Journal of Chemical Ecology, Vol. 15, No. 5, 1989

I D E N T I F I C A T I O N OF THE SEX P H E R O M O N E OF T H R E E Matsucoccus PINE BAST SCALES

GERALD

N. L A N I E R , 1 Y U N - T A I

SEUNG

QI, 2 J A N E T

R. W E S T , 1

C. P A R K , 3 F R A N C I S X. W E B S T E R , 1 a n d ROBERT M. SILVERSTEIN l

IState University of New York College of Environmental Science and Forestry Syraeuse, New York 13210 2Shanghai Institute of Entomology, Academia Sinica Shanghai, China 3Department of Entomology, Forest Research Institute Republic of Korea (Received April 28, 1988; accepted August 2, 1988) Abstraet--Matsucoccus resinosae in the United States, M. matsumurae in China, and M. thunbergianae in Korea use (2E, 4E)-4,6,10,12-tetramethyl2,4-tridecadien-7-one (1) (matsuone) as the primary component of their sex attractant pheromones. The structure was postulated from mass and NMR spectra and confirmed by synthesis of analogs 3, (2E,4E)-4,6,11,12-tetramethyl-2,4-tridecadien-7-one, and 4, (2E, 4Z)-4,6,11,12-tetramethyl-2,4-tridecadien-7-one. Both analogs were attractive to the males of M. resinosae in laboratory bioassays and to M. matsumurae in laboratory and field tests, but the 4Z analog (4) was much less so than the 4E analog (3) and had inhibitory effects at high concentrations. Dodecanol, isolated from aeration and solvent extracts of female M. resinosae, evoked characteristic wing-raising by pedestrian males, but the role of this response was not determined. Key Words--Matsucoccus resinosae, M. thunbergianae, M. matsumurae, sex pheromone, analog, dodecanol, Homoptera, Coccoidea, Margarodidae, (2E, 4E)-4,6,10,12-tetramethyl-2,4-tridecadien-7-one, matsuone.

INTRODUCTION Despite their great economic significance and diversity, research on semiochemical-mediated sexual behavior of scale insects and mealybugs (Coccoid e a ) h a s b e e n r e l a t i v e l y r e c e n t a n d l i m i t e d . T h e first r e p o r t o f a s e x a t t r a c t a n t 1645 0098-0331/89/0500-1645506.00/0 9 1989 Plenum Publishing Corporation

1646

LANIE~tET AL.

in the superfamily was for the red pine scale, Matsucoccus resinosae Bean & Godwin (Doane, 1966). Warthen et al. (1970) isolated the sex pheromone of the California red scale, Aonidiella aurantii (Maskell), and the chemical structures of the active compounds were described seven years later (Roelofs et al., 1977). To the present date, sex pheromone structures for three additional armored scales (Diaspididae) and two mealybugs (Pseudococcidae) have been reported. Three sex pheromone components have been identified from the San Jose scale, Quadraspidiotus perniciosus (Comstock), (Gieselmann et al., 1979b; Anderson et al., 1981), and two from the California red scale (Roelofs et al., 1977). Single component attractants were identified for the yellow scale, Aonidiella citrina (Coquillett), (Gieselmann et al., 1979a); the white peach scale, Pseudaulascaspis pentagona (Targioni-Tozzetti), (Heath et al., 1979); the Comstock mealybug, Pseudococcus comstocki (Kuwana), (Bierl-Leonhardt et al., 1980); and the citrus mealybug, Planococcus citri (Risso), (Bierl-Leonhardt et al., 1981). The multiple components of the San Jose scale and the California red scale are individually attractive and apparently not synergistic. The alcohol corresponding to the propanoate sex attractant of the white peach scale acts as a short-range sex stimulant but is not an attractant (Einhorn et al., 1983). Thus, for the purpose of bringing the sexes together, the multiple components are apparently redundant; effectively, all Coccoidea investigated to date respond to single components. Analogs of California red scale pheromones and the closely related yellow scale were not active (Roelofs et al., 1977, 1978, 1982; Anderson and Henrick, 1979; Gieselmann et al., 1980) while pheromone analogs of the white peach scale (Heath et al., 1979), the citrus mealybug (Bierl-Leonhardt et al., 1981), and the Comstock mealybug (Mori and Ueda, 1981; Uchida et al., 1981; BierlLeonhardt et al., 1982) showed somewhat inferior activity. Sex pheromones were structurally different (Roelofs et al., 1982) and not cross-attractive between Aonidiella species (Moreno et al., 1972) or Planococcus species (Tremblay and Rotundo, 1976). In contrast, cross-attraction was complete among M. resinosae, M. matsumurae (Kuwana) 4 (Young et al., 1984), and M. thunbergianae Miller and Park (Park et al., 1986), partial between M. matsumurae and M. massonianae Young and Hu (Young and Qi, 1983), and slight between M. massonianae and M. resinosae (Young et al. 1984). Pheromones ofM. matsumurae (Qi et al., 1983), M. josephi (Stemlicht et al., 1983), and M. resinosae (Park et al., 1986) have been isolated. We report the chemical structure of the primary component of the sex attractant pheromone isolated from M. resinosae in the United States, M. matsumurae in China, and M. thunbergianae in Korea. We also report identification of a compound from female 4McClure (1983) and Young et al. (1984) suggestedthat M. resinosae mightbe a junior synonym of M. matsumurae, but the names have yet to be formallyproposed.

