Group Title: relationship between insect mineral content and radiation sensitivity
Title: The Relationship between insect mineral content and radiation sensitivity
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Title: The Relationship between insect mineral content and radiation sensitivity
Physical Description: 137 leaves : ill. ; 28 cm.
Language: English
Creator: Levy, Richard, 1944-
Publication Date: 1971
Copyright Date: 1971
 Subjects
Subject: Insects -- Effect of radiation on   ( lcsh )
Radiation -- Toxicology   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Statement of Responsibility: by Richard Levy.
Thesis: Thesis (Ph. D.)--University of Florida, 1971.
Bibliography: Includes bibliographical references (leaves 128-136).
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00098203
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000422538
oclc - 37786589
notis - ACH0725

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The Relationship Between Insect
Mineral Content and Radiation Sensitivity















By

RICHARD LEVY















A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA
1971






























Copyright by

Richard Levy
1971















ACKNOWLEDGMENTS


Many people were involved in making this research possible.

First, and most importantly, I wish to thank the members of my

supervisory committee, Dr. Harvey L. Cromroy, Chairman, Dr. Richard E.

Bradley, Dr. Jerry F. Butler, Dr. Frank S. Blanton, and Dr. William G.

Eden for their continued guidance, encouragement, and numerous efforts

in my behalf. I am particularly grateful to Dr. Cromroy, who acted

as the main technical advisor in this research and who made possible

much of the technical assistance and financial support that the

research required. Special thanks are extended to Dr. Bradley,

Coordinator of the Faculty of Parasitology, for his time and effort

in making many of the financial arrangements which supported this

research.

I wish to acknowledge the technical assistance of Dr. James L.

Nation, and Miss Mary Ellen Smedley, who helped with various aspects

of this investigation.

I also wish to express my appreciation to the personnel of the

Insect Attractant, Basic Biology and Behavior Laboratory, the Insects

Affecting Man and Animals Laboratory for supplying most of the insects

used in the chemical analysis, and to Dr. S. Kramer of the University

of Florida Medical Center.







Very special thanks are extended to Dr. John A. Cornell, Depart-

ment of Statistics, who spent many hours preparing and analyzing the

data by computer.

I am especially grateful to my wife, Diane, for her continued

interest, encouragement, and support throughout this research.

This investigation was partly supported by NIH Training Grant

No. 1T01A100383-01 from the National Institute of Allergy and

Infectious Diseases.






















TABLE OF CONTENTS


Page


ACKNOWLEDGMENTS . . . . . .


LIST OF TABLES . . . . . .


LIST OF FIG S . . . . .


ABSTRACT . . . . . . .


CHAPTER


I INTRODUCTION . . . . .

II LITEPRTURE REVIEW . . . .


Principles of Atomic Absorption Spectroscopy


Atomic Absorption Analysis of Insect Tissue .


Radiation Sensitivity . . . . . .
General . . . . . . . .
Insect . . . . . . . .


Nuclear Indicators of Radiosensitivity .
Plant Nuclear Parameters . . .
Animal Nuclear Parameters . . .
Insect Nuclear Parameters . . .


Cytoplasmic Indicators of Radiosensitivity
Animal Cytoplasmic Parameters . ..
Insect Cytoplasmic Parameters . ..


Non-Cellular Parameters of Radiosensitivity


Interactions of Ionizing Radiation in Biological Systems.


Free-Radical Mechanism of Radiation Damage . . .


. . . iii


. . . viii


. . xiv


. . xv







3


3


4


7
7
8
. . 14
. . . 3



. . . 4


. . . 7
. . . 7



. . . 14
. . . 14
. . 15
. . 16


. . . 17
. . 17
. . . 18


. . . 19


. e . . .


. o
.
. .


. .
.







TABLE OF CONTENTS (Continued)


Page


CHAPTER

II (Continued)

Animal and Plant Minerals . . . . . . . .
The Significance of Minerals in Enzyme Systems .


Non-Enzymatic Functions of Minerals .
Iron . . . . . . . . .
Copper . . . . . . . .
Magnesium . . . . . . ...
Sodium . . . . . . .
Potassium . . . . . . . .


Insect Minerals . . . .
Iron . . . . .
Copper . . . .
Magnesium . . . .
Sodium and Potassium

III METHODS AND MATERIALS . .

Atomic Absorption Analysis
Insect Preparation .
Dehydration . . .
Ashing . . .
Mineral Determination

Radiation and LD50 Procedure

Data Analysis . . . .


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

Statistical Methodology . . . . . . . ..

LD50/24 Hour Predictor Models . . . . . . .
Stored-Product Feeders . . . . . . .
Organic-Plant Feeders . . . . . . . .
Blood-Feeders . . . . . . . . ....

LD50/28 Day Predictor Models . . . . . . .

LD 5/Mean-Mortality Predictor Models . . . . .

Mineral-Based LD50 Predictions . . . . . . .

Mechanisms of Death . . . . . . . . . .
LD50/24 hour Models . . . . . . . .
LD50/28 Day and LD50/Mean-Mortality Models . ..


. . 27
. . 27
. . 28
. . 29
. . 29
. . 30

. . . 31
. . 32
. . . 34
. . . 37
. . . 40

. . . 43


. . 53


. . . . . . 56









TABLE OF CONTENTS (Continued)


Page

CHAPTER

V CONCLUSION . . . . . . . . . . .. 102

APPENDIX .. ...... ... .. . .... ... ... . 104

LITERATURE CITED . . . . . . . . . . . 128

BIOGRAPHICAL SKETCH .. .. .. .. ... .. ..... . 137
















LIST OF TABLES


Table Page

1. Analysis of the simple regression model, y =
2.2147E12 (Cu/(Na x Mg) ) + 112.0665 representing
the LD50/24 hr radiation response for nineteen
species of insects. . . . . . . . . ... 63

2. Analysis of the multiple regression model, y =
68.4260 + 0.0061 (Na) + 3.4360E12 (Cu/(Na x Mg)2) +
0.06893 (K/Cu) + (-109.9400 (Cu/Fe)) representing the
LD50/24 hr radiation response for nineteen species of
insects.. . . . . . . . . . . . . 64

3. Analysis of the simple regression model, y =
244.5774 (Cu/Fe) + 172.0181 representing the LD50/
24 hr radiation response for six species of adult
storeo-product insects. . . . . . . . ... 66

4. Analysis of the multiple regression model, y =
424.5906 + (-0.3263 (Mg)) + (-46506.5039 (Cu/K)) +
589.8412 (Cu/Fe)representing the LD50/24 hr
radiation response for six species of adult stored-
product insects. . . . . . . . .. . . 67

5. Analysis of the multiple regression model, y =
308.3822 + (-7.0798 (Cu)) + 687.3172 (Cu/Fe) +
(-1229.1410 (Mg/K)) representing the LD50/24 hr
radiation response for six species of stored-
product insects. . . . . . . . .. . . 68

6. Analysis of the imple regression model, y = -27456.8938
(Cu/K) + 152.6571 representing the LD50/24 hr radiation
response for eight species of adult organic-plant
feeding insects. . . . . . . . .... . 70

7. Analysis of the simple regression model, y =
-6509.5691 (Cu/Na) + 155.6133 representing the LD50/
24 hr radiation response for eight species of adult
o-ganic-plant feeding insects. . . . . . ... 71







LIST OF TABLES (Continued)


Table Page

8. Analysis of the simple regression model, y =
.0054 (K) + 25.8061 representing the LD50/24 hr
radiation response for eight species of adult
organic-plant feeding insects. . . . . . . 72

9. Analysis of the simple regression model, y =
0.0166 (Na) + 51.3646 representing the LD50/24 hr
radiation response for eight species of adult organic-
plant feeding insects. . . . . . . . ... 73

10. Analysis of the multiple regression model, y =
110.2092 + 0.0087 (Na) + (-18940.1062 (Cu/K))
representing the LD5624 hr radiation response for
eight species of adult organic-plant feeding insects. . 74

11. Analysis of the multiple regression model, y =
92.6478 + 0.0031 (K) + (-3925.0502 (Cu/Na))
representing the LD50/24 hr radiation response for
eight species of adult organic-plant feeding insects. .. 75

12. Analysis of the simple regression model, y =
30.6599 (Mg/Na) + 171.5961 representing the LD50/24 hr
radiation response for five species of blood-feeding
insects . . . . . . . . . . . .. 77

13. Analysis of the simple regression model, y =
-0.0147 (Mg) + 172.2073 representing the LD50/24 hr
radiation response for five species of blood-
feeding insects. . . . . . . . .... . 78

14. Analysis of the multiple regression model, y =
224.1587 + (-1.2260 (Cu)) + (-0.0467 (Fe)) +
(-32.4359 (Mg/Na)) representing the LD50/24 hr
radiation response for five species of blood-
feeding insects. . . . . . . . .... . 79

15. Analysis of the multiple regression model, y =
187.2418 + (-0.0251 (Fe)) + (-37.9907 (Mg/Na))
representing the LD50/24 hr radiation response for
five species of blood-feeding insects. . . . ... 80

16. Analysis of the simple regression model, y =
-0.0024 (Mg + Na) + 11.4310 representing the LD50/
28 day radiation response for seven species of adult
insects. . . . . . . . . ... .... . 82






LIST OF TABLES (Continued)


Table Page

17. Analysis of the simple regression model y =
-0.3593 ((Fe x Na)/K) + 10.1094 representing the
LD50/28 day radiation response for seven species
of adult insects. . . . . . . . .... . 83

18. Analysis of the multiple regression model, y =
39.5350 + (-3009.0000 (Cu/K)) + (-0.0161 (Mg + Na))
+ 0.0001 (Fe x Na) representing the LD50/28 day
radiation response for seven species of adult
insects. . . . . . . . . ... ...... .84

19. Analysis of the multiple regression model, y =
47.1820 + (-7523.9000 (Cu/K)) + (-0.0173 (Mg + Na))
+ 2.2c'63E-5 (Fe x Na) + 1.3975 (Fe x Na)/K
representing the LD50/28 day radiation response for
seven species of adult insects. . . . . . ... 85

20. Analysis of the multiple regression model, y =
43.67/0 + (-5403.3000 (Cu/K)) + (-0.0180 (Mg + Na))
+ 0.0091 (Fe x Na) + 22.6400 ((Na + Mg)/K) repre-
senting the LD50/28 day radiation response for
seven species of adult insects. . . . . . ... 86

21. Analysis of the simple regression model, y =
499.7257 (Mg/K) +(-27.9200 representing the LD50/
mean-mortality radiation response for eight species
of adult insects. . . . . . . . .... . 88

22. Analysis of the multiple regression model, y =
-110.1670 + 887.5769 (Mg/K) + (-631.3156 (Cu/Mg)) +
5554.3929 (Fe/K) + 8.8102 (Na/Mg) representing the
LD50/mean-mortality radiation response for eight
species of adult insects. . . . . . . . .. 89

