INDUCTION OF LINEAR FURANOCOUMARINS IN CELERY, APIUM

GRAVEOLENS BY INSECT DAMAGE AND THElR EFFECTS ON LYGUS

LINEOLARIS AND THE PARASITOID PERSITENUS STYGICUS.

Pmsented to

The Faculty of Graduate Studies

of

The University of Guelph

by DIANE E. STANLEY-HORN

In partial fulfillment of requirements

for the degree of

Master of Science

June, 1999

The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distriiute or seU reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriete du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT

INDUCTION OF LINEAR FURANOCOUMARINS IN CELERY, APUM

GRAVEOLEUS BY INSECT DAMAGE AND THElR EFFECTS ON LYGUS

LINEOURIS AND THE PARASITOID PERlSTENUS SWGICUSa

Diane E. Stanley-Hom Advisor:

University of Guelph Professor M, K, Sears

Celery, Apium graveolens, produces three phototoxic linear furanocoumarins (LFCs) that can cause a photoàennatitic reactian in humans

in the presence of UV radiation. LFCs are toxic to numerous organisms and

are both constituüvely produced and induœd in response to several abiotic

and biotic stresses. Induction of LFCs by the tamished plant bug, Lygus lineolans UPB) and the cabbage looper, Tnchoplusia ni, and effects of LFCs on growth and development of TPB and the parasitoid Pemitenus stygicus were examined. 60th insects induced LFCs in celery, but concentrations in petioles rarely reached levels capable of causing photodermatitis.

Developmental rates of TPB were affected by ingestion of LFC-containing diet. However, other factors are likely to accaunt for the difierences obsewed between nymphs fed damaged and undarnaged celery tissue. The data suggest that other compounds in celery petioles may be of value to breeding programs involved in the development of insect-resistant genotypes. Acknowledgements

Iwould like to express my appreciation to the following people for their support and encouragement during the fuffillment of my degree. First, 1 would

Iike to thank Dr. Mark Sears for al1 of the advice and encouragement throughout this project and for always providing me with an alternative perspective. I would also like to thank Drs. Geny Stephenson and Rick Yada for their thoughtful advice and enthusiasm for my research. I am also grateful to the Ontario govemment for their financial support through an Ontario

Graduate Scholarship.

The completion of this project would not have been possible without the assistance of Linda Veldhuis, who willingly and patiently helped me battle the sometimes 'ternperamental' HPLC apparatus and to Marg Carter for her cornputer expertise. I would also like to thank al1 of my wonderful friends who made my experience at the University of Guelph so much fun: Simon

Lachance, Traœy Baute. Kurt Randall. Sarah Butler, Claudia Sheedy, Sarah

Rosloski, Laura VanEerd and the rest of the EVB gang. Finally. want to acknowledge my wondefil husband Greg for his love. patience and encouragement and my ever-supportive farnily. TABLE OF CON7ENTS .... Page Acknowledgements ...... m..m....i

Table of contents ...... ii

List of tables ...... iv List of figures ...... m...... m.m.....mmv List of appendices ...... vi Literature review ...... m...... 1

1. 1 Introduction...... 1 1.2 Toxicity of LFCs in humans ...... -2 1.3 Biosynthesis of LFCs ...... 3 1.4 Distribution of LFCs in Plants...... 6 1.5 LFCs as Inducible Resistance Factors ...... 10 1-51 Interactions with Pathogens...... 11 1.52 Interactions wifh lnsects ...... 12 1-6 Tritrop hic lnteractions ...... 16 1.7 The Celery-Pest Complex in Ontario ...... 17 1-71 The tamished plant bug, Lygus lineolaris (P . de B.) ...... 17 1-72 The ca bbage looper. Trichoplusia ni(Hu bner) ...... 21

Chapter 2: Induction of three phototoxic linear furanocoumarins celery. Apium graveolens L., in response to insect feeding damage

2.1 Abstract ...... 25 2.2 Introduction ...... ,.._...... 26 2.3 Objectives ...... 29 2.4 Materials and Methods ...... 30 2.41 Plant and insect sources ...... 30 2.42 Extraction and Analysis of LFCs ...... 31 2.43 Experimental set-up ...... 34 2.44 Statistical analysis ...... 38 2.5 Results ...... 38 2.51 Expt. 1 LFC induction in celery leaves by cabbage looper larvae ...... 38 2.52 Expt. 2 LFC induction in celery petioles by cabbage looper larvae ...... 39 2.53 Expt. 3 LFC induction in œbry leaves by TPB adub ...... 39 2.54 Expt .4 LFC induction in celery petioles by TPB adults ...... -41 2.55 Expt. 5 A wmparison of indudion in celery petioles between TPB adults and nymphs ...... 45 2.56 Expt .6 TPBinduced LFC production in fieldgrorni plants ...... 47 2.57 Expt. 7 Duration and extent of LFC induction following mechanical damage ...... 50 2.6 Summary of Results ...... 53 2.7 Discussion ...... 54 2.8 Recommendations for future research ...... 61

Chapter 3: An assessrnent of the impact of three phototoxic linear furanocoumarins in Apium gmveolens on growth and development of the parasitised and nort-pansitised nymphs of the tarnished ppkt bug (TPB). Lygw lineolaris (Palisot de Beauvob)

3.1 Abstract ...... 62 3.2 Introduction ...... 63 3.3 Objedive ...... 66 3.4 Materials and Methods ...... 67 3.41 Plant and diet sources ...... -67 3.42 lnsect sources ...... -68 3.43 Extradion and Analysis of LFCs ...... 69 3.44 Measurement of Growth Parameters...... 71 3.45 Experimental set-up ...... 72 3.46 Statistical Analysis ...... 74 3.5 Results ...... 74 3.51 Expt 1 Growth and development of parasitised and nonparaslised TPB fed damaged and undamaged celery tissue ...... 74 3.52 Expt. 2 Growth and development of parasitised and nonparasitised TPB on celery leaves, petioles and artificial diet containing LFCs ...... 77 3.53 Expt. 3 Growth and development of parasitised and nonparasitised TPB fed a semi-solid artificial diet containing LFCs ...... 82 3.6 Summary of Results ...... 84 3.7 Discussion ...... 85 3.8 Recommendations for Future Research ...... 91

Literature cited ...... m..mmmmmmmm ...... 92

iii LIST OF TABLES

Table 1. Mean weights (mg) of tamished plant bug nymphs fed a Iiquid artificial diet containhg various concentrations of LFCs ...... 81

Table 2. Mean weights of tarnished plant bug nymphs fed a semi-solid artificial diet containing various concentrations of LFCs ...... 83 Figure 1. Chernical structure of four linear furanocoumarins in celery: 1, psoralen; 2, xanthotoxin; 3, bergapten; 4, isophnpinellin...... 7

Figure 2. Induction of three linear furanocoumarins in celery petioles b cabbage looper larvae after 48 houn (48) or 72 hours (6). Data are means of six replicates. Sig nificant differences (ps0.05) between insect damaged and caged but undamaged petioles are indicated by an asterisk. Significant differences (~~0.05)over time are indicated using letters for each compound ...... 40

Figure 3. Induction of linear furanocoumarins in celery leaves by TPB adults. Density O refen to plants without insect-damaged leaflets. Densities 1 through 4 refer to plants with 1.2 or 4 insects in their insect-darnaged leaflet cages. Data are means of five replicates. Significant differenœs (pSO.05) between insectdamaged and caged but undamaged tissues for each compound are indicated by an asterisk. Xanthotoxin levels were greater in plants that contained insects in their treatment cages compared with plants that contained only cages ...... (ps0. 05)......

Figure 4. Induction of linear furanocoumarins in cekry petioles by TPB adults. Data are means of five replicates. Significant ciifferences (ps0.05) between insectdamaged and caged but undamaged tissues for each compound are indicated by an asterisk. Significant differences (ps0.05) in magnitude of induction between densities are indicated with letters for each compound...... 44

Figure 5. A cornparison of induction in celery petioles by TPB adults versus nymphs. Data are means of ten replicates. Significant differences (ps0.05) between damaged and undamaged tissue are indicated by an asterisk. Signifiant diflerenœs (ps0.05) between induction in different life stages are indicated using letten...... 46

Figure 6. LFC levels in field-grown plants artificially infested with TPB nymphs. LFC levels in heart tissue following infestation by 5 or 15 nymphs compared with caged but uninfested plants (A). LFC levels in randomly sarnpled and visibly damaged portions of petioles from artificially or naturally infested plants (B). Data are means of 7 to 10 repliates. Significant differences (psO.05) between levels or type of infestation are indicated by letten...... 49

Figure 7. Levels of linear furanocoumarins 2,4 and 7 days following mechanical damage of petioles (A) and leaves (B). PD= damaged petioles. PC= undamaged petioles. LD= damaged leaflets, LC= undamaged leafkts. Data are means of five replicates. Significant differenœs (~~0.05)over time are indicated using lette= for each compound.

Figure 8. Levels of linear furanocoumarins in petiolar tissue that was not damaged (PC) or was damaged (PD) on days O and 2 (A) or days O and 7 (6) or in leafiet tissue that was not darnaged (LC) or was damaged (LD) on days O and 2 (C) or on days O and 7 (D). Data are means of five replicates. Signiticant difrences (ps0.05) over the are indicated using letters for each compound, pooling treatment...... -52

Figure 9. Mean weights of non-parasitised (A) and parasitised (8) TPB nymphs fed damaged and undamaged celery tissue. Values are means of 4 replicates each cantaining six nyrnphs. Bars headed with the same letter are not significantly different (p0.05) for both parasitised and non-parasitised nymp hs...... -76

Figure 10. Mean weights of parasitised and non-parasitised TPB nyrnphs fed damaged or undamaged leaves or petioles. PETL = nymphs fed undamaged petioles, PETH= nymphs fed damaged petioles. LEAL= nymphs fed undamaged leaves. LEAH- nymphs fed damaged leaves. Bars headed by the same letter are not significantly diRerent (p>0.05)...... o...... o...... o...... o...... o...... o...... -79 LIST OF APPENDICES

Appendix 1. Summary of statistical analyses...... 1 O8

vii 1.1 lntraduction

Until the 1950's plant compounds that were not universally distributed in

plants and were not invohred in primary metabolism were generally considered to

be metabolic waste products. In a seminal paper, Fraenkel (1959) argued that

the 'raison d'être' of secondary compounds was to defend a plant against attack

by natural enemies. Since then, the number of plant carnpounds known to

exhibit resistance against natural enemies has risen drarnatically. Evidence for

induced resistance was fimt documented in 1972, with the publication of a paper

by Green and Ryan who demonstrated the induction of a proteinase inhibitor in

potatoes damaged by the Colorado potato beetle. Currently, increases in the

concentrations of sewndary plant compounds in response to insect feeding damage have been reported from over 100 plant-herbivore systems (Agrawal

1998) although a clear role for induced resistanœ has not been demonstrated in many cases. Application of induced plant resistance in agriculture is attractive because the metabolic costs of induced resistance responses are thought to be less than those of constitutively produced substances. A lower metabolic cost is incurred by plants whose resistance is augmented in response to attack by natural enemies and potential reductions in crop yield are minimized. For example, the evaluation of resistance and yield in tomatoes induced to produœ higher quantities of plant proteinase inhibitors is currentiy underway (Thaler

1999). However, some compounds that confer insect resistanœ in plants have detrimental effects in humans. These include immediately manifested toxic effects and long term health risks such as cancer. In an effort to enhance plant resistance, breeders may Ricidentally develop nisect-resistant plants that contain hamifut constitutive levels of natural toxins or plants with increased inducibility

(Ames et al. 1990). Data suggest that natural compounds account for almost

98% of the cancer risk fiom food (Hutt and MeniIl 1991) and Ames et al. (1987) cautioned that aithough only a few dozen of thousands of natural pesticides have been tested in animal bioassays, these compounds are being ingested in our diet at least 10 000 times more by weight than are man-made pesticides.

Secondary compounds in celery, Apium graveolens L. var. dulce (Miller) include a class of phenylpropanoid compounds called furanocoumarîns

(=psoralens). Three of the Iinear furanocoumarins (LFCs), psoralen, bergapten and xanthotoxin, can cause dematitis in people in the presence of UV Iight

(Scott et al. 1976, Ashwood-Smith et al. 1985. Berkely et al. 1986, Seligman et al. 1987, Ljunggren 3990, Aharoni et al. 1W6), exhibit rnutagenic and ciastogenic properties (Scott et al. 1976, Ashwood-Smith et al. 1980) and may be causally related to skin cancer (IARC 1983). LFCs are niducible (Beier and OeW 1983,

Lord et al. 1988, Dercks et al. 1990 Zangerl 1990, Ataga et al. 1993, Tnimble et al. 1994) and have been shown to confer resistance to many organisms in celery and other plants (Berenbaum 1978, Berenbaum 1981b, Lee and Berenbaum

1989, Zangeri 1990, Zangerl and Berenbaum 1990, Berenbaum et al. 1991).

The recent introduction of an insect-resistant variety of celery has resulted in an increased incidence of photodematitis in handlers because of high levels of

LFCs (8erkley et al. 1986, Arnes et al. 1990).

1.2 Toxicity of LFCs to humans

The concem about phototoxic linear furanocoumarins anses primarily out

of their ability to cause contact dematitis. commonly experienced by celery

scouts and harvesters and grocery store workers (Scott et al. 1976. lvie et al.

1981, Seligman et al. 1987, Ashwood-Smith et al. 1985, Berkely et al. 1986,

Ljunggren 1990, Finkelstein et al. 1994, Aharoni et al. 1996). LFC-induced

dermatitis is characterized by an erythematous, sunbum-like response that may

be accompanied by blisters followed by hyperpigmentation in the affected area

within seven to ten days (Seligman, 1987). Exposure to celery containing either

7pg LFCslg fresh tissue weight (Seligman et al. 1987) or 18pg LFCsIg fresh

tissue weight (Austad and Kavli 1983) is suffcient to cause chronic and acute

contact dematitis, respectively. in susceptible individuals (Scott et al. 1976).

One study, for example, found that 24% of individuals handling celery developed

contact dermatitis (Fleming 1990). Furanocoumanns can reach the skin not only

through direct contact but also through oral ingestion (Ljunggren 1990) and they

are not destroyed by cooking (Ivie et al. 1981). The phototoxic threshold dose of

xanthotoxin and bergapten ingested in food followed by UV exposure is not

reached under normal dietary habits. However, for celery root. for example, the

safety factor between a dietary dose and the phototoxic threshold dose is only 2- to 10-fold (Schlatter et al. 1991).

Simple coumanns and both linear and angular furanocoumarins fom monoadduds with DNA (primarily with pyrimidine base pairs). In the presence of

WB light (300 to 360nm) linear furanocoumarins are also capable of forming

diadducts with complementary strands of DNA resulting in permanent cross-

Iinkages between DNA strands (Grekin and Epstein 1981, Gruenert and Cleaver

1985) that can lead to sister chromatid exchange (Berenbaum 1991, Cortes et al.

1991). To a lesser extent photoaddition reactions occur with RNA (Wamer et al.

1995). The association constant with double helical DNA is as high for

xanthotoxin as for bergapten (Gruenet et al. 1985). In addition. absorption of

UV light results in an excited triplet-state furanocoumarin that can interact with

other biomolecules as well including unsaturated fatty acids (Specht et al. 1988),

and proteins (Berenbaum 1991). Finally, the excited furanocoumarin cm react

with ground state oxygen, resulting in the formation of singlet oxygen or hydroxy

and superoxide anion radicals leading to lipid peroxidation and enzyme

deactivation. ReactMty toward ground state oxygen is almost three times greater

for xanthotoxin than for bergapten (Berenbaum 1991).

Reactions with nucleic acids have been shown to be the basis for the

medicinal use of LFCs in ameliorating certain skin disorders such as psoriasis,

vitiligo, and disorders caused by Apolecia areata, Lichen planus and Mycosis

fungoides (Scott et al. 1976, Grekin and Epstein 1981). Akhough furanocoumarincontairiing plants have been used medicinally against skin disorders for thousands of years (Scott et al. 1976) there is some evidence

relating PUVA (psoralen plus UVA treatment) to basal cell and squamous cell carcinomas (Stem et al. 1979). In fact, the World Health Organization officially recognises LFCs as being causally related to skin cancer (IARC 1983). It should

be mentioned however that anti-mutagenic and antî-carcinogenic properties from

ingestion of dietary doses of furanocoumarins have also been described

(Edenharder et al. 1995). Given the amount of research documenting the toxic

effects of LFCs in human beings it is somewhat surprising that no regulations

exist in the United States (Tnimble pers. comm.) or Canada (McEwen pers. comm.) pertaining to acceptable levers of furanocoumarhs in celery.

1.3 Biosynthesis of LFCs

Furanocoumarins are composed of a benzopyran 2-one nucleus with a five member ring attached in either the 6,7 position (Iinear furanocoumarins) or the 7,8 position (angular furanocoumarins) (Berenbaum 1991). They are produced via the shikimic acid biosynthetic pathway beginning with the conversion of phenylalanine to trans-cinnamic acid by phenylalanine arnmonia lyase (PAL). Ortho-hydroxylation of trans-cinnamic acid yields

P'hydroxycinnamic acid, which is converted to its cis fom, the precunor to coumarin, in the presence of UV light. Altematively, trans-cinnamic acid may undergo parahydroxylation to yield p-coumaric acid (also a precursor to lignin) .

P-coumark acid may undergo Z'hydroxylation followed by conversion by 4- coumarate:CoAligase to 4-coumaryl CoA. This compound is intemediate in the biosynthesis of both flavonoids and phenylpropanoids. including 7- hydroxycoumann (umbelliferone). Umbelliferone is the precunor to both the angular and linear furanocoumarins. The production of the latter involves prenylation to fonn mamesin, followed by oxidative loss of the hydroxypropyl group in marmesin by 'psoralensynthase' to yield psoralen. Two enzymes, S-

adenosylmethionine: bergaptol O-methyltransferase and S-

adenosylmethionine:xanthotoxol Omethyltransferase generate bergapten (5-

methoxypsoralen) and xanthotoxin (8-methoxypsoralen), respectively.

