Field Response of Southern Pine Beetle Parasitoids to ... - Springer Link

Journal of Chemical Ecology. Vol. 23, No. 3. 1997

FIELD RESPONSE OF SOUTHERN PINE BEETLE PARASITOIDS TO SOME NATURAL ATTRACTANTS

B. T. SULLIVAN,* C. W. BERISFORD, and M. J. DALUSKY Department of Entomology University of Georgia Athens, Georgia 30602 (Received April 30, 1996; accepted November 10, 1996)

Abstract—Studies were performed to isolate and identify semiochemicals that mediate location of host-infested trees by parasitoids of the southern pine beetle (SPB), Dendroctonus frontalis. Bark or bolts removed from pines infested with SPB broods attracted significant numbers of the hymenopterous parasitoids Spathius pallidus and Roptrocerus xylophagorum to sticky traps placed in an active SPB infestation. Traps baited with the water distillate of SPB brood-infested bark also attracted both species of parasitoids. In contrast, a synthetic bait composed of 18 compounds identified from the headspace volatiles of attractive bark failed to trap parasitoids. The oxygenated and hydrocarbon components of the bark distillate were partitioned by silica gel liquid chromatography, and the resulting two fractions were tested in the field. Parasitoid attraction was greatest when both fractions were released from traps simultaneously. The hydrocarbon fraction, which failed to attract parasitoids, enhanced the weak attractiveness of the oxygenated fraction. Hence, it appears that no single compound is responsible for mediating SPB parasitoid host-tree location and that both oxygenated and hydrocarbon semiochemicals are involved in this process. Key Words—Parasitoid, bark beetle, Dendroctonus frontalis, Spathius pallidus, Roptrocerus xylophagorum, host location, semiochemical, water distillation, terpenes, fractionation.

INTRODUCTION

Numerous species of hymenopterous parasitoids are associated with the southern pine beetle (SPB), Dendroctonus frontalis Zimmermann (Coleoptera: Scoly*To whom correspondence should be addressed. 837 0098-0331/97/0300-0837$12.50/0 © 1997 Plenum Publishing Coiporation

838

SULLIVAN, BERISFORD, AND DALUSKY

tidae), the most destructive forest pest in the southeastern United States (Franklin, 1969; Berisford, 1980). Evidence indicates that these parasitoids may use olfactory cues to locate hosts and host habitats. Several SPB parasitoids have been reported to respond to traps baited with beetle pheromones, pine resin terpenes, or a combination of these (Camors and Payne, 1972; Dixon and Payne, 1980), and these odors may serve as cues in the location of host habitats, namely, infested stands (Payne, 1989). However, bark beetle parasitoids arrive on infested pines in the greatest numbers when their preferred host life stages, late-instar larvae and pupae, are most abundant (Camors and Payne, 1973; Dixon and Payne, 1979; Berisford et al., 1995). This period of maximum parasitoid arrival on host-infested trees generally follows the period of maximum beetle pheromone production and host-tree resin exudation (Sullivan et al., unpublished data), and it has been suggested that these compounds are likely not used as cues by parasitoids during selection of host-infested trees (Berisford and Franklin, 1969; Payne 1989). Susceptible brood stages are normally concealed within the bark, and, although heat perception (Richerson and Borden, 1972a, b) and tactile recognition (Ryan and Rudinsky, 1962) have been proposed as the host location mechanism for at least one species of bark beetle larval parasitoid, other evidence suggests that the cues used by bark beetle parasitoids in selecting infested trees and locating hosts are olfactory (Kudon and Berisford, 1980, 1981; Mills et al., 1991). Possible sources of parasitoid host-tree location cues include the pheromones of late-arriving SPB associates, odors produced by SPB larvae or their activities (Payne, 1989; Camors and Payne, 1973), as well as metabolites of yeasts and fungi introduced by the attacking beetles (Leufven and Birgersson, 1987; Birgersson and Leufvdn, 1988). Electroantennogram tests of the SPB parasitoids Coeloides pissodis (Ashmead) and Dinotiscus dendroctoni (Ashmead) have shown that both species possess olfactory receptors for a variety of beetle pheromones as well as host-tree odors associated with SPB larval development stages (Salom et al., 1991, 1992). The identities of compounds used by SPB parasitoids to locate individual trees infested with susceptible host stages are currently unknown. Previous efforts in our laboratory to produce a synthetic attractant as effective at attracting SPB parasitoids as naturally infested materials have been unsuccessful (Dalusky et al., unpublished data). Synthetic SPB parasitoid attractants are potentially important tools for furthering current knowledge of bark beetle-parasitoid interactions. Practically, these attractants might be employed to monitor parasitoid populations levels, to evaluate individual parasitoid species as potential biocontrol agents (Mills and Kriiger, 1988), or, possibly, to enhance levels of parasitism within active infestations (Lewis and Martin, 1990). The purpose of our study was to isolate and identify those compounds arising from infested trees that likely serve as host-tree location cues to foraging parasitoids. Three sets of experiments were conducted in pursuit of this goal. In the first, we identified

