In vitro hormonal stimulation of [14C]acetate incorporation by Heliothis armigera pheromone glands

Insect Biochem. Vol. 19, No. 1, pp. I-5, 1989

0020-1790/89 $3.00+0.00 Copyright © 1989 Pergamon Press pie

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IN VITRO HORMONAL STIMULATION OF

[14C]ACETATE INCORPORATION BY HELIOTHIS ARMIGERA PHEROMONE GLANDS V. SOROKER and A. RAFAELI Department of Entomology, Faculty of Agriculture, Rehovot 76-100, Israel (Received 25 February 1988; revised and accepted 12 July 1988)

Abstract--Isolated pheromone gland preparations of Heliothis armigera incorporate radioactivity from [z4C]sodium acetate at a linear rate for 24 h when incubated in a physiological saline. This incorporation is stimulated when methanolic or partially purified brain-complex extracts are present in the incubation medium and the stimulation is dose-dependent. The radioactive products extracted from the pheromone glands and media by hexane, revealed incorporation of radioactivity into a product exhibiting the same mobility as (Z)-l 1 hexadecenal, the main component of the pheromone of H. armigera, as analyzed by thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC). In addition, gas chromatographic (GC) analysis of the hexane soluble products after incubation in the absence of ['4C]sodium acetate revealed a significant stimulation of the concentration of (Z)-I 1 hexadecenal by brain-complex extracts. Key Word lndex: Heliothis armigera, hormone, pheromone, [14C]acetateincorporation,/n vitro pheromone glands

INTRODUCTION

MATERIALSAND METHODS

Ligation of female moths between the head and the thorax curtails pheromone production, and this is regained after injections of extracts of brain complexes (Raina and Klun, 1984; Oghuchi et al., 1985; Rafaeli and Soroker, 1989). The obvious question, when evidence is based on in vivo observations, is whether or not this gland comprises the true target organ for the neurohormone. Organ or tissue cultures are able to clarify the control mechanisms involved in the production and/or secretion of pheromone. The sex pheromone producing gland has been shown to be a modification of the intersegmental membrane between the eighth and ninth abdominal segments (Percy et al., 1971). White et al. (1972) studied the ultrastructural "cellular integrity" of the female gland during culture. Studies in vitro have hitherto relied mainly on behavioral data to detect pheromone (Srinivasan et al., 1986). Srinivasan et al. (1986) report their inability to detect sex pheromone by gas chromatography of extracts of cultures. On the other hand Jones and Berger (1978) demonstrated the incorporation of [1-14C] from sodium acetate into the pheromone of Trichoplusia ni in vivo as well as in vitro as analyzed both by gas chromatography and high pressure liquid chromatography. However, studies to date have not shown production in vitro of pheromone as a direct consequence of hormonal stimulation. This report presents evidence of a successful shortterm in vitro hormonal bioassay technique in which incorporation of radioactivity is stimulated and which may be used for the future study of the effect of brain hormone on pheromone gland preparations.

Animal~ Heliothis armigera larvae were raised on an artificial diet at a constant temperature of 25°C and a 14:10 fight:dark photoperiod (Rafaeli and Soroker, 1989). Abdominal tips, containing the eighth and ninth abdominal segments with the attached intersegmental membrane were removed under sterile conditions from 2-3 day old virgin femalesduring the first-third hour scotophase using a dim red light for illumination.

IB 19/I-A

In vitro incubation and analysis These pheromone glands were washed twice in sterile medium TC-199 (pH 7.2) (Difco Lab., U.S.A.) and then transferred individually to medium TC-199 containing 3-5 #Ci [1J4C]sodium acetate (56mCi/mmol; NEN, Boston, U.S.A.). They were incubated at 25°C in an incubator, maintaining the photoperiod. After the required incubation period the glands and media were extracted in 500/d hexane. Radioactivity in the hexane phase (400/~l aliquot) was counted in a Beckman scintillation counter after the addition of scintillant (Insta Gel, Packard) to each vial. Components of the pheromone gland extract, pooled from 4 to 7 incubations, were separated by thin layer chromatography (TLC) with a normal-phase silica gel plate (Merck, F254). The solvent system used was hexane-ethyl acetate (l:l). Standards were used to evaluate TLC separations. Triglycerides, (Z)-l 1 hexadecenol, (Z)-11 hexadecenal and (Z)-9 hexadecenoic acid were obtained from Sigma Chemical Co. (U.S.A.). R e values were obtained after detection of the samples by their u.v. absorption or 12 vapor exposure. Fractions on the TLC plate were cut and radioactivity was quantified by liquid scintillation counting after adding scintillation cocktail (Insta Gel, Packard). For HPLC analysis the extracted media and glands were pooled (equivalent to 4-6 pheromone glands). The hexane was evaporated to near dryness and the extract was reconstituted in the mobile 1

