of juvenile hormone synthesis in relation to vitellogenesis. J. Insect Physiol. 27, 203-210.
Johnson R. A. and Hill L. (1973) The activity of the corpora allata in the fourth and fifth larval instars of the migratory locust. J. Insect Physiol. 19, 1921-1932. Joly L. (1960) Functions des corpora allata chez Locusta migratoria L. These, Strasbourg. Joly P. (1972) Environmental regulation of endocrine activity of Acridids. Gen. Comp. Endocr. Suppl. 3, 459-465. Kiguchi K. and Riddiford L. M. (1978) A role of juvenile hormone in pupal development of the tobacco homworm, Manduca sexta. J. Insect Physiol. 24, 673-680.
Lanzrein B. (1979) The activity and stability of injected juvenile hormones (JH I, JH II and JH III) in last-instar larvae and adult females of the cockroach Nauphoeta cinerea. Gen. Comp. Endocr. 39, 69-78.
Lanzrein B., Gentinetta V., Abegglen H., Baker F. C., Miller C. A. and Schooley D. A. (1985) Titers of ecdysone, 20-hydroxyecdysone and juvenile hormone III through the life cycle of a hemimetabolous insect, the ovoviviparous cockroach Nauphoeta cinerea. Experientia 41,913-917.
Laufer H. and Borst D. W. (1983) Juvenile hormone and its mechanism of action. In Endocrinology of Insects (Ed. by Downer R. G. H. and Laufer H.), pp. 203-216. A. R. Liss, N.Y. Lawrence T. S., Beers W. H. and Gilula N. B. (1978) Transmission of hormonal stimulation by cell-to-cell communication. Nature 272, 501-506. Loveridge J. P. (1980) Cuticular water relations techniques. In Cuticle Techniques in Arthropods (Ed. by Miller T. A.), pp. 301-366. Springer, N.Y. Making P. (1958) Transpiration through the “Slifer’s patches” of Acrididae (Orthoptera). J. exp. Bioi. 48, 247-263.
Mitsui T. and Riddiford L. M. (1976) Pupal cuticle formation by Manduca sexta epidermis in vitro: patterns of ecdysone sensitivity. Dev. Biol. 54, 172-186. Morgan E. D. and Poole C. F. (1976) The pattern of ecdysone levels during development in the desert locust Schistocerca
gregaria. J. Insect Physiol. 22, 885-889.
Nickerson B. (1956) Pigmentation of hoppers of the desert locust (Schistocerca gregaria Forskal) in relation to phase coloration. Anti-Locust Bull. 24, l-34. Pener M. P. (1967) Effects of allatectomy and sectioning of the nerves of the corpora allata on oiicyte growth, male sexual behaviour, and colour change in adults of Schistocerca gregaria. J. Insect Physiol.
13, 665-684.
Pener M. P., Orsham L. and De Wilde J. (1978) Precocene II causes atrophy of corpora allata in Locusta migratoria. Nature 272, 35&353.
Richards G. (1981) Insect hormones in development. Biol. REFERENCES Bergot B. J., Schooley D. A. and De Kort C. A. D. (1981) Identification of JH III as the principal juvenile hormone in Lacusta migratoria. Experientia 37, 909-910. Brehelin M. and Aubry R. (1981) Correlative and experimental studies on blood ecdysteroid levels and integumental events in last instar larvae of Locusta migratoria (Insecta): results and critical evaluation. Brdl. Sot. zool. Fr. 107, 21-32. Gassier P. and Delorme-Joulie C. (1976) La differentiation imaginale du tegument chez le criquet pelerin, Schistocerca gregaria Forsk. III. Les differences phasaires et leur d&&minisme. Insectes sot. 23, 179-198. Chenevert R., Perron J. M., Paquin R. and Plante R. (1981)
Rev. 86, 501-549.
Riddiford L. M. (1978) Ecdysone induced change in cellular commitment of the tobacco hornworm Manduca sexra at the initiation of metamorphosis. Gen. Comp. Endocr. 34, 438-446.
