**Meet the editor**

Dr Farzana Perveen, Founder/Chairperson of the Department of Zoology at the Hazara University, Pakistan, obtained her BSc and MSc (Zoology: Entomology) from the University of Karachi, Pakistan, and her MAS from the Nagoya University, Japan. The Zoological Society of Pakistan awarded her a gold Medal for her PhD in Toxicology, obtained from the University of Karachi. She has,

among other, worked at the School of Agricultural Sciences, Nagoya University. Dr Perveen was the chairperson of the Department of Zoology, Kohat University of Science and Technology, Pakistan, and supervises BSc (Hons), MPhil, MS and PhD students. She has organized and attended numerous international and national seminars, conferences and workshops, as well as received multiple awards and fellowships. Dr Perveen has written more than 100 research papers and books, she is a member of international and national research societies, editorial boards and a book editor. Her fields of interest are Zoology, Agriculture, Integrated Pest Management, Toxicology and Genetics.

Contents

**Preface IX** 

A. C. Achudume

Farzana Perveen

Chapter 3 **Organophosphorus Insecticides** 

**Part 2 Vector Management 151** 

Alhaji Aliyu

Chapter 8 **Susceptibility Status** 

Chapter 7 **Vector Control Using Insecticides 153** 

Ronald Maestre Serrano

Chapter 1 **Insecticide 3** 

**Part 1 Insecticides Mode of Action 1** 

Chapter 2 **Chlorfluazuron as Reproductive Inhibitor 23** 

**and Glucose Homeostasis 63**  Apurva Kumar R. Joshi and P.S. Rajini

Chapter 5 **DDT and Its Metabolites in Mexico 99** 

Chapter 4 **The Toxicity of Fenitrothion and Permethrin 85**  Dong Wang, Hisao Naito and Tamie Nakajima

Chapter 6 **Presence of Dichlorodiphenyltrichloroethane (DDT)** 

Goran Gajski, Marko Gerić, Sanda Ravlić, Željka Capuder and Vera Garaj-Vrhovac

**in Croatia and Evaluation of Its Genotoxicity 117** 

**of** *Aedes aegypti* **to Insecticides in Colombia 163** 

Iván Nelinho Pérez Maldonado, Jorge Alejandro Alegría-Torres, Octavio Gaspar-Ramírez, Francisco Javier Pérez Vázquez,

Sandra Teresa Orta-Garcia and Lucia Guadalupe Pruneda Álvarez

## Contents

## **Preface XIII**



#### **Part 2 Vector Management 151**


Contents VII

Chapter 20 **The Conundrum of Chemical**

Allan T. Showler

Chapter 21 **Management of Tsetse Fly** 

**Boll Weevil Control in Subtropical Regions 437** 

**Using Insecticides in Northern Botswana 449** C. N. Kurugundla, P. M. Kgori and N. Moleele

**in Burkina Faso, West Africa: 10 Years' Surveys 479** K. R. Dabiré, A. Diabaté, M. Namountougou, L. Djogbenou,

**Cytochrome in Insecticide Resistance and Infection 503** 

**of Carbofuran and Pirimicarb Carbamic Insecticides 519** 

C. Wondji, F. Chandre, F. Simard, J-B. Ouédraogo,

**Part 4 Toxicological Profile of Insecticides 477** 

Chapter 22 **Trends in Insecticide Resistance in Natural Populations of Malaria Vectors** 

T. Martin, M. Weill and T. Baldet

Rute Félix and Henrique Silveira

Sonia Soloneski and Marcelo L. Larramendy

Chapter 23 **The Role of** *Anopheles gambiae* **P450** 

Chapter 24 **Genetic Toxicological Profile** 

Chapter 10 **Essential Plant Oils and Insecticidal Activity in** *Culex quinquefasciatus* **221**  Maureen Leyva, Olinka Tiomno, Juan E. Tacoronte, Maria del Carmen Marquetti and Domingo Montada


#### **Part 3 Pest Management 285**


Chapter 20 **The Conundrum of Chemical Boll Weevil Control in Subtropical Regions 437**  Allan T. Showler

VI Contents

Chapter 9 **Behavioral Responses of Mosquitoes to Insecticides 201**

Maureen Leyva, Olinka Tiomno, Juan E. Tacoronte, Maria del Carmen Marquetti and Domingo Montada

**by** *Bacillus thuringiensis* **subsp.** *israelensis* **239** Mario Ramírez-Lepe and Montserrat Ramírez-Suero

**and Insecticidal Activity in** *Culex quinquefasciatus* **221** 

**Cytochrome P450 Enzymes: Impact on Vector Control 265** 

**Deterrent and Toxic Metabolites Against Insects 287** 

**Pests in the Pacific Northwest of the United States 309**

**Gelechiidae) with Insecticides on Tomatoes 333**

**Flower Thrips and the Role of Insecticides 355**

**Against the Pear Psylla,** *Cacopsylla pyri* **L. 385**

**on Glassy-Winged Sharpshooter (***Homalodisca vitripennis* **)**

**the Prevention of Spread of** *Xylella fastidiosa* **in Grape 409**

Carlos García Salazar, Anamaría Gómez Rodas and John C. Wise

Theeraphap Chareonviriyaphap

Chapter 11 **Biological Control of Mosquito Larvae** 

Chapter 12 **Metabolism of Pyrethroids by Mosquito**

**Part 3 Pest Management 285** 

Silvia I. Rondon

Stefano Civolani

Chapter 19 **Use and Management** 

Pornpimol Rongnoparut, Sirikun Pethuan, Songklod Sarapusit and Panida Lertkiatmongkol

Chapter 13 **Bioactive Natural Products from Sapindaceae** 

Chapter 14 **Pest Management Strategies for Potato Insect** 

Chapter 15 **Management of** *Tuta absoluta* **(Lepidoptera,** 

Mohamed Braham and Lobna Hajji

Stuart R. Reitz and Joe Funderburk

Chapter 18 **Effects of Kaolin Particle Film and Imidacloprid** 

**(Hemiptera: Cicadellidae) Populations and**

**of Pesticides in Small Fruit Production 425** 

K.M. Tubajika, G.J. Puterka, N.C. Toscano,

Chapter 17 **The Past and Present of Pear Protection** 

J. Chen and E.L. Civerolo

Chapter 16 **Management Strategies for Western** 

Martina Díaz and Carmen Rossini

Chapter 10 **Essential Plant Oils** 

	- **Part 4 Toxicological Profile of Insecticides 477**

Preface

in biological control.

Agriculture is the mainstay of worldwide economy and the majority of urban and rural population of the world depends on it. Production of agricultural commodities is hindered by pest attacks. Sometimes the damage caused can be so severe that the economic yield of a crop is not possible. Insecticides are organic or inorganic chemical substances or mixtures of substances that can occur naturally or be synthesized, and are intended for preventing, destroying, repelling or mitigating the effect of any pest

Pesticides are divided to insecticides, fungicides, herbicides, rodenticides, acaricides and nematocides according to the organisms that they affect. There are various forms of insecticides; most are repellants or insect growth regulators used in agriculture, public health, horticulture or food storage. It is evident that insecticides have been used to boost food production to a considerable extent and to control disease vectors. Insecticides are used in various forms; from hydrocarbon oils, arsenical compounds, organochlorine, organophosphorus, carbamates, dinitrophenols, organic thiocynates, sulfur, sodium fluoride, pyrethroids and rotenone, to nicotine and bioactive natural products in solid or liquid form. These insecticides are highly toxic to pests and many others are relatively harmless to other organisms. Pests can respond to insecticides in

at least two different ways: behavioral action, namely avoidance and toxicity.

A bacterium Bti is applied successfully in biological control programs against mosquitoes and flies larvae all over the world. The study of each of its facets is addressed in this book and will open new perspectives to improve their effectiveness

Vector-borne diseases are a major contributor to the overall burden of diseases, particularly in tropical and sub-tropical areas, and a significant impediment to socioeconomic development in developing countries. Insecticides still provide the most promising countermeasures to control malaria, dengue hemorrhagic fever (DHF) and other arthropod-borne diseases. The knowledge about the mosquito's behavioral responses to particular chemicals is very important for the prioritization and design of appropriate vector prevention and control strategies. Today, the development of insecticide resistance in insect pests and disease vectors occurs worldwide and on an increasing scale. This phenomenon suggests that behavioral responses will likely play a significant role in how certain pesticides perform to interrupt human-vector contact

including avian, mammalian, crawling and flying insect pests.

## Preface

Agriculture is the mainstay of worldwide economy and the majority of urban and rural population of the world depends on it. Production of agricultural commodities is hindered by pest attacks. Sometimes the damage caused can be so severe that the economic yield of a crop is not possible. Insecticides are organic or inorganic chemical substances or mixtures of substances that can occur naturally or be synthesized, and are intended for preventing, destroying, repelling or mitigating the effect of any pest including avian, mammalian, crawling and flying insect pests.

Pesticides are divided to insecticides, fungicides, herbicides, rodenticides, acaricides and nematocides according to the organisms that they affect. There are various forms of insecticides; most are repellants or insect growth regulators used in agriculture, public health, horticulture or food storage. It is evident that insecticides have been used to boost food production to a considerable extent and to control disease vectors. Insecticides are used in various forms; from hydrocarbon oils, arsenical compounds, organochlorine, organophosphorus, carbamates, dinitrophenols, organic thiocynates, sulfur, sodium fluoride, pyrethroids and rotenone, to nicotine and bioactive natural products in solid or liquid form. These insecticides are highly toxic to pests and many others are relatively harmless to other organisms. Pests can respond to insecticides in at least two different ways: behavioral action, namely avoidance and toxicity.

A bacterium Bti is applied successfully in biological control programs against mosquitoes and flies larvae all over the world. The study of each of its facets is addressed in this book and will open new perspectives to improve their effectiveness in biological control.

Vector-borne diseases are a major contributor to the overall burden of diseases, particularly in tropical and sub-tropical areas, and a significant impediment to socioeconomic development in developing countries. Insecticides still provide the most promising countermeasures to control malaria, dengue hemorrhagic fever (DHF) and other arthropod-borne diseases. The knowledge about the mosquito's behavioral responses to particular chemicals is very important for the prioritization and design of appropriate vector prevention and control strategies. Today, the development of insecticide resistance in insect pests and disease vectors occurs worldwide and on an increasing scale. This phenomenon suggests that behavioral responses will likely play a significant role in how certain pesticides perform to interrupt human-vector contact

#### XIV Preface

while also reducing the selection pressure on target insects for developing resistance. Several factors are believed to play major roles in inducing pyrethroid resistance in mosquitoes. The most serious factor is the uncontrolled use of photo-stable pyrethroids. The relative resistance of mammals to pyrethroids is almost entirely attributable to their ability to hydrolyze the pyrethroids rapidly to their inactive acid and alcohol components, following direct injection into the mammalian CNS.

This book provides information on various aspects of pests, vectors, pesticides, biological control and resistance.

> **Farzana Perveen** Chairperson, Department of Zoology Hazara University, Garden Campus Mansehra Pakistan

**Part 1** 

**Insecticides Mode of Action** 

**1** 

*Nigeria* 

**Insecticide** 

A. C. Achudume

*Institute of Ecology and Environmental Studies* 

Insecticides are organic or inorganic chemicals substances or mixture of substances intended for preventing, destroying, repelling or mitigating the effect of any insect including crawling and flying insects which may occur naturally or is synthesized (pyrethroids) e.g. organic perfumed and hydrocarbon oil and pyrethrins. There are various forms of insecticides. Most of the synthesized insecticides are by their nature are hazardous on health under the condition in which it is used. Insecticides therefore, range from the extremely hazardous to those unlikely to produce any acute hazard. Most are repellants and or insect growth regulators used in agriculture, public health, horticulture, food storage or other chemical

It is evident that insecticides have been used to boost food production to a considerable extent and to control vectors of disease. However, these advantages that are of great economic benefits sometimes come with disadvantages when subjected to critical environmental and human health considerations. Many insecticides are newly synthesized

Insecticides have been used in various forms from hydrocarbon oils (tar oils), arsenical compounds, organochlorine, organ phosphorous compounds carbonates, dinitrophenols, organic thiocynates, sulfur, sodium fluoride, pyethroids ,rotenone to nicotine, in solid or liquid preparation. Interestingly, most of these have been withdrawn due to the deleterious effect of the substances. Analysis of these formulations, their by- products and residues had in the past aided objective re–evaluation and re-assessment of these substances on a benefit– risk analysis basis and their subsequent withdrawal from use when found to be dangerous to human health and the environment. The quality and sophistication of these analyses have grown and very minute quantities of these insecticides or their residues can be

The sequential organomentals and organometalloids insecticides are described in connection with the corresponding inorganic compounds. The highly toxic and recalcitrant compounds e.g. trichloro-bis-chlorophenyl ethane (DDT) and bis-chlorophenyl aqcetic acid (DDA) are formed unintentionally. The organic combination usually changes the absorption and distribution of a toxic metal and thus changes the emphasis of its effects, while the basic mode of action remains the same. The toxic effects of insecticides depend on the elements

**1. Introduction** 

substances used for similar purpose.

whose health and environmental implications are unknown.

**1.1 Inorganic and organ metal insecticides** 

analysed these days with a high degree of specify, precision and accuracy.

*Obafemi Awolowo UniversitY, Ile-Ife,* 

## **Insecticide**

## A. C. Achudume

*Institute of Ecology and Environmental Studies Obafemi Awolowo UniversitY, Ile-Ife, Nigeria* 

## **1. Introduction**

Insecticides are organic or inorganic chemicals substances or mixture of substances intended for preventing, destroying, repelling or mitigating the effect of any insect including crawling and flying insects which may occur naturally or is synthesized (pyrethroids) e.g. organic perfumed and hydrocarbon oil and pyrethrins. There are various forms of insecticides. Most of the synthesized insecticides are by their nature are hazardous on health under the condition in which it is used. Insecticides therefore, range from the extremely hazardous to those unlikely to produce any acute hazard. Most are repellants and or insect growth regulators used in agriculture, public health, horticulture, food storage or other chemical substances used for similar purpose.

It is evident that insecticides have been used to boost food production to a considerable extent and to control vectors of disease. However, these advantages that are of great economic benefits sometimes come with disadvantages when subjected to critical environmental and human health considerations. Many insecticides are newly synthesized whose health and environmental implications are unknown.

Insecticides have been used in various forms from hydrocarbon oils (tar oils), arsenical compounds, organochlorine, organ phosphorous compounds carbonates, dinitrophenols, organic thiocynates, sulfur, sodium fluoride, pyethroids ,rotenone to nicotine, in solid or liquid preparation. Interestingly, most of these have been withdrawn due to the deleterious effect of the substances. Analysis of these formulations, their by- products and residues had in the past aided objective re–evaluation and re-assessment of these substances on a benefit– risk analysis basis and their subsequent withdrawal from use when found to be dangerous to human health and the environment. The quality and sophistication of these analyses have grown and very minute quantities of these insecticides or their residues can be analysed these days with a high degree of specify, precision and accuracy.

#### **1.1 Inorganic and organ metal insecticides**

The sequential organomentals and organometalloids insecticides are described in connection with the corresponding inorganic compounds. The highly toxic and recalcitrant compounds e.g. trichloro-bis-chlorophenyl ethane (DDT) and bis-chlorophenyl aqcetic acid (DDA) are formed unintentionally. The organic combination usually changes the absorption and distribution of a toxic metal and thus changes the emphasis of its effects, while the basic mode of action remains the same. The toxic effects of insecticides depend on the elements

Insecticide 5

generating a bewildering variety of effects in mammals and insects, which although showing some analogies with those produced by other sodium channel toxins (Gray, 1985; Lazdunski et al., 1985) and with DDT (Narahashi, 1986), have many unique characteristics (Ray, 1982b). Thus, toxicity of pyrethroids is divided into two groups Table 1. Type 1 pyrethroids produce the simplest poisoning syndrome and produce sodium tail currents with relatively short time constants (Wright et al., 1988). Poisoning closely resembles that produced by DDT involving a progressive development of fine whole-body tremor, exaggerated startle response, uncoordinated twitching of the dorsal muscles, hyperexcitability, and death (Ray, 1982b). The tremor is associated with a large increase in metabolic rate and leads to hyperthermia which, with metabolic exhaustion, is the usual cause of death. Respiration and blood pressure are well sustained, but plasma noradrenalin, lactate, and adrenaline are greatly increased (Cremer and Servile 1982). Type 1 effects are generated largely by action on the central nervous system, as shown by the good correlation between brain levels of cismethrin and tremor (White et al., 1976). In addition to these central effects, there is evidence for repetitive firing in sensory nerves (Staatz-Benson and

**Type I Intermediate Type II**  Allethrin Cyhenothrin Cyfluthrin Barthrin Fenproponate Cyhalothrin Bioalethrin Flucythrinate Cypermethrin Cismethrin Deltamethrin Fenfluthrin Fenvalerate

Trans-fluorocyphenothrin Cis-fluorocyphenothrin

Table 1. I Acute toxicity of pyrethroids (Wright et al., 1988; Forshow and Ray, 1990).

The type 11 pyrethroid produces a more complex poisoning syndrome and act on a wider range of tissues. They give sodium tail currents with relative longterm constants (Wright, et al., 1988). At lower doses more suble repetitive behavior is seen (Brodie and Aldridge, 1982). As with type I pyrethroids, the primary action is on the central nervous system, since symptoms correlate well with brain concentrations (Rickard and Brodie, 1985). As might be expected, both classes of parathyroid produce large increases in brain glucose utilization (Cremer et al. 1983). A final factor distinguishing type 11 pyrethroids is their ability to depress resting chloride conductance, thereby amplifying any sodium or calcium effects

Intermediate signs representing a combination of type I and type 11 are produced by some pyrethroids. These appear to represent a true combination of the type I and 11 classes (Wright et al., 1983) and thus represent a transitional group. Evidence in support of this is given by measurement of the time constants of the sodium after potential produced by the

Hosko, 1986).

Kadethrin Permethrin Phenothrin Pyrethrin I Pyrethrin II Resmethrin Tetramethrin

(Forshaw and Ray, 1990).

that characterize it as inorganic or organmetal insecticides and on the specific properties of one form of the element or one of its components or merely on an inordinately high dosage. Some highly toxic elements such as iron, selenium, arsenic and fluorine are essential to normal development. The organometals and organometalloids are here described in connection with the corresponding inorganic compounds. Organic combination usually changes the absorption and distribution of a toxic metal and as a result changes the emphasis of its effects, but the basic mode of action remains the same.

The distinction between synthetic compounds and those of natural origin somewhat artificial. In practice, related compounds are assigned to one category or the other, depending on whether the particular compound of the group that was first known and used was of synthetic or of natural origin. For example, pyrethrum and later the naturally occurring pyrethrins were well known for years before the first synthetic parathyroid was made; as such, pyrethroid are thought of as variants of natural compounds, even though they have not been found in nature and are unlikely to occur.

#### **1.2 Pyrethrum and related compounds**

The insecticidal properties of pyrethrum flowers (chrysanthemum cinerarae- folum) have been recognized as insect powder since the middle of last century (McLaughlin 1973). In addition to their insect-killing activity, an attractive feature of the natural pyrethrins (pyrethrum) as insecticides was their lack of persistence in the environment and their rapid action whereby flying insects very quickly become incapacited and unable to fly. Prior the development of DDT, pyrethrum was a major insecticides for both domestic and agricultural use, despite its poor light stability. Development of synthetic pyrethroid with increased light stability and insecticidal activity allows it to be used as foliar insecticide while the natural pyrethrins are now used mainly as domestic insecticides.(Elliot, 1979).

#### **1.3 Mode of Action**

Pyrethrum and the synthetic pyrethroids are sodium channel toxins which, because of their remarkable potency and selection have found application in general pharmacology as well as toxicology (Lazdunski et al., 1985). Pyrethroids have a very high affinity for membrane sodium channels with dissociation constants of the order of 4x10-8 M (Sodeland,1985), and produce subtle changes in their function. By contrast, inexcitable cells are little affected by pyrethroids. The pyrethroids are thus referred to as open channel blockers.

#### **1.4 Metabolism**

The relative resistance of mammals to the pyrethriods is almost wholly attributable to their ability to hydrolyze the pyrethroids rapidly to their inactive acid and alcohol components, since direct injection into the mammalian CNS leads to susceptibility similar to that seen in insects (Lawrence and Casida, 1982). Metabolic disposal of the pyrethroids is very rapid (Gray et al., 1980), which means that toxicity is high by intravenous route, moderate by slower oral absorption, and often immeasurably moderate by slower oral absorption.

#### **1.5 Poisoning syndromes**

The pyrethroids are essentially functional toxins, producing their harmful effects largely secondarily, as a consequence of neuronal hyperexitability (Parker et al.1985). Despite this dependence on a relatively well-defined mode of action, the pyrethroids are capable of

that characterize it as inorganic or organmetal insecticides and on the specific properties of one form of the element or one of its components or merely on an inordinately high dosage. Some highly toxic elements such as iron, selenium, arsenic and fluorine are essential to normal development. The organometals and organometalloids are here described in connection with the corresponding inorganic compounds. Organic combination usually changes the absorption and distribution of a toxic metal and as a result changes the

The distinction between synthetic compounds and those of natural origin somewhat artificial. In practice, related compounds are assigned to one category or the other, depending on whether the particular compound of the group that was first known and used was of synthetic or of natural origin. For example, pyrethrum and later the naturally occurring pyrethrins were well known for years before the first synthetic parathyroid was made; as such, pyrethroid are thought of as variants of natural compounds, even though

The insecticidal properties of pyrethrum flowers (chrysanthemum cinerarae- folum) have been recognized as insect powder since the middle of last century (McLaughlin 1973). In addition to their insect-killing activity, an attractive feature of the natural pyrethrins (pyrethrum) as insecticides was their lack of persistence in the environment and their rapid action whereby flying insects very quickly become incapacited and unable to fly. Prior the development of DDT, pyrethrum was a major insecticides for both domestic and agricultural use, despite its poor light stability. Development of synthetic pyrethroid with increased light stability and insecticidal activity allows it to be used as foliar insecticide while the natural pyrethrins are now used mainly as domestic insecticides.(Elliot, 1979).

Pyrethrum and the synthetic pyrethroids are sodium channel toxins which, because of their remarkable potency and selection have found application in general pharmacology as well as toxicology (Lazdunski et al., 1985). Pyrethroids have a very high affinity for membrane sodium channels with dissociation constants of the order of 4x10-8 M (Sodeland,1985), and produce subtle changes in their function. By contrast, inexcitable cells are little affected by

The relative resistance of mammals to the pyrethriods is almost wholly attributable to their ability to hydrolyze the pyrethroids rapidly to their inactive acid and alcohol components, since direct injection into the mammalian CNS leads to susceptibility similar to that seen in insects (Lawrence and Casida, 1982). Metabolic disposal of the pyrethroids is very rapid (Gray et al., 1980), which means that toxicity is high by intravenous route, moderate by slower oral absorption, and often immeasurably moderate by slower oral absorption.

The pyrethroids are essentially functional toxins, producing their harmful effects largely secondarily, as a consequence of neuronal hyperexitability (Parker et al.1985). Despite this dependence on a relatively well-defined mode of action, the pyrethroids are capable of

pyrethroids. The pyrethroids are thus referred to as open channel blockers.

emphasis of its effects, but the basic mode of action remains the same.

they have not been found in nature and are unlikely to occur.

**1.2 Pyrethrum and related compounds** 

**1.3 Mode of Action** 

**1.4 Metabolism** 

**1.5 Poisoning syndromes** 

Insecticide 5

generating a bewildering variety of effects in mammals and insects, which although showing some analogies with those produced by other sodium channel toxins (Gray, 1985; Lazdunski et al., 1985) and with DDT (Narahashi, 1986), have many unique characteristics (Ray, 1982b). Thus, toxicity of pyrethroids is divided into two groups Table 1. Type 1 pyrethroids produce the simplest poisoning syndrome and produce sodium tail currents with relatively short time constants (Wright et al., 1988). Poisoning closely resembles that produced by DDT involving a progressive development of fine whole-body tremor, exaggerated startle response, uncoordinated twitching of the dorsal muscles, hyperexcitability, and death (Ray, 1982b). The tremor is associated with a large increase in metabolic rate and leads to hyperthermia which, with metabolic exhaustion, is the usual cause of death. Respiration and blood pressure are well sustained, but plasma noradrenalin, lactate, and adrenaline are greatly increased (Cremer and Servile 1982). Type 1 effects are generated largely by action on the central nervous system, as shown by the good correlation between brain levels of cismethrin and tremor (White et al., 1976). In addition to these central effects, there is evidence for repetitive firing in sensory nerves (Staatz-Benson and Hosko, 1986).


Table 1. I Acute toxicity of pyrethroids (Wright et al., 1988; Forshow and Ray, 1990).

The type 11 pyrethroid produces a more complex poisoning syndrome and act on a wider range of tissues. They give sodium tail currents with relative longterm constants (Wright, et al., 1988). At lower doses more suble repetitive behavior is seen (Brodie and Aldridge, 1982). As with type I pyrethroids, the primary action is on the central nervous system, since symptoms correlate well with brain concentrations (Rickard and Brodie, 1985). As might be expected, both classes of parathyroid produce large increases in brain glucose utilization (Cremer et al. 1983). A final factor distinguishing type 11 pyrethroids is their ability to depress resting chloride conductance, thereby amplifying any sodium or calcium effects (Forshaw and Ray, 1990).

Intermediate signs representing a combination of type I and type 11 are produced by some pyrethroids. These appear to represent a true combination of the type I and 11 classes (Wright et al., 1983) and thus represent a transitional group. Evidence in support of this is given by measurement of the time constants of the sodium after potential produced by the

Insecticide 7

The six known insecticidally active compounds in pryrethrum are esters of two acids and three alcohols. Specifically, pyrethrins l is the pyrethrolone ester of chrysanthermic acid, pyrethrin II is the pyrethrolone ester of pyrethria acid, cinerin l is the cinerolone ester of chrysanthemic acid, cinerin II is the cinerolone ester of pyrethric acid, jasmolin I is the jasmolone ester of chrysanthemic acid and jasmolin II is the jasmolone ester of pyrethric acid. There is much evidence indicating that the biological activity of these molecules

The six active ingredients are known collectively as pyrethrins; those based on chrysanthemic acid are called pyrethrin I, and those based on pyrethric acid are called pyrethrins II. Pyrethrins, generally combined with a synergist, are used in sprays and aerosols against a wide range of flying and crawling insects. Usually about 0.5% active pyrethrum principles are formulated. They are equally effective for control of head lice and

The insecticide 'Raid' belongs to a group of chemically stable pyrethrin, has widespread use in control of insects. Chemical stability, insecticide and organic phosphorus hydrocarbon have been shown to accumulate rapidly in tissues causing death and have profound effect on growth (Nebeker et al., 1994). Insecticide raid shows no observable effects on mortality and growth at lower test concentrations in rats. At higher concentration of 430 and 961 µg/g, survival decreased as concentration increases. In addition, mean total body weight of animals fed insecticides raid with concentrations of 430 and 961 µg/g were significantly decreased (P<0.05) than the controls. Conclusively, the higher the concentration of the insecticide Raid, the more hazardous it has on cell death (Achudume et al., 2008)( Table II). Bioaccumulation factor of insecticide Raid was observed in lipids, up to three times that of the feed at the first concentration and gradually decreases as the concentrations increase (Table III), whereas accumulation factor in the muscle (0.7), brain (0.5), and liver (0.3) was about the indicated number times that of the feed. At higher concentration of 961 µg/g, bioaccumulation factor decreased in the lipid to 1.2 and 0.6 in the muscle, 0.03 in the brain, and 0.08 in the liver. Using the mean of insecticide in feed, the tissues accumulate the insecticide in the following ascending order: brain < liver < muscle < lipid. Similarly, Table III indicates the estimated detectable levels of toxicity in rat tissues exposed to the insecticide Raid. The brain shows mild decrease in toxicity of the enzymes glucose-6-phosphatase and lactic acid dehydrogenase, whereas significant decreases were noticeable in the muscle and liver (Achudume et al. 2008). Long-term exposure of insecticide had been reported to result in systemic toxicity such that may impair the function of the nervous system and increase the risk of acute leukemia in children (Menegaux et al., 2006). Also, pesticides including organ phosphorus insecticides used against crawling and flying insects in homes have the potential of being carcinogens (Peter and Cherion, 2000). The adverse effect of insecticide Raid was demonstrated in a study by increase in alkaline phosphates activity in both plasma and liver which is a known measure of hepatic toxicity, and confirms "Raid" as a hepatotoxicant. The significant increase in alkaline phosphates activity (Table IV) may be due to hepatocellular necrosis which causes increase in permeability of cell membrane resulting in the release of this enzyme into the blood stream. The insecticide Raid significantly decreased reduced glutathione levels especially in the liver and this has implications for the ability of the animal to withstand oxidative stress. Studies have shown that GSH deficiency in cells is

depends on their configuration (Elliot, 1969, 1971).

flea in dogs and cats.

**1.7 Raid as insecticide** 

pyrethroids. Since pyrethroids appear to be essentially functional toxins, they produce few if any specific neuropathological effects.

Fig. 1. Pyrethrins of the form.

Fig. 2. Pyrethroids of the form.

#### **1.6 Identity, properties and uses**

The six known insecticidal active compounds in pyrethrum are esters of two acids and three alcohols. Insect powder made from "Dalmatian insect flower" (*Chrysanthemum cinerariaefolium*) is called pyrethrum powder or simply pyrethrum. The powder itself was formerly used as an insecticide, but now it is usually extracted. The six active ingredients are:


pyrethroids. Since pyrethroids appear to be essentially functional toxins, they produce few if

The six known insecticidal active compounds in pyrethrum are esters of two acids and three alcohols. Insect powder made from "Dalmatian insect flower" (*Chrysanthemum cinerariaefolium*) is called pyrethrum powder or simply pyrethrum. The powder itself was formerly used as an insecticide, but now it is usually extracted. The six active ingredients

any specific neuropathological effects.

Fig. 1. Pyrethrins of the form.

Fig. 2. Pyrethroids of the form.

are:

**1.6 Identity, properties and uses** 


The six known insecticidally active compounds in pryrethrum are esters of two acids and three alcohols. Specifically, pyrethrins l is the pyrethrolone ester of chrysanthermic acid, pyrethrin II is the pyrethrolone ester of pyrethria acid, cinerin l is the cinerolone ester of chrysanthemic acid, cinerin II is the cinerolone ester of pyrethric acid, jasmolin I is the jasmolone ester of chrysanthemic acid and jasmolin II is the jasmolone ester of pyrethric acid. There is much evidence indicating that the biological activity of these molecules depends on their configuration (Elliot, 1969, 1971).

The six active ingredients are known collectively as pyrethrins; those based on chrysanthemic acid are called pyrethrin I, and those based on pyrethric acid are called pyrethrins II. Pyrethrins, generally combined with a synergist, are used in sprays and aerosols against a wide range of flying and crawling insects. Usually about 0.5% active pyrethrum principles are formulated. They are equally effective for control of head lice and flea in dogs and cats.

#### **1.7 Raid as insecticide**

The insecticide 'Raid' belongs to a group of chemically stable pyrethrin, has widespread use in control of insects. Chemical stability, insecticide and organic phosphorus hydrocarbon have been shown to accumulate rapidly in tissues causing death and have profound effect on growth (Nebeker et al., 1994). Insecticide raid shows no observable effects on mortality and growth at lower test concentrations in rats. At higher concentration of 430 and 961 µg/g, survival decreased as concentration increases. In addition, mean total body weight of animals fed insecticides raid with concentrations of 430 and 961 µg/g were significantly decreased (P<0.05) than the controls. Conclusively, the higher the concentration of the insecticide Raid, the more hazardous it has on cell death (Achudume et al., 2008)( Table II).

Bioaccumulation factor of insecticide Raid was observed in lipids, up to three times that of the feed at the first concentration and gradually decreases as the concentrations increase (Table III), whereas accumulation factor in the muscle (0.7), brain (0.5), and liver (0.3) was about the indicated number times that of the feed. At higher concentration of 961 µg/g, bioaccumulation factor decreased in the lipid to 1.2 and 0.6 in the muscle, 0.03 in the brain, and 0.08 in the liver. Using the mean of insecticide in feed, the tissues accumulate the insecticide in the following ascending order: brain < liver < muscle < lipid. Similarly, Table III indicates the estimated detectable levels of toxicity in rat tissues exposed to the insecticide Raid. The brain shows mild decrease in toxicity of the enzymes glucose-6-phosphatase and lactic acid dehydrogenase, whereas significant decreases were noticeable in the muscle and liver (Achudume et al. 2008).

Long-term exposure of insecticide had been reported to result in systemic toxicity such that may impair the function of the nervous system and increase the risk of acute leukemia in children (Menegaux et al., 2006). Also, pesticides including organ phosphorus insecticides used against crawling and flying insects in homes have the potential of being carcinogens (Peter and Cherion, 2000). The adverse effect of insecticide Raid was demonstrated in a study by increase in alkaline phosphates activity in both plasma and liver which is a known measure of hepatic toxicity, and confirms "Raid" as a hepatotoxicant. The significant increase in alkaline phosphates activity (Table IV) may be due to hepatocellular necrosis which causes increase in permeability of cell membrane resulting in the release of this enzyme into the blood stream. The insecticide Raid significantly decreased reduced glutathione levels especially in the liver and this has implications for the ability of the animal to withstand oxidative stress. Studies have shown that GSH deficiency in cells is

Insecticide 9

Raid concentrations Alk pase GSH Glucose Tissue activity level level

In feed (µg/g) µgml-min-L mg/ml mg/g liver 430±20.2 Control 0.08±0.04 0.18±0.02 0.96±0.04

961.2±70.5 Control 0.09±0.05 0.19±0.05 0.96±0.52

Table 1. IV Effect of Raid concentrations in feed on hepatic enzyme activity, reduced

Data values are mean±SD \*Statistically significant p< 0.05

glutathione and glucose levels.

Fig. 3. Structure of rotenone.

Plasma 0.06±0.09 0.15±0.6 0.90±0.04

Control 0.08±0.04 0.18±0.02 0.94±0.01

Liver 0.06±0.02\* 0.15±0.01 1.05±0.12

Plasma 0.06±0.01 0.11±0.05 1.09±0.52 Control 0.08±0.08 0.19±0.02 0.96±0.06 Liver 0.05±0.08\* 0.09±0.03\* 1.66±0.04

associated with markedly decreased survival (Kohlmeier et al., 1997), thus, chemically stable lipid-soluble, organophosphorus insecticides are hazarddous to health through mechanisms including depletion of GSH (Menegaux et al., 2006).


Table 1. II mortality and growth of wistar rats exposed to different concentrations of "Raid".


Table 1. III Tissue total raid concentrations and bioaccumulation factors (BAF) in wistar rats.

associated with markedly decreased survival (Kohlmeier et al., 1997), thus, chemically stable lipid-soluble, organophosphorus insecticides are hazarddous to health through mechanisms

Table 1. II mortality and growth of wistar rats exposed to different concentrations of "Raid".

Raid Concentration in Wistar Rats (µg/g)a and Bioaccumulation Factor (BAF) Mean±SD Insecticide \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ "Raid" in Feed (µg/g) Lipid Muscle Brain Liver 00.0 - - - - 25.0±2.4 72.5± (2.9) 17.5(0.7) 12.5(0.5) 7.5(0.3) 54.0±9.2 86.4(1.6) 21.7(0.4) 16.4(0.3) 9.4(0.2) 108.2±12.5 172.8(1.6) 30.4(0.3) 19.5(0.2) 10.8(0.10) 216.2±14.6 280.8(1.3) 45.8(0.2) 22.9(0.1) 19.8(0.09) 430.0±20.6 324.0(0.8) 86.4(0.2) 25.8(0.06) 37.3(0.09) 961.2±70.5 1153.2(1.2) 576.6(0.6) 28.8(0.03) 76.9(0.08)

Table 1. III Tissue total raid concentrations and bioaccumulation factors (BAF) in wistar rats.

Mortality Means: SD body weight (g)

including depletion of GSH (Menegaux et al., 2006).

0.00 Nil 135=5.4 25.0=2.4 Nil 135=21.7 54.0=9.5 Nil 132=2.9 108.2=12.5 Nil 129=3.2 216.2=14.6 Nil 128=19.8 430.0=20.2 1 118=20.5 961.2=70.5 2 116=5.3

Means SD concentrations of insecticide "Raid" in feed

(Mg/g)


Data values are mean±SD \*Statistically significant p< 0.05

Table 1. IV Effect of Raid concentrations in feed on hepatic enzyme activity, reduced glutathione and glucose levels.

Fig. 3. Structure of rotenone.

