Diptera Culicidae): to Fenitrothion and Temephos In

Susceptibility of Culex quinquefasciatus Say Larvae

(Diptera Culicidae): to Fenitrothion and Temephos in

Rayyan Municipality, Qatar State

MURTADA OSMAN AHMED ELKAGAM Post graduate Diploma in Malariology (University of Gezira, 1999) Post graduate Diploma in Health Education (University of Gezira, 1998) B.sc. in Public and environmental Health (University of Khartoum, 1994)

A dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science In Medical Entomology and Vector Control Blue Nile National Institute for Communicable Diseases University of Gezira

August 2013

Susceptibility of Culex quinquefasciatus Say Larvae

(Diptera Culicidae): to Fenitrothion and Temephos in

Rayyan Municipality, Qatar State

MURTADA OSMAN AHMED ELKAGAM

Supervisor: Ustaz. Faisal Eltayeb Hassan

Co- supervisor: Dr. Hassan Ali Farag

August 2013

DEDICATION

To the memory of my late mother and father

To my son Mohammad with wishes for health and healing

Murtada

ACKNOWLEDGEMENT

First, all the thanks and gratitude to ALLAH for giving me the ability to do, and completion of this work.

I would like to express my sincere gratitude to Ustaz. Faisal Al-Tayeb, the supervisor for his guidance, supervision and encouragement throughout the stages of this research. Thanks also to Dr. Hassan Ali Farag (Pest control consultant, ministry of municipality, Qatar state), the co-supervisor for continued assistance in all phases of the research in the State of Qatar.

I express my thanks and gratitude to my wife Um Nada for her continuous help during all the stages of the study.

I would like to thank the Blue Nile National Institute for Communicable Diseases (BNNICD) for giving me the opportunity to conduct my research. Special thanks to the staff for their continued support.

Also thanks to my friends, brothers and sisters for their assistance during the study. And finally, I cannot forget to express my thank to anyone how extended hands during the study, without them this study would have not been possible.

Susceptibility of Culex quinquefasciatus Say Larvae Diptera: Culicidae to Fenitrothion and Temephos in Rayyan Municipality, Qatar State

MURTADA OSMAN AHMED ELKAGAM M.sc in Medical Entomology and Vector Control Blue Nile National Institute for Communicable Diseases, University of Gezira

ABSTRACT

Resistance monitoring should be an integral part of vector control programmes. The present study is to test Fenitrothion 50% and Temephos 10% against Culex quinquefasciatus larvae in a laboratory at Rayyan municipality, Qatar state. Different concentrations were taken separately, adding to the tested cups with 20 larvae and in a volume of water of 100 ml. then the mortality was recorded after 24, 48 hours. And analyzed by Probit. The results showed that the mortality of larvae in the summer was high than in the winter, due to the temperature, which represent the major factor that affect the survival of the larval stages of mosquitoes. Also, the appearance of resistance in vector mosquito larvae against the larvicide fenitrothion 50%, the maximum mortality value is (59.9%), which is less than (80%), the recommended value by WHO. The study recommends conducting a comprehensive entomological survey, in addition to continuing field surveillance for the development of larvicides resistance. Also, taking into account the seasonal changes in larval control strategy in Qatar.

اختبار حساسية يرقات بعوض الكيولكس الى مبيدي اليرقات فينيتروثيون وتيميفوس ببلدية الريان، دولة قطر

مرتضى عثمان أحمد الكجم

ماجستير الحشرات الطبية ومكافحة ناقالت االمراض

معهد النيل االزرق القومي لألمراض السارية – جامعة الجزيرة

ملخص الدراسة

TABLE OF CONTENTS

Topic Page No.

