Journal of Chemical Ecology, Vol. 20, No. 7, 1994

IDENTIFICATION AND FIELD EVALUATION OF Anomala octiescostata (COLEOPTERA: SCARABAEIDAE) SEX PHEROMONE 1

WALTER SOARES LEAL, 2'* MAKOTO HASEGAWA, 3 MASAAKI S A W A D A , 3 MIKIO ONO, 4 and YASUO U E D A 5 2Department of Insect Physiology and Behavior National Institute of Sericuttural and Entomological Science (NISES) 1-20hwashi, Tsukuba-shi 305 Japan 3Chiba Prefectural Agricultural Experiment Station 808 Daizenno-cho, Midori-ku, Chiba 266 Japan 4Fuji Flavor Co. Ltd. 3-5-8 Midorigaoka, Hamura-city, Tokyo 190-11 Japan 51baraki Agricultural Center, Agricultural Research Institute 3344 Kamikuni, Mito-shi 311-42 Japan (Received November 23, 1993; accepted February 15, 1994) Abstract--Using GC-EAD, the sex pheromone of the scarab beetle Anomala octiescostata was identified to be a 8:2 binary mixture of (R,Z)-5-(-)-(oct1-enyl)oxacyclopentan-2-one and (R,Z)-5-(-)-(dec-l-enyl)oxacyclopentan-2one. These semiochemicals have been also reported as sex pheromone constituents of other Anomala species, either geographically or seasonally isolated from A. octiescostata. Synthetic sex pheromone was highly attractive in the field; 0.1 mg captured significantly more males than two virgin females. Buried traps were significantly more attractive than those positioned at 30, 90, and 150 cm above the ground. In a dose-response test (0.1-100 mg), no saturation due to overdose of pheromone was observed, but in most cases, two dosages differing by 10-fold were not significantly different. Response of males to traps baited with different ratios of the two components was tested in two experiments with randomized blocks and Latin-square designs. Deviation from the natural ratio (g : 2) of sex pheromone did not significantly diminish the response of males. Peak flight activity of beetle was recorded at 9:0010:00 AM JST on sunny days in the end of April 1993.

Key Words--Coleoptem, Scarabaeidae, Anomala octiescostata,

(R,Z)-5-(-)(oct- 1-enyl)oxacyclopentan-2-one. (R,Z)-5-( -)-(dec- 1-enyl)oxacyclopentan-

*To whom correspondence should be addressed. ~Presented in part at the 10th Annual ISCE Meeting, July 31-August 4, 1993, Clearwater Beach, Florida. 1643 0098-0331/~10700- i 643507.00/0 © 1994 Plenum

Publishing Cm'porafion

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LEAL ET AL. 2-one, GC-EAD, Anomala cuprea, Anomala albopilosa sakishimana, flight activity, Latin square,

INTRODUCTION

The scarab beetle Anomala octiescostata Burmeister (Japanese name: hirataaokogane) is native to Japan and was originally recorded only in Kyushu Island in the south of the country. The adults appear in grass fields on sunny mornings during the months of April and May and have short seasonal flight activity. Due to the lack of a monitoring system, heavy outbreaks of the beetle may be overlooked. Although A. octiescostata was not previously recorded in the Kanto district--Gumma, Tochigi, Saitama, Ibaraki, Tokyo, Chiba, and Kanagawa (probably because it never reached pest status)--heavy infestations were recently detected in Chiba and Ibaraki prefectures. The insecticides registered for scarabs in Japan were not effective in controlling the beetle, and it reached large populations in a few years, showing the pattern of an exotic species, devoid of natural enemies, exploiting new habitats. As part of our project aimed at investigating the possibility of applying semiochemicals to the integrated pest management (IPM) of scarab beetles, we have recently successfully identified the sex pheromones of some economically important scarab pests in Japan (Leal et al., 1994), some of which are promising in mass trapping (Hasegawa et al., 1993). The alarming level of the A. octiescostata population in Chiba and Ibaraki prefectures prompted us to investigate the sex pheromone of A. octiescostata and evaluate its potential for field application. We report here on the identification and field testing of the sex pheromone. METHODS AND MATERIALS

