Gustatory responses of a specialist and a generalist grasshopper to terpenoids of Artemisia ludoviciana

Entomologia Experimentalis et Applicata 45: 37-46, 1987

(~: Dr W. Junk Pubhshers, Dordrecht - Printed in the Netherlands 37

Gustatory responses of a specialist and a generalist grasshopper to terpenoids of Artemisia ludoviciana

Michael H. Blust I & Theodore L. Hopkins Department o f Entomoloy, Kansas State University, Manhattan, KS 66506, U.S.A.; 1present address." 38 Green Mountain College, Poultney, VT 05764, U.S.A.

Accepted March 12. 1987

Key words." Grasshopper, Hypochlora alba, Melanoplus sanguinipes, Artemisia ludoviciana, Asteraceae, terpenoids, monoterpenes, sesquiterpene lactones, antifeedants, glandular trichomes, gustation

Abstract

Hypochlora alba (Dodge) is a specialist grasshopper that lives and feeds almost exclusively on the sage brush Artemisia ludoviciana Nutt., a plant mostly avoided by the generalist grasshopper Melanoplus sanguinipes (Fabr.). Analysis of leaves, seed heads, and glandular trichomes by gas chromatography-mass spectrometry revealed 1,8-cineole, camphor and borneol to be the major monoterpenes and achillin the major sesquiterpene lactone. These terpenoids increased over the growing season and were two to five times more concentrated in the seed heads than in the leaves. Gustatory choice tests showed a feeding stimulant(s) for H. alba to be present in extracts of A. ludoviciana and A. carruthii Wood, a closely related species that H. alba will feed upon, but not in a non-host species, A. filifolia Torr. This stimulant activity was found in a fraction containing primarily monoterpene hydrocarbons. Other fractions containing sesquiterpene lactones had antifeedant activity against M. sanguinipes. Tests with achillin showed the average foliar levels (ca. 2~ dry weight) to be above the rejection threshold of the generalist (0.5~ but below that of the specialist (4~ Reproductive tissue contained average levels greater (ca. 7~ than the rejection threshold of either species.

Introduction

Hypochlora alba is a highly specialized grasshopper which feeds almost exclusively on Louisiana sagewort, Artemisia ludoviciana (Knutson, 1982). This plant appears to be protected by both glandular and non-glandular trichomes and is fed upon very little by generalist grasshoppers such as the related Melanoplus sanguinipes (Mulkern et al., 1969). A. carruthii is a closely related species of sage-brush on which we have occasionally observed H. alba in west- ern Kansas. We have not found H. alba on Artemisia filifolia, A. tridentata Nutt., or A. frigida Willd. This model system has been the subject of several

previous studies emphasizing the physical defenses of the plant and adaptations of the specialist grasshopper to these defenses (Knutson, 1982; Smith & Grodowitz, 1983; Smith & Kreitner, 1983). We have recently found that the olfactory response (elec- troantennogram) of male/t, alba is much greater to total A. ludoviciana volatiles and individual monoterpenes than that of conspecific females or both sexes of M. sanguinipes (Blust & Hopkins, 1987).

This study examines the role of the terpenoid chemistry in the gustatory responses of specialist and generalist grasshopper species to A. ludoviciana and two closely related Artemisia species. By using

38

gustatory preference tests along with extraction, fractionation, and identification of terpenoid compounds in A. ludoviciana and related sage brush species, we have looked for feeding stimulants for the specialist H. alba and antifeedants for the generalist M. sanguinipes.

Materials and methods

Terpenoid identification. Terpenes from A. ludoviciana were tentatively identified from extracts and cold trapped volatiles using a Tracor 540 gas chro- matograph (GC) with flame ionization detector

(Blust & Hopkins, 1987). A 25 m BP-1 (SE-30 equivalent) vitreous silica 0.33 mm ID column was used with helium as the carrier at a head pressure of 6 psi. The temperature program was 50 ~ for 5 min to 275~ for 5 min at a rate of 10~ Confir- mation of structural identify of certain monoter-

pene components was obtained by capillary gas chromatography - mass spectrometry (GC-MS). Sesquiterpene lactones used as standards were iso- lated and identified from A. ludoviciana (Ohno et al., 1980).

