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STATE UNIVERSITY [LIB:ARIES“

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3 1293 00612 2588

LIBRARY
Michigan State
University

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

This is to certify that the

dissertation entitled

Characterization, Synergism and Inheritance of Resistance
to Azinphosmethyl, Carbofuran and Permethrin Insecticides
in the Colorado Potato Beetle (Coleoptera: Chrysomelidae)

presented by I

Philippos M. Ioannidis

has been accepted towards fulfillment
of the requirements for

Ph .0. degree in EDLQleng .

 

W¢W

Major prgfessor

Date 2 199

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

._... i 7‘.__.__ _.____ _.

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOD FINES return on or baton dds mo.

DATE DUE DATE DUE DATE DUE

 

JAN 0 s ‘99:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Afflrmdivo Action/Equal Opportunity Institution

 

 

CHARACTERIZATION, SYNERGISM AND INHERITANCE
OF RESISTANCE TO AZINPHOSMETHYL,
CARBOFURAN AND PERMETHRIN INSECTICIDES IN THE
COLORADO POTATO BEETLE
(COLEOP'I'ERA: CHRYSOMELIDAE)

By

Philippos M. Ioannidis

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Entomology

1990

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6405479614

ABSTRACT

CHARACTERIZATION, SYNERGISM AND INHERITANCE
OF RESISTANCE TO AZINPHOSMETHYL,
CARBOFURAN AND PERMETI-IRIN INSECTICIDES IN THE
COLORADO POTATO BEETLE
(COLEOPTERA: CHRYSOMELIDAE)

By

Philippos M. Ioannidis

Colorado potato beetle Leptinotarsa decemlineata (Say) were tested for
resistance to azinphosmethyl, car'bofuran, and permethrin by topical application
bioassays. The research was designed to characterize resistance (resistance
levels, cross-resistance, synergist responses, inheritance). Data indicated that
most populations in Michigan were heterogeneous for resistance to at least one
insecticide. There appeared to be three main patterns Of resistance in the field.
Pattern I: The resistance ratio was high for permethrin but low for the other
two insecticides. Pattern II: High levels of resistance to azinphosmethyl and
carbofuran and low to permethrin. Pattern III: Beetles were resistant to all three
insecticides.

Synergists studies were conducted to determine resistance mechanisms.
Beetles can use almost all the resistance mechanisms known for insects. The
effects Of the synergists piperonyl butoxide, DEF, and DEM indicated that mainly
mixed-function oxidase and esterases were involved in azinphosmethyl
resistance. Mixed-function oxidase and acetylcholinesterase were responsible
for carbofuran resistance. Another type Of mixed-function oxidase was

involved in permethrin resistance, plus two other mechanisms, esterases and

Philippos M. Ioannidis

the Kdr factor (knock-down resistance), which also gives cross-resistance to
DDT.

Synergists can be used in the field to improve insecticide effectiveness
and overcome resistance. Cross-resistance exists between aldicarb and
permethrin, between azinphosmethyl and carbofuran-imidan, between
carbofuran (acetylcholinesterase resistance) and thiodan probably linked genes.
NO cross-resistance was Observed in carbofuran and aldicarb. Use of mixtures
are not recommended; rotation of insecticides is preferable. Rotation Of crops is
absolutely necessary.

Resistance to carbofuran was inherited via a single, autosomal,
incompletely dominant factor resulting in acetylcholinesterase insensitivity.
Azinphosmethyl resistance appeared to be inherited mainly via two
incompletely dominant autosomal linked genes, with about a distance of 20
map units. The two main resistance factors involved in the highest level Of
resistance were mixed-function oxidase and esterases enzymes. These two
resistant factors were synergistic in effect when contained in the same
individual. Base line data and discriminating doses for assessment of resistance
were established for several insecticides. Strategies for management of
resistance in CPB are critical for continued control, resulting in long-term profit

and reduced pesticide applications.

To my sister

Your silent memory will

preserve your image

iv

pre

De]

Bis}

ACKNOWLEDGEMENTS

Conducting this research, including the academic experiences I
encountered throughout my studies at Michigan State University, has been
both challenging and rewarding. This experience has been the best it could
possibly be because of the tireless efforts and academic guidance of my
advisor, Dr. Edward Grafius. I sincerely appreciate all he has done for me,
including the financial support for this study.

I must share my gratitude with the other members Of my committee as
well. I thank Dr. Mark Whalon for his useful insights and for the stimulating
discussions we had, Dr. Robert Hollingwortthirector of the Pesticide
Research Center, for his valuable suggestions and contributions to this study
Obtained through the formal group meetings we had and through intellectual
discussions with him. I thank Dr. Donald Ramsdell for acting on my
committee, for the knowledge base I Obtained through his excellent class on
plant virology and for his suggestions and corrections on this dissertation.

I would also like tO thank 'Dr. Fumio Matsumura and Dr. Frank
Drummond, two of my previous committee members who transferred to
other universities, for their efforts.

I thank all of the staff members of Dr. Grafius' lab, both past and
present. I would also like to thank all of the faculty members and staff Of the
Department of Entomology, especially Dr. Fred Stehr. Thank you, Beth

Bishop for the collaboration and help during all these years. I would also like

to express my thanks to Ian Eschbach for helping with editions and typing this
dissertation.

Finally, it is necessary to express my appreciation to my employer, the
Hellenic Sugar Industry, not only for the financial support throughout my
studies, but also for the opportunity that was granted me to undertake this

degree.

vi

TABLE OF CONTENTS

List Of Tables ......................................................................................................... x
List Of Figures .................................................................................................. xiii
Introduction ......................................................................................................... 1
Chapter 1 - GENERAL METHODS AND MATERIALS ............................. 5
Field Monitoring for Resistance of the Colorado Potato Beetle ........ 6
Research Approach and Procedures ........................................................ 8
Colorado Potato Beetle Cultures .............................................................. 8
Experimental Conditions ........................................................................... 9
Topical Applications ................................................................................. 10
Detection of Resistance Using LDso Values
and Dose-Mortality Lines ................................................................ 10
Assessment of Mortality .......................................................................... 11
Standardization Of Insects for Testing ................................................... 11
Technical Information for Bioassays ..................................................... 12
Detection of Resistance Using the LTso's Values ............................... 14
Symptomological Observations ............................................................. 15
Selection for Insecticide Resistance ....................................................... 15
Introduction ................................................................................................ 15
Material-Methods ...................................................................................... 17
Selection Procedure A .............................................................................. 18
Selection Procedure B ............................................................................... 19

Chapter 2 - PATTERNS OF INSECTICIDE RESISTANCE TO
AZINPHOSMETHYL, CARBOFURAN, AND
PERMETHRIN IN THE COLORADO POTATO BEETLE

(COLEOPTERA: CHRYSOMELIDAE) ...................................... 21
Abstract ........................................................................................................ 22
Introduction ................................................................................................ 23
Materials and Methods ............................................................................. 24
Topical Application of Insecticides ........................................................ 25
Assessment of Mortality .......................................................................... 26
Results .......................................................................................................... 26
Discussion ................................................................................................... 29

vii

Chapter 3 - SYNERGIST STUDIES ................................................................ 35

Synergists ..................................................................................................... 36
Available Information about Synergists on Resistance of

the Colorado Potato Beetle ............................................................. 38
Use Of Insecticide Synergists to Indicate Resistance

Mechanisms - Research Approach and Procedures .................. 41
Piperonyl Butoxide (PB) ........................................................................... 43
MGK 264 ...................................................................................................... 44
DEF ............................................ 44
Diethyl Maleate (DEM) ............................................................................. 45
Chlorfenethol (DMC) ................................................................................ 46
Insecticide - Synergist Ratio ..................................................................... 47
Pyrethroid Synergism ............................................................................... 48
Mixed-Function Oxidases ........................................................................ 49
Results ......................................................................................................... 51
Conclusions for Synergist Studies ......................................................... 54
Different Types of Mixed Function Oxidases for Resistance

in Colorado Potato Beetle ............................................................... 59

Chapter 4 - SELECTION, INHERITANCE AND CHARACTERI-
ZATION OF CARBOFURAN RESISTANCE ....................... 65
Abstract ........................................................................................................ 66
Introduction ................................................................................................ 67
Materials and Methods ............................................................................. 68
Laboratory Techniques ............................................................................. 69
Field Selection ............................................................................................ 70
Laboratory Selection .................................................................................. 70
Resistance Mechanism Studies ................. 70
Inheritance Studies ................................................................................... 71
Results .......................................................................................................... 72
Chapter 5 - MECHANISMS AND GENETICS OF
AZINPHOSMETHYL RESISTANCE ...................................... 83

Abstract ........................................................................................................ 84
Introduction ........................................................................... ; .................... 85
Materials and Methods ............................................................................. 87
Insecticides and Synergist Used .............................................................. 87
Bioassay Tests - Measurements of Resistance ..................................... 87
Statistical Analysis ..................................................................................... 88
Establishment of Homozygous Strains ................................................. 89
Genetical Techniques ................................................................................ 9O
Reciprocal Crosses ..................................................................................... 91
Backcrosses and F2 ..................................................................................... 91
Discriminating Dose Technique ............................................................. 92
Results and Discussion .................................. ' ........................................... 9 2

viii

GENERAL DISCUSSION ................................................................................ 106
LIST OF REFERENCES .................................................................................... 112

APPENDIX I ....................................................................................................... 128

ix

Che

List of Tables

Chapter 1 — None
Chapter 2

Table 1. Pattern 1. Dose response data for eight populations
Of Colorado potato beetle with low resistance ratios for
azinphosmethyl and carbofuran, and moderate to high
resistance ratios for permethrin .................................................... 32

Table 2. Pattern II. Dose response data for populations of
Colorado potato beetle showing high resistance to
azinphosmethyl and carbofuran and low to moderate
resistance to permethrin ................................................................. 33

Table 3. Pattern III. Dose response data for five populations
of Colorado potato beetle showing high resistance to

all tested insecticides ........................................................................ 34
Chapter 3
Table 1. Synergism studies with Colorado potato beetle .................. 40

Table 2. Resistance and insecticide synergism of JP Macomb
population of the Colorado potato beetle to azin-
phosmethyl and carbofuran using topical application
bioassays .............................................................................................. 52

Table 3. Resistance and insecticide synergism of JP Macomb
population of Colorado potato beetle to permethrin
and DDT using tOpical application bioassays .............................. 53

Table 4. Synergism of azinphosmethyl of GRM Colorado
potato beetle strain from piperonyl butoxide or DEF
or DEMusing topical application bioassays ................................ 55

Table 5. Toxoicity and synergistic ratios Of azinphosmethyl
in mixtures with piperonyl butoxide (PB) or DEF or DEM
with two multiple highly resistant strains of Colorado
potato beetle, the Long Island and Macomb ............................... 58

Table 6. Three different cases Of resistance of the Colorado
potato beetle tO carbofuran. Synergistic studies with
piperonyl butoxide (PB) or DEF indicate possible
resistance mechanisms involved ................................................. 60

Table 7. Toxicity of azinphosmethyl, carbofuran and
permethrin on a strain Of Colorado potato beetle only
resistant to permethrin synergist ratios for mixtures of
permethrin and piperonyl butoxide (PB) or DEM ..................... 61

Table 7. Resistance levels and resistance mechanisms Of
different Colorado potato beetle strains for the resistance
to azinphosmethyl, carbofuran, permethrin and DDT
insecticides based on synergist studies ......................................... 63

Table 8. Resistance levels and resistance mechanisms Of
different Colorado potato beetle strains for the resistance
tO azinphosmethyl, carbofuran, permethrin and DDT
insecticides based on synergist studies ......................................... 63

Table 9. Cross resistance spectrum Of different resistant
strains Of Colorado potato beetle ................................................... 64

CHAPTER 4

Table 1. Resistance of the "Montcalm- C" strain of CPB to
carbofuran over four generations Of selection, beginning
with selection Of larvae In the field .............................................. 79

Table 2. Responses to carbofuran Of adults Of Colorado
potato beetle from the homozygous susceptibles (SS),
resistant (RR) parental strains, and the F1 (SS x R)
hybrids Of the reciprocal crosses .................................................... 80

Table 3. Colorado potato beetle percentage mortality from
carbofuran: Ratio expected if resistance is monofactorial
inherited and observed mortalities of susceptibles
by applying discriminating doses .................................................. 81

Table 4. Synergisms Of carbofuran and azinphosmethyl by
piperonyl butoxide (PB) and DEF in susceptible Vestaburg
(SS) and in two resistant strains to carbofuran with
different resistant mechanisms ..................................................... 82

xi

CHAPTER 5

Table 1. Toxicity of azinphosmethyl tO susceptibles (S),
resistance (R), parental strains and to F1 hybrids.
Synergism Of azinphosmethyl by piperonyl
butoxide (PB) with the F1 hybrids ................................................. 103

Table 2. Theoretical genotype expression in dihybrid crosses
and backcrosses with two autosomal resistance factors
to azinphosmethyl, the enzymes MFO's (AA) and
esterases (BB) ..................................................................................... 104

Table 3. Phenotype expressing Of resistance to azinphosmethyl

in the F2 progenies (F1 x F1) from two autosomal linked
factors to MFO's and esterases with 20 map distance. .............. 105

xii

List of Figures

CHAPTER 1 - none
CHAPTER 2 - none
CHAPTER 3 - none
CHAPTER 4

Figure 1. Dose-mortality responses to carbofuran Of adults of
susceptible (Vestaburg) and resistant (Montcalm-C)
strains Of Colorado potato beetle and their F1 Offspring,
as a result Of topical application bioassays ................................... 73

Figure 2. Dose mortality responses to carbofuran of
susceptible (Vestaburg-s), and resistant (Montcalm—CuR),
strains of Colorado potato beetle, and their F1 female
Offspring, and backcross offspring (BCl, ([Vestaburng}
females x Montcalm-C {R} males]) x Vestaburg males) ............ 75

Figure 3. Dose mortality responses to carbofuran of
susceptible (Vestaburg-s), and resistant (Montcalm-C-R),
strains of Colorado potato beetle, and their F1 female
offspring, and Offspring of two backcrosses to susceptible
males and females (Vestaburg) ...................................................... 76

CHAPTER 5

Figure 1. Dose mortality responses to azinphosmethyl Of
adults of Colorado potato beetle susceptible (Vestaburg
s) and resistant (Long Island-R) strains and F1 Offspring
(males and females). ....................................................................... 93

Figure 2. Dose mortality responses of adults Of Colorado
potato beetle susceptible (Vestaburg - s) and resistant
(Long Island-R) strains and F1 Offspring (males and
females) to azinphosmethyl and application Of
azinphosmethyl and piperonyl butoxide to F1
Offspring only .................................................................................... 94

xiii

Figure 3. Dose—mortality response to azinphosmethyl in adults
of susceptible (Vestaburg) and resistant (Long Island)
strains Of Colorado potato beetle and in their F1 Offspring
(Vestaburg females x L. Island (R) males) and in Offspring
of a backcross (BC1) of F1 females with Vestaburg males ........ 98

Figure 4. Dose-mortality response to azinphosmethyl in adults
of susceptible (Vestaburg) and resistant (Long Island)
strains of Colorado potato beetle and in their Offspring
of a second backcross (Vestaburg females x L. Island (R)
males and in third backcross .......................................................... 99

xiv

INTRO DUCTION

"The word resistance has come to be applied to any population, within
a species normally susceptible to a given insecticide, that is no longer
controlled by the insecticide in the area concerned" (Brown and Pal 1971).

Resistance arises as a result Of selection acting upon natural variation.
It is now clear that resistance is invariably "pre-adaptive". The idea that
resistance arises from natural variation does not preclude the occurrence of
new mutations after the insecticide has been applied (Wood 1981).

Resistance of insects to pesticides has a history since 1910 but its greatest
increase and strongest impacts have occurred during the last 40 years, after the
extensive use Of synthetic organic insecticides. Up to 1986, 447 species
(including most major pests) had been reported to be resistant to one or more
classes of chemicals (Georgiou 1986). It has been understood that resistance
may be caused by one of three mechanisms, acting singly or in combination,
decreased sensitivity at the site Of action, enhanced metabolic attack within the
insect, or decreased transport to the site Of action. Past experience and recent
studies on resistance modelling showed that reduction of selection pressure in
its broadest sense is a key factor in reduction of development of resistance.
Colorado potato beetles (CPB) Leptinotarsa decemlineata (Say) spread across
North America by the mid-1870's and started to increase in population in
Europe after the First World War with the first outbreak in Britain in 1901,
and since 1975 have been starting toward Asia (Hurst 1975). The significance

of this serious pest was described by Hurst in 1975 in a technical note as

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beetle
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follows: "The beetle can almost be regarded as a parent of the pesticide
industry, because it was largely to stop the devastation of the potato crops that
chemicals such as arsenates came into field use and were developed. The
beetle can claim to have brought a major European organization into being,
for the European and Mediterranean Plant Protection Organization
(henceforward abbreviated as EPPO) owed its origin to the committee formed
in the 1930's to try tO contain the progress Of the beetle across Europe."

The CPB colonizes and feeds on several cultivated crops - potato
(Solanum tuberosum L.), tomato (Igcopersion esculentum Mill) and eggplant
(Solanum melongena L.) and on a number of wild host plants: Buffalo bur
(Solanum rostratum Dunal), and common night shade (Solanum mm L.).
It is known that CPB has demonstrated to be a very adaptable insect (Groden
1986). The CPB can significantly reduce the yields of these crops and Often
completely defoliates them unless successful control measures are applied. It
has been speculated that herbivorous species which have frequently evolved
the capacity tO deal. with plant alkaloids, are in some sense preadapted to
dealing with the problems posed by dangerous chemicals in their
environment (Croft and Brown 1975). The Allelochemical alkaloids and
ketones are toxic to insects. The Leptine glycoalcaloids in Solanum
chalcoense are toxic to the CPB (Sinden et a1. 1986). Through the co-evolution
with the Solanaceous plants, it seems that the beetles have developed
effective mechanisms to overcome the alkaloids. This probably has facilitated
the development of resistance to insecticides.

The CPB has exhibited substantial resistance to almost all registered
insecticides in the eastern United States and has also started to become a
problem in the west. It has a remarkable propensity for resistance and it is

notorious for the speed with which it has developed resistance to all main

categori
year. 'I
the sut
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categories of insecticides, despite only two to three generations completed per
year. The CPB has required progressively less time to develop resistance to
the subsequently used insecticides: seven years tO DDT, five years to
azinphosmethyl, two for carbofuran, two for pyrethroids and one for
pyrethroids plus the synergist piperonyl butoxide (Georgiou 1986).

