Journal o f Chemical Ecology, Vol. 9, No. 1, 1983
AGGREGATION PHEROMONE COMPONENTS OF TWO SPECIES OF P I S S O D E S WEEVILS (COLEOPTERA: CURCULIONIDAE): Isolation, Identification, and Field Activity 1'2
D O N A L D C. B O O T H , 3 T H O M A S W. P H I L L I P S , 3 A L F C L A E S S O N , 4'5 R O B E R T M. S I L V E R S T E I N , 4 G E R A L D N. L A N I E R , 3 and J A N E T R. W E S T 4 3Department o f Environmental and Forest Biology and 4Department o f Chemistry State University o f New York College o f Environmental Science and Forestry Syracuse, New York 13210 5Biomedical Center, Uppsala University Uppsala, Sweden (Received February 17, 1982; revised April 8, 1982)
A b s t r a c t - - T w o related volatile compounds were identified from each of two species of Pissodes bark weevils and implicated as components of their aggregation pheromones. Grandisol (cis-2-isopropenyl-l-methylcyclobutaneethanol), and its corresponding aldehyde, grandisal, were isolated from males of both P. strobi and P. approximatus and were found in the abdomens and hindguts of the respective species. In field tests synthetic grandisol and grandisal together with odors from cut pine acted synergistically in attracting both sexes of P. approximatus. This response was similar to that elicited by male P. approximatus feeding on cut pine. Males and females of natural populations of 1". strobi were more responsive to caged males feeding on leaders of white pine than they were to leaders alone. The combination of grandisol, grandisal, and leaders was less attractive than males on leaders, but more attractive than leaders alone. From isolation of pheromone components at different times of the year, it was
1Research supported by grants from the National Science Foundation, The New York State Museum and Science Service, and a travel grant to A.C. from Stiffelsen Blanceflor Boncompagni-Lndod ovisi. 2Preliminary report presented at meeting of the Eastern Branch Entomological Society of America, Boston, Massachusetts, September 14-16, 1977. Abstract in J. AT. Y. Entomol. Soc. 85:167. In part from theses by DCB (Ph.D.) and TWP (M.S.).
0098-0331/83/0100-0001503.00/0 9 1983PlenumPublishingCorporation
BOOTH ET AL.
2
determined that males of both species produced grandisol and grandisal only at times when cohort females were reproductively mature. Key Words--Pissodes strobi, Pissodes approximatus, Coleoptera, Curculionidae, aggregation pheromone, grandisol, grandisal, Pinus strobus, white pine weevil.
INTRODUCTION
Bark weevils of the genus Pissodes Germar feed on conifer trees in the family Pinaceae and their larvae develop in the inner bark (phloem) of their hosts. Pissodes strobi (Peck), the white pine weevil, is notorious among forest insects for its deformation of pines and spruces throughout North America. The biology and habits of P. strobi have been extensively documented (Macaloney, 1930; Taylor, 1930; Belyea and Sullivan, 1959). In the spring the adults orient to the healthy terminal leader (top shoot) of a host tree, where they mate, feed, and oviposit. Larval feeding girdles the stem, killing old and new growth;one or more of the remaining lateral branches must assume dominance, resulting in a fork or crook in the stem that may render the tree useless for saw timber. The northern pine weevil, P. approxirnatus Hopkins, is not as economically important as P. strobi, although larvae may kill stressed trees and intense feeding by adults may be injurious to small trees (Finnegan, 1958). Adults of P. approximatus orient to cut or weakened trees to reproduce, and their larvae develop in the inner bark of this material. P. approxirnatus occurs on most pines and spruces throughout the northeastern and Lake states, and in the Canadian boreal forest west to the Rocky Mountains; P. strobi occurs in three somewhat distinct populations across North America, according to the preferred host of the region. In the east, P. strobi is found primarily on eastern white pine (Pinus strobus L.), in the Rocky Mountains in Engelmann spruce (Picea engelmannii Parry), and in the Pacific coastal region on Sitka spruce (Picea sitchensis Carr.) (Smith and Sugden, 1969). Finnegan (1958) reported mass flights ofP. approximatus in response to suitable host material (moribund pines) in Ontario. P. strobi forms aggregations on leaders early in its flight season, prior to increased dispersal and oviposition (Overhulser and Gara, 1975). The first evidence of an aggregation pheromone in Pissodes was reported by Booth and Lanier (1974). In that study, P. approximatus males confined on host material were more attractive to both sexes than females on host material or host material alone. Interestingly, P. strobi males caged on the same host material also attracted local P. approximatus. Subsequently, Booth (1978) demonstrated that P. strobi males caged on white pine leaders were attractive to conspecific males and females in the field; females feeding on leaders and leaders alone were not attractive. This paper reports the isolation and identification of volatile
PHEROMONE COMPONENTS OF
Pissodes
3
pheromone components from males of P. strobi and P. approximatus and discusses their significance for these species. METHODS AND MATERIALS
Weevil Populations. Weevils used in experiments were from natural populations within a 100-km radius of Syracuse, New York. P. strobi adults were obtained in an 8-year-old eastern white pine plantation in southeastern Cortland County, near Virgil, New York. Overwintered weevils were collected from the upper lateral branches of the previous year's brood trees as daily high temperatures approached 15-20 ~ C, usually in the second or third week of April. P. strobi used during winter months were obtained from naturally infested white pine leaders in August. The weevils were sexed according to the method of H a r m a n and Kulman (1966) and maintained on freshly cut white pine branches under ambient lighting in the spring or under a 16:8 (lightdark) photoperiod in winter. P. a_pproximatus adults were collected from the undersides of freshly cut red pine logs scattered on the forest floor within a 30-year-old red pine (Pinus resinosa Ait.) plantation at Heiberg Memorial Forest, near Tully, New York. Weevils used in the fall were obtained as they emerged from naturally infested pine logs brought into the laboratory. Collection, Isolation, and Identification of Pheromone Components. Volatiles from living Pissodes adults were collected by aeration and absorption on P o r a p a k Q (Waters Assoc., Framingham, Massachusetts) (Byrne et al., 1975). F r o m 50 to 150 weevils of each sex were aerated for 5-10 days in large vacuum desiccators while feeding on fresh cuttings of their respective host material: P. strobi on white pine leaders and P. approximatus on red pine branches. Aerations were carried out in the laboratory at 21 ~ C under ambient lighting in the spring or under a controlled 16:8 (light-dark) photoperiod in the fall or winter. Prior to aeration, the P o r a p a k was cleaned according to the method of Williams et al. (1981) and, following aeration, the volatiles were extracted with distilled pentane (5 ml/g). The extract was then dried over sodium sulfate and concentrated to 1-4 ml by fractional distillation in preparation for gas chromatography (GC) or bioassay. Hindguts or abdomens were dissected from the weevils and crushed in small amounts of distilled pentane; the slurry was sonicated and centrifuged, and the supernatant was used for GC analysis. Extracts were fractionated on a Varian 2700 c h r o m a t o g r a p h equipped with a flame ionization detector, a 99:1 effluent splitter, and a thermal gradient collector (Brownlee and Silverstein, 1968). Fractions were collected in 30.5 cm • 1.7 m m (OD) glass capillary tubes that were flame sealed and stored at - 3 0 ~ until used. A 5.4 m • 4 m m (ID) glass column packed with 4% Carbowax 20M on C h r o m o s o r b G 60-80 mesh was used under the following
4
BOOTH ET AL.