839

PARASITO1D RESPONSES

beetle-infested materials in the field that were likely to contain quantities of parasitoid-attractive volatile compounds. In the second, we used a water distillation procedure to produce a parasitoid-attractive crude extract from such beetleinfested materials. In the third, the chemical composition of the crude extract was analyzed using GC-MS, and tests were performed to determine whether the biologically active components of the extract were primarily oxygenated or hydrocarbon compounds.

METHODS AND MATERIALS

Sticky traps baited with SPB-infested materials or their extracts were used to assay parasitoid attraction in the field. Insects were trapped on Stikem Specialcoated hardware cloth cylinders (31 cm tall, 18 cm diam.) arranged concentrically around either infested bolts or trap bodies containing infested bark or extracts. Trap bodies consisted of cylinders of heavy-gauge, 1-cm-mesh black plastic netting (27 cm tall, 13 cm diam.) lined on their interior surfaces with four layers of cotton cheesecloth held in place by duct tape. The rationale behind this design was to provide a bole-like visual stimulus to parasitoids in the form of a dark, vertical shape and produce a more even diffusion of bait volatiles from the surface of the traps. Waxed, corrugated cardboard disks (13 cm diam.) were inserted into both ends of the trap bodies flush with the top and bottom of each cylinder. These disks served to maintain the trap shape, contain the enclosed bait, and attach the trap body to a pipe standard, which was inserted through holes cut into the center of each disk. Bolts or baited trap bodies were placed on 1.8-m-tall pipe standards within areas of an SPB infestation that contained beetle activity. Treatments were assigned at random to trap positions within blocks in a randomized complete block design. At any one time, two to four blocks were set up within a given SPB infestation, and traps were spaced a minimum of 5 m apart both within and between blocks. The total trapping time for a single replicate was 4 hr, and during that time traps were rotated within each block regularly so that each treatment spent an equal length of time at every position within the block. Trapping was normally performed during daylight hours on days with fair weather. Following trapping, sticky screens were removed from the trap standards, separated by sheets of wax paper, and returned to the laboratory. Parasitoids were removed from the screens by hand using a dissecting probe and cleaned in hexane before being stored in 70% ethanol for later identification and sexing. Experiment 1. Trapping assays were conducted in an active SPB infestation located in a mature, mixed stand of loblolly and shortleaf pine at Ft. Benning, Georgia, on June 7-15, 1995. Four candidate attractive sources were assayed in 12 complete blocks: (1) "late brood SPB bark" consisted of a trap body

840


filled with pieces of bark (total approximate surface area was 0.2 m2) taken from trees with both fourth instar SPB larvae mining in the corky bark and numerous parasitoids on the bark surface, presumably searching for oviposition sites; (2) "early brood SPB bark" was a trap body filled with bark taken from trees that contained predominantly early larval instar or egg stage brood SPB and had few or no parasitoids searching the bark surface; (3) "infested bolt" consisted of a ~25-cm-long, ~ 12-cm-diam. bolt cut from the mid- or lower bole of a pine that met the same criteria as the trees used in treatment 1; and (4) "blank" was an empty trap body presented as a check. "Bark" as used in treatments 1 and 2 and in the remainder of the experiments consisted of the outer corky bark with the phloem, cambium, and cork cambium tissues still attached. Bark was collected at a height of 1-3 m on the bole of each tree after loose outer bark flakes had been shaved off with a machete. Pieces of bark were removed by hand with a knife and placed into a trap body within 1 hour. Care was taken to prevent parasitoid adults from entering the trap bodies. The age of the beetle brood larvae was gauged in the field by the appearance of the larval galleries in the bark phloem: larvae were considered "early" only if they had not yet begun producing visible larval chambers in the phloem as described by Bridges et al. (1984). Bark used in each trap was collected from a different infested tree, and bolt baits used on consecutive trapping days were cut from a single tree. Experiment 2. Trapping was carried out on July 14-21, 1995, in an active SPB infestation in a mature, mixed loblolly and shortleaf pine stand located in the Oconee National Forest, Georgia. Four trap treatments were compared in eight complete blocks: (1) "bark extract" consisted of a trap body baited with the water distillate of late brood SPB-infested bark; (2) "synthetic bait" was a trap body baited with a synthetic parasitoid bait formulated using commercially available compounds; (3) "infested bark" consisted of a trap body filled with pieces of infested bark as distilled in treatment 1 above; and (4) "blank" was an unbaited trap body presented as a check. The bark extract used in treatment 1 above was obtained from pieces of loblolly pine bark as described in experiment 1, treatment 1, that were collected in the field, placed into resealable freezer bags, and kept frozen (-80°C) until extracted. Extractions were carried out using an apparatus designed for the distillation of small amounts of essential oils having a lower density than water (Guenther, 1948, pp. 316-319). In our distillations, a 4-liter Erlenmeyer flask heated on an electric hot plate/stirrer was used for boiling the extractable material, and an Allihn condenser was used to recondense volatilized oils and steam (Figure 1). Frozen bark (500 g) was ground for 30 sec at low speed in a Waring blender with deionized water (2-2.5 liters) and placed into the Erlenmeyer flask. The contents of the flask were heated to a steady boil, and the distillation was carried out for 3 hr after extract first began to accumulate in the oil trap. A

