COMPARATIVE STUDY OF PHEROMONE ...

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BILL S. HANSSON,1 and CHRISTER LOFSTEDT1,*. 1 Department of Ecology, Lund University. S-223 62 Lund, Sweden. 2Kutsaga Research Station.
Journal of Chemical Ecology, Vol. 25, No. 1, 1999

COMPARATIVE STUDY OF PHEROMONE PRODUCTION AND RESPONSE IN SWEDISH AND ZIMBABWEAN POPULATIONS OF TURNIP MOTH, Agrotis segetum

WENQI WU,1 C. B. COTTRELL,2 BILL S. HANSSON,1 and CHRISTER LOFSTEDT1,* 1 Department

of Ecology, Lund University S-223 62 Lund, Sweden 2Kutsaga Research Station Airport Ring Road, P.O. Box 1909 Harare, Zimbabwe

(Received October 28, 1996; accepted August 17, 1998) Abstract—Analysis of female sex pheromone gland extracts of the turnip moth {or common cutworm), Agrotis segetum, from Zimbabwe revealed three compounds previously identified as sex pheromone components in the Swedish population, namely (Z)-5-decenyl acetate (Z5-10:OAc), (Z)-7-dodecenyl acetate (Z7-12:OAc), and (Z )-9-tetradecenyl acetate (Z9-14:OAc). However, the proportions from the Zimbabwean population (1:0.25:0.03) differ from those in the Swedish population (1:5:2.5). In addition, gas chromatography-mass spectrometric (GC-MS) analysis of the Zimbabwean female gland extracts revealed a trace of (Z)-5-dodecenyl acetate (Z5-12:OAc). This compound has recently been identified as a fourth sex pheromone component for the Swedish population. Single-sensillum recordings from both Zimbabwean and Swedish populations showed the presence of two types of antennal receptors responding to either Z5-10:OAc or Z7-12:OAc. In Zimbabwean males the Z7-12:OAc receptor neuron appeared to be confined to the basal and medial thirds of the antennal branches, while in Swedish males it was distributed along the entire antennal branch. Dose-response curves of Z5-10: OAc or Z7-12: OAc specific receptor neurons from males of both populations showed similar response profiles, but the neurons of the Zimbabwean population showed higher maximal responses. In flight tunnel tests with Zimbabwean males, the three-component Zimbabwean blend of Z5-10:OAc, Z7-12:OAc and Z9-14:OAc elicited significantly greater responses than the Swedish blend, but not significantly greater than pheromone glands from calling Zimbabwean females. (Z)-5-decenol (Z5-10:OH), a constituent of gland extracts, exerted an antagonistic effect *To whom correspondence should be addressed.

177 0098-0331/99/0100-0177$16.00/0 © 1999 Plenum Publishing Corporation

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WU, COTTRELL, HANSSON, AND LOFSTEDT in the flight tunnel. In field tests conducted in Sweden, local males were preferentially attracted to local females, while in Zimbabwe preferential attraction to local females was less pronounced. Local response to the Swedish and Zimbabwean synthetic four-component blends mirrored the responses to the local females. Zimbabwean males are much more strongly attracted to Z5-10: OAc alone than are Swedish males and the high concentrations of Z7-12:OAc and/or Z9-14: OAc present in the Swedish blend reduced attraction of Zimbabwean males. This reduced attraction appears to be counteracted by the trace amounts of Z5-12:OAc found in the Swedish four-component blend. Addition of Z5-12: OAc to the three-component Zimbabwean blend did not, however, significantly increase the trap catches of Zimbabwean males. Key Words—Sex pheromone, turnip moth, common cutworm, Agrotis segetum, geographical population variation, receptor neurons, single-sensillum recordings, dose-response, flight tunnel, field tests.