conditions: 90 ~ for 4 min, raised to 215 ~ at 2~ 55 ml He/min, injector 140~ , detector 215 ~ . The collected compounds were further fractionated on a 6.0 m • 4 mm (ID) glass column (5% OV-225 on Chromosorb G 60-80 mesh, 80 ~ for 4 min, raised to 215 ~ at 2~ /min, 55 ml He/min, injector 140~ , detector 215~ Mass spectral electron impact (70 eV) data were obtained from a Finnegan GLC 9500. P r o t o n nuclear magnetic resonance ([1H]NMR) spectra were recorded (Varian XL-100 instrument) on 80/~g samples in 50 #1 C6D6 in the inner cell of a concentric tube. Infrared spectra were obtained on a Perkin-Elmer 621 instrument, fitted with a beam condenser, on 60 to 80-#g samples dissolved in CCh in a 4-~zl cavity cell. Synthetic Compounds. Synthetic (racemic) grandisol (cis-2-isopropenyl1-methylcyclobutaneethanol) was obtained from Chemical Samples Co. (division of Albany International, Inc.) and GC-purified for coinjection. The corresponding aldehyde of grandisol (grandisal) was obtained by oxidation with pyridinium chlorochromate in methylene chloride in the presence of sodium acetate (1 equivalent); reaction time was 80 min at room temperature. The reaction mixture was filtered through a 5 • 1.5 cm Florisil column, and after evaporation of the solvents the purity of the aldehyde was determined on a 3 m • 4 mm (ID) glass GC column (7% Carbowax on Chromosorb G 60-80 mesh) at 125~ C. The same synthetic procedure was used to provide aldehyde for field experiments. Field Assays. Bioactivity of putative pheromone components was tested in several trapping experiments in the field. P. approximatus experiments took place in mature red pine plantations near Tully, New York, while P. strobi tests were conducted in young white pine plantations near Virgil, New York. Two field tests of P. approximatus employed hardware cloth sticky traps placed on the ground (Booth and Lanier 1974), arranged in five randomized blocks of six traps each. The six treatments of the first test included: (1) male weevils caged on a red pine bolt, (2) the putative pheromone components grandisol and grandisal with a red pine bolt, (3) the same concentration of these compounds without red pine, (4) higher concentrations of these compounds without red pine, (5) solvent only (hexane), and (6) a red pine bolt alone. The second experiment tested the attractiveness of (1) male weevils on red pine, (2) grandisol and grandisal with red pine, (3) half as much of these compounds with red pine, (4) grandisal only with red pine, (5) grandisol only with red pine, and (6) a red pine bolt alone. The first P. approximatus test, conducted in the late summer, assayed those weevils which presumably eclosed in early summer and were currently reproductive (Zerillo et al., undated ms.); the second experiment tested the more active spring population. P. strobi tests, conducted in two consecutive spring seasons, employed hardware cloth sticky cylinders which surrounded screen-enclosed
P H E R O M O N E C O M P O N E N T S OF
eissodes
5
white pine leaders (Phillips 1981). Five replicates each of three different treatments (male P. strobi confined on leaders, grandisol and grandisal on a leader, and a leader alone) were established on trees picked at random within a one-acre plantation. In field tests of Pissodes species, synthetic pheromone components were released from snap-top polyethylene vials (Cole-Parmer Instrument Co.), and live weevils used in certain treatments were fieldcollected immediately prior to the tests.
RESULTS
Identification of Pheromone Components. Systematic GC fractionation of the extracts, monitored by laboratory bioassay of the fractions singly and in combination, was attempted (Silverstein, 1977). However, none of the laboratory olfactometers (Booth, 1978) provided consistent, interpretable results. Since the amounts of the fractions isolated from limited numbers of available weevils were too small for field testing, we turned to examining differences between chromatograms of extracts of male and female hindguts or abdomens. Two compounds that were present in the abdomens ofP. strobi males were absent in abdomens of females (31.5 and 51.0 min on the Carbowax 20M column). Each of these compounds were subsequently found in larger amounts in the more complex chromatogram of the Porapak extract of male P. strobi, from which they were collected and refractionated on the OV-225 column (40 and 48 min). The compound with the longer retention time was identified as grandisol (cis-2-isopropenyl-l-methylcyclobutaneethanol) (Figure la) from the mass, IR, and N M R spectra, and its identity was confirmed by coinjection with an authentic sample and by congruence of spectra (Tumlinson, 1969). We used the same techniques to identify the compound with the shorter retention time as the corresponding aldehyde, referred to here as grandisal (Hedin, 1977) (Figure lb). The same compounds were identified from hindgut and Porapak extracts
FIG. 1. Pissodes pheromone components: (a) grandisol, (b) grandisal.
6
BOOTH ET AL. TABLE 1. OCCURRENCE OF GRANDISOL AND GRANDISAL IN MALE P i s s o d e s a
Hindgut extracts (/~g/individual)
Aerations (/~g/weevil-hour) b
Species
Grandisol
Grandisal
Grandisol
Grandisal
P. s t r o b i P. a p p r o x i m a t u s
0.200 0.026
0.400 0.013
0.0033 0.0002
0.0074 0.0006
Presence of c o m p o u n d s determined by coinjection, a m o u n t s determined by GC comparison with known standards. bA weevil-hour is equal to the a m o u n t produced by one weevil in one hour of aeration.
from P. approximatus males. In neither species were the compounds detected in the other body parts of the male or in any of the extracts from females. The amounts of grandisol and grandisal occurring in the various extracts of male Pissodes, as determined by comparative GC, are presented in Table l; the occurrence of these compounds in males at different times of the year is given in Table 2. One striking difference between the two species of Pissodes is that, in both sampling methods (Table 1), P. strobi appears to produce both chemicals at a level ten times greater than that ofP. approximatus. Abdomen and hindgut extracts only reveal the compounds present within the insect at the time of dissection, but the extract from the Porapak aeration should represent the actual amounts and ratios of chemicals emitted by the insects through time. However, the results may not be quantitative because of uncertainties in collection and recovery efficiencies and in the stability of the aldehyde. In the Porapak extracts of the ratio of grandisol to grandisal produced by P. strobi is approximately 1 : 2, and that for P. approxirnatus is 1:3. Attempts to determine enantiomeric composition will be made when larger amounts of isolated grandisol and grandisal become available. Field Activity. Table 3 presents the data for two field tests of grandisol and grandisal with a population ofP. approximatus. The highest trap catch in both tests was on the treatment with male P. approximatus confined on a red pine bolt. However, the second treatment of each test, involving grandisol, grandisal, and a red pine bolt, attracted almost as many weevils as the male treatment. In the 1976 test, the two treatments with various amounts of grandisol and grandisal that did not include a red pine bolt caught significantly fewer weevils than that which included red pine, indicating synergism between odors from the host material and those from the synthetic chemicals. The two control treatments in this test, solvent (hexane) and a red pine bolt, caught similarly low numbers of weevils. In the 1977 test, the response to the higher levels of grandisol and grandisal was statistically
15 May 3 June
P. approximatus Hindgut Aeration
Aeration Abdomens Aeration Aeration
Field Field
Field Field Field Field
18 Oct. 21 July
11 June 3 Sept. 15 Dee. 24 Feb. l0 March
Date b
Hindgut Aeration
Aeration Aeration Aeration Aeration Aeration
Extract type
Condition
Extract type
a Occurrence of grandisol and grandisal was determined by GC coinjection with laboratory standards. b Refers to date aeration was completed or hindguts or abdomens dissected.