Dedication...... I

Acknowledgement...... II

Abstract (English & Arabic)...... III-IV

Table of Contents……………………………… V List of Abbreviations…………………………. VI

List of Figures………………………………… VII

List of Appendixes……..……………………. VIII

Chapter one, Introduction, Justification and 1 – 3 Objectives ……………………………..……… Chapter two, Literature Review ……………… 4 – 26

Chapter three, Materials and Methods ……….. 27 – 31

Chapter four, Results ………...……………….. 32 – 39

Chapter five, Discussions, Conclusion and 40 – 44 Recommendations ……………………………. References …………………………………… 45 – 51

Appendixes ………………………………….. A -

LIST OF ABBREVIATIONS

WHO World |Health Organization BNNICD Blue Nile National Institute for Communicable Diseases IVM Integrated Vector Management IGRs Insect Growth regulators Bti Bacillus thuringiensis israelensis TEPP Tetraethyl pyrophosphate OP Organophosphours AchE acetyl cholinesterase enzyme CNS central nervous system EC emulsifiable concentration GR granule WHOPES WHO Pesticide Evaluation Scheme EPA Environmental Protection Agency

LD50 Lethal dose to kill 50%

LC50 Lethal concentration to kill 50% ADI acceptable daily intake,: ARfD acute reference dose JH juvenile hormone CSIs chitin synthesis inhibitors FAO Food and Agriculture organization temp Temperature bw body weight

LIST OF FIGURES

Figure No. Topic Pages Efficacy of Fenitrothion 50% against Culex Figure. 1 quinquefasciatus larvae - test (1) showing 32 winter & summer curve (Jan&July2012)

Efficacy of Fenitrothion 50% against Culex Figure. 2 quinquefasciatus larvae - test (2) showing 33 winter & summer curve (Feb &Aug 2012) Efficacy of Fenitrothion 50% against Culex Figure. 3 quinquefasciatus larvae - test (3) showing 34 winter & summer curve (March & Sep 2012) Efficacy of temephos 10% against Culex Figure. 4 quinquefasciatus larvae - test (1) showing 35 winter & summer curve (Jan&July2012) Efficacy of temephos 10% against Culex Figure. 5 quinquefasciatus larvae - test (2) showing 36 winter & summer curve (Feb & Aug 2012) Efficacy of temephos 10% against Culex Figure. 6 quinquefasciatus larvae - test (3) showing 37 winter & summer curve (March & Sep 2012) Comparing the efficacy of fenitrothion and Figure. 7 temephos against Culex quinquefasciatus in 38

winter season March 2012 Comparing the efficacy of fenitrothion and Figure. 8 Temephos against Culex quinquefasciatus in 39

summer season sep 2012

LIST OF APPENDIXES

Topic Pages Effect of Temephos 10% against Culex quinquefasciatus larvae (Winter test) A Effect of Temephos 10% against Culex quinquefasciatus larvae (Summer test) B Effect of Fenitrothion 50% against Culex quinquefasciatus larvae (Winter test) C Effect of Fenitrothion 50% against Culex quinquefasciatus larvae (Summer test) D

Results of the efficacy test of each larvicides, E Fenitrothion 50% and Temephos 10%

Results of Culex quinquefasciatus larvae mortality for F each larvicide according to the exposure timE

Average temperature, Qatar climate, 2012 G

Probit analysis data, Fenitrothion 50% H - I

Probit analysis data, Temephos 10% J - K

Notable Arthropod-Borne Diseases L - M

Laboratory evaluation of the efficacy of larvicides N - EE

Qatar climate FF

CHAPTER ONE

INTRODUCTION

Up to date, there are about 3450 species of mosquitoes world-wide, out of these around 100 of are considered as vectors. The most important group, Family Culicidae, of biting insects belong to the order Diptera. Important vectors of several tropical diseases, including dengue, malaria, filariasis, Japanese encephalitis, yellow fever…etc.

Mosquitoes are known as vectors of several human diseases, and also cause environmental nuisances, due to their large geographical distribution. To adopt mosquito control strategy a detailed knowledge of the biology of the species is essential, knowing about the breeding sites of mosquitoes which can be used in the control program through targeting the larvae. Development of resistance to pesticides, in addition to the environmental concerns limit the use of some insecticides for mosquito control, since the discovery and identification the alternative tool for mosquito control, e.g.: biological control.

2- JUSTIFICATION Knowledge about insecticide resistance in target species is a basic requirement to guide insecticide use in mosquitoes control programme.

The most effective method of mosquito control in Qatar is "larviciding" i.e. The treatment of breeding sites where mosquito larvae are present, because Mosquitoes in the larval stage are an important goal and simple, where mosquitoes breed in water, and thus, it is easy to deal with them in this habitat. The World Health Organization (WHO) has called for the acceptance of a global strategic framework for integrated vector management (IVM); this entails increased emphasis on using all appropriate (practical and ecologically-sound) methods of vector control.

