EFFECTS OF DIFFERENT TYPES OF RADIATION 031'“: " '

VARIoILIs LIFE STAGES OF CEREAL LEAF BEETLE ;_i_;g_§_j5
OLIIema melanopus (Lmnaeus) 5: {5-5; 52155

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PATR'CK ANTH°NY BRENNAN """""" :52:

 

 

(flesh;

This is to certify that the
thesis entitled
EFFECTS OF DIFFERENT TYPES OF RADIATION
ON VARIOUS LIFE STAGES OF CEREAL LEAF
BEETLE, OuIema meIanoBus (Linnaeus)

presented by

Patrick Anthony Brennan

has been accepted towards fulfillment
of the requirements for

Ph . D ._ degree in _.E.D_t.Q[IJ.O_Lng

/
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Major professor
Dam- November I0, 1962

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ABSTRACT

EFFECTS OF DIFFERENT TYPES OF RADIATION
0N VARIOUS LIFE STAGES OF CEREAL LEAF
BEETLE, Oulema melanopus (Linnaeus)

by Patrick Anthony Brennan

Eggs, larvae and pupae were exposed to varying levels of beta
radiation. Observations were carried out to determine the effects
of exposure on hatching, larval development, pupation and eventual
adult emergence. In addition, pre—diapause adults were irradiated
with beta, gamma and X—rays. Following a period of cold treatment to
terminate diapause, a series of tests were initiated to determine
levels of induced sterility, effects on mating, feeding and longevity
of both irradiated males and females at the different treatment levels.
Effects on mating and induced sterility were established by pairing
irradiated males with untreated females. On the basis of the steril-
ity and mating data thus obtained mixing tests were set up in which
irradiated males were mixed with untreated males at ratios of 5:5 and
9:1 and paired with untreated females. Percentage hatch of eggs pro—
duced in these mixing tests was observed and the reduction as compared
with untreated male — untreated female matings was determined. In a
further series of mixing tests the untreated male was marked and his
relative participation in mating determined. From these mixing tests

with marked untreated males and the mating counts recorded for

 

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EFFECTS OF DIFFERENT TYPES OF RADIATION
ON VARIOUS LIFE STAGES OF CEREAL LEAF

BEETLE, Oulema melanopus (Linnaeus)
BY

Patrick Anthony Brennan

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Entomology

1967

 

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ACKNOWLEDGEMENTS

The author wishes to express his most sincere appreciation of
the support and encouragement provided by Dr. Gordon Guyer, Chairman,
Department of Entomology.

Special thanks are expressed to Dr. 0. K. Jantz for his un—
tiring help and guidance.

The author is also indebted to Dr. Ruppel, Dr. Hoopingarner,
Mr. Connin and many members of his staff.

Special appreciation is extended to the W. K. Kellogg Founda—
tion and to its director Dr. R. Mawby.

Appreciation is also extended to Dr. Bickert of the Agricultural
Engineering Department, to Dr. Yanders and Miss Catani at Michigan
State and to Dr. H. G. Olson of the Phoenix Memorial Laboratory at the

University of Michigan for their help in irradiation of material.

ii

 

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . 2
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . 18
RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . 34
Irradiation of Eggs . . . . . . . . . . . . . . . . . . . 34
Irradiation of Larvae . . . . . . . . . . . . . . . . . . 36
Irradiation of Pupae . . . . . . . . . . . . . . 45
Irradiation of Pre— —diapause Adults . . . . . . . 45
Mating Test with Pre— diapause Irradiated Males . . . . . 49
Sterility Test with Pre—diapause Irradiated Males . . . . 50
Feeding Test with Pre—diapause Irradiated Males . . . . . 6O
Mortality Test with Pre—diapause Irradiated Males . . . . 62
Mating Test with Pre—diapause Irradiated Females . . . . 73
Sterility Test with Pre—diapause Irradiated Females . . . 75
Feeding Test with Pre-diapause Irradiated Females . . . . 79
Mortality Test with Pre—diapause Irradiated Females . . . 77

Mating and Sterility Tests with Post-diapause
Irradiated Males . . . . . . 81
Feeding Test with Post— —diapause Irradiated Males . . . . 83
Mortality Test with Post- diapause Irradiated Males . . . 85
Mixing Tests with Pre—diapause Irradiated Males . . . . . 86
Mixing Tests with Post-Diapause Irradiated Males . . . . 89
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . 111

 

LIST OF TABLES

Table Page
1. Numbers of larvae which hatched from eggs exposed

to different levels of beta radiation . . . . . . . . . 34

2. Beta irradiation of 1—2 day old larvae . . . . . . . . . 36

3. Average weights and numbers of 5 day old larvae sur-
viving 10 days after exposure to beta radiation . . . . 36

4. Survival, pupation and emergence of beta irradiated
3rd instar larvae . . . . . . . . . . . . . . . . . . . 38

5. Damage rating on beta irradiated 3rd instar larvae . . . 39

6. Total estimated leaf consumption by beta irradiated
3rd instar larvae . . . . . . . . . . . . . . . . . . . 39

7. Weight increment, pupation and emergence of beta
irradiated early 4th instar larvae . . . . . . . . . . 41

8. Pupation and emergence of beta irradiated late 4th
instar larvae . . . . . . . . . . . . . . . . . . . . . 43

9. Percentage and range of emergence from beta irradiated
1-7 day old pupae . . . . . . . . . . . . . . . . . . . 46

10. Total matings, number of pairs which mated and mating
frequency amongst 10 individual pairings composed
of pre—diapause beta irradiated males with untreated
females . . . . . . . . . . . . . . . . . . . . . . . . 47

11. Total matings, numbers of pairs which mated and
mating frequency amongst 10 individual pairs
composed of pre—diapause gamma irradiated males
with untreated females . . . . . . . . . . . . . . . . 48

12. Total matings, number of pairs which mated and mating
frequency amongst 10 individual pairings composed
of pre—diapause X—ray irradiated males with untreated
females . . . . . . . . . . . . . . . . . . . . . . . . 48

13. Numbers of matings between groups of 10 irradiated
males paired with 10 untreated females . . . . . . . . 50

iv

 

 

LIST OF TABLES Cont.
Table Page

14. Egg production, percentage hatch and number of matings
resulting from 10 pre—diapause irradiated males
paired individually with untreated females . . . . . . 51

15. Egg production, percentage hatch and numbers of
matings between groups of 10 pre—diapause irradiated
males and 10 untreated females . . . . . . . . . . . . 52

16. Assessment of total damage to 10 leaves following
feeding by groups of 10 irradiated males for periods
of two days . . . . . . . . . . . . . . . . . . . . . . 61

17. Total damage ratings compiled by groups of 10 pre-
diapause irradiated males over an 8 day period . . . . 62

18. Accumulated percentage mortalities at 2—day
intervals among groups of 10 pre—diapause males
exposed to beta radiation . . . . . . . . . . . . . . . 64

19. Total percentage mortalities and average survival
in days in groups of 10 males exposed to beta
radiation prior to the initiation of diapause . . . . . 65

20. Total percentage mortalities and survival in days
in groups of 10 males exposed to gamma radiation
prior to the initiation of diapause . . . . . . . . . . 65

21. Accumulated percentage mortalities at two day inter—
vals among groups of 10 pre-diapause males exposed
to gamma radiation . . . . . . . . . . . . . . . . . . 66

22. Accumulated percentage mortalities at 2—day intervals
among groups of 10 pre—diapause males exposed to
X—ray radiation . . . . . . . . . . . . . . . . . . . . 67

23. Total percentage mortalities and survival in days in
groups of 10 males exposed to X—ray radiation prior
to the initiation of diapause . . . . . . . . . . . . . 68

24. Accumulated percent mortalities among groups of 10
males exposed to beta radiation pre—diapause . . . . . 68

25. Accumulated percent mortalities among groups of 10
males exposed to gamma radiation pre-diapause . . . . . 69

26. Accumulated percent mortalities among groups of 10
males exposed to X-ray radiation pre—diapause . . . . . 70

27. Positive observed matings between groups of 10 pre-
diapause beta irradiated females paired with 10
untreated males . . . . . . . . . . . . . . . . . . . . 73

 

LIST OF TABLES Cont.

Table

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

Page

Positive observed matings between groups of 10 pre—
diapause gamma irradiated females paired with 10
untreated males . . . . . . . . . . . . . . . . . . . . 74

Positive observed matings between groups of 10 pre-
diapause X-ray irradiated females paired with 10
untreated males . . . . . . . . . . . . . . . . . . . . 74

Egg production and hatching in groups of 10 pre—
diapause beta irradiated females paired with 10
untreated males . . . . . . . . . . . . . . . . . . . . 76

Egg production and hatching in groups of 10 pre-
diapause gamma irradiated females paired with
10 untreated males . . . . . . . . . . . . . . . . . . 76

Egg production and hatch in groups of 10 pre—
diapause X—ray irradiated females paired with
10 untreated males . . . . . . . . . . . . . . . . . . 77

Accumulated percentage mortalities at 2-day inter—
vals amongst groups of 10 pre—diapause beta
irradiated females . . . . . . . . . . . . . . . . . . 77

Accumulated percentage mortalities at 2 day inter—
vals among groups of 10 pre—diapause gamma
irradiated females . . . . . . . . . . . . . . . . . . 78

Accumulated percentage mortalities at 2—day inter—
vals among groups of 10 pre—diapause X—ray
irradiated females . . . . . . . . . . . . . . . . . . 78

Assessment of total damage to 10 leaves following
feeding by groups of 10 pre-diapause irradiated
females for periods of 2 days . . . . . . . . . . . . . 80

Total damage ratings compiled by groups of 10 pre-
diapause irradiated females over an 8-day feeding
period . . . . . . . . . . . . . . . . . . . . . . . . 81

Mating, egg production and percent hatch resulting
from pairing groups of 10 post-diapause males
irradiated at beta 2000 rads with 10 untreated
females . . . . . . . . . . . . . . . . . . . . . . . . 82

Assessment of total damage to 10 leaves following
feeding by groups of 10 post—diapause beta irradiated
males for periods of 2 days . . . . . . . . . . . . . . 84

vi

LIST OF TABLES Cont.
Table Page

40. Accumulated percentage mortalities at 2—day inter—
vals among groups of 10 post—diapause irradiated
males . . . . . . . . . . . . . . . . . . . . . . . . . 85

41. Total observed matings, egg production and percent
hatch from pre—diapause beta 1000 and 2000 rad
irradiated males mixed with untreated males at
ratios of 5:5 and 9:1 and paired with 10 untreated
females . . . . . . . . . . . . . . . . . . . . . . . . 86

42. Total observed matings, egg production and egg hatch
from pre-diapause gamma 1000 and 2000 rad irradiated
males mixed with untreated males at ratios of 5:5
and 9:1 and paired with 10 untreated females . . . . . 87

43. Total observed matings, egg production and egg hatch
from pre—diapause X—ray 1000 and 2000 roentgen
irradiated males mixed with untreated males at
ratios of 5:5 and 9:1 and paired with 10 untreated
females . . . . . . . . . . . . . . . . . . . . . . . . 87

44. Total observed matings, egg production and egg hatch
from post—diapause beta 2000 rad irradiated males
mixed with untreated males at a ratio of 9:1 and
paired with 10 untreated females . . . . . . . . . . . 9O

45. Egg production and percent hatch from post—diapause
beta 2000 rad irradiated males mixed with untreated
males at a ratio of 9:1 and paired with 1 untreated
female . . . . . . . . . . . . . . . . . . . . . . . . 91

46. No. of observed matings involving irradiated males
with irradiated females, irradiated males with the
untreated females, untreated male with irradiated
female, and untreated male with untreated female
following mixing of 9 males and 9 females irradiated
post—diapause with 2000 rads of beta radiation with
1 untreated male and 1 untreated female . . . . . . . . 92

47. Egg production and hatching percentage from pairings of
9 beta 2000 rad post—diapause irradiated males and 9
irradiated females mixed with 1 untreated male and 1
untreated female . . . . . . . . . . . . . . . . . . . 93

48. No. of observed matings involving irradiated males with
the untreated female and the untreated male with the
untreated female following mixing of 1 untreated male
and 1 untreated female with 9 males irradiated post—
diapause with 2000 rads of beta radiation . . . . . . . 96

vii

 

 

    

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viii

INTRODUCTION

Current methods of control for most of our major insect pests
have distinct disadvantages. Chemical control is expensive and recur-
rent and frequent applications have contributed substantially to the
accumulation of undesirable residues in our total environment. In
addition, the onset of resistance as a consequence of frequent ex-
posure has necessitated the introduction of broad spectrum insecticides
which have had considerable adverse effects on biological and inte-
grated control systems already in operation.

The release of sterile males offers a new technique for control
or even elimination of some of our outstanding insect problems. The
extent of current knowledge does not permit evaluation of the merits
and limitations of the method against the whole range of existing pests.
There are, undoubtedly, many instances in which the sterile male tech—
nique will not be feasible and there are many factors which must be
considered before a reasonable estimate of the potential of the tech—
nique can be made for any particular insect. The enormous variability
which exists in sensitivity of different insect species to radiation
coupled with differences in population densities etc. make it impera—
tive that each insect problem be examined individually before reaching

any decision on the likely outcome of releasing sterile males.

INTRODUCTION

Current methods of control for most of our major insect pests

have distinct disadvantages. Chemical control is expensive and recur-

rent and frequent applications have contributed substantially to the
accumulation of undesirable residues in our total environment. In
addition, the onset of resistance as a consequence of frequent ex—
posure has necessitated the introduction of broad spectrum insecticides
which have had considerable adverse effects on biological and inte—
grated control systems already in operation.

The release of sterile males offers a new technique for control
()1- even elimination of some of our outstanding insect problems. The
e:ctent of current knowledge does not permit evaluation of the merits
arlcl limitations of the method against the whole range of existing pests.
TTieare are, undoubtedly, many instances in which the sterile male tech—

ni<qtie will not be feasible and there are many factors which must be

corzszidered before a reasonable estimate of the potential of the tech—

nicliiea can be made for any particular insect. The enormous variability

Whi-C:11 exists in sensitivity of different insect species to radiation
COUI>31ed with differences in population densities etc. make it impera—
tive: that each insect problem be examined individually before reaching

any (decision on the likely outcome of releasing sterile males.

    

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LITERATURE REVIEW

The term radiation indicates a physical phenomenon in which
energy travels through space. Ionizing rays are so called because
their principal means of dissipation of energy in passage through
matter is the ejection of electrons from atoms in their path. When
an electrically neutral atom has lost one or more of its orbital
electrons the atom is left positively charged, that is, ionized.

Within a few years of their discovery, ionizing radiations
were recognized to induce dominant lethal effects in gametes, and be-
fore a score of years elapsed these effects were ascribed to chromosome
damage. The exact mechanism of radiation damage to the chromosome is
still far from being understood. Nuclear sensitivity to irradiation
gained general acceptance only after the intensified attention to in—
sects stimulated by Muller's 1927 Drosophila report. Previous to 1927
it had been reported repeatedly that germinal changes could be induced
by X—rays, but the work had been carried out in such a fashion that the
meaning of the data, as analyzed from a modern genetic standpoint, was
highly disputable and what were apparently the clearest cases gave
negative or contrary results on repetition. Lethal effects of radia—
tion have since been studied by a great number of authors on a great
variety of experimental materials. Lea (1946) has reviewed much of
this earlier literature. The work of Whiting (1946) has played a
major part in demonstrating that dominant lethals are indeed due to
damage to the chromosomes and not to cytoplasmic effects. In spite of
the mass of experimental data which had accumulated on radiation effects

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on Drosophila the evidence that the whole dominant lethal effect could
be explained in terms of chromosomal structural change was entirely
circumstantial. Whitings experiments were with the parasitic wasp
Habrobracon. The eggs of this wasp, if fertilized, develop into diploid
females; if they are not fertilized they develop into haploid (gyno-
genetic) males. An unfertilized egg which receives a few thousand
roentgens does not hatch, whether fertilized or not, subsequent to
irradiation. But on unfertilized egg which receives a much heavier
dose (something in the order of 3 x 104r) and is then fertilized by an
untreated sperm may develop into a haploid male in which the chromosomes
of the egg play no role. Evidently the dose of 3 x lOar leaves the
cytoplasm still capable of supporting development of the intact sperm
nucleus. This experiment demonstrates that the dominant lethal effect
obtained with smaller doses to the egg must therefore be due to damage
to the nucleus of the egg. It also demonstrates that damaged chromo—
somes are positively lethal to a cell and not merely negatively in—
effective, since a fertilized egg in which either egg or sperm has been
damaged by a moderate dose of radiation prior to fertilization does not
hatch, but an egg in which either egg or sperm chromosomes are absent,
or else have been rendered completely ineffective by a very heavy dose
of radiation, hatches as a haploid organism.

While the experiments of Whiting indicated that radiation effects
were located in the nucleus there is still considerable controversy as
to the exact mechanism involved. Chromosomal deletions, additions,
substitutions, inversions and point mutations are known to occur as a
result of exposure to radiation and it is conceivable that almost any

one of these could constitute a dominant lethal effect. Muller (1954)

    

 

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has shown that some dominant lethals at least are due to chromosomal
breakage followed by unequal partition at the next cell division,
while other workers such as Herskowitz_eg_al. (1962) and Lamb g£_al.
(1964) have verified the fact that chromosomal abnormalities are re—
sponsible for the great majority of unhatched eggs following exposure
to radiation. It was suggested by Vbn Borstel (1958) that dominant
lethality may be associated with interference with DNA synthesis. How-
ever, Vbn Borstel and Rekemeyer (1958) presented evidence that in both
Habrobracon and Drosophila if oocytes or spermatozoa are irradiated
the majority of eggs which fail to hatch die at an early stage. They
concluded that the early lethality cannot be due to chromosome break—
age and nondisjunction and proposed that it is due to damage to the
nucleus, but not necessarily to the chromosome.

Neither the target theory, which had Lea (1946) as its out-
standing exponent, nor the indirect mode of action, which supposes
that the effect of radiation is due to the formation of free radicals,
can adequately explain chromosomal aberrations. High doses can un-
doubtedly modify existing DNA enough to alter the template and thus
interfere with duplication. The law of Bergonie and Tribondeau (Bacq
and Alexander, 1961) states that ”the sensitivity of cells to irradia-
tion is in direct proportion to their reproductive activity and in—
versely proportional to their degree of differentiation." The higher
sensitivity depending on reproductive activity has been interpreted
on the basis of the type of damage incurred by the chromosomes as a
result of exposure to radiation. Chromosome breaks and subsequent
rejoinings result in the formation of acentric and dicentric strands.

