Journal of Chemical Ecology, Vol. 18, No. 12, 1992

THE ROLE OF LANIERONE IN THE CHEMICAL ECOLOGY OF Ips pini (COLEOPTERA: SCOLYTIDAE) IN CALIFORNIA

STEVEN

J. S E Y B O L D , j ' * S T E P H E N AIJUN KENNETH

A. TEALE, 3 DAVID

ZHANG, 4 FRANCIS Q. LINDAHL,

L. W O O D , 1

X. WEBSTER, 4

J R . , 2'5 a n d I S A O

KUBO I

tDepartment of Entomological Sciences ZGroup in Biostatistics, School of Public Health University of California at Berkeley Berkeley, California 94720 3Department of Environmental and Forest Biology 4Department of Chemistry College of Environmental Science and Forestry State University of New York Syracuse, New York 13210 (Received May 11, 1992; accepted August 10, 1992)

Abstract--Five doses of lanierone (2-hydmxy-4,4,6-trimethyl-2,5-cyclohexadien-l-one) were tested with one dose of enantiomefically pure [99.4% (4R)-(-)] ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) for activity as an aggregation pheromone of lps pini (Say) in California. The response of L pini to 1 mg/day ipsdienol + 20/~g/day lanierone was significantly greater than the response to ipsdienol alone, but the response pattern did not demonstrate a clear dose-response relationship. The response to the highest dose of lanierone (2 mg/day) was significantly lower than the response to ipsdienol alone. Ipsdienol attracted significantly more L pini than a male-infested log. Lanierone did not alter the percentage of male L pini responding to ipsdienol alone. Neither sex of L pini or Dendroctonus brevicomis LeConte from California produced detectable amounts of lanierone, but myrcene-aerated male D. brevicomis produced 97.8 %-(4S)-( +)-ipsdienol. The black-bellied clefid, Enoclerus lecontei (Wolcott) (Coleoptera: Cleridae) was attracted to lanierone when released with ipsdienol. Neither compound was attractive when released *To whom correspondence should be addressed. 5Current mailing address: Data Communication and Network Services, Information Systems and Technology, University of California at Berkeley, Berkeley, California 94720. 2305 0098-0331/92/1200-2305506.50/0 9 1992PlenumPublishingCorporation

2306

SEYBOLD ET AL. alone, proving synergism for the kairomoue of this predator. Lanierone did not influence the response of the predators Temnochila chlorodia (Mannerheim) (Coleoptera: Trogositidae) and Enoclerus sphegeus (F.) (Coleoptera: Cleridae), which were attracted to all treatments containing ipsdienol. Tomicobia tibialis Ashmead (Hymenoptera: Pteromalidae) responded in significantly greater numbers to the male-infested log than it did to ipsdienol or ipsdienol + 20 ~g/day lanierone. Key Words--Coleoptera, Scolytidae, Cleridae, Trogositidae, Pteromalidae, Ips pini, Dendroctonus brevicomis, Enoclerus lecontei, Enoclerus sphegeus, Temnochila chlorodia, Tomicobia tibialis, ipsdienol, lanierone, aggregation pheromone, kairomone.

INTRODUCTION

Ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) was identified as the aggregation pheromone of the pine engraver beetle, Ips pini (Say) (Coleoptera: Scolytidae), from populations in California (Birch et al., 1980) and New York (Stewart, 1975; Lanier et al., 1980). Prior to the identification of ipsdienol from I. pini, populations from California, Idaho, and New York were known to respond preferentially to their regionally specific pheromones (Lanier, 1972; Lanier et alo, 1972), yet populations from California (and Arizona) and Ontario were shown to be interfertile (Lanier, t972). In lab and field tests in California, (4R)-(-)-ipsdienol [95% (-)-lab, 97.8% (-)-field] was found to be attractive to L pini while the (4S)- ( + )-enantiomer [87 % ( + )-lab, 94.1% ( + )-field], which was inactive alone, interrupted the response to (-)-ipsdienol (Birch et al., 1980). In contrast, in New York, Lanier et al. (1980) found the maximum response to racemic ipsdienot and demonstrated synergism between the two enantiomers [91% ( - ) and 80% (+)] in a field bioassay. Raffa and Klepzig (1989) provided additional evidence that eastern populations of L pini prefer intermediate enantiomeric blends of ipsdienol when they showed maximum trap catches to 50 % ( - ) and 25% ( - ) in two separate field studies in Wisconsin. The high activity of (-)-ipsdienol in western populations was further documented in one population in southeastern British Columbia [98% (-)-ipsdienol, Miller, 1990] and in another population in California [99.9% (-)-ipsdienol, Seybold et al., 1992d]. Miller (1990) also demonstrated a preferential response to the racemate by three other populations in southern British Columbia. In a study of production of ipsdienol by individual I. pini, Miller et al. (1989) found that inter- and intrapoputational variation of enantiomeric composition of ipsdienol ranged from 91% ( - ) in California and 89% ( - ) in southeastern British Columbia to 43% ( - ) in New York and 34% ( - ) in southwestern British Columbia. Recently Teale and Lanier (1991) found considerable variability in the

ROLE OF LANIERONE IN

Ips

2307

response of a New York population to ipsdienol; in spring and early summer this compound had no activity. Subsequent investigations with the New York population resulted in the identification of lanierone (2-hydroxy-4,4,6-trimethyl2,5-cyclohexadien-l-one), which was shown to act as a synergist with synthetic racemic ipsdienol in the lab and in the field (Teale et al., 1991). In the present study we investigated a California population of L pini for the presence (or absence) and activity of lanierone to determine whether lanierone is a factor in the interpopulational specificity observed by Lanier et al. (1972). Because we have found that the black-bellied clerid, Enoclerus lecontei (Wolcott) (Coleoptera: Cleridae), is attracted to ipsdienol and lanierone and since E. leeontei is the principal predator of the western pine beetle, Dendroctonus brevicomis LeConte (Person, 1940), we also investigated a California population of D. brevicomis for the presence or absence of lanierone.

METHODS AND MATERIALS

Laboratory Studies. For analysis of lanierone production, L pini were collected on June 13, 1991 in ponderosa pine, Pinus ponderosa Laws., and Jeffrey pine, Pinus jeffreyi, Grev. & Balf. logging debris from area I of the field study site. For analysis of lanierone and ipsdienol production, D. brevicomis were collected on September 20, 1991 in a recently cut stem of P. ponderosa and by flight-trapping. The collection site was the University of California Blodgett Experimental Forest (El Dorado County, California). Insects were allowed to emerge from the infested material and stored at 4~ until utilized (Browne, 1972). Volatiles were collected separately from male and female L pini and D. brevicomis by introducing the insects individually into freshly cut logs of P. ponderosa. L pini males (741) and females (636) were introduced into the logs through holes drilled through the outer bark, secured in the artificial tunnels by screening, and allowed to feed in the phloem for 24 hr. After this feeding period, the logs (one set of three, 8-13 cm diameter • 28 cm length, for each sex) were placed into 19-liter silylated glass carboys (Sylon CT, Supelco Inc., Bellefonte, Pennsylvania) and aerated for 168 hr (July 12-19, 1991) with charcoalfiltered air (4 liters/rain). Volatiles from each carboy were trapped on 15 g Porapak Q (50/80 mesh) (Supelco Co., Bellefonte, Pennsylvania) (Byme et al., 1975) held in 25-cm • 2.4-cm (ID) glass columns. Each Porapak sample was extracted with 350 ml pentane. The same procedure was used with D. brevicomis males (166) and females (267), which were aerated October 15-22, 1991 (one set of two, 8- to 11-cm-diameter • 28-cm-length logs for each sex). Flight-trapped and newly emerged D. brevicomis of each sex were introduced into freshly cut P. ponderosa logs, allowed to feed for 48 hr, and recovered

2308

SEYBOLDET AL.