23. Analysis of the multiple regression model, y =
-36.7632 + 704.4908 (Mg/K) + (-833.3842 (Cu/Mg)) +
3454.4009 (Fe/K) + (-21.1603 (Mg/Na)) representing
the LD50/mean-mortality radiation response for
eight species of adult insects. . . . . . .. 90

24. Analysis of the multiple regression model, y =
-51.5398 + 457.4504 (Mg/K) + (-597.3484 (Cu/Mg)) +
5524.6261 (Fe/K) representing the LD50/mean-mortality
radiation response for eight species of adult insects.. 91









LIST OF TABLES (Continued)


Appendix
Table Page

1. Total body insect mineral content determined
by atomic absorption spectrophotometry . . ... 105

2. Predicted LD /24 hr radiation doses for 16
species of50insects based on the simple
regression model, y = Cu/(Na x Mg)2
representing 19 species of adult insects. . . .. 108

3. Predicted LD50/24 hr radiation doses for 16
species of insects based on the multiple
regression model, y = 68.4260 + 0.0061 (Na) +
3.4360E12 (Cu/(Na x Mg)2) + 0.0689 (K/Cu) +
(-109.9400 (Cu/Fe)) representing 19 species
of insects. . . . . . . . ... ..... 109

4. Predicted LD n/24 hr radiation doses for four
species of insects based on the simple
reZression model, y = 244.5774 (Cu/Fe) +
172.0181 representing six species of
stored-product insects. . . . . . . . ... 110

5. Predicted LD /24 hr radiation doses for four
species of insects based on the multiple
regression model, y = 424.5906 + (-0.3263 (Mg))
+ (-46506.5039 (Cu/K)) + 589.8412 (Cu/Fe) repre-
senting six species of stored-product insects ... 111

& Predicted LD50/24 hr radiation doses for four
species of insects based on the multiple
regression model, y = 308.3822 + (-7.0798 (Cu))
+ 687.3172 (Cu/Fe) + (-1229.1410 (Mg/K))
representing six species of stored-product insects. . 112

7. Predicted LD50/24 hr radiation doses for six
species of insects based on the simple
regression model, y = -27456.8938 (Cu/K))+
152.6571 representing eight species of organic-
plant feeding insects. . . . . . .... 113

& Predicted LD50/24 hr radiation doses for six
species of insects based on the simple
regression model, y = -6509.5691 (Cu/Na) +
158.6133 representing eight species of organic-
plant feeding insects. . . . . . . . ... 114








LIST OF TABLES (Continued)


Appendix
Table Page

9. Predicted LD /24 hr radiation doses for six
species of insects based on the simple
regression model, y = .0054 (K) + 25.8061
representing eight species of organic-plant
feeding insects. . . . . . . . . ... 115

10. Predicted LD /24 hr radiation doses for six
species of insects based on the simple
regression model, y = 0.0166 (Na) + 51.3646
representing eight species of organic-plant
feeding insects. . . . . . . . . ... 116

11. Predicted LDs0/24 hr radiation dese:s for six
species or insect based on the multiple
regression model, y = 110.2092 + 0.0087 (Na) +
(-18940.1062 (Cu/K)) representing eight
species of organic-plant feeding insects. . . .. 117

12. Predicted LD o/24 hr radiation doses for six
species of insects based on the multiple
regression model, y = 92.6478 + 0.0031 (K) +
(-3925.0502 (Cu/Na)) representing eight
species of organic-plant feeding insects. . . .. 118

13. Predicted LD 0/24 hr radiation doses for four
species of5 arthropods based on the simple
regression model, y = -30.6599 (Mg/Na) + 171.5961
representing five species of blood-feeding insects. . 119

14. Predicted LD50/24 hr radiation doses for four
species of arthropods based on the single
regression model, y = -0.0147 (Mg) + 172.2073
representing five species of blood-feeding insects. . 120

15. Predicted LD 0/24 hr radiation doses for four
species of arthropods based on the multiple
regression model, y = 224.1587 + (-1.2260 (Cu))
+ (-0.0467 (Fe)) + (-32.4359 (Mg/Na)) representing
five species of blood-feeding insects. . . . ... 121

16 Predicted LD50/24 hr radiation doses for four
species of arthropods based on the multiple
regression model, y = 187.2418 + (-0.0251 (Fe))
+ (-37.9907 (Mg/Na)) representing five species
of blood-feeding insects. . . . . . . ... 122









LIST OF TABLES (Continued)

Appendix
Table Page

17. Predicted LD /28 day radiation doses for 13
species of insects based on the simple
regression model, y = -0.0024 (Mg + Na) +
11.4310 representing seven species of adult
insects. . . . . . . . . . . . 123

18. Predicted LD /28 day radiation doses for 13
species of insects based on the multiple
regression model, y = 39.5350 + (-3009.0000
(Cu/K) + (-0.0161 (Mg + Na)) + 0.0001 (Fe x Na)
representing seven species of adult insects. ... . 124

19. Predicted LD /28 day radiation doses for 13
species of insects based on the multiple
regression model, y = 47.1820 + (-7523.9000
(Cu/K)) + (-0.0171 (Mg + Na)) + 2.2863E-5
(Fe x Na) + 1.3975 (Fe x Na)/K representing
seven species of adult insects. . . . . . . 125

20. Predicted LD50/mean-mortality radiation doses for
13 species of insects based on the simple
regression model, y = (499.7257 (Mg/K) +
(-27.9200) representing eight species of
adult insects. . . . . . . . .. . . 126

21. Predicted LD50/mean-mortality radiation doses for
13 species of insects based on the multiple
regression model, y = -110.1670 + 887.5769 (Mg/K)
+ (-631.3156 (Cu/Mg)) + 5554.3929 (Fe/K) +
8.8102 (Na/Mg) representing eight species of
adult insects. . . . . . . . . . . 127

















LIST OF FIGURES


figure Page

1. Optical system of Techtron Type AA-100 Atomic
Absorption Spectrophotometer. S S = optical
slits . . .. . . 6

2. LD50 doses for adult vertebrates, insects, and
unicellular organisms . . . . . . . .. 10

3. Effects of ionizing radiation on insects . . ... 12

4. Techtron Type AA-100 Atomic Absorption Spectro-
photometer . . . . . . . . ... . . 48

5. Instrument settings of Techtron Type AA-100 Atomic
Absorption Spectrophotometer . . . . . ... 50

6. Instrument settings and internal components of
Techtron Type AA-100 Atomic Absorption Spectro-
photometer . . . . . . . . ... . . 52

7. Cobalt-60 insect irradiator . . . . . . ... 55






Abstract of Dissertation Presented to the Graduate Council of the
University of Florida in Partial Fulfillment of the Requirements for
the Degree of Doctor of Philosophy


THE RELATIONSHIP BETWEEN INSECT
MINERAL CONTENT AND RADIATION SENSITIVITY



By

Richard Levy

December, 1971


Chairman: Harvey L. Cromroy
Major Department: Entomology and Nematclogy



Parts per million insect total body copper, iron, magnesium, sodium,

and potassium were analyzed by atomic absorption spectrophotometry to

determine if mineral content could be used as an effective biological

indicator for a species-specific dose of ionizing radiation.

Simple and multiple regression analysis of 22 species of adult

insects representing Anoplura, Coleoptera, Diptera, Hemiptera, Homoptera,

Lepidoptera, Orthoptera and Thysanura indicated that there was a

direct relationship between tissue-organ cation content and x-ray

or gamma radiation sensitivity as defined by the LD50/24
50/24 hour
LD50/28 day, and LD 50/mean-mortalitydose parameters.
50/28 day S0/mean-mortality

LD regression models were shown to be highly correlated
50/24 hour
to insect feeding preferences. Mechanisms of death were postulated as

being activated by oxidation and binding of mineral cations from high

doses of ionizing radiation, with the subsequent inhibition of the CNS,

and/or the inhibition of respiratory metabolism.







Analysis of LD and LD regression models
Analysis of LD50/28 day and LD50/mean-mortality regression models

appeared to indicate a species response to low doses of ionizing

radiation via free radical inhibition of an integrated series of

biological functions involving the CNS, respiratory system, circulatory

system, buffer systems, tyrosinase enzyme system, RNA synthesizing

system, and digestive-excretory systems. Several mechanisms for

radiation-induced delayed death were postulated.

Although no LD50 values were available for developing regression

models based on larvae, pupae, or nymphs, their mineral content did

demonstrate characteristic differences when compared to the adult

stages. Therefore, it would seem that specific mechanisms for charac-

terizing their radiation sensitivity according to life cycle stage

could be developed.

In all, 39 species of adults, 12 species of larvae, 5 species of

pupae, and 3 species of nymphs were analyzed by atomic absorption

spectrophotometry for insect total body Cu, Fe, Mg, Na, and K content

in parts per million.
















CHAPTER T


INTRODUCTION



Since 1935, when ionizing radiation was shown to produce mutations

in fruit flies, there has been considerable interest in the effects of

ionizing radiation on insects (74). In recent years, the successful

uses of ionizing radiation in the control of insect pests, as well as

the concern over the possible contamination of the biosphere by

radiation from nuclear reactors and possible nuclear disasters have

further stimulated study on radiation effects on insects (25-28).

Many attempts have been made to find biological indicators to

explain the nuclear and cytoplasmic effects of ionizing radiation

(12,79), but these have failed to account for the great variation in

the radiosensitivities across insect orders (25-29, 61, 87).

This research is aimed at determining whether a correlation exists

between the total body iron (Fe), copper (Cu), magnesium (Mg), sodium

(Na), and potassium (K) content of an insect species and the radiation

LD (dose required to kill 50% of a given population) of that same
50
species. The mineral content would, therefore, serve as an effective

biological predictor of radiosensitivity. The rationale for the

selection of these minerals was based on their important physiological




2



and biochemical roles in maintaining the normal homeostasis of organisms

(16,20,90). These cations were generally essential to the insects'

diet (39,51,52,96) and have been shown to be extremely important in

regulating several metabolic and nervous system functions (39,41,42,

101-105).

If a relationship between mineral content and LD50 could be estab-

lished, then the effect of a given radiation dose on the insect fraction

of an ecological food chain could be predicted. In addition, tentative

explanations of the mechanisms causing radiation protection and/or

radiation death in insects could be postulated.

















CHAPTER II


LITERATURE REVIEW



Principles of Atomic Absorption Spectroscopy



Since the development of commercial atomic absorption (= flame

photometry) instrumentation in the early 1960's, atomic absorption

spectroscopy has occupied an ever-increasing role in the clinical and

biological laboratory (16).

Atomic absorption spectroscopy can be simply defined as the absorp-

tion of radiant energy by atoms (16). This absorption and its quanti-

tative correlation with the concentration of metal ions originally

present in a sample solution served as the basis of analytical atomic

absorption spectrophotometry. The physical principles governing the

use of atomic absorption spectrophotometry can be briefly described

(16): Atoms of every element can absorb radiation at extremely narrow

wavelength bands which are characteristically different for every

element. For an element to be in a condition to absorb, its atoms

must be chemically unbound and in their minimum energy or ground state.

