Group Title: genetic study of apholate-resistance in Aedes aegypti (L.)
Title: A Genetic study of apholate-resistance in Aedes aegypti (L.)
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 Material Information
Title: A Genetic study of apholate-resistance in Aedes aegypti (L.)
Physical Description: 86 leaves : ill. ; 28 cm.
Language: English
Creator: Seawright, Jack Arlyn, 1941-
Publication Date: 1969
Copyright Date: 1969
 Subjects
Subject: Insect pests -- Control   ( lcsh )
Mosquitoes -- Control   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1969.
Bibliography: Includes bibliographical references (leaves 81-85).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Jack Arlyn Seawright.
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Bibliographic ID: UF00097780
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 - 000431392
oclc - 37600654
notis - ACJ0814

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A GENETIC STUDY OF APHOLATE-RESISTANCE

IN Aedes aegypti (L.)














By
JACK ARLYN SEAWRIGHT















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
1969






sYL/L/~


UNIVERSITY OF FLORIDA

3 1262 08552 2927














ACKNOWLEDGEMENTS


The author wishes to thank Dr. C. N. Smith, Dr. S. C. Schank,

Dr. P. B. Morgan, and Dr. F. S. Blanton for their assistance and

constructive criticism during the preparation of this manuscript.

My gratitude is extended to Dr. C. N. Smith for his interest and

advice and to Dr. S. C. Schank for providing the motivation that

made the author aware of the importance of cytogenetics in chemo-

sterilant research. Dr. P. B. Morgan provided advice on the

techniques for preparation of insect chromosomes, which was greatly

appreciated.

The author wishes to especially thank his wife, Rebecca, for

her understanding, support, and assistance in the preparation of

this manuscript.













TABLE OF CONTENTS


Page

IITRODUCTION . . . . . . . . . . . 2

REVIEW OF LITERATURE . . . . . . . .... .. 4

Resistance to Cnemosterilants . . . . . . 4

Apholate as a Chemosterilart. . . . . . . 7

Formal Genetics of Aedes aegypti. . . . . ... 12

Cytogenetics of Aedes aegypti . . . . . .. 14

Statistical Analysis of Resistance Data . . ... .17

METHODS AND MATERIALS. . . . . . . . . ... 18

Concentration-Sterility Curves with Apholate. ... 18

Loss of Resistance. . . . . . . . . ... 23

Concentration-Sterility Curves for Tepa and Metepa. 24

Cytogenetic Studies of Brain and Testes Squashes. . 25

Marker Strains. . . . . . . . .... . .28

Genetic Crosses . . . . . . . . ... . .29

RESULTS AND DISCUSSION . . . . . . . .... .30

Apholate Concentration-Sterility Experiments. ... .30

Cross-Resistance Experiments. . . . . . ... .46

Non-Selected Apholate-Resistant Strain. . . . ... 54

Genetic Crosses . . . . . . . .... .57

Cytogenetic Experiment. . . . . . . . . 61








Page

SUMMARY AND CONCLUSIONS ................... 73

APPENDIX . . . .. .. . . . . . . . . 76

LITERATURE CITED ............ ........... 80












LIST OF TABLES


Table Page

1. Basic schedule for rearing and handling Aedes aegypti
larvae, pupae, and adults during concentration-sterility
experiments . . . . . . . . ... ..... 22

2. Initial concentrations and resultant sterilities obtained
with apholate on males of resistant, susceptible, and F1
hybrid genotypes, treated from fourth larval instar until
pupation. . . . . . . . . ... ..... . 37

3. Initial concentrations and resultant sterilities obtained
with apholate on females of resistant, susceptible, and
F1 hybrid genotypes treated from fourth larval instar
until pupation. . . . . . . . . ... . 39

4. Summary of concentration-sterility data used to calculate
regression curve for the response of the resistant male
to apholate. . . . . . . . . . . 41

5. Summary of concentration-sterility data used to calculate
regression curve for the response of the resistant female
to apholate. . . . . . . . . ... . .. 41

6. Summary of concentration-sterility data used to calculate
regression curve for the response of the susceptible male
to apholate. . . . . . . . . . . . 42

7. Summary of concentration-sterility data used to calculate
regression curve for the response of the susceptible
female to apholate. . . . . . . . . ... .42

8. Summary of concentration-sterility data used to calculate
regression curve for the response of the Fl male
(susceptible female x resistant male) to apholate. ... 43

9. Summary of concentration-sterility data used to calculate
regression curve for the response of the Fl male
(resistant female x susceptible male) to apholate. ... 43

10. Summary of concentration-sterility data used to calculate
regression curve for the response of the F1 female
(resistant female x susceptible male) to apholate. ... . 44










11. Summary of concentration-sterility data used to calculate
regression curve for the response of the Fl female
(susceptible female x resistant male) to apholate.. .. 44

12. Summary of probit analyses of the data in Tables 4-11. . 45

13. Summary of data from the experiment conducted to
measure cross-resistance of the apholate-resistant
male to metepa. ... . . . .............. 45

14. Summary of data from the experiment to measure cross-
resistance of the apholate-resistant female to metepa. . 49

15. Summary of data from the experiment conducted to
measure cross-resistance of the apholate-resistant
male to tepa. .. . . . . . .... * * * 50

16. Summary of data from the experiment conducted to
measure cross-resistance of the apholate-resistant
female to tepa. .... . . . . . . . . 51

17. Summary of mortality in the apholate resistant and
susceptible strains after treatment with increasing
concentrations of metepa. . . . .... . . . 52

18. Summary of mortality in the apholate resistant and
susceptible strains after treatment with increasing
concentrations of tepa. . .... .. . . . . . 53

19. Summary of sterility in F2 progeny, treated with 20 ppm
of apholate of Cross A: F1 female (S sp blt female x
R wild male) x Fl male (S sp blt female x R wild male)
and Cross B: F1 female (R wild female x S sp blt male)
x Fl male (R wild female x S sp blt male). . .. .. . 58

20. Summary of sterility in backcross progeny, treated
with 20 ppm of apholate, of Cross C: F1 male (S blt
female x R wild male) x S blt female, Cross D: Fl
female (S blt female x R wild male) x S blt male,
Cross E: F1 female (R wild female x S blt male) x
S blt male, Cross F: F1 female (R female x S male) x
R male, and Cross G: Fl male (S female x R male) x
R female. . . . . . . ... . . . . 59


Page


Table













LIST OF FIGURES


Figure Page

1. Calculated regression lines for the sterility-
concentration experiments conducted with apholate
on the male genotypes. . . . . . . . ... 33

2. Calculated regression curves for the concentration-
sterility experiments conducted on the female
genotypes. . . . . . . . ... ..... .35

3. Regression lines visually fitted to data from the
experiments designed to measure cross-resistance
to tepa and metepa in the apholate-resistant (R)
male. . . . . . . . . ... ..... .48

4. Summary of data from the experiment to determine the
permanence of apholate resistance in the absence of
apholate selection pressure. . . . . . ... .56

5. Mitotic chromosomes from brain tissue of Aedes
aegypti. . . . . . . . . .. . . 63

6. Mitotic chromosomes from brain tissue of apholate-
susceptible strain of Aedes aegypti. . . . ... .65

7. Mitotic chromosomes from brain tissue of apholate-
susceptible strain of Aedes aegypti treated with
25 ppm of apholate during fourth larval instar... ... 68

8. Meiotic chromosomes from adult testes of Aedes
aegypti. . . . . . . . . ... ..... .70

9. ,Meiotic chromosomes from testes of Aedes aegypti. . 72


vii








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



A GENETIC STUDY OF APHOLATE RESISTANCE
IN Aedes aegypti (L.)


By

Jack Arlyn Seawright

December, 1969




Chairman: F. S. Blanton
Major Department: Entomology


An investigation was undertaken to study the mode of inheritance

of apholate resistance in Aedes aegypti (L). Sterility-concentration

curves were determined for each sex of the resistant, susceptible, and

Fl hybrid genotypes. The SC50 values were 48.30 for the resistant male,

2.26 for the susceptible male, 4.14 for the F1 male (resistant female x

susceptible male), and 19.50 for the F1 male (susceptible female x

resistant male); and 31.61 for the resistant female, 3.22 for the

susceptible female, 8.94 for the Fl female (resistant female x sus-

ceptible male), and 16.87 for the FI female (susceptible female x

resistant male). The results of genetic crosses indicated that apholate

resistance in A. aegypti was a quantitative trait with a marked paternal

influence on the resistance of F2 and backcross progeny.

There was no marked cross-resistance to tepa and metepa in the

apholate-resistant strain, but increased tolerances to both chemicals

were found. There was no loss of resistance through 9 generations after


viii








selection pressure was removed from a sub-colony of the apholate-

resistant strain.

The examinations of chromosome squashes were negative for

major chromosomal aberrations in the apholate-resistant strain.































A GENETIC STUDY OF APHOLATE RESISTANCE
IN Aedes aegypti (L.)













IIWTRODUCTIOfl


Since the introduction of large-scale use of insecticides for

control of insect pests, the most difficult problem in achieving

control has been the marked capacity of insects to develop resistance

to insecticides. The problems of dealing with a perpetual search for

newer and more efficient insecticides, as well as undesirability of

persistent chemical residues, required of entomologists the imagination

to devise new control measures. One of the most promising of recently

advanced control measures was the sterile-insect technique. The

utilization of this technique would involve releasing sterile insects

into a normal population or sterilizing the natural population. The

value of this control measure was demonstrated by the eradication of

the primary screwworm fly, Cochliomyia hominivorax, from the south-

eastern United States in 1957 by releasing millions of adult screwworm

flies sterilized by exposure to gamma-radiation in the pupal stage

(Baumnover, 1966).

