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Control of the biosynthetic pathway of
Sesamia nonagrioides
sex
pheromone by the pheromone biosynthesis activating neuropeptide
Esther Mas, Joan Llo`ria, Carme Quero, Francisco Camps, Gemma Fabria`s
*Department of Biological Organic Chemistry (IIQAB-CSIC), Jordi Girona, 18. 08034-Barcelona, Spain
Received 22 June 1999; received in revised form 28 December 1999; accepted 4 January 2000
Abstract
(Z)-11-Hexadecenyl acetate, the main pheromone component of Sesamia nonagrioides sex pheromone, is biosynthesized from palmitic acid by D11-desaturation followed by reduction and acetylation. Production of (Z)-11-hexadecenyl acetate is regulated by
the Pheromone Biosynthesis Activating Neuropeptide (PBAN). Transformation of (Z)-11-hexadecen-1-ol into the corresponding acetate is a target step for PBAN in the regulation of this biosynthetic sequence, thus being the first example of a PBAN-activated acetylation. The production of the minor component (Z)-11-hexadecenal is also stimulated by PBAN. The usefulness of pentafluoro-benzyloxime-derivatives for the analysis of aldehyde pheromone constituents by gas chromatography coupled to mass sepctrometry is also reported. 2000 Elsevier Science Ltd. All rights reserved.
Keywords:Sex pheromone biosynthesis; Acetylation; Aldehyde analysis; Moth; Neurohormone
1. Introduction
Biosynthesis of lepidopteran sex pheromones occurs from fatty acids by the combined action of desaturases, chain-shortening enzymes, reductases, acetyltransferases and oxidases (Roelofs and Bjostad, 1984). Whereas chain-shortening enzymes and desaturases are involved in modification of length and degree of unsaturation of the alkyl chain, respectively, reductases, acetyltransfer-ases and oxidacetyltransfer-ases are responsible for the type of func-tionality of the pheromone component.
Sex pheromone biosynthesis in many moth species is regulated by PBAN (Raina, 1993), a peptide hormone produced in the sub-esophageal ganglion complex. This
Abbreviations:GC–MS, gas chromatography coupled to mass trometry; SCAN–GC–MS, gas chromatography coupled to mass spec-trometry under SCAN; Z11-16:AL, (Z)-11-hexadecenal; Z11-16:OL, (Z)-11-hexadecen-1-ol; Z11-16:OAc, (Z)-11-hexadecenyl acetate; 16:Acid, hexadecanoic acid; d316:Acid, (16,16,16-2H3) hexadecanoic
acid; d9Z11-16:Acid, (13,13,14,14,15,15,16,16,16-2H9)
(Z)-11-hexade-cenoic acid; d3Z11-16:OAc, (16,16,16-2H3) (Z)-11-hexadecenyl
acet-ate; d9Z11-16:OAc, (13,13,14,14,15,15,16,16,16-2H9)
(Z)-11-hexade-cenyl acetate; PBAN, pheromone biosynthesis activating neuropeptide; SIM, selected ion monitoring; 13:OAc, tridecyl acetate.
* Corresponding author: Tel.:+34-3-4006100; fax:+34-3-2045904.
E-mail address:[email protected] (G. Fabria`s).
0965-1748/00/$ - see front matter2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 0 0 8 - 4
hormone has been isolated and sequenced from brains of Helicoverpa zea (Raina et al., 1989), Bombyx mori
(Kitamura et al. 1989, 1990) and Lymantria dispar
(Masler et al., 1994), and its sequence has been deduced from the encoding cDNA in Agrotis ipsilon(Duportets et al., 1998),H. assulta(Choi et al., 1998) andMamestra brassicae(Jacquin-Joly et al., 1998). In all these species, PBAN is a 33 or 34 amino acid peptide with an amidated C-terminus, in which the pheromonotropic activity lies in the C-terminal pentapeptide fragment FSPLR-NH2
(Raina and Kempe, 1990). Other peptides sharing this C-terminal sequence and thereby exhibiting pheromono-tropic activity have also been isolated fromPseudaletia separata (Matsumoto et al. 1990, 1992),B. mori (Imai et al., 1991) and cockroaches (Schoofs et al., 1993), in which they regulate coloration in phase polymorphism, induction of embryonic diapause and muscle contrac-tion, respectively.
