Journal of Chemical Ecology, Vol. 15, No. 4, 1989

IDENTIFICATION AND FIELD TESTING OF ADDITIONAL COMPONENTS OF FEMALE SEX PHEROMONE OF AFRICAN ARMYWORM, Spodoptera exempta (LEPIDOPTERA: NOCTUIDAE)

A.

C O R K , 1 J. M U R L I S , 2 a n d T .

MEGENASA

3

~Overseas Development Natural Resources Institute Central Avenue, Chatham Maritime Chatham, Kent ME4 4TB, U.K. 2Department of the Environment Romney House, 43 Marsham Street London SW1P 3PY, U.K. 3Desert Locust Control Organisation for East Africa P.O. Box 30023 Nairobi, Kenya (Received January 13, 1988; accepted June 29, 1988)

Abstract--Ovipositor washings from virgin female Spodoptera exempta (Walker) (Lepidoptera: Noctuidae) were analyzed by high-resolution gas chromatography (GC) linked to a male electroantennogram (EAG). GC retention times of the two major EAG responses observed were consistent with their assignment as (Z)-9-tetradecenyl acetate and (Z,E)-9,12-tetradecadienyl acetate, as previously identified. However, three other EAG responses were also noted that had GC retention times consistent with (Z)-9-tetradecenal, (27)_ 9-tetradecen-l-ol, and (Z)-ll-hexadecenyl acetate. The components were present in the ratio of 100 : 5 : 1.5 : 3.5 : 4, respectively. Further analysis of the ovipositor washings by GC linked to a mass spectrometer (GC-MS) confirmed these findings and indicated the presence of a sixth component consistent with (Z)- 11-tetradecenyl acetate present at 2 % of the major component. In field tests carried out in Kenya, (Z)-I 1-bexadecenyl acetate was the only newly identified component to enhance the catch of the original two-component mixture when presented in their natural ratio. The addition of (Z)-9tetradecen-l-ol reduced catch, while (Z)-9-tetradecenal and (Z)-I 1-tetradecenyl acetate had no apparent effect.

1349 0098-0331/89/0400-1349506.00/0 9 I989 Plenum Publishing Corporation

1350

CORK ET AL.

Key Words--Spodoptera exempta, Lepidoptera, Noctuidae, sex pheromone, (Z)-9-tetradecenal, (Z)-9-tetmdecenyl acetate, (Z)-I 1-tetmdecenyl acetate, (Z,E)-9,12-tetradecadienyl acetate, (Z)-9-tetradecen-l-ol, (Z)-11-hexadecenyl acetate.

INTRODUCTION

The African armyworm, Spodoptera exempta (Walker) (Lepidoptera: Noctuidae), is a serious but sporadic pest of graminaceous crops, including forage grasses, maize, wheat, rice, and millet, throughout Africa. It is also known to occur in Southeast Asia, Philippines, New Guinea, and Australia (Haggis, 1986). Beevor et al. (1975) identified two components of the female sex pheromone as (Z)-9-tetradecenyl acetate and (Z,E)-9,12-tetradecadienyl acetate in the ratio of 100:5. These were field tested in Kenya by Campion et al. (1976) and found to be highly attractive to male S. exempta, although the catches were significantly less than those with a light trap. S. exempta has been shown to be a migratory moth from both indirect (Brown et al., 1969) and direct evidence (Rose and Dewhurst, 1979; Riley et al., 1983; Rose et al., 1985). By tracking their progress using pheromone and light traps over many years and relating these data to weather movements, it is now possible to forecast when and where outbreaks of armyworm larvae may occur (Haggis, 1986). However, outbreaks do not take place throughout the year but appear to be seasonally restricted to coincide with rain-bearing wind movements, such as the intertropical convergence zone (Tucker and Pedgley, 1983; Haggis, 1986). In most years, there is a gap of two to four months after the rainy season in which few, if any, outbreaks are reported (Haggis, 1986). There do not appear to be major return migrations towards the equatorial region where the first seasonal outbreaks are recorded, and low density, nonmigrating populations of insects are thought to survive until more favorable conditions permit further population growth (Rose, 1979; Nyirenda, 1985). In order to study areas with low population levels, it is important to maximize the ability of the pheromone trap to attract and catch males of the target species. This is particularly important when closely related sympatric species, such as Spodoptera triturata are present and known to be attracted to a similar pheromone (Blair and Tannock, 1977; D.J. Chamberlain, personal communication). As part of this process, this paper describes a reexamination of the sex pheromone derived from adults of the gregarious-phase larvae (Faure, 1943) and the preliminary field evaluation of the new compounds found.