INTRODUCTION

The turnip moth (common cutworm of Africa), Agrotis segetum (Dennis and Schiffermuller) (Lepidoptera: Noctuidae), is a serious pest of many crops throughout Africa and most of Europe and Asia. Its sex pheromone has been previously identified as a blend of three components: (Z)-5-decenyl acetate (Z5-10:OAc); (Z)-7-dodecenyl acetate (Z7-12:OAc); and (Z)-9-tetradecenyl acetate (Z9-14:OAc) (Bestmann et al., 1978; Arn et al., 1980; Toth et al., 1980; Lofstedt et al., 1982). Recently another gland constituent, (Z)-5-dodecenyl acetate (Z5-12: OAc) (Lofstedt et al., 1986), was demonstrated to be both electrophysiologically and behaviorally active (Wu et al., 1995), and thus qualifies as a fourth sex pheromone component. Geographical variation in male responses to pheromone blends was first reported in field-trapping tests with several European populations of A. segetum (Arn et al., 1983). These tests showed that males from a French population were attracted not only to the ternary mixture but also to Z5-10: OAc alone. Variation in the production of, and response to, different three-component blends was further demonstrated in the laboratory by analysis of female extracts as well as by investigations of olfactory sensilla on male antennae from several European populations. The results revealed a correlation between the proportions of the three components and the number of their corresponding olfactory receptor neurons (Lofstedt et al., 1986; Hansson et al., 1990). A recent survey examined differences in the responses of male A. segetum to five different synthetic pheromone mixtures (one ternary, two binary and two single components) at 11 widely separated geographical locations in Europe, Asia, and Africa (Toth et al., 1992). At all localities in Eurasia as well as at the single north African site (Egypt), and despite more or less continuous vari-

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ation in local responses, the most attractive blend was always the ternary mixture (Z5-10: OAc, Z7-12: OAc, and Z9-14: OAc, 1:1:8). In contrast, at two southern African sites (Zimbabwe and South Africa), Z5-10: OAc alone was definitely the most attractive to local males. This suggested that these African populations had a markedly different pheromone system from Palaearctic populations. In addition to the geographical differences in the A. segetum pheromone system mentioned above, differences have also been described between the lepidopteran pheromone systems of sympatric putative sibling species, e.g., Diacrysia chrysitis and D. tutti (Lofstedt et al., 1994), between strains of the same species, e.g., Ostrinia nubilalis (Klun and cooperators, 1975; Kochansky et al., 1975; Roelofs et al., 1985), as well as between geographically separate populations of other species, e.g., Choristoneura rosaceana (Thomson et al., 1991), Zeiraphera diniana (Guerin et al., 1984), and Planotortrix excessana (Foster et al., 1986, 1989). The biological significance of geographical variation in the pheromone systems of populations of the same species is not understood, and the results of Toth et al. (1992) prompted us to study the pheromone system of A. segetum from Zimbabwe in greater detail and to compare it with that of the Swedish population. Hybrids between the Swedish and the Zimbabwean A. segetum have been established in our lab. The hybrid blend was also tested in the present study. In the present paper we report results from analyses of pheromone gland extracts, electrophysiological recordings of sensilla on male antennae, and behavioral investigations in a flight tunnel and in the field.

METHODS AND MATERIALS

Insects. A Zimbabwean culture of A. segetum was established in the Lund laboratory from pupae originating from Kutsaga Research Station, Harare, Zimbabwe. A Swedish culture of A. segetum was established from collections from southern Sweden and Denmark and maintained in the laboratory for over three years. Larvae of both populations were reared on a semisynthetic diet (Seshu Reddy and Davis, 1978) and were kept at 25°C on a 16-hr light-8-hr-dark cycle. Pupae were sexed based on the morphological difference at the ventral side of the last abdominal segment between males and females. Male pupae and adults were kept at 20°C with 14-hr light-10-hr dark photoperiod, while female pupae and adults were kept in a separate chamber at 23°C with a 16-hr light-8-hr dark photoperiod. First- to third-generation male Zimbabwean A. segetum were used for the electrophysiological and behavioral tests. Chemicals. Z5-10:OAc, Z5-12:OAc, Z7-12:OAc, Z9-14:OAc, and (Z)-5-decenol (Z5-10:OH) were obtained from our laboratory stock, initially

