Chemistry and function of a pheromone produced by the male of the southern armyworm Pseudaletia separata

J. InsectPhysiol.,1972,Vol. 18,pp. 781to 789. PergamonPress.

Printed in Great Britain

CHEMISTRY AND FUNCTION OF A PHEROMONE PRODUCED BY THE MALE OF THE SOUTHERN ARMYWORM PSEUDALETIA SEPARATA JOHN

R. CLEARWATER

Department of Botany and Zoology, Massey University, Palmerston North, New Zealand (Receiwed 17 August 1971) Abstract-The major component of extracts obtained from the male pheromone releasing organ of Pseudaletia separata is identified as benzaldehyde. Evidence is presented suggesting that this compound functions as an arrestant, facilitating mating by preventing the escape of the female. INTRODUCTION %IALES of several species of the Noctuidae possess tracts of fine evetiable scales known as ‘hair pencils’ (ELTRINGHAM,1925). These structures are everted a few seconds before copulation occurs (APLIN and BIRCH, 1968; BIRCH, 1970). Although no biological function has been proven, the fact that several volatile compounds have been identified from the hair pencils suggests that the function is one of chemical intraspecific communication (APLIN and BIRCH, 1968, 1970). The apparent lack of overt behavioural responses by female noctuids when stimulated by male produced pheromones has made the allocation of specific functions to specific compounds very difficult in this group (SHOREY, 1964; GRANT, 1970). Most of the complex molecules produced in minute amounts by insects for intraspecific communications are indetectable by the olfactory sensors of other phyla. The male produced pheromones for sexual communication are an exception, as many can be detected by the human nose. For example, Hepialus hectus is reported to smell of pineapple (DEEGENER,1902); Euploea raabnanthus of vanilla; Danaus plexippus of milkweed or red clover (BROWERet al., 1965); and Lethocerus indicens of cinnamon (CAILLOT and BOI~~ON,1954). The observation that males of Pseuakletia separata have a distinctive almond odour initiated this study of its pheromone. MATERIALS

AND METHODS

Experimental animals Moths were obtained either from a laboratory culture maintained maize seedlings, or were captured with a light trap.

on a diet of

Chemistry of the pheromone Crude extracts of the hair pencil were analysed on a gas chromatograph, As the only other extractable compounds were epicuticular waxes, which were expected to differ markedly from the pheromone in molecular weight and volatility, 781

782

JOHNR. CLEARWATER

the complex distillation and thin layer techniques normally necessary (GASTON et al., 1966) were dispensed with. Hair pencils were rapidly dissected from males and 10 to 50 placed in 1 ml of methylene chloride. These extracts were kept at 10°C until analysed. Three columns were used: (1) 3% cyclohexane dimethanol succinate on 60-80 mesh Chromosorb W. (2) 12% ethylene glycol succinate on 70-80 mesh Anakron A. (3) 15% Carbowax 20M on loo-120 mesh Gas chrom Q. A mass spectrum of these extracts was obtained with an AEI MS 902 mass spectrometer with an ionizing energy of 70 eV. The extract was concentrated, vaporized externally and admitted to the ion source at 100°C. Mass measurements were made at a resolving power of 12,000. The development of sexual maturity

Immediately after emergence, 34 unmated moths from a laboratory culture were distributed between three glass observation cages each of an internal volume of 2.8 x 1W cm3. A solution of loo/, sucrose was continuously available. The light intensity was less than 1 lx. Each cage was scored for the number of mated pairs, observations being taken at approximately hourly intervals between 06.00 and 12.00 hr midnight for 10 days. The dcerelopment of the pheromone An estimation of the concentration of benzaldehyde in the hair pencils of males during the period of adult activity was obtained with the methods used for identification of the pheromone. Four to five male moths were taken from a laboratory culture when they had reached the required age. The hair pencils of these moths were placed in 1 ml of methylene chloride and analysed on a Carbowax 20M column at 110°C. Function of the pheromone

