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Biological observations on the oriental rat flea, Xenopsylla cheopsis (Rothschild), with special studies on the effects of the chemosterilant, tris (1-aziridnyl) phosphine oxide

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Title:
Biological observations on the oriental rat flea, Xenopsylla cheopsis (Rothschild), with special studies on the effects of the chemosterilant, tris (1-aziridnyl) phosphine oxide
Creator:
Linkfield, Robert Loomis, 1930-
Publication Date:
Language:
English
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101 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Chromosomes ( jstor )
Egg production ( jstor )
Eggs ( jstor )
Female animals ( jstor )
Fleas ( jstor )
Larvae ( jstor )
Mating behavior ( jstor )
Ovarioles ( jstor )
Rats ( jstor )
Spermatozoa ( jstor )
Beneficial insects ( lcsh )
Dissertations, Academic -- Entomology and Nematology -- UF
Entomology and Nematology thesis Ph. D
Insect pests ( lcsh )
Insecticides ( lcsh )
Oriental rat flea ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1966.
Bibliography:
Includes bibliographical references (leaves 70-77).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Robert Loomis Linkfield.

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BIOLOGICAL OBSERVATIONS ON THE ORIENTAL RAT FLEA, Xenopsylla cheopis (ROTHSCHILD), WITH SPECIAL STUDIES
ON THE EFFECTS OF THE
CHEMOSTERILANT TRIS (1-AZIRIDINYL)
PHOSPHINE OXIDE






By
ROBERT LOOMIS LINKFIELD










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
December, 1966















ACKNOWLEDGEMENTS


The author wishes to express his sincere gratitude to the Chairman of his Supervisory Committee, Dr. J. T. Creighton, Department of Entomology, University of Florida, and the Co-Chairman, Dr. P. B. Morgan, United States Department of Agriculture, for their advice and assistance.

For their criticism of the manuscript, appreciation is also

expressed to committee members Dr. F. S. Blanton and Mr. W. T. Calaway, and to Dr. A. M. Laessle who read for Dr. Archie Carr.

Special thanks are due to Dr. C. N. Smith for the use of facilities at the U.S.D.A. Entomology Research Division Laboratory; to Dr. G. C. LaBrecque for his suggestions and encouragement; and to all the U.S.D.A. staff who assisted.

Dr. William Mendenhall, Chairman of the Department of Statistics,

assigned Mr. Marcello Pagano to aid inecertain statistical analyses; the Computer Center allotted 15 minutes of computer time. Their cooperation is gratefully acknowledged.

Last, but not least, appreciation goes to my wife, Ethel, who typed the preliminary copies of this manuscript and who gave affectionate encouragement throughout the course of this study.









ii
















TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS.. . . . . . . . . . . . . . . . . . . . . . . . . ii

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

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

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

REVIEW OF LITERATURE ........... . . . . . . . . . . . . 5

Chemosterilants . . . . . . . . . . .. . . . . . . . . . . . . . 5
Biology . ........ . . ....... . . . ... ...... 10
The adult . . . . . . . . . . . . . . . . . ... . . . . . . 10
Temperature and humidity . . . . . . . . . . . . . . . . 10
Light sensitivity . . . . . . . . . . . . . . . . . . . . 11
Feeding . . . . . . ... . . . . . . . . . . . . . . . 11
Mating .. . .... .. . . . ... . . .... . . 12
Miscellaneous . . . . . . . . . . . . . . . ... . . . . 13
Reproductive system .............. ..... . . . 13
Female . . . . . . . . . . . . . . . . . . . . . . . . . 13
Male . . . . . . . . . . . . . . . . . . . . . .... 15
The egg . . . . . . . ............ . . . . . . . . . . 15
Egg production .. ... . . . . . . . . . . . . . . . . . 15
Longevity . . . . . . . . . . . . . . . . . . . . . . . . . 18
The larva . . . . .. . . . . . . . . . . ... . . . . . . . 19
Prepupa and pupa ............. .... . . . . 20
Prepupa . . . ... ... .... . . . . . .... . . . . . 20
Pupa . . . . . . . . . . . . . . . . . . . . . .. . 21

METHODS AND MATERIALS '. . . . . . . . . . . . . . . . . ... . . 22

Rearing of X. cheopis . . . . . . . . . . . . . . . . . . . 22
Cytological techniques . . ...... . . . . ....... 22
Experimental design ................. ... . 24
Flea populations for experimental use . . . . . . . . .. . 24
Chemosterilant treatment . . . . . . . ... . .. , . 25
Sterility experiments . . . . . ... . . ... . . . . . . . 28
The emergence chamber ......... .. . . ..... . 33
Statistics . . . . . . . . . . . . . . . . . . . . . . . 33
Photography . . . . .. . . . . . . . . . . . . . . . . . 35









Page

RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 36

Biological Observations . ....... .. ....... . . . . 36
Ovarioles . . . . . . . . . . ... . . . . . . . . . . . . 36
Egg production . . . . . . . . . . . . . . . . . . . . . . . 36
Ratio of male to female .. . ................ 39
Behavior ................. . . . . . . . . ........... 39
Parthenogenesis .... . . . . . . . . . . .. . . . . . . . 46
General observations . ... . . . ... . . . . . . . . . 48
Life cycle . . . . . . . . . . . . . . . . . . . . . . .. 48
Larval and F1 adult emergence . . . . . . . . . . . . . . 48
Feeding site . ...... . . . .... . . . . . . . . . 48
Chemosterilization with Tepa .. . . . . . . . . . . .. ..... . 50
Egg hatch .... . . . . . . . . . . . . . 50
Natural sterility . . . . . . . . . . . . . . . . . . . . . 50
Egg breakage . . . . . . . . . . . . . . . . . . . . . . . . 50
Female ste ilization . . . . . ..... . . . . . . . . . 52
.5 mg/cm . . . . . . . . . . . . . . . . . . . . . . . . 52
10 mg/cm2 . . . . . . . . . . . . . . . . . . . . . . . . 53
Male sterilization . . . . . . . . . . . . . . . . . . . . . 53
5 mg/cm2 . . . . . . . . . . . . . . . . . . . . . . . . 56
10 mg/cm2 . . .. . . . . . . . . . . . . . . . . . . . . 56
15 mg/cm2 . . . . . . . . . . . . . 56
Inability to achieve total male sterility . . ........ 57 Treated females X treated males . . . . . . . . . . . . . . 59
Cytological effects of tepa . . . . . . . ... . . . . . . . 60
Field application . . . . . . . . . . . . . . . . . . . . . 67
Biology . . . . . ...... ........... . . . . 67
Chemosterilization . ...... . . . ... . . . . . . 67

S IARY . . . . . . ...... ... . . . . . . . . . . . . . . . .. ..... 68

REFERENCES CITE . . . . . . . . . . . . . . . . . . . . . . . . . 70

APPENDIX . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 78

BIOGRAPHICAL SKETCH . . . . . . . . . ... . . . . . . 99














iv















LIST OF TABLES


Table Page

1 Measurements of gonads of 30 male and 30 female X. cheopis
adults . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

2 Ratio of female to male in laboratory-reared and in fieldcollected X. cheopis adult populations . ........ ... 41

3 Production and development of eggs from virgin female X.
cheopis which had continuous access to blood meals . . . . . 47

4 Summary of results from experiments using tepa in varying
dosages and lengths of time for sterilization of X. cheopis
adults . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

5 Five untreated females mated with five untreated males . . 79

6 Five males treated with tepa (5mg/cm2 for 6 hrs.) and mated
with five untreated females . ............... 80

7-10 Ten untreated females mated with ten untreated males . . . . 81-84

11 Ten females treated with tepa (5mg/cm2 for 4 hrs.) and mated
with ten untreated males . ........ ........ . . 85

12 Ten females treated with tepa' (5mg/cm2 for 6 hrs.) and mated
with ten untreated males . ..... ............ 86

13-15 Ten females treated with tepa (10mg/cm2 for 4 hrs.) and mated
with ten untreated males . . . . ................ . .. 87-89

16-18 Ten males treated with tepa (5mg/cm2 for 6 hrs.) and mated
with ten untreated females .... .. ..... . . . . . .. 90-92

19 Ten males treated with tepa (10mg/cm2 for 4 hrs.) and mated
with ten untreated females . . ... . . . . . . . . . . 93

20-22 Ten males treated with tepa (10mg/cm2 for 6 hrs.) and mated
with ten untreated females ......... . . . . . . . . 94-96

23 Ten males treated with tepa (15mg/cm2 for 6 hrs.) and mated
with ten untreated females . . . ............. . 97


v









Table Page 24 Ten females treated with-tepa (5mg/cm2 for 6 hrs. and mated with ten nales treated with tepa at 10mg/cm for
4 hrs. ........ ............ ....... 98
















































vi















LIST OF FIGURES


Figure Page

1 Female ovarioles showing primary oocyte (A); oocytes in
various stages of development (B); germarial region (C);
and terminal filament (D). 130x . ...... ... . . . . . 14

2 Male. (A) testis; (B) sperm; (C) epididymis; (D) vas
deferens; (E) paired accessory glands. lllx . ...... 16

3 Vacuum aspirator used to sex adult fleas .. ... ... . 26 4 Pinning board used for treating filter paper strips . 27

5 VacucelR ice chest used to maintain darkness and humidity
for experimental mice and fleas in Mason jars. (A) black organdy cloth; (B) water compartment; (C) compartment for
Mason jars . . . . . ... . .. .. . . . ... ... . .. 30

6 Incubator. (A) whisper fan; (B) sponge and water container for maintaining humidity; (C) petri dishes containing eggs and growing larvae; CD) temperature-humidity
probe; (E) temperature-humidity recorder . ........ . 32

7 Adult emergence chambers . .... ... ..... . . . 34

8 Average and predicted daily egg production per X. cheopis
female from 12 experiments of 14 days each . ....... 37

9 Average egg production per female for 28 days based on that
of 10 females in one control experiment . ... . . . 40 10 Av :age egg production of mated and of virgin X. cheopis females which had continuous access to blood meals .. . 43 11 Bottom view of egg of X. cheopis adhering to bits of rat chow. 44x . . ......... ........ . ... .. . . 44

12 Top view of X. cheoois egg (A) which is stuck to the filter paper, and surrounded by bits of rat chow. 44x . . 45 13 Rate of egg-laying and percent emergence of larvae and F1 adults ................ . .... . .. . . . 49


vii









Figure Page

14 Ovarioles of fleas treated with tepa at 10 mg/cm2 for
4 hours. Note destruction of ovarioles, and vacuolation. 130x . ..... ........ . ... ........ . . 54

15 Spermatheca of a female flea treated with tepa at
10 mg/cm2 for 4 hours. Spermatheca head (A) contains
motile sperm. 120x . . .. . .............. . 55

16 Ovarioles of female fleas treated with tepa at 5 mg/cm2
for 6 hours. (A) remnant of o6cyte. 130x . ...... . . 61

17 Untreated female mitotic metaphase chromosomes (2n=18)
from the brain of the prepupa. 1667x .... . . . . . . 62

18 Metaphase chromosomes from tepa-treated larval brain.
(A) chromosome stickiness; (B) inter-arm chromatid exchange aberration. 1667x . . . . . .......... . . 64

19 Metaphase chromosomes from tepa-treated larval brain.
(A) chromosomal constriction. 1667x .. ........ . 65

20. Metaphase chromosomes from tepa-treated larval brain.
(A) chromosomal fragments; (B) isodiametric fragment;
(C) dicentric chromosome. 1667x . .. . . ..... .. 66


























vift















INTRODUCTION


The oriental rat flea, Xenopsylla cheopis (Rothschild), is

recognized as a pest all over the world (Buxton, 1941, and Atlas of Plague, 1952). It can be found on ship rats throughout the year (Newstead and Evans, 1921). Hirst (1927) gave the following account of its dispersal:

X. cheopis is able to develop apart from its hosts' nest, so
that it is capable of being much more easily dispersed in
material such as grain, the debris of which affords nourishment for the larvae. Thus grain not only serves as a means of transport for rats and fleas from place to place, but is the most suitable medium for the multiplication of the most
efficient insect vector of plague, X. cheopis, and the
principal carrier of both disease and flea, the grain-eating
Mus rattus.

According to Wu et al. (1936), plague, Pasteurella pestis (Lehman and Neumann), was first recorded in the sixth century A.D., starting in Egypt in 542, and finally spreading to Constantinople. This pandemic lasted for almost sixty years, and approximately 100,000,000 people died. In Europe in 1348, a plague pandemic, termed "the Black Death," killed 25,000,000 people (Herms, 1961). The plague pandemic which began in Hong Kong in 1894 was carried to many parts of the world through trade routes. Herms (1961) states that rats, infected by rat fleas, and transported in commercial goods, are the chief spreaders of the disease, with the plague likely to appear in a city "far removed from the original focus of infection."









From 1900 to 1952, cases of plague were recorded in thirty-nine countries (Atlas of Plague, 1952). In twenty-seven of these countries X. cheopis was one of the principal vectors. From 1945 to 1952, there werc i.irty-seven cases reported from the seaports of various countries.

Plague in the United States was first reported from San Francisco in 1900 (Herms, 1961, and Jellison, 1959). The epidemic ended in 1904, but reappeared in 1907. Other cities which have suffered from the disease include Seattle in 1907, New Orleans in 1912, several Gulf Coast cities in 1920, and Los Angeles in 1924.

The theory that rats in commerce are the principal carriers of

plague would seem to be confirmed bythe fact that most of the recorded plague.epidemics began in seaports.

At present, sylvatic plague is, endemic throughout the western

United States, and in the provinces of Alberta and Saskatchewan, Canada. It has been determined from thirty-eight species of rodents and lagomorphs. According to Meyer (1947), the most prolific carriers of plague are the Sciuridae. It was discovered in 1908 that plague was no longer confined to rats and rat fleas in North America, but had become established in the ground squirrel, Citellus beecheyi (Richardson) and in its fleas. McCoy (1910) reported on plague in ground squirrels in California. He warns:

In the suburbs of towns and cities, rats and squirrels come into-very close contact and it would seem very easy to have
the disease carried from the squirrels to the rats, and as
a result have a general infection of the cities.

X. cheopis is one of the vectors of murine typhus, Rickettsia mooseri Montiero, transmitted by fleas, with the rat and squirrel populations serving as reservoirs between epidemics. The cycle in this






3


instance is from animal to flea to animal to flea to --now and then-man. The body louse, Pediculus humanus humanus Linnaeus, carries European typhus directly to man, at the cost of fatal infection to the louse itself. The whole series of Rickettsial diseases, superficially so much alike, differ dramatically when considered epidemiologically. The transmission mechanism varies considerably from one disease to the other. There is no common vector, although piercing-sucking arthropods are invariably involved. There is even less uniformity in the degree to which an animal reservoir is necessary to keep the infection alive, and in whether or not the vector itself succumbs to the disease. Chemosterilant programs would seem to be ideally suited for work against all types of the Rickettsiae.

The tapeworm, Hymenolepis diminuta (Rudolphi), the larvae of which develop in X. cheopis and other fleas, can complete its life cycle in man or in any susceptible animal which might ingest the infected flea.

Up to the present time, X. cheopis has been controlled only through the use of insecticides or control of the rodent host. With the advent of radiation and, chemosterilants possible eradication of this and similar species can be envisaged.

No literature could be found relating tothis type of work, but literature concerning radiation and chemosterilants used on other species of insects was studied so that a basic, practical pattern of research could be planned.

Although radiation was considered as a possible effective means of sterilization, one would not like to release thousands of fleas in any given area. Since fleas are pestiferous whether or not they are diseased, there is definitely an advantage in setting up stations for






4


sterilizing rats. Before the initiation of such a program, however, it would. be essential to know what effects the chemosterilant might have on the fleas of the rat, especially whether or not feeding on the sterilized rat would in turn sterilize the flea. It was decided, therefore, to test the effects of the chemosterilant tepa on the Oriental rat flea.

X. cheopis, as is obvious from the preceding account, is still among the most important potential enemies of mankind. The basic purpose of this study, therefore,.was to determine if the flea can be sterilized, if its sterility and mortality doses are close, and how it is affected by the chemosterilant tepa.

Since the effective use of chemosterilants depends upon a thorough study of the species to be sterilized, the literature was reviewed, and careful observations were made concerning the biology of X. cheopis.















REVIEW OF LITERATURE

Chemosterilants


Radiation-imitating chemicals, termed "radiomimetic" by Dustin (1947), have been known for some years. It was not until the Second World War, however, with work on poison gases, that they were shown to be possible cancer cures (Alexander, 1960). The radiomimetic compounds, as in radiation, cause the following cellular effects: l1) mutation of genes and permanent changes in chromosomal structures;

(2) izerference with cell division processes which can cause death of the cell; (3) outright death of certain types of cells; C4) cancerous growths.

Fahmy and Fahmy (1958) found that alkylating agents (radiomimetic compounds) cause an amount of small deficiencies almost double those of mutagenically equivalent doses of X-radiation. These deficiencies are caused by failure in gene reproduction in situ. and not by chromosome breakage and reunion as in radiation. Alexander and Stacey (1958) suggest that the difference in biological action between radiation and radiozr.imetic compounds is that the latter have to diffuse into the cell making some of the DNA (deoxyribonucleic acid) molecules more accessible to the compound than are others.

A chemical which causes sexual sterility is called a chemosterilant. Weidhaas (1963) defines .it further:



5






6


---the term chemosterilant is restricted to compounds which
prevent production of sperm or ova, kill sperm or ova that have already been produced, or damage chromatic or genetic
material in the sperm or ova so that zygotes, if formed, do
not develop into mature progeny.

Two groups of chemical compounds which have shown great promise as sterilants of insects are the antimetabolites and the alkylating agents (LaBrecque, 1963 and 1965). An alkylating agent, the chemosterilant tepa causes replacement of a hydrogen atom with an alkyl group on an organic molecule. Its structural formula is the following:


0
H2 C I1 ,CH2
IN-P-N
H2 1 CH 2

H 2C--CH



According to Duvall (1960), tepa is very hygroscopic, being

"extremely soluble" --although unstable-- in water, as well as being "very soluble" in alcohol, in ether, and in acetone. Solutions of tepa and one of the three diluents kept at about 50C will remain comparatively stable for a week. Duvall reported the LD50 in mice to be 47.0 mg/kg/d by mouth, the maximum tolerated single dose being 75.0 mg/kg/d.

Hayes (1964) reported that rats injected intraperitoneally with a dosage of 0.2 mg/kg/d of tretamine (2,4,6-tris(i-aziridinyl)-S-triazine) remained sterile up to at least 8 weeks after the last dose. An intraperitoneal dosage of 0.05 mg/kg/d of tretamine caused sterility in male rats. Fertility returned 3-4 weeks after the last dosage. Sexual behavior of the sterilized male was normal, as was the number and motility of the sperm.






7


The compounds presently available are not very stable (Smith, 1963a, and Hayes, 1964), and more information is needed as to their toxicity and application before expanded programs can be undertaken (Smith, 1963b; Smith et al., 1964; and Barnes, 1964).

Mitlin et al. (1957) obtained sterilization in house flies by using mitotic poisons. Three of the four chemicals tested usually inhibited oviposition, and prevented ovarian growth.

In 1958, LaBrecque began screening chemical compounds for their chemosterilizing effects. Of 2,000 compounds initially tested, 5 caused sterility in the house fly, Musca domestica Linnaeus, when placed in its food.

By 1963, 2,000 chemicals had been tested, 40 of them causing

sterility, according to LaBrecque (1963). LaBrecque reported further in 1965 that 112 chemicals have been found to produce sterilant effects.

Several field tests have been conducted against the house fly with the chemosterilants tepa (trisCl-aziridinly)phosphine oxide), metepa (tris(2-methyl-l-aziridinyl)phosphine oxide), and apholate (2,2,4,4,6,6hexakis(l-aziridinyl)-2,2,4,4,6,6-hexahydro-l,3,5,2,4,6-triazatriphosphorine). In a refuse dump at Bahia Honda Key, Florida, LaBrecque et al. (1962) conducted the first field test using tepa against the house fly. As a result, the adult fly populations were reduced from 47 to zero per grid count within 4 weeks with the use of cornmeal baits containing

0.5% tep4. Female flies trapped at the dump were checked for egg. masses and viability. Egg masses had decreased from 100-10% within

4 weeks, and within 5 weeks egg viability had decreased to 1%. Metepa at 0.5% was applied in bait to droppings in a poultry house for control of the house fly CLaBrecque et al., 1963) with similar results.






8


In 1963, Gouck et al. conducted a test in a refuse dump at Pine isla: , Florida, using a cornmeal bait of 0.75% apholate. They obtained sporadic results probably due to introduction of fresh flies from garbage trucks. Nevertheless, a reduction of flies did occur from 68 per grid count to between 5 and 20 for the first 7 weeks..

When bait was made available continuously, the population then decreased to between 3 and 0 per grid count.

Morgan and LaBrecque (1962 and 1964b) reported on the effects of apholate, tepa, and metepa on the ovarian development of house flies. In general, these compounds were found to inhibit ovarian development. The chromatin of the nurse cell nuclei was clumped in irregular masses.

LaBrecque et al. (1966) achieved 99-100% sterility in the male

house fly at concentrations of hempa Chexamethylphosphoramide) as low as 0.25%. Female sterility was often as high as that of the male, although it varied.

Through the use of gamma radiation the screw-worm fly was eradicated from the island of Curacao, and from the southeastern United States. Recent research has been directed toward the use of chemosterilants to control this fly (Knipling, 1962). Chamberlain (1962) obtained sterility in adult screw-worm flies with apholate; however, he achieved only partial sterility in the pupae. In 1963, Crystal induced sterility in the same fly, with antimetabolites and with al.ylating agents. Of 29 compounds tested, 26 caused sterility when incorporated into the diet, and 12 by topical application. In 1965, he furi;er reported that the chemical N,N'-tetramethylenebis(1-aziridinecarbox..ide) was far superior to gamma radiation in sterilizing the screw-worm fly.









The yellow-fever mosquito, Aedes aegypti (Linnaeus), and the common malaria mosquito, Anopheles quadrimaculatus Say, can be sterilized with chemosterilants in either the larval or the adult'stage (Weidhaas et al., 1961; Weidhaas, 1962; Dame and Ford, 1964a; and Dame et al., 1964b,c). Apholate concentrations as low as 0.1%, when fed in the diet, reduced to zero the fertility of eggs laid by A. aegypti, while 0.5% completely eliminated egg fertility in A. quadrimaculatus.

When larvae of A. aegypti were treated with 10 p.p.m. of tepa, all of the emerging adult males, and almost all of the females, were sterile. Apholate at 10 p.p.m. was not effective. Residual tests demonstrated that tepa at 680 ug/ft2 gave complete sterility in both species of mosquito.

Glancey (1965) reported that, when fed in a honey solution, 0.5% hempa induced 100% sterility in female A. aegypti, and a 0.1% concentration produced 97% sterility in the male. He also found that residual deposits of 200-500 mg/ft2 for 4 hours produced 90% sterility in the male.

Apholate affects the female A. aegypti reproductive system almost the same as in house flies (Rai, 1964 and 1965). Rai stated that the egg f'ilicles of treated females were underdeveloped, and eventually degenerated; in some instances the follicular epithelium, eggs, and nurse cells degenerated.

Burden and Smittle (1963), testing twelve different chemosterilants on the German cockroach, Blatella germanica (Linnaeus), found that some of the compounds caused the oothecae to be deformed, resulting in delayed and/or reduced hatch. Smittle 1964) conducted tests with tepa on the reproductive organs and embryogenytof this cockroach. He found






10


lt 4 and 3 ug of tapa injected into adult male roaches produced complete sterility, although it inhibited neither mating nor sperm motility. Egg hatch was reduced 96% when 5 ug of tepa wasinjected into seventh-instar female nymphs. This chemosteriiant caused the male testes to atrophy, and the basal oocytes of females to be smaller than those of controls. Females mated with sterile males produced o6thecae; however, no hatch occurred.

