Action of pheromone biosynthesis activating neuropeptide on In vitro pheromone glands of Heliothis armigera females

J. Insect Physiol. Vol. 36, No. 9, pp. 641646, Printed in Great Britain. All rights reserved

1990 Copyright

0

0022-1910/90 $3.00 + 0.00 1990 Pergamon Press plc

ACTION OF PHEROMONE BlOSYNTHESIS ACTIVATING NEUROPEPTIDE ON IN VITRO PHEROMONE GLANDS OF HELIOTHIS ARMIGERA FEMALES A. RAFAELI,‘,* V. SOROKER,’B.

KAMENSKY’

and A. K. RAINA~

‘Department o:TEntomology, Faculty of Agriculture, The Hebrew University, Rehovot 76-100, Israel and %sect Chemical Ecology Laboratory, Agricultural Research Service, USDA, Beltsville, MD 20705, U.S.A. (Received 11 January 1990; revised 24 April 1990)

Abstract-Pheromone glands of Heliothis armigera were stimulated in vitro to incorporate r4C from the radioactive precursor sodium acetate, in the presence of synthetic pheromone biosynthesis activating neuropeptide (PBAN). When hexane extracts of the radioactive products were analysed by TLC and HPLC the radioactivity corresponded in retention time to the main pheromone component (Z)-1 1-hexadecenal. Maximal stimulation, as depicted by TLC analysis, was observed after 4 h of incubation. The PBAN response was shown to be dose dependent, maximal levels, as analysed by TLC, were obtained at a concentration of 5 pmol/gland. This response was shown to be mediated by CAMP. Key Word Zndex: Pheromone Biosynthesis glands; Heliothis armigera; cyclic-AMP

INTRODUCTION

Female moths attract conspecific males by the release of a sex pheromone. Research to date has shown that production of the pheromone in Heliothis spp. is under neuroendocrine control mediated by a hormone the pheromone biosynthesis activating neuropeptide (PBAN) (Raina et al., 1987; Raina et al., 1989; Rafaeli and !ioroker, 1989a). PBAN is produced in the suboesophageal ganglia and released via the corpora cardiaca into the haemolymph at the onset of scotophase (Raina and Menn, 1987). The H. zea PBAN has shown biological activity in a number of moths (Raina et al., 1989). We have previously demonstrated that stimulation of de nova pheromone biosynthesis occurs in in vitro pheromone glands cd H. armigera, in the presence of brain-complex extra.cts (Soroker and Rafaeli, 1989; Rafaeli et al., 1990). On the other hand no stimulation by brain extra.cts was observed in in vitro tissue cultures of pheromone glands of Trichoplusia ni and Argyrotaenia velutinana (Tang et al., 1989). Recent research on H. zea showed the necessity of the presence. of terminal abdominal ganglia in in vitro incubations for pheromone stimulation by brain extracts (Teal et al., 1989). Although we showed an

enhanced effect witb this ganglion, pheromone production per se did not require the presence of the ganglion (Rafaeli ei’ al., 1990). *To whom all correspondence

should he addressed.

Activating

Neuropeptide

(PBAN); pheromone

With the availability of synthetic H. zea PBAN (Raina et al., 1989) clarification of some of these conflicting reports is now possible. The question was whether the in vitro response, which was observed with brain-complex extracts by us, was indeed a result of PBAN or due to the action of other unknown factors present in these extracts.

MATERL4LS AND

METHODS

Insect culture H. armigera larvae were raised on an artificial diet at a constant temperature of 25°C and a 14: 10 light :dark cycle as reported previously (Rafaeli and Soroker, 1989). [‘“Cj-tcetate incorporation by pheromone gland preparations

The eighth and ninth abdominal segments with the attached intersegmental membrane containing the pheromone glands, were removed under sterile conditions from 2 to 3-day-old decapitated virgin females during the 8-10th hour photophase. The abdominal tips (referred heretofore as pheromone glands) were cleared of all internal tissues by squeezing between two forceps and washed twice in sterile TC-199 medium (Difco Lab., U.S.A.) to remove haemolymph residues. They were subsequently transferred individually to 100 ~1 TC-199 medium containing 2-3 PCi [r4C]acetate (56 mCi/mmol; NEN, Boston, U.S.A.) in 641

642

A.

RAFAEL1

and radioimmunoassay

a/.