V. SOROKERand A. RAFAELI phase containing the non-radioactive markers (Z)-11 hexadecenal (10/~g) and (Z)-9 hexadeeenoic acid (50/~g). HPLC analysis was on a Tracor LC pump 955 and a variable detector 970A using a RP-18 Lichrosorb column (5/~) (Merck). The mobile phase was 100% acetonitrile at a flow rate of I ml/min. Fractions were collected every 4 min into scintillation vials to which scintillant was added. Radioactivity was thus scanned throughout the elution profile. For brain-complex extracts, brains were dissected from females (2-3 days old) or males (2-5 days old), homogenized in 80% methanol, centrifuged for 2 min and the supernatant was removed and evaporated to dryness under a N2 stream. Partial separation of brain-complex extracts was performed on low pressure RP-18 cartridges (Seppak, Waters) as reported previously (Rafaeli and Soroker, 1989). Gas chromatographic analysis of (Z)-ll hexadecenal was performed as reported previously (Rafaeli and Soroker, 1989) using an OVI fused silica capillary column (50m, Mega, Italy). RESULTS

Time course of incorporation of radioactivity by pheromone gland preparations The i n c o r p o r a t i o n from [~4C]sodium acetate was assessed d u r i n g a 24 h period of incubation. A t several time intervals sample gland p r e p a r a t i o n s a n d media were extracted a n d the i n c o r p o r a t i o n level was determined. Table 1 shows t h a t after an initial lag period o f 3 h, the level of i n c o r p o r a t i o n of '4C from sodium acetate was linear. A t each time interval a control containing radioactive m e d i u m alone was also extracted a n d any c o n t a m i n a t i o n was thus followed t h r o u g h o u t the tested i n c u b a t i o n period. N o increase in radioactivity of such controls was observed indicating t h a t the increase in radioactivity was not due to c o n t a m i n a t i o n o f the media.

Effect of brain-complex extracts on incorporation of radioactivity by pheromone gland preparations Various b r a i n h o r m o n e sources were tested o n the level o f i n c o r p o r a t i o n of radioactivity by p h e r o m o n e glands in vitro. Table 2 shows t h a t b o t h male a n d

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Fig. 1. Dependence of the dose of female brain-complex extracts on the incorporation of ~4C from sodium acetate by in vitro pheromone gland preparations.

female brain-complex extracts significantly stimulated the n o r m a l level o f i n c o r p o r a t i o n by p h e r o m o n e gland p r e p a r a t i o n s after 4 h o f incubation. A percentage stimulation level relative to the control incorp o r a t i o n level was calculated. A l t h o u g h the control a n d h o r m o n e - t r e a t e d replicates were r u n concurrently, the different t r e a t m e n t s were p e r f o r m e d as separate experiments. The response to female brain-complex m e t h a n o l extracts was tested after 4 h o f i n c u b a t i o n at various c o n c e n t r a t i o n s of brains a n d a dose response relationship is s h o w n in Fig. 1.

Effect of light intensity on bioassay performance Table I. Time-dependence of incorporation in vitro of '4C from sodium acetate by pheromone gland preparations Total hexane Time '4C-incorporation extractable incubation level (cpm/gl) radioactivity (h) (mean ± SEM) n (cpm) 3.0 855 +__261.1 4 47 6.5 2012 + 494.0 5 51 9.5 7625 __.1004.0 5 69 24.0 29,895 + 13,168.0 4 59