Thaume-topoea pityocampa (Fabrias et al., 1995) and Manduca sexta (Fang et al., 1995) PBAN appears to act on the reduction of fatty acyl moieties.
The main component ofSesamia nonagrioidesfemale sex pheromone is (Z)-11-hexadecenyl acetate (Z11-16:OAc), with minor amounts of the corresponding alco-hol, (Z)-11-hexadecen-1-ol (Z11-16:OL) and aldehyde, (Z)-11-hexadecenal (Z11-16:AL) in 92:5:3 ratio (Sreng et al., 1985). Mazomenos also reported that dodecyl acetate was present in the pheromone gland extracts (Mazomenos, 1989). From these structures, we antici-pated that Z11-16:OAc would arise from D11
-desatu-ration of palmitic acid (16:Acid) followed by further reduction and acetylation. In this article we confirm this proposed biosynthetic sequence and provide data indi-cating that PBAN regulates this biosynthetic pathway with probable activation of the final acetylation step.
2. Experimental procedures
2.1. Insects
S. nonagrioides were supplied by the Centre UdL-IRTA, Lleida (Spain) as pupae. Pupae were sexed and kept under a 16-h light/8-h dark photoperiod at 25°C. The adults were provided with a 10% sucrose solution. Only virgin females (2-days old) were used in the experiments.
2.2. Chemicals
Bom-PBAN was obtained from Peninsula laboratories (Belmont, CA). Dimethylsulfoxide was obtained from Sigma (St Louis, MO) and pentafluorobenzylhydroxyl-amine hydrochloride was purchased from Aldrich (Milwaukee, WI). (16,16,16-2H
3) Hexadecanoic acid
(d316:Acid) was supplied by IC Chemikalien (Munich,
Federal Republic of Germany) and it was 99% pure. (13,13,14,14,15,15,16,16,16-2H
9) (Z)-11-Hexadecenoic
acid (d9Z11-16:Acid) was synthesized in our laboratory
following reported procedures (Navarro et al., 1997) and it was 99% pure (gas chromatography analysis of the methyl ester).
2.3. Treatments
To monitor the effect of PBAN on pheromone pro-duction, 3 h before the begining of darkness, insects were injected into the abdomen with 20µl of Meyer and Miller’s saline containing the amounts of PBAN indi-cated. Pheromone glands were dissected two hours after PBAN injection and the pheromone gland was extracted as described in the following section.
In the deuterium-labeling experiments, 4 h before the onset of the scotophase females were immobilized under
netting and putative precursors were topically applied to the gland as dimethylsulfoxide solutions (0.1 µl, 10
µg/µl). Controls received 0.1 µl of dimethylsulfoxide. One hour after the application, insects were freed and 20 µl of a PBAN solution (2.5 pmol/µl) in Meyer and Miller’s saline (Martinez and Camps, 1988) were injected into the abdomen. Controls were injected with 20 µl of saline. Pheromone glands were excised two hours after PBAN injection and the pheromone blend was extracted as described below.
2.4. Pheromone gland extraction
Unless otherwise stated, individual pheromone glands were extracted with a 30 µl hexane solution of tridecyl acetate (13:OAc, 1 ng/µl) for 1 h at 25°C.
2.5. Aldehyde derivatization
Groups of 5–7 pheromone glands were extracted with 100 µl of hexane. After 1 h at 25°C, hexane extracts were carefully evaporated almost to dryness under a gentle stream of nitrogen and the resulting residue was treated with a solution of pentafluorobenzylhydroxyl amine hydrochloride (5 µl, 1 mg/ml) in methanol. After 1.5 h at 25°C, the whole sample was injected into the chromatograph. Derivatization reactions with synthetic Z11-16:Al showed that more than 90% conversions were achieved with 1–50 ng samples using this protocol, as determined by gas chromatography coupled to mass spectrometry (GC–MS) analysis of samples using hex-adecane as internal standard.
