Journal of Chemical Ecology, Vol. 12, No. 4, 1986
SIGNIFICANCE OF PHOTOTOXIC PHYTOCHEMICALS IN INSECT HERBIVORY
G.H.N. T O W E R S Department of Botany University of British Columbia Vancouver, B.C., Canada V6T 2B1 (Received May 28, 1985; accepted August 13, 1985)
Abstract--The significance of the wide range of phototoxins, occurring in many plant species, with respect to their role in insect herbivory and on insects, is not fully understood. The types of compounds include polyacetylenes, quinones, furanocoumarins, tryptophan- and tyrosine-derived alkaloids and are distributed throughout some of the major families of flowering plants. Efforts are being made to determine the mechanisms of cellular toxicity of these compounds at the cellular and the organismal levels. Key Words--Furanocoumarin8, furanoquinolines, polyacetylenes, alkaloids, quinones, photosensitizers, photodynamic action, phototoxicity.
The effects on insects of phytochemicals which require light for the full expression of their toxicities was briefly reviewed recently with an emphasis of the role of photoactive polyacetylenes (Arnason et al., 1983). With our increasing understanding of the ecological importance of secondary plant chemicals, it is interesting now to examine more carefully the possible significance of these compounds, especially in relation to insect herbivory. This symposium is therefore most timely. In addition to photodynamic synthetic xanthene dyes (Carpenter et al., 1984), an increasing variety of photochemicals, including quinones, acetylenes, and alkaloids have been shown in recent years to be phototoxic to virus, bacteria, fungi, nematodes, and insects (Towers, 1984). The accumulation, in conspicuous quantities in plants, of these phytochemicals, in resin canals or glandular trichomes, suggests that they may play a role in deterring or destroying other organisms, including insects. These photoactive chemicals otherwise have no known metabolic or physiological functions in plants. For example, hyper813 0098-0331/86/0400-0813505.00/09 1986PlenumPublishingCorporation
814
TOWERS OH
0
OH
OH OH
OH
0
OH
icin (I), a quinone photosensitizer which accumulates in specialized glands in leaves of species of St. John's wort [Hypericum spp. (Clusiaceae)] and which is responsible for photodermatitis in grazing animals is not toxic to some species of beetles in the genus Chrysolina (Daloze and Pasteels, 1979). In fact, one species, C. brunsvicensis, stores hypericin (Rees, 1969). What role, if any, does this phototoxic chemical have in relation to other insects? We simply do not know. Phototoxic compounds or photosensitizers are chemicals which, when excited to a new electronic or electric state by absorption of a photon, react with other molecules in a given system. The resulting chemical changes in cells are often sufficiently severe to result in cell death. In many photoreactions of this type the excited photosensitizer transfers the excitation energy to oxygen which subsequently brings about an oxidation of another molecule. In this so-called photodynamic reaction, singlet oxygen, which may be the reactive species formed, may react with phospholipids, proteins, and sterols of plant membranes. For example, c~-terthienyl (II), a not uncommon thiophene product of acetylene metabolism in members of the Compositae or Asteraceae, is a very efficient singlet oxygen producer (Reyftmann et al., 1985). Photosensitizers like a-terthienyl damage cell membranes (Yamamoto et al., 1984). Other photosensitizers, with equal potential .for cellular destruction, penetrate cells to react with organelles including nuclei. Compounds of this type do not require oxygen. They form covalent adducts with bases in nucleic acids and may be termed photogenotoxic (Towers and Abramowski, 1983). There exist additional photosensitizers for which cellular targets have not yet been defined. Many polyacetylenes, or polyines as they are more correctly called, and their thiophene derivatives have recently been found to display phototoxicities.
