Journal of Chemical Ecology, Vol. 16, No. 6, 1990

ELECTROPHYSIOLOGICAL AND CHEMICAL ANALYSIS OF SEX PHEROMONE COMMUNICATION SYSTEM OF

THE MOTTLED UMBER, Erannis defoliaria (LEPIDOPTERA: GEOMETRIDAE)

B I L L S. H A N S S O N , l G A B O R S Z O C S , 2 F R A N K S C H M I D T , 3 W I T T K O

F R A N C K E , 3 C H R I S T E R L O F S T E D T , 1 and M I K L O S T O T H 2

t Department of Ecology Lund University

S-223 62 Lund, Sweden

2Research Institute for Plant Protection of the Hungarian Academy of Sciences Pf 102, H-1525 Budapest, Hungary

31nstitut fiir Organische Chemie der Universitdt Hamburg Martin-Luther-King-Platz 6

D-2000 Hamburg 13, Germany

(Received July 10, 1989; accepted October 20, 1989)

Abstraet--(Z,Z,Z)-3,6,9-nonadecatriene (Z3,Z6,Z9-19Hy) and (Z,Z)-3,9- cis-6,7-epoxy-nonadecadiene (Z3,Z9-cis-6,7-epo-19Hy) were identified in pheromone gland extracts from female Erannis defoliaria. The two compo- nents were found in a 1 : 3 ratio, with the main component, Z3, Z9-cis-6, 7- epo-19Hy present at an amount of about 1.5 ng per female. The components were identified by means of gas chromatography-mass spectrometry, gas chromatography-electroantennography and gas chromatography-single sen- sillum recordings. Single sensillum measurements on the male antenna showed two physiological types of sensitla. One type was characterized by a large spike amplitude cell responding to Z3,Z9-cis-6, 7-epoxy-19Hy and a small spike amplitude cell responding to Z3,Z6,Z9-19Hy. A second type responded only with a large spike amplitude cell to the epoxide, and this cell was inhib- ited by the triene. Of the two pheromone components, the epoxide gave the higher response in the EAG tests. Preliminary field tests support the identi- fication of the pheromone components. The epoxide was also found to be present in the extract of the pheromone gland of Colotois pennaria, and males of C. pennaria and Agriopis marginaria were trapped by the mixture of the identified compounds.

1887

0098-0331/90/0600-1887506,00/0 �9 1990 Plenum Publishing Corporation

1888 HANSSON ET AL.

Key Words--Erannis defoliaria (Z, Z, Z)-3, 6, 9-nonadecatriene, (Z, Z)-3,9- cis-6,7-epoxy-nonadecadiene, sex pheromone, identification, electroanten- nogram, single sensillum, peripheral inhibitor, Agriopis marginaria, Colo- tois pennaria, Lepidoptera, Geometridae.

INTRODUCTION

Adults of the mottled umber, Erannis defoliaria Clerck (Lepidoptera: Geome- tridae) are active at low temperatures in the late autumn. The female of this species, as in several other geometrids, is wingless. The female crawls up a tree trunk to emit the pheromone and attract conspecific males (Tvermyr, 1969). E. defoliaria is commonly found in most parts of Europe, where its larvae feed on foliage of almost any deciduous tree or shrub. It is particularly common on oak, birch, and hawthorn (Escherich, 1931), but sometimes attacks orchards, where it may cause considerable damage (Carter, 1982).

Sex attractants for male moths have been documented in numerous geo- metrids, but an actual pheromone has been described for only about 10 species (Am et al., 1986, and references therein). Straight-chain homoconjugated polyenic hydrocarbons with 17 or 19 carbon atoms, and epoxides derived from these, are the compounds most often identified as pheromone components in the Geometridae. Similar compounds have also been identified as pheromone components in Arctiidae and in a few genera of Noctuidae (see Arn et al., 1986).

We initiated the present work because E. defoliaria is regarded as an important pest in Northern and Middle Europe (Escherich, 1931; Kovacs, in Balas and Saringer, 1982), and also to expand the limited basic knowledge of geometrid pheromone communication systems.