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purchased from the Institute for Pesticide Research, Wageningen, The Netherlands, and were >99% pure with respect to geometrical isomers as determined by gas chromatography. For a single-sensillum screening test, 10Ug of each of the above compounds, diluted in 10 Ul hexane, was applied to a piece of filter paper (8x18 mm) that was inserted in a Pasteur pipet. For dose-response singlesensillum recordings, serial dilutions (10~3-102 Ug) of Z5-10: OAc, Z7-12: OAc, or Z5-10: OH in the 10 Ul hexane were used. The synthetic pheromone blends used for behavioral assays (Table 1) were checked by gas chromatography to ensure the purity and the ratios between components. The ratios of components in the hybrid blend were determined from females obtained from a cross between the Swedish and Zimbabwean A. segetum. In field tests, blends were applied to rubber septa in 50 Ul hexane to achieve the desired dosage. In flight tunnel assays, blends were applied to filter paper in 10 Ul hexane to achieve the desired dosage. In addition to the synthetic blends, pheromone glands from calling females and gland extracts also were tested in the flight tunnel. Analyses of Pheromone Gland Extracts. The terminal abdominal segments with the pheromone glands were dissected from 2- to 4-day-old females during the fourth to seventh hour of the scotophase. Glands were either used immediately for flight tunnel tests or extracted in hexane for 2 hr to be used later for chemical analyses and for flight tunnel assays (Zimbabwean females only). The female extracts were analyzed on a Hewlett Packard 5880A gas chromatograph equipped with a DB-Wax column (30 m x 0.25 mm ID, J&W Scientific, Folsom, California). Hydrogen was used as carrier gas. Samples were injected splitless. The injector temperature was 225°C and the split valve was opened 1 min after injection. The column temperature was maintained at 80°C for 2 min following the injection and then linearly increased to 230°C at a rate of 10°C/min. Gas chromatographic-mass spectrometric (GC-MS) analyses were performed by HP TABLE 1. DEFINITIONS OF SYNTHETIC BLEND CODES (SBC) iProportions

of components

SBC Z5-10:OAc Z5-12:OAc Z7-12:OAc Z9-14:OAc 1 2 3 4

5 6 7 8

1 1 1 1 1 1 1 1

0.1

Blend names

5 5

2.5 2.5

Swedish three-component blend Swedish four-component blend

0.25 0.25 0.25 1.61

0.03 0.03 0.92

Zimbabwean two-component blend Zimbabwean three-component blend Zimbabwean four-component blend Hybrid blend

0.1

0.1

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GC-MS with electron impact ionization (70 eV), equipped with a 5970b computer system, and interfaced with an HP 5890 GC. For the detection of pheromone components from female extracts, the GC-MS was operated in the selected ion monitoring mode. Selected ions were monitored in several groups and the groups were changed at preset times during the separation. These times were based on the retention times of the synthetic standards. The following diagnostic ions were chosen for the detection of pheromone compounds, Z5-10:OAc, m/z 138.20; Z5/Z7-12: OAc, m/z 166.20; Z9-14: OAc, m/z 194.20. Electrophysiological Recordings. Single-sensillum recordings were performed by using the tip-cutting technique (Kaissling, 1974; Van Der Pers and Den Otter, 1978). A male moth was secured in a plastic pipet, the tip of which had been cut to allow the head to protrude. The antennae were then fixed by dental wax and positioned to allow microelectrode access. A thin silver wire, serving as ground electrode, was inserted into the abdomen. A sensillum was cut by means of microscopic glass knives, and a recording electrode with a tip diameter of about 4 Um, filled with Beadle-Ephrussi Ringer was placed in contact with the cut surface of the sensillum. The antenna was continuously flushed with charcoal-filtered and moistened air delivered through a glass tube (ID 8 mm) at a speed of 0.5 m/sec, the outlet of which was about 10 mm from the antenna. The stimulus was injected into the airstream through an aperture (3 mm diameter) in the tube in a 0.5-sec puff by a stimulation device (Syntech, P.O. Box 1547, NL-1200 Hilversum, The Netherlands). Flight Tunnel Tests. Flight tunnel experiments were performed in a 2.5-mlong x 0.9-m-wide x 0.9-m-high Plexiglas flight tunnel as described by Lofstedt and Herrebout (1988). The flight tunnel conditions used were 21-23°C, 40-60% relative humidity, 0.3 m/sec wind velocity, and 0.6 lux light intensity. Two- to 3-day-old Zimbabwean male moths were transferred individually to 250-ml cylindrical screen cages in plastic cups before the initiation of the dark period and allowed to acclimate to the conditions in the tunnel room for 1 hr before they were tested 3-5 hr into the scotophase. Males were released individually into the plume from a cylindrical screen cage with the open end facing upwind. Six behavioral steps typically exhibited in the flight tunnel were recorded, taking flight (TF), orientation (Or), upwind flight 50 cm from the release cage in the plume (50 cm), upwind flight half the distance between the source and the release cage in the plume (HW), close approach within 10 cm to the source (190 cm), and source contact (SC). A male was considered to have contacted the source as soon as its antennae touched it. Males were tested once and then discarded. After testing, the release cages were heated at 200°C for 2 hr and the insect needles holding the filter paper dispensers were rinsed in acetone. Field Tests. Field tests were conducted with Lund II sticky traps (Anderbrant et al., 1989) placed in sugar beet fields in southern Sweden or by using

