Resistance to pyrethroid and organophosphate insecticides in the pink bollworm, Pectinophora gossypiella (Saunders).
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Authors Osman, Abdelgadir Ahmed.
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Resistance to pyrethroid and organophosphate insecticides in the pink bollworm, Pectinophora gossypiella (Saunders)
Osman, Abdelgadir Ahmed, Ph.D.
The University of Arizona, 1989
U·M·I 300 N. Zeeb Rd. Ann Arbor, MI 48106
RESISTANCE TO PYRETHROID AND ORGANOPHOSPHATE
INSECTICIDES IN THE PINK BOLL WORM,
Pectinophora gossypiella (Saunders)
by
Abdelgadir Ahmed Osman
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF ENTOMOLOGY
In Partial Fulfillment of the requirements For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
198 9 THE UNIVERSITY OF ARIZONA 2 GRADUATE COLLEGE
As members of the Final Examination Committee, we certify that we have read
the dissertation prepared by ABDELGADIR AHMED OSMAN
entitled RESTISTANCE TO PYRETHROID AND ORGANOPHOSPHATE INSECTICIDES IN THE PINK BOLLI.J"ORM, Pectinophora gossypiella (Saunders).
and recommend that it be accepted as fulfilling the dissertation requi~ement
for the Degree of Doctor of Philosophy
4 / 17 ! 89
Dr. Theo F. Watson Date 4 / 17 / 89 Date 4 / 17 / 89 Dr. 1. Irene Ter-ry Date nIl ..JL. L' 0, -.'" 4 / 17 / 89 KOV%?L C . ()~ ) Date 4 / 17 / 89 Dr. Paul Bartels Date
Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.
4 / 17 /89 Dissertation Director Dr. Thea F. Watson Date
- --_.-... _.... -.._- .. --_.-..... _------3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may ba granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the propsed use of the material is in the interests of scholarship. In all other instances, however, permission may be obtained from the :~thOr. ". 1
SIGNED: 'lf} btL rl .. ,\. ;& ,"------~!~-:::(~ 4
ACKNOWLEDGEMENT
It was a pleasure to have Dr. Theo F. Watson as my major professor and dissertation director. He provided me with guidance, moral and financial support. He also originated the idea of studying the susceptibility levels of the pink bollworm and selection for resistance. I would especially like to thank him for creating both, a pleasent working atmosphere for research and developing of new ideas, and an ideal professor-student relationship. I would like also to extend my sincere thanks to Drs. Leon Moore and Irene Terry of my major department for their valuable advice and constructive criticism; and Drs. Robert Briggs and Paul Bartels of my minor department for serving on my committee and critically reviewing the manuscript. I am also indebted to Dr. Allan Bartlett, the Geneticist of the Western Cotton Research Laboratory, USDA, Phoenix, for his valuable suggestions and continued support with regard to the genetic aspect of this study. I am also grateful to Lilian Mouge of the same department for furnishing me with the susceptible strain and providing a continuous supply of pink bollworm artificial diet. I extend my appreciation to Dr. David Blough for his advice regarding the statistical analysis of the results. Thanks are also due to Suzanne Kelly for offering and providing assistance in using the computer for graphs and probit analysis of the results. My special thanks are also extended to Justine Collins for providing the most needed last-minute help in formatting and printing the manuscript. As for the support provided by my friend Sakuntala Sivasupramaniam a word of "thanks" may fall way too short to express my gratitude. In fact she actively participated in this study from its inception to resolution. I would like also to recognize the fact that she literally put all her time and experience at my disposal. Such help is provided only by "true" friends. Finally, I would like to thank my parents, brothers and sisters for their moral support and their patiently, and lately impatiently, awaiting for a come back home ceremony. 5
TABLE OF CONTENTS
Page
LIST OF ILLUSTRATIONS. 7
LIST OF TABLES 8
ABSTRACT ... 10
INTRODUCTION 12
Research Objectives. 15
LITERA TURE REVIEW. . 17
Biology and Damage. 17 Management . . . . 19 Chemical Control. . 20 Organophosphorus Insecticides 21
Structure and Activity . . 22 Mechanism of Action: Enzyme Inhibition. 22 Metabolism . . 23 1.0xidation. . 23 2.Hydrolysis . 24 3.Conjugation. 25 Pyrethroid Insecticides. 25 Mode of Action . . 26 Effects on Peripheral Nerve Elements 27 Effects on the Central Nervous System. 28 Physiology of Knockdown 29 Structural Activity . 30 Metabolism . . . . . 31 Resistance ...... 32 Mechanisms of Resistance 34 Non-metabolic Resistance. 35 I.Reduced Penetration. 35 2.Target insensitivity. . 36 3.Genes Responsible for Non-Metabolic Resistance 38 Metabolic Resistance...... 39 I.Mixed Function Oxidases...... 40 2.Hydrolases and Glutathione S-Transferases 41 (i) Hydrolases...... 42 (ii) Glutathione S-Transferases . . . . . 42 3.Carboxylesterases ...... 43 4.Genes Responsible for Metabolic Resistance. 43 Selection for Resistance 44 Cross Resistance ...... 46
------6
TABLE OF CONTENTS Page
Synergism ...... 49 Genetics of Insecticide Resistance. 53 Relative Fitness . . 55
MA TERIALS AND METHODS 58
Insecticides and Synergists 58 Rearing of Pink Bollworm Strains. 58 Bioassay Procedures...... 59 Evaluation of Permethrin and Azinphosmethyl Resistance in Field Populations...... 61 Synergism of Permethrin...... 62 Reversion of Resistance to Permethrin in Field Strains. . . 62 Laboratory selection for Resistance to Permethrin and Azinphos- methyl...... 62 Selection at the Larval Stages 63 Selection at the Adult Stages. 63 Inheritance Study...... 64 Insect Strains ...... 64 Virgin Females...... 64 Bioassay and Genetic Crosses 65 Analysis of Data . 66 Pro bit Analysis. . 66 Synergism Tests . 66 Genetic Analysis. 66
RESUL TS AND DISCUSSION. 68
Susceptibility to Permethrin and Azinphosmethyl in Field Populations 68 Synergism of Permethrin...... 77 Reversion of Permethrin Resistance. . . 83 Selection for Resistance in Pink Bollworm 90 Selection with Azinphosmethyl. . . . 90 Selection with Permethrin. . . . ., .. .. 96 Inheritance of Resistance to Permethrin in a Laboratory Selected Strain ...... 103 Selection for Permethrin-Resistance . 103 Inheritance of Permethrin Resistance. 103
CONCLUSION. . . . . 114
REFERENCES CITED . 120 7
LIST OF ILLUSTRATIONS
Number Page
Dosage-mortality lines for azinphosmethyl on strains of pink bollworms collected from Safford and Yuma, relative to a standard laboratory strain ...... 70
2 Dosage-mortality lines for permethrin on five strains of pink bollworms collected from different locations, relative to a standard laboratory strain ...... 72
3 Synergism of permethrin by PBO and PBOjDEF combination in the Yuma (field) and laboratory susceptible strains of pink bollworm 79
4 Reversion of permethrin-resistance in pink bollworm (Marana strain) Following relaxation of insecticidal pressure ...... 85
5 Reversion of permethrin-resistance in pink bollworm (Yuma strain) following relaxation of insecticidal pressure...... 87
6 Dosage-mortality lines for an azinphosmethyl-selected strain of pink bollworm (Marana strain). Larvae subjected to selection ...... 93
7 Dosage-mortality lines for an azinphosmethyl-selected strain of pink bollworm (Marana strain). Adults subjected to selection ...... 95
8 Dosage-mortality lines for a permethrin-selectd strain of pink bollworm (Marana strain). Larvae subjected to selection...... 99
9 Dosage-mortality lines for a permethrin-selected strain of pink bollworm (Marana strain). Adults subjected to selection...... 101
10 Dosage-mortality lines for a permethrin-selected strain of pink bollworm (Yuma strain).Adults subjected to selection...... 105
11 Dosage-mortality lines for permethrin on susceptible and selected strains of pink bollworm and their Fl crosses...... 109
12 Dosage-mortality lines for permethrin on susceptible and selected strains of pink bollworm and their genetic crosses...... 11 I 8
LIST OF TABLES Number Page
Toxicity of azinphosmethyl to five strains of pink bollworm collected from different locations relative to a standard laboratory susceptible strain...... 69
2 Toxicity of permethrin to five pink bollworm strains collected from different locations relative to a standard laboratory susceptible strain...... 71
3 Dosage mortality data for synergism of permethrin by piperonyl butoxide(PBO) and DEF in the Yuma field and susceptible strains of pink bollworm ...... 78
4 Synergism of permethrin by piperonyl butoxide and DEF in the Yuma field and susceptible strains of pink bollworm...... 80
5 Reversion of permethrin resistance in pink bollworm (Marana strain) following relaxation of insecticidal pressure...... 84
6 Reversion of permethrin resistance in pink bollworm (Yuma strain) following relaxation of insecticidal pressure...... 86
7 Selection and progression of resistance to azinphosmethyl in the Marana strain of pink bollworm ...... 91
8 Dosage-mortality data for azinphosmethyl on adult pink bollworm upon selection for resistance at the 3rd larval stage (Marana strain) ...... 92
9 Dosage-mortality data for azinphosmethyl on adult pink bollworm upon selection for resistance at the adult stage (Marana strain) . . . .. 94
10 Selection and progression of resistance to permethrin in the Marana strain of pink bollworm...... 97
11 Dosage-mortality data for permethrin on adult pink bollworm upon selection for resistance at the 3rd larval ins tar (Marana strain) .. 98
12 Dosage-mortality data for permethrin on adult pink bollworm upon selection for resistance at the adult stage (Marana strain) ...... 100 9
LIST OF TABLES
Number Page
13 Dosage-mortality data for permethrin on adult pink bollworm upon selection for resistance at the adult stage (Yuma strain)...... 104
14 Dosage-mortality data for permethrin on susceptible and selected strains of pink bollworm and their genetic crosses ...... 108
15 Degree of dominance (D) of permethrin resistance in the selected strain of pink bollworm...... 110
16 Chi-square (X2) analysis of mortality data from Fl x susceptible and Fl x permethrin-resistant backcrosses of the pink bollworm. . . . . 112 10
ABSTRACT
Baseline data on susceptibility levels to azinphosmethyl and permethrin were generated on five field-collected populations of the pink bollworm,
Pectinophora gossypiella (Saunders), from Arizona and Southern California, relative to a standard susceptible laboratory strain. The field strains showed less than 2-fold resistance to azinphosmethyl but exhibited variable levels (1.3- to 18.3-fold) of resistance to permethrin. Resistance of pink bollworms to permethrin seems to be correlated with the pattern of insecticide-use prevalent in the localities studied. Strains from Yuma, Phoenix and Westmoreland exhibited highest levels of resistance to permethrin. Synergism of permethrin with an oxidase iI':ibitor, piperonyl butoxide (PBO), and an esterase inhibitor, S,S,S tributyl phosphorotrithioate (DEF), produced less than 2-fold synergism in the
Yuma strain. Results suggest that non-metabolic factor(s) may be involved in permethrin resistance of the Yuma field strain since neither PBO nor PBOjDEF combination suppressed resistance completely. It is possible that pink bollworm resistance is at least partially conferred by the kdr-gene.
Rearing of two field strains collected from Marana and Yuma under insecticide-free conditions resulted in reversion of resistance in four and five generations, respectively, to levels close to that found in the susceptible laboratory strain. Permethrin-resistance in these field strains is unstable and is apparently in its early phase of development. Monitoring of resistance in field strains should be performed preferably in the Fl generation.
Subsequently, selection studies were performed on both larval and adult stages to investigate the capacity of the pink bollworm to develop resistance in both life-t;tages. Selection of larvae with both azinphosmethyl and permethrin resulted in higher levels of resistance in larvae than in adults. Results suggest II
that azinphosmethyl possesses a low degree of selectivity for development of
resistance in pink bollworm adults. Fourteen to 16 generations of selection with
azinphosmethyl and permethrin produced ca. 2- and 9-fold resistance,
respectively, in the adult stage.
A laboratory-selected strain showing ca. 13-fold resistance was used in reciprocal crosses with a susceptible laboratory strain. The F 1 results suggested
that inheritance of permethrin resistance was autosomal and partially dominant.
Chi-square analysis of responses of backcross progeny indicated that resistance
seems to be conferred by a major gene under the influence of minor gene(s). 12
INTRODUCTION
Hundreds of species of insects, pathogens, rodents and weeds have developed resistance to pesticides as a consequence of basic evolutionary processes. Hence, the problem of resistance which was rarely encountered during the early 1950's, at least among insect pests, has greatly escalated such that fully susceptible populations are rarely seen in the 1980's (National
Research Council, 1986).
Since the first reported case of pesticide resistance by Melander in 1914, the incidence of resistance has proliferated exponentially such that it is an essential consideration in almost all pest control programs (Georghiou and
Mellon, 1983). Georghiou( 1986) reported that the occurrence of resistance was most acute among insects and mites and that at least 447 species had been reported to have developed resistance to one or more classes of insecticides.
Although the capacity to develop resistance to chemicals is universal, insecticides are uniquely powerful in selecting for resistance in almost every organism ranging from bacteria to mammals (Georghiou and Mellon, 1983).
The exponentially increasing cost of insecticide development, the dwindling rate of commercialization of pesticides and the frequent occurrence of cross and multiple resistance have brought insect pest resistance to as one of the most severe and challenging problems facing applied entomology today (Mectalf, 1980).
Furthermore, the present trend of using insecticides that are more selective and less hazardous to the environment seems to be partially hindered, at least at the present, by the previously adopted practice of using broad-spectrum and persistent insecticides. Evidently, the relatively rapid development of resistance to pyrethroid insecticides witnessed in some insects has been attributed to the previous use of DDT, dichloro diphenyl trichloroethane, which is known to 13 possesss a similar mode of action to pyrethroids (Miller and Adams, 1982) and to persist for over 12 years in soils (Buck et aI., 1983). Thus, despite the fact that synthetic pyrethroids possess many favorable characteristics, the potential for resistance in pyrethroids is a major limiting factor to their prolonged use.
Pyrethroids were first introduced in Arizona in 1977 (Jensen et aI., 1984) and have been extensively used in cotton ecosystems thereafter. In fact, the development of resistance by the tobacco budworm, Heliothis Virescens, to organophosphates, particularly to methyl parathion, O,O-dimethyl-O-4- nitrophenyl phosphorothioate, led farmers to depend heavily on pyrethroid insecticides (Sparks, 1981). Crowder et aJ. (1979) detected "tolerance" or low level cross resistance to pyrethroids in tobacco bud worm populations which had already been determined to be resistant to methyl parathion.
The pink bollworm, Pectinophora gossypiella (Saunders), is one of the most serious economic pests causing considerable damage to cotton production.
Carruth and Moore (1973) reported that serious secondary pest outbreaks result from numerous insecticide applications used in achieving economic control of the pink bollworm. Recently, variable levels of pink bollworm resistance to pyrethroids were detected by Bariola (1985).
Frisbie et aJ. (1986) mentioned that the development of resistance to key pesticides should be monitored and carefully managed to prolong the efficacy of these chemicals. Since chemicals will continue to be used in pest management programs, it is necessary to formulate regimes of insecticide use that concurrently retain insect pest populations at low density and prevent the evolution of resistance (Georghiou and Mellon, 1983). It is therefore imperative that a careful evaluation of the mechanism of resistance and the nature of genetic factors conferring resistance need to be examined in order to make 14 rational decisions in pest management. 15
Research Objectives
Organophosphorous insecticides have been used effectively since the 1960's as part of the pest management program to control adult pink bollworms. At present, both organophosphate and pyrethroid insecticides are being used to control the pink bollworm in infested areas of the U.S. cotton belt. Recent studies conducted in Arizona and California, have revealed that field populations are showing resistance to pyrethroids which were introduced only in the late
1970's (Haynes et ai., 1986; Bariola, 1985; Bariola and Lingren, 1984). Despite widespead interest in resistance management, there is a real scarcity of studies performed on monitoring and elucidation of mechanisms of resistance in the pink bollworm.
The objectives of this study were:
1. To elucidate whether there are significant differences in resistance to azinphosmethyl and permethrin among field populations of pink bollworm collected from Arizona and southern California. The results of these studies would help to visualize the spread of resistance among different areas based on geography, and history of insecticide use. Moreover, baseline data on susceptibility to azinphosmethyl and permethrin could be established.
2. To determine synergism of permethrin in both susceptible-laboratory and resistant-field (Yuma) strains. Both PBO and DEF, inhibitors of detoxifying enzymes, were used in this study. The results of this study would help us understand the nature of progression of permethrin resistance in the field population studied.
3. To determine the decline or reversion of resistance in field populations which have been released from selection pressure. Populations from Marana and 16
Yuma were reared under insecticide-free conditions in the laboratory to monitor the progression to susceptibility or a new "wild-type" state. Such studies will also help understand the degree of stability of resistance in these field strains.
4. To investigate the capacity of field strains of pink bollworm to develop resistance to azinphosmethyl and permethrin. Very often insecticide resistance studies focus on the life stage of the insect pest which is the target for chemical control, but according to Tabashnik et al. (1988) studies comparing development of resistance in different life stages could provide an insight into the resistance mechanisms involved and the ontogeny and evolution of resistance. Thus, selection was performed at the adult and larval stages using topical and contact toxicity methods, respectively. Moreover, knowledge of the effects of the insecticides used on larval as opposed to adult stages would be important in understanding the influence of biology and ecology on the current status of resistance in pink bollworm.
5. To elucidate the degree of dominance of resistance, whether resistance factor(s) was sex-linked or autosomal, and whether resistance is controlled by one (monogenic) or more than one (polygenic) loci.
-----~ - --~------17
LITERA TURE REVIEW
The pink bollworm, Pectinophora gossypiella (Saunders), primarily a mid and late-season cotton pest, is one of the most destructive pests of cotton in most of the cotton-producing countries in the world including the western cotton belt area of the United States (Roelofs, 1978). Busck (1917) reported that it ranks among the first half dozen most important insect pests of the world. It probably originated in India where it had been reported as a cotton pest since 1843 (Pearson, 1958). In Arizona, a pink bollworm infestation was reported as early as 1926; yet serious economic losses remained negligible until
1965 (Wene et aI., 1965a; Spears, 1968; Watson and Fullerton, 1969).