After the success of the screw'orm eradication program, con-

siderable interest developed in the possibility of sterilizing field

populations of insects, and also, the possibility of reducing the cost

of sterilization. Chemicals that would induce sexual sterility in

insects have received much attention, because it would in all proba-

bility be less expensive to use chemicals, and the mobility of the

sterilization equipment would be greater than with radiation equipment.








Also, the necessity for the mass rearing of insects for release could

be avoided if a chemical were applied directly to a natural field

population.

During the past decade, thousands of chemicals have been evalu-

ated for sterilant activity, and probably the most effective com-

pounds have been alkylating agents, particularly the aziridines.

Following the discovery of these chemosterilants, many re-

searchers speculated that insect populations would develop resistance

to the sterilizing effects of chemicals by Darwinian selection as in

insecticide resistance. Since entomologists had seen the development

of insecticide resistance to almost every class of chemical toxicant,

there was reason to expect resistance to chemosterilants. Indeed,

following experimental selection with alkylating agents, chemosteri-

lant resistance was subsequently developed in laboratory colonies of

Aedes aegypti (L.) (Hazard et al., 1964) and Musca domestic (L.)

(Abasa and Hansens, 1969).

Investigators at the Insects Affecting Man and Animals Research

Laboratory, United States Department of Agriculture, Gainesville,

Florida,induced resistance to the sterilizing effects of apholate,

in Aedes aegypti within a few generations after selection pressure

was initiated. (Hazard et al., 1964). This strain was made available,

and the investigation reported herein was undertaken to elucidate the

inheritance of this apholate resistance and to describe the resistant

strain in terms of the level of resistance and other aspects of

interest that existed in the strain.













REVIEW OF LITERATURE


Resistance to Chemosterilants

There have been only a few reports on the development of

chemosterilant resistance in insects, and all these have involved

the induction of resistance in laboratory strains under artificial

selection conditions. An excellent review on the use of chemicals

to induce sterility in field populations is presented by LaBrecque

(1968). However, since the chemosterilants have been used in the field

as experimental compounds only, there has been little opportunity for

field populations of insects to develop resistance.

The earliest report on chemosterilant resistance was made by

Hazard et al. (1964). These researchers reported a 4-to-5-fold

tolerance to apholate in A. aegypti following selection for 11

generations. The fourth larval instar was the stadium treated with

apholate. This apholate-selected strain is the one used during the

course of the investigation described herein. Patterson et al. (1967)

stated that this strain, since the report of Hazard et al. (1964), had

been maintained under selection pressure of 25 ppm of apholate and had

increased the level of resistance in the larval stage to 20-fold.

These researchers found only a 6-to-7-fold level of resistance in the

adult mosquitoes which were fed apholate in sugar water solutions.

They further demonstrated that no cross-resistance to tepa was present

in the strain, and only a 3-to-4-fold tolerance was found when larvae

were treated with metepa.










There have been two reports of metepa resistance induced in two

insect species. Sacca et al. (1966) reported an increased tolerance

to metepa which was applied topically to house flies, M. domestic.

However, they also noted that no resistance was observed when adult

house flies were treated topically with tepa and hempa. Klassen and

Matsumura (1966) found that metepa resistance was induced in A. aegypti

by applying selection pressure to fourth-instar larvae. There appeared

to be an improvement in the fertility of the selected strain when com-

pared to the parental strain. The parental strain produced a pre-

ponderance of non-embryonated eggs, as the hatching rate was usually

about 40%. They suggested that the selected strain laid only viable

eggs, which hardly seems reasonable and leads the author to consider

other possible mechanisms, i.e. increasing the vigor of the strains

by Darwinian selection via the treatment with metepa. These workers

further demonstrated that tissue homogenates of the metepa-resistant

strain detoxified 3 times as much metepa during incubation in the

presence of metepa for 30 minutes. The metepa determinations were

assessed by micro-chemical analysis for phosphorous.

Morgan et al. (1967) failed to develop resistance to apholate in

house flies when they subjected the adults continuously to concentra-

tions of apholate that produced less than 100% sterility. These

workers observed that a strain under selection with .01% of apholate

showed an increase in sterility from less than 10% in the F5 to 70%

in the F30, with a subsequent decrease in sterility to 20% in the F55

generation. The strain has maintained this level of sterility in a

more or less static manner to the F100 generation. They postulated







that the initial buildup of sterility was due to an accumulation of

genetic damage and presented cytological evidence that substantiated

this idea. Chromosome squash preparations yielded evidence of chro-

mosome stickiness, fragmentation, deletions, and the absence of an

autosomal homologue. In this same experiment, a colony of house flies

was selected with metepa in the adult food; because of reductions in

fecundity and fertility the strain died out in the F10 generation.

Observations similar to those of Morgan et al. (1967) were

made by George and Brown (1967); when they applied selection pressure

of hempa on larvae of A. aegypti, sterility increased until the strain

was lost in the F6 generation. They attributed the increased sterility

to inheritable recessive genetic defects which accumulated in the

strain due to inbreeding.

Abasa and Hansens (1969) reported a 5-fold increase in tolerance

to apholate in the house fly. They fed an apholate-sugar bait to

adults for 35 generations and first observed a tolerance of apholate

in the F15 generation. The selected strain showed a slight cross-

tolerance to metepa, but not to methotrexate.

The net analysis of laboratory chemosterilant selection applied

on A. aegypti and M. domestic indicates that the only development of

chemosterilant resistance of any magnitude was the apholate-resistant

strain of A. aegypti reported first by Hazard et al. (1964) and later

by Patterson et al. (1967). This was the only report of a species of

insect that could tolerate relatively high concentrations of a chemo-

sterilant and apparently maintain a reasonable level of fecundity and

fertility. The strain of A. aegypti reported as resistant to metepa

by Klassen and Matsumura (1966) had only a 3-to-4-fold level of








resistance, and most of the other chemosterilant selections have

resulted in a strain of insects more susceptible to the chemosterilant

than the parent strain. This apparent accumulation of genetic damage

was expected by many researchers and has been one of the basic con-

siderations in the argument that field populations of insects would

not develop resistance when treated with chemosterilants. There

have been very few large scale field experiments with chemosterilants

against native populations of insects, but Meifert et al. (1967) made

the observation that after two years of treating a population of

house flies on Grand Turk Island in the Bahamas, the fertility of

the flies on the island declined slightly, and no resistance developed.


Apholate as a Chemosterilant

The initial report on the use of apholate as a chemosterilant

was published by LaBrecque (1961). He found that apholate and two

other aziridine compounds induced sterility in both sexes of house

flies.

Since the discovery that apholate induced sterility in house

flies, 34 other species of insects and mites have been sterilized in

laboratory tests with apholate (LaBrecque, 1968). A review of re-

cent literature revealed an additional 7 species of insects which

have been sterilized with apholate. These species were Heliothis zea

(Boddie) and Heliothis virescens (F.) (Soto and Graves, 1967), Oulema

melanopus (L.) (Ezueh and Hoopingarner, 1967), Ostinia nubilalis

(Hubner) (Harding, 1967), Hypera postica (Gyllenhal) (McLaughlin and

Simpson, 1968), Bracon hebetor (Say) (Valcovic and Grosch, 1968), and

Epilachna varivestris Mulsant (Webb and Smith, 1968).







Apholate is an alkylating agent and a radiomimetic compound.

The five chief types of cytotoxic alkylating agents are mustard,

ethyleneimines, sulfonic esters, epoxides, and certain N-alkyl-N-

nitroso compounds. Apholate, by virtue of the six ethyleneimine

rings on the molecule, is in the second of the above general groups.

Alkylation is defined as the replacement of a hydrogen atom of a

molecule by an alkyl group, and alkylating agents have a markedly

varied structure and composition. The only chemical property that

distinguishes this heterogeneous group of compounds is the propensity

to alkylate electron-rich centers (Turner, 1968). Turner further

delineated the type reaction which is thought to occur during a

biological alkylation. The general scheme of the reaction is listed

below:

RY + HX Y ----R +---X ----H +

In the above reaction scheme, the rate depends upon the con-

centration of the alkylating agent and the target molecule as follows:

rate = f-RY_] -HX_

The ionization of the alkylating agent (RY) occurs at the same

time as the bond with the nucleophile (HX). Since alkylating agents

are electrophiles (electron loving), the nucleophiles with which they

react are polar compounds that donate an electron pair to the carbon

in the alkylating agent.

Therefore, the chief nucleophilic centers that might be a target

for alkylation are organic and inorganic anions, amino groups, and

sulfide groups. The range of possible nucleophile targets creates a

very complex problem as pertains to the elucidation of the critical








alkylations that could result in sterility. Since elucidation of the

mode of action of a chemosterilant would require not only the deter-

mination of which compound (s) is alkylated, but the site of alkyla-

tion, the wide range of nucleophile targets would make an investigation

of mode of action an insurmountable task.

Although a great deal of work has been done concerning the

cytological and cytogenetic aspects of chemosterilization (LaChance et al.,

1968), the author could find no literature pertaining to the use of

apholate and related aziridine chemosterilants for induction of visible

morphological mutants in insects. However, although ethyleneimine

demonstrated no sterilant activity in house flies (Borkovec et al.,

1964), it has been used as a mutagen (Lim and Snyder, 1968 and

Alexander and Glanges, 1965). These two reports dealt with induction

of translocations and recessive lethals, but also contained informa-

tion concerning the production of morphological mutants using

ethyleneimine for treatment of Drosophila melanogaster.

