Group Title: effect of cobalt 60 gamma rays on the biology of the eye gnat Hippelates Pusio Loew /
Title: The Effect of cobalt 60 gamma rays on the biology of the eye gnat Hippelates Pusio Loew /
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Title: The Effect of cobalt 60 gamma rays on the biology of the eye gnat Hippelates Pusio Loew /
Physical Description: v, 61 leaves : ill. ; 28 cm.
Language: English
Creator: Flint, Hollis Mitchell, 1938-
Publication Date: 1964
Copyright Date: 1964
 Subjects
Subject: Gamma rays -- Physiological effect   ( lcsh )
Entomology   ( lcsh )
Chloropidae   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1964.
Bibliography: Includes bibliographical references (leaves 55-60).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Hollis Mitchell Flint.
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Bibliographic ID: UF00097928
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000430667
oclc - 37423615
notis - ACJ0022

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THE EFFECT OF COBALT 60 GAMMA RAYS

ON THE BIOLOGY OF THE EYE GNAT

HIPPELATES PUSIO LOEW









By
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


June, 1964













ACKNOWLEDGMENTS


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

Page
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
Eggs.............................................. 32
Larvae ............ .. ............... .......... 32
Pupae............................................. 34
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


Table Page

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

Figure Page

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














INTRODUCTION


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

developed.

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.
1





2

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

reproductive organs.












LITERATURE REVIEW

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





4

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:





5

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





6

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.






7

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-

somes.

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,

1958).

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

8





9

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.


Experimental Procedure


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





10

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





11

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

15 diameters.


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

otherwise.


Histological Technique


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





12

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).


Statistical Methods


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





13

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-
hour adults.*

: 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



*3 replicates.
**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.






16

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






17

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

treated females.

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.






18

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


*3 replicates.
**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

pupal stage.







Mating Competitiveness


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.






20

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





21

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-

differentiated tissues.








Male Recovery


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





23

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**

Pupal stage
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

Adult stage

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





24

treatment, there was a great reduction in gonial cells which sub-

sequently were found to repopulate as age increased.


Controlled Mating


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.*

: Hatch
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.






25

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.


Egg Production


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


(O
0



0



0




-,

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(10
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H *)
4- *
. 0

od









cM









bl I
0-


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27

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

insects.

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






28

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

by checks.

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





29

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
36-hour adults.

: : 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






30

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
:Males Females
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.





31

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,

1956).

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





33

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.*

: Emergence
Dose : (%) : Cross**

0 57 UMx UF

500 52 UM x TF


1,500


2,500

3,500


TM x UF

UM x TF
TM x UF


H. pusio adults treated as


: lumber eggs :
:in 10 days :

49

129
106

144
99


*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

16,000 r.


Hatch


71

66
79

39
65


--


--


--

--






34

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
to emergence.*

: 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


*2 replicates.


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;






35

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


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






36

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.












0
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000 000 000 000 HH< H9H



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000 000 000c c0C,- 000 000


000 000 000
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38

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






39

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
untreated.



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.












D
4,










a.









SiV

r
^B1-- <'iFcA
^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
oocyte.


ovaries, each with an abnormal


b. Abnormal oocytes, arrow indicates area of undifferen-
tiated nurse cells.





























j


r


4---


.. 'c





44

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 Testes

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
emerence.


a. restes froa urtreated sale.
spermatogenesis.


Note distinct areas of


b, T~tes from aLle treated with 5,000 r au a adult 24 to
36 hours after emergence.

























a









h j. .




. ,


E ,<


rjv~ h

* Il /




ii~a~


r.






47

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

pupal stage.

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






48

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

pupal stage.

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
pupae

5,000 r on 48 459.3+7.32 174.5+3.28 580-350 220-110
adults


*7 days after emergence.


Chromosome Aberrations


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





49

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






50

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

the autosomes.

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
orcein-fast green.


















b. Chromosome figure from testis of insect treated
with 3,500 r as a late pupae. Arrow indicates a
broken chromosome.









9 '* '.i*
'*I'


~iI ..~..A


St


*"5
V.


pi


& I ;iiili
iW2













SUMMARY


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

cross.






54

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

period.

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.













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BIOGRAPHICAL SKETCH


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

of Philosophy.



June 18, 196..



Dean, College of Agriculture



Dean, Graduate School


Supervisory Committee:


kht 6 Lit 4u




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