PHEROMONE-MEDIATED BEHAVIOR of ADULT DOUGLAS-FIR TUSSOCK MOTHS, ORGYIA PSEUDOTSUGATA (Mcd.)

AN ABSTRACT OF THE THESIS OF

JAMES ARCHIE SWABYfor the degree of DOCTOR OF PHILOSOPHY

in ENTOMOLOGY, FORESTpresented on December 15, 1978

Title: PHEROMONE-MEDIATED BEHAVIOR OF ADULT DOUGLAS-FIR TUSSOCK MOTHS, ORGYIA PSEUDOTSUGATA (McD.)

(LEPIDOPTERA: LYMANTRIIDAE) Redacted for Privacy Abstract approved: / Redacted for Privacy merman

The sinuous flight behavior of male Douglas-fir tussock moths, Orgyia pseudotsugata, (McD.), is characterized by positive anemotaxis and positive chemoklinotaxis and varies depending on distance from the pheromone source, population density, and time of day. Visual cues play a minor role in close-range orientation to a pheromonesource, but males do respond to hdat energy equivalent to the internal body temperature of the females. A female may be simultaneously courted by two or more males, and a male may remain by the female after mating. Males and females can mate more than once, and there appears to be a minimum time- span which must elapse between consecutive matings by the same male. This probably limits males to one mating per evening. Egg viability is the same for single and multiple matings. Mated females wait for a period of time before laying their eggs and may interrupt oviposition to mate again. An unmated female may lay a portion of her eggs before mating or complete oviposition without copulating.

Comparisons of average elapsed courtship, copulation, pre- oviposition (the period between copulation and egg laying) and oviposition times for laboratory-reared and wild New

Mexico moths revealed that pre-oviposition is longer in laboratory moths; but courtship, copulation, and oviposition are the same.

Under the prevailing field study conditions, females lived up to seven days but their ability to attract males dropped after three days and the probability that they would mate dropped after two days. Attractancy of male flight is also influenced by temperature and the inhibitory effect of rainy weather. The decline in attractancy is due to a reduction in the amount of pheromone present and a loss of the ability to release the pheromone. Furthermore, at least 20% of the unmated females were not attractive. Pheromone-Mediated Behavior of Adult Douglas-fir Tussock Moths, Orgyia pseudotsugata (McD.) (Lepidoptera: Lymantriidae)

James Archie Swaby

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Completed December 1978

Commencement June 1979 APPROVED:

Redacted for Privacy

Professor of Entomology in charge of major

Redacted for Privacy

Resee5- En6mologist in charge of major

Redacted for Privacy

Chairman of Department of tomology

Redacted for Privacy

Dean of Graiduate School

Date thesis is presented December 15, 1978

Typed by Deanna L. Cramer for James Archie Swaby ACKNOWLEDGEMENTS

I am gratefully indebted to the many individuals who contributed advice and assistance during the course of this study. Appreciation is extended to Dr. Julius A. Rudinsky for accepting me into a graduate study program under his guidance and for the helpful comments he offered on writing the thesis. I am especially grateful to Dr. Gary E. Daterman whose support, guidance,and encouragement surpassed that required to fulfill his role as thesis director.

Special thanks are due Dr. Roger G. Petersen for serving as my minor professor and for providing valuable assistance in the statistical analysis. Thanks are also due Dr. David Faulkenberry for taking over the minor professor duties when Dr. Petersen left on sabbatical. I would also like to thank the other members of my graduate committee: Dr. D.

Michael Burgett, Dr. Lonne L. Sower, and Dr. Donald B.

Zabel who offered many useful comments and advice during the course of the study. Dr. Roy C. Beckwith, Dr. Burgett, Dr. Daterman, Dr. Rudinsky, and Dr. Sower are thanked for providing equipment. Finally, and most of all, I wish to express my sincere appreciation to my loving wife, Mary Ann, without whose sustaining encouragement, enduring patience, and personal sacrifice this would not have been possible.

Support for this research was provided by the United

States Department of Agriculture, Forest Service, Cooperative Research Agreement 16 USC 581; 581a-581i, Supplement Number 132. TABLE OF CONTENTS

Chapter a e

I INTRODUCTION 1 Research Objectives 2

II LITERATURE REVIEW 3 Taxonomy 3 History of Outbreaks 4 Biology and Life History 5 Sex Pheromones of Lepidoptera 9 Pheromone-Mediated Behavior in Insects. 12 Factors Affecting Pheromone-Mediated Behavior 23

III ORIENTATION OF MALES TO THE PHEROMONE SOURCE 31 Methods and Materials 31

Long-Distance Flight Orientation . 31 Short-Distance Flight Orientation. 33 Response to Color 35 Response to Different Shapes and Sizes 36 Response to Radiant Heat Energy 38 Measurement of Female Body

Temperature ...... 39 Response to Female Models plus Synthetic Pheromone, Synthe- tic Pheromone Alone and Live Females 40 Results and Discussion 42 Long-Distance Flight Orientation . 42 Short-Distance Flight Orientation. 45 Response to Color 45 Response to Different Shapes and Sizes 47 Response to Female Models plus Synthetic Pheromone, Synthe- tic Pheromone Alone and Live Females 47 Measurement of Female Body Temperature and Male Res- ponse to Radiant Heat Energy 51 Conclusions 52

IV MATING BEHAVIOR 54 Methods and Materials 54 Results and Discussion 55 Courtship Behavior 55 Table of Contents -- continued

Chapter Page

Copulatory Behavior 60 Pre-oviposition Behavior 62 Oviposition Behavior 62 Conclusions 65

V FACTORS AFFECTING PHEROMONE-MEDIATED BEHAVIOR 66 Methods and Materials 66 Results and Discussion 69 Conclusions 76

VI SUMMARY 78

REFERENCES CITED 82 LIST OF FIGURES

Figure Page

1 Photographs of a Douglas-fir tussock moth; (A) egg mass,(B) 1st instar larva,(C) 5th instar larva,(D) pupae,(E) female, and (F) male 6

2 The Douglas-fir tussock moth sex pheromone (Z)-6-heneicosene-11-one 8

3 Photograph of the Los Alamos field site 32

4 Drawings of the flightways used in (A) the mating behavior study and (B) all other studies 34

5 Photographs of (A) the triangular shaped traps used to test male response to different colors; (B) circular shaped traps used to test male response to size, shape and (C) heat, plus (D) the heating coil and (E) placement of the coil in the trap, and the (F) circular trap design used to

test male responses to different extracts. . . . 37

6 Photographs of the three types of trap baits used during the Los Alamos field studies: (A) female models plus synthetic pheromone, (B) synthetic pheromone alone, and (C) live females; and photographs of: (D) the triangular shape of a closed trap,(E) the placement of traps at the field site, and (F) a female used to study mating success of different age females 41

7 The sinuous flight pattern of male Douglas- fir tussock moths 43

8 Drawing of the reproductive organs of the female Douglas-fir tussock moth 57

9 Photographs of(A) an unfertilized egg mass,(B) a fertilized egg mass, and (C) an unfertilized egg mass that appears to be fertilized 64

10 Graphs of (A) the mean number of males caught per day in synthetic pheromone baited traps and (B) the number of hours of rain per day. . . 70 List of Figures -- continued

Figure Page

11 Graphs of the results of field studies on (A) the life expectancy and (B) the attractive life-span of unmated adult Douglas-fir tussock moth females, as well as (C) the effect of female age on mating success 71

12 Graph of the results of a laboratory study comparing extracts (hexane washes) of unmated Douglas-fir tussock moths reared under different temperature schedules 73

13 Graph of the results of a laboratory study comparing extracts obtained by soaking or washing the abdominal tips of unmated Douglas-fir tussock moth females in hexane 75

14 Graph of the results of the field study on the tendency of Douglas-fir tussock

moth females to lay unfertilized eggs . . . . 77 LIST OF TABLES

Table a e

1 Summary of the results of the study on the response of pheromone-stimulated male Douglas- fir tussock moths, Orgyia pseudotsugata

(McD.), to 21 combinations of seven colors. . . . 46

2 Summary of the results of the study on the response of pheromone-stimulated male Douglas- fir tussock moths, Orgyia pseudotsugata (McD.), to different sized or shaped visual models 48

3 Summary of the results of the study comparing the response of male Douglas-fir tussock moths, Orgyia pseudotsugata (McD.), to traps baited with synthetic pheromone plus a female model, synthetic pheromone alone, or a live female less than one day old 49

4 Comparison of elapsed times for different activities by singly mated laboratory reared and wild New Mexico Douglas-fir tussock moths, Orgyia pseudotsugata (McD ) 59 PHEROMONE-MEDIATED BEHAVIOR OF ADULT DOUGLAS-FIR TUSSOCK MOTHS, ORGYIA PSEUDOTSUGATA (McD.) (LEPIDOPTERA: LYMANTRIIDAE)

I. INTRODUCTION

The Douglas-fir tussock moth, Orgyia pseudotsugata

(McD.), is a serious defoliator of Douglas-fir, grand fir and white fir in western North America. Defoliation by the larvae either kills the tree, causes top kill or retards tree growth. The damaged trees may be so weakened that secondary insects kill them or cause top kill. Outbreaks normally last 1-4 years and are periodic, occurring at 8-15 year intervals. During outbreaks the caterpillars are easily found, but between high population periods the insect is virtually undetectable (Wickman et al.,1973a;

1973b). The recent identification and synthesis of the Douglas-fir tussock moth pheromone, (Z)-6-heneicosen-11- one, (Smith et al.,1975) has stimulated interest in its use in surveillance traps for detection and evaluation surveys as well as a control measure (Daterman and Sower, 1977;

Sower and Daterman, 1977). However, concern is being ex- pressed that progress in pheromone identification, synthesis, and application has greatly surpassed our basic understanding of the behavior and factors affecting the activities of insects responding to or releasing a pheromone

(Bartell, 1977; Doane, 1976; Koehler et al., 1977; 2 Wellington, 1976). Those concerns have prompted this study on the pheromone-mediated behavior of adult Douglas-fir tussock moths.

Research Objectives

The study objectives were:

1. Describe the pheromone-mediated behavior of adult

Doublas-fir tussock moths.

2. Identify major factors influencing the attractiveness

of female Douglas-fir tussock moths.

3. Identify major factors influencing the response of

male Douglas-fir tussock moths to the pheromone. 3

I/. LITERATURE REVIEW

The publication "Douglas-fir tussock moth, an annotated bibliography" by Campbell and Youngs (1977) cites 277 articles dealing directly or indirectly with the moth.

However, only three of those articles are on behavior and none of them deals extensively with the pheromone-mediated behavior. Nine additional articles discuss the use of the pheromone as a survey and control tool. The remaining articles deal with life history, biology, taxonomy, host relationships, outbreaks, control, and related material.

Taxonomy

The Douglas-fir tussock moth was first reported at

Summerdale (now Fishcamp), Mariposa County, California in

August 1906, by H.E. Burke (Eaten and Struble, 1957). At that time the insect was identified as Hemerocampa (Notolo- phus) oslari (Barnes). In 1916 W.B. Anderson reported finding the moth at Chase, B.C. (Anderson, 1919). Black- more (1919) identified Anderson's moths as Hemerocampa vetusta golosa Hy. Edw. Later, McDunnough (1921) classified both Anderson's and Burke's specimens as being a new species. He based his classification on the color of the larval tufts and the host plant preference.McDunnough then named the moth Hemerocampa pseudotsugata McD. Since then the genus was changed (based on the presence of one or 4 two pairs of spurs on the hind tibia) to Orgyia (Riotte, 1973) to make the accepted name Orgyia pseudotsugata (McD.).

Most recently, Ferguson (1978) has divided the species into

the three subspecies Orgyia pseudotsugata pseudotsugata

(McDunnough), Orgyia pseudotsugata morosa Ferguson, and

Orgyia pseudotsugata benigna Ferguson. All three sub-

species are considered collectively as the Douglas-fir tus-

sock moth in the remainder of this literature review; how-

ever, in all other sections of this thesis the name

Douglas-fir tussock moth refers specifically to the sub-

species Orgyia pseudotsugata benigna.

History of Outbreaks

As mentioned, outbreaks are cyclic, occurring every 8-

15 years. The history of 124 such outbreaks between 1916

and 1973 are reviewed by Buckhorn (1948), Sugden (1957),

Tunnock (1973), and Wickman et al. (1973a). The articles by Tunnock (1973) and Wickman et al. (1973a) also discuss

the outbreak pattern. There are three phases: release,

outbreak, and decline. The release phase lasts one year during which the larval population may multiply five to ten

times or more, thus increasing to outbreak levels. Several

years of inconspicuous buildup are probably required to

reach levels where such quick release can occur. The

second or outbreak phase is the period of most conspicuous

defoliation. Some outbreaks collapse at the end of this 5 second year, but frequently there isa large population increase in the third year. It is in the third year that the population declines. The decline is due to parasitism

and the nucleopolyhedrosis virus; the virus beingthe major

factor. Tree mortality is heavy the year aftersevere

defoliation and continues for an additionalyear after the population collapses. Most of the tree mortality is aided by bark beetles (Wickman, 1958).

Biology and Life History

Balch (1932) provided the first comprehensive review

of the biology and life history. That review was updated

by Wickman et al. (1973a). Edwards (1965) was the first to conduct laboratory studies on the activity rhythms of all

stages in the life cycle. Edward's study was followed by Manson's (1967) laboratory and field investigations of

larval behavior, then by Wickman's et al.(1975) work on the flight, attraction, and mating behavior.

In summary, the Douglas-fir tussock moth produces one

generation per year. The larvae hatch from eggs (Fig. 1A) in late spring or early summer after the host trees have begun their new growth. The tiny 3..2 mm gray caterpillars

are covered with long hairs that facilitate aerial disper-

son (Fig. 1B). Since the adult female (Fig. 1E) is wing-

less, aerial transport of the first and second instar

larvae is the major mode of population dispersal. The A B

C D

E F Figure 1. Photographs of a Douglas-fir tussock moth; (A) egg mass, (B) 1st instar larva, (C) 5th instar larva,(D) pupae, (E) female, and (F) male. 7 larvae go through five to seven instars with the males normally having five compared to six for the females. The early instars only feed on new foliage while later instars

(Fig. 1C) consume both new and old foliage. The first three instars eat the underside of the needle, leaving over half of the tissue. Later instars consume the entire needle. The upper third of the crown is defoliated first and the larvae deposit a loose silken web as they move from branch to branch. In heavy infestations, the brown tips, bare twigs, and dried needles are caught in the webbing, giving the tree a brown dead appearance. During a heavy infestation, the trees are completely defoliated and the caterpillars drop to the ground. They then crawl in search of food. They climb the first tree they come to regardless of whether the tree has been defoliated or not.