METHODS AND MATERIALS

Insects. Larvae hatched from overwintering eggs laid by adults collected in Budapest, Hungary, were reared on cherry leaves to pupation. Before the flight period, pupae were carefully removed from the soil of the rearing con- tainers, sexed, and subsequently stored on moistened filter paper in Petri dishes until eclosion. Emerging adults were collected daily and transferred to l-liter glass jars, where they were supplied with water on cotton wool. During their whole life cycle, insects were kept under outdoor conditions. Insects used in electrophysiological studies in Lund were transferred to Sweden as pupae.

Extracts. Females were observed to establish the time of calling and the presumed optimal time for pheromone gland extraction. To collect the phero- mone, the terminal segments of the abdomen of calling unmated females were

PHEROMONE OF Erannis defoliaria 1889

excised and extracted in a minimal amount of redistilled hexane for about 10 min.

Gas Chromatography. Extracts were analyzed by gas chromatography (GC) on a Hewlett Packard 5880 gas chromatograph equipped with a flame ionization detector (FID). Hydrogen was used as cartier gas. Two to four microliters of the extract were injected on a capillary column. The samples were run on a nonpolar DB1 column (30 m x 0.25 mm ID) (J&W Scientific, Folsom, Cali- fornia 96830) at an initial temperature of 100~ for 2 rain, and a temperature program of 15 ~ C/rain up to 240~ and on a medium polarity DB wax column (30 m x 0.25 mm ID) (J&W) at an initial temperature of 80~ for 2 min, and a program of 10~ up to 230~ On both columns, the equivalent chain lengths compared to a homologous series of straight-chain acetates (8 :OAc- 17 : OAc) were established (Swoboda, 1962).

The amount of pheromone components present in the females was estab- lished by comparison to a reference of known concentration.

A HP 5830 GC equipped with an effluent split and a DB1 column allowed simultaneous FID and electroantennographic detection (FID-EAD) (Am et al., 1975) and simultaneous FID and single sensillum detection (FID-SC)(Wad- hams, 1982; Van der Pers and L6fstedt, 1983). Hydrogen was used as carrier gas, and the effluent split ratio was approximately 1 : 1. The outlet for the EAD was placed in a purified airstream flowing over the antennal preparation at a speed of 0.5 m/sec. The electrophysiological methods were identical to the ones described below.

Mass Spectrometry. For structural determinations a VG 250 S mass spec- trometer (70 eV) linked to a HP 5890 gas chromatograph was used. Gas chro- matographic separation was achieved on a fused silica DB5 column (30 m • 0.25 mm ID; J&W), maintained for 3 min at an initial temperature of 60~ and then heated to 300~ at a rate of 4~

Synthesis. (Z, Z, Z)-3,6, 9-Nonadecatriene was prepared from linolenic acid and a 10-fold excess of propanoic acid by a mixed Kolbe electrolysis according to known methods (Conner et al., 1980). The crude product was purified by preparative gas chromatography on a 4m x 6-mm-ID column packed with OV- 101 on Chromosorb W-AWDMCS at 220~ The overall chemical purity was >98%.

(Z,Z,Z)-3,6,9-Nonadecatriene was converted to a mixture of the three corresponding monoepoxydienes by oxidation with m-chloroperbenzoic acid (Wong et al., 1985). The three isomers could be separated with hexane-diethy- lether at a ratio of 97:3 on a DB5 column (Merck) (30 m • 0.25 mm ID) maintained at an initial temperature of 60~ for 3 rain and then heated to 300~ at a rate of 3 ~ per minute. (Z, Z)-3,9-cis-6, 7-epoxynonadecadiene proved to be the first eluted epoxide, followed by (Z,Z)-3,6-cis-9, 10-epoxynonadeca- diene and (Z,Z)-6, 9-cis-3,4-epoxynonadecadiene, respectively. NMR spectra

1890 HANSSON ET AL.

and mass spectra were in accordance with those of the homologous C17 epox- ydienes (Millar et al., 1987). The purity of the produced (Z, Z)-3, 9-cis-6, 7- epoxynonadecadiene was 98 % with respect to the other positional isomers.

Electrophysiology. Electrophysiological recordings were made on excised male antennae. An antenna was placed with the base in a pipet electrode filled with Beadle-Ephrussi Ringer, and grounded via an Ag-AgC1 wire. For elec- troantennographic recordings (EAG), the distal tip of the antenna was placed in contact with a second electrode, similar to the indifferent electrode. The tip electrode was connected to a high-impedance DC amplifier with automatic baseline drift compensation.