Whereas coumarin and umbelliferone are present primarily in bound forrns

as glucosides and are released by p-glucosidase in response to tissue damage,

furanocoumarins are present primarily as aglycones. Furanocoumarins have

been found to be both developmentally regulated and produced in response to

biotic and abiotic elicitors from glucoside precursors and de novo (Berenbaum

1991).

1.4 Distribution of LFCs in Plants

More than 800 coumarins, including more than 300 simple coumarins have been identified in at least 70 plant families (Murray et al. 1982). Linear furanocoumarins (LFCs) have been isolated predominantly from species in the

Rutaceae and Apiaceae (= Apiaceae) (Murray et al. 1982, Berenbaum 1991).

Celery (Apiaceae) contains both linear and angular furanocoumarins. The main linear furanocoumarins in celery include the three phototoxic furanocoumarins, psoralen (P), xanthotoxin (8-methoxypsoralen) (X) and bergapten (5- methoxypsoralen) (8) (Berenbaum 1991) as well as isopimpinellin (5,8- dimethoxypsoralen), which is not photobiologically active (AshwoodSmith et al.

1992) (Fig. 1).

From a phytocentric perspective, the occurrence and relevanœ of interactions between LFCs and insect herbivores depend not only on their Figure 1. Chernical structure of four linear furanocoumarins in celery: . 1, psoralen; 2, xanthotoxin; 3. bergapten; 4, isopimpinellin. presence in particular taxa but more specifically, on their spatial and temporal distribution in the plant. Spatial considerations include their relative distribution in different plant parts and their histological location in tissues, whereas temporal considerations include ontogenetic changes and local or systemic induction by natural enemies and other stresses. '

Diawara et al. (1995) examined the relative distribution of LFCs in celery plant parts and found that leaves of the outer petioles contained significantly higher levels of the three phototoxic LFCs (44 pglg fresh tissue weight) than did the other plant parts, followed by leaves of the inner petioles (9.9pglg fresh tissue weight). No significant differenœs were found between the outer petioles. inner petioles, heart tissue or roots, al1 of which contained 0.9-3.6pg1g fresh tissue weight. These levels are similar to those determined by others for commercial celery cultivars (AshwoodSmith et al. 1985, Beier et al. 1983) though significant differences between cultivars (Brewer et al. 1995, Trumble et al. 1990) and inner and outer petioles (Diawara et al. 1996) have been reported. In general, levels observed in plants grown in the field are higher than those observed in plants grown in laboratory or greenhouse conditions and may fluctwte over the season

(Trumble et al. 1992, Diawara et al. 1995). In most studies, bergapten has been found to occur in highest concentrations, followed by xanthotoxin, but psoralen is often observed only in trace quantities (Trumble et al. 1990, Trumble et al. 1992,

Diawara et al. 1993a). However, other studies have found that xanthotoxin

(Beier, et al. 1983, and Surico et al. 1987) or psoralen (Diawara et al. 1992,

Trumble et al. 1990) occur in greatest quantities. The histological location of LFCs in celery tissues has not been detemined. Celery contains schizogenous canals scattered throughout the pen'cycle (Esau 1977). They are secretory and are thought to extend through the stem and foliage (Maksyrnowych and Ledbetter 1986). LFCs are thought to be restricted to schizogenous canals in seeds of celery (Bereobaum 1991) and accumulate prirnarily in petiolar and foliar canals in cow-parsnip, Heracleum lanatum Michx. (Apiaceae) (Camm et al. 1976). That cut petiolar surfaces, but not intact petioles, elicited a photodermatitic response in œlery handlers

(Seligman et al. 1987) also supports the idea that LFCs are contained in canals in celery petioles rather than on the surface. However, there is also evidence suggesting that LFCs occur in and on the surfaces of tissues as well. A study of several apiaceous and rutaœous species by Zobel and Brown (1990) revealed that a large proportion of each LFC was located on the leaf surface in most of the plants studied. In wmmon rue. Ruta graveolens L.. LFCs are present in the epidemal layer of both stems and leaves and in the mesophyll directly below the epidennis, while glands of leaves contain only traces of furanocoumarins. In fact, the cuticular layer contains 15.60% of the LFCs found in leaves (Zobel and

Brown 1989). The occurrence of bergapten and xanthotoxin in the surface wax of leaves of wild carrot, Daucus carota L., a plant containing only trace levels of

LFCs (Ivie et al. 1982) has also been reported (Ceska et al. 1986). Finally, Tang et al. (1 990) identified small quantities of psoralen, xanthotoxin. bergapten and isopimpinellin in samples of celery leaf volatiles suggesting that they may, in part, be extruded to the surface. Concentrations of linear furanocoumarins increase dramatically with plant

age between 8 and 18 weeks (Re& et al. 1997) with a subsequent decline in

bergapten concentrations in the last six to eight weeks before harvest (Trumble

et al. 1992). Significant decreases in levels of LFCs were also observed both in

and on senescing leaves of R. graveolens (Zobel and Brown 1991).

The nature of xanthotoxin induction was examined in parsnip. Pastinaca

sativa, L. foliage by Zangerf and Berenbaurn (1995) who concluded that

mechanical damage resulted in a rapid and localized response of short duration.

Induction was largely restricted to the damaged leaflet. though a small but significant increase was observed in the half of the terminal leafiet on the damaged side of the leaf. No differenœs were obsewed in the basal pair of leaflets. Fumer, rernoval of leaflets prior to damage did not affect the induced response suggesting that mobilization from distant sources did not occur. Lee

(1993) also found that in mechanically damaged parsnip roots, LFC induction was highly localised; levels in undamaged tissue were signficantly lower than those in damaged tissue separated by only 0.5cm. In celery, studies on LFC translocation have not been performed. The results of some studies suggest that psoralens, or a psoralen precursor or inducing factor, might be translocated based on high levels of one or more LFCs in apparently healthy areas of pathogen-infested tissues (Surico et al. 1987. Heath-Pagliuso et al. 1992.

Chaudhary et al. 1985). However, other researchers have found LFCs only in rotted areas of infected tissue (Wu et al. 1972). Also. the possibility that undetected fungal elicitors that migrated from the site of infection resulted in a localised response in apparently healthy tissue was not ~ledout.

1.5 LFCs as Inducible Resistance Factors

1.51 Interactions with pathogens

Numerous studies have shown that Iinear furanocoumarins act as

phytoalexins (Le. substances that increase in concentration in the presence of a

pathogenic agent). Induction occun following infection by viruses (Lord et al.

1988, Ataga et al. 19931, bacteria (Yu 1975, Ashwood-Smith et al. 1985,

Chaudhary et al. 1985, Surico et al. 1987) and fungi (Yu 1975, Desjardins et al.

1989a, Heath-Pagliuso et al. 1992, Cercauskas and Chiba 1991, Afek et al.

1995a). There is evidence that coumarins (Chang et al. 1997) and

furanocoumarins (Hudson et al. 1993, Scott et al. 1976) are active against RNA

and DNA viruses and other microbes (Scott et al. 1976. Young and Barth 1982).

A partial role for furanocoumarins in resistance against some fungal

pathogens has also been established. For example, Desjardins et al. (1989b)

tested 62 strains of Gibberella pulcans (anamorph Fusanum sarnbucinum) from

diseased plant tissue and soi1 debris for toleranœ to xanthotoxin (200 and 400

pglml). Only two of the 38 strains found in soi1 or soi1 debris were highly tolerant

(had growth that was more than 70% of the controls), though eight of the strains that did not show high levels of tolerance were highly tolerant to bergapten,

psoralen, coumatin, mamesin and umbelliferone. Other pathogens that are

deterred by high doses of LFCs are more susceptible to furanocoumarin

precursors. For example, both causative and correlative data indicate that

mamesin (the precusor to psoralen) and columbianetin (the precursor to the angular furanocoumarin, angelicin) confer a greater degree of resistance against several pathogens than do LFCs (Afek et al. 1994. Afek et al. 1995a, Afek et al.

1995b). i.52 Interactions with lnsects

Zangerl(199O) measured the induction of furanocoumarins in wild parsnip,

Pastinaca sativa L., in response to feeding damage by first instar cabbage looper larvae, Tnchoplusia ni (Hübner) and demonstrate& that bels of fne furanocoumarins (imperaton'n, xanthotoxin, bergapten, isopimpinellin and sphondin) were higher than those in undamaged leaflets after 24 hours. In celery, insect-induced production of the three phototoxic LFCs in celery was examined by Tnrmble et al. (1994). Severe feeding damage by the beet annywom, Spodoptera exigua (Hübner), resulted in induction of bergapten in petioles. However, the levels were well below those able to cause photodematitis. In leaves, constitutive and induced levels were above those capable of causing photodermatitis.

Research examining the effects of furanocoumarins on insects has been conducted for fewer than twenty species with the bulk of the studies on only 2 or

3 species (Berdegue et al. 1997). Most recent work has focused on resistance to

S. exigua, which is a serious pest of celery in California. where more than 50% of celery is grown in the United States (Ivey and Johnson 1987). Studies in which

LFCs are incorporated into artifidal diets suggest that LFCs exhibit antibiosis and antixenosis toward several species of insects. Antibiosis refers to those properties of a plant that detrimentally affect the life of a herbivore resulting in for example, reduced fecundity, decreased weight, increased development time and

increased mortality (Painter 1951) while antixenosis refers to properties that

result in nonpreference by an insect in a choice context (Kogan and Ortman

1978). However, those effects do not translate directly to resistance in plants.

In addition to binding to DNA and other cellular constituents, LFCs have been shown to bind to mixed-function oxidases (MFOs) in inseds. Both an unsubstituted furan ring and the position of methoxy groups are important for the inhibitory effects of LFCs against MFOs (Yajima and Munakata 1979,

Berenbaum 1978). Inhibition of MFOs by xanthotoxin is 100 times greater than that exhibited by psoralen, bergapten or isopimpinellin (Neal and Wu 1994).

UV-dependent and -independent decreases in survivorship in response to dietary additions of LFCs have been observed in several insects (Berenbaum

1978, Berenbaum et al. 1989, Diawara et al. 1993a) though LCx, values are often far greater than levels in found in many plants (e-g. 200-500 pgLFC Ig diet)

(Diawara et al. 1993a, Reitz and Trumble 1996). Sublethal effeds of LFCs include decreased larval and pupal weight and increased generation times.

Diawara et al. (1993a) found that ingestion of each of the three phototoxic LFCs resulted in decreased latval weight and increased generation times in S. exigua.

In other insects, the effacts of individual LFCs differ from one another (Lee and

Berenbaum 1989. Berenbaum et al. 1989). For example, Lee and Berenbaum

(1989) found that lanral T. ni had extended pupation times in response to the addition of psoralen to artificial diet while the addition of xanthotoxin resufted in decreased consumption and growth. Finally. a mixture of natural furanocoumarins obtained from an extract of pannip seeds was significantly

more detrimental to survivorship of H. zea than was xanthotoxin alone

(Berenbaum et al. 1991). In contrast. Diawara et al. (1993a) found that a

combination of the three phototoxic LFCs had antagonistic effects on

survivonhip. Only the corn bined effects of xanthotoxin and bergapten were

additive.

Finally, expenrnents in which insects were allowed to choose between two

or more food sources showed that LFCs may exhibit antixenosis at much lower

concentrations than those required to detrimentally affect growth and

development. Choice tests perfonned by Berdegue et al. (1997), revealed that

food preferences exhibited by S. exigua depended on the vanety and

concentration of LFCs. A combination of furanocoumarins similar to those found

in celery petioles caused preference for the control diet in neonates, whereas late

instars preferred a control diet only when the alternative contained a combination

of LFCs similar to that found in outer leaves. Similarly, Diawara et al. (1992).

found that S. exigua larvae show a preference for plants containing low levels of

LFCs (1.4-5.85pglg) compared with those containing high levels of LFCs (186-

326 wm- In contrast, the results of studies comparing different levels of LFCs in

various plants suggest that these compounds may play a role in resistance

against sorne species but that other compounds in the plant are likely involved in exhibiting antibiosis. Several studies have found that LFC levels are not directly correlated with resistance against insects though they appear to exhibit antibiosis in diet incorporation studies (Trumble et al. 1990, Diawara et al. 1992. Diawara et

al. 1993b). For example, an accession of Fool's watercress, Apium nodinomm

(1.) Lag., which contained low levels of LFCs (1 1.8pgfg petiolar tissue)

demonstrated effective antibiosis (100% rnortality) against a leafminer Linomyza

frifolii (Burgess) whib a cultivar of A. graveolens was more suitable for growth

but contained 33pg LFCs Ig petiolar tissue. Further, another Apium accession

containing 272pg LFCslg petiolar tissue supported high numbers of adult

leafminen qrumble et al. IWO).

In wild parsnip. the pattern and distribution of furanocoumarins strongly

supports the hypothesis that they are an evolved defense against insect

herbivory in which plants are optimally defended according to probability of insect

attack and tissue value (Berenbaum et al. 1986, Nitao 1990, Zangerl and

Berenbaum 1990, Zangerl and Banat 1992. Zangerl and Rutledge 1996,

Zangerl et al. 1997, Zangerl and Berenbaum 1998). Critics of the suggestion that

secondary compounds fundion to defend plants against natural enemies argue that many secondary cornpounds have multiple roles in the plant. The

importance of furanocoumarins and their precursors in celery and other plants

appear to reflect a more generalised resistance response. In addition to their

interactions with natural enemies, LFCs may function as germination autoinhibitors in some plants (Zobel and March 1993) or allelopathic compounds

(inhibiting germination of other plants) (Sinha-Roy and Chakraborty 1976,

Friedman et al. 1982, Bewick et al. 1984, Shilling et al. 1992, Kupidlowski et al.

1994). They are induced by numerous abiotic factors including sodium chloride (Yu 1975). cold temperatures (Beier and Oertli 1983. but see Chaudhary et al.

1985), acidic fog (Dercks et al. 1990) and copper sulphate treatment (Beier and

Oertli 1983). A number of studies have shown that LFCs are induced by UV Iight

(Beier and Oertli 1983, Zangerl and Berenbaum 1987, Zobel and Brown 1993.

McCloud and Berenbaum 1994) and that they may provide some protection to the plant against ultraviolet (Zobel and Brown 1993. Zobel et al. 1994). Because

LFCs appear to confer partial or complete resistanœ against numerous organisms and may have multiple roles in celery, the implications of their involvement in resistance against insect pests must be interpreted in light of the numerous interactions involved,

1.6 Tritrophic Interactions

One further aspect of the interactions between LFCs and insects that should be considered is the implications of host plant resistance for tritrophic interactions. An important aspect of biological control is the effect of plant secondary compounds on natural enemies. Parasitoids often find host-plant complexes more attractive than either their hosts or plants alone (Reed et al.

1995) and offen this results from the release of attractive plant volatiles upon damage by the host (Blaakmeer et al. 1994, Takabayashi et al. 1995).

Secondary compounds in plants can also affect growth and development of parasitoids via ingestion by their hosts (Fox et al. 1996). Reitz and Trumble

(1996) exposed larval T. ni to Copidosoma nondanum (Ashmead), a polyembryonic egg-larval parasitoid of plusiine Noctuidae and measured the effectsof four concentrations of LFCs incorporated into an artificial diet on both trophic levels. Hostnediated effects included delayed time until parasitoid egg

hatched by retarded growth of its host and decreased survival of parasiüzed

larvae compared with nonparasitized larvae. Reitz and Trumble (1997) also

found that increasing LÇC concentrations resulted in increased rnortality for both

larval S. exigua and a parasitoid, Atchyfas mamuratus (Townsend). Surviving

parasitoids, however, did not appear adversely affected.

9.7 The Celery-Pest Complex in Ontario

In Ontario, celery is grown primarily in the Bradford Marsh Region of the

Holland Marsh, Kettleby. The most important insect pests attacking celery in this

region are the aster leafhopper, Macrosteles fascihns (SM), aphids (e-g. Myzus

persicae Sulzer), and the tamished plant bug, Lygus lineolé~fs(Palisot de

Beauvois) (TPB) and pathogens including Fusanum oxyspomm f- sp. apii and

leaf blights. The cabbage looper, 7: ni, celery looper, Syngrapha falcifera (Kirby),

carrot weevil, Listronotus oregonensis (Leconte), nematodes and weeds are less

prevalent pests (Chaput 1993). TPB can cause complete crop loss in the Atlantic

provinces and is an important pest in Michigan (Bishop pers. comm., Kortier

Davis pers. comm.). 60th TPB and the cabbage looper are concems in Quebec

(Boivin et al. 1991).

1.71 The tamished plant bug, Lygus lineolaris (Palisot de Beauvios)

(Hemiptera, Y iridae)

The tamished plant bug overwinters as an adult in leaf litter, plant debris, hedge rows and brush piles. The adults are 6 to 6.5mm long and green or brown in colour with a characteristic yellow triangle rnarking on the wing. Oviposition usually occurs in early to rnid-May and lasts between 10 and 31 days (OMAFRA

1997). Gerber (1995) measured fecundity of female TPB for a field population in

Manitoba and found that the mean number of nyrnphs pet female ranged from

239 to 303. The female lays about 5 eggsl day, inserting them in the stems. petioles or midribs of leaves. and in buds and florets. The eggs hatch in 7-10 days and five nymphal instan develop over 12 to 34 days, depending on the temperature (OMAFRA 1997). Commonly. two generaüons occur per year.

Second-generation nymphs cause most of the damage in celery in Ontario

(Stewart and Khoury 1976).