The Douglas-fir tussock moth prefers Douglas fir, Pseudot- suga menziesii (Mirb.) Franco; grand fir, Abies grandis

(Dougl.) Lindl.; and white fir, Abies concolor, (Gord. and Glend.) Lindl.; but they will feed on a variety of other conifers when their preferred hosts are depleted. The Douglas-fir tussock moth begins pupating from late

July to the end of August. Pupation occurs inside a thin cocoon of silken webbing mixed with larvalhairs (Fig. ID).

These cocoons are usually scattered on the foliage and twigs on the upper part of the tree when the infestation is 8 light. But cocoons are also found on the lower portions when there is a heavy infestation.

The pupal stage lasts a minimum of 10 days, depending on temperature. Upon emerging, the wingless female (Fig. 1E) clings to her cocoon and rhythmically telescopes the terminal segments of her abdomen in and out. This behavior, termed "calling" is believed to facilitate the release of the pheromone by exposing the pheromone gland. The pheromone, Z- 6- heneicosen -l1 -one (Fig. 2), acts as a sex attractant, stimulating the male (Fig. 1F) to fly to the female.

CH3(CH (C 112)3C (C 112)9CH3 /C-C H 'H

Figure 2. The Douglas-fir tussock moth sex pheromone (Z)-6- heneicosene-11-one.

Male flight begins in the morning and extends into the even-

ing with peak flight activity between 1600 to 1700 hours

depending on environmental conditions. Peak mating activity occurs simultaneously with peak flight activity and mating

extends into the evening as males remain in attendance to 9

the females. After mating, the female laysa few to 350 eggs on her cocoon in a single mass of frothy, gelatinous

material containing many hairs from her body. This material

hardens to form a single mass ofeggs which overwinter. An unmated female may also lay unfertilizedeggs; a habit known as "spewing". Spewing is common among Lymantriids (Doane,

1968).

Sex Pheromones of Lepidoptera

The term pheromone was introduced by Karlson and

Liischer (1959). It was derived from the Greek pherein (to

transfer) and hormone (to excite). Pheromones are chemicals, either odors or taste substances, that are emitted by organisms into the environment where they functionas a mes-

sage to others of the same species (Shorey, 1976). As such, they are a subgroup of semiochemicals which are chemicals that may function as messages between organisms (Law and

Regnier, 1971). The subject of pheromones is extensively reviewed by Beroza (1970), Birch (1974), Jacobson (1972),

Shorey (1976), Shorey and McKelvey (1977), and Wood et al,

(1970).

Pheromones are divided into two major groups: releasers and primers. Releasers appear to act directly on the central nervous system to evoke an immediate but reversible change in behavior. Primers, on the other hand, trigger permanent physiological changes in the receiver (Wilson, 10 1963). A more recent classification divides pheromones into separate categories based on biological function

(Butler, 1967; 1970) or behavioral response (Shorey, 1973).

When classified on behavioral response insect pheromones release aggregation, trail-following, dispersion, sexual behavior, oviposition, alarm, and specialized behavior in the regulation of social insect colonies (Blum, 1969;

Jacobson, 1972; Shorey, 1973). The Douglas-fir tussock moth pheromone can be classified as a releaser that functions as a sex attractant, stimulating the male to fly to the female. As such, it is a sex pheromone. Sex pheromones are chemicals secreted by organisms of one sex that cause behavioral reactions in the opposite sex that facilitate mating (Shorey, 1976).

The phenomenon of olfactory attraction of insects to their mates has been observed for more than a century but until almost a decade ago behavioral studies were limited because the identity of the chemicals involved was unknown

(Roelofs, 1977). However, today we know the identity of over 100 active compounds affecting insect behavior,.most of which are either pheromones or allomones1/ (Wood, 1977).

Sex pheromones make up the largest portion of these identified compounds and probably half of the pheromone literature

1iFollowing Shorey (1976), "An allomone is a chemical or a mixture of chemicals that is released from one organism and that induces a response by an individual of another species; the response is adaptively favorable to the emitter." 11 deals with chemicals released by female moths which stimulate males to approach the females prior to mating (Shorey,

1976). Thirty different compounds have been identified from 50 different species of Lepidoptera in nine families.

Most of these compounds are unbranched chains of 12 to 21 carbon atoms having one or two points of unsaturation anda terminal functional group. Acetates comprise 60% of the 30 identified sex pheromones but epoxides, aldehydes, alcohols, isovalerates, and ketones also occur. The molecular weights range from 182 to 308, averaging 246.3. The double bonds are distributed from the third to the 13th carbon, but are most frequently on carbons 7, 9, and 11. Sixty percent of the double bonds are in the cis configuration. Ninety-three percent of the chains are 14, 16, or 18 carbons long

(Daterman, 1978; Shorey, 1976; Tamaki, 1977). The smallest is dodecadienol of the codling moth (Beroza et al., 1974;

Roloefs __et al., 1971) and the largest is (Z)-6-heneicosen- 11-one of the Douglas-fir tussock moth. The tussock moth pheromone is a 21 carbon ketone with the carbonyl on carbon eleven and a double bond at carbon six in a cis configuration (Fig. 2)(Smith et' al., 1975).

The number of identified sex pheromones is deceptive.

Recent studies indicate that a particular sex pheromone can be used by more than one species, especially in Lepidoptera

(Tamaki, 1977). Those findings indicate that the specificity of single compounds is low, and that more than one 12 compound might be involved. Presently, multiple component sex pheromones have been found in 15 lepidopterous species

(Tamaki, 1977). The synthetic sex pheromone of the Douglas- fir tussock moth is known to attract several other species

(Daterman et al., 1977). The multiple attractiveness along

with the fact that female baited traps capturemore males than synthetic pheromone baited traps have led Daterman

et al.(1976) to suggest that an additional, unidentified

compound is present in the natural sex pheromone of the

Douglas-fir tussock moth.

Pheromone-Mediated Behavior in Insects

Pheromone-mediated behavior in insects is reviewed by

Birch (1974), Shorey (1976), and Shorey and McKelvey (1977). More specifically, Bartell (1977) provides a review article on pheromone-mediated behavior in Lepidoptera. These publi- cations emphasize the remarkably diverse array of behaviors mediated by sex pheromones, either acting directly or indirectly to enhance the likelihood that mating will occur;

indirectly by stimulating the approach of the opposite sex and directly by stimulating courtship or copulatory responses. Furthermore, a single sex pheromone may stimulate more than one type of behavioral reaction in an insect.

These reactions frequently occur in a sequence or hierarchy of steps starting with activation of the insect, followed by orientation to the pheromone source, and culminating in 13 courtship and copulation (August, 1971;Bartell, 1977;

Bartell and Shorey, 1969b; Daterman, 1972;Doane, 1968; Shorey, 1964; 1973).

The first step, activation, is characterizedby "rest- lessness", wing vibration, elevation ofantennae, and groom- ing of antennae. A large number of species have been observed displaying these behaviors, primarily inthe laboratory, but it is doubtful that such activities playan important role in pheromone communication under natural conditions since the males are usually already flying whenthe females begin releasing their pheromone (Bartell, 1977).

The orientation of an insect to an odorsource is based on a complex medley of behavioral mechanisms that may operate separately or together, depending on the species and a number of physiological and environmental variables

(see factors affecting pheromone-mediated behavior,page

23)(Shorey, 1976). Furthermore, the behavioral mechanisms utilized for orientation to a distant odorsource differ from those employed at close range. The mechanisms for both long-distance and close-range orientation will becon- sidered after a review of our present knowledge concerning how insects respond.

An insect can respond to a stimulus, including a sex pheromone, in a number of ways. The initiation of movement may be nondirectional in nature, termed kinesis, or direc- tional, termed taxis. Kinesis reactions can be subdivided 14 into those in which the response is a change in the amount or speed of linear movement; starting, stopping, slowing down, or speeding up. This is termed orthokinesis as opposed to klinokinesis where the response is a change in the rate or frequency of nondirectional turning (Kennedy,

1977; Klopfer and Hailman, 1967).

Both orthokinesis and taxis are involved in orientation to a pheromone source. Pheromone stimulation may con trol the rapidity of locomotion. Furthermore, as the insect nears or arrives at the source, the rapidity may become inversely proportional to concentration resulting in arrestment of movement. Taxis, on the other hand, facili- tates orientation to the source and when coupled to orthokinesis, enables the insect to move towards the pheromone source (Shorey, 1976).

In flying insects, the mechanism of orientation is influenced by the characteristics of the air that serves as the medium of transport for the pheromone. Aerial dispersion of a chemical can occur by diffusion. Assuming that air is stationary, it is possible to conceive of a steep concentration gradient emanating from a pheromone source which would allow an insect to orient chemotactically to the source by the simultaneous comparison of the differing odor concentrations at two points in space. Any subsequent oriented turning would be termed chemotropo- taxis. But air is rarely static. Prevailing winds 15 cause air to flow and even in the absence of wind, air moves due to convective currents generated by the uneven heating of surfaces. Furthermore, except at very low velocities, air-flow is rarely laminar. Thus air is moving, and as it moves over an odor source, an elongate cloud or plume or odor molecules, termed an "odor-trail"

(Butler, 1970), is produced. This trail is not uniform and linear, but rather disrupted and filamentous. The plume consists of pulses of pheromone moving in the direction of the prevailing air-flow and laterally displaced by turbu- lence. The net result is that diffusion is of negligible importance in establishing pheromone gradients and chemo- tropotactic orientation is unlikely at distances greater than a few centimeters from the source. However, we do know that insects have chemotactic behavioral mechanisms enabling them to orient along these trails to locate a distant source (Morey, 1976).

A mere plausible mechanism for long-distance chemotactic orientation is termed chemoklinotaxis. This mechanism inVolves the movement of the entire body or part of the body much as the head or antennae in order to scan across the odor field. The insect then orients its body towards the area of highest oder concentration (Morey, 1976). The argument in favor of the chemoklinotactic mechanism is based on the similarity in the behavior of insects orienting to a pheromone source on land and in the air.Both are 16 characterized by a sequence of zigzagging turns. Observa- tion of this type of behavior has led to the hypothesis that both terrestrial and aerial insects sense when they are diverging laterally from the highest average pheromone concentration near the center of the plume whereupon they turn back towards the central axis. Such a chemotactic mechanism might function to continually re-orient the insects towards the central-axis of the plume. Shorey (1976) argues that it is difficult to understand how chemotropo- taxis could enable the observed lateral orientation when the organism is a considerable distance from the source since the odor-trail would be quite wide. However, it is likely that some form of chemoklinotaxis could operate to facilitate such lateral orientation.

Another chemotactic mechanism was proposed by Wright

(1958). This hypothesis takes into account the disrupted, filamentous nature of the plume. He proposed that an animal orienting to an odor source is guided by the frequency with which it encounters high density pulses of odor molecules. The animal turns in towards the central axis when the frequency of encounters is decreasing, but when they are increasing, he does not turn. It is generally agreed that Wright's hypothesis is plausible but needs to be tested (Shorey, 1976).

Limited experimental evidence supports the hypothesis that a chemotactic mechanism or a modified chemotactic 17 mechanism based on pulses is utilized for aerialtrail fol-

lowing (Farkas and Shorey, 1972; Shorey and Farkas,1973), but most authorities agree thatan insect cannot detect the direction of an odor source many metersor kilometers away by sensing a concentration gradient of molecules from the source. The shallowness and turbulent nature of the odor trail at such distances would be prohibitive. However, the insect might be stimulated to orient its body into the wind carrying the odor (Farkas and Shorey, 1974; Kennedy and

March, 1974; Schwinck, 1958; Shorey, 1973). This type of oriented movement is termed anemotaxis.

It is believed that flying insects sense the direction of their movement with regard to the wind by visual contact with the substratum. Thus if an insect sees that he is moving sideways with respect to the ground beneath him he senses that the wind is to his side and makes corrective movements to bring his body in line with the prevailing air-flow. He then sees that the terrain is moving front to back; therefore, he senses that he is flying into the wind

(Shorey, 1976). This visually based anemotaxis has been demonstrated in males of two moth species approaching sources of female sex pheromone (Kennedy and Marsh, 1974).

A flying insect can be expected to lose contact with an aerial odor-trail since the trail is so disjointed. The insect then might cease his upwind orientation and make back and forth crosswind maneuvers in a manner which 18

maximizes the likelihood of re-entering the trail. Such crosswind movements could also be visually coordinated

(Shorey, 1976). Such activity has been demonstrated in

several moth species when the males lost contact with the

odor-trail (Kennedy and Marsh, 1974; Traynier, 1968).

Olfactory orientation to a distant odor source probably involves both anemotaxis and chemotaxis operating

simultaneously with visual cues to enable the animal to

follow a disrupted, filamentous odor-trail to the pheromone

source. The role of chemotaxis, probably by chemoklino-

taxis, is to maintain the insect within the activespace?/

and the role of visually coordinated anemotaxis is to pro-

vide polarity (Shorey, 1976).

Prior to the proposal of anemotactic and chemotactic orientation mechanisms, investigators of moth behavior,

including Fabre (Teale, 1961), could not understand how an

olfactory system operated to enable males to approach dis-

tant odor sources. That uncertainty led to proposals of other mechanisms of distant communication, especially systems based on responses to radiant energy (Callahan, 1965;

1966), but there is presently no demonstrated evidence that

any message other than pheromone molecules emitted by

females is effective over long distances (Diesondorf et al.,

2 /The active space is the area within which the concentration of the odor stimulus is above the threshold for the appropriate behavioral response in the receiving organism (Bossert and Wilson, 1963). 19 1974). However, short-range responses to infrared radia-

tion have been demonstrated by Callahan (1966) inthe families Noctuidae, Sphingidae, and Lasiocampidae.

Close-range olfactory mechanisms may be coupled with responses to various portions of the electromagnetic

spectrum, including the infrared and visual portions. The

result being a medley of mechanisms which includeresponses to radiant energy emanating from the pheromone-emitting

female, visual orientation to nearby surfacesor to the

image of the female plus short-range anemotacticor chemo-

tactic orientation and arrestment of locomotion (Shorey,

1976).

