Parasitoids and Dipteran Predators Exploit Volatiles from Microbial Symbionts to Locate Bark Beetles

Author(s): Celia K. Boone, Diana L. Six, Yanbing Zheng, and Kenneth F. Raffa Source: Environmental Entomology, 37(1):150-161. 2008. Published By: Entomological Society of America DOI: http://dx.doi.org/10.1603/0046-225X(2008)37[150:PADPEV]2.0.CO;2 URL: http://www.bioone.org/doi/full/10.1603/0046-225X %282008%2937%5B150%3APADPEV%5D2.0.CO%3B2

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOnes Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.

BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

BIOLOGICAL CONTROLPARASITOIDS AND PREDATORS

Parasitoids and Dipteran Predators Exploit Volatiles from Microbial Symbionts to Locate Bark Beetles
CELIA K. BOONE,1 DIANA L. SIX,2 YANBING ZHENG,3
AND

KENNETH F. RAFFA1,4

Environ. Entomol. 37(1): 150161 (2008)

ABSTRACT Host location by parasitoids and dipteran predators of bark beetles is poorly understood. Unlike coleopteran predators that locate prey by orienting to prey pheromones, wasps and ies often attack life stages not present until after pheromone production ceases. Bark beetles have important microbial symbionts, which could provide sources of cues. We tested host trees, trees colonized by beetles and symbionts, and trees colonized by symbionts alone for attractiveness to hymenopteran parasitoids and dipteran predators. Field studies were conducted with Ips pini in Montana. Three pteromalid wasps were predominant. All were associated with the second and third instars of I. pini. Heydenia unica was more attracted to logs colonized by either I. pini or the fungus Ophiostoma ips than logs alone or blank controls (screen with no log). Rhopalicus pulchripennis was more attracted to logs colonized by I. pini than logs alone or blank controls. Dibrachys cavus was attracted to logs but did not distinguish whether or not they were colonized. Two dolichopodid predators were predominant. A Medetera species was more attracted to colonized than uncolonized logs and more attracted to logs than blank controls. It was also more attracted to logs colonized with the yeast Pichia scolyti than uncolonized logs, but attraction was less consistent. An unidentied dolichopodid was more attracted to logs colonized with I. pini, O. ips, and the bacteria Burkholderia sp., than to uncolonized logs. It was also attracted to uncolonized logs. Its responses were less consistent and pronounced than H. unica. These results suggest some parasitoids and dipteran predators exploit microbial symbionts of bark beetles to locate hosts. Overall, specialists showed strong attraction to fungal cues, whereas generalists were more attracted by plant volatiles. These results also show how microbial symbionts can have conicting effects on host tness. KEY WORDS Ips pini, parasitoid behavior, predator behavior, microbe interactions, semiochemicals

Bark beetles (Coleoptera: Curculionidae, Scolytinae) are subcorticolous herbivores that spend most of their life cycle in the phloem tissues of trees. Some species intermittently undergo large-scale population eruptions with signicant ecological and economical impacts. For example, the mountain pine beetle (Dendroctonus ponderosae Hopkins) has killed 8.5 million ha of lodgepole pine during the current outbreak in British Columbia (Safranyik and Carroll 2006). Environmental impacts of bark beetles are complex and can include alterations in forest structure, wildlife habitat, and succession (Franklin et al. 1987, Matsuoka et al. 2001, McMillin and Allen 2003). Bark beetles have complex relationships with microbial associates. These relationships vary in degree of specicity, evolutionary history, consistency of as1 Department of Entomology, University of Wisconsin, Madison, WI 53706. 2 Department of Ecosystem and Conservation Sciences, University of Montana, Missoula, MT 59812. 3 Department of Statistics, University of Wisconsin, Madison, WI 53706. 4 Kenneth F. Raffa, University of WisconsinMadison, Department of Entomology, 1630 Linden Dr., Madison, WI 53706 (e-mail: raffa@entomology.wisc.edu).

sociation, and functional role (Six and Paine 1999, Six 2003, Hofstetter et al. 2005). Some fungi contribute to beetle nutrition by concentrating nitrogen in the phloem (Goldhammer et al. 1990, Six and Paine 1998, Ayres et al. 2000) or providing a source of sterols (Bentz and Six 2006). Others may enhance the beetles ability to colonize living trees by assisting in overcoming their defenses (Solheim et al. 1993, Salle et al. 2005) or contributing to pheromone production (Brand et al. 1976). A diversity of bacteria and yeasts are also associated with bark beetles, both in their guts (Delalibera et al. 2005, Vasanthakumar et al. 2006) and on their exoskeletons (Six and Paine 1998, Lim et al. 2005). Effects of these symbionts on host beetles are largely unstudied, but some may assist in pheromone synthesis (Brand et al. 1975), nutrition (Hodges et al. 1968, Coppedge et al. 1995), or protection from antagonistic fungi (Cardoza et al. 2006). Microorganisms, including fungi, are known to produce a wide range of volatiles. Attraction to fungal volatiles is widespread among insects, including Diptera, Coleoptera, Collembola, Hymenoptera, and Lepidoptera, and occurs in a diversity of habitats such as trees, soil, and decaying organisms (Vet 1983, Bel-

0046-225X/08/01500161$04.00/0 2008 Entomological Society of America

February 2008

BOONE ET AL.: MICROBIAL ATTRACTION OF NATURAL ENEMIES

151

main et al. 2002). Volatile metabolites from fungi consist mostly of short chain alcohols and esters and mono- and sesquiterpenes (Hanssen 1993, Dorado et al. 2000), and production varies qualitatively and quantitatively among species (Sprechter and Hanssen 1983). In addition, there are pronounced differences between the volatile proles of lamentous ascomycetes such as Ophiostoma, Leptographium, and Ceratocystis, and yeasts such as Ambriostouzma, Ascordan, and Eremothecium (Lanza and Palmer 1977, Mironov et al. 1982). A diverse guild of natural enemies arrives at trees colonized by bark beetles. The most common are Coleoptera (Cleridae, Histeridae, Trogossitidae), Diptera (Dolichopodidae, Loncheaidae), and Hymenoptera (Pteromalidae, Braconidae) (Linit and Stephen 1983, Reeve 1997). Impacts of predators on beetle populations have been shown by long-term data sets and life tables (Schroeder and Weslien 1994, Reeve 1997, Erbilgin et al. 2002), exclusion experiments in the eld (Riley and Goyer 1986), and controlled laboratory assays (Nebeker 1989). A diverse guild of parasitoids is also associated with bark beetles (Amman 1984, Mills et al. 1991, Hougardy and Gregoire 2001, Feicht 2006); however, less is known about their impacts on beetle populations, and relatively few controlled experiments or population analyses have been conducted. Chemical cues are particularly important to natural enemies seeking cryptic prey (Turlings and Benrey 1998, DeMoraes and Mescher 1999). Coleopteran predators commonly exploit pheromones produced by adult bark beetles (Wood 1982), and some dipterans are attracted to combinations of tree volatiles and pheromones (Hulcr et al. 2005). Likewise, Tomicobia tibialis Ashmead, a parasitoid of adult Ips pini (Say), is attracted to host pheromones (Seybold et al. 1995). However, most parasitoids of bark beetles attack immature stages and therefore arrive long after pheromone emission has ceased. It is not known how these natural enemies locate hosts. Bark from infested trees remains attractive after beetles are removed, and larvae removed from bark do not elicit attraction, arrestment, or oviposition (Mills et al. 1991, Sullivan et al. 2000), indicating that additional factors are involved. A hydrocarbon fraction extracted from bark infested with Dendroctonus frontalis Zimmerman did not attract Roptrocerus xylophagorum (Ratzeburg) when presented alone, but enhanced attraction in combination with the oxygenated fraction (Sullivan et al. 1997, Sullivan and Berisford 2004). This suggests that multiple semiochemicals are involved in host location. The consistent association of symbiotic fungi with bark beetles in both time and space (Solheim 1992, Lieutier et al. 2004) and the association of preferred late instar hosts with specic fungi (Sullivan et al. 1997) make fungi potentially useful indicators of host presence to parasitoids. For example, parasitoids of the woodwasp Sirex noctilio Fabricius are attracted to fungal volatiles (Madden 1968). Ips pini (Say) is one of the most widely distributed bark beetles in North America. It normally is present

