Citation
Effect of the chemosterilant, Metepa, on the housefly, Musca domestica L.

Material Information

Title:
Effect of the chemosterilant, Metepa, on the housefly, Musca domestica L.
Creator:
Kung, Ku-Sheng, 1921-
Publication Date:
Language:
English
Physical Description:
190 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Dosage ( jstor )
Eggs ( jstor )
Female animals ( jstor )
Insects ( jstor )
Longevity ( jstor )
Mating behavior ( jstor )
Mortality ( jstor )
Permanence ( jstor )
Pupae ( jstor )
Spermatozoa ( jstor )
Dissertations, Academic -- Entomology and Nematology -- UF
Entomology and Nematology thesis Ph. D
Flies ( lcsh )
Insect pests -- Control ( lcsh )
Insect sterilization ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida, 1967.
Bibliography:
Includes bibliographical references (leaves 175-187).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Ku-Sheng Kung.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
This item is presumed in the public domain according to the terms of the Retrospective Dissertation Scanning (RDS) policy, which may be viewed at http://ufdc.ufl.edu/AA00007596/00001. The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator(ufdissertations@uflib.ufl.edu) with any additional information they can provide.
Resource Identifier:
029980674 ( ALEPH )
37760975 ( OCLC )

Downloads

This item has the following downloads:


Full Text














EFFECT OF THE CHEMOSTERILANT METEPA

ON THE HOUSEFLY, Musca domestica L.










By
KU-SHENG KUNG













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 August, 1967














ACKNOWLEDGMENTS


I would like to express my sincere gratitude to the chairman of my supervisory committee, Dr. J. T. Creighton, Department of Entomology, University of Florida, and cochairman, Dr. G. C. LaBrecque, United States Department of Agriculture, for their sound advice and generous assistance.

The valuable suggestions and encouragement from Dr. D. A. Roberts and Dr. B. J. Smittle of the supervisory committee and Dr. W. G. Eden, chairman of the Department of Entomology, are also greatly appreciated.

Special thanks are due to Dr. C. N. Smith for the

use of facilities at the U.S.D.A. Entomology Research Division Laboratory and to all the U.S.D.A. staff members for their assistance.

Last, but not least, I wish to extend my appreciation to my wife, Hung-yin, for her understanding and encouragement, which made this study possible.















TABLE OF CONTENTS


Page


ACKNOWLEDGMENTS ....... LIST OF TABLES. . . . . . . LIST OF FIGURES . . . . . .. INTRODUCTION. . . . . . . . . REVIEW OF LITERATURE. . . . .

Chemosterilants . . . . .
General Conception. .
Definition . . .
Principal Action.
Mode of Action of


. . . . . . 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0


. . . 0 . . . . 0
. . . . . .O .


. . .

. . .0

. . .


ii vi xii

1


0 0 . 5


. 0 ~ ~ ~ ~ 0 ~ ~
0 ~ 0 ~ ~ ~ 0 ~ 0 ~
. . . . . . . . . .
. . . . . . . . 0 ~


Two


Main Groups
. . . . . .


of Chemosterilants.


Important Chemosterilants . . .... Searching for New Chemosterilants . . . . Field Experiments in the Control of House


Flies by Chemosterilant Techni Structure, Physical and Chemical
Properties of Metepa. . . . . Specificity of Chemosterilants.


Biology . . . . . . . . . . . . . .
Name and Classification . . . .
Distribution. . . . . . . . . .
The Adult . . . . . . . . . . .
Emergence . . . . . . . . .
Feeding . . . . . .. . . .
Flight. . . . . . .....
Mating Behavior . . ....
Oviposition . . . . . . 0 Longevity . . . .. . . .
Sex Ratio . . . . . . . ..
The Egg . . . . . . . 0 .. . .
The Larva . . . . . . . . . .


iii


ques.


. . . . 9


. . . . . .

. 0 0 0 . . . . . .
. . . . .
. . . . . .

. . . . . .
. . . . . 0
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. .0
. . o o . . . . .












Breeding Places in Nature . . .
Rearing Medium in Laboratory .
Growth Rate and Instars . . .. The Pupa. . . . . . . . . . . . . .
Hibernation . . . . . . . . . . . .
Temperature, Humidity, and Light..
Temperature . . . . . . . . . .
Humidity. . . . . . . .. . .
Light . . . . . . . . . . . . .


. . . . .

. . . . .
. . . . .
. . . . .
. . . .
. . . . .
. . . . .
. . . . .
. . . . .


MATERIALS AND METHODS . . . . . . . . . . . . . . .

Technique of Rearing Stock House Flies. . . ...
Procedures for Testing, Rearing, and Observation.


Rearing and Observation Cages . . . . Sexing Virgin Females and Males . . . Keeping Same-Age Flies and Different-


. . .


Age Flies . . . . . . . .
Water and Fly-Food Supply . .
Solution and Injection . . .
Mating. . . . . . . . . . . .
Egging . . . .. . * . . . . ..
Egg Counting. . . . . . . . .
Hatching Counts . . . . . . .
Pupation Counts . . . . . ..
Criteria of Sterilization -Determining Sterility . . . Determination of Lethal Dosage . Determination of Sterility Dosage


. . . . . . .
. . . .6 . . .

* 0 0 S * 0 . . . . . . .
. . . . . .
. . . . .
. . . . .


S ~ ~ S S S S S ~ S S S S S ~ S S


Determination of the Permanence of Sterility.
Mating Competitiveness Test . . . . . . . ..
Rate of Chemosterilization of Sperm in Vivo .
Derivation of Survivorship Curves . . . . ..

RESULTS AND DISCUSSION. . . . . . . . . . . . .


Determination of Lethal Dosage. . .
Lethal Dosage of Male Flies . .
Lethal Dosage of Female Flies .
Comparisons . . . . . . . Determination of Sterility Dosage .


S ~ ~ S ~ S ~ ~ ~ S S ~ S S S . S ~ S S S S ~ ~ S


Pagie


. .










Pacge


Sterility Dosage of Male House Flies. . . . . 79
Individual Pair-Rearing and First Egging. 79 Collective Rearing and First Egging . . . 79
Collective Rearing and Average of
Four Successive Eggings . . . . . . . . 84
Sterility Dosage of Female House Flies. . . . 88
Female Sterilization up to Five Days Old. 88
Sterilization of Females Older than
Five Days . . . . . . . . . . . . . .. 102
Determination of the Permanence of Sterility. . . 107 Mating Competitiveness Test . . . . . . . . . . . 126
Rate of Chemosterilization of Sperm in Vivo . . . 135 Effects of Chemosterilant Metepa on Longevity . . 141
Longevity of House Flies in Each Treatment. . 142 Survivorship Curves in Each Treatment . . . . 142
Male House Flies . . . . . . . . . . . . 151
Female House Flies . ...... . .... 159

SUMMARY . . . . . . . . . . . . . . . . . . . . . 172

REFERENCES. . . . . . . . . . . . . . . . . . . . 175

BIOGRAPHICAL SKETCH. . .. . . . . .... . . 188













LIST OF TABLES


Table Page

1. Effects of Chemosterilant Metepa in Different
Dosages on Male House Flies of Different
Ages (Percentage of Mortality Based on
48-Hour Observation Period). . . . . . . . . 64

2. LD10, LD50, LD90, and Slope for Each Age of
Male Flies Estimated Directly from the
Curves on Logarithm-Probability Paper. . . . 67

3. Effects of Chemosterilant Metepa in Different
Dosages on Female House Flies of Different Ages (Percentage of Mortality Based on 48Hour Observation Period) . . . . . . . . . . 68

4. LD ILD5 'LD , and Slope for Each Age of
'emale-lies-9stimated Directly from the
Curves on Logarithm-Probability Paper. . . . 70

5. Effects of Metepa on Sterility of.Males of
Different Ages: Individual Pair Rearing
Method, Six Complete Replicates (Percentage
of Sterility Based on Number of Progeny
Reaching Pupal Stage from All Eggs of First
Egging in Each Replicate). . . . . . . . . . 80

'6. Effects of Metepa on Sterility of Males of
Different Ages: Collective Rearing Method,
5-10 Pairs of Flies, Two Complete Replicates (Percentage of Sterility Based on
Number of Progeny Reaching Pupal Stage from
a Sample of 100 Eggs of First Egging in
Each Replicate) . . . . . . ...... 82

7. Effects of Metepa on Sterility of Males of
Different Ages: Collective Rearing Method,










Table Pacre

5-10 Pairs of Flies, Two Complete Replicates (Percentage of Sterility Based on
Number of Progeny Reaching Pupal Stage
from a Sample of 100 Eggs of Four Consecutive Eggings in Each Replicate). . . . . . 85

8. Comparison of Three Different Treatments to
Determine the Relationship between Dosage
and Male Sterility . . . . . . . . . . . . 87

9. Effects of Metepa on Sterility of Females of
Different Ages: Collective-Rearing Method,
5 Pairs of Flies (10 Pairs at the 64 pg/
Female Dosage),Two Complete Replicates
(Percentage of Sterility Based on Number
of Progeny Reaching Pupal Stage from a
Sample of 100 Eggs of First Egging in Each
Replicate) . . . . . . . . . . . . . . . . 89

10. Effects of Metepa on Sterility of Females of
Different Ages: Collective-Rearing Method,
5 Pairs of Flies (10 Pairs at the 64 pg/
Female Dosage), Two Complete Replicates (Percentage of Sterility Based on Number
of Progeny Reaching Pupal Stage from a Sample of 100 Eggs of Four Consecutive
Eggings in Each Replicate) . . . . . . . . 90

11. Comparison of the Relationship between
Dosage and Female Sterility in the First
Egging and the Average of Four Consecutive
Eggings. . . . . . . . .. . . . . . . . 93

12. Estimated Minimum Dosage for 100 Percent
Sterility in Female Flies of Different Ages (Based on Collective Rearing and
Percentage of Pupation from a Sample of
100 Eggs per Egging) . . ...... . .. 94

13. Effects of Metepa on Sterility of One-DayOld Female Flies (Based on the Hatch of
Eggs). . . . . . . . . . . . . . . . . . 96


vii









Table Pae

14. Effects of Metepa on Sterility of Two-DayOld Female Flies (Based on the Hatch of
Eggs). . . . . . . . . . . . . . . . . 97

15. Effects of Metepa on Sterility of ThreeDay-Old Female Flies (Based on the
Hatch of Eggs) . . . . . . ..... . 98

16. Effects of Metepa on Sterility of FourDay-Old Female Flies (Based on the
Hatch of Eggs) . . . ......... 99

17. Effects of Metepa on Sterility of FiveDay-Old Female Flies (Based on the
Hatch of Eggs) . ......... . . . 100

18. Effects of Metepa on Sterility of Female
Flies Older than Five Days (Based on the
Percentage of Hatch of Eggs) . . . . . 103

19. Egging of 50-Day-Old Females Mated with 3-Day-Old Males (Mated on February 11,
1967) . . . . . . . . . . . . . . . . 105

20. Effects of Metepa on Permanence of Sterility of One-Day-Old Males Treated
by Means of Microinjection in the Pair
Rearing, Mating Group Series Test (Percentage of Sterility Based on
Number of Progeny Reaching Pupal Stage
from All Eggs in Each Egging after
First Mating). . . . . . . . . . . . .. 109

21. Effects of Metepa on Permanence of
Sterility of Two-Day-Old Males Treated by Means of Microinjection in the Pair
Rearing, Mating Group Series Test (Percentage of Sterility Based on
Number of Progeny Reaching Pupal Stage
from All Eggs in Each Egging after
First Mating). ........ . . . . 110


viii









Table Page

22. Effects of Metepa on Permanence of
Sterility of Three-Day-Old Males Treated
by Means of Microinjection in the Pair
Rearing, Mating Group Series Test (Percentage of Sterility Based on
Number of Progeny Reaching Pupal Stage
from All Eggs in Each Egging after
First Mating) . . . . . . . . . . . . . . 1ll

23. Effects of Metepa on Permanence of
Sterility of Four-Day-Old Males Treated
by Means of Microinjection in the Pair
Rearing, Mating Group Series Test (Percentage of Sterility Based on
Number of Progeny Reaching Pupal Stage
from All Eggs in Each Egging after
First Mating) . . . . . . . . . . . . . . 112

24. Effects of Metepa on Permanence of
Sterility of Five-Day-Old Males Treated
by Means of Microinjection in the Pair
Rearing, Mating Group Series Test
(Percentage of Sterility Based on
Number of Progeny Reaching Pupal Stage
from All Eggs in Each Egging after
First Mating) .. . . ...... .. . 113

25. Effects of Metepa on Permanence of Sterility
of One-Day-Old Males Treated by Means of Microinjection in the Pair Rearing, Consecutive Mating Series Test (Percentage of
Sterility Based on Number of Progeny
Reaching Pupal Stage from All Eggs in Each
Egging after Each Mating). . . . . . . . . 114

26. Effects of Metepa on Permanence of Sterility
of Two-Day-Old Males Treated by Means of Microinjection in the Pair Rearing, Consecutive Mating Series Test (Percentage of
Sterility Based on Number of Progeny
Reaching Pupal Stage from All Eggs in Each
Egging after Each Mating) . . . . . . * * 115









Table


27. Effects of Metepa on Permanence of Sterility
of Three-Day-Old Males Treated by Means of
Microinjection in the Pair Rearing, Consecutive Mating Series Test (Percentage of
Sterility Based on Number of Progeny
Reaching Pupal Stage from All Eggs in Each
Egging after Each Mating). . . . . . . . . 116

28. Effects of Metepa on Permanence of Sterility
of Four-Day-Old Males Treated by Means of
Microinjection in the Pair Rearing, Consecutive Mating Series Test (Percentage of
Sterility Based on Number of Progeny
Reaching Pupal Stage from All Eggs in Each
Egging after Each Mating). . . . . . . . . 117

29. Effects of Metepa on Permanence of Sterility
of Five-Day-Old Males Treated by Means of
Microinjection in the Pair Rearing, Consecutive Mating Series Test (Percentage of
Sterility Based on Number of Progeny
Reaching Pupal Stage from All Eggs in Each
Egging after Each Mating). . . . . . . . . 118

30. Effects of Metepa on Permanence of Sterility
of Males Treated by Means of Microinjection
in the Collective Rearing Method (Percentage of Sterility Based on Number of
Progeny Reaching Pupal Stage from a Sample
of 100 Eggs in Each Egging after Each
Consecutive Mating) . . . . . . . . . . . 119

31. Mating Competitiveness of One-Day-Old
Treated Male Flies Based on the Time Units 129

32. Mating Competitiveness of Two-Day-Old
Treated Male Flies Based on the Time
Units. . . . . . . . . . . . . . . . . . . 130

33. Mating Competitiveness of Three-Day-Old
Treated Male Flies Based on the Time
Units. . .. . . . . . . . . . . . . . . . 131


Page









Table

34. Mating Competitiveness of Four-Day-Old
Treated Male Flies Based on the Time
Units. . . . . . . . . . . . . . *.


35. Mating Competitiveness of Five-Day-Old
Treated Male Flies Based on the Time
Units. . . . . . . .. . . . . . .. . .

36. Rate of Metepa Chemosterilization of Sperm
in Vivo (Three-Day-Old Males Treated at


the Dosage of 8 pg/Male Fly, Crossed with Three-Day-Old Females Immediatel after Injection); Male Flies Were Injected August 8, 1966. . . . . ..


37. Longevity of Treated Male House Flies
without Female House Flies . . . . .

38. Longevity of Normal Male House Flies
without Female House Flies . . ..

39. Longevity of Normal Male House Flies
with Normal Female House Flies . . .

40. Longevity of Normal Male House Flies
with Treated Female House Flies. .

41. Longevity of Treated Male House Flies
with Normal Female House Flies . . .

42. Longevity of Treated Female House Flies
without Male House Flies . . . . ..

43. Longevity of Normal Female House Flies
without Male House Flies . . . . ..


y


. . . 136 . . . 143 . . . 144 . . . 145


S. . 146 . . . 148 . . . 149


. . .


PaSe


132


. 133


150
















Figure


1. Lethal-Dosage Curves for One-Day-Old Male
and Female Flies within 48 Hours ....

2. Lethal-Dosage Curves for Two-Day-Old Male
and Female Flies within 48 Hours . . ..

3. Lethal-Dosage Curves for Three-Day-Old
Male and Female Flies within 48 Hours .

4. Lethal-Dosage Curves for Four-Day-Old Male
and Female Flies within 48 Hours . . . .

5. Lethal-Dosage Curves for Five-Day-Old Male
and Female Flies within 48 Hours . . . .

6. Dosage-Sterility Curves for Different Ages
of Male House Flies Treated by Microinjection, Pair Rearing Method, from All
Eggs of First Egging . . . . . . . . .

7. Dosage-Sterility Curves for Different Ages
of Male House Flies Treated by Microinjection, Collective Rearing Method,
Using a Sample of 100 Eggs of First
Egging . . . . . . . . . . . .. . . . .

8. Dosage-Sterility Curves for Different Ages
of Male House Flies, Collective Rearing
Method, Using a Sample of 100 Eggs of
Four Consecutive Eggings in Each Replicate . . . . . . . . . . . . . . . .

9. Dosage-Sterility Curves for Different Ages
of Female House Flies, Collective
Rearing Method, Using a Sample of 100
Eggs at First Egging . . . . . . . .


LIST OF FIGURES


Page


* 71


S 72 . 73


S 74 . 75




S 81 S 83





. 86 . 91


xii








Figure


10. Dosage-Sterility Curves for Different Ages
of Female House Flies, Collective
Rearing Method, Using a Sample of 100
Eggs of Four Consecutive Eggings in Each
Replicate. . . . . . . . . . . . . . . . . 92

11. Relationship between Permanence of
Sterility and the Mating Time after
Injection of Male House Flies, Mating
Group Series Test (First Egging, All
Eggs Counted; Sterility Based on
Pupation). . . . . . . . . . . . . . . . . 121

12. Relationship between Permanence of
Sterility and the Mating Time after
Injection of Male House Flies, Consecutive Mating Series Test (First Egging,
All Eggs Counted; Sterility Based on
Pupation). . . . . . . . . . . . . . . . . 122

13. Permanence of Sterility of Male House Flies
Treated at Different Ages, Collective
Rearing Method, 5-10 Pairs of Flies
(Progeny Reaching Pupal Stage from a
Sample of 100 Eggs per Egging after Each
Consecutive Mating) . . . . . . . . . . .* 123

14. Survivorship Curves of Treated Male House
Flies without Female House Flies . . . . . 152

15. Survivorship Curves of Normal Male House
Flies without Female House Flies . . . . . 153

16. Survivorship Curves of Normal Male House
Flies with Normal Female House Flies . . . 155

17. Survivorship Curves of Normal Male House
Flies with Females Treated at Different Dosages according to Age: 70 pg/l-dayold; 50 pg/2-day-old; 45 pg/3-day-old;
30 pg/4-day-old; and 20 pg/5-day-old . . . 157


xiii


Page








Figure


18. Survivorship Curves of Treated Male House
Flies with Normal Female House Flies . . .


19. Survivorship Curves of Treated Female House
Flies without Male House Flies, at Different Dosages according to Age:
70 pg/l-day-old; 50 pg/2-day-old; 45 pg/
3-day-old; 30 pg/4-day-old; and 20 pg/
5-day-old . . . . . . . . . . . . .. 161

20. Survivorship Curves of Normal Female House
Flies without Male House Flies . . . . . . 162

21. Survivorship Curves of Normal Female House
Flies with Normal Male House Flies . . . . 164

22. Survivorship Curves of Normal Female House
Flies with Male House Flies Treated at the Dosage of 8 pg/Male Fly (Males and
Females of the Same Age Were Mated). . . . 168

23. Survivorship Curves of Treated Female House
Flies with Normal Male House Flies (Females Were Treated at Different
Dosages according to Age: 70 pg/l-dayold; 50 pg/2-day-old; 45 pg/3-day-old;
30 pg/4-day-old; and 20 pg/5-day-old). . . 170


xiv


Pggg


158













INTRODUCTION


The idea of using sterile male insects in a natural population was considered by E. F. Knipling as early as 1938 for possible control of the screw-worm fly, Cochliomvia hominivorax (Coquerel) (Lindquist, 1963). Knipling (1955, 1959, 1962, 1964, and 1966) has calculated and compared theoretical population declines of insect and other animal species subjected to a treatment which causes sterility as opposed to one that produces only direct kill, such as an

insecticide.

Research on materials and methods of producing

sterility in insects has progressed substantially during recent years, and the probability is good that it will be possible to produce reasonably competitive sterile insects of many species for release. Important new developments are being made in the insect attractant field. Effective methods of attracting insects by chemical or physical means might offer the possibility of integrating the attractant and sterility principles without the necessity of rearing and releasing insects. The trapped insects might be









sterilized and released, thus increasing the effectiveness of the trapping procedures (Knipling, 1964; Knipling & McGuire, 1966).

Two methods of achieving sterility have been investigated extensively: the use of X-rays or gamma rays from cobalt 60, as first investigated on the screw-worm by Bushland and Hopkins (1951, 1953), and the use of chemicals for sterilization, as reported by LaBrecque et al. (1960) and Smith et al. (1964). The data from preliminary field tests on the control of house flies are encouraging (LaBrecque et al., 1963a; Gouck et al., 1963b).

In recent years, the use of chemical agents as sexual sterilants for insects has attracted increasing interest and attention from entomologists. Three biological alkylating agents found to be effective insect chemosterilants were all aziridine derivatives (Borkovec, 1966). Tepa (aphoxide) and apholate were shown to be effective for house flies, Musca domestica Linnaeus (LaBrecque, 1961). Similarly, the methyl derivative of tepa, metepa, was reported by LaBrecque et al. (1963b) to sterilize house flies. Alkylating agents have also been used to sterilize two species of mosquitoes (Weidhass, 1962), the screw-worm fly









(Chamberlain, 1962), the German cockroach (Burden & Smittle, 1963), and other insects.

Various ways have been developed for evaluating ., chemosterilants in the laboratory, such as feeding technique (Gouck & LaBrecque, 1963, 1964), dipping technique (Piquett & Keller, 1962; Gouck, 1964; Chang & Borkovec, 1966a), injection technique (Chang & Borkovec, 1964), residual application (Meifert et al., 1963), and topical application (Gouck & LaBrecque, 1964; Chang et al., 1964). Each method has its own special advantages and disadvantages. A particular method is suitable for given types of behavior during certain developmental stages of a particular insect species, or for a special experiment design according to the purpose of the experiment.

This experiment was conducted using microinjection of a metepa water solution into the house fly. The purpose of this investigation was to seek solutions to the following problems:

A. To determine the lethal dosage (LD) in different sexes and different ages of house flies.

B. To determine the sterility dosage (SD) in different sexes and different ages of house flies.

C. To determine the permanence of sterility induced





4



in both males and females when subjected to dosages calculated to produce 100 percent sterility.

D. To derive survivorship curves for both males and females, individually and collectively, comparing treated house flies with normal ones.

E. To correlate toxicity with sterility and to consider other interrelationships.














REVIEW OF LITERATURE


Chemosterilants


General Conception


Definition

The term "chemosterilants" was first used in 1960 by LaBrecque. Chemosterilants may be defined as chemical compounds which reduce or entirely eliminate the reproductive capacity of an animal to which they are administered. Principal Action

Chemosterilants may affect one or both sexes of a

sexually reproducing species. Their action may be immediate or delayed; their effect may be temporary or permanent. Chemosterilants act on insects in three principal ways:

(1) by causing failure to produce ova or sperm, (2) by causing the death of sperm or ova after they have been produced; and (3) by inducing dominant lethal mutations or severely injuring the genetic material in the sperm and ova (Smith et al., 1964).








Mode of Action of Two Main Groups of Chemosterilants

The two groups of chemical compounds that have

shown the greatest promise as insect chemosterilants are the antimetabolites and the alkylating agents (LaBrecque, 1963, 1965; Smith et al., 1964).

Antimetabolites are a group of compounds that

inhibit the use of any of the products of metabolism by the treated organism. These chemicals interfere with the synthesis of nucleic acids (Borkovec, 1966). They are structural analogs of purines, pyrimidines, and folic acid (Ross, 1962; Jukes & Broquist, 1963). Most antimetabolic chemosterilants affect only the females of the species, particularly when administered to adult insects (Mitlin et al., 1957; LaBrecque et al., 19607 Crystal, 1963; Kilgore & Painter, 1962, 1964; Kilgore, 1965). Antimetabolites administered at a time when nucleic acid synthesis is occurring in many different tissues usually produce general symptoms rather than specific effects on the developing gonads (LaBrecque et al., 1960). The mode of action of antimetabolites in purine, pyrimidine, and folic acid analogs has been extensively investigated, and, in many instances, the precise steps in the metabolic process which they inhibit









are known (Montgomery, 1959; Timmis, 1962; Jukes & Broquist, 1963; Borkovec, 1966). Antimetabolites are considered to have less potential value as chemosterilants than alkylating agents.

Alkylating agents replace hydrogen in an organic

molecule with an alkyl group (Ross, 1962). In biochemistry, alkylation implies the introduction of a hydrocarbon radical,

often containing elements other than carbon and hydrogen, into a molecule under physiological conditions. Compounds capable of producing such reactions are referred to as alkylating agents (Borkovec, 1966). The principal classes of biological alkylating agents were reviewed in a monograph by Ross (1962). Hundreds of articles dealing with the mode of action of alkylating agents have been discussed by Timmis (1962), Wheeler (1962), and Kilgore (1965). The sterilizing activity of aziridines and other alkylating agents involves a similarity of action on a molecular level (Smith et al., 1964). When an alkylating agent replaces hydrogen in an organic molecule with an alkyl group within fundamental genetic material, the effect is similar to that produced by irradiation (Alexander, 1960).








Important Chemosterilants

The largest and most important group of biological

alkylating agents is the derivatives of aziridine (Borkovec, 1966), of which tepa [tris(l-aziridinyl)phosphine oxide, APO, aphoxide], tretamine [2, 4, 6-tris(l-aziridinyl)-striazine, triethylenemelamine, TEM], apholate (2, 2, 4, 4, 6, 6-hexahydro-, 2, 2, 4, 4, 6, 6-hexakis(l-aziridinyl)-l, 3, 5, 2, 4, 6-triazatriphosphorine], aphamide [N, N-ethylene bis (P, P-bis (1-aziridinyl) - N-methyl phosphinic amide], aphomide], and metepa [tris(2-methyl-l-aziridinyl)phosphine oxide, methaphoxide, MAPO] are the five best known, most highly active, and widely tested chemosterilants. Aphamide is primarily of historical importance, because its activity appears to be comparatively low in relation to the others.


Searching for New Chemosterilants


Mitlin et al. (1957) induced sterilization in house flies through the use of mitotic poisons. Three of the four chemicals tested usually inhibited oviposition and prevented ovarian growth. This work marked the beginning of largescale research in chemosterilants.

As early as 1958, LaBrecque began screening chemical










compounds for their chemosterilizing effects. Of 2000 compounds initially tested, five caused sterility in the house fly, Musca domestica Linnaeus, by feeding treatments (Linkfield, 1966). In 1960, LaBrecaue et al. tested 200 chemicals, 79 of which had some deleterious effect when added to the larval medium, but only ten of which affected development when combined with the adults' food. In 1962, LaBrecaue and Gouck tested 1100 compounds, twenty of which caused sterility in the adult house fly when administered in the food. In 1963, LaBrecque tested 2000 chemicals, 40 of them causing sterility (Linkfield, 1966). In 1965, LaBrecque reported that 112 chemicals had been found to produce sterilant effects, and this number continues to grow (Kohls et al., 1966).



Field Experiments in the Control of House
Flies by Chemosterilant Techniques


Several field tests have been conducted to control the house fly with the chemosterilants tepa, metepa, and apholate.

The first field test using tepa against the house fly was conducted in 1961 by LaBrecque et al. in a refuse dump at Bahia Honda Key, Florida; the adult fly populations









were reduced from 47 to 0 per grid count within four weeks with use of cornmeal bait containing 0.5 percent tepa. Female flies trapped at the dump were checked for egg masses and viability. The viability of egg masses had decreased from 100 to 10 percent (LaBrecque et al., 1962a). Metepa at

0.5 percent was applied to droppings in a poultry house in suburbs of Orlando for control of the house fly, with similar results (LaBrecque et al., 1963a).

Gouck et al. (1963b) conducted a test in a refuse

dump at Pine Island, Florida, using cornmeal bait containing

0.75 percent apholate. A reduction of flies from 68 per grid count to 5-20 occurred during the first seven weeks. When bait was made available continuously, the population decreased to 3 to 0 per grid count.


Structure, Physical and Chemical Properties of Metepa


Metepa, though usually much less effective than tepa (Murvosh et al., 1964a; Chang & Borkovec, 1964), still merits consideration because it is less toxic (Hayes, 1964), less hazardous to handle, and more stable than tepa (Beroza & Borkovec, 1964).

Metepa is a highly reactive tri-functional derivative









of phosphorous oxychloride and propylene imine. Its structural formula is as follows (Anonymous, 1962):


CH - CH 0 H - CH
N P N
/ I
CH 2 N7 CH 2
CH2 - CH
I
21
CH3
Ln3


The reactive functions of metepa are the three membered imine rings which open at the carbon-nitrogen bond to yield a wide variety of additional products.

The typical physical properties of metepa are as

follows: molecular weight 215; straw-colored liquid with a boiling point between 1180 and 1250C. at 1 nim.;Hg;specific gravity at 25oC. is 1.079; refractive index, n25D is

1.4798; completely soluble in water and common organic solvents (Anonymous, 1962).

The common form of the chemical is a liquid which contains a minimum of 92 percent metepa based on reactive imine assay and no more than 0.5 percent volatile material. Like all aziridine compounds, metepa rapidly loses reactivity in an even mildly acidic solution (Plapp et al., 1962; Beroza & Borkovec, 1964).









Metepa has been used successfully to sterilize the

stable fly (Harris, 1962), the house fly (LaBrecque et al., 1962a), the screw-worm (Gouck et al., 1963a), the gypsy moth (Collier & Downey, 1965), the pink bollworm (Ouye et al., 1965a), and many other insects (Smith & LaBrecque, 1967). The metabolic rate of a phosphorous-labeled sample of the chemosterilant metepa was investigated by Plapp et al. (1962). Adult house flies degraded 50 percent of large dosages of the chemical within two hours. The rates of degradation were similar in a susceptible fly strain and in two organophosphate-resistant strains. The stability of

0.05 molar metepa with respect to hydrolysis under alkaline and acid conditions was also determined.

Borkovec et al. (1964) determined that, in partially degraded solutions, the sterilizing activity was proportional to the content of intact tepa or metepa rather than to the total content of the aziridine function.

Chamberlain and Barrett (1964) determined that, with topical treatments, the male screw-worm fly (Cochliomvia hominivorax [Coquerel]) required 5.5 times as much metepa per gram of body weight as the male stable fly, Stomoxys calcitrans (Linnaeus), and the female screw-worm fly required 18 times as much as the female stable fly. The values for









feeding treatments of the screw-worm fly and stable fly were

3.9 and 6.2 times, respectively, for male and female.

House flies subjected to radioactive metepa residual deposits on glass lost 89 percent of their radioactivity within 24 hours. Because of the high degree of chemical activity of this compound, it is evident that the residual radioactivity in these insects does not represent unmetabolized metepa (Plapp et al., 1962; Dame & Schmidt, 1964a).


Specificity of Chemosterilants


The use of chemosterilants to sterilize an insect

can produce adverse effects, the magnitude of which will be

influenced by a number of factors. In a broad sense, specificity of chemosterilants encompasses a variety of selective activities of the compounds in different organisms, different organs or functions of each organism, different stages of development, and different modes of application (Borkovec, 1966).

There is wide variation in the susceptibility of

species, sexes, and developmental stages to both the lethal and the sterilizing effects of chemosterilization. Environmental factors such as temperature, humidity, pH value, food,








etc., can influence the results of the chemosterilants. The dose levels vary according to specificity, method of application, sex, species or strain, developmental stage, competitiveness, longevity, and environmental factors.

It is not necessary to cite individual papers in

this extensive literature, because they have been reviewed and listed as references in comprehensive publications. Anyone planning to work on insect chemosterilization should read the publications of Smith et al. (1964), Ascher (1964), Hayes (1964), Borkovec (1966), and Smith and LaBrecque (1967). Anyone planning work on house fly chemosterilization should read publications of LaBrecque and LaBrecque et al. (1960, 1961, 1962, 1963a, 1963b, 1965, 1966).

The following sections present the results of several studies on chemosterilization of the house fly.

The relationship between concentrations of metepa, apholate, and tepa in diet and degree of sterility induced in adult house flies showed that wider variation resulted than would be expected from similar tests with insecticides. The calculated sterility concentrations (SC50 and SC90) of metepa and apholate were similar; tepa sterilized at lower concentrations (Murvosh et al., 1964b).

LaBrecque et al. (1966) achieved 99 to 100 percent








sterility in the male house fly by feeding hempa (hexamethylphosphoramide) at concentrations as low as 0.25 percent. It was less effective against females.

Chang and Chiang (1964) studied the sterility effect

of thio-tepa on the house fly, M. domestica vicina Macquart. The feeding technique proved to be an effective and easy method. The use of 0.5 percent thio-tepa in milk powder (w/w) for two days, or 1 percent for one day, induced complete sterility; eggs were laid but these did not hatch.

Contact method and topical application also proved effective; 50 pg per fly was effective in reducing 90 percent of the reproductive potential. Pupae were most resistant. Third instar larvae were more sensitive than those in the first, and many morphological abnormalities appeared in the larval treatment. Two-day-old flies were almost as sensitive as newly emerged ones; however, four- and six-day-old flies were less sensitive.

Sterility was induced in the house fly, M. domestica, by dipping puparia containing pupae of different ages in apholate, tepa, and metepa at concentrations of 2.5 and 5 percent for 30 to 300 seconds. With all dipping periods and with both concentrations, apholate and metepa gave the most consistent sterility in flies emerging from puparia dipped









when pupae were one day old (Gouck, 1964). The dipping of one- and three-day-old pupae of house flies in solutions of hempa at 50 percent concentration in water for five minutes produced 100 percent sterility in both sexes (LaBrecque et al., 1966).

Male house flies sterilized by feeding a diet containing 1 percent apholate were as successful as normal males in competition for mates. The percentage of sterile eggs laid by females in cages containing normal and chemosterilized males was as high as, or higher than, would be expected from the ratio of sterile males present (LaBrecque et al., 1962a). Hempa at 1 percent in the flies' food did not impair mating competitiveness of males nor the mobility of the sperm (LaBrecque et al., 1966).

Morgan and LaBrecque (1962, 1964) reported that, in general, apholate, tepa, and metepa inhibit ovarian development in house flies. The chromatin of the nurse cell nuclei was clumped in irregular masses.

Tung (1965) treated the house fly, M. vicina, with thio-tepa in different solutions and for various durations. The results indicated that the sterility of female house flies was due to the degeneration of oogonia in the ovaries. The number of oogonia gradually decreased and degeneration








followed; finally, the ovary entirely atrophied. The degree of degeneration of oogonia was found to be proportional to the dosage of thio-tepa and the duration of treatment.

P32-labeled metepa was rapidly absorbed from a glass surface by both mosquitoes (Anopheles quadrimaculatus Say and Aedes aegypti [Linnaeus]) and house flies (Musca domestica Linnaeus). House flies and Anopheles cquadrimaculatus absorbed approximately 7 pg per insect during a four-hour exposure on a surface treated at 10 mg/ft2, whereas Aedes aegyqvpti picked up 2.5 pg. This intake resulted in a severe reduction of mating ability in mosquitoes, coupled with 99 percent sterility in house flies and A. aecypti males (Dame & Schmidt, 1964a).

When tepa uniformly labeled with C14 was injected

into the male house fly at the rate of 1 pg/fly, the radioactivity was transferred to female flies by copulation with treated males (Chang et al., 1966).