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commercial delta traps (PLA Insect Monitoring Systems, UK) in tobacco lands at Kutsaga Research Station in Zimbabwe. Red rubber septa (Thomas Scientific, USA) were used as dispensers. Three of the experiments were carried out in both Sweden and Zimbabwe and two others only in Zimbabwe. For convenience of description and reference, the different kinds of experiments are numbered and prefixed with "S" if performed in Sweden or "Z" if performed in Zimbabwe. The first two experiments (S1/Z1 and S2/Z2) were pairwise comparisons in which two traps, placed approximately 1 m apart, were used at each replicate site, with sites at least 23 m from one another. Trapped moths were counted and removed each day, or every other day, and at these times, the traps were switched in position to minimize position effects. For S1 (eight replicates) and Z1 (six replicates), each paired trap was loaded with one, same-aged virgin female (one Swedish and one Zimbabwean female per pair of traps). For S2 (24 replicates) and Z2 (15 replicates), each pair of traps was loaded with a septum carrying either synthetic blend code 2 or 7 (SBC 2 or SBC 7) (Table 1). The difference in the number of males trapped by each member of a pair of traps up to the last night on which both females were alive (S1 and Z1) or over five nights (S2 and Z2) was used as the response variable for analysis by a paired-sample Student t test. S1 and S2 were carried out from June 23 to July 1, 1994; Z1 from October 26 to November 10, 1994; and Z2 from January 23 to February 10, 1995. For the remaining experiments, more than two blends were tested at each replicate site. Traps at a site were approximately 25 m apart and arranged in a row perpendicular to the predominant wind direction. Sites were at least 60 m apart in Sweden or 100 m apart in Zimbabwe. When trapped males were removed, the traps at a site were moved one position in a standard direction. Experiments S3, Z3a, and Z3b compared six blends (SBCs 1, 2, 3, 5, 6, and 8). S3 was carried out from June 3 to 7, 1993; Z3a from October 11 to 30, 1993; and Z3b from November 7 to December 4, 1993. Five replicate sites were used for each experiment. Bait septa were renewed every five days. The response variate analyzed was the total catch for each blend at the replicate sites. Experiment Z4 compared five blends (SBCs 2, 3, 4, 6 and 7) at five sites using one set of baits exposed for five days between February 28 and March 5, 1994. The total number of males caught by each blend over the five days was analyzed. Experiment Z5 involved three blends. One Swedish blend, one Zimbabwean blend, and one Zimbabwean blend with Z5-12: OAc each were tested at four dosages (0.03-30 Ug in decadic steps). Dosage refers to the amount of Z5-10: OAc. The four dosages were tested at three rotating sites by using five sets of septa, each exposed for five nights, to give a total trapping period of 25 nights between March 5 and April 1, 1994. Fisher's exact test was performed in experiments S1, Z1, S2, and Z2. In experiments S3, Z3, S4, Z4, and Z5, analyses of variance of ln(x + 1) trans-

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formed catches and multiple comparisons between means were performed, with ANOVA followed by Fisher protected least significant difference (FPLSD) test for data which gave significant overall F values. In experiment Z3, the chi-square test of homogeneity was used to assess whether the two experiments Z3a and Z3b represent samples from the same population.

