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Suggested Pharmacophagy of the African Bushhopper Phymateus leprosus (Fabricius) (Pyrgomorphidae, Orthoptera) Uta Seibt. Gerhard Kasang* and Wolfgang Wickler Max-Planck-Institut für Verhaltensphysiologie. D-82319 Seewiesen, Federal Republic of Germany. E-mail: [email protected] * Author for correspondence and reprint requests Z. Naturforsch. 55c, 442-448 (2000); received December 13. 1999/March 7, 2000 Phymateus leprosus, Asclepiadaceae, Pharmacophagy The bushhopper Phymateus leprosus (Fabricius) in the field shows a special appetite for the milkweed Asclepias fruticosa. Asclepiadaceae, like Apocynaceae and Scrophulariaceae, contain cardiac glycosides. Raw and purified extracts of these plants phagostimulate larval and adult P. leprosus. We also screened natural and half-synthetic compounds found in those plant extracts. While saponins and sapogenins did not stimulate the animals, many cardiac glycosides and aglycones, offered on filter paper, proved to be phagostimulants.

Introduction is called “Bosstink Springkaan“ in South Africa Various butterflies, grasshoppers and aphids because of its repulsive smell. Larvae and adults feed on Asclepiadaceae, Euphorbiaceae, Scrophu of this species eat many different (including culti lariaceae, Apocynaceae, or Solanaceae which con vated) plants (Taylor, 1956; Smit, 1964, Bishop, tain secondary compounds, like pyrrolizidine alka 1940; Kevan, 1949; Steyn, 1962; Annecke and loids (PAs), cardenolides (CAs) and cardiac Moran, 1982), but particularly they like to feed on glycosides (CGs) which are noxious to many ver Asclepias milkweeds and some other plants that tebrates. The compounds are stored, either chemi contain CAs, CGs as well as genins (Abisch and cally unchanged or enzymatically converted, in tis Reichstein, 1962; Hegnauer, 1964). sues and special glands of the insects, protecting Cardenolides are common in plants of the fami them from predators. Typically, these poisonous lies Asclepiadaceae, Apocynaceae, and Scrophula insects show aposematic colouration (Brower, riaceae, which all seem to be eaten by some apose 1969; Duffey and Scudder, 1972; Rothschild and matic locusts and grasshoppers (Watt and Breyer- Reichstein, 1976; Malcolm, 1989; Frick and Wink, Brandwijk, 1962; Bernays and Chapman, 1978; 1995). The CGs from the defense glands of the Roth et al., 1994; Neuwinger, 1994). The CAs are insects and the secondary compounds of the plants glycosides of steroidgenins (aglycones, AGLs) they eat are of similar chemical structures. The with 23 C-atoms, but only a few special AGLs are CAs obtained from the insect bodies and from basic compounds to all the CAs that frequently their secretions show physiological effects similar occur in tropical and subtropical noxious plants. to compounds found in some arrow poisons of A f CAs of African noxious plants and insects have rican natives (Whitman, 1990: Neuwinger, 1994). been isolated and analyzed by various authors It has been assumed that also pyrgomorphid (Hesse and Reicheneder, 1936; Hesse et al., 1949; grasshoppers of the genus Phymateus store nox Reichstein, 1967; von Euw et al., 1967) who iden ious substances and become unpalatable to insec tified as main components two glycosides derived tivorous mammals and birds (Ebner, 1914; Swyn- from the CAs of calotropagenin: calotropin and nerton, 1915; Carpenter, 1938; Uvarov, 1977). The calactin; smaller amounts of calotoxin, uscharin, well-known bush locust or bush hopper P. leprosus uscharidin, and voruscharin were also found. Ac cording to Reichstein et al. (1968), Rothschild and Reichstein (1976), calotropin and calactin found in Abbreviations: CA, cardenolide; CG, cardiac glycoside; butterflies and grasshoppers stem from Calotropis AGL, aglycone = genin: PA, pyrrolizidine alkaloid. procera and related Asclepiadaceae.