Visual orientation to nearby objects has been observed

in a number of insect species. Male pink bollworm moths hover adjacent to vertical objects when stimulated by high concentrations of the female sex pheromone, presumably maximizing the likelihood that they will locate a female on a plant (Farkas et al., 1974).The high concentration of pheromone near the source also plays a role in visual orientation directly to the female image at close-range

(probably within a few centimeters of the releasing female)

(Daterman, 1972; Doane, 1968; Hidaka, 1972; Shorey and

Gaston, 1970; Traynier, 1968). This direct visual orientation is also influenced by the specificity of pheromones, since males of some species apparently are not able to visually detect the presence of a nearby female unless 20 stimulated by the appropriate odor (Shorey, 1976).

The high concentration of pheromone near the source also stimulates a mechanism which functions to slow down and eventually inhibit locomotion in flying insects. This mechanism involves a decrease in the rate of forward progress in proportion to the increasing concentration

(Bennett and Borden, 1971; Farkas and Shorey, 1974; Farkas et al., 1974; Traynier, 1968). Subsequent arrestment or inhibition of flight may occur once the male arrives at the source (Borden, 1967; Gara et al., 1965; Rudinsky, 1973;

Vita et al., 1964).

Chemotactic gradient following may also operate at distances less than a few centimeters from the pheromone source to steer the insect in the right direction. This orientation is promoted by wing fluttering and side to side turning while hovering or clinging to the substratum. The turning activity is considered to be advantageous since it allows the insect to sample the concentration of pheromone in many directions. Fluttering is viewed as being equivalent to the sniffing behavior of mammals in that it probably aids in detection of odor gradients by causing air to flow over the antennae (Shorey, 1976). Wing fluttering promotes close-range orientation another way in species with winged females. Fluttering by females, clinging to a surface and releasing a pheromone, sets up local air currents of up to

15 cm/sec a meter behind the female (Kaae and Shorey, 1972; 21 Shorey, 1973). Such an air-flow might enable the males to orient anemotactically to the releasing female.

The increasing pheromone concentration or thesecre- tion of additional components stimulates close-range behavioral patterns which promote two-way communication between the sexes once they have come together (Carde et al.,

1975). This type of close-range behavior varies greatly from species to species and includes one or more of the following: (1) attendance of the male in the vicinity of the female until she is ready to mate,(2) courtship behavior, (3) assumption or maintenance of the proper copulatory stance by the female, and (4) male copulatory behavior

(Shorey, 1976). As mentioned earlier, the sex pheromone may function to arrest locomotion in males but it may also stimulate a male to remain in attendance to a female until she is ready to mate. For example, certain female mosquitoes and mites release pheromones while they are still sexually immature.

These chemicals stimulate the males to remain by the female until she molts to the sexual stage (Shorey, 1976).

The sexual maturity of both the male and female may be established prior to their meeting, but this doesn't mean copulation will occur automatically. Often, a further ex- change of signals, referred to as courtship stimuli, is required to reduce the threshold for copulation (Shorey,

1976). Pheromones termed aphrodisiacs play a role in the 22 courtship behavior of male moths. The males of a number of species of moths have specialized structures referred to as scent brushes or wing pouches. These structures are associated with the production and release of the aphrodisiacs. The males display various behavioral patterns which allow them to apply the scent brushes to the female genitalia and antennae or expose her to the wing pouch secretions (Birch, 1970; Grant, 1970; 1974). In many species of noctuids there appears to be no overt female response to contact with scent brushes. In other families (Pyralidae,

Phycitiidae, Trotricidae, etc.) with wing pouches, the female responds by performing activities which permit the male to make genital contact (Bartell, 1977).

Unlike male moths, male butterflies do not orient along an aerial odor-trail to locate a distant female. Instead, the male sees a flying female and pursues her. He approaches and overtakes her. Then,while flying slightly ahead and above her, he employs his scent brushes to dis- tribute scent particles on her antennae. The female responds by landing (Brower et al., 1965; Brower and Jones,

1965; Myers and Brown, 1969; Pliske and Eisner, 1969). Female released sex pheromones may also directly stimulate the sterotyped behaviors exhibited by males during copulation (Shorey, 1976). Shorey (1964) has shown that the direction of copulatory attempts is oriented chemotactically in the cabbage looper moth. Furthermore, 23 indirect insemination can also be mediated by pheromones. For example, the males of the Collembolan species Sinella cuaviseta are stimulated to deposit their spermatophores in areas containing the female sex pheromone (Waldorf, 1974).

The females then pick up the spermatophores and deposit them in their genital tracts.

Factors Affecting Pheromone-Mediated Behavior

Mating behavior and the release of sex pheromones generally occur during the same time periods and under the same conditions (Shorey, 1974). Both environmental and physiological variables interact to promote sex pheromone communication when conditions are favorable for mating.

The influence of these variables on sex pheromone communication is reviewed by Shorey (1976).

The most studied environmental variables; light intensity, temperature, air velocity, and surrounding vegetation operate directly to control the occurrence and timing of sex pheromone behavior. The same variables function to inhibit or enhance the probability of mating by directly influencing both the male and female. Furthermore, light intensity and temperature also function indirectly on a daily or seasonal basis to modify the internal physiological clock that programs the timing of daily and seasonal pheromone behavior. This clock is probably entrained by the light/dark cycle, and functions to promote mating 24 during the day in some insects and at night, dusk, or dawn in others. In addition, the appropriate level of light

intensity may be important. Levels too high or too low may inhibit sex pheromone communication (Bartell and Shorey,

1969a; Carde and Roelofs, 1973; Shorey and Gaston, 1964;

Sower et al., 1970). Similarly, high and low temperature limits may restrict male response to the sex pheromone

(Aliniazee and Stafford, 1971; Batiste, 1970; Batiste et al., 1973a; 1973b; Collins and Potts, 1932; Karandinos,

1974). Presumably, the tendency is for the female to release her pheromone at temperatures favorable to male response (Sower et al., 1971b). The favorable temperature range varies according to the activity rhythm of the species; i.e., being higher for day active forms. Fluc- tuating temperatures may also be important. Carde and

Roelofs (1973) reported that decreasing temperatures interact with decreasing light intensity to stimulate a female

Holomelina immaculate moth to release her pheromone.

Pheromone communication in flying insects is affected by the prevailing wind speed. Sower et al. (1973c) point out that successful pheromone communication requires not only that the pheromone be transported by moving air to the vicinity of the responder, but also that the responder move to the emitter while uninterrupted release of the pheromone takes place. However, except for certain exceptions, the release of the pheromone occurs during brief periods, and 25 air velocity influences the length of these periods. Female cabbage looper moths for example, sense the prevailing wind velocities and adjust the duration of pheromone release accordingly (Kaae and Shorey, 1972). The length of con- tinuous emission is decreased from 20 minutes at low velocities (<0.1 m/sec) to 5 minutes at a velocity of 3 m/sec.

This reduction in the duration of pheromone release appears to be adaptive since it increases the potential distance of communication at low velocities, when the pheromone is transported more slowly downwind from the female. In addition many observations indicate that the ability of males to approach a releasing female is greatly impaired at low air velocities and velocities higher than the flight speed of the insect obviously prevent upwind orientation to a pheromone source (Casida et al., 1963; Farkas and Shorey, 1974; Kaae and Shorey, 1972; Karandinos, 1974; Soohoo and

Roberts, 1965; Sower et al., 1971a; 1973c). Thus there are practical restraints on successful communication distance, imposed by interactions between the wind velocity, duration of pheromone release, and male flight speed. These con- straints were used by Sower et al. (1973c) to devise the following formula for calculating the estimated maximum possible distance for successful communication:

X = vt(r-v)/r where X = communication distance (cm), t = duration of 26 uninterrupted release of pheromone (sec),r = approach speed of the males (cm/sec), andv = wind velocity (cm/ sec).

Pheromone communication may also be restricted bythe surrounding vegetation, particularly in monophagousspecies. The insect may limit pheromone communication to periods when they are in close proximity to their host plant

(Aliniazee, 1972; Sharma et al., 1971; Teetes and Randolph,

1970). This behavior may be due to: (1) the female limiting her release of the pheromone to when shesenses that she is on the proper host (Riddiford, 1967; Riddiford and

Williams, 1967a; 1967b);(2) the male not responding to the pheromone unless he senses he is in the right habitat;or

(3) the male and female being in the correct habitatany- way, having developed there or having been attracted to the area by plant-produced stimuli prior to reaching the stage where pheromone communication is employed. Restricting communication this way ensures that mating will occur in a vegetative habitat favorable to the survival of the species. Vegetation may also influence the vertical location of pheromone communication. It is generally assumed that the releasing females cling to foliage in the upper canopy. That assumption is based on the fact that males typically respond best to synthetic pheromone sources located in the top of the canopy (Aliniazee and Stafford,

1971; Kaae and Shorey, 1973; Miller and McDougall, 1973; 27 Saario et al., 1970; Sharma et al., 1971).

Interacting with the environmental variables just discussed, are various physiological variables. The interaction of these variables regulates when, where, and whether

sex pheromone communication and mating will occur. The physiological variables include the age of the insect, the internal clock that indicates the time of day or season, prior exposure to pheromone, prior mating and hormones

(Shorey, 1976).

With few exceptions the age at which an insect can participate in sex pheromone communication, either as a responder or releaser, is restricted to the sexually mature stage. This ensures that sex pheromone communication is synchronized with the physiological systems that regulate pre-copulatory behavior, copulation, and egg fertilization

(Fraenkel and Gunn, 1961; Happ and Wheeler, 1969; Payne et al., 1970; Shorey et al., 1968a; 1968b). In addition, several studies have shown the responsiveness of male

Lepidoptera may increase with age (Sekul and Cox, 1967;

Shorey and Gaston, 1965; Traynier, 1970). Finally, aging frequently accounts for reduced fecundity (Engelmann, 1970) due, in part, to reduced mating success. One of the factors contributing to reduced mating in whitemarked tussock moths was found to be related to decreased sex pheromone production and thus attractiveness (Grant, 1975; Percy et al., 1971). Grant and McCarty (1977) also found that the 28 pheromone releasing mechanism is altered withage in the white-marked tussock moth and is replaced by ovipositing behavior.

As mentioned, the sex pheromone-mediated behavior of insects is usually restricted to a certain time of day.

This daily rhythmicity has been extensively studied in moths where the female releases the sex pheromone and the male responds at a characteristic time that is controlled by a circadian rhythm (Carde and Roelofs, 1973; Fatzinger,

1973; Ohbayashi et al., 1973; Sanders and Lucuik, 1972;

Shorey and Gaston, 1965; Sower et al., 1970; 1971b; Tray- nier, 1970; Vick et al., 1973a). The length of time during each day when the males usually respond overlaps the period during which the females are releasing the pheromone; maximizing the likelihood that the two sexes will be brought together for mating (Fatzinger, 1973; Sower et al., 1971b). The successful culmination of sex pheromone communication (i.e. copulation) might temporarily or permanently reduce the likelihood of further premating behavior. This applies to both the male and female. For example, females of several monogamous insect species produce or release little or no additional sex pheromone once they have successfully mated (Banerjee, 1969; Bartell et al., 1969a;

Collins and Potts, 1932; Wharton and Wharton, 1957). This is in contrast to polygamous species which continue to produce and release pheromone prior to each mating (Shorey, 29 1976). Males on the other hand may have a refractory period following successful mating during which they will not respond to the sex pheromone (Shorey and Gaston, 1964).

Furthermore, even if successful mating does not occur after

the male responds to a sex pheromone he is likely to be

less responsive for some time as a result of the interaction of sensory adaptation and habituation. Olfactory sensory adaptation is defined by Shorey (1976) to be a transitory phenomenon where for a number of seconds or minutes following exposure to a chemical odor, the sensory neurons have an increased threshold for reaction to the same odor and thus a reduced tendency to relay the message to the central nervous system (Boeckh et al., 1965; Payne et al., 1970;

Schneider, 1969). Habituation, on the other hand, is a central nervous system phenomenon that causes an insect to be less responsive to a pheromone stimulus for many minutes or hours following a previous response, even though the previous response did not culminate in mating (Bartell and

Lawrence, 1973; Bartell and Roelofs, 1973; Shorey and

Gaston, 1964; Sower et al., 1973b; Traynier, 1970; Vick et al., 1973b).

Sex pheromone communication is also influenced by hormones. No apparent hormonal control of sex pheromone production is shown by species of short lived insects.

However, even in these species, hormones probably influence pheromone release (Riddiford and Williams, 1971). In other 30 insects, hormones have been shown to regulate pheromone production. The cyclic change in the amount of hormone secreted by the corpora allata of cockroaches, for example, controls a number of aspects of reproductive behavior, including pheromone production during the period when the female is receptive. When the females of certain saturniid moths sense that they are on the correct host or are at the right time in their circadian rhythm, the corpora cardiaca secretes hormones that stimulate the abdominal ganglia.

These ganglia respond by initiating the protrusion of the genitalia and the subsequent release of the sex pheromone

(Barth, 1961; 1962; 1965; 1968; 1970; Barth and Lester,

1973; Bell et al., 1970; Shorey, 1976). 31

III. ORIENTATION OF MALES TO THE PHEROMONE SOURCE

Methods and Materials

Long-Distance Flight Orientation

The long-distance flight behavior of male Douglas-fir

tussock moths was observed for three flightseasons. The

first observations were made from September 4to 18, 1975 near Fort Klamath, Oregon. The remaining flights were observed in New Mexico: first in Trigo Canyon from 11 to

23 August, 1976 then in Los Alamos between July 28and August 20, 1977. The Los Alamos site (Fig. 3) was located in a heavily infested area containinga 30-50 year old mixture composed of 90% white fir, and 10% ponderosa pine,

Pinus ponderosa Laws., at approximately 2,286m elevation.

