See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/230277820

Field responses of the Asian larch bark , subelongatus, to potential aggregation pheromone components: disparity between two populations in Northeastern

ARTICLE in SCIENCE · DECEMBER 2010 Impact Factor: 2.14 · DOI: 10.1111/j.1744-7917.2010.01375.x

CITATIONS READS 2 52

5 AUTHORS, INCLUDING:

Qing-He Zhang Bing-Zhong Ren Sterling International, Inc. Northeast Normal University

100 PUBLICATIONS 1,565 CITATIONS 22 PUBLICATIONS 107 CITATIONS

SEE PROFILE SEE PROFILE

All in-text references underlined in blue are linked to publications on ResearchGate, Available from: Qing-He Zhang letting you access and read them immediately. Retrieved on: 01 December 2015 Insect Science (2011) 18, 311–319, DOI 10.1111/j.1744-7917.2010.01375.x

ORIGINAL ARTICLE Field responses of the Asian larch , Ips subelongatus, to potential aggregation pheromone components: disparity between two populations in northeastern China

Li-Wen Song1,2, Qing-He Zhang3, Yue-Qu Chen2, Tong-Tong Zuo2 and Bing-Zhong Ren1 1School of Life Sciences, Northeast Normal University, Changchun, 2Institute of Forest Protection, Provincial Academy of Forestry Sciences, Changchun, China, 3Sterling International, Inc., Spokane, Washington, USA

Abstract Behavioral responses of the Asian larch bark beetle, Ips subelongatus Motsch. to three potential aggregation pheromone components, ipsenol (racemic or [−]- enantiomer), ipsdienol (racemic or [+]-enantiomer) and 3-methyl-3-buten-1-ol, were tested using partial or full factorial experimental designs in two provinces (Inner Mongolia and Jilin) of northeastern China. Our field bioassays in Inner Mongolia (Larix principis- rupprechtii Mayr. plantation) clearly showed that ipsenol, either racemic or 97%-(−)- enantiomer, was the only compound that significantly attracted both sexes of I. subelon- gatus, while all other compounds (singly or in combinations) were unattractive. There were no two- or three-way synergistic interactions. However, in Jilin Province (L. gmelini [Rupr.] Rupr. Plantation), all the individual compounds tested were inactive, except a very weak activity by 97%-(−)-ipsenol in 2004 when the beetle population was very high. While a combination of ipsenol and ipsdienol (racemates or enantiomerically pure natural enantiomers) showed a significant attraction for both sexes of I. subelongatus, indicat- ing a two-way synergistic interaction between these two major components, addition of 3-methyl-3-buten-1-ol to these active binary blend(s) did not have any effects on trap catches, suggesting that ipsenol and ipsdienol are the synergistic aggregation pheromone components of I. subelongatus in Jilin Province. It seems that 97%-(−)-ipsenol in Inner Mongolia or the binary blend of 97%-(−)-ipsenol and 97%-(+)-ipsdienol in Jilin Province are superior to their corresponding racemates, which might be due either to weak inhibitory effects of the antipode enantiomers or to reduced release rates of the active natural enan- tiomer(s) in the racemate(s). Our current bioassay results suggest that there is a strong geographical variation in aggregation pheromone response of I. subelongatus in northeast- ern China. Future research on the pheromone production and response of I. subelongatus from different regions in Northeast Asia will surely improve our understanding of the dynamic aggregation pheromone system of this economically important forest pest insect. Key words aggregation pheromone, enantiomers, geographical variation, Ips subelon- gatus, ipsdienol, ipsenol

Introduction Correspondence: Bing-Zhong Ren, School of Life Sciences, The Asian larch bark beetle, Ips subelongatus Motsch. Northeast Normal University, 5268 Renmin Street, Changchun 1860, is one of the most important pest in 130024, China. Tel: +86 431 85098200; email: renbz279@ larch plantations and natural stands (Larix spp.) in nenu.edu.cn northeast Asia (Zhang & Niemeyer, 1992). This bark