in pine forests in low numbers, but can be damaging when an accumulation of stressed trees or slash from harvesting operations supports a rapid increase in population. In the western United States, I. pini is typically bivoltine, with spring ights beginning in mid-April to early May, and summer ights occurring in late June to mid-July (Livingston 1979). Our objective was to determine whether hymenopteran parasitoids and dipteran predators of I. pini larvae use volatile cues produced by the beetles microbial symbionts to locate hosts under natural conditions. Materials and Methods Location All experiments were conducted at the same locations in a predominantly ponderosa pine, Pinus ponderosa C. Lawson, forest at the University of Montana Lubrecht Experimental Forest in Greenough, MT (4653.30 N, 11326.00 W). Experiment 1 Experiment 1 had three objectives: (1) determine the identity and sequence of arrival of parasitoids and dipteran predators during I. pini development; (2) identify microbial symbionts consistently associated with each beetle developmental stage, and (3) assess attraction of predators and parasitoids to trees with or without beetles and their natural complement of fungi. Experiment 1 was performed twice during 2002, once for each I. pini generation. The sampling periods were 20 May to 15 July, and 17 July to 28 August 2002. Treatments consisted of (1) log infested with I. pini and its natural complement of microbes, (2) log alone, and (3) blank control. To determine the initiation time of I. pini emergence ights and provide beetles for use in the experimental log treatments, we placed eightunit multiple funnel traps baited with synthetic I. pini pheromone lures near the study sites and monitored them throughout spring and summer. Two healthy ponderosa pine trees 15 cm diameter at 1.4 m height were felled and cut into 30-cm logs. On the same day, the ends of the logs were sprayed with 10% sodium hypochlorite solution to reduce contamination by extraneous fungi. The solution was allowed to dry, and the ends were sealed by dipping them in parafn wax. Logs were initially stored indoors to protect them from being colonized by wild beetles, assigned a treatment, screened, and placed in the eld. Treatment 1 was administered by introducing six male/female pairs into each of 12 logs, 14 16 d after tree felling. The beetles were introduced by drilling 3-mm-diameter holes 3 cm deep into the phloem, inserting one male and one female into each hole, and covering the holes with a 2.5 by 2.5-cm piece of screen. The logs were wrapped with aluminum screening (mesh size, 15 mm) to prevent entry by wild beetles. Logs used for treatment 2 were treated in the same manner but did not have beetles introduced. Treat-

152

ENVIRONMENTAL ENTOMOLOGY

Vol. 37, no. 1

ment 3 (control) consisted of an empty aluminum screen. Trap Design. The trap stand was a 2-m-long 1.3cm-diameter aluminum electrical conduit. Holes were drilled at right angles 5 cm from the top. Two 15-cm lengths of copper wire (6 awg) inserted through the holes provided support for treatment logs and sticky traps. Each conduit was inserted into a 2.0-cm-diameter support conduit in the ground, and the treatment was placed on the stand. Sticky traps consisted of 33-cm-long by 31-cm-wide pieces of aluminum hardware cloth (mesh size, 30 mm) coated with Tangle Trap. Each trap was wrapped around a screened treatment log, or in the case of a control, formed into a cylinder of the same size. The ends of the sticky screens were bound with small binder clips and secured to the copper wire supports with wire (22 gauge) and 4-cm alligator clips. Experimental Design and Sampling Procedure. Treatments were deployed in a randomized complete block design (RCBD) consisting of three sites with four blocks per site. Sites were established near areas with recent harvesting activities, considerable logging slash, and detectable populations of I. pini and natural enemies. Traps were arranged with 10 m between treatments, 100 m between blocks, and at least 500 m between sites. Sticky traps were collected and replaced with clean traps at 4-d intervals until adults emerged from logs in treatment 1. Treatments were rerandomized at each collection period. Insects were removed from sticky traps with a ne paint brush (Size 0) dipped in 100% Citrisolv to remove Tangle Trap and stored in 100% Citrisolv in 15-ml vials until identication. Hymenoptera and Diptera were identied using the following keys: Graham (1969), Krombein et al. (1979), McAlpine et al. (1981), and Bickel (1985). Taxonomic verication was performed by various experts identied in the acknowledgments. Voucher specimens of parasitoids were deposited at the UWMadison Department of Entomology Insect Research Collection. Isolation and Identication of Microbes Associated with Beetle Stages. A parallel set of logs with the same treatments outlined in experiment 1 was deployed 70 m from one study site. Subsamples were destructively sampled weekly from 29 May to 2 July (generation 1) and 24 July to 19 August (generation 2). The number of beetles in each developmental stage was recorded. Microbial samples were collected by streaking parental adults, eggs, larvae, pupae (or pupal chamber), and teneral adults across 2% malt extract agar (MEA). Boring dust (adult frass), larval frass, and phloem (4-mm cores) were also taken from tips of brood and parental adult galleries and egg niches and cultured individually onto MEA. Isolates were stored at 23C under natural light (15.5 L:8.5 D) for at least 10 d and visually inspected for the presence of fungi. By 10 d, cultures had begun to produce spores that aid in identication. Cultures that were not identied soon after 10 d were stored in a refrigerator until identications could be made. Ophiostoma species were identied directly from ini-

