~YSIOLOGIG~L AND CHEMICAL E COLOG Y Attraction of the Bark Beetle Parasitoid Rop~rocews xylophugorum (Hymenoptera: Pteromalidae) to Host-Associated Olfactory Cues
BRIAN T. SULLIVAN,’ EVA M. PETTERSSON,’ KATJA C. SELTMANN, AND C. WAYNE BERISFORD3
Environ. Entomol. 29(6): 1136-1151 (2066) ABSTRACT Studies were conducted to identify host location cues used by Roptrocerus xyloph- agorum (Batzeburg), a larval/pupal parasitoid of bark beetles. In Y-tube olfactometer bioassays, female rylophagorum were attracted to infested bark (i.e., phloem, cambium, and outer corky bark tissues) removed from bolts of loblolly pine, Pinus tueda L., colonized by the late instar larvae and pupae of the bark beetle Zps grundicollis Eichhoff (Coleoptera: Scolytidae). In contrast, bark taken from recently cut, uninfested bolts interrupted attraction to infested bark when these were presented together. Larval and pupal hosts isolated from infested bark were not attractive to parasitoids, whereas frass removed from the larval mines in infested bark was highly attractive. Bark from which hosts or both hosts and host frass were removed remained highly attractive. Bark sandwiches (fresh bark with the exposed surface pressed to glass microscope slides) infested with either third-instar or adult female 1. gt-an&x& were attractive to female parasitoids, whereas bark sandwiches with only mechanical damage to the phloem tissue were unattractive. A steam distillate of bark infested with host larvae was attractive to female R xyZophagorum, whereas a distillate of fresh pine resin was not attractive. Volatiles from the experimental baits were collected on Porapak Q and analyzed by coupled gas chromatography-mass spectrometry. Several compounds were identified that distin- guished baits with biological activity. These data show the importance of the complete host/plant complex for attraction of R. rylophagonrm to its host’s habitat and suggest a possible role for particular odors from uninfested host plant tissue in directing foraging parasitoids away from locations with few or no hosts.
KEY WORDS Roptrucetw rylophagorum, Ips grandiwl2is, Scolytidae, parasitoid host location, tritrophic interactions, semiochemicals
NUE*IEROUS STUDIES OF parasitoid-host interactions have and Tomicus piniperda (L.), and the southern pine demonstrated the role of semiochemicals in mediating beetle, Dendroctonusfiontul Zimmermann, the Dou- host and host habitat location by foraging female para- glas-fir beetle, D. pseudotsugae Hopkins, and the west- sitoids (Lewis et al. 1976; Vinson 1981,1984; Tumlin- em pine beetle, D. breoicomis LeConte (Bushing 1965, son et al. 1992; Godfray 1994; Vet et al. 1990). These Mills 1983). This parasitoid was established in Austra- semiochemical cues may arise from the host itself lia in the early 1980s as a potential biological control (Rice 1969, Mitchell and Man 1970, Stemlicht 1973)) agent for the introduced 1. grandicollis (Berisford and its products (Weseloh 1977, Cloutier and Bauduin Dahlsten 1989). R. xylophagam is among the most 1990, Ngi-Song and Oversholt 1997)) its food (Martin abundant parasitoid species found in association with et al. 1990, Ngi-Song et al. 1996, Takacs et al. 1997, bark beetle infestations (Berisford et al. 1971, Moser DeMoraes and Mescher 1999), or organisms found in 1971, Stephen and Dahlsten 1976, Goyer and Finger close association with the host (Madden 1968, Greany 1980, Ohmart and Voigt 1982, Langor 1991, Gara et et al. 1977, Dicke 1988, Thibout et al. 1993). al.1995). Roptrocems xylophugorum (Ratzeburg) is a larval/ Roptrocerus xybphugorum females parasitize hosts pupal idiobiont parasitoid of bark beetles (Coleoptera: concealed in bark by entering the galleries excavated Scolytidae) with an apparently holarctic distribution by adult bark beetles in the phloem tissue and drilling (Mills 1983, Samson 1984, Espelie et al. 1996). Its host through the gallery walls with their ovipositors (Dix range encompasses well over a dozen economically and Franklin 1981, Samson 1984). This parasitoid ap- important bark beetle species in Europe and North parently uses semiochemical cues to locate suscepti- America including the beetles Ips typogruph2c~ (L.) ble hosts. Female R. xylophugorum in the field are strongly attracted to host-infested bolts and bark as 1 Southern Research Station, USDA Forest Service, 2500 Shreve- well as distillates of host-infested bark; males show no port Highway, Pineville, LA 71360. such attraction (Sullivan 1997, Sullivan et al. 1997). In a Department of Chemical Ecology, Giiteborg University, Box 461, addition, there is evidence that semiochemicals play a SE-405 3.0 Gliteborg, Sweden. 3 Department of Entomology, University of Georgia, Athens, GA role in the location of, and discrimination among, 30602. particular host species or host life stages by this para-