Insecticide 11

Systemic insecticides are incorporated by treated plants. Insect ingest the insecticide

 Contact insecticides are toxic to insects by direct contact. Efficacy is often related to the quality of pesticide application in aerosols which often improve performance. Natural insecticides, such as pincotine, pyrethrum and neem extracts are from plants as

Organic insecticides are synthetic chemicals which comprise the largest numbers of

Insecticides are pesticides used to control insects many of these insecticides are very toxic to

Insecticides decompose readily so the residues do not accumulate on crops or in the soil. Insecticides include ovicides and larvicides used against the eggs and larvae of insects

The use of insecticides is believed to be one of the major factors behind the increase in agricultural productivity (McLaughlin,1973, van Emden and Pealall, 1996). Nearly all insecticides have the potential to significantly alter ecosystems; many are toxic to humans; and others are concentrated in the food chain (WHO 1962, 1972). Selected inorganic metals

Barium is an alkaline earth metal in the same group as magnesium, calcium, strontium and radium. It valence is two. All are water-and acid soluble compounds. They are poisonous. Barium carbonate is a rat poison. It is used in ceramics, paints, enamels, rubber and certain

Absorption, Distribution, Metabolism Excretion (ADME): Barium carbonate is highly insoluble in water. It is partially solubilized by acid in the stomach. The danger of the insecticide is through ingestion. Various barium compounds can cause pneumoconiosis. It is absorbed from gastrointestinal tracts of rat rapidly and completely. It is stored in bone and in other tissues (Hayes 1982, Castagnou et al 1957, Dencker et al., 1976, 83). Excretion takes

Mode of action: Barium stimulates striated cardiac and smooth muscle, regardless of the

Chromium is a metal somewhat like iron and separated in the periodic table by manganese. Only hexavalent chromium compounds (chromates) are important as pesticides. They are also the most toxic. Chromate is absorbed by the lung (Baetjer et al., 1959), gastrointestinal tract and skin. It is widely distributed in the liver, kidney, bone and spleen (Mackenzie et al 1958). Acute poisoning may produce death rapidly through shock or renal tubular damage

Mercury is toxic no matter what its chemical combination. It is widely distributed in the environment, and traces of it occur in food, water and tissues even in the absence of occupational exposure. Inhaled mercury vapour diffuses across the alveolar regions of the

Inorganic insecticides are manufactured with metals e.g. Heavy metals

insects and many others are relatively harmless to other organism except fish.

are discussed in the next section followed by individual insecticides organ metals.

place rapidly in urine and feces in 24hr ( Bauer et al. 1956).

while feeding on the plants.

defences against insects.

pesticides available.

respectively.

**2.1 Barium** 

plastics.

innervation.

**2.2 Chromium** 

**2.3 Mercury** 

and uremia (Steffee and Baetjer 1965).

Fig. 4. Nicotine, narnicotine and anabasine with two important metabolites of nicotine.

Some other studies confirm that glutathione deficiency is associated with impaired survival in HIV disease (Herzenbery et al., 1997). Glutathione may be consumed by conjugation reaction, which mainly involve metabolism of zenobiotic agent. However, the principle mechanisms of hepatocyte glutathione turnover are known to be by cellular efflux (Sies et al., 1978). Glutathione reducatase is a known defense against oxidative stress, which in turn needs glutathione as co-factors. Catalase is an antioxidant enzyme which destroys H2O2 that can form a highly reactive radical in the presence of iron as catalyst (Gutter ridge, 1995).

 Achudume et al., 2008 showed that bioaccumulation factor of insecticides raid was observed in lipid. Lipid peroxidation is a chemical mechanism capable of disrupting the structure and function of the biological membranes that occurs as a result of free radical attack on lipids. Some study confirms that insecticide raid increased lipid peroxidation, oxidative stress and hepatotoxicity due to reduced antioxidant system.

In addition, SOD is family of metalloid enzyme which is considered to be stress protein which decreases in response to oxidative stress (McCord, 1990). It is evident that decrease of SOD in the tissue is a confirmation of its protection from damage caused by insecticide Raid.

#### **2. Classes of insecticides**

 The classification of insecticides is done in several different ways (Hayes, 1982),(Heam 1973, Lehman, 1954, Martin and 1977).

Fig. 4. Nicotine, narnicotine and anabasine with two important metabolites of nicotine.

oxidative stress and hepatotoxicity due to reduced antioxidant system.

**2. Classes of insecticides** 

1973, Lehman, 1954, Martin and 1977).

Some other studies confirm that glutathione deficiency is associated with impaired survival in HIV disease (Herzenbery et al., 1997). Glutathione may be consumed by conjugation reaction, which mainly involve metabolism of zenobiotic agent. However, the principle mechanisms of hepatocyte glutathione turnover are known to be by cellular efflux (Sies et al., 1978). Glutathione reducatase is a known defense against oxidative stress, which in turn needs glutathione as co-factors. Catalase is an antioxidant enzyme which destroys H2O2 that can form a highly reactive radical in the presence of iron as catalyst (Gutter ridge, 1995). Achudume et al., 2008 showed that bioaccumulation factor of insecticides raid was observed in lipid. Lipid peroxidation is a chemical mechanism capable of disrupting the structure and function of the biological membranes that occurs as a result of free radical attack on lipids. Some study confirms that insecticide raid increased lipid peroxidation,

In addition, SOD is family of metalloid enzyme which is considered to be stress protein which decreases in response to oxidative stress (McCord, 1990). It is evident that decrease of SOD in the tissue is a confirmation of its protection from damage caused by insecticide Raid.

The classification of insecticides is done in several different ways (Hayes, 1982),(Heam


Insecticides are pesticides used to control insects many of these insecticides are very toxic to insects and many others are relatively harmless to other organism except fish.

Insecticides decompose readily so the residues do not accumulate on crops or in the soil. Insecticides include ovicides and larvicides used against the eggs and larvae of insects respectively.

The use of insecticides is believed to be one of the major factors behind the increase in agricultural productivity (McLaughlin,1973, van Emden and Pealall, 1996). Nearly all insecticides have the potential to significantly alter ecosystems; many are toxic to humans; and others are concentrated in the food chain (WHO 1962, 1972). Selected inorganic metals are discussed in the next section followed by individual insecticides organ metals.

## **2.1 Barium**

Barium is an alkaline earth metal in the same group as magnesium, calcium, strontium and radium. It valence is two. All are water-and acid soluble compounds. They are poisonous. Barium carbonate is a rat poison. It is used in ceramics, paints, enamels, rubber and certain plastics.

Absorption, Distribution, Metabolism Excretion (ADME): Barium carbonate is highly insoluble in water. It is partially solubilized by acid in the stomach. The danger of the insecticide is through ingestion. Various barium compounds can cause pneumoconiosis. It is absorbed from gastrointestinal tracts of rat rapidly and completely. It is stored in bone and in other tissues (Hayes 1982, Castagnou et al 1957, Dencker et al., 1976, 83). Excretion takes place rapidly in urine and feces in 24hr ( Bauer et al. 1956).

Mode of action: Barium stimulates striated cardiac and smooth muscle, regardless of the innervation.

#### **2.2 Chromium**

Chromium is a metal somewhat like iron and separated in the periodic table by manganese. Only hexavalent chromium compounds (chromates) are important as pesticides. They are also the most toxic. Chromate is absorbed by the lung (Baetjer et al., 1959), gastrointestinal tract and skin. It is widely distributed in the liver, kidney, bone and spleen (Mackenzie et al 1958). Acute poisoning may produce death rapidly through shock or renal tubular damage and uremia (Steffee and Baetjer 1965).

#### **2.3 Mercury**

Mercury is toxic no matter what its chemical combination. It is widely distributed in the environment, and traces of it occur in food, water and tissues even in the absence of occupational exposure. Inhaled mercury vapour diffuses across the alveolar regions of the

Insecticide 13

restricted its use as an insecticide to bait formulations (who, 1970). Sodium fluoride

Boric acid and borax have been used as an insecticide, both mainly for the control of cockroaches. Boric acid also is known as boracic acid and as orthoboric acid. Absorption from the gastrointestinal tract is rapid and virtually complete. Its peak concentration is in brain and less in other tissues. Boric acid is excreted unchanged in the urine (wong et al.,

Different groups of insecticide; derived from living organisms are entirely unrelated chemically and pharmacologically. They range from relatively simple alkaloids such as nicotine, with a molecular weight of only 162.2, through proteinaceous poisons to virulent living organism. They range in toxicity from harmless and fragile pheromones, which are

The distinction between synthetic compounds and those derived from living organisms is somewhat artificial. In practice, related compounds are assigned to one category or the other, depending on whether the particular compound of the group that was first known and used was of synthetic or of natural origin. For example, pyrethrum and later the naturally occurring pyrethriums were well known for years before the first synthetic pyrethroid was made; as a result, pyrethroids are thought of as various of natural compounds, even though they have not been found in nature and are unlikely to occur. By contrast, synthetic sodium fluoroacetate acquired a reputation as a rodenticids and was explored as a synthetic insecticide before it was realized that the potassium salt is the active

Perhaps the only unifying feature of the diverse array of poisons derived from living organism is the popular view that "natural" substances are harmless. On this matter of safety, an expert committee of the world health organisation pointed out that "all the most

The insecticidal properties of pyrethrum flowers (genus chrysanthemum) have been recognized since the middle of 1st century, when commercial sale of "insect powder" from Dalmatian pyrethrum flower heads began (McLaughlin, 1973). In addition to their insectkilling activity, their lack of persistence in the environment and rapid "knock down" activity whereby flying insects become uncoordinated and unable to fly makes it very useful. Pyrethrum used to be a major insecticide for both domestic and agricultural use despite its poor light stability. Its usefulness was extended by introduction of piperonyl butoxide and other compounds as synergists, which greatly reduced the unit cost of crop treatment. Development of synthetic pyrethroids with increased stability and insecticidal activity (Elliot 1977) reduced the use of pyrethrum. However, natural pyrethrins are now used mainly as domestic insecticides, while the synthetic pyrethroids represented 20-25% of the world foliar insecticide market in 1983 (Herve's 85) and the proportion is increasing

concentrates more in the plasma and liver and is excreted in urine.

**3.2 Insecticides derived from living organisms and other sources:** 

principal of a poisonous plant. Thus pyrethroids are discussed extensively.

poisonous materials so far know are, in fact, of natural origin" (WHO,1967).

**3. Miscellaneous elements** 

used as a chemical warfare agent.

**3.3 Pyrethrum and related compounds** 

**3.1 Boric acid** 

1964).

lung into the blood stream. Mercury vapour is a monatomic gas which is highly diffusible and lipid soluble (Berlin et al 1969a , Hush,1985). Once in the bloodstream mercury vapour enters the blood cells where it is oxidized to divalent inorganic mercury under the influence of catalase (Halbach and Clarkson 1978). Mercury is widely distributed with the highest concentrations in the kidney.

## **2.4 Thallium**

Thallium stands between mercury and lead in the periodic table, and compounds of these metals show marked similarities. All of them may produce immediate local irritation followed by delayed effects in various organs, notable the nervous system. Thallium sulphate has been more widely used as pesticide than any other compound of thallium. It has produced many cases of poisoning and serves as good example of the toxicity of thallium generally (Lund, 1956b).

Thallium is easily absorbed by the skin as well as by the respiratory and the gastrointestinal tracts. Thallium accumulates in hair follicles and much less in those in the resting phase. Excretion is slow and is entirely by urine in humans but in rats via faeces (Barclay et al., 1953, Lund, 1956a)

## **2.5 Lead arsenate**

Lead arsenate includes acid lead arsenate, dibasic lead arsenate, dilead orthoarsenate, diplumbic hydrogen arsenate, lead hydrogen arsenate and standard lead arsenate. Lead arsenate is used as an insecticide. it is used to control moths, leaf rollers and other chewing insects and in soil for the treatment of Japanese and Asian beetles in lawn. Absorption is generally via gastrointestinal. Dermal absorption is extremely small. Lead and arsenate are distributed separately in the body. lead is stored in highest concentration in the bone with much lower concentrations in soft tissues. Arsenic is stored in the liver and in some instances in the kidney at higher concentrations than those for lead (Fairhall and Miller, 1941. Lead is transferred to the fetus of animals humans (Heriuchi et al., 1959).

## **2.6 Antimony potassium tartrate**

This compound serves as a poison in baits to control insects, especially thrips, and as an emetic in bait to control rodents. Ingestion of the compound usually leads to repeated vomiting. Excretion is mainly urinary (Fairhall and Hyslop, 1947).

## **2.7 Sodium selenate**

Sodium selenate is an insecticide used in horticulture for control of mites, aphids and mealybugs. Various compounds of selenium are freely absorbed from the respiratory and gastrointestinal tracts. Dermal absorption is less important. Selenium is stored more in the liver, kidney, spleen, pancreas, heart and lung than in other organs (Underwood, 1977). Selenium is excreted chiefly in the urine but about 3-10% is metabolized and excreted by the lungs and through faecal excretion.

#### **2.8 Sodium fluoride**

Sodium fluoride is toxic to all forms of life. It has been used as an insecticide, rodenticide and herbicide and as fungicide for preservation of timber. Its toxicity to plants generally has

lung into the blood stream. Mercury vapour is a monatomic gas which is highly diffusible and lipid soluble (Berlin et al 1969a , Hush,1985). Once in the bloodstream mercury vapour enters the blood cells where it is oxidized to divalent inorganic mercury under the influence of catalase (Halbach and Clarkson 1978). Mercury is widely distributed with the highest

Thallium stands between mercury and lead in the periodic table, and compounds of these metals show marked similarities. All of them may produce immediate local irritation followed by delayed effects in various organs, notable the nervous system. Thallium sulphate has been more widely used as pesticide than any other compound of thallium. It has produced many cases of poisoning and serves as good example of the toxicity of

Thallium is easily absorbed by the skin as well as by the respiratory and the gastrointestinal tracts. Thallium accumulates in hair follicles and much less in those in the resting phase. Excretion is slow and is entirely by urine in humans but in rats via faeces (Barclay et al.,

Lead arsenate includes acid lead arsenate, dibasic lead arsenate, dilead orthoarsenate, diplumbic hydrogen arsenate, lead hydrogen arsenate and standard lead arsenate. Lead arsenate is used as an insecticide. it is used to control moths, leaf rollers and other chewing insects and in soil for the treatment of Japanese and Asian beetles in lawn. Absorption is generally via gastrointestinal. Dermal absorption is extremely small. Lead and arsenate are distributed separately in the body. lead is stored in highest concentration in the bone with much lower concentrations in soft tissues. Arsenic is stored in the liver and in some instances in the kidney at higher concentrations than those for lead (Fairhall and Miller, 1941. Lead is transferred to the fetus of animals humans (Heriuchi et al.,

This compound serves as a poison in baits to control insects, especially thrips, and as an emetic in bait to control rodents. Ingestion of the compound usually leads to repeated

Sodium selenate is an insecticide used in horticulture for control of mites, aphids and mealybugs. Various compounds of selenium are freely absorbed from the respiratory and gastrointestinal tracts. Dermal absorption is less important. Selenium is stored more in the liver, kidney, spleen, pancreas, heart and lung than in other organs (Underwood, 1977). Selenium is excreted chiefly in the urine but about 3-10% is metabolized and excreted by the

Sodium fluoride is toxic to all forms of life. It has been used as an insecticide, rodenticide and herbicide and as fungicide for preservation of timber. Its toxicity to plants generally has

vomiting. Excretion is mainly urinary (Fairhall and Hyslop, 1947).

concentrations in the kidney.

thallium generally (Lund, 1956b).

**2.6 Antimony potassium tartrate** 

lungs and through faecal excretion.

**2.7 Sodium selenate** 

**2.8 Sodium fluoride** 

**2.4 Thallium** 

1953, Lund, 1956a)

**2.5 Lead arsenate** 

1959).

restricted its use as an insecticide to bait formulations (who, 1970). Sodium fluoride concentrates more in the plasma and liver and is excreted in urine.

### **3. Miscellaneous elements**

#### **3.1 Boric acid**

Boric acid and borax have been used as an insecticide, both mainly for the control of cockroaches. Boric acid also is known as boracic acid and as orthoboric acid. Absorption from the gastrointestinal tract is rapid and virtually complete. Its peak concentration is in brain and less in other tissues. Boric acid is excreted unchanged in the urine (wong et al., 1964).

#### **3.2 Insecticides derived from living organisms and other sources:**

Different groups of insecticide; derived from living organisms are entirely unrelated chemically and pharmacologically. They range from relatively simple alkaloids such as nicotine, with a molecular weight of only 162.2, through proteinaceous poisons to virulent living organism. They range in toxicity from harmless and fragile pheromones, which are used as a chemical warfare agent.

The distinction between synthetic compounds and those derived from living organisms is somewhat artificial. In practice, related compounds are assigned to one category or the other, depending on whether the particular compound of the group that was first known and used was of synthetic or of natural origin. For example, pyrethrum and later the naturally occurring pyrethriums were well known for years before the first synthetic pyrethroid was made; as a result, pyrethroids are thought of as various of natural compounds, even though they have not been found in nature and are unlikely to occur. By contrast, synthetic sodium fluoroacetate acquired a reputation as a rodenticids and was explored as a synthetic insecticide before it was realized that the potassium salt is the active principal of a poisonous plant. Thus pyrethroids are discussed extensively.

Perhaps the only unifying feature of the diverse array of poisons derived from living organism is the popular view that "natural" substances are harmless. On this matter of safety, an expert committee of the world health organisation pointed out that "all the most poisonous materials so far know are, in fact, of natural origin" (WHO,1967).

#### **3.3 Pyrethrum and related compounds**

The insecticidal properties of pyrethrum flowers (genus chrysanthemum) have been recognized since the middle of 1st century, when commercial sale of "insect powder" from Dalmatian pyrethrum flower heads began (McLaughlin, 1973). In addition to their insectkilling activity, their lack of persistence in the environment and rapid "knock down" activity whereby flying insects become uncoordinated and unable to fly makes it very useful. Pyrethrum used to be a major insecticide for both domestic and agricultural use despite its poor light stability. Its usefulness was extended by introduction of piperonyl butoxide and other compounds as synergists, which greatly reduced the unit cost of crop treatment. Development of synthetic pyrethroids with increased stability and insecticidal activity (Elliot 1977) reduced the use of pyrethrum. However, natural pyrethrins are now used mainly as domestic insecticides, while the synthetic pyrethroids represented 20-25% of the world foliar insecticide market in 1983 (Herve's 85) and the proportion is increasing

Insecticide 15

Cypermethrin (R, S)-∞- cyano-3-pheno-xybenzyl-2, 2-dimethyl. There are eight isomeric forms. It was introduced commercially in 1977 as an emulsifiable concentrate to be used

Deltamethrin S-∞- cyano-3-phenoxybenzyl-(IR)-cis-3-(2, 2-dibromovinyl)2,2-dimethcyclopropane carboxylate. It is a single isomer. It is used against a wide range of insect pests. It produces a typical type II motor symptom in mammals (Barnes and verschoyle, 1974. Metabolism of deltamethrin involves rapid ester cleavages and hydroxylation (Shono eta al;

Fenproponate(∞-cyano-3-phenoxybenzyl-2,2,3,3-tetra-methylcyclopropanecarboxylate). There are eight isomerism forms. Fenpropathrin is another common name, was first developed by sumitomo and commercialized in 1980 as an emulsifiable concentrate to be used against a wide range of insect pests. Fenproponate produces intermediate or mixed

Fenvalerate (R,S)- ∞- cyano-3-phenoxy-benzyl (IR,IS)-2-(4-chlorophenyl)-3-methyl-1 butyrate. There are four isomeric forms. It should be noted that fenvalerate is not based on a cyclopropane ring structure. It was introduced commercially to be used against a wide range of insect pests fenvalrate produces typical type II motor symptoms in mammals

Phenothrin (3-phenoxybenzy-(IR,IS)-cis,trans-3(2-methylprop-1-enyl)-2,2 dimethyicyclopropane carboxylate). There are four isomeric forms. It is used as a domestic insecticide in a partially resolved mixture rich in the IR isomer (Sumithrin) and for grain protection. Phenothrin produces typical type 1 moto symptoms in mammals (Lawrence and Casida,

Rotenone-bearing plants have longed being used as a fish poison by many ancient different indigenous people, nut their use as an insecticide is probably more than a century old. Plants known to produce rotenone and other rotenoids belong to at least 68 species of the family Leguminosae, the same as that for peas and beans. The genera most exploited so far are Derris, native to southeast Asia, and lonchocarpus to south America (shepard 1951). Rotenone and other active principles often occur chiefly in the roots of rotenone bearing plants but may be in the leaves (as in *Tephrosia vogeli*), seeds (as in *Milletia pachycarpa*), or

Regardless of the genus or the particular part of the plant involved, the active constituents

Rotenone is (2R,6a 5,12a 5)-1,2,6,6a,12,12a-hexahydro-2-isopropenyl-8,9-dimethoxychromeno (3,4-b) furo(2,3-h) chromen-6-one. Its structure is depicted in fig. 3. Although

of rotenone-bearing plants may be extracted with ether or acetone as resin.

**4.3 Cypermethrin** 

**4.4 Deltamethrin** 

**4.5 Fenproponate** 

**4.6 Fenvalerate** 

**4.7 Phenothrin** 

1982)

(Verschoyle and Aldrige, 1980).

**4.8 Rotenone and related materials** 

bark (as in *Mundulea serica*).

1977).

against a wide range of insect pest (Elliot, 1977).

motor symptoms in mammals (Wright et al., 1988).

steadily. Thousands of new synthetic pyrethroids have been synthesized, some showing complete divergence from the original pyrethrins (casida et al., 1973). Table 2.1 and Table 2.2.

## **4. Mode of action**

Pyrethrum and the synthetic pyrethroid are sodium channel toxins which, because of their remarkable potency and selectivity, have found application in general toxicology (Lazdunski et al 1985). Their actions on the nerve membrane sodium channel are well understood. Pyrethroids have a very high affinity for membrane sodium channel, they have little effect on inactive sodium channels or close channels and produce subtle changes in their functions. After modification by prethroids, sodium channels continue in many of their normal functions, retaining their selectivity for sodium ions and link with membrane potential (Narahashi, 1986). The pyrethroids are thus known as open channel blockers. Detailed studies can be found in Narahishi 1986, Jacques et al., 1980, and Gray 1985.

## **4.1 Metabolism**

The relative resistance of mammals to the pyrethroids is almost wholly attributable to their ability to hydrolyze the pyrethroids rapidly to their inactive acid and alcohol components, since direct injection into the mammalian CNS leads to susceptibility similar to that observed in insects (Lawrence and Casida, 1982). Some additional resistance of homoeothermic organisms can be attributed to the negative temperature coefficient of action of the pyrethroids (Van den Bercken et al., 1973) which are thus less toxic at mammalian body temperature but the major effect is metabolic.

The metabolic pathways for the breakdown of the pyrethroids vary little between mammalian species but vary somewhat with structure. This literature has been ably summarized by Leahy (1985), and further references to the metabolism of specific pyrethroids are given in the sections on individual compounds. Generally pyrethrum and allethrin are broken down mainly by oxidation, whereas for the other pyrethroids ester hydrolysis predominates. These reactions can take place in both liver and plasma and are followed by hydroxylation and conjugation to glucuronides or sulphates, which are then excreted in the urine (Gray 1985).

#### **4.2 Individual insecticides**

Other known insecticides pyrethroids under organophosphates are listed below only selective ones are discussed.


Table 4. I Other known insecticides.

steadily. Thousands of new synthetic pyrethroids have been synthesized, some showing complete divergence from the original pyrethrins (casida et al., 1973). Table 2.1 and Table

Pyrethrum and the synthetic pyrethroid are sodium channel toxins which, because of their remarkable potency and selectivity, have found application in general toxicology (Lazdunski et al 1985). Their actions on the nerve membrane sodium channel are well understood. Pyrethroids have a very high affinity for membrane sodium channel, they have little effect on inactive sodium channels or close channels and produce subtle changes in their functions. After modification by prethroids, sodium channels continue in many of their normal functions, retaining their selectivity for sodium ions and link with membrane potential (Narahashi, 1986). The pyrethroids are thus known as open channel blockers.

Detailed studies can be found in Narahishi 1986, Jacques et al., 1980, and Gray 1985.

The relative resistance of mammals to the pyrethroids is almost wholly attributable to their ability to hydrolyze the pyrethroids rapidly to their inactive acid and alcohol components, since direct injection into the mammalian CNS leads to susceptibility similar to that observed in insects (Lawrence and Casida, 1982). Some additional resistance of homoeothermic organisms can be attributed to the negative temperature coefficient of action of the pyrethroids (Van den Bercken et al., 1973) which are thus less toxic at mammalian

The metabolic pathways for the breakdown of the pyrethroids vary little between mammalian species but vary somewhat with structure. This literature has been ably summarized by Leahy (1985), and further references to the metabolism of specific pyrethroids are given in the sections on individual compounds. Generally pyrethrum and allethrin are broken down mainly by oxidation, whereas for the other pyrethroids ester hydrolysis predominates. These reactions can take place in both liver and plasma and are followed by hydroxylation and conjugation to glucuronides or sulphates, which are then

Other known insecticides pyrethroids under organophosphates are listed below only

Permethrin

Phenothrin Prallethrin Resmethrin Tetramethrin Transfluthrin

2.2.

**4. Mode of action** 

**4.1 Metabolism** 

body temperature but the major effect is metabolic.

excreted in the urine (Gray 1985).

Cyhalothrin, Lambda-cyhalothrin

Table 4. I Other known insecticides.

**4.2 Individual insecticides** 

selective ones are discussed.

Allethrin Bifenthrin

Cypermethrin Cyfluthrin Deltamethrin Ftofenprox Fenvalerate

#### **4.3 Cypermethrin**

Cypermethrin (R, S)-∞- cyano-3-pheno-xybenzyl-2, 2-dimethyl. There are eight isomeric forms. It was introduced commercially in 1977 as an emulsifiable concentrate to be used against a wide range of insect pest (Elliot, 1977).

#### **4.4 Deltamethrin**

Deltamethrin S-∞- cyano-3-phenoxybenzyl-(IR)-cis-3-(2, 2-dibromovinyl)2,2-dimethcyclopropane carboxylate. It is a single isomer. It is used against a wide range of insect pests. It produces a typical type II motor symptom in mammals (Barnes and verschoyle, 1974. Metabolism of deltamethrin involves rapid ester cleavages and hydroxylation (Shono eta al; 1977).

#### **4.5 Fenproponate**

Fenproponate(∞-cyano-3-phenoxybenzyl-2,2,3,3-tetra-methylcyclopropanecarboxylate). There are eight isomerism forms. Fenpropathrin is another common name, was first developed by sumitomo and commercialized in 1980 as an emulsifiable concentrate to be used against a wide range of insect pests. Fenproponate produces intermediate or mixed motor symptoms in mammals (Wright et al., 1988).

#### **4.6 Fenvalerate**

Fenvalerate (R,S)- ∞- cyano-3-phenoxy-benzyl (IR,IS)-2-(4-chlorophenyl)-3-methyl-1 butyrate. There are four isomeric forms. It should be noted that fenvalerate is not based on a cyclopropane ring structure. It was introduced commercially to be used against a wide range of insect pests fenvalrate produces typical type II motor symptoms in mammals (Verschoyle and Aldrige, 1980).

#### **4.7 Phenothrin**

Phenothrin (3-phenoxybenzy-(IR,IS)-cis,trans-3(2-methylprop-1-enyl)-2,2 dimethyicyclopropane carboxylate). There are four isomeric forms. It is used as a domestic insecticide in a partially resolved mixture rich in the IR isomer (Sumithrin) and for grain protection. Phenothrin produces typical type 1 moto symptoms in mammals (Lawrence and Casida, 1982)

#### **4.8 Rotenone and related materials**

Rotenone-bearing plants have longed being used as a fish poison by many ancient different indigenous people, nut their use as an insecticide is probably more than a century old. Plants known to produce rotenone and other rotenoids belong to at least 68 species of the family Leguminosae, the same as that for peas and beans. The genera most exploited so far are Derris, native to southeast Asia, and lonchocarpus to south America (shepard 1951).

Rotenone and other active principles often occur chiefly in the roots of rotenone bearing plants but may be in the leaves (as in *Tephrosia vogeli*), seeds (as in *Milletia pachycarpa*), or bark (as in *Mundulea serica*).

Regardless of the genus or the particular part of the plant involved, the active constituents of rotenone-bearing plants may be extracted with ether or acetone as resin.

Rotenone is (2R,6a 5,12a 5)-1,2,6,6a,12,12a-hexahydro-2-isopropenyl-8,9-dimethoxychromeno (3,4-b) furo(2,3-h) chromen-6-one. Its structure is depicted in fig. 3. Although

Insecticide 17

The use of biological control agents has many potential advantages over chemical control, not least the possibility of high selectivity for the predators and other beneficial species. Several microorganisms or microbial products have been identified as potential insecticides (Miller et al, 1983). Most successful attempts have been directed against insects, as biological control of vertebrates has met with little success due to cross-infection problems. The world Health Organisation has investigated viruses, bacteria fungi and nematodes as potential insect control agents since all play a part in limiting the growth of natural insect

Viral insecticides are still in the experimental stage but many are under investigation, as reviewed by Miller et al., 1983. Bacterial insecticides represent the largest and widest used group and reviewed by Burges (1982) and Lysenko(1985). All of those used are sporeformers, since the spores can be readily stored in dried form and applied by conventional means as wettable powders or dusts. Many form a crystalline toxin within the spore which enhances their pathogenicity to insects. The most widely used is *Bacillus thuringiensis*. A closely similar bacterium, *Bacillus papilliae* has been used against Japanese beetle. It has the advantage that once spores are introduced into the environment the bacterial population is sustained by reinfection of the insect hosts, but the disadvantage that spore production requires expensive in-vivo production using insect pupae and is now of declining importance. It is highly specific, does not infect vertebrates, and despite production of a crystal toxin is nontoxic to mammals by repeated oral administration

Fungal insecticides are commercially produced for a variety of specific applications. Their importance in controlling natural insect populations has been recognized since 1834, Aschersonia has been used to control Floridian white fly on citrus since the early 1900s. Fungi have the advantages of forming a stable population in the insect environment and are capable of infection through the insect cuticle, not by ingestion as bacteria. A disadvantage is their susceptibility to widely used fungicides. Examples include *Beauveria basiana* is marketed as Boverin and used against Colorado beetle and corn borer in Russia and China. *Metarhizium anisopliae* was used against a range of insects as metaquino. *Hirsutella thompsoni*, is used to control citrus rust in the united states as myear and *Vecticillium lecani* is used as vertalec or mycotal for aphid control in united kingdom. Some fungi such as *Beauveria bassiana* produce toxins which may be involved in their pathogenicity. *Culicinomyces clavosporus* and *lagenidium giganteum* are mosquito pathogens

Nematate insecticides have been isolated from mosquito larvae at low natural population densities. They are reared in vivo, which is expensive, and there some resistant mosquito population Nametodes are tolerant of many insecticides and insect growth regulators and can be used in combined malaria control programs and are rapidly broken down by human

**5. Living organisms as pesticides** 

populations.

(Burges, 1982).

**5.2 Fungal insecticides** 

(Miller et al. 1983).

**5.3 Nematate insecticides** 

gastric juice (Gajana et. al; 1978).

**5.1 Viral insecticides** 

rotenone generally is considered to be the active ingredient in all resins isolated, the other constituents show considerable insecticidal activity (Metcalf, 1955).

Rotenone is readily oxidized and racemized in the presence of light and the process is accelerated in alkaline solution (Cheng et al., 1972). It is active as a nonsytemic pesticide against a wide variety of insects, arachnids and molluscs. Its rapid photodecomposition means that it is active only for about 1 week on plants or 2-6 days in water and this limits its commercial use though still finds use as a domestic garden insecticide.

Rotenone is a highly potent mitochondrial poison, blocking NADH oxidation, this property dominates its actions in animals (Heikkila et al., 1985).

Rotenone is metabolized rather effectively by the liver in isolated rat liver mitochondria, the aerobic oxidation of pyruvate is almost completely inhibited by rotenone (Haley 1978).

#### **4.9 Nicotine and related compounds**

Three closely related compounds (nicotine, nornicotine and anabasine fig4) were commonly used as insecticides, although only the most potent, nicotine, is now used to any extent. Nicotine is usually obtained from the dried leaves of nicotiana tabacum, but it also occurs in *N. rustica* and *Duboisia*, another genus of the solanaceae, and in three other taxonomically diverse general, namely Asclepia (Asclepidaceae), Equisetaceae (Equisetaceae), and Lycopodium (Lycopodiaceae); Nicotine (S-3-(1-methyl pyrrolidin-2 yl) pyridine) is used as nicotine sulphate as a stomach poison for leaf eating insects (Haigh and Haigh 1980). Nicotine is rapidly absorbed from all mucosal surfaces, including those of the mouth, gastro-intestinal tract, and lung. Since nicotine readily forms salts in acid solution, its penetration through biological membranes is strongly pH dependent (Schievelbein, 1982).

The metabolism of nicotine is highly complex and reviewed by Gorrod and Jenner (1975) and schievelbein (1982). Metabolism mainly by cytochrome P.450 linked microsomal oxidative pathways in the liver. Cotinine (Fig4) is major metabolite, which then undergoes further oxidation. Nicotine stimulates the action of acetylcholine at nicotinic receptors in the central nervous system, autonomics ganglia and some pheripheral nerves. It central actions result in tremor and convulsions, stimulation and then depression of ventilation and induction of vomiting by a direct action on the medulla. Ventilation is stimulated by peripheral actions on the aortic and carotid chemoreceptors, and adrenal catecholamine. Secretion is increased at low doses. Heart rate and blood pressure are largely dominated by sympathetic effects and show increases compounded by adrenal catecholamines. The gastrointestinal tract is dominated by parasympathetic effects and shows hypersecretion followed by block as well as increased tone and peristalsis. Death is usually a result of block of neuromuscular transmission in the respiratory muscles or a consequence of seizures. In addition to its action on cholinergic transmission, nicotine can act at noncholinergic sites and also activate receptors on sensory nerve endings and vagal C fibers (Martin, 1986).

The carcinogenic potential of tobacco is well established, but there is debate about the role of nicotine, which, although probably not carcinogenic itself can be converted to carcinogens such as N'-nitrosonornicotine and 4-(methyl-nitrosamino)-1-(3-pyriyl)-1-butanone. The metabolites cotinine and nicotine 1-N-oxide are not carcinogenic although they do produce hyperplasia of the bladder epithelium (Hoffmann et al., 1985).

rotenone generally is considered to be the active ingredient in all resins isolated, the other

Rotenone is readily oxidized and racemized in the presence of light and the process is accelerated in alkaline solution (Cheng et al., 1972). It is active as a nonsytemic pesticide against a wide variety of insects, arachnids and molluscs. Its rapid photodecomposition means that it is active only for about 1 week on plants or 2-6 days in water and this limits its

Rotenone is a highly potent mitochondrial poison, blocking NADH oxidation, this property

Rotenone is metabolized rather effectively by the liver in isolated rat liver mitochondria, the aerobic oxidation of pyruvate is almost completely inhibited by rotenone (Haley 1978).

Three closely related compounds (nicotine, nornicotine and anabasine fig4) were commonly used as insecticides, although only the most potent, nicotine, is now used to any extent. Nicotine is usually obtained from the dried leaves of nicotiana tabacum, but it also occurs in *N. rustica* and *Duboisia*, another genus of the solanaceae, and in three other taxonomically diverse general, namely Asclepia (Asclepidaceae), Equisetaceae (Equisetaceae), and Lycopodium (Lycopodiaceae); Nicotine (S-3-(1-methyl pyrrolidin-2 yl) pyridine) is used as nicotine sulphate as a stomach poison for leaf eating insects (Haigh and Haigh 1980). Nicotine is rapidly absorbed from all mucosal surfaces, including those of the mouth, gastro-intestinal tract, and lung. Since nicotine readily forms salts in acid solution, its penetration through biological membranes is strongly pH

The metabolism of nicotine is highly complex and reviewed by Gorrod and Jenner (1975) and schievelbein (1982). Metabolism mainly by cytochrome P.450 linked microsomal oxidative pathways in the liver. Cotinine (Fig4) is major metabolite, which then undergoes further oxidation. Nicotine stimulates the action of acetylcholine at nicotinic receptors in the central nervous system, autonomics ganglia and some pheripheral nerves. It central actions result in tremor and convulsions, stimulation and then depression of ventilation and induction of vomiting by a direct action on the medulla. Ventilation is stimulated by peripheral actions on the aortic and carotid chemoreceptors, and adrenal catecholamine. Secretion is increased at low doses. Heart rate and blood pressure are largely dominated by sympathetic effects and show increases compounded by adrenal catecholamines. The gastrointestinal tract is dominated by parasympathetic effects and shows hypersecretion followed by block as well as increased tone and peristalsis. Death is usually a result of block of neuromuscular transmission in the respiratory muscles or a consequence of seizures. In addition to its action on cholinergic transmission, nicotine can act at noncholinergic sites and also activate receptors on sensory nerve endings and vagal C

The carcinogenic potential of tobacco is well established, but there is debate about the role of nicotine, which, although probably not carcinogenic itself can be converted to carcinogens such as N'-nitrosonornicotine and 4-(methyl-nitrosamino)-1-(3-pyriyl)-1-butanone. The metabolites cotinine and nicotine 1-N-oxide are not carcinogenic although they do produce

hyperplasia of the bladder epithelium (Hoffmann et al., 1985).

constituents show considerable insecticidal activity (Metcalf, 1955).

commercial use though still finds use as a domestic garden insecticide.

dominates its actions in animals (Heikkila et al., 1985).

