J. Asia-Pacific Entomol. 3(1) : 33-40 (2000)

Identification of Volatiles Responsive to the Alder LeafBeetle (Agelastica coerulea) from its Host Plant,

the Japanese Alder (Alnus glutinosa)

Cheon Hae Jung, Kyeung Sik Han and Kyung Saeng Boo*

Abstract - Electroantennography (EAG) and gas chromatography coupled with electroantenno­graphic detection (GC-EAD) were used to identify volatiles from Alnus glutinosa attractive toAgelastica coerulea. Seven compounds (3-cyclohexene-I-methanol, 2-cyclohexen-l-01, 5-methyl-2­(l-methylethyl)-cyclohexanol, 5-alpha-androstan-17-beta-ol, 3-ethyl-3-hydroxy-androstan-17-one,(Z)-2-(9-octadecenyloxy)-ethanol, and hexadecanoic acid) showed responsiveness to the antenna ofA. coerulea. In bioassays, adults and larvae of A. coerulea were attracted to volatiles from A.glutinosa.

Key Words - Agelastica coerulea, Alder leaf beetle, Alnus glutinosa, Japanese alder, EAG, GC­EAD, Bioassay, Volatiles

Introduction

Host selection by insect hervibores comprises anumber of behavioral response to various stimuliassociated with host plants; stimuli may be olfac­tory, gustatory, visual, or mechanical (Visser, 1986).Plant odors have been considered of minor impor­tance in host selection for many insect species,especially those that perform as r-strategists (Kogan,1976). This view needs to be reconsidered becausesuch insects as aphids (Petterson, 1970; Petterson,1973; Chapman et al., 1981), white flies (Vaisham­payan et al., 1975), and rice brown plant hopper(Obata et al., 1983) possess the full equipment ofolfactory receptors and are attracted by host plantodors. It is generally assumed that sensitivity differsaccording to the host specialization: monophagousspecies should possess receptors tuned to volatilesthat are specific to their host plants, while poly­phagous insects would respond to a broader rangeof chemicals (Dickens, 1984; Vissser, 1986; Averillet al., 1988; Raguso et al., 1996).

There are many examples of insects being attract­ed to the odors of their host plants, both by flyingand by walking or crawling. Larvae of the desertlocust, Schistocerca gregaria (Kennedy et al., 1969;Moorhouse, 1971), and adult Colorado potato beetl­es, Leptinotarsa decemlineatta (De Wilde et al.,1969; Visser, 1976; Visser et al., 1977) and wing­less females of the aphid, Cryptomyzus korschelti,have been shown to walk upwind in the odor oftheir host plant in a wind tunnel (Visser et al., 1987).Plant chemicals, a result of the oxidative degrada­tion of leaf lipids, are commonly called green leafvolatiles and differ from plant to plant to some ex­tent. Although some insects have shown to be att­racted by a single green leaf chemical, this, of cour­se, cannot result in anything more than a very gen­eral attraction to vegetation. The Colorado potatobeetle, L. decemlineata, is attracted by the specificcombination of green leaf volatiles, trans-2-hexanal,cis-3-hexanyl acetate, cis-3-hexanyl acetate, cis-3­hexenol and trans-2-hexenol produced by potato.Crucifer- feeding insects are attracted by iso-thio­cyanates derived from the glucosinolates that char-

*To whom correspondence should be addressed. E-mail: ksboo@plaza.snu.ac.krSchool of Agricultural Biotechnology, Coil. Agric. Life Sci.. Seoul Nat!. Univ., Suwon 441-744, Korea. E-mail: ksboo@plaza.snu.ac.kr

-33-

34 J. Asia-Pacific Entomol. March 2000

acterize the plants chemically. Onion flies are attr­acted by volatile sulfur-containing compounds, suchas dipropyldisulfide (Bemays et al., 1994).