Resistance to DDT spread as early as 1953 to spots throughout the Red
River Valley (Post 1953). Hofmaster (1956) pointed out that there was
considerable evidence that DDT was not adequately controlling CPB on
potatoes on the Eastern shore of Virginia. Cutkomp (1958) confirmed
resistance Of CPB to DDT by determining the toxicity levels Of injected DDT to
adult beetles collected from two widely separated areas. The synergist
Chlorfenethol also used in combination with DDT did not produce any
detectable difference from DDT alone and he concluded that the results did
not provide any Clue as to the mechanisms Of the resistance. Harris et a1.
(1976) reported resistance to DDT and cyclodienes in Ontario, 240-fold to
Aldrin and 37-fold to Endrin. McDonald (1976) tested CPB from Southern
Alberta, Canada and found that 10 years after the DDT was) discontinued,
susceptibility (1966 23-fold) had returned to those levels recorded in 1957 and
mentioned that resistance to this compound was disappearing from this
species. Probably the above cases of resistance to DDT were the I<dr type, well
known today for its recessiveness. Tests with CPB from Quebec showed that a
strain was resistant to most insecticides and especially high for carbofuran
(>1600-fold), it was also resistant to DDT, Endrin, endosulfan,
azinphosmethyl, phosmet methidathion, methamidophos, phorate,
cloethocarb, and carbaryl (Harris et al. 1981). Now it is well documented that
populations of CPB with multiple mechanisms of resistance exist in many

places in the US.

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The Objective Of this work was to study some Of the factors which
constitute the whole phenomenon Of resistance. In the following chapters we
have described techniques and procedures including methods for laboratory
and field selection for resistance, with the insecticides azinphosmethyl,
carbofuran, and permethrin.

Field monitoring complemented with laboratory bioassays for
documentation Of resistance using laboratory and field collected strains were
also conducted. The data Obtained provided\baseline data for resistance from
the state Of Michigan and revealed different pgtterns Of resistance developed
in field populations. \

Synergists may be used to delay resistance development in addition tO
their use in overcoming established cases, and tO indicate resistant
mechanisms (Metcalf 1967). Studies were conducted with the well-known
insecticide synergists piperonyl butoxide, MGK 264, DEF, DEM, and
Chlorfenethol, to define enzymes involved in insecticide detoxification, and
to support biochemical analysis data.

Genetic principles and techniques have been of great value in
understanding basic mechanisms involved in the development, spread,
regression and inheritance of resistance to pesticides. We conducted
reciprocal crosses between susceptible and resistant strains and in addition,
the F1, F2 and several backcrosses, were successfully completed. All this
information can help significantly to design resistance management tactics.
Colorado potato beetle insecticide resistance is an urgent and alarming

problem which may result in considerable economic loss and even in the

discontinuation of potato production if allowed to go unchecked.

Chapter 1

General Methods and Materials

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Carbofuran

Field Monitoring for Resistance of the Colorado potato beetle

All Of the bioassays were conducted on insect populations collected
from individual fields at different locations. Most Of the populations were
summer adults (first generation) or of the F1 progeny collected just prior to
the application Of pesticides (about 1 week Old).

Synergism studies were applied directly to field resistant populations.
Resistance detection and monitoring was conducted as a basis for subsequent
resistance studies as well as to establish base-line data Of resistance for the
state of Michigan.

The insecticides mainly tested are some of the most commonly used in

Michigan for controlling potato beetles and belong to the four main categories

of pesticides.
INSECTICIDES
Azinphosmethyl Organophosphorothioate
Carbofuran Carbamate
Permethrin Pyrethroid
DDT Organochlorine

On the basis Of laboratory studies, carbofuran, chlorfenvinphos, and
azinphosmethyl were the bcést of registered materials as foliar sprays against a
population Of CPB of various stages (McClanahan 1975). Harris (1976) tested
40 insecticides against 3rd stage larvae and adults of a susceptible strain and he
reported that the most effective larvicides in descending order of efficacy
were Carbofuran > Endosulfan > Azinphosmethyl > Carbaryl > Phosmet >
Methamidophos. The most effective adulticides were Endosulfan >

Carbofuran > Azinphosmethyl > Phosmet > Carbaryl > Methamidophos. The

topic
1980)
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used (

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superiority of the compounds used in our studies has been also shown by
McDonald (1976) and Hare (1980).

For estimation of the Resistance Ratio (RR) we adopted the reported
topical toxicities for susceptible adult CPB from northern Maine (Forgash
1980). They were: 0.39 ug/ beetle for permethrin, 0.25 ug/ beetle for carbofuran,
and 0.3 ug/ beetle for azinphosmethyl. Susceptible adults from Idaho also had
toxicities Of insecticides in acetone solutions of equal or lower values tO those
mentioned above (Johnston et al. 1986). For DDT the value 10 ug/ beetle was
used (FAO 1974).

Permethrin was given an emergency registration for field use in
Connecticut in 1979, because field trials showed permethrin tO be the most
effective in controlling beetle populations after the development Of resistance
to carbamates and organophosphates (Hare 1980). It was very important to
include permethrin, a type I pyrethroid (Gammon et al. 1981, Scott et al. 1983)
in our studies for detection of cross-resistance to DDT. DDT was tested on
populations showing permethrin resistance. Specifically with permethrin we

performed the following work:

0 Standardized the bioassay technique for testing pyrethroids
against Colorado potato beetle,
Characterized permethrin resistance in Michigan,
Determined mechanism(s) involved in permethrin resistance
other than the Kdr factor which confers knockdown resistance
(which cannot be overcome by synergists).

The Kdr action is quantitative, since at higher dosages a normal
response is Observed and there is no recovery. Oppenoorth et a1. (1976)
concluded that any hypothesis on the resistance mechanism controlled by the

Kdr gene has to take into account the following:

synerg
outain
apphca
1
natural
would
suscept
detectio
means (

levels,

Researd

glflggggj

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USed ill 8

suitable $1

a) the Kdr factor affects the toxicity Of DDT and its analogues as well
as the pyrethroids, and

b) its action is quantitative since at higher dosages a normal
response is Observed.

From our studies and the control failures of applying pyrethroids plus
synergists after 1-2 years, it appears that the Kdr factor was well spread in
certain areas and may have been responsible for some repeated failures of
application Of pyrethroids.

Farnham (1977) suggested that since Kdr is a recessive character,
natural dilution of the population and the removal Of selection pressure
would help to greatly increase the number of heterozygotes which are
susceptible tO DDT or pyrethroids. There is, therefore, a need for early
detection Of this factor in a population, coupled with the use of an alternative
means of control in order to keep the frequency Of this gene at manageable

levels.

Research Approach and Procedures

Ir a lulur

Colorado potato beetle cultures were maintained in cages 50 x 50 x 70
cm located in walk-in environmental rooms maintained at 25i1'C, 50i10%
RH and a 16:8 L:D light regime.

Larvae and adults throughout their life span were reared on fresh
potato plants which had been planted in clay pots under standard techniques
used in greenhouses. Attempts were made to rear the beetles on the same

suitable susceptible variety of potato. This was essential for maintaining the

emeré
survi\
and s
condu.
Eggs h
cooling
leaves 1
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correct biotype populations. Differences in the resistance Of cultivars Of
potatoes tO CPB damage have been Observed (Mateeva-Radeva 1985).
Laboratory testing methods rarely simulate natural conditions. In our
experiments, however, we did not consider the role Of crop toxicity because
the larvae and adults fed on potato plants.

Boiteau (1983) described a method for synchronization Of CPB
emergence. Eggs can be held at 10°C for up to 12 days without affecting
survival. More than 90% of eggs collected daily over a period of up to 12 days
and stored at 10°C hatched in 1 day. Using the above technique, we
conducted bioassays on beetles (lab cultures) with the same approximate age.
Eggs were collected and stored daily. After 10 days eggs were removed from
cooling chambers. Hatched larvae were reared in petri dishes on potato
leaves for 3 days and were subsequently moved to whole plants in the cages
for growth and pupation. Useful information on longevity and fecundity of
the CPB and egg development and larvae development can be found in the

papers by Peferoen et a1. (1981), Logan et a1. (1985).

Experimental Conditions

All the bioassays were conducted in a second environmental room
maintained in identical conditions with the culture rooms. Attempts were
made to test one insecticide on several different strains simultaneously to
minimize the influence Of various factors affecting the bioassays. Therefore

the results were compared directly.

 

Chang
Insect
techni
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10

Tooieal Aoplieatiom
Deteetion of Reeietanee Qeing LDso Voluee and Doge-Mortality Lines

The standard bioassay technique for CPB (FAO 1974) was used. Some
changes have been made to the method as a result of preliminary work.
Insecticidal toxicity was assessed by topical application of 2 pl of solutions of
technical compounds in acetone. Applications of insecticides and synergists
were applied using a hand-Operated microapplicator to the second abdominal
sternite of adults and the lateral portion of the third abdominal segment of
larvae. All tests were conducted at 25:}:1 'C. For application Of the
compounds, the beetles were immobilized by holding them at the tip of a
plastic tube to which a vacuum was applied. Concentrations Of insecticides
were selected based on preliminary trials to give a range of 0-100% mortality.
TO Obtain this information, pesticides were applied in concentrations above
(10x), below (0.1x), and at (1x) the recommended field rates. For each bioassay
at least four concentrations were used. Control beetles were included with all -
the tests to which only acetone was applied.

Mortality values were corrected for natural mortality by Abbott's (1925)
formula. If control mortality was larger than 20%, we repeated the bioassay
(Busvine 1971). In most cases bioassays were performed at least twice.

Toxicity was measured by the log-probit analysis method (Finney 1971)
using the MSTAT-C microcomputer program (Eisensmith 1989).

11

Assessment of Mortality

The timing of the assessment Of mortality is extremely important.
There are differences between the insecticide groups. For azinphosmethyl
and carbofuran maximum mortality (90%) was Obtained within 3 days. For
permethrin, mortality is usually recorded within 24 hours. Since the
knockdown effect Of pyrethroids is well known and there is recovery in most
of the insects, for a multi-resistant population more than one assessment of
mortality was needed in order to obtain useful results. Mortality was

estimated as follows:

1/ 2 hour knockdown effect
3 days dead + knockdown effect
5 days dead + recovered

It was important to standardize the test procedures because the slope
and LDso for a particular population and insecticide can change considerably
with the application method, solvent used, sex ratio Of insects, time to
estimate mortality, temperature, age of insect, and so forth (Busvine 1971,
Winney 1973). Bioassays and tests with field beetles were undertaken either
directly, on the field material collected before sprays, or on the F1 colonized
generation. Since so many factors influence the validity of the bioassays, a
considerable amount Of time had been spent on designing appropriate

techniques with standard procedures. Great care must always be taken to

insure

respon
Techni

Probit
low X:
from t}

possibil

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12

insure that these variables are kept to a minimum when comparing the

response of different strains of CPB to insecticides.

Teehnical Information for Bioassays

 

Probit analysis: The responses Of each genotype (strain) must be linear with
low X2 values (X2 is a measure of the departure of the actual Observations
from the fitted probit-log concentration line). Otherwise the following

possibilities arise:

0 the population is genetically heterogeneous,
0 the test method is not sufficiently sensitive,
0 the insecticide may contain toxic impurities.

The shallower the slope, the greater was the variation in the susceptibility Of
the individuals of the population to the poison. The study of slope, LD90, and
LD10 would increase the value of the information given by the bioassay.

When the population exhibited homogeneous susceptible behavior, or
homogeneous resistance behavior, the dose ratio which was most appropriate
to use was the geometric series 1 : 2 :4 : 8 : 16 for estimating the LDso's.

With heterogeneous populations, which exhibited flat regression lines,
we found that it was best to use the ratio 1 : 3 : 9 : 27 : 81 for estimating the
LDso's. With heterogeneous populations it was best to use many doses (6 to 8)
and fewer replications per close when available beetles were limited.
Therefore, by inspecting the plotting Of‘ the Observed probits and the dose-
response curve shape, we identified the presence of two types of individuals

and we were able to estimate percentages Of susceptible and resistant beetles.

13

The ratio series 1 : 3 : 9 : 27 : 81 worked much better also with
pyrethroids. We noticed consistently that the regression lines for pyrethroids
were flatter than the ones for azinphosmethyl and carbofuran and observed
the same phenomenon working with pyrethroids on houseflies.
Occasionally, there are indications that the probit analysis represented by the
equation y = a + bx does not adequately express the relationship between
mortality probit and quantity Of pyrethroids applied topically (Ioannidis 1979).

Substances possessing similar mechanisms of action usually provide
parallel regression lines. Before making comparisons between LDso's of
insecticides or treatment, tests for parallelism Of the regression lines were
necessary. Also data did not always fit the sigmoid curve and care was taken
in the interpretation of the results, particularly with heterogeneous
populations. I

Carbofuran and DDT are quite insoluble in acetone when using large
amounts Of materials. This can result in some difficulty in the use Of topical
application in the laboratory. These compounds tended to crystallize on the
surface Of CPB when high doses were used. Such crystallization had a -
negative influence in the resulting toxicity since it reduced it. Thus any
resistance mechanisms that necessitate a large dose of these compounds are
likely to cause a disproportional increase in the LDso's (Brown et al. 1971). In
order to compare our results with the ones already published for carbofuran
and DDT, we decided to use acetone. We found the limit Of solubility for
carbofuran to be about 50 ug/ul and for DDT, 1'64 ug/ul. TO achieve
application Of larger doses for highly resistant strains a second drop was
applied to the same spot.

When resistance was evolving, the R gene conferring that resistance

was still at a low frequency of less than 10%. The LDso's of a field collected

14

sample could be similar to that Of a susceptible strain. The form of the dose

response curve was then the best indicator Of resistance.

Qeteett'on of Reeietanee going the LTso'e Voloee {lethal time)

The diagnostic dose, LT50, also was used when insufficient samples
were available to calculate a probit line and magnitude of resistance. In
testing for resistance, it was sometimes convenient to expose samples of
insects for different periods to the same concentration of insecticide, rather
than to different concentrations for a standard time. This procedure is
justified when it can be shown that the dose acquired by the insect is directly
proportional to the exposure time. Concentration x time is a constant (Wood
1981). For most Of the testing of unknown populations we gave a high dose
for each insecticide for estimation Of LTso's. Mortality was recorded in time
intervals such as 1/2 h, 1 h, 3 h, 9 h and 1 day. The applied dose for
azinphosmethyl was 200 ug/ beetle, the applied dose for permethrin was 20
ug/ beetle, and the applied dose for Carbofuran was 125 ug/ beetle. This
technique worked reasonably well and gave satisfactory results.

Before a final bioassay was conducted with an unknown population
and before setting the proper range of doses that were going to be used,
computation of LTso's with the above method gave a quick estimation Of the
level of resistance Of the population. Several tests were run with well knOwn
susceptible and resistant strains; therefore, standard values were established

for comparisons.

15

Observations on the type Of response Of insects after the topical
application with different insecticides (paralysis, motionless, recovery, very
active movement and lack of incoordination for certain time periods), gave
us some information on the mode Of action Of the poison (Matsumura 1985).
In addition, we Obtained information from the speed Of action Of the
insecticides. For example, toxicity Of azinphosmethyl was expressed after two
days Of application, while carbofuran killed extremely fast (within two hours).
Permethrin paralyzed the beetles in 20 minutes and there was good recovery
after 5 days when low doses Of less than 5 ug/ beetle were used. Beetles with

the Kdr resistant factor responded differently.

Selection for Insecticide Resistance

Iruin

Brown and Payne (1988) pointed out in the conclusion of their
excellent review paper Experimental Selection for Insecticide Resistance that
"a review Of the literature indicates that selection for insecticide resistance in
the laboratory has provided useful information about the levels of resistance
and the rates at which resistance to various insecticides has developed". In
the same study, the authors reviewed all the selection studies with the genera

Aedes Culex, Musca, and Heliothis and concluded that the rates at which

 

 

resistance development occurred were more rapid for DDT and permethrin

than the rates used for malathion, parathion, and other acetylcholinesterase

inhibitors.

16

It is now recognized that resistance is a preadaptive phenomenon and
that the insecticide acts as a powerful agent tO select those members Of the
populations that, for one reason or another, have the ability to survive
exposure. When the selection pressure is maintained over several
generations, substantial levels of resistance are Often rapidly attained (Crow
1960, Brown et a1. 1971). The idea that resistance arises from natural variation
does not preclude the occurrence Of new mutations after the insecticide has
been applied. There seems to be no reason to doubt that resistance mutations
occur naturally, from time to time, at the normal rate of 1--2x10'5 gametes or
less (Wood et a1. 1981).

Selection studies on the common green lacewing predator with
carbaryl, showed that larvae collected from widely separated areas of
California had different levels Of tolerance tO six insecticides. The most
tolerant larvae selected with carbaryl, exhibited more than 80% survival after
four generations, in contrast to three percent survival for the unselected
strain (Grafton-CardWell et al. 1986).

Selection for resistance to methyl parathion for 21 generations on a
field-collected strain of tobacco budworm, resulted in 31-fold greater resistance
to the insecticide than with an unselected strain. An estimate of heritability
of resistance indicated that 78% Of the variation was due to additive gene
action. The authors started the test with the progeny Of 20 insects that were
collected in the larval stage (Wolfenbarger et al. 1982). In another study on
selection for permethrin resistance in the tobacco budworm, approximately
500 larvae were collected from cotton fields. After 11 generations Of
continuous selection pressure the LDso increased 37—fold and also cross-
resistance to cypermethrin developed (Jensen 1984). Resistances of 33- and 27-

fold to fenvalerate and deltamethrin respectively, have been induced in

17

larvae Of the Egyptian cotton leafworm Spodoptera littoralis (Boyd) after
selection pressure for 23 successive generations (Riskallah 1983).

Most Of the kinds Of resistance developed under laboratory selection
pressure are probably special cases because what is not known in such cases is
the relative survivability Of these genotypes under field conditions.

Studies Of selection experiments indicate that it is preferable to begin
with large samples Of insects, gathered from several locations, increasing the
likelihood that genes for resistance are present in the initial selection (Roush
and McKenzie 1987, Brown and Payne 1988).

A major problem with selection studies is that the selection programs
have not had access to major resistance genes. The solution is to modify our
selection procedures generating resistant mutations or attempting the first
selection in the field, collecting the surviving ones and continuing the
selection in the lab. We applied the last technique with great success in our
studies, producing unique resistant strains which were utilized further in

synergistic, biochemical, and genetic experiments.
MW

The Colorado potato beetle is suitable for selection studies for several

reasons:
0 Techniques for rearing and maintaining lab cultures have been
developed, therefore, they are easy to rear.
0 CPB do very well under lab conditions.
0 CPB have developed resistance to all available insecticides.
0 CPB have a well-documented history of resistance in the fields.
0 CPB have a short life cycle. Under our lab conditions their life

span was 40 days and 7-8 generations per year could be obtained.

18

WW

Approximately 4000 individuals were collected from volunteer
potatoes covering a large area near Vestaburg, Michigan. Both larvae and
adults were collected from several locations at various times and mixed to
represent the range Of genetic potential Of natural populations. The total
number Of collected beetles was subdivided into four groups and selections

have been started at 80% mortality levels as follows:

Sub-population selection with azinphosmethyl
Sub-population selection with carbofuran

Sub-population selection with permethrin

IkOJNt-l

Sub—population unselected - control

Selection was applied only to adults at a level of 70-80% mortality.
Selection was applied first in two groups of beetles keeping a control batch,
just in case the selected group had died, to insure the continuation of the
interbreeding. Attempts were made for insecticides to be applied before
mating started, because if we delivered insecticides after mating had occurred,
susceptible sperm could be protected inside resistant females and survive to
the next generation.