0046-225X/00/1138-1151$02.00/0 0 2000 Entomological Society of America December 2000 SULLNAN ET AL.: HOST LOCATION BY xybphag~m 1139 sitoid. R. xybphagorum arrive on bark beetle colo- plied with charcoal-filtered and humidified air (-SO- nized trees and bolts in greatest numbers when sus- 70% BH). Airflow through each arm of the Y-tube was ceptible host life stages (i.e., late instar larvae and maintained at 4 cm/s (30 mllmin) by the positive pupae) are present (Berisford and Franklin 1969, pressure of an electric pump. Parasitoids were pre- Camors and Payne 1973, Stephen and Dahlsten 1976, vented from passing out of either arm of the Y-tube by Dixon and Payne 1979, Dix and Franklin 1981, Ohmart a screen barrier made of a disk of white cotton cloth and Voigt 1982). In choice experiments, R. xybphugo- ( 120 ct) held in place over the openings of each Y-tube rum were attracted significantly more to bolts infested arm by a Teflon O-ring. During testing, the bioassay with the same bark beetle species on which they had room was 26 rt 1°C and the only light source was a been reared than to bolts infested with an alternative, 15-W, red-filtered incandescent light bulb centered acceptable host species (Kudon and Berisford 1980). over the stem of the Y-tube. Although R. xylophago- The purpose of this study was to identify the bio- rum is a diurnal insect (Dix and Franklin 1981)) use of logical sources of the host and host habitat location the red-filtered light reduced the confounding effects cues of R. xybphugonrm and make an initial assess- of insect orientation to visual stimuli during bioassay ment of variables that might affect the production of trials. Trials were generally run between the sixth and cues and thus the efficiency of host finding by this twelfth hour of photophase. parasitoid. This study was undertaken as part of a Individual parasitoids released into the olfactome- larger effort to characterize and synthesize the host ter stem were recorded as choosing a given arm of the attractants for the parasitoids of the southern pine Y-tube when they spent >I5 s beyond a line posi- beetle, Dendroctonusfiontalis Zimmermann (Sullivan tioned 3 cm from the Y-intersection. Parasitoids that et al. 1997). failed to choose an arm in 5 min were recorded as nonresponders. The assignment of odor sources to Materials and Methods each arm of the olfactometer was reversed after every trial to eliminate directional bias. Parasitoids were not Insect Cultures. Adult R. xybphag~m were ob- reused, and Y-tubes were replaced with clean ones tained from a laboratory colony maintained at the after each trial, Bark extract and resin extract baits University of Georgia on 1. grundicollis-infested bolts were replaced after each trial but all other baits (i.e., of loblolly pine, Pinzrs taeda L. (Sullivan et al. 1999). bark, frass, and host insects) were exchanged with Adult parasitoids were collected daily as they emerged fresh material after the completion of each experi- from host-infested bolts held inside a saran-screen mental block (two replicates of each bait combination cage. These newly emerged parasitoids were housed within a given experiment). Between test days, Y- in mixed-sex groups in cotton-stoppered, 250 ml Er- tubes and all other olfactometer components poten- lenmeyer flasks packed loosely with Kimwipe (Kim- tially exposed to bait volatiles were washed with de- berly-Clark, Roswell, GA) and provisioned with tergent, rinsed with ethanol, and dried in an oven at honey, water, and a commercial diet (Eliminade, En- 120°C for at least I h. topath, Easton, PA.). Within 1 d after parasitoid col- Bioassay Experiments. R. xylophgorum females lection, the flasks were placed into a cold-temperature were subjected to 20 binary choice tests (Table 1). incubator at 8 + 1°C and aphotoperiod of 1410 (L:D) These tests were grouped into eight blocked experi- h. Flasks were removed from the incubator daily and ments consisting of up to four different such tests allowed to warm to room temperature for -30 min to performed in equal numbers of trials on the same days permit parasitoids to feed, take water, and mate. using the same pool of test subjects. This was done to One day before bioassays, 4- to 6-d-old, presumably eliminate the potentially complicating effects of be- mated female parasitoids were exposed to their host’s tween-day variation in parasitoid responsiveness microhabitat in a Plexiglas chamber (30 by 30 by 30 when comparing the results of component tests in an cm) enclosing pieces of bark infested with 1. grundi- experiment. On each day of trials, the tests within an collis larvae and pupae. Only those parasitoids that experiment group were performed consecutively in antennated larval frass or galleries and were arrested blocks of two to four replicates each, and the order of on the bark for >20 s were used in tests. This pre- the tests was randomized. conditioning was performed to improve overall para- Znfested and Uninfested Bark. In experiments 1-3, sitoid response rates to host-associated cues (Parra et parasitoids were assayed for their response to bark al. 1996, Riise et al. 1998). After exposure to the host/ (i.e., phloem, cambium, and outer corky bark tissues) plant complex, parasitoids were transferred to culture excised either from an uninfested pine bolt or from a flasks prepared as above and maintained at 26 -+- 1°C bolt infested with susceptible hosts. The uninfested and a photoperiod of 14:lO (L:D) h until bioassay. bark was excised from a bolt cut from a healthy loblolly Olfactometer Design and Methodology. The pine felled in the previous 7 d and stored at -4°C anemotaxis of walking R. xybphugorum in response to before use. The infested bark was removed from a various odor sources was tested in a Y-tube olfactom- loblolly pine log colonized 2-3 wk earlier in the lab- eter similar in design and operation to that of Stein- oratory by I. grandiwllis adults (Sullivan et al. 1999) berg et al. (1992). Each arm of a Pyrex glass Y-tube (4 and contained predominantly pupae and second and mm id.; stem 40 mm; arms 50 mm at a 135O angle to the third instars. Bark for either treatment was excised stem) was attached with Teflon tubing to a sealed glass ~30 min before the experiment and had its origins in odor source chamber (internal volume 30 ml) sup- bolts (80 2 20 cm by 15 + 5 cm diameter) from at least 1140 E NVIRONMEN T A L ENTOMOLOGY Vol. 29, no. 6