C 2010 The Authors 311 Journal compilation C Institute of Zoology, Chinese Academy of Sciences 312 L. W. Song et al. beetle not only infests weakened, wind-thrown, and an interesting question on a potential geographical vari- burned trees, but at high population densities, attacks ation in pheromone response of the Asian larch bark healthy larch trees, thereby causing significant tree mor- beetle. tality in this region (Zhang & Niemeyer, 1992). I. subelon- Our objectives were to: (i) field test behavioral func- gatus had been considered as a synonym of the European tionality of the three male-produced hindgut volatile larch bark beetle, I. cembrae Heer 1836, before 2000, compounds, (±)- or 97%-(−)-ipsenol, (±)- or 97%-(+)- especially by European forest entomologists (Postner, ipsdienol and 3-methyl-3-buten-1-ol, in the Asian larch 1974). However, a recent phylogenetic study using molec- bark beetle, I. subelongatus, in partial or full factorial ular (mitochondrial [mt]DNA-Cytochrome Oxidase 1 combinations in China; and (ii) determine if there is ge- [COI] sequence) characters by Stauffer et al. (2001) ographical variation in aggregation pheromone response on different populations of the larch bark beetle from between two populations of I. subelongatus in Inner Mon- Europe and Asia suggested that the larch bark beetle in golia and Jilin, northeastern China. Asia should be considered as a valid separate species, that is, I. subelongatus (Stauffer et al., 2001). The potential pheromone components of the Asian I. Methods and materials subelongatus was identified from the male hindgut sam- ples collected from Province, China, in 2000 by Field trapping gas chromatography–mass spectrometry (GC-MS) anal- ysis, with 100%-(−)-ipsenol and 96%-(+)-ipsdienol be- Six field trapping experiments were conducted during ing the major components and 3-methyl-3-buten-1-ol and the summers of 2004 and 2009 at two sites in northeastern several other bark beetle-related compounds as minor China. Site 1 (for both 2004 and 2009 tests) was a Larix components (Zhang et al., 2000). A detailed GC-EAD gmelini (Rupr.) Rupr. plantation stand (ca 45–50 years (electro-antennographic detection) analysis by the same old) in Jingyuetan Forest Farm (43◦47–48N, 125◦27E; research group found a very similar chemical compo- ca. 260 m elevation), Changchun, Jilin Province, China. sition of the key male-produced hindgut volatile com- Site 2 (for 2009 test only) was a 30-year-old larch planta- pounds from an Inner Mongolian population of I. subelon- tion stand of Larix principis-rupprechtii Mayr. (43◦35N; gatus, in northeastern China, although the enantiomeric 117◦31E; ca. 1 400 m elevation) at Huanggangliang For- compositions of major chiral compounds were not de- est Farm, Keshiketeng Qi, Inner Mongolia, approximately termined (Zhang et al., 2007a). Field trapping experi- 700 km west of Site 1. Eight-unit black funnel traps (Fuda ments in Inner Mongolia showed that the EAD-active, Plastic Co., Changchun, China) were used in all the ex- major male-hindgut component, racemic (±)-ipsenol, is periments. Traps were deployed in lines along the for- the only individual compound that significantly attracted est edges with 50 m (in 2004), and 10 m (in 2009) be- both sexes of I. subelongatus, while all other compounds, tween traps within each trap set/line (physical replicate), including two of the previously reported pheromone and with 100 m (in 2004) and 30 m (in 2009) between components of the European I. cembrae (ipsdienol and trap lines, respectively. All the synthetic aggregation 3-methyl-3-buten-1-ol) were unattractive (Zhang et al., pheromone candidates were purchased from Pherotech 2007a). (±)-Ipsdienol, 3-methyl-3-buten-1-ol or their bi- Inc. (now Contech Enterprises Inc., Delta, BC, Canada) nary blend had no synergistic or antagonistic effects as single component commercial dispensers with (±)- on I. subelongatus attraction when combined with (±)- /or 97%-(+)-ipsdienol (0.2 mg/day) and (±)-/or 97%- ipsenol (Zhang et al., 2007a). However, only the racemic (−)-ipsenol (0.4 mg/day) from the bubble caps and 3- ipsenol and ipsdienol were tested in their field bioas- methyl-3-buten-1-ol (not available [N/A] est. 0.5 mg/day) says, thus the behavioral functionality of the natural enan- from the plastic tube. These release rates were mea- tiomers remains unknown. In a recent pilot field testing sured by the manufacturer at 20◦C. The sexes of cap- in Changchun, Jilin Province, China, ca. 700 km east tured I. subelongatus were separated based on second of the Inner Mongolian site (Zhang et al., 2007a), Song and third elytral spine differences (Zhang & Niemeyer, (2005) indicated that either 97%-(−)-ipsenol or 97%- 1992). (+)-ipsdienol alone was inactive or at most weakly at- Experiment 1: Test of 97%-(−)-ipsenol, 97%-(+)- tractive, while the binary blend of 97%-(−)-ipsenol and ipsdienol and 3-methyl-3-buten-1-ol with first and sec- 97%-(+)-ipsdienol was strongly attractive to I. subelon- ond generations in Jilin in 2004. Experiment 1 covered gatus, suggesting a significant synergistic effect between both the first (April 19 to June 19, 2004) and second these two major male-specific hindgut compounds (Song, generations (June 10 to September 2, 2004) of I. sube- 2005). Such disparity between these two studies raised longatus dispersal flights at Site 1 (Jilin Province); and

