ELECTROPHYSIOLOGICALAND BEHAVIORAL RESPONSES OF

WALNUT HUSK FLIES TO WALNUT VOLATILES

A University Thesis Presented to the Faculty

of

California State University, Hayward

In Partial Fulfillment

of the Requirements for the Degree

Master of Science in Biological Sciences

By

William Hamersky

August, 1996 © Copyright by William Hamersky 1996

All Rights Reserved

ii ABSTRACT

Current monitoring methods for walnut husk flies (WHFs), Rhago/etis comp/eta Cresson, (Diptera: Tephritidae), a major pest of commercial walnuts, are ineffective at predicting the onset of damage, since the initiation of oviposition is determined by walnut phenology, and not fly population levels.

The purpose of this study was to initiate the development of a new monitoring

lure for WHFs by identifying walnut leaf volatiles that are kairomonally attractive to WHFs. One laboratory and three field experiments were

undertaken to test the hypothesis that walnut volatiles are selectively attractive to the flies.

Laboratory electroantennogram (EAG) experiments were performed to

record WHF responses to three groups of volatile compounds: seven

monoterpenes and four sesquiterpenes found in walnut leaves and husks, and

nine aliphatic green leaf volatiles. The aliphatic compounds elicited the

largest EAG responses, with 8 of 9 of the aliphatics ranked in the top 10 of 21

compounds tested. The monoterpenes and sesquiterpenes elicited weaker

absolute EAG responses, but in relation to their lower volatility, they were

assessed as strong EAG stimulants. Our interpretation of the EAG results

suggested that monoterpenes and sesquiterpenes should be included in field

attractancy tests.

iii A blend of monoterpenes and a blend of sesquiterpenes, similar in proportions to walnut leaf headspace concentrations, were field tested, with both blends being significantly more attractive than the control. The second field experiment was conducted to elucidate which components of a previously suggested male WHF attractant, angelica seed oil (ASOil)

(Angelica archangelica), are attractive. Distilled fractions of ASOil, along with crude ASOil were tested. Only one fraction was significantly attractive, a fraction composed predominantly of sesquiterpenes high in both alpha­ copaene (a known medfly, Ceratitis capitata, attractant) and caryophyllene, a sesquiterpene found in walnut headspace.

The final set of field experiments tested individual walnut headspace sesquiterpenes (caryophyllene, J3-farnesene, u-farnesene and germacrene D) and the one attractive ASOil fraction. Caryophyllene, a common plant volatile, was more attractive than any other test lure, including germacrene D, a more walnut-specific volatile.

This is the first report of a host-plant based volatile attractant for WHFs.

The attractant, caryophyllene, captured primarily male WHFs (-3:1, male:female). The impact of these studies may lead to a new WHF monitor­ ing tool that might be predictive of the onset of walnut damage by ovipositing females. The lead-time provided by a predictive lure will help walnut growers reduce WHF damage by accurately timing their spray applications, thus treating their orchards less often, and with less adverse environmental impact.

iv ELECTROPHYSIOLOGICAL AND BEHAVIORAL RESPONSES OF

WALNUT HUSK FLIES TO WALNUT VOLATILES

By

William Hamersky

Approved: Date:

v ACKNOWLEDGEMENTS

I would like to dedicate this thesis to my father,

who would have been very proud of me.

I would like to express my deepest gratitude to my major professor, advisor, mentor and friend, Dr. Sue Opp, who has been ever-optimistic, supportive, and steadfastly urged me onward. She always had time for me: to answer my questions (and my late night frantic phone calls), to give me good advice, and with tireless help, made this manuscript possible.

I would especially like to thank Christine, who has been with me my entire graduate career, encouraging me, editing first and second drafts, and accepting my favorite, soon-to-be-retired phrase, "I can't do that 'til my thesis is done!" I could not have done this without you.

I gratefully acknowledge Dr. Doug Light's patient tutelage in teaching me electrophysiological techniques, providing the necessary equipment and lab space (already somewhat crowded) and helping with all my field experiments. A veritable fountain of entomological information, he has been an inspiration to me.

vi And I would like to thank my friends and family. Thanks Mom for the late night company and the home-cooked meals. Thank you Drew, for the expert editing advice, and being an understanding friend. Thanks to Steve and Christine, who braved the Fremont and Hollister summer sun to help pluck walnut husk flies off sticky gooey traps.

And finally, I would like to express my thanks to all the researchers at

USDA Agricultural Research Service, Western Regional Research Center, at

Albany, CA for their help and support, especially R. A. Flath for his generous contribution of sesquiterpenes and angelica seed oil fractions. I am also indebted to the East Bay Regional Parks Department for allowing me to use the orchard at Ardenwood Historic Farm, and I would like to acknowledge Bill

Coates, UCCE Farm Advisor, for finding the sites in Hollister.

vii TABLE OF CONTENTS

Copyright page ii

Abstract iii

Acknowledgments vi

List of Tables xi

List of Figures xii

Chapter 1: Historical Literature Review 1

1.1 Introduction 1

1.2 Natural History of the Walnut Husk Fly 3

1.3 Historical Monitoring and Control Strategies 4

1.4 Present Monitoring and Control Strategies 8

1.5 Related Species 10

1.6 Walnut Husk Fly Behavioral Stimuli.. 13

1.7 Research Goals 14

1.8 References 16

Chapter 2: Olfactory Chemoreception 20

2.1 Introduction 20

viii 2.2 Materials and Methods 23

2.3 Results 26

2.4 Discussion 27

2.5 References 37

Chapter 3: Responses of Walnut Husk Flies to Walnut

Leaf Blends and Angelica Seed Oil Fractions

in Field Trials in Walnut Orchards 40

3.1 Introduction 40

3.2 Materials and Methods 43

3.3 Results 44

3.4 Discussion 46

3.5 References 58

Chapter 4: Responses of Walnut Husk Flies to Individual

Sesquiterpenes in Field Trials in Walnut Orchards 60

4.1 Introduction 60

4.2 Materials and Methods 61

4.3 Results 64

4.4 Discussion 65

4.5 References 78

ix Chapter 5: Conclusions, Practical Implications, and

Future Studies 80

5.1 Introduction 80

5.2 Olfactory Chemoreception 81

5.3 Walnut Husk Fly Responses to Walnut Leaf

Blends and Angelica Seed Oil Fractions 84

5.4 Walnut Husk Fly Responses to Individual

Sesquiterpene Test Lures 86

5.5 Ongoing and Future Studies 87

5.6 References 90

Appendix: Responses of Walnut Husk Flies to Individual

Green Leaf Volatiles in the Field 92

A.1 Introduction 92

A.2 Materials and Methods 92

A.3 Results 94

A.4 Discussion 94

A.5 References ,. 97

x LIST OF TABLES

Table Page

2.1 Comparison of walnut leaf volatiles present in steam

distillation vs. intact headspace analysis 33

2.2 Purity, source, control-corrected millivolt EAG responses,

and rankings of walnut husk fly responses to GLVs and

walnut leaf and husk headspace volatiles 35

3.1 Walnut leaf constituents with percentage found in

leaf headspace relative to volume needed to

create artificial blends 51

3.2 Distilled fractions of angelica seed oil, and their

major constituents including percentages of

u-copaene and caryophyllene 52

3.3 Mean daily capture for male WHF attracted to

walnut leaf volatiles in a walnut orchard

at Ardenwood, Fremont, CA 53

A.1 Field test results for individual green leaf volatiles:

group name, compound name, mean number

of flies captured, and ranking 96

xi LIST OF FIGURES

Figure Page

2.1 WHF responses to volatile compounds in an

electroantennogram bioassay 36

3.1 Comparison of male to female WHF captures on

traps baited with walnut leaf blends 54

3.2 Comparison of male to female WHF captures to

lures baited with ASOil fractions 55

3.3 Comparison of block 1 to block 2 male WHF

captures to lures baited with ASOil fractions 56

3.4 Comparison of block 1 to block 2 female WHF

captures to lures baited with ASOil fractions 57

4.1 Comparison of WHF captures on traps baited

with walnut leaf sesquiterpenes at two sites 74

4.2 Comparison, at the two sites, of male WHF captures

(by percentage of total) in relation to headspace

concentration of walnut sesquiterpenes 75

4.3 Percentage composition of the twelve headspace

volatiles found in walnut leaves 76

4.4 Comparison of male WHF captures to walnut

sesquiterpenes, by block, for the two sites 77

XII CHAPTER 1

Historical Literature Review

1.1 Introduction

The walnut husk fly, Rhago/etis comp/eta Cresson, is a small fly, native to the south-central United States, whose entire life cycle is associated with the Jug/ans genus of plants. The fly lives on both varieties of native California black walnut (Jug/ans ca/ifornica S. Watson): northern

California black walnut (J. c. var. hindsii), and southern California black walnut (J. c. var. californica). It is also a major indirect pest of commercially grown Persian or English walnuts (J. regia L.) (Boyce, 1934).

The walnut husk fly (WHF) was accidentally introduced into southern

California, probably in the mid 1920's, and was first found infesting several varieties of Persian walnuts in 1926 (Boyce, 1934). The fly spread rapidly but not uniformly throughout the state, and by the 1950's had appeared in

Oregon and Washington as well (Berlocher, 1984).

The WHF's continuous expansion into uninfested commercial walnut orchards in the state poses an ongoing problem, as California produces about 95% of the country's walnuts, with a 1994 value approaching one quarter billion dollars (Tippett, 1995). Reliance on and over-application of pesticides is a public concern, as the quantity of pesticides sprayed on walnut orchards for control of WHF has increased over the last twenty years 2

(W. Olsen, University of California Cooperative Extension (UCCE) Farm

Advisor, pers. comm.).

Walnut damage is initiated when female flies oviposit their eggs in the

husks of walnuts, and continues as larvae feed on the husk tissue.

Economic losses are due to three facets of larval husk feeding: (1) black staining of the walnut shell, (2) the damaged husk turns black absorbing

more sunlight, and (3) the damaged husk becomes too firmly attached to the walnut resulting in "sticktights". Stained shells lower the grade of that

particular load of walnuts. Darkened husks increase the temperature of

walnuts which can arrest nut development. Sticktights prevent the use of

automated de-hullers, machines that quickly and inexpensively remove the

intact walnut from the outer husk. All of these types of damage decrease nut

value and therefore reduce returns to walnut growers (Flint, 1987; Anon,

1966).

The time that damage occurs can also be a critical factor. For

example, in San Benito County, early season damage (June-July) can

substantially reduce walnut yield by 5-10%, resulting in rejection by a buyer if

damage is greater than 5% of the crop. While late-season damage (August­

September) usually does not result in tonnage loss of walnuts, some shell

staining may occur which can often be removed in the normal shell­

bleaching process (W. Coates, UCCE Farm Advisor, pers. comm.). 3

Pesticide treatments (e.g., malathion) have been developed for the control of WHF. However, the timing of these pesticide applications is the critical aspect to efficiently control WHF populations. Presently there are no effective methods of predicting when ovipositional damage to walnut husks is about to occur. Gravid females (with mature eggs) have been observed attempting to oviposit on early season immature walnuts, but were unsuccessful because the husks were too hard for the ovipositor to penetrate (Boyce, 1934). Eventually the husks "ripen" and become susceptible to attack when they soften enough for ovipositor penetration to occur. It is at this ovipositional period when pesticide applications are critical to the control of WHF.

1.2 Natural history of the walnut husk fly

The walnut husk fly, a univoltine insect, overwinters as a pupa in the ground beneath walnut trees, and emerges as an adult usually in late June to mid-July, but can emerge as early as mid-Mayor as late as mid-August.

Most pupae will eclose as adults the summer following larval development, but some pupae will remain in diapause for up to four years before emerging from the soil. The adults live for approximately one month and feed on honeydew, yeasts, plant sap, bacteria, etc. (Boyce, 1934), with females 4

requiring a nitrogen source (e.g., protein) to produce eggs (Tsiropoulos,

1978). The flies court and mate on developing walnuts with males establishing territories and attempting to mate with any females that alight.

When the walnut husks soften (Le., to allow penetration of the ovipositor), females will succeed in their ovipositional attempts by inserting 12-15 eggs at each oviposition site or "sting" (Boyce, 1934). These sites are the first direct and noticeable sign of walnut injury. Eggs hatch within five days, and the larvae feed on the inner husk tissue for three to five weeks, passing through three instar stages. When mature, the third instars feed through to the outer walnut husk surface, drop to the ground, burrow in, and overwinter as pupae.

1.3 Historical monitoring and control strategies

Many insect pests are monitored with semiochemical attractant traps to determine presence/absence and their population levels. When economic injury thresholds are reached, a treatment program is undertaken to control the pest. A brief discussion of the historical and current monitoring and control strategies for WHF follows.

As early as 1934, Boyce, in his classic monograph, "Bionomics of the walnut husk fly, Rhago/etis comp/eta", noted that WHF were attracted to the 5

volatile products of fermentation. The first monitoring traps designed to capture emerging WHF adults used an enamel pan filled with an attractant, a solution of glycine (an amino acid) and sodium hydroxide (Barnes and

Ortega, 1958). One of the volatiles released by both fermentation and the glycine-sodium hydroxide solution is ammonia. These early bait pans were replaced by less cumbersome dry, sticky traps, - quart-sized, white food cartons coated on the inside with a sticky material and baited with ammonium carbonate, an ammonia emitter. While not as effective as pan traps in capturing WHFs, these dry traps were much easier to use and service (Barnes and Ortega, 1958). A refinement of dry traps yielded fluorescent-yellow rectangle panel traps which were attractive because of similar light reflectance values to leaves, where most adult flies procure food

(Neilson and Wood, 1966; Prokopy, 1968). Originally developed for apple maggot flies, Rhagoletis pomonella Walsh, (Prokopy, 1975) these fluorescent-yellow traps were then used successfully on WHFs. In 1977,

Pherocon® AM (adult monitoring) yellow sticky panel traps baited with 2 g of ammonium carbonate were found to be more effective at capturing walnut husk flies than the food carton type trap (Riedl and Hoying, 1980b). Since then, traps like the Pherocon® AM continue to be the standard monitoring tool for WHF. 6

Historically, control of WHFs involved adulticides, such as the mineral desiccant cryolite used in the 1930's (Hislop et aI., 1981). Some of the early organophosphates such as parathion and malathion, applied with a food lure

(a protein hydrolysate bait such as Staley's No.7) were effective adulticides against WHFs (Barnes and Ortega, 1959; Nickel and Wong, 1966; Barnes and Madsen, 1980). In 1966, a systemic organophosphate ovicidellarvicide, phosphamidon, was introduced to the industry (Nickel and Wong, 1966).

With this insecticide, a grower could eliminate the protein hydrolysate bait because the chemical was applied directly to developing walnuts where the compound soaked into the husk, killing the eggs or young larvae. Concern about insecticidal resistance to some organophosphates favored the introduction of pyrethroids (e.g., fenvalerate and permethrin) which lack systemic action (Hislop et aI., 1981). Due to their higher costs, these have had only limited use in walnut orchards. Since that time, some of the organophosphates, in particular parathion and phosphamidon, have lost registration and are no longer legal for use against WHF in California.