Testes and ovaries of the eye gnat,'Hippelates pusio Loew, were reduced in size when treated with tepa, metepa, or apholate (Schwartz, 1965). The g..-,rium of the female ovarioles was the most severely affected; the germarial region of the male, void of spermatogonia, was almost collapsed. Sterility of 99-100% was obtained with 0.01% tepa,

0.1% metepa, and 0.5% apholate.

Tepa at 10 mg/ft2 affected bird malaria, Plasmodium gallinaceum Brumpt, in A. aegypti when the mosquito was subjected to the residue either before or after feeding on infected chicks (Altman, 1963). The chemical caused reductions in the number of infected mosquitoes, the mean oocyst count, and the rate of transmission.


Biology

Thi adult

Hirst (1926) was the first to report that emergence of X. cheopis fr:7. its cocoon is stimulated by vibration. The vibration can be mechanical, but breathing on the cocoons will also cause the adults to

emerge.

Temp-rature and humidity.--At lower temperatures, unfed adults live longer than at hi',er ones. Buxton (1948) concluded that at tempera::res from 24-320C, the adult flea could survive..









The life cycle of the flea wil ,vary according to the temperature and humidity. Hopkins (1935) reported that the life cycle could be acco:.plished in chout 36-63 days, the minimum being 42 days, at a :perature f 200C, and a relative humidity of 100%. Krishnamurthy et al. (i963) found that, at a temperature between 25-280C, and a relative hu..idity between 75-80%, the life cycle could be completed in 24-29 days.

The spiracles control the loss of water, according to Mellanby (1934), who found that in adult fleas exposed to 5% C02, the rate of water loss is doubled because the spiracles are permanently open. HKmidity is somewhat unimportant, then, since the flea at rest keeps most of its spi 'acles closed (Wigglesworth, 1935).

Buxto.n (1948) states that the adult flea is hardly affected by humidity, a : in its natural habitation the water loss would probably be made up at the next feeding. Smith (1951) found that high humidities cause adult female activity to increase to the extent that the female needs frequent blood meals.

Lighc sensitivity.--Adults of X. cheopis are photonegative.

(Mitzm-in, 1910, used the term "negatively heliotropic" while Pausch, 1962, described this condition as "negatively phototactic.") Edney (1945) reported that adults kept in the dark live slightly longer than those in the light. This is true even if the pre-adult stages have been passed in the dark. Smith (1951) states that fleas of the genus Xenopsylla are repelled by strong light, and that they are most active it. air which is saturated or nearly so.

Feedinc.--There is no difference in size of newly emerged adults which have not fed several times (Edney, 1945). Once 'fult fleas have






12


fed, they must continue to do so or else they will die (Hirst, 1923).

Sflea which has ingested a blood meal usually will not feed again

until the following day. The adults are persistent feeders, and will

. e feing even though they are engorged, to the extent that blood is excreted through the anus and falls down to the substrate. This habit (or behavior) is probably stimulated by adenosine triphosphate, which is found in the host's blood (Galun, 1966). The average Jult will engorge approximately 0.5 cc of blood at one feeding, according to Reports on Plague Investigations in India C1907). Maximum feeding was obtained with fleas 5-8 days old by Bar-Zeev and Sternberg (192). It was found that starved females ingest more blood than starved males. Both Mitzmain (1910) and Pausch (1962) say that the female must have a blood meal before copulating or laying eggs.

Fox et al. (1966) found that X. cheopis will feed on invertebrate as well ; on vertebrate blood. Placed on a Puerto Rican lizard, Anolis cristatellus cristatellus, the fleas began to feed either immediately or within 5 minutes. The longest life of an adult in the experiment was 36 days, while the minimum was 4 days. Under the conditions o'f this exrrime:. no progeny was obtained.

Sin-.--Suter (1964) described pictorially the copulatory

positions of male and female. He reported that before copulation the femal s need several blood :.eals; however, this is not true of the males which, in fact, can copulate immediately after emerging from the pupae, one male being able to mate with several females. Transfer of a arm takes place in about 10 minutes., Mating may take place on the host, in the r.;, or in the bedding.






13


s ~: ~ : cs.--S::ith (1951) stated that in unfed female fleas up tc one day oCl, there was no daily rhythm of activity.

Shi*e ,v and ,Naor (1964) reported that X. cheopis females from

5-10 days old were attracted more readily to male white rats than at any other age. In fact, for the first 3 days of their experiment the femaie'fleas were repelled.


Reprod:ucive system

Fe:ale.--In the adult female an ovary lies on either side of the mid-intestine. Each ovary consists of from 3-6 panoistic ovarioles; that is, ovarioles which lack specialized nutritive cells CFigure 1). (Among the holometabolous insects, the Siphonaptera is the only order which possesses the panoistic type of ovariole, according to Bonhag, 1958.) Normally the germarium in the ovariole contains many o6gonia. Here an occasional oogonial cell will undergo mitosis to form young oocytes. These odcytes move into the vitellarium or zone of growth

where prefollicular tissue surrounds them. This tissue then becomes the follicular epithelium of the primary oocyte. The primary oScytes, then, are separated from each other by an interfollicular zone of tissue. The o~cyte nucleus is called the germinal vesicle. Surrounding the ovariole is a thin outer epithelial sheath. Each ovariole has a terminal filament which unites to form the ligament of the ovary. The ligament is attached to the pericardial diaphragm.

The lateral oviducts from each ovary unite to form the common

oviduct which opens at the vagina located between the eighth and ninth abdominal sternites. The spermatheca lies below the rectum, and the spermat.hczal duct opens into the front wall of the vagina.






14











































Fig. 1.--Female ovarioles showing primary odcyte CA); o6cytes in various stages of development CB); germarial region C): and terminal filament (D). 130x.









In normal circumstances, the flea has onlyone spermatheca, but Sharma and Joshi (1961) have reportedi'an adult with two. The extra one was pear-shaped with no distinct head portion, whereas the other was of the normal hook or sickle shape.

Male.--The adult male hah one pair of oval-shaped testes which

have one layer of epithelial tissue [Wasserburger, 1961). At the base of each testis is a cup-like structure which contains a coiled duct or epididymis (Gunther, 1961). From this coiled 'duct comes the vas deferens which enters the seminal vesicle (Patton and Evans, 1929) or ejaculatory bulb (Gunther, 1961). It then enters the ductus ejaculatoris from which it proceeds to the aedoeagus. There are two pairs of accessory glands (one short and one long), which unite with the vas deferens near the testes (Figure 2). The egg

The egg of X. cheopis is oval, and when newly laid it is pearly white in color and slightly translucent. It is viscous, and adheres to the substrate (Mitzmain, 1910). The length is from 0.450 mm to

0.475 mm, and the width is 0.3 mm (Bacot and Ridewood, 1914, and Webster, 1929). There are 56.micropylar openings at the anterior end and 32 at the posterior end (Cameron, 1939). Egg production

Egg production in the female seems to vary. Patton and Evans

(1929) state that the female may lay from 2-6 or more eggs at a time, and that she is capable of laying from 300-400 eggs during a lifetime. Mitzmain (1910) reported that the majority of eggs obtained were laid on the first day, and that the average number was 6. In 1908, the






16








































Fig. 2.--Jale. CA) testis; CB) sperm; QC) epididymis; (D) vas deferens; CE) paired accessory glands. lllx.






17


Reort orn Plague Investilgaions in India reported that from 1-5 eggs t a zim;e are laid, one after the other; however, another of these rr;ots C(S12' indicated that the average nu; bur laid per wild flea in; r :.hly perio s for two and one half years ranged from 0.6-3.0. Their figure 'was based, however, on only one hour of egg-laying time. This report also states that the largest number of eggs was laid during the first hour after separation from the rat hosts.

In 1i35, Hopkins reported that with a "perch" Cresting material) t;.e average egg production per laying female was 3.8, while without a '"perch" it was 4.6. Pausch C1962) obtained an average of 2.2 eggs per fed female in his experiments.

Buxton (1948) found that fleas produced an abnormally.low number of eggs when fed on mice 9-11 days old; however, they laid more eggs when allowed to feed on adult mice for several hours before being placed on baby mice. He was of the opinion that the younger mice lack a sex hormone which stimulates egg production in the female flea. Rohschild, however, stated in 1965 that X. cheopis is not dependent on rat . x hormones. If the rat has been castrated, and its adrenal and pituitary glands removed, the flea fed upon it will still copulate and lay eggs, although it is not known if the eggs will produce progeny.

Luxton also found that higher temperature produces earlier egglaying and a greater quantity of progeny per day, and that progeny from parents fed on the adult mouse emerge sooner than those fed on the

Zaoy mouse.











uZ.ouh.s (c953) discovered that if X. cheonis adults were fed

cn a u..ea uig, zhe lived longer than those fed only once or

all. One ::.ale floa survived for 72 days; the maximum survival time wuas 153 days for a female which had had daily access to a rat. Burroughs concluded that:

The opportunity to obtain frequent blood meals had the
oroatest effect of any single factor in producing individual
-znc- iw-a fZeas.

This is also the opinion expressed by Leeson in 1936.

It is interestn to note that Bacot stated in 1914 that X. chersis may live for up to 10 months.

In 15-3, Hirst made reference to the fact that fleas held in glass tubes should.have some type of resting material. Hopkins (1935) conducted tests to determine if the use of resting material would prolong the life of theflea, nd whether or not it was conducive to egglaying. .e found that without a "perch" the average life for the male was 5.6 days and for the female 9.9 days. With a "perch," the average was 6.2 days for the male and 7.1 days for the female.

Some wild-c aght fleas, anesthetized with chloroform, had a

shorter mean longevity than did those not anesthetized. Crowding of larvae, which reduces adult longevity, also causes a reduction in the size of the adilts (Edney, 1947b).

Longevity of Fl adults is affected by the age of the parent fleas (Edney '7b). Those from parents less than 10 days old live longer thu. those frcm parents more than 11 days old.

Accordi..g to 7urroughs (1953), adults





19


opposite of Edney's conclusion (1947b) that:

Well developed, unfed, laboratory-bred adult fleas livelonger than those which have fed once before starvation,
and the latter live longer than those which have fed
several times.

X. cheopis, unlike many other parasitic insects, does not need to remain upon its host. A part of the adult life is spent in the nest of the host.


The larva

The larva hatches from the egg in from 2-10 days, depending on the temperature (Bacot and Ridewood, 1914, and Patton and Evans, 1929). The external morphology of the larval instars has been fully described by Bacot and Ridewood (1914), Henderson (1928), and Sikes (1930). The following general account of the larval stage is condensed primarily from writings of Bacot (1914), Bacot and Ridewood (1914), and Sikes

-(1930).

The newly hatched larva emerges from the egg with the aid of a hatching spine or egg-breaker which is located on the epicranium. It is about 1.4-2.0 mm long, cylindrical, semi-transparent, and about'0.22 mm thick. Living upon almost any kind of refuse, animal or vegetable, this larva will even feed on drops of dried blood which the adult flea has defecated.

It undergoes two molts during the active stage, and one in the

cocoon. While molting, it will lie straight, except in the third molt when it is bent to form a U-shape. In most instances the larva is very active; however, there are times when it will become quiescent and lie coiled up.




20


When the temperature is between 25-280C, and the relative humidity between 75-80%, the larval period is from 13-15 days

CKrishnamurthy et al., 1963).

The rearing media should not be wet, but the humidity should be between 75-90% for best larval growth. In 1949, Sharif reported that for the larva the critical humidity varies with the temperature. He demonstrated (1948a) that the chief source of water gain in the larva is through its food, and further concluded that the water requirement is slightly less for the male than for the female larva.

The nutritional requirements for the larva are simple according to Sharif (1948b). It can even live in the domestic rat burrow where the nutritive value of larval food is low. Pausch (1962) readily grew larvae on an artificial diet of 100 parts vitamin-free casein, 2 parts Wesson salt mixture, 1 part cholesterol and 9 vitamins.

Edney (1947b) reported that if too many larvae are reared together, even though there is sufficient food, the adult longevity is reduced. He also found that if larvae are reared in fine rather than course sand, the adults are unable to develop to their normal size. The fine sand abrades the larval epicuticle, causing water evaporation which cannot be regained even in high humidity. Prepupa and pupa

Prepupa.--Hopkins (1935) reported that the female has a shorter. larval period than the male. The fully grown larva is creamy white in color, and the alimentary canal is devoid of food. It then spins a cocoon of silk and debris. It is active at first, and any sudden stimulus or rise in temperature will cause it to leave the cocoon Cjellanby, 1933). Under normal circumstances the larva soon ceases






21


movement, and remains folded double within the cocoon. This is the prepupal stage.

If kept' at a temperature of 240C and a relative humidity of from 50-90%, the prepupa will gain weight by the absorption of water vapor from the air (Edney, 1947a). A relative humidity below 45% will kill it. The prepupa molts to the pupal stage in about 3.5 days.

,ua.--Under the rearing conditions presented by Krishnamurthy

et al. (1963), the pupal stage was from 6-8 days. Mellanby (1933) stated that, although the pupa is resistant to dry air, the prepupa is not, so that it is necessary to maintain a humidity of at least 60% or higher, along with a temperature of between 18-350C.

Normally the pupal period of the female is shorter than that of the male (Hopkins, 1935). Edney (1947a) found that adults which have emerged from ,pupae kept at higher humibities weigh more, have a higher percentage of water content, and live longer without food than those emerging from pupae kept at lower humidities.

Sharif (1935) discovered that the pupae of the fleas Nosopsyllus fasciatus (Bosc), Leptopsylla segnis (Schonherr), and Ceratophyllub gallinae (Schrank) have wing buds on the mesothorax; he could find none on X. cheopis.















METHODS AND MATERIALS


Rearing of X, cheopis

The Orlando strain of X. cheopis was used in all experiments. The fleas were mass-reared according to the methods of Smith and Eddy (1954) and of Gilbert (1964). Approximately 500 newly emerged adult fleas were put on a caged rat in a rearing pan containing approximately 1.5 liters of dry sand and 100 cc of powdered dog food. The rat was fed daily with pieces of dog food and apple. Approximately 21 days later it was removed, and the contents of the pan sifted through 8- and 16mesh screens to remove debris and cocoons. The sand containing eggs, larvae, and adults was then placed in an enamel pan and covered with organdy cloth. This pan was placed in an incubator where the temperature was 28�20C, and the relative humidity was 80�5%. The sand was sifted through the 16-mesh screen once a week for 3 weeks in order to obtain the cocoons.


Cytological techniques

Studies were undertaken to determine the effects of the chemosterilant tepa on flea chromosomes. Before proceeding with these studies, however; it was necessary to determine what tissue of the flea contained the most mitotic chromosomes, and at which stage of the flea's development. Many experiments proved that the best object for these studies was the supraoesophyageal ganglioh of the prepupa.


22






23

Under a dissecting microscope at 30X, the prepupa was dissected out of the cocoon with two pairs of extra-fine forceps in the modified Ringer's solution of Morgan and LaBrecque (1964a). The prepupa was then transferred to a deep-well slide containing Ringer's solution. The prothorax was held firmly with one pair of forceps while the head capsule was gently pulled away with the second pair. The supraoesophyageal ganglion, easily observed as two white oblong lobes attached to the prothoracic ganglion, was then severed from the ganglion with the forceps, and transferred for 10 minutes to a 1.0% hypotonic solution of sodium citrate. After being placed in modified Carnoy's fixative for 5 minutes, it was removed to a small drop of 45% acetic acid on a clean microscope slide which had been washed in acid alcohol. The tissue was covered with a siliconized cover slip.

Placed between the folds of a piece of filter paper (150 mm), the covered slide was then gently but firmly tapped over the tissue area 20 or 30 times with a moderately heavy rod. After this, the slide, still wrapped in filter paper, was squashed with a chromosome squash apparatus (Linkfield et al., in press), after which it could be quickly scanned under the phase microscope for.mitotic divisions.

The chromosome squash apparatus is based on the fulcrum principle, the pressure being supplied by a 25-pound lead weight. The slide containing the tissue to be squashed is placed under the pressure plate within a folded piece of filter paper. Using the apparatus at 166 pounds of force (216 lbs. psi for a 7/8" x 7/8" cover slip), excellent preparations were made.

The slide at this stage was placed on a piece of dry ice for a minimum of 30 minutes. Staining and permanent mounts were then made according to the technique of Morgan and LaBrecque (1964a).






24


To determine what cytogenetic effects tepa has on the somatic

chromosomes, a test was initiated using 20 larvae which were ready to pupate. A filter paper with a diameter of 9 cm was treated with tepa at 10 mg/cm2, and allowed to dry for about 16 hours. The larvae were placed on the treated filter paper for 4 hours. They were then removed to untreated paper for a 24-hour period. After this time, the supraoesophageal ganglions of several larvae were prepared according to the procedures previously described. Experimental design

CTo achieve maximum readability, the word "treat" in all its parts of speech is used only in reference to the use of the chemosterilant.)

Male laboratory white mice from 30-40 days old were used as hosts in all experiments.

A preliminary test showed the following procedures to be the most expedient for all experiments.

Flea populations for experimental use.--Prior to the treatments, newly emerged adult fleas were collected in a one-quart Mason jar containing a 9 cm filter paper upon which the fleas could rest. A piece of black organdy cloth was placed over the mouth of the jar before the rim was screwed on. The fleas were kept in the dark until they could be sexed, which was usually within 24 hours. Ether, U.S.P., was poured into a screw-top, ten-dram glass vial which contained a crumpled sheet of KimwhipeR paper, type 900-S. When not in ue, the vial was kept in an explosion-proof refrigerator.

On the second day, the adult fleas were sexed. The glass vial

containing ether was slowly lowered into.the Mason jar containing fleas, and the jar lid was screwed on tight until all activity had ceased,






25


after which time the vial was removed. The anesthetized fleas remained inactive for about 10 minutes. Approximately 30 fleas were poured into a plastic petri plate top which was then placed under a zoom binocular microscope and viewed at 10X. A suction apparatus (Fig. 3) was used to pick up the fleas. Male and female fleas were put into 'separate onequart Mason jars, each of which had a 9 cm filter paper placed on the bottom. The adult fleas were held in the dark for recovery from the anesthetic.

Chemosterilant treatment.--Whenever chemosterilant treatments were being conducted, rubber gloves were worn, and treatments were made in a negative-air-flow hood.

Filter paper strips (Fig. 4) used in all experiments were 7.3 cm2 similar to those reported in the World Health Organization Technical Report of 1963. Before treatment,,the paper strips were securely'stuck to a pinning board (Fig. 4). A small galvanized enamel pan was partially filled with cider vinegar (or 3% acetic acid), and a vial containing the chemosterilant,tepa dissolved in methanol was set in the pan. A pro-pipet was fitted to a 1 ml pipet, and 0.13-0.J6 ml of the chemosterilant solution was applied to the filter paper strips at a dosage of either 5, 10, or 15 mg/cm2

With a clean pipet, 0.13-0.16 ml of methanol was applied to all

control strips. Both groups of strips remained in the hood for 24 hours to dry.

After treatment of strips, cider vinegar was poured into the chemosterilant vial in order to deactivate the remaining active material before the vial was discarded. The pipet and all other reusable materials which came in contact with the chemosterilant were






26








































'Fig. 3.--Vacuum aspirator used to sex adult fleas.






27

































S4--Pinnin board used for treating filter er stris.







Fig. 4.--Pinning board used for treating filter paper strips.






28


thoroughly soaked in cider vinegar for 5 minutes before being washed in soapy water.

The treated filter papers were picked up with a pair of forceps, and placed in 18 x 150 mm test tubes. Twenty fleas were placed in each of 5 tubes, the open ends of which were covered with a piece'of white organdy cloth held in place by a cardboard collar. Depending on the type of reciprocal cross experiments to be conducted, either males or females were treated with the tepa. The fleas were held in the tubes for varying lengths of time (2, 4, or 6 hours).

After the required treatment time, the treated fleas were transferred to post-treatment test tubes each of which contained a clean filter paper strip. Controls were transferred to like tubes at the same time. The open end of the post-treatment test tube was placed below the treated one at an angle of about 40 degrees. The treated tube was gently tapped until the fleas fell into the clean tube. If the treated filter paper started to slide toward the clean tube, the tubes were separated and held upright while the treated paper resettled on the bottom, after which the procedure was repeated until all fleas were in the post-treatment tube. Sometimes a few fleas could not be dislodged from the paper strips by this method; however, when the tubes were held close to a light, the fleas would begin to move, at which time they would be tapped into the clean tube. These steps were taken. in order to avoid contamination of an aspirator.

Contaminated test tubes and filter paper strips were handled as previously described for contaminated material.

Sterility experiments.--Treated adult fleas were held in the dark for 24 hours, after which they were checked for mortality. Those not dead were used for determining which dosage effected sterility.






29


In initial sterility experiments, 5 male and 5 female fleas were used, but in subsequent testings it was decided to use 10 fleas of each sex. In order to demonstrate sterility, the following reciprocal crosses were made:

i. Untreated females X Untreated males

2. Untreated females X Treated males 3. Treated females X Untreated males

4. Treated females X Treated males

Mice confined in small, round, wire cages were lowered into onequart Mason jars with an unwound paper clip of the larger size, bent to hook at one end: The bottom of each jar contained a 9 cm filter paper, along with a strip of blotter paper to absorb the mouse's urine.

An equal number of both sexes of fleas was put into a labelled

Mason jar with the caged mouse. The jar was then placed in the larger side of a double-compartment VacucelR ice chest (Fig. 5). About two quarts of water were poured into the smaller compartment. Black organdy cloth was draped over the top of the ice chest in order to maintain darkness and as much humidity as possible.

The caged mouse was removed from the Mason jar daily, fed, placed in a freshly prepared jar, and returned to the chest. It should be noted that, when feeding the mouse,.care had to be taken not to lose fleas. The mouse cage was picked up with the paper clip hook in one hand, grasped with the fingers ofthe other hand, and quickly transferred to the new jar, with food being given to the mouse through a hole in one end of the cage before it was completely lowered into the jar. In most instances the fleas were around the tail area of the mouse, but a bulb-aspirator was 'kept at hand when making transfers in case.a flea would jump off.






30

































LA








Fig. 5.--VacucelR ice chest used to maintain darkness and humidity for experimental mice and fleas in Mason jars. (A) black organdy cloth; CB) water compartment; (C) compartment for Mason jars.





31


The primary reason for placing the mice in cages was to prevent them from eating the adult fleas, as was reported by Leeson C1936). Cameron (1939) stated that when fleas were first introduced upon a healthy mouse, it ate many of them.

The used Mason jars were taken to the binocular microscope to look for flea eggs. It was found best to removethe papers from the experimental jar before aspirating out any fleas to be transferred to the clean jars. In order to position the blotter and filter papers for removal from the jar, the paper clip hook was used, with extreme care being taken not to damage the eggs.

The blotter paper was removed with a pair of forceps and placed in a plastic petri dish cover 100 x 15 mm. It was then scanned under the binocular microscope at 10X. If eggs were found, a drop of modified Ringer's solution was put on the paper close to the egg, softening the paper and allowing the egg to be picked up with ease by the use of fine needle-nosed forceps. The egg is less likely to be damaged by this method, as some of the paper is removed with it.

After the top.of the blotter paperhad been scanned, the reverse side was checked. This same procedure was carried out on the filter paper, and on all droppings and debris left in the bottom of the jar.