C 2010 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 311–319 Aggregation pheromone components of larch bark beetle 313 tested a ternary blend of the two major male-produced Statistical analysis hindgut volatile compounds, 97%-(−)-ipsenol and 97%- (+)-ipsdienol (close to their natural enantiomeric com- Trap catch data were transformed by log(x + 1) to positions) plus 3-methyl-3-buten-1-ol, in comparison fit the assumption of homogeneity of variance for anal- with partial individuals, binary blends and two ternary ysis of variance (ANOVA). The means were compared blends with racemic replacements of single or both chi- by ANOVA followed by the Ryan–Einot–Gabriel–Welsh ral compounds. This ternary blend was also tested in all (REGW) multiple Q test (SPSS 16.0 for Windows, SPSS five experiments, and considered as a positive control Inc., Chicago, Il, US) at α = 0.05. Trap catch data, treatment. log(x + 1), from the treatments involved in the full fac- Experiment 2 Test of 97%-(−)-ipsenol, 97%-(+)- torial designs in Experiments 3–6 (i.e. all the single com- ipsdienol and 3-methyl-3-buten-1-ol with second genera- pounds and their possible binary and ternary blends) were tions in Jilin in 2004 (from June 10 to September 2, 2004; also analyzed by GLM univariate analysis (UNIANOVA; Site 1; Jilin). Experiment 2 was similar to Experiment 1 SPSS 16.0 for Windows) to determine if there were any in the ternary blend treatments, but focused more on the 2-way or 3-way synergistic interactions. The sex ratios of comparison with the three possible binary combinations. captured for the treatments within each experiment Experiment 1 had three physical replicates in a random- were compared with 95% binomial confidence intervals ized block design fashion, while seven sets (blocks) of (Byers & Wood, 1980). traps were deployed for Experiment 2. The traps in Exper- iments 1–2 were serviced every week, and their positions remained unchanged. Results Experiment 3 Full factorial test of 97%-(−)-ipsenol, 97%-(+)-ipsdienol and 3-methyl-3-buten-1-ol in Jilin in In Experiment 1, a total of 5 881 beetles were caught in 2009. Experiment 3 was carried out from May 8 to June three replicates during the whole flight season in 2004 24, 2009 (first generation), at Site 1 (Jilin Province) to in Jilin Province. During the first generation, the ternary determine the behavioral activity of the natural enan- blend consisting of the natural enantiomers of ipsenol and tiomers of the two major pheromone candidates, 97%- ipsdienol plus 3-methyl-3-buten-1-ol (positive control; (−)-ipsenol and 97%-(+)-ipsdienol, plus the achiral 3- unfilled bar) caught significantly more I. subelongatus methyl-3-buten-1-ol in a full factorial experiment design beetles than did any other treatments and the blank con- (i.e., all the single compounds and their possible binary trol (Fig. 1A). Replacing 97%-(−)-ipsenol and/or 97%- and ternary blends), plus three other ternary blend treat- (+)-ipsdienol with their corresponding racemates signif- ments with racemic replacements of either or both chiral icantly reduced trap catches; 97%-(−)-ipsenol alone was compounds. barely attractive, while 97%-(+)-ipsdienol was inactive Experiment 4 Full factorial test of (±)-ipsenol, (±)- (Fig. 1A). Trap catch data from the second generation ipsdienol and 3-methyl-3-buten-1-ol in Jilin in 2009. Ex- showed virtually the same pattern as the first genera- periment 4 was also conducted from May 8 to June 24, tion (Fig. 1B). In Experiment 2, a total of 5 924 beetles 2009, at the same site (Jilin province), ca. 50 m from exper- were trapped during seven replicates. The ternary blend iment 3, to test the behavioral functionality of the racemic with natural enantiomers of ipsenol and ipsdienol (pos- (±)-ipsenol and (±)-ipsdienol, plus 3-methyl-3-buten-1- itive control) again showed a strong attraction to both ol in a full factorial experiment design, plus three other sexes of I. subelongatus beetles (Fig. 2). Removal of ternary blend treatments with (−)- or (+)-enantiomeric 3-methyl-3-buten-1-ol from this active blend had no ef- replacements of either or both chiral compounds. Each ex- fects on trap catches. A binary blend of 97%-(−)-ipsenol periment had two replicates in a randomized block design and 3-methyl-3-buten-1-ol was slightly attractive, while fashion. The same treatments and experimental designs in the combination of 97%-(+)-ipsdienol and 3-methyl-3- Experiments 3 and 4 were repeated during the second gen- buten-1-ol was inactive (Fig. 2). eration (July 15–20, 2009) in Experiments 5 and 6, respec- Due to the extremely low population density and tively, at Site 2 in Inner Mongolia. In order to minimize cold/rainy weather in the early summer of 2009 at Site positional effects, traps together with dispensers within 1 (Jilin Province), only a few replicates were achieved each trap set in Experiments 3–6 (in 2009) were rotated during the first generation. In Experiment 3, a total of after each replicate (when ≥ 10–20 beetles were caught 137 beetles were captured during four replicates with in the best trap) in a randomized Latin-square design a mean sex ratio of 1.98 ± 0.58 : 1 (♀ : ♂) (no dif- (Byers, 1991). ferences among treatments). All individual compounds

C 2010 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 311–319 314 L. W. Song et al.