Extraction procedure. To isolate terpenoid compounds for GC analysis, foliage was field collected and oven dried at 50~ for 24 h. The leaves were finely chopped with a food grinder, followed by extraction with dichloromethane (approx. 125 g/l), filtered through Celite 545, and concentrated in a rotary evaporator using a 90~ water bath. The remaining solvent was removed under a nitrogen stream. The sample was weighed and diluted to a 10070 stock solution in chloroform.

A further purification of this extract made by the following method will hereafter be called the "oil". The solvent was entirely evaporated in a rotary evaporator with a 90 ~ water bath, and the residue then redissolved with 95~ ethanol while heating in the water bath. A 2070 lead acetate aqueous mixture was added in a 1:1 ratio to the ethanol to precipitate chlorophyll and some phenolic compounds. The mixture was filtered through Celite 545 and the fil- trate was placed in the rotary evaporator to remove the ethanol. The residue was dissolved in dich-

loromethane and partitioned against the remaining water in a separatory funnel. The dichloromethane portion was retained and the solvent removed in va- cuo as previously described leaving a yellowish- brown oil. This oil was weighed and dissolved in chloroform for a 1007o stock solution for gustatory tests.

Extract fractionation. Fractions of A. ludoviciana extract were prepared by liquid column chromatography, Silica gel was activated at 110 ~ for 18 h and 10 g were poured into a 1.2 cm diameter column in a 25 : 75 benzene : dichloromethane solution. After evaporation of the solvent approximately 1 g (10 ml of the 10% stock solution) was redissolved in 2 ml

of the initial chromatographic solvent and placed on the column. The solvent series used was benzene : dichloromethane in ratios of 1) 25 : 75 and 2) 5:95, followed by 3) 100% dichloromethane and dichloromethane: methanol in ratios of 4) 99: 1 and 5) 90: 10. All fractions were evaporated to dryness under a nitrogen stream, weighed, and redissolved in 10 ml of chloroform for use in gustatory tests.

Individual plant analysis. Individual A. ludoviciana plants were sampled 5 June, 9 July, 13 August, 11 September, and 25 November 1984. Plants were picked randomly in the field and placed in plastic bags. These were refrigerated within 1/2 h and extracted within 6 h. About one third of the total leaves were removed from the plant from all heights on the stem. These were cut into ca. 0.3 cm 2 pieces and

mixed. A 0.1 g portion was homogenized in a ground glass tissue grinder using ca. 4 ml of dichloromethane. The remainder of the cut leaves were weighed and oven dried to obtain wet/dry weight conversion factors for each plant. The leaf extract was filtered through glass wool and evaporated under a nitrogen stream to 0.4 ml for GC analysis. If reproductive parts were present, this same procedure was followed except no cutting of buds or flowers was needed before mixing.

Samples were analysed by GC using a Varian 3700 GC with flame ionization detector. The 1.8 m, 1.5 mm inside diameter, glass column was packed with 5~ SE-30 coated on 6 0 - 8 0 mesh Gas Chrome Q (Applied Science). The oven was temperature pro-

grammed from 7 0 - 2 0 0 ~ at 5 ~ with no ini-

tial temperature hold. Nitrogen was the carrier at a

rate of ca. 20 ml/min. Calculations of the compound

concentrations in terms of percent of plant part dry weight were based on the peak areas obtained from a Hewlett-Packard 3385 integrating recorder using camphene as an external standard.

Glandular trichomes. Seed heads that had been oven

dried at 50 ~ were shaken in a 80 mesh sieve. The pan below was placed under a binocular microscope and the heads of the glandular trichomes were picked up using the tip of an insect pin. A total of

500 gland heads were placed in 0.1 ml of dichloromethane for qualitative analysis by capillary

GC.