Insects. Beetles were first collected at a golf course, Chiba Springs Country Club, in Chiba Prefecture at the end of the flight season (May) in t992. In order to start a laboratory colony, eggs laid by the field-captured females were collected daily and transferred to wet sand in ice cream cups, which were kept at 25°C. After hatching, grubs were individually transferred to ice cream cups filled with humus and supplied with slices of sweet potato or carrot. These cups were kept at 25°C and the foodstuff was renewed weekly. Diapause termination of adults was achieved by chilling at 10°C for three to four months and then raising the temperature to 25°C. After emerging from diapause, adults were fed on cherry (Prunus donarium) leaves or on an artificial diet for wild silk moth (Nihon Chlorella Co.) at 25°C, 70% relative humidity, and 14L: 10D photoperiod.

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Pheromone Collection. The airborne volatiles from 97 field-collected female A. octiescostata fed on cherry leaves (or supplied only with syrup on pieces of cotton) were collected according to the method of Leal et al. (1992). The volatiles were extracted with hexane and, after concentration, the extracts were stored at - 4 0 ° C . Headspace volatiles of laboratory-raised beetles, in diapause or not, were also collected. Whole-body extracts of laboratory-raised virgin female or male beetles were obtained by washing in dichloromethane for 5 min. Isolation of Pheromone. Volatiles collected from the headspace of female beetles were separated on a silica gel column (Wako C-200) by successive elution with hexane-ether mixtures: 100 : 0, 95 : 5, 90 : 10; 80 : 20, 50 : 50, and 0: 100. Chromatographic and Mass Spectral (MS) Analyses. GC analyses were performed on Hewlett-Packard 5890, and mass spectra were recorded on a Hewlett-Packard 5891 mass selective detector equipped either with an HP-1 column (12 m x 0.2 mm; 0.33/~m) or a DB-wax column (30 m x 0.25 mm; 0.25 /~m), operated in splitless mode at 50°C for 1 min, programmed at 4°C/ min, held at this temperature for 1 min, programmed again at 10°C/min to 230°C and held at this temperature for 20 min [i.e., 50(1)-180(1)/4-230(20)/ 10]. Enantiomeric resolution of buibuilactone was achieved on a Chiraldex GTA column (20 m × 0.25 ram; 0.125 ram, Astec), operated in split mode at 115°C, using helium as carrier gas at 2 kg/cm 2 head pressure (flow rate of 4.4 ml/min). Gas Chromatography-Electroantennographic Detector (GC-EAD). The responses of A. octiescostata antennae were recorded with a previously described GC-EAD system (Leal et al., 1992, 1994). Antennae of beetles in diapause or not were used as the sensing elements. Synthesis. (R,Z)-5-(-)-(Oct-l-enyl)oxacyclopentan-2-one (buibuilactone) was synthesized as previously reported (Leal, 1991) and (R,Z)-5-(-)-(dec-1enyl)oxacyclopentan-2-one was prepared starting from protected D-ribose (Koseki et al., 1993). (S,Z)-5-(-)-(Oct-l-enyl)oxacyclopentan-2-one was synthesized by the method of Doolittle et al. (1980), starting from (S)-(+)-5-oxo-2-tetrahydrofurancarboxylic acid (Aldrich Chemical Company, Inc.). The optical purity of all synthetic pheromones was determined to be > 9 7 % ee by chiral chromatography (Leal, 1991). Wind Tunnel. The attractancy of the pheromone was preliminarily tested in a wind tunnel (2 m long, 30 cm ID) internally covered with wire mesh. Experiments were carded out from 9:00 A M to 4:00 PM in a clear room at 23°C and 60% relative humidity. Blank or synthetic sex pheromone containing filter paper was set 1.5 m away from groups of five insects placed in the downwind end of the tunnel and observed for 15 min at an airflow of 40 cm/sec. Field Evaluation. Tests on synthetic sex pheromone were conducted in two golf clubs, Chiba Springs Country Club (Chiba) and Edosaki Country Club