The plants of Alnus spp. were known to have abi­lity to fix nitrogen due to nitrogen-fixing bacteriadwelling on roots. Thus they have been widely im­planted as primary tree species for the purpose offertilizing soils in Korea. The alder leaf beetle(ALB), Agelastica coerulea, is the major pest insectfeeding on leaves of Alnus spp. such as Alnus gluti­nosa, Alnus japonica and Alnus hirsuta. ALBadults, over-wintered in the soil near host plants,crawl up host plants, after emergence in the spring,to feed on the leaf matrix primarily and females laytheir eggs on both sides of leaves late July. Hatchedlarvae grow up and mature larvae drop with damag­ed leaves to pupate on the ground. Adults 8-10 mmlong emerge in early August, habituate host plantsagain, sometimes dispersing to new plants and enterdiapause in late Sep. to early Oct (Choi, 1991). In­terestingly, emerging time of ALB adults in the spr­ing coincides with fully growing time of leaves ofAlnus spp. Their phenological coincidence may be aco-evolutionary process or insect's survival strategyto have a monopoly for the host plant. This workwas tried to identify chemicals, from their majorhost plant, the Japanese alder, A. glutinosa, attrac­tive to ALB and to examine the behavioral responseof ALB to these chemicals.

Materials and Methods

Experimental insectAgelastica coerulea (ALB) were mainly collected

from Alnus glutinosa during mid Jul. to late Sep.1998 at Suwon, Korea. Newly emerged ALB adultson other plants around A. glutinosa were also col­lected. They were maintained in plastic cages andgiven water with soaked cotton balls until experi­ments.

Plant materialsMature leaves of ALB's host plant, A. glutinosa

and non-host plants, Hibiscus syriacus and Corylusheterophylla var. mandshurica, were used for att­ractivity toward A. coeulea adults or larvae. Unda­maged mature leaves of A. glutinosa were immers-

ed in 200 ml hexane for 24 hrs, and filtered extractwas concentrated with a vacuum evaporator (EYE­LA, N-N series, Tokyo, Japan) to be re-dissolved inhexane. Extract was stored at refrigerator (4°C) untilbioassay for EAG and GC-EAD. Larva-fed leaveswere used as damaged leaves.

EAGALB antenna was cut at both ends and connected

to silver wire electrodes by conductive electrode gel(Spectra 360, Parker Laboratories, Inc., New Jersey,USA). In some cases only the basal end was cut tolengthen antenna's active state. The cut end wasconnected to a glass microelectrode by conductivegel. Filling solution was 1M KCI. The control stim­ulus was 10 ul hexane. Fresh cartridges were pre­pared just before each stimulation. The stimulatingchemical (2 sec. duration) was delivered into a puri­fied air stream flowing (2 liter/min) continuouslyover the preparation. The delivery system employ­ing a filter paper in a disposable pasteur pipetettecartridge has been described previously (Wadhamset al., 1982). Electronic signal was stored with soft­ware (EAG, Syntech, Hilversum, The Netherlands).In most cases an antenna was used only once, butsome of them used twice at one-hour interval atleast to give full time for recovery from possibleadaptation.

GCLeaf hexane extracts were separated with a DB­

225 capillary column fitted in Hewlett Packard6890 (Hewlett Packard, Wilmington, USA) orShimadzu gas chromatograph (Shimadzu, Kyoto,Japan). The oven temperature for the DB-225 col­umn (Alltech, Deerfield, USA) was maintained at60°C for 2 min, then programmed at 5°C/min to re­ach 200°C and maintained at 200°C for 20 min.Both instruments were fitted with a flame ionizationdetector and the carrier gas was He.

Coupled GC-EADFor the GC-EAD system, the effluent of the

capillary column in GC was divided into 1 : 1 in theoven to deliver simultaneously to the antennal pre­paration and the GC detector (Wadhams, 1990). Theseparation of leaf extract was achieved in ShimadzuGC (Shimadzu, Kyoto, Japan) equipped with a DB-

Vol. 3, No. I

Activated charcoal

J. Asia-Pacific Entomol.

Vacuum pump

Activated charcoal

35

Fig. I. Schematic diagram of the star-shaped olfactometer. Only one arm contained Alnus glutinosa leaves and other threearms were left vacant.

225 capillary column. The oven temperature for theDB-225 column was maintained at 60°C for 2 min,then programmed at 5°C/min to 200°C and main­tained at 200°C for 20 min. The antennal base wasconnected to the electrode gel and its distal end to aglass capillary microelectrode. Filling solution was1M KCI solution and Ag-AgCI electrode was used.GC-EAD gram was recorded with software (GC­EAD, Syntech, Hilversum, The Netherlands) whilemonitoring the response of ALB adults to effluents.

