THE EFFECT OF COBALT 60 GAMMA RAYS
ON THE BIOLOGY OF THE EYE GNAT
HIPPELATES PUSIO LOEW
HOLLIS MITCHELL FLINT
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
I should like to thank Dr. John T. Creighton, Head of the
Department of Entomology, and chairman of the supervisory committee,
for his guidance and interest during the period of graduate study.
Appreciation is also expressed to committee members Dr. Franklin
S. Blanton, Dr. William W. Smith, Dr. James A. Gregg, Dr. Germain C.
LaBrecque, and Dr. Alan D. Conger for their valuable suggestions. I
should also like to thank Dr. Carroll N. Smith for the use of the
facilities of the USDA Entomology Research Division laboratory, Dr.
Philip Morgan for advice and technical assistance with the cytological
studies, and Mr. Denver Johnson who operated the irradiator during the
test exposures. The period of graduate study was supported by a
National Defense Education Act fellowship.
My affectionate appreciation is given to my wife, Adela, who
typed the preliminary copies of this manuscript and who gave en-
couragement during the course of the study.
TABLE OF CONTENTS
ACKNOWLED ENTS................................................... ii
LIST OF TABLES ...................... ............................ iv
LIST OF FIGURES ...................................................... v
INTRODUCTION........... ............... ............... .. ....... 1
LITERATURE REVIEW.............. .............. ........ ........... 3
Radiosterilization of Insects.............................. 3
Morphological and Cytological Effects of Radiation.......... 5
MATERIALS AID METHODS ... ......................... ......... 8
Experimental Procedure................................... 9
The Cobalt-60 Source................................. ..... 11
Histological Technique ...................................... 11
Statistical Methods......... ..... .......................... 12
RESULTS AIJD DISCUSSIO ....................... ................... 14
The Effects of Radiation on the Biology of the Eye Gnat.... 14
The Sterilizing Dose................................ 14
Mating Competitiveness ......... .... ....... ........ 19
Male Recovery ..................... ......... .......... 22
Controlled Mating ............................... ..... 2
Egg Production................ ........ .......... .... 25
The Effects of Radiation on Stages of the Life Cycle... 28
Adults....................... ............... ... 28
Larvae ............ .. ............... .......... 32
Morphological and Cytological Effects...................... 34
The Ovaries.......................................... 35
The Testes........................................ .... 44
Chromosome Aberrations ............................... 48
SUMMARY ......................................................... 53
LITERATURE CITED................................................. 55
BIOGRAPHICAL SKETCH ........ ................................. 61
LIST OF TABLES
1. Fecundity and fertility of H. pusio treated as 24- to 36-
hour adults.................. .............. .... ... ....... 15
2. Fertility and fecundity of H. pusio treated as pupae 2
days prior to emergence...................................... 18
3. Competitiveness of H. pusio males treated with 5,000 r as
24- to 36-hour adults........................................ 19
4. Competitiveness of H. pusio males after treatment with
4,500 r as pupae 2 days prior to emergence.................... 20
5. Fertility recovery in males of H. pusio treated with gamma
radiation as adults and pupae .............................. 23
6. The effect of alternate crosses with untreated males and males
treated with 2,500 and 5,000 r on untreated females........... 24
7. Mortality of 50 H. pusio treated with 5,000 r as 24- to 36-
hour adults .......................... ............. .... 29
8. The LT-50 and LT-100 of H. pusio males and females irradiated
as 24- to 36-hour adults ..................................... 30
9. The emergence and fertility of H. pusio adults treated as last
instar larvae............................................. 33
10. Tahe mortality of 50 H. pusio treated as pupae 2 days prior to
emergence ..................... ..... 34
11. Effects of gamma radiation on the ovarioles of H. pusio
treated as pupae 2 days prior to emergence.................... 37
12. Testicular measurements of H. pusio treated with 4,500 r as
pupae 2 days prior to emergence, 5,000 r as 24- to 36-hour
adults, and untreated. ........................................ 48
LIST OF FIGURES
1. Egg production from untreated females of H. pusio and females
treated with 5,000 r as 24- to 36-hour adults in 3
replications .............. ......................................... 26
2. Ovaries of H. pusio treated with gamma radiation and untreated.. 41
3. Ovarian development of H. pusio after treatment with 3,500 r
as pupae 2 days prior to emergence.............................. 43
4. Testes from untreated and treated H. pusio 7 days after
emergence ...................... .. ........... ............... 46
5. Chromosomes of H. pusio......................................... 52
The eye gnat, Hippelates pusio Loew, belongs to the family
Chloropidae of the order Diptera. The taxonomy of the genus Hippelates
has been discussed by Aldrich (1929) and Sabrosky (1941). Hippelates
gnats have been linked with the mechanical transmission of yaws (Kumm
and Turner, 1936), conjunctivitis (Herms, 1926) and bovine mastitis
(Sanders, 1940). Early reference to the pestiferous habits of the eye
gnat was made by Schwarz (1895) who encountered large populations in
Alabama, Texas, and Florida. Members of the genera are found abundantly
throughout the southeastern and southwestern United States. They
have also been recorded in Central and South America. Several exten-
sive control programs have been initiated in southern California
where eye gnats have become particularly troublesome. Recent work on
chemical control has been performed by Dow and Willis (1959) and
Mulla (1963). An effective and practical means of control has not been
With the success of the screw-worm eradication project in
Florida (Knipling, 1960), interest has been directed toward the use of
the sterilized male technique for other insect pests. The use of steri-
lized insects is a form of control in which the males are sterilized
by either radiation or chemicals and released in large numbers to
compete with normal males for females in the natural population. Females
which mate with sterile males lay infertile eggs. Continued release of
sterilized male insects brings about a rapid reduction of the population.
Recent developments with compounds called chemical sterilants have
overshadowed the use of radiation for inducing sterility in insects.
But the use of radiation to sterilize insects may have advantages over
the use of chemicals (Von Borstel, 1960) in that radiation doses can
be controlled very accurately, affects only the desired insect species,
and does not cause dangerous contamination. Radiation sterilization
programs need not be concerned with insect resistance, residue hazards,
and detrimental effects on fish and wildlife.
Response to radiation in insects may be immediate as with cessa-
tion of growth (Grosch, 1956) or apparent after longer periods such as
shortening of the life span, reduction of fertility and fecundity, and
failure to react normally to the environment. Subtle changes occur in
the physiology and genetic transfer mechanism in the irradiated insect.
Research employing radiation effects have followed two main trends.
These include application of radiation to insect control and fundament-
al studies in genetics and cytology. The numbers of species used in
all radiation studies have been relatively few.
The author has been unable to find any literature on the effects
of radiation on H. pusio. The objective of this study is to provide
basic biological information concerning the effects of radiation on the
eye gnat. The studies reported here deal specifically with: (1) the
sterilizing dose for each sex treated in both the pupal and the adult
stage, (2) the effects of the sterilizing dose on male competitiveness
when treated in the pupal and the adult stage, (3) the lethal effects of
radiation on the stages of the life cycle, and (4) the determination of
the morphological and cytological effects of gamma radiation on the
Radiosterilization of Insects
The first worker to observe that insects could be sterilized by
radiation was Runner (1916) who was working with the cigarette beetle,
Lasioderma serricorne Fabricius. He observed that although mortality
was induced at higher dosages, fertility was lost with lower dosages
and suggested this approach would be a useful tool for the control of
the beetle. Subsequently, Muller (1927) demonstrated that heavy doses
of X-rays also induced gene mutations in Drosophila melanogaster Meigen.
He stated, "It has been found quite conclusively that treatment of the
sperm with relatively heavy doses of X-rays induces the occurrence of
true 'gene mutations' in a high proportion of the treated germ cells.
In addition to gene mutations, it was found that X-ray treatment caused
a high proportion of rearrangements in the linear order of the genes."
Recently, Bushland and Hopkins (1953) have shown that gamma rays produce
the same results when used at equal roentgen dosages.
Radiations which are deleterious to genetical material are of
2 kinds: the ionizing alpha, beta, and gamma rays, and non-ionizing
ultraviolet radiation. Despite the fact that X-rays and gamma rays
have a different origin they are precisely the same in their character-
istics and can be measured in the same units. The biological action of
radiation is most plausibly attributed to chemical changes resulting
from ionization (Catchside, 1943). Some general effects of induced
ionization on the cell are reduction of DNA synthesis, depolymeri-
zation of DNA, inhibition of mitosis, induction of mutations and
chromosomal breakage. The latter results in loss of pieces and trans-
locations (Wilson and Morrison, 1960).
Radiation may cause sterility in either of 2 ways (LaChance and
Bruns, 1963). The treatment may inhibit the formation and development
of mature sperm (fecundity ) or may not deter the production of these
cells per se but induce dominant lethal changes in the hereditary
material. The latter effect renders the gametes incapable of sus-
taining embryonic growth or causes death in the post-embryonic stages
(fertility). The dominant lethal mutation is of primary importance in
sterility studies with insects. In adults or in late stage pr.pae
where adult structure is nearly complete, cell division proceeds
slowly in the somatic tissues. Only in the gonads are mitosis and
meiosis proceeding rapidly. A dose of radiation that is tolerated by
the somatic cells selectively produces mutations in the germ cells. A
dominant lethal mutation produced in 1 parent overcomes the effect of
the corresponding gene from the other parent and the resulting zygote
dies. The irradiated chromosomes may also stick together so that
daughter cells do not receive the normal hereditary complement and these
gross structural changes are also classified as dominant lethal muta-
tions (Bushland, 1960). The dominant lethal mutations have been
classified into several types by Von Borstel and Rekemeyer (1959) and
Von Borstel (1959). The importance of dominant lethals is summarized
by Bartlett and Bell (1962) as follows:
An important aspect in the evaluation of radiation
damage in any species is the ability of that species to
perpetuate itself after irradiation. If the irradiated
adults are sterilized by a particular dose, then the
genetic consequences of irradiation are not of practical
importance. On the other hand, if the irradiation only
partially or insignificantly alters reproduction, then
genetic damage becomes a matter of concern.