Selection was conducted over successive generations by topical
application Of technical material of the insecticides in acetone solution. Pure
material must be used because some insecticides are themselves mildly

mutagenic and solvents and impurities as well may also increase mutation

rates (W 00d 1981).

19

Development Of resistance was monitored by calculating a dose-
mortality regression (probit analysis) in alternate generations. Details Of
application techniques and interpretation Of results have been given

previously.

li Pr eurB

Selection was started in the field and continued in the laboratory. We
began two selections of this type with carbofuran and permethrin. Details of
these selections are given in the following chapters (the selected populations
are named Montcalm-C and Montcalm-R). The results were impressive. The
advantage Of procedure B was that the initial selection was directed on a large
population, with a wider gene pool, therefore, resistant genes were more
likely to have been included in the selection.

It was useful to make comparisons between the two selection
procedures. The change in resistance levels can be calculated if information is
available about the initial frequency of R and S genes in a given population,
the population size, frequency of inbreeding, strength Of selection pressure,
number Of generations, fitness of R and S genes, etc. (Plapp et al. 1979).

During the project, we got valuable information about the biology of
CPB under insecticide selection pressure. Attempts were made to assess the
fitness disadvantage that results from resistance. Since we managed to create
resistant strains to carbofuran and azinphosmethyl (see Chapters 4 and 5)
using the selection Procedure 8, we stopped the continuation Of selections
with the sub-populations 1 and 2 which had not given any indication of

increase of resistance after being selected six times. Genetic heterogeneity

20

present in source colonies was probably insufficient for a resistance potential
to be expressed.

When reasonable levels Of resistance were Obtained we conducted
cross-resistance experiments to certain insecticides in order tO study the cross-
resistance patterns (Table 8, Chapter 3). A specific cross-resistance pattern can
Often provide information on the resistance mechanisms involved with an
insecticide. It was therefore important in determining which alternative
insecticides were most suitable for the control of the resistant populations,

and to design appropriate protection management tactics.

Chapter 2

Patterns of Insecticide Resistance to Azinphosmethyl,
Carbofuran and Permethrin in the
Colorado Potato Beetle
(Coleoptera: Chrysomelidae).

21

ABSTRACT:

Twenty-four field-collected and five laboratory strains Of Colorado
potato beetle, Leptinotarsa decemlineata (Say) were tested for resistance tO the
insecticides azinphosmethyl (organophosphate), carbofuran (carbamate), and
permethrin (pyrethroid) by the dose-mortality response method. Toxicity
was assessed by topical application. Log dose-probits Of the bioassays indicated
that most populations were heterogeneous for resistance to at least one
insecticide with shallow regression lines and high variability. Populations
with high permethrin resistance tended to have low levels Of resistance to
azinphosmethyl and carbofuran and vice versa. There appeared to be three
main patterns Of resistance in the field. Pattern I: the resistance ratio was
high for permethrin but low for the other two insecticides. Pattern 11: high
levels Of resistance to azinphosmethyl and carbofuran and low tO permethrin.
Pattern III: beetles were resistant tO all three insecticides and with cross-
resistance to DDT. Some negatively correlated resistance between permethrin
and azinphosmethyl-carbofuran may exist. The Observed patterns and
underlying resistance mechanisms are important for insecticide resistance

management in CPB.

Key words: Insecta, Colorado potato beetle, Insecticide resistance.

 

23

The Colorado potato beetle (CPB) is a major agricultural pest, in part
because Of its demonstrated ability to rapidly develop resistance to every
insecticide that has been used for its control (Forgash 1980). The CPB has been
shown to be resistant to all five major classes of insecticides, DDT,
cyclodienes, organophosphates, carbamates, and most recently pyrethroids
(Forgash 1985, Harris and Turnbull 1986).

Populations Of CPB have developed resistance to all nationally
registered insecticides and there are no insecticides presently available that
effectively control CPB in many parts Of the northeastern United States
(Ferro 1985). It took 7 years for the beetles tO develop resistance to DDT, the
first synthetic insecticide against which it was selected, 5 years for resistance to
azinphosmethyl, 2 years for carbofuran resistance, and 2 years for pyrethroid
resistance (Georghiou 1986). In Long Island, NY, fenvalerate lost effectiveness
in 2 years' time with a reported 600-fold resistance level. Although
combining fenvalerate with the synergist piperonyl butoxide reestablished
control, this combination failed in 1 year (Forgash 1985).

It is obvious from numerous reports of control failures, extensive
bioassays and synergistic studies Of different field-collected populations that
CPB has developed resistance tO a variety of insecticides in Michigan (Grafius
1986, Grafius et al. 1987, 1988, Ioannidis and Grafius 1988). To conserve the
usefulness of existing insecticides, to be able to develop chemical use
strategies, and to design resistance management tactics, it was necessary first
to study existing cross-resistance patterns Of insecticides. We were able to
isolate and retain in culture some of these strains, representing various steps
and types of resistance development for further biochemical (Ahammad-

Sahib et al. 1989) and genetics of resistance studies and testing of new

insecticides.

24

The Objectives of this study were: 1) to assess the current status Of
insecticide resistance in the Colorado potato beetle in Michigan, and to
establish base-line data for resistance; 2) to study the relationship between the
commonly-used insecticides azinphosmethyl, carbofuran and permethrin,
comparing toxicity data (LD50's, dosage-mortality curves) and Characterizing

arising patterns Of resistance to these insecticides.
Materials and Methods

Chemicals: The insecticides chosen for the bioassays were some of the
most effective and commonly used materials for controlling potato beetles in
Michigan and other areas (Hofmaster and Waterfield 1961, Forgash 1980, 1985,
Harris and Sveg 1976, McClanahan 1975, McDonald 1976) and represent the
three main categories of commercial insecticides: azinphosmethyl, an
organophosphate; carbofuran, a carbamate; and permethrin, a synthetic
pyrethroid. The insecticides (technical compounds) were azinphosmethyl
(90% purity) provided by Mobay Chemical Corporation, Kansas City, MO; '
carbofuran (98%) and permethrin (94.3%) provided by FMC Corporation,
Princeton, NJ.

Test Beetles: Twenty-four populations were tested during 1987-1988.
Bioassays were undertaken either directly on the field collected beetles before
foliage sprays, or on the F1 colonized generation, when beetles were collected
after foliage sprays. Thus, the tested individuals were representative of the
population genotypes. Collected beetles were kept at least 2 days, to ensure
that they were in good condition, before tests were conducted. Beetles before
and after treatment were maintained in cages (50x50x70cm) with potted potato

plants (RH 50% :t 10, temperature 25 1 1C, photophase 16:8 L:D).

25

Knockdown effect and mortality are sensitive to changes in physiological
conditions of the insects and bioassays were usually conducted at the same
time of day (2-5 pm).

Topical Application of Insecticides: The standard bioassay technique
for Colorado potato beetle (FAO 1974) was slightly modified. Instead of using
C02 to immobilize the adults, they were held on the tip of a glass tube to
which a vacuum was applied (3.3 mm diam.) briefly during treatment. This
avoided any possible side effects of C02 and possible obscuring Of early
symptoms of knockdown effect for permethrin. Toxicity was assessed by
topical application Of 2 ul Of solutions of the pure insecticide in acetone on
the second abdominal sternite Of the adult, with a hand operated
microapplicator. Doses were selected, based on preliminary trials, to give a
range of 10-100% mortality. For each bioassay, 4-7 doses were used to
determine a dose-mortality curve. Beetles treated with acetone were used as
controls in all tests. Mortality values were corrected for natural mortality by
Abbott's formula (Abbott 1925). If control mortality was larger than 20%, we
repeated the bioassays (Busvine 1971). In almost all cases, natural mortality
was less than 10%. If large amounts of beetles were available, tests were
repeated twice and the results were pooled. Toxicity was estimated by log-
probit analysis (Finney 1971, SAS 1985).

Carbofuran was quite insoluble in acetone when large amounts were
needed and often crystalized on the surface of the beetles at high
concentrations (larger than 50 ug/ul). Thus, any resistance mechanism that
necessitates a large dose of this compound is likely to give an inflated
estimate of the LDso (Brown and Pal 1971). However, to compare our results
with data already published for carbofuran, acetone was still used as solvent.

To achieve application of larger doses to highly resistant strains a second 2 ul

26

drop was applied to the same area on the beetle as soon as the first drop was
dry.

Assessment of Mortality: Timing of the assessment of mortality is
extremely important. There are differences between insecticide groups in the
speed of action. For azinphosmethyl and carbofuran, maximum mortality
was Obtained in 3 days. For permethrin, more than one assessment of
mortality was needed to draw useful conclusions. Knockdown effect is
common and there may be recovery of most of the insects in some
populations. Therefore, mortality for permethrin was estimated in half an
hour for knockdown effect, in 3 days for dead beetles + "knocked-down"
beetles, and in 5 days for dead + recovered beetles (final mortality). Recovery
several days after pyrethroid treatment has also been Observed by Grafius
(unpubl) in the field. In this study we report LDso's for permethrin mortality
at 5 days.

Resistance Ratio (RR): As susceptible strain LDso's for estimation of
resistance ratio (R) (Observed LDso/susceptible LDso), we adopted the
reported LDso topical toxicities for susceptible adults from Northern Maine;
0.3 ug/beetle for azinphosmethyl, 0.25 ug/beetle for carbofuran and 0.39
ug/beetle for permethrin (Forgash 1985). Idaho adults had similar LDso's
(Johnson and Sandvol 1986) In our study, the most susceptible populations

tested also had similar LDso's.

Results

Eight populations were susceptible to all three insecticides (RR 5 6).
Most of the tested field populations were to some extent resistant to

permethrin, with resistance ratios up to 110-fold. Log-dosage mortality curves

27

of bioassays for the majority of the examined populations indicated that
populations were heterogeneous with shallow regression slopes and high
variability. The variability and the shallow slopes would suggest that the next
step would be the rapid development of homogeneous resistance if the
populations were selected intensely.

Control by the recently introduced synthetic pyrethroids, especially
permethrin, is unsatisfactory in many areas (Grafius 1986, Ioannidis and
Grafius 1988). Populations with high permethrin resistance tended to have
low levels of resistance to azinphosmethyl and carbofuran and vice versa.
There appear to be three main patterns of resistance in the field. These
patterns reflect the underlying resistance mechanisms. Our results and the
proposed patterns are also supported by analyzing data reported by other
investigators from different geographic areas and chronological periods. 1

Pattern I: The resistance ratios for these populations were moderate (10
to 20 fold) to high (20 fold or more) for permethrin but low. for
azinphosmethyl and carbofuran (Table 1). This tended to occur in fields
where permethrin was frequently used, or where aldicarb (a soil applied'
insecticide) was used. The possible cross-resistance of aldicarb and
permethrin has also been discussed by Forgash (1985). Harris and Sveg (1976)
also reported results which fit this pattern; no evidence of resistance to
azinphosmethyl and carbofuran in four CPB populations, but resistance to
DDT and endosulfan. At this time, permethrin had not been introduced into
1 control programs but the similarities and cross resistance between DDT and
permethrin are well known (Sawicki 1973, Gammon et a1. 1981, 1985, Scott
and Matsumura 1983, Scott et a1. 1986).

Pattern II: The Opposite of pattern I was also observed; high levels of

resistance to azinphosmethyl and carbofuran and relatively low (less than 10

28

fold) resistance to permethrin (Table 2). The mechanisms responsible for
resistance to azinphosmethyl and carbofuran seem to be quite specific because
the resistance did not extend to permethrin. Forgash (1980), Hare (1980), and
Harris and Sveg (1981) reported similar relationships for resistant populations
of Colorado potato beetle. The resistance ratio in New Jersey for
azinphosmethyl was 100, for carbofuran 400, and for permethrin 5 (Forgash
1980). Connecticut River Valley populations showed resistance ratios of
8,266 for azinphosmethyl, 22,508 for carbofuran and 2.58 for permethrin. In
Quebec, in tests where formulated products were applied to potato plants,
beetles were resistant to azinphosmethyl and carbofuran and again susceptible
to permethrin (Harris and Sveg 1981).

. Our results suggest that at least one resistance mechanism is common
for azinphosmethyl and carbofuran and that this mechanism is determined
by the same or closely linked genes. In laboratory selection studies with
carbofuran, the beetles developed 360-fold resistance to carbofuran and 43-fold
cross-resistance to azinphosmethyl (without azinphosmethyl selection) after
four selections. Genetic analysis of resistance of this strain showed this
resistance .mechanism to be incomplely dominant and monofactorial in
inheritance (Grafius et al. 1988). Boiteau et al. (1987) also reported a high level
of resistance to carbofuran with cross resistance to organophosphates and low
resistance to permethrin and aldicarb.

Pattern III: Resistance to all tested insecticides was present in areas
with heavy spray pressure, using mixtures of different groups of chemicals,
10-15 sprays per season, and little or no crop rotation (Table 3). These beetles
were resistant to all three insecticides tested and showed high cross-resistance
to DDT. This may be the consequence of one Of the resistance mechanisms of

permethrin, the Kdr or knockdown resistance factor (Ioannidis and Grafius

 

29

1988). Multiple resistance populations are now established in many parts of
the United States, for example, Long Island, NY; Michigan; Virginia (Forgash
1985, Grafius 1987, Zehnder 1986).

Discussion

By studying and comparing the resistance patterns of Colorado potato
beetle in Michigan and in other areas, one can Observe that the rate of
development of resistance is much more rapid for carbofuran than for
azinphosmethyl. Populations were always either highly susceptible or highly
resistant to carbofuran. On the other hand, resistance to azinphosmethyl
varied a lot with the tested beetles (5 to SOC-fold). The same situation has
happened with azinphosmethyl and carbofuran resistance in the green rice
leafhopper in Asia (Hamma 1983).

The main resistance mechanisms for permethrin in Colorado potato
beetles, based on synergistic studies, were mixed function oxidases (MFO's)
and Kdr factor (Ioannidis and Grafius 1988). Silcox in 1985 showed that
oxidative detoxification was only one of the factors involved in CPB
pyrethroid resistance because synergistic studies with piperonyl butoxide was
unable to entirely overcome CPB pyrethroid resistance. In Pattern I, there was
no cross-resistance between permethrin and azinphosmethyl or carbofuran.
The MFO's responsible for permethrin resistance therefore appear to be
different from those responsible for azinphosmethyl and carbofuran
resistance. Further biochemical studies support this (Ahammad-Sahib et al.
1990). It has demonstrated that a field resistant strain (Long Island) which had
been further selected with azinphosmethyl shows high resistance to

azinphosmethyl, high resistance to carbofuran, and low resistance to

30

permethrin (Table 2). Synergistic and biochemical analysis with this strain
shows that mixed function oxidases were the main mechanisms for resistance
to azinphosmethyl for this strain (Ahammad-Sahib et al. 1990). Rose and
Brindley in 1984 working with carbamate resistant CPB strains concluded a
resistance mechanism in addition to MFO's, which is not sensitive to
piperonyl butoxide synergism, may be involved in CPB resistance to
carbamates. No cross-resistance between pyrethroids and carbamates has been
Observed in resistant diamondback moths (Cheng 1988). Cheng assumed that
the MFO's responsible for carbamate resistance are not involved in
pyrethroid resistance. Genetic analysis in houseflies has shown that there are
at least two genes involved in resistance by increasing oxidation (Tate et al.
1974, Plapp and Casida 1969). A gene on chromosome 2 causes oxidation to
aldrin, carbamates, organophosphates, and pyrethrins. A gene on
chromosome 5 causes oxidation to DDT, DDE, and diazoxon (the oxon form
of diazinon). A factor causing pyrethroid resistance on this chromosome may
represent the same gene (Oppenoorth 1985).

Useful recommendations can be drawn for CPB management by
monitoring resistance. Pattern I suggests the failure of control with
permethrin and indicates that a switch to a carbamate or organophosphate
should be appropriate. Pattern II suggests the opposite. Because
azinphosmethyl resistance gives resistance to phosmet it is not useful to use
these two organophosphates in mixtures or sequences. Cross resistance
between azinphosmethyl and carbofuran also occurs, at least in the tested
populations. .

Some negatively correlated resistance between permethrin and
azinphosmethyl-carbofuran may exist, where selection Of resistance by

pressure from permethrin leads to a corresponding selection of susceptibility

31

to azinphosmethyl and carbofuran and vice versa. This speculation is
supported by the observed patterns I and II and by selection studies in our
laboratory. Negatively correlated cross-resistance to a synthetic pyrethroid in
organophosphorus-resistant Tetranychus urticae (Koch) has been clearly
demonstrated; increasing azinphosmethyl resistance confering an increase in
fenvalerate susceptibility (Chapman and Penman 1979). A similar negatively
correlated cross-resistance has been noted between malathion and fenvalerate
in the resistant brown planthoppers _l\_l. paparvata, I_\l_. lugens Stal and
Laodelphax striatellus Fallen, two Of the most destructive pests of rice in
Japan (Miyata and Saito 1984).

If the negative correlated resistance is clearly demonstrated, then an
alternate use of the insecticides can be an effective strategy. Keiding (1963)
referred to possible reversal of resistance when insecticides are substituted by
another proper insecticide particularly in the early stages of detection of the
resistance. He discussed the factors influencing reversal of resistance to DDT,
BHC and diazinon in the housefly on farms in Denmark. Also, in the case of
negatively correlated resistance, the mixtures used in sprays must not consist
of a pair of compounds that display negatively correlated toxicity (Georghiou
1983).

Future studies on the mechanisms and cross-resistance linkages for
CPB will be necessary for a more complete understanding of the processes Of
resistance and potential resistance management tactics. Application of this
information to the management of CPB populations before resistance reaches

crisis proportions will result in long-term gains in CPB control and reduced

pesticide usage.

32

Table 1. Pattern I. Dose response data for eight populations of Colorado
potato beetle with low resistance ratios for azinphosmethyl and
carbofuran, and moderate to high resistance ratios for permethrin.