Table 1. Effect of PBAN on the total “‘C incorporation from sodium acetate by in vitro pheromone slands of Heliothisam&era females

the presence or absence of H. zea PBAN. Adipokinetic hormone of H. zea (Jaffe et al., 1986) was used as a control in some experiments. The glands were incubated at 25°C in an incubator under scotophase conditions. After 4 h of incubation (unless otherwise stated) the glands and media were extracted in hexane. Radioactivity in the hexane phase was counted in a Beckman scintillation counter after the addition of scintillant (Insta Gel, Packard) to each vial. Extraction CAMP

et

Treatment

CPM/gland

Control 1056+ 262.5 (5) PBAN (9 pmol) 3538 f 1548.5 (7)* Adipokinetic hormone (9 pmol) 1252+ 485.4 (7) (NS) Numbers represent means k SEM, figure in parentheses represent the number of replicates. *Significant at P c 0.05 by Wilkocson analysis.

of intracellular

radioactivity was quantified by liquid scintillation counting. HPLC analysis was performed on an LKB 2150 pump and a Linear UVis 200 variable detector using a RP-18 Lichrosorb column (5 p) (Merck). Marker components: (Z)-9-hexadecenoic acid, (Z)- 11-hexadecenal; (Z)- 11-hexadecenol and (Z)- llhexadecenyl acetate were eluted using a stepwise gradient of: 60% acetonitrile and distilled water to 70% at 2%/n& after 10min to 90 at 2%/min and after another 10 min to 100 at 2%/r&r at a flow rate of 2 ml/min. Fractions were collected every 0.5 min into scintillation vials to which scintillant was added.

Pheromone glands were removed from the incubation media after 10 min of incubation and homogenized in 100% ethanol. Intracellular CAMP concentrations were measured using a CAMP radioimmunoassay as reported previously (Rafaeli and Soroker, 1989b). Radioactive [‘HIcAMP was purchased from NEN (Boston, U.S.A.) and CAMP antiserum was purchased from Bio Makor (Israel). Chromatographic analyses

Components of the pheromone gland extract were separated by thin layer chromatography (TLC) with a normal-phase silica gel plate (Raedel-deHaen, DCKarten SIFf = 0.2 mm). The solvent system used was benzene : ether : ethanol : acetic acid (40 : 50 : 2 : 0.2). The markers used to evaluate TLC separations: Triglycerides, (Z)-1 1-hexadecenol, (Z)-1 l-hexadecenal and (Z)-9-hexadecenoic acid (not differing in Rf value from the presumed biosynthetic intermediate (Z)- 1I-hexadecenoic acid) were obtained from Sigma Chemical Co., (U.S.A.). Rf values were compared after detection of the samples by iodine vapour exposure. Fractions on the TLC plate were cut and

Statistical analysis

Data were subjected to Wilkocson two sample test or analysis of variance for normal distributions. In all cases the minimal number of observations was five. RESULTS

Effect of PBAN on the total 14C incorporation from sodium acetate into the hexane phase I

The prksence of PBAN (9 pmol) in the incubation medium was tested for total r4C incorporation into

incorporation Zll-16:Ald I 1600

I

-

1000 ZQ-16:Aold I

1

600 -

1

2

3

4

6

6

7

8

TLC fraction m

lL_

9 10 11 12 13 14 16 16 17 18 19 20

control

0

PBAN

Fig. 1. TLC separation of the hexane extractable products from single Bland extracts. Elution of (Z)-ll-hexadecenol occurred in fraction 8; (Z)-9-hexadeeanoic acid (Z9-16:Acid) in fraction 11; (Z)-ll-hexadeeenal (Zll-16:Ald) in fraction 14; and triglycerides in fraction 17. n =Control incubations; 0 = Incubations in the presence of PBAN (12 pmol/gland).

Action of PBAN 6000 [“Cl incorporatlon

643

tll-l&Ald

1

II

a

Z11-lerAo ZO-l&hid Zll-18:Ol

r-3

HPLC fraction m

Control

tZZI Braln

1200 [“Cl incorporation Zll-16:Ald

lOOO-

b

800 600 400

Lll-18:AG

200 0 1

4

7

10

13

16

19

22

26

23

31

34

HPLC fraction 0

PBAN

m

Control

Fig. 2. HPLC separation of the hexane extractable products from pooled gland extracts. Elution of (Z)-9-hexedecanoic acid (Z9-16:Acid) occurred in fraction 10; (Z)-ll-hexadecenol (Zl l-165:01) in fraction 16;(Z)-1 I-hexadeccnal (Zl l-16:Ald) in fraction 19;and (Z)-1 I-hexedeccnyl acetate (Zl l-16: AC) in fractions 25 as indicated. (a) Shows the stimulation by brain extracts W (1.2 equivalents/gland) as compared to control n incubations each of 9 pooled gland extracts; (b) shows the stimulation by PBAN 0 (14 pmol/gland) as compared to control n incubations each of 6 pooled gland extracts.