Feeding noxious substances from plants has chosen according to the water content of the been named “Pharmacophagy” (Haase 1892, material. Highly aqueous material was extracted 1893) and redefined recently by Boppre (1984) with methanol and/or distilled water. Repeatedly and Boppre et al. (1984) as a feeding behaviour frozen and thawed aqueous material was eluted that aims at specific secondary plant substances with polar solvents more efficiently than fresh which are then incorporated as drugs for special material. Fresh and highly aqueous plants gave purposes. best elution with MeOH and/or with HCC13. From To test for pharmacophagy ofP. leprosus, we ex dried or slightly aqueous materials the highest amined the feeding reactions of larvae and adults amounts of CAs were obtained with HCC13 - towards (1) different plants, particularly those MeOH or HCC13 - EtOH. containing CAs, and towards filter papers that were impregnated (2) with plant extracts, (3) with Solvents and reagents fractions of those extracts, and (4) with pure CAs. Short-chained alcohols are the best solvents for As controls we used filter papers that were empty the CAs and to extract them from plant material. or carried the solvents alone. Solvents Uvasol and Rotipur were highly pure for analytical and chromatographic purposes. Metha Materials and Methods nol, ethanol, chloroform, petrol ether (b.p. 3 0 - 60 °C), formamide and acetone were obtained Insects from Merck (Darmstadt), Carl Roth (Karlsruhe), Larval and adult P. leprosus came from Natal, and Sigma (Deisenhofen). Na2C 0 3 and Na2S 0 4 South Africa. Larvae, caught as 3rd or 4th larval (calcined) were used to purify and dry the solvents stage, were reared in insect breeding cages (30cm and extracts. Distilled or deionized water (Aquap- x 35cm x 25 cm) in a glasshouse. Adults were ob urificator, Miele, Gütersloh) was used for applica served in a large cage (160 cm x 156 cm x 210 cm). tions with aqueous and organic solvent mixtures. Room temperature was about 30 °C, the relative Standard solvents for chromatography (TLC) atmospheric humidity 40-6 0 % . Light period of were mixtures of ethylacetate - methanol (97: 3, the day was from 06.00 to 20.00 h, with artificial by vol.) and of chloroform, methanol, formamide light period added to the shorter natural daylight. (93; 6:1, by vol.). As basic food we used Chinese cabbage, oat flakes Due to osmotic interaction, distilled water and wheat shoots. eluted at least some CAs from intact plant tissue (suggesting that under natural conditions rain will Plants offered wash out some CAs, even more likely from dam aged plants). Aqueous alcohols with 20 to 30% Besides Asclepias, we offered various other water proved best for working up the CAs from plants (see Table I) that contain secondary sub fresh vegetable tissues. High-polar CGs as well as stances which are chemically related to the com less polar AGLs were eluted particularly well with pounds found in the grasshoppers’ preferred these solvents. As a disadvantage, MeOH also re food plants. solves a variety of other secondary substances. A mixture of HCC13 and MeOH proved a good Preparing plant extracts solvent for mildly polar CAs. From dried or Immediately after cutting in Africa some of the slightly aqueous materials CAs were best eluted fresh material was stored in methanol. Most of the with HCC13 - MeOH or HCC13 - EtOH. These material was dried at 20 °C and kept in deep-freez- molecules dissolve the outer waxy layers of the ing boxes and drug glasses. Fresh and dried leaves leaves, penetrate into the inner cell structures and and stems from different Asclepias species and destroy the fat and lipid-enriched membranes (for comparison) Nerium oleander from Central (Wagner, 1993). America were ground, pulverized and/or deeply frozen for extraction (Stahl and Schild, 1981; Extracts o f fresh leaves and stems Brower et al., 1982; Wagner et al., 1983; Wagner, Asclepias (physocarpa, syriaca, curassavica), N. 1993). Solvents as well as elution techniques were oleander, and D. stramonium