alive from the logs. Mid- and hindgut tissue was removed from fed and unfed insects and extracted for 2 hr in 200/zl pentane with 5 % ethyl acetate for analysis of lanierone production. D. brevicomis of each sex were also placed on glass wool in tightly sealed 19-liter glass carboys and incubated for 18 hr at room temperature with or without 200 #1 fl-myrcene (Byers, 1982). Mid- and hindgut tissue from these insects as well as from insects recovered from the Porapak Q aeration was removed and extracted for 24 hr in 200/xl diethyl ether for analysis of ipsdienol enantiomeric composition. Total guts dissected for ipsdienol analysis were as follows: unfed, no myrcene: 31 males, 44 females; unfed, myrcene: 78 males, 60 females; fed: 31 males; 37 females. The Porapak extracts of the L pini and D. brevicomis male and female aerations were analyzed for lanierone by GC-MS using multiple ion detection (m/z 109, 124, 137, 152) in a Finnigan model 4500 fitted with a 30-m SPB-1 capillary column (Supelco, Inc., Bellefonte, Pennsylvania), with the injection temperature set at 250~ The samples were run at 70 eV and the detection level for lanierone was - 0 . 5 ng. Porapak extracts of D. brevicomis male and female aerations were analyzed for ipsdienol by fractionation on HPLC (Seybold et al., 1992b,c). These fractions were assayed by GC for detection of ipsdienol (conditions below). The crude male D. brevicomis Porapak extract was also analyzed for the presence of ipsdienol by GC-MS using the conditions described above. Diethyl ether gut extracts of D. brevicomis were dried on sodium sulfate and filtered through sodium sulfate and glass wool before concentration to 20 /xl. Concentrated crude extracts were analyzed for ipsdienol enantiomeric composition by direct injection on capillary GC [Shimadzu GC-9A; column: 50 m Cydex-B (50 m x 0.22 mm ID x 0.25 /zm film), SGE Inc., Austin, Texas; He carrier gas; flow rate: 1.4 ml/min (2.7 kg/cm2), linear velocity: 24.2 cm/ sec, column temperature: 110~ isothermal, injector and detector temperatures: 250~ The presence of each enantiomer of ipsdienol on GC was verified by coinjection with authentic enantiopure ( + ) - and (-)-ipsdienol (Seybold et al., 1992a,c). Residue analysis of ipsdienol in membrane release devices was carried out by Consep Membranes Inc. (Bend, Oregon) using capillary GC. For analysis, each lure was cut into 1-cm strips and extracted in 10 ml acetone with 1 mg/ml hexadecane as internal standard. An aliquot of each extract (2 /xl) was analyzed by capillary GC [column: HP-5 (25 m x 0.2 mm ID x 0.33 #m film); He carrier gas; flow rate: 1 ml/min, linear velocity: 26.1 cm/sec; temperature program: 140~ for 5 min followed by 20~ to 220~ and held for 8 min; injection temperature: 250~ and detector temperature: 300~ Under these conditions, ipsdienol had a retention time of - 3 . 8 min and hexadecane had a retention time of - 9 . 5 min. Field Studies. A field study to determine the flight response of L pini and associated fauna to varying doses of lanierone released with a standard dose of

ROLE OF LAN1ERONE IN

lips

2309

ipsdienol was conducted between July 26 and August 3, 1991, in a recently thinned stand of P. ponderosa and P. jeffreyi located near Jelly Spring, Lassen County, California (19 km east of Hat Creek, Shasta County, California). This site is located 70 km southeast of that utilized by Birch et al. (1980) and Lanier et al. (1972) for L pini pheromone isolation and field testing. The 13-km 2 site was divided into three test areas, each separated by at least 2 km. Area I was located in the SE88 NE89 $26 T34N R6E, area II was located in the SW88 $23 T34N R6E, and area III was located in the NE88 S15 and NW88 $14 T34N R6E. In each area, eight-unit funnel traps (Pherotech Inc., Delta, British Columbia) (Lindgren, t983) and associated treatments were randomly assigned to positions located at least 100 m apart to minimize treatment interactions. This was a randomized block design, in which a block consisted of nine trap positions in a test area and a single day's trap catch. There were 24 blocks in the study (eight days at each of three test areas). Each day, the funnel traps were emptied, rebaited, and rerandomized between 7:30 and 11:30 AM. The treatments in the study were: (1) blank trap, (2) P. jeffreyi log infested with 20 male L pini, (3) synthetic ipsdienol (1 rag/day), (4) synthetic lanierone (20 #g/day), (5) ipsdienol (1 mg/day) + lanierone (0.1 /~g/day), (6)ipsdienol (1 mg/day) + lanierone (1 #g/day), (7) ipsdienol (1 rag/day) + lanierone (20 #g/day), (8) ipsdienol (1 rag/ day) + lanierone (100 #g/day), and (9) ipsdienol (1 mg/day) + lanierone (2 rag/day). Ipsdienol (neat) was released from membrane lures (Consep Membranes, Inc.), and lanierone (in hexane) was released from capped, 2.5-ml polyethylene vials (Kartell, Fisher Scientific). Each ipsdienol membrane lure was allowed to elute 1 mg for 24 hr, and then replaced with a fresh lure. The initial amount of racemic ipsdienol loaded in the device was 7.93 mg. The load was delivered volumetrically based on the specific gravity of pure ipsdienol (0.9) and adjusted for the chemical purity of the synthetic ipsdienol. Prior laboratory analysis 6 had shown that these lures release an average of 1.39 rag/day over a four-day period, 1.31 rag/day over a five-day period, and 1.02 mg/day over a seven-day period (conditions: 25~ + 1 ~ wind speed 0.3 m/sec, and relative humidity 20-30%). The daily release rate of ipsdienol during the study was determined from residue analysis of the used membrane lures. Each polyethylene vial was charged with a 100-#1 or 200-/zl hexane solution of lanierone in concentrations calibrated to give the desired release rates [100 #1 of 250 #g/ ml for 0.1 #g/day; 100 #1 of 500/zg/ml for 1 #g/day; 100 #1 of 1 mg/ml for 20 #g/day (two vials); 100/zl of 10 mg/ml for 100 #g/day; and 200 #1 of 50 mg/ml for 2 mg/day (two vials)]. The male-infested logs ( - 10 cm diameter • 12 cm length) were incubated for 36 hr prior to placement in the test and dissected following the test to determine how many males constructed feeding galleries. Throughout the course of the study, the mean maximum temperature 6Data on file at Consep MembranesInc., Bend, Oregon.

2310

SEYBOLD ET AL.

on the site was 32.7~ and the mean minimum temperature was 8.9~ with a range of 4.4~ to 36.1~ Because hexane was used as a solvent for lanierone, a second study was conducted to determine if the presence of hexane alone would influence the response of L pini and associated fauna to ipsdienol. This test was conducted on July 31, 1991 near area I of the lanierone dose study. The two treatments were ipsdienol (1 rag/day) and ipsdienol (1 mg/day) + hexane (100 ixl/day = 66 mgJday). Release devices were identical to those used in the lanierone dose study. Each treatment was replicated nine times. Because lanierone is a solid at room temperature, the effect of neat lanierone on the response of L pini to ipsdienol could not be tested. Chemicals. (-)-Ipsdienol [99.39% + 0.02% ( - ) , 97.86% + 0.61% chemical purity, mean of 10 injections + SE] used in field studies was purchased from Pherotech Inc. Technical grade /3-myrcene was purchased from Aldrich Chemical Co., Inc., Milwaukee, Wisconsin. Lanierone was synthesized in Syracuse (Teale et al., 1991). All solvents were HPLC grade. Statistical Analysis. Data from tests 1 and 2 were analyzed using a nonparametric randomized blocks procedure with aligned ranks to test for equality of all treatments (Lehmann, 1975, Chapter 6, pp. 270-273). Because we knew at the outset that our trap-catch data would not be normally distributed (trap catches are integer data), we chose to use this nonparametric analysis. Data were aligned by subtraction of the mean catch in each block. Selected pairwise comparisons of treatments were performed using the Wilcoxon signed-rank test (Lehmann, 1975, Chapter 3, pp. 123-132) and the Bonferroni method was used for pairwise comparisons to control overall error rate (procedurewise c~ = 0.05). Where results of tests are represented with an inequality sign, this indicates a one-sided test. Comparisons frequently involved the treatment ipsdienol (1 mg/ day) + lanierone (20/xg/day), because this represents an approximation of the ratio in which the two compounds occur in New York populations of L pini (Teale et al., 1991). In some cases, we performed one-sided pairwise comparisons in order to determine whether our data showed the same trends as reported for this insect in eastern North America (Teale et al., 1991). Regression analyses of percentage of male L pini and the number of E. lecontei responding on the logarithm of lanierone concentration were performed as described in Draper and Smith (1981, Chapter 1, pp. 8-33). In the regression analysis, a log [Y + 1] transformation was performed on the number of E. lecontei. An analysis of equality of proportions of male L pini caught for all treatments as well as pairwise comparisons of proportions of males caught for selected pairs of treatments were performed using Pearson's goodness-of-fit test. The Bonferroni method was also used to control overall error rate for these pairwise comparisons (procedurewise c~ = 0.05).