5 May 10 May 12 May 26 May
Date b
P. strobi
Species
Absent
Present
Laboratory Field
Field Laboratory Laboratory Laboratory Laboratory
Condition
TABLE 2. SEASONAL OCCURRENCE OF PHEROMONE COMPONENTS IN EXTRACTS OF PORAPAK Q AERATIONS, EXISED HINDGUTS, OR ABDOMENS OF MALE e i s s o d e s EXPOSED TO FIELD OR LABORATORY CONDITIONS PRIOR TO EXAMINATION" 9
BOOTH ET AL. TABLE 3. RESPONSE OF Pissodes approximatus TO GRANDISOL ( G O H ) , GRANmSAL ( G C H O ) , AND NATURAL ATTRACTANTS IN TWO FIELD TESTS
No. of weevils captured Date of Test
Treatments a'b
Females
Test 1: I0 Aug-17 Sept., 1976
4 Male P. approximatus plus red pine 8 mg GOH + 4 mg GCHO + red pine 8 mg GOH + 4 mg GCHO 14 mg GOH + 5 mg GCHO Solvent (25 #1 hexane) Red pine
Test 2: 11 May-6 July, 1977
5 Male P. approximatus plus red pine 2 8 m g G O H + 1 0 m g G C H O + redpine 14 mg GOH + 5 mg GCHO + red pine 5 mg GCHO + red pine 14 mg GOH + red pine Red pine
Males
Total c
27 27 7 14 1 3
18 10 3 2 6 1
45a 37a 10bc 16b 7bc 4c
142 109 54 22 5 8
21 30 21 6 4 0
163a 139a 75b 28c 9d 8d
a Red pine bolts were freshly cut, 12.7 • 10.2 cm; chemical baits were changed weekly. b Release rate of pheromone components was determined by cold trapping volatiles from a slow airstream and quantifying by GC: 8 mg GOH = 83 #g/day; 14 mg GOH = 174 #g/day; 28 mg GOH = 348 #g/day; 4 mg GCHO = 205 #g/day; 5 mg GCHO = 120 #g/day; 10 mg GCHO = 240 #g/day. CTotals followed by the same letter(s) are not significantly different (chi-square test for independence, P < 0.05).
TABLE 4. RESPONSE OF thssodes strobi TO GRANDISOL ( G O H ) WITH GRANDISAE ( G C H O ) , AND NATURAL ATTRACTANTS IN TWO FIELD TESTS
No. weevils captured Treatments a
Females
Males
Total b
Test 1: May 1 to June 27, 1980 3 males + leader 5 mg GOH + 10mg GCHO + leader leader
9 4 4
14 15 5
23a 19ab 9b
Test 2: April 30 to June 19, 1981 5 m g G O H + 1 0 m g G C H O + leader leader
10 0
9 9
19 9
a Chemical baits were changed biweekly. Release rate of pheromone components was determined gravimetrically: 5 mg GOH = 43 #g/day 10 mg GCHO = 80/zg/day. Totals m test 1 followed by the same letter are not significantly different (chi-square test for independence, P < 0.05). Differences between totals in test 2 were not significant. b
9
'
.
.
.
.
PHEROMONE COMPONENTS OF
Pissodes
9
similar to that for the male treatment; the response was nearly halved by halving the concentration of the chemicals. Grandisal with red pine was attractive, but attracted fewer weevils than when grandisol was included, indicating synergism between these two compounds. Grandisol with red pine was not attractive. Of the three treatments examined in the first P. strobi field test (Table 4), the most attractive was that with male P. strobi caged on an eastern white pine leader. The response to grandisol plus grandisal on a white pine leader was intermediate between the response to males on a leader and that to a leader control. The second P. strobi test was intended to be a repetition of the first; unfortunately, all of the male weevils on the male-baited leaders died early in the experiment and this treatment is not considered here. Responses to grandisol and grandisal on a leader and to the leader control were numerically identical to those of the first test, but not significantly different from each other. DISCUSSION
Our evidence indicates that two related volatile compounds produced by male P. approximatus serve as components of that species' aggregation pheromone and suggests that they serve in the same manner for P. strobi. Field response of P. approximatus was maximized when grandisol, grandisal, and host-associated odors were deployed together, indicating that the natural attractant is a multicomponent pheromone. The behavioral significance, if any, of each individual component is unknown at this time. As is the case for many bark and timber beetles (Scolytidae) (Borden and Stokkink, 1971) and the boll weevil (Hedin, 1977), pheromone production in P. approximatus (and presumably P. strobi) appears to be associated with the hindgut, as extracts of male bodies minus hindguts or abdomens revealed no traces of grandisol or grandisal. The different levels of pheromone release (Table I) may reflect a natural difference between the species, or perhaps simply different responses to the conditions of the aeration procedure. There are interesting similarities between the pheromone systems of P. strobi and P. approximatus and those reported for several other circulionid species. Tumlinson et al. (1969) reported that grandisol was one of four c o m p o n e n t s of the m a l e - p r o d u c e d boll weevil (Anthonomus grandis Boheman) aggregation pheromone. Although grandisal was never reported to have pheromonal activity for the boll weevil, this aldehyde was found in incubations of macerated male abdomens (Hedin, 1977). In addition, boll weevil pheromone compounds have been reported to be attractive to the pecan weevil Curculio caryae (Hedin et al., 1979) and the New Guinea sugarcane weevil, Rhabdoscelus obscurus (Chang and Curtis, 1972).
10
BOOTHET AL.