The effects on house flies of exposure for various periods of time (four, three, and two hours) to residues (1, 14, 30, and 60 days old) of tepa and metepa (250, 100, 50, 25, and 10 mg/ft2) on glass were studied by Meifert et al. (1963).

When newly emerged virgin adult house flies were









exposed for four hours to residues of hempa in glass jars (200 mg/ft2), only the male flies reached 100 percent sterility; the females never exceeded 38 percent. At dosages higher than 200 mg/ft2, the hempa residue acted as an adhesive, causing high mortality in the flies (LaBrecque et al., 1966).

Chang and Borkovec (1964) determined that tepa was

four times as effective as apholate and 12.5 times as effective as metepa in sterilizing male house flies by injection.

Hempa administered to male house flies by various methods produced the following results: injection with a dose of 40 pg/fly, 100 percent sterility; topical application with a dosage of 200 pg/fly, 100 percent sterility; and oral application of a 1 percent concentration in food, 99.9 percent sterility (Chang et al., 1964).

Two series of compounds related to tepa and hempa

were tested by Chang and Borkovec (1966) on male house flies (M. domestica Linnaeus) to determine the structure-activity relationship.









Biology


Name and Classification


Musca domestica is one of the best known and most used scientific names. For centuries the common name of M. domestica has been housefly (house fly, house-fly) in English-speaking countries. L. O. Howard (1911a, 1911b, 1911c) and his contemporaries applied the names "typhoid fly," "cholera fly," "dysentery fly," and "enteric fly," since typhoid fever and cholera were the most serious and widespread fly-borne diseases at that time.

According to present-day concepts of structure and taxonomy, the only form that has been generally recognized as biologically distinct is M_. domestica vicina Macquart, a subspecies tending to have a more tropical distribution than M. domestica domestica. The classification of M. domestica is as follows (West, 1951):

Kingdom: Animalia

Phylum: Arthropoda

Class: Hexapoda

Order: Diptera

Suborder: Cyclorrhapha (Athericera)

Series: Schizophora









Section: Muscoidea (Myodaria) Subsection: Calypteratae Family: Muscidae

Subfamily: Muscinae

Genus: Musca

Species: M. domestica Subspecies: M. domestica domestica Linnaeus
M. domestica vicina
Macquart

The subspecies M. domestica vicina differs from M.

d. domestica chiefly in having a more extensively orange abdomen, especially on tergites 1 to 3. The males differ further in having a somewhat narrower vertex, compared with the width of the compound eyes (Peffly & LaBrecque, 1956).


Distribution


The geographical distribution of M. domestica is usually considered world-wide. Graham-Smith (1914) stated:

Musca domestica is probably the most widely distributed
insect to be found; the animal most commonly associated with man, whom it appears to have followed over
the entire earth. It extends from the sub-polar regions
to the tropics, where it occurs in enormous numbers.









The Adult


Emergence

No special conditions are required for the emergence of adults from pupae. When the transformation has been completed, the fly pushes off the anterior end of the pupal case. Once its head is free, the fly crawls out of the puparium, at the same time extricating itself from the nymphal sheath, which remains as a lining to the empty case (West, 1951). Eversion of the ptilinum is accomplished by changes in blood pressure, and retraction by special muscles that do not persist in aged adults (Laing, 1935). The fly crawls rapidly about while its wings unfold and the exoskeleton proceeds to harden and darken. Finally, the ptilinum is withdrawn completely, leaving only the crescentic frontal "lunule" above the antennae to mark its previous location (West, 1951).

When the rearing room is maintained at 250C., the first adults emerge on the tenth day after hatching, the majority on the eleventh, and a few on the twelfth. Emergence is easily delayed by allowing pupal development to proceed at ambient rather than rearing-room temperature or by cool-storing the pupae (Bucher et al., 1948). At least 95 percent of the adults can emerge from the pupae under laboratory conditions









(Spiller, 1966). The author's experiment in the rearing room indicated that male flies come out first and that the size of the adult fly can be influenced by underfeeding in the larval stage.


Feeding

The house fly has shared man's food and developed

in his wastes and in those of his domestic animals since the world was young. House flies are almost omnivorous and breed in fermenting vegetable and animal matter and in other filth, without which they cannot exist, despite their high reproductive capability (West, 1951; Herms et al., 1961).

All day long their restless nature causes them to fly back and forth between the privy and the kitchen, between a wound that is infected and a fresh incision. When disease organisms are in the waste, the house fly carries them. This is what makes the species so dangerous and important.

Many older feeding techniques supplied liquid milk daily. Now this has been replaced by water and dry food (six parts granulated sugar, six parts nonfat dry milk, and one part powdered egg yolk) (LaBrecque et al., 1960). According to Dame and Fye (1964), who studied the feeding









behavior of house flies, on the dry baits 50 percent of the flies had fed by the twelfth to the sixteenth hour, while on the liquid bait 50 percent had fed by the fifth hour and 90 percent by the twelfth hour. Acree et al. (1959) showed that the response of house flies to sugar was apparently related to the relative humidity gradient between the bait source and the surrounding environment. Robbins et al. (1965) reported that both casein and yeast hydrolyzate contain feeding stimulants for the adult female house fly. The major active substance in yeast hydrolyzate is guanosine monophosphate, whereas several amino acids (leucine, lysine, isoleusine, and methionine) are the active compounds of casein. Solution in a phosphate buffer appears to be necessary for maximum activity.


Flight

Bishopp and LaakeJ(1921) carried out an extensive

series of flight experiments in Texas. Some 234,000 flies were captured, dusted with finely powdered red chalk or paint, and then liberated in the open fields. Within 24 hours, the house flies were captured an average of six miles distant from the point of release, and the maximum distance traveled was 13.14 miles by one female. Schoof and Siverly









(1954), using radioactive isotopes as markers, indicated that flies can fly as far as 20 miles from their source and that, under certain conditions, they may migrate in considerable numbers from one to four miles; the dispersion, however, is usually limited to a distance of 0.5 to two miles. Quarterman et al. (1954) suggested that in rural areas flies may move at random within an area eight to ten miles in diameter. Murvosh and Thaggard (1966) pointed out that an individual fly from the Gainesville laboratory strain appeared to move in a laboratory room at rates ranging from about 1.5 to six ft/sec, as clocked by a stop watch. West (1951) mentioned that if it were possible to stimulate a fly so that most of its flying time would be spent traveling in the same general direction, the distance traveled would be relatively enormous, perhaps hundreds of miles.

Hindle (1914) reported that fine weather and warm

temperatures were positive factors in encouraging dispersal, and that flies tend to travel much farther in the country than in town, where, of course, food and shelter are available on every hand. The time of day affected the tendency of flies to engage in dispersal flight.









Mating Behavior

Male flies are sexually aggressive and frequently mount females. Murvosh et al. (1964a) reported that the

mating behavior of the house fly is a complex phenomenon involving several factors:

(1) Both male and female must undergo a sexual

maturation time. The results of this study indicated that males and females will not mate for at least 16 and 24 hours, respectively, after emergence. Anesthesia prior to mating may have retarded the process somewhat, but this factor was not measured. Michelsen (1960), who did not anesthetize the flies prior to mating, reported that at 280C. males reached sexual maturity in 18 to 27 hours after emergence. Chang (1965a) studied the chemosterilization and mating behavior of male house flies; the results indicated that male and female house flies reached sexual maturity in 20 and 40 hours, respectively, after emergence. Mating may occur within 24 hours of emergence (Barber and Stanes, 1949).

(2) The male apparently has an instinctive drive to mate, which is not dependent upon the presence of the female

or a female odor.








(3) Males will attempt copulative strikes with males or certain inanimate objects but strike more readily and

more frequently at females.

(4) Preliminary evidence suggests the presence of some type of female sex attractant of low odor.

Actual copulation apparently never takes place in the air, though the seizure of the female by the male may occur in flight, after which the pair come rapidly to some surface; sometimes, however, if disturbed, a copulating pair may leave one surface and fly quickly to another (Murvosh et al., 1964a). The insemination process presumably begins as soon as copulation has been physically accomplished (Chang, 1965a). Murvosh et al. (1964a) found that after mating periods of one and two minutes no sperm were found in the female, but after three to five minutes some spermathecae contained a few sperm and some were completely filled.

Copulation time was studied using seven-day-old

virgin males mated with virgin females of the same age; of 61 mating pairs the shortest mating period was 44 minutes, whereas the longest extended to 96 minutes. The average time was 60 minutes. Hampton (1952), on the basis of more than 50 observations, stated that copulation lasts longer








than 30 minutes and may last two hours, but that the usual time is 1.5 hours. Sacca and Benetti (1960) reported, from 110 observations, a mean of 84 minutes, a maximum of 136 minutes, and a minimum of 59 minutes. Chang (1965a) reported that the average time flies remained in coitu was 56 minutes, with a range of 34 to 93 minutes and a standard

deviation of 12 minutes.

Mating behavior appeared the same under ultraviolet light as under normal light (Murvosh et al., 1964a). Also, house flies can mate in the absence of both ultraviolet and visible light (Rogoff, 1965). Male flies handicapped by removal of legs successfully mated with females (Chang, 1965a). Females of all strains mated more readily with males of their strain, whether or not the males were sterilized (Fye & LaBrecque, 1966). Chemosterilized males competed more successfully than normal males of the same strain when they were mated with females of that strain (LaBrecque et al., 1962; Fye & LaBrecque, 1966; LaBrecque et al., 1966). Radioactivity was transferred to female flies by copulation with treated males (Chang et al., 1966).

In a genetic study of the cross between red-brown and green-eyed house flies, Zimgrone et al. (1959) concluded that








the female usually mated only once and that fertilization sufficed for the entire egg-laying period. Only 2 percent of the females showed evidence of mating twice. Riemann et al. (1967) reported that most normally mated female house flies are monogamous, and none will mate more than a few times. The loss of sexual receptivity by females after

mating was caused primarily by the male seminal fluid, not mechanical stimulation or sperm.

When, in the laboratory, the subspecies Musca domestica domestica and M. domestica vicina were crossed, characteristics of the progeny were nearly those typical of the latter. In another cross (F1 and back-cross), the same

tendency was shown. Characteristics of M. domestica vicina are therefore considered dominant over those of M. domestica

domestica (Peffly, 1953).


Oviposition

The female fly walks over the material that is to

serve as food for her larvae, seeking crevices and cracks in

which the eggs may have a measure of protection. The female pushes her ovipositor as far into the crevice as possible to lay eggs. One egg or several may be laid in a single spot, but all will be hidden if circumstances permit. If the physical nature of the medium allows it, the fly crawls deep









into the coarser crevices to lay her eggs, so the eggs receive maximum protection from desiccation and actinic light (West, 1951).

Egg production in the female seems to vary. Herms. and James (1961) pointed out that from 75 to 150 eggs are deposited singly and piled up into masses, and that there are usually several such laying periods at intervals of three or four days. Female flies begin depositing eggs from nine to twelve days after emerging from the puparium. Lineva (1953), in studies on the physiological age of M. domestica females in Russia, found that a female that oviposited 20 times lived 62 days. Dunn (1923), reporting on his observations in Panama, stated that as many as 159 eggs may be deposited in one batch, and that one female may deposit as many as 20 batches, or a total of 2387 eggs, within 31 days after emergence. Hodge (1911) stated that

. . . a pair of flies beginning operations in April
may be progenitors, if all were to live, of
191,010,000,000,000,000,000 flies by August. Allowing one-eighth of cubic inch to a fly, this number would cover the earth 47 feet deep.

Harold (1965) recalculated that figure and decided that a layer of such a thickness would cover only an area the size of Germany, but that is still a lot of flies.

Spiller (1966) pointed out that neither adenine nor








yeast RNA improves egg laying. There are indications of undefined chemical or physical factors which facilitate high egg production. Possibly there are feeding stimulants (Robbins et al., 1965), the increased egg production reflecting

increased food intake. Among flies fed with the dry diet (LaBrecque et al., 1960), the eggs mature earlier than among those fed with other kinds of food and are laid on the fifth, sixth, or seventh day rather than on the ninth or tenth. Older female flies produce an increased proportion of infertile eggs (Callahan, 1962). Use of additional ammonium carbonate may increase the egg harvest at any one collection, but there is no evidence that total egg laying can be increased.


Longevity

A few adults die within a few days. Murvosh et al. (1964a) found that the longevity of the control flies was of surprisingly great duration, considering the reports in the literature. Room temperatures during the experiment ranged from 22.20C. (720F.) to 29.40C. (850F.), with a mean of 23.90C. (750F.); the relative humidity was 40 to 80 percent. This experiment showed that male flies lived an average of about 33 days, with a range of one to








71 days, whereas the females lived an average of 43 days, with a range of 1 to 99 days. Rockstein and Lieberman (1958) reported that, at 26.80C. and 45 percent relative humidity, the mean longevity of one strain was 17.4 days for males and 29.4 days for females. Varzandeh et al. (1954) reported the longevity of three susceptible strains of flies as 22.5, 32.3, and 30.8 days for females and 17.0, 23.4, and 23.0 days, respectively, for males.

Herms (1928), working in California, recorded an

average longevity of 30 days, with a maximum of 60 days in summer. Under experimental conditions during summer at Ithaca, New York, individual flies have been reared and kept

alive for 30 to 70 days.

Afifi and Knutson (1956), in a longevity study conducted for the National Association of Insecticide and Disinfectant Manufacturers, employing house flies with no history of insecticidal exposure maintained at 80oF. and 50 percent relative humidity, reported results as follows: treated parent, 17.1 days; untreated parent, 16.8 days; treated F2, 28.4 days; untreated F2, 27.1 days; treated F3, 23.7 days; and untreated F3, 23.9 days. The mean number of days from initial oviposition until all egg laying had ceased was as follows: treated parent, 22.4 days; untreated parent,









23.2 days; treated F2, 39.4; untreated F2, 38.6; treated F3, 30.7; and untreated F3, 30.4. They found no significant difference in results between treated and untreated flies.

The age of the parents has some effect on succeeding generations. Average length of adult life decreased when lines were bred exclusively from eggs deposited by young flies (Callahan, 1962). In another strain, continued use of

the last viable eggs decreased longevity and reduced fitness so that no more than three consecutive generations could be reared (Callahan, 1962). When eggs were always taken from females at the age of 20 to 30 days, the average longevity of the offspring was increased. Sex Ratio

In nature, the sex ratio is usually about one to one, but sometimes there is considerable departure from this norm. In the laboratory condition, Murvosh et al. (1964b) repdrted the sex ratio, based on 5233 individuals, to be 53.5 to 46.5; four years earlier, the sex ratio in a sample of 8700 flies from this same colony had been 50.6 to 49.4. Rogoff (1965) reported that the sex ratio, based on 16 samples of 100 pupae, was similar in jars of both the









control and full darkness series, and the results were within normal expectations.

Murvosh and Thaggard (1966), in ecological studies of the house fly, found an unusually high proportion of males in 21 samples of 4266 flies collected at various times from five kitchens on Mayaguana Island. The ratio was about

1.5 to one among 4266 Mayaguana flies. This was a highly significant deviation from the expected ratio of one to one mentioned above. They also reported that, in the same area, they had examined flies gathered from the hoods of vehicles in the carport outside their laboratory; each day for three days they counted 50 flies but noted only one female.

West (1951) reported that when the size of average

adults in a random sampling is small, the males will be more numerous, while if the average fly captured is of superior weight and size, this is never the case.

Herms (1928), while studying Lucilia sericata,

brought out the fact that an unfed larval population always yields a preponderance of males. He removed the larvae from food in lots of 100 at six-hour intervals, after allowing an initial feeding period of 30 hours; the longest feeding period allowed any lot was 96 hours. The longer feeding periods yielded a preponderance of female flies. Herms









felt that larval females required more nourishment than males for their development. This may also be true of the house fly, as indicated by the fact that the male flies usually emerge first. The female flies, after copulation, influenced by chemotropism, fly to the so-called "breeding places" for oviposition. The average longevity of the females is greater than for the males. The amount of nourishment in the "breeding places" is not always sufficient; therefore, to determine the sex ratio in the natural condition, the time, the place, the size of the sample, and the methods used should be carefully considered.


The Egg


The egg is pearly white in color and measures about

1 mm in length. The greatest diameter, which is near the posterior end, measures a little more than one-fourth of the length. Both ends are bluntly rounded, but the anterior is always more tapering. Along the dorsal surface of the egg are two distinct riblike thickenings.

The results of numerous investigations clearly demonstrate that excrement is one material upon which M. domestica habitually deposits its eggs. One or several eggs may be laid in a single spot, but all will be hidden if









conditions permit. Average egg size is proportional to body size (Spiller, 1966).

The time required for the completion of embryonic development varies greatly with temperature. Under warm summer conditions, the egg stage requires 8 to 12 hours (Herms & James, 1961). Melvin (1934) showed that both high and low temperatures tend to prolong the incubation period.


The Larva


Breeding Places in Nature

Though the larvae of house flies have reasonable

powers of migration, this activity is limited for the most part to the stage prior to pupation and has much more to do with the selection of a suitable site than with the seeking of materials for nourishment, since the mother fly deposits her eggs on carefully selected materials and there is usually no nourishment problem for her newly hatched offspring.

Hewitt (1914) recorded M. domestica as breeding in horse, cow, human, pig, rabbit, chicken, and other manures. Carrion, spoiled meat, offal of slaughterhouses, old broth, a boiled egg, rotting fowl feathers, decaying grain, cooked peas, boiled rice, barley malt, excreta-soiled straw, bread,









cake, milk, rotten peaches, plums, cherries, bananas, apricots, potatoes, potato peelings, cabbage, carrots, cucumbers, cantaloupe, and watermelon are common breeding places. In addition, the list includes such miscellaneous items as kitchen refuse, fermenting substances, sawdust and rags soiled with excrement, earth containing expectorated material, rubber, and snuff.

Horse manure is the favorite breeding ground of

the house fly. When horse manure mixed with straw or other refuse is piled in the open, it soon becomes heavily infested with maggots. At the present time, at least in developed areas, other breeding hosts may be vastly more important than excrement. Smith (1956), studying conditions in dairy barns in California, concluded that

. . . the fly breeding potential of these
enormous amounts of organic waste materials
(chicken and cow manure, garbage, etc.) is
far greater than it could possibly have been
back in the horse-and-buggy days ...
Perhaps the greatest numbers of house flies I have seen came from lima bean waste in the
field. I have seen nearly as great numbers
from celery waste, tomato, etc.


Rearing Medium in Laboratory

The Gainesville Laboratory of the Entomology

Research Division, Agricultural Research Service, U.S. Department of Agriculture, produced house flies for one year at









the rate of two million per week for use in release experiments. The methods used were similar to those described for this investigation, using the CSMA larval medium and a dry adult diet.

There are now no major difficulties in maintaining cultures. Though there is much literature pertaining to the artificial food of the larvae of the house fly, it is not relevant here. Insect Colonization and Mass Production by C. N. Smith (1966) provides a good background on this subject.

When larvae are reared at different densities on a standard medium, the numbers of pupae obtained are approximately a fixed proportion of the numbers of eggs used, but the size of individual pupae decreases as the rearing density increases (Spiller, 1966). It is very important in all experiments, especially those involving injection treatments, to keep conditions as uniform as possible and to provide quality control in the size of flies reared for testing. Growth Rate and Instars

The larvae, or maggots, molt twice and pass through three instars. All stages have twelve segments, of which the second, or postoral, segment is actually double, giving









a total of thirteen (Hewitt, 1914). There are no eyes, legs, antennae, or other appendages. In the first-instar larvae, only the two posterior spiracles are present; second- and third-instar larvae have both posterior and anterior spiracles.

First-instar larvae molt anywhere from 20 hours to four days after hatching; second-instar larvae require 24 hours to several days; third-instar larvae feed for three to nine days before undergoing pupation.

The rate of growth agrees fairly well with Przibram's rule (Teissier, 1931), which holds that in most insects an increase of 25 percent in the length of the rigid parts of the cuticle is achieved with each successive molt. Larsen and Thomsen (1940) made daily weighings during the preimaginal period and constructed weight curves to represent the rapidity of growth. First- and second-instar larvae were studied by Tao (1927). The third-instar larva grows to a size of 12 mm or a little more. The right and left anal lobes are useful in locomotion.


The Pupa


The third-instar larva goes to a suitable place,

somewhat cooler and drier than that required for its larval









stage, for its pupation; usually pupation occurs in its breeding place. In the laboratory, pupation occurs in the rearing medium. Under nearly ideal conditions, pupation begins early on the fifth day and is completed soon after the end of the sixth day.

The process of pupation consists of a general construction of the larva within its own integument so that the latter comes to form a puparium; the pupa case is formed by the last larval skin, within which the pupa is said to be coarctate. The pupal stage requires three to five days under suitable conditions; under adverse conditions several weeks may be required (West, 1951; Herms & James, 1961).


Hibernation


In cooler climates, under natural conditions, the continuous breeding of the house fly is interrupted by the winter season. Authorities differ in their opinions as to how the house fly hibernates. Hewitt (1914, 1915) held rather strongly that the house fly passes the winter only in the adult stage. Copeman (1914) maintained that, although the fact that adults can survive the cold is the principal factor in carrying the species through the winter, it is









possible that flies in the pupal stage also hibernate. Skinner (1915) stated that "houseflies pass the winter in the pupal stage and no other way."

The obvious conclusion is that the situation varies

from one locality and climate to another. There is no interruption in breeding under tropical conditions.


Temperature, Humidity, and Light


There are several important environmental factors

which influence the life process in the whole life cycle and which may accelerate, retard, or prevent entirely the fly's distribution, activity, growth, transformation, reproduction, and longevity.


Temperature

The influence of temperature on the speed of development of each stage of the house fly has been studied by many workers.

For each stage there is a maximum temperature above which development may not proceed, a minimum temperature at which activity also ceases, an optimum temperature at which growth, development, and normal activity proceed most satisfactorily, and a lethal temperature which causes the









death of the fly. Even within a given stage, these temperatures may vary according to sex or other factors.

Hewitt (1914) reported that two or three days may be required if the temperature remains as low as 10 C. (50oF.); between 15' and 200C. (590 to 680F.), hatching takes place usually within 24 hours after oviposition; between 250 and 350C. (770 to 950F.), from eight to twelve hours may suffice. Davidson (1944) demonstrated that variations in temperature are of great importance in determining the rate of embryonic development. At 37.2 C. (99oF.), the embryonic development may be completed in about 7.63 hours; at 150C. (590F.), it will be completed in about 51.45 hours; at 400c. (104oF.), about 8.05 hours; at 41.60C. (106.90F.),
0 0
only relatively few eggs hatch; at 42.8-C. (109 F.), no eggs can hatch.

Lorincz and Makara (1935) found that incubation required seven days at temperatures of 80 to 100C. (46.40 to 50 F.). At 200C. (68oF.), the period dropped to 22 hours, and at 300C. (86oF.) to 15 hours. At 400C. (104oF.), the eggs hatched 12 hours after oviposition.

Eggs of the house fly cannot survive at temperatures above 46.10 C. The actual lethal temperature is probably a little lower than this, especially under sustained exposure (West, 1951).









Jashi and Dnyansagar (1945) studied fly breeding in compost trenches in India and reported that, from April to June, when the temperature of the upper layer of rubbish ranged between 600 and 650C. (104o to 1490F.), few maggots or pupae could be found. During the rainy season, the temperature of this layer fell to 370 to 49oC. (98.60 to 1200F.) and many maggots and pupae were observed. Of course, during this period, there was much higher relative humidity. Puri (1943) states that, in general, high temperatures augment fly production by shortening the developmental period of the larvae, by hastening sexual maturation, and by stimulating mating and oviposition.

Kramer (1915) reported that development required only half as long at 300C. (800F.) as at-200C. (680F.). Kobayashi (1940) found that oviposition and larval development occurred in Japan at temperatures down to 150 to 160C. (590 to 60.8oF.). The same author (1935), working in Korea, showed that total development could take place in as brief a time as six days with the temperature at 280C. (82.40F.), but that this was extended to 41 days when temperatures ranged from 13 to 19 0C. (55.4o to 62.2oF.). Optimum conditions existed between 250 and 30 C. (77o to 86oF.), at which level development normally required from seven to twelve days.









The differential effect of the two types of media is very interesting. Except at extremes of high and low temperature, larvae reared on pig dung pupated sooner than those reared on horse or cow manure, and larvae reared on

pig dung attained greater size.

It was noticed that the activity of both second- and

third-instar larvae decreased markedly at temperatures above the optimum.

Feldman-Muhsam (1944b) reported that M. domestica vicina lived as long as 106 days in captivity at favorable temperatures; the flies died more quickly as temperatures rose.

Lodge (1918) reported that the optimum temperature for feeding of the house fly was from 38 to 48oC. (990 to 118 F.). The maximum temperature at which food was taken by the house fly was 550 to 580C. (1320 to 1360F.), and the
0 0 00
minimum was 10 to 13 C. (50 to 55 F.). Humidity

Feldman-Muhsam (1944a), who studied M. domestica vicina in Palestine, noted that cow manure dried very quickly, forming a crust; larvae and pupae could be found beneath, usually about four inches below the surface. The









same author (1944b) pointed out that in winter conditions are very different. Many larvae and pupae die because of excessive humidity. The same factor is believed to inhibit oviposition. Similar indications were observed during the present investigation.

Dakshinamurty (1948) showed that adult flies tended

to be most active when temperature was high and humidity low.

Katagai (1935), working in Formosa, reported flies

scarce from January through April, with a sudden increase in May, a decrease in June, and a second peak invariably associated with temperatures above 250C. (77oF.). Katagai noted that females were in the ascendancy between November and April, but males predominated during the remaining months of the year.


Light

The larvae of the house fly are more or less negatively phototropic. As a result, the larvae can avoid exposure to strong light with desiccation and seek a more suitable place.

Experiments using various colored light to attract the house fly were conducted by several workers (Awati, 1920; Parrott, 1927; Freeborn & Berry, 1935; Cameron,









1938, 1939; Harsham, 1946). They used different materials and different designs, and achieved somewhat different results, but all the results showed that the house flies were attracted in various numbers by various colors of light.

Ingle (1943) used blue light to attract flies to

screens for testing their reactions to various chemical substances. West (1951), citing the work of Kuzina, reported that M. domestica is active by night as well as by day, if the temperature is sufficiently high. No difficulty was experienced in catching flies in traps in the dark if the rooms were warm.

Rogoff (1965) demonstrated that house flies can

mate in darkness with neither ultraviolet nor visible light.













MATERIALS AND METHODS


The research work reported here was conducted in the Insects Affecting Man and Animals Research Laboratory, U.S. Department of Agriculture, Gainesville, Florida, from October 1965 to April 1967. Room temperature was maintained at 780 to 82 F. and relative humidity at 60 percent. However, the relative humidity was held at 50 percent in the rearing room for the stock house fly, Musca domestica Linnaeus. The Orlando regular colony of house flies (insecticide-susceptible strain) was used in all experiments.


Technique of Rearing Stock House Flies


The stock house flies were maintained by the following procedures (Insects Affecting Man and Animals Research Laboratory, Entomology Research Division, Agricultural Research Service, U.S.D.A., November 21, 1963).

House flies were collected by placing a paper cup containing week-old larval medium in a cage of breeding flies. The flies oviposited readily on this material and sufficient eggs were obtained in about two hours.









Larval medium was prepared by mixing 24 quarts of

Standard CSMA Fly Larval Media (Ralston Purina Company) with eight quarts of oat hulls and 18 to 20 quarts of water. About four quarts of medium were placed in a two-gallon glass jar lined with a polyethylene bag and about 2000 eggs were placed on the medium, after which the surface was lightly stirred to insure contact between the eggs and the medium. The jars were covered with a heavy cloth to prevent oviposition by flies that had escaped into the rearing room. The use of too many eggs in these containers would result in small adults due to larval overcrowding. Hatching occurred within eight hours and the jars remained undisturbed until pupation appeared complete. However, a careful watch was kept to see that the jars of larval medium did not become too hot during the accompanying fermentation. When a jar was warm enough to be uncomfortable to the touch, about one-half pint of water was added to the surface of the medium for cooling.

Pupation takes place near the surface of the medium. As soon as pupation appeared complete, the top few inches of the medium were removed, placed in a pan of water, and stirred. The medium sank to the bottom on standing and the pupae floated. The pupae were strained off, washed again,










allowed to dry, and placed in one-pint paper cups in colony cages.

When the pupae were introduced into the colony cages, a dry nutrient mix in three paper cups and water in two onepint paper cups fitted with wicks of paper toweling were added, to insure a proper supply of food and water to the emerging adults. Food and water were checked twice a week and replenished when necessary. The formula for the dry nutrient was as follows: six parts granulated sugar, six parts powdered nonfat dry milk, and one part powdered egg yolk.


Procedures for Testing, Rearing, and Observation


Rearing and Observation Cages


Two different sizes of cages were used throughout the experiments. Large cages were used for the testing of six or more pairs of flies, and the small ones for the testing of five pairs or less. In each cage were placed equal numbers of treated flies and flies of the opposite sex.

The large cage was constructed with an aluminum

frame, 10.5 inches long, six inches wide, and 9.75 inches high, covered with a cotton gauze tube which provided a









sleeve for the opening and was closed at the other end. Cages of this size and type were used for all tests of the

house flies in this laboratory.

The small cage was made using a 32-ounce wax-paper cup (5 inches high, with a base diameter of 4 inches and a top diameter of 4.75 inches), the bottom of which was cut off. The cup was covered with a cotton gauze tube, which provided a sleeve for the opening, and was closed at the other end.


Sexing Virgin Females and Males


A special sexing tool (suction apparatus) designed

by Dr. G. L. LaBrecque was used to pick up and separate the male and female flies. Flies were immobilized for manipulation with a continuous stream of carbon dioxide in a sexing well.

In the author's records, the shortest pre-mating period for house flies is not more than 12 hours. This phenomenon is not uncommon in this laboratory. It has been found advisable to sex the flies within four hours after emergence, and never after more than ten hours. On the other hand, it is equally unwise to sex flies just after their emergence, as the wings and other parts of the newly









emerged flies are not completely spread and hardened yet.

It is important to avoid sexing too many flies at one time. Carbon dioxide can have an adverse effect on longevity in house flies if the exposure is too prolonged or too intense. Overcrowding can also affect longevity, or influence the effects of chemosterilants, especially in the initial population. In the ordinary cage (10.5" x 6" x 9.75"), the number of house flies should not exceed 150, Overcrowded conditions can influence the fertility, mortality, longevity, and behavior of the house fly.


Keeping Same-Age Flies and DifferentAge Flies


Acquiring a sufficient number of flies of almost the same age for testing purposes requires a large number of pupae. Pupae were placed in an ordinary cage, and adults were removed to another cage at intervals of every one-half, one, or two hours during the emerging period, to keep the age differences within groups as small as possible.


Water and Fly-Food Supply


There was always an abundant food and water supply available for the flies. Each cage was furnished with fly










food in a paper cup as described above. Each cage was also furnished with a wax-paper cup containing cotton balls acting as wicks for distilled water; the amount of water needed depended upon the number of flies in the cage.

When the duration of the experiment was seven days or less, the water and food did not need to be added to or changed. For longer periods, as in experiments on longevity or successive egging tests, the old distilled water and fly food were replaced at periodic intervals. The water level in the water-supply cup was kept below the level of the cotton balls to protect the flies from accidental drowning.


Solution and Injection


The chemosterilant metepa was dissolved in distilled water at the desired concentrations. Each experiment or replicate used newly prepared metepa-water solution; it was considered unwise to use older metepa solutions for sterility or mortality tests.

The flies were injected under light CO2 anesthesia in the injection well. Injection was performed with a microinjector (Hamilton gas-tight syringe with a 30-gauge needle). The needle was inserted into the dorsal thorax through the cervix region by bending the head of the fly









slightly, taking care not to insert the needle too deeply into the thorax. The procedure was to inject one ul of the metepa solution into the house fly's body without moving or shaking the microinjector. After the needle was withdrawn, the head resumed its normal position, thus closing the puncture; bleeding after injection was seldom observed.


Mating


The treated flies were mated with normal flies of the opposite sex and appropriate ages. Both treated and normal flies were transferred directly to the observation cage by using a vial or petri dish in order to avoid the CO2 effect. In the pair-rearing test, as soon as the treated fly copulated with the normal fly, the pair was transferred to a separate cage. Occasionally, the mating time was recorded.

In the collective-rearing test, of course, it was

not necessary to transfer the mating pairs to another cage, nor was the mating time recorded. For convenience of catching or counting, a light was furnished outside the closed end of the cage. The light also served to keep the flies inside the rearing cage and to avoid escapes. It was also useful in feeding and other operations.









Egging


Flies isolated according to sex were treated and fed, then held for a certain specified period, after which the treated flies were mated with normal virgin flies of the opposite sex. After mating, suitable materials were used to collect the eggs. The old larval medium (one-half inch of moist CSMA medium in a souffle cup) or fresh larval

medium with a few drops of ammonium hydroxide added was used for stimulating the female fly to lay eggs.

Ovipositing behavior varied. Each female fly usually, though not always, laid one egg-mass in the packed medium in each egging; some laid two, three, or more eggmasses. Female flies usually do not lay eggs in a medium which is either too moist or too dry. For this reason, the

old medium which was used for egging was packed to form a slope, thus increasing its desirability as a site for oviposition and, especially in pairing tests, increasing the likelihood of getting all the eggs for complete counting.


Egg Counting


After three to four hours, the larval medium container was removed from the rearing cage, filled with water









and stirred with a small brush, causing the fly eggs to float to the surface. A small brush was used to break down the egg-masses and to transfer the eggs to small pieces of damp black cloth in a sample of 100 eggs from each cage. Since the fly eggs are white, they are readily visible and can be counted easily against the black background. The thinner and narrower this piece of black cloth is, the higher the pupation obtained.

In the collective rearing test, egg counting was by means of a sample. Since the virgin female can lay eggs without mating, and the mated female can lay a certain percentage of naturally infertile eggs, it is almost impossible to avoid variations between samples. Thorough stirring and mixing with a small brush was necessary before the eggs were transferred to the cloth, and a line-up arrangement of the sample eggs and careful separation were essential to avoid counting errors.

Since the shortest pre-hatching period of the house fly in the author's records was more than seven hours, no eggs were counted for the sterility test after the medium had been in the rearing cage for longer than that. The figure of 100 eggs per cage was selected to facilitate

counting, comparison, and further analysis of the data.










Hatching Counts


Two general methods of hatching counts were applied

(1) To count the hatching only, the eggs on the

black cloth were kept in a moistcondition within the petri dish for 24 to 48 hours after the egg counting and then examined by means of a binocular microscope. The egg shells showed the different characteristics in hatched and unhatched eggs. Usually, the unhatched egg maintains its original egg shape; however, this is not always true and can sometimes produce misleading results if these are based only on hatch percentage.

(2) To count the hatching when further information was desired, the egg cloth was placed on the top of the moist larval medium in a rearing container (usually a paper cup with a plastic bag) and covered with a small piece of cheesecloth, then maintained at 80 F. and 60 percent relative humidity for 24 to 48 hours. Here again, the egg hatch was examined by means of a binocular microscope and the counts recorded.


Pupation Counts


This step followed the same method as the second hatch count mentioned above. As the eggs hatched, the









larvae crawled from the cloth to the rearing medium in the container. The number of larvae that reached the pupal stage was determined by flotation. In the laboratory condition, pupation counts were conducted one week after

oviposition.

Various factors can influence the development of the larval stage. Moisture and temperature were the two most important factors concerned in all experiments. Ordinarily, pupation took place near the surface of the medium; however, pupation occasionally took place under the medium or at the bottom of the rearing container. The temperature was raised by the fermentation of the larval medium and often became unfavorable for larval development, especially in an overcrowded arrangement of the rearing containers in an incubation cabinet. For better results, the following suggestions are offered:

(1) Prepare the larval medium one day before using.

