Group Title: role of semiochemicals in the behavior of the horn fly, Haematobia irritans (L.), (Diptera: Muscidae) /
Title: The Role of semiochemicals in the behavior of the horn fly, Haematobia irritans (L.), (Diptera: Muscidae)
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 Material Information
Title: The Role of semiochemicals in the behavior of the horn fly, Haematobia irritans (L.), (Diptera: Muscidae)
Alternate Title: Haematobia irritans
Physical Description: xv, 212 leaves : ill. ; 28 cm.
Language: English
Creator: Bolton, Herbert Thomas, 1946-
Publication Date: 1980
Copyright Date: 1980
 Subjects
Subject: Horn fly -- Behavior   ( lcsh )
Horn fly -- Control   ( lcsh )
Pheromones   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 196-211.
Statement of Responsibility: by Herbert Thomas Bolton.
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00099374
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000014382
oclc - 06700010
notis - AAB7602

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THE ROLE OF SEMIOCHEMICALS IN THE BEHAVIOR OF THE
HORN FLY, Haematobia irritans (L.),
(DIPTERA: MUSCIAE





By

HERBERT THOMAS BOLTON


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY











UNIVERSITY OF FLORIDA


1980






























To Bryce and Adam B.

As long as the moon rises,
As long as the grass grows green,
As long as the river flows,
We will be friends,
We will live in peace.













ACKNOWLEDGMENTS


I wish to express my sincere appreciation to Dr. J.F. Butler,

chairman of my supervisory committee, for his guidance, advice, and

concern for my professional growth during this study.

To the Chief, Bureau of Medicine and Surgery, Department of the

Navy, I would like to express my deepest gratitude for the opportunity

to pursue this graduate study. I am especially indebted to Commander

J.A. Mulrennan, Jr., MSC, USN (retired) who on several occasions

assisted me in making this opportunity for study possible.

I gratefully acknowledge the continual assistance of the members

of my graduate committee:

Dr. D.A. Carlson, Insects Affecting Man and Animals Research

Laboratory, AR, SEA, USDA, for sharing his knowledge of insect pheromone

chemistry, for critically reviewing the chemical aspects of this study,

and for allowing me access to his laboratory facilities.

Dr. R.C. Littel, Department of Statistics for his discussions on

experimental design and statistical analysis of data.

Dr. J.L. Nation, Department of Entomology and Nematology, for his

discussions, particularly in the early stages of this study, on insect

pheromones and insect behavior.

Dr. R.H. Roberts, Insects Affecting Man and Animals Research

Laboratory, AR, SEA, USDA, for sharing his knowledge of horn fly

biology and behavior of Diptera throughout this study.







I also wish to thank the following individuals:

Dr. D.E. Weidhaas, Director, Insects Affecting Man and Animals

Research Laboratory AR, SEA, USDA, for generously allowing me to utilize

the facilities at that laboratory; Dr. D.G. Haile, Insects Affecting

Man and Animals Research Laboratory, AR, SEA, USDA, for assistance with

computer programming; Mr. W. Offen, Department of Statistics, for dis-

cussion concerning statistical analysis of data; Dr. N.C. Leppla,

Insect Attractants, Behavior, and Basic Biology Research Laboratory,

USDA, for providing access to the video camera equipment in his labora-

tory; and Dr. T.J. Walker, Department of Entomology, for reviewing

portions of this dissertation.

I extend special thanks to Ms. D. Simon, Mrs. T. Boyd, and Mr. W.

Smith for their assistance in the biological aspects of this study and

to Mr. K. Konya, Mrs. N. Chen-Langenmayr, and Ms. S. King for their

assistance in chemical aspects of this work.

To my wife, Margie, who simultaneously completed her own graduate

program, and my sons, Bryce and Adam, I am fondly indebted for their

support and affection.














TABLE OF CONTENTS


ACKNOWLEDGMENTS. ........... . . . . . . .

LIST OF TABLES ...... . . . . . . .

LIST OF FIGURES. . ... .. ... . . . . . . .

ABSTRACT . . . . . . . . . . . . .

CHAPTER

1 INTRODUCTION . . . . . . . . . . . .

2 LITERATURE REVIEW . . . . . . . . . .


Page

iii



xi

xiv


The Horn Fly . . .. . . .
History and Distribution ..
Bionomics . . . . . .
Mating . . . . .
Oviposition . . . .
Development . . . .
Feeding . . . . .
Longevity and seasonal occur
Host preference .. ....
Flight Behavior .. . ....
Dispersal . . .
Orientation to a host . .
Economic Damage .. . ....
Direct damage . . . .
Disease transmission ..
Control
Courtship and Mating Behavior of the D
General Considerations .. ...
Courtship and Mating Behavior of
The horn fly . . . .
The face fly . . . .
Fannia species . . . .
The house fly . . . .
The stable fly . . .
The tsetse flies . . .
Sex Pheromones of Diptera ...
General Considerations ...
Sex Pheromones of the Muscidae .


rence . .








iptera .

the Muscidae.








Page

The house fly . . . . .. . . 30
The face fly. ... ....... . ... 32
The stable fly. .... . ..... . .. 33
Fannia species . ........ . ... 34
The tsetse fly .. . . . . . . 35
The horn fly. .. . .... .. . . ... 36

3 THE COURTSHIP BEHAVIOR OF THE ADULT HORN FLY,
Haematobia irritans (L.), UNDER LABORATORY CONDITIONS 43

Abstract. . . . . . .. .. . . . . 43
Introduction. ... ....... ..... . 43
Materials and Methods . ... .. .. .. ..... 44
Standard Methods of Rearing and Maintaining
Horn Flies . . . . . . . . . 44
Methods for Observing Horn Fly Behavior. . . . 45
Standard Methods of Handling Horn Flies. .. .... 46
Experiments on Courtship Behavior of Untethered
Pairs . . . . . . . . . . . 47
Experiments on Female-Produced Stimuli Affecting
Courtship of Males .. . .. .. ... 47
Results . . . . . . . . . . 49
Preliminary Observation on Horn Fly Behavior in
the Laboratory . . . . . . .. .. .... 49
General Description of Courtship Behavior. .. . 50
Courtship Behavior of Untethered Pairs of Males
and Females. .. .. . . .. . . . 78
Courtship Behavior of Tethered Horn Flies. .... . 90
Discussion. ... ........... .. . ..... .100

4 A MATING STIMULANT PHEROMONE OF THE HORN FLY,
Haematobia irritans (L.): DEMONSTRATION OF BIOLOGICAL
ACTIVITY IN SEPARATED CUTICULAR COMPONENTS. .. ... 104

Abstract. .. . .. . . . . .. .... 104
Introduction. .. ... .. .. ... . . . 104
Materials and Methods .. . .. .... . 106
Results ............. . ..... . .. .... 110
Discussion. . .. .. . . . . .. . . .. 123

5 THE MATING STIMULANT PHEROMONE OF THE HORN FLY,
Haematobia irritans (L.): THE EFFECT OF VARYING
CONCENTRATION OI THE HIERARCHY OF COURTSHIP BEHAVIOR. 125

Abstract. . . . . . . .... .. . . 125
Introduction . . . . . . . . . . . 125
Materials and Methods . . . . . . . . 127
Results . . . . . . . . . . .. .. 131
Discussion. .. .. ... . . . . . .. 172










6 EVALUATION OF THE MATING STIMULANT PHEROMONE OF THE
HORN FLY, Haematobia irritans (L.), AS AN ATTRACTANT. . 174

Abstract. ................ . . 174
Introduction. .. . . . .... . . . . 174
Materials and Methods . .. ... .. . .... 175
Results .... ... ...... .. ..... . . 181
Discussion . . . . . . . . . . . 184

APPENDICES

A HORN FLY COURTSHIP DATA .. .. ... . . .. 188

B HIERARCHY OF COURTSHIP RESPONSE DATA. .... . . . 191

REFERENCES...... .. ....... . . ... 196

BIOGRAPHICAL SKETCH. .... ... . . .... .. 212














LIST OF TABLES


Table Page

2-1. Summary of sex pheromone research of the Muscidae .. . 37

3-1. Courtship parameters for untethered virgin male and
female pairs .. ..... ...... .. . . . 87

3-2. Courtship behavior of virgin pairs of H. irritans .. 88

3-3. Behavioral responses of virgin male horn flies to
tethered horn flies .... . . . .. .. .. .... 91

3-4. Behavioral responses of virgin and "mated" male horn
flies to tethered female horn flies .. .... ... ... 93

3-5. Behavioral responses of virgin and "mated" male horn
flies to tethered female horn flies .. . . ... .94

3-6. Hierarchy of courtship behavior of virgin male horn
flies to different treatments of tethered males and
females ............. . . . . . 96

3-7. Comparative hierarchy of courtship behavior of groups
of 1 or 5 virgin males. . . .. .. . . . 99

4-1. Hierarchy of courtship behavior of male H. irritans to
live and treated virgin male and female horn flies. . 112

4-2. Hierarchy of courtship behavior of male H. irritans to
T males treated with selected lipid exptracts .. .. 113

4-3. Hierarchy of courtship behavior of male H. irritans to
T males treated with selected lipid fractions . . .. 115

4-4. Hierarchy of courtship behavior of male H. irritans to
T males treated with hydrocarbon fractions. . . ... 117

4-5. Hierarchy of courtship behavior of male H. irritans to
T males treated with selected synthetic monoolefins . 118

4-6. Hierarchy of courtship behavior of male H. irritans to
T males treated with selected synthetic monoolefins . 119

4-7. Hierarchy of courtship behavior of male H. irritans to
T males treated with selected synthetic monoolefins . 121







Table


4-8. Hierarchy of courtship behavior of male H. irritans to
T males treated with selected synthetic monoolefins . 122

5-1. Quantification of total paraffins and monoolefins
recovered from hexane cuticular rinses of adult horn
flies . . ........... ......... 140

5-2. Quantification of major paraffins recovered from hexane
cuticular rinses of adult horn flies. .. . . ... 141

5-3. Quantification of the major monoolefins recovered from
hexane cuticular rinses of adult horn flies .. . . 150

5-4. Hierarchy of courtship behavior of adult male
H. irritans to hexane-washed T males treated with
selected lipid fractions .. .. .. .. ... .. 152

5-5. Hierarchy of courtship behavior of adult male
H. irritans to hexane-washed T males treated with
selected monoolefins and unbranched (normal)
paraffins .. .. ... .. ... .. . . . . 154

5-6. Hierarchy of courtship behavior of adult male
H. irritans to dead female H. irritans treated with
TZ)-9-tricosene .. .......... ...... 156

5-7. Parameters of profit analyses of elements of the
hierarchy of courtship response versus the dose of
a 1:1:1 mixture of (Z)-5-tricosene, (Z)-9-pentacosene,
and (Z)-9-heptacosene . . . .... .. ..... 171

6-1. The response of 1-2 day old male horn flies to doses of
(Z)-5-tricosene, (Z)-9-pentacosene, and (Z)-9-hepta-
cosene (1:1:1 mixture) tested in the olfactometer . . 182

6-2. The response of 1-2 day old male horn flies to dosages
of (Z)-5-tricosene, (Z)-9-pentacosene, and (Z)-9-
heptacosene (1:1:1 mixture) tested simultaneously in
the olfactometer. .. . . ... . . . . 183

6-3. Number of H. irritans collected in traps containing
5 mg of (ZJ-5-tricosene, (Z)-9-pentacosene, and (Z)-
9-heptacosene (1:1:1 mixture) versus control traps. .. 185

A-i. Percent of time spent by individual adult horn flies
in different activity categories during 5-minute
observation periods . ... ... .. .. . . 188

A-2. Courtship data for pairs of virgin male and virgin
female H. irritans. .. .. ... . . . . 189








Table


B-1. Hierarchy of courtship response of 5-7 day old virgin
male horn flies to hexane-washed T males treated with
varying doses of a 1:1:1 mixture of (Z)-5-tricosene,
(Z)-9-pentacosene, and (Z)-9-heptacosene . . .. . 191