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(Ibaraki) from April to May 1993. A survey for the occurrence of beetles in the autumn was done in Edosaki in 1993. Funnel JT traps (Japan Tobacco Inc.) were baited with synthetic sex pheromone incorporated into pellets (4-5 mm in diameter) made of a polyethylene-vinyl acetate copolymer. These were placed inside a pellet holder (Fuji Flavor Co.) and set 2 cm above the trap lip. The pheromone dosages in Ibaraki and Chiba were 1 and 10 mg of a 8 : 2 blend, respectively, unless otherwise mentioned. Experimental Design and Statistical Analysis. The experiments were done using two different designs: the candidate lures were replicated in randomized blocks in the Chiba and in Latin square in the Ibaraki experiments. Traps were set with an intertrap distance of 10 m and the pheromone dispenser set at 30 cm above the ground, unless otherwise mentioned. Capture data were transformed to log (x + 1) and differences between means were tested for significance by A N O V A using JMP software (Version 2) (SAS Institute, 1989). In this paper, treatments followed by the same letters are not significantly different at the 5 % level in the Tukey-Kramer honestly significant difference test.

RESULTS AND DISCUSSION

Identification of Pheromone. Volatiles collected from field-captured female beetles were stored until the emergence of the first laboratory-raised group. As A. octiescostata enters diapause as soon as the adult eclose from pupal stage, freshly eclosed male beetles could not be used in laboratory bioassay for monitoring the isolation of the sex pheromone because they did not display any sexual behavior. However, the antennae of diapausing males could be used in GC-EAD analyses. Two EAD-active peaks (ratio 8 : 2) were found in the female extract, the major one appeared at 33.62 and 24.77 min whereas the minor one gave R, at 37.07 and 30.57 min on DB-wax and HP-1 columns, respectively. The same EAD-active peaks were also detected in the volatiles collected from laboratory-raised virgin females after they were brought out of diapause. No trace of these two peaks was found in the volatiles collected either from females in diapause or males. The mass spectra and retention times in the two capillary columns of the two EAD-active peaks were identical to those of buibuilactone [(R,Z)-5-(-)-(oct-l-enyl)oxacyclopentan-2-one or (R,Z)-5-dodecen-4-olide] and japonilure [(R,Z)-5-(-)-(dec-l-enyl)oxacyclopentan-2-one or (R,Z)-5-tetradecen-4-olide]. The stereochemistry of the major component sex pheromone was determined by chiral chromatography (Leal, 1991). (R)-buibuilactone appeared at R, 65.54 min (k' = 81.03), whereas the S enantiomer appeared at 67.15 min (k' = 83.04, c~ = 1.02). The natural product from A. octiescostata was first separated by silica acid column in a hexane-ether (80 : 20) fraction before inject-

SEX PHEROMONE OF

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ing in the capillary column. It gave the same R, as synthetic (R)-buibuilactone, and no trace of its enantiomer was detected. Therefore, the major component sex pheromone was fully identified as (R,Z)-5-(-)-(oct-l-enyl)oxacyclopentan2-one. Nevertheless, it was not possible to determine analytically the absolute configuration of the minor component due to the small amount of natural japonilure. Buibuilactone and japonilure have been identified as constituents of the sex pheromone system of other scarab beetles, A. cuprea (Leal et al., 1993a) and A. albopilosa sakishimana (Leal et al., 1994). However, these species are geographically and/or seasonally isolated. A. cuprea and A. octiescostata have been recorded on the main island (Honshu) of the Japanese archipelago, but their flight activity is seasonally isolated. The former is active in summer, whereas the emergence of the latter occurs mainly in early spring. On the other hand, A. albopilosa sakishimana is geographically restricted to Okinawa. GC-EAD using A. octiescostata antenna as the sensing element gave the best performance of all scarab beetles investigated thus far (reviewed in Leal et al., 1994). The same antennae could be used for as long as 12 hr, and the response to the major sex response showed a signal-to-noise (S/N) ratio of 12. In the range tested, the same S/N ratio was recorded regardless of the amount of pheromone. Amounts below the detection limit of our flame ionization detector (FID) system ( < 0 . 1 ng) gave nearly the same response as that to 500 ng of synthetic sex pheromone. Whole-body extracts of female A. octiescostata generated such a small amount of the sex pheromone that they were not detected by FID; however their occurrence in the extracts was confirmed by EAD. Although the reproducibility of the response to the minor component was far better than that obtained with male A. cuprea antenna as the sensing element (Leal et al., 1993a), a few times the signal of the minor (but not the major constituent) was missed, although both compounds were detected by FID. Bioassay. The attractancy of the synthetic sex pheromone was first demonstrated in a wind tunnel. Males responded to 100 ng of a synthetic blend (8:2) by walking to and gathering near the pheromone source (89.6 ___ 7.9%; N = 5). No response was observed to control (0%, N = 8) or to filter paper loaded only with buibuilactone (0%, N = 2). Evaluation of Synthetic Sex Pheromone. Preliminary field experiments explored the possibility of using sticky traps because with them it would be easier to rerandomize the blocks. However, the fact that they become saturated with beetles within 30 min (Figure 1) prevented their use. Even JT funnel traps had to be emptied twice a day because they were completely loaded ( > 6000 beetles/trap) within 3 hr (8:00-11:00 AM) (Figure 1B). Height of Traps. On sunny days, A. octiescostata males emerge from the soil and appear on the ground, where they crawl and fly at low levels searching for females. Later they are also found feeding in the high part of plants around

