Chemical Stimulants of Leaf-Trenching by Cabbage Loopers ... · assayed a series of...

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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-TRENCHINGBY CABBAGE LOOPERS: NATURAL PRODUCTS,

NEUROTRANSMITTERS, INSECTICIDES, AND DRUGS

DAVID E. DUSSOURD1,∗

1Department of BiologyUniversity of Central ArkansasConway, 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 asPlantago lanceolata,are nottrenched. However, if exudate is applied to a looper’s mouth during feeding onP. lanceolata, the larva will often stop and cut a trench. Dissolved chemicals canbe similarly applied and tested for effectiveness at triggering trenching. Withthis assay, I have documented that lactucin from lettuce latex(Lactuca sativa),myristicin from parsley oil (Petroselinum crispum), and lobeline from cardi-nal flower (Lobelia cardinalis) elicit trenching. These compounds are the firsttrenching stimulants reported. Several other constituents of lettuce and parsley,including some phenylpropanoids, monoterpenes, and furanocoumarins had lit-tle or no activity. Cucurbitacin E glycoside found in cucurbits, another plantfamily trenched by cabbage loopers, also was inactive. Lactucin, myristicin, andlobeline all affect the nervous system of mammals, with lobeline acting specifi-cally as an antagonist of nicotinic acetylcholine receptors. To determine if cab-bage 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 includeddopamine, serotonin, the insecticide imidacloprid, and various drugs such asipratropium, apomorphine, buspirone, and metoclopramide. These results doc-ument that noxious plant chemicals trigger trenching, that loopers respond todifferent trenching stimulants in different plants, that diverse neuroactive chem-icals elicit the behavior, and that feeding deterrents are not all trenching stim-ulants. The trenching assay offers a novel approach for identifying defensiveplant compounds with potential uses in agriculture or medicine. Cabbage loop-ers 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]

2023

0098-0331/03/0900-2023/0C© 2003 Plenum Publishing Corporation

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

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

INTRODUCTION

The cabbage looper,Trichoplusia niHubner (Noctuidae), has a broad host rangethat includes over 20 families of plants (Eichlin and Cunningham, 1978; Sutherlandand Greene, 1984). Its catholic diet includes numerous crop plants, particularlycrucifers and lettuce, on which it is an important pest (Flint, 1987). Loopers are ableto feed on such diverse hosts, in part, because of their ability to disable defensiveplant canals by cutting a trench. A larva nibbles back and forth across a leaf, thuscreating a continuous line of bites that isolates a portion of the leaf on which itfeeds. Cabbage loopers are known to cut trenches in composites with latex canalssuch as lettuce (Asteraceae: Lactuceae), umbellifers with oil ducts such as parsley(Apiaceae), and cucurbits such as cucumber (Cucurbitaceae), which exude dropsof sticky phloem sap from damaged tissues (Dussourd and Denno, 1994). Cabbageloopers placed on cardinal flower,Lobelia cardinalis(Campanulaceae), also cuttrenches in this latex-bearing plant (Figure 1). Trenches sever secretory canalsand, thus, reduce the amount of exudate emitted distal to the trench at the looper’sfeeding site (Dussourd, 1999). Diverse plant species that lack exudates are nottrenched (Dussourd and Denno, 1994).

The goal of this study was to identify cues that cabbage loopers use to de-termine which plants to trench. Lettuce (Lactuca sativaL.), parsley (Petroselinumcrispum), cucumber (Cucumis sativusL.), and cardinal flower (Lobelia cardinalisL.) 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).LactucaandLobelialatex andCucumissap all become sticky upon exposure to air. Their adhesivenessshould be easily detected by feeding caterpillars; Lepidoptera larvae have bothmechanoreceptors and chemoreceptors on their maxillae and other mouthparts(Grimes and Neunzig, 1986; Chapman, 1995). However, cabbage loopers exposedto cucumber sap prevented from gelling still trenched, suggesting that anotherfactor triggers the behavior (Dussourd, 1997).

In this paper, I describe a test of the alternative hypothesis that noxious chem-icals in exudates elicit trenching. My approach was to place drops of dissolved

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CHEMICAL STIMULANTS OF LEAF-TRENCHING BY CABBAGE LOOPERS 2025

FIG. 1. Final instar cabbage looper cutting a trench across a leaf ofLobelia cardinalisbefore feeding on the distal tip to the left.