B-2. Number of trials in which specific elements in the
hierarchy of courtship response were observed when
virgin males were exposed to hexane-washed T males
treated with doses of a 1:1:1 mixture of (Z)-5-
tricosene, (Z)-9-pentacosene, and (Z)-9-heptacosene .. 195














LIST OF FIGURES


Figure Page

3-1. Male orienting to a tethered female .. . ... . 52

3-2. Male striking a tethered female from the rear of the
female. .. . . .. . . . . ... . . 55

3-3. Male striking a tethered female from a position at the
side of the female. ... ...... . . .. 57

3-4. Male's prothoracic tarsi rubbing a tethered female's
head when the male was in the most forward position
on the female's dorsum. .. .. . .. .. . . 59

3-5. Male backed on the dorsum of a tethered female and
attempting to copulate .. . .. ... . 61

3-6. Horn flies in copilo (view A) . . . . . . 63

3-7. Horn flies in copulo (view B) . . .. . . . 65

3-8. Horn flies in copulo (view C) .. . .. .. .. .. .... 67

3-9. Female dislodging a male at the termination of
copulation. ... ... .. ...... . . 70

3-10. Male which had arrested its movement on a tethered
female's dorsum .. ... ........ ... . 72

3-11. Male which had arrested its movement after an un-
successful copulatory attempt with a tethered female. . 74

3-12. Tethered female kicking at the male with one of her
metathoracic legs . . . . . . .... .. 77

3-13. Second male striking the dorsum of a male already
courting a tethered female. .. . . . .. . 80

3-14. Two males in a stack upon the dorsum of a tethered
female. . . . . . . . . . .... . 82

3-15. A group of males clustered about a tethered female. .. 84

3-16. Two males striking a tethered female. .. . .. ... 86








Figure Pa ge

3-17. Male copulating with a tethered, dead, winged female. . 98

5-1. Analytical gas chromatogram of total paraffins recovered
from cuticular rinses of virgin male 6-7 day old F
horn flies . . . . . . . . .. .. .. 133

5-2. Analytical gas chromatogram of total paraffins recovered
from cuticular rinses of virgin female 6-7 day old F
horn flies. .. . . . . . 135

5-3. Analytical gas chromatogram of total paraffins recovered
from cuticular rinses of field collected male W horn
flies . . . . . . . . .. . . . . 137

5-4. Analytical gas chromatogram of total paraffins recovered
from cuticular rinses of field collected female W horn
flies . . . .. . . ...... . . . 139

5-5. Analytical gas chromatogram of total monoolefins
recovered from cuticular rinses of virgin male 6-7 day
old F horn flies .. ... . ..... . 143

5-6. Analytical gas chromatogram of total monoolefins
recovered from cuticular rinses of virgin female 6-7
day old F horn flies. . . . . . . .. . 145

5-7. Analytical gas chromatogram of total monoolefins
recovered from cuticular rinses of field collected
male horn flies .. .. . . ... .. . . .. 147

5-8. Analytical gas chromatogram of total moncolefins
recovered from cuticular rinses of field collected
female W horn flies . . .. .. .. . . . .. 149

5-9. The relationship between the number of strikes per hour
and the dose of a 1:1:1 mixture of (Z)-5-tricosene,
(Z)-9-pentacosene, and (Z)-9-heptacosene on hexane-
washed T males. . ..... ... .. .. .. .. ..... .159

5-10. The relationship between the number of arrested move-
ments per hour and the dose of a 1:1:1 mixture of
(Z)-5-tricosene, (Z)-9-pentacosene, and (Z)-9-hepta-
cosene on hexane-washed T males .. . ... . 161

5-11. The relationship between the number of positioning on
the dorsum and the dose of a 1:1:1 mixture of (7)-5-
tricosene, (Z)-9-pentacosene, and (Z)-9-heptacosene on
hexane-washed T males .. .... . .. .. .. .. . 163

5-12. Probit analysis of the dose of a 1:1:1 mixture of
(Z)-5-tricosene, (Z)-9-pentacosene, and (Z)-9-hepta-
cosene applied to hexane-washed T males versus the
number of trials in which strikes were observed . .166







Figure Page

5-13. Probit analysis of the dose of a 1:1:1 mixture of
(Z)-5-tricosene, (Z)-9-pentacosene, and (Z)-9-
heptacosene applied to hexane-washed T males versus
the number of trials in which arrested movements
were observed. .. .. .... .... .. . . .. 168

5-14. Probit analysis of the dose of a 1:1:1 mixture of
(Z)-5-tricosene, (Z)-9-pentacosene, and (Z)-9-
heptacosene applied to hexane-washed T males versus
the number of trials in which positioning on the
dorsum were observed ......... . . .... .170

6-1. Schematic drawing of the olfactometer used in laboratory
bioassays. .. . . .. .. .. .. .. . .. . 178












Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy


THE ROLE OF SEMIOCHEMICALS IN THE BEHAVIOR OF THE HORN FLY,
Haematobia irritans (L.),(DIPTERA: MUSCIDAE)

By

Herbert Thomas Bolton

June 1980

Chairman: Jerry F. Butler
Major Department: Entomology and Nematology

The courtship behavior of mature, well-nourished horn fly,Haematobia

irritans (L.),mlales and females was similar to that of other muscids;

however, unlike other muscids during one element of the courtship se-

quence, the male rubs the female's head with its protarsi. The duration

of courtship and copulation was variable among pairs. Virgin males

courted tethered, virgin females with greater frequency than they

courted tethered, virgin males. Virgin males could readily differen-

tiate females from males and court females regardless of whether the

horn flies were live, dead, winged, or wingless.

Preliminary bioassays on the horn fly suggested that a mating

stimulant pheromone was involved in horn fly courtship behavior. Virgin

males readily courted live or dead females but rarely made mating strikes

upon live males, dead males, or females thoroughly washed with hexane.

Bioassays utilizing the response of virgin males to treated, virgin male

horn flies indicated that female cuticular hydrocarbons were responsible

for inducing male courtship behavior. Specifically, the female paraffin








and monoolefin fractions were biologically active when bioassayed alone

or in combination. Three synthetic monoolefins previously shown to be

the major components in the female monoolefin fraction were biologically

active in bioassays. The compounds (Z)-5-tricosene, (Z)-9-pentacosene,

and (Z)-9-heptacosene were each active, but greater male courtship

behavior was observed when these 3 compounds were bioassayed in com-

bination.

Florida Laboratory Strain (F) males and females and Florida Wild

Strain (W) (field-collected) males and females had similar analytical

gas chromatograms for total paraffins. The F and females had rela-

tively greater quantities of (Z)-b-tricosene, (Z)-9-pentacosene, and

(Z)-9-heptacosene than did F and W males; F and W males had relatively

greater quantities of (Z)-9-tricosene. Bioassays utilizing the response

of virgin males to treated, virgin male horn flies previously washed

with hexane indicated that (Z)-9-tricosene did not attenuate the court-

ship behavior of males toward dead females. Additionally, these bio-

assays demonstrated that with increasing doses of a 1:1:1 combination

of (Z)-b-tricosene, (Z)-9-pentacosene, and (Z)-9-heptacosene, the fre-

quency of successive elements of the courtship hierarchy of the male

was increased.

The combination of the 3 cuticular monoolefins previously demon-

strated to be active as a mating stimulant for male horn flies was tested

as an attractant to horn flies in laboratory olfactometer studies and in

simulated field trials conducted in outdoor screened enclosures. The

combination of (Z)-5-tricosene, (Z)-9-pentacosene, and (Z)-9-heptacosene

was attractive to virgin male horn flies in olfactometer trials; however,

in a simulated field trial, the combination was not attractive to either

sex at the dose tested.













CHAPTER 1

INTRODUCTION



Concomitant with the increased use of synthetic pesticides after

World War II was the decline in use of preventive practices for pest

control and the waning of interest in biological control studies.

Investigation of the long-term effects of pesticide use in the environ-

ment was minimal. As resistance to insecticides developed, public

awareness of the environmental impact of pesticide usage increased, and

anxiety about the acute and chronic effects of pesticides on man emerged,

pest control strategies came under scrutiny (Brand et al., 1979).

The concept of integrated pest management began to form as a

holistic approach to pest control utilizing alternative control methods

while maintaining pesticides as viable tools where appropriate. Among

alternative control methods, behavior modifying chemicals including

pheromones have been investigated more thoroughly as the chemical basis

of insect behavior has become firmly established. Practical applica-

tions of these chemicals include: trapping difficult to obtain species,

trapping to monitor or survey for specific pest species, luring insects

to areas createdd with pathogens or pesticides, mass trapping to suppress

pest populations, and disruption of insect communication (Brand et al.,

1979).

After the early success of the Southeastern Screwwormi Eradiation

Program, Knipling (1972) suggested that eradication of the horn fly was







feasible because this ectoparasite remained in close contact with

cattle and was, therefore, accessible to control. He proposed an

integrated control program involving insecticide treatments to reduce

low population numbers in the beginning of the horn fly season, followed

by release of sterile males to eliminate the remaining horn fly popula-

tion. Field trials with this general strategy have been handicapped by

difficulties in mass rearing the required numbers of horn flies and by

the abnormal behavior of released flies (Eschle et al., 1973; Graham

and Hourrigan, 1977). Chambers (1977) emphasized that the production

and perception of pheromones may be important in the reproduction of

many species; changes in the mating behavior of mass reared insects may

be indicative of a lack of adaptiveness critical to reproductive suc-

cess. Control of the quality of mass reared insects requires an under-

standing of the behavior of the insect paramount to their success in

the field. Laboratory evaluation of this behavior is needed to insure

competitiveness of mass reared individuals with their wild counterparts.

Presently, insect chemical communication is viewed as being more

complex than was originally envisioned. Ultimate use of this communi-

cation in pest management programs requires additional study of

chemically induced behavior under defined contexts and conditions

(Birch, 1978).

The purpose of this investigation was threefold: (1) to examine

and describe the courtship behavior of the horn fly in the laboratory;

(2) to determine if cuticular lipids were involved as seiniochemicals

(Nordlund and Lewis, 1976) in horn fly courtship; and (3) to investigate

candidate pheromones involved in horn fly courtship as possible




-3-


attractants or compounds which might influence the movement of horn

flies among animals in the field.

This dissertation consists of a literature review of the horn fly,

the sex pheromones of the Muscidae, and the courtship behavior of the

Muscidae as well as four chapters containing experimental work, written

in manuscript form, to be submitted for publication.













CHAPTER 2

LITERATURE REVIEW



The Horn Fly


History and Distribution


The horn fly, Haematobia irritans (L.), is an Old World species

and an obligate parasite of cattle as an adult. Although adult horn

fly populations on animals in Europe are usually low, the horn fly

rapidly became a serious pest of livestock in the United States after

its introduction with cattle imported into Philadelphia from Europe in

1885 or 1886 (Marlatt, 1910; Bruce, 1964; Graham and Hourrigan, 1977).

First recognized in 1887 by Riley (1889), the horn fly rapidly spread

to Michigan by 1892 (Davis, 1893), to California by 1893 (Bruce, 1964),

and to Hawaii by 1897 (James and Harood, 1969). By 1900, horn flies

were reported in most of the United States, Canada, and Puerto Rico

(Hargett and Goulding, 1962). Currently, the horn fly is cosmopolitan

in the New World with a general distribution from Venezuela to Canada

(Hargett and Goulding, 1962; Stone et al., 1965; Graham and Hourrigan,

1977).



Bionomics


rating. On the host, horn flies normally mate within 3 days of

eclosion with egg deposition beginning one day later (Bruce, 1942 and







1964; Harris et al., 1968; Schmidt, 1972). In the laboratory, females

begin to mate as much as 24 hours later than when held on the host

(Harris et al., 1968). The female horn fly appears to be monogamous,

but males can inseminate an average of 4.6 females (Harris et al., 1968).

Oviposition. Although the specific stimuli which induce egg

laying are not known (McClintock and Depner, 1954), adult female horn

flies leave their host to oviposit in freshly passed cattle droppings

or on the grass or soil beneath the dropping (McClintock and Depner,

1954; Bruce, 1942 and 1964; Harris, 1962; Sanders and Dobson, 1969).