II
PHOTOTOXICPHYTOCHEMICALSIN INSECTHERBIVORY
815
This large class of phytochemicals, characteristic of the Compositae (Asteraceae), Campanulaceae, Umbelliferae, and a number of other families of flowering plants (Bohlmann et al., 1973) include photosensitizers which have been shown to be phototoxic to many types of cells as well as to mosquito larvae (Arnason et al., 1981). On the other hand, none of the polyacetylenes of the Campanulaceae nor the characteristic acetylenes of the Umbelliferae and Araliaceae, namely falcarinone, falcarindione, falcarinol, or falcarindiol (III), appears to be phototoxic. Falcarindiol, however, in the leaf cuticle of the cultivated carrot, apparently stimulates oviposition in the female carrot fly, P s i l a rosae (Stadler and Buser, 1982). Concentrations of polyacetylenes in species of Compositae may be as high as 1% of the fresh weight, especially in young rhizomes at certain times of the year. The range of compounds usually differs between flowers, seeds, leaves, stems, and roots. Often the roots contain high levels. Is it possible that insect larvae, feeding on acetylene-rich roots or rhizomes, in the soil, may, on subsequent exposure to sunlight, either as larvae or as adults, suffer from phototoxicity? This has not been examined. Alpha-terthienyl, which has been studied most intensively, is so phototoxic to insect larvae that its potential as a commercial larvicide has been evaluated in stimulated pond trials. The cellular target of c~-terthienyl and of polyacetylenes appears to be membranes but not all of them require oxygen for their activity (McLachlan et al., 1984). Serious field studies remain to be done with respect to this large class of chemicals, many of which are demonstrably phototoxic to insects under laboratory conditions. Especially significant would be comparable analyses of their possible effects on phytophagous insects along the lines of the research on phototoxic furanocoumarins. The furanocoumarins, derivatives of phenylalanine, are characteristic of the Rutaceae and the Umbelliferae (Apiaceae). They also occur in members of the Leguminosae, Moraceae, Solanaceae, Pittosporaceae, Thymeleaceae, and Orchidaceae (Murray et al., 1982). Many of these tricyclic, planar compounds are phototoxic but perhaps the majority are not. Two nontoxic examples are 8hydroxypsoralen and isopimpinellin. Common plant species in which phototoxic linear furanocoumarins occur are given in Table 1. Many, but not all, of the biological effects of these much-studied compounds can be explained by photoinduced modification of DNA. The furanocoumarin intercalates in DNA and, on subsequent irradiation, reacts with pyrimidine bases to form covalently bound adducts. The photochemical reaction(s) is dependent on the presence of double bonds in the pyrone and in the furan rings. Some simple coumarins, however, e.g., 5,7-dimethyoxycoumarin are also
CH2=CH-~ H-(~C=C~2-CH-CH=CH-('CH2~)6-CH31 OH OH III
816
TOWERS TABLE 1. LINEARFURANOCOUMARINS
Psoralen (ficusin)
Ficus carica (Moraceae) Psoralea corylifolia (Leguminosae) Ficus carica Citrus bergamia (bergamot)(Rutaceae) Citrus limonum (lemon) (Rutaceae) Heracleum spp. (Umbeltiferae) Ammi majus (Umbelliferae) Seseli indicum (Umbelliferae) Petroselinum sativum (Umbelliferae) Ammi majus Angelica spp. (Umbelliferae) Pastinaca sativa (Umbelliferae) Ficus carica Ruta graveolens (Rutaceae)
5-Methoxypsoralen (bergapten)
8-Methoxypsoralen(xanthotoxin)
photosensitizers. The formation of bi-adducts by furanocoumarins occurs only with certain linear types and depends on nucleotide sequences, poly(dA-dT) 9 poly(dA-dT) sequence regions being the most favorable sites for intercalation and photocyclization. In the photoconjugation of furanocoumarins with pyrimidine bases there is no involvement of oxygen. It has been suggested, however, that with certain furanocoumarins such as angelicin (IV), reactive species of oxygen may play a major role in their carcinogenic effects (Joshi and Pathak, 1983). Plants usually store furanocoumatins as the photochemically active aglycones, the major sites of accumulation being oil channels or ducts present in leaves, stems, roots, and flower parts. They may also be found in the cuticle (Stadler and Buser, 1982). Many of them accumulate in roots, as with acetylene reservoirs in roots of Compositae, but their ecological importance as phototoxic defense compounds in not obvious as ultraviolet light does not penetrate more than a few millimeters below the soil surface. Why do they accumulate in roots? Insect predation of the leaves of umbellifers has been studied in some detail, and at least o n e species of butterfly, P a p i l i o p o I y x e n e , elaborates furanocoumatin detoxifying enzymes (Ivie et al., 1983). Some species of P a p i l i o actually appear to select species of umbellifers or members of the Rutaceae which con-
