Journal of Chemical Ecology, Vol. 15, No. I, 1989
MYRISTICIN, SAFROLE, A N D F A G A R A M I D E AS PHYTOSYNERGISTS OF X A N T H O T O X I N l
J.J. N E A L 2 Department of Entomology University of Illinois Urbana, Illinois 61801
(Received August 5, 1987; accepted November 24, 1987) Abstract--The methylenedioxyphenyl-containing (MDP) inhibitors of mixedfunction oxidase detoxification enzymes, myristicin, safrole, fagaramide, and isosafrole, occur with xanthotoxin or other toxic furanocoumarins in plants of the families Umbelliferae and Rutaceae. All four MDP compounds have a synergistic effect on the toxicity of xanthotoxin to Heliothis zea. Myristicin also increased the phototoxicity of xanthotoxin in the presence of UV light. The term phytosynergist is used to describe plant compounds that are present at concentrations producing no toxic effect by themselves but have a synergistic effect on cooccurring toxins. Key Words--Xanthotoxin, Heliothis zea, Lepidoptera, Noctuidae, myristicin, fagaramide, safrole, isosafrole, synergism, phytosynergist, phototoxicity.
INTRODUCTION The o c c u r r e n c e o f insecticide synergists in plants has led to speculation that their natural function m a y be s y n e r g i s m o f c o o c c u r r i n g toxins ( L a F o r g e and Barthel, 1944; K r i e g e r et al., 1971). This v i e w is supported by e x p e r i m e n t s in which the insecticide synergist myristicin (Figure 1), at levels present in plants, increased the mortality o f first-instar H e l i o t h i s z e a caterpillars fed xanthotoxin (Figure 1), a toxic f u r a n o c o u m a r i n o c c u r r i n g with myristicin in m a n y plants o f ~Taken in part from a thesis submitted to the Graduate College of the University of Illinois at Urbana-Champaign in partial fulfillment of the requirements for the degree of Doctor of Philosophy, June 1987. 2Present address: Purdue University, Department of Entomology, Entomology Hall, West Lafayette, Indiana 47907. 309 0098-0331/89/0100-0309506.00/0 © 1989 Plenum Publishing Coq~mtion
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o--/\ CH3
)
CH~
oAo
/
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III
NH
I
oAo
i/ CH2
IV
oAo
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FIG. 1. Xanthotoxin and plant compounds with a synergistic effect on its toxicity: xanthotoxin (I), fagaramide (II), isosafrole (III), myristicin (IV), and safrole (V).
the family Umbelliferae (Berenbaum and Neal, 1985). In this paper, compounds such as myristicin that are present at concentrations producing little or no toxic effect by themselves but which increase the toxicity of cooccurring toxins will be called phytosynergists. Myristicin, like many other compounds containing a methylenedioxyphenyl (MDP) substituent, is an inhibitor of mixed-function oxidases (MFOs) (Hodgson and Philpot, 1974). MFOs are detoxification enzymes that metabolize many xenobiotics (Hodgson, 1985) and are primarily responsible for the metabolism of xanthotoxin in at least two species of insects (Bull et al., 1984, 1986). The synergistic effect of myristicin on the toxicity of xanthotoxin to Heliothis zea is attributed to its inhibition of xanthotoxin metabolism, which increases the effective xanthotoxin concentration in the insect (Berenbaum and Neal, 1985). Xanthotoxin has two types of toxic action on insects, a " d a r k " toxicity (toxicity in the absence of UV-A light) and phototoxicity (increased toxicity in the presence of UV-A light) (Berenbaum, 1978), which act through different mechanisms (Murray et al., 1982). Both mechanisms are likely to be important under natural conditions; however, only the synergistic effect of myristicin on the dark toxicity of xanthotoxin has been measured (Berenbaum and Neal, 1985). If myristicin works by inhibiting the detoxification of xanthotoxin, however, its activity should be retained under a variety of environmental conditions and be independent of the mode of action of the toxin. Thus, myristicin should
PHYTOSYNERGISTS OF XANTHOTOXIN
3]1
also be a phytosynergist of xanthotoxin phototoxicity in the presence of UV-A light. Myristicin is not the only MDP-containing insecticide synergist that occurs with xanthotoxin in plant species. Phytochemical records show that safrole and apiole also occur with xanthotoxin in the Umbelliferae (Ceska et al., 1987; Hodisan et al., 1980; Murray et al., 1982; Hegnauer, 1973; Harborne, 1971), and fagaramide, asarinin, and sesamin occur with xanthotoxin in plants of the family Rutaceae (Thorns, 1911; Thoms and Thfimen, 1911; Hegnauer, 1973; Murray et al., 1982). Some rutaceous plants also contain safrole and isosafrole along with other toxic furanocoumarins (Chang, 1976; Hegnauer, 1973; Murray et al., 1982). Thus, myristicin is probably not the only plant compound to function naturally as a phytosynergist of xanthotoxin, and there are many toxins other than xanthotoxin that may occur with pbytosynergists. Three insecticide synergists that are MFO inhibitors, fagaramide, safrole, and isosafrole (Figure 1), were tested as synergists of xanthotoxin toxicity to Heliothis zea. In addition, the ability of phytosynergists to maintain activity independent of the mode of action of the toxin was studied by measuring the synergistic effect of myristicin on xanthotoxin in the presence as well as in the absence of UV-A light.