Droppings older than 10 minutes are unattractive unless the crust is

broken (Bruce, 1964). Greer and Butler (1973) demonstrated that horn

fly larvae could develop in cattle, bison, sheep, and horse manure;

however, larvae are believed to develop only in cattle manure in

nature (Deprer, 1961; Bruce, 1964). According to Bruce (1964), the

female produces 24 eggs in each of 15 batches during her lifetime. By

about as early as the third day after emergence, the female can spend

up to 10 minutes depositing 1-14 eggs before returning to a host.

Females are equally as active in egg laying during the day as during

the night (Sanders and Dobson, 1969; Kunz et al., 1970).

Development. Schmidt et al. (1972) noted a higher percent egg

hatch in more densely populated laboratory cages. In the field in

summer, eggs hatch within 16-24 hours and the newly emerged larvae crawl

into cracks and crevices in the manure. With drying of the manure,

larvae migrate to more moist portions of the dropping (Bruce, 1942 and

1964; McClintock and Depner, 1954). Within a 4 day period, the larvae

develop through 3 instars. The first instar, which is approximately

1.5 mm long by 0.25 mm wide, has no anterior spiracles but has heavily







pigmented knob-like posterior spiracles. At about 10.25 hours, molting

occurs to the slightly larger second instar which has anterior spiracles

with 4-6 branches. About 18-25 hours from the first molt, molting

occurs to the third instar which is approximately 6.5 mm long and 1.0 mm

wide and more highly pigmented than the first 2 instars (Bruce, 1964).

About 92.5 hours from hatch, larvae migrate to the underside of

the dropping or into the soil dependent on the relative moisture con-

tent of the soil and the dropping for pupation (Bruce, 1964; Escher,

1977). The pupal stage lasts about 5.5 days under field conditions.

Adults attempt to fly and locate a host about 1 hour after eclosion

(Bruce, 1964).

Kunz et al. (1970) reported the peak emergence of adults in central

Texas to range from 9-10 days after oviposition during the summer to

14-21 days after oviposition in September. Peak emergence varied by

3-4 days depending on whether the dropping was in the sun or shade.

Fewer horn flies emerged from droppings inhabited by other insects than

from droppings with low numbers of competing insects (Blume et al.,

1970). Droppings artificially covered within 5 minutes after deposition

to eliminate competition from other insects averaged 66.5 emerging flies

per dropping while uncovered droppings averaged 6.6 emerging flies

(Kunz et al., 1970). During the summer months in Florida, Wilkerson

(1974) and Greer (1975) reported an average of 8-19 emerging adults

per dropping.

Temperature and moisture greatly influence egg hatch and immature

development. Melvin and Beck (1931) and Melvin (1934) reported that

egg hatch occurred at 11.33 hours at 34.4 C and at 19 hours at 25-C with

the total developmental time from egg to the adult of 9.9 days at 30"C.




-7-


The egg to egg life cycle has been reported to vary from 9-12 days in

the field (Melvin and Beck, 1931; Bruce, 1942 and 1964; Hargett and

Goulding, 1962) to a maximum of 32 days in the laboratory (Depner,

1962). Recently, Wilkerson (1974) reported developmental times from

egg hatch to adult as follows: 8 days at 35.6-C; 8 days at 31.3"C;

9 days at 26.7'C; 11 days at 22.2'C; and 16 days at 17.8 C.

In both the field and the laboratory, the eclosion of horn flies

follows a circadian rhythm with variation influenced by temperature

(Harris et al., 1971; Hoelscher and Combs, 1971b). Females emerge as

much as 24 hours earlier than males (McClintock and Depner, 1954; Harris

et al., 1971; Hoelscher and Combs, 1971b). Glaser (1924) and Mohr

(1943) have reported sex ratios of 1:1 to 1:1.35.

Feeding. Both sexes are obligate blood feeders as adults. Al-

though early workers cited by Bruce (1942 and 1964) reported that horn

flies fed two or more times a day especially at dawn and dusk, later

investigations indicated that horn flies feed intermittently throughout

the day especially when interrupted by host movement. Utilizing an

electronic recording instrument, Harris and Miller (1969) established

that females feed an average of 12 times per day with feedings dis-

tributed evenly throughout the day. Each feeding lasted an average of

1.2 minutes; cumulatively, 14.3 minutes per day were occupied in feed-

ing. Harris and Frazar (1970) observed that males consumed only two-

thirds as much blood as females.

Lornrvi y and seasonal occurrence. Estimates of adult horn fly

longevity range from 28 days (McClintock and Depner, 1954) to 6-8 weeks

(Bruce, 1964): each estimate is highly influenced by ecological con-

ditions (Bruce, 1964). In the laboratory, Schmidt et al. (1972) found







that greater adult survival occurred in less densely populated cages

with the optimum density from 0.6-9.5 cm3 per fly.

Bruce (1942 and 1964) hypothesized that temperature limits the

presence or absence of horn fly populations while moisture determines

their abundance; warm, moist weather is favorable for horn fly longevity

while hot, dry weather and periods of low temperature are unfavorable.

Morgan (1964) emphasized that the micro-climate of the hair coat mantle

is equally important as is the macro-climate. In many areas of the

United States, adults are found continuously on cattle from spring

until late fall with possible population fluctuations throughout that

period (Wright, 1970). In Florida, horn flies are found on the host

year round; winter populations may average 15 flies per animal while

summer populations are as high as 1000 flies per animal (Butler, 1975).

In the temperate and cooler regions, horn flies diapause in winter

as either third instar larvae or pupae (Depner, 1962: Hoelscher et al.,

1967: Kunz et al., 1972; Hoelscher and Combs, 1971a). In the tropics

and subtropics, horn fly development occurs throughout the year

(Hoelscher et al., 1967). In central Florida diapause in the horn fly

has not been observed (Butler, 1975). Diapause in horn flies is be-

lieved to occur as the combined result of the changing photoperiod to

which adults are exposed and the temperature which the immatures ex-

perience (Depner, 1961 and 1962). Depner (1962) hypothesized that the

host may transmit a "UV" factor to the horn fly which in turn transmits

this factor to its offspring predisposing the pupae to diapause.

Host preference. Although adult horn flies essentially spend

their entire life upon cattle, other hosts such as sheep, goats, horses,

mules, dogs, deer, and humans are occasionally attacked (Bruce, 1942








and 1964) especially if a bovine host is not present (Graham and

Hourrigan, 1977). Horn flies are generally more numerous on black or

dark-colored animals than on light colored animals (Bruce, 1964) and

tend to rest on the dark areas of bi-colored animals (Burns et al.,

1962; Franks et al., 1964). Morgan (1964) hypothesized that these

differences in attraction are due to the flies seeking certain micro-

environmental temperature and humidity requirements. While each cow

in a herd can carry several hundred to 4,000 adult flies (Graham and

Hourrigan, 1977) and occasionally 10,000 flies (Bruce, 1942), bulls are

preferred with populations as high as 20,000 per animal (McClintock and

Depner, 1954; Pfadt, 1962). Bimonthly injection of 250 mg of testos-

terone propionate increased the attractiveness of steers, but weekly

injection of the same dose decreased attractiveness (Dobson et al.,

1970). Short-haired breeds or individuals are preferred hosts (McClin-

tock and Depner, 1954; Bruce, 1964), while Brahman cattle are less

attractive than European breeds (Tugwell et al., 1969).



Flight Behavior


Dispersal. Although early workers reported that horn flies re-

mained on the host continuously with the exception of the ovipositing

female (Bruce, 1938, 1942, and 1964; McClintock and Depner, 1954) and

those flies which were thought to be dislodged from the host at night

and unable to locate a new host (Eddy et al., 1962; Hargett and Goulding,

1962; Bruce, 1964), current investigations indicate that horn flies can

disperse over great distances from the host. Horn flies were captured

up to 8.1 km from release points by Eddy et al. (1969) while Tugwell

et al. (1966) found that adults could travel up to 14,100 m in







4 hours; females were found to move more than males while only 27 of

731 flies trapped on a pasture with 37 cows had been previously associ-

ated with those hosts (Tugwell et al., 1966). Greatest flight activity

occurred in the morning. On the other hand, Hoelscher et al. (1968)

found that fly movement was primarily nocturnal; movement of flies

between animals penned 91.4 m apart was reported. Studies by Kinzer

and Reeves (1974) indicated the following: 1) flies released in the

early morning and evening were more successful in locating a host than

those released during the day; 2) marked horn flies were recaptured in

10 hours from hosts located 11.8 km from the release point; 3) 68-77.

of the flies on one host left that animal within 11 hours; and 4) males

and females dispersed in equal numbers. Kinzer and Reeves (1974)

hypothesized that males and parous or nulliparous females have a strong

tendency to transfer between hosts. Investigation by Janes et al.

(1968), Hayes et al. (1972), and Wilkerson (1974) indicate that a sub-

stantial number of flies do migrate between herds of cattle.

Laboratory studies by Sauerman (unpublished data) using flight

mills demonstrated that recently emerged horn fly females can migrate

up to 3.9 km on energy reserves from the larval stage; adult flies

maintained on blood in the laboratory can fly more than 14.5 km. Al-

though the necessity for repeated blood feeding of the horn fly may

explain the tendency of the horn fly to seek a host (IcClintock and

Depner, 1954; Harris and Miller, 1969), it does not adequately explain

to several investigators the strong tendency that horn flies have to

transfer between hosts (Kinzer and Reeves, 1974; Hoelscher et al.,

1968).








Orientation to a host. After conducting a series of experiments

on the visual and olfactory responses of 2-3 day old horn flies col-

lected from the host and recently emerged, honey-fed horn flies, Hargett

(1962) and Hargett and Goulding (1962) concluded that vision was the

most important factor in aiding the horn fly in finding a host after

emergence. In addition, they found that horn flies: 1) responded

greatest to light filtered at 550 m, and shorter wavelengths; 2) were

attracted to areas of contrast; 3) were attracted to bovine hair and

extracts made from washing bovine hair; 4) probed in response to heat;

5) were negatively geotactic; and 6) showed a lack of humidity preference.

They hypothesized that recently closed flies utilize light stimuli and

negative geotaxis to begin flight. Areas of contrast in the fly's

visual field may override light stimuli; when a specific host comes

into view, vision directs the fly toward it. Kinzer and Reeves (1974)

suggested that horn fly dispersal and location of hosts over distance

were random in regard to wind direction, but directional in regard to

temperature, wind velocity, and humidity.

Kinzer et al. (1970) reported slight but statistically significant

attraction in laboratory olfactometer trials to cow odor and emanations

from a human arm. In other olfactometer studies, Nackley (1977) demon-

strated slight attraction of both males and females to the hydrocarbon

fraction of female cuticular lipids. Kinzer et al. (1978) developed

field olfactometers to measure the attractiveness of selected stimuli

to host seeking horn flies. Temperature and CO2 were the primary fac-

tors found to affect horn fly orientation.








Economic Damage


Direct damage. Stress from fly annoyance and blood loss, which

occur when horn fly populations are above economic thresholds, causes

significant loss in weight and milk production in cattle (Bruce, 1942 and

1964; McClintock and Depner, 1954; Hoelscher and Combs, 1971a). The

preferred feeding sites on animals with heavy infestations may develop

into open sores which are susceptible to screw-worm attack as well as

invasion by other parasites or diseases (Bruce, 1942 and 1964). The

daily consumption of 500 flies is about 7 ml of blood (Harris and

Frazar, 1970); extremely high horn fly populations can cause blood loss

which alone would reduce animal productivity (Butler, 1975). Reductions

in calf production have been reported (McClintock and Depner, 1954). A

one-fourth to one-half reduction in milk production in dairy herds not

protected from biting flies was demonstrated by Granett and Hansens

(1956).