An important component of current integrated pest management

(IPM) strategies involve biological control, which uses natural predators or parasitoids. There have been numerous unsuccessful attempts at biological control for WHF. In 1931-2, two braconid wasps, Opius tryoni Cam. and

O. humilis Silv., and one eulophid wasp, Tetrastichus giffardianus Silv., 7

were imported and released, but failed to become established. All three of these wasps are parasitoids of the Mediterranean fruit fly (medfly) (Ceratitis capitata Wiedemann). Several colonies of O. melleus Gahan, parasitoids of the apple maggot fly, were released in 1937, but none of the wasps were recovered. In 1951, several colonies of O. formosanus, an oriental fruit fly

(Dacus dorsa/is Hendel) parasitoid were imported and released, but also did not become established (Clausen, 1956).

More recently, Hagen et aL, (1982) have released numerous hymenopteran pupal and larval/pupal WHF parasites with limited success. A

1981 release of the braconid, Biosteres tryoni, a larval/pupal parasite of medflies, yielded emergence of only a few adults the following year (Hagen et aL, 1982). In 1982, two diapriid wasps, Psi/us (=Coptera) evansi and

Psi/us (=Coptera) occidentalis, which are pupal parasites, and one larval/pupal parasite, Biosteres sub/aevis were released (Hagen et aL, 1982).

B. sub/aevis, a natural parasite of walnut husk flies, is native to Texas. None of the Psi/us and only a few B. sub/aevis were recovered the next year. In

1983, they released the same three species as in 1982 with one additional pupal parasite, the pteromalid Nasonia vitripennis (Hagen et aL, 1983). No subsequent literature was available, so the further establishment of parasite populations is unknown at this time. 8

1.4 Present monitoring and control strategies

For the past twenty years, traps like the Pherocon® AM traps baited with ammonium carbonate (a source of ammonia) have been the primary method of monitoring WHF populations. Many walnut growers complain that current monitoring methods using yellow sticky panel traps are inadequate regarding timing, specificity, sensitivity and reliability. These traps confer no information on impending WHF egg-laying (i.e., the onset of damage) since female WHFs can be captured on yellow sticky panel traps up to 40-70 days before oviposition begins (Riedl and Hoying, 1980b). In orchards with low to medium levels of fly infestation, trap captures inform growers only of the absolute presence or absence of flies, without any indication of population levels with which to plan a treatment program. Growers with high fly populations rely on large increases in fly captures to gauge when to begin spray regimes. Orchards with low fly populations, however, never experience this increase in fly capture. Even experienced growers may not know when to treat these low level infestations, resulting in either spraying too often and wasting resources, or spraying too late and risking WHF damage. From this it would appear that an ideal trap-lure combination would capture only WHFs (specificity), just before oviposition begins (prediction, timing of damage), and work in orchards with all levels of infestation

(sensitivity and reliability). 9

Present control methods for WHF involve repeated bait spray applications of a mixture of a food lure (e.g., NuLure® , Miller Chemical and

Fertilizer Corp., Hanover, PA) plus pesticide (e.g., malathion). Only portions of trees are sprayed with this mixture. This not only reduces pesticide volume and lowers cost, but it prevents secondary outbreaks of spider mites, another walnut pest (the unsprayed canopies become refugium for spider mite predators, which usually keep the spider mite populations in check) (W.

Coates, UCCE Farm Advisor, pers. comm.). Following bait spray application, the adults are attracted to the food lure and ingest it along with the pesticide. While bait sprays are effective in killing adult flies, the

information from the monitoring traps is inadequate to help the growers decide when to spray. Instead of spraying once, just before oviposition begins, growers often miss this critical period - since it is so difficult to determine, and thus spray too early. Even multiple sprayings of orchards to

kill WHFs can lead to extensive crop damage, if not properly timed based on ovipositional data (J. Hasey, UCCE Farm Advisor, pers. comm.).

The active agent in bait sprays is usually lethal to flies for only a few days, while the lure (e.g., NuLure®) has been shown to lose it's attractiveness after only one day (Opp, unpub.), likely due to decreased

ammonia emission (Teranishi et aI., 1993). Since current bait sprays have

little residual effect, newly emerging adult flies have the opportunity to forage 10

for their natural sources of protein and thereby mature, court and mate, yielding new populations of WHFs. Since female flies can reach maturity and have viable eggs 10-20 days after emergence (Boyce, 1934), walnut growers are often advised to treat their orchards every two weeks during fly season. Although effective at killing adult flies, treating orchards every two weeks is excessive, especially since none of the sprayings may be timed optimally. Consequently, since WHF life expectancy in the field is

approximately one month (Boyce, 1934), many of the flies killed in the early treatments would not live long enough to cause damage.

1.5 Related species

Mediterranean fruit flies (medflies) are related tephritids that have

recently become pests in California. They are polyphagous, world-wide

pests, with over 250 known hosts (Hagen et aI., 1981). Monitoring and

control strategies for medflies differ from WHFs due to dissimilar natural

history (e.g., host choice, and mating system). Male medflies congregate in

leks, specific sites where males display and compete, and release sex

pheromones which attract female medflies. Medfly populations are

monitored with traps which use a food type lure (in McPhail traps), and/or

with Jackson traps that use as a lure, a synthetic mimic of natural 11

pheromones, Le., the parapheromone trimedlure. Control strategies for medflies in both Hawaii and California consist of combinations of mass releases of sterile males (SIT, sterile insect technique, a form of mate

"dilution") and bait spray applications of hydrolyzed protein plus malathion.

Some researchers think current monitoring traps for medflies are ineffective at documenting very low population levels (J. Carey, pers. comm.).

Researchers are attempting to develop more sensitive monitoring and detection traps for medflies using plant volatile attractants (Light et aL, 1988;

Jang et aL, 1989; Light and Jang, 1996) and others have increased male medfly captures with the addition of ammonium carbonate to trimedlure baited traps (Liquido et aL, 1993). Our group is researching some of these methodologies for relevancy to walnut husk flies.

In Greece, olive fruit flies (Dacus oleae Gmelin) are controlled by a mass trapping method that has several components. Researchers have developed a system that contains a food attractant, a feeding stimulant, a

male sex pheromone, a female aggregation pheromone (with additional

aphrodisiac qualities) and a hygroscopic material all on one small,

insecticide-soaked wooden board. These boards are effective for an entire

season, and the growers have reduced pesticide use by 99% per treatment

(Haniotakis et aL, 1991). This method, unfortunately, is not applicable to

walnut husk flies since no known pheromones for WHFs exist. 12

Monitoring of a closely related species, the apple maggot fly (AMF)

(Rhagoletis pomonella Walsh), a serious pest of apples throughout the eastern United States, California and Oregon, is being developed involving apple odors as attractants. Researchers have identified seven volatile esters attractive to apple maggot flies (Fein et aI., 1982), while others added one of those apple volatiles (butyl hexanoate) to traps and greatly increased capture rate over unbaited traps (Averill and Reissig, 1988). A combination of ammonium carbonate and butyl hexanoate significantly increased

R. pomonella trap capture over either ammonium carbonate or butyl

hexanoate alone (Duan and Prokopy, 1992).

Control of AMF in most commercial orchards in North America is

performed by ground spraying with an insecticide (azinphosmethyl or

phosmet) three to four times during July and August (Reissig, 1988).

Researchers are looking at more environmentally-sensitive methods of

controlling apple maggot fly, using 8 cm red spheres (apple mimics) baited with food lure and apple volatiles (Prokopy et aI., 1990a, 1990b).

As red spheres are apple mimics to AMF, green spheres might act as walnut mimics to WHF. The attractancy of green spheres to walnut husk

flies was discovered over fifteen years ago (Riedl and Hoying, 1980a). Our

research team is currently investigating the attractancy, to walnut husk flies,

of green spheres baited with various walnut volatiles. 13

1.6 Walnut husk fly behavioral stimuli

Visual and olfactory stimuli both affect the behavior of WHF. Unbaited yellow, rectangular panels are attractive to walnut husk flies all season, but green spherical shapes, which mimic ripening walnuts, are attractive only once oviposition has begun (Riedl and Hislop, 1985). Olfactory attractants for the WHF, such as fermenting substances (Boyce, 1934), glycine plus sodium hydroxide (Barnes and Osborn, 1958), basified protein hydrolysate baits (Teranishi et aL, 1993), ammonium carbonate (Liquido et aI., in press), and most recently, slow release ammonium carbonate (Teranishi et aL,

unpub.), all potentially mimic the release of ammonia from avian droppings which are likely food sources in nature.

As yet, there is no evidence of sex pheromones for WHF, and most

researchers believe an insect-produced chemical may not exist. Instead, it

has been hypothesized that WHF are responding to physical or chemical

cues of host condition (Riedl et aL, 1989). Kairomones, volatile chemicals

released by one species that benefits another, are produced by the walnut

husk as it ripens. These compounds may be detected by both male and

female WHF looking for appropriate mating and/or ovipositional sites,

although contact chemicals may also be involved (D. Light, pers. comm.).

Observations of WHF courtship and mating behaviors, which always occur

on or near developing walnuts (Opp et aL, 1996) support the hypothesis of 14

visual, physical or chemical walnut cues influencing fly behavior. We believe that changes in walnut volatiles may indicate to WHFs that the husk is soft enough for oviposition.

1.7 Research goals

The purpose of my research was to investigate the responses of WHF to volatile chemicals associated with developing walnuts (husks). The long­ term goal is to develop lures which can be used to predict when damage due to WHF oviposition is about to occur in walnuts. Being able to predict the occurrence of husk damage by female flies would permit California walnut growers to time their insecticide sprays more accurately, and therefore spray fewer pesticides and prevent unnecessary walnut damage.

In Chapter 2, I describe laboratory experiments in which

electroantennograms (EAGs, a research technique that can illuminate the

chemical nature of plant-insect interactions) were used to record WHF

responses to volatile compounds found in walnut leaf and husk headspace

and to other common compounds found in many green plants (green leaf

volatiles or GLVs). The purpose of these experiments was to screen

chemicals from walnuts and other plant sources which may influence WHF

behavior. 15

In Chapter 3, I discuss field studies where I tested the attractancy of different groups of compounds that showed EAG activity to WHFs. The two major groups of compounds found in walnut leaf and/or husk headspace, monoterpenes and sesquiterpenes, were tested as distinct blends in a walnut orchard in Fremont, California. In the same field setup, an historical

male WHF attractant, Angelica seed oil (ASOil) and distillate fractions of

ASOil were tested as well.

Chapter 4 describes my field experiments in two Northern California

sites, Fremont and Hollister, where I tested individual sesquiterpene lures for their attractancy to WHF.

In Chapter 5, I present conclusions, discuss the experiments, their

results, subsequent field experiments by my research team, and the

implications for future WHF control in walnut orchards.

In the Appendix I describe a field experiment using 8 individual GLVs

as test lures for WHFs in the Hollister orchard. 16

1.8 References

Anon. The fly is back stronger than ever! In Diamond Walnut News; 1966; pp. 16-17.

Averill, A. L.; Reissig, W. H. Specificity of olfactory responses in the tephritid fruit fly Rhagoletis pomonella. Entomologia Experimentalis et applicata 1988,47,211-222.

Barnes, M. M.; Madsen, H. F. The threat of the husk fly. In Diamond Walnut News; , 1980; pp. 13-14.

Barnes, M. M.; Ortega, J. C. Glycine-sodium hydroxide solution as an attractant for walnut husk fly. Journal of Economic Entomology 1958, 51, 532-534.

Barnes, M. M.; Ortega, J. C. Experiments with protein hydrolysate bait sprays for control of the walnut husk fly. Journal ofEconomic Entomology 1959,52,279-85.

Berlocher, S. H. Genetic changes coinciding with the colonization of California by the walnut husk fly, Rhagoletis completa. Evolution 1984,38,906-918.

Boyce, A. M. Bionomics of the walnut husk fly, Rhagoletis completa. Hilgardia 1934, 8,363-579.

Clausen, C. P. Biological control ofinsect pests in the continental United States; United States Department of Agriculture: Washington, D.C., June, 1956.

Duan, J. J.; Prokopy, R. J. Visual and odor stimuli influencing effectiveness of sticky spheres for trapping apple maggot flies Rhagoletis pomonella (Walsh) (Dipt., Tephritidae). J. Appl. Ent. 1992, 113, 271-279.

Fein, B. L.; Reissig, W. H.; Roelofs, W. L. Identification of apple volatiles attractive to the apple maggot, Rhagoletis pomonella. Journal of Chemical Ecology 1982, 8, 1473-1487.

Flint, M. L., Ed.; Integrated pest management for walnuts; 2nd ed.; University of California, 1987; Publication 3270, 96 pp. 17

Hagen, K. S.; Tassan, R L.; Fong, M. "Biological control of the walnut husk fly," Walnut Marketing Board, 1982.

Hagen, K. S.; Tassan, R L.; Fong, M. "Biological control of the walnut husk fly," Walnut Marketing Board, 1983.

Hagen, K. W.; Allen, W. W.; Tassan, R L. Mediterranean fruit fly: The worst may be yet to come. In California Agriculture; 1981; pp. 5-7.

Haniotakis, G.; Kozyrakis, M.; Fitsakis, T; Antonidaki, A. An effective mass trapping method for the control of Dacus oleae (Diptera: Tephritidae). Journal ofEconomic Entomology 1991, 84, 564-569.

Hislop, R. G.; Riedl, H.; Joos, J. L. Control of the walnut husk fly with pyrethroids and bait. In California Agriculture; 1981; 35, pp. 23-25.

Jang, E. B.; Light, D. M.; Dickens, J. C.; McGovern, T. P.; Nagata, J. T Electroantennogam responses of Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae) to trimedlure and its trans isomers. Journal of Chemical Ecology 1989, 15, 2219-2231.

Light, D. M.; Jang, E. B. Plant volatiles evoke and modulate tephritid behavior. In Fruit Fly Pests, A World Assessment oftheir Biology and Management; B. A. McPheron and G. J. Steck, Eds.; St. Lucie Press: Florida, 1996; 586 pp.

Light, D. M.; Jang, E. B.; Dickens, J. C. Electroantennogram responses of the Mediterranean fruit fly, Ceratitis capitata, to a spectrum of plant volatiles. Journal of Chemical Ecology 1988, 14, 159-180.

Liquido, N. J.; Teranishi, R; Kint, S. Increasing the efficiency of catching Mediterranean fruit fly (Diptera: Tephritidae) males in trimedlure­ baited traps with ammonia. Journal ofEconomic Entomology 1993, 86, 1700-1705.

Neilson, W. T. A.; Wood, F. A. Natural source of food of the apple maggot. Journal ofEconomic Entomology 1966,59,997-8.

Nickel, J. L.; Wong, T T. Y. Control of the walnut husk fly, Rhagoletis completa Cresson, with systemic insecticides. Journal ofEconomic Entomology 1966,59, 1079-1082. 18

Opp, S. B.; Spisak, S.; Telang, A; Hammond, S. Comparative mating systems of two Rhagoletis species; the adaptive significance of mate guarding. In Fruit Fly Pests, A World Assessment oftheir Biology and Management; B. A McPheron and G. J. Steck, Eds.; St. Lucie Press: Florida, 1996; 586 pp.

Prokopy, R J. Visual responses of apple maggot flies, Rhagoletis pomonella (Diptera:Tephritidae): Orchard studies. Entomologia Experimentalis et applicata 1968, 11,403-22.

Prokopy, R J. Selective new trap for Rhagoletis cingulata and R. pomonella flies. Environmental Entomology 1975, 420-4.

Prokopy, R J.; Christie, M.; Johnson, S. A; Rankin, K.; Donovan, C. Three years of the Massachusetts second stage apple IPM pilot project: blocks receiving apple maggot fly interception traps. Massachusetts Fruit Notes 1990a, 55,4-9.