METHODS AND MATERIALS
Chemicals. Myristicin was purchased from Saber Labs (Morton Grove, Illinois); fagaramide, safrole, and isosafrole from Aldrich Chemical Co. (Milwaukee, Wisconsin); and xanthotoxin from Sigma Chemical Co. (St. Louis, Missouri). Bioassays. Neonates of the corn earworm, Heliothis zea (Lepidoptera; Noctuidae), from a culture maintained by Dr. G. Waldbauer at the University of Illinois, were used for testing the oral toxicities of all allelochemicals and combinations of allelochemicals. H. zea was chosen for study because it is a generalist feeder, it is susceptible to the toxic effects of xanthotoxin, and because myristicin has a synergistic effect on xanthotoxin toxicity to this insect (Berenbaum and Neal, 1985). Chemicals were administered by incorporation into an artificial diet (Berenbaum and Neal, 1985) in which wheat germ is the only undefined ingredient. In order to ensure uniformity, xanthotoxin (dissolved in acetone) was mixed with the alphacel component of the diet and dried prior to its incorporation into the diet. Myristicin, fagaramide, safrole, and isosafrole were added to hot diet in acetone solution (1% of the total wet weight) prior to gelling. Neonates (30 per treatment) were placed individually on 200 mg diet in a 1.5-ml polypropylene test tube (Biorad, Richmond, California) and reared in a
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25°C incubator under cool, white, fluorescent lights (16 hr light-8 hr dark). When screened by the polypropylene tube, these lights delivered less than 0.2 mW/m 2 of either UV-A (290-400 nm) or UV-B (200-290 nm) light as measured by a UVX radiometer with UVX 36 and UVX 31 sensors (UVP Inc., San Gabriel, California), Caterpillars were monitored until they died or molted to second instar. Toxicity of the MDP compounds was first measured at 1000 ppm (weight/wet weight diet); concentrations at and above 1000 ppm have been recorded for some of the MDP compounds in plants (Lichtenstein and Casida, 1963; Shulgin, 1966). For compounds toxic at 1000 ppm, lower concentrations were tested until a no-effect level was found. To test for synergism, the toxicity of xanthotoxin alone was compared to its toxicity in the presence of MDP compounds. LCso values were determined from log dose/probit mortality plots (SAS Institute Inc., 1982). LCso values were considered significantly different if the 95% confidence intervals did not overlap. Synergistic ratios (SR) were calculated as the LC~0 of the toxin divided by the LCso of the toxin with synergist. For measuring the effects of myristicin-xanthotoxin combinations on H. z e a in the presence and absence of UV-A light at simulated natural levels, larvae were reared in 50-mm x 5-mm internal diameter Pyrex test tubes stoppered with a cotton applicator stick. Pyrex transmits solar UV wavelengths (290-400 nm) but eliminates nonsolar UV wavelengths. Natural UV-A light intensities were simulated using two Sylvania BLB bulbs that produced between 1700 and 1950 mW/m 2 UV-A light and between 0.4 and 0.7 mW/m 2 UV-B at the surface of the tubes as measured with a UVX radiometer; a 16 hr light-8 hr dark photoperiod was used. For the no-UV-light treatments, filters opaque to wavelengths less than 400 nm were used to exclude UV light. Toxicity was evaluated as described above.
