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

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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 ubiquinol
10
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:
H
2
O
2
+ Fe(III)-E H
2
O +O=Fe(IV)-E (1)
H
2
O
2
+ O=Fe(IV)-E H
2
O + 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 Ca
2+
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<16 h), the bugs from the Czech populations diapause and
virtually all individuals become brachypters, while under longer days the bugs do not
diapause and a fraction of population (014% in the populations of the Czech Republic)
becomes macropterous (Honk 1976, 1981). Macropterous individuals of the contemporary
populations of P. apterus are flightless (Socha and Zemek 2000a) and differ from
brachypters in various physiological and behavioral parameters, e.g. by a non-diapause type
of reproduction arrest (Socha and ula, 1996), lowered feeding and digestive enzyme
activities (Socha et al., 1998; ula et al., 1998), higher adipokinetic response (Socha et al.,
1999a) and enhanced walking and dispersal activities (Socha and Zemek, 2000b, 2003). The
rhythms in the walking activity of macropterous females were found to be diurnal (Maxov et
al., 2001) and correlated with the rhythms in mobilization of lipids from the fat body by
adipokinetic hormone and higher content of adipokinetic hormone in the CNS (Kodrk et al.,
2003).
The first AKH studies on P. apterus started in more than 10 years ago when the P.
apterus own AKH had not been identified. The first experiments studying difference of AKH
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 pGluLeu
AsnPheThrProAsnTrpNH
2
, 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-NH
2
. 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. LD
15
and LD
50
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, CaCl
2

Sodium carbonate, Na
2
CO
3

Sodium bicarbonate, NaHCO
3

Disodium hydrogen phosphate, Na
2
HPO
4

Sodium dihydrogen phosphate, NaH
2
PO
4

Potassium dihydrogen phosphate, KH
2
PO
4

Potassium hydrogen phosphate, K
2
HPO
4

Hydrochloric acid, HCl
Sulfuric acid, H
2
SO
4

Citric acid, C
6
H
8
O
7

Bicinchninic acid, BCA
Trichloroacetic acid, TCA
Trifluoroacetic acid, TFA
Acetonitrile, CH
3
CN
Copper sulfate, CuSO
4

Hydrogen peroxide, H
2
O
2

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)
Cys
1
-Pyrap-AKH (Sigma Genosys, Cambridge, UK)
Horseradish peroxidase, HRP (Vector Laboratories)
Pyrap-AKH, Polypeptide Laboratories s.r.o. (Prague, Czech Republic)


MATERIALS AND METHODS
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 CO
2
/H
2
O analyser (LI-COR Biosciences, Lincoln, NE)
Merck-Hitachi D-6000 chromatography system
Chromolith Performance RP-18e column




MATERIALS AND METHODS
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
MATERIALS AND METHODS
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.




MATERIALS AND METHODS
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, Ca
2+
and Mg
2+
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: C
9
H
6
C
l6
O
3
S
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.
MATERIALS AND METHODS
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: C
10
H
19
O
6
S
2
P
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.
MATERIALS AND METHODS
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 LD
15
and LD
50
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 LD
15
and LD
50
(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.
MATERIALS AND METHODS
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.
MATERIALS AND METHODS
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 Cys
1
-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 Cys
1
-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).
MATERIALS AND METHODS
29
ELISA Pyrap-AKH
Application of IgG overnight in 4
o
C, 100 l per well - diluted in coating buffer 1:2000
PREPARATION OF REQUIRED SOLUTIONS:
1. Coating buffer pH 9.6
Na
2
CO
3
- 0.424 g
NaHCO
3
- 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 : 9
Per 250 ml NaCl 21.91 g
Na
2
HPO
4
x 2 H
2
O 1.04 g
NaH
2
PO
4
x 2 H
2
O 0.37 g
To the working solution add Tween 20 to final
concentration 0.1%
Reaction is stopped by 50 l 0.5 M H
2
SO
4
OPD substrate - 100 l per well, 40 min at 37
o
C in darkness
Application of HRPS diluted in WB 1:500 100 l per well, 1 h at 37
o
C
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
o
C
Washing 3 x 200 l in WB
Blocking in 5% milk in WB 200 l per well, 2 h at 37
o
C
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 Na
2
HPO
4
5.68 g / 200 ml
12.15 ml A + 12.85 ml B + 4.25 mg OPD (pH 5.0) +
1.25 l 30 % H
2
O
2
5. 0.5 M H
2
SO
4
- 2.72 ml conc. H
2
SO
4
per 100 ml
Absorbance values were determined in a microtitre plate reader at 492 nm

Figure 13. Protocol for competitive ELISA.


MATERIALS AND METHODS
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 LD
15
(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 min
1
into the LI-7000 CO
2
/H
2
O
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 (V
CO2
)
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 h
1
bug
1
:
V
CO2
= (FE
CO2
FI
CO2
) x f
where f is the flow rate in l h
1
.
MATERIALS AND METHODS
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 LD
15
and LD
50
(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 H
2
O
2.
Degradation of H
2
O
2
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 H
2
O
2
.