This condition is generally achieved by vaporizing the sample in a

flame. The source of radiation is generally a hollow-cathode lamp,








whose cathode is made of the element being determined. The lamp

emits the characteristic line spectra of that element while the sample

absorbs energy only at a specific resonance line. The resultant effect

is a diminished resonance line caused by the absorption of the

vaporized sample in the flame.

The remaining spectral lines are removed by a monochromator which

has been adjusted to select a band of wavelengths around the resonance

line, and to reject all others. The photodetector would then "see"

only the diminished resonance line. A schematic of the optical system

is presented in Figure 1.

The results from an atomic absorption analysis would therefore be

consistent with Beer's Law which states that "the concentration of the

element in a sample is proportional to the absorbance (optical density)

in the flame" (16).

Analytical precision or reproducibility of 1% has been reported

for the atomic absorption technique when well-designed equipment was

used and elements could be determined in the optimum analytical range

(16).



Atomic Absorption Analysis of Insect Tissue



Relatively few researchers have used atomic absorption spectroscopy

for insect mineral determinations, although this technique has been exten-

sively used for many other organisms (16). Nation and Robinson (71),

using atomic absorption spectroscopy, have quantitatively determined the

parts per million Fe, Cu, Na, K, Mg, Ca, Zn, and Mn in whole adult honey

bees (Apis mellifera). They also determined the mineral concentrations































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in the separated body regions: head, thorax, and abdomen. In a

mineral salt requirement study, Stevens (96) determined the percent

Na, K, Mg, Fe, Cu, Ca, Zn, and Mn in the ash of whole adult and larvae

almond moths (Cadra cautella) with atomic absorption spectrophotometry.

No other literature was found using the atomic absorption technique

for insect mineral analysis.



Radiation Sensitivity



General


Two of the major parameters used in determining the effects of

ionizing radiation in multicellular organisms have been lethality and

life-span shortening (74,79). In practice, one can usually distinguish

between rapid killing (lethality) from massive radiation doses and a

delayed mortality (life-shortening) from substantially lower doses.

It is the belief of many researchers in the field of radiation biology

that the specific dose and dose-rate responses are related to nuclear

and/or cytoplasmic factors (12,79). Pizzarello and Witcofski (79, p. 216)

have stated that "any discussion of radiation lethality in cells must

compare the roles of the nucleus and cytoplasm as they contribute to

that end point. Since the metabolic activities of the cell occur

primarily in the cytoplasm, extensive damage there might be expected

to cause cellular death. But permanent lethal changes (serious damage

to genetic function and consequently to metabolism) must occur in the

nucleus. It must, therefore, be questioned: When the cell as a whole

is irradiated with enough energy to kill it, where has the immediate









lethal damage actually been produced -- in the nucleus or in the

cytoplasm, or in both?" An answer to this question would help

determine the causes) of death to the total organism and therefore

relate cellular and animal radiosensitivity.



Insect


Changes in an insect population following irradiation have most

frequently been expressed in terms of mortality and reproduction

(14,15,22,37,113,116). Most of the radiation sensitivity research on

insects has involved single species with such variable experimental

conditions, for example, dose, dose-rate, etc., that a useful comparison

of radiation doses in the literature has been difficult.

Considerable information is available on the radiation effects on

mammalian systems (7,12,79). Very little research on the physiological

effects of radiation on insect systems has been done, although it is

a well-known fact that many adult insects can withstand radiation

levels hundreds of times greater than the doses required to produce

death in mammals (74). Figure 2 illustrates comparative doses to kill

insects and other organisms, while Figure 3 compares the radiosensitivity

between insects.

The increased knowledge of the histopathology of vertebrate

irradiation has answered several questions on why adult insects are so

radiation tolerant (97). An adult vertebrate is dependent upon

mitotically active cells and relatively primitive tissue, whereas an

adult insect has no replaceable epithelium, no complicated mucous

membranes, and no true hematopoietic or lymphatic systems (97). The

insects body surface, or cuticle, is also known to be composed of



































Figure 2. LD doses for adult vertebrates, insects, and
50
unicellular organisms (3,24,46,47,74,113).




















INSECTS

Bracon
bee -
Tribolium
Drosophila


cockroach 10-


Log
Dose

R


-105-


-102-


UNICELLULAR ORGANISMS

- Paramecium

- amoeba
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VERTEBRATES
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non-living tissue. In addition, the internal structure of the adult

insect is chiefly composed of fat, muscle, and nerve tissue which are

known to be fairly radioresistant in mammals (97).

The only tissues known to undergo rapid division in insects are

the gonads, which are very sensitive to low doses of ionizing radiation

(74,97). DNA turnover in the insect midgut may also be affected by

ionizing radiation (28). Although much higher doses of radiation are

required to produce death, there is irreversible damage to the insect's

ability to reproduce (74).

The most generally accepted explanation for the radiation tolerance

of insects is based on the law of Bergonie and Tribondeau which states

that the sensitivity of cells to ionizing radiation is directly propor-

tional to their reproductive rate and inversely proportional to their

degree of differentiation (74,97).

After the insect hatches from the egg, very little cell division

occurs during larval life (74). Cell division and differentiation of

insect tissues occur mainly during embryonic development in the egg, so

that as a larva, growth occurs primarily by the enlargement of the cell

volume without an increase in cell numbers (74). Short bursts of

mitotic activity occur before molting and in some insects in the later

stages of pupation (74). Therefore, the adult insect owes its radiation

tolerance to its scarcity of mitotically active cells, with the immature

stages showing a much smaller degree of radiotolerance.




_ _


Nuclear Indicators of Radiosensitivity



Many nuclear and cytoplasmic parameters have been studied in an

attempt to locate the sites) of radiation damage in organisms (12,79).

The following nuclear parameters or indicators for determining radio-

sensitivity have been most widely used: nuclear volume, interphase

chromosome volume, DNA content, and ploidy (12).



Plant Nuclear Parameters


Plants, rather than animals, have been used most extensively as

test systems for many of the studies on nuclear parameters which affect

radiosensitivity (12). This is mostly due to the availability of plants

with wide ranges of meristematic nuclear volumes, chromosome numbers,

and ploidy (12). It can be presumed that similar factors are controlling

the radiosensitivity in both plants and animals (12).

The radiosensitivity of many species of plants has been determined

by Sparrow and co-workers (12,79,91-95). Using a regression analysis,

Sparrow has found marked correlations in plants between cellular

sensitivity based on LD50 and nuclear parameters such as nuclear

volume, interphase chromosome volume ( Nuclear volume ), chromosome
Chromosome number
numbers, and DNA content. These factors indicated that the nucleus was

a major site of radiation injury. Cromroy (25) has presented an

excellent review of the research dealing with nuclear indicators of

radiosensitivity in plants.


14








Animal Nuclear Parameters


Mammalian radiation sensitivity studies have dealt primarily with

cellular factors (7,25-28, 33,34). Bond and Sugahara (7) have presented

an excellent review of the literature concerning radiation LD50 doses

for numerous mammalian species.

Cromroy and co-workers (25-29) have used the nuclear volume and

interphase chromosome volume of the columnar epithelial cells of the

duodenal intestinal mucosa as a biological indicator to predict an

LD 5/30 day radiation dose. Their research on 11 species of mammals

indicated that the larger the interphase chromosome volume the more

resistant the mammal was to ionizing radiation, and consequently the

higher its LD50 This showed an inverse relationship to that estab-
50
lished for plants. A similar experiment relating interphase chromosome

volume to mean survival time was reported by Dunaway, et al. (33,34).

Dunaway,et al. (33) have also suggested a possible correlation between

the red blood cell count divided by the diameter of the intestine (2wr)

and LD50/30 day radiation dose as a rough predictor of mammalian radio-

sensitivity.

A relationship has been demonstrated between the nuclear volume

and the interphase chromosome volume of liver cells for three species

of amphibians to their LDS0 radiation dose (7). Their results

indicated a linear relationship between the interphase chromosome volume

and the LDs parameter.

Schubert (87) has reported several impressive correlations between

the LD radiation dose and the DNA content of mammals. Relationships
50
have also been shown to exist between sensitivity to ionizing radiation








and DNA content in avian, and yeast cells (79). In addition, nuclear

parameters such as nucleoli numbers, amount and distribution of

heterochromatin, and the location of the centromeres on chromosomes

have been correlated with the radiosensitivity of other organisms (12).



Insect Nuclear Parameters


Nuclear parameters have also been used in the determination of

insect radiosensitivity (12,25-29,61,79,91-95). A classical series of

experiments which demonstrated nuclear radiosensitivity in insects was

done by von Borstel and Rogers (8) and Whitney (115), who used the eggs

of the wasp Habrobracon. By separately irradiating the characteristically

polar nucleus and the cytoplasm of the wasp eggs, they were able to

determine the relative radiosensitivities of the two areas. These

comparative cytoplasmic and nuclear studies showed that significantly

lower doses were able to inactivate the nucleus when compared to the

cytoplasm.

The research of Brown and Nelson-Rees (11) on the mealybug

(Planococcus citri) further substantiated the importance of nuclear

damage, with specific reference to the role of the chromosomes. Their

research indicated that the genetic material in the nucleus was the

primary site of the fatal lesion.

The genome number (number of sets of chromosomes per cell) has

been extensively studied by Clark and Rubin (18) as a factor influencing

the radiosensitivity of insects. Based on a life-shortening parameter

for Hymenoptera, they found that haploid males which were produced

from unfertilized eggs were more radiosensitive than diploid females

which were produced from fertilized eggs. This was called the

phenomenon of haplo-diploidy.








Cromroy and co-workers (28,29) made an extensive study on 21

species of insects using the interphase chromosome volume of the

midgut epithelial cells as a biological indicator of radiosensitivity.

The results were similar to plants and indicated that the larger the

interphase chromosome volume the more sensitive the insect species

was to ionizing radiation.

Levy and Cromroy (61) have also attempted to relate the nuclear

volume of the ovarian nurse cells to the radiation dose required to

induce sterility in blood-feeding Dipterans.

Several cytogenetic studies have shovn the existence of unique

chromosome structures in different orders of insects and have indicated

a possible correlation between the location of the centromere and

radiosensitivity (28). Cromroy (28) has stated that the rate of DNA

turnover in the midgut of insects may be another factor to consider.



Cytoplasmic Indicators of Radiosensitivity



Animal Cytoplasmic Parameters


Numerous authors (12,79) have indicated the significance of certain

cytoplasmic characteristics in determining the relative radiosensitivities

of organisms. Duryee (35) determined that cytoplasmic factors were

correlated to the radiation-induced lethality of cells. His classical

transfer studies with amphibian eggs indicated that the irradiated

cytoplasm played an important role in the production of nuclear damage.

Research on the irradiation of Arabacia sperm and eggs (79) has

indicated the presence of certain cytoplasmic repair mechanisms.








