Journal of Chemical Ecology, Vol. 17, No. 1, 1991
LABORATORY AND FIELD TESTS WITH THE SYNTHETIC SEX PHEROMONE OF THREE M a t s u c o c c u s ~ PINE BAST SCALES
B.E. HIBBARD, 2 G.N. LANIER, 2'5 S.C. PARKS, 3 Y.T. QI, 4 F.X. W E B S T E R 2'* and R.M. SILVERSTEIN 2 2State University of New York College of Environmental Science and Forestry Syracuse, New York 13210 3Department of Forestry, College of Agriculture Chonnam National University Yongbong-dong, Kwangju 500-757 Republic of Korea 4Shanghai Institute of Entomology Academia Sinica Shanghai, China (Received May 10, 1990; accepted August 20, 1990)
Abstraet--Matsunone, (2E,4E)-4,6,10,12-tetramethyl-2,4-tridecadien-7one, is the primary sex pheromone of Matsucoccus resinosae, M. thunbergianae, and M. matsumurae. The synthetic compound was tested in the laboratory and in field tests with all three species, and significantly more males responded to synthetic matsuone than to controls. The attraction of synthetic matsuone to males in field tests was not significantly different from that of virgin females at the same matsuone release rate. An analog of matsuone previously shown to be biologically active was also tested in the laboratory with M. resinosae and 114. thunbergianae, and field tested with M. resinosae. The active analog required a dose approximately 100 times greater than matsuone to yield maximum laboratory bioassay response, and in field tests, attraction was not significantly different from controls at doses at which matsuone was significantly attractive. Key Words--Matsucoccus resinosae, M. thunbergianae, M. matsumurae, Coccoidea, Margarodidae, scale insects, pine bast scales, sex pheromone, (2E,4E)-4,6,10,12-tetramethyt-2,4-tridecadien-7-one, matsuone.
*To whom correspondence should be addressed. ~Homoptera: Coccoidea: Margarodidae. 5Deceased. 89 0098-0331/91/0100-0089506.50/0(c) 1991 Plenum PublishingCorporation
90
HIBBARD ET AL. INTRODUCTION
Pine bast scales in the genus Matsucoccus Cockerell are major pests of pine in France (Carle, 1968, 1974), Israel (Mendel et al., 1988), Korea (Park, 1988), China (Li et al., 1980), and the United States (Bean and Godwin, 1955; McClure, 1976, 1983b). Studies on the chemical communication of these insects began with Doane's (1966) report of a female-produced sex attractant for Matsucoccus resinosae Bean and Godwin. Sex pheromones have since been documented in M. matsumurae (Kuwana) (Qi et al., 1983), M. thunbergianae Millar and Park (Park et al., 1986), M. massonianae Young et Hu (Young and Qi, 1983), and M. josephi Bodenheimer and Harpaz (Sternlicht et al., 1983). Although a taxonomic revision has not been formally proposed, M. matsumurae and M. resinosae are almost certainly the same species (McClure, 1983a; Young et al., 1984). Park et al. (1986) reported the isolation of the sex pheromone of M. resinosae and the demonstration of its cross-attractiveness with M. thunbergianae. Spectral data available to Lanier et al. (1989) reduced the number of possible structures for this compound to four: 1, 2, 3 and 4 (Figure 1). On the basis of strong circumstantial evidence, Lanier et al. (1989) identified the pheromone ofM. resinosae, M. matsumurae, and M. thunbergianae as (2E,4E)-4,6,10,12tetramethyl-2,4-tridecadien-7-one (1, Figure 1), and gave it the trivial name matsuone. Although we were successful in synthesizing two analogs of this structure (3 and 4, Figure 1) and demonstrating biological activity with one of them (3), the compound we proposed as the true pheromone (1) in Lanier et al. (1989) was not available for testing. We recently synthesized matsuone (1) (see Figure 1) and its geometric isomer (2E,4Z)-4,6,11,12-tetramethyl-2,4-tridecadien-7-one (2) (Webster et al., 1990). We report here laboratory and field tests of three species of Matsucoccus pine bast scales with the two new synthetic compounds (1 and 2) and those analogs (3 and 4) tested by Lanier et al. (1989).
O
O
(1)
(2)
(3)
(4)
FIG. 1. Chemical structures of matsuone and the three analogs tested.
PHEROMONEOF Matsucoccus
91 METHODS AND MATERIALS
Field Sites and Insect Sources. Field experiments were conducted in Dutchess County, New York, U.S.A., from June 7 to September 15, 1989; in Tonggang County, Chonnam Province, Korea, from April 5 to 7, 1989; and the suburbs of Wu Xi City, China, from April 25 to 27, 1989 for M. resinosae, M. thunbergianae, and M. matsumurae, respectively. Infested branches of Pinus resinosa Ait., P. thunbergiana Franco, and P. tabulaeformis Carr. containing cocoons of M. resinosae, M. thunbergianae, and M. matsumurae, respectively, were placed in emergence cages similar to those described by Lanier et al. (1989). Males emerging from the cocoons were tested in laboratory behavioral bioassays. Laboratory Bioassays. The bioassay procedures used for M. resinosae and M. thunbergianae were those of Park et al. (1986) and Lanier et al. (1989). First, the test compound in 1/zl hexane in a micropipet was deposited just inside the tip of a medicine dropper. Excess hexane was expelled by depressing the bulb of the medicine dropper 20 times, and the dropper was positioned about 8 mm to one side of a pedestrian male. Puffs of air were delivered to the antennae of the pedestrian male by gently pressing the bulb at 1.5-sec intervals. Relative attraction was measured as the number of sides of a 4-cm-equilateral triangle along which a male would follow the retreating dropper tip. The highest response rating, three, indicated that the male followed the dropper for all three sides of the triangle, and the lowest response rating, zero, indicated no attraction. Incomplete following of the dropper along a side of the triangle was designated by the fraction of the side completed. For M. thunbergianae, males showing the response score of three to 2 x 10 -3 female equivalents (see Lanier et al., 1989) were tested, while with M. resinosae, the positive control was a live virgin female placed in the dropper tip. Bioassays with M. matsumurae in China used the method of Qi et al. (1983) and Lanier et al. (1989). Two filter paper disks of 1 cm diameter, each containing one of two materials or one material and a solvent blank, were placed into a 7-cm-diameter Petri dish. After the solvent had evaporated, 10 males were introduced and the Petri dish was covered. The behavior of males in the dish was observed, and the number of males on each of the disks was recorded after 10 min. Field Experiments. In Korea, the field experiments were performed in a P. thunbergiana forest with trees approximately 8 m tall. White plastic cards (10 x 14 cm) coated with Tanglefoot were affixed to horizontal bamboo poles 0.5 and 1.5 m above the ground. Traps were set in open areas of 15-40 m diam. and 7-20 m away from the trees. Treatments were assigned randomly on the poles so that each pole contained one replicate with different treatments spaced 1 m apart on the pole. Unbaited traps were placed in the field at 6 pro, April
92
HIBBARD ET AL.