The response of the female of P. separata to benzaldehyde was examined by means of an apparatus similar to that used by SHOFEY (1964) to demonstrate the anemotactic response of Trichoplusia ni. A glass tube 60 x 6 cm with lines dividing it into six equal lengths was capped with wire gauze ends. The light source was a Variac controlled incandescent bulb producing 0.5 lx. The air temperature was 19 to 21°C. A centrifugal fan produced an air flow down the tube. A steady introduction of benzaldehyde to the air flow was produced by a movable glass reservoir with an attached capillary. The concentration of benzaldehyde in the air flow was not measured. Ten virgin females were placed in the tube immediately following emergence, and experimental observations commenced three nights later. The experiment was carried out twice and the distributions noted in two ways. Initially, the distribution of the moths was noted every half hour from 07.00 to 10.00 hr. These readings, taken in the absence of benzaldehyde, served as a control. The benzaldehyde releaser was then incorporated into the air flow

CHEMISTFtY AND FUNCTION

OF A PHEROMONE OF THE SOUTHERN ARMYWORM

783

and a further

series of readings taken every 5 min between 10.05 and 11.30 hr. The second set of observations with more precise controls was carried out with a fresh experimental population under the same conditions. The experiment proceeded for 3 hr between 09.00 and 12.00 hr with readings taken every 5 min. During the first and last hour, benzaldehyde was not introduced and the positions of the moths during these times served as a control. Benzaldehyde was introduced between 10.00 and 10.55 hr.

RESULTS Chemistry of the pheromone Analysis of the hair pencil extract showed the presence of a peak, the behaviour of which on three columns was consistent with the presence of benzaldehyde (Fig. 1).

Column Carbowax 20M Carbowax 20M Ethylene glycol succinate Cyclohexane Dimethanol succinate

Temperature W)

Unknown (mm)

Standard benzaldehyde (mm)

100 96 100

91, 90 92,92 16, 17

91 18,18

100-101* 98

22 27, 29

15 26

* Unstable temperatures resulted in a considerable disparity between test and standard.

Addition of standard benzaldehyde to an extract which was then run on the cyclohexane dimethanol succinate column confirmed the fact that standard and unknown had the same retention time, as complete superposition of the peaks occurred. In addition to the majar peak, a smaller peak was observed with a retention of 75 mm on the Carbowax 20M column. This peak was not identified. The mass spectrum showed large peaks at 77, 105, and 106, typical of the disintegration pattern of benzaldehyde (MCCOLLUMand MAYERSON,1963) (Fig. 2). Radical (WI,)+ (C,H,C=O)+ (C,H,CHO) +

Table value 77.0391 105*0340 106W19

Experimental value 77.0391 1050339 106-0414

It was of interest that no peak at 122 (benzoic acid) was observed.

784

JOHN R. CLEARWATER

Carbowax 20 M

1~1 04%

Standard

2/A

50 H.f?/mt

Ethylene glycol succinate

Cyclobexone

dimetnanol slicctnale

;1 ii I

l-l

’

\ \r

111k --&A I%

i’.

.

2

I-II 5pLIIOHP/ml

Standard

5~1 I:100 Standard: unknown

1. GLC evidence for an identification with benzaidehyde. Carbowax 20M : Standard T = lOO”C, A = 3 x lo-lo; Extract T = 96”C, A = 1 x lo-lo. Ethylene glycol succinate: Standard T = lOO”C, A = 3 x 10-lo; Extract T = lOO”C, A = 1 x 10-10. Cyclohexane dimethanol succinate: T = lOO-lOl”C, A = 3200; Extract T = 100-lOl”C, 98°C; A = 40. Superposition T = 98”C, A = 40.

FIG.

.-CH,CI;5

C,HsCHO* CstpO+

Mass units

FIG. 2. Mass spectrum of extract (50 HP/ml).