tial isolations using morphological characteristics (Upadhyay 1981, Grylls and Seifert 1993) and DNA extraction and sequencing. For Ophiostoma, polymerase chain reaction (PCR) was performed using Bt2b and T10 primers to amplify a portion of the -tubulin gene. PCR products were sequenced on an ABI 3130X2 automated sequencer (Perkin-Elmer, Walthan, MA) at the Murdock Sequencing Facility at the University of Montana, Missoula, MT. Sequences were compared with sequences in the Sequence Match utility of GenBank using BLAST searches (http://ncbi.nlm.nih.gov/BLAST) to determine the closest match. Yeasts and bacteria were puried from initial isolates by dilution plating. A starting suspension was made by streaking a probe across the surface of an isolation plate and then swirling the probe in 1.0 ml sterile water. The suspension was vortexed briey. Dilutions of 101, 102, 103, and 104 of the original suspension were spread across the surface of MEA in 30 petri dishes and assessed for the growth of colonies after 24, 48, and 120 h. For each initial isolation, the relative prevalence of microbes was assessed from the dilution plate yielding the most distinct, nonoverlapping colonies after 48 h. Colonies in each plate were coded by morphology and counted. Pure cultures of the two most prevalent morphotypes over all isolations were produced using streak plates. The pure cultures were used for DNA extraction and sequencing. One of the two most common colony morphotypes was a bacterium. The partial 16S rRNA gene of this bacterium was amplied using the general primers 536f and 907r and PCR performed as in Holben et al. (2002). The Sequence Match utility of RDP II release nine (http://rdp.cme.msu.edu/) (Maidak et al. 1997) was used to determine the closest known relative. The second most common morphotype was a yeast. Its ITS region of rDNA was amplied using the primers ITS1-F and ITS4. The resulting sequence was compared with sequences in the Sequence Match utility of GenBank using BLAST searches (http://ncbi.nlm.nih. gov/BLAST) to determine the closest match. Experiment 2 Experiment 2 tested whether natural enemies are attracted to the predominant microbial symbionts of I. pini obtained in experiment 1. It was conducted once for each generation in 2003, with sampling periods of 17 June to 3 August and 428 August. Five healthy trees were felled and prepared as described in experiment 1. Based on results of isolations from parallel logs in experiment 1, the treatments during the rst ight were as follows: (1) blank control; (2) log alone; (3) log naturally infested with I. pini and its natural complement of microbes; (4) log inoculated with Ophiostoma ips (Rumbold) Nanff; (5) log inoculated with Burkholderia sp.; (6) log inoculated with Pichia scolyti (Phaff and Yoneyama); and (7) log with introduced I. pini adults. The same treatments were tested in generation 2, except treatment 7 was replaced with a log containing all three of the predominant microbes, O. ips, Burkholderia sp., and P. scolyti (six inoculation

February 2008

BOONE ET AL.: MICROBIAL ATTRACTION OF NATURAL ENEMIES

153

40

Parasitoids
To tal n o. Med etera sp.

Dipteran predators
60 50 40 30 20 10 0

To tal n o. un identi fied dol icho pod id

Total no. parasitoids

a.
30 20

b.

300 250 200 150 100 50 0

2002

10

Ju n -J un 22 -J un 2Ju l 12 -J ul 22 -J ul 1Au 11 g -A ug 21 -A ug 31 -A ug

-M ay

H. unica unica

R. pulchripennis pulchripennis

D. cavus cavus 70

24 -M ay 1Ju n 9Ju 17 n -J u 25 n -J un 3Ju 11 l -J u 21 l -J u 29 l -J ul 6A 14 u g -A 22 ug -A ug

Medetera sp.
Unidentified dolichopodid Unidentified dolichopodid

2-

23

12

20

To tal n o.Medetera sp.

c.

250

To tal n o. un iden tified do lich opo di d

60 50 40 30 20 10 0

d.

Total no. H. un ica

200 150 100 50 0

15

2003

10

19 -A

29 -A

20 -J

30 -J

Medetera sp. Medetera sp.

Unidentified dolichopodid Unidentified dolichopodid

Fig. 1. Seasonal abundance of hymenopteran parasitoids and dipteran predators arriving at traps containing various log, beetle, and symbiont treatments in western Montana during 2002 and 2003.

points each). Because we had inadequate numbers of adults in generation 1 to articially infest logs, we used naturally infested trees containing L2L3 larvae for treatment 3. No other insects were observed in these logs. The experimental design consisted of three sites with three blocks per site for generation 1 and three sites and four blocks per site for generation 2. Actively growing (10 d) cultures on 2% MEA were used for the treatments receiving microbes. The bark was smoothed slightly with a drawshave and sprayed with 70% ethanol. Sixteen evenly spaced 0.6-cm holes were drilled using a sterilized (70% ethanol) drill bit. A 0.6-cm-diameter plug of agar supporting a microbial culture was inserted into each hole using sterile forceps, the bark plug was replaced, and the hole was sealed with inert silicone sealant. Treatment 2 (inoculated control) received agar plugs but no microbes. Statistical Analyses Trap-catch data were analyzed as a nested block design tted with a Poisson regression using the function glmmPQL in the statistical package R (Ihaka and Gentleman 1996). The site effect was not signicant, so data were pooled across sites, analyzed according to RCBD using the glm function, and followed by mean contrasts. In 2002, the parasitoid data contained many zeros, resulting in numerical instability in R, so counts were ag-

gregated within sites across blocks for each treatment and analyzed according to RCBD tted with a Poisson regression using the genmod procedure in SAS (SAS Institute 2003). Because of strong temporal patterns (see Results), behavioral analyses of parasitoids were restricted to periods when they were abundant to avoid heteroskedascity caused by excessive zeroes: H. unica generation 1, L2; R. pulchripennisgeneration 1, L2; D. cavusgeneration 1, adults, generation 2, egg-L1. For Medetera sp. and the unidentied dolichopodid, generation effects were not signicant, so mean contrasts of treatment effects were performed on the pooled data of both generations. Experiment 2 was analyzed as in experiment 1. To account for unbalanced treatments between the rst and second generations, data were rst pooled by generation to compare treatments effects among all seven treatments in each generation. The data for both generations were pooled for comparisons among the rst six treatments only. Results Abundance and Seasonal Phenology of Parasitoids and Dipteran Predators and Their Association with I. pini Life Stages The most common bark beetle parasitoids in 2002 were the pteromalids Heydenia unica Cook and Davis

ug 16 -A ug 24 -A ug

ul

l Ju

Ju n

ug

ug

un

un

20 -J u

Ju

7J

Au

Ju

Ju 31 -

ul

ul

10 -J

30 -J

21 -

29 -

9-

15 -

23 -

8A

154

ENVIRONMENTAL ENTOMOLOGY

Vol. 37, no. 1

Table 1. Seasonal phenology of I. pini colonizing P. ponderosa logs in western Montana, 2002 Generation 1 Egg Larvae: L1 Larvae: L2 Larvae: L3 Generation 2 Egg Larvae: L1 Larvae: L2 Larvae: L3 Prepupae Pupae Adult: Teneral Adult: Mature 29 May 100 (5) 0 0 0 24 July 100 (55) 0 0 0 0 0 0 0 11 June 100 (31) 0 0 0 31 July 29 (10) 71 (25) 0 0 0 0 0 0 17 June 36 (19) 25 (13) 39 (20) 0 6 Aug. 0 0 33 (17) 67 (34) 0 0 0 0 25 June 0 0 100 (89) 0 13 Aug. 0 3 (2) 0 65 (39) 3 (2) 24 (14) 5 (3) 0 2 July 0 0 165 (15) 84 (81) 19 Aug. 0 0 0 3 (2) 2 (1) 7 (4) 85 (52) 3 (2)

Data represent percentage (N) associated with each stage. Generation 1: logs deployed 20 May; generation 2, logs deployed 17 July.