Prokopy, R J.; Johnson, S. A; O'Brien, M. T. Second-stage integrated management of apple arthropod pests. Entomologia Experimentalis et applicata 1990b, 54, 9-19.

Reissig, W. H. Management of the apple maggot in the eastern United States. In Ecology and Management ofEconomically Important Fruit Flies; M. T. Ali Niazee, Ed.; Special Report 830 Oregon State Univ. Agric. Exp. Sta., Corvallis, 1988; pp. 56-72.

Riedl, H.; Barnett, W. W.; Coates, W. W.; Coviello, R; Joos, J.; Olson, W. H. Walnut husk fly (Diptera: Tephritidae): Evaluation of traps for timing of control measures and for damage predictions. Journal ofEconomic Entomology 1989,82, 1191-1196.

Riedl, H.; Hoying, S. A Evaluation of trap designs and attractants for monitoring the walnut husk fly, Rhagoletis completa Cresson (Diptera: Tephritidae). Zeitschrift fur Angewandte Entomologie 1980a, 91, 510­ 520.

Riedl, H.; Hoying, S. A Seasonal patterns of emergence, flight activity and oviposition of the walnut husk fly in Northern California. Environmental Entomology 1980b, 9, 567-571. 19

Teranishi, R.; Kint, S.; Flath, R. A.; Light, D. M.; Opp, S. S.; Reynolds, K. M.; Hamersky, W. Protein hydrolysate components attractive to tephritids. Proceedings ofan International Symposium on management ofinsect pests: Nuclear and related molecular and genetic techniques 1993, 487-493.

Tippett, J. "1995 California walnut objective measurement survey results," California Agricultural Statistics Service, 1995.

Tsiropoulos, G. J. Holidic diets and nutritional requirements for survival and reproduction of the adult walnut husk fly. Journal ofInsect Physiology 1978, 24, 239-242. CHAPTER 2

Olfactory Chemoreception

2.1 Introduction

Flies use both visual and olfactory cues to interpret their environment.

The fly's most important olfactory organ is the antenna, which for most tephritids is composed of four different types of receptor sensilla, or hairs and pegs (Dickens et aI., 1988). One method available to researchers for examination of insect olfaction is the electroantennogram (EAG). An EAG is an electrophysiological recording of responses of an insect's antennal

receptor-neurons to a stimulus. EAGs may be used to evaluate the chemical nature of plant-insect interactions by measuring an insect's neuronal

response to different airborne plant volatiles and help screen for potential

plant kairomones (Le., plant volatiles that modify insect behavior, e.g.,

attractants).

Originally developed for lepidopterous species (Schneider, 1957), the electroantennogram technique has proven very successful for identification

of sex pheromones for pests such as codling moth (Roelofs et aI., 1971),

pink bollworm (Hummel et aI., 1973) and various moth pests (Roelofs et aI.,

1977). More recently, EAGs have been performed on some pestiferous tephritid fruit flies such as the medfly, Ceratitis capitata (Light et aI., 1992a), the oriental fruit fly, Dacus dorsalis (Light and Jang, 1987) and the apple

20 21

maggot fly, Rhago/etis pomonella (Frey et aL, 1992). However, no studies have been reported on the EAG responses of the walnut husk fly (WHF),

Rhago/etis comp/eta Cresson, to any substances.

Many chemical analyses of walnuts have been conducted, usually to qualify or quantify the different compounds that constitute walnut odor (e.g., steam distillation of walnut leaves (Nahrstedt et aL, 1981), and headspace analysis of chopped walnut meat (Clark and Nursten, 1977). These analyses involved destructive methods, in that the tissue (be it husk, leaf or meat) was peeled, cut, chopped and/or boiled in order to perform the analysis. No part of the plant remained intact, as a live WHF would encounter it. I was interested in the flies' responses to volatile compounds that exist in the field, on the trees, emanating from intact walnuts. To decide which compounds to test in the electroantennograms, the only appropriate analysis available to use as a guideline was from Buttery et aL, (1986) on freshly picked, intact walnut leaves. Even though no analyses were performed on the walnut husk headspace, I was able to use the Buttery et aL, (1986) leaf analysis since the leaves and husks of walnuts have reportedly similar chemical volatile constituents (R. Teranishi and R. G.

Buttery, USDA ARS WRRC, pers. comm.).

A comparison of the different compounds and concentrations of volatiles found in two different analyses of walnut leaves is shown in Table 22

2.1. From the table, it is apparent that very different leaf compositions are determined depending upon the isolation, entrapment and concentration method used, i.e., steam distillation vs. intact headspace. The leaf steam distillates had many more aliphatics, monoterpenes and hydrocarbons, whereas the headspace trapping of intact leaves has more sesquiterpenes,

but fewer constituents overall.

The EAG technique can be an efficient tool to differentiate insects'

relative chemoreceptivity among many compounds and uncover some

potential compounds to test as behavioral insect semiochemicals. Many

compounds have been found to be biologically and behaviorally active after

screening for the most responsive compounds through an EAG system

[(e.g., certain aliphatics to the alfalfa seed chalcid (Light et al., 1992b) ,

several green leaf volatiles (GLVs) to carrot flies (Guerin and Visser, 1980)

and sesquiterpenes to elm bark beetles (Millar et aI., 1986)]. I chose to

screen all the known and available walnut leaf and husk headspace volatiles

(WLHVs) (Buttery et aI., 1986) and some green leaf volatiles (Visser, 1979)

in an EAG preparation using walnut husk flies. The goal was to record

electroantennogram responses to compounds and assess the amplitude of

the EAGs with the hypothesis that compounds eliciting the largest EAG

amplitude might also elicit differential attraction in the field. This might

ultimately result in better lures for monitoring WHF. 23

2.2 Materials and Methods

WHF pupae were obtained from field-collected, infested walnuts harvested during summer 1992 in Ardenwood Historic Farm (East Bay

Regional Parks, Fremont, Alameda County, CA). The pupae were stored in a laboratory refrigerator for nine months at 5° C. To initiate adult development, pupae were placed in petri dishes in an incubator at 35° C,

50% relative humidity and a 16:8 Iightdark regime. After eclosion, the adult flies were placed in 25 x 25 x 25 cm Plexiglas® and screen cages in the laboratory and allowed to feed on a sugar and yeast hydrolysate mixture and on water ad libitum. EAG tests were conducted on two male and two female flies which were between 14 and 22 days old.

The electroantennogram setup followed that previously described

(Light et aI., 1988) with minor modifications. The test flies were restrained in a Plexiglas® holder in which the body and head were held immobile in a yoke and the antenna positioned to allow insertion of the microelectrodes.

To construct the microelectrodes, 1.0 mm diameter borosilicate glass microcapillary tubes (Haer, 30-30-1) were heat pulled in a microelectrode

puller (Browning-Flame, San Rafael, CA) to yield tips approximately 3

microns in diameter. Chlorided, silver wires were inserted into the

microcapillary tubes filled with insect saline solution (Kaissling's recipe, from

Roelofs, 1977). The protruding end of the silver wire was then connected to 24

a wire lead, thus completing one microelectrode. Two of these microelectrodes, a recording and a ground, were used for each WHF test animal. Using micromanipulators, the recording electrode was inserted into the distal portion of a WHF's third antennal segment, and the ground electrode was inserted into the fly's frons, just below the antennal attachment.

The electrodes were connected via a shielded preamplifier cable to a

Grass amplifier (Model P-16, Quincy, MA). The 100X amplified signal was displayed on a Tektronix 5113 oscilloscope, where each EAG stimulation deflection was measured in millivolts (mV) directly off the screen grid and recorded.

Chemists have identified twelve volatile compounds (plus three unidentified) from headspace analysis of walnut leaves (Buttery et aI., 1986).

Twenty-one compounds were tested in the EAG experiments (Table 2.2).

Although nine aliphatics (commonly called green leaf volatiles or GLVs) were used, only one, (Z)-3 hexenyl acetate, is found in walnut leaf/husk headspace (the other eight were used for continuity and comparison to other researchers' GLV electroantennogram results). Seven of eight known walnut leaf monoterpenes were used, because ~-pinene was not available. All four sesquiterpenes found in walnut leaf/husk headspace were used. 25

Most of the 21 compounds used were> 90% pure and were diluted with redistilled spectraphotometric grade pentane to yield 10% solutions

(100 ul of neat compound in 900 ul of pentane with lonox antioxidant added)

(Table 2.2). One microliter (1.0 ul) aliquots of each compound were pipetted onto individual 7 mm x 30 mm pieces of Whatman #1 filter paper which were held until the solvent evaporated, and then inserted into clean glass Pasteur pipettes. Pipettes for each compound were prepared immediately before each test EAG and were stored in a sealed container having negative air flow to reduce possible contamination.

During the testing, each fly's antenna was subjected to a constant stream (640 mllmin.) of charcoal filtered, humidified air from a compressed air tank. The air was routed through 7 mm diameter Teflon® tubing ending in disposable glass Pasteur pipettes 1.0 cm from the antenna. The air­ stimulus delivery system consisted of air flow through two hose lines regulated by a solenoid valve. One hose line allowed delivery of clean air to the antenna via the solenoid valve which, when activated, diverted the air flow through the second hose line. The second line terminated at an exchangeable pipette holding the evaporating test compound. A timer circuit regulated the solenoid valve activation for one second puff durations.

Captured and displayed on the oscilloscope screen were the time intervals immediately before, during, and after the puffing of the test compounds onto 26

the antenna. The EAG responses or downward negative polarity deflections,

caused by antennal receptor stimulation by the test compounds, were

captured and measured directly off the oscilloscope screen with an

interpretive accuracy of ± 0.05 mV. The order of presentation of test

compounds was random, and a period of at least 60 seconds of clean air

was given between test stimulations to allow the antennal receptors to

recover. Stimulations of the control (pipettes with only the pentane solvent

on filter paper) and the standard [(Z)-3 hexenyl acetate] were interspersed

every 5-10 test stimulations. The screen-recorded deflections were

"control-corrected" by the subtraction of the mV deflection caused by the

closest (or average) accompanying control stimulation. The mV responses

to test compounds were so adjusted to eliminate or minimize the response

due to mechanoreception and solvent chemoreception inherent in these odor

puff stimulations to antennae.

2.3 Results

The EAG results for the 21 compounds tested, using control-corrected

mean deflections, are shown graphically in Fig. 2.1 (with more detailed

information in Table 2.2). There were no statistically significant differences

in EAG responses due to sex (ANOVA: F=3.06, df=1, P=0.08), so the 27

responses of males and females were pooled together. Although there were statistically significant differences in the responses to the various compounds

(ANOVA: F=2.73, df=20, P=0.004), because of the small sample size, a multiple comparison test would not allow me to statistically differentiate between the compounds (Student-Newman-Keuls multiple comparison test;

P>0.05).

The compound eliciting the greatest EAG response (ranked #1) was

(E)-2 hexenal, a six carbon aliphatic aldehyde (Fig. 2.1). Eight of nine aliphatic compounds were in the top ten compounds which elicited the

largest EAG responses by the flies. Only two monoterpenoids, linalool and

(E)-beta-ocimene (ranked third and seventh, respectively), elicited EAG

responses greater than 0.20 mV. The remaining monoterpenes and all the sesquiterpenes together were ranked in the lower half of compounds tested.

2.4 Discussion

Due to time constraints and the beginning of the walnut husk fly field

season, EAGs were performed on only four individual flies. This number

was comparable to other dipteran EAG research (for carrot fly, n=6 (Guerin

and Visser, 1980). Thus, I felt some degree of confidence in the receptivity

trends that these results provide. 28

Eight of the top ten compounds eliciting the highest EAG amplitude responses were aliphatic - the relatively small, higher volatility six-carbon aldehydes, alcohols and acetates which are part of the "green leaf volatile

(GLV) complex" (Visser, 1979). Only one of the nine aliphatics, (Z)-3 hexenyl acetate, is found in walnut headspace. The other 11 monoterpenes and sesquiterpenes, all components of walnut leaf/husk headspace odor, are evenly distributed throughout the lower half of the rankings. Since almost

90% of the aliphatic GLVs tested are not found in intact and undamaged walnut leaves, it was surprising that the flies had greater responses to those compounds than to either the monoterpenes or the sesquiterpenes. One possible explanation is that, because the compound dilutions were based on volume and not moles or weight (i.e., equal volumes of each compound regardless of formula weight), more of the lighter molecular weight aliphatic molecules impacted the antenna (i.e., more molecules on, and evaporating from, the filter paper per air puff) than either the monoterpenes or sesquiterpenes. Since the EAG deflection displayed on the oscilloscope is a measurement of the total responses of the olfactory neurons located over the entire antennae, then the greater the concentration of molecules in a puff, the more likely a greater percentage of the responsive receptors would receive an adequate stimulation. This may have biased the stimulation rate and inflated the measured responses to the lighter compounds, in effect 29

diminishing the comparative responses of the antennae to the heavier monoterpene (10 carbons) and sesquiterpene (15 carbons) compounds that would be lower in concentration per stimulus puff. An alternative explanation

is that, for walnut husk flies, the electroantennogram procedure is either too

crude or incapable of accurately recording the responses of different types of

olfactory cells.

For WHFs, the ability to detect and discriminate among different

aliphatic compounds is important since some GLVs are also classified as

"damage volatiles" (Light et aI., 1988). Insect or mechanical damage to a

host and the resulting bacterial or fungal infections may make that walnut

either a poorer or better choice for either a courtship, mating, oviposition or

larval survival site. Unfortunately researchers have not yet identified the

constituents that make up walnut damage volatiles, so we cannot accurately

assess this question. In controlled experiments, however, traditional

chemistry procedures of grinding, blending, extracting and steam distilling of

walnut tissues, though more extreme in degree of damage, do liberate large

amounts of these GLV aldehydes, alcohols and acetates (Table 2.1).

Simply ranking WHF EAG responses by greatest to least response

tells the researcher little about the inherent attractiveness of a compound in

the field. The compounds tested followed a ranking that I believe was

influenced more by molecular weight and number of antennal impacts per 30

unit time than actual behavioral and/or physiological sensitivity. In the EAG rankings, the GLVs showed the highest response, but subsequent field studies using the same GLVs individually, resulted in low captures of flies and no statistically significant differences between any of the GLVs tested

(Two-way ANOVA: F=1.17, 8 df, P=0.32; Appendix). Apparently, WHFs can sense and discriminate GLVs as demonstrated through EAGs, but are not differentially attracted to individual GLVs in the field.

Although EAGs have been used as screening tools to identify bioactive compounds from long lists of possible attractants, this technique was not as successful with walnut husk flies. None of the WHF EAG

responses to any test compound far exceeded responses to any other test

compound. Deflections ranged from 0.024 - 0.332 mY; for comparison,

medfly responses to the standard hexan-1-ol were - 1.1 mV (Light et aI.,

1988, 1992a), moth responses to pheromones averaged 3 - 6 mV (Roelofs,

1979) while other moth amplitudes ranged from 2 - 15 mV (Van der Pers,

1981). It appears that walnut husk flies have broad chemoreceptivity to the

range in structural compounds tested, but lack selective responses, with

both males and females being similarly receptive.

More accurate analysis of walnut husk fly EAG responses might result

if one performed either dose response tests, or used molar or molal solutions

(1 mole of test compound in, respectively, 1 liter or 1000 g of solvent) in 31

setting up the test compounds. Dose response tests would allow the researcher to determine threshold levels of receptivity. Using the molar/molal methodology would allow similar numbers of molecules of the different compounds to impact the antennae, and so would yield truly comparable responses between compounds.