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FIG. 1. Response of A. octiescostata to synthetic sex pheromone. Beetles captured in 30 min in a sticky plate (A) and in 3 hr in a buried funnel trap (B).

golf courses. Therefore, we first investigated the effect of height on captures of the beetle. These experiments were carried out in Chiba (April 24-27) and Ibaraki (April 27-May 6). Catches in traps at 30 cm (Chiba) were significantly higher than those at 90 and 150 cm. On the other hand, experiments at Ibaraki demonstrated that buried traps (0 cm) captured significantly more beetles than those at 30 cm (Figure 2). By contrast, traps baited with virgin female Popillia japonica captured significantly more beetles when positioned at 28 cm than at 0, 56, or 112 cm (Klein et al., 1972). Catches of A. schonfeldti in synthetic sex pheromone-baited traps at 0 and 30 cm were not significantly different (Hasegawa et al., 1993). Interestingly, these three species display a very similar flight behavior (they all search for conspecific females at low levels); however, their patterns of response to sex pheromone-baited traps are different.

SEX P H E R O M O N E OF

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A. octiescostata Chiba 90.

a

60, Q,

30 e~

0 m 30

90

Height above ground

2000

150 (cm)

lbaraki a

1500

"~

m

tO00

5OO

0

Height above ground

30 (cm)

FIG. 2. Effect of the height of the traps on catches of A. octiescostata.

In practical applications, burying a trap would require more labor and would not always be possible. However, the fact that the lower the trap is positioned the better is the response of A. octiescostata to synthetic sex pheromone should be taken into consideration for the design of new "user-friendly" traps. Effect o f Dosage. Experiments were carried out in Chiba (May 5-8) and lbaraki (May 1-6) to determine the optimal dosage for capture of A. octiescostata with synthetic pheromone. A 10-fold increase in the amount of pheromone caused significantly better catches only from 0.1 to 1 mg (Chiba and Ibaraki). However, captures with 10 mg were not significantly different from 1 mg. Traps baited with 100 mg of the synthetic lure (Chiba) captured significantly more

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beetles than those with 1 mg (Figure 3). In the range tested, there was no saturation due to the overdose of sex pheromone. Attractancy of the synthetic sex pheromone was compared with that of two virgin female A. octiescostata (Chiba, May 3-13). Even 0.1 mg of the sex pheromone was a significantly better lure than two virgin female beetles. Catches were 18 times higher in traps having 1 mg of the synthetic sex pheromone than those baited with two virgin female beetles (Figure 4). We therefore recommend the use of 1 mg of synthetic sex pheromone in monitoring this species. Effect of Ratio of Two Components on Catches. The optimal ratio of two

Chiba a

600 500

ab

400 b 300 m e.,

200

IO~ 0

0.I

I

I 0

I00

Dosage (mg) Ibaraki

2000

a

1500

1000

~

SO0

0

0.I

I Dosage

I0 (rag)

FIG. 3. Captures of A. octiescostata with different dosages of synthetic sex pheromone

incorporated into plastic pellets.

SEX PHEROMONEOF A. octiescostata

1651

300

"o I I.