Figure 15. Catalase activity protocol.
MATERIALS AND METHODS
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.
MATERIALS AND METHODS
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 10
5
M
-1
cm
-1
was used). Amount of
TBARS was expressed in pmol l
-1
homogenate.
MATERIALS AND METHODS
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 KH
2
PO
4
: 8.51 g 62.5 ml distilled H
2
O
B: 1 M K
2
HPO
4
: 10.89 g 62.5 ml distilled H
2
O
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 H
2
O
0.1 M HCl (FW=36.45)
0.83 ml conc. HCl + 99.17 ml distilled H
2
O
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 10
5
M
-1
cm
-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 LD
15
(186.052 ng bug
-1
for endosulfan and 279.348 ng bug
-1
for
malathion) and LD
50
(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. LD
15
and LD
50
values for endosulfan and malathion in P. apterus 24 h after
exposure.
Endosulfan Malathion
LD
15
(ng) 186.052 279.348
LD
50
(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 bug
1
(close to LD
15
) with 80 pmol Pyrap-AKH bug
1
caused
a significant increase in mortality from 30.00 % up to 91.25 % (p=0.00). Similarly,
coinjection of 300 ng malathion bug
1
(close to LD
15
) with 80 pmol Pyrap-AKH bug
1
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 bug
1
(close to LD
50
) and malathion 450 ng bug
1
(close to LD
50
) were
applied. Coapplication of these doses with 80 pmol Pyrap-AKH bug
1
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

m
o
r
t
a
l
i
t
y

a
f
t
e
r

2
4

h
insecticide alone
insecticide + 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<0.01) were recorded after every
coapplication, except for malathion 300 ng + AKH (80 pmol).



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<0.01) in AKH levels were determined (Figure 19).
In case of lower dose (200 ng) AKH level increased from 3.051 (Ringer saline) up to 3.601
pmol per 1 CNS (p=0.0002) and in case of higher dose (250 ng) from 3.051 (Ringer saline)
up to 3.683 pmol per 1 CNS (p=0.0007).
Similarly, when malathion was applied, AKH levels also increased but only in case of
higher dose (450 ng) the increase was significant (p<0.05) (Figure 20). When lower dose
(300 ng) was applied the AKH level increased from 3.231 (Ringer saline) up to 3.389 pmol
per 1 CNS but this increase was not significant (p=0.266). However, when higher dose (450
ng) was applied the AKH level significantly increased from 3.231 (Ringer saline) up to 3.525
pmol per 1 CNS (p=0.043).
N M SD
Ringer saline (control for endosulfan) 12 3.051 0.360
Endosulfan 200 ng 15 3.601 0.300
Endosulfan 250 ng 15 3.683 0.471
Ringer saline (control for malathion) 12 3.231 0.403
Malathion 300 ng 15 3.389 0.321
Malathion 450 ng 15 3.525 0.318
RESULTS
38
**
**
2.0
2.5
3.0
3.5
4.0
4.5
Ringer saline endosulfan 200 ng endosulfan 250 ng
p
m
o
l

A
K
H

p
e
r

1

C
N
S

Figure 19. The effect of endosulfan (200 and 250 ng bug
1
) 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<0.01) were recorded after both
lower and higher dose of endosulfan applied.

*
2.0
2.5
3.0
3.5
4.0
4.5
Ringer saline malathion 300 ng malation 450 ng
p
m
o
l

A
K
H

p
e
r

1

C
N
S

Figure 20. The effect of malathion (300 and 450 ng bug
1
) 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<0.05) were recorded only after
higher dose of malathion applied.
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<0.01) (Figure 21). When lower dose (200 ng) was
applied the AKH level increased from 1.343 (Ringer saline) up to 1.541 fmol l
-1
haemolymph
but this increase was not significant (p=0.073). However, when higher dose (250 ng) was
applied the AKH level significantly increased from 1.343 (Ringer saline) up to 1.866 fmol l
-1

haemolymph (p=0.0004).
In case of malathion in both lower (300 ng) and higher (450 ng) dose applied
significant increase (p<0.01) in AKH levels were determined (Figure 22). In case of lower
dose (300 ng) AKH level increased from 1.343 (Ringer saline) up to 1.774 fmol l
-1

haemolymph (p=0.004) and in case of higher dose (250 ng) from 1.343 (Ringer saline) up to
2.338 fmol l
-1
haemolymph (p=0.00001).

N M SD
Ringer saline 6 1.343 0.184
Endosulfan 200 ng 7
1.541 0.175
Endosulfan 250 ng 5
1.866 0.122
Malathion 300 ng 4
1.774 0.129
Malathion 450 ng 4
2.338 0.124
RESULTS
40
**
0.0
0.5
1.0
1.5
2.0
2.5
Ringer saline endosulfan 200 ng endosulfan 250 ng
f
m
o
l

A
K
H

l
-
1

h
a
e
m
o
l
y
m
p
h

Figure 21. The effect of endosulfan (200 and 250 ng bug
1
) 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<0.01)
were recorded only after higher dose of endosulfan applied.