5, 1989. Treatments of 222/zg matsuone in 60/~1 benzene on rubber GC septa (Supelco Inc., Bellefonte, Pennsylvania), 60/xl benzene on rubber GC septa, and 20 newly emerging females in a 2 • 2 x 0.5-cm fine net cage were replicated 10 times: five each at 0.5 and 1.5 m above the ground. Emerging females used as baits were collected between 8:00 and 9:30 AM, April 6. Baits were attached with pins to the lower portion of the sticky traps between 9:30 and 10:00 AM, April 6. Those males that were caught on untreated traps in the early morning were removed as the baits were set. Counts of these traps were then made between 12:00 and 1:00 PM on both April 6 and 7 by removing the males from the traps during counting. In China, the field trapping experiments were conducted in a suburban forest of P. tabulaeformis. Three replications of the following six treatments were tested: 1, 10, and 40 /~g matsuone on filter paper, one and five newly emerged females, and a benzene control. The trap, a piece of sticky cardboard (28 x 23 cm) folded in the middle at a 60 ~ angle, was similar to that described by Lanier et al. (1989). Three 1-cm-diam. holes were cut into each flap, and a roll of filter paper (5 x 0.8 cm) that served as the pheromone dispenser was hung inside the trap. Traps were hung from pine branches at heights between 1.5 and 2 m. Distance between traps was greater than 20 m. In New York, traps placed 2.44 m high were positioned between 1 and 2 m from the nearest branches and were at least 1.5 m from each other within a small planting of P. resinosae. Most of the trees were widely spaced, so that the crown was visible to the ground. Since M. resinosae usually occurs in the crowns, this site was ideal for setting traps at reasonable heights. Tree height at the site ranged from 1.5 m to 20 m. Dewill elm bark beetle traps (Dewill Inc., Elmhurst, Illinois) were cut into thirds (sticky surface of 20 x 48 cm), formed into cylinders 15 cm in diameter with the sticky surface facing outward, and stapled around a vertical pole 2.44 m in height. The baits were protected from the sun by fastening them with wire approximately 2 cm within inverted plastic film canisters (3 cm diam. x 4.8 cm). The bait apparatus then was stapled to the pole so that it was situated inside the sticky cylinder. Trials with live females as a treatment were set up between 8:00 and 9:30 AM, and results were recorded after 1:00 PM. Trials without live females were set up between 1:30 and 5:30 PM, and results were recorded after several days. The first field experiment in New York tested the relative attraction of 25 /xg matsuone (1), 250/xg matsuone, 25 /xg 2, 250/xg 2, and a hexane control. Rubber GC septa served as the pheromone dispenser. Five replications of this experiment were set up on June 8 and dismantled on June 15. Ten more replications were set up on June 20 and dismantled on June 25. We next compared known quantities of matsuone with attraction to live adult females. Six replicates of 20 live females each (collected between 6:30 and 8:30 AM on August 15, 1989) were placed in fine netting and added to the
PHEROMONE OF
Matsucoccus
93
traps along with baits by 9:30 AM. Four doses of matsuone were chosen and replicated 10 times: 10, 50, 100, and 250 txg. Trap catch was recorded between 1:00 and 2:00 PM the same day. The pheromone dispenser for this experiment was a cotton dental wick (Healthco International, Boston, Massachusetts). The last experiment reported herein tested 200/~g each of matsuone (1), a hexane control, and 2, 3, and 4 on rubber GC septa. The experiment was set up September 15 and dismantled September 25. Release Rate Determination. The relative release rates of matsuone from rubber septa, dental cotton wicks, and filter paper were determined in the laboratory. Matsuone was added to the dispensers, and they were immediately placed in a 250-ml Erlenmeyer flask that was protected from UV radiation with aluminum foil. Each of the dispensers was tested with 250/zg of matsuone, and filter paper was also tested at 40/xg because this dose was effective for trapping males in China. There were four replicates of each dispenser at each dose tested. House vacuum pulled room air through activated carbon (6-14 mesh), past the dispensers, and through 0.6 g Porapak Q, on which matsuone was collected. Flow through the flask was held constant at 100 ml/min. Temperature was held constant at 20 _+ 2~ Volatiles were recovered from the Porapak with 4 ml of pentane. Extractions were made after 24, 48, and 72 hr for each dispenser, and also at 168 hr for rubber septa. The concentration of matsuone in the samples was determined with an HP 5890 gas chromatograph fitted with a 50-m methyl silicon capillary column (DB-1, Supelco, Inc., Bellefonte, Pennsylvania) with 1 ng//zl n-octadecane as an internal standard. Statistical Analysis. The statistical package SAS (SAS Institute, 1985) was used for data analysis. All field data were analyzed with one-way or two-way analysis of variance (ANOVA), then by Duncan's (1955) multiple-range test when significant F values were found in the ANOVA. The trap-catch data were subject to log (X + 1) or square root (X + 0.5) transformations as needed to meet the assumption of equal variance (Winer, 1971). Nonparametric procedures were required for analysis of laboratory bioassays with M. resinosae and M. thunbergianae. A rank transformation followed by the procedures above served this purpose (Conover and Iman, 1981; SAS Institute, 1985, p. 19).
RESULTS
Laboratory Bioassays. In bioassays with M. thunbergianae in Korea, responses of pedestrian males to all doses of matsuone tested except the lowest dose, 0.04 pg, were significantly higher (P < 0.05) than the control (Table 1). Maximum responses to matsuone were elicited by doses of 4.0 pg to 0.40 ng, while the dose required for maximum response to 3 was about 0.50 ng. Differences in attraction between the highest dose of 3 (0.50 ng) and doses of mat-
94
HIBBARD ET AL. TABLE 1. MEAN RESPONSES IN LABORATORY BIOASSAYS OF Matsucoccus
thunbergianae
Material Hexane Matsuone
Analog 3
MALES TO DILUTIONS OF MATSUONE AND ANALOG 3 a
Quantity (pg)
4.0 4.0 4.0 4.0 4.0 4.0 5.0 5.0 5.0 5.0
X • X • x x x • • x
10 2 10-' 10~ 10 ~ 102 103 10 -t 10~ 10 ~ 102
No. tested 24 24 24 24 24 24 24 24 24 24 24
Mean response + SE 0.06 0.21 1.08 2.88 2.73 2.79 2.04 0.15 0.42 1.98 2.69
+ 0.03 +_ 0.07 + 0.23 + 0.09 +__0.15 +_ 0.11 + 0.24 + 0.05 + 0. I0 + 0.22 + 0.15
E DE C A A A B E D B A
~Although the raw data are shown, statistical analysis was performed on rank transformed data. Significant differences are indicated by different letters after the mean.
suone from 4.00 pg to 0.40 ng were not significant. In bioassays with M. resinosae in the United States, responses to doses between 50.0 pg to 5.0 ng of matsuone, and between 5.0 ng and 50.0 ng of 3 were not significantly different from the positive control of a live female (Table 2). All doses of 2 and 4 elicited lower (P < 0.05) responses than a live female. All doses of matsuone and doses of 50.0 pg, 5.0 ng, and 50.0 ng of 2 gave greater responses than the hexane control. No dosage of 4 gave response significantly greater (P < 0.05) than the hexane control. For 2 and 3, responses to all doses from 50.0 pg to 50.0 ng were higher (P < 0.05) than to the hexane control. Laboratory bioassays in China utilizing the Petri dish bioassay indicate that M. matsumurae are significantly attracted to matsuone (Table 3). At all doses tested except the lowest dose (10 ng), more (P < 0.01) males were attracted to filter paper disks containing matsuone than to disks with benzene alone. Increasing the dosage of matsuone increased the number of males on the matsuone disk. In a choice test comparing 0.20/~g matsuone with 5 female equivalents (see Lanier et al., 1989) of crude extract, there was no significant difference between the number of males on the two filter paper disks. When comparing 0.20/zg matsuone in choice tests with 8.0/zg 3, significantly more males were found on the filter paper disk with 0.2/xg of matsuone. Field Tests. In field tests with M. thunbergianae in Korea, significantly more males were caught in traps baited with 222/zg matsuone 0.5 m from the ground than all other traps (Figure 2). Traps baited with 20 live females placed
PHEROMONE OF Matsucoccus
95
TABLE 2. MEAN RESPONSES IN LABORATORY BIOASSAYS OF Matsucoccus resinosae MALES TO DILUTIONS OF MATSUONE, MATSUONE ANALOGS, AND LIVE FEMALES a
Quantity Material Live females Hexane Matsuone
Analog 2
Analog 3
Analog 4
(pg)
1 female 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
• x x x x • x • • x
10 -~ 10o 10 ~ 102 103 104 10 L 10 ~ 10 t 102
X 10 3 >< 10 4
x x • x • • x • • • • •
10 - t 10 ~ 10 ~ 102 103 104 10 - I 10 ~ 10 ~ 102 103 104
No. tested 42 45 5 24 32 14 12 5 9 8 8 8 6 5 5 5 20 15 9 7 5 8 15 15 8 8
Mean response 3.00 0.10 1.70 1.53 2.57 2.71 2.71 1.60 0.00 0.44 0.95 0,69 t.92 0.67 0.00 0.50 1.43 1.85 2.78 2.25 0.00 0.50 0.00 0.23 0.06 0.00
+ 0.00 A _+ 0.04 HI + 0.52 CDE _+ 0.21 CDE _+ 0.16 ABC _+ 0.19 AB + 0.19AB +_ 0.51 CD + 0.00 I _+ 0.19 GHI _+ 0.32 FG _+ 0.19 FGH _+ 0.52 BCD + 0.11 EFG _+ 0.00 I _+ 0.34 GHI + 0.00 DEF 2:0.19 CDE _+ 0.06 AB + 0.00 ABC + 0.00 I + 0.44 GHI + 0.26 I +_ 0.36 HI _+ 0.00 I +_ 0.31 I
aAlthough the raw data are shown, statistical analysis was performed on rank transformed data. Significant differences are indicated by different letters after the mean.