Dosages required to produce more than 95 per cent dominant lethal
genes in gametes cause little impairment of the other biological
functions of insects. Thus, although aperm and ova remain alive and
the sperm retain full motility, the zygotes do not complete development.
With the sterile male technique it is not necessary that the female
be monogamous (Von Borstel, 1960) although the radiosterilized males
must compete with normal males in mating with females (Knipling, 1955).
In addition to studies on the effect of radiation on the
reproductive capabilities of insects, a great deal of research has been
conducted on the concentrations needed to produce lethality in adults.
The idea that an enormous dose must be used to irreversibly damage
adult insects applies only if immediate mortality is sought. lesser
concentrations will produce the same effect over a longer period of time.
Less than 10,000 r of X-rays or gamma rays can decrease the life span
significantly. The radio-sensitivity of a certain species is not constant
but varies with age, sex, and nutrition (Barter and Tuttle, 1957).
Morphological and iytological Effects of Radiation
The greatest amount of damage fruim radiation occurs in the repro-
ductive tissues. The functions of these tissues are partially or
completely destroyed at doses which are considerably lower than those
needed to produce gross functional changes in other body tissues.
LaChance and Bruns (1963) in their studies on o8genesis in Cochliomyia
hominivorax (oqouere) showed that the effect of irradiation on the
reproductive capacity of the female is largely dependent on the stage
of development of the ovarioles at the time of treatment. The
apparent failure of females to produce mature ova reflected the in-
ability of irradiated nurse cells to support normal vitellogenesis.
King and Sang (1959) suggested that an abnormal chromosomal complement
in irradiated nurse cells of Drosophila inhibited normal vitellogenesis.
Retardation of growth in the ovary is not due to a reduction in the
number of ovarioles which are retained in normal numbers but to morpho-
logical changes in the individual ovarioles (Erdman, 1960a; LaChance
and Bruns, 1963).
Ross and Cochran (1963) have made studies on irradiated ovaries
and testes of the cockroach Blattella germanica. Linnaeus. Their
results indicated that continued growth of the gonads was inhibited
when large nymphs were treated with gamma radiation. Annan (1955)
showed that abnormally small ovaries in irradiated adult Drosophila
were due to a process of atrophication. The exact cause of inhibition
of growth or atrophy of treated gonadal tissue is not known.
Histological techniques have been used to reveal destruction of
sensitive cell types in testes. Welshons and Russell (1957) demon-
strated that a period of temporary sterility in Drosophila after
treatment with 4,000 r was due to destruction of both secondary
spermatogonia and young spermatocytes. A subsequent return in fer-
tility was attributed to development of cells irradiated at a later
spermatogenic stage. Recovery of fertility was also observed by Grosch
and Sullivan (1954) in Habrobracon males irradiated with 3,300 r of X-rays.
Such recovery phenomena must be considered in control programs where
insects are released into the natural population.
Cytological studies have shown that visible changes in irradiated
chromosomes can be detected (Catchside, 1948; Lea, 1955). Koller and
Ahmed (1942) made cytological studies on the larval salivary gland
chromosomes of Drosophila pseudo-obscura Frolewa treated with 4,500 r
of X-rays and found structural changes in 40 per cent of the chromo-
Several useful techniques for the study of insect chromosomes
have been recently published (Breland, 1961; Oster and Balaban, 1963;
French et al., 1962). Chromosome numbers are known for about 400
species of Diptera, many of which are species of Drosophila (Boyes,
Several papers concerning the effects of radiation on insect
tissues have been reviewed by Grosch (1962).
MATERIALS AND METHODS
The strain of H. pusio utilized in these studies was from the
USDA Orlando laboratory colony. This colony was originally established
by Turner (1960) in 1958 from gnats collected in the vicinity of
Orlando, Florida. It was then transferred to the University of Florida,
Department of Entomology, where it was maintained for 3 years and is
now in the F85 generation.
A rearing procedure similar to that described by Jay (1961) was
used. When 5 to 10 thousand eggs had accumulated in a 1/2-gallon Mason
jar containing several thousand adult gnats they were collected by
adding approximately 1/2-pint of cool tap water directly through the
screen lid and swirling to loosen the eggs. The water was poured back
through the screen lid which retained the adult insects but allowed
the eggs to pass. The egg suspension was poured into a 1-gallon bain-
marie containing 3 quarts of larval rearing medium. The larval rearing
medium consisted of 5 parts of number 4 vermiculite (Zonolite Company),
1 part CSMA (Chemical Specialties Manufacturing Association) house fly
larval rearing medium and 2 parts of tap water. Tedion (Niagara Chemi-
cal Company) was added at 10 grams of a mixture containing 0.1 per cent
by weight to prevent infestation of the colony with mites (Mulla, 1958).
The bain-marie was covered with a piece of heavy cotton sailcloth held
in place with a large rubber band and maintained at 82" + 2" F. and
about 70 per cent relative humidity. Under these conditions, the life
cycle was completed in about 12 days. The insects were allowed to
emerge in a cage covered with 30 x 30 mesh plastic screen. The cage was
fitted with a 1-pint Mason jar at one end into which the positively
phototactic gnats moved when a black cloth was placed over the screen of
the cage. Food was provided on cotton dental rolls saturated with honey
and impaled on the sharp points of nails inserted through the wooden
frame of the emergence cage. Insects were collected from the emergence
cage and placed in a 1/2-gallon Mason jar which contained 1 or 2 cotton
rolls saturated with honey.
Insects were reared and tests conducted at 820 + 20 F. and 70
per cent relative humidity except where noted. Carbon dioxide was
used as an anesthetic wen handling the insects. Transfer and separa-
tion of sexes for testing purposes was done using a battery operated
device described by Schwartz (1964).
Containers for adult and pupal irradiation were 10-dram clear glass
shell vials fitted with plastic snap lids modified to accommodate 3/4-
inch 30 x 30 mesh screen lids. Insects were fed with pure honey in
these containers with a 1-inch section of a 1-m glass capillary tube
partially inserted through the screen lid. All test insects were provided
with food before and after irradiation.
Adult insects were held in 1-pint plastic food containers or in
1-pint Mason Jars unless otherwise noted. The lids of the plastic cages
contained a central 1/2-inch hole to accept a 1/2-inch cotton dental
roll and 2 marginal holes approximately 3/4-inch in diameter which were
covered by a coarse mesh linen cloth. The Mason jars were fitted with
30 x 30 mesh screen lids with a dental roll taped to the inner side of
the lid. .son jars were used in all mating aggressiveness tests and
the female insects were introduced into the jars 24 hours before the
male insects were added.
Pupae for irradiation were collected from larval rearing medium
by placing approximately 2 cups of medium in a gallon metal container
and adding 1/2-gallon of water. After a short interval only the pupae
and a small amount of media remained on the surface. The water was then
decanted through a 30 x 30 mesh screen which retained the pupae. The
pupae were then placed on blotting paper to dry. The dried material
was placed in a narrow mouth 1/2-gallon jar and subjected to an air
blast which removed the lighter media material and left the pupae.
The larvae to be irradiated were hand picked from the rearing
medium using a fine brush. They were then returned to the original
medium but not before it had been autoclaved at sufficient heat to
kill uncounted larvae and pupae. The medium was re-used because it was
found that survival of the test larvae was optimal when they were re-
turned to the original medium. The larvae were irradiated in the medium
contained in the 10-dram glass vials. The vials were then left uncapped
in an upright position in 1-pint Mason jars fitted with screen lids.
Adult food was provided on dental rolls when emergence began.
Eggs for irradiation were collected for testing by placing gravid
adults in 1/2-gallon jars for periods of 1 to 3 hours to allow oviposi-
tion to take place. One-half cup of water was then added to the
container and the accumulated eggs were loosened. The water and egg
suspension was then poured into a petri dish and 50 eggs, selected with
an eye dropper, were placed on dark colored blotting paper discs (1 x 1
inch) which had previously been boiled to remove excess dye. The qggs
were easily counted against the dark background. Each disc was
placed in a 10-dram glass vial for irradiation. After treatment, the
discs were retained in the capped vials for a period of 3 days from the
known oviposition date and then removed. The number of hatched eggs
were counted under a steroscopic microscope at a magnification of
The Cobalt-60 Source
The University of Florida Cobalt-60 irradiator (Teas, 1959) used
in the tests reported here contains 12 tubes loaded with Cobalt-60
wafers, and is submerged in a 13-foot water tank when not in the
operating position. In the operating position in air, the irradiator
is raised and dose rates determined by placing the objects to be
treated varying distances from the source. In the operating position,
the irradiator surrounds a galvanized iron can 8 inches in diameter and
21 inches high. Insects were treated in this central can at dose rates
varying from 3155 r per minute in December, 1961, to 2185 r per minute
in February, 1964. At doses of less than 1,000 r, specimens were placed
on the table surrounding the irradiator and irradiated with a dose rate
of about 70 r per minute to reduce error in exposure times. The dose
rate used in testing was from 2,600 to 2,135 r per minute unless noted
All dissections were made in Ringer's saline. Whole ovaries and
ovarioles were stained with aceto-carmine, squashed, and immediately
examined under a phase microscope at 150 diameters. Ovarioles were
separated by either gently mincing the ovary with the tips of
jeweler's forceps or by adding a cover slip and applying gentle pressure.
