Plant-insect coevolution and inhibition of acetylcholinesterase ...

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Journal of Chemical Ecology, Vol. 14, No. lO, 1988

PLANT-INSECT COEVOLUTION AND INHIBITION OF ACETYLCHOLINESTERASE

M.F.

RYAN

and O O N A G H

BYRNE

Department of Zoology University College Dublin Belfield, Dublin 4, Ireland (Received September 15, 1987; accepted March 15, 1988) Abstract--The theory of plant-insect coevolution provides for diffuse coevolution and the expectation that plants evolve broad-spectrum chemical defenses with which some insects coevolve by detoxifying and using the compounds as host-location cues. Specific biochemical modes of action have been assigned to relatively few such defense chemicals and one major class, the terpenoids, is investigated here. Six terpenoids inhibited the enzyme acetylcholinesterase (derived from electric eel) and elicited the appropriate in vivo effects of insect paralysis and mortality. The diterpene gossypol was a reversible uncompetitive inhibitor. Five monoterpenes, representing a range of functional groups, were reversible competitive inhibitors apparently occupying at least the hydrophobic site of the enzyme's active center. Such data suggest the involvement of acetylcholinesterase in the coevolved insect response to terpenoids. Key Words-Herbivory, pheromones, chemical defense, monoterpenes and diterpenes, insect paralysis and mortality, enzyme evolution, coevolution, acetylcholinesterase inhibition, plant-insect coevolution.

INTRODUCTION

T h e study o f c o e v o l u t i o n has g e n e r a t e d m u c h interest since Ehrlich and R a v e n c o i n e d the t e r m with the statement " o n e a p p r o a c h to what w e w o u l d like to call c o e v o l u t i o n is the e x a m i n a t i o n o f patterns o f interactions b e t w e e n two m a j o r groups o f o r g a n i s m s with a close and e v i d e n t e c o l o g i c a l relationship, such as plants and h e r b i v o r e s " (Ehrlich and R a v e n , 1964). A l t h o u g h a key influence on p r e s e n t - d a y studies o f c o e v o l u t i o n , Ehrlich and R a v e n w e r e not the first to r e c o g n i z e its existence. D a r w i n was aware o f the special relationships b e t w e e n 1965 0098-0331/88/1000-1965506.00/0 9 1988 Plenum Publishing Corporation

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insects and the plants they pollinated: " . . . thus can I understand how a flower and a bee might slowly become simultaneously or one after another, modified and adapted in the most perfect manner t o . . . each other" (Darwin, 1859). A comprehensive review of contributions subsequent to Darwin's would be rather inappropriate here, but the following seem especially significant. Janzen (1980) defined coevolution as when "an evolutionary change in a trait of the individuals of one population is followed by an evolutionary response by a second population to the change in the first." By this definition, the evolution of each trait is due to the others, a reciprocal condition that views plantinsect interactions as one to one. A more general definition is that coevolution has occurred when the interaction between two or more ecologically interacting species involves an adaptive response to genetic change in the other(s) (Futuyama, 1983). This is also less restrictive, allowing for the interaction of several and not specifically two species. Known as diffuse coevolution (Janzen, 1980), this occurs where many plants have evolved chemical and physical defenses against a diverse group of insects and also where many insects have acquired the ability to detoxify a wide range of plant chemicals (Futuyama, 1983). One consequence of the theory of diffuse coevolution is the expectation that plants would select a broad spectrum of defense, i.e., one or a few plant chemicals that would be antagonistic to a wide range of herbivorous insects. This is an economical measure, as the production of secondary chemicals is at the expense of other areas of metabolism. For example, tobacco plants especially rich in alkaloids are stunted, suggesting that energy allocated to the manufacture of nicotine has been abstracted from energy available for growth (Whittaker and Feeny, 1971). Also it would be metabolically impractical for any plant to evolve a new chemical against every insect species attacking it. Accordingly, some secondary plant chemicals should act as broad-spectrum insecticides, and indeed some have been used as such for centuries. The dried flower heads of Crysanthemum cinerariaefolium (Compositae) and C. coccineum contain pyrethrins, potent insecticides with a rapid knockdown action. A chemical class conspicuous among plant secondary compounds and containing chemicals inimical to insects are the terpenoids (Mabry and Gill, 1979). The cyclic monoterpene pulegone, an irritant commonly found in mint oils, deters feeding by the slug, Ariolimax dolichophallus (Rice) and by the fall army worm, Spodoptera frugiperda (Smith) and repels the German cockroach, Blattella germanica (L.) (Gunderson et al., 1985). Added to diet, it killed the larva of the fall armyworm and decreased pupation by the southern armyworm (Brattsten, 1983). Resistance of western red cedar wood, Thuja plicata (D.), to insect attack is attributed to the presence of monoterpenes, of which methyl thujate is toxic to larvae of the black carpet beetle, the furniture beetle, and the case-making beetle (Becker, 1963). Two other monoterpenes produced by westem red cedar, ~3-thujaplicin and -y-thujaplicin, are both highly insecticidal against