 

 

Strains Number of Slope 3; LDso's 95% CL. Resistance ratio
beetles S.E. ug / beetle lower upper RR
tested (N)
Montcalm 1988
Azinphosmethyl 74 4.12 i 1.26 0.79 0.53 1.33 2.60
Carbofuran 36 2.15 1 0.96 0.40 0.15 1.08 1.60
Permethrin 68 1.96 :1; 0.39 3.25 2.15 4.90 10.83
Bakera
Azinphosmethyl 108 2.28 i: 0.40 1.13 0.77 1.66 3.76
Carbofuran 72 3.88 i 0.84 0.80 0.61 1.03 9.20
Permethrin 60 4.99 -_t-_ 1.08 16.10 13.0 19.97 41.28
Leep
Azinphosmethyl 84 3.80 i 1.31 2.04 1.55 2.68 6.80
Carbofuran 78 1.55 :t 0.28 2.30 1.31 4.04 3.20
Permethrin 78 0.94 1 0.34 6.47 3.16 13.24 16.58
TIM
Azinphosmethyl 82 --- <1.56 --- --- <5.20
Carbofuran 80 3.20 i 1.44 0.47 0.34 0.67 1.88
Permethrin 76 1.28 i 0.37 5.19 3.05 8.83 13.30
Vestaburg IIa
Azinphosmethyl 96 2.79 1; 0.50 1.7 1.30 2.17 5.70
Carbofuran 96 3.09 i 0.74 0.2 0.16 10.27 1.00
Permethrin 76 1.73 :t 0.39 10.2 5.36 18.65 26.10
TV
Azinphosmethyl 118 1.82 -_t-_ 0.29 2.98 2.30 3.90 9.90
Carbofuran 110 1.30 i 0.32 0.19 0.09 0.49 1.00
Permethrin 98 3.20 :t. 0.59 12.53 9.79 16.03 32.12
Monroe ,
Azinphosmethyl 80 1.70 3; 0.37 1.82 1 .19 2.78 6.30
Carbofuran 80 2.39 :t 0.45 0.76 0.51 1.14 3.00
Permethrin 90 0.75 .t 0.28 11.03 8.42 14.50 28.30
SLF
Azinphosmethyl 64 --- <1.56 -— -—- 1.00
Carbofuran 69 0.78 i 0.32 0.29 0.05 1.96 1.00
Permethrin 75 1.59 5;, 0.50 21.93 10.25 46.90 56.23

 

a These strains are further maintained in our laboratory and are under selection with

permethrin.

 

 

la

33

Table 2. Pattern II. Dose response data for populations Of Colorado potato
beetle showing high resistance to azinphosmethyl and carbofuran
and low to moderate resistance to permethrin.

 

Strains Number of Slope i LD50's 95% CL. Resistance ratio
beetles S.E. ug / beetle lower upper RR
tested (N)

 

Long Island, NY (lab strain)a

Azinphosmethyl 96 2.5 :t 1.00 743.20 321.0 1714.00 2477.0

Carbofuran 80 1.41 :1; 0.42 47.80 27.6 82.20 191.0

Permethrin 90 2.71 i 0.45 3.70 2.8 5.00 9.5
GR Macomb

Azinphosmethyl 84 1.90 i 0.37 12.07 6.7 18.30 40.2

Carbofuran 86 --- >200.70 ----b --- >400.0

Permethrin 98 2.14 i 0.35 3.95 2.7 5.70 10.1
BL Macomb

Azinphosmethyl 92 2.07 _-4_- 0.35 10.72 7.6 15.20 35.7

Carbofuran 96 0.89 i 0.50 1574.60 32.8 --- >2000.0

Permethrin 100 1.85 :1; 0.28 2.70 1.8 3.90 6.9

 

a Long Island strain was under selection with azinphosmethyl in our laboratory.

b Not possible to estimate because of very high resistance levels (see Methods).

 

 

 

34

 

 

Table 3. Pattern III. Dose response data for five populations of Colorado
potato beetle showing high resistance to all tested insecticides.
Strains Number of Slope i LD50's 95% CL Resistance ratio
beetles (N) S.E. per/ beetle lower upper RR
JP Macomb 1987
Azinphosmethyl 98 0.80 i 0.18 36.28 14.7 89.3 120.3
Carbofuran 70 0.64: 0.27 21.98 3.6 131.8 87.9
Permethrin 80 1.05: 2.68 8.40 4.1 17.3 21.5
JP Macomb 1988a
Azinphosmethyl 102 0.79 i: 0.79 272.90 64.5 1146.0 907.0
Carbofuran 80 --- >400.00 -— —- >1000.0
Permethrin 87 0.95 i 0.44 42.40 8.4 213.3 108.7
U.B.
Azinphosmethyl 64 1.14 i 035 4.02 1.8 9.2 13.4
Carbofuran 69 --- >100.00 ---b -— >400.0
Permethrin 75 1.49 i 0.50 17.53 8.8 35.5 45.0
Shoe
Azinphosmethyl 69 1.95 i 0.41 7.22 4.7 11.0 24.1
Carbofuran 80 --- >100.00 , — — >400.0
Permethrin 94 0.73 :t 0 0.51 13.90 2.8 70.5 35.6
Mayer
Azinphosmethyl 96 2.67 5; 0 0.43 12.60 8.7 16.6 42.0
Carbofuran 96 --- >100.00 — — >400.0
Permethrin 96‘ 1.43 i 0 0.42 23.14 11.0 47.5 59.4

 

a This strain is now maintained in our laboratory.

b Not possible to estimate because Of very high resistance levels (see Methods).

Chapter 3

Synergist Studies

35

SYNERGISTS

One approach to elucidate the physiological mechanisms Of insecticide
resistance involves the use of synergists. Synergists are also of practical

importance to the entomologist (Metcalf 1967):

a) in the more economical or efficient control of insects by a mixture,
b) in increasing the spectrum Of activity of an insecticide,

C) in restoring the activity Of an insecticide against resistant strains.

Synergists have potential uses in insecticide resistance management as
analytical tools, control of resistant populations, delay Of resistance
development, and preservation of natural enemies (Raffa et al. 1983). But as
they have pointed out, the most promising use of synergists are as research
tools. Partly because of a failure in the above assumptions, the theoretical
potential Of synergists has been a commercial disappointment until now,
with the exception of pyrethroid-synergist aerosols. The extensive
investigation of synergism and synergists has led to a much better
understanding Of the mechanisms of detoxification in insects, Of the basic
biochemical processes involved in insecticide resistance.

A synergist will be defined as any chemical which in itself is not toxic
to insects as dosage used, but when combined with an insecticide, greatly
enhances the toxicity of the insecticide. This, of course, differs from
ootentiation, which occurs when two toxic materials applied together elicit a

response greater than that expected from the sum of the individual toxicants

(Kuhr 1976).

36

37

When tolerance or resistance is mainly metabolic, the synergist may
play a useful role in overcoming resistance even temporarily without loading
the environment with extensive use of toxicants.

Wilkinson (1968) described the role of insecticide synergism in
resistance problems. He pointed out that it becomes immediately obvious
that insecticide synergists constitute a group of compounds with considerable
theoretical potential for the control of a large number of resistant strains of
insects.

Although considerable variation is observed in the relative potency of
the synergists with different insecticides and in different species and strains of
insects, some degree of synergism can be expected to occur with all insects
which depend on microsomal oxidation for their survival after insecticide
exposure (Wilkinson 1971). A great amount of useful information on the
toxicology of an insecticide chemical has been and can be gained by
appropriate investigations with synergists. '

Regarding insecticide synergists and their mode of action, the reviews
by Casida (1970) and Wilkinson (1971) are very detailed and comprehensive. -
The synergist studies conducted were of great practical value because most of
them were applied directly on field resistant strains.

The use of insecticide synergists fits with IPM (Integrated Pest
Management) because mortality of the beneficial and non-target species will
be lower and the insecticide rates needed for the control of the pest will be
reduced. We can also use synergists to control resistant populations to some
extent. .

The use of PB and DEF as synergists with carbamates,
organophosphates, and pyrethroids has been well established with many

insecticides and species (Plapp et a1. 1967, Ishaaya et al. 1983, Liu et a1. 1984, Dai

38

et al. 1984, Hemingway et al. 1984). PB and DEF are widely used to indicate
which type of detoxification mechanism is involved (Attia 1980, McCord 1987,
Prabhaker 1988).

From the logdose-mortality regression equation apart from the
estimation of synergistic ratio (SR) at 50% mortality level, we could estimate
the SR in 90% mortality for the purpose of field efficacy. The SR90 is very
important since in most cases, 90% mortality is required for acceptable control
in the field.

The high synergistic effect of PB with azinphosmethyl, which we found
consistently with several populations, may prove useful in controlling
resistant populations and reducing the rate of selection of resistance in the
field or decreasing the frequency of resistant genes already present in field

populations.

It
.-
O
L-
O
C
.0
O
O
O
O

. . C
‘1 '. 1.. {.3‘1 .‘1..- l“: .I

basil:

Research that has been conducted with synergists on CPB is almost
exclusively with piperonyl butoxide (PB). Studies with other groups of
synergists have not been reported (Table 1) and synergistic studies with
azinphosmethyl are completely absent.

Forgash (1985) showed that PB reached a maximum effect when
applied 2 hours prior to pyrethroid treatment. In the same studies, rotenone
was synergized very well by PB suggesting that PB had two effects, i.e., it
increased penetration of rotenone and interferred with detoxification. This
interference could be through inhibition of MFO. In addition, PB was also
quite effective in combination with aldicarb or oxamyl but slightly so with

carbaryl and carbofuran. This is true, but in our study we found very good

39

synergism of carbofuran by PB also in strains with MFO's increasing activity.
It is well known that target insensitivity is another main resistant
mechanism for carbofuran (see details in Chapter 4). Increasing the PB
treatment from 10 ug to 40 ug greatly increased the degree of synergism of
aldicarb (aldicarb alone, LD50 = 14.0 ug/ beetle, aldicarb + IOPB = 4.50, aldicarb +
40PB = 0.31). It seems that the ratio 1 : 4, toxin : synergist, is the optimal. The
results with carbofuran are in agreement with our findings (low synergism
with PB) in some cases. Rose (1985) also found low synergism with PB and
carbofuran.

In the following table we have summarized all the synergist studies

available in the literature on research conducted on the CPB:

40

Table 1. Synergism studies with Colorado potato beetle.

 

 

 

Author/Synergists Insecticides Results
R.L. Rose Carbofuran A. Resistance of the New Jersey beetles
(1985) Carbamates (larvae) was less dependent upon
the enzymes monoxygenases
Piperonyl L % S = 29%
Butoxide
B. Significant differences between
resistant and susceptible Colorado
potato beetle larvae were not
observed in the penetration of
[14C] carbaryl.
C.A. Silcox, Permethrin Larvae and adults
et al. (1985) Fenvalerate
Piperonyl Also conducted However oxidative detoxicates is only one
Butoxide field experiments of the factors involved in CPB

pyrethroid resistance because PB was
unable to entirely overcome CPB
pyrethrins resistance.

During the experiments the beetles
used were field collected with small
resistance.

 

 

 

Forgash (1980) Carbofuran Only slight synergism
Azinphosmethyl
Piperonyl Chlorfenviphol None
Butoxide Aldicarb
Lindate
Phosmet
Carbaryl
Harris (1986) Fenvalerate 2:1 S-fold More toxic with R-strain
" 8:1 lZ-fold More toxic with R-strain
Piperonyl " 8:1 1.6-fold More toxic with S—strain
Butoxide Deltametrin 8:1 2.5-fold More toxic with S-strain

 

 

8:1 30-fold More toxic with R-strain

 

 

41

Lise 9f Insecticide Smgrgists to Indicate Resistance Mechanisms

Research Approach and Procedures

Synergist studies were conducted to complete the already available
information and to determine whether metabolic resistance mechanisms
were involved using lab resistant strains and different field collected
populations. Raffa and Priester (1983) emphasized that the simplest use of
synergism in resistance management is direct application to resistant
populations.

Instead of testing extensively one strain with all the available
synergists, we considered it would be better to test the well known synergists
(PB, MGK 264, DEF, DEM, Chlorfenethol) with different populations collected
from places with different spray histories. Therefore, we were able to carry
out comparisons to better elucidate the results, and to figure out different
patterns of resistance mechanisms.

Synergists used were:

 

Synergists Inhibition of

Piperonyl Butoxide (PB) . mixed-function oxidases
MGK 264 mixed-function oxidases
DEF esterases

DEM gluthathione-S transferase
Chlorfenethol (DMC) DDT dehydrochlorinase

References to the above synergists are given in the following pages.
To produce better effect in CPB, it was necessary to apply the synergist

in advance of the insecticide (two-hour pretreatment). The synergistic ratio

42

(SR) was calculated as the LD50 of the insecticide alone over the LD50 of the
mixture. It was considered that SR > 1 means we had synergism, and SR < 1
antagonism.

Two methods for applying synergists were used:

Method A: The use of ratio 1 : 1 or 1 : 2 or 1 : 4 (ratio insecticide :

synergism for example):

1:1 1:2 1:4
2:2 2:4 2:8
4:4 4:8 4:16
8:8 8:16 8:32

In the control process the largest dose of synergist was applied.

Method B: Keeping the synergist constant. Applying the largest dose of
synergist (x) which gives less than 10% mortality (preliminary work for
estimation of x was necessary). Therefore, we designed experiments as

follows:

Control
Synergist x
Insecticide a + x
Insecticide 2a + x
Insecticide 4a + x

Insecticide 8a + x

Since the tolerance of synergists by the beetles depends on the level of

resistance (the more resistant the beetles, the more synergist they can tolerate)

43

we adopted both the methods but favor the insecticide : synergist ratio
technique which gives more accurate results, particularly with moderate
levels of resistance. We used method B with highly resistant populations,
because it was technically impossible to apply 2000 ug/ beetle with one drop of
2 ul when the level of resistance to an insecticide is 500 ug/ beetle. After the
insecticide-synergist combination yields a presumed mechanism, specific
biochemical experiments were needed to confirm the resistance mechanisms.
It is worthwhile to note that a solid agreement between synergistic studies in

vivo and biochemical analysis in vitro were obtained in the case of the

 

Colorado potato beetle. Details about the synergists, techniques, and

considerations are given in the following passage.

Piperonyl Butoxide (PB)

Methyllenedioxyphenyl group. Mixed-function oxidase inhibitors
(Brown 1971). Studies with synergists such as PB and Sesamex have now
become an established and widely used technique for indicating the in 17229.
importance of oxidative degradation.

The methyllenedioxyphenyl (MDP) compounds are the most
important synergists from the standpoint of historical development, current
use, and effectiveness (Casida 1970). The best established mixed-function
oxidase (MFO) inhibitors are the MDP compounds. Through the use of
synergistic ratio the effects of PB provide a fairly precise indication of the
relative contribution of oxidative metabolism to the observed toxicity.

Sesamex, an MDP compound, is more active than the PB but it is not
used commercially because, in the presence of sunlight and moisture, it

decomposes to yield objectionable colored products. Generally, a highly

potent cholinesterase inhibitor and toxic compound such as carbofuran is
poorly synergized by PB (Casida 1970, Metcalf 1967). This is probably the case

with susceptible populations.

MQK 2Q

Alkylamines and Amides group. Mixed-function oxidases inhibitor.

The inhibition of MFO activity is not restricted to MDP compounds, it
also includes alkylamine derivatives such as MGK 264, and SKS 525-A. MGK
264's main use is as an insecticide synergist for pyrethroids (Worthing 1979).
Recently, Cochran (1987) demonstrated that resistance to pyrethroids and
bendiocarb in the German cockroach can be overcome by the use of PB. or
MGK 264 as synergists. The results were rather similar in terms of their
ability to reduce resistance. The optimum ratio of synergists to bendiocarb
was 1 : 3 and for Pyrethrin 1 : 10 (insecticide: synergist).

SKF 525-A and MGK 264 inhibits the conversion of thiophosphoryl
into phosphoryl by mammalian microsomes and cockroach guts but in -

houseflies SKF 525-A synergized azinphosmethyl.

12E

DEF (S,S,S-tributylphosphorothionite) is a plant defoliant that inhibits
esterases and oxidases (Plapp 1986). Data with _13.integpuitella larvae indicates
that DEF is probably an esterase synergist as well as an oxidase synergist (Attia
et al. 1980). A

Synergism by DEF is normally considered as indicative of the

involvement of esterases and/or glutathione S transferases in insecticide

45

metabolism (Hemingway and Georgiou 1984). Dimethyl and triphenyl
phosphate synergists, like DEF, behave as competitive inhibitors and would
have to be applied in the ratio of at least 1 mol inhibitor to 1 mol synergist
(Fest 1982).

The highest synergistic activity of DEF was obtained when it was given
to mice 6 hr before treatment with malathion; certain metabolic
transformation might be involved in the manifestation of the activity (Eto
1979). Moreover, against certain resistant insect strains DEF synergized to
some extent paraoxon, azinphosmethyl, the carbamate insecticides, and DDT.

Because DEF is a defoliant, mixtures of this synergist and insecticides
cannot be used in the field to overcome resistance due to esterases. We found
populations of CPB resistant partly due to esterase activity. Probably of
practical use are the mixtures with sprayable esterase inhibitors, such as
kitazin P, a systemic fungicide used to control Piricularia ma—ein rice
(Worthing 1979). Inhibition of carboxyesterase by kitazin P was the main
factor responsible for synergism between malathion and kitazin P against the
green rice leafhopper (Ozaki 1983). Hemingway and Georgiou (1984) used
kitazin P in synergistic and selection studies with Culex guinguefasciatus.

 

DEM is an inhibitor of glutathione s-transferase but can inhibit both
mixed-function oxidases and esterases. It is unfortunate that there are no
specific inhibitors of glutathione S-transferases (G-S-T) that can be tested in

vivo like the more specific inhibitors of enzymes as PB, DEF, and

 

Chlorfenethol. Therefore, negative results in synergism with DEM does not

necessme mean absence of glutathion S-transferase action.

46

DEM inhibits both MFO and transferase and Welling et al. (1985)
suggested that only if commonly used inhibitors of the MFO, like Sesamex
and PB, fail to synergize does it seem indicated to try DEM and analogs. It also
seems that thiono compounds are less synergized than their oxon analogs.

The importance of G-S-T as a possible resistance mechanism seems to
have been neglected due to the fact that the products of its action on
organophosphorous (OP) compounds are identical to those formed by other
enzymes. G-S-T is probably of more importance in OP metabolism in the
dealkylation of dimethyl phosphates and phosphorothionates (Oppenoorth
1976).

Chlorfenethol (DMC)

The compound 4,4'-dichloro-a-methylbenzhydrol used to have the
trademark name Dimite. The new trademark name is Chlorfenethol. It is a
non-systemic acaricide with a pronounced ovicidal activity, for use on fruit,
vegetables, and cotton at 50-75 g./100L (Worthing 1979). It is reported to act as
a synergist for DDT against DDT-resistant insects (Sumerford et al. 1951). The
ratio used was a water emulsion of 5% DDT and 1% DMC. Metcalf (1967)
summarized the investigation about the synergist DMC and analogs as
inhibitors of DDT dehydrochlorinase enzyme which is one of the
mechanisms of resistance to DDT (Oppenoorth 1984). DMC proved to be a

most effective synergist in resistant houseflies and mosquitos.

47

Insecticide-Synergist Ratig

Another factor that should be weighed in order to obtain maximum
insecticide synergism was the relative quantity of each ingredient needed for
treatment. Usually, the higher the synergist-insecticide ratio, the greater the
effectiveness of the combination. Eventually a maximum ratio is reached
beyond which additional synergist no longer enhances toxicity. Generally,
routine screening of synergists with carbamates is 1 : 5 to 1 : 10 insecticide-
synergist ratio (Kuhr 1976).

