Ring gland and prothoracic gland sensitivity to interspecific prothoracicotropic hormone extracts

J Comp Physiol B (1986) 156:767-771 Journal of Comparative ~,~,~,~,o,Bi~176

and Environ-

Physiology B " ~ Phys/ology

�9 Springer-Verlag 1986

Ring gland and prothoracic gland sensitivity to interspecific prothoracicotropic hormone extracts

Brian Roberts** and Lawrence I. Gilbert* Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514, USA

Accepted August 16, 1986

Summary. Using the techniques of intraspecific in vitro activation of prothoracic glands and ring glands by serial dilutions of prothoracicotropic hormone (PTTH) extracts from pupal Manduca sexta (Lepidoptera) and larval Sarcophaga bullata (Diptera), a dose-response of activation was observed for both species. In both species maximum activation was at 0.5 brain equivalents while the number of brain equivalents necessary for half maximal stimulation (EDso) was 0.20 for Manduca and 0.15 for Sarcophaga. When prothoracic glands or ring glands were challenged with interspecific PTTH extracts from a stage different from that of the gland donor, no dose-response of gland activation was observed. However, when M. sexta larval prothoracic glands were challenged by S. bullata larval PTTH extract, activation was observed. The dose-response profile fell midway between the dose-response curves obtained for the intraspecific assays. Thus, PTTH extract from one insect has the ability to activate the prothoracic glands of an insect representing another order.

Introduction

The prothoracicotropic hormone (PTTH) is a neu- ropeptide synthesized in the insect brain that is responsible for initiating the molting process in all insects thus far studied. This neurohormone acti- vates the prothoracic glands to increase their rate of ecdysone synthesis and release; ecdysone is then hydroxylated to 20-hydroxyecdysone by the fat body, gut, and other tissues. It is 20-hydroxyecdy-

* To whom offprint requests should be sent ** Present address: Department of Zoology, Monash Universi-

ty, Clayton, Victoria, 3168, Australia

sone, and perhaps ecdysone, that acts on the target tissues to elicit molting (see Gilbert et al. 1980).

Although the ecdysteroids appear to be the molting hormones of all arthropods (and perhaps some other invertebrates), and juvenile hormone is almost as well conserved, little is known of the generic specificity of PTTH action, although there is interspecific cross-reactivity among the prothoracicotropic hormone extracts and prothoracic glands of representatives of three superfamilies of Lepidoptera (Gilbert et al . /981; Agui et al. 1983). Since a sensitive assay for PTTH using prothoracic glands in vitro has been developed for Manduca sexta, (Bollenbacher et al. 1979; see Gilbert et al. 1981) and a comparable assay using the ring glands has been developed for the flesh fly, Sarcophaga bullata, (Roberts et al. 1984), interspecific studies have now been conducted. The present data sug- gest that the PTTH of representatives of two insect orders, Lepidoptera and Diptera, may share simi- larities in structure as well as function.

Materials and methods

Animals. Manduca sexta were reared on an artificial diet at 25_+ 0.5 ~ 40-50% RH, under a 16L: 8D photoperiod. Larvae were staged as described previously (Vince and Gilbert 1977; Goodman and Gilbert 1978).

Stocks of the ovoviviparous dipteran Sarcophaga bullata were reared under the same temperature and photoperiod re- gimes as M. sexta. The insects were maintained and staged according to methods described previously (Roberts and War- ren 1975; Roberts i976; Wentworth et al. I981).

Prothoracicotropic hormone (PTTH) assay. The in vitro activation of Manduca prothoracic glands by PTTH extracts is now firmly established and fully outlined elsewhere (Bollenbacher et al. 1979; Gilbert et al. 1981). Paired prothoracic glands from animals of known age were carefully excised and incubated separately in 25 t.tl of Grace's medium in the presence or absence of PTTH at 25 ~ for 2 h. Aliquots (10 lal) of culture medium were collected and the ecdysteroid content quantified by radio-

768 B. Roberts and L.I. Gilbert: Interspecific prothoracicotropic hormone

immunoassay (RIA) (Gilbert et al. 1977; Bollenbacher et al. 1979).

A protocol for the in vitro activation of S. bullata ring glands by PTTH extract has also been established (Roberts et al. 1984). Ring glands from larvae of known age were incubated in 10 gl of Grace's medium at 25 ~ for 2 h and aliquots (5 pl) were assayed by RIA as above. The PTTH in vitro assays for both species depend on the collection and extraction of suitably staged brains, Consequently, M. sexta brains and S. bullata brains (minus the ring gland complex) were collected at specified times, homogenized in Grace's medmm, boiled for 2 rain, centrifuged at 5,000 g for ~0 min and the supernatant (PTTH extract) collected and stored for serial dilution (Bolien- bacher et al. 1979). It should be noted that control tissues, such as nerve cord and muscle extracts are ineffective in stimulating the prothoradc glands of Manduca (Bollenbacher et al. 1979) or the ring glands of Sarcophaga (Roberts et al. 1984).

