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THE BIOLOGICAL ACTIVITY OF AMmmmggz‘oNf . ..
MOSQUITO LARVAE'AND; PUPAE AND OTHER -' f w  .  . : , -_
’ ARTHROPODS ' ~ . -   ,

Thesis for the'Degree of M. 3.3 7

MICHIGAN-STATE UNIVERSITY: A A -

- EDWARD DEAN CLARKE; JR, . '
"' 1976' j. ‘ ‘ *

 

ABSTRACT

THE BIOLOGICAL ACTIVITY OF AMINIMIDES ON
MOSQUITO LARVAE AND PUPAE AND
OTHER ARTHROPODS

BY
Edward Dean Clarke, Jr.

Aminimides or ammonio-amidates having the func-
tional group, Rl-g-N--+$-R2, have certain wetting and
emulsifying properties which render them as good anti-
bacterial agents. Because of their structural similarity
to aliphatic fatty acids, amines, and amides which have
antibacterial and insecticidal activity, their insecti-
cidal activity has been investigated in this thesis.

Of all the aminimides tested, all were found to

be ineffective against all terrestrial arthropods, and

the pupae of Aedes aegypti when topically applied.

 

Aminimides were most active against larvae and pupae of

Culex pipiens and Aedes aegypti when tested in water,

 

and activity was greatest when either acyl or alkyl sub-
stituent was C14 or C16’ Of the effective aminimides,
the ranking of their biological activity was strongly
correlated to the degree which they reduced the surface
tension of water. Since the aminimides as a class of

compounds have low mammalian toxicity and are effective

Edward Dean Clarke, Jr.

against mosquito larvae and pupae, it is considered that
they are as worthy of attention as the amines which have
been under investigation as a new group of mosquito larvi-

cides.

THE BIOLOGICAL ACTIVITY OF AMINIMIDES ON
MOSQUITO LARVAE AND PUPAE AND

OTHER ARTHROPODS

BY

Edward Dean Clarke, Jr.

A THESIS

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

MASTER OF SCIENCE

Department of Entomology

1976

To Mary Lou Clarke

ii

ACKNOWLE DGMENTS

I would like to thank Dr. A. W. A. Brown for his
patience, technical and financial aid without which I
could never have finished my M.S. program.

Dr. J. J. Kabara was also helpful with many tech-
nical aspects, financial aid, and had a great deal of
foresight.

My special gratitude is to Dr. T. M. Brown whose
friendship and technical aid I greatly value and would

like to acknowledge.

iii

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . 4
METHODS AND MATERIALS . . . . . . . . . . . 15
Test Organisms . . . . . . . . . . . . 15
Experimental Chemicals. . . . . . . . . . 17
Test Techniques . . . . . . . . . . . . 18
RESULTS . . . . . . . . . . . . . . . 26
DISCUSSION. . . . . . . . . . . . . . . 49
SUMMARY AND CONCLUSIONS . . . . . . . . . . 55
APPENDICES
APPENDIX
A. EXPERIMENTAL CHEMICALS AND THEIR PHYSICAL
PROPERTIES. . . . . . . . . . . . 57
LITERATURE CITED . . . . . . . . . . . . 62

iv

LIST OF TABLES
Table Page

I. Toxicity of topically applied aminimides to
Onc0peltus fasciatus nymphs . . . . . . . 27

 

II. Biological activity of aminimides against
Tribolium confusum larvae in treated flour . . 27

 

III. Biological activity of aminimides on Tetran -
chus urticae using the slide dip method . . . 28

 

IV. Concentration—mortality of biologically active
aminimides tested on larvae and pupae of
Culex pipiens and Aedes aegypti mosquitos . . 29

 

 

V. Concentration-mortality test data of biologi-
cally inactive aminimides on larvae and
pupae of Culex pipiens and Aedes aegypti
mosquitos . . . . . . . . . . . . . 32

 

 

VI. Summary of the biological activity of acyl,
alkyl, and ethoxylated aminimides on larvae
and pupae of Aedes aegypti and Culex pipiens
(21°C) . . . . . . . .. . . . . . . 36

 

 

VII. Effects of temperature and pH on the activities
of aminimides against larvae or pupae of
Aedes aegypti. . . . . . . . . . . . 43

 

VIII. Effects of aminimides used with carbaryl on
4th instar Culex pipiens larvae. . . . . . 43

 

IX. Effects of standard synergists used with amini-
mides on 4th instar larvae of Culex pipiens
and pupae of Aedes aegypti . . . . . . 44

 

X. Percent hatch of Aedes aegypti eggs laid on
aluminum panels after spray applications of
aminimides in 1% ethanolamine in distilled
water . . . . . . . . . . . . . . 44

 

XI. Biological activity of aminimides against adult
Aedes aegypti using impregnated filter-paper
technique . . . . . . . . . . . . . 45

 

Table Page

XII. Results of tOpically applied aminimides to
' Aedes aegypti pupae at a rate of 100
ug/pupae at 90% relative humidity . . . . 47

 

XIII. Adult after effect tests of M-3 on late 4th
instar larvae of Aedes aegypti . . . . . 47

 

XIV. Biological activity of aminimides tested
every third day against 4th instar Aedes
aegypti larvae . . . . . . . . . . 48

XV. Effects of aminimides on the surface tension
and their relation to larval and pupal
mortality in Aedes aegypti . . . . . . 48

 

XVI. The minimal inhibitory concentration (HQ/ml)
for long chain acyl aminimides tested on
microorganisms . . . . . . . . . . 51

vi

INTRODUCTION

Since the widespread use of second-generation
insecticides, principally the organochlorines, organ-
ophosphates and carbamates, widespread resistance has
been developed by a large number of insect pests (Brown,
1971). Many of these insect species have acquired resis-
tance to the control regime in a matter of 3 years. This
makes it likely that the usefulness of any new analogs of
the already developed insecticides will be of limited
value.

In order to maintain control of various insect
pests, insecticides having new and different modes of
toxic action and spectra of activity are urgently required.
Aminimides or ammonio-amidates represent a relatively new
1 - g - N -+é - R2, of biologically
active compounds. Because of their wetting properties and

functional group, R

emulsifying characteristics this group has been investi-
gated and found to have high antimicrobial activity by
Kabara et al. (1975a). Similar compounds (fatty acids,
amides and amines) have high activity as bactericides,
and are promising mosquito ovicides, larvicides and
pupacides. The potentialities of aminimides as mosquito
larvicides and pupacides is reported in this paper.

1

For a number of years fatty acids have been known
to have antimicrobial activity. Their structural relation-
ship to antimicrobial activity has been investigated by
Kabara et al. (1972a). On insects McFarlane (1968) found
that they caused deformations and inhibited growth when
applied to crickets in the nymphal stage. Curtis et a1.
(1970) assayed them against brine shrimp and found that
their toxicity varied with chain length. Saxena and
Thorsteinson (1971) tested fatty acids against larvae of
the yellow fever mosquito and found them to be toxic and
to affect moulting and metamorphosis.

Amides and amines were found by Kabara et al.
(1972b) to have antimicrobial activity. Mulla (1967)
discovered amides to be effective pupicides against several
species of mosquitos. Alexander and Beroza (1963) tested
aliphatic amides of cyclic amines and found several of
them to be good mosquito repellents.

Amines were evaluated against bacteria by Fuller
(1942), who found their activity varied with chain length
and structural groups. Kindler (1934) reported the role
of amines as chemotherapeutic agents.

Primary, secondary and tertiary amines were
assessed against barnacle larvae by Christie and Crisp
(1966) who found several of them to have good activity.

