in vitro activity of dipteran ring glands and activation by the prothoracicotropic hormone

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

54, 469-471 (1984)

In Vitro Activity of Dipteran Ring Glands and Activation Prothoracicotropic Hormone BRIAN ROBERTS,* *Department

LAWRENCE I. GILBERT,t

AND WALTER

by.the

E. BOLLENBACHERt

of Zoology, Monash University, Clayton, Victoria 3168, Australia, Biology, University of North Carolina, Chapel Hill, North Carolina

and PDepartment 27514

of

Accepted August 1, 1983 The relationship between hemolymph ecdysteroid titer, ring gland (RG) activity, and prothoracicotropic hormone (PTTH) activation of RG in vitro has been examined during the postfeeding larval, prepupal, and pupal stages of Sarcophaga bullata. Using the ecdysteroid radioimmunoassay (RIA), two significant peaks were recorded during the red spiracular stage and during the first few hours after the formation of the white prepupa and a third large peak 9 hr later. It is postulated that these increases in ecdysteroid titer are involved in the processes of pupariation, puparial tanning, and pupation, respectively. Ring glands isolated from Sarcophaga of known ages were incubated in vitro and the secreted ecdysone was quantified by RIA. Ring glands from early red spiracular stage larvae proved to be the most active and subsequent secretory activity of the RG oscillated every 4 hr with the oscillations gradually decreasing in amplitude. RG activity returned to a basal level 24 hr after formation of the white prepupa, about the time that the hemolymph ecdysteroid titer fell to its basal level. To demonstrate PITH activity in vitro, brains from 3- to 4-hr prepupae were chosen to activate ring glands from postfeeding larvae. Using a graded series of dilutions of PTTH extract it was shown that a dose-response relationship could be obtained for Sarcophaga similar to that demonstrated for the Manduca sexta PTTH-prothoracic gland system. In Sarcophaga maximal activation resulted in a IO-fold increase in ecdysone synthesis and secretion by ring glands stimulated with 0.5 brain eq. Half-maximal stimulation was attained with 0.2 brain eq of PTTH extract.

With the development of a specific, reproducible, and sensitive in vitro assay for the prothoracicotropic hormone (PTTH) of Munduca sextu (Bollenbacher et al., 1979), it has been possible to quantify the amount of neurohormone in the Munduca brain (Agui et al., 1979a; Bollenbacher and Gilbert, 1981), identify the neurosecretory cells (prothoracicotropes) responsible for its synthesis (Agui et al., 1979b), demonstrate the site of release of PTTH (Agui et al., 1979a), conduct comparative studies with the PTTH and prothoracic glands of other lepidopteran species (Gilbert et al., 1981; Agui et al., 1983), and purify one Munducu PTTH to homogeneity (W. Bollenbacher and L. I. Gilbert, unpublished observations). However, until now there have been no reports of comparable in vitro

assays on insects representing orders other than the Lepidoptera. Since PTTH is presumably the hormone responsible for initiating the molting process in all insects thus far studied, it was decided to devise an analogous in vitro assay for an insect taxonomically remote from Lepidoptera but which has been the subject of much endocrinological research. The fly Surcophugu bullutu was chosen for the following reasons. (1) It is a relatively large insect allowing the dissection of the endocrine glands with relative ease. (2) Dipterans have a fused retrocerebral complex (ring gland; King et al., 1966) rather than distinct corpora allata and prothoracic glands as found in Munducu. It was of interest to determine if ring glands reacted to PTTH in a manner similar to prothoracic

469 0016-6480/84 $1.50 Copyright Q 1984 by Academic Press, Inc. All rights of repnduction in any form reserved.

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GILBERT,

glands. (3) An important parameter in establishing the A4unduca PTTH assay was the knowledge that the only ecdysteroid secreted by the larval prothoracic glands was ecdysone (King et al., 1974). The Sarcophaga ring gland also secretes ecdysone (Bollenbacher et al., 1976). (4) Establishment of such an assay for a fly could form the basis for a similar assay for Drosophila with which one could probe the PTTH-ring gland system of brain and ring gland mutants. (5) Finally, we have a great deal of experience with Sarcophaga as an experimental insect and basic endocrinological data have already been generated (e.g., Wentworth et al., 1981). MATERIALS

AND METHODS

Animals. Breeding stocks of the ovoviviparous flesh fly S. bullata were reared and maintained at a constant temperature of 25 + 0.5” at 40-50% RH under a 16L:8D photoperiod. Larvae and adults were maintained and staged according to methods previously described (Roberts and Warren, 1975; Roberts, 1976; Wentworth er al., 1981). Collection and extraction dysteroid radioimmunoassay.