Such chromosomal abnormalities are of no consequence in the undividing

 

 

5
cell but once the process of cell division begins mechanical diffi-
culties arise. The acentric fragments are likely to be lost while the
dicentric chromosome may cause breakdown of the cell owing to the fact
that the two centromeres attempt to move to opposite poles at cell
division and the bridge thus formed interferes with normal functioning.
There is a considerable volume of well documented cytological evidence
that this essentially physical phenomenon does in fact occur.
Sonnenblick (1940) examined the developing embryo in an egg fertilized
by an irradiated sperm and noted the presence of clumped and broken
chromosomes. The question has arisen as to whether or not there is
any justification for the term dominant lethal with the implication
that a genetical effect is concerned. The arguments that the effect
is genetic have been presented by Muller (1940). The sperm is almost
entirely composed of chromatin, the volume of the head being about
equal to the combined volume of the chromosomes it contains. Thus
the action of radiations on sperm can hardly be on cytoplasm or nuclear
sap. Secondly, the proportion of female flies hatching from eggs
fertilized by irradiated sperm is reduced more than the proportion of
male flies, showing that the X—bearing sperm are more sensitive than
the Y—bearing sperm. If the action is a genetic one on the chromosome,
then this result is to be expected since fewer breaks are produced
in the Y chromosome which is genetically practically inert than in the
X. From the evidence presented there is little doubt that the effect
of radiation is on the chromosomes. According to Lea (1946) the evi-
dence strongly indicates that large deletions and asymmetrical inter—
changes behave as dominant lethals. However, Fano (1941) has shown

that calculations based on the frequencies found for the observable

'1'!“

 

 

 

6
types of aberration show that these aberrations are not frequent enough
to account for the whole of the dominant lethals.
Different organisms are tolerant of the genetic changes re-
sulting from exposure to radiation to a different degree. Lea (1946)

states that in Drosophila melanogaster the loss of even 5 percent of

 

the X chromosomes has a dominant lethal effect, while in maize the loss
of even a whole chromosome may be viable. The high level of resistance
to radiation present in insects as compared to mammals has long been
noted. This higher resistance is based on the fact that in insects
the only proliferative tissue present may be in the gonads. The over—
all extent of radiation damage to insects varies with the total dose
and the intensity with which it is delivered to the different groups
of the class. Mavor (1927), Henshaw and Henshaw (1932), Wharton and
Wharton (1959) and Grosch (1962) all report that radio sensitivity
tends to decrease with increasing maturity in insects. Day and Oster
(1963) have stated that the dose of X—rays or of gamma rays needed to
kill a mature insect is 100 times greater than that needed to kill a
mammal of comparative age. Clark and Mitchell (1952) have reported
that the LD50 for haploid embryos of Habrobracon increases from 200 r
during cleavage to about 7000 r over a 4—hour period. Differences in
other insects are even more dramatic. While 1—4 day old eggs of
Anobium and Xestobium can be killed by 400 r of gamma rays, doses of
48,000 to 68,000 r are necessary to kill mature eggs of Amobium, and
doses of above 32,000 r to kill mature eggs of Xestobium (Bletchly and
Fisher, 1957). In codling moth exposure of eggs to gamma irradiation
at levels of 2500 r resulted in a normal hatch while levels above

5000 r eliminated hatching (Hathaway 33.31:: 1966) studied the effects

 

 

 

7

of gamma radiation on eggs of Indian—meal and Angoumois moths. Larvae
hatched from eggs irradiated with 25000 r but these larvae were gener—
ally sluggish and died within a short time of hatching. Some hatch
occurred after treatment with 45,000 and 100,000 r but these larvae
were all inactive and died almost immediately. Exposure of 2—5 hour—
old eggs of the Queensland fruit fly, Strumeta tryoni (Froggalt) to
gamma at 1000 r resulted in a 3 percent larval emergence, while levels
of 2000 r and higher completely eliminated hatching (Macfarlane, 1966).
Tantawy (1966) reported that gamma irradiation at levels of 2000 r

applied to the eggs of Anopheles pharoensis resulted in the induction

 

of 100 percent dominant lethality. Tilton g£_a£. (1966) exposed eggs
of rice weevil, Sitophilus oryzae, lesser grain borer, the flour beetle,

Tribolium confusum, and cigarette beetle, Lasioderma serricorne, to

 

gamma radiation at levels of 13,200 to 100,000 r. No rice weevil
larvae emerged at any level but in the remaining species egg hatch
occurred although none of the emerged larvae reached the pupal stage.
Hoover 3£_a£. (1963) reported that no larvae emerged from rice weevil
eggs exposed to levels of 5000 r and higher of X—rays.

From the results just cited it is evident that enormous via—
bility exists between eggs of different species of insect with respect
to their susceptibility to radiation. The work of Clark and Mitchel
(1952) is particularly significant and would appear to indicate the
absolute futility of experiments involving exposure of eggs to levels
of radiation without being fully aware of the stage of development of
the egg. The old criterion of expressing the age of eggs at treatment

in terms of days is obviously no longer valid.

    

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The higher resistance to radiation found in larvae than in eggs
has already been referred to. The severity of the damage to larvae
varies with the total dose and to a lesser extent with the intensity
with which it is delivered. Hussey EEHEL- (1927 and 1932) observed
that sufficiently high doses will prolong the larval period in
Drosophila. The production of phenocopies was noted by Friesen (1936);
Epsteins (1939); Waddington (1942); Villee (1946) and Kroeger (1957).
Larval exposure which results in death during the subsequent pupal or
imaginal stages has been reported by Oster and Cicak (1958); Oster
(1959a,b and 1961); Ostertag and Muller (1959). Reduced fertility in
adults surviving from irradiated larvae has been reported by Moore
(1932) and Erdman (1961). Halberstaedter_g£_al. (1943) found that
doses in the range of 1,500 r for first instar larvae to 3500 r for
third—instar larvae caused appreciable killing of Drosophila larvae.
The development of irradiated Calliphora larvae into adults is
practically unaffected by doses below 1000 r, and irradiation of Culex
larvae with doses of about 3000 r or less is essentially without effect
on subsequent image formation. Macfarlane (1966) noted that, while
exposure levels of gamma radiation up to and including 10,000 r had
little effect on survival of larvae of the Queensland fruit fly, no
adults emerged at 2000 r or above. Pupation could be prevented by
dosages of between 20,000 and 40,000 r if the larvae were irradiated
the day after hatching but if treatment was delayed until the day be—
fore they normally would have pupated 80,000 to 160,000 r was required
to prevent pupation. Some Indian-meal moth larvae were able to pupate
after exposure to gamma radiation at levels as high as 100,000 r, but

none of these larvae become adults (Coghburn_gt_al., 1966). In rice

 

 

 

9
weevils exposure of larvae to gamma at levels above 5,000 r generally
resulted in failure of the larvae to develop into adults. Many first
instar larvae exposed to 2,500 r developed into the pupal and adult
stages but died before emergence from the kernels (Hoover $5.213, 1963).
Studies by Flint_g£'a£. (1966) on irradiation of boll weevil larvae
have shown that emergence from last-instar larvae receiving doses
ranging from 400 to 1600 r was not significantly different from con—
trols. Larvae receiving 2000 and 2400 r had reduced emergence and the
majority failed to pupate. Adult emergence was generally completed
whenever pupation occurred. Pupation and adult emergence were delayed
by all levels of treatment. This delay was most apparent at doses
above 1200 r where several additional days were required for adult
emergence. Although percentage emergence was unaffected by doses as
large as 1600 r, the life span of the subsequent adults was shortened
by smaller doses. Adults that emerged from larvae receiving doses of
1,600, 2000 and 2400 r were weak and appeared to be sluggish.
Henneberry g£_al: (1964) reported the effects of gamma radiation on
the Mexican bean beetle. Larvae were killed at doses of 4000, 8000
and 16000 r, those receiving 1600 r died within 14—16 hours of treat—
ment. Direct mortality of gypsy moth larvae is confined to individuals
exposed to doses of 20,000 r, but many treated at 10,000 r died after
transforming to pupae (Godwin gt al., 1964).

Bourgin and his co~workers (1956) in a detailed histological
study of irradiated Drosophila were able to delineate two categories
of damage: those which appear immediately (within 12 hours after
irradiation) and those which remain latent until the time when the

pupal stage should begin under normal circumstances. The former include

 

 

10
cell degeneration in tissues undergoing rapid division at the time of
treatment, whereas the latter are due to hormonal disturbances.
Baldwin and Salthouse (1959a,b) using the bloodsucking bug Rhodnius
were able to show that the appearance of radiation-induced damage could
be correlated with cell division.

Insect pupae are somewhat more resistant to radiation than
either larvae or eggs. However, some recent experiments have shown
that enormous variation exists between different species. Flint SE EL'
(1966) have shown that in boll weevil a dose slightly in excess of
2,400 r is lethal to pupae. Coghburn gt 3L. (1966) state that for
pupae of the Indian—meal and Angoumois grain moths the lethal dose is
in excess of 100,000 r. Tilton_g£_al. (1966) reported high mortality
of rice weevil, lesser grain borer, confused flour beetle, and cigarette
beetle pupae above 13,200 r. Godwin (1964) found that the mortality
dose for 9 day old gypsy moth pupae was 20,000 r, while Lewis and
Eddy (1964) reported that horn fly pupae succumbed at doses of 25,000 r.
Doses above 50,000 r were required to kill pupae of the Queensland
fruit fly (Macfarlane 1966). Pupae of the screw worm fly were not
seriously affected by doses of X—rays and gamma rays up to 20,000 r.
Pink bollworm pupae are capable of surviving doses in excess of 90,000 r
according to Oute §£_al: (1964).

The increased radioresistance upon reaching adulthood has been
reported in many insects. King (1955) found that doses of more than
60,000 r are needed to kill Drosophila adults and that some insects
can withstand doses above 100,000 r. Benschoter and Telich (1964)
reported that doses of 50,000 r killed only 50 percent of Mexican

fruit fly adults. According to Hathaway (1966) survival of codling

 

 

11
moth adults was normal after exposure to 50,000 r. Burgess and Bennett
(1966) found that a dose of 10,000 of X—rays caused no higher death
rate in treated adult male alfalfa weevils than occurred in controls.
The rice weevil, the boll weevil and the Mexican bean beetles are 3
insects in which the adult stage is relatively susceptible to radiation.
Flint_g£.al. (1966) reported that dosage levels of 4,800 r caused a
high level of mortality in male boll weevils, while Hoover_et_al. (1963)
discovered that the LD50 for rice weevil adults lies between 7,500 and
10,000 r. Henneberry 35.31: (1964) noted high mortality of Mexican
bean beetle adults at exposure levels of 4000, 8000 and 16000 r.

As indicated by Grosch (1962), radiation research featuring
insects is performed by investigators of two independed spheres of
interest. On the one hand are the geneticists who utilize the short
life cycles and large generations of insects. On the other hand are
the exterminators who are interested in alternative methods of insect
control. Much of the earlier work was concerned with the killing of
insects and food preservation. As indicated earlier, Muller in his
classical paper in 1927 mentioned both temporary and permanent steril—
ity induced in Drosophila as a result of exposure to radiation. While
recognition of the basic concept of induced sterility was by Muller,
its application as possible technique for controlling natural insect
populations must be accredited to E. F. Knipling of the U. S. Depart-
ment of Agriculture. As early as 1937 it occurred to Knipling that it
might be economically feasible to rear and release sterilized screw—
worm flies in sufficient numbers to exterminate the natural population.
In 1950 with encouragement from Muller who was then with the University

Of Tez<as research to explore the sterility concept was begun on

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12
screw—worm fly. Basic laboratory work was done by Bushland and Hopkins
(1951, 1953) and cyto— and histological work by Kaufman and Wasserman
(1957). This work indicated that a dosage of 5,000 roentgens of
ionizing radiation from an X—ray or gamma ray source applied to 7 day
old pupae would impart sterility to both sexes of emerging adults.
When normal females were mated with sterile males the normal females
laid only infertile eggs.

The first sterile male screw—worms were released on the islands
of Sanibel and Captiva, about 2 miles off the Florida coast in 1953.
The total area was 15 square miles and releases were carried out at
the rate of 100 sterile males per square mile per week giving a ratio
of sterile to naturally occurring males of approximately 4 to 1. The
naturaleopulation was reduced to a low level in 3 months but eradi—
cation was not achieved. It was discovered that adult screw worm
flies can fly up to 9 miles and re—infestation from the mainland was
occurring across the 2 mile stretch of water.

The next pilot experiment was begun in 1954 in Curacao in the
Netherlands Antilles. This island has an area of 170 sq. miles, with
40 miles of open sea isolating it from the nearest land. Releases of
400 sterile screw-worm flies per sq. mile per week were carried out.
This established a ratio of 3—4 sterile males for every male occurring
in the natural population. Before release 99 percent of the egg masses
deposited on animals at 11 test sites were fetile. During the first
week after release of sterile individuals 69 percent of the wild
females deposited infertile eggs. By the 4th week 79% of the egg
masses were sterile and by the 7th week no fertile eggs were found.

Complete eradication was obtained by the third month or after 3 or 4

 

 

13
generations had been exposed to sterile males. A full account of this
early work is given by Baumhover gt_al. (1955) and Knipling (1955) pre-
sented a discussion on the possibilities of insect control or eradica—
tion through the sustained release of sterile males in numbers exceeding
those in a normal population.

The spectacular results obtained on Curacao prompted the Animal
Disease Eradication Division of the U.S.D.A. in cooperation with the
Florida Livestock Board to initiate a large—scale program of sterile
screw—worm fly releases in Florida in 1958. The success of this pro-
gram has been reported by Bushland (1960) and Knipling (1960). The
cost of the program was $7 million with an estimated saving to cattle
growers in the Florida area of $140 million.

In 1962 a new screw—worm rearing plant was constructed at
Mission, Texas with a capacity to produce 100 million sterile screw-
worm flies per week. During that year there were 50,779 reported
cases of screw—worm infestation in livestock confirmed. By the end
of 1964 at a cost of $12 million the number of confirmed cases had
been reduced to 239 which represents 99.5 percent protection and a
total savings of close to $275 million.

The screw—worm success have inspired entomologists in various
parts of the world to consider similar methods for controlling other
pests. Quite a considerable volume of literature has accumulated on
the effects of radiation on different insect species. A survey of the
more recent literature illustrates the lack of uniformity which exists.
Burgess and Bennett (1966) reported the induction of almost total
sterility in adults of the alfalfa weevil, Hypera postica, at exposure

levels of 2000 r. Schmidt EEUEL' (1964) recorded a low level of hatching

14
in eggs from normal house-fly females mated with males which had been
exposed 2,850 r. Bushland and Hopkins (1951) exposed screw—worm adult
males to 5000 r and noted a hatch of less than 1 percent of egg pro—
duced following pairing of these males with untreated females. Ex-
posure of white pine weevil, Pissodes strobi, males to 5000 r resulted
in an 0.3 percent egg hatch (Jaynes 1957). Jaynes and Godwin (1957)
reported an 8—13 percent hatch in Pissodes strobi at the same level of
exposure. Park g£_al. (1958) noted that Tribolium confusum adults ex-
posed to 5000 r produced eggs 14 percent of which hatched. Terzian
and Stabler (1958) induced total sterility in Adgs aegypti adults by
exposure to 7500 r. Henneberry E£.EL- (1964) recorded the complete
absence of hatching amongst eggs of Mexican bean beetles which had been
exposed to 8000 r as adults. A similar instance of total sterility was
reported by Davis_g£_a£. (1959) following treatment of adults of

Anopheles qgadrimaculatus at levels of 8865 r. Flint g£_al, (1966)

 

found that 1 percent of boll weevil eggs hatched following treatment
of the adult males with 9000 r and increasing the dose to 15,000 r did
not result in 100 percent sterility. Steiner_g£_al. (1965) succeeded
in inducing total sterility in melon flies by exposing pupae to 9200 r.
Catcheside and Lea (1945) reported a hatching percentage of 1.1 after
exposure of Drosophila adults to 11,420 r. Doses of 12,000 r applied

to Anopheles quadrimaculatus pupae induced total sterility in emerging

 

adults (Schmidt_e£_al., 1964), while Tilton_e£_a£. (1966) found that a
dose of 25,000 r was necessary to sterilize adults of the rice weevil,
the lesser grain borer, the cigarette beetle and the confused flour

beetle. According to Walker and Brindley (1963) 1 percent of European

corn borer eggs will hatch after the adults have been subjected to a

15
dose of 32,000 r. Pink bollworm adults are sterilized after exposure
to 45,000 r (Oute e£_a£., 1964) and Hathaway (1966) reports an 0.4
percent hatch in codling moth eggs after exposure of the adults to
50,000 r.

In addition to screw—worm fly, the effectiveness of the sterile
male release technique has also been demonstrated against the melon
fly, 23325 curcurbitae, the oriental fruit fly, Daggs doraslio, the
Mediterranean fruit fly, Ceratitis capitata, and the Mexican fruit fly,
Anastreppa ludens. The successful eradication of melon fly from the
island of Rota has been reported by Steiner_e£_al. (1965). The results
of this program demonstrated decisively that a monogamous mating habit
is not an essential requirement for the success of the sterile male
technique. Steiner has estimated that polygamy does confer some advan-
tage on the untreated wild male, but all that is required is extension
of sterile releases until all wild flies are dead. The eradication
program on Rota, which has an area of 33 sq. miles, was begun in 1962.
Total elimination of fruit infestation was achieved within a year fol—
lowing release of 257 million flies irradiated as pupae with 9,500 r
from a cobalt 60 source. An interesting feature of the program was the
application of an insecticidal spray to about 20 of the principal fly-
producing sites before release of sterile insects was begun and the
consequent reduction of the natural population to a level where over-
flooding with sterile releases was more easily achieved.

First attempts to eradicate Mediterranean fruit flies from Rota
by Steiner were not successful. Nearly 10 million flies per week were
released but the target overflooding objective of 10 sterile for every

male occurring in the natural population was not achieved. According

16

to Christenson (1966) to adequately overflood population of the oriental
fruit fly it may be necessary to establish an initial ratio of sterile
to fertile flies of 25—50 to l. Releases of sterile oriental fruit
flies in 1964 quickly eliminated the natural population from all of
Guam. A typhoon in 1963 had destroyed many of the favored oriental
fruit fly host plants. Thus the natural population was reduced to an
extremely low level.

In addition to the successes already cited investigations are
progressing to determine the feasibility of applying the sterile male
technique for control of gypsy moth, Porthetria diapar, codling moth,

Carpocapoa pomonella, pink bollworm, Pectinophora ggssypiella, tsetse

 

 

fly, Glossima morsitans, boll weevil, Anthonomus grandis, cockchafer,
Melolontha vulgaris, and the vinegar fly, Drosophila melanogaster.

The prospects for success appear good for a number of these insect
species. Results obtained in experiments involving the boll weevil,
Anthonomus grandis, illustrate the problems which have arisen in a
number of instances. Flint SE EL' (1966) report the induction of 99
percent sterility in boll weevil adults exposed to 9000 r of X—rays
and increasing the dose to 15,000 r did not induce 100 percent steril—
ity. They further observed that a dose of 3200 r administered to
adults resulted in a barely acceptable survival level of 50 percent.
Coupled with this increase in mortality rate is the extremely serious
reduction in competitiveness amongst weevils which survive. This re—
duction has been estimated to be as high as 80 percent. Knipling
(1964) has discussed the difficulties which have arisen with induction
of sterility in the boll weevil and he concludes that the sterile male

technique may still be applicable to this insect especially where it is

l7

allied to other integrated systems of control. The air of pessimism
which in some quarters has replaced the early optimism generated by
the outstanding success with screw—worm fly is scarcely justifiable.
According to Knipling (1964) there are undoubtedly many instances in
which the sterile male technique will not be practical for controlling
or eliminating established populations of many of our destructive in-
sect species. Knipling emphasizes that the screw—worm successes were
achieved only after the accumulation of quite a considerable volume of
data by Bushland and others. Insects that have been or are now being
subjected to such basic research include the corn earworm, Heliothis

zea, Tobacco budworm, Heliothis vireacens, tobacco hornworm, Protoparce

 

sexta, sugar cane borer, Diatraea saccharalis, oriental fruit moth,
Grapholitha molesta, olive fruit fly, Dacus olece, Mediterranean flour
moth, Anagosta kuehniella, certain mosquitoes including Aedes and

Anopeles, and Queensland fruit fly, Dacus tryoni. Recent work with

 

codling moth (Hathaway, 1966) and European corn borer (Walker §£_gl,
1963) clearly indicates that once the basic essential knowledge has

been acquired considerable further progress is possible.