TPB is the principal mirid pest in the eastern and southem United States and is a serious pest across Canada. It is polyphagous, with at least 350 recorded hosts in the United States and Canada including 130 econornically important species (Young 1986). Represented among its host plants are 55 families in 30 of the 70 orders of angiosperms occurring in North Arnerica. TPB is also a predator on eggs and immatures of at least 10 families in 5 orders of insects and can consume live and dead conspecifïcs.

Like al1 species of Lygus, TPB feeds preferentially on either the developing reproductive organs (buds, flowers, and developing seeds) or on the apical meristem and leaf primordial tissue of plants (Strong 1970, Kuhlmann et al. 1998). Bath feeding and ovipositing occur on celery in Ontario. First- generation aduks emerge when celery is actively growing and females oviposit on young petioles as well as in the heart of the plant. Adult feeding and ovipositing results in cavities on the stalks that reduœ the market value of the plant. Both adults and nymphs cause damage. The latter occurs primarily on

leaves in the heart of the plant. When numerous nymphs are present, punctured

leaves in the heart of the plant turn black and decay (Hill 1032).

Tarnished plant bugs are lacerate and flush feeders. The stylets are used to lacerate a pocket of cells and flush out the contents with watery saliva, which is thought to be important in tissue necrosis (Laster et al. 1974). Though the composition of TPB saliva is unknown, the saliva of Hemiptera usually contains amylase (Hori 1970), cablase (Laurerna and Varis 1991) and may contain a polyphenol oxidase (PPO) and peroxidase (Miles 1972, but see Laurema and

Varis 1991). Also, mirids secrete pectin polygalacturonases (Strong 1970).

Besides enzymes, saliva of Lygus spp. contains amino acids, the suggested roles of which include, maintenance of proper pH, enhancement of salivary amylase activity and interference with protein denaturation by plant secondary compounds such as quinones. Different populations and three different species of Lygus differed greatly in the total content of amino acids in salivary glands

(Laurema and Varis 1991).

Because TPB is extremely polyphagous, it probably enwunters a large number of secondary plant compounds. However, little is known about how TPB detoxify, excrete, avoid or sequester compounds. Khattat and Stewart (1977) have examined diflerences in host plant suitability for TPB nymphs reared on different food plants in the laboratory. Nymphs reared on celery stalks experienced significantly higher mortality than those fed potato shoots, green or wax beans and pea or bean sprouts. Although the rnean number of eggs per female did not differ between celery and more suitable foods, the longevity of both females and males on celery was lower than that of bugs reared on potatoes, beans or sprouts. Although celery is damaged in the field, it is possible that it contains resistance factors that make it a less suitable food source for

TPB.

Monitorinq and Control

In Ontario, celery is monitored twice a week over the growing season by scouts who visually assess the plants. This involves physical contact with the foliage which increases exposure to LFCs if protective clothing is not wom. The economic threshold for TPB in Ontario is 0.2 inseds (adults or nymphs)lplant

(0.1 insects/plant duhg the last three weeks before harvest). Control. from early

May to mid-September involves the use of insecticides once an economic threshold is reached (Chaput, pers. comm.). In Quebec. most growers prevent

TPB damage by applying up to 8 insecticidal sprays over the season (Boivin et al. 1991). However, because chemical control of TPB is not practical in some crops (Day 1987) and insecticide resistance has been observed (Hollingsworth et al. 1997) the development of a better understanding of biological control of this pest is underway. A program to supplement the natural parasitism of Lygus populations in western Canada comrnenced in 1977. Parasitoids included

Peristenus digeoneutis Loan and P. stygicus Loan, which were first released in western Canada in 1978 (Craig and Loan 'i984). P. stygicus parasitises al1 stages of nymphs. usually laying one egg per host. but the later stages usually repel the wasp (Drea et al., 1973). The egg-larval stage lasts about 14 days after which the fully grown (fourth instar) larvae cuts a slit in the host's abdomen

(usually a fifth instar nymph) through which it emerges, then drops to the ground and spins a cocoon in which it pupates (Dolling 1991).

Little work been done on insect induction of LFCs in celery and no research has been published on the interactions between LFCs and a hernipteran, which exhibits a different type of feeding behaviour than do caterpillars. Because the responses of plants to insect feeding damage has been shown in other plant-insect systems to depend the type of insect damage

(Stout et al. 1994. Hlywka et al. 1994, Blaakmeer et al. 1994). an examination of the interactions between TPB and celery is of value. Also, a qualitative cornparison of the effects of TPB damage with that of an insect such as the cabbage looper, which exhibits a different type of damage, may provide valuable information about the nature of induction in celery.

1.72 The cabbage looper, Trichoplusis ni (Hubner) (Lepidoptera, Noctuidae)

The adult cabbage looper, is a mottled greyish-brown moth with a 38mm wing span and a small whitish figure eight marking on each wing. The rnoths can be seen during the day on the undersides of leaves. Female moths can lay 200-

350 eggs, usually ove? a 10-12 day period. Eggs are usually laid singly near the outer fringes of lower leaves and on plants not previously infested by loopers.

Larvae hatch about 3 to 6 days later and begin feeding immediately on the undersides of leaves. The larvae feed for 2 to 4 weeks before popating and rnay grow up to 40mm in the 5* and 6B instars Pupae are wrapped in silk and can be found in the undersides of lower leaves. The pupa is the ovemintering stage in the south but the moth does not ovenivinter in Canada or the North eastern

United States (Hutchison et al. 1997). Adult moths usually migrate to the

Northern United States and Canada from early July to late August. depending on weather and aimow patterns and there can be 1 to 3 generations per year in the

Northem United States depending on anival time and late summer temperatures

(Hutchison et al. 1997).

T. ni is also a polyphagous Ïnsect, feeding on over 80 host plants, including a number of econornically important species (Bin-Cheng 1994). In celery, larvae feed on the undenides of leaves and completely defoliate single stalks before moving to another (Jones and Granett 1982). Although young larvae skeletonise leaves, older larvae chew through veins. Damage to celery results both from holes in foliage and from the presenœ of fecal material.

The tamished plant bug differs from the cabbage looper in tems of their associations with celery in a number of ways, including their preferred feeding sites and the type of damage inflicted on the plant. Both the type of tissue attacked and the type of damage inflicted may be important factors in determining whether and the extent to which induction occurs and are important considerations in tens of assessing the risks associated with damage for humans.

As mentioned above, TPB feed preferentially on either the developing reproductive organs (buds, flowers, and developing seeds) or on the apical meristem and leaf primordia (Strong 1070, Kuhlmann et al. 1998) though, in celery, mature petioles and leaves are also attacked. Berenbaurn (1981b) suggested that TPB avoids linear furanocoumarins by feeding on seeds of wild parsnips, in which LFCs are restricted to oil glands. Constitutive levels of LFCs in celery hearts are low compared to those in other tissues and in other host plant species (Ceska et al. 1987. Zobel and Brown 1989, Diawara et al. 1995).

Though it is not known whether LFCs are inducible in the unexpanded leaves within celery hearts, they are not inducible in the unexpanded leaves of wild parsnip (Zangerl and Rutledge 1996). Thus, TPB feeding damage rnay not cause increased production of LFCs of a magnitude of concem to humans.

In contrast, lepidopteran larvae preferentially feed on the undersides of outer leaves and leave them skeletonized. This behaviour rnay allow for avoidance of furanocoumarÏns as has been suggested for parsnip foliage, where the bulk of LFCs are found in the veins (Zangerl 1990). That cabbage looper larvae rnay avoid secondary compounds has been dernonstrated by their ability to selectively trench plants as required to avoid plant exudates (Dussourd and

Denno 1994). However, veins rnay be avoided because of their toughness

(Berdegue and Trumble 1996). In either case, feeding damage rnay result in increases in LFCs in remaining skeletonized plant foliage, where constitutive levels are already high.

The type of cellular damage and chemical interactions between insects and plants rnay also influence the wounding response. TPB create lesions in the plant tissue. injecting enzymes in their saliva (Miles 1972) whereas cabbage loopers cut and grind food, removing plant material entirely. Although the chernical interactions between cabbage looper larvae and celery are unknown,

lepidopteran larvae often regurgitate on plant material while feeding .

Regu rg itant from larvae of the cornmon amywomi, Pseudalefia separata Walker,

is known to elicit plant volatiles, the composlion of which is dependent on the

life stage of the larvae. Because mechanical damage of parsnip foliage resulted

in an attenuated response compared with feeding darnage by cabbage loopen, it was suggested that larval regurgitant may play a role in the induction response in this plant as well (Zangerl 1990).

In this study, the ability of both cabbage looper larvae and tamished plant bug nymphs and adults to induce linear furanocoumarin production in celery was assessed. A qualitative cornparison was made between insects with different feeding behaviours, although the importanœ of quantitative changes were assessed in ternis of their proxirnity to levels capable of causing contact dematitis. In addition. the potential antibiotic effects of LFCs in both plant material and artificial diet were rneasured for the tarnished plant bug and one of its parasitoids, Peristenus stygicus Loan. Chapter 2: Induction of three phototoxic linear furanocoumarins in celery,

Apium graveolens L. in response to insect feeding damage.

2.1 Abstract

Herbivore-induced production of three phototoxic linear furanocoumarins

in celery, Apium graveolens L. was evaluated in the commercial cultivar 52-70HK

following darnage by the tamished plant bug, Lygus Iineolans (Palisot de

Beauvois) and the cabbage looper, Trichoplusia ni (Hübner). LFCs were

extracted from plant tissue and detected by HPLC. Both insects induced al1 three

LFCs in petioles, though concentrations rernained low. Total LFC levels that were high enough to cause chronic contact photodermatitis (27pglg LFCslg fresh tissue wt.) were reached following severe feeding damage by TPB in laboratory tests and in necrotic lesions of artificially infested field-grown plants exposed to

15 TPB nymphdplant LFCs were less inducible in leaves in ternis of the number of LFCs induced and the magnitude of induction. However, levels in leaves were constitutively high (9-22 pgLFCslg fresh tissue W.) and LFCs were induced in foliage containing low constitutive levels. lnduction of LFCs was predorninantly localized, but a somewhat systemic response was observed following either severe feeding damage or mechanical induction. Under current conventional control practiœs, the amount of feeding damage required to induce

LFCs to concentrations of concern are not reached because they exceed those that can cause unacceptable amounts of wsmetic damage to celery plants. 2.2 Introduction

The long-ten goals of reduœd reliance on pesticides through the

development of integrated pest management and the utilization of host plant

resistance are widely considered to be salutary (Panda and Khush 1991);

However, the fulfilment of these goals may result in the inadvertent selection for

higher levels of toxic natural compounds (Ames 1990).

Celery, Apium graveolens L. produœs three phototoxic Iinear furanocoumarins (LFCs), bergapten, xanthotoxin and psoralen, that have mutagenic, clastogenic (Scott et al. 1976) and carcinogenic (Stem et al. 1979) properties and are responsible for a photodermatitic reaction commonly experienced by celery handlers (Fleming 1990, Finkelstein et al. 1994).

Exposure to celery containing as Iittle as 7pg LFCslg fresh tissue weight

(Seligman et al. 1987) or more than 18pg LFCslg fresh tissue weight (Austad and

Kavli 1983) is sufficient to cause chronic and acute contact dermatitis, respectively in people. Handling diseased celery often results in increased risks of experiencing dermatitis (Seligrnan et al. 1987, Aharoni et al. 1996) because infection results in increased production of LFCs. induction of LFCs in celery also occurs in response to nurnerous other biotic and abiotic stresses (Yu 1975,

Beier and Oertli 1983, AshwoodSmith et al. 1985, Chaudhary et al. 1985,

Zangerl and Berenbaum 1987, Lord et al. 1988, Dercks et al. 1990. Cercauskas and Chiba, 1991, Heath-Pagliuso et al. 1992, Ataga et al. 1993, McCloud and

Berenbaum 1994). Although, LFC levels are measured in new celery breeding lines vrumble

et al. 1990), differenœs in inducibility are not tested. Thus, the development of

an understanding of the nature and magnitude of resistance responses involving toxic compounds is desirable as part of the development of safe and effective crop protection using host plant resistance. Few investigations have examined induction in response to herbivore damage in part because of the low tolerance for cosmetic darnage and frass in celery. However. severe feeding damage by

Spodoptera exigua (Hübner) has been shown to result in increases in the concentrations of LFCs in celery foliage to levels that may be of concern to handlen, though petiolar levels remain low (Trumble et al. 1994).

In Manitoba, Ontario, Eastern Canada and the North Eastern United

States, the tamished plant bug, Lygus Iineolafis (Palisot de Beauvois) (TPB) is an important pest of celery (Boivin et al. 1991, Kortier-Davis pers. corn., Bishop pers. corn., OMAFRA 1997). Lepidopteran pests, such as the cabbage looper,

Trichoplusia ni (Hübner), also occur. Because induction of secondary plant compounds rnay dHer qualitatively (Stout et al. 1994) and quantitatively (Hlywka et al. 1994, Hartley and Lawton 1991, Olson and Roseland, 1991), depending on both the type and amount of damage, and the type of tissue damaged (Zangerl and Rutledge 1996) an examination of induction by insect pests with different feeding behavioun may give a more complete understanding of the nature and implications of induction. In this study, concentrations of the three phototoxic

LFCs in celery were examined following exposure to feeding damage by the tarnished plant bug and the cabbage looper in order to assess the occurrence and magnitude of induction in difFerent tissues. 2.3 Objectives

1. To detemine if the three phototoxic linear furanocoumarins in

celery increase in concentration in response to feeding damage by the cabbage

looper and the tamished plant bug.

2. To determine if relative induction of the three LFCs dRers depending on the type of damage or plant tissue affected.

3, To determine if induced Ievels of LFCs return to constitutive levefs within 7 days following mechanical damage and if subsequent damage leads to increased induction. 2.4 Materials and Methods

2.41 Plant and lnsect Sources

Seeds of celery, cv Tali Utah 52-70HK. were obtained from Stokes Seeds

Inc. (Bufallo, NY). Plants were grown in a greenhouse under natural and artificial lighting (16:8hr (D:N) ) and given water as required and nutrient solution 2x per week (20:8:20 fertilizer solution). At ten weeks following seeding, plants were transplanted from plug trays into pots (12.7cm diameter) and given both nutrient solution as necessary and a calcium nitrate (200ppm) treatment twice per week.

Mature plants between the ages of 90 and 120 days were used in al1 experiments. Two to six weeks prior to the commencement of each experiment, mature standardized plants were moved to a controlled temperature growth chamber containing fulCspectrum Iights (approximately 125CL~l~m2at 340 nm) and uniform conditions of 24"-18°C dayfnight alternating temperature and a 16:8 hr. (D:N) photopenod.

In a field experiment, plants were grown in a greenhouse at the Bradford

Marsh Muck Crops Research Station, Kettleby, Ontario from Aprïl 1 to June 8,

1998 after which they were transplanted into the field. They received one micronutrient treatment containing boron, calcium and magnesium and were irrigated until the start of the experiment, on July 28, 1998. The field plot consisted of three rows of 100 plants each planted 15cm apart along the row and

60cm between rows.

Tamished plant bugs were obtained from a colony established at the

University of Guelph in January, 1997. The colony was maintained at 21°C with a 16:8 (LD) photoperiod and 55145% RH. Cages of adults contained potatoes, beans and lettuce as well as water-soaked dental wicks. Every three days, food was replaced and old food (containing eggs) was plaœd in 4L plastic buckets. Fresh lettuce, beans and water were given to developing nymphs every three days. The colony was periodically supplemented with field collected TPB adults to maintain genetic variability and augment the colony.

Cabbage loopers were obtained from a colony established at the

University of Guelph in September, 1997. The colony was maintained at 21°C with a 16:8 (L:D) photoperiod and 55%65% RH. Aduits were kept in 4L plastic buckets that had been haif filled with moistened vermiculite and were fed a Iiquid diet developed at AAFC, London (Lou Verdon, pers. comm.). Squares of cheesecloth, which served as oviposition substrates, were collected 5 and 8 days after moths begin to emerge. Surface sterilized eggs were placed into 1429 plastic cups containing artificial diet, made according to the proœdure used by

AAFC, London Ontario (Lou Vernon, pers. cornm.).

2.42 Extraction and Analvsis of LFCs

LFCs were extracted according to the procedure published by Tnimble et al. (1992). 7-bentyloxycoumarin, an interna1 standard, was synthesized by refluxing 7-hydroxycoumarin (1.62g. 1Ommol, Aldrich Chernical Co., Milwaukee,

WI), Na2C03 (5g) and benzyl bromide (29, ll.7mmol) in 100ml of acetone for

60hr under Np in a fumehoad. The mixture was then cooled and concentrated to dryness by rotary evaporation. The residue was taken up in water and extracted th ree times with chlorofom- Chlorofom extracts were combined, washed with NaCl solution, then wncentrated to dryness over Na2S04. The residue was

recrystallised from hot ethanol. The resulting cream coloured solid gave one

peak on HPLC analysis, and its composition was confinned by NMR analysis.

The caged portions of petiolar tissue (4.97 to 9.826) were excised from

frozen petioles and chopped finely with a dissecüng blade. Pieces were placed

in round-bottomed (20x1 16mm) cuiture tubes with 1Oml deionized water and

were spiked with 5p1 of the intemal standard (5pg 7-benzyloxycoumarin/2OpI

hexane:tetra hydrofuran (81: 1 9)). Samples were homogenized with a Polytron

tissue homogenizer (Kinematica, Switzerland) for 2.0 minutes. Toluene (1Oml)

was added to each tube, which was subsequently capped with aluminum foi1 and

vortexed for 1.5 minutes. The samples were then transferred to centrifuge tubes

and centrifuged for 40 minutes at 330g. The toluene layer of each sample was quantitatively transferred to 13x1 00mm culture tubes and evaporated to dryness on an N-evap at 40°C. A further 1-Oml of toluene was added and the samples were again evaporated to dryness to remove any traces of water by azeotropic distillation. The samples were reconstituted in 100p1 toluene and loaded ont0

Bakerbond spe solid-phase extraction columns (500mg of silica, J.T.Baker,

Mallinckrodt Baker Inc. NJ) which had been preconditioned with 4x2ml toluene.