Atkinson (1979) demonstrated field response to the deodar weevil, P. nemorensis Germar, to grandisol and grandisal in combination with a pine bolt, in much the same way as we have with P. approximatus. Recently, Fontaine and Foltz (1982) have shown that male P. nemorensis feeding on slash pine (Pinus ellioti Engelm.) were much more attractive to local weevils than feeding females or pine bolts alone, Preliminary studies (unpublished) show that P. nemorensis males produce grandisol and grandisal, and we shall investigate their function. P. approxirnatus and P. nemorensis are similar ecologically but have different distributions (northeastern and southeastern, respectively) and breed at different times of the year, Throughout the course of this study, several aerations and hindgut extractions of live weevils were conducted at different times of the year, but not all of the male extracts yielded grandisol and grandisal. It is apparent from Table 2 that males of P. strobi and P. approximatus that were producing pheromone components were collected in the spring from their respective natural breeding sites. These times coincide well with the periods of peak trap catches for these species, and with the times during which we have observed increased mating and oviposition in the field. In cases where the pheromone components were not found, the weevils were either used soon after eclosion and were not subjected to the same environmental stimuli as those collected in the field, or they were collected late in the breeding season. Those females collected with males that were producing pheromone (Table 2) were reproductively mature and oviposited readily on host material in the laboratory. Females of both P. strobi and P. approximatus apparently enter reproductive diapause in response to shortening day lengths while they break or avoid reproductive diapause during longer days of spring and early summer (ODell et al., undated ms., Zerillo et al., undated ms.). Male Pissodes will complete spermatogenesis within a week of eclosion and do not appear to have special environmental requirements for reproduction. Our data show that pheromone production in males concides with sexual maturity in females and suggest that pheromone production may be influenced by the same environmental stimuli that control reproductive biology in females. In the field tests of Pissodes pheromone activity, the most attractive treatments were the natural pheromone sources: males feeding on host material. We speculate that the natural pheromone of each species may incorporate an optimal blend of enantiomers of grandisol and grandisal. The synthetic grandisol and grandisal used in these experiments were racemic. Documentation now exists of enantiomer-based differences in the pheromone systems of two sympatrie species of Gnathotrichus ambrosia beetles (Borden et al., 1980) and in various species oflps bark beetles (for example Birch et al., 1980; Lanier et al., 1980). We are approaching the question of enantiomeric
PHEROMONE COMPONENTS OF Pissodes
11
composition of the aggregation pheromones in Pissodes by synthesizing the pure enantiomers and bioassaying various ratios. Acknowledgments--We extend our deep appreciation to P.A. Godwin and T.M. ODell of the USDA Forest Service, Northeast Forest Experiment Station, Hamden, Connecticut, for sharing their considerable knowledge of Pissodes biology with us and for providing several of their unpublished manuscripts. We thank Dr. John Henion, Cornell University, for the GC-MS data, and L. McCandless, ESF, for the [~H]NM R spectra. Mssrs. M. Angst, M. Evans, A. Grant, M. Griggs, R. Rabaglia, and D. Schiffhauer provided technical assistance in various aspects of this project. J.H. Tumlinson III, USDA Insect Attractants Laboratory, Gainesville, Florida, graciously provided us with a sample of authentic grandisol. We acknowledge, with many thanks, the critical reviews of the manuscript by J.H. Borden, Pestology Centre, Simon Fraser University, Burnaby, British Columbia, Canada; W.E. Burkholder, Department of Entomology, University of Wisconsin at Madison; and M.S. Fontaine, Entomology and Nematology Department, University of Florida at Gainesville.
REFERENCES ATKINSON, T.H. 1979. Bionomics of Pissodes nemorensis (Coleoptera: Curculionidae) in north Florida. PhD thesis. University of Florida, Gainesville. 100 pp. BELYEA,R. M., and SULLIVAN,C.R. 1956. The white pine weevil: A review of current knowledge. For. Chron. 32:58-67. BIRCH, M.C., LIGHT, D.M., WOOD, D.L., BROWNE, L.E., SILVERSTEIN, R.M., BERGOT, B.J., OHLOFF, G., WEST, J.R., and YOUNG,J.C. 1980. Pheromonal attraction and allomonal interruption of Ipspini in California by the two enantiomers of ipsdienol. J. Chem. EcoL 6:703-717. BOOTH, D.C. 1978. The chemical ecology and reproductive isolation of the white pine weevil, Pissodes strobi (Peck) and the northern pine weevil, P. approximatus Hopkins (Coleoptera: Curculionidae). PhD thesis. State University of New York, College of Environmental Science and Forestry, Syracuse. 100 pp. BOOTH, D.C., and LANIER, G.N. 1974. Evidence of an aggregating pheromone in Hssodes approximatus and P. strobi. Ann. Entomol. Soc. Am. 67:992-994. BORDEN, J.H., and STOKKINK, E. 1971. Secondary attraction in the Scolytidae. Can. Dept. Fish. and For. Inf. Rept. BC-X-57. 77 pp. BORDEN, J.H., HANDLEY, J.R., MCLEAN, J.A., SILVERSTEIN, R.M., CHONG, L., SLESSOR, K.N., JOHNSTON, B.D., and SCI~ULER, H.R. 1980. Enantiomer-based specificity in pheromone communication by two sympatric Gnathotrichus species (Coleoptera: Scolytidae). J. Chem. EcoL 6:445-456. BROWNLEE, R.G., and SILVERSTEIN, R.M. 1968. A micro-preparative gas chromatograph and a modified carbon skeleton determinator. Anal. Chem. 40:2077-2079. BYRNE, K.J., GORE, W.E., PEARCE, G.T., and SILVERSTEIN,R.M. 1975. Porapak Q collection of airborne organic compounds serving as models for insect pheromones. J. Chem. Ecol. 1:1-7. CHANG, V.C.S., and CURTIS, G.A. 1972. Pheromone production by the New Guinea sugarcane weevil. Environ. EntomoL 1(4):476-481. FINNEGAN, R.J. 1958. The pine weevil Pissodes approximatus Hopk., in southern Ontario. Can. EntomoL 90:348-354.
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FONTAINE,M. S, and FOLTZ,J.L. 1982. Field studies of a male-produced aggregating pheromone in the deodar weevil. Pissodes nemorensis Germar. Environ. Entomol. 11:881-883.