CHEMISTRY

AND FUNCTION

OF A PHEROMONE

OF THE SOUTHERN

Relationship between sexual maturity and concentration

785

ARMYWORM

ofpheromone

During the first and second night after emergence, feeding was frequent especially during the early hours, but no overt sexual behaviour could be observed. In all the three cages, extensive eversion of male genitalia was first observed on the third night, when the first successful mating was also noted. After reaching a peak, the mating frequency decreased rapidly (Fig. 3). Oviposition occurred 1 day after mating, the first eggs being laid on the fourth night.

Night

No.

FIG. 3. Mating frequency of P. separata. Thirty-four laboratory culture.

unmated

from

a

The development of the maximum benzaldehyde concentration in the hair pencil occurred on day 25 (Fig. 4). After very low concentrations immediately after emergence, the amount of benzaldehyde increased rapidly to a peak, after

-Column A --Column 8

Night

No.

FIG. 4. The development of pheromone in the hair pencil. Each point represents 8 to 10 hair pencils.

786

JOHN

R. CLEARWATER

which a slow decline was observed. Even at the end of adult activity, males retained 50 per cent of the pheromone that was observed on day 2-S. Function of the pheromone

The most striking point about the distributions was the tendency for the moths to aggregate in the first and last division of the tube (Fig. 5). Moths appeared to exhibit a distinct preference for resting on the gauze rather than the smooth glass

c

TO

c

10~00:1035 Eenzoldrhyde

9.00:9.55 Contml

c

11~00:11~55

COnrrOl

60

50 t

E ._ .p 5 5 f E

40

is

20

30

IO .i

0

123456

1234

Division No.

FIG. 5. The effect of benzaldehyde on the activity of the female. Ten unmated females (3 days old). Sum of distributions taken every 5 min over 1 hr.

walls of the tube. They would walk and fly through the middle of the tube but rarely stop there. The ratio of the sum of moths in the first and last compartments to the sum of the moths in the four middle compartments was considered to be an index of the activity of the experimental population. These ratios were compared with a Xs test with 1 d.f. For the first set of observations comparison of (a) and (b) ratios (Table 1): X2 =

6-56; o-02> P>O*Ol.

For the second set: (a) and (b) (b) and (c) (a) and (c)

x2 = 6.30; 0.02 > P > 0.01, x2 = 14.09; P P> 0.10.

This established that the control distributions did not differ significantly but there was a highly significant difference between the controls and the test at the 2 per cent level of significance. In the presence of benzaldehyde, females are less active.

AND FUNCTIONOF A PHEROMONE OF THE SOUTHERN ARMYWORM

CHEMISTRY

787

TABLE I-EFFECT OF BENZALDEHYDE ON FEMALES Division No. 1

2

3

4

5

6

2 (End divisions)

z (Mid divisions)

hr

13

7

6

5

3

24

37

21

hr

27

3

6

5

9

47

74

23

hr

66

2

3

7

12

30

96

24

hr

64

1

0

4

8

43

107

13

hr

40

3

3

7

1s

49

89

31

Experiment Control 07.00-10.00 Test 10.05-11.30 Control 09.00-09.55 Test 10.00-10.55 Control 11.00-11.55

I(a) I(b) II(a) III(b) III(c)

DISCUSSION The

preceding

extractable

chemical

evidence

from the hair pencil of

aldehyde from the Noctuidae in

proves

conclusively

that benzaldehyde

is

P. separata. This is the second report of benz-

; APLIN and BIRCH (1968) demonstrating

its presence

Leucania impura, L. conigera and Phlogophora meticulma. Other compounds

identified from male noctuids are Z-phenyl ethanol, 6 methyl-hept-5-en-2-one and the corresponding alcohol, 2 methyl butanoic acid, isobutyric acid, benzyl alcohol, and pinocarvone There

(APLIN

and BIRCH, 1970).

are at least two

sepmata.