For single sensillum measurements, an olfactory sensillum was cut by means of two glass knives, and a recording electrode was placed in contact with the cut surface of the sensillum. The recording electrode was a glass pipet with a tip diameter of about 4 t~m, filled with Ringer, thickened Ringer (PVP), and finally coated with a layer of Vaseline to prevent evaporation. The recording electrode was connected to a high-impedance AC amplifier (Hansson et al., 1987). In stationary recordings, 10/~g of a synthetic compound was applied to filter paper with a disposable 5-ml plastic syringe. Two milliliters of the syringe atmosphere was injected into a purified and moistened airstream flowing over the antenna at a speed of about 0.5 m/sec.

Field Tests. Preliminary field tests were conducted using sticky traps (Am et al., 1979) and established methods (T6th et al., 1987).

RESULTS

Chemical Analysis. The extract from E. defoliaria females was first inves- tigated by simultaneous FID-EAD. Gas chromatographic separation showed two compounds that elicted clear responses from the male antenna (Figure 1). Two FID-SC analyses showed an inhibition of the spontaneous activity by the first EAD active component, and a strong stimulation by the second EAD active component (Figure 2). On a DB1 column, the equivalent acetate chain lengths of the first and second active components were 1477 and 1639, respectively. When compared to available synthetic substances, the retention time of the first component coincided exactly with that of Z3,Z6,Z9-19Hy, and the second component matched the retention time of (Z, Z)-6, 9-nonadecadien-3-one, a sex pheromone component of Peribatodes rhomboidaria Den. & Schiff. (Buser et al., 1985). When analyzed on a DB wax column, component I again matched the retention time of Z3, Z6, Z9-19Hy. However, the second component did not coincide with the retention time of the ketone on this column.

Combined GC-MS analyses of the two EAD active peaks showed that the mass spectrum of the first component (M § -- 262) exactly matched that of

PHEROMONE OF Erannis defoliaria ! 891

II

160 200 240 o C

I p I T I I i I I I I I I I I I I l t 5 6 7 8 9 10 11 12 13 14 i5 16 17 18 min

FIG. 1 GC-FID chromatogram from analysis of a female Erranis defoliaria pheromone gland extract and corresponding EAD response from an antenna of a conspecific male. Two peaks (I and II) show EAD activity.

A

11.02 11.04 11.0S

11,16 t1.18 11,20 rnirl

.•• t 1122 11.24 11.26 min

1 6 0 170 1 8 0 1 9 0 2 0 0 210 ~ ,,i,Jj~,,,,i ............ ~,,,,i,,,,~,~,,,;~,,,, ;~i, i::, ,~

10 11 12 13 14 15 min . . . . . . . . . . . . . . . . . . . . . .

F[~. 2. GC-FID chromatogram from analysis of a female pheromone gland extract (A), and simultaneously recorded single sensillum response from a male olfactory sensillum in Erannis defoliaria (B). The retention time of Z3, Z6, Z9-19Hy and the simultaneous onset of the stimulation of the single sensillum preparation is shown by a white star. The retention time of and stimulation by Z3, Z9-cis-6, 7-epo-19Hy is shown by a black star.

1892 HANSSON ET AL.

Z3,Z6,Z9-19Hy (Becker et al., 1983). The mass spectrum of the second com- pound (M + = 278; C19H34 O according to high-resolution GC-MS) showed a base peak at m/z = 67 and diagnostic fragmentation with relatively intense signals in the lower mass region (Figure 3). Assuming close biogenetic relations between the two active compounds, the structure of the second component was more likely to be that of an epoxydiene than that of a ketodiene, since no char- acteristic acylium ion or product of a McLafferty rearrangement could be detected.

Of the three monoepoxydienes derived from Z3, Z6, Z9-19Hy, (Z, Z)-6, 9- cis-3,4-epoxynonadecadiene and (Z,Z)-3, 6-cis-9, 10-epoxynonadecadiene showed very different mass spectra when compared to the natural compound (Figure 3). However, the mass spectrum and gas chromatographic retention times (on three different columns) of synthetic (Z, Z)-3, 9-cis-6, 7-epoxynona- decadiene exactly matched the natural product. The enantiomeric configuration of the active pheromone component remains to be established. In each female about 1.5 ng of the main component (Z,Z)-3,9-cis-6,7-epoxynonadecadiene was found.