**
**
0.0
0.5
1.0
1.5
2.0
2.5
Ringer saline malathion 300 ng malathion 450 ng
f
m
o
l

A
K
H

l
-
1

h
a
e
m
o
l
y
m
p
h

Figure 22. The effect of malathion (300 and 450 ng bug
1
) 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 haemolymph SD. Significant differences (** p<0.01)
were recorded after both lower and higher dose of malathion applied.
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 CO
2
production are shown in Table 5.

Table 5. Mean values and standard deviations of CO
2
production in bugs determined 1, 3, 6
and 24 h after exposure to endosulfan and malathion.

Legend:
M mean value of CO
2
production (l h
1
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 CO
2
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 CO
2
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<0,05) in CO
2

production were recorded (Figure 23). The CO
2
production significantly increased compared
to Ringer saline 6 hours after application of endosulfan, and it continued to increase so 24
hours after application there was also significant difference compared to control. Compared
to AKH significant difference was recorded 1 and 24 hours after application of endosulfan.
When AKH was applied together with endosulfan changes in CO
2
production also occured,
but the highest increase was now recorded 1 hour after application. Significant differences
compared to Ringer saline, AKH and endosulfan alone were recorded 1, 3 and 6 hours after
application. Because of the high mortality after application of endosulfan together with AKH,
data for CO
2
production 24 hours after application are missing.
In case of malathion application statistically significant differences (p<0,05) in CO
2

production were also recorded (Figure 24). Application of malathion caused increase in CO
2
production, and the highest increase was recorded 1 hour after exposure. Although the CO
2
production then decreased toward control levels, significant differences compared to Ringer
saline were recorded after all 4 measurements (1, 3, 6 and 24 h). Significant difference
compared to AKH was recorded 3 and 6 hours after exposure to malathion. When AKH was
applied together with malathion, changes in CO
2
again occured and the highest increase in
production compared to other groups was recorded. As in application of malathion alone,
after initial increase the CO
2
production then decreased but still remained higher than control
till the end of the measurement. So 1 and 3 hours after application significant difference
compared to Ringer saline, AKH and malathion only was recorded, while 6 and 24 hours
after application significant difference compared to Ringer saline and AKH was recorded.
When AKH alone was applied increase in CO
2
production occured, but statistically
significant difference (p<0,05) compared to Ringer saline was recorded only 1 and 3 hours
after exposure.
RESULTS
43

bd
bd
cd
c
ad
acd
d
c
d
bd
ad
ab
abc
abc
abc
0
10
20
30
40
50
60
1 3 6 24
time of exposure (hours)
C
O
2

l

h
-
1

b
u
g
-
1

Ringer saline
AKH
endosulfan 200 ng
endosulfan 200 ng + AKH

Figure 23. The effect of endosulfan (200 ng bug
1
) and Pyrap-AKH (80 pmol bug
1
) 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<0.05) are indicated as follows:
a - Ringer saline versus any other group;
b - AKH versus any other group;
c - endosulfan versus any other group;
d - endosulfan + AKH versus any other group.






RESULTS
44

cd
bcd
cd
cd
d
ad
cd
cd
a
ad
abd
ab
ab
ab
abc
abc
0
10
20
30
40
50
60
1 3 6 24
time of exposure (hours)
C
O
2

l

h
-
1

b
u
g
-
1

Ringer saline
AKH
malathion 300 ng
malathion 300 ng + AKH

Figure 24. The effect of malathion (300 ng bug
1
) and Pyrap-AKH (80 pmol bug
1
) 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<0.05) are indicated as follows:
a - Ringer saline versus any other group;
b - AKH versus any other group;
c - malathion versus any other group;
d - malathion + AKH versus any other group.





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<0.01) in catalase activity was recorded compared to controls (Ringer saline only
and AKH only) (Figure 25). In case of lower dose applied (200 ng), catalase activity
significantly increased from 5.964 (Ringer saline) and 6.048 (AKH) up to 7.117 mol min
-1

(p=0.00 in both cases). In case of higher dose (250 ng), catalase activity significantly
increased from 5.964 (Ringer saline) and 6.048 (AKH) up to 7.161 mol min
-1
(p=0.00 in both
cases).
N M SD
Ringer saline 8 5.964 0.381
AKH (80 pmol) 8 6.048 0.246
Endosulfan 200 ng 8 7.117 0.265
Endosulfan 200 ng + AKH (80 pmol) 8 6.457 0.333
Endosulfan 250 ng 8 7.161 0.263
Endosulfan 250 ng + AKH (80 pmol) 8 6.479 0.363
Malathion 300 ng 8 7.139 0.303
Malathion 300 ng + AKH (80 pmol) 8 6.281 0.381
Malathion 450 ng 8 7.157 0.325
Malathion 450 ng + AKH (80 pmol) 8 6.334 0.389
RESULTS
46
When AKH was coapplied after endosulfan, a significant decrease (p<0.01) was
recorded compared to endosulfan alone, but a significant increase (p<0.05) compared to
both controls (Ringer saline only and AKH only) were recorded. In case of lower dose (200
ng) together with coapplication of AKH catalase activity decreased significantly from 7.117 to
6.457 mol min
-1
(p=0.001), and in case of higher dose (250 ng) together with coapplication
of AKH catalase activity decreased significantly from 7.161 to 6.479 mol min
-1
(p=0.001).
Compared to controls (Ringer saline only and AKH only) catalase activity increased
significantly after exposure to endosulfan (200 ng) together with AKH (80 pmol) from 5.964
(Ringer saline) and 6.048 (AKH) up to 6.457 mol min
-1
(p=0.02 in both cases), and after
exposure to endosulfan (250 ng) together with AKH (80 pmol) from 5.964 (Ringer saline) and
6.048 (AKH) up to 6.479 mol min
-1
(p=0.02 in both cases).