PHEROMONE OF PINE SCALES

164'7

M. resinosae that evokes a wing-raising response, but not taxis, by males of that species. Lastly, we present a comparison of laboratory bioassays of male M. resinosae response to analogs of its pheromone and to a crude extract of pheromone from M. thunbergianae females.

METHODS

AND MATERIALS

Chemistry of Pheromone and Analogs. In August 1982, 1984, and 1985, respectively, extracts of 3500, 17,000, and 26,500 female M. resinosae were fractionated as previously described (Park et al., 1986). A component that elicited attraction was in subfraction 3-5-4 and a component that evoked wingraising was in fraction 2. A unit-resolution, EI mass spectrum (70 eV) was obtained for the attractant on a Finnigan 4000 instrument fitted with a DB-1, 30-m capillary column, and for the wing-raising component on a Finnigan 3000 fitted with a 1-m OV-101 packed column. The tentative identification of the compound that elicited wing-raising was confirmed as dodecanol by coinjection with an authentic sample on a 6.1-m x 3-mm-ID glass column, 6% Carbowax 20 M and on a 3-m x 3-mm-ID glass column, 4% FFAP, and also by congruence of the mass spectrum with that of an authentic sample. A high-resolution mass spectrum on the sex attractant component was obtained on a Kratos MS 50TC spectrometer fitted with a 30m DB-1 column. The same components were also identified in the volatiles obtained by aerating the female M. resinosae for five days prior to extracting them; purified air (charcoal) was passed over the insects on crumpled filter paper in a flask, and the volatiles were collected on Porapak Q, from which they were extracted with pentane. An extract in 1 ml hexane of 400 female M. thunbergianae collected from infested Pinus thunbergiana in the southwestern extremity of Korea was used for cross-attraction bioassays against M. resinosae males (Park et al., 1986) and for coinjection with extracts of M. resinosae and M. matsumurae on a 30m DB-1 capillary column programmed, after 40 sec at 35~ to 200~ at 4~ min; a single sharp peak at 34.8 rain represented the attractant component common to the three samples. M. resinosae and M. thunbergianae extracts were also coinjected on a 50-m FFAP capillary column under the same conditions. In the spring of 1983, approximately 30,000 female M. matsumurae were collected from Pinus tabulaeformis in the suburbs of Wuxi, Jiangsu Province and in Haiyang County, Shandong Province. Purified air (molecular sieves) was passed over these insects for seven days, and the volatiles were trapped on Tenax (see Qi and Burkholder, 1982, for general procedure), which was then extracted with hexane. The active fraction was eluted from a silica gel column (35 x 0.6 cm, 200-320 mesh) with 2% ether in hexane, and the active subfrac-