In this study, repair of the radiation-damaged sperm only occurred

inside the egg cytoplasm.

Further evidence for cytoplasmic repair has been supplied by

experiments on multinucleated amoebae (12,79). These transplantation

experiments indicated that the cytoplasm, specifically the mitochondria,

was correlated to radiation sensitivity.

Additional research has indicated that cytoplasmic membranes may

be involved in radiation sensitivity (12,79). At high radiation doses,

membrane permeability of the mitochondria, endoplasmic reticulum, and

lysozymes has been shown to be inhibited with the resultant death of

the cell.

Specific cytoplasmic factors such as mitochondria number (79,87),

copper content (86-88), cytochrome oxidase levels (87), total cytoplasmic

cell volume (12), and the ratio of nuclear to cytoplasmic volume (12)

have also been shown to be directly related to the radiosensitivity

of mammalian species.



Insect Cytoplasmic Parameters


Very little research has been done on cytoplasmic indicators of

radiosensitivity in insects. Research similar to the Habrobracon

experiments was performed by Nakao (70) who irradiated silkworm eggs.

His work indicated a dose effect on the paternal chromosomes in the

egg cytoplasm.

Schubert (86,87) and Schubert and Westfast (88) have proposed an

interesting theory for determining the radiosensitivity of multicellular

organisms, including insects. In his Cu (I, II) peroxy theory Schubert








suggested that there should be a correlation between the radiosensitivity

of aerobic multicellular organisms to acutely lethal doses of radiation

and their total body copper content. The mechanism of damage is as

follows: Ionizing radiation produces organic peroxides which inhibit

the oxidative processes in the cytoplasm. This cytoplasmic effect is

assumed to involve an interference with the ability of the copper

oxidase enzymes from interacting with molecular oxygen due to the

radiation-induced oxidation of the cuprous component. Adverse reactions

in the cytoplasm then interact with the radiation-induced nuclear

damage to produce lethal effects.



Non-Cellular Parameters of Radiosensitivity



Although the reviewed results remain inconclusive, the effect

of radiation upon mammals, as well as insects, has been related to

age, stage of development, irradiation time, nutritional status,

oxygen tension, life-span, temperature (74), moisture content

(12,79), weight (28,791 phylogenetic classification, physical

activity (28), concentrations of protective agents, size, sex, and

specific target organs (79).



Interactions of Ionizing Radiation

in Biological Systems



Ionizing radiation may be produced by alpha particles or helium

nuclei, beta particles or high-speed electrons, neutrons, and

electromagnetic gamma or short-wave x-rays. Alpha particles, which









are too highly charged to penetrate tissues, and neutrons, which,

although extremely penetrating, have been shown to produce undesirable

chemical reactions in most biological substances. Neither has proven

useful for insect control (69). High-energy beta particles have been

used experimentally to kill powder post beetles in wood, confused

flour beetles in flour, and codling moth and potato tuberworm larvae in

plant tissues, but penetration was limited (69). Gamma and x-

irradiations are extremely penetrating but produce few ionizations per

centimeter of track, and insects are surprisingly resistant (69).

Such radiation can be produced from cobal -60 or from waste fission

products such as cesium-137, etc. (69).

Radiations produce their effects principally by the process of

ionization (the ejection of outer orbital electrons from atoms)

(12,79). Gamma and x-irradiation lose or transfer their energy in

matter by photoelectric absorption and compton scattering (12,79),

both of which result in the ejection of an orbital electron or by pair

production (12,79) which involves a change of the photon energy into

mass (a positive and a negative electron are formed).



Free-Radical Mechanism of Radiation Damage



Biological systems contain about 70-80% water. When their cells

and tissues are irradiated most of the energy will be transferred to

the water due to the greater number of target molecules encountered, in

contrast to the relatively few solute molecules. Chemical changes can

then occur if the energy from the ionizing ray or particle is trans-

ferred from the water to the important molecules within a cell (79).








Pizzarello and Witcofski (79, p. 85) have stated that "it is

obvious with respect to changes brought about in the molecules of which

cellular constituents are composed, that the interaction of ionizing

radiation and water, and the chemistry (the reactions possible) of

irradiated water, will be very important. The degree of change brought

about by radiation, in the constituents of the cell depend upon that

brought about in the molecules of which the cell is composed. The number

of abberations, for example, in the structure or function of chromosomes,

of mitochondria, or any organelle, depends upon how many molecules

within the )rganelle have been changed. The changes in these molecules

following irradiation are dependent primarily upon their interaction

with the products of the irradiated water, and, to a lesser but by no

means insignificant extent, upon the direct interaction of the molecules

with radiation." Therefore, the radiochemistry of water can be used tc

illustrate some of the generalizations for the possible interactions

of ionizing radiation in biological systems.

The interaction of ionizing radiation with biological systems

forms free radicals by the dissociation of water (12,79,85,112). The

following processes are involved in the initial interaction of

ionizing radiation with water (3,60): (e = electron; = unpaired

electron of free radical)


Excitation
ionizing
HOH radiation' H + OH . . . . . 1

Ionization
ionizing
HOH radiation (HOH)+ + e ......... 2









Followed by
H+ + e + H . . . . . . 3

or

HO + e H + OH ........... 4


and possibly
(HOH)+ H+ + OH ............ 5

Combination can result in the following postulated reactions (3,60):

H + OH HOH ............ 6

H + H --> H2, ............ 7

OH+ OH H2 ' ' '
OH + OH H20 ............ 8

or

*OH + OH ----- H20 + 0 0 .......... 9

and

O + 0 2 0 .......... 10

and with the presence of dissolved oxygen

*OH + OH + 0 H202 + 0 + 0 ..... 11

H + 0 OH 2 .H .......... 12

and possibly

0OH + 0OH H +0 + 0 ....... 13
2 2 22 2


Reactions 6 and 7 remove reducing agents while 8-13 produce

oxidizing agents. These oxidizing agents or the OH radical itself can

oxidize inorganic ions and/or organic compounds that are present in the

aqueous biological system (60).

Free radicals may interact with inorganic ions and produce changes

(12). With inorganic solutions, the primary reactions are reduction of

cations and anions by H radicals and oxidation of cations by OH -








and HO2 radicals and by H202. The general reactions for cations

(12) are: (C = cation; N = charge)


C+N + H C+(N-I) + H+

and/or

C+N + OH C+(N+I) + OH


The general reactions for anions (12) are: (A = anion; N = charge)


A-N + OH A-(N-l) + OH

and/or

A-N A-(N+1) + H+


In the presence of 02, the H radicals will be converted to

HO2 radicals and will oxidize rather than reduce materials (12).

Therefore, the net effect in a system containing oxygen is oxidation

of molecules. The extent of the reaction will depend upon the concen-

tration of the solute, the amount of oxygen in the solution, and the

presence of additional materials in solution (12). Since most inorganic

molecules are small, the indirect effect from ionizing radiation will

predominate (12).

Free radicals may also interact with the organic molecules in

cells and tissues and alter them (79). This can be illustrated by the

following reactions (79): (R = organic compound; = unpaired electron

of free radical)


HO + RH -- R + H20

and/or


RO (organic free radical) + H20 .


RH + HO *2









A drastic change in the chemistry of metabolism of the cell would

be expected if Rlwerean organic molecule essential to normal cellular

metabolism (79). In addition, H 02 could act as a metabolic inhibitor

if present in sufficient concentration (79).

It can generally be stated that the biological effects of ionizing

radiation on an organism will represent the efforts of that organism

to cope with the energy transferred to it after an interaction of

one of their atoms with an ionizing ray or particle. For any living

organism, this energy will be greater (79) than the system's normal

requirement for homeostasis and therefore will cause the standard

energy relationships to become unstable.



Animal and Plant Minerals



New methods for trace metal isolation and analysis have enabled

researchers to demonstrate the presence of a large number of elements

in plants and mammals (16). A number of these metals have been known

for several decades to possess biological activity, but the vast majority

have only recently received attention.

Elements are usually classified as essential or nonessential

although some metals such as Fe have been known to be considered in

both (16). Those present in mammals have been divided between macro and

trace elements (16). This division is somewhat arbitrary and would

depend on the sensitivity of the analytical methods used to detect the

minerals.

One of the most reliable techniques employed in much of the pioneering

research on trace elements has been emission spectroscopy (16).







Neutron activation analysis has extended the detection limits for many

metals to a few nanograms and anodic stripping voltammetry has

permitted the detection of 10'9M concentrations of certain metals (16).

No single analytical technique has been preferred for all analysis.

This is also true for atomic absorption spectrophotometry. Some elements

are ideally suited for atomic absorption analysis because of the high

sensitivity and freedom of interference from other elements. However,

others do not possess the required sensitivity for the analysis of

naturally occurring concentrations (16). In spite of all the analytical

advances for mineral analysis, there are still numerous cases of

disagreement as to the levels of metals present in biological tissues

(16). This has made it difficult to assign specific or even general

roles to these minerals.

Minerals do not always act alone in performing their biological

functions. There is sometimes an interelement dependence, and the

effects of one element may be dependent on the presence and concentra-

tions of another (16,20). The simultaneous actions of Na and K, as

well as other metals such as the alkaline earths, have been known to

be responsible for maintaining a proper balance in cellular metabolism

(16). These alkali metals have been said to exhibit an "ionic

strength effect" on enzyme systems containing other cations (16).

The mode of action of some minerals has been known, at least to a

certain extent. The most important role of minerals has been experi-

mentally shown to be as activators or deactivators of specific enzyme

systems (16,20,89,90). Some were also shown to be non-enzymatic in

function, either in addition to or instead of being enzyme-oriented

(16,20,89,90). Although many minerals have been shown to possess









in vitro activity toward enzyme or other systems, no known in vivo

activity or essentiality has been demonstrated (16).



The Significance of Minerals in Enzyme Systems


The entire physiology of an organism has been known to be dependent

on the complex biochemical changes which occur in their cells (89).

The large amounts of biochemical reactions occurring at the cellular

level are regulated by the 2,000 plus enzymes present in each cell (89).

These enzymes have been experimentally shown to be regulated or controlled

by one or more cations, especially the metallic ones (89).

SchUtte (89) has discussed the roles played by metallic ions in

living cells and has emphasized the fact that they form a cationicc

climate" in ihich interactions take place. He has also pointed out

the competition for specific sites on enzymes and substates which take

place at the sub-cellular level, and how a preferential absorption of

certain elements can either inhibit or induce enzyme activity, and,

therefore, alter the entire biochemistry of the cell or organism.

The numerous and complex ways in which metallic ions can influence

cellular metabolism were stated by Schutte (89, p. 33-34):

. around all the macromolecular structures in
the living cell hovers a cloud of metal ions, jostling
for position on the surface of the large molecules and
according to their numbers and characters, in the case
of enzymes, helping or hindering the movement of
molecules -- substates or products -- to and fro.