chemicals on the mouthparts of loopers feeding onPlantago lanceolataL.(Plantaginaceae), a plant that normally is not trenched. Loopers that receive dropsof lettuce latex (Lactuca serriola) or cucumber sap often stop feeding and cut atrench, responding as though the exudate is emitted by the plantain leaf(Dussourd, 1997). I tested if single chemicals similarly trigger trenching, andif different plants have the same or different trenching stimulants. Three naturalproducts that elicit trenching were identified; all three have been reported to affectthe nervous system of mammals. One of the compounds, lobeline, acts specifi-cally as an antagonist at nicotinic acetylcholine receptors (Dwoskin and Crooks,2001, 2002). Another of the trenching stimulants, myristicin, is thought to haveanticholinergic activity (Lavy, 1987; Pytte and Rygnestad, 1998). To determineif loopers respond selectively to compounds that affect acetylcholine synapses, Iassayed a series of neurotransmitters, insecticides, and drugs. These trials test ifcabbage loopers respond only to a few specific trenching stimulants from plantexudates, more generally to various compounds acting on acetylcholine recep-tors, or if diverse neuroactive chemicals elicit trenching. Cabbage loopers rou-tinely feed on plants in the Asteraceae and Apiaceae (Dussourd and Denno, 1994,and references therein), but members of the Campanulaceae, such asLobeliacardinalis, have not been previously reported as host plants (Sutherland andGreene, 1984). The final objective was to determine how frequently cabbage loop-ers trenchL. cardinalisand to test if the larvae can develop on this species.

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METHODS AND MATERIALS

Trenching Assay.Cabbage loopers from a laboratory culture were reared tothe final instar on potted plantain,Plantago lanceolata. Plantain does not emitexudate and is not trenched. Thus, the loopers used in this assay had no priorexperience with exudates or trenching. Early final-instars were deprived of foodfor 1–1.5 hr at 24–27◦C, then were allowed to walk onto the underside of anexcised plantain leaf held in a water pic to maintain turgidity. For consistencybetween trials, only flat mature leaves with a maximum width of∼1.2 to 2.2 cmwere used. When a larva initiated feeding, a drop of solution was carefully applieddirectly to the side of its mouth with a 5-µl Wiretrol micropipette. Each larvareceived a total of 1.5-µl solution dispensed in a sequence of four to six drops,with the exception that larvae tested with 60% acetone solutions typically receivedonly three drops because of rapid evaporation of solvent. Multiple droplets wereapplied to simulate the natural outflow of exudate that occurs when larvae severcanals while attempting to feed on plants with exudates. With innocuous solutions,such as water controls, larvae consumed the drops while continuing to feed withoutshowing any signs of disturbance. Drops were sucked into the mouth and swallowedduring normal feeding. With deterrent solutions, larvae usually stopped feedingimmediately, wiped their mouth vigorously on the leaf, opened and closed theirmandibles repeatedly, and then moved to a new feeding site. Each additional dropof solution was applied only after the larva recommenced feeding. I recorded larvalresponse to each droplet of solution and whether larvae interrupted feeding to cut atrench. For this study, a trench was defined as a continuous line of bites extendingat least 5 mm partially or completely across the leaf. For consistency betweentrials, all assays were performed by the author. Each caterpillar was tested onlyonce.

Whenever possible, chemicals were tested in water. Compounds with inad-equate solubility were dissolved in aqueous solutions of either 10% Tween 80(a surfactant) or the minimal concentration of acetone required (10–60%). BothTween 80 and acetone at these high concentrations deterred feeding by cabbageloopers and occasionally triggered trenching. The solutions were each tested with10 naive larvae, except for the two pesticides methomyl and methamidophos,which were extremely toxic to the cabbage loopers and were tested with onlyfive larvae each. Chemicals were tested at a standard dose of 50 nmol in 1.5µl(=33.3 mM). The only exceptions were lactucin and bergapten, which had poorsolubility in water and required unacceptably high concentrations of solvent todissolve at 33.3 mM. Instead, both were tested at 10 nmol in 1.5µl (=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; Sessaet al., 2000). Lactucin was isolated from the latex ofLactuca virosaL. (generously

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supplied by M. H. Bennett, University of London) and tested at both 5 and 10 nmolper 1.5µl to match concentrations reported in the latex of cultivated lettuce (Sessaet 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 havea widespread distribution in plants (Harborne and Baxter, 1993).

Parsley,Petroselinum crispum(Mill.) Mansf., produces a complex mixture ofphenylpropanoids, 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 twophenylpropanoids, myristicin and parsley apiole; three linear furanocoumarins,xanthotoxin, imperatorin, and bergapten; and two monoterpenes,α-pinene andγ -terpinene.

Lobelia cardinalisL. and the medicinal plantL. inflata L. (Indian tobacco)both contain the piperidine alkaloid, lobeline (Krochmal et al., 1972). These plantswere used by Native Americans and settlers in the United States for diverse medici-nal purposes, including as an emetic and respiratory stimulant; lobeline is believedto be the principal active compound (Stephens, 1980; Fodor and Colasanti, 1985;Kindscher, 1992). I tested if lobeline deters feeding by cabbage loopers and if ittriggers trenching.