The flea eggs were placed on a 3.8 cm black organdy cloth disc in a plastic petri dish,'which was then set in an incubator (Fig. 6). The incubator was kept at 26�20C, with a relative humidity of 86�2%. A whisper fan was installed to provide air circulation, and the humidity was maintained with a sponge partially immersed in distilled water in a plastic food container. The entire humidity unit was changed weekly, or sooner if necessary. The temperature and humidity were checked daily




















A








C r















Fig. 6.--Incubator. CA) whisper fan; (B) sponge and water container for maintaining humidity; (C) petri dishes containing eggs and growing larvae; (D), temperature-humidity probe; CE) temperature-humidity recorder.









with m e aid of an electric hygrometer indicator (Hygrodynamics, Inc.). The t.. te-hu :idit~~~ "- probe remained in the incubator throughout the resir g period.

es3 wr .Z:.ckld C. for hatch or 6 days. Larvae were pic.,e up :1. a very fine paint brush (#C0), and placed in a dated plastic npcrri 21:Ze containing sand and ground-up rat chow. The petri iaele, mar ,ca wit:. to dac.te on which the eggs were laid, was placed in the inc ao. .Rt the be inning of the third 'week an emergence chamber Was .1,i ce cra t; .ean media so that emerging adults could not sc a n3.

~~The aerzgnce chc ber.--The bottom was punched out of a one-pint car board ice cream container, and the top ridge trimmed off with a razor blade. The unridged top was then gently inserted into the petri dish containing larval medium and maturing larvae. The container was fastened to the petri plate with cellophane tape, and organdy cloth was s cured to the top with a rubber band. These containers made ideal adulz emergence cham-bers [Fig. 7). When adults began to emerge, the or jL.y cloth ;:as reoovc. and they were aspirated out of the chamber each day.

Statiftics.--The negative binomial transformation of Bartlett , 47) was us. :o stabilize .the variance of the original egg production data. The pooled "t" test of Snedecor [1962) was then used to

the dizorence in egg production between first and second weeks Control an. Cf c:uosterilant experiments.

-e aired 'ZJ" test of Menennhall C1964) was .ed to determine the

S ene in cr rnce ol larvae and F1 adults in control and chemozszril: &: e::peri:ne.ts. Percent sterility was figured acceding to the

-d of Crysail (Il6l .






34








































Fig. 7.--Adult emergence chambers





35



The predicted egg production curve was calculated using the formula y=K(l-e-Yt) where y= egg production; K= asymptotic constant; e= 2.7183; dr= growth constant; and t= time.

Photography. --Photomicrographs were made through an American

Optical Phasestar microscope with Ortho-illuminator. They were taken on 135 mm Panotomic X, Black and White film (ASA 32), and the green filter of the Ortho-illuminator was used.















RESULTS AND DISCUSSION

Biological Observations


Ovarioles

Wasserburger (1961) stated that he regularly found 5 ovarioles in each ovary of the female X. cheopis and Patton and Evans (1929) 4-6. While measuring the ovarioles and testes of the Orlando strain of the flea (Table 1), this author found ovaries with 3, 4, and 6 ovarioles each. Although none was found with 5 ovarioles, they could very well have been present in the Orlando strain. The variation in ovariole number could explain why there was some difference in egg production from one test to another, especially in one control test. There was no difference in overall egg production between the first and second weeks of 12 experiments Ct=1.657

Egg production

Figure 8 shows the daily egg production per female for 14 days, based on the average number of eggs counted daily from 120 females. Apparently all the primary obcytes are not mature at initial egg-laying. After about 4 days the ovarioles become synchronized and egg production increases probably close to its maximum. The mean daily egg production per female was 4.25�.21.

A growth curve (Fig. 8) was calculated using the least squares

method. It was based on the egg production per day in 12 experiments. The predicted daily egg production constant was 4.75 eggs per female.

.36











5


















* "t\~PREDICTED EGG PRODUCTION 1












0
e o e x p erimenPREDICTED EGG PRODUTION u..
























DAY






38














Table 1.--Measurements of gonads of 30 male and 30 female
X. cheopis adults.




Mean Range Standard Dimension iin mm n mm Deviation


MALE

Length of:
Right testis .62 .53-.70 .014 Left testis .63 .49-.70 .020 Width of:
Right testis .34 .32-.39 .007 Left testis .33 .28-.35 .007 FEMALE

Length of:
Ovariole .66 .53-.77 .015 Width of:
Primary oicyte .10 .07-.14 .008





39



The egg production per female from 10 females in a control experiment is shown in Fig. 9. This experiment, which was continued for double the initial 14 days, showed a higher egg production than any other. T.e increase is possibly due to the fact that almost all of these happened to be females with more ovarioles than the other experimental females. Nevertheless, the data are valid and informative.

It is interesting to note that the highest daily egg production was 12.6 eggs per female. These females laid 80 or more eggs-on 15 different days, while less than 80 eggs were laid on the other 13 days.


Ratio of male to female

The ratio of male to female adult fleas is very near 1:1. Bacot et al. (1914) proposed that in nature the females of X. cheopis are in excess of males by 15.05%. Their specimens were collected from host animals in the field. As can be seen from Table 2, their assumption is borne out in a calculation of the sex ratio in 2 of the author's experiments. Here all fleas were sexed and counted, and females were found to -e in excess of males by 13.66%. Apparently males remain on the host as often as do the females; during observations by this author, male fleas were found mainly on the host. The only time they were seen off the host was tXen they were with some of the females on the filter paper.


Behavior

Accor.ling to Wigglesworth (1964), the hormonal control of reproduction might be stimulated by the act of copulation, as in the cockroach, DiFloDtera punctata. In this species, mating appears to be nec-.- for the normal rate of egg growth. This might also be true of X. cheonis.









12






10








w






tu




4








2 4 6 8 10 12 14 16 18 20 22 24 26 28 DAY
Fig. 9.--Average egg production per female for 28 days based on that of 10 females in
one control experiment-





















. --Laio of fE aJ e to male in laborator*-rea ad and in field-colleczed
. :~o-:s adui populations.




PERCENTAGE OF

excess females
s ales over males Laboratory-reaeOda

53.46.80 13.66

Field-collectedb

0 46.50 15.05 ; :rgng - adlts in 2 of the author's Ioratory experiments.

20 fleas collected in several Egyptin 1>ai, and exr:ined by Bacot et al. (1914.






42


Norris (1954) found that the female Schistocerca gregaria must copulate in order to lay her full complement of eggs. Bacot (1914) reported that in his experiments no X. cheopis female laid any eggs without having fed first. He also stated that oviposition has always been preceded by several days of feeding.

Copulation, however, does not appear to be necessary for egglaying (Fig. 10). The stimulating factor for both copulation and egg-laying, then must be blood meals. Females which have not had a blood meal will neither copulate nor lay eggs. Nevertheless, virgin females will lay eggs at least.for a period of 14 days, and probably for longer if they have access to the necessary blood meals. The number of eggs produced decreases with time, but does not appear to cease completely.

There seems to be a difference in egg-laying behavior between mated and virgin females. Mated females will lay their eggs in clusters, or sometimes singularly, on or underneath the filter paper, on mouse feces, or on bits of rat chow. iln most instances these eggs will have bits of rat chow attached to them (Fig. 11). They are difficult to see (Fig. 12) without correct lighting. The surface of the egg has a sticky substance which, when dry, adheres the egg to the substrate, and bits of rat chow to the egg.

In view of the preceding observations, it is difficult not to

believe that the female fleas have actually placed bits of the rat chow around the newly laid eggs; as many of these covered eggs can be found several inches from the main area of debris. Even if they had fallen from the mouse onto the rat chow bits, it is unlikely that they could have ro led so far away and almost impossible for them to have adhered















MATED FEMALES













Lu
mJ


. 3

LU





2 - *
.



* VIRGIN FEMALES









1 2 3 4 5 6 7 8 10 11 12 13 14




I 2 374 5 8 9 10 11 12 13 14


Fig. l0.--Average egg production of mated and of virgin X. cheopis females which had
continuous access to blood meals.






44













































bits of rat chow. 44x.






45
































A












Fig. 12.--Top view of X. cheopis egg (A) which is stuck to the filter paper, and surrounded by bits of rat chow. 44x.






46


to the filter paper. Closer observation of egg-laying, and further research into this probable behavior, would be most interesting.

Eggs from virgin females do not have this sticky substance, since their eggs can easily be removed from the substrate with a dry paint brush or with forceps. No virgin females were observed off of the host even though mated females were often seen on or under the filter

paper.

Copious feeding causes extrusion of blood from the anus, providing nourishment for the larvae. The placing of food around the egg could be a behavior pattern for providing food for the emerging larvae, in which case the virgin female is unable to provide it. This behavior, as well as the production of the sticky substance, could be caused-by stimulation of the female collaterial glands during copulation. There is also the possibility that the male inseminates a substance during copulation which stimulates the female to initiate these behavior patterns.


Parthenogenesis

According to White C1964) there appears to be no literature

regarding parthenogenesis in the Siphonaptera (Aphaniptera) and in five other orders of insects.

In the virgin female egg production experiment, it is interesting to note that of 149 eggs produced in 14 days, 4 underwent partial embryonic development (Table 3). In one egg the head capsule was easily found, while in the others only setae were observed. These embryos did not hatch. Suomalainen C1962) called this rudimentary parthrOogentis; that is, when an unfertilized egg from a bisexual species starts to develop, it can be expected eventually to stop




















Table 3.--Production and development of eggs from
virgin female X. cheonis which had
continuous access to blood meals.




Daya Eggs Produced Embryos Formed Hatch


1 25 0 0 2 21 0 0 3 19 0 .0 4 17 1 0 5 18 1 0
6 7 0 0 7 6 0 0 8 5 1 0 9 10 1 0 10 4 0 0 11 2 0 0 12 3 0 0 13 5 0 0 14 7 0 0

Totals: 149 4 0


aEggs were first observed on the 3rd day after placing fleas on host.






48


development and die. Sometimes, however, an unfertilized egg will hatch and produce an adult; this is called tychoparthenogenesis (accidental parthenogenesis). In certain species of Drosophila the rate of parthenogenetically developing adults was 2/37,628 and 1/19,059 (Suomalainen, 1962).

Since it has been shown that embryonic development can take place in eggs laid by virgin X.' cheopis females, further experiments into the possibilities of tychoparthenogenesis should be initiated. General observations

Life cycle.--The author, under the conditions of his experiments, found that the life cycle could be accomplished in 15 to 21 days at a temperature of 26�20C, and a relative humidity of 86�2%. By 28 days emergence was complete. Each experiment took approximately 6 weeks to complete.

Larval and F1 adult emergence.--Figure 13 shows the daily percent total number of eggs deposited, and larval and F1 adult emergence from the 4 control experiments. The total larval emergence was 81.09%, while the F1 adult emergence was 90.12%.

Feeding site.--Bacot (1914) stated that rat fleas pick a special "point of vantage, X. cheopis making for the shoulders, neck and chest or for a spot beneath the forelegs." This author, however, observed that the fleas in all his experiments were usually to be found around the tail and 'scrotal area of the caged mouse. In fact, newly introduced fleas went directly to the posterior end. Additional studies would no doubt show whether or not this is the preferred feeding site. It is possible that, since mice usually clean themselves quite well around these areas, they eat many of their pests found there, and this is why









100 - 100
F, ADULTS




S* *. . .0 . ."' "
80 0 0 * . .0 0 0 . - 80
- * LARVAE 0* 0*
0U 0


*.. C
o *
UU
0 60 60 0

0


0 m





20 - 20
S 2 3 4 5 6 7 8 9 10 11 12 13 14








D40
z 0

I-"



20 -20 EGGS





1 2 3 4 5 6 7 8 9 10 11 12 13 14 DAY

Fig. 13.--Rate of egg-laying and percent emergence of larvae and F adults.






50


fleas on uncaged mice were found in the areas mentioned by Bacot, even though they prefer the other.


Chemosterilizationwith Tepa

Egg hatch

Eggs from female fleas which had been either treated, or crossed

with a treated male, were retained for 6 days, after which, if there had

nt no larval emergence, they were discarded. The control larvae

usually had emerged by the third day. Data on egg-laying and on larval and F_ adult emergence for all of the chemosterilant experiments and for the controls are summarized in Table 4. Tables 5 through 24 in the Appendix contain the results of each individual experiment. Natural sterility

The natural sterility obtained in 4 laboratory control experiments was 18.45% in the larvae and 10.14% in the Fl adults. Egg breakage

In an initial sterility experiment many eggs were broken in an effort to remove them from the filter paper. Since 34 eggs from the control alone were broken in the first 8 days, it was decided to try Ringer's solution in order to soften the filter paper and allow easier removal of eggs. On days 9-14 the solution was used. Only 6 eggs were broken during this 6-day period. It appeared that with Ringer's solution egg breakage would be reduced considerably and larval hatch improved. Therefore a statistical analysis using the paired ""' test wqs m e. Since Riinr's solutlsn was fond by this Analysis to improve hacch CZ=2.833 > Z05=1.645), it was used to remove eggs from the filter aperer In all experiments.











Table 4.--Summary of results from experiments using tepa in varying dosages and lengths of time for sterilization of X. cheonis adults.
Ten males and 10 females were used in each 14 day experiment.


RANGE AVERAGE NUMBER OF % Sterility
Based on
Treatment Eggs Emerging Eggs _ Emerging Erergence of

Dosage Contact Time
mg/cm2 hours Produced Surviveda Larvae F1 Adults Produced Surviveda Larvae F1 Adults Larvae FI Adults Treated female X untreated male:

5 4 3-45 3-45 1-38 0-21 22.00 21.00 16.00 8.00 69.20 84.00 5 6 4-24 4-24 0-14 0-11 6.00 6.00 4.00 2.00 - 98.20 99.28
10 4b 3-12 3-11 0-1 0-0 0.60 0.60 0.07 0.00 99.99 100.00 Treated male X untreated female:

5 6b 12-64 12-64 0-0.30 0-0.33 44.00 43.00 0.2 0.05 99.00 99.99
10 4b 6-60 6-57 0-9.00 0-4.00 39.00 37.00 4.0 0.80 85.30 99.50
10 6 13-59 13-59 0-1.30 0-0.67 42.00 42.00 0.2 0.10 99.20 99.99c
15 6 5-50 5-50 0-2.00 - 32.00 _32.00 0.1 - 99.99 Treated female (F) X treated male (M): S(F) 0-5 1-4 0 0 0.70 0.60 0 0 100.00 100.00 10 (M) 4
Untreated female X untreated male: CONTROLd 13-58 12-56 10-48 5-45 46.00 44.00 36.00 32.00 18.46 10.14 aSome eggs were found broken, or were broken in handling. Average of three replicates.

cExperiment was terminated 6 days after Day 14 because 100% sterility had not been achieved, and treated male mortality was 10% in the 24-hour post-treatment period.

dAverage of four replicates.





52



In the first hour after oviposition, the zygote is formed in the egg (Cameron,' 1939). For the first 8 days Cno Ringer!ssolution) the larval hatch was only 60%, while for the remaining 6 days Cwhen the solution was used) the larval hatch was 73.60%,. It is possible that maturation of the egg nucleus'and fusion of the pronuclei had been disrupted during removal from the filter paper without Ringer's solution. Female sterilization

5 mi/cm2.--Ten females treated with tepa at 5 mg/cm2 for 4 hours and mated with untreated males, had a decrease in egg production the first week t=3.53> t =2.447), while in the second week there was no .05
significant difference between egg production of the control fleas and of those treated (t=2.39 Z =1.645), while in the .05
second week there wqs little difference (Z=0.372 Z.05=1.645 in the first week; Z=4.560> Z.05 =1.645 in the second). , The larval sterility was
.05
calculated to be 69.20% and the Fl adult sterility, 84.00%.

As is obvious from these analyses, it is more accurate to base conclusions on the F1 generation rather than simply on larval hatch. Therefore, the criterion for sterility used in all of these studies was the emergence of Fl adults.

Bertram C1963) reported that thio-tepa (tris(l-aziridinyl)phosphine sulfide) caused reduced fertility and fecundity in female A. aegypti. Rai (1964) stated that eggs from female A. aegypti treated with apholate nust carry induced dominant lethals, since a low hatch was obtained. This is probably true of tepa-treated X. cheopis females as well.









Twenty females treated with tepa at 5 mg/cm2 for 6 hours, and mated with 20 untreated males, produced some eggs. In one of the experiments 10 females laid eggs for the first 2 days, after which time they did not lay again until the eighth day. Apparently, the primary obcytes were not, affected by the sterilant, but the secondary oOcytes were. At this dosage and contact time, it seems that the germarial region of the ovarioles was not affected, since they did begin to lay eggs again. These fe-ales were mated with untreated males, and larval hatch was observed from eggs laid on all days except the first.

in this experiment no F1 adults were produced from larvae which

emerged on the second and the tenth days. Nevertheless, F1 adults did emerge. from the eleventh through the fourteenth days of larval hatch. The larval sterility was 98.20%, and the Fl adult sterility was 99.28%.

10 mg/cm2.--Murray and Bickley (1964) had noted that vacuoles occurred in the ovaries of Culex p. quinquefasciatus when they were treated with apholate in concentrations of 15 ppm and higher.

The female X. cheopiscan be completely sterilized (100%) with tepa, using a dosage of 10 mg/cm2 for a contact treatment time of 4 hours ablee 4). As can be seen in Fig. 14, the ovarioles are completely destroyed. The remnants of the ovarioles are leathery, opaque, and white in color, and are vacuolated. These females had mated with untreated males; Fig. 15 shows a spermatheca with motile sperm in the head.


~ale sterilization

In the t'-eated males, 100% sterility was not obtained, although the dosage and treatment contact times were greater than those of the females. Nevertheless, 99.99% sterility was achieved (Table 4).






54'




















- - - ---- ---
























Fig. 14.--Ovarioies of fleas treated with tepa at 10 mg/cm2 for 4 hours. Note destruction of ovarioles, and vacuolation. 130x.






55














A

























Fig. 15.--Spermatheca of a female flea treated with tepa at 10 mg/cm2 for 4 hours. Spermatheca head (A) contains motile sperm. 120x.






56


The fact that the males were almost completely sterilized did not affect the egg production of the untreated females; larval hatch and F1 emergence, however, were greatly reduced.

S5 mg/cm2.--In an early experiment, 5 males were treated with tepa at 5 mg/cm2 for 6 hours, and mated with 5 untreated females. There was no significant difference in egg production between the control and treated for the 2-week experiment (t=0.0318 week; t=1.07 (t .05=2.417 for the second). The larval sterility, however, was 99.00%, and the F1 adult sterility was 99.99%.

Since the preceding test gave almost 100% sterility at a low

dosage, further tests were conducted at higher dosages to determine the

100% sterility level.

10 mg/cm2 .--The next test involved the treatment of 10 males with tepa at 10 mg/cm2 for a contact treatment time of 4 hours. The males were mated with 10 untreated females. It was reasoned that at a higher dosage, even though the contact time was only 4 hours, 100%.sterility could be achieved. This was notthe rsult, however, as is shown in Table.4. In this case the larval sterility was only 85.30% and thb F1 adult sterility was 99.50%.

In another experiment C3 replicates), using 10 males treated with tepa at 10 mg/cm2 for 6 hours and mated with 10 untreated females, complete sterility was still not obtained. Larval sterility did increase to 99.20%, and the F1 adult sterility to 99.99%.

15 mg/cm .--In view of the results of these tests, another was conducted using 10 males treated with tepa at 15 mg/cm2 for 6 hours. These

males were also mated with 10 untreated females: The larval sterility was 99.99%. This test was terminated 6 days after day 14 because 100%






57

sterility had not been achieved, and treated male mortality was 10% in the 24-hour post-treatment holding period. In the lower dosage experiments, no treated adult males died during the post-treatment period.

Inability to achieve total male sterility.--Several possibilities present themselves as to why the adult male flea cannot be made totally sterile. Wheeler (1962) states that the alkylating agents (of which tepa is one) can affect directly or indirectly the alkylation of nucleoproteins. Since the alkylation would interfere with mitosis, the rate and replication of deoxyribonucleic acid (DNA) could be altered. Also, DNA is most likely the primary site of alkylation and therefore the most sensitive. Kilgore (1965), working with house flies, reported that the alkylating agents "have a very pronounced effect on the metabolism of the nucleic acids." This is probably also true in X. cheopis, reducing the synthesis of DNA so that the egg cannot form lactic acid dehydrogenase.

In a personal communication (1966), Rothschild confirmed this

author's observation that in male flea testes, spermatogenesis is essentially complete when the male emerges from the pupa. At this.stage the testis has its full complement of sperm. There is no germarial region in the adult male. Since spermatogenesis is either completed or

almost completed upon emergence, the metabolism of the sperm in the testis is probably quite low. In other words, transport of nutrients and gas exchange are not actively taki g place, as they would be during spermatogenesis. If this is the case, the chemosterilant would probably have to diffuse through the epithelial layer of the testis, and penetrate the sperm heads.





58



Keiser et al. (1965) state that in chemosterilized fruit fly

males both the spermatogonia and the spermatocytes are destroyed, but that the spernatids which are beyond the last division continue to develc an- to mature.

It would seem that certain individual sperm have probably completed spermatogenesis, thus being unaffected by the alkylation of the DNA, while the remaining 99.99% of the sperm have been affected. This would cause the sperm inactivation described by LaChance (1967) and, therefore, the absence of pronuclear fusion, producing sterile eggs.

Sperm transferred by tepa-treated males during copulation is probably inactivated sperm or sperm which contains dominant lethal mutations. Asperry has certainly not taken place in such a case, since sperm were found motile in the spermatheca of the females at the termination of 14-day experiments.

Oviposition in these experiments was not affected when treated males were mated with untreated females. This condition was also reported by Mitlin et al. (1957) in the house fly. Bertram (1963) observed that even up to 32 days after treatment with thio-tepa tie males of A. aegypti had active sperm, and that these sperm were abundant in the untreated female spermathecae, Fahmy and Fahmy (1964) observed that u ;s.reated Drosophila females, mated to males treated with varying dosages of the chemosterilant tretamine (TEM), had a very high number of unhatched eggs. Rai (1964) reported that sperm of male A. aeg'ti treated with apholate produced dominant lethal mutations, since egg hatch was ext-emely low. The chemosterilant might cause a mutation which acts as a gametic lethal, or as an early zygotic lethal (Fahmy and F, ..y, 1964).




59






Apparently there are two types of sperm transferred by tepa-treated X. cheopis males. With the first (inactivated sperm), embryonic development does not take place, and the egg color remains essentially the same. With the second type of Sperm, however, embryonic development does take place, but the embryos cease growth at various stages of development, and die (dominant lethal mutation). In some of the eggs setae, head capsules, and segmentation of larvae can be seen. In others there appear to, behealthy larvae; however, they never emerge.

Dominant lethal mutations seem to affect not only the embryo, but the larvae as well. Upon emerging, larvae from the treated fleas in these experiments did not appear to differ from the control larvae. They were active, and readily burrowed into the larval media, as did the controls. Yet, as can be seen from Table 4, the percent sterility of F1 adults from larvae of treated males is higher than that of the emerging larvae. Therefore, some of these larvae were certainly affected. It was not determined at what stage of developmentthe larvae aied, but it would be interesting to know at exactly which stage death occurred. This could be done b' rearing the larvae in artificial media according to the method of Pausch (1962).


Treated females X treated males,

The.percent sterility obtained from the mating of treated fleas with untreated fleas indicated that more efficient results might be obtained when both sexes were treated with tepa. Therefore, 10 females treated with tepa at 5'mg/cm2 for 6 hours were mated With 10 males treated with tepa 10 mg/cm2 for 4 hours. The females laid a few





S60


eggs during the first 3 days of the experiment, after which no more were laid. No larvae were produced.

With such results, two factors seem to be involved. First, as can be seen in Fig. 16, the ovarioles were almost destroyed. The remnant of one oOcyte can be seen (A), ahd these ovarioles are also leathery, opaque, and white in color, as well 'as being vacuolated. Second, the sperm from the male must have been inactivated by tepa, since the treated male testes, as well as the spermathecae of the females, contained motile sperm.

Cytological effects of tepa

Since no work had been done previously on the cytological effects of radiation or chemosterilization of X. cheopis, it has been essential to incorporate descriptions and terminology of other authors on other insects in order to interpret the results on X. cheopis.