1648

LANIER ET AL,

tion was eluted from a Florisil column impregnated with 15% silver nitrate (35 x 0.6 cm, 120-150 mesh) with 10% ether in hexane. This active subfraction was concentrated, and aliquots were repeatedly injected on a 40-m x 0.3-mmID PEG capillary column at 158~ N 2 2 cc/min. The collected active peak (retention time 33 min) in 3.5 ml hexane gave a unit-resolution mass spectrum that was congruent with that obtained by GC-MS on subfraction 3-5-4 from M. resinosae (above). A GC-FTIR (vapor phase) was obtained on an IBM IR98 fitted with a DB-1 capillary. To obtain an NMR spectrum, the M. matsumurae sample was injected on a 1.5-m x 9-mm-ID glass column, 3% OV-101 on Chromosorb G 50/80, 140~ He 80 cc/min. The peak at 18-22 min was collected in a 25-cm x 1.7-mm-OD glass capillary under thermal gradient (Brownlee and Silverstein, 1968) and rinsed with 0.4 ml 100% C6D6 into a 5-mm-OD NMR tube. The NMR spectrum was obtained on a General Electric GN 500 spectrometer. Unit resolution mass spectra were obtained on two synthesized analogs (3 and 4) under the same conditions above for the primary attractant component. Compounds 3 and 4 were separated on a 2.4-m x 6-mm-ID glass column, 4% TCEP on Chromosorb G 60/80, 140~ N2 60 cc/min. 500-MHZ NMR spectra on 3 and 4 were obtained under the same conditions used for the isolated primary sex attractant component.

Preparing Dilutions of Synthetic Pheromone Analogs and Crude Female Extract. Two pheromone analogs were synthesized: a positional isomer of the M. resinosae pheromone component, 3, and its geometric isomer, 4. These compounds were prepared at concentrations of 5 ng in 1 txl hexane and 1 #g in 1 ixl hexane, respectively. The original dilution of compound 3 was further serially diluted so that each 1 #1 hexane dilution contained 0.5 ng, 0.05 ng, 5 pg, and 0.5 pg; the original dilution of compound 4 was also serially diluted to 0.1 Ixg, 0.01 ~g, 1 ng, 0.1 ng, and 0.01 ng in 1 tzl hexane. A crude pheromone extract was made by immersing in 1 ml of hexane 200 newly emerged M. thunbergianae females collected from a laboratory colony at Seoul on March 18, 1986. After three days of storage at room temperature, the crude extract was serially diluted to 10%, 1%, 0.1%, and 0.01%. The amount of extracted pheromone in 1 /xl of each dilution was 2 x 10 -2, 2 x 10 -3, 2 x 10 -4, and 2 x 10 .5 female equivalents (FE), respectively. The original crude extract and its hexane dilutions were stored at - 10 _+ 5 ~ Preparation of Males for Testing. Branches of red pine (Pinus resinosa) containing cocoons of M. resinosae were collected from Dutchess County, New York, in September 1987 and put in emergence cages made of cardboard boxes with three Petri dishes attached at one side of each cage. The cages were stored at 20 _+ 4~ under natural day light. Because the males show strong positive phototaxis, newly emerging males could be collected from inside the Petri dishes.