HARMAN, D.M., and KULMAN,H.M. 1966. A technique for sexing live white pine weevils, Pissodes strobi. Ann. Entomol. Soc. Am. 59:315-317. HEDIN, P.A. 1977. A study of factors that control biosynthesis of the compounds which comprise the boll weevil pheromone. J. Chem. Ecol. 3:279-289. HEDIN, P.A., PAYNE, J.A., CARPENTER,T.L., and NEEL, W. 1979. Sex pheromones of the male and female pecan weevil, Curculio caryae: Behavioral and chemical studies. Environ. Entomol. 8:521-523. LANIER, G.N., CLAESSON,A., STEWART,T., PISTON, J.J., and SILVERSTEIN,R.M. 1980. Ipspini: The basis for interpopulational differences in pheromone biology. J. Chem. Ecol. 6:677-687. MAcALONEY, H.J. 1930. The white pine weevil (Pissodes strobi Peck)--its biology and control. Bull. N.Y.S. Coll. For. 3:1-87. ODELL, T.M., GODWIN, P.A., and ZERILLO, R.T. (undated ms.) Factors contributing to reproductive diapause in the white pine weevil, Pissodes strobi (Peck). Unpublished manuscript. Forest Insect and Disease Laboratory. US Forest Service. Hamden, Connecticut. 24 pp. OVERHULSER,D.L., and GARA,R.I. 1975. Spring flight and adult activity of the white pine weevil, Pissodes strobi (Coleoptera: Curculionidae), on Sitka spruce in western Washington. Can. Entomol. 107:251-256. PHILLIPS, T.W. 1981. Aspects of host preference and chemically mediated aggregation in Pissodes strobi (Peck) and P. approximatus Hopkins (Coleoptera: Curculionidae). MS thesis. State University of New York, College of Environmental Science and Forestry, Syracuse. 94 pp. SILVERSTEIN, R.M. 1977. Complexity, diversity, and specificity of behavior-modifying chemicals: Examples mainly fro m Coleoptera and Hymenoptera, pp. 231-251, in H.H. Shorey and J.J. McKelvey, Jr. (eds.). Chemical Control of Insect Behavior. John Wiley, New York. SMITH, S.G., and SUGDEN,B.A. 1969. Host trees and breeding sites of native North American Pissodes bark weevils, with a note on synonomy. Ann. Entomol. Soc. Am. 62:146-148. TAVLOI~,R.L. 1930. The biology of the white pine weevil, Pissodes strobi (Peck), and a study of its insect parasites from an economic viewpoint. Entomol. Am. 9:167-246. TUMLINSON,J.R., lII. 1969. Isolation, identification and partial synthesis of the boll weevil attractant. Ph.D. thesis, Mississippi State University. State College. 76 pp. TUMLINSON, J.H., HARDEE, D.D., GUELDNER, R.C., THOMPSON, A.C., HEDIN, P.A., and MINVARD, J.P. 1969. Sex pheromones produced by male boll weevil: Isolation, identification, and synthesis. Science 166:1010-1012. WILLIAMS, H.J., SILVERSTHN, R.M., BURKHOLDER, W.E., KHORRAMSHAHI, A. 1981. Dominicalure 1 and 2: components of aggregation pheromone from male lesser grain borer Rhyzopertha dominica (F.) (Coleoptera: Bostriehidae). J. Chem. Ecol. 7:759-780. ZERILLO, R.T., ODELL, T.M., and GODWIN, P.A. (undated ms.) A description of the life history and behavior of the northern pine weevil, Pissodes approximatus Hopk. in Connecticut. Unpublished office report 4500FS-NE-2201. Forest Insect and Disease Laboratory. US Forest Service. Hamden, Connecticut. 6 pp.
AGGREGATION PHEROMONE COMPONENTS OF TWO SPECIES OF P I S S O D E S WEEVILS (COLEOPTERA: CURCULIONIDAE): Isolation, Identification, and Field Activity 1'2
D O N A L D C. B O O T H , 3 T H O M A S W. P H I L L I P S , 3 A L F C L A E S S O N , 4'5 R O B E R T M. S I L V E R S T E I N , 4 G E R A L D N. L A N I E R , 3 and J A N E T R. W E S T 4 3Department o f Environmental and Forest Biology and 4Department o f Chemistry State University o f New York College o f Environmental Science and Forestry Syracuse, New York 13210 5Biomedical Center, Uppsala University Uppsala, Sweden (Received February 17, 1982; revised April 8, 1982)
A b s t r a c t - - T w o related volatile compounds were identified from each of two species of Pissodes bark weevils and implicated as components of their aggregation pheromones. Grandisol (cis-2-isopropenyl-l-methylcyclobutaneethanol), and its corresponding aldehyde, grandisal, were isolated from males of both P. strobi and P. approximatus and were found in the abdomens and hindguts of the respective species. In field tests synthetic grandisol and grandisal together with odors from cut pine acted synergistically in attracting both sexes of P. approximatus. This response was similar to that elicited by male P. approximatus feeding on cut pine. Males and females of natural populations of 1". strobi were more responsive to caged males feeding on leaders of white pine than they were to leaders alone. The combination of grandisol, grandisal, and leaders was less attractive than males on leaders, but more attractive than leaders alone. From isolation of pheromone components at different times of the year, it was
1Research supported by grants from the National Science Foundation, The New York State Museum and Science Service, and a travel grant to A.C. from Stiffelsen Blanceflor Boncompagni-Lndod ovisi. 2Preliminary report presented at meeting of the Eastern Branch Entomological Society of America, Boston, Massachusetts, September 14-16, 1977. Abstract in J. AT. Y. Entomol. Soc. 85:167. In part from theses by DCB (Ph.D.) and TWP (M.S.).
0098-0331/83/0100-0001503.00/0 9 1983PlenumPublishingCorporation
BOOTH ET AL.
2
determined that males of both species produced grandisol and grandisal only at times when cohort females were reproductively mature. Key Words--Pissodes strobi, Pissodes approximatus, Coleoptera, Curculionidae, aggregation pheromone, grandisol, grandisal, Pinus strobus, white pine weevil.
INTRODUCTION
Bark weevils of the genus Pissodes Germar feed on conifer trees in the family Pinaceae and their larvae develop in the inner bark (phloem) of their hosts. Pissodes strobi (Peck), the white pine weevil, is notorious among forest insects for its deformation of pines and spruces throughout North America. The biology and habits of P. strobi have been extensively documented (Macaloney, 1930; Taylor, 1930; Belyea and Sullivan, 1959). In the spring the adults orient to the healthy terminal leader (top shoot) of a host tree, where they mate, feed, and oviposit. Larval feeding girdles the stem, killing old and new growth;one or more of the remaining lateral branches must assume dominance, resulting in a fork or crook in the stem that may render the tree useless for saw timber. The northern pine weevil, P. approxirnatus Hopkins, is not as economically important as P. strobi, although larvae may kill stressed trees and intense feeding by adults may be injurious to small trees (Finnegan, 1958). Adults of P. approximatus orient to cut or weakened trees to reproduce, and their larvae develop in the inner bark of this material. P. approxirnatus occurs on most pines and spruces throughout the northeastern and Lake states, and in the Canadian boreal forest west to the Rocky Mountains; P. strobi occurs in three somewhat distinct populations across North America, according to the preferred host of the region. In the east, P. strobi is found primarily on eastern white pine (Pinus strobus L.), in the Rocky Mountains in Engelmann spruce (Picea engelmannii Parry), and in the Pacific coastal region on Sitka spruce (Picea sitchensis Carr.) (Smith and Sugden, 1969). Finnegan (1958) reported mass flights ofP. approximatus in response to suitable host material (moribund pines) in Ontario. P. strobi forms aggregations on leaders early in its flight season, prior to increased dispersal and oviposition (Overhulser and Gara, 1975). The first evidence of an aggregation pheromone in Pissodes was reported by Booth and Lanier (1974). In that study, P. approximatus males confined on host material were more attractive to both sexes than females on host material or host material alone. Interestingly, P. strobi males caged on the same host material also attracted local P. approximatus. Subsequently, Booth (1978) demonstrated that P. strobi males caged on white pine leaders were attractive to conspecific males and females in the field; females feeding on leaders and leaders alone were not attractive. This paper reports the isolation and identification of volatile
PHEROMONE COMPONENTS OF
Pissodes
3
pheromone components from males of P. strobi and P. approximatus and discusses their significance for these species. METHODS AND MATERIALS
Weevil Populations. Weevils used in experiments were from natural populations within a 100-km radius of Syracuse, New York. P. strobi adults were obtained in an 8-year-old eastern white pine plantation in southeastern Cortland County, near Virgil, New York. Overwintered weevils were collected from the upper lateral branches of the previous year's brood trees as daily high temperatures approached 15-20 ~ C, usually in the second or third week of April. P. strobi used during winter months were obtained from naturally infested white pine leaders in August. The weevils were sexed according to the method of H a r m a n and Kulman (1966) and maintained on freshly cut white pine branches under ambient lighting in the spring or under a 16:8 (lightdark) photoperiod in winter. P. a_pproximatus adults were collected from the undersides of freshly cut red pine logs scattered on the forest floor within a 30-year-old red pine (Pinus resinosa Ait.) plantation at Heiberg Memorial Forest, near Tully, New York. Weevils used in the fall were obtained as they emerged from naturally infested pine logs brought into the laboratory. Collection, Isolation, and Identification of Pheromone Components. Volatiles from living Pissodes adults were collected by aeration and absorption on P o r a p a k Q (Waters Assoc., Framingham, Massachusetts) (Byrne et al., 1975). F r o m 50 to 150 weevils of each sex were aerated for 5-10 days in large vacuum desiccators while feeding on fresh cuttings of their respective host material: P. strobi on white pine leaders and P. approximatus on red pine branches. Aerations were carried out in the laboratory at 21 ~ C under ambient lighting in the spring or under a controlled 16:8 (light-dark) photoperiod in the fall or winter. Prior to aeration, the P o r a p a k was cleaned according to the method of Williams et al. (1981) and, following aeration, the volatiles were extracted with distilled pentane (5 ml/g). The extract was then dried over sodium sulfate and concentrated to 1-4 ml by fractional distillation in preparation for gas chromatography (GC) or bioassay. Hindguts or abdomens were dissected from the weevils and crushed in small amounts of distilled pentane; the slurry was sonicated and centrifuged, and the supernatant was used for GC analysis. Extracts were fractionated on a Varian 2700 c h r o m a t o g r a p h equipped with a flame ionization detector, a 99:1 effluent splitter, and a thermal gradient collector (Brownlee and Silverstein, 1968). Fractions were collected in 30.5 cm • 1.7 m m (OD) glass capillary tubes that were flame sealed and stored at - 3 0 ~ until used. A 5.4 m • 4 m m (ID) glass column packed with 4% Carbowax 20M on C h r o m o s o r b G 60-80 mesh was used under the following
4
BOOTH ET AL.