Bayreuther (1954) was the first to report the chromosome number in X. cheopis from meiotic division. The female flea has 2n=18 chromosomes with the sex chromosome being X1XlX2X2. The male flea has 2n=17 chromosomes with the sex chromosomes being trivalent XlX2Y, as in the Mantids.

The mitotic chromosomes of X. cheopis are J-shaped (atelopitic) or V-shaped (metacentric), in the terminology of White (1951). These shapes can be seen in Fig. 17.

Brains from treated larvae were examined for chromosomal aberrations; however, no changes were observed after 24 hours. Another sample, examined 48 hours after treatment, showed chromosomal aberrations.






61








































Fig. 46.--Ovarioles of female fleas treated with tepa at
5 mg/cm for 6 hours. (A) remnant of oicyte. 130x.






62


















,r












Fig.- 17-Utetdfmaemttcmtphs hoooe (2n=18 f ro th ranofte rpua 16r
;;r ;li i l





63


The terminology used by Rai C1964) in describing chromosomal

aberrations in A. aegypti is usually applied here to describe the mitotic effects.of tepa on the flea. He grouped the induced aberrations into three principal categories: physiological and structural aberrations, and miscellaneous effects which include induction of somatic polyploidy and probable interference with the normal replication mechanism.

In Fig.'18, two types of aberrations can be observed in the 4

chromosomes visible (all others having been squashed out of the field of view). The first, physiological, is chromosome stickiness. Stickiness is most pronounced at the chromosome ends. The second, structural, could not be found in Rai's descriptions; however, Catcheside'(1948) has diagranmed chromosome structural changes induced by radiation. According to his description this tepa-induced aberration would appear to be an inter-arm chromatid exchange aberration.

Chromosomal constriction seems to be visible in Fig. 19. This

condition might be due either to the ends sticking to each other, or to fusion at two broken ends.

In Fig. 20 there can be readily observed fragments similar to those shown by Purdon (1963), and there appears to be an isodiametric fragment similar to the type noted by Catcheside (1948). A dicentric chromosome is present. There appear to be many tecombinations, rather than deletions as photographed by Murray and Bickley (1964) with apholate on C. P. quinquefasciatus, and by Flint (1964) with radiation on H. pusio.

It would seem, then, that tepa'induces non-random breaks. As with apholate, tepa may also break the DNA core, and yet not completely break the chromosome matrix envelope.



























A B



















Fig. 18.--Metaphase chromosomes from tepa-treated larval brain. (A) chromosome stickiness; (a) inter-arm chromatid exchange aberration. 1667x.


















































Fig. 19.--Metaphase chromosomes from tepa-treated larval brain. (A) chromosomal constriction. 1667x.






66































/







ti





Pig. 20.--Metaphase chromosomes from tepa-treated larval
br4in. (A) chromosomal fragments; (B) isodiometric fragment;
(C) dicentric chromosome. 1667x.





67

Plapp et al. (i962) reported that with the chemosterilant p32 labelled methaphoxide (tris(2-methyl-l-aziridinyl)phosphine oxide), there was no difference in the metabolism of this compound in organophosphate-resistant and -susceptible house flies. This finding is supported by Wheeler (1i62) who states that as yet no mechanism of resistance to alkylating agents has been definitely established.


Field scil ication

Biology.--T e potential egg production of X. cheopis females can be determined using the results obtained from the predicted egg production curve in Fig. 8. After flea population counts have been made in the field, the size of future populations can be predicted. Thus, one would know when measures for control of the flea would be most appropriate for maximum effectiveness.

Using the rearing method described herein, one could determine the effectiveness of a chemosterilant by the percentage of F1 emergence from field-collected specimens.

Chemosterilization.--For practical field application of a sterility program, one essential factor is that the sterilized insects be able to mate with non-sterile individuals. Even though chemosterilant mortality would reduce a population, it would be better to have sterilized individuals live and mate with the non-sterile ones.

Now that sterility with tepa has been achieved in X. cheopis, it should be determined if this flea can become sterilized by feeding on tepa-fed rats or mice. If so, bait stations with the chemosterilant could be set up for the eradication of rodents and their ectoparasites.














SUMMARY


The female X. cheopis can be completely sterilized with tepa at a dosage of 10 mg/cm2 for a contact treatment time of 4 hours. The ovarioles are virtually destroyed with only remnants left. Treatment of females with a dosage of 5 mg/cm2 for 6 hours gave 99.28% sterility based on the emergence of F1 progeny.

Better than 99.99% sterility could not be achieved in males at dosages of 5, 10, or 15 mg/cm2 for 4 and for 6 hours.

When females treated for 6 hours with tepa at 5 mg/cm2 were mated w.ith males treated with tepa at 10 mg/cm2 for 4 hours, 100% sterility was achieved.

Larvae ready to pupate were treated with tepa at 10 mg/cm2 for 4 hours. Forty-eight hours after treatment, tepa-induced chromosomal aberrations were found in the larval brain. These aberrations appeared to be: Cl) chromosome stickiness; (2) inter-arm chromatid exchange;

(3) chromosomal constriction; (4) chromosomal fragments; (5) isodiometric fragments; and (6) dicentric chromosome. These aberrations were compared-with untreated larval brains in mitotic division.

It was found that the females of the Orlando strain of X. cheopis have 3, 4, or 6 ovarioles per ovary.

In 12 experiments lasting 14 days, the average number of eggs laid daily per female was 4.25�.21; .the highest was 12.6.

Blood meals are necessary for egg-laying.


68






69


Eggs of mated females are sticky and adhere to the substrate, as do bits of food. Mating appears to stimulate a gland in the female to produce this sticky substance which is lacking in the eggs of virgin females.

Four out of 149 eggs laid by virgin females underwent partial' embryonic development, but died before hatch Crudimentary parthenogenesis).
















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Bonhag, P. F. 1958. Ovarian structure and vitellogenesis in insects.
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APPENDIX






79











Table 5.--Five untreated females mated with five untreated males.




NUMBER OF


Eggs Emerging


Daya Produced Survivedb Larvae F1 Adults


1 5 5 3 3 2 23 19 11 1 3 23 21 15 10 4 25 21 12 1 5 30 21 15 5 6 26 24 '14 0 7 56 47 26 15 8 41 37 21 9 9 27 27 24 14 10 34 33, 26 21 11 39 38 16 10 12 42 42 38 38 13 31 31 21 20 14 30 26 20 18

Totals: 432 392 262 '165


aEggs were first observed on the 3rd day after placing fleas on host.

bSome eggs were found broken, or were broken in handling.-






80










Table 6.--Five males treated with tepa 5Smg/cm2 for 6 hrs.) and
mated with five untreated females.




NUMBER OF


Eggs Emerging


Daya Produced Survivedb Larvae F Adults


1 17 15 0 0 2 18 17 0 0 3 16 15 0 0 4 18 16 0 0 5 31 * 30 0 0 6 19 ' 15 0 0 7 34 34 1 1 8 38 36 0 0 9 28 28 0. 0 10 18 18 0 0 11 38 37 0 0 12 21 21 0 0 13 34 , 33 0 0 14 24 24 2 0

Totals: 354 339 3 1


aEggs were first observed on the 3rd day after placing "fleas on host.

bSome eggs were found broken, or were broken in handling.,






81










Table 7.--Ten untreated females mated with ten untreated males..




NUMBER OF


Eggs Emerging


Daya Produced Survivedb Larvae Fl Adults


1 9 9 9c 7 .2 22 21 18 18 3 30 ' 30 25 25 4 22 21 14 14 5 35 35 28d 27 6 19 19 14 11 7. 23 23 20 18 8 35 34 29 e 29 9 39 37 37 35 10 27 25 16 16 11 32 32 31 28 12 38 , 31 26 24 13 35 32 23 20 14 47 38 28 26

Totals: 413 387 318 298


aEggs were first observed on the 3rd day after placing fleas on host.

bSome eggs were found broken, or were broken in handling.

CTwo larvae died.

dCe larva died.






82











Table 8.--Ten untreated females mated with ten untreated males.




NUMBER OF'


Eggs Emerging


Daya Produced Survivedb Larvae F1 Adults


1 4 3 3c-2 29 29 24 20 3 80 70 59 51 4 48 39 28 17 5 49 49 32 28 6 51 51 41 35 7 41 41 38 34 8 22 20 15 14 9 32 32 28 .27 10 50 48 44 40 11 21 20 14 12 12 29 27 16 16 13 52 46 41 38 14 50 50 46 45

Totals: 558 525 429 '377


aEggs were first observed on the 3rd day after placing fleas on host.

Some eggs were found broken, or were broken in handling..

CAll larvae died.






84











Table 10.--Ten untreated females mated with ten untreated males.




NUMBER OF


Eggg Emerging


Daya Produced Survivedb Larvae F1 Adults


1 30 28 19 0 2 40 40 9 6 3 56 49 44 4 52 52 46 40 5 62 60 43 42 6 65 62 51 48 7 97 96 77 75 8 50 49 42 34 9 91 91 67 54 10 80 79 68 56 11 91 89 79 76 12 92 91 72 72 13 80 76 68 55 14 80 77 58 55

Totals: 966 946 748 657


aEggs were first observed on the 3rd day after placing fleas on host.

bSome eggs were found broken, or were broken in handling.'






83











Table 9.--Ten untreated females mated with ten untreated males.




NUMBER OF.


Eggs Emerging


Daya Produced Survivedb Larvae FI Adults


1 8 8 7 7 2 34 34 31 30 3 42 42 38 34 4 54 52 42 38 5 55 54 44c 40 6 40 36 26 23 7 62 62 57 54 8 53 53 43 40 9 36 36 30c 26 10 53 52 35 33 11 42 42, 34 32 12 51 51 38 37 13 28 28 27 27 14 54 51, 47 44

Totals: 612 601 499 465


aEggs were first observed on the 3rd day after placing ,fleas on host. bSome eggs were found broken, or were broken in handling.

COne larva died.






85











Table ll.--Ten females treated with tepa (5mg/cm2 for 4 hrs.) and
mated with ten untreated males.




NUMBER OF


Eggs Emerging

*a b Day Produced Survived Larvae F1 Adults


8 8 6 1
2 10 10 9 0 3 14 14 8 2 4 .3 3 1 1 5 6 6 4 0 6 38 37 24 6 7 22 22 18 2
19 19 15 13
9 26 25 22 5 10 39 37 28 21 11 23 22 19 15 12 24 23 -16 12 13 45 45 38 9 14 24 24 20 18

Totals: 301 295 228 105


aEggs were first observed on the 3rd day after placing fleas on host.
some eggs were found broken, or were broken in handling.






86'










Table 12.--Ten females treated with tepa (5mg/cm2 for 6 hrs.) and
mated with ten untreated males.




NUMBER OF


Eggs Emerging


Daya Produced Survivedb Larvae F Adults


4 4 0 0
2 22 22 14 0 3 0 0 0 0 4 0 0 0 0 5 0 0 0, 0 6 0 0 0 0 7 0 0 0 0 8 0 0 0 0
9 0 0 0 0 10 5 5 3 0 11 7 7 7 2 12 9 6 2 1 13 13 13 10 7 14 24 24 13 11

Totals: 84 81 49 21


aEggs were first observed on the 3rd day after placing fleas on host.

bSome eggs were found broken, or were broken in handling.-






87










Table 13.--Ten females treated with tepa (10mg/cm2 for 4 hrs.) and
mated with ten untreated males.




NUMBER OF


Eggs ' Emerging


Daya Produced Survivedb Larvae F1 Adults


1 5 0 0 2 12 11 1 0
3 5 5 1 0 4 3 3 1 0 5 0 0 0 0 6 0 0 0 0 7 0 0 0 0 8 0 0 0 0
9 0 0 0 0 10 0 0 0 0 11 0 0 0 0 12 0 0. 0 0 13 0 0 0 0 14 0 0 0 0

Totals: 25 24 3 0


aEggs were first observed on the 3rd day after placing fleas on host.
bSome eggs were broken, or were broken in handling.






88










Table 14.--Ten females treated with tepa (10mg/cm2 for 4 hrs.) and
mated with tcn untreated males.




NUMBER OF


Eggs Emerging


Day" Produced Survived Larvae F1 Adults


1 0 0 0 0 2 0 0 0 0 3 0 0 0 0 4 0 0 0 0 5 0 0 0 0 6 0 0 0 0 7 0 0 0 0 8 0 0 0 .0 9 0 0 0 0 10 0 0 0 0 11 0 0 0 0 12 0 0 0 0 13 0 0 0 0 14 0 0 0 0

Totals: 0 O 0 0


aControl fleas began egg production on the 3rd day after being placed on the host.






89










Table 15.--Ten females treated with tepa (10mg/cm2 for 4 hrs.) and
mated with ten untreated males'.




NUMBER OF '


Eggs Emerging


Day Produced Survived Larvae F Adults


1 0 0 0 0 2 0 0 0 0 3 0 '0 0 0 4 0 0 0 0 5 0 0 0 0
6 0 0 0 0 7 0 0 0 0 8 0 0 0 0 9 0 0 0 .0
10 0 0 0 0 11 0 0 0 0 12 0 0 0 0 13 0 0 0 0 14 0 0 0 0

Totals: 0 0 0 0


aControl fleas began egg production on the 3rd day after being placed on the host.






90










Table 16.--Ten males treated with tepa (5mg/cm2 for 6 hrs.) and'
mated with ten untreated females.




NUMBER OF


Eggs Emerging


Daya Produced Survivedb Larvae F1 Adults


1 7 6 0 0 2 24 24 2 0 3 36 36 0 0 4 56 56 1 1 5 57 56 1 0 6 30 30 0 0 7 37 37 0 0 8 53 53 0 0 9 64 64 1 0 10 40 39 0 0 11 56 56 0 0 12 45 45 0 0 13 53 53 0 0 14 50 50 0 0

Totals: 608 - 605 5 1

aEggs were first observed on the 3rd day after placing fleas on host.

bSome eggs were found broken, or were broken in handling..

















Table 17.--Ten males treated with tepa (5mg/cm2 for 6 hrs.) and"
mated with ten untreated females.




-NUMBER OF'


Eggs Emerging


Daya Produced Survivedb Larvae F1 Adults


10 10 0 0
2 31 31 0 0 3 30 30 1 0 4 30 28 0 0 5 64 62 0 0 6 30 29 0 0 7 54 54 0 0 8 40 39 0 0 9 55 55 0 0 10 36 35 .0 0 11 40 40 0 0 12 40 40 0 0 13 55 55 0 0 14 54 54 0 0

Totals: 569 562 1 0

a
Eggs were first observed on the 3rd day after placing fleas on host.
bSome eggs were found broken, or were broken in handling.-






92










Table 18.--Ten males treated with tepa (5mg/cm2 for 6 hrs.) and'
mated with ten untreated females.




NUMBER OF


Eggs Emerging


Day-a Produced Survivedb Larvae F1 Adults


1 19 19 1 0 2 27 27 0 0 3 .37 36 1 0 4 41 39 0 0 5 40 40 0 0 6 35 35 1 1 7 25 25 0 0
8 47 ' 47 0, 0
9 72 72 0 0 10 68 68 0 0 11 73 73 1 0 '12 70 70 0 0 13 46 46 0 0 14 54 54 0 0

Totals; 654 651 4 1


aEggs were'first observed on the 3rd day after placing fleas on host.

bSome eggs were found broken, or were broken in handling..




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PAGE 1

BIOLOGICAL OBSERVATIONS ON THE ORIENTAL RAT FLEA, Xenopsylla cheopis (ROTHSCHILD), WITH SPECIAL STUDIES ON THE EFFECTS OF THE CHEMOSTERILANT TRIS (1-AZIRIDINYL) PHOSPHINE OXIDE By ROBERT LOOMIS LINKFIELD 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 December, 1966

PAGE 2

ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude to the Chairman of his Supervisory Committee, Dr. J. T. Creighton, Department of Entomology, University of Florida, and the Co-Chairman, Dr. P. B. Morgan, United States Department of Agriculture, for their advice and assistance. For their criticism of the manuscript, appreciation is also expressed to committee members Dr. F. S. Blanton and Mr. W. T. Calaway, and to Dr. A. M. Laessle who read for Dr. Archie Carr. Special thanks are due to Dr. C. N. Smith for the use of facilities at the U.S.D.A. Entomology Research Division Laboratory; to Dr. G. C. LaBrecque for his suggestions and encouragement; and to all the U.S.D.A. staff who assisted. Dr. William Mendenhall, Chairman of the Department of Statistics, assigned Mr. Marcello Pagano to aid in.certain statistical analyses; the Computer Center allotted 15 minutes of computer time. Their cooperation is gratefully acknowledged. Last, but not least, appreciation goes to my wife, Ethel, who typed the preliminary copies of this manuscript and who gave affectionate encouragement throughout the course of this study. ii

PAGE 3

TABLE OF CONTENTS Page ACKNOWLEDGEMENTS. . . . ii LIST OF TABLES V LIST OF FIGURES "' . vii INTRODUCTION . . . . . 1 REVIEW OF LITERATURE . 5 Chemosterilants 1 . 5 Biology 10 The adult 10 Temperature and humidity 10 Light sensitivity 11 Feeding ' 11 Mating . . • 12 Miscellaneous . . . '. 13 Reproductive system 13 Female . . . 13 Male . ." . , , . 15 The egg , . i 15 Egg production 15 Longevity .18 The larva ............ V 19 Prepupa and pupa • 20 Prepupa 20 Pupa ................ * 21 METHODS AND MATERIALS ' . . '. 22 Rearing of X_. cheor>is 22 Cytological techniques 22 Experimental design 24 Flea populations for experimental use 24 Chemosterilant treatment , . • 25 Sterility experiments ' 28 The emergence chamber . 33 Statistics 33 Photography 35 iii

PAGE 4

Page RESULTS AND DISCUSSION 36 ! Biological Observations ... 36 Ovarioles . 36 Egg production 36 Ratio of male to female . ., 39 Behavior ' 39 Parthenogenesis .46 General observations ... 48 Life cycle 48 Larval and adult emergence 48 Feeding site 48 Chemosterilization with Tepa . . . . 50 Egg hatch ' • • 50 Natural sterility 50 Egg breakage 50 Female sterilization . . . 52 . 5 mg/cm ' * 52 10 mg/cm 2 53 Male sterilization 53 5 mg/cm 2 ......... 56 10 mg/cm 2 . . 56 15 mg/cm2 56 Inability to achieve total male sterility 57 Treated females X treated males . 59 Cytological effects of tepa . 60 Field application • 67 Biology 67 Chemosterilization • 67 SUMMARY , . 68 REFERENCES CITED ' * 70 . APPENDIX .......... .... 78 BIOGRAPHICAL SKETCH . . 99 iv

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LIST OF TABLES Table p age 1 Measurements of gonads of 30 male and 30 female X* cheopis adults s . . . 38 2 Ratio of female to male in laboratory-reared and in fieldcollected X_. cheopis adult populations ' . 41 3 Production and development of eggs from virgin female X. cheopis which had continuous access to blood meals . .~ . . . 47 4 Summary of results from experiments using tepa in varying dosages and lengths of time for sterilization of X. cheopis adults . '. 51 5 Five untreated females mated with five untreated males ... 79 6 Five males treated with tepa (5mg/cm 2 for 6 hrs.) and mated with five untreated females 80 7-10 Ten untreated females mated with ten untreated males .... 81-84 11 Ten females treated with tepa (5mg/cm for 4 hrs.) and mated with ten untreated males 85 12 Ten females treated with tepa 1 (5mg/cnr for 6 hrs.) and mated with ten untreated males 86 13-15 Ten females treated with tepa (lOmg/cm for 4 hrs.) and mated with ten untreated males \ . 87-89 16-18 Ten males treated with tepa (5mg/cm 2 for 6 hrs.) and mated with ten untreated females 90-92 19 Ten males treated with tepa (lOmg/cm for 4 hrs.) and mated with ten untreated females 1 93 20-22 • Ten males treated with tepa (10mg/cm 2 for 6 hrs.) and mated with ten untreated females ' 94-96 2 23 Ten ffiaies treated with tepa (15mg/cm for 6 hrs.) and mated with ten untreated females 97 v

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I Table • Page 2 24 Ten females treated with.tepa (5mg/cm for 6 hrs.} and mated with ten males treated with tepa at lOmg/cm for 4 hrs. . . . . . 98 vi

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LIST OF FIGURES Figure Page 1 Female ovarioles showing primary oocyte (A); oocytes in various stages of development (B) ; germarial region (C) ; and terminal filament (D) . 130x 14 2 Male. (A) testis; (B) sperm; (C) epididymis; (D) vas deferens; (E) paired accessory glands, lllx 16 5 Vacuum aspirator used to sex adult fleas 26 4 Pinning board used for treating filter paper strips ... 27 5 Vacucel ice chest used to maintain darkness and humidity for experimental mice and fleas in Mason jars. (A) black organdy cloth; (B) water compartment; (C) compartment for Mason jars 30 6 Incubator. (A) whisper fan; (B) sponge and water container for maintaining humidity; (C) petri dishes containing eggs and growing larvae; CD) temperature-humidity probe; (E) temperature-humidity recorder . . 32 7' Adult emergence chambers 34 8 Average and predicted daily egg production per X_. cheopis * female from 12 experiments of 14 days each 37 9 Average egg production per female for 28 days based on that of 10 females in one control experiment . . . 40 10 Av rage egg production of mated and of virgin X. cheopis ' females which had continuous access to blood meals .... 43 11 Bottom view of egg of X_. cheopis adhering to bits of rat chow. 44x . 44 12 Top view of X. cheopis egg (A) which is stuck to the filter paper, and surrounded by bits of rat chow. 44x . . 45 13 Rate of egg-laying and percent emergence of larvae and Fj adults ' 49 vii

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1 Figure Page 2 14 Ovarioles of fleas treated with tepa at 10 mg/cm for 4 hours. Note destruction of ovarioles, and vacuolation. 130x 54 15 Spermatheca of a female flea treated with tepa at 10 mg/cm 2 for 4 hours. Spermatheca head (A) contains motile sperm. 120x ..".'.'.< . . 55 < 16 Ovarioles of female fleas treated with tepa at 5 mg/cm 2 for 6 hours. (A) remnant of oocyte. 130x 61 17 Untreated female mitotic metaphase chromosomes (2n=18) from the brain of the prepupa. 1667x 62 18 Metaphase chromosomes from tepa-treated larval brain. (A) chromosome stickiness] (B) inter-arm chromatid exchange aberration. 1667x 64 19 Metaphase chromosomes from tepa-treated larval brain. (A) chromosomal constriction. 1667x 65 20 Metaphase chromosomes from tepa-treated larval brain. (A) chromosomal fragments; (B) isodiametric fragment; (C) dicentric chromosome. 1667x . . . i 66 viii

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INTRODUCTION The oriental rat flea, Xenopsylla cheopis (Rothschild), is recognized as a pest all over the world (Buxton, 1941, and Atlas of Plague, 1952). It can be found on ship rats throughout the year (Newstead and Evans, 1921). Hirst (1927) gave the following account of its dispersal: X. cheopis is able to develop apart from its hosts' nest, so that it is capable of being much more easily dispersed in material such as grain, the debris of which affords nourishment for the larvae. Thus grain not only serves as a means of transport for rats and fleas from place to place, but is the most suitable medium for the multiplication of the most efficient insect vector of plague, X_. cheopis , and the principal carrier of both disease and flea, the grain-eating Mus rattus . According to Wu et al. (1936), plague, Pasteurella pest is (Lehman and Neumann), was first recorded in the sixth century A.D., starting in i Egypt in 542, and finally spreading to Constantinople. This pandemic lasted for almost sixty years, and approximately 100,000,000 people died. In Europe in 1348, a plague pandemic, termed "the Black Death," killed 25,000,000 people (Herms, 1961). The plague pandemic which began in Hong Kong in 1894 was carried to many parts of the world through trade routes. Herms (1961) states that rats, infected by rat fleas, and transported in commercial goods, are the chief spreaders of the disease, with the plague likely to appear in a city "far removed from the original focus of infection." 1