 

 

 

MATERIALS AND METHODS

Irradiation of Immature Stages
of the Cereal Leaf Beetle
Eggs, larvae and pupae were exposed to beta radiation at
levels of 1000, 2000, 4000, 8000 and 16000 rads. The source utilized
was the 1,000,000 volt electron beam generator which delivers a dose

6

of 2.97 x 10 rad per minute at a distance of 47 centimeters from the

tube window.

Eggg: Eggs exposed in the first series of experiments were from
beetles which had been field—collected six months previously. These
beetles were caged on barley plants for two weeks after collection
and, upon the initiation of diapause, were stored in plastic cartons

at 3-50 C for 22 weeks to terminate diapause. The insects were then

0

placed on barley seedlings in the laboratory at a temperature of 24°
and subjected to a photoperiod of 16 hours. The pots of plants were
replaced at regular intervals to ensure a constant supply of food and
to enable the approximate age of eggs to be determined. Prior to
exposure to radiation the eggs were separated into two groups, 2—4 and
4—5 days old. In group 1, i.e., 2—4 day old eggs, small sections of
leaf with eggs attached were removed from the plants and placed on
moist tissue paper in petri dishes. Each petri dish contained a total
of fifty eggs and two dishes were included at each level of exposure.
In group 2 (the 4-5 day old eggs) treatment was carried out in petri

18

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19

dishes and also on eggs attached to leaves of plants growing in 4-inch
plastic pots. In this second age group two replicates of 50 eggs each
were exposed in petri dishes and two replicates of 50 eggs each on the
plants in pots. In all cases the lids of the petri dishes were removed
during treatment. The temperature of the exposure chamber was 180 C.
Immediately following treatment all material was returned to the
laboratory. The tissue paper in the petri dishes was maintained in a
moist condition by the daily addition of water and the plants in pots
were watered frequently.

Daily observations were carried out to determine the effects

of treatment on the rate of hatch and on the total percentage hatch.

Newly Emerged Larvae: In the first series of experiments with newly

 

emerged larvae the source of material was the same as that of the
treated eggs in the previous section. In the second series the larvae
which were treated had emerged from eggs which were deposited by spring
adults field-collected at Galien, Michigan in April 1966. Following
collection these adults Were taken to the laboratory and caged on
barley seedlings. Feeding and oviposition began immediately and the
pots of barley seedlings were changed at two day intervals.

In both series of experiments with newly emerged larvae post
oviposition handling was similar. After removal from the oviposition
cages the pots of plants with their complement of eggs were retained
in the laboratory at 24° C and 16 hours photoperiod. The plants were
watered at frequent intervals and in all cases hatching commenced

when the eggs were approximately 7 days old.

 

20

In the first series of tests the larvae were 1-2 days old at
the time of treatment. They were exposed to the varying levels of
beta irradiation while feeding on the plants on which the eggs were
originally laid. A total of 50 larvae were treated at each level of
exposure and one pot of 50 untreated larvae was incorporated as a con-
trol. The temperature of the exposure chamber was 18° C.

Following treatment, the larvae were transferred immediately to
new plants using camel—hair brush. The surface of the soil in each
pot was covered with a layer of fine silica sand and a clear glass
lamp chimney was placed over the plants. Mortality counts were taken
at two intervals during the subsequent development of the larvae. The
plants were replaced as became necessary and when the onset of pupa-
tion was indicated the soil in each pot was covered with a layer of
plaster of paris to prevent penetration of the prepupae down into the
soil. A 1/2 inch layer of fine silica sand was placed on top of the
plaster of paris to provide a pupation site and the chimney was then
replaced.

In addition to mortality and pupation counts an attempt was
made to determine the effects of radiation on larval development on
the basis of average weight gain. This estimation was carried out
eleven days after treatment. Surviving larvae were removed from the
plants and their adhering faecal coatings cleaned away by rolling them
gently on moist paper towelling with a camel-hair brush. The larvae
were dried by repeating the procedure on a dry section of towelling,
transferred to a small muslin sack, and weighed on a sensitive torsion
balance. Immediately after the weight had been recorded the larvae

were removed and the empty sack with its adherent moisture reweighed.

21
In this way an accurate estimation of average larval weight could be
attained.

In the second series of experiments 5 day old larvae derived
from eggs deposited by field-collected spring adults were used.
Handling was essentially similar to that for the 1—2 day old larvae
but in this instance the temperature of the exposure chamber was

27° C.

Third Instar Larvae: Eight to nine day old larvae from eggs laid by
beetles which had been field-collected as summer adults 6 months pre—
viously were reared on barley seedlings at a temperature of 24° C

and a photoperiod of 16 hours prior to treatment. Larvae were treated
while feeding on the leaves of plants grown in 4 inch pots. TWO repli-
cates of 40 larvae each were treated at each level of exposure, these
levels being 1000, 2000, 4000, 8000 and 16000 rads. In addition to
observations on mortality, pupation and emergence, an attempt was

made to estimate the effect of the different levels of exposure on
post treatment larval feeding. Amount of feeding based on percent
damage to the leaf surface was categorized as follows: 0 = no damage;
1 = less than 25 percent; 2 = 25—50 percent and 3 = 50 percent or more.
Since the number of leaves per pot was unequal in the different treat-
ments an attempt was made to calculate total leaf consumption for each
replicate. Numbers of leaves per pot falling into each category were
noted. An average percent damage was then estimated for each category.
For example, a figure of 12-1/2 percent was used for the 25 percent
category, it being assumed that the degree of damage was randomly dis-

tributed around this point. Similar figures taken for the remaining

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22

two categories were 37.5 and 62.5 percent. For the 50 percent and
above category 62.5 percent seemed the best estimate since 100 percent
damage was never encountered and therefore an upper limit of 75 percent
appeared acceptable. Multiplying the total number of leaves falling
into any one particular category by the average established for that
category gave an estimate of the total leaf destruction at that par—
ticular level of damage and summation of these estimates gave total

leaf consumption per pot.

Early Fourth Instar Larvae: Larvae treated in this experiment were

 

field—collected at Galien, Michigan on 24th June 1966. The method of
collection and subsequent handling was as follows. Oat stems on which
the larvae were feeding in the field were hand picked, placed on bar—
ley plants grown in aluminum trays, and transported to the laboratory.
Larvae of approximately the same age were selected for treatment and
subsequent development indicated that they were early fourth instar
larvae, the time of treatment being four days prior to the commence—
ment of pupation. The levels of exposure were 0, 500, 1000, 2000,
4000, 8000 and 16000 rads of beta irradiation and the temperature of
the exposure chamber was 27° C. Two replicates of 50 larvae each were
included at each level of treatment. The larvae were confined on pots
of barley seedlings prior to treatment by means of glass chimneys
placed over the leaves. The chimneys were removed during the period
of exposure and replaced immediately afterwards. Post treatment
handling was similar to that already described for other larvae. The
laboratory temperature was maintained near 24° C and the photoperiod

was 16 hours. The larvae were transferred to new plants immediately

 

23

following treatment by cutting off leaf sections with the larvae
attached and placing them on new leaves in pots covered by chimneys.
Ten larvae per replicate were weighed at this time and an average
larval weight at treatment was established and later compared with
average weights taken two days after treatment. Other post treatment
observations included pupation rates and eventual adult emergence.

In addition to the early fourth instar larvae, those collected
2 days prior to the commencement of pupation were selected for uni—
formity. Six replicates of 40 larvae each were then exposed at each
level. Data collected included pupation and adult emergence rates.

Since pupation was imminent, no weight records were taken.

Egpggg Pupae resulting from larvae reared in the laboratory were ex—
posed to beta radiation at levels of 500, 1000, 2000, 4000, 8000 and
16000 rads. A total of 500 pupae were exposed per treatment level in
5 replicates of 100 pupae each. In addition, 1000 untreated pupae
were incorporated as a control. Exposure was carried out in uncovered
plastic petri dishes with a chamber temperature of 27° C. The age of
pupae at treatment was 1—7 days. Following treatment the pupae were
returned to the laboratory and covered with a layer of sand to await

emergence of adults.

Irradiation of Cereal Leaf Beetle Adults

Pre-diapause Treatment: Adult beetles field—collected at Galien,

 

Michigan on July 5th, 1966, were taken to the laboratory and allowed
to feed on barley seedlings grown in 4 inch plastic pots. Two to 3

thousand insects were confined in cages approximately 17 inches square

 

24

containing five pots of barley plants per cage. The surface of the
soil in the pots was covered with plaster of Paris to facilitate re-
moval of the insects and the plants were replaced daily during the
feeding period. Feeding was continued for two weeks at which time
initiation of diapause was evident in many specimens. Feeding decreased
and beetles began to leave the plants and seek refuge in folded paper
towelling which had been placed in the top corners of each cage. The
insects were then removed from the cages, placed in plastic petri
dishes, and stored in a refrigerator at 5° C for 12 hours prior to
treatment. Groups of 1000 beetles were then exposed to beta, gamma
and X—ray radiation at levels of 500, 1000, 2000, 4000, 8000 and 16000 r.
Dosimetry for the beta and gamma treatments was in rads while that for
X—ray was in roentgens. The source utilized for the beta treatments
was the electron beam generator already described. The gamma source
was a 10,000 curie cobalt60 source with a dosage output of 700 k rad
per hour. The X—ray source was a G.E. Mamima 250 III operated at
250 KV and 15 m a delivering an acute dose of 150 roentgens per minute.

The insects exposed to gamma and X—ray radiation were confined
in the petri dishes with lids affixed during treatment. In the case
of those exposed to beta irradiation the lids of the petri dishes were
replaced by a fine muslin mesh. In addition, the dishes were placed
in a shallow tray of ice-water during treatment and an average tempera—
ture of 10° C was maintained. No ice-water bath was used during ex-
posure to gamma and X—ray due to the impracticability of maintaining a
constant temperature over the longer exposure periods required. Ex—
posure to gamma rays was carried out in a laboratory which was air

conditioned to maintain a constant temperature of 5° C. No manipulation

 

 

25
of the temperature was possible during the eray treatments and the
air temperature of the laboratory was monitored at 25° C. In addition
to the 1000 beetles treated at the various levels, a group of.1000
beetles was incorporated as a control for each of the three types of
radiation. Handling of untreated beetles duplicated that of treated
beetles.

Following treatment the irradiated beetles were stored in
plastic cartons in the refrigerator at a temperature of 3-5° C in
order to terminate diapause. The storage procedure was similar to
that utilized in the routine cereal leaf beetle rearing program at
Michigan State University.

Four plastic cartons, each containing 250 individuals, were
placed in cold storage at each treatment level. Of these two cartons
(i.e. 500 individuals) remained undisturbed during the period of cold
storage. The remaining 500 beetles at each treatment level were re-
moved from the refrigerator for a period which was sufficient to enable
the determination of sex by examining each beetle individually under
the dissecting microscope. Sex determination was based on the con-
cavity or convexity of the intercoxal plate — it being concave in males
and convex in females. This character is, apparently, the only sexual
dimorphism exhibited by this insect but with practice it enables deter-
mination of sex with 100 percent accuracy as verified by dissection.
Manipulation of individuals for examination was greatly facilitated by
utilyzing a vacuum pump to which a rubber tube was fitted. A small
conical-shaped plastic tube was placed on the end of the rubber tubing
and its dimensions were such that only the head and pronotum of the

insect were obscured while the greater portion of the thorax and the

 

    

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26
complete abdomen were exposed for examination. Sufficient vacuum was
used to maintain the insect in position. Excess suction effect and
subsequent damage to the insect was avoided by a side tube with an ad—
justable clamp. Using this technique sex determination was a simple
and rapid operation and the ill effects of handling were completely
eliminated. After determination of sex, male and female beetles were
placed in separate cartons and returned to the refrigerator for con—
tinuation of cold treatment. Previous experience had indicated that a
minimum period of 9 weeks at 3-5° C was required to terminate diapause.
In this instance a period of 11 weeks was allowed to elapse before

initiation of any of the tests described in the following sections.

Individual Pair Matings: Following a period of 11 weeks exposure to

 

cold treatment pair matings were set up at all levels of exposure for
each of the three types of radiation. Ten replicates of treated males
paired with individual untreated females were included at each exposure
level. Ten untreated single pairs were incorporated as a control for
each radiation type. Each pair was confined on barley seedlings grOWn
in pots and held at a temperature of 240 C and a photoperiod of 16
hours. Beginning on the second day after the insects had been placed
on the plants and for a further 30 consecutive days mating counts were
made three times daily. The 16 hour photoperiod extended from 8 a.m.
until midnight and the mating counts were taken at approximately 10 a.m.,
2 p.m. and 8 p.m. Mortality counts were taken daily and dead insects
were replaced by appropriate individuals from the supply retained in

the refrigerator.

.11.,

    

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s an elliiautuzis-mfi .'t.?'.' T-prmn : and: «I; 1': Jam'- 9
- : -. «it. an“ Ml,-

. in "I: . .- '.'| . I

. Ii. 12*

27

Mating Test with Multiple Pairs: In addition to the tests on individual

 

pairs already described a further series of mating tests were carried
out with irradiated males which had been subjected to cold treatment

for 13 weeks. In this instance ten irradiated males were paired with
ten untreated females in a single pot. One replicate was included at
each level of exposure for each of the three types of radiation. The
number of mating pairs per pot was recorded three times daily over an

11 day period. Every two days the plants were changed and the mortality

recorded. No dead insects were replaced in this experiment.

Sterility Test with Irradiated Males: Degree of sterility induced in

 

males at various treatment levels with each type of radiation was
determined from the individual and multiple pair matings already
described. The plants were changed at 3—4 day intervals in the individ—
ual pair matings and at tw0 day intervals for the multiple pairs.

These plants with their complement of eggs were retained in the labora—
tory at 24° C and 16 hours photoperiod in order that hatching percent—
ages could be determined. The plants were watered frequently and
examined daily for larval emergence. Newly hatched larvae were removed

from the leaves by means of a camel hair brush.

Feeding Test with Irradiated Males: These pre—diapause irradiated

 

males had been retained in the refrigerator at a temperature of 3—5° C
for a period of 15 weeks prior to commencement of the feeding test.
Groups of ten beetles were placed on barley seedlings grown in pots
and were covered with lamp chimneys the tops of which were sealed with

muslin mesh. Each pot contained a total of twenty leaves, surplus

 

 

28
leaves having been removed. All leaves were clipped to a height of
3 inches to insure a uniform quantity of available food per pot.

TWO replicates per treatment level were included — except X—ray
16000 r which had only one replicate. The test was run for eight con—
secutive days, the plants being changed and damage assessment carried
out at two day intervals.

Total leaf damage was assessed in the following manner. Ten
leaves were chosen at random from the twenty in each pot. The degree
of damage to each particular leaf was then rated on an ascending scale
of damage of from 0 to 4. The scale was:

0 — no damage

1 — 2—25% of leaf damaged
2 — 25—50% of leaf damaged
3 — 50—75Z " " "

4 — 75% and up " "

It will be noted that leaves exhibiting less than 2 percent
damage were recorded as being undamaged. This was done in order to
maintain equal intervals on the damage scale. However, a record of
all leaves showing less than two percent damage was kept. It was felt
that granting a rating of 1 to these leaves and their incorporation in
the total damage assessment would have presented an entirely false
picture. At the higher levels of treatment particularly quite a number
of leaves exhibited slight damage but granting them a rating equivalent
to that for leaves showing up to twenty-five percent damage appeared

to be completely unjustified.

Mini-i: uq sea-Align on!

.r‘:‘-'.-.. u.‘- and 221:1: '...'l alt}!!! 1 00“!

5D in! Ann FEW 1"sz srfl'

 

_..' . Eff-"J." "‘."131'1‘5-2

 

29

Irradiation of Adult Females: These field—collected, pre—diapause

 

irradiated females were placed in a refrigerator at 3-5° C for a period

of 17 weeks prior to initiation of the following tests.

Mating and Sterility Test: One group of ten females was selected at

 

each treatment level and placed on barley seedlings grown in pots
covered with glass lantern chimneys. Ten untreated males which had
been subjected to cold treatment for a similar period of time were
added to each pot. The insects were placed in the laboratory at a
temperature of 26—270 C and a photoperiod of 16 hours. Mating counts
were recorded three times daily over a 20—day period. The plants were
changed at two day intervals. Egg counts were recorded and the old
plants were retained in the laboratory in order to determine hatching
percentages. All hatched larvae were removed from the plants by means
of a small brush to facilitate estimation of subsequent hatchings.
Mortality records were taken at two day intervals for 16 days. Dead

insects were not replaced.

FeedingfiTest with Irradiated Females: The procedures utilized in the

 

feeding test with irradiated females were essentially similar to those

already described for irradiated males.

Post—diapause Treatment: The adult insects irradiated at the post—

 

diapause stage were field collected at Galien, Michigan in July, 1966
and following a 2 week feed—out period in the laboratory they were
placed in plastic cartons in a refrigerator at 3—5° C in order to ter—
minate diapause. After 14 weeks of cold treatment the insects were

removed from the refrigerator and their sex determined. Then they

30

were exposed to 2000 rads of beta radiation. During treatment the in—
sects were confined in plastic petri dishes the lids of which were re—
placed with fine mesh. During treatment and for a period of 30 minutes
prior to exposure the petri dishes were placed in a water bath at 60 C.
The air temperature of the exposure chamber was 25° C.

Following treatment the insects were returned to the laboratory
which was maintained at a temperature of approximately 270 C and a

photoperiod of 16 hours.

Mating and Sterility Test with Post—diapause Irradiated Males: TWO

 

groups of ten males which had been exposed to 2000 r of beta irradia-
tion at the termination of diapause were paired with ten untreated
females. Mating counts were taken three times daily over a period of
21 days for comparison with counts from ten untreated males paired
with ten untreated females. Mortality counts and egg production
figures were kept for the same period and hatching percentages were

subsequently determined.

 

Feeding Test with Post—diapause Irradiated Males: Post—diapause
irradiated males were subjected to a similar type of feeding test as
already described for pre-diapause irradiated insects. In this in-
stance the test lasted for 14 days. Damage was assessed at two day
intervals on the scale presented above. Mortality was recorded at two
day intervals and dead insects were replaced by irradiated males which
had been stored at 3-5° C since the time of treatment.

The level of exposure was 2000 r. TWO replicates of ten beetles
each were included and also two replicates Of 10 untreated males as

controls.

   

 

31

Mixing Tests

Test No. 1: In this test male insects which had been exposed to beta,
gamma and X—rays at levels of 1000 and 2000 r at the pre—diapause stage
were mixed with untreated males and then paired with untreated females.
The mixing ratios utilized in this experiment were:

5 irradiated males + 5 untreated males + 10 untreated females

9 " ” + l " ” + 10 " "
A single replicate was included at each level of mixing for both levels
of exposure and all three types of radiation.

The insects were allowed to feed on barley seedlings grown in
pots covered with glass chimneys. One control pot of ten untreated
males with ten untreated females was included for each of the three
types of radiation.

Mating counts were recorded three times daily over a period of
18 days. Daily replacement of both treated and untreated dead males
was carried out in order to maintain the initial ratios.

The plants on which the insects were feeding were changed at
two day intervals. Egg counts were recorded and newly hatched larvae
were removed from the leaves to facilitate accurate determination Of

hatching percentages.