conditions: 90 ~ for 4 min, raised to 215 ~ at 2~ 55 ml He/min, injector 140~ , detector 215 ~ . The collected compounds were further fractionated on a 6.0 m • 4 mm (ID) glass column (5% OV-225 on Chromosorb G 60-80 mesh, 80 ~ for 4 min, raised to 215 ~ at 2~ /min, 55 ml He/min, injector 140~ , detector 215~ Mass spectral electron impact (70 eV) data were obtained from a Finnegan GLC 9500. P r o t o n nuclear magnetic resonance ([1H]NMR) spectra were recorded (Varian XL-100 instrument) on 80/~g samples in 50 #1 C6D6 in the inner cell of a concentric tube. Infrared spectra were obtained on a Perkin-Elmer 621 instrument, fitted with a beam condenser, on 60 to 80-#g samples dissolved in CCh in a 4-~zl cavity cell. Synthetic Compounds. Synthetic (racemic) grandisol (cis-2-isopropenyl1-methylcyclobutaneethanol) was obtained from Chemical Samples Co. (division of Albany International, Inc.) and GC-purified for coinjection. The corresponding aldehyde of grandisol (grandisal) was obtained by oxidation with pyridinium chlorochromate in methylene chloride in the presence of sodium acetate (1 equivalent); reaction time was 80 min at room temperature. The reaction mixture was filtered through a 5 • 1.5 cm Florisil column, and after evaporation of the solvents the purity of the aldehyde was determined on a 3 m • 4 mm (ID) glass GC column (7% Carbowax on Chromosorb G 60-80 mesh) at 125~ C. The same synthetic procedure was used to provide aldehyde for field experiments. Field Assays. Bioactivity of putative pheromone components was tested in several trapping experiments in the field. P. approximatus experiments took place in mature red pine plantations near Tully, New York, while P. strobi tests were conducted in young white pine plantations near Virgil, New York. Two field tests of P. approximatus employed hardware cloth sticky traps placed on the ground (Booth and Lanier 1974), arranged in five randomized blocks of six traps each. The six treatments of the first test included: (1) male weevils caged on a red pine bolt, (2) the putative pheromone components grandisol and grandisal with a red pine bolt, (3) the same concentration of these compounds without red pine, (4) higher concentrations of these compounds without red pine, (5) solvent only (hexane), and (6) a red pine bolt alone. The second experiment tested the attractiveness of (1) male weevils on red pine, (2) grandisol and grandisal with red pine, (3) half as much of these compounds with red pine, (4) grandisal only with red pine, (5) grandisol only with red pine, and (6) a red pine bolt alone. The first P. approximatus test, conducted in the late summer, assayed those weevils which presumably eclosed in early summer and were currently reproductive (Zerillo et al., undated ms.); the second experiment tested the more active spring population. P. strobi tests, conducted in two consecutive spring seasons, employed hardware cloth sticky cylinders which surrounded screen-enclosed
P H E R O M O N E C O M P O N E N T S OF
eissodes
5
white pine leaders (Phillips 1981). Five replicates each of three different treatments (male P. strobi confined on leaders, grandisol and grandisal on a leader, and a leader alone) were established on trees picked at random within a one-acre plantation. In field tests of Pissodes species, synthetic pheromone components were released from snap-top polyethylene vials (Cole-Parmer Instrument Co.), and live weevils used in certain treatments were fieldcollected immediately prior to the tests.
RESULTS
Identification of Pheromone Components. Systematic GC fractionation of the extracts, monitored by laboratory bioassay of the fractions singly and in combination, was attempted (Silverstein, 1977). However, none of the laboratory olfactometers (Booth, 1978) provided consistent, interpretable results. Since the amounts of the fractions isolated from limited numbers of available weevils were too small for field testing, we turned to examining differences between chromatograms of extracts of male and female hindguts or abdomens. Two compounds that were present in the abdomens ofP. strobi males were absent in abdomens of females (31.5 and 51.0 min on the Carbowax 20M column). Each of these compounds were subsequently found in larger amounts in the more complex chromatogram of the Porapak extract of male P. strobi, from which they were collected and refractionated on the OV-225 column (40 and 48 min). The compound with the longer retention time was identified as grandisol (cis-2-isopropenyl-l-methylcyclobutaneethanol) (Figure la) from the mass, IR, and N M R spectra, and its identity was confirmed by coinjection with an authentic sample and by congruence of spectra (Tumlinson, 1969). We used the same techniques to identify the compound with the shorter retention time as the corresponding aldehyde, referred to here as grandisal (Hedin, 1977) (Figure lb). The same compounds were identified from hindgut and Porapak extracts
FIG. 1. Pissodes pheromone components: (a) grandisol, (b) grandisal.
