Ecdysiotropic activity in the lepidopteran hindgut—An update

Insect Biochem. Molec. Biol. Vol. 23, No. 1, pp. 25-32, 1993 Printed in Great Britain

0965-1748/93 $6.00 + 0.00 Pergamon Press Ltd

Ecdysiotropic Activity in the Lepidopteran Hindgut--An Update DALE B. GELMAN,*t BELGAUM S. THYAGARAJA,:~ ROBERT A. BELL*

The production of ecdysteroid by the insect prothoracic glands (PTGs) is initiated by an ecdysiotropin, prothoracicotropic hormone, which is produced by the brain and released into the hemolymph from its neurohemal organ. Recently we reported the discovery of another ecdysiotropin which is localized in the lepidopteran hindgut or proctodaeum. It is a small, heat-stable peptide which is resistant to freeze-thaw and to extraction with organic solvents. Based on size-exclusion HPLC, we now estimate its molecular weight to be 500-1500 Da. The hindgut ecdysiotropin stimulates the PTGs of Lymantria dispar to produce both ecdysone and 3-dehydroecdysone in a dose-dependent manner. Ecdysteroid production was maximum in the presence of 0.125 and 0.1 hindgut equivalents for Ostrinia nubilalis and L. dispar, respectively. Activity was detected throughout the pylorus and anterior intestine of the O. nubilalis hindgut. When proctodaea from 5th instar O. nubilalis were analyzed daily for ccdysiotropic activity, those from wandering larvae which had undergone gut purge were found to have the greatest concentration of ecdysiotropin. Cyclic A M P appears to act as a second messenger for the proctodaeal eedysiotropin as evidenced by the increased levels of cAMP present in PTGs incubated with hindgut extract. At doses which caused maximum stimulation, effects of brain and proctodaeal extracts were additive indicating that the two ecdysiotropins utilize separate receptors. Size exclusion HPLC of hemolymph obtained from prepupae that have experienced gut purge revealed the presence of an ecdysiotropin(s) whose molecular weight range is similar to that of the proctodaeal ecdysiotropin but not to that of the small form of brain PTTH. While the physiological function of the proctodaeal ecdysiotropin(s) is unknown, the discovery of such a peptide(s) is noteworthy in light of the reported production of ecdysteroids by isolated insect abdomens.

Ecdysiotropin Proctodaeum Ostrinia nubilalis

Lymantria dispar

Prothoracic glands Ecdysone

ecdysiotropic factor(s) in the hindguts of the European corn borer, Ostrinia nubilalis, and the gypsy moth, Until recently, the only known source of ecdysiotropin L y m a n t r i a dispar. These ecdysiotropins stimulate the in insects were paired neurosecretory cells located in the production of both ecdysone and 3-dehydroecdysone lateral and/or medial protocerebrum of the brain (Gibbs by the PTGs ofL. dispar. Westbrook et al. (1991), based and Riddiford, 1977; Agui et al., 1979; Mizoguchi on immunocytochemical studies, reported that the large et al., 1987). These ecdysiotropins, prothoracicotropic form of PTTH is present in the subesophageal conhormones (PTTH), are released into the hemolymph nectives, ventral nerve cord and proctodaeal nerves of from their neurohemal organ, and upon reaching the M . sexta as well as in the group III lateral neurosecretory prothoracic glands (PTGs), stimulate the production of cells and ventromedial neurosecretory cells of the brain. ecdysteroid. In lepidopterans, the ecdysteroid produced Zitfian et al. (1990) also have found evidence to support is for the most part 3-dehydroecdysone, which is con- the presence of ecdysiotropins in the ventral nerve cord. verted to ecdysone by a 3fl-forming-3-ketoecdysteroid These researchers (based on immunocytological studies) reductase (ketoreductase) in hemolymph (Warren et al., have reported the presence of a bombyxin-like molecule 1988; Kiriishi et al., 1990; Kelly et al., 1990) and then by in the thoracic ganglia and ventral median nerves of peripheral tissues to the physiologically active molting Galleria mellonella. hormone, 20-hydroxyecdysone (King, 1972). In 1991, The possible role(s) of abdominal centers in controlGelman et al. reported the presence of large amounts of ling molting and metamorphosis have for the most part received little or no attention despite the many reports of molting in headless pupae and isolated abdomens *Insect Neurobiology and Hormone Laboratory, Plant Sciences (Hsiao et al., 1975; Slama, 1975, 1983; Safranek et al., Institute, USDA, ARS, Beltsville,MD 20705, U.S.A. 1986; Sakurai et al., 1991) and the report by Beck and tAuthor for correspondence. Alexander (1964) of the existence of a hindgut factor SDepartment of Zoology, University of Maryland, College Park, MD 20742, U.S.A. (proctodone) which stimulates the onset of development INTRODUCTION