GC-Mass spectrometryA DB-225 capillary column fitted in a Hewlett

Packard 5890 GC was directly coupled to the massspectrometer and data system (JMS-AX505WA,Jeol, Tokyo, Japan). The ionization was by electronimpact at 70 eV and the experimental conditionswere the same as in gas chromatography.

BioassaysThe bioassays employed a Y-tube olfactometer for

ALB adults and a star-shaped olfactometer for ALBlarvae (Fig. 1). In the Y-tube olfactometer (stem: 7x 20 cm, arm: 5 x 7 ern) test, air was drawn throughactivated charcoal at the air flow rate of 2,500mllmin. ALB adults were used regardless of sexand recorded as a positive when they moved morethan 2 em from the bifurcation site in an olfacto­meter arm. As adults moved around very rapidly, itwas hard to check their movement at a fixed timeschedule. So, insects moving out of experimental

boundary were recorded as no response. After onetrial, the olfactometer tube was washed and theolfactometer arm for control was switched at eachtrial. To decide whether damaged plant leaves emitpeculiar chemicals attracting ALB adults, responseto damaged and intact A. glutinosa leaves werecompared.

Third instar larvae were used with the star-shapedolfactometer (radius: 5 em). After 30 min for initialadaptation, another 10 min was given for recoveringto a stable state. The air- flow rate was 500 mllmin.Individuals moved 3 em or longer into the arm fromthe inlet were counted as attracted to the chemical.Insects were excluded from counting when they didnot move at all. The inlet position was changedevery 2hrs.

After calculating the average and standard devia-

Table 1. Attraction of Agelastica coerulea larvae (3rd in­star) to Alnus glutinosa leaves in a star-shaped olfactome­ter (N = 15, Repeats = 2)

Time Mean no. larvae attracted(min) Control 1 Control 2 Leaves Control 3 NR

30 3.0a 2.5a 3.5a O.Ob* 6.060 0.5b 1.5b 1O.5a O.Ob 2.590 O.Ob O.Ob 1O.5a 0.5b 4.0

120 O.Ob O.Ob 1O.5a 0.5b 4.0

* Different letters within the same row denote significant differencebetween means (P< 0.05, DMRT). NR denotes 'no response' andcontrols mean 3 other arms, except for the treatment arm having leavesof the star shaped olfactometer.

36 J. Asia-Pacific Entomol. March 2000

Results

tion of data, we examined the significance of eachtreatment by Duncan's multiple range test or t-test(SAS, 1987).

Star-shaped olfactometer testALB larvae (3 rd instar) were attracted to A. glu­

tinosa leaves (Table 1). Once larvae were gatheredaround the inlet position of A. glutinosa leaves, theydid not move to other inlet sites for a long time.

Table 2. Response of A. coerulea adults attracted to A.

glutinosa leaves against air or other plant leaves in a Y­tube olfactometer. ALB choice behavior meant that theyhad an ability to differentiate host plant among many otherplants and ALB adults made a choice for host plantswithin 30 minutes

1) A. glutinosa vs. air (N = 13, R= 3)

V-tube olfactometer testALB adults were also attracted to A. glutinosa

leaves (Table 2-1) but did not discriminate betweenintact or damaged leaves of A. glutinosa (Table 2-4).While they fed on leaves of different Alnus spp. infields, they did not eat leaves of Corylus hetero­phylla, the same family of Betulaceae, and those ofHibiscus syriacus of family Malvaceae. ADB adultsdid not respond to leaves of the last two plant spec­ies in a Y-tube olfactomer test (Table 2-2, 2-3). Incontrast to the larval behavior staying at one inletsite once gathered in a star-shaped olfactomether,ALB adults showed an active searching behaviortoward or away from the olfactometer arm contain­ing A. glutinosa leaves. Considering the possiblesaturation of leaf volatiles in Y-tube olfactometerarms, it seemed that the decision making of theALB adults for choosing the attractive leaf could bewithin 20 min after introduction. In case of ALBadults collected from non-host plants, they did notdiscriminate a host plant from non-host plants(Table 3).ProbabilityTime (min) A. glutinosa Air

5103045

4.675.677.677.67

3.673.002.002.33

NSNS

P<0.05P<0.05

EAG testEAG test was conducted to examine whether

ALB adults respond to volatile chemicals from A.