1351

AFRICAN ARMYWORM PHEROMONE

METHODS

AND MATERIALS

lnsect Material. In order to carry out chemical and electrophysiological analyses, sixth-instar larvae were field collected in Kenya, reared on fresh grass, and allowed to pupate in bowls containing dry sandy soil. Pupae were dispatched by air to London, where they were sexed and maintained in an environmental cabinet on a reversed 12-hr light, 12-hr dark cycle, with temperatures alternating from 25 ~ to 22~ and a relative humidity of 70%. Adult moths were highly excitable and so were maintained with a 10% sucrose solution in groups of no more than 10 per container (Perspex, 28 x 15 • 9 cm) with tissue paper to discourage flying. Pheromone Collection. Ovipositor washings were prepared in hexane as described by Sower et al. (1973), using virgin female moths between 1 and 4 days old and usually between 8 and 9 hr into the dark phase. Volatiles from 2to 3-day old virgin female moths were collected on charcoal filters (5 rag) as previously described (Grob and Zurcher, 1976; Nesbitt et al., 1979; Tumlinson et al., 1982). Gas Chromatography (GC). GC analyses were conducted on a Carlo Erba Fractovap 4160 instrument fitted with a Grob split/splitless injector (220~ and flame ionization detector (220~ Fused silica capillary columns (25 m • 0.32 mm ID) were used coated with polar CP Wax 57CB (chemically bonded Carbowax 20 M; 0.21 /zm film thickness; Chrompack), highly polar OV275 (dicyanoallyl silicone; 0.2/zm film thickness; Chrompack), or nonpolar CP Sil 5CB (chemically bonded methyl silicone; 0.12/zm film thickness; Chrompack). Carrier gas was helium (linear velocity 40 cm/sec). All injections were made with the split valve closed for 40 sec onto a column held at 70~ for 2 min, then temperature programmed as indicated in the tables. Retention times of the compounds identified in the ovipositor washings, entrainments, and their synthetic analogs are summarized in Table 1. Temperature-programmed conditions were used that allowed the retention times to be converted to equivalent chain lengths (ECLs) relative to the retention times of straight-chain acetates; thus, for example, tetradecyl acetate = 14.00 (Harris and Habgood, 1966). Retention times of series of geometrical isomers and the natural compounds were usually obtained under isothermal conditions, so their retention times are quoted in minutes. Mass Spectrometry (GC-MS). Electron impact mass spectra were obtained on a Finnigan ITD 700 with open split interface to a Carlo Erba 5130 GC fitted with split/splitless injector (220~ and fused silica capillary column (25 m x 0.32 mm ID) coated with BP20 (chemically bonded Carbowax 20 M; 0.2/xm film thickness; S.G.E.). Carrier gas was helium (23 cm/sec). The oven tern-

1352

CORKET

AL.

TABLE 1. GC RETENTION TIMES OF PHEROMONE COMPONENTS OF Spodoptera exempta AND THEIR RELATIVE ABUNDANCE IN OVIPOSITOR WASHINGS

Retention times (ECLs) a Compound

CP Wax 57CB ~

CP Sil 5CB b

Relative composition

I Z9-14 : Aid II Z9-14 : Ac III Z11-14 : Ac IV Z, E9, 12-14:Ac V Z9-14 : OH VI Z11-16 : Ac

12.82 14.27 14.43 15.01 15.16 16.27

11.81 13.83 13.95 13.91 12.52 15.83

1.5 100 2 5 3.5 4

"Retention times in equivalent chain length units relative to the retention times of straight chain acetates. bOven temperature 70~ for 2 min then programmed at 20~ to 120~ and then at 4~ to 210~

perature was held at 70~ for 2 min, then programmed to 120~ at 20~ and then at 4~ to 220~ Electroantennography (EAG). Simultaneous recording of EAG responses from male antennae to GC column effluent were conducted essentially as described by Beevor et al. (1986), except that the indifferent microelectrode was inserted into an interstitial membrane at the proximal end of one flagellum and the recording electrode inserted into an interstitial membrane at the distal end of the other flagellum. Eluting compounds were deemed to have caused an EAG response if the amplitude of the response was at least 10% greater than the average of responses to fractions eluting before and after it. GC columns and operating conditions were as described above. EAG responses to synthetic compounds were tested by pulsing nitrogen through a Pasteur pipet containing 5 ng of the test compound applied in 2/zl of hexane, under conditions described by Beevor et al. (1986). Synthetic Chemicals. (Z,E)-9,12-Tetradecadienyl acetate was obtained from Food Industries Ltd. (U.K.) in 1978 and contained 99.4% Z,E, < 0 . 1 % E,Z, 0.2% Z,Z, and 0.36% E,E. The E,Z, E,E, and Z,Z isomers of 9,12-tetradecadienyl acetate were obtained from the Institute of Pesticide Research, Wageningen, The Netherlands. Wittig reaction with potassium tert-butoxide in THF between the triphenylphosphonium salt of 9-bromo-l-nonanol and (E)-2-pentenal gave a mixture of (Z,E)- and (E,E)-9,11-tetradecadienyt acetate. Similar reaction between the triphenylphosphonium salt of 8-bromo-l-octanol and (E)-2-hexenal gave the isomers of 8,10-tetradecadienyl acetate. These conjugated dienes could be iso-