Larval brain and testicular tissue were prepared for chromosome
studies by the technique of Morgan and laBrecque (in press). The
procedure is briefly outlined below. The dissected tissue was trans-
ferred to a drop of 1 per cent sodium citrate on a clean microscope
slide for 10 minutes and then to 45 per cent glacial acetic acid on
another slide. After 5 minutes a siliconized cover slip was added, a
piece of blotting paper was placed over the slide to absorb excess fluid
and heavy thumb pressure was applied in a straight line to avoid move-
ment of the cover slip and squash the preparation. The cover slip was
then tapped several times with a blunt instrument and the whole slide
placed on a cake of dry ice for 30 minutes. The slide was then placed
in 95 per cnt ethyl alcohol for 5 minutes. The slide was allowed to
drip excess alcohol and a drop of Gurr's natural orcein and fast green
stain applied while the slide was still wet. A cover slip was placed on
the slide and excess stain removed from the edges of the cover slip by
careful blotting. The slide was placed in the refrigerator overnight
and then examined under a phase contrast microscope using oil immersion
at a magnification of 970 diameters.
Chromosome number determinations were made using mounts prepared by
the Feulgen stain technique of Whiting (1950).
Abbott's (1925) formLula was used to adjust sterility to that
obtained in controls where probit analysis was used to calculate the
sterility dose (SD-99). Analysis of variance and "F" tests were
conducted by the methods of Snedecor (1961). The Chi square tests were
run following the procedures outlined by Freund and Williams (1961).
Calculated probit values and LD-50 figures were obtained from the
formula of Litchfield and Wilcoxon (1949). The term "significant
difference" is used in this paper to denote a probability level of 95
per cent and a highly significant difference indicates a probability
level of 99 per cent.
Replication as used in this paper indicates repetition of the
test with another generation of insects at another time. Thus one
replication is repeated independently of the other replications.
RESULTS AND DISCUSSION
The Effects of Radiation on the Biology of the Eye Gnat
Eye gnats were subjected to radiation treatment as pupae and
adults to determine the approximate sterilizing dose, mating com-
petitiveness of males, male recovery, mating effects, and egg
production. The lethal effects on egg, larval, pupal, and adult
stages of the life cycle were also tested. Preliminary tests indicated
that pupae which were to emerge within 2 days and adults at least 24
to 36 hours after emergence were the most suitable age of treatment
because of the low mortality and the insensitivity of the reproduct-
ive organs. In the literature, pupal age is generally given in units
of days after pupation. It was found by the author to be more accurate
to irradiate 2 days prior to the expected date of emergence. Only
insects emerging on the expected date were then used in evaluation of
results. In these studies, gnats were considered to be sterile when egg
hatch was reduced to 1 per cent or less.
The Sterilizing Dose
In tests conducted to determine the minimum level of radiation
needed to reduce the egg hatch to less than 1 per cent, 20 males were
exposed to each of 6 doses ranging from 2,500 to 5,000 r and were
crossed with 20 untreated females of the same age. The reciprocal
crosses were made using equal numbers of insects. The results are shown
in table 1.
Table 1.--Fecundity and fertility of H. pusio treated as 24- to 36-
: Sex treated : : watch
Dose in each cross** : Total eggs (%)
0 Both untreated 234 70.3
2,500 Male 252 16.3
Female 154 32.3
3,000 Male 269 6.0
Female 134 11.6
3,500 Male 219 4.6
Female 216 7.3
4,000 Male 228 2.3
Female 187 6.6
4,500 Male 209 1.0
Female 110 2.3
5,000 Male 216 0.6
Female 138 0.5
**20 pairs of insects per cross.
At 5,000 r, or twice the starting dose, the gnat fertility was
reduced to less than 1 per cent or a 26- to 65-fold decrease for males
and females respectively over the initial reduction at 2,500 r. There
was a significant difference between the fertility of males at each
level of radiation. Moreover, it was found that male fertility was
reduced about 5-fold at the 2,500 r level and female fertility was
reduced by only 1/2 that of untreated gnats. A significant difference
between the sterility induced in each sex was noted at the 2,500 r
dosage. The number of eggs produced by females treated with 2,500 r
was only about 1/2 that of untreated females.
Although there was no significant difference between the numbers
of eggs laid by females treated at the different dosages, there was
a significant difference in egg hatch. Fertility was decreased as the
dosage increased but not proportionately.
The dose needed to reduce egg hatch to 1/2 that of controls was
calculated to be approximately 2,200 r for males and 2,450 r for females.
The female gnat was about 1.09 times more resistant to irradiation than
male gnats at this level of fertility.
These results indicate a lower dose is required to sterilize the
eye gnat than some other .Diptera. Henneberry (1963) found that between
8,000 and 16,000 r was required to sterilize males of D. melanogaster
and over 4,000 r for the females. Lea (1955) and Hassett and Jenkins
(1952) have placed the sterilizing dose for Drosophila at approximately
8,000 r. Davis et al. (1959) showed that Anopheles quadrimaculatus Say
required between 8,865 and 12,900 r to sterilize both sexes. Some
other sterilizing doses for adult insects are: 5,000 r for the beetle,
Onthophagus texanus Schaeffer (Howden, 1957), 5,000 r for Trogoderma
sternale Jayne (Howden and Aurbach, 1958), 10,900 r for drone honey bees
(Lee, 1958), 30,000 r for the cattle grub, Ostrinia nubilialis (Hubner)
(Walker and Brindley, 1963) and 4,800 r for Habrobracon juglandis
Ashmead (Grosch and Sullivan, 1954).
Tests showed that eye gnats could be sterilized with lower dosages
when treated in the pupal stage than when treated as adults. Several
hundred pupae were treated with doses ranging from 500 to 4,500 r.
Twenty adults of each sex were collected from each dosage level as they
became available. Adult insects were crossed when 24 to 36 hours old
with untreated insects of the same age. The females were virgin at
this age since mating does not occur before approximately 36 hours
after emergence (Schwartz, 1964).
At the lowest dose of 500 r there was a 30 per cent decrease in
fertility with both sexes. At each subsequent level female fertility
was 2 to 5 times greater than male fertility. The reduction in
fertility was not proportional to dose. As shown in table 2, no viable
eggs were produced from matings with either treated sex at 4,500 r
as compared to the results obtained in the preceding test with adult
irradiation where 99 per cent sterility was found at 5,000 r. There
was no significant difference between egg production from untreated
females at each dosage level. Treated females crossed with untreated
males laid approximately normal numbers of eggs at 500 and 1,500 r. At
higher doses there was a 4- to 100-fold decrease in egg production from
The dose needed to reduce egg hatch to 1/2 that of control was
calculated to be approximately 850 r and 1,600 r for males and females,
respectively. The females were about 1.8 times more resistant at this
level of fertility.
These results compare closely to those obtained by Bushland and
Hopkins (1951) for C. hominivorax who found that males were sterilized
by 2,500 r when treated with X-rays as late pupae. Females required
5,000 r to induce sterility and produced greatly reduced numbers of
eggs at this dose.
Table 2.--Fertility and fecundity of H. pusio treated as pupae 2 days
prior to emergence.*
: Sex treated : : Hatch
Dose : in each cross** : Total eggs (%)
0 Both untreated 226 75.0
500 Male 171 55.3
Female 176 54.6
1,500 Male 211 17.1
Female 163 48.0
2,500 Male 181 4.3
Female 55 23.3
3,500 Male 153 3.7
Female 12 14.0
4,500 Male 168 0.0
Female 2 0.0
**20 pairs of insects per cross.
A comparison of the results of pupal and adult irradiation
obtained in this study showed that a level of less than 1 per cent
fertility was attained with both sexes when adults were treated with
5,000 r and pupae were treated with 4,500 r. At these doses, insects
treated in the pupal stage produced very few eggs while treatment of
adults reduced egg production to about 2/3 that of controls. As expected,
treatment of the male at either stage had little or no effect on egg
production in subsequent matings with untreated females. The SD-99 for
gnats irradiated as adults was found to be 4,550 r for males and 4,900 r
for females. The SD-99 for insects treated as pupae was 3,750 r for
males and 4,700 r for females. At this level of fertility, females were
1.07 times more resistant to irradiation when the gnats were treated as
adults and 1.24 times more resistant when gnats were treated in the
The effect of the sterilizing doses on male mating competitive-
ness was found to be dependent on whether the pupal or adult stage
was treated. Treated males were added in various ratios with un-
treated males to virgin females of the same age. The expected per cent
egg hatch was calculated from the egg hatch obtained in the control by
using the ratio of sterilized males to untreated males.
The results of tests with the gnats irradiated as adults,as shown
in table 3, indicate that the expected egg hatch was very close to the
observed egg hatch at all cross ratios. There was no significant
difference between the calculated egg hatch and the observed egg hatch.
The expected value was most closely reached with the 5:1:1 ratio.
Table 3.--Competitiveness of H. pusio males treated with 5,000 r as
24- to 36-hour adults.*
: Number : Hatch : Expected hatch
Cross ratio** : of eggs : (%) : (M)
0:1:1 247 77.3 --
1:1:1 322 34.0 38.3
2:1:1 327 27.6 25.8
3:1:1 310 22.6 19.3
5:1:1 410 7.5 7.2
1:0:1 257 0.0 0.0
*20 females per ratio, 3 replicates.
**Treated male:untreated male:untreated female.
The test was repeated except that in this instance the males
were treated in the pupal stage instead of the adult stage. Other
conditions remained unchanged. The results given in table 4 show
that the observed per cent egg hatch was not close to the expected
per cent hatch at any of the test ratios. The obtained hatches were
1.4 to 2.3 times greater than expected although a significant re-
duction in fertility was obtained in all ratios using treated males.
The expected reduction in fertility was most closely approached with
the 5:1:1 ratio. Reduction in fertility was not proportionate to the
numbers of treated males used.