PARASITOID RESPONSES

841

FIG. 1. Diagram of water distillation apparatus used to extract SPB-infested bark (after Guenther, 1948). Steam and oil volatilized from the flask of ground bark and boiling water are trapped in the water-cooled Allihn condenser, and the resulting condensate drips into the oil trap. The oil trap separates recondensed essential oils (they form a distinct, floating layer in the left-hand side of the oil trap) from recondensed steam while permitting the latter to reflux back into the boiling flask. The stopcock and drain allow accumulated oil extract to be removed from the still while it is in operation.

842


magnetic stirring bar was used to circulate the heated material in the flask. The extracted oil was collected into screw-top vials and stored in a freezer at —30°C. The synthetic bait consisted of 18 compounds identified by means of GCMS analysis of headspace volatiles of the parasitoid-attractive pine bark used in experiment 1, treatment 1, and the relative proportions of compounds employed in the blend were based on those found in the headspace samples. For these analyses, bark was collected in the field, placed into 1-qt heavy-gauge, resealable freezer bags, and transported and stored at —80°C until use. Prior to analysis the bags were allowed to equilibrate at 32°C, and then a 1-ml sample of air from within the bags was injected with a gas-tight syringe directly onto the GC-MS. Chirality of the identified compounds was not determined; however, racemic blends of compounds were incorporated in the bait whenever practical. The percentage composition of the synthetic bait by weight was as follows (all compounds were obtained from Aldrich Chemical Co., Milwaukee, Wisconsin, unless otherwise noted): 56% (±)-a-pinene, 1.8% (±)-camphene, 9.2% (-)-/3-pinene, 1.5% myrcene, 4.6% (±)-limonene, 0.93% p-cymene, 1.8% terpinolene, 0.83% (-)-fenchone (source: Fluka Chemical Corp., Ronkonkoma, New York), 2.3% camphor (source: Fluka), 0.19% pinocamphone (source: see text), 0.32% isopinocamphone (source: see text), 5.5% terpinen4-ol, 6.2% 4-allylanisole, 3.3% a-terpineol, 1.2% (-)-verbenone (source: Bedoukian Research Inc., Danbury, Connecticut), 0.14% (-)-borneol (source: Fluka), 1.3% ethanol (source: Aaper Alcohol, Shelbyville, Kentucky), and 1.3% acetone (source: J. T. Baker Inc., Phillipsburg, New Jersey). The purity of all compounds as measured by GC analysis was between 95% and 99%, with the exception of myrcene and camphene, which were 85% and 81% pure, respectively. Pino/isopinocamphone was obtained by the oxidation of isopinocampheol (source: Aldrich) with CrO3/H2SO4 (Jones reagent), and was added to the mixture in a 1:3 decane solution. For treatments 1 and 2, liquid bait (3 ml) was soaked into an exposed cellulose sponge (8.5 X 5.0 x 0.2 cm), which was suspended in the interior of a trap body. Sponge baits were prepared in the field immediately before the trapping was begun. The release rate of bait from the sponges was determined gravimetrically to be approximately 0.5 g/hr. Trap bodies not containing bark were lined with three paper towels moistened with deionized water to approximate the moisture released by freshly peeled bark. Experiment 3. The bark distillate of experiment 2 was fractionated using the procedures of Chamblee et al. (1991) as a general guide. Open column chromatographic separations were performed on a 18-cm x 1.6-cm-ID column dry packed with silica gel (30.9 g, Merck, grade 10180, 70-230 mesh, Aldrich). The column was preconditioned by rinsing sequentially with ethyl ether (100 ml), methylene chloride (100 ml), and pentane (100 ml). Following conditioning, bark extract (3 ml) collected as described above for experiment 2, treatment