AFRICAN ARMYWORM PHEROMONE

1353

merized to the thermodynamic equilibrium mixture of isomers by exposure to sunlight of a hexane solution containing a catalytic amount of iodine (cf. Beevor et al., 1986). Monounsaturated alcohols, acetates, and aldehydes were obtained by standard Wittig or acetylenic routes. The Z isomers used in field tests contained no more than 2 % of the corresponding E isomers. Field Tests. Field trials of synthetic pheromone mixtures were conducted in a mixture of cotton, maize, and fallow fields at the Cotton Research Station (Kibos, Kenya) in 1986 and 1987. Pheromone dispensers were white rubber septa (Aldrich, catalog No. Z10,072-2) impregnated with 0.1 ml of a hexane solution containing 1 mg of the pheromone blend and an equal weight of 2,6tert-butyl-4-methylphenol (BHT) as antioxidant. In 1986, sticky disk traps (60 cm diameter; Biological Control Systems Ltd., Treforest, Mid Glamorgan, U.K.) were used, fastened to a wooden Tshaped support 1.5 m above ground level. The pheromone dispenser was suspended at the center beneath a small inverted V-shaped roof to protect it from direct sunlight. Subsequent field work was conducted with funnel traps (8 cm diameter yellow funnel with white collecting box and green lid 3 cm above funnel rim; Biological Control Systems Ltd., U.K.), which were found to be as effective as disk traps and easier to maintain (Muftis et al., unpublished results). Each trap was fastened to an inverted L-shaped metal support 1.5 m from ground level. Lures were not changed during a trial. The traps were placed in circles containing one replicate of each treatment with at least 25 m between nearest neighbors and at least 100 m between replicates. Treatments were randomly assigned to each trap within a replicate and moved on one position clockwise each day, at which time the trap catch was recorded and discarded. The mean nightly catches were transformed to log (x + 1) to stabilize the variance, before subjecting them to analysis of variance. Differences between means were tested for significance at the 5 % level by Duncan's multiple-range test (DMRT).

RESULTS AND DISCUSSION

Structure Determination. Initial analyses of ovipositor washings by linked GC-EAG on polar CP Wax 57CB and OV-275 and nonpolar CP Sil 5CB columns confirmed the presence of (Z)-9-tetradecenyl acetate (Z9-14:Ac) and (Z,E)-9,12-tetradecadienyl acetate (Z, E9,12-14 : Ac) in the ratio of 20:1 as the major EAG active compounds, as described by Beevor et al. (1975). Pheromone yields of up to 200 ng of Z 9 - 1 4 : A c were obtained from the ovipositor washings, but 20 ng was more usual. The reasons for this difference were not investigated. Increasing the quantity of extract tested (equivalent to 80 ng of Z9-14 : Ac) and using temperature programmed runs to cover the range of reten-

1354

CORK ET AL,

tion times typical of lepidopteran pheromone components between decyl and eicosyl acetates, it became apparent that there were three additional EAG active compounds that had not been previously observed. Each of these compounds constituted 5 % or less of the major component, Z9-14 : Ac, by GC peak height. Relative GC retention times of these compounds in ECLs are tabulated in Table 1 together with those of a sixth compound detected by GC-MS and identified by comparison of GC retention times with synthetic analogs. For convenience, the compounds are designated I-VI on the basis of their elution order on a polar CP Wax 57CB column. Compound I displayed GC retention times characteristic of either a dodecenyl acetate or a tetradecenal. The absence of ions at m/z 61 (Me. COOH§ and m/z 166 ( M - 6 0 ) (Figure 1) from the MS, which are indicative of a dodecenyl acetate (Buser and Am, 1975; L6fstedt et al., 1982), suggested it was a tetradecenal isomer. This was supported by the presence of an ion at m/z 192, which could have arisen through the loss of water from a tetradecenal (Gudzinowicz et al., 1976) or a loss of CH3COOH from a tetradecadienyl acetate. The latter would be excluded on the basis of retention time. The position and geometry of the double bond were established as the (Z)-9 isomer by comparison of GC retention times with a range of synthetic analogs on polar CP Wax 57CB and nonpolar CP Sil 5CB columns (Table 2). The GC retention times (Table 1) and MS [m/z 61, 194 ( M - 6 0 ) ] (Figure 1) of compounds II and III indicated they were tetradecenyl acetate isomers. Comparison of their relative retention times with the complete range of tetradecenyl acetate isomers on polar CP Wax 57CB and the nonpolar CP Sil 5CB column with (Z)-12-tetradecenyl acetate as an internal standard (Nesbitt et al., 1986) showed that retention times of compound II were consistent only with those of Z9-14:Ac, as found by Beevor et al. (1975), and retention times of compound III were consistent only with those of (Z)-ll-tetradecenyl acetate (Z11-14: Ac). No EAG response was recorded at any time to Z11-14:Ac, presumably because it eluted just after the two major EAG-active compounds in the extract, namely, Z9-14:Ac on the polar columns and Z, E9,12-14:Ac (compound IV) on the nonpolar. Linked GC-EAG analyses with synthetic mixtures of these compounds in the same relative amounts also failed to yield a response to Z11-14 : Ac. The MS of compound IV was characteristic of a tetradecadienyl acetate ( M - 6 0 at m/z 192; rn/z 61) (Figure 1), and comparison of GC retention times of the isomers of 9,12-, 9,11-, and 8,10-tetradecadienyl acetates on polar CP Wax 57CB and nonpolar CP Sil 5CB columns confirmed the original assignment of Z, E9,12-14 : Ac (Beevor et al., 1975) (Table 3). Compound V had GC retemion times (Table 1) and MS (M-18 at m/z 194; no m/z 61) (Figure 1) consistent with a tetradecen-l-ol isomer. Comparison of its GC retention times with those of a range of synthetic tetradecen-1-

total ion I

hc

m/z

hc

_.._.~,

I~ ill

hc

_~/.jL._

VI

222

m

'194

192

_A 166

'

I

61

.

.

8B0 13 : 2S

.

.

t

.

.

.

.

!

.

.

9~

iS : 8 6

.

.

i

.

.

.

.

[

.

.

I Q~ 16 : 46

.

Ji

.

18 : 27

2 ~ : B'?