The four flies tested (two female, two male) showed no statistically significant differences between the sexes. This is not surprising, because for

EAG responses, it is common for tephritid flies to lack sexual dimorphism

(apple maggot fly, (Fein et aL, 1982), olive fruit fly, (Van der Pers et aL,

1984), or have only slight sexual dimorphism (medfly (Light et aL, 1988)).

Many bioactive compounds are large, more structurally complex monoterpenes and sesquiterpenes, not simple straight chain aliphatic molecules. Although the flies showed greatest EAG responses to the aliphatics, it is unlikely aliphatics would be attractants for at least two reasons: (1) eight of the nine aliphatics are absent from walnut leaf/husk headspace, so the chances are slim of a novel compound being a strong attractant, and (2) all nine are ubiquitous to many green plants, and so lack any olfactory host plant based uniqueness that an essentially monophagous insect would seemingly require. Being attracted to common aliphatics in the environment would confer no evolutionary selective advantage for WHF in either finding hosts or mates, unless the assessment of husk damage was 32

important for progeny survival. It seems more reasonable to think monoterpenes or sesquiterpenes might be important compounds for a WHF to cue in on, since in a field situation these compounds would be more limited in distribution than ubiquitous green leaf volatiles.

Although EAGs have been useful in the past and continue to be useful, this technique was not well suited to walnut husk flies. Low mV responses to compounds quite specific to walnuts, coupled with high mV responses to ubiquitous plant volatiles, may have led us to believe that the

GLVs might be attractive compounds. It was only later, when individual

GLVs and individual sesquiterpenes were field tested, that we discovered high EAG responses did not equate with attractiveness to the green leaf volatiles (Appendix). The caveat here, as it was twenty-five years ago

(Birch, 1971), is to always follow up electroantennograms with at least laboratory studies and preferably with field bioassays. 33

Compound Percent composition of walnut leaf volatiles

Steam distillation 1 Intact headspace 2 Aliphatic Z-3-hexenyl acetate nfa 2.00

Monoterpenes alpha-pinene 0.50 5.00 beta-pinene 1.00 11.00 camphene trace sabinene 0.20 7.00 myrcene trace 5.00 limonene 0.10 10.00 beta-phellandrene trace gamma-terpinene trace delta-3-carene trace (E)-beta-ocimene trace 12.00 unknown monoterpenes (2) 0.80 1,8 cineole 0.50 p-cymol 0.20 linalool 1.00 1.00 camphor 1.30 borneol 1.50 myrtenal 1.20 dihydrocarvone trace carvone 1.50 linalyl acetate 0.30 bornyl acetate 0.50

Table 2.1. Comparison of walnut leaf volatiles present in steam distillation vs. intact headspace analysis

1=(Nahrstedt et aI., 1981) 2=(Buttery et aI., 1986)

continued next page 34

Compound Percent composition of walnut leaf volatiles Steam distillation 1 Intact headspace 2 Sesquiterpenes caryophyllene 15.00 (E)-beta-farnesene 2.00 germacrene D 4.00 alpha-farnesene 13.00 unknown sesquiterpenes (3) 1.80 alpha-bisabolol trace oxygenated sesquiterpene 7.00

Other compounds eugenol 0.50 beta-damascone 2.00 beta-jonone 1.00 eicosane 0.30 unacosane 0.30 thymol 3.00 beta-eudesmol 21.00 isophytol trace unidentified compound 1.00 unidentified compound 2.00 unidentified compound 4.00

Hydrocarbons nonadecane 0.20 docosane 5.00 triacosane 2.00 tetracosane 2.50 pentacosane 15.00 hexacosane 0.10 heptacosane 4.50 nonacosane 1.50 triacontane trace Total number of compounds identified 41 12 Total percentage identified 78.3 87.00

Table 2.1, continued 35

1 Compound Purity Source Control-corrected mV responses 2 Mean.:tSEM Rank Males Females Aliphatics hexan-1-ol 98 A 0.30, 0.07 0.41, 0.38 0.289 +0.06 4 hexanal 98 A 0, 0.02 0.18, -0.09 0.025 +0.06 20 E-2-hexen-1-ol 97 A 0.3, 0.18 0.255, 0.33 0.265 +0.06 5 Z-3-hexen-1-ol 98 A 0.4, 0.15 0.404, 0.33 0.319 +0.06 2 E-2-hexenal 99 A 0.5, 0.2 0.303, 0.33 0.334 +0.06 1 E-2-hexenyl acetate 95 B 0.3, 0.03 0.352, 0.3 0.245 +0.06 6 Z-3-hexenyl acetate 98 C 0.25, 0.18 0.35, 0.14 0.230 +0.06 7 hexyl acetate 93 A 0.3, 0.05 0.4, 0.15 0.226 +0.06 8 2-heptanone 90 E 0.1 0.409, 0.08 0.172 +0.08 10

Monoterpenes R-(+)-limonene 99% A 0.17 0.13, 0.045 0.128 +0.07 13 S-(-)-limonene 96% A 0 0.14, 0.05 0.047 +0.07 18 linalool 97.30% A 0.3, 0.17 0.34, 0.44 0.314 +0.05 3 myrcene 65 D 0.27, 0.06 0.085, 0.093 0.128 +0.06 12 (E)-beta-ocimene 95% E 0.07 0.33, 0.28 0.187 +0.07 9 (R)-(+)-alpha pinene 97% A 0.01 0.027, 0.037 0.024 +0.01 21 (S)-(-)-alpha pinene 97% A 0.1, 0.027 0.025, 0.064 0.054 +0.01 17 sabinene 90% E 0.07 0.09, 0.181 0.104 +0.07 14

Sesquiterpenes caryophyllene 92.50% E 0.2, 0.003 0.11, 0.108 0.105 +0.06 15 (E)-beta-farnesene 91% E 0.05 0.015, 0.025 0.035 +0.07 19 germacrene D 99.40% E 0.4, 0.07 0.02, 0.144 0.158 +0.06 11 alpha-farnesene 93% E 0.2, -0.02 0.075, 0.14 0.098 +0.06 16

1 Sources of compounds: A, Aldrich Chemical Co.; B, CTC Organics; C, Tokyo Kasei; D, sample from R. G. Buttery, USDA ARS WRRC; E, sample from R. A. Flath, USDA ARS WRRC

2 Calculated by subtracting control (pentane) response from original deflection values (measured off oscilloscope screen)

Table 2.2. Purity, source, control corrected millivolt EAG responses, and rankings of walnut husk fly responses to GLVs and walnut leaf and husk headspace volatiles (n=4). E-2-hexenal ;:;.-=~:&:;;.-=X'"~~:;.: ::;'~~.1'~E;:~:~;'~:;'~S;S~;:;:!:;:!S;?'&.1'~:;~:::::::::::::~:~!:;;, ..~:::;:::;:;:;~~;.';S ..-=~ ;~~~:;:! ;:~:~:~~:&;.-=~w :'''-=~;:;:!:; ::?::;""",.,.:

E-2-h exen-1 -01 I:.:.:::.":~.:::-===.ta::::::::::::;":.~~~.:~.~:?'.t.t&:::&:: ...-&,,:~~:~:.·~:::~::::.:~.t.t,&i.:»i·::::::.·&6.."@":·:·: ~.:~»:::::::~::::.: '.::::>.s:.:.,,:»:-:.~ ';')o"...,N&.:;" ~.:~

E-2-hexenyl acetate I~:;::'»:>,,,·:·>&.%"<:l ::?,::?,;:;:;:;:;~:;::::::;~:;;;'O;:;'W.§.:;Z«":;:::::;:~:~~":.~ ';'::;:;~~;;;""';:::::::::!:!:::::::?;:;::'~ ·.,&:~.-=K::;"'-=:'::;"'''''-=::::;:;:''~ .•:~:;:!:;:;..,,-;;;..-=:;;;.-=:::?.~ Z-3-hexenyl acetate t;::,;:l>.':;;"·' ;:;:;::::::;:-;::::;:~:;:;:;::.~ ··;::::;K««::~.:::::::m::::~·::;·:::·~~ .;;:;:;::::; ~.';:-":::;:;:;:::;:?:::;:; ·.:,:.,,,.,6:'X«"·~ .;:;:;:;:;:; Aliphatics hexyl acetate

2-heptanone ",',:,&R'"",. ::?'~~?'?'?'?'?'?''"«;:;:~~~~?~~?'{U&~~:?????~:~:,"::?;~0;~~:~::~~~w.~ "~??~'~

hexanal m '.~'

"'C I r::: Iinalool ·:::::::::~Y"HY/.&:.~::::::::::::::::::::~:::~::*h~~&'h--:::~":.":.:::::::::::::.:::::::::du,,:::-:·:·:·;,::::::::::::::::~:~:::::::::::::::::::::::::::::-:::«..",,-'::::::::::WN":-)o"/Lu...::::::::-~:·,-::::"",,,,,,,,,»:-:::~,,::::::::::::::::::::::::::::::::::::::·:·:·:·:·:·:-:·:·:·::::6..::::i'::::::::::::$y"::»-hZ~.:: :.-::.-:::::::::;slli..--&...... \...... w ::J 0 c. (E) beta ocimene ::;::::::::::;::::;:;:;:~:::;"?;;::;.~:?::;.-::;::::;:;:?.;:;:;:::;:;:;::::::...o:;;.-;.~?.?"&; ::::::::::::::::::::&.-:"'-::::::::::::::~M ::?;:;'''!:::::::::?::?'E.1'/.:::::?::~S?::::~:~:~:::~:::m~:::;:;:: E 0 R (+) limonene ;X:::::::;'-:::::~":?'''''·''''''·'·: ~'Y"'::::::?'E.w~S::::::::~:::":;:::::;:?;:;:;:~~~ ~~:;:;::::; I U I myrcene Moinoterpenes sabinene ::::;x;ID...... ::::wffi:?=:=:?=:=:=::::::::::z='.:~:::::::~:.*:::: ::.-:::-:::;-:"'~:':::::.:&.":.:.:;:'-////M:-:::··-

(S) (-) alpha pinene ~'~~m::::?"ffff'«';;:::::::'o;::

S (-) Iimonene -..::::.;;;;:.".: (R) (+) alpha pinene

germacrene 0 ...... -:.:.:.:.:.:.:.'. :-... "':'.':-:' "7" •..:.: :.:.:-:.

caryophyllene '.. ~ ,.'.

alpha farnesene Sesquiterpen s (E) beta farnesene

o 0.05 0.1 0.15 0.2 0.25 0.3 0.35

Control-corrected mean EAG deflections in mV W 0'1

Figure 2.1 WHF responses to volatile compounds in an electroantennogram bioassay. 37

2.5 References

Birch, M. C. Intrinsic limitations in the use of electroantennograms to bioassay male pheromones in Lepidoptera. Nature 1971, 233, 57-58.

Buttery, R G.; Flath, R A.; Mon, T. R; Ling, L. C. Identification of Germacrene D in walnut and fig leaf volatiles. Journal ofAgricultural and Food Chemistry 1986,34,820-822.

Clark, R G.; Nursten, H. E. The sensory analysis and identification of volatiles from walnut (Juglans regia L.) headspace. Journal ofthe Science ofFood and Agriculture 1977,28,69-77.

Dickens, J. C.; Hart, W. G.; Light, D. M.; Jang, E. B. Tephritid olfaction: Morphology of the antennae of four tropical species of economic importance (Diptera: Tephritidae). Annals ofthe Entomological Society ofAmerica 1988, 81, 325-331.

Fein, B. L.; Reissig, W. H.; Roelofs, W. L. Identification of apple volatiles attractive to the apple maggot, Rhagoletis pomonella. Journal of Chemical Ecology 1982, 8, 1473-1487.

Frey, J. E.; Bierbaum, T. J.; Bush, G. L. Differences among sibling species Rhagoletis mendax and R. pomonella (Diptera: Tephritidae) in their antennal sensitivity to host fruit compounds. Journal of Chemical Ecology 1992, 18,2011-2024.

Guerin, P. M.; Visser, J. H. Electroantennogram responses of the carrot fly, Psila rosae, to volatile plant components. Physiological Entomology 1980,5,111-119.

Hummel, H. E.; Gaston, L. K.; Shorey, H. H.; Kaae, R S.; Byrne, K. J.; Silverstein, R M. Classification of the chemical status of the pink bollworm sex pheromone. Science 1973, 181, 873-875.

Light, D. M.; Jang, E. B. Electroantennogram responses of the oriental fruit fly, Dacus dorsalis, to a spectrum of alcohol and aldehyde plant volatiles. Entomologia Experimentalis et Applicata 1987,45,55-64. 38

Light, D. M.; Jang, E. B.; Dickens, J. C. Electroantennogram responses of the Mediterranean fruit fly, Ceratitis capitata, to a spectrum of plant volatiles. Journal of Chemical Ecology 1988, 14, 159-180.

Light, D. M.; Jang, E. B.; Flath, RA Electroantennogram responses of the Mediterranean fruit fly, Ceratitis capitata, to the volatile constituents of nectarines. Entomologia Experimentalis et applicata 1992a, 63, 13­ 26.

Light, D. M.; Kamm, J. A; Buttery, R G. Electroantennogram response of alfalfa seed chalcid, Bruchophagus roddi (Hymenoptera: Eurytomidae) to host- and nonhost-plant volatiles. Journal of Chemical Ecology 1992b, 18, 333-352.

Millar, J. G.; Zhao, C. H.; Lanier, G. N.; O'Callaghan, D. P.; Griggs, M.; West, J. R; Silverstein, R M. Components of moribund American Elm trees as attractants to elm bark beetles, Hylurgopinus rufipes and Scolytus multistriatus. Journal of Chemical Ecology 1986, 12, 583-608.

Nahrstedt, A; Vetter, U.; Hammerschmitt, F. J. Composition of the steam distillation product from the leaves of Juglans regia. Journal of Medicinal Plant Research 1981,42,313-331.

Roelofs, W. L. The scope and limitation of the electroantennogram technique on identifying pheromone components. In The evaluation ofbiological activity; N. R McFarlane, Ed.; Academic Press: New York, 1977;pp. 147-165.

Roelofs, W. L. Electroantennograms. Chemtech 1979, April, 222-227.

Roelofs, W. L.; Comeau, A; Hill, A; Milicevic, G. Sex attractant of the codling moth: characterizations with the electroantennogram technique. Science 1971,297-299.

Schneider, D. Elektrophysiologische Untersuchungen von chemo- und mechanorezeptoren der antenne des seidenspinners Bombyx mori L. Z. vergl. Physio/. 1957, 40, 8-41.

Van der Pers, J. N. C. Comparison of electroantennogram response spectra to plant volatiles in seven species of Yponomeuta and in the tortricid Adoxophyes. Entomologia Experimentalis et applicata 1981, 30, 181­ 192. 39

Van der Pers, J. N. C.; Haniotakis, G. E.; King, B. M. Electroantennogram responses from olfactory receptors in Dacus oleae. Entomologia Hellenica 1984,2,47-53.