ROLE OF LANIERONEIN Ips

2311

RESULTS

Laboratory Studies. The multiple ion detection mass spectra (m/z 109, 124, 137, 152) of the I. pin• male and female aerations revealed ipsdienol and no detectable level of lanierone in the mate extract, but neither compound in the female extract. Lanierone and ipsdienol were not detected in gut extracts from fed or unfed male or female D. brevicomis. Neither compound was detected in Porapak Q aeration extracts from male or female D. brevicomis. However, ipsdienol was present in gut extracts from male (but not female) insects that had been aerated with myrcene. The enantiomeric composition of the ipsdienol present in males was 97.79 • 0.04% ( + ) (mean of five injections ___ SE). Laboratory residue analysis of used membrane lures from the field study (Table 1) showed that the mean daily release rate of ipsdienol throughout the course of the study was 2.56 + 0.26 mg (grand mean of differences between day 0 and other days, nine measurements). This estimate of release rate should be viewed as a maximum estimate. The lures analyzed for day 0 of the study were not taken in the field at the time that the test was begun and then shipped for analysis. Rather, they were prepared in Berkeley and shipped to Bend for analysis. Thus, these devices had representative expected loads for day 0 and were prepared using the same equipment and materials as the other devices, but were not treated identically. FieM Studies. With the exception of one dose (2 mg/day), the ianierone dose study shows that L pin• responds in larger numbers to the combination of ipsdienol and lanierone than it does to ipsdienol alone (Figure 1). The response TABLE 1. QUANTITYOF IPSDIENOL REMAINING IN MEMBRANE LURES FIELD TESTED 19 km EAST OF HAT CREEK, CALIFORNIA, JULY 26-AuGuST 3, 1991

Day of study 0 1 2 3 4 5 5u 6 7 8 Hexane test.

Ipsdienol (mg) (mean • SE) 7.77 4.69 5.68 5.24 5.44 4.17 3.97 5.60 5.99 6.14

• • • • • • • • • •

0.05 0.18 0.07 0.12 0.09 0.26 0.07 0.14 0.10 0.05

Lures analyzed (N) 12 18 18 9 9 9 18 9 9 9

Field temperature (~ (minimum-maximum) 8.9-34.4 10.0-35.6 10.0-36.1 10.0-35.6 11.1-35.0 11.1-35.0 8.9-31.1 4.4-24.4 7.8-28.9

2312

SEYBOLD E T AL. 80

-,

6O u}

r

E

~4o D.

--"

17,2

20

T

_!_ Blank

Males

Lanlerone Ipsdlenol

0.1

1.0

20

100

2000

C o n c e n t r a t i o n of Lanierone f,ug/day)

FIC. 1. The effect of varying the release rate of lanierone with a constant release rate [1 rag!day, 99.4% ( - ) ] of ipsdienol on the mean number of Ips pini trapped 19 km east of Hat Creek, California, July 26-August 3, 1991. Male-infested logs, ipsdienol alone (l rag/day), lanierone alone (20/~g/day), and blank traps served as controls. Treatment means (per trap per day) given above bars represent treatments involved in pairwise comparisons. The following pairwise comparisons were tested: Comparison Ho: Ipsdienol HA: Ipsdienol Ho: Ipsdienol HA: Ipsdienot Ho: Ipsdienot HA: Ipsdienol

_> ipsdienol + 20/xg lanierone -< ipsdienol + 20/zg lanierone ipsdienol + 20/zg lanierone HA: Ipsdienol -< ipsdienol + 20/~g lanierone Ho: Ipsdienol + 20/~g tanierone _> male-infested log HA: Ipsdienol + 20 t~g lanierone < male-infested log Ho: Ipsdienol + 2 mg lanierone _< ipsdienol + 0.1 /~g lanierone HA: Ipsdienol + 2 mg lanierone > ipsdienol + 0.1 tzg lanierone

Type of test

Significant

Two-sided

No (P = 0.3209)

One-sided

No (P = 0.0202)

One-sided

No (P = 0.6942)

One-sided

No (P = 0.7242)

The test for equality of all treatments was not significant (P = 0.2400). The pairwise comparisons were analyzed using the Pearson's goodness-of-fit test and the Bonferroni method was used to control for overall error rate (procedurewise c~ = 0.05).

2314

SZVBOLDET AL.

The proportion of male L pini responding was not influenced by the presence of lanierone (Figure 2). Regression analysis of percentage of males on logarithm of concentration of lanierone [log (mg)] did not fit the model [R 2 = 0.0026, Y = - 0 . 3 7 (log X) + 45.17 (where Y = percentage of males trapped and X = mg lanierone), slope not significantly different from 0, P = 0.58, c~ = 0.05]. The percentage of male L pini responding to the male-infested log (44.3%) was not significantly different from the percentage of male L pini responding to (-)-ipsdienol (41.4%) (Figure 2). The addition of lanierone at 20/zg/day did not cause a significant increase in the percentage of male L pini responding to ipsdienol (45.7% vs. 41.4%) and the addition of 2 mg/day lanierone did not cause a significant increase over 0.1 #g/day lanierone (43.7% vs. 45.3 %). There was no significant difference between the responses of L pini to ipsdienol and to ipsdienol + hexane (mean response to ipsdienol _+ SE: 44.5 _+ 9.5; mean response to ipsdienol + hexane +_ SE: 48.3 + 10.2; two-sided Wilcoxon signed-rank test, P = 0.9528). Insect predators and parasitoids of L pini responded to various treatments in these studies. The proportion of each sex of these associates responding was not determined. None of these insects appeared to be attracted to lanierone alone. However, some were attracted to ipsdienol alone, and one, Enoclerus Iecontei, responded to the combination of these compounds. In selected paired comparisons, the responses of the predators Temnochila chlorodia (Mannerheim) (Coleoptera: Trogositidae) and Enoclerus sphegeus (F.) (Coleoptera: Cleridae) to ipsdienol + lanierone (20 #g/day) did not differ significantly from their responses to ipsdienol alone (Figures 3 and 4). The responses of both species to ipsdienol alone were significantly greater than they were to an unbaited trap. This study indicated a significant difference between the responses of T. chlorodia to ipsdienol and to the male-infested log (Figure 3). In this case, ipsdienol was more attractive than the male-infested log. The response of E. sphegeus to ipsdienol was not significantly different from the response to the male-infested log (Figure 4). E. lecontei exhibited a clear dose-dependent response to lanierone when delivered with ipsdienol (Figure 5). The response of this predator to ipsdienol + lanierone (20 txg/day) did not differ significantly from its response to the male-infested log, while the response to ipsdienol + lanierone (20/xg/day) was significantly greater than it was to a blank trap. The catch at traps baited with ipsdienol alone was not significantly different from the response to the blank trap. Although we could not analyze the comparison between the response to the blank trap and the response to lanierone (in hexane) alone, no E. lecontei were trapped coming to either treatment (Figure 5). The estimated slope in the regression analysis for E. lecontei was significant [R 2 = 0.52, log (Y + 1) = 0.17 (logX) + 0.69 (where Y = trap catch and X = mg lanierone), slope significantly different from O, t test, P = 0.0001, c~ -- 0.05].

ROLE OF L A N I E R O N E IN

[ps

2315

5

3.25

~4 UJ

"el

E ~2

0

Blank

Males

Lanlerone Ipsdlenol 0.1 %0 20 Concentration of Lanlerone f.ug/day)

100

2000

FIG. 3. The effect of varying the release rate of lanierone with a constant release rate [1 mg/day, 99.4% ( - ) ] of ipsdienol on the mean number of Temnochila chlorodia trapped 19 km east of Hat Creek, California, July 26-August 3, 1991. Ips pini maleinfested logs, ipsdienol alone (1 rag/day), lanierone alone (20 ~g/day), and blank traps served as controls. Treatment means (per trap per day) given above bars represent treatments involved in pairwise comparisons. The following pairwise comparisons were tested: Comparison Ho: Ipsdienol Ha: Ipsdienol Ho: Ipsdienol HA: Ipsdienol Ho: Ipsdienol Ha: Ipsdienol

_< blank > blank = male-infested log :~ male-infested log = ipsdienol + 20 #g lanierone :~ ipsdienol + 20 ~g lanierone

Type of test

Significant

One-sided

Yes (P < 0.0001)

Two-sided

Yes (P = 0.0026)

Two-sided

No (P = 0.3830)

The test for equality of all treatments was significant (P < 0.0001). The pairwise comparisons were analyzed using the Wilcoxon signed-rank test and the Bonferroni method was used to control for overall error rate (procedurewise c~ = 0.05).