at 0.5 m also caught more (P < 0.05) males than controls, but there was no significant difference between any treatments placed at 1.5 m. In China, traps baited with 10 and 40/xg of matsuone caught significantly more male M. matsumurae than control traps, traps with 1 female, and traps with 1 #g of matsuone (Figure 3). Differences between traps with 10 and 40/~g of matsuone and traps with five females were not significant, nor were differences between traps with 1 /xg of matsuone and control traps. Traps with one or five females caught significantly more males than control traps. In the United States, significantly more (P < 0.05) male M. resinosae were caught on traps baited with 250 #g of matsuone than those baited with 25
HIBBARD ET AL.
96
TABLE 3. MEAN RESPONSES OF m. matsumurae MALES IN CHOICE TESTS BETWEEN MATERIAL 1 AND MATERIAL 2 a Material 1 (ng matsuone)
Material 2
Mean response (material 1 - material 2)
10 20 50 100 200 400 800 1300 200 200
Benzene Benzene Benzene Benzene Benzene Benzene Benzene Benzene 5 ~male equivalents 8000 ng analog 3
2.00 3.67* 4.67* 6.67* 7.67* 8.33* 9.33* 9.67* 1.67" 5.33*
Significant differences are indicated by different letters after the mean. * = significantly different from zero (P < 0.01).
175
Z- 150 t~ o
100
"~ 75
E
d 5o z 25 0 Matsuone
20 females
Control
Treatment F~G. 2. Number of male M. thunbergianae caught in traps baited with 222 ~g matsuone on rubber septa, 20 live females, and a solvent control. Although raw data are shown, statistical analysis was performed on log (X + 1) -transformed data. Bars topped by different lowercase letters are significantly different (P < 0.05) as indicated by Duncan's (1955) numerical multiple-range test. Standard error bars are indicated.
/xg m a t s u o n e , 25 ~ g 2, 2 5 0 txg 2, o r a h e x a n e c o n t r o l ( F i g u r e 4); n o o t h e r s i g n i f i c a n t d i f f e r e n c e s w e r e f o u n d . I n d o s e - r e s p o n s e tests u s i n g c o t t o n d e n t a l w i c k s as t h e p h e r o m o n e d i s p e n s e r , t h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e b e t w e e n t r a p s b a i t e d w i t h 100 a n d 2 5 0 / x g m a t s u o n e , a n d t h e r e w a s n o d i f f e r e n c e b e t w e e n c o n t r o l t r a p s a n d t r a p s w i t h 2 0 l i v e f e m a l e s ( w e c a n n o t e x p l a i n this last result).
PHEROMONE OF
97
Matsucoccus 40
_E-
30
O5 O CO d3 20
E d 10
Z
Treatment FIG. 3. Number of male M. matsumurae caught in traps baited with matsuone on filter paper or live females. Bars topped by different lowercase letters are significantly different (P < 0.05) as indicated by Duncan's (1955) numerical multiple-range test. Standard error bars are indicated.
50
~4a t~ O 30 03 t~ 20
E d Z
lO
r
%
+'--
"4_
,v^
'/"
Treatment FIG. 4. Number of male M. resinosae caught in traps baited with matsuone, 2, or solvent on rubber septa. Although raw data are shown, statistical analysis was performed on square root (X + 0.5) -transformed data. Bars topped by different lowercase letters are significantly different (P < 0.05) as indicated by Duncan's (1955) numerical multiple-range test. Standard error bars are indicated.
98
HIBBARD ET AL.
All other differences were significant (Figure 5), including the difference between male catches on traps baited with 10 txg matsuone and control traps. A regression of the dose-response curve was significant (P < 0.05) with an R 2 of 50.9%. In a similar experiment in which all analogs were tested, more males (P < 0.05) were caught on traps baited with matsuone than traps baited with any analog. There was no significant difference between catches on traps baited with 2, 3, 4, and the hexane control (Figure 6). Release Rates. The release rate of matsuone from filter paper rolls and dental cotton wicks was significantly greater than that from robber GC septa (Supelco, Inc., Bellefonte, Pennsylvania) for each of the first three days (Table 4). Significantly more matsuone was released from cotton wicks than filter paper on day 2, but there was no significant difference on days 1 or 3. The release rate of 40/~g matsuone from filter paper was higher than that of 250/zg matsuone from rubber septa on day 1, but this difference was not significant on day 2 or day 3. The average release rate from rubber septa from days 4-7 was equivalent to that released from robber septa on day 2.
DISCUSSION
Park (1988) placed newly emerged M. thunbergianae females in a device designed to collect the female sex pheromone on filter paper. The filter paper was extracted with hexane, and 5 x 10 -3 female hour equivalents (FHE) yielded
14" "r'~
12'
'm
10.
0 69 0
8' 6
E z
4. 2, 0
Treatment
:%/%
F~6. 5. Number of male M. resinosae caught in traps baited with live females or different doses of matsuone on cotton dental wicks. Although raw data are shown, statistical analysis was performed on log (X + 1) -transformed data. Bars topped by different lowercase letters are significantly different (P < 0.05) as indicated by Duncan's (1955) numerical multiple-range test. Standard error bars are indicated.
PHEROMONE OF
Matsucoccus
99
10
03 8
0
6
E 4 d Z 2
Treatment FIO. 6. Number of male M. resinosae caught in traps baited with 200 ~g of matsuone and three analogs on rubber septa. Although raw data are shown, statistical analysis was performed on log (X + 1) data. Bars topped by different lowercase letters are significantly different (P < 0.05) as indicated by Duncan's (1955) numerical multiple-range test. Standard error bars are indicated.