The sample was rinsed Nice ont0 the column with 2xO.lml toluene. discarding the eluate. The cartridge was then eluted by gravity flow with 5% acetone in chloroform (3x0.5ml) discarding the first 0.5ml of eluate. The eluate containing the furanocoumarin fraction was collected and transferred to 13xl00mm culture tubes and wnœntrated to dryness using an Nevap. Samples were then

wrapped in foi1 and frozen until analysis by HPLC.

Frozen samples were thawed and vortexed for 30s in HPLC mobile phase

(hexane:tetrahydrofuran, 81:19). A 20pL aliquot was analysed by HPLC. HPLC

quantitation and detection was achieved using a Shimadzu model LC-GA high

performance liquid chromatograph with a Shimadzu SPD-M6A photodiode array

UV-VIS detector ai 290nm and quantifid on a Shimadzu SPD-M6A computer program (ver. 2.12) integrated to the detector. A Spheroclone silica column

(25cm x 4.6mrn1 5-pm parüde sire) with a 10rnmx4.6mm guard column with the same packing material was used (Phenomenex, Torrence, CA). The analytes were eluted isocratically with hexane:tetrahydrofuran (81:19, mked by the HPLC pump) at 1.5mVmin. at ambient temperature. All solvents were HPLC grade

(Aldrich Chemical Co., Milwaukee. WI and Fisher Scientific. Fair Lawn, NJ).

Relative retention times were as follows: ISTD. 1.00, psoralen, 1.2; bergapten,

1.5; xanthotoxin 2.5.

Calibration lines were generated using at least five concentrations of purified standards (Aldrich Chemical Co., Milwaukee. WI) spanning the range of interest. Calibration Iines for the three compounds were Iinear, with ? values of

0.99 or better. The calculated limits of detection and quantitation for each compound were as follows: psoralen, 0.01 5 and 0.049 pgiml. respectively; bergapten, 0.018 and 0.046 pglml respectively; and xanthotoxin, 0.0005 and

0 .O01 pglml respectively. Peak heights were used to calculate LFC concentrations. 2.43 Ex~erimentaiSet-UP

For the laboratory expenments split-plot designs were used in which plants were the whole-plot factor and the presenœ or absence of insects was the within plot factor. Thus, control and treatment cages occurred on each plant.

Experiment 1: LFC induction in celerv leaves bv cabbarie loo~erlarvae

Plant cages, made frorn 4L ventilated clear plastic jugs were supported by a lattice that hung under the lights and were plaœd over individual intact petioles matched for position from the heaR One control cage and one treatment cage were placed on each plant. Ten first instar cabbage loopers were allowed to feed for four days after which petioles were rernoved from the plant and frozen immediately. Before extraction, outlines of damaged and undamaged (control) leaflets matched for number and weight were traced ont0 transparent sheets for area quantification because the area lost to feeding damage was too small for detection by a leaf area meter.

Ex~eriment2: LFC induction in celetv ~etiolesbv cabbaae loo~erlarvae

One 50mm long plastic cage was placed on each of two outer petioles of each plant, serving as control and treatment cages. Cages were placed approximately 50mm below the first node. which occun approximately 350mm from the base of the petiole. Plants were uniform in size and caged petioles were rnatched for position from the heart. Cages were plaœd onto petioles by gently holding leaflets together and sliding the cage over the petiole until it rested below the lowest set of leaflets. Both openings of the cage were filled with foam pieces of about a one inch thickness that cushioned the petiole and allowed for ventilation of the cage. A single fourth instar was plaœd in each cage and allowed to feed for 48 or 72 hours, after which petioles were frozen immediately.

At the time of extraction, LFCS from the petiolar section enclosed by the cage were extracted and analysed for concentrations of the th ree phototoxic LFCs.

Experiment 3: LFC induction in celerv leaves bv TPB adults

Newly emerged male adult TPB were placed singly, in pairs or in groups of four into individual leaffet cages for a penod of seven days. Cages were composed of a Petri dish (50mm diam.) to which foam rings had been glued around the circumference. The lid and bottom were plaœd on either side of a leaflet such that the foam rested between them allowing for ventilation and cushioning of the leaflet. Cages were held together with elastic bands and supported by a lattice that hung under the Iights. After one week, insect damaged, caged but undamaged, and an uncaged leafiet from the same petiole were excised from each plant and frozen immediately until extraction.

Experiment 4: LFC induction in celerv ~etiolesbv TPB adults

Newly emerged male adult tarnished plant bugs were used in order to avoid oviposition damage. Two, four, six or eight TPB were placed in a petiolar cage (see expt 2) for seven days, after which petioles were frozen immediately until extraction. Mortality was recorded daily and aduits were replaœd upon death or escape.

Experiment 5: A com~arisonof induction in celerv ~etiolesbetween TPB adults and nvmphs

Outer petioles, paired for position on the plant, were exposed to either ten adult TPB or ten TPB nymphs by placing Ïnsects in clear plastic petiolar cages

(see expt 2). lnsects were allowed to feed for a period of one week and were

replaced daily upon death, escape or ernergenœ as adults. Each plant

contained two cages containing insects of the same stage and two control cages.

At the end of the exposure period. petioles were frozen imrnediately until

extraction of LFCs. Extracted petiolar samples included portions within cages as

well as petiolar samples above and below caged sections for plants exposed to

adult TPB.

Ex~eriment6: LFC TP B-induced LFC ~roduction in field-cirown ~lants

The experiment was set up as a completely randomized design wlh nine

replicates for each of three treatment conditions. Plexiglass cages (50cm x

35cm x 25cm) with cloth side panels were placed over individual whole field- grown plants standardized for size and amount of TPB damage already present.

Plants were randomly assigned to one of three treatment conditions and exposed to either five 3" instar TPB nymphs, ffieen 3d instar TPB nymphs or no TPB nymphs. Nymphs were aspirated ont0 the heart of the plant with the cage already in place and the soi1 was subsequently built up around the base of the cage to prevent escape. After 10 days. cages were removed and remaining nyrnphs were aspirated off of the plant as thoroughly as possible. A record was kept of the number of nymphs collected using three methods: scouting each plant, thoroughly examining the plant and taking the plant apart by cutting the crown and petioles at their bases. Four mature petioles were randomly selected from each plant. Tissue samples were transpoited back the University of Guelph on ice and frozen (-20°C) upon amval. Upon extraction, whole petioles from each sample were chopped with a razor and vîsibly damaged and randomly selected

49 samples were analysed. Samples from naturally infested plants of the same cohort, in an adjacent plot were taken seven days later and were analysed with the artificially infested plants. Finally, over a three week period prior to and during the experiment, naturally infested plants surrounding the caged plants were checked for TPB damage using the normal scouthg procedure of separating petioles and inspecting the plant center for 50 plants.

Ex~eriment7: Duration and extent of LFC induction followina mechanical damaae

Two petioles on each of 15 standardized plants were unifomily damaged.

The mechanical damage treatment consisted of piercing leaf and petiolar tissue, evenly along the entire length of petiole and through al1 leaves using a device made of 15 insect pins pushed through a plastic lid in a 1cm2 arrangement. Plant tissue was pierced two days and one day before being removed from the plant and placed in the buckets. Two petioles, including one damaged petiole, on each of five plants were sampled two days following the initial damaging event. The other damaged petiole was damaged again and sampled with another intact petiole five days after the initial damaging event. Another set of five plants was sampled four days following the initial damaging event. Finally, 7 days after the plants were damaged, a third set of five plants were sampled as above and a second damaged petiole was damaged again and sampled with an undamaged petiole on day 10. Sampled tissue was immediately frozen and upon extraction, a 29 leaf sample and 49 petiole sarnple was randomly chosen from each whole petiole-

2.44 Statistical Analvsis

Unless othewise stated, the results of ail induction experiments were analyzed using a muitivariate repeated measures ANOVA followed by independent univariate repeated measures ANOVAs for split-plot designs for each of the three compounds and their sum. Where analyses of residuals and

LEV MED tests revealed heterogeneity of variance, a variance stabilizing square root transformation was perfomed. Multiple comparisons were made using

Tukey's Studentised Range (HSD) test except in expeflment 7 where pre- planned contrasts were used (SAS. 1991). Summaries of the analyses are in

Appendix 1.

2.5 Results

2-51 Ex~eriment1 : LFC induction in celerv Ieaves bv cabbaae loo~erlarvae

The area consumed by laivae on damaged plants was 3.642 + 0.75% of the total leaf area (92.7 + 25.5cm2), and about 8.1 I 1.2 of 10 larvae were recovered from each plant. Linear furanocoumarins were analysed on a per weight basis (pg/g fresh tissue weight.) for comparison with results of other experiments. However, because linear furanocoumarins are localized primarily in veins in other plants (Zangerl and Bazar 1995), and because larvae skeletonise leaves, leaving veins relatively intact, calculations were also perfomed on a per area basis (riglcm2 intact tissue) so as not to over-estimate induction. Separate dependant t-tests for each compound and their sum revealed no significant differenœs (p>0.05) between damaged and undamaged

tissues by either calculation. Bergapten consistently occurred in highest amounts

in both damaged and intact leaves followed by xanthotoxin. then psoralen. Total

LFCs in intact leaves were 23.1 t 4.3 pg1g fresh tissue weight and 0.45 t 0.1

pg/cm2.

2.52 Ex~eriment2: LFC induction in celerv ~etiolesbv cabbarre loo~erlarvae

Across the periods, total petiolar levels of Iinear furanocoumarins in intact tissue were 0.4 + 0.1pglg fresh tissue weight and increases in bergapten. xanthotoxin and psoralen were 221%. 175% and 215% respectively (Fig. 2). The multivariate test was significant for the effect of feeding damage (al1 estimates

F3.7= 11-27, ps0.005). Univariate tests revealed that damaged petioles had higher levels of bergapten (Fis =6.03,ps0.05), xanthotoxin (Fivg=6.48, psO.05). psoralen = 6.15, ~~0.05)and total LFCs (Fis8= 7.84, ps0.005) than did undamaged petioles. Further, for bergapten (F1,l~ = 6.22, ps0.05) and xanthotoxin (Fi 1 = 5.27, pi0.05) LFC concentrations were greater after 72 houn than after 48 houn. Psoralen induction was significant only after 72 hours, (Fitg

= 8.87, psO.05) end there was significant variability in psoralen levels between plants (FlVg= 5.08, ps0.05).

2.53 Ex~eriment3: LFC induction in celen, leaves bv TPB adults

In cages wntaining one, two and four TPB, mortality rates of 25%, 70% and 55% occurred, respectively. However, insects were replaced daily upon death or escape to maintain approximately constant levels of feeding damage. lnsects were found rarely on the upper surfaces of leaves and were observed 1-0 am aged OControl 1 3 I

1 * * 0 -5 O - bergapten xanthotoxin psoralen to ta I t .-O uF CI b aC 3 O 2 -5 b œ* t t 2 0 O 1.5 O 1 h* J 0.5' x 01 bergapten x8n tîto toxln p 80 raien to t. I

Fig. 2 Induction of three linear furanocoumarins in celery petioles by cabbage looper larvae after 48 hours (A) or 72 hours (B). Data are means of six replicates. Significant differences (ps0.05) between insect damaged and caged but undamaged petioles are indicated by an astetisk Significant differenœs (ps0.05)over time are indicated using letters for each wmpound. often on the foam. Overall, levels of LFCs in undamaged leaflets were 9.a

1.Opg/g fresh tissue weight.

The multivariate test revealed that higher levels of bergapten occurred in

damaged tissues wmpared with undamaged tissues (al1 estimates, F3,9 = 6.79.

psO.05). Univariate analyses, which included data for uncaged leaves, revealed that bergapten occurred in greater-amounts in damaged leaflets than in caged or uncaged, undamaged leaflets (Fr= = 4.08. ps0.05). Also, a significant difference in density (FSml4= 4.59, ~~0.05)occuned, with levels of bergapten in plants exposed to four TPBldamaged leaflet being higher than those in plants exposed to one TPBldamaged leaflet (Fig. 3). Feeding damage by four insects resulted in an increase of bergapten by 214% compared with undamaged leaflets. A regression on the differences between treated and untreated leaflets revealed a significant Iinear increase in bergapten levels with increasing numbers of TPB

(FtVl3= 14.06, ps0.05). For xanthotoxin, although there was no effect of feeding damage, ciifferences between insect densities were found (F3,14= 4-09>ps0.05); sig nificantly higher levels of xanthotoxin were found in plants containing TPB- damaged leaflets compared with plants that contained caged and uncaged leafiets but no insects (F3,24= 4.53, psO.05). NO differences were observed for psoralen or total LFCs or between caged but undamaged or uncaged leafiets.

2.54 Experiment 4: LFC induction in celerv ~etiolesbv TPB adults

The mortality rate for TPB adults was approximately 30% in al1 conditions. lnsects were often observed standing on and feeding under the foam. In fact, the U U B X P 8 X P B X P LL A uncaged caged caged wiih tpb

Fig. 3 Induction of iinear furanocoumarins in celery leaves by TPB adults. Density O refers to plants without insed-damaged leafiets. Densities 1 through 4 refer to plants with 1, 2 or 4 insects in their insectdamaged leaflet cages. Data are means of five replicates. Significant differences (psû.05) between insect-damaged and caged but undamaged tissues for each wmpound are indicated by an asterisk Xanthotoxin levels were greater in plants that contained insects in their treatment cages compared with plants that contained only cages (ps0.05). area under the foam of petioles at the tops of cages containing insects showed

signs of necrosis for most plants. Total LFC levels in intact petioles were 0.2 k

0.04 pg LFClg fresh tissue weight The multivariate test was significant for

density (al1 estimates, e.g. Pillai's Trace Fsc8= 5.29, p~0.0005). and insect

darnage (ail estimates FSelr= 32.40, plO.OOO1). Univariate tests revealed that

insect damaged petioles had higher levels of al1 three LFCs cornpared with those

for undamaged petiofes (B. = 88.03. ps0.0001; X, = 53.07. p10.0001; P.

FlVis= 5.22, ps0.05; and total LFCs, 73.99, p~O.0001)(Fig. 4). DHerences

in levels of LFCs depended on the density of insects within a cage (B. F3,16=

3.45. ~~0.05,X, Fjtle = 3.77, ~10.05;P, F3,rs= 64.0, p~O.0001). Finally, for

bergapten, xanthotoxin and total LFCs, an interaction between insect damage and density was observed (B, F3,t6= 4.56. ps0.05; X, F3,16 = 3.32. psO.05; and total LFCs, Fais = 4.1 9. pS0.05). Induction of bergapten occurred at al1 densities but was greater for plants exposed to 6 or 8 insects compared with those exposed to two insects (~10.05). For example, induction by two and eight insects respectively, resulted in increases of 782% and 3138%. For xanthotoxin, induction occurred only for petioles exposed to 4, 6 or 8 insects and feeding damage by 6 insects resulted in xanthotoxin levels that were significantly greater than those observed in response to either 4 or 2 insects (ps0.05). Induction by two and six insects resulted in increases of 284% and 590%. respectively.

Finally, a regression analysis of the differences between treated and control tissues revealed a linear increase in induced levels of bergapten (F1~~=16.96, O LL BXPTBXPTBXPTBXPT 2 2tpblcag# am- wv -cage

Fig. 4. Induction of linear furanocoumarins in celery petioles by TPB adults. Data are means of five replicates. Significant differences (ps0.05) between insect-damaged and caged but undamaged tissues for each compound are indicated by an asterisk Significant differenœs (ps0.05) in magnitude of induction between densities are indicated with letters for each compound. 2.55 Ex~eriment5: A com~arisonof induction in celew ~etiolesbetween TPB adults and nvmphs

Mortality occurred at a rate of about 6% for bath adults and nymphs. Total

LFC levels in intact petioles were approximateiy 3.2 I 1.4pglg fresh tissue weight. The multivariate test revealed a significant difference between adult and nymphal TPB (F3,9= 14.41, p~O.0001), levels of feeding damage (FtVg= 3.78, ps0.05) and their interaction (F3s9= 8.48, p10.005) (Fig. 5). Total LFCs were induced in response to feeding damage by adults only (F1.12 = 10-37. p50.01).

There was also significant variability in the levels of LFCs between plants =

5.83, ps0.005). Induction of bergapten = 75.36, p~O.0001).was greater in response to feeding damage by adults than by nymphs (F1.14 = 7.23, p~0.05).

For example, feeding damage by adults and nymphs resulted in increases of

409% and 108%. respectively. Significant variability in petiolar LFC levels was observed for both stages, however, for adults, variability in bergapten levels was significant for undamaged petioles only (F43= 111.37, ps0.0001). lnsect feeding damage also caused significant increases in levels of xanthotoxin (Fl,l2= 33.41. ps0.0001). Again, significant variability in xanthotoxin levels between plants was observed in undamaged petioles only = 6.17, pd.005). A 133% increase in xanthotoxin was observed in adult-damaged tissue compared with a 484% increase in nymph-damaged tissue although the difFerenœ was accounted for by higher levels in control tissues of adult-damaged plants. No significant differences were observed for psoralen (p>0.05). B X P T B X P T Ad u Its Nymphs

Fig. 5. A cornparison of induction of linear furanocoumarins in celery petioles by TPB adults versus nymphs. Data are means of ten replicates. Significant differences (psO.05) between damaged and undamaged tissue are indicated by an asterisk. Significant differenœs (ps0.05) between induction in different life stages are indicated using lettem. LFC concentrations in samples of petiolar tissue above and below caged

sections from plants injured by adult TPB indicated that although no differences

were found within undarnaged petioles for bergapten or xanthotoxin. for both

damaged and undamaged - tissues, areas above cages had higher levels of

psoralen than areas within or below cages (F2,41= 23.44. p~0.0001). Further, in

insect-damaged tissue, both bergapten and xanthotoxin occurred in higher

concentrations within cages than above cages, and above cages than below

cages (B, FzWiz= 13.26, p10.001; X. F2,12 19-23. p~0.005). Finally, for

xanthotoxin only, higher levels of LFCs were found in samples above insect-

darnaged tissue compared with those above caged but undamaged tissue

= 4.58, ~~0.05).