of hemolymph

for

the ec-

At selected stages of larval-pupal development hemolymph samples were collected. For larvae prior to the white prepupal stage (WPP), the ventral anal papillae were cut and hemolymph was carefully expressed onto Paratilm. Immediately, 0.02 ml hemolymph was added to 0.20 ml of cold absolute methanol and extracted samples were stored at 4” until required. For samples of prepupal hemolymph, the puparium was pierced with a fine needle in the dorsal aspect of the second segment and carefully squeezed. This process usually results in the collection of clear hemolymph samples over the sampling period (O-24 hr). However, hemolymph samples were occasionally contaminated with other tissues (e.g., fat body) in which case they were either discarded or centrifuged at 3OOOgfor 15 min and the clear supernatant was used. Aliquots (0.1 ml) of the methanol extracts were quantified for ecdysteroids by radioimmunoassay (RIA) as described previously (Bollenbacher et al., 1979; Wentworth et al., 1981). At least five determinations were made for each titer point. In vitro ring gland assay for PTTH. Insects used as ring gland (RG) donors were placed on ice for approximately 20 min, covered with cold Grace’s insect tissue culture medium, slit postero-antero on the dorsal surface from the midregion up to the mouth hooks, and quickly pinned to expose the retrocerebral

AND BOLLENBACHER complex. RG from postfeeding larvae were excised by cutting aorta1 and tracheal connections and dissected free of surrounding tissues with extreme care so as not to touch the gland with dissecting instruments; cut tracheae provided natural devices for handling of the RG. White prepupal and prepupal samples were also collected and the antero-dorsal cuticle of the first three or four puparial segments was excised to expose the RG which were removed as noted above. Each RG was transferred to a O.Ol-ml drop of Grace’s medium located on a small Petri dish lid. This was then inverted as a hanging drop over a dish containing Grace’s medium to minimize evaporation. Incubations were at 25” for 2 hr. Brains (minus the RG) were excised from 3- to 4-hr prepupae, rinsed and homogenized in Grace’s medium, heat treated for 2 min, and centrifuged at 5OOOg for 10 min. The resulting supernatant was designated “PTTH extract.” Activation of the RG by PTTH was demonstrated with a dose-response protocol (Bollenbather et al., 1979). Activation was expressed as an activation ratio (A,), the amount of ecdysone synthesized by the experimental RG (+ PTTH) divided by the amount synthesized by the control RG (- PTTH) during the same period, or by the quantity of ecdysone synthesized by the experimental gland during the incubation period. Muscle and ventral ganglion complex extracts were prepared the same as were brain extracts, and were used as controls for studying tissue specificity of RG activation. After incubation of RG (? PTTH extract), 0.005-ml aliquots of the hanging drop were extracted with cold methanol and the ecdysone content of the supematant was quantified by RIA.

RESULTS Hemolymph ecdysteroid titer during late larval and prepupal development. Ecdyste-

roid levels in young postfeeding larvae (PFL) were extremely low (~0.02 ng/kl) but began to increase as the animals approached the red spiracular stage (RSS) (Fig. 1). Using the criterion of pigmentation in the area of the posterior spiracles, a process unique to the Sarcophagidae (Fraenkel, 1975), the RSS was carefully staged. Midway through the RSS (-2 hr) the ecdysteroid titer peaked at approximately 0.4 ng/ul hemolymph, but then dropped so that at the formation of the white prepupal stage (WPP, 0 hr) the level was only about 0.13 ng/pl hemolymph. The formation of the WPP provides an ideal

ACTIVATION +PFL

-10

-

-8

-6

RSS

-4

OF

RING

471

GLANDS

-P WPP

-2

0

2

4

6

8

10

12

14

16

18

20

22

24

FIG. 1. The hemolymph ecdysteroid titer during the postfeeding larval, prepupal, and pupal stages of S. bullara. PFL, postfeeding larva; RSS, red spiracular stage; WPP, white prepupal stage. Each datum point represents the mean (+ SEM) of at least five individual measurements.

time for synchronization of the population, and consequently, the ecdysteroid titer is plotted as a function of time before and after the WPP stage (Fig. I). Two hours following the formation of the WPP the hemolymph ecdysteroid titer increased to a level comparable to that of the midaged RSS. Shortly thereafter (4 hr post-WPP) the titer decreased again to 0.18 ng/$ hemolymph and remained at this level for several hours. It was during this period that apoIysis was observed in the anterior segment of the prepupae (Fig. 2). From 6 to 9 hr post-WPP the hemolymph ecdysteroid titer began to increase and by 10 hr it had increased dramatically, peaking at 1 ng/t.J hemolymph at 11 hr. By 13 hr the titer had decreased dramatically to 0.2 ng/kl hemolymph, after which time it dropped gradually to approximately 0.1 ng/l~,l hemolymph

FIG. 2. The apolysis space (arrowheads) is clearly evident in the early prepupal stage (5 hr) of S. bdlata.