Riddiford L. M. (1980) Interaction of ecdysteroid and iuvenile hormone in the regulation of larval growth and metamorphosis of the tobacco homworm. In-Progress in Ecdvsone Research (Ed. bv Hoffmann J. A.). DD. 409430. Els&ier-North Holland B. P., Amsterdam; . . Riddiford L. M. (1981) Hormonal control of epidermal cell development. Am. Zoof. 21, 751-762. Roseland C. R. and Riddiford L. M. (1980) Analysis of a cuticular spacing pattern after metamorphosis “in vitro” of larval integument. In Invertebrate Systems “In Vitro”
Differentiation of integumental areas (Ed. by Kurstak E., Maramorosch K. and Dubendorfer A.), pp. 117-123. Elsevier North Holland B. P., Amsterdam. Roussel J. P. (1977) L’action differentielle des hormones juveniles sur la metamorphose chez Locus& migratoria. J. Insect Physiol. 23, 1143-1150. Sbrenna G. and Antonelli M. (1977) Ultrastructure of the “fenestrae” specialized integumentary areas of the desert locust Schi.rtocerca gregaria Forsk. (Orthoptera, Acrididae). lni. J. Insect Morph. Embryol. 6, 265-275. Sbrenna G. and Sbrenna-Micciarelli A. (198Oa) Development and morphogenesis of the “fenestrae” specialized integumentary areas of the desert locust Schisrocerca gregaria (Orthoptera, Acrididae). Acta Embryol. Morph. Exp. 1, 199-215. Sbrenna G. and Sbrenna-Micciarelli A. (1980b) Juvenile hormone and pattern formation of “fenestrae” in Schistocerca gregaria (Orthoptera, Acrididae). Gen. Comp. Endocr. 40, 361.
Sbrenna G. and Sbrenna-Micciarelli A. (1983) Morphogenesis of the larval “fenestrae” during moulting in Schisiocerca gregaria Forskal (Orthoptera, Acrididae). Monitore 2001. ital. II, 71-W.
Schooley D. A., Baker F. C., Tsai L. W., Miller C. A. and Jamieson G. C. (1984) Juvenile hormones 0, I, and II exist only in L..epidoptera. In Biosynthesis, Metabolism and Mode of Action of Inuertebrate Hormones (Ed. by Hoffmann J. and Porchet M.), pp. 373-383. Springer, Heidelberg. Slifer E. H. (1951) Some unusual structures in Locusta migraroria migratorioides and their probable function as thermoreceptors. Proc. R. Sue. Ser. B. 138, 414437.
953
Slifer E. H. (1953) The pattern of specialized heat-sensitive areas on the surface of the body of Acrididae (Orthoptera). Part I: the males; part II: the females. Trans. Am. em. Sot. 79, 37-97. Szibbo C. M., Rotin D., Feyereisen R. and Tobe S. S. (1982) Synthesis and degradation of C 16 Juvenile hormone (JH III) during the final two stadia of the cockroach, Diploptera punctata. Gen. Camp. Endocr. 48, 25-32.
Tobe S. S., Ruegg R. P., Stay B. A., Baker F. C., Miller C. A. and Schooley D. A. (1985) Juvenile hormone titre and regulation in the cockroach Dipfoprera punctatu. Experientia 41, 1028-1034.
Truman J. W., Riddiford L. M. and Safranek L. (1974) Temporal patterns of response to ecdysone and juvenile hormone in the epidermis of the tobacco homworm, Manduca sexta. Dev. Biol. 39, 247-262.
Unnithan G. C., Nair K. K. and Syed A. (1980) Precoceneinduced metamorphosis in the desert locust Schbtocercu gregaria. Experientia 36, 135-136. Uvarov B. P. (1966) Grasshoppers and Locust. Vol. I. Anti-Locust Res. Centre, University Press, Cambridge. Wigglesworth V. B. (1952) Hormone balance and the control of metamorphosis in Rhodnius prolixus (Hemiptera). J. exp. Biol. 29, 62063 1.
Wigglesworth V. B. (1964) The hormonai regulation of growth and reproduction in insects. Adv. Insect. Physiol. 2, 247-336.
Williams C. M. (1953) Morphogenesis and the metamorphosis of insects. Harvey L.ect. 47, 126-155. Willis J. H. (1969) The programming of differentiation and its control by juvenile hormone in satumiids. J. Embryol. exp. Morph. 22, 2744.