Test No. 2: This and all subsequent mixing tests were made with in—
sects exposed to 2000 r of beta irradiation after the termination of
diapause by subjection to cold treatment. The mixing ratio in this

experiment was nine treated males plus one untreated male, these ten

 

 

 

32
males then being paired with ten untreated females. A total of nine
replicates was utilized in this experiment.
Mating was recorded 3 times daily over a period of 22 days.
Egg production and hatching records were also kept. Dead males were
replaced daily from a stock of irradiated males stored at 3-5° C
since the commencement of the experiment. Dead females were not re—

placed.

Test No. 3: The mixing ratio in this experiment was nine males
irradiated at beta 2000 r with one untreated male but in contrast to
the ten untreated females in the previous test only a single female

was included in the current test. Two replicates were included and

the test extended over a period of 21 days. The nine to one mixing
ratio of treated to untreated males was maintained by daily replacement

of dead males.

Test No. 4: Nine males and nine females which had been exposed to
2000 r of beta irradiation at the post diapause stage were mixed with
one untreated male and one untreated female. The untreated male and
female were marked with white paint and, in addition, the tip of one
elytron was clipped to facilitate identification in the event of re—
moval of the paint. Marking of the elytra with paint was completed in
such a fashion that the female could be identified even during the
process of mating. This was done by applying the paint to the outer
basal margin of each elytron.

Mating counts were taken three times daily over a period of 22

days and all dead insects were replaced on a daily basis.

33

Test No. 5: In this test nine post diapause irradiated males were
mixed with one untreated male and then paired with a single untreated
female. The tip of one elytron of the untreated male was clipped and
both elytra were marked with nail polish. Mating counts were made
three times daily over a period of 17 days and the activity of the
untreated male was thus recorded. Ten replicates were included and

dead insects were replaced on a daily basis.

Test No. 6: This mixing experiment was exactly similar to the one
just described except that in addition to the nine irradiated males
nine irradiated females were also included in each of the ten repli-
cates. The untreated male and the untreated female were both clipped
and marked with nail polish. Mating counts were taken three times

daily over a period of 17 days and dead insects were replaced each day.

 

RESULTS AND DISCUSSION

Irradiation of Eggs: The results of the hatching experiments on

irradiated eggs are presented in Table 1.

TABLE 1.--Numbers of larvae which hatched from eggs exposed to
different levels of Beta radiation

 

Exposure level in rads

 

 

 

Replicate
No. 0 1000 2000 4000 8000 16000
1 28 0 0 0 0 0
2 27 0 0 0 0 0
3 34 1 0 O 0 0
4 26 16 O 0 0 0
5 33 35 l 0 0 0
6 23 0 0 0 0 0
Totals 161 52 l 0 0 0

 

aEggs treated in replicates l & 2 were 2—4 days old; those in
replicates 3, 4, 5 & 6 were 4-7 days old.

b50 eggs per replicate.
The eggs in replicates 4 and 5 which were exposed to 1000 rads com—
menced hatching within 24 hours Of treatment. In the corresponding
controls hatching did not begin until three days after treatment.
This indicates that the eggs in both replicates 4 and 5 at the 1000

rad level of treatment were on the verge of hatching at treatment time.

34

 

 

35

The results in general indicate that exposure levels of 1000 rads
and up completely eliminate hatching except where treatment is carried
out just prior to the initiation of hatching.

The larvae which emerged from eggs treated at the 1000 rad level
appeared normal at the time of hatching and were retained in order
that their subsequent development might be observed and compared with
larvae from untreated eggs. These larvae were transferred to new
plants. Mortality counts were taken at frequent intervals and the
eventual numbers pupating were recorded. The single larva which
hatched in replicate number 3 at the 1000 rad level died within 24
hours of emergence. Of the 16 larvae in replicate number 4, all died
within 4 days of emergence, prior to pupation. A total of 35 larvae
hatched from the 50 eggs treated at 1000 rads in replicate 5. This
represents a hatch of 70 percent, an unusually high figure, the overall
percentage hatch for controls in this experiment being 57 percent, with
a range Of 46 to 68 percent. These larvae continued to develop nor—
mally and 17 eventually pupated. This represents a pupation figure of
48.6 percent. The corresponding pupation percentages in the 5 un-
treated controls were 14.3, 48.1, 11.8, 27.0 and 52.2 percent, respec-
tively.

A record was also kept of emergence of adult beetles from pupae
which had received 1000 rads of Beta radiation as eggs. Seventeen
days after the first pupae were formed at the 1000 rad level adult
emergence began and after two further days a total of 16 adults had

emerged from the original 17 pupae.

 

36

Irradiation of Newly Emerged Larvae: Results of observations on 1—2

 

day old larvae exposed to various levels of Beta radiation are pre-

sented in Table 2 and those on 5 day old larvae in Table 3.

TABLE 2.--Beta irradiation of 1—2 day old larvaea

 

Exposure level in rads

 

 

 

0 1000 2000 4000 8000 16000
No. surviving
after 7 days 46 40 23 ll 16 3
NO. surviving
after 11 days 29 30 0 2 0 3

 

Average larval
weight 11 days 10.4 8.3 9.0 8.6
after treatment

 

No. pupated 19 17 0 2 0 2

 

a .
50 larvae were included at each treatment level.

TABLE 3.-—Average weights in milligrams and numbers of 5 day old larvae
surviving 10 days after exposure to Beta radiation

 

Exposure in rads

 

 

Replicateb
No. 0 500 1000 2000
l 12.52 (40) 7.76 (30) 3.94 (19) O (0)
2 11.66 (40) 5.72 (27) 4.76 (34) 1.7 (8)
3 10.46 (34) 7.45 (40) 4.42 (23) O (O)
4 10.28 (38) 5.74 (32) 3.88 (27) O (0)

 

a . . .
Nos. in parenthes1s represent no. of larvae surv1v1ng 10 days
after treatment.

b
50 larvae were treated at each level of exposure.

   

 

 

37

As indicated in Table 2, mortality was extremely high above the
1000 rad level of exposure. Observations taken seven days after treat-
ment indicated apparent normal larval development at 1000 rads. Larvae
in treatments above this level were stunted. An interesting feature
of these larvae was the absence of the shiny black coating of mucous
and feces which appears to be characteristic of normally developing
larvae. However, the weighings taken eleven days after treatment in—
dicate that, while all levels of exposure reduce average larval weight,
this reduction is relatively slight in the case of larvae which actu—
ally survive to the pre—pupation stage. The numbers of individuals
which reached the pupa stage at the 4000 and 16000 rad levels are
obviously inadequate to permit any major interpretation but again it
is of interest to note that individuals which survived the initial
high rates of mortality did manage to pupate.

Results of observations on irradiated 5 day old larvae are
presented in Table 3. Only levels at which there was actual larval
survival are included in the table, that is, up to and including the
2000 rad level. Complete larval mortality occurred at the remaining
levels of 4000, 8000 and 16000 rads prior to completion of the ten day
post treatment period. At these higher levels of exposure post treat-
ment development appeared normal for the first three days followed by
complete cessation of feeding and the presence of numerous dead larvae
on the leaves.

At the end of the 10 day period the eight surviving larvae at
the 2000 rad level were definitely stunted. This observation is
verified by the average larval weight of only 1.7 mg. Larvae at the

500 and 1000 rad levels appeared normal and similar to controls

 

 

38
except in size. This retardation of growth was particularly evident
at the 1000 rad level but was also observed at the 500 rad level.
The results of the body weight estimations indicate that irradiation
of 5 day old larvae at levels of 500 and 1000 rads causes a reduction
in total body weight of 40.5 and 62.2 percent respectively as compared
with controls. An equivalent corrected figure for larvae irradiated

at the 2000 rad level was 84.9 percent reduction.

Irradiation of Third Instar Larvae: Observations included mortality

 

rates, feeding, percentage pupation and subsequent emergence of adults
at the various levels of exposure to beta radiation. These results
are presented in Tables 4, 5, and 6.

TABLE 4.——Surviva1, pupation and emergence of beta irradiated 3rd
instar larvae

 

Exposure level in rads

 

 

O 1000 2000 4000 8000 16000
Larval survival
4 days after 39 34 25 29 26 28
treatment 35 33 36 32 31 20
No. pupated 19 12 3 9 9 l
18 16 6 7 8 0
No. emerged 15 7 0 O 0 0
10 0 0 0 0 0

 

a40 larvae per replicate.

 

 

39

TABLE 5.——Damage rating on beta irradiated 3rd instar larvaea

 

 

 

 

 

 

 

Treatment EZAVZE No. Rated NO. Rated No. Rated
1n rads Rated 0 0—254 25-SOA 50—754
0 44 58 37 42
50 51 37 43
1000 128 93 43 24
94 75 53 33
2000 132 124 67 23
87 64 4o 27
4000 67 66 46 58
97 82 38 20
8000 151 74 41 34
133 102 64 28
16000 58 75 44 13
135 51 31 19

 

a4O larvae were included in each of the 2 replicates at each
level of exposure.

TABLE 6.--Total estimated leaf consumption by beta irradiated 3rd
instar larvaea

 

Exposure level in rads

 

 

O 1000 2000 4000 8000 16000
No. of leaves 47.4 42.8 55.0 61.8 45.9 34.0
consumed 47.1 49.9 39.9 37.0 54.2 29.9

 

aThe number of leaves per pot was not uniform and the total leaf
consumption was estimated by multiplying the number Of leaves in any
one particular category by the percentage damage already established
for that category.

 

40

Larval survival 4 days after treatment was reduced by all
levels of exposure to beta radiation, the effect being particularly
noticeable at the 16000 rad level. Visual Observations revealed the
presence of small and stunted larvae in at least one replicate at all
levels of exposure except in the controls. In replicate number 1 at
the 16000 rad level, 20 of the 28 larvae surviving 4 days after treat-
ment were of extremely small size. An additional feature of these
larvae was the complete absence of the normal black coating.

Pupation rates were significantly reduced at all levels of ex-
posure, particularly above 1000 rads. Adult emergence was completely
eliminated except in one replicate at the 1000 rad level.

The results of the estimations on larval feeding in the first
few days after treatment are presented in Tables 5 and 6.

The results presented in Tables 5 and 6 indicate that for at
least a period of 4 days after treatment exposure of 3rd instar larvae
to beta radiation at levels up to and including 8000 rads had no
effect on larval feeding. Even at the 16000 rad level of treatment
the amount of feeding was considerable. This was surprising, par—
ticularly as stunting of the larvae was noted at the end of the 4 day

period at the 16000 rad level of exposure.

Irradiation of Early Fourth Instar Larvae: The results of larvae

 

treated 4 days before pupation are given in Table 7. Average larval
weights are based on weighings of ten larvae per replicate and are
presented in milligrams.

Pupation rates were relatively high at all levels of exposure

except at 16000 rads. Adult emergence was drastically reduced at the

 

41

TABLE 7.-—Weight increment,a pupation, and emergence of beta irradiated
early fourth instar larvae 2C

 

Exposure level in rads

 

 

 

 

 

 

0 500 1000 2000 4000 8000 16000

No. pupated 4O 31 42 47 46 29 3

43 38 43 32 29 33 2
Percentage
pupation 83 69 85 79 75 62 5
Adult 13 11 2 0 0 0 0
emergence 13 16 3 O O O 0
Percentage
emergence 31.3 39.1 5.9 0 0 0 0
Average larval
weight 13.28 13.32 15.50 16.74 12.50 13.08 13.80
immediately 13.26 12.66 16.22 14.58 13.28 11.64 14.42

after treatment

 

Average larval
weight 2 days 18.70 17.60 16.56 17.06 15.96 14.30 13.48
after treatment 18.18 16.86 17.12 17.54 16.44 14.82 13.36

 

Percentage
weight 38.96 32.64 6.18 10.47 25.68 17.79 —4.88
increment

 

Percentage of
control wt. 100 93.4 91.3 93.5 87.8 78.9 72.7
attained

 

aWeights in milligrams.

b . .
50 larvae per repl1cate and two repl1cates at each level of
treatment.

C . .
The time of treatment was approx1mately 4 days pr1or to
pupation.

   

 

42
1000 rad level and at higher increments it was completely eliminated.
The figures presented for percentage larval weight increment at the
various exposure levels over the two day post treatment period are
hardly comparable since the average weights were not uniform originally.
From the controls it seems possible to assume that the maximum average
larval weight attainable is in the region of 18.5 mg. Naturally, the
nearer the average of any group approached this maximum at the time of
treatment, the smaller the ultimate weight increment will be. On this
basis the figures of 6.18 and 10.47% for the 1000 and 2000 rad level
respectively are not true reflections of the effects of exposure on
weight increment at these particular levels. At the other levels of
treatment the initial weight averages were essentially similar and the
figures presented are comparable. They show a progressive loss in the
rate of larval weight gain with increased dosage until finally at the
highest level of exposure there is an actual loss in weight over the
th day post treatment period. It was felt that expressing total
larval weight of the twenty larvae weighed at each treatment level as
a percentage of that of the twenty control larvae might provide a
better estimate of the effect of treatment and these estimates are
presented. All levels of treatment resulted in a reduction of the
average maximum weight attained, the effect being particularly notice-
able at the 8000 and 16000 rad levels.

A percentage weight increase of 38.96 over a two day period may
seem excessive for larvae at this stage of development, however
Schillinger (1967) has reported an increase of 76% over a three day
period. The larvae exposed in this radiation experiment were field

collected and had come from a poor level of nutrition as almost all

 

43
available food had already been devoured. These larvae were placed on
fresh plants immediately prior to treatment and maintained on a high

level of nutrition for the duration of the test period.

Irradiation of Late Fourth Instar Larvae: A total of 240 larvae were

 

treated at each level in six replicates of 40 larvae each and the stage
of treatment was approximately two days prior to pupation. The results

are presented in Table 8.

TABLE 8.——Pupation and emergence of beta irradiated late 4th instar

 

 

 

 

 

 

 

larvaea
Exposure level in rads

0 500 1000 2000 4000 8000 16000
Total treated 240 240 240 240 240 240 240
Total pupated 178 189 195 214 175 187 128
Percentage
pupation 74.2 78.7 81.3 89.2 72.9 77.9 53.3
Adult
emergence 79 48 38 6 4 l 0
Percentage
emergence 44.4 25.3 19.4 2.8 1.7 0.53 0

 

aThe time of treatment was approximately 2 days prior to
pupation.

Results in Table 8 show that a high rate of pupation occurred
at all levels up to and including the 8000 rad level. The percent
reduction at the 16000 rad level, though significant, was not exces—
sive. Adult emergence was significantly reduced by all levels of
treatment and the reduction was particularly severe above the 1000 rad

level. Examination of pupae from which adults failed to emerge revealed

 

44
that death of the pre—pupa occurred immediately or soon after con—
struction of the pupal chamber and in no instance did development
proceed beyond this point.

The results presented in Table 7 for irradiated early fourth
instar larvae and those in Table 8 for late fourth instar make an in—
teresting comparison. It is evident that at the 8000 and 16000 rad
levels, particularly the latter, the rate of pupation is greatly en-
hanced as treatment is delayed towards completion of the larval
feeding period and commencement of pupation. Eventual adult emergence,
as evidenced by the percentage emergence figures of 5.9 and 19.4 at
the 1000 rad level for early and late fourth instar larvae respectively,
is also increased by delay Of treatment. This effect is further
emphasized by the emergence of small numbers of adults at levels above
1000 rads in the larvae irradiated as late fourth instar while in early
fourth instar treated individuals levels above 1000 rads completely
eliminated adult emergence. However, that the substantial increase
in the numbers of individuals pupating as treatment is delayed is not
due to acquired increased resistance with age is indicated by the fact
that the great majority of larval which did manage to pupate succumbed
immediately or soon after completion of the pupation process. The
best possible explanation would seem to be that tissue damage as a
result of exposure to radiation requires a period of time to manifest
itself. This period as indicated by the results of exposures of early
and late fourth instar larvae would seem to be in excess of two days
and less than four. Tissue damage increases and manifests itself in
the death of the individual more quickly as the dosage level increases.

Thus, at the 16000 rad level particularly, larvae irradiated at the

 

 

 

 

45
early four instar stage died as larvae while those exposed as late
fourth instar survived the pupation process but no further development
took place before death ensued. As already stated, adult emergence
at the 1000 rad expsoure level showed an increase as exposure was de—
layed and, in addition, a small number of individuals emerged from
the later treatments at levels of 2000 and above whereas emergence was
completely eliminated at these levels in larvae receiving treatment at
an earlier stage. Both these facts are best explained on the basis
of recovery from radiation damage which is established and, presumably,
is more liable to occur in a relatively quiescent pupa than in an

actively feeding larva.

Irradiation of Pupae: The results of exposure of 1—7 day old pupae

 

to various levels of beta irradiation are presented in Table 9.

Emergence figures are presented in terms of total emergence
over all five replicates but the range of percentage emergence for
individual replicates is given in the tables.

All levels of exposure resulted in a significant reduction in
adult emergence. The degree of reduction was roughly similar at the
500, 1000, 2000 and 4000 rad levels while the effect was even more
drastic at the 8000 and 16000 rad levels.

Mating, Sterility, Feeding and Mortality Tests
with Pre—diapause Irradiated Adults

Mating Tests with Individual Pairs: Irradiated males were paired in—

 

dividually with untreated females. Each pair was confined separately.
Ten replicates were included at each level of treatment. Mating

counts were carried out three times daily over a period of thirty days.

42......

 

 

                  

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11191.2 ' {I

 

47

the results for beta, gamma and X—ray treated males are presented in

Tables 10, 11 and 12 respectively.

radiation resulted in reduced mating.

All levels of exposure to beta

TABLE 10.——Total matings, number of pairs which mated and mating fre—
quency amongst 10 individual pairings composed of pre—
diapause beta irradiated males with untreated females.

 

Exposure level in rads

 

 

 

 

0 500 1000 2000 4000 8000 16000
No. of matings
observed 73 44 14 31 9 0 5
No. of pairs
which actually 8 8 7 6 5 O 3
mated
NO. of matings 15 18 5 l6 4 O 3
between pairs 13 15 3 8 2 l
which actually 12 4 2 2 l 1
mated 12 2 l 2 1

12 2 l 2 l

4 l l l

4 1 l

1 l l

 

 

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-. -; i p.-
l

.-f.':': :..-.-‘

.33"th new»: at hula“: no]

I. 1' 1.0.91:- .Ivf--.5I!

 

48

TABLE ll.——Total matings, number of pairs which mated and mating fre—
quency amongst 10 individual pairs composed of pre—diapause
gamma irradiated male with untreated females

 

Exposure level in rads

 

 

 

 

0 500 1000 2000 4000 8000 16000
No. of matings
observed 24 46 32 15 23 2 4
No. of pairs
which actually 8 3 7 6 7 2 4
mated
No. of matings 8 23 14 5 7 l 1
between pairs 6 22 7 3 6 l l
which actually 3 1 5 2 4 l
mated 2 2 2 2 l

2 2 2 2

1 l 1 l

l l 1

1

 

TABLE 12.——Total matings, number of pairs which mated and mating fre-
quency amongst 10 individual pairings composed of pre-
diapause X-ray irradiated males with untreated females

 

Exposure level in roentgens

 

 

 

 

0 500 1000 2000 4000 8000 16000
No. of matings
observed 6 16 26 13 1 1 O
No. of pairs
which actually 2 2 3 l l 1 0
mated
No. of matings 4 15 18 13 l l 0
between pairs 2 l 5
which actually 3

mated

 

   

 

49

However, this reduction was not significant until levels of
treatment of 4000 rads and above were reached. At the 8000 and for
all practical purposes, at the 16000 rad levels mating was eliminated.

Exposure to gamma radiation at levels of 8000 and 16000 rads
almost completely eliminated mating. In contrast to the beta treat—
ments presented in Table 10, exposure at the 4000 rad level affected
neither the total number of Observed matings or the mating frequency.