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26

DALE B. GELMANet al.

in diapausing O. nubilalis. Ecdysteroid synthesis is known to occur in organs other than prothoracic glands including ovaries (Hagedorn et al., 1975), testes (Loeb et al., 1982; Gelman et al., 1989), epidermis (Delbecque et al., 1990) and possibly midgut (Sehnal and Zitnan, 1990). Synthesis by the ovaries and testes appears to be under the control of neurohormones produced in the brain, namely egg development neurosecretory hormone (Hagedorn et al., 1979), more recently referred to as ovarian ecdysteroidogenic hormones (Matsumoto et al., 1989), and testis ecdysiotropin (Loeb et al., 1987), respectively. Factors and/or mechanisms controlling ecdysteroid production by epidermis are unknown. Other ecdysteroid-synthesizing tissues may also be present in the abdomen. Sakurai et aI. (1991) have reported high levels of hemolymph ecdysteroid in isolated pupal abdomens of Manduca sexta, but even after an exhaustive search could not find sites of synthesis. Thus, the mechanisms involved in controlling molting in isolated abdomens as well as the source of the relatively high titers of ecdysteroid in these isolated abdomens are ripe areas for investigation. Because of their ability to control ecdysteroid production, ecdysiotropins play a prominent role in regulating molting and metamorphosis. Therefore, it was important to characterize the hindgut ecdysiotropin(s), and to investigate its source and physiological role(s) in the insect. In this paper, we report our results concerning the characterization of the hindgut ecdysiotropin(s), its localization, fluctuations during the 5th instar, interaction with brain PTTH and utilization of cyclic AMP as a second messenger.

MATERIALS AND M E T H O D S

Chemicals

Ecdysone, 20-hydroxyecdysone, pronase, NADPH and the molecular weight markers, carbonic anhydrase (29,000 Da), cytochrome c (12,400 Da), aprotinin (6500 Da), insulin chain B (3496 Da) and proctolin (648 Da) were purchased from Sigma Chemical Co. (St Louis, Mo, U.S.A.). Little gastrin (2126Da) was purchased from Peninsula Laboratories (Belmont, Calif., U.S.A.). Grace's medium was obtained from Gibco (Grand Island, N.Y., U.S.A.) and tritiated ecdysone was purchased from New England Nuclear Corp. (Boston, Mass., U.S.A.). The ecdysone antiserum was prepared by W. S. Bollenbacher (University of North Carolina, Chapel Hill, N.C., U.S.A.) from a hemisuccinate derivative of ecdysone (at the C-22 hydroxyl group) which had been coupled to thyroglobulin. Insect rearing and staging

European corn borer (Ostrinia nubilalis) eggs and medium were supplied by the Corn Insects Research Unit, USDA, ARS, Ankeny, Iowa. Insects were reared and staged as described in Gelman and Hayes (1982). Larvae were reared in 16 oz. plastic cups (Fort Howard

Cup Corp., Green Bay, Wis.) fitted with lids containing copper wire mesh. Insects destined to pupate were kept at 30° _+ I°C in a light,lark regimen of L : D 16'8, while those destined to diapause were maintained at a temperature of 26 _+ I°C and L:D 12:12. To synchronize experimental larvae, pharate 5th instars were transferred to 2-dram vials containing fresh medium. For the first 4 days of the last instar, larvae were selected based on their age in days post-ecdysis. Day-5 and -6 wandering larvae were divided into 2 groups, those in the process of gut purge were designated Out Brown (OB), and those that had completed gut purge were designated Out White (OW) and commonly referred to as prepupae. Extraction of tissue and in vitro bioassay for ecdysiotropin