Printed in Great
0022-1910/86 $3.00+0.00 Pergamon Journals Ltd
1986
Britain
ROLE OF THE JUVENILE HORMONE ON THE DIFFERENTIATION OF SPECIALIZED INTEGUMENTAL AREAS, “FENESTRAE”, IN SCHISTOCERCA GREGARIA (ACRIDIDAE: ORTHOPTERA) SBRENNA and ANNA MICCIARELLI-SBRENNA of Zoology, Via L. Borsari, 46-44100 Ferrara, Italy
GIOVANNI Institute
(Received 19 December
1985; revised 7 March 1986)
Ah&act---“Fenestrae” or “Slifer’s patches” are highly permeable integumentary areas of locusts, segmentally arranged in the adult abdominal terga of males and females. In S. gregaria they arise, after being newly programmed, from epidermal cells, and the developmental pattern proceeds during the last two larval instars. In order to study the mechanism by which the cells that form these areas respond to the hormonal stimuhzs to change commitment, 4th- and Sth-instar larvae were surgically allatectomizcd or treated with precocene. When performed within the first 24 h of the 4th-instar, allatectomy, whether surgical or chemical, leads to the appearance of precocious adults, with the integumentary areas on all terga. If removal of the corpora allata is done after that period, the precocious adults have smaller integumentary areas, and if the removal is delayed for 48 h, differentiation of the areas does not occur at all in the last terga. Topical application of juvenile hormone to the sites of the presumptive areas of the 4th-instar larvae, allatectomized during the first 12 h, reduces the specialized differentiation. The presence or absence of juvenile hormone commits the integumentary-area-forming cells to the deposition of larval or specialized
cuticle, respectively. The hormonal events during the larval instars and an asynchronism of the integumentary cellular cycle may explain the distribution of these integumentary areas. Key Word Index: Allatectomy, Slifer’s patches, specialized integumental pattern, Schisfocerca gregaria, precocious morphogenesis
INTRODUCI’ION The effect of juvenile hormone on epidermal cells has
been called “maintenance of the S~U~US quo” (Williams, 1953). In holometabolous insects levels of ecdysteroids and juvenile hormone in the different phases of development suggest that commitment of cells forming adult epidermally derived structures requires that the cells be exposed to ecdysteroids with no juvenile hormone (Mitsui and Riddiford, 1976; Kiguchi and Riddiford, 1978), and that the different structures require different and characteristic periods of time for changing their commitment (Truman et al., 1974). Some authors (Wigglesworth, 1964; Riddiford, 1980) hypothesize that this scheme could be valid also for the control of the metamorphosis of hemimetabolous insect. On the other hand, in these insects, experiments involving bioassay of haemolymph extracts or corpora allata (Wigglesworth, 1952; Joly, 1972; Johnson and Hill, 1973) lend weight to the suggestion that both the maintenance of larval characteristics and the metamorphosis are controlled in a similar way. In the median dorsal-zone of each abdominal tergum of adults of Schistocerca gregaria there is a pair of specialized integumentary areas that have been variously termed “fenestrae” (Slifer, 1951) and “Slifer’s patches” (Uvarov, 1966). Their cells differ 941
greatly from those of the surrounding integument and synthesize a cuticular layer composed of exocuticle alone, containing many large pore-canals (Sbrenna and Antoneili, 1977). This specialized integumentary area is involved in the active control of water content (Makings, 1968; Loveridge, 1980) and since the earlier terms “fenestra” and “Slifer’s patches” are not descriptive of their activity, we have called these structures as “highly permeable integumentary areas” (abbreviated to integumentary areas). The pattern of these areas in adults is reached gradually during the final-larval stages (Sbrenna and Sbrenna-Micciarelli, 1980a) indicating that the initial processes of this morphogenesis begin before the last-larval instar. This morphogenesis, which is realized in different successive stages, offers an excellent test-system for studying the action of ecdysteroids and juvenile hormone on integumental differentiation in hemimetabolous insects. Some preliminary results have already been published (Sbrenna and Sbrenna-Micciarelli, 1980b). In the present work we report and discuss the effect on morphogenesis of the integumentary areas when juvenile hormone deficiency is created by surgical removal of the corpora allata or by treatment with Precocene 2.
GIOVANNI SBRENNA and ANNA MICCIARELLI-SBRENNA
942 Table
I. Effect of surgical allatectomy on metamorphosis of 4th- and Sth-instar larvae of S. areaaria
Instar 5th
4th
Larval death
Time after ecdysis (hours)
Allatect. larvae
at next ecdysis
&I2 12-24
12 I7
0 0
0 0
I2 I7
6 6
&I2 12-24 2436 3648
27 I2 I9 8
2 I 3 I
25 II I4 4
0 0 2 3
8 6 6 4
*Sham-operated
Normal adults
Sham-operat. larvae*
larvae all moulted to normal adults.