6
BOOTH ET AL. TABLE 1. OCCURRENCE OF GRANDISOL AND GRANDISAL IN MALE P i s s o d e s a
Hindgut extracts (/~g/individual)
Aerations (/~g/weevil-hour) b
Species
Grandisol
Grandisal
Grandisol
Grandisal
P. s t r o b i P. a p p r o x i m a t u s
0.200 0.026
0.400 0.013
0.0033 0.0002
0.0074 0.0006
Presence of c o m p o u n d s determined by coinjection, a m o u n t s determined by GC comparison with known standards. bA weevil-hour is equal to the a m o u n t produced by one weevil in one hour of aeration.
from P. approximatus males. In neither species were the compounds detected in the other body parts of the male or in any of the extracts from females. The amounts of grandisol and grandisal occurring in the various extracts of male Pissodes, as determined by comparative GC, are presented in Table l; the occurrence of these compounds in males at different times of the year is given in Table 2. One striking difference between the two species of Pissodes is that, in both sampling methods (Table 1), P. strobi appears to produce both chemicals at a level ten times greater than that ofP. approximatus. Abdomen and hindgut extracts only reveal the compounds present within the insect at the time of dissection, but the extract from the Porapak aeration should represent the actual amounts and ratios of chemicals emitted by the insects through time. However, the results may not be quantitative because of uncertainties in collection and recovery efficiencies and in the stability of the aldehyde. In the Porapak extracts of the ratio of grandisol to grandisal produced by P. strobi is approximately 1 : 2, and that for P. approxirnatus is 1:3. Attempts to determine enantiomeric composition will be made when larger amounts of isolated grandisol and grandisal become available. Field Activity. Table 3 presents the data for two field tests of grandisol and grandisal with a population ofP. approximatus. The highest trap catch in both tests was on the treatment with male P. approximatus confined on a red pine bolt. However, the second treatment of each test, involving grandisol, grandisal, and a red pine bolt, attracted almost as many weevils as the male treatment. In the 1976 test, the two treatments with various amounts of grandisol and grandisal that did not include a red pine bolt caught significantly fewer weevils than that which included red pine, indicating synergism between odors from the host material and those from the synthetic chemicals. The two control treatments in this test, solvent (hexane) and a red pine bolt, caught similarly low numbers of weevils. In the 1977 test, the response to the higher levels of grandisol and grandisal was statistically
15 May 3 June
P. approximatus Hindgut Aeration
Aeration Abdomens Aeration Aeration
Field Field
Field Field Field Field
18 Oct. 21 July
11 June 3 Sept. 15 Dee. 24 Feb. l0 March
Date b
Hindgut Aeration
Aeration Aeration Aeration Aeration Aeration
Extract type
Condition
Extract type
a Occurrence of grandisol and grandisal was determined by GC coinjection with laboratory standards. b Refers to date aeration was completed or hindguts or abdomens dissected.
5 May 10 May 12 May 26 May
Date b
P. strobi
Species
Absent
Present
Laboratory Field
Field Laboratory Laboratory Laboratory Laboratory
Condition
TABLE 2. SEASONAL OCCURRENCE OF PHEROMONE COMPONENTS IN EXTRACTS OF PORAPAK Q AERATIONS, EXISED HINDGUTS, OR ABDOMENS OF MALE e i s s o d e s EXPOSED TO FIELD OR LABORATORY CONDITIONS PRIOR TO EXAMINATION" 9
BOOTH ET AL. TABLE 3. RESPONSE OF Pissodes approximatus TO GRANDISOL ( G O H ) , GRANmSAL ( G C H O ) , AND NATURAL ATTRACTANTS IN TWO FIELD TESTS
No. of weevils captured Date of Test
Treatments a'b
Females
Test 1: I0 Aug-17 Sept., 1976
4 Male P. approximatus plus red pine 8 mg GOH + 4 mg GCHO + red pine 8 mg GOH + 4 mg GCHO 14 mg GOH + 5 mg GCHO Solvent (25 #1 hexane) Red pine
Test 2: 11 May-6 July, 1977
5 Male P. approximatus plus red pine 2 8 m g G O H + 1 0 m g G C H O + redpine 14 mg GOH + 5 mg GCHO + red pine 5 mg GCHO + red pine 14 mg GOH + red pine Red pine
Males
Total c
27 27 7 14 1 3
18 10 3 2 6 1
45a 37a 10bc 16b 7bc 4c
142 109 54 22 5 8
21 30 21 6 4 0
163a 139a 75b 28c 9d 8d
a Red pine bolts were freshly cut, 12.7 • 10.2 cm; chemical baits were changed weekly. b Release rate of pheromone components was determined by cold trapping volatiles from a slow airstream and quantifying by GC: 8 mg GOH = 83 #g/day; 14 mg GOH = 174 #g/day; 28 mg GOH = 348 #g/day; 4 mg GCHO = 205 #g/day; 5 mg GCHO = 120 #g/day; 10 mg GCHO = 240 #g/day. CTotals followed by the same letter(s) are not significantly different (chi-square test for independence, P < 0.05).
TABLE 4. RESPONSE OF thssodes strobi TO GRANDISOL ( G O H ) WITH GRANDISAE ( G C H O ) , AND NATURAL ATTRACTANTS IN TWO FIELD TESTS
No. weevils captured Treatments a
Females
Males
Total b
Test 1: May 1 to June 27, 1980 3 males + leader 5 mg GOH + 10mg GCHO + leader leader
9 4 4
14 15 5
23a 19ab 9b
Test 2: April 30 to June 19, 1981 5 m g G O H + 1 0 m g G C H O + leader leader
10 0
9 9
19 9
a Chemical baits were changed biweekly. Release rate of pheromone components was determined gravimetrically: 5 mg GOH = 43 #g/day 10 mg GCHO = 80/zg/day. Totals m test 1 followed by the same letter are not significantly different (chi-square test for independence, P < 0.05). Differences between totals in test 2 were not significant. b
9
'
.
.
.
.
PHEROMONE COMPONENTS OF
Pissodes
9
similar to that for the male treatment; the response was nearly halved by halving the concentration of the chemicals. Grandisal with red pine was attractive, but attracted fewer weevils than when grandisol was included, indicating synergism between these two compounds. Grandisol with red pine was not attractive. Of the three treatments examined in the first P. strobi field test (Table 4), the most attractive was that with male P. strobi caged on an eastern white pine leader. The response to grandisol plus grandisal on a white pine leader was intermediate between the response to males on a leader and that to a leader control. The second P. strobi test was intended to be a repetition of the first; unfortunately, all of the male weevils on the male-baited leaders died early in the experiment and this treatment is not considered here. Responses to grandisol and grandisal on a leader and to the leader control were numerically identical to those of the first test, but not significantly different from each other. DISCUSSION
Our evidence indicates that two related volatile compounds produced by male P. approximatus serve as components of that species' aggregation pheromone and suggests that they serve in the same manner for P. strobi. Field response of P. approximatus was maximized when grandisol, grandisal, and host-associated odors were deployed together, indicating that the natural attractant is a multicomponent pheromone. The behavioral significance, if any, of each individual component is unknown at this time. As is the case for many bark and timber beetles (Scolytidae) (Borden and Stokkink, 1971) and the boll weevil (Hedin, 1977), pheromone production in P. approximatus (and presumably P. strobi) appears to be associated with the hindgut, as extracts of male bodies minus hindguts or abdomens revealed no traces of grandisol or grandisal. The different levels of pheromone release (Table I) may reflect a natural difference between the species, or perhaps simply different responses to the conditions of the aeration procedure. There are interesting similarities between the pheromone systems of P. strobi and P. approximatus and those reported for several other circulionid species. Tumlinson et al. (1969) reported that grandisol was one of four c o m p o n e n t s of the m a l e - p r o d u c e d boll weevil (Anthonomus grandis Boheman) aggregation pheromone. Although grandisal was never reported to have pheromonal activity for the boll weevil, this aldehyde was found in incubations of macerated male abdomens (Hedin, 1977). In addition, boll weevil pheromone compounds have been reported to be attractive to the pecan weevil Curculio caryae (Hedin et al., 1979) and the New Guinea sugarcane weevil, Rhabdoscelus obscurus (Chang and Curtis, 1972).