Cucurbits are well-known for the presence of bitter triterpenes named cucur-bitacins. The compounds occur throughout the plant including the leaves (Rehmet al., 1957; Guha and Sen, 1975), but are apparently absent from sap exudate(Tallamy, unpublished data, cited in McCloud et al., 1995). Early studies withsquash 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 stimulatefeeding by squash beetles and that trenching serves instead to reduce beetle expo-sure to sap exudate (McCloud et al., 1995). I used the cabbage looper bioassay totest if cucurbitacin E glycoside triggers trenching. The cucurbitacin sample waskindly provided by D. W. Tallamy, University of Delaware. The aglycone or glyco-side of cucurbitacin E occurs in diverse cucurbits including watermelon,Citrullusvulgaris (Rehm et al., 1957; Guha and Sen, 1975), a plant that cabbage looperstrench (Dussourd, unpublished observation).

Each natural product was tested independently in a randomized series witha solvent control (except for chlorogenic acid andγ -terpinene, which were testedalone). Fisher exact tests were used to compare the number of larvae cuttingtrenches in response to the test chemical and control.

The survey of natural products described earlier identified three compoundswith significant trenching activity. To verify activity, each of the compounds wasretested with a solvent control using a new cohort of cabbage loopers. Myristicinand lobeline were each tested with 20 larvae, whereas lactucin was tested with 10.

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To determine if trenching is triggered specifically by compounds acting onacetylcholine receptors, I used the cabbage looper assay to test the activity ofacetylcholine and several drugs that affect acetylcholine synapses. These com-pounds comprised an agonist for nicotinic cholinergic receptors, (−)-nicotine; anagonist for muscarinic cholinergic receptors, (+)-muscarine chloride; three antago-nists for nicotinic cholinergic receptors, decamethonium bromide, succinylcholinechloride, andD-tubocurarine chloride; an antagonist for muscarinic cholinergicreceptors, ipratropium bromide; and two anticholinesterases, pyridostigmine bro-mide and edrophonium chloride. The compounds were tested in random ordertogether with an aqueous control.

Insecticides acting on acetylcholine synapses were also tested. They includedthe 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 at-tacked 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 organophos-phorous insecticide, methamidophos. Both compounds are used to control cab-bage 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 sys-tem (Benson, 1993) and most sensory information is delivered to the CNS throughthe release of acetylcholine (Trimmer, 1995). To determine if cabbage loopersrespond only to chemicals that bind to acetylcholine receptors, I tested four addi-tional compounds that act as neurotransmitters, neuromodulators, and/or neurohor-mones: dopamine, serotonin, octopamine, and GABA. To test if loopers responddifferently to stimulants and inhibitors, as documented with other insects (Longand Murdock, 1983; Mullin et al., 1994; Cohen et al., 2002), I also assayed thefollowing drugs: R(−) apomorphine hydrochloride and fluphenazine dihydrochlo-ride, agonist and antagonist of dopamine receptors; buspirone hydrochloride andWAY-100635 maleate, agonist and antagonist of serotonin receptors; clonidineand metoclopramide, agonist and antagonist of octopamine receptors; muscimolhydrobromide and (-) bicuculline methobromide, agonist and antagonist ofγ -aminobutyric acid (GABA) receptors. Testing the 12 compounds required severaldays, a sufficiently long time that decomposition could occur if the compoundswere tested in a random sequence. Therefore, the 12 chemicals were tested one ata time.

Drug activities are based on studies with either insect (Benson, 1993; Roeder,1994; Burrows, 1996; Osborne, 1996; Aydar and Beadle, 1999) or mammalianmodels (Hardman and Limbird, 1996; Watling, 1998). Where information is

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available, most compounds are active in both groups. A notable exception isbicuculline, a diagnostic GABAA antagonist in vertebrates that is largely inac-tive 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 candevelop onLobelia cardinalis, 20 newly eclosed first instar larvae were placedin clip cages on newly mature leaves of pottedL. cardinalis that had not yetbolted. The clip cages were padded to provide a seal over the leaf surface withoutcompressing the latex canals (laticifers). Each larva was enclosed on a separateplant. As a control, 40 newly emerged first instars were offered excised leaves ofL. cardinalis cut in half to deplete the laticifers. Fresh leaves were provided asneeded, typically daily.

Cabbage loopers begin trenching in the second instar (Dussourd, 1993). Totest if trenching allows cabbage loopers to feed onL. cardinalis, 20 early thirdinstars 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 ofL. cardinalis.Larvae were checked daily for trenching and feeding.

In nature, newly eclosed larvae are not isolated in clip cages; they canroam freely to find suitable food. To test if unrestrained larvae can develop onL. cardinalis, I placed 20 newly eclosed first instars each on a separate non-bolted plant surrounded by a water moat to prevent dispersal. As a control, 40newly eclosed larvae were again offered excisedL. cardinalisleaves. Larvae in allLobeliaexperiments were held indoors at∼25◦C.