With carbaryl the ratio 1 : 5 gives the best synergism. The difference
between 1 : 5 and 1 : 10 was very small, when applying topically 26 different
methyllenedioxyphenyl compounds on Musca domestica (Hewlett et a1.
1967). In other studies with carbamates, the ratio 1 : 5 was used (Moorefield
1960, Fukuto et al. 1962, Plapp et al. 1967). Brattsten and Metcalf in 1970 and
1973 made an extensive and careful evaluation of synergistic effects of carbaryl
with PB (ratio 1 : 5). They tested 74 insect species representing 40 families, and
eight orders. In general, Coleoptera species, especially chrysomelidae, showed
high susceptibility and low synergistic ratio. This information was very
valuable for conducting comparisons since CPB is in the family
chrysomelidae.

With carbamates, maximum. inhibition of the enzyme system by
methyllenedioxyphenyl synergists (PB, Sesamex, Sulfoxide) occurs at a

carbamate-synergist ratio of 1 : 2 to 1 : 5 (Metcalf et al. 1966).

48

P hri rim

The importance of insect esterases in pyrethroid metabolism is that
esterases are likely to be of greater relative significance in detoxifying the
trans-isomers and oxidases with the cis-isomers (Iao et al. 1974). Chang and
Whalon (1985) found that pyrethroid esterases were very sensitive to
inhibition by DEF in the resistant predator mite Amblyseius Luigi; and
esterases hydrolyzed trans-Permethrin two times faster than cis-Permethrin.
Casida and Ruzo (1980) concluded that synthetic pyrethroids with primary
alcohols (permethrin) were mostly cleaved by esterases that were sensitive to
inhibition by a variety of organophosphorous compounds while those with
secondary alcohols (with a cyano group like cypermethrin, deltamethrin,
fenvalerate) were only slowly attacked by esterases. Similar results were
found by Dai et al. (1984). A follow-up survey surprisingly indicated that
brown planthopper 'was highly resistant to permethrin with only low or
moderate resistance to a-cyano containing pyrethroids.

The optimal ratio of mixtures of PB with pyrethroids for the control of
Diamond-back moth were 1 : I to 1 : 5 insecticide: PB (Liu et al. 1984).

NPC (I-napthyl N-propyl carbamate) (which is another inhibitor of
esterases apart from the classical DEF) applied to milkweed bugs was
essentially the same when applied simultaneously, 8 hours, or 24 hours prior
to the pyrethroids.

In some cases, the synergist is much more effective when it is applied
several hours before the insecticide. From a research standpoint, this is the

best way to study synergists because competitive uptake is eliminated.

49
Mixed-Function Qxidasgs

Since age, sex, and stage of development play an important role in
enzyme activity and the levels of MFO change dramatically between larvae
molting, it had been decided to conduct most of the bioassays with adults 1
week old and, when possible, testing the sexes separately depending on the
available number of individuals. Wilkinson and Brattstein (1973) have
summarized investigations on factors affecting microsomal activity (MFO),
such as age, sex, stage, strain differences, effect of nutrition and diet, and
pointed out that the problem is exacerbated by the fact that the entire activity
may occur within days or even hours.

Ahmad and Forgash (1975), working with gypsy moth, clearly
demonstrated that the increase in tolerance to carbaryl and diazinon with
larvae growth, as well as increase in PB efficacy, correlates with increases in
mixed-function oxidases in advanced instars. Tests were conducted using a 1 :
5 insecticide : synergist ratio.

A reduction of the fat-body fraction occurring in certain stages results
also in a reduction of the activating oxidation mechanisms which are
localized in the fat-body in favor of the detoxifying hydrolysis mechanisms.
The application of insecticides must, therefore, be directed against particularly
susceptible stages in the development of a pest (Fest 1982).

Because the microsomal enzymes are also responsible for the oxidative
activation of the organophosphorothionate insecticides P = S -9 P = O,
synergists often show an antagonistic effect on the toxicity. In our studies,
azinphosmethyl, despite the fact that it is a phosphorothionate, was
demonstrated to be synergized very well by PB and we have not observed any

antagonistic effect.

50

There are also reports of synergism of phosphorothionates
(fenitrothion, diazinon) by PB with the resistant moth Plodia interpunitella
(Attia 1980). O'Brien (1960) tried to elucide this paradox further in his studies
with the synergist SKF 525-A, an inhibitor of MFO. He found that SKF 525-A
synergized Guthion and Diazinon and inversely had an antagonistic effect
with parathion. This apparent contradiction, the synergistic and antagonistic
action, is well demonstrated by Sun et a1. (1967) who conducted experiments
using Sesamex (MFO inhibitor) as a synergist and several
organophosphorothionates or chlorinated insecticides. They pointed out that
these apparent differences between different phosphorothionates may be due
to the degree of stability and rate of penetration of P —) S and P —> O analogs as
well as to possible toxicity of some P = 5 compounds without converting to P
= O analogs.

The most important oxidase for xenobiotic metabolism is Cytochrome
P-450, a component of the microsomal mixed-function oxidase system. This
is called Cytochrome P-450 because an intense absorption at 450 nm appears as
a reaction of the reduced pigment with carbon monoxide (Eto 1979).

Recently there have been several reports about the existence of
multiple forms of P-450's. Wilkinson (1983) suggest that these are under
separate genetic control. Ronis et al. (1988), working with an insecticide
resistant housefly, was able to characterize four different isozymes of P-450. It
is interesting that two forms of P-450 were found in Drosoghila, the P-450A ~
59 kAa which was widespread among strains, whereas P-450 B-56 kAa was
expressed only in strains resistant to insecticides. Levi et al. in 1988 reported
that fenitrothion yields the corresponding fenitroxon by oxidative
desulfuration, an activation reaction, and 4-nitro-m-cresol by oxidative

dearylation, a detoxification reaction. They also found that four P-450

51

isozymes can be induced by phenobarbital in mice, with different effects in the

production of the oxon form of the fenitrothion.

Results

First results are given from the resistant field strain of beetle (collected
from the Macomb Co. area) in 1987.

Resistance to azinphosmethyl was found to be 81—fold (Table 2). The
population was heterogeneous, containing both susceptible and resistant
individuals, as indicated by the high X2 value. With the addition of PB,
resistance was reduced approximately 15-fold, indicating mixed-function
oxidase activity. The resistance ratios were calculated by dividing the LD50 of
an insecticide by the LD50 of the insecticide plus synergist. The response of
the beetles to azinphosmethyl plus PBO was homogeneous, a more uniform
level of resistance, as indicated by the low X2 value. This suggests that the
main mechanism for resistance to azinphosmethyl is mixed-function—
oxidase.

Actual resistance levels to carbofuran could not be determined for the
Macomb IP population because levels of technical grade material could not be
applied in high enough amounts to cause mortality. LD50 values were
estimated as approximately 1000 fold higher (Table 2). The synergism of
carbofuran with both PB and DEF was low. This lack of synergist activity plus
other observations discussed below suggest a change in the target site of the
insecticide as the mode of resistance.

With permethrin, a fairly effective synergistic ratio was found with PB

(6.5 times) (Table 3). With addition of the synergist PB returns the highly

 

52

Table 2. Resistance and insecticide synergism of IP Macomb population of the
Colorado potato beetle to azinphosmethyl and carbofuran using topical
application bioassays.

 

 

 

LDSO Synergism x2
(ug/beetle) Ratio (df 2)

azinphosmethyl 24.3a -- 7.0"

+ PB 1.6 14.9 0.6

+ DEF 122 2.0 1.1

+diethyl maleate 223 1.1 4.9“
carbofuran >1ooo,oa -

+ PB 1931 >5.0 6.24"

+ DEF 422.8 >2.0 _ 0.67

 

a Susceptible LDso: carbofuran = 0.2ug; azinphosmethyl 1.25ug.

“ Significantly different; this value is higher than figure at X2 for the 5%
level.

53

Table 3. Resistance and insecticide synergism of JP Macomb population of
Colorado potato beetle to permethrin and DDT using topical application
bioassays.

 

 

LD50 Synergism x2
(pg/beetle) Ratio (df2)
permethrin 16.80a " 1330*"
+ PB 2.50 6.90 1.90
+ DEF 6.20 2.70 6.30“

 

DDT >1000.oa - 0-45
+ PB 253.0 -- 3.60
+ DMC 393,0 - 6.30“

 

a Susceptible LDso: DDT = 10.0 pg; Permethrin 0.39 pg.

“ Significantly different; these values are higher than figure of X2 for the 5%
level.

'm Highly significantly different; these values are higher than figure of X2 for
the 1% level.

heterogeneous responses with permethrin alone to a homogeneous response
(low X2 value), also suggesting the involvement of mixed-function oxidases.
However, resistance of Colorado potato beetle to permethrin cannot be
explained sufficiently by the detoxification mechanisms using PB and DEF.
Resistance levels to DDT are also high for this population (Table 3).
Therefore, a possible additional resistance mechanism to permethrin is the
kdr factor (knock-down resistance). The LD50 for DDT was larger than 1000
ug/ beetle (Table 3). Bioassays with DDT and PB indicated that the synergistic
ratio was about 3.9 and for DDT plus Chlorfenethol (DMC) it was 1.2 The
permethrin-DDT cross resistance and the low synergism with DDT leads us to
the conclusion that another possible mechanism for these two insecticides
was probably the kdr factor. This may help explain populations that in some
cases were tolerant to pyrethroids when these materials were first used’in the

field.

Cl'f 'Ill'

The conclusions drawn are based on several field strains tested in our
laboratory. With all tested strains, the resistance to azinphosmethyl can be
reversed significantly by applying piperonyl butoxide, strong evidence that
mixed-function oxidases are primarily responsible for resistance to
azinphosmethyl.

The field populations resistant to azinphosmethyl were also highly
resistant to carbofuran. In one case 85-fold resistance to azinphosmethyl was

accompanied by more than 300-fold resistance to carbofuran.

55

Table 4. Synergism of azinphosmethyl of GRM Colorado potato beetle strain
from piperonyl butoxide or DEF or DEM using topical application bioassays.

 

Insecticide- LD50 Synergism x2
Synergist (uglbeetle) . Ratioa (3 M)
azinphosmethyl 14.26b 6.30"

azinphosmethyl

+ PB 0.65 21.9 0.65
azinphosmethyl

+ DEF 4.45 3.2 6.34"
azinphosmethyl

+ DEM 8.18 1.8 9.14“

 

a Synergistic ratio LD50 insecticide/ LD50 insecticide synergist.
b Resistant ratio 47.5,.LD50 = 0.3 for susceptibles.

" Significantly different; these values are higher than figure of X2 for the 5%
level.

56

In another field collected population (GRM) with a moderate level of
resistance ratio of 47.5 to azinphosmethyl the synergism with PB was
extremely high, returning the toxicity of azinphosmethyl back to susceptible.
The synergism with DEF and DEM was very low without affecting the
heterogenicity of the original population (Table 4). These results indicated
that MFO's were the only resistance mechanism. Synergist studies with two
highly resistant strains to azinphosmethyl, the Long Island (RR=380) and
Macomb (RR=186) indicated that beside the MFO's, esterases must be
involved in the resistance to azinphosmethyl (Table 5). It is interesting that
application of both the synergists (2 minute time interval) two hours before
the application of azinphosmethyl gave an extremely high synergistic ratio of
132, reducing the toxicity almost to susceptible levels, but the synergists
produced 20% mortality to the control.

For carbofuran, we found low synergism with piperonyl butoxide. We
also tested another inhibitor of mixed-function oxidase II, MGK 264. We did
not find any synergism. The synergism by DEF was also low. These results
suggest that acetylcholinesterase insensitivity was mainly responsible for
resistance to carbofuran. Further synergistic studies revealed three possible
cases of resistance to carbofuran can exist (Table 6): First, biochemical analysis
with the Long Island strain did not indicate the mechanism of
acetylcholinesterase to be present, and the resistance ratio (RR) to carbofuran
was 224.9-fold. Applications of carbofuran plus piperonyl butoxide reduced
the R to 15.85 (Table 4, Chapter 4). This was evidence that MFO's were the
main resistance mechanism in this strain. Second, with the IP strain almost
immune to the carbofuran and resistant as well to azinphosmethyl, the
carbofuran synergism by piperonyl butoxide had an intermediate SR less than

5, in contrast to the first (SR=14.9). Biochemical analysis with this strain

57

suggested that both the resistance mechanisms were present, MFO's and
acetylcholinesterase insensitivity. Thirdly, in the MNT-C selected strain for
resistance to carbofuran (600-fold), the synergism of carbofuran by piperonyl
butoxide was almost zero. Biochemical analysis confirms that only the
acetylcholinesterase insensitivity mechanism was present (Table 4, Chapter 4).
Rose and Brinley (1985) working on carbamates resistance with Colorado
potato beetle, did not find significant differences between susceptible and

resistant beetles in the penetration rates with 14C labeled carbaryl.

58

Table 5. Toxicity and synergistic ratios of azinphosmethyl in mixtures with
piperonyl butoxide (PB) or DEF or DEM with a susceptible and two multiple
highly resistant strains of Colorado potato beetle (Long Island and Macomb).

 

Pesticide and]

 

or Synergist LDso 95% Synergist
CPB Strain (1:4 ratio) (ug/beetle) c.l. Ratio
Arizona azinphosmethyl
(AZ) 1.46 0.86 - -1.85 -
AZ + PB 0.32 0.40 - 0.24 4.56

Long Island azinphosmethyl 556.00 306.00 - 921.00 -

AZ + PB ’ 27.60 20.20 - 36.90 20.10

AZ + DEF 63.10 39.90 - 99.70 8.80

AZ + DEM 288.70 132.00 - 617.00 1.90

AZ + PB + DEF 4.20 0.34 - 6.20 132.00
Macomb AZ 272.20 64.5 - 1133.00 -
(GRM)

AZ + PB 16.50 9.40 - 25.30 16.50

AZ + DEF 19.50 14.00

AZ + DEM 36.60 25.70 - 51.40 7.40

59

The Baker strain possesses a high level of resistance to permethrin
while it is susceptible to azinphosmethyl. and carbofuran (Table 7). The
synergism of permethrin by piperonyl butoxide was 22.87-fold, a strong
indication that mixed-function oxidases are involved in permethrin
resistance, and esterases based on the fact that synergism by DEF was 10.73-
fold. The absence of cross resistance to other insecticides leads us to believe
that in Colorado potato beetle more than one type of P-450 occurs. It is very
interesting that Baker strain shows cross-resistance to aldicarb (Table 9). This
explains the observation in the fields of control failures of permethrin where
aldicarb was used as a soil treatment.

The possibility of the existence of different kinds of MFO's in three
Colorado potato beetle strains was also biochemically supported. The levels

of resistance were as follows:

 

Azinphosmethyl ' Permethrin

Long Island (RL) High Low
Macomb (GRM) High High
Baker (RB) Susceptible High

In all three strains, the rnicrosome N ADPH oxidation representing the
MFO activity was about 50 to 100% greater than susceptibles. However, in the
strains RL and GRM, the oxidation enzyme aminopyrene N-demethylase was
about 100% greater while P-nitroanisole o-demethylase was 40% higher than
for the susceptible Arizona strain. Interestingly in the Baker strain

aminopyrene N-demethylase did not show any change, while the P-

60

Table 6. Three different cases of resistance of the Colorado potato beetle to
carbofuran. Synergistic studies with piperonyl butoxide (PB) or DEF indicate
possible resistance mechanisms involved.

 

 

 

LD50 Synergistic Resistance

Strain ug/ beetle Ratioa Mechanism
Long Island

carbofuran 47.3 --

carborufan + PB 3.2 14.9 MFO's

+ DEF 30.0 1.6

MN T-C

carbofuran 120.9

carbofuran + PB no - Ache.

carbofuran + DEF no -
IP Macomb

carbofuran >400.0 -

carbofuran +PB 198.0 >5.0 MFO's 6: Ache.

 

a Synergistic ratio: LD50 insecticide/LDso insecticide + synergist.

61

Table 7. Toxicity of azinphosmethyl, carbofuran and permethrin on Baker
strain of Colorado potato beetle only resistant to permethrin. Synergist ratios
for mixtures of permethrin and piperonyl butoxide (PB) or DEF.

 

 

Synergist

Insecticide LD50 95% Ratio

Synergism (pg/beetle) C.L. RRal
permethrin 24.48 8.90 - 67.37 -
permethrin + PB 1.07 0.79 - 1.44 22.87
permethrin + DEF 2.28 1.73 - 2.90 10.73
azinphosmethyl 0.77 0.49- 1.21 --
carbofuran 0.80 0.61 - 1.03 -

 

RR = LD50 permethrin/LDso permethrin + synergism
LDso for susceptible 0.39 ug/ beetle.

62

nitroanisole o-demethylase showed about 100% greater activity in
comparison with the Collins Road strain (susceptible strain), suggesting that
the MFO's types in RL and GRM are different compared to RB. (Amammad
Sahib and Whalon, unpublished data).

Finally, it seems that Colorado potato beetles can use all the available
resistance mechanisms to resist insecticides (Table 8). Therefore, according to
the type of insecticide and to different field selection pressures, beetles can
develop one or multiple resistance mechanisms. Physiological and
biochemical studies have been conducted i_n vitro which satisfactorily support

the above results (Ahammad Sahid et al. 1990).

63

Table 8. Resistance levels and resistance mechanisms of different Colorado
potato beetle strains for the resistance to azinphosmethyl, carbofuran,
permethrin and DDT insecticides based on synergist studies.

 

 

LD50 Resistance
Strains Insecticides ug/beetle RRal Mech.
Long azinphosmethyl 556.00 449.00 MFO's, Esterase
Island carbofuran 47.50 237.00 MFO's
permethrin 3.70 9.00 Esterase
JP azinphosmethyl 272.20 217.00 MFO's,Ach-ase,Esterase
Macomb carbofuran >400.00 >1000.00 MFO's,Ach-ase
permethrin 42.00 108.00 MFO's-P,Esterases,kdr
DDT >1000.00 MFO's-P, kdr
MN T-C azinphosmethyl 11.43 9.14 Ach-ase
carbofuran 120.95 605.00 Ach-ase
permethrin 10.00 8.30
Baker azinphosmethyl 1.13 1.00
carbofuran 0.80 4.00
permethrin 16.10 41.00 MFO's-P, Esterase

 

64

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Chapter 4

Selection, Inheritance and Characterization

of Carbofuran Resistance

65

ABSTRACT:

The objective was to select for resistance to carbofuran in a susceptible
field population of Colorado potato beetles, Leptinotarsa decemlineata (Say),
and assess the progression of resistance and characterize the inheritance and
possible mechanism of resistance. Initialselection in the field resulted in
over 99.9% mortality of treated larvae but high resistance in the survivors.
Subsequent laboratory selections at ca. 80% mortality increased resistance to >
IOO-fold by the fourth generation. Resistance appeared to be inherited via a
single, autosomal, incompletely dominant gene, resulting in decreased
acetylcholinesterase sensitivity. Other Michigan populations tested also
appeared to exhibit reduced cholinesterase sensitivity. Resistance to
carbofuran in a population from Long Island NY appears to involve
primarily mixed-function oxidase enzymes. The history of insecticide use
and resistance development probably affects which primary resistance

mechanism appears in response to carbofuran treatment.