PHEROMONE OF PINE SCALES

1649

Bioassays with Medicine Droppers. The bioassay procedures using medicine droppers were those of Park et al. (1986). Each test male was kept under a numbered, inverted, transparent plastic cup (ca. 20 ml) until tested. After each test, the male was covered with the same cup and kept for the next test. The stimulus was delivered to walking males, on a sheet of white paper, in puffs from a medicine dropper that had been charged with 1 ~1 of solution placed with a micropipet about 1 cm inside the tip. The dropper tip was positioned to one side of a walking male, about 8 mm from the antennae. Puffs of air were then forced from the medicine dropper by gently pressing the bulb at 1.5-sec intervals. The attraction was measured as degree of following towards the retreating dropper tip. Males that followed for one or more sides of a 4-cmsided equilateral triangle were given scores from 1 to 3, according to the number of triangle sides completed; males responding to the attractant but following less than one side were given a score of 0.5, and those who did not follow were given a score of 0. On each day of this experiment, between five and eight males were used at 5-rain intervals in successive tests of each dilution. A 1% dilution of the crude female extract (2 • 10 -3 FE) was used as a standard. Only actively walking males that had a response score of 3 to the standard were used. Males that became sluggish were replaced as needed to complete the test series. The bioassays were done at 22 +_ I~ 68 _+ 7% relative humidity, and 500-700 lux of fluorescent light between 8:00 AM and 12:30 PM from September 6 to 27, 1987. The number of stimuli varied among the days. Bioassays always proceeded from the lowest concentration to the highest, and the order for the tests with dilutions of crude extract, 3, and 4 was alternated each day. The data were analyzed in a completely randomized design and subjected to Duncan's multiple-range test. Dummy Female Experiments. Dummy females were made by rolling a 4 • 10-mm filter paper coated with rice paste to form a cylinder 4 mm long and 1.5 mm in diameter, similar in shape and size to a female. Each dummy female was treated with 1 ~1 of test solution, and held for 2 rain to allow the hexane to evaporate before the test. The dummy female was placed perpendicular to the path of the walking male, ca. 1 cm away, and the male behavior was observed. Bioassays were done at 22 _+ I~ 65 + 3% relative humidity, and 700 lux of fluorescent light between 10 AM and 3 PM from September 25 to October 8, 1987. Males were selected and replaced as in the medicine dropper bioassays, and allowed a rest of about 10 rain between tests. Laboratory and Field Bioassays of Compounds 3 and 4 Using M o matsumurae in China. The laboratory bioassay used was the Petri dish method described by Qi et al. (1983). The assay was conducted by placing two 1-cm pieces of filter paper, one containing the material to be tested, the other, an appropriate solvent blank, into a 7-cm-diameter Petri dish. After the solvent

1650

LANIER ET AL.

had evaporated, 10 males were admitted and the Petri dish was immediately covered. After a 2-min adaptation period, the number of males on the sample disk versus the number of males on the blank disk was counted every 2 min over a period of 20 min. The field experiments were conducted in the suburbs of Wuxi, PRC, from April 26 to 29, 1988. The trap consisted of a piece of cardboard (22 x 26 cm) that was folded so that the 26-cm side was bisected; the angle of the fold was 60 ~ Both sides of the trap were coated with Vaseline and a hole was cut (1 cm) in each flap. For these tests, a rolled piece of filter paper was utilized as the release device, and it was hung from the inside of the trap.

RESULTS AND DISCUSSION

Isolation and Identification of Active Compounds. M. resinosae pedestrian males demonstrated positive klinotaxis upon exposure to an air puff from a medicine dropper charged with solvent-or-aeration extracts of females (Park et al., 1986). While following the plume, males often raised their wings and antennae in a characteristic fashion. Bioassays of extract fractions showed that wing-raising and taxis were elicited by different compounds. Dodecanol was confirmed to be the principal component of the fraction that stimulated wingraising, and, in bioassays, synthetic dodecanol (1 txg in 1/A hexane) repeatedly elicited the typical wing-raising behavior. Bioassays of fractions after three successive GC fractionations placed the principal component of the sex attractant in subfraction 3-5-4 (Park et al., 1986). Spectral data suggested most aspects of the chemical structure but did not permit discrimination among possible structures 1-4. Synthesis of analogs 3 and 4 and comparison of their NMR spectra with that of the isolated attractant resulted in the assignment of structure 1 for the attractant. Identification of Pheromone Components. Unit-resolution GC-EI MS of the isolated attractant gave the following significant peaks (%): m/z 250 (M, 0.9), 141 (4.4), 123 (24), 109 (100), 81 (20), 67 (35), 57 (21), 55 (26), 43 (57), 41 (48). A GC-CI MS (methane) gave a base peak of m/z 251, thus confirming m/z 250 as the molecular ion. The high-resolution GC-MS gave a molecular ion at 250.2268 m/z (CI7H300, index of hydrogen deficiency of three). The molecule split into two fragments: 141.1275 m/z (10%, C9H170) and 109.0993 m/z (base, C8H13), the former containing a nonconjugated C = O group (GC-FTIR intense peak at 1747 cm-~), the latter containing two elements of unsaturation in the form of multiple bonds or rings, and possibly branching, so disposed as to stabilize the charge. A 500-MHz [~H]NMR spectrum, in deuterated benzene, and associated single-frequency decouplings provided the bulk of the data used in elucidating the structure(s). Although there were some impurities present in the isolated