TABLE 4. RELEASE RATE OF MATSUONE FROM PHEROMONE DISPENSERS AS MEASURED IN THE LABORATORY
Release rate (#g/day +_ SE) Dispenser type
Loadrate (/~g)
Rubbersepta Cotton wick Filter paper Filter paper
250 250 250 40
Day 1
Day 2
Day 3
1.50 + 0.60ef 0.41 + 0.03fg 0.12 _ 0.02g 12.52 _+ 2.84ab 8.93 + 0.29bc 1.98 _+ 0.29e 17.03 ___1.68a 3.95 + 0.80d 1.06 _+ 0.19ef 6.15 ___0.96d 1.09 _+ 0.90ef 0.25 _+ 0.10fg
Days 4-7 0.41 _+ O.09e
an average response of 2.2 in the same bioassay as used in the present study (5 x 10 -2 FHE gave an average of 3.0). This response is equivalent to the response of M. thunbergianae males to 4.0 pg of matsuone and M. resinosae males to 50.0 pg matsuone reported herein. If we assume that all of the sex pheromone was collected on the filter paper in Park (1988) and that matsuone is the only active compound, we can estimate the production of matsuone (based on the level of response) to be in the range of 0.8 ng/hr for M. thunbergianae and 10 ng/hr for M. matsumurae and M. resinosae. Because we determined the release rate of matsuone from the dispensers utilized in the present study (Table 4), we can compare the field trap catch of
100
HIBBARD ET AL.
synthetic matsuone to that of natural matsuone. The field tests in China were conducted for three days, so if we use the release rate of 40 t~g matsuone from filter paper from day 2 as an average, approximately 54 ng/hr of synthetic matsuone were released (Table 4). M. matsumurae females produce approximately 10 ng/hr, so the five females tested would have produced approximately 50 ng/ hr of natural matsuone (a conservative estimate given our assumptions). The number of male M. matsumurae caught on traps baited with five females and those baited with 40/~g matsuone on filter paper were not significantly different. In experiments in Korea with M. thunbergianae, septa were loaded with matsuone the afternoon before being applied to the traps, and the experiments were set up for two days, so it is again appropriate to use the release rate data from day 2. The release rate of matsuone from rubber GC septa on day 2 was approximately 17 ng/hr (Table 4), while the calculated release rate of 20 female M. thunbergianae would be approximately 16 ng/hr. Both traps baited with 20 females and traps baited with 222/zg synthetic lriatsuone on rubber GC septa caught significantly more males than control traps at 0.5 m in height. Traps baited with synthetic matsuone also caught significantly more males than traps baited with 20 females at 0.5 m (Figure 2), but this difference in trap catch was not significant when data from both heights (0.5 and 1.5 m) are pooled. If our calculation of the release rate of matsuone from these Matsucoccus spp. is correct, it appears that matsuone can account for all of the attraction to femalebaited traps. Demonstrating biological activity of the synthetic compound is crucial to verifying the identification of a pheromone. We have shown that synthetic matsuone is significantly attractive to three species of pine bast scales in laboratory and field tests, and we have shown that matsuone can account for all of the attraction of live females in the field. We have corroborated the conclusion of Lanier et al. (1989) that (2E,4E)-4,6,10,12-tetramethyl-2,4-tridecadien-7-one is the major sex pheromone of M. resinosae, M. thunbergianae, and M. matsumurae. Lanier et al. (1989) were correct in their prediction that matsuone would show much higher activity than its analogs. In laboratory bioassays with both M. resinosae and M. thunbergianae, the dose of 2 (the most active analog) required to produce a full response was approximately 100 times greater than that required for matsuone. In field trials with M. resinosae with 200/xg of all analogs present on rubber septa, only traps baited with matsuone caught significantly more males than the control. The final identification step is also the first step in the utilization of semiochemicals in integrated pest management. Synthetic pheromones have been produced for a number of other Coccoidea and have been successfully incorporated into management programs (for detection and monitoring) for the citrus mealybug, Planococcus citri (Risso) (Ortu and Delrio, 1982); the citrophilous mealybug, P. calceolariae Mask. (Rotundo et al., 1979); the San Jose scale,
PHEROMONE OF M a t s u c o c c u s
101
Quadraspidiotus perniciosus (Comstock) (Angerilli and Logan, 1986; Pfeiffer 1985); and the California red scale, Aonidiella aurantii (Maskell) (Ervin et al.,
1985; Moreno and Kennett, 1981, 1985; Sternlicht et al., 1981). Sex pheromones have even been used for mating disruption for the California red scale (Bar-Zakay et al., 1989). Synthetic matsuone produced by Webster et al. (1990) trapped significantly more male M. resinosae, M. thunbergianae, and M. matsumurae than control traps in the present experiments. The results from the present paper indicate that synthetic matsuone may be a valuable component in the management programs of M. resinosae, M. thunbergianae, and M. matsumurae. Acknowledgments--We thank Aijun Zhang for synthesizing the matsuone and 2 used in this study, Mr. Ray Vaughan from the Department of Environmental Conservation at Stonykill for help in locating field sites in New York, Mr. Randy Randazzo from the Sprout Lake Camp and Mr, Dick Conkline for cooperation in using their land for field sites in New York. We also thank Mr. Conkline for permission to down red pine trees for laboratory work. Mr. Hyung-Sun Kim from the Chonnam Forest Experiment Station assisted in the Korean field tests. Funding was provided in part by a grant from the National Science Foundation and the Department of Entomology, Forestry Research Institute of Korea.
REFERENCES
ANGERILLI,N.P.D., and LOGAN,D.M. 1986. The use of pheromone and barrier traps to monitor for San Jose scale (Homoptera: Diaspididae) phenology in the Okanagan Valley of British Columbia. Can. Entomol. 118:767-774. BAR-ZAKAY,I., PELEG, B.A., and HEFETZ, A. 1989. Mating disruption of the California red scale Aonidiella aurantii (Homoptera: Diaspididae). Hassadeh 70:1228-1231. BEAN, J.L., and GODWIN, P,A. 1955. Description and bionomics of a new red pine scale, Matsucoccus resinosae. For. Sci. 1:164-197. CARLE, P. 1968. M6thode d'obtention massive des pontes de Matsucoccus feytaudi Duc. (Coccoidea, Margarodidae) par pidgeage des femelles. Ann. Sci. For. 25:57-68. CARLE, P. 1974. Le ddpdrissement du pin mdsog6en en Provence. Ann. Sci. For. (Paris) 31:1-26. CONOVER, W.J., and IMAN, R.L. 1981. Rank transformations as a bridge between parametric and nonparametric statistics. Am. Stat. 35:124-t29. DOANE, C.C. 1966. Evidence fora sex attractant in females of the red pine scale. J. Econ. Entomol. 59:1539-1540. DUNCAN, D.B. 1955. Multiple range and multiple F tests. Biometrics 11:1-42. ERVIN, R.T., MORENO,D.D., BARITELLE,J.L., and GARDENER,P.D. 1985. Pheromone monitoring is cost effective. Calif. Agric., Sept.-Oct. 17-18. LANIER, G.N., QI, Y., WEST, J.R., PARK, S.C., WEBSTER, F.X., and SmVERSTEIN,R.M. t989. Identification of the sex pheromone of three Matsucoccus pine bast scales. J. Chem. Ecol. 15:1645-1659. LI, G., AHOQNG, L., HAN, R., Lm, X., and XIa, R. 1980. A study on the pine stem coccid Matsucoccus matsumurae Kuwara. Rep. Inst. For. Lianoing Prov. 9:1-27. McC•uRE, M.S. 1976. Colonization and establishment of the red pine scale, Matsucoccus resinosae (Homoptera: Margarodidae) in a Connecticut plantation. Environ. Entomol. 5:943-947. McCLuRE, M.S. 1983a. Temperature and host availability affect the distribution of Matsucoccus
102
HIBBARD ET AL.