No reports were found on the absorption and distribution of

apholate in insects. However, studies using the related aziridine

compounds, metepa and tepa, have been reported. Plapp et al. (1962)

found that larvae and adults of Culex tarsalis degraded metepa com-

pletely within 48 hours after treatment. Also, they found that house

flies injected with metepa degraded nearly 50% of the chemical within

2 hours. In another investigation, Chamberlain and Hamilton (1964)

found a rapid rate of degradation of metepa in the screwworm fly,

Cochliomyia hominivorax (Coquerel), and in the stable fly, Stomoxys

calcitrans (L.), and identified the principal metabolite as phosphoric







acid. Chang et al. (1966) noted that 50% of the tepa injected into

house flies had been degraded in 2 hours. Based on these studies on

degradation of aziridine chemosterilants, it is not unreasonable to

assume that a similar rapid degradation of apholate would occur in

insects.

Based on an investigation by Ristich et al. (1965), there

appeared to be a positive correlation between the per cent ethylenei-

mine in apholate analogs and per cent sterility induced in treated

house flies. A series of apholate-analogs substituted with chlorine

and dimethylamino groups on the aziridine rings and between the rings

were evaluated for sterilant activity. This work agreed with a later

paper by Crystal (1966), which showed that the presence of substituents

on the aziridine-ring carbon atoms reduced the sterilant activity of

the compound. These investigations indicated that the per cent

aziridine-ring in a compound was important for chemosterilization;

however, Glancey (1965) and George and Brown (1967) demonstrated

that A. aegypti (L.) was sterilized by treatment of the larvae with

hempa, a phosphoramide that is structurally and sterically related to

tepa but is not an alkylating agent. Therefore, in spite of the

assumption based on earlier work that the alkylating property was

necessary for sterilant activity, it apparently is not.

Kilgore and Painter (1964) reported that normal house fly eggs

had a low concentration of DNA initially after oviposition, but this

concentration increased rapidly prior to hatching. Sterile eggs from

females treated with apholate demonstrated no increase in DNA content.

They also found that lactate dehydrogenase activity increased rapidly








in developing eggs, but the activity in sterile eggs was not increased.

The authors suggested that ovarian damage, hormonal imbalance, or a

number of other metabolic abnormalities could have caused the effect

on DNA synthesis; however, based on their investigation no determina-

tion on direct or indirect effects was implied.

Mendoza and Peters (1968) found a significant reduction in a DNA

specific alkaline phosphatase in the ovaries of southern corn root-

worms following injection of apholate. They attributed the decrease

in activity to a direct inhibition of phosphatase by apholate; however,

it seems probable that other metabolic pathways were affected and in-

direct effects could have caused this inhibition.

Sharma and Rai (1969) observed a "deterioration" of brain tissue

and midgut epithelium in A. aegypti females which were treated with

apholate in the larval stage. Both tissues were severely affected,

and a lack of golgi bodies was noted in the midgut epithelium. Golgi

complexes are most prevalent and well developed in secretary cells,

and some evidence points to a role as a secretor of macromolecules

for these complexes. Also, golgi vacuoles develop into zymogen

granules, which contain and store the proteins produced by the

ribosomes (Mahler and Cordes, 1966). It appears, therefore,that a

reduction in enzymatic activity would probably occur in the midgut

epithelium. However, Rai and Sharma (in press) have demonstrated

that normal ovaries transplanted into the abdomens of chemosterilized

females developed normally. This indicated that either a considerable

repair of damaged cells occurred or those damaged cells continued to

function, possibly at a suboptimal level.










Turner (personal communication) has completed some preliminary

experiments concerning uptake and metabolism of apholate by larvae of

the apholate-resistant strain compared to the apholate-susceptible

strain. The susceptible strain showed an uptake of apholate almost

3 times greater than the resistant, and the rate of disappearance was

equal in both strains. The data collected by Turner indicated that

simple uptake is apparently a very important factor in apholate

resistance; however, the resistance can not be attributed to simple

accumulation alone. He used a concentration of 100 ppm of apholate

as a treatment for the larvae. This concentration is nearly 50

times greater than the SC50 for the susceptible strain reported by

Patterson et al. (1967), and based on the author's work exceeds the

SC90 for the resistant strain. Therefore, it is probable that the

high concentration used for treatment changed any difference that

existed between the two strains.

Maheswary (1968) found that ovarian alkaline phosphatase activity

was suppressed in both the apholate-resistant and susceptible strains

of A. aegypti following exposure of the larvae to apholate. However,

gross protein content of ovaries from the resistant strain was not

affected by apholate treatment. In contrast, protein content of

ovaries from the susceptible strain was severely reduced.


Formal Genetics of Aedes aegypti

A complete review of the status of the formal genetics of

A. aegypti was recently presented by Craig and Hickey (1967). They

recorded a total of 87 mutants that have been isolated; many of these

which involve color patterns were found from examinations of strains










of diverse geographical origin and others which involve structural

aberrations were obtained by inbreeding. Of the 87 mutants reported,

34 have been assigned to 3 linkage groups. All the mutants, except

those that represent polymorphisms from diverse areas, have arisen

spontaneously during inbreeding. A. aegypti demonstrates considerable

color variation in strains from diverse geographical origin and

apparently carries a great deal of concealed variability, even in

laboratory strains. Van deHey (1964) found a mutation load of 1.32

visible mutants per mosquito during inbreeding experiments with 4

strains of A. aegypti. He observed a higher rate of spontaneous

mutants during inbreeding than was observed following irradiation with

inbreeding.

Linkage data thus far collected indicate there is reason to

suspect the definitive linkage map in A. aegypti will be fairly short.

The total map distance of the three linkage groups at present is 110

units (43 + 37 + 30), and this led Craig and Hickey to speculate that

the maximum length may be approximately 150 units. Certainly, additional

linkage studies will lengthen the map. However, since Akstein (1962)

and Mescher and Rai (1966) observed a maximum of 2 chiasmata per

bivalent, this was further evidence to support a short map.

Since crossing over occurs in both sexes, and there has been no

standardized program of experimentation, linkage data from various

laboratories have been variable. Variations in map distances from

different laboratories remain unexplained; however, some progress has

been made in finding the reasons for these variations. According to

Craig and Hickey, age and sex have a marked influence on crossing









over; other sources of variation are the larval diet and the tempera-

ture of the larval medium. It appears that a uniform program of

experimentation needs to be adopted before many of the problems in

the variations of linkage data will be overcome.

The mechanism of sex determination in A. aegypti was determined

by McClelland (1962). He found that sex was determined either by a

small block of a chromosome or a single gene, with the male being

heterogametic. Gilchrist and Haldane (1947) postulated the same

mechanism for sex determination in Culex pipiens. There has been

speculation that the situation in Aedes and Culex mosquitoes is

analogous to that in some species of Chironomus where sex is deter-

mined by an inversion that occurs only in the males (Beerman, 1955;

Acton, 1957). A small-inversion mechanism would give results similar

to the M/m system, but unfortunately examination of the polytene

chromosomes in Aedes species has not been accomplished.



Cytogenetics of Aedes .agypti

In the past few years, marked progress has been made in the

cytogenetics of A. aegypti. Paralleling the development of the

formal genetics of A. aegypti, considerable interest has been shown

in mitotic and meiotic karyotype descriptions and the effects of

radiation and chemosterilants at the cellular level.

The first report concerning the mitotic karyotype of A. aegypti

was made by Carter (1918), who listed the chromosome number as 2n = 4,

based on examination of sectioned gonads. Sutton (1942) used salivary

gland preparations and indicated a diploid number of six. Then Rai









and Craig (1961) and Breland and Gassner (1961) gave a more detailed

description of the mitotic chromosomes based on observations of

squash preparations of larval brain tissue.

Based on size and location of centromere, Rai (1963a) described

the normal karyotype of A. aegypti. The karyotype is composed of 3

pairs of chromosomes designated I, II, and III. Based on measure-

ments on 10 mitotic, metaphase figures, the chromosomes averaged

5.4, 6.9, and 7.6 microns, respectively, in the brain tissue of larvae.

Mescher and Rai (1966) described spermatogenesis in A. aegypti.

They observed that primary spermatocytes were present from the be-

ginning of pupal life onward. Further observations were that usually

in pupae 2 to 4 hours old, most of the cells seen were in pachytene,

and that pachytene was of long duration and was seen in all prepara-

tions of pupal testes. The double nature of each homologue was clearly

visible in diplotene, with the strands held together, presumably, only

at points of chiasmata. Diakinesis was of short duration, during which

the chromosomes were in a highly contracted state. At metaphase I,

the number of chiasmata per bivalent ranged from 1-2, and the chromo-

somes moved away from their homologues in a non-synchronous fashion.

This manner of movement was not observed in anaphase II, where all

the chromosomes were observed to move together. The authors noted

that the greatest impediment to clear observation was chromosome

stickiness, particularly in metaphase I and anaphase I. Spermatids

were observed after the first third of pupal life, and mobile sperm

were seen by the time the males emerged. No reports pertaining to

o6genesis in A. aegypti were found.










Because of difficulties involving chromosome stickiness, it has

not been possible to map salivary chromosomes (Aldighieri, 1961,

Mescher, 1963). In these attempts to prepare the salivary polytene

chromosomes for observation, chromosomal stickiness resulted in

chromatic masses.

.Rai (1964) treated second-instar larvae of A. aegypti with 15 ppm

of apholate and examined squashes of brain tissue for the effects of

this treatment on somatic chromosomes. He observed that apholate

induced numerous aberrations, including stickiness, deletions, ring

chromosomes, dicentric chromosomes, and anaphase bridges. Although

he made no observations on gonadal tissue, similar damage in deve-

loping gametes could be expected to result in dominant lethality.