Biology and Damage
The pink bollworm female releases a sex phermone that attracts the males for mating. According to Little and Martin (1942) mating usually occurs approximately 24 hours after emergence and oviposition takes place 2 to 8 days later.
Females usually deposit 100 to 200 eggs but this varies with the season and environmental conditions (Philipp and Watson, 1971). The base of the boll seems to be the preferred site for oviposition (Little and Martin, 1942).
According to Noble (1969) eggs hatch in 4 to 5 days in midsummer; but Little and Martin (1942) reported that it may vary from 3 to 12 days. Larvae feed on both squares and bolls, but with special preference to susceptible, spongy, 2 to
3 week old bolls (Van Steenwyck, Ballmer and Reynolds, 1976). The larvae feed for a varying period of time (10 to 15 days) and complete their development within a single square or boll. In fact, Noble (1969) observed that the larval developmental rate is faster in squares than in bolls. 18
After feeding, the fourth larval instar may eat its way out of the boll, drop to the ground and pupate. Pupation normally takes place in the trash or in the soil and the duration of the pupal period varies from 6 to 14 days (Little and Martin, 1942). Diapausing larvae may remain inside one or two seeds spun together or exit the boll, and drop to the ground where they spin cocoons for overwintering. Temperature seems to be the primary factor governing pink bollworm multiplication rate since it affects the developmental time, immature mortality and fecundity (Pearson, 1958; Philipp and Watson, 1971)
The pink bollworm is most damaging when it feeds on cotton bolls and squares, however flowers can also be targets for attack especially during early season. Larvae feeding on squares spin webs resulting in rossetted blooms that prevent the bloom from opening completely. Feeding of larvae on squares and flowers often results in shedding of the reproductive structures (Noble, 1969;
Pearson, 1958).
Damage in bolls from larval feeding results in lower quality lint and seeds, as well as reduction in yield. Lint from heavily infested cotton is of lower grade caused by discoloration of the fiber and reduced fiber length and strength
(Fenton and Owen,1953; Little and Martin, 1942; Pearson, 1958). The cotton seed may be more severely damaged than the lint, accompanied by a loss in weight as well as reduction in the amount and quality of the oil produced (Little and
Martin, 1942; Fenton and Owen, 1953). Moreover, multiple infestations are very common especially late in the season and can render the late bolls practically valueless (Wene, Carruth and Telford, 1965b). Additional loss may be incurred from pink bollworm damage by secondary fungal infestation leading to decomposition of the bolls (Little and Martin, 1942; Noble,1969). 19
Management
The extensive use of chemicals on cotton, killing predators and parasites, has led to outbreaks of secondary pests such as the tobacco bud worm, Heliothis virescens (F.) and the cotton bollworm, Heliothis zea (Boddie). According to
Roelofs (1978) this has resulted in the search for alternative methods of control of the pink bollworm, a key pest of cotton. Moreover, the pink bollworm lays its eggs on the fruiting forms of cotton and the larvae bore into the flower buds or bolls immediately after hatching, rendering chemical control unsatisfactory and expensive (Stern, 1981).
It has long been known that the pink bollworm diapauses in the last larval ins tar , mainly in seeds and bolls remaining in the field after harvest. The place and time of diapause was used to suppress overwintering populations in Texas by modifications of some of the conventional cotton cultural practices
(Adkisson, 1962). Planting and harvesting the crop at a designated time and shredding and plowing up of the crop following harvest have greatly reduced the need for use of insecticides in controlling the pink bollworm in Texas
(Adkisson and Gaines, 1960) and in Arizona (Watson et aI., 1974; 1978).
According to Stern (1981) the success of the Texas program is largely dependent on legislative measures and "social" pressures.
Pheromones have been used in detecting the presence of new infestations and also as communication disruptants for control. Roelofs (1978) predicted that although insect resistance to this method of suppression could develop very slowly, the slow evolutionary changes in the pheromone system together with the option of increasing the rate of release or altering the chemicals in an air permeation program should deJay resistance for many years. 20
Chemical Control
It is well documented that insects are a major constraint in the economic production of cotton. According to Frisbie and Walker (1981): "The control or management of insects attacking the crop can best be described as a scenario of methodology ranging from a multiple-component fairly sophisticated management system to a single factor pesticide-oriented and somewhat inflexible scheme."
Frisbie and Walker (I 981) approached the use of insecticides on cotton insects from a historical perspective: Prior to the second World War, inorganic pesticides such as calcium arsenate were used. These were of limited effectiveness and were not totally accepted by the growers. With the development of the cheap and effective organochlorine compounds in the late
1940's the growers enthusiastically began to apply large amounts of DDT, BHC
(1,2,3,4,5,6-Hexachlorocyclohexane); toxaphene (Chlorinated camphene),aldrin
«I R,4S,5S,8R)- 1,2,3,4, I 0, I O-hexachloro- I ,4,4a,5,8,8a-hexahydro-1 ,4:5,8- dimethanonaphthalene), dieldrin «IR,4S,5S,8R)-1 ,2,3,4,1 0, 10 hexachloro-
I ,4,4a,5,8,8a,hexahydro-1 ,4:5,8-dimethanonaphthalene) and endrin «IR,4S,
5R,8S)1 ,2,3,4,10,10 hexachlorol ,4,4a,5,6, 7 ,8, 8a-octahydro-6,7-epoxy-l, 4:5,8- dimethanonaphthalene). In the excitement of these new developments, old philosophies anti management techniques were put to rest.
Unfortunately this euphoria lasted only 5 to 7 years, for by the mid 1950's insecticide resistance to chlorinated hydrocarbon insecticides had developed and the boll weevil, Anthonomus grandis (Boheman), again became the key pest. A solution to this problem came in the form of organophosphate and carbamate insecticides. Methyl parathion, azinphosmethyl (O,O-dimethyl S-[(4-oxo- I ,2,3- benzotriazin - 3( 4H) - yl)methyl] phosphorodithioate ), malathion (O,O-dimethyl
------21 phosphorodithioate of diethyl mercaptosuccinate or diethyl mercaptosuccinate,S ester with O,Odimethyl phosphorodithioate) EPN (O-ethyl O(4nitrophenyl) phenylphosphonothioate, or ethyl p-nitrophenyl thionobenzenephosphonate) and carbaryl (I-Naphthyl N-methylcarbamate) provided very effective control measures for the resistant boll weevil but were insufficient to control other key pests.
In the early 1960's the bollworm and the tobacco bud worm (TBW), had become resistant to both organochlorine and carbamate insecticides and were fast reaching key pest status. In response, growers used organophosphate insecticides in increasing amounts; consequently resistance began to develop to organophosphates by the late 1960's and early 1970's. Frisbie and Walker(1981), therefore concluded that in retrospect it is very evident that the unilateral use of insecticides has only given temporary relief in the history of cotton production and is therefore an unprofitable and unrealistic undertaking.
Organophosphate Insecticides
In 1911 it was brought to light that the combination of a phosphorous atom with four dissimilar substituents results in the formation of a chiral phosphorous center (Ohkawa,1982). It was soon evident that the biolological activity of the organophosphorous ester was largely determined by the chirality of the phosphorous atom. Gerhard Schrader initiated the pioneering work on organophosphates about 1934 in Germany. After the second World War, the large-scale synthesis of phosphoric acid esters as insecticides increased rapidly in association with their use in crop protection. The most important organophosphate insecticides marketed today are the trialkylphosphates, dialkylphosphates, phosphorothioates and phosphorodithioates (Matsumura, 1975). 22
O'Brien (1978) categorized the organophosphates into four classes, two of which
he called P(S) compounds and two P(O) compounds. He further mentioned that
although the P(S) compounds constitute a major class of insecticides in use, it
is the P(O) derivatives which interest the biochemist since the toxicity of the
P(S) compounds is invariably dependent on their conversion to P(O) derivatives.
Structure and Activity
It is imperative that all biologically active phosphoric acid esters possess
the "acyl" group. This rule links the structure with the biological activity of
organophosphates and denotes that phosphorylation of biologically important
targets should be the mechanism of insecticidal action (Fest and Schmidt, 1973).
All organophophorous compounds mimic the gross molecular shape of their
target enzyme acetylcholinesterase, and this structural complementarity with
their natural substrate is their most conspicuous feature (Matsumura, 1975). The
acti'/e center of acetylcholinesterase has two sites; the "anionic site" and the
"esteric site". According to Cleland (1963), O'Brien (1963), and Ohkawa (1982)
the primary parasympathomimetic effects of organophosphates can be attributed
partly or entirely to the phosphorylation of the serine residue at the active
site. Fukuto and Metcalf (1959) were of the opinion that the inhibitory effect is
also determined by physical and physiochemical properties.
Mechanism of Action: Enzyme inhibition
Unlike pyrethroids, organophosphates lack the selective toxicity between arthropods and mammals. Having neuromuscular junctions that are cholinergic in nature, mammals seem to be even more susceptible to organophosphate insecticides than arthropods. 23
In both mammals and arthropods, organophosphates disrupt nerve conduction by blocking the degradation of acetyl choline to acetic acid and choline, i.e., all organophosphates are anticholinesterases causing the accumulation of acetyl choline at the post synaptic membrane. In vertebrates, acetyl cholinesterase at the neuromuscular junction is one of the most important targets in poisoning. Studies with insects have shown that their neuromuscular junctions are not cholinergic, and synaptic transmission probably involves glutamate (O'Brien, 1978; Usherwood and MachiIi, 1968).
According to Fest and Schmidt (1973), the ganglia of insects are the seat of coordination and are comparable to the central nervous system of vertebrates. In general, acetylcholinesterase in insects appears to be entirely confined to the central nervous system (O'Brien, 1976, 1978). Although different in some aspects, mechanisms comparable to those in the vertebrates are said to be responsible for the toxic effects of organophosphates (Colhoun, 1963). The severe disruption of the cholinergic synapses caused by the toxic agents destroy the communication system leading to death.
Metabolism
Organophosphorous compounds undergo various metabolic reactions in living organisms. Compounds having similar structure are subject to common biotransformation reactions mediated largely by mixed-function oxidases, arylesterases, and glutathione S-transferases (Ohkawa, 1982).
I. Oxidation:
O~idation is one of the most important degradation reactions especially of the thiono-phosphates. Thiophosphoryl (P=S) esters are stereospecifically metabolized to the corresonding phosphoryl (P=O) esters in many organisms
------_._------24
(Ohkawa et aI., 1976). Contrary to expectations, this reaction enhances toxic action instead of resulting in detoxification (Fest and Schmidt, 1973). The P=S esters being poor an ti -cholinesterases, mixed-function oxidase-mediated
activation is necessary for intoxication by thiono-phosphates (Ohkawa, 1982). In
the vertebrates, these oxidation reactions occur in the liver microsomes (Brodie
and Gillette, 1958) whereas in insects they take place in the fat body.
According to Kilby (1963), functionally, the fat body is the liver of the insects;
serving as a depot for metabolic products and actively participates in metabolic
processes. Thus, microsomal oxidation may be an obligatory step in the
activation of a latent toxicant or may totally detoxify an insecticide in a single
step. An example of direct detoxication is the O-deethylation of
chlorfenvinphos, 2-chloro-(2,4-dichlorophenyl)-vinyl diethyl-phosphate, or 0,0- diethyl-0-1-(2' ,4'-dichloro-phenyI)-2-chlorovinylphosphate, whereas the oxidation of azinphosmethyl, desulfuration of parathion, O,O-diethyl 0-4-nitrophenyl phosphorothioate, and N -methyl hydroxylation of schradan, octamethylpyrophosphoramide, are classical examples of activation (Nakatsugawa and Dahm, 1962; Wilkinson, 1976).
2. Hydrolysis:
The detoxification of organophosphates by a variety of hydrolases is by far the most important mechanism of degradation of these toxicants (Fest and
Schmidt, 1973). Wilkinson (1976) stated that organophosphorus compounds
undergo enzymatic hydrolysis at either the ester or the acid anhydride bond.
According to "Shrader's rule" organophosphorus insecticides are usually constructed in such a way that one of the three ester groups is more susceptible to hydrolysis. Hence enzymatic hydrolysis proceeds with the removal 25 of the toxophoric phenol, enol, alcohol or mercaptan (Fest and Schmidt, 1973).
Krueger and Casida (I 961) investigated the in vitro hydrolysis of several organophosphorus insecticides in preparations from several insects and mammalian tissues. Their results indicated specificity of the hydrolytic enzymes to the phosphates since many of the phosphorothionates studied did not undergo hydrolysis. Wilkinson (1976) reported that later work by several researchers on insects and mammalian hydrolases yielded somewhat confusing results regarding the substrate specificity of the phosphotriester hydrolases.
3. Conjugation:
Conjugation reactions are energy dependent biosynthetic processes whereby natural or foreign compounds or their metabolites combine with readily available, endogeneous conjugating agents to form conjugants. Classic examples of conjugating agents include glucuronic acids, sulfate, acetyl, methyl, and glycine (Wilkinson, 1976).
Direct enzymatic attack on the substrate is a characteristic of the glutathione-S-hydrolases(GSH)-dependent transferase reaction. The importance of these enzymes in the metabolism of a large number of phosphorothionate and phosphate insecticides has been well documented (Wilkinson, 1976). Studies performed by Fukami and Shishido (1966) demonstrated the importance of GSH in the dealkylation of methyl and ethyl parathion by a soluble enzyme in rat liver and insect tissues. Since then many insecticides in various organisms have been shown to be degraded by GSH-dependent transferases (Wilkinson, 1976).
Pyrethroid Insecticides
Pyrethroids are synthetic analogues of pyrethrum which include
------_._------26 pyrethrins, cinerins and jasmoIin. Pyrethrum naturally occurs in the flowers of
Chrysanthemum spp., family Compositae (Matsumura, 1975). All pyrethroids are esters and are thus composed of an alcohol and an acid moiety. Crombie (1980) stated that there are six naturally occuring pyrethrins derived from combinations of two different acids and three different alcohols collectively referred to as "rethrins". These are pyrethrin I and II, cinerin I and II and jasmoIin I and II. Pyrethrin I is believed to be found in high concentrations of the aforementioned.
Pyrethroids are lipophilic compounds very active as contact and possibly stomach insecticides. They are highly selective, possessing an average selectivity factor of 4500 as compared to 16, 33, and 91 of carbamates, organophosphates, and organochlorines, respectively (Elliott and Janes, 1978). Comparatively, natural pyrethrins are more effective knockdown agents than synthethic pyrethroids although the latter are much more lethal to insects (Burt and
Goodchild, 1974).
Mode of Action
Elliott (1971) reported that the insecticidal action of pyrethroids is dependent upon important factors some of which are : rate of penetration, transport to the site of action, degree of susceptibility to detoxifying mechanisms and appropriate fitness of molecular structure at the sites of action. According to Miller and Adams (1982) and Narahashi (1976) the neurophysiological responses vary with the type of nerve element assayed, temperature, and the insecticide applied. These responses include negative after potentials, repetitive after discharges, suppression of action potential, block of impulse conductance or transmission at the neuromuscular junctions, and 27 spontaneous deplorization of the resting potential.
The primary studies on the mode of action of pyrethroid insecticides were
performed by Narahashi (l962a, 1962b, 1971). He reported that pyrethroids act directly on the nerve membrane producing symptoms of hyperexcitation. He also
reported that allethrin, (RS)-3-allyl-2-methyl-4-oxocyclopent-2-enyl(RS)- cis/trans chrysanthemate, modifies the axonal conduction to the central
nervous system of arthropods by changing the permeability of the nerve membrane to sodium and potassium ions. In a 1971 study he deduced a direct relationship between the blockage of axonal conduction and the paralysis of the poisoned insects. In fact, Camougis (l973) and Narahashi (l971) were in agreement on the mode of action of pyrethroids on invertebrates. They both believe that pyrethroids create changes in sodium and potassium currents that cause the malfunction of the nerve. The fact that synapses are affected at concentrations considerably lower than those effective on nerve fibers led
Narahashi (l976) to be~ieve that synapses are possibly the initial target site of pyrethroids. Different nerve elements, peripheral and central, have been assayed in order to elucidate the mechanism of action of pyrethroids.
Effects on Peripheral Nerve Elements:
Gammon (l978) found that when allethrin-treated cockroaches were subject to mechanical or electrical stimuli applied to the cerci, prolonged sensory responses were produced. In their studies on locusts, Clements and May (1977) determined that topical treatment with a variety of pyrethroid analogues resulted in prolonged firing in the crural nerve of the jumping leg and was restricted to sensory axons. Interestingly, the chordotonal sensory organ was found to be the source of the responses encountered. 28
According to Adams and Miller (1979), the availability of the dipteran flight motor systems has greatly facilitated in-vivo studies of pyrethroid action on motor elements. They deduced that the repetetive backfiring in the flight motor units occurred early during poisoning by some pyrethroids such as tetramethrin 3,4,5,6-tetrahydrophthalimidomethyl chrysanthemate, permethrin, 3-
(phenoxyphenyl) methyl (IRS) -cis,trans-3-(2,2-dichloroethenyl) -2,2-dimethyl cyclopropane carboxylate (approximately 60%trans, 40%cis isomers), cismethrin, and several DDT-type analogues and this effect was temperature correlated. No repetitive firing was observed when poisoned with deltamethrin, (S)-alpha- cyano-m-phenoxybenzyl (IR,3R)-3(2,2-dibromovinyl)2,2dimethylcyclopropane- carboxylate, kadethrin and fenvalerate, (RS)-alpha-cyano-3-phenoxybenzyl (Rs)-
2-( 4-Chlorophenyl)-3-methylbutyrate or cyano(3-phenoxyphenyl)methyl 4-chloro- alpha-( I-methylethyl)benzeneacetate or alpha-cyano-3-phenoxybenzyl 2-(4- chlorophenyl)-3-methylbutyrate. Salgado et aI., (1983) performed detailed studies on the toxic effect of pyrethroids on motor nerve terminals and the results seem to indicate that toxicity was correlated negatively with temperature and that repetitive firing was associated with knockdown.
Recent reports denote that changes in hormonal balance of insects could result from the action of pyrethroids on neurosecretory neurons. Orchard and
Osborne (1980) determined that the exposure of neurosecretory nerve endings of the stick insect Carausius morosius, to extremely low concentrations of pyrethroids resulted in a significant increase in firing activity.