To quantify the Manduea assay, an activation ratio (Ar) was obtained by dividing the quantity of ecdysteroid synthesized by the experimental gland (plus PTTH extract) by that synthesized by the contralateral control gland (minus PTTH extract). In the case of Sarcophaga ring glands, five individual ring glands from a specific stage were compared in an analogous manner since the individual ring glands differed in ecdysteroid basal synthetic capacity by only 7% (Roberts et al. 1984).

The relative responsiveness of different prothoracic glands or ring glands to PTTH extracts was assessed in a dose-depen- dent study by determining the amount of PTTH, expressed in brain equivalents, necessary to achieve maximum gland activation (A=,,). The effective dose of PTTH needed to achieve half-maximal activation of the prothoracic glands or ring glands (EDso), was then compared. The EDso has proven to be an accurate means of quantifying relative PTTH activity in brain extracts (Bollenbacher and Gilbert 1981; Agui et al. 1983).

Results

Intraspecific studies

The in vi t ro response o f Manduea day 0 pupa l prothoracic glands to Manduca day 0 P T T H extract was examined for subsequent compar i son purposes as it has been well s tudied previous ly (Bol lenbacher et at. 1979; see Gi lber t et al. 1981). Fig. 1 reveals tha t an A ~ o f a b o u t 4 was ob ta ined with 0.5 bra in equivalents o f P T T H extrac t while the EDso was 0.2 bra in equivalents . These values were in good ag reemen t wi th our previous data .

Ring glands f r o m pos t - feeding Sarcophaga larvae were studied in an ana logous m a n n e r using P T T H extracts p r epa red f r o m Sarcophaga prepupal brains , 3 -4 h af ter the f o r m a t i o n o f the white p repupae . Brains f r o m the lat ter stage possess po- tent P T T H activi ty (Rober t s et al. 1984). [For a discussion of the s taging o f Sarcophaga see Went - wor th el al. (1981). P repupa l tissues are here re- garded as larval tissues (see Discussion)]. The re- suits o f ring gland incuba t ion in the presence o f a g raded di lut ion series o f P T T H ext rac t show a typical dose- response (Fig. 2), s imilar to tha t o f

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Fig. 1. Dose-response of activation of Manduca pupal day 0 prothoracic glands by PTTH extract from Manduea pupal day 1 brains. Each point represents the mean (4-SEM) of 3-5 separate activation assays

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Fig. 2. Dose-response of activation of Sarcophaga ring glands from postfeeding larvae by PTTH extract from prepupal brains (3-4 h after white pre-pupal stage). Each point represents the mean (• of 5 separate activation assays. The Ar is pre- sented on the right abscissa

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the Manduca system. The A,,,~ of 9 was reached with 0.5 bra in equivalents while the EDso was 0.15 bra in equivalents . These da ta are basical ly identi- cal to those repor ted previously (Rober t s et al. 1984). H a v i n g reestabl ished the validi ty of the P T T H assay in the two insect systems, interspecific studies were init iated to de te rmine whether Man- duea P T T H extrac t could act ivate Sarcophaga ring glands and if Sarcophaga P T T H extract could s t imulate ecdysone synthesis by Manduca pro tho - racic glands.

Interspecific studies

First, Sarcophaga ring glands f r o m post - feeding larvae were chal lenged with P T T H extrac t f r o m Manduca day 1 pupal brains. A l though there was a g radua l increase in ecdys tero id synthesis with in- creasing dose, the typical s igmoidal dose- response curve (Figs. 1, 2) was not observed (Fig. 3) and

B. Roberts and L.I. Gilbert: Interspecific prothoracicotropic hormone 769

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Fig. 6. Summary of dose-response activation of ring glands and prothoracic glands by PTTH extracts. The graphs have been drawn with a common As 0 of 0.15 5 brain equivalents. Triangles denote Manduca pupal prothoracic glands activated by Man- duca PTTH extract (Fig. 1), circles denote Sarcophaga larval ring glands activated by Sarcophaga prepupal PTTH extract (see Fig. 2), squares denote Manduca larval prothoracic glands activated by Sarcophaga prepupal PTTH extract (see Fig. 5)

Fig. 3. Dose-response of activation of Sarcophaga ring glands from postfeeding larvae by PTTH extract from Manduca day 1 pupal brain

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Brain Equ iva len ts

Fig. 4. Dose-response of activation of day 0 Manduca prothoracic glands by PTTH extracts from Sarcophaga prepupal brains (3-4 h after white prepupal stage)

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Fig. 5. Dose-response of activation of Manduca fifth instar larval (day 3) prothoracic glands by PTTH extracts from Sarco- phaga prepupal brains (3-4 h after white prepupal stage)

the apparent Ar was only 2, and that at a level of 2 brain equivalents.