matsumurae (Kuwana) (Homoptera: Margarodidae) in Asia and North America. Ann. Entotool. Soc. Am. 76:761-765. McCLURE, M.S. 1983b. Population dynamics of pernicious parasite; density dependent vitality of the red pine scale. Ecology 64:710-718. MENDEL, Z., SHAPHIR, N., MADAR, Z., GOLAN, Y., SCHPETER, E., and ZEHAW, A. 1988. The effect of habitat and age of the Aleppo pine plantation on damage caused by Matsucoccus josephi. Hassadeh 68:2203-2207. MORENO, D.S., and KENNETT, C.E. 1981. Monitoring insect pest populations by trapping in California citrus orchards. Proc. Int. Soc. Citriculture 2:684-689. MORENO, D.S., and KENNETT, C.E. 1985. Predictive year-end California red scale (Homoptera: Diaspididae) orange fruit infestations based on catches of males in the San Joaquin Valley. J. Econ. Entomol. 78:1-9. ORTU, S., and DELRIO, G. 1982. Field trials with synthetic sexual pheromone of Planococcus citri (Rigso). Redia 65:341-354. PARK, S.C. 1988. Biology and pheromone-mediated behavior of Matsucoccus thunbergianae in Korea with reference to M. resinosae in the United States ((Homoptera: Coccoidea: Margarodidae). PhD thesis. State University of New York, Syracuse, New York. PARK,S,C., WEST,J.R., ABRAHAMSON,L.P., LANIER,G.N., and S1LVERSTEIN,R.M. 1986. Crossattraction between two species of Matsucoccus. Extraction, bioassay, and isolation of the sex pheromone. J. Chem. Ecol. 12:609-617. PFEIFFER, D.G. 1985. Pheromone trapping of males and prediction of crawler emergence for San Jose scale (Homoptera: Diaspididae) in Virginia apple orchards. J. Entomol. Sci. 20:351353. QI, Y., YANG, M., and ZHAO, F. 1983. A preliminary study of the sex pheromone of the Japanese pine bast scale Matsucoccus matsumurae (Kuwana) (in Chinese, English summary). Contrib. Shanghai Inst. Entomol. 3:21-28. ROTUNDO, G., TREMBLAY, E., and GIACOMETI1, R. 1979. Final results of mass captures of the citrophilous mealybug males (Pseudococcus calceolariae Mask.) (Homoptera: Pseudococcidae) in a citrus grove. Bull. Lab. Entomol. Agric. Portici 30:266-274. SAS Institute. 1985. SAS User's Guide: Statistics. SAS Institute, Cary, North Carolina. STERNLICHT,M., BAR-ZAKAY,I., SZlVOS,Y., and DUNKELBLUM,E. 1981. Prediction of infestation by and control of the California red scale, Aonidiella aurantii (Maskell), through use of its pheromone. Proc. Int. Soc. Citriculture 2:695-700. STERNLICHT,M., DUNKELBLUM,E., and GIESELMANN,M.J. 1983. Management of scale insects through utilization of their pheromone. Bet Dagan, Israel (unpublished). WEBSTER, F.X., ZHANG,A., PARK, S.K., and PRESTWlCH,G.D. 1990. Synthesis of matsuone, the sex pheromone of the red pine scale. J. Org. Chem. In press. WINER, B.M. 1971. Statistical Principles in Experimental Design, 2nd ed. McGraw-Hill, New York. YOUNG, B., and QI, Y. 1983. Application of sex pheromone to the identification of pine bast scales (Margarodidae, Coccoidea). Contrib. Shanghai Inst. Entomol. 3:273-278. (Chinese, English summary). YOUNG, B., MILLER, D.R., and MCCLURE, M.S. 1984. Attractivity of th female sex pheromone of Chinese Matsucoccus matsumurae (Kuwana) to males of M. matsumurae in Japan and to males of M. resinosae Bean and Godwin in the United States (Margarodidae, Coccoidea, Homoptera). Contrib. Shanghae Inst. Entomol. 4:1-20.
LABORATORY AND FIELD TESTS WITH THE SYNTHETIC SEX PHEROMONE OF THREE M a t s u c o c c u s ~ PINE BAST SCALES
B.E. HIBBARD, 2 G.N. LANIER, 2'5 S.C. PARKS, 3 Y.T. QI, 4 F.X. W E B S T E R 2'* and R.M. SILVERSTEIN 2 2State University of New York College of Environmental Science and Forestry Syracuse, New York 13210 3Department of Forestry, College of Agriculture Chonnam National University Yongbong-dong, Kwangju 500-757 Republic of Korea 4Shanghai Institute of Entomology Academia Sinica Shanghai, China (Received May 10, 1990; accepted August 20, 1990)
Abstraet--Matsunone, (2E,4E)-4,6,10,12-tetramethyl-2,4-tridecadien-7one, is the primary sex pheromone of Matsucoccus resinosae, M. thunbergianae, and M. matsumurae. The synthetic compound was tested in the laboratory and in field tests with all three species, and significantly more males responded to synthetic matsuone than to controls. The attraction of synthetic matsuone to males in field tests was not significantly different from that of virgin females at the same matsuone release rate. An analog of matsuone previously shown to be biologically active was also tested in the laboratory with M. resinosae and 114. thunbergianae, and field tested with M. resinosae. The active analog required a dose approximately 100 times greater than matsuone to yield maximum laboratory bioassay response, and in field tests, attraction was not significantly different from controls at doses at which matsuone was significantly attractive. Key Words--Matsucoccus resinosae, M. thunbergianae, M. matsumurae, Coccoidea, Margarodidae, scale insects, pine bast scales, sex pheromone, (2E,4E)-4,6,10,12-tetramethyt-2,4-tridecadien-7-one, matsuone.
*To whom correspondence should be addressed. ~Homoptera: Coccoidea: Margarodidae. 5Deceased. 89 0098-0331/91/0100-0089506.50/0(c) 1991 Plenum PublishingCorporation
90
HIBBARD ET AL. INTRODUCTION
Pine bast scales in the genus Matsucoccus Cockerell are major pests of pine in France (Carle, 1968, 1974), Israel (Mendel et al., 1988), Korea (Park, 1988), China (Li et al., 1980), and the United States (Bean and Godwin, 1955; McClure, 1976, 1983b). Studies on the chemical communication of these insects began with Doane's (1966) report of a female-produced sex attractant for Matsucoccus resinosae Bean and Godwin. Sex pheromones have since been documented in M. matsumurae (Kuwana) (Qi et al., 1983), M. thunbergianae Millar and Park (Park et al., 1986), M. massonianae Young et Hu (Young and Qi, 1983), and M. josephi Bodenheimer and Harpaz (Sternlicht et al., 1983). Although a taxonomic revision has not been formally proposed, M. matsumurae and M. resinosae are almost certainly the same species (McClure, 1983a; Young et al., 1984). Park et al. (1986) reported the isolation of the sex pheromone of M. resinosae and the demonstration of its cross-attractiveness with M. thunbergianae. Spectral data available to Lanier et al. (1989) reduced the number of possible structures for this compound to four: 1, 2, 3 and 4 (Figure 1). On the basis of strong circumstantial evidence, Lanier et al. (1989) identified the pheromone ofM. resinosae, M. matsumurae, and M. thunbergianae as (2E,4E)-4,6,10,12tetramethyl-2,4-tridecadien-7-one (1, Figure 1), and gave it the trivial name matsuone. Although we were successful in synthesizing two analogs of this structure (3 and 4, Figure 1) and demonstrating biological activity with one of them (3), the compound we proposed as the true pheromone (1) in Lanier et al. (1989) was not available for testing. We recently synthesized matsuone (1) (see Figure 1) and its geometric isomer (2E,4Z)-4,6,11,12-tetramethyl-2,4-tridecadien-7-one (2) (Webster et al., 1990). We report here laboratory and field tests of three species of Matsucoccus pine bast scales with the two new synthetic compounds (1 and 2) and those analogs (3 and 4) tested by Lanier et al. (1989).
O
O
(1)
(2)
(3)
(4)
FIG. 1. Chemical structures of matsuone and the three analogs tested.