Larvae were anesthetized in Petri dishes with gaseous carbon dioxide generated from dry ice. Proctodaea minus recta (pylorus and anterior intestine) (Drecktrah et al., 1966) and/or brains were removed and placed in Grace's medium for 20-60 min prior to being frozen at - 20°C. When needed, tissues were homogenized, extracted and incubated with PTGs that had been dissected from day-4 or -5 5th instar L. dispar as described in Gelman et al. (1991). To determine the ability of proctodaeal extracts prepared from O. nubilalis and L. dispar to stimulate the production of ecdysone and 3-dehydroecdysone by L. dispar PTGs, the triple incubation in vitro bioassay was utilized (Gelman et al., 1991). For all other experiments the triple incubation assay was modified to determine total ecdysteroid produced by the PTGs rather than individual amounts of ecdysone and 3-dehydroecdysone synthesized. For both types of assay PTGs were preincubated in Grace's medium for 2 h, rinsed in Grace's medium and then transferred to 25 #1 drops of fresh Grace's medium (controls) or Grace's medium containing the extract to be tested. After an additional 2 h of incubation, glands were removed. To determine total ecdysteroid produced by a stimulated PTG, 25 #1 of a ketoreductase preparation (described below) were added to the 25-/~1 drop of incubation medium. This enzyme converted the RIA-undetectable 3-dehydroecdysone to RIA-detectable ecdysone. After a third 1-h incubation, 30-pl aliquots were transferred to 6 x 50-ram tubes, the reaction was stopped with 100% methanol and the tubes were stored in the freezer prior to RIA. To determine the individual amounts of ecdysone and 3-dehydroecdysone produced, the 25/~1 drop (after a second 2 h of incubation) was divided into two 12-/~1 drops. Twelve microliters of the ketoreductase preparation were added to one drop (measured synthesis of ecdysone + 3-dehydroecdysone), Grace's medium to the other (measured synthesis of ecdysone). After an additional 1 h of incubation, 15-#1 aliquots were taken for RIA. In all cases, results were corrected to express ecdysteroid produced per gland. Because it was easy to obtain and had less than 20 pg/#l ecdysteroid (Gelman and Woods, 1983), hemolymph from 6- to 10-day old 5th instar diapause-bound larvae (2.0 ktl/100#l of Grace's

ECDYSIOTROPIC ACTIVITY IN THE HINDGUT medium made 0.2 mM with N A D P H ) served as the source of ketoreductase (unpublished results). Since medium in which maximally stimulated glands had been incubated contained similar amounts of RIA-detectable ecdysteroid after exposure to 0.25, 0.5 or 1.0#1 of hemolymph (added in 25 # 1 of Grace's medium/NADPH) (unpublished results), it was decided that 0.5 #1 of hemolymph would be sufficient to convert all the 3-dehydroecdysone produced during the second 2-h incubation to ecdysone.

27

equal portions of 200 #1 each. The experimental tube received 200#1 of the pronase preparation (4mg/ml Grace's medium, pH 7.2) while the control tube received 200 #1 of the same pronase preparation that had been placed in a boiling water bath for 2.5 min and centrifuged prior to the addition. The reaction was stopped by boiling, and after centrifugation and adjustment of the supernatants' pH to 6.0, they were tested in the in vitro P T G assay. Determination of c A M P levels in PTGs