RESULTS

Analyses of Pheromone Gland Extracts. Gas chromatographic analyses of female gland extracts from Zimbabwean and Swedish populations revealed three pheromone components: Z5-10: OAc, Z7-12: OAc, and Z9-14: OAc in clearly different proportions (Figure 1). The average ratio (±SEM) of the three compounds in 13 batches of Zimbabwean female gland extracts (the total number of glands in 13 batches was 100) was 1:0.25 ± 0.09:0.03 + 0.02, whereas the average ratio in the Swedish females was 1:4.8 ± 1.1:2.5 ± 1.0 from 20 extracts of one female gland each. GC-MS analyses on both Swedish and Zimbabwean female extracts revealed that at m/z 166.20 there was a trace peak with the same retention time as Z5-12: OAc. Electrophysiological Recordings. A total of 108 sensilla from antennae of male Zimbabwean A. segetum were examined by stimulation with Z5-10: OAc, Z7-12:OAc, Z9-14:OAc and Z5-10:OH. Two major types of sensilla were

FIG 1. GC-FID chromatogram of female pheromone gland extracts from two populations of Agrotis segetum; (A) Swedish, 1 female equivalent; (B) Zimbabwean, 3 female equivalents. Peak 1, Z5-10:OAc, 2, Z5-10:OH; 3, Z7-12:OAc; 4, Z7-12:OH; 5, Z9-14: OAc; 6, Z9-14: OH. *Internal standard Z8-13 : OAc.

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found. One type (86% of the sensilla examined) contained a receptor neuron responding to Z5-10:OAc with a large spike amplitude, together with another receptor neuron responding to Z5-10: OH with a small spike amplitude. Three of 36 of these sensilla responded also to Z5-12: OAc with a small spike amplitude. The other sensillum type (14% of sensilla examined) contained one neuron responding to Z7-12: OAc with a large spike amplitude. In Swedish males, of 30 sensilla examined, the percent of Z7-12:OAc-type sensilla was 23%. The Z7-12:0Ac-type sensilla in Zimbabwean male antennae were found in the basal and medial part of the antennal branch, while in Swedish male antennae the Z7-12: OAc-type sensilla could be found along the entire branch (Table 2). Dose-response curves obtained from male Zimbabwean and Swedish A. segetum receptor neurons when stimulated with Z5-10: OAc, Z5-10: OH, and Z7-12: OAc (Figure 2A-C) showed a similar response profile between the two populations. However the Zimbabwean receptors showed higher maximal response. Flight Tunnel Tests. In Zimbabwean A. segetum, the flight tunnel assays showed that a blend of Z5-10:OAc, Z7-12:OAc, and Z9-14:OAc at a 1:0.25:0.03 ratio elicited as many male responses as did the pheromone glands dissected from calling females. The response to this blend was significantly greater than the response to female extracts, which contained high amounts of Z5-10: OH and Z7-12: OH (Figure 3). The antagonistic effect of these alcohols was confirmed by a significant decrease in male response to the two-component blend with the addition of the combination of Z5-10: OH and Z7-12: OH or the addition of the female extract. Males from the Zimbabwean population were TABLE 2. SPATIAL DISTRIBUTION OF Two TYPES OF RECEPTORS ON LATERAL SIDE OF ANTENNAL BRANCHES OF ZIMBABWEAN AND SWEDISH MALE A. segetum POPULATIONS RESPONDING TO Z5-10 : OAc OR Z7-12 : OAc Sensillum types found (N) Zimbabwean populationa Antennal Region Basal

Medial Distal

Total found % in population a

Z5-10 : OAc

Z7-12 : OAc

Z5-10 : OAc

Z7-12 : OAc

35 29 29 93 86

11 4 0 15 14

12 5 6 23 77

3 2 2 7 23

Nine branches from seven males tested. Four branches from four males tested.

b

Swedish populationb

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FIG. 2. Dose-response curves of receptor neurons from Swedish and Zimbabwean male Agrotis segetum stimulated with Z5-10: OAc (A), Z5-10: OH (B), and Z7-12: OAc (C) (mean ± SEM, N = 10).

significantly more attracted to the Zimbabwean three-component blend (SBC 6) than to the Swedish three-component blend (SBC 1) or to Z5-10:OAc alone (Figure 4). As in the first flight tunnel experiment (Figure 3), the Zimbabwean two-component blend was also attractive (Figure 4).