A m e a n o f 2.4 Tomicobia tibialis A s h m e a d (Hymenoptera: Pteromalidae) (Figure 6) were trapped at male-infested logs each day while a m e a n o f less than 0.2 T. tibialis were trapped at lanierone, ipsdienol, or the c o m b i n a t i o n of these c o m p o u n d s . The response to the male-infested log was significantly greater than it was to the b l a n k trap, to ipsdienol, or to ipsdienol + lanierone (20 t~g/ day).

2316

S E Y B O L D ET A L . 1.2

1

c 0.8

g ~. 0.6

~ o

0.4

0.2

0

BIBnk

Males

Lanlerone Ipsdlenol 0.1 1.0 2O C o n c e n t r a t i o n o f L a n i e r o n e 01g/day)

100

2OO0

F~G. 4. The effect of varying the release rate of lanierone with a constant release rate [1 rag/day, 99.4% ( - ) ] of ipsdienol on the mean number of Enoclerus sphegeus trapped 19 km east of Hat Creek, California, July 26-August 3, 1991. I. pini male-infested logs, ipsdienol alone (1 mg/day), lanierone alone (20 /~g/day), and blank traps served as controls. Treatment means (per trap per day) given above bars represent treatments involved in pairwise comparisons. The following pairwise comparisons were tested: Comparison Ho: Ipsdienol HA: Ipsdienol Ho: Ipsdienol HA: Ipsdienol Ho: Ipsdienol HA: Ipsdienol

_< blank _> blank = male-infested log ,,s male-infested log = ipsdienol + 20 tzg lanierone r ipsdienol + 20 tzg lanierone

Type of test

Significant

One-sided

Yes (P = 0.0005)

Two-sided

No (P = 0.8557)

Two-sided

No (P = 0.2775)

The test for equality of all treatments was significant (P = 0.0010). The pairwise comparisons were analyzed using the Wilcoxon signed-rank test and the Bonferroni method was used to control for overall error rate (procedurewise oe = 0.05).

A total o f 40 Pityogenes carinulatus (LeConte) (Coleoptera: Scolytidae) were trapped during the study and 38 o f the specimens responded to treatments involving lanierone and ipsdienol. The test for equality of all treatments was significant (P = 0.002) for P. carinulatus and the response to ipsdienol + 0.1 #g/day lanierone (0.33 insects/trap/day) was significantly greater than the response to the blank trap (0 insects/trap/day)(P = 0.0072).

ROLE OF LANIERONEIN Ips 10

2317

-1

l "O

8 _.e

T

u]

0.88T

0.63

I

T BMnk

MaMs

Lanierone

Ipsdlenol

0.1

1.0

20

100

2000

Concentration of Lanierone (.ug/day)

FIG. 5. The effect of varying the release rate of lanierone with a constant release rate [1 rag/day, 99.4% ( - ) ] of ipsdienol on the mean number of Enoclerus lecontei trapped 19 km east of Hat Creek, California, July 26-August 3, 1991. I. pini male-infested logs, ipsdienol alone (1 rag/day), lanierone alone (20 Fzg/day), and blank traps served as controls. Treatment means (per trap per day) given above bars represent treatments involved in pairwise comparisons. The following pairwise comparisons were tested: Comparison Ho: lpsdienol = blank HA: Ipsdienol ~ blank Ho: Lanierone = blank HA: Lanierone :~ blank Ho: Ipsdienol + 20/xg lanierone HA: Ipsdienol + 20 txg lanierone Ho: Ipsdienol + 20/xg lanierone HA: Ipsdieno! + 20/zg lanierone

Type of test

_< blank >_ blank = male-infested log :/= male-infested log

Significant

Two-sided

No (P = 0.0833)

Two-sided

Could not test

One-sided

Yes (P < 0.0001)

Two-sided

No (P = 0.1786)

The test for equality of all treatments was significant (P < 0.0001). The pairwise comparisons were analyzed using the Wilcoxon signed-rank test and the Bonferroni method was used to control for overall error rate (procedurewise c~ = 0.05). T h e predators Enoclerus barri Knull (Coleoptera: Cleridae) and Othnius spp. (Coleoptera: Othniidae), and the associates Lasconotus subcostulatus Kraus (Coleoptera: Colydiidae), Corticeus spp. (Coleoptera: Tenebrionidae), Dendroctonus valens L e C o n t e (Coleoptera: Scolytidae), Pityophthorus jeffreyi B l a c k m a n (Coleoptera: Scolytidae), Aulonium spp. (Coleoptera: Colydiidae),

2318

SEYBOLD ET AL.

3., 1 3

2.38

m 2.5

E2

0.5 0.08 T

0

Blank

Males

0.04 T

T

T

Lanlerone Ipsdlenol 0.1 t.0 20 Concentration of Lanierone (Jug/day)

1(10

2O00

F ~ . 6. The effect of varying the release rate of lanierone with a constant release rate [1 mg/day, 99.4% ( - ) ] of ipsdienol on the mean number of Tomicobia tibialis trapped 19 km east of Hat Creek, California, July 26-August 3, 1991. Ips pini male-infested logs, ipsdienol alone (1 mg/day), tanierone alone (20 /zg/day), and blank traps served as controls. Treatment means (per trap per day) given above bars represent treatments involved in pairwise comparisons. The following pairwise comparisons were tested: Comparison Ho: Male-infestedlog HA: Male-infestedlog Ho: Male-infestedlog HA: Male-infestedlog Ho: Male-infestedlog HA: Male-infestedlog

_< blank >- blank -< ipsdienol >_ ipsdienol _< ipsdienol + 20 tzg lanierone >_ ipsdienol + 20/zg lanierone

Type of t e s t

Significant

One-sided

Yes (P < 0.0001)

One-sided

Yes (P < 0.0001)

One-sided

Yes (P < 0.0001)

The test for equality of all treatments was significant (P < 0.0001). The pairwise comparisons were analyzed using the Wilcoxon signed-rank test and the Bonferroni method was used to control for overall errOr rate (procedurewise c~ = 0.05).

Lyctocoris spp. (Hemiptera: Anthocoridae), and Cylistix spp. (Coleoptera: Histeridae) were trapped only in small numbers, and thus conclusions cannot be drawn about their responses to ipsdienol or lanierone. Dissection of the 24 male-infested logs used in this study revealed that a mean of 18.04 (range 15-20) males had constructed feeding galleries in each of these logs.

ROLE OF LANIERONE IN

Ips

2319

DISCUSSION

The California population of L pini that we studied behaves differently than the New York population in several ways. Ipsdienol elicits a proportionately higher level of response from California L pini than a male-infested log (Figure 1). However, New York L pini, in both lab and field studies, responds in significantly greater numbers to male-produced volatiles or a male-infested log than it does to ipsdienol (Teale et al., 1991). Teale et al. (1991) used a fieldstudy dose of ipsdienol (10 mg for a six-day test period) similar to that used in California (2.6 mg/day). Birch et al. (1980) discovered the importance of the (-)-enantiomer of ipsdienol to the field attraction of this California population of L pini. Response of L pini to 99.4% (-)-synthetic ipsdienol in our study may exceed the response to the male-infested log because of the higher release rate from the devices or because the optical purity of the synthetic compound exceeds that of a log infested with 20 male L pini [mean 98.2% ( - ) , range 96.6-98.9% ( - ) , 12 replicates]. The dose of (-)-ipsdienol released in 24 hr from these male-infested logs ranges from 0.7 to 1.9 rag/day (Seybold et al., 1992d). In California, L pini responds to ipsdienol at levels comparable to its response to a male-infested log from May through August (Seybold et al., 1992d), while in New York L pini responds to ipsdienol in greater numbers (although still lower than to the male-infested log) only after the beginning of August (Teale and Lanier, 1991). Similarly, in Wisconsin, Raffa (1991) reported trapping the most beetles late in the season, but the level of response relative to male-produced attractants was not tested. Nonetheless, in two of three years where the flight behavior of L pini was studied from spring through fall, Raffa (1991) demonstrated a response to ipsdienol in April and May. Wisconsin and New York populations are similar because they produce and respond to similar enantiomeric blends of ipsdienol (Seybold et al., unpublished; Raffa and Klepzig, 1989; Teale, 1990). Another difference between California and New York populations is that the response of California L pini to increasing doses of lanierone in the presence of ipsdienol does not demonstrate the clear dose-dependent response elicited from New York L pini (Figure 1 and Teale et al., 1991). In addition, the percentage of male L pini responding in California to the male-infested log is not significantly different from the percentage of male L pini responding to ( - ) ipsdienol alone. Adding lanierone to ipsdienol does not affect the proportion of males responding in California. In two different field studies, Birch et al. (1980) also found that the male to female sex ratio ofL pini responding to (-)-ipsdienol was not different from the sex ratio of L pini responding to a male-infested log. However, in New York, Teale et al. (1991) have found a positive relationship between the percentage of males in the responding population and the quantity

