interactions of adipokinetic hormones with insecticides

UNIVERSITY JOSIP JURAJ STROSSMAYER IN OSIJEK DEPARTMENT OF BIOLOGY

Graduate study of Biology

Mirna Velki

INTERACTIONS OF ADIPOKINETIC HORMONES WITH INSECTICIDES

Master thesis

Osijek, 2010.

BASIC DOCUMENTATION CARD

i

University of Josip Juraj Strossmayer in Osijek Master Thesis Department of Biology Graduate Study of Biology

Scientific area: Natural science Scientific field: Biology

INTERACTIONS OF ADIPOKINETIC HORMONES WITH INSECTICIDES

Mirna Velki

Thesis performed at: Department of Physiology, Institute of Entomology, esk Budjovice

Supervisor: Branimir K. Hackenberger, PhD, Assistant Professor Cosupervisor: Dalibor Kodrk, PhD, Associate Professor

Adipokinetic hormones (AKHs) are insect neuropetides controlling stress situations including those elicited by insecticide treatment. So the aim of this research was to investigate the interactions of AKH under stress conditions caused by insecticide treatment, including the effects on the mortality of Pyrrhocoris apterus. The bugs were treated with 2 insecticides endosulfan and malathion. Results showed that AKH cotreatment caused elevation of metabolism which could lead to faster penetration of insecticides into tissues and could be a reason for enhanced action of insecticides. Insecticide treatment caused significant increase of AKH content in CNS and haemolymph. Also, it was found that both endosulfan and malathion cause oxidative stress.

Number of pages: 65 Number of figures: 28 Number of tables: 8 Number of references: 87 Original in: English

Key words: Pyrrhocoris apterus, endosulfan, malathion, adipokinetic hormones, oxidative stress, metabolism

Date of the thesis defence: 28.06.2010.

Reviewers:

1. Enrih Merdi, PhD, Assistant Professor, Department of Biology, University of Josip Juraj Strossmayer in Osijek. 2. Branimir K. Hackenberger, PhD, Assisstant Professor, Department of Biology, University of Josip Juraj Strossmayer in Osijek. 3. Elizabeta Has-Schn, PhD, Associate Professor, Department of Biology, University of Josip Juraj Strossmayer in Osijek.

Thesis deposited in: Library of Department of Biology, University of Josip Juraj Strossmayer in Osijek.

ii

First, a special thanks goes to my mentor, doc. dr. sc. Branimir Hackenberger, for his useful advice and help that he provided during the writing of this Master's thesis. I also thank dr. sc. Sandri Stepi for her big help with completing this thesis.

I am immensely grateful to my comentor, doc. RNDr. Dalibor Kodrk, for providing me with the opportunity to make my Master's thesis at the Institute of Entomology in eske Budjovice, as well as for sharing with me his great experience and knowledge.

Besides my mentors, I would also like to thank all the members of the Department of Physiology at the Institute of Entomology in eske Budjovice, for their cordiality and hospitality.

I want to thank my family for their unconditional support throughout my college education.

Last, but not least, I thank all the people who have indirectly or directly helped in the realization of this thesis.

TABLE OF CONTENTS

iii

TABLE OF CONTENTS

1. INTRODUCTION............................................................................................................................... 1

1.1. ENVIRONMENTAL POLLUTION.............................................................................................. 1

1.2. PESTICIDES............................................................................................................................. 2

1.2.1. ORGANOPHOSPHORUS PESTICIDES.......................................................................... 2

1.2.2. ORGANOCHLORINE PESTICIDES................................................................................. 3

1.2.3. CARBAMATE PESTICIDES ............................................................................................. 3

1.2.4. PYRETHROID PESTICIDES ............................................................................................ 4

1.2.5. ENVIRONMENTAL FATE OF PESTICIDES .................................................................... 4

1.3. INSECTS AS MODEL ORGANISMS........................................................................................ 6

1.3.1. EFFECTS OF INSECTICIDES ......................................................................................... 7

1.3.2. INSECT CENTRAL NERVOUS SYSTEM (CNS) AND ENDOCRINE SYSTEM.............. 7

1.3.3. ADIPOKINETIC HORMONES (AKHS) .............................................................................. 8

1.4. OXIDATIVE STRESS................................................................................................................ 8

1.4.1. BIOMARKERS OF OXIDATIVE STRESS ...................................................................... 11

1.4.2. CATALASE (CAT)........................................................................................................... 12

1.4.3. PROTEIN CARBONYLS (PC) ........................................................................................ 12

1.4.4. THIOBARBITURIC ACID REACTIVE SUBSTANCES (TBARS).................................... 14

1.5. REVIEW OF RECENT STUDIES OF AKH AND Pyrrhocoris apterus AS A MODEL SPECIES................................................................................................................................ 15

2. RESEARCH OBJECTIVES ............................................................................................................ 19

3. MATERIALS AND METHODS ....................................................................................................... 20

3.1. CHEMICALS ........................................................................................................................... 20

3.2. INSTRUMENTS AND EQUIPMENT ....................................................................................... 21

TABLE OF CONTENTS

iv

3.3. EXPERIMENTAL INSECTS.................................................................................................... 22

3.3.1. TAXONOMY OF FIREBUG ............................................................................................ 22

3.3.2. BIOLOGY OF FIREBUG................................................................................................. 22

3.3.3. MAINTENANCE OF THE CULTURE.............................................................................. 23

3.4. CHARACTERISTICS OF INSECTICIDES.............................................................................. 24

3.4.1. ENDOSULFAN (GLOBAL E-35) ..................................................................................... 24

3.4.2. MALATHION (RADOTION E-50) .................................................................................... 25

3.5. INSECTICIDE TREATMENT .................................................................................................. 25

3.5.1. PRELIMINARY TEST FOR DETERMINATION OF MORTALITY .................................. 26

3.5.2. FINAL TEST FOR DETERMINATION OF MORTALITY ................................................ 26

3.6. HORMONAL TREATMENT .................................................................................................... 26

3.7. DISSECTION OF THE CNS ................................................................................................... 26

3.7.1. EXTRACTION OF AKH FROM CNS............................................................................... 27

3.8. HAEMOLYPMH SAMPLING................................................................................................... 28

3.8.1. EXTRACTION OF AKH FROM HAEMOLYPMH ............................................................ 28

3.9. QUANTIFICATION OF AKH BY COMPETITIVE ELISA......................................................... 28

3.10. METABOLIC RATE MEASURING........................................................................................ 30

3.11. OXIDATIVE STRESS............................................................................................................ 31

3.11.1. CATALASE MEASUREMENT....................................................................................... 31

3.11.2. PROTEIN CARBONYL DETERMINATION................................................................... 32

3.11.3. DETERMINATION OF LIPID PEROXIDATION ............................................................ 33

4. RESULTS........................................................................................................................................ 35

4.1. THE EFFECT OF AKH ON THE MORTALITY OF BUGS TREATED WITH ENDOSULFAN AND MALATHION ........................................................................................ 35

4.2. THE EFFECT OF ENDOSULFAN AND MALATHION ON AKH LEVEL IN CNS ................... 37

TABLE OF CONTENTS

v

4.3. THE EFFECT OF ENDOSULFAN AND MALATHION ON AKH LEVEL IN HAEMOLYMPH...................................................................................................................... 39

4.4. THE EFFECT OF AKH, ENDOSULFAN AND MALATHION ON CARBON DIOXIDE PRODUCTION ....................................................................................................................... 41

4.5. THE EFFECT OF AKH, ENDOSULFAN AND MALATHION ON OXIDATIVE STRESS........ 45

4.5.1. CATALASE ACTIVITY..................................................................................................... 45

4.5.2. PROTEIN CARBONYLS CONTENT............................................................................... 48

4.5.3. TBARS CONTENT .......................................................................................................... 49

5. DISCUSSION .................................................................................................................................. 51

6. CONCLUSIONS.............................................................................................................................. 57

7. REFERENCES................................................................................................................................ 58

8. APPENDIX ...................................................................................................................................... 65

INTRODUCTION

1

1. INTRODUCTION Advantages in organic synthetic chemistry led to development of various chemical compounds that are used in every day life, and these compounds can be found in food, water, air and soil. These chemicals which are foreign to the organisms (in the sense that they do not normally produce the compounds or consume them as part of their diet) are called xenobiotics. Xenobiotics include drugs, pesticides, food additives, polychlorinated biphenyls, dioxines, polycyclic aromatic hydrocarbons (PAHs), phenols, other industrial chemicals etc. During production and usage, these chemical compounds are imported into the environment and they become a part of all organisms.

1.1. ENVIRONMENTAL POLLUTION Environmental pollution is the addition of any substance (solid, liquid, or gas) or any form of energy (such as heat, sound, or radioactivity) that because of its chemical composition or quantity prevents the functioning of natural processes and produces undesirable environmental and health effects. There are two main classes of pollutants: those that are biodegradable (e.g. sewage), i.e. can be rendered harmless by natural processes and therefore cause no permanent harm if adequately dispersed or treated; and those that are non-biodegradable (e.g. heavy metals in industrial effluents, chlorinated hydrocarbons used as pesticides), which eventually accumulate in the environment and may be concentrated in food chains. Other forms of pollution in the environment include noise (e.g. from jet aircraft, traffic, and industrial processes) and thermal pollution (e.g. the release of excessive waste heat into lakes or rivers causing harm to wildlife). Recent pollution problems include the disposal of radioactive waste; acid rain; photochemical smog; increasing levels of human waste; high levels of carbon dioxide and other greenhouse gases in the atmosphere; damage to the ozone layer; and pollution of inland waters by agricultural fertilizers and sewage effluent. In general there are two types of pollution: 1. point source pollution - pollution that comes from a particular factory or outlet 2. diffuse pollution - pollution that comes from a number of sources, across large areas; for example - pesticides used on farms or gardens, vehicle exhaust fumes, household substances that go down the drain etc. One of the main sources of pollution is the deliberate release into the environment of substances such as pesticides. Pesticides are deliberately sprayed onto crops or agricultural land with the potential for exposure either via the crop itself or through contamination soil, water or air. The major problem of pesticide usage is persistence in the environment and an increase in concentration during passage through the food chain.