IV
PHOTOTOXIC PHYTOCHEMICALS IN INSECT HERBIVORY
"
R
817
0
RI R=H; RI=OCH 3 V
R=OCH3; RI=OCH 3
vI tain linear furanocoumarins as food sources (Berenbaum, 1981). The possible ecological importance of the furanocoumarins will be presented in later papers. The furanochromones khellin (V), and visnagin (VI), which are of polyketide origin, cooccur with furanocoumarins in species of Ammi (Umbelliferae) (Schonberg and Sinha, 1950; Spath and Gruber, 1941) and, like them, are phototoxic to bacteria and to fungi. They cause chromosomal damage to CHO cells in UV-A (Abeysekera et al., 1983). By analogy with the furanocoumatins (Kanne et al., 1982), khellin could be expected to react with a base such as thymine in a 2 + 2 photoaddition involving either the 2-3 or the 6-7 double bond of khellin and the 5 ' - 6 ' double bond of thymine. UV-A irradiation of a frozen aqueous solution of khellin and thymine in fact yields photoadducts including one between the 2-3 double bond of khellin and the 5 ' - 6 ' double bond of thymine (Abeysekera et al., 1983). The effects of these compounds on phytophagous insects has not been studied so far, nor have the polyketide benzofurans, 6-methoxyeuparin as well as encecalin and 7-hydroxyencecalin from species of Encelia, although these compounds have been shown to be phototoxic to bacteria and to fungi (Proksch et al., 1983). Some species in the Rutaceae, such as Skimmiajaponica, Dictamnus alba, and even species of Citrus, contain, in addition to appreciable concentrations of furanocoumarins such as 5- and 8-methoxypsoralens, photosensitizing furanoquinoline alkaloids, derivatives of anthranilic acid. These tricyclic planar compounds, such as dictamnine (VII) and skimmianine (VIII), intercalate in
VII
818
TOWERS
c,o OCH 3
VIII DNA in the dark and subsequently undergo photobinding in UV-A. Dictamnine binds preferentially to the same sites as 8-methoxypsoralen (Pfyffer et al., 1982). Dictamnine, as well as a number of other furanoquinolines and certain other alkaloids, is mutagenic and causes gross chromosomal abnormalities in CHO cells in UV-A (Towers and Abramowski, 1983). Their importance in relation to insect predation is unknown as is the significance of the cooccurrence of two biogenetically unrelated phototoxic compounds in one species. A second group of alkaloids which has been found to be photogenotoxic to CHO cells (Towers and Abramowski, 1983) and phototoxic to larvae of Aedes atropalpus is the/3-carbolines or harman compounds, e.g., harman (IX) (Arnason et al., 1983). These are very widely distributed, occurring in about 26 families of plants (Allen and Holmstedt, 1980). Additional tryptophan-derived alkaloids, such as 6-canthinone (X) and 5-methoxy-6-canthinone (XI) of Zanthoxylum (Rutaceae) and brevicolline (XII), an N-methylpyrolidine-substituted barman alkaloid which occurs in the sedge, Carex brevicollis, have also been shown to be phototoxic to bacteria, fungi, and CHO cells (Towers and Abramowski, 1983) but their effects on insects remain unstudied. Berberine, a wellknown phenylalanine-derived alkaloid is also phototoxic to mosquito larvae but
IX
X
OCH3
XI
819
PHOTOTOXIC PHYTOCHEMICALS IN INSECT HERBIVORY
XlI
OH
OH XIII
its mechanism of action is not known (Philogene et al., 1984). There are undoubtedly very many other alkaloids with this type of bioactivity awaiting research by chemical ecologists with an understanding of photobiology. The excreta of the silkworm (Bombyx mori), used in traditional Chinese medicine, contains cytostatic/cytotoxic porphyrins, including the phototoxic methyl esters of pheophorbides (Nakatani et al., 1981). The generation of singlet oxygen in light by excited states of certain porphyrins is responsible for their phototoxicity. How many kinds of phytophageous insects produce phototoxic frass? The ecological significance of phototoxic frass may be of interest in relation to microbial colonization of this material but is this phototoxicity fortuitous and is it really important ecologically? One can extend these questions to include the many other miscellaneous compounds of plant origin such as lachnanthocarpone (XIII), in species of Lachnanthes of the Hemodoraceae which are phototoxic to bacteria and even to vertebrates (Kornfeld and Edwards, 1972). How important are they to phytophagous insects? Some of these problems have been addressed in work reported in this symposium but much remains to be done; it seems to be clear from research so far that interesting and important new ecological concepts concerning the relations between insects, phytochemicals, and light remain to be discovered.