Weight losses to cattle due to horn fly damage have been evaluated

by comparing the weight change in insecticide treated animals to un-

treated animals. Reduction in weight gains up to 67V have been demon-

strated (Laake, 1946; Bruce and Decker, 1951; Cheng, 1958; Cutkomp and

Harvey, 1958; Koehler and Butler, 1976). In Nebraska, calves from cows

without horn fly populations gained 5.4-6.3 kg over calves from untreated

cows (Campbell, 1976). Recent work by Butler and Koehler (1977 and 1979)

in Florida demonstrated statistically significant differences in weight

gain from 10-727' as a result of residual sprays and dust bag control

treatments; treated animals gained 0.06-0.35 kg per day over untreated

animals with high fly populations.








Fly populations as low as 50 per side have been reported as being

economically damaging (Lofgren, 1970). In Florida, untreated animals

may carry an average of 800 flies per animal for up to 9 months of the

year (Butler and Koehler, 1979). Estimated losses in the United States

of over $197 million to the livestock industry have been estimated to

occur per year (Knipling, 1972; Graham and Hourrigan, 1977); other

estimates place the losses in control costs and production losses to

be in excess of S365 million annually (Anonymous, 1976). In Florida,

Butler (1975) estimated an annual primary market loss of 535,382,758

if no horn fly control was conducted in the state.

Disease transmission. Experimental evidence for disease trans-

mission by horn flies is minimal (McClintock and Depner, 1954; Greenberg,

1971 and 1973). But horn flies have been shown to transmit anthrax in

the laboratory. Morris (1918) obtained infections of anthrax in sheep

and guinea pigs fed upon for 1-3 minutes by horn flies which had pre-

viously fed upon guinea pigs infected with Bacillus anthracis; however,

horn flies were not shown to feed upon anthrax carcases naturally.

Glaser (1924) provided data indicating that Trypanosoma theileri could

be transmitted by H. irritans. Trypanosoma congolense and the polio-

virus have been reported uncommonly in association with horn flies

(Greenberg, 1971). The horn fly is additionally an intermediate host

of Stephanofilaria stelesi, a filarial nematode that causes skin lesions

on the underside of cattle (Hibler, 1966). McClintock and Depner (1951)

cited references indicating that the closely related genus, Lyperosia,

contains species suspected of transmitting trypanosomes; nevertheless,

horn flies themselves have not yet been implicated.








Hypothetically, the horn fly has a number of characteristics pro-

viding a high potential for disease transmission: they feed frequently

on cattle; feeding is interrupted in nature because of host response

to bites (Zumpt, 1973); and horn flies have been shown to move exten-

sively within a given herd (see section, "Flight Behavior"). Butler

et al. (1977) indicated that horn flies may be potential mechanical

vectors of diseases transmitted by contaminated mouthparts or infected

feces.

Control. Early efforts to control horn flies by scattering manure

or using various repellants were generally ineffective. Bruce (1938)

developed a moderately effective trap for horn flies which was situated

so that infested animals were forced to walk through the trap. Modern

residual insecticides applied in self-applicating devices such as back

and face rubbers, back oilers, and dust bags have proven to be effec-

tive. Insecticide treated collars, blocks, and ear tags are also

effective (Rogoff and Moxon, 1952; Lindquist and Hoffman, 1954).

Harvey and Brethour (1979) recently demonstrated that treatment of one

animal per herd with pyrethrin gave effective control of horn flies.

Of the many insect species known to prey upon or parasitize the horn

fly, the hymenopterous pupal parasites are the most numerous (Oepner,

1968; Greer, 1975; Escher, 1977); however, in natural situations they

do not appear to keep horn fly populations below economic levels.

Because current control of horn flies is completely dependent on

insecticides with no prospective alternatives available, Knipling

(1972) proposed that the feasibility of horn fly eradication be con-

sidered. He proposed two strategically timed insecticide applications

to reduce fly populations by 99"' followed by sterile fly releases for








6 generations totaling 300 released horn flies per animal. He pro-

jected a cost of $100 million dollars to achieve elimination of horn

flies from the United States, Canada, and Mexico. Sterile male re-

leases in west and central Texas (Kunz et al., 1974) were important

in showing that an isolated location was necessary to demonstrate

eradication. A 3 year integrated control program on the island of

Molokai in Hawaii using a combination of orally administered methoprene

and sterile male release apparently demonstrated that horn flies could

be eliminated from semi-isolated areas (Eschle et al., 1977). In

addition, this pilot study: 1) demonstrated difficulties in mass-

rearing of horn flies; 2) suggested problems in combining certain

control procedures; and 3) indicated that additional in-depth research

was necessary before any large control program could be undertaken

(Graham and Hourrigan, 1977).



Courtship and Mating Behavior of the Diptera

General Considerations

Richards (1927) provided one of the earliest reviews of insect

reproductive behavior from the perspective of sexual selection. He

described how the events in the sexual behavior of a species were thought

to be facilitated by a combination of gustatory, aural, visual, tactile,

or olfactory stimuli from either sex. According to Guhl and Schein

(1963) sexual behavior includes courtship and matiny activities. Court-

ship is "the premating behavior that stimulates one or both individuals

and initiates mating performance" (p. 191); mating is defined synonymous-

ly with copulation. Guhl and Schein (1968) emphasized that the point at

which courtship ends and mating begins can be difficult to determine.







Manning (1966) noted that the occurrence of courtship in insects

was varied and sporadic. For example, in the Muscidae and Calliphoridae,

males appeared to display little if any courtship behavior in compari-

son to the elaborate courtship displays of males of certain Droso-

philidae species. In many Muscid and Calliphorid species, males will

mount dark objects of an appropriate size. Even generalized visual

stimuli are not required since mating will occur in the dark. Also,

copulation with or rejection by a conspecific female can occur

within several seconds. Although this brief period between mounting

and attempted copulation did not seem to allow much time for the ex-

change of complex communication between the sexes, recent investigations

have demonstrated that Muscid males do perform a series of complex

courtship actions (Ewing, 1977). Bastock (1967) stated that arthropod

courtship was surprisingly complex, but was not evoked in any animal

with niechanical consistency.

Chapman (1969) and Matthews and Matthews (1978) have reviewed

general considerations of insect reproductive behavior. Parker (1978a)

listed many of the conventional encounter sites where males and females

meet before initiating courtship behavior. Anderson (1974) reported

that the mating activity of many arthropods of medical-veterinary

importance including many Diptera are closely associated with a host.

Of these arthropods, least is known about the mating behavior of the

Cyclorrhapha.

Although authors such as Richards (1927), Alexander (1964), Bastock

(1967), and Matthews and Matthews (1978) have attempted to consider

insect courtship behavior in an evolutionary perspective, there has

been a more recent attempt to evaluate insect reproductive behavior in








terms of modern sexual selection theory by a number of investigators

(Halliday, 1978; Parker, 1978b;Barrass, 1979; Blum and Blum, 1979;

Borgia, 1979; Otte, 1979). A noteworthy example of research conducted

with this theory in mind is a series of papers on the dung fly,

Scatophaga stercoraria, by Parker (1969, 1970a, 1970b, 1970c, 1970d,

1970e, 1971a, 1971b, 1974). Not only has this research provided experi-

mental data to develop models for sexual selection theory, but it also

represents an excellent example of a field study on the reproductive

behavior of a Dipteran species.



Courtship and eatingg Behavior of the Muscidae


The horn fly. Few details concerning the courtship or mating be-

havior of the horn fly, Haematobia irritans (L.), have been reported.

Bruce (1940, 1942) stated that mating flies had been observed in the

field as early as the second day after emergence. Hammer (1941) had

reported seeing copulating pairs of horn flies on the sides of cows;

however, he did not describe the courtship sequence preceding copula-

tion. Bruce (1964) noted that mating occurs on host animals and that

copulating pairs had been seen on vegetation near the host. Mating

pairs remained in copulation about 0.5 to 5.0 minutes. Anderson (1974)

listed Haematobia irritans as a species that mated on the host based on

the unpublished data from California of J.R. Anderson, E.C. Loomis, and

R. Fontaine. Courtship behavior was observed on cows but not in the

air around the host; males would jump or walk onto resting females and

if successful in copulating would remain in copulo with the female on

the host (Anderson, pers. comm., 1979, Division of Entomology and

Parasitology, University of California, Berkeley, CA). The data of








Harris et al. (1963) suggested that the horn fly is monogamous but males

inseminated an average of 4.6 females and up to as many as 8 females.

In the laboratory, mating occurred as early as 2 days after emergence;

most flies had mated by the end of the fourth day; however, when flies

were placed on a cow, mating began as early as 1 day after emergence,

and all flies had mated by the end of the second day.

The face fly. The mating habits and courtship behavior of the

face fly, Musca autumnalis DeGeer, have been investigated by a number

of researchers. Hammer (1941) noted that conspicuous objects in fields

such as a rock, cow, or cart were probable mating sites. Resting males

would catch females flying by. After ascending slightly as a couple,

they would fall to the ground to complete mating. Field observations

by Teskey (1969) confirmed Hammer's descriptions. Ode and Matthysse

(1967) observed mating pairs of face flies on the sunny sides of farm

houses in April after flies had emerged from hibernation. Female face

flies have been reported to mate once by Teskey(1960, 1969) and Jones

(1964) but more than once by Wang (1964). Killough and McClellan (1969)

observed that most females mate only once, but that those females

attempting to mate again had not obtained sperm during the first copu-

lation. Males mate repeatedly (Teskey,1969) with an average of 4 fe-

males (Lodha et al., 1970).

Most matings occurred between flies which were 3-7 days old (Wang,

1964; Teskey,1969; Lodha et al., 1970) but mating can begin 36 or 48

hours after pupal eclosion for males and females, respectively (Lodha

et al., 1970). Chaudhury and Ball (1974) noted that the period of

maximum attraction and insemination between mature virgin females (4-8

days old) and mature unmated males (5-6 days old) occurred around noon








and reached a minimum in the evening. Various authors have reported

the following times for the duration of copuation: 5 minutes-4 hours

(Wang, 1964); 60-85 minutes (Teskey, 1969); 44-76 minutes (Killough and

McClellan, 1969); and 25-135 minutes (Lodha et al., 1970). The average

copulation duration necessary for effective sperm transfer was 66.2

minutes; sperm transfer began after the first 4 minutes of copulation

(Lodha et al., 1970).

Teskey(1969) and Lodha et al. (1970) described some aspects of the

courtship behavior of face flies; however, Tobin and Stoffolano (1973b)

presented a detailed analysis of face fly courtship behavior and com-

pared the courtship behavior of both the face fly and house fly. In

a separate study, Tobin and Stoffolano (1973c) investigated how dif-

ferences in the courtship behavior of face flies and house flies may

play a role in preventing hybridization between these closely related

species.

Fannia species. The mating behavior of several species of the

genus Fannia has been described. Tauber (1968) reported details of

the courtship sequence of both Fannia femoralis Stein and F. canicularis

(L.). In several studies, Koyama (1962a, 1962b, 1974) has invesitaged

the sexual and physiological aspects of mating behavior of F. scalaris

Fabricius.

Male F. femoralis walk, jump, or fly onto the female's dorsum

beginning a sequence of precopulatory behavior similar to those of

F. canicularis; however, initial contact between the sexes of F. cani-

cularis can occur in flight (Tauber, 1968). Anderson (1974) cited his

unpublished data indicating that F. canicularis and F. femoralis mate

at the emergence sites on poultry ranches in California.








F. femoralis females mate only once unless the previous mating was

infertile. Both the length of courtship and the termination of copula-

tion appear to be controlled by the female. A non-receptive virgin or

previously mated female thwarted the advances of males by (1) kicking

with her legs; (2) arching her abdomen downward; (3) holding her wings

overlapped on her back; or (4) raising and rapidly moving her wings

(Tabuer, 1968).

F. scalaris Fabricius were observed swarming and mating under

willow trees by Koyama (1962a); only physiologically mature males dis-

played mating behavior. Although these males indiscriminately pursued

males and females of different ages, only mature males and middle-aged

virgin females copulated. Trial and error searching by males for fe-

males during precopulatory swarming appeared to be the primary way that

males located females (Koyama, 1962a, 1962b, 1974).