200

u~ o,a

en

10@

il I

b mg

2 VF

~

b'

0.1 mg 2 VF

FIG. 4. Comparative catches of A. octiescostata in traps baited with two virgin female beetles (2 VF) and 0.1 and 1 mg of synthetic sex pheromone.

components in the pheromone system of scarab beetles could not be experimentally determined for A. cuprea (Leal et al., 1993a) and Holotrichia parallela (Leal et al., unpublished). In order to rule out the possibility of error incurred by experimental design, we conducted experiments on the effect of the ratio of the two components on the catches of A. octiescostata not only in randomized blocks (as in the two cases cited), but also in a Latin-square design, which has been demonstrated to be more efficient than other designs for quantitative comparison of attractants (Perry et al., 1980). The former was done in Chiba (April 28-May 4) and the latter in Ibamki (April 23-28), where a larger area was available for the field tests. Neither in Latin-square nor in randomized-blocks experiments was a significant difference found in the response of male A. octiescostata to traps baited with the two components in the ratios of 9 : 1, 8 : 2, 7 : 3, and 6 : 4 (Figure 5). It has been long known that the optimum blend ratio of pheromone components of Lepidoptera is that which most closely approximates the natural ratio emitted by females. Behavioral responses diminish markedly even with slight deviations from this proportion (Baker, 1989). Deviation from the natural ratio (8 : 2) of sex pheromone emission by female A. octiescostata did not significantly diminish the attraction of males to traps in the field. One possible explanation is that the dosage of the synthetic bait was higher (Ibaraki, 1 mg; Chiba, 10 mg) than the level at which male beetles can discriminate different ratios of the sex pheromone. This hypothesis must await future field testing at lower dosages. The ratios of two components of other scarab sex pheromone systems have been demonstrated to change, according to the physiological condition of the female beetle (Leal et al., 1993a,b). That the ratio of buibuilactone/japonilure emitted by virgin A. cuprea changed considerably after mating and isolation

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300. a i

200 tO0

o

9:1 Ratio

8:2 o f the

7:3 two

6:4

components

lbaraki

1200'

.13 gk

--R

a

a

t

800

s. --

400

9:1 Ratio

:2 o f the

7:3 two

6:4

components

FIG. 5. Response ofA. octiescostata to traps baited with different ratios of buibuilactone and japonilure in two experimental designs: randomized blocks (Chiba) and Latin square (lbaraki).

was considered to be a selective advantage of virgin against mated females, giving the former a higher mating probability (Leal et al., 1993a). Therefore, it may be also possible that males of different physiological conditions respond to different ratios of the sex pheromone. Seasonal and Daily Flight Activity. Seasonal captures of A. octiescostata was investigated in Ibaraki with buried traps in a fairway. Catches reached a peak at the end of April 1993 (Figure 6), when over 10,000 beetles per trap

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10000 ~ ~ 8000 6000

ooo

April

May,

1993

FIG. 6. Seasonal flight activity of male A. octiescostata, as revealed by trap catches in Ibaraki Prefecture. A very small peak was also detected in autumn.

and day were captured. These catches reflect not only the high response of male A. octiescostata to sex pheromone, but also the level of beetle infestation at that

golf course. The highest flight activity of the beetle occurred during a holiday season in Japan, "golden week," when many golf competitions take place. In mid-September, flight activity by the beetles was observed by personnel of Edosaki Country Club. We set traps for one week (September 22-28) in three different places. One trap in a fairway (same height condition as for the data in Figure 6) captured a total of 111 beetles, whereas catches in traps at a tee positioned 50 cm above the ground numbered 66. Another trap at 150 cm above the ground captured 51 beetles. In other words, two peaks were found in the seasonal flight activity of A. octiescostata, the one in autumn being very small. Daily flight activity was investigated in Chiba (May 5, 1993) utilizing traps baited with 0.1, 1, 10, and 100 mg. The pattern of activity was almost the same (regardless of the dosage). Beetles were active from 7:00 AM to 3:00 PM, showing a peak of flight activity from 9:00 to 10:00 (Figure 7). The flight activity was affected by weather conditions. This experiment was conducted on a sunny day, but it become cloudy at about 11:00 AM and the temperature decreased, influencing the flight activity of A. octiescostata (decrease in catches; valley at - 11:00 AM). Wind was stronger at - 12:30 and - 1:00 PM, and this was reflected in the decrease in captures during this period (valley in between 12:00 and 1:00). In conclusion, male A. octiescostata are highly attracted to the synthetic sex pheromone system, which may be a valuable tool for monitoring and integrated pest management.