Effects on the Central Nervous System
In 1971, Narahashi reported that direct application of pyrethroid to the central nervous system of cockroaches led to an increase in spontaneous firing
------29 and bursts of action potential, and increased concentrations resulted in a block in spontaneous activity. Miller and Adams (1982) found that pyrethroids cause the uncoupling of flight motor neurons early in poisoning indicating that pyrethroids act on the central nervous system. Based on their findings, they concluded that pyrethroids not only act at the peripheral nervous system like
DDT, but can also affect the central nervous system, and that peripheral responses often tend to mask the more important central effects.
Physiology of Knockdown:
Mode of action of pyrethroids is closely linked with the mechanism of knockdown. Briggs et a\., (1974) found that knockdown increased with polarity up to a point, and then, when compounds are too polar there was a decrease in knockdown activity. They inferred that separate sites of action were present for knockdown and lethal action. On comparing the knockdown times of pen flies (having the gene pen for reduced penetration) to those without the gene,
Burt and Goodchild (1974) found that the pen flies were knocked down more slowly when pyrethroids were topically applied. There was no difference between the strains when the toxicant was injected. They concluded that: knockdo\ III site of action is central, knockdown may be the beginning of the toxic process and that the rate of penetration plays a minor role in ultimate toxicity.
Clements and May (1977) suggested that the physiological action inducing knockdown is dependent on molecular structure. Salgado et a\., (I983) observed that application of deltamethrin and fenvalerate at nano molar (nM) concentrations to a house fly larval neuromuscular preparation blocked neuromuscular transmission. Further investigations with deltamethrin led them to suggest that deItamethrin depolarizes nerves by modifying sodium channels. They 30 also found that knockdown resistant larvae were resistant to the depolarizing effect of deltamethrin indicating that the kdr gene modified the sodium channels rendering them less sensitive to pyrethroids. Clearly, there is lack of agreement on the mechanism of action of the pyrethroids.
Structural Activity
One of the major problems encountered with pyrethrum is its photo instability. This problem was overcome in synthetic pyrethroids (esters evolved from the natural pyrethrin I); and in 1973, Elliott et ai. synthesized a photostable synthetic pyrethroid, permethrin. The substitution of dihalovinyl group in the acid moiety and the incorporation of 3-phenoxybenzyl alcohols have improved the photostability of the compounds to metabolism and degradation (Miller and Adams, 1982). They further mentioned that the structural conformation of pyrethroids determines insecticidal activity and that the optically pure isomer often possesses far greater activity than the corresponding enantiomer.
According to Elliott (1977), all pyrethroids are Iipophylic and in this respect resemble chlorinated hydrocarbons and differ from most organophosphates and carbamates.
The fact that pyrethroid insecticides are effective at extremely low dosages and that minute changes in their structural configuration greatly alter their efficacy led Elliott et al. (1978) to conclude that a very specific interference with the biological system must be a part of their mechanism of action. According to Briggs et aI., (1976), the polarity of the molecule probably dictates the rate of penetration and the degree of susceptibility to the detoxifying agents. Clement & May (1977) suggest that pyrethroids that possess 31 an appropriate molecular configuration may be good knockdown agents and vice versa. If this hypothesis is correct, the authors argued, then polarity is not a critical factor for knockdown activity. They added that, obviously a compound that acts so rapidly to produce a knockdown must pass rapidly to the site of action but the site may not be very deep within the insect. Elliott and Janes
(1978) observed a more rapid knockdown of houseflies when pyrethroids were injected rather than when topically applied. These findings led to the assumption that pyrethroids act on the central nervous system rather than peripheral sites of the cuticle.
Metabolism
Chang and Kearns (1964) deduced from their studies on house flies that the major detoxication mechanism of pyrethrin I and cinerin I was due to oxidation and not ester hydrolysis. Studies performed by Elliott et al. (l972) on metabolism of pyrethrin I and allethrin in rats revealed that the methyl group of the acid side chain in both pyrethrin I and allethrin, and the pentadienyl side chain of pyrethrin I were subject to oxidation. They detected limited hydrolysis of allethrin but not of pyrethrin I.
Synthesis of more stable compounds in which changes were made in both the acid and the alcohol moieties were initated in order to overcome the rapid oxidation of natural pyrethrins. For example, in resmethrin, 5-benzyl-3- furylmethyl (IRS)-cis,trans-chrysanthemate or [{5-(phenylmethyl)··3- furanyl}methyl 2,2-dimethyl-3-(2-methyl-I-propnyl)cyclopropane carboxylate, the more stable 5-benzyl-3-furylmethyl alcohol is coupled to chrysanthemic acid, whereas in permethrin, the two methyl groups in the isobutenyl side chain of chrysanthemic have been replaced with chlorine atoms. According to De Vries
_._. __ .._. __ ._------32
(I979), the natural pyrethrins are less susceptible to hydrolysis primarily
because they are esters of secondary alcohols while in contrast, the 5-benzyl-3-
furylmethyl and 3-phenoxybenzyl alcohols are primary alcohols and can be more
readily hydrolysed. According to him it is therefore comprehend able that in
both insects and mammals, permethrin undergoes both hydrolytic and oxidative degradation.
Cypermethrin, alpha - cyano-3 - phenoxybenzyl(±)cis, trans,3 - (2,2 -dichloro
vinyl)-2,2-dimethyl cyclopropane carboxylate, decamethrin and fenvalerate
belong to a newer generation of pyrethroids possessing a cyano group in the a-carbon of the 3-phenoxy benzyl alcohols. Elliott (I 977) proposed that this
addition of the alpha-cyano group transforms the primary alcohol into secondary
alcohol, thus stablizing the ester bond either directly or through interference
with proper fit at the esteric site.
Based on the information they obtained from studying the hydrolytic and
oxidative metabolism of 44 different pyrethroids in a mouse liver microsomal
system, Soderland and Cas ida (I 977) divided pyrethroids into three groups with
respect to ease of detoxication. According to their classification the most
rapidly degraded compounds are the primary alcohol esters of trans-substituted chrysanthemic acids undergoing rapid hydrolysis and oxidation, while the x cyano-3-phenoxybenzyl esters are least susceptible to metabolic attack by either hydrolysis or oxidation.
Resistance
In the last several decades, many insect species have developed resistance to insecticides. Early examples include the resistance of San Jose scale to lime sulfur in 1914 and of California red scale, Anonidiella aurantii, to hydrogen
---~----~------33 cyanide in 1916. Today, insects display resistance to every class of insecticide available rendering resistance as one of the most challenging problems facing entomologists.
Resistance is heritable and is a consequence of basic evolutionary processes. It evolves as a result of natural selection acting upon variant populations (Crow, 1957; Brown and Pal, 1971) exemplifying the power and potential of adaptive evolution. This selection has greatly been enhanced by the farmers' high reliance on pesticide spraying which is probably a major cause of resistance (Georghiou, 1986). It has been observed that resistance to insecticides evolves more frequently in insect species characterized by a high reproductive rate i.e., a high potential rate of increase and a short life cycle resulting in more generations per unit time. These two criteria led farmers to use additional insecticidal treatments in order to suppress the pest populations. But this malpractice induced resistant individuals to dominate the populations and pass on their resistance genes to the succeeding generations through inheritance which is the most important characteristic of resistance (Brown and Pal, 1971).
Although resistance has a history of more than 75 years, its greatest impact had not been realized until the discovery and the extensive use of synthetic organic compounds during the last 40 years (Georghiou, 1986). In 1938, only seven species of insects and mites were reported resistant to DDT while in
1984, 447 species were reported resistant to all principal insecticide classes:
DDT, cyclodienes, organophosphates, carbamates and pyrethroids. Georghiou
(1986) reported that 97% of the resistant species were of agricultural or medical importance. Due to the very high selection pressure imposed on them resistance is frequently seen among orders of insects including major pests such as Diptera
(35% of the total), Lepidoptera (15%), Homoptera (10%) and Heteroptera (4%). 34
Moreover, he is of the opinion that the decline in the rate of increase in resistant arthropod species since 1980 could be attributed to the fact that most new cases of resistance to insecticides are noted in species that had earlier been recorded as resistant to previous pesticides. Therefore a count on number of different classes of insecticides to which each species is resistant would give us a more realistic assessment of the current trend. On considering classes of insecticides, 62% of the resistant species reported are resistant to cyclodiene insecticides, 52% to DDT, 47% to organophosphates and lower percentages in more recent introductions pyrethroids and carbamates. Although organophosphates were considered to be less efficient selectors of resistance in lieu of their shorter persistence, the high level of resistance attained in species to this class of insecticides could be attributed to their widespread use
(Georghiou, 1986).
Mechanisms of Resistance
As mentioned by Dauterman (1983), insects possess a complexity of mechanisms responsible for insecticide resistance that may vary from one species to the other and even among the strains of the same species. It is well known that in addition to biochemical mechanisms, penetration mechanisms, alterations in site sensitivity and behavioral avoidance patterns may also contribute to resistance. According to Fest and Schmidt (1983) genetically fixed tolerance seems to play a more significant role as a mechanism of resistance than the physiological mechanisms. But Dauterman (1983) reported that most workers agree that resistance to insecticides in insects is influenced mainly by genetically controlled physiological factors such as reduced penetration, diminished sensitivity of the target site, and enhanced detoxication. He was also
- ---~------_. ------_.------35 of the opinion that the structure of the insecticide and the stage of development of the insect determine the importance of each mechanism.
Resistance is often a complex phenomenon with several mechanisms operating together and joint action of two or more mechanisms in the same resistant strain is of common occurence. Also resistance is known to evolve more easily when more avenues are open. Sawicki (1970) demonstrated that in the house fly Musca domestica L. both reduced penetration of insecticide through the cuticle and increased degradation are causes of resistance. In the tobacco budworm all three factors: decreased penetration, increased detoxication, and decreased sensitivity at the site of action contribute towards resistance (Bull, 1981).
Non-Metabolic Resistance
I.Reduced Penetration:
The idea that reduced penetration of insecticides through the cuticle of insects may be a cause of resistance is not new. Sanchez and Sherman (1966) partly attributed resistance to DDT in a strain of tobacco bud worm to reduced penetration. Szeiez et aI., (1973), reported that rates of penetration of carbaryl, malathion, endrin, and DDT were more rapid and reached higher concentrations in internal tissues of tobacco bud worm larvae of the susceptible strain. In fact, even insecticides within the same group may vary in their capacity to penetrate through the cuticle of the same insect species. This phenomenon has been confirmed by Holden (1979) who reported that the penetration of permethrin (approximately 60% trans, 40% cis isomers) through the cuticle of the american cockroach, Periplaneta americana L. was significantly greater than that of cypermethrin. 36
It was elucidated by Plapp and Hoyer (1968) that when the decreased penetration factor was genetically combined with other resistance factors, the degree of resistance increased much beyond the level expected by a simple additive effect. Working with diazinon-selected strains of house flies, Sawicki
(l970) confirmed that the penetration-delaying factor alone confers little resistance; however, when combined with the desethylating factor resistance to many organophosphates is greatly increased in comparison to the effect of the desethylating factor alone. This indicates that interaction of both factors is greater against the thionates than against the corresponding phosphates.
According to Matsumura (1983) the existence of a penetration barrier at the target level (nervous system) has been demonstrated using dieldrin-resistant
German cockroaches. The kinetics of penetration into the nervous system would very probably be different from those of cuticular penetration. Matsumura (1983) postulates that penetration at the target site appears to be more important as a determinant of resistance with stable insecticides (eg. dieldrin) while cuticular penetration would play a significant role in confering resistance to easily metabolized insecticides such as malathion. He also stated that the mechanism of reduced penetration, its lack of specificity, and the effectiveness of such mechanism with stable insecticides require further investigations.
2. Target insensitivity:
The term "site insensitivity" was first coined to explain the knockdown resistance (kdr) factor which renders the nervous system less sensitive to DDT and pyrethroids (Wilkir.son,1983 ; Farnham, 1977). Matsumura (1983) explained that target or site insensitivity is the resistance caused by the reduction in the amount of toxicant reaching the "target" and reducing the sensitivity of this 37 site to the toxicant. In recent years, many studies have been performed on elucidating the mechanism of target site resistance. The availability of pure genetic stocks representing individual mechanisms has greatly contributed to the progress of these studies (Plapp, 1986).
Milani (1962) reported that knockdown resistance was fairly common among fly populations of the Latin district of Italy where the flies had been extensively treated with DDT. Cross resistance due to knockdown resistance extends to DDT, DDT analogues and to pyrethroids but not to cyclodienes or organophosphates. He concluded that the presence of the kdr gene in a population due to prior use of DDT leads to a decrease in the useful life of pyrethroids, but due to the recessive nature of the genes, natural dilution of the population after selection is removed would help to increase the number of susceptible heterozygotes. Intensive studies on house flies using DDT and pyrethroids were conducted by Chang and Plapp (1983a,b,c). These studies have revealed that resistant flies possessing kdr genes have a smaller number of target site receptors than susceptible ones. The studies also demonstrated that target site receptors of both susceptible and resistant strains have the same
binding affinity for DDT and pyrethroids. It was hence concluded that the difference between the two strains was 'strictly' quantitative. According to
Plapp (1986), decrease in number of receptor sites may be associated with target site resistance to DDTJpyrethroids and also cyclodienes. He proposed that decrease in receptor numbers probably makes it more difficult for the insecticide to reach the target site and thus confers resistance.
It has also been demonstrated that modification of the target enzyme, acetyl cholinesterase could result in resistance to organophosphates and carbamates (Dauterman and Hodgson, 1978). They mentioned that differences in 38 acetyl cholinesterase between susceptible and resistant strains of ticks, mites, house flies, and green leaf-hoppers have been reported. These differences rendered the acetyl cholinesterase (ACHE) of the resistant strain less susceptible to inhibition by cholinesterase inhibitors such as organophosphate and carbamate insecticides. In fact, Plapp (1986) reports resistance involving altered ACHE is perhaps the best understood resistance mechanism.
According to Matsumura (1983), site insensitivity as a mechanism of resistance is important and effective because it confers protection to the sensory system long enough for the poison to be eliminated via metabolism, excretion or storage in non-vital tissues.
3. Genes Responsible for Non-Metabolic Resistance:
Several studies have been conducted on house flies and have considerably added to our knowledge in understanding the genetics of resistance. The outcome of these studies has led to identifying the following resistance genes.
(i) Pen : It reduces the rate of penetration of insecticides. It is located on chromosome III and is inherited as a simple recessive. By itself, it seldom confers more than 3-fold resistance to any insecticide. It seems to be more important as an intensifier of other resistance genes and may consequently double the resistance levels (Sawicki and Farnham, 1968). Sawicki and Lord
(1970) found that flies with the pen factor had delayed knockdown accompanied by an increase in the amount of the insecticide remaining on the surface .
Previous studies (Forgash et aI., 1962) revealed that the tin factor was responsible for decreased penetration of organotin compounds in a parathion resistant strain of house fly. But, Sawicki and Lord (1970) determined that tin and pen factors are the same. 39
(ii) Kdr : It causes knockdown resistance to DDT and pyrethroids. Like pen, it is also located on chromosome III, but at a distinct locus. It is inherited in a recessive fashion and probably causes modification at the target site of action that leads to insensitivity. Like pen, it behaves as a modifier gene enhancing the effects of metabolic resistance mechanisms (Farnham, 19'77).
(iii) dld-r : It confers resistance to dieldrin and other cyclodienes. It is located on chromosome IV and is characterized by an incomplete recessive type of inheritance. Resistance to these insecticides also seems to involve changes at the target site.
(iv) ACHE : It confers resistance to organophosphate and carbamate insecticides.
It is located on chromosome II and exhibits a dominant type of inheritance.
According to Oppenoorth (1982) different allelles seem to vary in their capacity of confering resistance to organophosphate and carbamate insecticides.
Metabolic Resistance
Detoxication systems in insects have evolved over millions of years enabling them to survive naturally occuring toxicants, including the secondary plant products. Thus, as mentioned by Wilkinson (1983) "The development of insect resistance to insecticides through enhanced enzymatic detoxication should be considered as an accelerated version of the natural selection process which has been operating since life began. The insects were forewarned and forarmed to meet the challenges posed to them by the advent of modern synthetic insecticides."
Since metabolic factors generally play the most significant role in the overall biochemical defense system for developing resistance in insects, it is not surprising that the most studied aspect of resistance mechanisms is metabolic 40
resistance (Oppenoorth and Welling, 1976). According to Plapp and Wang (1983)
metabolic resistance to insecticides is most important in biodegradable
insecticides such as organophosphates and carbamates, and probably with
synthetic pyrethroids.
According to Plapp (1986) the phenomenon of enzyme induction is the key
to metabolic resistance. Previous studies ( Walker and Terriere, 1970) have
revealed that the classic insecticide DDT and certain cyclodienes act as inducers
of enzymes that breakdown a range of insecticides. Plapp (1984) described the
induction of different detoxifying enzymes, e.g., oxidases, esterases, and
glutathione transferases as being coordinate i.e., exposure to chemicals that
induce one detoxifying enzyme induce several. In this respect, the same author
stated that mixed function oxidases, glutathione transferases and DDT
dehydrochlorinase are coordinately induced in the house fly. Terriere and Yu
(1974) believed that there is a remote possibility that enzyme induction will
enhance resistance under field condition since it requires very high concentration of the inccticides.
1. Mixed Function Oxidages:
Mixed function oxidases (MFO) are ubiquitous in distribution, have had a long history as naturally occuring protectants, and are remarkable in their catalytic versatility towards many naturally occuring and synthetic insecticides
(Oppenoorth and Welling, 1976). The authors further state that the mixed function oxidases play a dominant role in determining the toxicity and biological activity of a given chemical, and as a result it is to be expected that organisms possessing high quantities of MFO's exhibit high tolerance to many chemicals.
Wilkinson (1983) also strongly supports the idea that MFO's are known to play 41
an important role in the development of resistance in many insects.
Since its discovery in 1967, increased levels of cytochrome P-450 have
been reported for a number of resistant house fly strains possessing high oxidase titers (Dauterman and Hodgson, 1978). According to Hodgson and
Kulkarni (1983), the high oxidase levels often associated with resistance are due to the microsomal P-450-dependent monoxygenase system. Many forms of P-450 are found in both susceptible and resistant insects and differences are seen in the qualitative characters of the complex of P-450's between strains.
