Journal of Chemical Ecology, Vol. 7, No. 6, 1981

ANTIFEEDANT

ACTION OF Z-DIHYDROMATRICARIA

ACID FROM SOLDIER BEETLES

(Chauliognathus

SPP.) 1

T H O M A S E I S N E R , 2 D A V I D HILL, 2 M I C H A E L G O E T Z , 3 SUBHASH JAIN, 3 DAVID ALSOP, 4 SCOTT CAMAZINE, 2 and J E R R O L D M E I N W A L D 3

2Section of Neurobiolog.v and Behavior Division of Biological Sciences, Cornel1 University, Ithaca, New York 14853 3Department of Chemistry, Cornell University 4Department of Biology, Queens College, Flushing, New York 11367 (Received January 5, 1981; revised March 2, 1981)

Abstract--The acetylenic acid, Z-dihydromatricaria acid (DHMA), previously isolated from the defensive secretion of Chauliognathus lecontei, and now shown to occur also in C. pennsvlvanicus, is a potent feeding deterrent to jumping spiders (Phidippus spp.). A simple bioassay with Phidippus is described, which is generally applicable to studies dealing with the isolation and evaluation of feeding deterrency of natural products from insects. By use of this assay, Phidippus were shown to be sensitive to as little as I #g D H M A , an amount equivalent to less than 2% of the D H M A content of C. pennsylvanicus. Key W o r d s - - D i h y d r o m a t r i c a r i a acid, antifeedant, acetylenic acid, Chauliognathus pennsylvanicus, Coleoptera, Cantharidae, soldier beetles, Phidippus, jumping spider, bioassay, defensive secretion, defensive behavior.

INTRODUCTION

An earlier paper of this series (Meinwald et al., 1968) dealt with the identification of an acetylenic acid, Z-dihydromatricaria acid (I; henceforth abbreviated DHMA), from the defensive secretion of a soldier beetle (Cantharidae: Chauliognathusleeontei). Although the beetle was shown to be 1Paper no. 67 of the series Defense Mechanisms of Arthropods. Paper no. 66 is: Goetz, M.A., Meinwald, J., and Eisner, T., Experientia. In press.

1149 00984)331/81! 1100-1149503.00:0 9 1981 Plenum Publishing Corporation

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EISNERET AL.

unacceptable to a diversity of predators (ants, carabid beetles, blue jays, grasshoppers mice), it was not determined whether the unacceptability was attributable to D H M A itself, since the compound was not isolated in amounts sufficient for testing. CH 3--CH---~CH--C~C--C------ C - - C H z - - C H 2--COOH I Working with another species of Chauliognathus (C. pennsylvanicus), we have now reinvestigated the defensive role of D H M A and were able to show by use of a bioassay with jumping spiders (Salticidae: Phidippus spp.), that the acid does indeed have potent antifeedant activity. We here present the results of this assay, together with data on D H M A isolation from C. pennsylvanicus, and observations on the defensive behavior of the beetle. METHODS AND MATERIALS

Chauliognathus pennsylvanicus is one of the commonest North American soldier beetles, ranging eastward of the Rocky Mountains and southward to Arizona. In the environs of Ithaca, New York, where our specimens were taken, the beetles sometimes aggregate by the hundreds in mid- to late summer on goldenrod (Solidago spp.). Jumping spiders coexist ecologically with soldier beetles, and as nonspecific aggressive insectivores are a major potential threat to Chauliognathus. The specimens of the two species studied by us, Phidippus audax and P. regius, had been maintained in the laboratory for a period of at least several weeks prior to testing. They were kept individually in plastic Petri dishes and fed mostly houseflies. The P. audax were taken from the same field sites as the Chauliognathus. Mass spectra were obtained using a Finnigan 3300 gas chromatographmass spectrometer coupled with a System Industries 150 computer. Nuclear magnetic resonance spectra were recorded at 90 MHz on a Varian EM 390 instrument. Chemical shifts (6) are reported in ppm downfield from internal tetramethylsilane. The H P L C analyses were conducted with a Waters model 6000 solvent delivery system and a Waters model 440 ultraviolet detector. Gas chromatographic analyses were carried out using a Varian Aerograph 2100 instrument. Ultraviolet spectra were recorded in methanol solution on a Cary 14 spectrophotometer. RESULTS

Glands and Defensive Behavior of C. pennsylvanicus. Dissection of freshly killed specimens of C. pennsylvanicus revealed that this beetle, like its congener C. lecontei (Figure I), has nine pairs of defensive glands. Eight pairs

ANTIFEEDANT ACTION

/ t

1151

,r

FIG. I. Chauliognathus lecontei, with prothoracic and abdominal defense glands indicated by outlines. The wings are shown cut away near their bases. The drawing applies as well to C. pennsylvanicus. are in the abdomen and consist of small pouches, enveloped by compressor muscles, opening near the posterolateral corners of the tergites. The ninth pair, seemingly devoid of surrounding muscles, has its openings in the pleural region beneath the pronotal shield. The defensive behavior of the beetles was studied by subjecting individuals to various types of disturbance in the laboratory. Typically, when a beetle was picked up by hand or in forceps, it emitted droplets of secretion from the gland openings (Figure 2A). The droplets were usually milky white in appearance, but occasionally turbid brown or almost clear. Localized stimulation tended to evoke responses from only a few glands at a time. This occurred, for example, when forceps were used to pinch single legs or antennae or restricted regions of the abdomen. The glands first discharged under such circumstances were almost invariably those closest to the site stimulated. It was also noted that the beetle frequently brought its legs into play to transfer secretion from the gland openings to the offending forceps. When, for example, a beetle was held by its front end, it flexed its abdomen forward beneath the body, emitted droplets of secretion from its glands, and then, if held persistently, proceeded to brush its hindlegs alternatively against secretion on the flanks of its abdomen (Figure 2B) and against the forceps. The latter were often visibly wetted as a result. The midlegs also occasionally took part in the brushing activities. Persistent disturbance commonly caused the beetles to regurgitate droplets of enteric fluid. As the animal struggled, the dark effluent seeped from the mouth onto legs and portions of the body, becoming mixed at times with secretion from the defensive glands. It was therefore occasionally this mixture, rather than pure glandular fluid, that was administered to the forceps by leg-dabbing.

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EISNER ET AL.

FIG. 2. (A) Close-up view of right flank of abdomen of C. pennsyivanicus, showing droplets of secretion emerging from defensive glands. (B) Comparable view, showing hindleg wiping against abdominal flank. Note side of abdomen wetted by smeared secretion. (Reference bars = 1 mm.)

Acceptability of C. pennsylvanicus to Jumping Spiders. Twenty individual C. pennsylvanicus, freshly collected in the field, were presented to single Phidippus in Petri dishes. The results are summarized in Figure 3. Each of the eight Chauliognathus offered to P. audax, the species of jumping spider collected from the same sites as the beetles, was rejected outright, and so were 7 of 12 beetles offered to P. regius. The spiders pounced on the beetles, held them briefly (less than 5 sec) and then released them. Droplets of secretion were sometimes emitted by the beetles during this period. The beetles were uninjured in the encounters and were lively when examined the next day.

ANT1FEEDANT

1153

ACTION

I0

Tesls w#h P oudox ....

6/2

R reg/us

~,~,:. :.X,I i:i:ii ~176176176

:i:i:i .%-,,

,,%~176

E/1

/to

/Io

N R~

RD

PE

E

FA TE FIG. 3. Fate of individual Chauliognathuspennsylvanicus offered to jumping spiders (Phidippus audax, P. regius). R1 = rejected immediately ( 5 sec); E = eaten.

Results (eaten versus rejected) for either the control or the 0.1 ~zg group differed significanlty (P < 0.0001) from the effect of the 10.0-pg dosage.

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EISNER ET AL,

Test substance is administered at a desired dosage of 1 #1 of solvent directly onto the fruit fly with a microsyringe. A few minutes are allowed for evaporation of solvent before a fly is given to a spider. The fly is suspended by inserting the hair loosely between its folded legs; it thus detaches readily from the tether when seized by the spider. For control purposes flies are also offered that are treated by topical addition to 1 #1 of solvent alone. We have used this bioassay for evaluation of antifeedant potency of a variety of chemical defensive agents from insects. We tested D H M A at three dosages (0.1, 1.0 and 10 #g; dichloromethane as solvent), with 10 flies per dosage, and an additional 10 control flies treated with dichloromethane alone. A total of 29 Phidippusregius were used; in cases where spiders were used more than once, consecutive tests were spaced at least one day apart. It is clear from the results (Figure 5), that even at a dosage as low as 1.0 #g, D H M A proved deterrent in 4 out of 10 tests. A dosage of I0 #g proved absolutely deterrent, while 0.1 #g was inactive. DISCUSSION

The defensive potential of D H MA appears established, certainly vis/~ vis jumping spiders. As little as 1 #g D H M A may be deterrent to a Phidippus, an a m o u n t equivalent to less than 2% of the total D H M A content of Chauliognathus and not much greater than the estimated average output (0.8 #g) of a single gland of the beetle. Whether D H M A is active also against other predators remains to be determined. We have preliminary evidence_ind!_catin_g that the compound is deterrent to some vertebrates (white-footed mice, Peromyscus leucopus) but not to others (Swainson's thrushes, Hylocichla

ustulata). In its defensive behavior, C. pennsylvanicus shares characteristics with other chemically protected arthropods. It is not unusual for these to have serially arranged glands and for the glands to discharge singly or in groups depending on the magnitude and degree of localization of an offense. Nor is it unusual for legs to be used in the spreading and administration of secretion or for the animal to disgorge enteric fluids when attacked. Leg-dabbing has been noted, for example, in tenebrionid beetles (Tschinkel, 1975; Eisner et al., 1974), Hemiptera (Remold, 1962) and opilionids (Eisner et al., 1971, 1977), while regurgitation under stress occurs in caterpillars, grasshoppers, and many other larval and adult insects (Eisner, 1970). Mixing enteric fluids with defensive secretion and administration of the mixture by leg-dabbing has been observed in certain opilionids (Eisner et al., 1971, 1977). One wonders about the origin of D H M A in Chauliognathus. ls the compound made by endogenous synthesis or do the beetles obtain it from an exogenous source? The latter alternative is at least within the realm of possibility since a variety of acetylenic compounds (including the methyl ester