Visser, J. H. Electroantennogram responses of the Colorado beetle, Leptinotarsa decemlineata to plant volatiles. Entomologia Experimentalis et applicata 1979,25,86-97. CHAPTER 3

Responses of Walnut Husk Flies to Walnut Leaf Blends and Angelica

Seed Oil Fractions in Field Trials in Walnut Orchards

3.1 Introduction

Electrophysiological studies of insect antennal responses to plant volatiles may be used as indicators of potential behavioral or kairomonal responses of insects. Yet, without further laboratory or field studies, physiological responses of insects to host plant volatiles may not be directly correlated with behavior. Based on previous electroantennogram (EAG) studies with walnut husk flies (WHF), Rhago/etis comp/eta Cresson, I had determined that various walnut (Jug/ans spp.) volatiles held promise for testing as attractant kairomones (see Chapter 2). Thus, I undertook this study to further elucidate the behavioral responses of WHF in the field to blends of walnut volatiles and other plant odors. Two field studies were conducted using: (1) blends of the different structural chemical groups found

in walnut leaf and husk headspace (WLHH), and (2) different fractions of angelica seed oil, ASOil, (Angelica archangelica, Apiaceae), an historical

WHF attractant (Barnes and Osborn, 1958).

The 12 identified volatile components of walnut leaf headspace

(Buttery et aI., 1986) can be separated into three chemical groups: (1)

40 41

aliphatics or green leaf volatiles (GLVs) (6-carbon straight chain compounds), (2) monoterpenes (10-carbon molecules composed of two isoprene units), and (3) sesquiterpenes (15-carbon molecules comprised of three isoprene units) (Table 3.1). Headspace-trapped walnut leaf odor is composed primarily of one aliphatic compound, seven monoterpenes, four sesquiterpenes, and three unidentified compounds (Buttery et aI., 1986).

These three groups may also be distinguished by molecular weight with the smaller compounds being lighter, so that by molecular weight, aliphatics < monoterpenes < sesquiterpenes.

(Z)-3 hexenyl acetate is a common compound and is the only aliphatic found in intact walnut leaf and husk headspace. It is ubiquitous to many green plants and is part ofthe "green leaf volatile (GLV) complex" (Visser,

1979). Monoterpenes and sesquiterpenes, as groups, contain many bioactive constituents, and it was decided to test each of these two groups as distinct blends. The two blends were produced by combining the individual compounds in the proportions found in walnut leaf headspace

(Table 3.1). Our reasoning for using these blends was to allow us to ascertain if the flies were responding to either the overall monoterpene and/or sesquiterpene components.

The rationale for using ASOil dates back to when Barnes and Osborn

(1958) tested certain compounds and crude oils that were reported attractive 42

to Mediterranean fruit flies (Ceratitis capitata) (Steiner et aL, 1957). Barnes and Osborn (1958) found WHF males also were attracted to angelica seed oil. Unfortunately, no research has been done since that time to elucidate how or why male walnut husk flies are attracted to angelica seed oil. In addition to pure ASOil, a fractional distillation of the oil was made available

(all were courtesy of R. A. Flath, USDA ARS WRRC, Albany, CA), and I chose to test some of the fractions for biological activity to WHF. A short path vacuum distillation of ASOil yielded six fractions, the middle four of which were used in this experiment (Table 3.2). The first fraction tested (i.e., the second cut from the distillation) was labeled ASO-75 and consisted mostly of monoterpenes. The second and third fractions tested, ASO-77 and

ASO-78, were predominantly sesquiterpenes. The final fraction tested was

ASO-79, comprised mostly of oxygenated compounds. Two of the fractions,

ASO-77 and ASO-78, differed mainly in the presence/relative absence respectively of a-copaene (11.1 % vs. 0.4%). This compound has shown some efficacy as an attractant for male medflies (Fornasiero et aL, 1969;

Jacobsen et aL, 1984; Flath et aL, 1994a, 1994b). 43

3.2 Materials and Methods

Field test with walnut leaf and husk blends

Walnut husk flies were trapped during summer 1993, in a desuetude, drought-stressed walnut orchard in Ardenwood Historic Farm in Fremont,

California (Alameda County), a part of the East Bay Regional Parks District.

Persian walnut trees (Juglans regia, var. 'Payne') of similar size, canopy and vigor were selected as "trapping-trees". Two replicate blocks were assigned of four trees each for a total of eight test trees. Trece® Pherocon AM NB

(adult monitoring, non-baited) yellow, sticky panel traps were hung in the trees at 2-2.3 m heights on the north side of each tree. Separate solutions for each of the three test lures [(Z)-3 hexenyl acetate, monoterpene blend and sesquiterpene blend] were prepared using the recipes in Table 3.1.

Aliquots (200 ul) of each solution were pipetted into individual 8 mm x 15 mm rubber septa, and hung via paper clips to the tops of each trap. A pentane control was used in each replicate. Every seven days the old test lures were removed, discarded, and replaced with new loaded septa (200 ul aliquots).

Traps were randomly rotated every second day within blocks to diminish positional effects. Adult flies were removed from traps daily, all flies were sexed, and the experiment ran for 9 days from 28 July to 06 August, 1993. 44

Field test with angelica seed oil fractions

As with the walnut leaf blend experiment, walnut trees of similar size, canopy and vigor were chosen. Two replicates were chosen of six trees each for a total of twelve test trees. Angelica seed oil (ASOil), four fractions

of ASOil (ASO-75, ASO-77, ASO-78 and ASO-79), and a pentane control

were used as lures. Test lures were diluted, prepared, hung and replaced as

in the walnut leaf blends experiment. All flies were sexed, and the

experiment ran for 9 days beginning 28 July and terminating 06 August,

1993.

Analysis

A one-way ANOVA on log (x+1) transformed data was performed to

determine differential fly capture by sex. Two-way ANOVAs were then

performed to test for effects of the different test lures, and to test for block

effects. 45

3.3 Results

Walnut leaf and husk blends

For every lure at Ardenwood (except the pentane control), significantly

more males than females (3.1:1) were captured (One-way ANOVA: F=37.6,

df=1, P«0.001) (Fig. 3.1).

Males showed significant differential attraction to the different

compounds with sesquiterpene and monoterpene lures being significantly

more attractive than either (Z)-3 hexenyl acetate or the pentane control

(Two-way ANOVA: F=8.23, df=3, P<0.001) (Table 3.3 and Fig. 3.1).

There were no significant block effects within the site (F=2.59, df=1,

P=0.112) and there was no significant interaction effect between lure and

block (F=1.85, df=3, P=0.146).

For females there were no statistically significant differences for any

tested parameter: lure, (Two-way ANOVA: F=0.403, df=3, P= 0.752),

block, (F=2.12, df=1, P= 0.150) or interaction effect (F=0.88, df=3, P=

0.452) (Fig. 3.1).

Angelica seed oil fractions

For every test lure, significantly more males than females (5.1:1) were

captured (One-way ANOVA: F=41.3, df=1, P«0.001) (Fig. 3.2). 46

For males, there was: (A) a significant block effect (Two-way ANOVA:

F=17.24, df=1, P«0.001) (Fig. 3.3), (B) a significant effect for the different test lures (F=6.56, df=5, P«0.001) (Fig. 3.3), but (C) there was no interaction effect between block and lure (F=0.95, df=5, P=0.451).

For females, the only significant difference was that the two blocks captured different numbers of flies (Two-way ANOVA: F=11.59, df=1,

P«0.001) (Fig. 3.4). There were no significant effects for test lures

(F=0.66, df=5, P=0.648) (Fig. 3.4) and no interaction effect between block and lure (F=0.99, df=5, P=0.425).

3.4 Discussion

Walnut leaf and husk blends

These field tests of walnut leaf and husk blends provided the first indication of sexual differences in WHF attractancy to host plant-derived volatiles (Fig. 3.1). In many previous studies of WHFs, trap captures were not sexed, thus numbers of males and females were lumped together (Riedl and Hoying, 1980a; Riedl et aI., 1981, 1989) even though a simple visual technique for sex determination has been known for over 35 years (Moffitt,

1958). Only a few studies separated data for number of males vs. females captured (Barnes and Osborn, 1958; Riedl and Hoying, 1980b; Riedl and 47

Hislop, 1985). Important information may have been overlooked by previous researchers if sexual dimorphisms in behavior were not analyzed.

It was surprising to find that female WHFs were not differentially attracted to any of the walnut leaf blends (Fig. 3.1). Since the females did not discriminate or have strong preferences among these odors, they apparently use other information and/or senses in their foraging behaviors, e.g., finding mating and oviposition sites. Both gravid and reproductively immature females were found on traps. Further studies are needed to determine if gravid and immature females differ in attraction to the dynamic progression in emitted walnut volatiles as walnuts seasonally mature

(R. Teranishi, pers. comm.).

Male WHFs showed marked differential attraction to the test volatiles, with the two artificial walnut leaf and husk blends capturing more flies than either the aliphatic or the pentane control. Although the mean daily capture rate of the sesquiterpene blend (11.1 flies) was higher, it was not statistically different from the capture rate of the monoterpene blend (7.8 flies)

(Table 3.3 and Fig. 3.1). Male walnut husk flies responded almost equally to both artificial blends, even though the seven monoterpenes comprise a greater proportion of walnut leaf and husk headspace (WLHH) (51%) than do the four sesquiterpenes (34%) (Table 3.1). The sesquiterpenes, by molecular weight, are the heaviest and the least volatile of all the tested 48

molecules that had been found in WLHH. In the electroantennogram experiments (Chapter 2), the aliphatics, the lightest and most volatile of the compounds tested, all yielded larger receptor responses than either the monoterpenes or sesquiterpenes. These walnut leaf blend experiments conducted in walnut orchards, in contrast, showed high male attractancy to the heavier and less volatile compounds. Since the evaporation rate (Le., molecules emitted per unit time) of the test volatiles was based on both the volumetric loading dose/septum and the compound's volatility (as with the

EAGs described in Chapter 2), then relatively more of the lower mass compound, the aliphatic (Z)-3 hexenyl acetate, was emitted from the lure's rubber septa formulation than were either the monoterpenes or the sesquiterpenes.

As this and other studies have shown, all electroantennogram experiments should be followed with behavioral bioassays (Birch, 1971) to demonstrate attractancy (or repellency), otherwise the EAG results may be misinterpreted.

Angelica seed oil fractions

In the ASOil experiment, as in the walnut leaf and husk blend experiment, more males than females were captured. For both male and female flies, there were significant differences between blocks for the 49

number of flies captured. This is not surprising since walnut husk flies are patchily distributed through their environment (S. B. Opp, pers. comm.).

Although we chose trees with similar canopy, size and robustness, this orchard had been abandoned (Le., not managed or watered) for many years.

There is always inherent individual variation in trees, which may be difficult or impossible to assess.

In the ASOil experiment, the fraction that captured the most male flies,

ASO-77, was high in sesquiterpenes and also contained a known medfly attractant, u-copaene (Fig. 3.2). This was an interesting discovery, since although the WHF and medfly are both tephritids, they are not particularly closely related (Foote et aI., 1993). Some researchers in California have been looking for a model species to replace the medfly in experiments.

Regulations for raising and experimenting with medflies are extremely rigid since the potential damage to California's agriculture industry from this imported pest could be on the order of hundreds of millions of dollars per year. If WHF and medfly prove to be similar in behavioral responses to host plant volatiles, opportunities for medfly research may exist in California using

WHF as a model.

ASOil contains a different array of monoterpenes and sesquiterpenes than walnut leaf and husk volatiles. A gas chromatograph-mass spectrometry (GC-MS) analysis of ASOil by Flath et al. shows that -88% of 50

the compounds are monoterpenes, while only -9% are sesquiterpenes

(Flath et a/., 1994a; Flath, USDA ARS WRRC, unpubl.). Although ASO-77, the sesquiterpene-rich ASOil fraction with a-copaene, was most attractive to

WHFs, there are many other sesquiterpenes in this fraction as well (e.g.,

1.7% caryophyllene). Anyone of those sesquiterpenes might have been attractive to the flies. Another fraction, ASO-78, also contained sesquiterpenes (with a reduced amount of a-copaene, 0.4%), but was not attractive to the flies.

In summary, the electroantennogram experiments from Chapter 2 suggested aliphatics as possible attractants due to their high EAG

responses. Other field data contradicted the results from the EAGs (see

Appendix). The aliphatics are all ubiquitous green leaf volatiles, and eight

out of the nine tested are not found in walnuts. The walnut volatiles field

experiments showed no statistically significant difference in attraction to the

sesquiterpene-vs. monoterpene-artificial blends. However, the ASOil field

experiments showed greatest attractancy to only one fraction, which

contained sesquiterpenes, not monoterpenes. The analysis of all the data

thus far suggests sesquiterpenes as potential male WHF attractants. The

next logical step was to test the individual constituents of the walnut

sesquiterpene blend in the field (see Chapter 4). 51

2 Chemical Group Compound name 1 Percentage in Volume per 3 walnut headspace 2.0 ml blend

monoterpene alpha-pinene 5 20 ul sabinene 7 28 ul beta-pinene 4 11 nfa 4 myrcene 5 20 ul limonene 10 40 ul (E) beta-ocimene 12 48 ul linalool 1 4 ul

monoterpene total percentage 51 Total volume of constituents for blend 204 ul

sesquiterpene caryophyllene 15 60 ul 5 (E) beta-farnesene 2 12 ul germacrene-D 13 78 ul alpha-farnesene 4 24 ul

sesquiterpene total percentage 34 Total volume of constituents for blend 174 ul

aliphatic (Z)-3 hexenyl acetate 2 200 ul

Total percent of 87 headspace identified 3 Unidentified 7 compounds Unaccounted for 6 Total 100%

1 All chemicals courtesy of R. A. Flath, USDA ARS WRRC, Albany, CA.

2 From Buttery et aI., 1986

3 The total constituents for each group was mixed with 1800 ul of pentane to yield - 2.0 ml of field solution.

4 Beta-pinene was not available, and so was not used in the monoterpene blend.

5 A technical error was discovered in which 60 ul, and not 90 ul of caryophyllene was used in the sesquiterpene blend.

Table 3.1 Walnut leaf constituents with percentage found in leaf headspace relative to volume needed to create artificial blends. 52

1 Fraction Code Constituents u-copaene 2 Caryophyllene 3 number 2nd cut ASO-75 monoterpenes 0.24 % 0.03 % (lightest-most volatile) 3rd cut ASO-77 sesquiterpenes 11.14 % 1.70 % with alpha-copaene 4th cut ASO-78 sesquiterpenes 0.38 % 0.22 % with reduced alpha- copaene 5th cut ASO-79 oxygenated <0.03 % n/a compounds (heaviest-least volatile) crude ASOil ASOil 0.87 % 0.14 %

All ASOil fractions courtesy of R. A. Flath, USDA ARS WRRC, Albany, CA

1 Order in which the samples were collected from the distillation

2 From Flath et. aI., 1994a

3 From Flath et. aI., unpubl.

Table 3.2 Distilled fractions of angelica seed oil, and their major constituents including percentages of u-copaene and caryophyllene. 53

Test lure used Mean daily capture 1 (:t SEM) sesquiterpene blend 11.11 (:t 1.93) a monoterpene blend 7.83 (:t 1.73) a

(Z)-3 hexenyl acetate 3.88 (± 0.98) b pentane control 2.50 (:t 0.71) b

All chemicals courtesy of R. A. Flath, USDA ARS WRRC, Albany, CA

1 Mean daily capture is the average number of flies caught per trap per day. Values followed by the same letter are not significantly different at a=O.05 (Student-Newman-Keuls mUltiple comparison test)

Table 3.3 Mean daily capture for male WHF attracted to walnut leaf volatiles in a walnut orchard at Ardenwood, Fremont, CA. 12

10 >- III "C ...CIl Q, Q, 8 III .=... CIl Q, .c -Cl ::;, 6 III U III CIl ii:... .cCIl 4 E ::;, c C III ::CIl 2

o

(Z)-3 hexenyl acetate Sesquiterpene blend Monoterpene blend Pentane Lure

Vl +;.