500 e A 400 1st generation-Jilin (April 19 to June 9, 2004; N=3) 300

200 d

/trap + SE cd 100 cd ab bc a 0 800 d 700 B 600 2nd generation-Jilin Ips subelongatus (June 10 to Sept. 2, 2004; N=3) 500 400 No. of 300 c 200 bc 100 b bc a a 0 +Id ±Id ±Id -Ie +Id -Ie Blank -Ie -Ie ±Ie MB MB MB MB

Fig. 1 Experiment 1. Mean captures of Ips subelongatus in traps baited with a ternary blend of 97%-(−)-ipsenol (-Ie), 97%-(+)- ipsdienol (+Id) and 3-methyl-3-buten-1-ol (MB) (as a positive control; unfilled bar), in comparison with partial individuals (+Id and –Ie), binary blend and two ternary blends with racemic replacements ([±]-ipsenol [±Ie] and/or [±]-ipsdienol [±Id]) of single or both chiral compounds, during first (A) and second (B) generations, April 19 to September 2, 2004, Jingyuetan Forest Farm, Jilin Province, China. An unbaited trap served as the negative control. Means within each generation followed by the same letter are not significantly different (ANOVA on Log[x+1], REGW multiple Q-test, P > 0.05).

500 d Expt 2 2nd generation-Jilin (June 10 to Sept. 2, 2004; N=7)

400 d /trap + SE 300

200

Ips subelongatus 100 c c No. of b a a 0 +Id +Id ±Id +Id +Id -Ie Blank -Ie ±Ie ±Ie -Ie MB MB MB MB MB

Fig. 2 Experiment 2. Mean captures of Ips subelongatus in traps baited with a ternary blend of 97%-(−)-ipsenol (-Ie), 97%-(+)- ipsdienol (+Id) and 3-methyl-3-buten-1-ol (MB) (as a positive control; unfilled bar), in comparison with three binary blends and two ternary blends with racemic replacements ([±]-ipsenol [±Ie] and/or [±]-ipsdienol [±Id]) of single or both chiral compounds, during the second generations, June 10 to September 2, 2004, Jingyuetan Forest Farm, Jilin Province, China. An unbaited blank trap served as the negative control. Means followed by the same letter are not significantly different (ANOVA on Log[x + 1], REGW multiple Q-test, P > 0.05).

C 2010 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 311–319 Aggregation pheromone components of larch bark beetle 315

20 Expt 3 1st generation-Jilin A (May 8–June 24, 2009; N=4) b

15 b

10 b ab 5 ab

/trap/visit + SE a aa a a a 0 30 d B Expt 5 2nd generation-Inner Mongolia 25 (July 15–20, 2009; N=22)

Ips subelongatus 20 cd c 15 cd c No. of c 10 b 5 aa aa 0 Blank +Id -Ie MB +Id +Id -Ie +Id ±Id +Id ±Id -Ie MB MB -Ie ±Ie ±Ie -Ie MB MB MB MB

Fig. 3 Mean captures of Ips subelongatus in traps baited with natural entantiomers of the two major pheromone candidates, 97%-(−)- ipsenol (-Ie) and 97%-(+)-ipsdienol (+Id), plus 3-methyl-3-buten-1-ol (MB) in a full factorial experiment design (i.e., all the individuals and their possible binary and ternary blends), plus three other ternary blend treatments with racemic replacements ([±]-ipsenol [±Ie] and/or [±]-ipsdienol [±Id]) of either or both chiral compounds, during the first generation at Jingyuetan Forest Farm, Jilin Province (A) (Experiment 3) and during the second generation at Huanggangliang Forest Farm, Keshiketeng Qi, in Inner Mongolia (B), 2009 (Experiment 5). An unbaited blank trap served as the negative control, and the ternary blend: +Id/–Ie/MB being as a positive control; unfilled bar. Means within each site followed by the same letter are not significantly different (ANOVA on Log[x + 1], REGW multiple Q-test, P > 0.05). and their two binary blends involving 3-methyl-3-buten- to both sexes of I. subelongatus adults, while all the indi- 1-ol were inactive; whereas the binary blend of 97%-(−)- viduals and other binary blends were inactive (Fig. 4A). ipsenol and 97%-(+)-ipsdienol was significantly attrac- There was a significant two-way synergistic interaction tive (Fig. 3A). There was a significant two-way syner- between racemic ipsenol and ipsdienol (UNIANOVA: gistic interaction between these two major components F = 7.341, P = 0.015). Addition of 3-methyl-3-buten- (UNIANOVA: F = 8.778, P = 0.007). The ternary blend 1-ol to this active binary blend seemed to reduce trap of 97%-(−)-ipsenol, 97%-(+)-ipsdienol and 3-methyl-3- catches somewhat, but the difference was not statistically buten-1-ol (positive control) also showed a strong attrac- significant (Fig. 4A; and no significant three-way inter- tion to I. subelongatus beetles, but was not different from action; UNIANOVA: F = 3.904, P = 0.07); furthermore, the active binary blend (Fig. 3A; and no significant three- this ternary blend was also not different from the blank, way interaction; UNIANOVA: F = 0.10, P = 0.754). due to the overall low replicate numbers and large within- Replacing 97%-(−)-ipsenol with its racemate from the ac- group variation. Two other ternary blends with 97%-(+)- tive ternary blend did not have any impact. Other ternary ipsdienol also showed significant trap catches, but were blends also caught considerable numbers of adult beetles, not significantly different from the active binary racemic but were not different from blank control traps. In Exper- blends of ipsenol and ipsdienol (Fig. 4A). iment 4, a total of 127 beetles were trapped during three In Experiment 5, a total of 1 971 beetles were caught replicates with a mean sex ratio of ca. 2.02 ± 0.41 : 1 during 22 replicates at Site 2 (second generation) in (♀ : ♂) (no differences among treatments; 95% binomial Inner Mongolia. The near enantiomerically pure, 97%- CI). Similar to Experiment 1, only the binary blend of (−)-ipsenol was the only compound that was signifi- racemic ipsenol and ipsdienol was significantly attractive cantly attractive to both sexes of I. subelongatus (Fig. 3B;

