Journal of Chemical Ecology, Vol. 12, No. 7, 1986
INTERSPECIFIC ACTIVITY OF SEMIOCHEMICALS AMONG SIBLING SPECIES OF Pissodes (COLEOPTERA: CURCULIONIDAE)
THOMAS
W.
PHILLIPS 1 and GERALD
N.
LANIER
Department of Environmental and Forest Biology State University of New York College of Environmental Science and Forestry Syracuse, New York 13210 (Received July 22, 1985; accepted November 12, 1985)
Abstraet--Pissodes strobi, P. approximatus, and P. nemorensis are sibling species of pine weevils that can hybridize in the laboratory but are presumed to be reproductively isolated in nature. Males of all three species produce the terpenoids grandisol and grandisal; these compounds serve as an aggregation pheromone for P. approximatus and P. nemorensis when deployed with odors from pine bolts. A series of field experiments examined the possibility of cross-attraction among the three species. Tests in New York and Florida found that parapatrieally distributed P. approximatus and P. nemorensis were crossattractive, but different photoperiodic conditioning was required for pheromone production in males of the two species. Long-day pheromone production (P. approximatus-type) was inherited in interspecific hybrids. Other tests showed that P. strobi males, or hybrid males from crosses of P. strobi with P. approximatus, were not attractive to sympatric P. approximatus. When the response of P. strobi was assessed to males of either P. strobi or P. approximatus confined on white pine leaders (the breeding site of P. strobi), no evidence of cross-attraction or pheromone activity was found; P. strobi were caught in equal numbers on P. strobi-baited leaders, P. approximatusbaited leaders, and unbaited leaders. Tests of interspecific interactions found that male P. strobi produce an allelochemical signal that interrupts the response of P. approximatus to its natural or synthetic aggregation pheromone. This interspecific response is apparently adaptive for members of both species (classified as an allomone-kairomone or synomone) because it may ultimately serve to prevent interspecific matings that would lower the fitness of the parents. Key Words--Pissodes, Coleoptera, Curculionidae, aggregation pheromone, cross-attraction, synomone, reproductive isolation, grandisol, grandisal. ~Present address: Dept. of Entomology and Nematology, 3103 McCarty Hall, University of Florida, Gainesville, FL 32611. 1587
oo98-o331/86/o7oo-15875o5.oo/o 9 1986Plenum PublishingCorporation
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PHILLIPSANDLANIER INTRODUCTION
Aggregation pheromones in Pissodes weevils were first reported by Booth and Lanier (1974), who found that males of P. approximatus Hopkins, the northern pine weevil, feeding on pine bolts would attract large numbers of conspecific males and females, while females feeding on pine bolts and pine bolts alone were not attractive. They also postulated that P. strobi (Peck), the white pine weevil, uses a male-produced aggregation pheromone. Booth et al. (1983) reported that males of P. approximatus produce two terpenoid compounds, grandisol (cis-2-isopropenyl-l-methylcyclobutaneethanol) and its corresponding aldehyde, grandisal, that, together with odor from cut pine logs, serve as an aggregation pheromone. They also found that males of P. strobi produce grandisol and grandisal, but repeated field tests have not convincingly documented attraction of P. strobi to these compounds (Booth, 1978; Phillips, 1981; Booth et al., 1983). Phillips and coworkers (1984) reported that male Deodar weevils, P. nemorensis Germar, also produce grandisol and grandisal and use them with host odors as an aggregation pheromone (cf. Fontaine and Foltz, 1982). No substantial quantitative differences in the production of these compounds among the three species, either in absolute amounts or relative concentrations, have been found (Silverstein and West, 2 unpublished data). Possible differences in enantiomeric composition of grandisol and grandisal released by these species have not been determined. We consider P. strobi, P. approximatus, and P. nemorensis to be sibling species because they are very similar morphologically but are presumed to be reproductively isolated in nature. These species are genetically very similar (Phillips, 1984), can hybridize with each other in the laboratory (Godwin and ODell, 1967; Smith, 1973), but differ ecologically and behaviorally. P. strobi breeds in one-year-old leaders of vigorous young pines, Pinus spp., and spruces, Picea spp., across North America (MacAloney, 1930; Smith and Sugden, 1969). P. approximatus is sympatric with P. strobi in northeastern North America, but breeds in the boles, branches, and root collars of weakened or recently cut pines and spruces (Finnegan, 1958). Breeding site separation between P. strobi and P. approximatus is strong; artificially produced hybrids display reduced reproductive fitness (Phillps and Lanier, 1983). If P. strobi and P. approximatus use aggregation pheromones that incorporate grandisol and grandisal, we predict that qualitative differences would prevent cross-attraction between the species. P. nemorensis also breeds in weakened or cut pines, but is distributed in southeastern North America and is presumed to be parapatric with P. approximatus (Baker, 1972; Hopkins, 1911). P. approximatus and P. nemorensis respond to the same synthetic pheromone (Booth et al., 1983; Phillips et al., 2R.M. Silversteinand J.R. West, Departmentof Chemistry,StateUniversityof New York College of EnvironmentalScienceand Forestry, Syracuse,New York 13210.
INTERSPECIFIC SEMIOCHEMICALS AMONG SIBLING WEEVIL SPECIES
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1984), and it is quite probable that they could be cross-attractive in nature. However, P. approximatus and P. nemorensis are apparently reproductively active in different seasons (Finnegan, 1958; Fontaine et al., 1983), so crossattraction in nature may never occur. Here we report a series of experiments in which we investigated the activity of pheromones and allelochemics among P. strobi, P. approximatus, and P. nemorensis. Using field tests with live weevils, we examined cross-attraction among species, the attractiveness of interspecific hybrids, and the interspecific interruption of pheromone activity. We will assess the importance of pheromones and allelochemics in reproductive isolation and host location, and we will discuss the evolution of semichemicals in these species.
METHODS
AND MATERIALS
Field experiments, described in detail below, were conducted in 1982, 1983, and 1985. Traps for assessing the response of either P. approximatus or P. nemorensis were sticky hardware-cloth cylinders placed directly on the forest floor (design of Booth and Lanier, 1974). Freshly cut pine bolts, about 15 x 25 cm, were enclosed in 30 x 45-cm fiberglass screen bags with or without male weevils and were placed inside the traps. Traps for P. strobi (one experiment) were 20 x 91-cm sticky hardware-cloth cylinders with the top ends covered by 20 x 20-cm sticky hardware-cloth squares. These traps were placed on poles supported by metal stakes and suspended over screen-enclosed (15 • 100-cm fiberglass screen sleeves) one-year-old leaders of 3- to 5-m tall eastern white pine trees, Pinus strobus L. Male Pissodes were then confined on leaders as attractive sources (Phillips, 1981). Because there are no diagnostic characters for adequately separating specimens of P. strobi, P. approximatus, and P. nemorensis, we initially employed three working hypotheses for the identification of trapped weevils based on geographic locality or ecological qualities of the field site and experiment. Weevils trapped in Florida (experiment 2, see below) were P. nemorensis based on distribution. Weevils flying to traps on white pine leaders (experiment 3) in open stands were considered P. strobi, while those caught in New York on ground traps containing cut logs (experiments 1, 4-7) were deemed P. approximatus. These assumptions are drawn from accumulated information on Pissodes behavior, ecology, karyology, and geographic distribution; unfortunately, there is no guarantee that they are true. Therefore, we challenged our designations by performing the discriminant analysis technique of Godwin et al. (1982) on subsamples of the trap-caught specimens (Phillips, 1984). Discriminant analysis corroborated the identification of P. strobi and P. approximatus by our a priori assumptions, but of weevils that could only be P. nemorensis based on geographic definition (experiment 2), a majority were identified
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PHILLIPS AND LANIER
as P. approximatus. The utility of the discriminant analysis technique is discussed elsewhere (Phillips, 1984; Phillips et al., 1986). Weevils used as attractive sources were either laboratory-reared or fieldcollected; unless otherwise indicated, all were reproductively mature. Females of the three species have certain environmental requirements for reproductive maturity and oviposition (ODell et al., unpublished; Atkinson, 1979; Fontaine et al., 1983). Although males of all three species will produce sperm within a few days of eclosion, Booth et al. (1983) indicated that males of P. strobi and P. approximatus will not produce pheromone unless they have experienced the same maturation period and conditioning required by females for reproduction. Lab-reared insects were manipulated to ensure reproductive and pheromona! activity (Phillips, 1981), while feral insects were collected on host material during the breeding season and were considered mature and pheromonally active. RESULTS
Experiment 1. Attraction of wild P. approximatus to males of P. approximatus, P. nemorensis, and P. strobi was assessed in a mature red pine, Pinus resinosa Ait., stand at Heiberg Memorial Forest (Cortland County, New York) between May 11, and June 18, 1982. This was chosen as the initial test because of the well documented pheromone behavior of P. approximatus (Phillips, 1981 ; Booth et al., 1983). All male weevils were laboratory-reared and conditioned prior to the test. P. approximatus and P. strobi were fed fresh cuttings of red and white pine, respectively, and held under ambient lighting in an insectary during April and May (increasing from 12 to 15 hr of light per day) at 2528~ P. nemorensis were fed red pine under 12:12 (light-dark) lighting at 23 ~ in a growth chamber for at least one month before the test. Experimental treatments consisted of five males of one species confined on a red pine bolt that was then placed in a trap; control traps included a bolt on which no weevils were caged. The four treatments were deployed in five completely randomized blocks of traps, with at least 15 m between traps and 20 m between blocks. Traps were checked weekly and live weevils or fresh bolts were replaced as needed. Traps baited with P. approximatus and P. nemorensis males caught significantly more weevils than those baited with P. strobi males, which caught no more weevils than uninfested bolts (Figure 1; analysis of variance (ANOVA) followed by Student-Newman-Keuls test, P < 0.05). There were no differences in the number of each sex responding to any treatment (t test, P < 0.05). There was no marked variation in weekly responses of weevils to P. approximatus and P. nemorensis traps (Figure 2), which indicates that P. nemorensis males continued to produce pheromone after five weeks away from their presumed reproductive photoperiod (12 : 12, light-dark).