The Trigo Canyon site was also located ina mixed stand of 60-80 year old and old growth Douglas-fir, white fir, and ponderosa pine at approximately 2,133 m elevation. The mixture was about 10% Douglas-fir and the remainder equally allocated to white fir and ponderosa pine. White fir, 40-60 years old, was the major (over 80%) species at the Fort Klamath site along with ponderosa pine. This site was at about 1,250 m elevation, and lightly infested. In addition to younger fir and pine, there were also scattered old growth fir and pine. 32

Figure 3. Photograph of the Los Alamos field site. 33 Short-Distance Flight Orientation

The short-range orientation was studied in the labora-

tory and field. All laboratory studies were conducted at

the Forestry Sciences Laboratory of the Pacific Northwest Forest and Range Experiment Station, USDA Forest Service,

in Corvallis, Oregon. The laboratory moths were reared using the procedures outlined by Thompson and Peterson

(1978). They were reared from egg masses collected in

Trigo Canyon, Media Dia Canyon, and Los Alamos, New Mexico.

All laboratory experiments were conducted in rooms equipped for temperature and photoperiod control. These rooms were used to house flightways (Fig. 4) designed to permit behavioral experimentation under controlled conditions. The specific design of the flightways used in each study varied but they all consisted of clear plastic tubes (100 cm long and 29 cm in diameter) connected to variable speed exhaust

fans. These fans were attached to screened access doors that fitted against the ends of the tubes to prevent the males from flying into the fans. The opposite ends were fitted with either a removable air-intake screen or boxes

(1 m3) with clear plastic walls and screened air-intake doors. These air-intake doors were screened on the outside and cardboard or screen trap supports of various designs were attached to the inside. These supports held the traps apart at desired distances and against the air-intake 34

Figure 4. Drawings of the flightways used in (A) the mating behavior study and (B) all other studies. AD, access door; AID air-intake door; AIS, air- intake screen, Ex, exhaust hose leading to variable speed exhaust fan; TB, transparent box, TT, transparent tube. 35 screen. The air-flow through the flightwayswas maintained at 6.7 cm/sec.

During the studies on short-range orientation, the

room temperature fluctuated between a mean low of 21°C and high of 24°C. A 14:10 hour light/dark photoperiod (scoto-

phase starting at 1800 hours) was maintained in theroom housing the flightways. Males were held in this room

through pupation and until they were one to two days old at which time 10-15 males were introduced into each flightway via the access doors (Fig. 4B). The males remaining in each flightway from previous introductions were not removed.

These introductions were made daily before noon. This was done in conjunction with the removal, cleaning, and replacement of traps. A randomly chosen pair of traps was placed in each flightway each day to test the response of pheromone-stimulated males to heat and different size, shape, and color stimuli. A 0.5 cm2(1.9 mg) piece of Hercon0wafer containing 0.019 mg (1%) of the synthetic sex pheromone was attached to the air-intake screen behind each trap.

Response to Color. Triangular traps 7.5 cm long and 9 cm wide were used to test the response of males to 21 combinations of seven different colors tested two at a time.

These traps were made of white, black, red, blue, brown, green, and yellow cardboard and lined withTanglefootO. Two different colored traps were fitted through triangular 36 holes in gray cardboard supports mounted on the insides of

the air-intake doors (Fig. 5A). The supports held the

traps 10 cm apart (19 cm between centers). The traps were checked daily to count the number of males caught and then

cleaned. After six days the traps were switched left to

right and tested six more days, bringing the total number of

replicates for each color combination to 12.After 12 days,

the color combination and synthetic baits were changed and another combination tested.

Response to Different Shapes and Sizes. The size and

shape studies utilized circular traps constructed from 946

cm3ice cream cartons with the top and bottom removed. The

cartons were painted black and lined withTanglefootO.

Two of these traps were fitted through circular holes cut

in black cardboard supports attached to the air-intake doors (Fig. 5B). The supports held the traps 10 cm apart

(21 cm between centers). Two black screws were fitted through the air-intake screens in such a way that the threaded ends were centered 2 cm from the trap openings.

Then 1 cm, 2 cm,4 cm, and 6 cm white circles, white triangles, white squares, white ellipses, and female models

alone or on a white ellipse background were attached to the ends of the screws in seven paired combinations. The 2 cm

circles, triangles, ellipses, and squares had the same sur-

face area. The number of males caught in each trap each day was recorded and the traps cleaned. After five days the 37

11V. q IMMO F

Figure 5. Photographs of (A) the triangular shaped traps used to test male response to different colors; (B) circular shaped traps used to test male response to size, shape and (C) heat, plus (D) the heating coil and (E) placement of the coil in the trap, and the (F) circular trap design used to test male response to different extracts. 38 objects were switched left to right and tested fivemore days, bringing the total number of replicatesper size or shape comparison to ten. The synthetic pheromone was changed between comparisons. The only exception to this procedure occurred when dried female models aloneor combined with an ellipse were compared to a white ellipseor empty trap. In these situations, five critical point dried females were positioned behind each trap on the air-intake screen along with the synthetic pheromone. The females were dried by serial emersion through 30, 50, 70, 85, and

100 percent acetone followed. by 30, 50, 70, 85, and 100 percent trichlorotriflouroethane rinsings. They were then deposited in a Preen 13 filled Bomar critical point dryer and brought to 900 psi at 38°C.Maintaining the 38°C temperature, the pressure was gradually reduced at 50 psi intervals down to 0 psi. The critical point for this system was 526 psi at 38°C.

Response to Radiant Heat Energy. Ice cream cartons (946 cm3) were also used to make the traps for the study of male response to heat. These traps were also painted black and lined withTanglefoot(i);however, one end was covered with black cardboard containing a 6 cm diameter hole offset to one side such that the outside edge of the hole was even with the inside edge of the trap. The other end of the trap was fitted with a removable screen. Only two of these traps were made and fitted through circular holes cut in 39

the screen attached to the outside and theblack cardboard

on the inside of the air-intake door (Fig. 5C). Once in position, the holes were 2 cm apart (8cm between centers). Next a coil of plastic tubing (Fig. 5D)was inserted

through a hole cut in the screened end ofone of the traps

(Fig. 5E). This coil was painted black and held 4cm in

from the center of the 6 cm opening. The coiled tube was attached by rubber tubing to a waterpump suspended in a tank of water. A heater was also suspended in the tank and adjusted to maintain the temperature in the center of the coil at 5 ± 2°C above the mean ambient air temperature during the peak period of male activity between 1800. and 2000 hours. The other trap remained empty. Synthetic pheromone was placed behind each trap. The traps were checked daily to record the number of males caught, then cleaned.After five days the coil was moved to the other trap and the experiment was repeated five more days to make a total of ten replications. This experiment differed from the others in that 10-50 males were introduced each day.

Measurement of Female Body Temperature. Accompanying the study on the response of males to radiant heat was an experiment designed to measure the internal body temperature of newly emerged female Douglas-fir tussock moths under natural conditions and compare those temperatures to the ambient air temperature. Female pupae were held at 27°C during the day and 16°C at night with a 14:10 hour light/ 40 dark photoperiod. Then 84 newly emerged females were attached to the tops, bottoms, and sides of Douglas-fir and white fir branches outside the Forestry Sciences Laboratory at 1000 hours on July 13 and 14, 1978.At 1600 hours the difference between their internal body temperature and ambient air temperature was measured using a Bailey Instru- ment model Bat-8 thermometer and two thermocouples.

Response to Female Models Plus Synthetic Pheromone,

Synthetic Pheromone Alone and Live Females. The Los Alamos field site (Fig. 3), described under long-distance flight orientation (page 31), was also used to study short-range flight orientation. The short-range response of males to female models was tested by placing critical point dried females (free of pheromone) in synthetic pheromone baited traps. These traps were constructed from 3.8 1 milk cartons with one side and both ends removed (Fig. 6A). The cartons were lined withTanglefootOcoated paper held in place with paper clips. The dried females were glued to strings attached to cardboard tags. One centimeter pieces of pheromone coatedMylar®plastic were pinned to the tags. The synthetic pheromone was released from a film of

impregnated resin on theMylar(D. The upper and lower limits of the release rate were 1.1 ± 0.5 (X ± SD) and

0.32 ± 0.14pg/cm2per day at 26°C (Sower and Daterman,

1977). The cardboard tags were held in the cartons with paper clips, and rubber bands were used to hold the cartons 41

Figure 6. Photographs of the three types of trap baits used during the Los Alamos field studies: (A) female models plus synthetic pheromone,(B) synthetic pheromone alone, and (C) live females; and photographs of: (D) the triangular shape of a closed trap,(E) the placement of traps at the field site, and (F)a female used to study mating success of different age females. 42

closed to form triangular traps (Fig. 6D). When the traps were closed, the female models were suspended 9.7 cm in

from either end. Twenty-five traps were made. These traps, 25 traps containing only the synthetic pheromone (Fig. 6B), and 25 containing live unmated females less than one day old (Fig. 6C) were randomly positioned 7.5 m apart at the

field site (Fig. 6E). The live female baited traps were made according to the procedure outlined in Chapter IV.

All the traps were checked daily to record the number of males caught and to replace theTanglefoot®. The experiment was repeated six times between July 28 and August 7.

Results and Discussion

Long-Distance Flight Orientation

The observed (n = 265) long-distance flight behavior

of male Douglas-fir tussock moths is illustrated in Figure

7. This three dimensional sinuous flight pattern is a re-

sult of positive anemotaxis and positive chemoklinotaxis.

The male flies into the wind while scanning across the odor

field with his antennae. This scanning is facilitated by movements of his entire body. When he perceives pheromone permeated air, he senses the areas of highest concentration

(possibly frequency of pulses) and orients along a concen-

tration gradient to the source. Similar zigzagging or

sinuous flight patterns have been observed in other moths

(Berisford and Brady, 1972; Brown, 1972; Doane and Carde, DECREASING RATE OF FORWARD PROGRESS

Figure 7. The sinuous flight pattern of male Douglas-fir tussock moths. 44 1973; Farkas and Shorey, 1972; Green, 1962; Hidaka, 1972). The sinuous flight pattern varied depending on population density and time of day. As stated, the males begin flying in the morning and continue into the evening with peak flight activity between 1600 to 1700 hours. Compara- tive observations on 60 males observed during the early portion of the daily flight, before the females began releasing the pheromone (before approximately 1500 hours), and 205 males later in the day, revealed that the males fly in a straighter line and at a faster rate of forward progress during the early hours than they do later when orienting to the sex pheromone. In addition the males do not orient to the contour of trees and branches during this early phase as they do later in the day. They are also less abundant during this early phase of the flight period.

The nature of the flight pattern also varies under different population densities. Observations of the arrival of 108 males at the pheromone source were obtained in a heavily infested stand and 15 in a lightly infested stand. In the low density population all 15 displayed the typical zigzagging flight pattern as they flew directly to or within a few centimeters of the pheromone source. As they approached the source, they reduced the rate of forward progress and increased the frequency while reducing the extent of zigzagging (Fig.. 7). This is in contrast to the behavior of the males in the high density situation who 45 rarely flew directly to the source (only 3 out of 108 flew

directly to or within a few centimeters of the pheromone

source). Instead, they exhibited the same variations in

the zigzagging pattern as they oriented to tree trunks or branches. They landed on a trunk or branch and began walk-

ing with frequent turning from side to side as they fanned

their wings. The males reversed direction frequently and attempted to mate with cocoons, mated or unmated females or

even other males. Males were observed to search among a group of releasing females for a period of time then fly

off without mating or simply stop all activity; these be-

haviors are believed to be due to habituation.

Short-Distance Flight Orientation

Response to Color. The results of the study on the

response of males to 21 combinations of seven colors are

summarized in Table 1. The mean percentiles of male catches per comparison ranged from 61.8% for white to

40.0% for yellow. The colors can be ranked from most to

least attractive as follows: white, brown, green, red,

blue, black, and yellow. However, statistical analysis based on a balanced incomplete block and analysis of variance revealed that male Douglas-fir tussock moths do

not show a statistically significant preference for any Table 1. Summary of the results of the study on the response of pheromone-stimulated male Douglas-fir tussock moths,Orgyia pseudotsugata(McD.), to21 combinations of seven colors. Percentage of Males Caught in Each Comparison Combination White Brown Green Red Blue Black Yellow

1 60.9 39.1 2 56.9 43.1 3 61.7 38.3 4 60.0 40.0 5 66.7 33.3 6 64.7 35.3 7 64.3 35.7 8 51.4 48.6 9 50.6 49.4 10 66.7 33.3 11 57.8 42.2 12 42.9 57.1 13 56.2 43.8 14 68.4 31.6 15 66.7 33.3 16 33.3 66.7 17 64.0 36.0 18 68.4 31.6 19 56.3 43.7 20 46.2 53.8 21 56.1 43.9 Total 370.9 329.9 313.0 309.7 302.4 234.0 240.1 Mean 61.8 55.0 52.2 51.6 50.4 39.0 40.0

/AnalysisAnalysis of variance indicated no significant difference between the means (P < 0.05) . 47 one of the colors. Therefore, it appears that visual response to colors plays a minor role in the close-range orientation to male Douglas-fir tussock moths.

Response to Different Shapes and Sizes. Table 2 summarizes the results of the laboratory study comparing the response of males to different shapes and sizes.

The males did not show a significant preference for any one of the sizes or shapes including the critical point dried females. This lack of preference for female models in the laboratory concurs with the findings of the 1977 field study which compared the response of males to female models plus synthetic pheromone, the synthetic pheromone alone, and live females (as presented in the next section).

Response to Female Models Plus Synthetic Pheromone,

Synthetic Pheromone Alone and Live Females.The results of the study comparing the response of male Douglas-fir tussock moths to traps baited with synthetic pheromone plus a female model, synthetic pheromone alone, and live females less than one day old are summarized in

Table 3. The mean number of males caught per day in traps containing pheromone and dried females was not significantly different from that of traps containing only pheromone, but there was a highly significant Table 2. Summary of the results of the study on the response of pheromone-stimulated male Douglas-fir tussock moths, Orgyia pseudotsugata (McD.), to different sized or shaped visual models. Number of Total number of Percentage of total Models compared males caught males caught number of males caught A B A A

1 cm circle 4 cm circle 67 73 140 47.9 52.1

1 cm circle 6 cm circle 44 41 85 51.8 48.2

2 cm circle triangle 66 48 114 57.9 42.1

2 cm circle square 88 77 165 53.3 46.7

2 cm circle ellipse 48 54 102 47.1 52.9

female on ellipse ellipse 52 52 104 50.0 50.0

female alone no model 106 113 219 48.4 51.6 Table 3. Summary of theresults of the study comparing the responseof male Douglas- fir tussock moths,'Orgyia pteudottugata (McD.) to traps baitedwith synthetic pheromoneplus a female model, synthetic pheromone alone,or a live female less thanone day old.