Table 4.--Competitiveness of H. pusio males after treatment with
4,500 r as pupae 2 days prior to emergence.*
: Number : Hatch Expected hatch
Cross ratio** : of eggs : (%) (%)
0:1:1 167 78.3 --
1:1:1 189 62.5 39.1
2:1:1 225 59.0 26.1
3:1:1 164 38.3 19.5
5:1:1 234 19.5 13.5
1:0:1 201 0.5 0.0
*20 females per ratio, 3 replicates.
**Treated male:untreated male:untreated female.
The results of the two competitiveness tests are generally in
accord with those obtained for other insects. Insects treated as adults
are usually found to be fully competitive. Henneberry and McGovern
(1963a) tested the effects of a sterilizing dose of 16,000 r on
competitiveness of D. melanogaster males and found them fully competi-
tive except at 1 ratio. Males of 0. nubilialls were shown to be
competitive after treatment with a sterilizing dose of 30,000 r
(Walker and Brindley, 1963).
Weidhaas and Schmidt (1963) reported males of Aedes aegypti L.
were not able to compete with untreated males after a dose of 10,000 r.
The same results were obtained by Davis et al. (1959) with males of
A. quadrimaculatus when treated with 11,820 r as 1-day-old pupae.
They noted that relatively large numbers of treated males had to be
introduced into a population to bring about reduction in fertility.
A field study using radiosterilized A. quadrimaculatus failed to reduce
natural populations, probably due to a lack of male competitiveness
based on irradiation effects and the use of a laboratory strain which
was highly selected (Dame and Schmidt, 1962; Weidhaas et al.,1962).
Steiner and Christenson (1956) and Christenson (1958) have reported
that gamma radiation adversely affected the mating competitiveness of
the oriental fruit fly, Dacus dorsalis Hendel. Treated males of
Anastrepha ludens Loew were also noncompetitive when treated as pupae
(Rhode et al., 1961). However, Bushland and Hopkins (1951) noted that
screw-worm flies were fully competitive when treated as late pupae.
Undoubtedly the effect of the sterilizing dose on male competitive-
ness is closely determined by the stage of maturity of the insect at
the time of irradiation. Late pupae have partially developed adult
structure while freshly pupated insects retain large amounts of un-
In tests with H. pusio males, significant recovery was found at
several dosage levels when gnats were treated in both the pupal and
adult stage. Gnats were irradiated as 24- to 36-hour adults with doses
ranging from 2,500 r to 5,000 r. The males were isolated from each
treatment level and further divided into 2 subgroups. One subgroup
at each dosage level was crossed immediately with untreated virgin
females. The second subgroup from each dosage level was held in a
10-drum vial for 18 days. After 18 days, these males were crossed with
virgin females which were 24 to 36 hours old. Ten pairs of insects were
used in all crosses. The tests were terminated 7 days after the crosses
had been made and egg hatch was determined. These same procedures were
repeated using males which had been treated in the pupal stage 2 days
prior to emergence.
The results shown in table 5 indicate that when males were
treated as pupae, there was significant recovery except at the 500 r
level. Egg hatch from the virgin females crossed with virgin males
which had been held 18 days after irradiation ranged from 3- to 7-fold
above that obtained from the virgin females which had been mated to the
freshly emerged irradiated males. The greatest recovery was shown at
the 2,500 r dosage where egg hatch went from approximately 7 per cent to
50 per cent. Recovery was found at the highest dosage of 4,500 r
although the insects were 93.5 per cent sterile.
Insects treated as adults had a significant recovery of fertility
at the 2,500 and 3,000 r levels. Thereafter, there was no statistical
difference between egg hatches from initial and delayed crosses. At
the 2,500 r dosage, fertility of the treated males increased approxi-
mately 2-fold during the second cross.
Table 5.--Fertility recovery in males of H. pusio treated with gamma
radiation as adults and pupae.*
Egg hatch (%) when mted at indicated interval : Chi
Dose 1-8 days 18-26 days :square**
0 77.5 72.0 0.84
500 48.o 56.0 2.73
1,500 12.5 44.0 157.29
2,500 5.3 35.5 372.27
3,500 3.5 14.0 171.56
4,500 0.0 6.5 109.00
0 72.5 73.0 0.44
2,500 18.5 35.5 258.00
3,000 3.5 12.5 46.31
3,500 4.5 3.5 0.45
4,000 1.0 1.5 1.00
4,500 0.5 2.0 5.00
5,000 0.5 1.5 4.00
*10 pairs of insects
**Chi square value at
per cross, 2 replicates.
.05 equals 5.991.
Results similar to those obtained by the author with gnats irradi-
ated as adults were observed by Grosch and Sullivan (1954) who
demonstrated permanent sterility in H. juglandis adults after treatment
with 4,800 r but at 3,300 r temporary sterility was obtained. Welshons
and Russell (1957) state that temporary sterility is due to depletion of
spermatogonia. The spermatogonial stage is especially sensitive to
irradiation. They treated Drosophila adults with 4,000 r and dissected
the testes at various intervals after treatment. At 48 hours after
treatment, there was a great reduction in gonial cells which sub-
sequently were found to repopulate as age increased.
A controlled mating test showed that multiple mating occurred
and that the reversal of treated and untreated males affected
viability of eggs from untreated females. Isolated females were mated
with single males which had been treated in the adult stage with
2,500 r or 5,000 r or were untreated. At the end of 9 days the order
of treated males and untreated males were reversed at each level of
radiation and the accumulated eggs were removed and held for hatch.
At the end of 16 days or 7 days after the second cross had been made,
the test was terminated and the second batch of eggs held for hatch.
A pair of insects in each of 5 cages were used in each test cross. The
test was replicated twice.
Table 6.-- The effect of alternate crosses with untreated males and
males treated with 2,500 and 5,000 r on untreated females.*
Dose :Order of crosses : Cross** :%)
2,500 First TM x UP 12.5
Second UM x UF 25.5
First UM x UF 72.5
Second 34 x UF 32.5
5,000 First TM x UF 11.5
Second UM x UF 32.5
First UM x UF 82.5
Second BI x UP 51.5
*5 isolated pairs per cross, 2 replicates.
**UM = untreated male, TM = treated male, UF = untreated female.
The sperm from the initial mating were apparently retained by the
female and subsequently diluted by sperm obtained in a second mating.
The resultant fertility of the gnat would then be due to the ratio of
untreated sperm to sperm bearing dominant lethal mutations. The ratio
of sperm after the second crossing appeared to be close to 1:1 in these
tests. At both 2,500 r and 5,000 r there was an approximate doubling
of fertility or reduction of fertility by 1/2, depending on the order
of crossing. These results are in accord with those obtained by
Henneberry and McGovern (1963b) with Drosophila and by Steiner and
Christenson (1956) with Dacus.
Fertility of female gnats was greatly reduced by treatment with
5,000 r. In a test designed to determine whether egg deposition from
treated insects was also reduced, adult insects were irradiated with
5,000 r and crosses were made using treated males with treated females,
untreated males with treated females, treated males with untreated
females, and untreated males with untreated females. Twenty insects of
each sex were used in each cross. Eggs were collected daily and counted.
Three replicates were made of the test.
There was no difference between egg production when females were
mated with treated or untreated males. The data were pooled across the
3 replicates. Egg production with treated and untreated females is
presented graphically in figure 1. Both treated and untreated females
began oviposition on the 5th day after emergence. Oviposition with
treated females was 2.3 and 1.6 times greater than from untreated
females on the 5th and 6th day, respectively. After the 6th day,
0 0 0 0 0 0 0 0 0
,-. C\J 0 o CO t-
H- H- H- H- H- u
untreated females produced greater numbers of eggs daily until the end
of the test. The average total egg production by untreated females was
17.1 eggs as compared to 14.8 eggs for the treated females, indicating
an approximate reduction of 12 per cent in egg production from treated
A further test on egg production was made using insects treated
as pupae with 2,500 and 5,000 r to compare the effects with adult
irradiation. Crosses were made using 10 pairs of gnats per vial.
Eggs were collected every 2 days and the test was terminated after 15
days. Two replicates were made.
From the results of the pupal irradiation test, no difference
was found between egg production of females mated to treated and un-
treated males as expected from the results of the sterility test.
The data were pooled and only the differences between treated and
untreated females were calculated. The untreated females produced
an average of 282 eggs per test. Females treated with 2,500 r produced
an average of 69 eggs per test and those treated with 5,000 r produced
an average of 7.5 eggs per test, or a 4-fold and 37-fold decrease from
untreated insects. Females treated with 2,500 r produced low numbers
of eggs continuously throughout the test; initial deposition began on
the 5th day with both treated and untreated females.
Egg production from irradiated insects has been studied with
several species. A linear decline in egg production has been found
with Tribolium castaneum Herbst (Bartlett and Bell, 1962) and H. juglandis
(Grosch and Sullivan, 1954) when females were treated with increasing
doses of radiation as adults. Egg production may be eliminated with
high doses such as 11,000 r with Habrobracon (Grosch, 1958) and 10,000 r
with Ae. aegypti (Terzian and Stabler, 1958). The lack of egg product-
ion in Drosophila after treatment with 10,000 r has been attributed
to destruction of germ calle (Ives et al., 1955).
Reduced egg production in insects is common with low doses
although the exact cause is not clear. Increased initial egg pro-
duction as found by the author in this study has not been commonly
observed with females treated as adults. However, Davis et al. (1959)
found the numbers of eggs deposited by A. quadrimaculatus females which
had received 1,500 and 2,500 r were almost twice the number deposited
King et al. (1956) found that egg deposition in Drosophila was
unchanged over a 35-day period after treatment with 2,000 r. King
(1957) noted that radiation treatment generally causes a slowing of
o8genesis. He observed that a reduction in total egg production of
treated females was due to inhibition of cell division and reduced
numbers of ovarioles and o8cytes per ovary in Drosophila.