Key words: Insecta, Colorado potato beetle, insecticide resistance, carbofuran,

acetylcholinesterase

 

66

67

Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), was one
of the very first pests to exhibit resistance to DDT (1952) and has subsequently
developed resistance to all synthetic insecticides on Long Island, New Jersey,
and other areas of the northeastern United States (Gauthier et al. 1981).
Apparently because of cross-resistance, insecticides often fail within 1 or 2
years of introduction (Forgash 1981, Grafius 1986). Michigan may be on the
geographic edge of severe problems. Within Michigan, potato beetle
populations range from virtual non-pest status to highly resistant to all major
insecticide groups (Ioannidis & Grafius 1990). This presents the opportunity
to collect and study populations in many different stages of resistance
development and to observe the development of resistance in individual
populations over time.

Insecticides have been relied upon for control of CPB since their very
first introduction. Selection for resistance can be reduced with IPM
techniques (e.g. Wright et al. 1987), but no effective natural enemies act on the
Colorado potato beetle in commercial situations (Harcourt 1964) and cultural
practices are often not practical, at least in the short term. Thus, effective
insecticide resistance management is essential for continued economic
control of CPB. Strategies for resistance management are dependent for their
success upon accurate understandings of resistance mechanisms, the genetics
and inheritance of resistance and gene flow between populations. However,
there is little information available on the mechanisms and genetics of
resistance (Ioannidis 8: Grafius 1988, Ahamaad et a1. 1990) or Colorado potato
beetle gene flow and dispersal (Caprio & Grafius 1990).

Carbofuran, a carbamate insecticide, was chosen for study because it is
highly effective for controlling Colorado potato beetles under normal

situations (e.g., Grafius et al. 1988), creating intense selection pressure.

68

Insecticide resistance to carbofuran in CPB appears to develop unusually
quickly - often within one growing season (Gauthier et al. 1981). Thus, an
understanding of insecticide resistance management strategies seems
essential for this material. Information might also be applicable to
management of resistance for other insecticides and CPB or for other pest
species.

The objectives were to select for resistance to carbofuran in a
susceptible field population of Colorado potato beetles and: 1) assess the
progression of resistance and 2) characterize the inheritance and possible

mechanism of the resulting resistance.
Materials and Methods

CPB populations. The initial field selection for carbofuran resistance
was conducted in 1987 at the Michigan State University Montcalm County
Potato Research Farm (Entrican MI). The CPB at the research farm were
susceptible to carbofuran (LDso = 0.63 ug/beetle) and organophosphate
insecticides (e.g., LD50 for azinphosmethyl = 1.27 ug/ beetle) and moderately
resistant to pyrethroids (e.g., LD50 for permethrin = 6.86 ug/ beetle). Beetles at
this site had been exposed to a range of commercial and experimental
insecticides in previous years, particularly endosulfan (a chlorinated
hydrocarbon), methamidophos (an organophosphate), and the carbamates,
carbaryl and aldicarb. To the best of our knowledge, no carbofuran had been
sprayed on the research farm prior to the study. Small plot studies of
carbofuran as a soil insecticide had been conducted previously, but these

never involved more than 0.3% of the potato acreage.

69

The laboratory-susceptible culture was started with ca. 300 individuals
collected from unsprayed volunteer potatoes next to a commercial farm near
Vestaburg MI (Montcalm Co.) in the summer of 1987 and kept in continuous
laboratory culture. This was the most susceptible population to carbofuran of
29 field populations tested (unpublished data). The LD50 for the Vestaburg
strain to carbofuran was 0.18 ug/ beetle and 1.24 ug/ beetle to azinphosmethyl,
with a homozygous response to both compounds. These values are in
agreement with those for susceptible strains reported by Forgash (1985) and
Johnston and Sandvol (1986). The Vestaburg strain has been regularly tested
to ensure continued susceptibility to carbofuran and shows no loss of vigor
due to inbreeding.

Laboratory techniques. Technical grade carbofuran (98% purity,
courtesty of FMC Corporation) in acetone was used for all laboratory
selections and assays. Adults were immobilized using a 3.3 mm diam.
vacuum hose and 2 ul of solution was placed on the underside of the
abdomen of each adult. Mortality was assessed 72 h post-treatment. Fresh
potato leaves from greenhouse plants were provided to the beetles daily
during the testing. For LDso determinations, at least four doses of carbofuran
with 16 beetles per dose (2 replications of 8 each), plus an untreated group
(acetone only), were used. In the back-cross tests up to eight doses were used
in order to help recognize any distinct plateau (Tsucamoto 1963). Results
were corrected for mortality in the controls (acetone alone - usually less than
10% mortality) using Abbott's (1925) formula and standard log-probit analyses
were performed (SAS Institute 1982). All laboratory rearing and experiments
were conducted at 25'Ci2'C, 16 h light : 8 h dark photophase. Adults were

tested ca. 1 week after emergence from pupation.

70

Field selection. Selection for carbofuran resistance was initiated in the
field on 16 July 1987. Carbofuran was applied at a 16 row x 168 m plot of
potatoes at 0.55 kg active ingredient per hectare using a commercial ground
insecticide sprayer. CPB was in the first generation larval stage at the time of
treatment and the pre-treatment density was estimated as 37 i 10 (mean :1: SE)
larvae per plant based on data from adjacent untreated plots (unpublished
data). The total number of CPB larvae treated in the field was approximately
488,400 (37 x 13,200 plants estimated in the plot). After treatment (24h) the
plot was searched visually for surviving larvae. All surviving larvae were
collected and returned to the laboratory.

Laboratory selection. A sample of the field-collected larvae was tested
for carbofuran resistance using a dose of 5 ug/ beetle (all survived.) The rest of
the larvae were fed untreated potato foliage and reared to the adult stage in
the laboratory. Progeny were reared to the adult stage and tested for LDso for
carbofuran as described above. The adults that survived the two highest
doses (12.5 and 2.5 ug/ beetle) were kept in culture. Progeny were reared to the
adult stage and a sample was again tested for LDso to carbofuran. The
untested adults were selected with carbofuran to cause ca. 80% mortality. This
process was repeated on the next 2 generations. These beetles were designated
as the "Montcalm-C" strain and maintained in culture and subjected to a dose
of 100 ug carbofuran/ beetle every generation to assure maintenance of
resistance.

Resistance mechanism studies. The synergists piperonyl butoxide
(PBO) and s,s,s,tributylphosphorothionate (DEF), a plant defoliant, were used
as indicators of possible resistance mechanisms. PB is a specific inhibitor of
mixed-function oxidase enzymes (MFO's) (Brown 1971, Casida 1970). DEF
inhibits esterases and MFO's (Plapp 1986). Synergists were applied at 100

71

ug/ beetle to Montcalm-C or 50 ug/ beetle to susceptible beetles, 2 h prior to
insecticide treatment of the beetles. 100 ug of PB caused no mortality to the
Montcalm-C beetles but more than 20% mortality to susceptible beetles. 50 ug
caused <10% mortality to susceptible beetles. DEF caused no mortality.

Resistance ratios with and without synergist were calculated from LDso
values for resistant and susceptible strains. Susceptibility of the Montcalm-C
strain to azinphosmethyl was also estimated using the standard LD50 method
and results were compared to LD50 values for the unselected (field-collected)
Montcalm Farm population.

Inheritance studies. Adults from the Montcalm-C and the Vestaburg
strains were sexed within 48 h after emerging from pupation. Male/ female
reciprocal crosses were conducted. Pairs were kept together until they began
mating (to insure that no male/ female errors had been made) and then
grouped with other pairs of the same cross in cages and fed potato foliage.
Adult offspring (F1 females and males) from the reciprocal crosses were tested
separately for estimation of log-probit lines. A subsample of the F1 adults
were treated with a discriminating dose (6.25 ug/ beetle) to be sure that there
were no accidents involving susceptibles. The survivors (all of them
resistant heterozygotes) were back-crossed (B1) to the susceptible strains and
adult progeny were tested for segregation of genotypes using both the
technique determination of dose-mortality lines for appearance of a distinct
plateau and with the discriminating dose technique, as before, and the ratio of
susceptibles to resistant beetles was calculated. (Tsucamoto 1963, Georgiou
1965, 1969, Hama and Iwata 1978). This process was continued for studying
the second backcross (B2) to Vestaburg males. Backcrossing was continued
with another susceptible strain (Arizona) to further confirm the segregation

of the genotypes and the monofactorial type of inheritance of this resistant

72

mechanism. The F2 adults (progeny of F1 x F1 crosses) were also tested.
Results. Initial field selection resulted in survival of ca. 80 larvae
(>99.90% mortality). Survivors occurred in groups of 5 to 10 larvae on a
plant, rather than as individual larvae, as might have occurred from random
effects of factors such as spray coverage. Analysis of adults developing from
the surviving larvae indicated that they were approximately 52 times more
resistant to carbofuran than the initial population (Table 1). Subsequent
laboratory selection increased the resistance ratio to 175 fold, within four
generations. Reports from commercial potato fields and on-farm resistance
monitoring also support the rapid change from carbofuran susceptible to
highly resistant, often within one growing season (Grafius et al. 1988).
Inheritance studies indicated that F1 offspring of resistant x susceptible
crosses were uniformly resistant. Resistance levels were nearly as high as
parental levels and showed no significant difference between sexes,
suggesting near complete dominance and autosomal inheritance (Fig. 1). The
degree of dominance ( A ) was estimated using the formula by (Stone 1968).

Values of A are presented in (Table 2).

A: 2X2-X1-X3
Xl-Xs

X1 ,X2 ,X3 are the log1o of the LDso's of:

X1 = Resistant,
X2 ' = Heterogenous,
X3 - Susceptible.

 

73

    

 

 

 

 

A.
6 ‘ Ves berg
F1 Females I ,I
'I
3
Ti
‘5
5 -
E
3 F1 Males ’
2 e'
n. / Montcalm-c
4 . .., . . ....., . ...., - f......, . “44....
.1 1 10 100 1000 10000
Dose (jig/MIG)
BI
5-
Vutaherg
.>.
i
‘6
2 5-1
73 F1 Illlu
E -
Montcalm-c
4 . ..., . . ....... . ......., .1nfiq
.1 1 10 100 1000
Done (pg/beetle)
Figure 1. Dose-mortality responses to carbofuran of adults of susceptible

(Vestaberg), and resistant (Montcalm-C) strains of Colorado potato
beetle and their F1 offspring, as a result of topical application bioassays.
A. Vestaberg males (ss) x Montcalm-C females (RR). B. Vestaberg
females (ss) x Montcalm-C males (RR).

74

The values of A can be:

A = 1 complete dominance
0<A<1 incomplete dominance
A = 0 intermediate
-1<A<0 incomplete recessivity
A = -1 complete recessivity

Values of 0.55 to 0.69 for A indicate incomplete dominance (Table 2).

F1 backcrosses to susceptible beetles and F1 x F1 crosses showed nearly
perfect Mendelian segregation, indicating a single gene or closely linked genes
(Figure 2, 3 and Table 3). The level of resistance to carbofuran of the MNT-C
strain was very high and ranges of the dosage-mortality curve for susceptibles
and resistant strain did not overlap (Figure 1). Therefore, it was possible to
draw effective discriminating doses between ($5) and (RR), (RS).

Cross-resistance and synergist experiments showed moderate cross-
resistance to azinphosmethyl and almost no synergism for carbofuran or
azinphosmethyl with either PB or DEF (Table 4). Lack of synergism and the
absence of low and intermediate levels of resistance in the Montcalm-C strain
or other populations from commercial fields in Michigan (Ioannidis and
Grafius 1989) suggests target site insensitivity. The extremely rapid mortality
of susceptible'beetles in response to carbofuran treatment (100% mortality in
<2 h) also suggests that detoxification enzymes would be of limited usefulness

unless they were present at very high levels. Preliminary data from in vitro

 

studies on the Montcalm-C strain and on another Michigan carbofuran-
resistant population showed altered acetylcholinesterase activity (Weirenga
and Hollingworth, unpublished data). It is interesting that this acetyl-
cholinesterase related resistant mechanism to carbofuran gives no cross
resistance to aldicarb (LDso = 2.36 pig/beetle). Aldicarb is an aliphatic

carbamate compared with carbofuran, an aromatic carbamate (Kuhr 1976).

75

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76

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Toxicity of aldicarb for the susceptible Vestaburg strain was LD50 =1.97
ug/ beetle.

The low levels of cross resistance to azinphosmethyl (Table 1) suggests
alteration of the target enzyme. If this acetylcholinesterase site insensitivity is
mainly a qualitative change, it should be possible to search for compounds
which will be more toxic or only toxic to resistant beetles and not to the
susceptible CPB or beneficial insects, like the green rice leafhopper where
alteration of the target enzyme is the main factor for resistance to carbamates
(Hama 1983). Alternation between compounds affecting different
acetylcholinesterase types could be an excellent control technique for this
specific kind of resistance.

Acetylcholinesterase insensitivity has been documented for several
other species (Devonshire and Moore 1984, Oppenoorth 1985). As in our case,
acetylcholinesterase insensitivity reported for carbamate resistance in the
green rice leafhopper, is mainly controlled by an incompletely dominant
autosomally inherited factor (Hama and Iwato 1978).

In contrast to the Michigan populations studied, a population from
Long Island NY, selected for azinphosmethyl resistance in our laboratory,
showed significant synergism of carbofuran by PB and a lower level of
resistance, probably indicating detoxification as the primary mechanism of
resistance (Table 4). 111171112 studies with this strain did not show any altered
acetylcholinesterase activity, but high P-450 oxidation activity (Ahamaad et al.
1990).

Historically, carbofuran was only used very late in the resistance
history on Long Island, after failure of numerous organophosphates and
carbamates (Forgash 1981). In Michigan, and specifically at the Montcalm site,

carbofuran resistance was selected much earlier in the resistance history. We

78

hypothesize that this difference in resistance history explains the different
mechanisms that have manifested themselves. At the time of selection,
detoxification enzymes in the Montcalm population were probably not near
the very high levels required to detoxify the rapidly-acting carbofuran, thus
leading to selection for target-site insensitivity.

Management of carbofuran resistance in CPB appears to be a difficult
proposition. The single gene dominant nature of the inheritance and high
selection pressure exerted by carbofuran treatment has resulted in the
occurrence of exceptionally rapid resistance. Resistant populations are, for all
practical purposes, immune to carbofuran treatment. The resistance factor
appears to be present at very low frequencies in many or perhaps all Michigan
populations, judging from the widespread and rapid buildup of resistance in
the field (Ioannidis 8: Grafius 1989). Rapid buildup of resistance to
insecticides is characteristic of mono-factorial inheritance (Wood and Bishop
.1981; Roush and Croft 1986). We do not know of any cases of continued
carbofuran use in commercial fields that have not resulted in rapid resistance.
Limited use of carbofuran (<once per CPB generation) and alternation of
carbofurantreatment with non-cross resistance materials (e.g., permethrin,
where effective, or aldicarb), along with non-chemical management tactics,

may increase the effective life of carbofuran in the field.

79

Table 1. Resistance of the "Montcalm-C" strain of CPB to carbofuran over four
generations of selection, beginning with selection of larvae in the field.

 

 

 

 

 

 

 

 

carbofuran azinphosmethyl1
Degree of Degree of

Selecting agent Gen. IDso heterogenity LDso heterogenity
carbofuran tested ug/beetle x2 (Af) [lg/beetle x2 (M)
before selection P 0.63 0.019(3) 1.27 3.9(2)
Selection (in field) F1 46.51 10.3 (6) 11.21 6.1(3)
99.9% level-larvae
Selection (in lab) F2 93.87 4.68(2) 9.10 83(3)"
70-80% level-adults
Selection (in lab) F3 120.95 352(2) 18.54 67(2)"
70-80% level-adults
Selection (in lab) F7 >100.00 — 11.43 1.1(2)
70—80% level-adults

 

 

 

 

 

 

1 Cross resistant to azinphosmethyl without selection.

“ Significantly different from expected (P<0.05).

 

80

Table 2. Responses to carbofuran of adults of Colorado potato beetle

from the homozygous susceptibles (SS), resistant (RR) parental strains,

and the F1 (58 x R) hybrids of the reciprocal crosses.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Degree
Confidence of
LDso Intervals Resistance domi-
Strain or cross Slope (SE) ug/beetle 95% Ratio 1 nance 2A
Vestaburg (55) 3.96 (0.81) 0.21 0.18 - 0.26 -
Arizona (55) 1.86 (0.47) 0.34 0.21- 0.55 -
MNT-C (RR) 1.39 (0.49) 120.95 49.20 - 297.90 575.90
F1 (550 x RRd' )Q 0.86 (0.36) 42.90 15.50 - 118.40 204.30 0.67
F1 (550 x RRd' )d' 0.52 (0.20) 29.30 8.00 - 107.90 139.50 0.55
F1 (550' x RRQ )d‘ 0.75 (0.21) 44.92 16.40 -122.80 213.90 0.69
F1 (586' x RRQ)Q 1.20 (0.23) 30.35 17.50 - 52.70 144.50 0.56

 

1 Resistance ratio determined by dividing the LD50 of the resistant strain by the
LD50 of the susceptible Vestaburg.

2 Degree of dominance formula Stone 1968 A = -1 complete recessiveness,
A = 0 intermediate, A = 1 complete dominance.

 

Table 3. Colorado potato beetle percentage mortality from carbofuran: Ratio
expected if resistance is monofactorial inherited and observed mortalities of

81

susceptibles (55) by applying discriminating doses.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Eprected Observed xi— ‘
Stages/Numbe Ratio Mort. 1 degree

Crosses Tested SS:RS:RR SS Indivds. of frdm,
FCToss to obtain F2

F1(vestad' x MNTCaQ» x F1 Larvae 98‘’1 1 :2: 1 23.5% 0.12
F: (vestaO‘ x MNTC‘Q) x F: Females 109a 1 :2: 1 23.8% 0.80
F1 (vestaO‘ x MNTCaQ) x F1 Males 75a 1 :2: 1 32.0% 1.97
1‘1 (“590' X MNTCW) X F1 Adult mix 160b 1 :2: 1 24.4% 0.03
Backcrosses series 1

B1: (vestQ x MNTd‘ )x vestO' Larvae 146a 1 : 1 :0 48.7% 0.11
B1: (vestQ x MNTd') x vestd' Females 95a 1 :1 :0 50.6% 0.01
B1: (vestQ x MNTd' )xvestd‘ Males 96a 1 :1 :0 48.9% 0.04
B2: B1 x vestd‘ Larvae 643 1 : 1 :0 62.5% 400'
B2: B1 x vestd' Females 80al 1 :1 :0 48.7% 0.05
B2: B1 x vestO' Males 123a 1 : 1 :0 43.9% 1.82
B3: 82 x ARad' Adult mix 54b 1 : 1 :0 64.8% 474'
B4: B3 x ARO‘ Adult mix 72a 1 :1 :0 52.8% 0.27
B5: B4 x ARd' Adult mix 84b 1 : 1 :0 44.0% 1.19
fickcrosses series 2

B1 (vestQ) x MNTO’) X vestd'

32 31x vest O) Larvae 64a 1 :1 :0 53.1% 0.25
32 BI x vest 0) Female 80a 1 :1 :0 52.5% 0.20
32 31 x vest 0) Males 803 1 :1 :0 55.0% 0.80
Backcrosses series 3 g

81 (vestO’ x MNT O) x vest 0 Adult mix 80$ 1 : 1 :0 47.5% 0.20
Bl (vestd' x MNT O) x vest (5‘ Adult mix 66b I 1 ; 1 :o I 42.4% l 1,51

 

 

‘ Values of X2 smaller than 3.84 (probability 0.05) considered to represent
satisfactory agreement between observed and expected results.

a Topical application bioassays (pooled results of all the doses within the

discriminating range, killing the 55).

b Segregation of susceptibles applying a distinct discriminating dose 3.12 [lg/beetle.