Using various strains of house flies, evidence has accumulated that carbamates appear to be metabolized almost exclusively by MFOs and as a result house flies have developed high levels of resistance to carbamates (Wilkinson,
197 I). He also stated that the results obtained with organophosphates are more complex and more difficult to predict. Studies using synergists have indicated
that MFOs are important in the resistance of various strains of insects to DDT,
pyrethrins, the carbamates, several organophosphates and even some of the
newer group of insecticides (Wilkinson, 1983).
2. Hydrolases and Glutathione S-Transferases:
The hydrolytic enzymes and glutathione S-transferase are responsible for organophosphorus insecticide resistance in some species and strains of insects
(Dauterman, 1983). He proposed that these mechanisms often act in concert with other resistance mechanisms such as MFOs, altered cholinesterases and reduced penetration and their importance is determined by the structure of insecticide, its concentration, and possibly the stage of development of the insect. 42
(i) Hydrolases:
In the thiono-phosphates (parathion series), the most important mechanism of resistance to the organophosphorus insecticides is the potentiation of enzymatic hydrolysis in the insect (Fest and Schmidt, 1973). They also
mentioned that resistance is not necessarily a result of selection but it may be
due to histological changes occuring during certain stages of the life cycle.
Early observations on organophosphate-resistant houseflies by Oppenoorth and
Asperen (1960) showed that the "aliesterases" originally present had been
transformed by gene mutation into "modified aliesterases" or phosphatases which could act as degrading enzymes for all organophosphates. According to
Dauterman (1983) detoxication of organophosphates by hydrolases is of rare occurrance and has been found in only a few resistant strains of houseflies and a few other insect species. He mentioned that hydrolases play a small role in resistance and are usually associated with other more important resistance mechanisms.
(ii) Glutathione S-Transferases:
Glutathione S- transferase mediated metabolism has been implied as a biochemical mechanism contributing to organophosphorus insecticide resistance.
Lewis (I 969) was the first to report that several house fly strains resistant to diazinon «O,O-Diethyl O-(2isipropyl-4-methyl-6-pyrimidinyl)phosphorothiote) and having a gene "a" for low aliesterase activity, also, possessed high glutathione s- transferase activity towards diazinon and diazoxon. In a later study, Lewis and Sawicki (1971) confirmed that certain house fly strains having low aliesterase and high phosphatase activity also possessed high glutathione S transferase activity. According to Dauterman and Hodgson (1978), studies have 43 indicated the presence of qualitative and quantitative differences in the enzyme from susceptible and resistant strains.
3. Carboxylesterases:
Unusually high levels of carboxylesterases have been recorded in several organophophorus resistant strains of a number of insect species (Dauterman and
Hodgson, 1978). According to Yasutomi (1983) detoxication by enzymes hydrolysing the insecticides is the main mechanism of OP resistance. He mentioned that malathion-resistant-strains of insects are characterized by increased carboxylesterase, which attack the unique weak points of this OP molecule. Dauterman and Hodgson (1978) reported that high levels of carboxylesterase were seen in several organophosphate resistant strains of a number of insect species when compared with their susceptible counterparts:
Culex tarsalis, HeIiothis virescens, and others. They proposed that the carboxylesterases of the resistant strains may be qualitatively and quantitatively different from those of the susceptible strains.
4. Genes Responsible for Metabolic Resistance:
In the foregoing, the different detoxifying enzymes associated with metabolic resistance to insecticides have been discussed. Busvine (1963) reported that organophosphate resistance is usually controlled by a single dominant gene although the physiology of resistance is of a complex nature. In the same year,
Keiding (1963) described diazinon resistance of Danish strains of the house fly and proposed that this resistance seemed to be controlled by two dominant genes. In recent years detailed genetic and biochemical studies have shown that resistance associated with all those processes is controlled mainly by genes
------44 located on chromosome II (Oppenoorth et aI., 1979) and inheritance seems to be intermediate to incompletely dominant. According to Plapp and Wang (1983) the most plausible deductions from these results would be that gene(s) on chromosome II are structural genes dealing with the changes in the biochemical process involved.
According to Plapp (1986), the major gene responsible for metabolic resistance induces the synthesis of the specific detoxifying enzyme. Such a gene has a qualitative effect and its product seems to be a receptor protein which recognizes and binds to insecticides followed by induction of enzyme synthesis.
The same author stated that this gene is inherited in a codominant fashion.
Selection for Resistance
The rate of development of resistance in insect populations has been evaluated in many cases using selection pressure under laboraory conditions. The results of these studies were summarized by Georghiou and Taylor (1976) as follows: "In most cases resistance evolves gradually at first, during a period of integration of various background ancillary factors in the genome of the population and subsequently at a faster rate to a maximal level which is dependent on the phenotypic expression of the R gene(s) in the resistant homozygote". The same authors believed that such studies generate the bioassay required for the determination of mechanisms of resistance, but they fall short in providing the information required for prognosis of the evolution of resistance under field conditions. Indeed, observations in the field have revealed that the rate of emergence of resistance varies not only among different species treated with the same insecticide but also among different populations of the same species (Bishop, 1981). 45
According to most population genetics theories, resistance in the field is
controlled by or 2 loci (Plapp, 1976). Whitten and McKenzie (1982) believed
that pesticide resistance usually has a simple genetic basis and that although
polygenic resistance may occur in nature, it is much more common in laboratory
trials. The same authors developed a conceptual model to explain this
difference. The model also identifies the conditions in the field which bring
about the selection of rare resistant alleles, the initial frequencies of which are
dependent on a balance between mutation and selection.
The review on inheritance of resistance by Crow (1957) gives no clear cut
answers concerning the number of loci involved in resistance. But since the
1960's a wealth of evidence has accumulated using more advanced techniques of
assays and data analysis. In a recent review by Roush and McKenzie (1987), it
was reported that available evidence suggests that resistance was due to allelic
variations at a single locus. Moreover, they mentioned that most of the
confusion regarding the number of genes responsible for resistance arises as a
result of laboratory selection studies, which usually lead to the development of
polygenic resistance. They ascribed the failure of laboratory strains to develop
monogenic resistance to genetic bottlenecks which are always present during
laboratory colonization, i.e., it is unlikely that any laboratory strain would
possess the major resistance alleles for pesticides that its an~estors had not
. been exposed to. Via(I986) felt that this dichotomy witnessed between
laboratory and field selection in the evolution of resistance indicates that it is
the very strong selection pressure imposed by the current regimes of pesticide
application and not any inbuilt bias which is reponsible for the genetic basis of
character variation. In contrast to laboratory selections where small genes are
accumulated over a long period of time (polygenically inherited resistance) field 46 selection regimes confer high levels of resistance under the control of simple
genes (Roush and McKenzie, 1987). They further mentioned a hypothesis that postulates the faster spread of monogenic resistance under field conditions.
Taylor and Georghiou(I982) believed that the newer methods of pesticide
application intent on lowering the effect of natural selection would cause
polygenic resistance to become more widespread.
This difference in the pattern of emergence of resistance may also be true
for reversion of resistance. Chen and Sun (1986) reported that when selection
pressure on larvae of the diamondback moth, Plutella xylostella, was relaxed,
pyrethroid and carbamate resistance persisted for a longer period of time than
organophosphorus resistance.
Cross Resistance
Investigating the mechanisms of resistance in house flies, Busvine (I 951)
determined that the Italian strain was resistant to both DDT and pyrethrins.
This phenomenon which was once described by the author as "surprising and
inexplicable" is commonly referred to in modern literature as cross resistance.
Today, it is generally accepted that pest populations that are already resistant
to one or more classes of pesticide compounds generally develop resistance more readily to new groups of chemicals having similar mode of action or metabolic pathways of detoxication (Georghiou, 1986).
Some degree of cross resistance is often witnessed between pesticides within the same class, and among classes depending on the mechanism of resistance. The intensity of the problem is clearly seen among the major classes of synthetic organic insecticides developed since World War II. All four classes are neurotoxins, but most insecticides have two sites of action in the nervous
---- -~-~------~--~------47 system: acetylcholinesterase for carbamates and organophosphates and nerve sensitivity for DDT and pyrethroids (Hammock and Soderlund, 1986). They mentioned that, genetic mechanisms altering these sites of action (gene kdr for
DDT and pyrethroids and "insensitive" ACHE for OP's and carbamates) resulted in cross resistance that renders entire classes of compounds ineffective.
Moreover, these mechanisms of resistance cannot be countered by the use of synergists.
It has been of great concern that since resistance to DDT is widespread it predisposes some insects to develop cross resistance to pyrethroids, thus shortening their effective life. In fact, Farnham and Sawicki (I976) believed that the rapid development of resistance to pyrethroids may have been facilitated by previous exposure to or by resistance to insecticides of unrelated groups. One year later Chadwick et aI., (1977) reported that a high level of
DDT -resistance in Aedes aegypti was associated with moderate levels of pyrethroid resistance. Despite the fact that DDT and pyrethroids belong to two different and chemically unrelated insecticide groups, they have some properties in common : a negative temperature coefficient, i.e., more toxic at lower temperatures, and poisoning the nerve in a similar manner. Moreover, the knockdown resistance gene ,Kdr, has been identified in several species as responsible for confering cross resistance to both DDT and pyrethroids
(Farnham, 1977; De Vries and Georghiou, 1980; Miller et aI., 1983).
According to Georghiou (I986), toxicological, genetic, and electrophysiological studies have shown beyond doubt that a semirecessive gene, kdr, is selected for ana provides protection against DDT as well as pyrethroid insecticides. He further states that when this gene is present, resistance to pyrethroids can often exceed 1000-fold in the kdr- homozygotes, in effect, 48
precluding their future use. The consequences would be more drastic in
developing countries where the use of pyrethroids directly succeeded that of
DDT than in the developed nations where several years of organophosphate and
carbamate use preceeded the use of pyrethroids (Georghiou, 1986).
Sawicki et al. (1984) showed that house flies selected with two organophosphates, malathion and trichlorfon, dimethyl (2,2,2-trichloro-l hydroxyethyl phospho nate), developed an esterase, E.0.33, that conferred only mild cross resistance to pyrethroids and the doses normally used continued to
be effective. However, use of pyrethroids on populations of flies which had
previously been exposed to DDT (kdr gene present at low frequencies), led to
the simultaneous selection of both kdr and the esterase, and the pyrethroids
were rendered ineffective. They concluded that the previous sequential use of
two unrelated classes of insecticides, DDT and organophosphates, led to the
failure of a third class (pyrethroids) by selecting for common mechanisms of
resistance. Frisbie and Walker (1981) reported that pests capable of several
degrees of cross resistance such as the tobacco budworm should be given special
attention since they are high risk candidates for the development of resistance
to pyrethroids.
Materials demonstrating negative cross resistance could be very valuable in
resistance management. Such materials cause decrease in resistance to other chemicals as resistance to them increases. Although this phenomenon has been documented in Homoptera (Ozaki, 1980), its benefits have not been proved under field conditions (Sawicki, 1981). According to Georghiou (1986) the ability of a population of organisms to accumulate many different mechanisms of resistance gives us an insight into the magnitude of the problem we face. He further mentioned that none of the known mechanisms found in field populations
~~~---~---~------~---- -~------49 excludes the evolution of any other mechanism, and no compound with negatively correlated resistance has been discovered which can be put to field use. Moreover, the problem is confounded further by the coexistence of several resistance mechanisms (multiresistance) which has become increasingly common among arthropod species. He reported that 17 insect species including key pests such as Heliothis Spp. and the Colorado potato beetle can resist all five insecticides classes, including the relatively recent pyrethroid insecticides. Hence the main weapon for countering this resistance is to develop alternative chemicals possessing structures unaffected by cross resistance besides giving special consideration to the sequence of use of insecticides (Georghiou and
Taylor, 1976).
In view of the fact that the majority of chemicals in use today have been adversely affected by resistance, Georghiou (1986) stated " The question may be posed, therefore, whether we have already selected in pests all the various oxidases, esterases, glutathiontransferases, dehydrochlorinases and other enzyme systems that may enable them to quickly evolve resistance to practically any toxicant that may be used against them. The answer will be provided in time by the pests themselves."
Synergism
The term "pesticide synergism" is usually used to describe the phenomenon where non toxic or negligibly toxic compounds have the capacity to enhance the toxicity of a pesticide. According to Davidson and Lyon (1979) the term
"synergism" as related to pesticides may be defined as " Joint action of two materials resulting in a total toxic effect greater than the sum of their toxic effects when applied separately." Synergists were initially developed to enhance and prolong the toxicity of pyrethrum insecticides, namely pyrethrin (Ware, 50
1982). Synergists have been mostly used to inhibit oxidases (e.g., piperonyl butoxide(PBO):a-{2-(2-Butoxyethoxy)ethoxy}-4,5-methyle-nedioxy -2-propyltoluene, an inhibitor of cytochrome P450 dependent monoxygenase system), esterases, e.g.
DEF:(S,S,S-Tributylphosphorotrithioate), and dehydrocholorinases (e.g., chlorofenethoI). Plapp (1986) is of the view that synergists have been used to inhibit specific detoxication enzymes or as antagonists (e.g., DE F) that successfully compete with the chemical for recognition by the receptor protein.
According to Yamamoto (1973) the synergistic effect is greatly influenced by the insect in question, insecticide formulation, insecticide/synergist ratio, method of adminstration, and choice of response which is usually kill or knockdown.
The mode of action of insecticides has aroused the interest of many researchers (Metcalf, 1967; Chang and Kearns, 1964; Wilkinson, 1971). Yamamoto
(1973) reported that synergists enhance the effectiveness of pyrethroids by reducing their rate of metabolism. Oxidases and esterases are the most significant detoxifying enzymes among insects. The choice of the optimum synergist to inhibit the detoxifying mechanism varies with the insect species
(Jao and Casida, 1974).
One of the major approaches to investigate and understand the physiological mechanisms of insecticide resistance involves the use of synergists which are known to act by inhibiting the detoxifying enzymes (Metcalf, 1967).
Scott and Georghiou (1986) demonstrated that the resistance mechanism responsible for high levels of permethrin resistance in the house fly, Musca domestica L., consisted of MFO-mediated detoxification (major mechanism), target site insensitivity and decreased cuticular penetration. According to Scott et al., (1986) insensitivity of the nervous system, also known as Kdr, is one of 51 the major mechanisms that causes evolution of resistance of pests to pyrethroids. The same authors believed that this mechanism may be responsible for the overall ineffectveness of PBO and DEF in reducing the level of resistance except to permethrin of the kdr-resistant strain of the southern house mosquito, Culex guinguefasciatus. A parallel study was conducted to investigate the role of PBO on pyrethroid resistant house flies. The results of this study revealed that metabolic degradation by the monooxygenase system plays a significant role in the resistance mechanism.
After approximately 5 years of annual exposure to pyrethroids,
Phyllonorycter biancardella, a pest of apples, showed a low level of resistance to permethrin and fenvalerate (4-6-fold), cross-resistance to cypermethrin; del tamethrin; cyflu thrin, cyano( 4-fl uoro-3 -phenoxyphenyl)methyl-3(2 ,2- dichloroethenyl)-2,2-dimethyl-cyclopropanecarboxylate and fluvalinate (a-RS,2R) fl u valina te[(RS) - al pha- c yano - 3 - p henox yb enzyl (R) - 2 -[ 2 - ch loro - 4-
(trifluoromethyl)anilino]-3-methyl-butanoate]( 4-17 -fold) and to DDT
(appoximately 39-fold) (Pree et aI., 1986). Resistance was not affected by the addition of different synergists such as PBO, DEF and triphenylphosphate(TPP) leading to the assumption that a non-metabolic knockdown resistance-type mechanism may be involved (Pree et aI., 1986). Chadwick et aI., (1977) also found that in the DDT-resistant BKK strain of A. aegypti, PBO augmented DDT to some extent, but a similiar effect was presesnt in the susceptible WRL strain indicating that metabolic oxidation may not be the main resistance mechanism with the Bangkok strain. A similar conclusion was reached by Byford et aI.,
(1985) when a synergism study was conducted on a cypermethrin resistant (35- fold) strain of Haematobia irritans. PBO and DEF yielded only 3.5-fold and 2.2- fold increase in toxicity, respectively. The results suggest an involvement of 52 another mechanism of resistance besides metabolism. Indeed, other mechanisms such as penetration and/or target site insensitivity may be involved.
From the foregoing it is clear that synergists have been generally designed and used in overcoming metabolic resistance mechanisms. These "typical" synergists are usually not efficient in countering target-site resistance. In recent years, there has been some progress in overcoming this deficiency. In the green rice leafhopper, Nephotettix cincticeps, resistance due to altered acetylcholinesterase, produced by N-methyl carbamates was suppressed by N propyl carbamates which were potent inhibitors of the altered enzyme
(Yamamoto et aI., 1983). Thus, a mixture of the two carbamates was found to suppress the resistance. Considering the target-site insensitivity to
DDT/pyrethroids and cyclodienes, this has proved to be the most difficult kind of resistance to deal with (Plapp, 1986). Plapp(1976) reported that resistance in the TBW to toxaphene and DDT could be dealt with by synergizing with chlordimeform, N-( 4-chloro-o-tolyl)-N ,N-dimethyl-formamidine. More recently,
Crowder et aI., (1984) determined that chlordimeform could be used to block pyrethroid resistance in TBW. Thus limited data are available to demonstrate that chlordimeform may be used to synergize and block the development of kdr type resistance, thus increasing the binding of synthetic pyrethroids to their target site proteins.
It has also been a challenge to many investigators to define the best insecticide/synergist ratio. In this respect, interesting results were obtained from a study on the interaction between synergists and permethrin in the adults of red flour beetle, Tribolium castaneum (Bodnaryk, Barker and Kudryk, 1984).
Chlordimeform:DEF (1:1 & 1:4) exhibit strong synergistic interaction whereas other pairs (chlordimeform : PBO, 1 : 4, or PBO : DEF 4: I) exhibited no
------" 53 synergism. A synergism study by Forgash (1985) showed that the toxicity of
fenvalerate, aldicarb, (2-methyl-2-(methylthio) propionaldehyde 0- methylcarbamoyl), and oxamyl, (methyl N'N'-dimethyl-N-{(methylcarbamoyl)oxy}
I-thiooxamimidate) to resistant populations of Colorado potato beetle,
Leptinotarsa decemlineata, was significantly enhanced by addition of PBO. In a different study (Silcox et aI., 1985) the same insect exhibited a lower level of resistance to both permethrin and fenvalerate when synergized by PBO.