Adult males exposed to X—rays at levels of 500, 1000 and 2000
roentgens mated more frequently than untreated males. Mating was
almost completely eliminated at exposure levels of 4000 roentgens and
above.

The results presented in Tables 10, 11, and 12 for beta, gamma
and X—ray irradiation can be compared although dosimetry for the beta
and gamma treatments was in rads, while that for the X—rays was in
roentgens. For all three types of radiation doses of 2000 r did not
significantly reduce mating. In both the beta and X—ray treatments
exposure levels of 4000 r did reduce mating significantly while at the
higher levels of 8000 and 16000 r mating was almost completely elimi—
nated. The gamma treatments differed from the other two types of
radiation in that mating was not impaired at the 4000 r level of ex—
posure. As was the case with beta and X—rays, exposure to gamma
radiation at levels of 8000 and 16000 almost completely eliminated

mating.

Mating Tests with Groups of Irradiated Males: In addition to the in—

 

dividual pair matings just described, one group of 10 irradiated males

was paired with 10 untreated females at each level of exposure for

 

“My.

 

 

50
each of the three types of radiation. Mating counts were made three
times daily for a period of eleven days. The total numbers of positive
matings observed at the various levels of treatment are presented in
Table 13.

TABLE l3.--Numbers of matings between groups of 10 irradiated pre—
diapause males paired with 10 untreated femalesa’

 

 

 

 

Type of

Radiation Control 500 1000 2000 4000 8000 16000
Beta 76 58 27 4O 0 l4 3
Gamma 65 68 77 40 28 4 4
X—ray 29 34 23 64 6 8 0

 

aA single replicate was included at each exposure level.

bDosimetry for beta and gamma was in rads, that for X—rays in
roentgens. The results in general are similar to those obtained in
the individual pair matings already described. Mating was not seri—
ously diminished by exposure to 2000 r. of any of the three types of
radiation. Treatment levels of 4000 r. and above of beta and X—ray
reduced mating to a very low level. Considerable mating took place
after exposure of males to 4000 r. of gamma radiation. As occurred
with beta and X—rays, exposure to gamma at levels of 8000 and
16000 r. almost completely eliminated mating.

Sterility Tests with Irradiated Males: Data concerning the level of

 

induced sterility in pre—diapause irradiated males were obtained from
the individual and multiple pair matings just described in which males
irradiated at various levels with beta, gamma and X—rays were paired
with untreated females. The results of the individual pair matings
are presented in Table 14 and those of the multiple pair matings in

Table 15.

   

 

51

TABLE 14.——Egg production, percentage hatch and number of matings
resulting from 10 pre—diapause irradiated males paired
individually with untreated femalesax

 

Exposure level

 

Control 500 1000 2000 4000 8000 16000

 

 

 

 

 

Beta 834 593 669 328 575 32 226
Total Eggs Gamma 121 414 268 491 270 76 233
X-ray 61 161 352 79 67 226 .376
Beta 35.4 32.7 15.5 2 7 0 5 0 0
Percentage Gamma 36.4 21 9 9.3 7 3 0 1.3 0
hatch X—ray 1.5 1 8 10.2 0 0 0 1.3
Beta 73 44 14 31 9 0 5
No. of Gamma 24 46 32 15 23 2 4
matings X—ray 6 16 26 13 1 1 0
Beta 11.4 13.5 47.8 1 .6 6 9 O 45 2
Eggs per Gamma 5.0 9.0 8 4 32.7 11 7 38 58 2
mating X—ray 10.1 10.0 13 5 6.0 67 0 22 6 0
Beta 4.0 4.4 7.4 0.3 0.3 0 0
Larvae per Gamma 1.8 2.0 0.8 2.4 0 0 5 0
mating X—ray 0.2 0.2 1.4 0 0 0 O

 

a , .
D051metry for the beta and gamma treatments was in rads; that
for the Xaray treatments was in roentgens.

b . . .
The figures in the columns represent the summation of the no.
of eggs and no. of matings etc. over the ten replicates at each level
of exposure.

 

 

52

TABLE 15.—-Egg production, percentage hatch and numbers of matings
between groups of 10 pre-diapause irradiated males and 10
untreated femalesa

 

 

Treatment level

 

Control 500 1000 2000 4000 8000 16000

 

 

 

Beta 514 340 431 304 396 502 236
No. of Gamma 300 573 420 211 339 143 67
eggs X—ray 179 451 270 190 290 210 0

Beta 37.4 17.6 17.4 2.3 0.8 0.2 0
Percent Gamma 31.7 17.6 22.6 4.3 0.9 0 7.4
hatch X—ray 37.5 27.7 20.0 4 7 5 9 0 0
No. of Beta 76 58 27 40 0 14 3
matings Gamma 65 68 77 40 28 4 4

X—ray 29 34 23 64 6 8 0

 

aDosimetry for beta and gamma was in rads; that for X—ray in
roentgens.

Beta; Eggs were produced in eight of the replicates included as con—
trols. A total of 834 eggs were produced (Table 14) the range in egg
production per female amongst the eight which did lay being 22—190.
The overall percentage hatch was 35.4, with a range of 7.5—47.9. No
direct correlation was found between mating and egg—production or
between mating and percentage egg hatch. The average number of eggs
per mating was 11.4 with a range of 5.2—33.5. The average number of
larvae per mating was 4.0, with a range of 0.9—15.0.

Four of the ten females paired with males which had been exposed
to 500 rads of Beta irradiation failed to produce eggs. Total egg
production for the six remaining females was 593 with a range of 22—170.
The overall viability was 32.7 percent, the range being 5.3—45.8. As

in the controls, egg production varied independently of the number of

   

 

53
observed matings. The average number of eggs per mating was 13.5 and
the range was 1.5—170.0. The average number of larvae per mating was
4.4 with a range of O.1-78.0.

At the 1000 rad level of exposure, eggs were produced in seven
of the ten replicates. Total egg production was 669 with a range per
female of 32—209. The overall hatching percentage was 15.5 with a
range of 13.-36.7. Once again egg production and percentage hatch
were uncorrelated with the number of observed matings. The average
number of eggs per mating was 47.8 with a range of 7.2—209. The aver—
age number of larvae per mating was 7.4 and the range was l.0—l8.0.

Six of the ten females at the 2000 rad level produced eggs.

The total number of eggs was 328 with a range per female of 17—125.
Hatching occurred in only two of the six replicates giving an overall
hatching figure of only 2.7 percent with a range of 0-7.l. The average
number of eggs per mating was 10.6 with a range of 2.7-62.5. The
number of larvae per mating was 0.3 on the average and the range was
0—2.5. Egg production and the numbers of observed matings varied
independently amongst the various replicates.

At the 4000 r level of treatment 575 eggs were produced with a
range per female of 1—282. Three larvae hatched at this level and all
were from the same female, giving an overall hatch of 0.5 percent.

The number of observed matings was severely reduced at this level of
exposure. Of two females which produced 282 and 169 eggs respectively
there was no record of them ever having mated. The average number of
eggs per mating was 63.9 with a range of 0.5—11.0. As already stated,
only three larvae hatched giving an overall average figure of 0.3

larvae per mating and a range of 0—3.

   

   

54

At the 8000 level of treatment eggs were produced in only one
of the ten replicates, the total being 32 eggs. No larvae hatched at
this level and no matings were observed.

Eight of the untreated females paired with males which had
been exposed to 16000 rads failed to produce eggs. The remaining two
females produced 214 and 12, giving a total of 226 eggs. No larvae
hatched at this level. Out of 900 observations made on the ten repli—
cates during a 30—day period only five positive matings were recorded.
Three of these concerned the pair which produced 214 eggs. There was
no record of the female which produced 12 eggs ever having mated. Thus 1

the average number of eggs per mating was 45.2 with a range of 0—7l.3.

Gamma: Eggs were produced in only four of the ten replicates included
as controls for this type of radiation. The total number produced was
121 with a range of 6-52. The percent hatch was 36.3 and the range
was 0—67.3. Egg production and mating were again independently vari—
able, one pair of insects for which there were ten positive mating
records failing to produce eggs. Percentage egg hatch and the fre—
quency of mating were apparently correlated. For one female which
produced 51 eggs, 17.6 percent of which hatched, there was only one
positive observed mating record. Another female which produced 52
eggs with a 67.3 hatching percentage had a record of seven positive
observed matings. The average number of eggs per mating was 5.0 with
a range of 0—51.0. The average number of larvae per mating was 1.8,
the range being 5.0—9.0.

At the 500 rad level of treatment as in the controls six of the

ten females failed to produce eggs. The remaining four females

     

 

55
produced a total of 414 with a range of 3—200. Hatching was extremely
variable. One female produced 200 eggs, 44 percent of which hatched
while an additional 140 eggs produced by another female failed to hatch.
The overall percent hatch was 21.9 with a range of 0—66.6. Egg pro—
duction and mating were apparently correlated. The two females which
produced 200 and 140 eggs had positive observed mating records of 23
and 22 respectively. The average number of eggs per mating was 9.0
with a range of 0-71. There were 2.0 larvae per mating on the average
and the range was 0—3.8.

Five of the ten females paired with males which had been ex— 1
posed to 1000 rads of gamma irradiation produced eggs. The total number
of eggs was 268 with a range per female of 4—101. Hatching in general
was low, the overall figure being 9.3 percent and a range per female
among the various replicates of 1.4—15.8. No definite trend of rela—
tionship between egg production and number of observed matings was
discernable. Similarly, there were no apparent correlations between
larval emergence and the mating history of the parent. The average
number of eggs per mating was 8.4, the range being 0.9—36.5. There
were 0.8 larvae per mating on the average and the range was 0.5—4.3.

At the 2000 rad level of treatment egg production was compara—
tively high and was attributable to six of the ten females. The total
number of eggs was 491 with a range per female of 7—191. Egg production
exhibited no particular trend in relation to mating activity. The
average number of eggs per mating was 32.7 with a range of 2.3-95.5.
Hatching was generally low, 7.3 percent, with a range of 7.1—13.9 for
individual replicates. The average number of larvae per mating was

2.4 with a range of O—9.4. The fact that mating and percentage hatch

   

56
were not correlated is emphasized by the absence of a positive mating
record for one female which produced a total of 115 eggs of which 13.9
percent hatched.

Exposure of males to 4000 rads of gamma irradiation and their
subsequent pairing with untreated females resulted in the production
of a total of 270 eggs. Egg—laying was confined to six of the ten
replicates with a range per female of 30—72. Mating and egg production
among females which did lay were unrelated. The average number of eggs
per mating was 11.7 with a range of 4.4-72.0. Hatching was completely
eliminated which would appear to indicate the induction of complete
male sterility at this level of treatment.

At the 8000 rad level of exposure egg production was seriously
reduced. Egg—laying occurred in only four of the ten females under
test giving a total of 76 eggs and a range per female of 1—37. One
egg hatched which represents a percentage hatch of 1.3. Mating was
nearly eliminated, only two positive matings being recorded. Thus
the average number of eggs per mating was 38.0 with a range of 9—37.

Considerable numbers of eggs were produced at the 16000 rad
level, the total being 233 and with a range per female of the six
which engaged in egg—laying of 7-107. Hatching was again entirely
eliminated. Mating was almost negligible, the four positive matings
recorded representing an average number of eggs per mating of 58.2

with a range of 7—107.

X—ray: Egg production in the untreated controls was extremely low.
Eight of the ten females failed to produce any eggs and a total of 61

with a range of 8—53 for the remaining two females. Only one larvae

 

 

57
hatched and this represented a hatching percentage of 1.5. Mating
was also limited, the female which produced 53 eggs having 2 recorded
positive matings and a further female which produced no eggs mated on
four occasions.

At the 500 r level of treatment only three of the ten females
produced eggs. The total number was 161, the range being 61-97.

Only three larvae hatched at this level representing a percentage
hatch of 1.8 with a range of 1.6—3.3. The absence of a correlation
between mating and egg production was illustrated by the fact that
there was no record of the female which produced 97 eggs having mated.
The average number of eggs per mating was 10.0 and there were 0.2
larvae per mating.

A total of 352 eggs were produced by the three females which
engaged in egg—laying at the 1000 level of treatment. The range per
female was 66—185. The total hatching percentage was 10.2 with a
range for individual females of 4.5—14.6. Egg-laying and mating
appeared related in that the three females which did lay eggs has
positive observed mating records while no mating was observed amongst
the females which failed to lay. Larval hatch was in proportion to
the number of observed positive matings. The average number of eggs
per mating was 13.5 with a range of 10.3—33.7. There were 1.4 larvae
per mating, the range here being 0.6—2.0.

Egg production was low at the 2000 r level, the total of 79
representing the output of three females with a range of 19—38 per
female. All of these eggs failed to hatch. Mating was confined to a

single replicate in which 19 eggs were produced. This gave a total

 

 

58
for that particular replicate of 1.5 eggs per mating, the overall total
being 6.0.

Small numbers of eggs were produced at the 4000 r level of ex-
posure. Only three females were involved in egg—laying giving a total
of 67 eggs with a range per female of 6—34. No larvae hatched at this
level of treatment and only one positive mating record was made over
the entire period.

Egg production was relatively high at the 8000 level, the total
output being 226 from three females. The range for female was 52-115.
No hatching occurred and, as at the 4000 r level, one positive mating
was recorded.

More eggs were produced at 16000 r than at any other level of
X—ray treatment. The total from six females was 376 with a range per
female of 2—115. Five of the 115 eggs produced by one female hatched.
This represented a percent hatch of 4.3 for this replicate with an
overall percentage hatch of 1.3. No matings were observed at this

level of treatment.

Male Sterility Test with Multiple Pairs: Ten irradiated males paired

 

with 10 untreated females were confined in a single pot at each level
of treatment. Egg production and hatching percentages during 25 days
are presented in Table 15. Mating counts made three times daily over
the first 11 days are included in the table.

The trends exhibited by these multiple pair mating are essen—
tially similar to those already illustrated by the individual pairs.
Egg producing and mating history are apparently not correlated. Egg

hatch is affected by levels of exposure of 500 and 1000 r but no

 

 

59
drastic reduction in percentage hatch occurs until males are exposed
to levels of 2000 r. The reduction at this level is quite dramatic
and indicates a high level of induced sterility.

The recordings of observed positive matings given in the table
cannot be directly compared with egg production since mating counts
were carried out only for a period of eleven days whereas the egg
counts covered a period of 25 days. However, these mating records
show conclusively that the considerable reduction in percent hatch at
the 2000 rad level for all three types of radiation is not due to the
inability of treated males to mate. The reduction in hatching per—
centage must therefore be due to induced sterility. With beta and
X—ray radiation levels of 4000 rads and above reduced either the
ability or the willingness to mate significantly. The degree of
sterility induced at these levels may be due to the absence of mating
rather than the impotency of produced sperm. A similar argument may
be used for gamma rays at levels of 8000 and 16000 rads. However, the
occurrence of numerous matings without subsequent high larval hatch
at the 2000 rad level for beta and X—ray and up to the 4000 rad level
for gamma rays would appear to validate the assumption of induced
sterility at these higher levels. It should be pointed out that a
hatching percentage of 7.4 at the gamma 16000 rad level of exposure
represents only five larvae which actually hatched, since only 67 eggs
were produced by the ten females at this level of exposure. Combining
this total egg production figure with that from the ten females paired
individually with males receiving 16000 rads of gamma irradiation and
expressing the five larvae as an overall percentage gives 1.7 percent

hatch.

   

60

FeedingfiTest with Pre—diapause Irradiated Males: The results of the

 

feeding test on irradiated males are presented in Table 16. Figures
in the columns represent the summation of damage of 10 individual
leaves rated separately. TWO replicates were incorporated at each
treatment level. Damage assessments were made at 2 day intervals over

a period of 8 days.

_Bg£a: Levels of exposure of 500 and 1000 r had no effect on male
feeding while levels of 2000 r resulted in a reduction of 42 percent
in total feeding. This reduction was found to be significant at the
95% level but not at the 99% with a t value of 2.834. Exposure levels
above 2000 r drastically reduced the ability or willingness of treated
insect to feed and the effect was scarcely more severe at the 16000 r
level than at the 4000 r level. A notable feature was the ascending
plane of nutrition exhibited by beetles exposed to 0, 500, and 1000 r
over the whole duration of the test. At the 2000 r level of exposure
the amount of feeding increased over the initial six days and then
showed a considerable decline over the final two day period. At the
4000, 8000 and 16000 r levels feeding during the initial two days of
testing compared favorably with that at the lower levels of exposure.
Subsequent ratings at these higher levels showed a steady decline and
in the final two days of testing zero ratings were recorded for all

three of these higher levels of exposure.

Gamma: Levels of exposure up to and including 2000 r did not signifi—
cantly reduce the ability of treated males to feed. At levels of 4000

and 8000 r considerable feeding occurred but the reduction was significant.

 

 

61

TABLE 16.——Assessment of total damage to 10 leaves following feedin
by groups of 10 irradiated males for periods of 2 daysa’

 

Exposure level

 

Control 500 ,1000 2000 4000 8000 16000

 

 

 

Rating 1 10 3 8 6 5 4 4
5 5 5 2 5 3 7
Beta 2 15 10 10 8 6 5 2
9 13 10 5 5 2 8
3 18 13 14 10 6 3 1
ll 14 12 10 2 0 3
4 15 15 14 9 0 0 0
17 26 26 8 0 0 0
Rating 1 12 10 ll 10 9 4 0
9 11 5 10 8 10 0
Gamma 2 20 12 11 10 10 9 0
13 16 9 10 9 10 0
3 20 18 18 ll 10 9 0
20 10 9 14 7 9 0
4 19 22 20 15 6 7 0
18 20 13 18 1 9 1
Rating 1 8 10 9 7 10 6 8
10 10 9 8 9 8
X—ray 2 17 10 10 10 10 10 10
15 10 9 10 10 10
3 10 18 17 14 9 10 3
3O 14 15 17 9 9
4 13 22 20 20 8 0 3
23 15 25 24 3 3

 

aThe scale of damage on which leaves were rated was 0 = damage;
1 = 2-25% of leaf damaged; 2 = 25—50% damaged; 3 = 50—75%; 4 = 75%
and up.

bDosimetry for beta and gamma was in rads; that for X—ray was
in roentgens.

 

 

62

TABLE l7.-—Tota1 damage ratings compiled by groups of 10 pre-diapause
irradiated males over an 8—day feeding period

 

Level of exposure

 

 

Control 500 1000 2000 4000 8000 16000
Beta 100 99 99 58 29 22 25
Gamma 131 119 96 98 59 67 1
X—ray 126 109 114 110 68 56 24

 

At the 16000 r treatment level feeding was almost eliminated. At all
levels up to 4000 r the degree of feeding showed an increase over the
duration of the test while at the 4000 r level itself the total feeding
rating for the final 2 days was less than fifty percent of what it had

been during the initial period of testing.

X—rays: All levels of exposure resulted in decreased feeding but the
effect was not significant until the 4000 r level of treatment was
reached. Even at the 4000, 8000 r exposure levels where the reduction
in feeding was significant at the 95 percent level it was not signifi—
cant at the 99 percent level. However, from Table 16 it will be noted
that at these latter three levels of exposure the damage rating as ex—
pressed for the last two days of testing shows a marked reduction from
that obtained for untreated insects and also for those exposed at
levels below 4000 r. From this it would appear valid to assume that
had the test been prolonged for a further period all levels of exposure

above 2000 r would have reduced feeding significantly.