843

1, was absorbed onto the column. The hydrocarbon fraction was eluted from the column with redistilled pentane (120 ml), and the oxygenated components were then eluted with redistilled methanol (130 ml). Additional methanol (30 ml) passed through the column as a check showed no traces of compound. The two fractions were concentrated to a convenient volume (10 ml) by using a Snyder distillation column. GC-MS analysis of the collected distillate revealed that less than 0.1% of either the hydrocarbon or oxygenated fractions were lost during the process of concentration. Hence 10 ml of each concentrated fraction was assumed to contain the same quantity of bark oil components as 3 ml of undiluted bark extract. Like concentrated fractions were combined to produce two stock extract fractions for use in all replicates of experiment 3. The composition of the two fractions as well as the unfractionated bark extract were determined by GC-MS analysis (see below). Trapping was carried out August 19-September 9, 1995, in the same SPB infestation utilized in experiment 2. For each treatment, baits were applied to cellulose sponges (10 X 10 X 0.4 cm) and suspended using wire inside a single trap body. Parasitoid responses to five trap treatments were compared within 10 complete blocks: (1) "bark extract" consisted of the water distillate (3 ml) of late brood SPB-infested bark produced and released in the same manner used above for experiment 2, treatment 1; (2) "hydrocarbon fraction" was the concentrated pentane eluted fraction (10 ml) of the bark distillate and redistilled methanol (10 ml) released from separate sponges; (3) "oxygenated fraction" was the concentrated methanol eluted fraction (10 ml) of the bark distillate and redistilled pentane (10 ml) released from separate sponges; (4) "both fractions" consisted of the hydrocarbon fraction (10 ml) and the oxygenated fraction (10 ml) released from separate sponges; and (5) "blank" consisted of redistilled pentane (10 ml) and methanol (10 ml) released from separate sponges. Statistical Analysis. Trap catch data for each species and sex as well as for species and sexes combined were normalized using the formula log,0(X +1) and subjected to a two-way analysis of variance using block and treatment as variables and the Student-Newman-Keuls (SNK) procedure for multiple pairwise comparisons (SigmaStat, Jandel Scientific, 1992-1994). All tests were performed with a = 0.05. Chemical Analysis. Bait compositions were analyzed on a Hewlett-Packard GCD G1800A GC-MS system equipped with a HP-FFAP fused-silica capillary column (Hewlett-Packard Corp., Palo Alto, California) (50 m X 0.2 mm ID; 0.33-mm film thickness). Bark extracts and concentrated fractions were diluted 1/50 in redistilled pentane prior to injection (1 fil). The temperature program was 32°C for 1 min, then 15°C/min to 75°C, then 6°C/min to 220°C for 12 min (use of two temperature ramps improved peak spacing and resolution); the flow rate of the helium carrier gas was 0.7 ml/min. Compounds were identified by their mass spectra and by retention time matches with known standards.

844


Percentage composition was calculated using peak integration areas of the total ion chromatograms; raw peak area percentages were corrected using response factors calculated by injecting known quantities of standards. For compounds with unknown response factors (ones for which no standard was available), response factors were assigned based on the compound's structural similarity to compounds with known response factors. RESULTS AND DISCUSSION

Ten species of recognized SPB parasitoids (Berisford, 1980) were caught on test traps during the course of the three experiments (Table 1). At both trapping sites the composition of species responding to traps baited with infested materials or their extracts was, overall, quite similar, and more than 80% of the parasitoids caught were Roptrocerus xylophagorum (Ratzeburg) and Spathius pallidus (Ashmead). However, these two species represented only 39% of the total trap catch on blank traps, and, in this regard, species composition of parasitoids recovered from blank traps differed significantly from that recovered from the infested material/extract baited ones (x2 test, P < 0.0001). These data

TABLE 1 . SPB PARASITOIDS CAUGHT IN FIELD-TRAPPING EXPERIMENTS Experiment 1 Parasitoid species

Braconidae Atanycolus sp. Cenocoelius sp. Coeloides pissodis (Ashmead) Dendrosoter sulcatus Muesbeck Spathius pallidus (Ashmead) Eupelmidae Eupelmus sp. Eurytomidae Eurytoma spp.