Experiments were designed to test w h e t h e r u n d e r n o r m a l l a b o r a t o r y light intensities the p h e r o m o n e glands c o n t i n u e d to respond to h o r m o n a l stimulation. A g r o u p o f p h e r o m o n e glands in vitro, rem o v e d u n d e r n o r m a l conditions as described in the methods, was i n c u b a t e d in the light or in the d a r k for 4 h in the presence a n d absence of male braincomplex extracts (1.67equivalents). The results (Table 3) show clearly t h a t u n d e r light conditions the

Table 2. Incorporation in vitro of radioactivity from sodium acetate into hexane extractable products by pheromone gland preparations of H. armigera Brain Incorporation level (cpm) % extract Control Stimulated Stimulation Male (Seppak, 716 + 158.3 1270 __.187.9 78 +_28.3 7 I equiv.) (P < 0.05) Male (800 MeOH, 455 + 78.9 932 +_225.5 110 + 48.2 8 2.7 equiv.) (P < 0.05) Female (80% MeOH, 2411 + 437.0 8005 + 2724.0 239 + 110.7 7 1.5 equiv.) (P < 0.05) Numbers represent mean + SEM, statistical significanceindicated in parentheses according to Wilcoxon's two-sample test.

Hormonal stimulation of []4C]acetate incorporation

3

Table 3. Effect of light intensity on the incorporation m vitro of t4C from sodium acetate by pheromone gland preparations of H. armlgera

Treatment Dark Light

Incorporation level(cpm) Control Stimulated 2663 _+580.1 (7)b 7109 _+1516.6 (8)a 2806 + 547.0 (7)b 4491 _+1518.4 (7)b

%

Stimulation 167 + 57.0 (8) 72 + 50.2 (7)

Numbers represent mean + SEM, figures in parentheses indicate number of replicates. Numbers followed by the different letters indicate a significant difference according to Wilcoxon's two-sample test.

Fractions coincident with the marker pheromone (Z)-I 1 hexadecenal (peak B) showed a significant increase in the level of radioactivity when exposed to TLC and HPLC separations of pheromone gland brain extracts (1.8-fold increase with respect to the preparation extracts control incorporation level). This response was particularly high when female Chromatography of the hexane extracts using pooled extracts from control and stimulated in vitro brain-complex extracts (1.5 equivalents) were tested pheromone gland incubations, was performed on a (Fig. 4, peak b) where it reached a total level of normal-phase TLC plate (Fig. 2). Fractions from 8 to 7999 cpm. In this experiment the control levels of 12cm were cut every 0.Scm and in all the other • incorporation into the corresponding peak b were positions fractions were cut every cm. A low level of equivalent to 574cpm (elution profile similar to incorporation was observed in control incubations. Fig. 3 and not included) thus revealing a 13-fold However, in the presence of female brain-complex increase incorporation into this fraction as a direct extracts (1.9 equivalents, 80% methanol) 4 distinct response to brain-complex extracts. In addition, feradioactive peaks were obtained (Rf 0.68; R r 0.61; Rr male brain-complex extracts stimulated the incorpo0.5 and R e 0.17). Co-chromatography of standard ration of radioactivity at a retention time equivalent non-radioactive compounds indicated that the peaks to the marker (Z)-9 hexadecenoic acid (Fig. 4, peak at l ~ 0.68 coeluted with triglycerides, that at Rf 0.61 a). An unknown compound with retention time of coeluted with (Z)-l 1 hexadecenal, and that at Rf 0.5 20 rain (Figs 3 and 4, peak c) was also observed. We coeluted with (Z)-I l hexadecenol. The fourth radio- also tested the total incorporation levels before injecactive peak did not correspond to the mobility of any ting the sample onto the HPLC column. In control incubations the total incorporation amounted to of the standards tested. Similar separations were performed on reverse- 718 cpm/pheromone gland and the stimulated level phase HPLC columns after stimulation with male reached 4351 cpm/pheromone gland. From these valbrain-complex extracts (l.5equivalents) (Fig. 3). ues the percentage of incorporation into peak b was level of stimulated ]4C incorporation is lower than the stimulated level incorporated under dark conditions.

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Fig. 2. Effect of female brain-complex extracts on the incorporation of radioactivity from [~4C]sodium acetate into hexane extractable products as separated by TLC using hexane:ethyl acetate (l:l) as the solvent system. PA = palmitic acid, ZI 1HDAL = (Z)-I 1 hexadecenal, Z11HDOL = (Z)-I 1 hexadecenol, TG = triglycerides.