RESULTS

Trenching Bioassays.Of the 12 natural products tested from potential hostplants of cabbage loopers, only 3 triggered significant trenching: the sesquiter-pene lactone, lactucin, from lettuce latex; the phenylpropanoid, myristicin, fromparsley oil; and the alkaloid, lobeline, fromLobelia(Table 1). Retesting the threecompounds 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), threefuranocoumarins, and two monoterpenes from parsley (Table 1). Cucurbitacin Eglycoside 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 com-pounds in Table 1 caused substantial mortality; with most chemicals, 100% of thelarvae survived to produce normal moths.

Acetylcholine did not affect looper behavior at the dose tested, but four ofthe eight drugs active at acetylcholine synapses elicited trenching (Table 2A).

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TABLE 1. CABBAGE LOOPERTRENCHING IN RESPONSE TONATURAL PRODUCTS

Solutiona % Trenchingb

Asteraceae:Lactuca1. 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) 030% Acetone control 0

3. Chlorogenic acidc (50 nmol/larva in water) 0Apiaceae: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) 010% Tween 80c control 0

6. Xanthotoxinc (50 nmol/larva in 60% acetone) 060% Acetone control 0

7. Imperatorine (50 nmol/larva in 60% acetone) 2060% Acetone control 0

8. Bergaptenf (10 nmol/larva in 60% acetone) 060% Acetone control 20

9. (1R)-(+)-α-Pinenef (50 nmol/larva in 10% Tween 80) 2010% Tween 80c control 0

10.γ -Terpinenef (50 nmol/larva in 10% Tween 80) 0Campanulaceae:Lobelia11. (-)-Lobeline hydrochloridef (50 nmol/larva in water) 70**

Water control 0Cucurbitaceae12. 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 = 10larvae/solution.

b Fisher’s exact tests compare the number of larvae cutting a trench inPlantago lanceolatain 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 controlsin different experiments have ever cut trenches (Dussourd, 1997, unpublisheddata). Furthermore, none of the thousands of cabbage loopers that I have raisedon Plantago lanceolatahave ever been observed to trench during rearing. Thefour compounds that triggered trenching included both agonists and antagonists

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FIG. 2. Percentage of cabbage loopers cutting a trench inPlantago lanceolataafter receiving10 nmol lactucin in a 50% acetone solution, 50 nmol myristicin in 10% Tween 80, or50 nmol lobeline in water. Each chemical was tested independently with a control, whichwas the same solution used to dissolve the test chemical. Lactucin and its control wereeach 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 exacttests, **0.01> P> 0.001, *** P < 0.001).

of acetylcholine receptors. The two anticholinesterases tested were both inactiveas trenching stimulants (as were the anticholinesterase insecticides, methomyland methamidophos, Table 2B). Nicotine caused one larva to regurgitate ontothe leaf; none of the other compounds caused overt poisoning. Larvae survivedtheir exposure to all compounds; 90–100% of the larvae produced moths for allcompounds tested.

Of the four insecticides tested, only imidacloprid triggered significant trench-ing (Table 2B). Two larvae showed clear signs of poisoning; one trenched half wayacross the leaf before falling off, regurgitating, and quivering with spasms. Oneof the loopers tested with cartap likewise completed only a partial trench beforebecoming immobilized and unresponsive. A second larva had just begun trenchingwhen it too was immobilized by cartap. All of the larvae tested with imidaclopridand cartap eventually resumed feeding and developed into adults of normal appear-ance. In contrast, both methomyl and methamidophos were fatal. Surprisingly, thelarvae scarcely responded to drops of these insecticides. Either the loopers wereunable to detect methomyl and methamidophos or the compounds were not de-terrent. Shortly after ingesting the entire dose, the larvae regurgitated, convulsed,fell from the leaf, and eventually became immobilized. The lack of larval responseeven to a lethal dose undoubtedly increases mortality when the insecticides areused against cabbage loopers on crops.

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TABLE 2. CABBAGE LOOPERTRENCHING IN RESPONSE TODRUGS

AND INSECTICIDES THATAFFECTACETYLCHOLINE SYNAPSES

Solutiona % Trenchingb

A. Drugs1. Acetylcholine chloridec 0

(−)-Nicotinec 30(+)-Muscarine chloridec 0Decamethonium bromided 40*Succinylcholine chloridec 0D-Tubocurarine chlorided 20Ipratropium bromidec 70**Pyridostigmine bromided 0Edrophonium chlorided 0Water control 0

B. Insecticides2. Imidaclopride in 30% acetone 40*

30% Acetone control 03. Cartap hydrochloridee 104. Methomyle 05. Methamidophose 06. Water control 0

a Numbers on left indicate solutions tested together in a randomizedsequence. All larvae received 50 nmol test chemical dissolved in 1.5-µlsolvent. Unless indicated otherwise, all chemicals were dissolved in water.N = 10 larvae/solution, exceptN = 5 for methomyl and for methami-dophos.