INTERSPECIFIC
SEMIOCHEMICALS
AMONG
,
i
,
SIBLING
,
9
WEEVIL
.
.
.
.
.
.
.
.
I
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SPECIES
J
Pn Ps control - - ] i
0
i
i
J
i
i
i
,
J
,
,
,
i
10 20 30 40 50 60 Total number of weevils trapped
FIG. 1. Total numbers of P. approximatus trapped in experiment 1, five traps per treatment. All traps include bolts of red pine. Treatment designations: Pa = five male P. approximatus, Pn = five male P. nemorensis, Ps = five male P. strobi, control = bolt with no weevils.
Experiment 2. A second experiment was conducted in northern Florida from November 12, to December 8, 1982, in which the response of P. nemorensis to males of P. approximatus and P. nemorensis was examined. P. approximatus males were laboratory-reared and conditioned under a 16 : 8 (lightdark) photoperiod at 23~ for one month prior to the test; P. nemorensis males were field-collected from split pine billet traps (Fontaine, 1981) and kept in an insectary no more than one week before the test. Five males of each species were caged on freshly cut bolts of slash pine, Pinus elliottii Engelm., and bolts
P_:aRproximatus
P_.~.nemorensis
16 14
lo
B
I
2
3
4
$
I
2
3
4
F
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WEEKS FIG. 2. Total numbers of weevils caught weekly on traps baited with either male P.
approximatus or male P. nemorensis in experiment 1; M = males, F = females.
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PHILLIPSANDLANIER
without weevils served as controls. Five completely randomized blocks, each containing the three treatments, were deployed in a young slash pine plantation west of Newberry, Florida, in Gilchrist County. The highest responses were to traps baited with males of P. approximatus and P. nemorensis (ANOVA, P < 0.10); only one weevil was caught on a control trap during the three-week period (Figure 3). These results complement those of experiment 1 and lead us to conclude that P. applvximatus and P. nemorensis are cross-attractive and utilize functionally identical aggregation pheromones. Experiment 3. To further investigate the possibility of cross-attraction between P. approximatus and P. strobi, sticky traps on eastern white pine leaders were used to determine the attractiveness of males of each species to P. strobi. Weevils used as pheromone sources for this test were field-collected from their natural breeding sites in May 1983; P. strobi from white pine leaders, and P. approximatus from red pine logs. Leaders on randomly selected 3- to 5-m tall white pines at a plantation near Harford, Cortland County, New York, were covered with screen sleeves in April to prevent attacks by native P. strobi. Five replicates each of the two male treatments and the blank leader control were distributed in a totally randomized design over the 2-hectare test area; five males per leader were used for the respective baited treatments. After a three-week period (May 23, to June 13, 1983) all traps had caught weevils, and there were no significant differences among treatments (ANOVA, P > 0.05; Figure 4). Although traps baited with males of either P. approximatus or P. strobi caught equal numbers of weevils, we cannot make any conclusions about cross-attraction. The large number of weevils on control traps
]
Pn
"E
1
Pa control 0
'10 2'0 3'0 Total number of weevils trapped
Fro. 3. Total number ofP. nemorensis trapped in experiment 2, five traps per treatment. All traps included bolts of slash pine. Treatment designations: Pn = five male P. nemorensis, Pa = five male P. approximatus, control = bolt with no weevils.
INTERSPECIFIC SEMIOCHEMICALS
i
;I
1593
AMONG SIBLING WEEVIL SPECIES
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Ps
I
Pa
I
control 0
'ib
' 2'0' 3'o ' 4'o
Total number of Weevi Is Trapped FIG. 4. Total numbers of P. strobi trapped in experiment 3, five traps per treatment. All traps included intact leaders of living white pines. Treatment designations: Ps = five male P. strobi, Pa = five male P. approximatus, control = leader with no weevils.
precludes any discussion of possible pheromone activity for either of the malebaited treatments. Experiment 4. This test examined the effects of site exposure and host tree species on the attractiveness of male P. strobi and P. approximatus to feral P. approximatus. Bolts of eastern white pine, rather than red pine, were used as host material. Sites were chosen at Heiberg Forest so that traps could be placed in a shaded, closed-canopy stand and at a nearby sunny, open canopy or open field area. Ground traps were deployed at five sun/shade sites as paired randomized blocks in an unbalanced design. Preferred host trees for both weevil species, i.e., red pine, white pine, and Norway spruce, Picea abies (Karst), were available at all sites (see Smith and Sugden, 1969). Site exposure was varied because each species is known to prefer certain types of sites: P. strobi predominantly attacks trees in sunny, open-growth stands (MacAloney, 1930), and P. approximatus oviposits on the undersides of logs, generally in shaded conditions (Finnegan, 1958; Hard, 1962). Weevils used as baits (five males per bolt) were field-collected; limited numbers of male P. approximatus prevented equal replication of treatments. The results after a four-week period (May 25, to June 27, 1983) indicated that the P. approximatus males were attractive when feeding on white pine bolts in either sunny or shaded conditions (Figure 5); traps baited with P. strobi males or bolts alone caught lower numbers of weevils in both shaded and sunny sites (ANOVA performed separately on sun and shade blocks, P < 0.05). These data are similar to those in experiment 1 in which P. strobi was on red pine bolts in a shaded site. T-test comparisons between the same treatments in sun and shade found no significant differences (P > 0.05).
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PHILLIPS AND LANIER
.... Pa
. . . ~ . . . . . ]. .
N
13
SUN
5
Pa
,
3
Ps
SHADE
5
Ps control
5
control 2
6
10
14
18
Mean number of weevils per trap
FIG. 5. Mean numbers of P. approxirnatus caught on traps in adjacent sun and shade blocks in experiment 4; N = number of traps per treatment. All traps included bolts of white pine, Treatment designations: Pa = five male P. approximatus, Ps = five male P. strobi, control = bolt with no weevils. Horizontal lines indicate standard errors of the means.
Experiment 5. The heritability of P. approximatus pheromone production in various types of hybrid males was examined in this experiment. Laboratory hybrids were obtained from the reciprocal crosses of P. approximatus with P. nemorensis and of P. approximatus with P. strobi. Pure laboratory strains of P. approximatus and P. nemorensis were also used; lab-reared P. strobi were not available for this test. Six different male-baited treatments and a control were deployed in an incomplete randomized block design at Heiberg Forest from June 6 to 28, 1983; red pine bolts were the host material, and five males were caged with each bolt. Limited numbers of progeny from various crosses prevented us from having equal sample sizes. All treatment weevils, including P. nemorensis and hybrids, were preconditioned in an insectary at least one month under the naturally occurring increasing spring photoperiod (14-15 hr of light) at 2 5 - 2 8 ~ Therefore, P. nemorensis males were not exposed to conditions for reproductive maturation, but rather to the conditions for reproductive maturation of P. approximatus and P. strobi. The largest catches were on P. approximatus-baited traps and the two P. approximatus x P. nemorensis hybrid treatments (Figure 6). P. nemorensis, both P. approximatus x P. strobi hybrids, and a blank bolt caught lower numbers of weevils. We presume that P. nemorensis males were not attractive because they were not conditioned for pheromone production. Like host selection behavior (Phillips and Lanier, 1983), pheromone production apparently breaks down in hybrids of P. approximatus and P. strobi. Significant differences oc-
1595
]NTERSPECIFIC SEMIOCHEMICALS A M O N G SIBLING WEEVIL SPECIES
N
Pa
'
1
6
'
Pn
5
Pa x Pn
5
Pn x P a
3
Pa x Ps
6
Ps x Pa
2
control
6 2
6
10
14
18
22
Mean number of w e e v i l s per t r a p [ZIG. 6. Mean numbers o f P . approximatus caught on traps in experiment 5; N = number of traps per treatment. All traps included bolts of red pine. Treatment designations: Pa = five male P. approximatus, Pn = five male P. nemorensis, P a x Pn = five male progeny from the cross of female Pa with male Pn, Pn x Pa = five male progeny from the cross of female Pn with male Pa, P a x Ps = five male progeny from the cross of female Pa with male P. strobi (Ps), Ps x Pa = five male progeny from the cross of female Ps with male Pa, control = bolt with no weevils. Horizontal lines indicate standard errors of the means.