Table 1. Summarized details of individual treatment pat* in Y-tube oUaetometer choice bioassaya of female R. zykqhgonun
Parasitoids Test Ar”Il Ar”l2 tested (n) 1.1 36 Cleanair Excised bark (21 cm”) from an uninfested pine bolt
1.2 36 Excised bark (21 cm’) from a pine bolt infested 2-3 Clean air wk with I. gnmdioollis (late larval and pupal brood present)
1.3 36 Excised bark (21 cma) from a pine bolt infested 2-3 Excised bark (21 cm”) from a” uninfested pine bolt wk with 1. grandiwllis (late larval and pupal brood present) 2 48 Excised bark (7 cm”) from a pine bolt infested 2-3 Excised bark (7 cm’) from a pine bolt infested 2-3 wk wk with I. grot~&wZZis (late larval and pupal brood with 1. grondicoZZis (late larval and pupal brood present) present) and excised bark (7 cm”) from a” uninfested loblolly pine bolt
3 42 Excised bark (7 cma) from a pine bolt infested 2-3 Excised bark (14 cm’) from a pine bolt infested 2-3 wk with 1. gmndicoZlis (late larval and pupal brood wk with I. grandicuZZis (late larval and pupal brood present) present)
4.1 32 Larval and pupal brood removed from bark (21 cm”) Clean air from a pine bolt infested 2-3 wk with 1. grcmd~llis
4.2 32 Clean air Bark (21 cm”) from a pine bolt infested 2-3 wk with 1. gra”dicoZZis, with larval and pupal brood removed 4.3 32 Larval and pupal brood removed from bark (21 cma) Bark (21 cma) from a pine bolt infested with 2-3 wk I. from a pine bolt infested 2-3 wk with I. grandicollis grandicollis, with larval and pupal brood removed
5.1 30 Frass removed from larval mines in bark (21 cm’) Clean air from a pine bolt infested 2-3 wk with 1. gnmdiwZZis
5.2 30 Clean air Bark (21 cm’) from a pine bolt infested 2-3 wk with I. grzm&coZZis, with brood and frass removed
5.3 30 Frass removed from larval mines in bark (21 cm%) Bark (21 cma) from a pine bolt infested 2-3 wk with I. from a pine bolt infested 2-3 wk with 1. grmulimllip gmndicollis, with brood and frass removed
6.1 32 Bark sandwich (21 cm”) artificially infested 3 d Clean air previously with 10 I. gmndtiZZis third-instar larvae 6.2 32 Bark sandwich (21 cm”) art&ially infested 3 d Excised bark (21cma) from a pine bolt infested 2-3 wk previously with 10 I. grcrn&coZZis third-instar larvae with 1. grandicollis (late larval and pupal brood present)
7.1 30 Bark sandwich (21 cm”) artificially infested 3 d Clean air previously with six I. gmndiwZ2~ adult females (insects removed immediately before bioassay)
7.2 30 Bark sandich (21 cm”) artificially infested 3 d Bark sandwich (21 cm’) arti&ially infested 3 d previously with six 1. gra&wZZis adult females previously with 10 I. gnrn&coZZis third-instar larvae (insects removed immediately before bioassay) (insects removed immediately before biossay)
7.3 30 Bark sandwich (21 cm’) receiving mechanical Clean air damage 3 d previously and immediately before test 7.4 30 Bark sandwich (21 cm2) receiving mechanical Bark sandwich (21 cm’) artificially infested 3 d damage 3 d previously and immediately before test previously with 10 I. grm&wZZis third-instar larvae (insects removed immediately before bioassay)
8.1 34 Steam distillate of loblolly pine bark infested with Neat isopropyl myristate (10 4) applied to Whatman larval and pupal z. grandioollis, diluted I:50 (by #l flter paper volume) in isopropyl myristatc (10 ~1) and applied to Whatman #I filter paper
8.2 34 Neat isopropyl myristate (10 pl) applied to Whatman Steam distillate of fresh loblolly pine resin, diluted 150 #I filter paper (by volume) in isopropyl myristate (10 d) and applied to Whatman #l filter paper
8.3 34 Steam distillate of loblolly pine bark infested with Steam distillate of fresh loblolly pine resin, diluted I:50 larval and pupal 1. grmuliwZZti, diluted 1:50 (by (by volume) in isopropyl myristate (10 ~1) and volume) in isopropyl my&ate (10 ~1) and applied applied to Whatman # 1 filter paper to Whatman #l filter paper
Tests designated with the same integer composed a single experimental block. Tests within a single experimental block wore performed in equal numbers of replicates on the same days “sing the same pool of parasitoids. The order in which the tests were performed on a given day was chosen at random. December 2000 SULLIVAN m AL.: HOST LOCATION BY R. zylophagorutn 1141 three different trees cut from the same stand. Parasi- given the same number of niches (i.e., 10) as the toids were presented a choice among uninfested bark larvae-infested pieces as well as =I5 diagonal scalpel (surface area, 21 cm”) versus clean air (experiment cuts (2 cm long) in the phloem tissue, but no insects Ll), infested bark (21 cm2) versus clean air (exper- were placed into the niches. After these preparations iment 1.2), uninfested bark (21 cm2) versus infested of the bark pieces, a clean, aseptic glass microscope bark (21 cm2) (experiment I.3), infested bark (7 cm2) slide (5 by 7 cm) was sealed to the surface of the versus uninfested bark (7 cm”), and infested bark (7 exposed phloem with binder clips. All bark manipu- cm2) presented together (experiment 2)) and infested lations were carried out with sterile instruments in a bark (7 cm2) versus a doubled quantity of infested clean environment. The sealed bark sandwiches were bark (I4 cm’) (experiment 3). then incubated for 3 d at ~28°C and 80-902 BH. In experiment 4, Immediately before the trials for experiment 7, all live female parasitoids were assayed for their response to or dead insects were removed from the infested bark either infested bark (as used in experiments I-3) with sandwiches by dissection, and the mechanical dam- susceptible host stages removed or the live, excised age-only sandwiches were likewise dissected to inflict hosts from this bark. The mean +- SD numbers and life a similar amount of damage as occurred in dissecting stages of hosts isolated from each 21-cm2 bark piece the larvae-infested pieces. Female adult I. grandicollis, used per trial were 0.19 * 0.39 first-instar larvae, 2.44 2 rather than males, were chosen for these tests because 2.42 second-instar larvae, 8.00 ? 