MATERIAL AND METHODS
Experimental animals
We used a strain of S. gregaria raised in our laboratory from eggs provided by the C.O.P.R. of London. The life history and rearing procedures have been described elsewhere (Sbrenna and Antonelli, 1977). We utilized 4th-instar nymphs at O-12, 12-24, 24-36 and 3648 h following ecdysis, and Sth-instar nymphs O-12 and 12-24 h after ecdysis. Extirpation of larval corpora allata
During the operation the larvae were anaesthetized with carbon dioxide; allatectomy was performed using Pener’s technique (persona1 demonstration). For the sham-operated insects, the same procedure was followed, but without removing the glands. Other intact insects of the same age were used as controls. The allatectomized, sham-operated and control insects were marked individually and observed daily and killed after metamorphosis from the 6th to the 20th day of adult life (Table 1). Juvenile hormone applications
Juvenile hormone 1 (Fluka A.G.) was dissolved in olive oil (10 pg/ ~1). Five pg were applied topically, with a Hamilton microlitre syringe, along the dorsai midline of the 7th abdominal tergum of carbon dioxide-anaesthetized 4th-instar larvae. Larvae were allatectomized 24 h prior to hormone application. After the application, the insects were kept in a glass jar with the same temperature and lighting conditions as the controls. Olive oil alone was applied to the controls. Precocene-2
Precocious adults
deposited on the surface of the Petri dishes (14 x 2 cm) at different concentrations (see Table 2) as described by Unnithan et al. (1980). Groups of 8 newly moulted 4th-instar nymphs were exposed to the precocene for 24 h at 35°C. After treatment the insects were transferred to rearing cages. Petri dishes for controls were prepared using acetone alone. The effects of the treatment were recorded daily. The animals were sacrificed and dissected after methamorphosis from the 6th to 20th day of adult life. Histological procedures
The terga from the 1st to the 8th abdominal segment of each insect were excised and then fixed in 2% glutaraldehyde in 0.1 M phosphate buffer at pH 7.0 or in 2% glutaraldehyde in 0.05 M cacodylate buffer at pH 7.2 with 4% sucrose. Some of these terga, with the muscles, tracheae and fat bodies removed, were mounted whole in balsam, after staining using one of the following dyes: Giemsa solution, Nile blue, Azure II or paracarmine (Chevreau et al., 1977). Others, after being fixed in glutaraldehyde, were post-fixed in 1% osmium tetroxide, dehydrated, and embedded in Epon-Araldite. The sections (1 pm thick) were stained with Azure II-methylene blue. Drawings of whole mounts of the integumentary areas, stained with Azure II, were made using a projection microscope (Reichert) and their surface areas were measured with a planimeter. The cell density was determined using three radomly chosen points of the integumentary areas of each insect. Cell counts were made on these areas. RESULTS
Normal development of the integumentary areas
treatments
Precocene 2, purchased from the Aldrich Chemical Co., was dissolved in spectra1 grade acetone and
Figure 1 shows a diagram of the developmental pattern of the specialized abdominal areas of S.
Table 2. Mode of application, time, and effect on metamorphosis of precocene treatment of 4th-instar larvae of S. gregaria Dose of precocene pg/cm’ Petri dishes (fig/larva) 45 pg/cm’ (865 pg/larva) 60 pgjcm’ (I 153 pg/larva)
Time after 3rd/4th instar ecdysis (hours)
Treated larvae
Precocious adult
Normal adult
Death’ during treatment
O-24
32
8(25%)
18(56.2%)
6(18%)
&24
40
16(40%)
14(35%)
10(25%)
Control with acetone
O-24
24
-
22(91.6%)
2(8.3%)
*The death occurred mostly during the 24 h of treatment.
Differentiation of integumental areas
m
sma33
lVNI9VWI
sIsAa33 w
0
m
-_-_ -
cu _
r
iv~dvi
943
944
GIOVANNI
SBRENNA
and ANNA MICCIARELLI-SBRENNA
P2
dd
1000 -
I
0
Control
q
V
(O-12 h
allat.
IV allat.10.12
ul
IV
Precoc.
h II
800-
;
5
m
m ;
400
2 :
200
0 2nd seg.
3rd seg.
4th
seg
5th
seg.
6th seg.
P2
7th seg.
I
99
1000
0 fzl q
8th seg.
Control V
allat.
(O-12
IV allat.lO-12
h 1 h )
: 800 CT Y m .;
600
g n m ;
400
: iii 200
L
0 d seg.
3rd seg.
4th seg.
5th seg.
6th seg.
7th seg.
8th seg.
Fig. 2. Effects of removal of the corpora allata or precocene-2 treatment on the area of abdominal integumentary areas according to abdominal segment. Each histogram represent the mean of 640 samples (+SD).
gregaria. These areas are not present during the first three larval instars: in the medial zone of each abdominal tergum there is only a heavily pigmented area called “cardiac band” (Nickerson, 1956). Differentiation begins during the 4th instar. During the mitotic period (36th hour) two or three small light-coloured zones appear, in the 2nd, 3rd and 4th terga, in the cardiac bands. In the hours that follow
these zones grow larger and then blend together to form two lighter strips parallel to the dorsal medial line. At about the 72nd hour differentiation ceases, before all the presumptive cells of the integumentary areas in these first terga have actually differentiated, a certain number of them (“peripheral presumptive cells”, Sbrenna and Sbrenna-Micciarelli, 1983) differentiate later in the next instar (5th). This inter-
Figs 3 and 4. Integumentary areas of a male control adult, present on the 3rd and 5th abdominal tergum respectively. Azure II-stained whole mounts (asterisk: tergal middle line). Bar = 0.5 mm. Fig. 5. Section of the highly permeable integument of a male control adult. Below the specialized exocuticle (ex) there is no endocuticle (ss: subcuticular space). Bar = 10 pm. Fig. 6. Whole mount of a female control integument. The integumentary area is outlined by the characteristic cuticular bulge (cb.). Bar = 0.15 mm. Fig. 7. Integumentary areas present on the 5th tergum of an allatectomized adult (deprived of corpora allata at O-12 h of the 5th instar). The edge of the areas are clearly delineated by the cuticular bulge (arrows) as the control (see Fig. 4). Fig. 8. Section of the highly permeable integument of an allatectomized adult (deprived of corpora allata at &I2 h of the 5th instar). The aspects of the specialized cuticle and epidermis are similar to HP integument of the control adult (see Fig. 5). Bar = IOpm.