curred among treatment means (ANOVA, P < 0.05), but any direct comparison of means is unreliable because of the unbalanced design of the experiment. Experiment 6. In this experiment we investigated the possibility that, in addition to simply being unattractive to P. approximatus, P. strobi males may actively prohibit the flight of P. approximatus to suitable host material. Freshly cut white pine bolts and field-collected male weevils (five per bolt) were used in ground traps in a mature red pine stand at Heiberg Memorial Forest. The response of P. approximatus to the following treatments, arranged in five completely randomized blocks, was assessed from May 10 to 22, 1985: five male P. approximatus on a bolt, five male P. strobi on a bolt, five male P. approximatus together with five male P. strobi on a bolt, and a bolt alone as a control. The highest responses were to traps baited with male P. approximatus, while the other three treatments caught significantly lower numbers of weevils (ANOVA followed by Student-Newman-Keuls test, P < 0.05; Figure 7). As in experiments 1 and 4, male P. strobi were not attractive to P. approximatus. However, it is quite clear from this experiment that male P. approximatus do not attract conspecifics when male P. strobi are confined with them on the same pine bolt. It is possible that male P. strobi produce a chemical signal that interrupts the response of P. approximatus to its natural pheromone, or that the
1596
PHILLIPS AND LANIER
Pa
control i
0
i
i
i
i
I
i
10 20 30 4'0 Total number of weevils trapped
Fie. 7. Total number of P. approximatus trapped in experiment 6, five traps per treatment. All traps contained bolts of white pine. Treatment designations: Pa = five male P. approximatus, Ps = five male P. strobi, Pa + o" o" Ps = five male P. approximatus with five male P. strobi, control = bolt with no weevils. presence of male P. strobi on the same bolt somehow suppresses pheromone production in male P. approximatus. Experiment 7. Our last field experiment examined the possible interruption of synthetic P. approximatus pheromone by male P. strobi. Ground traps with fresh white pine bolts were used in a mature red pine stand at Heiberg Memorial Forest from May 22 to June 21, 1985. Three treatments, deployed in five completely randomized blocks, were compared for their attractiveness to P. approximatus: synthetic racemic grandisol and grandisal with a pine bolt (P. approximatus pheromone), grandisol and grandisal with seven field-collected male P. strobi feeding on a pine bolt, and an unbaited pine bolt as a control. Racemic grandisol was obtained commercially and grandisal was synthesized from it (method of Booth et al., 1983). Grandisol (5 rag/bait) and grandisal (10 rag/ bait) were evaporated from rubber septa at the levels reported by Phillips and coworkers (1984). The addition of male P. strobi to traps baited with the aggregation pheromone of P. approximatus depressed the response of P. approximatus (Figure 8). The highest response was to traps baited with grandisol and grandisal, but this was reduced by almost half (ANOVA, Student-Newman-Keuls test, P < 0.10) in traps containing grandisol, grandisal, and male P. strobi; both these treatments caught significantly more weevils (P < 0.05) than the control. These data suggest that pheromone production by P. approximatus was not suppressed in experiment 6, but confirm that P. strobi males produce an allelochemical signal that blocks the pheromone response of P. approximatus. We believe that
1597
]NTERSPECIFIC SEMIOCHEMICALS A M O N G SIBLING WEEVIL SPEC1ES
GOH + GCHO "~ G O H + G C H O + ddPs
control
' lo' 2'o ' 3'o ' .'o ' so' ' 60 Total number of weevils trapped
'
FIG. 8. Total numbers of P. approximatus trapped in experiment 7, five traps per treatment. All traps contained bolts of white pine. Treatment designations: GOH + GCHO = pheromone bait of grandisol and grandisal, GOH + GCHO + c~ c~ Ps = pheromone bait of grandisol and grandisal with seven male P. strobi, control = bolt with no weevils or bait.
male P. strobi could not totally interrupt the response of P. approximatus in this experiment because the high levels of synthetic pheromone release (about 1.0 mg/day), compared to natural release rates from male weevils (about 20 ng/day; Booth, et al., 1983), probably overwhelmed the blocking odor produced by P. strobi.
DISCUSSION
We can draw four conclusions about weevil-produced semiochemicals in these sibling species: (1) P. approximatus and P. nemorensis are cross-attractive and apparently use functionally identical aggregation pheromones. (2) Different environmental stimuli (e. g., photoperiod) are prerequisite for pheromone production in P. approximatus and P. nemorensis; long-day pheromone production (P. approximatus-type) is heritable in interspecific hybrids. (3) P. strobi males are not attractive to P. approximatus, the reciprocal relationship is not clearly supported. (4) P. strobi males produce an allelochemical signal that can interrupt the response of P. approximatus to its natural or synthetic aggregation pheromone. If P. approximatus and P. nemorensis are biologically distinct species (i.e., no gene flow between them) that are geographically contiguous but potentially cross-attractive, there must be some mechanism preventing introgression at the zone of parapatry. The distributions of the two species are not well defined, and their designation as separate species has more of an historical rather than a biological meaning (see species descriptions by Germar, 1824; and Hopkins, 1911). There are no geological barriers that might separate their distributions, and suitable host conifers for both species are continuously distributed from
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PHILLIPSANDLANIER
north to south in the eastern United States. Seasonal isolation between P. approximatus and P. nemorensis may prevent hybridization in nature, despite the potential for cross-attraction. P. nemorensis is flying and reproductively active during the fall and winter months (Atkinson, 1979; Fontaine et al., 1983), while P. approximatus is active in the spring and summer. In another study (Phillips et al., 1986), we found that weevils responded to synthetic pheromone in both spring and fall seasons at one site in Virginia, which led us to question the validity of P. approximatus and P. nemorensis as separate species. As with reproductive development, pheromone activity is apparently dependent on environmental stimuli like photoperiod (experiment 5; Booth et al., 1983). Once the threshold level of conditioning for pheromone production has been crossed (e.g., about 30 days of short-day conditioning for P. nemorensis), the phenomenon cannot be reversed by a drastic change in conditions (experiment 1, Figure 2). This threshold phenomenon is similar to that found in studies of diapause with various insect species (Tauber and Tauber, 1976). Our finding that hybrid males of P. approximatus and P. nemorensis produce pheromone after longday conditioning (Figure 6) allows for a model of conspecificity that entails successful introgession of southern with northern populations. P. strobi males or interspecific hybrids are not attractive to P. approximatus (experiments 1, 4-6), despite the fact that males of both species produce the pheromone components grandisol and grandisal (Booth et al., 1983). The basis for this lack of cross-attraction lies in the fact that P. strobi males apparently produce one or more chemicals that block the response ofP. approximatus to its pheromone (experiments 6 and 7); the ability to produce this blocking odor is probably inherited in hybrids (experiment 5). Intra- and interspecific interactions in several species of insects are known to be mediated by enantiomeric specificity of semiochemicals (Silverstein, 1979). Since grandisol and grandisal are chiral, we might presume that the blocking phenomenon is imparted by species differences in enantiomeric blends of these compounds. However, we do not believe that the blocking phenomenon results from enantiomers of grandisol and grandisal because P. approximatus responds optimally (i.e., greater than or equal to the response to conspecific males) to racemic mixtures of these compounds. Theoretically, P. approximatus males may produce and respond to an enantiomeric blend of these compounds, or they may produce just one enantiomer of each (or a blend predominated by one enantiomer) and the other enantiomer is inactive. Even if P. strobi produced just one enantiomer each of grandisol and grandisal, it is unlikely that they would act allomonally and block the natural enantiomeric constitution of the P. approximatus pheromone. In other cases in which sympatric species display dual activity of enantiomeric semiochemicals, either separate enantiomers serve as a pheromone and an allomone for each species (e.g., ipsdienol for Ips pini and L paraconfusus; Birch et al., 1980), or a synergistic blend of enantiomers is required by one species for pheromonal and allomonal activity, while the other species uses just
INTERSPECIF[C SEM1OCHEMICALS AMONG SIBLING WEEVIL SPECIES
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one enantiomer as a pheromone (e.g., sulcatol for Gnathotrichus sulcatus and G. retusus; Borden et al., 1980). All possible and probable types of enantiomeric specificity are reviewed by Silverstein (1979). If our hypothesis above is correct, then male P. strobi must produce one or more other chemicals that block the response of P. approximatus to grandisol and grandisal. We have no evidence that P. strobi males produce an aggregation pheromone when feeding on white pine leaders (experiment 3; Phillips, 1981; Booth, 1978), and grandisol and grandisal, although produced by this species, are no more attractive to it than are white pine leaders (Booth et al., 1983; Phillips, unpublished data). It is possible that host location in P. strobi, which results in the finding of mates on suitable host leaders, is mediated primarily by host-related stimuli (e.g., Harris et al., 1983; VanderSar and Borden, 1977a,b). If the grandisol and grandisal produced by male P. strobi are qualitatively identical to the same compounds used as a pheromone by P. approximatus, then P. strobi would attract P. approximatus if it did not produce a blocking odor. We hypothesize that the evolution of a novel allelochemical signal in P. strobi was adaptive in the divergence and reproductive isolation of these two species. The blocking odor serves to prevent hybrid matings rather than to prevent competition for resources, a phenomenon known to occur in interspecific interactions of some bark beetles attacking the same host (Wood, 1982). P. strobi and P. approximatus use very different host resources. Any accidental interspecific matings would result in fertile hybrid progeny that would be unsuccessful in colonizing host material of either P. strobi or P. approximatus, thus reducing the fitness of both parents (Phillips and Lanier, 1983). P. approximatus aggregates on suitable host material in response to its aggregation pheronome; P. strobi aggregates on suitable host material via mechanisms that are not fully understood, and it produces a blocking odor to keep P. approximatus away. Because the blocking odor facilitates reproductive isolation and is adaptively favorable to individuals of both species, it could be referred to as an allomone-kairomone (Borden, 1977) or a synomone (Nordlund and Lewis, 1976). Acknowledgments--We appreciate the cooperation and assistance of John Foltz, University of Florida, Gainesville, in the execution of test 2. Sheri Rosenblum and Michael Griggs provided valuable technical assistance for these studies. F.X. Webster kindly performed the synthesis of the grandisal needed for experiment 7. We thank Robert Rabaglia and Stephen Teale for their careful reviews of the manuscript. This research was supported by grants from the National Science Foundation, DEB 82-01007 and BSR 84-15879. REFERENCES ATKINSON, T.H. 1979. Bionomics of Pissodes nemorensis (Coleoptera: Curculionidae) in North Florida. PhD thesis, University of Florida. Gainesville, Florida. 100 pp. BAKER, W.L. 1972. Eastern Forest Insects. U.S. Dept. of Agric. Forest Service Misc. Publ. No. 1175. 642 pp.