4.42 third-instar lar- females do not produce pheromones (Smith et al. vae, and 1.25 ? 1.25 pupae (11.88 + 5.00 total larvae 1993), and females will mine readily in bark in the and pupae). Bark was excised and hosts dissected ~30 absence of males. Some females laid eggs in the bark min before assay. Before the trials, the isolated hosts sandwiches, but none hatched before the bioassays. were mechanically cleaned with a camels-hair brush The mean t- SD numbers of living insects present in to remove all visible frass and plant material. Because the sandwiches at the end of the 3-d incubation were of the inherent difficulty of locating early-stage larvae as follows: larvae-infested sandwiches, 7.31 ‘-c 2.03 mining deeply in the bark, we were not successful in larvae, and 1.58 2 1.94 pupae; adult-infested sand- removing all hosts from the infested bark sections wiches, 5.07 + 2.46 adults (some adults abandoned the before the bioassays. However, additional, intensive bark sandwiches during the 3-d incubation). Parasi- dissection/examination of the bark following the bio- assays showed that we had removed >90% ofthe hosts toids were presented a choice among a larvae-dam- present within the tissue before testing. Parasitoids aged bark sandwich versus clean air (experiment 6.1), were presented a choice among isolated hosts versus a larvae-damaged bark sandwich versus a 3 by 7-cm clean air (experiment 4.1)) the bark from which these piece of larvae/pupae-infested bark from a bolt col- hosts were removed versus clean air (experiment 4.2)) onized 2-3 wk earlier by 1. grundicollis (experiment and isolated hosts versus their bark of origin (exper- 6.2), an adult-damaged bark sandwich versus clean air iment 4.3). (experiment 7.I), an adult-damaged bark sandwich Isolated Frass and Frass-Free Bark. In experiment 5, versus a larvae-damaged bark sandwich (experiment female parasitoids were assayed for their response to 7.2), an artificially damaged bark sandwich versus either infested bark (as used in experiments l-3) dis- clean air (experiment 7.3)) and an artificially damaged sected to remove host brood and host frass or the host bark sandwich versus a larvae-damaged bark sandwich larval frass collected from this bark. Clean, nylon- (experiment 7.4). bristled brushes were used to remove frass from the Distillates of Znfisted Bark or Fresh Resin. In exper- larval galleries of the bark pieces. The mean ? SD iment 8, female parasitoids were assayed for their fresh weight of frass used per trial was 104 ? 64 mg. response to either a steam distillate of bark infested Host brood and any adult frass present in the bark with I. grandicollis larvae or a steam distillate of fresh pieces were removed and discarded. Further dissec- pine resin. Fresh resin was collected from healthy tion/examination of the tested bark following the bio- loblolly pines by removing bark disks from tree trunks assays indicated that >95% of host brood and host frass with a cork borer and inserting screw-top vials into the (by weight) were removed before testing. Parasitoids openings. After -24 h, the vials were removed, were presented a choice among host larval frass versus capped, and stored at approximately -30°C before dis- clean air (experiment 5.1), the bark from which this tillation. The full, uncapped vials were placed into a frass was removed versus clean air (experiment 5.2), l,OOO-ml Erlenmeyer flask with 500 ml de-ionized wa- and larval frass versus its bark of origin (experiment ter and distilled according to Sullivan et al. (1997). 5.3). Tested baits consisted of these distillates diluted 1:50 Bark Sandwiches with Adult or Lm-val Hosts. In ex- in isopropyl myristate, an inert, nonvolatile oil. Blanks periments 6 and 7, female parasitoids were assayed for consisted of neat isopropyl my&state, and baits and their response to individual 3 by 7-cm pieces of fresh blanks were presented in the odor sample chambers as bark damaged by 10 third-instar I. grandicollis, by six a lo-/~1 aliquot applied to a 2 by Z-cm filter paper. adult female I. grandicollis, or by artificial manipula- Parasitoids were presented a choice among the distil- tion Bark pieces were infested by cutting evenly late of host-infested bark versus a blank (experiment spaced niches into the phloem tissue (-1 by 3 mm) 8.1), the resin distillate versus a blank (experiment and placing a single insect in each niche. The bark 8.2)) and the distillate of host-infested bark versus the pieces receiving the artificial damage treatment were resin distillate (experiment 8.3) 1142 ENVIRONMENTAL ENTOMOLOGY Vol. 29, no. 6