RESULTS In the absence of xanthotoxin, neither myristicin nor fagaramide Showed any toxicity at the highest dose tested. Both safrole and isosafrole were toxic at 1000 ppm, each killing 33% of the first-instar caterpillars. Safrole killed 10% at 700 ppm and was nontoxic at 400 ppm. Isosafrole killed 4 % at 400 ppm and was nontoxic at 100 ppm. Therefore, in tests for synergistic effects on xanthotoxin, myristicin and fagaramide were used at 1000 ppm, safrole at 400 ppm, and isosafrole at 100 ppm. Xanthotoxin has an LCso of 21,000 ppm (Table 1). The LCso values for combinations of xanthotoxin with myristicin, fagaramide, safrole, and isosafrole are 3200 ppm, 8300 ppm, 5900 ppm, and 8500 ppm, respectively. These values are all significantly different from the LCso of unsynergized xanthotoxin. Synergistic ratios are 6.5, 2.5, 3.5, and 2.5 for myristicin, fagaramide, safrole,
PHYTOSYNERG[STS OF XANTHOTOXIN
3 13
TABLE 1. SYNERGISM OF TOXICITY OF XANTHOTOXIN TO FIRST-INSTAR Heliothis zea BY MYRISTICIN, FAGARAMIDE, SAFROLE, AND ISOSAFROLE
Mortality (%)" Xanthotoxin (ppm in diet) 0 1000 2000 5000 10000 15000 20000 LCso 95% CL/' SR'
Control 0 NT 3 13 20 30 47 21,000 17,000-29,000
1000 ppm rnyristicin
1000 ppm fagaramide
0 23 43 67 97 NT NT 3,200 2,200-4,200 6.5
0 10 13 17 67 NT NT 8.300 6,800-11,000 2.5
400 ppm safrole 0 l0 37 40 77 NT NT 5,900 4,600-7,800 3.5
100 ppm isosafrole 0 20 13 37 57 NT NT 8,500 6,500-13,000 2.5
"N >- 30. NT = not tested. 1'95% CL = 95% confidence limit; LCso values are significantly different (P < 0.05) if the 95% confidence intervals do not overlap. 'SR = synergistic ratio.
and isosafrole, respectively (Table 1) but are not directly comparable because of differences in the dosages. In the presence of UV light, myristicin significantly increases the LCso of xanthotoxin 3.6-fold (Table 2). UV light also has a significant effect on the toxicity of xanthotoxin-myristicin combinations, increasing the LCs0 2.7-fold (Table 2).
DISCUSSION
Fagaramide, safrole, and myristicin all fit the definition of a phytosynergist; they have a synergistic effect on a cooccurring toxin at levels that are nontoxic. Isosafrole, which is also a synergist of xanthotoxin, may be a phytosynergist for other cooccurring toxins. These compounds do not necessarily act exclusively as phytosynergists in all plants that produce them. For example, levels of myristicin in some plant parts (Shulgin, 1966) have an insecticidal effect in some insects (Lichtenstein and Casida, 1963). However, insecticidal concentrations of these compounds are rare in plants compared to sublethal concentrations. The occurrence of these compounds at sublethal concentrations suggests a primary role as a phytosynergist.
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TABLE 2. SYNERGISMOF XANTHOTOXINTOXICITYTO FIRST-INSTARtteliothis zea BY COMBINATIONSOF MYRISTICIN (1000 ppm) AND UV-A LIGHT Mortality (%)" Xanthotoxin (ppm in diet)
No myristicin, UV-A light NT 17 40 43 50
100
250 500 1000 2500 LC.so 95 % CL
2,200 1300-15,000
Myristicin, no UV-A light
Myristicin with UV-A light
NT 10 20 47 70
30 47 57 63 70
1,600 1,300-2,100
600 0-1,300 Synergistic ratios
No myristicin/myristicin (UV-A present) No UV-A/UV-A (myristicin present)
3.6 2.7
"N >_ 30~ N T = not tested,
J'95 % CL = 95% confidence limit. LCso values are significantly different (P < 0.05) if the 95% confidence intervals do not overlap.