Alkyl secondary amines having C12 or longer were toxic

to houseflies, and the activity could be correlated to
chain length (Dahm and Kearns, 1941).
Many amines were evaluated against larvae of the

malaria mosquito, Anopheles quadrimaculatus, (U.S. Depart-

 

ment of Agriculture, 1947; King, 1954) and found to be
active. Mulla (1967) and Mulla et al. (1970) tested
various aliphatic primary amines, secondary amines, ter-
tiary amines, beta amines, diamines, and beta diamines
against larvae and pupae of several mosquito species;
good activity was found and chain length could be cor-
related with activity. Cline et a1. (1969) and Mulla and
Chaudhury (1968) tested aliphatic amines against mosquito
eggs and found them to be ovicidal. Mulla et a1. (1971,
1975) considered that certain amines were so promising in
mosquito control that field evaluations of them were made
against immature mosquitos, and certain formulations and
application techniques led to complete mosquito control.
Because of aminimides antimicrobial activity and
structural similarities to various fatty acids, their
potential as insecticides was therefore investigated and

is described in this thesis.

LITERATURE REVIEW

Fatty acids are materials that are inexpensive to
manufacture and have been found to have considerable bio-
logical activity. Their general formula is R—COOH. Maxi-
mum activity against gram-positive bacteria is reached
with lauric acid (C12) (Kabara et al., 1972a). The dienoic
derivatives of octadecanoic acid (C 18:2) were more active
than the monoenoic acid (C 18:1) which was more active than
the saturated acid (C18). In general esterification of the
polar end group (carboxyl) led to a compound that was less
active. Kabara et a1. (1973) found that the unsaturated
octadecanoates had maximum activity when the double bonds
were at the *2 and *8 positions, while for lauric acid
the greater effect was when the double bond was at the
*10 position.

Saturated fatty acids reach their maximum effects
against insects when their chain lengths are between C10
and C13. Saxena and Thorsteinson (1971) tested synthetic
“queen substance" and analogues on 4th-instar AgdggDaeggpti
larvae and found that the compound (9-oxo-2-decanoic acid)
had four effects, namely (1) caused rapid mortality in the

treated stage caused by "acute toxicity," (2) delayed

the moulting of larvae and (3) mechanically inhibited
metamorphosis as a result of nondetachment of the exuvia
during the larvae-pupal or pupal-adult molt and (4) bio-
logically inhibited the imaginal differentiation and thus
caused death. Increasing the chain length above C13
decreased the toxicity of saturated fatty acids, but
increased the toxicity of unsaturated fatty acids, with
linoleic and linolenic acids being the most toxic. The
presence of an oxo-group in a saturated or unsaturated C8
to C10 fatty acid remote from the carboxyl group reduced
the acute toxicity and increased the inhibition of metamor-
phosis.

McFarlane and Henneberry (1965) showed that fatty
acids when applied to the cuticle of the nymphal cricket

Gryllodes sigillatus (Walk) inhibited growth and reduced

 

wing development. Myristic and lauric acid methyl esters
of fatty acids were tested, and the effective ones were
methyl myristate and methyl stearate. When the fatty
acids were applied by addition to the diet, only lauric
acid inhibited growth and then when only applied at high
doses. McFarlane (1968) further studied the effects of
methyl palmitate and lauric acid when applied to nymphal

Gryllodes at different time-intervals. He concluded that

 

these compounds on insects probably affected either the

neuroendocrine system itself or else one of its sites of

action. He found that the effects were greatest on the
younger nymphs.

Levinson and Levinson (1973) tested the lipid
depressant ethyl-p-chlorophenoxyisobutyrate (ECPIB) on
Dermestes maculatus. When added to the diet ECPIB sup-
pressed fecundity and retarded growth. This activity

could eradicate the population of Dermestes maculatus so

 

treated within 2 or 3 generations. The activity of ECPIB
includes phagostimulation and increased fat metabolism
which can be neutralized by ingestion of oleate. When
oleic and linoleic acid were topically applied to the
larvae and pupae of the western spruce budworm (Choristo-
neura occidentalis) these agents abolished the production
of glucose-6-phosphatase or N-acetylglucosamine in pupae,
but had only small effects on their production in larvae.
This could have possibly been caused by increased lysosome
activity (Andrews and Miskus, 1972).

When fatty acids were assayed by Curtis et a1.
(1970) against brine shrimp larvae, it was found that the
most toxic compounds had a chain length of C10 to C13.
Unsaturated fatty acids were also tested and oleic,
linoleic, and linolenic were found to be the most toxic.

Ikeshoji and Mulla (1974) found that several
Z-alkyl fatty acids which occurred as a result of over-
crowding mosquito larvae (Ikeshoji and Mulla, 1970,

1974a,b) had mosquito larvicidal properties. Of the

compounds tested, the most active fatty acids were 2-ethy1-
octadecanoic acid, 3-methyloctadecanoic, and 2, 3-dimethyl-
octadecanoic acid. These chemicals never killed the larvae
in the same stadium as that when they were applied, but
death superceded immediately after the next ecdyis. The
2nd instar larvae, after treatment, were unmelanized in
their cuticles and head capsules. When treated before
pupation, the larvae drowned while pupating and their
cuticle was unmelanized. Final emergence of adults was
inhibited when early instar larvae were treated. These
compounds were also found to reduce larval heart-beat rates
and were also bacteriostatic (Ikeshoji and Mulla, 1970).
These substituted fatty acids possibly affect mosquito
larvae by penetrating deep into the epidermal cells where
the esterases for wax synthesis are located. Abrahamson
et a1. (1964) stated that the rates of esterification of
alkyl branched fatty acids, and the rate of hydrolysis

of their esters, are hindered when they are branched at

the *2 and *3 positions. Because of the steric hindrance
in enzymatic esterification, the mosquito larvae are
endangered by not being able to synthesize a sufficient
quantity of wax and triglycerides to meet the requirements
in the formation of a new cuticle. The esterified lipids
resulting from branched fatty acids do not provide a good
barrier against water penetration (Ikeshoji and Mulla,

1974b).

Amines (R—NHZ) and amides (R-CONHZ) having chain
lengths between C9 and C14 are the most active anti-
microbial agents (Kabara et al., 1972b; Fuller, 1942).
Amides are active against only gram-positive bacteria
while amines were active against gram—negative and gram-
positive bacteria. It was also found by Kabara et al.
(1972b) that mono-unsaturation failed to increase activity
as it does in fatty acids. Alexander and Beroza (1963)
reported that aliphatic amides of cyclic amines and tolyl

maleimides worked as repellents against Aedes aegypti

 

mosquitos.

Jensen and Liu (1963) tested primary, secondary,
and tertiary amines on influenza virus in tissue culture,
and found that they were inhibitory to the virus much the
same way as ammonia was. Silver and Kralovic (1969) tested
short chained triamines on bacteria, and found that they
"paralyzed" the permeability of the cell wall. It was
found that treated bacterial cells leaked potassium and
thiomethyl galactoside through their cell membrane. Many
years ago Dale (1920) pointed out that the effects of cer-
tain amines include certain physiological symptoms charac-
teristic of nicotine poisoning. The polyamines tested by
Tabor and Tabor (1964) neutralized, stabilized or labilized
the lipids and the membrane structure of ganglionic cells,
which is also a property of nicotine. Dahm and Kearns

(1941) tested alkyl secondary amines against adult

houseflies (Musca domestica) and found that maximum activity

 

11 t° C17'

Several amide derivatives of compounds related to

was achieved at chain lengths between C

farnesenic acid were tested by Cruikshank and Palmere (1971)
and were found to have various degrees of juvenile hormone

activity on Tenebrio molitor. Several amines structurally

 

related to juvenile hormone mimics were tested by Robbins
et a1. (1975) on a number of insect species. These com-
pounds exhibited juvenilizing affects on the tobacco horn-
worm Manduca sexta (L.), including formation of precocious
"fourth-instar prepupae" and abnormal pupae which appeared
to be prepupae-pupa intermediates. They also blocked
development in the early larvae stages, and.were lethal
when applied at the time of molting. Similar juvenilizing
and lethal effects were exhibited on larvae and pupae of

Aedes aegypti and Tribolium confusum. These compounds

 

were found to have reduced the cholesterol level of 80 -
85% to 5% of the total tissue sterols, and increased the
demosterol content from 1.0 - 0.5% to 50% (representing
total sterols of this insect). The toxicity and juvenile
effects were stated as being caused by inhibiting metamor-
phosis and the *24-sterol reductase system in larvae which
are involved in the conversion of plant sterols to choles-
terol.