the hexane extracta.ble products. It can be seen (Table 1) that the total counts are significantly higher than control incubations. Control incubations using H. zeu adipokinetic hormone (9 pmol) (also present in brain-complex extracts) caused no significant stimulation of t4C incorporation. Separation of the hexune extractable products result ing from PBAN stimulation

The hexane extractable products from in vitro pheromone gland incubations were separated on TLC. A sample separation is shown in (Fig. 1). PBAN significantly stimulated the incorporation of t4C into a fraction which corresponds in retention time to (Z)- 1 I -hexadecenal. Similarly, using reversed phase HPLC for separating the pheromone

components, both PBAN and brain extracts showed stimulation of incorporation which corresponds in elution time to (Z)-11-hexadecenal [Fig. 2(a), (b)]. Time course of PBAN stimulation

Using hexane extracts of total 14C incorporation [Fig. 3(a)] as well as TLC separations of single in vitro pheromone gland incubations [Fig. 3(b)] as the standard bioassay methods, we tested the time-course of PBAN stimulation. In both cases it was observed that peak stimulation occurs after 4 h. Dose-response

relationship

A dose-response study was performed using TLC separated hexane extracts of single gland incubations (Fig. 4). No significant increase in the incorporation

644

A. RAFAELI s

3500

e -

3000-

et al.

(6)

.; 2500.+ 2 o& 20002 0-

1?inn-

(7)

(6)

0

2

1

3

Time -++

(6)

7

--Y

"

Control

4

5

6

7

(hours) -+-

PBAN

b

0

1

2

4

3

5

6

7

Time (hours) *

Control

-+-

PBAN

Fig. 3. Time-course of PBAN stimulation of in vitro pheromone glands; (a) shows the effect on the total hexane extractable “C incorporated products from sodium acetate; (b) shows the effect of the corresponding incubation on the TLC separated (Z)-1 I-hexadecenal (Zl l-16:Ald) fraction. -O-= Control incubations; -•-= Incubations in the presence of PBAN (11 pmol/gland). Points depict means f SEM; figures in parentheses represent the number of replicates.

into the (2)-l I-hexadecenal fraction was observed at doses below 2 pmoles. Maximal response was observed at 5 pmol, and the response was not significantly different at higher doses. Intracellular CAMP levels

The glands PBAN (Table

intracellular CAMP levels of pheromone when incubated in the presence of 7pmol of showed a 230% increase over control glands 2). DISCUSSION

This report clearly demonstrates the action of synthetic H. zea PBAN on in vitro pheromone glands

and confirms our previous findings with methanolic extracts of brain-complex. Pheromone glands were stimulated to incorporate higher levels of radioactivity in the presence of PBAN. This was shown to be a specific response since another peptide hormone, H. zea adipokinetic hormone, usually present in the same methanolic extracts, did not initiate any stimulation. The response was also shown to be dosedependent. The optimal dose was between 3-5 pmol of PBAN per incubation reaching a maximal level at 5 pmol. Any further increases in the amount of hormone had no significant effect. The incubation time required for maximal stimulation was 4 h. These observations strengthen our previous hypothesis that the pheromone gland is a target tissue

645

Action of PBAN 5750

_

1

4600

T

Cl

T

4

0

8

12

T

16

20

PBAN Cpmoll

Fig. 4. Dose-response relationship of PBAN stimulation of the TLC separated (Z)-1 I-hexadecenal (ZII-16:Ald) fraction. Points represent means f SEM; the minimal number c,f replicates was 5. for PBAN. The conflicting data observed by other workers may be explained by differences in methodology. Tang et al. (1989) used larger doses of brainextracts as stimulant (30 brain equivalents/5 glands in 30/~1 saline; i.e. 1 brain/kl) and it is possible that such large doses of brain extracts may have had an inhibitory effect. In addition, smaller amounts of radioactive precursor were present in the medium (0.6 pCi/S glands in 30 ~1 saline) which may reduce the sensitivity of the bioassay. On the other hand, Teal et al. (1989) did n’ot use radioactive tracers in the in vitro incubations and analysed the small quantities of pheromone produced by gas chromatography thereby not detecting de novo biosynthesis of the pheromone components and only detecting the enhanced effect in the presence of the terminal abdominal ganglion. When we compare the dose-response curve of synthetic PBAN in vitro to that obtained in the in vivo bioassay (Raina et al., 1989) a close correlation can be observed. In the in vivo assay, maximal levels of stimulation were observed at a concentration of 4 pmol after which there was a drop and thereafter a saturation level was attained (Raina et al., 1989). When total 14Cincorporation was tested after stimulation by methanolic extracts of brain complexes Table