Figure 3.1 Comparison of male to female WHF captures on traps baited with walnut leaf blends 20

18

>- 16 III "C... III Co 14 Co ...~ -III 12 Co j;; Cl ::l 10 III U en III 8 ;:... III ,Q E 6 ::l l: l: III III 4 :E Males 2

0 pentane ASOil ASOil control ASOil fraction # ASOil fraction # fraction # 78 (complete) 77 75

ASOil fractions

VI VI

Figure 3.2 Comparison of male to female WHF captures to lures baited with ASOil fractions 25

20 >­ ...IV "Gl Q. Q. ...IV ... -Gl Q. :E til IV =CJ III ,~ ;:... Gl ~ =c c :ll :E

Block 1

pentane control ASOil fraction #77 ASOil fraction #78 ASOil fraction #79

ASOil (complete) ASOil fractions

V\ 0\

Figure 3.3 Comparison of block 1 to block 2 male WHF captures to lures baited with ASOil fractions 25

>­ III 't:l.. G.l C. C. jg.. G.l C. ~ Cl ::l III CJ III G.l is.. G.l ..Q E ::l C C III G.l ~

pentane control ASOil fraction #75 ASOil fraction #77 ASOil fraction #78 ASOil (complete) ASOil fractions

VI -.l

Figure 3.4 Comparison of block 1 to block 2 female WHF captures to lures baited with ASOil fractions 58

3.5 References

Barnes, M. M.; Osborn, H. T. Attractants for the walnut husk fly. Journal of Economic Entomology 1958,51,686 - 689.

Birch, M. C. Intrinsic limitations in the use of electroantennograms to bioassay male pheromones in Lepidoptera. Nature 1971, 233, 57-58.

Buttery, R G.; Flath, R A; Mon, T R; Ling, L. C. Identification of Germacrene 0 in walnut and fig leaf volatiles. Journal ofAgricultural and Food Chemistry 1986, 34, 820-822.

Flath, R A; Cunningham, R T; Mon, T. R; John, J. O. Additional male Mediterranean fruitfly (Ceratitis capitata Wied.) attractants from angelica seed oil (Angelica archangelica L.). Journal of Chemical Ecology 1994a, 20,1969-1984.

Flath, R A; Cunningham, R T; Mon, T R; John, J. O. Male lures for Mediterranean fruitfly (Ceratitis capitata Wied.): Structural analogs of u-copaene. Journal of Chemical Ecology 1994b, 20, 2595-2609.

Foote, R H.; Blanc, F. L.; Norrbom, A L. Handbook ofthe fruit flies (Diptera: Tephritidae) ofAmerica north ofMexico; Comstock Publishing Associates, a division of Cornell University Press: Ithaca and London, 1993, 571 pp.

Fornasiero, U.; Guitto, A; Caporale, G.; Baccichetti, F.; Musajo, L. Identficazione della sostanza attrattiva per i maschi della Ceratitis capitata, contenunuta nell'olio essenziale dei semi di Angelica archangelica. Gazetta Chmica Italiana 1969,99,700-710.

Jacobsen, M.; Uebel, E. C.; Lusbey, W. R; Cunningham, RT Essential oil yields medfly attractant. Chemical Engineering News 1984, Dec 17, 24.

Moffitt, H. R Rapid determination of sex in Rhagoletis completa Cresson. Journal ofEconomic Entomology 1958,51,551. 59

Riedl, H.; Barnett, W. W.; Coates, W. W.; Coviello, R; Joos, J.; Olson, W. H. Walnut husk fly (Diptera: Tephritidae): Evaluation of traps for timing of control measures and for damage predictions. Journal ofEconomic Entomology 1989, 82, 1191-1196.

Riedl, H.; Hislop, R Visual attraction of the walnut husk fly (Diptera: Tephritidae) to color rectangles and spheres. Environmental Entomology 1985, 14,810-814.

Riedl, H.; Hislop, R G.; Barnett, W. W.; Coates, W. W.; Fitch, L. B.; Joos, J. L.; Olson, W. H.; Profita, J. C.; Schreader, W. R New monitoring methods for the walnut husk fly. In California Agriculture; 1981; 35, pp.21-22.

Riedl, H.; Hoying, S. A. Evaluation of trap designs and attractants for monitoring the walnut husk fly, Rhagoletis completa Cresson (Diptera: Tephritidae). Zeitschrift fur Angewandte Entomologie 1980a, 91, 510­ 520.

Riedl, H.; Hoying, S. A. Seasonal patterns of emergence, flight activity and oviposition of the walnut husk fly in Northern California. Environmental Entomology 1980b, 9, 567-571.

Steiner, L. F.; Miyashita, D. H.; Christenson, L. D. Angelica oils as Mediterranean fruit fly lures. Journal ofEconomic Entomology 1957, 50 (4),505.

Visser, J. H. Electroantennogram responses of the Colorado beetle, Leptinotarsa decemlineata to plant volatiles. Entomologia Experimentalis et applicata 1979,25,86-97. CHAPTER 4

Responses of Walnut Husk Flies to Individual Sesquiterpenes

in Field Trials in Walnut Orchards

4.1 Introduction

Until recently, the only attractants used for monitoring walnut husk flies (WHF) in commercial walnut orchards were glycine-sodium hydroxide solutions, ammonia, and protein hydrolysate mixtures (e.g., Staley's Protein

Bait # 7 and NuLure) (Teranishi et aL, 1993; Barnes and Osborn, 1958).

The component most attractive to flies in many protein hydrolysate baits is primarily ammonia (Teranishi et aL, 1993). Glycine-sodium hydroxide baits that have aged for at least two weeks were more attractive than fresh glycine-sodium hydroxide baits (Barnes and Osborn, 1958) probably because of ammonia production (R. Teranishi, pers. comm.). While ammonia is a powerful attractant, it is also indiscriminate, capturing male and female walnut husk flies throughout the walnut growing season (Reynolds et aL, 1996). Thus, ammonia-based lures are not the desired specific monitoring lure for determining female walnut oviposition.

Analysis of electroantennogram (EAG) experiments performed in spring-summer 1993 on WHF showed that although the ubiquitous green leaf volatiles (GLVs) had the highest recorded EAG responses, the more walnut specific sesquiterpenes elicited EAG responses far greater than

60 61

predicted, based on their dosage and evaporation rates and thus warranted field attractancy tests (see Chapter 2). Additionally, in preliminary field

experiments, we investigated the difference in attractancy between blends of

monoterpenes and blends of sesquiterpenes, and the difference between

consecutive fractions of distilled Angelica seed oil (ASOil) (Angelica

archangelica, Apiaceae), a known attractant for male WHF (Barnes and

Osborn, 1958). These field behavioral experiments showed that the

sesquiterpene blend was the most attractive to WHF (see Chapter 3). The

same GLVs that were tested in the EAGs were eliminated as attractants

when field studies indicated no significant attraction of WHFs to any of the

GLV lures (Two-way ANOVA: F=1.17, 8 df, p=O.32; Appendix).

Based on previous experiments, we decided to test the hypothesis

that one or more walnut sesquiterpene leaf volatiles might be potent

attractants. Such attractants might serve as indicators of impending

oviposition in walnut husks by female WHFs. This would be vital information

to help walnut growers anticipate WHF egg laying to prevent WHF damage.

4.2 Materials and Methods

WHFs were trapped during summer 1993 at two locations in Northern

California: Ardenwood Historic Farm in Fremont (Alameda County), a part of 62

the East Bay Regional Parks District, and a commercial organic walnut orchard in Hollister (San Benito County). Four walnut leaf and husk sesquiterpenes were tested (a.-farnesene, 13-farnesene, caryophyllene, and germacrene D), along with a distilled fraction of angelica seed oil (ASO-77; see Chapter 3) and a pentane control (all supplied by R. A. Flath, USDA

ARS WRRC). All the compounds used were ~ 91 % pure. The compounds and ASO-77 were diluted with redistilled, spectraphotometric-grade pentane to a 10% solution (100 ul of compound in 900 ul of redistilled pentane, with lonox antioxidant added to each solution). Aliquots (200 ul) of each solution were pipetted into individual 8 mm x 15 mm rubber septa. Once the pentane solvent evaporated, the septa were attached via paper clips to the tops of

Trece@ Pherocon AM/NB (adult monitoring, non-baited) yellow, sticky panel traps. The traps were hung in the canopy of each tree at 2-2.3 m from the ground on the north side.

In both Ardenwood and Hollister, we had three replicate blocks of six trees per block, for a total of 18 test trees at each site. Certain Persian walnut trees (Juglans regia, variety 'Payne') were chosen as "trapping-trees" based on their similarity in size, vigor and canopy in their assigned block region of the orchard.

The Ardenwood traps were checked every one to two days and all

WHF were removed and sexed. The checked traps were then moved 63

sequentially to the next trapping-tree. Every seven days the old test lures were removed, discarded, and replaced with new loaded septa (filled with

200 ul aliquot of a diluted compound). Number of male and female WHF caught were converted to mean daily captures by dividing by the number of trapping days. This experiment ran for nearly five weeks, 06 August to 08

September, which is mid-to-Iate fly season.

The same methodology was used at the Hollister site, but the traps were checked every three to seven days. At each visit the flies were picked off the traps and sexed. Every seven days the traps were moved sequentially to the next trapping-tree, and the old lure septa were discarded and replaced with new, freshly loaded septa. Number of male and female

WHF caught per trap were converted to mean daily captures for comparison.

For data analysis, each trap check was considered one data point. This experiment ran for four weeks, 10 August to 07 September.

Analysis description

One-way ANOVA on log (x+1) transformed data was first used to test for differences in numbers of males vs. females captured. General linear model ANOVAs were then used to compare results from the different sites, to compare trap captures within the different blocks of each site, and to test for differential lure attractancy. 64

4.3 Results

Gender differences

Significantly greater numbers of male walnut husk flies were caught than females (F=83.16, df=1, P«O.001).

Site differences

The two sites were significantly different in the number of flies caught

(F=18.18, df=1, P

Ardenwood

For males only, there was significant differential attraction to the different lures (F=12.39, df=5, P ASOil fraction # 77> germacrene 0 > u-farnesene ~pentane control (Fig. 4.1). There were no significant differences in lure attractancy for females (F=1.06, df=5,

P=O.383) (Fig. 4.1).

There were significant block effects for both the males flies (F=42.91 , df=2, P

Hollister

For the Hollister site the results were similar. Males showed differential attraction to the lures (F=4.87, df=5, P<0.001). The rankings of the lures were slightly different however, with caryophyllene still the most attractive but the other compounds changed order (caryophyllene > ASOil fraction # 77 ~ germacrene 0 ~ pentane control ~ ~-farnesene ~ a-farnesene) (Fig. 4.1). Again no significant differences in attractancy to the different lures were found for females (F=0.46, df=5, P=0.805).

There were significant block effects for both male flies (F=8.07, df=2,

P<0.001) and female flies (F=10.94, df=2, P<0.001) (Fig. 4.4).

4.4 Discussion

Although sex ratios for WHF are near 1:1 for most of the fly season

(Boyce, 1934), significantly more males than females (3.6:1 in Ardenwood, and 2.3: 1 in Hollister) were caught on the sesquiterpene baited traps.

Almost 60% of all males captured were attracted to only two compounds, caryophyllene and ~-farnesene.

Caryophyllene, the most attractive lure to male WHF, is also the most

plentiful of any volatile found in walnut leaf headspace with a concentration

of 15% as analyzed by capillary GC-mass spectrometry (Fig. 4.3) (Buttery et 66

al., 1986). WHF males may be cueing in to the most prevalent volatile given

off by developing walnuts. However, the fact that the flies showed some

level of attraction to three of the four sesquiterpenes suggests a sensitivity to

many of the compounds found in walnuts.

The differences in magnitude in number of males captured between the Ardenwood and Hollister sites might be easy to explain since the sites

are very different. Ardenwood is an unmanaged orchard, receiving only

seasonal rain and no irrigation water. Hollister is an organically managed, well-maintained orchard that receives three or more deep summer waterings.

Ardenwood has many trees with dead or dying limbs in their canopies, and

some areas of the orchard are devoid of any living trees. Hollister has more

acres of trees, a contiguous canopy that is more continuous throughout the

orchard, and the trees appear healthier. Thus, higher fly capture occurred in

Ardenwood, where the trees appeared more stressed. Also, WHF

populations were reduced at Hollister due to insecticide overspray from

neighboring orchards.

The block differences within each site are also easily explained.

Walnut husk flies are known to be patchily distributed within orchards, a fact

which often confounds trap interpretation for growers. An important part of

my experimental design was to try to eliminate as many confounding

variables as possible. I attempted this by placing all traps in the same 67

compass quadrant (north side) of each tree and at the same height (2m).

Although I chose trees which appeared similar, there were apparently inherent differences between them. For orchards with medium to high walnut husk fly populations, it seems block effects are very difficult to abate.

The two study sites produced slightly different rankings of attractancy for the six test lures for male WHF. Caryophyllene was obviously, significantly and consistently the most attractive (ranked number one), but beyond that the rankings diverged (Fig. 4.2). There were three or four test lures significantly more attractive than the pentane control at Ardenwood, while only two test lures were significantly more attractive than pentane at

Hollister (Fig. 4.1, and Fig. 4.4). There are at least four plausible explanations for this result:

1. The trees at the Hollister site had a much earlier phenology and rate of

maturation than at Ardenwood. WHF emergence at Hollister began in

mid-May, while in Ardenwood flies emerged in late June. Because of

Hollister's advanced season, the walnuts also ripened sooner than at

Ardenwood. Thus, although we sampled these two sites at the same

chronological time (July to September), the samples were actually taken

at different phenological times, thus affecting fly age and responsiveness

to lures. It is possible that in Hollister we detected an attractancy shift in 68

WHF males, in the latter part of the season, away from l3-farnesene

(Fig. 4.2). More research is needed to fully understand these results.

2. Although the experiments were of similar duration (four weeks in Hollister

and almost five weeks in Ardenwood) there were only five sampling dates

in Hollister where traps were checked every three to seven days, while

there were 24 sampling dates in Ardenwood where traps were checked

every one to two days. Because of the fewer data points at Hollister, any

errors, problems, or differential weather patterns at the two sites could

have weighed more heavily on the results at Hollister.

3. Additionally, uncontrolled or "outside" influences over the field conditions

at Ardenwood were minimal but were much greater at Hollister. No

maintenance was ever performed on the trees at Ardenwood, and there

were no adjacent farms or orchards. The Hollister site had two or three

neighboring conventional walnut orchards that were not certified organic;

that is, they did use pesticides to control WHF. Prior to my experiment,

insecticide drift from aerial applications to these orchards did occur, and

accidental or intentional overspray into our "un-sprayed" areas was

suspected, thereby dramatically reducing the endemic WHF population

and their subsequent capture rate.

4. The fly captures at Hollister were much lower than at Ardenwood, so it is

possible that the rankings of the lures may have changed due to subtle 69

differences in the capture of a very small number of flies that resulted in

insignificance of statistical tests.