B e r e n b a u m and Neal (1985) p r o p o s e d that myristicin acts as an inhibitor o f xanthotoxin detoxification and thereby increases the effective concentration o f xanthotoxin in the insect. As predicted, myristicin is a synergist o f xanthotoxin in both the presence and absence o f near U V light. Phytosynergists may further e n h a n c e the effectiveness o f phototoxins against nocturnal feeders by e x t e n d i n g the length o f t i m e a p h o t o t o x i n remains in an insect. This could prev e n t elimination o f a p h o t o t o x i n prior to the next photophase. Little is k n o w n about the extent to which phytosynergists o c c u r in the plant k i n g d o m . W h i l e M D P c o m p o u n d s are widespread, most h a v e not been tested as inhibitors o f M F O s . T h e e c o l o g i c a l advantages to phytosynergists are as yet undefined. Possible benefits include a reduction o f the e n e r g y expenditure for defense or establishing a m o r e stable d e f e n s e ( B e r e n b a u m and N e a l , 1986). Such properties w o u l d be desirable in e c o n o m i c a l l y important crops where phytosynergists may h a v e practical benefits. Acknowledgments--Thanks to Dr. May Berenbaum for support, advice, and comments; Drs. David Seigler and Stanley Friedman for advice and comments; Ellen Heininger, Keywan Lee, Robert Marquis, James Nitao, Steven Passoa, Sherri Sandburg, and Arthur Zangerl for comments on the text; and Dr. Gilbert Waldbauer, Dr. Randy Cohen, and Nathan Schiff for Heliothis zea larvae. This research was supported by grants from the National Science Foundation (NSF BSR 8351407), and the Exxon Education Foundation to Dr. May Berenbaum.
PHYTOSYNERG1STS OF XANTHOTOXIN
3 15 REFERENCES
BERENBAUM,M. 1978. Toxicity of a furanocoumarin to armyworms: A case of biosynthetic escape from herbivores. Science 201:532-534. BERENBAUM, M., and NEAL, J.J. 1985. Synergism between myristicin and xanthotoxin, a naturally cooccurring plant toxicant. J. Chem. Ecol. 11 : 1349-1358. BERENBAUM, M., and NEAL, J.J. 1986. Interactions among allelochemicals and insect resistance in crop plants, pp. 416-430, b~ G.R. Waller (ed.). Allelochemicals: Role in Agriculture and Forestry. American Chemical Society Symposium Series No. 330 Washington, D.C. BULL, D.L., IVIE, G.W., BEIER, R.C., PRYOR, N.W., and OERTL[, E.H. 1984. Fate of photosensitizing furanocoumarins in tolerant and sensitive insects. J. Chem. Ecol. 10:893-911. BULL, D.L., IVIE, G.W., BEIER, R.C., and PRYOR, N.W., 1986. In vitro metabolism of a linear furanocoumarin (8-methoxypsoralen, xanthotoxin) by mixed-function oxidases of larvae of the black swallowtail butterfly and the fall armyworm. J. Chem. Ecol. 12:885-892. CESKA, O., CHAUDHARY,S.K., WARRINGTON,P.J., and ASHWOOD-SMITH,M.J. 1987. Photoactive furanocoumarins in fruits of some umbellifers. Phytochemist O' 26:165-169. CHANG, P.T.O. 1976. Phytochemical studies on Helietta parvifolia. Doctoral dissertation. University of Illinois Medical Center, Chicago, Illinois. HARBORNE, J.B. 1971. Flavonoid and phenylpropanoid pattern in the Umbelliferae, pp. 293-314, in V.H. Heywood (ed.). Biology and Chemistry of the Umbelliferae. Academic Press, London. HEGNAUER, R. 1973. Chemotaxonomie der Pflanzen, Band 6. Birkhauser-Verlag, Basel. Pages 174-239; 554-629. HODGSON, E. 1985. Microsomal mono-oxygenases, pp. 225-321. in G.A. Kerkut and L.I. Gilbert (eds.). Comprehensive Insect Physiology, Biochemistry and Pharmacology. Pergammon Press, New York. HODGSON, E., and PH]LPOT, R.M. 1974. Interaction of methylenedioxyphenyl (l,3-benzodioxole) compounds with enzymes and their effect on mammals. Drug Metab. Rev. 3:231-301. HODISAN,V., POPESCUH., and FAGARASAN,E. 1980. Studies on Anethunr graveolens. 