21 : 47

FIG. 1. Total ion chromatogram, m/z 40-300, and mass chromatograms of m/z 61, 71, 82, 166, 192, 194, and 222 of an extract corresponding to five female S. exempta abdominal tips. m/z 61 is an ion typical of acetates, while m/z 71 is typical of saturated hydrocarbons, and m/z 82 is typical of unsaturated straight-chain compounds. Ions at m/z 166, 194, and 222 are produced by loss of CH3COOH from dodecenyl, tetradecenyl, and hexadecenyl acetates, respectively, or loss of H20 from the corresponding alcohols. Similarly, ions at m/z 192 are characteristic of the loss of CH3COOH from tetradecadienyl acetates or H20 from tetradecenals and tetradecadienols. Pheromone components are labeled as in the text. Straight-chain hydrocarbons are labeled hc.

1356

CORK ET AL.

TABLE 2. RETENTION TIMES OF SYNTHETIC TETRADECENALSAND COMPONENT I

Retention times (min) Compound

CP Wax 57CB~

CP Sil 5CB~

Component I

22.41

20.02

14 : Aid

21.47

20.28

13-14 : Aid

22.87

20.25

Z12-14 : Aid E12-14 : Aid

23.67 23.14

20.91 20.58

Z11-14 :Ald E11-14 : Aid

22.88 22.58

20.41 20.32

Z10-14 : Aid El0-14 : Aid

22.60 22.31

20.21 20.20

Z9-14 : Aid E9-14 : Aid

22.41 22.27

20.02 20.06

Z8-14 : Aid E8-14 : Aid

22.24 22.19

19.94 19.99

Z7-14 : Aid E7-14 : Aid

22.20 22.12

19.83 20.02

Z6-14 : Aid E6-14 : Aid

22.19 22.13

19,88 20.00

Z5-14 : Aid E5-14 : Aid

21.97 22.02

19.83 20.00

Z4-14 : Aid

22.27

19.97

aOven temperature 70~ for 2 min then programmed at 4~

to 220~

o l i s o m e r s u n d e r i s o t h e r m a l c o n d i t i o n s on both p o l a r and n o n p o l a r c o l u m n s , u s i n g ( Z ) - 1 2 - t e t r a d e c e n - l - o l as an i n t e m a l standard, s h o w e d t h e m to be consistent o n l y with those o f ( Z ) - 9 - t e t r a d e c e n - l - o l ( Z 9 - 1 4 : O H ) (Table 4). G C retention t i m e s (Table 1) and M S [m/z 6 1 , 2 2 2 ( M - 6 0 ) ] (Figure 1) o f c o m p o u n d V I w e r e consistent with those o f a h e x a d e c e n y l acetate. All the h e x a d e c e n y l acetate i s o m e r s w e r e a v a i l a b l e for c o m p a r i s o n o f their G C retention t i m e s w i t h t h o s e o f c o m p o u n d V I e x c e p t (Z)- and ( E ) - 2 - h e x a d e c e n y l acetates. H o w e v e r , by a n a l o g y with the G C retention t i m e s o f tetradecenyl acetate isom e r s , w h i c h f o l l o w a similar pattern (Nesbitt et al., 1986), (Z)- and (E)- 2h e x a d e c e n y l acetates c o u l d be e x c l u d e d as candidates for c o m p o u n d VI. T h e G C retention t i m e s o f c o m p o u n d V I w e r e consistent only with those o f (Z)- 11hexadecenyl acetate ( Z 1 1 - 1 6 : A c ) on all three c o l u m n s u s e d (Table 5).

1357

AFRICAN ARMYWORM PHEROMONE

TABLE 3. RETENTION TIMES O13SYNTHETICTETRADECADIENYLACETATESAND COMPONENT IV Retention times (min) Compound

CP Wax 57CBa

CP Sil 5CBb

Component IV

28.50

26.10

Z,E9,12-14 : Ac E,Z9,12-14 : Ac Z,Z9,12-14 : Ac E,E9,12-14 : Ac

28.44 28.91 28.91 28.44

26.08 26.70 27.20 26.29

Z,E8,10-14 : Ac E,ZS, 10-14 : Ac Z,Z8,10-14 : Ac E,E8,10-14 : Ac

29.75 30.19 30.38 30.58

29.13 30.10 31.40 32.40

Z,E9,11-14 : Ac E,Z9,11-14 :Ac Z,Z9,11-14 : Ac E,E9,11-14 : Ac

30.05 30.32 30.47 30.79

29.95 31.17 31.88 32.85

~Oven temperature 70~ for 2 min then programmed at 4~ bOven temperature 70~ for 2 min then programmed at 20~

to 220~ to 130~

Analysis o f entrained volatiles from virgin females by G C - E A G and GCMS indicated that the pheromone released by the female was similar to that found in ovipositor extracts. Yields o f up to 50 ng/female/night of the major component Z 9 - 1 4 : Ac were collected. To ensure the entrainment apparatus was functioning, a synthetic mixture o f 100 ng o f each compound except Z 1 1 - 1 4 : A c was placed on aluminium foil. Recovery was between 50 and 80% for each compound in 4 hr. The relative E A G responses o f the six compounds identified in the female ovipositor washings at the 5-ng level were: I, 0.77 mV; II, 1.63 mV; III, 1.19 mV; IV, 2.46 mV; V, 1.0 mV; VI, 0.83 mV; and solvent blank, 0.6 mV. G C - M S also confirmed the presence o f a complete series of straight-chain saturated hydrocarbons (HC) in varying amounts from 21 HC to 29 HC, in the extracts (Figure 1). No E A G responses were ever observed to these compounds in linked G C - E A G analyses. Field Tests. In order to assess the effect o f individual compounds on male attractancy to pheromone traps, field trials conducted in 1986 compared the total mixture o f six compounds, in their naturally occurring ratio, with five-component mixtures in which either Z 9 - 1 4 : Aid, Z 9 - 1 4 : OH, Z 1 1 - 1 4 : Ac, or Z 1 1 -