82

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Chapter 5

Mechanisms and Genetics of

Azinphosmethyl Resistance

83

ABSTRACT:

Resistance to the organophosphate insecticide azinphosmethyl was
studied in the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say).
High levels of azinphosmethyl resistance appeared to be transmitted by
autosomal chromosomes. The mode of inheritance was incomplete in
dominance. Two main resistance factors were responsible for this level of
resistance: mixed-function oxidase enzymes (MFO's) and esterases. Esterase
enzymes were shown to have more activity in the Long Island resistant strain
than in Michigan beetles. Based on toxicity and synergism data with the
parental strains, the hybrids from the reciprocal crosses and on the offspring
of the backcrosses, it seemed that these two resistance factors were not simply
additive, but had synergistic action when contained in the same organism.
These beetles can exist just as well in the field. The rapid expansion of
resistance in the field and the fact that these highly resistant strains
performed very well under laboratory culture conditions indicate that there is
no reproductive disadvantage associated with them. The LDso of the
presumed AABB genotype was 556 pg azinphosmethyl/ beetle, for AAbb
(MFO's) 65 ug/ beetle, and for aaBB (esterases) 28 ug/ beetle. Therefore, the
synergistic action of these two resistance genes was six times more than the
additive result. Application of piperonyl butoxide as a synergist with
azinphosmethyl on the F1 hybrids partially suppressed the resistance while

also producing evidence of difactorial inheritance.

Key words: Insecta, Colorado potato beetle, insecticide resistance,

azinphosmethyl, genetics.

 

85

It is generally agreed that resistance to insecticides is controlled by
heredity and that transmission of the characters for resistance conforms to
Mendelian patterns (Crow 1960, Georgiou 1965, Tsukamoto 1983, Plapp 1986).
Resistance of the houseflies to either parathion or malathion is inherited
through single dominant gene pairs. The fact that the genes for parathion
resistance and those for malathion resistance lie on the same chromosome
indicates that these two types of gene are either alleles or closely linked (N guy

et al. 1960). Genetic studies on Dieldrin—resistance in Musca domestica L. and

 

M cuprina (Wied.) strongly indicate monofactorial inheritance (Guneidy
et a1. 1964). The same results have been found for Dieldrin-resistance in
houseflies by Georgiou et al. (1963). Houseflies are diploid organisms,
showing classical Mendelian inheritance characteristics as far as resistance is
concerned. Plapp (1983) pointed out that what is true for houselifes and other
higher Diptera in the way of resistance genetics is probably true for other
insects. That is, genetic mechanisms involved as probably ubiquitous and
rather specific. Malathion resistance in the Triboloium castaneum (Herbst) is
inherited as a simple autosomal semidominant trait (Beeman 1983). There
are also several reports on the inheritance of resistance, not only from the
well known genetic studies on flies of medical importance, but also on
agricultural pests and predators (Towgood and Brown 12962, Telford and
Brown 1964, Croft et al. 1976, Croft and Whalon 1983, Roush 1986).

Resistance to the organophosphate insecticide azinphosmethyl was
first reported in populations of the Colorado potato beetle (CPB) in 1971,
when azinphosmethyl started to show a decrease in activity in controlling
larvae in Long Island, NY farms. As a consequence, failure of
azinphosmethyl led to devastating losses in 1973 in Long Island, with the

beginning of resistance being reported in Virginia in 1974 (Gauthier et al.

86

1980). Topical toxicity of azinphosmethyl for a New Jersey population was 30
ug/ beetle, and application of piperonyl butoxide (PB) on this population did
not synergize azinphosmethyl (Forgash 1985). Indications are that for this
level of resistance, another resistance mechanism was responsible aside from
MFO's. Toxicity data from 24 different resistant populations in Michigan
indicated that resistance to azinphosmethyl can range from 5 to 600 ug/ beetle
(Ioannidis et al. 1990).

Synergistic and biochemical studies i_n vim and in Mg with a highly
homogeneously resistant strain to azinphosmethyl (LD50 = 556 ug/ beetle) and
with another Michigan field collected resistant strain to azinphosmethyl
(LDso - 358 ug/beetle) revealed that more than one resistance mechanism
contributed to this high level of azinphosmethyl resistance, due mainly to the
mixed function oxygenase (MFO's) and the esterases (Ahammad-Sahid et al.
1990).

Argentine et al. in 1989 reported that a strain of CPB from
Massachusetts was 435 times more resistant to azinphosmethyl than to a
susceptible strain. Based on synergist studies 11131119, they concluded that
esterases probably were involved in azinphosmethyl metabolism although
esterases could not be solely responsible for the observed resistance.

In our laboratory, the Long Island strain had been selected with
azinphosmethyl for six succeeding generations at 80% mortality level, before
being used in the crosses. This strain, after the selections, shows a
homogenous response, with steep log-dose mortality lines to the selecting
agent azinphosmethyl.

The objectives of this study were to: (1) investigate the mode of
inheritance of this high level of resistance of the Long Island strain, (2) to

determine whether the resistance to azinphosmethyl was carrying on

87

autosomal chromosomes and the existance of any kind of sex-linked
influence, and (3) to figure out from the backcross and F1 x F1 crosses how

many resistance factors were involved.

M rilnmhd

c" r'u

The insecticides used were technical grades of azinphosmethyl (90%
purity). The synergist, s,s,s,tributylphosphorotrithioate DEF (99%), was
provided by Mobay Corporation, Kansas City, MO. The piperonyl butoxide
(PB) (80%) was a gift of Mclaughlin Gormley King, Minneapolis, MN.

Bi -ma mn fri n

The successful identification of phenotypes depends to a large extent on
the proper choice of a sensitive bioassay method. We used a dose-response
test, the standard bioassay technique for CPB (FAO 1974). Some changes were ~
made in the method as a consequence of our experience with preliminary
work. The character being investigated cannot be seen, it can only be
recognized by challenging groups of beetles to a series of doses of insecticides,
i.e., quantal response methods. Conclusions on segregation of resistance in
progeny of various types of hybrids were made from data from dose-mortality
studies or by using distinct discriminating doses.

Treatments were conducted by t0pical application of 2 ug/ beetle on the
abdominal sternite. The insecticides were of technical grade and stock
solutions were made up in acetone. For the establishment of dose mortality

relationships, we used at least four doses. In the tests with backcrosses up to

88

nine concentrations were used because when the number of observed
mortality plots is not enough (for example only four to five plots), no distinct
plateau may be recognized even though the resistance is inherited in a simple

monofactorial system (Tsucamoto 1963).

Dosage-mortality lines for all parental F1, F2 and backcross populations
were determined by the probit analysis method of Finney (1971) using the
MSTAT computer program Eisensmith (1989). The chi-square (X2) value, a
measure of the departure of the actual observations from the fitted probit-log
dose line, was also calculated. The value of X2 was compared with tables for
this statistic for n-2 degrees of freedom (where n is the number of doses used
per bioassay). If the calculated value is found to be higher than the tabular X2
for the 5% level, this will be an indication of heterogeneity. '

To evaluate a genetic hypothesis, we need a test which can convert
deviations from expected values into the probability of such inequalities
occurring by chance. A particularly useful test is the chi-square (X2) method
when the probability is smaller than 0.05 (1 in 20), the difference between the
results observed and those expected is said to be significant (Snyder et al.

1985). The value of X2 is given by the formula:

x2 = (9m - ex1)2 + (Obz - exz)2
8X1 8X2

 

The above test was applied with the discriminating dose technique

which can separate two phenotypes: dead (susceptibles) and live (resistants).

89

In this formula, susceptibles refer to observed (Obl) and expected (exl)
whereas resistant refers to observed (Obz) and expected (exz).

The progeny from F2 and backcross generations is always
heterogeneous, with high x2 values because of the production of different
genotypes. Therefore straight log-probit lines are given only for the parental

and F1 hybrids.

E l'hmn fhmz ou ain
Vta ceil

This strain was collected from an unsprayed volunteer potato next to a
commercial farm near Vestaburg, MI. This has been interbreeding for 3 years
before the crosses started and its susceptibility has remained remarkably
stable. (LD50 = 1.25 ug azinphosmethyl/ adult and for larvae, 0.89). Dose-
mortality data has shown a straight line with a reasonably steep slope
indicating homogeneity (Table 1). Beetles were not affected by any

interbreeding depression.
L n Ila R i n

This field-collected strain was heterogeneously resistant to
azinphosmethyl when it was collected five years ago. After repeated selection
at 80% mortality and intercrossing for five generations, a resistant
homozygote had been produced. This gave rise to a steeper log-probit dose
line. This is clearly demonstrated by comparing the $10pes and X2 values in
the following regression equations. After the selections, b = 3.01 (steeper

slope) and x2 = 0.625 (very small), meaning a strong indication for

90

homogeneity was revealed. These beetles have been exposed to a dose of 200
pg azinphosmethyl/beetle for every generation to confirm maintenance of
resistance and homogeneity. The resistance ratio of this strain was 444-fold

and did not show any reproductive disadvantage.

Before selection

Heterogeneous y =1.80 + 1.18x, (x2 = 7.14 DP. 5) LD50 489 ug/ beetle

After selection

Homogenous (RR) y =-3.260 + 3.01x, (x2 = 0.625 DP. 5) LD50 556 ug/ beetle

Qgggfiggl techniques

Descriptions for these techniques and methods can be found in: Milani
1960, Georgiou 1965, Tsucamoto 1964, 1965, 1969, Plapp 1976, 1983.

The use of virgin females in crossing experiments is essential. The
maximum permissible period before the first emerged male reaches sexual
maturity depends on the temperature. Preliminary studies under our
laboratory conditions at 25' C show that the permissible period for separation
of sexes was less than 3 days after (emergence. For synchronization of the
emergence of adults we used the technique by Boiteau (1983). This facilitated
the bioassays and allowed testing beetles at approximately the same age, an
important intrinsic factor for accuracy and consistency.

Mass cross—mating (for all the experiments) was arranged by putting

together groups of about 20 males of one strain with the same number of

91

females of another strain. Beetles were maintained in the same way as has

been described in Chapter 1.

Rirlos

To determine the mode of resistance inheritance of azinphosmethyl,
reciprocal crosses were conducted between the Resistant and Susceptible
strains. Establishment of regression lines for the F-hybrids provided
information on the degree and type of resistance. The reciprocal crosses
were also used to determine whether resistance to azinphosmethyl was sex-
linked or autosomal. In the Colorado potato beetle the sex determination
mechanism is the xo type and the chromosome number is 2n = 34 + xo in the
males and 2n = 34 + xx in the females (Hsiao et al. 1983). In the males, the x
chromosome has no homologous pairing partner. Therefore, if any resistant
factor exists in the x chromosome of males, resistance occurs only to the
hybrid females from the cross SO x Rd .

For more confidence in the data, the interpretation of results was based
on the study of the complete dosage-mortality lines for the F2 and backcross
generations and, additionally, the discriminating dose technique was used to

confirm the results and separate the resistant phenotypes.

Wadi:

F1 progenies were backcrossed to the susceptible parent and dose-
mortality curves for the segregated phenotypes for offsprings were established
(Figure 2). Before each backcross, the F1 beetles were selected with a
discriminating dose for killing any accidentally involved susceptibles and

leaving the heterozygous resistant beetles alive.

92

Digeriminating dose technique

With the high resistance level and the incomplete dominance of the
F1's, this technique was convenient because effective discriminating doses
could be easily determined between the phenotypic susceptibles and the

resistants.

Results and Discussion

The high level of resistance is transmitted to the hybrids by either the
male or the female parent (Figure 1). Therefore, the resistance factors must be
located on the autosomes. The observed probit-log dose line was widely
separated and distinct from that of the susceptible strain, indicating
incomplete dominance. In both reciprocal crosses the females were slightly
more resistant than males. This portrays the possibility of slight cytoplasmic
or maternal influence with no sex linkage involvement. Of course, sex
linkage may also produce differences in the results of reciprocal crosses but in
those cases the phenotype can be predicted from the sex of the parents. With
regard to the Colorado potato beetle, female hybrids should show more
resistance only from the cross SQde as was previously explained. This,
however, was not the case. In the maternal effect, phenotype changes appear
because of differences in egg cytoplasm rather than differences in sex
chromosomes and often (although not always) affects both male and female
offspring equally. The characteristics of the probit-log dose lines of the

parental strains and of their F1 hybrids are given in Figure 1 and Table 1.

 

93

 

 

   

 

 

7 -
Offspring
Vestaburg females x L. Island males
Ihlu Fem-lee
Vnhberg (3) \ I.- ,-4’
e I
8 - \ I! I
:- ’ '
E
h I
O .1"
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c- , I
3 .o I
2 I" I”
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.’ 0’
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I’ c’
3 .-u--. v w....... . . ....... v . ....... . . .Ivru.
.1 1 10 100 1000 10000
Dose (pg/beetle)
B.
7 -
Offspring
VesMurg males x L. Island females
Von-bug (s) “m. m
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6 - \
2‘
= I
I I
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I I
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‘ _ , Long Island (R)
, x’
I r
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. 1 1 o 1 no 1 con 1 0000
Dose (pg/beetle)
Figure 1. Dose-mortality responses to azinphosmethyl of adults of Colorado potato

beetle susceptible (Vestaberg- s) and resistant (Long Island- R) strains
and F1 offspring (males and females). A. Vestaberg (5) females x Long
Island (Ft) males. B. Vestaberg (s) malesx Long Island (r) females.

 

94

 

     

 

 

   
   

 

7 .
Offspring
Offspring Vestaburg females x L Island males
Azinphosmethyl & PBO Mel Fe es
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. 1 10 100 1000 10000
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Figure 2. Dose-mortality responses of adults of Colorado potato beetle susceptible

(Vestaberg- s) and resistant (Long Island- R) strains and F1 offspring
(males and females) to azinphosmethyl and application of azinphosmethyl
and piperonyl butoxide to F1 offspring only. A. Vestaberg (3) females x
Long Island (R) males. 3. Vestaberg (5) males x Long Island (r) females.

95

Reciprocal crosses in preliminary studies with another field collected
susceptible population showed F1 hybrids with LDso's about the same as the
F1 hybrids from the crosses with Vestaburg (unpublished data). This strongly
supports the autosomal incomplete dominance of the inheritance of the
azinphosmethyl resistance.

Application of piperonyl butoxide on the F1 hybrids partially
synergized azinphosmethyl (Figure 2, Table 1) indicating that another
resistance factor exists. The synergism is less with the hybrids from the
$6 xRQ cross because the LDso's of 18.43 for males and 19.18 for females are
larger than the corresponding values from the cross SQ x Rd . It is
interesting that the level of resistance of the hybrids from 36 x RQ are
higher than the opposite cross (Figure 1, Table 1). Despite that, by considering
the confident limit values, there are no real significant differences.

The main factor for resistance to azinphosmethyl involves MFOs and
also esterases play an important role: these two enzymatic resistance
mechanisms give cross resistance to several other insecticides. This was
supported from synergistic studies with piperonyl butoxide and DEF with
different resistant strains (Chapter 3) and also from biochemical work with
two highly resistant strains (Ahammad-Sahid et al. 1990). Argentine (1989)
reported that esterase probably was the main factor for resistance to
azinphosmethyl, working with a 435-times resistant azinphosmethyl strain.
In their synergist studies, the dose of piperonyl butoxide used was the same
for susceptibles and resistants (5 ug/larva). In our opinion, based on
extensive synergist work in our laboratory, 5 ug/ larva is too small of a dose to
produce synergistic effects with resistant beetles. In our synergistic
experiments, we used stable doses of at least 50 ug/ larva and even 100

ug/ adult to the resistant strains, gave almost zero mortality. In the same

96

report, despite the fact that the backcrosses to susceptibles did not produce
distinct plateaus, Argentine et al. 1989 found that azinphosmethyl resistance
was largely monofactorial for their CPB, but its dominance or expressiveness
was influenced by other genes.

We have shown that the resistance to azinphosmethyl in the Long
Island strain of the CPB is inherited as two incomplete dominant genes.
Therefore, if the two genes independently segregated in the backcross, a
plateau at the 25% mortality level should be observed. If the two genes were
tightly linked, segregation should be exhibited as one factor with a distinct
plateau at the 50% mortality level. In our case, a very clear plateau can be
seen around the 40% level (Figure 3) indicating two distantly linked genes. If
more than two main resistant factors had been involved in the expression of
the resistance in the Long Island strain, the plateau in the backcrosses would
not have been present, but the plotted dose-mortality line would have been
straighter (no segregation) (Tsucamoto 1963, Snyder et al. 1985). The linkage
of the two resistant factors was supported also from the results of the F2
progenies. A discriminating dose of 3.12 ug/ beetle of azinphosmethyl applied
to the F2 progenies killed only the susceptibles. The estimated mortality was
13.9%. This was more in agreement with the expected mortality of 16% for
two linked genes with about a distance of 20 map units (Table 2), than the
value of 6.25% which should be expected for two independent segregating
genes. Furthermore, in a careful examination of the backcross lines in
(Figures 3 and 4) two plateaus can be shown at 40% and 60% mortality levels,
instead of 25% and 75% values as expected if two independent segregating
genes existed.

It is interesting that in all the backcrosses, the dose of 3.12 ug/beetle
(which kills only the susceptibles) gave a mortality around 40%, which was in

97

agreement with the percentage of susceptibles produced by two linked genes.
The evidence for difactorial mendelian inheritance is based on (1) the
presence of continuous distribution of the character of resistance in the F2 and
backcross progeny, and (2) the statistical agreement of the observed responses
to those which may be expected in case of difactorial inheritance with two
main linked genetic factors with around 20% recombination plus synergist
results. If other minor important genes contributed to this high level of
resistance they should be closely linked to these two main resistant factors
described above. Backcross to the resistant parent (F1 x Rd ) produced
genotypes with resistance between the F1 and the parent. Application of a
discriminating dose of 100 ug/beetle gave no mortality as it was expected
while 200 ug/ beetle killed about 50%. This was as expected with mendelian
inheritance.