Synergism increased a~ the insecticide : PBO ratio increased, but the adults were more susceptible to fen valerate than permethrin. Also the adults were approximately 3.39-fold more resistant than the larvae which may possess a comparatively less developed MFO detoxification system.
Although the use of insecticide synergists to manage resistance has attracted much interest, further studies are needed to evaluate their practical value. Plapp (1986) mentioned that the use of synergists to hinder the evolution of resistance would be determined by the presence or absence of another mechanism of resistance in the target population. He further mentioned that factors such as high cost, mammalian toxicity, formulation problems and high biochemical adaptivity encountered in some major pest insects have not been in favor of their use in pest management programs.
Genetics of Insecticide Resistance
As mentioned previously, during the process of coevolution, defenses and counter defenses have continuously been evolved in many living organisms.
Commonly encountered insect pests are not exempt from this general pattern of evolution whereby accumulation of heritable variation occurs through selection by environmental agencies (Georghiou, 1986). He considered the difference
------54 witnessed in artificially managed agroecosystems as being due to the nature of selection imposed by man in these systems. According to Plapp and Wang (1983)
" Exposure to insecticides has acted as a powerful selecting force, which concentrates the various preexisting genetic factors that confer resistance."
Thus, it is clear that the gene pool necessary to overcome the toxic effect of
pesticides is already present in the genetic make-up of the pest species at
various frequencies. (Georghiou, 1986).
Genes causing resistance to insecticides could be either structural or
regulatory. According to Plapp (1986), gene products that are targets for
pesticides such as enzymes, receptors and other cell compounds are sometimes
products of structural gene translations; and decrease in target site sensitivity
or increase in metabolism of pesticides could result from the mutation of these
genes. On the other hand, products of regulatory genes could control the time
and nature of expression of structural genes or recognize and bind to pesticides
controlling the induction of the appropriate detoxifying enzymes. Plapp and
Wang (1983) hypothesized that resistance is due to the interaction of both kinds of changes at least when one considers metabolic resistance to insecticides.
Plapp (1986) considered that especially at the population or subspecific level, the regulatory gene hypothesis provides a more likely model to explain changes in regulatory genes which are of major importance in insects. These changes may take place on a separate regulatory gene or at a regulatory site associated with structural genes which may be transcriptional, postranscriptionai, or translational in nature (Plapp and Wang, 1983). Levin
(1984) hypothesized that these changes encountered in regulatory genes provide an explanation to the genetic basis of adaptive variation in resistance development. Plapp (1986) supported Levin's hypothesis . He also felt that 55 available genetic and biochemical data reveal the existence of two types of regulatory genes: I. quantitative: all-or-none type inheritance; fully dominant or recessive and is similar in nature to some bacterial operons involving changes in the amount of protein synthesized. Separate protein products of distinct genes located adjacent to structural genes may be the site of variations and are therefore referred to as "near regulators" (Paigen, 1979), and 2. qualitative resistance showing codominant or intermediate pattern and present a mechanism involving changes in the nature of the protein synthesized, i.e., producing modifications in resistant insects.
Plapp (1984) postulated that changes in a single gene locus appears to regulate a variety of enzymes which are not adjacent to the enzymes whose activity it controls. Such regulators have been defined by Paigen (1979) as
"distant" regulators and are characterized by codominant inheritance.
Redundancy of resistant genes, often referred to as gene amplification, has been encountered in some insects (Georghiou, 1986). This phenomenon where resistant genes exist in mutiple copies has been reported in insect genera such as Myzus and Culex. According to Plapp and Wang (1983), such a genetic change, if it involves mutant structural genes would result in increased basal levels of enzyme activity in resistant strains with no loss in specific inducibility.
Relative Fitness
Studies on pesticide-resistant mutants generated in the laboratory and fitness experiments performed on laboratory-selected strains provide some information about alleles confering resistance and the fate of these populations
(NRC, 1986). They also mentioned that although estimation of genotypic fitness 56 is very difficult even in well controlled experiments, at least rough estimates of relative reproductive and survival rates of resistant and susceptible genotypes are necessary to consider increase in frequencies after pesticide treatment and decline once treatment is stopped or selection is relaxed. The NRC(1986) considers that it is necessary to obtain these estimates at different stages in the life cycle and under different concentrations of pesticides for resistant and susceptible genotypes. It is also necessary that investigations should be performed on susceptible and resistant genotypes isolated from natural sources and fitness studies should be performed under natural conditions since genetics of resistance in natural populations are probably different from those generated in short-term field studies (McKenzie et aI., 1982; Uyenoyama, 1986). The NRC
(1986) reported that laboratory studies performed under conditions closely approximating field conditions provide reliable estimation of toxicological dominance, but do not give accurate estimates of fitness differentials in the field. Crow (1957) mentioned that resistant genotypes should exhibit some fitness disadvantage in the absence of pesticides, although the difference may be small. Roush and McKenzie (I987) felt that it is necessary to determine the magnitude of such selective disadvantages, when present, if they are to be used in practice. They further described two methods commonly used to describe fitness disadvantages arising as an outcome of resistance: 1) measures the components of fitness, such as fecundity, development time, fertility, and mating competitiveness, for each genotype, and 2) termed the "population cage" follows alterations in genetic frequencies in replicable populations studied over a period of several discrete or overlapping generations, and this method is considered to be preferable over the former.
Undoubtedly, a thorough understanding of both the genetic factors and the
------57 mechanisms involved in pesticide resistance would provide us with the necessary information to manage the resistance problem. As of today, unfortunately, our comprehension of the basis of resistance in genetic terms is undoubtedly very low (Plapp and Wang, 1983; Georghiou, 1986).
------~------58
MATERIALS AND METHODS
Insecticides and Synergists
Two insecticides were used in this study:
1 )Technical grade permethrin [3(phenoxyphenyl)methyl-cis,trans-3-(2,2- dichloroethenyl)-2,2-dimethylcyclopropane carboxylate; Pounce, 98.4%, F.M.C.
Corp., Agricultural Chemical Division, Middleport, New York].
2)Technical grade azinphosmethyl [O,O-dimethyl S-(4-oxo-O-4-nitrophenyl
phosphorothioate); Guthion,91 %, Mobay Chemical Corp., Agricultural Chemical
Division, Kansas City, MO.].
The two synergists employed in the synergism study were:
1)Technical grade piperonyl butoxide [ PBO, 100%; Prentiss Drug and Chemical
Co. Inc. New York].
2)Technical grade DEF,94%, [S,S,S-tributyl phosphorotrithioate; Mobay Chemical
Co.].
All chemicals were prepared in redistiIIed acetone and refrigerated at 5° C.
Fresh preparations were made at two-monthly intervals.
Rearing of Pink bollworm Strains
The rearing procedure adopted in this study was adapted from the method used in the PBW rearing facility at the Western Cotton Research Center
(WCRC), Phoenix, Arizona. The rearing methodology was modified to suit small scale laboratory culturing.
Adult moths were held in 230 ml paper ice cream cups (S308 59
SweetheartR). These cups were stored in environators set at a temperature of
26 ± 1° C and 14:10 lightdark cycle. Humidity was also maintained at a constant level by the presence of free water in a plastic tray (40 x 27 x 9cm ).
The cardboard lids of these bioassay cups were replaced with wire-mesh lightly glued around the circumference. The moths were fed with 10% sucrose solution, diffused through cotton plugs (30 mm length, Johnson and JohnsonR ) inserted through an opening in the mesh-lined cover. These containers were used to hold pupae and adults and as bioassay chambers throughout the study.
The container lids were covered with squares of coarse white papers and weighted down with metal rings. The wax coating on the inside of the cups helped prevent oviposition on container walls. The paper squares served as oviposition sites and the 'egg sheets' were changed every 2 days. These sheets were stored in environators for 3 to 4 days, until the black head capsules of young larvae were visible inside the eggs.
The egg sheets were then transferred to 1.8 1. cardboard cartons containing artificial diet provided by the WCRC. Seven to 10 days later these cartons were placed in plastic boxes (40 x 27 x 9 cm) lined with a 3 to 4 cm layer of styrofoam, and held under laboratory conditions. This provided a substrate on which the fourth instar larvae could pupate upon cutting out of the cartons. Pupae were collected periodically and held in bioassay cups in environators to await adult emergence.
Bioassay Procedures
Adults: Assays were performed using 2 to 3- day-old moths of each population.
Preliminary tests indicated no significant differences in susceptibility between males and females and thus separation was not considered necessary.
------60
Serial dilutions of the insecticides (in acetone) were made on the basis of
wt/vol (active ingredient) and expressed as ug/ul. The symmetric design for
dose selection recommended by Finney (1971) was used in all studies. Four to
nine different concentrations of the insecticides were tested on 15 to 25
insects at a time. The entire procedure was replicated 3 to 4 times.
Adults were anaesthetized using a slow flow of carbon dioxide, just
sufficient to immobilize the moths during the dosing procedure. The moths were
positioned using a pair of forceps followed by application of 1 ul of the test
solution to the dorsum of the pro thorax. Topical applications were made using a
motor-driven microapplicator (Instrumentation Specialists Co.) fitted with a
calibrated syringe (Tuberculin, .25 ml). Treated insects were held in groups of
15 to 25 in the bioassay cups, provided with 10% sugar solution and returned to
the environators.
Mortality was recorded 48 hrs after treatment. Translocation (change of position) within 30 seconds following gentle probing with a blunt probe was used as a criterion for death. Control insects were immobilized with carbon dioxide and treated with acetone only.
Larvae: Third instar larvae weighing 13 ± 1 mg were used in this bioassay.
The bioassay chambers consisted of glass petri dishes (pyrexR, 10 x 75 mm), lined with one layer of filter paper ( WhatmanR No.1, 90 mm ). One- and- a half ml of the desired concentration was applied onto the filter paper lining the petri dish. This was immediately followed by slight tilting of the dish and slow rotating movements to ensure uniform coverage of the insecticide solution on the filter paper. Treated dishes were air-dried for 15 minutes prior to the placement of larvae in the dishes. Ten larvae per dish were used in range-
------~------61 finding studies and 20 to 30 larvae per dish were used at the time of pressuring. Two hours after exposure to the chemical, the larvae were transferred to cartons containing the media and the rearing procedure outlined previously followed.
Evaluation of Permethrin and Azinphosmethyl
Resistance in Field Populations
Five strains of pink bollworm collected from natural populations were employed to determine levels of susceptibility to permethrin and azinphosmethyl.
These strains were collected from widely separated localities of commericial cotton production in Arizona ( Safford, Marana, Phoenix, and Yuma ) and southern California (Westmoreland) and returned to the laboratory. Susceptible pink bollworm strains used in base line studies were obtained from the WCRC,
Phoenix, Arizona. This strain had been maintained on artificial wheat germ diet for 17 years and had not been exposed to insecticides during this period.
In August of 1986, a large number (approximately 4000) of infested cotton bolls was collected from each of the localities mentioned above. The infested bolls were evenly distributed on steel grids mounted on benches in greenhouses maintained at ca. 83° F. Metal trays lined with paper towels were placed under the racks to catch the emerging pink bollworm larvae as they dropped into the trays to pupate.
Pupae of the different strains were placed in separate bioassay cups and held in environators until adult emergence. Two to 3 day-old moths of this generation(Fo) were used for evaluation of resistance in the different localities. The standard topical bioassay procedure described previously was used. The field and laboratory strains were treated with permethrin and azinphosmethyl.
------62
Synergism of Permethrin
A synergism study was conducted on both the susceptible and the Yuma field strains to provide information on whether oxidative or hydrolytic pathways play a major role in resistance to permethrin. The synergists used were piperonyl butoxide (PBO), a mixed function oxidase (MFO) inhibitor and DEF, an inhibitor of hydrolytic esterases.
The synergists were mixed with reagent grade acetone to derive concentrations that were 5- and lO-fold higher than that of the insecticides (5:
I, PBO: permethrin and 5: 5: I, PBO: DEF: permethrin). Synergist dosages were topically applied to adult test insects one hour prior to the insecticidal treatment.
Reversion of Resistance to Permethrin in Field Strains
Two colonies of pink bollworm populations collected from Yuma and
Marana were established in the laboratory. The two strains were used for a parallel study on reversion of resistance to permethrin, and subsequent selections for resistance. Reversion of resistance was monitored every generation in the Marana strain using log dosage - probit mortality regression lines. Due to the difficulties encountered in establishing the Yuma strain in the laboratory such as Jow fecundity and viability, monitoring was initiated in the third generation. Assays were continued until a fairly constant level of susceptibility was maintained.
Laboratory Selection for Resistance to Permethrin and Azinphosmethyl
Both the Marana and Yuma strains were used in this study. The Marana 63 strain was selected for resistance to permethrin and azinphosmethyl at both larval and adult stages. On the other hand, the Yuma strain was selected for resistance to only permethrin, at the adult stage. The two strains had been reared in the laboratory for four generations prior to initiation of any insecticidal treatment.
Selection at the Larval Stages
The bioassay procedures (insecticidal film method) described for larvae
were used in this study. Two lines of the Marana colony were selected
separately with permethrin and azinphosmethyl. The progression of resistance in
larvae was noted by observing the percentage selection at different doses as the
study progressed. Ca. 50 to 90 percent selection pressure was applied to the
larvae during the period of study.
The development of resistance was monitored in adults using the topical
dosing procedure. For this purpose every two to three generations, a portion
of the larval population was not pressured and reared to adulthood. The
experiment was continued for a total of 14 and 16 generations for the Marana
azinphosmethyl and Marana-permethrin lines, respectively.
Selection at the Adult Stage
A parallel study on selection for resistance at the adult stage was conducted on the Marana strain. Both permethrin and azinphosmethyl were used as selection agents on this strain. The Yuma strain was selected with permethrin only. The resistance status was monitored every two to three generations throughout this study. Baseline toxicity levels were calculated at the initiation of selection. Early emerging adults were used to establish these lines
--- ._.-._------64
whereas late emerging adults were selected for resistance with a dosage
estimated to produce 70 to 80% mortality. The survivors served as parent stock
for the succeeding generations. In instances where baseline data were not
obtained, a range-finding study using 10 moths/dose was used. (Survivors at and
above the LOaD dose were added to the stock culture to give rise to succeeding
generations).
The selection phase of this experiment was continued for 15 generations
for the Yuma strain and 14 generations for the Marana strain using permethrin
and azinphosmethyl. The F13 generation of the Yuma strain and the Fe
generation of the Marana-permethrin strain were not selected due to reduced
colony size resulting from high mortalities in previous selections.
Inheritance Study
Insect Strains
The genetics of resistance to permethrin in pink bollworm was investigated
using the last permethrin - selected generation (FIS) of the Yuma strain. The susceptible strain obtained from the Western Cotton Research Center, Phoenix,
Arizona was used as the susceptible parent in these experiments.
Virgin Females
One of the essential requisites of any crossing experimemt is the use of
virgin females. Virgin females were obtained by sexing pupae according to the criteria outlined by Butt and Cantu (1962). The male and female pupae were separated and held in bioassay cups to await adult emergence.
------65
Bioassay and Genetic Crosses
Before proceeding with genetic crosses, susceptibility tests were conducted on the two parental strains (susceptible and Yuma- selected strain). Pupae of the two strains were sexed and tests were performed on adult males and females of both strains. Responses to permethrin produced insignificant differences in dosage-mortality data of the two sexes and consequently all subsequent bioassays were performed on unsexed moths. As a check against variations in procedures and alterations in toxicity of solutions, resistant and susceptible parent lines were bioassayed (using discriminatory doses) in all crosses.
Reciprocal Fl crosses were made by mass mating between susceptible and resistant strains. The resulting hybrid (Sf x Rm; Rf x Sm) progeny were reared to maturity for testing and further crossing.
A portion of the Fl progeny was used in assessment of dosage- mortality data. The remaining F 1 hybrid progeny(untreated) and the two parental strains were sexed at the pupal stage and appropriate crosses were made:
1) Fl x Fl cross giving rise to F2 generation (Sf x Rm) x (Sf x Rm) and 2)
Backcrosses to the susceptible and resistant parental strains-
(i) Backcrosses to susceptible parents: Sf x (Sf x Rm)m;
Sf x (Rf x Sm}m; (Sf x Rm)f x Sm; (Rf x Sm)f x Sm'
(ii) Backcrosses to resistant parents: Rf x (Sf x Rm)m;
(Sf x Rm)f x Rm'
The Rf x (Rf x Sm)m and the (Rf x Sm)f x Rm crosses were not conducted due to an insufficient number of Fl progeny obtained from the Rf x Sm crosses. 66
Analysis of Data
Probit Analysis
The results obtained from the dosage mortality experiments were evaluated using log dosage - probit mortality (Id-pm) regression lines described by Finney
(1971). LDso and LD9S values and their respective 95% confidence intervals, slopes, and standard errors were derived from the analysis. Separation of the
95% fiducial limits at the LDso values were used to indicate significant differences (P < 0.05). Resistance ratios (RR) were calculated by dividing the
LDso value obtained in the test population by that of either the susceptible or
Fl population. T -tests were used to test paired dosage mortality lines for parallelism (equal slopes, P < 0.01).
Synergism tests
The results of these studies were subjected to pro bit analysis and LDso,
LD95, 95% confidence intervals, slopes and standard errors were derived from it.
Using the LDso of synergized and unsynergized data of the susceptible and the
Yuma field strain, the following parameters were calculated: Resistance ratio
(RR), synergistic ratio (SR), synergistic difference (SD), and relative synergism ratio (RSR). Formulae used in calculations are given in Table 4.
Genetic Analysis
The dosage-mortality segregations obtained for the two Fl hybrid populations were compared to determine the autosomal or sex-linked nature of inheritence of resistance. The degree of dominance of permethrin resistance in the FI progeny was calculated using thA formulae given by Stone (1968) (Table
15). 67
Monofactorial inheritence of the resistant genes was tested by calculating chi-square values from the observed and expected mortalities of the Fl and backcross progenies. Expected mortalities were calculated assuming simple
Mendelian inheritance where backcross mortality at any given dose was the sum of half of the mortality of the Fl plus half of the mortality of the susceptible or resistant strain (Georghiou, 1969). Chi-square analysis was used to test the goodness of fit of observed and expected mortalities using the formulae described by Finney, 1971. (Table 16).