The Effects of Radiation on Stages of the Life Cycle
Adults.--One of the first observations made by the author was that
treatment of adults with levels of radiation below about 10,000 r caused
no apparent increased mortality over controls. It was apparent that
doses of about 5,000 r caused no increased mortality when administered
to either 24- to 36-hour adults or to pupae 2 days prior to emergence.
The test periods, 7 to 15 days, were not long enough to show the
effect of low doses on life span. Since these low doses produced
sterility, little effort was made to determine the effect of higher
doses on the life span. In a test made to compare the survival for
several weeks of both sexes after a dose of 5,000 r, 50 adult insects
of each sex were irradiated and 50 insects of each sex were held
untreated. Food was supplied as needed, clean 10-dram vials were
supplied once each week and mortality was recorded each week.
Temperature during this test was 76* + 5* F. and relative humidity
about 50 per cent. The results are given in table 7.
Table 7.--Mortality of 50 H. pusio treated with 5,000 r as 24- to
: : Mortality at indicated week :
Sex : Treatment : 1-5 : 6 : 7 : Total
Male 5,000 r 0 1 4 5
None 0 2 3 5
Female 5,000 r 0 0 1 1
None 0 0 0 0
The results indicate no increased mortality due to the radiation
treatment after 7 weeks.
The apparent low lethality of doses of approximately 5,000 r is
coconly observed among various insect species when treated as adults.
Cork (1957), working with T. confusum, found that 20,000 r killed all
the test beetles in 20 days whereas a chronic daily dose of 100 r or
a single dose of 3,000 r extended the life span by several per cent.
Be suggested that these results might be due to a general retardation
of the physiological processes, thus slowing the aging process, or to
stimulation of some repair mechanism with a resultant increase in the
ability to withstand normal damage to the system. Bushland and Hopkins
(1953) found that there was no apparent increase in mortality of
females of C. hominivorax irradiated with 5,000 r although fewer males
died in the controls when males were treated with the same dose. Ross
and Cochran (1963) showed no increase in mortality of B. germanica
when treated as adults with 3,200 r. However, Baxter and Tuttle
(1957) concluded that the life span of Drosophila was reduced pro-
portionally to dose when corrected for survival of the controls and
the same effect was demonstrated in Habrobracon by Clark (1961).
A test was conducted to determine what doses were necessary to
cause a reduction in life span and the level needed to cause death
within a few days. Forty insects of each sex were exposed to radiation
levels proceeding from 0 to 135,000 r in 15,000 r increments. Exposure
time at 135,000 r was 58 minutes and 4 seconds. Unmated insects were
held in 10-dram glass vials and food was provided as needed. Mortality
was recorded daily. Temperature and humidity were as in the preceding
test. The LT-50 and LT-100 for each sex at each dose level are given
in table 8.
Table 8.--The LT-50 and LT-100 of H. pusio males and females irradiated
as 24- to 36-hour adults.*
: Days after irradiation
Dose : LT-50 LT-100 LT-50 LT-100
0 61 66 64 94
15,000 29 41 24 54
30,000 15 21 17 31
45,000 11 14 11 17
60,ooo 7 9 8 12
75,000 5 7 6 8
90,000 5 6 5 7
105,000 4 4 4 5
120,000 4 4 4 4
135,000 3 4 3 4
*40 insects per treatment.
With the lowest dose, 15,000 r, the LT-50 for males and females
was reduced to approximately 1/2 that of untreated insects. No
difference occurs between the LT-50 and LT-l00 of males and females at
doses of 120,000 r and 135,000 r. Below 105,000 r the LT-100 of
females was always higher than that of males although the difference
decreased with increasing dose. The same statement was true of the
LT-50 except at 15,000 r where the LT-50 for males was higher. The
control gnats lived longer than the treated gnats. Doses greater
than 45,000 r produced an immediate comatose or moribund state which
lasted less than an hour except at 105,000 r and up where no recovery
of activity was observed. No insects were ever observed to feed at
doses of 105,000 r or higher. At these dosages the gnats were immobile
on the walls and floor of the cages and made no attempt to reach food.
At very high doses of radiation, the treated insect may become
moribund, as observed by the author at doses over 45,000 r. This
reaction was observed by Beidenthal (1945) who termed it "sluggish-
ness," and by Hassett and Jenkins (1952) and Sullivan and Grosch (1953).
Grosch applied the term "radiation induced lethargy." Insects treated
with doses sufficient to induce permanent lethargy will usually
survive longer than controls when both are kept under starvation con-
ditions. This is due to the inactivity of the treated insect (Grosch,
The use of very high doses has been suggested as a control measure
by Rassett and Jenkins (1952) who observed that 65,000 r could serve as
a quick knockdown dose for control of most insects. Bletchly and Fisher
(1957) found Lyctus and Anobium beetles could be controlled in situ by
a dose of 48,000 r.
Eggs.--Preliminary tests on the sensitivity of eggs at different
ages showed a wide variation in the sensitivity in that the more
mature eggs were insensitive while the freshly laid eggs were extremely
sensitive. Eggs 1 to 3, 24 to 36, and 47 to 49 hours from oviposition
were tested. The 1 to 3 and 24 to 26 hour eggs were irradiated with
100, 500, and 1,000 r at a dose rate of 68 r per minute plus a
control. The 47 to 49 hour eggs were irradiated with 4,000, 8,000,
and 12,000 r at a dose rate of 2,310 r per minute. Fifty freshly ovi-
posited eggs were placed on moist blotting paper at each level so that
development could proceed normally until treatment. The per cent
hatch was recorded and the LD-50 was calculated using probit analysis.
he LD-50 for 1- to 3-hour-old eggs was 126 r, for 24- to 36-hour-old
eggs 1,350 r, and for 47- to 49-hour-old eggs the LD-50 was 20,000 r.
Cole et al. (1959) gave the LD-50 as 136 r at 2 days for 1/2-
day-old house fly eggs, that of pupae as 15,000 r. Erdman (1960b)
found that treatment of 24-hour-old eggs of Habrobracon with 2,400 r
prevented the eclosion of any adult insects. These results are
essentially the same results obtained for H. pusio by the author.
Iaxrvae.--In a test using last instar larvae to observe the effects
of irradiation on this stage of the life cycle, larvae which had not
reached the prepupal stage were hand picked and placed on larval rearing
media contained in 1-pint jars. One hundred larvae were used per jar
at each level of radiation. An initial test had shown no emergence
after a dose of 5,000 r so the highest dose given was 3,500 r. A
dose rate of 68 r per minute was used. The jars were connected by
2-way lids to another pint jar and the emerging insects were collected
daily. Crosses were made using 10 insects of each sex when sufficient
insects were available. The results are compiled in table 9.
Table 9.--The emergence and fertility of
last instar larvae.*
Dose : (%) : Cross**
0 57 UMx UF
500 52 UM x TF
TM x UF
UM x TF
TM x UF
H. pusio adults treated as
: lumber eggs :
:in 10 days :
*100 larvae per treatment.
**UM = untreated male, UF = untreated female,
TF = treated female.
TI = treated male,
Emergence in the control was 57 per cent while 7 per cent emerged
at 3,500 r. Fertility was reduced in females but not in males at
1,500 r. Fecundity appeared to be low only in the control. Emergence
of gnats at 2,500 and 3,500 r was delayed 2 to 3 days beyond the un-
treated gnats and emergence was spread over a longer period of time.
These results compare favorably with those of Henneberry (1963)
who found 56 per cent survival in controls of larval irradiation tests
with Drosophila, and only 17 per cent survival to adult stage when larvae
were treated with 4,000 r. He also found no difference in the life span
of males and females irradiated as pupae or adults with doses up to
Pupae.--In a test to determine the effect of dose on pupal emer-
gence, 50 pupae were treated at each of 5 levels of radiation. Dose
rate was 3,130 r per minute, the temperature during rearing and up
till the time of the last eclosion was 70 t 5" F. The test was
replicated twice. The results are shown in table 10.
Table 10.--The mortality of 50 H. pusio treated as pupae 2 days prior
: Number emerged Average emergence
Dose : Test 1 : Test 2 (%)
0 45 47 92
1,000 45 49 94
5,000 40 49 89
10,000 29 36 65
15,000 15 27 42
20,000 4 2 6
There was no significant difference in gnat emergence between the
control and after treatment with 1,000 r and 5,000 r. At the 10,000 r
level there was a 30 per cent decrease in adult emergence as compared
to the untreated gnat emergence. The LD-50 dose was calculated by
probit analysis to be 12,000 r. Insects treated with 10,000 r and
higher doses were frequently found with the ptilinum extended and were
partially clear of the pupal case but were unable to complete emergence.
These were counted as not emerged.
Morphological and Cytological Effects
Studies of the cytological effect of gamma radiation on the
screw-worm fly (LaChance and Leverich, 1962; LaChance and Bruns, 1963),
the German cockroach (Ross and Cochran, 1963), Drosophila (King, 1957;
Cantwell and Henneberry, 1963) showed that morphological damage is
induced in insect gonads when they are treated with sterilizing
doses of radiation. This part of the study was undertaken to
correlate the observed effects of radiation on fecundity and fertility
with morphological and cytological damage to the gonads. The project
was divided into a study of the morphology of irradiated ovaries,
testes, and chromosomal aberrations.
The ovaries of treated and untreated insects were observed to
determine the effects of radiation on the whole ovary and the ovarioles.
Insects were treated as pupae with doses ranging up to 4,500 r. Approxi-
mately 10 insects were dissected at each treatment level. Dissections
were made 2, 5, and 8 days after emergence.