b Fisher’s exact tests compare the number of larvae cutting a trench inPlantago lanceolatain 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 trenchingstimulant. GABA had little affect on looper behavior at the dosage tested. The lowactivity of GABA is not surprising since GABA is known to stimulate feedingby diverse herbivores (Mullin et al., 1994). All drugs tested that affect dopamine,serotonin, and octopamine receptors elicited trenching, whereas only the antagonistof 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 oftenwiped solution off their mouthparts onto the leaf, sometimes repeatedly with eachapplication. The larvae frequently withdrew their head away from the leaf, then

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FIG. 3. Percentage of cabbage loopers that cut trenches inPlantago lanceolataleaves afterreceiving 50 nmol of the test chemical dissolved in 1.5-µl water. Each chemical was testedwith 10 final instar larvae. The chemicals included four neurotransmitters (dopamine, sero-tonin, octopamine, GABA), plus an agonist and antagonist of receptors stimulated by eachneurotransmitter. The three biogenic amines may act partly or primarily as neuromodula-tors or neurohormones in insects (Burrows, 1996). Asterisks indicate statistically greatertrenching 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. Somelarvae lowered their head to their legs to groom legs and mouthparts. Larvae oftenabandoned their feeding site and moved to another location on the leaf beforecontinuing to feed.

Compounds that triggered trenching were especially deterrent. For example,of the nine chemicals tested that affect acetylcholine synapses (Table 2A), thefour that triggered trenching were more deterrent than the five that did not. Larvaeexposed to the trenching stimulants were more likely to wipe their mouth on theleaf (9.5± 0.3 larvae/chemical; mean±1 SE) than larvae tested with the otherfive chemicals (1.8± 0.7, 0.05> P> 0.01, Mann–WhitneyU test). Likewise,larvae tested with the four trenching stimulants were more likely to withdraw andexhibit 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 feedingsites (6.8± 0.6 vs. 0.6± 0.4, 0.05> P> 0.01, Mann–WhitneyU ). Nearly all

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of the 132 loopers that trenched in this study exhibited signs of deterrence beforetrenching: 95% showed mouth-wiping, 98% chewing motions, 13% grooming,and 67% abandoning feeding sites (not including when larvae stopped feedingto trench). For chemicals tested in water, deterrency can be attributed directly tothe test chemical. Lobeline, for example, triggered mouth-wiping in 100% of the30 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 deter-rent solutions caused trenching. Xanthotoxin elicited vigorous repetitive mouth-wiping; 7 of 10 larvae left the leaf after receiving just one or two drops, but nonecut a trench. Excluding data for xanthotoxin, only 29 of 650 larvae tested in thisstudy walked off the leaf; larvae were reluctant to move onto the water pic andusually resumed feeding nearby. Xanthotoxin retested at a range of concentrationsagain was highly deterrent. Fewer larvae abandoned the leaf at lower xanthotoxindoses, but none trenched (Figure 4). Other deterrent solutions also had little orno activity as trenching stimulants. For example, bergapten in 60% acetone andterpinene 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%, andabandoning feeding sites in 50 and 60%; neither solution triggered trenching.

The naive larvae in this study required repeated contact with solutions beforethey initiated trenching. Only 6% started trenching after receiving the first drop,

FIG. 4. Effect of xanthotoxin on cabbage loopers. Each concentration of xanthotoxin wastested independently with a control, which was the solvent alone. Ten loopers were testedwith each solution; each received 1.5µl. Asterisks indicate a statistically significant differ-ence in the number of loopers abandoning the leaf comparing the xanthotoxin solution andcontrol (Fisher’s exact tests, **0.01> P > 0.001). Xanthotoxin was highly deterrent, butdid not trigger trenching at any of the concentrations tested.

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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 cutonly a short trench (52%) before continuing to feed. After encountering an addi-tional drop(s) of the test chemical, the larvae resumed trenching, as though theirfirst attempt to disrupt leaf canals had been inadequate and further trenching wasnecessary. Only 39% of the larvae cut a continuous trench across the entire leafthe first time that they trenched. This indicates that trenching is not a fixed actionpattern that is invariably carried through to completion on first attempt. On thecontrary, larvae appear to require continued stimulation to persevere at trenching.On plants with laticifers, trenching causes latex to flow onto the trench. Larvaepresumably encounter trenching stimulants in latex continuously until all laticiferscrossing the trench have been severed, at which point the larvae stop trenching andbegin to feed.

Cabbage Looper Survival on Lobelia.First instar cabbage loopers enclosedon pottedLobelia cardinalisleaves all died within 2–6 days (Figure 5). Only onereached the second instar and it died shortly thereafter. Larvae ate small pits inthe leaves, often causing latex to flow into the pits. Two larvae died with theirheads glued to the leaf by latex. Third instar larvae sleeved onL. cardinalisalsoperished. Eighteen of the 20 larvae cut one or more trenches, mostly across leaftips; however, the larvae did not feed beyond 40% of the trenches. When theseintact leaf tips were severed with scissors, latex oozed readily from all of themdocumenting that the trenches failed to sever all laticifers crossing the trench. Thirdinstars also ate small pits in the leaves and fed on the midrib. The larvae survivedup to 11 days (mean= 5 days), but none molted to the fourth instar.