820
TOWERS REFERENCES
ABEYSEKERA,B.F., ABRAMOWSKI,Z., and TOWERS, G.H.N. 1983. Genotoxicity of the natural furochromones, khellin and visnagin and the identification of a khellin-thymine photoadduct. Photochem. Photobiol. 38:311-315. ALLEN, J.R.F., and HOLMSTEDT, B.R. 1980. The simple 3-carboline alkaloids. Phytochemistry 19:1573-1982. ARNASON, T., SWAIN, T., WAT, C.K., GRAHAM,E.A., PARTINGTON, S., TOWERS, G.H.N., and LAM, J. 1981. Mosquito larvicidal activity of polyacetylenes from species in Asteraceae. Biochem. System. Ecol. 9:63-68. ARNASON, T., TOWERS, G.H.N., PHmOGENE, B.J.R., and LAMBERT, J.D.H. !983. The role of natural photosensitizers in plant resistance to insects. Am. Chem. Soc. 208:140-151. BERENBAUM, M. 1981. Patterns of furanocoumarin distribution and insect herbivory in the Umbelliferae: Plant chemistry and community structure. Ecology 62:1254-1266. BOHLMANN,F., BURKHARDT,T., and ZDERO, C. 1973. Naturally Occurring Acetylenes. Academic Press, London. CARPENTER, T.L., RESPIEIO, N.C., and HEITZ, J.R. 1984. Acute light-dependent toxicity of freeacid formulations of xanthene dyes to larval Culex pipiens quinquefasciatus Say (Diptera: Culicidae). Environ. Entomol. 13:1366-1370. DALOZE, D., and PASTEELS,J.M. 1979. Production of cardiac glycosides by chrysomelid beetles and larvae. J. Chem. Ecol. 5:63-77. IVIE, G.W., BULL, D.L., BEIER, R.C., PRYOR, N.W., and OERTLI, E.H. 1983. Metabolic detoxification: Mechanism of insect resistance to plant psoralens. Science 221:374-376. JOSHI, P.C., and PATHAK, M.A. 1983. Production of singlet oxygen and superoxide radicals by psoralens and their biological significance. Biochem. Biophys. Res. Commun. 112:638-646. KANNE, D.K., STRAUB, K., RAPAPORT, H., and HEARST, J.E. 1982. Psoralen-deoxyribonucleic acidphotoreaction. Characterization of the monoaddition products from 8-methoxypsoralen and 4,5',8-trimethylpsoralen. Biochemistry 21 :861-871. KORNFELD, J.M., and EDWARDS,J.M. 1972. An investigation of the photodynamic pigments in extracts of Lachnanthes tinctoria. Biochem. Biophys. Acta 286:88-90. MCLACHLAN, D., ARNASON,T., and LAM, J. 1984. The role of oxygen in photosensitization with polycetylenes and thiophene derivatives. Photochem. Photobiol. 39:177-182. MURRAY, R.D.H., MENDEZ, J., and BROWN, S.A. 1982. The Natural Coumarins: Occurrence, Chemistry, and Biochemistry. John Wiley & Sons, New York. NAKATAN1,Y., OUR1SSON,G., and BECK,J.P. 1981. Chemistry and biochemistry of Chinese drugs. VII. Cytostatic pheophytins from silkworm excreta and derived phototcytotoxic pheophorbides. Chem. Pharm. Bull. 29:2261-2269. PFYFFER, G.F., PFYFFER, B.U., and TOWERS, G.H.N. 1982. Monoaddition of dictamnine to synthetic double-stranded polydeoxyribonucleotides in UV-A and the effect of photomodified DNA on template activity. Photochem. Photobiol. 