~~~--~ ~-~-~~ ------68
RESULTS AND DISCUSSION
Susceptibility to Permethrin and Azinphosmethyl
in Field Populations
Results of probit analysis on response of PBW to azinphosmethyl are summarized in Table I and graphically presented in Fig. I. Based on 95% non overlapping confidence intervals for LD60s, all field collected populations were significantly different from the laboratory susceptible strain. Yet, no resistance ratio at 50% lethal dose (RRso), was greater than 2.00. In fact, the resistance ratios of the field strains ranged from 1.29 to 1.57 and the populations tested demonstrated only two levels of susceptibility to azinphosmethyl. As shown in
Table I, Safford, Marana and Westmoreland strains had significantly higher LDso values compared to those of Yuma and Phoenix strains. The fact that the
Safford strain showed the highest RR60 , although collected from an area historically known to have the least use of insecticides among the areas studied
(T.F.Watson and L.Moore, Univ. of Arizona, Personal Communication; 1989), may be due to the inherent genetic fitness of this strain. The regression lines of all strains, including the susceptible strain demonstrated relatively high slope values
(6.2 to 8.1). This may indicate that a high degree of genetic homogeneity exists in these populations ar,d seems to account for the high level of susceptibility towards azinphosmethyl.
In contrast to the results of azinphosmethyl, field populations from the monitored sites exhibited variable degrees of resistance to permethrin (Table 2 ;
Fig. 2.). In fact, the intensity of resistance to permethrin appeared to be variable among the PBW strains studied and was in the range of 1.29- to 18.25- fold. At the LDso, the field populations relative to the laboratory strain were 1.29-, 3.65-, 13.79-, 14.90- and 18.25-fold greater in the Safford, Marana, Yuma, 69
Table I. Toxicity of azinphosmethyl to five strains of pink bollworm collected from different locations relative to a standard laboratory susceptible strain.!L ------Strain Slope (SE)1/ LD501!1 95% C.I. LDg5 RRlI ------
Westmore. 7.52 (0.49) 27.35a 26.42-28.32 45.26 1.54
Marana 6.19 (0.52) 26.44a 25.38-27.55 48.76 1.49
Phoenix 8.09 (0.52) 23.97b 23.17-24.79 38.29 1.35
Safford 7.03 (0.49) 27.99a 26.98-29.04 47.97 1.57
Yuma 8.58 (0.54) 22.94b 22.24-23.66 35.66 1.29
Suscept. 6.23 (0.53) 17.79c 16.91-18.71 32.67 1.00
11 Data analyzed by computer probit analysis (Finney,1952) Y SE: Standard error J/ Lethal dosage in ug/g. LD50 values followed by the same letter are not significantly different when compared using 95% confidence intervals ~ RR: Resistance ratio = LD50 of Fx / LD50 of susceptible strain SAFFORD 951 1 " 90 M 80 0 70 R T 50 A L 30 I 20 T 10 V 5
0.001 0.01 0.1 1.0 DOSE (ug/moth) Fig.I. Dosage-mortality lines for azinphosmethyl on strains of pink bollworms collected from Safford and Yuma, relative to a standard laboratory strain.
o'-J 71
Table 2. Toxicity of permethrin to five pink bollworm strains collected from different locations relative to a standard laboratory susceptible strain.!! ------Strain Slope (SE)lI LDso~ 95% C.1. LDgS RR!I ------
Westmore. 7.53 (0.50) 22.63a 21.94-23.33 37.41 18.25
Marana 2.37 (0.22) 4.52c 4.02-5.10 22.44 3.65
Phoenix 5.94 (0.45) 18.48b 17.75-19.24 34.97 14.90
Safford 3.83 (0.26) 1.60d 1.49-1.72 4.31 1.29
Yuma 4.86 (0.41 ) 17.10b 16.33-17.91 37.30 13.79
Suscept. 4.09 (0.30) 1.24e 1.15-1.33 3.12 1.00
11 Data analyzed by computer probit analysis (Finney,1952) Y SE: Standard error J/ Lethal dosage in ug/g. LDso values followed by the same letter are not significantly different when compared using 95% confidence intervals ~ RR: resistance ratio = LDso of Fx / LDso of susceptible strain [AFFORD I 951 90 M eo 0 70 I SUSCEPT .--lI> R T 50 A L 30 I 20 T 10 Y 5 I T PHOENIX
o.OOt o.ot 0.1 1.0 DOSE (ug/moth) Fig.2. Dosage-mortality lines for permethrin on five strains of pink· bollworms collected from different locations, relative to a standard laboratory strain.
1'- 73
Phoenix and Westmoreland strains, respectively. The Westmoreland strain was
14.14-fold more resistant than the most susceptible field strain (Safford strain).
No significant differences in toxicities were observed between the Phoenix and the Yuma populations. Furthermore, the slopes of 1d-pm lines showed great variation in values ranging from 2.37 (Marana) to 7.53 (Westmoreland). The fact that the Marana strain exhibited the flattest slope among the populations tested may suggest that it is the most heterogenous strain.
The data collected from these studies verified that resistance to permethrin exists in all of the PBW populations surveyed. It also shows that azinphosmethyl seems to possess a low degree of selectivity for development of resistance and that development of high levels of resistance in field populations is unlikely to occur in the foreseeable future. It is interesting to note that azinphosmethyl has not been used in the Safford area(L.Moore, Personal
Communication, 1989). The higher level of tolerance exhibited by this strain to azinphosmethyl is too low to warrant any real concern. Thus, although azinphosmethyl has been extensively used in most areas since 1966, it is still recommended by the Arizona Cooperative Extension Service, (Moore et aI., 1988) as one of the primary insecticides for control of the PBW. In this respect,
Reagan (1980) reported that the extensive use of azinphosmethyl in pest management programs to control sugarcane borer, Diatraea saccharalis (F.), a serious pest of sugarcane in Louisiana for 20 years, did not induce resistance.
In a further investigation, Vines et al. (1984) stated that although records show that this pest had developed resistance even to the earliest compounds used for its control, resistance did not develop to azinphosmethyl under intense selection pressure. In field populations of Danish house fly , dimethoate resistance was unstable for many years until an upsurge of OP resistance occurred in 1970-1971 74 and became stable and widespread by 1973-1974 (Wood and Bishop, 1981). They attributed this increase in fitness of the resistant genotype to two factors: 1) the appearance and selection of modifier genes which reduce the iII-effects of the genes for resistance and 2) reconstruction of the genotype. Hence, the low level of "tolerance" to azinphosmethyl encountered in the PBW strains may be partially attributed to a deleterious effect of the genes responsible for resistance on the natural metabolic processes.
The data obtained suggest that a substantial level of resistance is developing among the PBW populations to permethrin. In this respect, resistance appears to be developing more rapidly in populations that, historically, have been exposed to a high level of intensive insecticidal pressure.
Permethrin is a relatively new insecticide that had been used on cotton pests including the PBW for about 10 years. Although pyrethroids have been widely used in all areas surveyed, the intensity of use has been highest in
Westmoreland, Yuma, and Phoenix areas, moderate in Marana, and lowest in
Safford(T.F. Watson, and L.Moore,Personal Communications, 1989). Recently, however, there have been reports of control failures with pyrethroids in
California (Haynes et aI., 1987). The fact that pyrethroids possess a chemical structure and a mode of action similar to that of DDT might have helped PBW populations previously exposed to DDT to confer cross resistance to pyrethroids.
Lowry and Tsao (1961) and Noble (1969) reported development of resistance to
DDT in PBW populations in Mexico and Texas, respectively. Due to instances of resistance to pYl'ethroids in other insects, coupled with the historic influence of insecticide resistance on profitable cotton production, it is not surprising, therefore, that cotton producers have been greatly concerned about the potential of insect pests for developing resistance to pyrethroids (Sparks, 1981). 75
The term "resistance" has been variously defined by different authors.
Roush (1980) stated that resistance is a genetic change which is usually acquired as a result of pesticide selection. Georghiou and Mellon (1983) defined resistance as a significant decrease in sensitivity of a population towards an insecticide leading to diminished control. Based on field observations, Wood and
Bishop (1981) reported that development of resistance to various insecticides varies not only among different species treated with the same insecticide, but even among populations of the same species.
Tabashnik et al. (1987) studied the inter- and intra-island variations in susceptibility of DDT, diazinon, permethrin, and fenvalerate in 10 diamondback moth field populations in Hawaii. They determined that some populations separated by less than 10 kilometers showed significant intra-island variations in susceptibility whereas variations among inter-island populations were insignificant. They believed that past selection by insecticides such as DDT and diazinon might have caused some cross resistance to permethrin. It is surprising that the diamondback moth, one of the most widely distributed insects in the world and having great dispersal abilities, exhibited local differentiation among populations separated by less than 10 kilometers. The results of their study might have been caused by a situation where gene flow was insufficient to overcome insecticide susceptibility between populations.
It is believed that the local and regional histories of pesticide-use could help in understanding observed differences in pattern of resistance in field populations (Georghiou, 1972, 1986). But, it was only in 1986 that Rosenheim and Hoy tested this hypothesis statistically. Working with Aphytis mel in us (De
Bach), a key biological control agent of the California red scale, the same authors investigated the role of past selection pressures (local and regional ) on 76 the development of intraspecific variations of pesticide resistance to five commonly used insecticides. Using multiple regression analysis, they concluded that the patterns of variability in responses and toxicity levels of different populations could be correlated to both local (in grove) and regional (county wide) histories of pesticide-use. They recommended that future studies on intra specific variations in susceptibility should incorporate analysis of past selection pressures together with the role of migration and host distribution.
As mentioned by Wood and Bishop (1981), resistance is not always an inevitable consequence of pesticide usage. They further reported that genetic and ecological factors seem to dictate the time required for resistance to be detected. Moffitt et al. (1988) found that resistance to diflubenzuron in the codling moth, Cydia pomonella (L.) appeared in a population which had not been previously exposed to related chemicals, while a population in another orchard was highly susceptible after 8 years of exposure to diflubenzuron. It is therefore not surprising that the population from Safford showed the least susceptibility to azinphosmethyI. Also, populations of Musca domestica in Danish farms showed variations in rate of evolution of resistance. For instance, resistance to some organophosphorus insecticides developed within only 1 year, to parathion in 9 years and to pyrethrum in over 21 years (Wood and Bishop,
1981). Roush and McKenzie (I987) suggested that the rate of development of resistance in the field is a function of the initial frequency, dominance of resistant alleles, relative fitness of the genotype and the population structure.
There has been a reluctance on the part of researchers to classify laboratory assessment on toxicity as "resistance", due to the fact that sometimes they are not directly associated with control failures in the field (Luttrell et aI., 1987). Working with field populations of Heliothis virescens, Plapp and
------77
Campanhola (1986) and Luttrell et aI., (1987) stated that the high LD50 values for pyrethroids in laboratory assessements could reflect loss of susceptibility and control failures under field conditions. In contrast, Dennehy et al. (1987) reported that the existance of propagate-resistant spider mites does not necessarily translate into failures of field performance. They also stated that ..
Variables of expression of any specific intensity of resistance under field treatment conditions, and the frequency of resistant phenotypes present at specific locations will influence whether reduced performance of the pesticide is observed under commericial application conditions."
Synergism of Permethrin
The Yuma field strain was used in synergism bioassays since it demonstrated relatively high levels of resistance(13.79-fold) to permethrin among the field strains studied. The results of these studies show the effect of PBO alone and in combination with DEF on the responses of PBW to permethrin
(Tables 3 and 4 and Fig. 3).
Comparisons of 95% confidence intervals of LD50 values showed that the mixture of synergists with both strains gave marginally higher but significant increases in permethrin toxicity (Table 3). Synergism of the susceptible strain gave significantly higher toxicity values. Moreover, the dual action of PBO and
DEF was more toxic than the use of PBO alone. The synergized treatments on the Yuma strain were not significantly different from each other although both were significantly more toxic than the unsynergized treatments.
Synergism ratio (SR) could be used to express the degree of synergism.
The study demonstrated synergistic effects « 2-fold) of PBO and (PBO + DEF)
------78
Table 3. Dosage mortality data for synergism of permethrin by piperonyl butoxide(PBO) and DEF in the Yuma field and susceptible strains of pink bollworm'!;
Susceptible strain Yuma field strain Treatment ------Slope LD50Y Slope LD50 (SE)'v (95% C.I.) (SE) (95% C.I.)
Permethrin 4.09 1.24c 3.12 4.86 17.IOb 37.30 (0.30) (l.l5-1.33) (0041 ) (16.33-17.91)
+PBO 3.89 1.00b 2.64 3.51 11.00a 32.38 (0.36) (0.91-1,08) (0.32) (10.16-11.92)
+PBO + DEF 5.59 0.74a 1.46 6.51 10.55a 18.88 (0.56) (0.70-0.79) (0.47) (10.02-11.09)
11 Data analyzed by computer probit analysis (Finney,1952) 'li SE: Standard error 'JJ Lethal dosage in ug/g. LD50 values followed by the same letter are not significantly different when compared using 95% confidence intervals
------79
Table 4. Synergism of permethrin by piperonyl butoxide and DEF in the Yuma field and susceptible strains of pink bollworm.!!
Susceptible strain Yuma field strain
Treatment RR~/ RSR2I
LD50 SR SD
Permethrin 1.24 17.10 13.79
+ PBO 1.00 1.24 0.24 11.00 1.55 6.10 11.00 1.25
+PBO + DEF 0.74 1.68 0.50 10.55 1.62 6.55 14.26 0.97
11 Data analyzed by computer probit analysis (Finney,1952) Y Lethal dosage in ug/ 'J/ SR: Synergism ratio (LD50 of unsynergized / LDso of synergized) 11 SD: Synergistic difference (LDso of unsynergized - LDso of synergized) ~ RR: Resistance ratio (LD50 of Yuma strain / LDso of susceptible strain) QJ RSR: Relative synergism ratio(RR of unsynergized / RR of synergized) S + PaD YUMA+PBO+llEF I I X 95 90 M 80 0 70 R T 50 A L 30 I 20 T 10 I /II VUMA+P80-; y 5
0.001 0.01 0.1 1.0 DOSE (ug/moth) 00 o Fig.3. Synergism of permethrin by PBO and PBO/DEF combination in the Yuma (field) and laboratory susceptible strains of pink bollworm. 81 in the resistant as well as the susceptible strains of PBW (Table 4). The addition of PBO caused 1.24- and 1.55-fold reduction while the coaddition of
PBO and DEF caused a 1.68- and 1.62-fold reduction in LD6o's of the susceptible and the Vuma strains, respectively. Based on the SR's, it appears that joint action of PBO and DEF results in a higher degree of synergism than the action of PBO alone, suggesting the involvement of esterases in the degradation of permethrin.
Resistance ratios (RR) and relative synergism ratios (RSR) for each treatment are shown in Table 4. The RSR values could be used as an overall index of synergistic efficiency. RSR values S 1.0 could be considered as an indication of no synergism and RSR > 1.0 to denote true synergistic effect between the susceptible and the resistant strains (Prabhaker et aI., 1988).
Accordingly, when permethrin was applied along with PBO the resistance ratio decreased to II-fold and gave an RSR value of 1.25 indicating true, but marginal synergistic effect. The RSR value of 0.97 (RR 14.26) obtained when
PBO/DEF combination was used indicates that DEF does not contribute to synergistic activity.
In summary, the results of this study show that oxidase-induced metabolic degradation played a significantly greater role in the resistance encountered in the Yuma strain relative to that present in the susceptible strain. Vet, the level of enhancement (RSR = 1.25) is apparently insufficient to account for the major difference in susceptibility between the two strains. The results also revealed that the coadminstration of oxidase and esterase inhibitors caused no difference in relative toxicity to the two strains (RSR = 0.97).
Resistance to pyrethroids in insects can be a result of increased metabolism by oxidative and hydrolytic degradation (Soderlund et aI., 1983) or
- ._-_._------.------82
knockdown resistance (kdr) also referred to as target site insensitivity (Miller
and Adams, 1982). Studies on effects of synergists on pyrethroid toxicity to
different pyrethroid-resistant insect species have shown that enhanced metabolic
detoxication contributes to resistance, but it is not a primary factor (Soderlund
et aI., 1983; Byford et aI., 1985; Sparks and Byford, 1988). As mentioned above,
the results indicate that neither oxidases nor esterases showed appreciable
inhibition of permethrin. The MFO system may only be partially responsible for
the permethrin resistance encountered in the Yuma strain. Byford et al. (1985)
reported that pyrethroid-resistant hornflies Haemotobia irritans (L.) are usually
cross-resistant to DDT and other pyrethroids but not to organophosphates and
carbamates. Thus, target site insensitivity may be the overriding factor
influencing resistance to pyrethroids. Although evidence from these studies need
to be validated with more detailed investigations, the permethrin-resistance
found in the Yuma field strain may be related to changes in the target site,
rendering it insensitive to pyrethroids. Keiding ( 1967) reported that, though the
DDT-selection pressure on the Danish houseflies was discontinued for up to 20
years, the same insect continued to exhibit resistance to DDT. Apparently, either the extensive use of DDT in the past or the recent wide scale use of
pyrethroids on cotton pests accounted for the permethrin resistance detected
recently in the field populations of the pink bollworm. It is of interest to note
that toxicity studies on the PBW population from Yuma showed signs of cross
resistance to cyfluthrin, a pyrethroid, but not to sulprofos,O-Ethyl 0(4-
(methylthio)phenyI)-S-propyl phosphorodithioate, an organophosphate (Watson and Osman, unpublished). Moreover, studies performed by Bull et aI., (1988) on
the TBW revealed that enhanced metabolic detoxication was not a contributing
factor during the initial stages of development of resistance but became more
------83 important at higher levels of resistance. Hence, it is diffi.cult to tell whether metabolic degradation will play a more significant role in permethrin-resistance of the Yuma population at a future date. However, based on the current trend of intensive use of pyrethroid insecticide in the cotton ecosystem, pyrethroid resistance in PBWs could become more serious in the future.