In contrast, most final instar cabbage loopers enclosed onL. cardinaliscuttrenches 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 ofLobeliacardinalis. Twenty larvae in the first instar, 20 in the third instar, and 10 in the fifth instarwere tested on intact leaves, whereas 40 first instar larvae were offered excised leaves.

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completely consumed the distal tips in 81% of them. Seven larvae pupated, butonly five adults emerged from the cocoons. The three larvae that failed to pupatefed beyond only 10 of their 20 trenches. The uneaten tips readily released latexwhen severed indicating that the larvae were often unsuccessful in deactivating thelatex system.

Excised leaves ofL. cardinaliswere 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 entireL. cardinalisplantsalso perished. However, four of the larvae reached the second instar by feedingextensively on senescent or damaged leaves, as well as by pit-feeding. The fourlarvae 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 severeddocumenting that some latex canals remained intact. As previously, the larvae onexcised leaves fed readily; 90% survived to pupate and 87.5% produced an adultmoth.

DISCUSSION

Isolated chemicals sufficed to elicit trenching by cabbage loopers. These com-pounds were active in the absence of other exudate components; notably, solutionsdid not have to be sticky to stimulate trenching. Effective stimulants included adiversity of structures (Figure 6) suggesting that either multiple receptors are in-volved in their detection or the receptors are broadly tuned. Three constituents ofpotential food plants caused significant trenching: lactucin from lettuce, myristicinfrom parsley, and lobeline fromLobelia. As described below, these compounds areknown or suspected to have toxic properties, suggesting that loopers cut trenchesspecifically to reduce their exposure to noxious substances during feeding.

Lactucin is classified as a sesquiterpene lactone, a diverse group of almost3500 reported structures known for cytotoxic, antibacterial, antifungal, and al-lelopathic properties (Picman, 1986). Several sesquiterpene lactones have docu-mented deterrency or toxicity to insect herbivores (Picman, 1986; Gershenzon andCroteau, 1991; Mullin et al., 1991). To my knowledge, lactucin has not been testedpreviously for its effects on insects. However, two other sesquiterpene lactonesreported 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 complexmixture that includes oxalate and sulfate conjugates (Sessa et al., 2000). Lactucinis only a minor component. It reaches a maximum level of∼2.8 mg/ml latex inbolted plants, whereas the total sesquiterpene lactone titer of these plants averages147.1 mg/ml latex (Sessa et al., 2000). Titers of sesquiterpene lactones in the latex

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

of wild Lactucaspecies, notablyL. virosa, can be much higher (Tamaki et al.,1995). In this study, lactucin triggered trenching at 0.92 mg/ml (=5 nmol/1.5µl),documenting that the compound is active at natural lactucin levels. Presumablythe abundant conjugates and derivatives of lactucin contribute to the trenchingactivity of Lactucalatex. Cabbage loopers feed extensively on composites in thefield, including cultivated and wild lettuce (L. sativa, L. serriola, L. canadensis)and other latex-bearing composites such asSonchus asper, Taraxacum officinale,andPyrrhopappus carolinianus(Dussourd and Denno, 1991, 1994). I have foundas many as 20 cabbage loopers on individual plants ofL. serriola andS. asper,which can be completely stripped of leaves. The loopers routinely cut trenches inthese species, apparently due specifically to the sesquiterpene lactones that theymust encounter often in these important food plants.

Cabbage loopers also cut trenches in multiple species of Apiaceae, includ-ing parsley, carrot, and parsnip (Dussourd and Denno, 1991; Zangerl and Bazzaz,1992). All three species produce myristicin (Lichtenstein and Casida, 1963;

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Kubeczka and Stahl, 1977; Porter, 1989; Hallstrom and Thuvander, 1997). Myris-ticin inhibits detoxification enzymes (mixed function oxidases) in caterpillars andthereby synergizes the activity of insecticides and natural toxins such as fura-nocoumarins (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 oftwo generalist caterpillars, the corn earworm and southern armyworm (Lichten-stein and Casida, 1963; Berenbaum and Neal, 1985). Myristicin levels vary greatlyin different parsley accessions, ranging from 0.1% to over 60% of the essentialoils extracted from whole plants (Simon and Quinn, 1988; Porter, 1989). Whenpresent at high concentration, myristicin is undoubtedly an important trenchingstimulant. Whether additional oil components such as the nonpolar sesquiterpenecaryophyllene also trigger trenching remains to be determined.