11. Chemical composition of essential oil from fruits. Contrib. Bot., Univ, Babes-Bolyai Cluj, Gradina Bot. pp. 263-266; Chem. Abstr. 94:136149d (1981). KRIEGER, R.I., FEENY, P.P., and WILKINSON, C.F. 1971. Detoxication enzymes in the guts of caterpillars: An evolutionary answer to plant defenses? Science 172:579-581. LAFORGE, F.B., and BARTHEL, W.F. 1944. A new constituent isolated from southern prickly-ash bark. J. Org. Chem. 9:250-253. LICHTENSTEIN, E.P., and CASIDA, J.E. 1963. Myristicin, an insecticide and synergist occurring naturally in the edible parts of parsnip. J. Agric. Food Chem. I 1:410-415. MURRAY, R.D.H., MENDEZ, J., and BROWN, S.A. 1982. The Natural Coumarins. John Wiley & Sons, New York. SAS INSTITUTEINC. 1982. SAS User's Guide: Statistics. SAS Institute lnc., Cary, North Carolina. Pages 287-292. SHULG]N, A.T. 1966. Possible implication of myristicin as a psychotropic substance. Nature 210:380-384. THOMS, H. 1911. 13bet die Konstitution des Xanthotoxins und seine Beziehungen zum Bergapten. Chem. Ber. 44:3325-3336. THOMS, H., and THOMEN, F. 191 i. l~/ber das Fagaramide, einen neuen stickstoffhattingen Stoff aus der Wurzelrinde yon Fagara xanthoxyloides Lain. Chem. Ber. 44:3717-3730.
MYRISTICIN, SAFROLE, A N D F A G A R A M I D E AS PHYTOSYNERGISTS OF X A N T H O T O X I N l
J.J. N E A L 2 Department of Entomology University of Illinois Urbana, Illinois 61801
(Received August 5, 1987; accepted November 24, 1987) Abstract--The methylenedioxyphenyl-containing (MDP) inhibitors of mixedfunction oxidase detoxification enzymes, myristicin, safrole, fagaramide, and isosafrole, occur with xanthotoxin or other toxic furanocoumarins in plants of the families Umbelliferae and Rutaceae. All four MDP compounds have a synergistic effect on the toxicity of xanthotoxin to Heliothis zea. Myristicin also increased the phototoxicity of xanthotoxin in the presence of UV light. The term phytosynergist is used to describe plant compounds that are present at concentrations producing no toxic effect by themselves but have a synergistic effect on cooccurring toxins. Key Words--Xanthotoxin, Heliothis zea, Lepidoptera, Noctuidae, myristicin, fagaramide, safrole, isosafrole, synergism, phytosynergist, phototoxicity.
INTRODUCTION The o c c u r r e n c e o f insecticide synergists in plants has led to speculation that their natural function m a y be s y n e r g i s m o f c o o c c u r r i n g toxins ( L a F o r g e and Barthel, 1944; K r i e g e r et al., 1971). This v i e w is supported by e x p e r i m e n t s in which the insecticide synergist myristicin (Figure 1), at levels present in plants, increased the mortality o f first-instar H e l i o t h i s z e a caterpillars fed xanthotoxin (Figure 1), a toxic f u r a n o c o u m a r i n o c c u r r i n g with myristicin in m a n y plants o f ~Taken in part from a thesis submitted to the Graduate College of the University of Illinois at Urbana-Champaign in partial fulfillment of the requirements for the degree of Doctor of Philosophy, June 1987. 2Present address: Purdue University, Department of Entomology, Entomology Hall, West Lafayette, Indiana 47907. 309 0098-0331/89/0100-0309506.00/0 © 1989 Plenum Publishing Coq~mtion
310
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oAo 0
o..~
o-.,.,,,.~o
o--/\ CH3
)
CH~
oAo
/
CHz
III
NH
I
oAo
i/ CH2
IV
oAo
// ell2
V
FIG. 1. Xanthotoxin and plant compounds with a synergistic effect on its toxicity: xanthotoxin (I), fagaramide (II), isosafrole (III), myristicin (IV), and safrole (V).