945
Figs 9 and 10. 3rd and 5th tergum of a precocious adult (allatectomized at &12 h of 4th instar). The integumentary areas have a reduced surface area and an irregular outline (see Figs 4 and 7). Azure IIstained whole mounts (asterisk: tergal middle line). Bar = 0.5 mm. Fig. 11. Section of highly permeable integument of a precocious adult similar to Figs 9 and 10. The cuticular bulge (cb) secreted by normal epidermis (EI) outlines the highly permeable integument of a 5th tergum as in the control adult (see Fig. 6). Bar = 10 pm. Figs 12 and 13. 3rd and 5th tergum of a male precocious adult (allatectomized at 2436 h of 4th instar). The integumentary areas of the 3rd tergum have reduced surface, whereas those of the posterior tergum (5th) are broken up into islets. Fig. 14. Greater
magnification
of the Fig. 13. The islets of integumentary area are consistently by a cuticular bulge (arrows). Bar = 0.25 mm.
marked
Figs 15 and 16. 3rd and 5th tergum of a male precocious adult (allatectomized at 3648 h of 4th instar). The anterior integumentary areas are very small and irregular, whereas the posterior ones do not differentiate. Respective bars = 0.5 mm and 0.2 mm. 946
Figs 17 and 18 Effects of topical application of juvenile hormone to 4th-instar larvae (allatectomized during the first 12 h after ecdysis). Islets of integumentary area are evident on the female 7th tergum (Fig. 17). The areas on the male 7th tergum are missing (Fig. 18). Respective bars = 0.5 mm and 0.2 mm. Figs 19 and 20. Whole mounts of 3rd and 5th tergum of male precocious adult after precocene-2 treatment during the 4th instar. The integumentary-area pattern is similar to that of a control and to a precocious adult surgically allatectomized during the first CL12h of the 4th instar (see Figs 3, 4, 9 and IO).
947
Differentiation of integumental areas ruption of the differentiation means that during the 4th instar, no lighter strips appear in the remaining terga (Sth-8th), but the cardiac bands are evident. After ecdysis to the 5th instar, pairs of lighter strips (called “larval integumentary areas”) appear in the 2nd, 3rd and 4th terga (Fig. 1). No differences in the larval form of the areas are present between males and females. Differentiation recommences during the 5th instar (the final larval instar) on about the 3rd day after ecdysis. From this moment both the peripheral presumptive cells, located around the lighter strips, and the cells forming the areas of the last four terga begin the differentiation process. After the next (imaginal) ecdysis, all the abdominal terga in both males and females have assumed the definitive pattern of integumentary areas (Figs 1, 3 and 4), and the normal epidexmal cells synthesize the endocuticular bulge (Sbrenna and Antonelli, 1977) between the normal integument and the integumentary areas (Fig. 6). The bulge, secreted during the first days of adult life, is missing in the “larval integumentary areas” of the 5th instar. Allatectomy during the 5th~larval instar Allatectomized Sth-instar larvae undergo methamorphosis on day 12-13. The removal of the corpora allata thus lengthens the final-larval instar by about 3 days. Morphologically the allatectomized insects resemble those described by Pener (1967). The adults do not take on the characteristic yellow colour which is observed in normal males reaching sexual maturation, and sexual behaviour and locomotor activity are less intense than in controls and shamoperated insects. Removal of the glands during the first hours of the instar (O-12 and 12-24 h) resulted in a small but statistically significant reduction in the sizes of the integumentary areas (Fig. 2) yet allatectomy did not modify the differentiation pattern of the areas and they were present on all the abdominal terga in their characteristic shape and appearance (Fig. 7). The integument is these areas presented a cuticle composed of exocuticle alone crossed by numerous straight, wide pore canals. Between the exocuticle and the specialized cells there was a wide subcuticular zone (Sbrenna and Antonelli, 1977), resembling that of control adults (Figs 5 and 8). Allatectomy during the 4th-larval instar O-I2-h old larvae In the controls and sham-operated insects the 4th-5th ecdysis occurs on about day 6 of the instar, and a last-instar larva emerges from the exuviae. When 4th-instar larvae were allatectomized within 12 h of ecdysis, the instar was lengthened by 34 days. Then they moulted to precocious adults (Pener et al., 1978) [Table 11. Removal of the corpora allata during the first O-12 h resulted in a reduction in the sizes of the integumentary areas (Fig. 2). The dimensions of these areas of the precocious adults was however much less than that of the control adults, especially in females, where the areas were roughly half the size of those in normal insects (Fig. 2). The cell density of the areas remains however unchanged: the 3rd tergum has
949