2320

SEYBOLOET AL.

of lanierone added to a fixed amount of ipsdienol. They also found that the percentage of male responders was highest (48%) to the male-infested log. In California, the percentage of males responding to the male-infested log (44.3 %) was not significantly greater than the percentage of males responding to ipsdienol and 20 #g/day lanierone (45.7%). There is a considerable difference between the enantiomeric composition of ipsdienol produced by New York and California populations of L pini. Birch et al. (1980) isolated ipsdienol from benzene extracts (Young et al., 1973) of California L pini male-produced frass, as well as from volatiles condensed in a liquid nitrogen trap. The enantiomeric composition of the ipsdienol from this isolation was not determined, but Stewart (1975) found that California L pini produce (-)-ipsdienol, and Miller et al. (1989) obtained a mean of 91% ( - ) from a population collected near Hat Creek, California. Lanier et al. (1980) reported the isolation of ipsdienol from male New York L pini by trapping volatiles on Porapak Q. Earlier, the enantiomeric composition of this ipsdienol had been found to be 35% ( - ) (Stewart, 1975). Miller et al. (1989) measured a mean of 43 % ( - ) from a population collected in New York. The isolations by Birch et al. (1980) and Lanier et al. (1980) were both guided by lab and field assays. In addition to the differences in the stereochemistry of ipsdienol, we have found that a California population of L pini does not produce detectable levels of lanierone. Based on the smallest detectable peak in the GC (FID) trace, lanierone production would have to be much less than 0.002% of the amount of ipsdienol produced by male L pini in California. This is one-hundredth of the amount of lanierone (0.2%, relative to ipsdienol) produced by I. pini in New York (Teale et al., 1991). The absence of lanierone in this California population of L pini and its presence in New York populations may be related to differences in the hosts (P. ponderosa vs. Pinus resinosa Ait.) from which volatiles were collected. Since Teale et al. (1991) did not find lanierone in Porapak extracts from female New York L pini in P. resinosa, it is unlikely that lanierone is present in the host itself. However, precursors for biosynthesis of lanierone may be present in P. resinosa, but not in P. ponderosa. It should be noted that the host species does not appear to affect ipsdienol production (including enantiomeric composition), which was unchanged when males from New York populations reared in P. resinosa were allowed to feed in P. ponderosa (Seybold et al., unpublished). Besides the interfertility of eastern (Ontario) and western (California and Arizona) populations of I. pini (Lanier, 1972), there are also some similarities in the flight behavior of western (California) and eastern (New York) populations. Neither responds to lanierone alone, and both appear to be inhibited at high lanierone concentrations [10 mg for a six-day test 7 period for eastern pop7Log[rag] doses in Teale et al. (1991) Figure 11 should read left to right, -3, -2, -1, 0, and 1.

ROLE OF LANIERONE IN

Ips

2321

ulations (Teale et al., 1991) and 2 mg/day for western populations (this study)]. Although western populations of L pini do not produce detectable amounts of lanierone, the ipsdienol-elicited flight response of L pini in California can be increased by its presence. This latter result would seem to suggest a relictual behavioral link between the eastern and western forms of L pini. Much remains to be learned about the evolutionary history of the pheromone variants of L pini [formerly L oregonis (Eichhoff) = western and I. pini (Say) = eastern; Hopping, 1964]. Based on glacial history and production and response profiles of enantiomeric blends of ipsdienol, Miller (1990) suggested that eastern populations of L pini originated from a southeastern refugium in the Appalachian Mountains, that eastern-like populations currently in British Columbia originated from a southwestern refugium, and that western populations in British Columbia derived from populations similar to those presently in northern California. However, there are other possible explanations for the origin of the pheromone types noted in British Columbia. These include introduction from eastern localities (J.W.A. Volney, personal communication); blending of eastern and western forms originating from eastern and western glacial refugia, respectively (D.E. Bright, personal communication); and post-Pleistocene colonization from the Beringia (= Yukon) refugium (D.E. Bright, personal communication). It is possible that an eastern population of L pini was introduced and established in British Columbia. Movement of insects in logs, timbers, and rough-wood crates by human transport is a well-known current problem (Ciesla, 1988), but it has probably also occurred during the past century. For example, there is an endemic population of I. calligraphus calligraphus in central California that probably originated from the southeastern United States and was present at least by 1914 (Herbert, 1916, Wood and Stark, 1968, and Lanier et al., 1991). The eastern form of L pini may have moved naturally into northwestern Canada from a southeastern refugium (Matthews, 1979) or from midcontinental refugia (Critchfield, 1985) by following the post-Pleistocene migration of jack pine, Pinus banksiana Lamb. P. banksiana is a rapidly colonizing species that invaded western Canada during the last 10,000 years where it occurs in a hybridization zone with P. contorta latifolia in northern Alberta and the southern Yukon Territory (Critchfield, 1957, 1985). P. contorta appears to have experienced a more sedentary post-Pleistocene colonization pattern, originating from refugia south of the Cordilleran glacial ice mass and, perhaps, in the Yukon (Critchfield, 1985). This zone of host hybridization prompts the question whether eastern and western forms of L pini also form a zone of sympatry in this region. The rapid migration of P. banksiana through Canada suggests that the eastern form of L pini may have become established in British Columbia in P. contorta latifolia prior to or contemporaneously with the advent of the western form,

2322

SEYBOLD ET AL.

which may have also utilized P. ponderosa, P. contorta murrayana, or P. contorta contorta as its refugial hosts. Ross (1955) also noted the transcontinental distribution of P. banksiana as a major factor in the evolution of the Neodiprion sawflies. Despite the continuity of genetic variation in P. banksiana across Canada, it is possible that it also occupied one or more western refugia during the Pleistocene (Critchfield, 1985). Similarly, host islands of P. contorta may have been available for L pini in ice-free corridors between the Cordilleran and Laurentide ice sheets (Critchfield, 1985). Miller (1990) discounted Beringia as a source of present-day I. pini in British Columbia based on the meager and highly localized modern-times collection record of L pini in coastal southeastern Alaska (Wood, 1982). However, in addition to indications that the host, P. contorta, may have been in a Yukon refugium (Critchfield, 1985), there is one record of L pini from interior Alaska (Bright, 1976). Moreover current distribution does not always reflect historical distribution [e.g., the scolytid, Carphoborus andersoni Swaine, occurs only in northern Alberta, the Northwest Territories, and Alaska, but fossils indicate its former presence in Minnesota, New York, and Ontario between 10,000 and 70,000 years ago (Bright, 1976; Wood, 1982; D.E. Bright, personal communication)]. Gahan (1938) and Reid (1957) documented the association of the solitary parasitoid wasp T. tibialis with L pini. T. tibialis has also been shown to respond to logs infested with male California five-spined ips, lps paraconfusus Lanier (Bedard, 1965) and to logs infested with male and not female I. pini (Rice, 1968). In a field study in California, Lanier et al. (1972) demonstrated that T. tibialis preferred California and Idaho male L pini-infested logs over New York male L pini-infested logs. In our study we have found that T. tibialis responded to logs infested with male I. pini, but not to ipsdienol, lanierone, or any combination of these compounds. This indicates that the kairomone that attracts T. tibialis to L pini probably involves other chemicals, although it may include ipsdienol, which is produced by males of both L paraconfusus and L pini (Silverstein et al., 1966; Birch et al., 1980). In the present study, 1". tibialis was often observed and collected from the white surface of the top of the funnel traps beneath the male-infested logs, but it was never observed or collected from this surface or near the release devices on traps baited with synthetic chemicals. Nonchemical behavioral stimuli associated with the presence of male frass, the pine log, and the attracted live bark beetles may also have contributed to the arrestment of T. tibialis on the surface of traps baited with male-infested logs. In contrast to its influence on L pini, lanierone had no effect on the responses of T. chIorodia or E. sphegeus to ipsdienol. As with California L pini, the response of these predators to the male-infested log appears to be fully explained by their response to ipsdienol. Despite many prior studies of bark beetle pheromones where T. chlorodia has been shown to respond to various natural and