(2) Keep a small distance between the rearing containers. Do not overcrowd these containers, especially during the first three days.

(3) Distribute three to five cups of water among the rearing containers on each shelf of the incubation cabinet.









(4) Maintain a slow air current around the rearing containers. Stationary air may overheat; strong air movement may dry out the larval medium.


Criteria of Sterilization -- Determining Sterility

In general, there are three criteria for determining

sterility: (1) egg-hatch percentages, (2) percentage of pupation, and (3) fecundity of second or later generations.

Each criterion has its advantages and disadvantages, depending on the purpose of the experiment. The results reported here are based primarily on the percentage of pupation.


Determination of Lethal Dosage


In the preliminary tests, the experiments on lethal dosage were conducted together with those on sterility dosage. Since the results of these preliminary tests indicated a wide range of difference between the sterility dosage and lethal dosage, it was decided to treat the lethal dosage independently.

Flies of both sexes, from one to five days old, were injected with the different concentrations of metepa-water solution, reared in different cages, and observed continuously










for 48 hours. The mortality of treated house flies was recorded by sex and by age for the different concentrations

of metepa solution injected. The dosages of metepawater solution injected in the preliminary tests were as follows: 0.25, 0.5, 1, 2, 4, 8, 12, 16, 20, 32, 40, 48, 64, 80, 96, 112, 120, 144, 160, 176, 200, 208, 240, and 280 pg/male or female house fly. The mortality at dosages below the rate of 40 pg/fly was not much different from that of the controls (injected with water only). Thus, only dosage levels at or above 40 pg/fly were selected for lethal dosage tests.


Determination of Sterility Dosage


The microinjection technique was used to determine the sterility dosage for flies of both sexes from one to five days old.

The procedures of this experiment were as follows: sexing virgin female and male flies keeping same-age flies as well as different-age flies; injections mating; egging and egg counts, hatching counts, and pupation counts; and finding the percentage of sterility.

In the preliminary test, both males and females were injected at the same dosage. The results showed that there










was a significant difference between the sexes, so the experiments were conducted separately by sex.

Based on the results of preliminary tests, the dosages for the sterility dosage tests were 0.25, 0.5, 1, 2, 4, and 8 pg/fly for males and 2, 4, 8, 16, 32, and 64 pg/fly for females.

The percentages of sterility were determined on the basis of first egging and successive eggings.

In male flies, both collecting-rearing methods and individual pair-rearing methods were used.

In female flies, collecting rearing under the dosages of 2, 4, 8, 16, 32, and 64 pg/fly were tested in two replicates, and individual pair-rearing tests were conducted at the following dosage rates: one-day-old-females, 70 pg/female; two-day-old females, 50 and 60 pg/fly; threeday-old females, 45 pg/fly; four-day-old females, 10, 15, 20, and 30 pg/fly; and five-day-old females, 5, 10, 15, and 20 pg/fly.

Six-, 19-, and 33-day-old female flies were injected with dosages of 1, 2, 4, 8, and 16 pg/fly, with five female flies in each treatment.









Determination of the Permanence of Sterility


Based on the results of the sterility dosage experiments, male and female flies of various ages were injected with the 100 percent sterility dosage. At this dosage, there was no statistical difference among male flies of different ages. All male flies from one to five days old injected at the dosage of 8 -pg/fly of. metepa-water solution showed 100 percent sterility, at least at first egging, when the injected male was mated with a normal virgin female one day after injection.

Both the collective-rearing method and the individual pair-rearing method were employed. Treated flies of both sexes and various ages were crossed -with normal flies of the opposite sex in different mating groups. The flies in the first group were mated one day after injection; consecutive matings were at six-day intervals. The sterility of the different mating groups and the sterility of the same group (first mating group) in different consecutive matings provided the basis for determining the permanence of sterility.









Mating Competitiveness Test


The mating competitiveness test employed in this

study was based on the time lapse between release and mating.

The treated and control flies were the same flies used for the permanence-of-sterility test.

Two observation cages were used for each mating of different mating groups, one for the treated males and the other for the controls. The female flies used for mating were identical in number, age, and brood. The females were placed in the cages first, the exact number of females based on the number of males to be tested. A petri dish was used to transfer the treated males and the controls into the cages. The petri dishes in both cages were opened simultaneously. The time between the release and mating for each pair in both cages was recorded.


Rate of Chemosterilization of Sperm in Vivo


Three-day-old male flies were injected with metepawater solution at the SD100 level under CO2 anesthetization and placed in a cage containing virgin females of the same age. As soon as the metepa-treated male copulated with a female, the pair was transferred to a separate cage. Both










injection time and the time copulation began were recorded. When the mating was over, the male was removed and destroyed. The sterility of the mated female, based on the pupation,

was determined.


Derivation of Survivorship Curves


Flies from one to five days old were injected with SD100 dosage. Both males and females were studied, individually and collectively. The different treatments for deriving survivorship curves were performed as follows:

Treated females + normal males Normal females + treated males

Normal females + normal males

Normal females

normal males

Treated females

treated males

All male flies, regardless of age, were injected at the rate of 8 pg/fly; female flies were injected with different dosages according to their ages. Mortality counts were made and recorded daily. Longevity was calculated and survivorship curves derived for each treatment on the basis of these counts.













RESULTS AND DISCUSSION


Determination of Lethal Dosage


A series of tests was performed to determine satisfactory ways to immobilize and inject flies during treatment.

Based on the preliminary tests, the different dosage rates were selected. Dosages of 40, 80, 120, 160, 200, and 240 pg/fly were used in all tests; dosages of 48, 112, 144, 176, 208, and 280 pg/fly were injected only in flies of certain ages. (See Table 1.)

Flies were injected at ages from one to five days. They were tested by sexes, in groups varying from 16 to 25 flies, with five replicates and a 48-hour observation

period for each treatment.

The observed relationships between mortality and

metepa concentration will be discussed in the following sections.


Lethal Dosage of Male Flies


The relationship between male mortality and the concentration of metepa-water solution is shown in Table 1.










Table l.--Effects of chemosterilant metepa in different
dosages on male house flies of different ages (percentage of mortality based on 48-hour observation period).


% of Mortality 1-Day-Old 2-Day-Old
Observed Corrected*Observed Corrected


40 48 80o 96 112 120 144 160 200 208 240

Control


5.00 5.00 16.67



52.50 63.33

65.00 93.24 100.00



100.00

2.00


3.06 3.06 14.97



51.53 62.58 64.29 93.10 100.00


3.75 16.25





65.00 93.33

100.00


100.00 100.00

3.75


0.00



12.99





63.63 93.07

100.00 100.00


*Abbott's correction applied.


J/Fly










Table l.--Extended


at 48 Hours
3-Day-Old 4-Day-Old
Observed Corrected Observed Corrected


1


8.75 6.41 27.50 25.67





88.75 88.46 98.75 98.72 00.00 100.00


7.50



18.25



65.00

67.50



94.11

98.75 100.00 100.00

3.33


5-Day-Old
Observed Corrected


7.50

22.50 48.33 90.00

92.50 95.00



97.64 100.00


4.31



15.43



63.79 66.38



93.91 98.71 100.00

100.00


3.40

19.13 46.08 89.56 94.78 94.78



97.54 100.00



100.00


100.00

2.50


100.00 100.00

4.17








The corrected mortality percentage attributable to metepa is derived from the total percentage of mortality by correcting for the deaths that occurred in the controls. This correction is calculated by Abbott's formula, as follows:


-' - C
P = x 100
100 - C

where P = the death rate (%) caused by the treatment,

P'= the total death rate (%) observed, and

C = the death rate (%) for the controls (i.e., not
associated with the treatment).

Dosage-mortality curves for male house flies were obtained by plotting the corrected mortality on logarithmprobability paper, verified by a Chi-square test. The dosage-mortality curves for different ages of male flies are shown in Figs. 1-5. The values of LD10, LD50, and LD90 and the slope were estimated directly from the curves, and those values are given in Table 2.


Lethal Dosage of Female Flies


Similarly, the relationship between female mortality and the concentration of metepa-water solution is shown in Table 3.

The dosage-response curves for female flies, based




















Table 2.--LDi; LD50, LD90, and slope for each age of male flies estimated directly from the curves on logarithmprobability paper.

Age of LD0 LD0 LD90
Fly Slope* (Days) (pg) (lg) (Wg)

1 64 102 162 6.55 2 78 108 152 8.41 3 54 91 153 5.49 4 52 82 130 6.48 5 44 70 112 6.45


Slope = 1/log LD84 - log LD50.










Table 3.--Effects of chemosterilant metepa in different dosages on female house flies of different ages (percentage of mortality based on 48-hour observation period).


1-Day-Old
Observed Corrected*


6.25 10.00 15.00 30.00 32.50 60.00 68.57

75.00 100.00


40 48 80

112 120 144

160 176 200

208 240

280 Control


3.27 7.14 12.30 27.78 30.35 58.73 67.57 74.21 100.00



100.00 100.00


% of Mortality
2-Day-Old
Observed Corrected


4.00 16.19 31.00



75.24


0



12.69 28.13



74.21


100.00 100.00 100.00 100.00



4.00


*Abbott's correction applied.


11g/Fly


100.00 100.00

3.08









Table 3.--Extended


at 48 Hours
3-Day-Old 4-Day-Old 5-Day-Old
Observed Corrected Observed Corrected Observed Corrected

6.67 4.28 8.75 6.41 11.67 6.32



35.00 33.33 31.76 30.01 38.33 34.59

37.50 35.89

45.00 43.58 70.00 69.23 83.33 82.32



83.52 83.09 83.52 83.09 89.41 88.77



100.00 100.00 100.00 100.00 100.00 100.00

100.00 100.00

100.00 100.00 100.00 100.00 100.00 100.00



2.05 2.05 5.71




70




on the corrected mortality, are also shown in Figs. 1-5. From the curves, the values for LD10, LD 0, and LD90 and 10 D0, and
the slope were determined and are given in Table 4.



Table 4.--LD10, LD50, LD90, and slope for each age of female flies estimated directly from the curves on logarithm-probability paper.

Age of LD0 50 LD5 LD90
Fly Slope (Days) (lg) (ug) (pg)

1 66 132 265 4.25 2 84 125 185 7.22 3 62 116 218 4.69 4 55 97 173 5.05 5 49 87 162 5.06


Comparisons


The data in Tables 1-4 show that the difference between males and females in each age are self-explanatory. The comparisons of the dosage-mortality curves between sexes and ages are seen in Figs. 1-5.

There is a significant difference in the mortality rates of male and female flies at dosages higher than








99.9 99.8

99.5

99
/

98 /
/




90

41
-d 80 /
I-I

70 /
0 o /
60

> 50 /
-P
40 -/

S30 /
4
o 20 /

0 / Male
-' 10
I Female
5 /


2 /

1 /
0.5
/
0.2 /
0 .1 . I I I I
30 40 80 120 160 200 300 Dose in micrograms per house fly (log. scale) Fig. l.--Lethal-Dosage Curves for One-Day-Old Male and
Female Flies within 48 Hours.








99.9
99.8

99.5 1

'99

98


95
~//


90 /
u

>1 /
S 80 / A/
70
60
60 /

50 .




40
30 30

20
o /
0I

S10 4'

U
U ~5/


2 ./ - Male
i 8_HoursFemale
1 /

0.5 I

0.2
0.1 ' I
30 40 80 120 160 200 300
Dose in micrograms per house fly (log. scale) Fig. 2.--Lethal-Dosage Curves for Two-Day-Old Male and
Female Flies within 48 Hours.








99.9 99.8

99.5

99

98
/
9S /

95
H/ m/
90
U/
>1 /
80. /

o/
70
o /
60./ 50 /
*4 /
40

30 30
0
20 /
0/

p 110/ "/Male
10 /
/Female
4 5


2

1 0.5

0.2
0 .i 1 1 I I I I
30 40 80 120 160 200 300
Dose in micrograms per house fly (log. scale)

Fig. 3.--Lethal-Dosage Curves for Three-Day-Old Male and
Female Flies within 48 Hours.








99.9
99.8

99.5 /
99

98


95
~//

90
~//

80
.r4 80/

70
o /
10 60

50 -/
40

4 30 /
0
20 .
o /
0

10 // Male
,, Female
U
5 /


2

1 /
0.5

0.2
0 . 1 1 , , I I I I 30 40 80 120 160 200 300 Dose in micrograms per house fly (log. scale) Fig. 4.--Lethal-Dosage Curves for Four-Day-'Old Male and
Female Flies within 48 Hours.








99.9 99.8 99.5

99 98


2/
1 ./

0.5
0.2
0.1
30 40 80 120 160 200 300 Dose in micrograms per house fly (log. scale) Fig. 5.--Lethal-Dosage Curves for Five-Day-Old Male and
Female Flies within 48 Hours.


/
/
/ / I
/
/
,//

' I /
//
/"

/,/ /









80 pg/fly, the mortality rate for males being higher at the same age and dosage. With concentrations lower than 80 pg/fly, the mortality rates were similar (see Tables 1 and 3).

On the basis of 48 hours' observation of both male and female flies, there was an apparent correlation between age and mortality rate. Within the age range of two to five days, the higher the dosage, the higher the rate of mortality. Within 48 hours, 100 percent mortality was achieved in both sexes at the rate of 200 pg/fly. It is possible that a shorter period of observation might reveal slight variations in these percentages, but it is believed that the results obtained would reflect the same tendency.


Determination of Sterility Dosage


Chemosterilants act on insects in three principal ways, as set forth in the review of the literature. The most interesting of these is inducing dominant lethal mutations or severely injuring the genetic material in the sperm an.d ova (Smith et al., 1964). Different developmental stages of the male gamete, such as spermatogonia, spermatocytes, spermatids, and spermatozoa, show a characteristic differential susceptibility to the effects of chemosterilants.









Wheeler (1962) stated that alkylating agents (e.g., metepa) can directly or indirectly affect the alkylation of nucleoproteins. Deoxyribonucleic acid (DNA) is most likely

the primary site of alkylation and therefore the most sensitive and the most subject to alteration.

Kilgore (1965), working with house flies, pointed

out that alkylating agents "have a very pronounced effect on the metabolism of the nucleic acids."

Keiser et al. (1965) reported that in chemosterilized male fruit flies both the spermatogonia and the spermatocytes are destroyed, but the spermatids which are beyond the last division continue to develop and mature.

Fahmy and Fahmy (1964) reported that untreated

Drosophila females mated to males treated with varying dosages of the chemosterilant tretamine (TEM) had a very high number of unhatched eggs. The chemosterilant might cause a mutation which acts as a gametic lethal, or as an early zygotic lethal, for the house fly. LaBrecque (Borkovec, 1966) reported that, where males chemosterilized with less than the minimum 100 percent sterility dosage were mated with normal females, the percentage of hatch of eggs obtained from second egging was often higher than the hatch of eggs laid shortly after mating. Linkfield (1966) reported that the









dominant lethal mutations seem to affect not only the embryo in fleas but the larva as well.

Most of the research on the physiological effects

of chemosterilants has been done on female insects, because the ovaries are usually partially developed in newly emerged adults, with the result that the interference of chemosterilants with their maturation and with oogenesis can be observed. The most often observed histopathological effect of chemosterilants on female house flies is the retardation or complete cessation of ovarian development (Morgan & LaBrecque, 1962, 1964; Tung, 1965). However, interference with ovarian growth and function is not a necessary condition for the induction of sterility, because certain compounds can sterilize fully developed eggs which have already been fertilized by an untreated male (Weidhaas, 1962). An effective female sterilant may have one of the following consequences: (a) no eggs are produced, or (b) eggs are laid but the zygote does not develop into a mature offspring. Both effects are frequently accompanied by observable morphological changes in the ovaries.

A series of preliminary tests was conducted to determine sterility dosage. The results of the preliminary tests showed that there was a significant difference between









the sexes, so the experiments were conducted separately according to sexes.


Sterility Dosage of Male House Flies


Among male house flies, regardless of age, 100 percent sterility was achieved through injection with a dose of

8 jg/fly -of metepa-water solution. Three different treatments were conducted to determine the sterility dosage in this experiment.


Individual Pair-Rearing and First Egging

The test of male sterility using the individual pairrearing method involved six replicates, from which all eggs were collected and counted. The results may be seen in Table 5. Dosage-response data were plotted on logarithmprobability paper, verified by a Chi-square test (Fig. 6).


Collective Rearing and First Eqging

Table 6 shows the results of a male sterility test employing the collective rearing : ithod, with five to ten pairs of flies tested in two complete replicates, and a random sample of 100 eggs from the first egging. Dosageresponse data are plotted in Fig. 7.







Table 5.--Effects of metepa on sterility of males of different ages: individual pair rearing method, six complete replicates (percentage of sterility based on number of progeny reaching pupal stage from all eggs of first egging in each replicate).


% of Sterility


1-day-old


2-day-old


3-day-old


4-day-old


5-day-old


13.11
(2.29)* 21.64 (11.64)
37.02 (28.99) 79.21 (76.56) 89.13 (87.74) 100.00
(100.00) 11.31


Control


12.30 (0.17)
22.37 (11.63)
38.97 (30.53) 81.91
(79.41) 94.81 (94.09) 100.00
(100.00) 12.15


12.18 (0.00) 23.67 (13.00) 49.01 (41.88) 82.93 (80.54) 97.76 (97.44) 100.00
(100.00)
12.26


15.72 (0.38)
26.16 (12.72) 57.60 (49.88) 82.91 (79.79) 97.90 (97.51) 100.00
(100.00) 15.40


15.65 (1.29) 26.02 (13.43) 56.26 (48.81) 80.00
(76.59) 99.68
(99.62) 100.00
(100.00) 14.54


show the corrected sterility by Abbott's formula.


Dosage
pg/Male


0.25


0.5 1.0


2.0 4.0


8.0


*Values in parentheses





81


99.9
+3
99.8

99.5

99

98 +2

95

90
o 0 w +1
>1
4 80

S70

Ao 60P0
50 >1 40
40
.r- 30

4J 20
tw-- -1
S10
W * 1-day-old a 2-day-old 5- 3-day-old
UA 4-day-old
2
2 * * 5-day-old

1 *
0.5

0.2
0.1 ' -3
0 . s I I I I 1 -
0.25 0.5 1 2 4 8 Dose in micrograms per male house fly (log. scale)

Fig. 6.--Dosage-Sterility Curves for Different Ages of Male
House Flies Treated by Microinjection, Pair
Rearing Method, from All Eggs of First Egging.








Table 6.--Effects of metepa on sterility of males of different ages: collective rearing method, 5-10 pairs of flies, two complete replicates (percentage of sterility based on number of progeny reaching pupal stage from a sample of 100 eggs of first egging in each replicate).


% of Sterility


I- y-old
11.00
(0.63)
32.00
(24.07) 3.J .12 (32.02;
63.20 (58.91) 96.80 (96.43) 100.00 (100.00)


2--day-old'
13.00 (4.65) 28.00 (21.09)
52.31 (47.74) 68.40 (65.37) 96.00 (95.61)
100.00
(100.00)


8.75


3-day-old
16.00 (8.45) 39.00 (33.51) 56.25 (52.32) 74.67 (72.39) 98.22 (98.06) 100.00
(100.00)


8.25


4-day-old

13.00 (0.00) 41.00 (27.43) 51.25
(40.07) 75.75 (70.17) 98.40 (98.03) 100.00
(100.00)


18.70


5-day-old
17.00 (0.00) 41.00 (27.37)


60.00 (50.71)
79.30 (74.52) 98.71
(98.41) 100.00
(100.00)


18.77


show the corrected sterility by Abbott's formula.


Dosage
pg/Male
0.25


0.5


1.0 2.0


4.0 8.0


Control


10.44


*Values in parentheses









99.9 +3
99.8 99.5

99

98 +2


95

90
U
w +1 >1 80
.p

S 70 0

o 6050 0
x


21 0 - A 440
30 �
.-

-P 20-1
%4
o 10 - * 1-day-old Sx 2-day-old
* 3-day-old
- 54-day-old a~* 5-day-old a 2 -2

1
0.5 r

0.2
0.1 A l -3
0.25 0.5 1 2 4 8
Dose in micrograms per male house fly (log. scale)

Fig. 7.--Dosage-Sterility Curves for Different Ages of Male
House Flies Treated by Microinjection, Collective Rearing Method, Using a Sample of 100 Eggs
of First Egging.









Collective Rearing and Average of Four Successive Eqqings

The male flies tested here are the same males as in the previous test. While the previous test used only the first egging to find sterility, in this instance results are based on the average of four successive eggings in each replicate (see Table 7). Again, dosage-response data were plotted on logarithm-probability paper (Fig. 8).

Based on Figs. 6-8, the relationship between dosage and male sterility in the three different treatments is summarized in Table 8.

The data developed under the first and second treatments were very similar. Obviously, because of the multiplicity of factors involved in the third treatment (Collective rearing, four successive eggings, a sample of 100 eggs for each egging), the rate of increase and decrease of the percentage of male sterility is highly changeable.

The dosages in the collective rearing method seem

low when compared with the data from the pair-rearing test. Though the pair-rearing treatment (first egging) appears more stable than the collective rearing (first egging), both methods can be used to determine male sterility dosage.

Different dosages produce different percentages of








Table 7.--Effects of motepa on sterility of males of different ages: collective rearing method, 5-10 pairs of flies, two complete replicates (percentage of sterility based on number of progeny reaching pupal stage from a sample of 100


eggs of four


consecutive eggings in each replicate).


/o% of Sterility


1-day-old


14.48 (2.57)* 29.33 (19.49) 32.39
(22.98)
70.00
(65.82)
97.23 (96.84) 99.67 (99.62) 12.22


2-day-old


24.76 (17.26)
33.86 (27.26) .48.70 (43.58) 74.54 (72.00) 9f.05 (97.86) 100.00 (100.00)
9.07


3-day-old


26.84 (15.87) 38.14
(28.29) 54.32 (46.09) 70.24
(63.59) 98.23 (94.38) 100.00 (100.00)
12.41


4-day-old

14.50
(0.00) 42.25 (29.31)
45.63 (33.44) 76.07 (70.71) 98.46 (98.11) 100.00 (100.00)
18.31


5-day-old

25.50 (9.10) 64.75 (56.99) 57.26 (47.85) 80.80
(76.57) 99.46 (99.34) 100.00
(100.00)
18.04


show the corrected sterility by Abbott's formula.


Dosage
pg/Male


0.25


0.5


1.0 2.0 4.0 8.0


Control


*Values in parentheses








99.9
L +3
99.8

99.5
99 / /"
. * .*+
98 / / / +2 .01
95
7-/ //
90 /.... .
90

80 .--- ~ *7+1
3 - / . 70 / .
(tie
mo -601 50 0

>1 400 40 * *...
4-)
30 - // ...1
20
44 ./ //
0 10 ' / ."...................... - a - l
10 . / 1-day-old 2-day-old
5 / -.-3-day-old
.*/ ..-4..... -day-old
S------- ----- 5-day-old -2
2 -2

1

0.5

0.2 -3
0.1 r I 1
0.25 0.5 1 2 4 8 Dose in micrograms per male house fly (log. scale)

Fig. 8.--Dosage-Sterility Curves for Different Ages of Male
Eouse Flies, Collective Rearing Method, Using a Sample of 100 Eggs of Four Consecutive Eggings
in Each Replicate.




Full Text

PAGE 1

EFFECT OF THE CHEMOSTERILANT METEPA ON THE HOUSEFLY, Musca domestica L. By KU-SHENG KUNG 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 August, 1967

PAGE 2

ACKNOWLEDGMENTS I would like to express my sincere gratitude to the chairman of my supervisory committee, Dr. J. T. Creighton, Department of Entomology, University of Florida, and cochairman, Dr. G. C. LaBrecgue, United States Department of Agriculture, for their sound advice and generous assistance. The valuable suggestions and encouragement from Dr. D. A. Roberts and Dr. B. J. Smittle of the supervisory committee and Dr. W. G. Eden, chairman of the Department of Entomology, are also greatly appreciated. Special thanks are due to Dr. C. N. Smith for the use of facilities at the U.S.D.A. Entomology Research Division Laboratory and to all the U.S.D.A. staff members for their assistance. Last, but not least, I wish to extend my appreciation to my wife, Hung-yin, for her understanding and encouragement, which made this study possible. ii

PAGE 3

TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . ii LIST OF TABLES vi LIST OF FIGURES x ii INTRODUCTION 1 REVIEW OF LITERATURE 5 Chemosterilants 5 General Conception 5 Definition 5 Principal Action 5 Mode of Action of Two Main Groups of Chemosterilants 6 Important Chemosterilants 8 Searching for New Chemosterilants 8 Field Experiments in the Controlof House Flies by Chemosterilant Techniques 9 Structure, Physical and Chemical Properties of Metepa 10 Specificity of Chemosterilants 13 Biology 19 Name and Classification 19 * Distribution 20 The Adult 21 Emergence 21 Feeding 22 Flight , 23 Mating Behavior 25 Oviposition 28 Longevity m 30 Sex Ratio 32 The Egg [ [ 34 The Larva 35 iii

PAGE 4

Pago Breeding Places in Nature 35 Rearing Medium in Laboratory 36 Growth Rate and Instars 37 The Pupa 38 Hibernation 39 Temperature, Humidity, and Light 40 Temperature 40 Humidity 43 Light 44 MATERIALS AND METHODS 46 J Technigue of Rearing Stock House Flies 46 Procedures for Testing, Rearing, and Observation. 48 Rearing and Observation Cages 48 Sexing Virgin Females and Males 49 Keeping Same-Age Flies and DifferentAge Flies 50 Water and Fly-Food Supply 50 Solution and Injection 51 Mating 52 Egging 53 Egg Counting 53 Hatching Counts 55 Pupation Counts 55 Criteria of Sterilization — Determining Sterility 57 Determination of Lethal Dosage 57 Determination of Sterility Dosage 58 Determination of the Permanence of Sterility. . . 60 Mating Competitiveness Test 61 Rate of Chemosterilization of Sperm in Vivo ... 61 Derivation of Survivorship Curves 62 RESULTS AND DISCUSSION 63 Determination of Lethal Dosage 63 Lethal Dosage of Male Flies 63 Lethal Dosage of Female Flies 66 Comparisons 70 Determination of Sterility Dosage 76 iv

PAGE 5

Page Sterility Dosage of Male House Flies 79 Individual Pair-Rearing and First Egging. 79 Collective Rearing and First Egging ... 79 Collective Rearing and Average of Four Successive Eggings 84 Sterility Dosage of Female House Flies. ... 88 Female Sterilization up to Five Days Old. 88 Sterilization of Females Older than Five Days 102 Determination of the Permanence of Sterility. . . 107 Mating Competitiveness Test 126 Rate of Chemosterilization of Sperm in Vivo . . . 135 Effects of Chemosterilant Metepa on Longevity . . 141 Longevity of House Flies in Each Treatment. . 142 Survivorship Curves in Each Treatment .... 142 Male House Flies. 151 Female House Flies 159 SUMMARY 172 REFERENCES 175 BIOGRAPHICAL SKETCH 188 v

PAGE 6

LIST OF TABLES Table Page 1. Effects of Chemosterilant Metepa in Different Dosages on Male House Flies of Different Ages (Percentage of Mortality Based on 48-Hour Observation Period) 64 2. LD 10 # LD5O' ^90 1 and Slope for Each Age of Male Flies Estimated Directly from the Curves on Logarithm-Probability Paper. ... 67 3. Effects of Chemosterilant Metepa in Different Dosages on Female House Flies of Different Ages (Percentage of Mortality Based on 48Hour Observation Period) 68 4. LD i 0 ' LD 5Q' ^90' and sl °P e for Eac h Age of Female Flies Estimated Directly from the Curves on LogarithmProbability Paper. ... 70 5. Effects of Metepa on Sterility of Males of Different Ages: Individual Pair Rearing Method, Six Complete Replicates (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from All Eggs of First Egging in Each Replicate) 80 > 6. Effects of Metepa on Sterility of Males of Different Ages: Collective Rearing Method, 5-10 Pairs of Flies, Two Complete Replicates (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from a Sample of 100 Eggs of First Egging in Each Replicate) 82 7. Effects of Metepa on Sterility of Males of Different Ages: Collective Rearing Method, vi

PAGE 7

Table Page 5-10 Pairs of Flies, Two Complete Replicates (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from a Sample of 100 Eggs of Four Consecutive Eggings in Each Replicate) 85 8. Comparison of Three Different Treatments to Determine the Relationship between Dosage and Male Sterility 87 9. Effects of Metepa on Sterility of Females of Different Ages: Collective-Rearing Method, 5 Pairs of Flies (10 Pairs at the 64 ug/ Female Dosage), Two Complete Replicates (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from a Sample of 100 Eggs of First Egging in Each Replicate) 89 10. Effects of Metepa on Sterility of Females of Different Ages: Collective-Rearing Method, 5 Pairs of Flies (10 Pairs at the 64 ug/ Female Dosage) , Two Complete Replicates (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from a Sample of 100 Eggs of Four Consecutive Eggings in Each Replicate) 90 11. Comparison of the Relationship between Dosage and Female Sterility in the First Egging and the Average of Four Consecutive Egg ing s 93 12. Estimated Minimum Dosage for 100 Percent Sterility in Female Flies of Different Ages (Based on Collective Rearing and Percentage of Pupation from a Sample of 100 Eggs per Egging) 94 13. Effects of Metepa on Sterility of One-DayOld Female Flies (Based on the Hatch of Eggs) 96 vii

PAGE 8

Table Page 14. Effects of Metepa on Sterility of Two-DayOld Female Flies (Based on the Hatch of Eggs) 97 15. Effects of Metepa on Sterility of ThreeDay-Old Female Flies (Based on the Hatch of Eggs) 98 16. Effects of Metepa on Sterility of FourDay-Old Female Flies (Based on the Hatch of Eggs) 99 17. Effects of Metepa on Sterility of FiveDay-Old Female Flies (Based on the Hatch of Eggs) 100 18. Effects of Metepa on Sterility of Female Flies Older than Five Days (Based on the Percentage of Hatch of Eggs) 103 19. Egging of 50-Day-Old Females Mated with 3-Day-Old Males (Mated on February 11, 1967) 20. Effects of Metepa on Permanence of Sterility of One-Day-Old Males Treated by Means of Microinjection in the Pair Rearing, Mating Group Series Test (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from All Eggs in Each Egging after First Mating) 21. Effects of Metepa on Permanence of Sterility of Two-Day-Old Males Treated by Means of Microinjection in the Pair Rearing, Mating Group Series Test (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from All Eggs in Each Egging after First Mating) 109 110 viii

PAGE 9

Table Page 22. Effects of Metepa on Permanence of Sterility of Three-Day-Old Males Treated by Means of Microinjection in the Pair Rearing, Mating Group Series Test (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from All Eggs in Each Egging after First Mating) Ill 23. Effects of Metepa on Permanence of Sterility of Four-Day-Old Males Treated by Means of Microinjection in the Pair Rearing, Mating Group Series Test (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from All Eggs in Each Egging after First Mating) 112 24. Effects of Metepa on Permanence of Sterility of Five-Day-Old Males Treated by Means of Microinjection in the Pair Rearing, Mating Group Series Test (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from All Eggs in Each Egging after First Mating) 113 25. Effects of Metepa on Permanence of Sterility of One-Day-Old Males Treated by Means of Microinjection in the Pair Rearing, Consecutive Mating Series Test (Percentage of ' Sterility Based on Number of Progeny Reaching Pupal Stage from All Eggs in Each Egging after Each Mating) 114 26. Effects of Metepa on Permanence of Sterility of Two-Day-Old Males Treated by Means of Microinjection in the Pair Rearing, Consecutive Mating Series Test (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from All Eggs in Each Egging after Each Mating) 115

PAGE 10

Table Page 27. Effects of Metepa on Permanence of Sterility of Three-Day-Old Males Treated by Means of Microinjection in the Pair Rearing, Consecutive Mating Series Test (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from All Eggs in Each Egging after Each Mating) 116 28. Effects of Metepa on Permanence of Sterility of Four -Day-Old Males Treated by Means of Microinjection in the Pair Rearing, Consecutive Mating Series Test (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from All Eggs in Each Egging after Each Mating) 117 29. Effects of Metepa on Permanence of Sterility of Five-Day-Old Males Treated by Means of Microinjection in the Pair Rearing, Consecutive Mating Series Test (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from All Eggs in Each Egging after Each Mating) 118 30. Effects of Metepa on Permanence of Sterility of Males Treated by Means of Microinjection in the Collective Rearing Method (Percentage of Sterility Based on Number of Progeny Reaching Pupal Stage from a Sample of 100 Eggs in Each Egging after Each Consecutive Mating) 119 31. Mating Competitiveness of One-Day-Old Treated Male Flies Based on the Time Units 129 32. Mating Competitiveness of Two-Day-Old Treated Male Flies Based on the Time Units 130 33. Mating Competitiveness of Three-Day-Old Treated Male Flies Based on the Time Units. . . 131 x

PAGE 11

Table Page 34. Mating Competitiveness of Four-Day-Old Treated Male Flies Based on the Time Units 132 35. Mating Competitiveness of Five-Day-Old Treated Male Flies Based on the Time Units 133 36. Rate of Metepa Chemosterilization of Sperm in Vivo (Three-Day-Old Males Treated at the Dosage of 8 yg/Male Fly, Crossed with Three-Day-Old Females Immediately after Injection) ; Male Flies Were Injected August 8, 1966 136 37. Longevity of Treated Male House Flies without Female House Flies 143 38. Longevity of Normal Male House Flies without Female House Flies 144 39. Longevity of Normal Male House Flies with Normal Female House Flies 145 40. Longevity of Normal Male House Flies with Treated Female House Flies. 146 41. Longevity of Treated Male House Flies with Normal Female House Flies 148 42. Longevity of Treated Female House Flies i, without Male House Flies 149 43. Longevity of Normal Female House Flies without Male House Flies 150 xi

PAGE 12

LIST OF FIGURES Figure Page 1. Lethal-Dosage Curves for One-Day-Old Male and Female Flies within 48 Hours 71 2. Lethal-Dosage Curves for Two-Day-Old Male and Female Flies within 48 Hours 72 3. Lethal-Dosage Curves for Three-Day-Old Male and Female Flies within 48 Hours. . . 73 4. Lethal-Dosage Curves for Four -Day-Old Male and Female Flies within 48 Hours 74 5. Lethal-Dosage Curves for Five-Day-Old Male and Female Flies within 48 Hours 75 6. Dosage-Sterility Curves for Different Ages of Male House Flies Treated by Microinjection, Pair Rearing Method, from All Eggs of First Egging 81 7. Dosage-Sterility Curves for Different Ages of Male House Flies Treated by Microinjection, Collective Rearing Method, Using a Sample of 100 Eggs of First Egging 83 8. Dosage-Sterility Curves for Different Ages of Male House Flies, Collective Rearing Method, Using a Sample of 100 Eggs of Four Consecutive Eggings in Each Replicate 86 9. Dosage-Sterility Curves for Different Ages of Female House Flies, Collective Rearing Method, Using a Sample of 100 Eggs at First Egging 91 xii

PAGE 13

Figure Page 10. Dosage-Sterility Curves for Different Ages of Female House Flies, Collective Rearing Method, Using a Sample of 100 Eggs of Four Consecutive Eggings in Each Replicate 92 11. Relationship between Permanence of Sterility and the Mating Time after Injection of Male House Flies, Mating Group Series Test (First Egging, All Eggs Counted; Sterility Based on Pupation) 121 12. Relationship between Permanence of Sterility and the Mating Time after Injection of Male House Flies, Consecutive Mating Series Test (First Egging, All Eggs Counted; Sterility Based on Pupation) 122 13. Permanence of Sterility of Male House Flies Treated at Different Ages, Collective Rearing Method, 5-10 Pairs of Flies (Progeny Reaching Pupal Stage from a Sample of 100 Eggs per Egging after Each Consecutive Mating) ' 123 14. Survivorship Curves of Treated Male House Flies without Female House Flies 152 15. Survivorship Curves of Normal Male House Flies without Female House Flies 153 16. Survivorship Curves of Normal Male House Flies with Normal Female House Flies ... 155 17. Survivorship Curves of Normal Male House Flies with Females Treated at Different Dosages according to Age: 70 yg/l-dayold; 50 ug/2-day-old; 45 Mg/3-day-old; 30 Mg/4-day-old; and 20 Mg/5-day-old ... 157 xiii

PAGE 14

Figure 18. Survivorship Curves of Treated Male House Flies with Normal Female House Flies . . . 19. Survivorship Curves of Treated Female House Flies without Male House Flies, at Different Dosages according to Age: 70 yg/l-day-old; 50 ug/2-day-old; 45 fig/ 3-day-old; 30 ug/4-day-old; and 20 ug/ 5-day-old 20. Survivorship Curves of Normal Female House Flies without Male House Flies 21. Survivorship Curves of Normal Female House Flies with Normal Male House Flies . . . . 22. Survivorship Curves of Normal Female House Flies with Male House Flies Treated at the Dosage of 8 yg/Male Fly (Males and Females of the Same Age Were Mated) . . . . 23. Survivorship Curves of Treated Female House Flies with Normal Male House Flies (Females Were Treated at Different Dosages according to Age: 70 ug/l-dayold; 50 ug/2-day-old; 45 ug/3-day-old; 30 Mg/4-day-old; and 20 yg/5-day-old') . . . xiv

PAGE 15

INTRODUCTION The idea of using sterile male insects in a natural population was considered by E. F. Knipling as early as 1938 for possible control of the screw-worm fly, Cochliomvia hominivorax (Coquerel) (Lindquist, 1963) . Knipling (1955, 1959, 1962, 1964, and 1966) has calculated and compared theoretical population declines of insect and other animal species subjected to a treatment which causes sterility as opposed to one that produces only direct kill, such as an insecticide . Research on materials and methods of producing sterility in insects has progressed substantially during recent years, and the probability is good that it will be possible to produce reasonably competitive sterile insects of many species for release. Important new developments are being made in the insect attractant field. Effective methods of attracting insects by chemical or physical means might offer the possibility of integrating the attractant and sterility principles without the necessity of rearing and releasing insects. The trapped insects might be

PAGE 16

sterilized and released, thus increasing the effectiveness of the trapping procedures (Knipling, 1964? Knipling & McGuire, 1966) . Two methods of achieving sterility have been investigated extensively: the use of X-rays or gamma rays from cobalt 60, as first investigated on the screw-worm by Bushland and Hopkins (1951, 1953) , and the use of chemicals for sterilization, as reported by LaBrecgue et al. (1960) and Smith et al. (1964). The da:a from preliminary field tests on the control of house flies are encouraging (LaBrecgue et al., 1963a; Gouck et al., 1963b) . In recent years, the use of chemical agents as sexual sterilants for insects has attracted increasing interest and attention from entomologists. Three biological alkylating agents found to be effective insect chemosterilants were all aziridine derivatives (Borkovec, 1966) . Tepa (aphoxide) and apholate were shown to be effective for house flies, Musca domes tic a Linnaeus (LaBrecgue, 1961). Similarly, the methyl derivative of tepa, metepa, was reported by LaBrecgue et al. (1963b) to sterilize house flies. Alkylating agents have also been used to sterilize two species of mosquitoes (Weidhass, 1962), the screw-worm fly

PAGE 17

3 (Chamberlain, 1962) , the German cockroach (Burden & Smittle, 1963), and other insects. Various ways have been developed for evaluating . chemosterilants in the laboratory, such as feeding tech' nique (Gouck & LaBrecque, 1963, 1964), dipping technique (Piquett & Keller, 1962; Gouck, 1964; Chang & Borkovec, 1966a), injection technique (Chang & Borkovec, 1964), residual application (Meifert et _al . , 1963), and topical application (Gouck & LaBrecque, 1964; Chang et_al., 1964). Each method has its own special advantages and disadvantages. A particular method is suitable for given types of behavior during certain developmental stages of a particular insect species, or for a special experiment design according to the purpose of the experiment. This experiment was conducted using microinjection of a metepa water solution into the house fly. The purpose of this investigation was to seek solutions to the following problems: A. To determine the lethal dosage (LD) in different sexes and different ages of house flies. B. To determine the sterility dosage (SD) in different sexes and different ages of house flies. C. To determine the permanence of sterility induced

PAGE 18

in both males and females when subjected to dosages calcu lated to produce 100 percent sterility. D. To derive survivorship curves for both males and females, individually and collectively, comparing treated house flies with normal ones. E. To correlate toxicity with sterility and to consider other interrelationships.