C 2010 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 311–319 316 L. W. Song et al.

25 A Expt 4 1st generation-Jilin c 20 (May 8–June 24, 2009; N=3) bc 15 bc 10 ab 5

/trap/visit + SE ab ab aaa a a 0 20 c Expt 6 2nd generation-Inner Mongolia B (July 15–20, 2009; N=22) 15 c c c bc

Ips subelongatus bc b 10 No. of

5

aa a a 0 Blank ±Id ±Ie MB ±Id ±Id ±Ie ±Id +Id +Id ±Id ±Ie MB MB ±Ie -Ie ±Ie -Ie MB MB MB MB

Fig. 4 Mean captures of Ips subelongatus in traps baited with racemates of the two major pheromone candidates, (±)-ipsenol (±Ie) and (±)-ipsdienol (±Id), plus 3-methyl-3-buten-1-ol (MB) in a full factorial experiment design (i.e., all the individuals and their possible binary and ternary blends), plus three other ternary blend treatments with (−)- or (+)- enantiomeric replacements (97%-[−]-ipsenol [-Ie] and 97%-[+]-ipsdienol [+Id]) of either or both chiral compounds, during the first generation at Jingyuetan Forest Farm, Jilin Province (A) (Experiment 4) and during the second generation at Huanggangliang Forest Farm, Keshiketeng Qi, in Inner Mongolia (B), 2009 (Experiment 6). An unbaited blank trap served as the negative control, and the ternary blend: +Id/–Ie/MB being a positive control; unfilled bar. Means within each site followed by the same letter are not significantly different (ANOVA on Log[x + 1], REGW multiple Q-test, P > 0.05).

UNIANOVA: F = 1 172, P < 0.0001). Addition of 97%- (±)-ipsdienol alone or their combination was unattrac- (+)-ipsdienol to the active 97%-(−)-ipsenol had no ef- tive, whereas (±)-ipsenol was significantly attractive to fect on trap catches (Fig. 3B; UNIANOVA: F = 0.003, both sexes of I. subelongatus (Fig. 4B; UNIANOVA: P = 0.958), while 3-methyl-3-buten-1-ol seemed to re- F = 1 562; P < 0.0001). All the binary combinations duce the attraction to 97%-(−)-ipsenol (UNIANOVA: and most of the ternary blends that contained 97%-(−)- F = 6.817; P = 0.01). Neither 97%-(+)-ipsdienol nor or (±)-ipsenol were significantly attractive, but they were 3-methyl-3-buten-1-ol alone, nor their binary blend was not different from (±)-ipsenol alone (Fig. 4B; no two-way attractive (Fig. 3B); whereas their ternary blend with or three-way interactions were detected; UNIANOVA: 97%-(−)-ipsenol (the positive control) showed no sig- F = 0.117 to 1.11; P = 0.125 to 0.732). However, combi- nificant difference in trap catches compared to 97%-(−)- nation of 97%-(+)-ipsdienol and 3-methyl-3-buten-1-ol ipsenol alone (Fig. 3B). The other three ternary blends seemed to reduce the trap catches slightly when added to also caught more beetles than did the blank control, but (±)-ipsenol (Fig. 4B). caught significantly lower than 97%-(−)-ipsenol alone (Fig. 3B). There were no significant differences in sex ratios (2.07 ± 0.42 : 1; ♀ : ♂) among treatments. In Ex- Discussion periment 6, a total of 1 853 beetles were caught during 22 replicates with a mean sex ratio of 2.11 ± 0.34 : 1 This is the first detailed field bioassay study on the be- (♀ : ♂) (no differences among treatments; 95% binomial havioral functionality of two major, near enantiomeri- CI). Similar to Experiment 5, 3-methyl-3-buten-1-ol or cally pure synthetic pheromone candidates (mimicking