(46), Rhopalicus pulchripennis Crawford (41), and Dibrachys cavus Walker (51). Most H. unica (84.8%) were captured during the rst generation (20 May to 15 July; peak, 1115 July; Fig. 1). Similarly, 75.6% of R. pulchripennis were caught during the rst generation (peak, 1115 July). D. cavus were more evenly distributed, with 47.1% captured in the rst generation and 52.9% in the second generation. Captures peaked during 2125 June and 2529 July. In 2003, the most abundant parasitoid was H. unica (107). No other species, including R. pulchripennis and D.cavus, were caught in appreciable numbers (40 individuals). The most common predators in 2002 were Dolichopodidae (2,256), consisting of 19.6% Medetera sp. (448) and 81.4% of an unidentied dolichopodid (1,808) (Fig. 1). Peak captures of Medetera sp. occurred 1115 July during I. pinis rst generation (11.6%), and 1014 August during the second (6.3%). Peak captures of the unidentied dolichopodid occurred 2125 July (5.1%) during the rst generation and 1014 August (13.2%) during the second generation. In 2003, 3,558 dolichopodids were captured, consisting of the same Medetera sp. (20.6%) and unidentied dolichopodid (79.4%) as in 2002. Peak captures of H. unica and the unidentied dolichopodid occurred 2731 July, and peak captures of Medetera sp. occurred 2529 June. During the second generation, peak captures of both Medetera sp. (14.2%) and the unidentied dolichopodid (16.5%) occurred 48 August. In 2002, I. pini larvae were present beginning 17 June during the rst generation and from 31 July to 19 August during the second generation (Table 1). Dur-

ing the second generation, the eggL1 stages of I. pini were present from 31 July to 12 August. The L2adult stages were present from 13 to 28 August. The extents to which various natural enemies were associated with I. pinis generations and life stages are shown in Table 2. Nearly all H. unica and R. pulchripennis were caught after the L1 stage. In contrast, the seasonal distribution of D. cavus mostly overlapped L2adult of I. pinis rst generation and eggL1 of I. pinis second generation. As with the hymenopteran parasitoids, dipteran predators were primarily associated with L2 and later (79.3% for Medetera sp. and 60.7% for the unidentied dolichopodid). In 2003, I. pini L2adults were present from 17 June to 31 July in the rst generation (Table 3). During this time, 85.1% of H. unica, 65.0% of Medetera sp., and 62.6% of the unidentied dolichopodid were captured. Ips pinis eggL1 stages were present from 31 July to 12 August, and L2adults were present from 13 to 28 August during the second generation. During the L2 adult stages, Medetera sp. comprised 20.8% and the unidentied dolichopodid comprised 20.9% of the total dipteran predators captured. Isolation and Identication of Microbes Associated with Beetle Stages Three microorganisms were consistently isolated from the P. ponderosa logs colonized by I. pini. O. ips was the most common associate of I. pini adults, being isolated from 89.7% of insects in the rst generation and 85.7% of insects in the second generation. The yeast, Pichia scolyti (Phaff and Yoneyama; 100% match to AY761155 in GenBank), and the bacterium Burkholderia sp. (95.4% match to AJ292641 in GenBank) often co-occurred with O. ips, but were not consistently associated with any specic I. pini developmental stages. Natural Enemy Responses to Sources of Volatiles Associated with I. pini Experiment 1: Plant Material With or Without I. pini. There were signicant sources of attraction for all three wasp species (Fig. 2a). Captures of H. unica were higher on I. pini--colonized logs than either uncolonized logs (P 0.0375) or blank controls (P 0.0001) and on uncolonized logs than blank controls (P 0.0001). R. pulchripennis captures were also signicantly higher on colonized logs than uncolonized logs

Table 2. Percentage (N) of hymenopteran parasitoids and dipteran predators associated with various life stages of I. pini in western Montana during 2002 Hymenopteran parasitoids Generation 1 2 Stage EggL1 L2adult EggL1 L2adult Dates 20 May to 13 June 14 June to 15 July 17 July to 2 Aug. 226 Aug. H. unica 2.2 (1) 82.6 (38) 0 15.2 (7) R. pulchripennis 0 75.6 (31) 0 24.4 (10) D. cavus 2.0 (1) 45.1 (23) 37.2 (19) 15.7 (8) Dipteran predators Medetera sp. 1.3 (6) 54.7 (245) 19.4 (87) 24.6 (110) Unidentied dolichopodid 0.01 (1) 13.5 (244) 39.3 (711) 47.2 (852)

February 2008

BOONE ET AL.: MICROBIAL ATTRACTION OF NATURAL ENEMIES

155

Table 3. Percentage (N) of H. unica, Medetera sp. and an unknown dolichopodid associated with various life stages of I. pini in western Montana during 2003 Generation 1 2 Stage EggL1 L2adult EggL1 L2adult Dates NA 17 June to 31 July 31 July to 12 Aug. 1328 Aug. H. unica NA 85.1 (91) 2.8 (3) 12.1 (13) Medetera sp. NA 65.0 (234) 14.2 (51) 20.8 (75) Unidentied dolichopodid NA 62.6 (870) 16.5 (230) 20.9 (290)

NA, not applicable.

(P 0.0001) or blank controls (P 0.0001). They were also attracted to uncolonized logs but only weakly (P 0.0414). D. cavus captures were higher on both colonized (P 0.0001) and uncolonized logs (P 0.0085) than blank controls. However, D. cavus did not distinguish between colonized and uncolonized logs (P 0.05). Logs colonized by I. pini attracted more Medetera sp. than did uncolonized logs (P 0.0001) and blank
15

controls (P 0.0001), and uncolonized logs attracted more than blank controls (P 0.0415; Fig. 2b). Uncolonized logs attracted more of the unidentied dolichopodid than colonized logs (P 0.0001) or blank controls (P 0.0001). However, colonized logs attracted more of this dolichopodid than did blank controls (P 0.0001). Experiment 2: Microorganisms. Logs colonized by L2L3 I. pini or the fungus O. ips were more attractive

a.
Mean no. parasitoids + SE
a

10

a a b
5

b b c
0

c R. pulchripennis b D. cavus

H. unica
100

Mean no. dipteran predators + SE

b.
80

a
60

40

a
20

c b c

Medetera sp. Log + I. pini Log

Unidentified dolichopodid Control

Fig. 2. Response (mean SE) of (a) hymenopteran parasitoids and (b) dipteran predators to P. ponderosa and P. ponderosa colonized by I. pini in western Montana. Means with the same letter are not signicantly different at P 0.05.