ROLE OF LANIERONE IN

Ips

2323

synthetic substrates, it has never been found to respond to ipsdienol alone [D. brevicomis-infested log (Vit6 and Gara, 1962); male L paraconfusus-infested log (Vit6 and Gara, 1962; Rice, 1969); synthetic exo-brevicomin (Bedard et al., 1969, 1980; Vit6 and Pitman, 1969); synthetic ipsenol and male L pini-infested log (Furniss and Livingston, 1979); and mixture of ipsenol, ipsdienol, and cisverbenol (Wood et al., 1968; Wood, 1970)]. The red-bellied clerid, E. sphegeus, is a principal predator of the mountain pine beetle, Dendroctonus ponderosae Hopkins (Stmble, 1942), and also preys on Ips spp. (Furniss and Carolin, 1977). Furniss and Livingston (1979) reported low numbers of E. sphegeus responding in Idaho to synthetic ipsenol and combined with logs infested with male I. pini, while Miller and Borden (1990) presented clear evidence from British Columbia that E. sphegeus is attracted to synthetic, racemic ipsdienol. In California we have confirmed the response of E. sphegeus to ipsdienol and further demonstrated that E. sphegeus will respond to almost optically pure (99.4%) (-)-ipsdienol. The black-bellied clerid, E. lecontei, is the principal predator of D. brevicomis (Person, 1940) and is known to aggregate rapidly on trees freshly attacked by D. brevicomis (Stephen and Dahlsten, 1976). It also preys on Ips spp. (Furniss and Carolin, 1977). Vit6 and Gara (1962) reported attraction of E. lecontei to P. ponderosa logs infested with any of five species of scolytids, including L paraconfusus and D. brevicomis. Wood et al. (1968) and Wood (1970) trapped E. lecontei responding to P. ponderosa logs infested with male L paraconfusus and to various mixtures of synthetic ipsenol, ipsdienol, and cis-verbenol. Lanier et al. (1972), Birch and Wood (1975), and Furniss and Livingston (1979) have reported response of E. lecontei to natural aggregation pheromones produced by male L pini. E. lecontei responded in a dose-dependent fashion to synthetic, racemic ipsdienol (Miller and Borden, 1990). In that study the pine monoterpene, /3-phellendrene, had no effect on the response of E. lecontei. In our study, E. lecontei did not respond to one release rate of 99.4% ( - ) ipsdienol or lanierone individually above control levels; however, together the two compounds represent a synergistic kairomone for a significant bark beetle natural enemy. This pattern of activity is similar to that discovered for L paraconfusus, where three terpene alcohols, including ipsdienol, were inactive individually, but together they elicited the attractant response (Silverstein et al., 1966; Wood et al., 1968). The dose-dependent response (Figure 5) stands in contrast to the response of L pini (Figure 1) to these two compounds, and to the results obtained by Miller and Borden (1990) in British Columbia. Indeed, E. lecontei in California responds to the ipsdienol-lanierone combination as/. pini does in New York (Teale et al., 1991). It would be interesting to determine the response pattern for E. lecontei to increasing doses or variable enantiomeric compositions of ipsdienol in the presence of a fixed dose of lanierone. Lanier et al. (1972) found that E. lecontei in California and Idaho exhibited

2324

SEYBOLD ET AL.

a strong preference for logs infested with male L pini from New York over those infested with males from California and Idaho. The strong response of E. lecontei to lanierone and ipsdienol, which are both produced by New York L pini, and the absence or very low amounts of lanierone in California L pini in this study are sufficient to explain the preference observed by Lanier et al. (1972). In this study, the lower response of E. lecontei to ipsdienol alone than to the male-infested log (Figure 5) may indicate the release of additional kairomonal compounds from the male-infested log. The strong response of E. lecontei to lanierone + ipsdienol may have evolved because of a close association between E. lecontei and eastern populations of I. pini, for which lanierone plays a major role in aggregation (Teale et ale, 1991). The current distribution ofE. lecontei is reported to range eastward from the Pacific coast to Ontario and Michigan (Person, 1940; Papp, 1960), and L pini is the principal bark beetle attacking pines in northeastern and north central North America. In contrast, E. sphegeus and T. chlorodia, neither of which discriminated between the combination of lanierone + ipsdienol and ipsdienol alone, have entirely western distributions (Person, 1940; Barron, 1971). T. chlorodia is replaced by the closely related T. virescens (F.) and T. acuta LeConte in eastern and southern North America (Barron, 1971). Another possibility is that the strong response of E. lecontei to lanierone + ipsdienol in western North America may have evolved in response to production of lanierone and ipsdienol by a western ancestral form of L pini that resembled its present-day eastern counterpart in this trait. Selection pressure exerted by E. lecontei on I. pini may have resulted in the loss or reduction of the biosynthetic capacity for lanierone production and a reduced response to lanierone and ipsdienol by the present population of L pini in California relative to other populations in North America. Miller et al. (unpublished) have found that the relative increase in response of California 1. pini to lanierone and ipsdienol compared to ipsdienol alone is the lowest of six populations tested in North America. It has been suggested that selection pressure by the predaceous clerid Thanasimus dubius (F.) results in a shift in response to the enantiomers of ipsdienol by populations of L pini in north-central North America (Raffa and Klepzig, 1989; Herms et al., 1991). In the same fashion, Payne et al. (1984) suggested that selection pressure by T. dubius on D. frontalis may result in a production-response shift in the enantiomeric composition of frontalin. Alternatively, Eo lecontei may have evolved a strong kairomonal response to ipsdienol and lanierone because these compounds are produced by other prey species in western North America. Male D. brevicomis have been reported to produce (+)-ipsdienol (Byers, 1982), and this compound was present in the extracts of hindgut tissue from males that were collected live from a pheromonebaited tree (Byers et al., 1984). In the latter study, the amount of ipsdienol was maximal when the males were exposed to elevated levels of oleoresin exuded

ROLE OF LANIERONE IN

[ps

2325

by the attacked tree (day 2, 80 ng/male). Ipsdienol remained detectable later in the colonization process (days 8 and 11, 5 and 7 ng/male, respectively). The high, but slightly delayed response of E. lecontei to an aggregation of D. brevicomis (Byers et al., 1984) may be explained by lanierone production in either or both sexes of D. brevicomis several days after arrival on the tree. This would also explain the increase in response of E. lecontei when a D. brevicomisinfested log is added to a male L paraconfusus log (Byers and Wood, 1980). Therefore, we hypothesized the presence of lanierone in D. brevicomis, but did not detect it. It is possible that lanierone is produced by D. brevicomis when both sexes are together in the standing tree or that it is produced by E. lecontei itself as an aggregation pheromone component whose activity is dependent on ipsdienol, a bark beetle-produced synergist. We have shown for the first time that the enantiomeric composition of ipsdienol produced by male D. brevicomis is 97.8% (+), which is slightly greater than that produced by the cohabiting I. paraconfusus from the same locality (Seybold et al., 1992b,c). This enantiomeric composition of ipsdienol may limit aggregations of D. brevieomis. Byers (1982) has shown that racemic ipsdienol interrupts response of D. brevicomis to synthetic aggregation pheromones. However, various enantiomeric blends of ipsdienol remain to be tested against the natural pheromone produced in trees under attack by D. brevicomis. This enantiomeric composition of ipsdienol would also interrupt aggregations of L pini in areas where L pini and D. brevicomis co-occur in P. ponderosa (e.g., eastern California, Oregon, Washington, Idaho, and southeastern British Columbia). The absence of male-produced ipsdienol in gut extracts of fed and unfed insects, as well as in the extract from the Porapak Q aeration may be a result of low myrcene content in the P. ponderosa logs used to feed D. brevicomis males. Byers et al. (1984) observed the correlation of ipsdienol production in male D. brevicomis with oleoresin exudation from P. ponderosa and myrcene is present in P. ponderosa oleoresin. Kohnle and Vit6 (1984) have classified the prey location strategies of bark beetle predators as generalist or specialist based on the number of kairomones used for prey location. Several members of the Trogositidae, including T. chlorodia, were grouped among the specialists (one kairomone), while members of the Cleridae were grouped among the generalists (several kairomones). However, we have demonstrated that, in addition to exo-brevicomin, T. chlorodia also responds to ipsdienol, which is produced by at least 21 species of Ips in North America (Seybold et al., 1992b) and by male D. brevicomis (Byers, 1982). E. leeontei and E. sphegeus, the principal predators of D. brevicomis and D. ponderosae, respectively, also respond to ipsdienol + lanierone and ipsdienol, respectively. In contrast to species-specific aggregation pheromones of their prey, these bark beetle predators have evolved the capacity to recognize multiple kairomone signals that are important components of the aggregation