The house fly. The courtship behavior of the house fly, Musca

domestic L., has been extensively studied by numerous investigators;

West (1951), Tobin (1972), and Colwell (1973) review many of the early

descriptive reports on house fly reproductive behavior. As well as

reviewing more current studies on house fly courtship, a number of

recent papers have presented detailed descriptions of house fly court-

ship (Tobin and Stoffolano, 1973a; Colwell and Shorey, 1975, 1976, and

1977) which were obtained using a combination visual observation as well

as still and motion picture photography.

A photographic analysis of house fly courtship by Tobin and

Stoffolano (1973a) provided a detailed description of the courtship

elements and time ranges for each element between virgin males and

virgin females. These females had been anesthetized prior to testing







and tethered in place with an insect pin placed through the thorax.

Barriers to hybridization caused by differences in courtship sequences

between house flies and stable flies were also investigated by Tobin

and Stoffolano (1973c).

Colwell and Shorey (1975) described the stereotyped courtship of

untethered, virgin house flies as follows: While vibrating his wings,

the male jumps or flies onto the dorsum of the female whose wings are

extended to a horizontal position and whose metathoracic legs are raised

behind her wings. As the male moves anteriorly on the female, he may

twist his head to one side and/or extend his proboscis. After reaching

down with his prothoracic legs and manipulating the female's prothoracic

legs, he moves backward on the female and attempts to copu-

late. Colwell and Shorey (1975) suggested that inconsistencies in

existing descriptions of house fly courtship may be due to genetic,

physiological, or environmental differences among strains. Their study

clarified many conflicting descriptions of the elements of courtship

by also emphasizing that variability exists in behavior displayed by

each sex during courtship and by suggesting how experimental conditions

can alter behavioral responses.

Experiments by Colwell and Shorey (1976),in which house flies were

reared without light, confined earlier reports that elimination of

visual stimuli impeded the rate of mating but did not prevent mating.

In addition, they reported that wingless males could still copulate

with females but that the courtship duration was lengthened. The time

to initiate copulation was greatly increased when both males and females

had various appendages amputated which hypothetically interfered with

the male's production or the female's reception of stimuli important








in the continuation of courtship. Murvosh et al. (1964) previously

had reported similar although less detailed findings.

In another investigation, Colwell and Shorey (1977) determined

that males could distinguish between tethered males and tethered females

even though a variety of visual, acoustical, and behavioral differences

between the sexes were eliminated. Female-produced chemicals) per-

ceived by both olfaction and contact chemoreception appeared to be

important in stimulating males to continue through the entire court-

ship sequence. The number of courtship responses between live males

and dead males increased with the age of the dead male while the number

of courtship responses between live males and dead females decreased

with the age of the dead female. At 20 days of age, no differences

existed between the number of courtship responses elicited by dead

males and females; therefore, Colwell and Shorey (1977) hypothesized

that early termination of courtship on normal males may be due, in part,

to inhibitory chemicals associated with males while continuation of

courtship on normal females may be due to excitatory chemicals which

are gradually lost after death.

Chabora (1978) reported reduced rates of courtship and mating in

a green-eyed, visually deficient mutant strain. Reciprocal crosses

between this deficient strain and a normal strain indicated that a

visual component appeared to be involved in the courtship of both males

and females. In addition, no evidence for a rare male advantage was

found which agreed with the previous findings of Childress and McDonald

(1973).

Zingrone et al. (1959) had established that most females are

monogamous with only 2.: of the females mating a second time. Cowan







and Rogoff (1968) demonstrated that individual males varied in their

mating responsiveness to control pseudoflies and pseudoflies treated

with benzene extracts of female flies. A greater number of flies

responded to treated pseudoflies than to controls. The activity of

individual responding flies to treated pseudoflies also occurred. The

response tendencies of males appeared to be inherited.

The stable fly. The mating behavior of the stable fly, Stomox s

calcitrans (L.),has been investigated in a number of separate studies

(Parr, 1962; Killough and McKinstry, 1965; Harris et al., 1966; Muhammed,

1975; Anderson, 1978). Field studies in Uganda by Parr (1962) indicated

that stable flies mated when each sex was 6 days old; yet Killough and

McKinstry (1965) reported that 1-day old males or females in the

laboratory could mate with 5-day old individuals of the opposite sex.

The data of Harris et al. (1966) showed that a few male and female

stable flies mated as early as 2 days after emergence; most had mated

by the fifth day. The female stable fly was reported to mate only

once, but the male mated as many as 9 females.

Muhammed (1975) compared and contrasted the sexual behavior of

laboratory stable flies with the descriptions made by Tobin and Stof-

folano (1973a) for the house fly. Of the seven generalized stages of

interactions which were observed between virgin males and virgin females,

he identified that head touching of the female by the male's proboscis while

the male positioned himself on female was the most important element

in stable fly courtship; this element occurred more frequently than

others and was reverted to and repeated if later events in the court-

ship sequence were disturbed. The "boxing" stage in house fly court-

shio described by Tobin and Stoffolano (1973a) in which the male's and







female's prothoracic legs touch was not observed in the stable

fly.

Anderson (1966) described basic aspects of stable fly court-

ship which were more fully elaborated upon in a recent report (Ander-

son, 1978). He described that the general mating behavior of properly

nourished, sexually mature stable flies observed in laboratory cages

was similar to the courtship behavior of the house fly and face fly as

described by other researchers. His descriptive account of the se-

quence of courtship events between aggressive, virgin males and recep-

tive and nonreceptive virgin females included details of how the male

positions himself on the female's dorsum and how the legs and wings of

each sex are positioned. No mention was made in this account of

touching the female's head by the male's proboscis as described by

Muhammed (1975).

By observing male and female pre-mating behavior in the field,

Bushman (pers. comm., 1978, Insects Affecting Man and Animals Research

Laboratory, USDA, Gainesville, FL) suggested that the initial male-

female contact in nature usually occurs in flight. This in-flight

contact precedes the courtship sequences described by most researchers

who observe mating behavior of caged laboratory flies. Muhammed (1975)

had noted that on occasion males did intercept females in flight in

laboratory observation cages. After the male and female fell to the

cage bottom, courtship behavior was resumed. Hanrer (1941) had also

described field observations of this phenomenon for a number of genera

including Stomoxvs.

The tsetse flies. Mellanby (1936) observed that mating occurred

rapidly in the laboratory when virgin male and female Glossina plpalis(R.D.)







were placed together. Often many males would court 1 female until 1

male succeeded in copulating with her. Males would then court the

next most attractive female while some females were left uncourted.

Squire (1951) described the general events occurring during the court-

ship of G. palpalis (R.-D.). A male would quickly mount a female who

had opened her wings. The male's hypopigium gripped the female's

abdomen and the claspers grasped the copulatory cushions on the female's

sixth segment. Disturbed pairs readily took flight with the female

essentially carrying the male along with her.

G. palpalis fuscipes males can seize females which are resting,

feeding, or flying. When copulating, the male is on the female's dor-

sum; her wings are slightly spread and bent. The tarsi of the pro-

thoracic legs grasp the female between the head and thorax, the male's

mesothoracic legs vary in position, but the male's metathoracic legs

trail behind or rest upon the female's wings. Before separating,

copulating pairs begin to move around (Buxton, 1955). Dean et al.

(1969) noted that virgin female G. morsitans orientalis Vanderplank

with partly opened wings passively accept males. Squire (1952) noted

that G. palpalis females were often seized by males in mid-air and

grasped with the claspers.

In G. morsitans orientalis, laboratory reared males are visually

attracted to moving objects and apparently can not distinguish females

until within 1-2 cm; females do not appear to seek out males. Squire

(1951) described the position of the male and female genitalia during

copulation of G. palpalis, and Pollock (1973) related the internal

anatomy of G. austeni Newstead females to copulatory process.







A number of investigators have described the means by which fe-

males reject advances by males. Mellanby (1936) stated that it appeared

impossible for males to copulate with a reluctant female G. palpalis

which had closed its wings and bent its abdomen downward. Squire

(1951) and Jordan (1958) observed that G. palpalis females repelled

male courtship attempts with vigorous wing movement or closed wings.

Mated females of Glossina morsitans orientalis also thwarted further

copulatory attempts by males by bringing the wings together, by curving

the abdomen downward, or by shaking the male off of her dorsum.

Nash (1955) reported that some G. palpalis pairs will mate within

24 hours of emergence. In G. morsitans orientalis the female's

willingness to accept males decreases as the number of successful

or unsuccessful copulatory attempts increases (Dean et al., 1969);

females copulating less than 45 minutes received no sperm. Rogers

(1973c) reported that the copulation duration of G. pallidipes Austen averaged

less than 26 minutes and decreased in duration slightly with older

females; other Glossina sp. rarely copulate less than 60 minutes.

The copulation duration of G. palpalis decreases with the age of the

female as follows: 1-3 day old, 2 hours; 4-9 days old, 75 minutes;

and 10 days old, less than 1 hour (Jordan, 1958).

Squire (1951) claimed that G. palpalis (R.-D.) females may be

mated many times; however, a single fertilization can provide the fe-

male with sufficient sperm for her lifetime (Buxton, 1955). Jordan

(1958) demonstrated that relating was most frequent with young G.

palalis females. Dame and Ford (1968) cited evidence for multiple

mating of both males and females of G. morsitans in the laboratory but

noted that the frequency of remating in nature was unknown. Rogers








(1973a) reported that of 109 female G. pallidipes caught on a bait

animal, 13 were previously inseminated but 2 females were only partly

inseminated. In the laboratory, 75 G. pallidipes males were repeatedly

mated; 32 males mated 1 time; 15 males mated 2 times; 3 males mated 3

times; and 1 male mated 4 times in 5 hours (Rogers, 1973b).

Foster (1976) reviewed the conflicting reports concerning the age

at which males of different Glossina species become sexually mature

and presented data demonstrating that blood feeding affects sexual

maturation differently in G. morsitans than in G. austeni. Tobe and

Langley (1978) reviewed general considerations of mating, receptivity,

and reproductive physiology of Glossina while Mulligan (1970) reviewed

general aspects of copulation and insemination of the genus.

Vanderplank (1948) reported on the sound produced during mating

of Glossina sp. Dean et al. (1969) demonstrated that elimination of

sound in mating pairs of G. morsitans orientalis by glueing the wings

and halteres did not alter mating success, but increased the time from

pairing of the sexes until copulation. Ultrasound components (30-70

kHz) produced by mating pairs of G. morsitans differ from feeding

sounds produced by each sex (Erickson and Moller, 1975). Rudrauf

(1977) reported that courting pairs of G. fuscipes fuscipes Newstead

emitted high-pitched frequency signals up to 80 kHz before and between

mating attempts; these signals were not detected from non-courting

pairs.







Sex Pheromones of Diptera


General Considerations


In 1971, Law and Regnier proposed the term semiochemical (GK.

simeon, a mark or signal) to describe those chemicals involved in the

chemical interaction between organisms. The tenn pheromone (GK.

pherein, to carry, and horman, to excite or to stimulate) was coined

by Karlson and Butenandt in 1959 to describe chemicals involved in

intraspecific interactions (Nordlund and Lewis, 1976). Within the last

5 years rapid progress has been made in practical research directed

toward the development of pheromones and other behavior-modifying

chemicals for insect pest management (Brand et al., 1979).

The use of pheromones by insects has been the most extensively

studied form of chemical communication (Young and Silverstein, 1975)

as is evidenced by the reviews on insect pheromones that have recently

been published in the form of books (Wood et al., 1970; Beroza, 1970;

Jacobson, 1972; Birch, 1974; Jacobson, 1975; Beroza, 1976; Inscoe and

Beroza, 1976; Shorey, 1976; Shorey and McKelvey, 1977) or review articles

(Shorey, 1973; Mayer and McLaughlin, 1975; Shorey, 1977; Roelofs, 1978;

Weaver, 1978; Brand, 1979). In particular, Young and Silverstein (1975)

provide an excellent review of important considerations in the biological

and chemical methodology in the study of insect communication. Seabrook

(1978) reviewed neurobiological contributions related to understanding insect

pheromone systems. Because insect pheromones can be common components

of the cuticular lipids, the review of insect waxes by Jackson (1976)

is informative.