In adult females, which emerged from the apholate-treated larvae,

oogonial maturation was almost completely inhibited. The ovarioles

remained underdeveloped and eventually degenerated. Only rarely

did eggs develop and these usually failed to hatch.

Aberrations similar to those observed by Rai were reported by

George and Brown (1967), when they treated fourth-instar larvae of

A. aegypti with hempa.

The aberrations induced in somatic chromosomes of A. aegypti by

apholate and hempa were basically the same as those induced by

x-irradiation (Rai, 1963b). Rai described the chromosomal aberrations

induced in brain cells by x-irradiation as similar to those induced

by apholate, and since he did not score the number of each class of

aberration observed, it was assumed that the x-irradiation and apholate

produced similar effects.










Statistical Analysis of Resistance Data

Resistance of insects to chemical toxicants is an invisible

character, and the most familiar means for investigating the in-

heritance of resistance is an interpretation of toxicological data.

Dosage-mortality curves give insight into the mode of inheritance.

Tsukamoto (1963) presented an excellent description of the use of

the log dosage-probit mortality curve in genetic investigations of

insecticide resistance. In a later paper Tsukamoto (1964) presented

a form of analysis of variance to determine the contribution of each

chromosome in inheritance studies of resistance. In a third paper

(1965), he described a statistical method for estimation of re-

combination values for insecticide resistance by elimination of errors

in analysis stemming from both differential viability and partial mani-

festation of the resistant genotype. This third paper by Tsukamoto

concentrated on a practical method to estimate overlapping phenotypes.












METHODS AND MATERIALS


The chemosterilants used in this investigation were apholate

/-2, 2, 4, 4, 6, 6 hexakis (1 aziridinyl) 2, 2, 4, 4, 6, 6 -

hexahydro 1, 3, 5, 2, 4, 6 triazatriphosphorine_, tepa /-tris

(1 aziridinyl) phosphine oxide_7, and metepa /-tris (2 methyl -

1 aziridinyl) phosphine oxide_7. Technical information pertaining

to chemical and physical properties of these three chemicals were

taken from Olin Mathieson Chemical Corporation (1964) and Kenaga (1966).

This information is listed in the appendix, pages 77-79.


Concentration-Sterility Curves With Apholate

In order to study the mode of inheritance of chemical resistance,

it is necessary to determine a concentration of chemical that will

distinguish the susceptible and resistant phenotypes. A preliminary

experiment was conducted to construct a concentration-sterility curve

for the susceptible mosquitoes. The apholate-susceptible strain was

used in these preliminary tests, because it was thought there would

probably be less variation in results obtained.

Approximately 150 first-instar larvae of the apholate-susceptible

strain of A. aegypti were placed in one liter of glass-distilled

water. A mixture of 2 parts liver powder:1 1 part yeast extract v/v



1. Obtained from Nutritional Biochemicals Corporation,
Cleveland, Ohio.









was provided as food and was added each day as needed. The larvae

required approximately one day to complete an instar and were usually

in the fourth instar early on the fifth day. The rearing room for

larvae and adults was maintained at 82 + 20 F and 70-80% relative

humidity. The technique for treatment in this experiment was a

modification of that described by Dame et al. (1964). Approximately

100 fourth-instar larvae of the apholate susceptible strain were ex-

posed in apholate solutions of 5, 10, 15, and 20 ppm until pupation.

After the first 6 hours of treatment, liver and yeast mixture was

added for food as described before. The pupae were usually collected

on the second and third days after treatment. The pupae were sexed

on the basis of size, the males being smaller than the females, and

placed in cages 12 x 12 x 6 inches for eclosion. Three days after

eclosion 25 treated males and 25 treated females were held in cages

with untreated mosquitoes of the opposite sex 3 days for mating.

Sugar cubes and water on cotton pads served as food during the period

for mating. On the sixth day after eclosion the treated females

and females mated to treated males were blooded on a guinea pig.

Two days following the blood meal, eggs were collected on wet filter

paper, which had been placed inside a jar, 3 1/2 inches in diameter x

3 1/2 inches high, half-filled with water. The eggs were allowed 5

days for maturation before hatching was initiated by flooding the

eggs with water, followed by placing the eggs in a vacuum for 30

minutes.

In the groups with treated males, egg samples were counted to

determine the per cent sterility and net per cent sterility was










calculated by Abbot's formula as follows:

NET % Sterility = Sterility in Sterility in
Treatment Control
100 Sterility in Control

A total count of the eggs laid by the treated females was

made in order to estimate the loss of fecundity induced by the

sterilant. Net per cent sterility was calculated by the following

formula:

NET % Sterility = 100(1-hf)

Where: h = the decimal frequency of hatch.

f = the decimal frequency of fecundity.

Since this technique gave reproducible results, it was adopted

for establishing concentration-sterility curves for the resistant

strain and Fl hybrids obtained from crossing the resistant and

susceptible strains.

Following the establishment of a treatment technique, experiments

were conducted on the resistant strain and the F1 hybrids to determine

a range of concentrations of apholate that would yield an estimate

of the SC50 of these genotypes. This was not necessary for the

susceptible strain, since a range had been determined in the pre-

liminary experiments.

One replication was conducted with each genotype at concentrations

of 5, 10, 15, and 20 ppm of apholate. The treatment and handling

schedule was consistent with the preliminary experiment. Induced

sterility was assessed in each sex by mating with untreated mosquitoes

from the susceptible strain.

The ranges of concentrations used to establish the SC50 values










for each sex of the resistant, susceptible, and F1 hybrid genotypes

ranged from 0.5 ppm to 80 ppm. Each concentration was replicated

3 5 times with 20 mosquitoes (selected at random from 100 adults

treated as larvae) per replicate. The basic schedule for rearing,

treating with apholate, and handling of the mosquitoes is listed in

Table 1. The females were placed in individual oviposition cups,

a four ounce squat cup lined with filter paper and half-filled with

water. Also, the treated males were mated to a single female in the

same size cup used for oviposition. These cups were covered by

plastic screen secured by a rubber band. In the early concentration-

sterility tests, an accurate account of the mortality and fecundity

of females mated to treated males was recorded, and was used as a

guideline for an approximation of the number of mosquitoes to be

used at each concentration in order to insure there would be enough

mated females to constitute a replication. Individual matings were

not done with the treated females, since the source of fertile sperm

was thought to be of little consequence. The females were mated en

masse in cages 12 x 12 x 6 inches.

The data collected from the concentration-sterility experiments

were analyzed by probit analysis as outlined in Finney (1952).

Concentrations used were transformed to logarithms and net per cent

sterilities were transformed to probits. A linear regression was

performed on the transformed data, and estimates of SC50 values,

slopes and fiducial limits of the SC50 values were calculated for

the genotypes tested.










Table 1. Basic schedule for rearing and handling Aedes aegypti
larvae, pupae, and adults during concentration-sterility
experiments.


Day Operation


0 Eggs were hatched and 100 first-instar larvae were
placed in 1 liter of glass-distilled water with liver
and yeast mixture as food.

1 Larvae in second instar. Food added.

2 Larvae in third instar. Food added.

3 Larvae in late third instar. Food added.

4 Fourth-instar larvae placed in apholate solutions.
Food added 6 hours after treatment initiated.

5 Food added.

6 Pupae (mostly males) collected.

7 Pupae collected..

8 Pupae collected. Adult males emerging.

9 Adults emerging.

10 Adult emergence complete.

11 Treated adults mated to untreated mosquitoes of
susceptible strain.

14 Females blooded on guinea pig.

16 Females placed in oviposition cups.

21 Eggs hatched in a vacuum and per cent sterility
recorded.










Loss of Resistance

The loss of insecticide resistance by both field and laboratory

populations of insects has been documented by many researchers.

Usually the loss in resistance occurred after several generations

during which the insects were not subjected to selection pressure by

exposure to the chemical, and the population demonstrated suscepti-

bility to concentrations of chemical to which it once was completely

refractory.

An experiment was conducted through 10 generations to determine

if any reduction in apholate resistance would occur in the resistant

strain. A sub-colony was established and reared without apholate

selection. Two groups of 100 larvae from each generation were

treated with 25 ppm of apholate in the manner described previously.

In addition to the two treated groups, a resistant control from the

generation being assayed was reared and the sterility assessed. Also,

two groups of 100 larvae from the susceptible strain were treated

with 25 ppm of apholate, and a third group from the susceptible group

was reared and the natural sterility was assessed. In each group,

sterility was assessed by mating to virgin mosquitoes of the sus-

ceptible strain. Only the male sterility was recorded to use as an

indication of any loss of resistance. Eggs were collected from the

females en masse on filter paper in a jar half-filled with water, and

samples of not less than 100 eggs were used for sterility counts.










Concentration-Sterility Curves For Tepa and Metepa

Other experiments were conducted to determine if cross-

resistance to two closely related aziridine chemosterilants was

manifest in the apholate-resistant colony. This experiment

paralleled the work reported by Patterson et al. (1967), with

the exception that the experiment described below assayed the

effect of tepa and metepa on each sex.

The rearing schedule in the preceding section was used in

these experiments. Approximately 100 fourth-instar larvae of

the apholate-resistant and apholate-susceptible strains were

treated at each concentration. Food was added 6 hours after

treatment had begun. Both strains were exposed to 10, 20, 40,

and 80 ppm of metepa and 2.5, 5.0, 10.0 and 20.0 ppm of tepa, with

3 replicates at each concentration. Since these two compounds

deteriorate rapidly at a pH below 6.5, the solutions were buffered

with 1 ml/liter of 1 M K2HPO4. As in the experiments with apholate,

treatment was continued until pupation. The pupae were rinsed and

transferred to glass-distilled water. Mortality counts of larvae,

pupae, and adults were recorded.