1358

CORK ET AL. TABLE 4. G C RETENTION TIMES OF SYNTHETIC TETRADECEN-1-OLS AND COMPONENT V

Retention times (min) Compound

CP Wax 57CB a

CP Sil 5CB b

Component V

32.34

27.75

14 : OH

28.33

30.23

13-14 : OH

34.31

28.73

Z12-14 :OH E12-14 :OH

38.44 35.77

32.02 30.51

Z11-14: OH E 11-14 : OH

34.36 33.30

29.48 29.13

Z10-14 :OH E l 0 - 1 4 : OH

33.28 32.02

28.66 28.57

Z9-14 : OH E9-14 : OH

32.30 31.82

27.75 28.13

Z8-14 : OH E8-14:OH

31.82 31.35

27.30 27.77

Z7-14 :OH E7-14:OH

31.31 31.08

26.95 27.66

Z6-14 : OH E6-14 : OH

31.30 31.04

27.00 27.47

Z5-14 : OH E5-14: OH

31.73 31.59

27.32 27.89

Z4-14 : OH E4-14 : OH

30.70 30.70

27.05 27.80

Z3-14 : OH

30.21

27.22

~Oven temperature 70~ bOven temperature 70~

for 2 min then programmed at 20~ for 2 min then programmed at 20~

to 135~ to 115~

16:Ac had been removed. Z9-14: Ac and Z,E9,12-14:Ac were present in all lures tested because they were assumed to be necessary for attraction. Removal of either Z9-14 : Aid or Z11-14 : Ac was found to have no effect on trap catch compared to the total mixture. However, removing Z 1 1 - 1 6 : A c significantly reduced catch and removing Z 9 - 1 4 : O H significantly increased catch (Table 6). The two-component mixture of Z 9 - 1 4 : A c and Z, E9,12-14 :Ac in their naturally occurring ratio of approximately 100:5, caught significantly more males than the six-component mixture, although it was significantly less attrac-

AFRICAN ARMYWORM PHEROMONE

1359

TABLE 5. G C RETENTION TIMES OF SYNTHETIC HEXADECENYL ACETATES AND COMPONENT VI

Retention times (min) Compound

CP Wax 57CB ~

OV 275 a

CP Sil 5 CB b

Component VI

29.39

21.67

30.90

16 : Ac

27.12

20.70

31.28

15-16 : Ac

31.55

23.02

31.03

Z14-16: Ac E14-16 : Ac

35.00 32.78

25.00 23.30

31.62 31.28

Z13-16 : Ac E13-16:Ac

31.40 30.38

22.80 21.80

31.18 31.05

Z12-16 : Ac E l 2 - 1 6 : Ac

30.35 29.50

22.25 21.35

31.04 30.99

Z11-16 : Ac E11-16:Ac

29.38 29.20

21.65 21.20

30.84 30.91

Z10-16 : Ac E l 0 - 1 6 : Ac

28.75 28.65

21.10 20.82

30.72 30.82

Z 9 - 1 6 : Ac E 9 - 1 6 :Ac

28.13 28.30

20.75 20.55

30.68 30.76

ZS-16 : Ac E 8 - 1 6 : Ac

27.85 28.02

20.55 20.30

30.52 30.68

Z 7 - 1 6 : Ac E 7 - 1 6 : Ac

27.47 27.88

20.40 20.15

30.50 30.68

Z 6 - 1 6 : Ac E 6 - 1 6 : Ac

27.25 27.76

20.20 20.15

30.44 30.70

Z 5 - 1 6 : Ac E 5 - 1 6 : Ac

27.45 27.90

20.23 20.15

30.53 30.69

Z 4 - 1 6 : Ac E4-16:Ac

26.63 27.88

20.00 20.00

30.49 30.77

Z 3 - 1 6 : Ac E 3 - 1 6 : Ac

27.35 27.60

19.90 19.65

30.64 30.72

aOven temperature 70~ bOven temperature 70~

for 2 min then programmed at 20~ for 2 min then programmed at 4~

tive than the five-component For comparative (Nyirenda, 14:Ac,

purposes,

to 120~ to 210~

mixture in which Z9-14 : OH had been removed. the "standard"

lure used throughout

East Africa,

1985, Haggis, 1986), a 100 : 7.5 mixture of Z9-14 : Ac and Z, E9,12-

was also included in the trial. Catches with this were not significantly

1360

CORK ET AL.