Total % of trapped catch

1 3 29

36 459

M/F

0.1 all F 0.3 all F 2.7 1:6.3 3.3 all F 42.7 1:458

Catch on blank

0 0 6 4 9

20

1.9

all F

3

7

0.7

allF

1

Pteromalidae Dinotiscus dendroctoni (Ashmead) Heydenia unica Cook & Davis Roptrocerus xylophagorum (Ratzeburg) Total

12 42 466 1075

1.1 a l l F 3.9 1:41 43.3 1:76.7 100.0 1:88.6

2 6 7 38


845

suggest that the superabundance of S. pallidus and R. xylophagorum relative to the other species caught on baited traps resulted from an overall stronger response to baits by these two species rather than a disproportion of their numbers in the local populations or an inherent selectivity in the basic trap design. Very few males of any parasitoid species were caught except for Heydenia unica Cook and Davis, and, in general, the sex ratio was strongly female-biased. Male parasitoid responses to treatment traps were not significantly different in any of the three experiments either when the parasitoid species were considered singly or grouped together. In previous studies, very few male parasitoids arrived on SPB-attacked trees when susceptible hosts were present (Camors and Payne, 1973; Dixon and Payne, 1980). Salom et al. (1991) suggested that male parasitoids of bark beetles remain on the trees from which they emerge in order to mate with emerging females and do not generally search for other infested trees. Adult males do not parasitize hosts and therefore probably do not respond to odor cues associated with host insects in the same manner as females. Experiment 1. Infested Materials. Total SPB parasitoid catch was greater for traps baited with any of the host-infested materials than for the blank, and bark infested with either early or late SPB brood stages attracted significantly

Experiment 2 Total % of tapped catch

M/F

Catch on Total % of blank trapped catch

M/F

1 0 5 8 68

0.4 0.0 1.8 2.9

F N/A 1:4 1:3

55.7 1:30.8

0 0 0 0 6

24.3

8

2.3 allF

1

6

5

1.5

1:4

0

0

0 9 1 17

3 46 143 280

0 0 5 7 191

10 38 79 343

0.0 N/A 0.0 N/A 1.5 allF 2.0 allF

2.9 all F 11.1 1:1.53 23.0 allF 100.0 1:15.6

Experiments 1-3 combined

Experiment 3

Catch on Total % o f blank trapped catch

1:67

1 N/A 1 1 1

2 3 39 51 718

2.1

all F

1

0.0

N/A

1.1 allF 16.4 1:1.09 51.1 1:142 100.0 1 : 10.4

M/F

allF allF 1:7.8 1:25.5 42.3 1:89.8

1 0 7 5 16

34

2.0 all F

5

0

12

0.7

1:12

1

0 4 2 11

25 126 688

1.5 allF 7.4 1:3.32

2 19 10 66

1698

0.1 0.2 2.3 3.0

Catch on blank

40.5 1:98.3 100.0 1:27.8

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more parasitoids than the infested bolt. The higher trap catch for bark-baited traps compared to bolt-baited traps was likely the result of the greater exposure of attractant-releasing surface area in the former. Total parasitoid catch was somewhat higher for traps baited with late brood SPB-infested bark than traps baited with earlier stages, but this difference was not significant. When the data were broken down by species and sex, only female R. xylophagorum and female S, pallidus showed a statistically significant response to traps baited with any of the SPB-infested materials (Figure 2). Females of these two species alone comprised 87% of the total trap catch on all baited (i.e., not blank) traps used in experiment 1. Although female 5. pallidus were attracted in significant numbers to both bark treatments, they showed a significant preference for bark containing late brood SPB as compared to bark infested with predominantly earlier developmental stages (Figure 2). Peak abundance of most parasitoids including S. pallidus has been shown to occur on SPB-infested trees when late-instar SPB larvae, the parasitoids' most frequently utilized host stage (Berisford, 1980), are present in large numbers (Camors and Payne, 1973; Dixon and Payne, 1980). However, previous studies have not established whether the cues that mediate this aggregation on suitable trees are arrestants or long- or short-range attractants. Our data suggest that 5. pallidus females are able to select materials infested with appropriate SPB host stages while in flight in that they showed a distinctly greater attraction to bark infested with the host stages that they are known to

FIG. 2. Mean numbers of female parasitoids caught in traps baited with SPB broodinfested materials. Within each species, means with the same letter are not significantly different at the P < 0.05 level, SNK test.