PAGE 19

REVIEW OF LITERATURE Chemosterilants General Conception Definition The term "chemosterilants" was first used in 1960 by LaBrecgue. Chemosterilants may be defined as chemical compounds which reduce or entirely eliminate the reproductive capacity of an animal to which they are administered. • Principal Action Chemosterilants may affect one or both sexes of a sexually reproducing species. Their action may be immediate or delayed; their effect may be temporary or permanent. Chemosterilants act on insects in three principal ways: (1) by causing failure to produce ova or sperm, (2) by causing the death of sperm or ova after they have been produced; and (3) by inducing dominant lethal mutations or severely injuring the genetic material in the sperm and ova (Smith et al. , 1964) .

PAGE 20

6 Mode of Action of Two Main Groups of Chemosterilants The two groups of chemical compounds that have shown the greatest promise as insect chemosterilants are the antimetabolites and the alkylating agents (LaBrecgue, 1963, 1965; Smith et al. , 1964). Antimetabolites are a group of compounds that inhibit the use of any of the products of metabolism by the treated organism. These chemicals interfere with the synthesis of nucleic acids (Borkovec, 1966) . They are structural analogs of purines, pyrimidines, and folic acid (Ross, 1962; Jukes & Broguist, 1963) . Most antimetabolic chemosterilants affect only the females of the species, particularly when administered to adult insects (Mitlin et al . , 1957; LaBrecgue et al., 1960; Crystal, 1963; Kilgore & Painter, 1962, 1964; Kilgore, 1965) . Antimetabolites administered at a time when nucleic acid synthesis is occurring in many different tissues usually produce general symptoms rather than specific effects on the developing gonads (LaBrecgue et al. , 1960) . The mode of action of antimetabolites in purine, pyrimidine, and folic acid analogs has been extensively investigated, and, in many instances, the precise steps in the metabolic process which they inhibit

PAGE 21

are known (Montgomery, 1959; Timmis, 1962; Jukes & Broquist, 1963; Borkovec, 1966) . Antimetabolites are considered to have less potential value as chemosterilants than alkylating agents. Alkylating agents replace hydrogen in an organic molecule with an alkyl group (Ross, 1962) . In biochemistry, alkylation implies the introduction of a hydrocarbon radical, often containing elements other than carbon and hydrogen, into a molecule under physiological conditions. Compounds capable of producing such reactions are referred to as alkylating agents (Borkovec, 1966) . The principal classes of biological alkylating agents were reviewed in a monograph by Ross (1962) . Hundreds of articles dealing with the mode of action of alkylating agents have been discussed by Timmis (1962) , Wheeler (1962) , and Kilgore (1965) . The sterilizing activity of aziridines and other alkylating agents involves a similarity of action on a molecular level (Smith et _al., 1964). When an alkylating agent replaces hydrogen in an organic molecule with an alkyl group within fundamental genetic material, the effect is similar to that produced by irradiation (Alexander, 1960).

PAGE 22

8 Important Chemosterilants The largest and most important group of biological alkylating agents is the derivatives of aziridine (Borkovec, 1966), of which tepa [tris (1-aziridinyl) phosphine oxide, APO, aphoxide], tretamine [2, 4, 6-tris (1-aziridinyl) -striazine, triethylenemelamine, TEM] , apholate [2, 2, 4, 4, 6, 6-hexahydro-, 2, 2, 4, 4, 6, 6-hexakis (1-aziridinyl) -1, 3, 5, 2, 4, 6-triazatriphosphorine] , aphamide [N, N-ethylene bis [P, P-bis (1-aziridinyl) N-methyl phosphinic amide] , aphomide], and metepa [tris (2-methyl-l-aziridinyl) phosphine oxide, methaphoxide , MAPO] are the five best known, most highly active, and widely tested chemosterilants. Aphamide is primarily of historical importance, because its activity appears to be comparatively low in relation to the others. Searching for New Chemosterilants Mitlin et al. (1957) induced sterilization in house flies through the use of mitotic poisons. Three of the four chemicals tested usually inhibited oviposition and prevented ovarian growth. This work marked the beginning of largescale research in chemosterilants. As early as 1958, LaBrecque began screening chemical

PAGE 23

9 compounds for their chemoster ilizing effects. Of 2000 compounds initially tested, five caused sterility in the house fl y» Musca domestica Linnaeus, by feeding treatments (Linkfield, 1966). In 1960, LaBrecque et al. tested 200 chemicals, 79 of which had some deleterious effect when added to the larval medium, but only ten of which affected development when combined with the adults' food. In 1962, LaBrecoue and Gouck tested 1100 compounds, twenty of which caused sterility in the adult house fly when administered in the food. In 1963, LaBrecque tested 2000 chemicals, 40 of them causing sterility (Linkfield, 1966) . In 1965, LaBrecque reported that 112 chemicals had been found to produce sterilant effects, and this number continues to grow (Kohls et al .. 1966) . Field Experiments in the Control of House Flies by Chemoster ilant Techniaues Several field tests have been conducted to control the house fly with the chemoster ilants tepa, metepa, and apholate . The first field test using tepa against the house fly was conducted in 1961 by LaBrecque et al. in a refuse dump at Bahia Honda Key, Florida; the adult fly populations

PAGE 24

10 were reduced from 47 to 0 per grid count within four weeks with use of cornmeal bait containing 0.5 percent tepa. Female flies trapped at the dump were checked for egg masses and viability. The viability of egg masses had decreased from 100 to 10 percent (LaBrecgue et al., 1962a). Metepa at 0.5 percent was applied to droppings in a poultry house in suburbs of Orlando for control of the house fly, with similar results (LaBrecgue et al. # 1963a). Gouck et _al. (1963b) conducted a test in a refuse dump at Pine Island, Florida, using cornmeal bait containing 0.75 percent apholate. A reduction of flies from 68 per grid count to 5-20 occurred during the first seven weeks. When bait was made available continuously, the population decreased to 3 to 0 per grid count. Structure, Physical and Chemical Properties of Metepa Metepa, though usually much less effective than tepa (Murvosh et al., 1964a ; Chang & Borkovec, 1964), still merits consideration because it is less toxic (Hayes, 1964), less hazardous to handle, and more stable than tepa (Beroza & Borkovec, 1964) . Metepa is a highly reactive tri-functional derivative

PAGE 25

11 of phosphorous oxychloride and propylene imine. its structural formula is as follows (Anonymous, 1962) : CH 3 CH 0 >CH CH. N P N / N \ CH 0 CH 2 I CH 3 The reactive functions of metepa are the three membered imine rings which open at the carbon-nitrogen bond to yield a wide variety of additional products. The typical physical properties of metepa are as follows: molecular weight 215; straw-colored liquid with a boiling point between 118° and 125°C. at 1 mm. ;Hg; specific gravity at 25°c. is 1.079; refractive index, n 25 D, is 1.4798; completely soluble in water and common organic solvents (Anonymous, 1962). The common form of the chemical is a liquid which contains a minimum of 92 percent metepa based on reactive imine assay and no more than 0.5 percent volatile material. Like all aziridine compounds, metepa rapidly loses reactivity in an even mildly acidic solution (Plapp et al . , 1962; Beroza & Borkovec, 1964) .

PAGE 26

12 Metepa has been used successfully to sterilize the stable fly (Harris, 1962), the house fly (LaBrecque et al.. 1962 a ), the screw-worm (Gouck et al., 1963a), the gypsy moth (Collier & Downey, 1965), the pink bollworm (Ouye et al.. 1965a), and many other insects (Smith & LaBrecque, 1967). The metabolic rate of a phosphorous-labeled sample of the chemosterilant metepa was investigated by Plapp et al . (1962) . Adult house flies degraded 50 percent of large dosages of the chemical within two hours. The rates of degradation were similar in a susceptible fly strain and in two organophosphate-resistant strains. The stability of 0.05 molar metepa with respect to hydrolysis under alkaline and acid conditions was also determined. Borkovec et al. (1964) determined that, in partially degraded solutions, the sterilizing activity was proportional to the content of intact tepa or metepa rather than to the total content of the aziridine function. Chamberlain and Barrett (1964) determined that, with topical treatments, the male screw-worm fly ( Cochliomvia hominivorax [Coguerel]) required 5.5 times as much metepa per gram of body weight as the male stable fly, Stomoxvs calcitrans (Linnaeus) , and the female screw-worm fly required 18 times as much as the female stable fly. The values for

PAGE 27

13 feeding treatments of the screw-worm fly and stable fly were 3.9 and 6.2 times, respectively, for male and female. House flies subjected to radioactive metepa residual deposits on glass lost 89 percent of their radioactivity within 24 hours. Because of the high degree of chemical activity of this compound, it is evident that the residual radioactivity in these insects does not represent unmetabolized metepa (Plapp et al., 1962; Dame & Schmidt, 1964a) . Specificity of Chemosterilants The use of chemosterilants to sterilize an insect can produce adverse effects, the magnitude of which will be influenced by a number of factors. In a broad sense, specificity of chemosterilants encompasses a variety of selective activities of the compounds in different organisms, different organs or functions of each organism, different stages of development, and different modes of application (Borkovec, 1966) . There is wide variation in the susceptibility of species, sexes, and developmental stages to both the lethal and the sterilizing effects of chemosterilization. Environmental factors such as temperature, humidity, pH value, food,

PAGE 28

14 etc., can influence the results of the chemosterilants . The dose levels vary according to specificity, method of application, sex, species or strain, developmental stage, competitiveness, longevity, and environmental factors. It is not necessary to cite individual papers in this extensive literature, because they have been reviewed and listed as references in comprehensive publications. Anyone planning to work on insect chemosterilization should read the publications of Smith et al . (1964) , Ascher (1964) , Hayes (1964) , Borkovec (1966) , and Smith and LaBrecque (1967) . Anyone planning work on house fly chemosterilization should read publications of LaBrecque and LaBrecque et al. (1960, 1961, 1962, 1963a, 1963b, 1965, 1966) . The following sections present the results of several studies on chemosterilization of the house fly. The relationship between concentrations of metepa, apholate, and tepa in diet and degree of sterility induced in adult house flies showed that wider variation resulted than would be expected from similar tests with insecticides. The calculated sterility concentrations (SC 5Q and SC 9Q ) of metepa and apholate were similar; tepa sterilized at lower concentrations (Murvosh et al.. 1964b) . LaBrecque et al. (1966) achieved 99 to 100 percent

PAGE 29

15 sterility in the male house fly by feeding hempa (hexamethylphosph or amide) at concentrations as low as 0.25 percent. It was less effective against females. Chang and Chiang (1964) studied the sterility effect of thio-tepa on the house fly, M. domestic a vicina Macguart. The feeding technique proved to be an effective and easy method. The use of 0.5 percent thio-tepa in milk powder (w/w) for two days, or 1 percent for one day, induced complete sterility; eggs were laid but these did not hatch. Contact method and topical application also proved effective; 50 pg per fly was effective in reducing 90 percent of the reproductive potential. Pupae were most resistant. Third instar larvae were more sensitive than those in the first, and many morphological abnormalities appeared in the larval treatment. Two-day-old flies were almost as sensitive as newly emerged ones; however, fourand six-day-old flies were less sensitive. * Sterility was induced in the house fly, M. domestica . by dipping puparia containing pupae of different ages in apholate, tepa, and metepa at concentrations of 2.5 and 5 percent for 30 to 300 seconds. With all dipping periods and with both concentrations, apholate and metepa gave the most consistent sterility in flies emerging from puparia dipped

PAGE 30

16 when pupae were one day old (Gouck, 1964) . The dipping of oneand three-day-old pupae of house flies in solutions of hempa at 50 percent concentration in water for five minutes produced 100 percent sterility in both sexes (LaBrecque et al. , 1966). Male house flies sterilized by feeding a diet containing 1 percent apholate were as successful as normal males in competition for mates. The percentage of sterile eggs laid by females in cages containing normal and chemosterilized males was as high as, or higher than, would be expected from the ratio of sterile males present (LaBrecque et a_l. , 1962a) . Hempa at 1 percent in the f lies ' food did not impair mating competitiveness of males nor the mobility of the sperm (LaBrecque et al. , 1966) . Morgan and LaBrecque (1962, 1964) reported that, in general, apholate, tepa, and metepa inhibit ovarian development in house flies. The chromatin of the nurse cell nuclei was clumped in irregular masses. Tung (1965) treated the house fly, M. vicina, with thio-tepa in different solutions and for various durations. The results indicated that the sterility of female house flies was due to the degeneration of oogonia in the ovaries. The number of oogonia gradually decreased and degeneration

PAGE 31

17 followed; finally, the ovary entirely atrophied. The degree of degeneration of oogonia was found to be proportional to the dosage of thio-tepa and the duration of treatment. 32 P -labeled metepa was rapidly absorbed from a glass surface by both mosquitoes ( Anopheles quadrimaculatus Say and Aedes aegypti [Linnaeus]) and house flies ( Musca dpmestica Linnaeus) . House flies and Anopheles quadrimaculatus absorbed approximately 7 ug per insect during a four-hour exposure on a surface treated at 10 mg/ft 2 , whereas Aedes aegypti picked up 2.5 Hg. This intake resulted in a severe reduction of mating ability in mosquitoes, coupled with 99 percent sterility in house flies and A. aegypti males (Dame & Schmidt, 1964a) . 14 When tepa uniformly labeled with C was injected into the male house fly at the rate of 1 yg/fly, the radioactivity was transferred to female flies by copulation with treated males (Chang et aJL., 1966). The effects on house flies of exposure for various periods of time (four, three, and two hours) to residues (1, 14, 30, and 60 days old) of tepa and metepa (250, 100, 50, 25, and 10 mg/ft ) on glass were studied by Meifert et ^1. (1963) . When newly emerged virgin adult house flies were

PAGE 32

18 exposed for four hours to residues of hempa in glass jars 2 (200 mg/ft ) , only the male flies reached 100 percent steril ity; the females never exceeded 38 percent. At dosages 2 higher than 200 mg/ft , the hempa residue acted as an adhesive, causing high mortality in the flies (LaBrecque et al., 1966). Chang and Borkovec (1964) determined that tepa was four times as effective as apholate and 12.5 times as effective as metepa in sterilizing male house flies by injection. Hempa administered to male house flies by various methods produced the following results: injection with a dose of 40 pg/fly, 100 percent sterility; topical application with a dosage of 200 pg/fly, 100 percent sterility; and oral application of a 1 percent concentration in food, 99.9 percent sterility (Chang et al., 1964). Two series of compounds related to tepa and hempa were tested by Chang and Borkovec (1966) on male house flies (M. domestica Linnaeus) to determine the structure-activity relationship.

PAGE 33

Biology Name and Classification Musca dome stic a is one of the best known and most used scientific names. For centuries the common name of M. dome stic a has been housefly (house fly, house-fly) in English-speaking countries. L. 0. Howard (1911a, 1911b, 1911c) and his contemporaries applied the names "typhoid fly," "cholera fly," "dysentery fly," and "enteric fly," since typhoid fever and cholera were the most serious and widespread fly-borne diseases at that time. According to present-day concepts of structure and taxonomy, the only form that has been generally recognized as biologically distinct is M. dome stic a vicina Macguart, a subspecies tending to have a more tropical distribution than M. dome stic a dome stic a . The classification of M. dome stic a is as follows (West, 1951): Kingdom: Animal i a Phylum: Arthropoda Class: Hexapoda Order: Diptera Suborder: Cyclorrhapha (Athericera) Series: Schizophora

PAGE 34

20 Section: Muscoidea (Myodaria) Subsection : Calypteratae Family: Muscidae Subfamily: Muscinae Genus : Musca Species: M. domes tica Subspecies: M. domestica domes tica Linnaeus M. domestica vicina Macquart The subspecies M. domestica vicina differs from M. d. domestica chiefly in having a more extensively orange abdomen, especially on tergites 1 to 3. The males differ further in having a somewhat narrower vertex, compared with the width of the compound eyes (Peffly & LaBrecque, 1956) . Distribution The geographical distribution of M. domestica is usually considered world-wide. Graham-Smith (1914) . stated: Musca domestica is probably the most widely distributed insect to be found; the animal most commonly associated with man, whom it appears to have followed over the entire earth. It extends from the sub-polar regions to the tropics, where it occurs in enormous numbers.

PAGE 35

21 The Adult Emergence No special conditions are required for the emergence of adults from pupae. When the transformation has been completed, the fly pushes off the anterior end of the pupal case. Once its head is free, the fly crawls out of the puparium, at the same time extricating itself from the nymphal sheath, which remains as a lining to the empty case (West, 1951) . Eversion of the ptilinum is accomplished by changes in blood pressure, and retraction by special muscles that do not persist in aged adults (Laing, 1935) . The fly crawls rapidly about while its wings unfold and the exoskeleton proceeds to harden and darken. Finally, the ptilinum is withdrawn completely, leaving only the crescentic frontal "lunule" above the antennae to mark its previous location (West, 1951) . When the rearing room is maintained at 25°C, the first adults emerge on the tenth day after hatching, the majority on the eleventh, and a few on the twelfth. Emergence is easily delayed by allowing pupal development to proceed at ambient rather than rearing -room temperature or by cool-storing the pupae (Bucher et al., 1948). At least 95 percent of the adults can emerge from the pupae under laboratory conditions

PAGE 36

22 (Spiller, 1966). The author's experiment in the rearing room indicated that male flies come out first and that the size of the adult fly can be influenced by underfeeding in the larval stage. . Feeding The house fly has shared man's food and developed in his wastes and in those of his domestic animals since the world was young. House flies are almost omnivorous and breed in fermenting vegetable and animal matter and in other filth, without which they cannot exist , despite their high reproductive capability (West, 1951; Herms et al . , 1961). All day long their restless nature causes them to fly back and forth between the privy and the kitchen, between a wound that is infected and a fresh incision. When disease organisms are in the waste, the house fly carries them. This is what makes the species so dangerous and important. Many older feeding techniques supplied liquid milk daily. Now this has been replaced by water and dry food (six parts granulated sugar, six parts nonfat dry milk, and one part powdered egg yolk) (LaBrecque et al . , 1960). According to Dame and Fye (1964) , who studied the feeding

PAGE 37

23 behavior of house flies, on the dry baits 50 percent of the flies had fed by the twelfth to the sixteenth hour, while on the liquid bait 50 percent had fed by the fifth hour and 90 percent by the twelfth hour. Acree et al. (1959) showed that the response of house flies to sugar was apparently related to the relative humidity gradient between the bait source and the surrounding environment. Robbins et al . (1965) reported that both casein and yeast hydrolyzate contain feeding stimulants for the adult female house fly. The major active substance in yeast hydrolyzate is guanosine monophosphate, whereas several amino acids (leucine, lysine, isoleusine, and methionine) are the active compounds of casein. Solution in a phosphate buffer appears to be necessary for maximum activity. Flight Bishopp and Laake (1921) carried out an extensive series of flight experiments in Texas. Some 234,000 flies were captured, dusted with finely powdered red chalk or paint, and then liberated in the open fields. Within 24 hours, the house flies were captured an average of six miles distant from the point of release, and the maximum distance traveled was 13.14 miles by one female. Schoof and Siverly

PAGE 38

24 (1954), using radioactive isotopes as markers, indicated that flies can fly as far as 20 miles from their source and that, under certain conditions, they may migrate in considerable numbers from one to four miles; the dispersion, however, is usually limited to a distance of 0.5 to two miles. Quarterman et al. (1954) suggested that in rural areas flies may move at random within an area eight to ten miles in diameter. Murvosh and Thaggard (1966) pointed out that an individual fly from the Gainesville laboratory strain appeared to move in a laboratory room at rates ranging from about 1.5 to six ft/sec, as clocked by a stop watch. West (1951) mentioned that if it were possible to stimulate a fly so that most of its flying time would be spent traveling in the same general direction, the distance traveled would be relatively enormous, perhaps hundreds of miles. Hindle (1914) reported that fine weather and warm temperatures were positive factors in encouraging dispersal, and that flies tend to travel much farther in the country than in town, where, of course, food and shelter are available on every hand. The time of day affected the tendency of flies to engage in dispersal flight.

PAGE 39

25 Mating Behavior Male flies are sexually aggressive and frequently mount females. Murvosh et _al . (1964a) reported that the mating "behavior of the house fly is a complex phenomenon involving several factors: (1) Both male and female must undergo a sexual maturation time. The results of this study indicated that males and females will not mate for at least 16 and 24 hours respectively, after emergence. Anesthesia prior to mating may have retarded the process somewhat, but this factor was not measured. Michelsen (1960) , who did not anesthetize the flies prior to mating, reported that at 28°C . males reached sexual maturity in 18 to 27 hours after emergence. Chang (1965a) studied the chemosterilization and mating behavior of male house flies; the results indicated that male and female house flies reached sexual maturity in 20 and 40 hours, respectively, after emergence. Mating may occur within 24 hours of emergence (Barber and Stanes, 1949) . (2) The male apparently has an instinctive drive to mate, which is not dependent upon the presence of the female or a female odor.

PAGE 40

26 (3) Males will attempt copulative strikes with males or certain inanimate objects but strike more readily and more frequently at females. (4) Preliminary evidence suggests the presence of some type of female sex attractant of low odor. Actual copulation apparently never takes place in the air, though the seizure of the female by the male may occur in flight, after which the pair come rapidly to some surface; sometimes, however, if disturbed, a copulating pair may leave one surface and fly quickly to another (Murvosh et _al., 1964a). The insemination process presumably begins as soon as copulation has been physically accomplished (Chang, 1965a) . Murvosh et .al. (1964a) found that after mating periods of one and two minutes no sperm were found in the female, but after three to five minutes some spermathecae contained a few sperm and some were completely filled. Copulation time was studied using seven-day-old virgin males mated with virgin females of the same age; of 61 mating pairs the shortest mating period was 44 minutes, whereas the longest extended to 96 minutes. The average time was 60 minutes. Hampton (1952), on the basis of more than 50 observations, stated that copulation lasts longer

PAGE 41

27 than 30 minutes and may last two hours, but that the usual time is 1.5 hours. Sacca and Benetti (1960) reported, from 110 observations, a mean of 84 minutes, a maximum of 136 minutes, and a minimum of 59 minutes. Chang (1965a) reported that the average time flies remained in coitu was 56 minutes, with a range of 34 to 93 minutes and a standard deviation of 12 minutes. Mating behavior appeared the same under ultraviolet light as under normal light (Murvosh et al. ( 1964a). Also, house flies can mate in the absence of both ultraviolet and visible light (Rogoff , 1965) . Male flies handicapped by removal of legs successfully mated with females (Chang, 1965a) . Females of all strains mated more readily with males of their strain, whether or not the males were sterilized (Fye & LaBrecque, 1966) . Chemosterilized males competed more successfully than normal males of the same strain when they were mated with females of that strain (LaBrecque et al., 1962; Fye & LaBrecque, 1966; LaBrecque et al . , 1966) . Radioactivity was transferred to female flies by copulation with treated males (Chang et al . , 1966) . In a genetic study of the cross between red-brown and green-eyed house flies, Zimgrone et al. (1959) concluded that

PAGE 42

28 the female usually mated only once and that fertilization sufficed for the entire egg-laying period. Only 2 percent of the females showed evidence of mating twice. Riemann et al. (1967) reported that most normally mated female house flies are monogamous, and none will mate more than a few times . The loss of sexual receptivity by females after mating was caused primarily by the male seminal fluid, not mechanical stimulation or sperm. When, in the laboratory, the subspecies Musca domes tica dome stic a and M. domestic a vicina were crossed, characteristics of the progeny were nearly those typical of the latter. In another cross (F 1 and back-cross) , the same tendency was shown. Characteristics of M. dome stic a vicina are therefore considered dominant over those of M. domestica dome stic a (Peffly, 1953) . Oviposition The female fly walks over the material that is to serve as food for her larvae, seeking crevices and cracks in which the eggs may have a measure of protection. The female pushes her ovipositor as far into the crevice as possible to lay eggs. One egg or several may be laid in a single spot, but all will be hidden if circumstances permit. If the physical nature of the medium allows it, the fly crawls deep

PAGE 43

29 into the coarser crevices to lay her eggs, so the eggs receive maximum protection from desiccation and actinic light (West, 1951) . Egg production in the female seems to vary. Herms . and James (1961) pointed out that from 75 to 150 eggs are deposited singly and piled up into masses, and that there are usually several such laying periods at intervals of three or four days. Female flies begin depositing eggs from nine to twelve days after emerging from the puparium. Lineva (1953) , in studies on the physiological age of M. domestica females in Russia, found that a female that oviposited 20 times lived 62 days. Dunn (1923), reporting on his observations in Panama, stated that as many as 159 eggs may be deposited in one batch, and that one female may deposit as many as 20 batches, or a total of 2387 eggs, within 31 days after emergence. Hodge (1911) stated that ... a pair of flies beginning operations in April ' may be progenitors, if all were to live, of 191,010,000,000,000,000,000 flies by August. Allowing one-eighth of cubic inch to a fly# this number would cover the earth 47 feet deep. Harold (1965) recalculated that figure and decided that a layer of such a thickness would cover only an area the size of Germany, but that is still a lot of flies. Spiller (1966) pointed out that neither adenine nor

PAGE 44

30 yeast RNA improves egg laying. There are indications of undefined chemical or physical factors which facilitate high egg production. Possibly there are feeding stimulants (Robbins et al., 1965), the increased egg production reflecting increased food intake. Among flies fed with the dry diet (LaBrecque et al., I960), the eggs mature earlier than among those fed with other kinds of food and are laid on the fifth, sixth, or seventh day rather than on the ninth or tenth. Older female flies produce an increased proportion of infertile eggs (Callahan, 1962) . Use of additional ammonium carbonate may increase the egg harvest at any one collection, but there is no evidence that total egg laying can be increased. Longevit y A few adults die within a few days . Murvosh et al . (1964a) found that the longevity of the control flies was of surprisingly great duration, considering the reports in the literature. Room temperatures during the experiment ranged from 22.2°C. (72°F.) to 29.4°C. (85°F.) , with a mean of 23.9°C. (75°F.); the relative humidity was 40 to 80 percent. This experiment showed that male flies lived an average of about 33 days, with a range of one to

PAGE 45

31 71 days, whereas the females lived an average of 43 days, with a range of 1 to 99 days. Rockstein and Lieberman (1958) reported that, at 26.8°C. and 45 percent relative humidity, the mean longevity of one strain was 17.4 days for males and 29.4 days for females. Varzandeh et al. (1954) reported the longevity of three susceptible strains of flies as 22.5, 32.3, and 30.8 days for females and 17.0, 23.4, and 23.0 days, respectively, for males. Herms (1928) , working in California, recorded an average longevity of 30 days, with a maximum of 60 days in summer. Under experimental conditions during summer at Ithaca, New York, individual flies have been reared and kept alive for 30 to 70 days. Afifi and Knutson (1956) , in a longevity study conducted for the National Association of Insecticide and Disinfectant Manufacturers, employing house flies with no history of insecticidal exposure maintained at 80°F. and 50 percent relative humidity, reported results as follows: treated parent, 17.1 days; untreated parent, 16.8 days; treated P 2 , 28.4 days; untreated F 2 , 27.1 days; treated F 3 , 23.7 days; and untreated F 3 , 23.9 days. The mean number of days from initial oviposition until all egg laying had ceased was as follows: treated parent, 22.4 days; untreated parent,

PAGE 46

32 23.2 days; treated F 2 , 39.4; untreated F 2 , 38.6; treated F,, 30.7; and untreated F , 30.4. They found no significant difference in results between treated and untreated flies. The age of the parents has some effect on succeeding generations. Average length of adult life decreased when lines were bred exclusively from eggs deposited by young flies (Callahan, 1962) . In another strain, continued use of the last viable eggs decreased longevity and reduced fitness so that no more than three consecutive generations could be reared (Callahan, 1962) . When eggs were always taken from females at the age of 20 to 30 days, the average longevity of the offspring was increased. Sex Ratio In nature, the sex ratio is usually about one to one, but sometimes there is considerable departure from this norm. In the laboratory condition, Murvosh et aJL. (1964b) reported the sex ratio, based on 5233 individuals, to be 53.5 to 46.5; four years earlier, the sex ratio in a sample of 8700 flies from this same colony had been 50.6 to 49.4. Rogoff (1965) reported that the sex ratio, based on 16 samples of 100 pupae, was similar in jars of both the

PAGE 47

33 control and full darkness series, and the results were within normal expectations. Murvosh and Thaggard (1966) , in ecological studies of the house fly, found an unusually high proportion of males in 21 samples of 4266 flies collected at various times from five kitchens on Mayaguana Island. The ratio was about 1.5 to one among 4266 Mayaguana flies. This was a highly significant deviation from the expected ratio of one to one mentioned above. They also reported that, in the same area, they had examined flies gathered from the hoods of vehicles in the carport outside their laboratory; each day for three days they counted 50 flies but noted only one female. West (1951) reported that when the size of average adults in a random sampling is small, the males will be more numerous, while if the average fly captured is of superior weight and size, this is never the case. Herms (1928) , while studying Lucilia sericata , brought out the fact that an unfed larval population always yields a preponderance of males. He removed the larvae from food in lots of 100 at six -hour intervals, after allowing an initial feeding period of 30 hours; the longest feeding period allowed any lot was 96 hours. The longer feeding periods yielded a preponderance of female flies. Herms

PAGE 48

34 felt that larval females required more nourishment than males for their development. This may also be true of the house fly, as indicated by the fact that the male flies usually emerge first. The female flies, after copulation, influenced by chemotropism, fly to the so-called "breeding places" for oviposition. The average longevity of the females is greater than for the males. The amount of nourishment in the "breeding places" is not always sufficient; therefore, to determine the sex ratio in the natural condition, the time, the place, the size of the sample, and the methods used should be carefully considered. The Egg The egg is pearly white in color and measures about 1 mm in length. The greatest diameter, which is near the posterior end, measures a little more than one-fourth of the length. Both ends are bluntly rounded, but the anterior is always more tapering. Along the dorsal surface of the egg are two distinct riblike thickenings. The results of numerous investigations clearly demonstrate that excrement is one material upon which M. domes tica habitually deposits its eggs. One or several eggs may be laid in a single spot, but all will be hidden if

PAGE 49

35 conditions permit. Average egg size is proportional to body size (Spiller, 1966) . The time required for the completion of embryonic development varies greatly with temperature. Under warm summer conditions, the egg stage requires 8 to 12 hours (Herms & James, 1961) . Melvin (1934) showed that both high and low temperatures tend to prolong the incubation period. The Larva Breeding Places in Nature Though the larvae of house flies have reasonable powers of migration, this activity is limited for the most part to the stage prior to pupation and has much more to do with the selection of a suitable site than with the seeking of materials for nourishment, since the mother fly deposits her eggs on carefully selected materials and there is usually no nourishment problem for her newly hatched offspring. Hewitt (1914) recorded M. domestic a as breeding in horse, cow, human, pig, rabbit, chicken, and other manures. Carrion, spoiled meat, offal of slaughterhouses, old broth, a boiled egg, rotting fowl feathers, decaying grain, cooked peas, boiled rice, barley malt, excreta-soiled straw, bread,