Total number Average number of Number of samples of males males caug4t per Bait (traps) caught trap1.-/ Female model plus synthetic pheromone 150 2,564 17.1-a/ Synthetic pheromone alone 150 2,315 15.4.!/

Live model 25 666 26.64-b/

1/Averageswith the same letter are not significantly different; those with different letters are significantly different (P < 0.025). 50 difference (P < 0.025) between themean number caught in the live female baited traps and either ofthe other two

types. The question of why live female baitedtraps

consistently catch more males than the syntheticsex pheromone remains unanswered. The results of the

preceding experiments on short-distance orientationrule

out visual orientation to preferred colorsor shapes,

including the image of the female body. This leaves acoustic, further olfactory, or other components of the

electromagnetic spectrum as the remainingsources of

stimulation to which the male might orient. The possibility of additional components of the sex pheromone has and continues to be investigated, and acoustic stimulation

appears to be a rather remote possibility (Daterman, per-

sonal communication). Therefore, further studies were restricted to investigating other components of the electro-

magnetic spectrum outside the visual region. It was hypothesized that the female Douglas-fir tussock moth could

raise her body temperature above ambient air temperature to

an extent sufficient to stimulate oriented behavior in the male. To test this the difference between the body temperature of the females and ambient air temperature was

measured. This temperature difference was then compared to

the temperature difference used to test the response of males to radiant heat energy, as described on page 38. The results are presented below. 51 Measurement of Female Body Temperature and Male Res- ponse to Radiant Heat Energy. The results of the measure- ments of the difference between the body temperature of the females and ambient air temperature revealed a difference range of 0.2 °C to 6.2°C above ambient, averaging 2.4°C

(ambient air temperature being 33.6°C). This is compared to 5 ± 2 °C above ambient air temperature of the heat element used to test male response to heat; thus a range of

3-7°C. The results of the study testing male response to that heat element showed that a larger proportion (158 out of 248 captured males) were caught in the trap containing the heat element and pheromone; the difference being highly significant (P < 0.01 by chi-square test of significance).

In addition, the majority of males (63 out of 90) caught in the pheromone control trap were caught on the side nearest the pheromone and heat element trap and may have also been orienting to the heat element. The sinuous nature of the flight pattern combined with the close proximity (2 cm apart) of the trap openings (Fig. 5C) may have promoted the capture of males in the pheromone control trap even though they were orienting to the heat element. The demonstrated preference towards the heat element and the fact that the female can raise her body temperature above ambient air temperature indicate that heat energy emitted by female Douglas-fir tussock-moths plays an 52 important role in the pheromone-mediated close-range orientation to the female and may be an important factor in explaining the consistent superiority of live female baited traps over synthetic pheromone baited traps.

Conclusions

The pheromone-mediated long-distance flight behavior of male Douglas-fir tussock moths is characterized by positive anemotaxis and positive chemoklinotaxis. The resulting zigzagging flight pattern (Fig. 7) varies depending on the population density and time of day, being straighter with a faster rate of forward progress and not oriented to the contour of tree trunks or branches until the females begin releasing. In a low density population the males fly directly to or within a few centimeters of a releasing female as opposed to a high density situation where they orient to a tree trunk or branch rather than to the pheromone source. In either situation, the male reduces his rate of forward progress and increases the frequency while reducing the extent of zigzagging prior to landing. In high density situations the male walks along the tree trunk or branch while fanning his wings and turns from side to side. He may reverse direction and will attempt to mate with other males, cocoons and mated or unmated females.

After searching among a group of releasing females for a period of time, a male may fly off or simply stop all 53 activity; this is due to habituation.

Visual cues play a minor role in the pheromone- mediated close-range orientation of male Douglas-fir tussock moths. The males do not have a color, size, or shape preference, not even to the female body image. However, they do respond to heat energy 3 -7 °C above ambient air temperature. This response to heat is believed to play an important role in the pheromone-mediated behavior of male Douglas-fir tussock moths when close to the female and may be an important factor in explaining the consistent superiority of live female baited traps over synthetic sex pheromone baited traps. 54

IV. MATING BEHAVIOR

Methods and Materials

Fifty-two observations of Douglas-fir tussock moth mating behavior were made. Both laboratory and field studies were conducted. When possible, quantified information was obtained by recording the elapsed time (±0.01 min)

for courtship, copulation, pre-oviposition (the period between copulation and egg laying), and oviposition. In addition, twelve multiple mated females were preserved in alcohol for dissection to confirm the transfer of more than one spermatophore, and egg masses from both multiple and single matings were incubated.

The mating behavior was studied in the laboratory during the summer of 1976. The moths were reared from egg masses collected in Trigo Canyon, New Mexico. During pupation, they were subjected to a 12:12 hour photoperiod with

21°C days and 15.6°C nights. Newly emerged females (1-72 hours old) were left clinging to their cocoons on the air- intake screen of the flightway (Fig. 4A); and males, 24-96 hours old, were introduced into the flight chamber via the access door. Their activities were then observed. During the study the room conditions were maintained at 21°C and

12:12 hour photoperiod with the scotophase starting at 1800 hours. This method was used to obtain 33 observations. 55 The other 19 observations were obtained between

August 11 and 23, 1976 in Trigo Canyon, New Mexico; therefore, the same population base was used for both laboratory and field studies. The latter studies were accomplished by searching the lower branches of Douglas-fir and true fir trees for unmated females. This searching activity was conducted in an area with a high population density approximately two miles up the canyon, and was carried out each day before active flight began. Unmated females were watched until the male(s) arrived, at which time data col- lection began.

Results and Discussion

Courtship Behavior

Courtship begins when the male arrives and starts fanning his wings. The wings are rapidly elevated and depressed from approximately a 90' angle dorsally to a 12° angle ventrally. These angles vary according to the position of the male with respect to the female, cocoon, and surrounding foliage. The male's antennae are held out in front of the head and parallel to each other and the dorsum during wing fanning. While fanning, the male gropes along the female's body with the terminal segments of his abdomen.

To accomplish this, he bends his abdomen from side to side and dorso-ventrally. During this searching activity, his claspers are open and the aedeagus is extended. The male 56 concentrates his probing around the area of the female's

ostium bursae (Fig. 8); however,as stated, a male may direct his searching activity towards other parts of the

female's body, the cocoon, foliage, or another male. The male continues to probe the female until his aedeagus is in the immediate vicinity of the ostium bursae (Fig. 8); at

which time he stops fanning his wings and usually spreads

his antennae. The wings remain elevated at 55' -90' angle

while the abdomen is twisted from side to side. It is assumed that this twisting promotes penetration of the

aedeagus into the female's ductus bursae (Fig. 8). The male may pause several times during courtship but will con-

tinue fanning and probing until he successfully enters the

female. The male's courtship behavior is completed once he has penetrated the female and has firmly gripped her with his claspers.

Two or more males will simultaneously court the same

female. Nineteen of the 52 observations were characterized by simultaneous courtship by two or more males. Of the 19,

six occurred under field conditions on different days. In

such instances the courtship activities of the unsuccessful male(s) continues after a successful one has penetrated the

female. A rival usually does not displace a copulating male even though the intruder continues his activities for

several minutes. The rival(s) usually leaves after a few minutes but may become quiescent and remain near the 57

SP CoB

CeB DB

Figure 8. Drawing of the reproductive organs of the female Douglas-fir tussock moth. AG, accessory gland; AGD, accessory gland duct; CD, common duct of accessory glands; CeB, cervix bursae; CoB, cor- pus bursae; DB, ductus bursae; DS, ductus semina- lis; DSp, ductus spermatheca; 0, ovariole, OB, ostium bursae; OC, oviductus communis; OL, oviductus lateralis; 0o, oocytes; OP, ovipore; OV, ovipositor, R, rectum, RAG, reservoir of the accessory glands; SC, spermathecal chamber; SG, spermathecal gland; SP, spermatophore; TF, terminal filament; V, vagina. 58 copulating pair. The quiescent state is believed to be

due to habituation. This rivalry was reported by Wickman et al. (1975) as was the male's trait of remaining by the

female for a period of time after mating.

During courtship, the female clings to her cocoon. Her antennae are either held out to the side or pressed back against the thorax, and she does not exhibit discerni- ble courtship behavior. Wing, leg, antennae, and abdomen movements do occur, but it is believed that these activities

are efforts to maintain her position on the cocoon. How- ever, a female may twist and telescope her abdomen towards

the male's abdomen; these movements appear to promote con-

tact between the aedeagus and the ostium bursae (Fig. 8).

These observations support those of Shorey (1976). He re- ports that the female sex pheromone is often such an impor-

tant stimulus of premating behavior that the remainder of

the female's body is not necessary.

The elapsed time for courtship was the same (P < 0.05) for laboratory and field moths (Table 4). The combined courtship time for the two populations ranged from 0.03 to 1.17 min, and averaged 0.43 min. However, this courtship time and the timing of all other behavioral activities reported in this thesis were recorded under certain temperature conditions (i.e. 21C in the laboratory), and the results will vary with different temperatures. Table 4. Comparison of elapsed times for different activities by singly mated laboratory reared and wild New Mexico Douglas-fir tussock moths, Orgyia pseudotsugata (McD.).

Elapsed time for:

Moth Courtship Copulation Pre-oviposition Oviposition Population Range Average Range Average Range Average Range Average

Laboratory .1 reared 0.03-1.17 0.48- 13.24-61.13 31.57b 34.71-186.11 106.50c 112-177 143e

Wild b New Mexico 0.03-0.84 0.31a 13.56-52.3 27.82 28.53-93.2 52.38d 178-181 179e

2 Pooled 0.03-1.17 0.43 13.24-61.13 30.38 112-181 151

1 The averages within each column having the same letter were not significantly different (P < 0.05) according to t-tests.

2 The ranges and averages for the two populations were combined when the separate averages were not significantly different. 60 Copulatory Behavior

Once the male has successfully inserted his aedeagus into the ostium bursae (Fig. 8), the couple becomes quiescent. Their body positions vary due to factors such as the orientation of the male to the cocoon, female, or foliage, and the female's position with regard to her cocoon and the surrounding foliage. However, the male's ventral surface is typically against the female's side or her venter. The latter arrangement may occur when the female is hanging on the end of the cocoon with her ventral, abdomenal surface exposed. The male's abdomen is bent ventrally and usually

to the side. He clings to the cocoon, female, foliage or all three. In the situation where he is clinging only to the female, she may display movements of her wings,

antennae, legs, and/or abdomen. The role these movements play is not clear, but they are believed to be attempts to maintain her hold. However, most females remain motionless with their antennae drawn back along their thorax, and

parallel to their backs. The male usually holds his antennae dorsally at a 20°-30 angle to the long axis of the body, and back at approximately a 10 angle to the dorsum.

His wings are typically held parallel to his dorsum or

slightly lowered with the underwings partially visible or

completely concealed when viewed from above. When dis-

turbed by the activities of another male, a copulating male

folds his wings over his abdomen in a tent-like fashion, 61.

completely concealing the underwings, and presses his

antennae against his dorsum.

The elapsed time for copulation was the same (P <

0.05) for laboratory and field moths (Table 4). The combined copulation times for the two populations ranged from

5.9 to 42.56 min, and averaged 24.94 min. To obtain a measure of mating time, the courtship and copulation times were summed for singly mated pairs under laboratory and

field conditions. The mating period for laboratory moths ranged from 11.89 to 42.74 min, averaging 26.79 min, while

the wild population averaged 23.24 min and ranged from

13.62 to 32.22 min. The combined range for laboratory and

field populations was 11.89 to 42.74 min and averaged

25.76 min. So mating lasts about 26 minutes. Copulation ends when the male begins his separation

behavior. He separates from the female by fanning his wings, twisting and telescoping his abdomen, and pushing

his legs against the female, her cocoon or the foliage.

Once separated, the male may remain by the female for a

period of time, but most males fly off immediately. The

female does not appear to take an active role in separa-

tion, and remains quiet for some time (termed the pre-

oviposition period) before she starts laying eggs. 62 Pre-oviposition Behavior

The quiescent period of pre-oviposition was significantly (P < 0.05) longer for the laboratory reared females than for the wild population. Pre-oviposition for the laboratory females ranged from 34.71 to 186.11 min, averaging 106.5 min compared to a range of 28.53 to 93.21 min and an average of 52.38 min for the wild females

(Table 4). All 52 females observed exhibited a pre-oviposition stage. These results differ from the observations of Wickman et al. (1975) who reported females laying eggs immediately after mating.

Oviposition Behavior

The female begins oviposition by crawling along the cocoon until she is in a position which enables her to touch the cocoon with the end of her abdomen. At the same time, the terminal abdominal segments are protracted to form an "ovipositor". She moves her abdomen in and out, from side to side, and dorsal-ventrally to deposit the eggs.

These abdominal movements rub off body hairs which lodge in the liquid cementing material secreted during egg laying. The elapsed egg laying time ranged from 112 to 180 min

(Table 4). The average time for oviposition by laboratory reared moths did not differ significantly (P < 0.05) from that of the wild females.. The average time for oviposition was 151 min or about 2-1/2 hours. Wickman et al. (1975) 63 reported oviposition lasting from 45-360 min.

Three females, including two wild females, interrupted their egg laying to mate again. In addition, a total of 17 out of 52 females mated more than once, and 4 of the 17 occurred under natural conditions. Furthermore, females were noted mating as many as four times, and dissections confirmed the transfer of more than one spermatophore (Fig.

8). Males were also observed to mate up to four times, but there appears to be a minimum time-span which must elapse between consecutive matings by the same male. Females, on the other hand, can mate again immediately. The wild population of males is probably restricted to one mating per night due to this recovery time. Shorey and Gaston (1964) reported a similar restriction in the cabbage looper moth.

The proportion of fertilized eggs per egg mass was the same

(P < 0.05) for masses layed by females which had mated once and those that mated more than once. These observations of multiple mating in females, including ovipositing females, concur with the findings of Wickman et al. (1975). Finally, a virgin female may lay a portion of her eggs before mating or complete oviposition without copulating.

