Chemical Stimulants of Leaf-Trenching by Cabbage Loopers: Natural Products, Neurotransmitters, Insecticides, and Drugs

P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style ﬁle version June 28th, 2002

Journal of Chemical Ecology, Vol. 29, No. 9, September 2003 (C 2003)

CHEMICAL STIMULANTS OF LEAF-TRENCHING BY CABBAGE LOOPERS: NATURAL PRODUCTS, NEUROTRANSMITTERS, INSECTICIDES, AND DRUGS

DAVID E. DUSSOURD1,

1Department of Biology University of Central Arkansas Conway, Arkansas, USA 72035

(Received December 28, 2002; accepted April 30, 2003)

Abstract—Larvae of the cabbage looper, Trichoplusia ni (Lepidoptera: Noctu- idae), often transect leaves with a narrow trench before eating the distal section. The trench reduces larval exposure to exudates, such as latex, during feeding. Plant species that do not emit exudate, such as Plantago lanceolata, are not trenched. However, if exudate is applied to a looper’s mouth during feeding on P. lanceolata, the larva will often stop and cut a trench. Dissolved chemicals can be similarly applied and tested for effectiveness at triggering trenching. With this assay, I have documented that lactucin from lettuce latex (Lactuca sativa), myristicin from parsley oil (Petroselinum crispum), and lobeline from cardinal flower (Lobelia cardinalis) elicit trenching. These compounds are the first trenching stimulants reported. Several other constituents of lettuce and parsley, including some phenylpropanoids, monoterpenes, and furanocoumarins had little or no activity. Cucurbitacin E glycoside found in cucurbits, another plant family trenched by cabbage loopers, also was inactive. Lactucin, myristicin, and lobeline all affect the nervous system of mammals, with lobeline acting specifically as an antagonist of nicotinic acetylcholine receptors. To determine if cabbage loopers respond selectively to compounds active at acetylcholine synapses, I tested several neurotransmitters, insecticides, and drugs with known neurolog- ical activity, many of which triggered trenching. Active compounds included dopamine, serotonin, the insecticide imidacloprid, and various drugs such as ipratropium, apomorphine, buspirone, and metoclopramide. These results doc- ument that noxious plant chemicals trigger trenching, that loopers respond to different trenching stimulants in different plants, that diverse neuroactive chemicals elicit the behavior, and that feeding deterrents are not all trenching stimulants. The trenching assay offers a novel approach for identifying defensive plant compounds with potential uses in agriculture or medicine. Cabbage loopers in the lab and field routinely trench and feed on plants in the Asteraceae and

To whom correspondence should be addressed. E-mail: [email protected]

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Apiaceae. However, ﬁrst and third instar larvae enclosed on Lobelia cardinalis (Campanulaceae) failed to develop, even though the third instar larvae attempted to trench. Trenching ability does not guarantee effective feeding on plants with canal-borne exudates. Cabbage loopers must not only recognize and respond to trenching stimulants, they must also tolerate exudates during the trenching procedure to disable canalicular defenses.

Key Words—Trichoplusia ni, lactucin, myristicin, lobeline, Lactuca sativa, Petroselinum crispum, Lobelia cardinalis, plant–insect interactions, plant defense, laticifer, insect behavior.

INTRODUCTION

The cabbage looper, Trichoplusia ni Hübner (Noctuidae), has a broad host range that includes over 20 families of plants (Eichlin and Cunningham, 1978; Sutherland and Greene, 1984). Its catholic diet includes numerous crop plants, particularly crucifers and lettuce, on which it is an important pest (Flint, 1987). Loopers are able to feed on such diverse hosts, in part, because of their ability to disable defensive plant canals by cutting a trench. A larva nibbles back and forth across a leaf, thus creating a continuous line of bites that isolates a portion of the leaf on which it feeds. Cabbage loopers are known to cut trenches in composites with latex canals such as lettuce (Asteraceae: Lactuceae), umbellifers with oil ducts such as parsley (Apiaceae), and cucurbits such as cucumber (Cucurbitaceae), which exude drops of sticky phloem sap from damaged tissues (Dussourd and Denno, 1994). Cabbage loopers placed on cardinal flower, Lobelia cardinalis (Campanulaceae), also cut trenches in this latex-bearing plant (Figure 1). Trenches sever secretory canals and, thus, reduce the amount of exudate emitted distal to the trench at the looper’s feeding site (Dussourd, 1999). Diverse plant species that lack exudates are not trenched (Dussourd and Denno, 1994). The goal of this study was to identify cues that cabbage loopers use to determine which plants to trench. Lettuce (Lactuca sativa L.), parsley (Petroselinum crispum), cucumber (Cucumis sativus L.), and cardinal flower (Lobelia cardinalis L.) are classified in three different orders (Angiosperm Phylogeny Group, 1998). They share the presence of canals that emit exudate, but the canals (laticifers, oil ducts, and phloem) differ greatly in anatomy and chemical composition (Fahn, 1979; Metcalfe and Chalk, 1983; Dussourd and Denno, 1991). Lactuca and Lobelia latex and Cucumis sap all become sticky upon exposure to air. Their adhesiveness should be easily detected by feeding caterpillars; Lepidoptera larvae have both mechanoreceptors and chemoreceptors on their maxillae and other mouthparts (Grimes and Neunzig, 1986; Chapman, 1995). However, cabbage loopers exposed to cucumber sap prevented from gelling still trenched, suggesting that another factor triggers the behavior (Dussourd, 1997). In this paper, I describe a test of the alternative hypothesis that noxious chemicals in exudates elicit trenching. My approach was to place drops of dissolved P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

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FIG. 1. Final instar cabbage looper cutting a trench across a leaf of Lobelia cardinalis before feeding on the distal tip to the left. chemicals on the mouthparts of loopers feeding on Plantago lanceolata L. (Plantaginaceae), a plant that normally is not trenched. Loopers that receive drops of lettuce latex (Lactuca serriola) or cucumber sap often stop feeding and cut a trench, responding as though the exudate is emitted by the plantain leaf (Dussourd, 1997). I tested if single chemicals similarly trigger trenching, and if different plants have the same or different trenching stimulants. Three natural products that elicit trenching were identified; all three have been reported to affect the nervous system of mammals. One of the compounds, lobeline, acts specifically as an antagonist at nicotinic acetylcholine receptors (Dwoskin and Crooks, 2001, 2002). Another of the trenching stimulants, myristicin, is thought to have anticholinergic activity (Lavy, 1987; Pytte and Rygnestad, 1998). To determine if loopers respond selectively to compounds that affect acetylcholine synapses, I assayed a series of neurotransmitters, insecticides, and drugs. These trials test if cabbage loopers respond only to a few specific trenching stimulants from plant exudates, more generally to various compounds acting on acetylcholine receptors, or if diverse neuroactive chemicals elicit trenching. Cabbage loopers routinely feed on plants in the Asteraceae and Apiaceae (Dussourd and Denno, 1994, and references therein), but members of the Campanulaceae, such as Lobelia cardinalis, have not been previously reported as host plants (Sutherland and Greene, 1984). The final objective was to determine how frequently cabbage loopers trench L. cardinalis and to test if the larvae can develop on this species. P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

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METHODS AND MATERIALS

Trenching Assay. Cabbage loopers from a laboratory culture were reared to the final instar on potted plantain, Plantago lanceolata. Plantain does not emit exudate and is not trenched. Thus, the loopers used in this assay had no prior experience with exudates or trenching. Early final-instars were deprived of food for 1–1.5 hr at 24–27C, then were allowed to walk onto the underside of an excised plantain leaf held in a water pic to maintain turgidity. For consistency between trials, only flat mature leaves with a maximum width of 1.2 to 2.2 cm were used. When a larva initiated feeding, a drop of solution was carefully applied directly to the side of its mouth with a 5-l Wiretrol micropipette. Each larva received a total of 1.5-l solution dispensed in a sequence of four to six drops, with the exception that larvae tested with 60% acetone solutions typically received only three drops because of rapid evaporation of solvent. Multiple droplets were applied to simulate the natural outflow of exudate that occurs when larvae sever canals while attempting to feed on plants with exudates. With innocuous solutions, such as water controls, larvae consumed the drops while continuing to feed without showing any signs of disturbance. Drops were sucked into the mouth and swallowed during normal feeding. With deterrent solutions, larvae usually stopped feeding immediately, wiped their mouth vigorously on the leaf, opened and closed their mandibles repeatedly, and then moved to a new feeding site. Each additional drop of solution was applied only after the larva recommenced feeding. I recorded larval response to each droplet of solution and whether larvae interrupted feeding to cut a trench. For this study, a trench was defined as a continuous line of bites extending at least 5 mm partially or completely across the leaf. For consistency between trials, all assays were performed by the author. Each caterpillar was tested only once. Whenever possible, chemicals were tested in water. Compounds with inadequate solubility were dissolved in aqueous solutions of either 10% Tween 80 (a surfactant) or the minimal concentration of acetone required (10–60%). Both Tween 80 and acetone at these high concentrations deterred feeding by cabbage loopers and occasionally triggered trenching. The solutions were each tested with 10 naive larvae, except for the two pesticides methomyl and methamidophos, which were extremely toxic to the cabbage loopers and were tested with only five larvae each. Chemicals were tested at a standard dose of 50 nmol in 1.5 l (=33.3 mM). The only exceptions were lactucin and bergapten, which had poor solubility in water and required unacceptably high concentrations of solvent to dissolve at 33.3 mM. Instead, both were tested at 10 nmol in 1.5 l(=6.7 mM). Bioassay of Natural Products, Neurotransmitters, Insecticides, and Drugs. Natural products tested with the trenching assay included the sesquiterpene lactone, lactucin, found in the latex of wild and cultivated lettuce (Tamaki et al., 1995; Sessa et al., 2000). Lactucin was isolated from the latex of Lactuca virosa L. (generously P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