The adults, both males and females, were mated en masse to

virgin untreated mosquitoes of the susceptible strain. The females

were allowed to feed on a guinea pig and eggs were collected en

masse on filter paper lining a glass jar, which was half-filled

with water. Samples of not less than 200 eggs were hatched to

assess sterility. For purposes of comparing the sterility induced

in the resistant and susceptible strains, the results from the males










were plotted on log-concentration probit-sterility graphs, and

SC50 values were estimated for the resistant and susceptible

genotypes.


Cytogenetic Studies of Brain and Testes Squashes

Since the initiation of selection for apholate resistance,

the resistant colony has been selected for over seventy genera-

tions with 25 ppm of apholate. In view of this rigid selection

with a mutagenic chemical, it was suspected genetic damage could

probably be detected by examination of chromosome squashes.

Preparation of chromosome squashes for mosquito larval brain was

described by Rai (1963a) and Breland (1961), and squash preparation

of mosquito pupal testes was described by Mescher and Rai (1966).

The techniques used in this investigation were a combination of

desirable procedures selected from the methods of those authors

above.

Rapid cell divisions in the brain of a mosquito occur

intermittently, and the selection of the stage for studying mitosis

in the life cycle was important. Usually the brain undergoes

considerable growth preceding a larval moult, and the prepupal

stage of the last larval instar was chosen for the present study,

because the brain is much larger and more easily dissected than at

earlier stages. The prepupa can be recognized by the presence of

the developing pupal trumpets in the thoracic region.

With the aid of a low-power (about 20X) dissecting microscope,

the head was removed from the body and placed in a hypotonic solution

of 1% sodium citrate for 30 minutes. As reported by Morgan and









La Brecque (1964), this caused the cells to swell and produced more

uniform squashes. The head was then placed in Carnoy's fixative

(1 part 95% ethanol: 1 part glacial acetic acid) for 1 minute.

The fixative caused the brain to change from a colorless, highly

refractive body to an opaque white body which facilitated the

dissection of brain tissue from the digestive tract.

After fixation, the head was placed in a drop of Belar's

saline on a slide, and the brain was dissected out of the head

capsule. The head capsule was teased open with minute pins,

which are ordinarily used for mounting small insects. One needle

was placed in the mouth to hold the head in place. The second

needle was inserted into the mouth and pushed anteriorly to cause

the head capsule to split. The brain lies transversely between the

eyes across the dorsal side of the head capsule. After the capsule

was split, it was necessary to severe the nerves that connect to

each eye and the circumcentric commisures that pass ventrally

around the digestive tract.

The brain was transferred to a drop of aceto-orcein stain

for ten minutes. Then the stain was washed off the slide with 45%

acetic acid. A cover slip was placed over the brain, and the slide

was squashed with the aid of a mechanism developed by Linkfield

et al. (1967). Good, uniform preparations were obtained using

225 pounds of pressure per square inch. Slides were sealed with

finger-nail polish and remained usable for approximately one month.

Testes were dissected from pupae, which were nearly ready to

eclose, and from adults less than 24 hours old, and squashes were

made for examination of meiosis.










The pupae selected were dark and easily distinguished, because

the formation of adult scales was easily seen through the cuticle.

The abdominal section was severed from the body and placed in

hypotonic sodium citrate for 15 minutes. Then the whole abdomen

was fixed in Carnoy's fixative for 1 minute. The sausage-shaped

testes, which lie in the dorsolateral region of the sixth abdominal

segment, were dissected from the abdomen in Belar's saline, and

stained for 10 minutes in aceto-orcein stain. The stain was washed

off the slide with 45% acetic acid. A coverslip was placed over

the testes and the slide was squashed as described previously with

200 pounds of pressure per square inch.

Testes from adults were prepared as in the manner described

above. The testes were dissected from the body by grasping the

terminal segment of the male with a pair of fine forceps and

applying a gentle, smooth pull. The mosquito was held in place by

a needle held behind the thorax. The testes were left attached to

the terminal segment, which served as a handle, during transfers

from slide to slide.

Squashes were made of the brain and testes of mosquitoes from

the susceptible strain, the resistant strain, and the Fl hybrids from

crosses of those two strains. Preparations were also made of the

brain of larvae of the susceptible and resistant strains treated

with 25 ppm of apholate.

The preparations were examined with a phase-contrast micro-

scope, and photomicrographs were taken with a 3 1/4 x 4 1/4 inch

Polaroid camera and a 35 mm camera. The Polaroid film used was









T:,pe 107/ASA 3000 black and white, and 35 mm films used were

Panatomic-X (ASA 32) and Agfa Isopan (ASA 25). The Polaroid

prints were used immediately for comparing the chromosomes from

different cells, and the 35 mm exposures were retained for a

permanent record.


Marker Strains

A multiple mutant marker strain called RED EYE was received

from Dr. G. B. Craig of Notre Dame University, Notre Dame, Indiana;

however, this strain had low rates of fecundity and fertility and,

therefore, was not satisfactory for a sterility investigation. The

following is a description of this RED EYE strain and strains derived

from outcrossing this strain followed by selection of desired genotypes

in the F2 progeny.

RED EYE: This strain was homozygous for red eye, spot,

and black-tarsi.

Red eye (re) was reported by McClelland (1966) as a sex-linked,

recessive mutant that was expressed as a deep blood red eye color.

It can be detected in larvae, pupae, and adults.

Spot (sp) was described by Craig and Van deHey (1962) as an

autosomal, recessive gene in linkage group II. This mutant displays

a marked sexual dimorphism. In the male, it is expressed by an

absence of the spot normally found on each lateral, abdominal tergite,

but in the female, the spots are enlarged and extend to the postero-

lateral corners of the tergite.

Black-tarsi (blt) was also isolated by Craig and Van deHey (1962).

It is an autosomal, recessive gene in linkage group III and is









expressed in both sexes by a reduction of the band of white scales

found on the basal portion of most of the tarsal segments.

From the RED EYE strain, the following strains were selected

from the F2 progeny of a cross of RED EYE males to wild type females.

sp-blt: Homozygous for spot and black-tarsi.

sp: Homozygous for spot.

bit: Homozygous for black-tarsi.

Due to a failure in the heating equipment, the sp-blt and sp

strains were lost, but subsequently recovered by inbreeding the bit

strain.


Genetic Crosses

The backcross and F2 progeny from the crosses of the apholate-

resistant (R) and apholate-susceptible (S) genotypes listed below

were assayed for fertility with 20 ppm of apholate by the method

developed during the course of the concentration-sterility experiments.


F2 Crosses

Cross A: F1

Fl

Cross B: Fl

Fl


Backcrosses

Cross C: Fl

Cross D: F1

Cross E: Fl

Cross F: F1

Cross G: F,


female (S sp bit female x R wild male) x

male (S sp bit female x R wild male)

female (R wild female x S sp bit male) x

male (R wild female x S sp bit male)




male (S bit female x R wild male) x S bit female

female (S bit female x R wild male) x S bit male

female (R wild female x S bit male) x S bit male

female (R female x S male) x R male

male (S female x R male) x R female












RESULTS AND DISCUSSION


Apholate Concentration-Sterility Experiments

The sterility data collected during the preliminary concentra-

tion-sterility experiments are presented in Tables 2 and 3. The

results listed in Table 2 were from treated male groups, and Table 3

contains the data from the treated female groups. From these experi-

ments, it was apparent that differences existed between the response

of the reciprocal FI hybrid males. Also, the highest concentration

tested had only a small effect on the resistant males and females.

The differences between the Fl hybrid male groups were sur-

prising, and suggested that the resistance was dominant in the male

and additive or recessive in the female. The data for the female

groups did not appear to be linear, but this could have been attributed

to the methods of collecting eggs from the females en masse. When

a group of females laid their eggs in a common place, more eggs were

deposited on the water and would not hatch.

The results of those experiments conducted to determine SC50

values for the resistant male, resistant female, susceptible male,

and susceptible female are presented in Tables 4, 5, 6, and 7, re-

spectively. The results of the experiments on the Fl hybrid males

and females are presented in Tables 8, 9, 10, and 11. The SC50

values, slopes of the lines, and fiducial limits for SC50 values are

presented in Table 12. These data are presented graphically in the










form of calculated regression lines in Figures 1 and 2 for the male

and female genotypes, respectively. It was of interest that the

slopes of the lines for the resistant male and female genotypes

were over twice as great as the slopes of the lines for the susceptible

male and female genotypes. This would indicate that the resistance

was at a stable level and most likely would not increase. Patterson

(1969, personal communication) indicated that selection applied to

the resistant strain at 50 ppm and 100 ppm of apholate for several

generations failed to increase the resistance.

Examination of the data from the individual egg counts revealed

that only a few individuals of the susceptible strain, particularly

the females, were partially fertile at the higher concentrations

tested. Also, this was found in the Fl hybrids, particularly in the

Fl female (susceptible male x resistant female) genotype. Approximately

10% of the supposedly susceptible females demonstrated a degree of

resistance comparable to the Fl female (resistant male x susceptible

female); however, none of the susceptible males tested showed a marked

degree of resistance.

