Journal of Chemical Ecology, Vol. 18, No. 3, 1992

IDENTIFICATION PHEROMONE

XXX.

CLEARWlNG

AND

SYNTHESIS

SEX PHEROMONE MOTH

OF INSECT OF MULBERRY

Paradoxecia pieli

LIEU 1

TAN ZHONG-XIN, 2 LIN GUO-QIANG, 2'* LIU HAN-QUAN, 2 PU GUAN-QIN, 3 DU HE-MING, 3 MAO JIAN-PIN, 3 MENG LIAN-ZHONG, 4 and WU CAI-HONG4 2Shanghai Institute of Organic Chemistry Academia Sinica 345 Lingling Rd., 200032, Shanghai, China 3Su Zhou College of Sericulture 215151, Jiang Su, China 4Department of Biology Peking University 100871, Beijing, China (Received April 15, 1991; accepted October 31, 1991) Abstract--(E,Z)-3,13-Octadecadienyl acetate (la) and (E,Z)-3,13-octadecadien-l-ol (2a) were identified from the sex pheromone gland of the virgin female mulberry clearwing moth Paradoxecia pieli L., by GC analysis, EAG, SCR survey, and field bioassay. One female equivalent contained 250 ng of l a and 30 ng of 2a. In the field tests, 100/~g of synthetic l a was attractive to male moths of the species. Key Words--Insect sex pheromone, mulberry clearwing moth, Lepidoptera, Sesiidae, Paradoxeciapieli L. (E,Z)-3,13-octadecadien-yl acetate, (E,Z)-3,13octadecadien-l-ol, single cell recording, EAG.

INTRODUCTION

The mulberry clearwing moth, Paroxloxeciapieli L. (Sesiidae),is distributedin Jiang Su, Zhe Jiang, Si Chuan and Gui Zhou provinces in China. The larvae *To w h o m correspondence should be addressed. lIdentificationand Synthesis of Insect Pheromone XXIX. Sex Pheromone of the Vine Tree Borer Parathrene RegalisB. Guo Guang-zhong, Liu Han-quan, Lin Guo-qiang, W u Cai-hong, Meng Lian-zhong, Organic Cl~mistry(CH.), 504, 1990. 419 0098-0331/92/0300-0419506.50/09 1992PlenumPublishingCorporation

420

TAN ET AL. TABLE 1. POSSIBLECHEMICALSTRUCTUREOF SEX PHERONOMEOF SESIIDAEa 13 3 n-C4H9CH= CH(CH2)sCH= CH(CHz)2OR R = Ac la lb lc ld

E,Z Z,E

Z,Z E,E

R = H 2a 2b

E,Z

Z,E 2c Z,Z 2 d E,E

aE,Z describes the E form of configuration at A3 and the Z form at AI3. Similar meanings apply to Z,E, Z,Z and E,E. damage mulberry trees by making holes in the twigs. In the most severe situations, 30% of the twigs and leaf sprouts could be killed. The larvae and nymphs remain inside the twigs, therefore, it is difficult to kill this insect by spraying with insecticides. Because the insect has only one generation each year, it may be possible to use a mass-trapping technique with insect sex pheromone as a control. The sex pheromone of Sesiidae has been identified as 3,13-octadecadien1-ol, or its acetate, with few exceptions (Zhang et al., 1986). In our previous reports, the major sex pheromones of the poplar twig clearwing moth, Paranthrene tabaniformis Rott, (Zhang et al., 1986); the poplar pole clearwing moth, Sphecia siningensis Hsu (Guo et al., 1990a); and the vine tree borer, Paranthrene regalis Bulter, (Guo et al., 1990b) were identified as (E,Z)-3,13-octadecadien-l-ol (2a), (Z,Z)-3,13-octadecadien-l-ol (2e), and (E,Z)-3,13octadecadien-l-ol (2a), 5 respectively (Table 1). Here we report the identification of the sex pheromone of the mulberry clearwing moth. METHODS AND MATERIALS

Pheromone Collection. Twigs in which either nymphs or the last instar larvae were located were removed from mulberry trees in Ru Guo County in late May, and they were stored in the laboratory under normal conditions. Care was taken to keep adequate humidity by occasionally watering some pieces of cotton placed adjacent to the twigs. Adults were sexed immediately upon emergence. The ovipositors of 1- to 2-day-old virgin female moths (altogether 25 virgin females were collected for use) were removed and dipped in acetyl acetate. 5(E,Z)-3,13-Octadecadien-1-ol and its Z,Z-isomer were abbreviatedas (E,Z)-3,13-18 : OH and (Z,Z)3,13-18 :OH, respectively. Similar abbreviations were used for other OH isomers and their acetate isomers.