Christie and Crisp (1966) tested aliphatic amines

on larvae of the barnacle Elminius modestus and found that

 

10

primary amines of C8 to C14 chain length were the most
active, while secondary and tertiary amines were active up

to a chain length of C They concluded that amines act

16'
upon metazoans in a different way or at a different site
of action than the majority of organic substances. From
their work on barnacles, they pointed out that the amines
they tested exhibit four characteristics of compounds that
are physically toxic, these being (1) reversible narcosis,
(2) similar toxic effect with wide variations in time of
exposure, (3) little dependence on temperature, and
(4) similar toxicity measured on the thermodynamic scale.
Aliphatic amines were tested by Mulla (1967)

against larvae and pupae of the southern house mosquito

Culex pipiens quinquefasciatus (Say) and he found that

 

lauryl, palmityl, t-stearyl-t-behenyl, oleyl, tallow,

tall oil, and coco oil primary aliphatic amines were more
toxic to larvae than pupae. Monoalkyl amines were more
active than dialkyl amines and secondary amines were
ineffective against larvae or pupae. The tertiary amines
had little activity against larvae or pupae, while the
alpha diamines showed the greatest activity of any group
evaluated. Primary beta-amines were more effective against
larvae than pupae, while the amides tested were more toxic
to pupae than larvae. The tertiary beta-diamines were

more active against larvae, and the beta-diamines were most

active against larvae and pupae when their chain was greater

11

than C11. When put in amine test solutions, the larvae
immediately became hyperactive and began to curl up and
touch their mouthparts to the anal gills and siphon. The
larvae wriggle to the surface but soon after reaching the
surface they repeated their past behavior. They also can-
not rest at the surface as the untreated organisms can.

Peak mortality was reached more rapidly than in the case

of conventional insecticides. Pupae were also rapidly
affected when exposed to lethal concentrations. The pupae
would often snap their abdomens without darting from one
place to another. Normal pupae dart after snapping their
abdomens once, but those treated with amines had to snap
several times before they moved. The treated pupae also
became sluggish and did not respond to moving shadows, and
shortly thereafter became paralyzed and remained on the air-
water interface. Adults emerging from treated solutions

had abnormal wings, lost their body- and wing-scales, and
many were unable to take flight. The duration of the larvae
and pupae stages was also lengthened by some of these
aliphatic amines.

The biodegradable cationic surface-active agents
produce symptoms on mosquito larvae and pupae unlike those
observed in poisoning induced by organochlorine, organo-
phosphorus, or carbamate insecticides. Their course of
gross symptomatology is similar to that produced by

treatment with petroleum oils, and when tested against

12

organophophate-resistant mosquito larvae (Mulla et al.,
1970) there was no cross-resistance. Mulla (1967) stated
that lowering of the surface tension of water did not seem
to be a primary factor in their biocidal activity since
some good surface-active agents tested did not kill larvae
and pupae at much higher rates than those at which some
of the aliphatic amines were effective.

Several theories as to the mode of action of amines
on mosquito larvae and pupae were advanced by Mulla (1967),
namely: (1) because of the substantive properties of
aliphatic amines they probably change physical, electrical,
and hydrophobic-hydrophilic characteristics of the cuticle;
(2) the lipophilic portion of the aliphatic amines dissolves
or disrupts the epidermal layer of larvae and pupae result-
ing in nutrient, chemical, and water imbalance accounting
for the quick-acting power of the amines; (3) the aliphatic
amines may interfere with or disrupt the membrane of the
anal gills, thus resulting in salt and water imbalance, or
they may disrupt or change the functional integrity of
tracheae; (4) there may be interference in hormonal balance
or amino-acid metabolism as evidenced by the abnormal
eclosion of adults, appearance of abnormal structures,
and shedding of scales in the emerging adult. Mulla also
stated that from gross observations of symptoms, damage
and death due to physical action seemed plausible; however,

the type of physical action involved seemed to be complex

l3

and may be followed by metabolic and chemical changes
resulting in death, delayed development, or morphogenetic
changes in the immature stages of the mosquitos.

Soaps and emulsifying agents can affect aquatic
insects in several ways. The lipophilic side chain can
attach itself to the waxy epicuticle which is responsible
for prevention of water migration in and out of the insect
(Wigglesworth, 1965) and can cause slight cuticular dis-
ruptions, while the hydrophilic portion can provide for
migration of water into the insect (Wigglesworth, 1945;
Beament, 1945). With the waxy layer thus affected, the
insect is rendered more permeable to both water and
insecticides.

Soaps and emulsifying agents are lethal to mosquito
larvae and pupae by lowering the surface tension of the air-
water interface. This causes the pupae or larvae to lose
attachment of their respiratory trumpets to the surface film
(Christophers, 1960). Early works by Russell and Rae (1941)
give the range of 27 to 36 dynes/cm as being fatal to mos-
quito larvae and pupae, without the time-period it took to
cause this mortality. Senior-White (1943) gave his test
findings on mortality in relation to concentrations of
various soaps versus time constants, but did not quantify
the lowering of the surface tension in dynes/cm. Manzelli
(1941) notes that some of the effects of reduced surface

tension on mosquito life—stages are that emerging adults

l4

and mosquito egg rafts sink. Whereas mosquito larvae are
sclerotized only in the head capsule and anal siphon, the
pupae have a cuticle that is entirely heavily sclerotized
(Clements, 1963). Mosquito larvae, especially early instars
resist submersion by being able to respire dissolved oxygen
through their cuticle and anal papillae, but submerged
pupae burn up the oxygen reserve in their tissue, become
heavier than water and drown (Christophers, 1960). Because
of the difference between larval and pupal cuticles, pupae
are much more susceptible to lowering of the surface tension
of the air-water interface.

Aminimides or ammonio-amidates are a relatively new
group of fatty-acid derivatives. They are surface-active
agents and exist as bipolar ions.l Aminimides derived from
C12 to C18 possess interesting wetting prOperties and
emulsification characteristics (McKillip et al., 1973).
These compounds were tested by Kabara et al. (1975a) and
Kabara and Haitsma (1975b) and found to be very active
against gram-positive bacteria and yeast organisms. Their
activity against gram-negative bacteria is low or absent.

-+'

9
The authors found that the aminimides, R -C-N - N-R , were
I

l 2
most active when the acyl (R1) or alkyl (R2) sides had a
chain length of C14 or C16' Among the aminimides they
tested, the unsaturated compounds tended to be more active

than the saturated ones.

METHODS AN D MATERIALS

Test Organisms

 

The insects used in evaluating the biological
activity of aminimides were: the large milkweed bug

Oncopeltus fasciatus (Dallas), the confused flour beetle

 

Tribolium confusum, the yellow fever mosquito Aedes aegypti,

 

 

the common house or bird mosquito Culex pipiens pipiens,

 

and the common orchard or two-spotted mite Tetranychus

 

urticae.

The milkweed bug colony employed was a compound
strain: individuals from Michigan, North Carolina, and
California were interbred and selected for eating sun-
flower seeds. The strain was raised on sunflower seeds
and distilled water, and kept in gallon jars at 30°C and
60-70% relative humidity. Only 5th-instar nymphs were
used for susceptibility tests.