The fiducial limits of the susceptible female and Fl female

(susceptible male x resistant female) genotype barely overlapped, and

the fiducial limits of the Fl hybrid female genotypes overlapped con-

siderably. Theoretically, the F1 hybrid female genotypes should be

identical, and the latter overlap was expected. The large differences

in slope and SC50 values are unexplainable in terms of monofactorial

inheritance; however, the SC50 values and position of the two curves

(in relation to resistant and susceptible female genotypes) indicated






























Figure 1. Calculated regression lines for the sterility-concentration
experiments conducted with apholate on the male genotypes
listed below:


0 apholate-resistant (R).
A apholate-susceptible (S).
F1 male (S female x R male).
A F1 male (R female x S male).




























































10 20 50


Log Concentration (ppm)


6.0








H

5.0 a


Pc






























Calculated regression curves for the concentration-sterility
experiments conducted with apholate on the female genotypes
listed below:


apholate-resistant (R).
apholate-susceptible (S).
Fl female (S female x R male).
F1 female (R female x S male).


Figure 2.
























































1 I I 1 1 111


I 1


1 I i


100


Log Concentration (ppm)


6.0








*H

5.0 c
-p
*i-i
,
0
-.4
P.





4.0


.I I










an incomplete dominance (additive effect) of the resistance. There

was approximately a 10-fold resistance to the effects of apholate in

the resistant female genotype.

The sterility induced in the 4 male genotypes tested was not

as complicated as the data from the female experiments. The sus-

ceptible male and F1 male (susceptible male x resistant female), with

SC50 values of 2.26 and 4.14, were virtually the same in terms of

susceptibility to apholate. There was approximately a 24-fold level

of resistance to apholate in the resistant male. The SC50 for the

resistant male was 16.7 ppm greater than the resistant female, but

any difference between these two genotypes could have been exaggerated

due to the two different methods for calculating sterility. The total

reduction in fecundity was taken into account in the resistant female,

but no allowance was taken for sperm mortality or inactivation in

calculating the net sterility in the resistant male. If it is assumed

that in addition to dominant lethals in the form of deletions, chromo-

some breaks resulting in acentric fragments and dicentric chromosomes,

and other aberrations, there also occurred damage of another nature

that resulted in less motile sperm, then the data from the male

genotypes and female genotypes are not comparable for differences.

The greater tolerance of apholate exhibited by the susceptible female

over the susceptible male was probably due to the 10% of the females

mentioned previously that were partially refractory to apholate.

The SC50 value for the F1 male (resistant male x susceptible

female) was 19.5 ppm, which was about 10 times the SC50 of the





















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*









Table No. 4.


Summary of concentration-sterility data used to calculate
regression curve for the response of the resistant male
to apholate.


Concentration of No. No. Eggs To Net
Apholate (ppm) Eggs* Hatched % Sterility Sterility

Check 2034 1979 2.7

25 1566 1353 13.6 11.40

30 1189 922 22.5 20.30

35 1313 946 28.0 26.00

45 1431 904 36.8 35.00

50 1127 507 55.1 53.80

60 822 286 65.2 64.20

80 1174 132 88.8 88.40


Total of 3 replicates.


Table No. 5. Summary of concentration-sterility data used to calculate
regression curve for the response of the resistant female
to apholate.

Concentration of No. No. Eggs % Eggs Fecundity % Net
Apholate (ppm) Eggs* Hatched Hatched Frequency Sterility

Check 1924 1839 95.6 1.00

20 1602 1392 86.9 .83 28.0

30 1445 1092 75.6 .75 43.3

35 1279 921 72.0 .66 52.5

45 961 536 55.8 .50 72.1

60 692 271 39.1 .36 86.0


* Total of 3 replicates.









Table No. 6.


Summary of concentration-sterility data used to calculate
regression curve for the response of the susceptible
male to apholate.


Concentration of No. No. Eggs % Net
Apholate (ppm) Eggs* Hatched % Sterility Sterility

Check 5952 5591 6.1

.5 3299 2713 17.8 12.5

1.0 5208 3888 25.4 20.5

2.0 4931 2502 49.3 46.0

4.0 5086 1778 65.1 62.8

6.0 4524 713 84.3 82.3

8.0 4633 452 90.3 89.3


Total of 3 replicates.


Table No. 7. Summary of concentration-sterility data used to calculate
regression curve for the response of the susceptible
female to apholate.

Concentration of No. No. Eggs % Eggs Fecundity % Net
Apholate (ppm) Eggs* Hatched Hatched Frequency Sterility

Check 1934 1845 95.4 1.00

1 2142 1504 70.2 1.00 25.4

2 1730 1306 75.5 .89 29.6

4 1280 1001 80.7 .66 53-4

8 860 588 71.7 .44 68.1

12 586 252 45.1 .30 86.4

16 357 184 54.0 .18 90.1

20 264 72 28.6 .13 96.2


* Total of 3 replicates.









Table No. 8.


Summary of
regression
susceptiblel


concentration-sterility data used to calculate
curve for the response of the Fl male
e female x resistant male) to apholate.


Concentration of No. No. Eggs % Eggs % Net
Apholate (ppm) Eggs* Hatched Hatched % Sterility Sterility

Check 1368 1264 92.4 7.6 --

4 1185 1007 84.9 15.1 8.1

8 2799 1974 70.5 29.5 23.7

12 1588 1026 64.6 35.4 30.1

16 2380 1162 51.2 48.8 44.6

24 1297 527 40.6 59.4 56.1



* Total of 3 replicates.


Table No. 9. Summary of concentration-sterility data used to calculate
regression curve for the response of the Fl male
(resistant female x susceptible male) to apholate.

Concentrations of No. No. Eggs % Eggs % Net
Apholate (ppm) Eggs* Hatched Hatched % Sterility Sterility

Check 2541 2436 95.8 4.2

2 2188 1638 74.8 25.2 21.9

4 3204 1683 52.5 47.5 45.2

8 3197 743 23.2 76.8 75.8

12 3402 602 17.7 82.3 81.5

16 1359 39 2.8 97.2 97.1


* Total of 3 replicates.








Table No. 10.


Summary of
regression
(resistant


concentration-sterility data used to calculate
curve for the response of the Fl female
female x susceptible male) to apholate.


Concentration of No. No. Eggs % Eggs Fecundity % Net
Apholate (ppm) Eggs* Hatched Hatched Frequency Sterility

Check 1876 1674 89.2 1.00

4 1470 1325 90.1 .78 30.8

8 1292 1092 84.5 .68 42.6

12 1102 754 68.4 .58 60.4

16 970 646 66.6 .52 65.4


* Total of 3 replicates.


Table No. 11. Summary of concentration-sterility data used to calculate
regression curve for the response of the Fl female
(susceptible female x resistant male) to apholate

Concentration of No. No. Eggs % Eggs Fecundity % Net
Apholate (ppm) Eggs* Hatched Hatched Frequency Sterility

Check 1310 1016 77.5 1.00 22.5

8 1134 944 83.2 .86 28.5

16 779 534 68.5 .59 59.6

24 591 222 37.5 .45 83.2

32 359 169 47.1 .27 87.3


* Total of 3 replicates.








Table 12. Summary of probit analyses

SC 5
Genotype (ppm)

FI female (S female x R male) 16.87

F1 female (R female x S male) 8.94

S female 3.22

R female 31.61

FI male (S female x R male) 19.50

Fl male (R female x S male) 4.14

S male 2.26

R male 48.30


Table 13. Summary of data from the experiment conducted to measure
cross-resistance of the apholate-resistant male to metepa.

Concentration of No. No. Eggs % net
Strain Metepa (ppm) Eggs* Hatched Hatch Sterility

Susceptible Check 600 588 98.0 --
male
10 723 546 75.5 22.9

20 684 354 51.7 47.2

40 642 99 15.4 84.3

80 600 15 2.5 97.4


Resistant
male


Check

10

20

40

80


588

693

621

627

750


537

618

507

470

435


91.3

89.2

81.6

74.9

58.0


2.3

10.6

18.0

36.5


* Total of 3 replicates.


of the data in Tables 4-11.

b Fiducial Limits (P=.95)
(Slope) Lower Upper

4.10 14.5 19.5

1.55 4.1 19.3

1.76 1.8 5.6

3.47 27.4 36.4

2.01 9.5 39.9

3.02 3.2 5.4

1.98 1.4 3.4

4.47 45.2 51.6










susceptible male. The fiducial limits did not overlap 'itri any; of the

other 3 male genotxpes, and tie data showed a definite paternal effect.

From these data it appeared the resistance was expressed as an incom-

plete dominant (additive effect) and was linked with the sex: locus.

Eased on the fiducial limits (P = .95) of the SC50 values and

the log concentration-probit sterility curves (Figures 1 ard 2), a

concentration that would separate the resistant male from the sus-

ceptible male genotypes was selected at 20 ppm of apholate. The data

from the female genotypes indicated that the selection of a concentra-

tion to separate the resistant from susceptible was impossible; there-

fore, it was decided to use the males for determining the mode of in-

heritance of the resistance.


Cross-Resistance Experiments

It was reasonable to expect that the apholate-resistant strain

of A. aegypti would demonstrate resistance to other aziridine com-

pounds. The data collected in the experiments to determine the

sterility induced by tepa and metepa in the resistant and susceptible

strains are presented in Tables 13, 14, 15 and 16. The net per cent

sterilities from the treated male groups were graphed (Figure 3) and

estimates of the SC50 values were taken from this graph. The data

indicated a 5-fold tolerance of metepa and a 3-fold tolerance of tepa

by the resistant males. The SC50 value for metepa on the resistant

male might not be valid, because it was necessary to make a consider-

able extrapolation beyond the actual data.

The data from the female groups (Tables 14 and 16) indicated

that the resistant female was more tolerant of metepa and tepa than































Figure 3. Regression lines visually fitted to data from the experiments
designed to measure cross-resistance to tepa and metepa in
the apholate-resistant (R) male.