2.56 Experirnent 6: TPB-induced LFC production in field-arown plants

Light readings for caged plants were approximately 45% of those for

uncaged plants. Recovery of nyrnphs from the different treatrnent conditions were as follows: controls, O.7kl.3 TPB (median = O), 5 nymphslcage, 3.9I 2.4

TPB (median = 4.5) and 15 nymphslcage, 723.7 TPB (median = 7). Initial scouting of plants by separating petioles and obsenring the centre of the plant

resulted in recovery of 14% of the total nymphs eventually recovered through thorough observation. Of the recovered nymphs, 58% in the highest infestation condition were S* instars and 47% in the low infestation condition were 5th instars. There were several 3" instars that were probably already on the plant as first instars or eggs at the commencement of the experiment. Most petioles showed signs of damage and several of those exposed to 15 nymphs had symptoms of blackheart. The percent of naturally infested plants wntaining at least some TPB damage was approximately 44.4 k 14.8%.

In a random sample of 10 naturally infested plants, 48.58 I21.58 % of the petioles were damaged per plant with each petiole sustaining about 102% damage by area. Comparison of heart, damaged petiolar sections and randomly selected petiolar sections in the three artificial infestation conditions revealed that only bergapten levels differed between conditions (F- = 7.24, p~0.005)with tissue exposed to 15 TPB nymphs containing higher levels than tissue exposed to 5 nymphs which in tum contained higher levels lhan control tissues: Visibly damaged tissues also had higher levels of bergapten and xanthotoxin than either randomly sampled or heart tissues (FZv37= 5.62. p

(F2.21 = 4.96. ~~0.05)(Fig. 6).

Further, a comparison of visibly damaged and random petiolar tissues in caged versus uncaged plants that were untreated and exposed to natural infestations of TPB showed that naturally infested plants had levels of bergapten that were not significantly different (p>0.05) from those of caged plants exposed to either 15 or 5 nymphs but higher than caged control plants (F3,25=8.331 ps0.001). Across conditions. levels of bergapten and xanthotoxin were higher in Fig. 6. Levels of linear furanocoumarins in field-grown celery plants aftïficially infested by TPB nymphs. LFC levels in heart tissue following infestation with 5 or 15 nymphs cornpared with caged but non-infested plants (A)- LFC levels in randomly sampled and visibly damaged partions of petioles ftorn artifiaally or naturally infested plants (B). Data are means of 7 to 10 replicates. Significant differences (psû.05) betwaen levels or type of infestation are indicated by letters. visibly darnaged tissue compared with randomly sampled tissue (F1,25=16.98,

p10.0005 and F1,23=7-42.ps0-05, respectively).

2.57 Experiment 7 Duration and extent of LFC induction followina mechanical

damane

The multivariate test revealed that the mechanical damage treatment

caused significant induction of LFCs in petioles (al1 estimates, F3,r2= 3.50,

ps0.05). This was accounted for by induction of bergapten (F1,14=10.60, p10.01),

and induction of xanthotoxin approached significance (Fivl4= 4.40. p=0.0546).

No treatment effect was obsewed for LFC levels in the leaves (p>0.05).

Univariate tests showed that significantly higher levels of bergapten (Fivs8=

171-75. p~0.0001)~xanthotoxin (Fits8= 78.3. p~0.0001),psoralen = 86.28,

ps0.0005) and total LFCs = 248.94, ps0.0001) were found in leaves

compared to petioles. Four contrasts were performed cornparing levels of LFCs

between 2 days and either 4, 5 or 7 days and between 7 and 10 days. The level

of significance for pre-planned contrasts aRer Bonferroni adjustment was

aM=O.OI 3. Total LFCs were greater in damaged petioles (F1,18 = 9.82, p

no apparent effect of the damaging treatment, a decrease in xanthotoxin levels was obsenred in both control and treated tissues from 2 to 4 days (Fi,is = 8.09, p10.01) (Fig. 7). Petiolar levels of xanthotoxin increased from 7 to 10 days

= 9.00, peO.01) in both damaged and undamaged tissues (Fig. 8). Between plants, significant variability was found in leaves bergapten (F1o,zi = 1.71, psO.O1). Fig. 7. Levels of Iinear furanocoumarins 2.4 and 7 days following mechanical damage of petioles (A) and leaflets (8). PD = damaged petioles, PC = undamaged petioles, LD = damaged leaflets, LC = undamaged leaflets. Data are means of five replicates. Significant differences (p~O.013)are indicated using letters for each compound. A 10 - Q Bergapten ~Xhnthotoxin~Psonlen 8 -.

Fig. 8. Levels of linear furanocoumarins in petiolar tissue that was not damaged (PC) or was damaged (PD) on days O and 2 (A) or days O and 7 (B) or in leaflet tissue that was not damaged (LC) or was damaged (LD) on days O and 2 (C) or on days O and 7 (O). Data are means of five replicates. Significant differences (psO.013) over time are indicated using letters for each compound, pooling treatrnents. 2.6 Summary of Results from the Induction Experiments:

O Feeding damage by either the cabbage looper or the tamished plant bug

induces the three phototoxic linear furanocoumarins in celery tissue.

Generally, bergapten, which occurs in the highest constitutive amounts is the

most inducible, followed by xanthotoxin, then psoralen.

*3 In leaves, feeding by ten first instar cabbage looper larvae over four days did

not induce LFCs and feeding damage by TPB for seven days resulted in

induction of bergapten at the highest level of feeding darnage only.

*' *' In petioles, al1 three LFCs were induced either by a single fourth instar larvae

feeding for three days or 4, 6 or 8 TPB feeding over 7 days. Lower levels of

feeding damage resulted in induction of bergapten and xanthotoxin only.

*:* *:* LFC induction in response to nymphs was significantly less than that

observed in response to adults. However, induction of bergapten by nymphs

in field plants resulted in levels of LFCs capable of causing chronic contact

dermatitis in visibly damaged areas of petioles and approached those levels

in randomly sampled petiolar tissue and heart tissues of damaged plants. + In laboratory studies, the constitutive and induced levels in leaves reached those capable of causing chronic contact dennatitis and sometimes reached

levels able to cause acute contact dermatitis. lnduced levels of LFCs in

petioles were high enough to cause dennatitis in one experiment only, when

ten adult TPB were allowed to feed for seven days.

*:* *:* Results of several experiments suggest that systemic induction of LFCs may

occur. 2.7 Discussion

Feeding damage by cabbage looper larvae and TPB nymphs and adults

which exhibit different behaviours resulted in induction of the three phototoxic

LFCs in celery petioles with no overall differences occumng in the pattern of

induction elicited by the two pests; bergapten was the most inducible followed by

xanthotoxin, then psoralen. The levels of LFCs in petioles did not reach those

capable of causing contact dermatitis Ri people, in most laboratory expenments

or in randomly sampled portions of artificially infested field plants. These results are similar to those observed by Tnimble et al. (1 994) who found that celery damaged by ten 3" instar lanrae of S. exigua (that were allowed to complete development on the plant before analysis) had petiolar levels of LFCs that reached only 3pglg fresh tissue weight. In the present study, severe feeding damage &y TPB adults (expt. 5) caused induction of LFCs to concentrations well above those able to cause chronic contact dermatitis. Levels of LFCs in visibly damaged portions of field grown plants also contained levels of LFCs that were capable of causing chronic contact dermatitis.

The levels of damage used in the artificial infestation experiment are similar to what may occur on a given plant prior to spraying (Chaput, pers. cornm.). Although the economic threshold is much lower, the actual number of nymphs on a plant at the threshold may be much higher than 1 TPB in 5 or ten plants because no accurate scouting methods are available for TPB. However, under current conventional control practices, the amount of feeding damage able to induce LFCs to concentrations of concem are not reached because they

exceed those that cause unacœptable levels of cosmetic damage.

Interestingly, nymphs caused an attenuated induction response compared

with that of adults. The possibility that adults simply fed more than nymphs

cannot be ruled out because the amount of damage caused by each life stage

was not quantified. However. plant responses to insects have been shown to

differ depending on the life stage of the insect in other systerns caused by

differences in regurgitant or saliva (Takabayashi et al. 1995). In addition,

differences in salivary content between Ife stages have been observed in at least

one mirid; certain proteinases occur in the juveniles and in female but not male

adults of Mins dolabratus L. salivary glands (Miles 1972). Only male adults were

used in the present experiments to avoid oviposition damage to the plant. Saliva

of TPB is thought to be involved in the toxic effect of TPB feeding damage on plants (Laster and Merideth 1974) and salivary factors of mirids have been implicated in induction of phenylalanine ammonia lyase (PAL) (an enzyme commencing the production of furanocoumarins, flavonoids and phenolics) in birch (Hartley and Lawton, 1991). Thus. developmental differences in saliva may account for the observed differences between stages.

Constitutive and induced levels of LFCs in leaves were above those capable of causing chronic contact dematitis and on occasion, above those capable of causing acute contact dermatitis. Leaves were not inducible, except in response to severe feeding damage by TPB adults on plants that had relatively low constitutive levels of LFCs. In fact. leaves did not appear to be as inducible as petioles in ternis of the variety of LFCs that were induœd, the percent

induction and the levels of damage required for induction. The lack of induction

in leaves in response to feeding damage by first instar cabbage looper larvae

may have been a result of insuficient damage. In contrast, Zangerl(1990) found

that mechanical damage causing 2% tissue loss in parsnip foliage resulted in

significant induction of LFCs and that this response was less than that

experienced in response to neonate cabbage looper larvae after feeding for only

24 hours (Zangerl 1990). The discrepancy in results between species may be a

consequence of genetic differenœs between these two species.

The difference in inducibility of leaves versus petioles is consistent with

Optimal Defense Theory, one of the prominent theories describing the

distribution and production of plant secondaiy compounds. The theory predick that tissues with high value or probability of attack by natural enemies should

have high levels of constitutively produced defenses and low inducibility whereas tissues with less value or a lower probability of attack should have low levels of constitutively produced defenses and high inducibility (McKey 1979). The distribution and inducibility of LFCs in wild parsnip strongly supports this theory

(Nitao 1990, Zangerl and Rutledge 1996)

Finally, the results of the field experiment provide the fint documentation of induction in celery heart tissue; exposure of plants to 15 TPB resulted in increased levels of bergapten in hearts compared with unexposed plants. In contrast, Zangerl and Rutledge (1996) found that LFCs were not inducible in unexpanded leaves of wild parsnip. The resulh of several expenments suggest that LFCs may undergo

systemic induction. Translocation was measured directly in the experiment

comparing induction in adults versus nymphs. Levels of xanthotoxin above TPB

treatment cages were higher than those above control cages while tissue below

damaged petiolar tissue did not contain levels that were difFerent from those in

tissue below control cages. Some acropetal induction of xanthotoxin in wild

parsnip has been shown to occur although induction was found to be highly

localized and mainly restricted to the damaged leaflet (Zangerl and Berenbaum,

199411995). In aintrast, the data from the present study also provide

circumstantial evidence for induction of LFCs in leaflets opposite the damaged

leaflets and in uncaged leaflets subtending the insect-damaged leafiets. Across

insect densities, plants that contained TPB in some of their cages had higher

foljar levels of xanthotoxin than plants that did not experienœ insect damage.

Also, the interaction between the presence and number of insects was not

significant for bergapten reflecting the increasing trend in bergapten levels in

control cages with increasing densities of insects.

Finally, in the recovery and sensitization experiment, fluctuations in levels

of xanthotoxin following damage occurred in both the damaged and undamaged

petioles. Foliar xanthotoxin levels decreased in darnaged and undamaged tissues from 2 to 4 days, possibly indicating early induction in both damaged and

undamaged tissue by day 2 with a subsequent retum to constitutive levels.

Zangerl and Berenbaum (1995) observed induction of xanthotoxin 6 hours following mechanical damage. with maximum concentrations reached after 24 hours in wild pannip. A second damaging on day 7 resulted in increases of xanthotoxin in damaged and undamaged petioles by day 10. Numerous additional fluctuations in both damaged and undamaged tissues were significant for al1 three LFCs before Bonferroni adjustment for the number of contrasts and may be revealed by repeating the experiment with a greater number of rep l icates.

lncreased levels in completely separate petioles, though apparent in the mechanically damaged plants was not obvious in the insect damaged plants and may reflect differences in the type or extent of damage. Mechanical damage occurred along the entire length of petiole and on al1 of its leaflets, whereas insect darnage was localized. Experiments measuring translocation in parsnip also used highly localized insect or mechanical damage (Zangerl 1990, Lee

1993, Zangerl and Berenbaum 199411995). However, differences between plant responses to mechanical damage and insed damage in tens of the magnitude of response (Baldwin 1988, Zangerl 1990, Baldwin 1991, Hartley and Lawton

1991, Olson and Roseland 1991) and the type and spatial extent of response

(Stout et al. 1996) have been shown to occur in other plants.

Further research is required to clearly establish the nature of LFC induction in celery. LFCs may have been translocated from their site of synthesis in damaged tissues, to remote locations (separate petioles). However, because

LFCs are normally present as lipophilic aglycones (Berenbaum 1991) and the rnetabolic cost associated with translocation of Iipophilic substances through the phloern is high (Genhenzon 1994) 1 is unlikely that LFCs were translocated directly. Furanocoumarins are not normally found in the phloem (Camm et al.

1976), although Berenbaum (1995) reported that xanthotoxin was taken up by cut stems of Heracleum lanatum-

Alternatively, induction in remote tissues may have involved the translocation of a hydrophilic precursor or other endogenous cue; cell wall fragments (Ryan et al. 1986, Baldwin 1991), simple phenolic acids and hydroxamic acid glucosides (Genhenzon, 1994) wound hormones (Hartley and

Lawton 1991) or wound-induced signals such as action potentiak (Hartley and

Lawton, 1991) are suspected or known to be involved in systemic responses in other plants. Finally, volatile cues have also been show to be involved in induction (Hartley and Lawton 1991, Schoonhoven et al. 1998). For example,

Olson and Roseland (1991) found that insectdamaged sunflower foliage produced increased levels of two coumarins in damaged plants within a few days and in completeiy separate undamaged plants affer one week. They suggest that methyl jasmonate or some other volatile may have caused induction in adjacent plants. Volatile complexes in plants are known to change following wounding (Schoonhoven et al. 1998). Also, ethylene is a well-known wound- induced signal (Hartley and Lawton, 1991). Quite possibly, more than one factor is involved in the induced response. Given the rapidity of induction in experiment

7 and the fact that celery vasculature occurs in relatively distinct petiolar modules, volatile elicitors are likely candidates for the observed effects.

However, the results of the experiment measuring induction in leaves suggest that an endogenous cue was responsible for induction within petioles because adjacent plants would presumably have responded to a volatile elicitot. In the

latter case, plant volatiles may have been contained somewhat by the foliar

cages.

In summary, insect feeding damage elicits a rapidly induœd response

involving the increase of one or more phototoxic LFCs in hearts. petioles and

leaves of celery. Localized damage results in a predorninantly localized

response that is qualitatively similar regardless of whether the plant in exposed to

chewing or to laceratemush feeding damage but quantitatively different

depending on the stage of TPB. Induction of LFCs by insects alone did not

increase the risks of experiencing contact photodematitis, except under severe

conditions of feeding damage. The contribution of insect elicited induction is

small compared ta that which occurs in response to other factors such as

pathogen infection. Yet, plants at the edges of fields near alternative hosts rnay

be more prone to such damage and several factors increase the risks associated with insect induction of LFCs. Celery is exposed to numerous elicitors throughout the growing season and both foliage and outer petioles are removed at harvest, which may trigger a damage-induced response. Also. post-harvest

increases in LFCs are common (Beier and Oertli 1983) and healthy areas of insect-damaged and pathogen-infected plants (Surico et al. 1987, Heath-

Pagliuso et al. 1992) may contain induced levels of LFCs. 2.8 Recommendations for Future Research

Future research should attempt to describe the temporal and spatial boundaries of the induced response in celery because there may be concems of contacting or ingesting increased LFC concentrations in apparently healthy tissue of damaged plants. Systemic induction may depend on the type and amount of damage, with low levels of damage eliciting only a localized response. The extent and nature of induction may also depend on the cultivars învolved and the environmental conditions of the plant. Because the production of LFCs is elicited by numerous stresses, an investigation of the sirnultaneous or sequential stimulation by more than one factor should be conducted to assess rïsks of low levels of insect damage in an agricultural context. An evaluation of organically grown celery rnay provide a useful situation for detemining the effects of natural elicitors over a growing season. Finally, because insect feeding causes cosmetic damage, the rnost likely increase in acceptance of insect damage will be for infestations early in the season because the plant may subsequently outgrow the visible symptoms. An evaluation of the effects of early damage on subsequent levels of LFCs and subsequent herbivore attack will be an important step in evaluating the risks and benefits of these compounds as part of an induœd resistance response. Chapter 3. An assessrnent of the impact of three phototoxic linear furanocoumarins in Apium gmveolens on growth and development of parasitised and non-pansitised nymphs of the tarnished plant bug (TPB),

Lygus lineolaris (Palisot de Beauvois).