2.6. Analytical methods
To determine the effect of PBAN on pheromone pro-duction, the extracts were analyzed by capillary gas chromatography using a Fisons gas chromatograph (8000 series) equipped with a non-polar Hewlett Packard HP-1 capillary column (30 m×0.20 mm I.D.) The fol-lowing temperature program was used: from 100 to 220°C at 5°C/min and then to 260°C at 10°C/min after an initial delay of 1 min.
Pentafluorobenzyloxime-derivatives were analyzed by GC–MS using a Fisons gas chromatograph (8000 series) coupled to a Fisons MD-800 mass selective detector. The system was equipped with a non-polar Hewlett Packard HP-1 capillary column (30 m×0.20 mm I.D.) using the following temperature program: from 100 to 250°C at 5°C/min and then to 280°C at 10°C/min after an initial delay of 2 min. Analyses were conducted under the SCAN mode. The same procedure was used to ana-lyze Z11-16:AL in pheromone extracts.
monitoring (SIM) mode. The ions selected were: 13:OAc, 168 (M-60); Z11-16:OAc, 222 (M-60); (16,16,16-2H
3) (Z)-11-hexadecenyl acetate (d3
Z11-16:OAc), 225 (M-60); (13,13,14,14,15,15,16,16,16-2H 9)
(Z)-11-hexadecenyl acetate (d9Z11-16:OAc), 231
(M-60).
3. Results and discussion
Pheromone titers of S. nonagrioides fluctuate diur-nally with the highest titers occurring at the sixth hour of the scotophase (Babilis and Mazomenos, 1992). No pheromone is produced during the photophase (Babilis and Mazomenos, 1992). This photoperiodicity of phero-mone production is probably controlled by a brain factor similar to PBAN. In agreement with this assumption, PBAN injection into whole S. nonagrioides females stimulates sex pheromone production in the photophase. Furthermore, as shown in Fig. 1, this effect was dose-dependent. The minimum dose tested that elicited a sig-nificant response was 12.5 pmol. Although maximum stimulation was achieved with 200 pmols/gland, there was not statistical difference in the amounts of phero-mones produced with 50, 100 and 200 pmol. A PBAN dose of 100 pmol/insect was considered suitable for further experiments.
To determine the effects of PBAN on the production of Z11-16:AL, 12–15 pheromone glands from insects that had been injected with either saline or PBAN in the photophase were pooled and the GC–MS analysis of these extracts were performed under SCAN (SCAN– GC–MS). Whereas no Z11-16:AL was detected in extracts from insects injected with saline, both the reten-tion time and mass spectrum of putative Z11-16:AL present in extracts from PBAN-injected females were identical to those of a synthetic sample of Z11-16:AL
Fig. 1. Dose-dependence of PBAN-induction of sex pheromone pro-duction inS. nonagrioidesfemales. The doses of PBAN injected into whole females in the photophase are given in the abcissa and the amounts of Z11-16:OAc extracted are depicted in the ordinate. Data are mean±SEM of seven individual replicates. Different letters denote statistical difference atP,0.05.
(data not shown). The identity of Z11-16:AL was also confirmed by SCAN–GC–MS analysis of samples obtained from pools of 5–7 glands derivatized with pen-tafluorobenzyl hydroxylamine hydrochloride (van Kuijk et al., 1986). In our search for aldehyde derivatives hav-ing characteristic and intense fragments for the efficient and reliable identification of pheromone aldehydes, we found that the pentafluorobenzyl oximes (van Kuijk et al., 1986) were very suitable, since they are easily pre-pared and they give abundant and characteristic frag-ments in their mass spectra. These fragfrag-ments arise from cleavage at the benzylic position to give both, the tropil-ium ion (m/z181, base peak) and ions corresponding to R–CH=N–O+ and R–CH=N+. These last fragments are of diagnostic value for structural characterization of the parent aldehyde, R–CHO. As shown in Fig. 2, the mass spectra of both natural and synthetic materials were identical. Diagnostic ions are those atm/z252 and 236, corresponding to fragments C4H9–CH=CH–(CH2)9–
CH=N–O+ and C4H9–CH=CH–(CH2)9–CH=N+,
respect-ively.