Experimental insects. Adults of two species of grasshoppers were used in this investigation. The special-

ist, H. alba, was collected from A. ludoviciana in a field ca. 8 km west of Brookville, Kansas. It was

maintained in a rearing room at 28 ~ and fed on fresh cut host plants and water until tested. The

generalist, M. sanguinipes was from a non- diapausing laboratory colony that was reared in a

greenhouse and fed seedling rye grass and ground Purina R guinea-pig pellets and water. Experimental

insects were placed in a cage with water only for 24 h prior to testing and held at 28 ~

Disc tests. Gustatory bioassays were conducted using nitrocellulose membrane discs (Schleicher & Schuell, 2.5 cm diameter) (Bristow et al., 1979). The

test substances were dissolved in chloroform since dichloromethane caused the discs to deform. Plastic

cages (7.3 cm by 5.0 cm by 3.5 cm) were used to con-

fine the grasshoppers with 2 discs, a test and control (solvent) treatment. The discs were pinned on insect pins and an index card dot, from a paper punch, was pinned beneath the disc for support. These discs were pinned through the cage bot tom into a styrofoam board. Two grasshoppers were added per

cage, and sexes were never mixed in a cage. Some tests used one grasshopper per cage when insufficient numbers were available. The cages were alternated male and female on the board if both sexes were used in the test. The boards with the attached cages were

39

placed in a lighted growth chamber for 24 h at 28 ~

The grasshoppers were then removed and the disc

areas were measured using a Lycore leaf area meter.

Differences between areas eaten from control and test discs were analysed by paired t-tests (p < 0.05).

Results

Terpenoid chemistry. A. ludoviciana contains a vari-

ety of terpenoid compounds contributing to its rich secondary chemistry. A representative gas chro-

matogram of an A. ludoviciana extract is shown in

Fig. 1. Identification information on the numbered peaks is given in Table 1. The monoterpenes

1-8,cineole, camphor and borneol, and the sesquiterpene lactone achillin, account for more than 50~ of

this sample. These compounds were usually the major components extracted by our procedure. A.

ludoviciana has a large number of glandular trichomes on the leaves and within the buds (Smith

& Kreitner, 1983). Analysis of the material in the trichome heads showed that both the major monoterpenes and sesquiterpene lactones were

found in ca. the same ratios in the glands as in the foliage extracts (Fig. 1).

Sampling of vegetative and reproductive plant

parts during a growing season revealed a large amount of variation between individual plants, with certain compounds varying in concentration much

more than others (Fig. 2). Achillin, for example, was

not found or was in trace amounts of 11 of the 26 plants sampled during the season. Plants not

producing achillin were found throughout the growing season. In the same way, some plants did not pro-

duce much camphor and others produced little borneol. 1,8-Cineole concentrations were more

consistent between plants. In spite of this variation some consistencies were apparent (Fig. 2). The

reproductive plant parts always had terpenoid concentrations two to five times greater than the vegeta-

tive parts. Concentrations of terpenoids in the reproductive tissue reached almost 15070 of its dry weight. Achillin alone reached levels of 5 - 1 0 % of the tissue dry weight. In the vegetative tissue, most of the compounds appeared to have lower concentrations in the young plants (June) and reached levels

40

11

1(

11

A

22

22

B 20

1314

2 56 I 21

I l l I I l I I I I I I I I I

r

R e t e n t i o n t i m e ( m i n u t e s )

I tO

Fig. 1. Capillary gas chromatograms of (A) glandular trichomes and (B) extract from Artemisia ludoviciana foliage.

in July that remained steady for the remainder of the season. Terpenoids appeared to decline in the reproductive tissue as the plants senesced.

The extract was distributed among the five column chromatographic fractions as 38, 16, 3, 21, and 21~ by weight, of the eluted material. The total weight of the fractions was 1.16 g. The slightly greater total than the original amount of extract is probably due to some solvent being retained when the fractions were weighed. Gas chromatograms of each of the five fractions (Blust, 1985) showed that most of the monoterpenes, including all of the monoterpene

hydrocarbons were eluted in fraction one. Fraction two contained most of the remaining monoterpenes including the majority of 1,8-cineole and borneol. Fraction three contained the remaining amounts of 1,8-cineole and borneol as well as the major portions of some unidentified monoterpenes. Fraction four included the majority of the sesquiterpene lactones and fraction five included the remaining sesquiterpene lactones and other unidentified compounds.