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supplied by M. H. Bennett, University of London) and tested at both 5 and 10 nmol per 1.5 l to match concentrations reported in the latex of cultivated lettuce (Sessa et al., 2000). I also tested two phenylpropanoids, caffeic acid and chlorogenic acid, both of which reportedly occur in lettuce latex (Tibbitts and Read, 1976) and have a widespread distribution in plants (Harborne and Baxter, 1993). Parsley, Petroselinum crispum (Mill.) Mansf., produces a complex mixture of phenylpropanoids, coumarins, and terpenoids (MacLeod et al., 1985; Knogge et al., 1987; Porter, 1989). These compounds occur at least partly within the oil ducts (Hegnauer, 1971; Stahl-Biskup and Wichtmann, 1991; Wu and Hahlbrock, 1992; Reinold and Hahlbrock, 1997). I tested a representative series that included two phenylpropanoids, myristicin and parsley apiole; three linear furanocoumarins, xanthotoxin, imperatorin, and bergapten; and two monoterpenes, -pinene and -terpinene. Lobelia cardinalis L. and the medicinal plant L. inflata L. (Indian tobacco) both contain the piperidine alkaloid, lobeline (Krochmal et al., 1972). These plants were used by Native Americans and settlers in the United States for diverse medicinal purposes, including as an emetic and respiratory stimulant; lobeline is believed to be the principal active compound (Stephens, 1980; Fodor and Colasanti, 1985; Kindscher, 1992). I tested if lobeline deters feeding by cabbage loopers and if it triggers trenching. Cucurbits are well-known for the presence of bitter triterpenes named cucurbitacins. The compounds occur throughout the plant including the leaves (Rehm et al., 1957; Guha and Sen, 1975), but are apparently absent from sap exudate (Tallamy, unpublished data, cited in McCloud et al., 1995). Early studies with squash beetles suggested that trenching functions to prevent cucurbits from in- creasing cucurbitacin levels in response to damage (Carroll and Hoffman, 1980; Tallamy, 1985). Subsequent work revealed that cucurbitacins actually stimulate feeding by squash beetles and that trenching serves instead to reduce beetle exposure to sap exudate (McCloud et al., 1995). I used the cabbage looper bioassay to test if cucurbitacin E glycoside triggers trenching. The cucurbitacin sample was kindly provided by D. W. Tallamy, University of Delaware. The aglycone or glycoside of cucurbitacin E occurs in diverse cucurbits including watermelon, Citrullus vulgaris (Rehm et al., 1957; Guha and Sen, 1975), a plant that cabbage loopers trench (Dussourd, unpublished observation). Each natural product was tested independently in a randomized series with a solvent control (except for chlorogenic acid and -terpinene, which were tested alone). Fisher exact tests were used to compare the number of larvae cutting trenches in response to the test chemical and control. The survey of natural products described earlier identified three compounds with significant trenching activity. To verify activity, each of the compounds was retested with a solvent control using a new cohort of cabbage loopers. Myristicin and lobeline were each tested with 20 larvae, whereas lactucin was tested with 10. P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

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To determine if trenching is triggered specifically by compounds acting on acetylcholine receptors, I used the cabbage looper assay to test the activity of acetylcholine and several drugs that affect acetylcholine synapses. These compounds comprised an agonist for nicotinic cholinergic receptors, ()-nicotine; an agonist for muscarinic cholinergic receptors, (+)-muscarine chloride; three antagonists for nicotinic cholinergic receptors, decamethonium bromide, succinylcholine chloride, and D-tubocurarine chloride; an antagonist for muscarinic cholinergic receptors, ipratropium bromide; and two anticholinesterases, pyridostigmine bromide and edrophonium chloride. The compounds were tested in random order together with an aqueous control. Insecticides acting on acetylcholine synapses were also tested. They included the chloronicotinyl compound, imidacloprid, and the nereistoxin analog, cartap. Imidacloprid is currently the largest-selling insecticide worldwide (Nauen et al., 2001); it acts as an agonist of nicotinic acetylcholine receptors (Matsuda et al., 2001) and is commonly used to control sucking insects on lettuce and other crops attacked by cabbage loopers (Palumbo et al., 1997; Thomson, 1998). Cartap also tar- gets the nicotinic acetylcholine receptor, but at a different site (Casida and Quistad, 1998). I also tested the carbamate insecticide, methomyl, and the organophosphorous insecticide, methamidophos. Both compounds are used to control cabbage looper populations on lettuce and other vegetable crops (Anonymous, 1989; Thomson, 1998). Carbamate and organophosphorous insecticides inhibit acetyl- cholinesterase (Cremlyn, 1991). In insects, acetylcholine is a major neurotransmitter in the central nervous system (Benson, 1993) and most sensory information is delivered to the CNS through the release of acetylcholine (Trimmer, 1995). To determine if cabbage loopers respond only to chemicals that bind to acetylcholine receptors, I tested four additional compounds that act as neurotransmitters, neuromodulators, and/or neurohormones: dopamine, serotonin, octopamine, and GABA. To test if loopers respond differently to stimulants and inhibitors, as documented with other insects (Long and Murdock, 1983; Mullin et al., 1994; Cohen et al., 2002), I also assayed the following drugs: R() apomorphine hydrochloride and fluphenazine dihydrochlo- ride, agonist and antagonist of dopamine receptors; buspirone hydrochloride and WAY-100635 maleate, agonist and antagonist of serotonin receptors; clonidine and metoclopramide, agonist and antagonist of octopamine receptors; muscimol hydrobromide and (-) bicuculline methobromide, agonist and antagonist of - aminobutyric acid (GABA) receptors. Testing the 12 compounds required several days, a sufficiently long time that decomposition could occur if the compounds were tested in a random sequence. Therefore, the 12 chemicals were tested one at a time. Drug activities are based on studies with either insect (Benson, 1993; Roeder, 1994; Burrows, 1996; Osborne, 1996; Aydar and Beadle, 1999) or mammalian models (Hardman and Limbird, 1996; Watling, 1998). Where information is P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

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available, most compounds are active in both groups. A notable exception is bicuculline, a diagnostic GABAA antagonist in vertebrates that is largely inactive against GABA responses in insects (Benson, 1993; Aydar and Beadle, 1999). Sources of chemicals are indicated in Tables 1 and 2 and in Figure 3. Survival of Cabbage Loopers on Lobelia. To test if cabbage loopers can develop on Lobelia cardinalis, 20 newly eclosed first instar larvae were placed in clip cages on newly mature leaves of potted L. cardinalis that had not yet bolted. The clip cages were padded to provide a seal over the leaf surface without compressing the latex canals (laticifers). Each larva was enclosed on a separate plant. As a control, 40 newly emerged first instars were offered excised leaves of L. cardinalis cut in half to deplete the laticifers. Fresh leaves were provided as needed, typically daily. Cabbage loopers begin trenching in the second instar (Dussourd, 1993). To test if trenching allows cabbage loopers to feed on L. cardinalis, 20 early third instars and 10 early fifth instars were each sleeved on separate nonbolted plants. The larvae were reared to the third or fifth instar on excised leaves of L. cardinalis. Larvae were checked daily for trenching and feeding. In nature, newly eclosed larvae are not isolated in clip cages; they can roam freely to find suitable food. To test if unrestrained larvae can develop on L. cardinalis, I placed 20 newly eclosed first instars each on a separate nonbolted plant surrounded by a water moat to prevent dispersal. As a control, 40 newly eclosed larvae were again offered excised L. cardinalis leaves. Larvae in all Lobelia experiments were held indoors at 25C.