Mortality of Pre—diapause Irradiated Males: The individual pair and

 

multiple pair matings in which males exposed to varying levels of beta,

 

 

63
gamma and X—rays were mated with untreated females after a period of
cold treatment provided data on the ability of irradiated males to
survive. In the individual pair tests mortality counts were recorded
and dead insects were replaced daily over a period of 31 days. In the
multiple pair matings records were made at 2 day intervals over a
period of 25 days.

The results of these various mortality tests are presented in
Tables 18, 19, 20, 21, 22, 23, 24, 25 and 26. With the exception of
the 500 rad level of exposure, the results in general show increased
mortality as the dosage increases. At the 4000, 8000 and 16000 rad
exposure levels 100 percent mortality was attained in 30, 24, and 12
days, respectively. Mortality at the 500 rad level was higher up to
the 12 day period than at any other level of exposure. This would
seem to suggest that mortality at the 500 rad level is due to some
factor other than exposure to radiation. At the 1000 rad level the
total mortality occurring over the 30 day period was twice that of
the control. Increasing the dosage to 2000 rads resulted in a further
doubling of the percentage mortality. Further increase of dosage
beyond the 2000 rad level caused complete mortality, the interval
necessary to achieve it being proportionate to dosage increment.

As indicated earlier, in these individual pair matings dead
insects were replaced on a daily basis and the total number of replace-
ments carried at the various levels of exposure reflects the dif—
ferential mortalities which occurred over the 31 day period. These

figures are presented in Table 19 in the form of total percentages.

 

 

 

64

TABLE 18.——Accumulated percentage mortalities at 2 day intervals
among groups of 10 pre—diapause males exposed to beta

 

 

 

radiation

Period Exposure levels in rads
digs 0 500 1000 2000 4000 8000 16000
2 10 0 10 10 0 10 10
4 10 20 10 20 0 20 30
6 10 50 10 20 20 20 30
8 10 60 10 30 20 30 40
10 10 70 10 30 30 30 60
12 10 8O 10 3O 30 30 100
14 10 80 20 40 40 60 100
16 10 80 20 50 40 80 100
18 10 80 20 60 40 90 100
20 20 80 20 60 40 90 100
22 20 80 3O 70 50 90 100
24 20 80 40 7O 60 100 100
26 20 80 4O 80 80 100 100
28 20 90 40 8O 90 100 100

30 20 90 4O 90 100 100 100

 

   

65

TABLE l9.—-Total percentage mortalities and average survival in days
in groups of 10 males exposed to beta radiation prior to
the initiation of diapausea

 

Exposure levels in rads

 

0 500 1000 2000 4000 8000 16000

 

Total percent
mortality 40 140 40 130 160 150 290

Average survival
in days 21 6 l7 9 10 8 6

 

a
Following treatment the insects were placed in cold storage
for a period of 11 weeks prior to testing.

TABLE 20.——Tota1 percentage mortalities and survival in days in groups
of 10 males exposed to gamma radiation prior to the initia-
tion of diapause

 

Exposure level in rads

 

O 500 1000 2000 4000 8000 16000

 

Total percent
mortality in 30 80 70 70 160 150 240
31 days

Average survival
in days 20 13 14 11 8 9 6

 

a . .

The insects were subjected to cold treatment for 11 weeks fol-
lowing treatment and dead insects were replaced daily once testing
began.

 

 

66

TABLE 21.——Accumulated percentage mortalities at 2 day intervals among
groups of 10 pre-diapause males exposed to gamma radiation

 

Exposure levels in rads

 

Period in

 

days 0 500 1000 2000 4000 8000 16000
2 0 10 10 30 0 0 V 10
4 0 20 10 40 20 0 10
6 0 20 10 40 20 0 30
8 0 20 20 40 30 0 4O

10 O 20 30 50 4O 30 50
12 10 30 3O 50 50 40 70
14 10 30 30 50 60 40 90
16 10 30 30 50 60 40 100
18 20 30 30 50 60 40 100
20 30 30 30 50 60 40 100
22 3O 40 30 50 90 50 100
24 30 40 40 50 9O 80 100
26 30 50 50 50 100 100 100
28 30 50 50 50 100 100 100

30 30 50 50 50 100 100 100

 

 

67

TABLE 22.--Accumu1ated percentage mortalities at 2 day intervals among
groups of 10 pre—diapause males exposed to X—ray radiation

 

Exposure levels in roentgens

 

Period in

 

days 0 500 1000 2000 4000 8000 16000
2 0 0 0 O 0 10 0
4 O O 10 0 10 20 10
6 0 0 10 0 10 20 10
8 20 0 10 O 10 20 10

10 20 10 10 0 10 30 20
12 20 20 10 0 10 30 60
14 20 20 10 10 20 30 80
16 20 20 10 10 20 40 90
18 20 20 10 10 30 40 100
20 20 20 10 10 40 60 100
22 20 20 20 10 50 70 100
24 20 20 30 10 60 80 100
26 20 20 30 10 70 90 100
28 20 20 30 10 80 100 100

30 2O 20 3O 10 80 100 100

 

68

TABLE 23.——Total percentage mortalities and survival in days in groups
of 10 males exposed to X—ray radiation prior to the initia—
tion of diapausea

 

 

Exposure levels in roentgens

 

0 500 1000 2000 4000 8000 16000

 

Total percent
mortality 20 30 50 10 110 120 230

Average survival
in days 22 21 18 26 ll 9 7

 

a . . .

Follow1ng treatment the insects were subjected to cold treat—
ment for a period of 11 weeks and dead insects were replaced on a
daily basis once testing began.

TABLE 24.——Accumulated percent mortalities among groups of 10 males
exposed to beta radiation pre—diapause

 

Exposure levels in rads

 

Period in

 

days 0 500 1000 2000 4000 8000 16000
2 0 O 30 10 6O 0 30
4 10 10 40 10 7O 10 3O
6 10 20 40 10 70 20 40
8 20 20 40 40 80 60 60

10 50 40 60 50 100 90 90
12 70 50 60 50 100 100 100
14 70 50 60 50 100 100 100
16 80 60 60 80 100 100 100
18 80 60 60 80 100 100 100
20 80 60 6O 80 100 100 100
22 100 70 60 80 100 100 100
24 100 70 60 80 100 100 100

25 100 70 80 80 100 100 100

 

 

69

TABLE 25.-—Accumulated percent mortalities among groups of 10 males
exposed to gamma radiation pre—diapause

 

Exposure levels in rads

 

Period in

 

days 0 500 1000 2000 4000 8000 16000
2 0 0 20 20 0 80 40
4 10 30 20 30 20 90 60
6 10 4O 20 30 20 9O 60
8 20 50 30 30 40 . 90 70

10 50 6O 50 30 90 100 100
12 50 7O 50 50 100 100 100
14 50 7O 50 50 100 100 100
16 50 80 60 60 100 100 100
18 50 80 60 60 100 100 100
20 50 80 60 60 100 100 100
22 70 100 80 60 100 100 100
24 70 100 80 60 100 100 100

25 70 100 90 70 100 100 100

 

   

70

TABLE 26.-—Accumulated percent mortalities among groups of 10 males
exposed to X—ray radiation pre—diapause

 

Exposure levels in roentgens

 

Period in

 

days 0 500 1000 2000 4000 8000 16000
2 0 0 0 0 10 20 20
4 O 10 0 0 40 20 70
6 0 3O 0 20 70 50 70
8 10 40 0 20 70 50 70

10 10 50 0 30 90 90 100
12 10 50 0 50 90 100 100
14 10 50 0 50 90 100 100
16 20 50 0 50 90 100 100
18 20 50 0 50 90 100 100
20 20 50 0 50 90 100 100
22 20 60 20 60 90 100 100
24 20 60 20 60 90 100 100

25 20 70 30 70 90 100 100

 

   

 

71

The data presented in Table 19 afford a better estimate of the
effects of exposure of adult males to beta radiation at the pre-
diapause stage. Ignoring the 500 rad level of treatment once again,
the pattern of survival is that up to and including the 1000 rad level
mortality was comparable to that in the control. The average survival
in days of untreated males was 21 days and at the 1000 rad level it
was 17 days. Increasing the level of exposure to 2000 rads resulted
in a substantial increase in mortality and a lowering of the average
period of survival to 9 days. At exposure levels above 2000 rads
mortality was further increased. At the 16000 rad exposure the average
survival was 6 days which represents a reduction of 71 percent as com—
pared with the control.

The results of the mortality observations on males exposed to
gamma radiation are presented in Tables 20 and 21.

The results in Tables 21 and 22 indicate that exposure to gamma
ration resulted in increased mortality and reduced the average period
of survival considerably. Exposure to 2000 rads of gamma reduced
survival in days by almost one half and a further reduction of almost
50 percent occurred when the level of exposure was increased to 16000
rads. However, from Table 21 it is evident that at the 2000 rad level
the percentage of the 10 original beetles which died during the 31 days
was 50 percent as compared with 100 percent mortality at the 4000,
8000, and 16000 rad levels of exposure.

Mortality data on male beetles exposed to varying levels of
X—rays are presented in Tables 22 and 23. Survival of males exposed
to X—rays compared favorably with that of untreated males up to and

including the 2000 roentgen level of exposure. At the 4000 roentgen

   

 

72
level the total percentage mortality showed a highly significant in-
crease and survival as measured in days was reduced to exactly half
that in controls. At the 16000 roentgen level mortality increased
enormously and survival period was reduced by a factor of more than
2/3.

In addition to the observations just described from the individual
mating tests, addition data on survival of males exposed pre—diapause
to vary levels of beta, gamma and X-rays were provided by the mating
tests with multiple pairs. These results are presented in Tables 24,
25 and 26.

In contrast with the results for beta irradiated males presented
in Table 18 a high mortality among untreated males occurred in the
current test. A 100 percent mortality was attained in the untreated
beetles, while the figures for the 500, 1000 and 2000 rad levels were
70, 80, and 80 percent respectively. Mortality at the 2000 rad level
was in general not significantly different from that in the control.

A 50 percent mortality figure was attained in both treatments on the
10th day after the test began. At the 4000, 8000 and 16000 rad levels
100 percent mortality occurred at the 12th, 14th and 16th day respec—
tively. Thus, at these higher treatment levels mortality was high
initially and total mortality occurred at an early stage.

Mortalities occurring in gamma irradiated males paired in groups
of 10 with untreated females are presented in Table 25. At exposure
levels of 500, 1000 and 2000 rads the trends are rather indefinite and
all that can be stated is that exposure at these levels resulted in a
higher initial mortality than occurred in the control. At the 4000,

8000, and 16000 rad levels mortality rate increased significantly, 100

 

73
percent mortality occurring at 12, 10, and 10 day, respectively for
the three higher levels of exposure.
From the mortality data presented in Table 26 for pre—diapause
males exposed to X—ray radiation the only valid inference would appear
to be that exposure level of 4000 roentgens and above have a signifi—

cant depressing effect on the ability of irradiated males to survive.

Mating Test with Irradiated Females: Irradiated females were paired
with untreated males. Each treatment level was represented by a group
of ten selected females retained in one pot. After the addition of
the untreated males mating counts were carried out three times daily
over a period of 20 days.

The results are presented in Tables 27, 28 and 29. In each
table the period for which the test was run is broken down into 4-day
intervals.

TABLE 27.——Positive observed matings between groups of 10 pre—diapause
beta irradiated females paired with 10 untreated males

 

Exposure level in rads

 

Period in

 

days Control 500 1000 2000 4000 8000 16000
1—4 2 6 O 2 0 O 0
5—8 5 5 6 5 4 0 0
9—12 11 13 l 10 3 4 1
13—16 14 21 1 5 0 5 10
17—20 19 12 l 2 2 8 6

 

Totals 51 57 9 24 9 l7 l7

 

   

 

74

TABLE 28.——Positive observed matings between groups of 10 pre—diapause
gamma irradiated females paired with 10 untreated males

 

Exposure level in rads

 

Period in

 

 

days Control 500 1000 2000 4000 8000 16000
1—4 8 9 4 3 5 6 0
5—8 35 21 19 ll 4 7 4
9—12 30 26 19 23 3 3 2
13—16 12 12 7 7 5 1 0
17-20 11 3 3 . 3 2 0 0
Totals 96 71 52 47 l9 l7 6

 

TABLE 29.-—Positive observed matings between groups of 10 pre-diapause
X—ray irradiated females paired with 10 untreated males

 

Exposure level in roentgens

 

Period in

 

 

days Control 500 1000 2000 4000 8000 16000
1-4 3 10 7 2 4 0 1

5—8 17 26 8 7 9 7 6

9—12 13 32 26 27 13 6 1
13-16 12 18 16 28 9 3 2
17—20 8 4 8 10 4 1 2

Totals 53 90 65 74 39 17 12

 

2333: Exposure of adult female beetles to beta irradiation at levels
of 1000 r and above seriously reduced mating. This reduction may re—
flect a diminution in either their willingness or ability to mate or,
alternatively, it may signify their reduced attractiveness for the

untreated male. An exposure level of 500 r had no adverse effect on

 

75
mating.and, in fact, the total number of matings observed at this level

was higher than in the control.

Gamma: All levels of exposure to gamma irradiation reduced matings
between treated females and untreated males. This reduction increased
with the dosage increment. However, no serious effect was observed
until the 4000 r level of exposure was reached. The 1600 r level of
treatment eliminated mating for all practical purposes. The data as
presented in the table do not entirely indicate the true reduction in
mating vigor at the various levels of exposure as no replacements were
carried out during the period of testing and no account is taken of

the differential mortalities which occurred.

X—ray: Levels of exposure of 8000 and 16000 r seriously reduced
matings between treated females and untreated males. At levels of
500, 1000 and 2000 r the number of matings recorded was higher than

in the control.

Sterility Test with Irradiated Females: Egg production records were

 

kept for the entire duration of the previously described 20 day mating
test between untreated males and females exposed to various levels of
beta, gamma and X-ray irradiation. The total egg production figures
together with their subsequent hatching percentages are presented in
Tables 30, 31, and 32.

Exposure of adult females to beta, gamma and X—ray irradiation
at levels of 2000 r and above nearly eliminated egg production. Treat—

ment levels of 1000 r for all three types of irradiation reduced egg

   

76
production considerably and resulted in extremely low hatching per-
centages. Exposure levels of 500 r did not appreciably reduce egg
production but did decrease hatching. The percentage reduction in
egg hatch at 500 r of beta, gamma and X—ray as compared with the con—
trols was 18.9, 17.7 and 43.0 respectively.

TABLE 30.--Egg production and hatching in groups of 10 pre—diapause
beta irradiated females paired with 10 untreated males

 

Exposure level in rads

 

 

Control 500 1000 2000 4000 8000 16000
Total eggs 210 343 119 l 0 O 0
Total larvae 126 141 4 O 0 0 0
Percentage
hatch 60.0 41.1 3.4 0 O 0 0

 

TABLE 31.-—Egg production and hatching in groups of 10 pre-diapause
gamma irradiated females paired with 10 untreated males

 

Exposure level in rads

 

 

Control 500 1000 2000 4000 8000 16000
Total eggs 514 335 110 8 0 0 0
Total larvae 240 97 7 0 0 0 0

Percentage
hatch 46.7 29.0 6.4 0 0 O 0

 

   

77

TABLE 32.——Egg production and hatch in groups of 10 pre—diapause
X—ray irradiated females paired with 10 untreated males

 

Exposure level in roentgens

 

 

Control 500 1000 2000 4000 8000 16000
Total eggs 457 381 22 l 0 0 0
Total larvae 296 83 1 0 0 0 0
Percentage
hatch 64.8 21.8 4.5 0 0 0 0

 

Mortality Test with Irradiated Females: Female mortalities were re—

 

corded at two day intervals for the first sixteen days of the 20 day
mating and sterility test on females exposed to various levels of beta,
gamma and X—ray irradiation. The results are presented in Tables 33,
34 and 35. The figures in the various columns represent the total
accumulated percentage mortalities at the specified periods.

TABLE 33.——Accumulated percentage mortalities at 2 day intervals
amongst groups of 10 pre—diapause beta irradiated females

 

Exposure level in rads

 

Period in

 

days Control 500 1000 2000 4000 8000 16000
2 O 0 0 20 50 80 33.3
4 0 0 0 3O 80 100 66.6
6 0 0 0 40 80 100 100
8 10 0 10 70 80 100 100
10 30 10 10 70 100 100 100
12 3O 10 20 70 100 100 100
14 40 20 20 70 100 100 100

16 40 30 30 70 100 100 100

 

   

 

78

TABLE 34.——Accumu1ated percentage mortalities at 2 day intervals among
groups of 10 pre—diapause gamma irradiated females

 

Exposure level in rads

 

Period in

 

days Control 500 1000 2000 4000 8000 16000
2 0 0 O 20 4O 20 57

4 10 10 30 20 9O 40 71

6 20 40 50 20 100 60 86

8 30 50 50 30 100 70 100
10 40 70 70 40 100 100 100
12 6O 70 7O 70 100 100 100
14 80 80 80 70 100 100 100
16 9O 90 100 80 100 100 100

 

TABLE 35.——Accumulated percentage mortalities at 2 day intervals among
groups of 10 pre—diapause X—ray irradiated females

 

Exposure level in roentgens

 

Period in

 

days Control 500 1000 2000 4000 8000 16000
2 0 O 20 O 30 3O 30

4 0 O 20 10 30 50 4O

6 10 0 20 20 70 50 50

8 20 0 20 0 80 80 9O
10 20 0 30 3O 80 100 90
12 30 10 30 40 80 100 100
14 30 20 50 50 80 100 100

16 30 20 60 50 90 100 100

 

 

79

Bega: Exposure levels of 2000 r and above of beta irradiation greatly
increased the mortality rate of exposed females. At the 4000, 8000 and
16000 r levels total mortality occurred at 10, 4 and 6 days respectively.
Exposure to 2000 r resulted in a 70 percent mortality in the first

eight days of testing and no subsequent deaths occurred during the suc—
ceeding eight days. At the 500 and 1000 r levels total mortality over

the entire 16 day period was less than in the control.

Gamma: In the control and at the various treatment levels a high
level of mortality had occurred at the end of the sixteen day period.
At the 4000, 8000 and 16000 r levels of exposure total mortality

occurred in 6, 10 and 8 days respectively.

X—ray: Treatment levels of 1000 r and above noticeably increased the
total mortality over the sixteen day period. Mortality was particu—

larly high at the 4000, 8000 and 16000 r levels of exposure.

Feeding Test with Irradiated Females: The results of the feeding test

 

carried out on irradiated females are presented in Table 36.
The figures in the columns represent the summation of damage
of ten individual leaves rated separately for each level of treatment

on the damage scale presented in the materials and methods section.

   

 

80

TABLE 36.——Assessment of total damage to 10 leaves following feeding
by groups of 10 pre~diapause irradiated females for
periods of 2 daysaa

 

Exposure,level in rads

 

Control 500 1000 2000 4000 8000 16000

 

 

 

Rating 1 12 3 1 4 l 0 0

9 6 0 2 3 2 0

Beta 2 28 7 ll 9 6 7 0
27 11 ll 8 10 10 0

3 24 19 27 15 8 8 0

33 26 19 8 12 9 0

4 15 20 22 9 4 0 0

18 11 19 5 2 3 0

Rating 1 9 3 4 O O O 0

8 l l 0 l O 0

Gamma 2 34 20 18 4 9 6 4
25 20 10 3 4 2 2

3 30 25 17 6 10 8 2

29 26 19 15 7 6 1

4 24 20 12 11 3 0 O

21 18 15 17 4 4 0

Rating 1 6 7 10 6 8 0 0

5 8 7 6 5 4 0

X—ray 2 24 22 27 25 19 7 6
27 26 20 22 20 9 6

3 30 26 28 25 21 7 4

37 25 28 19 24 12 5

4 18 28 25 l9 l4 6 0

23 22 23 l7 14 O O

 

aThe scale of damage on which leaves were rated was 0 = no
damage; 1 = 2—25% of leaf damage; 2 = 25—50% damaged; 3 = 50—751;
4 = 75% and up.

bDosimetry for beta and gamma rays was in rads; that for
X—rays was in roentgens.