PAGE 50

36 cake, milk, rotten peaches, plums, cherries, bananas, apricots, potatoes, potato peelings, cabbage, carrots, cucumbers, cantaloupe, and watermelon are common breeding places. In addition, the list includes such miscellaneous items as kitchen refuse, fermenting substances, sawdust and rags soiled with excrement, earth containing expectorated material, rubber, and snuff. Horse manure is the favorite breeding ground of the house fly. When horse manure mixed with straw or other refuse is piled in the open, it soon becomes heavily infested with maggots. At the present time, at least in developed areas, other breeding hosts may be vastly more important than excrement. Smith (1956) , studying conditions in dairy barns in California, concluded that . . . the fly breeding potential of these enormous amounts of organic waste materials (chicken and cow manure, garbage, etc.) is far greater than it could possibly have been back in the horse-and-buggy days. ... Perhaps the greatest numbers of house flies I have seen came from lima bean waste in the field. I have seen nearly as great numbers from celery waste, tomato, etc. Rearing Medium in Laboratory The Gainesville Laboratory of the Entomology Research Division, Agricultural Research Service, U.S. Department of Agriculture, produced house flies for one year at

PAGE 51

37 the rate of two million per week for use in release experiments. The methods used were similar to those described for this investigation, using the CSMA larval medium and a dry adult diet. There are now no major difficulties in maintaining cultures. Though there is much literature pertaining to the artificial food of the larvae of the house fly, it is not relevant here . Insect Colonization and Mass Production by C. N. Smith (1966) provides a good background on this subject. When larvae are reared at different densities on a standard medium, the numbers of pupae obtained are approximately a fixed proportion of the numbers of eggs used, but the size of individual pupae decreases as the rearing density increases (Spiller, 1966) . It is very important in all experiments, especially those involving injection treatments, to keep conditions as uniform as possible and to provide quality control in the size of flies reared for testing. Growth Rate and Instars The larvae, or maggots, molt twice and pass through three instars. All stages have twelve segments, of which the second, or postoral, segment is actually double, giving

PAGE 52

38 a total of thirteen (Hewitt, 1914). There are no eyes, legs, antennae, or other appendages. In the first-instar larvae, only the two posterior spiracles are present; secondand third-instar larvae have both posterior and anterior spiracles. First-instar larvae molt anywhere from 20 hours to four days after hatching; second-instar larvae require 24 hours to several days; third-instar larvae feed for three to nine days before undergoing pupation. The rate of growth agrees fairly well with Przibram's rule (Teissier, 1931) , which holds that in most insects an increase of 25 percent in the length of the rigid parts of the cuticle is achieved with each successive molt. Larsen and Thomsen (1940) made daily weighings during the preimaginal period and constructed weight curves to represent the rapidity of growth. Firstand second-instar larvae were studied by Tao (1927) . The third-instar larva grows to a size of 12 mm or a little more. The right and left anal lobes are useful in locomotion. The Pupa The third-instar larva goes to a suitable place, somewhat cooler and drier than that required for its larval

PAGE 53

39 stage, for its pupation; usually pupation occurs in its breeding place. In the laboratory, pupation occurs in the rearing medium. Under nearly ideal conditions, pupation begins early on the fifth day and is completed soon after the end of the sixth day. The process of pupation consists of a general construction of the larva within its own integument so that the latter comes to form a puparium; the pupa case is formed by the last larval skin, within which the pupa is said to be coarctate. The pupal stage requires three to five days under suitable conditions; under adverse conditions several weeks may be required (West, 1951; Herms & James, 1961) . Hibernation In cooler climates, under natural conditions, the continuous breeding of the house fly is interrupted by the winter season. Authorities differ in their opinions as to how the house fly hibernates. Hewitt (1914, 1915) held rather strongly that the house fly passes the winter only in the adult stage. Copeman (1914) maintained that, although the fact that adults can survive the cold is the principal factor in carrying the species through the winter, it is

PAGE 54

40 possible that flies in the pupal stage also hibernate. Skinner (1915) stated that "houseflies pass the winter in the pupal stage and no other way. " The obvious conclusion is that the situation varies from one locality and climate to another. There is no interruption in breeding under tropical conditions. Temperature, Humidity, and Light There are several important environmental factors which influence the life process in the whole life cycle and which may accelerate, retard, or prevent entirely the fly's distribution, activity, growth, transformation, reproduction, and longevity. Temperature The influence of temperature on the speed of development of each stage of the house fly has been studied by many workers . For each stage there is a maximum temperature above which development may not proceed, a minimum temperature at which activity also ceases, an optimum temperature at which growth, development, and normal activity proceed most satisfactorily, and a lethal temperature which causes the

PAGE 55

41 death of the fly. Even within a given stage, these temperatures may vary according to sex or other factors. Hewitt (1914) reported that two or three days may be required if the temperature remains as low as 10°C. (50°F.); between 15° and 20°C. (59° to 68°F.), hatching takes place usually within 24 hours after oviposition; between 25° and 35°C. (77° to 95°F.), from eight to twelve hours may suffice. Davidson (1944) demonstrated that variations in temperature are of great importance in determining the rate of embryonic development. At 37.2°C. (99°F.), the embryonic development may be completed in about 7.63 hours; at 15°C. (59°F.), it will be completed in about 51.45 hours; at 40°C. (104°F.), about 8.05 hours; at 41.6°C. (106. 9°F.), only relatively few eggs hatch; at 42.8°C. (109°F.), no eggs can hatch. Lorincz and Makara (1935) found that incubation required seven days at temperatures of 8 to 10°C. (46.4° to 50°F.). At 20°C. (68°F.), the period dropped to 22 hours, and at 30°C. (86°F.) to 15 hours. At 40°C. (104°F.), the eggs hatched 12 hours after oviposition. Eggs of the house fly cannot survive at temperatures above 46.1°c. The actual lethal temperature is probably a little lower than this, especially under sustained exposure (West, 1951) .

PAGE 56

42 Jashi and Dnyansagar (1945) studied fly breeding in compost trenches in India and reported that, from April to June, when the temperature of the upper layer of rubbish ranged between 60° and 65°C. (104° to 149°F.), few maggots or pupae could be found. During the rainy season, the temperature of this layer fell to 37° to 49°C. (98.6° to 120°F.) and many maggots and pupae were observed. Of course, during this period, there was much higher relative humidity. Puri (1943) states that, in general, high temperatures augment fly production by shortening the developmental period of the larvae, by hastening sexual maturation, and by stimulating mating and oviposition. Kramer (1915) reported that development required only half as long at 30°c. (80°F.) as at -20°C. (68°F.). Kobayashi (1940) found that oviposition and larval development occurred in Japan at temperatures down to 15° to 16°c. (59° to 60.8°F.). The same author (1935), working in Korea, showed that total development could take place in as brief a time as six days with the temperature at 28°C. (82.4°F.), but that this was extended to 41 days when temperatures ranged from 13° to 19°C. (55.4° to 62.2°F.). Optimum conditions existed between 25° and 30°C. (77° to 86°F.), at which level development normally required from seven to twelve days.

PAGE 57

The differential effect of the two types of media is very interesting. Except at extremes of high and low temperature, larvae reared on pig dung pupated sooner than those reared on horse or cow manure, and larvae reared on pig dung attained greater size. It was noticed that the activity of both secondand third-instar larvae decreased markedly at temperatures above the optimum. Feldman-Muhsam (1944b) reported that M. domestic a vicina lived as long as 106 days in captivity at favorable temperatures; the flies died more quickly as temperatures rose . Lodge (1918) reported that the optimum temperature for fe -ding of the house fly was from 38° to 48°C . (99° to 118°F.) . The maximum temperature at which food was taken by the house fly was 55° to 58°C. (132° to 136°?.), and the o o o o minimum was 10 to 13 C. (50 to 55 F . ) . V Humidit y Feldman-Muhsam (1944a) , who studied M. domes tic a vicina in Palestine, noted that cow manure dried very quickly, forming a crust; larvae and pupae could be found beneath, usually about four inches below the surface. The

PAGE 58

44 same author (1944b) pointed out that in winter conditions are very different. Many larvae and pupae die because of excessive humidity. The same factor is believed to inhibit oviposition. Similar indications were observed during the present investigation. Dakshinamurty (1948) showed that adult flies tended to be most active when temperature was high and humidity low. Katagai (1935) , working in Formosa, reported flies scarce from January through April, with a sudden increase in May, a decrease in June, and a second peak invariably associated with temperatures above 25°C. (77°F.) . Katagai noted that females were in the ascendancy between November and April, but males predominated during the remaining months of the year. Light The larvae of the house fly are more or less negatively phototropic. • As a result, the larvae can avoid exposure to strong light with desiccation and seek a more su it able pi ace . Experiments using various colored light to attract the house fly were conducted by several workers (Awati, 1920; Parrott, 1927; Freeborn & Berry, 1935; Cameron,

PAGE 59

45 1938, 1939; Harsham, 1946). They used different materials and different designs, and achieved somewhat different results, but all the results showed that the house flies were attracted in various numbers by various colors of light. Ingle (1943) used blue light to attract flies to screens for testing their reactions to various chemical substances. West (1951), citing the work of Kuzina, reported that M. domestica is active by night as well as by day, if the temperature is sufficiently high. No difficulty was experienced in catching flies in traps in the dark if the rooms were warm. Rogoff (1965) demonstrated that house flies can mate in darkness with neither ultraviolet nor visible light.

PAGE 60

MATERIALS AND METHODS The research work reported here was conducted in the Insects Affecting Man and Animals Research Laboratory, U.S. Department of Agriculture, Gainesville, Florida, from October 1965 to April 1967. Room temperature was maintained at 78° to 82°F. and relative humidity at 60 percent. However, the relative humidity was held at 50 percent in the rearing room for the stock house fly, Musca domestica Linnaeus. The Orlando regular colony of house flies (insecticide-susceptible strain) was used in all experiments. Technique of Rearing Stock House Flies The stock house flies were maintained by the following procedures (Insects Affecting Man and Animals Research Laboratory, Entomology Research Division, Agricultural Research Service, U.S.D.A., November 21, 1963). House flies were collected by placing a paper cup containing week-old larval medium in a cage of breeding flies. The flies oviposited readily on this material and sufficient eggs were obtained in about two hours. 46

PAGE 61

47 Larval medium was prepared by mixing 24 quarts of Standard CSMA Fly Larval Media (Ralston Purina Company) with eight quarts of oat hulls and 13 to 20 quarts of water. About four quarts of medium were placed in a two-gallon glass jar lined with a polyethylene bag and about 2000 eggs were placed on the medium, after which the surface was lightly stirred to insure contact between the eggs and the medium. The jars were covered with a heavy cloth to prevent oviposition by flies that had escaped into the rearing room. The use of too many eggs in these containers would result in small adults due to larval overcrowding. Hatching occurred within eight hours and the jars remained undisturbed until pupation appeared complete. However, a careful watch was kept to see that the jars of larval medium did not become too hot during the accompanying fermentation. When a jar was warm enough to be uncomfortable to the touch, about one-half pint of water was added to the surface of the medium for cooling. Pupation takes place near the surface of the medium. As soon as pupation appeared complete, the top few inches of the medium were removed, placed in a pan of water, and stirred. The medium sank to the bottom on standing and the pupae floated. The pupae were strained off, washed again.

PAGE 62

48 allowed to dry, and placed in one-pint paper cups in colony cages . When the pupae were introduced into the colony cages, a dry nutrient mix in three paper cups and water in two onepint paper cups fitted with wicks of paper toweling were added, to insure a proper supply of food and water to the emerging adults. Food and water were checked twice a week and replenished when necessary. The formula for the dry nutrient was as follows: six parts granulated sugar, six parts powdered nonfat dry milk, and one part powdered egg yolk. Procedures for Testing. Rearing, and Observation Rearing and Observation Cages Two different sizes of cages were used throughout the experiments. Large cages were used for the testing of six or more pairs of flies, and the small ones for the testing of five pairs or less. In each cage were placed equal numbers of treated flies and flies of the opposite sex. The large cage was constructed with an aluminum frame, 10.5 inches long, six inches wide, and 9.75 inches high, covered with a cotton gauze tube which provided a

PAGE 63

sleeve for the opening and was closed at the other end. Cages of this size and type were used for all tests of the house flies in this laboratory. The small cage was made using a 32-ounce wax-paper cup (5 inches high, with a base diameter of 4 inches and a top diameter of 4.75 inches), the bottom of which was cut off. The cup was covered with a cotton gauze tube, which provided a sleeve for the opening, and was closed at the . other end. Sexing Virgin Females and Males A special sexing tool (suction apparatus) designed by Dr. G. L. LaBrecgue was used to pick up and separate the male and female flies. Flies were immobilized for manipulation with a continuous stream of carbon dioxide in a sexing well. In the author's records, the shortest pre-mating period for house flies is not more than 12 hours. This phenomenon is not uncommon in this laboratory. It has been found advisable to sex the flies within four hours after emergence, and never after more than ten hours. On the other hand, it is egually unwise to sex flies just after their emergence, as the wings and other parts of the newly

PAGE 64

50 emerged flies are not completely spread and hardened yet. It is important to avoid sexing too many flies at one time. Carbon dioxide can have an adverse effect on longevity in house flies if the exposure is too prolonged or too intense. Overcrowding can also affect longevity, or influence the effects of chemosterilants , especially in the initial population. In the ordinary cage (10.5" x 6" x 9.75"), the number of house flies should not exceed 150, Overcrowded conditions can influence the fertility, mortality, longevity, and behavior of the house fly. Keeping Same-Age Flies and DifferentAge Flies Acquiring a sufficient number of flies of almost the same age for testing purposes requires a large number of pupae. Pupae were placed in an ordinary cage, and adults were removed to another cage at intervals of every one-half, one, or two hours during the emerging period, to keep the age differences within groups as small as possible. Water and Fly-Food Supply There was always an abundant food and water supply available for the flies. Each cage was furnished with fly

PAGE 65

51 food in a paper cup as described above. Each cage was also furnished with a wax-paper cup containing cotton balls acting as wicks for distilled water; the amount of water needed depended upon the number of flies in the cage. When the duration of the experiment was seven days or less, the water and food did not need to be added to or changed. For longer periods, as in experiments on longevity or successive egging tests, the old distilled water and fly food were replaced at periodic intervals. The water level in the water-supply cup was kept below the level of the cotton balls to protect the flies from accidental drowning. Solution and Injection The chemoster ilant metepa was dissolved in distilled water at the desired concentrations. Each experiment or replicate used newly prepared metepa-water solution; it was considered unwise to use older metepa solutions for sterility or mortality tests. The flies were injected under light COj anesthesia in the injection well. Injection was performed with a microinjector (Hamilton gas-tight syringe with a 30-gauge needle) . The needle was inserted into the dorsal thorax through the cervix region by bending the head of the fly

PAGE 66

52 slightly, taking care not to insert the needle too deeply into the thorax. The procedure was to inject one ul of the metepa solution into the house fly's body without moving or shaking the microinjector . After the needle was withdrawn , the head resumed its normal position, thus closing the puncture; bleeding after injection was seldom observed. Mating The treated flies were mated with normal flies of the opposite sex and appropriate ages. Both treated and normal flies were transferred directly to the observation cage by using a vial or petri dish in order to avoid the C0 2 effect, in the pair-rearing test, as soon as the treated fly copulated with the normal fly, the pair was transferred to a separate cage. Occasionally, the mating time was recorded. In the collective-rearing test, of course, it was not necessary to transfer the mating pairs to another cage, nor was the mating time recorded. For convenience of catching or counting, a light was furnished outside the closed end of the cage. The light also served to keep the flies inside the rearing cage and to avoid escapes. It was also useful in feeding and other operations.

PAGE 67

53 Egging Flies isolated according to sex were treated and fed, then held for a certain specified period, after which the treated flies were mated with normal virgin flies of the opposite sex. After mating, suitable materials were used to collect the eggs. The old larval medium (one-half inch of moist CSMA medium in a souffle cup) or fresh larval medium with a few drops of ammonium hydroxide added was used for stimulating the female fly to lay eggs. Ovipositing behavior varied. Each female fly usually, though not always, laid one egg-mass in the packed medium in each egging; some laid two, three, or more eggmasses. Female flies usually do not lay eggs in a medium which is either too moist or too dry. For this reason, the old medium which was used for egging was packed to form a slope, thus increasing its desirability as a site for ovippsition and, especially in pairing tests, increasing the likelihood of getting all the eggs for complete counting. Egg Counting After three to four hours, the larval medium container was removed from the rearing cage, filled with water

PAGE 68

54 and stirred with a small brush, causing the fly eggs to float to the surface. A small brush was used to break down the egg-masses and to transfer the eggs to small pieces of damp black cloth in a sample of 100 eggs from each cage. Since the fly eggs are white, they are readily visible and can be counted easily against the black background. The thinner and narrower this piece of black cloth is, the higher the pupation obtained. In the collective rearing test, egg counting was by means of a sample. Since the virgin female can lay eggs without mating, and the mated female can lay a certain percentage of naturally infertile eggs, it is almost impossible to avoid variations between samples. Thorough stirring and mixing with a small brush was necessary before the eggs were transferred to the cloth, and a line-up arrangement of the sample eggs and careful separation were essential to avoid counting errors. Since the shortest pre-hatching period of the house fly in the author's records was more than seven hours, no eggs were counted for the sterility test after the medium had been in the rearing cage for longer than that. The figure of 100 eggs per cage was selected to facilitate counting, comparison, and further analysis of the data.

PAGE 69

55 Hatching Counts Two general methods of hatching counts were applied: (1) To count the hatching only, the eggs on the black cloth were kept in a moist condition within the petri dish for 24 to 48 hours after the egg counting and then examined by means of a binocular microscope. The egg shells showed the different characteristics in hatched and unhatched eggs. Usually, the unhatched egg maintains its original egg shape; however, this is not always true and can sometimes produce misleading results if these are based only on hatch percentage. (2) To count the hatching when further information was desired, the egg cloth was placed on the top of the moist larval medium in a rearing container (usually a paper cup with a plastic bag) and covered with a small piece of cheesecloth, then maintained at 80°F. and 60 percent relative humidity for 24 to 48 hours. Here again, the egg hatch was examined by means of a binocular microscope and the counts recorded. Pupation Counts This step followed the same method as the second hatch count mentioned above. As the eggs hatched, the

PAGE 70

56 larvae crawled from the cloth to the rearing medium in the container . The number of larvae that reached the pupal stage was determined by flotation. In the laboratory condition, pupation counts were conducted one week after oviposit ion . Various factors can influence the development of the larval stage. Moisture and temperature were the two most important factors concerned in all experiments. Ordinarily, pupation took place near the surface of the medium; however, pupation occasionally took place under the medium or at the bottom of the rearing container. The temperature was raised by the fermentation of the larval medium and often became unfavorable for larval development, especially in an overcrowded arrangement of the rearing containers in an incubation cabinet. For better results, the following suggestions are offered: (1) Prepare the larval medium one day before using. (2) Keep a small distance between the rearing containers. Do not overcrowd these containers, especially during the first three days. (3) Distribute three to five cups of water among the rearing containers on each shelf of the incubation cabinet .

PAGE 71

57 (4) Maintain a slow air current around the rearing containers. Stationary air may overheat; strong air movement may dry out the larval medium. Criteria of Sterilization — Determining Sterility In general, there are three criteria for determining sterility: (1) egg-hatch percentages, (2) percentage of pupation, and (3) fecundity of second or later generations. Each criterion has its advantages and disadvantages, depending on the purpose of the experiment. The results reported here are based primarily on the percentage of pupation. Determination of Lethal Dosage In the preliminary tests, the experiments on lethal dosage were conducted together with those on sterility dosage. Since the results of -chese preliminary tests indicated a wide range of difference between the sterility dosage and lethal dosage, it was decided to treat the lethal dosage independently. Flies of both sexes, from one to five days old, were injected with the different concentrations of metepa -water solution, reared in different cages, and observed continuously

PAGE 72

58 for 48 hours. The mortality of treated house flies was recorded by sex and by age for the different concentrations of metepa solution injected. The dosages of metepawater solution injected in the preliminary tests were as follows: 0.25, 0.5, 1, 2, 4, 8, 12, 16, 20, 32, 40, 48, 64, 80, 96, 112, 120, 144, 160, 176, 200, 208, 240, and 280 ug/male or female house fly. The mortality at dosages below the rate of 40 ug/fly was not much different from that of the controls (injected with water only) . Thus, only dosage levels at or above 40 ug/fly were selected for lethal dosage tests. Determination of Sterility Dosage The microinjection technigue was used to determine the sterility dosage for flies of both sexes from one to five days old. The procedures of this experiment were as follows: sexing virgin female and male flies; keeping same-age flies as v^H as different-age flies; injection; mating; egging and egg counts, hatching counts, and pupation counts; and finding the percentage of sterility. In the preliminary test, both males and females were injected at the same dosage. The results showed that there

PAGE 73

59 was a significant difference between the sexes, so the experiments were conducted separately by sex. Based on the results of preliminary tests, the dosages for the sterility dosage tests were 0.25, 0.5, 1, 2, 4, and 8 Mg/fly for males and 2, 4, 8, 16, 32, and 64 Mg/fly for females. The percentages of sterility were determined on the basis of first egging and successive eggings. In male flies, both collecting-rearing methods and individual pair-rearing methods were used. In female flies, collecting rearing under the dosages of 2, 4, 8, 16, 32, and 64 fig/fly were tested in two replicates, and individual pair -rearing tests were conducted at the following dosage rates: one-day-oldfemales, 70 ug/female; two-day-old females, 50 and 60 Mg/fly; threeday-old females, 45 Mg/fly; four-day-old females, 10, 15, 20, and 30 Mg/fly; and five-day-old females, 5, 10, 15, and 20 Mg/fly. Six-, 19-, and 33-day-old female flies were injected with dosages of 1, 2, 4, 8, and 16 Mg/fly, with five female flies in each treatment.

PAGE 74

60 Determination of the Permanence of Sterility Based on the results of the sterility dosage experiments , male and female flies of various ages were injected with the 100 percent sterility dosage. At this dosage, there was no statistical difference among male flies of different ages. All male flies from one to five days old injected at the dosage of 8 yg/fiLy of . metepa-water solution showed 100 percent sterility, at least at first egging, when the injected male was mated with a normal virgin female one day after injection. Both the collective-rearing method and the individual pair-rearing method were employed. Treated flies of both sexes and various ages were crossed with normal flies of the opposite sex in different mating groups. The flies in the first group were mated one day after injection; consecutive matings were at six-day intervals. The sterility of the different mating groups and the sterility of the same group (first mating group) in different consecutive matings provided the basis for determining the permanence of sterility.

PAGE 75

Mating Competitiveness Test The mating competitiveness test employed in this study was based on the time lapse between release and mating. The treated and control flies were the same flies used for the permanence-of-sterility test. Two observation cages were used for each mating of different mating groups, one for the treated males and the other for the controls. The female flies used for mating were identical in number, age, and brood. The females were placed in the cages first, the exact number of females based on the number of males to be tested. A petri dish was used to transfer the treated males and the controls into the cages. The petri dishes in both cages were opened simultaneously. The time between the release and mating for each pair in both cages was recorded. Rate of Chemosterilization of Sperm in Vivo Three-day-old male flies were injected with metepawater solution at the SD 10Q level under C0 2 anesthetization and placed in a cage containing virgin females of the same age. As soon as the metepa-treated male copulated with a female, the pair was transferred to a separate cage. Both

PAGE 76

62 injection time and the time copulation began were recorded. When the mating was over, the male was removed and destroyed. The sterility of the mated female, based on the pupation, was determined. Derivation of Survivorship Curves Flies from one to five days old were injected with SD^qq dosage. Both males and females were studied, individually and collectively. The different treatments for deriving survivorship curves were performed as follows: Treated females + normal males Normal females + treated males Normal females + normal males Normal females normal males Treated females treated males All male flies, regardless of age, were injected at the rate of 8 Mg/fly; female flies were injected with different dosages according to their ages. Mortality counts were made and recorded daily. Longevity was calculated and survivorship curves derived for each treatment on the basis of these counts.

PAGE 77

RESULTS AND DISCUSSION Determination of Lethal Dosage A series of tests was performed to determine satisfactory ways to immobilize and inject flies during treatment Based on the preliminary tests, the different dosage rates were selected. Dosages of 40, 80, 120, 160, 200, and 240 Mg/fly were used in all tests; dosages of 48, 112, 144, 176, 208, and 280 Mg/fly were injected only in flies of certain ages. (See Table 1.) Flies were injected at ages from one to five days. They were tested by sexes, in groups varying from 16 to 25 flies, with five replicates and a 48-hour observation period for each treatment. The observed relationships between mortality and metepa concentration will be discussed in the following sections . Lethal Dosage of Male Flies The relationship between male mortality and the concentration of metepa-water solution is shown in Table 1. 63

PAGE 78

64 Table 1. — Effects of chemosterilant raetepa in different dosages on male house flies of different ages (percentage of mortality based on 48-hour observation period) . L 3 /Pi V % of Mortality 1-Day•Old 2-D ay-Old Observed Corrected* Observed Corrected 40 5.00 3.06 3.75 0.00 48 5.00 3.06 80 16.67 14.97 16.25 12.99 96 112 51 5"} 120 63.33 62.58 65.00 63.63 144 65.00 64.29 160 93.24 93.10 93.33 93.07 200 100.00 100.00 100.00 100.00 208 240 100.00 100.00 100.00 100.00 Control 2.00 3.75 *Abbott's correction applied.

PAGE 79

65 Table 1. — Extended at 48 Hours 3-Day-Old 4-Day-01d 5-Day-01d fVH cptvp H coy r\ 7.50 4.31 8.75 6.41 7.50 3.40 22.50 19.13 18.25 15.43 27 . 50 25.67 48.33 46.08 •• 90.00 89.56 65.00 63 .79 • 92 50 94 78 67.50 66.38 88.75 88.46 95.00 94.78 94.11 93.91 98.75 98.72 97.64 97.54 98.75 98.71 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 3.33 2.50 4.17

PAGE 80

66 The corrected mortality percentage attributable to metepa is derived from the total percentage of mortality by correcting for the deaths that occurred in the controls. This correction is calculated by Abbott's formula, as follows: "5 1 — C x 100 100 c where P = the death rate (%) caused by the treatment, P'= the total death rate (%) observed, and C the death rate {%) for the controls (i.e., not associated with the treatment) . Dosage-mortality curves for male house flies were obtained by plotting the corrected mortality on logarithmprobability paper, verified by a Chi-sguare test. The dosage-mortality curves for different ages of male flies are shown in Figs. 1-5. The values of LD 1Q , LD 5Q , and LD 9Q and the slope were estimated directly from the curves, and those values are given in Table 2. > Lethal Dosage of Female Flies Similarly, the relationship between female mortality and the concentration of metepa-water solution is shown in Table 3. The dosage-response curves for female flies, based

PAGE 81

67 Table 2. — LD. , LD..,-, LD Q _, and slope for each age of male «lu yu flies estimated directly from the curves on logarithmprobability paper. Age of Fly (Days) LD io (pg) LD 50 (ug) LD 90 (»g) Slope* 1 64 102 162 6.55 2 78 108 152 8.41 3 54 91 153 5.49 4 52 82 130 6.48 5 44 70 112 6.45 * Slope = 1/log LD Q . log LD

PAGE 82

68 Table 3. — Effects of chemoster ilant metepa in different dosages on female house flies of different ages (percentage of mortality based on 48-hour observation period) . % of Mortality • ug/Fly l-Day-01d 2-Day-01d Observed Corrected* Observed Corrected 40 6.25 3.27 4.00 0 48 10.00 7.14 80 15.00 12.30 16.19 12.69 112 30.00 27.78 120 32.50 30.35 31.00 28.13 144 60.00 58.73 160 68.57 67.57 75.24 74.21 176 75.00 74.21 200 100.00 100.00 100.00 100.00 208 240 100.00 100.00 100.00 100.00 .280 100.00 100.00 Control 3.08 4.00 Abbott's correction applied.

PAGE 83

69 Table 3. — Extended at 48 Hours 3 -Day-Old 4-Day-OId 5-Day-Old Observed Corrected Observed Corrected Observed Corrected 6.67 4.28 8.75 6.41 11.67 6.32 35.00 33.33 31.76 30.01 38.33 34.59 37.50 35.89 45.00 43.58 70.00 69.23 83.33 82.32 83.52 83.09 83.52 83.09 89.41 88.77 100.00 100.00 100.00 100.00 " 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 2.05 2.05 5.71

PAGE 84

70 on the corrected mortality, are also shown in Figs. 1-5. From the curves, the values for LD.,-, LD_-, and LD_. and 10 d0 90 the slope were determined and are given in Table 4. Table 4. — LD 10 ' LD 50' LD 90' and slo P e for each a 9" e of female flies estimated directly from the curves on logarithm-probability paper. Age of Fly (Days) M 10 (ug) LD 50 (ug) LD 90 (pg) Slope 1 66 132 265 4.25 2 84 125 185 7.22 3 62 116 218 4.69 4 55 97 173 5.05 5 49 87 162 5.06 Comparisons The data in Tables 1-4 show that the difference between males and females in each age are self-explanatory. The comparisons of the dosage-mortality curves between sexes and ages are seen in Figs. 1-5. There is a significant difference in the mortality rates of male and female flies at dosages higher than

PAGE 85

71 H ra 0 n >i +J >H H H * •p •H H (0 P U o S m o 0) CP A3 •P c
PAGE 86

72 rH (D U to >i +j H •H 8 « p 5-1 O g o u 99.9 99.8 |_ 99.5 99 98 95 90 80 70 60 50 40 30 20 10 5 2 1 0.5 0.2 0.1 30 40 80 120 160 200 300 Dose in micrograms per house fly (log. scale) Fig. 2. — Lethal-Dosage Curves for Two-Day-Old Male and Female Flies within 48 Hours.

PAGE 87

Fig. 3. — Lethal-Dosage Curves for Three-Day-Old Male and Female Flies within 48 Hours.

PAGE 88

74 99.9 99.8 I / / 99.5 _ / 0.2 0 .1 I 1 ! ! ' ' | 30 40 80 120 160 200 300 Dose in micrograms per house fly (log. scale) Lethal-Dosage Curves for Four-Day-Old Male and Female Flies within 48 Hours.

PAGE 89

75 0) rH 10 U W >t +> •H H 'i-i •a § >1 4J •H H ro +J n o e O OJ CP (0 4J C 0) CJ M /? 04 99.9 99.8 99.5 99 98 95 90 80 70 60 50 40 30 20 10 5 2 1 0.5 0.2 0.1 Male Female 30 40 80 120 160 200 300 Dose in micrograms per house fly (log. scale) Fig. 5. — Lethal-Dosage Curves for Five-Day-Old Male and Female Flies within 48 Hours. i I

PAGE 90

76 80 Hg/fly# the mortality rate for males being higher at the same age and dosage. With concentrations lower than 80 pg/fly, the mortality rates were similar (see Tables 1 and 3). • On the basis of 48 hours' observation of both male and female flies, there was an apparent correlation between age and mortality rate. Within the age range of two to five days, the higher the dosage, the higher the rate of mortality. Within 48 hours, 100 percent mortality was achieved in both sexes at the rate of 200 ug/fly. It is possible that a shorter period of observation might reveal slight variations in these percentages, but it is believed that the results obtained would reflect the same tendency. Determination of Sterility Dosage Chemosterilants act on insects in three principal ways, as set forth in the review of the literature. The most interesting of these is inducing dominant lethal mutations or severely injuring the genetic material in the sperm and ova (Smith et al. , 1964). Different developmental stages of the male gamete, such as spermatogonia, spermatocytes, spermatids, and spermatozoa, show a characteristic differential susceptibility to the effects of chemosterilants.

PAGE 91

77 Wheeler (1962) stated that alkylating agents (e.g., metepa) can directly or indirectly affect the alkylation of nucleoproteins . Deoxyribonucleic acid (DNA) is most likely the primary site of alkylation and therefore the most sensitive and the most subject to alteration. Kilgore (1965) , working with house flies, pointed out that alkylating agents "have a very pronounced effect on the metabolism of the nucleic acids." Keiser et _al. (1965) reported that in chemosterilized male fruit flies both the spermatogonia and the spermatocytes are destroyed, but the spermatids which are beyond the last division continue to develop and mature . Fahmy and Fahmy (1964) reported that untreated Drosophila females mated to males treated with varying dosages of the chemosterilant tretamine (TEM) had a very high number of unhatched eggs. The chemosterilant might cause a mutation which acts as a gametic lethal, or as an early zygotic lethal, for the house fly. LaBrecgue (Borkovec, 1966) reported that, where males chemosterilized with less than the minimum 100 percent sterility dosage were mated with normal females, the percentage of hatch of eggs obtained from second egging was often higher than the hatch of eggs laid shortly after mating. Linkfield (1966) reported that the

PAGE 92

78 dominant lethal mutations seem to affect not only the embryo in fleas but the larva as well. Most of the research on the physiological effects of chemosterilants has been done on female insects, because the ovaries are usually partially developed in newly emerged adults, with the result that the interference of chemosterilants with their maturation and with oogenesis can be observed. The most often observed histopathological effect of chemosterilants on female house flies is the retardation or complete cessation of ovarian development (Morgan & LaBrecque, 1962, 1964; Tung, 1965) . However, interference with ovarian growth and function is not a necessary condition for the induction of sterility, because certain compounds can sterilize fully developed eggs which have already been fertilized by an untreated male (Weidhaas, 1962) . An effective female sterilant may have one of the following consequences: (a) no eggs are produced, or (b) eggs are laid but the zygote does not develop into a mature offspring. Both effects are frequently accompanied by observable morphological changes in the ovaries. A series of preliminary tests was conducted to determine sterility dosage. The results of the preliminary tests showed that there was a significant difference between

PAGE 93

79 the sexes, so the experiments were conducted separately according to sexes. Sterility Dosage of Male House Flies Among male house flies, regardless of age, 100 percent sterility was achieved through injection with a dose of 8 ' ug/fly _of 1 metepa-water solution. Three different treat ments were conducted to determine the sterility dosage in this experiment. Individual Pair-Rearing and First Egging The test of male sterility using the individual pair rearing method involved six replicates, from which all eggs were collected and counted. The results may be seen in Table 5. Dosage-response data were plotted on logarithmprobability paper, verified by a Chi-sguare test (Fig. 6) . Collective Rearing and First Egging Table 6 shows the results of a male sterility test employing the collective rearing method, with five to ten pairs of flies tested in two complete replicates, and a random sample of 100 eggs from the first egging. Dosageresponse data are plotted in Fig. 7.

PAGE 94

80 0 "0 0 rH •rl £ o > H3 0 1 •rH Q) r3 >1 TJ CO CD fO C (3 »D •H ,Q c 1 •H in >i .. +> tr> W -H C O H •H Cn-rl tn TI (0 M CP rH O 0) 0 •P -P 1 •P >1 . w PS U TJ O 0 •H 1 m MH Q) •rl >1 4J •H •a HI rH rH 0 0 V— 1 •H 0 u t7> ^ 1 u 0 >i .p (0 1-i ft rH CO 'a (0 " — ' i — i • i 6 _ r-t (0 m co UJ o o 4-1 0; <— 0 -P 0 TS 10 J-i u >i u ti t .rl rH r-. 0 •H rH 0) ft 1 rH ft On q) >1 •H CD f3 rl rl H •P TJ (1) W | •P CD CM W -P rH 0) IQ G "A 0 ft a s »c (0 0 rH ft 0 0 0 a 1 ! +J x >i (0 E w o Ti | >H « G rH 0 TJ iH 0 w ,C >1 •P +> c U CD CD m 0 H C ft 1 -H 1 rl m • H3 0 if) O 0 H u CD rH (D o ra rH H re £ •9 CO \ 9 0 CP :-i ft c Q 3. m a\ cn CO vD rH o 00 CM o CD CM o »N ou o m 1 ft vu o O IT) m rH n Cj o Oi o o H CM rH in CO r> a* o o iH rH rH CN w 1 — 1 i rn / — \ CJ C-J CO rH / — \ Oi u ] w CJ _, o CM (7i r*> O O LO rH CM rH in CO P>cn O O rH rH rH f — — CO P** o r — 1 UJ 1 ft O o 1—1 c o O rf\ OJ i n U ) C_J r* \ cx o CO m o\ rH CM o O O CM H CM rH ps CO CO rH rH rH a\ O o" in to VD Ci m CO o o o rH o cx H CO o H o o CM rH cn rH CO CO CO CTt cn o o rH rH rH CM in ex CM o o CO as o o o o rH rCO CO o o rH H rH o o H O * CN O o CO o u +1 0 u

PAGE 95

81 Dose in micrograms per male house fly (log. scale) Fig. 6. — Dosage-Sterility Curves for Different Ages of Male House Flies Treated by Microinjection, Pair Rearing Method, from All Eggs of First Egging.