2326

SEYBOLD ET AL.

p h e r o m o n e o f different prey species. Vit6 and Gara (1962) o b s e r v e d the response o f these three predators to five scolytid species. T h e ability to locate aggregations o f several bark beetle species occurring throughout its geographic range increases the size o f the possible food base for the predator and thus increases the probability o f survival and reproduction. T h e strong response o f E. lecontei to ipsdienol and lanierone released at 2 rag/day and the inhibition o f L pini response at this dose suggest a novel control strategy using behavioral chemicals. Nascent aggregations o f L pini could be treated with a proportionately high dose o f lanierone c o m p a r e d to ipsdienol, which w o u l d m i n i m i z e further bark beetle aggregation while recruiting additional predators. T o g e t h e r with lanierone, the use o f ( + ) - i p s d i e n o l , a strong inhibitor for L pini (Seybold et al., 1992d) and possibly for D. brevicomis (Byers, 1982), m a y also decrease the probability o f further bark beetle aggregation. Acknowledgments--We dedicate this study to the memory of the late Gerald N. Lanier, a pioneer in insect chemical ecology, who devoted much of his scientific career to unravelling the pheromone biology of lps pini. We would like to thank P. Shea, USDA Forest Service, Davis, California, for providing the facilities for the field portion of our study and J. Tupy, N. Lewis, and J. Davis for field and laboratory assistance. J. Tupy critically reviewed the manuscript. J. Gillespie, M. Banfield, D. Bestwick, E. Christensen, and K. Hayes of Consep Membrane, Inc., Bend, Oregon, carried out the formulation and analysis of ipsdienol. We thank D. Bright for stimulating and helpful discussions regarding the evolution of L pini and for identifying scolytid associates of L pini. C. Jordan, Department of Entomological Sciences, University of California at Berkeley, provided assistance with preparation of graphics. This research was supported by the University of California at Berkeley, National Science Foundation Dissertation Improvement Grant BSR-8914625 to S.J.S. and D.L.W., and USDA-CSRS Grant 8802317 to F.X.W.

REFERENCES

BARRON, J.R. 1971. A revision of the Trogositidae of America north of Mexico (Coleoptera: Cleroidea). Mem. Entomol. Soc. Can. 75:1-143 BEDARD,W.D. 1965. The biology of Tomicobia tibialis (Hymenoptera: Pteromalidae) parasitizing Ips confusus (Coteoptera: Scolytidae) in California. Contrib. Boyce Thompson Inst. 23:77-82. BEDARD,W.D., TILDEN,P.E., WOOD, D.L., SILVERSTEIN,R.M., BROWNLEE,R.G., and RODIN, J.O. 1969. Western pine beetle: Field response to its sex pheromone and a synergistic host terpene, myrcene. Science 164:1284-1285. BEDARD,W.D., WOOD,D.L., TILDEN,P.E., LINDAHL,K.Q., SILVERSTEIN,R.M., and RODIN,J.O. 1980. Field responses of the western pine beetle and one of its predators to host- and beetleproduced compounds. J. Chem. Ecol. 6:625-641. BIRCH, M.C., and WOOD, D.L. 1975. Mutual inhibition of the attractant pheromone response by two species of Ips (Coleoptera: Scolytidae). J. Chem. Ecol. 1: 101-113. BIRC~, M.C., LIGr~T, D.M., WOOD, D.L., BROWNE, L.E., SILVERSTEIN,R.M., BERGOT, B.J., OHLOFF, G., WEST, J.R., and YOUNG,J.C. 1980. Pheromonal attraction and allomonal interruption of Ips pini in California by the two enantiomers of ipsdienol. J. Chem. Ecol. 6:703717.

ROLE OF LANIERONEIN Ips

2327

BRIGHT, D.E. 1976. The Insects and Arachnids of Canada, Part 2. The Bark Beetles of Canada and Alaska. Canada Department of Agriculture Publication 1576, 241 pp. BROWNE, L.E. 1972. An emergence cage and refrigerated collector for wood-boring insects and their associates. J. Econ. Entomol. 65:1499-1501. BYERS, J.A. 1982. Male-specific conversion of the host plant compound, myrcene, to the pheromone, (+)-ipsdienol, in the bark beetle, Dendroctonus brevicomis. J. Chem. Ecol. 8:363-371. BYERS, J.A., and WOOD, D.L. 1980. Interspecific inhibition of the response of the bark beetles, Dendroctonus brevicomis and Ips paraconfusus, to their pheromones in the field. J. Chem. Ecol. 6:149-164. BYERS, J.A., WOOD, D.L., CRAIG, J., and HENDRY, L.B. 1984. Attractive and inhibitory pheromones produced in the bark beetle, Dendroctonus brevicomis, during host colonization: Regulation of inter- and intraspecific competition. J. Chem. Ecol. 10:861-877. BYRNE, K.J., GORE, W.E., PIERCE, G.T., and SILVERSTEIN,R.M. 1975. Porapak-Q collection of airborne organic compounds serving as models for insect pheromones. J. Chem. Ecol. 1 : 1-7. CIESLA, W.M. 1988. Pine bark beetles: A new pest management challenge for Chilean foresters. J. For. 86:27-31. CRITCHFIELD,W.B. 1957. Geographic variation in Pinus contorta. Maria Moors Cabot Foundation Publication No. 3. 118 pp. CRITCHFIELD,W.B. 1985. The late Quaternary history of lodgepole and jack pines. Can. J. For. Res. 15:749-772. DRAPER, N.R., and SMITH, H. 1981. Applied Regression Analysis. John Wiley & Sons, New York, 709 pp. FURNISS, M.M., and LIVINGSTON, R.L. 1979. Inhibition by ipsenol of pine engraver attraction in northern Idaho. Environ. Entomol. 8:369-372. FURN1SS, R.L., and CAROLIN,V.M. 1977. Western forest insects. USDA Forest Service Miscellaneous Publication No. 1339, 654 pp. GAHAN, A.B. 1938. Notes on some genera and species of Chalcidoidea (Hymenoptera). Proc. Entomol. Soc. Wash. 40:219-223. HERBERT, F.B. 1916. Biological notes on certain western species of the genus lps (DeGeer). USDA Bureau Entomology and Plant Quarantine Unpublished Report, 44 pp. HERMS, D.A., HAACK,R.A., and AYRES, B.D. 1991. Variation in semiochemical-mediated preypredator interaction: Ips pini (Scolytidae) and Thanasimus dubius (Cleridae). J. Chem. Ecol. 17:515-524. HOPPING, G.R. 1964. The North American species in group IV and V ofIps DeGeer. Can. Entomol. 96:970-978. KOHNLE, U., and VITfi, J.P. 1984. Bark beetle predators: Strategies in the olfactory perception of prey species by clerid and trogositid beetles. Z. Angew. Entomol. 98:504-505. LANIER, G.N. 1972. Biosystematics of the genus lps (Coleoptera: Scolytidae) in North America. Hopping's Groups IV and X. Can. Entomol. 104:361-388. LANIER, G.N., and BURKHOLDER,W.E. 1974. Pheromones in speciation of Coleoptera, pp. 161189, in M.C. Birch (ed.). Pheromones. North Holland, Amsterdam. LANIER, G.N., BIRCH, M.C., SCHMITZ, R.F., and FURNISS, M.M. 1972. Pheromones of Ips pini (Coleoptera: Scolytidae): Variation in response among three populations. Can. Entomol. 104:1917-1923. LANIER, G.N., CLAESSON,A., STEWART,T., PISTON, J., and SILVERSTEIN, R.M. 1980. Ips pini; The basis for interpopulational differences in pheromone biology. J. Chem. Ecol. 6:677-687. LANIER,G.N., TEALE,S.A., and PAJARES,J.A. 1991. Biosystematics of the genus Ips (Coleoptera: Scolytidae) in North America: Review of the Ips calligraphus group. Can. Entomol. 123:11031124. LEHMANN, E.L. 1975. Nonparametrics: Statistical Methods Based on Ranks. Holden-Day, San Francisco, 457 pp.