The wealth of living forms is reflected at the
cellular and sub-cellular level by a vast number of
possible molecular interactions, among which the
relatively indestructible metal ions appear to have
been exploited fully in a directive capacity quickening
or slowing the rate of structural change of the more
evanescent carbon compounds, helping to provide these
metabolic bridges and feed-backs.








Non-Enzymatic Functions of Minerals


The most important role of metallic ions is obviously in controlling

many enzyme systems, although these elements can also function in other

ways. Metal ions have been shown to function as catalysts without

being associated with enzyme systems (16,20,89,90). In addition,

metallic ions have been shown to be capable of catalyzing reactions in

organic systems, and are definitely known to exist in living tissue,

but there is virtually no available information to show the extent

these catalytic reactions take place in living cells (16,20,89,90).

The catalytic properties of metallic ions have been shown to be

increased and made more specific when they were bound to certain

proteins, specifically the metallo-enzyme complexes (89). However,

these ions were shown to be capable of catalyzing the reactions by

themselves (89).

The following general discussion on tne importance and distribution

of mineral cations in plants and animals will be based entirely on

the 5 elements studied in this dissertation.



Iron


Fe was the first trace or minor element recognized as being

necessary for both plants and animals (16). Most of the Fe in mammalian

systems has been found in complex form bound to proteins, either as

porphyrin or heme compounds, especially hemoglobin of blood erythrocytes

or myoglobin of muscles (16,20,90). The remaining concentration was

shown to be stored in the liver, parts of the cytochrome and catalase









enzyme systems, and in low concentrations in blood plasma where it was

bound to transferring (16,20,90). Free, inorganic Fe was negligible (16).

Among organs and tissues of the body, the liver and spleen usually

rank highest in Fe content, followed by the kidney, heart, skeletal

muscles, pancreas, and brain (16,20,90).



Copper


Cu was first identified in biological material by Bucholtz in 1816

and Meissner in 1817 who found it in plants (20). It was found to be

a normal constituent of human organs by Devergie in 1840 (16). Since

then scientists and physiologists have been investigating its function

in brain, kidney, liver, heart, blood, and other tissues, and have founj

it to play a complex role in many body functions (16,20,90). So far

as is known, Cu can be found in every form of animal or plant life in

minute amounts (16,20).

In man, Cu has been found to be equally divided in blood between

cells and plasma (16,20,90). Investigations of the Cu content in the

organs of several animals have shown that the metal was located in all

parts of the animals, but mainly concentrated in the brain, kidney, liver,

and heart (16,20,90).

A variety of Cu-protein compounds have been isolated from plant

and animal tissues, several of which were enzymes with oxidative

functions. Tyrosinase, laccase, uricase, cytochrome oxidase, ascorbic

acid oxidase, and others, are believed to be Cu compounds (16). Cu,

in close association with Fe, has been shown to have an important

function in the formation of hemoglobin in red blood corpuscles (16,29,90).








Cu has also been found to be a normal constituent of hemocyanin, the

respiratory pigment in the blood of certain marine invertebrates,

which serves the same function as hemoglobin in mammals (16,20,38,64).



Magnesium


Mg has been known to be a component in the porphyrin moiety of

chlorophyll (16) and therefore would be important in the process of

photosynthesis in green plants. Since the discovery that Mg was

responsible for activating the enzyme, alkaline phosphatase, other

researchers have shown that dozens of other enzymes require Mg, at least

in vitro (16). These included phosphatases, oxidation-reduction

enzymes, kinases, synthesizing enzymes, phosphorylating enzymes, and

dehydration enzymes. There is also the probability that Mg is bound

to the phosphate groups of many of these enzymes (16).

Mg, as v.ell as K, was shown to be concentrated in the intracellular

spaces of soft tissue and has been found in cell mitochondria where

many enzymes occur (16,90).



Sodium


Na is considered to be the major cation of extracellular fluids,

its intracellular concentration being very low (16,20,90). No one

specific function has been attributed to Na (16). It has been known

to be essential for the proper functioning of mammalian tissue (16,

20,90). The different concentration between the extra- and intra-

cellular fluids has been shown to be important in maintaining the

electrical potential of muscles (16,20,90). Na is also a primary








determinant of body fluid osmolarity and is therefore involved in the

regulation of fluid volume (16,20,90). The presence of Na in cell

nuclei and in mitochondria has indicated that many enzyme systems

function in a Na environment (16,20).

Na has also been found in plants and has been shown to be

important for proper tissue functioning (16,20).



Potassium


K has been known to play an essential role in animal and plant

metabolism (16,20). K is classified as the major intracellular base

ion and has been shown to be an activator of specific enzymes (16,20,90).

It therefore plays a predominantly intracellular role which contrasts

with the distribution of Na.

K has been shown to have an important function in the enzymatic

breakdown of glucose (16,20). It was shown to activate the enzymatic

catalysis of the phosphorylation of sugar and the enzyme, pyruvic

phosphoferase, which is important in the formation of ATP (16,20).

Enzymes of the Krebs cycle are known to exist as complexes associated

with the mitochondria (16,20). K which is required by the cell

mitochondria for the production of ATP in the Krebs cycle, has been

shown to be bound to these cellular structures (16). Na has been

shown to play an antagonistic role to K and inhibits the enzymes

activated by K (16,20), that is, there is a balance between the two

ions.

Various pathological processes have been known to involve the

loss of cellular K (16,20,90). The accompanying transfer of Na from








the extracellular fluid in the cell could lead to disruption or

inhibition of the enzymatic machinery.

The rate of amino acid utilization for protein regeneration has

also been shown to depend on cellular stores of K (16,20,90). K has

also been demonstrated as an important mineral for proper muscle

functioning. Energy production of muscle cells and the mechanism of

contraction were functions shown to be related to the intracellular

K concentration in the extracellular fluids bathing the muscle cells

(16,20,90).

There has been no evidence to prove that K is stored (16.) In

addition, the ion has been shown to be involved in the acid-base

equilibria in the body (16,20,90).

Although the function of K in plants is not known, it has been

shown to be absolutely essential for proper growth, and appears to be

involved in the synthesis of proteins and carbohydrates (16,20).



Insect Minerals



A number of different minerals, some only as traces, are consumed

and are present in the tissues of an insect. A dietary source of macro

(Mg, Na, K, etc.) and trace or micro (Fe, Cu, etc.) inorganic ions

has been shown to be essential for insects, but relatively little

work on salt requirements has been carried out because traces of

elements are often present as impurities in other dietary factors

(2,39). The balance between the respective components of a diet is

of primary importance since it relates to metabolic processes (52).









There is general agreement among researchers that the ash of

insects contains Na, Mg, Ca, Zn, Cu, Al, Si, P, As, S, Cl, Mn, Fe,

(1) and Mo (J. L. Nation, personal communication, 1971). All or some of

these minerals may influence the chemical reactions taking place in the

tissues, but it has been difficult to determine whether some of the

major and/or trace elements are utilized specifically, as well as their

active concentrations (39). Chapman (13) has stated that minerals are

important in the insect for the maintenance of an ionic balance

suitable for the activity of living cells, as co-factors of some enzyme

systems, and as integral parts of others.

Quantitative and qualitative mineral cation studies on insects

have, for the most part, been limited to the hemolymph, and the rectal

and malpighlan tubule fluids (13,38,39,117). These studies have

mainly been concerned with Mg, Na, and K due to their known importance

in maintaining a normal internal homeostasis. The wide variations in

hemolymph mineral concentration among the insect orders have also been

of interest to many researchers. Very little quantitative data is

available on whole body or specific tissue-organ mineral content for

insects.

The following discussion on insect minerals will be limited to the

S cations studied in this dissertation, although several others have

been shown to be important in insect metabolism (39,41,42).



Iron


Quantitative data on Fe content in insect tissues is scarce. The

metabolism of Fe has been described by Bowen (9,10), Waterhouse (108,109),

and Waterhouse and Day (110) for several species of insects. Their extensive









research on Lucilia cuprina larvae indicated that there was a small

zone in the center of the midgut where Fe was absorbed and that

specific cells in this area were responsible for the Fe uptake. The

accumulation of what may be thought of as reserve stores of heavy

metals in the insect midgut has been known for many other insect

species (13).

In describing the Ba and Mn metabolism of social wasps, Bowen (9)

postulated that Fe follows the same metabolic route common to all

divalent cations.

Fe has been shown to be present in t'e hemolymph of some insects,

as a structural component of hemoglobin (13) in the digestive secretions

ofPopilliajaponica larvae (51), and in the cytochromes of insects as

an Fe-porphyrin ring complex (13,41).

Audioraciographic Fe-59 studies by Poulson and Bowen (82) with

four species of Drosophila larvae have shown that some tissues contain

higher Fe concentrations in the nucleus than others. Fe-59 research

on salivary glands, midgut, malpighian tubules, and rectum have

indicated that Fe-59 molecules were not bound to the chromosomes, but

were localized in the nucleoplasm (77).

All insects are capable of synthesizing cytochromes which are

known to be essential in respiration (13). The various cytochromes

differ in the forms of their heme grouping (13,41). They are usually

present in small amounts, but in the flight muscles, the concentration

is high enough to produce a reddish-brown color (13).

Only a few insects living in conditions of low oxygen tension

have been shown to contain hemoglobin, and these were colored red by

the pigment showing through the integument (13). Hemoglobin has also








been shown to serve a respiratory function (13,41). In Chironomus

larvae the hemoglobin was found to be in solution in the blood, while

in the larvae of Gasterophilis it was concentrated in the fat body

(13).

Bilins, a linear Fe-porphyrin arrangement, are formed by the

opening out of ring porphyrins as a results of oxidation and are typically

blue or green (13). In Chironomus the bilins from the hemoglobin of the

larvae were shown to accumulate in the fat of the adult and impart a

green color to the newly emerged adult fly (13). The pericardial cells

of Rhodnius also became green when bilins derived from injested

hemoglobin were accumulated (13). Rhodnius has also been shown to

store Fe from the hemoglobin in the gut epithelium and bilins in the

nephrocytes '13), which may represent a type of storage excretion.



Copper


Comar and Bronner (20) have presented quantitative Cu analysis of

whole adult, larval, andpupal insects, as well as the hemolymph of the

Southern armyworm and the horse bottle fly. An excellent literature

review on invertebrate Cu metabolism was presented by Dethier (32).

Many of the following references were extracted from his extensive

report.

Analyses of Cu by Melvin (65),0kamoto et al. (75,76), Waterhouse

(109), and Clare (17) have shown the Cu distribution in insect tissues.

They found that the alimentary canal and malphigian tubules contained

the largest concentration of Cu while the muscles, cuticle, blood,

gonads, and foregut were less concentrated.