EFF" -_|.’lJ_ _-

   

 

81

TABLE 37.—-Total damage ratings compiled by groups of 10 pre—diapause
irradiated females over an 8 day feeding period

 

Exposure levels in rads

 

 

Control 500 1000 2000 4000 8000 16000
Beta 166 103 110 60 46 38 0
Gamma 180 133 96 56 38 26 9
X—ray 170 164 168 139 125 45 21

 

Exposure of pre—diapause female beetles to beta, gamma and
X—rays reduced feeding after diapause had been terminated by sub—
jecting the insects to a period of cold treatment. The degree of
suppression increased with the dosage increment. In the beta and gamma
treatments all levels of exposure resulted in a significant reduction
in feeding. The percentage reduction at the 2000 rad level of treat—
ment was 64 and 69 percent for beta and gamma, respectively. The
X—ray treatments were different from the beta and gamma in that only
at the highest exposure levels of 8000 and 16000 roentgens was the

reduction significant.

Tests with Post Diapause Irradiated Beetles

Mating and Sterility Test: Ten males which had been exposed to 2000 r

 

of beta irradiation were paired with ten untreated females. Two
replicates were used and a control of ten untreated males with ten
untreated females was also incorporated. Mating counts were recorded
three times daily over a twenty—two day period and records of egg
production and percentage hatch were kept. The results are presented

in Table 38.

 

 

82

TABLE 38.——Mating, egg production and percentage hatch resulting from
pairing groups of 10 post—diapause males irradiated at
beta 2000 rads with 10 untreated females

 

 

Treatment No. of matings No. of eggs Percentage hatch
Control 102 649 38.5
Beta 2000 r 76 524 9.0
Beta 2000 r 73 702 13.9

 

Exposure of post diapause adults to 2000 r of beta irradiation
decreases the ability of treated males to mate when paired with un—
treated females. Egg production from untreated females was not
impaired by mating them with irradiated males. Egg hatch was con—
siderably reduced, the percentage reduction in the two replicates being
76.6 and 63.9 respectively.

It is interesting to compare mating counts and hatching per—
centages from post diapause irradiated males with the results presented
in Tables 14 and 15 for pre~diapause irradiated adults. The mating
counts are not directly comparable since the counts with pre—diapause
males were made over a period of eleven days while those with post
diapause adults cover a period of twenty two days. Substituting post
diapause counts for the first eleven days of the twenty two day period
enables direct comparisons to be made. The eleven day counts for the
two replicates of post diapause treated males are 54 and 60 respectively,
while the total from Table 15 for pre—diapause treatment males is 40.
This would indicate that pre—diapause treatment has a more adverse
effect on mating vigor than does treatment at the post—diapause stage.
The hatching percentages presented for eggs produced as a result of

matings between untreated females and males exposed to 2000 r of beta

 

 

83
irradiation post—diapause are 9.0 and 13.9 percent. The equivalent
figure from Table 14 for eggs from untreated females mated to males
receiving 2000 r of beta irradiation pre—diapause is 2.7 percent. The
differential development of the male reproductive organs at the time
of treatment offers a possible explanation for this discrepancy. In—
dications are that the post diapause male has a full complement of
sperm while this probably is not true at the initiation of diapause.
Thus the explanation may lie in the fact that sperm which is fully
formed at the time of exposure is less susceptible than sperm which

is produced from radiation damaged tissue.

Feeding Test with Post Diapause Irradiated Males: Male insects which

 

had been subjected to a period of cold treatment in order to terminate
diapause were exposed to 2000 r of beta irradiation. Following treat-
ment a feeding test was carried out over a period of 14 days, damage
being assessed on the basis of percentage leaf destruction at two day
intervals. TWO replicates of ten males each were included and a
further two replicates of ten untreated males each were incorporated
as controls. The results are presented in Table 39.

The results of the feeding test on post-diapause irradiated
males indicate a complete absence of adverse effects on feeding. Re—
sults of feeding tests with pre—diapause irradiated beetles were
presented in Table 16. Tests in this instance covered a period of
eight days. Techniques of damage rating and provision of food material
etc. were alike in both tests. Taking the first four ratings from
Table 39 for post—diapause irradiated males allows a direct comparison

of effects on feeding when treatment is carried out at the pre—and

 

 

 

84
post—diapause stage. Expressing the total damage ratings over the
eight day period for pre— and post—diapause irradiated beetles as
percentages of their respective controls provides the most accurate
comparison. Thus the pre—diapause males subjected to 2000 r of beta
irradiation amassed a total damage rating which was only 64.4 percent
of that for the controls. The equivalent figure for post—diapause
irradiated males was 109.9 percent. It is therefore evident that,
while exposure of post—diapause males to beta irradiation at levels
of 2000 r has no adverse effect on feeding, treatment with similar
quantities of irradiation at the pre—diapause stage causes a signifi—
cant reduction in feeding.
TABLE 39.——Assessment of total damage to 10 leaves following feeding

by groups of 10 post—diapause beta irradiated males for
periods of 2 daysa’

 

 

 

Control I Control II Beta 2000 I Beta 2000 II

Rating 1 5 10 11 10
2 16 22 21 28

3 23 23 25 31

4 24 18 18 22

5 23 15 17 14

6 23 23 21 11

7 23 18 17 14

Totals 137 129 130 130

 

aThe scale of damage on which leaves were rated was 0 = no
damage; 1 = 2—25% of leaf damaged; 2 = 25—50% damaged; 3 = 50—75%;
4 = 75% and up.

b . . .
The figures in the columns represent the summation of damage
for 10 leaves rated separately.

   

 

85

Mortality Test with Post Diapause Irradiated Males: Male beetles

 

which had been exposed to 2000 r of beta irradiation were paired with
untreated females and mortality counts were taken daily over a period
of 21 days. Two replicates of ten males each were included at the
2000 r level and, in addition, a control of ten untreated males.

The results are presented in Table 40 as percent mortalities
occurring at two day intervals over the twenty two day period.

TABLE 40.-—Accumu1ated percentage mortalities at 2 day intervals
among groups of 10 post—diapause irradiated males

 

Period in

 

days Control Beta 2000 r Beta 2000 r
2 O 20 0
4 10 40 0
6 10 40 10
8 10 40 10

10 10 50 30
12 30 60 40
14 30 70 50
16 60 70 70
18 80 7O 70
20 80 70 80
22 90 70 80

 

Male mortality over the entire twenty two day period was higher
in the control than in either of the two groups of insects exposed to
2000 r of beta irradiation. However, up to and including the sixteenth

day of testing, mortalities were consistently higher in both treatments

 

86
than in the control. This is reflected in the number of days at which
a fifty percent mortality occurred which was 15 days in the control

and 9 and 12 days in treatments of 2000 r.

Mixing Test 1

Males which had been irradiated at the initiation of diapause
were mixed with untreated males in ratios of 5:5 and 9:1 and paired
with untreated females. Levels of exposure were 1000 and 2000 rads
for beta and gamma and 1000 and 2000 roentgens for X—rays. Mating
counts were recorded 3 times daily over a period of 18 days and egg
production and subsequent hatch were also noted. The results are pre—
sented in Tables 41, 42, and 43.

TABLE 41.-~Total observed matings, egg production and percent hatch
from pre—diapause beta 1000 and 2000 rad irradiated males

mixed with untreated males at ratios of 5:5 and 9:1 and
paired with 10 untreated femalesa’

 

 

No. of No. of Percent

Treatment Mixing ratio matings eggs hatch
Control 10du + 109u 39 318 33.0
Beta 1000 r 50*R + 50“ + 109UL 31 29 48.3
Beta 1000 r 90“ + 10“ + 10s;u 20 154 18.2
Beta 2000 r So‘R + 50*“ + 109u 48 277 60.7
Beta 2000 r 9o’R + 10“ + 109;L1 43 281 12.5

 

aMating and egg production records were taken over 18 days.

bIrradiated males are designated as dR
Untreated males are designated as d“
Untreated females are designated as Q“

87

TABLE 42.——Total observed matings, egg production and egg hatch from
pre—diapause gamma 1000 and 2000 rad irradiated males mixed
with untreated males at ratios of 5:5 and 9:1 and paired with
10 untreated femalesa’

 

 

No. of No. of Percent

Treatment Mixing ratio matings eggs hatch
Control lOd’u + 109u 72 14 57.1
Gamma 1000 r 5aR + 5s“ + 109u 65 59 16.9
Gamma 1000 r 96R + 1au + 109u 66 39 20.5
Gamma 2000 r st + 5d” + 109u 98 172 22.1
Gamma 2000 r 9dR + 1au + 109u 114 179 12.3

 

aMating and egg production records were taken over 18 days.

bIrradiated males are designated as dR
Untreated males are designated as d”
Untreated females are designated as 9“

TABLE 43.—~Total observed matings, egg production and egg hatch from
pre—diapause X—ray 1000 and 2000 roentgen irradiated males
mixed with untreated males at ratios of 5:5 and 9:1 and
paired with 10 untreated femalesa’

 

 

No. of No. of Percent

Treatment Mixing ratio matings eggs hatch
Control 10a“ + 109u 32 9 44.4
X—ray 1000 r SdR + 5s“ + 109u 73 169 27.2
X—ray 1000 r 9eR + is“ + 109u 48 261 33.7
X—ray 2000 r SdR + 5s“ + 109u 94 193 12.4
X—ray 2000 r 9eR + 1du + 109u 82 345 7.0

 

aMating and egg production records taken over 18 days.

bIrradiated males designated as dR
Untreated males designated as d“
Untreated females designated as Qu

 

 

88

Baga: The mating counts presented in Table 42 represent recordings
taken three times daily over a period of 18 days. The number of
matings observed at the 5:5 and 9:1 of Beta 2000 was actually higher
than in the control. Egg production was extremely low at the 5:5
mixing ratio of the 1000 r level. Otherwise, egg production was not
significantly depressed as a result of mixing. Egg hatch at the 5:5
ratio was higher at both the 1000 and 2000 r levels than in the control.
The reduced hatch at the 9:1 mixing ratio at both the 1000 and 2000 r
levels would appear to indicate a dilution effect of viable sperm with
that from irradiated males. The inclusion of ten females — one for
every male — eliminates competition effect and thus it would appear
valid to interpret the low hatching percentage at the 9:1 mixing ratio
of the 2000 r level of exposure on the basis of sperm dilution. The

33 percent hatch presented for the control is low. An overall hatching
figure for untreated male — untreated female matings in all experiments
was near 50 percent. On this basis the 12.5 percent hatch at the 9:1
mixing ratio of the 2000 r level would be a reduction of 75 percent in

hatching potential.

Gamma; The number of matings observed at the 9:1 mixing ratio of the
2000 r level of exposure was higher than in the control or in any of

the other treatments. This would again demonstrate the absence of
adverse effects on mating vigor at the 2000 r level of exposure. Egg
production was extremely low in the control and also showed considerable
depression in both levels of mixing at the 1000 r level. Egg hatch was
significantly reduced by all levels of mixing. At the 9:1 mixing ratio

of the 2000 r treatment level the percent reduction in hatchability as

   

 

89
compared with a 50 percent figure for untreated male-untreated female

matings was 75.4 which corresponds with the figure at the 9:1 ratio of

Beta 2000 r.
X—ray: Observed matings and egg production were reduced in untreated

male—untreated female matings. All levels of mixing at both the 1000
and 2000 r levels of treatment resulted in reduced hatching. This was-
particularly evident at the 2000 r level. The 7.0 percent hatch at
the 9:1 mixing ratio of the 2000 r exposure level represents 86 per—
cent reduction in hatchability as compared with a 50 percent figure

for untreated.

Mixing Test 2

This and all subsequent mixing tests were made with insects
exposed to 2000 rads of beta radiation after termination of diapause
by subjection to cold treatment. The results are presented in Table
44. Mixing ratios of 9 beta 2000 r irradiated males with one untreated
male paired with 10 untreated females did not reduce the capacity or
willingness to mate. Neither was egg production significantly impaired.
Hatching was considerably reduced with a range of 5.0 to 25.1 percent
over the nine replications as compared with a percentage hatch of 46.9
for untreated male—untreated female matings. The overall percentage
hatch for the nine replications was 13.3 percent. This represents a
percentage reduction in hatching potential of 71.6 and can be attrib—
uted to sperm dilution effect alone since ten females, or one for
every male, were included in each replicate. Assuming total sterility

and full competitiveness on the part of irradiated males one would

   

 

90

TABLE 44.——Total observed matings, egg production and egg hatch from
post—diapause beta 2000 rad irradiated males mixed with
untreated males at a ratio of 9:1 and paired with 10
untreated femalesa

 

 

No. of No. of
matings eggs Percentage hatch
Control 101 649 46.9
Rep. No. l 123 495 10.5
2 123 489 7.9
3 140 519 10.6
4 88 719 15.3
5 52 579 5.0
6 77 762 13.0
7 71 126 9.5
8 84 602 25.1
9 113 710 23.0

 

aMating and egg production records were taken over 22 days.

expect a 90 percent reduction in the overall hatching figure since

this was the ratio of treated to untreated males employed. The failure
to attain 90 percent reduction could possibly be due to the lack of
induction of total sterility at the 2000 r level of exposure or to re—
duced mating vigor as a consequence of treatment. The data presented
in Table 44 indicates that overall mating vigor is not impaired at the
2000 r level and, furthermore, even in replicates where mating counts
were low as compared with control this was not reflected in the per—
centage hatch eventually attained. Table 30 shows clearly that 2000 r
of beta irradiation administered to post diapause males does not induce

total sterility. In two replicates in which ten males exposed to 2000 r

 

91
were paired with ten untreated females the percent reduction in hatch
was 76.6 and 63.9. This provides an average of 70.3 percent which is
almost identical with the level of reduction in the current mixing ex-
periments. From this it would appear valid to infer that a nine to
one mixing ratio of treated to untreated males and pairing them with
ten untreated females results in a percentage hatch which reflects
the level of sterility induced in the treated males and that the

presence of one untreated male is entirely nullified.

Mixing Test 3

In contrast to the test just described, only a single female
was included in each replicate of the current test. The results are
presented in Table 45.

TABLE 45.——Egg production and percent hatch from post diapause beta

2000 rad irradiated males mixed with untreated males at a
ratio of 9:1 and pair with 1 untreated femalea’

 

 

Treatment Mixing ratio No. of eggs Percent hatch
Beta 2000 r 9o'R + 10“ + 19“ 97 6.2
Beta 2000 r 96‘R + 10'“ + 19“ 0 o

 

aEgg production records were taken over 21 days.
bIrradiated males designated as dR
Untreated males designated as d“
Untreated females designated as 9U
Eggs were produced in only one of the two replicates and the
hatching figure was 6.2 percent. The previously described series of
mixing experiments in which ten untreated females were included in

place of the single female in the present test gave an average hatching

   

 

92
figure over nine replications of 13.3 percent with a range of 5.0 to

25.1 percent.

Mixing Test 4

In this test nine males and nine females exposed to 2000 rads
of beta radiation after the termination of diapause were mixed with
one untreated male and one untreated female. The untreated male and
the untreated female were marked with white paint. The numbers of

matings concerning the various possible combinations are presented

 

in Table 46 and egg production and percent hatch in Table 47.

TABLE 46.——No. of observed matings involving irradiated males with
irradiated females, irradiated males with the untreated
female, untreated male with irradiated female, and
untreated male with untreated female following mixing of
9 males and 9 females irradiated post—diapause with 2000
rads of beta radiation with 1 untreated male and 1 un—
treated femalea’

 

Mating combinations

 

 

Replicate 6R with 9R 6R with 9U d“ with 9R du with 9“
l 98 6 8 0
2 112 l l 0
3 108 4 6 0

 

a
Irradiated males designated as dR; irradiated females as 9R;
untreated males as d“; untreated females as 9”.

bMating records were taken 3 times daily over 22 days.

 

 

93

TABLE 47.——Egg production and hatching percentages from pairings of
9 beta 2000 rad post—diapause irradiated males and 9
irradiated females mixed with 1 untreated male and 1
untreated femalea’

 

Mixing Ratio: 90’R + 99R + lo“u + 19u

 

 

Replicate No. No. of eggs Percentage hatch
l 43 14.0
2 5 0
3 39 2.6

 

a . . . .
Irradiated males deSignated as dR; irradiated females as 9R;
untreated males as d“; untreated females as 9“.

bEgg production records were taken over a period of 22 days.

As expected the majority of observed matings were between
treated males and treated females. Over all three replicates this
combination of treated male with treated female constituted 92.4 per—
cent of all matings observed. Treated male with untreated female and
untreated male with treated female matings comprised 3.2 and 4.3 of
the total observed matings respectively. No mating between the un—
treated male and the untreated female was observed.

Egg production was low and the validity of presenting a per—
centage hatching figure from such a low number of eggs which were pro—
duced by only three females is questionable. As already indicated,
total sterility is not induced by the exposure of post diapause males
to 2000 r of beta irradiation. Pairings between males treated at this
level and untreated females resulted in a percentage hatch with a
range of 9.0—13.9. Hence the 14.0 percent hatching figure obtained in

replicate number one of this mixing experiment is not incompatable

   

94
with what would be expected to occur in the absence of matings between
the untreated male and the untreated female. However, the fact that
no such matings were observed is no indication that they did not
actually occur. Again it must be emphasized that the mating observa—
tions carried out in this and other experiments consisted of three spot
checks at three different periods over an entire daily photo period of
sixteen hours. The overall hatching figure for the three-replicates
was 8.0 percent. This compares reasonably favorably with the figure of
6.2 in the previous series of tests which were exactly similar except
for the inclusion of nine females irradiated at the beta 2000 r level.
The test was designed basically to determine the effect of incorporating
nine irradiated females in addition to the nine irradiated males. It
was felt that exposure to 2000 r of beta irradiation might adversely
effect the total volume of sperm produced by irradiated males. As
already indicated, 92.4 percent of all observed matings involved
irradiated males with irradiated females. Previous tests with irradiated
females showed that females exposed to 2000 r of beta irradiation do
not produce eggs and hence sperm transferred in the process of irra—
diated male — irradiated female matings is essentially wasted. How—
ever, an 8 percent hatch from eggs produced by the untreated female
indicates that sufficient sperm is still produced by irradiated males
to maintain the necessary dilution ratio. The conclusions that sperm
production is not affected as a result of exposure to 2000 r of beta
irradiation and that the effectiveness of the sterility technique is
not impaired by failure to separate males and females would appear to
be valid. Hence on a practical basis the irradiation at levels of

2000 r of males and females together and their release assuming a

   

95
fifty—fifty sex ratio in sufficient numbers to establish a ratio of
nine irradiated males for every male occurring in the natural popula—
tion would seem to be equally effective with regard to percentage re—
duction as the much more tedious process of sexing enormous numbers of
beetles and the release of males only. There is of course the factor
of additional damage inflicted by the release of vast numbers of use—
less females to be considered. Table 29 indicates that feeding by
females exposed to 2000 r of beta irradiation is only in the region
of 36 percent of that in untreated females. The equivalent figure for
males from Table 16 at the same level of exposure is 58 percent.
Fortunately in the case of cereal leaf beetle the majority of the
damage is inflicted by the larvae and adult feeding has been deter—
mined to be of little consequence. Nevertheless, it ought to be borne
in mind that release of nine irradiated males and nine irradiated
females for every male in the natural population will result in an

increase of 846 percent in adult inflicted damage.