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FIG. 3. Upwind behavioral responses of male Zimbabwean A. segetum in a flight tunnel to different pheromone stimuli [including Zimbabwean female extracts (3FE) and Zimbabwean female glands (2FE)]. The behaviors are: TF, take off; Or, orientation; 50 cm, moths flying upwind 50 cm from the release point in the plume, HW, moths flying half of the distance from the release point to the source; 190 cm, moths flying from the release point to 10 cm downwind of the source; SC, source contact. Different letters indicate values significantly different at the 95% confidence level, according to the method of adjusted significance levels for proportions (Ryan, 1960). Blends tested are independent of those in Table 1.

Field Tests. Three trapping experiments (numbered 1-3) were carried out in both Sweden and Zimbabwe; two further experiments (4 and 5) were carried out only in Zimbabwe. Experiments S1 and Z1 tested the relative attractiveness of same-aged vir-

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FIG. 4. Upwind behavioral responses of male Zimbabwean A. segetum in a flight tunnel to synthetic blends of components. Abbreviations and statistical analyses as in Figure 3, and blend proportions as in Table 1.

gin Swedish and Zimbabwean stock females to local males. In Sweden, Swedish females trapped 95% of the total catch of local males. In Zimbabwe, Swedish females caught only 30% (Table 3, A). The differences in the proportions of males caught by the two types of females at the two localities are statistically significant (Fisher's exact test, two-tailed P = 1.84 x 10-14), indicating a differential response of local males to local vs foreign females. The mean difference (S - Z) in catch between members of the pairs of females tested was significant in Sweden (paired t test, two-tailed P - 0.011) but not in Zimbabwe (P = 0.258) (Table 3, B). The difference in the degree of selectivity exercised by local males for local females was not due solely to the smaller number of pairs tested in Zimbabwe but to a real difference in behavior (supported by an examination of the number of pairs falling into different attractiveness categories (Fisher's exact test, two-tailed P = 0.003; Table 3, C).

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TABLE 3. RELATIVE ATTRACTIVNESS OF PAIRS OF SAME-AGED VIRGIN FEMALES, ONE SWEDISH (S), ONE ZIMBABWEAN (Z), IN FIELD TESTS IN SWEDEN AND ZIMBABWE Total catch (%) Locality

Swedish females

Zimbabwean females

A. Total catch of males, percentages in parentheses Sweden Zimbabwe

53 (95) 21 (30)

3 (5) 49 (70)

B. Analyses of differences (S-Z) in male catches by pairs Sweden Zimbabwe

Pairs

Mean Diff.

SE

t

P (2-tailed)

8 6

6.25 -4.67

1.81 3.66

3.45 1.28

0.0107 0.2580

Attractiveness category

C. Frequencies of pairs in different relative attractiveness categories Sweden Zimbabwe

S >Z

S=Z

SZ

S=Z

S P > 0.01). Experiments S3, Z3a, and Z3b tested the relative attractiveness of six pheromone component blends: SBC 1, 2, 3, 5, 6, and 8 (Table 1). In Zimbabwe the same experiment was carried out on two different occasions (designated experiments Z3a and Z3b). A chi-square test of homogeneity of the frequencies obtained in the two Zimbabwean experiments suggests that these two data sets do not represent random samples from the same population (P = 0.005). In Sweden, the most attractive blend was the Swedish four-component blend (SBC 2); however, responses to SBC 1, 6, and 8 were not significantly different from the response to SBC 2 (Figure 5). SBC 3 (Z5-10: OAc alone) was the least attractive one in Sweden. In Zimbabwe, the Zimbabwean three-component blend (SBC 6) consistently caught most male moths, while the Swedish threecomponent blend (SBC 1) and Zimbabwean two-component blend (SBC 5) were the least attractive (Figure 5). The results showed the high relative attractiveness of Z5-10: OAc alone (SBC 3) for Zimbabwean males as well as the very strong

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FIG. 5. Field trapping of male A. segetum in Sweden and in two separate trials in Zimbabwe (a and b) to six synthetic pheromone blends (experiments S3 and Z3) (N = 5). The same letters within each series indicate values that are not significantly different according to ANOVA [ln(x + 1) transformation] followed by the FPLSD test (P < 0.05).

discrimination by Zimbabwean males between the Swedish three- (SBC 1) and four-component (SBC 2) blends. In two experiments conducted only in Zimbabwe, five blends (SBC 2, 3, 4, 6, and 7 (Table 1) were tested (experiment Z4), while three blends (SBC 1, 6, and 7) were each tested at four dosages (experiment Z5). Although the

