Insect Biochem., Vol. 8, pp. 389 to 391 0020-1700/78/1001--0389502.00/0 © Pergamon Press Ltd. 1978 Printed in Great Britain

VOLATILE CONSTITUENTS OF THE TARNISHED PLANT BUG*, ~',

R. C. GUELDNERI § and W. L. PARROTT 2

1Chemistry Research and 2Host Plant Resistance Research, Boll Weevil Research Laboratory, Sci. Educ. Admin., USDA, Mississippi state, MS 39762, U.S.A.

(Received 17 November 1977)

Abstract--Analyses of the steam-distillable oils of male and female tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois), with an integrated gas chromatography-mass spectrometry system showed that most of the compounds were esters. Hexyl butyrate and (E)-2-hexenyl butyrate were the major constituents and comprised 60--90To of the total oil. (E)-2-Hexenyl butyrate is present in larger quantities in females than in males.

Key Word Index: Tarnished plant bugs, esters, scent gland, sex pheromone

I N T R O D U C T I O N

THE TARNISHED plant bug, Lygus lineolaris (Palisot de Beauvois), damages cot ton by feeding on growing terminals and fruiting buds (SCALES and FURR, 1968). I f insecticides are used to control the plant bugs, the parasites and predators of a second economic cotton pest, the tobacco budworm, Heliothis virescens (Fabricius), are killed. Therefore, methods o f control that do not require the use of pesticides are desirable,. and methods are needed for monitoring the build up of populations of tarnished plant bugs.

Female tarnished plant bugs are attractive to male bugs in the field (SCALES, 1968). This attraction may he due to the volatile compounds present in the scent glands. For example, a number of species o f t h e superfamilies, Coreoidea and Penta tomoidea (WATERHOUSE et aL, 1961; WATERHOUSE and GILBY, 1964; BLUM e ta / . , 1961; GILBY and WATERHOUSE, 1965; CHOUDHURI et al., 1970) were found to contain alcohols, aldehydes, acids, esters, ketones, and hydrocarbons, with unbranched chains (sometimes unsaturated) of two to thirteen carbon atoms. Our objective in the present study was to identify the source of this attraction in the tarnished plant bug so that it could be used as a survey tool to determine population levels. We presumed that the attractant would he composed of volatile organic substances.

M A T E R I A L S AND M E T H O D S

Collection of insects

The adults used in the study were reared continuously on a diet of green beans, Phaseolus vulgaris L. (PARROrT et aL, 1975). The laboratory culture was maintained at a

* Lygus lineolaris (Palisot de Beauvois). ~'In cooperation with Mississippi Agricultural and

Forestry Experiment Station, Mississippi State, MS 39762. Received for publication.

:[: Mention of a proprietary product in this paper does not constitute an endorsement of this product by the USDA.

§Present address: Southern Grain Insects Research Laboratory, Science and Education Administration, USDA, Tifton, GA 31794.

temperature of 27°C and 50% r.h. and a photophase of 13.5 hr. The stock culture was derived from insects collected in October 1969 in Oktibbeha County, Mississippi. The average weights of males and females were 5.93 mg and 6.05 rag, respectively, based on the total weight of 100 insects of each seX.

The insects were anaesthetized with carbon dioxide or frozen and then sexed, counted, and stored in pentane or dichloromethane at - 20°C until the analyses.

Isolation of volatile oils

The stored insects and the solvent in which they were stored were steam distilled to remove the volatile components. The aqueous distillate was extracted five times with pentane or dichloromethane. The extractions were dried over anhydrous sodium sulphate and concentrated in vacuo to a small volume (5-10 ml) that was further concentrated under a stream of nitrogen.

Analysis by gas chromatography-mass

Spectrometry (GC-MS). The total oils and fractions of the oils eluted sequentially from a Florisil chromatographic column with pentane and pentane diethyl ether (95:5, 90:10, 80:20, all v/v) were introduced into an integrated gas chromatograph-mass spectrometer (Hewlett-Packard Model 5930A). Gas chromatographic profiles were obtained with a Hewlett-Packard Model 5711A gas chromatograph and a 76.2-m × 0.762-mm i.d. stainless steel capillary tube prepared and coated with OV-17 according to the procedure of MON (1971). The same column was used to introduce the samples into the mass spectrometer.

The GC profiles were obtained with a flame ionization detector (FID) at a nitrogen flow through the capillary column of 5 ml/min. After injection of the sample, the column was held at 80°C for 8 rain, programmed from 80-180°C at 4°C/rain, and then held at 180°C until the termination of the GC run. The same temperature programme and flow rate of the helium was used during mass spectral andlysis. Relative concentrations of the components were obtained by measuring peak height x retention time and normalizing to 100% (GRANT and CLAar,.E, 1971). Fragment ion values were compared with mass spectral data from CORNU and MASSOT (1966), McLAr~RTV (1966) and ST~r, n4AGi~N et al. (1969).