PHEROMONEOF Matsucoccus
91 METHODS AND MATERIALS
Field Sites and Insect Sources. Field experiments were conducted in Dutchess County, New York, U.S.A., from June 7 to September 15, 1989; in Tonggang County, Chonnam Province, Korea, from April 5 to 7, 1989; and the suburbs of Wu Xi City, China, from April 25 to 27, 1989 for M. resinosae, M. thunbergianae, and M. matsumurae, respectively. Infested branches of Pinus resinosa Ait., P. thunbergiana Franco, and P. tabulaeformis Carr. containing cocoons of M. resinosae, M. thunbergianae, and M. matsumurae, respectively, were placed in emergence cages similar to those described by Lanier et al. (1989). Males emerging from the cocoons were tested in laboratory behavioral bioassays. Laboratory Bioassays. The bioassay procedures used for M. resinosae and M. thunbergianae were those of Park et al. (1986) and Lanier et al. (1989). First, the test compound in 1/zl hexane in a micropipet was deposited just inside the tip of a medicine dropper. Excess hexane was expelled by depressing the bulb of the medicine dropper 20 times, and the dropper was positioned about 8 mm to one side of a pedestrian male. Puffs of air were delivered to the antennae of the pedestrian male by gently pressing the bulb at 1.5-sec intervals. Relative attraction was measured as the number of sides of a 4-cm-equilateral triangle along which a male would follow the retreating dropper tip. The highest response rating, three, indicated that the male followed the dropper for all three sides of the triangle, and the lowest response rating, zero, indicated no attraction. Incomplete following of the dropper along a side of the triangle was designated by the fraction of the side completed. For M. thunbergianae, males showing the response score of three to 2 x 10 -3 female equivalents (see Lanier et al., 1989) were tested, while with M. resinosae, the positive control was a live virgin female placed in the dropper tip. Bioassays with M. matsumurae in China used the method of Qi et al. (1983) and Lanier et al. (1989). Two filter paper disks of 1 cm diameter, each containing one of two materials or one material and a solvent blank, were placed into a 7-cm-diameter Petri dish. After the solvent had evaporated, 10 males were introduced and the Petri dish was covered. The behavior of males in the dish was observed, and the number of males on each of the disks was recorded after 10 min. Field Experiments. In Korea, the field experiments were performed in a P. thunbergiana forest with trees approximately 8 m tall. White plastic cards (10 x 14 cm) coated with Tanglefoot were affixed to horizontal bamboo poles 0.5 and 1.5 m above the ground. Traps were set in open areas of 15-40 m diam. and 7-20 m away from the trees. Treatments were assigned randomly on the poles so that each pole contained one replicate with different treatments spaced 1 m apart on the pole. Unbaited traps were placed in the field at 6 pro, April
92
HIBBARD ET AL.
5, 1989. Treatments of 222/zg matsuone in 60/~1 benzene on rubber GC septa (Supelco Inc., Bellefonte, Pennsylvania), 60/xl benzene on rubber GC septa, and 20 newly emerging females in a 2 • 2 x 0.5-cm fine net cage were replicated 10 times: five each at 0.5 and 1.5 m above the ground. Emerging females used as baits were collected between 8:00 and 9:30 AM, April 6. Baits were attached with pins to the lower portion of the sticky traps between 9:30 and 10:00 AM, April 6. Those males that were caught on untreated traps in the early morning were removed as the baits were set. Counts of these traps were then made between 12:00 and 1:00 PM on both April 6 and 7 by removing the males from the traps during counting. In China, the field trapping experiments were conducted in a suburban forest of P. tabulaeformis. Three replications of the following six treatments were tested: 1, 10, and 40 /~g matsuone on filter paper, one and five newly emerged females, and a benzene control. The trap, a piece of sticky cardboard (28 x 23 cm) folded in the middle at a 60 ~ angle, was similar to that described by Lanier et al. (1989). Three 1-cm-diam. holes were cut into each flap, and a roll of filter paper (5 x 0.8 cm) that served as the pheromone dispenser was hung inside the trap. Traps were hung from pine branches at heights between 1.5 and 2 m. Distance between traps was greater than 20 m. In New York, traps placed 2.44 m high were positioned between 1 and 2 m from the nearest branches and were at least 1.5 m from each other within a small planting of P. resinosae. Most of the trees were widely spaced, so that the crown was visible to the ground. Since M. resinosae usually occurs in the crowns, this site was ideal for setting traps at reasonable heights. Tree height at the site ranged from 1.5 m to 20 m. Dewill elm bark beetle traps (Dewill Inc., Elmhurst, Illinois) were cut into thirds (sticky surface of 20 x 48 cm), formed into cylinders 15 cm in diameter with the sticky surface facing outward, and stapled around a vertical pole 2.44 m in height. The baits were protected from the sun by fastening them with wire approximately 2 cm within inverted plastic film canisters (3 cm diam. x 4.8 cm). The bait apparatus then was stapled to the pole so that it was situated inside the sticky cylinder. Trials with live females as a treatment were set up between 8:00 and 9:30 AM, and results were recorded after 1:00 PM. Trials without live females were set up between 1:30 and 5:30 PM, and results were recorded after several days. The first field experiment in New York tested the relative attraction of 25 /xg matsuone (1), 250/xg matsuone, 25 /xg 2, 250/xg 2, and a hexane control. Rubber GC septa served as the pheromone dispenser. Five replications of this experiment were set up on June 8 and dismantled on June 15. Ten more replications were set up on June 20 and dismantled on June 25. We next compared known quantities of matsuone with attraction to live adult females. Six replicates of 20 live females each (collected between 6:30 and 8:30 AM on August 15, 1989) were placed in fine netting and added to the
PHEROMONE OF
Matsucoccus
93
traps along with baits by 9:30 AM. Four doses of matsuone were chosen and replicated 10 times: 10, 50, 100, and 250 txg. Trap catch was recorded between 1:00 and 2:00 PM the same day. The pheromone dispenser for this experiment was a cotton dental wick (Healthco International, Boston, Massachusetts). The last experiment reported herein tested 200/~g each of matsuone (1), a hexane control, and 2, 3, and 4 on rubber GC septa. The experiment was set up September 15 and dismantled September 25. Release Rate Determination. The relative release rates of matsuone from rubber septa, dental cotton wicks, and filter paper were determined in the laboratory. Matsuone was added to the dispensers, and they were immediately placed in a 250-ml Erlenmeyer flask that was protected from UV radiation with aluminum foil. Each of the dispensers was tested with 250/zg of matsuone, and filter paper was also tested at 40/xg because this dose was effective for trapping males in China. There were four replicates of each dispenser at each dose tested. House vacuum pulled room air through activated carbon (6-14 mesh), past the dispensers, and through 0.6 g Porapak Q, on which matsuone was collected. Flow through the flask was held constant at 100 ml/min. Temperature was held constant at 20 _+ 2~ Volatiles were recovered from the Porapak with 4 ml of pentane. Extractions were made after 24, 48, and 72 hr for each dispenser, and also at 168 hr for rubber septa. The concentration of matsuone in the samples was determined with an HP 5890 gas chromatograph fitted with a 50-m methyl silicon capillary column (DB-1, Supelco, Inc., Bellefonte, Pennsylvania) with 1 ng//zl n-octadecane as an internal standard. Statistical Analysis. The statistical package SAS (SAS Institute, 1985) was used for data analysis. All field data were analyzed with one-way or two-way analysis of variance (ANOVA), then by Duncan's (1955) multiple-range test when significant F values were found in the ANOVA. The trap-catch data were subject to log (X + 1) or square root (X + 0.5) transformations as needed to meet the assumption of equal variance (Winer, 1971). Nonparametric procedures were required for analysis of laboratory bioassays with M. resinosae and M. thunbergianae. A rank transformation followed by the procedures above served this purpose (Conover and Iman, 1981; SAS Institute, 1985, p. 19).