35:793-797. PHILOGENE, B.J.R., ARNASON, J.T., TOWERS, G.H.N., ABRAMOWSKI,Z., CAMPOS, F., CHAMPAGNE, D., and McLACHLAN, D. 1984. Berberine: A naturally occurring phototoxic alkaloid. J. Chem. Ecol. 10:115-123. PROKSCH, P., PROKSCH,M., TOWERS, G.H.N., and RODRIGUEZ,E. 1983. Phototoxic and insecticidal activities of chromenes and benzofurans from Encelia (Asteraceae). J. A~at. Prod. 46:331334. REES, C.J.C. 1969. Chemoreceptor specificity associated with choice of feeding site by the beetle Chrysolina brunsvicensis on its food plant Hyperieum hirsutum, pp. 565-583, in J. De Wilde and L.M. Schoonhoven (eds.). Insect and Host Plant. North-Holland Publishing, Amsterdam. REYFTMANN, J.P., KAGAN,J., SANTUS,R., and MORLIERE,P. 1985. Excited state properties of ~terthienyl and related molecules. Photochem. Photobiol. 41:1-7.
PHOTOTOXIC PHYTOCHEMICALSIN INSECT HERBIVORY
82 1
SCHONBERG,A., and SINHA,A. 1950. Khellin and allied compounds. J. Am. Chem. Soc. 72:16111616. SPATH, E., and GRUBER, W. 1941. Die Konstitution des Visnagins aus Ammi visnaga. Berichte 74:1492-1500. STADLER,E., and BUSER, H.R. 1982. Proceedings of the 5th International Symposium on InsectPlant Relationships. Wageningen, 1982, Pudoc, Wageningen, Netherlands. TOWERS, G.H.N. 1984. Interactions of light with phytochemicals in some natural and novel systems. Can. J. Bot. 62:2900-2911. TOWERS, G.H.N., and ABRAMOWSKI,Z. 1983. UV-mediated genotoxicity of furanoquinoline and of certain tryptophan-derived alkaloids. J. Nat. Prod. 46:576-581. YAMAMOTO, E., MACRAE, W.D., GARCIA, F.J., and TOWERS, G.H.N. 1984. Photodynamic hemolysis caused by a-terthienyl. Planta Med. 50:124-127.
SIGNIFICANCE OF PHOTOTOXIC PHYTOCHEMICALS IN INSECT HERBIVORY
G.H.N. T O W E R S Department of Botany University of British Columbia Vancouver, B.C., Canada V6T 2B1 (Received May 28, 1985; accepted August 13, 1985)
Abstract--The significance of the wide range of phototoxins, occurring in many plant species, with respect to their role in insect herbivory and on insects, is not fully understood. The types of compounds include polyacetylenes, quinones, furanocoumarins, tryptophan- and tyrosine-derived alkaloids and are distributed throughout some of the major families of flowering plants. Efforts are being made to determine the mechanisms of cellular toxicity of these compounds at the cellular and the organismal levels. Key Words--Furanocoumarin8, furanoquinolines, polyacetylenes, alkaloids, quinones, photosensitizers, photodynamic action, phototoxicity.