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2 i From 1900 to 1952, cases of plague were recorded in thirty-nine countries (Atlas of Plague, 1952). In twenty-seven of these countries —• cheopis wa s one of the principal vectors. From 1945 to .1952, there were /..irty-seven cases reported from the seaports of various countries. Plague in the United States was first reported from San Francisco in 1900 (Herms, 1961, and Jellison, 1959). The epidemic ended in 1904, but reappeared in 1907. Other cities which have suffered from the disease include Seattle in 1907, New Orleans in 1912, several Gulf Coast cities in 1920, and Los Angeles in 1924. The theory that rats in commerce are the principal carriers of plague would seem to be confirmed by the fact that most of the recorded plague, epidemics beganin seaports. At present, syl vatic plague is, endemic throughout the western United States, and in the provinces of Alberta and Saskatchewan, Canada. It has been determined from thirty-eight species of rodents and lagomorphs. According to Meyer (1947), the most prolific carriers of plague are the Sciuridae. It was discovered in 1908 that plague was no longer confined to rats and rat fleas in North America, but had become established in the ground squirrel, Citellus beecheyi (Richardson) and in its fleas. McCoy (1910) reported on plague in ground squirrels in California. He warns: In the suburbs of towns and cities, rats and squirrels come intovery close contact and it would seem very easy to have the disease carried from the squirrels to the rats, and as a result have a general infection of the cities. —• c heopis is one of the vectors of murine typhus, Rickettsia ir.ooseri Montiero, transmitted by fleas, with the rat and squirrel populations serving as reservoirs between epidemics. The cycle in this

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3 instance is from animal to flea to animal to flea to --now and then — man. The body louse, Pediculus humanus humanus Linnaeus, carries i European typhus directly to man, at the cost of fatal infection to the louse itself. The whole series of Rickettsial diseases, superficially so much alike, differ dramatically when considered epidemiologically. The transmission mechanism varies considerably from one disease to the other. There is no common vector, although piercing-sucking arthropods are invariably involved. There is even less uniformity in the degree to which an animal reservoir is necessary to keep the infection alive, and in whether or not the vector itself succumbs to the disease. Chemosterilant programs would seem to be ideally suited for work against all types of the Rickettsiae. The tapeworm, Hymenolepis diminuta (Rudolphi) , the larvae of which develop in X. cheopis and other fleas, can complete its life cycle in man or in any susceptible animal which might ingest the infected flea. Up to the present time, X_. cheopis has been controlled only through the use of insecticides or control of the rodent host. With the advent of radiation and chemosterilants possible eradication of this and ' similar species can be envisaged. No literature could be found relating to this type of work, but literature concerning radiation and chemosterilants used on other species of insects was studied so that a basic, practical pattern of research could be planned. i Although radiation was considered as a possible effective means of sterilization, one would not like to release thousands of fleas in any given area. Since fleas are pestiferous whether or not they are diseased, there is definitely an advantage in setting up stations for

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4 i sterilizing rats. Before the initiation of such a program, however, it would be essential to know what effects the chemosterilant might have on the fleas of the rat, especially whether or not feeding on the sterilized rat would in turn sterilize the flea. It was decided, therefore, to test the effects of the chemosterilant tepa on the Oriental rat flea. X. cheopis , as is obvious from the preceding account, is still among the most important potential enemies of mankind. The basic purpose of this study, therefore, was to determine if the flea can be sterilized, if its sterility and mortality doses are close, and how it is affected by the chemosterilant tepa. Since the effective use of chemosterilants depends upon a thorough study of the species to be sterilized, the literature was reviewed, and careful observations were made concerning the biology of X. cheopis .

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REVIEW OF LITERATURE Chemosterilants Radiation-imitating chemicals, termed "radiomimetic" by Dustin CI 94 7 ) , have been known for some years. It was not until the Second World War, however, with work on poison gases, that they were shown to be possible cancer cures (Alexander, 1960). The radiomimetic compounds, as in radiation, cause the following cellular effects: CL) mutation of genes and permanent changesin chromosomal structures; (2) interference with cell division processes which can cause death of the cell; (3) outright death of certain types of cells; (4) cancerous growths. Fahmy and Fahmy (1958) found that alkylating agents (radiomimetic compounds) cause an amount of small deficiencies almost double those of mutagenically equivalent doses of X-radiation. These deficiencies are caused by failure in gene reproduction in situ, and not by chromosome breakage and reunion as in radiation. Alexander and Stacey (1958) suggest that the difference in biological action between radiation and radipr;.imetic compounds is that the latter have to diffuse into the cell making some of the DNA (deoxyribonucleic acid) molecules more accessible to the compound than are others. A chemical which causes sexual sterility is called a chemosterilant. Weidhaas (1963) defines it further: 5

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6 — the term chemosterilant is restricted to compounds which prevent production of sperm or ova, kill sperm or ova that have already been produced, or damage chromatic or genetic material in the sperm or ova so that zygotes, if formed, do not develop into mature progeny. Two groups of chemical compounds which have shown great promise as sterilants of insects are the antimetabolites and the alkylating agents (LaBrecque, 1963 and 1965). An alkylating agent, the chemosterilant tepa causes replacement of a hydrogen atom with an alkyl group on an organic molecule. Its structural formula is the following: 0 According to Duvall (1960), tepa is very hygroscopic, being "extremely soluble" --although unstable-in water, as well as being "very soluble" in alcohol, in ether, and in acetone. Solutions of tepa and one of the three diluents kept at about 5°C will remain comparatively stable for a week. Duvall reported the LD^ in mice to be 47.0 mg/kg/d by mouth, the maximum tolerated single dose being 75.0 mg/kg/d. Hayes (1964) reported that rats injected intraperitoneal ly with a dosage of 0.2 mg/kg/d of tretamine (2,4,6-tris(l-aziridinyl)-5-triazine) remained sterile up to at least 8 weeks after the last dose. An intraperitoneal dosage of 0.05 mg/kg/d of tretamine caused sterility in male rats. Fertility returned 3-4 weeks after the last dosage. Sexual behavior of the sterilized male was normal, as was the number and motility of the sperm.

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The compounds presently available are not very stable (.Smith, 1963a, and Hayes, 1964), and more information is needed as to their toxicity and application before expanded programs can be undertaken (Smith, 1963b; Smith et al., 1964; and Barnes, 1964). Mitlin et al. (1957) obtained sterilization in house flies by using mitotic poisons. Three of the four chemicals tested usually inhibited oviposition, and prevented ovarian growth. In 195S, LaBrecque began screening chemical compounds for their chemosterilizing effects. Of 2,000 compounds initially tested, 5 caused sterility in the house fly, Muse a domestica Linnaeus, when placed in its food. By 1963, 2,000 chemicals had been tested, 40 of them causing sterility, according to LaBrecque (1963). LaBrecque reported further in 1965 that 112 chemicals have been found to produce sterilant effects. Several field tests have been conducted against the house fly with the chemosterilants tepa (tris Cl-aziridinly)phosphine oxide), metepa Ctris(2-methyl-l-aziridinyl)phosphine oxide), and apholate (2,2,4,4,6,6hexakis(l-aziridinyl)-2,2,4,4,6,6-hexahydro-l,3,5,2,4,6-triazatriphosphorine) . In a refuse dump at Bahia Honda Key, Florida, LaBrecque et al (1962) conducted the first field test using tepa against the house fly. As a result, the adult fly populations were reduced from 47 to zero per grid count within 4 weeks with the use of cornmeal baits containing Q.5% tepa. Female flies trapped at the dump were checked for egg. masses and viability. Egg masses had decreased from 100-10% within 4 weeks, and within 5 weeks egg viability had decreased to 1%. Metepa at 0.5% was applied in bait to droppings in a poultry house for control of the house fly QLaBrecque et al., 1963) with similar results.

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s In 1965, Gouck et al. conducted a test in a refuse dump at Pine Island, Florida, using a cornmeal bait of 0.75% apholate. They obtained sporadic results probably due to introduction of fresh flies from garbage trucks. Nevertheless, a reduction of flies did occur from 6S per grid count to between 5 and 20 for the first 7 weeks.When bait was made available continuously, the population then decreased to between 5 and 0 per grid count. Morgan and LaBrecque (1962 and 1964b) reported on the effects of apholate, tepa, and metepa on the ovarian development of house flies. In general, these compounds were found to inhibit ovarian development. The chromatin of the nurse cell nuclei was clumped in irregular masses. LaBrecque et al . (1966) achieved 99-100% sterility in the male house fly at concentrations of hempa (hexamethylphosphoramide) as low as 0.25%. Female sterility was often as high as that of the male, although it varied. Through the use of gamma radiation the screw-worm fly was eradicated from the island of Curacao, and from the southeastern United States. Recent research has been directed toward the use of chemosterilants to control this fly (Knipling, 1962). Chamberlain (1962) obtained sterility in adult screw-worm flies with apholate; however, he achieved only partial sterility in the pupae. In 1963, Crystal induced sterility in the same fly, with antimetabolites and with alkylating agents. Of 29 compounds tested, 26 caused sterility when incorporated into the diet, and 12 by topical application. In 1965, he further reported that the chemical N,N'-tetramethylenebis(l-aziridinecarboxamide) was far superior to gamma, radiation in sterilizing the screw-worm fly.

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9 The yellow-fever mosquito, Aedes aegypti (Linnaeus), and the common malaria mosquito, Anopheles quadrimaculatus Say, can be sterilized with chemosterilants in either the larval or the adult stage (tfeidhaas et al., 1961; Weidhaas, 1962; Dame and Ford, 1964a; and Dame et al . , 1964b, c) . Apholate concentrations as low as 0.1%, when fed in the diet, reduced to zero the fertility of eggs laid by A. aegypti , while 0.5% completely eliminated egg fertility in A. quadrimaculatus . When larvae of A. aegypti were treated' with 10 p. p.m. of tepa, all of the emerging adult males, and almost all of the females, were sterile. Apholate at 10 p. p.m. was not effective. Residual tests demonstrated that tepa at 680 ug/ft^ gave complete sterility in both species of mosquito. Glancey (1965) reported that, when fed in a honey solution, 0.5% hempa induced 100% sterility in female A. aegypti , and a 0.1% concentration produced 97% sterility in the male. He also found that residual deposits of 200-500 mg/ft for 4 hours produced 90% sterility in the male. Apholate affects the female A. aegypti reproductive system almost the same as in house flies (Rai, 1964 and 1965). Rai stated that the egg f-Ilicles of treated females were underdeveloped, and eventually degenerated; in some instances the follicular epithelium, eggs, and i nurse cells degenerated. Burden and Smittle (1963), testing twelve different chemosterilants on the German cockroach, Blatella germanica (Linnaeus) > f 0U nd that some of the compounds caused the obthecae to be deformed, resulting in delayed and/or reduced hatch. Smittle (1964) conducted tests with tepa on the reproductive organs and embryogeny of this cockroach. He found

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I 1 10 that 4 and 5 ug of ' tepainjected into adult male roaches produced complete sterility, although it inhibited neither mating nor sperm motility. Egg hatch was reduced 96% when 5 ug of tepa was, injected into seventh-instar female nymphs. This chemosteriiant caused the male testes to atrophy, and the basal oocytes of females to be smaller than those of controls. Females mated with sterile males produced oothecae; however, no hatch occurred. Testes and ovaries of the eye gnat , ' Hippelates pusio Loew, were reduced in size when treated with tepa, metepa, or apholate (Schwartz, 196i). The gc-..;arium of the female ovarioles was the most severely affected; the germarial region of the male, void of spermatogonia, was almost collapsed. Sterility of 99-100% was obtained with 0.01% tepa, 0.1% metepa, and 0.5% apholate. Tepa at 10 mg/ft affected bird malaria, Plasmodium gallinaceum Brumpt, in A. aegypti when the mosquito was subjected to the residue either before or after feeding on infected chicks (Altman, 1963). The chemical caused reductions in the number of infected mosquitoes, the mean oocyst count, and the rate of transmission. Biology The adult , , . Hirst (1926) was the first to report that emergence of X_. cheopis from its cocoon is stimulated by vibration. The vibration can be mechanical, but breathing on the cocoons will also cause the adults to emerge . Temperature and humidity . --At lower te/.peratures, nfed adults live longer than at higher ones. Buxton (1948) concluded that at temperatures from 24-32°C, the adult flea could survive...

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The life cycle of the flea will c vary according to the temperature and humidity. Hopkins (1955) reported that the life cycle could be accoir.pl i shed in about 56-65 days, the minimum being 42 days, at a temperature of 20°C, and a relative humidity of 100%. Krishnamurthy et al. (1965) found that, at a temperature between 25-28°C, and a relative humidity between 75-80%, the life cycle could be completed in 24-29 days. The spiracles control the loss of water, according to Mellanby (1954), who found that in adult fleas exposed to 5% C0 2 , the rate of water loss is doubled because the spiracles are permanently open. Humidity is somewhat unimportant, then, since the flea at rest keeps most of its spiracles closed (Wigglesworth, 1955). Buxton (194S) states that the adult flea is hardly affected by humidity, and in its natural habitation the water loss would probably be made up at the next feeding. Smith (1951) found that high humidities cause adult female activity to increase to the extent that the female needs frequent blood meals. Light sensitivity . --Adults of X_. cheopis are photonegative-. (Mitzm_m, 1910, used the term "negatively heliotropic" while Pausch, 1962, described this condition as "negatively phototactic .") Edney (1945) reported that adults kept in the dark live slightly longer than those in the light. This is true even if the pre-adult stages have been passed in the dark. Smith (1951) states that fleas of the genus Xenonsylla are repelled by strong light, and that they are most active in air which is saturated or nearly so. Feeding . --There is no difference in size of newly emerged adults which have not fed several times (Edney, 1945). Once adult fleas have

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I 12 fed, they must continue to do so or else they will die (Hirst, 1923). A flea which has ingested a blood meal usually will not feed again until the following day. The adults are persistent feeders, and will zinue feeding even though they are engorged, to the extent that blood is excreted through the anus and falls down to the substrate. This habit (or behavior) is probably stimulated by adenosine triphosphate, which is found in the host's blood (Galun, 1966). The i average adult will engorge approximately 0.5 cc of blood at one feeding, according to Reports on Plague Investigations in India (1907). Maximum feeding was obtained with fleas 5-8 days old by Bar-Zeev and Sternberg (1962). It was found that starved females ingest more blood than starved males. Both Mitzmain (1910) and Pausch (1962) say that the female must have a blood meal before copulating or laying eggs. Fox et al. (1966) found that X_. cheopis will feed on invertebrate as well s on vertebrate blood. Placed on a Puerto Rican lizard, Anolis cristatellus cristateilus, the fleas began to feed either immediately or within 5 minutes. The longest life of an adult in the experiment was 36 days, while the minimum was 4 days. Under the conditions of this experiment no progeny was obtained. 4:x ing . — Suter (1964) described pictorially the copulatory positions of male and female. He reported that before copulation the females need several blood ..eals; however, this is not true of the males which, in fact, can copulate immediately after emerging from the pupae, one male being able to mate with several females. Transfer of S] jrm takes place in about 10 minutes. Mating may take place on the host, in the nest, or in the bedding.

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13 Mlscellangdus, . — Smith (1951) stated that in unfed female fleas up to one day old, there was no daily rhythm of activity. Shuiov and Naor (1964) reported that X_. cheopis females from 5-10 days old were attracted more readily to male white rats than at any other age. In fact, for the first 3 days of their experiment the female' fleas were repelled. Reproductive system , Female . --In the adult female an ovary lies on either side of the mid-intestine. Each ovary consists of from 3-6 panoistic ovarioles; that is, ovarioles which lack specialized nutritive cells (Figure %), (Among the holometabolous insects, the Siphonaptera is the only order which possesses the panoistic type of ovariole, according to Bonhag, 1958.) Normally the germarium in the ovariole contains many oogonia. Here an occasional oogonial cell will undergo mitosis to form young oocytes. These oocytes move into the vitellarium or zone of growth where prefollicular tissue surrounds them. This tissue then becomes the follicular epithelium of the primary oocyte. The primary oocytes, then, are separated from each other by an inter follicular zone of tissue. The oocyte nucleus is called the germinal vesicle. Surrounding the ovariole is a thin outer epithelial sheath. Each ovariole has a terminal filament which unites to form the ligament of the ovary. The ligament is attached to the pericardial diaphragm. The lateral oviducts from each ovary unite to form the common oviduct which opens at the vagina located between the eighth and ninth abdominal stemites. The spermatheca lies below the rectum, and the Spermathecal duct opens into the front wall of the vagina.

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14 Fig. 1. --Female ovarioles showing primary oocyte CA] ; oocytes .in various stages of development (B) ; germarial region (C) : and terminal filament (D) . 130x.

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15 In normal circumstances, the flea has onlyone spermatheca, but Sharma and Joshi (1961) nave reportedi 1 an adult with two. The extra one was pear-shaped with no distinct head portion, whereas the other was of the normal hook or sickle shape. Male. — The adult male has one pair of oval -shaped testes which have one layer of epithelial tissue (Wasserburger , 1961). At the base of each testis is a cup-like structure which contains a coiled duct or epididymis (Gunther, 1961). From this coiled duct comes the vas deferens which enters the seminal vesicle (Patton and Evans, 1929) or ejaculatory bulb (Gunther, 1961). It then enters the ductus ejaculatoris from which it proceeds to the aedoeagus. There are two pairs of accessory glands (one short and one long) , which unite with the vas deferens near the testes (Figure 2) . The egg The egg of X. cheopis is oval, and when newly laid it is pearly white in color and slightly translucent. It is viscous, and adheres to the substrate (Mitzmain, 1910). The length is from 0.450 mm to 0.475 mm, and the width is 0.3 mm (Bacot and Ridewood, 1914, and Webster, 1929). There are 56micropylar openings at the anterior end and 32 at the posterior end (Cameron, 1939). Egg production Egg production in the female seems to vary. Patton and Evans (1929) state that the female may lay from 2-6 or more eggs at a time, and that she is capable of laying from 300-400 eggs during a lifetime. Mitzmain (1910) reported that the majority of eggs obtained were laid on the first day, and that the average number was 6. In 1908, the

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16 Fig. 2.^-Male, (A), testis; (B) sperm; QC) epididymis; (D) vas deferens; (E) paired accessory glands, lllx.

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Report on Plague Investigations in India reported that from 1-5 eggs at a time are laid, one after the other; however, another of these reports (1912) indicated that the average number laid per wild .flea in monthly periods for two and one half years ranged from 0.6-3.0. I Their figure vas based, however, on only one hour of egg-laying time. This report also states that the largest number of eggs was laid during the first hour after separation from the rat hosts. In 1^35, Hopkins reported that with a "perch" (Vesting, material) the average egg production per laying female was 3.8, while without a "perch" it was 4.6. Pausch (1962 ) obtained an average of 2.2 eggs per fed female in his experiments. i Buxton (1948) found that fleas produced an abnormally low number of eggs when fed on mice 9-11 days old; however, they laid more eggs when allowed to feed on adult mice for several hours before being placed on baby mice. He was of the opinion that the younger mice lack a sex hormone which stimulates egg production in the female flea. 1 Rothschild, however, stated in 1965 that X_. cheopis is not dependent on rat sex hormones. If the rat has been castrated, and its adrenal and pituitary glands removed, the flea fed upon it will still copulate and lay eggs, although it is not known if the eggs will produce progeny. Luxton also found that higher temperature produces earlier egglaying and a greater quantity of progeny per day, and that progeny from parents fed on the adult mouse emerge sooner than those fed on the baby mouse.

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18 Longevity Burroughs Ci^53) discovered that if X_. cheopis adults were fed — iiy on a guinea pig, the/ lived longer than those fed only once or apt a,t all. One male flea survived for 72 days; the maximum survival time was 153 days for a female which had had daily access to a rat. Burroughs concluded that: The opportunity to obtain frequent blood meals had the greatest effect of any single factor in producing individual long-lived fleas.. i This is also the opinion expressed by Leeson in 1936. It is interesting' to note that Bacot stated in 1914 that X. cheep is may live for up to 10 months. 1 . In 192,3, Hirst made reference to the fact that fleas held in' glass tubes should, have some type of resting material. Hopkins (1935) conducted tests to determine if the use of resting material would prolong the life of the flea, and whether or not it was conducive to egglaying, .a found that without a "perch" the average life for the male was -5.6 days and for the female 9.9 days. With a "perch," the average was 6.2 days for the male and 7.1 days for the female. Some wild-caught fleas, anesthetized with chloroform, had a shorter mean longevity. than did those not anesthetized. Crowding of larvae, which reduces adult longevity, also causes a reduction in the size of the adults (Edney, 1947b) . Longevity of adults is affected by the age of the parent fleas (Edney, :;K7b). Those from parents less than 10 days old live longer than those from parents more than 11 days old. According tc burroughs (1955), adults which have continuous access to blood meals will live longer than unfed individuals. This is the

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19 opposite of Edney's conclusion (1947b) that: Well developed, unfed, laboratory-bred adult fleas live longer than those which have fed once before starvation, and the latter live longer than those which have fed several times. X. cheopis , unlike many other parasitic insects, does not need to remain upon its host. A part of the adult life is spent in the nest of the host. The larva The larva hatches from the egg in from 2-10 days, depending on the temperature (Bacot and Ridewood, 1914, and Patton and Evans, 1929). The external morphology of the larval instars has been fully described by Bacot and Ridewood (1914), Henderson (1928), and Sikes (1930). The following general account of the larval stage is condensed primarily from writings of Bacot (1914), Bacot and Ridewood (1914), and Sikes (1930). • The newly hatched larva emerges from the egg with the aid of a hatching spine or egg-breaker which is located on the epicranium. It is about 1.4-2.0 mm long, cylindrical, semi-transparent, and abouf 0.22 mm thick. Living upon almost any kind of refuse, animal or vegetable, this larva will even feed on drops of dried blood which the adult flea has defecated. It undergoes two molts during the active stage, and one in the cocoon. While molting, it will lie straight, except in the third molt when it is bent to form a U-shape. In most instances the larva is very active; however, there are times when it will become quiescent and lie coiled' up.

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20 When the temperature is between 25-28°C, and the relative humidity between 75-80%, the larval period is from 15-15 days CKrishnamu -thy et al., 1963). The rearing media should not be wet, but the humidity should be between 75-90% for best larval growth. In 1949, Sharif reported that for the larva the critical humidity varies with the temperature. He demonstrated (1948a) that the chief source of water gain in the larva is through its food, and further concluded that the water requirement is slightly less for the male than for the female larva. The nutritional requirements for the larva are simple according to Sharif (1948b) . it can even live in the domestic rat burrow where the nutritive value of larval food is low. Pausch (1962) readily grew larvae on an artificial diet of 100 parts vitamin-free casein, 2 parts Wesson salt mixture, 1 part cholesterol and 9 vitamins. Edney (1947b) reported that if too many larvae are reared together, even though there is sufficient food, the adult longevity is reduced. He also found that if larvae are reared in fine rather than course sand, the adults are unable to develop to their normal size. The fine sand abrades the larval epicuticle, causing water evaporation which cannot be regained even in high humidity. Prepupa and pupa Prepupa. —Hopkins (1935) reported that the female has a shorter larval period than the male. The fully grown larva is creamy white in color, and the alimentary canal is devoid of food. It then spins a cocoon of silk and debris. It is active at first, and any sudden stimulus or rise in temperature will cause it to leave the cocoon (Melianby, 1933). Under normal circumstances the larva soon ceases

PAGE 29

21 movement, and remains folded double within the cocoon. This is the prepupal stage. If kept at a temperature of 24°C and a relative humidity of from 50-90%, the prepupa will gain weight by the absorption of water vapor from the air (Edney, 1947a). A relative humidity below 45% will kill it. The prepupa molts to the pupal stage in about 3.5 days. Pupa . — Under the rearing conditions presented by Krishnamurthy et al. (1963), the pupal stage was from 6-8 days. Mellanby (1933) stated that, although the pupa is resistant to dry air, the prepupa is not, so that it is necessary to maintain a humidity of at least 60% or higher, along with a temperature of between 18-35°C. Normally the pupal period of the female is shorter than that of the male Qfopkins, 1935). Edney (1947a) found that adults which have emerged from pupae kept at higher humidities weigh more, have a higher percentage of water content, and live longer without food than those emerging from pupae kept at lower humidities. Sharif (1935) discovered that the pupae of the fleas Nosopsyllus fasciatus (Bosc) , Leptopsylla segnis (Schonherr) , and Ceratophyllus gallinae (Schrank) j^ ave wing |, uds on tlie me sothorax; he could find none on X< cheopis .