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the larva of the old-house borer, Hylotrupes bajulus (Cerambycidae), a common pest of structural timber in Europe (Becker, 1963). The diterpene gossypol, an abundant diterpene in the lysigenous glands of cotton, decreases weight gain in larvae of the tobacco budworm and pink bollworm (Hedin et al., 1983) (for many other examples of terpenoid-insect interactions, see Mabry and Gill, 1979). However, specific biochemical modes of action have been assigned to few such compounds. A common structural feature of terpenoids is their hydrocarbon skeleton, which in turn confers upon them a common property, hydrophobicity. Many hydrophobic compounds are associated with protein deactivation and enzyme inhibition, and one enzyme particularly susceptible to hydrophobic interactions is acetylcholinesterase (Hansch and Deutsch, 1966), present in the neuromuscular junction. This enzyme also functions in the peripheral sensory nervous system of the insect antenna (Sanes et al., 1977). The present report quantifies the toxicity of a range of plant terpenoids to a nonadapted insect and provides what seems to be the first evidence that they are reversible inhibitors of acetylcholinesterase.

METHODS

AND MATERIALS

Compounds that inhibit or inactivate acetylcholinesterase (ACHE), cause acetylcholine to accumulate at the cholinergic site. This produces continuous stimulation of cholinergic nerve fibers throughout the central and peripheral nervous system, followed by paralysis and death (Corbett et al., 1984). Accordingly, we used an in vivo assay established as appropriate to quantify insect mortality and an in vitro assay for AChE inhibition. The following five monoterpenes, representing a range of functional groups, were selected for investigation: citral (aldehyde), pulegone (ketone), linalool (alcohol), (-)-bornyl acetate (ester), and cineole (ether); for comparison a single diterpene gossypol (alcohol) was included (Figure 1). As these monoterpenes are characteristic constituents of plant leaves and as gossypol is associated with pigment glands of cotton seed, the test insect, TriboIium castaneum, a pest of stored grain, may be viewed as a nonadapted species. The insecticide-susceptible Tribolium colony was derived from stocks originally supplie.d by the University of Chicago. Insect Mortality. Mortality was assessed by an F.A.O. contact method devised to measure the resistance of agricultural insect pests to insecticides (Anon., 1970) with the modification that acetone served as solvent in place of Risella oil. Usually four concentrations of each test compound were used within a range established by previous experiment as eliciting mortality (for specific concentrations see Figure 2). There were two replicates of each treatment and

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/~~COOCH3

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FIG. 1. Configurations of the five monoterpenes and the diterpene (gossypol) selected for investigation: citral is a mixture of geranial (a) and neral (b).

of the control, i.e., solvent only. Solutions were pipetted in 0.5-ml aliquots onto filter papers (5.5 cm diam.) that were allowed to dry (approx. 1 min). Then batches of 20 beetles, prestarved for 24 hr, were transferred to each treated paper where they were confined by plastic arenas, sealed on top by glass plates, that were placed in an incubator (28~ Mortality was estimated from knockdown, i.e., the inability of the insect, after 5 hr in the arena, to stand or walk after a gentle push forwards with a forceps. Correction for control mortality was made using the Abbott formula (Abbott, 1925), PT = [(Po - Pc)/100 Pc] X 100, where PT is corrected percentage mortality, Po is observed mortality, and Pc is control mortality. LCso values (concentrations eliciting 50 % mortality), were derived by interpolation from lines representing dosage-mortality trends drawn on logarithmic probability paper; such lines were constructed using the regression formula for best fit.