Mixing Test No. 5

Mixing Ratio: Nine post—diapause irradiated males plus one
untreated male paired with one untreated female. The untreated male
was marked with nail polish and mating records were taken three times
daily over a 17 day period. The results are presented in Table 48.

As indicated in the table, the results over the ten replicates
were extremely variable. The overall percentage of matings involving
the untreated male with the untreated female was 19.1 percent. Hence
the number of matings between treated males and the untreated female

constituted 80.9 percent of all observed matings. The expected values

   

 

96

TABLE 48.-—No. of observed matings involving irradiated males with
the untreated female and the untreated male with the
untreated female following mixing of 1 untreated male and
1 untreated female with 9 males irradiated post—diapause
with 2000 rads of beta radiationa’

 

Z between du

 

d‘R with 9“ 0’“ with 9“ + 9“
Replicate No. 1 l6 0 0
2 7 6 46.2
3 l4 1 6.7
4 10 3 23 1
5 l4 0 0
6 12 l 7 7
7 12 3 20.0
8 8 2 20.0
9 10 6 37.5
10 11 5 31.3

 

a . .
Irradiated males deSignated as dR; untreated males as d“;
untreated females as 9”.

bMating records taken 3 times daily over 17 days.

for untreated male—untreated female and treated male—untreated female
matings on the basis of the nine to one mixing ratio were 10.0 and 90.0
percent respectively. The actual figures observed represent a decrease
of slightly less than ten percent in mating vigor in males receiving
2000 r of beta irradiation. This comparatively slight reduction was
not entirely unexpected. As will be discussed later, differential
mortalities did occur during the course of the experiment and dead
insects were replaced on a daily basis from a supply retained in the

refrigerator at a temperature of 38—400 F. Previous experience

   

 

97
indicated that a minimum period of 4—5 days is required before a male
removed from storage at such temperatures attains his full mating
capacity. Consequently even immediate replacement of a dead irradiated
male effectively reduced the mixing ratio from nine to one to eight to
one for a period of 4—5 days and this will eventually be reflected in
any assessment of mating behavior as an apparent reduction in overall

mating vigor of irradiated individuals.

Mixing Test 6

Nine post—diapause irradiated males and 9 irradiated females

 

were mixed with 1 untreated male and 1 untreated female. Both the
untreated male and the untreated female were clipped and marked with
nail polish. The results of the mating counts taken 3 times daily
over a period of 17 days are presented in Table 49.

Radiated male—untreated female and untreated male—untreated
female pairings comprised 51 of a total of 592 observed matings. These
51 matings consisted of 84.3 percent between radiated males and un-
treated females and 15.7 percent between untreated males and untreated
females. The expected values for these two types of mating combina-
tions were 90 and 10 percent respectively on the basis of the nine to
one mixing ratio. The actual figure of 84.3 percent which was obtained
represents a loss in competitiveness or mating vigor of 5.7 percent
on the part of irradiated males. The figure obtained in the previously
described experiment where irradiated females were not included was

9.1 percent depression.

   

98

TABLE 49.——No. of observed matings involving the irradiated males with
irradiated females, irradiated males with the untreated
female, untreated male with irradiated females, and un—
treated male with untreated female following mixing of 1
untreated male and 1 untreated female with 9 males and 9
females irradiated post—diapause with 2000 rads of beta
radiationa,b

 

Mating combinations

 

Replicate # o'R with 9R 03 with 9“ d“ with 9R 0‘“ with 9“

 

1 . 71 l 7 1
2 65 6 1 l
3 27 6 8 2
4 62 0 8 1
5 43 6 6 0
6 28 7 6 0
7 37 l 9 1
8 50 5 4 0
9 36 9 12 2
10 62 2 9 O

 

a . . . .
Irradiated males deSignated as dR; irradiated females as 9R;
untreated male as d”; untreated female as 9“.

bMating records taken 3 times daily over 17 days.

At this point it would seem opportune to consider further the
role of irradiated females incorporated into this experiment and their
effect on the application of the sterile technique in general. In
mixing test number IV already described it was stated that the inclusion
of nine irradiated females was apparently in no way detrimental to the
effectiveness of the technique. This assumption is vindicated in the

present series of mixing experiments and indications are that the

 

   

99
inclusion of nine irradiated females is beneficial at least to some
extent in minimizing the effect of reduced mating vigor in irradiated
males. The overall reduction in vigor in this series of experiments
in which nine irradiated females were incorporated was 5.7 percent as
compared with 9.1 percent in the previous tests where no irradiated
females were included. The incorporation of irradiated females which
are incapable of producing eggs and contributing to the succeeding
generation could conceivably affect the untreated male in tw0 ways.
First, these useless matings will tax his powers to produce sperm in
large quantities and secondly they will apparently reduce his ability
to take advantage of the reduced mating vigor of irradiated males. The
results obtained in the first series of mixing experiments with post
diapause irradiated males indicated less than full participation by
the single untreated male where ten untreated females were included.
The current series of tests show that the diminished effectiveness of
the untreated male is not due to his inability to compete for and to
mate with the untreated female. In fact these mating tests showed
that untreated male—untreated female matings were 5.7 percent higher
than the expected value. On the basis of the mating ratios established
in this current series of tests one would expect an average hatch of
17.5 percent to have occurred in mixing experiment number one. The
actual figure obtained was 13.3 percent and the reduction of 4.2 per—
cent from the expected value can only be attributed to the inability
of a single male to produce sperm in sufficient quantities to maintain
a nine to one ratio of irradiated to viable sperm in ten females. Thus
the utilization of irradiated males and irradiated female in equal

numbers has a two—fold advantage. First, the presence of the

 

 

100
irradiated females reduces overall hatch by a factor of 4.2 percent
and second, they reduce the proportion of untreated male—untreated
female matings by 3.4 percent. This will ultimately reflect on the
number of irradiated males necessary for release for eradication pur—
poses. It must, however, be balanced against the additional damage
inflicted on the host crop by the release of large numbers of irra—
diated females.

At this point it is well to consider further the role of the
untreated male and to attempt to determine the maximum contribution he
is capable of making in terms of viable sperm. As indicated in
Table 30 the hatching percentages for two groups of ten males exposed
to 2000 r beta irradiation at the post diapause stage and paired with
ten untreated females was 9.0 and 13.9 percent with an average of 11.5
percent. Assuming an average hatching figure of 50 percent in untreated
male—untreated female matings this would indicate that 23 percent of
the sperm from beta 2000 r exposed males are viable while the figure
for untreated males is 100 percent viability. Assuming equal output
of sperm from treated and untreated individuals, the nine irradiated
males contribute .9 of the total sperm while that from the untreated
constitutes .1. Thus we can arrive at a total viable sperm content
from all sources where a nine to one ratio of treated to untreated
males is maintained. That for the nine treated males is the percentage
viable sperm multiplies by .9 which equals 23 x .9 or 20.7 percent.

It is presumed that sperm from untreated males is 100 percent viable.
Therefore the contribution of viable sperm provided by the untreated
male in the nine to one mixing ratio is .1 x 100 or 10 percent. This

gives a total viable sperm content of 30.7 percent as a result of a

101
nine to one mixing ratio. Assuming again a 50 percent hatching figure
as normal one would expect a hatching figure of 15.4 percent in mixing
experiments where the untreated male participated to his full poten—
tial. The actual figure over the nine replications in this series of
mixing experiments was 13.3 percent. A number of conclusions appear
valid on the basis of these results. Firstly, male beetles exposed to
2000 r of beta radiation are fully competitive with untreated males
as regards mating vigor. Secondly, sperm production and the ability
of these sperm to penetrate the egg are not impaired by exposure to
2000 r. The actual hatching figure of 13.3 percent as compared with
an expected figure of 15.4 percent indicates slightly less than full
participation on the part of the untreated male. As already stated,
the inclusion of ten females eliminates competition for the female as
a factor in these experiments. Conversely, however, the inclusion of
ten females may overtax the capabilities of the single untreated male,
albeit only slightly, and this may explain the less than expected
participation.

Any attempted irradication program involves the release into
the natural population of nine irradiated males for every naturally
occurring male. The ratio of released males to naturally occurring
females is similarly nine to one. Two factors play a role in popula—
tion reduction — sperm dilution and competition for the female. The
previously described series of experiments incorporating ten untreated
females was designed to eliminate the competitive effect and conse—
quently to enable an estimation to be made of the reduction effect due
to sperm dilution alone. The resultant reduction over a series of

nine replicates was 71.6 percent. The present series of tests involving

 

102
a single female paired with nine irradiated and one untreated male
incorporates both sperm dilution and competition effects. Hence it
should be possible to proportion the reduction and attribute it on a
percentage basis to its respective causes. The present series of
tests were designated to facilitate this proportionment. The total
percentage egg hatch in this test was 6.2 which represents an overall
reduction of 87.6 percent as compared with normal. As already stated,
the percentage reduction due to sperm dilution is 71.6 percent which
gives up a figure of 16 percent reduction attributable to competition
effect. With tests involving a single female there is unquestionably
a reduction effect on egg production due to the presence of such a high
male-female ratio and also as a result of excessive handling. The
assessment of sperm dilution effect is undoubtedly valid as it covers
a series of nine replications and egg production was not impaired.
Whether or not the attempt to determine the combined effects of sperm
dilution and competition on the basis of a single female which de—
posited a total of 97 eggs is valid must remain open to some degree

of doubt.

 

 

SUMMARY

Eggs of cereal leaf beetle were found to be extremely sensitive
to all levels of beta radiation and hatching was completely eliminated
except where treatment was delayed until hatching was imminent. Mor—
tality of newly emerged larvae exposed to beta radiation was extremely
high above 1000 rads. Average larval weight was reduced by exposure
but in individuals which survived the initial high mortality rate the
reduction was relatively slight and pupation did occur. Irradiation
of slightly older larvae resulted in 100 percent mortality at exposure
levels of 4000, 8000 and 16000 rads. At the lower levels of treatment
the reduction in average larval weight following radiation was much
more severe than in newly emerged larvae. Irradiation of older larvae
demonstrated that percentage pupation increases as treatment is delayed.
Fifty three percent of late 4th instar larvae exposed to 16000 rads
succeeded in pupating when treatment was delayed until 2 days prior
to pupation. When larvae were irradiated with 16000 rads 4 days prior
to pupation only 5 percent pupated.

Reduced adult emergence from exposed pupae was evident at all
levels of treatment. The degree of reduction was similar at all levels
except the 8000 and 16000 rad levels where it was even more severe.

Observations on pre—diapause adults exposed to beta, gamma and
X—rays indicated that 4000 r of beta and X—rays and 8000 r of gamma
radiation reduced mating in irradiated males. Some reduction was also

103

   

104

evident at the lower levels of exposure but the effect was not signifi—
cant. Matings between irradiated males and normal females indicates a
high level of sterility induced at exposure levels of 2000 r and above
but even the highest exposure level of 16000 r did not induce 100 per—
cent sterility. Egg production in normal females mated to irradiated
males compared favorably with that from normal matings. Exposure levels
above 2000 r seriously diminished feeding in irradiated males. Ex—
posure of pre-diapause females to levels of 2000 r almost completely
eliminated egg production in all three types of radiation. In the
first of a series of two tests on the effects of radiation on survival
of pre—diapause irradiated males exposure levels of 2000 rads of beta
and gamma reduced survival in terms of days by approximately 1/2 and
a further reduction occurred at higher levels. Untreated males sur—
vived for 21 days on the average. Irradiation with 2000 rads of beta
and gamma radiation resulted in an average survival of 9 and 11 days,
respectively. Exposure to 2000 roentgens of X—rays in this series of
tests had no effect on survival but increasing the dosage to 4000 r
cut survival time in half. In a second series of tests exposure to
2000 r of beta, gamma and X—rays had no significant effect on male
survival but higher exposures of all three types of radiation resulted
in a marked increase in mortality.

Mating tests with post—diapause beetles exposed to 2000 r of
beta indicated some reduction in mating as a result of treatment.
The level of induced sterility resulting from exposure to 2000 rads
of beta radiation at the post—diapause stage was less than when
treatment was carried and before the initiation of diapause. Post—

diapause exposure at 2000 rads had no effect on feeding of treated

 

 

 

105

males while the same level of exposure caused a 42 percent reduction
in male feeding when treatment was at the pre—diapause stage. Total
mortality over a 22 day period was higher in one group of untreated
males than in two replicates of 10 post—diapause males irradiated at
2000 rads of beta. However, a 50 percent mortality figure occurred
in 15 days in untreated beetles as compared with 9 and 12 days in
those exposed to 2000 rads of beta at the post-diapause stage.

Mixing tests in which pre—diapause males exposed to 1000 and
2000 r of beta, gamma and X—rays were mixed with untreated males at
ratios of 5:5 and 9:1 and then paired with untreated females yielded
results which varied with the type of radiation. In the beta treat—
ments mixing ratios of 5:5 at both the 1000 and 2000 r level resulted
in a higher percentage hatch than occurred in untreated male — untreated
female matings. The 9:1 mixing ratio at the 2000 r level resulted in
an overall reduction in hatching potential of 75 percent. In the gamma
treatments all levels of mixing at both the 1000 and 2000 r level re—
sulted in reduced hatching, the effect being more pronounced at the
9:1 mixing ratio. This ratio at the 2000 r level reduced hatching by
75.4 percent as compared with the control. This figure is almost
identical with the percent reduction in the beta treatment at the same
level of exposure and mixing ratio. A similar trend was observed in
the X—ray treatment, the percentage reduction in hatching potential
resulting from a mixing ratio of 9:1 at the 2000 roentgen level of
exposure being 86 percent. In a further series of mixing tests in
which 9 beta 2000 irradiated males were mixed with one untreated male
and then paired with 10 untreated females eggs were produced in con—

sicerable numbers and the hatching range was 5.0 to 25.1 percent with

 

.1.“! .,.:I

 

106
an average of 13.3 percent. This represents a reduction in hatching
of 71.6 percent. Mixing tests in which only one female was included
resulted in a hatching percentage of 6.2. The inclusion of 9 irradiated
females resulted in an 8.3 percent hatch. An attempt to determine the
mating involvement of the untreated male was carried out. Nine beta
2000 irradiated males were mixed with 1 untreated male and 1 untreated
female which were marked for identification. Mating counts revealed
that irradiated males participated in 80.9 percent of matings as
against an expected value of 90 percent on the basis of the 9:1 mixing
ratio. In a further test in which irradiated females were included
84.3 percent of matings involving the untreated female were with
irradiated males. On the basis of these two tests the reduction in

mating vigor of irradiated males is in the region of 6 to 10 percent.

 

   

CONCLUSION

Any attempt to evaluate the potential of the sterile male
technique for control or eradication of cereal leaf beetle must of
necessity be tentative. It is perhaps best to approach such an evalua-
tion on the basis of the guide—lines set down by Knipling (1964) as
prerequisites for the success of the technique. These requirements
are:

1. Availability of a method of inducing sterility without serious
adverse effects on mating behavior and competitiveness.

2. Practicability of rearing the insect in sufficient numbers.

3. Quantitative information must be available on natural population
densities at low levels in the population cycle.

4. Possession of a practical way of reducing natural populations to
levels manageable with sterile insects.

5. Information on the rate of population increase as a guide for
determining the necessary rate of overflooding with sterile insects.

6. Cost of current methods of control plus the losses caused by the
insect must be higher than the cost of reducing the natural popula—
tion plus the cost of rearing and releasing the required number of
sterile insects.

7. If complete control cannot be maintained because of reinfestations
by migrating insects, or new introductions, the cost of maintaining
complete control by continuing sterile insect releases must be

107

   

108
favorable in relation to the cost for current methods of control,
plus the additional losses caused by the insect.

8. There would be justification for employing the sterile insect
release method, even if it were more costly than current methods
of controlling or eradicating insect populations, if it provides
advantages in overcoming hazards to man and his environment.

9. Sterile insects to be released must not cause undue losses to
crops or livestock or create hazards for man that outweigh the
benefits of achieving or maintaining population control.

The situation with regard to induction of sterility in adult
cereal leaf beetle males is presented in Table 14. Percentage egg
hatch at exposure levels of 2000 r for beta, gamma and X—rays was 2.7,
7.3, and 0.0 respectively. However as indicated in the table increasing
exposure to 16000 r did not insure 100 percent sterility. The levels
of sterility induced at 2000 r of beta and X—rays would appear to be
adequate. Increasing the level of exposure to beta and X—rays to
4000 r is not feasible since mating was significantly reduced at this
level. The 7.3 percent hatch at 2000 rads of gamma radiation may be
less than satisfactory. In view of the fact that 4000 rads of gamma
radiation did not reduce mating in exposed males an increase to 4000 r
is possible for this type of radiation. The reduced percentage hatch
in mixing experiments consisting of males exposed to 2000 r and in—
corporated at a ratio of 9 irradiated males to 1 untreated and the
analysis of percentage mating involvement of these treated males in—
dicated the loss of competitiveness as a result of exposure is in the
region of 5 to 10 percent. This reduction is certainly not excessive
and can undoubtedly be compensated for by a release ratio of 10 or 11

to l.

   

109

Any discussion on competitiveness of irradiated males must con—
sider mortality factors which arise as a consequence of exposure. It
ought to be remembered that in the mixing experiments carried out dead
insects were replaced on a daily basis. It has already been noted that
peak mating activity occurs only after an initial feeding period of
5 days in adult males. Whether or not weekly replacement of dead in—
sects would prove extremely detrimental to the success of the technique
must remain a matter for speculation. Conversely, it can be argued
that the confinement of the insects on the plants by means of glass
chimneys may well prove to be advantageous to the untreated male.

As regards rearing the situation with cereal leaf beetle cannot
be stated to be anything other than unsatisfactory. Some progress
towards obtaining a synthetic diet has been made but at this point it
simply is not possible to state when mass rearing will be possible.

Quantitative data on natural population densities is similarly
lacking and information on the rate of population increase as a guide
for determining release numbers is simply not available. Reduction of
the natural population to a level that is manageable is presumably
possible by the use of properly timed insecticidal sprays. Reductions
of 90—95 percent are no doubt possible. The cost of current control
methods is not excessive but such treatments are contributing to the
accumulation of undesirable residues in our total environment. The
value of the crop and maximum damage attainable must be considered.
This probably lies in the range of 25—50 percent damage at normal
population levels. With regard to cost, cereal leaf beetle is in a
rather unique position. It is of comparative recent introduction and

its distribution, while reasonably extensive, is but a fraction of its

   

110
ultimate potential. Thus the expenditure of sums for its eradication
or curtailment which are out of all proportion to the extent of its
current damage would undoubtedly be economically justifiable.

The reduction in competitiveness as a result of exposure to
levels of irradiation necessary to induce a satisfactory degree of
sterilization was found to be in the region of 5—10 percent. Mortality
at similar levels in two series of tests was separately estimated as
a reduction of 50 percent in survival time in the first test and as
not significantly different from controls in the second test. A 50
percent reduction in longevity still allows a survival period of 11—12
days which should at least be adequate on the basis of the weekly
releases envisaged in any release program. In general, the series of
tests carried out to determine induced sterility levels and effects
on competitiveness and survival of irradiated males would appear to
indicate that, provided mass rearing on artificial media can be
achieved, the sterile male technique has a definite potential for

eradication of cereal leaf beetle.

 

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