RESULTS

Trenching Bioassays. Of the 12 natural products tested from potential host plants of cabbage loopers, only 3 triggered significant trenching: the sesquiterpene lactone, lactucin, from lettuce latex; the phenylpropanoid, myristicin, from parsley oil; and the alkaloid, lobeline, from Lobelia (Table 1). Retesting the three compounds with new cohorts of larvae confirmed their activity (Figure 2). Com- pounds with little or no activity included the two phenylpropanoids from lettuce (caffeic acid and chlorogenic acid), plus a phenylpropanoid (parsley apiole), three furanocoumarins, and two monoterpenes from parsley (Table 1). Cucurbitacin E glycoside from cucumber also did not trigger trenching at 50 nmol/larva (Table 1), nor at four lower concentrations tested in water (0.02, 0.2, 2, 20 nmol/larva; 10 larvae/concentration; Dussourd, unpublished data). None of the plant compounds in Table 1 caused substantial mortality; with most chemicals, 100% of the larvae survived to produce normal moths. Acetylcholine did not affect looper behavior at the dose tested, but four of the eight drugs active at acetylcholine synapses elicited trenching (Table 2A). P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

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TABLE 1. CABBAGE LOOPER TRENCHING IN RESPONSE TO NATURAL PRODUCTS

Solutiona % Trenchingb

Asteraceae: Lactuca 1. Lactucin (10 nmol/larva in 50% acetone) 60* Lactucin (5 nmol/larva in 50% acetone) 60* 50% Acetone control 10 2. Caffeic acidc (50 nmol/larva in 30% acetone) 0 30% Acetone control 0 3. Chlorogenic acidc (50 nmol/larva in water) 0 Apiaceae: Petroselinum 4. Myristicinc (50 nmol/larva in 10% Tween 80) 70** 10% Tween 80c control 0 5. Parsley apioled (50 nmol/larva in 10% Tween 80) 0 10% Tween 80c control 0 6. Xanthotoxinc (50 nmol/larva in 60% acetone) 0 60% Acetone control 0 7. Imperatorine (50 nmol/larva in 60% acetone) 20 60% Acetone control 0 8. Bergapten f (10 nmol/larva in 60% acetone) 0 60% Acetone control 20 9. (1R)-(+)--Pinene f (50 nmol/larva in 10% Tween 80) 20 10% Tween 80c control 0 10. -Terpinene f (50 nmol/larva in 10% Tween 80) 0 Campanulaceae: Lobelia 11. (-)-Lobeline hydrochloride f (50 nmol/larva in water) 70** Water control 0 Cucurbitaceae 12. Cucurbitacin E glycoside (50 nmol/larva in 10% Tween 80) 0 10% Tween 80c control 10

a Numbers on left indicate solutions tested together in a randomized sequence; N = 10 larvae/solution. b Fisher’s exact tests compare the number of larvae cutting a trench in Plantago lanceolata in response to chemicals and solvent controls. c Source of chemical: Sigma. d Source of chemical: Indoﬁne. e Source of chemical: ICN. f Source of chemical: Aldrich 0.05 > P > 0.01; **0.01 > P > 0.001.

Only two of these showed statistically greater activity than the water control; however, even low levels of trenching suggest biological activity for these water- soluble compounds since none of >100 cabbage loopers tested with water controls in different experiments have ever cut trenches (Dussourd, 1997, unpublished data). Furthermore, none of the thousands of cabbage loopers that I have raised on Plantago lanceolata have ever been observed to trench during rearing. The four compounds that triggered trenching included both agonists and antagonists P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style ﬁle version June 28th, 2002

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FIG. 2. Percentage of cabbage loopers cutting a trench in Plantago lanceolata after receiving 10 nmol lactucin in a 50% acetone solution, 50 nmol myristicin in 10% Tween 80, or 50 nmol lobeline in water. Each chemical was tested independently with a control, which was the same solution used to dissolve the test chemical. Lactucin and its control were each tested with 10 larvae, whereas the other solutions were all tested with 20 larvae. Asterisks indicate statistically greater trenching response than the control (Fisher’s exact tests, **0.01 > P > 0.001, ***P < 0.001).

of acetylcholine receptors. The two anticholinesterases tested were both inactive as trenching stimulants (as were the anticholinesterase insecticides, methomyl and methamidophos, Table 2B). Nicotine caused one larva to regurgitate onto the leaf; none of the other compounds caused overt poisoning. Larvae survived their exposure to all compounds; 90–100% of the larvae produced moths for all compounds tested. Of the four insecticides tested, only imidacloprid triggered signiﬁcant trenching (Table 2B). Two larvae showed clear signs of poisoning; one trenched half way across the leaf before falling off, regurgitating, and quivering with spasms. One of the loopers tested with cartap likewise completed only a partial trench before becoming immobilized and unresponsive. A second larva had just begun trenching when it too was immobilized by cartap. All of the larvae tested with imidacloprid and cartap eventually resumed feeding and developed into adults of normal appear- ance. In contrast, both methomyl and methamidophos were fatal. Surprisingly, the larvae scarcely responded to drops of these insecticides. Either the loopers were unable to detect methomyl and methamidophos or the compounds were not deterrent. Shortly after ingesting the entire dose, the larvae regurgitated, convulsed, fell from the leaf, and eventually became immobilized. The lack of larval response even to a lethal dose undoubtedly increases mortality when the insecticides are used against cabbage loopers on crops. P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style ﬁle version June 28th, 2002

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TABLE 2. CABBAGE LOOPER TRENCHING IN RESPONSE TO DRUGS AND INSECTICIDES THAT AFFECT ACETYLCHOLINE SYNAPSES

Solutiona % Trenchingb

A. Drugs 1. Acetylcholine chloridec 0 ()-Nicotinec 30 (+)-Muscarine chloridec 0 Decamethonium bromided 40* Succinylcholine chloridec 0 D-Tubocurarine chlorided 20 Ipratropium bromidec 70** Pyridostigmine bromided 0 Edrophonium chlorided 0 Water control 0 B. Insecticides 2. Imidaclopride in 30% acetone 40* 30% Acetone control 0 3. Cartap hydrochloridee 10 4. Methomyle 0 5. Methamidophose 0 6. Water control 0

a Numbers on left indicate solutions tested together in a randomized sequence. All larvae received 50 nmol test chemical dissolved in 1.5-l solvent. Unless indicated otherwise, all chemicals were dissolved in water. N = 10 larvae/solution, except N = 5 for methomyl and for methamidophos. b Fisher’s exact tests compare the number of larvae cutting a trench in Plantago lanceolata in response to chemicals and solvent controls. c Source of chemical: Sigma. d Source of chemical: ICN. e Source of chemical: Chem Service. 0.05 > P > 0.01; **0.01 > P > 0.001.

Cabbage loopers also responded to chemicals that bind to other neuroreceptors (Figure 3). Dopamine and serotonin both deterred feeding and triggered trenching. Octopamine was mildly deterrent to some larvae, but was inactive as a trenching stimulant. GABA had little affect on looper behavior at the dosage tested. The low activity of GABA is not surprising since GABA is known to stimulate feeding by diverse herbivores (Mullin et al., 1994). All drugs tested that affect dopamine, serotonin, and octopamine receptors elicited trenching, whereas only the antagonist of GABA receptors was active (Figure 3). No toxic responses were noted; 90–100% of the larvae developed into moths in all treatments. Many of the chemicals tested were deterrent to the loopers. Larvae often wiped solution off their mouthparts onto the leaf, sometimes repeatedly with each application. The larvae frequently withdrew their head away from the leaf, then P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style ﬁle version June 28th, 2002

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FIG. 3. Percentage of cabbage loopers that cut trenches in Plantago lanceolata leaves after receiving 50 nmol of the test chemical dissolved in 1.5-l water. Each chemical was tested with 10 ﬁnal instar larvae. The chemicals included four neurotransmitters (dopamine, serotonin, octopamine, GABA), plus an agonist and antagonist of receptors stimulated by each neurotransmitter. The three biogenic amines may act partly or primarily as neuromodulators or neurohormones in insects (Burrows, 1996). Asterisks indicate statistically greater trenching response than the aqueous control (Fisher’s exact tests, **0.01 > P > 0.001, ***P < 0.001). Superscripts after the chemical names indicate the source of the chemical: 1ICN, 2Sigma, 3RBI.

moved their mandibles together and apart repeatedly as though chewing. Some larvae lowered their head to their legs to groom legs and mouthparts. Larvae often abandoned their feeding site and moved to another location on the leaf before continuing to feed. Compounds that triggered trenching were especially deterrent. For example, of the nine chemicals tested that affect acetylcholine synapses (Table 2A), the four that triggered trenching were more deterrent than the five that did not. Larvae exposed to the trenching stimulants were more likely to wipe their mouth on the leaf (9.5 0.3 larvae/chemical; mean 1 SE) than larvae tested with the other five chemicals (1.8 0.7, 0.05 > P > 0.01, Mann–Whitney U test). Likewise, larvae tested with the four trenching stimulants were more likely to withdraw and exhibit chewing motions (9.0 0.7 versus 1.8 0.8 larvae, 0.05 > P > 0.01), to groom (2.5 0.9 vs. 0.2 0.2, 0.05 > P > 0.01), and to abandon feeding sites (6.8 0.6 vs. 0.6 0.4, 0.05 > P > 0.01, Mann–Whitney U). Nearly all P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