A R male treated with tepa.
A S male treated with tepa.
o R male treated with metepa.
0 S male treated with metepa.




























































5 10 20 50


Log Concentration (ppm)


48

















6.0










5.0 4
tp
*H





o
40






4.0


100









Table No. 14.


Summary of data from the experiment to measure cross-
resistance of the apholate-resistant female to metepa.


Concentration of
Strain Metepa (ppm)


No. Eggs
in Sample*


No. Eggs % % Net
Hatched Hatch Sterility


Susceptible
female


Check

10


600

630

678

657

360


513

384

171


98.0 --

80.0 17.4

75.6 22.8

58.4 40.4

47.5 51.5


Resistant
female


Check


600

600

6oo

648

600


495

417

495

333

387


82.5

69.5 16.9


82.5


0.0


51.4 37.7

64.5 14.8


* Total of 3 replicates.









Table No. 15. Summary of data from the experiment conducted to measure
cross-resistance of the apholate-resistant male to tepa.


Strain

Susceptible
male


Concentration of
tepa (ppm)

Check

2.5

5.0

10.0

20.0


No.
Eggs*

600

561

600

600

600


No. Eggs
Hatched

582

216

108

60

0


Hatch

97.0

38.5

18.0

10.0

00.0


o Net
Sterility


60.3

81.4

89.7

100.0


Resistant
male


* Total of 3 replicates.


Check

2.5

5.0

10.0

20.0


600

600

609

600

600


516

441

252

60

0


86.0

73.5

41.4

10.0

00.0


14.5

51.8

88.4

100.0










Table No. 16.


Summary of data from the experiment conducted to measure
cross-resistance of the apholate-resistant female to
tepa.


Concentration of No. No. Eggs % % Net
Strain Tepa (ppm) Eggs* Hatched Hatch Sterility

Susceptible Check 600 582 97.0 --
female
2.5 615 477 77.6 20.0

5.0 621 447 71.9 25.8

10.0 612 339 55.4 42.8

20.0 no eggs -- -- 100.0


Resistant Check 639 537 84.0
female
2.5 600 474 79.0 5.9

5.0 654 546 83.5 0.6

10.0 600 360 60.0 28.6

20.0 no eggs -- -- 100.0


* Total of 3 replicates.










Table No. 17.


Summary of mortality in the apholate-resistant and
susceptible strains after treatment with increasing
concentrations of metepa.


Metepa Male Susceptible Strain Resistant Strain
Concentration of No. No. Net % No. No. Net %
Metepa (ppm) Treated* Dead Mortality Treated Dead Mortality


300

300

300

300

300


1 .3

0 --

28 9.3

18 6.0

60 20.0


Metepa Female


300

300

300

300

300


0

13 4.3

11 3.7

67 22.3

65 21.6


* Total of 3 replicates.


Check


300


300

300


2.0

19.0

27.0


Check


300

300

300

300

300


2.0

1.7










Table No. 18.


Summary of mortality in the apholate-resistant and
susceptible strains after treatment with increasing
concentrations of tepa.


Tepa Male
Concentration of
Tepa (ppm)

Check

2.5

5.0

10.0

20.0


Susceptible
No. No.
Treated* Dead

300 10

300 28

300 48

300 67

300 262


Strain
Net %
Mortality

3.3

9.3

16.0

22.3

87.3


Resistant Strain
No. No. Net %
Treated Dead Mortality

300 29 9.7

300 17 5.7

300 30 1o.o

300 150 50.0

300 243 80.9


Tepa Female

Check

2.5

5.0

10.0

20.0


* Total of 3 replicates.


300

300

300

300

300


10

52

34

55

288


3.3

17.4

11.3

18.3

96.1


300

300

300

300

300


36

26

19

110

250


12.0

8.7

6.3

36.6

83.5


I










the susceptible female. A reduction in fecundity was noticed to

correspond to increasing concentration in the females treated with

both tepa and metepa; however, the reductions in the tepa treatments

were more severe than the metepa treatments.

Mortality data collected during these experiments are presented

in Tables 17 and 18 for metepa and tepa, respectively. Mortality

as a result of the metepa treatments was low; however, a greater

mortality was noticed in the resistant female compared to the sus-

ceptible female.

Mortality in the tepa treatments was quite severe, and no major

differences existed between strains. At 20 ppm there were only a

few individuals that survived to become adults. Most of the mortality

was seen in the pupal stage and emerging adults. The dead adults

were apparently very weak and never left the surface of the water.

Larval mortality was negligible in all treatments, but at the highest

concentrations of both compounds, some larvae required nearly a week

to complete the fourth instar and pupate. These pupae invariably

died.


Non-Selected Apholate-Resistant Strain

The expected loss of resistance after termination of apholate

selection in the resistant strain was not observed through 9 genera-

tions. Figure 4 summarizes the data from this experiment in terms

of per cent hatch observed in each generation. The level of resistance

in the non-selected colony increased compared to the regular apholate-

resistant colony and maintained a per cent hatch from a minimum of

15% to a maximum of 30% greater than the selected colony. Closer






























Summary of data from the experiment to determine the
permanence of apholate-resistance in the absence of apholate
selection pressure. Each point on the graph is the average
of two replicates.


* non-selected apholate-resistant strain.

0 assay groups from the non-selected strain
treated with 25 ppm apholate.

A apholate-resistant strain treated each
generation with 25 ppm apholate.


Figure 4.
















































Fl F2 F3 F4 F F6


Generation


100


90


80


70


60
:h
50


40


30


20


10










examination of the data indicated that this difference could have

resulted from a loss of genetic damage. This statement is based on

the data from the Fl generation, which placed the assay group of

resistant males almost half-way between the regular apholate-selected

colony and the non-selected colony in the parent generation. In the

succeeding 3 generations, there was an apparent increase in the

resistance level, but then a decrease occurred in the F5. The

lowest per cent hatch occurred in the F6 generation, but this

deviation from the other data could have possibly resulted from

the temperature of the rearing room, which was 50 F lower than

usual and delayed pupation by 1 day. This extra day of treatment

probably increased the sterility induced.

Instead of losing apholate resistance, the non-selected colony

became more tolerant of apholate and after 9 generations maintained

a higher per cent hatch when treated with apholate than the regular

apholate-resistant colony. The non-selected resistant strain also

increased in per cent hatch above the 90% level and maintained that

level for the duration of the experiment. Males of a susceptible

colony maintained as a control were completely sterilized each

generation by the apholate treatment.


Genetic Crosses

The results from the F2 crosses and backcrosses are presented in

Tables 19 and 20, respectively. These data did not fit any genetic

models for 3 genes or less, and since there are only 3 linkage groups

in A. aegypti, a quantitative type of inheritance was probably involved

in apholate resistance. There was a definite paternal effect on the









Table Dio. 19.


Swumiary of sterility in F2 progeny, treated with 20 ppm
of apholate, of Cross A: Fl female (S s blt female x
R i.rld male) x Fl male (S sp blt female x R wild male)
and. Cross B: Fi female (R wild female x S so bit male)
: F1 male (R wild female x S sa bit male).


Cross Marker Phenotype No. Sterile No. Fertile Total


A + + 61 178 239

+ bit 20 57 51

sp + 14 37 77

sp blt 5 4 9


Total 100 276 376


E + + 89 0 89

+ bit 22 2 24

sp 49 2 51

sp bit 13 0 13


Total 173 4 177









Table No. 20.


Summary of sterility in backcross progeny, treated with
20 ppm of apholate, of Cross C: Fl male (S blt female x
R wild male) x S blt female, Cross D: FI female (S blt
female x R wild male) x S blt male, Cross E: Fl female
(R wild female x S blt male x S blt male, Cross F:
FI female (R female x S male) x R male, and Cross G:
Fl male (S female x R male) x R female.


Cross Marker Phenotype No. Sterile No. Fertile Total


C + 66 72 138

bit 69 72 141


Total 135 144 279


D + 78 24 102

blt 81 12 93


Total 159 36 195


E + 147 12 159

blt 126 6 132


Total 273 18 291


F + 108 84 192

G + 5 174 179










resistance of the males, and apparently the gene (s) on the se:x:

chromosome had a greater effect when coupled to the male locus.

This was easily seen when comparing the number of resistant males

of Crosses A to B and C to D.

Although it was quite clear that resistance was manifest in

more males that bai a resistant male ancestor in the parent cross,

as opposed to those with susceptible male ancestors, the mechanism

for such a phenomenon was not clear. An expression of the resistance

as a dominant when coupled with the male locus did not fit the data,

as obviously the resistance was manifest in repulsion (Cross D) in

some individuals and absent (Crosses A and C) in coupling in many

individuals. The large number, about 50%, of sterile males in Cross

C indicated tnat the resistance was certainly not due to a single

dominant gene and linkage data with blt as a marker in Cross C and

w-ith 5p and blt as markers in Cross A clearly showed that the resistant

phenotype was not exclusively dependent on either of linkage groups

2 and 3. Therefore, a rather complicated mode of inheritance appeared

to be involved. Perhaps differential expressivity of the resistant

genotype was involved, but certainly this would have been expressed

in the concentration-sterility curves established for the F1 males,

whereas it was not. Therefore, the expression of apholate resistance

appears to be complicated and quantitative in nature with more than

3 genes involved. The sex chromosome derived from the male parent

probably had a major role in the resistant phenotype, but the exact

nature and mechanism of this role was not evident from the data.