TABLE 6. CATCHES OF MALE S. exempta MOTHS IN TRAPS BAITED WITH COMBINATIONS OF PHEROMONE COMPONENTS IN NATURALLY OCCURRING RATIOS AND STANDARD LURE OF 100 : 7.5, Z 9 - 1 4 : Ac to Z,E9,12-14 : Ac (4 REPLICATES, 28 NIGHTS)

Pheromone component (/zg) Z9-14:Ald

Z9-14:Ac

Zll-14:Ac

Z, E9,12 - 14.'Ac

Z9-14:OH

Zll-16:Ac

Mean catch/ trap/nighta

15 0 15 15 15

1000 1000 1000 1000 1000

20 20 0 20 20

50 50 50 50 50

35 35 35 0 35

50 50 50 50 0

22c 18cd 17cd 51a 15d

0 0

1000 1000

0 0

50 75

0 0

0 0

40b 46ab

aActual mean catches/trap/night; means followed by the same letter are not significantly different at the 5 % level by DMRT using log(x + 1) transformed data.

different from those of either the two-component mixture with the natural ratio of compounds or the five-component mixture without Z9-14: OH, although the latter did catch 10% more moths (Table 6). Because of the antagonistic nature of Z9-14 : OH, it was not included in field trials conducted in 1987. Table 7 shows the results from a trial in which individual components were removed from the resulting five-component mixture. The most dramatic result was obtained by the removal of Z, E9,12-14: Ac, which resulted in no catch at all. Differences between catches for the other TABLE 7. CATCHES OF MALE S. exempta MOTHS IN TRAPS BAITED WITH COMBINATIONS OF FIVE OF THE COMPOUNDS IDENTIFIED COMPARED TO Two-CoMPONENT MIXTURE (4 REPLICATES, 24 NIGHTS)

Pheromone component (txg) Z9-14 : Aid

Z9-14 : Ac

ZI 1-14 : Ac

Z, E9,12 - 14:Ac

Z11-16 : Ac

Mean catch/ trap/night"

15 15 15 15 0 0

1000 1000 1000 1000 1000 1000

20 20 20 0 20 0

50 50 0 50 50 50

50 0 50 50 50 0

11.3a 10.5a 0b 13.1a 12.7a 9.8a

a Actual mean catches/trap/night; means followed by the same letter are not significantly different at the 5 % level by DMRT using log(x + 1) transformed data.

1361

AFRICAN ARMYWORM PHEROMONE

TABLE 8. CATCHES OF MALE S.

exempta MOTHS IN TRAPS BAITED WITH T w o -

AND FIVE-

COMPONENTBLENDSWITHDIFFERENTLEVELS OF Z,E9,12-14 : AC (4 REPLICATES,24 NIGHTS) Pheromone component(t~g) Z9-14 :Ald

Z9-14 : Ac

Zll-14:Ac

Z,E9,12-14: Ac

Zll-16:Ac

Meancatch/trap/night~

15 15 15

1000 1000 1000

20 20 20

50 75 100

50 50 50

15.3a 15.5a 9.8b

0 0 0

1000 1000 1000

0 0 0

50 75 100

0 0 0

12.8b 12.8b 6.7c

~'Actual mean catch/trap/night; means followed by the same letter are not significantlydifferent at the 5% level by DMRT using log(x + 1) transformeddata.

treatments were not significant, although in each case mixtures without Z 1 1 16 : Ac caught fewer moths. The 1986 trials had also indicated that the 1 0 0 : 7 . 5 mixture of Z 9 - 1 4 : Ac and Z, E9,12-14 : Ac may be more attractive than the 100 : 5 mixture. In order to check this and provide a further comparison between the five-component blend and the two-component blend a trial was undertaken in which 5, 7.5, or 10 % Z, E9,12-14 :Ac was added to the two- and five-component mixtures (Table 8). In each case the five-component mixture caught significantly more moths, confirming previous results. There was no difference in catch between those with mixtures containing 5 or 7.5 % Z, E9,12-14:Ac, although the addition of 10% reduced catch significantly in both cases. The only newly identified compound to show any potential for increasing attractancy in the field from the 1986 field tests was Z 1 1 - 1 6 : A c . In order to confirm this, the " s t a n d a r d " two-component mixture was compared with mixtures in which between 2.5 and 10% Z 1 1 - 1 6 : A c had been added. In each case, the addition of Z 1 1 - 1 6 : Ac increased catch significantly above that of the twocomponent mixture, although the actual amount added did not seem to be important (Table 9).

CONCLUSION

This reassessment of the female sex pheromone of the African armyworm,

Spodoptera exempta, has confirmed the presence of Z 9 - 1 4 : A c and z, E g , 1 2 14 : Ac, in the ratio of 100 : 5 (Beevor et al., 1975). In addition, four new com-

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TABLE 9. CATCHES OF MALE S. e x e m p t a MOTHS IN TRAPS BAITED WITH STANDARD Two-CoMPONENT MIXTURE COMPARED TO THOSE WITH Z 1 1 - 1 6 : A c ADDED

(4 REPLICATES,20 NICHTS) Pheromone component (#g) Z9-14:Ac

Z,E9,12-14 :Ac

1000 1000 1000 1000

75 75 75 75

Zll-16:Ac 0 25 50 100

Mean catch~trap~night a 4.2a 5.9b 5.7b 6.4b

~Actual mean catches/trap/night; means followed by the same letter are not significantly different at the 5% level by DMRT using log(x + 1) transformed data.