the family Umbelliferae (Berenbaum and Neal, 1985). In this paper, compounds such as myristicin that are present at concentrations producing little or no toxic effect by themselves but which increase the toxicity of cooccurring toxins will be called phytosynergists. Myristicin, like many other compounds containing a methylenedioxyphenyl (MDP) substituent, is an inhibitor of mixed-function oxidases (MFOs) (Hodgson and Philpot, 1974). MFOs are detoxification enzymes that metabolize many xenobiotics (Hodgson, 1985) and are primarily responsible for the metabolism of xanthotoxin in at least two species of insects (Bull et al., 1984, 1986). The synergistic effect of myristicin on the toxicity of xanthotoxin to Heliothis zea is attributed to its inhibition of xanthotoxin metabolism, which increases the effective xanthotoxin concentration in the insect (Berenbaum and Neal, 1985). Xanthotoxin has two types of toxic action on insects, a " d a r k " toxicity (toxicity in the absence of UV-A light) and phototoxicity (increased toxicity in the presence of UV-A light) (Berenbaum, 1978), which act through different mechanisms (Murray et al., 1982). Both mechanisms are likely to be important under natural conditions; however, only the synergistic effect of myristicin on the dark toxicity of xanthotoxin has been measured (Berenbaum and Neal, 1985). If myristicin works by inhibiting the detoxification of xanthotoxin, however, its activity should be retained under a variety of environmental conditions and be independent of the mode of action of the toxin. Thus, myristicin should
PHYTOSYNERGISTS OF XANTHOTOXIN
3]1
also be a phytosynergist of xanthotoxin phototoxicity in the presence of UV-A light. Myristicin is not the only MDP-containing insecticide synergist that occurs with xanthotoxin in plant species. Phytochemical records show that safrole and apiole also occur with xanthotoxin in the Umbelliferae (Ceska et al., 1987; Hodisan et al., 1980; Murray et al., 1982; Hegnauer, 1973; Harborne, 1971), and fagaramide, asarinin, and sesamin occur with xanthotoxin in plants of the family Rutaceae (Thorns, 1911; Thoms and Thfimen, 1911; Hegnauer, 1973; Murray et al., 1982). Some rutaceous plants also contain safrole and isosafrole along with other toxic furanocoumarins (Chang, 1976; Hegnauer, 1973; Murray et al., 1982). Thus, myristicin is probably not the only plant compound to function naturally as a phytosynergist of xanthotoxin, and there are many toxins other than xanthotoxin that may occur with pbytosynergists. Three insecticide synergists that are MFO inhibitors, fagaramide, safrole, and isosafrole (Figure 1), were tested as synergists of xanthotoxin toxicity to Heliothis zea. In addition, the ability of phytosynergists to maintain activity independent of the mode of action of the toxin was studied by measuring the synergistic effect of myristicin on xanthotoxin in the presence as well as in the absence of UV-A light.