Histochemical studies of Cu in the sheep blowfly, Lucilia cuprina

by Waterhouse (109) have shown that in a region of the larval midgut

there were two small bands of cuprophilic or Cu-absorbing cells, lie

also showed that the malpighian tubules contained Cu and that no Cu

was present in the pericardial and ventral nephrocytes, muscles, foregut,

and food. The cuprophilic cells were later shown to contain esterases

and cytochrome oxidase, with mitochondria scattered throughout the

area (13). Results of Waterhouse's experiments (109) showed that

certain cells of the midgut were selectively concerned with absorption

of Cu and that the malpighian tubules were concerned with its elimina-

tion. Poulson (80) and Poulson and Bowen's (81) study of 40 species of

Drosophila has confirmed the presence of special Cu-absorbing cells

in the midgut of insects.

In another study, Poulson,ct al. (83) fed a Cu-64 solution to

fruit flies. The Cu was shown to accumulate in the intestinal tract

and malpighian tubules mainly in a form that could not be demonstrated

by histochemical technique. He concluded that Cu-uptake followed two

distinct pathways, one for ionic forms, and the other for bound forms.

Melvin (65) and Clare (17) have analyzed exuviae, and egg cases

of several species of insects and found appreciable concentrations of

Cu. From the results, they concluded that the insects were exhibiting

a type of storage excretion.

In another experiment, Waterhouse (109) determined the Cu content

and distribution in the developmental stages of Lucilia cuprina. He

found variations in the egg, larva, and adult stages. The insect egg

shell was found to contain the greatest concentration of Cu, with the

whole egg, newly hatched larva, and the adult with approximately the

same Cu content.









Clare (17) has found an increase in the Cu concentration in the

cuticle, muscles, and fat body when a Cu-enriched diet was fed to

Periplaneta americana and Blatella germanica. This indicated that more

Cu was absorbed into the hemolymph and distributed to these tissues.

Waterhouse (109) indicated that the Cu content was regulated when

increased levels were administered to various insects. Analysis of

Cu in insects has suggested that a large percentage of it is firmly bound

in organic combination (32). Dethier (32) has stated that at least

one-third of the total Cu concentration was in this form, specifically,

as a structural component of the enzyme, tyrosinase.

Sussman (98) has extensively reviewed the functions of tyrosinase

and found that four separate enzymes, dopaoxidase, catechol oxidase,

diphenolase, and polyphenolase, actually make up a tyrosinasee complex."

He has also postulated the occurrence of other copper-protein oxidases

in insects. Bodine and co-workers (6) have suggested that protyrosinase,

an inactive form of tyrosinase which was found in grasshopper eggs and

mealworm larvae, actually belonged to the tyrosinasee complex." Danneel

(30) has also described a dopa-phenolase-catecholase system in

Drosophila.

The freshly moulted insect cuticle is known to be initially soft

and colorless, but then rapidly darkens and hardens. Pryor (84) was

the first to prove that these changes in the ootheca of a cockroach and

the cuticle of a blowfly larva resulted from the quinone tanning of a

protein. Researchers have demonstrated that these Cu-phenols arise

from the oxidation of blood tyrosine by tyrosinase (31,40,106).

It has also been suggested that as in vertebrates there may be

some relationship between Cu and Fe metabolism, but nothing is known








of this relationship in insects (36,78). Attempts have been made to

show that tyrosinase is of major importance in insect metamorphosis

and also as a terminal oxidase in the respiration of intact insects

(32). Although Cu has been speculated to be part of a respiratory

pigment, no such pigment has been isolated from insects (78) even

though experiments have indicated that tyrosinase did increase the

rate of oxygen uptake in damaged insect tissues (98). Heller (48)

has suggested that tyrosinase oxidizes certain substrates which are

then reduced by cozymase as part of the respiratory system.

Several authors (5,21,49) have also proposed that tyrosinase in

the hemolymph and corpuscles of various insects, by acting upon the

phenolic products of protein breakdown, assist in the removal of

these toxic materials. Cu in the hemolyipn, mostly completed to

tyrosinase, h;s been quantitatively determined (32).

Excessive but sublethal amounts of Cu in the diet were shown to

induce marked morphological abnormalities in Periplaneta americana and

Blatella germanica. Clare (17) demonstrated that roach wings became

curled and more brittle than normal. In addition, the roaches, as

well as blowflies, exhibited a marked increase in water content.



Magnesium


Quantitative estimates of Mg have mainly been limited to the

insect hemolymph, where the Mg concentration has usually been found to

be relatively high (13,38). In phytophagous insects this was shown to

reflect the high level of Mg obtained in the diet, since it is a

constituent of chlorophyll (13). In many Lepidoptera larvae the Mg








concentration has been found to fall when they stop feeding, although

a fairly large concentration still occurred (13). It is a general

consensus among researchers that all insects tend to concentrate this

metal (13).

The concentration of Mg has been found to vary greatly, depending

on the insect species, and stage of development. In Phasmida, Mg has

been found to almost replace Na (13).

Adult Lepidoptera have been generally found to contain less Mg in

the hemolymph than the larvae (13). Other orders have been shown to

be roughly similar in Mg levels in both larval and adult stages, while

others have been found to resemble the Lepidoptera (13).

Several insect enzymes have been shown in vitro to require the

Mg 2+ as a co-factor for enzyme activation. The following enzymes have

been found: Trehalose-6-phosphatase, L-ketoglutarate dehydrogenase,

acetate-activating enzyme, succinic dehydrogenase, glucose-6-phosphate

dehydrogenase, 6-phosphogluconate dehydrogenase, hexokinase, isocitric

dehydrogenase, phosphofructokinase, malic enzyme, inorganic phosphatase,

oxalocetic carboxylase, phospohydrolase, L-glycero-phosphate oxidase,

luciferase, apyrase, adenosine triphosphatase, adenylate kinase and

nucleotide dephosphorylating enzymes (39,41). Mg has also been

proposed as being a structural component of adenosine triphosphatase,

making up a Mg-ATP coenzyme complex (39,41).

The products of the various metabolic reactions have been isolated

and identified by in vitro experimentation (41). Although the specific

enzymes have not been isolated, they are assumed to be present to

catalyze the formation of the products and are thought to resemble the

vertebrate enzymes in structure and function (41). There is presently








no data of value to suggest Mg 2+ activation in any other insect

enzyme system (41), although the enolase, pyruvic kinase, and 3-

phosphoglyceric-l-kinase enzymes are assumed to be the same in all

animal tissues, and,therefore, would require Mg for activation (39,41).

A model to explain the mechanism by which Mg 2+ differentially

influences chromosomal RNA synthesis has been proposed by Lezzi (62).

He has found that Mg 2+ induced, in isolated salivary gland nuclei of

two species of Chironomids, a specific enlargement of one Balbiani ring,

while Na or K did not. This specific enlargement was presumed to

indicate an increase in RNA synthesis in this chromosome region.

The digestive secretions of Popillia japonica were analyzed and

were found to contain several cations including Mg, K, Na, and Fe

(51). It was postulated that these basic cations acted as a buffer

system to neutralize the large concentration of acidic anions.

It has also been proposed that Mg was involved in the ionic

mechanism of nerve transmission (13). It has been suggested that Mg

can substitute for the extremely low concentrations of Na in many

herbivorous insects and, therefore, maintain the proper ionic balance

for neural conduction. In other insects high concentrations of Mg

were found to block neural conduction.

Mg concentration has also been shown, in vitro and in vivo, to

be important in maintaining and regulating the insect heartbeat (58).

Alone, and in combination with other ions, it was shown to cause

stimulation or paralysis of the heart muscle in several insect species.









Sodium and Potassium


Na and K will be discussed together since the effect of one is

usually dependent on the concentration of the other. Like Mg,

quantitative determinations of Na and K have mainly been limited to the

insect body fluids where they have been shown to function in regulating

internal osmolarity and acid-base equilibria (39,41). Nervous system

analysis has also favored these 2 minerals (100-105).

Na has been found to be the most abundant cation in the insect

hemolymph, but in Lepidoptera, Hymenoptera, and some Coleoptera there

were relatively small amounts and it contributed only 10% of the total

osmolar concentration (13). The absolute concentration of K was found

to be usually lower than that of Na, ranging from 2-100% of the total

osmolar concentration (13).

The Na/K ratio has been shown to vary considerably among the insect

orders. High values for the Na/K ratio (Na/K 2 40) have been found to

occur in Odonata, Orthoptera, Diptera, and some Coleoptera, while in

the phytophagous Coleoptera, Hymenoptera, and Lepidoptera the Na/K

ratio was considerably lower, even less than one (13). It has been

suggested that these last orders evolved with the angiosperms, which

contained relatively little Na, and the adaptation of the insects to a

lower Na/K ratio reduced the amount of regulation which was necessary

to maintain a fairly constant Mg concentration in the hemolymph C13).

It was shown the Na and K, as well as Mg, were absorbed from the

midgut and rectum into the hemolymph by a process of active and/or

passive absorption (13,100). The rate of absorption and final concen-

trations was found to be dependent on the concentration of anions and

the insect environment-aquatic or terrestrial. No storage of these ions

was shown to take place in the epithelium or midgut tissues.









The importance of Na and K concentration in the normal conduction

of a nerve impulse in an insect has been shown to be dependent on the

nerve cell's ability to transport these ions across its plasma membrane

(50,53,56,104).

Na and K levels have been determined in the abdominal and thoracic

ganglia of the ventral nerve chord in several Orthopterans (mainly

cockroaches and locusts) to determine the cation fluxes occurring

between the hemolymph and nervous system (100-105). These ionic changes,

as observed by changes in the Na to K ratio, were shown to be related

to the permeability of the continuous cellular and fibrous membrane

which enveloped the nervous system (73,107). This membrane, the perilemma,

was found to serve a protective function by limiting the entry of Na+

and K as well as acetylcholine molecules into the underlying nervous

tissue (104,107).

Na and K have been shown to be involved in the normal functioning of

the dorsal vessel in various insect species (58). It was shown that

regulation of the heart beat, that is, paralysis or stimulation could

be affected by changes in the Na and K concentration on the heart

muscle.

K levels in the hemolymph were also shown to affect the general

activity of locusts (13). A low concentration of K in the hemolymph

was shown to raise the muscle resting potential. As a result, the

action potential arising from the stimulation was higher and the insect

would jump farther and more often than when the K level was high. The

K concentration in the hemolymph was also found to increase substantially

before moulting and was presumed to be responsible for much of the

inactivity occurring at this time (13,55). Various concentrations of








Na and K were demonstrated to stimulate or inhibit the opening and

closing of the respiratory spiracles in several insects (54).

Several enzymes have been shown to require Na+ and/or K as a

co-factor for activation. In vitro experimentation of insect tissues

have shown that pyruvic kinase, 6-phosphogluconate dehydrogenase, and
+
glucose-6-phosphate dehydrogenase required K while glycogen phosphorylase

was found to require both Na+ and K (39,41).