Sesquiterpenes as a group are important components of plant-insect interactions. Each of the sesquiterpenes tested is biologically active in at least one insect group: caryophyllene is attractive to boll weevils (Minyard et aI., 1969) and also to green lacewings (Flint et aI., 1979); germacrene D is a cockroach pheromone component (Tahara et aI., 1975), and more recently has been shown to be attractive to pickleworm moths (Peterson et aI., 1994);

13-farnesene is an aphid alarm pheromone component (Wohlers, 1981;

Wohlers and Tjallingii, 1983); and u-farnesene is an attractant for codling moth larvae (Sutherland and Hutchins, 1972).

Caryophyllene is a compound found in many plants whereas germacrene D is rarer in the plant kingdom. Germacrene D is found in the

Apiaceae (Erigeron, Eupatorium, Chrysanthemum), the Pinaceae (Pinus,

Pseudotsuga), the Moraceae (Ficus), and the Juglandaceae (Juglans)

(Tahara et aI., 1975). It was surprising thatgermacrene D did not induce

high WHF responses since it is the second most common component of walnut odor (13% of headspace), only slightly less common than

caryophyllene (15%) (Fig. 4.3), and is more specific to walnuts than the other

sesquiterpenes tested. 70

In Chapter 3 I discussed the different ASOil fractions and their attractancy to WHFs. An interesting observation is that the fraction most attractive, ASO-77, also contained the highest concentrations of both a-copaene (11.14%) and caryophyllene (1.70 %) (see Chapter 3, Table 3.2).

If a-copaene is the WHF attractant in crude ASOil (which it most likely is,

D. Light, pers. comm.), then the increase in a-copaene content from the

crude oil (0.87%) to the amount in ASO-77 (11.14%) would make the fraction

more attractive to WHFs. Additionally, the level of caryophyllene in ASO-77

(1.70%) is much greater than in the crude ASOil (0.14%). Thus, a-copaene

and caryophyllene might be independent attractants or possibly co­

attractants.

An interesting observation made during these experiments was the

almost exclusive attraction of WHFs to walnut leaf and husk volatile lures.

Few other insects were found on these traps, such as the walnut-infesting

codling moth. Other experiments I performed in the same orchards, using

traps baited with ammonia lures, showed indiscriminate attraction with high

numbers and many species of insects caught. This specificity of WHF

captures on traps baited with walnut volatiles supports the idea of host­

specific attractancy, in addition to providing easier trap maintenance.

It is known that male WHF wait on developing walnuts to court and

attempt copulation with females that alight (Opp et aI., 1996), and so it is 71

understandable that males would be attracted to the volatiles from walnuts.

The literature is contradictory on sexually-dimorphic attractancy of ammonia to walnut husk flies. Some studies have indicated that males and females are equally attracted to ammonia lures (Barnes and Osborn, 1958), while other studies claim females are more strongly attracted to ammonia baits than males (Teranishi et aL, 1993). Regardless of the contradictions, the question remains: why are female WHF not attracted to the tested walnut volatiles? We do not know what other undiscovered olfactory or other modality cues attract and bring females to susceptible walnuts, and it is these females that sting and initiate damage on walnuts.

My research indicates that female orientation to walnuts is probably not based solely on olfactory cues. Others have suggested that visual cues

(i.e., color and shape) are important in the attraction of WHF. However, in those studies, WHFs captured on traps were not separated by sex (Riedl and Hislop, 1985; Riedl et aL, 1989) or the researchers did not find any sexual differences in trap color preferences (Riedl and Hislop, 1985). Riedl and Hislop (1985) found that both male and female walnut husk flies were equally attracted to yellow traps early in the season and also to green spheres later in the season. Riedl et aL (1989) later found that while yellow, sticky panel traps baited with ammonium carbonate were attractive to WHF throughout the season, unbaited green spheres (more closely mimicking 72

walnuts) only became attractive to the flies close to when oviposition began.

Thus, previous studies gave little indication that male WHFs would be attracted to walnut volatiles.

A GC-MS analysis of the volatiles of walnut husks showed that the same compounds were present in both husks as in leaves and in approximately the same concentrations (R. Teranishi, pers. comm.).

However, it is not known if there are changes in the concentrations of the volatiles over time as the walnut husks ripen. This is a very important point because WHFs may be cueing into subtle adjustments in both the presence/absence and concentrations of the different volatiles. Female

WHFs are not readily found on walnut husks, nor sting or oviposit in husks until a certain chemically undefined maturation and phenology of the husk is reached. Males may be attracted to developing walnuts because these will be the sites of copulation and of oviposition. A thorough investigation of the phenology of the volatiles from ripening walnuts and their susceptibility states is needed to form a correlation with the temporal phasing when oviposition attempts begin and when they become successful. Plant phenology has been shown to affect insect attraction to plant volatiles in other insects. For example, the western corn rootworm is more responsive to volatiles from senescing portions of corn silk than younger silk (Abou­

Fakhr et aI., 1996). Any changes or shifts in compound concentrations from 73

walnuts entering their sting susceptibility phase might lead to the development of a lure for WHF that is more specific to the timing of oviposition. Different blends of sesquiterpene lures that mimic the volatile

pattern of the ripening walnuts might result in more efficacious trapping of

WHF of potentially either sex.

We have seen that male WHFs are strongly attracted to one

sesquiterpene component of walnut leaf and husk headspace,

caryophyllene, while female flies show little response to this volatile. The

developing walnuts are used by males as sites to court and mate with

females, and are the food source for the next generation of walnut husk flies.

Current monitoring methods that use yellow, sticky panel traps baited with

ammonium carbonate are adequate population density indicators, but they

are ineffective predictors of oviposition and the onset of damage to the

walnut crop (Riedl and Hoying, 1980; Riedl et aI., 1981, 1989). Further

research with sesquiterpene lures, either individually or in blends, may yield

a lure capable of predicting female oviposition (Opp et aI., unpubl.), and

allow walnut growers to spray their orchards less often and with greater

efficacy. 35

,::~ >- 30 III "C ...<» Q. 25 Q. ...III ... -<» Q. 20 .c -Cl ::::l III tJ 15 CIl u.. J: ::... 10 <» .c E ::::lc 5 C III <» :E 0

Males caryophyllene beta-farnesene Ardenwood 3 ASOil fraction #77

Males Compound Hollister 1

--.l ~

Figure 4.1 Comparison of WHF captures on traps baited with walnut leaf sesquiterpenes at two sites 40

35 "C _cu'" 0 U l: 30 ~:8 ~ l/l-"' 25.!!liil!! "' u cu E l:o ..Q, 20 iiju~ o ~ ':I :: fa ~ 15 0 C cu cu cu l/l "'enu 10 C ..8- CU"C ~ l: 5 ~ "' o caryophyllene

% of walnut leaf headspace Compound Hollister males

Ardenwood Site males

...... Vl

Figure 4.2 Comparison, at the two sites, of male WHF captures (by percentage of total) in relation to headspace concentration of walnut sesquiterpenes ""IOp"""" 1~~' ~~~'" """""" .'::::::::~ I~ ~~J~!f!'~'ii"i~t"i\~\~;,\!

beta-farnesene

'tl (E) beta-ocimene § -I!lI',',""""',.,.,...... &. myrcene § m""""""""""""""",,·,·,·,...... (.) beta-pinene

limonene

sabinene

alpha-pinene

linalool

(Z)-3 hexenyl acetate

o 2 4 6 8 10 12 14 16

Percentage of total headspace

-J 0\

Figure 4.3 Percentage composition of the twelve headspace volatiles found in walnut leav.es (from Buttery et aI., 1986) 35

30 l/) 11.>- §~ eu ...eu ;;Q. E Q. ~ ...eu­ ,Q ... E eu c.c::;, Q. c Cl III ::;, eu III :: l.l

ASOil fraction #77

Compound

Site and replicate number

Hollister 3

-J -J

Figure 4.4 Comparison of male WHF captures to walnut sesquiterpenes by block, for the two sites 78

4.5 References

Abou-Fakhr, E. M.; Hibbard, E. 8.; Jewett, D. K.; Bjostad, L. B. Electroantennogram responses of western corn rootworm (Coleoptera: Chrysomelidae) adults in relation to maize silk morphology and phenology. Environmental Entomology 1996,25,430-435.

Barnes, M. M.; Osborn, H. T. Attractants for the walnut husk fly. Journal of Economic Entomology 1958,51,686 - 689.

Boyce, A M. Bionomics of the walnut husk fly, Rhagoletis completa. Hilgardia 1934, 8, 363-579.

Buttery, R G.; Flath, R A; Mon, T. R; Ling, L. C. Identification of Germacrene D in walnut and fig leaf volatiles. Journal ofAgricultural and Food Chemistry 1986,34,820-822.

Flint, H. M.; Salter, S. S.; Walters, S. Caryophyllene: an attractant for the green lacewing. Environmental Entomology 1979, 8, 1123-1125.

Minyard, J. P.; Hardee, D. D.; Gueldner, R C.; Thompson, A C.; Wiygul, G.; Hedin, P. A Constituents of the cotton flower bud. Compounds attractive to the boll weevil. Journal ofAgricultural and Food Chemistry 1969, 17, 1093-7.

Opp, S. 8.; Spisak, S.; Telang, A; Hammond, S. Comparative mating systems of two Rhagoletis species; the adaptive significance of mate guarding. In Fruit Fly Pests, A World Assessment oftheir Biology and Management, 8. A McPheron and G. J. Steck, Eds.; St. Lucie Press: Florida, 1996; 586 pp.

Peterson, J. K.; Horvat, R J.; Elsey, K. D. Squash leaf glandular trichome volatiles: Identification and influence on behavior of female pickleworm moth [Diaphania nitidalis (StolL)] (Lepidoptera: Pyralidae). Journal of Chemical Ecology 1994, 20, 2099-109.

Reynolds, K.; Opp, S. 8.; Moen, M.; Denham, K. Mark recapture studies of walnut husk flies attracted to food-based lures. In Fruit Fly Pests, A World Assessment oftheir Biology and Management; B. A. McPheron and G. J. Steck, Eds.; St. Lucie Press: Florida, 1996; 586 pp. 79

Riedl, H.; Barnett, W. W.; Coates, W. W.; Coviello, R; Joos, J.; Olson, W. H. Walnut husk fly (Diptera: Tephritidae): Evaluation of traps for timing of control measures and for damage predictions. Journal ofEconomic Entomology 1989,82,1191-1196.

Riedl, H.; Hislop, R Visual attraction of the walnut husk fly (Diptera: Tephritidae) to color rectangles and spheres. Environmental Entomology 1985, 14,810-814.

Riedl, H.; Hislop, R G.; Barnett, W. W.; Coates, W. W.; Fitch, L. B.; Joos, J. L.; Olson, W. H.; Profita, J. C.; Schreader, W. R New monitoring methods for the walnut husk fly. In California Agriculture; 1981; 35, pp. 21-22.

Riedl, H.; Hoying, S. A. Evaluation of trap designs and attractants for monitoring the walnut husk fly, Rhagoletis completa Cresson (Diptera: Tephritidae). Zeitschrift fur Angewandte Entomologie 1980, 91, 510­ 520.

Sutherland, O. R W.; Hutchins, R F. N. u-farnesene, a natural attractant for codling moth larvae. Nature 1972,239, 170.

Tahara, S.; Yoshida, M.; Mizutani, J.; Kitamura, C.; Takahashi, S. A sex stimulant to the male American cockroach in the compositae plants. Agricultural and Biological Chemistry 1975,39, 1517-1518.

Teranishi, R; Kint, S.; Flath, R A.; Light, D. M.; Opp, S. B.; Reynolds, K. M.; Hamersky, W. Protein hydrolysate components attractive to tephritids. Proceedings ofan International Symposium on management ofinsect pests: Nuclear and related molecular and genetic techniques 1993, 487-493.

Wohlers, P. Effects of the alarm pheromone (E)-J3-farnesene on dispersal behaviour of the pea aphid Acyrthosiphon pisum. Entomologia Experimentalis et applicata 1981, 29, 117-124.

Wohlers, P.; Tjallingii, W. F. Electroantennogram responses of aphids to the alarm pheromone (E)-J3-farnesene. Entomologia Experimentalis et applicata 1983,33,79-82. CHAPTER 5

Conclusions, Practical Implications, and Future Studies

5.1 Introduction

This concluding chapter is divided into sections based on the three primary experiments conducted for this thesis. In these sections I discuss the experimental results, and where appropriate, the impact on agricultural monitoring of walnut husk flies (WHFs). The final section is a discussion of ongoing and future research.

The original purpose of my research was to perform preliminary experiments that might identify walnut leaf volatiles that are kairomonally attractive to WHFs. These represent the first steps to reach the ultimate goal of developing a lure that would be predictive of onset and occurrence of walnut damage. Since there are numerous volatiles that comprise walnut leaf and husk headspace, it would have been impractical to initially field test all the compounds for attractancy. Instead I tried to screen the volatiles for potential olfactory importance to WHFs by using electroantennograms (EAGs) (Chapter

2). The results, after interpretation, suggested that terpenes, both monoterpenes and sesquiterpenes, might be relatively good candidates for attractants.

80 81

A field test of a blend of sesquiterpenes vs. a blend of monoterpenes demonstrated no statistically significant difference between them, but both blends captured more flies than the controls (Chapter 3). Additionally, we field tested angelica seed oil (ASOil), an historical WHF attractant, and different distilled fractions of ASOil. Only one fraction of ASOil, predominantly composed of sesquiterpenes, was attractive. None of the other fractions, nor the crude ASOil, were more attractive than the controls (Chapter 3). Since it appeared that walnut leaf volatile sesquiterpenes were biologically active, we decided to test, in the final experiments of the season, individual sesquiterpene lures for their attractancy to the flies (Chapter 4).

5.2 Olfactory Chemoreception

Our original purpose for using EAGs was as a receptivity screening tool to test WHFs by recording their antennal responses to known walnut leaf volatiles. Compounds eliciting high EAG activity would then be field tested for attractancy to the flies.

For closely related tephritid species, researchers have recorded EAG responses and successfully correlated them with attractancy, e.g., apple maggot fly (AMF) (Frey et aI., 1992), medfly (Light et aI., 1992; Light and

Jang, 1996) and oriental fruit fly (Light and Jang, 1987; Light and Jang 82

unpubl.; Jang and Light, 1991). Conversely, some EAG studies found lack of attractancy to some compounds that elicited high EAG responses, e.g., medfly (Light et aI., 1988, 1992). As there have been no prior reported EAGs performed on WHFs, I chose to test responses of WHFs to walnut leaf volatiles in an EAG olfactory bioassay.

In the EAGs, I tested WHFs with all the monoterpenes and sesquiterpenes reported in walnut leaf headspace plus a standard set of nine green leaf volatiles. The EAG results from the flies, although showing statistically significant differences between compounds, had much lower measured responses than other reported tephritid EAG research «O.4mV).

Without detailed analysis, the EAG results would have suggested that the smallest molecules (the aliphatics or green leaf volatiles, GLVs) might have been the best candidates for attractants. This seemed counter-intuitive, since most (8 of 9) of the GLVs tested were not part of the walnut headspace and might not be present in the WHF's immediate environment. The question, then, is why would the flies respond to GLVs?

Light et aI., (1988) suggested that medflies and other insects have a large sensory investment in detecting GLVs, which could be related to fundamental needs, i.e., foraging for water or food and finding shelter. Since some GLVs are also classified as damage volatiles, attractancy may not be the appropriate response for WHFs. WHFs prefer intact hosts, and GLVs 83

might indicate a damaged host, which may be a poor choice for female oviposition.