Surprisingly, furanocoumarins were ineffective trenching stimulants, eventhough these compounds occur in the oil ducts (Camm et al., 1976; Wu andHahlbrock, 1992; Reinold and Hahlbrock, 1997) and are toxic to diverse her-bivores (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 stronglydeterrent (Figure 4). Parsley apiole also was inactive at the dosage tested, despiteits structural similarity to myristicin. Cabbage loopers clearly do not trench in-discriminately in response to all allelochemicals. A further example is providedby cardenolides, which are extremely deterrent and toxic to cabbage loopers, butdid not cause trenching when tested at 25µg/larva (=33 nmol/larva for digitoxin;Dussourd and Hoyle, 2000).

Little information is available on how lobeline affects insects, although it hasbeen reported to deter feeding by a ctenuchid caterpillar and honeybees (Wink andSchneider, 1990; Detzel and Wink, 1993). Mammalian herbivores (cows, sheep,goats) that feed on variousLobeliaspecies suffer negative effects that can includesluggishness, hemorrhage, and coma; overdoses of lobeline produce similar effects(Dollahite and Allen, 1962; Kingsbury, 1964).Lobelia cardinaliscontains 7 mglobeline/g dry weight, which is comparable to levels in the medicinal plant,L.inflata (Krochmal et al., 1972). Overdoses of lobeline orL. inflata in humanscause 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 fromthe loopers even though cucurbitacins are intensely bitter to humans (Metcalf etal., 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 oncabbage looper feeding (Tallamy et al., 1997). Trenching activity in cucumber sap

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has been traced not to cucurbitacins, but to a polar fraction (Dussourd, 1997) thatcontains at least two active components. One of the components has been partiallycharacterized as anN-alkyl pyridinium salt (Capron, Wiemer, and Dussourd, un-published data). Lettuce, parsley,Lobelia, and cucumber thus contain strikinglydifferent trenching stimulants: a sesquiterpene lactone, phenylpropanoid, and twoalkaloids; there is no common releaser for trenching in these diverse plants. Thesolvents used to dissolve less polar compounds in this study sometimes also elicitedtrenching, 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 (Dussourdand Denno, 1994). However, loopers tested with the trenching assay had not pre-viously experienced exudates. In addition, the larvae received only 4–6 dropletsof solution, unlike loopers on intact plants which contact exudates repeatedly dur-ing trenching (Dussourd, 1999). It is also unlikely that lactucin, myristicin, andlobeline are the only trenching stimulants in lettuce, parsley, andLobelia. L. car-dinalis, for example, contains multiple piperidine alkaloids (Gupta and Spenser,1971; Krochmal et al., 1972), in addition to lobeline. Although not all allelochem-icals were tested, this study documents that isolated chemicals at biologically rele-vant concentrations suffice to elicit trenching and provides a method for screeningadditional compounds for trenching activity.

Of the four insecticides tested, imidacloprid was the least harmful for theloopers and the most effective at triggering trenching. Imidacloprid is commonlyused to control sucking insects, fleas, and beetles; it is generally less effectiveagainst caterpillars (Thomson, 1998). Methomyl and methamidophos were bothlethal at the dosage used, whereas cartap caused temporary immobility in all lar-vae. Cartap is convertedin vivo to nereistoxin, which blocks cholinergic synaptictransmission 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 tubocu-rarine, a constituent of curare arrow poison (Hardman and Limbird, 1996), andnicotine, a contact insecticide from tobacco used to kill insect pests for over 300years (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 blowfliesand cockroaches, octopamine and octopamine agonists delivered by injectionor by ingestion increase feeding, but octopamine antagonists decrease feeding(Long and Murdock, 1983; Cohen et al., 2002). Likewise, applying agonists forGABA/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,

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and sometimes reacted differently to compounds with similar modes of action. Forexample, octopamine was inactive at the dosage tested, whereas the octopamineagonist, clonidine, elicited trenching in 5 of 10 larvae (Figure 3). The similar ac-tivity of agonists and antagonists suggests that neurotransmitters and drugs triggertrenching by stimulating peripheral chemoreceptors and not through direct contactwith the CNS following absorption from the gut. This conclusion is supported bythe rapid response of some larvae; for example, one looper initiated trenching afterreceiving just a single drop of dopamine.

The three natural trenching stimulants, lactucin, myristicin, and lobeline, allhave been reported to affect the nervous system of mammals. Latex from wildLactucaspecies (lactucarium) has been used medicinally as a sedative and ananalgesic for hundreds of years (Stevens, 1948; Crellin and Philpott, 1990). Lab-oratory studies with mice suggest that lactucin is at least partly responsible forits activity (Schenck, 1966; Gromek et al., 1992). Myristicin injected in variousmammals causes behavioral depression, ataxia, and disorientation (Truitt et al.,1961; Florio et al., 1972; Truitt, 1979). Higher doses in monkeys lead to respira-tory arrest (Truitt et al., 1961). Lobeline has multiple pharmacological effects. Itacts as a potent antagonist atα3β2 andα4β2 neuronal nicotinic receptor subtypes(Dwoskin and Crooks, 2001). Lobeline also alters dopamine storage and utilizationapparently by inhibiting dopamine uptake by synaptic vesicles and by stimulatingthe release of dopamine from the vesicles into the cytosol (Dwoskin and Crooks,2002). Interestingly, with all three trenching stimulants, plant species containingthe 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 treatmentfor 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 systemof cabbage loopers, although none have been tested for neurotoxicity to insects.