Extraction of hemolymph to determine the presence of ecdysiotropin(s) Ostrinia nubilalis prepupae were anesthetized as described above. The first abdominal proleg was removed and hemolymph was collected with a micropipette and placed in a 1.5-ml polypropylene microcentrifuge tube containing 500 #1 of ice cold methanol: acetic acid:water: thiodiglycol (90:9:1:0.1). Forty microliters of hemolymph were placed in each tube and a total of 25 tubes was prepared. After centrifugation for 5 min at 16,000g and 4°C, in an Eppendorf table top centrifuge, supernatants were transferred to new tubes and stored in the freezer at - 2 0 ° C . Prior to size exclusion HPLC, tubes were dried on a Savant Speed Vac Concentrator (Forma Scientific, Marietta, Ohio) and reconstituted in 1.0 ml of 15% acetonitrile/0.1% T F A - H 2 0 . The extract was put onto a conditioned C18 Sep-Pak cartridge (Waters Associates, Milford, Mass.), and the active material was eluted with 80% acetonitrile/0.1% T F A - H 2 0 and concentrated to 200#1 prior to size exclusion HPLC. R/A The ecdysteroid RIA was performed using the methods of Borst and O'Connor (1972) and Bollenbacher et aI. (1975) as described in Gelman and Woods (1983). Tritiated ecdysone (63.5Ci/mmol) was used as the radioactively labeled antigen and 20-hydroxyecdysone (50 5000 pg) was utilized as the standard ecdysteroid. Results were converted to ecdysone equivalents (divided by a factor of 1.3) based on the relative affinity of the antiserum for the two ecdysteroids. Size exclusion H P L C Proctodaea were extracted in ice cold 40% acetonitrile/ 0.1% TFA. Hemolymph was extracted as described above. Extracts of proctodaea or hemolymph were subjected to size exclusion chromatography as described in Gelman et al. (1992) and as described briefly in the legend to Fig. 2. Fractions were dried, reconstituted in Grace's medium and tested in the triple incubation in vitro P T G bioassay. Treatment with pronase Treatment with pronase was performed as described in Gelman et al. (1992). Briefly, active fractions from the size exclusion column were reconstituted in 400 #1 of Grace's medium (pH 7.2) and then divided into two

PTGs that had been incubated for varying periods of time (1-14rain) either in Grace's medium (controls), Grace's medium containing 0.125 equivalents of crude proctodaeal extract or Grace's medium containing a given fraction from the size exclusion column, were sonicated in 200 #1 of ice cold 50% ethanol. Homogenates were centrifuged at 16,000g and 4°C for 5 min. Precipitates were washed with an additional 200 #1 of 50% ethanol and combined supernatants were dried in a Speed Vac. Samples were reconstituted in 250 #1 of 0.05 M sodium acetate buffer (pH 4.75) and analyzed for cAMP by the method of Kingan (1989). Briefly, samples and standards were acetylated and 5 # 1 aliquots were pipetted in duplicate into wells of a 96-well plate that previously had been coated with cAMP conjugate and blocked with normal goat serum. Control wells received buffer only. Forty five microliters of additional buffer were added to each well followed by 50#1 of primary antibody. Peroxidaseconjugated goat anti-rabbit IgG was used as the second antibody, and 3,Y,5,5'-tetramethylbenzidine and H202 were used to develop the color. The reaction was stopped with 1.0 M H3PO 4. Femtomoles of cAMP were determined using an E L I S A plate reader coupled to the program "Soft Max" (Kingan, 1989).

RESULTS

Synthesis of ecdysone and 3-dehydroecdysone by PTGs incubated with proctodaeal extracts Ecdysone and 3-dehydroecdysone were synthesized in a dose-dependent manner by day-5 5th instar L. dispar PTGs incubated with proctodaeal extracts from both O. nubilalis [Fig. I(A)] and L. dispar [Fig. I(B)]. Maximum stimulation was observed at 0.1 and 0.125 proctodaeal equivalents, respectively. The ratio of 3-dehydroecdysone: ecdysone varied depending on the proctodaeal dose, but at maximum stimulation, this ratio was higher (6.4:1) for L. dispar than for O. nubilalis (2.7:1). To insure that results were not confounded by the possible presence of ecdysteroids in proctodaeal tissue, all proctodaeal extracts were sampled and subjected to ecdysteroid RIA. In all cases, ecdysteroid levels were found to be undetectable or insignificant. Size exclusion H P L C Size exclusion H P L C performed on proctodaeal extracts from O. nubilalis revealed that ecdysiotropic