In general, insects can be highly selective and sensitive to sex

pheromones (Young and Silverstein, 1975). But unlike the sex pheromones

of Lepidoptera, those of the Diptera are usually not a sufficient

stimulus by themselves to evoke male copulatory behavior without simul-

taneous visual stimuli (Shorey, 1973). The ability of Dipterous female

sex pheromones to attract males from a distance is low. Males tend to

aggregate in the vicinity of females due to other stimuli such as

environmental conditions (Shorey, 1973). Anderson (1974) pointed out

that for all arthropods of medical and veterinary importance including

the Diptera, pheromones do not play a major role in bringing the sexes

together, but rather function in recognition of the sexes once they

have met at a given site.

In his review of the behavioral responses of Diptera to pheromones

and other semiochemicals, Fletcher (1977) reported that most pheromones

appear to be complex blends of compounds. Tamaki (1977) addressed the

problem of complexity, diversity, and specificity of pheromones and

semiochemicals in Diptera and Lepidoptera. When considering species

associated with a host, Young and Silverstein (1975) suggested that

synergism of a pheromone may involve compounds emanating from the host.

Several species of Dipteran pests of fruit and vegetable crops

have been controlled with baits consisting of an attractant and pesti-

cide or with attractant baited traps (Brand, 1979; Roelofs, 1979). Of

the Dipteran pests that directly affect man or animals, pheromones have

been identified and field-tested in only a few cases. The house fly

pheromone, muscalure, is one of only two pheromones presently registered

by EPA for use in pest control (Brand, 1979; Roelofs, 1979).







Sex Pheromones of the Muscidae


The house fly. The report by Carlson et al. (1971) that (Z)-9-trico-

sene, commonly called muscalure, was a sex attractant pheromone of the

house fly, Musca domestic L., represented the first isolation, identi-

fication, and synthesis of a sex pheromone from the family Muscidae.

Since that report, sex pheromones, primarily mating stimulant compounds,

of other species in the family have been reported as summarized in

Table 2-1.

A number of independent studies had preceded the identification of

muscalure. Rogoff et al. (1964) demonstrated the presence of a house

fly sex pheromone by the attraction of males to females in olfactometer

trials and by the stimulation of males to mate with pseudoflies treated

with extracts of female flies. These studies were confirmed by Murvosh

et al. (1965) while Mayer and Thaggard (1966) provided additional data

in support of the presence of a sex attractant. Mayer and James (1971),

Silhacek et al. (1972a and 1972b), and Voaden et al. (1972) accomplished

partial isolation of the active compound from the non-polar lipid

fraction. After the identification of muscalure as the sex attractant

by Carlson et al. (1971), Rogoff et al. (1973) confirmed that muscalure

also functioned as a mating stimulant for male house flies.

Several later investigations indicated that (Z)-9-tricosene was not

highly specific and was not the only natural compound responsible for

releasing the sexual behavior of male house flies. A number of struc-

tural analogs of muscalure tested by Carlson et al. (1974) were as

active as muscalure. In 1972, Mansingh et al. reported active members

of a series of C19 to C25(Z)-9-alkenes which induced and maintained








high excitement and mating behavior of male house flies. The greatest

activity demonstrated was for a ratio of (Z)-9-tricosene (70 ) and

(Z)-9-heneicosene (30Y,) which were hypothesized to be a mating stimu-

lant and attractant, respectively; however, these findings could not be

confined by Carlson et al. (1974). The enhancement of mating strike

activity by the combination of certain methyl-branched C27 and C29

paraffins of female origin was documented by Uebel et al. (1976).

Carlson et al. (1974) suggested that the low specificity of Dipteran

sex pheromones such as nuscalure versus sex pheromones of Lepidoptera

may be characteristic of sex pheromones that are not highly potent.

In field evaluations, Carlson and Beroza (1973) found that musca-

lure increased house fly catches in several types of fly traps. Unex-

pectedly, both females and males were attracted to muscalure baited

traps indicating that muscalure might act as an aggregation pheromone

for both sexes. Morgan et al. (1974), while testing (Z)-9-tricosene in

the field in sugar bait formulations, also found that muscalure was an

attractant for both sexes. A field study in open poultry houses by

Mitchell et al. (1975) demonstrated that the addition of (Z)-9-tricosene

to baits in fly traps increased the capture of house flies 2 to 14 times

over traps without (Z)-9-tricosene in the bait. The sex ratios of flies

caught in both treated and untreated traps was similar.

Uebel et al. (1978d) reported that the total non-hydrocarbon frac-

tion of female house flies may be active at 100 and 200 ig in stimu-

lating males to copulate with treated pseudoflies. Isolation and

identification of three major non-hydrocarbon fractions led to the

discovery that two of these compounds were as active or more active at

100 and 200 i:g in pseudofly tests than was (Z)-9-tricosene. The







authors hypothesized that the unsaturated ketone, (Z)-14-tricosen-

10-one, and the epoxide, (cis)-9,10-epoxytricosane, were involved in

the sex pheromone complex of the house fly.

The face fly. Attraction and mating stimulation of male flies by

a volatile sex pheromone extracted from mature female face flies (Musca

autumnalis De Geer) was reported by Chaudhury et al. (1972). The

pheromone was tentatively thought to be an unsaturated hydrocarbon.

Uebel et al. (1975a) isolated and identified the active components of

the pheromone including the straight-chain monoalkenes (Z)-14-nonacosene,

(Z)-13-nonacosene, and (Z)-13-heptacosene. Pheromone activity was

bioassayed by counting mating strikes made by virgin males upon other

virgin males treated with 100 jg, 200 11g, or 300 ,g of selected mono-

olefins. Treated males were impaled on pins taped to soda straws which

were introduced into quart Mason jars holding a set number of virgin

males. The number of strikes made upon these treated males versus the

strikes upon an impaled female served as the basis for evaluating

pheromone activity. Both male and female adults were shown to have

about equal quantities of these active cuticular compounds; however,

males had higher concentrations of saturated hydrocarbons, especially

heptacosane and nonacosane, which reduced the activity of the previously

listed alkenes. Because females had a lower proportion of saturated

hydrocarbons and a higher proportion of active unsaturates than did males,

the authors proposed that males may have a means to differentiate

the sexes.

Using the bioassay described above, Sonnet et al. (1975) evaluated

combinations of synthetic compounds, originally detenrined in male and

female face fly cuticular washes, along with other selected synthetic







compounds in an attempt to optimize the activity of the most active

natural compound (Z)-14-nonacosene. No combination was found to be

more active than (Z)-14-nonacosene although several inhibitory compounds

were identified. Sonnet et al. (1975) mistakenly reported that Uebel

et al. (1975a) had found nonacosene and heptacosene to be inhibitory;

in fact, Uebel et al. (1975a) had reported that nonacosane and heptaco-

sane were inhibitory.

The stable fly. Using a combination of bioassay techniques to

investigate possible pheromones of the stable fly, Stomoxys calcitrans

(L.), Muhammed et al. (1975) reported that trans and cis olefins were

the mating stimulant compounds for males. Female polyolefins were re-

ported as a sex attractant for males. Uebel et al. (1975b) found that

a number of saturated and unsaturated female hydrocarbons were the

mating stimulant for the male. The active saturates included mono- and

dimethyl-substituted hentriacotanes and tritriacontanes while (Z)-9-

hentriacontene (Z)-9-tritriacontene, and methyl-branched hentriacon-

tenes and tritriacontenes were the main active unsaturates. Sonnet

et al. (1977) continued work on the saturated hydrocarbons by synthe-

sizing and testing several dozen methyl-branched and 1,5-dimethyl-

branched alkanes. Bioassay results indicated that the 15-methyl and

15,19-dimethyltritriacontanes had the greatest mating stimulant

activity.

Both Uebel et al. (1975b) and Sonnet et al. (1977) used the Mason

jar bioassay which measured strikes upon treated, impaled males as was

previously described for the face fly studies conducted by Uebel et al.

(1975a). Treatment dosages of 50, 100, 150, or 200 og were tested.

Sonnet also used a bioassay designed by Harris et al. (1976) in which







strikes of male stable flies upon treated male house flies were mechani-

cally recorded. Bioassays by Sonnet et al. (1979) utilized hexane-rinsed

male house flies as the pseudofly rather than male face flies to avoid

interference in bioassays due to the natural polyolefin present on male

face flies.

Additional work by Sonnet et al. (1979) indicated that the stable

fly sex pheromone which induces male copulatory behavior was a complex

of compounds which were most active in combination. Principal components

included (Z)-9-hentria- and tritriacontenes; 13-methyl-1-hentri- and

tritriacontenes as well as an array of methyl-branched hentria and

tritriacontanes, with 11- and 15-methyl-substituted compounds being

the most active.

Fannia species. Uebel et al. (1975c) identified (Z)-9-pentacosene

as the hydrocarbon which stimulated the male little house fly, Fannia

canicularis (L.), to mate. This compound represented the major portion

of the cuticular hydrocarbons of the female and appeared to be more

specific in eliciting male copulatory response than did the sex phero-

mones of other Muscidae (Uebel et al., 1977). Unbranched monoolefins

of 31 and 33 carbon length were described by Uebel et al. (1978a) as

active mating stimulant compounds from female F. pusio (Wiedemann). A

major C31 monoolefin, (Z)-11-hentriacontene, found in the female but

not in the male, was found to be the most active of synthetic compounds

bioassayed. The same compound was the most active male mating stimu-

lant of F. femoralis (Stein) tested by Uebel (1978b). The addition of

female alkanes increased the activity of tested C31 monoolefins. The

primary difference in the sex pheromone makeup of F. pusio versus F.

femoralis appears to be the synergistic action of female alkanes







occurring in F. femoralis but apparently not in F. pusio. Males of

either species will, however, attempt to copulate with females of the

other species if the two sexes are placed together in a confined

environment.

The bioassays utilized in these studies on species of Fannia were

all conducted as described by Uebel et al. (1975a) with treatment dosages

of 100 and 200 ug. Only mating strikes were recorded in these bioassays.

Males displaying other courtship behavior were physically removed from

the treated pseudofly.

In simulated field trials, the respective mating stimulant phero-

mone of F. canicularis, (Z)-9-pentacosene, and F. pusio, (Z)-1l-hentria-

contene, were moderately effective in attracting males to pheromone

treated baits. The mating stimulant of F. femoralis, (Z)-ll-hentria-

contene, was ineffective in attracting either sex of that species

(Uebel et al., 1978c).

The tsetse fly. Langley et al. (1975) reported evidence for the

existence of a sex-recognition pheromone in the tsetse fly, Glossina

morsitans morsitans (Westwood). Located in the non-polar cuticular

lipid fraction of the female, the pheromone appeared to be active only

at short distances. The active component was hypothesized to be a long

chain hydrocarbon with a carbon number of C31 to C38. Carlson et al.

(1978) later isolated, identified, and synthesized three sex recognition

components which are naturally produced by the females and which stimu-

late males to initiate sexual behavior upon contact with the female.

The compounds 15,19-dimethylheptatriacontane, 17,21-dimethylheptatria-

contane, and 15,19,23-trimethylheptatriacontane were reported to be the

least volatile and most stable pheromones reported to date. Field




-36-


testing of these compounds in Tanzania confirmed the stimulatory activity

of the most active isomer on wild males and established increased

efficiency of currently-used survey methods when supplemented with

pheromone (Dr. D. Carlson, pers. comm., 1978, Insects Affecting Man

and Animals Research Laboratory, USDA, Gainesville, FL).

The horn fly. Mackley (1977) extracted, purified, and examined

horn fly lipids for possible pheromone activity as attractants or con-

tact stimulants. Olfactometer studies demonstrated a low order of

attraction of about 1.7x of sexually mature female horn flies to the

total monoolefin fraction than to blank controls. Contact stimulation

assays indicated that horn fly courtship behavior was, in part, chemi-

cally mediated; however, no active compounds were identified because of

the lack of a suitable bioassay.










Table 2-1. A summary of sex pheromone research on the Muscidae.