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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 lps pini in California by the two enantiomers of ipsdienol. J. Chem. Ecol. 6:703717. 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, New York. 100 pp. BOOTH, D.C., and LANIER, G.N. 1974. Evidence of an aggregating pheromone in Pissodes approximatus and P. strobi. Ann. Entomol. Soc. Am. 67:992-994. BOOTH, D.C., PHILLIPS,T.W., CLAESSON,A., SILVERSTEIN,R.M., LANIER,G.N., and WEST, J.R. 1983. Aggregation pheromone components of two species of Pissodes weevils (Coleoptera: Curculionidae): Isolation, identification, and field activity. J. Chem. Ecol. 9:1-12. BORDEN, J.H. 1977. Behavioral responses of Coleoptera to pheromones, altomones, and kairomones, pp. 169-198, in H.H. Shorey and J.J. McKelvey (eds.). Chemical Control of Insect Behavior. Wiley, New York. BORDEN, J.U., HANDLEY,J.R., MCLEAN, J.A., SILVERSTEIN,R.M. CHONG, L., SLESSOR,K.N., JOHNSTON, B.D., and SCHULER,H.R. 1980. Enantiomer based specificity in pheromone communication by two sympatric Gnathotrichus species (Coleoptera: Scolytidae). J. Chem. Ecol. 6:445-456. FINNEGAN, R.J. 1958. The pine weevil Pissodes approximatus Hopkins in sourthern Ontario. Can. Entomol. 90:340-354. FONTAINE, M.S. 1981. Reproductive ecology of the deodar weevil, Pissodes nemorensis (Coleoptera: Curculionidae), in north Florida. MS thesis, University of Florida. Gainesville, Florida. 100 pp. FONTAINE, M.S., and FOLTZ, J.L. 1982. Field studies of a male-released aggregation pheromone in Pissodes nemorensis. Environ. Entomol. 11:881-883. FONTAINE, M.S., FOLTZ, J.L., and NATION, J.L. 1983. Reproductive anatomy and seasonal development of the deodar weevil, Pissodes nemorensis (Coleoptera: Curculionidae) in north Florida. Environ. Entomol. 12:687-691. GERMAR,E.F. 1824. Insectornm species novae aut minus cognitae, descriptionibus illustratae. Vol. 1. pp. 316-319. GODWIN, P.A., and ODELL, T.M. 1967. Experimental hybridization of Pissodes strobi and P. approximatus (Coleoptera: Curculionidae). Ann. Entomol. Soc. Am. 60:55-58. GODWIN,P.A., VALENTINE,H.T., and ODELL, T.M. 1982. Identification of Pissodes strobi, P. approximatus and P. nemorensis (Coleoptera: Curculionidae) using descriminant analysis. Ann. Entomol. Soc. Am. 75:599-604. HARD, J.S. 1962. Bionomic ivestigations of the northern pine weevil, Pissodes approximatus Hopkins (Coleoptera: Curculionidae) MS thesis, New York State College of Forestry. Syracuse, New York. 59 pp. HARRIS, L.J., BORDEN, J.H., PIERCE, H.D., JR., and OEHLSCHLAGER,A.C. 1983. Cortical resin monoterpenes in Sitka spruce and resistance by the white pine weevil, Pissodes strobi (Coleoptera: Curculionidae). Can. J. For. Res. 13:350-352. HOPKINS,A.D. 191 l. Contributions toward a monograph of the bark-weevils of the genus Pissodes. U.S. Dept. of Agric. Entomol. Bull. 20 (Tech. Ser.). Part 1. MACALONEY, H.J. 1930. The white pine weevil (Pissodes strobi Peck) its biology and control. Bull. N.Y. State Coll. For. 3:1-87. NORDLUND, D.A., and LEWIS, W.J. 1976. Terminology of chemical releasing stimuli in intraspecific and interspecific interactions. J. Chem. Ecol. 2:211-220. ODELL, T.M., GODWIN, P.A., and ZER1LLO,R.T. (unpublished ms.). The ethological relationship of Pissodes strobi (Peck) and P. approximatus Hopkins under restricted field conditions. Un-
INTERSPECIFIC SEMIOCHEMICALS AMONG SIBLING WEEVIL SPECIES
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published office report 4500-FS-2201. Forest Insect and Disease Laboratory. U.S. Forest Service. Hamden, Connecticut. 14 pp. 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, NY. 94 pp. PHILLIPS, T.W. 1984. Ecology and systematics of Pissodes sibling species. PhD thesis, State University of New York College of Environmental Science and Forestry. Syracuse, New York. 204 pp. PHILLIPS, T.W., and LANIER,G.N. t983. Biosystematics of Pissodes Germar (Coleoptera: Curculionidae): feeding preference and breeding site specificity in P. strobi and P. approximatus. Can. Entomol. 115:1627-1636. PHILLIPS, T.W., WEST, J.R., FOLTZ, J.L., SILVERSTEIN, R.M., and LANIER, G.N. 1984. Aggregation pheromone of the deodar weevil, Pissodes nemorensis (Coleoptera: Curculionidae): Isolation and activity of grandisol and grandisal. J. Chem. Ecol. 10:1417-1423. PHILLIPS, T.W., TEALE, S.A., and LANIER, G.N. 1986. Biosystematics of Pissodes Germar (Coleoptera: Curculionidae): Seasonality, morphology, and synonymy of P. approximatus and P. nemorensis. Can. Entomol. Submitted. SILVERSTEIN,R.M. 1979. Enantiomeric composition and bioactivity of chiral semiochemicals in insects, pp. 133-146, in F.J. Ritter (ed.). Chemical Ecology: Odour Communication in Animals. Elsevier/North Holland, Amsterdam. SMITH, S.G. 1973. Chromosomal polymorphism and interspecific relationships in Pissodes weevils: Additional cytogenetic evidence of synonymy. Can. J. Genet. Cytol. 15:83-100. SMITH, S.G., and SucI~N, B.A. 1969. Host trees and breeding sites of native North American Pissodes bark weevils, with a note on synonymy. Ann. Entomol. Soc. Am. 62:146-148. TAUBER, M.J., and TAUBER, C.A. 1976. Insect seasonality, diapause maintenance, and postdiapause development. Annu. Rev. Entomol. 21:81-107. VANDERSAR, T.J.D., and BORDEN, J.H. 1977a. Visual orientation of Pissodes strobi Peck (Coleoptera: Curculionidae) in relation to host selection behavior. Can. J. Zool. 55:2042-2049. VANDERSAR,T.J.D., and BORDEN, J.H. 1977b. Role of geotaxis and phototaxis in the feeding and oviposition behavior of overwintered Pissodes strobi. Environ. Entomol. 6:743-749. WOOD D.L. 1982. The role of pheromones, kairomones, and allomones in the host selection and colonization behavior of bark beetles. Annu. Rev. Entomol. 27:411-446.