C 2010 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 311–319 Aggregation pheromone components of larch bark beetle 317 their natural enantiomeric compositions), 97%-(−)- alone in 2004 when the beetle population was very high ipsenol and 97%-(+)-ipsdienol (Zhang et al., 2000) in (Fig. 1A/B). However, combination of ipsenol and ips- the Asian larch bark beetle, I. subelongatus. dienol (racemates or enantiomerically pure [mimicking Our field testing at Huanggangliang Forest Farm (Site their natural enantiomers]) showed a significant attrac- 2) in Inner Mongolia clearly showed that major male- tion to both sexes of I. subelongatus, indicating a strong hindgut component, ipsenol (97%-[−]- or [±]-), is the two-way synergistic interaction between these two major only individual compound that significantly attracts both components (Fig. 3A, Fig. 4A, and the UNIANOVA re- sexes of I. subelongatus, while all the other compounds sults). Addition of 3-methyl-3-buten-1-ol to these active (singly or in combinations) were unattractive (Fig. 3B binary blend(s) did not show any affects on trap catches, and Fig. 4B). Using a subtractive method (Byers, 1992), further supported by our UNIANOVAanalysis that there is removal of ipsenol from the attractive synthetic blends no three-way interaction among these tested compounds. virtually eliminated attraction of I. subelongatus, while Data from Experiment 1 also indicated no significant vari- subtraction of each of the other compounds had no ef- ations in pheromone response between first and second fect. Our data fully supports a recent report by Zhang generations (Fig. 1A/B). Both our partial (in 2004) and full et al. (2007a) of a field bioassay in a larch (L. principis- factorial (2009) design experiments suggest that ipsenol rupprechtii Mayr.) plantation at Huamugou Forest Farm and ipsdienol are the synergistic aggregation pheromone (42◦24–52N; 116◦40–117◦36E; 1 500 m elevation), components of I. subelongatus in Jilin Province. No di- Keshiketeng Qi, Inner Mongolia, approximately 120 km rect field trapping comparison between the racemic and southwest of the current Site 2. No effort was made to di- enantiomerically pure binary blends of ipsenol and ips- rectly compare the trap catches between 97%-(−)-ipsenol dienol was conducted; however, the ternary blend of 97%- and (±)-ipsenol within the same experiment; however, (−)-ipsenol, 97%-(+)-ipsdienol and 3-methyl-3-buten-1- based on the trap catches of several shared ternary blend ol was significantly more attractive than other racemate(s) treatments between Experiments 5 and 6, one might con- of the ternary blends in several cases, suggesting that the fidently conclude that these two experiments were car- binary blend consisting of their natural enantiomers might ried out at the same larch stand with a relatively uni- be superior to their racemic binary blend (Figs. 1 and 2). form beetle population (see Figs. 3B and 4B). Therefore, Such disparity might be due either to inhibitory effects indirect comparison between 97%-(−)-ipsenol and (±)- of the antipode enantiomers or to reduced release rates ipsenol from the two experiments might be valid. During of active enantiomers in the racemates. Therefore, di- the same experimental period, the almost natural enan- rect field comparison between the enantiomerically pure tiomer percentage of 97%-(−)-ipsenol (determined based binary blend and the racemate is needed to further un- on hindgut samples taken from Liaoning Province, China; derstand the aggregation pheromone system of the Jilin [Zhang et al., 2000]) seemed to catch more beetles than population. did its racemate, with a medium level of effect size (Co- No chemical analysis on the pheromone production of hen’s d = 0.517) (Cohen, 1988). This effect might be due the local I. subelongatus population from Jilin Province either to a weak inhibitory affect of the antipode (+)- has been conducted yet; however, Zhang et al. (2000) iden- enantiomer or to a reduced release rate of (−)-ipsenol in tified 100%-(−)-ipsenol and 96%-(+)-ipsdienol as ma- the racemate. Our current data plus the previous report by jor male hind-gut components and 3-methyl-3-buten-1-ol Zhang et al. (2007a) indicate that ipsenol is the key, if not and several other bark beetle-related compounds as minor the sole, aggregation pheromone component for I. sube- components of the Asian I. subelongatus samples col- longatus populations in Inner Mongolia, China. No data lected from Dahuofang Forest Farm (41◦52N; 123◦55E) is yet available on the natural enantiomeric composition in Liaoning Province, China, ca. 250 km southwest of of ipsenol or ipsdienol for Inner Mongolian populations. the Jilin site (both areas have very similar forest struc- Therefore, further study on this subject and a direct field tures/landscapes, weather conditions and beetle phenol- comparison between (−)- and (±)-ipsenol is needed to ogy). Thus, similar pheromone production and response fully understand the aggregation pheromone system of patterns between populations from these two neighboring the Inner Mongolian populations. locations are expected, although a comparative labora- Surprisingly, our field trapping results from 2004 and tory/field study is needed. 2009 at a L. gmelini (Rupr.) Rupr. plantation of Jingyue- Our field bioassay results, especially the comparative tan Forest Farm, in Changchun, Jilin Province, showed a trapping experiments conducted in 2009 from two loca- quite different behavioral response pattern by I. subelon- tions, suggest that there is a strong geographical variation gatus. All the individual compounds tested were inactive in aggregation pheromone response of the Asian larch alone, except a very weak activity by 97%-(−)-ipsenol bark beetle, I. subelongatus in northeastern China. These