It has been postulated that Na and K may be involved in the mechanisms)

of nuclear activation (39,41). Experimental evidence has suggested that

the ratio of the concentration of Na and K may determine whether

genes were active or quiet in Chironomid salivary gland chromosomes. By

stepwise alteration of this ratio it was possible to induce a series of

puffs in geim, loci in a sequence which paralleled the natural sequence

in the period from late larval to middle pupal life. This has suggested

that ecdysone and juvenile hormone may not act directly on the

chromosomes, but may exert their effects in altering the Na/K ratio of

the nucleus.

The in vivo and in vitro uptake of Na-22 from the hemolymph into

cell nuclei of larval salivary glands was measured and compared. The

uptake curves were discussed in relation to recent findings on the

induction of puffs in polytene chromosomes by inorganic ions (63).

















CHAPTER III


METHODS AND MATERIALS



Atomic Absorption Analysis



Insect Preparation


Twenty-two species of insects representing 8 orders were obtained

from coloni.; at the USDA Laboratories,l Department of Entomology and

Nematology, and the J. Hillis Miller Medical Center at the University

of Florida, Gainesville, Florida. Insects in the larval, pupal,

nymphal, and adult stages were analyzed for mineral content (see Appendix

Table 1), although only adults were incorporated into the statistical

models. Adult male and female insects were pooled together from several

independent samples to obtain a sufficient tissue weight for a mineral

analysis. In several instances, it was necessary to combine different

age groups to obtain the necessary weight.

The insects were maintained in holding cages or jars from 12-24

hours without food to assure that their guts would be voided and that

the elemental analysis would be based entirely upon tissue-organ



Insects Affecting Man and Animals Research Laboratory; Insect
Attractants, Behavior, and Basic Biology Research Laboratory.








mineral concentrations and not undigested food in the gastro-

intestinal system.

All the insects with the exception of Musca domestic and one

sample of Attagenus megatoma were heat-killed in Blue-M oven main-

tained at 80-85 C. The other two species were freeze-killed at

-18 C and stored until analysis.



Dehydration


The atomic absorption mineral analysis was based on milligrams of

element per kilogram of dry body tissue or parts per million (71). A

dry weight analysis was used to reduce the individual weight variations

caused by moisture content, and therefore assure a greater degree of

quantitative accuracy and experimental reproducibility (J. L. Nation,

personal communication, 1971).

The dehydration time varied with the insect group, size and

quantity usually being the determining factors. Time ranges were deter-

mined for the different insect orders by removing several samples from

the oven and weighing then at 12, 24, 36, 48, 60, and 72 hour intervals.

When the weight loss reached an equilibrium point (indicating complete

dehydration) the insects were removed from the oven and placed in a

drying desiccator containing silica gel and DrieriteR (CaSO4). The

samples were then weighed on a Mettler type 1115 balance placed in

labelled crucibles, and transferred to aThermolyne type 1500 furnace

to be ashed.








Ashing


The furnace temperature was slowly raised to 400-5000 C over a

4 hour period and maintained there for 12-18 hours. When the furnace

had cooled, the samples were removed and 0.5 ml 2N HNO3 was added to

each crucible. The samples were placed back into the furnace and slowly

dried at under 1000 C for one hour. The temperature was again raised

to 400-5000 C and held there for 4-5 hours to complete the ashing

procedure. Mien the oven had cooled to 700 C the samples were removed.

The appearance of a uniformly white or grey colored ash was used as

the deciding criterion as to whether the Jnsect tissue was sufficiently

ashed. If the ash appeared blotched and/or lumpy, 0.5 ml 2N HNO was

added and the samples) re-ashed for 3-4 hours at 4000 C.

Solution of the ash was effected by adding 1.0 ml lN HC1 to each

crucible. Solutions of the ash for samples of greater than 0.5 g dry

body weight ;ere made up to a volume of 10.0 ml with deionized water.

A final ash volume of 5.0 ml was used when the dry sample material

weighed less than 0.5 g. Volumetric flasks and pipettes were used in

preparing the standards and samples for a mineral analysis.

In a few instances the ash did not completely go into solution with

the addition of the 1.0 ml 1N HC1 even though the external appearance

seemed uniformly grey or white. When this occurred, the HC1 was

evaporated in an oven at 800 C, 0.5 ml 2N HNO3 added, and the samples

re-ashed for 2-3 hours at 300-4000 C.








Mineral Determination


A Techtron model AA-100 Atomic Absorption Spectrophotometer

(see Figure 4) was used in all mineral determinations with air-

acetylene gas mixtures. The standard machine settings for the wave-

length, gain, lamp position, lamp current, and air and acetylene flow

rate (see Figures 5 and 6) varied for each mineral to be determined.

The internal arrangement of the machine is also shown in Figure 6.

The ideal ranges of concentrations for determining the various elements

in parts per million were as follows: K, 1-8; Na, .2-.8; Mg, .2-.8;

Cu, 1-8; and Fe, 1-8 (71).

A series of three to four working standards per mineral were

prepared each day prior to an analysis frwia Fisher Chemical Company

1000 ppm shelf standards. The stock solutions were prepared August,

1970, and refrigerated until use (J. L. Nation, personal communication,

1971). Except for Cu, the standard stock solutions were prepared in

deionized water (71). Because Cu was determined in undiluted samples,

standards were prepared in 0.1 N HC1 (71). The standards were graded

to correspond to the ideal absorption range for each mineral. A

dilution range for sample determination was 1:200 1:500 for Mg, K,

and Na, and 1:5 1:10 for Fe. No dilution was used for Cu. Three

determinations were made for each mineral in the sequence: standards-

samples-standards-samples-standards-samples (71). After each deter-

mination of a standard or sample, deionized water was aspirated through

the sample intake valve to verify a return of the meter reading to

100% transmission, and adjustment made when necessary (71).


































4
C)

0

0

0

4O
U)
r)
0
0
0,

0




-'-
4-)









0
*H




O




41)


0








to
+-C




48











O
I-
0
I-
0










I-








O
0



z

















Lu





































Figure 5. Instrument settings of Techtron Type AA-100
Atomic Absorption Spectrophotometer (57).






































LAMP CURRENT, LAMP POSITIONING AND COARSE GAIN CONTROLS.


GAS CONTROLS, NEBULIZER AND BURNER ADJUST.




































Figure 5. Instrument settings of Techtron Type AA-100
Atomic Absorption Spectrophotometer (57).








































LAMP CURRENT, LAMP POSITIONING AND COARSE GAIN CONTROLS.


GAS CONTROLS, NEBULIZER AND BURNER ADJUST.


. .. A








Results were either recorded on a Varicord model 43 chart recorder

or read directly from the meter. After completing a series of five

mineral determinations, the mean absorbance for each standard was

graphically plotted against its corresponding concentration in parts per

million (71). The parts per million of the specific mineral in the

sample were then read directly off the corresponding graph. Graphic

plots of absorbances over the standard ranges were linear for Fe, Cu,

and K, and slightly curvilinear for the Mg and Na. All glassware was

cleaned in a dichromate-sulfuric acid solution and rinsed in deionized

water (71).

The total body mineral concentration for each insect species was

calculated with the following formula:



S parts per million of
Insect total body sample x specific mineral
mineral content in in solution
parts per million = milligram dry body x 1000
weight of insect tissue



These values were then used in the final correlation analysis.



Radiation and LD Procedure
50


Mixed groups of adult male and female insects were irradiated at

the Insects Affecting Man and Animals USDA Laboratory with a cobalt-

60 source (see Figure 7) at a dose rate of 1200 R/min, or with a 300

KVP Maxitron x-ray therapy unit with 1 nun of aluminum filtration at a

dose rate of 2.16 kR/min, to obtain an LD50/24 hour dose for a species

population (25-29). Three replications per point were obtained for







































Figure 7. Cobalt-60 insect irradiator.








1


A


-__


.....-- --









verification of the LD50 dose. Insects were maintained under maximal

survival conditions after irradiation and mortality counts made at

24 hours.

The LD50 data for bedbugs, bodylice, houseflies, and silver fish

was taken from Cole, et al. (19), who used a cobalt-60 source with a

dose rate of 1000 R/min. The LD50/28 day and mean mortality doses were

extrapolated from the radiation literature (19,22,23,67,68,72,99).



Data Analysis



An IBM 360 computer was used to devise simple and multiple

regression statistical models using the method of least squares for

analysis. The computer programs were formulated using APL language by

the Department of Statistics, University o- Florida, Gainesville,

Florida. By manipulating the mineral data, it was possible to obtain

several linear models that showed an excellent correlation between

insect mineral content and radiation sensitivity as defined by our

LD50/24 hour, LD50/28 hour day, and LD50/mean mortality dose parameters.

To test the strength of the relationships, R and r-coefficients,

as well as "t" and F-statistics were calculated (66). The standard

error for each predicted or expected LD50 was also determined (66).

Curvilinearity in the models was also tested (J. A. Cornell, personal

communication, 1971). Due to the small samples and great variations

across insect orders in the models, a significance level of at least

.10 was used to indicate an adequate correlation between mineral content

and insect radiation sensitivity.


















CHAPTER IV


RESULTS AND DISCUSSION



Statistical Methodology



Several statistical models were formulated which showed a direct

and/or indirect relationship between insect mineral cation concen-

tration and radiation sensitivity as defined by one of the parameters

selected.

The insect total body Fe, Cu, Mg, Na, and K were obtained by

atomic absorption spectrophotometric analysis of the insect tissues.

Linear models were formulated by computer manipulation of the five

minerals in the form of ratios and/or whole numbers. No model was

observed to be curvilinear in response. The statistical methodology

is presented initially so that the results will be meaningful to the

reader.

The data was analyzed by the method of least squares and

resulted in the formulation of simple and multiple regression equations

or models which would serve to predict a species-specific radiation

dose when the mineral content of that species was known. Mineral cation

content would then serve as an effective biological indicator of insect

radiation sensitivity.








Simple regression analysis of the data was based on the slope

equation for a straight line, y = a(x) + b (26), where y = expected

or predicted LD50 radiation dose in rads or Roentgens, a = slope,

x = mineral content in parts per million (ppm), and b = intercept.

The coefficient of correlation, r, was calculated with each

simple regression model to indicate the strength of the linear relation-

ship between the two variables, y and x (66). When the coefficient of

correlation was used, a value of zero would indicate no correlation at

all, while the limiting values +1 or -1 would indicate either a perfect

positive or negative correlation between y and x.

A positive correlation indicated by a +r would show that the

higher the concentration of a specific mineral or ratio of minerals as

defined by x in the simple regression equation, the greater the radiation

tolerance of an insect species. A negative correlation, -r, for a

simple regression model would indicate that the greater the concen-

tration of minerals in the insect tissues, the more sensitive the insect

would be to ionizing radiation, hence, an inverse relationship.

A significance level of .05 or .10 was chosen for all regression

analyses so that comparisons could be made as to the validity of the

different models. Since the statistical models were based on small

samples, the "t"-statistic was chosen as the final criterion as to the

usefulness of a simple regression model (66). The "t"-statistic was

calculated with each simple regression formula to indicate the

probability of obtaining a valid LD50 response from a given set of

mineral data.