3.1 Abstract

Linear furanocoumarins, secondary metabolites present in celery, Apium graveolens, have been shown to confer antibiotic and antkenoüc resistanœ against several insect herbivores and several of their parasitoids. The effects of the linear furanocoumarins, psoralen, bergapten and xanthotoxin on the growth and development of the tamished plant bug. Lygus lindans, and one of its parasitoids, Penstenus stygicus. were assessed through diet incorporation and exposure to mechanically damaged and undamaged celery tissue.

Concentrations of diet incorporated LFCs that normally occur in celery had contradictory effects on the developmental rates of parasitised and non- parasitised nymphs depending on diet consistency. LFCs alone did not account for the effects observed in nymphs fed celery tissue. Paraslised nymphs fed undamaged foliage weighed more and developed more quickly than those fed damaged foliage or petioles but experienced earlier mortality. Similarly, non- parasitised nymphs fed undamaged foliage experienced earlier mortality but those fed darnaged tissue gained most weight. Damaging petioles, however, did not affect the growth and developrnent of parasitised nymphs but resulted in decreased weight gain and rates of development for non-parasitised nymphs.

The relevance of potential induction of other resistanœ compounds is discussed. 3.2 Introduction

lncreases in the production of secondary metabolites in response to insect

herbivory have often been shown to enhance resistance by reducing suitability of plants to herbivores and pathogens (Tallamy and Raupp 1991, Zangerl, 1990,

Stout et al. 1996, Agrawal 1998). Herbivore-induced production of furanocoumarins resulting in enhanced resistance has been demonstrated in wild parsnip following attack by 1. ni (Zangerl, 1990). LFCs have been shown to exhibit both antibiotic (Diawara et al. 1993a. Berenbaum et al. 1991, Lee and

Berenbaum 1989) and antixenotic (Berdegue et al 1997) effects against insects fed LFC-containing artificial diets. Survival tends to be high for both polyphagous and oligophagous insects following ingestion of LFCs (Diawara et al. 1993a.

Reitz and Tnirnble 1996) but decreased larval and pupal weights and increased generation times are wmmonly observed (Diawara et al. 1993a. Lee and

Berenbaum 1989, Berenbaurn et al. 1989). Sublethal effects cm confer resistance in a number of ways. For example, decreased weight often leads to reduced fitness in the adult stage of an insect (Herrns and Mattson 1992).

Most research investigating the interactions between LFCs and insects has been conducted using lepidopteran larvae (Berdegue et al. 1997) primarily because representatives of this group have become specialized on wild LFC- containing umbellifers (Berenbaum 1990) and because they include major pest species of celery in the United States. The few hemimetabolous insects known to feed on LFC-containing plants are thought to avoid the compounds by feeding selectively on tissues that contain low concentrations and by avoiding schizogenous canals that store LFCs (Berenbaum 1990). In the North Eastern

United States and Canada, the tamished plant bug, a hemimetabolous and polyphagous insect, is one of the greatest insect pest problems in celery.

Whether TPB avoids LFCs by feeding preferentially on the heart and inner surfaces of inner petioles is unknown. However, meristematic tissue often contains low levels of secondary metabolites (Hems and Mattson 1992) and celery contains low levels of LFCs relative to wiid species (Ceska et al. 1987).

Thus, 1 is possible that these compounds confer some resistanœ against this pest.

The sucœss of plant resistance depends not only on the expression of antibiosis, but also on the expression of antixenosis and interactions between plant resistance factors and natural enemies. If resistance leads to increased movement on the plant, for exarnple, an insect is often more likely to be observed and attacked by natural enemies (Bemays 1991). Plants rnay also provide chernical cues for natural enemies and influence the quality and survivorship of their prey or hosts (Price 1986). Parasitoids may be attracted to host-plant complexes but not hosts alone (Condit and Cates 1982, Reed et al. 1995) sometirnes as a result of plant volatiles that are induced and released in the presence of host regurgitants (Blaakmeer et al. 1994, Takabayashi et al. 1995).

Secondary compounds in host plants can also have an effect on the growth and development of parasitoids (Fox et al. 1996). LFCs have been shown to exhibit host-mediated detrimental effects and direct effects on the growth and development of parasitoids of insects fed LFCcontaining diets (Reitz

and Trumble 1996, Reitz and Trumble 1997).

Since host-plant resistance and biological control are important aspects of

integrated pest management, corn bining these tactics by assessing the suitability

for both plant pests and their natural enemies is desirable. Biological control of

TPB by parasitoids is being emphasized to reduce damage by this pest in other crops partly in response to the development of insecticide resistanœ (eg.

Hollingsworth et al., 1997) and to increase the compatibility between TPB control and IPM programs developed for other pests (Day, 1987). To detemine whether

LFCs found in celery plants affect the growth and development of TPB and one of its parasitoids, P. stygicus. nymphs were exposed to damaged and undarnaged celery tissue and to LFCs that had been incorporated into artificial diet. 3.3 Objective

1. To assess whether phototoxic linear furanocoumarins in celery affect growth

and development of the tarnished plant bug and one of its parasitoids, P.

stysicus by:

a) measuring differences in weight gain. developmental rate, survival and

adult longevity of parasitised and nonparasitised tamished plant bugs fed

diets containing LFCs and

b) measuring differences in emergence and wasp size of P. stygicus whose

hosts have been fed diets containing LFCs. 3.4 Materials and Methods

3.41 Plant and Diet Sources

Seeds of celery, Apium graveoiens L. cv Tall Utah 52-70HK (Stokes

Seeds Inc.) were grown in a greenhouse under natural and artficial lighting

(16:8hr (L:D) and given water as required and nutrient solution twice per week

(20:8:20 NPK solution). Ten weeks after seeding, plants were transplanted from plug trays into pots (12.7c.m diam.) and given both nutrient solution as necessary and a calcium nitrate (2OOppm) treatment twice per week. Mature plants between the ages of 90 and 120 days were used in al1 experiments. Two to six weeks prior to the commencement of each experiment mature standardized plants were moved to a controlled temperature growth chamber containing full- spectrurn lights (approximately 125p~/cm2at 340nm) and uniform conditions of

24O-18OC daylnight altemating temperature and a 16:8 hr. (L:D) photoperiod.

Damaaina Treatment

Leaf and petiolar tissue was pierced evenly and along the entire length of petiole and through all leaflets, using a device containing an array of ten insect pins covering a 1cm2 area. Plant tissue was pierced two days and one day before entire petioles were removed by making a neat cut at their base with a razor

(expt 1) or 50mm petiolar sections and single leaflets were removed (expt 2).

Artificial Diet

Artificial diet #3 (Vandenant 1967) was made as a single batch up to the addition of linseed oil and LFCs were incorporated at this stage. LFCs were weighed and thoroughly mixed in the appropriate amount of linseed oil. then mixed well into the diet at about 60°C under constant stimng. 5ml glass vials were filled with the

diet, then covered with parafilm. The vials were inverted and placed in holes in

the Iids of 429 plastic containers (see below), resting on a piece of nylon that was

stretched under the lid. Nymphs fed on the diet by piercing the parafilm through

the nylon (Vanderzant 7967). Due to leakage of the diet into the containers from

piercing of the parafilm by nyrnphs, the diet was rnodified in the third experhent

adding 15mls agar/IOOrnls diet following compleüon of diet formulation. This

allowed for a semi-solid wnsistency. The resulting diet was fed to nymphs as

1-5gm portions wrapped in a strip of aluminum so that two sides were exposed.

3.42 lnsect Sources Tamished plant bugs were obtained from a colony established at the

University of Guelph in January, 1997. The colony was maintained at 21°C with

a 16:8 (L:D) photoperiod and 55%-65% RH. Cages for adults contained

potatoes, beans and lettuce as well as water-soaked dental wicks. Every three

days, food was replaced and old food (containhg eggs) was placed in 4L plastic

buckets. F resh lettuce, beans, potatoes and water were given to developing

nymphs every three days. The colony was periodically supplemented with field

collected TPB adults to maintain genetic variability and augment the colony.

P. stygcus wasps were obtained from a colony established at the

University of Guelph. Adult wasps were maintained at 21°C with a 16:8 (LD) photoperiod and 55%-65% RH. Wasps were fed a dilute honey solution that was smeared on the mesh over their cages. Parasitisation

Late second instar and early third instar TPB nymphs were collected from the colony and haif were randomly selected for parasitisation. Nymphs were parasitised by enclosing about ten nymphs in a container with a mated female wasp and removing them individually by aspiration as they were parasitised.

Parasitised and non-parasitised nymphs were kept in buckets containing beans, lettuce and potatoes at 2I0Cfor four days (expts. 1 and 2) or five days (expt 3).

3.43 Plant Extraction and Analvsis of LFCs

LFCs were extracted according to the procedure published by Tnimble et al. (1992). 7-Benzyloxycoumarin, an intemal standard, was synthesised by refluxing 7-hydroxycoumarin (1.62g1 l0mmol. Aldrich Chemical CO.),Na2C03 (59) and benzyl bromide (29, Il-7mmol) in 100ml of acetone for 60hr under N2 in a fumehood. The mixture was then cooled and concentrated to dryness by rotary evaporation. The residue was taken up in water and extracted three times with chIoroforrn. Chlorofom extracts were cornbined, washed with brine, then concentrated to dryness over NazSO4. The residue was recrystallised from hot ethanol. The resulting cream coloured solid gave one peak on HPLC analysis, and its composition was confinned by NMR analysis.

Plant material (approx. lg leaffet samples or 2g petiolar samples) was chopped with a dissecting blade and placed in round-bottamecl (2Ox116mm) culture tubes with lOml deionized water and were spiked with 5pl of the intemal standard (5pg 7-bentyloxycouma~n/2OpI hexane:tetrahydrofuran (8 1: 1 9)).

Samples were homogenized with a Polytron tissue homogenizer (Kinematica, Switzerland) for 2.0 minutes. Toluene (10ml) was added to each tube that was subsequently capped with aluminum foi1 and vortexed for 1-5 minutes. Samples were then transferred to centrifuge tubes and œntrifuged for 40 minutes at 3309.

The toluene layer of each sample was quantitatively transferred to 13xl00mm culture tubes and evaporated to dryness on an N-evap at 40°C. A further 1-0ml of toluene was added and the samples were again evaporated to dryness to remove any traces of water. Sarnples were reconstituted in 100pl toluene and

Ioaded ont0 Bakerbond spe solid-phase extraction columns (500rng of silica,

J.T.Baker, Mallinckrodt Baker Inc. NJ) which had been preconditioned with 4x2ml toluene. The sample was rinsed Nice ont0 the column with 2xO.lml toluene, discarding the eluate. The cartridge was then eluted by gravity flow with 5% acetone in chlorofomi (3x0.5ml) discarding the first 0Sml of eluate. The eluate containing the furanocoumarin fraction was collected and transferred to

13x100mm culture tubes and concentrated to dryness using an Nevap.

Samples were then wrapped in foi1 and frozen until analysis by HPLC.

Frozen samples were thawed and vortexed for 30s in HPLC mobile phase

(hexane:tetrahydrofuran, 81:19). A 20pL aliquot was analysed by HPLC. HPLC quantitation and detection was achieved using a Shimadzu mode1 LC-GA high performance liquid chromatograph with a Shimadzu SPD-M6A photodiode array

UV-VIS detector at 290nm and quantified on a Shimadzu SPD-M6A cornputer program (ver. 2.12) integrated to the detector. A Spheroclone silica column

(25cm x 4.6mm. 5-pm particle sire) with a lOmmx4.6mm guard column with the same packing material was used (Phenomenex, Torrence, CA). The analytes were eluted isocratically with hexane:tetrahydrofuran (81 :19, mixed by the HPLC purnp) at 1.5mWrnin. at arnbient temperature. All solvents were HPLC grade

(Aldrich Chemical Co., and Fisher Scientific, Fair Lawn, NJ). Relative retention times were as follows: ISTD, 1.00, psoralen, 1.2; 5-methoxypsoralen, 1.5; 8- methoxypsoralen 2.5.

Calibration Iines were generated using at least five concentrations of purified standards (Aldrich Chemical Co.) spanning the range of interest.

Calibration lines for the three mmpounds were linear, with ? values of 0.99 or better. The Iirnits of detection and quantitation, calculated for each compound, were as follows: psoralen, 0.015 and 0.049 pg/ml, respectively; bergapten, 0.018 and 0.046 pglml. respectively; and xanthotoxin, 0.0005 and 0 .O01 pglml, respectively. Peak heights were used to calculate LFC concentrations.

3 44 Measurement of Growth Parameters

Experiments were conducted in a controlled temperature growth chamber, containing full-spectrum lights (approximately 45wlcm2 at 340nrn through the growth cup Iids) and uniform condlions of 21°-180C dayhight alternating temperature and a 16:8 hr. (L:D) photoperiod. In al1 experiments, weights of tarnished plant bug, developmental stages and survivorship were recorded every hnro days (expt. 1) or three days (expts. 2 and 3) and tissue or diet were replaced.

Where possible, weight gain was also adjusted for changes in development by comparing average weight for each nyrnph within a given instar. In addition, emergence of parasitoid larval and adults was recorded. In expt. 1. aduit size (as assessed by head width) was also rneasured upon death using a microscope fitted with a micrometer. Sex ratios of parasitoids were not anafysed because of low nurnbers of emerging wasps and because ratios change wnstantly in the field making interpretation difficult (Dolling 1991). Longevity and fecundity of adult tpb were recorded for expt. 1 only since total numbers of insects were too low for analysis.

3.45 Ex~erimentalSet-UD

Ex~eriment1: Growth and devero~rnent of ~arasitisedand un~arasitised tarnished plant bua nymohs fed damacred and undamaaed celeiv tissue.

Expt. 1 was conducted from February gm to March lgm, 1998. Petioles. including leaf material, were neatly cut at their bases and were placed in pairs

(one intact and one damaged) in 4L buckets containing nutrient solution. A plastic jug was placed over the top of the petiote and was suspended from a wooden lattice that hung just beneath the Iights. Cloth was attached to the open mouth of the jug and an elastic band closed the cloth around the petiole between the lowest and the second lowest set of paired leaflets. A group of six parasitised or unparaslised 3" instar nymphs were plaœd in each of four 4L clear plastic jugs. Every two days, celery tissue was weighed and replaced and nymphs were collected as a group and assessed for the following: developmental stage, weight, survival, and larval or adult parasitoid emergence. Adult non- parasitised tarnished plant bugs were placed in 4L white plastic buckets containing potatoes, lettuce and beans and their longevity and fecundity were recorded. LFCs were extracted from random samples of mechanically damaged and undamaged leaflet and petiolar tissue samples following exposure to insects. Experiment 2: Growth and development of ~arasitised and non-~arasitised tarnished plant bug nvm~hson celerv- leaves. ~etiolesand artificial diet containina LFCs.

Expt. 2 was conducted from August 18'k until Odober 15*. 1998. Nymphs were reared in 429 clear plastic containers with ventilated lids that contained a thin layer of agar on the bottom covered by a perforated sheet of filter paper to control humidity. Prior to parasitoid emergence, a small amount of moistened vermiculite was added to cages as a pupation site, in a small cavity cut out of the agar. Nymphs were either fed a single damaged or undamaged celery leaflet

(approximateiy 0.5gm). a damaged or undamaged celery petiole section

(approximately 3gm) or liquid artificial diet (5mls) to which LFCs had been added.

Diet treatments contained LFCs in the following combinations: Opglgm (control),

0.75pg1gm bergapten + 0.5pgfgm xanthotoxin + Opg/gm psoralen (low dose),

5pgfgm bergapten + 5pg/gm xanthotoxin + 0.5pgIgm psoralen (intermediate dose) or 37.5pgfgm bergapten + 25pgfgm xanthotoxin + 2.5pgIgm psoralen (high dose). Combinations were chosen based on documented values of LFCs found in cultivars of the 52-70 series and other diet incarportation experiments (e-g.

Diawara et al. 1995, Berdegue et al. 1997). Plant tissue and diet were replaced every two days and tissue was weighed before and after TPB exposure.

Ex~eriment3: Growth and development of ~arasitisedand non-~arasitised tarnished la nt bua nvmphs fed a semi-solid artificial diet containina LFCs.

Expt. 3 was conducteci from September 29'" until October lgM, 1998.

Nymphs were reared in 429 clear plastic containers with ventilated lids that contained a thin layer of agar on the bottom covered by a perforated sheet of

waxed paper. Diet treatrnents contained LFCs in the following combinations:

Opglgm (control), 3pg/gm bergapten + 2pglgrn xanthotoxin + Opglgm psoralen

(low dose), 24Ciglgm bergapten + 21pg1gm xanthotoxin + 2Ciglgm psoralen

(intermediate dose) or 37.5Ciglgm bergapten + 31.5pg/gm xanthotoxin + 3pgIgm

psoralen (high dose). Plant tissue and diet were replaced every three days and

tissue was weighed before and after TPB exposure.

3.46 Statistical Analvsis Changes in weight and developmental stages were analysed using

ANOVAs for repeated measures followed by profile transformations (that

compared adjacent levels of time) and, where appropriate, Tukey's studentised

range test (SAS, 1991). LFC levels were analysed using repeated measures

ANOVAs for a split-plot design. DHerences in survivorship were measured using

a factorial ANOVA as days to mortality for the latter two experiments. In the firs? experïment where groups of nymphs were measured together, changes in percent survival over time were compared among treatrnents using an ANOVA for repeated measures. DHerences in days to emergenœ of parasitoid larvae and adults andlor percent ernergence were analysed using ANOVAs or 1-tests.

Where subsequent analyses further elucidated those described by significant main effects and interactions, the significanœ of subsequent analyses are reported.

3.5 Results

3.51 Ex~eriment1 : Growth and develo~mentof ~arasitisedand un~arasitised tarnished dant bua nvm~hsfed damaaed and undarnaaed celerv tissue. The damaging treatment resulted in induction of bergapten only in leaves and petioles (F1,15=7.09. psO.05). BO# xanthotoxin and bergapten occurred in higher levels in leaves than petioles (FIvl5=5.59, ~~0.05and F1,15=7-72, p~0-05, respectively), however, psoralen occurred in levels below the lhlof quantitation.