Species


Musca domestic L.
(house fly)


Compound(s)


(Z)-9-tricosene


7:3 ratio of
(Z)-9-tricosene
and
(Z)-9-heneicosene

(Z)-9-tricosene


(Z)-9-tricosene


(Z)-9-tricosene
and analogs



(Z)-9-tricosene


(Z)-9-tricosene


Experimental
Bioassay



ol factoleter


olfactometer


pseudofly


field trial-
baited traps

olfactometer,
pseudofly


Type of
Pheromone or
Induced Behavior



sex attractant
for males

mating stimulant
and sex attrac-
tant for males


mating stimulant
for males

attractant for
males and females

sex stimulant as
well as sex
attractant for
ina les


field trial attractant for
males and females


field trial


attractant for
males and females


Citation


Carlson, Mayer, Silhacek, James,
Beroza, and Bierl, 1971

Mansingh, Steele, Smallman,
Meresz, and Mozogai, 1972



Rogoff, Gretz, Jacobson, and
Beroza, 1973

Carlson and Beroza, 1973


Carlson, Doolittle, Beroza,
Rogoff, and Gretz, 1974



Morgan, Gilbert, and Fye,
1974

Mitchell, Tingle, and Carlson,
1975










Table 2-1. Continued


Species


Musca domestic L.
(continued)


Compound(s) Experimental
Bioassay


(Z)-9-tricosene
plus methyl- and
dimethyl-branched
C27 to C29 paraf-
fins of female
origin

(Z)-14-tricosen-
10-one and
cis-9,10,epoxy-
tricosane


pseudofly


pseudofly


Type of
Pheromone or
Induced Behavior



mating stimulant
for males


stimulated copu-
latory responses
from males


Citation


Uebel, Sonnet, and Miller,
1976


Uebel, Schwarz, Lusby, Miller,
and Sonnet, 1978d


Musca autunnalls
De Geer
(face fly)


female face fly,
unsaturated
hydrocarbons

(Z)-14-nonacosene
(Z)-13-nonacosene
(Z)-13-heptacosene

(Z)-14-nonacosene


ol factometer,
female face
fly models

treated male
face flies


sex attractant and
sex stimulant for
males

mating stimulant
for males


treated male mating stimulant
face flies for males


Chaudhury, Ball, and Jones,
1972


Uebel, Sonnet, Miller, and
Beroza, 1975a


Sonnet, Uebel, and Miller,
1975










Table 2-1. Continued


Experimental
Bioassay


Type of
Pheromone or
Induced Behavior


Stolmoxys
calcitrans (L.)


female polyolefin
fraction


(E) and (Z)
olefins


saturated hydro-
carbons: mono-
and dimethyl-
substituted
hentria- and
tritriaconLanes

unsaturated hydro-
carbons: (Z)-9-
hentriacontene
(C31); (Z)-9-tri-
triacontene (C33);
methyl-branched
hentria- and
tritriacontenes


olfactometer,
pseudofly,
activity meter

ol factometer,
pseudofly,
activity meter

treated male
stable flies


treated male
stable flies


sex attractant
for males


mating stimulant
for males


mating stimulant
for males


Muhammed, Butler, and Carlson,
1975


Uebel, Sonnet, Bierl, and
Miller, 1975b


mating stimulant
for males


Species


Compound(s)


Citation










Table 2-1. Continued


Compound(s)


Type of
Experimental Pheromone or Citation
Bioassay Induced Behavior


Stomoxfys
calcitrans (L.)
-continued)


Fannia
canicularis (L.)
Little house fly)


11 ,15-dimethylhen
triacontane; 15-
methyltritriacon-
tane; 15,19-di-
methyltritriacon-
tane

(Z)-9-hentriacon-
tene; (Z)-9-tri-
triacontene; 13-
methyl-1-hentria-
contene; 13-
methyl-l-tritria-
contene


treated male
stable flies,
male house
flies with
recording
device

male house
flies rinsed
with hexane


(Z)-9-pentacosene pseudofly


(Z)-9-pentacosene pseudofly


mating stimulant
for males


mating stimulant
for males









mating stimulant
for males

mating stimulant
for males


Sonnet, Uebel, Harris, and
Miller, 1977


Sonnet, Uebel, Lusby, Schwarz,
and Miller, 1979









Uebel, Menzer, Sonnet, and
Miller, 1975c

Uebel, Sonnet, Menzer, Miller,
and Lusby, 1977


Species









Table 2-1. Continued


Species


Fannia pu'io
(Wiedenrann)





Fannia femoralis
(Stein)


Fannia canicularis


F. pusio
(Wiedemann)


Compound(s)


unbranched C31 to
C33 mono-olefins
especially (Z)-
11-hentriacontene



C31 to C33 mono-
olefin especially
(Z)-ll-hentria-
contene

species' respec-
tive mating
stimulants as
listed above


Experimental
Bioassay


pseudofly






pseudofly


simulated
field
trial-baited
traps


F. femoralis
(STeT r)


Type of
Pheromone or
Induced Behavior



mating stimulant
for males





mating stimulant
for males



slight attractant
for males

slight attractant
for males

not attractive to
males


Citation


Uebel, Schwarz, Menzer, and
Miller, 1978a





Uebel Schwarz, Miller, and
Menzer, 1978b



Uebel, Schwarz, Sonnet, Miller,
and Menzer, 1978c


Glossina morsitans
(Wes twod)
(tsetse fly)


pseudofly mating stimulant
for males


Langley, Pimley, and Carlson,
1975


long chain
hydrocarbons










Table 2-1. Continued


Experimental
Bioassay


Type of
Pheromone or
Induced Behavior


Glossina morsitans
(westwtoo rT
(tsetse fly)
(continued)


Haematobia irritans
(horn fy)
(hor-n fly)


15,19-dimethyl- pseudofly
heptatriacontane;
17,21-dimethyl-
heptatriacontane;
15,19,23-trimethyl-
heptatriacontane


horn fly
circular
monoolefins


olfactometer


sex stimulation
and recognition
by males


attractant for
females


Carlson, Langley, and Huyton,
1978


Mackley, 1977


Species


Compound(s)


Citation














CHAPTER 3

THE COURTSHIP BEHAVIOR OF THE ADULT HORN FLY,
Haematobia irritans (L.),UNDER
LABORATORY CONDITIONS



Abstract


The courtship behavior of mature, well-nourished horn fly males and

females was similar to that of other muscids; however, unlike other

muscids during one element of the courtship sequence, the male rubs the

female's head with its protarsi. The duration of courtship and copula-

tion was variable among pairs. Virgin males courted tethered, virgin

females with greater frequency than they courted tethered, virgin males.

Virgin males could readily differentiate females from males and court

females regardless of whether the horn flies were live, dead, winged,

or wingless.



Introduction


Research on the horn fly, Haematobia irritans, has provided informa-

tion on many facets of the biology, behavior, and autecology of this

species; however, descriptive literature on horn fly courtship is meager.

Hammer (1941) reported observing copulating pairs on host animals in the

field. Bruce (1964) made similar field observations and noted that

mating pairs remained in copulo about 0.5-5.0 minutes. Anderson (1974)

listed H. irritans (L.) as a species that mated on the host. The data








of Harris et al. (1968) suggested that most horn fly matings occurred

between 2-4 days after emergence under laboratory conditions; however,

when flies were reared on a host, most flies had mated by the end of the

second day. The data of Harris et al. (1968) also suggested that the

female horn fly was monogamous.

The basic objectives of this study were to describe the courtship

behavior of the horn fly in the laboratory, to obtain a quantitative

measure of the behavioral responses occurring between the sexes during

courtship, and to investigate selected courtship stimuli which may be

important to the male when courting the female.



Materials and Methods


Standard Methods of Rearing and Maintaining Horn Flies


Horn flies used in these studies were from the laboratory colony

maintained at the University of Florida (Florida Laboratory Strain, F)

as described by Greer (1975). Adults were reared at a tempera-

ture of 27 2 2'C and a relative humidity of 70 10 ; continuous lighting

for the colony was provided by 6 Westinghouse F40C', (40 W) Cool White

fluorescent bulbs. Adults could obtain blood meals at will through the

cage top from gauze-covered pads of absorbant surgical cellulose (Doe-

skin Products, Inc., New York, NY) soaked with bovine blood treated with

0.1 gm of Kanamycin 30,000 units of Iycostatin and 3.75 qm of sodium

citrate per liter. Blood pads on adult cages were changed twice daily.

As adults, flies were considered virgin if they had been sexed

within 24 hours of emergence. Flies lightly anesthetized with carbon

dioxide were sexed and placed in groups of about 50 flies in 140 ml







clear plastic specimen containers fitted with screen tops. For some

experiments individual pupae were placed in 140 ml clear plastic speci-

men containers. The emerged fly was kept isolated from other flies

until testing. In other experiments, male and female flies of a specific

age were obtained from dated cages of adults. Because these flies had

been held together as mixed sexes for at least 8 days, these flies were

considered to be mated when used in experiments. Virgin flies between

the ages of 3-9 days were considered sexually mature on the basis of

preliminary laboratory observation as well as the data of Harris et al.

(1968), Gale (1977), and Mackley (1977).


Methods for Observing Horn Fy Behavior

Visual observation, video tape analysis, still photography, and

motion picture photography were used to obtain information on horn fly

courtship. Visual observation was initially used to observe general

aspects of horn fly behavior in laboratory colony rearing cages and

was used in later experiments to count the frequency of specific ele-

ments of horn fly courtship. All observations were made at ambient

laboratory conditions (25 2"C, RH 70 20 ) on the bioassay apparatus

described on page 108.

Photographic techniques were used to obtain more detailed informa-

tion on horn fly courtship. Courting flies were video taped with a

Panasonic TV camera equipped with a S2 110 RND 11.5-110 mm lens

(1:2.3) and a +2 accessory close-up lens. Scotch :1BU-40 and MBU-60

video cassettes were analyzed at normal speed and with pause/still

frame advance on a JVC video cassette recorder, Model CR-6060 U. Black

and white photographs and color slides were taken with a Pentax'" ESII

35 mm camera with extension tubes and a Takumar' 100 mm macro lens.








Electronic flash gave an exposure speed of 1/1000 sec. Motion pictures

of horn fly courtship sequences were taken with a Fujica XC-100 super

8 mm camera fitted with a Takumar 100 nmm macro lens and accessory

close-up lens on Fuji color movie film (ASA 25 and 200). Film speeds

from 18-72 fps and a variable shutter control allowed single frame

exposures of 1/40-1/640 sec. Films were viewed at several speeds on an

editor-viewer so that individual frames could be analyzed.

The arenas for photography were either a plastic chamber

(25 x 20 x 5 mm) fitted with a glass front or a 140 ml clear plastic

specimen container fitted with a glass front. Variable back-lighting was used

to obtain the proper lighting for the exposures listed above.

Standard Methods of Handling Horn Flies


For photographic purposes or experiments on close-range courtship

behavior, horn flies were handled in 2 general ways. In some studies,

males and females were provided access to blood until transferred

without CO2 anesthesia to the observation container or photographic

arena. Both sexes were free to move about in the container for the

duration of the test. In other studies, individual flies were fixed

or "tethered" in one location in the observation container or photo-

graphic arena. These flies had access to blood until they were "tethered."

Under light CO2 anesthesia, the ventral surface of the abdomen of a fly

to be tethered was glued to the head of an insect pin with Weldwood

Contact Cement (U.S. Plywood, Kalamazoo, MI). The pin was inserted

into an 8 mm thick, white polyethylene foam sheet (Bernel Foam Products

Co., Inc., Buffalo, NY). The excess pin was cut off so that the fly

was in a normal standing position with its tarsi in contact with the








foam substrate. Tethered in this manner, flies were able to move

all of their appendages. Male horn flies would readily court tethered

females. Dead flies for experiments were obtained by freezing the flies at

-150C and thawing them before testing.