a*b*c**
a*b*c**
a**b** a**b**
0
1
2
3
4
5
6
7
8
Ringer saline AKH (80 pmol) endosulfan 200 ng endosulfan 200 ng +
AKH (80 pmol)
endosulfan 250 ng endosulfan 250 ng +
AKH (80 pmol)
c
a
t
a
l
a
s
e

a
c
t
i
v
i
t
y

m
o
l

m
i
n
-
1

Figure 25. Catalase activity in P. apterus measured 3 h after exposure to insecticides and
insecticides together with AKH; presented results are mean values of catalase activity SD.
Significant differences (* p<0.05, ** p<0.01) are indicated as follows:
a - Ringer saline versus any other group;
b - AKH versus any other group;
c - endosulfan versus endosulfan + AKH within the same dose.



RESULTS
47
Similar results were recorded using malathion. In both malathion doses (lower 300 ng
and higher 450 ng dose), significant increase (p<0.01) in catalase activity was recorded
compared to controls (Ringer saline only and AKH only) (Figure 26). In case of lower dose
(300 ng), catalase activity significantly increased from 5.964 (Ringer saline) and 6.048 (AKH)
up to 7.139 mol min
-1
(p=0.00 in both cases). In case of higher dose (450 ng), catalase
activity significantly increased from 5.964 (Ringer saline) and 6.048 (AKH) up to 7.157 mol
min
-1
(p=0.00 in both cases).
When AKH was coapplied after malathion, a significant decrease (p<0.01) was
recorded compared to malathion only, but there were no significant changes recorded
compared to controls (Ringer saline only and AKH only). In case of lower dose (300 ng)
coapplied with AKH catalase activity decreased significantly from 7.139 to 6.281 mol min
-1

(p=0.0003), and in case of higher dose (450 ng) coapplied with AKH catalase activity
decreased significantly from 7.157 to 6.334 mol min
-1
(p=0.0007).

c**
c**
a**b** a**b**
0
1
2
3
4
5
6
7
8
Ringer saline AKH (80 pmol) malathion 300 ng malathion 300 ng +
AKH (80 pmol)
malathion 450 ng malathion 450 ng +
AKH (80 pmol)
c
a
t
a
l
a
s
e

a
c
t
i
v
i
t
y

m
o
l

m
i
n
-
1

Figure 26. Catalase activity in P. apterus measured 3 h after exposure to insecticides and
insecticides together with AKH; presented results are mean values of catalase activity SD.
Significant differences (* p<0.05, ** p<0.01) are indicated as follows:
a - Ringer saline versus any other group;
b - AKH versus any other group;
c - malathion versus malathion + AKH within the same dose.

RESULTS
48
4.5.2. Protein carbonyls content

Protein carbonyl content was measured 24 hours after exposure to insecticides but
since no significant increase in carbonyls was found (in both lower and higher dose applied),
the experiment was repeated after 48 hours of exposure (Table 7). Repeated experiment
was conducted only with lower doses of insecticides because the prolongation of exposure
increased mortality to almost 100 %. In each sample 8 bugs were used for the measurement
and t-test was employed to determine the statistically siginificant difference between the
samples. Results obtained after 48 hours of exposure are presented in Figure 27.

Table 7. Mean values and standard deviations of protein carbonyls content measured 48 h
after exposure to endosulfan and malathion.

Legend:
M mean value of protein carbonyls content (nmol mg
-1
proteins)
SD standard deviation

0
20
40
60
80
100
120
Ringer saline endosulfan 200 ng malathion 300 ng
p
r
o
t
e
i
n

c
a
r
b
o
n
y
l
s

n
m
o
l

m
g
-
1

p
r
o
t
e
i
n
s

Figure 27. Protein carbonyl content in P. apterus measured 48 h after exposure to
insecticides; presented results are mean values of protein carbonyls content SD. No
significant differences between experimental groups (endosulfan and malathion) and control
(Ringer saline) were determined.
N M SD
Ringer saline 8 77.677 17.058
Endosulfan 200 ng 8 86.770 27.857
Malathion 300 ng 8 78.097 15.154
RESULTS
49
Again, no significant differences between control (Ringer saline) and experimental
groups exposed to endosulfan (p=0.44) and malathion (p=0.96) were found. Since no
differences were determined, experiments with AKH coapplication were not conducted.

4.5.3. TBARS content

Results of TBARS content, measured 24 hours after exposure to insecticides, are
shown in Table 8. For measurement of TBARS content 21 (22) bugs were used in each
sample and t-test was employed to determine the statistically siginificant difference between
the samples.