The confused flour beetle colony was a mixed strain
obtained from Dr. Robert Mills at Kansas State University
consisting of stock from Sacramento (B-25), San Bernadino
(B-166) and Davis (B-28) California. They were raised on
a diet of ball-milled yeast and whole wheat flour. Ovi-
position was obtained by adding adult beetles to whole

wheat flour in 1 gallon jars, and after 1 week the adults

15

16

were sifted out. When the larvae were 2 weeks old they
were used for testing the activity of the compounds. They
were held at 30°C and 60-70% relative humidity while being
tested.

The orchard mites were obtained from a greenhouse
culture of mixed origin maintained by Dr. B. A. Croft at
Michigan State University. Mites were raised on young pea
plants at 30°C and 80% relative humidity. Only 1- or 2-day
old females of uniform age were used in tests.

The yellow fever mosquito was the Rock strain
obtained from the Department of Biology, University of
Notre Dame. The larvae were reared in enamel-coated pans
at 30°C. Adults were raised in collapsible aluminum cages
held at 30°C and 70-80% relative humidity. Larvae were
fed on guineapig barley pellets and adults were maintained
on raisins and 10% sugar solution. Anesthetized guinea-
pigs were the source of blood meals for the Aedes aegypti
mosquitos, being exposed in dimmed light. For testing,
only 4th-instar larvae and pupae less than one day old were
used. The mosquito eggs used were stored in mason jars
containing 20 ml of distilled water to maintain a high
relative humidity and were used for tests after they had
been stored for 3-18 days.

The Culex pipiens colony originated from sewage

 

ponds at Belding, Michigan in 1973. They were raised in

a fashion similar to that employed on Aedes aegypti larvae

 

17

and adults, the only differences being that the larvae

were fed on dry barley pellets and ground Purina Cat ChowR

and the adults were given blood meals from chickens. Only

4th-instar larvae and less than one day-old pupae were used

for testing.

Experimental Chemicals

 

The aminimide compounds tested here were synthesized
by The Ashland Chemical Company, Dublin, Ohio. The func-
- +
tional group is Rl-g-N - N-Rz. Exemplifying one of the six

active acyl aminimides, the structure of M-3 is,

9 ' +933 QH

C-N - N - CH -CH-CH . M-57 is one of the three

14 CE 2 3

active long chain alkyl aminimides tested, and its structure
9H3 9 $H3 9H

is, H2C=C - C-N - N -CH2-CH-(CH2)13-CH3. One of the seven

CH
ethoxylated acyl amigimides which had a fair degree of

CH3(CH2)

activity is M—116, its structural formula is,

9 9H3 9“

) -é—N - N - CH -CH-CH +30. Ethoxylated amini-

2 12 CH3 2 3

mides were exposed to ethylene or propylene oxide at the

- 9H3

molar ratios given in Appendix A. M-6, CH3-¢-SOZN -+2 - CH3
H3

was the only sulfonated aminimide tested, while M-ll,

CH3-(CH

0 .
CF3-CF2-C- N-N+(CH3)3, was the only fluorinated aminimide
tested. Several quaternary aminimides were also tested,

of the ones tested M-31 had a structural formula of,

H 9 CH OH
+§ 3 I - a +3 .
fl-CH - -CH -C-N N -CH -CH-(CH ) -CH . These
2C1- CH 2 CH 2 2 13 3
3 3

compounds exist as bipolar ions and their chemistry has

been investigated by Timpe (1972) and McKillip et a1.

18

(1973). They possess wetting properties and emulsifying
characteristics, and behave like non-ionic surfactants
with relation to cloud point (Kameyama et al., 1968,
1969). They exhibit a Kraft Point phenomenon similar to
that exhibited by ionic surfactants (Corkill, 1970).

The aminimides used were recrystallized 5 or 6
times and were chromatographically pure. Several of their
solubility constants are listed with their structural
formulae in Appendix A. Kabara and Haitsma (1975b) found
that on the laboratory CF-l mice, aminimides were toxic
at 200-400 mg/kg when interperitonially injected, and

2,000-4,000 mg/kg when given orally.

Test Techniques

 

Fifth instar nymphs of the large milkweed bug were
anesthetized with CO2 and the test compounds were topically
applied to their abdomens as a 10% solution in acetone.

The application of l microliter containing 100 micrograms
of the compound was made with an ISCO applicator and a
Hamilton 250-microliter syringe fitted with a 27-gauge
needle. The solvent was allowed to evaporate and then the
bugs were placed in pint sealers with water and sunflower
seeds. Mortality counts were taken 24 hours later. Con-
trols consisted of bugs treated with l microgram of acetone.

Two-week-old larvae of the confused flour beetle

were used in the effectiveness tests for this species.

19

The candidate compounds were stirred into 20 grams of
whole wheat flour and 40 m1 of methylene chloride was
added. This was stirred and mixed for 2 minutes in a
round-bottom flask. The solvent was removed on a RotavaporR
(Buchi) distillation apparatus. The treated flour was then
placed on a sheet of aluminum foil and allowed to dry over-
night. Controls consisted of methylene chloride alone and
malathion. The flour was divided into two vials and
inoculated with 25 two-week-old larvae (into each vial)
and held at 30°C for 3 weeks when the percentage mortality
was estimated from the number of adults that failed to
emerge. I

Tests on the two—spotted mite were performed accord-
ing to the ESA slide-dip method for susceptibility levels
(Anonymous, 1968). Two pieces of double-sided sticky tape
were attached to a glass microscope slide. A piece of
filament tape was placed, sticky side up, on top of the
double-sided sticky tape. Twenty young female mites of
uniform age were placed on their backs using a fine camel-
hair brush. The slides were dipped and stirred in the
acaricide solution for 5 seconds, taken out and allowed
to drain for 15 minutes by placing them on their edge.
Control tests were made with a known acaricide (Fundal)
and also with the solvent (acetone) alone. The treated

mites were stored for 24 hours at 30°C and 80-90% relative

20

humidity. Mites were judged dead if on being probed by
a camel-hair brush they failed to move their legs.
Effectiveness tests of the aminimides were made on

Culex pipiens and Aedes aegypti mosquito larvae and pupae

 

 

according to the WHO Standard Method for susceptibility
testing (World Health Organization, 1970). Only early-
4th-instar larvae and less than 1-day-old pupae were used
in susceptibility testing. Lots of 25 larvae or pupae

were placed in 50-ml beakers containing 25 ml of distilled
water. Into 600-m1 beakers were placed 225 ml of distilled
water. At least 3 different concentrations each in dupli-
cate were employed in each test. The solutions of the com-
pounds were prepared to have a concentration of 100 ppm and
were then serially diluted down. One ml of the compound

in ethanol solution was pipetted into the 225-ml water in
600-ml beakers and stirred with a glass rod. The most
dilute solutions were pipetted first and the most concen-
trated last. Controls were run with malathion (a known
insecticide) and with the solvent (ethanol or acetone).
After the compounds were pipetted they were allowed to
equilibrate for 15 minutes. The mosquito larvae or pupae
were added by tipping the contents of the small beakers
into the large beakers. The temperature of the water
during the tests was 21-23°C. After a 24-hour period,
mortality counts were taken. Larvae and pupae that were

scored as dead were those that failed to move if probed,

21

were discolored or were unable to rise to the surface or
showed unnatural positions, tremors, uncoordination, rigor
or failure to respond to shadows. The concentrations
sought and chosen were those that gave partial mortality,
and where possible three of them were used in multiples of
two. If more than 10% of the larvae in the controls died
or pupated, or more than 10% of the pupae tested subse-
quently emerged, the tests were rerun.

Pupae of Aedes aegypti were treated with aminimides

 

by topical application, the method being similar to that
used on the milkweed bug. The main difference was in the
amount of solvent used. The aminimides were diluted in
water instead of ethanol or acetone and were applied at

a rate of 100 ug/pupae. Three replicates, each of 10
pupae were used, along with 2 control replicates. The
pupae were placed on damp #2 Whatman filter paper and
topically treated by means of an ISCO applicator; 15
minutes later they were placed in 100 m1 of distilled
water and held for 24 hours, when mortality counts were
taken. All replicates were held until emergence to deter-
mine whether adult emergence was affected.

Three aminimide compounds (M-8, M-l8, and M-20)
were tested to determine their longer-term effects on
Aedes aegypti larvae. Testing was similar to the WHO
Standard Method, the difference being that the volume

of water was increased to 500 ml. Four replicates using

22

4th-instar larvae were used on each solution. The test
solutions were reinoculated every third day with fresh
larvae, which were held for 24 hours before being recorded
and removed. The concentration of 10 ppm was used, as
being between the LC50 and LC90 of the candidate compounds.
To determine the possible after-effects on the
adult mosquitos, after the treated larvae had pupated and
emerged, three concentrations of M-3 were employed. These

tests were similar to the WHO method but the Aedes aegypti

 

larvae were held in the treatment solutions for up to five
days. After 24 hours mortality counts were taken and sur-
vivors were held to determine the number of emergent adults.
Ovicidal tests were run according to the technique
used by Cline et a1. (1969) and Mulla and Chaudhury (1968).

Aedes aegypti eggs were collected on #2 gauge roughened

 

aluminum panels (9 x 2.4 cm.) taped into 400 or 600 m1
beakers. Eggs used were between 3 and 18 days old. A 1%
ethanolamine emulsifier was used with 0.4% of aminimide

in distilled water. Controls were of 1% ethanolamine alone
and a 0.4% solution of malathion. Exactly 2.5 mls. of
solution were used per replicate and over 300 eggs per
replicate were employed. Two replicates were used for each
compound tested or control. A Potter Spray Tower was used
to apply the compounds. The solutions were sprayed on the
aluminum panels to the point of runoff (25 mls.) and then

placed in beakers where they were stored for 24 hours at

23

29°C and at 60-70% relative humidity. A hatching medium
of larval water was used to stimulate hatching and after
24 hours in this medium at 28°C hatch counts were recorded.
The panels were removed from the hatching medium and
allowed to dry before being counted. A WILD Heerburg
stereoscope microscope (6-50X) was used to count the
mosquito eggs. Intact eggs and partially emerged larvae
were scored as dead eggs.

Aminimides were tested for their effect on adult

Aedes aegypti by dissolving them in oil and impregnating

 

them into filter paper. Solutions were made using the test
chemicals and a 5:1 mixture of chloroform and olive oil,
applied to Whatman #1 filter papers by means of a needle
and syringe and allowed to dry overnight in a ventilated
hood. The treated filter paper was fitted into a plexi-
glass cylinder which had a screen over one end and a
removable slide at the other. The adult female Aedes to
be used in the test were first aspirated into a holding
cylinder where they were counted, and then lots of 25 were
placed in each cylinder (2 replicates per solution per
concentration) and held for 24 hours at 28°C and 80%
relative humidity before mortality counts were taken.
Malathion solution, and the olive oil-chloroform solvent
alone, were used as controls.

Aminimides were tested as synergists for carbaryl

against 4th-instar Culex pipiens larvae according to the

 

24

WHO method, and the solutions being made up in a ratio of
1:10 (carbaryl to aminimide). Controls consisted of ethanol,
carbaryl, and carbaryl plus piperonyl butoxide.

In tests to see whether the aminimides were syner-
gized by standard synergists (piperonyl butoxide, sesamex,

and triphenylphosphate) against 4th-instar Culex pipiens

 

larvae, a 1:1 ratio (aminimide to synergists) was employed
for the mixture: the controls consisted of ethanol, of
the M-8 aminimide alone, and each of the three synergists
alone at their highest concentration (when used in the 1:1
mixture). The aminimide M-3 was also tested against Aedes
aegypti pupae with the synergists triphenyl phosphate,
TOCP, and piperonyl butoxide at a ratio of 1:1 (M-3 to
synergist).

The aminimide M—3 was tested against Aedes aegypti

 

larvae at 3 different pH's by means of phosphate buffers.
In addition M-3 and M-8 were tested against Aedes aegypti
larvae and pupae at an elevated temperature of 28°C.

To determine whether the reduction in surface
tension caused by the addition of aminimides to water was
a factor in their biological activity, tests were run at
different concentrations of different aminimides (of a
homologous series) to determine a possible correlation
between reduced surface tension and larvae or pupae mor-
tality. The hanging-ring method on a Model #20 Fisher

Tensiometer was employed. The apparent surface tension

25

was read from the tensiometer and the true surface tension
was then determined by using a correction factor chart.

Distilled water was used as a test standard.

RESULTS

Aminimides had little or no toxic effect on

Oncopeltus fasciatus (Table I), Tribolium confusum

 

 

(Table II), or on Tetranychus urticae (Table III).

 

The aminimides that had LC50 values of less than

40 ppm on larvae or pupae of Aedes aegypti or Culex pipiens

 

 

are given in Table IV. Aminimides failing to be active

on mosquito immatures are listed in Table V along with the
concentrations at which they were tested. Table VI re-
classifies the aminimides according to acyl or alkyl families
of different chain lengths and summarizes their activities
accordingly. Three of the most active aminimides tested
(M-3, M-8, and M-20) were sufficiently active to kill both
larvae and pupae at concentrations between 1 and 10 ppm
(Table IV). With the compounds M-3, M-4, M-18, M—20, M-57,
M-71, M-108, M-116, M-122, and M-124 the activity was con-
siderably greater against the pupae than against the larvae.
For both mosquito strains, M-4 had very little effect on
the larvae but was somewhat toxic to the pupae. M-2, M-3,
M-8, M-20, M-57, M-108, M-ll6, M-122, and M-124 were more

effective than malathion on the pupae of Culex pipiens

 

and M-3, M-8, M-18, M-20, M-57, M-116, and M-122 were

26

27

TABLE I. Toxicity of topically applied aminimides to
Oncopeltus fasciatus nymphs.

 

 

 

Compound Dose (ug/nymph) % Mortality

M-4 100 0
M-8 100 0
M-18 100 0
M-20 100 0
M-7l 100 0
N,N dimethyl

dodecylamide 100 45
Malathion 100 100
Control (Acetone) --- 0

 

TABLE II. Biological activity of aminimides against
Tribolium confusum larvae in treated flour.

 

 

 

Compound (1% by Weight) % Mortality
M-4 . 8
M—8 6
M-18 0
M-20 2
M-7l 2
N,N dimethyl dodecylamide 28
Malathion 100

Control (Methylene chloride) 0

 

28

TABLE III. Biological activity of aminimides on Tetranychus

 

urticae using the slide dip method.

 

 

Compounds % Mortality at 1% Concentration*
M-4 56
M-8 44
M-18 62
M-20 16
Fundal+ 100

Control (acetone, '
water 1:1) 0

 

*
Held for 24 hours

+Held for 48 hours

29

 

 

 

 

 

m o m m o.H ml:
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o o o o mm.m an:
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mm mm ooa mm mm mu:
m o m o o.H «I:
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30

 

O O O N O.H OHHIZ
mm O mm mH O.m OHHIz
Om Om OO OO m.NH OHHIS
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N n O m O.m OOHIS
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m O m m m.NH Hun:
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O O N O mN0.0 hmlz
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OOH On OOH Om mm hmlz
m NH O m O.m ONIz
mm mm OOH Oh OH omuz
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m O O O O.H Oan
mm m O O O.m OHIZ
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mmmmmmmmmmemq mNMMMNmmmmwmmq Afimmv cOHumHucmocoo pcsomsou

 

(NMHHmuuoz w

 

AwmscHuaouv .>H mamme

31

 

 

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.NuHHmuuoz m

 

lowscHucooO .>H momma

32

 

 

 

 

 

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O mm mun:

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m mm OHIS

OH om OHIS

Om OOH OHIS

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O mm OI:

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m OOH H12

mommmMmMMMmen ONMMMNWMWMQWMH Heady GOHumupcmosOU vasomfioo

SMHHmunoz w
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Hpmmmmm mmomm pom mconHm meso no madam tam mm>an do mwows

IHGHEM w>HuomcH SHHMOHOOHOHQ mo must pmmu muHHmuHoEIGOHumuucwogoo .> mqmfia

33

 

 

 

O m.~H Owns
O mm men:
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mm m.mH Hmuz
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Om OOH Hmnz
311:“... Jan... .33 E .233:
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AUOHZHHHSOUV .> 3&8

34

 

 

 

OH ON mNan

ON OO ONHIS

NN OOH ONHIE

Om ON mNHIz

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OH ON ON MNHIz

mv ON OO MNHIZ

OO OO OOH MNHIS

m m ON HNHIz

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mN OH OOH HNHIS

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O om mOuz

O ON NOIS

N OO NOIz

N OOH NOIZ

OH O ON vng

«m O OO wmlz

OO O OOH vmlz

mammma Hmmwwmq mummmwmmmmwmmq Hammv coHumuucmocoo vasomfioo
muHHmuuoz w

AcmssHucoov .> mHmNB

35

 

 

OH O mN mmHIz
Om OH om mmle
OO NH OOH mmHIz
O m.NH Nmle
O ON NmHIS
O om NMHIZ
mmmsm mm>HMH mmmsm wm>HMH
.mmmmmmm .m. .MMNNWMM xm Hammv GOHumuucwosoo vasomfioo

 

suHHmuuos O

 

HooscHuaoov .> mHmHB

36

 

£2 £2 dz dz OUHEHHDMH
mcHEMngpmmxouwxnumuHanmsHO OHuz
«2 «z «z «z mOHsHuwmum mcHsm HmnumsHua mmHuz
mm.N m.m m.N 0.0 OOHEHuHEHmm mcHEm HmaumEHHB OI:
O.Hm m.hm O.nH O.HN mOHsHumHnms qusm HanumsHue On:
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Hz «2 «2 <2 mOHEHHSMH msHEm
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woman mm>HMH madam wm>umq

vasomaou

 

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lame 3.3 ES 33

 

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msmHmHm meso can Humammm mmowd mo wwmsm pom mm>HMH co mooHEHcst
wOHMmeonum pom .meHm .Hwom mo wuH>Huom HMOHOOHOHQ may no mumafism .H> mqmma

 

 

37

 

 

 

 

 

 

0.0m m.mm O.Hm O.Nm mpHaHumom
mcHEMkumHnwsaxonthnlNIHmnumEHQ thz

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HmuHsHmmmxouumauNIHHOumaHO ONHuz

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mcHEmomUmxmsmeHUNSINIHmnumEHQ Omlz

ON.O m.m mm.O 0.0 OUHEHHMHUMthHOE
OCHEoomUmuumuhxouomslNIHMSHOEHQ ONuz

«z «z «z m2 OOHEHHSHOOHMBHOE
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mopHEHGHEm meHm GHmco mcoq .m

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mcHsmHsnummxouwmsumuHmaumsHa NOHIE

mm.m Hm O.m OH mOHsHuHsHmm
maHamHNOummxouOHnumunnumsHo OHuz

mmmsm mm>umq mwmsm mm>umq
mcmHmHm mesU Hummmmm moumd GOHumHuommo HMUHEmsu vasomeou
Hammvomoq HEOOOOOOH

mescHucooO .H> mamma

38

 

 

 

 

 

 

O.v oz w.m mm omv+ onEHuHEHom
HmmoummxonomsumnHmsumsHO OmHuz
Hz oz oz Hz onw+ wOHEHuHEHom
HmmoummxouomgumuHsnumsHO mNHuz
O.H Om O.H hm omOH+ mOHsHuHusm
HmmoummxouomnlmlesquHQ NNHIE
«z «z 42 oz omOH+ onsHuHsHmm
HumouosxouomoumuHsauwaHa HNH-s
«z Hz «2 dz omO+ wOHEHuHEHom
HumoummxouomsumuHmnuman ONHuz
0.0 O.HH N.> 0.0H omO+ wOHEHpmHHmE
HumonmsxouomnuNnHmsuman OHHuz
m.NH 0.0H m.NH O.HN omO+ onEHHaoH
HmmoumxxonwmsumnHNaumeHo OOH:z
mthEHaHEo Hhoo owuloxosuw aHono mcoq .0
womam wo>HoH womam wo>HoH
mameHm waaU Hummmwo mwowm aOHumHHomwo HooHEwso oaaomeoo
Hammoomoa Asmmvomoa
AUOaGHuGOUV . H> mam»;

39

auH>Huoo oz u oz
.1

 

 

 

 

 

 

 

mO.H H5. O.O O. Hammaumo
OH Omo. O.N mNH. GOHnuoHoz
Hz oz <2 dz wuowHo EaHpom
oz «2 £2 £2 waHEonowoon
O.OH O.O m.hH O.O moHemHaomooo HmsumsHOnz.z
wwoaoumnam Homuaou .o

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wameHm waao Hummmwo mwowd aoHumHHomwQ Hoowfiwnu caaomfiou

EOE 83 E3 88

Atwaawuaoov .H> wands

40

more effective than carbaryl was on the pupae of Aedes
aegypti. The significance of these activities is that
these compounds on the whole were more toxic to the pupal
stage than to the larval stage.

When treated at acute toxic levels (not less than
the LCSO), the larvae died much more rapidly with the
aminimides than with the malathion or carbaryl controls.
They showed adverse symptoms within the first half hour,
and after three hours 50% were dead. Their diving reactions
to light were much impaired and they had a difficult time
maintaining their contact with the air-water interface.
White fluffy material, probably the peritrOphic membrane
(Abedi and Brown, 1961) was excreted through the anus when
the treatment concentration was at 10 ppm or greater. The
larvae would often curl over as if trying to bite off this
material. One to three hours after death the larvae were
slightly swollen and in a markedly extended and relaxed
state, with some discolorations on the dorsal surface. Of
the aminimide compounds, the pupicidal activity was
greatest if the chain length was Cl6 on either the amine
(alkyl) or amide (acyl) side. The C14 aminimides displayed
some activity but usually only at greater concentrations.
Of the alkyl aminimides tested M-20 (C14) had LC50 values
for larvae and pupae between 1 and 10 ppm.

Exposed pupae were affected even more rapidly than

the larvae. Instead of spending most of the time at the

41

water surface, they would lie on the bottom of the beaker.
After several hours of exposure they could rise to the
surface only with great difficulty. The pupae in most
cases could not maintain themselves at the air-water inter-
face, and had to snap many times in order to regain the
surface. Many abnormalities were noted in the treated
pupae. When treated only in the pupal stage the pupal
wing-pads, mouthparts, and thoracic air case was disrupted.
If treatment was begun at the late larvae to pupal stage,
the pupae were formed with mouthparts and wing-pads stick-
ing straight out instead of being glued to the thorax, and
they were untanned. The treated pupae were examined under
a light microscope to determine if any cuticular entrance
could be discerned, but the pupal cuticle, although abnormal
in appearance had no disruptions or other detectable points
of entry. As with the larvae, the aminimides having the
chain length of C16 were the most active on the pupae,
followed by those having a C14 chain length.

The ethoxylated aminimides evaluated for their

activity against Culex pipiens and Aedes aegypti larvae

 

 

and pupae were found to be less effective than their parent
compounds (Table VI).
Raising the temperature from 21°C to 28°C only

had a minimal effect on the aminimides' toxicity to Aedes

42

aegypti larvae and pupae (Table VII). The aminimides were
more toxic when tested at higher pH than in acidic
solutions.

When aminimides were tested as synergists for
carbaryl (Table VIII) they failed to show the synergizing
characteristics of piperonyl butoxide, but showed an inde-
pendent joint action. This combination follows the equation

Pc = Pa + Pb - Pan where:

Pc = proportion of animals killed by the combi-

nation of poison a and b,
P = proportion killed by poison a,
P = proportion killed by poison b,

P P = proportion killed by both a and b (from

Bliss, 1939).

When the standard synergists were added to the
aminimides (Table IX) there was no true synergism. The
aminimides' toxicity was apparently enhanced but not
significantly.

When tested by standard methods the aminimides

failed to have any ovicidal effects on Aedes aegypti eggs

 

(Table X).

Aminimides dissolved in oil impregnated into filter
paper (Table XI) did not kill adult female Aedes aegypti;

thus they showed no contact toxicity.

43

TABLE VII. Effects of temperature and pH on the activities
of aminimides against larvae or pupae of Aedes

 

 

 

 

aegypti.
LC50(ppm)
Aedes aegypti
Larvae Pupae
M-3 (21°C) 1.65
M-3 (28°C) 1.60
M-8 (21°C) 9.0
M-8 (28°C) 8.0
M-3 (pH 6.5) 1.8
M-3 (pH 7.0) 1.65
M-3 (pH 7.5) 0.625

 

TABLE VIII. Effects of aminimides used with carbaryl on
4th instar Culex pipiens larvae.

 

 

 

 

Compounds LC50(ppm PB) LC50(ppm carbaryl)
Carbaryl .71
Piperonyl Butoxide (PB) 20
Compounds in mix (10:1
carbaryl)

PB .14
M-4 .44
M-8 .28
M-18 .34
M-20 .41
M-31 .45
M-54 .54
M-62 .43
M-71 .50

M-125 .54

 

44

TABLE IX. Effects of standard synergists used with amini-
mides on 4th instar larvae of Culex pipiens and
pupae of Aedes aegypti.

 

 

 

LC50(ppm aminimide)

 

A. M-8 used on larvae of Culex

 

 

pipiens
M-8 13.5
M-8 + Piperonyl Butoxide (PB) 8.6
M-8 + Sesamex (1:1) 9.2
M-8 + Tri-phenyl phosphate (TPP) 6.8
B. M-3 used on Aedes aegypti pupae
M-3 2.2
M-3 + Tri-O-Tolyl PhOSphate (1:1) 1.25
M-3 + TPP (1:1) 2.6
M-3 + PB (1:1) 1.45
Control % Mortality at 20 ppm
TOCP 0
PB 2
Sesamex 0
TPP 0

 

TABLE X. Percent hatch of Aedes aegypti eggs laid on alumi-
num panels after spray applications of aminimides
in 1% ethanolamine in distilled water.

 

 

Compounds % Hatch No. Eggs Tested
M-4 95.2 878
M-8 86.1 748
M-18 93 785
M-ZO 95.2 415
M-7l 94.5 835
M-ll6 58.3 1,105
M-120 81.9 856
Malathion 94.6 687

Control (1% ethanol amine
in distilled water) 94.2 639

 

45

TABLE XI. Biological activity of aminimides against adult
Aedes aegypti using impregnated filter-paper

 

 

 

technique.
Test Compounds Mortality (5% Test Compound)
M-l 1/25, 2/25
M-2 6/25, 3/25
M-3 0/25, 1/25
M-l32 0/25, 0/25
Malathion 25/25, 26/26

Olive Oil ' 0/25, 2/25

 

46

When M-3 was topically applied to the pupae, the
aminimide had no effect, in contrast to malathion which
caused 100% kill at the same concentration (Table XII).

In the adult after-effect tests (Table XIII)
using M-3 at 10 ppm, 55% of the larvae were killed and
adults failed to emerge. At 5 ppm the larval mortality was
greater than 50%, but at 1 ppm was nearly all the adults
emergenced.

Water containing M-18 and M-20 at 10 ppm remained

toxic to Aedes aegypti larvae for 12 days, whereas M-8

 

(10 ppm) was only effective for 5 days (Table XIV).
Measurements of the surface tension (dynes/cm)
for comparison with the biological activity obtained
showed that aminimides toxicity to Aedes aegypti pupae
was in direct proportion to which the surface tension of
the water had been lowered (Table XV). When the surface
tension values (X-axis) were plotted against the percent
mortalities (Y-axis) a definite negative correlation was
made evident, and the correlation coefficient obtained by
calculation (r=-.78) was of a high value. The most effec-
tive compound M-3 (chain length C16) lowered the surface
tension the most, while M-2 (chain length C14) which was
nearly as effective, reduced the surface tension nearly

as much.

47

 

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DISCUSSION

The common feature among the test species to which

the aminimides were non-toxic, i.e., Onc0peltus fasciatus

 

nymphs, Aedes aegypti adults, Tribolium confusum larvae

 

 

 

and pupae, and young Tetranychus urticae, is that they are
all terrestrial organisms. In their type and spectrum of
activity the aminimides have the greatest similarity to
the various amines and amides tested by Mulla (1967)

which are also organo-nitrogen compounds whose maximum
biological activity is reached against aquatic organisms
and life-stages.

Had the biological action of aminimides been
exerted on some target enzyme, then it is likely that
their activity would have been synergized by one or more
of the synergists tested (Table IX). However, since none
of these enzyme synergists were effective when used with
the aminimides, it is likely that these compounds have a
non-enzymatic mode of action.

Of the 4 criteria advanced by Christie and Crisp
(1966) for compounds that exert their toxic effects by
physical action (reversible narcosis, little dependence on
temperature, toxicity independent of time of exposure
and similar toxicity on the thermodynamic scale), the

49

50

aminimides demonstrate the first three. When removed
from the test solutions, the affected larvae soon
recovered. Increasing the temperature of the test
solutions from 21°C to 28°C had only minimal effects
on aminimides' toxicity to mosquito larvae or pupae.

Mulla (1967), however, concluded that amines have
a more complex mode of action on mosquito larvae and pupae,
including a change in the pr0perties of the cuticle and
possible interference with hormonal or amino-acid metabo-
lism. He ruled out the possibility of the toxicity of
amines being simply due to lowering of surface tension at
the air-water interface, but did not take any surface
tension readings of any of the amine test solutions.
Kabara and Haitsma (1975b) noted that all surface-active
agents, which are efficient bactericides, have been found
to possess a marked ability to reduce surface tension
although the converse is not necessarily true. Further—
more an increase in the chain length of the carbon chain
from C6 to C16 progressively increases the surface activity
of the compound with maximum activity being reached at C14
or C16' Table XVI (from Kabara et al., 1975a) shows the
antimicrobial activity of several series of homologous
acyl aminimides.

The measurements made on the reduction in surface
tension of a homologous series of aminimides (M—1, M-2,

M-3 and M-4) verifies the statements of Kabara and Haitsma

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51

 

 

 

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.H>N mqmfla

52

(1975b) on surface tension. When tested at 10 ppm the

acyl derivatives lowered the surface tension in proportion
to their chain length (Table XV). At C12 (M-l) the surface
tension was lowered from 73.0 dynes/cm (distilled water)

to 68.1 dynes/cm, Cl4 (M-2) lowered the surface tension to
47.4 dynes/cm, C16 (M-3) lowered the surface tension to
39.7 dynes/cm, while C18 (M—4) only lowered the surface
tension to 59.6 dynes/cm. ChristOphers (1960) has stated
that compounds which reduce the surface tension make good
pupicides by causing the pupae to lose attachment to the
air-water interface and drowning. The high value of the
correlation coefficient (r = -.78) for the aminimides
verifies the relation between 24-hour mortality of mosquito
pupae and reduced surface tension. Furthermore these
aminimides had no lethal effect when tOpically applied to

pupae of Aedes aegypti, nor did they cause any abnormal

 

adult emergence. For substances which reduce the surface
tension of the air-water interface, LC50 values can be
stated either by their concentration (ppm) or by their
surface tension in dynes/cm.

A succession of cuticular abnormalities was
observed when treatment was maintained through the larvae
and pupal stages by aminimides. The cuticular abnormali-
ties were most frequent with acyl or alkyl aminimides
having chain lengths of C14 and C16' The air bubble

responsible for the hydrostatic balance of mosquito pupae

53

in water (Christophers, 1960) is formed by the cementing
together of the wing-pads, mouthparts, and thorax enclosing
a small air pocket. Since it is at C1; and C16 that the
aminimides have their peak emulsifying properties, it is
therefore theoretically possible that aminimides emulsify
off the cement layer, disrupting the air bubble and causing
the pupae to lose their hydrostatic balance. The cuticular
abnormalities evident in the larval-pupal stage may derive
from the fact that it is just before or just after molting
that the cement is poured over the wax surface (Wigglesworth,
1965). This would give the aminimides a "window" or time
period where they could compete with the insect for the
cement. At high concentrations aminimides could conceivably
prevent enough cement from reaching the cuticle to allow
the formation of a properly formed pupal integument. The
abnormalities that occur in the pupal-adult stage at lethal
concentrations may have a similar explanation. The emerging
adult cannot maintain itself on the air-water interface
because of the reduced surface tension (Manzeli, 1943).
The resulting "wetting“ of the untanned and unhardened
new adult cuticle would result in the loss of its cement
to the aminimide solution, preventing the development of a
normal integument and also drowning the adult mosquito.
Aminimides possess potential as good mosquito
larvicides and pupicides. Being more effective pupicides

than carbaryl or malathion and having lethal effects on

54

mosquito larvae, and in the transition between larvae and
pupae, and between pupae and adults, their effectiveness
and specificity renders them potential mosquito control
agents. Since mosquito larvae feed on bacteria (Chris-
tophers, 1960), the bactericidal qualities of aminimides
would have a further negative-growth effect on mosquito
larvae. The unhinging effect of the spiracular apparatus
at the air-water interface caused by reducing the surface
tension is a very specific type of activity which would be
useful in combatting mosquitos of medical importance that
are resistant to standard insecticides. The low toxicities
of these compounds (200-400 mg/kg) allow therefore to be
easily handled. Aliphatic amines, which have toxicities
and exert symptoms similar to those observed with amini-
mides, have been tested in California (Mulla et al., 1970;
Mulla and Darwazeh, 1971, 1975) at rates between 0.25 and
1.0 lbs./acre, and gave good control of mosquito larvae.
Because these compounds are most active in water,
their effects on other types of aquatic metazoans should be
tested. Their effects on such pests as nematodes or pro-
tozoa, and their effectiveness when field-tested against
mosquito immatures should be investigated. The possible
use as wetting agents for standard insecticides is another

way these compounds could be used.

S UMMARY AND CONCLUS IONS

1. Of 35 aminimides tested in water against larvae

and pupae of the mosquitos Aedes aegypti and Culex pipiens,

 

 

3 of them killed both larvae and pupae of either species
at concentrations between 1 and 10 ppm. In 2 of these the

acyl substituent was of chain length C and in the third

16’
the alkyl substituent was of chain length C14. Ten of them

killed pupae at doses lower than the larvicides now in use.

2. The aminimides were found to be ineffective

against terrestrial arthropods such as Tetranychus urticae,

 

Oncopeltus fasciatus, Aedes aegypti adults, and Tribolium

 

 

 

confusum. They were also inactive against pupae of Aedes

aegypti when topically applied in a test chamber.

3. Of the effective aminimides, the ranking of
their biological activity was strongly correlated to the
degree to which they reduced the surface tension of water.
The symptoms shown by the mosquito larvae and pupae in
water treated with aminimides strongly suggests that
these compounds acted as physical poisons preventing the
insects from maintaining contact with the air-water

interface.

55

56

4. Since the aminimides as a class of compounds
are of low toxicity to higher animals, and since certain
of them are active against pupae as well as larvae, it
is considered that they are as worthy of attention as
the amines which have recently been under investigation

as a new group of mosquito larvicides.

APPENDICES

APPENDIX A

EXPERIMENTAL CHEMICALS AND THEIR

PHYSICAL PROPERTIES

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57

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59

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60

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61

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LITERATURE CITED

LITERATURE CITED

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Alexander, B. H. and M. Beroza. 1963. Aliphatic amides of
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Andrews, T. L. and R. P. Miskus. 1972. Some effects of
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Bliss, C. I. 1939. The toxicity of poisons applied

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62

63

Brown, A. W. A. 1971. Pest resistance to pesticides. In
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Christie, A. O. and D. J. Crisp. 1966. Toxicity of ali-
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Christophers, S. R. 1960. Aedes aegypti, The Yellow Fever

 

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Clements, A. N. 1963. The Physiology of Mosquitoes.
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Cline, R. E., D. P. Wilton and R. W. Fay. 1969. Aliphatic
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Corkill, J., K. Gemmell, J. Goodman and T. Walker. 1970.
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Cruickshank, P. A. and R. M. Palmer. 1971. Terpenoid
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Curtis, R. F., D. T. Coxon and G. Levett. 1974. Toxicity
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brine shrimp (Artemia salina) Fd. Cosmet. Toxicol.

 

12:233-235.
Dahm, P. A. and C. W. Kearns. 1941. The toxicity of alkyl
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34:462-466 .

64

Dale, H. H. 1920. John HOpkins Hospital Bull. 31:22.

Fahmy, M. A. and H. T. Gordon. 1965. Selective synergism
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Fuller, A. T. 1942. Antibacterial action and chemical
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House, H. L. and J. S. Barlow. 1960. Effects of oleic and
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Hueck, H. J., D. M. M. Adema and J. R. Wiegmann. 1966.
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Ikeshoji, T. and M. S. Mulla. 1970. Overcrowding factors
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Ikeshoji, T. and M. S. Mulla. 1974a. Overcrowding factors
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Ikeshoji, T. and M. S. Mulla. 1974b. Overcrowding factors
of mosquito larvae: Activity of branched fatty acids

against mosquito larvae. Env. Ent. 3:487-491.

Jensen,

Kabara,

Kabara,

Kabara,

Kabara,

Kabara,

65

E. M. and P. C. Liu. 1963. Inhibitory effect of
simple aliphatic amines on influenza virus in
tissue culture. Proc. Soc. Exp. Biol. Med.
112:456-459.

J. J., D. M. Swieczkowski, A. J. Conley and J. P.
Truant. 1972a. Fatty acids and derivatives as
antimicrobial agents. Antimicrob. Ag. and Chemo.
2(1):23-28.

J. J., A. J. Conley and J. P. Truant. 1972b.
Relationship of chemical structure and anti-
microbial activity of alkyl amides and amines.
Antimicr. Ag. and Chemo. 2:492-498.

J. J., A. J. Conley and D. M. Swieczkowski. 1973.
Antimicrobial action of isomeric fatty acids on

Group H Streptococcus. J. of Med. Chem. 16:1060-

 

1063.

J. J., W. J. McKillip and E. A. Sedor. 1975a.
Aminimides: I. Antimicrobial effect of some long-
chain fatty acid derivatives. JAOCS, in press.

J. J. and G. V. Haitsma. 1975b. Aminimides: II.
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