INTRODUCTION

2

In some cases the means of exposure is determined by the nature of the toxic substance. There are several types of exposure: 1. Intentional ingestion for example taking drugs and food additives, or alcohol and cigarettes. 2. Occupational exposure this is chronic, continual exposure; via inhalation or skin contact. 3. Environmental exposure in this type of exposure effluents from factories

contaminate both immediate and more distant environments (seas and oceans or the atmosphere in other countries). This form of exposure is usually chronic. Environmental exposure is also important in relation to pesticides contaminating air, water, soil and food.

4. Accidental poisoning this is usually acute rather than chronic exposure; drugs, pesticides, household products and natural poisons may all be involved in this type of exposure. 5. Intentional poisoning. Pollution of our environment has become an increasing with the development of industry and agriculture and with the increase in population. That is not to say that pollution did not exist before the nineteenth century. However, pollution on the current scale started during the Industrial Revolution. Consequently air, water and soil have all suffered pollution (Timbrell, 1995).

1.2. PESTICIDES Pesticides are chemical substances with synthetic or biological origin that are used for preventing, destroying, repelling, growth regulating, and generally for control of any kind of pest. Usage of pesticides has a wide application in agriculture, forestry and public health. Pesticides can be classified in many ways. Depending on target groups of organisms they can be divided on insecticides, herbicides, acaricides, rodenticides, nematocides, limacides, avicides, fungicides. Based on the chemical structure and mode of action, classification is more complex. The most important groups of pesticides (based on chemical structure and mode of action) include organophosphates, carbamates, pyrethroids and organochlorine pesticides (Hayes and Laws, 1991).

1.2.1. Organophosphorus pesticides Organophosphate pesticides are synthetic in origin and are normally esters, amides, or thiol derivatives of phosphoric, phosphonic, phosphorothioic, or phosphonothioic acids. These pesticides can be absorbed by all routes, including inhalation, ingestion, and dermal absorption. The toxicological effects of the organophosphorus pesticides are almost entirely

INTRODUCTION

3

due to the inhibition of acetylcholinesterase in the nervous system, resulting in respiratory, myocardial and neuromuscular transmission impairment (Stenersen, 2004). The main target organs are the nervous system, respiratory tract and cardiovascular system. They are mostly used as insecticides to control insect vectors which are found in food and commercial crops, and infestations in domestic and commercial buildings, and in man or domestic animals. In this group of pesticides belong acephate, chlorpyrifos, diazinon, dimethoate, malathion, parathion, pirimiphos-methyl, temephos etc.

1.2.2. Organochlorine pesticides Organochlorine pesticides are hydrocarbon compounds containing multiple chlorine substitutions. There are four main types of these pesticides dichlorodiphenylethanes, cyclodienes, chlorinated benzenes and cyclohexanes. All share a similar pair of carbon rings, one ring being heavily chlorinated. They are mostly used as insecticides. These pesticides are hydrophobic, lipophilic and extremely stable. Once in the environment they are subject to global deposition processes and bioaccumulate in the food chain. Toxicity appears to be via disruption of neural function and specific disturbances vary by chemistry. Mode of action is by blocking the ion channels (Na-K channels), blocking Na+-K+ ATPase, binding for receptors of sex hormones, and their metabolites form DNA adducts (Stenersen, 2004). Since biotransformation of organochlorine pesticides is a complex process involving mediation via xenobiotic-metabolizing phase I (cytochrome P450 (CYP)) and conjugating phase II enzymes they also cause the induction of CYP enzymes (Routti et al., 2009.). Representative compounds in this group include DDT, methoxychlor, dieldrin, chlordane, toxaphene, lindane, endosulfan etc. Introduced in the 1940s, they were widely used in agriculture and pest control until research and public concern regarding the hazards of their use led to restrictions and bans. But despite that, world-wide use of non-targeted organochlorine pesticides like endosulfan and lindane continues and DDT is still employed in developing countries, primarily for mosquito and malaria control. In Republic of Croatia since 2009 none of these pesticides has authorization for usage.

1.2.3. Carbamate pesticides This is a large group of synthetic pesticides that are used in agriculture as insecticides, fungicides, herbicides, nematocides, or sprout inhibitors. In addition, they are used as biocides for industrial or other applications and in household products. A potential use is in public health vector control. All carbamates are substituted carbamide acid ester and three classes of carbamate pesticides are known. The carbamate ester derivatives, used as insecticides (and nematocides), are generally stable and have a low vapour pressure and low water solubility. The carbamate herbicides (and sprout inhibitors) have structure with

INTRODUCTION

4

aromatic and/or aliphatic segment. And carbamate fungicides contain a benzimidazole group. Carbamates are effective insecticides by virtue of their ability to inhibit acetylcholinesterase (AChE). They can also inhibit other esterases (Stenersen, 2004). The carbamylation of AChE is unstable, and the regeneration of the enzyme is relatively rapid compared with a phosphorylated enzyme. Thus, the carbamates are less dangerous with regard to human exposure than organophosphorus pesticides but most of them are extremely toxic to Hymenoptera. Carbofuran, fenoxycarb, carbaryl, ethienocarb and fenobucarb are some of carbamate pesticides.

1.2.4. Pyrethroid pesticides Pyrethroid pesticides are synthetic analogues of pyrethrins, which are natural chemicals found in chrysanthemum flowers (Chrysanthemum cinerariefolium and Chrysanthemum cocineum). They are used to control a wide range of insects in public and commercial buildings, animal facilities, warehouses, agricultural fields, and greenhouses. They are also applied on livestock to control insects. Their mode of action is interference with transmission of nerve impulses these chemicals prolong sodium channel opening when a nerve cell is depolarized (Stenersen, 2004). They also cause oxidative stress and enhanced production of free radicals, and also enhance estrogen activity. Generally, they are not persistent in the environment due to their rapid degradation within days to several months. In this group belongs cypermethrin, permethrin, resmethrin, tetramethrin etc.

1.2.5. Environmental fate of pesticides When pesticides enter into the environment, they are distributed in four main compartments water, air, soil and living organisms. Many processes affect what happens to pesticides in the environment (Figure 1).

Figure 1. Environmental fate of pesticides.

INTRODUCTION

5

These processes include:

1) ADSORPTION Adsorption is the binding of pesticides to soil particles. The amount of pesticide adsorbed to the soil varies with the type of pesticide, soil, moisture, soil pH, and soil texture. Pesticides are strongly adsorbed to soils that are high in clay or organic matter. They are not as strongly adsorbed to sandy soils.

2) TRANSFER processes that move the pesticide away from the target site; these include volatilization, spray drift, runoff, leaching, absorption and crop removal. Volatilization is the process of solids or liquids converting into a gas, which can move away from the initial application site. Pesticides volatize most readily from sandy and wet soils. Hot, dry, or windy weather and small spray drops increase volatilization. Spray drift is the airborne movement of spray droplets away from a treatment site during application. Drift can damage nearby sensitive crops or can contaminate crops ready to harvest. Drift may also be a hazard to people, domestic animals, or pollinating insects. Excessive drift also reduces the pesticide applied to the target and can reduce the effectiveness of a treatment. Runoff is the movement of pesticides in water over a sloping surface. Runoff can also occur when water is added to a field faster than it can be absorbed into the soil. Pesticides may move with runoff as compounds dissolved in the water or attached to soil particles. Leaching is the movement of pesticides in water through the soil. The factors influencing whether pesticides will be leached into groundwater include characteristics of the soil and pesticide, and their interaction with water from a rain-event such as irrigation or rainfall. Groundwater may be contaminated if pesticides leach from treated fields, mixing sites, washing sites, or waste disposal areas. Absorption is the uptake of pesticides and other chemicals into plants or microorganisms. Most pesticides break down once they are absorbed. Pesticide residues may be broken down or remain inside the plant or animal and be released back into the environment when the animal dies or as the plant decays. Crop removal through harvest or grazing may remove pesticide residues.

3) BREAKDOWN and DEGRADATION Degradation is the process of pesticide breakdown after application. Pesticides are broken down by microbes, chemical reactions, and light or photodegradation. This process may take anywhere from hours or days to years, depending on environmental conditions and the chemical characteristics of the pesticide. Pesticides that break down quickly generally do

INTRODUCTION

6

not persist in the environment or on the crop. However pesticides that break down too rapidly may only provide short-term control. Microbial breakdown is the breakdown of chemicals by microorganisms such as fungi and bacteria, and chemical breakdown is the breakdown of pesticides by chemical reactions in the soil. In case of photodegradation, the pesticides are decayed by sunlight, and all pesticides are susceptible to photodegradation to some extent

1.3. INSECTS AS MODEL ORGANISMS A model organism is an animal, plant or microbe that can be used to study certain biological processes. The history of model organisms began with the idea that certain organisms can be studied and used to gain knowledge of other organisms or as a control (ideal) for other individuals of the same species. Characteristics of good model organisms are that they breed quickly; they are widespread and readily available for use in experiments; they are easily maintained in laboratory; the biology of model organisms is well known; and they are sensitive towards toxicant or some other factor that is investigated. Insects are good models when the mechanisms underling evolution and speciation are studied there are more than one million species and their diversity and distribution is amazing. Besides that, insects are prevalent in almost all types of habitats and biotopes so it is important to conduct intensive research about their ecology, morphology and physiology. Since insect often live close to people, urban and also agricultural areas, they are exposed to many synthetic chemicals like pesticides i.e. insecticides. Although the main targets of insecticides are pest insects, many other neutral and beneficial insects are also affected. Beneficial insects play an important role in reducing and controlling populations of both plant and insect pests by acting as predators or parasitoids to these detrimental organisms, they also act as pollinators or produce useful products, and can also serve as food. Neutral and beneficial insects are very diverse and belong to different orders Hemiptera, Neuroptera, Coleoptera, Diptera, Hymenoptera etc. So in order to investigate the effect of insecticide on non-target insect species, first it is necessary to determine which model insect will be used in the research. Today many insect species are used as model organisms fruit fly, cockroaches, locusts, grasshoppers, lepidopterans, midges etc. A representative of Heteroptera, the firebug Pyrrhocoris apterus, is also often used as model organism. The importance of the firebug as experimental tool for biological research expanded continually. One of the main reasons of its wide use is easy laboratory breeding, and also a lot of morphological, physiological, biochemical, endocrinological, genetics, behavioral etc. data. Due to those facts the firebug was selected as a model species for this study.