INTERSPECIFIC ACTIVITY OF SEMIOCHEMICALS AMONG SIBLING SPECIES OF Pissodes (COLEOPTERA: CURCULIONIDAE)
THOMAS
W.
PHILLIPS 1 and GERALD
N.
LANIER
Department of Environmental and Forest Biology State University of New York College of Environmental Science and Forestry Syracuse, New York 13210 (Received July 22, 1985; accepted November 12, 1985)
Abstraet--Pissodes strobi, P. approximatus, and P. nemorensis are sibling species of pine weevils that can hybridize in the laboratory but are presumed to be reproductively isolated in nature. Males of all three species produce the terpenoids grandisol and grandisal; these compounds serve as an aggregation pheromone for P. approximatus and P. nemorensis when deployed with odors from pine bolts. A series of field experiments examined the possibility of cross-attraction among the three species. Tests in New York and Florida found that parapatrieally distributed P. approximatus and P. nemorensis were crossattractive, but different photoperiodic conditioning was required for pheromone production in males of the two species. Long-day pheromone production (P. approximatus-type) was inherited in interspecific hybrids. Other tests showed that P. strobi males, or hybrid males from crosses of P. strobi with P. approximatus, were not attractive to sympatric P. approximatus. When the response of P. strobi was assessed to males of either P. strobi or P. approximatus confined on white pine leaders (the breeding site of P. strobi), no evidence of cross-attraction or pheromone activity was found; P. strobi were caught in equal numbers on P. strobi-baited leaders, P. approximatusbaited leaders, and unbaited leaders. Tests of interspecific interactions found that male P. strobi produce an allelochemical signal that interrupts the response of P. approximatus to its natural or synthetic aggregation pheromone. This interspecific response is apparently adaptive for members of both species (classified as an allomone-kairomone or synomone) because it may ultimately serve to prevent interspecific matings that would lower the fitness of the parents. Key Words--Pissodes, Coleoptera, Curculionidae, aggregation pheromone, cross-attraction, synomone, reproductive isolation, grandisol, grandisal. ~Present address: Dept. of Entomology and Nematology, 3103 McCarty Hall, University of Florida, Gainesville, FL 32611. 1587
oo98-o331/86/o7oo-15875o5.oo/o 9 1986Plenum PublishingCorporation
1588
PHILLIPSANDLANIER INTRODUCTION
Aggregation pheromones in Pissodes weevils were first reported by Booth and Lanier (1974), who found that males of P. approximatus Hopkins, the northern pine weevil, feeding on pine bolts would attract large numbers of conspecific males and females, while females feeding on pine bolts and pine bolts alone were not attractive. They also postulated that P. strobi (Peck), the white pine weevil, uses a male-produced aggregation pheromone. Booth et al. (1983) reported that males of P. approximatus produce two terpenoid compounds, grandisol (cis-2-isopropenyl-l-methylcyclobutaneethanol) and its corresponding aldehyde, grandisal, that, together with odor from cut pine logs, serve as an aggregation pheromone. They also found that males of P. strobi produce grandisol and grandisal, but repeated field tests have not convincingly documented attraction of P. strobi to these compounds (Booth, 1978; Phillips, 1981; Booth et al., 1983). Phillips and coworkers (1984) reported that male Deodar weevils, P. nemorensis Germar, also produce grandisol and grandisal and use them with host odors as an aggregation pheromone (cf. Fontaine and Foltz, 1982). No substantial quantitative differences in the production of these compounds among the three species, either in absolute amounts or relative concentrations, have been found (Silverstein and West, 2 unpublished data). Possible differences in enantiomeric composition of grandisol and grandisal released by these species have not been determined. We consider P. strobi, P. approximatus, and P. nemorensis to be sibling species because they are very similar morphologically but are presumed to be reproductively isolated in nature. These species are genetically very similar (Phillips, 1984), can hybridize with each other in the laboratory (Godwin and ODell, 1967; Smith, 1973), but differ ecologically and behaviorally. P. strobi breeds in one-year-old leaders of vigorous young pines, Pinus spp., and spruces, Picea spp., across North America (MacAloney, 1930; Smith and Sugden, 1969). P. approximatus is sympatric with P. strobi in northeastern North America, but breeds in the boles, branches, and root collars of weakened or recently cut pines and spruces (Finnegan, 1958). Breeding site separation between P. strobi and P. approximatus is strong; artificially produced hybrids display reduced reproductive fitness (Phillps and Lanier, 1983). If P. strobi and P. approximatus use aggregation pheromones that incorporate grandisol and grandisal, we predict that qualitative differences would prevent cross-attraction between the species. P. nemorensis also breeds in weakened or cut pines, but is distributed in southeastern North America and is presumed to be parapatric with P. approximatus (Baker, 1972; Hopkins, 1911). P. approximatus and P. nemorensis respond to the same synthetic pheromone (Booth et al., 1983; Phillips et al., 2R.M. Silversteinand J.R. West, Departmentof Chemistry,StateUniversityof New York College of EnvironmentalScienceand Forestry, Syracuse,New York 13210.
INTERSPECIFIC SEMIOCHEMICALS AMONG SIBLING WEEVIL SPECIES
1589
1984), and it is quite probable that they could be cross-attractive in nature. However, P. approximatus and P. nemorensis are apparently reproductively active in different seasons (Finnegan, 1958; Fontaine et al., 1983), so crossattraction in nature may never occur. Here we report a series of experiments in which we investigated the activity of pheromones and allelochemics among P. strobi, P. approximatus, and P. nemorensis. Using field tests with live weevils, we examined cross-attraction among species, the attractiveness of interspecific hybrids, and the interspecific interruption of pheromone activity. We will assess the importance of pheromones and allelochemics in reproductive isolation and host location, and we will discuss the evolution of semichemicals in these species.
METHODS
AND MATERIALS
Field experiments, described in detail below, were conducted in 1982, 1983, and 1985. Traps for assessing the response of either P. approximatus or P. nemorensis were sticky hardware-cloth cylinders placed directly on the forest floor (design of Booth and Lanier, 1974). Freshly cut pine bolts, about 15 x 25 cm, were enclosed in 30 x 45-cm fiberglass screen bags with or without male weevils and were placed inside the traps. Traps for P. strobi (one experiment) were 20 x 91-cm sticky hardware-cloth cylinders with the top ends covered by 20 x 20-cm sticky hardware-cloth squares. These traps were placed on poles supported by metal stakes and suspended over screen-enclosed (15 • 100-cm fiberglass screen sleeves) one-year-old leaders of 3- to 5-m tall eastern white pine trees, Pinus strobus L. Male Pissodes were then confined on leaders as attractive sources (Phillips, 1981). Because there are no diagnostic characters for adequately separating specimens of P. strobi, P. approximatus, and P. nemorensis, we initially employed three working hypotheses for the identification of trapped weevils based on geographic locality or ecological qualities of the field site and experiment. Weevils trapped in Florida (experiment 2, see below) were P. nemorensis based on distribution. Weevils flying to traps on white pine leaders (experiment 3) in open stands were considered P. strobi, while those caught in New York on ground traps containing cut logs (experiments 1, 4-7) were deemed P. approximatus. These assumptions are drawn from accumulated information on Pissodes behavior, ecology, karyology, and geographic distribution; unfortunately, there is no guarantee that they are true. Therefore, we challenged our designations by performing the discriminant analysis technique of Godwin et al. (1982) on subsamples of the trap-caught specimens (Phillips, 1984). Discriminant analysis corroborated the identification of P. strobi and P. approximatus by our a priori assumptions, but of weevils that could only be P. nemorensis based on geographic definition (experiment 2), a majority were identified
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PHILLIPS AND LANIER
as P. approximatus. The utility of the discriminant analysis technique is discussed elsewhere (Phillips, 1984; Phillips et al., 1986). Weevils used as attractive sources were either laboratory-reared or fieldcollected; unless otherwise indicated, all were reproductively mature. Females of the three species have certain environmental requirements for reproductive maturity and oviposition (ODell et al., unpublished; Atkinson, 1979; Fontaine et al., 1983). Although males of all three species will produce sperm within a few days of eclosion, Booth et al. (1983) indicated that males of P. strobi and P. approximatus will not produce pheromone unless they have experienced the same maturation period and conditioning required by females for reproduction. Lab-reared insects were manipulated to ensure reproductive and pheromona! activity (Phillips, 1981), while feral insects were collected on host material during the breeding season and were considered mature and pheromonally active. RESULTS
Experiment 1. Attraction of wild P. approximatus to males of P. approximatus, P. nemorensis, and P. strobi was assessed in a mature red pine, Pinus resinosa Ait., stand at Heiberg Memorial Forest (Cortland County, New York) between May 11, and June 18, 1982. This was chosen as the initial test because of the well documented pheromone behavior of P. approximatus (Phillips, 1981 ; Booth et al., 1983). All male weevils were laboratory-reared and conditioned prior to the test. P. approximatus and P. strobi were fed fresh cuttings of red and white pine, respectively, and held under ambient lighting in an insectary during April and May (increasing from 12 to 15 hr of light per day) at 2528~ P. nemorensis were fed red pine under 12:12 (light-dark) lighting at 23 ~ in a growth chamber for at least one month before the test. Experimental treatments consisted of five males of one species confined on a red pine bolt that was then placed in a trap; control traps included a bolt on which no weevils were caged. The four treatments were deployed in five completely randomized blocks of traps, with at least 15 m between traps and 20 m between blocks. Traps were checked weekly and live weevils or fresh bolts were replaced as needed. Traps baited with P. approximatus and P. nemorensis males caught significantly more weevils than those baited with P. strobi males, which caught no more weevils than uninfested bolts (Figure 1; analysis of variance (ANOVA) followed by Student-Newman-Keuls test, P < 0.05). There were no differences in the number of each sex responding to any treatment (t test, P < 0.05). There was no marked variation in weekly responses of weevils to P. approximatus and P. nemorensis traps (Figure 2), which indicates that P. nemorensis males continued to produce pheromone after five weeks away from their presumed reproductive photoperiod (12 : 12, light-dark).
INTERSPECIFIC
SEMIOCHEMICALS
AMONG
,
i
,
SIBLING
,
9
WEEVIL
.
.
.
.
.
.
.
.
I
Pa
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1591
SPECIES
J
Pn Ps control - - ] i
0
i
i
J
i
i
i
,
J
,
,
,
i
10 20 30 40 50 60 Total number of weevils trapped
FIG. 1. Total numbers of P. approximatus trapped in experiment 1, five traps per treatment. All traps include bolts of red pine. Treatment designations: Pa = five male P. approximatus, Pn = five male P. nemorensis, Ps = five male P. strobi, control = bolt with no weevils.
Experiment 2. A second experiment was conducted in northern Florida from November 12, to December 8, 1982, in which the response of P. nemorensis to males of P. approximatus and P. nemorensis was examined. P. approximatus males were laboratory-reared and conditioned under a 16 : 8 (lightdark) photoperiod at 23~ for one month prior to the test; P. nemorensis males were field-collected from split pine billet traps (Fontaine, 1981) and kept in an insectary no more than one week before the test. Five males of each species were caged on freshly cut bolts of slash pine, Pinus elliottii Engelm., and bolts
P_:aRproximatus
P_.~.nemorensis
16 14
lo
B
I
2
3
4
$
I
2
3
4
F
5
WEEKS FIG. 2. Total numbers of weevils caught weekly on traps baited with either male P.
approximatus or male P. nemorensis in experiment 1; M = males, F = females.
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PHILLIPSANDLANIER
without weevils served as controls. Five completely randomized blocks, each containing the three treatments, were deployed in a young slash pine plantation west of Newberry, Florida, in Gilchrist County. The highest responses were to traps baited with males of P. approximatus and P. nemorensis (ANOVA, P < 0.10); only one weevil was caught on a control trap during the three-week period (Figure 3). These results complement those of experiment 1 and lead us to conclude that P. applvximatus and P. nemorensis are cross-attractive and utilize functionally identical aggregation pheromones. Experiment 3. To further investigate the possibility of cross-attraction between P. approximatus and P. strobi, sticky traps on eastern white pine leaders were used to determine the attractiveness of males of each species to P. strobi. Weevils used as pheromone sources for this test were field-collected from their natural breeding sites in May 1983; P. strobi from white pine leaders, and P. approximatus from red pine logs. Leaders on randomly selected 3- to 5-m tall white pines at a plantation near Harford, Cortland County, New York, were covered with screen sleeves in April to prevent attacks by native P. strobi. Five replicates each of the two male treatments and the blank leader control were distributed in a totally randomized design over the 2-hectare test area; five males per leader were used for the respective baited treatments. After a three-week period (May 23, to June 13, 1983) all traps had caught weevils, and there were no significant differences among treatments (ANOVA, P > 0.05; Figure 4). Although traps baited with males of either P. approximatus or P. strobi caught equal numbers of weevils, we cannot make any conclusions about cross-attraction. The large number of weevils on control traps
]
Pn
"E
1
Pa control 0
'10 2'0 3'0 Total number of weevils trapped
Fro. 3. Total number ofP. nemorensis trapped in experiment 2, five traps per treatment. All traps included bolts of slash pine. Treatment designations: Pn = five male P. nemorensis, Pa = five male P. approximatus, control = bolt with no weevils.
INTERSPECIFIC SEMIOCHEMICALS
i
;I
1593
AMONG SIBLING WEEVIL SPECIES
i
]
Ps
I
Pa
I
control 0
'ib
' 2'0' 3'o ' 4'o
Total number of Weevi Is Trapped FIG. 4. Total numbers of P. strobi trapped in experiment 3, five traps per treatment. All traps included intact leaders of living white pines. Treatment designations: Ps = five male P. strobi, Pa = five male P. approximatus, control = leader with no weevils.
precludes any discussion of possible pheromone activity for either of the malebaited treatments. Experiment 4. This test examined the effects of site exposure and host tree species on the attractiveness of male P. strobi and P. approximatus to feral P. approximatus. Bolts of eastern white pine, rather than red pine, were used as host material. Sites were chosen at Heiberg Forest so that traps could be placed in a shaded, closed-canopy stand and at a nearby sunny, open canopy or open field area. Ground traps were deployed at five sun/shade sites as paired randomized blocks in an unbalanced design. Preferred host trees for both weevil species, i.e., red pine, white pine, and Norway spruce, Picea abies (Karst), were available at all sites (see Smith and Sugden, 1969). Site exposure was varied because each species is known to prefer certain types of sites: P. strobi predominantly attacks trees in sunny, open-growth stands (MacAloney, 1930), and P. approximatus oviposits on the undersides of logs, generally in shaded conditions (Finnegan, 1958; Hard, 1962). Weevils used as baits (five males per bolt) were field-collected; limited numbers of male P. approximatus prevented equal replication of treatments. The results after a four-week period (May 25, to June 27, 1983) indicated that the P. approximatus males were attractive when feeding on white pine bolts in either sunny or shaded conditions (Figure 5); traps baited with P. strobi males or bolts alone caught lower numbers of weevils in both shaded and sunny sites (ANOVA performed separately on sun and shade blocks, P < 0.05). These data are similar to those in experiment 1 in which P. strobi was on red pine bolts in a shaded site. T-test comparisons between the same treatments in sun and shade found no significant differences (P > 0.05).
1594
PHILLIPS AND LANIER
.... Pa
. . . ~ . . . . . ]. .
N
13
SUN
5
Pa
,
3
Ps
SHADE
5
Ps control
5
control 2
6
10
14
18
Mean number of weevils per trap
FIG. 5. Mean numbers of P. approxirnatus caught on traps in adjacent sun and shade blocks in experiment 4; N = number of traps per treatment. All traps included bolts of white pine, Treatment designations: Pa = five male P. approximatus, Ps = five male P. strobi, control = bolt with no weevils. Horizontal lines indicate standard errors of the means.
Experiment 5. The heritability of P. approximatus pheromone production in various types of hybrid males was examined in this experiment. Laboratory hybrids were obtained from the reciprocal crosses of P. approximatus with P. nemorensis and of P. approximatus with P. strobi. Pure laboratory strains of P. approximatus and P. nemorensis were also used; lab-reared P. strobi were not available for this test. Six different male-baited treatments and a control were deployed in an incomplete randomized block design at Heiberg Forest from June 6 to 28, 1983; red pine bolts were the host material, and five males were caged with each bolt. Limited numbers of progeny from various crosses prevented us from having equal sample sizes. All treatment weevils, including P. nemorensis and hybrids, were preconditioned in an insectary at least one month under the naturally occurring increasing spring photoperiod (14-15 hr of light) at 2 5 - 2 8 ~ Therefore, P. nemorensis males were not exposed to conditions for reproductive maturation, but rather to the conditions for reproductive maturation of P. approximatus and P. strobi. The largest catches were on P. approximatus-baited traps and the two P. approximatus x P. nemorensis hybrid treatments (Figure 6). P. nemorensis, both P. approximatus x P. strobi hybrids, and a blank bolt caught lower numbers of weevils. We presume that P. nemorensis males were not attractive because they were not conditioned for pheromone production. Like host selection behavior (Phillips and Lanier, 1983), pheromone production apparently breaks down in hybrids of P. approximatus and P. strobi. Significant differences oc-
1595
]NTERSPECIFIC SEMIOCHEMICALS A M O N G SIBLING WEEVIL SPECIES
N
Pa
'
1
6
'
Pn
5
Pa x Pn
5
Pn x P a
3
Pa x Ps
6
Ps x Pa
2
control
6 2
6
10
14
18
22
Mean number of w e e v i l s per t r a p [ZIG. 6. Mean numbers o f P . approximatus caught on traps in experiment 5; N = number of traps per treatment. All traps included bolts of red pine. Treatment designations: Pa = five male P. approximatus, Pn = five male P. nemorensis, P a x Pn = five male progeny from the cross of female Pa with male Pn, Pn x Pa = five male progeny from the cross of female Pn with male Pa, P a x Ps = five male progeny from the cross of female Pa with male P. strobi (Ps), Ps x Pa = five male progeny from the cross of female Ps with male Pa, control = bolt with no weevils. Horizontal lines indicate standard errors of the means.
curred among treatment means (ANOVA, P < 0.05), but any direct comparison of means is unreliable because of the unbalanced design of the experiment. Experiment 6. In this experiment we investigated the possibility that, in addition to simply being unattractive to P. approximatus, P. strobi males may actively prohibit the flight of P. approximatus to suitable host material. Freshly cut white pine bolts and field-collected male weevils (five per bolt) were used in ground traps in a mature red pine stand at Heiberg Memorial Forest. The response of P. approximatus to the following treatments, arranged in five completely randomized blocks, was assessed from May 10 to 22, 1985: five male P. approximatus on a bolt, five male P. strobi on a bolt, five male P. approximatus together with five male P. strobi on a bolt, and a bolt alone as a control. The highest responses were to traps baited with male P. approximatus, while the other three treatments caught significantly lower numbers of weevils (ANOVA followed by Student-Newman-Keuls test, P < 0.05; Figure 7). As in experiments 1 and 4, male P. strobi were not attractive to P. approximatus. However, it is quite clear from this experiment that male P. approximatus do not attract conspecifics when male P. strobi are confined with them on the same pine bolt. It is possible that male P. strobi produce a chemical signal that interrupts the response of P. approximatus to its natural pheromone, or that the
1596
PHILLIPS AND LANIER
Pa
control i
0
i
i
i
i
I
i
10 20 30 4'0 Total number of weevils trapped
Fie. 7. Total number of P. approximatus trapped in experiment 6, five traps per treatment. All traps contained bolts of white pine. Treatment designations: Pa = five male P. approximatus, Ps = five male P. strobi, Pa + o" o" Ps = five male P. approximatus with five male P. strobi, control = bolt with no weevils. presence of male P. strobi on the same bolt somehow suppresses pheromone production in male P. approximatus. Experiment 7. Our last field experiment examined the possible interruption of synthetic P. approximatus pheromone by male P. strobi. Ground traps with fresh white pine bolts were used in a mature red pine stand at Heiberg Memorial Forest from May 22 to June 21, 1985. Three treatments, deployed in five completely randomized blocks, were compared for their attractiveness to P. approximatus: synthetic racemic grandisol and grandisal with a pine bolt (P. approximatus pheromone), grandisol and grandisal with seven field-collected male P. strobi feeding on a pine bolt, and an unbaited pine bolt as a control. Racemic grandisol was obtained commercially and grandisal was synthesized from it (method of Booth et al., 1983). Grandisol (5 rag/bait) and grandisal (10 rag/ bait) were evaporated from rubber septa at the levels reported by Phillips and coworkers (1984). The addition of male P. strobi to traps baited with the aggregation pheromone of P. approximatus depressed the response of P. approximatus (Figure 8). The highest response was to traps baited with grandisol and grandisal, but this was reduced by almost half (ANOVA, Student-Newman-Keuls test, P < 0.10) in traps containing grandisol, grandisal, and male P. strobi; both these treatments caught significantly more weevils (P < 0.05) than the control. These data suggest that pheromone production by P. approximatus was not suppressed in experiment 6, but confirm that P. strobi males produce an allelochemical signal that blocks the pheromone response of P. approximatus. We believe that
1597
]NTERSPECIFIC SEMIOCHEMICALS A M O N G SIBLING WEEVIL SPEC1ES
GOH + GCHO "~ G O H + G C H O + ddPs
control
' lo' 2'o ' 3'o ' .'o ' so' ' 60 Total number of weevils trapped
'
FIG. 8. Total numbers of P. approximatus trapped in experiment 7, five traps per treatment. All traps contained bolts of white pine. Treatment designations: GOH + GCHO = pheromone bait of grandisol and grandisal, GOH + GCHO + c~ c~ Ps = pheromone bait of grandisol and grandisal with seven male P. strobi, control = bolt with no weevils or bait.
male P. strobi could not totally interrupt the response of P. approximatus in this experiment because the high levels of synthetic pheromone release (about 1.0 mg/day), compared to natural release rates from male weevils (about 20 ng/day; Booth, et al., 1983), probably overwhelmed the blocking odor produced by P. strobi.
DISCUSSION
We can draw four conclusions about weevil-produced semiochemicals in these sibling species: (1) P. approximatus and P. nemorensis are cross-attractive and apparently use functionally identical aggregation pheromones. (2) Different environmental stimuli (e. g., photoperiod) are prerequisite for pheromone production in P. approximatus and P. nemorensis; long-day pheromone production (P. approximatus-type) is heritable in interspecific hybrids. (3) P. strobi males are not attractive to P. approximatus, the reciprocal relationship is not clearly supported. (4) P. strobi males produce an allelochemical signal that can interrupt the response of P. approximatus to its natural or synthetic aggregation pheromone. If P. approximatus and P. nemorensis are biologically distinct species (i.e., no gene flow between them) that are geographically contiguous but potentially cross-attractive, there must be some mechanism preventing introgression at the zone of parapatry. The distributions of the two species are not well defined, and their designation as separate species has more of an historical rather than a biological meaning (see species descriptions by Germar, 1824; and Hopkins, 1911). There are no geological barriers that might separate their distributions, and suitable host conifers for both species are continuously distributed from
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north to south in the eastern United States. Seasonal isolation between P. approximatus and P. nemorensis may prevent hybridization in nature, despite the potential for cross-attraction. P. nemorensis is flying and reproductively active during the fall and winter months (Atkinson, 1979; Fontaine et al., 1983), while P. approximatus is active in the spring and summer. In another study (Phillips et al., 1986), we found that weevils responded to synthetic pheromone in both spring and fall seasons at one site in Virginia, which led us to question the validity of P. approximatus and P. nemorensis as separate species. As with reproductive development, pheromone activity is apparently dependent on environmental stimuli like photoperiod (experiment 5; Booth et al., 1983). Once the threshold level of conditioning for pheromone production has been crossed (e.g., about 30 days of short-day conditioning for P. nemorensis), the phenomenon cannot be reversed by a drastic change in conditions (experiment 1, Figure 2). This threshold phenomenon is similar to that found in studies of diapause with various insect species (Tauber and Tauber, 1976). Our finding that hybrid males of P. approximatus and P. nemorensis produce pheromone after longday conditioning (Figure 6) allows for a model of conspecificity that entails successful introgession of southern with northern populations. P. strobi males or interspecific hybrids are not attractive to P. approximatus (experiments 1, 4-6), despite the fact that males of both species produce the pheromone components grandisol and grandisal (Booth et al., 1983). The basis for this lack of cross-attraction lies in the fact that P. strobi males apparently produce one or more chemicals that block the response ofP. approximatus to its pheromone (experiments 6 and 7); the ability to produce this blocking odor is probably inherited in hybrids (experiment 5). Intra- and interspecific interactions in several species of insects are known to be mediated by enantiomeric specificity of semiochemicals (Silverstein, 1979). Since grandisol and grandisal are chiral, we might presume that the blocking phenomenon is imparted by species differences in enantiomeric blends of these compounds. However, we do not believe that the blocking phenomenon results from enantiomers of grandisol and grandisal because P. approximatus responds optimally (i.e., greater than or equal to the response to conspecific males) to racemic mixtures of these compounds. Theoretically, P. approximatus males may produce and respond to an enantiomeric blend of these compounds, or they may produce just one enantiomer of each (or a blend predominated by one enantiomer) and the other enantiomer is inactive. Even if P. strobi produced just one enantiomer each of grandisol and grandisal, it is unlikely that they would act allomonally and block the natural enantiomeric constitution of the P. approximatus pheromone. In other cases in which sympatric species display dual activity of enantiomeric semiochemicals, either separate enantiomers serve as a pheromone and an allomone for each species (e.g., ipsdienol for Ips pini and L paraconfusus; Birch et al., 1980), or a synergistic blend of enantiomers is required by one species for pheromonal and allomonal activity, while the other species uses just
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one enantiomer as a pheromone (e.g., sulcatol for Gnathotrichus sulcatus and G. retusus; Borden et al., 1980). All possible and probable types of enantiomeric specificity are reviewed by Silverstein (1979). If our hypothesis above is correct, then male P. strobi must produce one or more other chemicals that block the response of P. approximatus to grandisol and grandisal. We have no evidence that P. strobi males produce an aggregation pheromone when feeding on white pine leaders (experiment 3; Phillips, 1981; Booth, 1978), and grandisol and grandisal, although produced by this species, are no more attractive to it than are white pine leaders (Booth et al., 1983; Phillips, unpublished data). It is possible that host location in P. strobi, which results in the finding of mates on suitable host leaders, is mediated primarily by host-related stimuli (e.g., Harris et al., 1983; VanderSar and Borden, 1977a,b). If the grandisol and grandisal produced by male P. strobi are qualitatively identical to the same compounds used as a pheromone by P. approximatus, then P. strobi would attract P. approximatus if it did not produce a blocking odor. We hypothesize that the evolution of a novel allelochemical signal in P. strobi was adaptive in the divergence and reproductive isolation of these two species. The blocking odor serves to prevent hybrid matings rather than to prevent competition for resources, a phenomenon known to occur in interspecific interactions of some bark beetles attacking the same host (Wood, 1982). P. strobi and P. approximatus use very different host resources. Any accidental interspecific matings would result in fertile hybrid progeny that would be unsuccessful in colonizing host material of either P. strobi or P. approximatus, thus reducing the fitness of both parents (Phillips and Lanier, 1983). P. approximatus aggregates on suitable host material in response to its aggregation pheronome; P. strobi aggregates on suitable host material via mechanisms that are not fully understood, and it produces a blocking odor to keep P. approximatus away. Because the blocking odor facilitates reproductive isolation and is adaptively favorable to individuals of both species, it could be referred to as an allomone-kairomone (Borden, 1977) or a synomone (Nordlund and Lewis, 1976). Acknowledgments--We appreciate the cooperation and assistance of John Foltz, University of Florida, Gainesville, in the execution of test 2. Sheri Rosenblum and Michael Griggs provided valuable technical assistance for these studies. F.X. Webster kindly performed the synthesis of the grandisal needed for experiment 7. We thank Robert Rabaglia and Stephen Teale for their careful reviews of the manuscript. This research was supported by grants from the National Science Foundation, DEB 82-01007 and BSR 84-15879. REFERENCES ATKINSON, T.H. 1979. Bionomics of Pissodes nemorensis (Coleoptera: Curculionidae) in North Florida. PhD thesis, University of Florida. Gainesville, Florida. 100 pp. BAKER, W.L. 1972. Eastern Forest Insects. U.S. Dept. of Agric. Forest Service Misc. Publ. No. 1175. 642 pp.