PAGE 96

82 CD .3 •P U CD 4H rH 0 O 0) u tr> (3 P .. C CO CD H , o •p S •ri P H •H rl W 0) i -rl CD loo tr> cu o U P ft (0 H 4-1 -rl O "W M 4-1 (D O g CO 3 CT> C CO cd 4-i m p ; ; o a g co Cr> (3 o H fO CD O T3 0) CO ro rH >1 *J rl rH •rl 'rl o •p >o +J •H rH •H M CD P CO m o •-a rH o ! >i r •a i in n3 rH o I >1 CO rH o I >1 rd I ro H O i >l m •j i CN rH 0 I >1 a) CD rH co \ o tr> Q ao o o rH O CN H rH c O o O o co c U 1 <— > i> o r— 1 o o o o cu rH V." CN o in o o H rH rH r-» o o o ro LO U J . — , o O o o CN o < — i o >r-N 00 o rH rH o m o CO CO o o CO H cn LO l> C\ cr> o o rH r— 1 r 1 1 o LO O rH LO o» CN ! f^l li ) o o ro i rn j t N O 10 CD ro CN CN CO CD O O CO rH ro ro in in CTl C5^ o O rH rH " " o ID o H o O rH O O LO O o o N M » J c_> i o / — i. o 1 ^ 0~ . XT ro 1 — i ro LO /— % V— ' rn w rH w CM CN LO o cn <^ o O — H rH •:; — o ro O >> CN CN o rH o O O o c o O rH O CN C". CO o o • • • • « • • • • • • rH o <• CN ro 00 o o o rH CO ro ro VO m 00 00 o o rH rH rH -0 CN IT, o o rH O I CN o o CO o u •p c o u

PAGE 97

83 +3 +2 . +1 . -1 -2 -3 0.25 0.5 1 2 4 8 Dose in micrograms per male house fly (log. scale) Fig. 7. — Dosage-Sterility Curves for Different Ages of Male House Flies Treated by Microinjection, Collective Rearing Method, Using a Sample of 100 Eggs of First Egging.

PAGE 98

84 Collective Rearing and Average of Four Successive Eggings The male flies tested here are the same males as in the previous test. While the previous test used only the first egging to find sterility, in this instance results are based on the average of four successive eggings in each replicate (see Table 7) . Again, dosage-response data were plotted on logarithm-probability paper (Fig. 8) . Based on Figs. 6-8, the relationship between dosage and male sterility in the three different treatments is summarized in Table 8. The data developed under the first and second treatments were very similar. Obviously, because of the multiplicity of factors involved in the third treatment (collective rearing, four successive eggings, a sample of 100 eggs for each egging) , the rate of increase and decrease of the percentage of male sterility is highly changeable. The dosages in the collective rearing method seem low when compared with the data from the pair-rearing test. Though the pair-rearing treatment (first egging) appears more stable than the collective rearing (first egging) , both methods can be used to determine male sterility dosage. Different dosages produce different percentages of

PAGE 99

85 0) P 0 CO M-J rH O o u n GJ Cn IB 4J c m V 1 +> •ri H H Vi a; -:-> U C o o o rH m o QJ cn o It +J C c u Vl rH • ft — e (0 re U •H s 0 w vi rj U •H rH -P ft (0 CJ1 ft cn Cn g C « CO c •H H *w o ro ft to C 0) rjn 0 M ft Vi 3 > •H +J S3 U 0] (0 G o u 0 0 VI V! «H 4J •i-I 0 0 O S IQ ft ! n rn O c Cn •o 0 ! a n 10 0 -.-> r 0 u o 0) •H 0 DQ 4-1 ro W I met A >i • +J cn G li QJ H ri rx -2 ro c (0 o P &< Vl u >1 4J H r* I •H V: C -P CO 0 r 0 rH G : >i IQ r O I m O i >1 ro "O l rn H O I >. ro T3 CN O (1) rH rj> rc O rji Q =L o o in rH in a r> cn in in o vo CO P» c\ cn cn cn o o o o rH rH O O in o <3< rC0 CO CN r-i r> cn CN rH co r> If) in h CN CO CO CO CN CO CO ^ CN r> r> CO Pcn cn o o O o co cn CO \o cn cn cn cn cn cn o CO H ro CO O O rH O O
PAGE 100

86 99.9 0.2 0.1 • 1-day-old 2-day-old 3-day-old 4-day-old 5-day-old +3 +2 +1 -1 -2 -3 0.25 0.5 1 2 4 8 Dose in micrograms per male house fly (log. scale) Fig. 8. — Dosage-Sterility Curves for Different Ages of Male Eouse Flies, Collective Rearing Method, Using a Sample of 100 Eggs of Four Consecutive Eggings in Each Replicate.

PAGE 101

87 0 rH o «~ C\ CP Q a. o C 3. w — o ^» h tr> Q 3. W *» (0 •h WHO) 0 En >i r3 O O ft CP rH ^ < ro S DO +1 £ QJ e +> ra M Eh 01 o ro CN co to 0 4-1 •H CP u to > •rl c H a .-3 0^ o o CN rH CO o in o rH CM • • # * » • CO CO cn CN CO CM CO rH in o O o V • • I • • • CM CO CN CN CN in O in CM CN CM CN o H CO O O • • • * « • rH rH o rH rH O in in in CO o CM CN rco CO rCN CN CO rH • • • • • » o o O O o o in 0 +> o o 4H 0 ai H a* .-3 cn Cr> c c •H -H Jh 0) (0 0 O Jh +J > rH •r| U 0 a) rH W rH tj> 0 CT> CJ <1) CN a > •H n o Q) U U CO w en cr> o +J o rH HH cn 0 •rl CD CO cu (0 -rl •P cn cn C G •rl -H rH CP j> 0 Cn cn U CD O 0 c rH in

PAGE 102

88 sterility. The sterility of male flies varies in direct proportion to the dosage. Among male flies up to five days old, there was no significant difference in the 100 percent sterility dosage. Sterility Dosage of Female House Flies The results of the preliminary tests showed that the sterility dosage in female flies is much higher than that for males of the same age. These tests also showed that there were significant differences according to age. This experiment was split into the following two groups: Female Sterilization up to Five Days Old Collective rearing treatment . — Based on the results of the preliminary tests, dosages of 2, 4, 8, 16, 32, and 64 ug/female ' fly were selected for testing. Five to ten pairs of flies were tested at each dosage; two complete replicates were performed. The results of these experiments are shown in Tables 9 and 10; the dosage-response data were plotted on logarithm-probability paper (Figs. 9 and 10). The relationships between dosage and female sterility, as revealed by these two tests, are compared in Table 11. Clearly, the higher the dosage, the higher the percentage

PAGE 103

89 o i S u P a) r-i » 0 0 CP ro to .. o cr. CP a) f3 rH m •a e d) m n N. w a o c (0 C »H tP •H CP CP c 10 g a n r3 o >i rH 4-> > -' •H H 01 •H t) SH 'H a) i— i p m n o ft 0 n O f0 p ft i to I ro "0 H O I >i (0 •w i vX ID H IT) g OJ (1) CP fa f3 X w CP O a. Q ' o CP CCP O ID O o o o" O o" CO CO If o ID m O o o o O o CN oo <' CO H o o o o O o CO tCP CO CP CP o o o o o o rH H r-i rH rH rH ^ — CO o CO o o o o O o CO 00 id vD ID o o o o O o CO crif) <' CN r» r> o o o o O o CM VD CD CO CP CP o o o o o o H r-i H 1 — 1 rH rH rH — CO in CO CO H rH O o CP ID o CO 00 r*^ r> in O o o cc c m <^ o 00 CO o o CM r-i in s r* Co CO CA CP o o rH rH rH o o CO ID in rH O o r-i o CO r<' co i—; i—; o o o V£ CO o cn \o H o VD <£> o o CO r-i r-i m m CO CO CP CP o o rH rH * r — S t> o 00 if) CM r-i rCP CO o CM o o c\ 10 CN O CM rCO r> CN o CN o CN M O CO rH CP CP CO ^ > rH H m ID CO CO CN CO VO r-i
PAGE 104

90 I 0 CP O 2 c CD 4-> -H iH ,£ rH » O O (0 U (!) 0) id o to >i ftJ 03 o c cd CD 13 CD (0 CD O «H rH >4 .p rfl ft co c £ ., ^ ©one M m O -H 0) \ tr>H 1 C 0) •H ,Q CM O CD CO 4-> 0 I o > u c iH +J TJ 10 CO CO CD C e CD CO O ^ CD p CO CO u i O H ft +J H -H ^ CD •H ^ H O H H 0) •H CO 4-1 CO n oj ca cr> rj -iH 4J H *H CD w m o o CI O O O C'H 03 fC CO 4-1 «H ft 5-1 CD *H C O 4-> .C -P U 4-> (3 g CD CD O O 4H H W H tJ> ft 1 C CD CD • U m O Cd CD 4-> H CD 4-1 CO U CD rH rH CD ft TJ g ft 0 3 cj ft c .a B3 > •rl +J H H •H 54 CD •P W

1 id IT) -0 >1 Id 1 H O ! O ro H O ! >1 03 r O ! CN «0 .-; C ! >1 rd r w ! H tr> o a O v£> VD CO cm rin cn Lfi en CM CTi in cm OO ^ o o\ o in o CO Li o o oo H in ^ CM rH rH ^ CN co en vo in rcr> oo r~~ o o o ^t* 00 o ^ CM o o o o 00 a\ 00 rin a\ en cr. a\ CT> CTi o o o o r«X) oo in co i-l cm o >h o m o o o m in cm o o CN CO Si* rH CN CO rin oo c\ CTi co en o\ in o cm o in vd o rH CTi CTi v£> CTi ^ CO O O O O CTi O CN — m co in "tf LO co CTi CTi in co rrH co in o ^ in r^in o o o o o rH rH O O O O 00 O CN rH cn ^ CN vo in VD CO co co CTi C^ CN CTi h m o o <7\ rH r» in co in cr> o CN co in rH CTt 00 CN CN rH — ' CN H CO CO m sj 1 cn co co oo CO o rH CN 00 in CO in CM o o co O O CO rH rH in CN CO CN o o o O O CN rH rH CN CO o u 4-> o u

PAGE 105

91 Dose in micrograms per female house fly (log. scale) Fig. 9. — Dosage-Sterility Curves for Different Ages of Female House Flies, Collective Rearing Method, Using a Sample of 100 Eggs at First Egging.

PAGE 106

92 I i i i i i i 1 2 4 8 16 32 64 Dose in micrograms per female house fly (log. scale) Fig. 10. — Dosage-Sterility Curves for Different Ages of Female House Flies, Collective Rearing Method, Using a Sample of 100 Eggs of Four Consecutive Eggings in Each Replicate.

PAGE 107

93 CO o rH r\ >M IN l * i 1 N IN % If) in r\ U f~H V — ^ r-\ * — i r-\ t-J ID n"\ w . — | v — 1 f-\ r-\ V-/ rr\ U> 0 — \_J .-— -> w Q CO ( | i — i CO /— \ 'l. — vO n vO U 1 W — rn LO n /—\ V—/ , | CO W CM CM r-i rH o o O m in O O o rH ID CO o CN Q • • • • • • • o CM IT) CO rH n 0 •H in i rH CM n rH CO lf> cnrH ii <: to e o Em oo -P C o £ 4id o Eh C cn •rH CP 0 4-> u m o 00 o o o r-i UH o 81 rH ft en c •iH CP , s. cn c •H C) Cn > cn •iH 0) n 10 M o u S, 0 EJ DQ n Cn 10 cu e o •rl o +J H <" •H 0 »H 0 CD H o ft CP 10 M 00 0) > <

PAGE 108

94 of sterility. It is equally clear from this table that there is a direct relationship between age and percentage of sterility at any given dosage. The dosage required to produce 100 percent sterility in female flies varies according to age. The minimum dosages are estimated in Table 12. Table 12. — Estimated minimum dosage for 100 percent sterility in female flies of different ages (based on collective rearing and percentage of pupation from a sample of 100 eggs per egging) . Age of Fly (Days) Estimated Minimum Dosage for 100% Sterility . . (ug/f emale fly) First Egging Four Cons ecu tive Eggings 1 >64 >64 2 >32 to <64 >32 to <64 3 >32 to <64 >32 to <64 4 > 8 to <16 >32 to <64 . 5 > 8 to <16 >32 to <64 The chemosterilant metepa can influence not only the percentage of female sterility but also the female's fecundity potential; the total number of eggs laid by treated females was much reduced, especially at the higher dosages.

PAGE 109

95 The degree of development of the reproductive system was restricted. After high dosage, the ovaries of treated females were atrophied; this phenomenon is clearly observable by dissecting the treated female under a binocular microscope. The treated females showed some degree of restoration of fertility, but in most cases this restoration was limited to young flies treated at low dosages. Pair -rearing treatment . — Based on the information in the previous section, dosages were selected for the individual pair -rearing tests; the results appear in Tables 13-17. Obviously, the one-day-old females injected at the rate of 70 ug/fly in pair-rearing treatments could not lay any eggs; the treatment produced permanent 100 percent sterility (Table 13) . Two-day-old females treated at dosages of 50 and 60 Mg/fly, mated with normal males six days after injection and egged one day after mating, could lay eggs, but no eggs could hatch. When these flies were mated 12 days after injection and egged the next day, some of the eggs hatched. When mating took place 26 days after injection, only a few eggs were laid and they were infertile. The total number of eggs laid decreased with increased

PAGE 110

96 c 0 fl) to id fl n Q) •H H »H O rH (0 e 0) 0 I >1 m i (D C O IH 0 n cp 0) H 0 >i P fl •H H •H M 0 +J u c 0 ft 0) 4J 0) g 0-. (0 4-> o 0 -H (0 p DO CP CP w 0 O o 2 CP fl a in cp CP w q •rH CP CP w CP c c CO CP CP w CP c C -H CN CP CP H CP c •H CP CP w cp o w C M +» >i •rl (D IC (0 4-> -P r> -H c c c c 0 0 0 0 u u u u CN CN

PAGE 111

97 C o •0 0) W (0 (0 (1) •H rH m r-ri r" r•4-' n in CP W H rc h) n CPCF ,c c tj -p •H H m< CP HH W 0 • 0 2 Cn G H CT> » n Cr cn CP c H G •H CM Cn •0 Cn 0) W 0 -p f3 K * CT> 0 -P c CO H 1 H Cn 0 Cn W T5 Cn a) to G -p >i r| O U 13 -p -p i Cn H (TJ fa « \ O Cn a a. m O w H O 2 0< co o o m \ W o o n <7i O O
PAGE 112

98 3 -a 0) a n q no •H rO CO cn CP w o o S3 4-) B in cn CP w +1 If C •H cn Cn W — Cn ro cn Cn w CO Cn Cn c W c •rl CN Cn •0 Cn 0) W .c 0 -p (0 X * «p +> Cn 0 (0 C H •rl t Cn 0 Cn 55 W H3 Cn CD G S-i -P to •H CD CO S P -P 0) (0 rC >H s-i 'D 2 (0 ^ w CD >i Cn H re tn to S. O Cn Q aO (0 u • -rl O rO 53 ft O CO m cn CO \ in CO CN ^" CO co in m in CN m CN cn CN CN CN in rH H o 00 o rH O o> o m O o CO CN CN m in rH rH CO CO in in rH rH rH rH CN CN i—i rH H rH 0 0 0 0 m H m rl in !-. in m -p •P 4J v p c c G c 0 0 0 0 CJ U U u CN CN CN CN CN CN

PAGE 113

99 C o •H n CO CT C7 a VH 0 o 2; X Ui CJ B W "0 c c; 03 EC «w o o •H CP CP w cp 'O C U -H co cp CP w c •H cm cn CP w CP c •H CP CP w ^1 0 -p (0 01 CP -P •rt Q) (0 £h 2 ID Q CD >i CP H O CP Q a. O co H • -H O co h in CO CP CN rH in co h o cp CN CO VO i— I H tH r— I r-l H H rl H H m o o H CN CO o G o u CP O rH CO o« O rH in o CP W O CP CO [*VO rH VO rH o rH m co co r-» omcN H O H m H COH in VO rH OOOOH OOP rO O rH r* r» r» r» co co r-{ r-i r4 r4 CN CN CO CO CO CN CN CN moo rH CN CO rH H rH 0 0 0 U O U O O U +> CO +) H CN +J C C C 0 0 0 o cj CNCNCNinCN CN CN CN CN ojCNCNCNCN (N CM CNCNrH

PAGE 114

100 g o CO W tO CO o •H H VH 0 rH a e CO m •a rH 0 I >1 •a I Q) > •H UH IW o 10 tJI CP CO * 0 P rC •H O P rc •H p -p CO c o ro cu o P (1) e m o w p o a w I I • 0) H (0 CO p 0 n CJ w 0) ,C u +J (0 EC Uh O CP g •H CP CP w ^ 5? U -H co tr> CP w CP c •H CP CP w CP G •H CP CP w -P rc cp a) P Eh 03 10 Q 0) >i CP H A3 b W \ O CP Q 3VW O w H O f0 53 o« V0 H in CM CO > CO o o o m WW O O O cm CP CP \ CO CP CM CP co o O o W\ W m o o o h rH VD CO VD CO CP CM O ^ n o d r» in h o vo fN N VO H H O H H H w\ w w\ w o o o o r> o o cp o co r» cm co rH rH in r» \f n h > o H H d VD CTi w w\ rH Tj< o o in CM CO rvo G o u in o m o H rH CM 0 rH p 6 o o m o o P g o o CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM CM W CP c •H CP CP 0) > •-H •P 3 0 0) W G 0 u CP G •H •P ro & rH 01 p (D >i rrj tj w G o • W •C rH co a g > o cj IP +J P G •rH Ph >i (0 ffl t0 Tf £ I CO CP > G -H •rH m CP CP -P 0) CO p 'a (0 CO p 8, o p Si

PAGE 115

101 dosage. Time was also an important factor. The number of eggs increased with each subsequent egging. Treated females laid the largest number of eggs 13 days after injection, after which the number decreased. No eggs were laid after 13 days by females treated at the rate of 60 Mg/fly (Table 14) . Three-day-old females in any mating group injected at the rate of 45 Mg/fly were found to lay as many or more eggs than the controls at the first egging; only a few eggs of the second egging of the first mating group (five days after injection) can hatch (Table 15) . Four-day-old females treated with a dosage of 30 Mg/fly laid as many eggs or more than the controls at the first egging, within 11 days after injection, but the number of eggs was rapidly reduced thereafter. Females treated with a dosage of less than 20 Mg/fly laid fertile eggs within 11 days after injection (Table 16) . Five-day-old females injected at the dosage of 20 Mg/fly laid infertile eggs. Females treated with concentrations of 15, 10, or even 5 Mg/fly, mated with normal males, can lay sterile eggs within four days after injection (Table 17) . For comparisons of the dosage-sterility relationship,

PAGE 116

102 time is one of the main factors. Delays in mating after injection or in egging after mating can alter the results. Both the collective-rearing and pair-rearing methods show the same results. The dosages required for 100 percent female sterility vary according to age. Both methods can be used for determining the dosage and permanence of female sterility. The results from the first egging are more stable; therefore, it is suggested that tests on chemosterilization be limited to first egging results. s Sterilization of Females Older than Five Days This experiment involved virgin female flies six days old (sexed December 14, 1966) , 19 days old (sexed December 1, 1966), and 33 days old (sexed November 17, 1966) The dosages used were 1, 2, 4, 8, and 16 ug/' fly. Five females per small cage were used for each dosage, injected on December 20, 1966, mated with normal eight -day-old male flies December 22, 1966, and egged December 23 and 28, 1966. The sterility determinations are based on the egg hatch percentage. The results of this experiment are shown in Table 18. Here again, the data show varying susceptibility to metepa -water solution according to age. It is possible that either the water or mechanical injury or both can affect

PAGE 117

103 vo VO cn cn G rH •rl en • cn oo H cn •C • C U CN O O •p a) H -h +j OHO •rl £ U 0 -p CO p 0 H H (1) -P VO VD cn cn G rH •H Cn « tn ci w cn +j • w O H „ m m 0 W Cn • cn o w 53 * >i -O rC MH CJ O -P «p O U cn • Cn 0 w S3 O >i Cn iH O Cn Q =L O « w 0) *H (0 CnH Q < fa w o tj* in in o i-h r» o O I s ^ CO h vO p d ci in o o o r*» cn o O W O ^J 1 r-cm CM ^ H CI 00 ^ ^ VD rH o m in in in m in cn o vo cn CN iH co o vo o m co O ^J* VD rH ^ (N o o O cn co rH CT> CO cn m co in co ih H C) rl ci in ^ CN ^ in CN O C) VD 00 CN o u 4J G 0 U VD vO CN rH CTi VD o o cn CO 00 rH rH o o m co cn in O O O CN ^ O O O H ro o vo in CO CN t-i t-i r-i rH 00 00 O H * VO 00 Tj" CN H rH 0 S-t •p G 0 O o o o o o o o o o o o o VD 00 ^ CN rH 0 U •P G o CJ

PAGE 118

104 the female fly sterility; physiological change may also be a factor. These questions, however, cannot be answered on the basis of currently available data. Lineva (1953) reported that a female that oviposited 20 times lived 62 days; Dunn (1923) reported that one female may deposit as many as 21 batches or a total of 2387 eggs in 31 days after emergence; Callahan (1962) reported that the older female flies produced an increased proportion of infertile eggs. These workers studied house flies of different strains in different geographical regions; their results are not necessarily applicable here. The following test was conducted using the same strain and under the same conditions as the previous tests Eleven 50-day-old normal female flies were mated with the same number of three-day-old normal males in a large cage; as soon as the mating process started, the pair was transferred to a separate small cage and observed. The result (Table 19) show that female flies at the age of 50 days can mate with males normally. Actually, 50 days is much beyond the mean lifespan of the normal female house fly. Among the eleven pairs, one female died during mating; one female died after mating but before the first egging; and one female was engaged in the mating process for over three days, but she laid no eggs. A total of 8 of

PAGE 119

105 Table 19. — Egging of 50-day-old females mated with 3-day-old males (mated on February 11, 1967) . Pair 1st Egging Feb. 14 % of 2nd Egging Feb. 18 % of No. No. of Eggs No. of Pupae Sterility No. of No. of SterEggs Pupae ility 1 63 38 39.69 Female died after 1st egging 2 47 36 23.41 Female died after 1st egging 3 66 27 59.10 Female died after 1st egging 4 51 32 37.26 40 27 32.50 5 Female died during mating 6 9 4 55.54 62 51 17.75 7 11 5 54.55 remade aieu alter 1st egging 8 30 21 30.00 38 15 60.53 9 v Female died before 1st egging 10 Mating process lasted over three days, but no eggs were laid. 11 62 39 37.10 Female died after 1st egging

PAGE 120

106 Table 19. — Extended 3rd Egging Feb. 21 % of No. of No. of SterEggs Pupae ility 4th Egging Feb. 25 % of No. of No. of SterEggs Pupae ility Female died before 3rd egging 102 98 3.02 85 74 12.95

PAGE 121

107 the 11 pairs laid eggs. The average number of eggs in the first egging was less than that of normal females between the ages of six and 15 days; on the other hand, the percentage of sterility in the first egging was much greater. These results support the conclusions of Callahan (1962) , but the percentage of sterility in subsequent eggings can be reduced nearly to normal if the female still lives. Some females, at least under laboratory conditions, can lay eggs at the age of 64 days. Determination of the Permanence of Sterility A study of the permanence of male sterility was conducted by means of microinjection with metepa -water solution, at a dosage level close to the minimum required to induce 100 percent male sterility, as determined by previous sterility tests. The criterion used here was based on pupation. Five to ten replicates were performed in both collective-rearing and individual pair-rearing treatments. In the pair-rearing treatment, there were two differ ent series. In the first series, the treated males as well as the controls were divided into mating groups ; the males of the first group were mated one day after injection, the

PAGE 122

108 other groups at six-day intervals thereafter. The results of this series are presented in Tables 20-24. The second series employed the males which had been used in the first mating groups of the first series, who were then consecutively mated with normal females at six -day intervals. The results of this series are presented in Tables 25-29. The results of the collective-rearing treatments are summarized in Table 30. Based on the data in Tables 20-24, the relationship between the percentage of male sterility and the time (or duration) between injection and first mating is plotted in Fig. 11. Similarly, the data in Tables 25-29 are plotted in Fig. 12, showing the relationship between the percentage of sterility and the number of subsequent matings. The data from the collective-rearing test are plotted in Fig. 13. This experiment resulted in the following conclusions : (1) The injection of male flies with a dosage of 8 • „ug/fly induced only temporary 100 percent sterility. (2) in the mating group series, Fig. 11 shows the percentage of sterility to be inversely proportional to the length of time between injection and mating. (3) in the consecutive mating series, the percentage

PAGE 123

109 & tJ 4H 0) O -P (0 CD +> O (0 0) i -P i O !8 C P H P G P Oi rH (3 c 4J rH rH 0) 4J ft (0 (3 0 CD U a ft CO H Oi'H -cm 01 -H HOD H t8 p O 13 0 m e e I c o H (8 a CO Cn ft O (1) ft (8 4-> "H ft 0 c H CD E o H 4J trlO 0 4-1 o 4J 0 0 4H 4-1 . c n O c . •H i P •H rH •H u a 0 P c •H u o> u o> H U rrt c (N p TO 0) rH Eh •v. -p 1 i i~l •H i H 0) rH p 0 W 1 . M V-r-s O 1 P II t *rH /— « U VP r 1 VJ rr\ W P a (A rH p <8 CD u Eh 1 fa C b -3 •H Q 0) p UH H (8 0 m 2 2 m tn to 0 C ft •h p • P o O 18 rl Z 2 O VJ t • rsi l N ^HH r— 4 O o rH co C o ro o ro • • • • • « o o vO H o o Cn CD CO rH rH CO IT) o O 01 • • • • • CO rH 00 cn CN rH rH * o o O 01 CO to ro o o CM 01 o o in t • # • • • t t • • o o en C71 o. CN 01 o o o cn CO l> CO t> 00 *> rH H — ' — ' in CN CO in VD o CN VD VD CN p TJ •0 rC n C h P rH (N ro rC in

PAGE 124

110 ft U 1 r* rrf U-l •M •rl VU W m ft! CD G) /I u5 (J 1 u To r fM 1 1 I 1 t \ V c rrt 05 a) o\ w O 0 H u ro •n e & U) Tf u 1 H u> 0 00 1 0) >i P n it (0 i CD •H s M p CD H 05 4-1 UH 0 ft 0 3 CP >1 0 m P P p •rl CP w •rl P (1) P 05 U-l o 0) o c 6 s H u id 9 u >1 c a CP o ft O U o G O . T3 CD 05 10 .a >i •p •rl H •rl P a p u H •H U-l rl P m (0 >1 p •w rH -rH P CD P w U-l o f — I r\ \J CD CP u i •p •rH c I A U ) CP u ri %/ M TJ 'O U 0) vO CO p rH ID t O CP P c« Eh CP C •rH CP CP w C CM CP q •rH CP CP w p u r-l o H p c o o •0 rH <* rH CN O o CO in in in in CN CN • • • • • • m rH IN 00 CP 00 in 00 m in VD o o CP. VD CO O rH in t • • • • • • rH O CN CP rH 00 CN VD CN CN rH /VI IN 1 rH — • 00 CP CM O O rH CN rH VO rCO O o rH CM rH CP rCP o 00 CM x* CO O o CP in in rH CO CP CO CN CP o o CP cn 00 r* t> rH o o VD t> m CO vD H VO r> CN o CM o o CP CP CP CO CO CO vO VO VO in rH rH CO in CP m in in CM in rCN 00 CO CO CP CO p •0 r3 r3 rC m c u +» P rH CM CO <* m VO

PAGE 125

Ill >1 fi 0 . UH m QJ u 4-) n 01 cp ro +J C 1 M ro d) "0 P I a to p "H p o c •rl CP CP (J .C O r3 a> c •H 05 cp CP ro g VM ft +j p •rl CP iH •rl CP P C a) -h •P -P to ro o 0 u C e rl •P <0 e •rl P o ro ro ft ft w H •rl u ro C ro £ 8, c o 0) fit (0 +) ft 0) •p 5 rl C o O -P O •P CJ o in IH w I I B •rl O M U •H E CM «H CN O rl a) p 0) *rl ri rd >i c o f ft IH 0 H ffi c o «a i p •rl Q) 01 -rl H C P ,Q (0 ! •P •rl rH •rl rl 0) P M 0 _j n VJ P CP •P t— C •H o Cr> O CP w fj rC Q) P P (O O p Eh iH r* u p CP p t/-I •rl r. o CP u CP r.i W -0 u p ro (0 p EH CJ 9 rH CP W 10 in < a g •H en CP rH >i fa C fa id •rl Q a) +J 4-1 rH o ro 0 ro H a m cp o) o c ft • +> O O nJ U a a o CO rH rH O in VD CN P • • • CP P in CP rH c c rH CN rH •rl 0 CP o CP w CN O o O t> 0 VD o o in VD c •p • • • • • t CN ro CP. o o CO n 0 CP o o CO CO P ^ — rH rH Eh * * ro CN CN * * in CN in CO CO o m • • CO rH w ^ — *— * * — » •0 O CP o rVD in CN ro in 4J Cl> o o in in CO VD CO •<* in ro CP 05 p • • # • • • • • • • • • rH m o o CP CP VD in ro CN VD in ro (1) o o CP CP CP CP cn p rH rH — Eh — in VD in VD CN CN in CO CN -P 05 rH n3 G CN •a c CN CP a •rl P ro a •0 p P -P ro in

PAGE 126

U' rri \J r* r-« 0 n_i *>1 m 1 1 n u 1 M 4J IT1 H C (1) A) f-n U' 0 v. M m IT* u 10 si r*— rj to m rn •0 (11 (1) a) QJ r 1 w ifl E (0 QJ H M n VJ c •rl cp H UJ c H u M M m ai fl) (0 QJ +j c i H M c •fl QJ 8. IB CP Q) Oh 0 0) rH c M CP w fl U QJ CU QJ -~ E £ r4 10 QJ ff< w •P U-l CP : 1 ). 0 c fa TO fl\ C a QJ j i rH u U-l — rH 0) (0 0 fl t C o XI s: X •H u 4-> c V rn QJ G P •n 0 U C (1) •H 73 yj 0 QJ U-( V) w U ID •H -a 1 e • >. n U-l •P CN 0 •H CP in QJ 01 H 0 c a, rH c V4 H XI a QJ « P 0 fl a) 4J 0 rd £ a £ 0 112 o rH CP ID ,—1 • • • o ro CN CN O o rH rH r* O o rro CP • t • • • • o o CP CP CN o o CP CP CP rH rH — rH CO o [-. CP t • • rH VO m rH * o O o n o o o o in • • t • • o o o o CO 00 o o o o CP CP rH rH rH rH IT) in in in rH ,-1 rH 4-> >d T) 05 c M rH m m • • rH •<* • CO O CN rH 00 CN 3 • • * o « m UJ o «M 0) rj 4J 4J o n S >i ,Q 00 o >, rH 4J • • •rl rH rH •rl u QJ 4J (11 i in o # • * CD CN * 4-> CP 00 O a) M u o CJ QJ * 4-1 rH n fl 0 rH ,c •rl (0 fl r* in > 01 fl QJ 01 01 QJ fl CP ro 4J CN CN c QJ QJ rH fl fl E X) c QJ •rl 4-> fl 01 QJ QJ M 3 4-1 rH CO 0 r * X rC 4-» 4J

PAGE 127

113 .0 +» 0 CO M +J 0} rO -P C (1) QJ rH m e rH •a — rH 0 +j 1 to >i c •H CP CP a) .c u to -H •H H VH o >1 •p •rl (0 o H cp s M m qj CP (0 • p r-* •rl &> k C 0) »rl P -P to m o to c •H 4J 04 CO B 4J CP 03 CP a •rH U 1 c QJ CP o •rH 4H rH 0) *J HH to a u 04 0 0) rC <4H to •P 0 a) c 4J •rl rH CJ QJ 6 on m •rl 0 Ct c to CJ c p •n 0 o q U 0) •rl VM O CJ ItH n a U (0 f •rH .0 1 B • >1 «w •P CN 0 li OJ a •rH rH a >H 8 (0 CJ ' 5-1 W CJ P ID QJ W C •P CN to *4H QJ w M xo o Ij r-i i) g p> • rH o CP r j CP w T3 +J O to •P (0 CJ rH Eh a) QJ rH CP 10 (0 < CJ £ •rl n QJ CP rH > fa C (X4 (0 •rl P 0) P *W rH (0 o (0 s £ m cp w oca. •H 3 • O o a W 55 S O o o o o CN in fO CO rH rH • • t vo CO «• ^» n CN CO CN rH CO CN « • • # • • o rH CO rH CO 00 in CO cn CT> rH in in 00 in • • • • • • • rH CO CTi CO rH CO rH CN rH CN rH rH O tn o VO CN 00 O CO O in rH CP CO CN CO CTi en rH VO VO • o CP CTi rH cr. CN m rH 00 o o cn CT> CTi cn CO CO r» P* VO P* p* rH «— » (N O o 00 00 rrH O VO • • • • • • • CO CO CN CN P* CN CN rH rH o o o o CO CO rH CO vo r> CN C\ CM rH m oo co in o ro o o o o o o o t-i r-i H rH CP 00 C\ CT> CO CM 0> 0> CO ^J00 00 rH 00 00 in vo VO CN CO m CN VO o CO vo VO CO 4J C CM T3 rH CO rC 4J rC 4J in H-» VO 00 00 P» CN a\ co vo vo CM p p*

PAGE 128

114 «0 ai •p ta u P c ,Q Cn cn (0 cn -p ri a) -p H (11 01 0 H (I) H 0 *H rH 1 U rtJ >i CD (0 w g 13 0 I Cn n 0) c «w O 4-1 0) rt cn E « • p ^ id n 0i c O >i > 4J »H •H H -H M (1) u •P C o o to o o G (0 s £ H c 0 Cn m en cj C (0 •W CD £ -on C HI 4J h to m U fl c 0) 01 o M >h rl ro ft ft m o o re +j n ft _ CD H E c 3 o «W -H O -P u n Q) C (0 H rtj «w o A W O >i £ "H cu +» y CD E -p u c O I m «w -h cm O h o (0 4-1 C (0 a Eh E O o rH >i -P H rH H M 0 •P W «P 0 * rH Cn 4J •H o ^ * Cn w ,c (1) •p •p (0 (1) v. PI C_i L 1 rH o rH 0 r ) Cn W •0 •O M +J co ra a) H E-i rH Cn C •H Cn Cn w «a c CM iH tjl Cn W P to rH rH 0 SH 4J C O O 0 cu •p cu M a> Cn w •w w Cn rH > C fa -j •H P J +J «4H iTJ O s mo cn o a cj c tQ > -r4 • C -H +1 O 0 4J (3 2 U 3 S CO cn 00 o o o o • • o o o o rH rH o CO cn oo in o oo • • (T> 00 cn cn o CO o o o o • • o o o o H rH in ro O « in o CM CN • • cn cn cn cr> o m CO rH CN O • • cn cn CM cn • CM CM VD CM CN VO • t Tj< CN cn cn CO m CN t CM CO rcn rH r~cn vD o •' • o cn cn co o CO o CM CN 00 h r» • • rH CO cn co o CO CO co cn • • 00 VD CO CO vD O CM vD CM cn vD 00 H CO • •
PAGE 129