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FIG. 6. Field trapping of male A segetum in Zimbabwe to test the effect of Z5-12: OAc (experiment Z4) (N = 5). The same letters within each series indicate values that are not significantly different according to ANOVA [ln(x +1) transformation] followed by the FPLSD test (P < 0.05).

response was highest to the Zimbabwean four-component blend (SBC 7) in Z4 and at three of four doses in Z5, the responses to SBC 7 were never significantly different from the responses to the Zimbabwean three-component blend (SBC 6) (Figures 6 and 7). In experiment Z4, Z5-10: OAc alone (SBC 3) and Z5-10: OAc plus Z5-12: OAc (SBC 4) were clearly the least attractive (Figure 6). In experiment Z5, the optimum catches for all three blends were at a dose of 3Ug of Z5-10: OAc in field trapping in Zimbabwe (Figure 7).

DISCUSSION

The present study confirms that a Zimbabwean population of A. segetum has pheromone-emitting and -receiving characteristics that are distinctly different from those of European populations (Toth et al., 1992). The pheromone characteristics of the Zimbabwean population are, however, rather different from those reported in the original survey. This discrepancy may involve the proportions of components used by Toth et al. (1992) for their ternary mixture. Gas chromatographic analyses of Zimbabwean female gland extracts showed that the three major components (Z5-10: OAc, Z7-12: OAc, and Z9-14: OAc) are in the proportions 1:0.25 :0.03 as against the proportions 1:4.8:2.5 found in gland extracts of Swedish females. The Swedish proportions are in agreement with those reported previously (Lofstedt and Odham, 1984; Lofstedt et al., 1985b).

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FIG. 7. Field trapping of male A. segetum in Zimbabwe with four different doses of three blends (Experiment Z5) (N = 5). Dosage refers to the amount of Z5-10: OAc. Same letters within each series indicate values that are not significantly different based on ANOVA [ln(x + 1) transformation] followed by the FPLSD test (P < 0.05).

In addition, female gland extracts from both populations show traces of a fourth component with a retention time corresponding to Z5-12:OAc. This component has been reported previously (Lofstedt et al., 1986) and has been shown to be both electrophysiologically and behaviorally active in Swedish A. segetum (Wu et al., 1995). In flight tunnel assays, Zimbabwean males were significantly more attracted to the Zimbabwean three-component blend than to the Swedish three-component blend or Z5-10: OAc alone. Field pairwise comparison tests suggested that Swedish males have a stronger selectivity for local females and blends than do Zimbabwean males for local females and blends. Zimbabwean males do, however, select markedly against the Swedish three-component blend or the hybrid blend in both flight tunnel and field tests. The difference between these two blends and Zimbabwean blends is the proportions of the second and third components (Z7-12: OAc and Z9-14: OAc, respectively). The higher proportions of one or both of these compounds in the Swedish blend seems to be responsible for their decreased attractiveness to Zimbabwean males. Nevertheless, the presence of quite small amounts of either one or both of the second and third components in the Zimbabwean three-component blend significantly enhances its attractiveness over Z5-10: OAc alone. When Toth et al. (1992) first reported the existence of populations of A. segetum with different charac-