472

ROBERTS,

GILBERT,

AND BOLLENBACHER

24 hr after the WPP stage. Of particular interest in this titer was that each peak was approximately 3 hr in duration and, except for the absence of a peak at 6 hr post-WPP, the peaks occurred approximately every 4 hr. To determine if the fluctuations in the hemolymph ecdysteroid titer were due principally to the activity of the RG, we examined the ability of the RG from selected developmental stages to synthesize ecdysone in vitro. RG activity in vitro. The activity of the RG, i.e., rates of ecdysone synthesis, was assessed in vitro during the pre- and postWPP stages. During most of the PFL the synthesis of ecdysone by the RG during the 2-hr incubation period was minimal (0.2 ng) (Fig. 3), but as the larvae approached the RSS, synthesis increased reaching a maximum of 5.4 ng during the first third of the RSS (- 3 hr). This proved to be the maximal rate of secretion observed in this study. Following this initial increase, ecdysone synthesis by the RG oscillated, with three additional significant peaks of synthesis of decreasing amplitude occurring at approximately 4-hr intervals. By 21 hr postWPP, gland activity had returned to the -

PFL

-aRSS+

low, early PFL levels. The significant peaks of RG activity at - 3, 1, and 9 hr correlated temporally with the three peaks in the hemolymph ecdysteroid titer (Fig. 1). By contrast, at the time of increased RG activity at 3 hr post-WPP, there was no increase in the hemolymph ecdysteroid titer. Irrespective of this single discrepancy between RG activity and ecdysteroid titer, it appears that RG activity contributes to fluctuations in the hemolymph ecdysteroid titer at - 3, 1, and 9 hr, perhaps as a consequence of activation by PTTH. In addition, these data provide information essential for developing-an in vitro system with which direct activation of RG by PTTH could be demonstrated. Activation of RG by PTTH in vitro. To demonstrate direct activation of RG by PTTH a protocol similar to that used with Manduca (Bollenbacher et al., 1979) was employed. Based on RG activity in vitro PFL glands were selected for activation by PTTH since they were minimally active (Fig. 3), yet were at a stage where they should be developmentally competent to respond to PTTH. The dipteran RG structure is complex (King et al., 1966) when

WPP 4

6-

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-6

-6

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0

2

4

6 Age

6

IO

12

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16

I6

20

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(hr)

FIG. 3. Ecdysone secretion by RG in vitro. RG were obtained from the postfeeding larval, prepupal, and pupal stages of S. bulluta. PFL, postfeeding larva; RSS, red spiracular stage; WPP, white prepupal stage. Each datum point represents the mean (2 SEM) of at least five individual measurements. The amounts of ecdysone noted are those secreted in two hours (see Materials and Methods).

ACTIVATION

OF

compared to the neuroendocrine system of other orders as it is a distinct structure composed of paired prothoracic glands, the corpus allatum and the corpus cardiacurn. Although it is possible to dissect the RG into two halves and thereby produce left and right prothoracic glands, this procedure was not attempted in the present study because King et al. (1966) have shown that the prothoracic gland components of the RG are continuous in the dorsal aspect of the RG, and damage to cells would occur if they were separated. Such damage to glands used in an in vitro bioassay should be avoided. Consequently, a paired gland protocol (Bollenbacher et al., 1979) for demonstrating activation of RG could not be used. Instead, glands from one group of animals served as controls (incubated in the absence of PTTH) and glands from another group served as the experimentals (incubated in the presence of PTTH). That this approach was acceptable was demonstrated by showing that individual rates of ecdysone synthesis by 25 RG from PFL varied by only 7%, a value comparable to the acceptable 10% variability observed between members of a pair of Munduca prothoracic glands (Bollenbacher et al., 1979). Brains (minus RG complex) from 3- to 4-hr prepupae were chosen to demonstrate if PTTH extract directly activates ecdysone biosynthesis by the RG. At this stage the hemolymph ecdysteroid level (Fig. 1) was rising and RG synthetic activity (Fig. 3) was high, but not maximal. Using a dose-response protocol, activation of the RG by the PTTH extract was observed over a range of 0.03 to 2.0 brain eq. Activation was achieved and the RG response was similar to that obtained with lepidopteran prothoracic glands (Bollenbather et al., 1979; Agui et al., 1983). Gland activity ranged from an unactivated basal level of 0.6 ng/2 hr to an activated level of 6.6 ng/2 hr with saturating amounts of P’I?H extract. An activation ratio (A,), obtained by dividing the ecdysone synthe-