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of the 132 loopers that trenched in this study exhibited signs of deterrence before trenching: 95% showed mouth-wiping, 98% chewing motions, 13% grooming, and 67% abandoning feeding sites (not including when larvae stopped feeding to trench). For chemicals tested in water, deterrency can be attributed directly to the test chemical. Lobeline, for example, triggered mouth-wiping in 100% of the 30 larvae tested (vs. 0% for water controls), 100% chewing motions (vs. 3%), 17% grooming (vs. 0%), and 83% abandoning feeding sites (vs. 0% for water). Furthermore, 10 of the 30 larvae walked off the leaf after receiving lobeline. Although solutions that elicited trenching were all deterrent, not all deterrent solutions caused trenching. Xanthotoxin elicited vigorous repetitive mouth- wiping; 7 of 10 larvae left the leaf after receiving just one or two drops, but none cut a trench. Excluding data for xanthotoxin, only 29 of 650 larvae tested in this study walked off the leaf; larvae were reluctant to move onto the water pic and usually resumed feeding nearby. Xanthotoxin retested at a range of concentrations again was highly deterrent. Fewer larvae abandoned the leaf at lower xanthotoxin doses, but none trenched (Figure 4). Other deterrent solutions also had little or no activity as trenching stimulants. For example, bergapten in 60% acetone and terpinene in 10% Tween 80 caused mouth-wiping, respectively, in 90 and 100% of the larvae, chewing movements in 100 and 90%, grooming in 80 and 30%, and abandoning feeding sites in 50 and 60%; neither solution triggered trenching. The naive larvae in this study required repeated contact with solutions before they initiated trenching. Only 6% started trenching after receiving the ﬁrst drop,

FIG. 4. Effect of xanthotoxin on cabbage loopers. Each concentration of xanthotoxin was tested independently with a control, which was the solvent alone. Ten loopers were tested with each solution; each received 1.5 l. Asterisks indicate a statistically signiﬁcant differ- ence in the number of loopers abandoning the leaf comparing the xanthotoxin solution and control (Fisher’s exact tests, **0.01 > P > 0.001). Xanthotoxin was highly deterrent, but did not trigger trenching at any of the concentrations tested. P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style ﬁle version June 28th, 2002

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32% after the second, 31% after the third, and 31% after the fourth to sixth drops. Even then, larvae often cut one or more veins first (9% of 132 trenchers) or cut only a short trench (52%) before continuing to feed. After encountering an additional drop(s) of the test chemical, the larvae resumed trenching, as though their first attempt to disrupt leaf canals had been inadequate and further trenching was necessary. Only 39% of the larvae cut a continuous trench across the entire leaf the first time that they trenched. This indicates that trenching is not a fixed action pattern that is invariably carried through to completion on first attempt. On the contrary, larvae appear to require continued stimulation to persevere at trenching. On plants with laticifers, trenching causes latex to flow onto the trench. Larvae presumably encounter trenching stimulants in latex continuously until all laticifers crossing the trench have been severed, at which point the larvae stop trenching and begin to feed. Cabbage Looper Survival on Lobelia. First instar cabbage loopers enclosed on potted Lobelia cardinalis leaves all died within 2–6 days (Figure 5). Only one reached the second instar and it died shortly thereafter. Larvae ate small pits in the leaves, often causing latex to flow into the pits. Two larvae died with their heads glued to the leaf by latex. Third instar larvae sleeved on L. cardinalis also perished. Eighteen of the 20 larvae cut one or more trenches, mostly across leaf tips; however, the larvae did not feed beyond 40% of the trenches. When these intact leaf tips were severed with scissors, latex oozed readily from all of them documenting that the trenches failed to sever all laticifers crossing the trench. Third instars also ate small pits in the leaves and fed on the midrib. The larvae survived up to 11 days (mean = 5 days), but none molted to the fourth instar. In contrast, most final instar cabbage loopers enclosed on L. cardinalis cut trenches readily and fed beyond the trenches. The 10 larvae trenched 68 leaves and

FIG. 5. Survival of cabbage loopers enclosed with either intact or excised leaves of Lobelia cardinalis. Twenty larvae in the first instar, 20 in the third instar, and 10 in the fifth instar were tested on intact leaves, whereas 40 first instar larvae were offered excised leaves. P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

2036 DUSSOURD

completely consumed the distal tips in 81% of them. Seven larvae pupated, but only ﬁve adults emerged from the cocoons. The three larvae that failed to pupate fed beyond only 10 of their 20 trenches. The uneaten tips readily released latex when severed indicating that the larvae were often unsuccessful in deactivating the latex system. Excised leaves of L. cardinalis were highly acceptable for cabbage loopers. Fully 97.5% of the 40 loopers pupated in just 12–14 days from egg hatch (mean = 12.4 days) and all pupae produced an adult (Figure 5). The newly emerged cabbage loopers released on entire L. cardinalis plants also perished. However, four of the larvae reached the second instar by feeding extensively on senescent or damaged leaves, as well as by pit-feeding. The four larvae cut trenches across 18 leaves, but fed successfully on only four of them. The remaining 14 leaves all released latex readily when their tips were severed documenting that some latex canals remained intact. As previously, the larvae on excised leaves fed readily; 90% survived to pupate and 87.5% produced an adult moth.

DISCUSSION

Isolated chemicals sufficed to elicit trenching by cabbage loopers. These compounds were active in the absence of other exudate components; notably, solutions did not have to be sticky to stimulate trenching. Effective stimulants included a diversity of structures (Figure 6) suggesting that either multiple receptors are in- volved in their detection or the receptors are broadly tuned. Three constituents of potential food plants caused significant trenching: lactucin from lettuce, myristicin from parsley, and lobeline from Lobelia. As described below, these compounds are known or suspected to have toxic properties, suggesting that loopers cut trenches specifically to reduce their exposure to noxious substances during feeding. Lactucin is classified as a sesquiterpene lactone, a diverse group of almost 3500 reported structures known for cytotoxic, antibacterial, antifungal, and al- lelopathic properties (Picman, 1986). Several sesquiterpene lactones have documented deterrency or toxicity to insect herbivores (Picman, 1986; Gershenzon and Croteau, 1991; Mullin et al., 1991). To my knowledge, lactucin has not been tested previously for its effects on insects. However, two other sesquiterpene lactones reported from lettuce latex, lactucopicrin and 8-deoxylactucin (Price et al., 1990), are known to deter feeding by locusts (Rees and Harborne, 1985). The sesquiterpene lactones in the latex of cultivated lettuce occur as a complex mixture that includes oxalate and sulfate conjugates (Sessa et al., 2000). Lactucin is only a minor component. It reaches a maximum level of 2.8 mg/ml latex in bolted plants, whereas the total sesquiterpene lactone titer of these plants averages 147.1 mg/ml latex (Sessa et al., 2000). Titers of sesquiterpene lactones in the latex P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

CHEMICAL STIMULANTS OF LEAF-TRENCHING BY CABBAGE LOOPERS 2037

FIG. 6. Chemicals that trigger trenching include the sesquiterpene lactone, lactucin (1), from lettuce latex; the phenylpropanoid, myristicin (2), from parsley oil; the alkaloid, lobeline (3) from Lobelia; the neurotransmitters, dopamine hydrochloride (4) and serotonin hydrochloride (5); the insecticide, imidacloprid (6); and several drugs that stimulate or inhibit neural receptors, such as R() apomorphine hydrochloride (7) and buspirone hydrochloride (8).

of wild Lactuca species, notably L. virosa, can be much higher (Tamaki et al., 1995). In this study, lactucin triggered trenching at 0.92 mg/ml (=5 nmol/1.5 l), documenting that the compound is active at natural lactucin levels. Presumably the abundant conjugates and derivatives of lactucin contribute to the trenching activity of Lactuca latex. Cabbage loopers feed extensively on composites in the field, including cultivated and wild lettuce (L. sativa, L. serriola, L. canadensis) and other latex-bearing composites such as Sonchus asper, Taraxacum officinale, and Pyrrhopappus carolinianus (Dussourd and Denno, 1991, 1994). I have found as many as 20 cabbage loopers on individual plants of L. serriola and S. asper, which can be completely stripped of leaves. The loopers routinely cut trenches in these species, apparently due specifically to the sesquiterpene lactones that they must encounter often in these important food plants. Cabbage loopers also cut trenches in multiple species of Apiaceae, including parsley, carrot, and parsnip (Dussourd and Denno, 1991; Zangerl and Bazzaz, 1992). All three species produce myristicin (Lichtenstein and Casida, 1963; P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