Paradoxecia pieli

PHEROMONE

421

The extract was then filtered over glass wool and the clean filtrate was concentrated, sealed in a glass tube (ID 1.0 mm), and stored in a freezer until needed for EAG survey, GC analysis, and field tests. The antennae of 1- to 2day-old unmated male moths were cut off according to the published paper for EAG bioassays (Roelofs et al., 1971). GC Analysis. GC 102 instrument (Shanghai Analytical Instrument Factory) was employed for GC analysis under two conditions: condition A--glass column 5 m x 3 mm ID, packed with 3% OV-1 on 80-100 mesh Gas Chrom Q, column temperature 210~ condition B--glass column 5 m x 3 mm ID, packed with 5% DEGS on 80-100 mesh Gas Chrom Q column temperature 180~ Single Cell Recording (SCR). The electrophysiological recording technique used was essentially the same as that applied by Kaissling and Thorson (1980). Each antenna was cut off from a male moth that had emerged two days before. One capillary (about 10/~m diameter) was placed over the end of the sensillum trichodeum olfactory hair where the tip had been removed to record the potential and nerve impulses of the receptor cell. The stimulus source was a piece of filter paper treated with 0.01/~g of the test compound. In our case, this was the minimum amount used for SCR so that the effect of possible impurity isomers could be diminished. FieM Bioassays. Field tests have been conducted since 1985 in Jiang Su Province during the adult flight season. Water traps were placed on the ground; each trap was made from a large bowl (25 cm diam.) filled with water and a lure. The lure was fastened with a piece of wire and placed in the middle of the bowl 3 cm above the water surface. Each lure was charged with either 100 /zg or 200/~g of the synthetic chemical using rolled filter paper or polyethylene hollow tubes as dispensers. The crude extract was also employed as the natural lure for comparison under the same conditions. Chemicals. All geometric isomers of 3,13-octadecadien-l-ol (2a-d) and their acetates (la-d) were synthesized at purities of over 95% as previously reported by Zhang et al. (1985). Satisfactory spectroscopic data (IR, [IH]NMR, MS) were obtained with all the above compounds.

RESULTS AND DISCUSSION

EAG Survey. Among the eight compounds ( l a - d and 2a-d) tested using the EAG survey technique on the mulberry clearwing moth, (E,Z)-3,13-18 : OAc (la) gave the highest response, and (E,Z)-3,13-18:OH (2a) exhibited the next highest in comparison with the untreated control. The EAG responses and calculated response index are shown in Table 2. The results indicate that the pheromone system might consist of several components with (E,Z)-3,1318:OAc (la) as the major component.

422

TAN ET AL.

Field Bioassays. The eight compounds ( l a - d and 2a-d) were bioassayed individually in the field (Table 3). Only (E,Z)-3,13-18:OAc (la) caught significant numbers of male moths; the other seven compounds ( l b - d and 2a-d) were not active during five days of testing in 1985. The preliminary test also showed that the addition of either (Z,Z)-3,13-18:OAc (lc) or (E,Z)-3,1318 : OH (2a) to (E,Z)-3,13-18 : OAc (la) gave no obvious increase in the number of male moths captured. Therefore, only (la) was used in the subsequent tests. GC Analysis. The crude washing from female sex gland (0.4-1 FE) was performed on GC on either column A (nonpolar column) or column B (polar column). For comparison, four synthetic acetates (la-d) and their corresponding alcohols (2a-d) were performed on GC under the same conditions. There were TABLE 2. E A G RESPONSES OF MULBERRY CLEARWING MOTH, Paradoxecia pieli L., TO ALL GEOMETRIC ISOMERS OF 3,13-OCTADECADIEN-1-O1 (2) AND THEIR ACETATES (1) EAG responses (mv) Chemicals