We have successfully completed six generations of backcrossing with
the susceptible strain Vestaburg followed by mild selection with
azinphosmethyl. The surviving heterozygotes of the 6th backcross progeny
(after giving a discriminating dose which kills the susceptible aabb and the
Aabb, aabB heterozygous beetles) were mass-inbred. The resulting progeny
were under selection to produce a homozygous resistant strain. Therefore,
the genetic background of the resistant strain became essentially equivalent to
the 98.44% of the susceptibles. The new strain can be utilized for further
genetic studies and biochemical work.

Based on synergist studies with the parental resistant strain, the F1 and
backcross progenies, it was possible to estimate the amount of resistance given
by each allele to azinphosmethyl (Table 2). The AABB stands for: AA for
MFO's and BB for esterases, the two main enzymatic resistance mechanisms.

Therefore, these two linked genes in the backcross with the susceptibles, and

98

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100

in the F2, yielded the genotypes presented in Table 2. The phenotypic
expression of resistance is presented in Table 3 based on the confidence limits
of the corresponding LD50 values.

The procedure of backcrossing while selecting for resistance to one
compound is a very effective way of isolating a single gene if one exists (Crow
1957). Since we have estimated the discriminating doses of the LDso's which
kill the corresponding genotypes (Table 2) it would be possible, by applying
the following technique, to separate the two main resistance mechanisms,
establishing different pure strains with one of the resistant factors. Although
genetic markers are unknown as yet for the Colorado potato beetle, the
specific inhibitor of MFO's (piperonyl butoxide) can be used like a marker.
Progeny from the backcross with the susceptible are maintained in pairs and
eggs collected, keeping them separated from eggs from other pairs. A
discriminating dose of 3.12 ug azinphosmethyl/beetle can be applied which
kills only the susceptible aabb. After five days the beetles again receive the
application of piperonyl butoxide 20 ug/ beetles followed after two hours with
the same discriminating dose of azinphosmethyl. Only the pairs with the
Aabb genome should be dead; therefore, we raise only the eggs which were
produced from the beetles before they died. The new beetles will interbreed;
therefore these must have only the A_ genotype. If the same procedure is
repeated with application of azinphosmethyl with piperonyl butoxide on the
new beetles, all the pairs should die, which is clear evidence for a pure strain.

The same technique can be applied using DEF, which inhibits esterases
and is not a specific inhibitor of but probably also inhibits oxidation (Attia et
a1. 1980, Plapp 1986). The above procedure should necessarily be repeated
several times to purify the strain with only the esterase resistance

mechanisms. It is also possible that DEF inhibits MFO activity slightly in the

101

Colorado potato beetle, since microsomal preparations from beetles treated
with DEF show only a small reduction in ability to oxidize NADPH
(Ahammad-Sahid et a1. 1990).

A potential marker with the Colorado potato beetle is limited only to a
white-body color mutant larva, which has been established. This trait has
proved to be stable for at least nine generations (Boiteau 1980). This mutant
trait is recessive to the wild type and is due to single autosomal genes (Hsiao
1983). It is interesting that we have noticed this mutant only in the Vestaburg
strain selected for permethrin resistance in our laboratory. Attempts to
establish a colony by interbreeding albino beetles were not successful. We
noticed rare mating between them, while albinos mated successfully with
normal ones. Further attention and examination may raise some useful
correlation to the permethrin resistance.

It is interesting that there are reports for inheritance of resistance of
organophosphates in other species concluding that more than one gene is
involved in the resistance. Harris et a1. (1970) early in 1961, reported that two
sets of alleles affect the inheritance of malathion resistance in housefly, -
exhibiting incomplete duplicate dominance epistasis. Oppenoorth (1959) in
genetic studies with the diazinon resistant housefly found that one gene is
responsible for low esterase activity and responsible for part of the resistance
and at least one other resistance factor is present (MFO's, Oppenoorth 1985).

The genetics of malathion resistance in two strains of the flour beetle
Tribolium castfianeum (Herbst) show that resistance in one strain was
polygenic for the other one, the resistance was due to a dominant allele at a
single autosomal focus (Wool et al. 1982). Raymond et al. (1987) reported that
the number of additive and independent genes contributing to chlorpyrifos

resistance in Culex pipiens (L.) was at least two and perhaps more, and they

 

102

had found in previous studies the presence of an insensitive acetyl
cholinesterase and a modified oxidative detoxification. It is very interesting
that one of the strains in our laboratory, the I-P strain of Colorado potato
beetle, possesses both of the above resistance mechanisms. This has been
demonstrated by the use of synergists which were unable to eliminate the
total level of resistance and from biochemical studies (Ahammad-Sahid et al.
1990, Wierenga and Hollingworth - unpublished data).

It is obvious now that several resistance mechanisms might be
involved in different strains. Therefore, to obtain specific knowledge for
resistance mechanisms for each gene it is necessary to select special resistant
strains in which only a single resistance gene will be involved.

These results and results of future studies will provide essential
information for new approaches in the control of the CPB and to better
elucidate the insecticide resistance problem which should be considered as
one of the subjects of population genetics (Roush et al. 1986). A better
understanding of the detailed genetic mechanisms should help us to confront
the Colorado potato beetle, one of the most threatening pests in the US. and

other parts of the world for potato plantations.

103

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104

Table 2. Theoretical genotypic expression in dihybrid crosses and backcrosses
with two autosomal resistance factors to azinphosmethyl, the enzymes MFO's

(AA) and esterases (BB).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Two Linked Genes W0 independent genes
Genotype LDso's [20% recombination [independent segregation
F1 x F1 uglbeetle expected ratios] expected ratios]

AABB 556.00 16.00% 6.25%

AABb - 8.00% 12.50%

AaBB - 8.00% 12.50%

AaBb 144.001 34.00% 25.00%

AAbb 65.002 1.00% 6.26%

aaBB 28.003 1.00% 6.25%

Aabb - 8.00% 12.50%
a—abTB 13.004 8.00% 12.30%

aabb 1.250 16.00% 6.25%

Backcross to

susceptible

AaBb 40.00% 25.00%

Aabb 10.00% 25.00%

BEST; 10.00% 25.00%

aabb 40.00% 25.00%

 

 

1 Mean of F1 offsprings of the reciprocal crosses to azinphosmethyl.
2 Resistance of the resistant strain to azinphosmethyl plus DEF synergist from

Table 4.

3 Resistance of the resistant strain to azinphosmethyl plus PB synergist from

Table 4.

4 Mean of F1 offsprings of the reciprocal crosses to azinphosmethyl plus PB

from Table 1.

 

 

105

Table 3. Phenotype expressing of resistance to azinphosmethyl in the F2
progenies (F1 x F1) from two autosomal linked factors for MFO's and esterases

with 20 map distance.

 

 

 

 

 

 

 

 

 

 

 

 

Theoretical

Phenotypic range Fraction of
Number of alleles of resistancea F2
Phenotypes for resistance ug/beetle %
AABB 4 Highly resistant <402.00 - 710.00 16
AABb, AaBB 3 Highly resistant 300.00- 400.00b 16
AaBb 2 Intermediate 80.00- 290.00 2
AAaa,aaBB 2 Medium 40.00- 80.00C 34
Aabb,aabB 1 Low 3.70- 41.00d 16
aabb 0 Susceptible 1.01- 1.49 16

 

a The 95% confidence limits from the corresponding LDso's (Table 2).
b Arithmetical estimation.
C Application of azinphosmethyl with PB or DEF on the resistant parents.
d Application of azinphosmethyl with PB on the F1 offsprings.

 

General Discussion

106

General Discussion

Past experience and recent studies on resistance modeling show that

reduction of selection pressure in its broadest sense is a key factor for slowing

the development of resistance.

An important requirement for developing strategies for avoiding or

delaying the development of resistance is the study of the factors which

influence the evolution of these phenomena. These factors are classified as

follows (Georgiou and Taylor 1986):

A.

Genetic: (1) frequency of R alleles; (2) number of R alleles; (3)

dominance of R alleles; (4) penetrance, expressivity; interactions of R
alleles; (5) past selection of other chemicals; (6) extent of integration of
R genome with fitness factors.

Biological:

 

a. Biotic potential: (1) generation turn-over; (2) offspring per
generation; (3) monogamy/ polygamy; parthenogensis.

b. Behavioral: (1) isolation; mobility; migration; (2)
monophagy/polyphagy; fortuitous survival; refugia.

Operational:

a. The chemical: (1) chemical nature of pesticide; (2) relationship to
earlier used chemicals; (3) persistence of residues; formulation.

b. The application: (1) application threshold; (2) selection

threshold; (3) life stage(s) selected; (4) mode of application; (5)
space-limited selection; (6) alternating selection.

Factors in the genetic and biological categories are inherent qualities of

the species and, therefore, are beyond management. However, knowledge of

how these contribute to selection pressure is important. In the following

discussion some factors are summarized from the information obtained

through this study which can help in designing control tactics. '

107

108

Use of synergists in these practices and particularly piperonyl butoxide
can significantly increase the toxicity of certain insecticides, since it is well
documented now that mixed-function oxidases is the main defense
mechanism of the Colorado potato beetle.

The high synergistic effect of PB with azinphosmethyl, which we have
found consistently with several populations, may prove useful in controlling
resistant populations and reducing the rate of selection of resistance in the
field or decreasing the frequency of resistant genes already present in field
populations.

Since DEF is a defoliant, mixtures of this synergist and insecticides
cannot be used in the field to overcome resistance due to esterases. We have
found resistant populations of CPB partly due to esterase activity. Post-
harvest treatment or non-crop site treatment may be possible. Probably of
practical use are the mixtures with sprayable esterase inhibitors, such as
kitazin P, a systemic fungicide used to control Piricularia oryzae in rice
(Worthing 1979), in combination with insecticides (Ozaki 1983).

As was mentioned before apart from the estimation of synergistic ratios
(SR) at 50% mortality level, we can estimate the SR in 90% mortality for the
purpose of field efficacy. The SRgo is very important since in most cases, 90%
mortality is required for acceptable control. Argentine et a1. 1989 reported that
permethrin resistance to Colorado potato beetle has a recessive character.
From our studies and the control failure of applying pyrethroids plus
synergists after 1-2 years, it appears that the Kdr (knockdown-resistance) factor
was well spread in certain areas and may well be responsible for some
reported failures of application of pyrethroids.

Two types of resistance to permethrin exist. There are cases in the field

where the Colorado potato beetle populations developed resistance to

109

permethrin relatively fast. This is typical when resistance is dominant or
semidominant (enzymatic resistant mechanism), where the RR homozygotes
and Rr heterozygotes are able to survive the application of normal rates of
insecticides. If, on the other hand, the resistance is recessive (Kdr), the
population remains susceptible until the quantitatively smallest fraction of rr
homozygotes appear.

Farnham (1977) suggested that because Kdr is a recessive character,
therefore natural dilution of the population and the removal of selection
pressure would help to increase greatly the number of heterozygotes which
are susceptible to DDT or pyrethroids. There is, therefore, a need for early
detection of this factor in a population, coupled with an alternative means of
control in order to keep the frequency of this gene at manageable levels.

There are indications and some evidence that between the insecticides
azinphosmethyl and carbofuran on one side and permethrin on the other
side, negative correlated resistance exists, where selection of resistance by
pressure from insecticide A leads to a corresponding selection of susceptibility
to insecticide B, and vice versa. Therefore, when A is no longer effective in
in controlling the CPB, a switch to B may give control again for a few years.
Alternate useof insecticides A and B can be repeated. Georgiou (1963)
suggested that the mixtures used in sprays must not consist of a pair of
compounds that display negatively correlated toxicity. Examples of negatively
correlated resistance are few in the literature; there are probably more
undocumented cases. We tried to document further this hypothesis for CPB
with selection studies, by searching the literature for cases of resistance to
CPB, and, mostly, by conducting bioassays directly to field collected
populations. Therefore, some very useful conclusions were drawn, affecting

recommendations for the use of mixtures of azinphosmethyl and permethrin

110

or carbofuran and permethrin with resistant populations, a tactic used so far
by some farmers. Valuable information for resistance management tactics
were obtained (Chapter 2).

Carbofuran kills extremely fast, (<1 hour), compared with
azinphosmethyl which may take 3 days to express its toxicity. Carbofuran is
profoundly more toxic to the larva than adults, therefore less amount of
chemical can be applied to the larvae stages.

Some fungicides affect the development of the CPB. Hare (1984)
reported suppression of CPB on solanaceous crops with a copper-based
fungicide. Therefore fungicides might be chosen not only for their ability to
control plant diseases, but also for their capacity to help manage the beetles.

Migratory flight of Colorado potato beetles is merely a particular
prolonged flight which, it seems, is ontogenetically controlled and adapted to
occur at a certain stage in adult development (Johnson 1967). Generally, CPB
are pests with relatively low dispersal tendencies. Therefore, insecticide
pressures favor the development of resistance and cultural practices are very
important. Crop rotation is fundamental to solve and retard resistant
problems particularly with Colorado potato beetle, since we now know that
the resistant mechanisms are inherited almost as dominant genes and
migration of susceptibles does not seem to be a factor.

Detection and monitoring of resistance is fundamental. During this
study, discriminating doses have been established for several insecticides,
therefore they can be used effectively to determine resistant portions in field
populations.

Dose-mortality lines have been calculated for more than 30

populations from different areas of Michigan and can be used to track

111

resistance over time and make comparisons with development of resistance
in other areas and even other countries.

One of the principles for resistance management is to apply the least
insecticide amount on the population to conserve susceptibility. It must be
enough to eliminate the susceptibles (SS) and heterozygotes (R5), in the case
of permethrin resistance. Because of the recessiveness of permethrin
resistance, this is easier than for azinphosmethyl and carbofuran, where
resistance is inherited as incompletely dominant.

Application of mixtures of insecticides is another resistance
management technique. Information in the literature is contradictory. The
idea behind this technique is that it is difficult for individuals to carry more
than one resistant mechanism. We believe that the use of mixtures in
resistance management of the Colorado potato beetle is very risky. Field
observations and our laboratory studies with several different multiple
resistance strains suggests that beetles have a propensity to develop resistance
and resistant genes preexist in the populations, in significant frequencies.
Rotation of insecticides seems more advantageous since our information for
resistance patterns and cross resistance, essentially increases. Insecticides
where resistance depends on the interaction of several resistance factors
should be avoided or used last, since these give a broader spectrum of cross-
resistance, particularly for those in which resistance development comes
from enzymatic mechanisms.

For estimation of resistance levels to a certain insecticide comparisons
should be made with populations that recently were controlled by this
insecticide. It used to be that resistance in field-collected strains as compared
with susceptible strains that had been kept in isolation for long periods of

time in an insecticide-free environment. This may have given wrong

112

estimations. Field populations have acquired some degree of resistance, and
the genomes of these populations adapted to new agricultural environments
under the continuous pressure of insecticides used. Development of
resistance is a natural process and must be considered as an evolutionary
phenomenon from the genetic consideration point of view.

Resistance management strategies are currently being adopted for the
Colorado potato beetle in an attempt to extend the potential and usefulness of
the insecticides, but for how long? The design of an integrated pest
management system for this threatening pest is a necessity.

Oppenoorth 1985 mentions that as entomologists and toxicologists, our
knowledge of resistance mechanisms is increasing, but the development and
molecular genetics of resistance has just been touched. Whether chemical
control of a majority of pest species will remain possible seems difficult to

predict at present.

List of References

LIST OF REFERENCES

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Ahammad-Sahib, K.I., R. Hollingworth, M. Whalon, P. Ioannidis and E.
Grafius. 1989. The role of mixed function oxidase and other metabolic
enzymes in insecticide resistance in Colorado potato beetle. Pestic.
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Ahman, S. and AJ. Forgash. 1975. Toxicity of carbaryl and diazinon to gypsy
moth larvae: changes in relation to larval growth. J. Econ. Entomol.
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Apperson, CS. and GP. Georgiou. 1975. Mechanisms of resistance to
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153-157.

 

 

Argentine, ].A., Clark, ].M. and D.N. Ferro. 1989. Genetics and synergism of
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Beeman, R.W. 1983. Inheritance and linkage of malathion resistance in the
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Boiteau, G. 1983. Synchronization of Colorado potato beetle (Coleoptera:
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Boiteau, G., R.H. Parry and CR. Harris. 1987. Insecticide resistance in New
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Brattsten, LB. and R.L. Metcalf. 1970. The synergistic ratio of carbaryl with

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115

Brattsen, LB. and RL. Metcalf. 1973. Synergism of carbaryl toxicity in natural
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Brown, A.W.A. and R. Pal. 1971. Insecticide resistance in arthropods. erd
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Busvine, ].R. 1971. Techniques for testing insecticides, Sec. Ed. C.A.B.,
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Caprio, M. and E. Grafius. 1990. Effects of light, temperature, and feeding
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Casida, ].E. 1970. Mixed-function oxidase involvement in the biochemistry of
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Casida, LE. and LO. Ruzo. 1980. Metabolic chemistry of pyrethroid
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Chang, GK. and M.E. Whalon. 1985. Hydrolysis of permethrin by pyrethroid
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Appendix 1

128

APPENDIX 1

Record of Deposition of Voucher Specimens*

The specimens listed on the following sheet(s) have been deposited in
the named museum(s) as samples of those species or other taxa which were
used in this research. Voucher recognition labels bearing the Voucher
No. have been attached or included in fluid-preserved specimens.

Voucher No.: 1990-02

 

Title of thesis or dissertation (or other research projects):

Characterization, Synergism and Inheritance of Resistance to
azinphosmethyl, carbofuran and permethrin insecticides in the
Colorado potato beetle (ColeOptera: Chrysomelidae)

Museum(s) where deposited and abbreviations for table on following sheets:
Entomology Museum, Michigan State University (MSU)

Other Museums:

none

Investigator's Name (3) (typed)
Philippos M. Ioannidis

 

 

 

Date March 26, 1990

*Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in
Nbrth America. Bull. Entomol. Soc. Amer. 24:141-42.

Deposit as follows:

Original: Include as Appendix 1 in ribbon copy of thesis or
dissertation.

Copies: Included as Appendix 1 in copies of thesis or dissertation.
Museum(s) files.
Research project files.

This form is available from and the Voucher No. is assigned by the Curator,
Michigan State University Entomology mseum.

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Voucher Specimen Data

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“mafimuum
maummn oumuoa oumuoaoo
A>mmv mpmmcfiasmomu mmumuOCAuamm
-w:r=e-if,.-;---su. .i1
160- :ou_mcai1 can now: no pouuoH~0o :oxmu umguo uo mmaomam
e r r m m e .m % mcaewomam poC camp floss;
rOdellapWS
epeinuupm g
hettddUV.ag
wdiOAAPNLE

 

 

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