pounds, Z9-14 : Aid, Z11-14: Ac, Z9-14 : OH, and Z11-16 : Ac, were characterized from virgin female ovipositor washings and volatiles. These compounds were present at 1.5, 2, 3.5, and 4 % of the major component, respectively. Field tests confirmed that Z9-14 :Ac and Z, E9,12-14 : Ac are essential for attractiveness and showed that addition of Z l l - 1 6 : A c at 2.5-10% of the major component could significantly increase catches. Using a 1 mg loading of pheromone, the other compounds identified had no effect at the naturally occurring level except Z9-14 :OH, which significantly reduced catch. The study was conducted with the intention of maximizing trap catch for use in the East African monitoring system without incurring high cost. For this reason, compounds of commercial purity were used. Z9-14:Ac currently employed in the network contains approximately 2 % of the E isomer, which could have an effect on behavior. Similarly, it was important that the effective lifetime of the lures was closely related to those used in the regional monitoring system, although the loading used may not have been optimal for testing the effects of the minor components. Z9-14 : Ac, Z11-14 : Ac, Z9-14 : OH, and Z, E9,12-14 : Ac are commonly found in the sex pheromones of Noctuidae and the genus Spodoptera in particular (Am et al., 1986). However, Z9-14:Ald and Z11-16:Ac have only previously been identified in North American species, such as Spodoptera frugiperda (Mitchell et al., 1985; Tumlinson et al., 1986), and Z11-16:Ac in Spodoptera eridania (Teal et al., 1985) and Spodoptera sunia (Bestmann et al., 1988). The apparent lack of effect of Z9-14 : Aid and Z11-14 : Ac on trap catch, at the levels tested, does not necessarily mean they do not influence insect behavior. Pheromones are generally considered to be species specific (Card6 and Baker, 1984), so where a number of closely related sympatric species have

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p h e r o m o n e c o m p o n e n t s in c o m m o n , it m a y be anticipated that the other, often m i n o r , c o m p o n e n t s c o u l d be i n v o l v e d in deterring cross-attraction (Grant et al., 1988; L i n n e t al., 1987). Spodoptera triturata is s o m e t i m e s caught in S. exempta traps, and so it is c o n c e i v a b l e that, in o r d e r to ensure species specificity, all the c o m p o n e n t s presently identified, e x c e p t perhaps Z 9 - 1 4 : O H , will h a v e to be included in the lures. W i n d - t u n n e l studies and further field trials are n o w in progress in an attempt to elucidate the possible role o f the m i n o r c o m p o n e n t s in inter- and intraspecific c h e m i c a l c o m m u n i c a t i o n . Acknowledgments--The authors would like to thank Dr. D.J.W. Rose, Dr. L.D.C. Fishpool, Mr. W.W. Page, and Mr. C.F. Dewhurst, ODNRI, for their invaluable comments and assistance with field work; Mr. W.Y.K. Malinga, Director, Cotton Research Station, Kibos, Kenya, for providing the facilities for field testing; Miss F. Casci, ODNRI, for assistance with field testing; Miss S.M. Green, ODNRI, for advice and assistance with statistical analyses; Miss J.L. Mullings, ODNRI, for technical assistance with the laboratory studies; and Dr. D.R. Hall and Dr. D.G. Campion for critical review of the manuscript. This study was partially supported by EEC DG XII contract TSD.A.205UK(H). Insects were imported under MAFF license No PHF.748/147. REFERENCES ARN, H., TOTH, M., and PR1ESNER,E. 1986. List of sex pheromones of Lepidoptera and related attractants. OILB-SROP/IOBC-WPRS, France. BEEVOR, P.S., HALL, D.R., LESTER, R., POPPI, R.G., READ, J.S., and NESBITT,B.F. 1975. Sex pheromones of the armyworm moth, Spodoptera exempta (Walker). Experientia 31:22-23. BEEVOR, P.S., CORK, A., HALL, D.R., NESBITT, B.F., DAY, R.K., and MUMFORD,J.D. 1986. Components of female sex pheromone of cocoa pod borer moth, Conopomorpha cramerella. J. Chem. Ecol. 12:1-23. BESTMANN,H.J., ATTYGALLE,A.B., SCHWARZ,J., VOSTROWSKY,O., and KNAUF,W. 1988. Identification of sex pheromone components of Spodoptera sunia Guenre (Lepidoptem: Noctuidae). J. Chem. Ecol. 14:683-690. BLAIR, B.W., and TANNOCK,J. 1977. A possible pheromone for Spodoptera triturata (Walker) (Lepidoptera: Noctuidae). Rhod. J. Agric. Res. 15:225-226. BROWN, E.S., BETTS,E., and RAINEY,R.C. 1969. Seasonal changes in distribution of the African armyworrn, Spodoptera exempta (Walker) (Lepidoptera: Noctuidae), with special reference to eastern Africa. Bull. Entomol. Res. 58:661-728. BUSER, H.R., and ARN, H. 1975. Analysis of insect pheromones by quadrupole mass fragmentography and high resolution gas chromatography. J. Chromatogr. 106:83-95. CAMPION, D.G., ODIYO, P.O., MUSHI, A.M., HALL, D.R., LESTER,R., and NESBITT,B.F. 1976. Field tests with the synthetic sex pheromone of the African armyworm, Spodoptera exempta (Wlk). C.O.P.R. Misc. Rep. 25:1-4. CARDI~,R.T., and BAKER,T.C. 1984. Sex communication with pheromones, pp. 355-383, in W.T. Bell and R.T. Card6 (eds.). Chemical Ecology of Insects. Chapman and Hall, London. FAURE,J.C. 1943. Phase variation in the armyworm, Laphygma exempta. (Wlk.). Sci. Bull. Dep. Agric. For. Un. S. Afr. No. 234:17. GRANT, A.J., O'CONNELL, R.J., and HAMMOND,A.M., JR. 1988. A comparative study of pheromone perception in two species of noctuid moths. J. Insect Behav. 1:75-96. GROB, K., and ZURCHER,F. 1976. Stripping of organic trace substances from water. Equipment and procedure. J. Chromatogr. 117:285-294.