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teristics in southern and eastern Africa, they characterized them as being most strongly attracted to Z5-10: OAc alone. However, the component ratio (1:1:8) used for comparison deviated widely from what is now known of the pheromone composition of the Zimbabwean population. In light of the present experiments, the high amounts of one or both of Z7-12: OAc and Z9-14: OAc would be expected to have an antagonistic effect on Zimbabwean male attractivity. The effect of the fourth component (Z5-12: OAc) is particularly interesting. When added to Z5-10:OAc, it significantly decreased attractivity for Zimbabwean males. In contrast, when added to the Zimbabwean three-component blend to produce the four-component blend, it did not statistically increase attractiveness even though an examination of the catches does suggest some enhancement. In further striking contrast, when added to the Swedish three-component blend to produce the Swedish four-component one, it caused a statistically significant increase in attractiveness for Zimbabwean males, bringing the four-component Swedish blend virtually into the ranges of attractiveness (for Zimbabwean males) of the Zimbabwean three-component blend and even the Zimbabwean fourcomponent blend. Apparently, the presence of small amounts of the fourth component (Z5-12: OAc) in some way overcomes the loss of attractivity to Zimbabwean males caused by the presence of the higher concentrations of one or both of the second (Z7-12: OAc) and third (Z9-14: OAc) component in the Swedish blends. In the total absence of the second and third components, the fourth component in high concentrations reduced attractivity for Zimbabwean males. Although it was not possible to demonstrate, by single sensillum recordings, a definite difference in the proportions of Z5-10:0Ac-type receptor neurons to Z7-12: OAc-type receptors on Swedish as against Zimbabwean male antennal branches, there was a suggestion that in Zimbabwean males the latter type of neurons was confined to the basal and medial thirds of the antennal branches, whereas it was present along the whole branch length in Swedish males. The overall proportions of Z7-12: OAc-type sensilla are similar to those previously reported (Lofstedt et al., 1982, 1986). It has earlier been reported that different physiologically defined sensillum types are present at certain locations on the male antennae. In Agrotis exclamationis, receptor cells in the medially situated sensilla responded to totally different pheromone components from receptor neurons in the lateral sensilla (Hansson et al., 1986). In Spodoptera littoralis sensilla specifically tuned to Z9,E11-14: OAc were evenly distributed over the ventral antennal surface, while the second sensillum type containing one neuron responding to Z9,E12-14:OAc and another neuron responding to Z9-14:OH was only found along the lateral part of the antennal segments (Ljungberg et al., 1993). Likewise, in Trichoplusia ni sensilla tuned to Z9-14: OAc and 12: OAc were reported to be located in the middle subsegments of the antenna away from the lateral margins and within a row of hairs (Todd et al., 1993). In the present study, we found that the Z7-12: OAc sensillum type was situated in the basal and

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medial antennal branches on the male antennae of Zimbabwean turnip moths. If the number of sensilla and the distribution of particular sensillum types are proved to be consistent among individuals from one population, it will be possible to construct a map illustrating precisely the location of different sensilla on the male antennae of each populations. This accomplishment will be of great use for further studies of a particular sensillum type. The antagonistic effect of Z5-10: OH has previously been demonstrated in the Swedish population (Lofstedt et al., 1985a). The present experiments demonstrate a significant reduction in the behavioral responses observed in flight tunnel assays when this compound was present in female gland extracts or in active synthetic blends and also that it is a behavioral antagonist for the Zimbabwean population. Although Z5-10: OH was found in the female gland extracts in both populations, it is not known whether it is emitted by the female glands during calling. It has been shown in the present study that the glands themselves are not antagonistic to males. Analyses of airborne volatile collections from glands therefore need to be done in the future. Taking into account the relatively large amounts of the second and third components in the Swedish blends that can apparently be "neutralized" for the Zimbabwean male by a relatively small amount of the fourth component, it seems unlikely that the sensory integration occurs at the level of the primary receptor neurons. Integration is much more likely to reside in the central nervous system at levels where the sensory stimuli from the different neurons are coordinated. Antennal lobe neurons that respond only to the blend, and not to any single component, as well as neurons that recognize the differences in ratios between pheromone components were found in A. segetum (Wu et al., 1996). These results indicate that pheromone discrimination between the two populations can be partially achieved at the antennal lobe level. Differences in the ratios of homologous acetates in the pheromones of different geographical populations have also been reported for Zeiraphera diniana (Guerin et al., 1984) and Planotortrix exessana (Foster et al., 1986,1989), but their evolutionary interpretation is not yet understood. From our present study, there is no evidence for the idea that interpopulational differences have been brought about by reproductive character displacement resulting from the presence of species using similar pheromone signals. It is interesting, in this context, that the sympatric and sporadically numerous pest species Chrysodeixis acuta was more attracted to the Zimbabwean four-component blend of the sympatric Zimbabwean population than it was to the Swedish four-component blend. Acknowledgments—We thank Anna Tunlid and Erling Jirle for technical assistance, Jun Wei Zhu for help with the GC-MS operation, and Siana LaForest for comments on an earlier version of the manuscript. This study was supported by grants from the Swedish research councils NFR and SJFR, The Bank of Sweden Tercentenary Foundation, and the Knut and Alice Wallenberg Foundation.

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