Table 8. Mean values and standard deviations of TBARS content measured 24 h after
exposure to endosulfan, malathion and AKH.

Legend:
M mean value of TBARS content (pmol l
-1
homogenate)
SD standard deviation

The results (Figure 28) show that exposure to both endosulfan (200 ng) and
malathion (300 ng) caused significant increase (p<0.01) in TBARS content compared to
controls (Ringer saline only and AKH only). In case of endosulfan (200 ng), TBARS content
significantly increased from 4.885 (Ringer saline) and 4.873 (AKH) up to 5.096 pmol l
-1

homogenate (p=0.0001 i.e. p=0.00). Similarly, in case of malathion (300 ng), TBARS content
significantly increased from 4.885 (Ringer saline) and 4.873 (AKH) up to 5.188 pmol l
-1

homogenate (p=0.00 in both cases).
When malathion (300 ng) was applied together with AKH (80 pmol) significant
increase (p<0.01) in TBARS content was also recorded compared to controls (Ringer saline
only and AKH only) from 4.885 (Ringer saline) and 4.873 (AKH) up to 5.089 pmol l
-1

homogenate (p=0.00 in both cases). Although when malathion was applied together with
AKH decrease in TBARS content was recorded compared to treatment to malathion only,
this decrease was not significant (p=0.061).
There is no data for TBARS content after coapplication of endosulfan (200 ng) with
AKH (80 pmol) because of the very high mortality.
N M SD
Ringer saline 21
4.885 0.183
AKH (80 pmol) 22
4.873 0.195
Endosulfan 200 ng 21
5.096 0.295
Malathion 300 ng 21
5.188 0.229
Malathion 300 ng + AKH (80 pmol) 21
5.089 0.246
RESULTS
50

**
**
**
3.5
3.7
3.9
4.1
4.3
4.5
4.7
4.9
5.1
5.3
5.5
Ringer saline AKH (80 pmol) endosulfan 200 ng malathion 300 ng malathion 300 ng +
AKH (80 pmol)
T
B
A
R
S

p
m
o
l

l -
1

h
o
m
o
g
e
n
a
t
e

Figure 28. TBARS content in P. apterus measured 24 h after exposure to insecticides and
insecticides with AKH; presented results are mean values of TBARS content SD.
Significant differences (** p<0.01) were recorded after exposure to endosulfan (200 ng),
malathion (300 ng) and malathion (300 ng) + AKH (80 pmol) compared to controls (Ringer
saline only and AKH only). In case of coapplication of AKH after malathion treatment no
significant changes compared to malathion alone were determined.
DISCUSSION
51
5. DISCUSSION

Stress defense mechanisms in insects can be induced by various stressors such as
X-ray radiation, chemical injury, infections etc. and also pesticides. The stress reactions and
defensive behavior of insects have been commonly studied in the context of individual
organisms and their responses at the level of cells, hormones and biochemical pathways.
The previous research conducted on the mechanisms of the influence of insecticides
indicated disturbances in the hormonal balance. These resulted in the insects death when
the disturbances caused irreparable damages in organism (Ivanovi and Jankovi-Hladni,
1991). The following more detailed investigations showed that various stressors, including
insecticides, cause specific changes in the activity of neurosecretory neurons and synthesis
of different neurohormones such as bioamines, ecdysiotropins, ecdysiostatins,
allatoregulatory neuropeptides and adipokinetic neurohormones (Peri-Mataruga et al., 2006;
Kodrk, 2008).
The insecticides endosulfan and malathion, used in this study, are applied against a
number of insect pests and mites in agriculture, greenhouses and gardens, as well as for the
public vector control. Bioactivation of malathion is necessary for it to exert its toxic effect
primarily inhibition of AChE. Bioactivation is primarily mediated by cytochrome P450
enzymes, which create the active metabolite malaoxon (Costa, 2008; Roberts, 1998). This
active metabolite is more toxic than the parent malathion. The species selectivity of
malathion toward insects is primarily the result of a deficiency in detoxifying
carboxylesterases by these organisms, which then allows malathion to undergo oxidation to
form the highly toxic analog malaoxon (Cohen, 1984; Matolczy et al., 1988). Malathion has
low mammalian toxicity because a carboxyl esterase that can use malathion as a substrate is
abundant in the mammalian liver. This specific carboxyl esterase is not present in insects,
and this is the reason for the favorable selectivity index of this pesticide (Stenersen, 2004). In
case of endosulfan, the primary acute effect is on the nervous system (it is neurotoxic to both
insects and mammals). Endosulfan belongs to cyclodiene group of chlorinated hydrocarbons,
but it is slightly more reactive, more readily metabolized, and less prone to bioaccumulation
compared with other cyclodienes such as aldrin and dieldrin (Guerin and Kennedy, 1992). In
particular, endosulfan has a relatively reactive cyclic sulfite diester group (Van Woerden,
1963) and, as a consequence, its environmental persistence is lower than that of other
cyclodienes, although still higher than that of many other insecticides. Endosulfan is a
stereoisomer mixture of alpha () and beta () isomers in the ratio 7:3 (Hayes and Laws,
1991), and both isomers are metabolized into endosulfan sulfate (shows similar acute toxicity
to the parent compound).
DISCUSSION
52
Besides the target pests, many other non-target organisms are also affected by
usage of these insecticides. The present work showed that both endosulfan and malathion
elicit mortality in the P. apterus adults within 24 hours of treatment, although the LD
15
and
LD
50
values were lower for endosulfan (Table 1) indicating stronger effect on mortality
compared to malathion. This difference in values can be explained by different structure and
mode of action of these insecticides since endosulfan is an organochlorine and malathion
organophosphoruous insecticide. Furthermore, based on acute toxicity, endosulfan is
classified as highly toxic (Category I), and acute toxicity of malathion as moderately toxic
(Category II) so this result was expected. In addition, when LC
50
values of endosulfan and
malathion for other insects (e.g. mosquitoes and midges) are compared, it can be noticed
that LC
50
values are again lower for endosulfan compared to malathion (e.g. Culex fatigans:
LC
50
(endosulfan) = 142.5 g/l, LC
50
(malathion) = 750.7 g/l; Chironomus riparius: LC
50