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GUDZINOWICZ, B.J., GUDZINOWICZ, M.J., and MARTIN, H.F. 1976. Fundamentals of Integrated GC-MS. Part II. Mass Spectroscopy. Marcel Dekker, New York. HAGGIS, M.J. 1986. Distribution of the African armyworm, Spodoptera exempta (Walker) (Lepidoptera: Noctuidae), and the frequency of larval outbreaks in Africa and Arabia. Bull. Entomol. Res. 76:151-170. HARRIS, W.E., and HABGOOD, H.W. 1966. Programmed Temperature Gas Chromatography. John Wiley & Sons, New York. LINN, C.E., JR., CAMPBELL,M.G., and ROELOFS,W.L. 1987. Pheromone components and active spaces: What do moths smell and where do they smell it? Science 237:650-652. LOFSTEDT, C., VANDER PERS, J.N.C., LOFQVIST,J., LANNE, B.S., APPELGREN,M., BERGSTROM, G., and THELIN, B. 1982. Sex pheromone components of the turnip moth, Agrotis segetum. Chemical identification, electrophysiological evaluation and behavioural activity. J. Chem. Ecol. 8:1305-1321. MITCHELL, E.R., TUMLINSON,J.H., and MCNEIL, J.N. 1985. Field evaluation of commercial pheromone formulations and traps using a more effective sex pheromone blend for the fall armyworm (Lepidoptera: Noctuidae). J. Econ. Entomol. 78:1364-1369. NESB1TT, B.F., BEEVOR, P.S., LESTER, R., DAVIES, J.C., and SESHU REDDY, K.V. 1979. Components of the sex pheromone of the spotted stalk borer, Chilo partellus. (Swinhoe) (Lepidoptera: Pyralidae): Identification and preliminary field trials. J. Chem. Ecol. 5:153-163. NESBITT, B.F., BEEVOR, P.S., CORK, A., HALL, D.R., DAVID, H., and NANDAGOPAL,V. 1986. The sex pheromone of sugarcane stalk borer, Chilo auricilius. Identification of four components and field tests. J. Chem. Ecol. 12:1377-1387. NYIRENDA, G.K.C. 1985. Persistent populations of males of the African armyworm, Spodoptera exempta (Walker) (Lepidoptera: Noctuidae), in Malawi. Bull. Entomol. Res. 75:405-415. RILEY, J.R., REYNOLDS, D.R., and FARMERY,M.J. 1983. Observations on flight behaviour of the armyworm moth, Spodoptera exempta, at the emergence site using radar and infra-red optical techniques. Ecol. EntomoL 8:395-418. ROSE, D.J.W. 1979. The significance of low density populations of the African armyworm, Spodoptera exempta. Phil. Trans. R. Soc. London B287:393-402. ROSE, D.J.W., and DEWHURST, C.F. 1979. The African armyworm, Spodoptera exempta--congregation of moths in trees before flight. Entomol. Exp. Appl. 26:346-348. ROSE, D.J.W., PAGE, W.W., DEWHURST,C.F., RILEY, J.R., REYNOLDS, D.R., PEDGLEY,D.E., and TUCKER, M.R. 1985. Downwind migration of the African armyworm moth, Spodoptera exempta, studied by mark-and-recapture and by radar. Ecol. Entomol. 10:299-313. SOWER, L.L., COFFELT,J.A., and VICK, K.W. 1973. Sex pheromone: A simple method of obtaining relatively pure material from females of five species of moths. J. Econ. Entomol. 66:12201222. STEINBRECHT,R.A. 1982. Electrophysiological assay of synthetic and natural sex pheromones in the African armyworm moth, Spodoptera exempta. Entomol. Exp. Appl. 32:13-22. TEAL, P.E.A., MITCHELL, E.R., TUMLINSON,J.H., HEATH, R.R., and SUGIE, H. 1985. Identification of volatile sex pheromone components released by the southern armyworm, Spodoptera eridania (Cramer). J. Chem. Ecol. 11:717-725. TUCKER, M.R., and PEDCLEY, D.E. 1983. Rainfall and outbreaks of the African armyworm, Spodoptera exempta (Walker) (Lepidoptera: Noctuidae). Bull. Entomol. Res. 73:195-199. TUMLINSON, J.H., HEATH, R.R., and TEAL, P.E.A. 1982. Analysis of chemical communications systems of Lepidoptera, pp. 1-25, in B.A. Leonhardt and M. Beroza (eds.). Insect Pheromone Technology: Chemistry and Applications. ACS Symposium Series 190. American Chemical Society, Washington, D.C. TUMLINSON, J.H., MITCHELL, E.R., TEAL, P.E.A., HEATH, R.R., and MENGELKOCH,L.J. 1986. Sex pheromone of fall armyworm, Spodopterafrugiperda (J.E. Smith). Identification of components critical to attraction in the field. J. Chem. Ecol. 12:1909-1926.