2) A. glutinosa vs. Hibiscus syriacus (N =15, R =3)

Time (min) A. glutinosa H. syriacus Probability

5 5.00 1.33 NS10 5.00 0.66 P< 0.0520 8.00 0.33 P< 0.0530 8.67 1.00 P< 0.01

3) A. glutinosa vs. Corylus sp. (N = 15, R=3)

Table 3. Number of A. coerulea adults, collected fromnon-host plants, attracted to A. glutinosa leaves againstother plant leaves in a Y-tube olfactometer. Newly emerg­ed ALB adults could not recognize A. glutinosa as hostplants and did not discriminate A. glutinosa from otherplants

1) A. glutinosa vs. Corylus sp. (N = 10, R= 3)

Corylus heterophyllaTime (min) A. glutinosa var. Probability

mandshurica

Time(min) A. glutinosa

Corylus heterophyllavar.

mandshuricaProbability

5102030

4.005.337.338.00

0.432.000.000.67

NSNS

P<O.OIP<0.05

5102030

1.672.001.672.33

1.671.331.331.67

NSNSNSNS

4) A. glutinosa leaves damaged vs. undamaged (N = V20, 2) A. glutinosa vs. H. syriacus (N = 10, R = 3)R=4)

Time (min) Damaged Undamaged ProbabilityTime (min) A. glutinosa H. syriacus Probability

5 6.00 4.75 NS 5 2.33 1.00 NS

10 6.50 5.00 NS 10 2.00 2.00 NS

20 8.00 5.50 NS 20 2.33 2.33 NS

30 9.25 5.75 NS 30 1.67 3.00 NS

Vol. 3, No.1 J. Asia-Pacific Entomol. 37

Table 4. Identified chemicals from hexane extracts of A. glutinosa leaves by GC-Mass spectroscopy and EAG amplitudefor some of them responded by A. coerulea adults

No. Retention time (min) Chemicals EAG(mv)

1234

567

89

10

11

1213

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

2.72

2.93

4.32

5.09

6.16

6.56

6.91

8.24

9.49

10.96

11.12

11.25

13.54

13.78

14.31

14.55

15.49

16.39

16.53

17.70

18.63

19.80

20.55

22.76

23.48

24.31

.26.47

27.00

27.29

27.56

28.79

32.94

33.85

34.89

37.50

40.86

3-cyclohexene-l-methanol

1,3, 5-cycloheptatriene, methyl benzene

1, 3-dimethyl benzene

1,4-dimethyl benzene

propanoic acid

2-cyclohexen-l-01

2,6-dimethyl-5-heptenal

2-methyl-2-hexanol

cyclohexane methyl pivalate

(R)-(-)-(2)-14-methyl-8-hexadecen-l-01

benzaldehyde

nonanol

2-hydroxybenzaldehyde

cyclopenta-l, 3-cyclopropa-l, 2-benzene

benzene acetaldehyde

cyclobutal 1,2,3, 4-dicyclopentene

(Z, 2)-9, 12-octadecadienoyl chloride

5-methyl-2-( I-methylethylj-cyclohexanol

4, II, II-trimethyl-8-methylene-bicyclo-7, 2 undec-4-ene

3,7-dimethyl- (E)-2,6-octadien-l-01

IH-cyclopenta-l, 3-cyclopropa-l, 2-benzene,Naphthalene

l-bromo-5-heptadecene, 9-eicosyne

2, 4-methano-2H-indeno-l, 2-b:5, 6-b'bisoxirene

2-methoxy-4- (2-propenyl)-phenol

4-(2,6,6-trimethyl-l-cyclohexen-I-yl)-3-buten-2-one

5-alpha-andorstan-17-beta-ol

(-)-spathulenol

3-ethyl-3-hydroxy-androstan-17-one

hexadecanal

(2)- 2-(9-octadecenyloxy )-ethanol

santalol

2H-benzocyclohepten-2-one

3,7, 11, 15-tetramethyl, (R) - (R), (R) - (E) -2-hexadecen-l-01

hexadecanoic acid

platambin

heptacosane

0.89

1.38

1.52

1.04

0.98

1.09

0.82

glutinosa leaves or not. ALB female adults showedhigh response to hexane extracts of A. glutinosaleaves (Fig. 2).