contain highly polar 444 U. Seibt et al. • Pharmacophagy of the African Bushhopper water-soluble glycosides. Plant material was ex with 100 ml HCCI3 - EtOH (3:2, by vol.). Washing tracted for 7 days at 20 °C (1) with distilled water, with 40 ml of 1 m Na2C 0 3 solution removed all (2) with chloroform - methanol (HCC13- MeOH; acidic components from the HCCI3 extract. After 2:1, by vol.), (3) with methanol purissimum washing with 50 ml water and drying with 20 g (MeOH). Na2S 0 4 the purified extract contained most of For all batches it was necessary to decant and the CAs. filter the raw extracts, then to concentrate the clear solutions at 60 °C by vacuum (Rotavapor, Feeding tests Biichi). Thereafter these extracts were kept and The following substances were tested: stored at 4 °C in flasks. AGLs of Scrophulariaceae: digitoxigenin, digoxigenin, gitoxigenin, 3-deoxy-digitoxigenin, gitoxi- Extracts o f dried leaves and stems genin-3-acetate; Plant material ofA. physocarpa (from S. Africa) AGLs of Apocynaceae: oleandrigenin and ouaba- and Nerium oleander was dried at 100 °C for 24 genin. hours in a drying oven (Memmert). Then 10 g of CGs of Scrophulariaceae: digitoxin, digoxin, gi- that material was ground to dust (Pulverisette, toxin and acetyl-digitoxin. Meliert) and shaken at 20 °C with 100 ml aqueous CGs of Apocynaceae: oleandrin, acovenoside A, MeOH. The suspensions were kept in darkness for ouabain, helveticoside, neriifolin, convallatoxin 2 days at 20 °C. After filtration under vacuum and and strophanthidin. washing the residue repeatedly with 1 liter aque Control Sapogenins and Saponinswere: diosgenin, ous MeOH the solvent volume was reduced and yamogenin, hecogenin, gitogenin, digitonin, he- concentrated exactly to 500 ml. deracoside C, ginsenoside R b l, escinol (Merck and Roth). Preparative purification and fractionation o f CAs Test compounds were obtained from pharma ceutical companies that isolated CGs and AGLs The extracts from A. physocarpa, A. syriaca, and from plants. N. oleander obtained with different solvents con The prepared plant extracts were dropped onto tained a mixture of CAs (Abisch and Reichstein, 4 cm2 large filter papers. The impregnated papers 1962; Hegnauer,1964; Rothschild et a/.,1970; were colour-labeled and fixed to branches and Seiber et al. 1982). The crude plant extracts ob brushwood in the test cages, easily accessible for tained with aqueous ethanol contained a variety the animals. The number of animals available for of other secondary substances. the tests varied. The animals were constantly ob served for some hours and then controlled daily, Liquid-Liquid partition o f CAs from and the feeding results recorded. A. physocarpa During feeding tests, wheat shoots where con tinuously offered ad lib. to ensure that the bush- The crude extract of 10 g of dry material (pow hoppers never had to feed from test papers due to dered leaves, stems and branches with about 1% hunger. Empty control papers as well as papers of blossoms, seeds, and seed capsules) was dis with solvents alone remained untouched, proving solved in 500 ml MeOH -H20 (4:1, by vol.) and that the animals needed phagostimulants and did evaporated in vacuo, the dry residue taken up with not consume filter paper per se. Test substances 200 ml acetone, MeOH, and H20 (2:2:1, by vol.). that were not eaten were regarded as not to stimu The solution was extracted exhaustingly 5 times late feeding. with 100 ml petrol ether (b.p. 4 0 -6 0 °C). This li quid-liquid partition removed the fats and lipid Results and Discussion compounds from the aqueous solution almost completely. Then the petrol ether-fraction was re As the test substances do not evaporate, the in extracted with 50 ml aqueous MeOH. The main sects possibly could not smell, and choose from a fraction of M e0H -H 20 was eluted 4 times with distance. They instead had to respond to any sub 100 ml HCCI3. Extraction was finally completed stance upon encounter. Direct observation proved U. Seibt et al. • Pharmacophagy of the African Bushhopper 445