**4.9 Nicotine and related compounds** 

dependent (Schievelbein, 1982).

fibers (Martin, 1986).

## **5. Living organisms as pesticides**

The use of biological control agents has many potential advantages over chemical control, not least the possibility of high selectivity for the predators and other beneficial species. Several microorganisms or microbial products have been identified as potential insecticides (Miller et al, 1983). Most successful attempts have been directed against insects, as biological control of vertebrates has met with little success due to cross-infection problems. The world Health Organisation has investigated viruses, bacteria fungi and nematodes as potential insect control agents since all play a part in limiting the growth of natural insect populations.

## **5.1 Viral insecticides**

Viral insecticides are still in the experimental stage but many are under investigation, as reviewed by Miller et al., 1983. Bacterial insecticides represent the largest and widest used group and reviewed by Burges (1982) and Lysenko(1985). All of those used are sporeformers, since the spores can be readily stored in dried form and applied by conventional means as wettable powders or dusts. Many form a crystalline toxin within the spore which enhances their pathogenicity to insects. The most widely used is *Bacillus thuringiensis*. A closely similar bacterium, *Bacillus papilliae* has been used against Japanese beetle. It has the advantage that once spores are introduced into the environment the bacterial population is sustained by reinfection of the insect hosts, but the disadvantage that spore production requires expensive in-vivo production using insect pupae and is now of declining importance. It is highly specific, does not infect vertebrates, and despite production of a crystal toxin is nontoxic to mammals by repeated oral administration (Burges, 1982).

#### **5.2 Fungal insecticides**

Fungal insecticides are commercially produced for a variety of specific applications. Their importance in controlling natural insect populations has been recognized since 1834, Aschersonia has been used to control Floridian white fly on citrus since the early 1900s. Fungi have the advantages of forming a stable population in the insect environment and are capable of infection through the insect cuticle, not by ingestion as bacteria. A disadvantage is their susceptibility to widely used fungicides. Examples include *Beauveria basiana* is marketed as Boverin and used against Colorado beetle and corn borer in Russia and China. *Metarhizium anisopliae* was used against a range of insects as metaquino. *Hirsutella thompsoni*, is used to control citrus rust in the united states as myear and *Vecticillium lecani* is used as vertalec or mycotal for aphid control in united kingdom. Some fungi such as *Beauveria bassiana* produce toxins which may be involved in their pathogenicity. *Culicinomyces clavosporus* and *lagenidium giganteum* are mosquito pathogens (Miller et al. 1983).

#### **5.3 Nematate insecticides**

Nematate insecticides have been isolated from mosquito larvae at low natural population densities. They are reared in vivo, which is expensive, and there some resistant mosquito population Nametodes are tolerant of many insecticides and insect growth regulators and can be used in combined malaria control programs and are rapidly broken down by human gastric juice (Gajana et. al; 1978).

Insecticide 19

Cremer, J.E and Seville, M.P. 1982-Comparative effects of two Pyrethroids, deltamethrin and

Cremer, J.E., Cunningham, V.J. and Seville, M.P. 1983. Relationship between extraction and

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## **6. Conclusion**

Given the enthusiasm of the proponents of biological insect control and the limited role that these agents play in current pest control may be perhaps surprising. There are however, a number of difficulties in sustaining a usefully large population of the control agent on crops, or in the case of mosquitoes at the water surface, and in agriculture difficulties associated with the very high host specificity of some agents. More fundamental problems are the potential risk from replicating agents which can increase in the environment and the possibility of transfer of toxin encoding genes from invertebrate to vertebrate bacteria or viruses. It is clear, however, that current experience with biological control agents is very encouraging and that they can be expected to play an important part in integrated pest control programs in the future (Laird, 1985).

While animals as well as humans may be adversely affected mainly by ingestion of the active ingredients, the effect of propellant chemical cannot be ignored. Inflammatory activation might be an important mechanism underlying toxicity effects in the tissue (Mense et al., 2006). The role of propellant in the toxicity of insecticide Raid may not be cleared. A comprehensive assessment of the risk associated with environmental use of insecticide Raid was determined in various tissues as it affects the basal biochemical molecules of cells (Achudme et al., 2008).

## **7. References**


Given the enthusiasm of the proponents of biological insect control and the limited role that these agents play in current pest control may be perhaps surprising. There are however, a number of difficulties in sustaining a usefully large population of the control agent on crops, or in the case of mosquitoes at the water surface, and in agriculture difficulties associated with the very high host specificity of some agents. More fundamental problems are the potential risk from replicating agents which can increase in the environment and the possibility of transfer of toxin encoding genes from invertebrate to vertebrate bacteria or viruses. It is clear, however, that current experience with biological control agents is very encouraging and that they can be expected to play an important part in integrated pest

While animals as well as humans may be adversely affected mainly by ingestion of the active ingredients, the effect of propellant chemical cannot be ignored. Inflammatory activation might be an important mechanism underlying toxicity effects in the tissue (Mense et al., 2006). The role of propellant in the toxicity of insecticide Raid may not be cleared. A comprehensive assessment of the risk associated with environmental use of insecticide Raid was determined in various tissues as it affects the basal biochemical molecules of cells

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(J.E Casida, ed), pp 3-15. Academic Press, New York and London.

comm., ed), vol.3 pp. 393-415. Pergamon Oxford.

Counc; Molvern, Worcestershire, England.

and the 1 methyl-4-phenylpridium ion after stereotoxic administration to rats:implication for the mechanism of 1-methyl-4-phenyl-1,3,3,6-tetrahydropyridine

pyrethroid insecticides" (J.P. Leahey, ed) Taylor & Francis, London and

Absorption, transportation, deposition and excretion of lead 6. The lead contents in

Pyrethroids with the Na+ channel in mammalian neuronal cells in culture. Biochim.

Renaud, J., Schmid, A. and Vigne, P. 1985 Markers of membrane ionic channels In "Vascular Neuroeffector Mechanisms (J.A Bevan et al. Eds) Elsevier,

Wales, 1952-71, Br. J. Ind. Mect 30, 253 -258

toxicity. Neurosci. LeH. 62, 389-394.

fluids.J.Appl Toxicol 5,327-332.

Biophys. Acta 600, 882-897.

Philadelphia.

letter, 26,67-75.

Amsterdam.

219, 715-721.

Drug Off. 18, 3-13

pharmacol. Toxicol 12,251-259.

Reay, and P.N.B. Usherwood, eds), pp 36-40. Ellis Horword, Chichester, U.K.


**2** 

Farzana Perveen

*Mansehra Pakistan* 

*Chairperson, Department of Zoology Hazara University, Garden Campus* 

**Chlorfluazuron as Reproductive Inhibitor** 

Benzoyl phenyl ureas (BPUs) inhibit chitin synthesis during growth and development in insects and act as moult disruptors, therefore, they have been called insect growth regulators (Wright and Retnakaran, 1987; Binnington and Retnakaran, 1991). IGRs, such as dimilin, are effective against a considerable range of insect larvae and adults in a variety of situations. The compound disrupts the moulting process by interfering with chitin synthesis. Research on the different aspects of dimilin as a chitin synthesis inhibitor, toxicant, ovicide, disrupting adult emergence, and residual effects have been done with various insect species, e.g., Jakob, 1973; James, 1974; Qureshi et al.,1983; Naqvi and Rub, 1985; Ganiev, 1986; Khan and Naqvi, 1988; Gupta et al., 1991; Tahir et al., 1992; Nizam, 1993. Several modes of action have been reported for these pesticides. For example, phagodeterrents and repellents (Abro et al., 1997), chitin synthesis inhibition (Hajjar and Casida, 1979), growth inhibition and abnormal development (Hashizume, 1988), ovicidal action (Hatakoshi, 1992), insecticidal effects on the reproductive system (Chang and

Chlorfluazuron (Atabron) is a benzyl phenyl urea (BPU) chitin-synthesis inhibitor (CSI) and insect growth regulator (IGR) is formed by Ishihara Sangyo Kaisha, Japan. Some important details concerning the insecticide chlorfluazuron are given below (provided by

Chemical name : [1-{3,5-dichlor-4-(3-chlor-5-trifluoromethyl-2-pyridyloxy)

phenyl}-3-(2,6-ifluorobenzoyl) urea] (IUPAC nomenclature)

Borkovec, 1980) and neurotoxic effects on insect behaviour (Haynes, 1988).

**1. Introduction** 

**1.1 Chlorfluazuron** 

Ishihara Sangyo Kaisha, Japan) (Perveen, 2005):

Common name : Chlorfluazuron (proposed to ISO) Other names : Atabron or Helix or Aim

Code number : IKI-7899, CG-112913, pp-145

Appearance : Crystalline solid at 20 C

Odor : Odorless

Source : Ishihara Sangyo Kaisha Ltd., Tokyo, Japan

Formulation type : 5% w/w (Emulsifiable concentration: EC)

**1.1.1 Salient physical and chemical properties (Perveen, 2005)** 

Wright, C.D.P., Forshaw, P.J. and Ray, D.E. 1988 Classification of the actions of two Pyrethroid insecticides in the rat, using the trigeminal reflex and skeletal muscle as test systems. Pestic. Biochem, Physiol. 30, 79-86.

## **Chlorfluazuron as Reproductive Inhibitor**

## Farzana Perveen

*Chairperson, Department of Zoology Hazara University, Garden Campus Mansehra Pakistan* 

## **1. Introduction**

22 Insecticides – Pest Engineering

Wright, C.D.P., Forshaw, P.J. and Ray, D.E. 1988 Classification of the actions of two

test systems. Pestic. Biochem, Physiol. 30, 79-86.

Pyrethroid insecticides in the rat, using the trigeminal reflex and skeletal muscle as

Benzoyl phenyl ureas (BPUs) inhibit chitin synthesis during growth and development in insects and act as moult disruptors, therefore, they have been called insect growth regulators (Wright and Retnakaran, 1987; Binnington and Retnakaran, 1991). IGRs, such as dimilin, are effective against a considerable range of insect larvae and adults in a variety of situations. The compound disrupts the moulting process by interfering with chitin synthesis. Research on the different aspects of dimilin as a chitin synthesis inhibitor, toxicant, ovicide, disrupting adult emergence, and residual effects have been done with various insect species, e.g., Jakob, 1973; James, 1974; Qureshi et al.,1983; Naqvi and Rub, 1985; Ganiev, 1986; Khan and Naqvi, 1988; Gupta et al., 1991; Tahir et al., 1992; Nizam, 1993. Several modes of action have been reported for these pesticides. For example, phagodeterrents and repellents (Abro et al., 1997), chitin synthesis inhibition (Hajjar and Casida, 1979), growth inhibition and abnormal development (Hashizume, 1988), ovicidal action (Hatakoshi, 1992), insecticidal effects on the reproductive system (Chang and Borkovec, 1980) and neurotoxic effects on insect behaviour (Haynes, 1988).

## **1.1 Chlorfluazuron**

Chlorfluazuron (Atabron) is a benzyl phenyl urea (BPU) chitin-synthesis inhibitor (CSI) and insect growth regulator (IGR) is formed by Ishihara Sangyo Kaisha, Japan. Some important details concerning the insecticide chlorfluazuron are given below (provided by Ishihara Sangyo Kaisha, Japan) (Perveen, 2005):


#### **1.1.1 Salient physical and chemical properties (Perveen, 2005)**


Chlorfluazuron as Reproductive Inhibitor 25

hanging pots, they can reach the plants below by "parachuting" down on silken threads. The overall colour of the later-stages of the larvae can vary from light to dark brown, and the body is strongly speckled with tiny white spots. Initially, when larvae grow become a translucent green with a dark thorax. The young larvae are smooth-skinned with a pattern of red, yellow, and green lines, and with a dark patch on the mesothorax. Larvae initially eat only the flesh of their food leaves, leaving the veins intact. Later, as they grow, they eat

Many populations are extremely resistant to pesticides and, if they become well established, can be exceptionally difficult to control. In these cases, it is important that a comprehensive treatment programme is implemented, incorporating a range of reliable control methods, including physical destruction of insects. The *S. litura,* as it is the most common to be encountered in a UK nursery, but the larvae and adults of all noctuids are similar in appearance and are difficult to tell apart without laboratory examination (Khuhro et al., 1986). Larvae become brown with three thin yellow lines down the back, one in the middle and one on each side. A row of black dots runs along each side, and a conspicuous row of dark triangles decorates each side of the back. The last-instar larva is very dark, with four prominent yellow triangles on the mesothorax. When disturbed, the larvae curl into a tight spiral with the head protected in the centre. Larvae further develop characteristic markings on their backs. These include: a square of four yellow spots, each on a black patch, located just behind the head; a further pair of black patches just behind these, and another pair of black patches towards the end of the larva; typically, there are three orange-brown lines, punctuated with dashes of black and yellow along the back of the body. Depending on the background colour, these markings may be more evident on some larvae than others; the larvae ultimately grow up to 4.5 cm long; larvae are nocturnal, and during the day can be found at the base of plants or under pots. The feeding activity of young larvae causes "windows" in the leaves, while older larvae can completely defoliate plants if present in large numbers. Stems, buds, flowers and fruits may also be damaged. The larvae burrow into the soil below the plant for several centimeters and pupate there without a cocoon. As they do so, they produce a quantity of fluid, and drown in this if they pupate in captivity in an empty glass jar. They pupate successfully if 0.5 cm of sand is provided in the container. In January in Melbourne, the pupal stage lasts three weeks, but larvae that pupate at the end of summer emerge the following spring. The red-brown pupae are up to 2 cm long. The thoracical, ventral knobs found on covering of pupa (Khuhro et al., 1986). Adult moths with brown colour are up to 2 cm long with a wingspan of approximately 4 cm. The fore-wings are brown, with a large number of pale cream streaks and dashes and, when the adults are newly emerged, there may be a violet tint to the fore-wing. The hind-wings are a translucent white, edged with brown. The hind-wings are silvery white. It has a wingspan of about 4.0 cm. The males but not the females have a blue-grey band from the apex to the inner margin of each forewing. The pheromones of this species (specific sex-attractant scents used by females to attract males) have been elucidated. As the adults are nocturnal, light or pheromone traps should be used for monitoring purposes. Seek assurance from suppliers that plants are free from this pest as part of any commercial contract: carefully inspect new plants and produce on arrival, including any packaging material, to check for eggs and

whole leaves, and even flowers and fruit (Khuhro et al., 1986).

larvae and for signs of damage (Khuhro et al., 1986).

Early notification of the presence of this pest, will allow rapid implementation of a comprehensive treatment programme, and will help eradicate it quickly from nursery. Established outbreaks are very damaging and difficult to eradicate. Various methods of


#### **1.2 Spodoptera litura**

The *S. litura* is found in most of the Caroline and in the South Pacific Island including American Samoa. It also occurs in the northern two thirds of Australia. The moth is also widespread throughout India and recognized as quarantine pest in EU legislation. It is present in Mediterranean Europe and Africa. It is the most commonly intercepted in the UK, on imported ornamentals and their products. *Spodoptera litura* is also a destructive pest of subtropical and tropical agriculture, and has the potential to be a serious pest of glasshouse crops in northern Europe. It was found as feeding on impatience on Victoria Peak on Hong Kong Island and readily switched to (western) lettuce (Etman and Hooper, 1979). In 1974, Etman and Hooper initiated an investigation into the radiobiology of *S. litura*, and reported that it was a significant pest of cotton in the Ord River region, Australia (Etman and Hooper, 1979). Its larvae are a major cosmopolitan pest of a wide range of crops (Skibbe et al., 1995). Matsuura and Naito (1997) reported that *S. litura* causes serious widespread damage to many agricultural crops in the far southern of the Central Japan every year. They hypothesized that adult *S. litura* immigrate into Japan from overseas every year by longdistance migration. The larvae are destroyed many economically important crops such as *Gossypium hirsutum* L., *Brassica oleracea* L., *Spinacea oleracea* L., *Trifolium alexandrinum* L., *Medicago sativa* L., *Arachis hypogaea* L., *Phaseolus aureus* Roxb., *Phaseolus vulgaria* L. and *Nicotiana tabacum* L. during different seasons throughout the year in Pakistan (Younis, 1973). Their larvae eat nearly all types of herbaceous plants. Some examples of plants are: tobacco, *Nicotiana tabacum* L.; tomatoes, *Lycopersicum esculentum* Mill.; cauliflower, *Brassica botrytis* L.; beetroot, *Beta vulgaris conditiva* L.; silver beet (swiss chard) *Beta vulgaris cicla* (L.); peanuts, *Arachis hypogaea* L.; beans, *Phaseolus vulgaris* L.; banana, *Musa* paradisiaca L.; strawberry, *Fragaria* vesca L.; apple, *Malus pumila* Mill.; lettuce, *Lactuca sativa* L.; zinnia, *Zinnia elegans*  Jacq.; dahlia, *Dahlia pinnata* Cav.; ape, *Alocasia macrorrhiza* (L.); geranium, *Pelargoniumx zonale*; St. John's lily, *Crinum asiaticum* L.; mangrove lily, *Crinum pedunculatum* (Fragrant); leek, *Allium porrum* (Leek); horsetail she oak, *Casuarina equisetifolia* L.; *Fuchsia* and many other garden plants (Baloch and Abbasi, 1977). Several common names have been used for *S. litura*, for example defoliator cutworm, oriental leafworm, cluster caterpillar and common cutworm. The larvae are quite polyphagous for example eat all types of herbaceous plants and have been reaching the status of international pest. In 1968, a panel convened by the International Atomic Agency listed species of *S. litura* as a pest on which basic and applied research was needed in order to evaluate the potential of the sterile insect release method for control (Anonymous, 1969).

Eggs of *S. litura* are laid in batches, on plants and other surfaces such as pots, benches or glasshouse structures. Eggs are normally laid in the irregular furry masses covered with orange-brown hairs giving them a "felt-like" appearance on the underside of a leaf of a food plant similar to the egg of the brown locust, *Locustana pardalina* (Walk.) (Matthee, 1951). On hatching, larvae (caterpillars) are 2–3 mm long with white bodies and black heads and are very difficult to detect visually. If they emerge from eggs laid on glasshouse structures or

Stability : No detectable decomposition over at least 3 months at 50 C

The *S. litura* is found in most of the Caroline and in the South Pacific Island including American Samoa. It also occurs in the northern two thirds of Australia. The moth is also widespread throughout India and recognized as quarantine pest in EU legislation. It is present in Mediterranean Europe and Africa. It is the most commonly intercepted in the UK, on imported ornamentals and their products. *Spodoptera litura* is also a destructive pest of subtropical and tropical agriculture, and has the potential to be a serious pest of glasshouse crops in northern Europe. It was found as feeding on impatience on Victoria Peak on Hong Kong Island and readily switched to (western) lettuce (Etman and Hooper, 1979). In 1974, Etman and Hooper initiated an investigation into the radiobiology of *S. litura*, and reported that it was a significant pest of cotton in the Ord River region, Australia (Etman and Hooper, 1979). Its larvae are a major cosmopolitan pest of a wide range of crops (Skibbe et al., 1995). Matsuura and Naito (1997) reported that *S. litura* causes serious widespread damage to many agricultural crops in the far southern of the Central Japan every year. They hypothesized that adult *S. litura* immigrate into Japan from overseas every year by longdistance migration. The larvae are destroyed many economically important crops such as *Gossypium hirsutum* L., *Brassica oleracea* L., *Spinacea oleracea* L., *Trifolium alexandrinum* L., *Medicago sativa* L., *Arachis hypogaea* L., *Phaseolus aureus* Roxb., *Phaseolus vulgaria* L. and *Nicotiana tabacum* L. during different seasons throughout the year in Pakistan (Younis, 1973). Their larvae eat nearly all types of herbaceous plants. Some examples of plants are: tobacco, *Nicotiana tabacum* L.; tomatoes, *Lycopersicum esculentum* Mill.; cauliflower, *Brassica botrytis* L.; beetroot, *Beta vulgaris conditiva* L.; silver beet (swiss chard) *Beta vulgaris cicla* (L.); peanuts, *Arachis hypogaea* L.; beans, *Phaseolus vulgaris* L.; banana, *Musa* paradisiaca L.; strawberry, *Fragaria* vesca L.; apple, *Malus pumila* Mill.; lettuce, *Lactuca sativa* L.; zinnia, *Zinnia elegans*  Jacq.; dahlia, *Dahlia pinnata* Cav.; ape, *Alocasia macrorrhiza* (L.); geranium, *Pelargoniumx zonale*; St. John's lily, *Crinum asiaticum* L.; mangrove lily, *Crinum pedunculatum* (Fragrant); leek, *Allium porrum* (Leek); horsetail she oak, *Casuarina equisetifolia* L.; *Fuchsia* and many other garden plants (Baloch and Abbasi, 1977). Several common names have been used for *S. litura*, for example defoliator cutworm, oriental leafworm, cluster caterpillar and common cutworm. The larvae are quite polyphagous for example eat all types of herbaceous plants and have been reaching the status of international pest. In 1968, a panel convened by the International Atomic Agency listed species of *S. litura* as a pest on which basic and applied research was needed in order to evaluate the potential of the sterile insect release method for

Eggs of *S. litura* are laid in batches, on plants and other surfaces such as pots, benches or glasshouse structures. Eggs are normally laid in the irregular furry masses covered with orange-brown hairs giving them a "felt-like" appearance on the underside of a leaf of a food plant similar to the egg of the brown locust, *Locustana pardalina* (Walk.) (Matthee, 1951). On hatching, larvae (caterpillars) are 2–3 mm long with white bodies and black heads and are very difficult to detect visually. If they emerge from eggs laid on glasshouse structures or

Melting point : 222.0 – 223.9 C (decomposes after melting)

Vapor pressure : <10 – 8 pa, <10-10 Torr at 20 C

Volatility : Relatively non-volatile

Specific gravity : 1.4977 at 20 C

**1.2 Spodoptera litura** 

control (Anonymous, 1969).

hanging pots, they can reach the plants below by "parachuting" down on silken threads. The overall colour of the later-stages of the larvae can vary from light to dark brown, and the body is strongly speckled with tiny white spots. Initially, when larvae grow become a translucent green with a dark thorax. The young larvae are smooth-skinned with a pattern of red, yellow, and green lines, and with a dark patch on the mesothorax. Larvae initially eat only the flesh of their food leaves, leaving the veins intact. Later, as they grow, they eat whole leaves, and even flowers and fruit (Khuhro et al., 1986).

Many populations are extremely resistant to pesticides and, if they become well established, can be exceptionally difficult to control. In these cases, it is important that a comprehensive treatment programme is implemented, incorporating a range of reliable control methods, including physical destruction of insects. The *S. litura,* as it is the most common to be encountered in a UK nursery, but the larvae and adults of all noctuids are similar in appearance and are difficult to tell apart without laboratory examination (Khuhro et al., 1986). Larvae become brown with three thin yellow lines down the back, one in the middle and one on each side. A row of black dots runs along each side, and a conspicuous row of dark triangles decorates each side of the back. The last-instar larva is very dark, with four prominent yellow triangles on the mesothorax. When disturbed, the larvae curl into a tight spiral with the head protected in the centre. Larvae further develop characteristic markings on their backs. These include: a square of four yellow spots, each on a black patch, located just behind the head; a further pair of black patches just behind these, and another pair of black patches towards the end of the larva; typically, there are three orange-brown lines, punctuated with dashes of black and yellow along the back of the body. Depending on the background colour, these markings may be more evident on some larvae than others; the larvae ultimately grow up to 4.5 cm long; larvae are nocturnal, and during the day can be found at the base of plants or under pots. The feeding activity of young larvae causes "windows" in the leaves, while older larvae can completely defoliate plants if present in large numbers. Stems, buds, flowers and fruits may also be damaged. The larvae burrow into the soil below the plant for several centimeters and pupate there without a cocoon. As they do so, they produce a quantity of fluid, and drown in this if they pupate in captivity in an empty glass jar. They pupate successfully if 0.5 cm of sand is provided in the container. In January in Melbourne, the pupal stage lasts three weeks, but larvae that pupate at the end of summer emerge the following spring. The red-brown pupae are up to 2 cm long. The thoracical, ventral knobs found on covering of pupa (Khuhro et al., 1986). Adult moths with brown colour are up to 2 cm long with a wingspan of approximately 4 cm. The fore-wings are brown, with a large number of pale cream streaks and dashes and, when the adults are newly emerged, there may be a violet tint to the fore-wing. The hind-wings are a translucent white, edged with brown. The hind-wings are silvery white. It has a wingspan of about 4.0 cm. The males but not the females have a blue-grey band from the apex to the inner margin of each forewing. The pheromones of this species (specific sex-attractant scents used by females to attract males) have been elucidated. As the adults are nocturnal, light or pheromone traps should be used for monitoring purposes. Seek assurance from suppliers that plants are free from this pest as part of any commercial contract: carefully inspect new plants and produce on arrival, including any packaging material, to check for eggs and larvae and for signs of damage (Khuhro et al., 1986).

Early notification of the presence of this pest, will allow rapid implementation of a comprehensive treatment programme, and will help eradicate it quickly from nursery. Established outbreaks are very damaging and difficult to eradicate. Various methods of

Chlorfluazuron as Reproductive Inhibitor 27

chlorfluazuron is particularly suited to integrated pest management programmes. Although chlorfluazuron has contact toxicity at higher rates, the major route of toxicity to insects is ingestion, and it has no root, systemic or foliar translaminar activity. Like other BPUs, chlorfluazuron is believed to disrupt chitin formation and, thus, kills the insects when they moult. This mode of action necessarily means that it is effective only against immature insects and that it is relatively slow actions. When higher dosages of chlorfluazuron were applied to newly ecdysed fifth-instar larvae, it had a devastating effect on the *S. litura* population by killing them during larval, pupal and adult stages (Hashizume, 1988). In insect pest management, the purpose of research is to maintain the pest population below the economic injury level. The mode of action of chlorfluazuron, as a CSI is known to some extent. However, but the knowledge of its effects on reproduction are rare. Insect structure and physiology may vary considerably during growth and development, with certain stages being more susceptible to insecticides than others. For example, the cuticle varies in its composition during larval development and this has been related to changes in IGR susceptibility. The activity of various insecticides detoxifying enzymes, such as MFO, glutathione S-transferase and epoxide hydrase also fluctuate during the life cycle of an insect (Yu, 1983). For this purpose newly ecdysed fifth-instar larvae and newly ecdysed pupae were selected for the treatments for the present research. The main objective of the present research is to determine the effects of sublethal doses (LD10: 1.00 ng larva-1; 0.12 ng female pupa-1; 1.23 ng male pupa-1 or LD30: 3.75 ng larva-1) of chlorfluazuron on the reproduction (e.g., fecundity, fertility and hatchability) when ha been apply to newly

ecdysed fifth-instar larvae and newly ecdysed pupae of *S. litura* (Perveen, 2000a).

Experiments were conducted with *Spodoptera litura* (F.) (Lepidoptera: Noctuidae) taken from a stock that was established from eggs obtained from Aburahi Laboratory of Shionogi Pharmaceutical (Koga-Shiga-Pref., Japan). The larvae of *S. litura* were reared in the laboratory under controlled conditions on the artificial diet Insecta LF® (Nihon Nohsankohgyo, Kanagawa, Japan). The rearing temperature was maintained at 251 °C, with a L16:D8 hour photoperiod and 50-60% r.h. To facilitate observations, the dark period was set from 06:00 to 14:00 hours. Adults were fed on a 10% sucrose solution soaked in cotton. The eggs, which were laid on Rido® cooking paper (Lion, Tokyo, Japan), were collected every 3rd day and kept in 90 ml plastic cups (4 cm in diameter: 4×4 cm high) for hatching under the

Sublethal doses, LD10 (1.00 ng larva-1; 0.12 ng female pupa-1; 1.23 ng male pupa-1) or LD30 (3.75 ng larva-1) were applied to newly ecdysed fifth-instar larvae and newly ecdysed pupae. These LD10 and LD30 values were calculated based on interpret alone of the results of the toxicity data of larval tests at adult emergence. The treated and untreated insects, at all developmental stages including fifth- and sixth-instar larvae, pupae and adults, were weighed separately, on different developmental days, using an analytical balance (Sartorius Analytical AC-2105, Tokyo, Japan) to a precision of 0.001 mg, to determine the effect of chlorfluazuron on the body weight. The duration of each developemental stage was also

**2.1 Experimental procedures** 

same environmental conditions (Perveen, 2000a).

**2.1.2 Chlorfluazuron and its application** 

strictly recorded (Perveen, 2000a).

**2.1.1 Insect rearing** 

control of *S. litura* have been investigated. Biologically, it has been controlled by the nematode, *Steinernema carpocapsae* (Weiser) and parasitoid fly, *Exorista japonica* (Townsend). A baculovirus has also been used. Resistant species of plants are also grown to save the crops from this pest. Resistant tomatoes are most commonly cultivated (Khuhro et al., 1986).

## **2. Effects of chlorfluazurn on reproduction of** *Spodoptera litura*

Reproductive inhibition induced by BPUs has been reported the most widely when applied to adults or eggs of insect pests rather than to application to larvae or pupae (Fytizas, 1976). When these compounds were applied to females, males or both sexes of insect pests, BPUs induced a variety of effects on reproduction; they caused a decrease in fecundity, fertility and/or hatchability. It has been reported that treatment of adult insect pests with diflubenzuron disrupts the secretion of adult cuticle (Hunter and Vincent 1974; Ker, 1977), and the production of peritrophic membrane in the grasshopper, *Locusta migratoria* (L.) (Clark et al., 1977) and the meal worm, *Tenebrio molitor* L. (Soltani, 1984; Soltani et al., 1987*).*  In addition, topical treatments of male and female adult boll weevils, *Anthonomous grandis* Boheman; stable flies, *Stomoxys calcitrans* (L.) and *M. domestica* with TH-6040 [{N-(4 chlorophenyl)-N-26-difluorobenzoyl}urea] caused significant reduction of egg fertility and hatchability. It also causes inhibition in the fecundity of female adults of several species of insect pests (Holst, 1974; Taft and Hopkins, 1975; Crystal, 1978; Hajjar and Casida, 1979; Otten and Todd, 1979). Diflubenzuron applied to adult females caused a decrease in fecundity in the Mexican bean beetle, *Epilachna varivestis* Mulsant and Colorado potato beetle, *Leptinotarsa decemlineata* Say (Holst, 1974), and adversely affected egg viability in *St. calcitrans* and *M. domestica* (Wright and Spates, 1976). When 2 day-old female adults of the Japanese beetle, *Oryzae japonica* Willemse, were starved for 6 hours, and then allowed to consume 500 µg a.i. of diflubenzuron over another 6 hours, the fecundity of the treated females, in term of number of eggs laid per pod, was significantly decreased from controls. In controls, most pods gave an egg hatch of 82.5% but a hatch of only 8.5% hatched in the treated females (Lim and Lee, 1982). Similarly, treating eggs with diflubenzuron caused reduction in hatching in the mosquitoes, *Culex pipiens* Say, *C. quinquefascialus* Say (Miura et al.*,* 1976), the almond moth, *Ephestia cautella* (Walk.) (Nickle, 1979), the two-spotted lady beetle, *Adalia bipunctata* (L.) and the seven-spotted lady beetle, *Coccinella septempunctata* L. (Olszak, 1994). Reports of inhibition of reproduction when larvae or pupae (instead of adults or eggs) were treated with BPUs are rare and little literature is available (Madore et al., 1983). Brushwein and Granett (1977), working with the spruce budworm, *Choristoneura fumiferana* (Celemens), demonstrated that certain moult-inhibiting IGRs such as EL-494 (Eli Lilly and Co., New York, USA) fed to sixth-instar larvae, caused reproductive failure in adults surviving after the larval treatment. Therefore, in this research newly ecdysed fifthinstar larvae and newly ecdysed pupae of *S. litura* were used as test materials. Chlorfluazuron, a comparatively new IGR and BPU that was discovered by Ishihara Sangyo Kaisha Ltd., Tokyo, Japan, that has been developed and sold commercially as Atabron, Helix and Aim in many countries, including Japan in cooperation with Novelties Co. Ltd, ICI-AGRO and Ciba Geigy. It is a relatively highly active chitin synthesis inhibitor and it is, therefore, an effective treatment for the control of major lepidopteran insect pests in crops such as cotton, fruits, tea, vegetables and where insect resistance to conventional insecticides is becoming a serious problem. Chlorfluazuron exhibits no activity against important beneficial insects (Haga et al., 1992). The highly selective insecticidal activity of

control of *S. litura* have been investigated. Biologically, it has been controlled by the nematode, *Steinernema carpocapsae* (Weiser) and parasitoid fly, *Exorista japonica* (Townsend). A baculovirus has also been used. Resistant species of plants are also grown to save the crops from this pest. Resistant tomatoes are most commonly cultivated (Khuhro et al., 1986).

Reproductive inhibition induced by BPUs has been reported the most widely when applied to adults or eggs of insect pests rather than to application to larvae or pupae (Fytizas, 1976). When these compounds were applied to females, males or both sexes of insect pests, BPUs induced a variety of effects on reproduction; they caused a decrease in fecundity, fertility and/or hatchability. It has been reported that treatment of adult insect pests with diflubenzuron disrupts the secretion of adult cuticle (Hunter and Vincent 1974; Ker, 1977), and the production of peritrophic membrane in the grasshopper, *Locusta migratoria* (L.) (Clark et al., 1977) and the meal worm, *Tenebrio molitor* L. (Soltani, 1984; Soltani et al., 1987*).*  In addition, topical treatments of male and female adult boll weevils, *Anthonomous grandis* Boheman; stable flies, *Stomoxys calcitrans* (L.) and *M. domestica* with TH-6040 [{N-(4 chlorophenyl)-N-26-difluorobenzoyl}urea] caused significant reduction of egg fertility and hatchability. It also causes inhibition in the fecundity of female adults of several species of insect pests (Holst, 1974; Taft and Hopkins, 1975; Crystal, 1978; Hajjar and Casida, 1979; Otten and Todd, 1979). Diflubenzuron applied to adult females caused a decrease in fecundity in the Mexican bean beetle, *Epilachna varivestis* Mulsant and Colorado potato beetle, *Leptinotarsa decemlineata* Say (Holst, 1974), and adversely affected egg viability in *St. calcitrans* and *M. domestica* (Wright and Spates, 1976). When 2 day-old female adults of the Japanese beetle, *Oryzae japonica* Willemse, were starved for 6 hours, and then allowed to consume 500 µg a.i. of diflubenzuron over another 6 hours, the fecundity of the treated females, in term of number of eggs laid per pod, was significantly decreased from controls. In controls, most pods gave an egg hatch of 82.5% but a hatch of only 8.5% hatched in the treated females (Lim and Lee, 1982). Similarly, treating eggs with diflubenzuron caused reduction in hatching in the mosquitoes, *Culex pipiens* Say, *C. quinquefascialus* Say (Miura et al.*,* 1976), the almond moth, *Ephestia cautella* (Walk.) (Nickle, 1979), the two-spotted lady beetle, *Adalia bipunctata* (L.) and the seven-spotted lady beetle, *Coccinella septempunctata* L. (Olszak, 1994). Reports of inhibition of reproduction when larvae or pupae (instead of adults or eggs) were treated with BPUs are rare and little literature is available (Madore et al., 1983). Brushwein and Granett (1977), working with the spruce budworm, *Choristoneura fumiferana* (Celemens), demonstrated that certain moult-inhibiting IGRs such as EL-494 (Eli Lilly and Co., New York, USA) fed to sixth-instar larvae, caused reproductive failure in adults surviving after the larval treatment. Therefore, in this research newly ecdysed fifthinstar larvae and newly ecdysed pupae of *S. litura* were used as test materials. Chlorfluazuron, a comparatively new IGR and BPU that was discovered by Ishihara Sangyo Kaisha Ltd., Tokyo, Japan, that has been developed and sold commercially as Atabron, Helix and Aim in many countries, including Japan in cooperation with Novelties Co. Ltd, ICI-AGRO and Ciba Geigy. It is a relatively highly active chitin synthesis inhibitor and it is, therefore, an effective treatment for the control of major lepidopteran insect pests in crops such as cotton, fruits, tea, vegetables and where insect resistance to conventional insecticides is becoming a serious problem. Chlorfluazuron exhibits no activity against important beneficial insects (Haga et al., 1992). The highly selective insecticidal activity of

**2. Effects of chlorfluazurn on reproduction of** *Spodoptera litura*

chlorfluazuron is particularly suited to integrated pest management programmes. Although chlorfluazuron has contact toxicity at higher rates, the major route of toxicity to insects is ingestion, and it has no root, systemic or foliar translaminar activity. Like other BPUs, chlorfluazuron is believed to disrupt chitin formation and, thus, kills the insects when they moult. This mode of action necessarily means that it is effective only against immature insects and that it is relatively slow actions. When higher dosages of chlorfluazuron were applied to newly ecdysed fifth-instar larvae, it had a devastating effect on the *S. litura* population by killing them during larval, pupal and adult stages (Hashizume, 1988). In insect pest management, the purpose of research is to maintain the pest population below the economic injury level. The mode of action of chlorfluazuron, as a CSI is known to some extent. However, but the knowledge of its effects on reproduction are rare. Insect structure and physiology may vary considerably during growth and development, with certain stages being more susceptible to insecticides than others. For example, the cuticle varies in its composition during larval development and this has been related to changes in IGR susceptibility. The activity of various insecticides detoxifying enzymes, such as MFO, glutathione S-transferase and epoxide hydrase also fluctuate during the life cycle of an insect (Yu, 1983). For this purpose newly ecdysed fifth-instar larvae and newly ecdysed pupae were selected for the treatments for the present research. The main objective of the present research is to determine the effects of sublethal doses (LD10: 1.00 ng larva-1; 0.12 ng female pupa-1; 1.23 ng male pupa-1 or LD30: 3.75 ng larva-1) of chlorfluazuron on the reproduction (e.g., fecundity, fertility and hatchability) when ha been apply to newly ecdysed fifth-instar larvae and newly ecdysed pupae of *S. litura* (Perveen, 2000a).