Multiple regression analysis represented a multi-dimensional

figure based on the linear equation, y = b0 + b (x ) + b2(x2) + b3 (x3)

+ . b (x ) (66), where y = the expected or predicted LDs0 in rads








or Roentgens, b . b = the estimated parameters (LD50 and minerals)
n 50
of the model, and xl x = total body insect mineral content in

parts per million (ppm).

The multiple regression prediction equation was based upon a number

of variables, xl, x2, x3 x and was obtained by the method of

least squares in exactly the same manner as that employed for the simple

linear models (66).

Multiple regression analysis was used to explain a "combined

effect" resulting from the interaction of several independent mineral

variables represented by xl, x2, x3, . x in the form of ratios and or
n
whole numbers. These values, when analyzed together in a single equa-

tion, could then be used to predict a species-specific radiation lethality

dose in a given time period.

The coefficient of multiple determination, R, was used to test the

slope or linear strength of each multiple regression model and was

represented as being equivalent to r. Therefore r = R for means of

comparison (J. A. Cornell, personal communication, 1971).

The F-statistic at a .05 or .10 significance level was used to

analyze the validity of a multiple regression model and to determine

the limits of its prediction capability. In some special cases there

was a degree of ambiguity between the relationship of a high linear

coefficient value, R, for a model that produced excellent LD50

predictions, and a poor F-statistic (see Tables 15, 19, 20). When this

occurred all models significant at the .05 and .10 levels as well as

models which were not significant at the .10 level were shown for

reasons of comparison.









The standard error (SE) was calculated for each predicted or

estimated LDSO in both the simple and multiple regression models to

place bounds for error, plus or minus, around each predicted radiation

dose (J. A. Cornell, personal communication, 1971).

A statistical range of error, 10-15%, should be expected in the

LD50 values experimentally obtained by x and/or gamma irradiation of

the insects, since there is an error in measuring actual radiation

exposure inherent in the ion chambers currently available (H. L. Cromroy,

personal communication, 1971).

The major problem encountered was the use of models which

consisted of non-representative numbers of insect species. The results

could be misleading or misinterpreted if one did not consider the following

relationships between the test statistics (J. A. Cornell, personal

communication, 1971):



(1) t = rf

(2) F = t2


(3) t = [n-2] r
IJ-r2


where t = "t"-statistic for a simple regression model, N = number of

species in a model, n-2 = degrees of freedom, and r = coefficient of

correlation;



(4) F = [N (K+1)] R2
K (1-R2)

where F = F-statistic for a multiple regression model, N = number of

species in a model, K = number of parameters (mineral variables) in a

model, N (K+l) and K = degrees of freedom.








(5) Null hypothesis (I0): a = 0 (slope = 0)
Alternative hypothesis (H ): a / 0

for simple regression analysis by
t-test at the .05 or .10 level


(6) Null hypothesis (HO): b . bn = 0
Alternative hypothesis (H ): b . / 0
an
for multiple regression analysis
by F-test at the .05 or .10 level


Acceptance of H0 is in effect the rejection of the predictor

equation based on the test statistic. Rejection of Ho is acceptance

of H1 and therefore is a measure of the validity of the predictor
a
equation.

By studying these formulae, one can better understand the relation-

ship between the test statistics and therefore understand the validity

or inadequac, of a model (see Tables 15, 19, 20). In most cases this

was seen to be related to the number of species and/or number of mineral

parameters in a model.



LD50/24 Hour Predictor Models



Nineteen species representing Anoplura, Diptera, Coleoptera,

Hemiptera, Homoptera, Hymenoptera, and Orthoptera were available for

deriving a model based on a 24 hour radiation exposure. The 24 hour x

and/or gamma radiation doses, as well as the total body mineral content,

were calculated for each species and a correlation analysis was performed

to test the relationship between insect mineral content and radiation

sensitivity.








The primary objective was to derive a single equation that could

be used as an accurate predictor or biological indicator of radiation

sensitivity across the insect orders, once the mineral content for a

particular species was known.

The predicted LD50/24 hour radiation doses were based on the

calculated slope or linear regression formula, y = 2.2147E12 (x) +

112.0065, where x = Cu/(Na x Mg)2 in parts per million (see Table 1).

Although the "t"-statistic for the 19 insect species was significant,

the slope equation did not account for as much variation between

species as expected.

Additional analyses were performed on the 19 species in an attempt

to improve on the prediction capability of the simple regression model.

Multiple regression analysis of the data revealed the predictor

equation, y = b0 + b1 (Na) + b2 (Cu/(Na x Mig) 2) + b3 (K/Cu) + bq (Cu/Fe),

where b = 68.4260, b = 0.0061, b = 3.4360E12, b = 0.0689, and
0 1 2 3
b = -109.9400 (see Table 2).

The results of the simple and multiple regression models failed

to account for the large species variation between the observed and

expected LD50 radiation doses. In an attempt to reduce the variation,

the 19 species were sub-divided into categories based on feeding

preference, since it is known that minerals are ingested and accumulated

in the insect tissues in relation to their diet (39,52).

The following sub-divisions were chosen: (a) stored-product feeders,

(b) organic-plant feeders, and (c) blood-feeders. The individual insects

in the 19 species model that pertained to a specific feeding group were

then separated and analyzed under one of the above sub-divisions.




63







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0l 90 mo 0~0 E U
SCM o o oz 9- r- 0 0
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v v U iC 0 L \0 E HM M Z- t- ) a l E A
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CH C:( E *H 1 D
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3 -H P0 n3 Ue rt (U 3C CI

C) U IC IC -H U +J I) IC 4 -) r C r +
IC O IICI IC cE njl IC IC


*rU U -I P-I Ha rtIC1- 4 CU 4


P.. cH H C) C CiC IC ^ H C:H
4. ) i -Ei U I L O U H 0U E

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CO0E-i E-.







Stored-Product Feeders


Analysis of six species of Coleoptera, which were reared on grain

and/or flour, revealed a great improvement in the predicted LD5 doses

when compared to the values obtained in the 19 species model.

A simple linear model, y = 244.5774 (Cu/Fe) + 172.0181, was

derived (see Table 3). Multiple regression analysis of the six stored-

product insects produced two models for predicting a 24 hour radiation

lethality response based on mineral content. The following two linear

equations were derived:


(1) y = b + b (Mg) + b (Cu/K) + b3 (Cu/Fe), where

b = 424.5906, b = -0.3263, b = -46506.5039, and b = 589.8412
0 1 2 3
(Table 4);


(2) y = b + b (Cu) + b (Cu/Fe) + b (Mg/K), where

b = 308.3822, b = -7.0798, b 687.3172, and b = -1229.1410
0 1 2 3
(Table 5).




Organic-Plant Feeders


Eight species of laboratory-reared insects, which fed on organic

or decayed plant matter, were analyzed to test the validity of the diet-

based sub-division. Only one true plant-feeder was presented in the

models due to a lack of LD50 data as well as a lack of sufficient tissue

weight to represent this group. The simple and multiple regression

models were based on Orthoptera, mainly cockroaches, although Diptera

and Thysanura were also represented.













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The method of least squares produced four simple regression

equations which showed a high degree of linear correlation between

insect mineral content and insect radiation sensitivity. The following

four formulae and associated statistics were derived:


(1) y = -27456.8938 (Cu/K) + 152.671 (Table 6),

(2) y = -6509.5691 (Cu/Na) + 158.6133 (Table 7),

(3) y = 0.0054 (K) + 25.8061 (Table 8),

(4) y = 0.0166 (Na) + 51.3646 (Table 9).


Results obtained by multiple regression analysis showed a marked

improvement over the prediction capability of the simple regression

models. Two predictor equations were derived which indicated a strong

relationship between mineral content and insect radiation sensitivity.

The following are the equations that were formulated:


(1) y = bg + b1 (Na) + b2 (Cu/K), where

b = 110.2092, b = 0.0087, and b = -18940.1062 (Table 10);


(2) y = bg + b1 (K) + b2 (Cu/Na), where

b = 92.6478, b = 0.0031, and b = -3925.0502 (Table 11).
0 1 2




Blood-Feeders


The final sub-division based on feeding preference was the blood-

feeders. Since the mineral content of one species of Anoplura was

obtained from a mixed sample of nymphs and adults, it was decided that

separation of these two stages would be advisable in the final correlation

analysis. The result was a model based on five species which represented






















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three orders: Anoplura, Diptera, and Hemiptera. The following two

models were obtained by simple regression analysis:


(1) y = -30.6599 (Mg/Na) + 171.5961 (Table 12);

(2) y = -0.0147 (Mg) + 172.2073 (Table 13).


Analysis based on a combination of minerals in the form of

fractions and whole numbers produced two multiple regression formulas

that could be used to predict a species-specific LD50/24 hour radiation

dose. The following multiple regression equations or models were

formulated:


(1) y= b + bI (Cu) + b2 (Fe) + b (Mg/Na), where

b = 224.1558, b = -1.2260, b = -0.0467, and b = -32.4359
0 1 2 3
(Table 14);


(2) y = b + b (Fe) + b (Mg/Na), where

b = 187.2418, b = -0.0251, b = -37.9907 (Table 15).
0 1 2




LD50/28 Day Predictor Models



Seven species of adult insects representing two orders, Coleoptera

and Orthoptera, were analyzed by simple and multiple regression analysis

to determine if the total body mineral content for an insect species was

related to an LD50/28 day radiation mortality dose.

Correlation analysis of the seven species resulted in the following

two simple regression predictor equations:



















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(1) y = -0.0024 (Mg + Na) + 11.4310 (Table 16);

(2) y = -0.3593 ((Fe x Na)/K) + 10.1094 (Table 17).


Multiple regression analysis of the LD50/28 day data resulted in

the formulation of three models. The prediction capability of these

models for determining an LD 50/28 day radiation dose was shown to be

superior to those obtained from the simple regression models. The

following models were derived:


(1) y = bg + bi (Cu/K) + b2 (Mg + Na)

b = 39.5350, b = -3009.0000, b
0 i 2
(Table 18);


(2) y = b + b (Cu/K) + b (Mg + Na)
0 1 2
b ((Fe x Na)/K), where
4
b = 47.182, b = -7523.900, b =
0 1 2
an, b = 1.3975 (Table 19);
4

(3) y = b + b (Cu/K) + b (Mg + Na)
0 1 2
b ((Na + Mg)/K), where

b = 43.6770, b = -5403.3000, b
and b = 22.6400 2
and b = 22.6400 (Table 20).
4


+ b3 (Fe x Na), where

= -0.0161, and b = 0.0001
3



+ b (Fe x Na) +
3


-0.0173, b = 2.2863 E-5,
3



+ b (Fe x Na) +



= -0.0180, b = 0.0001,
3


LD50/Mean-Mortality Predictor Models


Simple and multiple regression models were derived for determining

an LD50 radiation dose based on the mean-mortality (mean-life expectancy)

for eight species of laboratory-reared insects. Insects representing the





82


















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