156
20

ENVIRONMENTAL ENTOMOLOGY
a

Vol. 37, no. 1

Mean no. H. unica + SE

15 ab 10 bc 5 d 0 cd cd d

sp .

La rv ae )

C on tr ol

de ri a

P. s

I. pi ni (

Fig. 3. Response (mean SE) of H. unica to P. ponderosa, P. ponderosa colonized by I. pini, P. ponderosa inoculated with microbial symbionts, and blank controls. Experiments conducted during the L2-adults stages of I. pinis rst generation (17 June to 3 August). Means with the same letter are not signicantly different at P 0.05. Treatments containing I. pini were administered either as logs infested with larvae or logs exposed to adults that subsequently produced brood.

to H. unica than any other treatment (Fig. 3), with the exception that the O. ips and Burkholderia sp. treatments were not statistically different. Arrival by H. unica was equivalent to logs colonized by L2L3 I. pini and O. ips. Logs colonized with O. ips caught more H. unica than did logs alone (P 0.0001) or blank controls (P 0.0003). Captures on logs colonized with Burkholderia sp. were higher than on blank controls but did not differ from uncolonized logs. Logs colonized with P. scolyti or I. pini adults did not capture more H. unica than either uncolonized logs or blank controls. Overall, logs colonized by P. scolyti caught more Medetera sp. than uncolonized logs (P 0.0377) or blank controls (P 0.0001; Fig. 4a). No other treatments were more attractive than logs alone. Medetera sp. was more attracted to all treatments containing logs than blank controls (P 0.05). However, responses varied among experiments associated with the two generations of I. pini (Fig. 4, b and c). Overall, logs colonized by I. pini (all stages), O. ips, and Burkholderia sp. were more attractive to the unidentied dolichopodid than uncolonized logs or controls (Fig. 5a). This species was also more attracted to all treatments containing logs than blank controls during both generations of I. pini (P 0.05; Fig. 5, b and c). Like Medetera sp., however, responses by this y varied between experiments. Discussion These results support the hypotheses that some parasitoids and dipteran predators locate bark beetles by orienting to volatile cues associated with host development (Sullivan et al. 1997, 2000, Pettersson et al. 2001), and in particular, that some species are attracted to volatiles emitted by the beetles fungal symbionts (Sullivan and Berisford 2004). Different parasitoid species vary in their behaviors. For example, H.

B ur kh ol

unica and R. pulchripennis are more attracted to I. pini colonized plant tissue than uncolonized plant tissue. Moreover, the arrival of H. unica at colonized logs can be explained largely by attraction to cues from microbial symbionts, especially O. ips. D. cavus is likewise attracted to ponderosa pine, but in contrast to the other wasps, its attraction is not increased by I. pini colonization. The identities of the attractive volatiles are unknown, but work with Rhopalicus tutela (Walker) suggests that oxygenated monoterpenes, such as those released by I. typographusinfested trees, are involved (Pettersson 2001, Pettersson and Boland 2003). Consistent with the role of fungi in parasitoid attraction, ophiostomatoids metabolize terpenoid compounds (Hanssen 1993). Like the parasitoids, some dipteran predators were attracted to volatiles associated with host plants; plants colonized by I. pini and its microbial symbionts. Medetera sp. and the unidentied dolichopodid were more attracted to plant tissue, and each showed some additional attraction to logs colonized by fungi. However, their responses were less consistent and pronounced than those of H. unica. Overall, the stronger attraction of H. unica and R. pulchripennis to I. pini larvae and/or associated microbes, and the stronger attraction of D. cavus and predators to plant volatiles, support the view that specialist natural enemies exploit more explicit infochemicals than generalists (Steidle and van Loom 2003). For example, the host range of H. unica is limited to bark beetles in Dendroctonus, Ips, Scolytus, Phloesinus, and Polygraphus (Krombein et al. 1979). Most, if not all, species in these genera are associated with Ophiostoma or closely related fungi (Paine et al. 1997). Thus, volatiles from these fungi combined with tree compounds such as -pinene (Camors and Payne 1972) may provide a reliable signal for detecting hosts. Furthermore, H. unica seems to be highly specialized on a particular stage

I. pi ni (

Ad ul ts )

Lo g

O .i

co

ly ti

ps

February 2008
8

BOONE ET AL.: MICROBIAL ATTRACTION OF NATURAL ENEMIES

a

157

a. Pooled
6 4 d 2 0 bc

ab bc c

sp .

ol

ps

ni

Lo

tr

pi

.i

on

ria

I.

Mean no. Medetera sp. + SE

b. Generation 1
6 4 bc 2 0 c a ab ab

ur

kh

ol

P. s

de

co
a a

rv ae )

tr ol

.i ps

Lo

co

ol

P. s

i(

de

in

kh

I. p

ur

c. Generation 2
3

a ab ab ab

2 c 1 0

bc

bc

I.

pi ly ti sp co ria P. s C om bi ne d m ic ro be s .

lts )

ol

Lo

tr

Ad u

on

.i

ps

i(

Fig. 4. Response (mean SE) of Medetera sp. to P. ponderosa, P. ponderosa colonized by I. pini, P. ponderosa inoculated with microbial symbionts, and blank controls (a) pooled across I. pinis generations, (b) generation 1, and (c) generation 2. Means with the same letter are not signicantly different at P 0.05. Treatments containing I. pini were administered either as logs infested with larvae, logs exposed to adults that subsequently produced brood, or both techniques (data pooled).

of development. For example, 84% of H. unica arriving at trees colonized by D. frontalis were associated with third-instar larvae (Camors and Payne 1972), similar to our observations that they are mostly associated with later developmental stages of I. pini (Table 2). Likewise, although R. pulchripennis parasitizes many bark beetle species, it is limited to

ur kh o

I. p

in

the Scolytinae. It showed the highest relative attraction to colonized versus uncolonized tissue, although there were insufcient numbers in 2003 to test whether this was caused by microorganisms. In contrast, D. cavus is polyphagous, attacking many insect orders and spiders (Graham 1969, Krombein et al. 1979). Similarly, most dolichopodids are rel-

ld e

ni

(A du

on

La

ria

lts )

sp

ly

ti

ly

ti

158
30

ENVIRONMENTAL ENTOMOLOGY
a ab

Vol. 37, no. 1

a. Pooled
b
20

c
10

sp

Lo

O .i

on

ria

Mean no. unidentified dolichopodid + SE

de

30

b. Generation 1
ab
20

ab bc

ur

kh

ab bc c

10

ol

ps

ol

P. ol yt

sc

I.

sp

rv ae

Lo

on

O .i

(L a

de

P.

ni

kh

pi

ur

I.