Insects

sequently

phenyl

possible

are unable

metabolic

to synthesize

alanine is an essential component

The first possibility

involves the modification

pathway

producing

the quinones

the food

ingested

subsequently

during

modified

If benzaldehyde to expect

between

in

P.

con-

may contain cinnamic

from the

Alternatively acid which

is

of this species it would be reasonable

the mating

frequency

and the appearance

of

in the hair pencils. of benzaldehyde activity.

decrease.

ring,

of the diet (GILMOUR, 1965).

essential for sclerotin production.

is the male pheromone

overt

development

benzene

of one of the intermediates

the larval period

concentration sexual

for benzaldehyde

to form benzaldehyde.

a relationship

benzaldehyde

origins

the conjugated

It appears highly significant that the highest is observed on the day preceding the initiation of

The form of the mating frequency and benzaldehyde graphs is similar, both rapidly reaching a peak followed by a gradual

SHOREY and GASTON (1965)

demonstrated

attained the ‘maximum of 1 pg of pheromone gence whilst the maximum

mating frequency

that females

of

T. ni first

on the second night following occurred

approximately

emer-

24 hr later

(SHOREY, 1964; SHOREY et al., 1968). These workers also report that there is no great decrease in pheromone concentration with increasing age, and suggest that this is because T. ni mates more than once during its lifetime. In contrast, the quantity of the pheromone decreases rapidly after reaching a peak in Bombyx mori and

Porthetria dispm; as these two species mate only once, a continuing

high

788

JOHN R. CLEARWATER

pheromone level would have no biological significance (COLLINS and POTTS, 1932 ; KARLSONand BUTENANDT,1959). Before a compound can be identified as a pheromone, a specific ethological response must be elicited when the compound is present in physiological quantities. BUTENANDTand HECKER(1961) demonstrated that synthetic trans-lo-cislZhexadecadien-l-01 elicited the circling dance from males of B. mori when present at concentrations of lo-l2 pg/ml of air establishing this compound as the pheromone of this species. By contrast, the suggestion that 2-2-dimethyl-3-isopropylidene-cyclopropyl propionate is the pheromone of Periplaneta awicana is unacceptable, as the synthesized compound is now known to be completely inactive in stimulating the male cockroach (JACOBSONand BEROZA,1965). None of the many chemicals so far identified from the hair pencils of noctuids has been shown to possess a specific effect on the female. The change in distribution of females of P. separata when in the presence of benzaldehyde indicates that the effect of this compound on the female is to inhibit movement. This hypothesis is in consonance with earlier observations that males successfully mated only with females that did not attempt to move away as the male approached (Clearwater, unpublished results). The observation by BIRCH (1970), that females of P. meticulosa (Noctuidae) with antennae removed typically took flight as soon as a courting male made contact, adds further support. It is suggested that an approaching male of P. separata stimulates the female in two ways. The visual stimulus of an approaching object is likely to initiate a general escape reaction. The chemical stimulus that is simultaneously presented is postulated to inhibit this reaction. A similar interpretation is proposed by BROWEF~ et al. (1965) for Danaus g&&us berenice. The female queen butterfly avoids the visual stimulus presented by the pursuing male by initiating an escape flight which is sustained and vigorous. As the male overtakes and ‘hair pencils’ the female, it slows and descends to the ground. Males deprived of hair pencils actively pursue females and engage in the same aerial manmuvres but after having caused their mates to alight, fail to induce them to remain on the ground (PLISKE and EISNER, 1969). The male pheromones of P. separata and D. g. berenike can thus be considered arrestant pheromones according to the terminology of DJZTHIER et al. (1960). Benzaldehyde has previously been reported in insects as a defensive secretion. Aquatic dytiscid beetles secrete this compound, possibly as an antibacterial agent (GILMOUR, 1965). Benzaldehyde is also the major component of the defensive secretion of the ant Vero~~sor pergander’ (BLUM et al., 1969). Consequently, APLIN and BIRCH (1968) speculate that this secretion may serve to deter rival males. The fact that the males disseminate the pheromone when approaching a female and not when approached by males while in copula render this supposition unlikely. REFERENCES APLIN R. T. and BIRCH M. C. (1968) Pheromones from the abdominal brushes of male noctuid Lepidoptera. Nature, Lond. 217, 1167-1168.