PHEROMONE

1651

OF PINE SCALES

sample, the following peaks were easily discerned: 8 0.76 (d, 3H), 0.81 (d, 3H), 0.85 (d, 3H), 1.14 (d, 3H), 1.60 (d, 3H), 1.68 (s, 3H), 2.40 (t, 2H), 3.29 (m, 1H), 5.25 (d, 1H), 5.50 (m, 1H), and 6.01 (d, 1H). These absorptions account for 24 of the 30 protons and allow us to make certain tentative assumptions. The molecule contains six methyl groups, three of which are quite far upfield and are probably associated with purely aliphatic surroundings; two of them at 8 1.60 and 1.68 are probably olefinic methyls, and one of them has an intermediate chemical shift. The compound contains three olefinic protons that are probably associated with two different double bonds. Furthermore, to account for the downfield doublet at 8 6.01, the two double bonds are assumed to be Conjugated. Most of the structure can be assembled on the basis of the decoupling experiments. Irradiation of the methyl doublet at 8 1.14 collapsed the multiplet at 6 3.29 to a doublet. Irradiation of the multiplet at 8 3.29 collapsed the doublets at 8 1.14 and 8 5.25 to singlets. Since this spin system is closed (no other proton couplings), and since the downfield chemical shift of the methine multiplet at 83.29 cannot be explained solely on the basis of being allylic, the proton is probably next to the C--O group; thus we draw the fragment shown in Scheme 1, which contains a trisubstituted olefin. 3.29

O

5.25

SCHEME

1.

Irradiation of the doublet at 6 6.01 collapsed the multiplet at 8 5.50 to a quartet. Termination of this end of the chain was deduced by irradiating at 8 5.50; the doublet at 8 1.60 collapsed to a singlet. Since the two double bonds, one of which is trisubstituted while the other is disubstituted, are conjugated, the other olefinic methyl group at 6 1.68 must be on C-4. This information allowed us to piece together completely one end of the molecule, including the carbonyl group (Scheme 2). The terminal double bond can be assigned the t r a n s configuration on the basis of the large (17 Hz) coupling constant between the two olefinic protons. The stereochemistry of the trisubstituted double bond cannot be determine from this information. 5 I.!4 6 3.29 I

Ill O

"

5 1.68 I

I H s 525

H S 5.50

13 H ~ 6.01

SCHEME 2.

1 16~

1652

L A N I E R E T AL.

:O.

:O:

+

+

I R--C=O

m/e 141

+

~

C+/,..~....... m/e 109

Fro. 1. Cleavage in the mass spectrum to give base peak.

The mass spectrum indicated that the main cleavage was adjacent to the carbonyl group to give, as the base peak, a hydrocarbon fragment containing a highly stabilized tertiary carbocation (m/z 109, C8H13, Figure 1). The remainder of the molecule is represented by the peak at m/z 141 (C9H170 , Figure 1). The /3,y-unsaturated carbonyl system accounts for all of the "functional groups" because subtraction of the formula for this fragment, C9H130 , from the molecular formula, C17H30O, gives CsH17--a saturated hydrocarbon chain. This chain, R in Figure 1, contains three methyl groups, each of which is adjacent to a single proton since each shows a doublet in the NMR spectrum. Individual irradiation of the methyl doublet at 6 0.81 and at 6 0.85 caused a partial collapse of the multiplet at 6 1.58, indicating that the hydrocarbon chain terminates with an isopropyl group. Only placement of the methyl group represented by the doublet at 6 0.76 remains. Unfortunately, the important methine proton adjacent to this methyl group is obscured by impurities. Therefore, the evidence can only reduce the number of possible structures to four, if one ignores the stereochemistry of the two chiral centers. The four structures are shown in Figure 2.

o

o

1

2

3

4

o

FIG. 2. The four structures permitted by the spectral data.