that they did not serially visit and memorize sev Accepted food plants eral papers to finally return to the most promising one. Instead, the animals seemed to move ran Our caged larvae and adults accepted all the domly in the cage. In doing so, 90.4% of the test plants offered, though not all parts of them readily papers had clearly been visited between day 1 and (see Table I). Nerium oleander was rarely eaten, 7 of exposure (Fig. 1). Untouched papers re probably due to the hardness of the leaves. The mained exposed to the animals for up to 70 days. animals just nibbled at Hedera helix and Taxus A test substance was considered to be accepted baccata. Both in the field and in our cages, the and thus active as a phagostimulant if at least 15% animals did not exclusively feed from plants that of the respective test paper had been consumed. contained noxious secondary substances. (The

Table I. Feeding response ofP.leprosus towards various plants. * = greenhouse grown; + = accepted; P = only nibbled.

Species (Family) Substances contained Offered Response parts

Asclepias fruticosa (Asclepiadaceae)* Gomphoside, Afroside, leaves + Calotropin, about 20 Cardenolides Asclepias syriaca (Asclepiadaceae)* Syriogenin, Syrioside, leaves + Syriobioside about 8 Cardenolides Convallaria majalis (Convallariaceae) Convallatoxin, 38 Glycosides leaves + from 8 Genins Cussonia s. spicata (Araliaceae) Jamaicine - Andirinaglucoside leaves + Vincetoxicum hirundinaria (Asclepiadaceae) Vincetoxin, a-Amyrin, leaves + Triterpene, ß-Sitosterin, Sterines, various Cardenolide Glycosides Digitalis purpurea (Scrophulariaceae) Purpureaglycoside A + B, about 10 leaves + Cardenolides and Glycosides Eupatorium cannabium (Asteraceae) Euparin, Echinatin Pyrrolizidine- leaves + alkaloids, Eupatoriopicrin Hedera helix (Araliaceae) Flederacoside B + C, Germacren, leaves P //edera-Saponins, Sesquiterpenes Nerium oleander (Apocynaceae) Oleandrin, Digistroside. leaves + Stropeside, about 28 Cardenolide glycosides Polygonatum multiflorum (Convallariaceae) Homoserinlactone, Chelidonic- leaves + acid, Steroid- saponines Sambucus nigra (Caprifoliaceae) a-Sambunigrin, cyanogenic leaves + Glycosides, Saponines stems + Senecio fuchsii (Asteraceae) Fuchsisenecin, Senecionin, leaves + Jacobin, Pyrrolizidine alkaloids stems + Senecio nemorensis (Asteraceae) Senecionin, Jacobin, Pyrrolizidine leaves + alkaloids flowers + stems + Solidago canadensis (Asteraceae) Quercetin, Catechine, Tannin, leaves P Triterpenic acid. Saponines, Flavonoids Taxus baccata (Taxaceae) Taxins, alkaloids leaves P Veratrum album (Melanthiaceae) Protoveratrin A+B, Germerin, leaves + Steroid alkaloids 446 U. Seibt et al. ■ Pharmacophagy of the African Bushhopper

Table II. Plant extracts accepted for feeding byPleprosus (at least 4 individuals tested).

Solvent h 2o MeOH CHC1,/ MeOH

Food plant Dry n n Dry n n Dry n n weight samples samples weight samples samples weight samples samples mg/ml offered accepted mg/ml offered accepted mg/ml offered accepted

Asclepias 0.820 2 2 0.740 26 29 0.630 19 13 syriaca Asclepias 0.125 2 2 0.105 6 5 0.240 8 8 fruticosa Asclepias 0.150 3 2 0.175 5 5 0.205 5 5 curassavica Datura 0.055 2 0 0.035 2 0 0.040 3 1 stramonium Nerium 0.530 2 2 0.645 10 10 0.775 6 6 oleander

HCCI3 MeOH from Datura was nibbled at (Ta- ble II).

Natural and half-synthetic CAs as phagostimulants As can be seen from Table III, the CGs and cardiac AGLs offered in various concentrations on filter paper proved to be phagostimulants; excep tions are ouabain, 3-deoxy-digitoxigenin and ouabagenin. None of the saponins and sapogenins were accepted. Calotropis procera (Asclepiadaceae) contains Exposition (days) the following CGs: calotropin, calactin, calotoxin, uscharidin, uscharin, voruscharin, proceroside, and Fig. 1. Cumulative percentages of 292 baited filter paper probes that were accepted (at least 15% consumed) and ascleposide. The CAs contained also small had thus been visited during exposition days 1 to 7. amounts of the genins calotropagenin, uzarigenin, coroglaucigenin and corotoxigenin. Feeding was stimulated by: (1) Most AGLs of plant substances are listed according to Roth et Scrophulariaceae and Apocynaceae. (The hy al., 1994). The cabbage tree, Cussonia s.spicata, is droxyl group at C-3 position seems important for included in the list because in Transvaal we ob the stimulating effect of AGLs and CGs. The only served a group of Phymateus larvae eating the exception found is 3ß-acetyl-strophanthidin.) The leaves CGs of Asclepiadaceae, Scrophulariaceae and Apocynaceae were as stimulating as their AGLs. (2) A mixture of the glycosides from Convalla- Phagostimulants in plant extracts ria majalis as well as pure convallatoxin; also the Secondary substances eluted with distilled water pure glycosides of Asclepiadaceae, Scrophularia from fresh A. curassavica, A. syriaca, and N. olean ceae and Apocynaceae. der; as well as those substances eluted with MeOH The sapogenins and saponins as well as the ter from A. physocarpa and N. oleander clearly stim penoid escinol did not stimulate feeding in P. le- ulated feeding behaviour. Aqueous as well as prosus. methanolic extracts of Datura stramonium were The results satisfy Haase's (1893) criterion of not eaten. However, an extract obtained with pharmacophagy for Phymateus leprosus. Still un U. Seibt et al. ■ Pharmacophagy of the African Bushhopper 447

Table III. Cardiac glycosides, half-synthetic cardiac aglycones, saponins and their genins (offered in various concen trations) accepted for feeding byP. leprosus.