Each ovariole consisted of a terminal filamnt at the distal end,
followed by the germarium, a tapered moniliform series of 2 to 4
follicles and, at the basal end, a pedicel. The development of the
primary follicles of the eye gnat was divided into 5 stages by Schwartz
(1964) and consisted briefly of the following: stage "a", no oicyte
visible in the primary follicle; stage "b", o8cyte visible but occupy-
ing less than 1/2 the volume of the follicle; stage "c", o6eyte
occupying 51 to 75 per cent of the follicle; stage "d", 76 to 100 per
cent o8cyte formation; and stage "e", egg fully developed with chorionic
pattern and/or micropyle visible. This classification scheme was used
to evaluate the effects of irradiation on obcyte development in this
investigation. In the author's study, the oScyte was observed to enlarge
as the nurse cells atrophied. The second follicle of the ovariole did
not undergo o8cyte development until the primary follicle had discharged
its mature egg. The second follicle then proceeded to develop as did
the first follicle. A third and fourth follicle are usually present
but did not develop until the preceding follicle had discharged its
egg. The germarium contained the gonial cells which develop into
obcytes and nurse cells within the follicles.
A comparison of treated and untreated ovarioles was made by
examining the primary follicle and recording its stage of development.
The second follicular cell was scored as being normal, abnormal, or
absent. Normal indicates the follicle was at stage "a" and appeared
the same as the controls at that age. The term abnormal was applied
to second follicles which were markedly smaller, irregular in shape
and/or showed loss of normal cell structure. When the second follicle
could not be differentiated from other tissue it was scored as absent.
The gemarium was scored as normal if it resembled the control of
the same age. A germarium was noted as abnormal if it had lost its
characteristic shape or cell structure. At the higher dosages used,
the germarium was sometimes not distinguishable from other tissues
and in these cases it was scored as absent. Provision was also made
in the table for recording additional follicles, properly termed third,
fourth, or fifth follicles. If any more than the first and second
follicles were present, the ovary was scored for additional follicles.
The results are given in table 11.
r-Ic 000 000
0\--\0 t-- -*0 r- r-10 0 000 000
000 000 000 000 HH< H9H
ooo ooo ooo co Cr- ooo oo
000 000 000c c0C,- 000 000
000 000 000
000 000 000
U C'O u
H o0 m
oOq a 0\ 00 0o
H- nH HHHnn
HMCO 000 000
\D CU r-I 0\D co
0MO OO 0
H00 0 c H
r 00 000
cO" r-4 090O\ 000
c0 U\c c\ rUNo C ur\co Cu iN\co 0 IcO C NLrcCO
o W1 r
0 8 8 08
a ooa\ QQQ 2o, o\,
0Oi O 000 O n r
ooo ooo ooo oCo a 0 9
ooo ooo 000 t-0 000 000
0 OM 0
00 0 -
The ovaries of untreated gnats 2 days after emergence were all
found to have stage "a" follicular development and in 90 per cent of
the ovarioles a third follicle could be distinguished besides the
second follicle and germarium. All the ovarioles in each ovary
appeared in the same stage of development. After 5 days, over 1/2
the ovaries contained primary follicles with fully mature eggs and
only 1 adult was found with stage "a" oocytes. This was probably due
to oviposition and subsequent development of the second follicles.
After 8 days, approximately 1/2 the ovaries had apparently discharged
their mature eggs and the second follicles were in the early stages of
o6cyte development. At least 50 per cent of all untreated ovaries
were found at 5 and 8 days to have ovarioles with 3 or more follicles.
Gnat pupae treated with 500 r showed o8cyte development very
similar to untreated insects. The only difference observed between
treated and untreated ovaries was a wider variation in the developing
stages of the follicles. With 2-day-old adults, some ovaries contained
nearly mature eggs. The remaining follicles and germaria showed no
variation from normal. At 1,500 r there was an apparent reduction in
the number of third follicles and 2 of the second follicles were found
to be abnormal. First follicle development appeared normal at 2 days.
Three 8-day-old adults showed loss of nurse cell differentiation in
their developing follicles. Some ovaries contained follicles in
several stages of development or with a reduced number of ovarioles,
a condition not observed previously by the author.
At 2,500 r a large proportion of the ovaries were classified as
malformed. These ovaries had ovarioles which showed loss of cellular
differentiation in all follicles. That proportion of the ovaries which
were not malformed contained o8cytes in an advanced stage of develop-
ment but these oScytes contained no distinct nurse cells. These were
classified by size alone. Some ovaries which showed development
contained as few as 2 distinguishable ovarioles. With this exposure
the germarium was usually present but in many cases was abnormal in
shape, being only a terminal clump of tissue.
Pupae treated with 3,500 and 4,500 r showed an almost complete
lack of follicular or ovarian development as adults. At the 3,500 r
level, 5-day-old adults had partially developed eggs which had no
nurse cell differentiation. They had only 1 to 3 developed ovarioles
per ovary. At 4,500 r no remnant of an ovariole was found. Figures 2
and 3 show ovarian damage obtained by irradiation of the pupal stage
with these dosage levels.
The damage observed in ovaries of adults treated as pupae
explains the egg production and fertility from gnats treated with the
same doses in preceding tests. Females treated with 3,500 and 4,500 r
laid an average of less than 1 egg per female in the egg production
test and in the present test dissected ovaries showed complete lack of
differentiation into normal structures at these doses.
Similar results were obtained by LaChance and Bruns (1963) with
C. hominivorax. These workers treated 5-day-old pupae with 4,000 r
and found that primary follicles remained undeveloped and secondary
follicles failed to develop or were atrophied. At 2,000 r they found
retarded growth, but nurse cells, second follicles and germaria were
generally present. They suggest that failure of treated females to
produce mature ova reflects an inability of nurse cells to support
Figure 2.--Ovaries of H. pusio treated with gaima radiation and
a. Untreated ovaries with stage "b" oocytes.
b. Treated ovaries from insect irradiated with
4,500 r as a pupae 2 days prior to emergence.
Note complete lack of normal ovariole development.
^p *1 r *
Figure 3.--Ovarian development of H. pusio after treatment with
3,500 r as pupae 2 days prior to emergence.
a. Arrows indicate
ovaries, each with an abnormal
b. Abnormal oocytes, arrow indicates area of undifferen-
tiated nurse cells.
normal vitellogenesis. In the author's study of H. pusio ovarioles,
nurse cells were found to be abnormal at doses as low as 1,500 r in
that the nurse cells failed to differentiate into visible cells.
In studies with the German cockroach, Ross and Cochran (1963)
found that insects treated as nymphs with doses from 3,200 to 6,400 r
produced ovaries in the adult stage which appeared to have ceased
growth at the time of irradiation. The dose of 3,200 r effectively
sterilized female roaches. A dose of 1,600 r caused a high proportion
of ovarioles to be abnormal in structure.
The normal testis has been described by Schwartz (1964) as a
pear-shaped body having a knob-like apical end and a broad base where
it attaches to the vas deferens. The testis is divided into several
distinct zones of spermatogenesis which are visible in untreated testes
(see figure 4). Basally, there is a darkened zone of mature sperm or
transformation zone. This zone of mature sperm fills approximately
1/2 the untreated testis by the seventh day.
The effect of irradiation was observed on the testes of males
treated as 24- to 36-hour adults or treated in the pupal stage 2 days
prior to emergence. The sterilizing dose of 4,500 r for pupal irradi-
ation and 5,000 r for adult irradiation was used. Testicular measure-
ments were made from males 7 days + 1 day after emergence. The test
gnats were from the same generation. Approximately 25 males were
examined for each treatment. Measurements were made on both testes
from each male using an ocular scale and the measurements converted to
microns with an accuracy of 10 microns. Each observation in the table
is from a single testis.
Figure 4.--Trtes from untreated and treated F. ltSO 7 days aiter
a. restes froa urtreated sale.
Note distinct areas of
b, T~tes from aLle treated with 5,000 r au a adult 24 to
36 hours after emergence.
h j. .
* Il /
The length and width of the testes from males treated in the
pupal stage was significantly different from untreated testes. The
smallest testis examined in this study was from a male treated in
the pupal stage which had the dimensions of 250 x 112 microns in length
and width respectively. The largest testis was from a male treated
in the adult stage which had a length and width of 580 x 210 microns.
In males treated as adults or pupae, the darkened zone of mature
sperm extended the full length of the testes and gave the entire
testis an opaque appearance. Schwartz (1964) observed a similar effect
from treatment of H. pusio with chemosterilants.
The shapes of testes from males treated as pupae were markedly
different than those from either untreated males or males treated as
adults. The whole testis had a shriveled and knobby appearance, the
apical tip of the testis was often reflexed on the main axis of the
testis at about 90". The difference in size between 2 testes in an
individual did not vary appreciably but occasionally a more reduced
testes was found on one side. Several individual testes were seen with
patchy areas of pigmentation. The areas without pigmentation appeared to
be transparent. This effect was observed only in males treated in the
Testes from males treated as adults were somewhat longer than
those from untreated males although widths were approximately the same.
The shape of the treated testis was more oblong than the untreated
testis due to slightly increased length. The testes of the gnats
treated in the adult stage were perceptibly knobby. The apical tip
was straight or slightly reflexed as observed in untreated testes. The
distortion of the apical tip of the testes from males treated as adults
was less severe than that observed in testes from males treated in the
Testes from males treated in both stages of development with the
sterilizing dose revealed active sperm and sperm bundles upon squashing
of the whole testis.
Table 12.--Testicular measurements of H. pusio treated with 4,500 r
as pupae 2 days prior to emergence, 5,000 r as 24- to 36-
hour adults, and untreated.*
: Number : Dimensions in microns + SE : Range
Treatment :observed: Length : Width : Length : Width
None 46 428.0+5.22 181.5f2.86 530-330 220-150
4,500 r on 50 380.2+6.20 132.0+3.50 470-280 170-90
5,000 r on 48 459.3+7.32 174.5+3.28 580-350 220-110
*7 days after emergence.