The unfertilized egg masses can usually be distinguished

from fertilized masses. Fertilized eggs are cemented to-

gether along with numerous body hairs in a geometric manner

(Fig. 9B). This is in contrast to the interrupted, 64

Figure 9. Photographs of (A) an unfertilized egg mass, (B)a fertilized egg mass, and (C) an unfertilized egg mass that appears to be fertilized. 65 haphazard deposition of unfertilized eggs (termed "spew-

ing"); characterized by far fewer eggs and body hairs (Fig.

9A). However, it is not always possible to determine

fertility by the outward appearance of the egg mass (Fig.

9C).

Conclusions

A female may be simultaneously courted by two or more

males, and a male may remain by the female after mating.

Mating lasts about 26 minutes. Males and females can mate more than once and there appears to be a minimum time-span

which must elapse between consecutive matings by the same

male, probably restricting the male to one mating per night.

About an hour elapses before the female begins laying her

eggs, and it takes her approximately 2-1/2 hours to finish

ovipositing. However, the elapsed time for the various mating, preovipesiting, and ovipositing behaviors may vary

depending on temperature. An unmated female may lay a por-

tion of her eggs before mating or complete oviposition without copulating. Egg viability is the same for single and multiple matings. Comparisons of average elapsed courtship, copulation, pre-oviposition, and oviposition times

for laboratory reared and wild New Mexico moths reveal that

pre-oviposition is longer in laboratory moths; but court-

ship, copulation and oviposition are the same. 66

V. FACTORS AFFECTING PHEROMONE-MEDIATED BEHAVIOR

Methods and Materials

Both laboratory and field studies testing the effects of certain factors on the pheromone-mediated behavior of the Douglas-fir tussock moth were conducted. The field studies were conducted between July 28 and August 20, 1977 at the Los Alamos, New Mexico field site (Fig. 3). A hygrothermograph was set up at this site and supplemented by direct weather observations.

Two methods were used to study the effects of age and weather on the attractiveness and mating success of wild

Douglas-fir tussock moth females. The first consisted of collecting female pupae at the field site and transporting them to the field laboratory where they finished pupating.

Upon emerging, females less than twenty-four hours old were attached to cardboard tags by taping their cocoons to the tags. The tags were then covered with screen and attached to 1.9 2. milk cartons. These cartons had one side and both ends removed and were lined with Tanglefoot coated paper. The paper was held in place with paper clips, and rubber bands were used to hold the cartons closed to form triangular traps (Fig. 6C). This trap design pre- vented the females from mating while exposing them to the weather and male flight. It also permitted daily. 67 observations and replacement of theTanglefoot®with a minimum of disturbance to the female. One hundred and ninety-one traps were made and attached with wire twists to the lower branches of trees approximately 7.5 m apart along with the synthetic sex pheromone baited traps described in Chapter III (Fig. 4B). Each trap was checked daily to see if the female had layed eggs ("spewed"), had attracted any males, and was still alive.

The second method consisted of holding the newly emerged females in the field laboratory until they reached certain ages then attaching them to fir trees by tying their cocoons to the foliage (Fig. 6F). Thirty-six females were put out on each of four days such that there were six less than 1 day old, six 1 day old, six 2,3, 4, and 5 days old. Thus a total of 144 females were used. Each female was checked daily to see if she was still on her cocoon and if she had mated. The egg masses were incubated to confirm successful mating. The laboratory studies were conducted between June 8 and July 15, 1978. Females were held under a 14:10 hour light /dark photoperiod (scotophase starting at 1800) and either a 21:4°C light/dark or 27:16°C light/dark temperature schedule as pupae and adults. When the females had reached certain adult ages, they were subjected to pheromone gland washing. An extraction procedure similar to that described by Sower et al.(1973a) was used to obtain 10 sec pheromone 68 gland washes in 5 ml of hexane. The abdominal tips, containing the glands, were then deposited in a separate vial containing 5 ml of hexane. Fifty females were extracted for each of seven age classes and two temperature schedules bringing the total to 700 females. The 28 different extract samples were stored at -50°C until they could be tested.

To test the response of males to the extracts, samples equivalent to one female (0.01 ml) were applied to fiberglass wicks on the screened lids of black circular traps.

These traps were made from 1.9 1 ice cream cartons with centrally located 4 cm diameter holes cut in the bottoms.

The resulting traps were painted black and lined with

Tanglefoot®. Each trap was fitted through a single hole cut in the screens attached to the inside and outside of the air-intake door (Fig. 5F). The number of males caught in each trap was checked prior to 1100 hours each day, then the traps were cleaned and the flightways vacuumed clean of all old males. Twenty-five new males, 1-2 days old, were then introduced into each flightway. These males had pupated under the same conditions as those used in the close-range orientation studies described in Chapter III.

The males were left in the flightways until 1800 hours at which time randomly chosen extract traps were inserted into the flightways, and the appropriate extracts applied to the fiberglass wicks. Each of the 21 extracts of interest were tested three times bringing the total to 63. 69 The average temperature profile in the room during the study fluctuated between 22-30°C, and a 14:10 hour light/ dark photoperiod was maintained with the scotophase starting at 1800 hours.

Results and Discussion

The Los Alamos field studies revealed the effect of rain on the pheromone-mediated flight behavior of male

Douglas-fir tussock moths. As Figure 10 illustrates, the magnitude of the male flight decreases as the number of hours of daily rain increases. Male flight is most severely restricted when rainy weather extends through the peak flight period.

In addition to establishing that the males do not fly in the rain, the rainy weather also limited data analysis on the 191 female baited traps. The analysis was further restricted due to incomplete records since it was impos- sible to observe all 191 females from emergence to death.

For example, data on the female life expectancy was limited to 43 females. The data are summarized in Figure 11A. The females lived up to seven days; however, their attractive life was not that long. Figure 11B illustrates that only

37% of the four day old females remained attractive as compared to 78% of the three day old females. Furthermore, the results of the study designed to test the effect of 70

A a. 4 w20- u. I- 0ig ig um a. di 1 0-

Z 4.1 Z 4v 0 1111 n

I III II II 11111 II I I I II III

.1101I.

I I I I I I I I I I I I I I I I I I i I I I I 0) Tr o Csi el 0) .-- 01 I I I I I t- Co CO CO CO DATE Figure 10. Graphs of (A) the mean number of males caught per day in synthetic pheromone baited traps and (B) the number of hours of rain per day. 100 100 90 90 80 E 70 >u au 0 50 0Zi 1.1, 40 40

Z^ 30 30 U No, 20 20

10 10 -

2 3 4 5 6 7 8 5 TIME (DAYSI TIME IS DAYS) ADULT AGE (DAYS

Figure 11. Graphs of the results of field studies on (A) the life expectancy and (B) the attractive life-span of unmated adult Douglas-fir tussock moth females, as well as (C)the effect of female age on mating success. 72 aging on mating success (Fig. 11C) revealed that only 29% of the two day old females compared to 54% of the one day

old females successfully mated. Therefore, even though the population of wild females studied lived up to seven days, their ability to attract males dropped after three days,

and the probability that they would successfully mate

dropped after one. These findings are in agreement with

those of Percy et al. (1971). They found that female whitemarked tussock moths were most attractive on the day

of emergence and attractancy declined thereafter. The poorest responses were obtained with females more than two

days old. Here again the results may vary under differing

temperature conditions.

The effect of aging was further studied in the labora-

tory by varying the temperature to which the females were

exposed. The data are summarized in Figure 12. Temperature

does affect the attractive lifespan of the female; females held on a 27:16°C light/dark temperature schedule showed a decline in attractancy after seven days compared to three days for females held under a 21:4°C schedule. These re-

sults were not expected since it was hypothesized that the lower temperature schedule would reduce female activity; thereby, extending the attractive life. However, the re-

sults do show that temperature and age can combine to affect pheromone-mediated behavior. It is further speculated that 73

12 11

10- sei 9 4 8 O of W 7 0. cm 6 z 4 5 V 4 4 3 2

1<2 3<4 5 <6 7 <8 9 <10 11 <12 13<14 FEMALE ADULT AGE (DAYS) Figure 12. Graph of the results of a laboratory study comparing extracts (hexane washes) of unmated Douglas-fir tussock moths reared under different temperature schedules. 74 the- attractive life- span, may vary between populations an4 geographic locations.

The laboratory studies also provide some insights into the mechanisms by which attractancy is diminished. Figure 13 summarizes the results from the comparison of male attraction to extracts from different age females obtained by washing or soaking the abdominal tip in hexane. Attrac- tancy to both dropped after the females were seven days old indicating a decline in the amount of pheromone present.

The difference in the attractiveness of the two extracts after day seven implies that the female loses the ability to release the remaining pheromone, but statistical analysis (t-tests) reveals that none of the paired means (paired by age) are significantly different. However, observations do indicate that older females do not exhibit the pheromone releasing behavior (i.e. "calling behavior"). Therefore, the decline in attractancy with age is due to a reduction in the amount of pheromone present and a loss of the ability to release the pheromone. These conclusions are supported by Grant's (1977) observations on the whitemarked tussock moth. He found that the reduced mating success of older females is related to decreased pheromone production and replacement of calling behavior by "spewing".

The Los Alamos studies also provide information on the tendency of females to lay unfertilized eggs ("spewing") as discussed in Chapter IV. Twenty-six or 60.5% of the 43 75

12 11 10 9 >- 4 0 8

MI 7 Q .

4 5 V 4 4 cW 3 2 1

1<2 3<4 5<6 7 <8 9 <10 11 <12 13<14 FEMALE ADULT AGE (DAYS) Figure 13. Graph of the results of a laboratory study comparing extracts obtained by soaking or washing the abdominal tips of unmated Douglas- fir tussock moth females in hexane. 76 females observed from emergence to death spewed. In addition, considering all females that spewed during the study,

55% were 4-5 days old before they layed eggs.

Finally, it has generally been assumed that all adult females will attract males and mate. The results of the Los Alamos study on the attractiveness of unmated female revealed that 20% (16 out of 79) of the females did not attract any males. This nonattractancy may vary between populations and geographic location as well as under different weather conditions, but the results do not support the assumption that all adult females attract males and mate.

Conclusions

The wild female population studied lived up to seven days but their ability to attract males dropped after three days and the probability that they would successfully mate dropped after one day. Attractancy is also influenced by temperature, and the decline in attractancy is due to a reduction in the amount of pheromone present and a loss of the female's ability to release the pheromone. Twenty percent of the females studied were not attractive and males do not fly in the rain. Finally, 60.5% of the unmated females layed infertile eggs, and 55% waited until they were 4-5 days old before laying infertile eggs. 77

25 -

20 0 cQ 0 15 D O . 00

1 2 3 4 5 6 TIME (DAYS)

Figure 14. Graph of the results of the field study on the tendency of Douglas-fir tussock moth females to lay unfertilized eggs. 78

V/. SUMMARY

Both laboratory and field studies on the pheromone- mediated behavior of the Douglas-fir tussock moth were conducted. The long-distance flight orientation of males to the pheromone source was observed over three field seasons.

In addition, the short-distance flight orientation of pheromone-stimulated males to visual and heat stimuli was studied in the field and laboratory. Laboratory and field observations of mating behavior as well as studies on the effects of certain factors influencing pheromone-mediated behavior were also conducted.

The pheromone-mediated long-distance flight behavior of male Douglas-fir tussock moths is characterized by positive anemotaxis and positive chemoklinotaxis. The resulting zigzagging flight pattern varies depending on the population density and time of day, being straighter with a faster rate of forward progress and not oriented to the contour of tree trunks or branches until the females begin releasing. In low density populations the males fly directly to or within a few centimeters of a releasing female as opposed to a high density situation where they orient to a tree trunk or branch rather than to the pheromone source. In either situation, the males reduce their rate of forward progress and increase the frequency while reducing the extent of zigzagging prior to landing. In 79 high density situations the males walk along the tree trunk or branch while fanning their wings and turn from side to side. They may reverse direction and will attempt to mate with other males, cocoons, and mated or unmated females.

After searching among a group of releasing females for a period of time, a male may fly off or simply stop all activity.

Visual cues play a minor role in the pheromone- mediated close-range orientation of male Douglas-fir tussock moths. The males do not have a color, size, or shape preference, not even to the female body image. However, they do respond to heat energy 3-7°C above ambient air temperature. This response to heat is believed to play an important role in the pheromone-mediated behavior of male Douglas-fir tussock moths when close to the female and may be an important factor in explaining the consistent superiority of live female baited traps over synthetic sex pheromone baited traps.

Once the male locates a female he begins his mating behavior. With regard to the mating behavior, it was found that a female may be simultaneously courted by two or more males, and .a male may remain by -the female after mating.

Mating lasts about 26 minutes. Males and females can mate more than once and there appears to be a minimum timespan which must elapse between consecutive matings by the same male, probably restricting the male to one mating per 80 night. Egg viability is the same for single and multiple matings. About an hour elapses before the female begins laying her eggs, and it takes her approximately 2-1/2 hours to finish ovipositing. An unmated female may lay a portion of her eggs before mating or complete oviposition without copulating. It is not always possible to distinguish between fertilized and unfertilized egg masses. Comparisons of average elapsed courtship, copulation, pre-oviposition, and oviposition times for laboratory reared and wild New

Mexico moths reveal that pre-oviposition is longer in laboratory moths; but courtship, copulation, and oviposition are the same. One high density population of wild females lived up to seven days, but their ability to attract males dropped after three days and the probability that they would successfully mate dropped after one. day. Attractancy is also influenced by temperature, and the decline in attractancy is due to a reduction in the amount of pheromone present and a loss of the ability to release the pheromone. At least 20% of the unmated females studied were not attractive; a factor which is compounded by the inhibitory effect of rain on male flight. Finally, 60.5% of the unmated females layed infertile eggs, and 55% of those were 4-5 days old before they layed.

These findings are of importance to our understanding of the population dynamics of the Douglas-fir tussock moth. 81 Fecundity is related to the number of females, the number of eggs per female, the sex ratio, and the number of generations per year. Any factor influencing these is going to affect our ability to predict population trends.

These studies show that actual fecundity cannot be deter- mined by simple numerical counts. For example, a mere estimate of the number of adult females will not suffice since not all females are attractive. In addition, the combined influences of female age and rainy weather can operate to reduce the probability that an attractive female will mate. Furthermore, a mere calculation of the number of eggs per female is not adequate. The fact that females lay unfertilized eggs affects fecundity by reducing the number of eggs per female that are potentially available for fertilization. Therefore, what is needed is an estimate of the number of eggs available for fertilization per attractive female that mates, plus attention to the influences of environmental and physiological factors such as age, rain, population density, and geographic location. 82

REFERENCES CITED

AliNiazee, M. T., and E. M. Stafford, 1971. Evidence of a sex pheromone in the omnivorous leaf roller, Platynota stultana (Lepidoptera; Tortricidae): Laboratory and field testing of male attraction to virgin females. Ann. Entomol. Soc. Am. 64:1330-1335.