METHODS AND MATERIALS
Chemicals. Myristicin was purchased from Saber Labs (Morton Grove, Illinois); fagaramide, safrole, and isosafrole from Aldrich Chemical Co. (Milwaukee, Wisconsin); and xanthotoxin from Sigma Chemical Co. (St. Louis, Missouri). Bioassays. Neonates of the corn earworm, Heliothis zea (Lepidoptera; Noctuidae), from a culture maintained by Dr. G. Waldbauer at the University of Illinois, were used for testing the oral toxicities of all allelochemicals and combinations of allelochemicals. H. zea was chosen for study because it is a generalist feeder, it is susceptible to the toxic effects of xanthotoxin, and because myristicin has a synergistic effect on xanthotoxin toxicity to this insect (Berenbaum and Neal, 1985). Chemicals were administered by incorporation into an artificial diet (Berenbaum and Neal, 1985) in which wheat germ is the only undefined ingredient. In order to ensure uniformity, xanthotoxin (dissolved in acetone) was mixed with the alphacel component of the diet and dried prior to its incorporation into the diet. Myristicin, fagaramide, safrole, and isosafrole were added to hot diet in acetone solution (1% of the total wet weight) prior to gelling. Neonates (30 per treatment) were placed individually on 200 mg diet in a 1.5-ml polypropylene test tube (Biorad, Richmond, California) and reared in a
312
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25°C incubator under cool, white, fluorescent lights (16 hr light-8 hr dark). When screened by the polypropylene tube, these lights delivered less than 0.2 mW/m 2 of either UV-A (290-400 nm) or UV-B (200-290 nm) light as measured by a UVX radiometer with UVX 36 and UVX 31 sensors (UVP Inc., San Gabriel, California), Caterpillars were monitored until they died or molted to second instar. Toxicity of the MDP compounds was first measured at 1000 ppm (weight/wet weight diet); concentrations at and above 1000 ppm have been recorded for some of the MDP compounds in plants (Lichtenstein and Casida, 1963; Shulgin, 1966). For compounds toxic at 1000 ppm, lower concentrations were tested until a no-effect level was found. To test for synergism, the toxicity of xanthotoxin alone was compared to its toxicity in the presence of MDP compounds. LCso values were determined from log dose/probit mortality plots (SAS Institute Inc., 1982). LCso values were considered significantly different if the 95% confidence intervals did not overlap. Synergistic ratios (SR) were calculated as the LC~0 of the toxin divided by the LCso of the toxin with synergist. For measuring the effects of myristicin-xanthotoxin combinations on H. z e a in the presence and absence of UV-A light at simulated natural levels, larvae were reared in 50-mm x 5-mm internal diameter Pyrex test tubes stoppered with a cotton applicator stick. Pyrex transmits solar UV wavelengths (290-400 nm) but eliminates nonsolar UV wavelengths. Natural UV-A light intensities were simulated using two Sylvania BLB bulbs that produced between 1700 and 1950 mW/m 2 UV-A light and between 0.4 and 0.7 mW/m 2 UV-B at the surface of the tubes as measured with a UVX radiometer; a 16 hr light-8 hr dark photoperiod was used. For the no-UV-light treatments, filters opaque to wavelengths less than 400 nm were used to exclude UV light. Toxicity was evaluated as described above.
RESULTS In the absence of xanthotoxin, neither myristicin nor fagaramide Showed any toxicity at the highest dose tested. Both safrole and isosafrole were toxic at 1000 ppm, each killing 33% of the first-instar caterpillars. Safrole killed 10% at 700 ppm and was nontoxic at 400 ppm. Isosafrole killed 4 % at 400 ppm and was nontoxic at 100 ppm. Therefore, in tests for synergistic effects on xanthotoxin, myristicin and fagaramide were used at 1000 ppm, safrole at 400 ppm, and isosafrole at 100 ppm. Xanthotoxin has an LCso of 21,000 ppm (Table 1). The LCso values for combinations of xanthotoxin with myristicin, fagaramide, safrole, and isosafrole are 3200 ppm, 8300 ppm, 5900 ppm, and 8500 ppm, respectively. These values are all significantly different from the LCso of unsynergized xanthotoxin. Synergistic ratios are 6.5, 2.5, 3.5, and 2.5 for myristicin, fagaramide, safrole,
PHYTOSYNERG[STS OF XANTHOTOXIN
3 13
TABLE 1. SYNERGISM OF TOXICITY OF XANTHOTOXIN TO FIRST-INSTAR Heliothis zea BY MYRISTICIN, FAGARAMIDE, SAFROLE, AND ISOSAFROLE
Mortality (%)" Xanthotoxin (ppm in diet) 0 1000 2000 5000 10000 15000 20000 LCso 95% CL/' SR'
Control 0 NT 3 13 20 30 47 21,000 17,000-29,000
1000 ppm rnyristicin
1000 ppm fagaramide
0 23 43 67 97 NT NT 3,200 2,200-4,200 6.5
0 10 13 17 67 NT NT 8.300 6,800-11,000 2.5
400 ppm safrole 0 l0 37 40 77 NT NT 5,900 4,600-7,800 3.5
100 ppm isosafrole 0 20 13 37 57 NT NT 8,500 6,500-13,000 2.5
"N >- 30. NT = not tested. 1'95% CL = 95% confidence limit; LCso values are significantly different (P < 0.05) if the 95% confidence intervals do not overlap. 'SR = synergistic ratio.