Experiments on Courtship Behavior of Untethered Pairs


To observe the courtship behavior of laboratory horn flies, a 4-6

day old virgin male and a 4-6 day old virgin female, each isolated from

other flies since emergence, were placed together in a 140 ml clear

plastic observation container. The frequencies of the following specific

courtship elements were recorded: 1) strike (+), an attempt by the

virgin male to get onto the dorsum of the female; 2) arrested movement

(++), after striking the female, the responding male remained in con-

tact with the female but did not continue with additional elements of

the courtship sequence; 3) positioning on the dorsum (+++), after

striking the female, the responding male positioned itself on the

female and placed its genitalia below the end of the female's abdomen;

and 4) copulation (+-- +). In addition, certain aspects of these

courtship sequences were timed. If copulation did occur, the female

was separated from the male and examined to determine if sperm had been

transferred. Spermatheca were removed, transferred to a drop of Ringer's

saline, and examined for sperm at 100x and 450x both before and after

squashing.



Experiments on Female-Produced Stimuli Affecting Courtship of Males


To assess how horn fly males might differently court males versus

females, the behavioral responses of groups of 35 virgin males were








observed toward both virgin, tethered males and females. During each of

4 consecutive 15-minute periods 4 elements of courtship behavior were

recorded: touch, whenever a male contacted a tethered fly; strike,

when a male attempted to get onto the dorsum of a tethered fly; posi-

tioning on dorsum, when a male moved backward on the dorsum of the

tethered fly and assumed a position for mating; and copulatory attempt,

whenever a male placed the ventral portion of the tip of his abdomen

against the posterior region of the tethered fly. Tethered flies were

randomly assigned to one of 6 predetermined positions on a piece of

8 mm thick, white polyethylene foam circle which fit snugly into the

bottom of a 1000 ml glass beaker. The positions of the tethered flies

described a circular pattern approximately 7 cm in diameter with each

tethered fly facing the center of the beaker.

Under light CO2 anesthesia, 35 males were transferred to a 140 ml

plastic specimen container and given 1 hour to recover from CO2 before

testing began. withoutt anesthesia, these 35 males were transferred to

the beaker containing the tethered flies. The beaker was covered with

a clear plastic film containing several dozen air holes. After four

15-minute periods, all flies were discarded. With these same procedures,

the courtship behavior between males and females of different ages (3

day old versus 8-9 day old) or females of different mating status

(virgin versus mated) was also investigated. Each set of data was

analyzed as a split-plot in time analysis of variance. Appropriate F

tests or Duncan's multiple range testswere used as tests of significance.

Further experiments to evaluate the importance of female-produced

stimuli were conducted by observing the courtship of groups of five 3-6

day old virgin males to both tethered males and females with combinations








of the following treatments: live, dead, winged, and wingless. Flies

to be tested were sexed and held in groups until the proper age for

testing. Flies for each treatment were tethered as previously de-

scribed; dead flies were obtained by exposing flies briefly to -15C

temperature. Wings were removed with forceps. The frequency of response

to treatments was evaluated by observing if courtship behavior was dis-

played toward the tethered fly within a 3-minute period after the clear

plastic specimen container holding the virgin males was placed over

the tethered fly. The first observed courtship behavior was scored as

follows: 1) strike (+); (2) arrested movement (++); 3) positioning on

dorsum (+++); and 4) copulation (++++). For each treatment, thirty 3-minute

trials were performed. The frequency data were analyzed utilizing the

chi-square test for contingency tables (Steele and Torrie, 1960;

Ostle, 1963).



Results


Preliminary Observation on Horn Fly Behavior in the Laboratory


Visual observation of horn fly behavior in laboratory rearing cages

containing several thousand flies or in 120 ml clear plastic cages con-

taining a single pair of virgin flies provided several preliminary

findings. Although these observations were qualitative in nature, an

overview was obtained of the general activity patterns of horn flies

under laboratory conditions (Appendix A-i).

The activity patterns of these flies did change from emergence

through several days of age. In particular, the beginning of courtship

behavior was not evident until approximately 2.5 days after emergence.







Although few copulating pairs were observed after the fourth day, males

continued to court females which became increasingly resistant to male

advances and either positioned themselves in locations in the cage

which appeared to minimize contact with males or displayed specific

rejection behavior. This behavior will be addressed in the next section.



General Description of Courtship Behavior


The courtship behavior of mature, well-nourished H. irritans males

and females was similar to that described for other species in the

family Muscidae. The following description of horn fly courtship is

based on the composite information gained from visual and photographic

observations of pairs of courting horn flies in which the female was in

some cases tethered and in other cases untethered.

A male horn fly would often orient to and directly approach a female

several fly lengths away or at other times would encounter females by

seemingly random movement. Orientation to the female was particularly

noticeable when the male was on the same surface as the female (Figure

3-1). Males would then walk, jump, or fly into the dorsum of the fe-

males. In each of these methods of mounting the female, the male's

wings were in motion. Initial courtship contact between the sexes while

both were in flight was rarely seen. The direction and angle of approach

as well as the landing position of the male on the female was variable;

therefore, the bocy parts of each sex which initially made contact

during a given strike varied considerably. The male usually approached

the female from the rear. But a male which did not land facing the

same direction as the female would quickly reorient itself so that it




















Figure 3-1. Male (on right) orienting to a tethered female.






























































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L








faced the same direction as the female. Figure 3-2 shows a male striking

from the rear of the female and Figure 3-3 shows a male striking from

the side of the female. In these strikes the protarsi and mesotarsi

touched the female before the metatarsi. However, in one video tape

sequence, initial contact occurred when the male's metathoracic legs

first touched and then grasped the female's abdomen.

When a male landed on the female, each of the female's wings be-

came extended at approximately a 45" angle to her body. The male began

to raise himself so that his body was above and parallel to the female's

dorsum. The male moved forward until his head was over the female's

thorax,then slightly raised his abdomen so that the angle between his

body and the female's dorsum approached 45. When the male was in this

most forward position on the female, he used his prothoracic tarsi to

rub the female's head (Figure 3-4). As he stopped rubbing the female's

head and moved backward on the female, the male placed his prothoracic

tarsi at the bases of the female's wings and his mesothoracic legs

along the sides of the anterior portion of the female's abdomen. As

the male continued to move backward, he slightly arched his abdomen

downward past the tip of the female's abdomen and attempted to make

genital contact (Figure 3-5) by grasping the slightly extended ovi-

positor of the female. Simultaneously, the male positioned his meta-

thoracic legs firmly beneath the female's abdomen.

Figures 3-6 to 3-8 show several views of horn fly pairs in copulo.

The male's prothoracic tarsi grasped the female at the wing bases; the

metathoracic legs held the female laterally at the anterior of the

female's abdomen; the metathoracic legs were held approximately parallel

to each other beneath the female's abdomen. All of the female's legs





















Figure 3-2. Male striking a tethered female from the rear of
the female.

















' I
I


T0





















Figure 3-3. Male striking a tethered female from a position at
the side of the female.







7-A


K





















Figure 3-4. Male's prothoracic tarsi rubbing a tethered female's
head when then the male was in the most forward
position on the female's dorsum.




























ILK





















Figure 3-5. Male backed on the dorsum of a tethered female and
attempting to copulate.




















Figure 3-6. Horn flies in copulo (view A).




















Figure 3-7. Horn flies in copulo (view B).






























Figure 3-8. Horn flies in copulo (view C).









































* -


^'







were in contact with the substrate. While in copulo, the pairs

generally remained motionless except for some pairs in which the female

occasionally walked about or cleaned herself with her metathoracic legs.

If pairs in copulo were disturbed, the female with the male in position

would briefly walk about or take flight. Males were not apparently

distracted by this movement. Copulation usually was ended as the

female became more active and made movements such as rapid wing vibra-

tion or kicking with the metathoracic legs to dislodge the male

(Figure 3-9).

The movement of the male's wings continued throughout most of the

courtship sequence until the male was in position to make a copulatory

attempt. Males unable to copulate on an initial attempt often repeated

earlier elements of the courtship sequence while on the female's dorsum.

The male would often move forward again, rub the female's head and

back while positioning himself for another copulatory attempt. Other

males would stop their wing movement and rest motionless on the female's

dorsum (Figure 3-10) until either resuming courtship or flying off.

Other males would back off of the female and remain motionless about

1 fly length from the female. Some males would remain in this posi-

tion facing the female's abdomen for up to several minutes before

resuming courtship or flying off. Other males behaved similarly but

kept one or two of their prolegs on the female's abdomen or wings

(Figure 3-11). This resting behavior on or near the female was desig-

nated arrested mioverment.

Tethering the female versus having the female free to move about

appeared to increase the frequency of arrested movement (++) shown by

the male. However, the arrested movement of the male on the female





















Figure 3-9. Female dislodging a male at the termination of
copulation.







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XV


I





















Figure 3-10. Male which had arrested its movement on a tethered
female's dorsum.





















Male which had arrested its movement after an
unsuccessful copulatory attempt with a tethered
female. The male had backed off of the female's
dorsum but had kept its prothoracic legs on the
female's wings.


Figure 3-11.








was also seen during the interaction of pairs in the laboratory rearing

cages in which both sexes were free to move about. Although males were

often prevented from actively continuing courtship because of the

female's resting position or defensive behavior, some males were ob-

served to rest on the female's dorsum for approximately 30 seconds to

1 minute before either flying off or resuming courtship. In addition,

the qualitative comparison of data from several experiments suggested

that when virgin females were tethered they were less likely to copulate

in a given period of time than were virgin, untethered females. The

frequency of copulation will be discussed for these experiments of

concern.

Whether tethered or untethered, females displayed considerable

defensive behavior. The advances of sexually aggressive males were

often thwarted by the female's choice of a relatively protected resting

position or by her defensive movements. In the observation container,

the photographic arena, or rearing cage, untethered females often rested

along the inside edges of the cage, particularly the top edge, which

prevented strikes of males from behind and appeared to minimize the

females' contact with males. A female could also stop courtship

activity by 1) curling her abdomen downward while holding her over-

lapped wings high above her body; 2) kicking with her legs, especially

the metathoracic legs (Figure 3-12); 3) rapidly vibrating her wings;

4) flying or moving away from the male; or 5) by turning their body to

put the male at her side. Each of these defensive movements could be

used singly or in combination preventing the male from effectively

mounting the female or effectively positioning himself on the female

to make a copulatory attempt.





















Figure 3-12. Tethered female kicking at the male with one of her
metathoracic legs.








Male interaction with other males was occasionally observed.

Males would strike other untethered males in the observation con-

tainer but would rarely strike a tethered male. Males would also

readily strike the dorsum of a male already courting a tethered female

(Figure 3-13) forming a stack of males upon a tethered female (Figure

3-14). On a number of occasions groups of males would cluster about

a tethered female with each male attempting to grasp the female (Figure

3-15); usually this resulted in no male being able to effectively court

the female. At other times, males did not cluster about the tethered

female, but actively competed for the female (Figure 3-16). Often 1

male could thwart or block the attempts of other males to dislodge him

from his position on the female. But at other times, males were

successful in dislodging another male off of the female's dorsum.



Courtship Behavior of Untethered Pairs of Males and Females


The observation of courting pairs of 4-6 day old, virgin horn

flies permitted the quantification of several parameters of courtship

(Tables 3-1, 3-2, A-2). Considerable variability existed among pairs

in the time required from when the pairs were placed together until a

given element of the courtship interaction occurred. The mean time

(n = 19) between placing the pair together until the first strike

occurred was 7 min 47 sec (Table 3-1), but individual values ranged

from 15 sec to 30 min 2 sec. Likewise, the time from the first strike

until copulation began ranged from 12 sec to 25 min with a nean of 6

min 52 sec. The total time from placing the pairs together to the

beginning of copulation ranged from 2 min to 33 min 30 sec with a mean





















Figure 3-13. Second male striking the dorsum of a male already
courting a tethered female.














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4 *:r;


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X; ;i
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u-l'
Sr
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~5 ~" 1


:r,.





















Figure 3-14. Two males in a stack upon the dorsum of a tethered
female.



























Is





















Figure 3-15. A group of males clustered about a tethered
female.





















Figure 3-16. Two males striking a tethered female.




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