#### **2.1 Experimental procedures**

#### **2.1.1 Insect rearing**

Experiments were conducted with *Spodoptera litura* (F.) (Lepidoptera: Noctuidae) taken from a stock that was established from eggs obtained from Aburahi Laboratory of Shionogi Pharmaceutical (Koga-Shiga-Pref., Japan). The larvae of *S. litura* were reared in the laboratory under controlled conditions on the artificial diet Insecta LF® (Nihon Nohsankohgyo, Kanagawa, Japan). The rearing temperature was maintained at 251 °C, with a L16:D8 hour photoperiod and 50-60% r.h. To facilitate observations, the dark period was set from 06:00 to 14:00 hours. Adults were fed on a 10% sucrose solution soaked in cotton. The eggs, which were laid on Rido® cooking paper (Lion, Tokyo, Japan), were collected every 3rd day and kept in 90 ml plastic cups (4 cm in diameter: 4×4 cm high) for hatching under the same environmental conditions (Perveen, 2000a).

#### **2.1.2 Chlorfluazuron and its application**

Sublethal doses, LD10 (1.00 ng larva-1; 0.12 ng female pupa-1; 1.23 ng male pupa-1) or LD30 (3.75 ng larva-1) were applied to newly ecdysed fifth-instar larvae and newly ecdysed pupae. These LD10 and LD30 values were calculated based on interpret alone of the results of the toxicity data of larval tests at adult emergence. The treated and untreated insects, at all developmental stages including fifth- and sixth-instar larvae, pupae and adults, were weighed separately, on different developmental days, using an analytical balance (Sartorius Analytical AC-2105, Tokyo, Japan) to a precision of 0.001 mg, to determine the effect of chlorfluazuron on the body weight. The duration of each developemental stage was also strictly recorded (Perveen, 2000a).

Chlorfluazuron as Reproductive Inhibitor 29

with an untreated male, the fecundity was 1462353 (LD10♀U♂) and 1266237 (LD30♀ <sup>U</sup>♂), respectively. When the male was treated with the LD10 and mated with an untreated female, the fecundity was 1407334 (U♀LD10♂). However, in the same cross when the male was treated with LD30 instead of the LD10 (U♀LD30♂), the fecundity was 1270215. When both sexes were treated with the LD10 (LD10♀LD10♂), it was 1330295 and when both sexes were treated with the LD30 concentration (LD30♀LD30♂), it was 1331 295. In all the crosses, the fecundity was significantly reduced when compared with the control cross (U♀U♂) (Table 1)

<sup>U</sup>♀U♂ 30 2250198a 1984208a 88.46.6a LD10♀U♂ 30 1462353b 1010315b 68.911.8b <sup>U</sup>♀LD10♂ 28 1407334b 688317c 48.317.1c LD10♀LD10♂ 30 1330295b 643265c 48.614.2c LD30♀U♂ 29 1266237b 828206b 65.811.4b <sup>U</sup>♀LD30♂ 30 1270215b 36155d 28.811.2d LD30♀LD30♂ 29 1331295b 33121d 27.19.1d aLD10: 1.00 ng larva-1; LD30: 3.75ng larva-1; U: untreated (control); ♀: female adults; ♂: male adults; n:

bNumber of eggs oviposited during the whole life of female adult were counted (fecundity) and from

cData were analyzed using one way ANOVA (Concepts, 1989) at *P*<0.0001). Means within columns followed by different letters indicate significant differences by Scheffe`s *<sup>F</sup>*-test (Scheffe, 1953) at 5%. dHatchability % values were normalized by arcsin transformation before statistical analysis (Anderson

Table 1. Effects of sublethal doses of chlorfluazuron on fecundity, fertility and hatchability after topical application of newly ecdysed-fifth instar larvae of *Spodoptera litura* (Source:

When the LD10 of chlorfluazuron was applied to newly ecdysed pupae and the resulting adults were paired, the control fecundity was 2170175. It was not significantly reduced (P<0.02) compared with the larval treatment. The fecundity in the treated pupal cross (LD10♀U♂) was 64083. It was not significantly reduced (P<0.02) compared with the same cross with treated larvae. In the pupal treatment, the fecundity was suppressed to a similar degree in the crosses LD10♀U♂, U♀LD10♂, LD10♀ LD10♂. This reduction was significant (P<0.0001) when compared with the control cross (Table 1) (Perveen, 2000a). There was no significant reduction (P<0.02) between the larval and pupal treatments with respect to

The mean fertility of females was 1984208 larvae when both the male and female were untreated, i.e. the control (U♀U♂) (Table 1). When the female was treated with either the LD10 or LD30 and mated with an untreated male, the fertility was (LD10♀U♂: 1010315; LD30♀ <sup>U</sup>♂: 828206, respectively), i.e., significantly reduced compared with the control cross (U♀ <sup>U</sup>♂). When the male was treated with the LD10 and mated with an untreated female, the fertility (U♀ LD10♂: 688317) was significantly reduced compared with the crosses, i.e.,

Fertilityb,c (meanSD) Hatchability%c,d (meanSD)

(meanSD)

(Perveen, 2000a).

Mating pairsa

number of pairs used

and McLean 1974).

Perveen, 2000a).

(femalemale) na Fecundityb,c

the eggs number of hatched that larvae were counted (fertility).

fecundity (Tables 1 and 2) (Perveen, 2005).

#### **2.1.3 Mating**

After both larval and pupal treatments, females and males that emerged between 2 and 8 hour (most adults emerged in the dark photoperiod) on the same day were collected at 0200–1000 hour. These adults were considered as 0 day old and paired just before the dark photoperiod (12 hour old) of the next day. Each female and male pair was kept separately in a plastic cup (430 cm3; height: 8.0 cm; diameter: 9.5 cm) for the whole life-span. The cup was padded with Rido cooking paper on its wall and with a disc of 70 mm filter paper on the bottom. The pairs were fed throughout their life by cotton wool soaked in 10% sugar solution in small plastic cups. All *S. litura* were examined daily (Perveen, 2000a).

To determine the effects of sublethal doses of chlorfluazuron on reproductivity, seven different mating combinations of female and male crosses were established. These were: (1) Untreated female mated with untreated male (U♀U♂); (2) LD10-treated female mated with untreated male (LD10♀U♂); (3) Untreated female mated with LD10-treated male (U♀LD10♂); (4) LD10-treated female mated with LD10-treated male (LD10♀LD10♂); (5) LD30-treated female mated with untreated male (LD30♀U♂); (6) Untreated female mated with LD30-treated male (U♀ LD30♂); (7) LD30-treated female mated with LD30-treated male (LD30♀ LD30♂). For the fecundity, fertility and hatchability experiments, 15–30 pairs were used for each cross. Eggs were laid on the cooking paper after 24 hours and were collected during 0800–1000 hour, cut out and kept in cups (90 cm3) for hatching. Eggs laid were hatched within 84 hours. Observation of oviposition continued until the death of female. Four days after each collection of eggs, the fecundity, fertility and hatchability of the laid eggs were assessed. After the natural death of females, the spermatophores were separated from the bursa copulatrix with a fine forceps in 0.9% NaCl (Saline or Ringer's solution: Barbosa, 1974) under the binocular microscope (10magnification) (Olympus Co. Ltd., Tokyo, Japan) (Perveen, 2000a).

#### **2.1.4 Data analysis**

Data for the effects of sublethal doses of chlorfluazuron on reproductivity and viability were analyzed using analysis of variance, one way ANOVA (Concepts, 1989; Minitab, 1997; Walpol and Myers1998) at P<0.0001 and Scheffe's F-test (multiple range) (Scheffe, 1953) at 5%. Hatchability percentage values were normalized by arcsin transformation before statistical analysis (Anderson and McLean, 1974).

#### **2.2 Results**

When the LD10 (1.00 ng larva-1; 0.12 ng female pupa-1; 1.23 ng male pupa-1) and the LD30 (3.75 ng larva-1) of chlorfluazuron were applied to newly ecdysed fifth-instar larvae or newly ecdysed pupae, it was observed that the fecundity of the resulting adults and the fertility and hatchability of their eggs, was significantly reduced {P<0.0001 (for larval treatment); P<0.05 (for pupal treatment)}, compared with untreated adults, but no significant differences were observed between larval and pupal treatments (P<0.02) (Tables 1 and 2). When chlorfluazuron was applied to newly ecdysed fifth-instar larvae at sublethal doses, the number of eggs oviposited by a treated females mated with an untreated male (T♀U♂) was suppressed to the same degree as an untreated female mated with a treated male (U♀T♂) or a treated female mated with a treated male (T♀T♂) (Table 1). The mean female fecundity was 2250198 eggs when both male and female were untreated (control), i.e., (U♀U♂), (Table 2). When the female was treated either by the LD10 or LD30 and mated

After both larval and pupal treatments, females and males that emerged between 2 and 8 hour (most adults emerged in the dark photoperiod) on the same day were collected at 0200–1000 hour. These adults were considered as 0 day old and paired just before the dark photoperiod (12 hour old) of the next day. Each female and male pair was kept separately in a plastic cup (430 cm3; height: 8.0 cm; diameter: 9.5 cm) for the whole life-span. The cup was padded with Rido cooking paper on its wall and with a disc of 70 mm filter paper on the bottom. The pairs were fed throughout their life by cotton wool soaked in 10% sugar

To determine the effects of sublethal doses of chlorfluazuron on reproductivity, seven different mating combinations of female and male crosses were established. These were: (1) Untreated female mated with untreated male (U♀U♂); (2) LD10-treated female mated with untreated male (LD10♀U♂); (3) Untreated female mated with LD10-treated male (U♀LD10♂); (4) LD10-treated female mated with LD10-treated male (LD10♀LD10♂); (5) LD30-treated female mated with untreated male (LD30♀U♂); (6) Untreated female mated with LD30-treated male (U♀ LD30♂); (7) LD30-treated female mated with LD30-treated male (LD30♀ LD30♂). For the fecundity, fertility and hatchability experiments, 15–30 pairs were used for each cross. Eggs were laid on the cooking paper after 24 hours and were collected during 0800–1000 hour, cut out and kept in cups (90 cm3) for hatching. Eggs laid were hatched within 84 hours. Observation of oviposition continued until the death of female. Four days after each collection of eggs, the fecundity, fertility and hatchability of the laid eggs were assessed. After the natural death of females, the spermatophores were separated from the bursa copulatrix with a fine forceps in 0.9% NaCl (Saline or Ringer's solution: Barbosa, 1974) under the binocular microscope (10magnification) (Olympus Co. Ltd., Tokyo, Japan)

Data for the effects of sublethal doses of chlorfluazuron on reproductivity and viability were analyzed using analysis of variance, one way ANOVA (Concepts, 1989; Minitab, 1997; Walpol and Myers1998) at P<0.0001 and Scheffe's F-test (multiple range) (Scheffe, 1953) at 5%. Hatchability percentage values were normalized by arcsin transformation before

When the LD10 (1.00 ng larva-1; 0.12 ng female pupa-1; 1.23 ng male pupa-1) and the LD30 (3.75 ng larva-1) of chlorfluazuron were applied to newly ecdysed fifth-instar larvae or newly ecdysed pupae, it was observed that the fecundity of the resulting adults and the fertility and hatchability of their eggs, was significantly reduced {P<0.0001 (for larval treatment); P<0.05 (for pupal treatment)}, compared with untreated adults, but no significant differences were observed between larval and pupal treatments (P<0.02) (Tables 1 and 2). When chlorfluazuron was applied to newly ecdysed fifth-instar larvae at sublethal doses, the number of eggs oviposited by a treated females mated with an untreated male (T♀U♂) was suppressed to the same degree as an untreated female mated with a treated male (U♀T♂) or a treated female mated with a treated male (T♀T♂) (Table 1). The mean female fecundity was 2250198 eggs when both male and female were untreated (control), i.e., (U♀U♂), (Table 2). When the female was treated either by the LD10 or LD30 and mated

solution in small plastic cups. All *S. litura* were examined daily (Perveen, 2000a).

**2.1.3 Mating** 

(Perveen, 2000a).

**2.2 Results** 

**2.1.4 Data analysis** 

statistical analysis (Anderson and McLean, 1974).

with an untreated male, the fecundity was 1462353 (LD10♀U♂) and 1266237 (LD30♀ <sup>U</sup>♂), respectively. When the male was treated with the LD10 and mated with an untreated female, the fecundity was 1407334 (U♀LD10♂). However, in the same cross when the male was treated with LD30 instead of the LD10 (U♀LD30♂), the fecundity was 1270215. When both sexes were treated with the LD10 (LD10♀LD10♂), it was 1330295 and when both sexes were treated with the LD30 concentration (LD30♀LD30♂), it was 1331 295. In all the crosses, the fecundity was significantly reduced when compared with the control cross (U♀U♂) (Table 1) (Perveen, 2000a).


aLD10: 1.00 ng larva-1; LD30: 3.75ng larva-1; U: untreated (control); ♀: female adults; ♂: male adults; n: number of pairs used

bNumber of eggs oviposited during the whole life of female adult were counted (fecundity) and from the eggs number of hatched that larvae were counted (fertility).

cData were analyzed using one way ANOVA (Concepts, 1989) at *P*<0.0001). Means within columns followed by different letters indicate significant differences by Scheffe`s *<sup>F</sup>*-test (Scheffe, 1953) at 5%. dHatchability % values were normalized by arcsin transformation before statistical analysis (Anderson and McLean 1974).

Table 1. Effects of sublethal doses of chlorfluazuron on fecundity, fertility and hatchability after topical application of newly ecdysed-fifth instar larvae of *Spodoptera litura* (Source: Perveen, 2000a).

When the LD10 of chlorfluazuron was applied to newly ecdysed pupae and the resulting adults were paired, the control fecundity was 2170175. It was not significantly reduced (P<0.02) compared with the larval treatment. The fecundity in the treated pupal cross (LD10♀U♂) was 64083. It was not significantly reduced (P<0.02) compared with the same cross with treated larvae. In the pupal treatment, the fecundity was suppressed to a similar degree in the crosses LD10♀U♂, U♀LD10♂, LD10♀ LD10♂. This reduction was significant (P<0.0001) when compared with the control cross (Table 1) (Perveen, 2000a). There was no significant reduction (P<0.02) between the larval and pupal treatments with respect to fecundity (Tables 1 and 2) (Perveen, 2005).

The mean fertility of females was 1984208 larvae when both the male and female were untreated, i.e. the control (U♀U♂) (Table 1). When the female was treated with either the LD10 or LD30 and mated with an untreated male, the fertility was (LD10♀U♂: 1010315; LD30♀ <sup>U</sup>♂: 828206, respectively), i.e., significantly reduced compared with the control cross (U♀ <sup>U</sup>♂). When the male was treated with the LD10 and mated with an untreated female, the fertility (U♀ LD10♂: 688317) was significantly reduced compared with the crosses, i.e.,

Chlorfluazuron as Reproductive Inhibitor 31

97.8%, respectively, for the larval and pupal treatments. The larval-treated cross, LD10♀U♂, was 68.9%, which is not significantly reduced (P<0.02) than the same cross of the pupal treatment in which the hatchability was 66.5%. The hatchability was reduced 48.7% and 52.7% in the U♀LD10♂ cross, respectively, for the larval and pupal treatments. In the LD10♀LD10♂ cross, it was reduced to 48.6% and 49.3%, respectively, for these treatments. There was no significant reduction (P<0.02) between larval and pupal treatments with

When chlorfluazuron was applied to newly ecdysed fifth instars at sublethal doses, LD10 (1.00 ng larva-1) or LD30 (3.75 ng larva-1), it was observed that the fecundity of resulting adults as well as the hatching rate of their eggs was suppressed. The hatching rate of eggs oviposited by an untreated female mated with a treated male was suppressed to the same degree as that ofeggs oviposited by a treated female mated with a treated male. However, Madore et al. (1983) studied the effects when different concentrations of sublethal doses of the UC-62644 (chlorfluazuron-25) fed to sixth instar larvae of spruce budworm. Homologous crosses between adults of the 0.01, 0.025 and 0.034 ppm treatments showed 0, 69 and 97% reduction, respectively, in the numbers of eggs laid per 30 pairs of moths when compared with control. Emam et al. (1988) reported the fecundity of *S. littoralis* adults decreased significantly from 977.64 eggs in control to 421.75 eggs, a decrease of about 56%, for adults feeding 10% honey solution containing 0.5 p.p.m. chlorflufluazuron. The corresponding fertility inhibition amounted to 32%. In the present case the fertility was significantly different when only the female was treated or only the male was treated. It is obvious from the results that the fertility and hatchability were affected more when the male was treated in comparison with the female, as also reported by Abro et al. (1997), who found that males were more sensitive to insecticides than females, when five concentrations of

cyhalothrin and fluvalinate were tested against fourth instar larvae of *S. litura*.

reasons for the sublethal effects of chlorfluazuron on reproductivity and viability.

To clarify the sublethal effects of chlorfiuazuron on reproductivity of common cutworm, *Spodoptera litura*, experiments were conducted under laboratory conditions. Reduction in the body weight was observed in the larvae and pupae when treated with a sublethal dose (LD30: 3.75 ng larva-1) and in the adults when treated with sublethal doses (LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1) as newly ecdysed fifth instar larvae of *S. litura*, although the number of matings per female and life span of adult females and males remained unaffected by the same treatments. When sublethal doses were applied only to females or only to males, or both sexes, the average fecundity reduction was up to 35–44%. When only females were treated with sublethal doses, fertility was reduced by 49–58%; when only males were treated fertility was reduced by 65–81% and when both sexes were treated, fertility was reduced by 68±83%. Hatchability was reduced by 22–26% when only females were treated, by 44–66% when only males were treated and by 45–72% when both sexes were treated with LD10 or LD30 doses as newly ecdysed fifth instars. The results from these observations suggest that the fecundity was reduced to a similar degree when only females or only males or both sexes were treated with LD10 or LD30 doses as newly ecdysed fifth instars. However the fertility and hatchability were affected more when only males were treated with LD10 and much more when treated with LD30. Currently, work is in progress to find out the main

respect to hatchability (Tables 1 and 2) (Perveen, 2000a).

**2.3 Discussion** 

**2.4 Conclusion** 

LD10♀U♂ and LD30♀U♂. However, in the same cross when the male was treated with the LD30 instead of LD10, the fertility was (U♀ LD30♂: 368155) significantly lower than the <sup>U</sup>♀LD10♂ cross. When both sexes were treated with the LD10, the fertility was (LD10♀LD10♂: 643265) not significantly different from the U♀ LD10♂ cross. Similarly, when both sexes were treated with the LD30, the fertility was (LD30♀LD30♂: 333121) not significantly different from the U♀LD30♂ cross (Table 1) (Perveen, 2000a).


aLD10: 0.12 ng female pupa-1; 1.23 ng male pupa-1; n: number of pairs used bNumber of eggs oviposited (fecundity) the during whole life of female adults were counted and from the number of eggs that hatched larvae were counted (fertility).

cData were analyzed using one-way ANOVA (Concepts, 1989) at P < 0.001. Means within a column followed by different letters indicate significant differences according to Scheffe's F-test (Scheffe, 1953) at 5%.

dHatchability% values were normalized by arcsin transformation before statistical analysis (Anderson and McLean 1974).

Table 2. Effects of a sublethal doses of chlorfluazuron on fecundity, fertility and hatchability after topical application of newly ecdysed pupae of *Spodoptera litura* (Source: Perveen, 2005).

When the LD10 of chlorfluazuron was applied to newly ecdysed pupae and resulting adults were paired, the fertility of the control cross, U♀U♂ was 2123177, which was not significantly reduced (P<0.02) than that in the larval treatment. In the same way, with the LD10♀U♂ cross, the fertility was 109079, which was not significantly reduced (P<0.02) than the same cross in the larval treatment. The fertility was reduced 61 –65% in the U♀LD10♂ cross, which was significantly reduced (P<0.02) than the LD10♀U♂ cross with a fertility reduction of 46–49%. The fertility of the U♀LD10♂ cross was not significantly reduced (P<0.02) than the LD10♀LD10♂ cross, which was reduced 65–68% for both larval and pupal treatments. There were no significant reductions (P<0.02) between larval and pupal treatments with respect to fertility (Tables 1 and 2). The mean hatchability during a female life-span was 88.4(6.6)% when both male and female were untreated, i.e. the control (U♀U♂) cross (Table 1). When the female was treated, either by the LD10 or LD30 and mated with an untreated male, the hatchabilities were 68.9 (11.8)% (LD10♀U♂) and 65.8(11.4)% (LD30♀U♂), respectively significantly reduced compared with the control cross. When the male was treated with the LD10 and mated with an untreated female, the hatchability, 48.3(17.1)% (U♀LD10♂) was significantly reduced than the LD10♀U♂ and LD30♀U♂ crosses. However, in the same cross when the male was treated with LD30, instead of LD10, hatchability, 28.8(11.2)% (U♀LD30♂), significantly reduced than the U♀LD10♂ crosses. When both sexes were treated with LD10, the hatchability was 48.6(14.2)% (LD10♀LD10♂), not significantly different from the U♀LD10♂ cross. Similarly, when both sexes were treated with LD30, the hatchability was 27.1(9.1)% (LD30♀LD30♂), which was not significantly different from the U♀LD30♂ cross (Table 3.1). The hatchability for the U♀U♂ cross was 88.4% and respect to hatchability (Tables 1 and 2) (Perveen, 2000a).

97.8%, respectively, for the larval and pupal treatments. The larval-treated cross, LD10♀U♂, was 68.9%, which is not significantly reduced (P<0.02) than the same cross of the pupal treatment in which the hatchability was 66.5%. The hatchability was reduced 48.7% and 52.7% in the U♀LD10♂ cross, respectively, for the larval and pupal treatments. In the LD10♀LD10♂ cross, it was reduced to 48.6% and 49.3%, respectively, for these treatments.

## **2.3 Discussion**

30 Insecticides – Pest Engineering

LD10♀U♂ and LD30♀U♂. However, in the same cross when the male was treated with the LD30 instead of LD10, the fertility was (U♀ LD30♂: 368155) significantly lower than the <sup>U</sup>♀LD10♂ cross. When both sexes were treated with the LD10, the fertility was (LD10♀LD10♂: 643265) not significantly different from the U♀ LD10♂ cross. Similarly, when both sexes were treated with the LD30, the fertility was (LD30♀LD30♂: 333121) not significantly different

> Fertilityb,c (meanSD)

Hatchability

%c,d

from the U♀LD30♂ cross (Table 1) (Perveen, 2000a).

the number of eggs that hatched larvae were counted (fertility).

na Fecundityb,c (meanSD)

<sup>U</sup>♀U♂ 15 2170175a 2123177a 97.8a LD10♀U♂ 15 164083b 109079b 66.5b <sup>U</sup>♀LD10♂ 15 158075b 82749c 52.3c LD10♀LD10♂ 15 152476b 75151c 49.3c

aLD10: 0.12 ng female pupa-1; 1.23 ng male pupa-1; n: number of pairs used bNumber of eggs oviposited (fecundity) the during whole life of female adults were counted and from

cData were analyzed using one-way ANOVA (Concepts, 1989) at P < 0.001. Means within a column followed by different letters indicate significant differences according to Scheffe's F-test (Scheffe, 1953)

dHatchability% values were normalized by arcsin transformation before statistical analysis (Anderson

Table 2. Effects of a sublethal doses of chlorfluazuron on fecundity, fertility and hatchability after topical application of newly ecdysed pupae of *Spodoptera litura* (Source: Perveen, 2005). When the LD10 of chlorfluazuron was applied to newly ecdysed pupae and resulting adults were paired, the fertility of the control cross, U♀U♂ was 2123177, which was not significantly reduced (P<0.02) than that in the larval treatment. In the same way, with the LD10♀U♂ cross, the fertility was 109079, which was not significantly reduced (P<0.02) than the same cross in the larval treatment. The fertility was reduced 61 –65% in the U♀LD10♂ cross, which was significantly reduced (P<0.02) than the LD10♀U♂ cross with a fertility reduction of 46–49%. The fertility of the U♀LD10♂ cross was not significantly reduced (P<0.02) than the LD10♀LD10♂ cross, which was reduced 65–68% for both larval and pupal treatments. There were no significant reductions (P<0.02) between larval and pupal treatments with respect to fertility (Tables 1 and 2). The mean hatchability during a female life-span was 88.4(6.6)% when both male and female were untreated, i.e. the control (U♀U♂) cross (Table 1). When the female was treated, either by the LD10 or LD30 and mated with an untreated male, the hatchabilities were 68.9 (11.8)% (LD10♀U♂) and 65.8(11.4)% (LD30♀U♂), respectively significantly reduced compared with the control cross. When the male was treated with the LD10 and mated with an untreated female, the hatchability, 48.3(17.1)% (U♀LD10♂) was significantly reduced than the LD10♀U♂ and LD30♀U♂ crosses. However, in the same cross when the male was treated with LD30, instead of LD10, hatchability, 28.8(11.2)% (U♀LD30♂), significantly reduced than the U♀LD10♂ crosses. When both sexes were treated with LD10, the hatchability was 48.6(14.2)% (LD10♀LD10♂), not significantly different from the U♀LD10♂ cross. Similarly, when both sexes were treated with LD30, the hatchability was 27.1(9.1)% (LD30♀LD30♂), which was not significantly different from the U♀LD30♂ cross (Table 3.1). The hatchability for the U♀U♂ cross was 88.4% and

Mating pairsa (femalemale)

at 5%.

and McLean 1974).

When chlorfluazuron was applied to newly ecdysed fifth instars at sublethal doses, LD10 (1.00 ng larva-1) or LD30 (3.75 ng larva-1), it was observed that the fecundity of resulting adults as well as the hatching rate of their eggs was suppressed. The hatching rate of eggs oviposited by an untreated female mated with a treated male was suppressed to the same degree as that ofeggs oviposited by a treated female mated with a treated male. However, Madore et al. (1983) studied the effects when different concentrations of sublethal doses of the UC-62644 (chlorfluazuron-25) fed to sixth instar larvae of spruce budworm. Homologous crosses between adults of the 0.01, 0.025 and 0.034 ppm treatments showed 0, 69 and 97% reduction, respectively, in the numbers of eggs laid per 30 pairs of moths when compared with control. Emam et al. (1988) reported the fecundity of *S. littoralis* adults decreased significantly from 977.64 eggs in control to 421.75 eggs, a decrease of about 56%, for adults feeding 10% honey solution containing 0.5 p.p.m. chlorflufluazuron. The corresponding fertility inhibition amounted to 32%. In the present case the fertility was significantly different when only the female was treated or only the male was treated. It is obvious from the results that the fertility and hatchability were affected more when the male was treated in comparison with the female, as also reported by Abro et al. (1997), who found that males were more sensitive to insecticides than females, when five concentrations of cyhalothrin and fluvalinate were tested against fourth instar larvae of *S. litura*.

There was no significant reduction (P<0.02) between larval and pupal treatments with

#### **2.4 Conclusion**

To clarify the sublethal effects of chlorfiuazuron on reproductivity of common cutworm, *Spodoptera litura*, experiments were conducted under laboratory conditions. Reduction in the body weight was observed in the larvae and pupae when treated with a sublethal dose (LD30: 3.75 ng larva-1) and in the adults when treated with sublethal doses (LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1) as newly ecdysed fifth instar larvae of *S. litura*, although the number of matings per female and life span of adult females and males remained unaffected by the same treatments. When sublethal doses were applied only to females or only to males, or both sexes, the average fecundity reduction was up to 35–44%. When only females were treated with sublethal doses, fertility was reduced by 49–58%; when only males were treated fertility was reduced by 65–81% and when both sexes were treated, fertility was reduced by 68±83%. Hatchability was reduced by 22–26% when only females were treated, by 44–66% when only males were treated and by 45–72% when both sexes were treated with LD10 or LD30 doses as newly ecdysed fifth instars. The results from these observations suggest that the fecundity was reduced to a similar degree when only females or only males or both sexes were treated with LD10 or LD30 doses as newly ecdysed fifth instars. However the fertility and hatchability were affected more when only males were treated with LD10 and much more when treated with LD30. Currently, work is in progress to find out the main reasons for the sublethal effects of chlorfluazuron on reproductivity and viability.

Chlorfluazuron as Reproductive Inhibitor 33

larva-1) were applied to newly ecdysed fifth-instar larvae same mentioned in Section 2.1.2. To determine the effects of chlorzuazuron on the ovaries, control and treated batches of insects, collected from the fifth day after pupation to the seventh day after adult emergence, were used, depending on the experiment requirements. Ovaries were dissected from these insects in Ringer's solution under a binocular microscope (10magnifcation: Nikon, Nippon Kogaku, Tokyo, Japan), and the lengths of pedicle, vitellarium, and germarium of each ovariole were measured. The number of mature oöcytes in each of the ovarioles was also counted. The Ringer's solution was removed, and the freshly dissected ovaries were placed in a small covered container that had been preweighed. The dissected ovaries were weighed on an analytical balance (AC-205, Sartorius Analytical, Tokyo, Japan) and kept in the same container in the oven for 24 h at 62±1 °C for evaporation of water. The dried ovaries were

The procedure to stain the nuclei for cell density from the germarium during female adults required age was adapted from the method described by He (1994). First, the germarium of the female were removed and kept on a microscope slide and carefully crushed with micro forceps until it was extended and roughly evenly distributed over the slide. Second, several drops of 3:1 methanol-acetic acid were introduced to the slide to fix the preparation for 15 min and then the excess fixing solution was absorbed with a filter paper after the fixation. Third, several drops of a 2–5% Giemsa solution dissolved by Sorensen-Gomori buffer solution (monobasic and dibasic sodium phosphate, 0.07 M, pH 6.8) were introduced to the slide for 10–30 min to stain the preparation. Then after the staining, the slide was washed gently and carefully and dried in the air. Finally, the air-dried preparation was checked with

The length and width of the basal oöcytes were measured from the 5th d after pupation to 0 day after adult emergence in control and treated insects. Oöcyte measurements were made on three to four basal oöcytes per pair of ovaries that were taken from 9-10 insects. Measurements were made with a graduated slide under the phase contrast microscope at 400magnification (BH2, Olympus, Tokyo, Japan). The size of basal oöcytes was calculated by the formula used by Loeb et al*.* (1984) for the size of a prolate spheroid, 4/3π(ab2), where *a* is the radius of the long and *b* is the short dimension of the same oöcyte (Perveen and

The thickness of the follicular epithelium of basal oöcytes was observed by making a parafilm microtomy conducted according to the method described by Yoshida (1994). Basal oöcytes were fixed in Carnoy's solution for 3 hour, washed in 70% ethanol for 2 hour, and dehydrated in an alcohol series 70, 80, 90, and 95% and twice in 100%, followed by a benzene and ethanol solution (1:1), each for 30 min. Incubation was done three times at 60 °C in benzene and paraffin (1:1), and then in parafin only, each for 30 min. Five-mm microtome sections were cut into a rolling ribbon. It was stained in xylene I (10 min), xylene II (5 min), followed by an ethanol series of 100, 90, 80, and 70%, each for 5 min, Mayer's hematoxylin for 15 min, and washed under running water. Microtome sections were mounted in 1% eosin (10 min), distilled water (2 sec), followed by an alcohol series 50, 70, 80, and 90%, and twice in 100% (each for 1-5 min), xylene and 100% ethanol (1:1; 5 min), xylene I (5 min), xylene II (5 min). Finally, the sections were embedded on microscopic graduated slide in a drop of Canada balsam. The microscopic graduated slide was covered with a glass cover slip. The thickness of follicular epithelium was measured under a phase

a phase-contrast microscope at 20magnification (Perveen and Miyata, 2000).

reweighed (Perveen and Miyata, 2000).

**3.1.2 Histology** 

Miyata, 2000).

## **3. Effects of chlorfluazurn on female reproductive system of** *Spodoptera litura*

In many insects oviposition requires the development of the ovary, egg maturation, mating and, in some insects, feeding of the females. Ovarian development, which includes oöcyte growth and vitellogenesis, is under the hormonal control, of either juvenile hormone or ecdysteroid (Engelmann, 1979). In many insects, juvenile hormone (JH) regulates the biosynthesis and uptake of vitellogenin by the oöcytes. Among Lepidoptera, e.g., the tobacco hawkmoth, *Manduca sexta* L. (Sroka and Gilbert, 1971; Nijhout and Riddiford, 1974) and the large white butterfly *Pieris brassica* L. (Karlinsky, 1963 and 1967; Benz, 1969), juvenile hormone is required for full development of the ovaries in adults, whereas in the silkworm *Bombyx mori* L. (Chatani and Ohnishi, 1976), giant silk moth, *Hyalophora cecropia* (L.) (Williams, 1952; Pan, 1977), ailanthus silkmoth, *Samia cynthia* (Drury) (Takahashi and Mizohata, 1975) and ricemoth, *Corcyra cephalonica* (Stainton) (Deb and Chakarvorty, 1981) ovarian development occurs as part of adult development initiated by ecdysteroid. Juvenile hormone or juvenile hormone analogue (JHA) application at a critical period, however, induces abnormal development of the ovary as well as other tissues, although juvenile hormone analogues can replace natural juvenile hormone in regulating oöcyte maturation (Nomura, 1994). In the normal state, the ovary develops during one day before and after eclosion in the presence of juvenile hormone (as described above) and a haemolymph factor stimulates the ovary to start oviposition. When S-71639 was applied to pupae, it inhibited adult emergence when a relatively a high dose was applied. If adults did emerge, they could not oviposit through inhibition of the haemolymph factor, hatchability was also reduced (Hatakoshi and Hirano, 1990). The effects of diflubenzuron on fecundity resulted from treatment of adult females by contact or ingestion (Leuschner, 1974; Fytizas, 1976). When *O. japonica*, adults females were fed diflubenzuron, it retarded the maturation of oöcytes (Lim and Lee, 1982). In *T. molitor*, diflubenzuron reduced mealworm longevity (Soltani et al., 1987), the number of oöcytes per ovary, the duration of the oviposition period and the fecundity (Soltani, 1984). Diflubenzuron, topically applied (0.5 µg insect-1) to codling moth, *Cydia pomonella* L. on pupal ecdysis, inhibited the growth and development of oöcytes. It delayed adult emergence and caused a decrease in both the thickness of the follicular epithelium and the size of the basal oöcytes during pupal development. On the other hand, the size of basal oöcytes, the protein content per ovary and the number of oöcytes per ovary recorded in newly emerged adults were significantly reduced by the diflubenzuron treatment. These results, together with observations in several other species, indicated that the reduction in fecundity and egg viability was probably due to interference by diflubenzuron with vitellogenesis (Soltani and Mazouni, 1992). Under laboratory conditions, effects of topical application of sublethal doses of chlorfluazuron (LD10: 1.00 ng larva-1 or LD30: 3.75 ng larva-1) on newly ecdysed fifth-instar on fecundity, fertility and hatchability have been investigated. Thus, it is investigated the causes of the decrease in these parameters. To obtain more information, sublethal doses of chlorfluazuron topically have been applied to newly ecdysed fifth-instar larvae of *S. litura* and the effects on female reproductive system during ovarian development and oögenesis have been observed.