I

II

III

IV

Index a

(E,Z)-3,13-18 : OAc (la) (Z, E)-3,13-18 : OAc (lb) (Z,Z)-3,13-18: OAc (lc) (E,E)-3,13-18 : OAc (ld) (E,Z)-3,13-18: OH (2a) (Z,E)-3,13-18 : OH (2b) (Z,Z)-3,13-18 :OH (2e) (E, E)-3,13-18 : OH (2d)

1.0 0.5 0.2 0.2 0.3 0.3 0.2 0.3

1.0 0.3 0.1 0.1 0.8 0.3 0.2 0.1

1.0 0.5 0 0.2 0.8 0.1 0.3 0.1

1.0 0.43 0.1 0.17 0.63 0.23 0.23 0.17

1.0 0.3 0 0 0.5 0.1 0.1 0

CK

0.2

0.2

0.1

0.17

0

abased on the response value (in mv) of (E,Z)-3,13-18:OAc (la) as 1.0 and that of check CK as Zel"O.

TABLE 3. FIELD ATTRACTION TESTS OF SYNTHETIC COMPOUNDS TO MALE MOTHS a

Compounds 3,13-18:OAc (1)

Male moths captured

3,13-18:OH (2)

E,Z

Z,E

Z,Z

E,E

E,Z

Z,E

Z,Z

E,E

CK

33

1

1

1

1

0

1

0

0

aDate: June 29-July 5, 1985; place: Campus of Su Zhou College of Sericulture; dosage: each lure contained 200 #g of the test compound.

Paradoxecia pieli PHEROMONE

423

two diagnostic peaks from the crude washing that appeared on column A with retention times of 11.3 min (peak I) and 16.0 min (peak II), respectively, and two peaks on column B with retention times at of 15.2 min and 18.9 min. The retention times for 2 a - d under the same conditions were 11.3 min on column A and 18.9, 20.0, 20.9, and 18.1 min on column B. For l a - d , the retention time were 16.0 min on column A and 15.2, 15.0, 15.8, and 14.6 min on column B (see Table 4). Thus the above data showed that the natural sex pheromone consisted of 2a, which appeared on GC as peak I, and l a , as peak II (Table 4). Calculation of the peak areas on GC with the standard compounds revealed that 1 FE contained 30 ng (peak I) and 250 ng (peak II). Therefore, 250 ng of l a and 30 ng o f 2a was obtained from the crude washings of the female's sex pheromone gland. In Figure 1, receptor potentials and nerve impulses were recorded from an olfactory hair on the male moth antenna of Paradoxecia pieli. Each cell is activated by only one pheromone component (distinguishable by the amplitude of the nerve impulses). The stimuli were ( E , Z ) - 3 , 1 3 - 1 8 : O A c (Figure 1A) and (Z,Z)-3,13-18:OAc (Figure 1B). Each response is AC- and DC-amplified (upper and lower trace, respectively). The single cell recording (Figure 1) from the mulberry clearwing moth indicated that each sensilla trichodeum contained two receptor cells. The nerve impulses came from receptor cells that could be distinguished from each other by their amplitudes. One type of receptor cell producing the larger spikes was

TABLE 4. RETENTIONTIME OBTAINEDFROM NATURALSEX PHEROMONEAND FROM SYNTHETICCHEMICALSl a - d AND2a-d Retention time (min) Samples

Condition A

Condition B

(E,Z)-3,13-18 :OAc (la) (Z,E)-3,13-18:OAc (lb) (Z, Z)-3,13-18 : OAc (le) (E,E)-3,13-18 :OAc (ld) (E, Z)-3,13-18: OH (2a) (Z,E)-3,13-18: OH (2b) (Z, Z)-3,13-18 :OH (2c) (E, E)-3,13-18 : OH (2d)

16.0 16.0 16.0 16.0 11.3 11.3 11.3 11.3

15.2 15.0 15.8 14.6 18.9 20.0 20.9 18.1

n-18 OAc

17.8

11.0

Peak I

11.3

18.9

Peak II

16.0

15.2

Natural sex pheromone

T A N ET A L .