R E S U L T S AND D I S C U S S I O N

Those compounds found in the tarnished plant bug by mass spectrometry are listed in Table 1. Some that

389

390 R. C. GUELDNER AND W. L. PARROTT

Table 1. Volatile compounds in the tarnished plant bug and mass spectral evidence

Molecular Masses of fragment ions in order Compound name weight of abundance

Esters *Ethyl butyrate

*Butyl acetate

*(E)-2-Hexenyl acetate

Ethyl-2-hexenoate

Butyl butyrate

*Ethyl hexanoate

*Hexyl acetate

Hexenyl butyrate }

*Hexyl butyrate

*Ethyl myristate

Other

(E)-2-Hexenal

(Z)-3-Hexen-l-ol

Phenyl acetaldehyde

Indole

Benzothiazole

Usually 60-80~o of the total oil

C ~ s alcohol

116

116

142

142

144

144

144

170

172

256

98

100

120

117

135

236

236

222

43,29,27,71,41,42,39,28,88,60

43,56,27,73,29,39,42,28,55,44

43A1,67,39,27,55,29,82,57,54

55,29,27,41,39,97,99,73,68,42

43,41,71,56,27,89,42,29, 55,84

43,29,27,41,88,28,42,60,39,55

43,56,41,42,55,27,29,61,28,39

43,71,41,55,27,67,39,29,54,82

43,4131,27,56,89,29,42,55,39

43,41,57,29,55,28,88,27,71,69

41,29,39,27,55,42,69,57,43,84

43,41,67,27,39,28,55,29,82,57

91,28,39,65,29,41,92,43,63,51

117,90,89,88,63,28,58,39,118,116

135,108,69,45,63,82,49,39,38,37

43,57,41,29,28,39,55,165,27,180

43,41,28,57,29,55,27,39,69,67

41,43,55,57,29,28,69,82,27,39

* Confirmed by mass spectrum of authentic sample.

were tentatively identified (marked with an asterisk) were confined by obtaining spectra for authentic compounds. Esters were qualitatively ~ and quantitatively the predominant class of compounds. All esters had even-numbered unbranched alcohols and acids.

When the males and females in a sample of bugs held, for three days on fresh green beans were compared, differences between sexes were minimum for any given date except for hexyl and hexenyl butyrate. However, other samples gave different gas chromatographic profiles, a variability that appears to be a characteristic of this culture of tarnished plant bugs and also of Oncopeltusfasciatus (Dallas) (LENER, 1967, 1969). Although storage and handling may affect the gas chromatographic profile, the variability was present even when tarnished plant bugs (chilled for 5 min) were put directly into hot water (nearly boiling) in the steam distillation apparatus to minimize continuing enzymatic action. (Once the volatile oils were isolated from the bug carcases, they were stable and gave the same profile for at least six weeks.) As an example of the variability experienced in

one sample, butyl butyrate comprised about 400/0 of the total oil; in another, none was detected.

The amount of hexenyl butyrate in the male was always less than the amount in the female by a factor of five to twenty. This variation results from the changing amount in the female. In the male, the ratio of hexenyl butyrate to hexyl butyrate (the major component for males) remained constant at about l:10. In females the major component fluctuated between hexenyl butyrate and hexyl butyrate, but the ratio was about 1:1.

Excision of the thorax (containing the scent gland) showed it to be the richest source of compounds. Extracts from the head showed only traces of Compounds, and extracts from the abdomens showed a slightly higher concentration. However, the similarity between the gas chromatographic profiles of the abdomen and thorax indicated that much of the oil obtained from extraction of the abdomen was caused by contamination during excision of the thorax. These results, plus the fact that nymphs (mixed sexes) of the tarnished plant bug do not contain either hexyl or hexenyl butyrate at the fifth (last) instar, lend support

Volatile esters of plant bugs 391

to the suggestion of GUPTA (1961) that the scent glands of Heteroptera (Hemiptera) are used for defence by the nymphs and for both defence and sexual identification by the adults. However, in the nymphal stage, the scent glands do not always contain significant amounts of volatile compounds.

The esters found in the tarnished plant bug may not be accumulated from the food source since green beans, when steam-distilled, yield fifty times less oil than do the bugs on a weight per weight basis, and the infra red spectrum of the oil showed no ester carbonyl. It seems likely that the esters are synthesized de novo, and the even number of the carbon chains indicates biosynthesis from acetate.

One of the giant water bugs, Lethocerus indicus (LePeletier and Serville) was shown by BUTENANDT and TAM (1957) to contain (E)-2-hexenyl acetate and by DEVAKUL and MAARSE (1964) tO contain (E)-2- hexenyl butyrate in addition. Also, the scent gland of this species is much larger in the male than in the female. In Oncopeltus fasciatus (Dallas), the tubular glands of the scent gland complex are larger in the male than in the female (GAMES and STADDON, 1973), and the sexual dimorphism is expressed chemically: the scent released onto the peritreme consists of 10-30% acetates in the male, but the scent released on the peritreme by the female is made up almost entirely of aldehydes. The case of the tarnished plant bug may be similar, that is, the sexual dimorphism may be expressed by the relative amounts of hexenyl butyrate contained in the male and female. Hexenyl butyrate seems likely to be an important component of the sex pheromone.

No bioassay data have been obtained for Hemiptera that show a particular compound or combination of compounds is a sex pheronome. We also were unsuccessful in preliminary trials in demonstrating sex pheromone activity for a few of the compounds identified from the insect.

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