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Fig. 4. Effect of female brain-complex extracts on the incorporation of radioactivity from [~4C]sodium acetate into (Z)-I1 hexadecenal. The elution profile shows the retention times of markers (a), (Z)-9 hexadecenoic acid (50 #g) and (b), (Z)-ll hexadecenal (10#g). The cross-hatched histograms depict the level of radioactivity in the corresponding I ml fractions (see text for HPLC conditions).

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Fig. 3. Effect of male brain-complex extracts on the incorporation of radioactivity from [14C]sodium acetate into (Z)-11 hexadecenal. The top graph shows the elution profile containing marker components (A), (Z)-9 hexadecenoic acid (50#g) and (B), (Z)-ll hexadecenal (10/~g). The bottom graph depicts the corresponding radioactivity present in the collected 1 ml fractions (see text for HPLC conditions).

therefore calculated as 16% in the control incubations and 37% when brain-complex extracts were present.

GC separations of pheromone gland preparation extracts Further verification of the presence of (Z)-I 1 hexadecenal in incubations subjected to brain-complex extracts was obtained by gas chromatographic analysis of the hexane extractable products in the absence of radioactive precursors. Hexane extracts of the incubation medium alone and incubation medium

containing brain-complex extracts, in the absence of pheromone glands, were run under the same GC conditions to verify that no interference in detection of (Z)-I 1 hexadecenal occurs. The presence of higher concentrations of (Z)-I l hexedecenal were observed in hexane extracts of pheromone gland incubations which were stimulated by female brain-complex methanol extracts (1.2 equivalents) (Table 4).

Table 4. (Z)-I 1 hexadccenal concentrations in the hexane extractable products after in vitro incubation of pheromone glands in the absence and presence of female brain-complex methanol extracts as quantified by gas chromatographic analysis

Treatment Control (7 h incubation) Female brain-complex extact (1.1 equiv./gland, 7 h incubation)

(Z)-I 1 hexadecenal concentration (ng/gland/incubation) 0.62 + 0.496a (8) 11.95 _+4.708b (14)

Numbers represent mean + SEM, figures in parentheses indicate number of replicates. Statistical significance P < 0.01 according to Wilcoxon's two-sample test, P < 0.05 according to Student's t -test.

Hormonal stimulation of [~4C]acetate incorporation DISCUSSION A hormonal controlling influence on the production of pheromone by H. armigera female sex pheromone glands was suggested from our previous in vivo studies (Rafaeli and Soroker, 1989). However, injections of brain-complex extracts into an insect suffers in that evidence is indirect and the same overall effect may be produced by acting through a mediating target tissue. Evidence for our hypothesis was observed by gas chromatographic analysis of hexane extracts of in vitro incubations in the absence of radioactivity. The significant stimulation by braincomplex extracts observed suggests strongly that (Z)-I 1 hexadecenal is produced by pheromone glands and is under the control of a neurohormone. In this report we also demonstrate that the braincomplex extracts stimulate the total level of ~4C incorporation from sodium acetate by acting directly on the pheromone gland preparations in vitro. This provides evidence that pheromone glands are capable of de novo synthesis of hexane extractable products which are subject to the dose-dependent stimulatory influence of brain-complex extracts. Since acetate is involved in fatty acid biosynthesis, being the general precursor of acetyl CoA, the findings that incorporation of radioactivity from acetate is stimulated by brain-complex extracts have some implications regarding the regulation of fatty acid biosynthesis in the pheromone gland. It is interesting to note that the hormonal stimulation is only observed when the experiments were performed under dim red light illumination. It may be that pheromone gland preparations lose their responsiveness to hormonal stimulation in vitro when exposed to light. On the other hand the induced hexane extractable products may undergo chemical degradation at higher light intensities due to photo-oxidation. The in vitro hormonal response observed in this study revealed the stimulation of products showing the same mobility as (Z)-I 1 hexadecenal, as observed by TLC and HPLC analysis. Using similar TLC and HPLC separation techniques Ando et al. (1988) showed incorporation of topically applied ~4Clabeled precursors into the pheromone fraction of the silkworm moth. The percentage incorporation levels into the pheromone fraction in our study amounted to 16% in control incubations and 37% in stimulated incubations. These levels are considerably higher than the levels obtained with topically applied precursors (Ando et al., 1988), in which optimum conditions resulted in 1.5%. Therefore the in vitro study reported here provides sufficient levels of incorporation for further biosynthetic studies. In addition, the present study showed a stimulation of the production of fractions showing the same mobility as (Z)-I 1 hexadecenol, as observed by TLC analysis. The hypothetical pathway of aldehyde pheromone components from the fatty acyl moieties has been proposed to be via the oxidation of the corresponding alcohol (Morse and Meighen, 1986). This may explain the presence of (Z)-I 1 hexedecenol in the hexane extractable products resulting from brain-complex stimulation. In the HPLC separations a peak was observed which corresponded to the mobility of (Z)-9 hexadecenoic acid. The HPLC