#### **3.1 Experimental procedure**

#### **3.1.1 Ovary measurement**

Experimental *S. litura* were rared in the same way as mentioned in Section 2.1.1. Sublethal doses, LD10 (1.00 ng larva-1; 0.12 ng female pupa-1; 1.23 ng male pupa-1) or LD30 (3.75 ng larva-1) were applied to newly ecdysed fifth-instar larvae same mentioned in Section 2.1.2. To determine the effects of chlorzuazuron on the ovaries, control and treated batches of insects, collected from the fifth day after pupation to the seventh day after adult emergence, were used, depending on the experiment requirements. Ovaries were dissected from these insects in Ringer's solution under a binocular microscope (10magnifcation: Nikon, Nippon Kogaku, Tokyo, Japan), and the lengths of pedicle, vitellarium, and germarium of each ovariole were measured. The number of mature oöcytes in each of the ovarioles was also counted. The Ringer's solution was removed, and the freshly dissected ovaries were placed in a small covered container that had been preweighed. The dissected ovaries were weighed on an analytical balance (AC-205, Sartorius Analytical, Tokyo, Japan) and kept in the same container in the oven for 24 h at 62±1 °C for evaporation of water. The dried ovaries were reweighed (Perveen and Miyata, 2000).

#### **3.1.2 Histology**

32 Insecticides – Pest Engineering

In many insects oviposition requires the development of the ovary, egg maturation, mating and, in some insects, feeding of the females. Ovarian development, which includes oöcyte growth and vitellogenesis, is under the hormonal control, of either juvenile hormone or ecdysteroid (Engelmann, 1979). In many insects, juvenile hormone (JH) regulates the biosynthesis and uptake of vitellogenin by the oöcytes. Among Lepidoptera, e.g., the tobacco hawkmoth, *Manduca sexta* L. (Sroka and Gilbert, 1971; Nijhout and Riddiford, 1974) and the large white butterfly *Pieris brassica* L. (Karlinsky, 1963 and 1967; Benz, 1969), juvenile hormone is required for full development of the ovaries in adults, whereas in the silkworm *Bombyx mori* L. (Chatani and Ohnishi, 1976), giant silk moth, *Hyalophora cecropia* (L.) (Williams, 1952; Pan, 1977), ailanthus silkmoth, *Samia cynthia* (Drury) (Takahashi and Mizohata, 1975) and ricemoth, *Corcyra cephalonica* (Stainton) (Deb and Chakarvorty, 1981) ovarian development occurs as part of adult development initiated by ecdysteroid. Juvenile hormone or juvenile hormone analogue (JHA) application at a critical period, however, induces abnormal development of the ovary as well as other tissues, although juvenile hormone analogues can replace natural juvenile hormone in regulating oöcyte maturation (Nomura, 1994). In the normal state, the ovary develops during one day before and after eclosion in the presence of juvenile hormone (as described above) and a haemolymph factor stimulates the ovary to start oviposition. When S-71639 was applied to pupae, it inhibited adult emergence when a relatively a high dose was applied. If adults did emerge, they could not oviposit through inhibition of the haemolymph factor, hatchability was also reduced (Hatakoshi and Hirano, 1990). The effects of diflubenzuron on fecundity resulted from treatment of adult females by contact or ingestion (Leuschner, 1974; Fytizas, 1976). When *O. japonica*, adults females were fed diflubenzuron, it retarded the maturation of oöcytes (Lim and Lee, 1982). In *T. molitor*, diflubenzuron reduced mealworm longevity (Soltani et al., 1987), the number of oöcytes per ovary, the duration of the oviposition period and the fecundity (Soltani, 1984). Diflubenzuron, topically applied (0.5 µg insect-1) to codling moth, *Cydia pomonella* L. on pupal ecdysis, inhibited the growth and development of oöcytes. It delayed adult emergence and caused a decrease in both the thickness of the follicular epithelium and the size of the basal oöcytes during pupal development. On the other hand, the size of basal oöcytes, the protein content per ovary and the number of oöcytes per ovary recorded in newly emerged adults were significantly reduced by the diflubenzuron treatment. These results, together with observations in several other species, indicated that the reduction in fecundity and egg viability was probably due to interference by diflubenzuron with vitellogenesis (Soltani and Mazouni, 1992). Under laboratory conditions, effects of topical application of sublethal doses of chlorfluazuron (LD10: 1.00 ng larva-1 or LD30: 3.75 ng larva-1) on newly ecdysed fifth-instar on fecundity, fertility and hatchability have been investigated. Thus, it is investigated the causes of the decrease in these parameters. To obtain more information, sublethal doses of chlorfluazuron topically have been applied to newly ecdysed fifth-instar larvae of *S. litura* and the effects on female reproductive system during ovarian development and oögenesis have been observed.

Experimental *S. litura* were rared in the same way as mentioned in Section 2.1.1. Sublethal doses, LD10 (1.00 ng larva-1; 0.12 ng female pupa-1; 1.23 ng male pupa-1) or LD30 (3.75 ng

**3. Effects of chlorfluazurn on female reproductive system of** *Spodoptera* 

*litura*

**3.1 Experimental procedure 3.1.1 Ovary measurement** 

The procedure to stain the nuclei for cell density from the germarium during female adults required age was adapted from the method described by He (1994). First, the germarium of the female were removed and kept on a microscope slide and carefully crushed with micro forceps until it was extended and roughly evenly distributed over the slide. Second, several drops of 3:1 methanol-acetic acid were introduced to the slide to fix the preparation for 15 min and then the excess fixing solution was absorbed with a filter paper after the fixation. Third, several drops of a 2–5% Giemsa solution dissolved by Sorensen-Gomori buffer solution (monobasic and dibasic sodium phosphate, 0.07 M, pH 6.8) were introduced to the slide for 10–30 min to stain the preparation. Then after the staining, the slide was washed gently and carefully and dried in the air. Finally, the air-dried preparation was checked with a phase-contrast microscope at 20magnification (Perveen and Miyata, 2000).

The length and width of the basal oöcytes were measured from the 5th d after pupation to 0 day after adult emergence in control and treated insects. Oöcyte measurements were made on three to four basal oöcytes per pair of ovaries that were taken from 9-10 insects. Measurements were made with a graduated slide under the phase contrast microscope at 400magnification (BH2, Olympus, Tokyo, Japan). The size of basal oöcytes was calculated by the formula used by Loeb et al*.* (1984) for the size of a prolate spheroid, 4/3π(ab2), where *a* is the radius of the long and *b* is the short dimension of the same oöcyte (Perveen and Miyata, 2000).

The thickness of the follicular epithelium of basal oöcytes was observed by making a parafilm microtomy conducted according to the method described by Yoshida (1994). Basal oöcytes were fixed in Carnoy's solution for 3 hour, washed in 70% ethanol for 2 hour, and dehydrated in an alcohol series 70, 80, 90, and 95% and twice in 100%, followed by a benzene and ethanol solution (1:1), each for 30 min. Incubation was done three times at 60 °C in benzene and paraffin (1:1), and then in parafin only, each for 30 min. Five-mm microtome sections were cut into a rolling ribbon. It was stained in xylene I (10 min), xylene II (5 min), followed by an ethanol series of 100, 90, 80, and 70%, each for 5 min, Mayer's hematoxylin for 15 min, and washed under running water. Microtome sections were mounted in 1% eosin (10 min), distilled water (2 sec), followed by an alcohol series 50, 70, 80, and 90%, and twice in 100% (each for 1-5 min), xylene and 100% ethanol (1:1; 5 min), xylene I (5 min), xylene II (5 min). Finally, the sections were embedded on microscopic graduated slide in a drop of Canada balsam. The microscopic graduated slide was covered with a glass cover slip. The thickness of follicular epithelium was measured under a phase

Chlorfluazuron as Reproductive Inhibitor 35

ovarian weight and dry ovarian weight in newly emerged adults when compared with the controls (Table 3). Significant reductions were not observed in fresh body weight (P=0.0567), fresh ovarian weight (P=0.7788) and dry ovarian weight (P=0.5757), when the LD10 and LD30 treatments were compared. Similarly, ratios of fresh ovarian/fresh body weight (31.0%), dry ovarian/fresh ovarian weight (28.0%) and dry ovarian/fresh body weight (9.0%), were not

> DOWa,b (MSD) mg

C 30 25511.6a 81.19.2a 23.61.4a 31.52.9a 28.92.4a 9.20.7a LD10 30 2293.2b 72.03.4b 20.01.4b 31.21.5a 28.11.2a 9.00.8a LD30 30 2245.9b 70.94.2b 19.91.3b 31.32.4a 28.00.5a 9.10.8a

aC: control; T: trearment; LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; n: number of insects used; FBW: fresh body weight; FOW: fresh ovarian weight; DOW: dry ovarian weight; % R: percent ratio bData were analyzed using one-way ANOVA (Concepts, 1989) at P<0.001. Means within a column followed by different letters indicate significant differences according to Scheffe's F-test (Scheffe, 1953)

Table 3. Effects of sublethal doses of chlorfluazuron on the ovarian and body weight of newly emerged adults after topical application to newly ecdysed fifth-instar larvae of

Ovaries are small on the 8th day after pupation. From the 8th day after pupation to the day before adult emergence, ovarian weight slowly increased; after that it increased sharply until the day of adult emergence, and then, increased gradually until the 2nd day after adult emergence, when it reached maximum (12019.4 mg). Then, in the controls it decreased gradually until the 7th day after adult emergence. The pattern of changes in fresh ovarian weight in the LD10– or LD30–treated females was similar as observed in the controls during various developmental days of pupae and adults. The fresh ovarian weight was significantly reduced on the 8th day after pupation (P<0.0001); on the 9th day after pupation (P<0.0001); on the 1st day after adult emergence (P<0.0003); on the 2nd day after adult emergence (P<0.0001); on the 3rd day after adult emergence (P<0.0001); on the 4th day after adult emergence (P<0.0001); on the 5th day after adult emergence (P<0.0001); on the 6th day after adult emergence (P<0.0001); on the 7th day after adult emergence (P<0.0001) in the LD10- or LD30-treated females compared with the controls, but no significant reduction was observed (P=0.0979–0.970) between the LD10- or LD30-treated females during ovarian

In newly emerged the LD10– or LD30–treated adults, the total length of the ovariole was significantly reduced (P<0.0001) compared with the control, but there were no significant reductions (P=0.0508) between the LD10– or LD30–treatments. In the LD10– or LD30–treated insects, the germarium (immature oögonia) was significantly longer (P<0.0001) than that of the pedicle (fully mature ova) and the vitellarium (under developing oöcytes) compared with the controls in which the vitellarium was significantly longer (P<0.0001) than the germarium and pedicle (Figure 3; Table 4) (Perveen and Miyata, 2000; Perveen, 2011).

% R=FOW /FBWa,b (MSD)

% R=DOW /FBWa,b (MSD)

% R=DOW /FOWa,b (MSD)

significantly different (Table 3) (Perveen and Miyata, 2000).

FOWa,b (MSD) mg

*Spodoptera litura* (Source: Perveen and Miyata, 2000).

development (Figure 2) (Perveen and Miyata, 2000).

FBWa,b (MSD) mg

Ta na

at 5%.

contrast microscope (BH2, Olympus, Tokyo, Japan) at 400magnifcation (Perveen and Miyata, 2000).

## **3.1.3 Data analysis**

Data were analyzed using analysis of variance, one way ANOVA (Concepts, 1989) at *P*<0.01 and Scheffe's *F*-test (Scheffe, 1953) at 5%.

#### **3.2 Results**

The morphology of the adult female reproductive system of *S. litura* is shown in Figure 1.

Fig. 1. The morphology of the female reproductive system of *S. litura*: A: corpus bursae; B: signum, C: ductus bursae; D: ostium bursae; and E: diverticulum of bursa copulatrix; F: ductis seminalis; G: spermathecal gland; H: utriculus I: lagena of spermatheca; J: ductus receptaculi; K: accessory gland (paired); L: accessory gland reservoir (paired); M: vestibulum; N: calyx of the unpaired oviductus communis; O: one of four ovarioles of ovary (paired); P: papillae anales; Q: rectum; X: corpus, Y: collum, and Z: frenum of spermatophores; (Source: Etman and Hooper, 1979).

#### **3.2.1 Effects on ovarian development**

Sublethal doses of chlorfluazuron (LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1), applied to newly ecdysed fifth-instar larvae significantly (P<0.0001) reduced the body weight, fresh

contrast microscope (BH2, Olympus, Tokyo, Japan) at 400magnifcation (Perveen and

Data were analyzed using analysis of variance, one way ANOVA (Concepts, 1989) at *P*<0.01

The morphology of the adult female reproductive system of *S. litura* is shown in Figure 1.

Fig. 1. The morphology of the female reproductive system of *S. litura*: A: corpus bursae; B: signum, C: ductus bursae; D: ostium bursae; and E: diverticulum of bursa copulatrix; F: ductis seminalis; G: spermathecal gland; H: utriculus I: lagena of spermatheca; J: ductus receptaculi; K: accessory gland (paired); L: accessory gland reservoir (paired); M:

vestibulum; N: calyx of the unpaired oviductus communis; O: one of four ovarioles of ovary

Sublethal doses of chlorfluazuron (LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1), applied to newly ecdysed fifth-instar larvae significantly (P<0.0001) reduced the body weight, fresh

(paired); P: papillae anales; Q: rectum; X: corpus, Y: collum, and Z: frenum of

spermatophores; (Source: Etman and Hooper, 1979).

**3.2.1 Effects on ovarian development** 

Miyata, 2000).

**3.2 Results** 

**3.1.3 Data analysis** 

and Scheffe's *F*-test (Scheffe, 1953) at 5%.

ovarian weight and dry ovarian weight in newly emerged adults when compared with the controls (Table 3). Significant reductions were not observed in fresh body weight (P=0.0567), fresh ovarian weight (P=0.7788) and dry ovarian weight (P=0.5757), when the LD10 and LD30 treatments were compared. Similarly, ratios of fresh ovarian/fresh body weight (31.0%), dry ovarian/fresh ovarian weight (28.0%) and dry ovarian/fresh body weight (9.0%), were not significantly different (Table 3) (Perveen and Miyata, 2000).


aC: control; T: trearment; LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; n: number of insects used; FBW: fresh body weight; FOW: fresh ovarian weight; DOW: dry ovarian weight; % R: percent ratio bData were analyzed using one-way ANOVA (Concepts, 1989) at P<0.001. Means within a column followed by different letters indicate significant differences according to Scheffe's F-test (Scheffe, 1953) at 5%.

Table 3. Effects of sublethal doses of chlorfluazuron on the ovarian and body weight of newly emerged adults after topical application to newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen and Miyata, 2000).

Ovaries are small on the 8th day after pupation. From the 8th day after pupation to the day before adult emergence, ovarian weight slowly increased; after that it increased sharply until the day of adult emergence, and then, increased gradually until the 2nd day after adult emergence, when it reached maximum (12019.4 mg). Then, in the controls it decreased gradually until the 7th day after adult emergence. The pattern of changes in fresh ovarian weight in the LD10– or LD30–treated females was similar as observed in the controls during various developmental days of pupae and adults. The fresh ovarian weight was significantly reduced on the 8th day after pupation (P<0.0001); on the 9th day after pupation (P<0.0001); on the 1st day after adult emergence (P<0.0003); on the 2nd day after adult emergence (P<0.0001); on the 3rd day after adult emergence (P<0.0001); on the 4th day after adult emergence (P<0.0001); on the 5th day after adult emergence (P<0.0001); on the 6th day after adult emergence (P<0.0001); on the 7th day after adult emergence (P<0.0001) in the LD10- or LD30-treated females compared with the controls, but no significant reduction was observed (P=0.0979–0.970) between the LD10- or LD30-treated females during ovarian development (Figure 2) (Perveen and Miyata, 2000).

In newly emerged the LD10– or LD30–treated adults, the total length of the ovariole was significantly reduced (P<0.0001) compared with the control, but there were no significant reductions (P=0.0508) between the LD10– or LD30–treatments. In the LD10– or LD30–treated insects, the germarium (immature oögonia) was significantly longer (P<0.0001) than that of the pedicle (fully mature ova) and the vitellarium (under developing oöcytes) compared with the controls in which the vitellarium was significantly longer (P<0.0001) than the germarium and pedicle (Figure 3; Table 4) (Perveen and Miyata, 2000; Perveen, 2011).

Chlorfluazuron as Reproductive Inhibitor 37

LVbc

(MSD)mm

LG bc (MSD)mm R P:V:G (MSD)mm

Treatmentsa

Hooper 1979).

at 5%.

n1 n2 TLObc

(Source: Perveen and Miyata, 2000).

(Perveen and Miyata, 2000).

(MSD)mm

LPbc

(MSD)mm

Control 10 80 104.85.1a3 33.53.4a 45.24.4a 26.14.2a 32:43:25 LD10 9 72 91.55.5b 22.15.0b 30.52.1b 39.02.7b 24:33:43 LD30 9 72 88.79.6b 20.44.4 28.66.7b 40.08.9b 23:32:45

aLD10, 1.00 ng larva-1; LD30, 3.75 ng larva-1; n1: number of insects used; n2: number of ovariole measured; TLO: total length of ovariols; LP: length of pedicle; LV: length of vitellarium; LG: length of germarium; RP:V:G: Ratio of pedicle:vitellarium:germanium bIn Spodoptera litura, the paired ovaries are composed of 8 ovarioles. Four ovarioles are found on each side of the body cavity, forming several loops. Each ovarioles differentiated into 3 parts: (1) pedicle (fully matured eggs), (2) vitellarium (oöcytes and trophocytes), (3) germanium (oögonia) (Etman and

cData were analyzed using one-way ANOVA (Concepts, 1989) at P<0.0001. Means within a column followed by different letters indicate significant differences according to Scheffe's F-test (Scheffe, 1953)

Table 4. Effects of sublethal doses of chlorfluazuron on ovarian development in newly emerged adults after topical application to newly ecdysed fifth instars of *Spodoptera litura* 

When ratios of the length of the pedicle, vitellarium and germarium were compared, they were 32:43:25 for the controls, 24:33:43 for the LD10 and 23:32:45 for the LD30. There was a significant reduction when the %ratios of the LD10 and LD30 were compared with the controls, but there was significant reduction between the LD10 and LD30 treatments (Table 4). When ovarian maturation was observed untreated females had mature ova with an occasional one or two being absorbed (solid ova) in the ovarioles. In the LD10–treated females, the spacing in the ovarioles and the absorption of ova different from the control. In LD30–treated females, besides the spacing and absorption , sometimes only immature ova (germarium) were found in the ovarioles (data is not presented) (Perveen and Miyata, 2000). Mature ova were not observed in the pupae during the 2nd day before adult emergence, but a few mature ova were found the day before adult emergence. The number of mature ova sharply increased until the 1st day after adult emergence, and then gradually increased until the 2nd day after adult emergence. The maximum number of mature ova 7252.0 was found on the second day after adult emergence. On the same day, the number of mature eggs was significantly reduced (P<0.0001) in the LD10– or LD30–treated females as compared with the controls, but no significant reduction was observed (P=0.0984) between the LD10 and LD30 treatments. From the 2nd day to the 7th day after adult emergence, absorption of mature ova started gradually in the controls, and the LD10– or LD30–treated females. The pattern of maturation of ova in ovaries was similar in the controls, LD10 and LD30 treatments (Figure 4)

The cell density, expressed as number of nuclei per mm2, was determined at various days during sexual maturation in the germaria of the controls, LD10– or LD30–treated females (Table 4.3). In the controls, on the 2nd day of adult emergence, the density was 16369.17 nuclei mm2-1. The cell density increased until the 3rd day, when it was 18298.87 nuclei mm2-1 and decreased thereafter. On the 4th day of adult emergence, the cell density was 132356.20 nuclei mm2-1. In the LD10– or LD30–treated insects, the patterns of the cell density change in the germarium were the same as in the controls but the values were significantly (P<0.01) lower in the LD10– treated females and more were significantly decreased in the LD30–treated females compared with the controls, during the days the 2nd, 3rd and 4th of adult female development. There

Fig. 3. A comparison of ovarian morphology of newly emerged adult *Spodoptera litura*, in A: untreated (control); B: treated with the LD10 dose and C: treated with the LD30 dose of chlorfluazuron. Bars in photographs indicate 100 m (Source: Perveen, 2011).

Fig. 2. Effect of sublethal doses of chlorfluazuron on ovarian weight during different developmental days (post pupal and 1 to 7 day after adult emergence). For control (○; n = 10), LD10 (1.00 ng larva-1) treated (□; n = 9), and LD30 (3.75 ng larva-1) treated (Δ; n = 9) after topical application to newly ecdysed fifth instars of *Spodoptera litura*. Data were analyzed using one-way ANOVA (Concepts, 1989) at *P*<0.0001 and Scheffe's *F*-test (Concepts, 1989) at

Fig. 3. A comparison of ovarian morphology of newly emerged adult *Spodoptera litura*, in A: untreated (control); B: treated with the LD10 dose and C: treated with the LD30 dose of

chlorfluazuron. Bars in photographs indicate 100 m (Source: Perveen, 2011).

5%. Vertical bars indicate SD; (Source: Perveen and Miyata, 2000).


aLD10, 1.00 ng larva-1; LD30, 3.75 ng larva-1; n1: number of insects used; n2: number of ovariole measured; TLO: total length of ovariols; LP: length of pedicle; LV: length of vitellarium; LG: length of germarium;

RP:V:G: Ratio of pedicle:vitellarium:germanium bIn Spodoptera litura, the paired ovaries are composed of 8 ovarioles. Four ovarioles are found on each side of the body cavity, forming several loops. Each ovarioles differentiated into 3 parts: (1) pedicle (fully matured eggs), (2) vitellarium (oöcytes and trophocytes), (3) germanium (oögonia) (Etman and Hooper 1979).

cData were analyzed using one-way ANOVA (Concepts, 1989) at P<0.0001. Means within a column followed by different letters indicate significant differences according to Scheffe's F-test (Scheffe, 1953) at 5%.

Table 4. Effects of sublethal doses of chlorfluazuron on ovarian development in newly emerged adults after topical application to newly ecdysed fifth instars of *Spodoptera litura*  (Source: Perveen and Miyata, 2000).

When ratios of the length of the pedicle, vitellarium and germarium were compared, they were 32:43:25 for the controls, 24:33:43 for the LD10 and 23:32:45 for the LD30. There was a significant reduction when the %ratios of the LD10 and LD30 were compared with the controls, but there was significant reduction between the LD10 and LD30 treatments (Table 4). When ovarian maturation was observed untreated females had mature ova with an occasional one or two being absorbed (solid ova) in the ovarioles. In the LD10–treated females, the spacing in the ovarioles and the absorption of ova different from the control. In LD30–treated females, besides the spacing and absorption , sometimes only immature ova (germarium) were found in the ovarioles (data is not presented) (Perveen and Miyata, 2000). Mature ova were not observed in the pupae during the 2nd day before adult emergence, but a few mature ova were found the day before adult emergence. The number of mature ova sharply increased until the 1st day after adult emergence, and then gradually increased until the 2nd day after adult emergence. The maximum number of mature ova 7252.0 was found on the second day after adult emergence. On the same day, the number of mature eggs was significantly reduced (P<0.0001) in the LD10– or LD30–treated females as compared with the controls, but no significant reduction was observed (P=0.0984) between the LD10 and LD30 treatments. From the 2nd day to the 7th day after adult emergence, absorption of mature ova started gradually in the controls, and the LD10– or LD30–treated females. The pattern of maturation of ova in ovaries was similar in the controls, LD10 and LD30 treatments (Figure 4) (Perveen and Miyata, 2000).

The cell density, expressed as number of nuclei per mm2, was determined at various days during sexual maturation in the germaria of the controls, LD10– or LD30–treated females (Table 4.3). In the controls, on the 2nd day of adult emergence, the density was 16369.17 nuclei mm2-1. The cell density increased until the 3rd day, when it was 18298.87 nuclei mm2-1 and decreased thereafter. On the 4th day of adult emergence, the cell density was 132356.20 nuclei mm2-1. In the LD10– or LD30–treated insects, the patterns of the cell density change in the germarium were the same as in the controls but the values were significantly (P<0.01) lower in the LD10– treated females and more were significantly decreased in the LD30–treated females compared with the controls, during the days the 2nd, 3rd and 4th of adult female development. There

Chlorfluazuron as Reproductive Inhibitor 39

In the controls, the basal oöcytes were tiny on the 5th day after pupation, but increased sharply until the 8th day, after which they increased slowly until adult emergence. The maximum size of the basal oöcytes on the day of adult emergence was significantly reduced (P<0.0002) in the LD10– or LD30–treated females, but there was no significant difference (P=0.9976) between LD10– and LD30–treated females (Figure 5) (Perveen and Miyata, 2000).

Fig. 5. Effect of sublethal doses of chlorfluazuron on size of basal oöcytes during different developmental days (5 to 9 day after pupation pupae and newly emerged adults). For control (○), LD10 (1.00 ng larva-1) treated (□), and LD30 (3.75 ng larva-1) treated (Δ) after topical application to newly ecdysed 5th instars of *Spodoptera litura*. Data were analyzed using one-way ANOVA (Concepts, 1989) at *P*<0.0002 and Scheffe's *F*-test (Scheffe, 1953) at

The thickness of the follicular epithelium of the basal oöcytes gradually increased and reached a maximum on the 8th day after pupation, after which it sharply declined. On the 8th day after pupation, it was significantly reduced (P<0.005) in the LD10– or LD30–treated females when compared with control females, but there was no significant difference (P=0.8686) between the LD10 or LD30 treatments. The reduction was approximately 34% in the LD10– and 39% in the LD30–treated females. The patterns of the development of the follicular epithelium of basal oöcytes were not similar in the LD10– or LD30–treated females compared with the controls. However, pattern was similar between the LD10– and LD30– treated females. In the LD10– or LD30–treated females, the follicular epithelium reached maximum on the 9th day after pupation, and then declined. The development of the follicular epithelium was delayed by one day in the LD10– or LD30–treated females compared with the controls. On the 9th day, it was thicker in the LD30–treated females and thickest in

5%. Verticle bars indicate SD (n = 9-10); (source: Perveen and Miyata, 2000).

**3.2.2 Effects on oöcytes development** 

was also a significant reduction (P<0.05) in the cell density between the LD10– and LD30– treated females during adult development (Table 5) (Perveen, 2011).

Fig. 4. Effect of sublethal doses of chlorfluazuron on number of mature eggs in the ovaries during different developmental days (post pupal and 1 to 7 day after adult emergence). For control (○; n = 13), LD10 (1.00 ng larva-1) treated (□; n = 11), and LD30 (3.75 ng larva-1) treated (Δ; n = 13) after topical application to newly molted fifth instars of *Spodoptera litura*. Data were analyzed using one-way ANOVA (Concepts, 1989) at *P*<0.0001 and Scheffe's *F*-test (Scheffe, 1953) at 5%. Vertical bars indicate SD; (Source: Perveen and Miyata, 2000).


aLD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; n1: number of insects used; n2: number of ovariole measured

bThe age of female adults was taken from the day of adult emergence.

cData were analyzed using one-way ANOVA (Concepts, 1989) at P<0.01. Means within a column followed by different letters indicate significant differences according to Scheffe's F-test (Scheffe, 1953) at 5%.

Table 5. Effects of sublethal doses of chlorfluazuron on the cell density in the germarium after topical application to newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2011).

was also a significant reduction (P<0.05) in the cell density between the LD10– and LD30–

Fig. 4. Effect of sublethal doses of chlorfluazuron on number of mature eggs in the ovaries during different developmental days (post pupal and 1 to 7 day after adult emergence). For control (○; n = 13), LD10 (1.00 ng larva-1) treated (□; n = 11), and LD30 (3.75 ng larva-1) treated (Δ; n = 13) after topical application to newly molted fifth instars of *Spodoptera litura*. Data were analyzed using one-way ANOVA (Concepts, 1989) at *P*<0.0001 and Scheffe's *F*-test (Scheffe, 1953) at 5%. Vertical bars indicate SD; (Source: Perveen and Miyata, 2000).

Cell density number of nuclei (mm2)-1 in the germarium

during female adults age (MSD)b,c

Treatmentsa n1 n2 2 day-old 3 day-old 4 day-old Control 5 10 16369.17a 18298.87a 132356.20a LD10 5 10 157042.50b 175349.91b 12359.50b LD30 5 10 14898.60c 16447.68c 108961.42c

aLD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; n1: number of insects used; n2: number of ovariole

cData were analyzed using one-way ANOVA (Concepts, 1989) at P<0.01. Means within a column followed by different letters indicate significant differences according to Scheffe's F-test (Scheffe,

Table 5. Effects of sublethal doses of chlorfluazuron on the cell density in the germarium after topical application to newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source:

bThe age of female adults was taken from the day of adult emergence.

measured

1953) at 5%.

Perveen, 2011).

treated females during adult development (Table 5) (Perveen, 2011).

#### **3.2.2 Effects on oöcytes development**

In the controls, the basal oöcytes were tiny on the 5th day after pupation, but increased sharply until the 8th day, after which they increased slowly until adult emergence. The maximum size of the basal oöcytes on the day of adult emergence was significantly reduced (P<0.0002) in the LD10– or LD30–treated females, but there was no significant difference (P=0.9976) between LD10– and LD30–treated females (Figure 5) (Perveen and Miyata, 2000).

Fig. 5. Effect of sublethal doses of chlorfluazuron on size of basal oöcytes during different developmental days (5 to 9 day after pupation pupae and newly emerged adults). For control (○), LD10 (1.00 ng larva-1) treated (□), and LD30 (3.75 ng larva-1) treated (Δ) after topical application to newly ecdysed 5th instars of *Spodoptera litura*. Data were analyzed using one-way ANOVA (Concepts, 1989) at *P*<0.0002 and Scheffe's *F*-test (Scheffe, 1953) at 5%. Verticle bars indicate SD (n = 9-10); (source: Perveen and Miyata, 2000).

The thickness of the follicular epithelium of the basal oöcytes gradually increased and reached a maximum on the 8th day after pupation, after which it sharply declined. On the 8th day after pupation, it was significantly reduced (P<0.005) in the LD10– or LD30–treated females when compared with control females, but there was no significant difference (P=0.8686) between the LD10 or LD30 treatments. The reduction was approximately 34% in the LD10– and 39% in the LD30–treated females. The patterns of the development of the follicular epithelium of basal oöcytes were not similar in the LD10– or LD30–treated females compared with the controls. However, pattern was similar between the LD10– and LD30– treated females. In the LD10– or LD30–treated females, the follicular epithelium reached maximum on the 9th day after pupation, and then declined. The development of the follicular epithelium was delayed by one day in the LD10– or LD30–treated females compared with the controls. On the 9th day, it was thicker in the LD30–treated females and thickest in

Chlorfluazuron as Reproductive Inhibitor 41

weight, dry ovarian/fresh ovarian weight, and dry ovarian/fresh body weight were the same among the newly emerged control, LD10 or LD30 treated females, indicating a reduction in the body, fresh, and dry ovarian weights with the same degree of reduction in treated females and controls. However, Soltani and Pickens and DeMilo (1977) reported that 0.5 mg DFB, when topically applied at pupal ecdysis to *Cydia pomonella* (L.), did not cause the ovarian weight be significantly reduced (*P*<0.05) between control and treated newly emerged adult females. Nor did Hatakoshi (1992) observe any signifcant reduction (*P*<0.05) in the ovarian weight between control and treated last day of pupae to third day after adult emergence of *S. litura,* when 0.3 ng pyriproxyfen was topically applied at pupal ecdysis. The differences in results observed in these experiments may be related to the pesticides, the kind of insect used and their developmental stages. *Spodoptera litura* has paired ovaries that branch into four polytrophic meroistic ovarioles located on the ventral side of the body cavity, making several loops of ovarioles, with all basal oöcytes developing simultaneously each ovariole is differentiated into tree portions according to the developmental stages of the oöcytes: (1) the yellowish green pedicle, where fully matured ova are stored; (2) the reddish orange vitellarium, which contains the developing oöcyte and trophocyte follicles which undergo accumulation of yolk proteins, and choriogenesis; and (3) the whitis germarium, which contains oögoia, from which germ cells proliferate and follicles are formed. Similar observations were reported by Riakhel and Dhadialla (1992) and Etman and

As in other Lepidopterous species, the ovaries of *S. litura* start to differentiate, and develop at the pupal stage. Indeed, in controls, the thickness of follicular epithelium of basal oöcyte reached its maximum size on the eighth day after pupation. This coincided with the start of follicular epithelium resorption. Histological examination on *S. litura* showed that topical application of sublethal doses of chlorfuazuron to newly ecdysed ffth-instar larvae affected growth and development of oöcytes during pupal and adult stages by affecting size and thickness of follicular epithelium (Figures 5 and 6). However, in *C. pomonella,* (in controls) the basal oöcytes reached their maximum size 7 day after pupation. In this insect, this coincided with the start of follicular epithelium resorption. Hence, a 0.5-mg dose of DFB applied topically to newly ecdysed pupae affected the growth and development of oöcytes by causing a decrease in both the thickness of the follicular epithelium and size of basal oöcytes during the pupal development (Soltani and Mazouni, 1992). Lim and Lee (1982) reported that 2-d-old adult females of *O. japonica,* starved for 6 h and consumed 500 mg (AI) of DFB with two maize discs. The females were found to have retarded ovarian development, caused by a delay of oöcytes development, and an increased percentage of oöcytes resorption. This caused a decrease in fecundity and egg viability of the females. However, significant reduction was not observed either in the number of ovarioles or in the length of basal oöcytes in treated insects. Differences in these results might be a result of the use of different BPUs. Also, the doses used by Lim and Lee (1982) were very high compared

with those used in present study or in the Soltani and Mazouni (1992) experiments.

In newly emerged treated adults, the germarium was much longer than the pedicle and vitelarium as compared with the controls in which the vitelarium was longer than the germarium and pedicle (Table 4). This shows that maturation of oöcytes was delayed in treated adult females as compared with the controls. The maximum thickness of the follicular epithelium of basal ooctes was observed on the 9 day after pupation in treated females, whereas it was on the 8 d after pupation in the controls (Figure 6). Subsequent to the 8th or 9th day, resorption of follicular epithelium started in control and treated females,

Hooper (1979), which were confirmed here.

LD10–treated females than in control females, but significant differences were not observed (P=0.7611) among these three groups, i.e. controls, LD10– and LD30–treated females (Figure 6) (Perveen and Miyata, 2000).

Fig. 6. Effect of sublethal doses of chlorfluazuron on thickness of follicular epithelium of basal oöcytes during different developmental days (5 to 9 days after pupation and newly emerged adults). For control (○), LD10 (1.00 ng larva-1) treated (□), and LD30 (3.75 ng larva-1) treated (Δ) after topical application to newly ecdysed 5th instar larvae of *Spodoptera litura*. Data were analyzed using one-way ANOVA (Concepts, 1989) at *P*<0.005 and Scheffe's *F*-test (Scheffe, 1953) at 5%. Vertical bars indicate SD (n = 9-10); (Source: Perveen and Miyata, 2000).