GC-EAD measurementGC-MS spectroscopy result showed that leaf

hexane extracts contained several derivatives of so­called green leaf volatiles (Table 4). ALB adult fe­males responded to 9 compounds and among them,2 compounds, with retention times of about 31 and39 min, were not identified by gas mass spectro­scopy (Fig. 3).

38 J. Asia-PacificEntomol. March 2000

leaf extracthexane

b

L..---__'-----_

:> 2EorCJlc ., ..11. =CJlQ) ea: 0

air

*Different letters denote significant difference between means (p < 0.05, DMRT)

Fig. 2. Electroantennographic response of Agelastica coerulea adult female to hexane extract of A. glutinosa leaf (N = 2, R=6).

:> 3

18

II

45.00 50.00

Fig. 3. Parallel FlD and EAD chromatograms obtained from hexane extracts of A. glutinosa leaf. Arrows indicate respon­sive nine chemicals, 7 chemicals were identified by GC-MS chromatography but the other 2 chemicals (RT. ca. 31 and 39min, each) were not identified.

Discussion

In generall, a specialist hervibore can locate itshost plant species by sensing peculiar plant odors.Green leaf volatiles resulting from oxidation of leaflipids are known to key chemicals alluring an insectto plants. One or combination of several chemicalsmay trigger a series of attractive behavior. Althoughthe electroantennographic response of ALB adultsshowed that they responded to leaf hexane extractsand that GC-EAD experiments showed 9 com­pounds to be active in stimulating their antennae,but we have not determined which compound is themost important chemical or what combination is?Also we do not know whether ALB adults werestimulated to feed on and/or to oviposit. So basedon chemicals identified through GC-MS analysis,

the behavior test in field should be carried out in thefuture to measure degree of attractiveness and toobserve real behavioral response. Duke (1992) re­ported four chemicals, alnusfoliediolone, delta­amyrenone, 3-beta-hydroxyglutin-5-en, and L-orni­thine from A. glutinosa leaf, but we could not iden­tify the same chemicals from our extracts. SinceALB adults do eat the leaves of different plantspecies belonging to the same genus, Alnus, therecould be a common component (s) among Alnusspp. But existence of more ALB adults on A. gluti­nosa than other Alnus spp. indirectly tells therecould be a food preference chemical in A. glutinosa.

The process that newly emerged adults find theirhost plants may be innate or acquired by experi­ences. In the case of ALB adults, determination ofpupation site of larvae adhering to falling leavesseems to depend on a random process. While col-

Vol. 3, No.1 J. Asia-Pacific Entomol. 39

lecting ALB adults, we found the ALB adults resi­ding on non-host plants around its host plant spec­ies. The Y-tube olfactometer test of the ALB adultscollected from non-host plants showed they couldnot discriminate between the host and non-hosts. Inspite of emergence of some adults at other plants,they seem to be able to find out their host plants andcontinue their life cycle successfully. How is it pos­sible? When adults and larvae share the same foodplants like A. coerulea, host plants can act as rende­zvous sites where the both sexes meet and copulate.Whereas adults explore different food sources,odors of oviposition sites are attractive to femalesas well as males. The European corn borer, Ostrinianubilaris, males were trapped with phenylacetal­dehyde, a corn silk odor constituent (Cantelo et al.,1979). Male Rhagoletis pomonella flies is attractedto the odor of apples (Fein et al., 1982). In polyga­mous bark beetle species, males primarily select thehost trees and females follow, lured by the interplayof pheromone and host odors (Wood, 1982). Insectlearning is another subject complicating a problemof elucidating attractive chemicals, so another ex­perimental scheme will be required in the near fu­ture.

On the other hand, green leaf volatiles can bedisruptive semiochemicals to beetles (Dickens et al.,1992; Wilson et al., 1996; Borden et al., 1997).Green leaf volatiles were patented as "inhibitors"of pine beetle aggregation pheromones demonstra­ting that hexanal and I-hexanol were repellents tothe southern pine beetle, Dendroctonus frontalis;the small southern pine engraver, Ips avulsus andthe eastern fivespined ips, I. Grandicolis (Dickens etal., 1993, 1995). 1- hexanol has recently been de­monstrated to be a multifunctional pheromone pro­duced by male Pityogenes knechteli, another pinebark beetle (Savoie et al., 1998). Consequently, theresponse of insects to green leaf volatiles eventuallydepends on insect species themselves.

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(Received December 23, 1999; Accepted February 15,2000)