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RYAN AND BYRNE

Enzyme Assays. Inhibition of AChE was assessed by the Ellman (1961) assay. Monoterpenes were dissolved in 5 ml absolute alcohol and made up to 50 ml in 0.1 M phosphate buffer, pH 8, to give a stock solution of 0.09 M. Gossypol is a paste at 0.09 M so the stock solution was 0.009 M. Serial dilution yielded the assay concentrations With the result that the concentration of ethanol never exceeded 2 %, which we experimentally assessed as inhibiting the enzyme by less than 4% or negligible. The substrate was acetylthiocholine iodide and the color reagent was 5,5'-dithiobis-2-nitrobenzoic acid (DTNB). Each assay was duplicated, and a typical assay mixture contained 40 /~1 substrate, 20 #1 DTNB, 1 ml terpene solution, and 100 ~1 electric eel AChE (0.5 unit). Hydrolysis was measured at 25~ in a Pye Unicam SP8-100 spectrophotometer at 412 nm, and all test and control assays were corrected by blanks for nonenzymic hydrolysis. Chemicals. Citral (99 % pure), pulegone (85 %), linalool (99 %), (-)-bornyl acetate (97%), and cineole (99%) were supplied by Aldrich, Gillingham, Dorset, England. Gossypol, AChE (c-3389, electric eel), DTNB, and acetylthiocholine iodide were supplied by Sigma, Poole, Dorset, England.

RESULTS AND DISCUSSION

All six terpenes killed T. castaneum, and this mortality was dose-responsive (Figure 2). Pulegone was the most potent (LCs0, 0.12 x 104 ppm) followed by gossypol (0.45 x 104), citral (1.5 x 104), linalool (2.5 x 104), (-)-bornyl acetate (2.7 x 104), and cineole (4.3 x 104). It was evident that beetles became paralysed prior to death. All six terpenoids reversibly inhibited ACHE. Gossypol was an uncompetitive inhibitor, as indicated by decreasing inhibition associated with decreasing substrate concentrations and by the parallelism of the Dixon plot (Figure 3). The five monoterpenes were competitive inhibitors, as indicated by increasing inhibition associated with decreasing substrate concentration and by the intersections in the Dixon plots (Figure 3). Inhibition constants (Ki) were: cineole 2.5 x 10 -2 mM; pulegone 8.5 x 10 -~ mM; gossypol 1.5 raM; linalool 5.5 mM; citral 7.0 mM, and (-)-bornyl acetate 21.3 mM. The present evidence from both in vivo and in vitro experiments consistently indicates that the terpenoids tested are effective inhibitors of acetylcholinesterase: specifically, they paralyzed and killed a nonadapted insect and also inhibited electric eel ACHE. There is little doubt that terpenoids may act as plant defense compounds or allomones, but this seems to be the first demonstration that a relevant biochemical-physiological mechanism is inhibition of cholinesterase. As the five monoterpenes tested represent five distinct functional groups, such inhibition may be a widespread property of monoterpenes.

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FIG. 3. Dixon plots derived from the inhibition of acetylcholinesterase by the six terpenoids. In each plot the concentrations of substrate (acetylthiocholine iodide) are 0.066 mM (O - - O) and 0.5 mM (11 - - i ) . The additional substrate concentration tor gossypol is 0.2 mM (A - - &).