PAGE 30

METHODS AND MATERIALS Rearing of X. cheopis The Orlando strain of )C. cheopis was used in all experiments. The fleas were mass-reared according to the methods of Smith and Eddy (1954) and of Gilbert (1964). Approximately 500 newly emerged adult fleas were put on a caged rat in a rearing pan containing approximately , 1.5 liters of dry sand and 100 cc of powdered dog food. The rat was fed daily with pieces of dog food and apple. Approximately 21 days later it was removed, and the contents of the pan sifted through 8and 16mesh screens to remove debris and cocoons. The sand containing eggs, larvae, and adults was then placed in an enamel pan and covered with organdy cloth. This pan was placed in an incubator where the temperature was 28±2°C, and the relative humidity was 80±5%. The sand was I sifted through the 16-mesh screen once a week for 3 weeks in order to obtain the cocoons. Cytological techniques Studies were undertaken to determine the effects of the chemosterilant tepa on flea chromosomes. Before proceeding with these studies, however, it was necessary to determine what tissue of the flea contained the most mitotic chromosomes, and at which stage of the flea's development. Many experiments proved that the best object for these studies was the supraoesophyageal ganglion of the prepupa. 22

PAGE 31

23 Under a dissecting microscope at 30X, the prepupa was dissected out of the cocoon with two pairs of extra-fine forceps in the modified Ringer's solution of Morgan and LaBrecque (1964a) . The prepupa was then transferred to a deep-well slide containing Ringer's solution. The prothorax was held firmly with one pair of forceps while the head capsule was gently pulled away with the second pair. The supraoesophyageal ganglion, easily observed as two white oblong lobes attached to the prothoracic ganglion, was then severed" from the ganglion with the forceps, and transferred for 10 minutes to a 1.0% hypotonic solution of sodium citrate. After being placed in modified Carnoy's fixative for 5 minutes, it was removed to a small drop of 45% acetic acid on a clean microscope slide which had been washed in acid alcohol. The tissue was covered with a siliconized cover slip. Placed between the folds of a piece of filter paper (150 mm), the covered slide was then gently but firmly tapped over the tissue area 20 or 30 times with a moderately heavy rod. After this, the slide, still wrapped in filter paper, was squashed with a chromosome squash l apparatus (Linkfield e t al., in press), after which it could be quickly scanned under the phase microscope for mitotic divisions. The chromosome squash apparatus is based on the fulcrum principle, the pressure being supplied by a 25-pound lead weight. The slide containing the tissue to be squashed is placed under the pressure plate within a folded piece of filter paper. Using the apparatus at 166 pounds of force (216 lbs. psi for a 7/8" x 7/8" cover slip), excellent preparations were made. The slide at this stage was placed on a piece of dry ice for a minimum of 30 minutes. Staining and permanent mounts were then made according to the technique of Morgan and LaBrecque (1964a).

PAGE 32

24 To determine what cytogenetic effects tepa has on the somatic chromosomes, a test was initiated using 20 larvae which were ready to pupate. A filter paper with a diameter of 9 cm was treated with tepa 2 at 10 mg/cm , and allowed to dry for about 16 hours. The larvae were placed on the treated filter paper for 4 hours. They were then removed to untreated paper for a 24 -hour period; After this time, the supraoesophageal ganglions of several larvae were prepared according to the procedures previously described. Experimental design (To achieve maximum readability, the word "treat" in all its parts of speech is used only in reference to the use of the chemosterilant .) Male laboratory white mice from 30-40 days old were used as hosts in all experiments. A preliminary test showed the following procedures to be the most expedient for all experiments. Flea populations for experimental use . --Prior to the treatments, newly emerged adult fleas were collected in a one-quart Mason jar containing a 9 cm filter paper upon which the fleas could rest. A piece of black organdy cloth was placed over the mouth of the jar before the rim was screwed on. The fleas were kept in the dark until they could be sexed, which was usually within 24 hours. Ether, U.S. P., was poured into a screw-top, ten-dram glass vial which contained a crumpled sheet n of Kimwhipe paper, type 900-S. When not in use, the vial was kept in an explosion-proof refrigerator. On the second day, the adult fleas were sexed. The glass vial containing ether was slowly lowered into. the Mason jar containing fleas, and the jar lid was screwed on tight until all activity had ceased,

PAGE 33

25 t after which time the vial was removed. The anesthetized fleas remained inactive for about 10 minutes. Approximately 30 fleas were poured into a plastic petri plate top which was then placed under a zoom binocular microscope and viewed at 10X. A suction apparatus QPig. 3) was used to pick up the fleas. Male and female fleas were put into separate onequart Mason jars, each of which had a 9 cm filter paper placed on the bottom. The adult fleas were held in the dark for recovrry from the anesthetic. Chemosterilant treatment . --Whenever chemosterilant treatments were being conducted, rubber gloves were worn, and treatments were made in a negative-air-flow hood. 2 Filter paper strips (Fig. 4) used in all experiments were 7.3 cm , similar to those reported in the World Health Organization Technical Report of 1963. Before treatment , the paper strips were securely stuck to a pinning board (Fig. 4). A small galvanized enamel pan was partially filled with cider vinegar (or 3% acetic acid), and a vial containing the chemosterilant ,tepa dissolved in methanol was set in the pan. A pro-pipet was fitted to a 1 ml pipet, and 0.13-0.16 ml of the chemosterilant solution was applied to the filter paper strips at a 2 dosage of either 5, 10, or 15 mg/cm . With a clean pipet, 0.13-0.16 ml of methanol was applied to all control strips. Both groups of strips remained in the hood for 24 hours to dry. After treatment of strips, cider vinegar was poured into the chemosterilant vial in order to deactivate the remaining active material before the vial was discarded. The pipet and all other reusable materials which came in contact with the chemosterilant were

PAGE 34

26 •Fig. 3.— Vacuum aspirator used to sex adult fleas,

PAGE 35

Fig. 4. --Pinning board used for treating filter paper strips

PAGE 36

I 28 thoroughly soaked in cider vinegar for 5 minutes before being washed in soapy water. The treated filter papers were picked up with a pair of forceps, and placed in 18 x 150 mm test tubes. Twenty fleas were placed in each of 5 tubes, the open ends of which were covered with a piece of white organdy cloth held in place by a cardboard collar. Depending on the type of reciprocal cross experiments to be conducted, either males or females were treated with the tepa. The fleas were held in the tubes for varying lengths of time (2, 4, or 6 hours). After the required treatment time, the treated fleas were transferred to post -treatment test tubes each of which contained a clean filter paper strip. Controls were transferred to like tubes at the same time. The open end of the post -treatment test tube was placed below the treated one at an angle of about 40 degrees. The treated tube was gently tapped until the fleas fell into the clean tube. If the treated filter paper started to slide toward the clean tube, the tubes were separated and held upright while the treated paper resettled on the bottom, after which the procedure was repeated until all fleas were in the post -treatment tube. Sometimes a few fleas could not be dislodged from the paper strips by this method; however, when the tubes were held close to a light, the fleas would begin to move, at which time they would be tapped into the clean tube. These steps were taken in order to avoid contamination of an aspirator. Contaminated test tubes and filter paper strips were handled as previously described for contaminated material. Sterility experiments . — Treated adult fleas were held in the dark for 24 hours, after which they were checked for mortality. Those not dead were used for determining which dosage effected sterility.

PAGE 37

29 In initial sterility experiments, 5 male and 5 female fleas were used, but in subsequent testings it was decided to use 10 fleas of each sex. In order to demonstrate sterility, the following reciprocal crosses were made: , X. Untreated females X Untreated males 2. Untreated females X Treated males 3. Treated females X Untreated males 4. Treated females X Treated males Mice confined in small, round, wire cages were lowered into onequart Mason jars with an unwound paper clip of the larger size, bent to hook at one end: The bottom of each jar contained a 9 cm filter paper, along with a strip of blotter paper to absorb the mouse's urine. An equal number of both sexes of fleas was put into a labelled Mason jar with the caged mouse. The jar was then placed in the larger n side of a double-compartment Vacucel ice chest (Fig. 5). About two quarts of water were poured into the smaller compartment. Black organdy cloth was draped over the top of the ice chest in order to maintain darkness and as much humidity as possible. The caged mouse was removed from the Mason jar daily, fed, placed in a freshly prepared jar, and returned to the chest. It should be noted that, when feeding the mouse, -care had to be taken not to lose fleas. The mouse cage was picked up with the paper clip hook in one hand, grasped with the fingers of the other hand, and quickly transferred to the new jar, with food being given to the mouse through a hole in one end of the cage before it was completely lowered into the jar. In most instances the fleas were around the tail area of the mouse, but a bulb-aspirator was kept at hand when making transfers in case a flea would jump off.

PAGE 38

30 Fig. 5. — Vacucel ice chest used to maintain darkness and humidity for experimental mice and fleas in Mason jars. (A) black organdy cloth; QB) water compartment; (C) compartment for Mason jars. ' i

PAGE 39

The primary reason for placing the mice in cages was to prevent them from eating the adult fleas, as was reported byLeeson (1936). Cameron (1939) stated that when fleas were first introduced upon a i healthy mouse, it ate many of them. The used Mason jars were taken to the binocular microscope to look for flea eggs. It was found best to remove, the papers from the experimental jar before aspirating out any fleas to be transferred to the clean jars. In order to position the blotter and filter papers for removal from the jar, the paper clip hook was used, with extreme care being taken not to damage the eggs. The blotter paper was removed with a pair of forceps and placed in a plastic petri dish cover 100 x 15 mm. It was then scanned under the binocular microscope at 10X. If eggs were found, a drop of modified Ringer's solution was put on the paper close to the egg, softening the paper and allowing the egg to be picked up with ease by the use of fine needle-nosed forceps. The egg is less likely to be damaged by this method, as some of the paper is removed with it. After the top. of the blotter paper had been scanned, the reverse side was checked. This same procedure was carried out on the filter paper, and on all droppings and debris left in the bottom of the jar. The flea eggs were placed on a 3.8 cm black organdy cloth disc in a plastic petri dish, which was then set in an incubator (Fig. 6). The incubator was kept at 26±2°C, with a relative humidity of 86±2%. A whisper fan was installed to provide air circulation, and the humidity was maintained with a sponge partially immersed in distilled water in a plastic food container. The entire humidity unit was changed weekly, or sooner if necessary. The temperature and humidity were checked daily

PAGE 40

32 Fig. 6. — Incubator, (A) whisper fan; (B) sponge and water container for maintaining humidity; (C) petri dishes containing eggs and growing larvae; (D) temperature-humidity probe; (E) temperature-humidity recorder.

PAGE 41

53 with the aid of an, electric hygrometer indicator (Hygrodynamics, Inc.). The temperature -humidity probe remained in the incubator throughout the testing period. The eggs vers , checked dai / for hatch for 6 days. Larvae were picked up with a very fine paint brush (jf£d) , and placed in a dated plastic petri plate containing sand and ground-up rat chow. The petri plate, narked with the date on which the eggs were laid, was placed in the incthator. At the beginning of the third week an emergence chamber was placed ever the rearing media so that emerging adults could not escaoe. The e'.'.erger.ce chamber . --The bottom was punched out of a one-pint cardboard ice cream container, and the top ridge trimmed off with a razor blade. The unridged top was then gently inserted into the petri dish containing larval medium and maturing larvae. The container was fastened to the petri plate with cellophane tape, and organdy cloth was s -cured to the top with a, rubber band. These containers made ideal adult emergence chambers (Fig. 7). When adults began to emerge, the or:,_.-.dy cloth was removed and they were aspirated out of the chamber each day. Statistics . — The negative binomial transformation of Bartiett (1C47) was us.:, to stabilize the variance of the original egg production data'. The pooled "t" test of Snedecor G-S62) was then used to iest the difference in egg production between first and second weeks of control and of chemosterilant experiments. The paired -Z" test of Mendenhall (1964) was v:ed to determine the di :ence in emergence of larvae and F-^ adults in control and chemosterilant experiments. Percent sterility was figured according to the . ?
PAGE 42

34 t Fig. 7.— Adult emergence chambers

PAGE 43

35 The predicted egg production curve was calculated using the formula y=KCl-e~ yt ) where y= egg production; K= asymptotic constant; e= 2.7183; ?= growth constant; and t= time. Pho t o g r aphy . Pho t om i cro g r aph s were made through an American r Optical Phasestar microscope with Ortho-illuminator. They were taken on 155 mm Panotomic X, Black and White film (ASA 32), and the green filter of the Ortho-illuminator was used.

PAGE 44

RESULTS AND DISCUSSION Biological Observations Ovarioles Wasserburger (1961) stated that he regularly found 5 ovarioles in each ovary of the female X. cheopis and Patton and Evans (1929) 4-6. While measuring the ovarioles and testes of the Orlando strain of the flea (Table 1), this author found ovaries with 3, 4, and 6 ovarioles each. Although none was found with 5 ovarioles, they could very well have been present in the Orlando strain. The variation in ovariole number could explain why there was some difference in egg production from one test to another, especially in one control test. There was no difference in overall egg production between the first and second weeks of 12 experiments £=1.657
PAGE 46

38 Table l, --Measurements of gonads of 30 male and 30 female X. cheopis adults. j Dimension Mean in mm Range in mm Standard Deviation MALE Length of: Right testis .62 Left testis .63 .53-. 70 .49-. 70 .014 .020 Width of: Right testis .34 Left testis .33 .32-. 39 .28-. 35 .007 .007 FEMALE Length of: Ovariole .66 Width of: ' Primary oocyte .10 .53-. 77 .07-. 14 .015 .008 I

PAGE 47

• ^.y. " 39 BffVThe egg production per female from 10 females in a control experiment is shown in Fig. 9. This experiment, which was continued for double the initial 14 days, showed a higher egg production than any other. The increase is possibly due to the fact that almost all of these happened to be females with more ovarioles than the other experimental females. Nevertheless, the data are valid and informative. It is interesting to note that the highest daily egg production was 12.6 eggs per female. These females laid 80 or more eggs on 15 different days, while less than 80 eggs were laid on the other 13 days. Ratio of male to female The ratio of male to female adult fleas is very near 1:1. Bacot et al. (1914) proposed that in nature the females of X_. cheopis are in excess of males' by 15.05%. Their specimens were collected from host animals in the field. As can be seen from Table 2, their assumption is borne out in a calculation of the sex ratio in 2 of the author's experiments. Here all fleas were sexed and counted, and females were found to be in excess of males by 13.66%. Apparently males remain on the host as often as do the females; during observations by this author, male fleas were found mainly on the host. The only time they were seen off the host was when they were with some of the females on the filter paper. Behavior According to Wigglesworth (1964), the hormonal control of reproduction might be stimulated by the act of copulation, as in the cockroach, Diploptera punctata . In this species, mating appears to be nee^d for the normal rate of egg growth. This might also be true of X_. cheopis .

PAGE 48

3WW3J aad sooi

PAGE 49

Table 2. --Ratio of female to male in laboratory-reared and in field-collected X. Ciieotds adult populations. PERCENTAGE OF j excess females Females Males over males Laboratory-rear ed a 53.20 46.80 13 66 • ; Field-collected b 55.50 46.50 15 05 "755 emarging F-j_ adults in 2 of the author's laboratory ' experiments. ^3,439 fleas collected in several Egyptian locales, and examined by Bacot et al. ^1914],.

PAGE 50

42 Morris (1954) found that the female Schistocerca gregaria must copulate in order to lay her full complement of eggs. Bacot (1914) reported that in his experiments no X< cheopis female laid any eggs without having fed first. He also stated that oviposition has always been preceded by several days of feeding. Copulation, however, does not appear to be necessary for egglaying (Fig. 10). The stimulating factor for both copulation and egg-laying, then must be blood meals. Females which have not had a blood meal will neither copulate nor lay eggs. Nevertheless, virgin females will lay eggs at least for a period of 14 days, and probably for longer if they have access to the necessary blood meals. The number of eggs produced decreases with time, but does not appear to cease completely. There seems to be a difference in egg-laying behavior between mated and virgin females. Mated females will lay their eggs in clusters, or sometimes singularly, on or underneath the filter paper, on mouse feces, or on bits of rat chow, in most instances these eggs will have bits of rat chow attached to them (Fig. 11). They are difficult to see (Fig. 12) without correct lighting. The surface of the egg has a sticky substance which, when dry, adheres the egg to the substrate, and bits of rat chow to the egg. In view of the preceding observations, it is difficult not to believe that the female fleas have actually placed bits of the rat chow aTOiind the newly laid eggs; as many of these covered eggs can be found several inches from the main area of debris. Even if they had fallen from the mouse onto the rat chow bits, it is unlikely that they could have rolled so far away and almost impossible for them to have adhered

PAGE 51

4 5 » « j % I a "0 cd o H ^* s I/, o i ~ ' cd £ o w •H P. O o x: u x'l c •H M h •H > o "O cd tj o> *j cd £ tt-l O (3 o H I/) 4-> — i O cd 3 o TJ £ 0 ^ -o ft O o M .-1 M ,£> o o a> H W) al 1/1 1/1 o > o < o — o o 3 C 04 00 •H +J O u 21VVV3d 23d SOQ3

PAGE 52

44 Fig. 11. — Bottom view of egg of X_. cheopis adhering to bits of rat chow. 44x.

PAGE 53

45 Fig. 12. --Top view of X. cheopis egg (A) which is stuck to the filter paper, and surrounded by bits of rat chow. 44x.

PAGE 54

46 i to the filter paper. Closer observation of egg-laying, and further I research into this probable behavior, would be most interesting. Eggs from virgin females do not have this sticky substance, since their eggs can easily be removed from the substrate with a dry paint brush or with forceps. No virgin females were observed off of the host even though mated females were often seen on or under the filter paper. Copious feeding causes extrusion of blood from the anus, providing nourishment for the larvae. The placing of food around the egg could be a behavior pattern for providing food for the emerging larvae, in which case the virgin female is unable to provide it. This behavior, as well as the production of the sticky substance, could be caused by stimulation of the female collaterial glands during copulation. There is also the possibility that the male inseminates a substance during copulation which stimulates the female to initiate these behavior patterns. Parthenogenesis , According to White (1964) there appears to be no literature regarding parthenogenesis in the Siphonaptera (Aphaniptera) an d in five other orders of insects. In the virgin female egg production experiment, it is interesting to note that of 149 eggs produced in 14 days, 4 underwent partial embryonic development (Table 3) . In one egg the head capsule was easily found, while in the others only setae were observed. These embryos did not hatch. Suomalainen C 1962 ) called this rudimentary parthenogenesis; that is, when an unfertilized egg from a bisexual speci <>s starts to develop, it can be expected eventually to stop

PAGE 55

47 iaoie 3. --Production and development of eggs from virgin female X. cheopis which had continuous access to blood meals. tv ^ **a Day Eggs Produced Embryos Formed Hat en 1 £• O 1 u 0 21 • 0 n 3 19 0 .0 4 17 1 0 5 18 1 0 6 7 :£'y$§J|v . 0.$|||§|p 0 7 6 0 0 8 ' 5 1 0 9 10 1 0 10 4 0 0 11 2 , 0 0 12 3 0. 0 13 5 0 0 14 7 0 0 Totals: 149 4 0 a Eggs were first observed on the 3rd dayafter placing fleas on host.

PAGE 56

48 development and die. Sometimes, however, an unfertilized egg will hatch and produce an adult; this is called tychoparthenogenesis (accidental parthenogenesis) . In certain species of Drosophila the rate of parthenogenetically developing adults was 2/37,628 and 1/19,059 (Suomalainen, 1962). Since it has been shown that embryonic development can take place in eggs laid by virgin X.' cheopis females, further experiments into the possibilities of tychoparthenogenesis should be initiated. General observations Life cycle . --The author, under the conditions of his experiments, found that the life cycle could be accomplished in 15 to 21 days at a temperature of 26±2°C, and a relative humidity of 86±2%. By 28 days emergence was complete. Each experiment took approximately 6 weeks to complete. Larval and F-[ adult emergence . --Figure 13 shows the daily percent total number of eggs deposited, and larval and F-^ adult emergence from the 4 control experiments. The total larval emergence was 81.09%, while the F^ adult emergence was 90.12%. Feeding site .—Bacot (1914) stated that rat fleas pick a special "point of vantage, X_. cheopis making for the shoulders, neck and chest or for a spot beneath the forelegs." This author, however, observed that the fleas in all his experiments were usually to be found around i the tail and scrotal area of the caged mouse. In fact, newly introduced fleas went directly to the posterior end. Additional studies would no doubt show whether or not this is the preferred feeding site. It is possible that, since mice usually clean themselves quite well around these areas, they eat many of their pests found there, and this is why

PAGE 57

«-"-C-^ S302 }o zZZVt.V.i IVIOI %

PAGE 58

50 fleas on uncaged mice were found in the areas mentioned by Bacot, even though they prefer the other. , Chemosterilization with Tepa Egg hatch , , Eggs froir female fleas which had been either treated, or crossed with a treated male, were retained for 6 days, after which, if there had ': n no larval emergence, they were discarded. The control larvae usually had emerged by the third day. Data on egg-laying and on larval and P. adult emergence for all of the chemosterilant experiments and for the controls are summarized in Table 4. Tables 5 through 24 in the Appendix contain the results of each individual experiment. Natural sterility The natural sterility obtained in 4 laboratory control experiments was 18.45% in the larvae and 10.14% in the adults. Egg breakage In an initial sterility experiment many eggs were broken in an effort to remove them from the filter paper. Since 34 eggs from the control alone were broken in the first 8 days, it was decided to try Ringer's solution in order to soften the filter paper and allow easier removal of eggs. On days 9-14 the solution was used. Only 6 eggs were broken during this 6-day period. It appeared that with Ringer's solution egg breakage would be reduced considerably and larval hatch improved . Therefore a statistical analysis using the paired "7." test was !;.a< «. Sincr Rinf.er's so.luf-ion Was fountl by this myolysis to improve hatch (2=2.833 ^> Z ^,.=1.645) , it was used to remove eggs from the filter paper in ail experiments.