Gustatory choice tests with H. alba and M. sanguinipes. Extracts from A. ludoviciana applied to

Table 1. Terpenoids identified from Ar tem&ta ludovwtana.

Peak Compound Retennon ID

number 1 identification time 2 source 3

1 unknown (MW 136) 10.91 c

2 unknown (MW 136) II 15 c

3 a- thujene 11.47 c

4 a-pinene 11.66 abcde

5 camphene 11.99 abcde

6 unknown (MW 136) 12.50 c

7 l-~3-pinene 12.61 c

8 myrcene 12.81 abcde

9 unknown (MW 126) 13.23 c

10 p-cymene 13.50 abcd 11 1,8-cineole 13.70 abcde

12 Artemisia ketone 14.10 cd

13 unknown (MW 154) 14.39 c

14 a- thujone 15.00 c

15 unknown (MW 150) 15.32 cd

16 camphor 15.79 abcde

17 borneol 16.30 abcde

18 bornyl acetate 18.31 abcd

19 sesquiterpene (MW 204) 21.10 c

20 sesquiterpene (MW 204) 21.68 c

21 vulgarin 29.15 ab f

22 achillin 30.05 ab f

23 parishin C 31.35 ab f

matncar in 4 32.90 ab f

artecanin 33.17 ab f

citronellal d

linalool d

pinocarvone d

terpinen-4-ol d

estragol d

cis-pinene-2-ol d

chrysanthone d geraniol d

J Peak numbers used for Fig. 1.

2 BP-I capillary column (see text for conditions).

3 Letters indicate several analyses done by or for the authors

and additional literature as follows: a. retention time SE-30

packed column, b. retention time BP-I capillary column,

c. mass spec. (USDA), d. mass spec. (DSIR) New Zealand,

e. Stangl & Gregor (1980), f. Ohno et al., 1980. 4 11,13 dehydrodesacetylmatricarm.

membrane discs at concentrations approximating those of the plant (6.3% dry weight) stimulated H. alba to feed on the treated discs almost exclusively (Table 2). Similar results were obtained with extracts of A. carruthii, a species of sage closely related to A. ludoviciana and an alternate host for Ho alba in

41

some parts of its range. However, no significant differences between control and test discs were found when extract from A. filifolia, a non-host sage plant, was used. Oils from the two host species were also stimulatory to the specialist while oil from the non-host was not. Although H. alba fed on the membrane discs with the host plant extracts, the number of insects responding to plant fractions and individual compounds was generally quite low, indicating a lack of all the necessary stimulants to evoke a stronger response.

Gustatory choice tests with fractions from A. lu-

doviciana showed only the first of the five fractions to have any stimulatory activity (Table 2). Addition- al testing with fractions one and two confirmed the activity of fraction one and showed inconsistent results with fraction two. Gustatory choice tests of the major monoterpenes, 1,8-cineole, camphor and borneol at a range of concentrations, failed to show any stimulatory activity with H. alba.

Since achillin was found to be a major component of most A. ludoviciana plants, extensive gustatory testing was done with this compound. No evidence of feeding stimulation or rejection was found at levels up to 2% dry weight. Above this level, at 4% dry weight, antifeedant activity was observed. Although these tests indicate that achillin is not a feeding stimulant, the evidence of antifeedant activity is weak due to the reluctance of H. alba to feed on untreated discs. This resulted in a low numbers of insects responding in many of the tests. Although some additional testing of achillin antifeedant activity was done with H. alba using sucrose treated discs, no real improvement in response was noted.