RING

473

GLANDS

sized by RG plus PTTH by that synthesized by RG minus PTTH (control), revealed a maximum activation of 10 (Fig. 4). Halfmaximal activation of the RG (A, = 5.5) was obtained with an ED,, of 0.2 brain eq of PTTH extract. The specificity of RG activation by the PTTH extract was demonstrated by conducting identical dose-response studies with extracts of abdominal ganglionic mass and muscle. Neither was capable of activating the RG in vitro (Fig. 4), indicating that activation was specific for a factor present in the brain extract, probably PTTH. Although highly suggestive of PTTH activation, final proof must await purification of PTTH from the Sarcophaga brain. DISCUSSION

In the course of this investigation several important aspects of the endocrinology of the Diptera have been noted. First, it appears that the PTTH-RG system of Surcophaga functions in vitro in a manner analogous to the PTTH-prothoracic gland system of Manduca. Second, the oscillations in the hemolymph ecdysteroid titer and in vitro RG activity are similar in both the Diptera and some Lepidoptera. This fluctuating ecdysteroid titer and RG activity have not been observed previously during the RSS and early prepupal stages of Diptera (Shaaya and Karlson, 1965; Ohtaki et al., 1968; Barritt and Birt, 1970; Seligman et a/., 1977; Berreur et al.? 1979; Briers and De Loof, 1980; and Wentworth et al., 1981). However, similar fluctuations over a longer period have been observed for the larval-pupal development of Culpodes ethlius (Dean et al., 1980). The physiological significance of these repeated peaks is not clear, but based upon characteristic morphological and behavioral changes that occur during this developmental period, these peaks may play important roles in Sarcophaga development. The first surge in the ecdysteroid titer

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AND BOLLENBACHER

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FIG. 4. The activation of RG excised from postfeeding larvae by PTTH extracts of prepupal brains (0). Ganglion extract (0) and muscle extract (A) were used as controls. Each datum point represents the mean (?SEM) of the amount of ecdysone secreted for five separate activation assays. The A, (activation ratio) is the ratio of the amount of ecdysone secreted by RG (+ PTTH) to that secreted by control RG ( - PTTH).

that occurs several hours prior to the WPP stage could be involved in the development of the RSS. This developmental stage is characterized by the progressive tanning of the cuticle between and around the posterior spiracles, a phenomenon exclusive to the Sarcophagidae (Zdarek and Fraenkel, 1972). The sclerotization of the posterior spiracles and the spiracular plates of the third instar larva may be analogous to the preecdysial tanning of the mandibular mouth hooks and anterior and posterior spiracles of Sarcophaga, which are presumably elicited by 20-hydroxyecdysone (Roberts et al., 1982). In addition to a possible role in tanning, the first ecdysteroid peak may be involved in initiating pupariation, since the WPP stage occurs approximately l-2 hr after this peak. Some years ago, it was shown that ecdysteroids elicited pupariation (Shaaya and Karlson, 1965; Berreur and Fraenkel, 1969), and the data reported here support these findings. However, this interpretation does not include the findings of Sivasubramanian et al. (1974) (see Fraenkel, 1975) that suggest the existence of a puparial tanning factor and an anterior retraction factor which elicit pupariation and puparial tanning. Perhaps these factors act in conjunction with ecdysteroids.