PAGE 59

51 0) (0 60 BO U pi «« J3 3 .3 T3 0) 3 •P 3 •a «* ai o d 0)OJ to a <3 O i 41

o (> o> o o o o o o O O Io o o o o o o o no H -O o o o o O O no N « O 0* fi d o m «* H Nf N rl co n^ co 1/} CM P I n) o> H H o o o o> o n n o« on lo t> o 0> (ffl o> o> M O M H d*dd o o o o o o o o co rc-i oi o o o o o o o o 3 CUM CO ^ CO 01 p to u co o d o o o o o CO O CO o iiicA \n \n io ei cm no co J> IB 01 M H O O O o o o p o NO 01 H s •a o> •p • a I p § X ai •a 0) p hi o> n •p i I. O NO N*f O o o o o o 3 o o I 1 NO * NO Nf NO NO NO Ij* T3 8 2 o X> 01 X3 o CO P^ •H 3 <*4 o o> > c 0) 0) X) o c -o 5 M 01 •p to i>? o o 01 J3 ,3 « • O NO-JJ ID c c E & 0) rO "" 0> to I * -p 0) S 8. 0) e n U 3 01 43 B-4

to IS to 01 >

PAGE 60

52 i In the first hour after oviposition, the zygote is formed in the egg (Cameron,' 1939) . For the first 8 days (no Ringer's solution) the larval hatch was only 60%, while for the remaining 6 days (when the solution was used) the larval hatch was 73.60%. It is possible that maturation of the egg nucleus and fusion of the pronuclei had been disrupted during removal from the filter paper without Ringer's solution. Female sterilization 5 mg/cir. .--Ten females treated with tepa at 5 mg/cm^ for 4 hours and mated with untreated males, had a decrease in egg production the first week (t=3.53> t =2.447), while in the second week there was no v .05 significant difference between egg production of the control fleas and of those treated (t=2.39 Z =1.645), while in the .05 second week there was little difference (Z=0.372 <^Z =1.645). The emergence of the F^ adult generation, however, was significantly reduced in both the first and second weeks (Z=5.556> Z ^=1.645 in the first week; Z=4.56Q^> Z =1.645 in the second). , The larval sterility was calculated to be 69.20% and the ? 1 adult sterility, 84.00%. As is obvious from these analyses, it is more accurate to base conclusions on the generation rather than simply on larval hatch. Therefore, the criterion for sterility used in all of these studies was the emergence of F^ adults. Bertram (1963) reported that thio-tepa (tris (l-aziridinyl)phosphine sulfide) caused reduced fertility and fecundity in female A. aegypti . Rai (1964) stated that eggs from female A. aegypti treated with apholate must carry induced dominant lethals, since a low hatch was obtained. This is probably true of tepa-treatcd cheopis females as well.

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53 Twenty females treated with tepa at 5 mg/cm for 6 hours, and mated with 20 untreated males, produced some eggs. In one of the experiments 10 females laid eggs for the first 2 days, after which time they did not lay again until the eighth day. Apparently, the primary oocytes were not affected by the sterilant, but the secondary oocytes were. At this dosage and contact time, it seems that the germarial region of the ovarioles was not affected, since they did begin to lay e t^s again. These females were mated with untreated males, and larval hatch was observed from eggs laid on all days except the first. In this experiment no F adults were produced from larvae which emerged on the second and the tenth days. Nevertheless, F l adults did emerge . from the eleventh through the fourteenth days of larval hatch. The larval sterility was 98.20%, and the adult sterility was 99.28%. 2 10 mg/cm .--Murray and Bickley (1964) had noted that vacuoles occurred in the ovaries of Culex p_. quinquefasciatus when they were treated with apholate in concentrations of 15 ppm and higher. The female X_. cheopis can be completely sterilized (100%) with tepa, using a dosage of 10 mg/cm 2 for a contact treatment time of 4 hours (Table 4). As can be seen in Fig. 14, the ovarioles are completely destroyed. The remnants of the ovarioles are leathery, opaque, and white in color, and are vacuolated. These females had mated with untreated males; Fig. 15 shows a spermatheca with motile sperm in the head. 'i .'iale sterilization In the treated males, 100% sterility was not obtained, although the Gosage and treatment contact times were greater than those of the females. Nevei I '•.closs, 99.99% sterility was achieved (Table 4).

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Fig. 1^.--Ovari'oies of fleas treated with tepa at 10 rag/cm* for 4 hours. Note destruction of ovarioles, and vacuolation. I30x. . •

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55 Fig. 15.--Spermatheca of a female flea treated with tepa at 10 mg/qn* for 4 hours. Spermatheca head (A) contains motile sperm. 120x.

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56 The fact that the males were almost completely sterilized did not affect the egg production of the untreated females; larval hatch and F^ emergence, however, were greatly reduced. , 5 mg/cm 2 . — In an early experiment, 5 males were treated with tepa at 5 mg/cm 2 for 6 hours, and mated with 5 untreated females. There was no significant difference in egg production between the control and treated for the 2-week experiment (t=0.0318
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57 sterility had not been achieved, and treated male mortality was 10% in the 24-hour post -treatment holding period. In the lower dosage experiments, no treated adult males died during the post -treatment period. Inability to achieve total male sterility . — Several possibilities present themselves as to why the adult male flea cannot be made totally sterile. Wheeler (1962) states that the alkylating agents (of which tepa is one) can affect directly or indirectly the alkylation of nucleoproteins. Since the alkylation would interfere with mitosis, the rate and replication of deoxyribonucleic acid (DNA) could be altered. Also, DNA is most likely the primary site of alkylation and therefore the most sensitive. Kilgore (1965), working with house flies, reported that the alkylating agents "have a very pronounced effect on the metabolism of the nucleic acids." This is probably also true in X. cheopis , reducing the synthesis of DNA so that the egg cannot form lactic acid dehydrogenase. In a personal communication (1966), Rothschild confirmed this . author's observation that in male flea testes, spermatogenesis is essentially complete when the male emerges from the pupa. At this -stage the testis has its full complement of sperm. There is no germarial region in the adult male. Since spermatogenesis is either completed or almost completed upon emergence, the metabolism of the sperm in the testis is probably quite low. In other words, transport of nutrients I and gas exchange are not actively taking place, as they would be during spermatogenesis. If this is the case, the chemosterilant would probably have to diffuse through the epithelial layer of the testis, and penetrate the sperm heads.

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Keiser et al. (1965) state that in chemosterilized fruit fly males both the spermatogonia and the spermatocytes are destroyed, but that the spermatids which are beyond the last division continue to develop and to mature. It would seem that certain individual sperm have probably completed spermatogenesis, thus being unaffected by the alkylation of the DXA, while the remaining 99.99% of the sperm have been affected. This would cause the sperm inactivation described by LaChance (1967) and, therefore, the absence of pronuclear fusion, producing sterile eggs. Sperm transferrer by tepa-treated males during copulation is probably inactivated sperm or sperm which contains dominant lethal mutations. Aspermy has certainly not taken place in such a case, since sperm were found motile in the spermatheca of the females at the termination of 14-day experiments. Oviposition in these experiments was not affected when treated males were mated with untreated females. This condition was also reported by Mitlin et al. (1957) in the house fly. Bertram (1963) observed that even up to 32 days after treatment with thio-tepa the males of A. aegypti had active sperm, and that these sperm were abundant in the untreated female spermathecae, Fahmy and Fahmy (1964) observed that ur.:reated Drosophila females, mated to males treated with varying dosages of the chemosterilant tretamine (TEM) , had a very high number of unmatched eggs. Rai (1964) reported that sperm of male A. aegypti treated with apholate produced dominant lethal mutations, since e^g hatch was extremely low. The chemosterilant might cause a mutation which acts as a gametic lethal, or as an early zygotic lethal (Fahmy and Fa"....y, 1964). .

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59 Apparently there are two types of sperm transferred by tepa-treated X_. cheopis males. With the first (inactivated sperm), embryonic development does not take place, and the egg color remains essentially the same. With the second type of sperm, however, embryonic development does take place, but the embryos cease growth at various stages of development, and die (dominant lethal mutation). In some of the eggs setae, head capsules, and segmentation of larvae can be seen. In others there appear to, be healthy larvae; however, they never emerge. Dominant lethal mutations seem to affect not only the embryo, but the larvae as well. Upon emerging, larvae from the treated fleas in these experiments did not appear to differ from the control larvae. They were active, and readily burrowed into the larval media, as did the controls. Yet, as can be seen from Table 4, the percent sterility of adults from larvae of treated males is higher than that of the emerging larvae. Therefore, some of these larvae were certainly affected. It was not determined at what stage of development ,the larvae died, but it would be interesting to know at exactly which stage death occurred. This could be done by rearing the larvae in artificial media according to the method of Pausch (1962). Treated females X treated males , The percent sterility obtained from the mating of treated fleas with untreated fleas indicated that mor,e efficient results might be obtained when both sexes were treated with tepa. Therefore, 10 females treated with tepa at 5 mg/cm 2 for 6 hours were mated with 10 males treated with tepa 10 mg/cm 2 for 4 hours. The females laid a few t

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eggs during the first 3 days of the experiment, after which no more were laid. No larvae were produced. With such results, two factors seem to be involved. First, as can be seen in Fig. 16, the ovarioles were almost destroyed. The remnant of one oocyte can be seen (A) , and these ovarioles are also leathery, opaque, and white in .color, as well as being vacuolated. Second, the sperm from the male must have been inactivated by tepa, since the treated male testes, as well as the spermathecae of the females, contained motile sperm. 1 Cytological effects of tepa Since no work, had been done previously on the cytological effects of radiation or chemosterilization of X_. cheopis , it has been essential to incorporate descriptions and terminology of other authors on other insects in order to interpret the results on X. cheopis . Bayreuther (1954) was the first to report the chromosome number in X_. cheopis from meiotic division. The female flea has 2n=18 chromosomes with the sex chromosome being X 1 X 1 X 2 X 2 . The male flea has 2n=17 chromosomes with the sex chromosomes being trivalent X^Y, as in the Mantids. , The mitotic chromosomes of X. cheopis are J-shaped (atelomitic) or V-shaped (metacentric) f i n t ne terminology of White (1951). These shapes can be seen in Fig. 17. Brains from treated larvae were examined for chromosomal aberrations; however, no changes were observed after 24 hours. Another sample, examined 48 hours after treatment, showed chromosomal aberrations.

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62 Fig. 17. — Untreated female mitotic metaphase chromosomes (2n=18) from the brain of the prepupa. 1667x.

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63 The terminology used by Rai (1964) 'in describing chromosomal aberrations in A. aegypti is usually applied here to describe the mitotic effects. of tepa on the flea. He grouped the induced aberrations into three principal categories: physiological and structural aberrations, and miscellaneous effects which include induction of somatic polyploidy and probable interference with the normal replication mechanism. In Fig. '18, two types of aberrations can be observed in the 4 , chromosomes visible (all others having been squashed out of the field of view). The first, physiological, is chromosome stickiness. Stickiness is most pronounced at the chromosome ends. The second, structural, could not be found in Rai's descriptions; however, Catcheside (1948) has diagrammed chromosome structural changes induced by radiation. According to his description this tepa-induced aberration would appear to be an inter-arm chromatid exchange aberration. Chromosomal constriction seems to be visible in Fig. 19. This condition might be due either to the ends sticking to each other, or to fusion at two broken ends. In Fig. 20 there can be readily observed fragments similar to those shown by Purdon (1963) , and there appears to be an isodiametric fragment similar to the type noted by Catcheside (1948). A dicentric chromosome is present. There appear to be many recombinations, rather than deletions as photographed by Murray and Bickley (1964) with apholate on £. p_. quinquef asciatus , and by Flint (1964) with radiation on H. pusio . It would seem, then, that tepa induces non-random breaks. As with apholate, tepa may also break the DNA core, and yet not completely break the chromosome matrix envelope. i

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64 Fig. 18. --Metaphase chromosomes from tepa-treated larval brain. (A) chromosome stickiness; (B) inter-arm chromatid exchange aberration. 1667x.

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65 Fig. 19 . — Metaphase chromosomes from tepa-treated larval brain. (A) chromosomal constriction. 1667x.

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66 Fig. 20. — Metaphase chromosomes from tepa-treated larval brain. (A) chromosomal fragments; (B) isodiometric fragment; (C) dicentric chromosome. 1667x.

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67 Piapp et al. (1962 j reported that with the chcmosteriiant , labelled methaphoxide (tris (2-methyl-l-aziridinyl)phosphine oxide), there was no difference in the metabolism of this compound in organophosphate-resistant and -susceptible house flies. This finding is supported by Wheeler (1^62) who states that as yet no mechanism of resistance to alkylating agents has been definitely established. Field application Biology . — The potential egg production of X. cheopis females can be determined using the results obtained from the predicted egg production curve in Fig. 8. After flea population counts have been made in the field, the size of future populations can be predicted. Thus, one would know when measures for control of the flea would be most appropriate for maximum effectiveness. Using the rearing method described herein, one could determine the effectiveness of a chemosterilant by the percentage of F^ emergence from field-collected specimens. Chemosterilization . --For practical field application of a sterility program, one essential factor is that the sterilized insects be able to mate with non-sterile individuals. Even though chemosterilant mortality would reduce a population, it would be better to have sterilized individuals live and mate with the non-sterile ones. Now that sterility with tepa has been achieved in X. cheopis , it should be determined if this flea can become sterilized by feeding on tepa-fed rats or mice. If so, bait stations with the chemosterilant could be set up for the eradication of rodents and their ectoparasites.

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SUMMARY The female X. cheopis can be completely sterilized with tepa at 2 a dosage of 10 mg/cm for a contact treatment time of 4 hours. The ovarioles are virtually destroyed with only remnants left. Treatment of females with a dosage of 5 mg/cm 2 for 6 hours gave 99.28% sterility based on the emergence of F 1 progeny. Better than 99.99% sterility could not be achieved in males at dosages of 5, 10, or 15 mg/cm 2 for 4 and for 6 hours. When females treated for 6 hours with tepa at 5 mg/cm 2 were mated Kith males treated with tepa at 10 mg/cm 2 for 4 hours, 100% sterility was achieved. Larvae ready to pupate were treated with tepa at 10 mg/cm 2 for 4 hours. Forty-eight hours after treatment, tepa-induced chromosomal aberrations were found in the larval brain. These aberrations appeared to be: (I) chromosome stickiness; (2) inter-arm chromatid exchange; (3) chromosomal constriction; (4) chromosomal fragments; (5) isodiometric fragments; and (6) dicentric chromosome. These aberrations were compared with untreated larval brains in mitotic division. It was found that the females of the Orlando strain of X. cheopis have 3, 4, or 6 ovarioles per ovary. In 12 experiments lasting 14 days, the average number of eggs laid daily per female was 4.251.21; the highest was 12.6. Bleed meals are necessary for egg-laying. 68

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69 E~gs of mated females are sticky and adhere to the substrate, as do bits of food. Mating appears to stimulate a gland in the female to produce this sticky substance which is lacking in the eggs of virgin females. Four out of 149 eggs laid by virgin females underwent partial* embryonic development, but died before hatch (rudimentary parthenogenesis) .

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REFERENCES CITED Alexander, P. 1960. Radiationimitating chemicals. Sci. Amer. 202(1): 99-108. Alexander, P., and K. A. Stacey. 1958. Comparison of the changes produced by ionizing radiations and by the alkylating agents: evidence for a similar mechanism at the molecular level. Part IV. Ann. N.Y. Acad. Sci. 68:1225-1237. Altman, R. M. 1963. The effects of tepa on Plasmodium gallinaceum in Aedes aegypti . Am. J. Hyg. 77 (3) :221-227. Atlas of Plague: 1952. American Geographical Society of New York; issued with geographical review. 42(4). Bacot, A. W. 1914. LXIX. A stud;of the bionomics of the common rat flea and other species associated with human habitations, with special reference to the influence of temperature and humidity at various periods of the life history of the insect. J. Hyg. Plague Suppl. Ill: 447-654. Bacot, A. W. , and W. G. Ridewood. 1914. Observations on the larvae of fleas. Parasitol. 7:157-175. Bacot, A:, G. F. Petri, and R. E. Todd. 1914. The fleas found oh rats and other rodents, living in association with man, and trapped in the towns, villages and Nile boats of upper Egypt. J. Hyg! 14:498-508. Barnes, J. M. 1964. Toxic hazards and the use of insect chemosterilants. Trans. Roy. Soc. Trop. Med. Hyg. 58:327-332. Bar-Zeev, M. , and S. Sternberg. 1962. Factors affecting the feeding of fleas QCenopsylla cheopis Rothsch.) through a membrane. Ent. Exp. Appl. 5:60-6S. Bayreuther, K. 1354. Die chromosomen der Flohe (Aphaniptera) . Die Naturwissenchaften. 41:26. Bertram, D. S. 1963. Observations on the chemosterilant effect of an alkylating agent, thio-tepa, on wild-caught Anopheles gambiae var. melas (Theo.) in Gambia, West Africa, and on laboratory-bred A. g. gambiae Giles and Aedes aegypti (1.). Trans. Roy. Soc. Trop.~Med. Hyg. 57 C5) : 322-335. 70

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71 Bonhag, P. F. 1958. Ovarian structure and vitellogenesis in insects. Ann. Rev. Entomol. 3:137-160. Burden, G. S., and B. J. Smittle. 1963. Chemosterilant studies with the German cockroach. Fla. Ent. 46:229-234. Burroughs, A. L. 1953. Syl vatic plague studies. X. Survival of rodent fleas in the laboratory. Parasitol. 43:229-234. Buxton, P. A. 1941. The recorded distribution of certain fleas. Bull. Ent. Res. 32:119-122. 1948. Experiments with mice and fleas. Parasitol. 39:119-124. Cameron, D. 1939. The embryological development of the Asiatic rat flea (Xenopsylla cheopis Rothschild). Thesis. Cornell Univ. 52 pp. Catcheside, D. G. 1948. Genetic effects of radiations. Adv. in, Genetics. 2:271-358. Chamberlain, W. F. 1962. Chemical sterilization of the screw-worm. J. Econ. Entomol. 55(2) :240-248 . Crystal, M. M. 1963. The induction of sexual sterility in the screwworm fly by antimetabolites and alkylating agents. J. Econ. Entomol. 56(4) :468-473. 1965. First efficient chemosterilants against screw-worm flies (Diptera: Calliphoridae) . J. Med. Entomol. 2 (3) :317-319. Dame, E. A., and H. R. Ford. 1964. Chemosterilization and its permanency in mosquitoes , Nature. 201 (4920) :733-734 . i Dame, D. A., D. B. Woodard, and H. R. Ford. 1964a. Chemosterilization of Aedes aegypti (L.) by larval treatments. Mosq. News. 24(1): 6-14. Dame, D. A., D. B. Woodard, H. R. Ford, and D. E. Weidhaas. 1964b. Field behavior of sexually sterile Anopheles quadrimaculatus males. Mosq. News. 24(1): 6-14. Dustin, P. 1947. Some new aspects of mitotic poisoning. Nature. 159 794-797. Duvall, L. R. I960., Agent data summary. Tris (l-aziridinyl)phosphine oxide (TEPA) . Cancer Chemotherapy Reports. 8:156-175. Edney, E. B. 1945. Laboratory studies on the bionomics of the rat fleas, Xenopsylla brasiliensis Baker and X. cheopis (Roths.). I. Certain effects of light, temperature, and humidity on the rate of development and on adult longevity. Bull. Ent. Res. 35:399-416*

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72 Edney, E. B. 1947a. Laboratory studies on the bionomics of the rat fleas, Xenopsylla brasiliensis Baker and X_. cheopis (Ttoths.)» II. Water relations during the cocoon period. Bull. Ent. Res. 38:263-280. 1947b. Laboratory studies on the bionomics of the rat fleas, Xenopsylla brasiliensis Baker and X. cheopis (Roths.). III. Further factors affecting adult longevity. Bull. Ent. Res. 38: 389-404. Fahmy, 0. G., and M. J. Fahmy. 1958. Discussion. Part II. Biological effects of alkylating agents. Ann. N. Y. Acad. Sci. 68: 736-749. 1964. The chemistry and genetics of the alkylating chemosterilants. II. From a symposium on chemosterilants in pest and vector control. Trans. Roy. Soc. Trop. Med. Hyg. 58 (4) :318-326. .. Flint, H. M. 1964. The effect of Cobalt 60 gamma rays on the biology of the eye gnat Hippelates pusio Loew. Ph.D. Dissertation. Univ. of Florida. t Fox, I., R. I. Fox, and L. G. Bayona. 1966. Fleas fed onlizards in the laboratory in Puerto Rico. J. Med. Ent. 2:395-396. Galun, R. 1966. Feeding stimulants of the rat flea, Xenopsylla cheopis Roth. Life Sci. 5:1335-1342. Gilbert, I. H. 1964. Laboratory rearing of cockroaches, bed-bugs, human lice and fleas. Bull. W.H.O. 31:561-563. Glancey, B. M. 1965. Hempa as a chemosterilant for the yellow-fever mosquito Aedes Aegypti (h.) (Diptera: Culicidae). Mosq. New. 25: 392-396. Gouck, H. K. , D. W. Meifert, and J. B. Gahan. 1963. A field experiment with apholate as a chemosterilant for the control of house flies. J. Econ. Entomol. 56(4) :445-446. Gunther, K. K. 1961. Funktionell-anatomische Untersuchung des mannlichen Kopulations-apparates der Flohe unter besonderer Berucksichtigung seiner postembryonal en Entwicklung (Siphonaptera) . Deutsche Entomol. Zeit. 8:258-349. Hayes, W. J. 1964. The toxicology of chemosterilants. Bull. Wld. Hlth. Org. 31:721-736. Henderson, J. R. 1928. A note on some external characters of larvae of Xenopsylla cheopis . Parasitol. 20:115-118. Herms, W. B. 1961. Medical Entomology. The Macmillan Co., N.Y. 643 pp.

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73 Hirst, L. F. 1923. On the transmission of plague by fleas of the genus Xenopsylla . Indian J. Med. Res. 10:789-820. 1926. Researches on the parasitology of plague. Ceylon J. Sci. Sect. D. 1:155-455, 1927. Researches on the parasitology of plague. Part II. Ceylon J. Sci. Sect. D. 1:277-455. Hopkins, G. H. E. 1935. Some observations on the bionomics of fleas in East Africa. Parasitol. 27:480-488. Jellison, W. L. 1959. Fleas and disease. Ann. Rev. Ent. 4:389-414. Keiser, J., L. F. Steiner, and H. Kamasaki. 1965. Effects, of chemosterilants against the oriental fruit fly, melon fly, and Mediterranean fruit fly. J. Econ. Entomol. 58:682-685. Kilgore, W. W. 1965. Biochemistry of insect sterilants. Abstract of papers. 150th meeting of Amer. Chem. Soc. p. 26A. Knipling, E. F. 1962. Potentialities and progress in the development of chemo sterilants for insect control. J. Econ. Entomol. 55(5): 782-786. Krishnamurthy, B. S., S. N. Ray, and G. C. Joshi. 1963. Note on technique used in rearing and maintaining a colony of the oriental rat-flea ( Xenopsylla cheopis) . W.H.O. EBL/working paper 19/63. LaBrecque, G. C. 1963. Chemo sterilants for the control of houseflies. Adv.. in Chem. 41:42-46. 1965. Chemo sterilants for the control of insects. Proc. XII Int. Congr. Ent., London. 1964. pp. 515-516. LaBrecque, G. C. , C. N. Smith, and D. W. Meifert. 1962. A field experi ment in the control of house flies with chemo steril ant baits. J. Econ. Entomol. 55(4) :449-451 . LaBrecque, G. C, D. W. Meifert, and R. L. Fye. 1963. A field study on the control of house flies with chemo steril ant techniques. J. Econ. Ent. 56(2) :150-152. LaBrecque, G. C. , P. B. Morgan, D. W. Meifert, and R. L. Fye. 1966. Effectiveness of hempa as a house fly chemosterilant . J. Med. Ent. 3:40-43. LaChance, chapter in LaBrecque, G. C, and C. N. Smith. 1967. Insect chemosterilants. Appleton-Century-Crofts Ci n manu script ) . Leeson, H. S. 1932. The effect of temperature and humidity upon the survival of certain unfed rat fleas. Parasitol. 24:196-2Q9.