A study was undertaken to (1) define the somatic number of chromo-
somes in H. pusio, and (2) determine if there was morphological damage
to the chromosomes with the sterilizing doses. It was the objective of
this work to relate the chromosomal damage to the reproductive performance
of the insect induced by the sterilizing dose.
The untreated larval brain was selected for chromosome number
studies. Last instar larvae were taken from the laboratory colony and
squash preparations were made of the brain tissue. The brain was found
to be dorsally located about 1/4 the body length from the anterior end.
It was identified by the bilobed structure and milky opaque color which
differentiated it from surrounding tissue.
Chromosome figures were readily obtained in the larval brain
preparations using both the orcein-fast green stain technique of
Morgan and LaBrecque (in press) and the Feulgen staining procedure
of Whiting (1950). Slides prepared by the Feulgen method showed the
chromosome number to be 2N = 8. Four distinct pairs of homologous
chromosomes were observed.
The phenomenon of somatic pairing was consistently encountered.
Somatic pairing is characterized by a close association between homo-
logous chromosomes during mitotic divisions and is commonly observed
in Diptera (LaChance, 1964). At least 25 preparations were stained by
the orcein-fast green method but in no case was a full chromosome
complement of 8 observed. Instead there were 3 pairs of chromosomes
which were somatically paired plus an additional chromosome which
stained identically to the others but which had no observable hoeolog.
From comparisons with the Feulgen prepared slides it was determined that
there were actually 2 homologous chromosomes, 1 of which could not be
shown with the orcein-fast green stain. As shown in the Feulgen prep-
arations, these ehromosoes appear morphologically identical and it is
suggested by the author that they are the sex chromosomes. Thus, no
morphologically differentiated heterochromosomes (XY) were observed
although differences in stain characteristics were found.
LaChance (1964) studied the chromosomes of the horn fly, Haematobia
irritans (L.), and the stable fly, Stomoxys calcitrans (L.), and found
the diploid (2N) chromosome number to be 10 in both species. He found
no morphologically differentiated heterochromosomes using aceto-orcein
stain and concluded the sex chromosomes were not distinguishable from
Preparations were made from the testes from treated pupae and
adults. Pupae were treated 2 days prior to emergence and the testes were
removed 0 to 12 hours after emergence. Adults were treated 1 to 36
hours after emergence and the testis removed immediately. The
dissected testes were stained using the orcein-fast green method which
produced darkly stained chromosomes.
Initial trials using doses of 4,500 r on pupae and 5,000 r on
the adults produced slides with no or very few chromosome figures.
The dose was reduced to 2,500 r for both the pupae and the adults, and
the same dissection schedule was maintained. There was an increase in
number of chromosome figures although the chromosome figures from
treated testes were never found in numbers approaching those on slides
from the larval brain. Damaged chromosomes were not frequently en-
countered but figures were found where chromosome breakage was evident.
In such cases the chromosomes were fragmented and the pieces scattered
so that individual chromosomes could not be recognized. Many more
chromosomal patterns were observed from insects treated in the pupal
stage, probably due to greater activity in cell division.
Figure 5.--Chromosomes of H. pusio.
a. Untreated larval brain tissue stained with
b. Chromosome figure from testis of insect treated
with 3,500 r as a late pupae. Arrow indicates a
9 '* '.i*
& I ;iiili
1. The dose required to sterilize insects treated as 24- to
36-hour adults was determined to be approximately 5,000 r for both
males and females. When insects were treated as pupae 2 days prior to
emergence, the sterilizing dose was found to be approximately 4,500 r
for both sexes.
2. Males treated as adults with the sterilizing dose were found
to be fully competitive with untreated males for untreated virgin
females. Males treated in the pupal stage were partially competitive
with untreated males for virgin females at the ratios and doses tested.
3. A partial recovery of fertility was observed in males after
18 days when treated as pupae at doses up to and including 4,500 r.
Males treated as adults recovered partial fertility at doses of 2,500
and 3,000 r. Beyond 3,000 r a permanent sterility was induced.
4. In multiple mating tests, females initially crossed with males
treated with 2,500 and 5,000 r produced a high percentage of infertile
eggs. Upon subsequent substitution of untreated males, fertility was
increased. Females crossed initially to untreated males produced a
high percentage of fertile eggs but on subsequent substitution of treated
males, the percentage of egg hatch was reduced. These results indicate
the females to be polygamous and a carry-over of sperm from the initial
5. Females treated with 4,500 r as pupae 2 days prior to adult
emergence did not produce eggs. Females treated as 24- to 36-hour
adults with 5,000 r produced initially greater numbers of eggs but
total production during the 15-day test period was reduced as compared
to untreated females. Untreated females appeared to exhibit 3 peaks
of oviposition but did produce eggs continuously over the 15-day test
6. The lethal effects of radiation were determined for each sex
treated as adults with doses up to 135,000 r which produced 100 per cent
mortality in 4 days. A dose of 5,000 r caused no increased mortality
in either sex during a T-week test period.
7. The LD-50 dose for pupae treated 2 days prior to emergence was
found to be 12,000 r. No significant difference was found in emergence
of pupae treated with 5,000 r as compared to controls.
8. The lethal effect of radiation was tested on larvae and eggs.
Last instar larvae were very susceptible to a dose of 3,500 r as
measured by adult emergence but showed resistance to doses of 2,500 r
and lower. Eggs which were 1 to 3 hours old had an LD-50 of 126 r.
After 47 to 49 hours of development, the eggs had an LD-50 of 20,000 r.
9. A cytological study of ovarioles was conducted to determine the
state of development in untreated insects at 2, 5, and 8 days when
compared to those irradiated with various doses as pupae. The effects
of radiation on ovarian growth were the loss of ovariole differentiation
at 3,500 r and 4,500 r and a loss of nurse cell organization in the
developing oocytes at lower doses.
10. The chromosomes of treated and untreated insects were studied
and abnormalities were observed in those treated with the sterilizing dose.
The 2N chromosome number is 8.
Abbott, W. S. 1925. A method of computing the effectiveness of an
insecticide. J. Econ. Entomol. 18: 265-267.
Aldrich, J.. M. 1929. Notes on the synonomy of Diptera, No. 3. Proc.
Ent. Soc. Wash. 31(2): 32-36.
Annan, M. E. 1955:. X-ray induced impairment of fecundity and fertility
of Drosophila robusta. J. Heredity 46: 177-182.
Bartlett, A. C., and A. E. Bell. 1962. The effect of irradiation on
the reproduction in two strains of Tribolium castaneum Herbst.
Radiation Research 17: 864-877.
Baxter, R. C., and L. W. Tuttle. 1957. Life span shortening in
irradiated Drosophila. Radiation Research (abstract) 7: 303.
Bletchly, J. D., and R. C. Fisher. 1957. Use of gamma radiation for
the destruction of wood boring insects. Nature 179: 670.
Boyes, J. W. 1958. Chromosomes in classification of Diptera. Prec.
Tenth Internatl. Congr. Entomol. (Montreal, 1956) 2: 899-906.
Breland, 0. P. 1961. Studies on the chromosomes of mosquitoes. Ann.
Entomol. Soc. Am. 54: 360-375.
Bushland, R. C. 1960. Male sterilization for the control of insects.
In "Advances in Pest Control Research" edited by R. L. Metcalf,
Interscience Publ. Inc., N. Y. Vol. 3: 1-25.
Bushland, R. C., and D. E. Hopkins. 1951. Experiments with screw-worm
flies sterilized by X-rays. J. Econ. Entomol. 44: 725-731.
Bushland, R. C., and D. E. Hopkins. 1953. Sterilization of screw-worm
flies with X-rays and gamma rays. J. Econ. Entomol. 46: 648-656.
Cantwell, G. E., and T. J. Henneberry. 1963. The effect of geaia
radiation and apholate on the reproductive tissues of Drosophila
melanogaster Meigen. J. Insect Pathol. 5: 251-264.
Catchside, D. G. 1948. Genetic effects of radiation. Advances in
Genetics. 2: 271-367.
Christenson, L. D. 1958. Recent progress in the development of pro-
cedures for eradicating or controlling tropical fruit flies. Proc.
Tenth Internatl. Congr. Entomol. (Montreal, 1956) 3: 11-16.
Clark, M. M. 1961. Some effects of X-irradiation on the longevity in
Habrobracon females. Radiation Research 15: 515-519.
Cole, M. M., G. C. laBrecque, and G. S. Burden. 1959. Effects of gamma
radiation on some insects affecting man. J. Econ. Entomol. 52:
Cork, J. M. 1957. Gamma radiation and longevity of the flour beetle.
Radiation Research 7: 551-557.
Dame, D. A., and C. H. Schmidt. 1962. The importance of competitive-
ness of radiosterilized males in mosquito control programs. Proc.
Forty-ninth Annual Meeting, N. J. Mosq. Exterm. Assoc., p. 165-168.
Davis, A. N., J. B. Gahan, D. E. Weidhaas, and C. N. Smith. 1959.
Exploratory studies with gamma radiation for sterilization and
control of Anopheles quadrimaculatus. J. Econ. Entomol. 52: 868-870.
Dow, R. P., and M. J. Willis. 1959. Evaluation of insecticides for
the control of Hippelates pusio in the soil. J. Econ. Entomol.
Erdman, H. E. 1960a. Adult longevity as a sensitive criterion of
radiation-induced damage wn 24-hour Habrobracon (Hymenoptera)
embryos are X-rayed. J. Econ. Entomol. 53: 971-972.
Erdman, H. E. 1960b. Divergence between lethal doses and sterilizing
doses of X-rays with progressive development in Habrobracon
females. Nature 186: 254-255.
French, W. L., R. H. Baker, and J. B. Kitzmiller. 1962. Preparation
of mosquito chromosomes. Mosquito News 22(4): 377-383.