115 fQ •O Q) +J ro Q> U •P n i ro I O .* P M-l o >1 -p •H H •H rl 0) •p (0 «H O o o c c (0 E M c o ro ft 4-1 0) & «u o (0 p u 0 m «P w I I W 0) •H rl •H 4-> u 0 n c o o (0 . CP^» ro cp p n c •H -P ro B ft o ro cp o C H id u cp ro »n G •H a i -p •H H a) P (0 c id a) «p g o 4> rH rH -H 5l CO 4J w m o CP c •rH CP CP w •o u cn CP G •H CP CP w c CN CP q •rl CP CP w 4-1 CO 0 u 4-1 G O U 13 QJ -P 0) n 13 CO 4J ro 0) u Eh 0 0) rH CP to ro < CO e •rl IT, co CP rH G tu ro -H a o 4-1 «P rH ro o ro £ x I UH O 01 o o o c w > -w • G -rl +» o o 4-i ro CO CP VU Cn r(N 00 VD • • t • • in IT) m CO rH cn vD vD in rH rH CN CN rH CN cn in in VD in in • • • • • • • • • • CP CP in <* rH rH cn CN in CP CP CP CP CP CP C\ CO CTi CO rVD rH rH rH CN CP vD CO CO CP O • • • • • • • H vD CN CN c cn CN ™ Vir* o o CP CP rH CN cn rH CO rH o o VD VD CO cn H O CN in CO CP CP o o co if) CN CN O cn CN CP cn r>rH o o CP CP cn CP CP CP cn CO 00 CO CO CO rH H CN rH « cn o o o o • • o o o o cn CP in CN cn CN CP • 4

4J rH CN cn m VD r^ 4-1

PAGE 130

116 A •0 o •P ro a> u P (0 (1) H ro e rH o TJ I 0) 0) p ,c •p ip o >1 .p •H H •rl P QJ -P w IP o 0) o c c ro s P c •H cn cn ro tr> 4J C a o p & p 01 QJ P tfi 0) •P P QJ W CP c •H P ro e a > •H -P U (1) w 0 QJ CJ ro 0) c H CP id s p ip 0) tr> • +j cn C •rl -P go (0 ft e a* c •H » O cn ro " 0) c •P P d) P P •P 03 ft P (1) p m >i c 1 1 H P 1 S •P • rH i|H •H CM O P QJ 0) (/) P H C « (B ro CJ e 0 •P •P rH •H P 0) •P W ip 0 CP C •P CP Cn W T3 C fN c •H CP CP w p o p p c o u O -P ro QJ p Eh O u p c 0 u n3 0J P ro QJ u 6* QJ CT> CO < QJ ^» •rl W CT> H > c h ro H Q p «P w ro o S QJ r-i ro i GJ &4 QJ rH ro 2 ip o o 25 I o o n c 0 u > •rl +J cn C •P P ro S m m CM in rH rH t • • • • o cn rH rH CN CM CM o . — * m in o m o o O in in in • • • • • • • • • • o o rH cn <* cn cn cn cn o o cn CO co CO cn cn rH rH • — rH in in • • • • • CO o o rH CN rH CN rH — * ^— n o o" o o V0 CO in rH VD o o o O CM CO rH rin O o o o O CM rH rH 00 CO o o o o cn cn 00 rH rH H rH •« — ' »-» • in o rH in CM CN in 00 CM •P m rH C CM P CO p H-> m

PAGE 131

117 cp >i c & Q) -P CP CP "O (0 CP V -P CO P c (0 0) .C i p ft} 10 0) T3 (0 I P O «rl «P +) <1) (0 CP g CP u C M-l O >i > 4J "H •H rH •H P O 0 o co co «p O 0) o c •P CD CO C •H H +J ra m | e ft ,C U CP (0 o c o O -P ,c p » O CD CP (0 -P G CD *P h p id O <0 c g p ft g 0 >1 c co cn 0 u p •H 5 ft ft vp 0) o ,C (0 -P P ft V CO C rQ P -P g 0 § o •H +> w fli T3 •n co C CO •P (0 vp O ;Q «P P W O >i 1 *H +) I g -P U-i 0 p u CO c 0 00 «P 1 *J •P rH •P P CO P W IP o as rH 0 CP P a •P •p c CP 0 CP u w •0 c 0) CN +J (0 CO p rH 0 CP p c -P •p c CP 0 CP u w rP CO CO iH +J id 0 p Eh co • fa C fa ra •P Q 0) +J «P rH m o ICS 2 a i r\ V_r> 1 A U 1 u» i i s-\ w lA U f i t t • • • C-> IN l N »n o O CN in CN r» CO o o O ro cn vo CN o • t • • • • • • * • o o cn CO CO m cn cn o o Cn i cn ro rH • • • • • •p rH ro in VD rH rH ro * ro CN CN teri CO O o" o O CN in rH ro in rH O o o O cn in CN 00 cn CO •0 • • • • • • • • • te o o o o 00 00 VO o o o o cn cn cn cn cn cn u rH rH rH iH re in m in m ro CN in cn CN 1 0 cn 0 a CQ o c > -p • c •P 4-» 4J Xi •0 .C 0 0 P fl) CO a p +> 25 u 3 2 H CN ro rC P in

PAGE 132

118 £ CD o p u •P •P c ra q) ,c d)UO P (0 03 «— G CD -H H 4J (0 03 CO -P 13 0> cd 0) CD rH H rH >i P «J fO CD (0 o I 1 •H cd tn P > G 4H : H B o -P Oi O 03 C rl >i > •r) +1 ID (S •H CJ P CD (1) 03 -P G CO O 4-4 O a) u c ft g & rG o Oi 03 oca> O -rl C P » CJ CD o> id +j c a) 4-1 H M (0 P Q IS ^1 tj< G CD (0 p E u u •rl . ft G *j o cd o rC (3 +J ft P -rl g 0) e c c CD -rl CJi Di O Oi p cd P CD C 0 4-4 -H O 4J U 03 CD 11 4J -o O) O G 03 CD -rl (0 4-1 o A »h n w u I •P -P 01 4-1 CN O H •r4 P 0) CD 03 -P rH G 03 rtf 0) 4H Eh fl ° 4-> •H H •r-4 P •H o o> CJ Oi W -a •O cd P +> ITS CD P Eh 0) Oi to << CD •H 03 01 rH >i G Pd (0 Q +J 4-1 w rO O s CD H id g CD fa cd rH i 4-1 O 0 0) 03 0) • G > O O-H it S3 U -P 2 c H -p CO vD • CO VO • VD Oi rH 0 O m rH CO r» VD P rH o rH CO VD Oi Oi •P • • • • • • • c G rH 01 OI CO OI CN GO •rl 0 rH CI rH CN CN CN Oi u cn r*4 O O cn 01 in CN 01 Oi in CN CN •0 cd O o r» o OI in rH 01 CO GO GO Oi c P CO CN flj O o IT) ro CO CN n CO Oi VD CO a> O o Oi Oi Oi 01 01 CO CO CO 00 Oi 01 p rH rH Eh rH m VD o o o o 0 CO VD 01 rH o CO p • • • • • t • Oi p 00 CM ro in CN CO G G CO CN ro CN CN n •P 0 Oi u Oi * K *— * o o CO in CN o rH rH VD ro in r» •P o o OI Oi 01 01 01 OI 01 OI 01 Oi Oi u rH rH * — Eh in vo vo CN CO in CN vo ro o ro VD ro CN -p T3 »0 £1 03 G P 4-1 H CN ro in p vo -P

PAGE 133

119 Table 30. — Effects of metepa on permanence of sterility of males treated by means of microinjection in the collective rearing method (percentage of sterility based on number of progeny reaching pupal stage from a sample of 100 eggs in each egging after each consecutive mating) . Consecutive Mating _ 7° of (Days l-Day-Old 2-Day-01d after In— — — . , Treated Control Treated Control section) 1st 100.00 1.00 100.00 4.00 (2 days) (100.00)* (100.00) 2nd 99.00 4.77 100.00 37.00 (6 days) (98.94) (100.00) 3rd 91.00 1.24 96.00 21.00 (11 days) (90.88) (94.93) 4th 65.00 85.00 (20 days) Average 96.67 21.96 98.67 36.75 (95.73) (97.89) Values in parentheses show the corrected sterility by Abbott's formula.

PAGE 134

120 Table 30. ^-Extended Sterility , 3-Day-Old 4-Day-Old 5-Day-Old Treated Control Treated Control Treated Control 100.00 13.00 100.00 21.0 100.00 2.00 (100.00) (100.00) (100.00) 100.00 8.00 100.00 5.00 100.00 20.00 (100.00) (100.00) (100.00) 45.00 100.00 20.0 100.00 22.00 (100. 00)i (100.00) 61.00 93.00 37.0 97.23 68.00 (88.00) (91.34) 100.00 31.75 97.67 20.75 99.26 28.00 (100.00) (97.05) (98.97)

PAGE 135

121 Days for mating after injection Fig. 11. — Relationship between Permanence of Sterility and the Mating Time after Injection of Male House Flies, [Mating Group Series Test (First Egging, All Eggs Counted; Sterility Based on Pupation) .

PAGE 136

122 100 7 13 19 25 31 Days for mating after injection Fig. 12. — Relationship between Permanence of Sterility and the Mating Time after Injection of Male House Flies, Consecutive Mating Series Test (First Egging, All Eggs Counted; Sterility Based on Pupation) .

PAGE 137

123 100 90 +> •H H •H * U 0 •P (Q «H 0 0) CP S 8Q u u 0) 01 70 »— » — A— « o 0 e o K— r1day-old 2day-old 3day-old 4day-old 5 day-old 1 2 6 11 20 Days for mating after injection Fig. 13. — Permanence of Sterility of Male House Flies Treated at Different Ages, Collective Rearing Method, 5-10 Pairs of Flies (Progeny Reaching Pupal Stage from a Sample of 100 Eggs per Egging after Each Consecutive Mating) ..

PAGE 138

124 of sterility was inversely proportional to the number of matings of the same treated male. Both the mating group and the consecutive mating series indicate that a certain number or percentage of sterile sperm of the treated males can regain fertility within their own reproductive system. (Continuous mating, however, could result in a shortage of sperm. Chang et al. (1966) reported that there are no sperm to be transferred from the male flies to the females if the males have been continuously mated more than three times.) It seems, however, that this is not the only source of this phenomenon, because all the males in the mating group series were mating for the first time after injection, and they still showed a degree of fertility. Are there any sperm that can be produced after the chemical sterilization effect is over? This is possible, but cannot be determined without further study. (4) Comparing the data in Tables 20-29, the percentage of sterility in the first egging appeared to be higher than that in the second egging. This phenomenon is more significant in the mating group series than in the consecutive mating series. However, in both series, the fertility phenomenon was most clearly represented among males treated at two days of age (Tables 21 and 26) .

PAGE 139

125 Therefore, the fertility of the sperm can be regained not only within the male reproductive system but also after the sperm have been transferred into the female's reproductive system (in the spermatheca) . (5) The rate of restoration of fertility could be affected by various factors, but age seems to be of primary importance. The evidence indicates that the younger the male fly at the time of treatment, the greater the subseguent restoration of fertility. The duration of the condition of 100 percent sterility at the dosage level of 8 ug/male fly in oneand two-day-old treated males, in both the mating group and the consecutive mating series, and five-day-old treated males in the consecutive mating series was not more than one week; threeand fourday-old treated males in the consecutive mating series and fourand five-day-old treated males in the mating group series maintained 100 percent sterility for a week or a little more. Under the collective-rearing method (Table 30) , 100 percent sterility in one-day-old treated male flies lasted a week or less? older treated males remained sterile more than a week. Flies injected at the age of four or five days

PAGE 140

126 can sustain sterility up to 11 days after injection. Moreover, the results of the rate of metepa chemosterilization of sperm in the bodies of house flies tested indicated that some females, at least, mated with treated males at 44 to 175 minutes after injection, maintained the sterile sperm in their own bodies for a period of 22 to 34 days and laid 100 percent sterile eggs only. The permanence of female sterility was considered earlier in this paper (see Tables 13-17) . Since house flies, both male and female, are only temporarily sterilized by microinjection at the minimum SD 10Q dosage of metepa, time is a primary factor. For comparison tests, time for injection (age of fly), time for mating (after injection) , and time for egging (after mating) should be considered. Mating Competitiveness Test • In attempting to control the house fly by chemosterilizing techniques, one important aspect to be considered is the sexual competitiveness of chemosterilized males (Smith et .al., 1964). Obviously, if the mating abilities of the sterilized and unsterilized males are at the same level, then they have equal opportunities to be mating partners.

PAGE 141

127 Many factors can influence mating behavior. All flies should be treated under identical conditions. Mating competitiveness can be determined in a variety of ways: (1) By comparison of the number of successful mating s during the lifespan or within a certain period. (2) By comparing the actual egg hatch or pupation with the theoretically expected effects, based on the known ratio of treated to untreated males mated with normal females (LaBrecgue et aJL . , 1962a); this is the most widely used technigue at the present time. (3) By direct observation of the mating of limited numbers at a time, with treated males marked with dye, radioisotope technigues, or other appropriate methods (Murvosh et al. , 1964a) . (4) By comparison of the mortality rate of females confined with aggressive males (Baumhover, 1965); if the aggressiveness and competitiveness of sterilized males were reduced by the chemical, the longevity of the females with which they were confined would be increased. (5) By comparison of the duration between the time released and the time mated under the same conditions but in separate cages, with the same number of flies. The average

PAGE 142

128 duration can be used as a criterion to determine which is the faster mating partner. It is reasonable to conclude that the faster the mating, the stronger the aggressiveness or competitiveness. This is the method used here. The male flies treated were from one to five days of age. Each male fly was given an injection of metepawater solution at the SD 10Q level (8 ug/'flylr. controls were injected with distilled water. The results are presented in Tables 31-35. There were, altogether, 27 different mating groups observed, five to ten pairs of flies in each group compared with the same number of control pairs. There was a highly significant difference between the treated males and the controls. In 19 of the 27 mating groups (70.37 percent), the treated males, eleven days old or younger, consistently proved to be faster mating partners than the controls. This was true of all five mating groups of one-dayold treated males. The age of the treated male in the fifth (last) mating group was 26 days, which is greater than the mean longevity of the male fly, indicating that one-day-old treated males maintain excellent mating competitiveness throughout their lives (see Table 31) . With the two-day-old treated males, only in the

PAGE 143

129 Table 31. — Mating competitiveness of one-day-old treated male flies based on the time units. Mating Group 1st 2nd 3rd 4th 5th Age of flies Male 2 8 14 20 26 (days) Female 5 6 5 6 6 Time after injection 1 "7 1 "5 lJ (days) No. of pairs Treated 10(6) * 10 (6) 5(3) 5(5) 5(5) observed Control 10(6) 10(6) 5(3) 5(5) 5(5) Percentacre of mated pairs Treated 20 30 20 20 20 in first Control 10 20 20 0 0 minute. Time for matTreated 1-3-6** 1-1.83 1-4.33 1-19.4 1-31.4 ing after -3 -10 -60 -75 crossed (minutes) Control 1-4.17 1-2.67 1-^7.67 9-25.8 2-36-75 -8 -7 -20 -60 Values in parentheses show the actual pairs to be counted in the test. **The first and last numbers show the range of the time between release and mating of the actual pairs to be counted; the middle numbers are the mean.

PAGE 144

130 P p in o u tH -P C •rl •P S •a jh co T3 C CM •P in co oo CM in m m o\ in CO 5f a) 0 id , e nJ 0) 8 fa W 0 •H «W (0 O TJ 0) co in CM co H U 4) QH «W -rl M (0 4J > O CJ • 1 1 *rt w • • rn • rH (1) /rt r* co ro CO in rH O i I * VP IU CO | iH Q) in m o o CN I CN | s s -p 0) CO ri CO I • rH •n J \ Cj co co (N rH W (U o o •41 | o n in in r1 rH rH i rH CO (0 r< fl) i_J g n r \ \J j ) cJ rM | | rf CO CO^ o | 1) , — j in in o o CM (N rn Ui t. -rl •H i o. r* /ii i nl r* in in CO rH ,— J t| jj o o | 1 O (0 m m CM rH CM CO Q) •*» t) r* rrt r*H w w j 1 m (U 1 1 00 S G 5 § 00 00 00 | • rn r 1 in vo in o o o o 1 o 1 CN »> 0) o H rH CO rH rH CM O rl ^-J r< « rM U C J J CJ 4J * CO rn FA r-« Wi O O t rn ii H H rH • o (1) ri in rH in _e CO (TJ O O o o | CM I n< >j l \ •H rH H CO rH rH 1 rH | r* »-t _j rn n\ w rH] It W rrt «< rH Tf rH rrt rt rt ii rt QJ 0 C) w V r-H rri \ rl JJ ti M u i) fit il +' <-< i 1 rt IT 1 tr" Eh r \ . I r* rl SZ IU , , •« +J 1 rH 0 MH n 0 P r^ 0 H CO rrj •H 0 Sh a & cn M Cn •rl u * C f0 •p !H c H IJH 4H 0 3 o u
PAGE 145

131 Table 33. — Mating competitiveness of three-day-old treated male flies based on the time units. Mating Group 1st 2nd 3rd 4th 5th Age of flies Male 4 10 16 22 28 (days) Female 4 5 5 7 5 Time after injection 1 7 13 19 25 (days) No. of pairs Treated 10(8) * 10(9) 7(4) 5(5) 5(4) observed Control 10(8) 10(9) 7(4) 5(5) 5(4) Percentage of mated pairs Treated 30 40 14.28 20 0 in first Control 10 40 14.28 0 20 minute -, Time for matTreated 1-4.75**, 2.25 1-7.75 1-69-16.75 ing after -6 -4 -20 21 -30 crossed (minutes) Control 1-5.36 1-2.78 1-121-7.4 1-16.75 -16 -11 25 -20 -45 Values in parentheses show the actual pairs to be counted in the test. **The first and last numbers show the range of the time between release and mating of the actual pairs to be counted; the middle numbers are the mean.

PAGE 146

132 Table 34. — Mating competitiveness of four-day-old treated male flies based on the time units. Mating Group 1st 2nd 3rd 4th Age of flies (days) Male Female 5 5 11 55 17 5 23 7 Time after injection (days) 1 7 13 19 No. of pairs observed Treated Control 10(9) * 10(9) 8(7) 8(7) 7(4) 7(4) 5(5) 5(5) Percentage of mated pairs in first minute Treated Control 30 30 37.5 25.0 14.28 28.57 0 20 Time for mating after crossed (minutes) Treated Control 1-2.2** -5 1-2.54 -5 1-2.71" -9 1-3.43 -9 1-4.75 -13 1^2.75 -7 1-2655 1-17.8 -60 Values in parentheses show the actual pairs to be counted in the test. **The first and last numbers show the range of the time between release and mating of the actual pairs to be counted; the middle numbers are the mean.

PAGE 147

133 ,C +J CM »> r» ,c jj VD ro VD ro rv § H u ,G VD P O 1T\ iin CO (h •H •P 2 in ^4 CN u CO r» CO H 13 C CM VD CM H 4J (0 rH VO in w r( 01 w fO rH ro l «H ro 0 o ro in ro in CN ro rH G) C «— » •POM »H -H >i (0 +) rO O U Oj OJ w Eh "H • 01 Ui (0 [*•» in W ro rH i 1 01 •Tl • | rH c\ w ro ro in VD C\ w 01 w c V — ' i 1 1 i-« r4 in in CN «cH XI rH JJ c U") C) 1 CM n w • • If) ro rri \j •4— ' • • W r™i rH fl\ 01 nj w-U >T XT I i CTv I ro _n |M r — i CM M rn Ui 1 1 P4 01 H u vO ri w Cs 9 VD •§ U 1 • Mr* 2 ro ro i r~~l ^— ' CM 01 »-< Q i 1 00 i in in in CN -» If) 5 r-< CO 00 1 ^ rH A-* o 1 I fO Ui u Vj" XI • VD in >l 1 1 rH > n\ i i r— | i —J n s u M r\ rM rt r»i ni w rn _Q u • r/i Ui r* -X VW rn ui 4c ro (0 hi rH rH 1 • rO QN-H SH 0) C A 13 +> M +> •H (I) -P 04 CO 0 +» •0 3 P VH > u Q> m VH OJ £ rO O U oj "0 •H •P r0 (0 -H g o « 0 rO C •H C n c 15 0 04 g •H E •H u ro

PAGE 148

134 sixth (last) mating group (when the flies were 33 days old) were the males in the control group faster mating partners than the treated males. In other words, the two-day-old treated males can maintain their mating aggressiveness or competitiveness for long periods, even much longer than the mean longevity of normal male flies (see Table 32) . In the three-day-old treated males in the fifth (last) mating group (when the males were 28 days old) , there was no difference in the rate of mating between the treated males and the controls. The treated males at ages younger than 28 days were the faster mating partners when compared with the controls at the same ages (see Table 33). In the four-day-old flies in the third mating group or thereafter (when the flies were 17 days old or older) , the treated males were the slower mating partners when compared with the controls (see Table 34) . The results from fourth (24 days old) to seventh (the last, 42 days old) mating groups among five-day-old flies showed that the treated flies of this age are the faster mating partners only when the ages of the house flies are less than 24 days (see Table 35) . LaBrecque et al. (1962^ reported that male house flies sterilized by feeding on a diet containing 1 percent

PAGE 149

135 apholate were as successful as normal males in competition for mates, and also pointed out that the percentage of sterile eggs laid by females in cages containing normal and chemosterilized males was as high or higher than would be expected from the ratio of sterile males present. Since then, many studies have been carried out on different species, chemosterilants, and applications, following the same pattern as that used by LaBrecque et a_l. (1962a) to determine mating competitiveness. The results of several studies on house flies and other species indicated that the treated males were actually more competitive than the untreated males . Based on the results obtained here, the metepasterilized males, under the minimum dosage for 100 percent sterility, have been shown to be faster mating partners, at least when they are younger than 11 days old. Rate of Chemosterilization of Sperm in Vivo Three-day-old male flies were injected with a dosage of 8 ug/fly (SD 1Q0 ) of metepa-water solution and mated with female flies of the same age. The results of this experiment are given in Table 36.

PAGE 150

136 Table 36. — Rate of metapa chemosterilization of sperm in vivo (three-day-old males treated at the dosage of 8 pg/ male fly, crossed with three-day-old females immediately after injection) ; male flies were injected August 8, 1966. Fly Time from 1st Egging Auq. 10 % of 2nd Egging Aua. 16 % of No. Injection to Mating (Min . ) No. of Eggs No. of Pupae Sterility No. of Eggs No. of Pupae Sterility 1 9 . 120 0 100.00 132 2 98.49 2 20 136 10 92.65 180 28 84.45 3 37 80 0 100.00 93 0 100.00 4 -.41 100 0 100.00 32 0 100.00 5 43 65 0 100.00 79 1 98.75 6 44 63 0 100.00 118 0 100.00 7 60 72 0 100.00 101 0 100.00 8 73 89 0 100.00 114 0 100.00 9 75 162 0 100.00 59 0 100.00 10 76 94 0 100.00 119 0 100.00 11 107 > 128 0 100.00 49 0 100.00 12 162 117 0 100.00 113 0 100.00 13 175 121 0 100.00 35 0 100.00

PAGE 151

137 Table 36.-nExtended 3rd Egging q£ 4th Egging % Qf 5th Egging % Qf Auc t23 ster _ Auc T30 st er SePt. 12 ster _ No. of No. of J*7? No. of No. of JJ£ y No. of No. of Eggs Pupae 1 1 y Eggs Pupae Eggs Pupae 1 1 y 39 2 94.87 36 5 86.11 25 5 80.00 53 0 100.00 62 2 96.77 14 1 92.86 18 0 100.00 81 0 100.00 87 0 100.00 44 0 100.00 78 0 100.00 25 0 100.00 95 0 100.00 72 0 100.00 88 0 100.00 66 0 100.00 58 0 100.00

PAGE 152

138 The rate of metepa chemosterilization of sperm in the male house fly is rather fast. Among the 13 injected male flies, the shortest time after injection that mating occurred was nine minutes; the female mated to this male was 100 percent sterile in the first egging, based on the pupation. For the second, third, and fourth eggings, the sterility was 98.4, 94.87, and 86.11 percent, respectively. The second interval between injection and mating was 20 minutes. The female mated to this injected male produced 92.65 percent sterility in the first egging, based on the pupation; 84.45 percent sterility in the second egging; and 80.00 percent sterility in the third egging. According to the records of this. investigation, based on pupation, there were no fertile eggs produced in the first egging by those females which mated with the treated males within 37 minutes after injection. Further, there was no fertility when those females mated with the male 43 minutes after injection. However, in the other male sterility test, when the copulating time was two or more days after injection, partial restoration of male fertility occurred in the second or third egging. Some sterile sperm can regain fertility after the

PAGE 153

139 sperm are transferred into the females during certain periods, so the degree of sterility can be reduced somewhat according to duration, dosage, and other possible conditions which have been discussed in the section concerning permanence of sterility. Chang (1965a) , in studying the rate of tepa chemosterilization of sperm in male house flies by microinjection technique with a dosage level of one yg/three-day-old male, reported that the shortest time between injection and copulation was 15 minutes; the sterility was 31 percent, based on egg hatching. The time for 50 percent sterility was 23 minutes and for 100 percent sterility about 200 minutes (by extrapolation) . Because of the differences in chemosterilants , dosage, and the criteria for determining sterility, it is impossible to compare the results of others with those achieved here. The distribution of the metepa is rapid and general within insects, as indicated by Dame and Schmidt (1964a, 1964b) . The rate of metepa cheraosterilization of sperm in male house flies is rather fast. Although the C0 2 anesthesia prior to mating may have retarded the process, in this study the time for 50 percent sterility between injection and mating (three-day-old males, COanesthesia,

PAGE 154

140 microinjection with 8 fig/fly metepa-water solution, 26.7°C. [80°F.], 60 percent relative humidity) was less than nine minutes. In addition, the duration of copulation and the time of sperm transfer of house flies should be considered. Chang (1965a) indicated that the duration of copulation of house flies ranges from 34 to 93 minutes, with a mean of 56 minutes and a standard deviation of 12 minutes. Murvosh et al . (1964a) reported that the duration of copulation ranges! from 44 to 96 minutes, with a mean of 60 minutes. Sacca and Benetti (1960) reported that the range is 59 to 136 minutes, and the mean is 84 minutes. Hampton (1952) pointed out that copulation lasts longer than 30 minutes and may last two hours, but the usual time is 1.5 hours. For the time of sperm transfer, Chang (1965a) stated that the insemination process presumably starts as soon as copulation has been physically established. Murvosh et al.. (1964a) pointed out that the sperm transfer does not occur during the first two minutes. Therefore, it is possible that the metepa can affect the sperm during the copulation process of males. Whether those sperm can regain fertility because of being transferred at the beginning of the duration of copulation (shortage of affecting t ime) or because of other factors

PAGE 155

141 cannot be decided; also, for the same reason, the actual affecting time may be longer than the time between injection and mating which occurred in this test. Effects of Chemosterilant Metepa on Longevity The longevity of chemosterilized insects, particularly the males, is also of importance (Smith et aJL., 1964) . Murvosh et _al. (1964b) reported that house flies fed with dry food containing metepa and apholate at 1 percent (w/w) substantially shortened the lifespan; more than 90 percent of the males survived the first ten days; by the tenth day, the greater part of the sexual activity of the males would already have been accomplished. From this point, some reduction of longevity can be tolerated. Many factors can influence the longevity of the house fly. Some research work on this subject has been described in the review of literature. For this experiment, flies (both sexes, individually and collectively) were injected with SD dosaqe at aqes 100 3 from one to five days, with two to four replicates for each treatment. Each treatment involved 73 to 322 flies; altogether there were 4375 males and 4793 females treated and

PAGE 156

142 observed. Mortality counts were recorded daily. Based on the mortality, the longevity of house flies in each treatment was calculated and the survivorship curves for each treatment derived. Longevity of House Flies in Each Treatment The longevity of house flies following each treatment is summarized in Tables 37-43. Because the one-day-old flies were injected one day after their emergence, and the records were taken one day after injection, to the actual mean of the longevity should be added at least one day; for the same reason, two, three, four, and five days, respectively, should be added to the means of those two-, three-, four-, and five-day -old flies treated. Tables 37-43 show only the lifespan after injection. Survivorship Curves in Each Treatment Just as the lethal and sterility dosages for house flies are significantly different between sexes, this difference is equally pronounced with regard to longevity. Consequently, the survivorship curves are derived separately.

PAGE 157

143 O -H 10 • Q* +J o 4J O W Q tT> (0 (0 >, i CJ>H nj fa w \ O t7> Q 3L O CO CJ H (B IP fa Q « w CN CM ro ro CN CO ro CN CM ro ro CN H CO CM rCM CN CO ro cn rH in VO rH ro CN CTi o CO VD * • • • • CO CO CO en vo m CN o VO o CO CN ro CO 00 o • • • • # in in H H rH iH rH CO in m H in ro m CO 00 CO CO 00 iH cn ro Tj" m

PAGE 158

144 rH n O 'H 10 • CU 4J O Q) ro D. U •D O OJ 4J 0) ro 0 rH "0 M 10 c o P 10 "O flj >i c ro +j W D h ro > Q (D — E — D 10 E >i •in ro X Q ro 2 E — 3 10 E >i •3 ro C Q •H ^ s «W (Q 0 >i >. ro CP < CM CM rH VD CD CO >£> rr» rH rH rH rH cc ro cd CP CN CP CD rH in m in r* • • • • in I s CD CD CP CP CO OJ o r*> ro >^ CM in ^ ro rCO O rH CD 0) Q > — co (N in ro
PAGE 159

145 0 -H W rH O • Qi 4J O Q) (0 25 OS O O o 2 ro n o •H •O (0 w a g >i •H> nj X Q ro & 6 — c a en a x w O a a) «h m 5> fa Q CM (N VO CO 10 r» ro r» vO o m 00 CM O 00 t * VO vo VO H CM ro ro ro r» OJ CM m CM vD vD VD CM 00 CM CO <• O in in r ro >i rH o 00 r» ro VD k ro in CM VD vD ro ro o vD a> Q ro O o in ro rin in • • • • • • • • • • H rH CM o o 00 O CO CM CO CM ro CM ro rH ro rH CM r» m ro vo CM CD CD G a rH rH rH rH rH OS 0 d CD 05 CD 0) 05 g rH g rH B rH g rH g CD (C ro CD 05 CD 05 CD VH g VH g «w E l« g 4-1 m

PAGE 160

146 O H 01 H CO • Dh +> O CO (0 2 U 4-4 o o oi +i a) m • -H (U Ohm a fa a o •H P (0 T3 c (0 (0 Q CO «~ 03 (0 >i M f0 CO Q 0 ^ P 01 ' >1 S g CO ^— ' s H I «J 3 £ •H «— » s c (0 01 0) CJ CP X H ro CO 01 W o CT> Q a. O oi >i >i CP fa Q 3 ° CN 3 ^j00 CTi CI O H H CM CN CN CN vo cn cr> o ifl in m co h co CO <* cn o VD H co ^ in h co co ^ in CO CM H ^ in CN H H H CN H CO. H CN CN CO CN in vo CO CO CM co rH H H H cn CO vO o VD rH rH o O o cn CO rcn O H cn cn r» CN H cn r» CN CN 00 in CO o CN CN CN 00 CO • • • • • • • • • t • • Cn cn m VO cn CN VO CN H H CO m H CO O VO w • o CN cn in vO cn CN CN VO CO CN CO CN CN CO • • t • • • • • t • t • cn 00 H VD vO in o CO H H H H H H H H H H CN O VO CO CO a) 0 CO o (0 cn g 3e ag ag ag 3. g a* o o o in in o 55 S5 m a vo S5 H 53 CM

PAGE 161

147 (N IT) vO cn CN H <* (N VO 00 00 rH 00 CN CO in 00 VO o • • • • t • 00 m CN VO rH in in cn rH r» CO m 00 cn rH rH o rin 00 rH CN CN o cn in VO in • • • • • • CO o cn rH o rH rH rH H r-l H CN in CO 00 cn «* cn cn cn in cn in vO H 0) 0 rH H m s E (U 0) VH Q) VH rH \ H \ (0 cn cd cn S a. E a. o o cn a h 0) rH i 0) m h N U cn E oi o rH E c m in

PAGE 162

148 CN CN CN CN CN cn on in 00 in CN CN VO n rH rH in m CO ro rH rH rH rH rH rH rH rH rH rH ro rH C0 in rH rH VO c\ O VO rH in rH CN o VO 00 o o rrH 9t CN CN 00 m en rf CN o CO m rrH vo rH vo CN 00 rH rH rH CN rH rH ro o ro CO 00 r> r» G\ 00 in CO r CM CN o o CT> tr> rH o ro in O r> o VO co rH m 00 H rH ro H CN rH CN CN rH CN oo CO CO o o CO CN in in VO ro VO 00 VO ro in ro H H rH VO rH rH rH CN rH rH CO CO s e \ CD 3. * 00 52! c; rH ro oo 0) 0 CD CD rH
PAGE 163

149 O 'H tQ rH Q CP w P f3 0) Q fa g ? C Q •rH O 0) >i o tr> Q a. «P ^-s O u Odd CP fa P CM o CM o o CM CM CM CM CM in in o o H N W CO CM CM CM CO CM VD VO CP CM — ' in in CO CM ,—| CM CO ,— i rH r-| r-l rH CO r1 CO VO CO rH in m CM CM in CO cn CO CO CP rH vO m p» co CO CM o CM • • • • • # • • I CM co CO CO CM CO rH H rH rH rH rH rH rH CM VO CO in rH CP VO CM r-i cr. VO CP CO r> VO VO in VD o rH CM CP CP VO o CO CP rH CO CM 00 VO • • • • • • • t • CM CO rH CO CM vO in CM rH rH rH CM rH rH CM rH CO in O in CO CM VD CM VO VO in vo in vO VO vO o o H CM m

PAGE 164

150 uh I O -ri ia rH QJ • ft 4J o

4J 0) CO Q fl) ^ (0 >i l-i n> 0 Q 5 (0 a a •5 a O (0 >1 >1 o h m Cn &« Q CM CM CN CN CN CN O o CN CN co CN in cn rH rin CN o o CN CO o CN o CN CO ro o in in «£> in CN O o • • • • • <* m rH rH rH rH VD o o rH CO m in in cn in cn CO rH CN t # • • • CO CO rH co CO CO CO CO cn

PAGE 165

151 Male House Flies The male house flies in this experiment were divided into five different groups: treated male house flies only; normal male house flies only; normal male house flies with normal females; normal males with treated females; and treated males with normal females. Treated male house flies . — The range and mean of the longevity of house flies and their standard deviation are presented in Table 37, the survivorship curves in Fig. 14. The curves show that the lifespan of male house flies after injection is related to their ages at time of treatment. As in the lethal dosage tests, the younger the male flies at injection, the greater the longevity. Regardless of age at time of treatment, the longevity of the treated males is less than that of normal males. Normal male house flies . — In this group, the variations in longevity are much smaller than in any other group (see Table 38) . The curves in Fig. 15 are much closer than in the other figures. Normal males with normal females . — The variations in longevity in this group are greater than in the previous

PAGE 166

152 • — • — • — • 1day-old 2day-old 3 day-old 4day-old 5 day-old 10 20 50 14.— 30 '40 Time in days . Survivorship Curves of Treated Male House Flies 60

PAGE 167

153 •H .C (0 u o > > u 3 W «H O 0) &> m •p c
PAGE 168

154 one. The mean longevity of this group (Table 39) is shorter than that of normal males alone. The survivorship curves (Fig. 16) show that the longevity of male house flies is somewhat related to their age at time of mating. Particularly at the age of five days (the upper age limit considered here) , among male flies mated with normal females of the same age, longevity was shortened considerably. Normal males with treated females . — Because the females were treated in different dosages (based on the SD 10Q in different ages of female flies) , the factors shortening the longevity of the male flies in this group are complex. The survivorship curves (Fig. 17) show the same tendency as those in Fig. 16. The data in Table 40 show that, among males of the same age, longevity is interrelated with the longevity of the treated females which were confined in the same cage. Treated males with normal females . — The data in Table 41 and the curves in Fig. 18 show that there are three subgroups which can be considered: oneand two-day-old male flies; threeand five-day-old male flies; and fourday-old male flies. The average longevity of males in this group is the shortest in the entire experiment.