Surprisingly, M. sanguinipes showed strong gustatory stimulation to whole plant extracts of both A. ludoviciana and A. carruthii (Table 2). Unlike H. alba however, the generalist was also stimulated by the extract of A. filifolia. The oil of A. ludoviciana containing the sesquiterpene lactones showed strong antifeedant activity at the same concentration which showed feeding stimulation in H. alba.

Gustatory tests with M. sanguinipes using the five fractions showed distinct antifeedant activity in fractions 4 and 5 while fractions 1, 2 and 3 were all stimulatory (Table 2). Achillin and the other sesquiterpene lactones were eluted in fractions four

42

i1 i1 [ ] Vegetative

iI I June July Aug. Sept. Nov. June July Aug. Sept. Nov. June July Aug. Sept. Nov.

TOTAL SIrSQUITERPs IJ, CTONE~S ACHILLIN TOTAL MONOTERPIrNES 2.0 2.0 2.0

~.6 7-~ Vegetative 1.6 Vegetative t.6

Reproductive ~ , . 4 ,.4 7- ,.4

~ .| .6

t-" .2 .2 t -T-- ~ .2

0 o 0 June July Aug. Sept. Nov. June July Aug. Sept. Nov. June July Aug. Sept. Nov.

1.8-CINEOLE CAMPHOR BORNEOL

Fig. 2. Comparison of Artemisia ludoviciana terpernoid concentrations (percent of dry weight) during growing season, and between vegetative and reproductive plant parts. Sampling dates and numbers of samples (n): 5 June (n=6), 9 July (n=10), 13 Aug. (n=5), 11 Sept. (n=5), 25 Nov. (n=5).

and five. Further testing using purified achillin confirmed the antifeedant activity of this compound against the generalist grasshopper. While no significant activity was found at levels of 0.25~ dry weight and below, levels of 0.5% up to 10~ all showed highly significant antifeedant activity (Table 2). Further testing was done using achillin at this threshold level of 0.5~ by adding sucrose to test and control discs. When sucrose was added at a 0.1~ level, achillin was still significantly repellent although the difference between feeding on test and control discs was diminished. After raising sucrose to a 0.5% level, no significant differences were observed between control and test discs (Table 2).

Individual monoterpenes were also tested using M. sanguinpes. These results showed highly variable

and mostly insignificant activity. 1,8-Cineole showed significant but not strong antifeedant activity at levels above 1~ dry weight (P = 0.025, n=19) while higher levels of camphene, about 5~ dry weight showed significant activity as a feeding stimulant (P = 0.05, n = 20).

Discussion

Our investigations of the monoterpenes in A. ludoviciana confirmed the presence of several including 1,8-cineole, camphor and borneol as previously reported (Stangl & Greger, 1980; Thakur & Singh, 1979). In addition we found a number of other monoterpenes including some, such as thujane

43

Table 2. G u s t a t o r y cho ice tests us ing Artemzsta ex t rac t s , oils, f r a c t i ons , a n d the se squ i t e rpene l a c tone achi l l in . Resul ts expressed as

a v e r a g e a m o u n t o f f eed ing (cm 2) o n c o n t r o l ( C O N T . ) a n d T E S T discs , a n d s ign i f i cance (SIG.) o f p a i r e d t- tests , n l / n 2 = n u m b e r o f

g r a s s h o p p e r s r e s p o n d i n g / n u m b e r tes ted.

C o n c e n t r a t i o n H. alba M. sangumipes

(% d r y weight )