847

parasitize most frequently. This finding supports the hypothesis that S. pallidus aggregation on trees infested with susceptible host stages is mediated by an attractant that can be detected by these parasitoids at some distance (at least a few centimeters) away from its source. Female R. xylophagorum also responded in significant numbers to both bark baits; however, this species appeared to differ from S. pallidus in its treatment preferences (Figure 2). The proportions of 5. pallidus and R. xylophagorum responding to the two bark treatments differed significantly (x2 test; P < 0.0001), suggesting that each species was responding maximally to cues that differed quantitatively or qualitatively. Unlike S. pallidus, R. xylophagorum did not show a preference for bark containing late-instar SPB larvae, (Figure 2). This was a somewhat surprising result since both of these species are reported to parasitize the same host stages (Franklin, 1969; Moore, 1972), and one study has reported that peak abundance of these two species on infested trees occurs approximately at the same time, that is, when the predominant SPB life stages in infested trees are late larvae and pupae (Dixon and Payne, 1980). However, Camors and Payne (1973) reported that arrival of R. xylophagorum on SPBinfested trees preceded that of S. pallidus by approximately one week, and one study that examined the two sexes of R. xylophagorum separately indicated that peak arrival of females occurs when early-instar larvae are the predominant SPB life stage (Dix and Franklin, 1981). Although evidence suggests R. xylophagorum normally parasitizes the same brood stages as S. pallidus, we hypothesize that an earlier arrival on SPB-infested trees might offer some benefits to R. xylophagorum females. This species enters into bark beetle galleries and oviposits onto hosts while they are still in the phloem tissue (Berisford et al., 1970); in contrast, all other species of SPB parasitoids oviposit through the outer bark, which has been shown to act as a physical barrier to parasitization of beetle larvae that are deep within the bark tissues (Goyer and Finger, 1980; Gargiullo and Berisford, 1981) or that have not yet tunneled into the outer bark layers to pupate (Payne, 1980). Hence, R. xylophagorum have physical access to SPB hosts at an effectively earlier time than the other parasitoid species, and response to host-associated semiochemicals or other cues that are specific to this earlier time period might allow this parasitoid to take greater advantage of its unique access to hosts. Based on these results, pine bark infested with larval SPB brood appeared to be a good candidate material for extraction of parasitoid-attractive volatiles since: (1) it was attractive when separated from the rest of the tree bole to at least two species of parasitoids, implying that volatile attractants were present in the infested bark tissues, and (2) bark could be readily collected, transported, and manipulated in the laboratory. Experiment 2: Bark Extract and Synthetic Bait. Only traps baited with either the bark extract or the infested bark trapped significantly more parasitoids than

848


the blank, and there was no significant difference in the total number of parasitoids caught by these two attractive trap baits. As with experiment 1, only female R. xylophagorum and female S. pallldus showed a statistically significant response to any of the baits when the trap catch data were broken down by parasitoid species and sex (Figure 3). The responses of these two species to the different treatments were essentially identical, and together they represented the majority (79%) of parasitoids caught on baited traps during the experiment. The data for experiment 2 demonstrate that a water-distilled extract of SPBinfested bark is an effective SPB parasitoid attractant and is capable of luring the same composition of parasitoid species as the unextracted, infested bark. These findings support the hypothesis that the attractiveness of trees and bark infested with susceptible host stages is an olfactory-based phenomenon and suggest that the water distillation process can isolate, at least to some degree, semiochemicals mediating parasitoid host-tree location. It is difficult to estimate whether there was any change in activity of the extracted mixture caused by the extraction process itself, since the bark-baited traps and the extract-baited traps were likely releasing volatiles at very different rates. It should be noted, however, that the water distillation process used to produce the bark extract does not accumulate water-soluble compounds, and the absence of such compounds may have influenced the extract's biological activity. In addition, water distil-

FIG. 3. Mean numbers of female parasitoids caught in traps baited with a water-distilled extract of SPB brood-infested bark, a synthetic bait, and controls. Within each species, means with the same letter are not significantly different at the P < 0.05 level, SNK test.