Electrophysiology. The activity of the synthetic pheromone components was tested by both EAG and single sensillum experiments. FID-EAD runs with 5 ng of the synthetic substances showed a strong EAG response to both of them. The component elicting the strongest response was the Z3, Z9-cis-6, 7-epo-19Hy.

The single sensillum measurements, made on 12 individual sensilla, revealed two physiological types of sensilla. In type I, a large spike amplitude cell responded to the epoxide and a small spike amplitude cell responded to the triene. In the type II sensillum, only one cell responded. It responded to the epoxide with a large spike amplitude cell, but was inhibited by the triene com- ponent (Figures 2 and 4). A small cell could be seen also in the type II receptor, but it was not stimulated by any of the pheromone components.

Field Tests. Preliminary field trapping experiments performed in Hungary resulted in substantial trap catches of male mottled umber moths with a mixture of the epoxide and the triene (Table 1).

In the course of the field tests, males of another geometrid, Colotois pen- naria L., were caught with the same bait (Table 1). Supplementary FID-EAD and GC-MS analyses proved Z3, Z9-cis-6, 7-epo-19Hy to be present in female pheromone gland extracts of C. pennaria as well. This species also is regarded as a member of the pest complex of autumn-flying geometrids (Balas and Sar- inger, 1982). More detailed results conceming the identification of the phero- mone of this species will be published elsewhere.

In another field test, conducted in the early spring of 1989, in Hungary, high numbers of a spring-flying geometrid, Agriopis marginaria F, a close rel- ative of E. defoliaria, were captured (Table 1). A. marginaria is regarded as a potential orchard pest in Central Europe (Balas and Saringer, 1982).

P H E R O M O N E O F Erannis defoliaria

A I00 �9

1893

8

50-

I00-

50 70

0

90 ii0 130 150 170 190 210 230 250 270 m/z

0

50 70 90 ii0 130 150 170 190 210 230 250 270 m/z

C 100-

I [%]

50-

0

50 70 90 llO 130 150 170 190 210 230 250 270

FIG. 3. Electron impact mass spectra (70 eV) of (Z,Z)-6,9-cis-3,4-epoxy-nonadeca- diene (A), (Z, Z)-3,9-cis-6, 7-epoxy-nonadecadiene (B), and (Z, Z)-3, 6-cis-9, 10-epoxy- nonadiene (C).

Type l

epoxide

,t.., . . . . . . .

............. ~Rl~lrllT=rF'lFm,r"'r~ . . . . ~ , .Ac

triene AC

" - " ' ~ ' " ~ ~ ~ DC

epoxide AC

Type II

triene

i i

AC

i

FIG. 4. Single sensillum responses from two physiologically different types (I and II) of male pheromone sensitive sensilla to the two identified pheromone components Z3,Z6,Z9-19Hy and Z3,Z9-cis-6, 7-epo-19Hy. The horizontal black bar indicates the stimulation time (1 sec).

TABLE 1. FIELD CATCHES OF MALE GEOMETRIDS IN TRAPS BAITED WITH MIXTURE OF Z3, Z9-cis-6,7-epo- 19Hy AND Z3,26, 29-19Hy rN PRELIMINARY TESTS IN HUNGARY a

Total number of males caught Z3,Z6,Z9-

19Hy Z3, Z9-cis-6,7-epo-19Hy Erannis Agriopis Colotois (/zg) (/~g) defoliaria marginaria pennaria

0 0 1 0 0 30 100 123 98 54

"Tests conducted from November 1, 1988 to March 13, 1989, near Budapest in six replicates.