ANTIFEEDANT ACTION

1157

of DHMA) occur in fungi and higher plants (Sr 1963). Among the many Compositae known to contain the compounds are species of Solidago (Gibbs, 1974), the genus that includes the goldenrods on which our beetles were most commonly found in large numbers. But whether portions of plants containing acetylenic compounds are actually ingested by Chauliognathus (and whether as a consequence their oral effluent also contains the substances) remains unknown. Extraction of whole Chauliognathusyielded ca. 2-5 times more D H M A than the beetles seemed capable of ejecting from their glands. This in itself was indicative ofextraglandular presence of DHMA, which was confirmed by the finding of D H M A in the blood of the beetles. One could easily envison such systemic D H M A having supplementary defensive potential. Chauliognathus do not bleed spontaneously (as do meloid and other beetles that "reflex bleed"), but they bleed when injured and can readily survive the loss of legs or antennae in the laboratory. Defensive use of blood could therefore be a reality for the beetles in nature. Interestingly, it is not unusual to find a Chauliognathus in the field, particularly later in the season, with a leg or portion of an antenna missing. The fact that D H M A plays a protective role in soldier beetles offers at least some support to the notion that acetylenic compounds also serve for defense in the plants that contain these substances. However, experimental evidence to that effect is lacking. The bioassay with Phidippus is potentially broadly applicable. The spiders are easily reared and maintained, are sensitive to a wide range of chemicals, and are reliable in performance. In the two species used by us, sensitivity varies little from individual to individual. We are currently using the assay in studies of a number of defensive chemicals from insects and have found it particularly useful in efforts to "home in" on active chemicals present in complex mixtures, as for example in whole insect extracts. For such purposes we simply fractionate the mixture by extraction with different solvents, test the individual fractions with Phidippus, and then proceed by subfractionation and testing of subfractions, until activity is found to be restricted to one or more pure compounds, which can then be identified. Given the sensitivity of the spiders and the fact that activity ratings of test samples can often be obtained on the basis of only 10 individual Drosophila offerings, very little material may be used in an assay. The testing procedure therefore lends itself to the study of samples which by virtue of rarity of source organism may be available in only small quantities. Acknowledgments--Study supported by NIH grants AI-02908 and AI-12020, NSF grant PC M 77-15914, Hatch grant NYC- 191406, and a fellowship from the Fonds National Suisse de la Recherche Scientifique to M. Goetz. We thank Karen Hicks and Gerald Eidens for technical assistance, and Art Kluge for doing the UV assay of D H M A in the "milked" sample of Chauliognathus secretion.

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EISNER, T. 1970. Chemical defense against predation in arthropods, pp. 157-217, in E. Sondheimer and J.B. Simeone (eds.) Chemical Ecology. Academic Press, New York. EISNER,T., KLUGE,A.F., CARREL,J.C. and MEINWALD,J. 1971. Defense ofa phalangid: Liquid repellent administered by leg dabbing. Science 173:650-652. EISNER,T., ANESHANSLEY,D., EISNER,M., RUTOWSKI,R., CHONG, B., and MEINWALD,J. 1974, Chemical defense and sound production in Australian tenebrionid beetles (Ade/ium spp.) Psyche 81:189-208. EISNER,T., JONES,T.H., HICgS, K., SILBERGLIED,R.E., and MEINWALD,J. 1977. Quinones and phenols in the defensive secretions of neotropical opilionids, J. Chem. Ecol. 3:32t-329. GlanS, R.D. 1974. Chemotaxonomy of Flowering Plants, Vol. I, p. 89. McGill-Queeffs University Press, Montreal. MEINWALD, J., MEINWALD,Y.C., CHALMERS,A.M., and EISNER,T. 1968. Dihydromatricaria acid: Acetylenic acid secreted by soldier beetle. Science 160:890-892. REMOLD,H. 1962. Uber die hiologische Bedeutung der Duftdr/isen bei Landwanzen (Geocorisae). Z. Vergl. Physiol. 45:636-694. SORENSEN,N.A. 1963. Chemical taxonomy of acetylenic compounds, pp. 219-252, in T. Swain (ed.). Chemical Plant Taxonomy. Academic Press, New York. TSCHINKEL, W.R. 1975. A comparative study of the chemical defensive system of tenebrionid beetles. Defensive behavior and ancillary features. Ann. Entomol. Soc. Am. 68:439-453.