1653

PHEROMONEOF PINE SCALES

To complete the structure assignment, a synthetic scheme was devised, the essential features of which (1) utilize either of the two primary bromides (5 and 6, Scheme 3) thus enabling us to pinpoint the uncertain position of the final methyl group; and (2) provide both geometric isomers during the formation of the trisubstituted double bond.

@

Br

~

5

B

r 6

SCHEME 3.

We now have enough information to identify the isolated attractant, having synthesized 3 and 4. Although the methyl group is in the wrong position, the synthesis did allow us to assign the correct methyl position and the stereochemistry of the trisubstituted double bond as trans by NOE NMR experiments with both analogs 3 and 4. Irradiation of the methyl group connected to the trisubstituted olefin in 3 gave no NOE enhancement while irradiation of the corresponding methyl group in 4 gave an expected NOE enhancement. The same type of enhancement was noted in a similar olefinic system by Takeda et al. (1982). A similar /3,3,-unsaturated ketone system in the antibiotic 9-methylstreptimidone 7 also assisted in assigning the stereochemistry (Scheme 4) (Becker and Rickards, 1979). The greater biological activity of the E analog 3 over that of the Z analog 4 may be an additional, albeit nonrigorous, argument for the 4E configuration of the isolated attractant (see below). o

O

OH O

7

8

O

SCHEME 4.

The hydrocarbon portion of the pheromone molecule has been synthesized and analyzed. A 500-MHz[~H]NMR spectrum of the ketone/benzyl ether, 8, closely matches the upfield portion of the [~H]NMR spectrum of the pheromone. We are now converting compound 8 into the racemic pheromone, 1, whose systematic name is (2E, 4E)-4,6,10,12-tetramethyl-2,4-tridecadien-7-one and to which we assign the trivial name matsuone. Details of the synthesis of racemic compounds 1-4 will be reported elsewhere, and elucidation of the stereochemistry at C-6 and C-10 will be undertaken for compounds 1 and 2.

1654

LANIER ET AL.

Bioassays of Sex Pheromone Analogs 3 and 4. Males of M. resinosae were attracted by 2 • 10 -4 FE of the M. thunbergianae female crude extract in the medicine dropper bioassay. The mean response score approximates those of M. resinosae and M. thunbergianae to 3 x 10 - 4 FE ofM. resinosae female crude extract (Park et al., 1986). At concentrations of 0.05 ng and higher, compound 3 elicited response by male M. resinosae equivalent to that of 2 • 10 - 4 FE of extract of M. thunbergianae females, while the index of response to 4 at a concentration 200 times higher (0.01 /zg)was, at most, 50% of that of 3 (Table 1). The threshold concentration was 5 pg for 3 and about 0.1 ng for 4, although the mean value of response scores at this concentration was not significantly greater than that of the hexane control. A decrease in the attractiveness of 4 at concentrations higher than 0.01/zg (Table 1) was associated with apparent irritation (movement sideways from the dropper tip or momentary cessation of walking, lowering the body and/or raising wings). In the dummy female experiments, male behavior was classified into four categories: (1) males that showed no interest, by passing by or walking less

TABLE 1. MEAN RESPONSES OF Matsucoccus resinosae MALES TO DILUTIONS OF PHEROMONE ANALOGS AND CRUDE EXTRACT OF

Material

Hexane Crude Extract Compound 3

Compound 4

M. thunbergianae FEMALES a

Quantity ~

No. tested

Mean response"