INTRODUCTION

7

1.3.1. Effects of insecticides Insecticides are substances of chemical or biological origin that control insect populations. Such substances are mainly used to control pests that infest cultivated plants and crops or to eliminate disease-carrying insects in specific areas. Insecticide treatment represents a severe stress for targeted insects, and their requirements in these situations are essentially similar to combat and reduce the immediate stress problems. Some insecticides are stomach poisons, some inhalation poisons, and others contact poisons. Based on the mode of action some insecticides kill insects, some destroy the insect's ability to reproduce or prevent the insect from growing, and while other ones work as repellents probably acting on some hormones. Insecticides may also affect neurons or muscle systems, and cause disturbances in endocrine system that affect metabolism, homeostasis or development. Subsequently, changes in central nervous system and endocrine system will demonstrate the endpoints of insecticide effect.

1.3.2. Insect central nervous system (CNS) and endocrine system The nervous system receives, transmits and integrates information about the internal and external environments and determines behavior. The insect CNS consists of a brain and a series of ganglia connected to a ventral nerve cord. The CNS is anatomically and functionally closely related to the endocrine system (Figure 2) that consists of brain neurosecretory cells, corpora cardiaca (CC), corpora allata (CA), prothoracic glands and isolated endocrine cells in ganglia and gut (Klowden, 2007).

Figure 2. Schematic diagram of the neuroendocrine complex of P. apterus: 1 protocerebrum; 2 lobi optici; 3 suboesophageal ganglion; 4 hypocerebral ganglion;

5 frontal ganglion; 6 nervi corporis cardiaci; 7 corpora cardiaca; 8 corpus allatum; 9 aorta

INTRODUCTION

8

An important source of insect hormones in certain developing periods is also gonads. The CC are major neuroendocrine structures attached to the brain: they store and secrete neurohormones synthesized by brain neurosecretory cells, but also contain intrinsic neurosecretory cells that synthesize and secrete neurohormones. Corpora allata secrete juvenile hormone and are attached to the corpora cardiaca. The prothoracic glands are the main source of ecdysteroids and other endocrine cells produce hormones with various functions (Keeley, 2010)

1.3.3. Adipokinetic hormones (AKHs) Insect metabolism, especially its energetic component, is predominantly controlled by adipokinetic hormones (AKHs), which are synthesized, stored and released by neurosecretory cells of CC (Gde et al., 2003). They are okta-, nona- or decapeptides with both termini blocked. N-terminus is blocked by pyroglutamate residue and C-terminus is amidated. AKHs control insect intermediary metabolism by operating as typical stress hormones. Although the major function of AKHs is the control of insect metabolism, these peptides are pleiotropic, with a number of actions attached to their metabolic role (Kodrk, 2008). Generally, they behave as typical stress hormones by stimulating catabolic reactions (mobilization lipids, carbohydrates and/or certain amino acids), making energy more available, while inhibiting synthetic reactions. They mobilize entire energy reserves to combat the immediate stress problems and suppress processes that are momentarily less important and could, if allowed to continue, even draw on the mobilized energy (Kodrk et al., 2010a). Dozens of AKHs have been identified from all main insect orders so far (Gde et al., 1997; Gde et al., 2003) including Heteroptera, where a number of various AKH were identified (Kodrk et al., 2010). For various studies of AKH characteristics a representative of Heteroptera the firebug Pyrrcohoris apterus is commonly used. Two AKHs Pyrap-AKH and Peram-CAH-II were recently identified in this species (Kodrk et al., 2000; Kodrk et al., 2002b). The firebug appears to be a suitable model for the study of mutual interactions of pesticides with the insect endocrine system, especially with AKHs, because there is a lot of information available on its physiology, biochemistry and genetics.

1.4. OXIDATIVE STRESS Oxidative stress occurs when the production of potentially destructive reactive oxygen species (ROS) exceeds the bodies own natural antioxidant defenses, resulting in organism damage or even in its death. This imbalance can result from a lack of antioxidant capacity caused by disturbance in production, distribution, or by an over-abundance of ROS from an environmental or behavioral stressor. If not regulated properly, the excess ROS can damage a cells lipids, protein or DNA, inhibiting natural function.

INTRODUCTION

9

ROS are products of normal cellular metabolism (Figure 3). Most of the body's energy is produced by the enzymatically controlled reaction of oxygen with hydrogen in oxidative phosphorylation occurring within the mitochondria during oxidative metabolism. During those enzymatic cascades, free radicals are formed (Valko et al., 2007).

Figure 3. Major pathways of reactive oxygen species generation and metabolism.

ROS are small, highly reactive, oxygencontaining molecules like hydrogen peroxide, hydroxyl radical and superoxide anion. Also, transition metals like Fe or Cu, although required for certain enzymatic functions, exacerbate oxidative damage by catalyzing the conversion of hydrogen peroxide into highly reactive hydroxyl radical. ROS have many effects on cell metabolism such as role in apoptosis, induction of host defense genes and mobilization of ion transport systems, redox signaling etc. (Rada et al., 2008). But if not effectively and rapidly removed from the cells, ROS generated in oxidative metabolism inflict damage on all classes of macromolecules and can ultimately lead to cell death. ROS can damage a wide range of macromolecules and have harmful effects they can damage DNA, cause oxidations of polydesaturated fatty acids in lipids (lipid peroxidation), cause oxidations of amino acids in proteins, inactivate specific enzymes by oxidation of co-factors etc. (Figure 4).

INTRODUCTION

10

Figure 4. Regulation of cellular processes in response to oxidative stress: ROS may induce cell damage or may initiate a cascade of adaptive signaling mechanisms

that lead to proliferation, differentiation, adaptation or apoptosis.

Apart from the above mentioned endogenous sources the ROS can be created by acting of various exogenous sources. Those sources include exposure to environmental pollutants such as emission from automobiles and industries, consumption of alcohol in excess, cigarette smoking, contact with asbestos, exposure to ionizing radiation, and bacterial, fungal or viral infections. Pesticides may also cause generation of ROS, which may lead to oxidative stress, indicating the role of ROS in pesticide toxicity (Yang et al., 1996). Antioxidant systems are involved to counteract the toxicity of reactive oxygen species (Figure 5). These systems that tend to inhibit oxyradical formation include the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione-dependent peroxidase (GPX) and glutathione reductase (GR) which are critically important in the detoxification of radicals to nonreactive molecules (Van der Oost et al., 2003). Also, numerous low-molecular-weight antioxidants, such as GSH, -carotene (vitamin B), ascorbate (vitamin C), -tocopherol (vitamin E) and ubiquinol10 have been described (Lopez-Torres et al., 1993). Under normal conditions these antioxidants protect the cells and tissues from oxidative damage.

INTRODUCTION

11

Figure 5. Antioxidant pathways.

What are the cellular defense strategies against oxidative damage by ROS? Main among them is the enzyme SOD. This enzyme scavenges superoxide radicals by catalyzing the conversion of two of these radicals into hydrogen peroxide and molecular oxygen. The oxidized form of the enzyme is reduced by superoxide to form oxygen. The reduced form of the enzyme, formed in this reaction, then reacts with a second superoxide ion to form peroxide, which takes up two protons along the reaction path to yield hydrogen peroxide. The hydrogen peroxide formed by SOD and by other processes is scavenged by CAT, a ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. SOD and CAT are remarkably efficient, performing their reactions at or near the diffusion-limited rate. Other cellular defenses against oxidative damage include the antioxidant vitamins, vitamins E and C. Because it is lipophilic, vitamin E is especially useful in protecting membranes from lipid peroxidation. The importance of the cell's defense against ROS is demonstrated by the presence of SOD in all aerobic organisms (Stegeman et al., 1992).

1.4.1. Biomarkers of oxidative stress Ecotoxicological biomarkers are defined as biochemical, cellular, histological or physiological changes that can be measured in tissue samples or body fluids or at the organism level, and they provide early evidence about exposure and/or effects of one or more pollutants (Kurelec et al., 1993). Biomarkers are used as indicators of a biological state. Many biomarkers have been developed to evaluate oxidative stress. Since many antioxidant enzymes and low-molecular-weight antioxidants participate in removal of ROS, by measuring their activities and content it is possible to evaluate oxidative stress.