15

c. Generation 2
10

a ab ab ab

b
5

I.

pi

ni ol yt C om bi ne d m ic ro be s i

ol

Lo

tr

lts

(A du

O .i

ps

on

ria

sp

ol

de

ni

pi

Fig. 5. Response (mean SE) of the unidentied dolichopodid to P. ponderosa, P. ponderosa colonized by I. pini, P. ponderosa inoculated with microbial symbionts, and blank controls (a) pooled across I. pinis generations, (b) generation 1, and (c) generation 2. Means with the same letter are not signicantly different at P 0.05. Treatments containing I. pini were administered either as logs infested with larvae, logs exposed to adults that subsequently produced brood, or both techniques (data pooled).

atively generalist predators or scavengers and occur in diverse habitats (Coulibaly 1993). The numbers of known parasitoids of bark beetles obtained in this study were quite low, being only 2% that of our captures of predaceous ies. Consistently more predators than parasitoids of I. pini are captured in the eld, regardless of region or the trapping method used (Raffa and Dahlsten 1995, Aukema et al.

2000, Dahlsten et al. 2004). Furthermore, studies with North American and European bark beetles have shown relatively low mortality attributable to parasitoids (Amman 1984, Weslien and Schroeder 1999, Hougardy and Gregoire 2001, Feicht 2006). Overall, these results suggest that parasitoids are probably insufcient to exert much population regulation or selective pressure on I. pini under natural conditions.

B ur kh

I.

ol

P.

sc

(A du

ria

sc

lts

tr

ol yt i

ni

pi

tr

ps

ol

February 2008

BOONE ET AL.: MICROBIAL ATTRACTION OF NATURAL ENEMIES

159

These results add to our understanding of how microbial associates of bark beetles can have multiple and varying effects on their vectors (Klepzig and Six 2004, Kopper et al. 2004). For example, O. ips can increase I. pini brood production (Kopper et al. 2004), but can also have an indirect negative effect on its host by attracting natural enemies. From the natural enemys perspective, O. ips offers the most reliable signal of I. pini larvae, because it is the most frequent associate, is present with host larvae, and occurs in all, or almost all, trees colonized by I. pini (Klepzig et al. 1991). The amount and location of the host tree that microorganisms colonize may inuence the strengths and roles of their signals. For example, O. ips often colonizes almost all of the phloem and much of the sapwood, whereas bacteria and yeast often occupy only restricted microhabitats along the beetles gallery. Thus, O. ips may produce a stronger signal and be important in long distance host location, whereas bacteria and yeast may be more important in short distance location.

Acknowledgments
The assistance of the staff of Lubrecht Experimental Forest, University of Montana, especially H. Goetz and F. Maus, is deeply appreciated. M. Phau, C. Paterson, T. Benson (Department of Ecosystem and Conservation Science, University of Montana, Missoula, MT), and K. Kieler (Department of Entomology, UW, Madison, WI) provided technical assistance. S. Krauth (Department of Entomology, UW, Madison, WI), E. Grissel, USDA Research Entomologist, U.S. Museum of Natural History, Smithsonian Institute (Pteromalidae); J. Luhman, Biological Control Scientist and Adjunct Assistant Professor, University of Minnesota, Department of Entomology (Braconidae); M. W. Gates, USDA Systematic Entomology Research Scientist, Smithsonian Institute (Eulophidae); and M. Yoder, PhD Candidate, Texas A&M University, Department of Entomology (Diapriidae), assisted with insect identication. We thank S. Adams (Division of Biological Services, University of Montana, Missoula, MT) for identifying the bacterial isolate used in this project. J. Zhu (Department of Statistics, UWMadison, Madison, WI) provided statistical advice. We thank the anonymous reviewers for helpful critiques of this paper. This work was supported by USDA NRI (2003-3502-13528), the National Science Foundation (DEB0314215), and UWMadison College of Agricultural and Life Sciences.

References Cited
Amman, G. D. 1984. Mountain pine beetle (Coleoptera: Scolytidae) mortality in three types of infestations (Dendroctonus ponderosae, Pinus contorta). Environ. Entomol. 13: 184191. Aukema, B. H., D. L. Dahlsten, and K. F. Raffa. 2000. Exploiting behavioral disparities among predators and prey to selectively remove pests: Maximizing the ratio of bark beetles to predators removed during semiochemically based trap out. Environ. Entomol. 29: 651660. Ayres, M. P., R. T. Wilkens, J. J. Ruel, M. J. Lombardero, and E. Vallery. 2000. Nitrogen budgets of phloem-feeding bark beetles with and without symbiotic fungi. Ecology 81: 21982210.

Belmain, S. R., M.S.J. Simmonds, and W. M. Blaney. 2002. Inuence of odor from wood-decaying fungi on host selection behavior of deathwatch beetle, Xestobium rufovillosum. J. Chem. Ecol. 28: 741754. Bentz, B. J., and D. L. Six. 2006. Ergosterol content of fungi associated with Dendroctonus ponderosae and Dendroctonus rupennis (Coleoptera: Curculionidae, Scolytinae). Ann. Entomol. Soc. Am. 99: 189 194. Bickel, D. J. 1985. A revision of the Nearctic Medetera (Diptera: Dolichopodidae). USDA Tech. Bull. 1692: 1109. Brand, J. M., J. W. Bracke, A. J. Markovetz, D. L. Wood, and L. E. Browne. 1975. Production of verbenol pheromone by a bacterium isolated from bark beetles. Nature (Lond.) 254: 136 137. Brand, J. M., J. W. Bracke, L. N. Britton, A. J. Markovetz, and S. J. Barras. 1976. Bark beetle pheromones: production of verbenone by a mycangial fungus of Dendroctonus frontalis. J. Chem. Ecol. 2: 195199. Camors, F. B., and T. L. Payne. 1972. Response of Heydenia unica (Hymenoptera: Pteromalidae) to Dendroctonus frontalis (Coleoptera: Scolytidae) pheromones and a host-tree terpene. Ann. Entomol. Soc. Am. 65: 3133. Cardoza, Y. J., K. D. Klepzig, and K. F. Raffa. 2006. Bacteria in oral secretions of an endophytic insect inhibit antagonistic fungi. Ecol. Entomol. 31: 636 645. Coppedge, B. R., F. M. Stephen, and G. W. Felton. 1995. Variation in female southern pine beetle size and lipid content in relation to fungal associates. Can. Entomol. 127: 145154. Coulibaly, B. 1993. Dolichopodidae (Diptera) in the biological control of certain insects harmful to forest ecosystems. Insect Sci. Appl. 14: 85 87. Dahlsten, D. L., D. L. Six, D. L. Rowney, A. B. Lawson, N. Erbilgin, and K. F. Raffa. 2004. Attraction of Ips pini (Coleoptera: Scolytinae) and its predators to natural attractants and synthetic semiochemicals in northern California: Implications for population monitoring. Environ. Entomol. 33: 1554 1561. Delalibera, I., J. Handelsman, and K. F. Raffa. 2005. Contrasts in cellulolytic activities of gut microorganisms between the wood borer, Saperda vestita (Coleoptera: Cerambycidae), and the bark beetles, Ips pini and Dendroctonus frontalis (Coleoptera: Curculionidae). Environ. Entomol. 34: 541547. DeMoraes, C. M., and M. C. Mescher. 1999. Interactions in entomology: plant-parasitoid interaction in tritrophic systems. J. Entomol. Sci. 34: 3139. Dorado, J., F. W. Claassen, T. A. van Beek, G. Lenon, J.B.P.A. Wijnberg, and R. Sierra-Alvarez. 2000. Elimination and detoxication of softwood extractives by white-rot fungi. J. Biotech. 80: 231240. Erbilgin, N., E. V. Nordheim, B. H. Aukema, and K. F. Raffa. 2002. Population dynamics of Ips pini and Ips grandicollis in red pine plantations in Wisconsin: Within- and between-year associations with predators, competitors, and habitat quality. Environ. Entomol. 31: 10431051. Feicht, E. 2006. Frequency, species composition and efciency of Ips typographus (Col., Scolytidae) parasitoids in infested spruce forests in the National Park Bavarian Forest over three consecutive years. J. Pest Sci. 79: 3539. Franklin, J. F., H. H. Shugart, and M. E. Harmon. 1987. Tree death as an ecological process. BioScience 37: 550 556. Goldhammer, D. S., F. M. Stephen, and T. D. Paine. 1990. The effect of the fungi Ceratocystis minor (Hedgecock) Hunt, var. barrasii Taylor, and SJB-122 on reproduction of