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74 1936. Further experiments upon the longevity of Xenopsylla cheOpis Roths. (Siphonapter) . Parasitol. 28:403-409. Linkfield, R. L. , P. B. Morgan, and C. G. Haugh. 1967. A new chromosome squash apparatus. Ann. Ent. Soc. Am. (in press). McCoy, G. W. 1910. Bubonic plague in ground squirrels. N. Y. Med. J. 92:652-655. t Mellanby, K. 1933. The influence of temperature and humidity on the pupation of Xenopsylla cheopis . Bull. Ent. Res; 24:197-202. 1934. The site of loss of water from insects. Proc. Roy. Soc. Lond. Ser. B. 116:139-149. Mendenhall, W. 1964. Introduction to Statistics. 305 pp. Wadsworth Pub. Co. Meyer, K. F. 1947. The prevention of plague in the light of newer knowledge. Ann. N. V. Acad. Sci. 48:429-467. Mitlin, N., B. A. Butt, and T. J. Shortino. 1957. Effect of mitotic poisons on house fly oviposition. Physiol. Zool . 30C2) :133 i 136. Mitzmain, M. B. 1910. General observations on the bionomics of the rodent and human fleas. Treasury Dept. Pub. Hlth. Marine-Hosp. Serv. U. S. Pub. Hlth. Bull. No. 38. 34 pp. Morgan, P. B., and G. C. LaBrecque. 1962. The effect of apholate on the ovarian development of house flies. J. Econ. Entomol. 55: 626-628. j . • 1964a. Preparation of house fly chromosomes. Ann. Ent. Soc. Amer. 57:794-795. 1964b. Effect of tepa and metepa on ovarian development of house flies. J. Econ. Entomol. 57:896-899. Murray, W. S., and W. E. Bickley. 1964. Effects of apholate on the southern house mosquito; Culex quinquefasciatus Say. Univ. of Maryland. Bull. A134. 37 pp. Newstead, R. , and A. M. Evans. 1921. Report on rat-flea investigations. Ann. Trop. Med. § Parasitol. 15:287-300. Norris, M. J. 1954. Sexual maturation in the desert locust Schistocerca gregaria Forskal. Anti-locuSt Bull. 18:1-44. Patton, (t. S., and A. M. Evans. 1929. Insects, ticks, mites and venemous animals. Parts 1 and 2. H. R. Grubb, Ltd. Pausch, R. D. 1962. The nutrition of the larva of the oriental rat flea, Xenopsylla cheopis Cloths.) with additional notes on its bionomics. Ph.D. Dissertation. Univ. of Illinois.

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75 Plapp, Jr., F. W., W. S. Bigley, G. A. Chapman, and G. W. Eddy. 1962. Metabolism of methaphoxide in mosquitoes, house flies and mice. J. Econ. Entomol.' 55:607-613. Purdon, C. E. 1963. Genetic effects of radiations. Acad. Press. New York. 173. pp. Rai, K. S. 1964. Cytogenetic effects of chemosterilants in mosquitoes. II. Mechanism of apholate-induced changes in fecundity and fertility of Aedes aegyjgti (L. ) . Biol. Bull. 127 (1) : 119-131 . 1965. Cytogenetics of chemosterilant induced sterility in the mosquito Aedes aegy_p_ti_ (L.). Int. Congr. Ent. Proc. 12:255-256. Reports on Plague Investigations in India. 1907. XV. Further observations on the transmission of plague by fleas with special reference to the fate of the plague bacillus in the body of the rat flea (P. cheopis) . J. Hyg. 7:395-397. 1908. XXIX. Observations on the bionomics of fleas with special reference to P_. cheopis . J. Hyg. 8:236-259. 1912. LV. Observations on flea breeding in Poona. J. Hyg. 12:300325. Rothschild, M. 1965. Fleas. Sci. Amer. 213:44-53. Schwartz, P. H. 1965. Effects of apholate, metepa and tepa on reproductive tissues of Hippelates pusio Loew. J. Invert. Path. 7:148-151. Sharif, M. 1935. On the presence of wing buds in the pupa of Aphaniptera. Parasitol. 27:461-464. 1948a. The water relations of the larva of Xenopsylla cheopis * (Siphonaptera) . Parasitol. 39:148-155. 1948b. Nutritional requirements of flea larvae, and their bearing on the specific distribution and host preferences of the three Indian species of Xenopsylla (Siphonaptera). Parasitol. 38:253-263. 1949. Effects of constant temperature and humidity on the development of the larvae and the pupae of the three Indian species of Xenopsylla (Insecta: Siphonaptera). Phil. Trans. B. -233:581-635. Sharma, M. I. D. , and G. C. Joshi. 1961. An abnormal form of female rat flea, Xenopsylla cheopis Roths. Nature. 191:729. Shulov, A., and D. Naor. 1964. Experiments on the olfactory responses and nost-specificity of the oriental rat flea (Xenopsylla cheopis) (Siphonaptera: Pulicidae): Parasitol. 54:225-231. Sikes, E. K. 1930.' Larvae of Ceratophyllus wickhami and other species of fleas. Parasitol. 22:242-259.

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76 Smith, A. 1951. The effect of relative, humidity on the activity of the tropical rat flea, Xenopsylla cheopis , (Roths.) . (Siphonaptera) Bull. Ent. Res. 42:585-600. Smith, C. N. 1963a. Prospects for vector control through sterilization procedures. Suppl. Bull. Wld. Hlth. Org. 29:99-106. 1963b. Chemosterilants as a potential weapon for insect control,. Adv. in Chem. 41:36-41. ' Smith, C. N. , and G. W. Eddy. 1954. Techniques for rearing and handling body lice, oriental rat fleas, and cat fleas. Bull. Wld., Hlth. Org. 10:127-137. Smith, C. N. , G. C. LaBrecque, and A. B. Borkovec. 1964. Insect chemosterilants. Ann. Rev. Ent. 9:269-284. Smittle, B. J. 1964. The effects of tepa on the embryogeny and reproductive organs of the German cockroach, Blatella germanica (L.). Ph.D. Dissertation. Rutgers Univ. 87 pp. Snedecor, G. W. 1962. Statistical methods applied to agriculture and biology. 5th ed. Iowa State Univ. Press, Ames. 534 pp. Suomalainen, E. 1962. Significance of parthenogenesis in the evolution of insects. Ann. Rev. Entomol. 7:349-366. Suter, Von P. R. 1964. Biologie von Echidnophaga gallinacea (Westw.) and Vergleich mit andern Vernal lenstypen bei Flohen. Acta Tropica. 21:193-238. Wasserburger, H. J. 1961. Beitrage zur Histologic und mikroskopischen Anatomie von Xenopsylla cheopis Rothschild. Deutsche Entomologisch zeitschrift. 8:373-416. Webster, W. J. 1929. The anatomy of the Indian Xenopsylla larvae. Indian J. Med. Res. 17:90-93. Weidhaas, D. E. 1962. Chemical sterilization of mosquitoes. Nature. 195:786-787. , Weidhaas, D. E. 1963. Highlights of research on chemosterilization of mosquitoes. Proc. and Papers 31st Ann. Conf. Calif. Mosq. Contr. Assoc. pp. 21-23. Weidhaas, D. E., H. R. Ford, J. B. Gahan, and C. N. Smith. 1961. Preliminary observations on chemosterilization of mosquitoes. N. J. Mosquito Extermin. Assoc. Proc. 48:106-109. Weidhaas, D. E., C. H. Schmidt, and E. L. Seabrook. 1962. Field studies on the release of sterile males for the control of Anopheles quadrimaculatus . Mosq, News. 22:283-291.

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77 Wheeler, G. P. 1962. Studies related to the mechanisms of action of cytotoxic alkylating agents: a review. Cancer Res. 22:1334-1349. White,. M. J. D. 1951. Cytogenetics of Orthopteroid insects. Adv. in Genetics. 4:267-330. 1964. Cytogenetic mechanisms in insect reproduction. Roy. Entomol. Soc. Insect Reproduction Symposium No. 2:1-12. Wigglesworth, V. B. ,1935. The regulation of respiration in the flea, Xenopsylla cheopis (Roths.) (Pulicidae) . Proc. Roy. Soc. Lond. Ser. B. 118:397-419. 1964. The hormonal regulation of growth and reproduction in insects. Adv. in Insect Physiol. 2:247-336. Wu, L. T., J. W. H. Chun, R. Pollitzer, and,C. Y. Wu. . 1936. Plague, a manual for medical and public health workers. Shanghai: Weishengshu National Quar. Serv. 547 pp.

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APPENDIX

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79 Table 5. — Five untreated females mated with five untreated males. NUMBER OF Eggs Emerging Day a Produced Survived 0 Larvae Fj^ Adults 1 5 5 3 3 2 23 19 11 1 3 23 21 15 10 4 25 21 12 S : ' 1 5 30 21 15 5 6 26 24 14 0 7 56 47 26 15 8 41 37 21 . 9 9 27 27 24 14 10 34 , 33 , 26 21 11 39 38 16 10 12 42 v 42 38 38 13 31 31 21 20 14 30 26 20 18 Totals: 432 392 262 165 a Eggs were first observed on the 3rd day after placing fleas on host. °So;r»e eggs were found broken, or were broken in handling.-

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80 Table 6.— Five males treated with tepa C5mg/cm 2 for 6 hrs.) and mated with five untreated females. NUMBER OF Eggs Emerging b Day* Produced Survived 13 Larvae F Adults 0 0 0 0 0 0 1 0 0 0 0 0 0 0 'Eggs were first observed on the 3rd day after placing 'fleas on host. Some eggs were found broken, or were broken in handling.1 17 15 0 2 18 17 0 3 16 15 0 4 • 18 16 0 & .. •>>';;•:'. 31 30 0 19 1 15 ' 0 7 34 .34 1 8 38 36 , 0 9 28 28 0 10 1 18 U 18. , . 0 11 38 37 0 12 21 21 0 13 34 33 0 14 24 24 2 Totals: 354 339 3

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81 Table 7. — Ten untreated females mated with ten untreated males. , NUMBER OF Eggs , • Emerging Day a Produced Survived^ Larvae F. Adults n\ 1 9 9 9 C 7 ij^i .2 22 21 18 • 18 3 3Q 30 25 25 4 V* 22 21 14 14 5 35 ' 35 28 d 27 6 19 • 19 14 11 7. ' 23 23 20 18 8 35 34 29 29 9 39 37 37 35 10 27 25 16 16 11 52. " 32 31 28 12 38 31 26 24 13 35 32 23 20 14 47 38 ' 28 26 * Totals: 413 387 318 298 Eggs were first observed on the 3rd day after placing fleas on host. Some eggs were found broken, or were broken in handling. Two larvae died. Gr.e larva died.

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82 I Table 8. --Ten untreated females mated with, ten untreated males. NUMBER OF Eggs Emerging Day Produced Survived 15 Larvae F Adults 1 4 3 3 C 2 . 29 29 24 20 3 y80 ' ' , 70 59 51 '4 48 . . 39 28 17 5 49 49 32 28 6 51 51 41 35 7 ' " 41 41 i 38 34 8 22 20 15 14 9 32 32 28 .27 10 50 48 44 40 11 21 20 14 12 12 29 27 16 16 13 52 46 41 38 14 50 50 46 45 Totals: 558 525 429 377 a Eggs were first observed on the 3rd day after placing fleas on host. Some eggs were found broken, or were broken in handling.. All ^arvae died.

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84 Table 10. — Ten untreated females mated with ten untreated males. NUMBER OF Eggs' Emerging 3. "h Da/ Produced Survived , Larvae F-, Adults 1 30 28 19 0 40 40 9 6 3 c o 56 49 44 4 52 52 46 40 5 62 60 43 42 ' 6 ' 65 62 51 48 7 97 96 77 75 8 50 49 42 vv.34 9 91 91 67 54 10 . 80 ; 79 68 56 11 91 , 89 79 76 12 92 91 72 72 13 80 76 68 55 14 80 77 58 55 Totals': 966 946 748 657 a Eggs were first observed on the 3rd day after placing fleas on host. b Some eggs were found broken, or were broken in handling.-

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83 Table 9. --Ten untreated females mated with ten untreated males. NUMBER OF Eggs Emerging Day Produced Survived 15 Larvae F, Adults 1 8 8 7 2 34 34 31 30 3 42 42 38 34 4 54 52 42 38 5 55 54 44 c 40 6 . 40 36 23 7 • 62 62 57 54 8 53 53 43 40 9 36 36 30 c 26 10 53 52 35 33 11 ' 42 ) 42 34 32 12 51 51 38 37 13 28 28 27 27 14 54 51 47 44 Totals: 612 601 499 465 Eggs were first observed on the 3rd day after placing fleas c "Some eggs were found broken, or were broken in handling. c 0ne larva died.

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85 ' 2 Table 11. — Ten females treated with tepa (5mg/cm for 4 hrs.) and mated with ten untreated males. NUMBER 0? ' Eggs Emerging Day a Produced Survived^ Larvae F, Adults 4-1 \C 8 8 6 1 2 10 10 9 0 3 14 14 8 " 2 4 3 3 1 1 5 6 , 6 4 0 6 38 37 24 6 7 22 22 18 2 19 19 15 • 13 9 26 25 22 5 10 39 37 28 21 11 23 22 19 15 12 24 23 16 12 13 45 45 38 9 14 24 24 20 18 Totals: 301 295 228 105 Eggs were first observed on the 3rd day after placing fleas on host. Some eggs were found broken, or were broken in handling. v

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86' Table 12.— Ten females treated with tepa C5mg/cm for 6 hrs.) and mated with, ten untreated males. NUMBER OF ' Eggs Emerging r, a. Day • Produced Survived Larvae , ? l Adults 4 4 " 0 0 2 22 22 14 0 3 0 0 0 0 4 0 0 0 0 5 0 0 Vf o 0 6 0 0 0 ' 0 7 0 0 0 0 8 0 0 «»• 0 9 0 0 0 0 10 5 5 3 0 11 7 '•' 7 7 2 12 . 9 6 2 1 ' 13 13 13 10 7 14 24 24 13 11 Totals: 84 81 49 : 21 a Eggs were first observed on the 3rd day after placing fleas on host. Seme eggs were found broken, or were broken in handling.-

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87 2 1 Table 13. --Ten females treated with tepa C i0m g/ cra f° r 4 hrs.) and mated with ten untreated males. NUMBER OF Eggs Emerging Day a Produced Survived Larvae F. Adults 1 5 5 0 0 2 12 11 1 0 3 5 5 1 0 4 3 3 ,1 0 5 0 0 0 0 6 . 0 0 0 0 7 0 0 0 0 8 0 0 0 , 0 9 ' 0 0 0 0 10 0 0 0 0 11 0 0 0 0 12 $$&*o 0 0 0 13 0 0 0 0 14 0 0 0 Totals: 25 24 3 0 a Eggs were first observed on the 3rd day after placing fleas on host, ^Some eggs were broken, or were broken in handling.

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88 Table' 14. --Ten females treated with tepa (10mg/cm 2 for 4 hrs.) and mated with, ten untreated males. NUMBER OF ' Eggs Emerging Day a Produced Survived Larvae F 1 Adults 1 0 0 0 0 2 0 0 0 0 3 0 0 0 0 4 0 0 0 0 5 0 0 0 0 6 0 0 0 7 o 0 0 0 8 o . 0 ' 0 9 0 0 0 0 10 0, • 0 0 0 11 0 0 0 0 12 0 0 0 0 K 13 0 0 • -V"-/ 0'.'*'./ 0 14 0 0 0 0 Totals: 0 0 0 a Control fleas began egg production on the 3rd day after being placed on the host.

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89 Table 15. --Ten females treated with tepa (10mg/ cnl for 4 hrs.) and mated with ten untreated males'. NUMBER OF Eggs Emerging Da/ Produced Survived Larvae F Adults 1 o 0 0 0 2 ?A .... . 0..';, vr 0 0 0 3 0 ' 0 0 0 4 o 0 0 0 5 0 0 0 0 6 0 0 0 0 M?' 7 0 0 0 0 8 0 0 0 0 9 0 : o 0 ' ' . 0 10 0 0 0 11 0 0 0 0 12 0 0 0 0 ' * 13 0 0 0 S:, 0 14 0 0 0 0 Totals: 0 0 0 } 0 Control fleas began egg production on the 3rd day after being placed on the host.

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90 2 Table 16. --Ten males treated with, tepa (5mg/cm for 6 hrs.) and mated with ten untreated females. NUMBER OF ' Eggs Emerging Day a Produced Survived* 3 Larvae Fj Adults r 7 6 0 0 2 24 24 2 0 5 36 36 0 0 4 56 56 1 1 5 57 56 1 0 6 30 30 o : 0 7 : 'i r \ 37 37 0 0 8 53 53 0 , 0 9 64 64 1 0 10 40 • > 39 0 0 11 56 56 0 0 12 45 45 0 0 * 13 53 53 Q 0 14 50 50 0 0 Totals: 608 605 5 ' 1 a Eggs were first observed on the 3rd day after placing fleas on host. Some eggs were found broken, or were broken in handling.-

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91 I Table 17.— Ten males treated with tepa (5mg/cm 2 for 6 hrs.) and mated with ten untreated females. NUMBER OF Eggs Emerging Produced Survived* 3 Larvae F x Adults • 4 . ' '. .»* J. 10 10 0 0 31 31 0 0 3 30 ' 30 1 0 4 30 28 0 0 5 64 62 0 0 6 30 29 . 0 f\ 0 7 : 54 54 0 0 8 40 39 0 . 0 9 55 55 0 0 10 36 35 , 0 0 11 40 40 0 0 12 40 40 0 0 * 13 55 55 0 0 14 54 54 0 0 Totals: 569 562 1 r ! 0 Eggs were first observed on the 3rd day after placing fleas on host. Some eggs were found broken, or were broken in handling.-

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92 2 ' Table 18. — Ten males treated with tepa (5mg/cm for 6 hrs.) and mated with ten untreated females. NUMBER OF ' Eggs Emerging Da/ Produced Survived 0 i Larvae F, Adults 1 M 19 19 1 0 2 27 27 0 0 3 37 36 ' 1 0 4 41 39 0 0 5 40 40 0 0 6 35 35 1 1 7 25 25 0 0 8 47 ' 47 0 . 0 • 9 72 72 0 0 10 68 68 0 0 11 73 73 1 0 12 70 70 0 0 13 46 ' 46 0 0 14 54 54 0 0 Totals; 654 651 4 1 i i a Eggs were first observed on the 3rd day after placing fleas on host. °Some eggs were found broken, or were broken in handling.-

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93 Table 19. --Ten males treated with tepa (lOmg/cm for 4 hrs.) and crossed with ten untreated females. NUMBER OF Eggs Emerging Day a Produced Survived* 5 Larvae Adults 1 6 6 0 0 2 38 38 1 0 3 39 34 2 0 26 23 1 0 5 43 ' 43 3 0 6 60 ' 55 7 0 7 52 46 6 1 8 35 33 i 5 «, o." '^m% 9 33 32 2 0 10 ' 57 . j 33 , 3 o 1 11 39 39 7 4 12 57 57 7 3 * 13 40 40 9 0 14 47 44 8 3 Totals: 552 , 523 61 11 ^Eggs were first observed on the 3rd day after placing fleas on host. Some eggs were found broken, or were broken in handling.'

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94 Table 20. --Ten males treated with tepa (lOmg/cm for 6 hrs.) and' mated with ten untreated females. NUMBER OF Eggs Emerging Day a Produced Survived* 5 Larvae , Fj^ Adults 18 18 0 0 2 41 40 0 0 3 22 21 0 0 4 25 25 , 0 0 5 40 40 3 2 49 49 0 0 7 41 41 0 0 8 43 43 0 o iy. 9 . 21 21 0 10 38 38 0 o •'. 11 33 ' 33 • 0 0 12 27 .' 27 ' , 0 0 13 ' 32 32 0 0 14 26 26 0 0 Totals.: 456 454 3 • 2 i a Eggs were first observed on the 3rd day after placing fleas on host. Some eggs were found broken, or were broken in handling..

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95 Table 21. — Ten males treated with tepa (lOmg/cm^ for 6 hrs.) and mated with ten untreated females. NUMBER OF Eggs Emerging Day a Produced Survived* 3 Larvae F, Adults 1 5 5 0 0 2 30 29 0 0 3 65 65 0 0 4 48 48 ' 0 0 5 47 47 o 0 6 73 73 0 0 7 49 49 0 0 8 48 48 0 • 0 9 44 44 1 0 10 33 i 33 0 0 11 18 18 0 0 12 30 30 1 1 13 48 48 0 0 14 55 55 0 0 Totals: 593 592 2 1 Eggs were first observed on the 3rd day after placing fleas on host. 'Some eggs were found broken, or were broken in handling.'

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96 Table 22. — Ten males treated with tepa (lOmg/cm^ for 6 hrs.) and mated with ten untreated females. NUMBER OF ' Eggs Emerging Day a Produced Survived* 3 Larvae Adults I 16 16 1 2 30 29 0 0 3 32 32 1 0 4 48 47 0 5 54 54 1 0 6 47 47 0 0 7 69 68 1 1 8 55 53 0 0 9 49 49 0 0 10 22 , 21 , 0 0 11 55 55 0 0 12 63 63 0 o V 13 • 78 78 0 0 14 96 96 0 0 Totals: 714 708 4 J," ? 1. ^t,^ Eggs were first observed on the 3rd day after placing fleas on host. Some eggs were found broken, or were broken in handling.-

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97 Table 23. — Ten males treated with tepa (15mg/cm for 6 hrs.) and mated with ten untreated females. NUMBER OF Eggs Emerging Day* Produced Survived Larvae F 1 Adults c 1 5 5 0 2 28 28 0 3 33 31 0 4 • • 36 34 0 5 45 45 ' 0 6 50 50 1 7 . }± 38 38 . 0 8 31 31 1 9 35 35 0 10 20 20 0 11 29 29 0 12 32 32 0 13 33 33 0 14 37 37 0 Totals: 452 448 2 Eggs were first observed on the 3rd day after placing fleas on host. ^Some eggs were found broken, or were broken in handling. Experiment was terminated 6 days after Day 14 because 100% sterility had not been achieved, and treated male mortality was 10% in the 24-hour post-treatment holding period.

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98 Table 24. — Ten females treated with tepa (5mg/cm for 6 h'rs.} and mated with ten males treated with tepa at lOmg/cnr for 4 hrs. NUMBER OF Eggs Emerging Daya Produced Survived^ 5 Larvae Fj Adults 1 1 1 0 5 4 0 0 3 4 3 0 0 4 . o 0 . 0 . 0 5 • Q ' 0 0 0 6 o 0 0 0 7 0 0 • 0 0 8 0 0 0 o , 9 * 0 0 . 0 0 10 0 0 0 0 11 0 0 0 0 12 0 0 0 0 13 0 0 0 0 14 o 0 0 ; o Totals: 10 8 t 0 a Eggs were first observed on the 3rd day after placing fleas on host. Some eggs were found broken, or were broken in handling.

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BIOGRAPHICAL SKETCH Robert Loomis Linkfield was born on October 28, 1930, at Jamaica, Long Island, New York. In 1949, after graduation from Bayside High School, he began a two-year course of study at Long Island Agricultural and Technical Institute, from which he received the degree of Associate of Applied Science in 1951. From then until 1953 he served with the United States Army as a Sound Ranging Specialist (GR8) , with eleven months in Korea during the police action there. In 'June of 1956 he received the degree of Bachelor of Science from the University of Georgia, and was awarded the degree of Master of Science (Entomology) in June of 1957, after which he was employed by the United States government. After a year with the Agency for International Development, for which he. worked as junior overseas officer trainee, he was transferred in 1959 to the United States Department of Agriculture, Agricultural Research Service, Regional Insect Control Project, with which he was associate entomologist for a year, and entomologist-in-charge for three years in Iran and" Libya. With the help of a graduate assistantship at the J. Hiilis Miller Medical Center .here he is in charge of pest control for the medical science complex, he enrolled in the Graduate School at the University of Florida. From September 1963 to the present he has worked toward the degree of ' Doctor of Philosophy. 99

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100 Married to the former Ethel Skelton, he is the father of a daughter. He is a member of the Society of the Sigma Xi; Alpha Zeta; Phi Sigma; Newell Entomological Society; Florida Entomological Society; and the Washington Entomological Society.

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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 l degree of Doctor of Philosophy. i December, 1966 Dean, College of Agriculture i Dean, Graduate School Supervisory Committee: