IN Y1YQ STUDIES OF SUSPECTED MECHANISMS OF DDT-RESISTANCE
IN BLATTELLA GERM.ANICA (L.)
by
George Lawrence Rolof son
Thesis submitted to the Graduate Faculty of the
Virginia Polytechnic Institute
in partial fulfillment for the degree of
DOCTOR OF PHILOSOPHY
in
Entomology
APPROVED: Donald G. Cochran
James McD. Grayson Mary H. Ross
Ryland E. Webb David A. West
Blacksburg, Virginia
May 1968 TABLE OF CONTENTS
Page I. INTRODUCTION , ...... 1 II. LITERATURE REVIEW ...... 3 Early Insecticide Resistance •• ...... 3 Development of DDT • ...... 4 Development of DDT Resistance in Houseflies . . . . 6 DDT-Resistance in Cockroaches and Other Insects 13 Inheritance of DDT Resistance ...... 18
Mode of Action of DDT ...... ~ . . . . 23 DDT Synergism by Sesamex • • • • ...... 35 III. METIIODS AND MATERIALS ...... 42 Cockroach Strains • • • • • • 42 Treatment Procedure ...... 43
Sample Extraction and Cleanup it • • • • • • • • 44 Quantitation of DDT and Metabolites ...... 47 Thin Layer Chromatography ...... 48 IV. RESULTS AND DISCUSSION • • • ...... 50
Toxicological Date • . . . • • • if • • • 50 DDT Recovery , • • • • • • • • ...... 52 Penetration ...... 52 Detoxication • ...... • • 66 Excretion • • • • • • • • • • • • • • • • • • • 102
Combined Effects • • • ~ • • ~ j • • • • • ~ • ~ • • • • 122
ii iii
Page v. STJMMARY 133
VI. REFERENCES CITED . . .. 135 VII. VITA. 154 ACKNOWLEDGEMENTS The writer wishes to express his appreciation to Dr. Donald
G. Cochran for his helpful criticisms and suggestions throughout the duration of this program. Appreciation is also extended to Dr.
Jack L. Bishop for his helpful suggestions in the early part of this work and to Dr. James McD. Grayson for his continuous thoughtful encouragement. The writer is grateful to Drs. Cochran, Graysoni Ross, Webb and West for their critical reading of this manuscript and to Professor Rodney Young for the use of his laboratory and equipment. Appreciation is also extended to Mrs. Jean Dickinson, Mr. J. E. Dunwoody, and Mr. Ty Ku who provided valuable consultation throughout this study.
Finally, the writer would like to acknowledge his laboratory assistants, Mrs. Frank Marshall and Mrs. Herbert Thomas, for their faithful work in this project. Recognition is due to Mrs. John Proco for typing this manuscript.
iv LIST OF ABBREVIATIONS 1. DDT - 2,2-bis-(p-chlorophenyl)-1,1,1-trichloroethane. 2. DDE ... 2,2-bis-(p-chlorophenyl)-1,1-dichloroethylene.
3. ODA - di(p-chlorophenyl) acetic acid. 4. DBP ... 4,4·' ·dichlorobenzophenone. 5. DDD (TDE) - 2,2-bis-(p-chlorophenyl)-1,1-dichloroethane.
6. dicofol - 2,2-bis-(p-chlorophenyl)-1,1,1-trichloroethanol.
7. sesamex (sesoxane) - acetaldehyde 2·(2-ethoxy-ethoxy) ethyl 3:4 methylene-dioxyphenyl acetal. 8. F-DMC - bis-(p-chlorophenyl)-trifluoromethyl-carbinol. 9. NAD - oxidized nicotinamide adenine dinucleotide. 10. NADPH - reduced nicotinamide adenine dinucleotide phosphate. 11. DMC • bis•(p-chlorophenyl)•methyl-carbinol.
v LIST OF FIGURES Figure Page 1. Penetration of 8 ug. of DDT in male and female susceptible- strain German cockroaches ...... • 56 2. Penetration of 8 ug. of DDT in resistant and susceptible male German cockroaches • ...... • • 58 3. Penetration of ·30 ug. of DDT in resistant and susceptible
German male cockroaches • t • I • e f I e f + • • t t f • • • 60 4. Penetration of 30 ug. of DDT in VPI•DDT-strain male and female German cockroaches ...... • • • 62 5. Penetration of 30 ug. of DDT in male and female Landsthul- strain German cockroaches ...... • • 64 6. Penetration of three DDT treatment levels in male VPI- DDT-strain German cockroaches • • • • • • • • • • • • • • • • 67 7. Penetration of two DDT treatment levels in male Landsthul• strain German cockroaches . . . . . - ...... • • • • 69 8. Penetration of 8 ug. of DDT in susceptible-strain male cockroaches pretreated with two different levels of
sesamex • • • • • • • • • • • t • • • • • • • • • - • • • • • 71 9. Penetration of 30 ug. of DDT in VPI-DDT•strain male German cockroaches following pretreatment with three levels of sesamex • • • • • • • • • • • • • • • • • • • • •.• 73 10. Penetration of 30 ug. of DDT in Landsthul•strain male German cockroaches following pretreatment with three
levels of sesamex • • • • • • • • • • • • • • • • • • • • • • • 75
vi vii
Figure Page
11. Conversion of 8 ug. of DDT to dicofol by susceptible•strain male and female German cockroaches ...... 79 12. Conversion of 8 ug. of DDT to dicofol by male VPI•DDT- and susceptible~strain German cockroaches • . . • • • • • • . . . 81 13. Conversion of 30 ug. of DDT to dicofol in males of two resistant and one susceptible strain of German cockroaches 83 14. Conversion of 30 ug. of DDT to dicofol by male and female
VPI•DDT•strain German cockroaches . • • • • • • • • • • • • • 87 15. Conversion of 30 ug. of DDT to dicofol by male and female
Landsthul-strain German cockroaches • • • • • • • • • • • • • 89
16. Conversion of the penetra~ed dose from three different DDT treatments to dicofol by VPI-DDT•strain male German cockroaches • • • • • • ...... 91 17. Actual dicofol produced by VPI•DDT•strain male German
cockroaches following treatment wi~h three different dosage levels of DDT • • • • • • • • • • • • • • • • • • • · • 93
18. Conversion of 8 ug. of DDT to dicofol by susceptible•
strain male German ~ockroaches in the presence of two
levels of sesamex • • • • • ...... Ill • • • • • • • • • 96 19. Conversion of 30 ug. of DDT to dicofol by VPI•DDT•strain male German cockro•ches following pretreatment with three
different levels o~ sesamex • • • • • • • • • , • • • • • • • 98 viii
Figure
20. Conversion of 30 ug. of DDT to dicofol by Landsthul~strain male German cockroaches following pretreatment with three different levels of sesamex • • • • • • • • • • • • • • • • • 100 21. Excretion of DDT by male and female susceptible-strain
German cockroaches following treatment with 8 ug. of DDT
per insect • • • • • • • • • • • • • • • • • • • • • • • • • • 104 22. Excretion of DDT by male and female VPI•DDT•strain German cockroaches following a treatment of 30 ug. of DDT per
insect • • • • • • • • • • • • • • • • • • • • • • • • • • • • 106 23. Excretion of .DDT by male and female Landsthul-strain German.
cockroaches following a 30 ug. treatment of DDT per insect • • 108 24. Excretion of DDT by VPI-DDT- and susceptible-strain male
German cockroaches following treatment with 8 ug. of DDT
per insect • • • • • • • • • • • • • • • • • • • • • • • • • • 111 25. Excretion of DDT following a 30 ug. DDT treatment by two resistant• and one susceptible-strain of male German
cockroaches • • • • • • • • • • • • • • • • • • • • • • • •• 113 26. Excretion of DDT by VPI•DDT-strain male German cockroaches after treatment with three different dosage levels of
DDT . . ·• • • • • • • • • • • • • • • • • • • • • • • • .. • • 115 27. Actual ug. of DDT excreted by male VPI .. DDT•strain German cockroaches following DDT treatments of three different dosage
levels • • • • • • • • • • • • • • • • • • • • • • • • • • • 118 ix
Figure
28. Excretion of DDT by susceptible-strain male German cock-
roaches following treatment with 8 ug. of DDT in combination
with two levels of sesamex • • • • • • • • • • • • • • • • • 120 29. Excretion of DDT by VPI-DDT-strain male German cockroaches
following treatment with 30 ug. of DDT in three different
combinations with sesamex • • • • • • • • • • • • • • • • • 123 30. Excretion of DDT by Landsthul-strain male German cockroaches
following treatment with 30 ug. of DDT in three different
combinations with sesamex • • • • • • • • • • • • • • • • • 125 31. Internal DDT concentrations in VPI•DDT•strain male German
cockroaches following three different treatments of DDT • • 128 LIST OF TABLES Table Page I. Six-day mortality counts in susceptible-strain male German cockroaches following treatment with 8 ug. of DDT alone and
in combination with several levels of sesamex (Ses.) ••• • • 51 II. A typical thin-layer chromatographic confirmatory analysis for DDT and dicofol in internal and excreta sample fractions • 53 III. Penetration of several dosage levels of DDT in resistant and susceptible German cockroaches in presence and absence of
sesamex (Ses.) • • • • • • • • • • • • • • • • • • • • • • • • . 54 rv. Detoxication of several dosage levels of DDT in resistant and susceptible German cockroaches in presence and absence of
sesamex {Ses.) • • • • • . • • • • • • • • • • • • • • • • • • 78 V. Excretion of DDT by resistant and susceptible German cock• roaches following treatment with several dosage levels of
DDT in presence and absence of sesamex (Ses.) • • • • • • • • 103 VI. Internal concentrations of DDT in resistant and susceptible German cockroaches following three different treatment levels
and 127 in presence absence of sesamex (Ses.) • • • • • • • • • • • LIST OF TABLES IN APPENDIX. A Table Page I. DDT and dicofol recovered from resistant and susceptible German cockroaches after treatment with 8 ug. of DDT per
insect . . . . . • • • • • • • • • • • • • • • • • • • • • • 156 II. DDT and dicofol recovered from susceptible German cock·
roaches after treatment with sesamex and 8 ug. of DDT per
insect • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1S7 III. DDT and dicofol recovered from resistant and susceptible German cockroaches after treatment with 30 ug. of DDT • • • • 158 rv. DDT and dicofol recovered from resistant VPI-DDT-strain German cockroaches after treatment with sesamex and 30 ug. of DDT per insect • • • • • • • • •. • • • • • • • • • • • • • 159 v. DDT and dicofol recovered from resistant Landsthul-strain German cockroaches after treatment with 30 ug. of DDT per
insect • • • • • • • • • • • • • • • • • • • • ...... •• 160 VI. DDT and dicofol recovered from resistant Landsthul-strain
German cockroaches ~fter treatment with sesamex and 30 ug.
of DDT per insect ••••. • ••••••••••••••• • • 161 VII. DDT and dicofol recov-ered from resistant German cockroaches
after treatment with 150 ug. of DDT per insect • • • • • • • • 162
xi I. INTRODUCTION
The development of resistance to insecticides by insects has been under intense investigation for about twenty years. This area is probably one of the most active areas of entomological research at the present time. The development of DDT resistance by the house fly, Musca domestica (L.), alerted entomologists to the problems they were to be confronted with in their efforts to control insect populations.
DDT was the first successful modern-day synthetic organic insecticide.
When it became obvious that insects could develop a tolerance for this insecticide in their environment, it also became apparent that a similar situation could develop for other synthetic materials used extensively in insect control programs. This has, in fact, been observed to occur in the case of the synthetic cyclodiene, organic phosphate, and carba- mate insecticides, to mention only the most common examples.
In the case of a few insect· species, the mechanism of resistance to DDT and to certain other toxicants has been successfully determined.
For example, the DDT-resistance of certain house fly strains can be nearly completely attributed to their ability to detoxify this compound to the non-toxic derivative, DDE (Lovell and Kearns 1959). Reduced penetration and increased excretion have been similarly related to the
DDT resistance level of other insects (O'Brien 1967). In most cases, however, efforts to determine the DDT resistance mechanism have been less successful. Many investigators have suggested that it resides at the site of toxic action of this insecticide. Current evidence indicates that the site of action is on or near the nerve membrane,
- 1 - - 2 -
but the precise mechanism of DDT action is not yet known (O'Brien
1967). The German cockroach, Blattella germanica (L.), has been shown
to develop laboratory and field resistance to DDT and other insecticides
(Grayson 1951 and Heal~ al. 1953). It has also been shown that DDT-
resistant German cockroaches are capable of metabolizing DDT to the non•
toxic metabolite, dicofol (Kelthane), by a microsomal oxidation process
(Agosin ~al. 196la).
Considering these facts together with the results of recent sesamex
synergism studies on insecticide metabolism, further investigation of
DDT resistance in this insect seemed appropriate. Therefore, the .
objectives of this study were as follows:
1. To compare the rates of penetration, metabolism, and excretion ·
of DDT between resistant and susceptible strains of the
German cockroach.
2. To determine the effect of the synergist sesamex on DDT
metabolism in the German cockroach. II. LITERATURE REVIEW
Early Insecticide Resistance
The first report of the development of insecticide resistance in insects was published in 1914 by Melander who observed the San Jose' scale, Aspidiotus perniciosus (Comst.) to be increasingly more difficult to kill. He showed that in certain localities this scale insect was apparently becoming resistant to lime-sulfur sprays after regular treatment over an extended period of time. Two years later Quayle
(1916) reported a strain of the California red scale, Aonidiella aurantii (Mask.) on citrus trees in Southern California, to be far more difficult to control with cyanide fumigation than the general population of red scale in most other localities. He also noted resistance to HCN in the black scale, Saissetia oleae (Bern.). Quayle at first thought that this was due to some defect in the fumigation procedure. He was later able to show that the individuals which were alive after one HCN fumigation were more resistant to a second fumigation than individuals which had not been previously exposed
(Quayle 1922). These reports were substantiated by Woglum (1925).
Boyce (1928) showed similar results using adult Drosophila melanogaster
(Meig.), when he developed strains in the laboratory which were resistant to HCN fumigation.
The early observations of resistance were not limited to the
Coccidae or to the effects of contact sprays or gases. Hough (1928,
1934) called attention to the difference in the ability of larvae of
- 3 - - 4 -
two strains of the codling moth, Carpocapsa pomonella (L.), to enter
apples sprayed with lead arsenate. He showed that larvae from the
Grand Valley of Colorado demonstrated a marked superiority over larvae
from Virginia in their capacity to enter sprayed apples. These
investigations demonstrated the existence of different strains of the codling moth and provided support for the developing theory that resistance was due to the occurrence of individuals within a species which differed from other individuals in some aspect of their biology.
Morphological differences were either very slight or completely absent.
Several other investigators also provided support for this theory
(Painter 1930; Thorpe 1931; Smith 1941).
In 1942iZnipling reported on the acquired resistance to phenothiazine by larvae of the primary screwworm, Cochliomyia hominivorax (Coq.), and in 1947, Whitnall and Bradford reported the resistance of the one host blue tick, Booehilus decoloratus Koch, to arsenic.
The preceeding examples represent a brief outline of the develop• ment of resistance in insects to some of the earlier insecticides. A more complete review of this subject was presented in papers by Quayle
(1943), Babers (1949), and Browri (1951).
Develoement of DDT
DDT was first synthesized and described in 1874 by Otto Zeidler.
He reacted anhydrous chloral with chlorobenzene in the presence of concentrated sulfuric acid and showed chemically pure DDT to be a crystalline solid, practically colorless and odorless, rather stable, and insoluble in water, but soluble in most organic solvents. The insecticidal properties of DDT were discovered in 1936 by Paul Muller, - 5 -
a chemist in the Basle, Switzerland laboratories of J. R. Geigy, A. G.
Initially, the insecticidal use of this material was covered by a
number of patents, the first application being filed in Switzerland
in 1940. This and other information on the early development of DDT
is presented in articles by Annand ~ .!!_. (1944) and Cristol and Haller
(1945). A detailed listing of the foreign literature on DDT was
prepared by Roark (1945).
The first patent governing the use of DDT in the United States
was awarded to Muller in 1943. One of the more accurate accounts of
DDT's introduction into this country was published by Froelicher (1944). Draize --et al. (1944) published some of the first reports on toxicological · studies of DDT, and some of the results of early insecticidal tests by workers in the Bureau of Entomology and Plant Quarantine were published
by Annand~ al. (1944). These and other results indicated that DDT was extremely active against a wide range of insect pests.
In the toxicological studies of DDT it became desirable to gain
some knowledge of its metabolic fate. White and Sweeney (1945) reported
that the DDT metabolite in rabbit urine, following DDT ingestion, was
DDA. Neal ~ .!!_. (1946) reported excretion of the same metabolite in
the urine of a human subject.
The need for a sensitive method of detection and determination of
DDT soon became obvious. In 1945, Schecter ~ al. reported the
development of such a technique. Their procedure depended upon in•
tensive nitration of DDT to polynitro derivatives and the production
of intense colors upon addition of methanolic sodium methylate to a - 6 -
benzene solution of the nitration products. This method replaced
the less sensitive chlorine determination methods of detection which
had been used previously. It became known as the Schecter•Haller
reaction, after Schecter and one of his coworkers, and was used exten•
sively for the following 10 to 15 years in the detection of DDT and
related compounds. It has now been replaced by more.sensitive gas
chromatographic analyses.
Development of DDT Resistance in Houseflies
In reviewing early DDT resistance in insects, Decker and Bruce
(1952a) stated that, "It is no doubt significant that housefly resistance to DDT was first recognized and reported in Europe, where
DDT came into commercial use in 1944 and was used extensively by civil and military personnel in 1945." In 1947, Sacca attributed the
failure of DDT to control houseflies in Italy to the presence of a variety of housefly which had become resistant to DDT. That same year
Wiesmann (1947) reported the presence of housefly resistance to DDT
in Sweden. These represented the first reports of DDT resistance in
insect populations, and it soon became evident that DDT-resistance in houseflies was occurring throughout the world following several years of intensive use of DDT in fly control programs. Working in the United
States, Lindquist and Wilson (1948) reported the development of resistance
in a strain of houseflies after the exposure of 14 consecutive generations
to dosages of DDT which allowed only a small percentage survival. Barber
and Schmitt (1948) reported the first occurrence of a wild strain of DDT•
resistant housefly in this country in 1948. The following year Keiding - 7 -
and Van Deurs (1949) recorded the development of a DDT-resistant
strain of the housefly in Denmark and March and Metcalf (1949b)
reported housefly control with DDT in Southern California to be
unsuccessful.
With the failure of DDT residual sprays to effectively control houseflies due to the development of DDT-resistance by these flies, other organic insecticides came into use in control programs. The most effective of these were benzene hexachloride (BHC) and dieldrin.
However, March and Metcalf (1949a, 1950) soon reported the presence of resistance to these two compounds in houseflies. One year later
BHC resistance was reported in houseflies in Egypt (Gahan and Weir
1950).
These reports of resistance in the housefly stimulated research on the comparative morphology and physiology of DDT-resistant an4 non- resistant strains. Wiesmann (1947) had earlier reported morphological and physiological differences between a DDT-resistant strain of housefly from Sweden, and a non-resistant laboratory strain from
Switzerland. His resistant strain showed greater cuticular pigmenta- tion, stiffer tarsal bristles, shorter and wider tarsal segments, and a lower susceptibility to immobilization by heat and cold. March and
Lewallen (1950) were not able to show that the morphological differences between the DDT-resistant and non-resistant housefly strains they had reported earlier were correlated in any way to resistance.
In 1950, Sternburg ~ al. and Perry and Hoskins, working inde- pendently, showed DDT-resistant houseflies to be capable of converting - 8 - absorbed DDT to its non-toxic metabolite, DDE. This process involved
removal of HCl from DDT and became known as s and co-workers showed that resistance was not caused failure of DDT to cuticle and they were unable to detect the presence of the conversion mechanism in non-resistant On
Hoskins indicated that the conversion did occur in flies, but at a very low level. They also suggested that the conversion process be in nature since heat killed were not of the conversion.
These reports were the rst accounts of DDT in insects in which the of the end product was reasonab certain. In a subsequent publication that year, Sternburg and Kearns (1950) indicated that the major site of DDT detoxification in the adult was in cuticle-hypoderm. reported that susceptible houseflies possessed a weak ability to form DDE when fed DDT but no DDE was detected in this strain
Using c14-labeled DDT, Lindquist ( found resistant and susceptible strains flies capable metabolizing absorbed DDT at the same rate. Hoffman reported that
were able to detoxify DDT, and Lindquist
(195lb) showed most of a topical dose of DDT to be distributed through• out the cuticle rather than in the internal organs.
Winteringhan1 al. (1951) confirmed the work of Perry
Hoskins (1950) using the bromine analog of DDT but interpreted the results to mean that the metabolism was not enough to account - 9 ...
for the successful resistance of the flies. Busvine (1951) observed
the resistance of different fly strains to not always be of the
same type. He concluded that DDE formation did not fully account
for the DDT resistance, and that an additional defense mechanism must be postulated. Winteringham ,!E. al. (1951) summed up the
general consensus of opinion prevalent at the time by saying that,
"although an attractive possibility, the role of metabolism in the
resistance of flies to DDT is not clear."
In this manner two opposing theories concerning the mechanism
of DDT-resistance in houseflies evolved. The following several years
brought supporting evidence for each theory. Ferguson and Kearns
(1949) had earlier found milkweed bugs, Oncopeltus fasciatus (Dall.),
injected with 100 ug. of DDT, to be able to metabolize 80 to 100
per cent of the toxicant within 90 minutes. The extracts of these bugs were non-toxic to houseflies, and colorimetric and ultraviolet
spectro-analyses failed to show the presence of DDE or DDA. Since a metabolite of DDT was not identified in this work, it was used as
evidence that metabolism, especially to DDE, was not a major factor
in DDT-resistance in insects.
It was noted by Bruce (1949) that certain resistant strains of houseflies required one to two days longer for the change from egg
to adult. In confirming this observation March and Lewellen (1950)
showed the average life cycle of a highly resistant fly strain to be 14.l days compared with 13.9 days for their susceptible strain. - 10 -
Pimentel -et -al. (1951) reported longer larval periods for DDT- resistant strains than for a susceptible strain, the increase being
roughly proportional to the resistance. They also observed DDT-
resistant flies from larvae which pupated the first 48 hours to be
less resistant to DDT than flies from larvae which pupated the last
48 hours in a given larval population. No differences were found
between resistant and susceptible strains in number of eggs, hatching
period, fertility, length of pupal period, pupal weight, sex ratio,
preovipositional period or length of adult life. McKenzie and Hoskins
(1954) culminated these reports by splittingamoderately resistant
strain of houseflies into an early substrain (first half to pupate) and
a late substrain (last half to pupate). They were able to show a 6
to 8 fold increase in resistance to DDT in the late substrain after 15
generations with no exposure to DDT.
In a study of DDT absorption and metabolism in resistant houseflies,
Winteringham (1952) discounted the importance of metabolism when he
observed that lower amounts of DDT were absorbed with higher topical
doses and that DDE formation decreased with increased dose absorbed.
Babers and Pratt (1953) substantiated the work of Winteringham and
further concluded that increased ability to dehydrochlorinate an
insecticide was not the only requirement for resistance. They cited
the work by Brown and Rogers (1950) which showed DDT-resistant house-
flies to be resistant also to the effects of such materials as 1, 1,
dianisylneopentane which contains no chlorine but which produces the
tremors and paralysis characteristic of DDT. In a review of the
physiology of DDT resistance Chadwick (1952) likewise questioned the - 11 - significance of dehydrochlorination of DDT as a cause of resistance.
Bettini (1948) excluded the effects of possible differences in external morphology on DDT resistance when he observed DDT-resistant
£lies to maintain their resistance when the insecticide was injected into the thorax. Perry and Hoskins (1950) supported their contention that DDE formation was an important aspect of the resistance mechanism in houseflies by using the DDT synergist, piperonyl cyclonene. They showed that this synergist markedly increased the toxicity of DDT to DDT-resistant flies while at the same time largely prevented the formation of DDE. Perry ~ al. (1953) further supported the metabolism theory by proposing an index for measuring the degree of DDT resistance in various strains of flies. They suggested the index figure correspond to the percentage conversion of absorbed
DDT to DDE. Support for this theory also came from Tahori and Hoskins
(1953) who presented evidence that the metabolism of absorbed DDT appeared to proceed by the route: DDT to DDE to X (an unidentified metabolite). Sternburg --et al. (1953) solidified the metabolism theory when they reported that a crude enzyme preparation from DDT-resistant houseflies could dehydrochlorinate DDT under the proper conditions while no such results could be found using susceptible flies. A year later, in 1954, Sternburg ~ .§!l. reported the partial purification of this enzyme and proposed the name "DDT-dehydrochlorinase." They still were unable to measure activity of this enzyme in. susceptible flies. - 12 -
Several thorough literature reviews and summaries concerning
resistance of insects to insecticides had appeared in the literature by this time (Babers and Pratt 1951; Harrison 1952; Hess 1952; March
1952; and Smith 1955).
Although opposition to the DDT metabolism theory had not disappeared completely (LeRoux and Morrison 1954; Smyth and Roys 1955; and Kerr et al. 1957), the evidence which appeared in the literature in the
late 1950's and early 1960's seems to have established metabolic degradation of DDT to DDE by the enzyme DDT-dehydrochlorinase as a major factor in DDT-resistance of houseflies. Using DDT synergists,
Moorefield and Kearns (1955) were able to inhibit in vivo metabolism of DDT to DDE in DDT-resistant houseflies thereby potentiating the action of DDT. No such potentiation was evident in susceptible
strains. Hadaway (1956) confirmed the findings of Perry and Hoskins
(195lb), who showed susceptible as well as DDT-resistant flies to be capable of detoxifying DDT. Moorefield (1956) further purified
DDT-dehydrochlorinase from houseflies and Moorefield and Kearns
(1957) quantitated the dehydrochlorination of DDT in the larval, pupal
and adult stages of flies. DDE production was not observed in
egg homogenates. However, they were able to show that the dehydro- chlorination mechanism increased rapidly, paralleling larval growth
and then decreased sharply at the time of pupation. The level of dehydrochlorination at pupation was then maintained throughout adult
life. The finding, that larvae of a given strain were more tolerant of DDT than adults of the same strain, was not new information
(Sternburg and Kearns 1950), but the fact that this was correlated - 13 -
with corresponding changes in the level of DDT-dehydrochlorinase
presented additional evidence in support of the role of detoxification
in DDT resistance. Lipke and Kearns (1958) demonstrated the presence
of DDT-dehydrochlorinase in susceptible houseflies, published infor- mation on isolation, chemical properties and spectrophotometric
assay of the enzyme (Lipke and Kearns 1959a), and reported on its
substrate and cofactor specificities (Lipke and Kearns 1959b).
During these years numerous reviews of the physiological basis of
insecticide resistance were published (Metcalf 1955b; Winteringham
and Barnes 1955; Hoskins and Gordon 1956; Brown 1957, 1960; Chadwick
1957; Perry 1958, 1960a, 1960b).
DDT~Resistance in Cockroaches and Other Insects
In the many studies of DDT metabolism conducted in the process of detennining the role of DDE fonnation in the DDT-resistance of houseflies, several authors postulated that other metabolites of DDT were being fonned. Sternburg ~ .!J:.. (1950) and Perry and Hoskins (1950) were unable to account for all of the applied dose in tenns of DDT and DDE and suggested that other metabolites, not responding
to the analytical techniques used, were also being fonned. Perry
and Hoskins (1951b) could account for only 39 percent of the original
topical dose in surviving resistant flies five days after treatment.
Tahori and Hoskins (1953) and Babers and Pratt (1953) obtained evidence
that both susceptible and resistant flies produced an unknown metabolite in the metabolism of DDT. This unknown metabolite generally
became known as metabolite X. In each of these cases the poor
recovery of applied dose was used as evidence for the production of
other metabolites of DDT. Terriere and Schonbrod (1955) proposed 14 - that a water-soluble conjugate was being formed in DDT-resistant and
-susceptible flies. However, Perry ~ al. (1955) using radioactive
DDT made a quantitative study of the products of DDT metabolism in resistant houseflies and concluded that the very small amounts of metabolites other than DDE produced after extended intervals did not appear to be vital to the fly's survival.
Studies of DDT-resistance and metabolism in insects other than houseflies and the subsequent identification of metabolites other than DDE proved to be highly valuable in evaluating the overall resistance pnenomenon in insects.
In 1951, Grayson reported the first evidence showing the German cockroach capable of developing resistance to DDT. He indicated that after seven generations of laboratory selection for survival to treatments of DDT or BHC the male and female cockroaches showed
DDT-resistance factors of 2.3 and 14.3, respectively, over a susceptible strain. Only a slight degree of resistance to BHC was evident. Two years later Grayson (1953) reported that after twelve generations of selection, the DDT-resistance factors had risen to 22 and 198 for the males and females in this strain of cockroaches. He also showed that the females of the treated strain produced fewer nymphs and the egg cases were smaller than in the untreated strain. Later that year, the first report of a field-developed resistant strain of the German cockroach appeared in the literature, Heal il al. (1953). Found in an area where chlordane had been used in control programs, this strain was shown to be extremely resistant to chlordane (over 100 - 15 -
fold) and significantly resistant to lindane (10 to 12 fold) and to
DDT (5 to 6 fold). In further work with the German cockroach, Babers
and Roan (1953) showed a strain weakly resistant to DDT (5 to 6 fold),
to absorb more DDT in 48 hours than a susceptible strain and at the
same time dehydrochlorinate more of the absorbed DDT. Both strains were suspected of metabolizing absorbed DDT to some other unknown metabolite.
Munson and Gottlieb (1953) showed a high correlation between
DDT-resistance in American cockroaches and their lipid content and
Butts et al. (1953) gave evidence that a water-soluble conjugated compound composed of a derivative of DDT and another fragment, perhaps carbohydrate in nature, was directly related to the detoxification mechanism of DDT in American cockroaches.
Pielou and Glasser (1952), in conducting laboratory selection
for DDT-resistance in a parasitic wasp, Macrocentrus ancxlivorus, confirmed earlier evidence that female insects developed greater resistance than males, and Cochran (1955) showed this to be the case in American cockroaches also. Lindquist and Dahm (1956) reported the presence of DDT, DDE, and three unidentified metabolites in excreta of the adult female Madeira roach, Leucophaea maderae (F.). They identified only DDE as a DDT metabolite in fifth instar European corn borer larvae, Pyrausta nubilalis (Hbn). Noted earlier was the report by Ferguson and Kearns (1949) that DDT was not metabolized in substantial quantities into DDE, DDA, or DBP in the large milkweed bug. They suggested that an unknown metabolite was being formed. - 16 -
Sternburg and Kearns (1952a) reported on the metabolism and absorption of DDT by various insects which were normally resistant to DDT. Their results showed the differential grasshopper, Melanoplus differentialis Thomas, capable of degrading oral and topical dosages of DDT to DDE as did the red-legged grasshopper, M. femur-rubrum DeGeer, a related species. The Mexican bean beetle, Epilachna varivestis
Mulsant, degraded oral and topical dosages of DDT to DDE and further converted DDE to an unidentified compound, while Argyrotaenia velutinana
Walker, the red-banded leaf roller, degraded topical and oral doses of DDT to DDE. Sternburg and Kearns considered the conversion of DDT to DDA, though a principle DDT metabolic route in vertebrates, to be of minor importance in insects. They thought the one report of DDA formation in a strain of resistant housefly (Sternburg and Kearns
1950) to probably be the result of a fault in the extraction process.
Chattoraj and Kearns (1958) confirmed the presence of DDT-dehydro- chlorinase in the Mexican bean beetle.
Hoskins and Witt (1958) studied the metabolism of DDT in thirty species of insects and showed the metabolism to fall into three classes: (a) absorbed DDT remained largely unchanged and could be recovered, (b) much of the DDT was converted to DDE,and (c) the chief products of metabolism did not respond to the Schecter•Haller test and therefore were not DDE or DDA. Perry and Buckner (1958) reported that a DDT-resistant strain of the human body louse,
Pediculus humanus humanus, could metabolize DDT to a non-toxic water-soluble compound, while Chattoraj and Brown (1960) showed DDE production in susceptible strains of Aedes aegypti (L.) to be about one-sixth that of DDT-resistant strains. - 17 -
A development of major importance in the understanding of DDT
metabolism in insects was the discovery in 1959 by Tsukamoto that the
major DDT metabolite of Drosophila melanogaster (Meig.) was dicofol;
the polar hydrol-type derivative of DDT. He supported this claim
with paper and column chromatographic data and with ultraviolet
spectrophotometric evidence. In a subsequent publication (1960)
Tsukamoto presented evidence showing resistant flies capable of
producing more dicofol than susceptible flies and he concluded that
the increased metabolism in resistant flies was a cause and not a
consequence of resistance. Using c14-DDT, Menzel~ .21· (1961) confinned these observations. In further studies using various DDT
analogues, Tsukamoto (1961) was able to show the presence of the
unsubstituted hydrogen atom in the alkane moiety ~o be essential for
the DDT to dicofol conversion in Drosophila. He also reported
positive evidence of the reaction occurring in Drosophila virilis,
German and American cockroaches, and houseflies.
Agosin and coworkers (196la) confirmed this work by successfully
preparing a microsomal enzyme system capable of converting DDT to a
dicofol-like metabolite in the German and American cockroaches and in
·the housefly. Dinamarca and co-workers (1962) reported that five metabolites of DDT were formed in nymph and adult male Triatoma
infestans. DDE was shown to be the major metabolite produced by
nymphs and one metabolite had the same chromatographic identity as
dicofol. These workers postulated that the production of the more
polar metabolites involved oxidative processes independent of the mechanism of DDE formation. These observations were confirmed by - 18 -
Agosin g al. (1964) who further postulated that the production of
polar metabolites of DDT in Triatoma infestans followed the sequence
DDT --- dicofol --- metabolite no. 2. Inheritance of DDT Resistance
As the problems relating to insect resistance to insecticides
became evident, more attention was directed toward the study of how
the resistance mechanism was passed from one generation to the next.
In a review of arthropod resistance to chemicals, Hoskins and
Gordon (1956) suggested that since insecticides had not been shown
to be mutagenic agents, the changes undergone by a population under
exposure to an insecticide must result either from mutations occurring
for other reasons or, more likely, from the spread of genetic characters already present in a small part of the original population.
Painter (1930) and Thorpe (1931) hinted at this latter possibility in their earlier discussions concerning the existence of individuals within wild populations which differed from the main portion of the population. Dobzhansky (1951) suggested that the gene for resistance in red scale populations (Quayle 1916, 1922) probably arose by mutation and was maintained by conditions of fumigation. He also
related the insecticide resistance variability of a population to the
typical genetic variation found in populations and suggested that this variation made up the raw materials of evolution. Smith (1941)
considered it probable that resistant mutants appeared from time to
time in any area of dense populations, but that development of
resistance did not always occur because of lack of selection. Brown
(1951) suggested that insect species were probably heterozygous for - 19 -
many genes thus providing a range of genotypes from which an insecticide
could select the more resistant individuals.
Hough (1928, 1934) crossed two strains of codling moth which
differed in their ability to enter apples sprayed with lead arsenate,
and showed the first generation to be intermediate between the parents
in ability to enter sprayed apples. Backcross tests showed progeny to
be intermediate in resistance between the hybrid F1 and the parental strain. This work represented the first report of investigations
involving the genetic mechanism of inheritance of insecticide
resistance. Dickson (1941) showed the resistance of the California
red scale to be inherited as a sex-linked factor, a report later confinned by Yust --et al. (1943). In one of the early reports dealing with inheritance of DDT•
resistance in houseflies, Bruce and Decker (1950) concluded that
autosomal multiple-gene inheritance was probably involved. One year
later, in 1951, Harrison seemed to contradict this conclusion when
she reported that resistance to knockdown by DDT was inherited as a
single recessive gene. That same year Busvine observed that resistance in different strains was not always of the same type.
Harrison later clarified this somewhat conflicting evidence by
suggesting (Harrison 1952) and later showing evidence (Harrison
1953) that resistance to knockdown and to kill by DDT were inherited
separately in the housefly. Johnston.!:!.!!• (1954) found no evidence of sex-linked inheritance of DDT-resistance in houseflies, but they did conclude that the resistance factor was cytoplasmic. - 20 -
In a review of the genetic aspects of selection for resistance,
Crow (1952) suggested that a continuous but accelerating rise in resistance probably indicates that resistance in insects is due to several genetic factors. He also maintained that in the absence of the insecticide there would be a reversion of the resistance level, but that it would probably not be rapid enough to be helpful from the standpoint of insect c.ontrol. Decker and Bruce (1952b) also reviewed the acquisition of resistance by insects.
Early reports on DDT-resistance in houseflies referred to a general hardiness in resistant flies with the resistance extending from DDT to other insecticides (Lindquist and Wilson 1948 and
Wilson and Gahan 1948), or, to a specific resistance to DDT and very closely related compounds ·(Barber and Schmitt 1948). Some reports
(Gahan and Weir 1950 and Pratt and Babers 1950) indicated that cross• resistance in DDT-resistant houseflies was of an intermediate nature.
It was soon evident that the chlorinated hydrocarbon insecticides could be divided into two groups on the basis of housefly tolerance.
One group consisted of DDT and related compounds such as methoxychlor and TDE, the other group was comprised of lindane, heptachlor, aldrin, dieldrin and chlordane.
Decker and Bruce (1952a) reported that houseflies already selected for DDT-resistance could be selected for lindane-resistance in fewer generations than the susceptible flies. Busvine (1954) confirmed this report and concluded that when resistance to any member of one group is developed, some degree of resistance to other members - 21 -
of that group also appears, but the flies remain more or less susceptible
to the compounds of the other groups. Supporting evidence for these
observations came from several workers. Sternburg and Kearns (1956)
showed that DDT-dehydrochlorinase was not involved in lindane metabolism in houseflies, and Oppenoorth (1959) presented evidence
that houseflies resistant to gann:na-BHC had only a low level of resistance to organo-phosphorus compounds. Mengle and Casida (1960)
reported that exposure to either phosphates or carbamates might result in high level resistance to chlorinated hydrocarbon insecticides, whereas the reverse situation does not hold, although DDT-resistant
flies develop organo-phosphorus resistance faster than normal flies.
In 1956, Lovell and Kearns showed that houseflies were capable of inheriting the ability to synthesize DDT-dehydrochlorinase. Later, using a single fly assay for this enzyme, they were able to show a quantitative relationship between the presence of DDT-dehydrochlorinase and resistance to DDT (Lovell and Kearns 1959).
More recent genetic studies of the resistance mechanisms in house-
flies have been directed primarily toward isolating and identifying resistance genes through the study of their linkage relationships. The gene responsible for knockdown-resistance was located on the second chromosome and the gene responsible for the presence of DDT-dehydro-
chlorinase was found to be on the fifth chromosome. A third factor on chromosome three appears to be responsible for an unknown type of
DDT-resistance. DDT-dehydrochlorinases of different activities and properties have been found in different strains of flies. Grigolo - 22 - and Oppenoorth (1966) reviewed and confirmed many of these findings.
They stated that the resistance mechanism of the knockdown gene was still unknown, but speculated on an altered site of action of DDT.
In 1952, Cochran et al. reported the first observations on the inheritance mechanism of DDT-resistance in a resistant strain of the
German cockroach developed in the laboratory (Grayson 1951). The mechanism appeared to be complex in nature, involving chromosomal and extra-chromosomal factors. In 1953, Heal ..!:...!:. al. showed a field collected strain of German cockroaches to be highly resistant to chlordane and significantly resistant to lindane and DDT. Resistance to chlordane in this strain was confirmed by several workers (Fisk and Isert 1953; Grayson 1954; and Butts and Davidson 1955) and cross- resistance was shown to.extend to dieldrin (Fisk and Isert 1953 and
Butts and Davidson 1955) and to heptachlor (Butts and Davidson 1955) but not to diazinon (Fisk and Isert 1953). Clarke and Cochran (1959) showed that chlordane resistant cockroaches possessed no cross-resistance to insecticides of other chemical groups. The same was found to be true of DDT-resistant cockroaches. As with houseflies, cross- resistance within each of these two groups was evident.
A review of cockroach resistance was presented in 1960 by
Grayson. Several years later Cochran (1965) summarized the more recent findings in genetic studies of cockroach resistance. Data indicated that DDT-resistance was inherited as a simple Mendelian autosomal recessive with the probable involvement of modifiers as indicated by F2 and backcross results. F1 hybrids of resistant and susceptible parents were slightly more resistant than the susceptible parent. - 23 -
Further results indicated that aldrin resistance was inherited primarily as a simple Mendelian autosomal semi-dominant trait. DDT- resistance and aldrin-resistance were apparently not linked.
Good reviews of the resistance problem in insects and some aspects of its inheritance are presented by the following workers:
Metcalf (1955b), Crow (1957), Moorefield (1958a) and Brown (1960).
Mode of Action of DDT
In an early review of the development of DDT, Cristal and Haller
(1945) cited the detailed studies of Lauger, Martin, and Muller (1944) which led to the discovery of the insecticidal properties of DDT.
According to Cristal and Haller, these workers concluded that an insecticide which killed by contact must have at least two components - a toxic portion and a carrier portion to transport the toxic material to the site of action in an insect. These workers also indicated the bis(p-chlorophenyl) grouping to be toxic while the CH-CC1 3 group was the lipid soluble carrier group. In another early study on the insecticidal action of DDT and its analogues, Martin and Wain (1944) indicated that the CH-CC13 group was associated with insecticidal activity since DDE was inactive as an insecticide. These authors thought the toxicity of DDT was due to the HCl simultaneously produced in DDE formation. This theory however was later nullified by Brown and Rogers (1950) when they showed the tremors and paralysis character- istic of DDT poisoning to also be produced by 1, 1-dianisylneopentane, a chlorine-free compound similar in structure to DDT. They also showed - 24 -
a DDT-resistant housefly strain to be resistant to the effects of this compound.
Smith (1945) published the results of some of the first studies of the effects of DDT poisoning in mammals. His data indicated DDT to be quite toxic, its effects cumulative, and its principal actions to be on the central nervous system and the liver.
In determining the toxicity of DDT to representatives of various animal phyla, Richards and Cutkomp (1946) observed animals with chitinous exoskeletons to be especially susceptible. These authors concluded that
DDT was selectively concentrated by chitinous cuticles by means of adsorption phenomena. Although Lord (1948) questioned the exact mechanism involved, he was able to confirm that DDT and its analogues were readily sorbed by chitin from colloidal suspension and that the rate of sorption depended upon the concentration of the suspension and the surface area of the chitin. No significant correlation could be shown between the sorption of DDT analogues on chitin and their toxicity.
Hurst (1940) had earlier shown the inner chitin layer of the insect cuticle to be relatively permeable to both polar and nonpolar compounds while the outer lipoid layer was relatively impermeable to polar compounds. He further showed the permeability of the lipoid layer to polar compounds possessing weak dissociation properties to be enhanced by the presence of nonpolar substances. Wigglesworth (1941) substantiated these observations.
O'Brien (1967) strongly contests these reports, maintaining that - 25 - penetration of DDT in various animal phyla is not related to differential integument permeability or to polarity phenomenon. He speculates that metabolism and mode of application of DDT have a great influence on the susceptibility of different animals to this compound.
With the appearance of DDT-resistance in housefly populations, the hypothesis that this resistance was the result of decreased permeability of the cuticle was suggested by Wiesmann (1947). This was soon dis• proved however when the rate of penetration of DDT was shown to be the same in resistant and susceptible strains (Winteringham --et al. 1951 and Sternburg et al. 1950) and when resistance flies were observed to maintain their resistance to DDT when it was injected into the hemolymph
(Bettini 1948; March and Metcalf 1949b; and Busvine 1951). On the other hand, the high degree of resistance to DDT shown by Melanoplus femur-rubrum is probably a result of decreased penetration of DDT through the cuticle, since DDT was shown to be much more toxic when injected into the hemolymph of this insect (0 1 Brien 1967). Likewise, the high level of resistance to DDT shown by larvae of the Khapra beetle,
Trogoderma granarium, is also attributed to reduced cuticular penetra- tion (Winteringham 1952). The cuticular wax of this insect has been shown to dissolve DDT at a slower rate than that observed in other insects
(Pradhan!:£ 2.1.• 1952). In a series of early reports on the usefulness of DDT as an insecticide (Annand~ !1· 1944), better DDT activity was observed to - 26 - be associated with lower temperatures. This observation was confirmed by many other investigators in the following years (Lindquist .!il'., .!b.·
1945; Potter and Gillham 1946; Hoffman and Lindquist 1949; Hoffman~ al.
1949; Guthrie 1950; Woodruff 1950; Vinson and Kearns 1952). Fan~ .!b.· (1948) observed highly susceptible arthropods to have either a positive or a negative temperature coefficient depending on the DDT concentration.
At high concentrations this coefficient was positive and at lower concentrations it was negative. The lower concentrations of DDT were shown to penetrate the cuticle more effectively at low temperatures and a positive temperature coefficient was always observed following injection, regardless of the concentration. From these results it was concluded that the negative temperature coefficient at low DDT concentrations was a property of the cuticle. These and other observations made it apparent that the direction of the temperature coefficient of insecticidal activity was influenced by the test species, life stage, and method of administration of the toxicant.
Later, however, Roth .!il'., al. (1953) presented evidence indicating
DDT to be absorbed more rapidly at 90 degrees F. than at 70 degrees in the housefly. Yamasaki and Ishii (1954) substantiated these results using the American cockroach and concluded that neither the penetration of DDT through the cuticle nor the detoxification of DDT within the insect body could account for the negative temperature coefficient mechanism. Yamasaki and Ishii suggested that the susceptibility of the nerve to DDT was the physiological factor responsible for the negative temperature coefficient of the action of DDT. - 27 -
A publication by Yeager and Munson (1945) reported the results from one of the earliest studies of the effects of DDT on the insect nervous system. These workers showed that in the American cockroach rather high DDT concentrations acted more readily on motor nerve fibers than on sensory fibers and caused repetitive discharges of nerve impulses somewhere in the motor fibers. The following year, 1946, Roeder and
Weiant presented evidence which suggested that comparatively low concentrations of DDT acted primarily on peripheral sensory receptors.
They concluded from their observations that in the American cockroach the tremors characteristic of DDT poisoning were due to an intense patternless bombardment of the motor neurones by trains of impulses originating ih sensory endings. They later confirmed these findings in a subsequent report (Roeder and Weiant (1948). Tobias and Kollros
(1946) confirmed these observations in the American cockroach and further postulated that low doses of DDT excite motor fibers by impulses which enter the ganglion from afferent nerves and traverse the reflex arc, while high doses act directly on the motor nerves.
A good review of the DDT poisoning symptoms in the American cockroach was included in this report. Also in 1946 Bodenstein presented evidence from Drosophila,which indicated that DDT acted on peripheral nerves and not on muscle or myoneural junctions while Welsh and Gordon (1946) suggested that DDT was absorbed at or near the surface of the nerve axon and acted as a barrier to normal calcium interaction with the axon surface. Supporting data were presented in a later publication by these authors working with crayfish (Gordon and Welsh 1948). - 28 -
In 1947 Welsh and Gordon summarized the efforts which had been made toward the elucidation of the mechanism of action of DDT with the
following statement: "The characteristic effect of DDT on arthropod motor axons is a multiplication of nerve impulses occurring in a DDT•
treated region; a single nerve impulse arriving at the treated region
gives rise to a prolonged volley of impulses." These authors felt
the primary action of DDT to be physical, rather than chemical and
said it probably involved the adsorption of the insecticide on the
lipid surface of the axon. In 1951 Roeder and Weiant reported evidence
supporting this theory. They suggested that DDT was immediately bound at the surface of sensory axons and that the time which elapsed between
application of DDT and the appearance of nerve impulse trains represented
the time required for DDT to be absorbed in the lipoid layer underlying
the axon surface.
In 1953 Pratt and Babers (1953a) reported one of the earliest
attempts to correlate the effects of DDT on the nervous system of houseflies with the degree of DDT-resistance observed in that insect.
After observing the leg tremors which resulted from the direct appli-
cation of DDT solutions to the exposed thoracic ganglion, they concluded
ganglionic tissue of DDT-resistant flies to be less susceptible to DDT
poisoning than similar tissue in DDT-susceptible flies.
The mechanism of production of bioelectric potentials in nerve
and muscle tissues is probably best expalined by the sodium theory
proposed by Hodgkin (1951). This theory suggests that the resting - 29 - membrane is readily permeable to potassium and chloride ions and that a high internal concentration of potassium is maintained in the nerve fiber by a similar high internal concentration of impermeable anions.
An active sodium pump mechanism maintains a low sodium ion concentration inside the resting nerve fiber. A large increase in permeability to sodiu..~ occurs when the nerve fiber is depolarized by a flow of electrical current from an immediately adjacent area of the nerve. The resulting rapid entry of sodium ions into the nerve causes a reversal in the potential difference across the nerve membrane thus providing current for the subsequent depolarization of adjacent areas of resting nerve.
Yamasaki and Narahashi (1957a) proposed that DDT affects the nervous function of axons by changing the mechanism of ionic transfer across the membrane. They suggested that DDT changes the ionic permeability of the nerve membrane either by affecting the active metabolism of the nerve or by a more direct physical action on the nerve membrane. In a subsequent publication, Yamasaki and Narahashi
(1957b) related DDT poisoning to an increase in negative after-potential.
Several years later they showed the increased negative after-potential to be caused by the suppression of potassium ion efflux from the axon
(Narahashi and Yamasaki 1960).
Several good reviews on mode of action of insecticides had appeared in the literature by this time which included sections on the mode of action of DDT (Metcalf 1955a; Kearns 1956; Martin 1956; Dahm 1957). - 30 -
O'Brien and Matsumura (1964) referred to a review on charge transfer complexes by Szent-Gyorgyi (1960) in attempting to establish a molecular basis for the effects of DDT on nerve tissue. According to Szent-Gyorgyi, only one electron is transferred from a donor molecule to an acceptor under conditions of charge transfer as opposed to the usual two-electron transfer in oxidoreduction situations. Also, in charge transfer, the two molecules involved usually stay together.
O'Brien and Matsumura suggested that the high electron affinity of DDT and its low biological reactivity made this compound capable of forming such charge transfer complexes with a component of the axon thereby 14 destablizing it. In observing the uptake of C -DDT on whole or homogenated n~rve cords of the American cockroach, these workers showed two plateaus which suggested sequential saturation of two nerve components. This bound DDT was then shown to elute from a Sephadex column with a small amount of organic matter. A diethylamino- ethyl cellulose column was used to separate this radioactive DDT into two fractions, one associated with little organic matter and the second associated with much more. The only evidence for charge transfer formation was the detection of a new shoulder of ultraviolet absorption in the 245-270 millmicron range. O'Brien and Matsumura felt that the formation of charge transfer complexes helped explain the somewhat unique action of DDT on the nerve axon itself whereas most compounds affecting the nervous system act at the synapse by interferring with the transmitter mechanism. Matsumura and O'Brien (1966a, 1966b) presented further evidence supporting their contention that DDT formed charge transfer complexes with components of the insect nerve and they related - 31 - this to an interferenc·e with potassium ion efflux from the nerve. As in their previous work, bound DDT was shown to pass through a Sephadex column while unbound DDT was retained.
However, Hatanaka ~al. (1967) cast some doubt upon the validity of the Sephadex column technique for demonstrating complex formation between DDT and nerve components. They showed that DDT would pass through a Sephadex column following incubation with non-nervous tissues, with detergent, and with non-toxic analogs of DDT. These authors suggest that emulsifiers, which include some natural tissue components, are responsible for the formation of DDT micelles of sufficient size to elute rapidly through a Sephadex column. They did not disprove the concept of complex formation with these data but they contended that the content of natural emulsifiers in nerve tissue is probably ample to account for the observed behavior on Sephadex.
Also in 1967 Hayashi and Matsumura showed nerve components of homogenized heads of German cockroaches resistant to DDT to have less binding capacity with DDT than similar preparations from susceptible cockroaches. Although the implication here is that the resistant cockroaches are possibly using this decreased binding capacity to their advantage, the correlation of this phenomenon with the observed
DDT resistance level in these cockroaches must await identification and characterization of the nerve components.involved.
Many of the early investigations into the mode or site of action of DDT attempted to relate DDT poisoning to an essential metabolic process or to the inhibition of a particular enzyme. Tobias ~ al.
(1946) reported the free acetyl choline of American cockroach and house• fly central nervous systems to increase about 200 per cent during the - 32 -
late stages of DDT poisoning and esterase activity to not be effected.
Calhoun (1959) confirmed this work. Merrill ~ al. (1946) reported
a 90 per cent reduction of total body glycogen and glucose after DDT
poisoning in the American cockroach, but felt that these changes were
not critical to the cause of death. Lord (1949) felt that death in
DDT poisoned Oryzaephilus surinamensis was a result of the increased
activity caused by the DDT. Several groups of workers observed a two
to five fold increase in oxygen consumption following DDT treatment of
various insects but could not relate it directly to a toxic mechanism
(Ludwig 1946; Buck and Keister 1949; Hoffulan and Lindquist 1949;
Lord 1949; and Micks and Murthy 1961). Fullmer and Hoskins (1951).
showed DDT to cause an increase in the respiration rates of DDT•
resistant and -susceptible houseflies but that the increase was much
less in the resistant flies.
Babers and Pratt (1950) reported cholinesterase activity in the heads of DDT-resistant houseflies to be significantly lower the first
five days after emergence than similar activity in susceptible flies.
However, this difference could not be confirmed in a subsequent
investigation by these authors using the same strains (Pratt and Babers
1953b). They showed considerable variation in the cholinesterase
activity of six susceptible strains of flies.
Sacktor (1950, 1951) reported inhibition of cytochrome oxidase
in housefly brei by DDT and methoxychlor, and Johnston (1951) found
that DDT and DDE produced from 70 to 90 per cent in vitro inhibition of
rat heart succinoxidase and cytochrome oxidase but had no effect on
succinic dehydrogenase. - 33 -
Sternburg and Kearns (1952b) discovered the blood of DDT poisoned insects to contain toxic compounds not derived from and unrelated to
DDT. These unidentified compounds in themselves were apparently capable of producing violent reactions in nerve tissues.
Gunther et ~· (1954) postulated an interesting theory on the mechanism of action of DDT-type compounds in an attempt to explain the insecticidal activity of DDT against larvae of Culex quinguefasciatus
Say. They suggested that the DDT-type molecule was able to fit into or onto an apoenzyme or other protein and be held tightly, thus inhibiting the normal activity of that protein. In this light, close fitting and tightly held molecules would be the better insecticides. They further suggested that the insecticidal activity of the DDT-type compounds would be directly related to their van der Waals' attractive forces for the protein involved. This theory has failed to gain much support.
In 1960, Kearns (1960) presented a good review of some of the efforts to associate insecticidal activity with metabolic function.
Also in 1960, Agosin £.!:. !1_. were able to show DDT and DDE to inhibit triosephosphate dehydrogenase in adult male Triatoma infestans but not in nymphs. Using specific antibody techniques they showed that although these two enzymes catalyzed similar reactions, they were not identical to each other. This was an important observation since adult males were later shown to be quite susceptible to DDT while nymphs were relatively resistant (Agosin £.!:. !1_. 196lb). These were the first reports of a positive correlation between the action of DDT on metabolic enzyme systems and DDT-resistance. In this latter publication these - 34 - workers also showed that eight enzymes of the glycolytic and pentose phosphate pathways were inhibited by DDT in adult male specimens while only two enzymes were inhibited in the nymphs.
In 1963 Agosin et al. showed that DDT accelerated the reactions of the pentose-phosphate pathway in !• :i.nfestans nymphs and they indicated this activation was caused by an increased synthesis of NAD-kinase.
Earlier attempts to demonstrate an inductive role of DDT in relation
to DDT-dehydrochlorinase and DDT-resistance had been unsuccessful
(Moorefield 1959). Ilevicky ~al. (1964) confirmed Agosin £S_ al. and presented additional evidence that the increased activity of NAD- kinase was an inductive effect rather than a case of enzyme activation.
They also showed that a minimal internal concentration of 15 ug. of
DDT was required for increased NAD-kinase activity. The enzyme was also induced with DDD, but not with DDE or kelthane. These authors postulated that DDT treatment induces a change in enzyme activity in l• infestans which alters its physiological capabilities. They further suggested that these changes might be significant in relation to DDT- resistance.
In 1967, Ilivicky and Agosin reported on the effects of DDT on the supply of reduced glutathione required for the dehydrochlorination of DDT in T. infestans. These authors presented evidence which indicates that DDT causes an increase in the synthesis of enzymes responsible for biosynthesis and degradation of glutathione. The net effect was apparently an increase in turnover of glutathione by
DDT with little change in levels of tissue glutathione. It was - 35 - suggested that suitable levels of reduced glutathione would be maintained by the glutathione-reductase system which requires NADPH, the NADPH in turn being supplied by the pentose-phosphate pathway whose activity is increased in the presence of DDT.
DDT SYNERGISM BY SESAMEX
Metcalf (1967), in a review on the mode of action of insecticide synergists, defined synergism as"··· the substantially more than additive toxic or pharmacological action of two substances used together "
The earliest recorded use of a synergist or activating agent with an insecticide was that of Freeborn and Regan (1932) who were able . to show that pine oil increased the effectiveness of a pyrethrin spray used to control flies on dairy cattle. Subsequent to this report,
Pearson (1935) showed this activating property to also be evident when pine oil was used with derris and Rotenone. Following these accounts, other workers reported the use of activating agents in common insect sprays (Weed 1938 and Pierpont 1939).
In 1940, Eagleson reported that sesame oil acted as a synergist when combined with pyrethrins and rotenone. He observed that the synergistic effect reduced the necessary concentration of insect toxin required to produce 100 per cent mortality of houseflies and it prolonged the paralysis of the insects. The sesame oil alone was non• toxic to the flies. Eagleson did not find the enhancement in toxicity of pyrethrum insecticides to be a property of other vegetable oils.
Haller and his co-workers (1942a) fractionated sesame oil by molecular distillation and from the two most active fractions isolated sesamin, a - 36 - compound exhibiting marked synergism with pyrethrins. By a systematic examination of compounds similar to sesamin, these co-workers made the very important discovery that the intact 3,4-methylenedioxyphenyl group is essential for sesamin 1 s synergistic activity (Haller £E_ .!l•
1942b, 1942c). Realizing the importance of the methylene dioxyphenyl group in relation to synergism, other workers initiated studies on the effects of various compounds containing this group on pyrethrins (Gertler and Haller 1943; Harvill --et al. 1943; and Synerholm and Hartzell 1945).
Sesame oil was not the only synergist source being investigated at this time (Hartzell and Scudder 1942; Gertler £E_ .!l· 1943; Gersdorff and Gertler 1944) but it was beginning to appear to be one of the more important sources. In addition, World War II greatly reduced the supply and consequently increased the cost of pyrethrins. As a result, a vast amount of effort was expended toward finding materials which would synergize pyrethrins, thereby decreasing the amount of this insecticide required for effective insect control (Prill and Synerholm 1946;
Prill £E_ al. 1946, 1947; Wachs 1947; Schroeder £E_ al. 1948; Synerholm
~ al. 1948).
Haller and co-workers (1942a) had recognized that considerable synergestic activity remained after the removal of sesamin from sesame oil, as had subsequent workers (Parkin and Green 1944). Beroza (1954) undertook to identify any compound or compounds in the sesame oil other than sesamin that exerted any appreciable synergism with pyrethrins in order that the synergistic action of sesame oil might - 37 - be fully accounted for. By the systematic entomological examination of silicic acid column fractions of sesame oil two pyrethrum synergists, sesamin and sesamolin, were found to account for practically all the synergistic activity of the oil. Sesamolin, which had not been known to be synergistic, was about five times as active as sesamin and, even though usually present in smaller amount than sesamin, it was believed to account for most of the synergistic activity in sesame oil. The chemical structure of sesamolin and sesamin were subsequently identified
(Beroza 1955; Erdtman and Pelchowicz 1955; and Haslam and Haworth 1955).
The intense synergistic properties of sesamolin established it as one of the most potent natural pyrethrum synergists. Furthermore, this work indicated that the ultimate in synthetic synergists had not yet been attained, since sesamolin was far more effective a synergist for natural pyrethrins than the best commercial synergist. This information suggested that other derivatives of 3,4-methylenedioxyphenyl should be tested for synergism. Beroza (1956) and Gersdorff et al.(1956) reported on the preparation and test results of 66 such compounds. Many of these compounds proved to be active and one, sesoxane, was thought to show commercial promise. Beroza and Barthel (1957) published a useful review of their data on the relation between chemical structure and synergistic activity of methylenedioxyphenyl compounds. Prill and
Smith (1955) substantiated these finding by showing that replacement of the methylene by an ethylene group in the methylenedioxyphenyl moiety of several highly synergistic compounds destroyed their synergistic activity. Moore and Hewlett (1958) also substantiated - 38 -
Beroza's work by showing, through chemical structure and synergistic activity tests, that of the candidate pyrethrum synergists tested, sesoxane exhibited the highest potency.
In 1960, Sun and Johnson made the important observation that the amino or amido group in almost all members of vinyl phosphates, vinyl phosphonates, and nonvinyl phosphates was related to unusually high synergistic action with sesoxane against h9useflies. In biological systems the primary and secondary amines were known to be susceptible to . oxidation by amine oxidases with the liberation of ammonia. Sun and
Johnson considered it reasonable then to suspect that the amino or amido groups of the vinyl phosphates and phosphonates might also be susceptible to certain biological oxidations. Thus, Sun and Johnson postulated that the apparent high increase in toxicity of these phosphates was due to the inhibition of such oxidation by pyrethrin synergists.
As supporting evidence for this theory, they were able to show that sesamex (sesoxane) inhibited the oxidation of methyl parathion and stabilized its oxygen analog, methyl paraoxon. They also showed that sesamex inhibited the oxidation of the chlorinated hydrocarbon insecticides aldrin and heptachlor to their generally more toxic epoxide forms, dieldrin and heptachlor epoxide. Brooks and Harrison
(1963a, 1963b) substantiated these results by showing that sesamex enhanced the toxicity of a number of cyclodiene compounds on houseflies.
These authors felt that sesamex was protecting both forms of the compounds against enzymatic detoxication resulting in increased insecticidal material in the insect. A DDT-sesamex combination showed only a slight - 39 - increase in toxicity with houseflies (Sun and Johnson 1960). These results along with later reports concerning the nature of certain metabolites of the carbamate insecticide Sevin (Dorough ~ !.!.• 1963 and Dorough and Casida 1964), helped explain the observed synergistic activity of sesoxane with Sevin (Moorefield 1958b and Eldefrawi .!:.!:, al.
1959).
It was the general opinion of workers in the field at that time
that synergists prevented the detoxication of pyrethrins in insects
(Metcalf 1955a). Most authors considered enzymic hydrolysis, rather
than enzymic oxidation as the possible cause for detoxication of pyrethrins. On the basis of their data Sun and Johnson (1960) speculated that pyrethrins were detoxified by biological oxidations and that the synergism produced by pyrethrin-synergist combinations was due to the inhibition of such oxidation.
Several workers reported on the role of hydroxylation in the metabolism of toxic materials in insects (Fouts and Brodie 1956 and
Agosin ~ .§1. 196la). Morello (1964)) using various activators and inhibitors, was able to show hydroxylation of DDT to play a part in
DDT resistance exhibited by Triatoma infestans nymphs. Philleo ~ al.
(1965) showed that sesamex was capable of inhibiting the hydroxylation of naphthalene in houseflies, a biological oxidation earlier shown to be of importance in the metabolism and excretion of this compound (Arias and Terriere 1962). They presented data which indicated this inhibition was competitive in nature. Philleo .!:.!:, .§1. suggested that the diversity - 40 -
of action shown by methylene-dioxyphenyl compounds seemed to indicate
that these inhibitors interferred with some process such as utilization of oxygen, which is common to a variety of oxidations.
In 1966, Grigolo and Oppenoorth reported an unknown detoxication mechanism located on chromosome number 3 of the DDT-resistant housefly
to be overcome with sesamex. Earlier, Eldefrawi ~ al. (1959) had found
sesamex to be a synergist for DDT on Gennan cockroaches.
Perry and Hoskins (1950, 195la, 195lb) were the first to report
the finding of a material which enhanced the effectiveness of DDT against resistant strains of houseflies. All previous studies on synergistic action with DDT had been made with supposedly normal insects and no relation between resistance and synergism had been reported. These authors showed that piperonyl cyclonene, a pyrethrin synergist, greatly increased the insecticidal action of DDT with resistant houseflies when application was made topically with acetone solutions or by spraying kerosene solutions. Only a slight effect was shown with a DDT-susceptible strain of flies. Perry and Hoskins indicated that this synergistic action was not due to increased penetration in the presence of piperonyl cyclonene. They observed
that increased ability to convert absorbed DDT to its non-toxic ethylenic derivatii· 1 DDE, was characteristic of the resistant strain and that piperonyl cyclonene largely prevented this conversion.
Sumerford ~ al. (1951) thought it possible that the mechanism of DDT resistance developed by the insect could be interrupted by a - 41 - compound structurally related to DDT, and more especially by an analog which shared some of its physical properties. This, in fact, was borne out by the fair degree of synergistic activity provided for DDT by its p, p'-difluorine analog, DMC (Riemschneider 1949). Sumerford and co• workers (1951) observed DMC to be an extremely active DDT synergist against resistant houseflies. March £!:. al. (1952) also reported several effective synergists for DDT and closely related compounds.
Later, Tahori (1955), Bergman~ al. (1955), and Cohen and Tahori (1957) showed F·DMC to be a very active DDT synergist against houseflies. III. METHODS AND MATERIALS
Adult German cockroaches were treated with varying levels of DDT, alone and in combination with sesamex, and held at room temperature in petri dish cages containing food and water for various exposure intervals. Three replications were included for each treatment and strain. Each replication contained 10 cockroaches.
The cockroaches, which had been rinsed to remove the external
DDT, ware homogenized and extracted to recover internal DDT and metabolites. The excreta pellets in the petri dish cages were extracted to recover excreted DDT and metabolites. Following cleanup, the DDT and metabolites from these fractions were identified and quantitated by gas-liquid chromatography and the identifications were verified by thin-layer chromatography.
Cockroach Strains
Two DDT-resistant strains and one susceptible strain of the German cockroach were used in this study. The highly resistant VPI•DDT• strain was developed in this laboratory by Grayson (1951), and the slightly less resistant Landsthul-strain was field collected in
Germany. The resistance levels of these strains were maintained by periodic selection for survival to DDT exposure administered by a dipping technique previously described by Grayson (1954). The susceptible-strain has been maintained a number of years in this laboratory with no known exposure to insecticides other than the small amounts of DDT, TDE, DDE, and dicofol recently found in the cockroach food source (Rolofson and Bishop 1967).
.. 42 - - 43 -
Dosage-mortality data were obtained from DDT dipping tests with
adult male and female cockroaches of these three strains. The cockroaches were dipped in water suspensions of DDT prepared from 75 per cent wettable powder. Mortality counts were made after six days. The data were plotted on log-probit paper and lines were fitted to the points by the method of least squares described by Bliss (1935).
Treatment Procedure
One to two week old adult cockroaches were anesthetized with carbon
dioxide and placed in a holding chamber equipped with a carbon dioxide
delivery tube. Each cockroach was removed from this chamber, treated with a known quantity of DDT in acetone solution, and placed in the petri-dish cages. The treatment was applied to the ventral thoracic
surface of the insect at the base of the mesothoracic coxae. The hand-operated microapplicator-syringe apparatus delivered a 1.8 ul
drop of solution. The calculated treatment levels used in this study were 8, 30 and 150 ug. of DDT per insect. The 8 and 30 ug. treatments were from solutions which contained these doses in a 1.8 ul volume.
The 150 ug. treatment was administered in the form of five 30 ug. doses with an interval of several minutes between each dose.
A 0-hour recovery of DDT from cockroaches treated with the three
dosage levels was quantitated and considered to be the actual applied
dose for each treatment. On this basis, the applied dose for the 8 ug.
DDT treatment was 6.59 ug. of DDT, for the 30 ug. DDT treatment was 24.55
ug. of DDT, and for the 150 ug. DDT treatment was 122.77 ug. of DDT per
insect. - 44 -
The treated cockroaches remained in the petri-dish cages for exposure periods of 3, 6, 12, 24, 48, and 72 hours. At the end of the exposure period the petri-dishes were placed in a freezer for later analysis.
In the synergism studies the sesamex applications were made 45 minutes prior to the DDT at the same location using the same applicator.
1. Apparatus
a. Microapplicator -- (Dutky and Fest).
b. 1 cc. tuberculin syringe with 27 gauge needle bent at 90
degree angle (Fisher Scientific Co.).
2. Reagents
a. Acetone -- redistilled (b.p. 56.1°c).
b. p,p1 ·DDT (99.9%) -- (Geigy Chem. Corp., Ardsley, N.Y.).
c. Sesamex -- (Shulton, Inc., New York 20, N.Y.).
Sample Extraction and Cleanup
The sample extraction and cleanup procedures are essentially those of Moats (1963) with the exception that the 60/100 mesh Florisil was deactivated by adding 3.5 ml. of water per 100 g of Florisil.
1. External -- the non-penetrated DDT was recovered by the following procedure.
a. Swirl the 10 cockroaches of each replicate for 10 seconds
in 10 ml. of a mixture of petroleum ether and methylene
chloride (80:20 v/v), referred to hereafter as the extraction
solvent.
b. Add this rinse to a 25 g Florisil column. - 45 -
c. Rinse the cockroaches two at a time with the aid of
forceps for 10 seconds in two 7 ml. portions of extraction
solvent and add these rinses to the Florisil column. Rinse
the wash beakers between each pair of cockroaches.
d. After the entire replicate is rinsed and the rinses have
been added to the Florisil column, elute the column with
250 ml. of extraction solvent and collect the eluate in
a 500 ml. erlenmeyer flask labelled External.
2. Internal -- to recover the internal DDT and metabolites the
following procedure was employed:
a. Place the 10 rinsed cockroaches from each replicate in a
small (90 ml.) Omni mixer grinding cup containing 10 grams
of Florisil.
b. Submerge grinding cup in ice and grind for 2 minutes at a
rheostat setting which allows 70-80 per cent full power.
c. Allow preparation to sit undisturbed for 5 minutes after
grinding.
d. Add the resulting cockroach-Florisil mixture to the top
of a 25g Florisil column and elute with 250 ml. of extraction
solvent.
e. Collect eluate in a 250 ml. erlenmeyer flask labelled Internal.
3. Excreta -- the excreted DDT and metabolites were collected in the
following manner: - 46 -
a. Using a spatula, scrape the excreta pellets from the exposure
dish into a mortar containing a pinch of Florisil.
b. Rinse the exposure dish with small amount of extraction solvent
and add to the mortar.
c. Grind mortar contents thoroughly with a pestle and rinse
resulting slurry onto a 15g Florisil column.
d. Elute column with 200 ml. of extraction solvent and collect
eluate in a 250 ml. erlenmeyer flask labelled Excreta.
4. Evaporation -- the flasks containing the external, internal, and
excreta fractions were each connected to a Snyder column and
evaporated to dryness under vacuum in a 30-50° water bath.
Each sampke was redissolved in hexane and transferred to graduated
centrifuge tubes for gas-chromatographic quantitation.
5. A12par a tus
a. Omni-mixer (Ivan Sorvall, Inc., Norwalk, Conn.).
b. Chromatographic columns -- 500 x 21 mm o.d.
c. Vacuum evaporator -- a vacuum manifold was connected with a
stopcock to a Snyder column, 250 x 19 mm o.d., containing
three glass antisurging marbles.
d. Water bath -- 30-50° C.
6. Reagents
a. Aluminum oxide -- (M. Woelm, Eschwege, Germany. Distributed by
Alupharm Chemicals, New Orleans, La.).
b. Florisil -- (Floridan Co., Hancock, W. Va.).
0 c. Hexane ..... redistilled (b.p. 67 C.). - 47 -
d. Methylene chloride -- redistilled (b.p. 38°c.).
e. Petroleum ether -- redistilled (b.p. 30-50°c.) and purified
through activated Aluminum oxide column.
guantitation of DDT and Metabolites
A 2 ft. 8 in. by 1/4 in. o.d. glass column was packed with 10%
Dow 200-500 on acid-washed 45/60-mesh Chromosorb P. The column temperature was 215 0 c. and the detector temperature was 243 0 C. The carrier gas was nitrogen with a flow rate of 60 cubic centimeters
per minute.
Stock solutions of DDT, DDE, and dicofol dissolved in hexane were
used as reference standards. A standard was injected before and
after each series of three treatment replicates to account for variation in detector response. Each sample peak was quantitated by
comparing its peak area with the average peak area of the corresponding
compound in the two standards.
1. Apparatus
a. Microtek Mt-220 gas chromatograph -- equipped with a nickel-
source electron-capture detector (Microtek Instruments Corp.,
Austin, Texas).
2. Standards
a. p,p'•DDT (99.9%) (Geigy Chem. Corp., Ardsley, N.Y.).
b. p,p 1 -DDE (99.8%) (Geigy Chem. Corp., Ardsley, N.Y.). c. p,p'•Dicofol (100'7o) .... (Rohm and Haas, Philadelphia 5, Penn;.). - 48 -
Thin Layer Chromatography
The methods used for the thin-layer chromatographic confirmatory analyses were essentially those described' by Kovacs (1963). A brief description of this procedure follows:
1. Apparatus
a. Standard Brinkman model applicator, mounting board, and other
accessories. (Brinkman Instruments, Inc., Great Neck, L.I.N.Y,).
b, 8 x 8 inch double strength window glass.
c. Chromatographic chamber and Whatman No. 3 liner, 22 x 26 cm. d. Spray bottle.
2. Reagents
a. Adsorbent -- aluminum oxide G with 10 per cent Caso4 binder. (Manufactured by E. Merck, Darmstadt, West Germany. Dis-
tributed by Brinkman Instruments, Inc., Great Neck, L.I.N.Y.).
b. Developing solvent -- n-heptane and acetone (98:2 v/v).
c. Chromogenic Agent -- dissolve .1 g AgNo3 in 1 ml. water. add 10 ml. of 2-phenoxyethanol (Eastman Organic Chemicals,
Rochester, N.Y.), dilute to 200 ml. with acetone, reagent
grade, add a small drop of 30% H2o2, and mix.
3. Preparation of Adsorbent Lay~r
a. Weight 30g Al203 into a flask and add 65 ml. distilled water. b. Mix gently for 50-60 seconds and apply in:unediately to clean
dry plates using the applicator.
c. Allow plates to dry in place on the mounting board for 15
minutes and then place in a ll0°c. oven for one hour. Allow
to cool and store in desiccating cabinet. - 49 -
4. Preparation of Standards and Samples
a. DDT, DDE, and dicofol standards were prepared alone and in
combination at a concentration of 5 ug./ul of hexane.
b. The samples prepared for gas-chromatographic quantitation were
evaporated to a volume which contained a concentration sufficient
for detection.
5. Sample Spotting
Using a 50 ul Hamilton syringe, .5-1 ug. of each standard - alone and in combination - were spotted.one inch from the bottom edge of
the plate at 3/4 in. intervals. Each sample was spotted in a similar manner.
6. Development of Plates
a. Develop the chromatograms in the chromatographic tank using
n-heptane-acetone (98:2 v/v) as the mobile phase.
b. Remove the plates from the chamber when the solvent front has
risen 10 cm. above the spotting line. Allow to dry for 5
minutes in a hood.
7. Spraiing the Plates
Spray the developed plates evenly with the chromogenic agent and allow to dry in the hood for 5 minutes.
8. Exposure
Expose the plates to ultraviolet light at a distance of 10 cm.
for 15-30 minutes. IV. RESULTS AND DISCUSSION
Toxicological ~
The Lc50 values (in grams of actual DDT·per liter) calculated for the three cockroach strains used in this study are as follows: VPI•
DDT males = 60.0, females = greater than 60.0; Landsthul males =
40.0, females = greater than 60.0; susceptible males = 0.064, females
= 0.080. These figures indicate that a high degree of resistance to
DDT exists in the VPI-DDT and Landsthul strains. The maximum effective
dipping concentration was previously found to be 60 grams of DDT per
liter (Cochran, Grayson, and Ross, personal communication). Since
this solution imparts only 20 to 30 per cent mortality in resistant
females, the Lc 50 values in these cases are only approximations. The 8 ug. DDT treatment caused 30 per cent mortality in susceptible male cockroaches after six days. In combination with several levels of
the synergist sesamex, an increase in mortality was observed with this
DDT treatment, the increase being proportional to the amount of sesamex
used (Table I). This 8 ug. DDT treatment caused no mortality in the
VPI-DDT-strain of resistant cockroaches.
The 30 ug. DDT treatment allowed only a 24 hour measurement of
penetration, detoxication, and excretion in susceptible-strain males
before a high level of mortality occurred. Even at 24 hours the
susceptible males may have entered a state of intoxication. Neither
the 30 ug. nor the 150 ug. DDT treatments produced mortality in the two
resistant cockroach strains. Similarly, no mortality was observed at
any level in the sesamex•DDT combinations used against the resistant
cockroach strains.
- 50 - - 51 ...
Table I • Six-day mortality counts in susceptible-strain male German cockroaches following treatment with 8 ug. of DDT alone and in combination with several levels of sesamex (Ses.).
Treatment Mortality (ug. /insect) %
8 DDT 30 16 Ses. + 8 DDT 48 40 Ses. + 8 DDT 80 80 Bes. + 8 DDT 94 - 52 -
DDT RECOVERY
Preliminary tests indicated that dicofol was the major metabolite of DDT degradation in the resistant and susceptible cockroach strains used in this study. The small amounts of DDE found in the various sample fractions were shown to never exceed the DDE level of the applied
DDT. Overall recovery of the applied dose ranged from 87.3 to 102.9 per cent with an average recovery of 95.6 per cent. The recovery variation within each treatment indicated that production of metabolites other than dicofol did not occur at a detectable level. Furthermore, gas chromatographic and thin-layer confirmatory analyses gave no indication of the production of metabolites other than dicofol by these cockroach strains. This finding disagrees with Hoskins !!:£ al. (1958) who reported the production of several relatively polar unidentified metabolites of
DDT by the German cockroach. The results of a typical thin-layer analysis are presented in Table II.
The external, internal, and excreta DDT·and the internal and excreta dicofol quantitated for the various treatments and exposure intervals are presented in Appendix A, along with recovery data. Similar data obtained from cockroaches which had been pretreated with various levels of sesamex are also included in this appendix.
Penetration
The portion of the applied DDT which penetrated the cockroach cuticle was derived for each treatment exposure interval by combining the internal and excreta DDT with the internal and excreta dicofol. The resulting quantities are presented in Table III along with the per cent of applied dose which each represents. - 53 ..
Table II. A typical thin-layer chromatographic confirmatory analysis for DDT and dicofol in internal and excreta sample fractions.
Sample Spotted Rf Values
DDT standard .4
Dicofol standard .125
Combined DDT and dicofol standards .125, .4
72 hour VPI-DDT-strain male excreta .125, .4
72 hour Landsthul-strain male internal .125, .4 Table III. Penetration of several dosage levels of DDT in resistant and susceptible German cockroaches in presence and absence of Sesamex (Ses.). The number in parentheses represents the per cent of applied dose penetrated.
Susceptible-Males Susceptible- VPI-DDT- Females Males B DDT Ib Ses • + S DDT 40 Ses. + 8 DDT 30 DDT 8 DDT B DDT Hour DDT DDT DDT DDT DDT DDT after penetrated penetrated penetrated penetrated penetrated penetrated treatment ~ug. /insect) ~ug./insectl ~ug. /insect) ~ug./insect) {ug./insect) {ug./insect) 3 1.45 (22.0) .77 (11.7) .78 (11.8) 2.65 (10.8) • 95 (14.4) 1.16 (17.6) 6 1.88 (28.5) .83 (12.6) .so (12.1) 3.87 (15.8) 1.19 (18.1) 1.31 (19.9) 12 2.02 (30.7) 2.07 (31.4) 1.80 (27 .3) 4.29 (17.5) 1.52 (23.1) 1.53 (23.2) 24 1.76 (26.7) 1.56 (23.7) 1.75 (26.6) 4.28 (17.4) 1.65 (25.0) 1.74 (26.4) 48 1.63 (24.7) 1.54 (23.4) High Mortality 1.70 (25.8) 1.76 (26. 7) 1.81 (27.5) 1.35 (20.5) High Mortality 1.67 (25.3) 1.47 (22.3) VI 72 ~ VPI-DDT- VPI-DDT-Males Females 30 DDT 30 Ses. + 30 DDT 60 Ses. + 30 DDT 90 Ses. + 30 DDT 150 DDT 30 DDT 3 3.51 (14.3) 2.68 (10.9) 3.02 (12.3) 3.76 (15.3) 19.42 (15.8) 4.47 (18.2) 6 4.45 (18.1) 3.24 (13.2) 4.22 (17.2) 3.66 (14.9) 17.93 (14.6) 4.40 (17 .9) 12 4.61 (18.8) 5.99 (24.4) 4.85 (19.8) 6.03 (24.6) 28.45 (23.2) 4.67 (19.0) 24 S.81 (23.7) 7.02 (28.6) 6.43 (26.2) 6.88 (28.0) 48.76 (39.7) 7.20 (29.3) 48 12.18 (49.6) 11.59 (47.2) 10.70 (43.6) ll .. 65 (47.5) 56.56 (46.1) 8.71 (35.5) 72 10. 77 (43. 9) 10. 43 ( 42. 5) 10.93 (44.5) 12.16 (49.5) 63.54 (51.8) 8.65 (35.2) Landsthul- Landsthul-Males Females 3 3.62 (14.7) 3.13 (12.7) 3.36 (13.7) 3.39 (13.8) 17.09 (13.9) 5.25 (21.4) 6 4.31 (17.6) 5.65 (23.0) 3.53 (14.4) 3.92 (16.0) 19.69 (16.0) 4.43 (18.1) 12 4.24 (17.3) 3.61 (14.7) 3.16 (12. 9) 4.81 (19.6) 26.77 (21.8) 4.35 (17.7) 24 4.71 (19.2) 6.60 (26.9) 7.10 (28.9) 6.42 (26.2) 40.39 (32.9) 5.41 (22.0) 48 11.43 (46.6) 11.58 (47.2) 11.55 (47 .O) 11.86 (48.3) 55.53 (45.2) 6.98 (28.4) 72 11.37 (46.3) 11.02 (44.9) ll.59 (47.2) 11.71 (47.7) 82.35 (67.l) 7.73 (31.5) - 55 -
Figure 1 shows the rate of penetration of the 8 ug. DDT treatment
in male and female susceptible-strain cockroaches. It is apparent that
penetration of this treatment dose of DDT is not affected by the sex of
this strain. Figure 2 shows the penetration of the 8 ug. DDT treatment
in VPI·DDT- and susceptible-strain males. Although penetration was
slightly less at the 72 hour interval in the resistant strain, it
appears that penetration of this dosage level in these two strains is
quite similar. Penetration of the 30 ug. DDT treatment in male cock·
roaches of the two resistant strains and the susceptible strain was
also observed to be very similar (Figure 3). At this treatment level the
susceptible-strain cockroaches were unable to survive longer than 24
hours. However, their penetration pattern was nearly identical to
those of the two resistant strains up to that time period. If penetration
of DDT were a major factor in the DDT-resistance phenomenon of these
cockroaches, we probably would have observed a greater difference
between the penetration rates of the resistant and susceptible
cockroaches following treatment with identical dosages of DDT.
Apparently, penetration of DDT is not involved in the DDT-resistance mechanism of the resistant cockroach strains used in this study.
Penetration of the 30 ug. DDT treatment appeared to be somewhat
slower in the VPI-DDT-strain females than in the males at the 48 and
72 hour time intervals (Figure 4). A similar trend was observed in the
Landsthul•strain cockroaches (Figure 5). This somewhat lower rate of
penetration of DDT shown by resistant strain females could help to - 56 -
Figure 1. Penetration of 8 ug. of DDT in male and female ~usceptible-strain German cockroaches. - 57 -
0 0 N,....
{/) QJ (/) ...... QJ r-1 ~ cu QJ ;:E: i:i..
c 0 oc oO """ IJI 0I ,-.. (/) l-1 .....,,.c:: DI 0I Q)
•r-1a E-t
Q) l-1 ::I {/) 0 ~ ~
co N """
N \ o"\ ...... c"0 o"
0 IO 0 0 ("') N N r-1 - 58 -
Figure 2. Penetration of 8 ug. of DDT in resistant and susceptible male German cockroaches. '1;j (!) ~ ('iS 35 M ~ (!) ij 30 Cle
5
3 6 12 24 48 72 Exposure Time (hrs.) - 60 •
Figure 3. Penetration of 30 ug. of DDT in resistant and susceptible male German cockroaches. 50 o~
-~-~ • ··~--=--~a•
"O 3 6 12 24 48 72 Exposure Time (hrs.) - 62 - Figure 4. Penetration of 30 ug. of DDT in VPI•DDT-strain in.ale and female German cockroaches. - 63 .. 0 CJ) (L) CJ) .-I (L) .-I ~ co (l) ::E: Pt-I E-1 E-1 Q A Q Q I I H H P-4 Pol > > QC) Cl I I ..::r 0 iC ...... en l-1 I I ..c:: 0 i< (L) -s •.-1 I I E-i (1) "'4 ;:I CJ) 0 ~ IJ::I ..:!' N 0 0 0 0 lf'I M N .... - 64 - Figure 5. Penetration of 30 ug. of DDT in male and female Landsthul•strain German cockroaches. - 65 ... 0 0 tll QJ ti) .-1 QJ .-1 <\'! m :::.:: µ.. .-1 .-1 :I ::I ,.c:: ,.c:: -I.I -I.I tll tll '"d 't:I i:: ra lil ,..:i ,..:i 00 0 0 ...... I I ...... 0 0 . tll H I I 6 0 0 .~ I I E-1 QJ H ::I ti) 0 i::i.. >cl i::r:I ...... 0 a N 00 11 co 0 /' 0 0 0 0 0 0 LI"\ -.r M N r-1 - 66 - explain the higher degree of resistance to DDT found in this sex. In each resistant strain the rate of penetration was nearly the same in both sexes up to about 30 hours after treatment. After this time the rate of penetration increased at a much faster rate in the males than in the females. Figure 6 shows the penetration of DDT from three different DDT treatment levels in VPI-DDT resistant males. These data show that the penetration of DDT is directly proportional to the concentration of the applied dose. A similar relationship is shown between the 30 ug. and 150 ug. DDT treatment levels by Landsthul-strain males (Figure 7). Pretreatment of the susceptible-strain male cockroaches with two levels of sesamex had no great effect on penetration of the 8 ug, DDT treat- ment. Although the penetration of DDT in the presence of sesamex appeared to be somewhat slower at the 3 and 6 hour time intervals, the three curves are in very close proximity after 12 hours have elapsed (Figure 8). High mortality in the cockroaches pretreated with 40 ug. of sesamex prevented the collection of data at the 48 and 72 hour time intervals for this treatment. Sesamex pretreatments had no apparent effect on penetration of the 30 ug. DDT treatment in the resistant VPI-DDT- and Landsthul-strains (Figures 9 and 10, respectively). Detoxication The detoxication of DDT to the non-toxic metabolite dicofol in resistant and susceptible cockroaches was measured by quantitating the dicofol present in the internal and excreta fractions for each treatment - 67 - Figure 6. Penetration of three DDT treatment levels in male VPI-DDT•strain German cockroaches. - 68 .. 0 0 H H Q H Q Q Q Q Q . . b.O . b.O ;:; b.O ;:; ;:; 0 0 I/'\ 00 ("') ...i 00 I I I 0 0 • ..:t -+c 0 0 ...... t1.I $-1 I I I ....,.,..c: -it D 0 (I) e •.-1 l I j H (l) 1-1;j t1.I 0p. ~ µ:::i 0 I I i 0 0 0 0 0 0 0 00 ...... \() I/'\ ("') N ...i - 69 - Figure 7. Penetration of two DDT treatment levels in male Landsthul-strain German cockroaches. - 70 - 0 00 H H -.::t A § A . • . Cl) 0.0 0.0 -1-1 ::I ::I ..._,,,~ 0 0 C'I') ll"l ..... ~ .... .H QJ 1-1 I ::I 0 Cl) i< 0 p., :i< I I i:r.:I i< 0 I I -.::t 0 1' N 0 \ 0 b 0 0 0 0 0 0 0 0 0 ("') ..... 0\ 00 ...... '° II'\ -.::t N - 71 - Figure 8. Penetration of 8 ug. of DDT in susceptible-strain male cockroaches pretreated with two different levels of sesamex. 'tj Q) .µ 35 al 1-t .µ Q) ~p. 30 ~ --.....8 _-a a- 25 ...... • • N i j 0 I f- p. 20 I • ~ " \H 0 .. -I .µ 15 I II -a-a- 8 ug. DDT ~ 16 ug. Sesamex + 8 ug. DDT u 10 Hor0 i I -o-o--·-·-40 ug. Sesamex + 8 ug. DDT 5 3 6 12 24 48 72 Exposure Time (hrsv) - 73 - Figure 9. Penetration of 30 ug. of DDT in VPI•DDT•strain male German cockroaches following pretreatment with three levels of sesamex. 50 0 ~: 'Q • ~ 40 ,,..as ;µ 11) ~ c:i.. ------.------~-- 3 6 12. 24 48 72 Exposure Time (hrs.) - 75 - Figure 10. Penetra~ion of 30 ug. of DDT in Landsthul-strain male Ge~an cockroaches following pretreatment with three 1¢vels of sesamex. 50 I f30 • 't:I I #/ 3 6 12 24 48 72 Exposure Time (hrs.) - 77 - dose at every time interval (Appendix A). The portion of the penetrated dose which was converted to dicofol was determined by combining the internal and excreta dicofol for each treatment exposure period. These results are presented in Table IV. Figure 11 shows the conversion of the penetrated 8 ug. DDT dose to dicofol in susceptible-strain male and female cockroaches. This figure indicates that the conversion mechanism is indeed functional in non-resistant cockroaches. The conversion rates of the two sexes are generally similar, but the female cockroaches appear to be able to convert DDT to dicofol at a slightly faster rate than the males after 48 and 72 hours. The somewhat higher Lc 50 value for these females may be partially attributable to this factor. The resistant VPI-DDT• strain male cockroaches also detoxify the 8 ug. DDT dose at a faster rate than the susceptible-strain males (Figure 12). Although the two conversion rates are quite similar up to the 24 hour time interval, the resistant strain converted 19.7 per cent of the penetrated dose to dicofol after 72 hours exposure while the susceptible strain converted only 11.6 per cent by that time. Detoxication of the 30 ug. DDT treatment by the males of the two resistant strains was nearly identical. They both converted approximately 15 per cent of the penetrated DDT dose to dicofol after 72 hours (Figure 13). The susceptible-strain conversion curve shown in this figure was identical with those of the resistant strains for the first 6 hours, but then the conversion rate reached a level of only about half that exhibited by the resistant strains Table IV • Detoxication of several dosage levels of DDT in resistant and susceptible German cockroaches in presence and absence of sesamex (Ses.). The number in parentheses represents the per cent of penetrated dose converted to dicofol. Susceptible- VPI-DDT- Susceetible-Males Females Males 8 DDT 16 Ses. + 8 DDT 40 Ses. + 8 DDT· 30 DDT 8 DDT 8 DDT Hour dicofol dicofol dicofol dicofol dicofol dicofol after produced produced produced produced produced produced treatment {ug./insectl {ug./insect~ {ug. /insecq {ug./insect) {ug./insectl {ug./insect) 3 .04 (2.8) .01 (1.3) .01 (1.3) .03 (1.1) .03 (3.2) .01 (0.9) 6 .12 (6.4) .02 (2.4) .02 (2.5) .32 (8.3) .04 (3.4) .09 (6.9) 12 .18 (8.9) .09 (4.3) .04 (2.2) .40 (9.3) .09 (5.9) .17 (11.l) 24 .27 (15.3) .20 (12.8) .05 (2.9) .34 (7.9) .19 (11.5) .26 (14.9) 48 .19 (11.7) .15 (9.7) High Mortality .26 (15.3) .28 (15.9) 72 .21 (11.6) .08 (5.9) High Mortality .25 (15.0) .29 (19.7) -...) VPI-DDT- °' VPI-DDT-Males Females 30 DDT 30 Ses. + 30 DDT 60 Ses. + 30 DDT 90 Ses. + 30 DDT 150 DDT 30 DDT 3 .07 (2.0) .01 (0.4) .01 (0.3) .01 (0.3) .09 (0.5) .01 (0.2) 6 .42 (9.4) .03 (0.9) .02 (0.5) .01 (0.3) .23 (1.3) .06 (1.4) 12 .68 (14.8) .08 (1. 3) .07 (1.4) .04 (0. 7) .46 (1.6) .18 (3.9) 24 .84 (14.5) .29 (4.1) .27 (4.2) .24 (3.5) .98 (2.0) .84 (11. 7) 48 1.51 (12.4) • 92 (7. 9) • 71 (6.6) .42 (3.6) 1.52 (2.7) 1.63 (18.7) 72 1.66 (15.4) .82 (7.9) .77 (7.0) .65 (5.3) 1.33 (2.1) 1. 73 {20. O) Landsthul- Landsthul-Males Females 3 .08 (2.2) .01 (0.3) .01 (0.3) .01 (0.3) .06 (0.4) .04 (0.8) 6 .39 (9.0) .10 (1.8) .03 {0.8) .01 (0.3) .18 (0.9) .17 (3.8) 12 .56 (13.2) .06 (1. 7) .02 (0.6) .02 (0.4) .51 (1. 9) .35 (8.0) 24 .64 (13.6) .44 (6.7) .30 (4.2) . .;25 (3. 9) 1.27 (3.1) .47 (8.7) 48 1.40 (12.2) .90 (7 .8) .82 (7.1) .62 (5.2) 1.58 (2.8) 1.54 (22.1) 72 1.65 (14.5) 1.09 (9.9) .88 (7 .6) .85 (7 .3) 2.54 (3.1) 1.87 (24.2) - 79 - Figure 11. Conversion of 8 ug. of DDT to dicofol by susceptible• strain male and female German cockroaches. M 0 4-1 20 0 C) •.-1 't) 18 0 .µ 'ti Figure 12. Conversion of 8 ug. of DDT to dicofol by male VPI-DDT- and susceptible-strain German cockroaches. - 82 .. 0 0 \'/) QJ ...... Cll Cl) ::E:! QJ .-I OJ Cll r-1 ::i::: ...... a E-1 +.> A i:i.. A co 0 0 .:::i' 0I 0I ...... Cl). 1-i ..c: 0I 0I -~ ...... l I E-1 (I) 1-1 :::I en 0 Ac :< "'1 .:::i' 00 N N .-I - 83 - Figure 13·. Conversion of 30 ug. of DDT to dicofol in males of two resistant and one susceptible strain of German cockroaches...... 0 ~ 0 20 u •..! "O 0 18 .u "O ~ j I I I 3 6 12 24 48 72 Exposure Time (hrs.) - 85 - at the 12 and 24 hour .periods. Mortality began to occur shortly after 24 hours. The lower conversion rates shown by susceptible males at the 12 and 24 hour time intervals are evidently not due to reduced penetration since the penetration rates in the three strains were shown to be nearly identical up to 24 hours (Figure 3). This smaller conversion of DDT to dicofol in susceptible cock- roaches may have been caused by a physiological state of intoxication occurring prior to death, or to inherant differences in the strains. In any case, it seems probable that a much greater difference in dicofol production would have been observed between the resistant and susceptible strains at the 8 ug. and 30 ug. DDT treatment levels if detoxication to dicofol were a major factor responsible for the large difference in resistance levels exhibited by these strains. As was observed in the susceptible-strain cockroaches, the VPI-DDT• strain females also convert DDT to dicofol at a slightly faster rate than do the males of this strain (Figure 14). At 72 hours the females converted 20 per cent of the penetrated DDT dose to dicofol as compared to 15.4 per cent conversion by the males. The Landsthul•strain males and females showed similar trends (Figure 15). Here again, this may be partially responsible for the higher degree of resistance observed in resistant females. The DDT conversion rates in the resistant strains show quite different trends between sexes. The rate of conversion in the resistant males increases rapidly up to 12 hours, after which only a - 86 - slight increase in rate occurs. In the females, however, a more gradual increase in conversion rate proceeds for 48 hours before leveling off. Similar, though less obvious differences are seen in the susceptible strain cockroaches. Perhaps the lower treatment dose effected the trends in this case. The differences observed in the conversion curves between sexes may be related to the fat body content of these cock- roaches. The faster initial conversion rates observed in the males are possibly a result of a higher concentration of free DDT present in the body fluids. In the females, the DDT could initially be stored in the fat body tissue and gradually released and converted to dicofol. Apparently the conversion trends are not directly effected by the penetration trends since exactly opposite patterns are observed (Figures 4 and 5). Figure 16 shows the conversion of the penetrated dose from three different DDT treatments to dicofol by VPI-DDT-strain male cockroaches. The highest rate of conversion was with the 8 ug. DDT treatment which reached a 19.7 per cent conversion at the 72 hour time interval. The lowest rate of conversion was with the 150 ug. DDT treatment where only a 2.1 per cent conversion occurred. These trends are, of course, related to the penetrated doses of the respective treatments which were shown in Figure 5. When the actual amounts of dicofol produced at these treatment levels are compared (Figure 17) it appears that the production of dicofol reaches a maximum level which is not exceeded in the presence of additional DDT. Similar trends were shown by Landsthul• strain males. - 87 - Figure 14. Conversion of 30 ug. of DDT to dicofol by male and female VPI-DDT-strain German cockroaches. - 88 - 0 0 Cl) (]) C/) ...... (j) m .-1 i:tl ~ ::0:: ~ E-4 E-1 A A A A I I H H j:l.f Al > > (X) 0 0 ...:I" 0I 0I • Cl.I -1-1 ..c: 0I 0I -s(]) •rl I I E-4 (]) 1-1 ::i rn 0 c. x: ~ ...:I" 0 C'I 0 C'I 0 (X) N ...... - 89 - Figure 15. Conversion of 30 ug. of DDT to dicofol by male and female Landsthul-strain Germ.an cockroaches. 26 24 J _-0 .-f 0 l,j..j 0 22 0 or-I 't:I .3 20 't:I 18 EJ..I QJ > g 16 0 Cl) Vl \0 0 14 _n_ 0 't:I o- / ------0 ] 12 ~ I'll J..I ~ 10 i:: Cl) D c:i. I -a ~ 8 0 ~ i:: Cl) 6 0 Landsthul Males J..I -o-o- Cl) Poi 4 -o-o- Landsthul Females 2J 3 6 12 24 48 72 Exposure Time (hrs.) - 91 - Figure 16. Conversion of the penetrated dose from three different DDT treatments to dicofol by VPI·DDT• strain male German cockroaches. -0-0- 8 ug. DDT .--i -o-o- 30 ug. DDT 0 4-1 20 0 0 -*-*-150 ug. DDT 0 -.-! "O 18 0 .j,J "O Cl) 16 0 .j,J I-! LO -D Cl) 0 0 ~ 14 0 I 0 l..O QJ 12 N !I) 0 "O 0------"O 10 -----.------·------.------,- 3 6 12 24 48 72 Exposure Time (hrs.) - 93 - Figure 17. Actual dicofol produced by VPI•DDT-strain male German cockroaches following treatment with three different dosage levels of DDT. - 94 - 0 0 co ..;:t ...... en M ...... a N ...... 0 LI'\ 0 LI'\ N • .-l. .-l. . - 95 - The conversion of DDT to dicofol in resistant and susceptible cockroaches is inhibited by pretreatment with sesamex, a known inhibitor of microsomal oxidations. Figure 18 shows that in susceptible-strain males, a 2:1 ratio of sesamex to DDT decreased conversion to 2.9 per cent of the penetrated dose at this time interval. These reductions in dicofol production in susceptible-strain males correlate well with the observed increase in six-day mortality shown to occur with sesamex- DDT combinations in this strain (Table I). Apparently, the rate of detoxication of the 8 ug. DDT treatment by susceptible-strain males is sufficient to provide partial protection from the toxic action of DDT. When the detoxication process is inhibited with sesamex, more DDT is evidently available to "enter" the DDT site of action and increased mortality results~ Figures 19 and 20 show the effects of three levels of sesamex on dicofol production by male cockroaches of VPI-DDT- and Landsthul- strains, respectively, following treatment with 30 ug. of DDT. Each increasing concentration of sesamex causes a greater reduction in dicofol production. In VPI-DDT•strain males at the 72 hour interval a 3:1 ratio of sesamex to DDT reduced DDT conversion to dicofol from 15.4 per cent to 5.3 per cent of the penetrated dose. In Landsthul-strain males at the same time period the 3:1 sesamex-DDT combination reduced DDT con• version from 14.5 per cent to 7.3 per cent of the penetrated dose. Unlike the susceptible-strain, no increased mortality was observed with any of the sesamex-DDT combinations. This evidence indicates that even - 96 - Figure 18. Conversion of 8 ug. of DDT to dicofol by susceptible• strain male German cockroaches in the presence of two levels of sesamex. -a-o- 8 ug. DDT .-I I 16 ug. Sesamex + 8 ug. DDT iE 20 --·-·- 0 -o-o- 40 ug. Sesamex + 8 ug. DDT 0 I or-! ""Cl 18 0 ,µ "t:l Q) 16 ,µ 1-1 I a 3 6 12 24 48 72 Exposure Time (hrs.) - 98 - Figure 19. Conversion of 30 ug. of DDT to dicofol by VPI- DDT-strain male German cockroaches following pretreatment with three different levels of sesamex. -o-o- 30 ug. DDT .-1 I 30 ug. Sesamex + 30 ug. DDT 0 --·~·- '-1-1 0 -a-a- 60 ug. Sesamex + 30 ug. DDT .,...0 "O 90 ug. Sesamex + 30 ug. DDT 0 ::J .µ -*-*- "O (1) .µ 16 I -0 1-1 0 (1) 6 0 14 E:0 0 --o (1) en 121 \0 0 \0 "O I "O 10"" (l) 0 .µ I tll !-{ .µ 8 Q} • • m 0 p.. 6 '-1-1 0 .µ -* t:l 4 Q) 0 0 ~i- *-- 1-1 2 Q) ~ M-2=-==f~ ------I 3 6 12 24 48 72 Exposure Time (hrs.) - 100 - Figure 20. Conversion of 30 ug. of DDT to dicofol by Landsthul• strain male German cockroaches following pre- treatment with three different levels of sesamex. -0------0- 30 ug. DDT .-1 0 I 30 ug. Sesamex + 30 ug. DDT 4-1 20 0 -·-·- () -a-o- 60 ug. Sesamex + 30 ug. DDT ..-{ "d 18 ~ 0 90 ug. Sesamex + 30 ug. DDT .µ -*-*- "d OJ 16 .µ k OJ -0 ~ 14 o- 0 0 ----. OJ ----0 I-' {I) 12 0 0 I-' "d lo "d 10 OJ .µ Cl! --· k .µ OJ __ i i::; 81 - _-a OJ I -· c:i.. 6 I I / 4-1 0 .µ ------Q 4 OJ 0 1-1 21 0 OJ ~ ,,,.,.,..•---- P-t .{ef.-::tr. .,. I I -----.- .------.------.- 3 6 12 24 48 72 Exposure Time (hrs.) - 102 - though the detoxication mechanism is inhibited by a factor of 1/2 to 2/3 in the resistant strains, the increased level of unchanged DDT caused by this inhibition has no noticeable effect on the resistant cockroaches. These observations add support to the theory that detoxication of DDT is not of great importance in the resistance of these Gennan cockroaches to DDT. Apparently, the site of action of DDT in resistant cockroaches remains protected from the toxic action of DDT in some, as yet unknown, manner. Excretion The excretion of unchanged DDT by resistant and susceptible German cockroaches following several different DDT treatments was measured to see if a relationship exists between the various levels of DDT resistance in these strains and the excretion of penetrated DDT. The quantitated excreta DDT for the various treatments and expo• sure intervals is presented in Table V. Figure 21 shows the rate of DDT excretion in susceptible-strain male and female cockroaches following treatment with 8 ug. of DDT. After 72 hours the males excreted 25.4 per cent of the penetrated dose, while the females excreted only 14.4 per cent. Similar trends, but at a strikingly higher level, were observed in the resistant strains following a 30 ug. treatment of DDT (Figures 22 and 23). These differences in excretion between sexes may be a result of an increased ability on the part of female cockroaches to store DDT in their abundant fat body tissues. Table v . Excretion of DDT by resistant and susceptible German cockroaches following treatment with several dosage levels of DDT in presence and absence of sesamex (Ses.}. The number in parentheses represents the per cent of penetrated dose excreted as DDT. S1,1sceptible- VPI-DDT- SusceEtible-Males Females Males 8 DDT 16 Ses. + 8 DDT 40 Ses. + 8 DDT 30 DDT 8 DDT 8 DDT Hour DDT DDT DDT DDT· DDT DDT after excreted excreted excreted excreted excreted excreted treatment {ug./insect} {ug ./insect} {ug./insect) {ug. /insecti {ug./insect} (ug./inse_ct) 3 .06 (4.1) .26 (33.8) .19 (24.4) .28 (10.6) .09 (9.5) . 03 (2. 6) 6 .20 (10.6) .19 (22.9) .17 (21.3) .49 (12.7) .10 (8.4) .05 (3.8) 12 .22 (10.9) .25 (12.1) .37 (20.6) .66 (15.4) .20 (13.2) .16 (10.S) 24 .21 (11.9) .26 (16.7) .40 (22.9) 1.32 (30.8) .19 (11.5) .28 (16.1) 48 .31 (19.0) . 20 (13. 0) High Mortality .26 (15.3) .40 (22. 7) I-' 72 .46 (25.4) .28 (20. 7) High Mortality .24 (14.4) .40 (27 .2) 0 w VPI-DDT- VPI-DDT-Males Females 30 DDT - 30 Ses. + 30 DDT 60 Ses. + 30 DDT 90 Ses. + 30 DDT 150 DDT 30 DDT 3 .10 (2.8) .35 (13.1) .39 (12.9) .34 (9.0) 7 .20 (37 .1) .64 (14.3) 6 .38 (8 .5) .54 (16.7) .28 (6.6) .40 (10.9) 3 .43 (19 .1) .66 (15.0) 12 1.21 (26.2) • 78 (13 .0) .36 {7 .4) .67 (11.1) 10.02 {35.2) .82 (17 .6) 24 2.00 (34.4) 1. 51 (21. 5) 1.92 {29.9) 1.94 (28.2) 15.10 (31.0) 2.88 {40.0) 48 6.22 (51.1) 7.12 {61.4) 6.57 (61.4) 7.67 (65.8) 27.03 (47.8) 3.52 (40.4) 72 6. 62 (61.5) 7.29 {69.9) 7 .38 {67 .5) 8.23 {67.7) 43.43 (68.4) 3.99 (46.1) Landsthul- Landsthul-Males Females 3 .09 (2 .5) .58 (18.5) .28 (8.3) .47 (13.9) 5.54 (32.4) .37 (7.0) 6 .48 (11.1) • 68 {12. 0) .38 {10.8) .41 (10.5) 6.11 (31.0) .41 (9.3) 12 1.40 {33 .0) .61 (16.9) .41 (13. 0) .46 (9 .6) 9.55 {35.7) .84 (19.3) 24 1.82 (38. 6) 2 .16 {32. 7) 1.68 (23.7) 1.59 (24.8) 13 .27 {32. 9) 1.45 (26 .8) 48 6.04 (52.8) 8.23 (71.1) 7.24 (62.7) 7.85 (66.2) 26.32 (47 .4) 2.31 {33.1) 72 7.47 (65.7) 8.19 {74.3) 8.14 (70.2) 8.55 (73.0) 58 .26 (70. 7) 3.32 (42.9) - 104 - Figure 21. Excretion of DDT by male and female susceptible- strain Gennan cockroaches following treatment with 8 ug. of DDT per insect. E-1 -o-o- Susceptible Males § m -0-0- Susceptible Females ell 35 "d I.LI ./..I I.LI ""'0x 30 3 6 12 24 48 72 Exposure Time (hrs.) - 106 - Figure 22. Excretion of DDT by male and female VPI-DDT-strain German cockroaches following a treatment of 30 ug. of DDT per insect. - 107 - 0 0 co D 0 ..;j' f/) {fl• 0I 0I ..;j' 0 0 N 0I 0I I I N o~ ..... \ 0, '° 0 0 ~ 0 0 0 0 0 0 0 0 00 ll"I ..;j' C"") N ..... " "° .r.aa s-e pa4a.I::>xa a sop pa41u4auad ;to :iua::> .lad - 108 - Figure 23. Excretion of DDT by male and female Landsthul• strain Gennan cockroaches following a 30 ug. treatment of DDT per insect. E-1 80 ... -o-o- l.andsthul Males ~ ~ UI I -o-o- Landsthul Females ct! 70 "d QJ .u I --o QJ i..i 0 60 >: QJ v .-0 fl) I 0 50 "d ...... 0 "d l.D Cl) .u ~ -o ell ~ 40 .u o______-o. QJ j d -o QJ p.. 30 IH 0 .u d 20 i~o Cl) CJ $-f Cl) 1/0 P-r 10 Pr1 0 3 6 12 24 48 72 Exposure Time (hrs.) - 110 - Increased storage would leave less free DDT available to the excretion mechanism. Figure 24 shows the excretion of DDT by VPI-DDT- and susceptible- s train males following the 8 ug. DDT treatment. Although the resistant strain excretes DDT at a slightly faster rate, the two strains appear to be nearly equal in their excretion capacities at this dosage level. Excretion of the 30 ug. DDT treatment by males of the three strains is shown in Figure 25. The DDT excretion curves for the two resistant strains are nearly identical. The excretion curve for the susceptible• strain males in this figure is very similar to those of the resistant strains up to the time that mortality occurs. These results seem to indicate that excretion of unchanged DDT is not of great importance in the DDT-resistance mechanism of these cockroaches. It appears, however, that excretion does play an important role in ridding the cockroaches of the penetrated dose following DDT treatment, provided they can remain alive. Figure 26 shows that following the 8 ug. DDT treatment 27.2 per cent of the penetrated dose was excreted as unchanged DDT by VPI-DDT-strain males 72 hours after treatment. Following the 30 ug. DDT treatment these cockroaches excreted 61.5 per cent of the pene• trated dose at this time interval, a substantial 34.3 per cent increase in excretion rate. However, following the 150 ug. treatment the excretion rate after 72 hours increased only 6.9 per cent beyond that of the 30 ug. treatment to a level of 68.4 per cent excretion. These results would seem to indicate that a maximum excretion rate was being approached with the higher treatment level. On the other hand, when the actual ug. of DDT excreted at each dosage level are compared, a much - 111 - Figure 24. Excretion of DDT by VPI-DDT- and susceptible-strain male German cockroaches following treatment with 8 ug. of DDT per insect. - 112 - (/) Ul• 1-1 I .J:: aI 0 -~ I I •.-! E-t Q) 1-1 ::I Ul 0 ~ i:z;:i 0 ...:!' IN co' a'-\, o '"'ab 0 0 ("") N - 113 - Figure 25. Excretion of DDT following a 30 ug. DDT treatment by two resistant- and one susceptible-strain of male German cockroaches. E-l 80 Q Q w ti! 70 'O Q) ,µ 0 Q) ""0 ~o ;.: 6J l ------.---- . ---r· 3 6 12 24 48 72 Exposure Time (hrs.) - 115 - Figure 26. Excretion of DDT by VPI-DDT-strain male German cockroaches after treatment with three different dosage levels of DDT. 150 ug. DDT 901 -o-o- -o-o- 30 ug. DDT E-f A 80 -i 8 ug. DDT A -*-*- I'll a:! 70, "Cl ~O (JJ .u ,...(JJ I _.,.,,.- --D 0 60 K lll (JJ I'll 0 "d 501 I-' ~g I-' "d Cl' l1I .u a:!,... 40 .u 0 different relationship is observed between the various treatments (Figure 27). At the 72 hour exposure interval the VPI-DDT-strain males excreted 0.4 ug. of unchanged DDT following the 8 ug. DDT treatment, 6.62 ug. following the 30 ug. treatment, and 43.43 ug. following the 150 ug. treatment. These figures represent a 108 per cent increase in excretion of actual DDT per insect when the treatment level is increased from 8 ug. to 150 ug. of DDT. Similar excretion patterns were observed with Landsthul-strain males following the 30 ug. and 150 ug. DDT treatments. Obviously, the excretion mechanism of these resistant cockroaches is very adaptable and extremely efficient in removing penetrated DDT from these insects. Its activity is most noticeable in the presence of high DDT concentrations. Evidence presented here indicates that the excretion mechanism of the susceptible cockroaches would have a similar capability if death could be avoided. The effect of sesamex on DDT excretion by susceptible-strain males following an 8 ug. DDT treatment is shown in Figure 28. At the 3 hour exposure interval with both of the sesamex-DDT combinations, a sharp increase in DDT excretion was observed. After 12 hours of exposure the 2:1 sesamex-DDT excretion curve assumed the same general pattern shown with the 8 ug. DDT treatment alone. The 5:1 sesamex-DDT excretion curve however remained at a rather high level until death occurred. Sesamex appears to have an unstablilizing effect on the excretory mechanism of susceptible-strain male cockroaches. This phenomenon is apparently not directly related to the DDT concentration since a gradual excretion pattern was exhibited by this strain following - 118 - Figure 27. Actual ug. of DDT excreted by male VPI-DDT-strain German cockroaches following DDT treatments of three different dosage levels. - 119 - 0 0 «> 0 0 ...::;t m. -1-1 E-1 i:-.. E-1 Cl Cl Cl e A Cl A . . . ~ 00 00 g,o. •r-1 ::I ::I E-1 «> 0 0 \1) C""I U"I 1-1 ..... ::I U.l 0 p. x I I I ~ -+:. 0 0 ...::;t I I I 0 0 N i' 0 0 I I I 0 ..... - 120 - Figure 28. Excretion of DDT by susceptible-strain male German cockroaches following treatment with 8 ug. of DDT in combination with two levels of sesamex. -D-0- 8 ug. DDT -0-0- 16 ug. Sesamex + 8 ug. DDT E--1 A 40 ug. Sesamex + 8 ug. DDT A -*-*- (l,l a;j 'O 35 QJ ,µ I 0 (!) $.! 0 >:: 30 (!) (!) (l,l 0 0 I-' N """d I-' 3 6 12 24 48 72 Exposure Time (hrs.) - 122 - a 30 ug. DDT treatment (Figure 25). No data were collected for sesamex combinations with the 30 ug. DDT treatment against the susceptible strain because the level of toxication from the 30 ug. of DDT alone was quite high in this strain. Certainly, a higher state of intoxication is present in the susceptible males treated with the sesamex-DDT dombinations, since increased mortality has been related to these treatments (Table I). Increased activity and excitability are usually associated with such a physiological state resulting from DDT poisoning. If this increased activity could result in an increase in excretion, the effect of sesamex on the excretion of susceptible- strain males could perhaps be explained. No attempt was made here, to elucidate this apparent effect of sesamex on excretion by susceptible males. Figures 29 and 30 show the excretion of the 30 ug. DDT treatment by VPI-DDT- and Landsthul-strain males, respectively, following pre- treatment with three levels of sesamex. The sesamex treatments appear to cause a slight general increase in excretion after 48 and 72 hours, but no gross effect is evident in these resistant strains. COMBINED EFFECTS The amount of internal DDT found in the cockroaches following the various DDT treatments gives an indication of the combined effectiveness of the detoxication and excretion processes. The internal DDT was quantitated for the various treatments and exposure intervals and this information is presented in Table VI. It is readily seen from these data that the penetrated dose is substantially reduced by the detoxication and excretion processes, primarily the latter. Figure 31 shows the - 123 - Figure 29. Excretion of DD1' by Vl:'.L-JJJJT-stra1n male German cockroaches following treatment with 30 ug. of DDT in three different combinations with sesamex. H 80 § r/l "1 'd Cl) .µ 70] Cl) *::::::::::::::-: ~i 1-1 0 ~8- --o :.: 60 (!) r/l 0 50 'd ...... 'd N (!) ~~ +" .IJ I Q3 1-1 40 .IJ (!) Q 3 6 12 24 48 72 Exposure Time (hrs.) - 125 - Figure 30. Excretion of DDT by Landsthul-strain male German cockroaches following treatment with 30 ug. of DDT in three different combinations with sesamex. gE-1 80 Ol tl1 • D 'O Q.) 701 -* .j..J ·- :::::::::::=:--o OJ ~!- H 0x 60 Q,I Q,I Cll 0 ...... 'O 50 ~ /~o N o~ 'O 3 6 12 24 48 72 Exposure Time (hrs.) Table VI . Internal concentrations of DDT in resistant and susceptible Gennan cockroaches following three different treatment levels in presence and absence of sesamex (Ses.). The number in parentheses represents the per cent of penetrated dose. Susceptible VPI-DDT- SusceEtible-Males Females Males 8 DDT 16 Ses. + 8 DDT 40 Ses. + 8 DDT. 30 DDT 8 DDT 8 DDT Hour Internal Internal Internal Internal Internal Internal after DDT DDT DDT DDT DDT DDT treatment {ug. /insect} {ug. /insect~ {ug./insect} {u~./insect} {ug. /insect} {ug./insect) 3 1.35 (93.1) 0.5 (64. 9) 0 .58 (74 .4) 2 .34 (91.4) 0.83 (87 .4) 1.12 (96. 6) 6 1.56 (83.0) 0.62 (74. 7) 0.61 (76.3) 3.06 (79.1) 1.05 (88.2) 1.17 (89.3) 12 1.62 (80.2) 1.73 (83.6} 1.39 (77.2) 3.23 (75.3) 1.23 (80.9) 1.20 (78.4) 24 1.28 (72. 7) 1.10 (70.5) 1.29 (73. 7) 2 .62 (61.2) 1.26 (76.4) 1.20 (69.0) 48 1.13 (69.3) 1.19 (77.3) High Mortality 1.18 (69.4) 1.08 (61.4) ...... 72 1.14 (63.0) 0.99 (73.3) 1.18 (70.7) 0.78 (53.1) N> High Mortality -...J VPI-DDT- VPI-DDT-Males Females 30 DDT 30 Ses. + 30 DDT 60 Ses. + 30 DDT 90 Ses. + 30 DDT 150DDT 30 DDT 3 3.34 (95.2) 2.32 (86.6) 2. 62 (86 .8) 3.41 (90.7) 12.13 (62.5) 3.82 (85.5) 6 3.65 (82.0) 2.67 (82.4} 3.92 (92.9} 3.25 (88.8) 14. 2 7 (7 9 . 6) 3.68 (83.6) 12 2.72 (59.0) 5.13 (85.6) 4.42 (91.1) 5.32 (88.2) 17.97 (63.2) 3.67 (78.6) 24 2.97 (51.1) 5.22 (74.4) 4.24 (65.9) 4.70 (68.3) 32 .68 (67 .0) 3 .48 (74. 5) 48 4.45 (36.5) 3.55 (30.6) 3.42 (32.0) 3.56 (30.6) 28.01 (49.5) 3.56 (49.4) 72 2 .49 (23 .1) 2.32 (22.2) 2.78 (25.4) 3.28 (27.0) 18.78 (29.6) 2.98 (34.5) Landsthul- Landsthul-Males Females 3 3.45 (95.3) 2.54 (81.2) 3.07 (91.4) 2.91 (85.8) 11.49 (67 .2) 4.84 (92.2) 6 3.44 (79.8) 4.87 (86.2) 3.12 (88.4) 3.50 (89.3) 13.40 (68.1) 3.85 (86.9) 12 2.28 (53.8} - 2. 94 (81.4} 2.73 (86.4} 4 .33. (90. 0) 16.71 (62.4) 3.16 (72.6} 24 2.25 (47.8) 4.00 (60.6) 5.12 (72.l} 4.58 (71.3) 25.85 (64.0) 3.49 (64.5) 48 3.99 (34.9) 2.45 (21.2) 3.49 (30.2) 3.39 (28.6) 27.63 (49.8) 3.13 (44.8) 72 2.25 {19.8) 1. 74 (15 .8) 2.56 (22.2) 2.31 (19.7) 21.55 (26 .2) 2.54 (32.9) - 128 - Figure 31. Internal DDT concentrations in VPI-DDT-strain male German cockroaches following three different treatments of DDT. - 129 - 0 E'-1 E'-1 E'-1 Q Q i:::i Q. i:::i. Q. 00 bO 00 ;:l ;:l ;:l c:o 0 0 <"'") l/"\ .-l I I I 0 0 it co I I I ..:t 0 Cl it ...... • I I I rn '-"..d'"' (JJ ....El E--t (JJ M ::I Cl.I 0 ~ f:i:l ..:t N N ..... 0 0 N ..... - 130 - relative effectiveness of these processes in removing the penetrated DDT dose following treatment with three dosage levels in the VPI-DDT resistant males. The processes are apparently more effective at the higher dosage levels although some of the early effects at the 150 ug. level may be artifact. It is interesting to note that the internal DDT concentration in resistant females is consistently higher than that of the resistant males (Table VI) which is perhaps a result of greater DDT storage in the female fat body. In general, the internal DDT concentrations substantiate the trends established for penetration, metabolism, and excretion in this study. The evidence presented here is sufficient to indicate that none of the following known DDT resistance mechanisms fit the facts for the German cockroach. 1. Detoxication of DDT to DDE by dehydrochlorinase as exemplified by certain DDT-resistant housefly strains. No metabolism to DDE was detected in this study. This mechanism can be inhibited by DMC. DMC has no effect on roach resistance to DDT. 2. Detoxication of DDT to dicofol as occurs in DDT-resistant Drosophila. Dicofol is produced by both susceptible and resistant cockroaches, but the level of metabolism is quite low. One would expect that high levels of metabolism in the resistant strain would be necessary if this were the major resistance mechanism. In addition, sesamex greatly inhibits dicofol production in susceptible and resistant strains, but does not eliminate or even lower resistance. - 131 - 3. Differences in penetration and/or excretion of DDT are often mentioned but seldom proved as resistance mechanisms. The evidence presented here clearly indicates that the penetration and excretion patterns exhibited by susceptible and resistant strains of cockroaches are very similar. In addition, large amounts of unchanged DDT were present in the tissues of the resistant insects at the end of each experiment in spite of the efficient excretion process. It seems quite unlikely that this would occur if excretion were the major defense mechanism. Clearly, individuals of the resistant strains can tolerate much higher internal concentrations of DDT than can their susceptible counterparts. On the other hand, the unknown resistance mechanism of houseflies which imparts resistance to knockdown or paralysis by DDT does have the following striking similarities to the resistance mechanism present in cockroaches: 1. The resistant cockroaches are not knocked down or paralyzed by DDT. 2• The synergists which inhibit metabolic degradation of DDT do not effect the knockdown of resistant cockroaches. A similar situation was observed in houseflies with synergists which inhibit conversion of DDT to DDE and DDT to an unknown product, probably dicofol1 (Grigolo and Oppenoorth 1966). - 132 - 3. The DDT-resistance factor of German cockroaches and the factor for resistance to knockdown in houseflies are both inherited as simple Mendelian autosomal recessive traits (Cochran 1965 and Harrison 1953, respectively). Grigolo and Oppenoorth (1966} suggested that the mechanism responsible for resistance to knockdown in houseflies may alter the site of action of DDT in that insect. The evidence presented in this study is also consistent with a mechanism of DDT resistance in the German cockroach being associated with the site of action of DDT. If the charge-transfer complex formation theory of O'Brien and Matsumura (1964} can be substantiated as the mode of action of DDT, then the results reported by Hayashi and Matsumura (1967) may be an indication of the actual mechanism of DDT resistance in German cockroaches and perhaps in other insects. Under this hypothesis the resistance mechanism possibly involves a reduced binding capacity of resistant insect nerve tissue for the .DDT molecule as compared to susceptible insect nerve tissue. Iden~ification of the nerve components to which DDT becomes bound must be made before this possible mechanism of DDT resistance can advance beyond the theoretical stage. V. SUMMARY l. Resistance to DDT in three strains of the German cockroach has been studied in .!.!! ~ tests. 2. Penetration of DDT does not appear to be responsible for the difference in resistance levels of the three cockroach strains, since trends are nearly identical in-so-far as they were determined. 3. Sesamex pretreatment had no effect on penetration of DDT- 4. Susceptible as well as resistant cockroaches are capable of converting DDT to dicofol. It appears that susceptible cock• roaches are able to accomplish this conversion at a somewhat slower rate than resistant cockroaches until intoxication occurs. 5. Female cockroaches of all three strains convert DDT to dicofol at a faster rate than their respective males. 6. Dicofol production reaches a maximum whereafter an increase in penetrated DDT does not increase dicofol production. 7. Sesamex pretreatments substantially inhibit the conversion of DDT to dicofol in all three cockroach strains resulting in increased mortality in the susceptible strain only. 8. Excretion of unchanged DDT does not appear to be directly related to the resistance levels observed in the resistant strains. 9. The excretory mechanism is capable of removing much of the penetrated DDT provided the insect can survive the treatment. 10. Excretion of DDT was consistently higher in male than in female cockroaches. 11. Sesamex appeared to increase the excretion of DDT in susceptible males but not in resistant males. - 133 .. - 134 - 12. The detoxication- and especially the excretion-mechanisms are very efficient in removing penetrated DDT from the insect, particularly at high treatment levels. 13. Resistant cockroaches can withstand much higher internal DDT concentrations than susceptible cockroaches. 14. It is proposed that the DDT resistance mechanism in German cockroaches is similar to the unknown mechanism responsible for re1iatance to knockdown in houseflies. VI. REFERENCES CITED Agosin1 M., A. Neghme, and N. Scaramelli. 1960. Non-identity of triosephosphate dehydrogenase of adult male and nymph development stages of Triatoma infestans. Nature 186: 1052-53. Agosin, M., D. Michaeli, R. Miskus, s. Nagasawa, and W. M. Hoskins. 196la. A new DDT-metabolizing enzyme in the German cockroach. J. Econ. Entomol. 54: 340-42. Agosin, M., N. Scaramelli, and A. Neghme. 196lb. Intermediary carbohydrate metabolism of Triatoma infestans (Insecta; Hemiptera). I. Glycolytic· and pentose phosphate pathway enzymes and the effect of DDT. Comp. Biochem. Physiol. 1= 143-59. Agosin, M., N. Scaramelli, M. L. Dinamarca, and L. Aravena. 1963. Intermediary carbohydrate metabolism in Triatoma infestans. II. The metabolism of cl4_glucose in Triatoma infestans nymphs and the effect of DDT. Comp. Biochem. Physiol. ~: 311-20. Agosin, M., A. Morello, and N. Scaramelli. 1964. !artial characteri• zation of the in vivo metabolites of DDT-c 1 in Triatoma infestans. J:-E~ Entomol. 57: 974-77. Annand, P. N., ~al. 1944. Tests conducted by the Bureau of Entomology and Plant Quarantine to appraise the usefulness of DDT as an insecticide. J. Econ. Entomol. 37: 125-59. Arias, R. o. and L. c. Terriere. 1962. The hydroxylation of naphthalene-1-c14 by house fly microsomes. J. Econ. Entomol. 55: 925-29. Babers, F. H. 1949. Development of insect resistance to insecticides. u.s. Bur. Ent. and Plant Quarant. E-776. Babers, F. H. and J.J. Pratt, Jr. 1950. Studies on the resistance of insects to insecticides. I. Cholinesterase in house flies (Musca Domestica L.) resistant to DDT. Physiol. Zool. 23: 58-63. Babers, F. H. and J. J. Pratt, Jr. 1951. Development of insect resistance to insecticides. II. A critical review of the literature up to 1951. u.s. Bur. Ent. and Plant Quarant• E-818. Babers, F. R. and J. J. Pratt, Jr. 1953. Resistance of insects to insecticides. The metabolism of injected DDT. J. Econ. Entomol. 46: 977-81. .. 135 - - 136 - Babers, F. H. and c. C. Roan. 1953. The dehydrochlorination of DDT by resistant cockroaches. J. Econ. Entomol. 46: 1105. Barber, G. W. and J. B. Schmitt. 1948. House flies resistant to DDT residuals sprays. New Jersey Agr. Expt. Sta. Bul. 742. Bergman, E. D., A. s. Tahori, A. Kaluszyner, S. Reuter. 1955. A new group of DDT synergists. Nature 176: 266-67. Beroza, M. 1954. Pyrethrum synergists in sesame oil. Sesamolin, a potent synergist. J. Amer. Oil Chem. Soc. 11: 302-05. Beroza, M. 1955. The structure of sesamolin and its stereo chemical relationship to sesamin, asarinin and pinoresinol. J. Amer. Chem. Soc. lJ_: 3332-34. Beroza, M. 1956. 3-4 methylenedioxyphenoxy compounds as synergists for natural and synthetic pyrethrins. J. Agr. Food Chem. !±= 49-53. Beroza, M. and W. F. Barthel. 1957. Chemical structure and activity of pyrethrin and allethrin synergists for control of the housefiy. J. Agr. Food. Chem • .2,: 855-59. Bettini, S. 1948. DDT-resisting flies. Riv. Parassitol. .2,: 137-42. (Chem. Abstracts only). Bliss, C. I. 1935. The calculation of the dosage-mortality curve. Ann. Appl. Biol. 22: 134-67. Bodenstein, D. 1946. Investigation on the locus of action of DDT in flies (Droso2hila). Biol. Bull.: 90: 148-57. Boyce, A. M. 1928. Studies on the resistance of certain insects to hydrocyanic acid. J. Econ. Entomol. 21: 715-20. Brooks, G. T. and A. Harrison. 1963a. Metabolism and toxicity of the 'cyclodiene' insecticides. Biochem. J. 87: 5P-6P. Brooks, G. T. and A. Harrison. 1963b. Relations between structure, metabolism and toxicity of the 'cyclodiene' insecticides. Nature 198: 1169·71. Brown, A. W. A. 1951. Appearance of insecticide•resistant strains. Insect Control by Chemicals. 759-63. John Wiley & Sons, New York. Brown, A. W. A. 1957. Insecticide-resistance and Darwinism. Botyu•Kagaku 22: 277-82. - 137 - Brown, A. W. A. 1960. The resistance problem, vector control, and WHO. Misc. Publ. Ent. Soc. Amer. £: 59-67. Brown, H. D. and E. F. Rogers. 1950. The insecticidal activity of 1,1-dianisylneopentane. J. Amer. Chen. Soc. 72: 1864-65. Bruce, W. N. 1949. Latest report on fly control. Pests .!l: 7, 28. Bruce, W. N. and G. c. Decker. 1950. House fly tolerance for insecticides. Soap. 26: 122. Buck, J, B. and M. L. Keister. 1949. Respiration and water loss in the adult blowfly, Phormia regina, and their relation to the physiological action of DDT. Biol. Bull. 21.: 64-81. Busvine, J. B. 1951. Mechanism of resistance to insecticide in houseflies. Nature 168: 193-95. Busvine, J. R. 1954. Houseflies resistant to a group of chlorinated hydrocarbon insecticides. Nature 174: 783-85. Butts, J. S., S. C. Chang, B. E. Christensen & c. H. Wang. 1953. DDT dEtoxification product in American cockroaches. Science 117: 699. Butts, W. L. and R. H. Davidson. 1955. The toxicity of five organic insecticides to resistant and non-resistant stratus of Blattella gennanica (L.). J. Econ. Entomol. 48: 572-74. Chadwick, L. E. 1952. The current status of physiological studies on DDT resistance. Amer. Jour. Trop. Med. Hyg. 1: 404-11. Chadwick, L. E. 1957. Progress in physiological studies of insecticide resistance. Bull. Wld. Hlth. Org. -.16: 1203-18. Chattoraj, A. N. and A. W. A. Brown. 1960. Internal DDE production by normal and DDT-resistant larvae of Aedes aegyPti. J. Econ. Entomol. ,21: 1049-51. Chattoraj, A. N. and c. W. Kearns. 1958. DDT-dehydrochlorinase activity in the Mexican bean beetle. Bull. Entomol. Soc. Amer. ~: 95. Clarke, T. H. and D. G. Cochran. 1959. Cross-resistance in insecticide-resistant strains of the German cockroach, Blattella germanica (L.). Bull. Wld. Hlth. Org. 20:. 823-33. Cochran, D. G. 1955. Differential susceptibility of the sexes and developmental stages of the American cockroach to several insecticides. J. Econ. Entomol. ~: 131-33. - 138 - Cochran, D. G. 1965. Insecticide resistance factors and genetic linkage in Blattella germanica (Dictyoptera). Proc. XII Int. Congr. Ent. 240. Cochran, D. G., J.M. Grayson, and M. Levitan. 1952. Chromosomal and cytoplasmic factors in transmission of DDT resistance in the German cockroach. J. Econ. Entomol. 45: 997-1001. Cohen, S. and A. S. Tahori. 1957. Mode of action of di-(p-chloro- phenyl)-(trifluoromethyl)-carbinol, as a synergist to DDT against DDT-resistant houseflies. J. Agr. Food Chem. ,2: 519-23. Colhoun, E. H. 1959. Acetylcholine in Periplaneta americana (L.). III. Acetylcholine in roaches treated with tetraethyl pyro• phosphate and DDT. Can. J. Biochem. Physiol. 37: 259-72. Cristol, S. J and 11. L. Haller. 1945. The chemistry of DDT - a review. Chem. Eng. News 23: 2070-75. Crow, J. F. 1952. Some genetic aspects of selection for resistance. Bull. Natl. Research Council, Publ. 219: 72-75. Crow, J. F. 1957. Genetics of insect resistance to chemicals. Ann. Rev. Entomol. £: 227-46. Dahm, P. A. 1957. The mode of action of insecticides exclusive of organic phosphorus compounds. Ann. Rev. Entomol. £: 247-60. Decker, G. C. and W. N. Bruce. l952a. House fly resistance to chemicals. Amer. Journ. Trop. Med. Hyg. l: 395-403. Decker, G. C. and W. N. Bruce. 1952b. House fly resistance to chemicals. Bull. Natl. Research Council, Publ. 219: 25-31. Dickson, R. C. 1941. Inheritance of resistance to hydrocyanic acid fumigation in the California red scale. Hilgardia 13: 515-22. Dinamarca, M. L.,1~· Agosin, and A. Neghme. 1962. The metabolic fate of C •DDT in Triatoma infestans. Exptl. Parasitol. 12: 61-72. - 139 - Dobzhansky, T. 1951. Genetics and the origin of species. 3rd Ed. Columbia Univ. Press, New York. 94-5. Dorough, H. W., N. C. Leeling, and J. E. Casida. 1963. Nonhydrolytic pathway in metabolism of N-methyl-carbamate insecticides. Science 140: 170-71. Dorough, H. W. and J. E. Casida. 1964. Nature of certain carbamate metabolites of the insecticide Sevin. J. Agr. Food Chem. 12: 294-304. Draize, J. w., G. Woodard, O. G. Fitzhugh, A. A. Nelson, R. B. Smith, Jr., and H. o. Calvery. 1944. Summary of toxicological studies of the insecticide DDT 2,2-(p-chlorophenyl)l,l,1- trichloroethane. Chem. Eng. News 22: 1503-04. Eagleson, C. 1940. Oil synergist for insecticides. U.S. Patent No. 2,202,145. Official Gazette 514: 274. Eldefrawi, M. E., R. Miskus, W. M. Hoskins. 1959. Resistance to Sevin by DDT- and parathion-resistant houseflies and sesoxane as Sevin synergist. Science 129: 898-99. Erdtman, H. and z. Pelchowicz. 1955. Structure of sesamolin and configuration of sesamin. Chemistry and Industry. 567. Fan, H. Y., T. H. Cheng, and A. G. Richards. 1948. The temperature coefficients of DDT action in insects. Physiol. Zool. 21: 48-59. Ferguson, W. c. and c. w. Kearns. 1949. The metabolism of DDT in the large milkweed bug. J. Econ. Entomol. 42: 810-17. Fisk, F. W. and J. A. Isert. 1953. Comparative toxicities of certain organic insecticides to resistant and non-resistant strains of the German cockroach, Blattella germanica (L.). J. Econ. Entomol. 46: 1059-62. Fouts, J. R. and B. B. Brodie. 1956. On the mechanism of drug potentiation by Iproniazid (a-Isopropyl-1-Isonicotinyl Hydrazine). J. Pharm. Exptl. Therap. 116: 480-84. Freeborn, s. B. and w. M. Regan. 1932. Fly sprays for dairy cows - a progress report. J. Econ. Entomol. 25: 167-74. Froelicher, v. 1944. The story of DDT. Soap. 20: 115, 117, 119, 145. - 140 - Fullmer, O. H. and W. M. Hoskins. 1951. Effects of DDT upon the respiration of susceptible and resistant house flies. J. Econ. Entomol. 44: 858-70. Gahan, J. B. and J. M. Weir. 1950. House flies resistant to benzene hexachloride. Science J1l: 651-52. Gersdorff, W. A. and S. I. Gertler. 1944. Pyrethrum synergists. Soap. 20: 123, 125. Gersdorff, W. A., P. G. Piquett, and M. Beroza. 1956. Comparative synergistic effects of synthetic 3,4-methylenedioxyphenoxy compounds in pyrethrum and allethrin fly sprays. J. Agr. Food. Chem. !!:_: 858-62. Gertler, S. I., J. H. Fales, and H. L. Haller. 1943. Fly spray kill. Soap. 19: 105, 107. Gertler, S. I. and H. L. J. Haller. 1943. u.s. Patent No. 2,326,350. Official Gazette 553: 421. Gordon, R. T. and J. H. Welsh. 1948. The role of ions in axon surface reactions to toxic organic compounds. J. Cell. Comp. Physiol. 31: 395-419. Grayson, J. M. 1951. Response of the German cockroach to sublethal concentrations of DDT and benzene hexachloride. J. Econ. Entomol. 44: 315-17. Grayson, J, M. 1953. Effects on the German cockroach of twelve generations of selection for survival to treatments with DDT and benzene hexachloride. J. Econ. Entomol. 46: 124-27. Grayson, J, M. 1954. Differences between a resistant and a non- resistant strain of the German cockroach. J. Econ. Entomol. 47: 253-56. Grayson, J.M. 1960. Insecticidal resistance and control in cock• roaches. Misc. Publ. Ent. Soc. Amer. II: 55-8. Grigolo, A. and F. J. Oppenoorth. 1966. The importance of DDT• dehydrochlorinase for the effect of the resistance gene Kdr in the housefly Musca domestica L. Genetica 37: 159·70. - 141 - Gunther, F. A., R. C. Blinn, G. E. Carman and R. L. Metcalf. 1954, Mechanisms of insecticidal action. The structural topography theory and DDT-type compounds. Arch. Biochem. Biophys. 50: 504-5. Guthrie, F. E. 1950. Effect of temperature on toxicity of certain organic insecticides. J. Econ. Entomol. 43: 559-60. Hadaway, A. B. 1956. Cumulative effect of sub-lethal doses of insecticides on house flies. Nature 178: 149-50. Haller, H. L., E. R. McGovran, L. D. Goodhue, and W. N. Sullican. 1942a. The synergistic action of sesamin with pyrethrum insecticides. J. Org. Chem. Z: 183-84. Haller, H. L., F. B. LaForge, and w. N. Sullivan. 1942b. Some compounds related to sesamin: their structures and their synergistic effect with pyrethrum insecticides. J. Org. Chem. l: 185-88. Haller, H. L., F. B. LaForge, and w. N. Sullivan. 1942c. Effect of sesamin and related compounds on the insecticidal action of pyrethrum on house flies. J. Econ. Entomol. 35: 247•48. Harrison, C. M. 1951. Inheritance of resistance to DDT in the housefly, Musca domestica L. Nature 167: 855-56. Harrison, c. M. 1952. The resistance of insects to insecticides. Trans. Roy. Soc. Trop. Med. Hygiene 46: 255-63. Harrison, C. M. 1953. DDT-resistance and its inheritance in the house fly. J. Econ. Entomol. 46: 528-30. Hartzell, A and H. I. Scudder. 1942. Histological effects of pyrethrum and an activator on the central nervous system of the housefly. J. Econ. Entomol. 35: 428-33. Harvill, E. K., A. Hartzell, and J. M. Arthur. 1943. Toxicity of piperine solutions to houseflies. Contrib. Boyce Thompson Instit. 13: 87-92. Haslam, E. and R. D. Haworth. 1955. The constitution of sesamolin. J. Chem. Soc. P. 827-33. Hatanaka, A., B. D. Hilton, and R. D. O'Brien. 1967. The apparent binding of DDT to tissue components. J. Agr. Food Chem. 15: 854-57. Hayashi, M. and F. Matsumura. 1967. Interactions of DDT with the nervous system of the resistant and susceptible German cockroaches. Nature 1!1= 1510-12. - 142 - Heal, R. E., K. B. Nash, and M. Williams. 1953. An insecticide· resistant strain of the German cockroach from Corpus Christi, Texas. J. Econ. Entomol. 46: 385-6. Hess, A. D. 1952. The significance of insecticide resistance in vector control programs. Amer. Journ. Trop. Med. Hyg. 1: 371-88. Hodgkin, A. L. 1951. The ionic basis of electrical activity in nerve and muscle. Biol. Rev. 26: 339-409. Hoffman, R. A. and A. W. Lindquist. 1949. Effect of temperature on knockdown and mortality of house flies exposed to residues of several chlorinated hydrocarbon insecticides. J. Econ. Entomol. 42: 891-93. Hoffman, R. A., A. R. Roth, and A. W. Lindquist. 1949. Effect of air temperature on the i~secticidal action of some compounds on the sheep tick and on migration of sheep tick on the animal. J. Econ. Entomol. 42: 893-96. Hoffman, R. A.~ A. R. Roth, and A. W. Lindquist. 1951. Effect on house flies of intennittent exposures to small amounts of DDT residues. J. Econ. Entomol. 44: 734-36. Hoskins, w. M. and H. T. Gordon. 1956. Arthropod resistance to chemicals. Ann. Rev. Entomol. 1: 89-122. Hoskins, W. M. and J.M. Witt. 1958. Types of DDT metabolism as illustrated in several insect species. Proc. X Int. Congr. Ent. .£: 151-56. Hoskins, W. M., R. Miskus, and M. E. Eldefrawi. 1958. The biochemistry of DDT resistance in insects. Seminar on the susceptibility of insects to insecticides. Panama,-Pan-Amer. Health Organ. 239-53. Hough, W. s. 1928. Relative resistance to arsenical poisoning of two codling moth strains. J. Econ. Entomol. 21: 325-29. Hough, W. s. 1934. Colorado and Virginia strains of codling moth in relation to their ability to enter sprayed and unsprayed apples, J. Agr. Res. 48: 533-53. Hurst, H. 1940. Permeability of insect cuticle. Nature 145: 462-63. Ilivicky, J. and M. Agosin. 1967. Glutathione to turnover in . Triatoma infestans treated with 2,2-bis-(p•chlorophenyl) •l,1,1-trichloroethane. Exptl. Parasitol. 1.Q.: 345·56. - 143 - Ilevicky, J., M. L. Dinamarca, and M. Agosin. 1964. Activity of NAD·kinase of nymph Triatoma infestans upon treatment with DDT and other compounds. Comp. Biochem. Physiol. J:l: 291-301. Johnston, C. D. 1951. The in vitro effect of DDT and related compounds on the succinoxidase"'""System of rat heart. Arch. Biochem. Biophys. 31: 375-82. Johnston, E. F., R. Bogart, and A. W. Lindquist. 1954. The resistance to DDT by houseflies. Some genetic and environmental factors, J. Heredity 45: 177-82. Kearns, c. w. 1956. The mode of action of insecticides. Ann. Rev. Entomol. ,1: 123-48. Kearns, c. W. 1960. Biochemical aspects of resistance. Misc. Publ. Ent. Soc, Amer • .f: 113-17. Keiding, J. and H. VanDeurs. 1949. DDT-resistance in house-flies in Denmark. Nature 163: 964-65. Kerr, R. W., D~ G. Venables, W. J. Roulston, and H. J. Schnitzerling. 1957, Specific DDT-resistance in houseflies. Nature 180: 1132-33. Knipling, E. F. 1942. Acquired resistance to phenothiazine by larvae of the primary screwworm. J. Econ. Entomol. 35: 63-4. Kovacs, M. F. Jr. 1963. Thin-layer chromatography for chlorinated pesticide residue analysis. J. Ass. Off. Anal. Chem. 46: 884-93. Lauger, P., H. Martin, and P. Muller. 1944. Uber Konstitution und toxische Wirkung von naturlichen und neuen synthetischen insektentotenden Stoffen. Helvetica Chim. Acta 27: 892-928. LeRoux, E. J. and F. O. Morrison. 1954. The adsorption distribution and site of action of DDI4in DDT-resistant and DDT-susceptible house flies using carbon labelled DDT. J. Econ. Entomol. 47: 1058-66. Lindquist, D. A. and P. A. Dahm. 1956. Metabolism of radioactive DDT by the Madeira roach and European corn borer. J. Econ. Entomol. 49: 579-84. - 144 - Lindquist, A. W. and A. G. Wilson. 1948. Development of a strain of houseflies resistant to DDT. Science 107: 276. Lindquist, A. W., H. G. Wilson, H. O. Schroeder, and A. H. Madden. 1945. Effect of temperatures on knockdown and kill of house• flies exposed to DDT. J, Econ. Entomol. 38: 261-64. Lindquist, A. W., A. R. Roth, W.W. Yates, J. S. Butts, and R. A. Hoffman. 195la. Use of radioactive tracers in studies of penetration and metabolism of DDT in houseflies. J. Econ. Entomol. 44: 167-72. Lindquist, A. w., A. R. Roth, R. A. Hoffman, and J. s. Butts. 195lb. The distribution of radioactive DDT in house flies. J. Econ. Entomol. 44: 931w34. Lipke, H. and C. W. Kearns. 1958. Specificity of house fly DDT• dehydrochlorinase. Bull. Entomol. Soc. Amer. !'!:: 95. Lipke, H. and C. W. Kearns. 1959a. DDT-dehydrochlorinase. I. Isolation, chemical properties, and spectophotometric assay._ J. Biol. Chem. 234: 2123-28. Lipke, H. and c. W. Kearns. 1959b. DDT-dehydrochlorinase. II. Substrate and cofactor specificity. J. Biol. Chem. 234: 2129-32. Lord, K. A. 1948. The sorption of DDT and its analogues by chitin. Biochem. J. 43: 72-8. Lord, K. A. 1949. The effect of insecticides on the respiration of Oryzaephilus surinamensis: An attempt to compare the speeds of action of a number of DDT analogues. Ann. Appl. Biol. 36: 113-38. Lov~ll, B. J. and C. W. Kearns. 1956. Inheritance of ability to synthesize DDT-dehydrochlorinase and its relationship to DDT- resistance in the house fly. Bull. Ent. Soc. Amer. 2: 17-18. Lovell, J. B. and C. W. Kearns. 1959. Inheritance of DDT-dehydro- chlorinase in the house fly. J. Econ. Entomol. 52: 931-35. Ludwig, D. 1946. The effect of DDT on the metabolism of the Japanese beetle, Popillia japonica Newman. Ann. Entomol. Soc. Amer. 39: 496-509. McKenzie, R. E. and W. M. Hoskins. 1954. Correlation between the length of the larval period of Musca domestica L. and resistance of adult flies to insecticides. March, R. B. 1952. Summary of research on insects resistant to insecticides. Bull. Natl. Research Council, Publ. l!.2.: 45 ... 53. - 145 .. March, R. B. and L. L. Lewallen. 1950. The comparison of DDT- resistant and non-resistant house flies. J. Econ. Entomol. 43: 721-22. March, R. B. and R. L. Metcalf. 1949a. Development of resistance to organic insecticides other than DDT by houseflies. J. Econ. Entomol. 42:990. March, R. B. and R. L. Metcalf. 1949b. Laboratory and field studies of DDT resistant houseflies in Southern California. Bull. Calif. State Dept. Agr. 38: 93-101. March, R. B. and R. L. Metcalf. 1950. Insecticide-resistant flies• Soap. 1.§.: 121. March, R. B., R. L. Metcalf, and L. I. Lewallen. 1952. Synergists for DDT against insecticide-resistant house flies. J. Econ. Entomol. 45: 851-60. Martin, H. 1956. The chemis~ry of insecticides. Ann. Rev. Ent. 1: 149-66. Martin, H. and R. L. Wain. 1944. Insecticidal action of DDT. Nature 154: 512-13. Matsumura, F. and R. D. O'Brien. 1966a. Absorption and binding of DDT by the central nervous system of the American cockroach. J. Agr. Food Chem. 14: 36-39. Matsumura, F. and R. D. O'Brien. 1966b. Interactions of DDT with components of American cockroach nerve. J. Agr. Food Chem. 14: 39-43. Melander, A. L. 1914. Can insects become resistant to sprays? J. Econ. Entomol. 7: 167-73. Mengle, D. c. and J. E. Casida. 1960. Biochemical factors in the acquired resistance of houseflies to organophosphate insecticides. J. Agr. Food Chem • .§.: 431-7. Menzel, D. B., s. M. Smith, R. Miskus, and W. M. Hoskins. 1961. The metabolism of cl4 labelled DDT in the larvae, pupae, and adults of Drosophila melanogaster. J. Econ. Entomol. 54: 9-12. Merrill, R. s~, J. Savit, and J.M. Tobias. 1946. Certain biochemical changes in the DDT poisoned cockroach and their prevention by prolonged anesthesia. Jour. Cell. Comp. Physiol. 28: 465•76. - 146 .. Metcalf, R. L. 1955a. Organic insecticides, their chemistry and mode of action. Interscience, New York. Metcalf, R. L. 1955b. Physiological basis for insect resistance to insecticides. Physiol. Rev. 35: 197-232. Metcalf, R. L. 1967. Mode of action of insecticide synergists. Ann. Rev. Entomol. 12: 229-256. Micks, D. W. and M. R. v. Murthy. 1961. The effects of DDT on oxidative metabolism in susceptible and DDT-resistant Aedes aegypti. J. Econ. Entomol. ,2i: 461-5. Moats, w. A. 1963. One-step chromatographic cleanups of chlorinated hydrocarbon pesticide residues in butterfat. II. Chromatography on florisil. J. Ass. Off. Anal. Chem. 46: 172-6. Moore, B. P. and P. s. Hewlett. 1958. Insecticidal synergism with the pyrethrins: studies on the relat.ionship between chemical structure and synergistic activity in the 3:4-methylenedio~y phenyl compounds. J. Sci. Food.Agric. 2: 666-72. Moorefield, H. H. 1956. Purification of DDT-dehydrochlorinase from resistant house flies. Contrib. Boyce Thompson Instit. 18: 303-10. Moorefield, H. H. 1958a. The origin of DDT resistance in the house fly. Contrib. Boyce Thompson Instit. 19: 403-9. Moorefield, H. H. 1958b. Synergism of the carbamate insecticides. Contrib. of the Boyce Thompson Instit. 19: 501-7. Moorefield, Herbert H. 1959. Mechanisms of resistance to chlorinated hydrocarbons. Can. J. Biochem. Physiol. 11.= 1099-1103. Moorefield, H. H. and C. W. Kearns. 1955. Mechanism of action of certain synergists for DDT against resistant house flies. J. Econ. Entomol. 48: 403-6. Moorefield, H. H. and c. W. Kearns. 1957. Levels of DDT-dehydro- chlorinase during metamorphosis of the resistant house fly. J. Econ. Entomol. 50: 11-13. Morello, A. 1964. Role of DDT-hydroxylation in resistance. Nature 203: 785-6. Muller, P. 1943. Devitalizing composition of matter. U.S. Patent No. 2,329,074. Official Gazette 554: 167-8. Munson, S. c. and Marvin I. Gottlieb. 1953. The differences between male and female American roaches in total lipid content and in susceptibility to DDT. J. Econ. Entomol. ~: 798-802. - 147 - Narahashi, T. and T. Yamasaki. 1960. Mechanism of increase in negative after-potential by Dicophanum (DDT) in the giant axons of the cockroach. J. Physiol. 152: 122-40. Neal, P.A., T. R. Sweeney, S. s. Spicer, and W. F. von OeHinger. 1946. The excretion of DDT (2,2-bis-(p-chlorophenyl)-1,1,1• trichloroethane) in man, together with clinical observations. Public Hlth. Rpts. 61: 403-9. O'Brien, R. D. 1967. Insecticides, action, and metabolism. Academic Press, New York. O'Brien, R. D. and F. Matsumura. 1964. A new hypothesis of its mode of action. Science:J!t.2.: 657-8. Oppenoorth, F. J. 1959. Resistance patterns to various organophosphorus insecticides in some strains of houseflies. Entomologia Experimontalis Et Applicata 1= 216-23. Painter, R. H. 1930. The biological strains of Hessian fly. J. Econ. Entomol. 23: 322-6. Parkin, E. A. and A. A. Green. 1944. Activity of pyrethrins by sesame oil. Nature 154: 16-17. Pearson, A. M. 1935. The role of pine oil in cattle fly sprays. Delaware Agr. Expt. Sta. Bul. 196: 5-63. Perry, A. S. 1958. Factors associated with DDT-resistance in the house fly, Musca domestica L. Proc. X Int. Cong. Entomol. 1: 157-72. Perry, A. s. 1960a. Biochemical aspects of insect resistance to the chlorinated hydrocarbon insecticides. Misc. Publ. Entomol. Soc. Amer. II P. 119-37. Perry, A. s. 1960b. Metabolism of insecticides by various insect species. J. Agr. & Food Chem. ~: 266-72. Perry, A. s. and A. J. Buckner. 1958. Biochemical investigations on DDT-resistance in the human body louse, Pediculus humanus humanus. Amer. J. Trap. Med. Hyg. l: 620-26. Perry, A. s. and W. M. Hoskins. 1950. The detoxification of DDT by resistant houseflies and inhibition of this process by piperonyl cyclonene. Science 11:1: 600-1. Perry, A. S. and W. M. Hoskins. 195la. Synergistic action with DDT toward resistant houseflies. J. Econ. Entomol. 44: 839-50. Perry, A. s. and w. M. Hoskins. 195lb. Detoxification of DDT as a factor in the resistance of house flies. J. Econ. Entomol. 44: 850-7. - 148 - Perry, A. s., R. W. Fay, and A. J. Buckner. 1953. Dehydrochlorination as a measure of DDT-resistance in house flies. J. Econ. Entomol. 46: 972-6. Perry, A. S., J. A. Jensen, and G. W. Pearce. 1955. Colorimetric and radiometric determinations of DDT and its metabolites in resistant houseflies. J. Agr. Food Chem. 3: 1008-11. Philleo, W.W., R.R. Schonbrod, and L. C. Terriere. 1965. Methylene- dioxyphenyl compounds as inhibitors of the hydroxylation of naphthalene in houseflies. J. Agr. Food Chem. 13: 113-5. Pielou, D. P. and R.F. Glasser. 1952. Selection for DDT resistance in a beneficial insect parasite. Science 115: 117-8. Pierpont, R. L. 1939. Terpene ethers in pyrethrum and rotenone fly sprays. Delaware Agr. Expt. Sta. Bul. 217: 59 pp. Pimentel, D., J. E. Dewey, and H. H. Schwardt. 1951. An increase in the duration of the life cycle of DDT-resistant strains. of the house fly. J. Econ. Entomol. 44: 477-81. Potter, c. and E. M. Gillham. 1946. Effects of atmospheric environment, before and after treatment, on the toxicity to insects of contact poisons. Ann. Appl. Biol. 33: 142-59. Pradhan, s., M. R. G. K. Nair, s. Krishnaswami. 1952. Lipoid solubility as a factor in the toxicity of contact insecticides. Nature 170: 619-20. Pratt, J. J., Jr., and F. H. Babers. 1950. Cross tolerances in resistant houseflies. Science 112: 141-2. Pratt, J. J., Jr. and Frank H. Babers. 1953a. Sensitivity to DDT of nerve ganglia of susceptible and resistant house flies. J. Econ. Entomol. 46: 700-2. Pratt, J. J., Jr. and Frank H. Babers. 1953b. The resistance of insects to insecticides. Some differences between strains of house flies. J. Econ. Entomol. 46: 864-9. Prill, E. A. and W. R. Smith. 1955. Comparison of ethylenedioxyphenyl and methylenedioxyphenyl compounds as extenders for pyrethrins. Contrib. Boyce Thompson Instit. 18: 187-92. Prill, E. A., A. Hartzell, J, M. Arthur. 1946. Insecticidal thio ethers derived from safrole, isosafrole, and other aryl olefins. Contrib. Boyce Thompson Instit. 14: 127-50. Prill, E. A., A. Hartzell, and J.M. Arthur. 1947. Some cyclic acetals containing the 3,4-methylenedioxyphenyl radical and their effectiveness against houseflies. Contrib. Boyce Thompson Instit. 14: 397-403. - 149 - Prill, E. A. and M. E. Synerholm. 1946. Report on some miscellaneous methylene dioxyphenyl compounds tested for synergism with pyrethrum in fly sprays. Contrib. Boyce Thompson Instit. 14: 221-7. Quayle, H. J. 1916. Are scales becoming resistant to fumigation. California University Jour. Agr. 1: 333-4. Quayle, H. J. 1922. Resistance of certain scale insects in certain localities to hydrocyanic acid fumigation. J. Econ. Entomol. 15: 400-4. Quayle, H. J. 1943. The increase in resistance in insects to insec- ticides. J. Econ. Entomol. 36: 493-500. Richards, A. G. and L. K. Cutkomp. 1946. Correlation between the possession of a chitinous cuticle and sensitivity to DDT. Biol. Bull. 90: 97-108. Riemschneider, R. 1949. Increasing the initial toxicity and period of action of p,p'-DDT. Z. angew. Entomol. · 31: 431-40. (Chem .. Abstracts only). Roeder, K. D. and E. A. Weiant. 1946. The site of action of DDT in the cockroach. Science 103: 304-6. Roeder, K. D. and E. A. Weiant. 1948. The effect of DDT on sensory and motor structures in the cockroach leg. Jour. Cell. Comp. Physiol. 32: 175-86. Roeder, K. D. and E. A. Weiant. 1951. The effect of concentration, temperature and washing on the time of appearance of DDT- induced trains in sensory fibers of the cockroach. Ann. Entomol. Soc. Amer. 44: 372-80. Roark, R. C. 1945. A second list of publications on DDT. U.S. Bur. Ent. and Plant Quarant. E-660. Rolofson, G. L. and J. L. Bishop. 1967. DDT and related compounds in dog food and CSMA media used in insect rearing. J. Econ. Entomol. 60: 1186-87. Roth, A. R., A. W. Lindquist, and L. c. Terriere. 1953. Effect of temperature and the activity of house flies on their absorption of DDT. J. Econ. Entomol. 46: 127-30. Sacca, G. 1947. DDT-resisting flies. Riv. parassitol • .§.: 127-8. (Chem. Abstracts only). - 150 - Sacktor, B. 1950. A comparison of the cytochrome oxidase activity of two strains of house flies. J. Econ. Entomol. 43: 832-38. Sacktor, B. 1951. Some aspects of respiratory metabolism during metamorphosis of normal and DDT-resistant houseflies. Biol. Bull. 100: 229-43. Schechter, M. S., S. B. Soloway, R. A. Hayes, and H. L. Haller. 1945. Colorimetric determination of DDT. J. Indust. Eng. Chem., Analy. Ed. 11: 704-9. Schroeder, H. 0., H. A. Jones and A. W. Lindquist, 1948. Certain compounds containing the methylenedioxyphenyl group as synergists for pyrethrum to control flies and mosquitoes. J, Econ. Entomol. 41: 890-94. Smith, H. S. 1941. Racial segregation in insect populations and its significance in applied entomology. J. Econ. Entomol. 34: 1-13. Smith, J. N. 1955. Detoxication mechanisms in insects. Biol. Rev. 30: 45.5-75. Smith, M. I. 1945. Further studies on the pharmacologic action of 2, 2 bis. (p-chlorophenyl) 1, 1, 1 trichloroethane (DDT). Public Hlth. Rpts. 60: 289~301. Smyth, T., Jr. and C. c. Roys. 1955. Chemoreception in insects and the action of DDT. Biol. Bull. 108: 66-76. Sternburg, J. and C. W. Kearns. 1950. Degradation of DDT by resistant and susceptible strains of house flies. Ann. Entomol. Soc. Amer. 43: 444-58. Sternburg, J. and c. W. Kearns. 1952a. Metabolic fate of DDT when applied to certain naturally tolerant insects. J. of Econ. Entomol. 45: 497-508. Sternburg, J, and c. w. Kearns. 1952b. The presence of toxins other than DDT in the blood of DDT-poisoned roaches. Science 116: 144-7. Sternburg, J, and C. W. Kearns. 1956. Pentachlorocyclohexene, an intennediate in the metabolism of lindane by house flies. J, Econ. Entomol. 49: 548-52. Sternburg, J., c. W. Kearns, and W. N. Bruce. 1950. Absorption and metabolism of DDT by resistant and susceptible house flies. J. Econ. Entomol. 43: 214-9. - 151 - Sternburg, J., C. W. Kearns, and H. Moorefield. 1954. DDT-dehy- drochlorinase, an enzyme found in DDT-resistant flies. J, Agr. Food Chem. ~: 1125-30. Sternburg, J., E. B. Vinson, and C. W. Kearns. 1953. Enzymatic dehydrochlorination of DDT by resistant flies. J. Econ. Entomol. 46: 513-5. Sumerford, W. T., M. B. Goette, K. D. Quarterman, and S. L. Schenck.. 1951. The potentiation of DDT against resistant houseflies by several structurally related compounds. Science 114: 6-7. Sun, Y. P. and E. R. Johnson. 1960. Synergistic and antagonistic actions of insecticide-synergist combinations and their mode of action. J. Agr. Food Chem. ~: 261-6. Synerholm, M. E. and A. Hartzell. 1945. Some compounds containing the 3,4-methylene-dioxyphenyl group and their toxicities toward houseflies. Contrib. Boyce Thompson Instit. 14: 79. Synerholm, M. E., A. Hartzell, and V. Cullmann. 1948. Pyrethrin extenders. Contrib. Boyce Thompson Instit. 15: 35-45. Szent•Gyorgyi, A. 1960. Introduction to a submolecular biology. Academic Press, New York. ·.L·anori, A. ;:;. .L~.:>.:>. uiary1-trinuoromethyl-carbinols as synergists for DDT against DDT-resistant house flies. J. Econ. Entomol. 48: 638-42. Tahori, A. S. and W. M. Hoskins. 1953. The absorption, distribution and metabolism of DDT in DDT-resistant houseflies. J. Econ. Entomol. 46: 302-6. Terriere, L. c. and R. D. Schonbrod. 1955. The excretion of a radioactive metabolite by house flies treated with carbon14 labeled DDT. J. Econ. Entomol. 48: 736-9. Thorpe, w. H. 1931. Biological races in insects and their significance in evolution. Ann. Appl. Biol. 18: 406-14. Tobias, J. M. and J. J. Kollros. 1946. Loci of action of DDT in the cockroach Periplaneta americana. Biol. Bull. 91: 247-55. Tobias, J.M., J. J. Kollros, and J. Savit. 1946. Acetylcholine and related substances in the cockroach, fly and crayfish and the effect of DDT. Jour. Cell. Comp. Physiol. 28: 159-82. Tsukamoto, M. 1959. Metabolic.fate of DDT in Drosophila melanogaster. I. Identification of a non•DDE metabolite. Botyu-kagaku £!t: 141-51. .. 152 - Tsukamoto, M. 1960. Metabolic fate of DDT in Drosophila melanogaster. II. DDT-resistance and kelthane production. Botyu-Kagaku 25: 156-62. Tsukamoto, M. 1961. Metabolic fate of DDT in Drosophila melanogaster. III. Comparative studies. Botyu-Kagaku 12_: 74-87. Vinson, E. B. and C. W. Kearns. 1952. Temperature and the action of DDT on the american roach. J. Econ. Entomol. 45: 484-96. Wachs, H. 1947. Synergistic insecticides. Science 105: 530-531. Weed, A. 1938. New insecticide compound. Soap. 14: 133, 135. Welsh, J. H. and H. T. Gordon. 1946. The mode of action of DDT. Fed. Proc. 5: 112. Welsh, J. H. and H. T. Gordon. 1947. The mode of action of certain insecticides on the Arthropod nerve axon. J. Cell. Comp. Physiol. 30: 147-171. White, W. C. aod T. R. Sweeney. 1945. The metabolism of 2,2 bis (p-chlorophenyl) 1,1,1-trichloroethane (DDT). I. A metabolite from rabbit urine, di (p-chlorophenyl) acetic acid; its isolation, identification, and synthesis. Public Hlth. Rept. 60: 66-71. Whitnall, A. B. M. and B. Bradford. 1947. An arsenic-resistant tick and. its control with ga!Illllexane dips. Bull. Entomol. Res. 38: 353-72. Wiesman, R. 1947. Untersuchungen uber das physiologische Verhalten von Musca domestica (L.) verschiender Provenienzen. Mitt. Schweiz. Erit. Gesell. 20: 484-504. (Chem. Abstracts only). Wigglesworth, V. B. 1941. Permeability of insect cuticle. Nature 147: 116. Wilson, H. G. and J. B. Gahan. 1948. Susceptibility of DDT-resistant houseflies to other insecticidal sprays. Science 107: 276-7. Winteringham, F. P. W. 1952. Metabolism of DDT by resistant house flies. Bull. Natl.• Research Council Publ. 219: 61-4. Winteringham, F. P. W. and J.M. Barnes. 1955. Comparative response of insects and mannnals to certain halogenated hydrocarbons used as insecticides. - 153 - Winteringham, F. P. W., P. M. Loveday, and A. Harrison. 1951. Resistance of houseflies to DDT. Nature 167: 106-7. Woglum, R. S. 1925. Observations on insects developing inununity to insecticides. J. Econ. Entomol. 1..§.: 593-7. Woodruff, N. 1950. Interactions between temperature and toxicity of four insecticides applied by injection. J. Econ, Entomol. 43: 663-9. Yamasaki, T. and T. Ishii. 1954. Studies on the mechanism of action of insecticides. VIII. Effects of temperature on the nerve susceptibility to DDT in the cockroaches. Botyu-Kagaku 12,: 39-46. Yamasaki, T. and T. Narahashi. 1957a. Effects of oxygen lack, metabolic inhibitors, and DDT on the resting potential of insect nerve. Botyu-Kagaku 22: 259-76. Yamasaki, T. and T. Narahashi. 1957b. Increase in the negative after-potential of insect nerve by DDT. Botyu•Kagaku 22: 296-3Q4. Yeager, J. F. and S. C. Munson. 1945. Physiological evidence of a site of action of DDT in an insect. Science 102: 305-7 • • ust, H. R., H. D. Nelson and R. L. Busbey. ,1943. Comparative susceptibility of two strains of California red scale to HCN, with special reference to the inheritance of resistance. J. Econ. Entomol. 36: 744-9. The vita has been removed from the scanned document - 155 - APPENDIX A - 156 - Table I. DDT and dicofol recovered from resistant and susceptible Gennan cockroaches after treatment with 8 ug. of DDT per insect. Susceptible-Strain Males Recovery of Hour a:tter External Internal Excreta a:e:elied dose treatment DDT dicofol DDT dicofol DDT ug. % 3 5.22 0.04 1.35 0.06 6.67 101.2 6 4.83 0.12 1.56 0.20 6.71 101.8 12 4.68 0.18 1.62 0.22 6.70 101.7 24 4.51 0.26 1.28 0.01 0.21 6.27 95.l 48 4.25 0 .18 1.13 0.01 0.31 5.88 89.2 72 4.23 0.19 1.14 0.02 0.46 6.04 91.7 Susce:etible-St:rain Females 3 5.54 0.03 0.83 0.09 6.48 98.4 6 5.29 0.04 1.05 0.10 6.47 98.l 12 4.99 0.09 1.23 0.20 6.51 98.7 24 4.75 0.19 1.26 0.01 0.19 6.40 97.l 48 4.34 0.24 1.18 0.02 0.26 6.04 91.7 72 4.29 0.24 1.18 0.01 0.24 5.96 90.4 VPI-DDT-Strain Males 3 5.36 0.01 1.12 0.03 6.52 98.9 6 5.16 0.09 1.17 0.05 6.47 98.2 12 5.08 0.17 1.20 0.16 6.61 100.3 24 4.49 0.25 1.20 0.01 0.28 6.23 94.5 48 4.40 0.25 1.08 0.03 0.40 6.16 93.4 72 4.39 0.26 0.78 0.03 0.40 5.86 88.9 - 157 - Table II. DDT and dicofol recovered from susceptible German cockroaches after treatment with sesamex and 8 ug. of DDT per insect. 16 ug. Sesamex + 8 ug. DDT Recovery of Hour after External Internal Excreta aE12lied dose treatment DDT di co fol DDT dicofol DDT ug. % 3 5.79 0.01 0.50 0.26 6.56 99.5 6 5.52 0.02 0.62 0.19 6.35 96.4 12 4.62 0.09 1.73 0.25 6.69 101.5 24 4.73 0.19 1.10 0.01 0.26 6.29 95.4 48 4.74 0.14 1.19 0.01 0.20 6.28 95.3 72 4.67 0.07 0.99 0.01 0.28 6.02 91.4 40 ug. Sesamex + 8 ug. DDT 3 5.80 0.01 0.58 0.19 6.58 99.8 6 5.58 0.02 0.61 0.17 6.38 96.8 12 4.68 0.04 1.39 0.37 6.48 98.3 24 4.71 o.os 1.29 0.01 0.40 6.46 98.0 48 High Mortality 72 High Mortality - 158 - Table III. DDT and dicofol recovered from resistant and susceptible German cockroaches after treatment with 30 ug. of DDT. VPI-DDT-Strain Males Recovery of Hour after External Internal Excreta aEE lied dose treatment DDT dicofol DDT dicofol DDT ug. % 3 21.75 0.06 3.34 0.01 0.10 •25.26 102.9 6 20.52 0.38 3.65 0.04 0.38 24.97 101. 7 12 20.43 0.62 2. 72 0.06 1.21 25.04 102.0 24 19.02 0.76 2.97 0.08 2.00 24.83 101.1 48 11.94 1.36 4.45 0.15 6.22 24.12 98.2 72 11.61 1.48 2.49 0.18 6.62 22.38 91.2 VPI-DDT-Strain Females 3 19.68 0.01 3.82 0.64 24.15 98.4 6 18.64 0.06 3.68 0.66 23.04 93.8 12 18.14 0.17 3.67 0.01 0.82 22.81 92.9 24 18.04 0.73 3.48 0.11 2.88 25.24 102.8 48 15.75 1.38 3.56 0.25 3.52 24.46 99.6 72 r5.96 1.42 2.93 0.31 3.99 24.61 100.2 SusceEtible-Strain Males 3 21.61 0.03 2.34 0.28 24.26 98.8 6 20.69 0.22 3.06 0.10 0.49 24.56 100.0 12 18.40 0.35 3.23 0.05 0.66 22.69 92.4 24 19.96 0.32 2.62 0.02 1.32 24.24 98.7 48 High Mortality 72 High Mortality - 159 - Table IV. DDT and dicofol recovered from resistant VPI-DDT-strain German cockroaches after treatment with sesamex and 30 ug. of DDT per insect. 30 ugo Sesamex + 30 ug. DDT Recovery of Hour after External Internal Excreta aE12lied dose treatment DDT dicofol DDT dicofol DDT . us;• fo 3 21.59 0.01 2.32 0.35 24.27 98.9 6 20.20 0.02 2.67 0.01 0.54 23.44 95.5 12 16.95 0.07 5.13 0.01 0.78 22.94 93.4 24 15.20 0.28 5.22 0.01 1.51 22.22 90.5 48 12.75 0.79 3.55 0.13 7.12 24.34 99.1 72 12.71 0.66 2.32 0.16 7.29 23.14 94.3 60 ug. Sesamex + 30 ug. DDT 3 20.95 0.01 2.62 0.39 23.97 97.6 6 18.73 0.01 3.92 0.01 0.28 22.95 93.5 12 17 .16 0.06 4.42 0.01 0.36 22.01 89.6 24 15.97 0.26 4.24 0.01 1.92 22.40 91.2 48 13.36 0.61 3.42 0.10 6.57 24.06 98.0 72 12.54 0.62 2.78 0.15 7.38 23.47 9c; c. 90 ug. Sesamex + 30 ug. DDT 21.27 0.01 3.41 0.34 2.).U.,j .LU~.U 6 19.30 0.01 3.25 0.004 0.40 22.96 93.5 12 16.39 0.04 5.32 0.003 0.67 22.42 91.3 24 15.27 0.23 4. 70 0.01 1.94 22.15 90.2 48 12.26 0.35 3.56 0.07 7.67 23.91 97.4 72 12.05 0.55 3.28 0.10 8.23 24.21 98.6 - 160 .. Table V. DDT and dicofol recovered from resistant Landsthul-strain Gennan cockroaches after treatment with 30 ug. of DDT per insect. Landsthul-Strain Males Recovery of Hour after External Internal Excreta aEElied dose treatment DDT dicofol DDT dicof ol DDT ug. lo 3 21.33 0.06 3.45 0.02 0 .. 09 24.95 101.6 6 20.46 0.33 3.44 0.06 0.48 24. 77 100.9 12 19.62 0.50 2.28 0.06 1.40 23.86 97.2 24 19.83 0.57 2.25 0.07 1.82 24.54 100.0 48 12.05 1.13 3.99 0.,27 6.04 23.48 95.6 72 11. 76 1.27 2.25 0.38 7.47 23.13 94.2 Landsthul-Strain Females 3 18.49 0.04 4.84 0.37 23.74 96.7 6 18.79 0.17 3.85 0.004 0.41 23.22 94.6 12 18.19 0.31 3.16 0.04 0.84 22.54 91.8 24 16.84 0.36 3.49 0.11 1.45 22.25 90.6 48 1-5.36 1.29 3.13 0.25 2.31 22.34 91.0 72 14.43 1.35 2.54 0.52 3.32 22.16 90.3 - 161 - Table VI. DDT and dicofol recovered from resistant Landsthul-strain Gennan cockroaches after treatment with sesamex and 30 ug. of DDT per insect. 30 ug. Sesamex + 30 ug. DDT Recovery of Hour after External Internal Excreta a12Elied dose treatment DDT dicofol DDT dicof ol DDT ug. lo 3 19.5 0.01 2.54 0.58 22.63 92.2 6 17.90 Oo09 4.87 0.01 0.68 23.55 95.9 12 18.57 0.05 2.94 0.01 0.61 22.18 90.3 24 16.68 0.40 4.00 0.04 2.16 23.28 94.8 48 10 .. 58 0.74 2.45 0.16 8.23 22.16 90.3 72 10.40 0.88 1. 74 0.21 8.19 21.42 87.3 60 ug. Sesamex + 30 us. DDT 3 19.24 0.01 3.07 0.28 22.60 92.1 6 19.09 0.03 3.12 0.38 22.62 92.l 12 18.96 0.02 2.73 0.003 0.41 22.12 90.1 24 15.78 0.27 5.12 0.03 1.68 22.88 93.2 48 11.16 0.66 3.49 0.16 7.24 22. 71 92.5 72 10.56 0.67 2.57 0.21 8.14 22.15 90.2 90 ug. Sesamex + 30 ug. DDT 3 19.38 0.01 2.91 0.47 22. 77 92.7 6 18.90 0.01 3.50 0.41 22.82 93.0 12 18.79 0.02 4.33 0.46 23.60 96.1 24 15.89 0.23 4.58 0.02 1.59 22.31 90.9 48 12.46 0.56 3.39 0.06 7.85 24.32 99.1 72 10.54 . 0.65 2.31 0.20 8.55 22.25 90.6 - 162 - Table VII. DDT and dicofol recovered from resistant German cockroaches after treatment with 150 ug. of DDT per insect. VPI-DDT-Strain Males Recovery of Hour after External Internal Excreta aEE lied dose treatment DDT dicofol DDT dicof ol DDT ug. % 3 97.56 0.08 12.13 0.01 7.20 116.98 95.3 6 96.37 0.19 14.27 0.04 3.43 114.30 93.1 12 86.11 0.41 17.97 0.05 10.02 114.56 93.3 24 70.80 0.91 32.68 0.07 15.10 119.56 97.4 48 61.81 1.31 28.01 0.21 27.03 118.37 96.4 72 54.06 1.08 18.78 0.25 43.43 117.60 95.8 Landsthul-Strain Males 3 98.74 o.os 11.49 0.01 5.54 115.83 94.3 6 95.44 0.15 13.40 0.03 6.11 115.13 93.8 12 88.97 0.47 16. 71 0.04 9.55 115.74 94.3 24 71.81 1.21 25.85 0.06 13.27 112.20 91.4 48 59.17 1.50 27.63 0.08 26.32 114. 70 93.4 72 41·.32 2.17 21.55 0.37 58.26 123.67 100.7 lN .Y1YQ STUDIES OF SUSPECTED MECHANISMS OF DDT•RESISTANCE IN BLATTELLA GERMANICA (L.) by George Lawrence Rolofson Abstract The rates of penetration, metabolism, and excretion of DDT have been studied !.!!. ~ in resistant and susceptible strains of the Gennan cockroach, Blattella germanica (L.). The cockroaches were exposed to various DDT treatments for intervals ranging from 3 to 72 hours. DDT and its metabolites were identified and quantitated in external, internal, and excreta fractions using gas-liquid chroma- tography. The identifications were verified by thin-layer chromatography. It has been shown that DDT penetration is nearly identical for the three cockroach strains used in this study (two resistant and one susceptible strain). For this reason penetration is not thought to be related to the DDT resistance mechanism in this insect. Susceptible and resistant cockroaches are both capable of converting DDT to dicofol which was the only metabolite observed. The conversion rate is somewhat faster in the resistant strains than in the susceptible strain but in no case exceeds 20% of the absorbed dose. Dicofol production reaches a maximum which is not exceeded in the presence of additional DDT. Female cockroaches of all three strains convert DDT to dicofol at a faster rate than their respective males. The inhibition of dicofol production by the synergist sesamex occurs at a high level in all strains, but ts in mortali in the strain Th , metabolism does not seem to be a or resistance mechanism. Excretion of unchanged DDT is apparently not re to the observed resistance levels. The excretion mechanism is, however, le of removing much of the penetrated DDT provided the insect can survive the treatment. Excretion of DDT was consistently higher male than in The combined effects of the detoxication and excretion mechanisms are extremely efficient in removing penetrated DDT from this insect, particularly at higher treatment levels. Never-the-less, resistant cockroaches have and can apparently withstand much higher internal concentrations of DDT than susceptible cockroaches. The Du1-rei:;.1.:>1.ance mechanism of these cockroaches appears to be similar in nature to the unknown mechanism responsible for resistance to knockdown (Kdr) or paralysis by DDT in houseflies. This mechanism may alter the site of action of DDT and result in a reduced binding capacity of resistant insect nerve tissue for the DDT molecule as compared to susceptible insect nerve tissue.