160

ENVIRONMENTAL ENTOMOLOGY

Vol. 37, no. 1

the southern pine beetle, Dendroctonus frontalis Zimmerman (Coleoptera, Scolytidae). Can. Entomol. 122: 407 418. Graham, M.W.R. de V. 1969. The Pteromalidae of northwestern Europe (Hymenoptera: Chalcidoidea). British Museum of Natural History, London, UK. Grylls, B. T., and K. A. Seifert. 1993. A synoptic key to species of Ophiostoma, and Ceratocystiopsis, pp. 261268. In M. J. Wingeld, K. A. Seifert, and J. F. Webber (eds.), Ceratocystis and Ophiostoma: taxonomy, ecology, and pathology. APS Press, St. Paul, MN. Hanssen, H. P. 1993. Volatile metabolites produced by species of Ophiostoma and Ceratocystis, pp. 117126. In M. J. Wingeld, K. A. Seifert, and J. F. Webber (eds.), Ceratocystis and Ophiostoma: taxonomy, ecology, and pathology. APS Press, St. Paul, MN. Hodges, J. D., S. J. Barras, and J. K. Maudlin. 1968. Amino acids in inner bark of loblolly pine as affected by the southern pine beetle and associated micro-organisms. Can. J. Bot. 46: 146772. Hofstetter, R. W., J. B. Mahfouz, K. D. Klepzig, and M. P. Ayres. 2005. Effects of tree phytochemistry on the interactions among endophloedic fungi associated with the southern pine beetle. J. Chem. Ecol. 31: 539 560. Holben, W. E., P. Williams, S. M. Saarinen, and J.H.A. Apajalhti. 2002. Phylogenetic analysis of intestinal microora indicates a novel Mycoplasma phylotype in farmed and wild salmon. Microbial Ecol. 44: 175185. Hougardy, E., and J. C. Gregoire. 2001. Bark beetle parasitoids population surveys following stem damage in spruce stands in the Vosges region (France). Integr. Pest Manage. Rev. 6: 163138. Hulcr, J., M. Pollet, K. Ubik, and J. Vrkoc. 2005. Exploitation of kairomones and synomones by Medetera spp. (Diptera: Dolichopodidae), predators of spruce bark beetles. Eur. J. Entomol. 102: 655 662. Ihaka, R., and R. Gentleman. 1996. R: a language for data analysis and graphics. J. Comput. Graph. Stat. 5: 299 314. Klepzig, K. D., and D. L. Six. 2004. Bark beetle-fungal symbiosis: context dependency in complex associations. Symbiosis 37: 189 205. Klepzig, K. D., K. F. Raffa, and E. B. Smalley. 1991. Association of an insect-fungal complex with red pine decline in Wisconsin. For. Sci. 37: 1119 1139. Kopper, B. J., K. D. Klepzig, and K. F. Raffa. 2004. Components of antagonism and mutualism in Ips pini-fungal interactions: Relationship to a life history of colonizing highly stressed and dead trees. Environ. Entomol. 33: 28 34. Krombein, K. V., P. D. Hurd, P. D. Smith, and B. D. Burks. 1979. Catalogue of Hymenoptera in America North of Mexico. vol. I. Symphyta and Apocrita (Parasitica). Smithsonian Institution Press, Washington, DC. Lanza, E., and J. K. Palmer. 1977. Biosynthesis of monoterpenes by Ceratocystis moniliformis. Phytochemistry 16: 15551560. Lieutier, F., A. Yart, H. Ye, D. Sauvard, and V. Gallois. 2004. Variations in growth and virulence of Leptographium wingeldii Morelet, a fungus associated with the bark beetle Tomicus piniperda L. Ann. For. Sci. 61: 4553. Lim, Y. W., J. J. Kim, M. Lu, and C. Breuil. 2005. Determining fungal diversity on Dendroctonus ponderosae and Ips pini affecting lodgepole pine using cultural and molecular methods. Fungal Divers. 19: 79 94. Linit, M. J., and F. M. Stephen. 1983. Parasite and predator component of within tree southern pine beetle (Coleoptera: Scolytidae) mortality. Can. Entomol. 115: 679 688.