INTRODUCTION

12

1.4.2. Catalase (CAT) Catalase (CAT) is a heme containing redox enzyme present in peroxisomes of nearly all aerobic cells. This enzyme serves to protect the cell from the toxic effects of hydrogen peroxide by catalyzing its decomposition into molecular oxygen and water without the production of free radicals. The protein exists as a dumbbell-shaped tetramer of four identical subunits, each containing a heme prosthetic group at the catalytic center. The very rigid, stable structure of CAT is resistant to unfolding, which makes them uniquely stable enzymes that are more resistant to pH, thermal denaturation and proteolysis than most other enzymes. This enzyme breaks down hydrogen peroxide by a two-stage mechanism in which hydrogen peroxide alternately oxidizes and reduces the haem iron at the active site. The chemistry of catalase catalysis has not been precisely solved yet, but the following has been proposed. The catalytic process occurs in two already stages: H2O2 + Fe(III)-E H2O +O=Fe(IV)-E (1) H2O2 + O=Fe(IV)-E H2O + Fe(III)-E (2) where Fe-E represents the iron center of the heme attached to the rest of the enzyme (E). In the first step, one hydrogen peroxide molecule oxidizes the haem to an oxyferryl species. In the second step, a second hydrogen peroxide molecule is used as a reductant to regenerate the enzyme, producing water and oxygen. Much of the hydrogen peroxide that is produced during oxidative cellular metabolism comes from the breakdown of one of the most damaging ROS, namely the superoxide anion radical. As earlier mentioned, superoxide is broken down by SOD into hydrogen peroxide and oxygen. Peroxisomes participate in the metabolism of fatty acids. Hydrogen peroxide is a byproduct of fatty acid oxidation. Also, white blood cells produce hydrogen peroxide to kill bacteria. In both cases CAT prevents the hydrogen peroxide from harming the cell itself. It also helps prevent the conversion of hydrogen peroxide to hydroxyl radicals, potentially dangerous molecules that can attack and even mutate DNA. CAT is one of the enzymes that form the first line of defense against free radicals therefore its regulation depends mainly upon the oxidant status of the cell. However, there are other factors involved in their regulation, including the enzyme-modulating action of various hormones such as growth hormone, prolactin and melatonin.

1.4.3. Protein carbonyls (PC) Proteins can become modified by a large number of reactions involving ROS. Various forms of ROS are well known to promote non-specific protein oxidation, with negative effects on protein structure and function. Protein oxidation can involve cleavage of the polypeptide chain, modification of amino acid side chains, and conversion of the protein to derivatives that are highly sensitive to proteolytic degradation.

INTRODUCTION

13

Carbonyl derivatives are formed by a direct metal catalyzed oxidative (metal catalyzed oxidation, MCO) attack on the amino-acid side chains of proline, arginine, lysine, and threonine. In addition, carbonyl derivatives on lysine, cysteine, and histidine can be formed by secondary reactions with reactive carbonyl compounds on carbohydrates (glycoxidation products), lipids, and advanced glycation/lipoxidation end products (Figure 6).

Figure 6. Carbonylation and derivatization of a protein amino-acid side chain.

The quantitatively most important products of the carbonylation reaction are glutamic semialdehyde from arginine and proline, and aminoadipic semialdehyde from lysine (Requena et al., 2003). Compared to other oxidative modifications, carbonyls are relatively difficult to induce and in contrast to, for example, methionine sulfoxide and cysteine disulfide bond formation, carbonylation is an irreversible oxidative process (Dalle-Donne et al., 2003b). Thus, a cell must rid itself of carbonylated proteins by degrading them. Different sensitive methods have been developed for the detection and quantification of protein carbonyl groups and most of these involve derivatization of the carbonyl group with 2,4-dinitrophenylhydrazine and subsequent immunodetection of the resulting hydrazone using monoclonal or polyclonal antibodies (Levine, 2002).

INTRODUCTION

14

The action of reactive oxygen species on proteins has been widely demonstrated to increase the formation of carbonyl groups. Moreover, carbonyl stress may be due to the damaging effect of various monodicarbonyls such as malondialdehyde (MDA) and 4-hydroxy-2,3-nonenal (HNE) and of hypochlorous acid (whose production is catalyzed by myeloperoxidase in neutrophils) on proteins. MDA and HNE are end-products derived from peroxidation of polyunsaturated fatty acids and related esters. In contrast to free radicals, aldehydes are relatively stable and therefore able to diffuse within or out of the cell and to attack targets distant from the site of original free-radical-initiated events. These aldehydic molecules are considered ultimate mediators of toxic effects elicited by oxidative stress occurring in biological materials (Meagher et al., 2000). Measurement of MDA levels in plasma or serum provides a suitable in vivo index of lipid peroxidation and represents a non-invasive biomarker of oxidative stress often clinically employed to investigate radical-mediated physiological and pathological conditions (Meagher et al., 2000).

1.4.4. Thiobarbituric acid reactive substances (TBARS) Besides the protein carbonyls formation, generated ROS can cause one more biochemical alteration lipid peroxidation. If high concentration of ROS is generated, they can attack polyunsaturated fatty acids in the cell membrane leading to a chain of chemical reactions called lipid peroxidation or the oxidation of polyunsaturated fatty acids (Figure 7).

Figure 7. Basic reaction sequence of lipid peroxidation.

INTRODUCTION

15

As a fatty acid is broken down, aldehydes are formed (Halliwell and Chirico, 1993). Among the aldehydes, malondialdehyde (MDA) is the major product of lipid peroxidation. The most common method used to assess lipid peroxidation is based on reaction of MDA with the 2-thiobarbituric acid (TBA). The resulting products modify the physical characteristics of biological membranes, and have impact on membrane Ca2+ transport, as also on molecules connected on membranes such as proteins and cholesterol. Peroxidized membranes become rigid and lose permeability and integrity (Ursini et al., 1991).

1.5. REVIEW OF RECENT STUDIES OF AKH AND Pyrrhocoris apterus AS A MODEL SPECIES The firebug, Pyrrhocoris apterus, is a common Palaearctic phytophagous species with core distribution in the Mediterranean area and eastern and central Asia. In central Europe this species can be found at the bases of linden (Tilia spp.) trees whose seeds serve as a food. It is a convenient model insect for which detailed information on its biology is already available (Socha, 1993). P. apterus is a typical representative of the wing-polymorphic insects which have evolved flightlessness (Honk, 1995; Socha and Zemek, 2000a) and changed the modus of their movement from flight to walking (Socha and Zemek, 2000b). This bug produces individuals of long-winged (macropterous) and short-winged (brachypterous) morphs (Socha, 1993). In this bug, both reproductive diapause and wing polymorphism are controlled by photoperiod and temperature (Hodek 1968; Honk 1976). Development of macropterous morph is controlled by a recessive allele whose expression is favoured by a long-day photoperiod and a high temperature (Honk, 1976, 1981). Under short-day conditions (photophase

INTRODUCTION

16

characteristics between long-wing (macropterous) and short-winged (brachypterous) morphs used the Locusta migratoria AKH the Locmi-AKH-I. The adipokinetic response, expressed as an increase of haemolymph lipids after injection of the Locmi-AKH-I was assessed in relation to age, wing dimorphism and type of reproductive arrest (Socha et al., 1999a). These two wing morphs can be distinguished by specific behavioral, biochemical and physiological features, including the differences in their adipokinetic response. They might represent important features associated with different roles of two wing morphs in the life of this species. On another paper a hypothesis whether AKH can affect locomotor activity in the flightless bug P. apterus was tested (Socha et al., 1999b). Experimental bugs were injected with hormone and control received only Ringers saline. Each treated female was immediately transferred into the activity monitoring unit, and its locomotor activity was recorded. The results revealed higher locomotor activity in AKH treated bugs than those injected with saline only. Later on, two own AKHs were isolated and characterized from P. apterus (Kodrk et al., 2000; Kodrk et al., 2002b). Pyrap-AKH is an octapeptide with the sequence pGluLeuAsnPheThrProAsnTrpNH2, and this peptide was the first identified adipokinetic hormone described in a representative species of the suborder Heteroptera. The second adipokinetic hormone found in P. apterus is Peram-CAH-II. This is also an octapeptide with the sequence pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2. This peptide differed from the Pyrap-AKH by one amino acid in position 3. Topical application and/or injection of the peptide induced lipid mobilization, but was inactive in mobilization of carbohydrates. A quantitative study of adipokinetic hormone of P. apterus was conducted by means of enzyme-linked immunoassay (ELISA) (Goldsworthy et al., 2002). The ELISA measures as little as 20 fmol of Pyrap-AKH. Tested against a range of synthetic peptides, the assay had a high sensitivity for peptides containing the C-terminal motif FTPNWamide. The amounts of Pyrap-AKH in the brain, corpora cardiaca, suboesophageal ganglia, and fused thoracic and abdominal ganglionic mass were very small, with only the corpora cardiaca containing appreciable levels of the hormone (cca. 4 pmol per bug). Measurements of circulating titres of AKH in P. apterus are only possible in the ELISA described in this paper by using pooled samples of haemolymph, and after preliminary clean-up of the haemolymph samples. The AKH-content of the corpora cardiaca/corpus allatum complex can be estimated by calibrating the hyperlipaemic response to tissue extracts against the doseresponse data of (Kodrk et al., 2000) for Pyrap-AKH in Pyrrhocoris. The data presented in this research for the determination of the Pyrap-AKH-content using an ELISA, suggest a value of about 4 pmol per pair of corpora cardiaca.