and isosafrole, respectively (Table 1) but are not directly comparable because of differences in the dosages. In the presence of UV light, myristicin significantly increases the LCso of xanthotoxin 3.6-fold (Table 2). UV light also has a significant effect on the toxicity of xanthotoxin-myristicin combinations, increasing the LCs0 2.7-fold (Table 2).
DISCUSSION
Fagaramide, safrole, and myristicin all fit the definition of a phytosynergist; they have a synergistic effect on a cooccurring toxin at levels that are nontoxic. Isosafrole, which is also a synergist of xanthotoxin, may be a phytosynergist for other cooccurring toxins. These compounds do not necessarily act exclusively as phytosynergists in all plants that produce them. For example, levels of myristicin in some plant parts (Shulgin, 1966) have an insecticidal effect in some insects (Lichtenstein and Casida, 1963). However, insecticidal concentrations of these compounds are rare in plants compared to sublethal concentrations. The occurrence of these compounds at sublethal concentrations suggests a primary role as a phytosynergist.
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TABLE 2. SYNERGISMOF XANTHOTOXINTOXICITYTO FIRST-INSTARtteliothis zea BY COMBINATIONSOF MYRISTICIN (1000 ppm) AND UV-A LIGHT Mortality (%)" Xanthotoxin (ppm in diet)
No myristicin, UV-A light NT 17 40 43 50
100
250 500 1000 2500 LC.so 95 % CL
2,200 1300-15,000
Myristicin, no UV-A light
Myristicin with UV-A light
NT 10 20 47 70
30 47 57 63 70
1,600 1,300-2,100
600 0-1,300 Synergistic ratios
No myristicin/myristicin (UV-A present) No UV-A/UV-A (myristicin present)
3.6 2.7
"N >_ 30~ N T = not tested,
J'95 % CL = 95% confidence limit. LCso values are significantly different (P < 0.05) if the 95% confidence intervals do not overlap.
B e r e n b a u m and Neal (1985) p r o p o s e d that myristicin acts as an inhibitor o f xanthotoxin detoxification and thereby increases the effective concentration o f xanthotoxin in the insect. As predicted, myristicin is a synergist o f xanthotoxin in both the presence and absence o f near U V light. Phytosynergists may further e n h a n c e the effectiveness o f phototoxins against nocturnal feeders by e x t e n d i n g the length o f t i m e a p h o t o t o x i n remains in an insect. This could prev e n t elimination o f a p h o t o t o x i n prior to the next photophase. Little is k n o w n about the extent to which phytosynergists o c c u r in the plant k i n g d o m . W h i l e M D P c o m p o u n d s are widespread, most h a v e not been tested as inhibitors o f M F O s . T h e e c o l o g i c a l advantages to phytosynergists are as yet undefined. Possible benefits include a reduction o f the e n e r g y expenditure for defense or establishing a m o r e stable d e f e n s e ( B e r e n b a u m and N e a l , 1986). Such properties w o u l d be desirable in e c o n o m i c a l l y important crops where phytosynergists may h a v e practical benefits. Acknowledgments--Thanks to Dr. May Berenbaum for support, advice, and comments; Drs. David Seigler and Stanley Friedman for advice and comments; Ellen Heininger, Keywan Lee, Robert Marquis, James Nitao, Steven Passoa, Sherri Sandburg, and Arthur Zangerl for comments on the text; and Dr. Gilbert Waldbauer, Dr. Randy Cohen, and Nathan Schiff for Heliothis zea larvae. This research was supported by grants from the National Science Foundation (NSF BSR 8351407), and the Exxon Education Foundation to Dr. May Berenbaum.
PHYTOSYNERG1STS OF XANTHOTOXIN
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