PHEROMONE OF Erannis defoliaria 1895

DISCUSSION

With regard to pheromone composition, the only well-investigated geo- metrid occurring in Europe is Operophtera brumata L., the winter moth. Inde- pendently, Roelofs et al. (1982) and Bestmann et al. (1982) reported a pheromone consisting of a single component (Z,Z,Z)-l,3,6,9-nonadecate- traene. While in O. brumata, Alsophila pometaria HalT. (Wong et al., 1984), and Sabulodes caberata Guen6e (McDonough et al., 1986) the pheromone seems to consist of hydrocarbons only, other species produce mixtures of hydro- carbons and their corresponding oxygen-containing derivatives. The sex pher- omone of P. rhomboidaria is a mixture of Z3,Z6,Z9-19Hy and (Z,Z)-6,9- nonadecadien-3-one (Buser et al., 1985), while Boarmia selenaria Schiff. pro- duces (Z, Z)-6, 9-cis-3,4-epoxynonadecadiene instead of the ketone (Becker et al., 1987). In the present study the pheromone of E. defoliaria showed the same dual principle as it consisted of the nonadecatriene found in the former species, along with a new epoxide, Z3,Z9-cis-6, 7-epo-19Hy, as the oxygen-containing component.

The single sensillum recordings reported in this paper are the first pub- lished on geometrid male responses to the female sex pheromone. One of the sensillum types encountered (type I) showed a type of response that is often encountered in various moth orders (Den Otter, 1977; Hansen, 1984; Priesner et al., 1984; Hansson et al., 1987): a large spike amplitude cell responding to the main pheromone component in the female pheromone gland extract and a small spike amplitude cell responding to the minor component. However, a second type of sensillum was discovered. This type of sensillum (type II) responded with a large spike amplitude to the main component, but was inhib- ited by the minor component. As far as we know, this phenomenon has not been observed in the relationship between pheromone components at the periph- eral reception level. It has been shown that plant substances can inhibit olfactory cells, as in the interaction between (Z)-I 1-tetradecenyl acetate and geraniol in male Yponomeuta evonymellus L. (Yponomeutidae) pheromone receptors (Van Der Pers et al., 1980), but the specific action of one pheromone component inhibiting the response to another is new. This gives the moth a second source of infonxlation about the exact proportions of the pheromone components. The type II cell could enable the male to assess the concentrations of the components in the pheromone blend emitted by the female very exactly. Too much epoxide would give a response that is too strong, and too much triene would decrease the response. A delicate balance between the two components would be needed to elicit the "r ight" frequency of action potentials in the olfactory cell.

It is interesting to note that Z3,Z9-cis-6,7-epo-19Hy, in common with many other components of moth pheromones, is not a species-specific com- pound. We also identified it as a sex pheromone component of C. pennaria and

1896 HANSSON ET AL.

found it to be attractive to A. marginaria, which indicates that this compound may be a generally occurring pheromone component among geometrids. Field tests have shown that males o f other geometrid species also were attracted to this type o f compound. Wong et al. (1985) showed in an extensive field-trap- ping and electroantennographic study that males of 17 geometrids were caught in traps baited with different mixtures of triene hydrocarbons and diene epox- ides. However, the enantiomeric configuration has not been established and might be an important factor in the species specificity of the sex pheromones in these species.

To our knowledge, Z3, Z9-cis-6, 7-epo-19Hy has not been found in the sex pheromone of any other species; neither has there been any published demon- stration of its field activity towards male Lepidoptera. However, its shorter homolog, (Z,Z)-3, 9-cis-6, 7-epoxy-heptadecadiene has been reported to catch male Semiothisa delectata Hulst. in mixtures with the parent hydrocarbon (Z ,Z ,Z ) -3 , 6, 9-heptadecatriene (Millar et al., 1987), and in S. ulsterata Pear- sail its occurrence in the female pheromone gland extract has been demonstrated by comparison of retention times and electrophysiological activity of peaks (Millar et al., 1987.

We conclude that the mixture of Z3, Z6, Z9-19Hy and Z3,Z9-cis-6, 7-epo- 19Hy offers a useful basis for optimization of a practically applicable attractant to catch males of E: defoliaria. Further studies o f the species specificity of attractant mixtures and determination of the absolute configuration of the natural epoxide are in progress.

Acknowledgments--The authors wish to thank the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and Grant OTKA 1012 of the Hungarian Academy of Sciences as well as the Swedish research councils NFR, FRN, and SJFR for financial support, and Dr. Roger Finlay for correcting the English.

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PHEROMONE OF Erannis defoliaria 1897

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