INTRODUCTION

17

Effect of topical application of Pyrap-AKH on P. apterus was investigated in order to determine the relationship between the stimulatory activity of the Pyrap-AKH injection on locomotor activity and the possible simultaneous effect of injury stressor (due to injection) on this process (Kodrk et al., 2002a). Both injection and topical application increased the levels of lipids in the haemolymph and stimulated locomotor activity. Comparison of two different methods of Pyrap-AKH application on P. apterus showed that AKH can stimulate the bug's locomotion independently of a potential injury stressor. However, the positive correlation between the hyperlipemia and the effect on locomotion holds only for injection, suggesting that a stressing factor caused by injection could play a role in the appearance of the complex response to the adipokinetic hormone. When the injury stressor is absent (such as in topical application) the responses are slower. It seems evident that the different rates of lipid mobilization in response to injection and topical application are primarily caused by different dynamics of the titers of AKH in the body, but one could be speculated that this phenomenon might also be related to the effect of injury stress. The results with the topical application of Pyrap-AKH on macropterous females, especially its delayed stimulatory effect on locomotion, indicated that the whole process may be mediated through the metabolic pathway supplying the fuels necessary for enhanced walking activity, probably via mobilization of lipids (Socha et al., 1999b). A neuromodulatory effect of the Pyrap-AKH in this process is highly unlikely. Developmental and diel changes of both P. apterus AKHs in CNS and haemolymph were also investigated (Kodrk et al., 2003). Significant differences among developmental changes of the AKH content in CNS of three experimental groups of bugs were determined. The highest amount of AKHs was revealed in macropterous, intermediate in reproductive brachypterous, and the lowest one in diapausing brachypterous bugs. There is a continuous increase in the amounts of these hormones with the age in all experimental groups. The AKH content in CNS of macropterous bugs fluctuates during a 24 h cycle of 18 h light : 6 h dark photoperiod with the peak occurring during the photophase (c. 17 h). Diel rhythm of AKH content in the CNS of macropterous bugs is positively correlated with diel rhythm in adipokinetic response of the fat body to AKH. These results indicated that regulation of haemolymph lipid concentration is more complex in this species and involves at least two mechanisms, i.e. changes in the release (or storage) pattern of AKH in the CC, and changes in the number and/or sensitivity of available AKH receptors in the fat body cells. A question arose, if AKHs could play their role in stress situations which are not directly related to mobilization and following consumption of energy. To verify this hypothesis the stress situation was elicited by application of insecticides. The effect of an insecticide, permethrin, on the titer of two adipokinetic hormones in the central nervous system (CNS) and haemolymph of the P. apterus was tested (Kodrk et al., 2005). This study proved the positive effect of the insecticide permethrin on the titer and ratio of AKHs in adults, and

INTRODUCTION

18

contributed to a better understanding of endocrinological response of insects to insecticides. The increase of AKH in haemolymph after the treatment was 6- to 8-fold in macropters, only about 2.5-fold in reproductive brachypters, and nil in diapausing brachypters. The low response of diapausing brachypterous adults to insecticide treatment probably resulted from the lowered level of their metabolism (ula et al., 1998) and from the higher resistance of a diapausing organism to the insecticide doses used. In another paper (Kodrk et al., 2010a) an effect of the interactions pyrethroid vs. AKH on physiological processes in P. apterus body were studied. The results showed that co-injection of permethrin with Pyrap-AKH induced a significant 2.3 fold increase in the bug mortality compared to the insecticide alone. Also, injections of permethrin elicited significant increase of the AKH level in CNS and the haemolymph. These results indicated an involvement of AKH in stress response to permethrin. The enhanced effect of insecticide by AKH treatments probably results from the stimulatory role on bug metabolism: the carbon dioxide production was increased after the permethrin treatment, and also after the permethrin plus AKH co-treatment, compared to control. The elevation of metabolism could intensify the permethrin action by its faster penetration into tissues and by stimulation of biochemically active cells, and could be a reason for enhanced action of permethrin after its co-treatment with Pyrap-AKH. By using paraquat (PQ), a bipyridilium herbicide, the in vivo effects on oxidative stress in P. apterus were investigated (Veea et al., 2007). Result showed that PQ treatment did result in significantly enhanced carbonyl contents in hemolymph, but interestingly co-injection of Pyrap-AKH with PQ decreased their levels to those found in control groups. Also surprising was that Pyrap-AKH injection alone did not change the carbonyl contents to those below control values. This indicates possibly that stressor action is needed for AKH to potentiate the response as in case of phenoloxidase activity (Goldsworthy et al., 2002) or lipid stores mobilization rate after injection or topical application of external AKH (Kodrk et al., 2002a).

RESEARCH OBJECTIVES

19

2. RESEARCH OBJECTIVES

Since adipokinetic hormones (AKHs) are insect neuropetides controlling stress situations including those elicited by insecticide treatment, the main goal of the research presented in this Master Thesis was to investigate the AKH characteristics under stress conditions elicited by an insecticides endosulfan and malathion in a model species Pyrrhocoris apterus.

Research objectives: to determine mortality i.e. LD15 and LD50 values for endosulfan and malathion; to determine whether exposure to endosulfan and malathion together with AKH

cotreatment causes increased mortality compared to exposure just to insecticide alone;

to determine whether exposure to endosulfan and malathion causes changes in total AKH content in CNS and haemolymph;

to determine a level of metabolism by means of a carbon dioxide production after exposure to insecticides endosulfan and malathion, to Pyrap-AKH, and to Pyrap-AKH together with the insecticides;

to determine whether exposure to endosulfan and malathion causes oxidative stress in P. apterus.

Based on the previous research on effect of a insecticide against the firebug P. apterus (Kodrk et Socha, 2005; Kodrk et al., 2010a), which showed interactions between AKH and pyrethroid insecticide, the aim of this research was to determine whether insecticides from other chemical classes (organochlorine and organophosphorus insecticides) will also possess effects on physiology and endocrinology of firebug P. apterus.

MATERIALS AND METHODS

20

3. MATERIALS AND METHODS

3.1. CHEMICALS

For preparation of this Master Thesis, chemicals used for proccesing of samples and all necesarry measurements were:

Methanol Ethanol Ethyl acetate Sodium hydroxide, NaOH Sodium chloride, NaCl Potassium chloride, KCl Calcium chloride, CaCl2 Sodium carbonate, Na2CO3 Sodium bicarbonate, NaHCO3 Disodium hydrogen phosphate, Na2HPO4 Sodium dihydrogen phosphate, NaH2PO4 Potassium dihydrogen phosphate, KH2PO4 Potassium hydrogen phosphate, K2HPO4 Hydrochloric acid, HCl Sulfuric acid, H2SO4 Citric acid, C6H8O7 Bicinchninic acid, BCA Trichloroacetic acid, TCA Trifluoroacetic acid, TFA Acetonitrile, CH3CN Copper sulfate, CuSO4 Hydrogen peroxide, H2O2 Polysorbate 20, Tween 20 5 % skim milk Ortho-phenylenediamine, OPD Ethylenediaminetetraacetic acid, EDTA 2,4-dinitrophenylhydrazine, DNPH 2-thiobarbituric acid, TBA Butylated hydroxytoluen, BHT Guanidine hydrochloride Streptomycin sulfate Biotin Long Arm Maleimide (BLAM) (Vector Laboratories, Peterborough, UK) Cys1-Pyrap-AKH (Sigma Genosys, Cambridge, UK) Horseradish peroxidase, HRP (Vector Laboratories) Pyrap-AKH, Polypeptide Laboratories s.r.o. (Prague, Czech Republic)


21

3.2 INSTRUMENTS AND EQUIPMENT

For preparation of this Master Thesis next instruments and equipment were used: Laboratory glassware Glass jars, 0.25 l and 0.5 l Dissection instruments Hamilton syringe, 10 l (Hamilton, Reno, Nevada) Plastic test tubes, 2 ml, Eppendorf Adjustable micropipettes, Nichipet Adjustable micropipettes, Eppendorf Refrigerator Freezer, -20 C Freezer, -80 C Water bath Thermobox Microscope, Olympus Vortex mixer Magnetic mixer Potter-Elvehjem homogeniser Mechanical Stirrer, Heidolph RZR 2021 Ultrasonic homogeniser (Bandelin Sonopuls HD 2070) Ultrasonic homogeniser (4710 series, Cole-Pharmer Instrument Co., Chicago, IL,

USA) Microtitre plates (Gama, Ceske Budejovice, Czech Republic) Microtitre plates (high binding Costar, Corning Incorporated, Corning, NY, USA) Scale, Ohaus Explorer Centrifuge, Hettich EBA 21 Centrifuge, Hettich EBA 12 R Vacuum concentrator/centrifugal evaporator (Jouan RC 1022) Vibrating platform shaker (Heidolph Titramax 1000) ELISA Reader (SpectraMax 340PC) BioTek Synergy4 Hybrid Multi-Mode Microplate Reader Heios Thermospectronic UV-spectrophotometer v4.55 LI-7000 CO2/H2O analyser (LI-COR Biosciences, Lincoln, NE) Merck-Hitachi D-6000 chromatography system Chromolith Performance RP-18e column


22

3.3. EXPERIMENTAL INSECTS

3.3.1. Taxonomy of firebug (Pyrrhocoris apterus) Kingdom: Animalia animals Phylum: Arthropoda arthropods Class: Insecta insects Subclass: Pterygota winged insects Superorder: Exopterygota Order: Hemiptera true bugs, cicadas, hoppers, aphids and allies hemiptera Suborder: Heteroptera true bugs Family: Pyrrhocoridae red bugs Genus: Pyrrhocoris firebugs Species: Pyrrhocoris apterus firebug

Figure 8. Pyrrhocoris apterus.

3.3.2. Biology of the firebug (Pyrrhocoris apterus) This bug belongs to superorder Exopterygota in which insects undergo a simple or incomplete metamorphosis. The life cycle includes just three stages egg, nymph and adult. During the nymph stage, gradual change occurs until the nymph resembles the adult. Only the adult stage has functional wings. P. apterus also belongs to the order Hemiptera whose main characteristic is possession of mouthparts where the mandibles and maxillae have evolved into a proboscis, sheathed within a modified labium to form a "beak" or "rostrum" which is capable of piercing tissues (usually plant tissues) and sucking out the liquids typically sap (Kendall, 2010). Pyrrhocoridae is a family of insects with more than 300 species world-wide. A common species in Europe is the firebug. The firebug, P. apterus (Figure 8), is


23

a black and red European bug the size of a finger-nail. Their diet consists primarily of seeds from linden trees and mallows. The firebug belongs to a dimorphic species, which produces adults of the long-winged (macropterous) and short-winged (brachypterous) morphs (Seidenstcker, 1953). Most adults are short-winged, though a few long-winged individuals may occur in any population (up to 14 %). They occur in woodland margins and clearings, and grassy scrubby places, with bare ground and suitable hibernation sites. They are widespread in southern and central Europe. This bug is a good model organism for experiments and it is readily reared in the laboratory in glass jars, on a diet of water and dry linden seeds, and with crumpled paper as a substitute for the leaf litter (Carlisle et al., 1966).