RESULTS
Laboratory Bioassays. In bioassays with M. thunbergianae in Korea, responses of pedestrian males to all doses of matsuone tested except the lowest dose, 0.04 pg, were significantly higher (P < 0.05) than the control (Table 1). Maximum responses to matsuone were elicited by doses of 4.0 pg to 0.40 ng, while the dose required for maximum response to 3 was about 0.50 ng. Differences in attraction between the highest dose of 3 (0.50 ng) and doses of mat-
94
HIBBARD ET AL. TABLE 1. MEAN RESPONSES IN LABORATORY BIOASSAYS OF Matsucoccus
thunbergianae
Material Hexane Matsuone
Analog 3
MALES TO DILUTIONS OF MATSUONE AND ANALOG 3 a
Quantity (pg)
4.0 4.0 4.0 4.0 4.0 4.0 5.0 5.0 5.0 5.0
X • X • x x x • • x
10 2 10-' 10~ 10 ~ 102 103 10 -t 10~ 10 ~ 102
No. tested 24 24 24 24 24 24 24 24 24 24 24
Mean response + SE 0.06 0.21 1.08 2.88 2.73 2.79 2.04 0.15 0.42 1.98 2.69
+ 0.03 +_ 0.07 + 0.23 + 0.09 +__0.15 +_ 0.11 + 0.24 + 0.05 + 0. I0 + 0.22 + 0.15
E DE C A A A B E D B A
~Although the raw data are shown, statistical analysis was performed on rank transformed data. Significant differences are indicated by different letters after the mean.
suone from 4.00 pg to 0.40 ng were not significant. In bioassays with M. resinosae in the United States, responses to doses between 50.0 pg to 5.0 ng of matsuone, and between 5.0 ng and 50.0 ng of 3 were not significantly different from the positive control of a live female (Table 2). All doses of 2 and 4 elicited lower (P < 0.05) responses than a live female. All doses of matsuone and doses of 50.0 pg, 5.0 ng, and 50.0 ng of 2 gave greater responses than the hexane control. No dosage of 4 gave response significantly greater (P < 0.05) than the hexane control. For 2 and 3, responses to all doses from 50.0 pg to 50.0 ng were higher (P < 0.05) than to the hexane control. Laboratory bioassays in China utilizing the Petri dish bioassay indicate that M. matsumurae are significantly attracted to matsuone (Table 3). At all doses tested except the lowest dose (10 ng), more (P < 0.01) males were attracted to filter paper disks containing matsuone than to disks with benzene alone. Increasing the dosage of matsuone increased the number of males on the matsuone disk. In a choice test comparing 0.20/~g matsuone with 5 female equivalents (see Lanier et al., 1989) of crude extract, there was no significant difference between the number of males on the two filter paper disks. When comparing 0.20/zg matsuone in choice tests with 8.0/zg 3, significantly more males were found on the filter paper disk with 0.2/xg of matsuone. Field Tests. In field tests with M. thunbergianae in Korea, significantly more males were caught in traps baited with 222/zg matsuone 0.5 m from the ground than all other traps (Figure 2). Traps baited with 20 live females placed
PHEROMONE OF Matsucoccus
95
TABLE 2. MEAN RESPONSES IN LABORATORY BIOASSAYS OF Matsucoccus resinosae MALES TO DILUTIONS OF MATSUONE, MATSUONE ANALOGS, AND LIVE FEMALES a
Quantity Material Live females Hexane Matsuone
Analog 2
Analog 3
Analog 4
(pg)
1 female 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
• x x x x • x • • x
10 -~ 10o 10 ~ 102 103 104 10 L 10 ~ 10 t 102
X 10 3 >< 10 4
x x • x • • x • • • • •
10 - t 10 ~ 10 ~ 102 103 104 10 - I 10 ~ 10 ~ 102 103 104
No. tested 42 45 5 24 32 14 12 5 9 8 8 8 6 5 5 5 20 15 9 7 5 8 15 15 8 8
Mean response 3.00 0.10 1.70 1.53 2.57 2.71 2.71 1.60 0.00 0.44 0.95 0,69 t.92 0.67 0.00 0.50 1.43 1.85 2.78 2.25 0.00 0.50 0.00 0.23 0.06 0.00
+ 0.00 A _+ 0.04 HI + 0.52 CDE _+ 0.21 CDE _+ 0.16 ABC _+ 0.19 AB + 0.19AB +_ 0.51 CD + 0.00 I _+ 0.19 GHI _+ 0.32 FG _+ 0.19 FGH _+ 0.52 BCD + 0.11 EFG _+ 0.00 I _+ 0.34 GHI + 0.00 DEF 2:0.19 CDE _+ 0.06 AB + 0.00 ABC + 0.00 I + 0.44 GHI + 0.26 I +_ 0.36 HI _+ 0.00 I +_ 0.31 I
aAlthough the raw data are shown, statistical analysis was performed on rank transformed data. Significant differences are indicated by different letters after the mean.
at 0.5 m also caught more (P < 0.05) males than controls, but there was no significant difference between any treatments placed at 1.5 m. In China, traps baited with 10 and 40/xg of matsuone caught significantly more male M. matsumurae than control traps, traps with 1 female, and traps with 1 #g of matsuone (Figure 3). Differences between traps with 10 and 40/~g of matsuone and traps with five females were not significant, nor were differences between traps with 1 /xg of matsuone and control traps. Traps with one or five females caught significantly more males than control traps. In the United States, significantly more (P < 0.05) male M. resinosae were caught on traps baited with 250 #g of matsuone than those baited with 25
HIBBARD ET AL.
96
TABLE 3. MEAN RESPONSES OF m. matsumurae MALES IN CHOICE TESTS BETWEEN MATERIAL 1 AND MATERIAL 2 a Material 1 (ng matsuone)
Material 2
Mean response (material 1 - material 2)
10 20 50 100 200 400 800 1300 200 200
Benzene Benzene Benzene Benzene Benzene Benzene Benzene Benzene 5 ~male equivalents 8000 ng analog 3
2.00 3.67* 4.67* 6.67* 7.67* 8.33* 9.33* 9.67* 1.67" 5.33*
Significant differences are indicated by different letters after the mean. * = significantly different from zero (P < 0.01).
175
Z- 150 t~ o
100
"~ 75
E
d 5o z 25 0 Matsuone
20 females
Control
Treatment F~G. 2. Number of male M. thunbergianae caught in traps baited with 222 ~g matsuone on rubber septa, 20 live females, and a solvent control. Although raw data are shown, statistical analysis was performed on log (X + 1) -transformed data. Bars topped by different lowercase letters are significantly different (P < 0.05) as indicated by Duncan's (1955) numerical multiple-range test. Standard error bars are indicated.
/xg m a t s u o n e , 25 ~ g 2, 2 5 0 txg 2, o r a h e x a n e c o n t r o l ( F i g u r e 4); n o o t h e r s i g n i f i c a n t d i f f e r e n c e s w e r e f o u n d . I n d o s e - r e s p o n s e tests u s i n g c o t t o n d e n t a l w i c k s as t h e p h e r o m o n e d i s p e n s e r , t h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e b e t w e e n t r a p s b a i t e d w i t h 100 a n d 2 5 0 / x g m a t s u o n e , a n d t h e r e w a s n o d i f f e r e n c e b e t w e e n c o n t r o l t r a p s a n d t r a p s w i t h 2 0 l i v e f e m a l e s ( w e c a n n o t e x p l a i n this last result).
PHEROMONE OF
97
Matsucoccus 40
_E-
30
O5 O CO d3 20
E d 10
Z
Treatment FIG. 3. Number of male M. matsumurae caught in traps baited with matsuone on filter paper or live females. Bars topped by different lowercase letters are significantly different (P < 0.05) as indicated by Duncan's (1955) numerical multiple-range test. Standard error bars are indicated.
50
~4a t~ O 30 03 t~ 20
E d Z
lO
r
%
+'--
"4_
,v^
'/"
Treatment FIG. 4. Number of male M. resinosae caught in traps baited with matsuone, 2, or solvent on rubber septa. Although raw data are shown, statistical analysis was performed on square root (X + 0.5) -transformed data. Bars topped by different lowercase letters are significantly different (P < 0.05) as indicated by Duncan's (1955) numerical multiple-range test. Standard error bars are indicated.