2. Stimulation of intracellular CAMP levels by PBAN

Treatment Control PBAN (7 pmol)

fmol CAMP/gland 300 +

50 (8)

700 f 80 (8)*

Numbers represent means f SEM, figures in parentheses represent the number of replicates. *Significantly different at P c 0.0004 by Student’s t-test.

(Soroker and Rafaeli, 1989) maximal activation was obtained at 0.5 brain equivalents. According to calculations based on in vivo evidence this brain-extract dose corresponds to the concentration of 3-5 pmol (Raina et al., 1989) which is also the maximal dose obtained in this study. Subsequent TLC and HPLC analyses were also consistent with our previous findings in which brain-complex extracts stimulated the production of a compound coeluting with the marker pheromone component, (2)-l 1-hexadecenal (Soroker and Rafaeli, 1989) which was subsequently observed using preparative radio-GC (on a Carbowax 20M column) to coelute with the pheromone component (Rafaeli et al., 1990). In addition, intracellular CAMP levels were stimulated as a direct consequence of PBAN stimulation. This finding supports our previous hypothesis that CAMP may be the mediator in the PBAN stimulation of in vitro pheromone glands whereby we showed an increase in intracellular CAMP levels as a result of brain-extract stimulation. In addition, the physiological response was mimicked by 8-bromo-CAMP, forskolin and isobutyl-methylxanthine whereas no stimulation by 8-bromo-cGMP was obtainded (Rafaeli and Soroker, 1989b). Therefore all the data suggests that CAMP may be the physiological second messenger. Further investigations on the physiological effect of PBAN on the pheromone biosynthetic pathway are currently in progress. research was supported by a fund for basic research administered by the Israel Academy of Science.& Humanities to A. Rafaeli. We thank Oma Shaul and Pablo Chercasky for technical assistance.

Acknowledgements-This

REFERENCES Jaffe H., Raina A. K., Riley C. T., Fraser B. A., Holman G. M., Wagner R. M., Ridgway R. L. and Hayes D. K. (1986) Isolation and primary structure of a peptide from the corpora cardiaca of Heliothis zea with adipokinetic activity. Biochem. Biophys. Res. Commun. 135, 622-628. Rafaeli A. and Soroker V. (1989a) Effect of diel-rhythm and brain hormone on pheromone production in two Lepidopteran species. J. them. Ecol. 15, 447-455. Rafaeli A. and Soroker V. (1989b) CAMP mediation of the hormonal stimulation of 14C-acetate incorporation by Heliothis armigera pheromone glands in vitro. Mol. Cell. Endocr. 65,43-48. Rafaeli A., Soroker V., Klun J. and Raina A. K. (1990) Stimulation of de nova pheromone biosynthesis by in vitro pheromone glands of Heliothis spp. In Insect Neurochemistry and Neurophysiology (Edited by Borkovec A. B. and Masler E.). Humana Press, Clifton, N.J. Raina A. K. and Menn J. J. (1987) Endocrine regulation of oheromone oroduction in Lepidoptera. In Pheromone Biochemisfry-(Edited by Prestwich G. D. and Blomquist G. J.). DD. 159-174. Academic Press, Orlando, Fla. Raina A. K., Jaffe H., Klun J. A., Ridgway R. L. and Hayes D. K. (1987) Characteristics of a neurohonnone that controls sex pheromone production in Heliofhis zeu. J. Insect Physiol. 33, 809-814. I.

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Tang J. D., Charlton R. E., Jurenka R. A., Wolf W. A., Phelan P. L., Sreng L. and Roelofs W. L. (1989) Regulation of pheromone biosynthesis by a brain hormone in two moth species. Proc. nurn. Acad. Sci. U.S.A. 86, 1806-1810. Teal P. E. A.. Tumlinson J. H. and Oberlander H. (1989) Neural regulation of sex pheromone biosynthesis in Heiiothis moths. Proc. natn. Acad. Sci. U.S.A. 86, 2488-2492.