10
BOOTHET AL.
Atkinson (1979) demonstrated field response to the deodar weevil, P. nemorensis Germar, to grandisol and grandisal in combination with a pine bolt, in much the same way as we have with P. approximatus. Recently, Fontaine and Foltz (1982) have shown that male P. nemorensis feeding on slash pine (Pinus ellioti Engelm.) were much more attractive to local weevils than feeding females or pine bolts alone, Preliminary studies (unpublished) show that P. nemorensis males produce grandisol and grandisal, and we shall investigate their function. P. approxirnatus and P. nemorensis are similar ecologically but have different distributions (northeastern and southeastern, respectively) and breed at different times of the year, Throughout the course of this study, several aerations and hindgut extractions of live weevils were conducted at different times of the year, but not all of the male extracts yielded grandisol and grandisal. It is apparent from Table 2 that males of P. strobi and P. approximatus that were producing pheromone components were collected in the spring from their respective natural breeding sites. These times coincide well with the periods of peak trap catches for these species, and with the times during which we have observed increased mating and oviposition in the field. In cases where the pheromone components were not found, the weevils were either used soon after eclosion and were not subjected to the same environmental stimuli as those collected in the field, or they were collected late in the breeding season. Those females collected with males that were producing pheromone (Table 2) were reproductively mature and oviposited readily on host material in the laboratory. Females of both P. strobi and P. approximatus apparently enter reproductive diapause in response to shortening day lengths while they break or avoid reproductive diapause during longer days of spring and early summer (ODell et al., undated ms., Zerillo et al., undated ms.). Male Pissodes will complete spermatogenesis within a week of eclosion and do not appear to have special environmental requirements for reproduction. Our data show that pheromone production in males concides with sexual maturity in females and suggest that pheromone production may be influenced by the same environmental stimuli that control reproductive biology in females. In the field tests of Pissodes pheromone activity, the most attractive treatments were the natural pheromone sources: males feeding on host material. We speculate that the natural pheromone of each species may incorporate an optimal blend of enantiomers of grandisol and grandisal. The synthetic grandisol and grandisal used in these experiments were racemic. Documentation now exists of enantiomer-based differences in the pheromone systems of two sympatrie species of Gnathotrichus ambrosia beetles (Borden et al., 1980) and in various species oflps bark beetles (for example Birch et al., 1980; Lanier et al., 1980). We are approaching the question of enantiomeric
PHEROMONE COMPONENTS OF Pissodes
11
composition of the aggregation pheromones in Pissodes by synthesizing the pure enantiomers and bioassaying various ratios. Acknowledgments--We extend our deep appreciation to P.A. Godwin and T.M. ODell of the USDA Forest Service, Northeast Forest Experiment Station, Hamden, Connecticut, for sharing their considerable knowledge of Pissodes biology with us and for providing several of their unpublished manuscripts. We thank Dr. John Henion, Cornell University, for the GC-MS data, and L. McCandless, ESF, for the [~H]NM R spectra. Mssrs. M. Angst, M. Evans, A. Grant, M. Griggs, R. Rabaglia, and D. Schiffhauer provided technical assistance in various aspects of this project. J.H. Tumlinson III, USDA Insect Attractants Laboratory, Gainesville, Florida, graciously provided us with a sample of authentic grandisol. We acknowledge, with many thanks, the critical reviews of the manuscript by J.H. Borden, Pestology Centre, Simon Fraser University, Burnaby, British Columbia, Canada; W.E. Burkholder, Department of Entomology, University of Wisconsin at Madison; and M.S. Fontaine, Entomology and Nematology Department, University of Florida at Gainesville.
REFERENCES ATKINSON, T.H. 1979. Bionomics of Pissodes nemorensis (Coleoptera: Curculionidae) in north Florida. PhD thesis. University of Florida, Gainesville. 100 pp. BELYEA,R. M., and SULLIVAN,C.R. 1956. The white pine weevil: A review of current knowledge. For. Chron. 32:58-67. BIRCH, M.C., LIGHT, D.M., WOOD, D.L., BROWNE, L.E., SILVERSTEIN, R.M., BERGOT, B.J., OHLOFF, G., WEST, J.R., and YOUNG,J.C. 1980. Pheromonal attraction and allomonal interruption of Ipspini in California by the two enantiomers of ipsdienol. J. Chem. EcoL 6:703-717. BOOTH, D.C. 1978. The chemical ecology and reproductive isolation of the white pine weevil, Pissodes strobi (Peck) and the northern pine weevil, P. approximatus Hopkins (Coleoptera: Curculionidae). PhD thesis. State University of New York, College of Environmental Science and Forestry, Syracuse. 100 pp. BOOTH, D.C., and LANIER, G.N. 1974. Evidence of an aggregating pheromone in Hssodes approximatus and P. strobi. Ann. Entomol. Soc. Am. 67:992-994. BORDEN, J.H., and STOKKINK, E. 1971. Secondary attraction in the Scolytidae. Can. Dept. Fish. and For. Inf. Rept. BC-X-57. 77 pp. BORDEN, J.H., HANDLEY, J.R., MCLEAN, J.A., SILVERSTEIN, R.M., CHONG, L., SLESSOR, K.N., JOHNSTON, B.D., and SCI~ULER, H.R. 1980. Enantiomer-based specificity in pheromone communication by two sympatric Gnathotrichus species (Coleoptera: Scolytidae). J. Chem. EcoL 6:445-456. BROWNLEE, R.G., and SILVERSTEIN, R.M. 1968. A micro-preparative gas chromatograph and a modified carbon skeleton determinator. Anal. Chem. 40:2077-2079. BYRNE, K.J., GORE, W.E., PEARCE, G.T., and SILVERSTEIN,R.M. 1975. Porapak Q collection of airborne organic compounds serving as models for insect pheromones. J. Chem. Ecol. 1:1-7. CHANG, V.C.S., and CURTIS, G.A. 1972. Pheromone production by the New Guinea sugarcane weevil. Environ. EntomoL 1(4):476-481. FINNEGAN, R.J. 1958. The pine weevil Pissodes approximatus Hopk., in southern Ontario. Can. EntomoL 90:348-354.
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FONTAINE,M. S, and FOLTZ,J.L. 1982. Field studies of a male-produced aggregating pheromone in the deodar weevil. Pissodes nemorensis Germar. Environ. Entomol. 11:881-883.
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