4.040cells/mm2 compared to 4.070 cells in control adults; and the 5th tergum has 5.560 cells/mm2 compared to of 5.670 observed in the controls. The normal integument of the precocious adults was found to be cytologically different from that of control adults, due to the presence of pigment granules in. the epithelial cells. Also these precocious adults had quiescent integumentary glands as previously described by Cassier and Delorme-Joulie (1976). On the contrary in all the abdominal terga of both male and female precocious adults there were integumentary areas resembling those in the control adults in shape and appearance (Figs 9 and 10). The cuticle and also the specialized cells resembled those of the specialized areas of normal adults and were separated from the epidermal cells with a very clearcut endocuticular boundary (Fig. 1I). 12-24-h old larvae The precocious adults formed after allatectomy of 12-24-h old, 4th-instar larvae had specialized areas on all the abdominal terga. This delayed removal of the corpora allata did not influence the differentiation of the presumptive cells. The integumentary areas of the anterior terga were like those present in precocious adults obtained from 4th-instar larvae which were deprived of their glands on the 12th hour of life. By contrast the integumentary areas of the last terga had a very irregular outline and, instead of lying within the areas as they normally do, the trichoid sensilla remained outside them. 24-36-h old larvae After, allatectomy of 24-36-h old, 4th-instar larvae, most formed precocious adults. The specialized areas of the first three terga of these precocious adults differentiated normally, but those of the last terga were much smaller or broken up into tiny islets (Figs 12 and 13). The cells of these islets however resembled those of the controls, with regularly spaced nuclei, clear-cut cell boundaries and the characteristic stellate shape. These small areas could be distinguished because their borders were consistently marked by an endocuticular thickening (Fig. 14). In males the integumentary areas of the 4th tergum were also smaller, and sometimes one of the two areas is broken up into small round areas. 3648-h old larvae When 36-48-h old, 4th-instar larvae were allatectomized, only half formed precocious adults. The remainder formed larvae (Table 1). The specialized areas of the first terga of the precocious adults differentiated normally but were very small and irregular. They resembled the lighter strips (larval integumentary areas) of 5th~instar controls but these areas were bounded by endocuticular border (Fig. 15). On the contrary the areas of the four posterior terga did not differentiate. At this level of the abdomen all the cells appeared identical to those of normally developing epidermal cells before the onset of differentiation (Fig. 16). EJect of juvenile hormone on 4th -I’nstarallatectomized larvae We have investigated the juvenilizing activities of the juvenile hormone using juvenile hormone I.
950
GIOVANNI SBRENNAand ANNA MICCIARELLI-SBRENNA
Hormones 1 and 11 are more active than hormone III in many bioassays (Roussel, 1976; Lanzrein, 1979). Fourth-instar larvae, allatectomized O-12 h after ecdysis and treated 24 h after the operation with juvenile hormone, moulted into precocious adults on the 10th day following ecdysis. The morphogenetic response of the integumentary areas to topical treatment was different in the two sexes, the females being less sensitive to the hormone than the males. In females, the specialized areas appeared on all the abdominal terga, although the areas on the 7th tergum (i.e. where the hormone was applied) and on the 8th were smaller or irregularly shaped and lobed. These areas however were always clearly identifiable, due to the endocuticular thickening marking their boundaries (Fig. 17). In the males the specialized areas on the 7th (juvenile hormone-treated) and 8th terga were missing (Fig. 18) or present in the form of small islets which were often difficult to distinguish. Effect of precocene-2
treatment in the 4th instar
Precocene-2 treatment of 4th-instar larvae of S. gregaria induces some insects to moult to precocious adults whereas others moult to normal Sth-instar larvae and then to morphogenetically normal adults (Table 2). These effects are in accord with the results of Unnithan et al. (1980) and Chenevert et al. (1981). The larvae moulting to precocious adults moult 4-5 days later than the controls and have the specialized areas on all the abdominal terga. In females the integumentary areas of the anterior terga have the same shape as those of the controls, whereas in the posterior terga the outline, marked by the endocuticular border, is irregular. In males the integumentary areas of the 4th tergum were often composed of small islets, and those of the terga behind of the 4th, though having the same basic inverted-L shape as in the controls, often had irregular outlines (Fig. 20). The surface area of these regions in the males treated with precocene was about half that of normal adult males and smaller than that of precocious adults obtained by surgical allatectomy (Fig. 2). After treatment with precocene, the cell density in the integumentary areas resembled that of both allatectomized and normal adults (3.910 cells/mm2 for the 3rd tergum and 5.270 cells/mm’ for the 5th tergum). DISCUSSION
Allatectomy and morphogenetic pattern of the integumentary areas
The presence of specialized areas on all the terga of the precocious adults of S. gregaria signifies that, in the 9-10 days which elapse between the moment of allatectomy and the successive ecdysis, there is differentiation not only of the presumptive integumentary area cells of the first terga (2nd, 3rd and 4th) as in the controls (Fig. I), but also of the integumentary area cells present in the last terga (5th, 6th, 7th and 8th). Thus the morphogenetic pattern of the areas which during normal development extends over two instars may occur more rapidly, within a single instar.