3.3.3. Maintenance of the culture A stock cultures of the firebug, P. apterus (L.) (Heteroptera, Insecta), established from wild populations collected at esk Budjovice (Czech Republic), were used in preparation of this Master Thesis. Nymphs and adults of the short-winged (brachypterous) morph were kept in 0.5 l glass jars (Figure 9) in a mass culture (approximately 40 specimens per jar) and reared at constant temperature of 26 1 C under long-day conditions (18 h light : 6 h dark). They were supplied with linden seeds and water ad libitum, which were replenished twice weekly. Freshly ecdysed adults were transferred to small 0.25 l glass jars (females and males separately) and kept under the same photoperiodic, food and temperature regimes at which they had developed.

Figure 9. Cultures of the firebug Pyrrhocoris apterus.


24

3.4. CHARACTERISTICS OF INSECTICIDES Formulations of insecticides used in this experiment were for commercial usage. Besides the active substance, formulation also contains other ingredients like solvens, surface active compounds, carriers and antioxidans which improve insecticide properties for storage, handling and usage. The active substance is chemical compund that has specifical effect on the target organism.

3.4.1. Endosulfan (Global E-35) Endosulfan (Figure 10) is an insecticide with contact and gut action that belongs to a group of organochlorine cyclodiene pesticides. Global E-35 (produced by: Chromos Agro d.d.) is commercial name of endosulfan preparation used in this experiment. It is used against a wide variety of insects and mites. It acts as neurotoxin by inhibiting GABA receptors at synapses, Ca2+ and Mg2+ ATPase, and acetylcholinesterase, and also works as an endocrine disruptor (EXTOXNET PIP, 1996; IPCS, 2010).

CHEMICAL NAME: 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3 benzadioxathiepin 3-oxide

TRADE NAME: Global E-35 CHEMICAL CLASS: chlorinated hydrocarbon MOLECULAR FORMULA: C9H6Cl6O3S REALTIVE MOLECULAR MASS: 406.96 APPEARANCE: pure endosulfan is a colourless crystal, technical grade is a yellow-brown

colour MELTING POINT: 79 100 C (technical endosulfan) WATER SOLUBILITY: 0.33 mg/l, 22 C SOLUBILITY IN OTHER SOLVENTS: s. in toluene and hexane

Figure 10. Structure of endosulfan.


25

3.4.2. Malathion (Radotion E-50) Malathion (Figure 11) is one of the most often used organophosphate insecticide in the world. Radotion E-50 (produced by: Herbos) is commercial name of malathion preparation used in this experiment. It is a nonsystemic organophosphorous insecticide and acaricide with contact, gut and respiratory action. Malathion is metabolically converted to its structurally-similar metabolite, malaoxon, in insects (oxidative desulfurization) and mammals (hydrolsyis by carboxylesterase). Malaoxon inhibits an important nervous system enzyme acetylcholinesterase (EXTOXNET PIP, 1996; IPCS, 2010).

CHEMICAL NAME: diethyl (dimethoxy thiophosphorylthio) succinate TRADE NAME: Radotion E-50 CHEMICAL CLASS: organophosphate MOLECULAR FORMULA: C10H19O6S2P REALTIVE MOLECULAR MASS: 330.36 APPEARANCE: technical malathion is a clear, amber liquid at room temperature MELTING POINT: 2.85 C WATER SOLUBILITY: 145 mg/l SOLUBILITY IN OTHER SOLVENTS: v.s. in most organic solvents

Figure 11. Structure of malathion.

3.5. INSECTICIDE TREATMENT Insects were treated with different doses of insecticides endosulfan and malathion by injecting from 5 1000 ng solved in 2 l Ringer saline. This volume was injected by using a Hamilton syringe (10 l; Hamilton) through the metathoracal-abdominal intersegmental membrane to the thorax of the experimental bug.


26

3.5.1. Preliminary test for determination of mortality Preliminary test was done to define doses for final test in order to determine the mortality. The experimental bugs were treated individually by injection into the haemocoel with 5, 100, 200, 300, 500 and 1000 ng insecticide. In each dose 10 individuals were used (both male and female bugs were used). Mortality was determined after 24 hours of exposure (by determining the number of died and living bugs), and the results were used for determining which doses should be used in the final test.

3.5.2. Final test for determination of mortality After results obtainted from preliminary test, doses for final test were determined. The experimental bugs were treated individually by injection into the haemocoel with 225, 250 and 275 ng of endosulfan, and with 450, 475 and 500 ng of malathion. In each dose about 20 individuals were used (both male and female bugs were used). Mortality was determined after 24 hours of exposure. The LD15 and LD50 were determined by a probit analysis from the number of bugs that died during 24 hours after insecticide treatment.

3.6. HORMONAL TREATMENT Insecticide doses close to the LD15 and LD50 (200 and 250 ng for endosulfan, and 300 and 450 ng for malathion, respectively) were used for analyses of the insecticide interaction with the P. apterus AKH. Pyrap-AKH (dominant member of the two P. apterus AKHs, Pyrap-AKH and Peram-CAH-II), custom synthesized by Polypeptide Laboratories s.r.o. (Prague, Czech Republic), was dissolved in 20% methanol in Ringer saline. The experimental bugs (both male and female bugs were used) were injected with insecticide and cotreated with the hormonal solution (80 pmol in 2 l of the solution), using the methods described above. A 30 minutes delay between the treatments was maintained for recovery of the bugs from the stress of the first injection. Similarly, control bugs were treated with Pyrap-AKH only. Results were evaluated 24 hours later.

3.7. DISSECTION OF THE CNS After exposure to insecticides for 24 hours bugs were dissected (ten-day old brachypterous females were used). Central nervous system, which consists of brain with CC and CA attached, was dissected under microscope by using the scissors and forceps. First, the head together with first thoracic segment was cut off. Then the first thoracic segment together with the remains of other organs was removed and the internal part of the head capsule was washed by Ringer saline to move pieces of fat body and tissue debris. Finally the brain with CC and CA attached was taken out of the head and stored in freezer until the extraction of AKH.


27

3.7.1. Extraction of AKH from CNS Methanolic extract (80% methanol) of the brain with CC and CA attached was used for determination of the AKH content in the CNS by means of a competitive ELISA. Extraction was done in two steps (Figure 12). In the first step, 200 l of 80 % methanol was added to each sample (1 CNS) and the tissue was homogenized with ultrasonic homogenizator. After centrifuging (1 min, 18 000 rpm) supernatant was separated with micropipette and the pellet was re-extracted once more. Both supernatants were combined and evaporated to dryness in vacuum concentrator; the pellets were thrown away. The residues of the evaporation were dissolved in washing buffer (without Tween) and used for determination of AKH by the competitive ELISA.

Figure 12. Protocol for extraction of AKH from CNS.


28

3.8. HAEMOLYMPH SAMPLING After exposure to insecticides for 24 hours, the haemolymph was collected from ten-day old brachypterous females which were used for this experiment. The top of the antenna was cut off with scissors and by gentle squeezing the bugs body, haemolymph leaked out. The haemolymph was then collected with micropipette and stored in an eppendorf tube in freezer until the extraction of AKH.

3.8.1. Extraction of AKH from haemolymph For determination of the endogenous AKH titre in the haemolymph by ELISA, some pre-purification steps were essential. Briefly, haemolymph samples were extracted in 80 % methanol and after centrifugation the supernatants were evaporated to dryness (as previously described). The residues of the evaporation were dissolved in 0.11 % TFA (trifluoroacetic acid), applied to a solid phase extraction cartridge (Sep Pak C18, Waters), and eluted in 60 % acetonitrile. The eluent was subjected to HPLC analysis on a Merck-Hitachi D-6000 chromatography system using a Chromolith Performance RP-18e column. Fractions eluting between 6.25 8.00 minutes were subjected to competitive ELISA. Retention times of the two P. apterus synthetic adipokinetic peptides Pyrap-AKH and Peram-CAH-II were 7.08 and 7.15 minutes, respectively. The efficiency of recovery of haemolymph AKH during the extraction procedure was checked by adding 500 fmol of Pyrap-AKH to 20 l samples of haemolymph before the extraction. The recovery of AKH was checked using ELISA and estimated from five separate parallel measurements.

3.9. QUANTIFICATION OF AKH BY COMPETITIVE ELISA A competitive ELISA was used for determination of total AKH content in Pyrrhocoris apterus CNS and haemolymph (Goldsworthy et al., 2002) (Figure 13). Briefly, rabbit antibodies were raised commercially against Cys1-Pyrap-AKH (Sigma Genosys, Cambridge, UK) and the resulting antibody recognised well both the Pyrap-AKH and the Peram-CAH-II. A biotinylated probe was prepared from Cys1-Pyrap-AKH using Biotin Long Arm Maleimide (BLAM, Vector Laboratories, Peterborough, UK). The ELISA comprised pre-coating of a 96-well microtitre plates (Figure 14) (high binding Costar, Corning Incorporated, Corning, NY, USA) overnight with the antibody IgG preparation in coating buffer (IgG dilution 1:2000; 100 l per well). After blocking (non-fat dried milk), test samples (1/4 CNS or 20 l of haemolymph) were added to specific wells, followed by the biotinylated probe (100 fmol per well), both in an assay buffer. After the competition for the binding sites on the IgG bound to the plates a streptavidin conjugated with HRP (horseradish peroxidase, Vector Laboratories), diluted 1:500 was added to each well. All of the above mentioned steps were terminated by washing (with phosphate washing buffer).