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900

30

300

l0

,oot 0 ".o.' .o.' .o.' o ' o ' o "

....

. o ,o ' o . o. o.o.~ . ~ " o ' o . o.O. O. O

0

0

' ' ''7'7'7'7"7'7"7'7"7'7'7'7 Time (h)

Ft~. 7. Daily flight activity o f male A. octiescostata in relation to the ambient temperature. Data were recorded at a golf club in Chiba on a sunny day. Acknowledgments--We are indebted to the personnel of Edosaki and Chiba Springs country clubs for their cooperation and assistance with these studies. We are grateful to Komei Osada and Sakae Onozawa (Edosaki), Tetsuya Takahashi (Chiba), and Satoshi Yamanaka (SDS Biotech, Japan) for their help in gathering field data.

REFERENCES T.C. 1989. Sex pheromone communication in the Lepidoptera: New research progress. Experientia 45:248-262. DOOLrr'rLE, R.E., TUMLINSON,J.H., PROVEAUX,A.T., and HEATH, R.R. 1980. Synthesis of the sex pheromone of the Japanese beetle. J. Chem. Ecol. 6:473-485. HASEGAWA, M., LEAL, W.S., and SAWADA, M. 1993. Field evaluation of Anomala schonfeldti Ohaus (Coleoptera: Scarabaeidae) synthetic sex pheromone. J. Chem. Ecol. 19:1453-1459. KLEIN, M.G., LAND, T.L., JR., and LAWRENCE,K.O. 1972. The influence of height of exposure of virgin female Japanese beetle on the captures of males. Environ. Entomol. 1:600-601. KOSEKI, K., EBATA, T., KADOKURA,H., KAWAKAMI,H., ONO, M., and MATSUSHITA,H. 1993. Synthesis of sex pheromones of the Japanese beetle and cupreous chafer beetle. Tetrahedron 49:5961-5968. LEAL, W.S. 1991. (R,Z)-5-(-)-(Oct-l-enyl)oxacyclopentan-2-one, the sex pheromone of the scarab beetle Anomala cuprea. Naturwissenschafien 78:521-523. LEAL, W.S., MOCHIZUK[,F., WAKAMURA,S., and YASUDA,T. 1992. Electroantennographic detection of Anomala cuprea sex pheromone. Appl. Entomol. Zool. 27:289-291. LEAL, W.S., SAWADA,M., and HASEGAWA,M. 1993a. The scarab beetle Anomala cuprea utilizes the sex pheromone of Popillia japonica as a minor component. 3. Chem. Ecol. 19:1303-1313. LEAL, W.S., SAWADA,M., MATSUVAMA,S., KUWAHARA,Y., and HASEGAWA,M. 1993b. Unusual periodicity of sex pheromone production in the large black chafer Holotrichia parallela. J. Chem. Ecol. 19:1381-1391. LEA]., W.S., KAWAMURA,F., and ONo, M. 1994. The scarab beetle Anomala albopilosa sakishiBAKER,

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mana utilizes the same sex pheromone blend as a closely related and geographically isolated species, Anomala cuprea. J. Chem. Ecol. 20:1667-1676. PERRY, J.N., WALL, C., and GREENWAV, A.R. 1980. Latin square designs in field experiments involving sex attractants. Ecol. Entomol. 5:385-396. SAS INSTITUTE. 1992. Software for Statistical Visualization on the Apple ® Macintosh. JMI~ Introductory Guide. SAS Institute, Inc., Cary, North Carolina.

Identification and field evaluation ofAnomala octiescostata (Coleoptera: Scarabaeidae) sex pheromone.

Using GC-EAD, the sex pheromone of the scarab beetleAnomala octiescostata was identified to be a 8:2 binary mixture of (R,Z)-5-(-)-(oct-1-enyl)oxacycl...
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