Significantly the total surface area of the integumentary areas of precocious adults was always less than that of the controls. The reduction of these areas seems to be isometric in both sexes since the irregular pattern seen in both anterior and posterior terga is the same as that of the controls (Fig. 2). Thus, like the wings of allatectomized individuals of Locusta (Joly, 1960) the integumentary areas are smaller, as a portion of the development preceding metamorphosis has been omitted. The marked size reduction of the integumentary areas in females seems to be the logical consequence of the fact that in normal development the females of S. gregaria locust are always larger than the males (surface area 1.582 mm* for females and 1.219 mm* for males, see in Slifer, 1953). Surgical allatectomy performed approx 36 h into the penultimate instar clearly showed that cell reprogramming in this system occurs over a period of time, as has been found in other hormonally controlled developmental systems (Riddiford, 1978; Lawrence et al., 1978). The outward appearance and the difference in surface areas of the islets in precocious adults shows that the differentiation process starts with 2 or 3 small centres within the presumptive areas and then gradually involves the surrounding cells (Fig. 14). In addition, the competence of the cells to differentiate into integumentary-area cells decreases along the anterior-posterior axis of each tergum in a manner similar to that observed in Manduca by Truman et al. (1974). In both 4th instars allatectomized at 36 h and those allatectomized at O-12 h and given juvenile hormone 24 h later, the integumentary areas or islets with larger surface areas are always located anteriorly. Juvenile hormone and dtyerentiation control
Differentiation of presumptive cells of the integumentary areas is sensitive to changes in the humoral environment and responds to them in predictable ways. When the corpora allata have been removed or inactivated at the beginning of the next to the last larval instar, early differentiation of the integumentary areas can be observed. The presence of corpora allata in situ starting from 3648 h of the 4th instar interferes with the morphogenesis (Fig. 21, upper part). Likewise the topical administration of juvenile hormone on well-defined presumptive areas after surgical allatectomy alters the morphogenesis of the corresponding integumentary areas. Our results can thus be summarized as follows: (a) with juvenile hormone present (corpora allata active), the presumptive cells located in the cardiac bands of the terga are not capable of modifying their fate, (b) when the hormone is lacking (corpora allata surgically removed or inactivated chemically), the presumptive cells lose their capacity to synthesize larval cuticle, become committed to forming integumentary areas with a specialized cuticle which appear in the next moult. This scheme,is compatible with what is known in other insects regarding the change of commitment by other true epidermal cells (Richard, 1981; Riddiford, 1981; Laufer and Borst, 1983) when ecdysteroids are increasing in absence of juvenile hormone.
Differentiation
951
of integumental areas
+davs
Ial is
II iis
1
2
3
4
5
6
7
0
;;34.-
5
davs
Al adult cell normal integum
larval cell normal integum.
cs ,1;5!
anterior morphogenesis pulse c**********
LARVAL -
H.P.l.As
B) H.Rl. areas - forming cells of 2nd. 3rd and 4th tergum
NO morphogenesis
anterior peripher morphogenesis *+*++*********a
c> 9 lmllllm
NO morphogenesis
posterior morphogenesis
*****c+*****c* H.P.1. areas -forming cells of 5th,6th,7th and 6th tergum
specialized H.PI.A. cells of 2nd. 3rd,end 4th tergum
speclellzed H.P.I.A. cells of 5th,6th, ‘Ith,snd 8th tergum
Fig. 21. Summary of the chronological sequence of hormone-dependent events in differentiation of integumentary areas. (A) The epidermal cells of the normal abdominal integument, during 5th instar, undergo adult differentiation in concomitance with the ecdysteroid pulse and with absence of juvenile hormone (adult reprogrammation). (B) Some cells that form integumentary areas of the anterior terga (bl), at the beginning of the 4th instar (perhaps in concomitance with an ecdysteroid pulse and during the gradual increase in juvenile hormone undergo their differentiation in specialized cells (morphogenesis pulse). The remainders (b2) differentiate during the 5th instar (periferal morphogenesis). (C) The cells that form integumentary areas of posterior terga differentiate only in concomitance with the hormonal variations present in the 5th instar (posterior morphogenesis). Ekdysteroids (---) and juvenile hormone (. . .) patterns from Morgan and Poole (1976), Injeyan and Tobe, 1981.