The second ecdysteroid peak occurred several hours after the WPP stage when the puparium was of orange-red coloration, a time just before apolysis of the anterior segments of the animal. In addition, this increase immediately preceded the brown coloration of the puparium that represents cuticular sclerotization, which is initiated by molting hormone (Karlson and Sekeris, 1976). Therefore, this second peak may be responsible for puparium sclerotization as well. The third and largest hemolymph ecdysteroid peak occurred 11 hr after the WPP stage and is temporally similar to those previously reported for whole body extracts of Sarcophaga (Briers and De Loof, 1980; Wentworth et al., 1981) and for other Diptera (e.g., Redfern, 1983; see Richards, 1981). It is presumably important for completion of apolysis, secretion of the pupal cuticle, and completion of puparial tanning. The analysis of ecdysone biosynthetic activity of RG in vitro indicates that fluctuating RG activity contributes significantly to the hemolymph ecdysteroid titer, but that correlations may not exist for all of the surges in ecdysteroid titer. Although the peaks in RG activity and ecdysteroid titer differed quantitatively, there were excellent temporal correlations at - 3, 1, and 9 hr.

ACTIVATION

OF RING GLANDS

The quantitative discrepancy between RG activity and ecdysteroid titers was perplexing initially, but recent studies with Manduca prothoracic glands in vitro indicate that apparent gland activity in vitro and titers of hemolymph ecdysteroids do not always agree quantitatively (Ciancio, Gunnar, Bollenbacher, and Gilbert, unpublished). For Manduca PG this difference appears to involve the in vitro conditions used for incubating the glands, in that the system lacks a hemolymph factor(s), presumably a lipoprotein carrying the sterol substrate for ecdysone biosynthesis, that contributes substantially to the regulation of gland activity in situ. The possibility exists that similar factors are present in Sarcophaga hemolymph. Thus, the apparent activity of the RG in vitro would be quantitatively less than their activity in situ. The principal temporal difference between in vitro RG activity and ecdysteroid titer is the peak of RG activity at 4-5 hr which is not accompanied by a concomitant increase in the ecdysteroid titer. In Drosophila a similar lack of correlation has recently been reported between RG activity in vitro and the ecdysteroid titer of whole animal extracts (Redfern, 1983). This could be due to the in vitro conditions (see above), alternative sources of ecdysone, differential rates of ecdysteroid catabolism (Young, 1976), conjugation, and excretion. In the case of the present study, hemolymph samples rather than whole body extracts were analyzed and the additional possibility exists that ecdysteroids were sequestered by other tissues (e.g., fat body) from the hemolymph. The oscillations in the synthesis of ecdysone by RG in vitro and the apparent resulting oscillatory fluxes in the ecdysteroid titer were only detected because RG activity and ecdysteroid titer were determined at hourly intervals. This unexpected finding poses new questions about how RG activity ultimately regulates development, and suggests that the endocrine control of

475

insect postembryonic development is more complex than previously thought. There could be a neuroendocrinological basis for these oscillations involving the brain and the mode of PTTH release. In this case, PTTH release could be occurring in a pulsatile manner driving the oscillations in RG activity and hormone titer. This possibility is supported by the recent finding of pulsatile release of PTTH in Manduca during larval-pupal development (Bollenbacher and Gilbert, 1981; Bollenbacher, unpublished). [See Krieger (1979) for analogous findings with CRF, ACTH, and corticosteroids in mammals.] The low basal activity of PFL RG proved to be ideal for demonstrating dose-dependent PTTH activation of the glands in vitro. The maximum activation achieved was greater than that noted for PTTH-activated prothoracic glands of Munduca, but was comparable to the maximum activation of Bombyx and Mamestra prothoracic glands by their respective PTTHs (Agui et al., 1983). The RG A, of 5.5 obtained with an ED,, of 0.2 Sarcophaga brain eq was reasonably similar to that obtained for the comparable lepidopteran systems. These data, and the finding that neither muscle nor nervous tissue (excluding the brain) extracts elicited RG activation, indicate that the stimulatory factor in these studies is PTTH. This cannot be demonstrated unequivocally until the Sarcophaga PTTH is purified and characterized. Not only should this assay be of value in probing the endocrinology of the dipteran endocrine system, and perhaps in purifying the Sarcophaga PTTH, but it has laid the foundation for an investigation of the analogous system in Drosophila and, thus, the possibility of using genetic probes for answering basic questions in insect neuroendocrinology. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health to L. I. Gilbert (AM-301 18) and W. E. Bollenbacher (NS-18791).

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GILBERT,

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(1974). Nature and role of proteinaceous hormonal factors acting during puparium formation in flies. Biol. Bull. Woods Hole 147, 163- 185. Wentworth, S. L., Roberts, B., and O’Connor, J. D. (1981). Ecdysteroid titres during postembryonic development of Sarcophaga bullata (Sacrophagidae: Diptera). J. Insect Physiol. 27, 436-440.

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