28

D A L E B. G E L M A N et al. A

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F I G U R E 1. Production of 3-dehydroecdysone and ecdysone by PTGs from day-5 5th instar, L. dispar incubated with proctodaeal extracts from O. nubilalis (A) and L. dispar (B). PTGs (day 5, 5th instar) were incubated in vitro with proctodaeal extracts as described in Materials and Methods. Ecdysone production was measured by RIA. Since the ecdysone antiserum used in the RIA does not detect 3-dehydroecdysone, a portion of the incubation medium was treated with a ketoreductase-containing hemolymph preparation prior to preparation for RIA. Results are expressed in terms of pg total ecdysone equivalents produced by a single PTG. Each point is the mean of at least 5 separate determinations. Standard errors not shown were smaller than the size of the d a t u m point. 3-DehydroE = 3-dehydroecdysone.

activity eluted primarily in fractions 34-37 (Fig. 2). This corresponds to a molecular weight range of approx. 5001500 Da. When fractions 34-37 were combined, dried, reconstituted in Grace's medium and tested in the in vitro PTG bioassay, ecdysteroid synthesis was stimulated in a dose-dependent manner (Fig. 3). Maximum stimulation was observed at 0.25 proctodaeal equivalents. Therefore, this dose was utilized in experiments designed to test the effects of pronase (a broad-spectrum protease) on the ecdysiotropic activity of pooled fractions 34-37. Activity of pronase-treated samples (as compared to controls) was reduced to 6.0% (Fig. 4) indicating that the ecdysiotropin present in these fractions was peptidic. Determination of cyclic A M P levels in PTGs stimulated by proctodaeal extracts from O. nubilalis When PTGs were incubated with crude proctodaeal extract (0.25 tissue equivalents) for 1-t4 min, maximum production of cyclic AMP was observed at 2min [Fig. 5(A)]. Cyclic AMP levels fell rapidly with increased incubation times and reached control levels in glands incubated for 10 rain. Fractions 33-38 also were tested individually to determine their ability to stimulate cyclic AMP production. At 2.0 tissue equivalents (based on the

F I G U R E 2. Molecular weight range of ecdysiotropin(s) extracted from proctodaea of O. nubilalis prepupae. Fifty proctodaea from prepupae that had experienced gut purge were extracted and subjected to size exclusion H P L C as described in Materials and Methods. The extract was injected onto two Waters (Waters Associates, Milford, Mass.) H P L C size exclusion Protein Pak 125 columns (7.5 x 300 m m ) arranged in tandem and preceded by a T S K SWXL progel guard column (4.0cm x 6.0) (Supelco Inc., Bellefonte, Pa). The eluting solvent was 40% acetonitrile/0.1% T F A - H 2 0 and the flow rate was 1 ml/min. Fractions of 600 #1 were collected in 1.5-ml polypropylene microcentrifuge tubes. Fractions were dried and reconstituted in Grace's medium. Two proctodaeal equivalents (based on concentration of crude extract) were tested in triplicate in the in vitro P T G (day 4, 5th instar) bioassay. Results are expressed in terms of pg total ecdysone equivalents produced by a single P T G upon stimulation by a given fraction. Molecular weight markers used are described in the "Chemicals" section of Materials and Methods. Results are from a single run; however, duplicate experiments gave similar values.

concentration of the crude extract) and an incubation time of 2.0 min, fractions 35 37 showed considerable activity while 34, although active in the PTG bioassay, stimulated only low levels of cyclic AMP production [Fig. 5(B)]. Ecdysteroid synthesis by PTGs incubated with proctodaeal extracts prepared from day 1-5/6 5th instars When assayed at 0.125 tissue equivalents, crude proctodaeal extracts from OW O. nubilalis larvae were considerably more active than extracts prepared from 4000

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F I G U R E 3. D o s ~ r e s p o n s e curve for the low molecular weight ecdysiotropin(s). Fractions 34-37 (Fig. 2) were pooled, dried and reconstituted in Grace's medium. Aliquots containing 0.032~).5 O. nubilalis proctodaeal equivalents (based on concentration of crude extract) were tested in the in vitro P T G (day 5, 5th instar) assay. Results are expressed in terms of pg total ecdysone equivalents produced by a single PTG. Each point represents the mean of at least 5 separate determinations. Error bars represent standard errors of the mean.