Overall, LFC levels were very low; total levels in intact foliar tissue were only

3.8+0.7pg/g fresh tissue weight while intact petioles contained approximately

1.3+0.3pg/g fresh tissue weight. Decreases in tissue weight over the two day exposure period was variable. Overall, water loss was 33.2% for damaged tissue and 20.1% for undamaged tissue. A regression of nyrnphal weight over time with tissue loss revealed a signifiant positive relationship for non- parasitised nymphs fed damaged tissue (Fivz3=5.18, piO.05) and approached significance for non-parasitised nymphs fed undamaged tissue (F1,23= 3.176, p=0.088).

Parasitised nymphs fed undamaged celery gained significantly more weight than those in al other treatment conditions from day 4 to day 6 (a difference that was maintained until day 8, after which parasitoids emerged)

(F1,12=29.02, ps0.0005) (Fig. 9). Between day 2 and day 4. parasitised -nyrnphs fed damaged tissue developed faster than nymphs in al1 other conditions

(F1,12=7.54, pS0.05) and from day 4 to day 6, paraslised nymphs fed damaged or undamaged tissue developed more quickly than non-parasitised nymphs

(F1,r2=7.76,pS0.05). I~~~rn~hsfed damaged tissue ONymphs fed intact tissue] 5

5 a a 4 3 - hh Em ergence 2 1 -

O ;- 8 I l 1 *

Sampling day

Fig. 9. Mean weights of nongarasitised (A) and parasitised (B) TPB nymphs fed damaged and undamaged celery tissue. Values are means of 4 replicates each containing six nymphs. Bars headed with the same letter are not significantly different (p>0.05) for both parasitised and non-parasitised nymphs. Survival of nymphs was high but variable and mortality did not differ

among conditions (14.7k 6.7%) (~~0.05).The number of surviving adults in the

non-parasitised group was 16 of 24 for nymphs fed damaged tissue and 19 of 24

for nymphs fed intact tissue. with a sex ratio of approximately 50-50 in each

condition. Fecundity was not analysed because emergence occurred over a 7d

period and adults began to die within 5d of the last emerging adult. thus low

numbers of second generation nyrnphs were recovered (

Parasitoid larvae from all surviving nymphs except one emerged on day

10 and day 12. The total number of larvae emerging did not dmer between the two groups; 17 (71%) larvae emerged from nymphs fed damaged tissue and 16

(66%) from nyrnphs fed undamaged tissue. Of those parasitoids that emerged, only 8 from nymphs fed intact tissue and 5 from those fed damaged tissue emerged as adults. There were no dineremes in adult size between the two groups as measured by head width (p>0.05).

3.52 Ex~erirnent2: Growth and develo~mentof ~arasitisedand non-~arasitised tarnished ~lantbuci nvm~hson celerv leaves, ~etiolesand artificial diet containina LFCs.

The damaging treatment resülted in induction of bergapten in petioles only

(FItli = 6.93. ps0.05). The levels of LFCs in both petioles and leaves were surprisingly high cornpared with those found in other experiments; leaves contained 43.3e.6pg total LFCs Igm fresh tissue weight and petioles contained

13.W 1.6~total LFCslpg fresh tissue weight. Psoralen ocairred in measurable quantities but did not dHer in concentration between leaves and petioles. However, leaves contained higher levels of bergapten and xanthotoxin than petioles (Fl,tt = 70.28, ps0.0005 and F1,to = 15.89, ps0.005 respedively).

A greater decrease in tissue weight was also observed compared with that recorded in expt. 1; damaged leaves lost approximately 47.3% of their water content over a 2 day period wmpared with 34.6% for undamaged leaves and

20.2% for petioles in either condition. The containers were humid and condensation was often wiped from theirwalls, thus nymphs were not deprived of water. There was no relationship between weight gain in nymphs and weight loss in tissue for any of the treatrnents.

Although al1 nymphs gained weight (F3,,t1=70.72, psO.0001), the rate of growth depended on both the treatment and the tissue (al1 estimates, F3,41=

3.45, ps0.05). No overall differences in weight gain were observed between parasitised and non-parasitised nymphs (Fig . 1O). Non-parasitised nymphs fed darnaged leaves gained more weight than those fed damaged petioles

(F3,24=5.07, psO.01) especially between day g and day 12 (FlS21=14.26. ps0.001) when insects were in the fflh instar and adult stages. When weight gain was adjusted for developmental stage, the observed differences were still significant for the ffih instar and adult stages. For parasitised nymphs, an initial difference in weight gain from day O to day 3 was obsewed; nymphs fed intact petioles or damaged leaves gained more weight than those fed damaged petioles or intact leaves (Ft,1~3.94.p~0.05). However, from day 6 to day 9 (prior to emergence on days 9 and 12), nymphs fed intact leaves gained more weight than those in al1 other conditions (F3~t7=7.97,ps0.005). Day 6

OPETL P €TH LEAL LEAH

N onparasitised P arasitised Day 9

aPETL PETH LEAL LEAH

Nonparasitised P arasitised

Day 12

PETH rnLEAL Em ergence

N onparasitised P arasitise d

Fig. 10. Mean weights of parasitised and non-parasitised TPB nymphs fed damaged or undamaged leaves or petioles. Values are means of 5-10 nymphs. PETL = nymphs fed undamaged petioles, PETH= nymphs fed damaged petioles, LEAL= nymphs fed undamaged leaves, LEAH= nymphs fed damaged leaves. Bars headed by the same letter are not significantly different (ps0.05). Both parasitised and non-parasitised nymphs had shorter survival time

when reared on intact leaves than on any other food (F3.70 = 4.1 6, pSO.01)-

A corn parison of survival until emergence (day 9) revealed that parasitised

nymphs suffered earlier mortality than non-parasitised nymphs (FIJo' 5.65.

~~0.05).Non-parasitised nymphs fed damaged petioles experienced earlier

mortality than those fed damaged leaves (FJSs = 3.79. p~0.05) Larval emergence was quite low and did not d-fler between conditions; only 12 of 37 larvae from parasitised hosts emerged, due to high mortality of nymphs. Of the five larvae ernerging from nymphs reared on intact petioles, only 2 emerged as adults, while al1 four larvae emerging from nymphs fed damaged petioles successfully emerged as adults. No adults emerged from cornons of larvae whose hosts had been fed leaves.

Artificial Diets

This experiment was terminated on day 10 because of high mortality due to leaking of the Iiquid artiicial diet into the growth cup through tears in the parafilm created by nyrnph feeding. However, data analysis for surviving nymphs revealed some interesting differences between treatrnents. Between day 6 and day 9, nymphs fed the control diet gained significantly more weight than those fed diets containing the low or medium dose of LFCs whether or not nymphs were parasitised (F3s23=3.16, psO.05). In fact, nymphs fed the low and intemediate doses often lost weight between day 6 and day 9. By day 9, parasitised nymphs weighed significantly more than non-parasitised nymphs

(FI ,23=5.68,ps0.025) (Table 1). Developrnental differences were also observed Table 1. Mean weights (mg) of tamished plant bug nymphs fed a liquid artificial diet containing various concentrations of LFCs. LFCs Control Low Medium High (pg/g diet) O 1-25 10.5 65 Non-parasitised nymphs D~Y mean mean mean Mean O 0.7 0.7 0.7 0.7 3 1.O 0.8 0.7 0-7 6 1.O 1-4 1.1 1-0 9 1.7' 1.ob 1.ob 1.3ab Parasitised nymphs D~Y Mean mean mean Mean O 0-7 0-6 0.5 0.5 3 0.8 0-9 0.6 1 -2 6 1.4 1-0 1-6 1-6 9 2Sa 1.4b 1.ob 1.8ab

Wthin each parasitisation condition, letters indicate significantly difteren increases in weight between means. Across time parasitised nymphs weighed significantly more than non-parasitised nymphs (ps0.05). with nymphs fed the control diet developing faster than those on the diet containing the intermediate dose of LFCs (F3,n=5.31, p~0.01)and parasitised nymp hs developing faster than non-parasitised nyrnphs (FI ,23=14-02. p~0.005)

(Table 1). When weights were adjusted for developmental stage, a difFerent pattern emerged, with parasitised fourth instars fed a high dose of LFCs weighing more than those fed a low dose (F3,i8=3.4,ps0.05). Thus, differenœs in weight gain between nyrnphs reared on control and treated diets were largely accounted for &y faster rates of development for nymphs fed the control diet. Suivival of nymphs was not analyzed because the artificial diet was the single largest mortality factor. The experiment was teminateci before emergenœ occurred.

3.53 Experirnent 3: Growth and devefo~rnentof ~arasitisedand non-~arasitised tarnished ~lantbua nvm~hsfed a semi-solid artficial diet containincl LFCs.

Analysis of weight gain from day O to day 6 revealed that parasitised nymphs weighed more = 27.36, p10.0001) and developed more quickly

(F1,68=3.97,ps0.05) than non-parasitised nymphs. Between treatrnents. from day O to day 3. a significantly greater increase in weight was observed for nymphs reared on diets containing the low and intermediate doses of LFCs compared with those exposed to control diet or the diet containing the highest dose of LFCs (FSvse= 7.88, p10.001) (Table 2). However, when weight gain was adjusted for development, there were no differences in the weights of fourth or fifth instars (p>0.05). Thus, the differences in weight gain are largely accounted for by differences in developmental rates. Table 2. Mean weights (mg) of tamished plant bug nymphs fed a semi-solid artificial diet containing various concentrations of LFCs. LFCs Control Low Medium High (pg/g diet) O 4.6 47 72 Non-parasitised D~Y mean mean mean Mean O i-5 1.3 1.3 1.5 3 1-7" 1.8b 2.2b 1 ,7b 6 2.8 2.2 2.1 2.1 9 2.3 2-0 2.0 2-1 Parasitised D~Y Mean mean mean O 2.1 2.1 2.2 3 2.5" 2.gb 3.0" Nat tasteci 6 2.7 3.1 3.0 9 3.1 3, f

Within each parasitisation condition, letters indicate signifïcantly different increases in weight between means. Across time parasitised nymphs weighed significantly more than non-parasitised nymphs (ps0.05). Mortality was high and did not differ among nymphs fed dmerent diets

(p>0.05). Mortality ranged from 40% to 73% of nyrnphs across diet treatments.

Time to larval emergenœ also did not differ. In each treatment. 5 to 7 lanrae emerged from parasitised nymphs. Of those that emerged. cocoons were only found from 4 of the larvae that emerged from nymphs fed the low LFC diet and 3 of the larvae that emerged from nymphs fed the intemediate LFC diet. Wasps emerged from al1 four cocoons of larvae whose hosts had been fed the diet containing low levek of LFCs and two from those whose hosts had been the diet containing the medium dose. One of the cocoons in the latter condition contained a partially developed adult All larvae emerged on day 9 and day 12.

3.6 Summary of Resulîs

-3 Mechanically damaging plants caused induction of bergapten in leaves and

petioles of plants containing low constitutive levels of LFCs (expt. 1) and in

petioles of plants containing high constitutive levels of LFCs (expt 2).

*:* *:* Weight gain in parasitised nymphs fed undamaged petioles with attached

leaflets (expt 1) or undamaged leaflets (expt. 2) was significantly greater than

that for parasitised nymphs in al1 other treatrnents.

0:- Parasitised nymphs developed more quickly than non-parasitised nymphs.

4+ lnitially (day O to 3) parasitised nymphs fed intact petioles or damaged leaves

gained more weight than those fed damaged petioles or intact leaves.

*:* *:* Non-parasitised nymphs fed damaged leaves gained more weight than those

fed damaged petioles.

03 All nymphs had a shorter survival time when fed intact leaves No differences in time to parasitoid emergenœ were observecl.

From day 6 to 9. nymphs fed a liquid artificial diet without LFCs gained more

weight and developed more quickly than those fed diet containing the low or

medium doses of LFCs.

From day O to 3 TPB nymphs fed a semi-solid diet containing low or

intemediate doses of LFCs gained more weight than those fed the control

diet or a diet containing a high dose of LFCs.

Differences in weight gain for nymphs fed artificial diet were largely accounted

for by differences in developmental rates.

Parasitised nyrnphs fed artificial diet weighed more and developed more

quickly than non-parasitised nymphs.

3.7 Discussion

Mechanically damaging celery tissue resulted in changes in food quality that affected weight gain, developmental rate and survivorship of both parasitised and non-parasitised TPB nymphs. However, the data indicate that LFCs alone are not likely to be responsible for the observed changes. The differences in

LFC levels across experiments and in different tissues were far greater than those observed between damaged and undamaged tissues. In fact. no induction was observed in fdiage in experiment 2. In addition, the differences in growth and development of nyrnphs reared on celery tissue were not consistent with those observed for nymphs reared on artificial diet.

LFC-containing diets were found to affect developmental rates of TPB nyrnphs compared with those of nymphs fed control diets, even at low doses. The results of the first artificial diet study revealed a decrease in weight and

developmental rates for nymphs fed the diet containing low or intermediate levels

of LFCs compared with those fed the control diet by day 9. In contrast. the

results of the second artificial diet experiment revealed an eariy increase in

weight gain and developmental rates of nymphs fed low or intermediate doses of

LFCs compared with nymphs fed the control diet. That consumption of LFCs

rnay decrease Vie rate of development of TPB is consistent with the observations

of othen for lepidopteran larvae (Berenbaum 1978, Berenbaum et al. 1989,

Diawara et al. 1993a. Reband Trumble 1996).

Similar dnerences in developmental rates were observed for doses

ranging from 1.25 to 47pg LFCslg diet. These doses spanned the range that

occurred in celery tissue, which may provide a partial explanation as to why

similar effects did not occur in nymphs fed plant tissue differing in LFC levels.

That nymphs reacted similarly to the diet containing a high dose of LFCs and the

control diet suggests that the compounds may not have been evenly distributed

throughout the diet and were avoided by the insect. Levels in diets were verified

by HPLC, however, greater variability in levels occurred with increasing doses of

LFCs. Thus, LFCs rnay have occurred in small hydrophobie pockets in the diet that could have been avoided by nymphs. lmprovements in the artificial diet technique and cornparison with tissue lacking LFCs, or difFering only in LFC levels is desitable in order to further elucidate their effects. Comparison of growth on celety tissues wiai that on lettuce or green beans (which do not contain LFCs) show that slowei development occurred on celery under the same growth conditions (data not shown) which is consistent with the results of the artificial diet experiments.

No differenœs were obsewed between the responses of parasitised and non-parasitised nymp hs to LFCs in artificial diet at the concentrations tested.

LFCs did not affect time to parasitoid emergence but the number of larvae and adults that emerged may have been too low to detect any differences.

Parasitised and non-parasitised nymphs responded similarly to LFCs and parasitised nymphs weighed more than non-parasitised nymphs, which is consistent with the findings of others for parasitised nymphs in the field (Dolling

1991). It was not possible to extricate the effects of LFCs on survival from the negative effects of the arüficial diet itself; no differenœs were observed between conditions and mortality rates were high. The artificial diet developed for TPB is not commonly used due to higher mortality cornpared with growth on plant material. Future attempts to use the solid diet should include the development of a improved method of delivery such as the use of a thin covering over the diet.

In addition, the incorporation of LFCs on a membrane (not possible with a liquid diet bot a common procedure for solid diets) would allow for the use of higher doses of LFCs because the amount would not be limited to that which mixed well in the linseed oil. -

Results for the experiments using damaged and undamaged celery tissue suggest that factors other than LFCs affect growth and development of these insects. Parasitised nymphs fed undamaged tissue (expt 1) or foliage (expt 2) experienced greater weight gain and developmental rates than nyrnphs in al1 other conditions prior to emergenœ. Because parasitised nymphs normally weigh more than non-parasitised nyrnphs, the data suggest that feeding on damaged foliage inhibits an expected increase in growth. This may have occurred as a resuit of decreased cansumption rather than decreased efficiency of conversion of food to biomass; hyperphagy has been shown to account for increased weig ht gain in parasiüsed larvae cornpared with non-parasitised larvae in other systems (Reb and Trurnble 1996).

No differences were observed in percent emergence, time to emergence for P. stygicus larvae and wasps, or wasp size. Thus, mechanically damaging the host plant either resulted in direct effects on the laival stage of the parasitoid or in detrimental effeds in TPB nymphs resultnig from the combination of damaged tissue and parasitisation but not either condition alone. Reitz and

Trumble (1996) found that an LFCcontaining diet affected only the larval stage of the parasitoid Copidosoma floridanum, exposed through one of its hosts, T. ni

Because the efficiency of food conversion from mirid to braconid is very high

(Dolling, 1991). it would not be surprising to find that detrimental compounds in the diet of their hosts affect larval parasitoids directly in this system as well.

Damaged foliage was more suitable than intact foliage for non-parasitised nymphs in ternis of both weight gain and survival. While no significant differences in LFC content were observed between damaged and undamaged tissue, other factors such as water stress may explain these resuits. Water- stressed plants often have higher concentrations of free amino acids such as proline (Fukutoku and Yamada 1982, Zangerl and Berenbaum 1998) and soluble carbohydrates (Haglund 1980, Fukutoku and Yamada 1982) which have ken shown to serve as a favourable resource for insect herbivores- Water deficits

also lead to changes in secondary compounds that may affect herbivores

(Gershenzon 1984). Differences in water content cannot explain the reduced weight gain and developmental rates of non-parasitised nymphs fed damaged petioles compared with intact petioles. Possibly, other induced compounds were involved in a resistance response.

A number of studies have found that LFCs do not wholly account for effects observed in growth and development of insects fed LFC-containing plants or diets (Trumble et al. 1990, Diawara et al. 1992, Diawara et al. 1993b).