conditions in this study do not discriminate between isomers or slight changes in the position of double bonds such that (Z)-9 hexadecenoic acid would elute together with (Z)-I 1 hexadecenoic acid [(Z)-I 1 hexadecenoic acid was unavailable to us]. It is thus clearly possible that the observed radioactive compound, produced as a result of stimulation by female braincomplex extracts, represents the complementary fatty acid moieties of (Z)-ll hexadecenal and/or (Z)-9 hexadecenal, that is, (Z)-I 1 hexadecenoic acid and/or (Z)-9 hexadecenoic acid. The presence of similar complementary fatty acid moieties of the pheromone components have been demonstrated previously in other lepidopterans (Bjostad and Roelofs, 1984a, b; Ando et al., 1988). We are currently investigating the chemical identity of all the radioactive products produced by brain-extract stimulation. Acknowledgements--The research was funded by a BARD

Grant No. US 784-84. We thank R. Ben-Zaken for her dedicated attention to our Heliothis armigera insect culture and her technical assistance.

REFERENCES

Ando T., Hase T., Funayoshi A., Arima R. and Uchiyama M. (1988) Sex pheromone biosynthesis from 14C-hexadecanoic acid in the silkworm moth. Agric. Biol. Chem. 52, 141-147. Bjostad L. B. and Roelofs W. L. (1984a) Sex pheromone biosynthetic precursors in Bombyx mori. Insect Biochem. 14, 275-278. Bjostad L. B. and Roelofs W. L. (1984b) Biosynthesis of sex pheromone components and glycerolipid precursors from sodium [l-~4C]acetate in redbanded lcafroUer moth. J. chem. Ecol. 10, 681-691. Dunkelblum E., (3othilf S. and Kehat M. (1980) Identification of the sex pheromone of the cotton bollworm Heliothis armigera, in Israel. Phytoparasitica 8, 209-211. Jones I. F. and Berger R. S. (1978) Incorporation of(l-14C) acetate into cis-7-dodecen-1-ol acetate, a sex pheromone in the Cabbage looper (Trichoplusia hi). Envir. Ent. 7, 666-669. Ohguchi Y., Tatsuki S., Usui K., Arai K., Kurihara M., Uchiumi K. and Fukami J. (1985) Hormone-like substance present in the cephalic organs of the female moth, Chilo suppresalis (Walker) (Lepidoptera: Pyralidae) and controlling sex pheromone production. Jap. J. appl. Ent. Zool. 29, 265-269. Percy J. E., Gardiner E. J. and Weatherston J. (1971) Studies of physiologically active arthropod secretions. VI. Evidence for a sex pheromone in female Ortgyia leucostigma (Lepidoptera: Lymantriidae). Can. Ent. 103, 706--712. Rafaeli A. and Soroker V. (1989) Influence of diel rhythm and brain hormone on pheromone production in two Lepidopteran species. J. chem. Ecol. In press. Raina A. K. and Klun J. A. (1984) Brain factor control of sex pheromone production in the female corn earworm moth. Science 225, 531-533. Srinvasan A., Coffelt J. A., Norman P. and Williams B. (1986) Sex pheromone gland of the navel orangeworm, Amyelois transitella (Lepidoptera: Pyralidae). Location, bioassay and in vitro maintenance. Fla Ent. 69, 169-174. White M. R., Amborski R. L., Hammond A. M. Jr and Amborski (3. F. (1972) Organ culture of the terminal abdominal segment of an adult female Lepidopteron. In Vitro 8, 30-36.