C 2010 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 311–319 318 L. W. Song et al. two sites are only 700 km apart, but they represent two ford et al., 1990), and Pseudips mexicanus (Hopkins) quite different bio-geographical regions (with many dif- (Seybold, 1992; Savoie et al., 1998). However, the mecha- ferences in their flora and fauna), with the Jilin site being nisms involved in such geographical variations are not yet a lower elevation plateau landscape and L. gmelini as the clearly understood. A comparative DNA (mtDNA-COI key host species, whereas the Inner Mongolian sites (in the sequence) analysis study on five populations of the Asian current study and in Zhang et al. [2007a]) are two isolated I. subelongatus collected from all the four provinces in nearby forest farms in the middle of a desert grass land northeastern China is underway (F.Lakatos, pers. comm.). at relatively high elevation, and L. principis-rupprechtii is Future research on the pheromone productions and re- the key host species (Zhang et al., 2007a), largely isolated sponses of this beetle species from different regions in from other populations of I. subelongatus. Therefore, the northeast Asia will surely enrich our understanding of evolution of I. subelongatus population in this part of In- the aggregation pheromone systems, and ultimately, pro- ner Mongolia might have been different from other popu- vide critical information needed for the development of lations in northeastern China, and the Asian part of Russia aggregation pheromone attractant systems optimized for or Japan. In fact, an Inner Mongolian I. duplicatus popu- various geographical regions for monitoring and/or mass lation from a local spruce natural reserve, ca. 50 km from trapping of this economically important forest pest insect either site, showed significant differences in both aggre- (Schlyter et al., 2001b). gation pheromone production and behavioral responses from its European counterparts (Zhang & Niemeyer, 1992; Schlyter et al., 2001a, 2001b; Zhang et al., 2007b), Acknowledgments which was recently further supported by a compara- The technical support of colleagues at Huanggangliang tive genetic structure study with approximately 0.8% Forest Farm, Inner Mongolia and Jingyuetan Forest Farm, DNA sequence divergence (COI gene) (Lakatos et al., Jilin, China is highly appreciated. We thank Dr. J. A. By- 2007). ers (USDA-ARS) and Prof. Fredrik Schlyter (SLU, Swe- Interestingly, the pheromone response pattern in the den) for reviewing an earlier version of this manuscript. Jilin population is somewhat similar to a closely related This study was supported by grants from Jilin Provin- sibling species, the European larch beetle I. cembrae, with cial Department of Science and Technology (Contract No. a 4.3% difference in mtDNA sequences from the Asian 20020213 and 20070571). I. subelongatus (Stauffer et al., 2001). For I. cembrae, a three-component synergistic pheromone blend consist- ing of ipsenol, ipsdienol, and 3-methyl-3-buten-1-ol was References first reported by Stoakley et al. (1978). In a later GC- MS study on aeration samples of I. cembrae-infested Berisford, C.W., Payne, T.L. and Berisford, Y.C. (1990) Ge- larch logs, another monoterpene alcohol, amitinol, was ographical variation in response of southern pine beetle identified which significantly enhanced field response (Coleoptera: Scolytidae) to aggregating pheromones in labo- to the previously reported aggregation pheromone blend ratory bioassays. Environmental Entomology, 19, 1671–1674. (Kohnle et al., 1988). The natural enantiomeric compo- Byers, J.A. (1991) Basic algorithms for random sampling sitions of ipsenol and ipsdienol from the European I. and treatment randomization. Computers in Biology and cembrae were 95%-(−)-ipsenol and 95%-(+)-ipsdienol Medicine, 21, 69–77. (Francke & Vite, 1983), similar to those found from Asian Byers, J.A. (1992) Optimal fractionation and bioassay plans I. subelongatus (Zhang et al., 2000). However, the absence for isolation of synergistic chemicals: the subtractive- of amitinol and inactiveness of 3-methyl-3-buten-1-ol in combination method. Journal of Chemical Ecology, 18, 1603– the Asian populations provide further support that there 1621. are two distinct species of larch bark beetles (Stauffer Byers, J.A. and Wood, D.L. (1980) Interspecific inhibition of the et al., 2001). response of the bark beetles, Dendroctonus brevicomis and In moth pheromone communication signals, both quan- Ips paraconfusus, to their pheromones in the field. Journal of titative and qualitative intraspecific differences have been Chemical Ecology, 6, 149–164. found across geographic regions (Groot et al., 2009; Cohen, J. (1988) Statistical Power Analysis for the Behavioral Grant et al., 2009). Geographical variations in aggre- Sciences, 2nd ed. Lawrence Earlbaum Associates, Hillsdale, gation pheromone responses and/or production were NJ, 553 pp. also reported in several North American bark beetle Francke, W. and Vite, J.P. (1983) Oxygenated terpenes in species, such as Ips pini (Seybold, 1992; Seybold et al., pheromone systems of bark beetles. Zeitschrift fur¨ Ange- 1995; Miller et al., 1997), Dendroctonus frontalis (Beris- wandte Entomologie, 96, 146–156.