However, the involvement of synergists such as myristicin, safrole and fagaramide (themselves, inhibitors of mixed-function oxidases) (Berenbaum and

Neal 1985, Neal 1989) are usually not considered end may confound results.

Another compound thought to be invohred in celery resistance to insects is the pthalide, sedanenolide (Meade et al. 1994). In a study of celery petiolar volatiles,

Macleod and Arnes (1998), found that the major pthalide present was sedanenolide, accounting for 28.1 % of the celery volatiles. However, levels of pthalides are highly variable and depend on genotype and growth conditions

(e-g. Van Wassenhove et al. 1990). Coumarin, which occurs in a bound fonn as trans-o-glucosyloxycinnamic acid. is also released from the leaf surface upon tissue wounding and rnay confer partial resistance (Panda and Khush, 1995).

Other potential candidates include chlorogenic acid which is found in numerous plants including those of the Apioideae and has ben shown to exhibit toxic effects in the corn earwom, Heliothis zea (Boddie) (Isman and Duffy, 1982).

Of interest, however, is the finding that in wild parsnip, mechanical damage results in the induction of furanocoumarins and myristicin only (Zangerl et al. 1997, Zangerl and Berenbaum 1998). Possibly, this is also the case for celery and differences in resistance can be accounted for by the release of compounds already present in undamaged tissue, synergists and precursors of furanocoumarins (Afek et al. 1994, Afek et al. 1995) and nutritional variables.

The mechanisms of resistance in celery are as yet unknown and require further study.

Damaged petioles are less suitable for non-parasitised nymphs but equally suitable to parasitised nymphs compard with intact tissue. Thus, other compounds induced in the petioles may exhibit antibiosis against non-parasitised

TPB only. Currently, efforts are underway to develop insect-resistant genotypes containing low levels of LFCs in order to enhance control of lepidopteran pests such as S. exigua (Diawara et al. 1996). Breeding programs may also be able to select for genotypes that have low levels of LFCs but maintain resistance to insects such as TPB. It must be remembered, however, that while LFCs may not confer resistance against adapted pests, they may play a role in resistance against non-adapted species and efforts to further minimize their occurrence in commercial crops may lead to unanticipated increases in the presence of other pest species. 3.8 Recommendations for Future Research

The development of celery cultivars with enhanœd resistanœ to TPB and other insects that is not based on the production of the three phototoxic LFCs will be invaluable to celery growers in Ontario and elsewhere. These compounds did not appear to detrimentally affect growth and development of TPB; difFerences observed in diet incorporation studies did not translate to differenœs observed in tissue studies. However, there rnay be other important ways in which TPB interacts with these and related compounds. A examination of antixenotic effects of LFCs on TPB, for example, may be fortuitous because such effects have been found to occur at concentrations that are far lower than those resulting in antibiosis in other herbivores (Berdegue and Trumble 1997). Finally, because

TPB is a very polyphagous pest, and because diet incorporation studies are valuable in the study of insect-plant interactions, the continued development of a suitable artificial diet will be beneficial to elucidate TPB responses to LFCs and othet secondary compounds. The addition of agar to fom a semi-solid diet improved the incorporation experiment because as lacerate and flush feeders,

TPB tear their food, which resulted in leakage of the Iiquid diet. TPB feeding was not deterred by the addition of agar. but the diet caused the nymphs to stick to the sides of the containers after feeding. Further improvements in delivery and incorporation of substances will improve the diet. Literature Cited

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Table1 . Summary of the Analysis of Varianœ measuring induction of LFCs in celery petioles by cabbage looper lanrae.

Table 2. Surnmary of the Analysis of Variance measuring induction of LFCs in celery leaves by tamished plant bug adults.

Table 3. Summary of the Analysis of Variance measuring induction of LFCs in celery petioles by tamished plant bug adults

Table 4. Summary of the Analysis of Variance measuring indudion of LFCs in celery petioles by tamished plant bug adults and nymphs.

Table 5. Summary of the Analysis of Vanance measuring induction of LFCs in field-grown celery plants artificially infested with tamished plant bugs.

Table 6. Summary of the Analysis of Variance measuring induction of LFCs in mechanically damaged celery plants Mer4,5,7 w 10 days.

Table 7. Summary of the repeated measures Analysis of Variance measuring weight gain in TPB nymphs fed damaged or undamaged celery petioles with leaves intact.

Table 8. Summary of the repeated measures Analysis of Variance measuring weight gain in parasitised (A) and non-parasitised (B) TPB nymphs fed damaged or undamaged celery petioles or leaflets.

Table 9. Sumrnary of the repeated rneasures Analysis of Variance measuring weight gain in parasitised and non-parasitised TPB nymphs fed LFCs incorporated into a liquid artificial diet.

10. Table 10. Summary of the repeated measures Analysis of Variance measuring weight gain in parasitised and non-parasitised TPB nymphs fed LFCS incorporated into a semi-solid artificial diet Table 1. Summary of the Analysis of Variance measwing induction of LFCs in celery petioles by cabbage looper larvae. Source DF Sumof Mean Square F value Pr> F Squares Univariate Tests Bergapten Model Tirne Error (Plant(lÏme)) Bug BugTime Error Corrected Total Xanthotoxin Model Time Error (Plantmme))

Emor Corrected Total Psoralen Modet Time Error (Plantmme)) Bug BugTime Error Corrected Total Total Model Time Error (Plantfime)) Bug BugTime Error Corrected Total 22 5.121 Note: The Multivariate ANOVA was signifimnt for bug, al1 estimates F3,?=11.27, ps0,005 Table 2. Surnmary of the Analysis of VaMnce measuring induction of LFCs in celery leaves by tamished plant bug adults. Source OF Sumof Mean Square F value Pr> F

Univariate Tests Bergapten Mode! Density Eror (Plant(Density)) Treatment Densityqreatment Error Corrected Total Xanthotoxin Model Density Erro r (Plant(Density)) Treatment DensityTreatment Error Corrected Total Psoralen Mode1 Density Erro r (f lant(Density)) Treatment Density'Bug Error Corrected Total Total Model Density Erro r (Plant(Density)) Treatment

Error 29 79.847 2.753 Corrected Total 47 133.331 Note: The Multivariate ANOVA was significant for treatment, ail estirnates F3,9= 6.79, pû.05- The density by treatment interaction approached significance, Pillai's Trace Fea= 2.4029, p=0.065 Table 3. Summary of the Analysis of Variance measurïng induction of LFCs in celery petioles by tamished plant bug adults. Source DF Sumof Mean Square F value Pr, F Squares Univariate Tests Bergapten Model Density Emor (Plant(Density)) Bug Density'Bug Emor Corrected Total Xanthotoxin Model Density Emor (P la nt(De nsity)) Bug Density'Bug Emor Corrected Total Psoralen Model Densiîy Emor (Plant(Density)) Bug Denslty*Bug Error Corrected Total Total Model Density Emor (Plant(Density)) Bug Density'Bug Error

Corrected------Total------Note: The Multivanate ANOVA was signifiant for density, Pillai's Trace F9.48~5.29, ps0.00005 and bug, al1 estimates FSbl4=32.40, p~O.000001 Table 4. Summary of the Analysis of Variance measuring induction of LFCs in celery petioles by tamished plant bug adults and nymphs. Source DF Sumof Mean Square F value Pr> F

Univariate Tests Bergapten Model Sage ' Plant (Stage) Bug Stage'Bug Plant(Stage)*Bug Erro r Corrected Total Xanthotoxin Modef Stage Plant(Stage) Bug Stag e*Bug Plant (Stage)*Bug Error Corrected Total Psoralen Model Stage Plant(Stage) Bug Stage'Bug Plant (Stage)*Bug Error Corrected Total Total Model Stage PIant(Stage) Bug Stage*Bug Plant (Stage)*Bug Error Corrected Total 31 1955.055 Faluesfor Stage (the between plot factor) are calculated using- an MS Emr value based on the MS for ~lant(aage)(Sas. 1991) Note: The Multivanate ANOVA was significant for stage, al1 estimates F3,s=14.41, pSO-0001. for bug, al1 estimates F3,p3.79, p10.05 and their interaction, F3.p 8.48, pdI.005, Table 5. Summary of the Analysis of Variance measuring induction of LFCs in field-grown celery plants artificially infested with tamished plant buas. Source DF Sum of Mean Square F value Pr, F Squares Univanate Tests Bergapten Model ~ond' Plant (Cond) Tissue CondTssue Ertor Corrected Total Xanthotoxin Model ~ond' Plant (Cond) Tissue CondTssue Error Corrected Total Psoralen Model cond' Plant (Cond) Tissue CondTssue Error Corrected Total Total Model cond' Plant (Cond) Tissue CondTssue Error Corrected Total 57 41.596 values for Stage (the between plot factor) are calculated using an MS Error value based on the MS for Plant(Stage) (Sas, 1991) Table 6. Summary of the Analysis of Variance measuring induction of LFCs in mechanically damaged celery plants after 4. 5, 7 or 10 days. Source OF Sumof Mean Square F value Pr, F

Univariate Tests Petioles Bergapten Model ~a y' Plant (Day) Treatment Error Corrected Total Cont rasts 2vs4 2vs 5 2vs 7 7 vs 10 Xant hotoxin Model ~ay' Plant (Day) Treatment Erro r Corrected Total Contrasts 2 vs 4 2 vs 5 2 vs 7 7vs 10 Psoralen Model ~a y' Plant (Day) Treatment Error Corrected Total Contrasts 2 vs 4 2 vs 5 2 vs 7 7 vs 10 Total Modei ~ay' Plant (Day) Treatment Error Correded Total Contrasts 2 vs 4 2 vs 5 2 vs 7 7vs 10 Univariate Tests Leaves Bergapten Model ~a y' Plant (Day) Treatment Emr Corrected Total Contrasts 2vs4 2vs 5 2vs 7 7vs 10 Xanthotoxin Model ~ay' Plant (Day) Treatment Error Corrected Total Contrasts 2vs 4 2vs 5 2vs 7 7vs 10 Psoralen Modei ~ay' Plant (Day) Treatrnent Error Corrected Total Contrasts 2 vs 4 2vs 5 2vs7 7vs 10 Total Model ~a y' Plant (Day) Treatrnent Error Corrected Total Contrasts 2 vs 4 2vs 5 2vs7 7vs 10 2.690 2.690 0.86 0.367 F values for Day (the between subjects factor) are calarlated us in^ an MS Enor value besed on the MS for Plant(Stage) (Sas, 1991) Table 7. Summary of the Repeated Measures Analysis of Variance measuring weight gain in TPB nymphs fed damaged or undamaged celery petioles with leaves intact. Source DF Surnof Mean Square F value Pr> F Squares Between Su bjects Cond 1 2.779 2.n9 21 .O3 0,0006 Treat 1 0.91 1 0.91 1 6.89 0,022 CondTreat 1 4.087 4,087 30.93 0,0001 Error 12 1.586 0-132

Source DF Som of Mean F value Pr> F Adj RF Squares Square G-G H-F Within Subjeds Time 4 37-761 9.140 284.83 9.E-33 8.E-21 7.E-32 Time * Cond 4 0.938 0.235 7.08 0.0002 0.002 0.002 fime * Treat 4 1.384 0.346 10.44 4.E-06 0-0002 5.E-06 Time * Cond * Treat 4 2.374 0.593 17-91 5.E-09 3.E-06 8.E-O9 Error 48 1.591 0.033 Greenhouse-Geisser G = 0.61 O6 Huynh-Feldt E = 0,9699

Source DF Sumof Mean Square F value Pr, F Squares Profile Analysis Constrast 1-~ay O to 2 Mean 1 4.363 4.363 72.21 2.E.46 Cond 1 0.0008 0.0008 0.01 0.907 Treat 1 0.079 0,079 1..31 0.274 Cond*Treat 1 0-056 0,056 0.92 0.355 Error 12 0.073 0.060 Contrast 2 Day 2 to 4 Mean 1 2.142 2-942 1O1 -61 3.E-07 Cond 1 0.00003 0,00003 0.00 0.970 Treat 1 0.0008 0,0008 0.04 0,845 CondTreat 1 0.003 0.003 0.1 7 0,692 Error 12 0.253 0.021 Contrast 2 Day 4 to 6 Mean 1 6.265 6.265 116-75 2.E-07 Cond 1 0.987 0,987 18.39 0.001 Treat 1 1.206 1.206 22-49 0.0005 CondTreat 1 1.558 1-558 29.02 0.0002 Error 12 0.644 0-054 Contrast 3 Day 6 to 8 Mean 1 2.734 2,734 26.74 0-00023 Cond 1 0.054 0.051 0.53 0.481 Treat 1 0.015 0.01 S 0.1 5 0-703 CondTreat 1 0.003 0,003 0.03 0.876 Erro r 12 1.227 0.1 02 Table 8. Summary of the Repeated Measures Analysis of Varianœ measuring weight gain in parasitised (A) and non-parasitised (6) TPB nymphs fed damaged or undamaged celer'petioles or leaflets. A. Non-parasitised nymphs Source DF Sum of Mean Square F value Pr> F

8etween Subjeds Treat 3 13-105 4.368 5.07 0.007 Error 24 20.695 0.862

Source DF Sum of Mean Fvaiue Pr> F Adj P>F Squares Square G-G H-F Within Subjects TÎme 4 66,437 16.609 76.99 4.E-29 2.E-21 4.E-27 Time * Treat 12 7.109 0.592 2.75 0.003 0.009 0,004 Error 96 20-711 0.216 Greenhouse-Geisser E = 0.7175 Huynh-Feldt E = 0.9272 B. Parasitised nymphs Source OF Sum of Mean Square F value Pr> F Squares Between Subjects Treat 3 4.1 38 1.379 2.1 8 0.128 Error 17 10,761 0.633

Source OF Sum of Mean Fvalue Pr, F Adj P>F Squares Square G-G H-F Within Subjects Time 3 12.829 4.276 52.14 1.E-15 2.E-12 1.E-15 fime 'Treat 9 2.775 0.308 3-76 0-001 0.003 0.001 Error 51 4.182 0.082 GreenhouseGeisser E = 0.7708 Huynh-Feldt E = 1-0568 B. Parasitised Nymphs Source DF Sumof Mean Square F value Pr> F Squares Profile Analysis Constrast 1 Day O to 3 Mean 1 2.965 2.765 34.06 2.E.-O5 Treat 3 1,029 0.343 3-94 0.265 Error 17 1.480 0.087 Contrast 2 Day 3 to 6 Mean 1 3.557 3.557 16.30 0.0009 Treat 3 1.058 0.353 1:62 0.222 Error 17 3.708 0.21 8 Contrast 2 Day 6 to 9 Mean 1 1.146 .1.146 7-98 0.01 2 Treat 3 3.434 1 .I4S 7.97 0.0016 Error 17 2.441 0.1U Table 9. Summary of the Repeated Measures Analysis of Variance measuring weight gain ni parasitised and nonparasitised TPB nymphs fed LFCs incorporated into a Iiquid artificial diet Source DF Sumof Mean Square F value Pr, F Squares Between Subjeds Cond 1 1,561 1.561 3-82 0,063 Treat 3 2-903 0,967 2.37 0,097 Cond * Treat 3 1,422 0.474 1-16 0,346 Error 23 9-391 0.408

Source DF Surn of Mean Fvalue Pr, F Adj BF Squares Square GG H-F Within Subjeds Tirne 3 9-713 3.238 17.36 2-E-08 4.E-07 2.E-O8 Time 'Cond 3 0,531 0.1 77 0-95 0,422 0.406 0.422 Time * Treat 9 3.252 0.361 1-94 0,061 0,081 0.061 Time * Treat 'Cond 9 1.335 0.1 48 0.79 0,622 0.596 0.622 Error 69 12.872 0-187 Greenhouse-Geisser s = 0.7859 Huynh-Feldt c = 1-1 479 Source DF Surnof Mean Square F value Pr> F Squares Profile Analysis Constrast 1 Day O to 3 Mean Cond Treat Cond 'Treat Error Constrast 1 Day 3 to 6 Mean Cond Treat Cond 'Treat Error Contrast 2 Day 6 to 9 Mean Cond Treat Cond Treat Error 23 12.280 0.534 Table 10. Summary of the Repeated Measures Analysis of Variance measuring weight gain in parasitised and non-parasitised TPB nymphs fed LFCs incorporated into a semi-solid artificial diet. Source DF Sumof Mean Square F value Pr> F Squares Between Subjects Cond 1 35.575 35.575 27-36 2.E-06 Treat 3 2.41 1 0.804 0.62 0.606 Cond 'Treat 2 0.370 0-185 0.14 0.868 Error 68 88,415 1.300

Source DF Sumof Mean Fvalue Pr> F Adj P% Squares Square G-G H-F Wthin Subjects Time 2 14.340 7-170 65-18 1-E-20 4.E-48 2.E-20 Time Tond 2 0.066 0-033 0.30 0-741 0,715 0.739 Tirne * Treat 6 1.810 0.302 2.74 0.015 0.019 0.015 Time * Treat Cond 4 0.468 0,117 1-06 0.376 0.373 0.376 Error 136 14.959 0.110 GreenhouseGeisser 6 = 0.8921 Huynh-Feldt E = 0.9954 Source DF Sumof Mean Square F value Pr> F

Profile Analysis Constrast 1 Day O to 3 Mean 1 15.618 15.618 108.68 1. E-15 Cond 1 0.107 0.1 07 0.74 0.391 Treat 3 3.398 1 -133 7.88 0.001 Cond * Treat 2 0.742 0.371 2.58 0.83 Error 68 9.772 0-1U Contrast 2 Day 3 to 6 Mean 1 1.332 f .332 5.06 0.028 Cond 1 0.091 0.091 0.35 0.558 Treat 3 0.311 0.1 03 0.39 0.758 Cond Treat 2 0.660 0.330 1.25 0.292 Error 68 17.892 0.263