The effects on insects of phytochemicals which require light for the full expression of their toxicities was briefly reviewed recently with an emphasis of the role of photoactive polyacetylenes (Arnason et al., 1983). With our increasing understanding of the ecological importance of secondary plant chemicals, it is interesting now to examine more carefully the possible significance of these compounds, especially in relation to insect herbivory. This symposium is therefore most timely. In addition to photodynamic synthetic xanthene dyes (Carpenter et al., 1984), an increasing variety of photochemicals, including quinones, acetylenes, and alkaloids have been shown in recent years to be phototoxic to virus, bacteria, fungi, nematodes, and insects (Towers, 1984). The accumulation, in conspicuous quantities in plants, of these phytochemicals, in resin canals or glandular trichomes, suggests that they may play a role in deterring or destroying other organisms, including insects. These photoactive chemicals otherwise have no known metabolic or physiological functions in plants. For example, hyper813 0098-0331/86/0400-0813505.00/09 1986PlenumPublishingCorporation
814
TOWERS OH
0
OH
OH OH
OH
0
OH
icin (I), a quinone photosensitizer which accumulates in specialized glands in leaves of species of St. John's wort [Hypericum spp. (Clusiaceae)] and which is responsible for photodermatitis in grazing animals is not toxic to some species of beetles in the genus Chrysolina (Daloze and Pasteels, 1979). In fact, one species, C. brunsvicensis, stores hypericin (Rees, 1969). What role, if any, does this phototoxic chemical have in relation to other insects? We simply do not know. Phototoxic compounds or photosensitizers are chemicals which, when excited to a new electronic or electric state by absorption of a photon, react with other molecules in a given system. The resulting chemical changes in cells are often sufficiently severe to result in cell death. In many photoreactions of this type the excited photosensitizer transfers the excitation energy to oxygen which subsequently brings about an oxidation of another molecule. In this so-called photodynamic reaction, singlet oxygen, which may be the reactive species formed, may react with phospholipids, proteins, and sterols of plant membranes. For example, c~-terthienyl (II), a not uncommon thiophene product of acetylene metabolism in members of the Compositae or Asteraceae, is a very efficient singlet oxygen producer (Reyftmann et al., 1985). Photosensitizers like a-terthienyl damage cell membranes (Yamamoto et al., 1984). Other photosensitizers, with equal potential .for cellular destruction, penetrate cells to react with organelles including nuclei. Compounds of this type do not require oxygen. They form covalent adducts with bases in nucleic acids and may be termed photogenotoxic (Towers and Abramowski, 1983). There exist additional photosensitizers for which cellular targets have not yet been defined. Many polyacetylenes, or polyines as they are more correctly called, and their thiophene derivatives have recently been found to display phototoxicities.
II
PHOTOTOXICPHYTOCHEMICALSIN INSECTHERBIVORY
815
This large class of phytochemicals, characteristic of the Compositae (Asteraceae), Campanulaceae, Umbelliferae, and a number of other families of flowering plants (Bohlmann et al., 1973) include photosensitizers which have been shown to be phototoxic to many types of cells as well as to mosquito larvae (Arnason et al., 1981). On the other hand, none of the polyacetylenes of the Campanulaceae nor the characteristic acetylenes of the Umbelliferae and Araliaceae, namely falcarinone, falcarindione, falcarinol, or falcarindiol (III), appears to be phototoxic. Falcarindiol, however, in the leaf cuticle of the cultivated carrot, apparently stimulates oviposition in the female carrot fly, P s i l a rosae (Stadler and Buser, 1982). Concentrations of polyacetylenes in species of Compositae may be as high as 1% of the fresh weight, especially in young rhizomes at certain times of the year. The range of compounds usually differs between flowers, seeds, leaves, stems, and roots. Often the roots contain high levels. Is it possible that insect larvae, feeding on acetylene-rich roots or rhizomes, in the soil, may, on subsequent exposure to sunlight, either as larvae or as adults, suffer from phototoxicity? This has not been examined. Alpha-terthienyl, which has been studied most intensively, is so phototoxic to