(endosulfan) = 100 g/l; LC
50
(malathion) = 297.9 g/l; Pteronarcys californicus: LC
50

(endosulfan) = 10.6 g/l; LC
50
(malathion) = 39.2 g/l) (PAN Pesticide Database).
Very important result of the present study is that the coapplication of endosulfan and
malathion with Pyrap-AKH significantly increased the mortality of the bugs (Figure 18). Since
endosulfan alone had stronger effect on mortality, it was assumed that in combination with
AKH will again cause higher mortality. This assumption was confirmed in case of
coapplication of endosulfan with AKH when the stronger effect on mortality was recorded
compared to malathion (i.e. in application of lower dose of endosulfan with AKH mortality
increased from 30.00 % up to 91.25 %, and in application of lower dose of malathion with
AKH it increased from 25.00 % up to 36.25 %). In both endosulfan doses applied (200 ng
and 250 ng) mortality significantly increased almost up to 100 %. However, when AKH was
coapplied with malathion the increase in mortality was significant only in case of the higher
dose (450 ng). This increase in mortality can be explained by probable Pyrap-AKH induced
unspecific increase of the metabolic intensity in the experimental bugs documented by the
increase of carbon dioxide production. The increased metabolic rate could intensify both
endosulfan and malathion action by its faster penetration into tissues and by more intensive
effects on biochemically active cells. Those experiments showed that application of
endosulfan, malathion and Pyrap-AKH caused significant changes in carbon dioxide
production. Both endosulfan and malathion caused elevation of metabolism, although the
trends were different. Malathion immediately caused increase in carbon dioxide production
which decreased after that towards the control production (Figure 24), while in case of
endosulfan carbon dioxide production increased significantly above control production later
compared to malathion (Figure 23). When Pyrap-AKH alone was applied, increase in carbon
dioxide production was also recorded and the trend was similar as in case of malathion.
When endosulfan and malathion were coapplied together with Pyrap-AKH, the highest
DISCUSSION
53
increase in carbon dioxide production was recorded i.e. the production after coapplication
exceeded production after application of both insecticides only and Pyrap-AKH only. Since
coapplication of Pyrap-AKH with both insecticides caused elevation in metabolism and also
increase in mortality, interaction between these substances can be characterized as
potentiation. Potentiation is an interaction between substances when an effect due to two
substances with different modes of action (and only one is toxic) is greater than expected
from the effects of the individual components. In this case the Pyrap-AKH potentiated the
effect of both used insecticides and caused increase in mortality, but effect on endosulfan
was more evident.
Since AKHs are typical stress hormones, their level in the insect body increases
under stress conditions. The first demonstration of insecticide effect on the AKH level was
investigated on locusts when poisoning with deltamethrin. The insecticide caused large
increase in the concentrations of AKH in haemolymph 20 minutes after treatment (Candy,
2002). Later on, an involvement of AKHs in the response of P. apterus adults to insecticide
was demonstrated in the concrete pyrethroid permethrin treatment (Kodrk and Socha, 2005;
Kodrk et al., 2010a). For determination of AKH levels in CNS and haemolymph in the
present study ten-day old brachypterous females were used because of the higher AKH level
and other AKH characteristics (Socha et al., 1999a). Injection of both endosulfan (Figure 19)
and malathion (Figure 20) induced a significant increase in AKH levels in the CNS. Again the
stronger effect of endosulfan was recorded compared to malathion significant increase was
recorded in both lower (200 ng) and higher (250 ng) endosulfan dose, and only in higher
(450 ng) malathion dose. Since CC in CNS serves as a storage of AKH, it was important to
determine whether this increase in AKH level was also present in haemolymph. The results
showed that a prolonged insecticide effect (24 hours) led to an increase in AKH level not only
in the CNS but also in the haemolymph (Figure 21 and 22). Elevated AKH titers in the
haemolymph can be simply explained by increased release of AKH from CC. Increase in the
AKH level within an insects body is obviously connected with the triggering of a sequence of
stress-protective events (Kodrk, 2008).
Recently, it was proven that defects induced by oxidative stress (deterioration of
proteins caused by protein carbonylation, decrease in glutathione levels, attenuation of total
antioxidant activity in haemolymph) are reduced by the injection of exogenous AKH into the
insect body (Veea et al., 2007; Kodrk et al., 2007). These facts indicate that there is a
feedback regulation between an oxidative stressor and AKH actions, and that AKHs might be
involved in the activation of an antioxidant protection mechanism. Similar results were
obtained in this study. Catalase activity was measured 3 hours after exposure to insecticides
alone and insecticides together with AKH cotreatment. Insecticide alone caused significant
increase in catalase activity, but in case of application of insecticide together with AKH a
DISCUSSION
54
significant decrease was recorded compared to insecticide alone (Table 6). This indicates
two phenomenons first, both endosulfan and malathion caused oxidative stress in P.
apterus; and second, AKH reduced catalase activity caused by oxidative stress. The
induction of oxidative stress and increased formation of ROS was expected because for both
endosulfan and malathion is well-known that promote formation of free radicals. And since
free radical-scavenging enzymes superoxide dismutase, catalase and glutathione peroxidase
are the first line of cellular defense against oxidative injury, the induction of catalase after
application of these insecticides was also expected. One can speculate that AKH affects a
production of hydrogen peroxide by unknown mechanism, and therefore the activity of