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Kubeczka and Stahl, 1977; Porter, 1989; Hallstrom and Thuvander, 1997). Myris- ticin inhibits detoxification enzymes (mixed function oxidases) in caterpillars and thereby synergizes the activity of insecticides and natural toxins such as furanocoumarins (Lichtenstein and Casida, 1963; Berenbaum and Neal, 1985; Neal, 1989). When tested by itself, myristicin is poisonous to some fly, aphid, beetle, and caterpillar species (Lichtenstein and Casida, 1963; Lichtenstein et al., 1974; Marston et al., 1995; Srivastava et al., 2001), but does not increase mortality of two generalist caterpillars, the corn earworm and southern armyworm (Lichten- stein and Casida, 1963; Berenbaum and Neal, 1985). Myristicin levels vary greatly in different parsley accessions, ranging from 0.1% to over 60% of the essential oils extracted from whole plants (Simon and Quinn, 1988; Porter, 1989). When present at high concentration, myristicin is undoubtedly an important trenching stimulant. Whether additional oil components such as the nonpolar sesquiterpene caryophyllene also trigger trenching remains to be determined. Surprisingly, furanocoumarins were ineffective trenching stimulants, even though these compounds occur in the oil ducts (Camm et al., 1976; Wu and Hahlbrock, 1992; Reinold and Hahlbrock, 1997) and are toxic to diverse herbivores (Berenbaum, 1990; Reitz and Trumble, 1996). Xanthotoxin mixed in ar- tificial diet at 0.24% fresh mass prevents growth of cabbage loopers (Zangerl, 1990). Nevertheless, xanthotoxin did not elicit trenching, although it was strongly deterrent (Figure 4). Parsley apiole also was inactive at the dosage tested, despite its structural similarity to myristicin. Cabbage loopers clearly do not trench in- discriminately in response to all allelochemicals. A further example is provided by cardenolides, which are extremely deterrent and toxic to cabbage loopers, but did not cause trenching when tested at 25 g/larva (=33 nmol/larva for digitoxin; Dussourd and Hoyle, 2000). Little information is available on how lobeline affects insects, although it has been reported to deter feeding by a ctenuchid caterpillar and honeybees (Wink and Schneider, 1990; Detzel and Wink, 1993). Mammalian herbivores (cows, sheep, goats) that feed on various Lobelia species suffer negative effects that can include sluggishness, hemorrhage, and coma; overdoses of lobeline produce similar effects (Dollahite and Allen, 1962; Kingsbury, 1964). Lobelia cardinalis contains 7 mg lobeline/g dry weight, which is comparable to levels in the medicinal plant, L. inflata (Krochmal et al., 1972). Overdoses of lobeline or L. inflata in humans cause dizziness, stupor, tremors, paralysis, convulsions, coma, and death (Arena, 1970; Duke, 1985; Dwoskin and Crooks, 2002). Cucurbitacin E glycoside was the only compound characteristic of the Cucur- bitaceae that I tested. When dissolved in water, it elicited minimal reaction from the loopers even though cucurbitacins are intensely bitter to humans (Metcalf et al., 1980) and have deterrent or toxic effects on many herbivores (Tallamy et al., 1997). However, cucurbitacin B applied to leaf disks had no significant effects on cabbage looper feeding (Tallamy et al., 1997). Trenching activity in cucumber sap P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

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has been traced not to cucurbitacins, but to a polar fraction (Dussourd, 1997) that contains at least two active components. One of the components has been partially characterized as an N-alkyl pyridinium salt (Capron, Wiemer, and Dussourd, unpublished data). Lettuce, parsley, Lobelia, and cucumber thus contain strikingly different trenching stimulants: a sesquiterpene lactone, phenylpropanoid, and two alkaloids; there is no common releaser for trenching in these diverse plants. The solvents used to dissolve less polar compounds in this study sometimes also elicited trenching, but only at much higher concentrations than the natural products (e.g., 60% acetone vs. 6.7 mM lactucin). Even with the most active stimulants, less than 100% of the larvae trenched, whereas final-instar cabbage loopers isolated on intact plants of prickly lettuce (Lactuca serriola) or on other laticiferous composites invariably trench (Dussourd and Denno, 1994). However, loopers tested with the trenching assay had not previously experienced exudates. In addition, the larvae received only 4–6 droplets of solution, unlike loopers on intact plants which contact exudates repeatedly during trenching (Dussourd, 1999). It is also unlikely that lactucin, myristicin, and lobeline are the only trenching stimulants in lettuce, parsley, and Lobelia. L. cardinalis, for example, contains multiple piperidine alkaloids (Gupta and Spenser, 1971; Krochmal et al., 1972), in addition to lobeline. Although not all allelochemicals were tested, this study documents that isolated chemicals at biologically rele- vant concentrations suffice to elicit trenching and provides a method for screening additional compounds for trenching activity. Of the four insecticides tested, imidacloprid was the least harmful for the loopers and the most effective at triggering trenching. Imidacloprid is commonly used to control sucking insects, fleas, and beetles; it is generally less effective against caterpillars (Thomson, 1998). Methomyl and methamidophos were both lethal at the dosage used, whereas cartap caused temporary immobility in all larvae. Cartap is converted in vivo to nereistoxin, which blocks cholinergic synaptic transmission and rapidly immobilizes insects (Sattelle, 1985). Of the 21 neurotransmitters and drugs tested, 13 caused trenching (Table 2A, Figure 3). Many of the active compounds are potent neurotoxins such as tubocurarine, a constituent of curare arrow poison (Hardman and Limbird, 1996), and nicotine, a contact insecticide from tobacco used to kill insect pests for over 300 years (Cremlyn, 1991). Both agonists and antagonists of cholinergic, dopaminer- gic, and serotonergic receptors were deterrent and caused the loopers to trench. Other insects are more specific in their responses. For example, in blowflies and cockroaches, octopamine and octopamine agonists delivered by injection or by ingestion increase feeding, but octopamine antagonists decrease feeding (Long and Murdock, 1983; Cohen et al., 2002). Likewise, applying agonists for GABA/glycine receptors to flower disks stimulates feeding by western corn root- worms, whereas GABA/glycine antagonists deter feeding (Mullin et al., 1994). Cabbage loopers, in contrast, often responded the same to agonists and antagonists, P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

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and sometimes reacted differently to compounds with similar modes of action. For example, octopamine was inactive at the dosage tested, whereas the octopamine agonist, clonidine, elicited trenching in 5 of 10 larvae (Figure 3). The similar activity of agonists and antagonists suggests that neurotransmitters and drugs trigger trenching by stimulating peripheral chemoreceptors and not through direct contact with the CNS following absorption from the gut. This conclusion is supported by the rapid response of some larvae; for example, one looper initiated trenching after receiving just a single drop of dopamine. The three natural trenching stimulants, lactucin, myristicin, and lobeline, all have been reported to affect the nervous system of mammals. Latex from wild Lactuca species (lactucarium) has been used medicinally as a sedative and an analgesic for hundreds of years (Stevens, 1948; Crellin and Philpott, 1990). Lab- oratory studies with mice suggest that lactucin is at least partly responsible for its activity (Schenck, 1966; Gromek et al., 1992). Myristicin injected in various mammals causes behavioral depression, ataxia, and disorientation (Truitt et al., 1961; Florio et al., 1972; Truitt, 1979). Higher doses in monkeys lead to respiratory arrest (Truitt et al., 1961). Lobeline has multiple pharmacological effects. It acts as a potent antagonist at 32 and 42 neuronal nicotinic receptor subtypes (Dwoskin and Crooks, 2001). Lobeline also alters dopamine storage and utilization apparently by inhibiting dopamine uptake by synaptic vesicles and by stimulating the release of dopamine from the vesicles into the cytosol (Dwoskin and Crooks, 2002). Interestingly, with all three trenching stimulants, plant species containing the compounds are smoked or consumed for their purported hallucinogenic, nar- cotic, or euphoric effects (Truitt et al., 1961; Huang et al., 1982; Duke, 1985; Foster and Tyler, 1999). Lobeline is currently being investigated as a treatment for methamphetamine abuse (Harrod et al., 2001; Dwoskin and Crooks, 2002) and has been marketed as a treatment for tobacco smoking (Fodor and Colasanti, 1985). Presumably the three trenching stimulants also affect the nervous system of cabbage loopers, although none have been tested for neurotoxicity to insects. Neurotoxins, including many alkaloids, are abundant in plants (Murdock et al., 1985; Hartmann, 1991; Wink, 1998). The toxins are sometimes sequestered in latex canals, such as opium alkaloids found in poppy latex (Bernath, 1998) and heart poisons (cardenolides) in milkweed latex (Dussourd and Hoyle, 2000). Neurotransmitters, including acetylcholine, dopamine, and serotonin, also have a widespread distribution in plants (Roshchina, 2001), and sometimes occur at high concentration (Steelink et al., 1967; Oliver et al., 1991). In poppy latex (Roberts et al., 1983), dopamine concentrations reach 2.66 mg/ml (=17.4 mM), close to the levels that elicited trenching in this study (33.3 mM). Despite their ability to trench, cabbage loopers were unable to develop on intact Lobelia cardinalis. Early instar larvae ate pits in the leaves, but failed to develop on potted plants, except on senescent or damaged leaves. Third and ﬁfth instar loopers cut trenches, but only the larger larvae were able to feed effectively. P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style ﬁle version June 28th, 2002