#### **3.3 Discussion**

Topical application of sublethal doses of chlorfluazuron (LD10: 1.00 ng per larva or LD30: 3.75 ng per larva) on newly ecdysed fifth instars had an effect on the reproduction of *S. litura* by reducing fecundity, fertility, and hatchability (Perveen, 2000a). Thus, this study was conducted to establish the causes of the reduction in these parameters. It was found that topical appication of sublethal doses of chlorfuazuron had an affect on ovarian development and oögenesis by decreasing the weight of ovaries during postpupal and adult developmental days of LD10 or LD30 treated females. The basic factors responsible for the reduction in ovarian weight were reduction in the length of different parts of the ovarioles, decrease in the number of mature ova, reduction in the size of basal oöcytes and thickness of their follicular epithelium, and reduction in protein content of ovarian constituents as compared with the controls. In this study, topical application of sublethal doses of chlorfuazuron signifcantly reduced the ovarian weight to that of the controls in postpupal and adult developmental days (Figure 2; Table 4.3). The ratios of fresh ovarian/fresh body

LD10–treated females than in control females, but significant differences were not observed (P=0.7611) among these three groups, i.e. controls, LD10– and LD30–treated females (Figure 6)

Fig. 6. Effect of sublethal doses of chlorfluazuron on thickness of follicular epithelium of basal oöcytes during different developmental days (5 to 9 days after pupation and newly emerged adults). For control (○), LD10 (1.00 ng larva-1) treated (□), and LD30 (3.75 ng larva-1) treated (Δ) after topical application to newly ecdysed 5th instar larvae of *Spodoptera litura*. Data were analyzed using one-way ANOVA (Concepts, 1989) at *P*<0.005 and Scheffe's *F*-test (Scheffe, 1953) at 5%. Vertical bars indicate SD (n = 9-10); (Source: Perveen and Miyata,

Topical application of sublethal doses of chlorfluazuron (LD10: 1.00 ng per larva or LD30: 3.75 ng per larva) on newly ecdysed fifth instars had an effect on the reproduction of *S. litura* by reducing fecundity, fertility, and hatchability (Perveen, 2000a). Thus, this study was conducted to establish the causes of the reduction in these parameters. It was found that topical appication of sublethal doses of chlorfuazuron had an affect on ovarian development and oögenesis by decreasing the weight of ovaries during postpupal and adult developmental days of LD10 or LD30 treated females. The basic factors responsible for the reduction in ovarian weight were reduction in the length of different parts of the ovarioles, decrease in the number of mature ova, reduction in the size of basal oöcytes and thickness of their follicular epithelium, and reduction in protein content of ovarian constituents as compared with the controls. In this study, topical application of sublethal doses of chlorfuazuron signifcantly reduced the ovarian weight to that of the controls in postpupal and adult developmental days (Figure 2; Table 4.3). The ratios of fresh ovarian/fresh body

(Perveen and Miyata, 2000).

2000).

**3.3 Discussion** 

weight, dry ovarian/fresh ovarian weight, and dry ovarian/fresh body weight were the same among the newly emerged control, LD10 or LD30 treated females, indicating a reduction in the body, fresh, and dry ovarian weights with the same degree of reduction in treated females and controls. However, Soltani and Pickens and DeMilo (1977) reported that 0.5 mg DFB, when topically applied at pupal ecdysis to *Cydia pomonella* (L.), did not cause the ovarian weight be significantly reduced (*P*<0.05) between control and treated newly emerged adult females. Nor did Hatakoshi (1992) observe any signifcant reduction (*P*<0.05) in the ovarian weight between control and treated last day of pupae to third day after adult emergence of *S. litura,* when 0.3 ng pyriproxyfen was topically applied at pupal ecdysis. The differences in results observed in these experiments may be related to the pesticides, the kind of insect used and their developmental stages. *Spodoptera litura* has paired ovaries that branch into four polytrophic meroistic ovarioles located on the ventral side of the body cavity, making several loops of ovarioles, with all basal oöcytes developing simultaneously each ovariole is differentiated into tree portions according to the developmental stages of the oöcytes: (1) the yellowish green pedicle, where fully matured ova are stored; (2) the reddish orange vitellarium, which contains the developing oöcyte and trophocyte follicles which undergo accumulation of yolk proteins, and choriogenesis; and (3) the whitis germarium, which contains oögoia, from which germ cells proliferate and follicles are formed. Similar observations were reported by Riakhel and Dhadialla (1992) and Etman and Hooper (1979), which were confirmed here.

As in other Lepidopterous species, the ovaries of *S. litura* start to differentiate, and develop at the pupal stage. Indeed, in controls, the thickness of follicular epithelium of basal oöcyte reached its maximum size on the eighth day after pupation. This coincided with the start of follicular epithelium resorption. Histological examination on *S. litura* showed that topical application of sublethal doses of chlorfuazuron to newly ecdysed ffth-instar larvae affected growth and development of oöcytes during pupal and adult stages by affecting size and thickness of follicular epithelium (Figures 5 and 6). However, in *C. pomonella,* (in controls) the basal oöcytes reached their maximum size 7 day after pupation. In this insect, this coincided with the start of follicular epithelium resorption. Hence, a 0.5-mg dose of DFB applied topically to newly ecdysed pupae affected the growth and development of oöcytes by causing a decrease in both the thickness of the follicular epithelium and size of basal oöcytes during the pupal development (Soltani and Mazouni, 1992). Lim and Lee (1982) reported that 2-d-old adult females of *O. japonica,* starved for 6 h and consumed 500 mg (AI) of DFB with two maize discs. The females were found to have retarded ovarian development, caused by a delay of oöcytes development, and an increased percentage of oöcytes resorption. This caused a decrease in fecundity and egg viability of the females. However, significant reduction was not observed either in the number of ovarioles or in the length of basal oöcytes in treated insects. Differences in these results might be a result of the use of different BPUs. Also, the doses used by Lim and Lee (1982) were very high compared with those used in present study or in the Soltani and Mazouni (1992) experiments.

In newly emerged treated adults, the germarium was much longer than the pedicle and vitelarium as compared with the controls in which the vitelarium was longer than the germarium and pedicle (Table 4). This shows that maturation of oöcytes was delayed in treated adult females as compared with the controls. The maximum thickness of the follicular epithelium of basal ooctes was observed on the 9 day after pupation in treated females, whereas it was on the 8 d after pupation in the controls (Figure 6). Subsequent to the 8th or 9th day, resorption of follicular epithelium started in control and treated females,

Chlorfluazuron as Reproductive Inhibitor 43

being enclosed in a common membrane called the scrotum. The testes lie dorsally and appear to be held in place by trachea and strands of basement membrane-like material (Amaldoss, 1989). Although reports of several layers surround the testes of lepidopteran have been made, a single capsule and follicular layers are present in *S. litura* (Amaldoss, 1989). Chase and Gilliland (1972) described the *tunica externa* and *interna* as nothing more than basement membranes over the capsule and follicular layers. The intra-follicular layer is divided into eight incomplete compartments in the tobacco leafminer, *Phthorimaea operculella*  (Zeller). This layer is similar to that in the larger canna leafroller, *Calpodes ethlius* (Stoll), and tobacco moth, *Ephestia elutella* (Hübner). It also bears the pigments responsible for the bright

A characteristic feature of the testicular follicles is the presence of large cells or a nucleated mass of protoplasm in the apex of the germarium. This is known as an apical cell or versonian cell. This is the region where there are successive stages of development of the germ cells occur. The upper part contains the primary spermatogonia and is known as the germarium. This is followed by a region called the zone of growth. The region or zone of growth is where spermatogonia multiply and usually become encysted. The maturation zone, where maturation takes place follows. Finally, is the zone of transformation where the spermatocytes develop into spermatids (spermiogenasis) completing spermatogenesis (Amaldoss, 1989). Two distinct types of spermatozoa are produced in the Lepidoptera: eupyrene (nucleated) spermatozoa which can fertilize the egg; and apyrene (anucleated) which are smaller and completely lacking in nuclear material, and do not appear to play any role in activation of the eggs (Doncaster, 1911; Goldschmidt, 1916). The eupyrene sperm can easily be counted in the male tract because they remain in bundles until they are transferred during mating, but the apyrene sperm are dispersed shortly after they leave the testis. Like eupyrene sperm, the apyrene sperm are produced in large numbers, usually contributing over half the sperm complement, and are transferred to the females with the eupyrene sperm during mating. It was thought that the apyrene sperm did not appear to play any role in activation of the eggs (Friedlander and Gitay, 1972). Their function has remained unclear ever since their discovery by Meves (1902), although several hypotheses concerning the function of the apyrene sperm have been proposed (Silberglied et al*.,* 1984). Holt and North (1970 b) proposed that, in the cabbage looper, *Trichoplusia ni* (Hüebner), apyrene sperm might aid the transport of eupyrene sperm from the male reproductive tract to the female reproductive tract. Katsuno (1977 a) reported that the apyrene sperm in the *B. mori*, might facilitate the migration of eupyrene sperm through the cellular barrier, separating the testis from the efferent ducts. Gage and Cook (1994) reported that nutritional stress seriously affected the number and size of eupyrene and apyrene sperm production in the Indian meal moth, *Plodia interpunctella* (Hübner). However, sperm development in Lepidoptera takes place in the larvae (Munson, 1906; Machida, 1929; Garbini and Imberski, 1977). Usually, spermatogenesis starts in the late larval instars and proceeds on a schedule well correlated with the insect's metamorphosis. Studies *in vitro* and *in vivo* indicated that high titre of juvenile hormone inhibits spermatogenesis, and that sperm mitosis and meiosis require sufficient ecdysteroid titre. During the post-embryonic development of eupyrene and apyrene sperm bundles, when the insect is going to pupation, the juvenile hormone titre declines (Leviatan and Friedlander, 1979). Other factors have also been reported to promote spermatogenesis *in vitro* and *in vivo* (Dumser, 1980 a). In adult males *S. litura*, both apyrene and eupyrene sperm appear in bundles in the testis, but in the vas deferens only the eupyrene sperm were still in bundles, as reported for *B. mori* (Katsuno 1977 b), *T. ni* (Holt

yellow-coloured testes. It is clear that spermatogenesis persists in the adult testis.

respectively. When ovarian maturation was scored, as depicted in Figure 4.4, a maximum number of matured oöcytes were found in the second day after adult emergence in the controls. From this day, resorption of mature oöcytes started. The chlorfuazuron-treated females showed the same pattern of mature oöcyte resorption up to the seventh day after adult emergence as in the controls. However, Hatakoshi (1992) reported that when 0-day pupae of female *S. litura* were topically treated with pyriproxyen (0.3 ng per pupa), few or no mature oöcytes were found in newly emerged females, but controls had mature oöcytes with one occasionally being resorbed. The maturation of insect eggs dependent, among other factors, on the materials taken up from the surrounding hemolymph (Telfer et al., 1981), and by materials synthesized by the ovary in situ (Indrasith et al.,1988). These materials include proteins, lipids, and carbohydrates, all of which are required for the embryogenesis (Kunkel and Nordin 1985, *Kanost* et al., 1990). Difubenzuron also caued a decrease in ovarian protein content in *C. pomonella* (Soltani and Mazouni, 1992). Decrease in the ovarian protein content suggests an interference of BPUs with vitellogenesis. It has been reported that DFB could affect ecdysteroid secretion from other organs, such as the epidermis, in *T. molitor* (Soltani, 1984), ovaries in *C. pomonella* (Soltani et al., 1989a; 1989b), and the concentration of hemolymph constituents in *T. molitor* (Soltani, 1990). Future studies should clarify the biochemical mechanism. Moreover, this work dose not clarify why signiÞcant differences were not observed (*P*<0.0001) between effects of LD10 (1.00 ng larva-1) and LD30 (3.75 ng larva-1) treated females, although LD30 dose was much higher than LD10 dose. Further studies are needed to obtain more knowledge about the effects of chlorfuazuron on oögenesis. Currently, the biochemical mechanism involved has been explored.

#### **3.4 Conclusion**

Sublethal doses of chlorfiuazuron (LD10: 1.00 ng larva-1 or LD30: 3.75 ng larva-1) topically applied on newly ecdysed fifth instars of *S. litura* significantly reduced ovarian weight and number of mature eggs in pupae and adults, compared with those of the controls. The ratios of fresh ovarian/fresh body weight, dry ovarian/fresh ovarian weight, and dry ovarian/fresh body weight were the same among controls, LD10, and LD30 treated newly emerged adults. In treated adults, the germarium was significantly longer than the pedicle and vitelarium compared with those of the controls, whereas in controls the vitelarium was significantly longer than the germarium and pedicle. This indicates a delayed of maturation of ovarioles in treated cutworms. These doses also disrupt growth and development of oöcytes by significantly affecting the size of basal oöcytes and thickness of follicular epithelium. The maximum size of basal oöcytes recorded on the day of adult emergence was significantly reduced in LD10 or LD30 treated females, compared with those of the controls. The thickness of the follicular epithelium of basal oöcytes reached to a maximum in the controls on the 8th day and in treated females on the ninth day after pupation. The effects of chlorfiuazuron on ovarian development and oögenesis are presumed to be responsible for the reduction in fecundity caused by sublethal exposure to chlorfiuazuron.

## **4. Effects of sublethal doses of chlorfluazurn on male reproductive system of**  *Spodoptera litura*

The deep yellow-coloured testes of *S. litura* are distinctly paired in larvae and they appear as a single round organ in adults. The testes of *S. litura* resemble those of other lepidopterans,

respectively. When ovarian maturation was scored, as depicted in Figure 4.4, a maximum number of matured oöcytes were found in the second day after adult emergence in the controls. From this day, resorption of mature oöcytes started. The chlorfuazuron-treated females showed the same pattern of mature oöcyte resorption up to the seventh day after adult emergence as in the controls. However, Hatakoshi (1992) reported that when 0-day pupae of female *S. litura* were topically treated with pyriproxyen (0.3 ng per pupa), few or no mature oöcytes were found in newly emerged females, but controls had mature oöcytes with one occasionally being resorbed. The maturation of insect eggs dependent, among other factors, on the materials taken up from the surrounding hemolymph (Telfer et al., 1981), and by materials synthesized by the ovary in situ (Indrasith et al.,1988). These materials include proteins, lipids, and carbohydrates, all of which are required for the embryogenesis (Kunkel and Nordin 1985, *Kanost* et al., 1990). Difubenzuron also caued a decrease in ovarian protein content in *C. pomonella* (Soltani and Mazouni, 1992). Decrease in the ovarian protein content suggests an interference of BPUs with vitellogenesis. It has been reported that DFB could affect ecdysteroid secretion from other organs, such as the epidermis, in *T. molitor* (Soltani, 1984), ovaries in *C. pomonella* (Soltani et al., 1989a; 1989b), and the concentration of hemolymph constituents in *T. molitor* (Soltani, 1990). Future studies should clarify the biochemical mechanism. Moreover, this work dose not clarify why signiÞcant differences were not observed (*P*<0.0001) between effects of LD10 (1.00 ng larva-1) and LD30 (3.75 ng larva-1) treated females, although LD30 dose was much higher than LD10 dose. Further studies are needed to obtain more knowledge about the effects of chlorfuazuron on oögenesis. Currently, the biochemical mechanism involved has been

Sublethal doses of chlorfiuazuron (LD10: 1.00 ng larva-1 or LD30: 3.75 ng larva-1) topically applied on newly ecdysed fifth instars of *S. litura* significantly reduced ovarian weight and number of mature eggs in pupae and adults, compared with those of the controls. The ratios of fresh ovarian/fresh body weight, dry ovarian/fresh ovarian weight, and dry ovarian/fresh body weight were the same among controls, LD10, and LD30 treated newly emerged adults. In treated adults, the germarium was significantly longer than the pedicle and vitelarium compared with those of the controls, whereas in controls the vitelarium was significantly longer than the germarium and pedicle. This indicates a delayed of maturation of ovarioles in treated cutworms. These doses also disrupt growth and development of oöcytes by significantly affecting the size of basal oöcytes and thickness of follicular epithelium. The maximum size of basal oöcytes recorded on the day of adult emergence was significantly reduced in LD10 or LD30 treated females, compared with those of the controls. The thickness of the follicular epithelium of basal oöcytes reached to a maximum in the controls on the 8th day and in treated females on the ninth day after pupation. The effects of chlorfiuazuron on ovarian development and oögenesis are presumed to be responsible for

**4. Effects of sublethal doses of chlorfluazurn on male reproductive system of** 

The deep yellow-coloured testes of *S. litura* are distinctly paired in larvae and they appear as a single round organ in adults. The testes of *S. litura* resemble those of other lepidopterans,

the reduction in fecundity caused by sublethal exposure to chlorfiuazuron.

explored.

**3.4 Conclusion** 

*Spodoptera litura*

being enclosed in a common membrane called the scrotum. The testes lie dorsally and appear to be held in place by trachea and strands of basement membrane-like material (Amaldoss, 1989). Although reports of several layers surround the testes of lepidopteran have been made, a single capsule and follicular layers are present in *S. litura* (Amaldoss, 1989). Chase and Gilliland (1972) described the *tunica externa* and *interna* as nothing more than basement membranes over the capsule and follicular layers. The intra-follicular layer is divided into eight incomplete compartments in the tobacco leafminer, *Phthorimaea operculella*  (Zeller). This layer is similar to that in the larger canna leafroller, *Calpodes ethlius* (Stoll), and tobacco moth, *Ephestia elutella* (Hübner). It also bears the pigments responsible for the bright yellow-coloured testes. It is clear that spermatogenesis persists in the adult testis.

A characteristic feature of the testicular follicles is the presence of large cells or a nucleated mass of protoplasm in the apex of the germarium. This is known as an apical cell or versonian cell. This is the region where there are successive stages of development of the germ cells occur. The upper part contains the primary spermatogonia and is known as the germarium. This is followed by a region called the zone of growth. The region or zone of growth is where spermatogonia multiply and usually become encysted. The maturation zone, where maturation takes place follows. Finally, is the zone of transformation where the spermatocytes develop into spermatids (spermiogenasis) completing spermatogenesis (Amaldoss, 1989). Two distinct types of spermatozoa are produced in the Lepidoptera: eupyrene (nucleated) spermatozoa which can fertilize the egg; and apyrene (anucleated) which are smaller and completely lacking in nuclear material, and do not appear to play any role in activation of the eggs (Doncaster, 1911; Goldschmidt, 1916). The eupyrene sperm can easily be counted in the male tract because they remain in bundles until they are transferred during mating, but the apyrene sperm are dispersed shortly after they leave the testis. Like eupyrene sperm, the apyrene sperm are produced in large numbers, usually contributing over half the sperm complement, and are transferred to the females with the eupyrene sperm during mating. It was thought that the apyrene sperm did not appear to play any role in activation of the eggs (Friedlander and Gitay, 1972). Their function has remained unclear ever since their discovery by Meves (1902), although several hypotheses concerning the function of the apyrene sperm have been proposed (Silberglied et al*.,* 1984). Holt and North (1970 b) proposed that, in the cabbage looper, *Trichoplusia ni* (Hüebner), apyrene sperm might aid the transport of eupyrene sperm from the male reproductive tract to the female reproductive tract. Katsuno (1977 a) reported that the apyrene sperm in the *B. mori*, might facilitate the migration of eupyrene sperm through the cellular barrier, separating the testis from the efferent ducts. Gage and Cook (1994) reported that nutritional stress seriously affected the number and size of eupyrene and apyrene sperm production in the Indian meal moth, *Plodia interpunctella* (Hübner). However, sperm development in Lepidoptera takes place in the larvae (Munson, 1906; Machida, 1929; Garbini and Imberski, 1977). Usually, spermatogenesis starts in the late larval instars and proceeds on a schedule well correlated with the insect's metamorphosis. Studies *in vitro* and *in vivo* indicated that high titre of juvenile hormone inhibits spermatogenesis, and that sperm mitosis and meiosis require sufficient ecdysteroid titre. During the post-embryonic development of eupyrene and apyrene sperm bundles, when the insect is going to pupation, the juvenile hormone titre declines (Leviatan and Friedlander, 1979). Other factors have also been reported to promote spermatogenesis *in vitro* and *in vivo* (Dumser, 1980 a). In adult males *S. litura*, both apyrene and eupyrene sperm appear in bundles in the testis, but in the vas deferens only the eupyrene sperm were still in bundles, as reported for *B. mori* (Katsuno 1977 b), *T. ni* (Holt

Chlorfluazuron as Reproductive Inhibitor 45

spermatogonia; (2) primary spermatocytes; (3) secondary spermatocytes; (4) spermatids; (5) elongated cysts with maturing sperm and (6) bundles with fully matured sperm. The length and width of sperm bundles were measured with a calibrated ocular micrometer a phase

Data were analyzed using analysis of variance, one way ANOVA (Concepts, 1989) at

The structural morphogenesis was seen in sixth-instar larvae of *S. litura* during development. When fifth-instar larvae ecdysed into newly sixth-instar larvae (N: 0 day), larvae remained unchanged for upto 2 hour. Then, they changed to a slender surface during 1st day (S). After that, they changed to being puffy during 2nd day (early-last-instar stage: P). Then, they changed to digging stage during 3rd day (mid-last-instar stage: D). Then, they changed to early burrow during 4th day (pre-late-last-instar stage: B1). After that, it changed to late burrow during 5th day (post-late-last-instar stage: B2). The morphogenesis for phase variations used for convenient of observations during experiments is given in Table 6. The morphology of the adult male reproductive system of *S. litura* is shown in Figure 7 (Perveen,

> Name of the phasesa

stage) 3rd

early burrowed (pre late last instar stage) 4th

late burrowed (post late last instar stage) 5th

aSymbols for phase variations observed during developmental days of sixth-instar larvae were used for

Table 6. Structural morphogenesis during five developmental days of sixth-instar larvae of

0 N newly ecdysed 0–2 1 S slender surface 1st 2 P puffy (early last instar stage) 2nd

3 D digging (mid last instar

Duration (hour day-1)

contrast microscope at 400magnification (Perveen, 2000b).

*P*<0.0001 and Scheffe's *F*-test (Scheffe, 1953) at 5%.

Symbols for phase variationsa

**4.1.3 Data analysis** 

**4.2 Results** 

2000b).

Developmental category (days)

4 B1

5 B2

the convenience of observations.

*Spodoptera litura* (Source: Perveen, 2005).

and North, 1970 a) and the army worm, *Pseudaletia separata* (Walk.) (He, 1994). The testes of early larvae contain a large number of spermatogonial cells near the outer border of the follicles. There is a preponderance of spermatocytes containing primary spermatocytes during the penultimate and early last-instar larvae. Secondary spermatocytes are present in the early last-instar larvae, persisting through to the middle of the last-instar larvae. They then begin to differentiate into spermatids. In the process of elongation and maturation of spermatids, the spermatocytes assume an elliptical shape. The sperm bundles formed as a result of maturation of the spermatids are seen abundantly in adults. Spermiogenesis is, however, not synchronous, and spermatozoa in various stages of differentiation can be detected in the testes of freshly emerged adults (Sridevi et al*.,* 1989a). In the pupa, the apyrene sperm bundles emerge from the testicular follicle into the vas efferens earlier than the eupyrene sperm bundles and the bundles separate when they pass through the basement membrane of the testis (Katsuno, 1977b). The apyrene spermatozoa migrate from the vas efferens into the seminal vesicle through the vas deferens during the pupa (Katsuno, 1977c). In the post-pupal period, however, the eupyrene sperm bundles and apyrene spermatozoa migrate simultaneously through the same way (Katsuno, 1977d). The effects of chlorfluazuron have been examined on male reproductive system during testicular development and spermatogenesis when sublethal doses have been topically applied to newly ecdysed fifth-instar larvae of *S. litura*.

#### **4.1 Experimental procedure 4.1.1 Histology of testis**

Testes from newly molted sixth instar larvae to 5-day-old virgin adult males (treated and control), were dissected in 0.9% of NaCl under a binocular microscope. The length and width of each testis were measured by the same procedure as used for the oöcytes measurement. Testis volume was calculated for larval testes using the formula 4/3π (lengthwidth2), assuming that the testis is a prolate spheroid (Loeb et al*.,* 1984). For the fused pupal and adult testes, the formula 4/3πr3 was used, with r as the radius of the globular gonad. The treated and control weight and sheaths thickness of testes were measured by the same procedure used for the ovaries as described above (Perveen, 2000b). The thickness of treated and control testes sheaths or vas deferenti of untreated and treated relevant stages of insect was observed by making a parafilm microtomy conducted according to the method used by Yoshida (1994) and the procedure to stain the nuclei of

sperm was adapted from the method by He (1994) (Perveen, 2000b).

#### **4.1.2 Spermatogenesis**

A staining method was used for determining number of the cysts, eupyrene and apyrene sperm for treated and control (He et al., 1995). First, the testis was transferred to a microscopic grid slide (each square= 1mm2) and crushed until it was evenly distributed on the slide. Secondly, several drops of methanol-acetic acid solution (3 : 1; v/v) were added to the slide to fix the reparation for 15 min, and the excess fixing solution was absorbed with filter paper. Third, several drops of 2-5% Giemsa solution dissolved in Sorensen-Gomori buffer solution (monobasic and dibasic sodium phosphate, 0.07 M, pH 6.8) were added to the slide to stain the preparation for 10-30 min. The slide was washed with water and air dried after staining. Finally, the air-dried preparation was observed for counting of bundles and cysts under a phase contrast microscope at 20mignification. Cysts were classified into the following six developmental stages as described by Chaudhury and Raun (1966): (1) spermatogonia; (2) primary spermatocytes; (3) secondary spermatocytes; (4) spermatids; (5) elongated cysts with maturing sperm and (6) bundles with fully matured sperm. The length and width of sperm bundles were measured with a calibrated ocular micrometer a phase contrast microscope at 400magnification (Perveen, 2000b).

## **4.1.3 Data analysis**

Data were analyzed using analysis of variance, one way ANOVA (Concepts, 1989) at *P*<0.0001 and Scheffe's *F*-test (Scheffe, 1953) at 5%.

## **4.2 Results**

44 Insecticides – Pest Engineering

and North, 1970 a) and the army worm, *Pseudaletia separata* (Walk.) (He, 1994). The testes of early larvae contain a large number of spermatogonial cells near the outer border of the follicles. There is a preponderance of spermatocytes containing primary spermatocytes during the penultimate and early last-instar larvae. Secondary spermatocytes are present in the early last-instar larvae, persisting through to the middle of the last-instar larvae. They then begin to differentiate into spermatids. In the process of elongation and maturation of spermatids, the spermatocytes assume an elliptical shape. The sperm bundles formed as a result of maturation of the spermatids are seen abundantly in adults. Spermiogenesis is, however, not synchronous, and spermatozoa in various stages of differentiation can be detected in the testes of freshly emerged adults (Sridevi et al*.,* 1989a). In the pupa, the apyrene sperm bundles emerge from the testicular follicle into the vas efferens earlier than the eupyrene sperm bundles and the bundles separate when they pass through the basement membrane of the testis (Katsuno, 1977b). The apyrene spermatozoa migrate from the vas efferens into the seminal vesicle through the vas deferens during the pupa (Katsuno, 1977c). In the post-pupal period, however, the eupyrene sperm bundles and apyrene spermatozoa migrate simultaneously through the same way (Katsuno, 1977d). The effects of chlorfluazuron have been examined on male reproductive system during testicular development and spermatogenesis when sublethal doses have been topically applied to

Testes from newly molted sixth instar larvae to 5-day-old virgin adult males (treated and control), were dissected in 0.9% of NaCl under a binocular microscope. The length and width of each testis were measured by the same procedure as used for the oöcytes measurement. Testis volume was calculated for larval testes using the formula 4/3π (lengthwidth2), assuming that the testis is a prolate spheroid (Loeb et al*.,* 1984). For the fused pupal and adult testes, the formula 4/3πr3 was used, with r as the radius of the globular gonad. The treated and control weight and sheaths thickness of testes were measured by the same procedure used for the ovaries as described above (Perveen, 2000b). The thickness of treated and control testes sheaths or vas deferenti of untreated and treated relevant stages of insect was observed by making a parafilm microtomy conducted according to the method used by Yoshida (1994) and the procedure to stain the nuclei of

A staining method was used for determining number of the cysts, eupyrene and apyrene sperm for treated and control (He et al., 1995). First, the testis was transferred to a microscopic grid slide (each square= 1mm2) and crushed until it was evenly distributed on the slide. Secondly, several drops of methanol-acetic acid solution (3 : 1; v/v) were added to the slide to fix the reparation for 15 min, and the excess fixing solution was absorbed with filter paper. Third, several drops of 2-5% Giemsa solution dissolved in Sorensen-Gomori buffer solution (monobasic and dibasic sodium phosphate, 0.07 M, pH 6.8) were added to the slide to stain the preparation for 10-30 min. The slide was washed with water and air dried after staining. Finally, the air-dried preparation was observed for counting of bundles and cysts under a phase contrast microscope at 20mignification. Cysts were classified into the following six developmental stages as described by Chaudhury and Raun (1966): (1)

sperm was adapted from the method by He (1994) (Perveen, 2000b).

newly ecdysed fifth-instar larvae of *S. litura*.

**4.1 Experimental procedure 4.1.1 Histology of testis** 

**4.1.2 Spermatogenesis** 

The structural morphogenesis was seen in sixth-instar larvae of *S. litura* during development. When fifth-instar larvae ecdysed into newly sixth-instar larvae (N: 0 day), larvae remained unchanged for upto 2 hour. Then, they changed to a slender surface during 1st day (S). After that, they changed to being puffy during 2nd day (early-last-instar stage: P). Then, they changed to digging stage during 3rd day (mid-last-instar stage: D). Then, they changed to early burrow during 4th day (pre-late-last-instar stage: B1). After that, it changed to late burrow during 5th day (post-late-last-instar stage: B2). The morphogenesis for phase variations used for convenient of observations during experiments is given in Table 6. The morphology of the adult male reproductive system of *S. litura* is shown in Figure 7 (Perveen, 2000b).


aSymbols for phase variations observed during developmental days of sixth-instar larvae were used for the convenience of observations.

Table 6. Structural morphogenesis during five developmental days of sixth-instar larvae of *Spodoptera litura* (Source: Perveen, 2005).

Chlorfluazuron as Reproductive Inhibitor 47

compared with the controls. The weight and size of testes were significantly reduced (P<0.001) in the LD10-treated and more significantly reduced (P<0.0001) in LD30-treated males compared with the controls from newly ecdysed sixth-instar larvae to the 5th day after adult emergence. Testes reached their maximum size in treated males (LD10: 4.161.54 mm3 and 8.00.83 mg; LD30: 2.791.00 mm3 and 4.881.05 mg; n= 10, respectively) on the same day as the controls. The patterns of development of the testes with respect to the volume and weight were the similar in the controls and the LD10- or LD30-treated males (Figures 8a

considered as testes pair equivalent; (Source: Perveen, 2000b).

Fig. 8. Effects of sublethal doses of chlorfluazuron (LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1) on the testis volume (a) and weight (b) of *Spodoptera litura* during newly ecdysed sixth-instar larvae to 5th day after adult emergence; controls: O; LD10: □; LD30: Δ; data analyzed using one-way ANOVA (Concepts, 1989) at *P*<0.0001 and Scheffe`s *F*-test (Scheffe, 1953) at 5%; vertical bars: SD; N, S, P and D: larval (Table 5.1), P: pupal and A: adult developmental days; n = 10 for each point; paired larval testes and fused single pupal or adult testis were

The thickness of the testis sheath gradually and then sharply increased until the 4th day of moulting of sixth-instar larvae. It decreased when the two larval testes fused on the 5th day (last-day) of the sixth-instar larvae; it again increased and reached to a maximum [(60.51)10-2 mm; n=10] on the 0 day of pupation and gradually declined in the newly emerged adults. The thickness remained constant until the 2nd day after adult emergence. Sublethal doses rapidly disrupted the development of testis by significantly decreasing (P<0.0001) the thickness of the testes sheath as compared with that of the controls. This reduction occurred from newly ecdysed sixth-instar larvae to the 0 day of pupation in the LD10-treated and the 1st day after pupation in the LD30-treated males. The thickness of the

and b) (Perveen, 2000b).

Fig. 7. The morphology of the male reproductive system of *Spodoptera litura*: A: testis; B: seminal vesicle (paired); C: vas deferens (paired); D: accessory glands (paired); E: ductus ejaculatorious duplex; F: primary segment of ductus ejaculatorious simplex; G: muascular area; H: area of frenum formation; and I: area of collum formation of the cuticular secondary segment of the ductus ejaculatorious simplex; J: caecum of aedeagus; K: aedeagus (Source: Etman and Hooper, 1979).

#### **4.2.1 Effects on testicular development**

The testes of *S. litura* show the three dimentional measureable structure. Each mature testis consists of four follicles or lobes, each separated by an inner layer of sheath cells. An outer sheath cell layer further surrounds all follicles. In sixth-instar larvae, the volume and weight of the testes gradually then rather sharply increased until the 4th day of sixth-instar larvae. The volume weight decreased when two larval testes fused on the 5th day (last day) after moulting of sixth-instar larvae. They again sharply increased in size, reached a maximum (6.211.31 mm3 and 11.940.42 mg, n=30, respectively) on the 0 day of pupation and gradually declined until the 5th day after adult emergence. Sublethal doses of chlorfluazuron rapidly disrupted the development of testes by decreasing the volume and weight of testes

Fig. 7. The morphology of the male reproductive system of *Spodoptera litura*: A: testis; B: seminal vesicle (paired); C: vas deferens (paired); D: accessory glands (paired); E: ductus ejaculatorious duplex; F: primary segment of ductus ejaculatorious simplex; G: muascular area; H: area of frenum formation; and I: area of collum formation of the cuticular secondary segment of the ductus ejaculatorious simplex; J: caecum of aedeagus; K: aedeagus (Source:

The testes of *S. litura* show the three dimentional measureable structure. Each mature testis consists of four follicles or lobes, each separated by an inner layer of sheath cells. An outer sheath cell layer further surrounds all follicles. In sixth-instar larvae, the volume and weight of the testes gradually then rather sharply increased until the 4th day of sixth-instar larvae. The volume weight decreased when two larval testes fused on the 5th day (last day) after moulting of sixth-instar larvae. They again sharply increased in size, reached a maximum (6.211.31 mm3 and 11.940.42 mg, n=30, respectively) on the 0 day of pupation and gradually declined until the 5th day after adult emergence. Sublethal doses of chlorfluazuron rapidly disrupted the development of testes by decreasing the volume and weight of testes

Etman and Hooper, 1979).

**4.2.1 Effects on testicular development** 

compared with the controls. The weight and size of testes were significantly reduced (P<0.001) in the LD10-treated and more significantly reduced (P<0.0001) in LD30-treated males compared with the controls from newly ecdysed sixth-instar larvae to the 5th day after adult emergence. Testes reached their maximum size in treated males (LD10: 4.161.54 mm3 and 8.00.83 mg; LD30: 2.791.00 mm3 and 4.881.05 mg; n= 10, respectively) on the same day as the controls. The patterns of development of the testes with respect to the volume and weight were the similar in the controls and the LD10- or LD30-treated males (Figures 8a and b) (Perveen, 2000b).

Fig. 8. Effects of sublethal doses of chlorfluazuron (LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1) on the testis volume (a) and weight (b) of *Spodoptera litura* during newly ecdysed sixth-instar larvae to 5th day after adult emergence; controls: O; LD10: □; LD30: Δ; data analyzed using one-way ANOVA (Concepts, 1989) at *P*<0.0001 and Scheffe`s *F*-test (Scheffe, 1953) at 5%; vertical bars: SD; N, S, P and D: larval (Table 5.1), P: pupal and A: adult developmental days; n = 10 for each point; paired larval testes and fused single pupal or adult testis were considered as testes pair equivalent; (Source: Perveen, 2000b).

The thickness of the testis sheath gradually and then sharply increased until the 4th day of moulting of sixth-instar larvae. It decreased when the two larval testes fused on the 5th day (last-day) of the sixth-instar larvae; it again increased and reached to a maximum [(60.51)10-2 mm; n=10] on the 0 day of pupation and gradually declined in the newly emerged adults. The thickness remained constant until the 2nd day after adult emergence. Sublethal doses rapidly disrupted the development of testis by significantly decreasing (P<0.0001) the thickness of the testes sheath as compared with that of the controls. This reduction occurred from newly ecdysed sixth-instar larvae to the 0 day of pupation in the LD10-treated and the 1st day after pupation in the LD30-treated males. The thickness of the

Chlorfluazuron as Reproductive Inhibitor 49

\*SSb (MSD)

1st C 13 244718a 489335a 139718a 0.00.0 0.00.0 0.00.0 LD10 11 19607b 39207b 11206b 0.00.0 0.00.0 0.00.0 LD30 10 13206c 26415b 7544c 0.00.0 0.00.0 0.00.0 3rd C 13 13118a 42809a 31468a 0.00.0 0.00.0 0.00.0 LD10 11 10505b 34307b 25207b 0.00.0 0.00.0 0.00.0 LD30 10 7075c 23117c 16897c 0.00.0 0.00.0 0.00.0 5th C 13 4378a 35745a 47177a 0.00.0 0.00.0 0.00.0 LD10 11 3425b 28707b 37806b 0.00.0 0.00.0 0.00.0 LD30 10 2246c 19338c 25469c 0.00.0 0.00.0 0.00.0

aTS: treatment stages; T: treatments; n: number of males used; C: control; LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; Sg: spermatogonia; PS: primary spermatocytes; SS: secondary spermatocytes; St: spermatids; ECMS: elongated cysts with mature sperm; ESB: eupyrene sperm bundles; larvae ecdysed usually

bData were analyzed using one-way ANOVA (Concepts, 1989) at P<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s F-test (Scheffe, 1953) at 5%.