insect larvae that its potential as a commercial larvicide has been evaluated in stimulated pond trials. The cellular target of c~-terthienyl and of polyacetylenes appears to be membranes but not all of them require oxygen for their activity (McLachlan et al., 1984). Serious field studies remain to be done with respect to this large class of chemicals, many of which are demonstrably phototoxic to insects under laboratory conditions. Especially significant would be comparable analyses of their possible effects on phytophagous insects along the lines of the research on phototoxic furanocoumarins. The furanocoumarins, derivatives of phenylalanine, are characteristic of the Rutaceae and the Umbelliferae (Apiaceae). They also occur in members of the Leguminosae, Moraceae, Solanaceae, Pittosporaceae, Thymeleaceae, and Orchidaceae (Murray et al., 1982). Many of these tricyclic, planar compounds are phototoxic but perhaps the majority are not. Two nontoxic examples are 8hydroxypsoralen and isopimpinellin. Common plant species in which phototoxic linear furanocoumarins occur are given in Table 1. Many, but not all, of the biological effects of these much-studied compounds can be explained by photoinduced modification of DNA. The furanocoumarin intercalates in DNA and, on subsequent irradiation, reacts with pyrimidine bases to form covalently bound adducts. The photochemical reaction(s) is dependent on the presence of double bonds in the pyrone and in the furan rings. Some simple coumarins, however, e.g., 5,7-dimethyoxycoumarin are also
CH2=CH-~ H-(~C=C~2-CH-CH=CH-('CH2~)6-CH31 OH OH III
816
TOWERS TABLE 1. LINEARFURANOCOUMARINS
Psoralen (ficusin)
Ficus carica (Moraceae) Psoralea corylifolia (Leguminosae) Ficus carica Citrus bergamia (bergamot)(Rutaceae) Citrus limonum (lemon) (Rutaceae) Heracleum spp. (Umbeltiferae) Ammi majus (Umbelliferae) Seseli indicum (Umbelliferae) Petroselinum sativum (Umbelliferae) Ammi majus Angelica spp. (Umbelliferae) Pastinaca sativa (Umbelliferae) Ficus carica Ruta graveolens (Rutaceae)
5-Methoxypsoralen (bergapten)
8-Methoxypsoralen(xanthotoxin)
photosensitizers. The formation of bi-adducts by furanocoumarins occurs only with certain linear types and depends on nucleotide sequences, poly(dA-dT) 9 poly(dA-dT) sequence regions being the most favorable sites for intercalation and photocyclization. In the photoconjugation of furanocoumarins with pyrimidine bases there is no involvement of oxygen. It has been suggested, however, that with certain furanocoumarins such as angelicin (IV), reactive species of oxygen may play a major role in their carcinogenic effects (Joshi and Pathak, 1983). Plants usually store furanocoumatins as the photochemically active aglycones, the major sites of accumulation being oil channels or ducts present in leaves, stems, roots, and flower parts. They may also be found in the cuticle (Stadler and Buser, 1982). Many of them accumulate in roots, as with acetylene reservoirs in roots of Compositae, but their ecological importance as phototoxic defense compounds in not obvious as ultraviolet light does not penetrate more than a few millimeters below the soil surface. Why do they accumulate in roots? Insect predation of the leaves of umbellifers has been studied in some detail, and at least o n e species of butterfly, P a p i l i o p o I y x e n e , elaborates furanocoumatin detoxifying enzymes (Ivie et al., 1983). Some species of P a p i l i o actually appear to select species of umbellifers or members of the Rutaceae which con-
IV
PHOTOTOXIC PHYTOCHEMICALS IN INSECT HERBIVORY
"
R
817
0
RI R=H; RI=OCH 3 V
R=OCH3; RI=OCH 3
vI tain linear furanocoumarins as food sources (Berenbaum, 1981). The possible ecological importance of the furanocoumarins will be presented in later papers. The furanochromones khellin (V), and visnagin (VI), which are of polyketide origin, cooccur with furanocoumarins in species of Ammi (Umbelliferae) (Schonberg and Sinha, 1950; Spath and Gruber, 1941) and, like them, are phototoxic to bacteria and to fungi. They cause chromosomal damage to CHO cells in UV-A (Abeysekera et al., 1983). By analogy with the furanocoumatins (Kanne et al., 1982), khellin could be expected to react with a base such as thymine in a 2 + 2 photoaddition involving either the 2-3 or the 6-7 double bond of khellin and the 5 ' - 6 ' double bond of thymine. UV-A irradiation of a frozen aqueous solution of khellin and thymine in fact yields photoadducts including one between the 2-3 double bond of