Collection and Analysis of Volatiles. Bark baits (as preference (Fig. 1, experiment 3). Although more used in experiments 1, 6, and 7) were placed in the parasitoids selected the olfactometer arm baited with olfactometer odor source chambers, and an electric isolated hosts over the blank arm, the numbers of vacuum pump drew charcoal-filtered air for -90 min insects responding to the arm baited with isolated at 30 ml/min through the chambers and into a column hosts was too low to indicate a significant level of (PTFE tubing; 4 cm by 3 mm i.d.) packed with Porapak attraction (Fig. 2, experiment 4.1). Infested bark re- Q (0.1 g; 50-80 mesh; Waters Associates, Milford, mained highly attractive to parasitoids after nearly all MA). Twelve different pieces of bark (originating hosts had been removed, and parasitoids strongly pre- from two trees, six pieces derived from each) of each ferred the odors of this material over odors of the treatment were sampled, and aerations were per- isolated hosts (Fig. 2, experiment 4.2, 4.3). Both host formed at 26 ? 1°C. Within 1 h after completion of the larval frass and the bark fragments from which this aerations, columns were extracted with 1.2 ml redis- frass had been removed were highly attractive, and tilled pentane, and the extracts were stored at approx- parasitoids exhibited no preference for either of these imately -80°C in glass vials with Teflon lined tops. materials (Fig. 2, experiment 5.1-5.3). Additionally, the distillates bioassayed in experiment Bark sandwiches infested 3 d with third-instar 1. 8 were diluted I/ 1,000 in redistilled pentane and both grandicollis larvae were attractive to R. rylophagorum these and the Porapak extracts were spiked with in- females, and, with 23 of 32 insects responding, this ternal standards (100 pg undecane and 20 pg ethyl parasitoid did not show a significant preference be- caprate). Samples of the above mentioned extracts tween these larvae-infested bark sandwiches and bark and dilutions (1 ~1) were analyzed on a Hewlett- excised from I. grundicollis-colonized bolts (Fig. 3, Packard GCD 1800A coupled gas chromatograph- experiment 6.1,6.2). Bark sandwiches infested 3 d with mass spectrometer (GC-MS) fitted with a Hewlett- female, adult 1. grandicollis were likewise attractive, Packard Innowax column (60 m by 0.25 mm i.d., 0.50 and with 27 of 30 insects responding, there was no pm film thickness). Compounds were identified by significant preference observed for bark sandwiches their mass spectra and by retention time matches infested with either adults or larvae (Fig. 3, experi- when identified standards were available. Compounds ment 7.1,7.2), Odors from bark sandwiches receiving identified by mass spectra alone are noted in Table 2. simulated host damage had no discernible activity Response curves were calculated for identified com- (Fig. 3, experiment 7.3, 7.4). pounds for which pure standards were available, and Roptrocerus xylophugorum females were attracted the volatile compoundsin the samples were quantified to the steam distillate of bark infested with larvae and by their ion abundances relative to those of the in- pupae of I. grandicolh (Fig. 4, experiment 8.1). In ternal standards. Compounds for which no pure stan- contrast, the distillate of fresh pine resin was not sig- dard was available were quantified using the standard nificantly attractive (Fig. 4, experiment 8.2,8.3). curve of a structurally similar compound. Analysis of Volatiles. Forty-eight compounds were Statistical Analysis. The null hypothesis that the isolated and quantified in the aerations of bark baits, parasitoids showed no preference for either olfactom- and 31 of these compounds quantitatively distin- eter arm (i.e., a hypothesized response proportion of guished the bait treatments paired in the Y-tube ol- 5050) was tested either with a G-test for goodness- factometer bioassays (Table 2). Aerations ofbark from of-fit (when >25 insects responded) or by calculating bolts infested with larval- and pupal-stage I. grandi- the cumulative probability of the observed propor- collis brood had significantly higher quantities of 21 tions plus “all worse” outcomes using a binomial prob- compounds than aerations of bark from uninfested ability table (Sokal and Rohlf 1995). Quantities of pine bolts. These included three hydrocarbon mono- volatiles present in the Porapak extracts were com- terpenes (a-fenchene, p-cymene, and a-p-dimethyl- pared among bark treatments with a one-way analysis styrene) , I2 oxygenated monoterpenes (fenchone, ofvariance (ANOVA) on ranks ((Y = 0.05) followed by linalool, camphor, isopinocamphone, pinocarvone, a Tukey test for all-pairwise comparisons (SPSS 1997). terpinen-4-01, myrtenal, trcn.s-pinocarveol, a-terpin- eol, bomeol, myrtenol, and p-cymen-8-01)) two “green leaf volatiles” (Visser et al. 1979, Bemays and Chap- Results man 1994) (I-hexanol and 3-hexen-l-01)) two nonter- Bioassay Experiments. Female R. xyZo$ugorum pene aromatics (styrene and benzaldehyde), and two were attracted to bark removed from bolts infested unknown compounds. With the exception of 3-hexen- with 1. grandicollis larvae and pupae, and strongly l-01 and one unidentified compound, all of these com- preferred the odors of infested bark over those of pounds were also present in higher concentrations in uninfested bark (Fig. 1, experiment 1.2, 1.3). Parasi- the steam distillate of infested bark than the steam toids were not attracted to the odors of fresh, unin- distillate of fresh resin, Ten compounds were present fested bark (Fig. 1, experiment 1.1). Parasitoids in significantly higher quantities in the aerations of the strongly preferred the odors of host-infested bark pre- bark sandwiches damaged by third-instar I. grandicoZ- sented alone over these odors presented in combina- Zis larvae than sandwiches damaged mechanically. tion with the odors of fresh bark (Fig. 1, experiment These included four oxygenated monoterpenes (pi- 2). Nearly twice as many parasitoids chose the olfac- nocamphone, camphor, isopinocamphone, and bome- tometer arm baited with a doubled surface area of 01)) one hydrocarbon sesquiterpene (o-cedrene), two host-infested bark, however this was not a significant nonterpene aromatics (styrene and benzaldehyde) , Table 2. Volatile compounds identihd from bait treatmen@ tested in Y-olfaetmneter bioaseays of R. .syrOphgonun