Neurotoxins, including many alkaloids, are abundant in plants (Murdocket al., 1985; Hartmann, 1991; Wink, 1998). The toxins are sometimes sequesteredin 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 awidespread distribution in plants (Roshchina, 2001), and sometimes occur at highconcentration (Steelink et al., 1967; Oliver et al., 1991). In poppy latex (Robertset al., 1983), dopamine concentrations reach 2.66 mg/ml (=17.4 mM), close to thelevels that elicited trenching in this study (33.3 mM).

Despite their ability to trench, cabbage loopers were unable to develop onintact Lobelia cardinalis. Early instar larvae ate pits in the leaves, but failed todevelop on potted plants, except on senescent or damaged leaves. Third and fifthinstar loopers cut trenches, but only the larger larvae were able to feed effectively.

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Excised leaves, in contrast, were highly acceptable, presumably because excisiondepressurizes 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 thatcan include larval stage (Zalucki et al., 2002) and prior damage (Lewis, 1984).

In summary, the results reported herein support the hypothesis that cabbageloopers cut trenches specifically to deactivate secretory canals in host plants andthereby reduce their ingestion of poisons that may often include neurotoxins.However, trenching ability alone is inadequate to permit feeding. Trenchers mustalso be able to tolerate high levels of exudate chemicals during trenching andlower levels during subsequent feeding. The known toxicity of the three identifiedtrenching stimulants suggests that cabbage loopers use host toxins specifically ascues for trenching.

Many questions remain. Are lactucin, myristicin, and lobeline poisonous tocabbage loopers, and do these compounds affect their nervous system? Lactucinis known to occur in the latex ofLactuca(Sessa et al., 2000). Is lobeline sim-ilarly sequestered inLobelia latex, and is myristicin stored in the oil ducts ofPetroselinum? How much of these compounds do cabbage loopers ingest duringtrenching and during subsequent feeding on distal tissues with depleted canals?Why do some feeding deterrents trigger trenching, whereas others do not? Do thediverse compounds that elicit trenching all stimulate the same taste receptor(s)?Cabbage loopers are classified in the plusiine tribe Argyrogrammatini (LaFontaineand Poole, 1991) together with other trenching species that includeEnigmogrammabasigeraand the soybean looper, Pseudoplusia includens. These three species dif-fer substantially in host range. Cabbage loopers, for example, cannot develop onundamagedLobelia cardinalis, butE. basigeracut trenches and develop rapidly topupation on intact plants in both lab and field (Dussourd, unpublished observation).Likewise, soybean loopers, Pseudoplusia includens,develop poorly onLactucaserriola, 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, andLobelia all have canal systems that deliver exudateswith 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 severveins or cut trenches before feeding (Dussourd and Denno, 1991; Dussourd, 1993and references therein; Becerra, 1994; McCloud et al., 1995; Jolivet, 1998; Clarkeand Zalucki, 2000; Becerra et al., 2001). Given that over 20,000 species of plantscontain latex or resin canals (Farrell et al., 1991; Lewinsohn, 1991), that up to7 species of vein cutters have been reported per plant species (Dussourd and

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Denno, 1991), and that some latex-bearing species are attacked by over 125species of leaf-feeding insects (Basset and Novotny, 1999), the true number ofinsects exhibiting these behaviors undoubtedly numbers in the thousands. Plantsmanipulated with vein cuts or trenches include members of over a dozen families(Dussourd, 1993). The insect behaviors provide a convenient assay for identify-ing 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 stimulatetrenching by soybean loopers (Pseudoplusia includens), what chemicals in pa-paya and cassava elicit trenching by the cassava hornworm (Erinnyis ello), andwhat substances in milkweed latex trigger vein severance by monarchs (Danausplexippus). The results reported here suggest that trenching stimulants are likelyto be important chemical defenses for plants, not just harmless cues. Vein cut-ting and trenching assays, thus, have the potential to direct the isolation of novelstructures with insecticidal properties and possible applications in agriculture ormedicine.

Acknowledgments—Thanks to M. H. Bennett for providing lactucin; to D. W. Tallamy for pro-viding 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 anonymousreviewers for insightful comments on earlier versions of the manuscript; to K. Poole and W. L. Roelofs(Geneva Experiment Station) for repeatedly supplyingT. nieggs; to H. Dukat, M. M. Atwell, and M. R.Helmus for assistance in the greenhouse and lab; and to the University of Central Arkansas ResearchCouncil for financial support.

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