98
HIBBARD ET AL.
All other differences were significant (Figure 5), including the difference between male catches on traps baited with 10 txg matsuone and control traps. A regression of the dose-response curve was significant (P < 0.05) with an R 2 of 50.9%. In a similar experiment in which all analogs were tested, more males (P < 0.05) were caught on traps baited with matsuone than traps baited with any analog. There was no significant difference between catches on traps baited with 2, 3, 4, and the hexane control (Figure 6). Release Rates. The release rate of matsuone from filter paper rolls and dental cotton wicks was significantly greater than that from robber GC septa (Supelco, Inc., Bellefonte, Pennsylvania) for each of the first three days (Table 4). Significantly more matsuone was released from cotton wicks than filter paper on day 2, but there was no significant difference on days 1 or 3. The release rate of 40/~g matsuone from filter paper was higher than that of 250/zg matsuone from rubber septa on day 1, but this difference was not significant on day 2 or day 3. The average release rate from rubber septa from days 4-7 was equivalent to that released from robber septa on day 2.
DISCUSSION
Park (1988) placed newly emerged M. thunbergianae females in a device designed to collect the female sex pheromone on filter paper. The filter paper was extracted with hexane, and 5 x 10 -3 female hour equivalents (FHE) yielded
14" "r'~
12'
'm
10.
0 69 0
8' 6
E z
4. 2, 0
Treatment
:%/%
F~6. 5. Number of male M. resinosae caught in traps baited with live females or different doses of matsuone on cotton dental wicks. Although raw data are shown, statistical analysis was performed on log (X + 1) -transformed data. Bars topped by different lowercase letters are significantly different (P < 0.05) as indicated by Duncan's (1955) numerical multiple-range test. Standard error bars are indicated.
PHEROMONE OF
Matsucoccus
99
10
03 8
0
6
E 4 d Z 2
Treatment FIO. 6. Number of male M. resinosae caught in traps baited with 200 ~g of matsuone and three analogs on rubber septa. Although raw data are shown, statistical analysis was performed on log (X + 1) data. Bars topped by different lowercase letters are significantly different (P < 0.05) as indicated by Duncan's (1955) numerical multiple-range test. Standard error bars are indicated.
TABLE 4. RELEASE RATE OF MATSUONE FROM PHEROMONE DISPENSERS AS MEASURED IN THE LABORATORY
Release rate (#g/day +_ SE) Dispenser type
Loadrate (/~g)
Rubbersepta Cotton wick Filter paper Filter paper
250 250 250 40
Day 1
Day 2
Day 3
1.50 + 0.60ef 0.41 + 0.03fg 0.12 _ 0.02g 12.52 _+ 2.84ab 8.93 + 0.29bc 1.98 _+ 0.29e 17.03 ___1.68a 3.95 + 0.80d 1.06 _+ 0.19ef 6.15 ___0.96d 1.09 _+ 0.90ef 0.25 _+ 0.10fg
Days 4-7 0.41 _+ O.09e
an average response of 2.2 in the same bioassay as used in the present study (5 x 10 -2 FHE gave an average of 3.0). This response is equivalent to the response of M. thunbergianae males to 4.0 pg of matsuone and M. resinosae males to 50.0 pg matsuone reported herein. If we assume that all of the sex pheromone was collected on the filter paper in Park (1988) and that matsuone is the only active compound, we can estimate the production of matsuone (based on the level of response) to be in the range of 0.8 ng/hr for M. thunbergianae and 10 ng/hr for M. matsumurae and M. resinosae. Because we determined the release rate of matsuone from the dispensers utilized in the present study (Table 4), we can compare the field trap catch of
100
HIBBARD ET AL.
synthetic matsuone to that of natural matsuone. The field tests in China were conducted for three days, so if we use the release rate of 40 t~g matsuone from filter paper from day 2 as an average, approximately 54 ng/hr of synthetic matsuone were released (Table 4). M. matsumurae females produce approximately 10 ng/hr, so the five females tested would have produced approximately 50 ng/ hr of natural matsuone (a conservative estimate given our assumptions). The number of male M. matsumurae caught on traps baited with five females and those baited with 40/~g matsuone on filter paper were not significantly different. In experiments in Korea with M. thunbergianae, septa were loaded with matsuone the afternoon before being applied to the traps, and the experiments were set up for two days, so it is again appropriate to use the release rate data from day 2. The release rate of matsuone from rubber GC septa on day 2 was approximately 17 ng/hr (Table 4), while the calculated release rate of 20 female M. thunbergianae would be approximately 16 ng/hr. Both traps baited with 20 females and traps baited with 222/zg synthetic lriatsuone on rubber GC septa caught significantly more males than control traps at 0.5 m in height. Traps baited with synthetic matsuone also caught significantly more males than traps baited with 20 females at 0.5 m (Figure 2), but this difference in trap catch was not significant when data from both heights (0.5 and 1.5 m) are pooled. If our calculation of the release rate of matsuone from these Matsucoccus spp. is correct, it appears that matsuone can account for all of the attraction to femalebaited traps. Demonstrating biological activity of the synthetic compound is crucial to verifying the identification of a pheromone. We have shown that synthetic matsuone is significantly attractive to three species of pine bast scales in laboratory and field tests, and we have shown that matsuone can account for all of the attraction of live females in the field. We have corroborated the conclusion of Lanier et al. (1989) that (2E,4E)-4,6,10,12-tetramethyl-2,4-tridecadien-7-one is the major sex pheromone of M. resinosae, M. thunbergianae, and M. matsumurae. Lanier et al. (1989) were correct in their prediction that matsuone would show much higher activity than its analogs. In laboratory bioassays with both M. resinosae and M. thunbergianae, the dose of 2 (the most active analog) required to produce a full response was approximately 100 times greater than that required for matsuone. In field trials with M. resinosae with 200/xg of all analogs present on rubber septa, only traps baited with matsuone caught significantly more males than the control. The final identification step is also the first step in the utilization of semiochemicals in integrated pest management. Synthetic pheromones have been produced for a number of other Coccoidea and have been successfully incorporated into management programs (for detection and monitoring) for the citrus mealybug, Planococcus citri (Risso) (Ortu and Delrio, 1982); the citrophilous mealybug, P. calceolariae Mask. (Rotundo et al., 1979); the San Jose scale,
PHEROMONE OF M a t s u c o c c u s
101
Quadraspidiotus perniciosus (Comstock) (Angerilli and Logan, 1986; Pfeiffer 1985); and the California red scale, Aonidiella aurantii (Maskell) (Ervin et al.,
1985; Moreno and Kennett, 1981, 1985; Sternlicht et al., 1981). Sex pheromones have even been used for mating disruption for the California red scale (Bar-Zakay et al., 1989). Synthetic matsuone produced by Webster et al. (1990) trapped significantly more male M. resinosae, M. thunbergianae, and M. matsumurae than control traps in the present experiments. The results from the present paper indicate that synthetic matsuone may be a valuable component in the management programs of M. resinosae, M. thunbergianae, and M. matsumurae. Acknowledgments--We thank Aijun Zhang for synthesizing the matsuone and 2 used in this study, Mr. Ray Vaughan from the Department of Environmental Conservation at Stonykill for help in locating field sites in New York, Mr. Randy Randazzo from the Sprout Lake Camp and Mr, Dick Conkline for cooperation in using their land for field sites in New York. We also thank Mr. Conkline for permission to down red pine trees for laboratory work. Mr. Hyung-Sun Kim from the Chonnam Forest Experiment Station assisted in the Korean field tests. Funding was provided in part by a grant from the National Science Foundation and the Department of Entomology, Forestry Research Institute of Korea.
REFERENCES
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