29

ELISA Pyrap-AKH

Application of IgG overnight in 4o C, 100 l per well - diluted in coating buffer 1:2000

PREPARATION OF REQUIRED SOLUTIONS:

1. Coating buffer pH 9.6

Na2CO3 - 0.424 g

NaHCO3 - 0.504 g per 100 ml

2. Washing buffer pH 7.5 - 10 mM PBS buffer, stock

solution 10x conc., working solution to solve 1 : 9Per 250 ml NaCl 21.91 g

Na2HPO4 x 2 H2O 1.04 g

NaH2PO4 x 2 H2O 0.37 g To the working solution add Tween 20 to final

concentration 0.1%

Reaction is stopped by 50 l 0.5 M H2SO4

OPD substrate - 100 l per well, 40 min at 37 oC in darkness

Application of HRPS diluted in WB 1:500 100 l per well, 1 h at 37oC

Washing 3 x 200 l in washing buffer WB

50 l Ag solution ( CNS) + 50 l 100 fmol BLAM, both in WB without Tween

Final volume 100 l/well - incubation 1 h at 37 oC

Washing 3 x 200 l in WB

Blocking in 5% milk in WB 200 l per well, 2 h at 37 oC

Washing 3 x 200 l in washing buffer (WB)

Washing 6 x 200 l in WB

PREPARATION OF REQUIRED SOLUTIONS:

3. Blocking buffer - 2 % BSA or 5 % skim milk in

washing buffer

4. OPD (ortho-phenylenediamine) substrate

A - 0.1 M citric acid 3.84 g / 200 ml

B - 0.2 M Na2HPO4 5.68 g / 200 ml

12.15 ml A + 12.85 ml B + 4.25 mg OPD (pH 5.0) +

1.25 l 30 % H2O2

5. 0.5 M H2SO4 - 2.72 ml conc. H2SO4 per 100 ml

Absorbance values were determined in a microtitre plate reader at 492 nm

Figure 13. Protocol for competitive ELISA.


30

Finally, freshly prepared OPD (orthophenylenediamine) reagent was added and then the reaction was stopped by 0.5 M sulphuric acid. The absorbance values were determined in ELISA reader at 492 nm. Two columns of each plate always contained a dilution series of synthetic Pyrap-AKH (standard) which allowed the construction of a competition curve and estimation of the AKH content of the unknown samples. Positive (BLAM +) and negative (BLAM -) controls were also included.

Figure 14. Microtitre plate.

3.10. METABOLIC RATE MEASURING Male bugs were exposed to insecticide doses close to the LD15 (200 ng for endosulfan and 300 ng for malathion) and then used for metabolic rate measuring (Kodrk et al., 2010a). A flow-through respirometry system was used to measure the rate of carbon dioxide production of the experimental bugs. Air is pushed through a chamber with the analysed insects in this system at a flow rate of 60 ml min1 into the LI-7000 CO2/H2O analyser (LI-COR Biosciences, Lincoln, NE), which is interfaced with a computer. The carbon dioxide measurement is based on the difference of infrared radiation passing through two gas cells. A reference gas flows continuously within the reference cell, and its signal is subtracted from the sample cell signal in order to get rid of any variation in the ground signal. The output of the analyser is proportional to the difference in absorption between the two cells. A group of ten bugs was measured (0), 1, 3, 6 and 24 hours after treatment for a period of 20 minutes. Only living individuals were used for the analysis. Data were analysed by data acquisition software (Sable Systems, Las Vegas, NV). The carbon dioxide production (VCO2) was calculated from fractional concentrations of carbon dioxide going into (FI) and coming out of (FE) the respirometry chamber, using an equation according to Withers (1977) and expressed in l h1 bug1:

VCO2 = (FECO2 FICO2) x f where f is the flow rate in l h1.


31

3.11. OXIDATIVE STRESS Effects of insecticides endosulfan and malathion on oxidative stress were examined by measuring 3 biomarkers of oxidative stress catalase activity, protein carbonyls content and amount of thiobarbituric acid reactive substances. Both male and female bugs were exposed to insecticide doses close to the LD15 and LD50 (200 and 250 ng for endosulfan, and 300 and 450 ng for malathion respectively) and then used for analysis.

3.11.1. Catalase measurement Catalase activity was measured spectrophotometrically at 240 nm according to Claiborne (1985) (Figure 15). After exposure to insecticides (3 hours) and insecticides together with AKH cotreatment (3 hours), whole bugs were homogenized in sodium phosphate buffer, and homogenates were centrifuged in order to gain the S9 fraction. Reaction mixture comprised of phosphate buffer and supernatant, and the reaction was initiated by the addition of H2O2. Degradation of H2O2 was measured on 240 nm during 30 seconds, and the activity of catalase was expressed in mol min-1 using the extinction coefficient of 42.6 M-1 cm-1 for H2O2.

Figure 15. Catalase activity protocol.


32

3.11.2. Protein carbonyl determination Protein carbonyls (PC) were quantified after their reaction with 2,4-dinitrophenylhydrazine (DNPH) (Levine et al., 1990) (Figure 16).

Figure 16. Protocol for protein carbonyls determination.


33

After exposure to insecticides for 48 hours, whole bugs were homogenized in potassium phospahte buffer, centrifuged and the supernatants were treated with 28 % TCA (trichloroacetic acid) and centifuged again. Supernatants were discarded and the pellets were mixed with 7 mM solution DNPH in 2 M HCl (sample) or with 2 M HCl only (blank). After incubation in dark at 37 C for 1 hour, samples and blanks were centrifuged and supernatants were casted. Pellets were rinsed 3x with ethanol:ethyl acetate (1:1 v/v) solution and after last centifugation pellets were dissolved in 6 M guanidine-HCl solution. The carbonyls were quantified at 370 nm in a microtitre plate reader and extinction coefficient of 20 000 M-1 cm-1 was used. Amount of carboyl proteins was expressed in nanomoles per miligram proteins in guanidine-HCl solution.

3.11.3. Determination of lipid peroxidation Lipid peroxidation was determined spectrophotometrically by the formation of thiobarbituric acid reactive substances (TBARS) (Rice-Evans et al., 1991) (Figure 17). TBARS determination was performed 24 hours after exposure to insecticides and insecticides together with AKH cotreatment. Whole bugs were homogenized in potassium phospahte buffer, centrifuged and the supernatants were treated with 28 % TCA (trichloroacetic acid) and centifuged again. Supernatants were then mixed with same volume of TBA reagent and blanks contained water instead of the supernatant. TBA reagent consisted of saturated TBA solution in 0.1 M HCl, and BHT was added in reagent to avoid tissue peroxidation during heating. Mixture was then incubated in water bath (on 95 C) for 1 hour. After cooling on ice mixture was centrifuged. Absorbance of supernatant was measured at 535 nm (extinction coefficient of 1.56 x 105 M-1cm-1 was used). Amount of TBARS was expressed in pmol l-1 homogenate.


34

THIOBARBITURIC ACID REACTIVE SUBSTANCES

(TBARS)

PREPARATION OF REQUIRED SOLUTIONS

50 mM K-phosphate buffer (pH 7.0)

for 0.1 M K-phosphate buffer (pH 7.0):

A: 1 M KH2PO4: 8.51 g 62.5 ml distilled H2O

B: 1 M K2HPO4: 10.89 g 62.5 ml distilled H2O

38.5 ml A + 61.5 mL B fill up till 1 l with distilled

H2O (900 ml)

saturated TBA solution

0.67% 2-thiobarbituric acid (TBA): 0.67 g + 100 ml

distilled H2O

0.1 M HCl (FW=36.45)

0.83 ml conc. HCl + 99.17 ml distilled H2O

10 mM butylated hydroxytoluen (FW=220.4)

0.2204 g BHT + 100 ml 96% ethanol

preparation of TBA reagent:

TBA reagent contains saturated TBA solution in 0.1

M HCl and 10 mM BHT previously soluted in

ethanol; pH is adjusted at 2.5 with NaOH)

100 ml TBA + 100 ml 0.1 M HCl + 1 ml BHT

- supernatant is mixed with same volume of TBA reagent

- blank contains water instead of supernatant

- tubes with mixtures are incubated in water bath (95 C)

for 1 h

- cooling on ice

- CENTRIFUGE OF MIXTURE - 10 min; 5 000 g

- supernatant is used for measurement of TBARS

absorbance at 535 nm

(molar extinction coefficient = 1.56 x 105 M-1cm-1)

(amount of TBARS is expressed in pmol l-1

homogenate)

TISSUE HOMOGENIZATION

- whole bugs were used

- phosphate buffer; ratio 1/5 w/v

- bug tissue with phosphate buffer is homogenized

in Potter-Elvehjem homogenisator for 10 seconds

- in further procedure supernatant (S9) is used

- 100 l aliquot (S9) is mixed with 200 l 28 % TCA

(trichloroacetic acid) vortex

CENTRIFUGE

10 min; 5 000 g

SUPERNATANT is used

for measurement of

TBARS

CENTRIFUGE

15 min; 9 000 g; 4 C

S9

Figure 17. Protocol for TBARS determination.

RESULTS

35

4. RESULTS

4.1. THE EFFECT OF AKH ON THE MORTALITY OF BUGS TREATED WITH ENDOSULFAN AND MALATHION

After determination of LD15 (186.052 ng bug-1 for endosulfan and 279.348 ng bug-1 for malathion) and LD50 (237.448 ng bug-1 for endosulfan and 452.564 ng bug-1 for malathion) (Table 1) by a probit analysis from the number of bugs that died during 24 hours after insecticide treatment, doses close to these were used for analysis of the effect of AKH on mortality of insects treated with endosulfan and malathion. For this purpose, the test was conducted in 4 replicates each containing 20 bugs. After calculating the percentage of mortality for each replicate (Table 2), the t-test was employed in order to determine statistically siginificant differences between the samples.