Livingston, R. L. 1979. The pine engraver, Ips pini (Say), in Idaho: Life history, habits and management recommendations. Idaho Dept. Lands, Forests, Insects, Disease Conditions Report No. 79 3. Madden, J. L. 1968. Behavioral responses of parasites to the symbiotic fungus associated with Sirex noctilio. Nature (Lond.) 218: 189 190. Maidak, B. L., G. J. Olsen, N. Larsen, R. Overbeek, M. J. McCaughey, and C. R. Woese. 1997. The rdp (ribosomal database project). Nucleic Acids Res. 25: 109 11. Matsuoka, S. M., C. M. Handel, and D. R. Ruthrauff. 2001. Densities of breeding birds and changes in vegetation in an Alaskan boreal forest following a massive disturbance by spruce beetles. Can. J. Zool. 79: 1678 1690. McAlpine, J. F., B. V. Peterson, G. E. Shewell, H. J. Teskey, J. R. Vockeroth, and D. M. Wood. 1981. Manual of Nearctic Diptera. vol. I. Canadian Government Publishing Centre, Hull, Quebec. McMillin, J. D., and K. K. Allen. 2003. Effects of Douglas r beetle (Coleoptera: Scolytidae) infestations on forest overstory and understory conditions in western Wyoming. West. N. Am. Nat. 63: 498 506. Mills, N. J., K. Kruger, and J. Schlup. 1991. Short-range host location mechanisms of bark beetle parasitoids. J. Appl. Entomol. 111: 33 43. Mironov, V. A., M. I. Tsibulskaya, and M. T. Yanotovsky. 1982. Synthesis of monoterpenes by the ascomycete Eremothecium ashbyi. Prikl Biokhim. Mikrobiol. 18: 343345. Nebeker, T. E. 1989. Bark beetles, natural enemies, management selection interactions, pp. 71 80. In D. L. Kulhavy and M. C. Miller (eds.), Potential for biological control of Dendroctonus and Ips bark beetles. School of Forestry, Stephen F. Austin State University, Nacogdoches, TX. Paine, T. D., K. F. Raffa, and T. C. Harrington. 1997. Interactions among scolytid bark beetles, their associated fungi, and live host conifers. Annu. Rev. Entomol. 42: 179 206. Pettersson, E. M. 2001. Volatiles from potential hosts of Rhopalicus tutela, a bark beetle parasitoid. J. Chem. Ecol. 27: 2219 2231. Pettersson, E. M., and W. Boland. 2003. Potential parasitoid attractants, volatile composition throughout a bark beetle attack. Chemoecology 13: 2737. Pettersson, E. M., E. Hallberg, and G. Birgersson. 2001. Evidence for the importance of odour-perception in the parasitoid Rhopalicus tutela (Walker) (Hym., Pteromalidae). J. Appl. Entomol. 125: 293301. Raffa, K. F., and D. L. Dahlsten. 1995. Differential responses among natural enemies and prey to bark beetle pheromones. Oecologia (Berl.) 80: 556 569. Reeve, J. D. 1997. Predation and bark beetle dynamics. Oecologia (Berl.) 112: 48 54. Riley, M. A., and R. A. Goyer. 1986. Impact of benecial insects on Ips spp. (Coleoptera: Scolytidae) bark beetles in felled loblolly and slash pines in Louisiana. Environ. Entomol. 15: 1220 1224. Safranyik, L., and A.L. Carroll. 2006. The biology and epidemiology of the mountain pine beetle in lodgepole pine forests, pp. 3 66. In L. Safranyik and B. Wilson (eds.), The mountain pine beetle: a synthesis of its biology, management and impacts on lodgepole pine. NRC, CFS, PFC, Victoria, Canada. Salle, A., R. Monclus, A. Yart, J. Garcia, P. Romary, and F. Lieutier. 2005. Fungal ora associated with Ips typographus: frequency, virulence, and ability to stimulate the host defense reaction in relation to insect population levels. Can. J. For. Res. 35: 365373.

February 2008

BOONE ET AL.: MICROBIAL ATTRACTION OF NATURAL ENEMIES

161

SAS Institute. 2003. SAS 9.1.3. SAS Institute, Cary, NC. Schroeder, L. M., and J. Weslien. 1994. Reduced offspring production in bark beetle Tomicus piniperda in pine bolts baited with ethanol and alpha-pinene, which attract antagonistic insects. J. Chem. Ecol. 20: 1429 1444. Seybold, S. J., T. Ohtsuka, D. L. Wood, and I. Kubo. 1995. Enantiomeric composition of ipsdienol: a chemotaxonomic character for North American populations of Ips spp. in the pini subgeneric group (Coleoptera: Scolytidae). J. Chem. Ecol. 21: 9951016. Six, D. L. 2003. A comparison of mycangial and phoretic fungi of individual mountain pine beetles. Can. J. For. Res. 33: 13311334. Six, D. L., and T. D. Paine. 1998. Effects of mycangial fungi and host tree species on progeny survival and emergence of Dendroctonus ponderosae (Coleoptera: Scolytidae). Environ. Entomol. 27: 13931401. Six, D. L., and T. D. Paine. 1999. Phylogenetic comparison of ascomycete mycangial fungi and Dendroctonus bark beetles (Coleoptera: Scolytidae). Ann. Entomol. Soc. Am. 92: 159 166. Solheim. H. 1992. The early stages of fungal invasion in Norway spruce infested by the bark beetle Ips typographus. Can. J. Bot. 70: 15. Solheim, H., B. Langstrom, and C. Hellqvist. 1993. Pathogenicity of the bluestain fungi Leptographium wingeldii and Ophiostoma minus to Scots pine: effect of tree pruning and inoculum density. Can. J. For. Res. 23: 1438 1443. Sprechter, E., and H. P. Hanssen. 1983. Distribution and strain-dependent formation of volatile metabolites in the genus Ceratocystis. Antonie van Leeuwehoek 49: 493 499. Steidle, J.L.M., and J. A. van Loom. 2003. Dietary specialization and infochemical use in carnivorous arthropods: testing a concept. Entomol. Exp. Appl. 108: 133148.

Sullivan, B. T., and C. W. Berisford. 2004. Semiochemicals from fungal associates of bark beetles may mediate host location behavior of parasitoids. J. Chem. Ecol. 30: 703 717. Sullivan, B. T., C. W. Berisford, and W. J. Dalusky. 1997. Field response of southern pine beetle parasitoids to some natural attractants. J. Chem. Ecol. 23: 837 856. Sullivan, B. T., E. M. Pettersson, K. C. Seltmann, and C. W. Berisford. 2000. Attraction of the bark beetle parasitoid Roptrocerus xylophagorum (Hymenoptera: Pteromalidae) to host-associated olfactory cues. Environ. Entomol. 29: 1138 1151. Turlings, T. C., and B. Benrey. 1998. Effects of plant metabolites on the behavior and development of parasitic wasps. Ecoscience 5: 321333. Upadhyay, H. P. 1981. A monograph of Ceratocystis and Ceratocystiopsis. University of Georgia Press, Athens, GA. Vasanthakumar, A., I. Delalibera, J. Handelsman, K. D. Klepzig, P. Schloss, and K. F. Raffa. 2006. Characterization of gut-associated microorganisms in larvae and adults of the southern pine beetle, Dendroctonus frontalis Zimmerman. Environ. Entomol. 35: 1710 1717. Vet, L.E.M. 1983. Host-habitat location through olfactory cues by Leptopilina clavipes (Hartig) (Hymenoptera: Eucoilidae), a parasitoid of fungivorous Drosophila: the inuence of conditioning. Neth. J. Zool. 33: 225248. Weslien, J., and L. M. Schroeder. 1999. Population levels of bark beetles and associated insects in managed and unmanaged spruce stands. For. Ecol. Manag. 115: 267275. Wood, D. L. 1982. The role of pheromones, kairomones, and allomones in the host selection behavior of bark beetles. Annu. Rev. Entomol. 27: 411 446. Received for publication 17 August 2007; accepted 18 October 2007.