catalase necessary for reduction of hydrogen peroxide is lower. In case of malathion,
coapplication of AKH decreased the catalase activity to control levels (Figure 26). Similarly,
in case of endosulfan, coapplication of AKH decreased the catalase activity but there was
still significant increase in catalase activity compared to control (Figure 25) which may be
due to stronger effect of endosulfan. Interestingly, AKH alone did not cause significant
changes compared to control (Ringer saline) indicating that stressor action is needed for
AKH activation and activation an antioxidant protection mechanism.
Oxidative stress was also evaluated by determining the level of thiobarbituric acid
reactive species (TBARS) as a marker of lipid peroxidation. Peroxidation of lipids can disturb
the assembly of the membrane, causing changes in fluidity and permeability, alterations of
ion transport and inhibition of metabolic processes (Nigam and Schewe, 2000). Results
obtained after determination of TBARS levels (24 hours after exposure) were similar to
results for catalase activity. Again, both endosulfan and malathion induced oxidative stress
and caused significant increase in TBARS content compared to control. Also AKH alone did
not cause significant changes compared to control (Ringer saline) (Table 8). But when AKH
was coapplied with malathion, the TBARS content decreased but not significantly (Figure
28). This insignificant change, compared to significant decrease in catalase activity after
coapplication of AKH, may be due to longer time of exposure (in case of TBARS 24 hours
and in case of catalase activity 3 hours). Longer time of exposure could cause more
damaging effects and therefore same amount of AKH would not be enough to cause
significant decrease in TBARS levels. This means that AKH still may have affected the
activation of an antioxidant protection mechanism but insufficiently to cause significant
difference. So, the discrepancies can be solved by application of bigger amount of AKH or by
different exposure time.
The third investigated biomarker of oxidative stress was the protein carbonyl content.
Studies on protein oxidation have been greatly facilitated by the availability of a simple,
robust, and accurate method to quantify protein oxidation: the carbonyl assay (Levine et al.,
1994). The introduction of carbonyl groups into proteins can be triggered by different ROS or
DISCUSSION
55
secondary by-products of oxidative stress and can arise at different sites and by different
mechanisms (Stadtman and Levine, 2003; Dalle-Donne et al., 2006). Determination of
protein carbonyls in this study did not give positive result. Even after repeated experiment
with prolonged time of exposure (from 24 to 48 hours) determination of protein carbonyl
content still did not result in significant difference compared to control (Figure 27). Carbonyls
are relatively difficult to induce and might therefore indicate a more severe oxidative stress
(Dalle-Donne et al., 2003a). Considering that and the fact that that levels of TBARS after
oxidative stress increase much earlier than the protein carbonyls levels, it can be assumed
that even the prolonged exposure time (48 hours) was too short to induce formation of
significantly higher levels of protein carbonyls. But since the prolongation of exposure causes
increase in mortality, experiment with lower doses and repeated exposure to insecticides
should be conducted.
Considering all the above data, it appears that the probable explanation for the
enhanced effect of insecticides by AKH treatments is the stimulatory role of this neuropeptide
on insect metabolism.
Insect neuropeptides are important bioregulators of a number of aspects of insect
physiology and behavior because they influence virtually all aspects of insect life such as
growth and development, intermediary metabolism and homeostasis, stress response,
muscle movement, reproduction and behavior (Gde, 2004). Although a lot of information is
available on their primary structures, synthesis, structureactivity relationship, mode of
action, release, receptor binding and degradation (Van der Horst et al., 2001; De Loof, 2008;
Nassel, 2002; Gde et al., 1997), the possible use of these potent molecular messengers
with respect to putative insect control has been reviewed very briefly (Gde and Goldsworthy
2003; Kodrk and Socha, 2005; Bede et al., 2007). It is usually supposed that neuropeptides
are not practical as pest insect control agents owing to their susceptibility to degradation and
inability to penetrate the insect cuticle, enter the insect circulatory system and reach the
target receptor sites (Nachman et al., 1993). However, it was found that some
neuropeptides, especially the AKHs, were capable of influencing insect metabolic functions
when applied topically to the body surface. It is known that AKHs occupy a key position in the
possible modulation of physiological events (Gde et al., 1997; Gde et al., 2003; Kodrk,
2008). In P. apterus, the topical application of Pyrap-AKH dissolved in 20% methanol
affected the metabolic functions in the same way as injection of the hormone (Kodrk et al.,
2002a; Kodrk et al., 2002b). Similar results were obtained also in Gryllus bimaculatus
(Lorenz et al., 2004), where the Grybi-AKH was topically applied solved in 2-propanol. It is
obvious that the organic solvents aid penetration of the AKHs through the cuticular waxes.
However, this feature appears to be interesting for the possible use of some neuropeptides
as potential candidates, or supporting agents, for insect pest control. The present study
DISCUSSION
56
describes an interesting feature of AKH intensification of the effect of an insecticide by
coapplication of a peptide hormone. Adults of P. apterus are shown to be more sensitive to
equivalent doses of endosulfan and malathion after AKH treatment, such that a lower amount
of the insecticide could potentially be used to achieve the same effect.
Whether peptides such as AKH could be used in future as suitable agents for the
enhanced control of economically important insect pests remains to be an open question.
CONCLUSIONS
57
6. CONCLUSIONS