C o n t . Test Sig.1 n 1 /n2 C o n t . Test Sig. n l / n 2

A. ludovtclana

6.3 E X T R A C T 0 .00 0 .42 + + + + 6 2 / 8 0 0 .20 2.87 + + + + 2 0 / 2 0

6.3 O I L 0 .09 0 .32 + + + + 17 /39 0 .75 0.11 16 /20

A. carruthti

6.3 E X T R A C T 0.03 0 .56 + + + + 3 4 / 3 6

6.3 O I L 0 .10 0.31 + + + + 13 /40

A. filifoha

6.3 E X T R A C T 0.25 0 .67 NS 6 / 8

6.3 O I L 0 .14 0 .17 NS 6 / 2 0

A. ludovlctana f r ac t i ons 2

0 .14 2.71 + + + + 2 0 / 2 0

0 .44 1.49 + + + + 2 0 / 2 0

First set

F R 1 0 .00 0 .49 + + 3 / 2 0 1.23 2 .70 + + + + 18 /18

FR 2 N F 0 / 2 0 0 .52 2 .49 + + + + 18 /18

ER 3 N F 0 / 2 0 0 .19 1.23 + + + + 18 /18

F R 4 NE 0 / 2 0 1.04 0 .17 17 /18

FR 5 NE 0 / 2 0 1.74 0.61 18/18

Second set

FR I 0.03 0 .16 + + + + 6 2 / 8 0

F R 2 0 .12 0 .39 + + + 8 / 2 0

FR 2 0 .26 0.05 NS 11 /20

Achilhn

10 0 .62 0.13 2 0 / 2 0

4 1.06 0 .00 3 / 2 0 0 .27 0.05 16 /20

2 0 .23 0.11 NS 2 / 2 0 1.22 0 .16 2 0 / 2 0

1 0 .33 0.18 - - 5 / 2 0 0 .75 0 .46 3 1 / 4 0

0.5 0 .23 0 .12 NS 8 / 2 0 1.01 0.31 19 /20

0.25 0 .29 0 .34 NS 3 / 2 0 0 .06 0 .13 NS 11 /20

0 .12 0 .15 0 .66 NS 3 / 2 0 0 .57 0 .59 NS 3 0 / 4 0

0 .06 0.01 0.45 + + 2 / 2 0 0 .10 0 .08 NS 8 / 2 0

Achillin + 0.1 Suc rose

0 .5 0.81 0 .63 7 8 / 8 0

Achillin + 0.5 Suc rose

0.5 1.75 1.73 NS 8 0 / 8 0

" + a n d - " ind ica t e d i r ec t ion o f p r e f e r ence t o w a r d test subs t ance . " + + o r - - " s ign i f i can t at P < 0 . 1 . " + + + o r - - -

s ign i f i can t a t P < 0 . 0 5 . " + + + + o r . . . . " s ign i f i can t a t P < 0 . 0 2 5 . N F = no feeding .

2 C o n c e n t r a t i o n p r o p o r t i o n a l to o r ig ina l ex t rac t tests.

44

derivatives and artemisia ketone, previously report-

ed from related species of Artemisia (Stangl & Greger, 1980; Thakur & Singh, 1979). The identification of sesquiterpene lactones from A. ludoviciana

was reported by Ohno et al. (1980). Of these, we found achillin to be predominant sesquiterpene lactone in most individual plants analyzed.

The isolation of the terpenoids from the glandular trichomes is consistent with investigations on other Artemisia species. Kelsey & Shafizadeh (1980) observed that all of the sesquiterpene lactones and some of the monoterpenes are stored in the glandular trichomes in Artemisia nova. In a review of glandular trichomes, Kelsey et al. (1984) stated that the isoprenoids are the most common metabolites in the glandular trichomes of flowering plants. They reported concentrations of terpenoids from glands may range between 0 and 20~ of the plant dry

weight. We found up to 15~ of the dry weight of A. ludoviciana can be monoterpenes and sesquiterpene lactones, particularly in the flowering parts of the plant. In A. nova Kelsey & Shafizadeh (1980) reported sesquiterpene lactone levels in the foliage of 2 to 3.5%. This matches well with our measurements of 1 to 3070 for A. ludoviciana. The levels of monoterpenes in foliage reported forA. novao f 1 to 2%, and 0.5 to 407o for A. tridentata (Kelsey et al., 1982) also match our measurements of ca. 1~ for A. ludovi-