Reversion of Permethrin Resistance
The purpose of this study was to help understand the pattern of reversion of permethrin-resistance in field populations, in the total absence of exposure to insecticides. Two field populations were used in this study: the
Yuma strain (13.06-fold) and the Marana strain (3.65-fold). Results are presented in Tables 5 and 6, respectively. Corresponding dosage-mortality lines showing reversion patterns are seen in Figs. 4 and 5. Both populations exhibited similar trends in their fairly quick reversion to 'stable' levels of susceptibility.
Both the Marana and the Yuma populations continued to approach susceptibility levels and by the F4 generation had lost ca. 56 and 80%, respectively, of their resistance. In fact, no significant fluctuation occurred beyond the F5 generation in the Marana strain whereas the Yuma strain lost
85% of its resistance by the same generation and retained that level through the end of the study (F7 generation). Although the two strains reached low and fairly stable levels of susceptibility, they never attained the susceptible level of the laboratory strain. At the beginning of the study the slope of the Yuma field strain was steeper than that of the Marana field strain, indicating heterogenity of the Marana strain. Also the Yuma field strain was more resistant than that of the Marana field strain. These two factors may explain
....-._._._---_. __ ._ ...._- ... _._ ...._------84
Table 5. Reversion of permethrin resistance in pink bollworm (Marana strain) following relaxation of insecticidal pressure.!! ------Gen.a! Slope (SE)~ LD50!1 95% C.I. LD95 RR2I ------
Fo 2.37 (0.22) 4.52d 4.02-5.10 22.44 1.00
Fl 2.18 (0.34) 3.71cd 3.14-4.32 21.13 0.82
F2 2.92 (0.37) 3.06bc 2.46-3.45 11.20 0.68
F3 3.67 (0.43) 2.78b 2.52-3.03 7.79 0.62
F4 3.45 (0.43) 1.99a 1.76-2.20 5.96 0.44
F6 3.42 (0.42) 2.07a 1.85-2.29 6.27 0.46
11 Data analyzed by computer probit analysis (Finney,1952) Y Filial generation Jj SE: Standard error 11 Lethal dosage in ug/g. LD50 values followed by the same letter are not significantly different when compared using 95% confidence intervals 'J/ RR: Resistance ratio = LD50 of Fx / LD50 of Fo
~.-..- ..-~~-----.-----~---~.---~------F3 i - .F1 % 95 90 M 80 0 70 R T 50 A L 30 I 20 T 10 Y 5
0.001 0.01 0.1 1.0 DOSE (ug/MOTH)
FigA. Reversion of permethrin-resistance in pink boIl worm (Marana strain) foIlowing relaxation of insecticidal pressure. CP U1 86
Table 6. Reversion of permethrin resistance in pink bollworm (Yuma strain) following relaxation of insecticidal pressure.!! ------Gen.Y Slope (SE)~ LDsoil 95% C.1. LOgS RR2I ------
Fo 4.86 (0.41) 16.20a 15.47-16.97 35.34 1.00
Fs 2.40 (0.34) 6.37b 5.28-7.37 30.89 0.39
F4 3.40 (0.37) 3.21c 2.87-3.58 9.76 0.20
Fs 3.14 (0.42) 2.36d 2.07-2.62 7.97 0.15
F7 3.70 (0.40) 2.44d 2.22-2.67 6.79 0.15
1/ Data analyzed by computer probit analysis (Finney,1952) V Filial generation 'JJ SE: Standard error ~ Lethal dosage in ug/g. LDso values followed by the same letter are not significantly different when compared using 95% confidence intervals ~ RR: resistance ratio = LDso of Fx / LD50 of Fo x 95 90 M 80 o 70 R T 50 A L 30 I 20 T 10 Y 5
0.001 0.01 0.1 1.0 DOSE (ug/MOTH) Fig.S. Reversion of permethrin-resistance in pink bollworm (Yuma strain) following relaxation of insecticidal pressure.
co '-.I 88
the fact that the slopes of the Id-pm regression lines of reversion of the
Marana strain did not produce noticeable changes beyond the F2 generation of
this study. As for the Yuma strain, due to the shortage of insects in the Fl
and F2 generations no regression lines were drawn for these two generations
which accounted for approximately 61 % reduction of resistance. The only
significant change in the slope beyond the Fs generation was observed in the
F7 generation; no significant changes were observed among the slopes of the
F4, Fs and F7 generations. With reference to house flies, Metcalf (1955) and Crow (1957) reported
that the reversion of resistance in populations that are returned to insecticide free conditions is a characteristic of partially resistant and not homozygously
resistant strains. This was further investigated by Scott and Georghiou (1985) who found that no reversion of resistance to permethrin occurred in a highly resistant strain even after 10 generations of complete absence of selection
pressure. Also, the study performed on another dipteran, Aedes aegypti, by
Abedi and Brown (1960), clearly demonstrated this concept. The strain employed in their study rapidly gained DDT -resistance under laboratory selection pressure,
but quickly reverted to susceptibility upon relaxation of selection pressure in the initial generations. Further selection, however, restored development of resistance to ca. 200-fold by the Fg generation. Relaxation of pressure in later generations produced a relatively low degree of reversion and it was therefore concluded that a comparatively stable level of resistance had developed. Based on the above-mentioned laboratory studies and as may be expected, the Yuma and Marana field populations used in this study were not homozygous for the resistance genes.
Working with field strains of two-spotted spidermite, Tetranychus urticae 89
Koch, Flexner et aI. (1988) reported that the apparently unstable nature of oraganotin (OT) resistance causes some reversion to occur between the last application of OT in 1 year and the first application in the following year. Also
OT resistance was found to fluctuate during the growing season, increasing after each application and decreasing when the selection pressure was relaxed.
Wood and Bishop (1981) mentioned that it is common that insecticides produce a temporary loss of fitness. Georghiou (1972) postulated that coadaptation accounts for observations that resistance tends to revert during the 'early' stages of selection, but become more stable at later stages of the selection process. Roush and Plapp (I982) found that homozygously diazinon resistant (RR) strains of houseflies possess 57 to 89% of the reproductive potential relative to that of the heterozygous strain (RS) and also to that of the homozygously susceptible (SS) strain, suggesting that the effect of the resistant genes on reproduction was recessive. Abedi and Brown (1960) also attributed the reversion to susceptibility after the 'initial' phase of selection of
Aedes aegypti to the lower biotic potential of the resistant individuals. They also postulated that once resistance is stabilized, the RR individuals are no longer at a disadvantage under insecticide-free condition. The initial phase of laboratory colonization of the Yuma strain (F1 and F2 generations) showed noticeably low fecundity and egg-hatchability indicating that the population was at a disadvantage under insecticide-free laboratory conditions. On the other hand, Sawicki et al. (1980) reported that even highly resistant clones of Myzus persicae lost resistance abruptly upon relaxation of selection pressure.
Bauernfeind and Chapman (1985) found that resistance could remain stable in clones for long periods (10-30 generations), but once reversion started, they returned to complete susceptibility within only four generations.
------~---~------90
Selection For Resistance in Pink BolIworm
Selection with Azinphosmethyl
Continuous selection of both larval and adult pink bolIworms of the
Marana strain with azinphosmethyl produced less than two-fold "resistance" in adult moths by the end of the F 14 generation. The dosage-mortality data of the
Fl generation were used as reference points to follow the progression of
resistance.
Table 7 shows the selection and progression of resistance in the larvae
using contact-toxicity methods. Larvae were selected for resistance at a
selection pressure of 53 to 94% mortality. Based on the selecting dose,
resistance ratio apparently rose to about 7-fold by the Fa generation and to 15-
fold by the end of the experiment (F14 generation). Selection was conducted at the larval stage (Table 7) and the corresponding dosage-mortality values
obtained on the adult moths are presented in both Table 8 and Fig. 6. Clearly,
resistance developed very slowly in adults and only 1.93 RR was obtained by the
Fl4 generation. It is interesting to note that though selection on larvae
produced less than 2-fold resistance in adults, ca. IS-fold resistance was
developed in larvae during the same period.
As seen in Table 9 and Fig. 7, selection for azinphosmethyl resistance at
the adult stage at ca. LDao produced only I.60-fold resistance by the F14
generation. The highest resistance ratio (1.94). was observed in the F12
generation. It is therefore not surprising that even after more than 20 years of field exposure to azinphosmethyl (Watson or Moore, Univ. of Arizona, Personal
Communication) the pink bolIworm has not yet exhibited a significant level of resistance. 91
Table 7. Selection and progression of resistance to azinphosmethyl in the Marana strain of pink bollworm.
Generation Dose(ugjul) % selection pressure!!
F1 0.3 - 0.5 72
F2 0.3-0.7 94
F3 0.4-0.6 53
F4 0.5-1.5 75
F5 0.7-2.0 91
F6 1.0-2.5 59
F7 1.6 69
F8 2.5-3.0 82
F9 2.0-3.0 65
FlO 3.0-3.5 71
FI I 3.0-4.5 92
F12 3.0-4.0 83
FI3 4.5-5.0 79
F14 4.0-6.0 90
F15 6.0 82 ------l/Larvae selected for resistance using the dish treatment procedure 92
Table 8. Dosage-mortality data for azinphosmethyl on adult pink boll- worm upon selection for resistance at the 3rd larval stage (Marana strain).!! ------Gen.£! Slope (SE)l!i LDso!! 95% C.1. LDgS RR~.1 ------
FI 7.23 (0.88) 21.05a 19.96-22.07 35.54 1.00
F3 7.71 (0.96) 21.73a 20.61-22.78 35.51 1.03
Fs 5.44 (0.65) 22.01a 20.63-23.27 44.14 1.05
F7 4.17 (0.67) 29.90bc 27.50-32.64 74.10 1.42
Fg 5.15 (0.67) 26.66b 24.95-28.25 55.61 1.27
F12 4.97 (0.68) 34.07c 31.60-36.45 73.03 1.62
FI4 6.13 (0.85) 40.60d 38.44-43.24 75.30 1.93
11 Data analyzed by computer probit analysis (Finney,1952) Y Filial generation 'JJ SE: Standard error 11 Lethal dosage in ug/g. LDso values followed by the same letter are not significantly different when compared using 95% confidence intervals $J RR: Resistance ratio = LDso of F x / LDso of F 1 F5 F9 % 95 I I I 90 M 80 0 70 R T 50 A L 30 I 20 T 10 Y 5
0.001 0.01 0.1 1.0 DOSE (ug/MOTH)
Fig.6. Dosage-mortality lines for an azinphosmethyl-selected strain of pink bollworm (Marana strain). Larvae subjected to selection. 1.0 W 94
Table 9. Dosage-mortality data for azinphosmethyl on adult pink bollworm upon selection for resistance at the adult stage (Marana strain).!! ------Gen.Y Slope (SE)~ LDsoil 95% C.I. LDgS RR'v ------
Fl 7.23 (0.9 I) 20.93a 19.81-21.98 35.34 1.00
Fg 6.82 (0.70) 22.30a 21.25-23.39 38.87 1.07
Fs 7.96 (0.03) 27.38b 26.22-28.63 44.08 1.31
F7 6.25 (0.81 ) 25.75b 24.14-27.11 47.22 1.23
Fg 9.33 (0.07) 26.39b 25.40-27.40 39.60 1.26
F12 5.59 (0.84) 40.70d 38.60-43.47 80.16 1.94
F14 5.06 (0.64) 33.44c 31.37-35.48 70.72 1.60
.v Data analyzed by computer probit analysis (Finney,1952) Y Filial generation Y SE: Standard error !I Lethal dosage in ug/g. LDso values followed by the same letter are not significantly different when compared using 95% confidence intervals ~ RR: Resistance ratio = LDso of Fx / LDso of Fl % 95 90 M 80 o 70 R T 50 A l 30 I 20 T 10 Y 5
0.001 0.01 0.1 1.0 DOSE (ug/MOTH) Fig.7. Dosage-mortality lines for an azinphosmethyl-selected strain of pink
bollworm (Marana strain). Adults subjected to selection. ~ c..n 96
Selection with Permethrin
The selection and development of resistance to permethrin in larvae through 15 generations is presented in Table 10. Based on the selecting dose a ca. 25-fold increase in the resistance ratio was observed by the end of the study (FIG)' By the FlO generation only ca. 8-fold increase in resistance ratio had been obtained. However, the rate of development of resistance was higher during the latter part of the selection study. Parallel monitoring of development of resistance in adults produced 9.74-fold increase in resistance in the FIG generation (Table II and Fig. 8). The highest level of resistance attained in this study was in the F14 generation (13.22 fold). The progression of resistance was somewhat gradual during most of the selection process. However by the F14 generation, the resistance ratio increased ca. 3.5 fold relative to the F12 generation. The slope obtained in Fs was significantly lower than those obtained in F12 through FIG generations, apparently indicating an increase in homogenity in later generations.
The results generated on selection for permethrin resistance in adult PBWs aTe presented in Table 12 and Fig.9. The development of resistance for the most part, seemed to be spaced evenly during the course of this study. By the F 14 generation, 8.84-fold increase in resistance was attained. Examination of slope values indicate that an apparently heterogeneous population used at the beginning of the study tended to become relatively more homozygous as selection progressed. It may be worthwhile to mention that larval selection produced ca. 25-fold resistance in larvae by the end of the study, as opposed to a maximum of 8.84-fold increase obtained on selection at the adult stage.
Wood and Bishop (1981) reported that in closed laboratory populations, the evolution of insecticide resistance should be directly related to the selection 97
Table 10. Selection and progression of resistance to permethrin in the Marana strain of pink bollworm.
Generation Dose(ug/ul) % selection pressure!.!
Fl 0.05-0.2 64
F2 .08-0.2 67
F3 .10-0.25 61
F4 .15-0.25 92
F5 .15-0.25 57
F6 0.25 63
F7 .30-0.4 71
F8 0.50 76
F9 .40-1.0 83
FlO 1.00 58
Fll 1.00-2.0 89
FI2 1.50 81
F13 1.00-2.0 76
F14 1.50-3.0 68
F15 3.00 73
11 Larvae selected for resistance using the dish treatment procedure
------98
Table 11. Dosage-mortality data for permethrin on adult pink bollworm upon selection for resistance at the 3rd larval instar (Marana strain).!! ------Gen.V Slope (SE)1!I LD60il 95% C.1. LDg6 RR2I ------
FI 3.45 (0.42) 1.97a 1.75-2.18 5.89 1.00
Fs 3.19 (0.35) 2.29ab 2.07-2.5 37.52 1.16
Fs 2.98 (0.44) 2.50b 2.20-2.81 8.95 1.27
F7 3.00 (0.46) 3.37c 2.92-3.77 11.92 1.7 I
Fg 3.27 (0.53) 7.87d 7.08-8.80 25.01 3.99
FI2 5.53 (0.55) 7.52d 7.10-7.98 14.93 3.82
FI4 4.56 (0.69) 26.04f 14.20-28.47 59.78 13.22
FI6 4.18 (0.49) 19.1ge 17.56-20.84 47.52 9.74
11 Data analyzed by computer probit analysis (Finney,1952) Y Filial generation Y SE: Standard error Y Lethal dosage in ug/g. LD60 values followed by the same letter are not significantly different when compared using 95% confidence intervals 'JJ RR: Resistance ratio = LD60 of Fx / LD60 of Fl 95 % Fi 90 F3 M 80 F5 a 70 R 50 " T A I F7-L//.. / /~ ,t. F16 L 30 I " 20 " T 10 Y 5
0.001 0.01 0.1 1.0 DOSE (ug/MOTH)
Fig.8. Dosage-mortality lines for a permethrin-selectd strain of pink bollworm (Marana strain). Larvae subjected to selection. I.D I.D 100
Table 12. Dosage-mortality data for permethrin on adult pink bollworm upon selection for resistance at the adult stage (Marana strain).!! ------_._------Gen.~ Slope (SE)!I LDGo!j 95% C.I. LDg5 RRY ------
Fl 3.45 (0.42) 1.96a 1.74-2.16 5.85 1.00
Fs 2.57 (0.35) 3.l7b 2.71-3.63 13.87 1.62
F5 2.64 (0.49) 2.45ab 2.10-2.77 10.31 1.25
F7 2.70 (0.37) 5.03c 4.33-5.65 20.48 2.57
Fg 3.22 (0.39) 7.04d 6.26-7.76 22.84 3.59
F12 5.03 (0.52) 14.82e 13 .80-15.92 31.46 7.56
F14 5.16 (0.56) 17.33f 16.23-18.51 36.12 8.84
!I Data analyzed by computer probit analysis (Finney,1952) Y Filial generation Y SE: Standard error 11 Lethal dosage in ugjg. LDGO values foHowed by the same letter are not significantly different when compared using 95% confidence intervals 'J../ RR: Resistance ratio = LDGO of Fx j LDGO of Fl
------~------X 95 90 M 80 0 70 I R --/// // U-F12 T 50 r /// / / /¥J Ft.c A L 30 I 20 T to y 5
0.001 0.01 0.1 1.0 DOSE (ug/MOTH) Fig.9. Dosage-mortality lines for a permethrin-selected strain of pink bollworm (Marana strain). Adults subjected to selection...... o...... 102 pressure. According to Roush and McKenzie (1987) the rate of change in allele frequency in a closed population is dependent on the initial allele frequency, dominance and the relative fitness of different genotypes. It is expected that in laboratory selection studies, resistance would develop gradually at first and later at a faster rate (Georghiou and Taylor, 1977). The response shown by the PBW larvae relative to selection by azinphosmethyl and permethrin conformed to this pattern of resistance. Although only moderate levels of resistance were reached by the end of these studies, resistance attained (in larvae) by the Fa generation was followed by a more rapid inci'ease in later generations. Jensen et a1. (1984) observed a sudden rise in permethrin-resistance of the tobacco budworm larvae in the FlO generation. Selection studies performed on houseflies by De Vries
(1979) and Scott and Georghiou (1985) showed that resistance rose rapidly to high levels when the strains were subject to a high selection pressure. The development of resistance in PBW adults was more gradual as compared to that seen in the larvae.
