Pheromone biosynthetic pathways in the moths Helicoverpa zea and Helicoverpa assulta

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Abstract

Sex pheromones of many Lepidopteran species have relatively simple structures consisting of a hydrocarbon chain with a functional group and usually one to several double bonds. The sex pheromones are usually derived from fatty acids through a specific biosynthetic pathway. We investigated the incorporation of deuterium-labeled palmitic and stearic acid precursors into pheromone components of Helicoverpa zea and Helicoverpa assulta. The major pheromone component for H. zea is (Z)11-hexadecenal (Z11-16:Ald) while H. assulta utilizes (Z)9-hexadecenal (Z9-16:Ald). We found that H. zea uses palmitic acid to form Z11-16:Ald via Δ11 desaturation and reduction, but also requires stearic acid to biosynthesize the minor pheromone components Z9-16:Ald and Z7-16:Ald. The Z9-16:Ald is produced by Δ11 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. The Z7-16:Ald is produced by △9 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. H. assulta uses palmitic acid as a substrate to form Z9-16:Ald, Z11-16:Ald and 16:Ald. The amount of labeling indicated that the Δ9 desaturase is the major desaturase present in the pheromone gland cells of H. assulta; whereas, the △11 desaturase is the major desaturase in pheromone glands of H. zea. It also appears that H. assulta lacks chain-shortening enzymes since stearic acid did not label any of the 16-carbon aldehydes.

Introduction

Sex pheromones of many Lepidopteran species are acyclic, fatty acid-derived compounds, 12–18 carbons in chain length with an oxygenated functional group and one to three double bonds. Specific enzymes found in pheromone glands of female moths are involved in key steps of the pheromone biosynthetic pathway. The pathway generally includes fatty acid synthetases, desaturases, limited chain-shortening enzymes, and functional modification of the carbonyl carbon to make species-specific pheromone blends. Biosynthetic pathways in several moth species, since the first work in Argyrotaenia velutinana (Bjostad and Roelofs, 1981), have been studied and reviewed (Roelofs and Bjostad, 1984, Bjostad et al., 1987, Morse and Meighen, 1987, Wolf and Roelofs, 1989, Jurenka and Roelofs, 1993, Tillman et al., 1999). Specific desaturases in lepidopteran species play a predominant role in producing unsaturated intermediates from saturated fatty acids of various chain lengths.

In the Helicoverpa and Heliothis genera all species investigated so far use (Z)11-hexadecenal (Z11-16:Ald) as their major sex pheromone component, except Helicoverpa assulta which utilizes (Z)9-hexadecenal (Z9-16:Ald) as the major sex pheromone component (Arn et al., 2001). A minor component found in the oriental tobacco budworm, H. assulta, is Z11-16:Ald (Sugie et al., 1991, Cork et al., 1992). The minor components of H. zea include (Z)7-hexadecenal (Z7-16:Ald), Z9-16:Ald, and hexadecenal (16:Ald) and are found at less than 10% of the level of the major component (Klun et al., 1980). The major and minor components are most likely made from precursors of 16- or 18-carbon saturated fatty acids and have similar biosynthetic steps to formation of the Z11-16:Ald as shown in H. zea (Teal and Tumlinson, 1986, Jurenka et al., 1991).

The biosynthesis of all components has been confirmed to be under regulation by a pheromone biosynthesis activating neuropeptide (PBAN) (Raina and Klun, 1984, Choi et al., 1998). PBAN is produced in the brain–subesophageal ganglion complex (Br-SEG), and can be released into the hemolymph to stimulate the pheromone gland to produce pheromone. The first neuropeptide was determined to be a 33-amino acid peptide in H. zea (Raina et al., 1989), and additionally identified and characterized in other moths (Tillman et al., 1999). Physiological action of PBAN on pheromone biosynthesis in several moths has been proposed to involve an enzymatic step or steps in or prior to fatty acid biosynthesis (Soroker and Rafaeli, 1989, Tang et al., 1989, Jurenka et al., 1991, Jacquin et al., 1994). In other moths, PBAN apparently is regulating the process of acyl-group reduction (Ozawa et al., 1993, Fabriàs et al., 1994, Fang et al., 1995). Recently it was shown that PBAN stimulation of the pheromone biosynthesis occurred, in part at least, by activation of the acetyl transferase in Sesamia nonagrioides (Mas et al., 2000).

In the present study, we determined which precursor fatty acid is utilized to produce major and minor pheromone components in pheromone gland cells of H. assulta and H. zea by monitoring labeled precursor incorporation. We were especially interested in determining how the Z9-16:Ald was formed in H. assulta. This compound could be made through a Δ9 desaturase acting on palmitic acid (16:acid) or through a Δ11 desaturase acting on stearic acid (18:acid) and then chain-shortened to palmitoleic acid (Z9-16:acid). We were also interested in determining how the minor components of both species were made and how PBAN controls pheromone biosynthesis.

Section snippets

Insects

Larvae of H. assulta and H. zea were reared on an artificial diet at 25±1 °C under a light:dark cycle (L/D), 16:8. Pupae were sexed and allowed to emerge separately. A sucrose solution (2%) was provided to adults. Virgin female adults in their third or fourth photophase were used throughout this study.

Materials

Deuterium labeled fatty acids used included: (16,16,16-D3)-palmitic acid (D3-16:Acid) (ICON Services Inc., NJ) and (18,18,18-D3)-stearic acid (D3-18:Acid) (CDN Isotopes Inc., Pointe-Claire,

Results

Sex pheromone titers in both moths were significantly increased above the control values after injection of PBAN during the photophase (Fig. 2, Fig. 3). Females produce little pheromone during the photophase in both species. Administration of PBAN shortly after the application of the deuterium-labeled precursors to the gland helped ensure incorporation into the pheromone.

Fig. 1 illustrates typical results found with the labeling studies. When D3-16:acid was utilized, the most abundantly labeled

Discussion

In the present study we investigated two moths, H. assulta and H. zea, to determine which precursor fatty acid is utilized to produce their pheromone components. The biosynthetic pathway of Lepidopteran insects generally includes fatty acid synthesis, desaturases, limited chain-shortening enzymes, and functional modification of the carbonyl carbon to make species-specific blends (Tillman et al., 1999). In H. zea the biosynthetic pathway of the major pheromone component Z11-16:Ald occurs through

Acknowledgements

This research was in part supported by Research Grant No. IS-2978-98R to R.J. from BARD, The United States–Israel Binational Agricultural Research and Development Fund, Grant No. 2001-35302-10882 to R.J. from CSAEES-USDA-NRI, and by the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, project 6578. This study was also supported in part by “The Brain Korea 21 Project” in Korea.

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