Table 1. LD15 and LD50 values for endosulfan and malathion in P. apterus 24 h after exposure.

Endosulfan Malathion LD15 (ng) 186.052 279.348 LD50 (ng) 237.448 452.564

Table 2. Mean values and standard deviations of bug's mortality after exposure to insecticides only and insecticides together with AKH.

Insecticide Insecticide only

(N=4) Insecticide + AKH (80 pmol)

(N=4) M SD M SD

Endosulfan 200 ng 30.00 4.08 91.25 2.50 Endosulfan 250 ng 57.50 2.89 98.75 2.50 Malathion 300 ng 25.00 7.07 36.25 8.54 Malathion 450 ng 46.25 10.31 73.75 7.50

Legend: M mean value of bugs mortality SD standard deviation

The results show (Figure 18) that, compared to application of insecticide only, coinjection of 200 ng endosulfan bug1 (close to LD15) with 80 pmol Pyrap-AKH bug1 caused a significant increase in mortality from 30.00 % up to 91.25 % (p=0.00). Similarly, coinjection of 300 ng malathion bug1 (close to LD15) with 80 pmol Pyrap-AKH bug1 also

RESULTS

36

caused increase in mortality, but not significantly from 25.00 % up to 36.25 % only (p=0.089). A similar picture was found in the same experiment when a higher doses of endosulfan 250 ng bug1 (close to LD50) and malathion 450 ng bug1 (close to LD50) were applied. Coapplication of these doses with 80 pmol Pyrap-AKH bug1 caused significant increase in mortality for both endosulfan and malathion. In the case of endosulfan application mortality increased from 57.50 % up to 98.75 % (p=0.00), and in the case of malathion application from 46.25 % up to 73.75 % (p=0.005). Control mortality after injection of the Ringer saline only and Pyrap-AKH only was negligible (0 % for Ringer saline and 1.25 % for Pyrap-AKH).

**

**

**

0

20

40

60

80

100

endosulfan 200 ng;endosulfan 200 ng+AKH

endosulfan 250 ng;endosulfan 250 ng+AKH

malathion 300 ng;malathion 300 ng+AKH

malathion 450 ng;malathion 450 ng+AKH

% o

f mo

rtalit

y a

fter

24 h

insecticide aloneinsecticide + AKH

Figure 18. Increase in mortality in P. apterus after coapplication of 80 pmol Pyrap-AKH bug-1 compared to application of insecticide only; presented results are mean values of the percentages of mortality SD. Significant differences (** p

RESULTS

37

4.2. THE EFFECT OF ENDOSULFAN AND MALATHION ON AKH LEVEL IN CNS

Results of AKH levels in the bugs CNS determined 24 hours after exposure to endosulfan and malathion are shown in Table 3. AKH levels were determined in 15 bugs per experimental group (12 per control group) and t-test was employed to determine the statistically siginificant difference between the samples.

Table 3. Mean values and standard deviations of AKH level in bug's CNS determined 24 h after exposure to endosulfan and malathion.

Legend: M mean value of AKH level (pmol per 1 CNS) SD standard deviation

The results revealed that in both lower (200 ng) and higher (250 ng) dose of endosulfan applied significant increase (p

RESULTS

38

**

**

2.0

2.5

3.0

3.5

4.0

4.5

Ringer saline endosulfan 200 ng endosulfan 250 ng

pmo

l AK

H pe

r 1

CNS

Figure 19. The effect of endosulfan (200 and 250 ng bug1) injected into the haemocoel of P. apterus on the level of AKHs in CNS 24 h after treatment; presented results are mean values of the level of AKH in CNS SD. Significant differences (** p

RESULTS

39

4.3. THE EFFECT OF ENDOSULFAN AND MALATHION ON AKH LEVEL IN HAEMOLYMPH

Results of AKH levels in the bugs haemolymph determined 24 hours after exposure to endosulfan and malathion are shown in Table 4. For determination of AKH levels 20 l of haemolymph was collected per sample, and after the results were obtained the t-test was employed to determine the statistically siginificant difference between the samples.

Table 4. Mean values and standard deviations of AKH level in bug's haemolymph determined 24 h after exposure to endosulfan and malathion.

Legend: M mean value of AKH level (fmol l-1 haemolymph) SD standard deviation

The results revealed that in both lower (200 ng) and higher (250 ng) dose of endosulfan applied increase in AKH levels was recorded, but only in case of higher dose (250 ng) the increase was significant (p

RESULTS

40

**

0.0

0.5

1.0

1.5

2.0

2.5

Ringer saline endosulfan 200 ng endosulfan 250 ng

fmo

l AK

H l

-1

hae

mo

lym

ph

Figure 21. The effect of endosulfan (200 and 250 ng bug1) injected into the haemocoel of P. apterus on the level of AKHs in haemolymph 24 h after treatment; presented results are mean values of the level of AKH in haemolypmh SD. Significant differences (** p

RESULTS

41

4.4. THE EFFECT OF AKH, ENDOSULFAN AND MALATHION ON CARBON DIOXIDE PRODUCTION

The carbon dioxide production was monitored as a marker of the metabolic intensity in the experimental bugs. Results from CO2 production are shown in Table 5.

Table 5. Mean values and standard deviations of CO2 production in bugs determined 1, 3, 6 and 24 h after exposure to endosulfan and malathion.

Legend: M mean value of CO2 production (l h1 bug) SD standard deviation

Time of exposure (h) M SD

Intacted 0 14.040 2.556 1 17.580 3.620 3 17.061 4.467 6 15.738 3.680

Ringer saline

24 14.290 3.423 1 27.042 6.798 3 21.629 4.206 6 17.960 3.409

AKH (80 pmol)

24 18.033 3.182 1 16.478 3.862 3 19.268 5.199 6 21.024 6.671

Endosulfan 200 ng

24 27.653 7.346 1 42.717 12.083 3 39.543 5.942 6 36.473 5.907

Endosulfan 200 ng + AKH (80 pmol)

24 - -

1 32.134 8.199 3 29.272 8.035 6 25.936 5.968

Malathion 300 ng

24 20.574 5.495 1 42.041 10.427 3 35.466 7.800 6 29.485 8.240

Malathion 300 ng + AKH (80 pmol)

24 23.433 6.405

RESULTS

42

A group of ten bugs was used for measurement of CO2 production (0), 1, 3, 6 and 24 hours after treatment. For this purpose, the test was conducted in 5 replicates and only living individuals were used for the analysis. There is no data for CO2 production 24 hours after coapplication of endosulfan (200 ng) with AKH (80 pmol) because of the very high mortality. After measurement one-way ANOVA and Tukeys multiple comparison test were employed to determine the statistically siginificant difference between the samples. Application of endosulfan, malathion and Pyrap-AKH caused significant changes in carbon dioxide production. Malathion and Pyrap-AKH caused increased dioxide production which in time decreased towards control production. Endosulfan caused different trend the carbon dioxide production increased significantly above control production later compared to malathion and Pyrap-AKH. When endosulfan and malathion were applied together with Pyrap-AKH the highest increase in carbon dioxide production was recorded. In case of endosulfan application statistically significant changes (p

RESULTS

43

bdbdcd

c

ad

acd

d cd

bd

ad

ab

abc

abc

abc

0

10

20

30

40

50

60

1 3 6 24

time of exposure (hours)

CO2

l h-1

bu

g-1

Ringer salineAKHendosulfan 200 ngendosulfan 200 ng + AKH

Figure 23. The effect of endosulfan (200 ng bug1) and Pyrap-AKH (80 pmol bug1) both injected into the haemocoel of P. apterus on carbon dioxide production 1, 3, 6 and 24 h after treatment; presented results are mean values of carbon dioxide production SD. Significant differences (p

RESULTS

44

cdbcd cd

cdd

ad

cd

cd

a

adabd

abab

ab

abc

abc

0

10

20

30

40

50

60

1 3 6 24time of exposure (hours)

CO2

l h-1

bu

g-1

Ringer salineAKHmalathion 300 ngmalathion 300 ng + AKH

Figure 24. The effect of malathion (300 ng bug1) and Pyrap-AKH (80 pmol bug1) both injected into the haemocoel of P. apterus on carbon dioxide production 1, 3, 6 and 24 h after treatment; presented results are mean values of carbon dioxide production SD. Significant differences (p

RESULTS

45

4.5. THE EFFECT OF AKH, ENDOSULFAN AND MALATHION ON OXIDATIVE STRESS

4.5.1. Catalase activity

Results of catalase activity measurement are shown in Table 6. Activity was measured in 8 bugs per sample and t-test was employed to determine the statistically siginificant difference between the samples. Significant differences between control and experimental treatments were recorded in all groups after 3 hours of exposure. After injection of insecticide only, significantly higher increase of catalase activity was recorded compared to co-injection of insecticide together with AKH. There were no significant differences between groups exposed to different concentrations of the same insecticide nor between groups exposed to different insecticides, i.e. endosulfan and malathion.

Table 6. Mean values and standard deviations of catalase activity measured 3 h after exposure to AKH, endosulfan and malathion.

Legend: M mean value of catalase activity (mol min-1) SD standard deviation

In both endosulfan doses (lower 200 ng and higher 250 ng dose), a significant increase (p

RESULTS

46

When AKH was coapplied after endosulfan, a significant decrease (p

RESULTS

47

Similar results were recorded using malathion. In both malathion doses (lower 300 ng and higher 450 ng dose), significant increase (p

RESULTS

48

4.5.2. Protein carbonyls content

Protein carbonyl content was measured 24 hours after exposure to insecticides but since no

interactions of adipokinetic hormones with insecticides

Documents

masters thesis

department of physiology

thesis defence

library of department

insecticides endosulfan

assisstant professor

interactions of akh

institute of entomology