Mean + SE quantity (fig) volatiles collected from 21 cm2 pieces of loblolly pine bark during a 90-min aeration Bark sandwich damaged by: Compound ClaSS Bark excised from bolts: Steam distillates (w/PI) Artificial Infested 2-3 wk by 1. g?72nd~Zlis larvae I. gmndimzzis adults Uninfested Fresh pine Larvae- manipulation I. gnlndiwllis resin infested bark Tricyclene HM 0.30 0.13ab” 0.075 0.012a 0.48 0.34&l 0.67 0.08bc 1.2 0.k 3.6 2.1 a-Pinene HM 36. 12.0a 14. 2.0a 22. 5.0a 12x10’ 12.ob 18x10’ Mob 45x 10’ 31x10’ a-Fenchene HM 0.056 0.046a 0.003 O.OOZa 0.020 0.013a 0.006 0.006a 1.6 0.4b 0.050 2.1 Camphene HM 0.97 0.46ab 0.19 0.04a 1.4 l.lab 1.7 0.2bc 4.2 0.7c 9.6 9.2 HeZi”alb GLV 2.3 0.8b 2.8 0.8b 0.97 0.36b NDa 0.50 0.24ab ND 0.47 pinen. HM 15. 6.ab 2.7 1.3a 6.1 3.9ah 23. 7.oab 43. 21.ob 57. 15x10’ Myrcene HM 2.8 l.lab 0.57 0.16a 3.9 3.oab 4.7 o.4b 6.4 2.ob 18. 25. a-Phellandrene HM 0.009 0.005ab 0.092 (Continued) 1144 ENVIRONMENTAL ENWMOL~GY Vol. 29, no. 6 and three compounds of uncertain chemical classifi- cation. Aerations of the bark sandwiches damaged by female adult I. grundicollis had significantly lower quantities of a single hydrocarbon monoterpene (p- cymene) than aerations of bark sandwiches damaged by larvae. Aerations of bark sandwiches damaged by third-instar I. differed significantly from aerations of bark fragments from bolts infested with larval and pupal-stage 1. grundicollis. Relative to the latter, the former had significantly lower quantities of eight hydrocarbon monoterpenes (tricyclene, ol-pinene, cu-fenchene, camphene, cz-terpinene, li- monene, p-cymene, and terpinolene) and three oxy- genated monoterpenes (fenchone, terpinen-4-01, and a-terpineol) and had significantly higher quantities of a single unknown compound. Discussion Our data show that the cues that attract R. xybph agorum to its host’s habitat arise from the interaction between the host insect and the tissues of its host plant, whereas odors from isolated hosts or uninfested tree tissue apparently play a limited and secondary role in host habitat location. Attraction of parasitoids to pieces of fresh, excised bark was stimulated after 3 d of feeding by host larvae, whereas such barkincubated for the same period and receiving only artificial dam- age showed no evidence of being attractive (experi- ments 6.1, 7.3, 7.4). Hosts isolated from highly attrac- tive, infested bark were not appreciably attractive when presented alone, and parasitoids unfailingly chose the olfactometer arm baited with bark from which >90% of the hosts had been removed over the olfactometer arm baited with the hosts themselves (experiment 4). These findings reflect previous ob- servations that isolated host larvae and pupae will neither arrest nor induce oviposition behaviors in fe- male R. xylophugorum that come in direct physical contact with them (Samson 1984; B.T.S., unpublished data), Host location cues that originate directly from the bodies of host insects are probably quite rare in nature, likely because of strong natural selection against hosts producing such cues (Vet et al. 1991, Tumlinson et al. 1992, Vet and Dicke 1992, Godfray 1994). Conversely, infested bark remained highly at- tractive despite the removal of hosts (experiment 4.2)) and host-depleted bark did not differ significantly from bark with hosts left in place (experiment 1.2) in the proportion of parasitoids responding to the bark- baited olfactometer arm (P = 0.1, 2 test). Similar findings occurred in field studies in which female A. 3 I Idd& xylophegorum were attracted to bolts cut from pines infested by larval D.fiontulis despite the removal of all bark and hosts, and this parasitoid responded in similar numbers both to this host-free xylem tissue and to the infested bark from which it had been separated (Sul- livan 1997). Female FL xylophugorum did not discriminate be- tween host frass and the surrounding, host-damaged plant tissue in which this frass had been deposited, hence our data do not indicate one of these as the December 2000 SULLIVAN m AL.: HOST LOCATION BY R. xylophugorum 1145 CHOICES BY FEMALE R. XYLOPHAGORUM IN A Y-TUBE OLFACTOMETER No Response Exp. 1.1 Fresh, uninfested bark [0] 89.9% [32] Exp. 1.2 *** mlleanair(2] 41.7% [I51 Bark infested with host Exp. 1.3 * * * larvae & pupae [23] Fresh, uninfested bark [0] 38.1% [13] Exp.2 *** Bark infested with host Ba$ infested with host larvae & pupae, larvae & pupae [36] and fresh, uninfested bark [7] 10.4% [5] I I Exp. 3 Bark infested with host 2 x Bark infested with larvae & pupae [8] host larvae & pupae [I 51 45.2% [I91 I 1 1 I I I ! I 1 I I I I I I I I I 80 70 60 50 40 30 20 10 0 10 20 30 40 50 60 70 80 Percent Response Fig. 1. Responses of individual walking R. xyZ&zgmum females in a Y-tube olfactometer to pieces of bark excised from lobllolly pine bolts either uninfested or infested 2-3 wk earlier by 1. grandiwllis adults (brood predominantly in the late larval and pupal stages of development). Raw numbers