AlliNiazee, M. T. 1972. Sex pheromone studies with the omnivorous leaf roller, Platynota stultana (Lepidop- tera: Tortricidae): Effects of viii-Eainvironmental factors on attraction of males to the traps baited with virgin females. Ann. Entomol. Soc. Am. 65:958- 961.

Anderson, W. B. 1919. Notes on the tussock moth, Hemero- campa, vetusta gulosa Hy. Edw. Agric. Gaz. Can. 6:139.

August, C. J. 1971. The role of male and female pheromones in the mating behavior of Tenebrio molitor. J. Insect. Physiol. 17:739-751.

Balch, R. E. 1932. The fir tussock moth (Hemerocampa pseudotsugata MCD.). J. Econ. Entomol. 25:1143-1148.

Banerjee, A. C. 1969. Sex attractants in sod webworms. J. Econ. Entomol. 62:705-708.

Bartell, R. J. and H. H. Shorey. 1969a. A quantitative bioassay for the sex pheromone of Epiphyas postuittana (Lepidoptera) and factors limiting male responsiveness. J. Insect Physiol. 15:33-40.

. 1969b. Pheromone concentrations required to elicit successive steps in the mating sequence of males to the light-brown apple moth, Epiphyas postuittana. Ann. Entomol. Soc. Am. 62:1206-1207.

Bartell, R. J. and L. A. Lawrence. 1973. Reduction in responsiveness of males of Epiphyas postuittana (Lepidoptera) to sex pheromone following previous brief pheromonal exposure. J. Insect. Physiol. 19: 845-855.

Bartell, R. J. and W. L. Roelofs. 1973. Inhibition of sexual response in males of the moth Arvyrotaenia velutinana by brief exposure to synthetic pheromone or geometrical isomer. J. Insect Physiol. 19:655-661. 83 Bartell, R. J. 1977. Behavioral responses of Lepidoptera to pheromones. In "Chemical Control of Insect Be- havior Theory and Application" (H. H. Shorey and J. J. McKelvey, Jr., eds.), pp. 201-213. John Wiley and Sons, New York.

Barth, R. H., Jr. 1961. Hormonal control of sex attractant production in the Cuban cockroach. Science 133: 1598-1599.

. 1962. The endocrine control of mating behavior in the cockroach Byrsotria fumigata (Guerin). Gen. Comp. Endocrinol. 2:53-69.

1965. Insect mating behavior: Endocrine control of a chemical communication system. Science 149: 882-883.

. 1968. The comparative physiology of reproductive process in cockroaches. Part 1. Mating behavior and its endocrine control. Adv. Reprod. Physiol. 3: 167-207.

. 1970. Pheromone-endocrine interactions in insects. Mem. Soc. Endocrinol. 18:373-404.

Barth, R. H., and L. J. Lester. 1973. Neurohormonal control of sexual behavior in insects. Ann. Rev. Entomol. 18:445-472.

Batiste, W. C. 1970. A timing sex-pheromone trap with special reference to codling moth collections. J. Econ. Entomol. 63:915-918. Batiste, W. C., W. H. Olson, and A. Berlowitz. 1973a. Codling moth: Influence of temperature and daylight intensity on periodicity of flight in the field. J. Econ. Entomol. 66:883-892.

. 1973b. Codling moths: Diel periodicity of catch in synthetic sex attractant vs. female-baited traps. Environ. Entomol. 2:673-676.

Bell, W. J., and R. H. Barth, Jr. 1970. Quantitative effects of juvenile hormone on reproduction in the cockroach Bytsotria fumigata. J. Insect Physiol. 16:2302-2313.

Bennett, R. B. and J. H. Borden. 1971. Flight arrestment of tethered Dendroctonus pseudotsugae and Trypodendron lineatum (Coleoptera: Scolytidae) in response to olfactory stimuli. Ann. Entomol. Soc. Am. 64:1273-1286. 84

Berisford, C. W. and U. E. Brady. 1972. Attraction of Nantucket pine tip moth males to the female sex pheromone. J. Econ. Entomol. 65:430-433.

Beroza, M. 1970. "Chemicals Controlling Insect Behavior." Academic Press, New York. 170 pp.

Beroza, M., A. Bierl, and H. R. Moffit. 1974. Sex pheromones: (E,E)-8, 10-dodecadien-1-01 in the codling moth. Science, 183:89-90.

Birch, M. C. 1970. Structure and function of the pheromone-producing brush-organs in males of Phiogophora meticulosa (L.)(Lepidoptera: Noctuidae). Trans. R. Entomol. Soc. London 122:277-292.

, ed. 1974. "Pheromones."Am. Elsevier, New York. 461 pp.

Blackmore, E. H. 1919. Report of Provincial Museum of Natural History, Victoria, B. C., for 1918. 16 pp.

Blum, M. S. 1969. Alarm pheromones. Ann. Rev. Entomol. 14:52-80.

Boeckh, J., K. E. Kaissling, and D. Schneider. 1965. Insect olfactory receptors. Cold Spring Harbor Symp. Quant. Biol. 30:263-280.

Borden, J. H. 1967. Factors influencing the response of 1Es confusus (Coleoptera: Scolytidae) to male attractant. Can. Entomol. 99:1164-1193.

Bossert, W. H., and E. 0. Wilson. 1963. The analysis of olfactory communication among animals. J. Theor. Biol. 5:443-469.

Brower, L. P. and A. M. Jones. 1965. Precourtship interaction of wing and abdominal sex glands in male Danaus butterflies. Proc. R. Entomol. Soc. London (A) 10:117-151.

Brower, L. P., J. V. Z. Brower, and F. P. Cranston. 1965. Courtship behavior of the queen butterfly, Danaus gilippus berenice (Cramer). Zoologica (N.Y.) 50:1-40.

Brown, L. N. 1972. Mating behavior and life habits of the sweet-bay silk moth (Callosamia carolina). Science 176:73-75. 85 Buckhorn, W. J. 1948. Douglas-fir tussock moth history and extent of outbreaks. USDA For. Insect Lab., Port- land, Oreg. 4 pp. Unpubl.

Butler, C. G. 1967. Insect pheromones. Biol. Rev. Cambridge Phil. Soc. 42:42-87.

. 1970. Chemical communication in insects: Be- havioral and ecologic aspects. Adv. chemoreception 1:35-78.

Callahan, P. S. 1965. Intermediate and far infrared sensing of nocturnal insects. Part I. Evidences for far infrared (FIR) electromagnetic theory of communication and sensing in moths and its relationship to the limiting biosphere of the corn earworm. Anns. Entomol. Soc. Am. 58(5):727-745.

1966. Electromagnetic communication in insects-elements of the terrestrial infrared environment including generation, transmission, and detection by moths. pp. 156-181 In pest control by chemical, biological, genetic, and physical means - a symposium. U.S. Dep. Agr. ARS 33-110. 115 pp.

Campbell, R. W. and L. C. Young. 1977. Douglas-fir tussock moth: An annotated bibliography. Review Draft Douglas-fir Tussock Moth Research and Development Pro- gram. USDA Forest Service and Cooperative State Research Service. Portland, Oregon. 115 pp.

Card4, R. T., and W. L. Roelofs. 1973. Temperature modi- fication of male sex pheromone response and factors affecting female calling in Holomelina immaculata (Lepidoptera: Arctiidae).Can. Entomol. 105:1505-1512.

Cards, R. T., T. C. Baker, and W. L. Roelofs. 1975. Be- havioral role of individual components of a multi- chemical attractant system in the oriental fruit moth. Nature 253:348-349.

Casida, J. E., H. C. Coppel, and T. Watanbe. 1963. Puri- fication and potency of the sex attractant from the introduced pine sawfly, Diprion similis. J. Econ. Entomol. 56:18-24.

Collins, C. W., and S. F. Potts. 1932. Attractants for the flying gypsy moths as an aid in locating new infestations. USDA, Dept. Agr., Tech. Bull. 336:1- 43. 86 Daterman, G. E. 1972. Laboratory bioassay for sex pheromone of the European pine shoot moth, Rhyacionia buoliana. Ann. Entomol. Soc. Am. 65:119-123.

Daterman, G. E., L. J. Peterson, R. C. Robbins, and R. G. Smith. 1976. Laboratory and field bioassay of the Douglas-fir tussock moth pheromone (Z)-6-heneicosen- 11-one. Env. Entomol. 5:1187-1190.

Daterman, G. E., R. L. Livingston, and R. G. Robbins. 1977. How to identify tussock moths cLught in pheromone traps. USDA Combined Forest Pest Research and Development Program. Agr. Handbook No. 517.

Daterman, G. E., and L. L. Sower. 1977. Douglas-fir tussock moth pheromone research using controlled-release systems. From the. 1977 International Controlled Re- lease Pesticide Symposium Proceedings, August 22, 23, and 24. Portland, Oregon. p. 68-77.

Daterman, G. E. 1978. The role of pheromones in forestry. In "Forest Insect Survey and Control, a Syllabus." (J. A. Rudinsky, ed.) 322-339 pp. OSU Book Store, Inc., Corvallis, Oregon.

Diesendorf, M., G. Stange, and A. W. Snyder. 1974. A theoretical investigation of radiation mechanisms of insect chemoreception. Proc. R. Soc. London, Ser. B. 185:33 -49..

Deane, C. C. 1968. Aspects of mating behavior of the gypsy moth. Ann. Entomol. Soc. Am. 61:768-773.

Doane, C. C., and R. T. Carde. 1973. Competition of gypsy moth males at a sex-pheromone source and a mechanism for terminating searching behavior. Environ. Entomol. 2:603-605.

Doane, C. C. 1976. Flight and mating behavior of the gypsy moth. In"Perspectives in Forest Entomology" (J. F. Andersonand H. K. Kaya, eds.) p. 127-148. Academic Press,San Francisco.

Eaton, C. B. and G. R. Struble. 1957. The Douglas-fir tussock moth in California. Pan-Pac Entomol. 33:105- 108.

Edwards, S. K. 1965. Activity rhythms of Lepidopterous defoliators. III. The Douglas-fir tussock moth, Orgyia pseUdotsuItta (McDunnough)(Liparidae). Can. J. Zool. 43:673-681. 87

Engelmann, F. 1970. "The Physiology of Insect Reproduc- tion." Perganon Press, Oxford. 307 pp.

Farkas, S. R., and H. H. Shorey. 1972. Chemical trail- following by flying insects: A mechanism for orienting to a distant odor source. Science 178:67-68.

. 1974. Mechanisms of orientation to a distant pheromone source. In "Pheromones" (M. C. Birch, ed.), pp. 81-85. Am. Elsevier, New York.

Farkas, S. R., H. H. Shorey, and L. K. Gaston. 1974. Sex pheromones of Lepidoptera. Influence of pheromone concentration and visual cues on aerial odor-trail following by males of Pectinophora gossypiella. Ann. Entomol. Soc. Am. 67:633-638.

Fatzinger, C. W. 1973. Circadien rhythmicity of sex pheromone release by Dioryctria abietella (Lepidop- tera: Pyralidae (Phycitinae) and the effect of a diel light cycle on its precopulatory behavior. Ann. Entomol. Soc. Am. 66:1147-1153.

Ferguson, D. C. 1978. "The Moths of American North of Mexico." E. W. Classey Limited and the Wedge Entomo- logical Research Foundation. 70-75 pp.

Fraenkel, G. S. and D. L. Gunn. 1961. "The Orientation of Animals." Dover, New York. 376 pp.

Gara, R. I., J. P. Vite, and H. H. Graner. 1965. Manipu- lation of Dehdroctohus frontalis by use of a population aggregating pheromone. Contrib. Boyce Thompson Inst. 23:55-66.

Grant, G. G. 1970. Evidence for a male sex pheromone in the noctuid Trichoplusia ni. Nature 227:1345-1346.

. 1974. Male sex pheromone from the wing gland of the Indian meal moth, Plodia interpunctella (Hbn.) (Lepidoptera: PhycitidaeT7nperientia 30:917-918.

. 1975. Extraction and bioassay of female sex pheromone of white-marked tussock moth, Orgyia leucostigMa (Lepidoptera: lymantriidae). Can. Ent. 107:303-309.

Grant, G. G., and L. McCarty. 1977. Effect of age on calling behavior and mating success of whitemarked tussock moths. Can. Bimonth. Res. Notes. 33:2-3. 88

Green, G. W. 1962. Flight and dispersal of the European pine shoot moth, Rhyacionia buoliana (Schiff.). I. Factors affecting flight and the flight potential of females. Can. Entomol. 94:282-299.

Happ, G. W., and 3. Wheeler. 1969. Bioassay, preliminary purification, and effect of age, crowding, and mating on the release of sex pheromone by female Tenebrio

molitor. Ann. Entomol. Soc. Am. 62:846-85 .

Hidaka, T. 1972. Biology of Hyphantria cunea Drury (Lepidoptera, Arctiidae) in Japan. XIV. Mating Behavior. Appl. Entomol. Zool. 7:116-132.

Jacobson, M. 1972. "Insect Sex Pheromone." Academic Press, New York. 381 pp.

Kaae, R. S. and H. H. Shorey. 1972. Sex pheromones of noctuid moths. XXVII. Influence of wind velocity on sex pheromone releasing behavior of Trichoplusia ni females. Ann. Entomol. Soc. Am. 65:436-440.

. 1973. Sex pheromones of Lepidoptera. 44 Influence of environmental conditions on the location of pheromone communication and mating in Pectinophora gossypiella. Environ. Entomol. 2:1081-1084.

Karandinos, M. G. 1974. Environmental conditions and sex activity of Synanthedon pictips in Wisconsin, monitored with virgin female pheromone traps.Environ. Entomol. 3:431-438.

Karlson, P., and M. Lttscher. 1959. "Pheromones": A new term for a class of biologically active substances. Nature (London) 183:55-56.