2 • 10 5 FE a 2 x 10 4 F E 0.5 pg 5.0 pg 50.0 pg 0.5 ng 5.0 ng 10.0 pg 0.1 ng 1.0 ng 10.0 ng 0.1 /~g 1.0/~g

23 15 15 28 28 28 15 21 5 21 21 21 21 21

0.04 E 0.27 DE 2.33A 0.13 DE 0.70 BCD 2.54 A 2.80 A 2.74 A 0.20 D E 0.57 CDE 0.95 BC 1.21 B 0.93 BC 0.63 BCD

~Response scores: 0 = no following; 0.5 = followed dropper for less than one full test triangle; 1, 2, or 3 = number of triangle sides completed. bEach in 1 /xl hexane. "Means followed by the same letter are not significantly different (P < 0.05, Duncan's multiplerange test). dFemale equivalent.

PHEROMONE OF PINE SCALES

1655

than two female body lengths on the dummy females, (2) those that lingered on dummy females by walking on them for more than two female body lengths, (3) those that showed obvious copulation attempts, and (4) those that, upon contact with a dummy female, showed apparent irritation by retreating a short distance sideways or backwards. A male that avoided contact with dummy females often lowered its body or raised its wings. All males or those that showed avoidance reaction either walked away or returned to the dummy females and repeated the avoidance behavior. Actively walking males, both in the presence and absence of pheromone, held their antennae parallel to the long axis of the body in lateral view and about 90 ~ from each other in dorsal view. Wings were usually positioned flat, and the tips were dragged when walking; the costal margins of the wings made about a 90 ~ angle with each other. The posture of the antennae or wings did not change until the male contacted the female or other substances bearing sufficient pheromone. This was in contrast with the behavior of the citrus mealybug (Gravitz and Willson, 1968) and the yellow scale (Moreno et al., 1972), where the posture of male antennae was changed before touching the females. When a male contacted and climbed on the female, the antennae closed to an angle of 0-30 ~ and were lifted 30-80 ~. The wings also were often lifted 30-60 ~. The male attempted a copulation thrust by bending the aedeagus downward, either immediately or after orienting his body to that of the female. This posture was maintained until copulation was completed. Table 2 shows the results of the dummy female tests. Most males made copulatory attempts or lingered on dummy females treated with 2 x 10 .3 FE of crude extract. Some males avoided dummy females bearing 2 • 10 -2 F E - a concentration 100 times stronger than that which elicited strong response in the medicine dropper assay (Table 1). On the contrary, avoidance behavior was not observed when males were presented with dummy females treated with 5 ng of 3, which is also 100 times stronger than what is required for a strong response in the medicine dropper assay. Males presented with dummy females treated with 4 did not attempt copulation and either showed no response or even avoided them~ Although our female extract was made at 10 AM, some of the newly emerged females probably copulated before they were collected and immersed in hexane. Males of M. thunbergianae that showed avoidance behavior to the same crude extract dilutions used in this experiment, did not avoid dummy females bearing 5 x 10 .2 FE of extract made from newly emerged virgin females (Park, 1988). This suggests that mated females produce a chemical that inhibits male copulatory behavior (perhaps this is the role of dodecanol). Equal parts of compounds 3 and 4 on dummy females evoked lingering and copulatory attempts by males. However, when 4 was increased relative to 3, most males avoided the dummy females. Although the number of males tested was too small for statistical significance, and since male physiology

1656

LANIER ET AL.