Cytogenetic Experiment

Examination of chromosomes from the larval brain and adult

testes divulged only one aberration in the resistant strain from

the normal karyotype seen in the susceptible strain. The single

aberration is shown in Figure 5c in the form of a deletion or

absence of stainable nucleic acid in one arm of chromosome III

(according to Rai, 1963a). This deletion, however, was observed

only in one cell in this particular squash, with other cells on the

slide normal. The number of brain squashes examined were 36, 27, 20,

and 20 for the resistant, susceptible, Fl (susceptible female x

resistant male), and Fl (resistant female x susceptible male)

genotypes, respectively. Figures 5b and 5d are representative of

the brain mitoses at metaphase examined in the apholate-resistant

mosquitoes. Figures 6c, 5a, 6d, and 6a show mitotic prophase, meta-

phase, anaphase and telophase from the susceptible strain. The lack

of observable aberrations corresponded to the data from the non-

selected resistant strain. Those data indicated a loss of genetic

damage by an increase in per cent hatch after selection was terminated.

Major aberrations such as translocations would have persisted in the

population for several generations'(Curtis, 1968). Terminal breaks

and relatively large deletions that produced no dominant effects would

have also persisted for some time. However, the per cent hatch in the

non-selected resistant strain increased to a value comparable to

normal hatch in A. aegypti that were never exposed to apholate in one

generation.

































Figure 5. Mitotic chromosomes from brain tissue of Aedes aegypti.


a. Apholate-susceptible strain.

b. Apholate-resistant strain.

c. Apholate-resistant strain; note apparent deletion.

d. Apholate-resistant strain.








63







....... ... ..




...............






























Mitotic chromosomes from brain tissue of apholate-susceptible
strain of Aedes aegypti.

a. Normal telophase.

b. ,Metaphase from mosquito treated with 25 ppm apholate
during fourth larval instar.

c. Normal prophase.

d. Normal anaphase.


Figure 6.













Figures 6b and 7a-7d show the chromosomal damage caused by

treatment of the susceptible strain with 20 ppm of apholate.

Chromosomal stickiness and a loss of normal shape and form are seen

in Figures 6b, 7a, and 7c. In Figure 7d, the chromosomes appeared

to have constrictions which were not normal. Normal meiotic

figures from adult squashes are shown in Figures 8a-9d. No more

than 2 chiasmata per bivalent were observed, which agreed with

Mescher and Rai (1966). Figures 9a and 9b demonstrate the diffi-

culties encountered in working with the pupal testes for squashes.

The chromosomes in metaphase I and anaphase I adhered to form a

single chromatic mass. It was impossible to distinguish individual

chromosomes, and at the first observation it appeared as though

the mosquitoes had been accidentally exposed to apholate.

In accordance with Mescher and Rai (1966), the earliest stage

at which chromosomes became clearly visible was pachytene. They

attributed this lack of visible leptotene and zygotene stages to

somatic pairing of chromosomes in the mitotic division preceding

meiosis. Pachytene apparently is of long duration relative to other

stages in A. aegypti and was present in all individuals examined in

much greater numbers than the other stages.































Figure 7. Mitotic chromosomes from brain tissue of apholate-susceptible
strain of Aedes aegypti treated with 25 ppm of apholate
during fourth larval instar.














V*































Figure 8. Meiotic chromosomes from adult testes of Aedes aegypti:
a., b., and d., diplotene; c. diakinesis. Note maximum
of two chiasmata per bivalent.











































C*
'. . 7* i
p Ar
#th~

lb


II.
p I I, -
0~r
B
* *


V0


* .4
SJr '
D
S~


ii


I I


a.~i
~J4.


.































Figure 9. Meiotic chromosomes from testes of Aedes aegypti:

a. and b., chromosomes prepared from pupal testes, which
appear as a single chromatic mass,

c. prophase II from adult testis.

d. metaphase II from adult testis.







.1


Ot


'1ri

p.ii
0E
SI
p
U-


A *


4*1
Q


I
S*

'4$>)
r
^w
OsIF f-


Ir qli 4
l t







I, .
-V 'L
Pik"* "^ ;



I.. ^ ^"













SUMMARY AND CONCLUSIONS


Chemosterilant resistance has been of concern to entomologists

since the initial discoveries that certain compounds would effectively

render insects sterile. Many researchers were familiar with the

problems encountered in development of insecticide resistance by

field populations of insects, and this prompted numerous groups of

investigators to attempt to induce chemosterilant resistance in insects

under experimental conditions in the laboratory. Resistance to

apholate was subsequently developed in Aedes aegypti (L). This investi-

gation was undertaken to elucidate the mode of inheritance of the

apholate resistance in the species mentioned above, to determine

whether any cross-resistance to tepa and metepa was evident in the

apholate strain, and to study the permanence of the resistance through

succeeding generations upon removal of selection pressure. In addition,

a study was made on the chromosomes of the apholate-resistant strain

to determine whether the mutagenic agent, apholate, had induced large

deletions, terminal breaks, and translocations in the population,

which had been exposed to apholate for more than 70 generations.

The mosquitoes were exposed to the chemosterilant in the larval

stage. Sterility-concentration curves were established for each sex

of the resistant, susceptible, and F1 hybrid genotypes, and SC50

values were calculated by probit analysis for each genotype. The SC50

values were 48.30 for the resistant male, 2.26 for the susceptible










male, 4.1i for the Fl male (resistant female x susceptible male), and

19.50 for the Fl male (susceptible female x resistant male); and

31.61 for the resistant female, 3.22 for the susceptible female,

8.9h for the F1 female (resistant female x susceptible male), and

16.87 for the F1 female (susceptible female x resistant male).

From the concentration-sterility data, 20 ppm of apholate was

selected as a concentration to discriminate the resistant male and

F1 male (susceptible female x resistant male) genotypes from the

susceptible male and F1 male (resistant female x susceptible male)

genotypes. Progeny from genetic crosses were subjected to 20 ppm

of ap'iolate for bioassay in order to assess resistance. The results

of the crosses indicated that apholate resistance in A. aegypti was

a quantitative trait, with the additional complication of a marked

paternal influence on the resistance of F2 and backcross progeny.

There was no marked cross-resistance to tepa and metepa present

in the apholate-resistant strain, but there were increased tolerances

of 5-fold to metepa and 2.5-fold to tepa in the resistant males.

Estimates of the tolerance levels of tepa and metepa were gained by

examining probit sterility-log concentration graphs of the data

collected from treated males of the resistant and susceptible strains.

There was no loss of resistance through 9 generations after

selection pressure was removed from a sub-colony of the apholate-

resistant strain. The resistance level, which was based on per cent

hatch, actually increased during the first 4 generations, but then it

decreased and remained fairly stable through the ninth generation.










The examination of chromosome squashes of the apholate-resistant

mosquitoes for major chromosomal aberrations were negative, except

for an apparent deletion found in a single cell of one individual.

The lack of major aberrations substantiated the rate of disappearance

of an inherent sterility of about 20% in 2 generations after apholate

selection was terminated.

It is suggested that future investigations use an approach to

the selection of genetic crosses to yield information concerning

the heritability of the trait.




































APPENDIX










APHOLATE


Chemical Name: 2,2,4,4,6,6-hexakis (l-arizidinyl) 2,2,4,4,6,6-

Hexahydro-1,3,5,2,4,6,-triazatriphosphorine.

Other Designations: SQ 8388

ENT 26316

OM 2174


Structural Formula:


P
N


Where R is:


Physical Properties:

White Powder

M. P. 1500 C

Molecular formula C12H24N9P3

Molecular weight 387.4

Approximate solubility (% weight)

Water: 33%

Ethanol: 15%

Chloroform: 31%










APHOLATE (Cont. 'd)

Approximate solubility (% weight) (Cont.'d)

Methanol: 14%

Acetone, xylene, mineral oil, hexane: 1%

Stability: Moisture, high temperature, low pH, proteinaceous

materials and highly sorptive carriers cause poly-

merization.


Chemical Name: tris

Other Designations:


METEPA

(2-methyl-l-aziridinyl) phosphine oxide.

Metaphoxide

Methyl Aphoxide

ENT 50003

M APO


Structural Formula:


R P


Physical Properties:

Molecular formula:

Molecular weight:


-R Where R is






C9H18N30P

214.9


TEPA

Chemical Name: tris (l-aziridinyl) phosphine oxide.

Other Designations: Aphoxide

APO

ENT 24915










TEPA (Cont.d)

Structural Formula:


0


R ---P -R


R


Where R is


CH2


Physical Properties:

Molecular formula:

Molecular weight:


C6HI2N30P

172.9




































LITERATURE CITED














LITERATURE CITED


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BIOGRAPHICAL SKETCH


Jack Arlyn Seawright was born September 9, 1941, at Ware Shoals,

South Carolina. In 1959, he was graduated from Ware Shoals High

School. He received the degree of Bachelor of Science with a major

in Biology from Clemson University in January, 1964. In December,

1965, he received the degree of Master of Science with a major in

Entomology from Clemson University. From August, 1965, he has been

enrolled at the University of Florida in pursuit of the degree of

Doctor of Philosophy with a major in Entomology.

Jack Arlyn Seawright is married to the former Mattie Rebecca

Medlin and is the father of a son, David Arlyn, and a Daughter,

Amy Lauren. He is a member of Gamma Sigma Delta and the Entomological

Society of America.









This dissertation was prepared under the direction of the

chairman of the candidate's supervisory committee and has been

approved by all members of that committee. It was submitted to

the Dean of the College of Agriculture and to the Graduate Council,

and was approved as partial fulfillment of the requirements for the

degree of Doctor of Philosophy.


December, 1969





ean, College of Agriculture





Dean, Graduate School






Supervisory Committee:




Chairman






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