ECDYSIOTROPIC ACTIVITY IN THE HINDGUT

29

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FIGURE 6. Ecdysteroid synthesis by PTGs incubated with proctodaeal extracts prepared from various-aged 5th instar O. nubilalis. PTGs (day 4, 5th instar) were incubated in vitro in 25-#1 drops containing 0.125 tissue equivalents proctodaeal extract (see Materials and Methods). Results are expressed in terms of total ecdysone equivalents produced by a single PTG. Each point represents the mean of at least 8 separate determinations. OW, day 5-6 prepupae that have completed gut purge; OB, day 5 6 wandering larvae that have not completed gut purge.

younger larvae (Fig. 6). Since the wet weights of proctodaea dissected from larvae 2-6 days post-ecdysis were the same (0.45 ___0.02 mg/proctodaeum), there was no need to correct for differences in mass. However, since the wet weight of proctodaea from day-1 5th instars was 0.34 mg/proctodaeum, activity for this day was 1.4 times that shown, making it equivalent to the activity of a day-2 hindgut. A dose-response curve was generated for hindgut ecdysiotropic activity on each day of the 5th instar. These results revealed that when assayed at 0.25 equivalents (data not shown), activity was approx. 2 times greater in OW proctodaea than in all others sampled. At 0.5 equivalents ecdysiotropic activity of proctodaeal extracts prepared from younger larvae had increased to 86% of that found in OW insects (data not shown).

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FIGURE 4. Effect of prouase on ecdysiotropic activity of the low molecular weight ecdysiotropin(s). From a pooled sample of fractions 34 37 (Fig. 2), 200#1 containing 0.5 tissue equivalents per 25 #1 of proctodaeal extract were treated with pronase or boiled pronase (control) as described in Materials and Methods. Aliquots (0.25 proctodaeal equivalents) were tested in the in vitro PTG (day 5, 5th instar) assay. Results are expressed in terms of total ecdysone equivalents produced by a single PTG. Each bar represents the mean of 4 separate determinations _+ SE.

A

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FRACTION FIGURE 5. cAMP production by PTGs incubated with crude proctodaeal extract (A) or with fractions from the size exclusion column (B). (A) Day 5, 5th instar PTGs were incubated for varying times from 1 to 14 min in 25-#1 drops containing 0.25 tissue equivalents of crude proctodaeal extract. Glands were extracted in 50% ethanol:water and cAMP levels were determined by an ELISA technique as described in Materials and Methods. Each point represents the mean of at least 6 separate determinations. Control values (10 fmol/gland) were not subtracted from experimental values. Standard errors not shown were smaller than the size of the datum point. (B) Sufficient sample was aliquotted from each of fractions 33-38 from the size exclusion column (Fig. 3) to prepare 25-#1 drops of incubation medium containing 2.0 tissue equivalents of proctodaeal extract (based on concentration of crude extract prior to HPLC). Samples were dried and reconstituted in Grace's medium. PTGs were incubated in quadruplicate for 2 min. Extraction and cAMP determination was performed as in (A). Each point represents the mean of at least 4 separate determinations.

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FIGURE 7. Ecdysteroid synthesis by PTGs incubated with extracts prepared from various portions of the O. nubilalis proctodaeum. Crude extracts were prepared as described in Materials and Methods. Sufficient proctodaeal tissue was extracted so that the concentration of each portion in the incubation mixture was equal to 0.125 tissue equivalents of total proctodaeum (pylorus plus anterior intestine)/ 25 #1. Results are expressed in terms of total ecdysone equivalents produced by a single PTG (day 4, 5th instar). Each point represents the mean of at least 8 separate determinations + SE.

30

DALE B. GELMAN et al. 6000-

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q do 6 6 (5 FIGURE 8. Combined effect of O. nubilalis proctodaeai and brain extracts on ecdysteroidproduction by PTGs (day 4, 5th instar). Brain and proctodaeal extracts singly or in combination were prepared and tested as described in Materials and Methods. Expression of results is as in Fig. 7.