Freund, J. E., and F. J. Williams. 1l61. Modern Business Statistics.
Prentice Hall, New Jersey. 539 p.
Grosch, D. S. 1956. Induced lethargy and the radiation control of
insects. J. Econ. Entomol. 50: 438-440.
Grosch, D. S. 1958. The quantitative alterations in Habrobracon
fecundity induced by COX0 exposures. Radiation Research 9: 123-124.
Grosch, D. S. 1962. Entomological aspects of radiation as related to
genetics and physiology. Ann. Rev. of Ent. 7: 81-106.
Grosch, D. S., and R. L. Sullivan. 1954. The quantitative aspects of
permanent and temporary sterility induced in female Habrobracon by
X-rays and B-radiation. Radiation Research 1: 294-320.
Hassett, C. C., and D. W. Jenkins. 1952. Use of fission products for
insect control. Nucleonics 10: 42-46.
Heidenthal, C. 1945. The occurrence of X-ray dominant lethal mutations
in Habrobracon. Genetics 30: 197-205.
Henneberry, T. J. 1963. Effects of gamma radiation on the fertility and
longevity of Drosophila melanogaster. J. Econ. Entomol. 56: 279-281.
Henneberry, T. J., and W. L. McGovern. 1963a. Effects of gamma radiation
on mating competitiveness and behavior of Drosophila melanogaster
males. J. Econ. Entomel. 56: 739-740.
Henneberry, T. J., and W. L. McGovern. 1963b. Some effects of gamma
radiation on fertility of Drosophila melanogaster and viability
of sperm after multiple matings of males. J. Econ. Entomol.
Berms, W. B. 1926. Hippelates flies and certain other pests of the
Coachella Valley, California. J. Econ. Entomol. 19: 692-695.
Howden, H. F. 1957. Investigations on sterility and deformities of
Onthophagus induced by gamma radiation. Ann. Entomol. Soc. Am.
Howden, H. F., and S. I. Auerbach. 1958. Some effects of ga
radiation on Trogoderma sternale Jayne. Ann. Entomol. Soc. Am.
Ives, P. T., R. S. Heilman, and H. H. Plough. 1955. The LDcO for
gamma radiation in Drosophila melanogaster. Genetics 4 577.
Jay, E. G. 1961. laboratory and field studies on the ecology of
Hippelates pusio Loew. (Diptera: Chloropidae) and related species.
Master's Thesis, University of Florida.
King, R. C. 1957. The cytology of the irradiated ovary of Drosophila
Lmelanogaster. Exptl. Cell Research 13: 545-552.
King, R. C., J. B. Darrow, and N. W. Kaye. 1956. Studies of different
classes of mutations induced by radiation in Drosophila melano-
gaster females. Genetics. 14: 890-900.
King, R. C., and J. H. Sang. 1959. Oogenesis in adult Drosophila
melanogaster. VIII. The role of folic acid in oogenesis. Growth
Knipling, E. F. 1955. Possibilities of insect control or eradication
through the use of sexually sterile males. J. Econ. Entomol.
Knipling, E. F. 1960. The eradication of the screw-worm fly. Sci.
Am. 203: 54-61.
Koller, P. C., and I. A. R. S. Ahmed. 1942. X-ray induced structural
changes in chromosomes of Drosophila pseudo-obscura. J. Genet.
Kumm, H. W., and T. B. Turner. 1936. The transmission of yaws from
man to rabbits by an insect vector, Hippelates pallipes Loew.
Am. J. Trop. Med. 16: 245-262.
LaChance, L. E. 1964. Chromosome studies in three species of Diptera
(Muscidae and Hypodermatidae). Amn. Entomol. Soc. Am. 57: 69-73.
LaChance, L. E., and S. B. Bruns. 1963. Oogenesis and radiosensitivity
in Cochliomyia hominivorax (Diptera: Calliphoridae). Biol. Bull.
LaChance, L. E., and A. P. Leverich. 1962. Radiosensitivity of de-
veloping reproductive cells in female Cochliomyia hominivorax.
Genetics 47: 721-735.
Lea, D. E. 1955. Action of radiation on living cells. 2nd ed.
Cambridge University Press, Cambridge, England. 416 p.
Lee, W. R. 1958. The dosage response curve for radiation induced
dominant lethal mutations in the honey bee. Genetics. 43: 480-492.
Litchfield, J. T., and F. Wilcoxon. 1949. A simplified method of
evaluating dose-effect experiments. J. Pharmacol. Exptl.
Therap. 96: 94-113.
Morgan, P. B., and G. C. LaBrecque. In press. Preparation of house
fly (Musca domestic L.) chromosomes. Ann. Entomol..Soc.-Am.
Mulla, M. S. 1958. Control of mites in laboratory culture of the eye
gnat Hippelates collusor Townsend. J. Econ. Entomol. 50: 461-462.
Mulla, M. S. 1963. Activity of new insecticides against adults of
Hippelates collusor and H. pusio. J. Econ. Entomol. 56: 47-50.
Muller, H. J. 1927. Artificial transmutation of the gene. Science
Oster, I. I., e.nd G. Balaban. 1963. A modified method for preparing
somatic chromosomes. Drosophila Information Service. Vol. 37:
Rhode, R. H., F. Lopez, F. Eguisa, r;nd J. Telich. 1961. Effect of
gamma radiation on the reproductive potential of the Mexican
fruit fly. J. Econ. Entomol. 54: 202-203.
Ross, M. H., and D. G. Cochran. 1963. Some early effects of ionizing
radiation on the German cockroach, Blattella germanica. Ann.
Entomol. Soc. Am. 56: 256-261.
Runner, G. A. 1916. Effect of roentgen rays on the tobacco, or
cigarette beetle. J. Agr. Research 6: 383.
Sabrosky, C. W. 1941. The Hippelates flies or eye gnats: Preliminary
notes. Canad. Ent. 73: 23-27.
Sanders, D. A. 1940. Musca domestic and Hippelates flies, vectors of
bovine mastitis. Science 92: 286.
Schwartz, P. H. 1964. Reproduction and chemical sterilization of the
eye gnat, Hippelates pusio Loew. Doctoral Dissertation. Univer-
sity of Florida.
Schwarz, E. A. 1895. The Hippelates plague in Florida. Insect Life.
Snedecor, G. W. 1961. Statistical Methods Applied to Agriculture and
Biology. 5th Edition. The Iowa State University Press, Ames. 534 p.
Steiner, L. F., and L. D. Christenson. 1956. Potential usefulness of
the sterile fly release method in fruit fly eradication programs.
Proc. Hawaii Acad. Sci. 1955-56.
Sullivan, R. L., and D. S. Grosch. 1953. The radiation tolerance of
an adult wasp. Nucleonics 11: 21-23.
Teas, H. J. 1959. A multipurpose agricultural cobalt-60 irradiator.
Florida Agricultural Experiment Stations Journal Series No. 874
Terzian, L. A., and N. Stabler. 1958. A study of some effects of gamma
radiation on the adults and eggs of Aedes aegypti. Biol. Bull.
Turner, E. R. 1960. Certain aspects of the biology and control of
the eye gnat Hippelates pusio Loew. Master's Thesis, University
of Florida. 73 p.
Walker, J. R., and T. A. Brindley. 1963. Effect of X-ray exposure on
the European corn borer. J. Econ. Entomol. 56: 522-525.
Weidhaas, D. E., and C. H. Schmidt. 1963. Mating ability of male
mosquitoes, Aedes aegypti L. sterilized chemically or by gamma
radiation. Mosquito News 23: 32-34.
Weidhaas, D. E., C. H. Schmidt, and W. F. Chamberlain. 1962. Research
on radiation in insect control. Radioisotopes and Radiation in
Entomology. International Atomic Energy Agency, Vienna, p. 257-265.
Welshons, W. J., and W. L. Russell. 1957. The effect of X-rays on
Drosophila testes and a method for obtaining spermatogonial
mutation rates. Nat. Acad. Sci. 43: 608-613.
Whiting, A. R. 1950. A modification of the Schmuck-Metz whole-mount
technique for chromosome study. Stain Tech. 25(1): 21-22.
Wilson, G. B., and J. H. Morrison. 1960. Cytology. Reinhold Pub-
lishing Corp., New York. 194 p.
von Borstel, R. C. 1959. On the nature of dominant lethability
induced by radiation. Atti. Assoc. Gen. Ital. 5: 35-50.
von Borstel, R. C. 1960. Population control by release of irradiated
males. Science 131: 878, 880-882.
von Borstel, R. C., and M. L. Rekemeyer. 1959. Radiation-induced and
genetically contrived dominant lethality in Habrobracon and
Drosophila. Genetics 44: 1053-1074.
Hollis Mitchell Flint was born May 28, 1938, at Miami, Florida.
He was graduated from Mainland High School in Daytona, Florida, in
1956. In June, 1960, he received the degree of Bachelor of Science
from Stetson University, Deland, Florida. In 1960 he enrolled in the
Graduate School of the University of Florida. He held a National
Defense Education Act fellowship from 1961 to 1964 in the Department
of Entomology. From 1960 until the present time he has pursued his
work toward the degree of Doctor of Philosophy.
Hollis M. Flint is married to the former Adela Dale Sheerer.
He is a member of the Entomological Society of America, the American
Association for the Advancement of Science, the Beta Beta Beta
biology honorary society and the Newell Entomological Society.
This dissertation was prepared under the direction of the
chairman of the candidate's supervisory committee and has been proved
by all members of that committee. It was submitted to the Dean of the
College of Agriculture and to the Graduate Council, and was approved
as partial fulfillment of the requirements for the degree of Doctor
June 18, 196..
Dean, College of Agriculture
Dean, Graduate School
kht 6 Lit 4u