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Excised leaves, in contrast, were highly acceptable, presumably because excision depressurizes latex canals, thereby reducing larval ingestion of latex chemicals. These results illustrate the difficulty of defining an insect’s host range. Plant suit- ability depends not just on allelochemical content, but also on diverse factors that can include larval stage (Zalucki et al., 2002) and prior damage (Lewis, 1984). In summary, the results reported herein support the hypothesis that cabbage loopers cut trenches specifically to deactivate secretory canals in host plants and thereby reduce their ingestion of poisons that may often include neurotoxins. However, trenching ability alone is inadequate to permit feeding. Trenchers must also be able to tolerate high levels of exudate chemicals during trenching and lower levels during subsequent feeding. The known toxicity of the three identified trenching stimulants suggests that cabbage loopers use host toxins specifically as cues for trenching. Many questions remain. Are lactucin, myristicin, and lobeline poisonous to cabbage loopers, and do these compounds affect their nervous system? Lactucin is known to occur in the latex of Lactuca (Sessa et al., 2000). Is lobeline similarly sequestered in Lobelia latex, and is myristicin stored in the oil ducts of Petroselinum? How much of these compounds do cabbage loopers ingest during trenching and during subsequent feeding on distal tissues with depleted canals? Why do some feeding deterrents trigger trenching, whereas others do not? Do the diverse compounds that elicit trenching all stimulate the same taste receptor(s)? Cabbage loopers are classified in the plusiine tribe Argyrogrammatini (LaFontaine and Poole, 1991) together with other trenching species that include Enigmogramma basigera and the soybean looper, Pseudoplusia includens. These three species differ substantially in host range. Cabbage loopers, for example, cannot develop on undamaged Lobelia cardinalis,butE. basigera cut trenches and develop rapidly to pupation on intact plants in both lab and field (Dussourd, unpublished observation). Likewise, soybean loopers, Pseudoplusia includens, develop poorly on Lactuca serriola, a highly acceptable host for cabbage loopers (Tune and Dussourd, 2000). Do these three plusiine species differ in their tolerance of trenching stimulants, resulting in the observed variation in host range? Finally, why do cabbage loopers fail to trench plants lacking exudates? Lactuca, Petroselinum, and Lobelia all have canal systems that deliver exudates with concentrated allelochemicals directly to the mouthparts of biting herbivores. Do plants not trenched lack effective trenching stimulants or are stimulants present, but available at too low a concentration or counterbalanced by phagostimulants? The cabbage looper is just one of over 100 insect species known to sever veins or cut trenches before feeding (Dussourd and Denno, 1991; Dussourd, 1993 and references therein; Becerra, 1994; McCloud et al., 1995; Jolivet, 1998; Clarke and Zalucki, 2000; Becerra et al., 2001). Given that over 20,000 species of plants contain latex or resin canals (Farrell et al., 1991; Lewinsohn, 1991), that up to 7 species of vein cutters have been reported per plant species (Dussourd and P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

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Denno, 1991), and that some latex-bearing species are attacked by over 125 species of leaf-feeding insects (Basset and Novotny, 1999), the true number of insects exhibiting these behaviors undoubtedly numbers in the thousands. Plants manipulated with vein cuts or trenches include members of over a dozen families (Dussourd, 1993). The insect behaviors provide a convenient assay for identifying compounds in these plants that are especially signiﬁcant for insect herbivores, indeed of such importance to have selected for sophisticated behavioral coun- termeasures. One wonders, for example, what compounds in mulberry stimulate trenching by soybean loopers (Pseudoplusia includens), what chemicals in pa- paya and cassava elicit trenching by the cassava hornworm (Erinnyis ello), and what substances in milkweed latex trigger vein severance by monarchs (Danaus plexippus). The results reported here suggest that trenching stimulants are likely to be important chemical defenses for plants, not just harmless cues. Vein cutting and trenching assays, thus, have the potential to direct the isolation of novel structures with insecticidal properties and possible applications in agriculture or medicine.

Acknowledgments—Thanks to M. H. Bennett for providing lactucin; to D. W. Tallamy for providing cucurbitacin E glycoside; to D. F. Wiemer, M. Capron, M. H. Bennett, H. Eichenseer, and J. L. Frazier for helpful advice; to K. C. Larson, E. A. Bernays, M. R. Helmus, J. A. Murray, and anonymous reviewers for insightful comments on earlier versions of the manuscript; to K. Poole and W. L. Roelofs (Geneva Experiment Station) for repeatedly supplying T. ni eggs; to H. Dukat, M. M. Atwell, and M. R. Helmus for assistance in the greenhouse and lab; and to the University of Central Arkansas Research Council for ﬁnancial support.

REFERENCES

ANGIOSPERM PHYLOGENY GROUP 1998. An ordinal classification for the families of flowering plants. Ann. MO. Bot. Gard. 85:531–553. ANONYMOUS 1989. Crop Protection Chemicals Reference. Chemical and Pharmaceutical Press, Paris. ARENA, J. M. 1970. Poisoning: Toxicology, Symptoms, Treatments. Charles C. Thomas, Springfield, Illinois. AYDAR, E. and BEADLE, D. J. 1999. The pharmacological profile of GABA receptors on cultured insect neurones. J. Insect Physiol. 45:213–219. BASSET, Y. and NOVOTNY, V. 1999. Species richness of insect herbivore communities on Ficus in Papua New Guinea. Biol. J. Linn. Soc. 67:477–499. BECERRA, J. X. 1994. Squirt-gun defense in Bursera and the chrysomelid counterploy. Ecology 75:1991–1996. BECERRA, J. X., VENABLE, D. L., EVANS, P.H., and BOWERS, W.S. 2001. Interactions between chemical and mechanical defenses in the plant genus Bursera and their implications for herbivores. Am. Zool. 41:865–876. BENSON, J. A. 1993. The electrophysiological pharmacology of neurotransmitter receptors on lo- cust neuronal somata, pp. 390–413, in Y. Pichon (ed.). Comparative Molecular Neurobiology. Birkhäuser, Basel, Switzerland. BERENBAUM, M. R. 1990. Evolution of specialization in insect–umbellifer associations. Annu. Rev. Entomol. 35:319–343. P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

CHEMICAL STIMULANTS OF LEAF-TRENCHING BY CABBAGE LOOPERS 2043

BERENBAUM, M. and NEAL, J. J. 1985. Synergism between myristicin and xanthotoxin, a naturally cooccurring plant toxicant. J. Chem. Ecol. 11:1349–1358. BERNATH, J. (ed.) 1998. Poppy: The Genus Papaver. Harwood Academic, Amsterdam. BURROWS, M. 1996. The Neurobiology of an Insect Brain. Oxford University Press, Oxford. CAMM, E. L., WAT, C., and TOWERS, G. H. N. 1976. An assessment of the roles of furanocoumarins in Heracleum lanatum. Can. J. Bot. 54:2562–2566. CARROLL,C.R.andHOFFMAN, C. A. 1980. Chemical feeding deterrent mobilized in response to insect herbivory and counteradaptation in Epilachna tredecimnotata. Science 209:414– 416. CASIDA,J.E.andQUISTAD, G. B. 1998. Golden age of insecticide research: Past, present, or future? Annu. Rev. Entomol. 43:1–16. CHAPMAN, R. F. 1995. Chemosensory regulation of feeding, pp. 101–136 in R. F. Chapman and G. de Boer (eds.). Regulatory Mechanisms in Insect Feeding. Chapman and Hall, New York. CLARKE,A.R.andZALUCKI, M. P. 2000. Foraging and vein-cutting behaviour of Euploea core corinna (W. S. Macleay) (Lepidoptera: Nymphalidae) caterpillars feeding on latex-bearing leaves. Aust. J. Entomol. 39:283–290. COHEN,R.W.,MAHONEY, D. A., and CAN, H. D. 2002. Possible regulation of feeding behavior in cockroach nymphs by the neurotransmitter octopamine. J. Insect Behav. 15:37–50. CRELLIN,J.K.andPHILPOTT, J. 1990. Herbal Medicine Past and Present. II: A Reference Guide to Medicinal Plants. Duke University Press, Durham, North Carolina. CREMLYN, R. J. 1991. Agrochemicals: Preparation and Mode of Action. Wiley, New York. DETZEL, A. and WINK, M. 1993. Attraction, deterrence or intoxication of bees (Apis mellifera) by plant allelochemicals. Chemoecology 4:8–18. DOLLAHITE,J.W.andALLEN, T. J. 1962. Poisoning of cattle, sheep, and goats with Lobezla and Centaurium species. Southwest. Vet. 15:126–130. DUKE, J. A. 1985. CRC Handbook of Medicinal Herbs. CRC Press, Boca Raton, Florida. DUSSOURD, D. E. 1993. Foraging with finesse: Caterpillar adaptations for circumventing plant defenses, pp. 92–131, in N. E. Stamp and T. M. Casey (eds.). Caterpillars: Ecological and Evolutionary Constraints on Foraging. Chapman and Hall, New York. DUSSOURD, D. E. 1997. Plant exudates trigger leaf-trenching by cabbage loopers, Trichoplusia ni. Oecologia 112:362–369. DUSSOURD, D. E. 1999. Behavioral sabotage of plant defense: Do vein cuts and trenches reduce insect exposure to exudate? J. Insect Behav. 12:501–515. DUSSOURD, D. E. and DENNO, R. F. 1991. Deactivation of plant defense: Correspondence between insect behavior and secretory canal architecture. Ecology 72:1383–1396. DUSSOURD,D.E.andDENNO, R. F. 1994. Host range of generalist caterpillars: Trenching permits feeding on plants with secretory canals. Ecology 75:69–78. DUSSOURD,D.E.andHOYLE, A. M. 2000. Poisoned plusiines: Toxicity of milkweed latex and cardenolides to some generalist caterpillars. Chemoecology 10:11–16. DWOSKIN,L.P.andCROOKS, P. A. 2001. Competitive neuronal nicotinic receptor antagonists: A new direction for drug discovery. J. Pharmacol. Exp. Ther. 298:395–402. DWOSKIN,L.P.andCROOKS, P. A. 2002. A novel mechanism of action and potential use for lobeline as a treatment for psychostimulant abuse. Biochem. Pharmacol. 63:89–98. EICHLIN,T.D.andCUNNINGHAM, H. B. 1978. The Plusiinae (Lepidoptera: Noctuidae) of America north of Mexico, emphasizing genitalic and larval morphology. US Dept. Agric. Tech. Bull. 1567:1–122. FAHN, A. 1979. Secretory Tissues in Plants. Academic Press, New York. FARRELL, B. D., DUSSOURD, D. E., and MITTER, C. 1991. Escalation of plant defense: Do latex and resin canals spur plant diversification? Am. Nat. 138:881–900. FLINT, M. L. 1987. Integrated Pest Management for Cole Crops and Lettuce. University of California, Publication No. 3307. P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

2044 DUSSOURD

FLORIO, V.,FUENTES, J. A., ZIEGLER, H., and LONGO, V.G. 1972. EEG and behavioral effects in animals of some amphetamine derivatives with hallucinogenic properties. Behav. Biol. 7:401–414. FODOR,G.B.andCOLASANTI, B. 1985. The pyridine and piperidine alkaloids: Chemistry and pharmacology, pp. 1–90, in S. W. Pelletier (ed.). Alkaloids: Chemical and Biological Perspectives, Vol. 3. Wiley, New York. FOSTER, S. and TYLER, V. E. 1999. Tyler’s Honest Herbal: A Sensible Guide to the Use of Herbs and Related Remedies. Haworth Herbal Press, New York. GERSHENZON, J. and CROTEAU, R. 1991. Terpenoids, pp. 165–219, in G. A. Rosenthal and M. R. Berenbaum (eds.). Herbivores: Their Interactions with Secondary Plant Metabolites. Academic Press, New York. GRIMES,L.R.andNEUNZIG, H. H. 1986. Morphological survey of the maxillae in last-stage larvae of the suborder Ditrysia (Lepidoptera): Mesal lobes (Laciniogaleae). Ann. Entomol. Soc. Am. 79:510–526. GROMEK, D., KISIEL, W., KLODZINSKA, A., and CHOJNACKA-WOJCIK, E. 1992. Biologically active preparations from Lactuca virosa L. Phytother. R. 6:285–287. GUHA, J. and SEN, S. P. 1975. The cucurbitacins—A review. Plant Biochem. J. 2:12–28. GUPTA,R.N.andSPENSER, I. D. 1971. Biosynthesis of lobinaline. Can. J. Chem. 49:384–397. HALLSTROM, H. and THUVANDER, A. 1997. Toxicological evaluation of myristicin. Nat. Toxins 5:186– 192. HARBORNE,J.B.andBAXTER, H. 1993. Phytochemical Dictionary: A Handbook of Bioactive Com- pounds from Plants. Taylor and Francis, Washington, DC. HARDMAN,J.G.andLIMBIRD, L. E. (eds.) 1996. Goodman and Gilman’s The Pharmacological Basis of Therapeutics. McGraw-Hill, New York. HARROD, S. B., DWOSKIN,L.P.,CROOKS, P. A., KLEBAUR, J. E., and BARDO, M. T. 2001. Lobeline attenuates d-methamphetamine self-administration in rats. J. Pharmacol. Exp. Ther. 298:172– 179. HARTMANN, T. 1991. Alkaloids, pp. 79–121, in G. A. Rosenthal and M. R. Berenbaum (eds.). Her- bivores: Their Interactions with Secondary Plant Metabolites. Vol. I: The Chemical Participants. Academic Press, New York. HEGNAUER, R. 1971. Chemical patterns and relationships of Umbelliferae, pp. 267–277, in V. H. Heywood (ed.). The Biology and Chemistry of the Umbelliferae. Academic Press, New York. HUANG, Z. J., KINGHORN, A. D., and FARNSWORTH, N. R. 1982. Studies on herbal remedies. I: Analysis of herbal smoking preparations alleged to contain lettuce (Lactuca sativa L.) and other natural products. J. Pharm. Sci. 71:270–271. JOLIVET, P. 1998. Interrelationship Between Insects and Plants. CRC Press, New York. KINDSCHER, K. 1992. Medicinal Wild Plants of the Prairie: An Ethnobotanical Guide. University Press of Kansas, Lawrence, Kansas. KINGSBURY, J. M. 1964. Poisonous Plants of the United States and Canada. Prentice-Hall, Englewood Cliffs, New Jersey. KNOGGE, W., KOMBRINK, E., SCHMELZER, E., and HAHLBROCK, K. 1987. Occurrence of phytoalexins and other putative defense-related substances in uninfected parsley plants. Planta 171:279–287. KROCHMAL, A., WILKEN, L., and CHIEN, M. 1972. Lobeline content of four Appalachian Lobelias. Lloydia 35:303–304. KUBECZKA,K.H.andSTAHL, E. 1977. On the essential oils from the Apiaceae (Umbelliferae). II: The essential oils from the above ground parts of Pastinaca sativa. Planta Med. 31:173–184. LAFONTAINE,J.D.andPOOLE, R. W. 1991. Noctuoidea, noctuidae (part), in R. B. Dominick et al. (eds.). The Moths of America North of Mexico, Fasc. 25.1. Wedge Entomological Research Foundation, Washington, DC. LAVY, G. 1987. Nutmeg intoxication in pregnancy: A case report. J. Reprod. Med. 32:63–64. LEWINSOHN, T. M. 1991. The geographical distribution of plant latex. Chemoecology 2:64–68. P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style ﬁle version June 28th, 2002

CHEMICAL STIMULANTS OF LEAF-TRENCHING BY CABBAGE LOOPERS 2045

LEWIS, A. C. 1984. Plant quality and grasshopper feeding: Effects of sunflower condition on preference and performance in Melanoplus differentialis. Ecology 65:836–843. LICHTENSTEIN,E.P.andCASIDA, J. E. 1963. Myristicin, an insecticide and synergist occurring naturally in the edible parts of parsnips. J. Agric. Food Chem. 11:410–415. LICHTENSTEIN,E.P.,LIANG,T.T.,SCHULZ, K. R., SCHNOES, H. K., and CARTER, G. T. 1974. Insecticidal and synergistic components isolated from dill plants. J. Agric. Food Chem. 22:658–664. LONG,T.F.andMURDOCK, L. L. 1983. Stimulation of blowfly feeding behavior by octopaminergic drugs. Proc. Natl. Acad. Sci. USA 80:4159–4163. MACLEOD, A. J., SNYDER, C. H., and SUBRAMANIAN, G. 1985. Volatile aroma constituents of parsley leaves. Phytochemistry 24:2623–2627. MARSTON, A., HOSTETTMANN, K., and MSONTHI, J. D. 1995. Isolation of antifungal and larvicidal constituents of Diplolophium buchanani by centrifugal partition chromatography. J. Nat. Prod. 58:128–130. MATSUDA, K., BUCKINGHAM, S. D., KLEIER, D., RAUH, J. J., GRAUSO, M., and SATTELLE, D. B. 2001. Neonicotinoids: Insecticides acting on insect nicotinic acetylcholine receptors. Trends Pharmacol. Sci. 22:573–580. MCCLOUD, E. S., TALLAMY, D. W., and HALAWEISH, F. T. 1995. Squash beetle trenching behaviour: Avoidance of cucurbitacin induction or mucilaginous plant sap? Ecol. Entomol. 20:51–59. METCALF, R. L., METCALF, R. A., and RHODES, A. M. 1980. Cucurbitacins as kairomones for dia- broticite beetles. Proc. Natl. Acad. Sci. USA 77:3769–3772. METCALFE,C.R.andCHALK, L. 1983. Anatomy of the Dicotyledons, Vol.II. Clarendon Press, Oxford. MULLIN, C. A., ALFATAFTA, A. A., HARMAN, J. L., EVERETT, S. L., and SERINO, A. A. 1991. Feed- ing and toxic effects of floral sesquiterpene lactones, diterpenes, and phenolics from sunflower (Helianthus annuus L.) on western corn rootworm. J. Agric. Food Chem. 39:2293–2299. MULLIN, C. A., CHYB, S., EICHENSEER, H., HOLLISTER, B., and FRAZIER, J. L. 1994. Neuroreceptor mechanisms in insect gustation: A pharmacological approach. J. Insect Physiol. 40:913–931. MURDOCK, L. L., BROOKHART, G., EDGECOMB, R. S., LONG, T. F., and SUDLOW, L. 1985. Do plants “psychomanipulate” insects? pp. 337–351, in P. A. Hedin (ed.). Bioregulators for Pest Control. ACS Symposium Series 276. American Chemical Society, Washington, DC. NAUEN, R., EBBINGHAUS-KINTSCHER, U., ELBERT, A., JESCHKE, P., and TIETJEN, K. 2001. Acetyl- choline receptors as sites for developing neonicotinoid insecticides, pp. 77–105, in I. Ishaaya (ed.). Biochemical Sites of Insecticide Action and Resistance. Springer-Verlag, Berlin. NEAL, J. J. 1989. Myristicin, safrole, and fagaramide as phytosynergists of xanthotoxin. J. Chem. Ecol. 15:309–315. OLIVER, F., AMON, E. U., BREATHNACH, A., FRANCIS, D. M., SARATHCHANDRA, P., KOBZA BLACK, A., and GREAVES, M. W. 1991. Contact urticaria due to the common stinging nettle (Urtica dioica)—Histological, ultrastructural and pharmacological studies. Clin. Exp. Derm. 16:1–7. OSBORNE, R. H. 1996. Insect neurotransmission: Neurotransmitters and their receptors. Pharmacol. Ther. 69:117–142. PALUMBO, J., MULLIS, C., JR., REYES, F., and AMAYA, A. 1997. Evaluation of foliar insecticide approaches for aphid management in head lettuce, pp. 171–177, in N. F. Oebker (ed.). 1997 Vegetable Report. University of Arizona Press, Tucson, Arizona. PICMAN, A. K. 1986. Biological activities of sesquiterpene lactones. Biochem. Syst. Ecol. 14:255–281. PORTER, N. G. 1989. Composition and yield of commercial essential oils from parsley. 1: Herb oil and crop development. Flav. Frag. J. 4:207–219. PRICE, K. R., DUPONT, M. S., SHEPHERD, R., CHAN, H. W., and FENWICK, G. R. 1990. Relationship between the chemical and sensory properties of exotic salad crops—Coloured lettuce (Lactuca sativa) and chicory (Cichorium intybus). J. Sci. Food Agric. 53:185–192. PYTTE, M. and RYGNESTAD, T. 1998. Nutmeg intoxication—A case report. Tidsskr Nor Laegeforen 118:4346–4347. P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