In order to determine the exact mode of action of insecticides, their effects on target
and non-target insects, as well as stress reactions and defense mechanisms of insects after
insecticide treatment it is necessary to conduct research on model organisms. Since insect
metabolism is predominantly controlled by AKHs which behave as typical stress hormones, it
was important to investigate the interactions between AKH and insecticides. For that purpose
this study was conducted on firebug (Pyrrhocoris apterus) which was exposed to insecticides
endosulfan and malathion. Essential conclusions of this research are:

1. Both endosulfan and malathion elicit mortality in the P. apterus adults within 24 hours of
treatment by injection. Obtained LD
15
and LD
50
values are lower for endosulfan.

2. Coapplication of endosulfan and malathion with Pyrap-AKH significantly increase the
mortality of the bugs. Again, endosulfan has stronger effect on mortality compared to
malathion.

3. Application of endosulfan, malathion and Pyrap-AKH cause significant changes in
carbon dioxide production. Malathion and Pyrap-AKH cause increase in dioxide production
which in time decreases towards control production. Endosulfan cause 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 is recorded.

4. Injection of both endosulfan and malathion induce a significant increase in AKH levels in
the CNS and haemolymph. Elevated AKH titers in the haemolymph are present due to
increased release of AKH from CC.

5. Both endosulfan and malathion cause oxidative stress in P. apterus. These insecticides
cause significant increase in catalase activity and TBARS content. Obtained negative result
for protein carbonyls content is probably due to too short time of exposure.

6. When Pyrap-AKH was coapplied with insecticides, catalase activity significantly
decrease. This indicates that there is a feedback regulation between an oxidative stressor
and AKH actions, and that AKHs might be involved in the activation of an antioxidant
protection mechanism.
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APPENDIX
65
8. APPENDIX

Figure 1. Environmental fate of pesticides
http://www.agf.gov.bc.ca/pesticides/c_2.htm

Figure 2. Schematic diagram of the neuroendocrine complex of P. apterus
Socha R., Hodkov M. M. 1989. The absence of corpus allatum in prothetelic adultiforms
in a strain of Pyrrhocoris apterus (Heteroptera, Pyrrhocoridae). Acta Entomologica
Bohemoslovaca 86(3): 167171.

Figure 3. Major pathways of reactive oxygen species generation and metabolism
Aitken R. J., Shaun D. R. 2008. Antioxidant systems and oxidative stress in the testes.
Oxid Med Cell Longev. 1: 1524.

Figure 4. Regulation of cellular processes in response to oxidative stress
Haddad Critical Care 2002 6: 481.

Figure 5. Antioxidant pathways
http://www.redlabs.be/red-labs/our-science/oxidative-stress.php

Figure 6. Carbonylation and derivatization of a protein amino-acid side chain
Nystrm T. 2005. Role of oxidative carbonylation in protein quality control and
senescence. The EMBO Journal 24: 13111317.

Figure 7. Basic reaction sequence of lipid peroxidation
Young I. S., McEneny J. 2001. Lipoprotein oxidation and atherosclerosis. Biochemical
Society Transactions 29: 358362.

Figure 8. Pyrrhocoris apterus
http://farm4.static.flickr.com/3176/2738273721_ec11488947.jpg

Figure 9. Structure of endosulfan
http://www.pesticideinfo.org/Detail_Chemical.jsp?Rec_Id=PC35085

Figure 10. Structure of malathion
http://www.pesticideinfo.org/Detail_Chemical.jsp?Rec_Id=PC32924