Localization of ecdysiotropic activity in the hindgut of O. nubilalis Using the in vitro bioassay, various portions of the hindgut were assayed for ecdysiotropic activity. Sufficient proctodaeal tissue was extracted so that 0.125 tissue equivalents of total proctodaeum (pylorus plus anterior intestine) was used for each portion. Stimulation of ecdysteroid synthesis was similar for all portions tested (Fig. 7) indicating that the ecdysiotropin(s) is located throughout the pylorus and anterior intestine.

Combined effect of O. nubilalis proctodaeal and brain extracts on ecdysteroid production by PTGs Brain and proctodaeal extracts were prepared at twice the desired concentration. Equal amounts of each were combined or individual extracts were diluted two times so that the same extracts were utilized to test individual 3

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FIGURE 9. Molecular weight range of hemolymph ecdysiotropin(s). One milliliter of hemolymphfrom OW larvae was extracted, Sep-Paked and concentrated to 200 # 1as described in Materials and Methods. The extract was subjected to size exclusion HPLC as described for Fig. 2. One hundred fifty microliters of each fraction (20-37) were dried, reconstituted in 95 ~tl of Grace's medium and tested in triplicate in the in vitro PTG (day 4, 5th instar) assay. Results are expressed in terms of total ecdysone equivalents produced by a single PTG upon stimulation by a given fraction (if losses were minimal, contained the equivalent of 67y1 of hemolymph). Size exclusion results for a proctodaeal extract (Fig. 2) are included for comparison. While results shown are from a single HPLC run, duplicate experiments gave similar values.

and combined effects, t-Tests; (one-directional, P < 0.05) were used to determine whether ecdysteroid production by PTGs incubated in the presence of a combined preparation of proctodaeal (HG) and brain (BR) extract (0.25 HG/2.0 BR; 0.25 HG/1.5 BR; 0.125 HG/2.0 BR; 0.125 HG/1.5 BR) was significantly greater than ecdysteroid production by PTGs incubated in the presence of either proctodaeal or brain extract alone. At doses which caused maximum stimulation of PTGs (1.5-2.0 brain equivalents and 0.125-0.25 proctodaeal equivalents), effects of brain and proctodaeal extracts were additive (Fig. 8). As expected, at lower doses, stimulatory effects of brain and proctodaeal extracts also appeared to be additive (Fig. 8).

Molecular weight range of hemolymph ecdysiotropin(s) Extracts of hemolymph prepared from OW O. nubilalis larvae were subjected to size exclusion chromatography. Since hemolymph from OW larvae contains considerable amounts ofecdysteroids (Gelman and Woods, 1983) and since these elute in fractions 38 and 39 (unpublished results), only fractions 20-37 were tested in the in vitro PTG bioassay. Fractions 35-37 possessed considerable ecdysiotropic activity corresponding to a molecular weight range of 500-1000 Da (Fig. 9). No activity was detected in the molecular weight range of the small form of brain PTTH (1500-3000 Da). DISCUSSION Our results confirm the presence of ecdysiotropic activity in proctodaeal extracts of both L. dispar and O. nubilalis first reported by Gelman et al. (1991). The original decision to examine proctodaeal extracts for the presence of ecdysiotropic factors was based on Beck and Alexander's findings (1964) concerning the presence of proctodone, a factor present in the anterior proctodaeum which is capable of stimulating a molt in diapausing larvae. In our experiments, both ecdysone and 3-dehydroecdysone were produced in a dose-dependent manner by stimulated PTGs (Fig. 1). Maximum stimulation was achieved at similar doses for L. dispar (0.1 tissue equivalents) and O. nubilalis (0.125 tissue equivalents) although the ratio of 3-dehydroecdysone to ecdysone produced was 2.4 times greater for L. dispar extracts. Intact PTGs do have some ketoreductase activity (Gelman et al., 1991) which would account for the production of ecdysone. However, it is difficult to explain why PTGs stimulated with L. dispar extracts produce considerably less ecdysone than PTGs incubated with O. nubilalis extracts. A difference in the level of ketoreductase in the two extracts could not be responsible since excess ketoreductase is added prior to the last 1 h of incubation. Based on results from size exclusion H P L C (Fig. 2), the ecdysiotropin(s) in O. nubilalis proctodaeal extracts has a molecular weight in the range of 500-1500 Da. These results contradict previous reports (based on molecular weight determinations with Amicon filters) of higher molecular weights (Gelman et al., 1991). Of