ciana foliage. However, we observed monoterpene levels to be up to 5~ in the reproductive parts compared to a reported 0.2 to 1~ for A. nova. Higher terpenoid concentrations in the reproductive tissue was also observed by Thakur & Singh (1979) in dis- cussing the production of oil inA. pallens, common- ly known as "davana". They state that the oil content of the flower heads is 1.4~ compared to 0.3~ in the leaf and stem. We observed levels of terpenoids almost five times more concentrated in the reproductive tissues of A. ludoviciana than in the foliage. Achillin alone averaged almost 7~ of the reproductive tissue dry weight with total sesquiterpene lactones accounting for 9-10070. The levels of terpenoids in the reproductive tissue was highest in the developing buds and decreased after flowering and during seed dispersal. The decline may represent loss of material as the heads of the glandular trichomes often stick to the seeds.

The foliage showed an increase in terpenoids from early growth in June to July and remained at those levels until leaf senescence. Monoterpenes appeared to maintain levels of slightly over 1070 throughout the

season, however many individual monoterpenes appeared to increase from early season levels while other unidentified monoterpenes present early in the season decreased. This trend was also noted by Kel- sey et al. (1982) in a study of Artemisia tridentata.

They observed that monoterpenes and crude terpenoids were lowest in the spring and reached a maxi- mum at flowering.

The results of the gustatory tests show differences between the specialist and generalist species. The specialist H. alba was stimulated only by the total lipid extracts of its two known host species, A. lu-

doviciana and A. carruthii, and their oils, but not by the extract or oil of a related sage plant, A. filifolia.

These data indicate the presence of an extractable feeding stimulant or mixture of stimulants from the host plants that remains in the oil after refinement. The generalist M. sanguinipes however was stimulated by the extracts of all three species indicating the presence of a more general feeding stimulant. When the extract was refined to the oil, the latter showed strong antifeedant activity against M. sanguinipes.

This indicates the removal of the general feeding stimulant and/or the concentration of antifeedant compounds as a result of the refining process.

When the extract was.fractionated, only the first fraction containing primarily the monoterpene hydrocarbons definitely stimulated H. alba to feed on the discs, while the remaining fractions showed little activity. Other compounds may have been present in this fraction which were not volatile in the GC, or which were not identified. M. sanguinipes was stimulated by the first three fractions, which contained the monoterpenes, but repelled by the last two fractions, which contained the sesquiterpene lactones. Sesquiterpene lactones have been implicated as insect antifeedants (Gershenzon et al., 1985; Mabry & Gill, 1979; Smith et al., 1983; Stipanovic, 1983).

The dominance of achillin in the plant's terpenoid chemistry and its storage in the glandular trichomes prompted further testing of this compound. Achillin had antifeedant activity against both species of

grasshopers but the specialist had a higher threshold (4%) than the generalist (0.5%) when achillin was

tested without sucrose. At the 0.5% level, M. sanguinipes was still repelled with 0.1% sucrose added to test and control discs, but a level of 0.5~ sucrose was sufficient to overcome the antifeedant activity. The 4% threshold for H. alba is above the average level of achillin (2~ found in the foliage, but below that found in the reproductive tissue (7%). Field and laboratory observations have failed to show any evidence of H. alba feeding on reproductive tissue. The 0.5% threshold for M. sanguinipes is well below the average level found in both foliage and reproductive tissue. We have also observed that M. sanguinipes is deterred from feeding by a sesquiterpene lactone from sunflowers (Gershenzon et al., 1985).

When an insect such as H. alba evolves a monpha-

gous relationship with one species of plant over most of its range, it must obtain some adaptive advantage. Possible explanations include minimizing competi- tion from other herbivorous insects, adaptations to the specific microhabitat and host phenology, including morphological and behavioral crypsis, reduced metabolic cost of detoxification and diges- tive systems, and protection from predators by se- questering plant secondary chemicals as toxins or repellents (Feeny, 1973; Beck & Schoonhoven, 1980). 11. alba may have gained one or more of the above advantages by specializing on A. ludoviciana. Its cryptic color and behavior probably aid in escaping predation. It also appears to have minimized compe- tition as few other insects feed on this sage. Another possible advantage H. alba has gained is protection from gut parasites. Malameba locustae is a pro- tazoan parasite of Acrididae. Taylor & King (1937) attempted to infest many species of grasshoppers with this parasite. H. alba was one of only two species that did not become infested. This disease invad- ed our laboratory colony at one time and killed most of the generalist M. sanguinipes but did not appear to affect H. alba. With the large amounts of monoterpenes and sesquiterpene lactones in the diet, it is possible that the host plant phytochemicals are providing protection to the grasshopper against gut parasites. Sesquiterpene lactones have been reported to have antimicrobial activity against a number of organisms including pathogenic pro- tozoans (Ivie & Witzel, 1983).