Table 7. Effect of sublethal doses of chlorfluazuron on spermatogenesis in the testes of sixthinstar larvae during 1st, 3rd and 5th days of development after topical application to newly

When different developmental stages of the cysts were observed in testis during spermatogenesis in newly ecdysed pupae, on the 5th and 10th day after pupation, the number of spermatogonia, primary and secondary spermatocytes, were decreased. However, the spermatids and elongated cysts with mature sperm gradually increased in controls. Eupyrene sperm bundles were not present on the 0-5 day-old pupae. However, they were found on the 10th day after pupation (mean: 10023.0 numbers). The pattern of spermatogenesis was the same in the controls, LD10- and LD30-treated male pupae. However, the developmental stages of the sperm were significantly reduced (P<0.001) in the LD10-treated and even more significantly reduced (P<0.0001) in the LD30-treated males

Different developmental stages of cysts were observed in testes during spermatogenesis in newly emerged, 1 and 2 day-old adults. The spermatogonia were not present in newly emerged and 1 day-old adults. Primary spermatocytes were not found in 1 and 2 day-old adults, but they (mean: 1744.0 number) were found in newly emerged adults. The secondary spermatocytes, spermatids and elongated cysts with mature sperm were present, but gradually decreased in number in the controls. Eupyrene sperm bundles gradually increased in number in the controls. The pattern of spermatogenesis was the same in the controls, LD10- and LD30-treated male pupae. However, the stages of sperm development were significantly reduced (P<0.001) in the LD10- and even more significantly reduced (P<0.0001) in LD30-treated males as compared with the controls (Table 9) (Perveen, 2000b).

St (MSD) ECMS (MSD) ESB (MSD)

TSa Ta na \*Sgb

(MSD)

during 0200 to 0800 hour and collected between 0800 to 1000 hour

compared with the controls (Table 8) (Perveen, 2000b).

ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b).

\*PSb (MSD)

testes sheath in chlorfluazuron-treated males reached a maximum [(5.90.67)10-2 mm] in the LD10-treated males on the 1st day and [(5.91.1)10-2 mm] in the LD30-treated males on the 2nd day after pupation whereas, in the controls, it was on the 0 day of pupation. This result shows that attainment of the maximum thickness of the testes sheath was delayed by one day in LD10- and by two days in LD30-treated males compared with the controls. However, no significant reduction was observed in the maximum thickness of the testes sheath among the control and LD10- or LD30-treated males. The developmental pattern of the testes sheath in the LD10- or LD30-treated males was similar to that of the controls (Figure 9) (Perveen, 2000b).

Fig. 9. Effects of sublethal doses of chlorfluazuron (LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1) on the thickness of testis sheath of *Spodoptera litura* during newly ecdysed sixth-instar larvae to 5th day after adult emergence; controls: O; LD10: □; LD30: Δ; data analyzed using one-way ANOVA (Concepts, 1989) at *P*<0.0001 and Scheffe`s *F*-test (Scheffe, 1953) at 5%; vertical bars: SD; S, P, D and B1: larval (Table 5.1), P: pupal and A: adult developmental days; n = 10 for each point; paired larval testes and fused single pupal or adult testis were considered as a testes pair equivalent; (Source: Perveen, 2000b).

#### **4.2.2 Effects on spermatogenesis**

When the different developmental stages of cysts were observed in testes during spermatogenesis on the 1, 3 and 5 day-old sixth-instar larvae, the number of spermatogonia, primary and secondary spermatocytes was significantly reduced (P<0.001) in the LD10- and even more significantly reduced (P<0.0001) in the LD30-treated males as compared with those of controls. Spermatids, elongated cysts with mature sperm and eupyrene sperm bundles were not found in the controls and LD10- or LD30-treated larval testes (Table 7) (Perveen, 2000b).

testes sheath in chlorfluazuron-treated males reached a maximum [(5.90.67)10-2 mm] in the LD10-treated males on the 1st day and [(5.91.1)10-2 mm] in the LD30-treated males on the 2nd day after pupation whereas, in the controls, it was on the 0 day of pupation. This result shows that attainment of the maximum thickness of the testes sheath was delayed by one day in LD10- and by two days in LD30-treated males compared with the controls. However, no significant reduction was observed in the maximum thickness of the testes sheath among the control and LD10- or LD30-treated males. The developmental pattern of the testes sheath in the LD10- or LD30-treated males was similar to that of the controls (Figure 9)

Fig. 9. Effects of sublethal doses of chlorfluazuron (LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1) on the thickness of testis sheath of *Spodoptera litura* during newly ecdysed sixth-instar larvae to 5th day after adult emergence; controls: O; LD10: □; LD30: Δ; data analyzed using one-way ANOVA (Concepts, 1989) at *P*<0.0001 and Scheffe`s *F*-test (Scheffe, 1953) at 5%; vertical bars: SD; S, P, D and B1: larval (Table 5.1), P: pupal and A: adult developmental days; n = 10 for each point; paired larval testes and fused single pupal or adult testis were considered as

When the different developmental stages of cysts were observed in testes during spermatogenesis on the 1, 3 and 5 day-old sixth-instar larvae, the number of spermatogonia, primary and secondary spermatocytes was significantly reduced (P<0.001) in the LD10- and even more significantly reduced (P<0.0001) in the LD30-treated males as compared with those of controls. Spermatids, elongated cysts with mature sperm and eupyrene sperm bundles were not found in the controls and LD10- or LD30-treated larval testes (Table 7)

a testes pair equivalent; (Source: Perveen, 2000b).

**4.2.2 Effects on spermatogenesis** 

(Perveen, 2000b).

(Perveen, 2000b).


aTS: treatment stages; T: treatments; n: number of males used; C: control; LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; Sg: spermatogonia; PS: primary spermatocytes; SS: secondary spermatocytes; St: spermatids; ECMS: elongated cysts with mature sperm; ESB: eupyrene sperm bundles; larvae ecdysed usually during 0200 to 0800 hour and collected between 0800 to 1000 hour

bData were analyzed using one-way ANOVA (Concepts, 1989) at P<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s F-test (Scheffe, 1953) at 5%.

Table 7. Effect of sublethal doses of chlorfluazuron on spermatogenesis in the testes of sixthinstar larvae during 1st, 3rd and 5th days of development after topical application to newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b).

When different developmental stages of the cysts were observed in testis during spermatogenesis in newly ecdysed pupae, on the 5th and 10th day after pupation, the number of spermatogonia, primary and secondary spermatocytes, were decreased. However, the spermatids and elongated cysts with mature sperm gradually increased in controls. Eupyrene sperm bundles were not present on the 0-5 day-old pupae. However, they were found on the 10th day after pupation (mean: 10023.0 numbers). The pattern of spermatogenesis was the same in the controls, LD10- and LD30-treated male pupae. However, the developmental stages of the sperm were significantly reduced (P<0.001) in the LD10-treated and even more significantly reduced (P<0.0001) in the LD30-treated males compared with the controls (Table 8) (Perveen, 2000b).

Different developmental stages of cysts were observed in testes during spermatogenesis in newly emerged, 1 and 2 day-old adults. The spermatogonia were not present in newly emerged and 1 day-old adults. Primary spermatocytes were not found in 1 and 2 day-old adults, but they (mean: 1744.0 number) were found in newly emerged adults. The secondary spermatocytes, spermatids and elongated cysts with mature sperm were present, but gradually decreased in number in the controls. Eupyrene sperm bundles gradually increased in number in the controls. The pattern of spermatogenesis was the same in the controls, LD10- and LD30-treated male pupae. However, the stages of sperm development were significantly reduced (P<0.001) in the LD10- and even more significantly reduced (P<0.0001) in LD30-treated males as compared with the controls (Table 9) (Perveen, 2000b).

Chlorfluazuron as Reproductive Inhibitor 51

(P<0.0001) in LD30- treated adults compared with the controls. When males were treated with the LD10 or LD30, the ratio of eupyrene to apyrene sperm bundles was not significantly changed; apyrene sperm bundle comprised about half of the total sperm complement (Table

Control 30 4893546a 4697520a 51.1:48.9 LD10 30 3920426b 3763466b 51.0:48.9 LD30 30 2641161c 2386271c 52.5:47.5 aLD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; T: treatments; n: number of males used; NESB: number of eupyrene sperm bundle; NASB: number of apyrene sperm bundle; ESB: eupyrene sperm bundle; ASB:

bData were analyzed using one-way ANOVA (Concepts, 1989) at P<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s F-test (Scheffe, 1953) at 5%. Table 10. Effect of sublethal doses of chlorfluazuron and comparison of the number of eupyrene and apyrene sperm bundles in the testis of newly emerged unmated adults after topical application to newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen,

Developmental Ta na Size (dm in m)

Spermatogonia (S) Control 10 7.10.7a 6–8

stages of sperm (MSD)b ranges (min–max)

 LD10 10 5.90.8b 5–7 LD30 10 4.10.9c 3–5 Primary Control 10 15.10.7a 14–16 spermatocytes (D) LD10 10 13.01.3b 11–14

 LD30 10 11.41.5c 10–48 Secondry Control 10 31.01.8a 28–33 spermatocyte (B2) LD10 10 29.21.6b 27–31

LD30 10 25.01.2c 24–27

 LD10 10 2.90.87b 2–4 LD30 10 1.90.7c 1–3

aLD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; T: treatments; n: number of males used; for S, D and B2 refer to Table 5.1; P10: ten day old pupae bData were analyzed using one-way ANOVA (Concepts, 1989) at *<sup>P</sup>*<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s *F*-test (Scheffe, 1953) at 5%. Table 11. Effect of sublethal doses of chlorfluazuron on the size of various developmental stages of sperm observed in the testes of sixth-instar larvae and puape after topical

application to newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b).

Spermatids (P10) Control 10 4.10.9a 3–5

NASBb (MSD) Ratios (%)= ESB:ASB

10).

apyrene sperm bundle

2000b).

Ta na NESBb

(MSD)


aTS: treatment stages; T: treatments; n: number of males used; C: control; LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; Sg: spermatogonia; PS: primary spermatocytes; SS: secondary spermatocytes; St: spermatids; ECMS: elongated cysts with mature sperm; ESB: eupyrene sperm bundles; pupation occurred usually during 0200 to 0800 hour and collected between 0800 to 1000 hour.

bData were analyzed using one-way ANOVA (Concepts, 1989) at P<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s F-test (Scheffe, 1953) at 5%.

Table 8. Effect of sublethal doses of chlorfluazuron on spermatogenesis in the testes of pupae during 1st, 5th and 10th days of development after topical application to newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b).


aTS: treatment stages; T: treatments; n: number of males used; C: control; LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; M: mean; Sg.: spermatogonia; PS: primary spermatocytes; SS: secondary spermatocytes; St.: spermatids; ECMS: elongated cysts with mature sperm; ESB: eupyrene sperm bundles; adults emerged usually between 2300 to 0200 hour and and between collected 0800 to 1000 hour. bData were analyzed using 1-way ANOVA (Concepts, 1989) at P<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s F-test (Scheffe, 1953) at 5%.

Table 9. Effect of sublethal doses of chlorfluazuron on spermatogenesis in the testes of adults during 0, 1st and 2nd day of development after topical application to newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b).

In the testis of newly emerged LD10- treated adults, the number of eupyrene and apyrene sperm bundles was significantly reduced (P<0.001), and even more significantly reduced

1st C 13 2626ac 17475a 27107a 36706a 3498a 0.00.0 LD10 11 2056b 14004b 21706b 29407b 2805b 0.00.0 LD30 10 1425c 9436b 14625c 19804c 189 5c 0.00.0 3rd C 13 1757a 10478a 20104a 48055a 69711a 0.00.0 LD10 11 1404b 8405b 16103b 38055b 5605b 0.00.0 LD30 10 945c 5666c 10853c 25937c 3774c 0.00.0 5th C 13 864a 4365a 6983a 29696a 35813a 0.00.0 LD10 11 703b 3504b 5606b 26604b 33393b 0.00.0 LD30 10 474c 2363c 3775c 17923c 22634c 0.00.0 aTS: treatment stages; T: treatments; n: number of males used; C: control; LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; Sg: spermatogonia; PS: primary spermatocytes; SS: secondary spermatocytes; St: spermatids; ECMS: elongated cysts with mature sperm; ESB: eupyrene sperm bundles; pupation occurred usually

bData were analyzed using one-way ANOVA (Concepts, 1989) at P<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s F-test (Scheffe, 1953) at 5%. Table 8. Effect of sublethal doses of chlorfluazuron on spermatogenesis in the testes of pupae during 1st, 5th and 10th days of development after topical application to newly

> SSb (MSD)

0 C 13 00a 1744a 5243a 10484a 20975a 48933a LD10 11 353b 1407b 3853b 8403b 16806b 39204b LD30 10 242c 943b 2592c 5666c 11323c 26412c 1st C 13 00a 00a 1746a 6113a 10476a 69025a LD10 11 143b 283b 981b 4905b 8403b 55304b LD30 10 92c 192c 662c 3304c 5662c 37285c 2nd C 13 0.00.0 0.00.0 433a 2194a 4376a 80376a LD10 11 0.00.0 0.00.0 353b 1753b 3505b 64405b LD30 10 0.00.0 0.00.0 242c 1184c 2364c 43384c aTS: treatment stages; T: treatments; n: number of males used; C: control; LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; M: mean; Sg.: spermatogonia; PS: primary spermatocytes; SS: secondary spermatocytes; St.: spermatids; ECMS: elongated cysts with mature sperm; ESB: eupyrene sperm bundles; adults emerged

St (MSD)

ECMS (MSD)

ESB (M SD)

SSb (MSD)

St (MSD)

ECMS (MSD)

ESB (MSD)

PSb (MSD)

TSa Ta na Sgb

TSa Ta na Sgb

(MSD)

during 0200 to 0800 hour and collected between 0800 to 1000 hour.

(MSD)

ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b).

usually between 2300 to 0200 hour and and between collected 0800 to 1000 hour.

fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b).

bData were analyzed using 1-way ANOVA (Concepts, 1989) at P<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s F-test (Scheffe, 1953) at 5%. Table 9. Effect of sublethal doses of chlorfluazuron on spermatogenesis in the testes of adults during 0, 1st and 2nd day of development after topical application to newly ecdysed

In the testis of newly emerged LD10- treated adults, the number of eupyrene and apyrene sperm bundles was significantly reduced (P<0.001), and even more significantly reduced

PSb (MSD)



aLD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; T: treatments; n: number of males used; NESB: number of eupyrene sperm bundle; NASB: number of apyrene sperm bundle; ESB: eupyrene sperm bundle; ASB: apyrene sperm bundle

bData were analyzed using one-way ANOVA (Concepts, 1989) at P<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s F-test (Scheffe, 1953) at 5%.

Table 10. Effect of sublethal doses of chlorfluazuron and comparison of the number of eupyrene and apyrene sperm bundles in the testis of newly emerged unmated adults after topical application to newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b).


aLD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; T: treatments; n: number of males used; for S, D and B2 refer

to Table 5.1; P10: ten day old pupae bData were analyzed using one-way ANOVA (Concepts, 1989) at *<sup>P</sup>*<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s *F*-test (Scheffe, 1953) at 5%.

Table 11. Effect of sublethal doses of chlorfluazuron on the size of various developmental stages of sperm observed in the testes of sixth-instar larvae and puape after topical application to newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b).

Chlorfluazuron as Reproductive Inhibitor 53

In the vas deferens of male pre-adult controls, the mean number of eupyrene sperm bundles was 10229, but no sperm bundles observed in the LD10- or LD30-treated males of the same

In newly emerged control males, the mean number of eupyrene sperm bundles was 1002116, and in LD10-treated adult males, 23 4.9. In LD30-treated adults male there was no sperm bundles were observed. Moreover, in 1 day-old LD10-treated adult males, the number of eupyrene sperm bundles was significantly (P<0.001) reduced and more significantly (P<0.0001) reduced in LD30-treated males compared with the controls (Table 14) (Perveen,

In the testis and vas deferens of newly emerged, LD10-treated males, the total number of eupyrene sperm bundles was significantly reduced (P<0.001) and more significantly reduced (P<0.0001) in LD30-treated males had no sperm bundles in the vas deferens

<sup>l</sup>30 4893546a 1002116a 5791640a LD10 30 3920426b 234.9b 3943425b LD30 30 2641161c 00c 2641161c aLD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; T: treatments; n: number of males used; NESB: number of eupyrene sperm bundle; NASB: number of apyrene sperm bundle; ESB: eupyrene sperm bundle; ASB: apyrene sperm bundle; TNESB: total number of eupyrene sperm bundle bData were analyzed using one-way ANOVA (Concepts, 1989) at P<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s *F*-test (Scheffe, 1953) at 5%.

Table 14. Effect of sublethal doses of chlorfluazuron on the total number of eupyrene sperm bundles in the testis and vas deferens of newly emerged adults after topical application to

Topical application of sublethal doses of chlorfuazuron (LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1) has an effect on reproduction of *S. litura* by reducing the fecundity, fertilityand hatchability. Fecundity was reduced to a similar degree (35±44%) when females, males or both sexes were treated with LD10 or LD30. Fertility was reduced by 42% or 52% when females were treated with LD10 or LD30, respectively, and by 60% or 63%, respectively, when males or both sexes were treated with LD10. Fertility was reduced by 78% or 80% when males or both sexes were treated with LD30. The hatchability was reduced by 20% or 23% when females were treated with LD10 or LD30, respectively, and by 37% or 39%, respectively, when males or both sexes were treated with LD30. Hatchability was reduced by 55% or 56% when males or both sexes were treated with LD30 (Perveen, 2000a). Thus, this study was conducted to determine the causes of the reduction in these reproductive parameters. Topical application of sublethal doses of chlorfuazuron has an effect on testis development by decreasing the volume and weight of testes and its sheath thickness, and also on spermatogenesis by decreasing the number of cysts, eupyrene and apyrene bundles during different times in development. Reduction in the testes volume and weight might be caused

newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b)

NASB in vasa deferensa,b (MSD)

TNESB (testis+ vasa deferens)a,b (MSD)

age (Table 13) (Perveen, 2000b).

compared with the controls (Table 14) (Perveen, 2000b).

(MSD)

Ta na NESB in testisa,b

2000b).

Contro

**4.3 Discussion** 

The size of the spermatogonia, primary spermatocytes, secondary spermatocytes and spermatids was significantly reduced (P<0.001) in LD10-treated and more significantly reduced (P<0.0001) in LD30-treated insects compared with the controls (Table 11) in newly emerged adults (Perveen, 2000b).

In newly emerged adults, the width and length of elongated cysts with mature sperm, eupyrene and apyrene sperm bundles were significantly reduced (P<0.001) in LD10- and more significantly reduced (P<0.0001) in LD30- treated insects compared with the controls (Table 12) (Perveen, 2000b).


aLD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; T: treatments; n: number of males used bData were analyzed using one-way ANOVA (Concepts, 1989) at *<sup>P</sup>*<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s *F*-test (Scheffe, 1953) at 5%.

Table 12. Effect of sublethal doses of chlorfluazuron on size of various developmental stages of sperm observed in the testes of newly emerged adults after topical application to newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b).


aLD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; T: treatments; n: number of males used bData were analyzed using one-way ANOVA (Concepts, 1989) at *<sup>P</sup>*<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s *F*-test (Scheffe, 1953) at 5%.

Table 13. Effect of sublethal doses of chlorfluazuron on the number of eupyrene sperm bundles in the vas deferens during different developmental days of adults after topical application to newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b)

The size of the spermatogonia, primary spermatocytes, secondary spermatocytes and spermatids was significantly reduced (P<0.001) in LD10-treated and more significantly reduced (P<0.0001) in LD30-treated insects compared with the controls (Table 11) in newly

In newly emerged adults, the width and length of elongated cysts with mature sperm, eupyrene and apyrene sperm bundles were significantly reduced (P<0.001) in LD10- and more significantly reduced (P<0.0001) in LD30- treated insects compared with the controls

Developmental Ta na length (m)b width (m)b

stages of sperm MSD Ranges

sizes of various developmental stages

Elongated Control 10 45.52.4a (42–50) 40.02.0a (35–42) spermatocytes LD10 10 42.21.7b (40–44) 37.21.3b (35–39) LD30 10 40.00.8c (39–41) 35.21.6c (34–38)

Eupyrene sperm Control 10 98.04.4a (92–105) 33.21.9a (30–36) bundles LD10 10 94.71.4b (93–97) 31.31.4b (29–33) LD30 10 91.51.0c (90–93) 29.11.2c (28–31)

Apyrene sperm Control 10 24.91.2a (23–27) 16.11.7a (14–19) bundles LD10 10 94.71.4b (21–25) 14.21.3b (12–16) LD30 10 20.30.9c (19–22) 12.21.2c (11–14)

aLD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; T: treatments; n: number of males used bData were analyzed using one-way ANOVA (Concepts, 1989) at *<sup>P</sup>*<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s *F*-test (Scheffe, 1953) at 5%.

ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b).

(MSD)

Pre-adultb

Ta na Eupyrene sperm bundles in vas deferens

Control 30 10229a 1002116a 2513407a LD10 30 0b 234.9b 1621159b LD30 30 0b 0c 108075c

aLD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; T: treatments; n: number of males used bData were analyzed using one-way ANOVA (Concepts, 1989) at *<sup>P</sup>*<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s *F*-test (Scheffe, 1953) at 5%. Table 13. Effect of sublethal doses of chlorfluazuron on the number of eupyrene sperm bundles in the vas deferens during different developmental days of adults after topical application to newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b)

Table 12. Effect of sublethal doses of chlorfluazuron on size of various developmental stages of sperm observed in the testes of newly emerged adults after topical application to newly

> Newly emerged adultb (MSD)

(min–max) MSD Ranges

(min–max)

One day old adultb (MSD)

emerged adults (Perveen, 2000b).

(Table 12) (Perveen, 2000b).

In the vas deferens of male pre-adult controls, the mean number of eupyrene sperm bundles was 10229, but no sperm bundles observed in the LD10- or LD30-treated males of the same age (Table 13) (Perveen, 2000b).

In newly emerged control males, the mean number of eupyrene sperm bundles was 1002116, and in LD10-treated adult males, 23 4.9. In LD30-treated adults male there was no sperm bundles were observed. Moreover, in 1 day-old LD10-treated adult males, the number of eupyrene sperm bundles was significantly (P<0.001) reduced and more significantly (P<0.0001) reduced in LD30-treated males compared with the controls (Table 14) (Perveen, 2000b).

In the testis and vas deferens of newly emerged, LD10-treated males, the total number of eupyrene sperm bundles was significantly reduced (P<0.001) and more significantly reduced (P<0.0001) in LD30-treated males had no sperm bundles in the vas deferens compared with the controls (Table 14) (Perveen, 2000b).


aLD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1; T: treatments; n: number of males used; NESB: number of eupyrene sperm bundle; NASB: number of apyrene sperm bundle; ESB: eupyrene sperm bundle; ASB:

apyrene sperm bundle; TNESB: total number of eupyrene sperm bundle bData were analyzed using one-way ANOVA (Concepts, 1989) at P<0.0001. Means within columns followed by different letters are significantly different by Scheffe`s *F*-test (Scheffe, 1953) at 5%.

Table 14. Effect of sublethal doses of chlorfluazuron on the total number of eupyrene sperm bundles in the testis and vas deferens of newly emerged adults after topical application to newly ecdysed fifth-instar larvae of *Spodoptera litura* (Source: Perveen, 2000b)

#### **4.3 Discussion**

Topical application of sublethal doses of chlorfuazuron (LD10: 1.00 ng larva-1; LD30: 3.75 ng larva-1) has an effect on reproduction of *S. litura* by reducing the fecundity, fertilityand hatchability. Fecundity was reduced to a similar degree (35±44%) when females, males or both sexes were treated with LD10 or LD30. Fertility was reduced by 42% or 52% when females were treated with LD10 or LD30, respectively, and by 60% or 63%, respectively, when males or both sexes were treated with LD10. Fertility was reduced by 78% or 80% when males or both sexes were treated with LD30. The hatchability was reduced by 20% or 23% when females were treated with LD10 or LD30, respectively, and by 37% or 39%, respectively, when males or both sexes were treated with LD30. Hatchability was reduced by 55% or 56% when males or both sexes were treated with LD30 (Perveen, 2000a). Thus, this study was conducted to determine the causes of the reduction in these reproductive parameters. Topical application of sublethal doses of chlorfuazuron has an effect on testis development by decreasing the volume and weight of testes and its sheath thickness, and also on spermatogenesis by decreasing the number of cysts, eupyrene and apyrene bundles during different times in development. Reduction in the testes volume and weight might be caused

Chlorfluazuron as Reproductive Inhibitor 55

caused by the delay of spermatogenesis. When males or both sexes are treated on the first day of pairing, few or none mate. However, seven to nine pairs mate in control or female treated crosses. During the next day, seven to 10 pairs mate in 10±13 pairs of seven combinations of crosses (Perveen, 2008). These results suggest that the delay of the first mating is caused by the delayed spermatogenesis. The effect of chlorfuazuron on testicular development and spermatogenesis is one of the factors responsible for the reduction in fecundity, fertility and hatchability caused by the sublethal doses of chlorfuazuron. More work is in progress to determine the biochemical mechanism of these effects in *S. litura*.

The physiological mechanism of action of chlorfluazuron describes here on testicular development and spermatogenesis when sublethal doses (LD10: 1.00 ng larva-1 or LD30: 3.75 ng larva-1) are applied topically to the cuticle of newly ecdysed fifth instars of *Spodoptera litura*. These doses disrupt the growth and development of testes by decreasing the volume and weight of testes and thickness of testes sheath as compared with that of the controls. Additionally, such doses disrupt spermatogenesis by reducing the number and size of eupyrene and apyrene sperm bundles in the testis. Very few or no eupyrene sperm bundles are observed in vas deferens of pre- and newly ecdysed adults compared with controls. This result shows that the transfer of sperm bundles from testes to vas deferens is delayed in treated males. The effects of chlorfluazuron on testicular development and spermatogenesis are thought to be one of the factors responsible for the reduction in fecundity, fertility and

Abro, G. H., Memon, A. J. and Syed, T. S. (1997). Sublethal effects of cyhalothrin and fluvalinate on biology of *Spodoptera litura* F. Pakistan J. Zool. 29: 181–184. Anderson, V. L. and McLean, R. L. (1974). Design of experiments: a realistic approach.

Amaldoss, G. (1989). Ultrastructure and physiology of the ductus ejaculatorius duplex and

Anonymous, (1969). Summary of panel meeting in "Sterile male technique for eradication or

Baloch, S. H. and Abbasi, Q. D. (1977). Investigation of the ecology and biology of the

Barbosa, P. (1974). Dissecting fluids and saline solutions. *In*: "Manual of basic techniques in insect' histology". (Mass, A. D. ed.), Oxford Publisher, London. p. 243. Bargers, A. and Raw, F. (1967). Soil biology. Academic Press. New York. pp. 53. Beeman, R.

Benz, G. (1969). Influence of mating, insemination and other factors on oÖgenesis and

oviposition in the moth *Zeiraphera diniana*. J. Ins. Physiol. 15: 55–71.

control of harmful insect". Intl. Atom. Ener. Com. Vienna. pp. 80

seminal vesicle of male *Spodoptera litura* (Fabricius) (Lepidoptera: Noctuidae). Proc.

cutworm (Noctuidae: Lepidoptera) in the Hyderabad region. First Annu. Res.

W. (1982). Advances in mode of action of insecticides. Ann. Rev. Entomol. 27: 253–

hatchability caused by sublethal doses of chlorfluazuron.

Indian Acad. Sci. (Anim. Sci.) 98 (6): 405–418.

Report, Pakistan Sci. Found. Islamabad. p. 80.

Dekker, New York*.* p. 70.

**4.4 Conclusion** 

**5. References** 

281.

by a reduction in the number of cysts, number and size of sperm bundles, thickness of testes sheath, and/or the reduction in protein content of testes constituents. When females are treated with LD10 of chlorfuazuron, no signifiant reduction in the inseminated eupyrene sperm number is observed compared with controls. However, LD10 and LD30 treatment of males significantly reduces (65.8% and 88.6%) the number of inseminated eupyrene sperm. Moreover, no significant (P<0.0001) differences in the reduction are observed on the inseminated eupyrene sperm number when males or both sexes are treated either with LD10 or LD30 (Perveen, 2008). Therefore, the main cause of the reduction in the fecundity, fertility and hatchability is a decrease in inseminated eupyrene sperm numbers. In larval *S. litura*, the bright yellow-coloured testes are distinctly paired, reniform and situated between the 5th and 6th abdominal segments. Each of the lateral testicular lobes is made up of four follicles. The testicular lobes are enclosed within two thick, double-layered peritoneal sheaths. Each of these sheaths comprises two layers of epithelial cells. The external sheath is made up of lightly stained cuboidal cells resting on a basement membrane. This forms a common envelope to all the follicles of the testis and is penetrated by tracheal branches. The inner sheath is made up of more darkly stained elliptical cells, within which muscle fibres are distinguisable. Ingrowths from this sheath in the form of double-walled septa penetrate between the follicles to separate them from one another. The testes sheath reaches its maximum thickness at day 0 of pupation in the controls, whereas in LD10- treated males maximum thickness occurs later at 1 day. In LD30-treated males maximum thickness occurs at 2 day after pupation. Difubenzuron affects ecdysteroid secretion from the epidermis in Tenebrio molitor (Soltani, 1984), ovaries of *Cydia pomonella* (Soltani et al*.,* 1987; Soltani et al*.,*  1989b) and also the concentrations of haemolymph constituents in *T. molitor* (Soltani, 1992). Ecdysteroids have been reported to stimulate spermatogenesis in many insect species (Dumser, 1980b; Gelman and Hayes, 1982). In Mamestra brassica (Shimizu et al*.,* 1985), *Heliothis virescens* (Loeb, 1986) and *L. dispar* (Loeb et al*.,* 1988) testes synthesize ecdysteroids. The treatment with chlorfuazuron effects ecdysteroid production by the testis sheet remains to be investigated. Gelman and Hayes (1982) and Gelman et al. (1988) observed in *Ostrinia nubilalis*, the size and weight of separate testes paired. Topical application of the sublethal dose, LD10 of chlorfuazuron significantly reduces (P<0.0001) the weight and size of testes, and this is even greater in LD30-treated males. However, the topical application of similar sublethal doses of chlorfuazuron significantly reduced (P<0.0001) ovarian weight in postpupal and developing pharate adult females as compared with that of the controls (Perveen and Miyata, 2000). However, significant differences are not observed in ovarian weight when adult females are treated with LD10 or LD30 doses. The maturation of insect testes depends, among other factors, upon the materials that are taken up from the surrounding haemolymph and by materials synthesized by the testes *in situ*. These materials include protein, lipid and carbohydrate, all of which are required for development of the genital tract (Kunkel and Nordin, 1985; Kanost et al*.,* 1990). In newly emerged males from LD10- or LD30-treated larvae the eupyrene and apyrene sperm bundles are significantly (P<0.0001) smaller in size and number compared with those of controls. Spermatozoa descend regularly from the testis through the vas deferens into the seminal vesicles, which fill with eupyrene sperm bundles with cysts and individual apyrene sperm. Yoshida (1994) also reported that the number of eupyrene sperm bundles in the testis and vas deferens of newly emerged treated (LD10) males is reduced by 36%, and the initiation of sperm movement from testis to seminal vesicle was delayed. The present results show that initiation of sperm movement from testis to vas deferens is delayed after chlorofuazuron treatment and this is caused by the delay of spermatogenesis. When males or both sexes are treated on the first day of pairing, few or none mate. However, seven to nine pairs mate in control or female treated crosses. During the next day, seven to 10 pairs mate in 10±13 pairs of seven combinations of crosses (Perveen, 2008). These results suggest that the delay of the first mating is caused by the delayed spermatogenesis. The effect of chlorfuazuron on testicular development and spermatogenesis is one of the factors responsible for the reduction in fecundity, fertility and hatchability caused by the sublethal doses of chlorfuazuron. More work is in progress to determine the biochemical mechanism of these effects in *S. litura*.

#### **4.4 Conclusion**

54 Insecticides – Pest Engineering

by a reduction in the number of cysts, number and size of sperm bundles, thickness of testes sheath, and/or the reduction in protein content of testes constituents. When females are treated with LD10 of chlorfuazuron, no signifiant reduction in the inseminated eupyrene sperm number is observed compared with controls. However, LD10 and LD30 treatment of males significantly reduces (65.8% and 88.6%) the number of inseminated eupyrene sperm. Moreover, no significant (P<0.0001) differences in the reduction are observed on the inseminated eupyrene sperm number when males or both sexes are treated either with LD10 or LD30 (Perveen, 2008). Therefore, the main cause of the reduction in the fecundity, fertility and hatchability is a decrease in inseminated eupyrene sperm numbers. In larval *S. litura*, the bright yellow-coloured testes are distinctly paired, reniform and situated between the 5th and 6th abdominal segments. Each of the lateral testicular lobes is made up of four follicles. The testicular lobes are enclosed within two thick, double-layered peritoneal sheaths. Each of these sheaths comprises two layers of epithelial cells. The external sheath is made up of lightly stained cuboidal cells resting on a basement membrane. This forms a common envelope to all the follicles of the testis and is penetrated by tracheal branches. The inner sheath is made up of more darkly stained elliptical cells, within which muscle fibres are distinguisable. Ingrowths from this sheath in the form of double-walled septa penetrate between the follicles to separate them from one another. The testes sheath reaches its maximum thickness at day 0 of pupation in the controls, whereas in LD10- treated males maximum thickness occurs later at 1 day. In LD30-treated males maximum thickness occurs at 2 day after pupation. Difubenzuron affects ecdysteroid secretion from the epidermis in Tenebrio molitor (Soltani, 1984), ovaries of *Cydia pomonella* (Soltani et al*.,* 1987; Soltani et al*.,*  1989b) and also the concentrations of haemolymph constituents in *T. molitor* (Soltani, 1992). Ecdysteroids have been reported to stimulate spermatogenesis in many insect species (Dumser, 1980b; Gelman and Hayes, 1982). In Mamestra brassica (Shimizu et al*.,* 1985), *Heliothis virescens* (Loeb, 1986) and *L. dispar* (Loeb et al*.,* 1988) testes synthesize ecdysteroids. The treatment with chlorfuazuron effects ecdysteroid production by the testis sheet remains to be investigated. Gelman and Hayes (1982) and Gelman et al. (1988) observed in *Ostrinia nubilalis*, the size and weight of separate testes paired. Topical application of the sublethal dose, LD10 of chlorfuazuron significantly reduces (P<0.0001) the weight and size of testes, and this is even greater in LD30-treated males. However, the topical application of similar sublethal doses of chlorfuazuron significantly reduced (P<0.0001) ovarian weight in postpupal and developing pharate adult females as compared with that of the controls (Perveen and Miyata, 2000). However, significant differences are not observed in ovarian weight when adult females are treated with LD10 or LD30 doses. The maturation of insect testes depends, among other factors, upon the materials that are taken up from the surrounding haemolymph and by materials synthesized by the testes *in situ*. These materials include protein, lipid and carbohydrate, all of which are required for development of the genital tract (Kunkel and Nordin, 1985; Kanost et al*.,* 1990). In newly emerged males from LD10- or LD30-treated larvae the eupyrene and apyrene sperm bundles are significantly (P<0.0001) smaller in size and number compared with those of controls. Spermatozoa descend regularly from the testis through the vas deferens into the seminal vesicles, which fill with eupyrene sperm bundles with cysts and individual apyrene sperm. Yoshida (1994) also reported that the number of eupyrene sperm bundles in the testis and vas deferens of newly emerged treated (LD10) males is reduced by 36%, and the initiation of sperm movement from testis to seminal vesicle was delayed. The present results show that initiation of sperm movement from testis to vas deferens is delayed after chlorofuazuron treatment and this is

The physiological mechanism of action of chlorfluazuron describes here on testicular development and spermatogenesis when sublethal doses (LD10: 1.00 ng larva-1 or LD30: 3.75 ng larva-1) are applied topically to the cuticle of newly ecdysed fifth instars of *Spodoptera litura*. These doses disrupt the growth and development of testes by decreasing the volume and weight of testes and thickness of testes sheath as compared with that of the controls. Additionally, such doses disrupt spermatogenesis by reducing the number and size of eupyrene and apyrene sperm bundles in the testis. Very few or no eupyrene sperm bundles are observed in vas deferens of pre- and newly ecdysed adults compared with controls. This result shows that the transfer of sperm bundles from testes to vas deferens is delayed in treated males. The effects of chlorfluazuron on testicular development and spermatogenesis are thought to be one of the factors responsible for the reduction in fecundity, fertility and hatchability caused by sublethal doses of chlorfluazuron.