ECDYSIOTROPIC ACTIVITY IN THE HINDGUT

course it is possible that these higher molecular weight moieties were destroyed upon exposure to organic solvents and thus were undetectable in current experiments. Since all other known ecdysiotropins (PTTHs) have higher molecular weights (Bollenbacher et al., 1984; Nagasawa et al., 1986; Masler et al., 1986; Maruyama et al., 1988; Gelman et al., 1993), the proctodaeal ecdysiotropin(s) appears to be a unique molecule. Since it is inactivated upon treatment with pronase (Fig. 4), we can conclude that this low molecular weight ecdysiotropin is peptidic. Cyclic AMP was produced in PTGs exposed to both crude proctodaeal extracts [Fig. 5(A)] and semi-purified extracts [Fig. 5(B)] indicating that this cyclic nucleotide probably acts as a second messenger for the proctodaeal ecdysiotropin(s). Smith et al. (1984) have reported similar involvement of the cAMP system in PTTH's stimulation of ecdysteroid production by M . sexta PTGs. In our experiments, cAMP levels in PTGs were highest after 2 min of incubation and then fell to lower levels with increasing incubation times, probably due to the action of a phosphodiesterase which destroys cAMP by converting it to ATP. In similar experiments, Smith et al. (1986) report that cAMP can be detected in PTTHstimulated larval glands but not in pupal glands unless phosphodiesterase inhibitors are added to the incubation medium. That fraction 34 from the size exclusion HPLC did exhibit ecdysiotropic activity but did not stimulate the production of cAMP in PTGs, implies that the hindgut extracts contain more than one ecdysiotropin and that these ecdysiotropins may utilize different second messenger systems. Experiments in which active size exclusion fractions were combined and subjected to further purification by reverse phase HPLC revealed the presence of at least two groups of fractions possessing ecdysiotropic activity (unpublished results). Ecdysiotropic activity is located throughout the pylorus and anterior intestine of the hindgut (Fig. 7), and may actually be associated with the extensive nerve network that covers the proctodaeum, but at this time, its source is unknown. Purification of the ecdysiotropin will allow us to generate antibodies which, with the help of immunocytochemistry, should allow us to localize the peptide. While ecdysiotropic activity was present in the O. nubilalis hindgut throughout the 5th instar, it was present in greatest concentration in the prepupal proctodaeum (Fig. 6). It is at this time that hemolymph ecdysteroid titers begin to rise in preparation for the pupal molt (Gelman and Woods, 1983). The physiological role of the hindgut ecdysiotropin(s), if any, in the regulation of molting and metamorphosis is not known. However, an ecdysiotropin whose molecular weight range is the same as that of the hindgut ecdysiotropin was identified in the hemolymph of prepupae (Fig. 9). It is noteworthy that although hemolymph of prepupae contains a 500-1000 Da ecdysiotropin, no ecdysiotropic activity was detected in size exclusion fractions corresponding to the molecular weight of the small form of

31

O. nubilalis brain PTTH (Gelman et al., 1993). It will be necessary to purify and sequence the proctodaeal and hemolymph low molecular weight ecdysiotropin(s) to determine if they share the same identity. If so, there would be support for the view that the hindgut ecdysiotropin serves an endocrine function. The increased levels observed in OW proctodaea (Fig. 6), then, would more likely be due to increased synthesis and release rather than an accumulation due to the shutdown of release at this time. If the hindgut ecdysiotropin is released into the hemolymph and causes in vivo stimulation of the PTGs, it is significant that at levels resulting in maximum activation of PTGs, the effects of brain and hindgut ecdysiotropins were additive (Fig. 8). These results suggest that the two peptides work through separate receptors. Such synergistic activity would allow for a more finely tuned regulatory system for the control of ecdysteroid production by PTGs.

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Acknowledgements--We wish to thank A. A. Khalidi for technical

assistance and S. D. Beck, D. L. Dahlmann and C.-M. Yin for their critical readings of the manuscript. Mention of a commercial product does not imply endorsement of the U.S. Department of Agriculture.