khellin and the 5 ' - 6 ' double bond of thymine (Abeysekera et al., 1983). The effects of these compounds on phytophagous insects has not been studied so far, nor have the polyketide benzofurans, 6-methoxyeuparin as well as encecalin and 7-hydroxyencecalin from species of Encelia, although these compounds have been shown to be phototoxic to bacteria and to fungi (Proksch et al., 1983). Some species in the Rutaceae, such as Skimmiajaponica, Dictamnus alba, and even species of Citrus, contain, in addition to appreciable concentrations of furanocoumarins such as 5- and 8-methoxypsoralens, photosensitizing furanoquinoline alkaloids, derivatives of anthranilic acid. These tricyclic planar compounds, such as dictamnine (VII) and skimmianine (VIII), intercalate in
VII
818
TOWERS
c,o OCH 3
VIII DNA in the dark and subsequently undergo photobinding in UV-A. Dictamnine binds preferentially to the same sites as 8-methoxypsoralen (Pfyffer et al., 1982). Dictamnine, as well as a number of other furanoquinolines and certain other alkaloids, is mutagenic and causes gross chromosomal abnormalities in CHO cells in UV-A (Towers and Abramowski, 1983). Their importance in relation to insect predation is unknown as is the significance of the cooccurrence of two biogenetically unrelated phototoxic compounds in one species. A second group of alkaloids which has been found to be photogenotoxic to CHO cells (Towers and Abramowski, 1983) and phototoxic to larvae of Aedes atropalpus is the/3-carbolines or harman compounds, e.g., harman (IX) (Arnason et al., 1983). These are very widely distributed, occurring in about 26 families of plants (Allen and Holmstedt, 1980). Additional tryptophan-derived alkaloids, such as 6-canthinone (X) and 5-methoxy-6-canthinone (XI) of Zanthoxylum (Rutaceae) and brevicolline (XII), an N-methylpyrolidine-substituted barman alkaloid which occurs in the sedge, Carex brevicollis, have also been shown to be phototoxic to bacteria, fungi, and CHO cells (Towers and Abramowski, 1983) but their effects on insects remain unstudied. Berberine, a wellknown phenylalanine-derived alkaloid is also phototoxic to mosquito larvae but
IX
X
OCH3
XI
819
PHOTOTOXIC PHYTOCHEMICALS IN INSECT HERBIVORY
XlI
OH
OH XIII
its mechanism of action is not known (Philogene et al., 1984). There are undoubtedly very many other alkaloids with this type of bioactivity awaiting research by chemical ecologists with an understanding of photobiology. The excreta of the silkworm (Bombyx mori), used in traditional Chinese medicine, contains cytostatic/cytotoxic porphyrins, including the phototoxic methyl esters of pheophorbides (Nakatani et al., 1981). The generation of singlet oxygen in light by excited states of certain porphyrins is responsible for their phototoxicity. How many kinds of phytophageous insects produce phototoxic frass? The ecological significance of phototoxic frass may be of interest in relation to microbial colonization of this material but is this phototoxicity fortuitous and is it really important ecologically? One can extend these questions to include the many other miscellaneous compounds of plant origin such as lachnanthocarpone (XIII), in species of Lachnanthes of the Hemodoraceae which are phototoxic to bacteria and even to vertebrates (Kornfeld and Edwards, 1972). How important are they to phytophagous insects? Some of these problems have been addressed in work reported in this symposium but much remains to be done; it seems to be clear from research so far that interesting and important new ecological concepts concerning the relations between insects, phytochemicals, and light remain to be discovered.
820
TOWERS REFERENCES
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SCHONBERG,A., and SINHA,A. 1950. Khellin and allied compounds. J. Am. Chem. Soc. 72:16111616. SPATH, E., and GRUBER, W. 1941. Die Konstitution des Visnagins aus Ammi visnaga. Berichte 74:1492-1500. STADLER,E., and BUSER, H.R. 1982. Proceedings of the 5th International Symposium on InsectPlant Relationships. Wageningen, 1982, Pudoc, Wageningen, Netherlands. TOWERS, G.H.N. 1984. Interactions of light with phytochemicals in some natural and novel systems. Can. J. Bot. 62:2900-2911. TOWERS, G.H.N., and ABRAMOWSKI,Z. 1983. UV-mediated genotoxicity of furanoquinoline and of certain tryptophan-derived alkaloids. J. Nat. Prod. 46:576-581. YAMAMOTO, E., MACRAE, W.D., GARCIA, F.J., and TOWERS, G.H.N. 1984. Photodynamic hemolysis caused by a-terthienyl. Planta Med. 50:124-127.