PAGE 169

155 •H Xi n u o > > U P w «h o 0) 8, M 0 100 90 V iU t ...... 1-day 2day3day4day.*—.5-dayold old old old old 10 20 . " . 30 4( Time in days Fig. 16. — Survivorship Curves of Normal Male House Flies with Normal Female House Flies.

PAGE 170

Fig. 17. — Survivorship Curves of Normal Male House Flies with Females Treated at Different Dosages according to Age: 70 ug/l-day-old; 50 Mg/ 2-day-old; 45 fig/3-day-old; 30 ug/4-day-old; and 20 yg/5-day-old.

PAGE 171

157 •M S H o > > H 3 01 •H O 0) % •P C •»-»< Lis* 10 20 30 Time in days 40 50

PAGE 172

158 100k ...... 1 -day-old _»_« 2 -day-old o—c-.-o , 3-day-old i.--i>.^...> 4 -day-old 5 -day-old I 10 2030 40 50 60 70 Time in days • Fig. 18. — Survivorship Curves of Treated Male House Flies with Normal Female House Flies.

PAGE 173

159 Female House Flies As with the males, the female house flies treated in this experiment were divided into five groups. Treated female house flies . — The data in Table 42 definitely show that the longevity of treated female house flies varies according to the dosage of metepa injected; the higher the dosage, the shorter the lifespan. Because the SD 1Q0 of female flies varies according to the age of the flies, the survivorship curves in Fig. 19 involved two factors: the age of the females and the dosage of metepa. The longevity of one-day-old females injected with 70 ug/fly is very close to that of the two-day-old female injected with 50 ug/fly. The longevity of five-day-old females injected with 20 ug/fly is very close to that of the four-day-old female injected with 30 ug/fly. The three-day-old female injected with 45 ug/fly can survive much longer than those previously mentioned. Normal female house flies . i — The survivorship curve of one-day-old females in Fig. 20 shows some difference from the others. It may be due to this sample size still not being large enough, or to the different brood, or to other reasons. However, as a whole, the survivorship curves are

PAGE 174

Fig. 19. — Survivorship Curves of Treated Female House Flies without Male House Flies, at Different Dosages according to Age: 70 fig/l-day-old; 50 Mg/2-day-old; 45 yg/ 3-day-old; 30 yg/4-day-old? and 20 Mg/ 5-day-old.

PAGE 175

161 100.1day-old 2day-old 3day-old 4day-old 5day-old 10 20 30 40 Time in days 50 60

PAGE 176

162 -10 1day-old 2day-old 3day-old 4 day-old « » •« 5-day-old 0 £>-.. 10 20 30 40 Time in days Fig. 20. — Survivorship Curves of Normal Female House Flies without Male House Flies.

PAGE 177

163 closely related, and the data in Table 43 show only slight differences. Normal females with normal males . — The average longevity of normal females with normal males is shorter than that of normal females without males. The curves in Fig. 21 show that the older the female at the time of first mating, the shorter the lifespan (see Table 39) . Normal females with treated males . — The higher the dosage injected, the shorter the longevity (see Table 40). Comparing the results of this group with the group of treated females without males, it is clear that the longevity of treated females can be influenced by mating with normal males; the reduction in longevity affects not only the treated female but the normal male as well. Treated females with normal males . — When one or both of the partners in mating has been treated, the copulation process differs slightly from the normal condition; very often the mating pair cannot separate, and they finally die together. In addition to the general ideas which were mentioned in each group, some specific characteristics of survivorship

PAGE 178

164 1 -day-old *— *— k— x 2-day-old o-.-^.-o—o 3-day-old 4 day-old 5day-old 10 20 50 60 30 , 40 Time in days Fig. 21. — Survivorship Curves of Normal Female House Flies with Normal Male House Flies. 70

PAGE 179

165 curves are self-explanatory. It is possible to compare each treatment in specific cases according to the survivorship curves. The information can be obtained from the same or different curves in the same figure or in different figures. Since the survivorship curves were derived from the mortality counts, so every point on the survivorship curves can indicate the mortality as well. The number of days required for 50 percent survival (= 50 percent mortality) is, in most cases, very close to the mean of the longevity. Any two points on the same curve clearly indicate the percentage of survivorship (100 percent survivorship = percent mortality) and the number of days (period) between these two points. Any two successive points indicate the death rate for that particular day and treatment. The first and last points of each curve show the range of longevity in that particular case. Taking, as an example, the survivorship curves, 80 percent of one-day-old treated males (injected with 8 pg/v . fly) can live more than 15 days without females, while the five-day-old treated male without females lives only slightly more than five days (Fig. 14) ; 80 percent of one-day-old normal males without females can live more than 18 days (Fig. 15) , while the one-day-old normal males with normal females live more than 15 days (Fig. 16) ; and 84 percent of one-

PAGE 180

166 day-old treated females . (70 tfg/fly) without males can live more than one day (in other words, 16 percent of them can die within one day) (Fig. 23) . Much more information can be derived from the survivorship curves presented in this paper. According to the survivorship curves (Figs. 14-23) of all treatments, the following significant facts can be pointed out: (1) The longevity of the normal virgin female house fly is longer than that of the normal virgin male (Figs. 15 and 20) . (2) The longevity of the mated normal males (normal males with normal females) is shorter than the virgin normal males (normal males without females) ; the variation of survivorship curves of mated males is greater than that of the virgin males (Figs. 15 and 16). similarly, the longevity of the mated females (normal females with normal males) is shorter than that of the virgin females; the variation of survivorship curves of mated females is greater than that of virgin females (Figs. 20 and 21). (3) The longevity of male and female house flies can be influenced by injection of the minimum dosage of SD 10Q of metepa-water solution (Figs. 14, 15, 19, and 20). The slopes

PAGE 181

Fig. 22. — Survivorship Curves of Normal Female House Flies with Male House Flies Treated at the Dosage of 8 ng/Male Fly (Males and Females of the Same Age Were Mated) .

PAGE 182

168 I 90~% V fork 80 TO, \V 1-day-old SSvJSIS *-*-*-< 2 -day-old \ \ o..^..^.^ 3-day-old ^..jl-.j^.a 4 -day-old 70 *£VY\\ t-"-*»* 5 -day-old n n k 60 > •H > 3 (0 H 50 « m P . . c 40
PAGE 183

Fig. 23. — Survivorship Curves of Treated Female House Flies with Normal Male House Flies (Females Were Treated at Different Dosages according to Age: 70 pg/l-day-old; 50 ug/2-day-old; 45 Mg/3-day-old; 30 yg/ 4-day-old; and 20 ug/5-day-old) .

PAGE 184

170 100 s •s u o > •H > 3 0) M-l O o a) o u
PAGE 185

171 of survivorship curves of oneor two-day-old injected flies are much greater at the start than those of the untreated flies (Figs. 14, 18, 19, 23). (4) The longevity of house flies can be influenced by age at time of first mating; the older the flies are at time of mating, the shorter is the longevity (Figs. 16 and 21) . Longevity also can be influenced by mating with injected flies of the opposite sex; the longevity of normal females (or males) mated with normal males (or females) is greater than that of normal females (or males) mated with injected males (or females) (Figs. 17 and 22) . (5) Among flies up to five days old that have been injected with a minimum dosage of SD 10Q of metepa, the variation of survivorship curves for males is smaller than that for females (Figs. 14 and 19); they experience the shortest lifespans among all treatments of both sexes if mated with normal opposite sex flies, and the variation of survivorship curves for such injected females is much smaller than that for such injected males (Figs. 18 and 23) .

PAGE 186

SUMMARY Microinjection technique is the best method for determining the lethal dosage, sterility dosage, and other quantitative evaluations of chemosterilant activity, if the chemosterilant is water-soluble. There is a significant difference in mortality between male house flies and females at dosages higher than 80 Mg/fly; the mortality of male flies was higher than that of females of the same age at the same dosage. The mortality of both sexes is related to age; among flies older than two days, the older the flies at time of treatment, the higher the mortality. One hundred percent mortality was achieved within 48 hours at the rate of 200 Mg/fly. The sterility dosage for female house flies up to five days old is much higher than for males of the same age. At dosages below the level of SD , the percentaqe of 100 sterility is increased by increasing the dosage. The minimum dosage required to induce 100 percent sterility in male flies is 8 Mg/fly; there is little difference with regard to age up to five days; the permanence of sterility at this dosage is about a week. 172

PAGE 187

173 Different ages of female flies display different susceptibility to the metepa-water solution. The dosage for 100 percent sterility in one-day-old female flies is over 64 fig/fly (collective rearing) and below 70 ug/fly (pair rearing) , while the SD^qq for the five-day-old is a little more than 8 pg/fly (collective rearing) , 5 pg/fly (pair rearing egged at fourth day after injection), and 15 Mg/fly (pair rearing egged within nine days after injection) . For comparisons of the dosage-sterility relationship in house flies, time is one of the main factors; delays in the time for injection (age of the fly) , for mating after injection, or for egging after mating could alter the results. The results from the first egging are more stable than those from other consecutive eggings; therefore, it is suggested that only results of first egging be considered for chemosterilizing tests. The 50 go and the SD 10, 50,90 and their slopes were calculated for both sexes at different ages. In the laboratory condition, the female can mate at the age of 50 days; the average number of eggs is smaller and the percentage of sterility in the first egging is much greater when compared with the normal female flies at ages

PAGE 188

174 of 6 to 15 days, but this result could be changed in the second egging or thereafter. A female can lay eggs at the age of 64 days. Among male flies injected with a dosage of 8 fig/fly, only temporary 100 percent sterility could be obtained; the percentage of sterility of treated males decreased in an inverse proportion to the duration of time between injection and mating, or the number of matings and eggings. The sterile sperm can become fertile either in the male reproductive system or after they have been transferred into the female's reproductive system; the degree of restoration of fertility varies according to the age of the male fly at time of treatment; the younger the fly, the greater the restoration of fertility. The metepa-sterilized males (8 Mg/fly) proved to be faster mating partners when compared with the controls; three-day-old sterilized males achieved 50 percent sterility with less than nine minutes between injection and mating. The longevities and survivorship curves of the house flies in different treatments were calculated and derived, and their relations were carefully compared.

PAGE 189

REFERENCES Acree, F., Jr., P. L. Davis, S. F. Spear, G. C. LaBrecgue, & H. G. Wilson. 1959. Nature of attractant in sucrose fed on by house flies. J. Econ. Entomol . 53: 415-20. Afifi, S. E. D., & H. Knutson. 1956. Reproductive potential, longevity, and weight of house flies which survived one insect icidal treatment. J. Econ. Entomol. 49 (3): 310-13. Alexander, P. 1960. Radiationimitating chemicals. Sci. Amer. 202: 99-108. Anonymous. 1962. MAPO, a reactive trifunctional imine. Interchemical Corp., New York. 30 pp. Ascher, K. R. S. 1964. A review of chemosterilants and oviposit ion-inhibitors in insects. World Rev. Pest Control, 3, 7. Awati, P. R. 1920. Bionomics of house flies . II. Attraction of houseflies to different colors. Indian J. Med. Res. 7: 553-59. Barber, G. W., & E. B. Stanes. 1949. The activities of house flies. J. N. Y. Entomol. Soc. 52: 203-14. Baumhover, A. M. 1965. Sexual aggressiveness of male screwworm flies measured by effect on female mortality. J. Econ. Entomol. 58: 544-48. Beroi.a, M., & A. B. Borkovec. 1964. The stability of tepa and other aziridine chemosterilants. J. Med. Chem. 7: 44-49. Bishopp, F. C, & E. W. Laake. 1921. Dispersion of flies by flight. J. Agric. Res. 21: 729-66. Borkovec, A. B. 1966. Insect chemosterilants. J. Wiley, New York. 133 pp. 175

PAGE 190

176 Borkovec, A. B. , S. C. Chang, & A. M. Limburg. 1964. Effect of pH on sterilizing activity of tepa and metepa in male house flies. J. Econ. Entomol. 57: 815-17. Bucher, G. E. , J. W. MacB. Cameron, & A. Wilkes. 1948. Study on the house fly ( Musca domestica L.) . II. The effects of low temperatures on laboratory reared puparia. Can. J. Res. D26: 25-56. Burden, G. S., & B. J. Smittle. 1963. Chemosterilant studies with the German cockroach. Florida Entomol. 46: 229-34. Bushland, R. C. , & D. E. Hopkins. 1951. Experiments with screw-worm flies sterilized by X-rays. J. Econ. Entomol. 44 (5) : 725-31. Bushland, R. C. , & D. E. Hopkins. 1953. Sterilization of screw-worm flies with X-rays and gamma rays. J. Econ. Entomol. 46 (4) : 648-56. Callahan, R. F. 1962. Effects of parental age on life cycle of the house fly, Musca domestica Linnaeus (Diptera: Muscidae) . J. N. Y. Entomol. Soc. 70: 150-58. Cameron, J. W. M. 1938. The reactions of the house fly, Musca domestica L. to light of different wavelengths. Can. J. Res. D16: 307-42. Cameron, J. W. M. 1939. Reaction of house flies to light of different wave-lengths. Nature 143: 208. Chamberlain, W. F. 1962. Chemical sterilization of the screw-worm. J. Econ. Entomol. 55: 240*-48. Chamberlain, W. F., & G. C. Barrett. 1964. A comparison of the amounts of metepa required to sterilize the screw-worm fly and stable fly. J. Econ. Entomol. 57: 267-69. Chang, J. T. P., & Y. C. Chiang. 1964. Studies on insect chemosterilants. III. The sterilizing effect of thio-tepa on the common house fly, Musca domestica

PAGE 191

177 vicina Macq. . Acta Entomol. Sinica 13 (5) : 679688. Chang, S. C. 1965a. Chemosterilization and mating behavior of male house flies. J. Econ. Entomol. 58: 669-72. Chang, S. C. 1965b. Improved bioassay method for evaluating the potency of chemosterilants against house flies. J. Econ. Entomol. 58: 796. Chang, S. C. , & A. B. Borkovec. 1964. Quantitative effects of tepa, metepa, and apholate on sterilization of male house flies. J. Econ. Entomol. 57: 488-90. Chang, S. C. , & A. B. Borkovec. 1966a. Determination of tepa residues on chemosterilized Mexican fruit flies. J. Econ. Entomol. 59: 102-104. Chang, S. C, & A. B. Borkovec. 1966b. Structure-activity relationship in tepa and hempa analogs. J. Econ. Entomol. 59: 1359-62. Chang, S. C. , A. B. Borkovec, & C. W. Woods. 1966. Fate of tepa uniformly labeled with C 14 in male house flies. J. Econ. Entomol. 59: 937?-944. Chang, S. C. , P. H. Terry, & A. B. Borkovec. 1964. Insect chemosterilants with low toxicity for mammals. Science 144: 57r58. Collier, C. W. , & J. E. Downey. 1965. Laboratory evaluation of certain chemosterilants against the gypsy moth. J. Econ. Entomol. 58: 649-51. * Copeman, S. M. , & E. E. Austen. 1914. Repts. Local Govt. Bd. Publ. Health & Med. Subjects, n.s. (I. Do house flies hibernate? 6-26) . Crystal, M. M. 1963. The induction of sexual sterility in the screw-worm fly by antimetabolites and alkylating agents. J. Econ. Entomol. 56 (4) : 468-73. Dakshinamurty, S. 1948. The common house fly, Musca domes tica L. and its behavior to temperature and humidity. Bull. Entomol. Res. 39: 339-57.

PAGE 192

178 Dame, D. A., & R. L . Fye. 1964. Studies on feeding behavior of house flies. J. Econ. Entomol. 57 (5): 776-77. Dame, D. A. , & C. H. Schmidt. 1964a. Uptake of metepa and its effect on two species of mosquitoes ( Anopheles quadrimacultus . Aedes aeqypti ) , and house flies (Muse a dome stic a ) . J. Econ. Entomol. 57 (1): 77-81. 32 Dame, D. A., & C. R. Schmidt. 1964b. P -labeled semen for mosquito mating studies. J. Econ. Entomol. 57 (5): 669-72. Davidson, J. 1944. On the relationship between temperature and rate of development of insects at constant temperatures. J. Anim. Ecol. 13: 26-28. Dunn, L. H. 1923. Observations on the oviposition of the house fly, Musca dome stic a in Panama. Bull. Entomol. Res. 13: 301-5. Fahmy, 0. G., and M. J. Fahmy. 1964. The chemistry and genetics of alkylating chemosterilants . Trans. Roy. Soc. Trop. Med. Hyg. 58: 318-326. Feldman-Muhsam, B. 1944a. A note on the conditions of pupation of Musca dome stic a vicina (Diptera) in Palestine, and its application. Proc. R. Entomol. Soc. London, Ser. A, Gen. Entomol. 19: 139-40. Feldman-Muhsam, B. 1944b. Studies on the ecology of the Levant house fly, Musca dome stic a vicina Macq. Bull. Entomol. Res. 35: 53-67. Freeborn, S. B., & L. J. Berry. 1935. Color preferences of the house fly, Musca dome stic a L. J. Econ. Entomol. 28: 913-16. Fye, R. L., H. K. Gouck, & G. C. LaBrecque. 1965. Compounds causing sterility in adult house flies. J. Econ. Entomol. 58: 446-48. Fye, R. L., & G. C. LaBrecque. 1966. Sexual acceptability of laboratory strains of male house flies in competition with wild strains. J. Econ. Entomol. 59 (3): 538-40. Fye, R. L., G. C. LaBrecque, & H. K. Gouck. 1966. Screening tests of chemicals for sterilization of adult house flies. J. Econ. Entomol. 59: 485-87.

PAGE 193

179 Gouck, H. K. 1964. Chemosterilization of house flies by treatment in the pupal stage. J. Econ. Entomol. 57: 239-41. Gouck, H. K. , M. M. Crystal, A. B. Borkovec, & D. W. Meifert. 1963a. A comparison of techniques for screening chemosterilants of house flies and screw-worm flies. J. Econ. Entomol. 56: 506r9. Gouck, H. K. f & G. C. LaBrecque. 1963. Studies with compounds affecting the development of house fly larvae. Agric. Service Publ., 33-87. Gouck, H. K. , & G. C. LaBrecque. 1964. Chemicals affecting fertility in adult house flies. J. Econ. Entomol* 57: 663-64. Gouck, H. K. , D. W. Meifert, & J. B. Gahan. 1963b. A field experiment with apholate as chemosterilant for the control of house flies. J. Econ. Entomol. 56: 445-46. Graham-Smith, G. S. 1914. Flies in relation to disease: non-bloodsucking flies. Cambridge, Eng. Hampton, U. M. 1952. Reproduction in house fly (Musca domestica L. ) . Proc. R. Entomol. Soc. London (A) 27: 29-32. Harold, 0. 1965. The natural history of flies. W. W. Norton, New York. 324 pp. Harris, R. L. 1962. Chemical induction of sterility in the • stable fly. J. Econ. Entomol. 55: 882-86. Harsham, A. 1946. Debunking a color theory. Food Indust. 18: 851-984. Hayes, W. J., Jr. 1964. The toxicology of chemosterilants. Bull. World Health Organ. 31: 721-36. Herms. W. B. 1928. The effect of different quantities of food during the larval period on the sex ratio and size of Lucilia sericata Meigen and Theobaldia incidens Thorn. J. Econ. Entomol. 21 t 720-29.

PAGE 194

180 Herms, W. D . , & M. T. James. 1961. Medical entomology. Macmillan, New York. 643 pp. Hewitt, G. G. 1914. The house fly, Musca domestica Linn., its structure, habits, development, relation to disease and control. Cambridge University Press, London, Hewitt, G. G. 1915. Notes on the pupation of the house fly ( Musca domestica ) and its mode of overwintering. Can. Entomol. 47: 73-78. Hindle, E. 1914. The flight of the house fly. Proc. Cambridge Phil. Soc. 17: 310-13. Hodge, C. F. 1911. How you can make your home, town or city flyless. Nature and Culture 3: 9-23. Howard, L. 0. 1911a. The house fly, disease carrier: an account of its dangerous activities and the means of destroying it. New York & London. Howard, L. 0. 1911b. House flies. U.S. Dept. Agric, Farmer's Bull. 459. Howard, L. 0. 1911c. Flies as carriers of infection. Science (n.s.) 34: 24-25. Ingle, L. 1943. An apparatus for testing chemotropic responses of flying insects. J. Econ. Entomol. 36: 108-10. Jashi, K. G., & V. R. Dnyansagar. 1945. Some observations on fly breeding in compost trenches. Indian Med. Gaz. 80: 358-61. Jukes, T. H., & H. P. Broquist. 1963. Sulfonamides and folic acid antagonists, in R . M . Hochster & J. H. Cuastel (Eds.), Metabolic inhibitors, Vol. I. Academic Press, New York. Pp. 481-534. Katagai, T. 1935. Seasonal fluctuation of the numbers of Musca domestica L. in city of Taihoku. (In Japanese.) Tokyo Ijishimshi, No. 2929: 1218-23. Keiser, I., L. F. Steiner, & H. Kamasaki. 1965. Effect of chemosterilants against the Oriental fruit fly, melon fly, and Mediterranean fruit fly. J. Econ. Entomol. 58: 682-685.

PAGE 195

181 Kilgore, W. W. 1965. Biochemistry of insect sterilants. Abstract of papers, 150th meeting of Amer. Chem. Soc, p. 264. Kilgore, W. W. , & R. R. Painter. 1962. The effect of 5-f luorouracil on the viability of house fly eggs. J. Econ. Entomol. 55: 710r-12. Kilgore, W. W. , & R. R. Painter. 1964. Effect of the chemosterilant apholate on the synthesis of cellular components in developing house fly eggs. Biochem. J. 92: 353?-57. Knipling, E. F. 1955. Possibilities of insect control or eradication through the use of sexually sterile males. J. Econ. Entomol. 48: 459r-62. Knipling, E. F. 1959. Sterile-male method of population control. Science 130: 902 r-4. Knipling, E. F. 1962. Potentialities and process in the development of chemosterilants for insect control. J. Econ. Entomol. 55: 782-86. Knipling, E. F. 1964. The potential role of the sterility method for insect population control with special reference to combining this method with conventional methods. Agric. Res. Service Publ., 33-98. Knipling, E. F. 1966. Some basic principles in insect population suppression. Bull. Entomol. Soc. Amer. 12: 7. Knipling, E. F. , & J. U. McGuire, Jr. 1966. Population models to test hypothetical effects of sex attractants used for insect control. U.S. Dept. Agric. Inf. Bull. 308. 20 pp. Kobayashi, H. 1935. The influence of temperature upon the development of larvae of Musca domestica . Trans. Dynam. Develop. 10: 385-95. Kobayashi, H. 1940. Passing winter in flies. (In Japanese.) Rept. Jap. Assoc. Adv. Sci. 15: 233-36.

PAGE 196

182 Kohls, R. E. / A. J. Lemin, & P. W. O'Connell. 1966. New chemosterilants against the house fly. J. Econ. Entomol. 59: 745-46. Kramer, S. D. 1915. The effect of temperature on the life cycle of Musca domes tica and Culex pipiens . Science 41: 874-77. LaBrecque, G. C. 1961. Studies with three alkylating agents as house fly sterilants. J. Econ. Entomol. 54: 684-89. LaBrecque, G. C. 1963. Chemosterilants for the control of house flies. Adv. Chem. Ser. 41: 42-46. LaBrecque, G. C. 1965. Status of chemosterilization. Intern. At. Energy Agency Tech. Rept. Ser. 44: 46. LaBrecque, G. C. , P. H. Adcock, & C. N. Smith. 1960. Tests with compounds affecting house fly metabolism. J. Econ. Entomol. 53: 802-805. LaBrecque, G. C. , & H. K. Gouck. 1963. Compounds affecting fertility in adult house flies. J. Econ. Entomol. 56: 476. LaBrecque, G. C, & J. C. Keller (Eds.).1965. Advances in insect population control by the sterile-male technique. Intern. At. Energy Agency Tech. Rept. Ser. 44: 79 pp. LaBrecque, G. C. , D. W. Meifert, & R. L. Fye. 1963a. A field study on the control of house flies with chemosterilant techniques. J. Econ. Entomol. 56: 150-52. LaBrecque, G. C. , D. W. Meifert, & H. K. Gouck. 1963b. Effectiveness of three 2-methylaziridine derivatives as house fly chemosterilants. Florida Entomol. 46: 7-10. LaBrecque, G. C. , D. W. Meifert, & C. N. Smith. 1962a. Mating competitiveness of chemosterilized and normal house flies. Science 136: 388-89.

PAGE 197

183 LaBrecque, G. C. # P. B. Morgan, D. W. Meifert, & R. L. Fye. 1966. Effectiveness of hempa as a house fly chemosteriiant. J. Med. Entomol. 3: 40-43. LaBrecque, G. C. , C. N. Smith, & D. W. Meifert. 1962b. A field experiment in control of house flies with chemosterilant baits. J. Econ. Entomol. 55: 44951. Laing, J. 1935. On the ptilinum of the blow fly ( Calli phora erythrocephala ) . Quart. J. Micro. Soc. 77: 497-521. Larsen, E. B. , & M. Thomsen. 1940. The influence of temperature on the development of some species of Diptera. Repr. fr. Videntsk. Medd. fra. Dansk Naturh. For en. Bd. 104. Lindquist, A. W. (Ed.). 1963. Insect population control by the sterile-male technique. Tech. Rept. Series No. 21, Intern. At. Energy Agency, Vienna. 59 pp. Lineva, V. A. 1953. On the physiological age of female house flies, Musca domestica L. (Diptera, Muscidae) . (In Russian.) Entomol. Obozr. 33: 161-73. Linkfield, R. L. 1966. Biological observations on the Oriental rat flea, Xenopsylla cheopis (Rothschild) with special studies on the effects of the chemosterilant Tris (1-aziridinyl) phosphine oxide. Ph.D. dissertation, university of Florida. Lodge, 0. G. 1918. An examination of the sense-reactions of flies. Bull. Entomol. Res. 9: 141-51. Lorincz, F., & G. Makara. 1935. Observations on fly control and the biology of the house fly. L. 0. N. Health Org., C. H./Hyg. rur./E. H. 5. Geneva (multi graph) . Meifert, D. W. , R. L. Fye, & G. C. LaBrecque. 1963. Effect on house flies of exposure to residual applications of chemosterilants. Florida Entomol. 46 (2) : 16168.

PAGE 198

184 Melvin, R. 1934. Incubation period of eggs of certain Muscoid flies at different constant temperatures. Ann. Entomol. Soc. Amer. 27: 406-10. Michelsen, A. 1960. Experiments on the period of maturation of the male house fly, Musca domestica L. Oikos Hi 250-64. Mitlin, N., B. A. Butt, & T. J. Shortino. 1957. Effect of miotic poisons on house fly oviposition. Physiol. Zool. 30: 133-36. Montgomery, J. A. 1959. The relation of anticancer activity to chemical structure. Cancer Res. 19: 447. Morgan, P. B. , & G. C. LaBrecque. 1962. The effect of apholate on the ovarian development of house flies. J. Econ. Entomol. 55: 626-28. Morgan, P. B. , & G. C. LaBrecque. 1964. Effect of tepa and metepa on ovarian development of house flies. J. Econ. Entomol. 57: 896-99. Murvosh, C. M. , R. L. Fye, & G. C. LaBrecque. 1964a.. Studies on the mating behavior of the house fly, Musca domestica L. Ohio J. Sci. 64 (4): 264-71. Murvosh, C. M. , G. C. LaBrecque, & C. N. Smith. 1964b. Effect of three chemosterilants on house fly longevity and sterility. J. Econ. Entomol. 57 (1) : 89-93. Murvosh, C. M. , G. C. LaBrecque, & C. N. Smith. 1965. Sex attraction in the house fly, Musca domestica L. Ohio J. Sci. 65 (2) : 68-71. Murvosh, C. M. , & C. W. Thaggard. 1966. Ecological studies of the house fly. Ann. Entomol. Soc. Amer. 59 (3) : 533-47. Ouye, M. T. , R. S. Garcia, & D. F. Martin. 1965a. Sterilization of pink boll-worm adults with metepa. J. Econ. Entomol. 58: 1018t20. Ouye, M. T. , H. M. Graham, R. S. Garcia, & D. F. Martin. 1965b. Comparative mating competitiveness of metepa-

PAGE 199

185 sterilized and normal pink boll-worm males in laboratory and field cages. J. Econ. Entomol . 58: 927-29. Parrott, P. J. 1927. Progress report on light traps for insect control. New York. Peffly, R . L. 1953. Crossing and sexual isolation of the Egyptian forms of Musca domestica (Diptera, Muscidae) Evolution 7: 65-75. Peffly, R . L., & G. C. LaBrecgue. 1956. Marking and trapping studies on dispersal and abundance of Egyptian house flies. J. Econ. Entomol. 49: 214-17. Piquett, P. G., & J. C Keller. 1962. A screening method for chemosterilants of house flies. J. Econ. Entomol. 55: 261-62. Plapp, F. W., W. S. Bigley, G. A. Chapman, & G. W. Eddy. 1962 Metabolism of methaphoxide in mosquitoes, house flies and mice. J. Econ. Entomol. 55: 607-13. Puri, I. M. 1943. The house-frequenting flies, their relation to disease and their control. Health Bull. 31, 2nd ed. Simla, India. Quarterman, K. D. , J. W. Kilpatrick, & W. Mathis. 1954. Fly dispersal in a rural area near Savannah, Georgia. J. Econ. Entomol. 47: 413-19. Riemann, J. G., D. J. Moen, and B. J. Thorson. 1967. Female monogamy and its control in house flies. Insect Physiol. 13: 407-18. Robbins, W. E., M. J. Thompson, R. T. Yamamoto, & T. J. Shortino. 1965. Feeding stimulants for the female house fly, Musca domestica Linnaeus. Science 147: 628-30. Rockstein, M., & H. M. Lieberman. 1958. Survival curves for male and female house flies ( Musca domestica L.), Nature 181: 787-88. Rogoff, W. M. 1965. Mating of the house fly, Musca domes tica L., in monitored darkness. J. Med. Entomol. 2 (1): 54-56.

PAGE 200

186 Rogoff, W. M. , A. D. .Beltz, J. C. Johnson, & F. W. Plapp. 1964. A sex pheromone in the house fly, Musca domestica L. J. Insect Physiol. 10: 239-46. Ross, W. C. J. 1962. Biological alkylating agents. Butterwor ths , London . Sacca, G. , & M. P. Benetti. 1960. Ricerche spermimentali sulla maturita e sul comportamento sessuale di Musca domestica L. Rc. Inst. Superiore Sanita 23: 423-32. Schoof, H. P., & R. F. Siverly. 1954. Multiple release studies on the dispersion of Musca domestica at Phoenix, Arizona. J. Econ. Entomol. 47: 830-88. Skinner, H. 1915. How does the house fly pass the winter? Entomol. News 26: 263-64. Smith, A. C. 1956. Fly prevention on dairy operations. Calif. Vector Views 3: 57, 59-60. Smith, C. N. (Ed.). 1966. Insect colonization and mass production. Academic Press, New York. 618 pp. Smith, C. N. , G. C. LaBrecque, & A. B. Borkovec. 1964. Insect chemosterilants. Ann. Rev. Entomol. 9: 269-84. Smith, C. N. , & G. C. LaBrecque. 1967. Insect chemosterilants. Appleton-Century-Crofts, New York. In press. Spiller, D. 1963. Procedure for rearing house flies. Nature 199: 405. Spiller, D. 1966. House flies, in C. N. Smith (Ed.), Insect colonization and mass production. Academic Press, New York. Pp. 203-25. Tao, S. M. 1927. A comparative study of the early larval stages of some common flies. Am. J. Hyg. 7: 735-61. Teissier, G. 1931. Growth measurements. Trav. Stat. Biol. Roscoff 9: 29-238.

PAGE 201

187 Timmis, G. M. 1962. Antagonists of purine and pyrimidine metabolites and of folic acid. Adv. Cancer Res. 6: 369-401. Tung, B. 1965. A preliminary observation on the mechanism of sterilization of the house flies ( Musca vicina Macguart) treated with thiotepa. ACTA Entomol. Sinica 14 (3) : 250-56. Varzandeh, M. , W. N. Bruce, & G. C. Decker. 1954. Resistance to insecticides as a factor influencing the biotic potential of the house fly. J. Econ. Entomol . 47: ; 129-34." Weidhaas, D. E. 1962. Chemical sterilization of mosguitoes, Nature 195 (4843) : 786-87. West, L. S. 1951. The house fly. Comstock Publishing Co., New York. 584 pp. Wheeler, G. P. 1962. Studies related to the mechanism of action of cytotoxic alkylating agents: a review. Cancer Res. 22: 1334-49. Zimgrone, L. D. , W. N. Bruce, & G. C. Decker. 1959. A mating study of female house fly. J. Econ. Entomol. 52: 236.

PAGE 202

BIOGRAPHICAL SKETCH Ku-sheng Kung was born in Wu-chin, Kiangsu, China, on April 3, 1921. He received his primary education at Wu-chin and his secondary education in Foochow, Fukien, China. In the fall of 1940, after one year's teaching work at Nin-hwa, Fukien, he entered Fukien Provincial College of Agriculture, and he received his B.S. degree with major in entomophytopathology in July 1944. He was admitted to the Graduate School of the University of Wisconsin in Madison in April 1956, from which, in July 1958, he received his M.S. degree with major in entomology and plant pathology. He was appointed an assistant in the Fukien Provincial College of Agriculture (1944-1947) , and instructor (1947-1951), associate professor (1951-1955), and professor (1955-1956) in the Taiwan Provincial College of Agriculture, Taichung, Taiwan, China. In 1958 the Taiwan Provincial College of Agriculture became Chung Hsing University, and Kusheng Kung was named chairman of the Department of Agricultural Education at that institution, serving in that capacity 188

PAGE 203

189 until 1961, when he became chairman of the newly established Department of Entomology. Ku-sheng Kung came to the University of Florida in October 1964 under the Fulbright Fund as a visiting professor in the Department of Entomology. In October 1965 he applied for and obtained an assistantship and entered the Graduate School of the University of Florida to begin work toward the Ph.D. degree with major in entomology. Ku-sheng Kung is married to the former Miss Hungyin Lei, and is the father of four children. He is a member of the Society of Alpha Zeta, Phi Sigma, Entomology Society of China, Taiwan Entomo-Phyto-Pathological Society, Agricultural Society of China, and the Agricultural Educational Society of China.

PAGE 204

This dissertation was prepared under the direction of the chairman of the candidate's supervisory committee and has been approved by all members of that committee. It was submitted to the Dean of the College of Agriculture and to the Graduate Council, and was approved as partial fulfillment of the requirements for the degree of Doctor of * Philosophy. August 12, 1967 Dean, College of Agriculture Dean, Graduate School Supervisory Committee t Co-Chairman fa