Although many GLVs have been shown not to be individually attractive, they are capable of synergism with either pheromones (Light et aI., 1993) or attractants, e.g., ammonia (Hamersky and Light, unpubl.; Light and Jang,

1996). While researchers have shown that GLVs can induce high EAG responses in oriental and Mediterranean fruit flies (Light and Jang, 1987;

Light et aI., 1988) ,I have shown that GLVs are not attractive to WHFs in the field (see Appendix).

The electroantennogram results gave us no clear path to take in terms of field trials (see Discussion section in Chapter 2), although by factoring in molecular weight differences and resulting volatility differences, the sesquiterpene responses may have been relatively greater (on a molar basis) than actually recorded. We decided to test the attractancy to WHFs of blends of monoterpenes, blends of sesquiterpenes and one representative GLV.

As for the appropriateness of using electroantennograms as a future screening tool, my conclusion is that EAG methodology is not well suited for walnut husk flies. 84

5.3 Walnut Husk Fly Responses to Walnut Leaf Blends

and Angelica Seed Oil Fractions

Apple maggot flies (AMFs) are oligophagous insects whose primary hosts are apples, so it was appropriate that they were tested for attractancy to apple volatiles (Fein et aI., 1982). Since AMFs have only a few host plants, olfactory specificity has evolved in the flies (Frey and Bush, 1990). I applied that same principle, that oligophagous insect pests are usually sensitive to volatiles from their primary host, to WHFs. I could find no literature describing experiments where WHFs were tested for behavioral responses to walnut leaf and husk volatiles (WHLVs). I decided to test the attractancy of WLHVs to

WHFs in the field.

Since WH LV headspace contains only three families of volatile compounds -- aliphatics, monoterpenes, and sesquiterpenes - it was straightforward to employ experiments testing separate blends of each family of compounds. The results showed that while the sesquiterpene blend resulted in higher mean trap captures, the capture rate was not statistically significantly greater than the monoterpene blend's rate. The aliphatic group was no more attractive than the control. These results suggested that both sesquiterpenes and monoterpenes are attractive to WHFs.

Experiments using a male WHF attractant, angelica seed oil (ASOil), discovered 38 years ago (Barnes and Osborn, 1958), contributed more details 85

regarding attractancy. We used different distilled fractions of ASOil, separated roughly into the same three families as the walnut leaf volatiles.

We found that one distillate, a fraction composed primarily of sesquiterpenes, was more attractive than any other fraction. This fraction had a high proportion of a medfly attractant, a-copaene, and a smaller amount of caryophyllene. The other fractions, including another sesquiterpene fraction lacking a-copaene and caryophyllene, were no more attractive than the solvent control to WHFs.

Aggregating the results from the electroantennograms, the walnut blend studies, and the ASOil fieldwork, we concluded it must be one or more sesquiterpenes that are attracting the flies. Only four sesquiterpenes have been identified in walnut headspace, and although a-copaene is another sesquiterpene, it is not found in walnut leaf and husk headspace. Therefore, our final experiment was to field test the attractancy of the walnut specific sesquiterpenes to WHFs at two different sites (Section 5.4). Ongoing studies with sesquiterpene blends are discussed in Section 5.5.

5.4 Walnut Husk Fly Responses to Individual Sesquiterpene Test Lures

Caryophyllene was the most attractive of all the sesquiterpenes tested.

The two walnut orchards where testing occurred however, had different 86

phenologies in that Hollister was approximately one month more advanced than Ardenwood. This might help explain the finding that the second most attractive compound differed by site (Le., p-farnesene ranked #2 at

Ardenwood, but #4 at Hollister). This is interesting because if WHFs detect and respond to the odors of walnuts as they ripen (as we think they do), we might have documented a shift in attractancy between orchards in different phenological states. Support for this post-hoc hypothesis could be obtained by documenting when stings begin. Unfortunately, since I did not record the onset of oviposition by the flies in the two orchards, I was not able to analyze my data temporally and correlate it with either lure attractancy shifts or oviposition.

The following summer (1994) the team repeated and expanded the experiments at one of the original sites and in three additional commercial orchards (Light and Opp, unpubL; Opp et aL, 1995). Yellow panel traps and green spheres baited with ammonium carbonate were tested against yellow panel traps and green spheres baited with caryophyllene. While the ammonium carbonate baited traps caught the most WHFs, the green spheres baited with caryophyllene caught predominantly males with the peak of male capture coinciding with female egglaying, in most cases. (Opp et aL, 1995).

Two sets of experiments, performed during two distinct fly seasons, replicated the finding that caryophyllene is the first host-based attractant found 87

for male WHFs. The 1994 studies (with experiments to be repeated in summer 1996) demonstrated that peak fly capture on caryophyllene-baited green spheres may predict female WHF oviposition. These are first steps in helping to unravel this insect-plant interaction. Once that relationship is better understood, the task might be far easier to find a WHF attractant which will predict walnut damage.

5.5 Ongoing and Future Studies

My fieldwork in 1993, and the team's research in 1994, were performed in both an abandoned walnut orchard and a commercial organic orchard where no insecticides were sprayed during the experiments. Later fieldwork by the research team (1994-present) has been, and continues to be, performed in traditional walnut orchards where insecticides are used. A strength in using commercial walnut orchards for research is the direct applicability to other orchards. The utility of commercial orchards for experiments has one major drawback in that the experiment can end quite suddenly if the grower decides to spray for WHFs (and in fact this has occurred numerous times).

Even with the inherent problems, efficacy tests in commercial walnut orchards have substantiated caryophyllene as an attractant for male WHF 88

that, most of the time, is predictive of female WHF oviposition (Opp et aL,

1995). Future experiments are planned to resolve the correlation of male attraction to this lure and female oviposition'.

Insects, and flies especially, are often spatially clumped in distribution.

The patchiness of insect populations in croplands seems to be common knowledge among farm advisors. This patchiness concept can also be extended to the distribution of flies on individual trees. The microclimates at the treetop and at the canopy bottom are different. Trap placement within a tree canopy is important for pest monitoring, since Opp et aL (1995) determined that caryophyllene-baited traps placed high in the canopy (5m) caught significantly more flies than traps low in the canopy (2m). Additionally, they found that the higher traps caught peak numbers of males at about the same time as female oviposition began.

The caryophyllene lure that Opp et aL, (1995) used is only one of several walnut leaf and husk volatiles. Since headspace analyses have been published on walnut leaves (Buttery et aL, 1986), but not yet on ripening walnut husks, I have only R. Teranishi's personal communication claiming that the volatile constituents are primarily the same from leaf to husk. Since he has not published or provided details, such as percentages of the different compounds, additional trapping of volatiles, followed by gas chromatograph- 89

mass spectrometry (GC-MS) procedures need to be performed on the chemicals of ripening walnuts as they change with plant phenology.

This is in fact, a current research goal of the USDA, ARS, WRRC

(Albany, CA) team effort, (Light et al.) to capture the WLVH by headspace trapping from intact walnuts in the field at weekly or more frequent intervals throughout the growing season. This will provide a baseline of WLVH concentration changes throughout the growing season. Testing the individual sesquiterpene/blend lures should be concurrent with headspace trapping, and occur in the same orchard. Correlation of WHF captures with changes in walnut volatile percentages can then be thoroughly investigated. By using different sesquiterpene blend concentrations, in addition to individual sesquiterpenes, researchers might be able to document a shift in attractancy,

(for example, from a lure high in p-farnesene to one with a lower concentration) and correlate it to a point in husk phenology when stings begin.

Continued testing of sesquiterpene lures, when linked with proper trap placement and walnut phenology, should bring into development the next generation of walnut husk fly lures, those that will be predictive of the onset of walnut damage. 90

5.6 References

Barnes, M. M.; Osborn, H. T. Attractants for the walnut husk fly. Journal of Economic Entomology 1958,51,686 - 689.

Buttery, R G.; Flath, R A; Mon, T. R; Ling, L. C. Identification of Germacrene D in walnut and fig leaf volatiles. Journal ofAgricultural and Food Chemistry 1986,34,820-822.

Fein, B. L.; Reissig, W. H.; Roelofs, W. L. Identification of apple volatiles attractive to the apple maggot, Rhagoletis pomonel/a. Journal of Chemical Ecology 1982, 8, 1473-1487.

Frey, J. E; Bierbaum, T. J.; Bush, G. L. Differences among sibling species Rhagoletis mendax and R. pomonel/a (Diptera: Tephritidae) in their antennal sensitivity to host fruit compounds. Journal ofChemical Ecology 1992, 18,2011-2024.

Frey, J. E; Bush, G. L. Rhagoletis sibling species and host races differ in host odor recognition. Entomologia Experimentalis et applicata 1990, 57, 123-131.

Jang, E 8.; Light, D. M. Behavioral responses of female oriental fruit flies to the odor of papayas at three ripeness stages in a laboratory flight tunnel (Diptera: Tephritidae). Journal ofInsect Behavior 1991,4,751­ 761.

Light, D. M.; Flath, R A; Buttery, R G.; Zalom, F. G.; Rice, R E; Dickens, J. C.; Jang, E. B. Host-plant green-leaf volatiles synergize the synthetic sex pheromones of the corn earworm and codling moth (Lepidoptera). Chemoecology 1993, 4, 145-152.

Light, D. M.; Jang, E B. Electroantennogram responses of the oriental fruit fly, Dacus dorsalis, to a spectrum of alcohol and aldehyde plant volatiles. Entomologia Experimentalis et Applicata 1987,45,55-64.

Light, D. M.; Jang, E B. Plant volatiles evoke and modulate tephritid behavior. In Fruit Fly Pests, A World Assessment oftheir Biology and Management; B. A McPheron and G. J. Steck, Eds.; St. Lucie Press: Florida, 1996; 586 pp. 91

Light, D. M.; Jang, E. B.; Dickens, J. C. Electroantennogram responses of the Mediterranean fruit fly, Ceratitis capitata, to a spectrum of plant volatiles. Journal of Chemical Ecology 1988, 14, 159-180.

Light, D. M.; Jang, E. B.; Flath, R. A. Electroantennogram responses of the Mediterranean fruit fly, Ceratitis capitata, to the volatile constituents of nectarines. Entomologia Experimentalis et applicata 1992, 63, 13-26.

Opp, S. B.; Reynolds, K.; Olson, 8.; Buchner, R.; Pickel, C.; Reil, W. "Development of new lures for walnut husk fly based on host plant volatiles," Walnut Research Reports 1995; Walnut Marketing Board, 1995. APPENDIX

Responses of Walnut Husk Flies to Individual Green Leaf

Volatiles in the Field

A.1 Introduction

Green leaf volatiles (GLVs) are ubiquitous, aliphatic compounds found

in most green plants (Visser, 1979). Eight common GLVs were used in the electroantennogram (EAG) experiments (Chapter 2), and 7 of those GLVs were ranked in the top 10 compounds that induced the greatest antennal

responses from walnut husk flies (WHFs). To further understand the

relationship between GLVs and WHFs, field tests were performed in Hollister

to test the attractancy of these compounds individually to WHFs. I field tested

those green leaf volatiles that were used in the EAG experiments (all the

6-carbon aldehydes, acetates and alcohols).

A.2 Materials and Methods

This experiment tested the attractiveness of individual green leaf

volatiles to WHFs. The field methodology was similar to both walnut leaf

volatile experiments discussed in Chapters 3 and 4 and is only briefly

described here. One block of nine trees in the Hollister orchard was chosen

92 93

for similar size, canopy and robustness of its trees. Pherocon® AM NB yellow, sticky panel traps were hung at 2-2.3 m heights on the north side of each tree. Individual glass microcapillary tubes (1.0 mm inside diameter x

30 mm long and sealed at one end) were filled ca. half full with one of eight volatiles for a total of nine tubes (8 different GLVs plus 1 empty control) (Table

A.1) and were taped to the upper edge of the traps.

The alcohol and acetate groups of volatiles are inherently stable, so these tubes remained on the traps for the duration of the experiment. The aldehydes, however, are oxygen-sensitive, and change into acids which reduce the output of remaining aldehyde volatiles. To minimize this effect and

~eep the aldehyde output more steady, these aldehyde lure tubes were changed weekly. The old aldehyde tubes were removed and replaced with fresh compound in new tubes.

The traps were checked every three to eight days, with all flies removed, placed on data sheets and sexed. The traps were randomly rotated through the nine trees once per week. Trap captures were standardized to number of flies caught per trap per day. The experiment ran for 6 weeks from

03 August to 07 September 1993. 94

Analysis

General linear model, two-way analyses of variance (ANOVAs) were used on log (x+1) transformed data to test for differences in numbers of males vs. females captured, and to test for effects of the different GLV test lures.

A.3 Results

The only statistically significant difference found was that more males than females were captured (2.1:1) (Two-way ANOVA; F=9.33, df=1,

P=O.003). There were no statistically significant differences for: (1) any of the test lures used, i.e., no test lure was more attractive than any other (F=1.273,

df=8, P=O.268) (Table A.1) or (2) any interaction effect between sex and lure

(F=O.095, df=8, P=O.999).

A.4 Discussion

Even though no statistically significant differences were seen in the

number of WHFs captured with the lures, more than twice as many males than females were captured on the traps. Sexual dimorphism for lure

attractancy has been a common theme through my research, and is shown 95

again for individual GLVs. Males appear to be sensing the environment on a more olfactory basis than females.

The lack of differential attractancy to these GLVs reinforces the caveat

stated earlier that electroantennogram results must be followed by field work to prove behavioral responses. The EAG responses to the aliphatics (GLVs)

were ranked in eight of the top ten positions, but in the field, there was no

significant attraction greater than controls to these same compounds.

Of all the GLVs tested, only (Z)-3 hexenyl acetate is present in the

headspace of walnut leaves collected in spring and summer (Buttery et aI.,

1986). All others are part of the GLV complex of compounds found in many

green plants.

The finding that more male than female flies were captured is

consistent with almost all the lures tested, including monoterpene blends,

sesquiterpene blends, individual sesquiterpenes, and also individual green

leaf volatiles. The low capture rates may indicate that WHFs are not tuning in

to these smaller more ubiquitous compounds, but are responding to more

host-specific compounds. 96

Group Compound Mean number flies caught Rank per trap per day (± SEM) * Alcohols (Z)-3-Hexen-1-ol 0.854 (±0.391) 7

Hexan-1-ol 1.431 (±0.391) 3

(E)-2-Hexen-1-ol 1.163 (±0.391) 6

Aldehydes

(E)-2-Hexenal 1.193 (±0.391) 5

Hexanal 1.639 (±0.391) 2

Acetates

(Z)-3-Hexenyl acetate 1.274 (±0.391) 4

Hexyl acetate 1.806 (±0.391) 1

(E)-2-Hexenyl acetate 0.625 (±0.391) 8

Control

Empty tube 0.465 (±0.552) 9

Table A.1 Field test results for individual green leaf volatiles: group name, compound name, mean number of flies captured, and ranking.

*There were no statistically significant differences in capture rate between any test lures, and the male to female sex ratio was 2.1 :1. 97

A.5 References

Buttery, R G.; Flath, R A.; Mon, T. R; Ling, L. C. Identification of Germacrene D in walnut and fig leaf volatiles. Journal ofAgricultural and Food Chemistry 1986, 34, 820-822.

Visser, J. H. Electroantennogram responses of the Colorado beetle, Leptinotarsa decemlineata to plant volatiles. Entomologia Experimentalis et applicata 1979,25,86-97.