TABLE 2. BEHAVIOR OF Matsucoccus resinosae MALE PRESENTED WITH DUMMY FEMALES BEARING PHEROMONE ANALOGS OR CRUDE EXTRACT OF

M. thunbergianae FEMALES No. showing behavior ~' Material

Quantity ~

No. tested

N

L

C

A

Crude extract

2 x 10 3 FE c 2 x 10 -2 FE 0.5 ng 5.0 ng 0.1 /zg 1.0/~g

8 15 13 7 6 14 2 15

1 2 2 1 4 5 0 4

3 5 4 1 0 0 1 2

4 6 7 5 0 0 1 1

0 2 0 0 2 9 0 8

Compound 3 Compound 4 Mixture 1a Mixture 2 e

Each in 1/~1 hexane. hN = no response; L = lingering; C = copulatory attempt; A = avoidance (see text for description of behavior). ' Female equivalent. a C o m p o u n d 3 - c o m p o u n d 4 - h e x a n e = 1 : 1 : 8. e Compound 3 - c o m p o u n d 4 - h e x a n e = 1 : 3 : 6.

affecting copulatory behavior should be studied further, it is apparent that pheromone analogs may elicit inhibitory as well attractive behaviors. In laboratory bioassays with M. matsumurae in China, compound 3 was significantly more attractive than compound 4 at all doses (Table 3). Qi et al.

TABLE 3. MEAN RESPONSES OF Matsucoccus matsumurae TO DILUTIONS OF PHEROMONE ANALOGS 3 AND 4

Material

Dose (/zg)

Compound 3

1.3 2.6 5.3 8.0 16.0 26.0 1.3 8.0 16.0

Compound 4

X + SE ~ 27.7 52.7 53.7 54.7 77.6 81.0 2.7 6.7 13.0

+ 7.03a _+ 9.30b _+ 7.66b _+ 1.76b _+ 9.13c _+ 5.27d + 3.51e + 3.51f + 3.04g

a M e a n s followed by the same letters are not significantly different as determined by the WilcoxonMann-Whitney rank-sum test.

PHEROMONE OF PINE SCALES

1657

TABLE 4. MEAN TRAP CATCHES OF Matsucoccus matsumurae MALES TOTRAPS BAITED WITH COMPOUNDS 3 AND 4

Material

Dose (/zg)

Compound 3

16 56 32

Compound 4 Control

X _+ SE w 2.67 17.33 1.33 0.33

_+ 1,76a -L-_6.33b + 7.03a,c + 1.76c

"Means followed by the same letters are not significantly different as determined by the WilcoxonMann-Whitney rank-sum test.

(1983) found that 16/zg of compound 3 elicited a response comparable to 5 FE in laboratory bioassays. In field tests in China, only the 56-tzg dose of compound 3 attracted significantly more males than the controls; both 32 /zg of compound 4 and 16/zg of compound 3 were equivalent in attractiveness to the controls (Table 4). Analogs have not been found previously to reduce catches of flying male scales on traps (Roelofs et al., 1977; Gieselmann et al., 1980) or close range attraction by walking males (Heath et al., 1979). It is not known whether some of these coccoid pheromone analogs previously reported have inhibitory effects on close range attraction or copulatory behavior. Based on our results and previous reports of partial activity of coccoid pheromone analogs, we expect the synthetic pheromone (1) of M. resinosae to show much higher activity than its analogues; these studies are in progress. Acknowledgments--We are greatly indebted to the following: Ellen Gallagher, Michael Griggs, Alan Jones, Lorene Lanier, Juan Pajares, Carl Palm, Stephen Teale, Alison Teale, Shaofu Xu, Wen-jun Fu, and Fang-gui Zhao (the latter three at the Shanghai Institute of Entomology) for scale collections, extraction, and aerations; Timothy Wachs (Comell University) for the GCFTIR and GC-MS, and Hazel Jennison for GC-MS; Dr. Michael Kolor and Donald Rizzo (both from General Foods Inc.) for high-resolution mass spectra; Gregory Hefron for high-resolution NMR spectra; and Sang kyu Park for his contribution to the synthesis of the analogs. C. Palm and S. Teale assisted with manuscript preparation and data analysis. We are grateful to the New York Department of Environmental Conservation for use of facilities at Ninhin Mountain and assistance in locating M. resinosae infestations.

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Identification of the sex pheromone of threeMatsucoccus pine bast scales.

Matsucoccus resinosae in the United States,M. matsumurae in China, andM. thunbergianae in Korea use (2E, 4E)-4,6,10,12-tetramethyl-2,4-tridecadien-7-o...
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