of insects responding/not responding are shown in brackets. Bars denoted by asterisks indicate a significant preference for that treatment (***, P CHOICES BY FEMALE R. XYLOPHAGORUM IN A Y-TUBE OLFACTOMETER No Response I Exp. 4.1 Host larvae 8 pupae removed from infested bark [6] Clean air [I] 78.1% [25] Exp. 4.2 Clean air [0] Infested bark with *** hosts removed [24] 25.0% [8] Host larvae & pupae removed Exp. 4.3 from infested bark [0] Host larval frass removed Exp. 5.1 l * * from infested bark [23] Clean air [0] 23.3% [7] Clean air [0] Infested bark with hosts * l l Exp. 5.2 & frass removed [I 91 36.7% [II] Host larval frass removed Infested bark with hosts Exp. 5.3 from infested bark [12] & frass removed [I 0] 26.7% [8] I 90 80 70 60 50 40 30 20 10 0 10 20 30 40 50 60 70 80 90 Percent Response Fig. 2. Responses of individual walking R. xylophugorum females in a Y-tube olfactometer to 1. grmdicollis larvae and pupae, larval frass, or the loblolly pine bark from which these were removed. Raw numbers of insects responding/not responding are shown in brackets. Bars denoted by asterisks indicate a significant preference for that treatment (***, P CHOICES BY FEMALE R, XYLOPHAGORUM IN A Y-TUBE OLFACTOMETER 1 No Response Exp. 6.1 Bark sandwich with 40.6% [I 3] *** larvae mining 3 d [I91 Clean air [0] I I Exp. 6.2 Bark sandwich with Larvae-infested bark from 28.1% (91 larvae mining 3 d [8] “naturally” colonized bolt [15] I I Exp. 7.1 23.3% [7] Exp. 7.2 10.0% [3] Bark sandwich (3 d-old) with Exp. 7.3 mechanical damage 63.3% [I91 Bark sandwich (3 d-old) with Exp. 7.4 ii*** 26.7%[8] / mechanical damage only [0] , I I 90 80 70 60 50 40 30 20 10 0 IO 20 30 40 50 60 70 80 Percent Response Fig. 3. Responses of individual walking R. zyZq/argorum females in a Y-tube olfactometer to bark excised from an 2. gmndicollis-infested bolt or bark sandwiches damaged by either feeding I. grandiwlh larvae, feeding female adults, or artificial manipulation. Raw numbers of insects responding/not responding are shown in parentheses. Bars denoted by asterisks indicate a significant preference for that treatment (***, P CHOICES BY FEMALE R. XYLOPHAGORUM IN A Y-TUBE OLFACTOMETER No Response Exp. 8.1 *** *~solventblank~llDistillate of bark infested with 44.1% [I51 Exp. 8.2 Solvent blank [0] 91.2% [31] I * * Distillate of bark Infested with Distillate of fresh Exp. 8.3 i, grandcollrs larvae [I I] pine resin [I] 64.7% [22] I I I I I I I I I I 1 I 70 60 50 40 30 20 10 0 IO 20 30 40 Percent Response Fig. 4. Responses of individual walking R. q!ophagorum females in a Y-tube olfactometer to diluted steam distillates of either bark infested with I. grandicoZ2i.s larvae or fresh loblolly pine resin. Raw numbers of insects responding/not responding are shown in parentheses. Bars denoted by asterisks indicate a signiEcant preference for that treatment (**, P CO.01; ***, P Hunt, D.W.A., J. H. Borden, B. S. Lindgren, and G. Gries. Payne, T. L. 1989. Olfactory basis for insect enemies of al- 1989. The role of autoxidation of a-pinene in the pro- lied species, pp. 55-69. In D. L. Kulhavy and M. C. Miller duction of pheromones of Den&octonus ponderosae (Co- [ eds.], Potential for biological control of Dendroctontu leoptera: Scolytidae). Can. J. For. Res. 19: 1275-1282. and Ips bark beetles. University of Texas Press, Austin. Kudon, L. H., and C. W. Berisford. 1980. Influence ofhrood Pettersson, E. M., B. T. Sullivan, P. Anderson, C. W. Beris- hosts on host preferences of bark beetle parasites. Nature ford, and G. Birgersson. 2000. Odor perception in the (Lond.) 283: 288-290. bark beetle parasitoid Roptrocerus rylophagorum (Ratze- Langor, D. W. 1991. Arthropods and nematodes co-occur- burg) (Hymenoptera: Pteromalidae) exposed to host- ing with the eastern larch beetle, Dendrocton~ simplex associated volatiles. J. Chem. Ecol. (in press). (Col.: Scolytidae) , in Newfoundland. Entomophaga 36: Reid, R. W. 1958. The behaviour of the mountain pine bee- 303-313. tle, Denukoctonus monticolue Hopk., during mating, egg L.eufi&, A., and G. Birgersson. 1987. Quantitative variation laying, and gallery construction. Can Entomol. 9: 505-509. of different monoterpenes around galleries of Ips typogra- Rice, R. E. 1969. Response of some predators and parasites phus (Coleoptera: Scolytidae) attacking Norway spruce. of Ips con* (Let.) (Coleoptera: Scolytidae) to olfac- Can. J. Bot. 65: 1038-1044. tory attractants. Contrib. Boyce Thompson Inst. 24: 189- Leufvkn, A., G. Bergstrom, and E. Falsen. 1988. Oxygenated 194. monoterpenes produced by yeasts, isolated from Ips ty- R&e, U.S.R., W. J. Lewis, and J. H. Tumlinson. 1998. Spec- pograp~us (Coleoptera: Scolytidae) and grown in phloem ificity of systemically released cotton volatiles as attract- medium. J. Chem. Ecol. 14: 353-362. ants for specialist and generalist parasitic wasps. J. Chem. Leufv&, A. 1991. Role of microorganisms in spruce bark Ecol. 24: 303-319. beetle-conifer interactions, pp. 467-483. In P. Barbosa, Samson, P. R. 1984. The biology of Ruptrocerus zybphago- V. A. Krischik, and C. G. Jones [ eds.], Microbial media- rum (Hym.: Torymidae), with a note on its taxonomic tion of plant-herbivore interactions. Wiley, New York. status. Entomophaga 29: 287-298. Lewinsohn, E., M. Gijzen, and R. Croteau. 1991. Defense Smith, M. T., S. M. Salom, and T. L. Payne. 1993. The south- mechanisms of conifers. Plant Physiol. 