2328

SEYBOLD ET AL.

LINDGREN, B.S~ 1983. A multiple funnel trap for scolytid beetles (Coleoptera). Can. Entomol. 115:299,302. MATHEWS,J.V. 1979. Tertiary and quaternary environments: Historical background for an analysis of the Canadian insect fauna, pp. 31-86, in H.V. Danks (ed.). Canada and its Insect Fauna. Memoirs of the Entomological Society of Canada, No. 108. MILLER, D.R. 1990. Reproductive and ecological isolation: Community structure in the use of semiochemicals by pine bark beetles (Coleoptera: Scolytidae). PhD thesis. Simon Fraser Univel~ity, Burnaby, British Columbia. 166 pp. MmLER, D.R., and BORDEN, J.H. 1990. /3-Phellandrene: Kairomone for pine engraver, Ips pini (Say) (Coteoptera: Scolytidae). J. Chem. Ecol. 16:2519-2531. MILLER, D.R., BORDEN,J.H., and SLESSOR,K.N. 1989. Inter- and intrapopulation variation of the pheromone, ipsdienol produced by male pine engravers, Ips pini (Say) (Coleoptera: Scolytidae). J. Chem. Ecol. 15:233-247. PAPP C:S. 1960. The Cleridae of North America. Part I. The geographical distribution of Cleridae of North America, north of the Panama Canal. Bull South. Calif. Acad. Sci. 59:76-88. PAYNE, T.L., DICKENS, J.C., and RlCHERSON, J.V. 1984. Insect predator-prey coevolution via enantiomeric specificity in a kairomone-pheromone system. J. Chem. Ecol. 10:487-492. PERSON,H.L. 1940~ The clerid Thanasimus lecontei (Wolc.) as a factor in the control of the western pine beetle. J. For. 38:390-396. RAFFA, K.F. 1991. Temporal and spatial disparities among bark beetles, predators, and associates responding to synthetic bark beetle pheromones: Ipspini (Coleoptera: Scolytidae) in Wisconsin. EnvirOn. Entomol. 20:1665-1679. RAFFA, K.F., and KLEPZlO, K.D. 1989. Chiral escape of bark beetles from predators responding to a bark beetle pheromone. Oecologia 80:566-569. RE~D, R.W. 1957. The bark beetle complex associated with lodgepole pine slash in Alberta Part II--Notes on the biologies of several hymenopterous parasites. Can. Entomol. 89:5-8. RICE, R.E. 1968. Observations on host selection by Tomicobia tibialis Ashmead (Hymenoptera: Pteromalidae). Contrib. Boyce Thompson Inst. 24:53-56. RICE, R.E. 1969. Response of some predators and parasites of lps confusus (LeC.) (Coleoptera: Scolytidae) to olfactory attractants. Contrib. Boyce Thompson Inst. 24:189-194. Ross, H.H. 1955. The taxonomy and evolution of the sawfly genus Neodiprion. For. Sci. 1:196209~ SEYBOLD, S.J., YOKOKAWA,Y., KUBO, I., TUpY, J.L., and WOOD, D.L. 1992a. Analytical resolution of the enantiomers o f the aggregation pheromone components of Ips spp. (Coleoptera: Scolytidae) by direct injection on capillary gas chromatography. J. Chromatogr. In manuscript. SEYBOL.D, S.J., OHTSUKA,T., WOOD, D.L., and KUBO, I. 1992b. The enantiomeric composition of ipsenol and ipsdienol from selected species in the subgeneric groups of Ips (Coleoptera: Scolytidae). J. Chem. Ecol. In manuscript. SEYBOLD, S.J., YOKOKAWA,Y., CHAUDHURI,S.K., KUBO, I., OHTSUKA, T., NOMURA, M., and WOOD, D.L. 1992c. Preparative resolution and confirmation of absolute configuration of enantiopure ipsdienot. Tetrahedron Asymmetry. In manuscript. SEYBOLD, S.J., WOOD, D.L., OHTSUKA,T., NOMURA,M., KUBO, I., and SCHULTZ, M.E. 1992d. The response of the pine engraver, lps pini (Say) (Coleoptera: Scolytidae) and its insectan associates to the pure enantiomers of ipsdienol in California. J. Chem. Ecol. In manuscript. SmVERSTEIN,R.M., RODIN, J.O., and WOOD, D.L. 1966. Sex attractants in frass produced by male Ips confusus in ponderosa pine. Science 154:509-510. STEPHEN, F.M., and DAttLSTEN, D.L. 1976. The arrival sequence of the arthropod complex following attack by Dendroctonus brevicomis (Coleoptera: Scolytidae) in ponderosa pine. Can. Entomol. 108:283-304. STEWART, T.E. 1975. Volatiles isolated from lps pini: Isolation, identification, enantiomeric corn-

ROLE OF LANIERONEIN [ps

2329

position, biological activity, and the enantiomeric composition of other insect pheromone alcohols and bicyclic ketals. MSc thesis. College of Environmental Science and Forestry, SUNY, Syracuse, New York, 135 pp. STRUBLE, G. R. 1942. Biology of two native coleopterous predators of the mountain pine beetle in sugar pine. Pan-Pac. Entomol. 18:97-107. TEALE, S.A. 1990. Ecology and evolution of pheromone communication in lps pini (Coleoptera: Scolytidae). PhD thesis. SUNY, Syracuse, New York. 109 pp. TEALE, S.A., and LANIER, G.N. 1991. Seasonal variability in response of lps pini (Coleoptera: Scolytidae) to ipsdienol in New York. J. Chem. Ecol. 17:1145-1158. TEALE, S.A., WEBSTER, F.X., ZHANG, A., and LANIER, G.N. 1991. Lanierone: A new pheromone component from lps pini (Coleoptera: Scolytidae) in New York. J. Chem. Ecol. 17:11591176. VITI~, J.P., and GARA, R.I. 1962. Volatile attractants from ponderosa pine attacked by bark beetles (Coleoptera: Scolytidae). Contrib. Boyce Thompson Inst. Pl. Res. 21:251-273. VIT~, J.P., and PITMAN, G.B. 1969. Insect and host odors in the aggregation of the western pine beetle. Can. Entomol. 101:113-117. WOOD, D.L. 1970. Pheromones of bark beetles, pp. 301-316, in D.L. Wood, R.M. Silverstein, M. Nakajima (eds.). Control of Insect Behavior, Academic Press, New York. WOOD, D.L., and STARK,R.W. 1968. The life history oflps ealligraphus (Coleoptera: Scolytidae) with notes on its biology in California. Can. Entomol. 100:145-151. WOOD, D.L., BROWNE, L.E., BEDARD,W.D., TILDEN, P.E., SILVERSTE1N,R.M., and RODIN, J.O. 1968. Response of lps confusus to synthetic sex pheromones in nature. Science 159:13731374. WOOD, S.L. 1982. The bark and ambrosia beetles of North and Central America (Coleoptera: Scolytidae), a taxonomic monograph. Great Basin Nat. No. 6, 1359 pp. YOUNG, J.C., BROWNLEE, R.G., RODIN, J.O., HILDEBRAND, D.N., SILVERSTEIN,R.M., WOOD, D.L., BIRCH, M.C., and BROWNE,L.E. 1973. Identification of linalool produced by two species of bark beetles of the genus lps. J. Insect Physiol. 193:1615-1622.