It is evident from the results of these studies that resistance developed more rapidly in larvae than in adult PBWs. This difference appears more clearly when azinphosmethyl was used as a selecting agent. Also, as shown in the results, development of resistance in larvae did not produce similar levels of resistance in adults. Studies performed on Aedes aegypti also demonstrated that the relationship between adult and larval resistance to DDT is unclear (Wood,
1981). The same author reviewed the work on this subject and reported that although adult resistance has not been developed in the absence of larval resistance, the reverse was observed in some instances. Laboratory selection of adult Aedes aegypti with DDT produced resistance in larvae as well as in adults
( Margham and Wood, 1974). Wood and Bishop (1981) believe that the variability 103 in the time taken to develop resistance among populations of the same species and different species is dictated by genetic as well as ecological factors.
The results show that larval selection produced resistance in larvae as well as in adults. It is therefore fortunate that chemical control in the field is directed against the adults, and the larvae, being inside the bolls, are naturally protected from direct exposure to insecticides. In light of these facts, it is clear that if not for this ecological aspect of the PBW, resistance might have been a much more severe problem than it is today.
Inheritance of Resistance to Permethrin
in a Laboratory-Selected Strain
Selection for Permethrin-Resistance
Dosage-mortality data generated from the selection studies on the Yuma field strain are shown in Table 13 and graphically presented in Fig. 10.
Significantly higher LD50 values were obtained as selection progressed and
12.73-fold resistance was reached by the F15 generation. Relaxation of selection pressure in F15 did not result in a significant reversion of resistance in the subsequent FIG generation. Although the strain was moderately resistant by the termination of the study, examination of the slope values showed that the population was tending to become more homozygous. This apparently homozygous strain was used in the inheritance studies.
Inheritance of Permethrin Resistance
The results of bioassays of permethrin on the susceptible strain, permethrin-resistant strain (selected Yuma strain), and their crosses are shown 104
Table 13. Dosage-mortality data for permethrin on adult pink bollworm upon selection for resistance at the adult stage (Yuma strain).!! ------Gen.Y Slope (SE)Y LDsoY 95% C.I. LDgS RR~/ ------
FI 3.41 (0.35) 3.21a 2.90-3.56 9.75 1.00
Fg 3.32 (0.38) 7.41b 6.53-8.27 23.20 2.31
F6 2.60 (0.34) 6.39b 5.35-7.34 27.42 1.99
Fa 3.25 (0.43) 9,41c 8.40-10,45 30.16 2.93
FlO 4.87 (0.56) 18.17d 16.85-19.53 39.57 5.66
FI2 8.57 (0.97) 33.01e 31.82-34.25 51.38 10.28
F13 not selected
F15 7.26 (0.89) 40.85f 39.13-42.61 68.84 12.73 End of selection
FI6 7.39 (0.26) 38.8lf 36.34-41.52 64.78 12.09 ------11 Data analyzed by computer pro bit analysis (Finney,1952) Y Filial generation 'JJ SE: Standard error ~ Lethal dosage in ug/g. LDso values followed by the same letter are not significantly different when compared using 95% confidence intervals ~ RR: Resistance ratio = LDso of Fx / LDso of FI 95 " 90 M 80 I / ff/ I I~Ft2 0 70 R T 50 A l 30 I F3 I 20 T to y 5
0.001 0.01 0.1 1.0 DOSE (ug/MOTH) Fig.lO. Dosage-mortality lines for a permethrin-selected strain of pink' bollworm (Yuma strain}.Adults subjected to selection...... o U1 106 in Table 14. There was no significant difference in the LDGO and the slope values of the two reciprocal F 1 generations. These results strongly suggest that permethrin resistance is controlled by autosomally inherited factor(s). It is evident that the possibility of maternal inheritance is ruled out. The two Fl crosses (Sf x Rm ; Rf x Sm) produced progeny with resistance ratios of 10.99 and 9.80, respectively (Table 14). The LDGO values obtained in the reciprocal
Fl generations were closer to that of the resistant than the susceptible parents indicating that resistance is conferred by a partially dominant gene(s) (Fig.! I ).
Stone's (1968) formula was used to calculate the degree of dominance as presented in Table 15.
The Fl back-crosses to the susceptible and resistant parents produced offspring that displayed plateaus around 40 to 60% mortality (Fig. 12). Chi square analysis of the backcross results presented in Table 16 indicates that resistance to permethrin may be controlled by one major gene with contributory influences from additional factors (minor genes).
Progeny of backcrosses to both susceptible and resistant parents showed deviations from expected mortality. The Fl back-cross to the susceptible parents exhibited lower-than-expected mortality at low concentrations as opposed to higher-than-expected mortalities at higher concentrations. Of the eight experimental points obtained, five showed significant deviations from the expected values. Similarly, of the seven experimental points obtained in the Fl backcross to the resistant parents three points showed significant difference in mortality at low concentrations. Malcom and Wood (1982) obtained similar results when Aedes aegypti was selected for knockdown resistance to permethrin. They reported that resistance was under the control of one major gene but it is influenced by additional factors. Thus it is not certain that 107 resistance to permethrin in PBW is conferred by a single gene. From these studies, inheritence of permethrin resistance in PBW(adults selected), is apparently autosomal, incompletely dominant and probably conferred by one major gene with additional factors influencing resistance.
Extensive studies have been performed to elucidate patterns of resistance
in dipterans, whereas very limited work has been done on lepidopterans (Payne
et aI., 1988). In most or maybe all instances, resistance-conferring gene or
genes have been determined to be autosomal. Liu et al. (1981) found that
fen valerate resistance in the diamondback moth was polygenic and inherited by
incompletely recessive autosomal factors. Studies on the tobacco budworm showed that resistance to methyl parathion (Whitten, 1978) and methomyl (Roush and Wolfenbarger, 1985) was inherited as a single, incompletely dominant, autosomal factor. Recent studies on TBW conducted by Payne et al. (1988) revealed that resistance to permethrin was monofactorial, incompletely recessive and autosomal. They mentioned that the gene responsible for resistance was probably conferred by target site insensitivity.
Based on the data obtained on PBW, resistance appears to be conferred by an incompletely dominant factor(s). Halliday and Georghiou (1985) hypothesized that deviations from expected mortality observed in backcross data may be due to minor genes for resistance being found on separate loci and segregating independently of the major gene(s). Payne et aI., (1988) explained that differences from expected mortality would indicate that the major factor was actually more recessive than indicated by the data, and that the minor gene(s) could be more dominant or intermediate in expression. The fact that a broad plateau was obtained in their backcross data led the authors to conclude that the minor unlinked genes were not important in permethrin resistance. In
------108
Table 14. Dosage mortality data for permethrin on susceptible and selected strains of pink bollworm and their genetic crosses.!} ------Strain or crossll Slope (SE) LD60l!./ 95% C.I. LDg6 RRiI ------
Susceptible P. 4.09 (0.30) 1.24 1.15-1.33 3.12 1.00
Selected P. 7.26 (0.89) 40.85 39.13-42.61 68.84 32.94
F 1(R f x Sm)2I 4.23 (0.48) 12.15 11.18-13.20 29.77 9.80
F1(Sf X Rm) 4.49 (0.40) 13.63 12.79-14.53 31.67 10.99
F2 0.89 (0.15) 6.04 4.25-8.45 427.02 4.87
BACKCROSSES:
(Rf x Sm)m x Sf 2.77 (0.37) 3.94 3.52-4.39 15.45 3.18
(Rf x Sm)f x Sm 3.17 (0.38) 3.63 3.22-4.12 11.99 2.93 (Sf x Rm)m x Sf 3.18 (0.25) 3.78 3.53-4.06 12.47 3.05
(Sf x Rm)f x Sm 2.93 (0.24) 4.29 3.97-4.65 15.61 3.46
(Sf x Rm)m x Rf 2.62(0.29) 20.98 19.02-23.15 88.99 16.92
(Sf x Rm)f x Rm 2.33(0.38) 20.09 16.81-23.19 102.33 16.20 ------
1/ Data analyzed by computer probit analysis (Finney,1952) Y P:Parent; S:Susceptible; R:Resistant; m:Male; f:Female 'J.J Lethal dosage in ug/g. LD60 values followed by the same letter are not significantly different when compared using 95% confidence intervals Y RR: Resistance ratio = LD50 of Fx / LD50 of parent susceptible strain §j S:susceptible; R:resistant; f:female; m:male
------95 " 90 M 80 a 70 R T 50 A L 30 I 20 T 10 Y 5
0.001 0.01 0.1 1.0 DOSE (ug/MOTH) Fig.ll Dosage-mortality lines for permethrin on susceptible and selected strains of pink bollworm and their Fl crosses.
o~ ~ 110
Table 15. Degree of dominance (D) of permethrin resistance in the selected strain of pink bollworm.!!
Dosage( ug/ g)
0.37 0.31
0.50 0.45
liD: Degree of dominance = 2 X2 - XI--=...Xs (Stone, 1968). Xl - X3
Xl = log dosage of selected strain. X2 = log dosage of FI hybrid. X3 = log dosage of susceptible strain.
YS:susceptible; R:resistant; f:female; m:male % 95 90 M ISO o 70 R T 50 A L 30 I 20 T 10 Y 5
0.001 0.01 0.1 1.0 DOSE (ug/moth) Fig.l2 Dosage-mortality lines for permethrin on susceptible and selected strains of pink bollworm and their genetic crosses...... 112
Table 16. Chi-square (X2) analysis of mortality data from Fl x susceptible and Fl x permethrin-resistant backcrosses of the pink bollworm. ------Fl x S!l Fl x RaJ ------Dose ObslU ExpY X 2 'Qj Dose Obs Exp X2
(ug/moth) (ug/adult) ------0.020 7 36 28.21* 0.10 25 9 20.68*
0.025 20 42 15.89* 0.15 31 20 5.43*
0.030 25 45 12.93* 0.20 43 31 4.11 *
0.040 45 49 0.45 0.30 50 46 0.58
0.060 51 52 0.02 0.40 58 61 0.14
0.070 61 53 2.19 0.50 74 75 0.01
0.080 77 54 18.17* 0.60 89 87 0.20
0.100 83 59 18.27*
Total X2 96.93* 32.46* df 7 6
1) S:Susceptible parent
Y R:Resistant parent
Y Observed % mortalities
~ Expected % mortalities based on simple Mendelian inheritance (1:1 ,SS/RS, RR/RS); df=l.
~ Individual and total chi-square values tested for significance at P=0.05 113 contrast, the results of the studies on PBW showed greater deviations from expected mortalities in backcross responses indicating that the degree of dominance of the major gene may be less than observed and/or the minor genes may have a greater influence on resistance.
It is recognized that the field situation is more conducive for selection of monogenic (single major gene) resistance (Wood and Bishop, 1981). Also Whitten and McKenzie (1982) stated that pesticide resistance usually has a simple genetic basis in the field as opposed to the laboratory-selected strains in which resistance is inherited in a more complex polygenic manner. The confusion over the number of genes involved in resistance obtained in laboratory studies, was attributed to selective channels of variation (Roush and McKenzie, 1987). They also mentioned that nearly all laboratory regimes select within an existing, common phenotypic distribution, resulting in polygenic inheritance patterns, in contrast to field programs which are designed to kill all individual pests. It is therefore possible that permethrin resistance in the laboratory-selected PBW strain was conferred by more than one gene. 114
CONCLUSION
Results of the surveillance studies indicated that the pink bollworm populations used in the study, from geographically isolated locations showed very low levels of tolerance to azinphosmethyl and variable degrees of resistance to permethrin. Wood and Bishop (1981) mentioned that there were no reports of resistance in tsetse fly after 25 years of exposure to chemicals and hence insecticide usage does not inevitably result in resistance. However, Brown
(1977) is of the opinion that in the long run there is little hope that any species will remain free of resistance. Therefore, although pink bollworm populations have not exhibited a marked resistance to azinphosmethyl after approximately 2 decades of exposure and that there is no imminent danger of resistance in the foreseeable future although one can reasonably expect resistance at some point in time if continued use of the product occurs.
The low to moderate levels of permethrin-resistance obtained among the populations of the localities studied show the capacity of the pink bollworm of becoming resistant to permethrin. As mentioned by Wood and Bishop (1981) it is difficult to interpret the results of laboratory bioassays in the absence of information concerning response of the strain before selection was begun.
According to Bull (I 98 1) development of resistance could be inferred with more certainty if field control failures are substantiated by laboratory bioassay. The pattern of resistance observed in the localities studied appears to be historically related to the intensity of the insecticidal control measures practiced therein.
The results of this study could be used as baseline data for future studies on pink bollworm in these localities.
The synergism results show that less than two-fold resistance in the Yuma
--_._------_.- ---_. ---_.... ------115
strain were synergized when PBO was used, both alone and in combination
with DEF. These results suggest that permethrin resistance in the field
population studied is at least partly related to non-metabolic factors. Reduced
penetration has been reported as a resistance factor in a number of insects (
Oppenoorth and Welling, 1976), but it is usually related to the presence of other resistance factors, primarily detoxication. Many studies on permethrin
resistance in other insect pests have revealed the importance of a kdr mechanism. Hence there is a possibility that pink bollworm resistance is at least
partly conferred by the kdr-gene.
When the Marana and the Yuma field strains were released from selection
pressures, they lost resistance to permethrin and reverted to susceptibility in
four and five generations, respectively. The toxicity values obtained in the first
generation of laboratory rearing dropped and by the second and third
generations fell to significantly low values. Thus it is clear that evaluation of
resistance in the pink bollworm should be done preferably on the Fl generation.
Martinez-Carrillo and Reynolds (1983) reached the same conclusions with regard
to dosage mortality studies on TBW. Moreover, it has been recommended that
standard tests on laboratory-reared larvae should not be done beyond the F2
generation (Annonymous, 1969).
During the initial stages of development, pesticide resistance is often
. associated with deficiencies in fitness, vigor, behavior and reproductive potential
rendering the resistant populations more susceptible to other control methods
(NRC, 1986). Therefore a knowledge of stability of resistance can be valuable since populations which are at the early stages of developing resistance could
be controlled by using other insecticides or management practices selectively
until resistance is reduced. Thus, the results of this study are encouraging since 116 resistance to permethrin in field populations appears at its initial stage of development.
Laboratory selection studies on field-collected strains produced low levels of azinphosmethyl resistance and moderate levels of resistance to permethrin in adults. Keiding (1986) mentioned that the predictive values of experiments on laboratory selections are very limited since the type and rate of resistance development in laboratory-selected strains could be quite different from what is witnessed in field populations. Hence, these differences seen between selection for resistance in the laboratory and under field conditions necessitate that such studies should be performed on an initially large field-collected strain which would possibly include the major resistance genes (Roush and McKenzie, 1987;
NRC, 1986). The NRC (1986) further mentions that continuous selection of strains in the laboratory for many generation is of limited value in resistant management programs.
The field strains employed in these studies were selected in both, larval and adult stages. Azinphosmethyl- and permethrin-selected larvae and adults developed low and moderate levels of resistance to azinphosmethyl and permethrin, respectively, in adult stages. Observations using % selection dose
(Table 7) indicated that larval selection with azinphosmethyl resulted in a higher level of resistance in larvae than in adults whereas larval selection with permethrin produced a somewhat parallel development of resistance between the two stages.
According to Wood and Bishop (1981) differences in resistance-mechanisms may account for the differential levels of resistance observed in larval and adult stages. As mentioned by Tabashnik et al. (1988) understanding the effect of insecticides on nontarget stages is important in understanding mechanisms 117 of resistance, and the development and evolution of resistance . It is of interest here to note that the NRC (1986) recommended that pesticides should be used against life stages of pests which are less prone to developing resistance. They reported that the rate of development of resistance could be lowered by directing insecticides against stages such as the adults and early larval ins tars since the later instars have greater enzymatic activity and fall into the higher risk category (prone to resistance).
Unlike with most other lepidopterous pests, control measures on the pink bollworm are directed against the adult moth due to biological and ecological reasons. The oviposition characteristics of the female are such that eggs are laid principally between the boll and the calyx and thus naturally protected from ovicides. Moreover, the fact that larvae older than the first instar lie within bolls provides protection to the larvae from pest control agents.
Therefore, it is evident that although the results of these studies show that the larvae are apparently capable of developing resistance, especially with azinphosmethyl, they are not selected under field situations. Also as suggested by Roush and McKenzie (1987) studies on pesticide resistance that are based on the fundamentals of ecological genetics, provide a better understanding of the evolutionary changes and hence provide practical contributions to pest management programs. The impact of population biology, migration capabilities, presence or absence of refugia, reproductive potential and dominance/recessive nature of the resistant alleles could determine the rate of the development of resistance. Using computer-simulated models, Georghiou and Taylor (1977) examined and quantified the influence of various genetic and biological factors on the evolution of resistance. They determined that population mobility and influx of susceptible migrants into treated areas could substantially slow down
------118 the rate of development of resistance. Bariola et aI.(I973) found that the flight range of overwintered pink bollworm adults extends over an area 56 kms. It seems that these biological and ecological factors discussed above have been responsible for the slow evolution of resistance in the pink bollworm relative to other lepidopterous pests.
Although genetic investigations on the PBW had been undertaken since
1967 (Bartlett,1989), there have been no studies reported to date on inheritance of pesticide resistance on the PBW. The results of inheritance studies indicated that permethrin resistance in the pink bollworm is conferred by autosomal, partially dominant gene(s). The bioassays on backcross progeny did not produce unequivocal evidence that resistance was determined by a single gene. It is possible that one major gene is involved, but the influence of minor genes is apparent from the results of the chi-square analysis.
Georghiou and Taylor (I 977) determined that population size was greatly influenced by the dominance of the resistance alleles and that a recessive gene requires approximately twice the time taken by a dominant gene to attain economic status. The results of this study show that the gene(s) that confer resistance are partially dominant, yet the biological/ecological features of the pink bollworm have apparently played a major role in slowing down the development of resistance. These studies were performed on a laboratory selected strain. It has been reported that very often selection regimes conducted under laboratory conditions lead to development of polygenic resistance whereas a more simple monogenic inheritence is found in fie If! populations. Hence, it may not be correct to directly extrapolate these results to inheritance of resistance in field strains.
From these studies it could be concluded that further investigations should 119 be performed to elucidate the importance of biology and ecology in relation to insecticide resistance in the PBW. Such studies would help formulate improved insecticide use patterns, and better pest management practices. Moreover, very limited literature is available, at present, on insecticide resistance in PBW.
Subsequently, continuous monitoring for insecticide susceptibility would be useful in detecting any alterations in susceptibility levels, especially to widely used insecticides. 120
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