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RYAN AND BYRNE

An apparent anomaly is that the order of potency for the compounds differs according to whether mortality or inhibition is assayed; for example, pulegone, the most potent as an insecticide, ranks second as an AChE inhibitor. It is relevant that vertebrate and insect AChE may differ in their susceptibility to insecticides (Hollingworth et al., 1967). Also it is widely recognized that in vitro Ki values are a rather poor indicator of insecticidal potencies, as they cannot reflect differential abilities of compounds to penetrate cuticle and gut barriers or their differential solubilities in insect hemolymph: each factor influences migration rates to the cholinergic sites (Corbett et al., 1984). For example, three carbamates gave Ki values against housefly brain AChE of 4 x 10 - 4 mM, 2 x 10 -I mM and 5 • 10 -2 mM, respectively, representing a 500-fold range; their potencies against housefly using topical application were 50, 500, and 100/xg/g, respectively, or a 10-fold range (Kolbezen et al., 1954). Another study ascertained no correlation between LDs0 values of carbamate insecticides and in vitro Ki values with honeybee AChE (Abdel-Raof et al., 1977). Accordingly, Ki values are taken here as adequate to establish compounds as AChE inhibitors but without the expectation of a direct relationship with toxicity. Nevertheless, it is noteworthy that m-nitrophenyl-N-methylcarbamate has a Ki value of 2.0 • 10 -1 mM (Kolbezen et al., 1954) as compared with 8.5 x 10 -~ mM for pulegone. Also, pulegone's LCso potency is 0.12 • 10 4 ppm as compared with LCso values ranging from 0.007 to 0.027 • 10 4 ppm for the commercial insecticide and cholinesterase inhibitor, malathion, using the same assay and various susceptible strains of T. c a s t a n e u m (Champ and CampbellBrown, 1970). Pulegone's potency, 4.4- to 17.1-fold less than this insecticide's, clearly indicates that its action as an AChE inhibitor is relevant to its function as a plant defense compound; the same rationale may be applied to the other terpenoids tested. Gossypol is an uncompetitive inhibitor of ACHE, i.e., it binds not to the enzyme but to the enzyme-substrate complex thus preventing product formation. The five monoterpenes, however, are competitive inhibitors, i.e., they compete with the substrate for its active center on the enzyme. The interaction of the substrate (acetylcholine) with AChE's active center is usually represented as involving three subsites, the anionic, esteratic, and hydrophobic sites (Figure 4). As none of the five monoterpenes possesses a charged region and only one possesses a carbonyl group, this suggests by elimination that they take effect mainly by hydrophobic binding. Their configuration reinforces this, as it is consistent with a good fit to the enzyme's hydrophobic site (Figure 4). The in vivo insecticidal activity of various methyl carbamate and organophosphate insecticides is closely correlated with the extent of their hydrophobic bindings to AChE (Hansch and Deutsch, 1966). Despite the ability of these terpenoids to inhibit cholinesterase and kill a nonadapted insect, they are not deleterious to all insect species. Specifically,

1973

PLANT INSECT COEVOLUT1ON

0

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FIG. 4. Diagrammatic representation of the active center of acetylcholinesterase (ACHE) indicating the anionic, hydrophobic, and esteratic sites together with the structural formula of acetylcholine (ACh). At bottom is a representation of ACh hydrolysis involving the carbonyl group of ACh and the enzyme's esteratic site: OH represents a hydroxyl group on a serine residue and HA represents a hydrogen donor (after Corbett et al., 1984). As competitive inhibitors, all five monoterpenes must bind to the enzyme's active center. At top is the configuration of pulegone with two CH groups designated to indicate the precise fit possible with the enzyme's hydrophobic site: the structures of the other four monoterpenes are also consistent with such binding.

linalool, a constituent o f pine, oils o f Ceylon, cinnamon, sassafras, etc., is an attractant for the silkworm (Brattsten, 1983). ( - ) - B o r n y l acetate is a host-location cue for the carrot fly larva, and it seems particularly relevant that strong and statistically significant attraction was elicited by a concentration on filter paper of 9.8 • 104 ppm (Ryan and Guerin, 1982); this concentration was associated with 97 % mortality o f Tribolium. Thus, the present data are not assignable to concentration effects. ( + ) - B o r n y l acetate is a mimic of the sex

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RYANANDBYRNE

pheromone of the American cockroach (Periplanetaamericana) (Manabe and Nishino, 1983). Citral, a constituent of citrus fruits, is also an alarm and defense pheromone of ants but an attractant and assembly pheromone for the honeybee worker (Wilson, (1971). The foregoing effects are consistent with the principles of diffuse coevolution but the compounds probably were detoxified by recipients before use as attractants. Detoxifying enzymes of insects belong to the general categories of oxidases, hydrolases, transferases, and reductases (Ahmad et al., 1986). Insect success in detoxifying cholinergic inhibitors, as evidenced by strains with developed resistance to organophosphate and carbamate insecticides, is assigned to specific enzymes. One is a modified carboxyesterase that apparently lost most of its esterase activity, acquiring phosphatase activity instead (Sawicki, 1973); another is gluthathione-S-transferase (Lewis, 1969). In addition, AChE has evolved in resistant strains to be more slowly inhibited than AChE in susceptible ones: 1.2- to 6-fold less in organophosphate-resistant strains and 17- to 1570-fold less in carbamate-resistant strains (Hemingway and Georghiou, 1983). The process by which detoxified terpenoids subsequently become attractants is not easy to visualize, but the involvement of AChE in antennal sensory transmission seems a further reason for considering a role for this enzyme in plant-insect coevolution as mediated by terpenoids.