45

R~sum~

Rdponses de deux orthoptkres, Fun spdcialiste, l'autre gdndraliste, aux terp~nes dArtemisia ludoviciana Nutt. (Asteracae)

Hypochlora alba Dodge, orthoptbre sp6cialiste, con- somme presque exclusivement A. ludoviciana, plante produisant des terp6nes en grandes quantit6s dans ses trichomes glandulaires. Cette plante n'est pas consomm6e par des orthopteres g6n6ralistes, comme Melanoplus sanguinpes Fabr. Uanalyse d'extraits et de fractions de la plante en chromatographie en pha- se gazeuse associ6e fl la spectrom6trie de masse a r6- v616 de nombreux monoterpbnes (1,8-cin6ole, camphre, born6ol et autres) et une grande quantit6 d'achilline, lactone sesquiterp6ne. Ces terpbnes 6taient 2/~ 5 lois plus concentr6s dans les organes reproducteurs de l'armoise que darts ses organes v6g6- tatifs. La concentration de terpbnes dans les feuilles

augmente de la plante jeune jusqu'fl la maturit6. Les plantes ont pr6sent6 une grande variabilit6 en concentrations relative et totale en terpbnes. Uinfluence des terpenes darts la r6gulation du comportement alimentaire du sp6cialiste et du g6n6raliste a 6t6 exa- min6e par des tests de choix gustatif sur des disques de membrane. Des extraits de A. ludaviciana et de A. carruthii Wood,- espbce tr6s voisine que H. alba peut consommer-, contenaient un (ou plusiers) pha-

gostimulants de H. alba, tandis que A. filifolia Torr., esp6ce non consomm6e, n'en contenait pas. Cette ac- tivit6 stimulatrice 6tait 61u6e darts la moins polaire des 5 fractions, qui contenait principalement les hydrocarbones monoterp6nes. La prise de nourritu-

re de M. sanguinipes a 6t6 stimul6e par des extraits des 3 armoises examin6es, et par les 3 premi6res fractions de A. ludoviciana, tandis que ses 2 dernibres fractions, contenant les lactones sesquiterpbnes, ont pr6sent6 une action phagodissuasive. Des tests avec l'achilline, principale lactone sesquiterp6ne, ont montr6 que sa concentration moyenne dans les feuilles (2% du poids frais) 6tait sup6rieure au seuil de rejet du g6n6raliste (0,5 %), mais inf6rieure fl celui du sp6cialiste (4%). La concentration moyenne des organes reproducteurs (7%) 6tait sup6rieure au seuil de rejet des de0x esp6ces.

46

Acknowledgements

The authors thank Dr. Harry Young, DSIR, Auck- land, New Zealand; and Dr. Ralph Howard, U.S. Grain Marketing Research Laboratory, USDA-ARS, Manhattan, KS 66502 for assistance in obtaining the mass spectra of the head space volatiles from A. ludoviciana; and Drs. Tom J. Mabry and Jonathan Gershenzon, University of Texas, Austin, TX, for providing monoterpene and the sesquiterpene lactone standards and advice on extraction procedures. Supported in part by National Science Foundation grant No. DEB 7905482 and Kansas Agricultural Experiment Station Project No. 548. Contribution No. 87-31-J from the Kansas Agricultural Experi- ment Station.

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Gustatory responses of a specialist and a generalist grasshopper to terpenoids of Artemisia ludoviciana