Kennedy, J. S., and D. Marsh. 1974. Pheromone-regulated anemotaxis in flying moths. Science 184:999-1001. Kennedy, J. S. 1977. Olfactory responses to distant plants and other odor sources. In "Chemical Control of Insect Behavior Theory and Application." (H. H. Shorey and J. J. McKelvey, Jr., eds.), pp. 67-91. John Wiley and Sons, New York.

Klopfer, P. H., and J. P. Hailman. 1967. "An Introduction to Animal Behavior."Prentice-Hall Inc., New Jersey. 297 pp.

Koehler, C. S., J. J. McKelvey, W. L. Roelofs, H. H. Shorey, R. M. Silverstein, and D. L. Wood. 1977. Advancing towards operational behavioral-modifying 89 chemicals. In "Chemical Control of Insect Behavior Theory and Applicatidn." (H. H. Shorey and J. J. McKelvey, Jr., eds.),- pp. 115-400. John Wiley and Sons, New York.

Law, J. H. and F. E. Regnier. 1971. Pheromones. Ann. Rev. Biochem. 40:533-548.

Mason, R. R. and J. W. Baxter. 1967. Studies of the biology and behavior of the Douglas-fir tussock moth, 1966. USDA For. Serv. Pac. Northwest For. and Range Exp. Stn., Corvallis, Oregon. 28 pp. Unpubl.

McDunnough, J. C. 1921. New British Columbia tussock moth, Hemerocampa pseudotsugata McD. Can. Entomol. 53:53-56.

Miller, C. A. and G. A. McDougall. 1973. Spruce budworm moth trapping using virgin females. Can. J. Zool. 51:853-858.

Myers, J., and L. P. Brown. 1969. A behavioral analysis of the courtship pheromone receptors of the Queen butterfly, Dataus gilippus berenice. J. Insect Physiol. 15:2117-2130.

Ohbayashi, N., T. Yushima, H. Noguchi, and Y. Tamaki. 1973. Time of mating and sex pheromone production and release of Spodopteraclitura (F.)(Lepidoptera: Noctuidae). Konchu 41:389-395.

Payne, T. C., H. H. Shorey, and L. K. Gaston. 1970. Sex pheromones of noctuid moths: Factors influencing antennal responsiveness in males of Trichoplusia ni. J. Insect Physiol. 16:1043-1055.

Percy, J. E., E. J. Gardiner, and J. Weatherston. 1971. Studies of physiologically active arthropod secretions VI. Evidence for a sex pheromone in female Orgyia leucostigMa (Lepidoptera: Lymantriidae). Can. Ent. 103:706-712.

Pliske, T. E., and T. Eisner. 1969. Sex pheromone of the queen butterfly: Biology. Science 164:1170-1172.

Riddiford, L. M. 1967. Trans-2-hexenal mating stimulant of polyphemus moths. Science 158:139-141.

Riddiford, L. M. and C. M. Williams. 1967a. Volatile principle from oak leaves: Role in sex life of the polyphemus moth. Science 155:589-590. 90

Riddiford, L. M., and C. M. Williams. 1967b. Chemical signaling between moths and host plant. Science 156: 541.

1971. Role of the Corpora cardiaca in the behavior of saturniid moths. I. Release of sex pheromone. Biol. Bull. 140:1-7.

Riotte, J. C. E. 1973. Summary and discussion of studies on North American species of the genus Orgyia (Lepidoptera: Lymantriidae). Ent. Zeits. 63(14): 159-162.

Roelofs, W. L., A. Cameau, A. Hill, and G. Milicevic. 1971. Sex attractant of the codling moth: Character- ization with electroantennogram technique. Science 174:297-299.

Roelofs, W. L. 1977. An overview-the evolving philoso- phies and methodologies of pheromone chemistry. In "Chemical Control of Insect Behavior Theory and Application". (H. H. Shorey and J. J. McKelvey, eds.). pp. 287-297. John Wiley and Sons, New York.

Rudinsky, J. A. 1973. Multiple functions of the Douglas- fir beetle pheromone 3- methyl -2- cyclohexene -1 -one. Environ. Entomol. 2:579-585.

Saario, C. A., H. H. Shorey, and L. K. Gaston. 1970. Sex pheromones of noctuid moths. XIX. Effectof environmental and seasonal factors on captures ofmales of Trichoplusia ni in pheromone-baited traps. Ann. Entomol. Soc. Am. 63:667-672.

Sanders, C. J., and G. S. Lucuik. 1972. Factors affecting calling by female eastern spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae). Can. Entomol. 104:1751-1762.

Schneider, D. 1969. Insect olfactions: Deciphering system for chemical messages. Science 163:1031-1037.

Schwinck, L. 1958. A study of olfactory stimuli in the orientation of moths. Proc. Int. Congr. Entomol. 10th, 1956 Vol. 2:577-582.

Sekul, A. A. and H. C. Cox. 1967. Response of males to the female sex pheromone of the fall army worm Spodoptera frugiperda (Lepidoptera: noctuidae): A laboratory evaluation. Ann. Entomol. Soc. Am. 60: 691-693. 91

Sharma, R. K., R. E. Rice, H. T. Reynolds, andH. H. Shorey. 1971. Seasonal influence and effect of trap location on catches of pink bollworm males in sticky traps baited with hexalure. Ann. Entomol. Soc. Am. 64:102-105.

Shorey, H. H. 1964. Sex pheromones of noctuid moths. II. Mating behavior of Trichoplusia ni (Lepidoptera: Noctuidae) with special reference to tare role of the sex pheromone. Ann. Entomol. Soc. Am. 57:371-377.

Shorey, H. H. and L. K. Gaston. 1964. Sex pheromones of noctuid moths. III. Inhibition of male responses to the sex pheromone in Trichoplusia ni (Lepidoptera: Noctuidae). Ann. Entomol. Soc. Am. 57:775-779.

1965. Sex pheromones of noctuid moths. V. Circadian rhythm of pheromone-responsiveness in males of Autographa californica, Heliothis virescens, Spodoptera exigna, and Trichoplusia ni (Lepidoptera: Noctuidae). Ann. Entomol. Soc. Am. 5lf:597-600.

Shorey, H. H., S. U. McFarland, and L. K. Gaston. 1968a. Sex pheromone of noctuid moths. Changes in pheromone quantity, as related to reproductive age and mating history, in females of seven species of noctuidae (Lepidoptera). Ann. Entomol. Soc. Am. 61:372-376.

Shorey, H. H., K. L. Morin, and L. K. Gaston. 1968b. Sex pheromones of noctuid moths. XV. Timing of development of pheromone-responsiveness and other indicators of reproductive age in males of eight species. Ann. Entomol. Soc. Am. 61:857-861.

Shorey, H. H. and L. K. Gaston. 1970. Sex pheromones of noctuid moths. XX. Short-range visual orientation by pheromone-stimulated males of Trichoplusia ni. Ann. Entomol. Soc. Am. 63:829-832.

Shorey, H. H. 1973. Behavioral responses to insect pheromones. Ann. Rev. Entomol. 18:349-380.

Shorey, H. H. and S. R. Farkas. 1973. Sex pheromones of Lepidoptera. 42. Terrestrial odor-trail following by pheromone-stimulated males of Trichoplusia ni. Ann. Entomol. Soc. Am. 66:1213-1214.

Shorey, H. H. 1974. Environmental and physiological control of insect sex pheromone behavior. In "Phero- mones" (M. C. Birch, ed.). pp. 62-80. Am. Elsevier, New York. 92 Shorey, H. H. 1976. "Animal Communication by Pheromones." Academic Press, New York. 167 pp.

Shorey, H. H. 1976. Interactions of insedts with their chemical environment. In "Chemical Control of Insect Behavior Theory and Application." (H. H. Shorey and J. J. McKelvey, Jr., eds.). pp. 1-5. John Wiley and Sons, New York.

Shorey, H. H., and J. J. McKelvey, Jr. 1977. "Chemical Control of Insect Behavior Theory and Application." John Wiley and Sons, New York. 414 pp.

Smith, R. G., G. E. Daterman, and G. D.Daves, Jr. 1975. Douglas-fir tussock moth: Sex pheromone identification and synthesis. Science 188:63-64.

SooHoo, C. F., and R. J. Roberts. 1965. Sex attraction in Rhopaea (Coleoptera: Scarabaeidae). Nature (London) 205:724-725.

Sower, L. L., H. H. Shorey, and L. K. Gaston. 1970. Sex pheromone of Noctuid moths XXI. Light: dark cycle regulation and light inhibition ofsex pheromone release by female Trichoplusia ni. Ann. Entomol. Soc. Am. 63:1090-1092.

Sower, L. L., L. K. Gaston, and H. H. Shorey. 1971a. Sex pheromones of Noctuid moths. XXVI. Female release rate, male response threshold, and communication distance of TrichoplUsia ni. Ann. Entomol. Soc. Am. 64:1448-1456.

Sower, L. L., H. H. Shorey, and L. K. Gaston. 1971b. Sex pheromones of noctuid moths. XXV. Effects of temperature and photoperiod on circadian rhythms ofsex pheromone released by females of Trichoplusia ni. Ann. Entomol. Soc. Am. 64:448-492.

Sower, L. L., J. A. Coffelt, and K. W. Vick. 1973a. Sex pheromone: A simple method of obtaining relatively pure material from females of five species of moths. J. Econ. Entomol. 66:1220-1222.

Sower, L. L., J. S. Long, K. W. Vick, and J. A. Coffelt. 1973b. Sex pheromone of the angounois grain moth: Effects of habituation on the pheromone response of the male. Ann. Entomol. Soc. Am. 66:991-995.

Sower, L. L., R. S. Kaae, and H. H. Shorey. 1973c. Sex pheromones of lepidoptera: XLI. Factors limiting potential distance of sex pheromone communication in 93

Trichoplusia ni. Ann. Ent. Soc. Am. 66:1121-1122.

Sower, L. L., and G. E. Daterman. 1977. Evaluation of synthetic sex pheromone as a control agent for Douglas-fir tussock moths.Env. Ent. 6:889-892.

Sugden, B. A. 1957. A brief history of outbreaks of Douglas-fir tussock moth, Hemerocampa pseudotsugata McD., in British Columbia. Entomol. Soc. B. C. Proc. 54:37-39.

Tamaki, Y. 1977. Complexity, diversity, and specificity of behavior-modifying chemicals in lepidoptera and diptera. In "Chemical Control of Insect Behavior Theory and. Application". (H. H. Shorey, and J. J. McKelvey, Jr., eds.) 253-285 pp. John Wiley and Sons, New York.

Teale, E. W. 1961. "The Insect World of J. Henre Fabre". Dodd, Mead, New York. 333 pp.

Teetes, G. L., and N. M. Randolph. 1970. Color preference and sex attraction among sunflower moths. J. Econ. Entomol. 63:1358-1359.

Thompson, G. L., and L. J. Peterson. 1978. Rearing the Douglas-fir tussock moth. USDA Combined Forest Pest Research and Development Program. Agr. Handbook No. 520.

Traynier, R. M. M. 1968. Sex attraction in the mediter- ranean flour moth Anagasta kthniella: Location of the female by male. Can. Entomol. 100:5-10.

. 1970.. Habituation of the response to sex pheromone in two species of Lepidoptera with reference to a method of control. Entomol. Exp. Appl. 13:179- 181.

Tunnock, S. 1973. The Douglas-fir tussock moth in the northern region - A cartographic history of the outbreaks from 1928 to 1973. USDA For. Serv.,Div. State and Priv. For., Missoula, Mont. Report 73-27. 18 pp.

Vick, K. W., P. C. Drummond,and J. A. Coffelt. 1973a. TrogoderMa inclutum andT. glabrum: Effects of time of day on production ofYemale pheromone, male res-

ponsivenegs, and mating. Ann. Entomol. Soc. Am. 66: 1001-1004. ,94 Vick, K. W., P. C. Drummond,L. L. Dower, and J. A. Coffelt. 1973b. Sex pheromone habituation: The effects of habituationon the pheromone response level of Trogoderma inclusum(Coleoptera: Dermesti- dae). Ann. Entomol. Soc. Am. 66:667-670.

Vita, J. P., R. I. Gara, andH. D. Van Scheller. 1964. Field observations on theresponse to attractants of bark beetles infesting pines. Contrib. Boyce Thomp- son Inst. 22:39-50.

Waldorf, E. S. 1974. Sex pheromone in the springtail, Sinella cUrviseta. Environ. Entomol. 3:916-918.

Wellington, W. G. 1976. Applying behavioral studies in entomological problems. In "Perspectives in Forest Entomology" (J. F. Anderson, andH. K. Kaya, eds.), pp. 87-97. Academic Press, San Francisco.

Wharton, M. L., and D. R. A. Wharton.1957. The production of sex attractant substanceand of oothecae by the normal and irradiated AmericanCockroach, Periplaneta Americana L. J. Insect Physiol. 1:229- 239.

Wickman, B. E. 1958. Mortality of white fir following defoliation by the Douglas-fir tussockmoth in California, 1957. USDA For. Serv. Res. Note 137. 4 pp. Calif. For. and Range Exp. Stn., Berkeley, Calif.

Wickman, B. E., R. R. Mason, and C. G.Thompson. 1973a. Major outbreaks of the Douglas-fir tussockmoth in Oregon and. California. USDA For. Serv. Gen. Tech. Rep. PNW-5. 18 pp. Pac. Northwest For. and Range Exp. Stn., Portland, Oregon.

Wickman, B. E., G. C. Trostle, and P. E. Buffman. 1973b. Douglas-fir tussock moth. USDA For. Serv. For. Pest Leaflet 86. 6 pp (revised).

Wickman, B. E., R. R. Mason, and H. G. Paul. 1975. Flight, attraction, and mating behavior of the Douglas-fir tussock moth in Oregon. Environ. Entomol. 4:405-408.

Wilson, E. 0. 1963. Pheromones. Sci. Amer. 208:100-114.

Wood, D. L., R. M. Silverstein, andM. Nakajima. 1970. "Control of Insect Behavior byNatural Products." Academic Press, New York. 345pp. 95

Wood, D. L. 1977. Manipulation of forest insect pests. In "ChemicalControl of Insects Theory and Applica- tion" (H. H.Shorey, and J. J. McKelvey, Jr., eds.). pp. 369-384. John Wiley and Sons, New York.

Wright, R. H. 1958. The olfactory guidance of flying insects. Can. Entomol. 90:81-89.