C 2010 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 311–319 Aggregation pheromone components of larch bark beetle 319

Grant, G.G., Millar, J.G. and Trudel, R. (2009) Pheromone iden- genus Ips (Coleoptera: Scolytidae). PhD thesis. University of tification of Dioryctria abietivorella (Lepidoptera: Pyralidae) California. 355 pp. from an eastern North American population: geographic vari- Seybold, S.J., Ohtsuka, T., Wood, D.L. and Kubo, I. (1995) The ation in pheromone response. Canadian Entomologist, 141, enantiomeric composition of ipsdienol: A chemotaxonomic 2, 129–135. character for North American populations of Ips spp. in the Groot, A.T., Inglis, O., Bowdridge, S., Santangelo, R.G., Blance, pini subgeneric group (Coleoptera: Scolytidae). Journal of C., Lopez,¨ J.D., Vargas, A.T., Could, F. and Schal, C. (2009) Chemical Ecology, 21, 995–1016. Geographic and temporal variation in moth chemical commu- Song, L.-W. (2005) Primary research on semiochemicals of Ips nication. Evolution, 63(8), 1987–2003. subelongatus M.–Analysis on bioactivity and application of Kohnle, U., Vite,´ J.P., Erbacher, C., Bartels, J. and Francke, W. synthetic lures. Masters Thesis. Northeast Normal University. (1988) Aggregation response of European engraver beetles of 40 pp. the genus Ips mediated by terpenoid pheromone. Entomologia Stauffer, C., Kirisits, T., Nussbaumer, C., Pavlin, R. and Wing- Experimentalis et Applicata, 49, 43–53. field, M.J. (2001) Phylogenetic relationships between the Eu- Lakatos, F., Grodzki, W., Zhang, Q.H. and Stauffer, C. (2007) ropean and Asian eight spined larch bark beetle populations Genetic comparison of Ips duplicatus (Sahlberg, 1836) (Coleoptera, Scolytidae) inferred from DNA sequences and (Coleoptera, Scolytidae) populations from Europe and Asia. fungal associates. European Journal of Entomology, 98, 99– Journal of Forestry Research, 12, 345–349. 105. Miller, D.R., Gibson, K.E., Raffa, K.F.,Seybold, S.J., Teale, S.A. Stoakley, J.T., Bakke, A., Renwick, J.A.A. and Vite,´ J.P. (1978) and Wood, D.L. (1997) Geographic variation in response of The aggregation pheromone system of the larch bark beetle pine engraver, Ips pini, and associated species to pheromone, Ips cembrae Heer. Zeitschrift fur¨ Angewandte Entomologie, lanierone. Journal of Chemical Ecology, 23, 2013–2031. 86, 174–177. Postner, M. (1974) Scolytidae, Borkenkafer.¨ Die Zhang, Q.-H., Birgersson, G., Schlyter, F.and Chen, G.-F.(2000) Forstschadlinge¨ Europas (ed. W. Schwenke), pp. 334– Pheromone components in the larch bark beetle, Ips cembrae, 481. Paul Parey, Hamburg. from China: Quantitative variation among attack phases and Savoie, A., Borden, J.H., Pierce, H.D.J., Gries, R. and Gries, individuals. Journal of Chemical Ecology, 26, 841–858. G. (1998) Aggregation pheromone of Pityogenes knechteli Zhang, Q.-H. and Niemeyer, H. (1992) Morphological char- and semiochemical-based interactions with three other bark acteristics for sexing living adults of Ips cembrae (Heer) beetles. Journal of Chemical Ecology, 24, 321–337. (Col., Scolytidae). Journal of Applied Entomology, 114, 403– Schlyter, F., Svensson, M., Zhang, Q.-H., Knizek, M., Krokene, 409. P., Ivarsson, P. and Birgersson, G. (2001a) A model for peak Zhang, Q.-H., Schlyter, F., Chen, G. and Wang, Y. (2007a) Elec- and width of signaling windows: Ips duplicatus and Chilo trophysiological and behavioral responses of Ips subelongatus partellus pheromone component proportions – Does response to semiochemicals from its hosts, nonhosts and conspecifics have a wider window than production? Journal of Chemical in china. Journal of Chemical Ecology, 33, 391–404. Ecology, 27, 1481–1512. Zhang, Q.-H., Schlyter, F., Liu, G.T. and Birgersson, G. (2007b) Schlyter, F., Zhang, Q.-H., Liu, G.T. and Ji, L.Z. (2001b) A Electrophysiological and behavioral responses of Ips dupli- successful case of pheromone mass-trapping of the bark beetle catus to aggregation pheromone in Inner Mongolia, China: Ips duplicatus in a forest island, analysed by 20-year time- amitinol as a potential pheromone component. Journal of series data. Integrated Pest Management Review, 6, 185–196. Chemical Ecology, 33, 1303–1315. Seybold, S.J. (1992) The role of chirality in the olfactory- directed aggregation behavior of pine engraver beetles in the Accepted June 3, 2010

C 2010 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 311–319