The last objective of this work was to investigate the effect of PBAN on the sex pheromone biosynthetic
Fig. 3. Proposed biosynthetic pathway ofS. nonagrioidessex phero-mone and step activated by PBAN.
way of S. nonagrioides sex pheromone. As previously reported in other species (Jacquin et al., 1994; Jurenka et al., 1991), the mass labeling experiments reported in this article show that Z11-16:OAc, the main component of S. nonagrioides sex pheromone, is biosynthesized from palmitic acid by D11-desaturation followed by
reduction and acetylation (Fig. 3). To investigate the step(s) activated by PBAN in this species, incorporation of label from a series of tracers into Z11-16:OAc was monitored in the photophase. When d316:Acid was
administered, labeled Z11-16:OAc was only detected in females that had been injected with PBAN (Table 1). Similar results were also found in treatments with d9
Z11-16:Acid and d9Z11-16:OL, which gave rise to labeled
d9Z11-16:OAc only in females treated with PBAN. This
last result is in contrast with those reported in other spec-ies (Bestmann et al., 1987; Dunkelblum et al., 1989; Jur-enka and Roelofs, 1989; Teal and Tumlinson, 1987; Zhao et al., 1995), in which alcohol acetylation is rather unspecific and occurs throughout the photoperiodic cycle. Additionally, this result indicates that inS. nonag-rioides, PBAN stimulation of the biosynthesis occurs, at least in part, by activation of the acetyl transferase. This is the first example of activation of acetyl transferase by PBAN in a sex pheromone biosynthetic pathway. In the other species so far investigated, PBAN appears to act on a step prior to fatty acid synthesis (Jurenka et al., 1991; Tang et al., 1989) or by activation of the reduction
Table 1
Effect of PBAN on the incorporation of labeled precursors into Z11-16:OAc
Z11-16:OAc (relative amounts/gland)a
Natural Labeled
Tracer Saline PBAN Saline PBAN
d316:Acid ,1 106.6±31.5 n.d. 8.2±1.8
d9Z11-16:Acid ,1 86.1±30.3 n.d. 6.4±1.7
d9Z11-16:OL ,1 100.2±26.6 n.d. 9.7±3.6
aRelative amounts of Z11-16:OAc were determined in the GC–MS
chromatograms from the ratios between ions 222/182, 225/182 and 231/182, where ions 222, 225, 231 and 182 correspond to natural Z11-16:OAc, d3Z11-16:OAc, d9Z11-16:OAc and tridecyl acetate,
respect-ively. Results are expressed as means±S.E. with 7–9 individual repli-cates. Saline, saline-injected females; PBAN, PBAN-injected females; n.d., not detected.
of the last acyl precursor (Fabrias et al., 1994; Fang et al., 1995; Ozawa et al., 1993).
Although mass-labeling experiments were not perfor-med, it is reasonable to assume that Z11-16:AL is biosynthesized from Z11-16:Acid, either by direct reduction of Z11-16:Acid or by oxidation of Z11-16:OL. As for the first possibility, fatty acyl reductases that give rise to aldehydes have been reported in plants (Vioque and Kolattukudy, 1997), although such enzymes are heretofore unprecedented in insect pheromone glands. Several articles have reported on the biosynthesis of pheromone aldehydes from the corresponding alcohols (Morse and Meighen, 1984; Teal and Tumlinson, 1987), or even from the acetates (Morse and Meighen, 1984). Therefore, in the light of the existing literature, the biosynthesis of Z11-16:AL by oxidation of Z11-16:OH seems more plausible. This assumption is currently under investigation in our laboratories using the pen-tafluorobenzyloxime-derivative.
Acknowledgements
We thank CAICYT (grant AGF98-0844), EC (grant FAIR CT-96-1302), Generalitat de Catalunya (grant 1997SGR-00021) for financial support and SEDQ S.A. for a pre-doctoral fellowship to E.M.
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