424

A

iildlllllhlhllln!~lblhtllli I dh ,|hit it tit II i l l ~ l t l U ~ l l t H inll~l"~ll I l l |~1 I | ~ ' '

P '"ql

.

"-

_

I. i ~ r

-

J

B 2mvI

Fie. 1. Single cell recording.

sensitive to (E,Z)-3,13-18:OAc (la) (Figure 1A); the other, producing smaller spikes, was reactive to (Z,Z)-3,13-18 : OAc (lc) (Figure 1B). Each type of cell was specifically responsive and sensitive to a particular pheromone component. In general, the nerve impulses could be recorded by dosages of 0.01 #g. The cells also responded to (E,Z)-3,13-18: OH (2a) and (Z,Z)-3,13-18: OH (2c) but exhibited lower responses than those to (E,Z)-3,13-18:OAc (la) and (Z,Z)-3,13-18:OAc (lc). No response was observed to (Z,E)-3,13-18:OAc (lb), (E,E)-3,13-18:OAc (ld), (Z,E)-3,13-18:OH (2b), and (E,E)-3,1318:OH (2(!). CONCLUSION The data obtained from GC analysis field bioassays, EAG survey, and SCR strongly suggest that (E,Z)-3,13-18 : OAc (la) is the major sex pheromone component of the mulberry clearwing moth, Paradoxecia pieli L., and that (E,Z)3,13-18:OH (2a) also was found in the pheromone gland, la and 2a averaged 250 ng and 30 ng, respectively, in each of the sex pheromone glands (1 FE). In the field test, only l a was attractive to male moths. This compound has been used for monitoring populations of this insect since 1985. The addition of 2a to la did not increase significantly captures of male mulberry clearwing moths. The behavioral function of 2a has not yet been established. SCR investigation showed clearly that each sensilla trichodeum contained two kinds of receptor cells. One kind responded to (E,Z)-3,13-18:OAc (la) and produced the larger spikes; the other was sensitive to (Z,Z)-3,13-18: OAc (le) and released the smaller spikes. Further work is needed to understand the reason for the receptor

Paradoxecia pieli PHEROMONE

425

cells' response to ( Z , Z ) - 3 , 1 3 - 1 8 : O A c p h e r o m o n e gland.

( l c ) , w h i c h was not found in the sex

REFERENCES Gvo, G.Z., Wu, P.H., LIN, G.Q., LI, Z.Y., ZHANG,X.H., LIE, H.Q. 1990a. Identification and synthesis of insect pheromone XXVIII. Sex pheromone of poplar pole clearwing moth Sphecia siningensis Hsu. J. Chem. Ecol. 16:819-820. Guo, GUAN-ZHONG,LIU, H.-QUAN, LIN, Guo-QIANG,WU, CAI-HONG,MEN, LIAN-ZHONG.1990b. Identification and synthesis of insect pheromone XXIX. Sex pheromone of the vine tree borer Paranthrene regalis B. Org. Chem. (Ch.) 1990:504-506. KA1SSLING,K.E., and TnORSON,J. 1980. Insect olfactory sensilla. Structural, chemical and electrical aspects of the functional organization, pp. 261-283, in Receptors for Neurotransmitters, Hormones and Pheromones in Insects. Elsevier/North-Holland, Amsterdam. ROELOFS,W.L., and CARPIT, A. 1971. Sex pheromone perception--electroantennogram responses of the red-banded leafroller moth. J. Insect Physiol. 17:1969-1982. ZHANG,X.H., GUO, G.Z., LIN, G.Q., Wu, Y.W., Wu, P.H., LI, Z.Y., WEI, K. 1986. Studies on identification and syntheses of insect pheromone XXII. Sex pheromone of twig clearwing moth, Paranthrene tabaniformis Rott. Structure and synthesis. J. Chem. Ecol. 12:1263-1271.