2046 DUSSOURD

REES,S.B.andHARBORNE, J. B. 1985. The role of sesquiterpene lactones and phenolics in the chemical defense of the chicory plant. Phytochemistry 24:2225–2231. REHM, S., ENSLIN, P. R., MEEUSE, A. D. J., and WESSELS, J. H. 1957. Bitter principles of the Cu- curbitaceae. VII: The distribution of bitter principles in this plant family. J. Sci. Food Agric. 8:679–691. REINOLD, S. and HAHLBROCK, K. 1997. In situ localization of phenylpropanoid biosynthetic mRNAs and proteins in parsley (Petroselinum crispum). Bot. Acta 110:431–443. REITZ,S.R.andTRUMBLE, J. T. 1996. Tritrophic interactions among linear furanocoumarins, the herbivore Trichoplusia ni (Lepidoptera: Noctuidae), and the polyembryonic parasitoid Copidosoma floridanum (Hymenoptera: Encyrtidae). Environ. Entomol. 25:1391–1397. ROBERTS,M.F.,MCCARTHY, D., KUTCHAN, T. M., and COSCIA, C. J. 1983. Localization of enzymes and alkaloidal metabolites in Papaver latex. Arch. Biochem. Biophys. 222:599–609. ROEDER, T. 1994. Biogenic amines and their receptors in insects. Comp. Biochem. Physiol. 107C:1–12. ROSHCHINA, V. V. 2001. Neurotransmitters in Plant Life. Science Publishers, Enfield, New Hampshire. SATTELLE, D. B. 1985. Acetylcholine receptors, pp. 395–434, in G. A. Kerkut and L. I. Gilbert (eds.). Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol. 11: Pharmacology. Pergamon Press, New York. SCHENCK, G. 1966. Pharmacology of nitrogen-free bitter principles of Lactuca virosa: Destruction of these substances during extraction and drying, pp. 112–117, in G. S. Sidhu, I. K. Kacker, P. B. Sattur, G. Thyagarajan, and V. R. K. Paramahamsa (eds.). CNS Drugs: A Symposium Held at the Regional Research Laboratory Hyderabad, India, January 24–30, 1966. Sree Saraswaty Press, Calcutta, India. SESSA, R. A., BENNETT, M. H., LEWIS, M. J., MANSFIELD, J. W., and BEALE, M. H. 2000. Metabolite profiling of sesquiterpene lactones from Lactuca species. J. Biol. Chem. 275:26877– 26884. SIMON,J.E.andQUINN, J. 1988. Characterization of essential oil of parsley. J. Agric. Food Chem. 36:467–472. SRIVASTAVA, S., GUPTA, M. M., PRAJAPATI, V., TRIPATHI, A. K., and KUMAR, S. 2001. Insecticidal activity of myristicin from Piper mullesua. Pharm. Biol. 39:226–229. STAHL-BISKUP, E. and WICHTMANN, E. M. 1991. Composition of the essential oils from roots of some Apiaceae in relation to the development of their oil duct systems. Flav. Frag. J. 6:249– 255. STEELINK, C., YEUNG, M., and CALDWELL, R. L. 1967. Phenolic constituents of healthy and wound tissues in the giant cactus (Carnegiea gigantea). Phytochemistry 6:1435–1440. STEPHENS, H. A. 1980. Poisonous Plants of the Central United States. Regents Press of Kansas, Lawrence, Kansas. STEVENS, W. C. 1948. Kansas Wild Flowers. University of Kansas Press, Lawrence. SUTHERLAND,D.W.S.andGREENE, G. L. 1984. Cultivated and wild host plants. US Dept. Agric. Tech. Bull. 1684:1–13. TALLAMY, D. W. 1985. Squash beetle feeding behavior: An adaptation against induced cucurbit defenses. Ecology 66:1574–1579. TALLAMY,D.W.,STULL, J., EHRESMAN,N.P.,GORSKI, P. M., and MASON, C. E. 1997. Cucurbitacins as feeding and oviposition deterrents to insects. Environ. Entomol. 26:678–683. TAMAKI, H., ROBINSON,R.W.,ANDERSON, J. L., and STOEWSAND, G. S. 1995. Sesquiterpene lactones in virus-resistant lettuce. J. Agric. Food Chem. 43:6–8. THOMSON, W. T. 1998. Agricultural Chemicals Book. I: Insecticides, Acaricides and Ovicides. Thom- son, Fresno, California. TIBBITTS,T.W.andREAD, M. 1976. Rate of metabolite accumulation into latex of lettuce and proposed association with tipburn injury. J. Am. Soc. Hort. Sci. 101:406–409. TRIMMER, B. A. 1995. Current excitement from insect muscarinic receptors. TINS 18:104–111. P1: ILT Journal of Chemical Ecology [joec] PP930-joec-470202 August 20, 2003 16:32 Style file version June 28th, 2002

CHEMICAL STIMULANTS OF LEAF-TRENCHING BY CABBAGE LOOPERS 2047

TRUITT, E. B., JR. 1979. The pharmacology of myristicin and nutmeg, pp. 215–222, in D. H. Efron, B. Holmstedt, and N. S. Kline (eds.). Ethnopharmacologic Search for Psychoactive Drugs. Raven Press, New York. TRUITT, E. B., JR., CALLAWAY, E., BRAUDE, M. C., and KRANTZ, J. C., JR. 1961. The pharmacology of myristicin: A contribution to the psychopharmacology of nutmeg. J. Neuropsychiatry 2:205–210. TUNE, R. and DUSSOURD, D. E. 2000. Specialized generalists: Constraints on host range in some plusiine caterpillars. Oecologia 123:543–549. WATLING, K. J. 1998. The RBI Handbook of Receptor Classiﬁcation and Signal Transduction. RBI, Natick, Massachusetts. WINK, M. 1998. Modes of action of alkaloids, pp. 301–326, in M. F. Roberts and M. Wink (eds.). Alkaloids: Biochemistry, Ecology, and Medicinal Applications. Plenum, New York. WINK, M. and SCHNEIDER, D. 1990. Fate of plant-derived secondary metabolites in three moth species (Syntomis mogadorensis, Syntomeida epilais, and Creatonotos transiens). J. Comp. Physiol. B 160:389–400. WU, S. and HAHLBROCK, K. 1992. In situ localization of phenylpropanoid-related gene expression in different tissues of light- and dark-grown parsley seedlings. Z. Naturforsch. 47c:591–600. ZALUCKI,M.P.,CLARKE, A. R., and MALCOLM, S. B. 2002. Ecology and behavior of ﬁrst instar larval Lepidoptera. Annu. Rev. Entomol. 47:361–393. ZANGERL, A. R. 1990. Furanocoumarin induction in wild parsnip: Evidence for an induced defense against herbivores. Ecology 71:1926–1932. ZANGERL,A.R.andBAZZAZ, F. A. 1992. Theory and pattern in plant defense allocation, pp. 363–391, in R. S. Fritz and E. L. Simms (eds.). Plant Resistance to Herbivores and Pathogens: Ecology, Evolution and Genetics. University of Chicago Press, Chicago, Illinois.