When Manduca day 0 pupal prothoracic glands were incubated in the presence of PTTH extract from Sarcophaga prepupal brains, again a "physi- ological" dose-response was not obtained (Fig. 4). Indeed, if there was any possible activation, it occurred at 12.5 brain equivalents, and although this could be expected considering the difference in size between the Manduca and Sarcophaga brains the

shape of the curve is not indicative of hormonal activation.

Since the above studies involved assay glands from animals at one stage and PTTH extract from brains of animals at another stage (i.e. larval vs pupal), and since there are developmental changes in the ratio of the two forms of Manduca PTTH (O'Brien et al. 1986), we tested glands and extract from analogous stages in an interspecific study. The PTTH extract was from Sarcophaga prepupae (3-4 h after white puparium formation) and the prothoracic glands from day 3, fifth instar Man- duca larvae. In this case, a typical PTTH prothoracic gland dose-response curve was generated (Fig. 5) revealing an Am, x of 6.5 with 3.12 brain equivalents and an EDso of 0.6 brain equivalents. As can be seen in Fig. 6, these data fall between those obtained in the intraspecific studies for Man- duca and Sarcophaga. Sarcophaga nerve cord and muscle tissue extracts did not stimulate ecdysteroid synthesis in prothoracic glands indicating that the response to Sarcophaga PTTH extract is not a non- specific effect.

D i s c u s s i o n

The intraspecific studies (Manduca PTTH extract on Manduca prothoracic glands; Sarcophaga PTTH extract on Sarcophaga ring glands) corrobo- rate our previous findings (Bollenbacher et al.

770 B. Roberts and L.I. Gilbert: Interspecific prothoracicotropic hormone

1979; Roberts et al. 1984) and will not be discussed further. The data indicate that ring glands from Sareophaga larvae are not activated by Manduca pupal PTTH extract and that Manduea pupal prothoracic glands are not activated by PTTH extracts from Sarcophaga post-feeding larvae. However, the latter does stimulate Manduca larval prothoracic glands in a manner typical of hormonal activation. Before speculating on possible reasons for this 'developmental specificity' it is important to defend the term 'larval' PTTH extract for material extracted from the brain of Sarcophaga prepupae. During the last larval instar of Manduca the large ecdysteroid peak on day 7 (Bollenbacher et al. 1981) elicits apolysis which is followed by the secretion of pupal cuticle and 2 to 3 days later by pupation. The stage between apolysis and pupation is known as the pharate pupa or prepupa. On the other hand, Sarcophaga is a cyclorrhaphous dipteran, and approximately halfway through the third and final instar, larvae leave the food to become post-feeding larvae. They then empty their crops and seek a place for pupariation. Cyclorrha- phous fly pupae are unique among all insects; they lie inside a barrel-like structure, the puparium, which is formed from the cuticle of the last larval instar. The process of puparium formation in- volves the irreversible retraction of the first three larval segments, the contraction of the body and the concurrent shrinkage of the larval cuticle (Fraenkel and Bhaskaran 1973). When these events have been completed the insect is known as the white prepupa. At 25 ~ these processes take place within 30 rain, and the white prepupa has become an important distinguishable stage in timing developmental events. A few minutes after the white prepupa is formed the larval cuticle begins to hard- en and darken, and this form is termed the prepupa. It must be emphasized that the prepupal period is, therefore, an extension of the larval period as unlike the case of Manduca, apolysis and pupal cuticle secretion have not yet occurred (Wentworth et al. 1981 ; Roberts et al. 1984). Thus, in a physio- logical sense, we feel justified in concluding that larval PTTH extracts from Sarcophaga are capable of activating larval Manduca prothoracic glands.

The observation that PTTH extract from one insect order is capable of stimulating the prothoracic glands of an insect representing another order has not been made previously although cross-reactivity has been noted among insects representing three superfamilies of a single order (Lepidoptera) (Agui et al. 1983). This implies some conservation of the PTTt t molecule(s) during evolution. How- ever, it must be stated that we have dealt with

crude preparations of PTTH and there is the dis- tinct possibility that the cross-effectiveness observed here would not occur if pure Sarcophaga PTTH was used to challenge the Manduca prothoracic glands. In addition, the data do not explain the failure of the interspecific studies when different developmental stages are used for assay glands and PTTH extract. This may be due to the ex- istence of more than one form of PTTH, the ratio between them determining the major function at a particular developmental stage. In the case of Manduca, there are two forms of PTTH (Bollen- bacher et al. 1984; see Bollenbacher and Gilbert 1981, and Gilbert et al. 1981), the ratios change during development (O'Brien et al. 1986) and the sensitivity of the prothoracic glands to PTTH changes depending on developmental state (Bowen et al. 1984). Research is continuing to determine the possible relationship between dipteran and le- pidopteran PTTH and whether any of the above noted possibilities will explain the present results.

Acknowledgements. We thank Shelia King for secretarial assis- tance and Dr. W.E. Bollenbacher for advice. This work was supported by grant AM-30118 from the National Institutes of Health.

References

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Ring gland and prothoracic gland sensitivity to interspecific prothoracicotropic hormone extracts