849

lation is known to cause some chemical changes in extracted essential oils (Guenther, 1948). In contrast to the bark extract, our synthetic bait was not attractive to parasitoids either when species and sexes were considered individually or grouped together (Figure 3). This result was somewhat surprising since most of the major chemical constituents present in attractive bark extract were included in the synthetic mixture (Table 2), the relative proportions of these compounds were roughly similar in both baits, and both were released at the same rate. These observations suggest that no individual compound in the synthetic bait is responsible for the observed attraction of parasitoids to the crude extract of infested bark and that possibly: (1) critical constituents present in the bark extract were absent from the synthetic blend, (2) the relative proportions of components in the synthetic blend were not those required to trigger parasitoid attraction, or (3) the particular enantiomers of chiral compounds presented in the synthetic bait were not the biologically active ones. Experiment 3: Fractionated Bark Extract. Analysis of the products of the silica gel chromatography by GC-MS revealed nearly complete separation of the bark extract into hydrocarbon and oxygenated fractions. Some 4-allylanisole (9.3% of the total) eluted with the pentane fraction, but otherwise all identifiable compounds in the pentane fraction were hydrocarbons, while all those in the methanol fraction were oxygenated (Figure 4; Table 2). Among the five treatments tested, total parasitoid catch was greatest for traps baited with either the unfractionated bark extract or both the hydrocarbon and the oxygenated bark extract fractions simultaneously, and these treatments caught significantly greater numbers of parasitoids than traps baited with either the hydrocarbon or the oxygenated fractions alone. Traps baited with the oxygenated fraction alone were somewhat attractive, while the hydrocarbon fraction alone appeared to be unattractive to parasitoids. The fractionation process itself did not appear to substantially alter the attractiveness of the extracted components of the bark, since traps baited with both fractions caught a similar total number of parasitoids as traps baited with the original, unfractionated bark extract. When the trap catch data for experiment 3 were categorized by species and sex, only female R. xylophagorum and female S. pallidus showed a significant response to any of the baited treatments (Figure 5). Responses of female R. xylophagorum and female S. pallidus to the treatment baits were very similar, except that female S. pallidus were attracted in somewhat greater numbers to traps baited with both extract fractions than those baited with the unfractionated extract, while female R. xylophagorum found both treatments nearly equally attractive. This difference may be the result of disparities in the relative release rates of components from the whole extract baits when compared to the fractionated baits.

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SULLIVAN, BERISFORD, AND DALUSKY TABLE 2. CHEMICAL COMPOSITION OF BARK EXTRACT USED IN EXPERIMENT 3

Peak

Compound name

ID"

1 1 3 4 5 6 7 & 9 10 11

Tricyclene a-Pinene of-Fenchene Camphene 0-Pinene Myrcene a-Phellandrene 1,4-Cineol a-Terpinene Limonene /3-Phellandrene Eucalyptol •y-Terpinene p-Cymene Terpinolene Fenchone p,a-Dimethylstyrene Linalool Camphor Isopinocamphone Fenchyl Alcohol Bornyl Acetate Terpitien-4-ol Caryophyllene Myrtenal frans-Pinocarveol Isobomeol 4-Allylanisole a-Humulene a-Terpineol Borneol Verbenone Piperitone Carvone Myrtenol p-Cymen-8-ol m-Myrtanol Unidentified Total

MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS MS, RT MS, RT MS.RT MS, RT MS, RT MS MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT MS, RT

12

13 14 15 16

17 18 19 20 21 22 23 24

25 26 27 28 29 30 31

32 33 34

35 36 37

Type of compound''

Wt % in whole bark extract

HM HM HM HM HM HM HM OM

0.46 54.73 1.00 1.65 9.77 1.96 0.06 0.04 0.18 3.18 0.44 0.05 0.23 0.47 1.73 0.25 0.61 0.05 0.96 0.23 0.37 0.07 6.99 1.26 0.10 0.23 0.11 3.31 0.32 5.69 0.63 0.56 0.05 0.07 0.17 0.32 0.04 1.67 100.00

HM HM HM OM HM HM HM OM HM OM OM OM OM OM OM HS OM OM OM OM

HS OM OM OM&P OM OM OM&P OM OM

Present in synthetic baitc

C Y C Y Y Y N N N Y N N N

Y Y Y N N Y Y

N N Y N N N N

Y N Y Y Y N N N N N

°MS = identification by mass spectrometry; RT = retention time matching that of known standards. ''HM = hydrocarbon monoterpene; HS = hydrocarbon sesquiterpene; OM = oxygenated monoterpene; P = SPB pheromone. CY = included in formulation of synthetic bait; N = not present in synthetic bait; C = present in synthetic bait as a contaminant.