Sec. plant Compounds Hg / 4 cm2 Formula Molecular n n substances ([ig / 4 cm2 filter paper) filter paper weight samples samples offered accepted*)

Cardiac Acovenoside A 50; 1000 C30H46O9 550.7 2 2 glycosides Convallatoxin 5 -1 0 0 0 C29H42O 10 550.6 42 42 Digitoxin 5 0 -1 0 0 0 C4] Hft40i3 764.9 12 12 a/ß-Acetyl-digitoxin 1 -5 0 0 C43 H^ftO ] 4 807.0 8 8 Digoxin 1 0 0 -2 0 0 C4iH64Oi4 780.9 3 2 Gitoxin 2 0 0 -5 0 0 C41H64Oi4 780.9 3 3 Helveticoside 100; 200 C29H42O9 534.6 2 2 Neriifolin 5 0 -5 0 0 C30H46O8 534.7 5 5 Oleandrin 5 0 -1 0 0 0 c 32h 48o 9 576.7 8 8 Ouabain 50-1000 c 29h 44o ,2 584.6 6 0 K-Strophantoside 100; 1000 c 42h 64o 19 873.0 2 2 CAs (Calotropis procera) 5 -1 0 0 0 22 22 CAs (Convallaria majalis) 5 -1 0 0 0 8 6

Cardiac Digitoxigenin 5 0 -1 0 0 0 C23H34O4 374.5 9 8 aglycones 3-Deoxy-digitoxigenin 200-500 C23H3403 358.5 3 0 Digoxigenin 250; 500 c 23h 34o 5 390.5 2 2 3ß,12ß-Diacetyl-digoxigenin 250; 500 c 27h 38o 7 474.6 2 2 Gitoxigenin 2 0 0 -5 0 0 c 23h 34o 5 390.5 3 1 3ß-Acetyl-gitoxigenin 250; 500 c 25h 36o 6 432.6 2 1 Oleandrigenin 5-250 c 25h 36o 6 432.6 9 7 Ouabagenin 5 0 -1 0 0 0 c 23h 34o 8 438.5 6 0 Strophanthidin 500; 1000 c 23h 32o 6 404.5 2 2 3ß-Acetyl-strophanthidin 100; 250 C25H3407 446.5 2 2

Saponins & Diosgenin 1 0 -1 0 0 0 C27H4203 414.6 6 0 Sapogenins Yamogenin 1 0 -1 0 0 0 C27H4203 414.6 6 0 Hecogenin 1 0 -1 0 0 0 C27H4204 430.6 6 0 Gitogenin 1 0 -1 0 0 0 C27H4404 432.6 6 0 Ginsenoside Rbl 10-1000 C54H920 23 1109.3 6 0 Hederacoside C 10-1000 C59H960 26 1221.6 6 0 Digitonin 50; 2000 C56H92029 1229.3 2 0 Escinol 10-1000 C54H840 23 1101.2 6 0

*) Acceptance was not related to the compound concentration. published data (by Kasang et al.) show that the ful suggestions, to Prof. Dr. Erwin Beck (Bayre various cardenolides offered were in fact incorpo uth) and Rainer Bussmann (Tübingen) for sending rated by the animals into their repugnatorial secre Asclepias from Mount Kenya. We thank tion. They are thus pharmacophageous according Mr.Greuert (Merck AG) for providing sapogenins to the criteria outlined by Boppre (1984). and Dipl.-Biol. Ilse Wickler for technical assis tance in plant and hopper cultivation. We grate Acknowledgements fully acknowledge Erna Roth for technical and The authors are grateful to Prof. Dr. Michael chemical assistance. Boppre for providing fresh plants as well as help 448 U. Seibt et al. • Pharmacophagy of the African Bushhopper

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