A c k n o w l e d g m e n t s - I s is a pleasure to thank Michael A. Raftery, California Institute of Technology, Pasadena, and E. O. Wilson, Harvard University, for kind and stimulating sabbatical hospitality to M F R in 1982 and 1986, respectively.

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CHAMP, B.R., and CAMPBELL-BROWN, M.J. 1970. Insecticide resistance in Australian TriboIium castaneum (Herbst) 1. A test method for detecting insecticide resistance. J. Stored Prod. Res. 6:53-70. CORBETT, J.R., WRIGHT, R., and BAILLIE, A.C. 1984. The Biochemical Mode of Action of Pesticides, 2nd ed. Academic Press, New York. DARWIN, C. 1859. On the Origin of Species. John Murray, London. EHRLICrf, P.R., and RAVEN, P.H. 1964. Butterflies and plants: A study in coevolution. Evolution. 18:586-608. ELLMAN,G.L., COURTNEY,K.D., ANDRES,V., JR., and FEATHERSTONE, R.M. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7:8895. FUTUYAMA, D.J. 1983. Evolutionary interactions among herbivorous insects and plants, pp. 20723l, in D.J. Futuyama and M. Slatkin (eds.). Coevolution. Sinauer Associates, Sunderland, Massachusetts. GUNDERSON, C.A,, SAMUELIAN,J.H. EVANS, C.K., and BRATTSTEN,L.B. 1985. Effects of the mint monoterpene, pulegone, on Spodoptera eridania (Lepidoptera: Noctuidae). Environ. Entomol. 14:859-862. HANSCH, C., and DEUTSCH, E.Q. 1966. The use of substituent constants in the study of structureactivity relationships in cholinesterase inhibitors. Biochim. Biophys. Acta 126:117-128. HEDIN, P.A., JENKINS, J.N., COLLUM, D.H., WHITE, W.H., and PARROTT, W.L. 1983. Multiple factors in cotton contributing to resistance to the tobacco budwol-m, Heliothis virescens F., pp. 347-365, in P.A. Hedin (ed.). Plant Resistance to Insects, Syrup. Ser. No. 208. American Chemical Society Washington, D.C. HEMINGWAY,J., and GEORGHIOU,G.P. 1983. Studies on the acetylcholinesterase of Anopheles albimanus resistant and susceptible to organophosphate and carbamate insecticides. Pestic. Biochem. Physiol. 19:167-171. HOLLINGWORTH, R.M., FUKVTO, T.R., and METCALF, R.L. 1967. Selectivity of sumithion compared with methylparathion. Influence of structure on anticholinesterase activity. J. Agric. Food Chem. 15:235-24l. JANZEN, D.H. 1980. When is it coevolution? Evolution 34:611-612. KOLBEZEN, M.J., METCALF, R.L., and FUKUTO, T.R. 1954. Insecticidal activity of carbamate cholinesterase inhibitors. J. Agric. Food Chem. 2:864-870. LEWIS, J.B. 1969. Detoxification of Diazinon by subcellular fractions of Diazinon-resistant and susceptible houseflies. Nature 224:917. MABRY, T.J., and GILL, J.E. 1979. Sesquiterpene lactones and other terpenoids, pp. 501-537~ in G.A. Rosenthal and D.H. Janzen (eds.). Herbivores, Their Interaction with Secondary Plant Metabolites. Academic Press, New York. MANABE, S., and NISnINO, C. 1983. Sex pheromonal activity of (+)-bomyl acetate and related compounds to the American cockroach. J. Chem. Ecol. 9:433-448. RYAN, M.F., and GUERIN, P.M. 1982. Behavioural responses of the carrot fly larva, Psila rosae, to carrot root volatiles. Physiol. Entomol. 7:315-324. SANES, J.R., PRESCOTT, D.J., and HILDEBRAND, J.G. 1977. Cholinergic neurochemical development of normal and deafferented antennal lobes during metamorphosis of the moth, Manduca sexta. Brain Res. 119:389-402. SAWICKI,R.M. 1973. Recent advances in the study of the genetics of resistance in the housefly, Musca domestica. Pestic. Sci. 4:501-512. WHITTAKER, RIH., and FEENY, P.P. 1971. Allelochemics: Chemical interactions between species. Science 171:757-770. WILSON, E.O. 1971. The Insect Societies, Harvard University Press, Cambridge, Mass.