96: 44-49. em pine bark beetle guild: an historical review of the Lewis, W. J., R. L. Jones, H. R. Gross, Jr., and D. A. Nordlund research on the semiochemical-based communication 1976. The role ofkairomones and other behavioral chem- system of the five principal species. Bulletin 93-4. Vir- icals in host finding by parasitic insects. Behav. Biol. 16: ginia Agricultural Experiment Station, Virginia Polytech- 267-289. nic Institute and State University, Blacksburg, VA. Madden, J. L. 1968. Behavioral responses of parasites to the Sokal, R. R., and F. J. Rohlf. 1995. Biometry, 3rd ed. Free- symbiotic fungus associated with Sirex Noctilio. Nature man, New York. (Land.) 218: 189. SPSS. 1997. SigmaStat User’s Guide, version 2.0, Chicago, Martin, W. R., Jr., D. A. Nordlund, and W. C. Nettles, Jr. IL. 1990. Response of parasitoid Eucelatoria bryani to se- Steinberg, S., M. Dicke, L.E.M. Vet, and R. Wanningen. lectedplant materialin an olfactometer. J. Chem. Ecol. 16: 1992. Response of the braconid parasitoid Cotesiu (=Ap- 499 -508. unteks) glomeratu to volatile infochemicals: effects of Mills, N. J. 1983. The natural enemies of scolytids infesting bioassay set-up, parasitoid age and experience and baro- conifer bark in Europe in relation to the biological control metric flux. Entomol. Exp. Appl. 63: 163-175. Stephen, F. M., and D. L. Dahlsten. 1976. The arrival se- of Dendroctcmus spp. in Canada. Biocontrol News Inf. 4: 305-328. quence of the arthropod complex following attack by Mitchell, W. C., and R.F.L. Man. 1970. Response of the Dendroctonvs breviwmis (Coleoptera: Scolytidae) in female southern green stink bug and its parasite ponderosa pine. Can. Entomol. 108: 283-304. Sternlicht, M. 1973. Parasitic wasps attracted by the sex Trichupodu pennipes to male stink bug pheromone. J. pheromone of their coccid host. Entomophaga 18: 339- Econ. Entomol. 64: 856-859. 342. Moser, J. C. 1971. Relative abundance of southern pine bee- Sullivan, B. T. 1997. The chemical ecology of host habitat tle associates in east Texas. Ann. Entomol. Sot. Am. 64: location by larval parasitoids of the southern pine beetle, 72-77. Dendroctonus~tulis Zimmermann: Olfactory cues and Ngi-Song, A. J., W. A. Oversholt, P.G.N. Njagi, M. Dicke, J. N. their possible sources. Ph.D. dissertation, University of Ayertey, and W. Lwande. 1996. Volatile infochemicals Georgia, Athens. used in host and host habitat location by Cotesiaflnvipes Sullivan, B. T., C. W. Berisford, and M. J. Dalusky. 1997. Cameron and Cotesia sesamine (Cameron) (Hym- Field response of southern pine beetle parasitoids to enoptera: Braconidae), larval parasitoids of stemborers some natural attractants. J. Chem. Ecol. 23: 837-856. on Graminae. J. Chem. Ecol. 22: 307-323. Sullivan, B. T., K. C. Seltmann, and C. W. Berisford. 1999. A Ngi-Song A. J.,and W. A. Oversholt. 1997. Host location and simple continuous-rearing technique for the bark beetle acceptance by Cotesiujbzvipes Cameron and C. sesamiae parasitoid, Roptwcaus zylophagorum (Ratzeburg). J. En- (Cameron) (Hymenoptera: Braconidae), parasitoids of tomol. Sci. 34: 260-264. African gramineous stemborers: role of frass and other Takacs, S., G. Gries, and R. Gries. 1997. Semiochemical- host cues. Biol. Control 9: 136-142. mediated location of host habitat by Apanteles carpati Ohmart, C. P., and W. G. Voigt. 1982. Temporal and spatial (Say) (Hymenoptera: braconidae) a parasitoid of clothes arrival patterns of Ips pkxtographus m&timu.s (Co- moth larvae. J. Chem. Ecol. 23: 459-472. leoptera: Scolytidae) and its insect associates on freshly Thibout, E., J. F. Guillot, and J. Auger. 1993. Microorgan- felled Pinus radiatu in California. Can. Entomol. 114: isms are involved in the production ofvolatile kairomones 337-348. affecting the host seeking behaviour of Diadrumus pul- Parra, J.R.P., S. B. Vinson, S. M. Gomes, and F. L. Consoli. chellus, a parasitoid of Acrolepiopsis assectellu. Physiol. 1996. Flight response of Ifabrobracon hebetur( Say) (Hy- Entomol. 18: 176-182. menoptera: Braconidae) in a wind tunnel to volatiles Tumlinson, J. H.,T.C.J. Turlings, and W. J. Lewis. 1992. The associated with infestations of Epheatiu kuehnielkz Zeller semiochemical complexes that mediate insect parasitoid (Lepidoptera: Pyralidae) . Biol. Control 6: 143-150. foraging. Agric. Zool. Rev. 5: 22-252. December 2000 SULLIVAN ET AL.: HOST LOCATION BY R. xybphagorum 1151 Vet, L.E.M., W. J. Lewis, D. R. Papaj, and J. C. van Lenteren. In W. J. Bell and R. T. Carde [ eds. 1, Chemical ecology of 1990. A variable -response model for parasitoid foraging insects. Chapman 8r Hall, London. behavior. J. Insect Behav. 3: 471-490. Visser, J. H., S. Van Straten, and H. Maarse. 1979. Isolation Vet, L.E.M., F. L. Wtickers, and M. Dicke. 1991. How to and identification of volatiles in the foliage of potato, hunt for hiding hosts: the reliability-detectability prob- SoZunum tuberosum, a host plant of the Colorado beetle, lem in foraging parasitoids. Neth. J. Zool. 41: 202-213. L.eptinotama decemlineatu. J. Chem. Ecol. 5: 13-25. Vet, L.E.M., and M. Dicke. 199.2. Ecology of infochemical Weseloh, R. M. 1977. Behavioral responses of the parasite, Apanteles melanoscelus, to gypsy moth silk. Environ. En- use by natural enemies in a tritrophic context. Annu. Rev. tomol. 5: 1128-1132. Entomol. 37: 141-172. Whitman, D. W., and F. J. Eller. 1990. Parasitic wasps orient Vinson, S. B. 1981. Habitat location, pp. 51-78. In D. A. to green leaf volatiles. Chemoecology 1: 69-76. Nordland, R. L. Jones, and W. J. Lewis [eds.], Semio- chemicals: their role in pest control. Wiley, New York. Reoeioed fi publication 20 March 2000; accepted 26 July Vinson, S. B. 1984. Parasitoid-host relationship, pp. 205-236. 2000.