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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 lps pini in California by the two enantiomers of ipsdienol. J. Chem. Ecol. 6:703717. 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, New York. 100 pp. BOOTH, D.C., and LANIER, G.N. 1974. Evidence of an aggregating pheromone in Pissodes approximatus and P. strobi. Ann. Entomol. Soc. Am. 67:992-994. BOOTH, D.C., PHILLIPS,T.W., CLAESSON,A., SILVERSTEIN,R.M., LANIER,G.N., and WEST, J.R. 1983. Aggregation pheromone components of two species of Pissodes weevils (Coleoptera: Curculionidae): Isolation, identification, and field activity. J. Chem. Ecol. 9:1-12. BORDEN, J.H. 1977. Behavioral responses of Coleoptera to pheromones, altomones, and kairomones, pp. 169-198, in H.H. Shorey and J.J. McKelvey (eds.). Chemical Control of Insect Behavior. Wiley, New York. BORDEN, J.U., HANDLEY,J.R., MCLEAN, J.A., SILVERSTEIN,R.M. CHONG, L., SLESSOR,K.N., JOHNSTON, B.D., and SCHULER,H.R. 1980. Enantiomer based specificity in pheromone communication by two sympatric Gnathotrichus species (Coleoptera: Scolytidae). J. Chem. Ecol. 6:445-456. FINNEGAN, R.J. 1958. The pine weevil Pissodes approximatus Hopkins in sourthern Ontario. Can. Entomol. 90:340-354. FONTAINE, M.S. 1981. Reproductive ecology of the deodar weevil, Pissodes nemorensis (Coleoptera: Curculionidae), in north Florida. MS thesis, University of Florida. Gainesville, Florida. 100 pp. FONTAINE, M.S., and FOLTZ, J.L. 1982. Field studies of a male-released aggregation pheromone in Pissodes nemorensis. Environ. Entomol. 11:881-883. FONTAINE, M.S., FOLTZ, J.L., and NATION, J.L. 1983. Reproductive anatomy and seasonal development of the deodar weevil, Pissodes nemorensis (Coleoptera: Curculionidae) in north Florida. Environ. Entomol. 12:687-691. GERMAR,E.F. 1824. Insectornm species novae aut minus cognitae, descriptionibus illustratae. Vol. 1. pp. 316-319. GODWIN, P.A., and ODELL, T.M. 1967. Experimental hybridization of Pissodes strobi and P. approximatus (Coleoptera: Curculionidae). Ann. Entomol. Soc. Am. 60:55-58. GODWIN,P.A., VALENTINE,H.T., and ODELL, T.M. 1982. Identification of Pissodes strobi, P. approximatus and P. nemorensis (Coleoptera: Curculionidae) using descriminant analysis. Ann. Entomol. Soc. Am. 75:599-604. HARD, J.S. 1962. Bionomic ivestigations of the northern pine weevil, Pissodes approximatus Hopkins (Coleoptera: Curculionidae) MS thesis, New York State College of Forestry. Syracuse, New York. 59 pp. HARRIS, L.J., BORDEN, J.H., PIERCE, H.D., JR., and OEHLSCHLAGER,A.C. 1983. Cortical resin monoterpenes in Sitka spruce and resistance by the white pine weevil, Pissodes strobi (Coleoptera: Curculionidae). Can. J. For. Res. 13:350-352. HOPKINS,A.D. 191 l. Contributions toward a monograph of the bark-weevils of the genus Pissodes. U.S. Dept. of Agric. Entomol. Bull. 20 (Tech. Ser.). Part 1. MACALONEY, H.J. 1930. The white pine weevil (Pissodes strobi Peck) its biology and control. Bull. N.Y. State Coll. For. 3:1-87. NORDLUND, D.A., and LEWIS, W.J. 1976. Terminology of chemical releasing stimuli in intraspecific and interspecific interactions. J. Chem. Ecol. 2:211-220. ODELL, T.M., GODWIN, P.A., and ZER1LLO,R.T. (unpublished ms.). The ethological relationship of Pissodes strobi (Peck) and P. approximatus Hopkins under restricted field conditions. Un-
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published office report 4500-FS-2201. Forest Insect and Disease Laboratory. U.S. Forest Service. Hamden, Connecticut. 14 pp. 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, NY. 94 pp. PHILLIPS, T.W. 1984. Ecology and systematics of Pissodes sibling species. PhD thesis, State University of New York College of Environmental Science and Forestry. Syracuse, New York. 204 pp. PHILLIPS, T.W., and LANIER,G.N. t983. Biosystematics of Pissodes Germar (Coleoptera: Curculionidae): feeding preference and breeding site specificity in P. strobi and P. approximatus. Can. Entomol. 115:1627-1636. PHILLIPS, T.W., WEST, J.R., FOLTZ, J.L., SILVERSTEIN, R.M., and LANIER, G.N. 1984. Aggregation pheromone of the deodar weevil, Pissodes nemorensis (Coleoptera: Curculionidae): Isolation and activity of grandisol and grandisal. J. Chem. Ecol. 10:1417-1423. PHILLIPS, T.W., TEALE, S.A., and LANIER, G.N. 1986. Biosystematics of Pissodes Germar (Coleoptera: Curculionidae): Seasonality, morphology, and synonymy of P. approximatus and P. nemorensis. Can. Entomol. Submitted. SILVERSTEIN,R.M. 1979. Enantiomeric composition and bioactivity of chiral semiochemicals in insects, pp. 133-146, in F.J. Ritter (ed.). Chemical Ecology: Odour Communication in Animals. Elsevier/North Holland, Amsterdam. SMITH, S.G. 1973. Chromosomal polymorphism and interspecific relationships in Pissodes weevils: Additional cytogenetic evidence of synonymy. Can. J. Genet. Cytol. 15:83-100. SMITH, S.G., and SucI~N, B.A. 1969. Host trees and breeding sites of native North American Pissodes bark weevils, with a note on synonymy. Ann. Entomol. Soc. Am. 62:146-148. TAUBER, M.J., and TAUBER, C.A. 1976. Insect seasonality, diapause maintenance, and postdiapause development. Annu. Rev. Entomol. 21:81-107. VANDERSAR, T.J.D., and BORDEN, J.H. 1977a. Visual orientation of Pissodes strobi Peck (Coleoptera: Curculionidae) in relation to host selection behavior. Can. J. Zool. 55:2042-2049. VANDERSAR,T.J.D., and BORDEN, J.H. 1977b. Role of geotaxis and phototaxis in the feeding and oviposition behavior of overwintered Pissodes strobi. Environ. Entomol. 6:743-749. WOOD D.L. 1982. The role of pheromones, kairomones, and allomones in the host selection and colonization behavior of bark beetles. Annu. Rev. Entomol. 27:411-446.