## **5. References**


Chlorfluazuron as Reproductive Inhibitor 57

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**3** 

*India* 

**Organophosphorus Insecticides** 

*Food Protectants and Infestation Control Department, Central Food Technological* 

The modern world has heavily thrived on the revolution in agricultural practices that have culminated in tremendous boost in agricultural productivity. Pesticides are perhaps one of the most important and effective strategies of the green revolution. Pesticides are the only class of toxic substances that are intentionally released into the environment for achieving greater good, a decision that far outweighs their toxicological concerns. Organophosphorus insecticides (OPI) are one of the most extensively used classes of insecticides. Chemically they are derivatives of phosphoric (H3PO4), phosphorous (H3PO3) or phosphinic acid (H3PO2) (Abou-Donia, 2003). The OPI were initially introduced as replacements for the much persistent organochlorine insecticides (Galloway & Handy, 2003). With systemic, contact and fumigant action, OPI find use as pest control agents in various situations. OPI are extensively used in agricultural practices for protecting food and commercial crops from various types of insects. In addition, OPI are also used in household situations for mitigating menacing pest varieties. They are not very stable chemically or biochemically and are degraded in soil, sediments and in surface water. Perhaps, it is this instability of these agents that has lead to their widespread and indiscriminate use, which has exposed animal and human life to various forms of health hazard. The increase in their use has lead to wide range of ecotoxicological problems and exposure to OPI is believed to be major cause of

Huge scientific body of evidence suggests that OPI exposure is a major toxicological threat that may affect human and animal health because of their various toxicities such as neurotoxicity, endocrine toxicity, immunotoxicity, reproductive toxicity, genotoxicity and ability to induce organ damage, alterations in cellular oxidative balance and disrupt glucose homeostasis. Indeed, the data on residue levels of OPI in various sources reported from India does create a huge cause for concern regarding their toxic effects. Samples of raw and bottled water were reported to be contaminated with various OPI residues, some of which were much higher than recommended levels (Mathur et al., 2003). Sanghi et al. (2003) have reported OPI residue levels in breast milk samples in India. Based on the levels of OPI residues, it has been speculated that infants may consume 4.1 times higher levels of malathion than the average daily intake levels recommended by the World Health Organisation. Similarly, human blood samples were reported to be contaminated with residues of monocrotophos, chlorpyrifos, malathion and phosphamidon (Mathur et al.,

**1. Introduction** 

morbidity and mortality in many countries.

**and Glucose Homeostasis** 

Apurva Kumar R. Joshi and P.S. Rajini

*Research Institute (CSIR lab), Mysore,* 


## **Organophosphorus Insecticides and Glucose Homeostasis**

Apurva Kumar R. Joshi and P.S. Rajini *Food Protectants and Infestation Control Department, Central Food Technological Research Institute (CSIR lab), Mysore,* 

*India* 

## **1. Introduction**

62 Insecticides – Pest Engineering

Yoshida, K. (1994). Studies on the physiological effects of chlorfluazuron: with special

Thesis, Graduate School of Agricultural Sciences, Nagoya University. p. 69. Younis, M. (1973). Losses of cotton crop due to insect pests. *Proc. Crop. Produc. Sem. Organ.* 

Yu, S. J. (1983). Age variation in insecticide susceptibility and detoxification capacity of fall

armyworm (Lepidoptera: Noctuidae) larvae. J. Econ. Entomol. 76:

*ESSO (Pak.) Ltd*. p. 110.

reference to reproductive effects of chlorfluazuron on *Spodoptera litura*. M. Sc.

The modern world has heavily thrived on the revolution in agricultural practices that have culminated in tremendous boost in agricultural productivity. Pesticides are perhaps one of the most important and effective strategies of the green revolution. Pesticides are the only class of toxic substances that are intentionally released into the environment for achieving greater good, a decision that far outweighs their toxicological concerns. Organophosphorus insecticides (OPI) are one of the most extensively used classes of insecticides. Chemically they are derivatives of phosphoric (H3PO4), phosphorous (H3PO3) or phosphinic acid (H3PO2) (Abou-Donia, 2003). The OPI were initially introduced as replacements for the much persistent organochlorine insecticides (Galloway & Handy, 2003). With systemic, contact and fumigant action, OPI find use as pest control agents in various situations. OPI are extensively used in agricultural practices for protecting food and commercial crops from various types of insects. In addition, OPI are also used in household situations for mitigating menacing pest varieties. They are not very stable chemically or biochemically and are degraded in soil, sediments and in surface water. Perhaps, it is this instability of these agents that has lead to their widespread and indiscriminate use, which has exposed animal and human life to various forms of health hazard. The increase in their use has lead to wide range of ecotoxicological problems and exposure to OPI is believed to be major cause of morbidity and mortality in many countries.

Huge scientific body of evidence suggests that OPI exposure is a major toxicological threat that may affect human and animal health because of their various toxicities such as neurotoxicity, endocrine toxicity, immunotoxicity, reproductive toxicity, genotoxicity and ability to induce organ damage, alterations in cellular oxidative balance and disrupt glucose homeostasis. Indeed, the data on residue levels of OPI in various sources reported from India does create a huge cause for concern regarding their toxic effects. Samples of raw and bottled water were reported to be contaminated with various OPI residues, some of which were much higher than recommended levels (Mathur et al., 2003). Sanghi et al. (2003) have reported OPI residue levels in breast milk samples in India. Based on the levels of OPI residues, it has been speculated that infants may consume 4.1 times higher levels of malathion than the average daily intake levels recommended by the World Health Organisation. Similarly, human blood samples were reported to be contaminated with residues of monocrotophos, chlorpyrifos, malathion and phosphamidon (Mathur et al.,

Organophosphorus Insecticides and Glucose Homeostasis 65

protein carbonylation, depletion of cellular antioxidant pools and alterations in enzymatic antioxidant status appear to be chief mechanisms of OPI-induced oxidative stress that often results in pathophysiological changes and organ damage. Several studies have demonstrated usefulness of antioxidant intervention in alleviating oxidative stress and pathophysiological changes induced by OPI (Kamath et al., 2008, Soltaninejad & Abdollahi, 2009). These studies lend unequivocal support to view that oxidative stress mediates as one

**3. Organophosphorus insecticides and glucose homeostasis: mechanistic** 

In addition to neurotoxicity and oxidative stress, alterations in glucose homeostasis often culminating hyperglycemia is increasingly being reported as characteristic outcome of OPI toxicity. Meller et al., (1981) have described two cases of human subjects who were hospitalized with many complications including hyperglycemia. With no pseudocholinesterase detected, patients were given pralidoxime (AChE activator), which improved their condition and normalized hyperglycemia. Investigations revealed that they may have been exposed to malathion sprayed in their area. This case presents a classic case of hyperglycemic outcome following exposure to OPI as patients also exhibited miosis and muscle twitching. Numerous experiments have been conducted with experimental animals that reveal hyperglycemia as a characteristic outcome of OPI poisoning. A recent review by Rahimi & Abdollahi (2007) provides an exhaustive account of investigations revealing

There are certain characteristic features of alterations induced by OPI in glucose homeostasis. In cases of exposure to single dose of OPI, hyperglycemia appears to set in rapidly and peak changes are often followed by a trend of normalization. High dose of diazinon has been reported to cause hyperglycemia in mice that follows a trend of normalization (Seifert, 2001). Acute exposure of rats to malathion resulted in hyperglycemia with peak increase occurring at 2.2h after administration followed by decrease after 4h (Rodrigues et al., 1986). A similar case of reversible hyperglycemia has been reported by Lasram et al., (2008) following administration of a single dose of

Biochemical changes associated with hyperglycemia serve as useful tools to understand etiology of OPI-induced hyperglycemia. Malathion has been reported to cause hyperglycemia in fasted rats. Interestingly, these hyperglycemic responses were not associated with hepatic glycogen depletion. The reversible phase of hyperglycemia was associated with increased glycogen deposition in liver, indicating that glucose may have come from gluconeogenesis (Gupta, 1974). Malathion induced hyperglycemia was associated with AChE inhibition in pancreas. More importantly, the trend of reversibility coincided with spontaneous reactivation of inhibited AChE (Lasram et al., 2008), indicating involvement of AChE-inhibition in hyperglycemia. Increase in blood glucose induced by sub chronic exposure of rats to malathion has been reported to be associated with increased glycogen phosphorylase and phosphoenolpyruvate carboxykinase activities, indicating involvement of both glycogenolytic and gluconeogenic processes. Increase in blood glucose levels induced by sub chronic exposure of rats to acephate has been reported to be associated with decrease in hepatic glycogen content (Deotare &

of the chief mechanisms of OPI toxicity.

hyperglycemia in cases of OPI exposure.

malathion to rats.

Chakrabarti, 1981).

**insights** 

2005). Thus, OPI present a realistic environmental threat that could affect various facets of human health.

## **2. Toxicity of organophosphorus insecticides**

The toxicity of active OPI is attributed to their ability to inhibit acetylcholinesterase (AChE, choline hydrolase, EC 3.1.1.7), an enzyme that catalyses the hydrolysis of the neurotransmitter acetylcholine (ACh), leading to cholinergic stress as a result of stimulation of muscarinic and nicotinic ACh receptors (Fukuto, 1990; Sogorb & Vilanova, 2003; Abou-Donia, 2003). The inhibition of AChE by an OPI takes place via a chemical reaction in which the serine hydroxyl moiety (of the active site) is phosphorylated. The phosphorylated enzyme is highly stable and, depending on the groups attached to the central 'P' atom of the OPI molecule, may be irreversibly inhibited.

There are several factors that determine the toxicity of OPI. Important of these are route and levels of exposure, structure of the substance and its interaction with the biotransformation/detoxification system of the body. The metabolic fate of OPI is basically the same in insects and animals. Following absorption, the distribution of OPI is variable. Blood half-lives are usually short, although plasma levels are in some cases maintained for several days. OPI undergo extensive biotransformation, which is complex and involves several metabolic systems in different organs, with simultaneous oxidative biotransformation at a number of points in the molecule, utilizing the cytochrome P-450 isoenzyme system. Metabolism occurs principally by oxidation, hydrolysis by esterases, and by transfer of portions of the molecule to glutathione. Oxidation of OPI may result in more or less toxic products. Most mammals have more efficient hydrolytic enzymes than insects and, therefore, are often more efficient in their detoxification processes. Numerous conjugation reactions follow the primary metabolic processes, and elimination of the phosphorus-containing residue may be via the urine or faeces. Some bound residues remain in exposed animals. Binding seems to be to proteins, principally, since there are limited data showing that incorporation of residues into DNA (Eto, 1974).

#### **2.1 Neurotoxicity**

Based on structure-function relationships, OPI are essentially neurotoxicants. Most important of their neurotoxicities is their 'cholinergic toxicity', which is a consequence of acetylcholinesterase (AChE) inhibition by OPI leading to accumulation of ACh and cholinergic stress. Signs of cholinergic toxicity include miosis, muscle fasciculation, excessive glandular secretions, nausea and vomiting (Namba, 1971). In addition, OPI are known to exert two other forms of neurotoxicities- Organophosphorus ester-induced delayed neurotoxicity (OPIDN) and Organophosphorus ester-induced chronic neurotoxicity (OPICN). OPIDN is a neurodegenerative disorder characterized by delayed onset of prolonged ataxia and upper motor neuron spasticity as a result of single or multiple exposures. OPICN refers to other forms of neurotoxicity that is distinct from both cholinergic toxicity and OPIDN. OPICN is characterized by neuronal degeneration and subsequent neurobehavioral and neuropsychological consequences (Abou-Donia, 2003).

#### **2.2 Oxidative stress**

Numerous studies provide evidence for the propensity of OPI to disrupt oxidative balance leading to oxidative stress (Soltaninejad & Abdollahi, 2009). Increased lipid peroxidation,

2005). Thus, OPI present a realistic environmental threat that could affect various facets of

The toxicity of active OPI is attributed to their ability to inhibit acetylcholinesterase (AChE, choline hydrolase, EC 3.1.1.7), an enzyme that catalyses the hydrolysis of the neurotransmitter acetylcholine (ACh), leading to cholinergic stress as a result of stimulation of muscarinic and nicotinic ACh receptors (Fukuto, 1990; Sogorb & Vilanova, 2003; Abou-Donia, 2003). The inhibition of AChE by an OPI takes place via a chemical reaction in which the serine hydroxyl moiety (of the active site) is phosphorylated. The phosphorylated enzyme is highly stable and, depending on the groups attached to the central 'P' atom of the

There are several factors that determine the toxicity of OPI. Important of these are route and levels of exposure, structure of the substance and its interaction with the biotransformation/detoxification system of the body. The metabolic fate of OPI is basically the same in insects and animals. Following absorption, the distribution of OPI is variable. Blood half-lives are usually short, although plasma levels are in some cases maintained for several days. OPI undergo extensive biotransformation, which is complex and involves several metabolic systems in different organs, with simultaneous oxidative biotransformation at a number of points in the molecule, utilizing the cytochrome P-450 isoenzyme system. Metabolism occurs principally by oxidation, hydrolysis by esterases, and by transfer of portions of the molecule to glutathione. Oxidation of OPI may result in more or less toxic products. Most mammals have more efficient hydrolytic enzymes than insects and, therefore, are often more efficient in their detoxification processes. Numerous conjugation reactions follow the primary metabolic processes, and elimination of the phosphorus-containing residue may be via the urine or faeces. Some bound residues remain in exposed animals. Binding seems to be to proteins, principally, since there are limited data

Based on structure-function relationships, OPI are essentially neurotoxicants. Most important of their neurotoxicities is their 'cholinergic toxicity', which is a consequence of acetylcholinesterase (AChE) inhibition by OPI leading to accumulation of ACh and cholinergic stress. Signs of cholinergic toxicity include miosis, muscle fasciculation, excessive glandular secretions, nausea and vomiting (Namba, 1971). In addition, OPI are known to exert two other forms of neurotoxicities- Organophosphorus ester-induced delayed neurotoxicity (OPIDN) and Organophosphorus ester-induced chronic neurotoxicity (OPICN). OPIDN is a neurodegenerative disorder characterized by delayed onset of prolonged ataxia and upper motor neuron spasticity as a result of single or multiple exposures. OPICN refers to other forms of neurotoxicity that is distinct from both cholinergic toxicity and OPIDN. OPICN is characterized by neuronal degeneration and subsequent neurobehavioral and neuropsychological consequences (Abou-Donia, 2003).

Numerous studies provide evidence for the propensity of OPI to disrupt oxidative balance leading to oxidative stress (Soltaninejad & Abdollahi, 2009). Increased lipid peroxidation,

**2. Toxicity of organophosphorus insecticides** 

showing that incorporation of residues into DNA (Eto, 1974).

OPI molecule, may be irreversibly inhibited.

human health.

**2.1 Neurotoxicity** 

**2.2 Oxidative stress** 

protein carbonylation, depletion of cellular antioxidant pools and alterations in enzymatic antioxidant status appear to be chief mechanisms of OPI-induced oxidative stress that often results in pathophysiological changes and organ damage. Several studies have demonstrated usefulness of antioxidant intervention in alleviating oxidative stress and pathophysiological changes induced by OPI (Kamath et al., 2008, Soltaninejad & Abdollahi, 2009). These studies lend unequivocal support to view that oxidative stress mediates as one of the chief mechanisms of OPI toxicity.

## **3. Organophosphorus insecticides and glucose homeostasis: mechanistic insights**

In addition to neurotoxicity and oxidative stress, alterations in glucose homeostasis often culminating hyperglycemia is increasingly being reported as characteristic outcome of OPI toxicity. Meller et al., (1981) have described two cases of human subjects who were hospitalized with many complications including hyperglycemia. With no pseudocholinesterase detected, patients were given pralidoxime (AChE activator), which improved their condition and normalized hyperglycemia. Investigations revealed that they may have been exposed to malathion sprayed in their area. This case presents a classic case of hyperglycemic outcome following exposure to OPI as patients also exhibited miosis and muscle twitching. Numerous experiments have been conducted with experimental animals that reveal hyperglycemia as a characteristic outcome of OPI poisoning. A recent review by Rahimi & Abdollahi (2007) provides an exhaustive account of investigations revealing hyperglycemia in cases of OPI exposure.

There are certain characteristic features of alterations induced by OPI in glucose homeostasis. In cases of exposure to single dose of OPI, hyperglycemia appears to set in rapidly and peak changes are often followed by a trend of normalization. High dose of diazinon has been reported to cause hyperglycemia in mice that follows a trend of normalization (Seifert, 2001). Acute exposure of rats to malathion resulted in hyperglycemia with peak increase occurring at 2.2h after administration followed by decrease after 4h (Rodrigues et al., 1986). A similar case of reversible hyperglycemia has been reported by Lasram et al., (2008) following administration of a single dose of malathion to rats.

Biochemical changes associated with hyperglycemia serve as useful tools to understand etiology of OPI-induced hyperglycemia. Malathion has been reported to cause hyperglycemia in fasted rats. Interestingly, these hyperglycemic responses were not associated with hepatic glycogen depletion. The reversible phase of hyperglycemia was associated with increased glycogen deposition in liver, indicating that glucose may have come from gluconeogenesis (Gupta, 1974). Malathion induced hyperglycemia was associated with AChE inhibition in pancreas. More importantly, the trend of reversibility coincided with spontaneous reactivation of inhibited AChE (Lasram et al., 2008), indicating involvement of AChE-inhibition in hyperglycemia. Increase in blood glucose induced by sub chronic exposure of rats to malathion has been reported to be associated with increased glycogen phosphorylase and phosphoenolpyruvate carboxykinase activities, indicating involvement of both glycogenolytic and gluconeogenic processes. Increase in blood glucose levels induced by sub chronic exposure of rats to acephate has been reported to be associated with decrease in hepatic glycogen content (Deotare & Chakrabarti, 1981).

Organophosphorus Insecticides and Glucose Homeostasis 67

Table 1. Blood glucose, acetylcholinesterase (AChE) and reduced glutathione (GSH) levels in

Further, DM also caused significant pancreatic damage as reflected by increased amylase (2- 3 folds) and lipase (20 & 38%) activities in serum (**Fig 2**). These changes were sharply paralleled by significant damage in pancreatic milieu. There was a dose-related elevation in ROS levels in pancreas of treated rats. While the increase at the lower dose was 66%, a dramatic (150%) increase was evident at the higher dose. Concomitantly, a dose-related increase in TBARS (lipid peroxidation index) levels was observed in the pancreas of DM treated rats. There was 2.5 and 3.7 fold increase in TBARS level at lower and higher doses of DM respectively (**Fig. 3**). Activities of selected antioxidant enzymes were significantly elevated in the pancreas of treated rats compared to that of control rats. (**Table 2**) (Kamath & Rajini, 2007). These results are in accordance with the study of Hagar et al., (2002) who had earlier reported increased blood glucose levels and hyerinsulinemia with concomitant histochemical and ultramicrostructural changes in pancreas of rats following chronic

Fig. 2. Changes in pancreatic damage markers in rats induced by Dimethoate after 30 days

Comparison of control and other groups (*P* < 0.01), Comparison of DM1 with DM2 (*P* <

(DM1: 20 mg/kg b.w/d; DM2: 40 mg/kg b.w/d). Values are mean SEM (n=6); \*

0 85.33 ± 3.85 91.33 ± 2.41 4.96 ± 1.47 1.11 ± 0.02 20 87.34 ± 5.23 105.28 ± 3.57a 2.94 ± 1.75 0.99 ± 0.05a 40 85.00 ± 5.30 138.67 ± 5.70,b 0.43 ± 0.21a,b 0.91 ± 0.07a,b

(nmoles substrate hydrolyzed /min/mg protein)

GSH (mg/g tissue)

Treatment Blood glucose (mg/dl) AChE

Initial Final

b Comparison of DM (20mg /kg b.w.) group with DM (40 mg/kg b.w.) group

pancreas of rats administered oral doses of Dimethoate (DM) for 30 days.

Dimethoate (mg/kg b.w.)

Values are mean SEM (n=6);

exposure to dimethoate.

0.01)

a Comparison of control and other groups;

#### **3.1 Pancreatic dysfunctions**

Acute pancreatitis is also a well known complication of OP poisoning (Dressel et al., 1979; Frick et al., 1987; Hsiao et al., 1996), and epidemiological findings indicate that the incidence of pancreatitis is high in OPI intoxication based on various pathophysiological reports (Gokalp et al., 2005). The precise mechanisms underlying OPI-induced pancreatitis are still undefined, although it is believed to involve obstruction of pancreatic ducts and /or enhanced reactive oxygen species (Dressel et al., 1982; Sevillano et al., 2003, Sultatos, 1994). Involvement of oxidative stress following acute exposure to OPI has been reported recently (Banerjee et al., 2001) and it has been demonstrated unequivocally that lipid peroxidation is one of the molecular mechanisms involved in OPI-induced cytotoxicity (Akhgari et al., 2003; Ranjbar et al., 2002; Abdollahi et al., 2004b).

In view of the above, we attempted to understand the potential of repeated oral doses of dimethoate (DM) (at 20 and 40mg/kg b.w/d for 30days; doses corresponding to 1/20 and 1/10LD50 values) to cause alterations in glucose homeostasis and the associated biochemical alterations in pancreas of rats. We observed distinct signs of glucose intolerance among rats administered DM (**Fig. 1**) at time points at which un-treated rats showed normal glucose tolerance after an oral glucose load (3g/kg b.w.). We also observed that DM at both doses caused significant increase in blood glucose levels with concomitant inhibition of acetylcholinesterase activity and depletion of reduced glutathione contents in pancreas (**Table 1**) (Kamath & Rajini, 2007).

Fig. 1. Oral glucose tolerance at the end of 30 days in control (CTR) and Dimethoate (DM) treated rats.


Values are mean SEM (n=6);

66 Insecticides – Pest Engineering

Acute pancreatitis is also a well known complication of OP poisoning (Dressel et al., 1979; Frick et al., 1987; Hsiao et al., 1996), and epidemiological findings indicate that the incidence of pancreatitis is high in OPI intoxication based on various pathophysiological reports (Gokalp et al., 2005). The precise mechanisms underlying OPI-induced pancreatitis are still undefined, although it is believed to involve obstruction of pancreatic ducts and /or enhanced reactive oxygen species (Dressel et al., 1982; Sevillano et al., 2003, Sultatos, 1994). Involvement of oxidative stress following acute exposure to OPI has been reported recently (Banerjee et al., 2001) and it has been demonstrated unequivocally that lipid peroxidation is one of the molecular mechanisms involved in OPI-induced cytotoxicity (Akhgari et al., 2003;

In view of the above, we attempted to understand the potential of repeated oral doses of dimethoate (DM) (at 20 and 40mg/kg b.w/d for 30days; doses corresponding to 1/20 and 1/10LD50 values) to cause alterations in glucose homeostasis and the associated biochemical alterations in pancreas of rats. We observed distinct signs of glucose intolerance among rats administered DM (**Fig. 1**) at time points at which un-treated rats showed normal glucose tolerance after an oral glucose load (3g/kg b.w.). We also observed that DM at both doses caused significant increase in blood glucose levels with concomitant inhibition of acetylcholinesterase activity and depletion of reduced glutathione contents in pancreas

Fig. 1. Oral glucose tolerance at the end of 30 days in control (CTR) and Dimethoate (DM)

**3.1 Pancreatic dysfunctions** 

Ranjbar et al., 2002; Abdollahi et al., 2004b).

(**Table 1**) (Kamath & Rajini, 2007).

treated rats.

a Comparison of control and other groups;

b Comparison of DM (20mg /kg b.w.) group with DM (40 mg/kg b.w.) group

Table 1. Blood glucose, acetylcholinesterase (AChE) and reduced glutathione (GSH) levels in pancreas of rats administered oral doses of Dimethoate (DM) for 30 days.

Further, DM also caused significant pancreatic damage as reflected by increased amylase (2- 3 folds) and lipase (20 & 38%) activities in serum (**Fig 2**). These changes were sharply paralleled by significant damage in pancreatic milieu. There was a dose-related elevation in ROS levels in pancreas of treated rats. While the increase at the lower dose was 66%, a dramatic (150%) increase was evident at the higher dose. Concomitantly, a dose-related increase in TBARS (lipid peroxidation index) levels was observed in the pancreas of DM treated rats. There was 2.5 and 3.7 fold increase in TBARS level at lower and higher doses of DM respectively (**Fig. 3**). Activities of selected antioxidant enzymes were significantly elevated in the pancreas of treated rats compared to that of control rats. (**Table 2**) (Kamath & Rajini, 2007). These results are in accordance with the study of Hagar et al., (2002) who had earlier reported increased blood glucose levels and hyerinsulinemia with concomitant histochemical and ultramicrostructural changes in pancreas of rats following chronic exposure to dimethoate.

Fig. 2. Changes in pancreatic damage markers in rats induced by Dimethoate after 30 days (DM1: 20 mg/kg b.w/d; DM2: 40 mg/kg b.w/d). Values are mean SEM (n=6); \* Comparison of control and other groups (*P* < 0.01), Comparison of DM1 with DM2 (*P* < 0.01)

Organophosphorus Insecticides and Glucose Homeostasis 69

Studies undertaken by several researchers to investigate the mechanisms mediating hyperglycemic effects of OPI have mainly focused on the involvement of cholinergic stress and adrenal functions. We have extensively studied the mechanistic involvement of adrenals in glucotoxicity of OPI in rats mainly under acute and short-term exposure regimes. The rationale for studying adrenal involvement emerged from the typical hyperglycemic behaviour of single dose (oral) of two OPI-acephate and monocrotophos. Single dose of acephate and monocrotophos elicited rapid and transient hyperglycemia after administration. Both OPI were administered to overnight-fasted rats at 1/10 doses of their LD50 (LD50; acephate-1400mg/kg b.w., monocrotophos-18mg/kg b.w.). As depicted in **Fig. 4**, both acephate and monocrotophos induced reversible hyperglycemia with peak occurring at 2h after exposure. Acephate induced peak hyperglycemia at 2h (87%), which tended to normalize thereafter and attained near-control values at 8h after administration (Joshi & Rajini, 2009). Similarly, monocrotophos also induced rapid hyperglycemia with peak occurring at 2h (103%). Interestingly, monocrotophos induced hyperglycemia exhibited steep reversibility compared to acephate, with normalization occurring at 6h (Joshi & Rajini, 2010). This trend observed in the present study is consistent with other reports, which demonstrated reversible hyperglycemia in experimental animals following OPI administration. While Malathion has been reported to cause reversible hyperglycemia in rats (Gupta, 1974; Rodrigues et al., 1986; Seifert, 2001; Lasram et al., 2008), acute exposure to

diazinon induced reversible hyperglycemia in mice (Seifert, 2001).

Fig. 4. Time-course of blood glucose levels in rats administered a single oral dose of

Based on the above results, we reasoned that the reversible hyperglycemia could be triggered by transient changes in the hormonal milieu of glucose homeostasis. Adrenals are an important part of the endocrine system and play a key role in glucose homeostasis by secreting glucocorticoid and amine hormones. Glucocorticoid hormones (GCs) (mainly cortisol in man and corticosterone in rodents) are secreted by the adrenal cortex under the control of hypothalamic-pituitary-adrenal axis. Glucocorticoid hormones, along with other key hormones, act to maintain blood glucose levels within narrow limits (Andrews & Walker, 1999). GCs, glucagon and epinephrine raise blood glucose by inhibiting glucose

02468 Time (h)

Acephate Monocrotophos

acephate (140mg/kg b.w.) and monocrotophos (1.8mg/kg b.w.).

**3.2 Adrenal involvement** 

0

30

60

% change from control

90

120

Fig. 3. Extent of lipid peroxidation and ROS levels in pancreas of control (CTR) and Dimethoate treated rats (DM1: 20 mg/kg b.w/d; DM2: 40 mg/kg b.w/d). Values are mean SEM (n=6); \* Comparison of control and other groups (*P* < 0.01), Comparison of DM1 with DM2 (*P* < 0.01).

Several studies have demonstrated pancreatitis after exposure to OPI (Dressel et al., 1979; Moore & James, 1988; Hsiao et al., 1996). Increase in the serum lipase and amylase activities reported by us clearly indicates that DM results in a state of pancreatic damage. Increased serum lipase activity has also been reported after administration of methidathion, an OPI (Mollaoglu et al., 2003). These results agree with earlier reports of acute pancreatitis in humans after accidental cutaneous exposure to DM (Marsh et al., 1988) and increase in amylase activity reported in dogs after diazinon administration (Dressel et al., 1982). Together, these studies clearly indicate that OPI possess propensity to elicit structural and functional alterations in pancreatic milieu that may be associated with disruptions in euglycemic conditions. From these studies, it may be argued that OPI may present a great threat to pancreatic functions in human beings and such threats may have far-reaching consequences on gluco-regulation in human beings.


1units/mg protein; 2µmol/min/mg protein; 3nmol/ min/ mg protein; 4µmol/ min / mg protein

Values are mean SEM (n=6) a Comparison of control (CTR) and other groups;

b Comparison of DM1 (DM: 20mg /kg b.w/d) group with DM2 (DM: 40 mg/kg b.w/d) group

Table 2. Antioxidant enzyme activities in pancreas of rats administered oral doses of Dimethoate for 30 days.

#### **3.2 Adrenal involvement**

68 Insecticides – Pest Engineering

Fig. 3. Extent of lipid peroxidation and ROS levels in pancreas of control (CTR) and

with DM2 (*P* < 0.01).

consequences on gluco-regulation in human beings.

Values are mean SEM (n=6) a Comparison of control (CTR) and other groups;

Dimethoate for 30 days.

Group Enzyme Activity

Dimethoate treated rats (DM1: 20 mg/kg b.w/d; DM2: 40 mg/kg b.w/d). Values are mean SEM (n=6); \* Comparison of control and other groups (*P* < 0.01), Comparison of DM1

Several studies have demonstrated pancreatitis after exposure to OPI (Dressel et al., 1979; Moore & James, 1988; Hsiao et al., 1996). Increase in the serum lipase and amylase activities reported by us clearly indicates that DM results in a state of pancreatic damage. Increased serum lipase activity has also been reported after administration of methidathion, an OPI (Mollaoglu et al., 2003). These results agree with earlier reports of acute pancreatitis in humans after accidental cutaneous exposure to DM (Marsh et al., 1988) and increase in amylase activity reported in dogs after diazinon administration (Dressel et al., 1982). Together, these studies clearly indicate that OPI possess propensity to elicit structural and functional alterations in pancreatic milieu that may be associated with disruptions in euglycemic conditions. From these studies, it may be argued that OPI may present a great threat to pancreatic functions in human beings and such threats may have far-reaching

SOD1 CAT2 GPX3 GR3 GST4

CTR 26.42 ± 2.2 9.38 ± 0.31 27.18 ± 5.24 17.50 ± 1.60 0.03 ± 0.004 DM1 42.72 ± 0.38a 10.24 ± 0.32 25.23 ± 3.89 19.72 ± 2.03 0.04 ± 0.003a DM2 56.23 ±1.18a,b 15.44 ± 0.51a,b 13.85 ± 2.20a.b 25.30 ± 1.30a,b 0.06 ± 0.003a,b

1units/mg protein; 2µmol/min/mg protein; 3nmol/ min/ mg protein; 4µmol/ min / mg protein

b Comparison of DM1 (DM: 20mg /kg b.w/d) group with DM2 (DM: 40 mg/kg b.w/d) group

Table 2. Antioxidant enzyme activities in pancreas of rats administered oral doses of

Studies undertaken by several researchers to investigate the mechanisms mediating hyperglycemic effects of OPI have mainly focused on the involvement of cholinergic stress and adrenal functions. We have extensively studied the mechanistic involvement of adrenals in glucotoxicity of OPI in rats mainly under acute and short-term exposure regimes. The rationale for studying adrenal involvement emerged from the typical hyperglycemic behaviour of single dose (oral) of two OPI-acephate and monocrotophos. Single dose of acephate and monocrotophos elicited rapid and transient hyperglycemia after administration. Both OPI were administered to overnight-fasted rats at 1/10 doses of their LD50 (LD50; acephate-1400mg/kg b.w., monocrotophos-18mg/kg b.w.). As depicted in **Fig. 4**, both acephate and monocrotophos induced reversible hyperglycemia with peak occurring at 2h after exposure. Acephate induced peak hyperglycemia at 2h (87%), which tended to normalize thereafter and attained near-control values at 8h after administration (Joshi & Rajini, 2009). Similarly, monocrotophos also induced rapid hyperglycemia with peak occurring at 2h (103%). Interestingly, monocrotophos induced hyperglycemia exhibited steep reversibility compared to acephate, with normalization occurring at 6h (Joshi & Rajini, 2010). This trend observed in the present study is consistent with other reports, which demonstrated reversible hyperglycemia in experimental animals following OPI administration. While Malathion has been reported to cause reversible hyperglycemia in rats (Gupta, 1974; Rodrigues et al., 1986; Seifert, 2001; Lasram et al., 2008), acute exposure to diazinon induced reversible hyperglycemia in mice (Seifert, 2001).

Fig. 4. Time-course of blood glucose levels in rats administered a single oral dose of acephate (140mg/kg b.w.) and monocrotophos (1.8mg/kg b.w.).

Based on the above results, we reasoned that the reversible hyperglycemia could be triggered by transient changes in the hormonal milieu of glucose homeostasis. Adrenals are an important part of the endocrine system and play a key role in glucose homeostasis by secreting glucocorticoid and amine hormones. Glucocorticoid hormones (GCs) (mainly cortisol in man and corticosterone in rodents) are secreted by the adrenal cortex under the control of hypothalamic-pituitary-adrenal axis. Glucocorticoid hormones, along with other key hormones, act to maintain blood glucose levels within narrow limits (Andrews & Walker, 1999). GCs, glucagon and epinephrine raise blood glucose by inhibiting glucose

Organophosphorus Insecticides and Glucose Homeostasis 71

phenomenal increase in liver glycogen levels. The data presented above clearly demonstrates co-existence of hypercorticosteronemia and induction of liver gluconeogenesis enzyme activities with hyperglycemia in OPI treated rats, indicating that OPI may trigger induction of liver gluconeogenesis machinery as result of hypercorticosteronemia, leading to

Plasma corticosterone \* 30.9±3.4a 55.0±2.5b 44.0±2.7b Adrenal cholesterol\*\* 26.5±1.4a 15.6±0.56b 12.5±0.47b Blood glucose \*\*\* 101.6±4.6a 182.4±5.2b 142.7±5.2c Liver G6Pase# 90.14±4.38a 171.93±5.61b 112.84±4.18c Liver TAT ## 14.28±1.34a 26.31±0.87b 23.7±0.48b Hepatic glycogen\$ 316.2±34.90a 325.3±29.12a 1145.0±27.92b *(*Joshi and Rajin*i,* 2009*)*

Plasma corticosterone \* 36.62±1.2a 73.82±3.8b 45.65±1.8a Blood glucose \*\* 95.2±1.8a 194.8±3.7b 121.3±1.9c Liver TAT # 15.86±0.8a 32.27±1.2b 26.87±1.8c Hepatic glycogen## 213.8±49.2a 216.4±21.1a 925.7±27.6b *(*Joshi and Rajini, 2010*)*

Indeed, role of adrenals in glucotoxicity of OPI has been explored earlier. Matin et al., (1989) earlier demonstrated that single dose diazinon (OPI) caused hyperglycemia and induction of liver gluconeogenesis enzymes in normal rats while these changes did not manifest in adrenalectomized rats, indicating the involvement of adrenals in the glucotoxicity of diazinon. Our attempts to study the adrenal and glycemic effects of acephate and monocrotophos revealed that two compounds, which exhibit anticholinesterase property, elicited similar effects. Thus, the effects raised question whether the adrenal and glycemic effects are mediated through the anticholinesterase property of OPI. To address the question, we studied the extent of AChE inhibition elicited by monocrotophos at 2 and 4h

\* µg/dl; \*\* mg/g tissue ; # tyrosine aminotranferase (nmol/min/mg protein); ## µg/g tissue

Table 4. Biochemical effects of monocrotophos (1.8mg/kg b.w.) in rats

At time interval after administration 0h 2h 6h

At time interval after administration 0h 2h 4h

hyperglycemia.

Data analyzed by ANOVA followed by Tukey Test (n=6)

Data analyzed by ANOVA followed by Tukey Test (n=6)

## tyrosine aminotranferase (nmol/min/mg protein); \$ µg/g tissue

Table 3. Biochemical effects of acephate (140mg/kg b.w.) in rats