CHEMISTRYANDFUNCTIONOF A PHEROMONE OF THE SOUTHERN ARMYWORM

789

APLIN R. T. and BIRCH M. C. (1970) Identification of odorous compounds from male Lepidoptera. Experientiu 26, 1193-l 194. BIRCH M. C. (1970) Pre-courtship use of abdominal brushes by the nocturnal moth Phlogophora meticulosa (Lepidoptera: Noctuidae). Anim. Behav. 18, 310-316. BLUM M. S., P~DOVANIF., CURLEY A., and HAWK R. E. (1969) Benzaldehyde: a defensive secretion of a harvester ant. Camp. Biochem. Physiol. 29, 461-465. BROWERL. P., BROWERJ. V. Z., and CRANSTONF. P. (1965) Courtship behaviour of the queen butterfly Danaus gilippus berenice (Cramer). Zoologica, N. Y. 50, l-39. BUTENANDTA. and HECKERE. (1961) Synthese des Bombykols, des Sexual-Lockstoffes des Seidenspinners, und seiner geometrischen Isomeren. Angew Chem. 73, 349-353. CAILLOT Y. and BOI~~ON C. (1954) Diveloppement larvaire du Belostome (Lethocerus indicus Lep.) Insecte Hemipthre, Hydrocoryse, Cryptocerate. Annls Sci. nut. (Zool.) (11) 16, 51-64. COLLINS C. W. and POTTS S. F. (1932) Attractants for the flying gypsy moths as an aid in locating new infestations. Tech. Bull. U.S. Dep. Agric., 336, pp. l-43. DEEGENERP. (1902) Das Duftorgan von Hepiulus hectus. Z. wiss. Zool. 71, 276-295. DETHIER V. G., BROWNEL. B., and SMITH C. N. (1960) The designation of chemicals in terms of the responses they elicit from insects. J. econ. Ent. 53, 134-136. ELTRINGHAMH. (1925) On the abdominal brushes in certain male noctuid moths. Trans. ent. Sot. Lond., 1925, l-5. GA~TON L. K., FUKUTO T. R., and SHOREY H. H. (1966) Sex pheromones of noctuid moths-IX. Isolation techniques and quantitative analysis for the pheromones with special reference to that of Trichoplusia ni (Lepidoptera: Noctuidae). Ann. ent. Sot. Am. 59, 1062-1066. GILMOURD. (1965) The Metabolism of Insects. Oliver 6 Boyd, Edinburgh. GRANT G. G. (1970) Evidence for a male sex pheromone in the noctuid Trichoplusia ni. Nature, Lond. 227, 1345-1346. JACOBSONM. and BEROZAM. (1965) American cockroach sex attractant. Science, Wash. 147, 748-749. KARLSONP. and BUTENANL-JT A. (1959) Pheromones (ectohormones) in insects. A. Rev. Ent. 4, 39-58. MCCOLLUM J. D. and MEYER~~N S. (1963) Organic ions in the gas phase-X. Decomposition of benzaldehyde under electron impact. J. Am. them. Sot. 85, 1739-1741. PLISKET. E. and EISNERT. (1969) Sex pheromone of the Queen butterfly: biology. Science, Wash. 164, 1170-1172. SHOREY H. H. (1964) Sex pheromones of noctuid moths-II. Mating behaviour of Trichoplusia ni (Lepidoptera: Noctuidae) with special reference to the role of the sex pheromone. Ann. ent. Sot. Am. 57, 371-377. SHOREYH. H. and GASTONL. K. (1965) Sex pheromones of noctuid moths-VII. Quantitative aspects of the production and release of pheromone by females of Trichoplusia ni (Lepidoptera: Noctuidae). Ann. ent. Sot. Am. 68, 604-608. SHOREYH. H., MCFARLANDS. U., and GA~TONL. K. (1968) Sex pheromones of noctuid moths-XIII. Changes in pheromone quantity as related to reproductive age and mating history in females of seven species of Noctuidae (Lepidoptera). Ann. ent. Sot. Am. 61, 372-376.