The scheme also agrees with Wigglesworth’s gestion (1952) according to which the
sug-
“premetamorphic” imaginal differentiation occurs only during brief periods of juvenile hormone absence that supposedly exist in larval instars. Unfortunately accurate measurements of change in juvenile hormone synthesis in hemimetabolous insects during larval life are not numerous (Szibbo et al., 1982; Tobe er al., 1985; Lanzrein et al., 1985) and some of the reported identifications should be treated with caution (see e.g. Bergot et al., 1981; Schooley et al., 1984 for comments on this matter). However in support of our scheme (a) the gradual increase of juvenile hormone
synthesis during the first days of the 4th instar both of Diploptera (Szibbo et al., 1982) and S. gregaria (Injeyan and Tobe, 1981) and (b) also the ecdysteroid pulse present at the beginning of the penultimate instar of Nauphoeta (Lanzrein et al., 1985) are to be held in consideration. Cellular competence and imaginal dlferentiation
It is generally agreed that imaginal differentiation commences when the presumptive cells have acquired competence (Laufer and Borst, 1983). During the final (4th/5th) larval moult the abdominal epidermis and the presumptive cells of the integumentary areas
952
GIOVANNI SBRENNA and
in the 5th-8th segments continue to secrete larval cuticle whereas the presumptive cells of the anterior integumentary areas (abdominal segments 2nd4th) undergo their specialized differentiation (Fig. 1). One explanation for these differing responses might be that the cells of the integumetary areas differ in their sensitivity to juvenile hormone, as found in Nuuphoeta (Lanzrein, 1979). Although we have no experimental evidence to exclude this, we suggest, instead, that some cells (“anterior peripheral presumptive cells”, Sbrenna and Sbrenna-Micciarelli, 1983) and cells forming the integumentary areas of Sth-8th segments during the 4th instar are not committed to differentiation to integumentary areas. In various other morphogenetic systems regulated by ecdysteroids (the eye of S. cynthiu, see Willis, 1969; gin-trap region of Manduca see Riddiford, 1978; the intrasegmental pocks see Roseland and Riddiford, 1980; integument of Locusta see Brehelin and Aubry, 1981) the response of the cells has also been observed to be asynchronous. In larvae of S. gregaria asynchronism in the cellular cycle (Sbrenna, unpublished observations) may come into play to regulate the reprogramming of epiderrnal cells. Thus, not all of the presumptive cell population present at the beginning of the 4th instar is able to start differentiation. Only a certain number of cells, depending on their abdominal tergal position, can acquire competence to form integumentary areas, then lose the possibility of larval cuticle synthesis at about 36-48 h, differentiating themselves into larval integumentaryarea cells. The other presumptive cells, both in these anterior terga and in the posterior ones become competent to differentiation during the pre-apolysis period of the 5th instar. In this way, after the imaginal ecdysis the specialized areas reach their final pattern. Figure 21 shows a schematic summary of progress of developmental programme of the integumentary areas in two last-instar larvae of Schistocerca.
Some results of this report support this hypothesis although further investigations are necessary for the solution of these problems. Hormone applications and in vitro cultures are now underway to explore the conditions and hormonal requirements of these different tergal segments. authors wish to thank Professor Lynn M. Riddiford for her critical reading of the manuscript and Professor G. Colombo for his continuous encouragement during the course of this work. Acknowledgements-The
ANNA MICCIARELLI-SBRENNA
Morphogenetic effect of precocene I and II on Schistocerca gregaria (Forsk.). Experientia 37, 32-33. Chevreau J., Bellot J. and Cabanier M. J. (1977) Formuiaire de Techniques Histologiques. Maloine S. A., Paris, Injeyan H. S. and Tobe S. S. (1981) Phase polymorphism in Schistocerca gregaria: assessment of juvenile hormone synthesis in relation to vitellogenesis. J. Insect Physiol. 27, 203-210.
Johnson R. A. and Hill L. (1973) The activity of the corpora allata in the fourth and fifth larval instars of the migratory locust. J. Insect Physiol. 19, 1921-1932. Joly L. (1960) Functions des corpora allata chez Locusta migratoria L. These, Strasbourg. Joly P. (1972) Environmental regulation of endocrine activity of Acridids. Gen. Comp. Endocr. Suppl. 3, 459-465. Kiguchi K. and Riddiford L. M. (1978) A role of juvenile hormone in pupal development of the tobacco homworm, Manduca sexta. J. Insect Physiol. 24, 673-680.
Lanzrein B. (1979) The activity and stability of injected juvenile hormones (JH I, JH II and JH III) in last-instar larvae and adult females of the cockroach Nauphoeta cinerea. Gen. Comp. Endocr. 39, 69-78.
Lanzrein B., Gentinetta V., Abegglen H., Baker F. C., Miller C. A. and Schooley D. A. (1985) Titers of ecdysone, 20-hydroxyecdysone and juvenile hormone III through the life cycle of a hemimetabolous insect, the ovoviviparous cockroach Nauphoeta cinerea. Experientia 41,913-917.
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