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FIG. 4. Total ion chromatogram (TIC) of water-distilled extract of SPB brood-infested loblolly pine bark (a) and TICs of fractions of this extract produced on a silica gel column: the pentane-eluted (hydrocarbon) fraction (b) and the methanol-eluted (oxygenated) fraction (c). The labeled compounds are listed in Table 2.

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FIG. 4. Continued.

FIG. 5. Mean numbers of female parasitoids caught in traps baited with components of a water-distilled bark extract fractionated using liquid chromatography. Within each species, means with the same letter are not significantly different at the P < 0.05 level, SNK test.

PARAS1TOID RESPONSES

853

It appears that no single compound was responsible for the observed parasitoid attraction to bark extract-baited traps and that compounds from both the hydrocarbon and oxygenated chemical classes were being used simultaneously during orientation by the two responding species of parasitoids. Electroantennogram studies on two other species of SPB parasitoids found that they possess receptors for a large number of host insect and tree-produced compoundsincluding both hydrocarbon and oxygenated monoterpenes (Salom et al., 1991, 1992). In foraging for hosts, the SPB parasitoids may be responding to a complex blend of odors associated with trees infested with susceptible stages of beetle brood, and presentation of the total natural blend of odors may be necessary to elicit maximal parasitoid attraction. The hydrocarbon fraction, which comprised 78% of the original bark extract, consisted largely of hydrocarbon terpenes normally found in fresh loblolly pine resin (Paine et al., 1987; Coyne and Keith, 1972). Although the hydrocarbon fraction failed to attract parasitoids on its own, when released with the oxygenated fraction it produced a fourfold increase in the number of parasitoids attracted by its counterpart; hence the hydrocarbon components appear to be acting as synergists in the whole bark extract. Seventy percent of the hydrocarbon fraction was the monoterpene a-pinene, which is a known synergist of the attractants of numerous conifer-infesting scolyttds and some of their natural enemies (Borden, 1982; Chdnier and Philogehe, 1989; Schroeder and Lindelow, 1989). In addition, a-pinene was found to be attractive to the SPB parasitoid Heydenia unica Cook and Davis when presented in the field using a sleeve olfactometer (Camors and Payne, 1972). The oxygenated fraction, which was significantly attractive to parasitoids on its own, represented merely 22% of the components in the unfractionated bark extract and consisted almost entirely of oxygenated monoterpenes (Table 2). It has been found that the overall levels of oxygenated monoterpenes arising from the boles of some bark-beetle-attacked conifers increases as an attack progresses (Leufven and Birgersson, 1987; Birgersson and Bergstrom, 1989), and the presence of large numbers of searching parasitoids on SPB-infested trees has been found to coincide with the occurrence of such elevated levels of oxygenated monoterpenes (Birgersson et al., 1992). Increased concentrations of airborne oxygenated monoterpenes may signal the presence of susceptible hosts to foraging parasitoids, and the observed parasitoid responsiveness to the oxygenated fraction is in agreement with this hypothesis. Two known SPB pheromones, myrtenol and verbenone (Payne, 1980), were present in this fraction in relatively small amounts (Table 2) and may have played a role in the observed attraction. Further experiments will be necessary to identify the precise blend of chemical constituents in the crude bark extract that are responsible for mediating SPB parasitoid host-tree location. The bark used to produce the attractive extract

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contained biomass from bark beetle brood, associated arthropods, symbiotic microbes, as well as the host-tree; hence the attractive compounds may have their origin in any one or several of these biological sources. Another important goal for future research will be to define the biological and chemical events that trigger the release of critical olfactory cues from bark-beetle-infested trees and are responsible for the characteristic timing of parasitoid arrival. Acknowledgments—The authors wish to thank the following individuals for their assistance with this work: Nick LeCroy, Trich Jackson, Joe Petty, Richard Garland, Jay Smith, and Susan Kring provided general technical assistance. Dr. Karl Espelie, Dr. Goran Birgersson, and Dr. Gary DeBarr reviewed the manuscript prior to submission, and Dr. Eric Grissell, Dr. Paul Marsh, and Dr. Michael Schauff of the Systematic Entomology Laboratory, USDA, and Dr. Mike Sharkey provided identifications for trapped parasitoids. Bob Fowler, Bob Griffith (USDA Forest Service, Oconee National Forest), and Bob Larimore (DNR, Ft. Benning Military Reservation) located field sites for the research, and Jeff Gillman advised us on water distillation procedures. Special thanks to the USDA Forest Service, Oconee National Forest, and the Ft. Benning Department of Natural Resources for their cooperation in providing us with field sites and other resources. This research was funded by USDA SEA grant 90-37250-5267.

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