Vector-borne diseases are an increasing cause of death and human suffering worldwide. Efforts to control these diseases have been focused on the use of conventional insecticides, but the insect resistance (whether physiological, biochemical, or behavioral) to insecticides is now an immense practical problem. Resistance arises through the over-use or misuse/ abuse of insecticides against a pest species and results in the selection of resistant forms of the pest and the consequent evolution of populations that are resistant to that insecticide.

3. Objectives Population of mosquitoes are a useful tool to provide qualitative and quantitative understandings of influences of vector larval interventions on diseases transmission.

3.1. General Objective Laboratory test of the Susceptibility of Culex quinquefasciatus (Say) larvae to Fenitrothion 50% and Temephos10% in Rayyan municipality, Qatar State.

3.2. Specific Objectives

3.2.1. To identify the efficacy of Fenitrothion 50% and Temephos 10% against Culex quinquefasciatus larvae. 3.2.2. To detect the presence of resistant in mosquito larval population (Culex quinquefasciatus, Say) 3.2.3. To compare the efficacy of the two larvicides with regard to the winter and summer season.

CHAPTER TWO LITERATURE REVEW

INTRODUCTION

Mosquito life cycles and behaviors, as well as mosquito-borne pathogen transmission have been the subject of intense research for the past 70 years, yet further study is still needed about the ecology of mosquito population dynamics and control in relation to land use and land change, specifically urbanization. It is, therefore, necessary to assess the biological and non-biological factors influencing mosquito control programs in urban areas to assure they are engaging in appropriate activities that lead to the control of pathogen transmission and a reduction in mosquito populations (Mulla et al., 2003) and (Chen et al., 2006). Mosquito larvae have a well-developed head with mouth brushes used for feeding, a large thorax with no legs and a segmented abdomen. Larvae breathe through spiracles located on the eighth abdominal segment, or through a siphon, and therefore must come to the surface frequently. The larvae spend most of their time feeding on algae, bacteria, and other micro-organisms in the surface micro layer (Mulla et al., 2003). 2.1. 1. BIOLOGY Larvae develop through four instars, after which they metamorphose into pupae. At the end of each instar, the larvae molt, shedding their exoskeleton to allow for further growth (Mulla et al., 2003).

2.1.2. CHEMICAL CONTROL Although insecticides are invaluable in preventing and controlling damage to agricultural products and to the health of man and animals, they are not without side-effects on the environment and its biota. There

14 is a critical need to find and develop new agents and products for the control of this and other important species of mosquitoes. Insect growth regulators (IGRs) are now increasingly used to control Aedes and other mosquito larvae. These compounds have unique modes of action, and are often selective and do not persist in the environment. Such attributes are desirable when dealing with the problem of pest and insecticides resistance (Mulla et al, 2003). Pyriproxyfen is a new generation of IGR. It is a juvenile hormone analogue (JHA) and a relatively stable aromatic compound. functions as an insecticide by overloading the hormonal system of the target insect, ultimately affecting its egg production, brood care and other social interactions, and inhibiting its growth (Invest et al., 2008). Bacillus thuringiensis commonly referred to as B.t. is the microorganism that produces chemical toxic to insets, which was registered in the United States for the use as pesticide in 1961 and reregistered 1998. Bacillus thuringiensis subsp. israelensis (Bti) is one of the most famous spore forming bacterium, able to produce specific insecticidal toxins during sporulation. The toxin is a combination of six main proteins aggregated into a solid crystal encased in the bacterial cell, exhibiting acute toxicity towards dipteran insects, such as larval mosquitoes and black flies. Today, this bacterium is widely used for the preparation of commercial bio-insecticides used in insect control programs (Gill et al., 2008).

2.3. MOSQUITOES

A mosquito is a small biting insect having a cylindrical body, a prominent proboscis, a pair of wings, three pairs of legs and a pair of straight palps (Dheer et al., 2008).

Mosquitoes have a worldwide distribution and can occur at elevations as high as 4,300 m and below sea level in mines. They are abundant in the tropics, and almost unbelievably large swarms of them occur in the Artic (Lieutenant et al., 2001)

Many mosquito species are vectors of pathogens that cause widespread human diseases. This medically significant role makes these insects the center of research, the aim of which is to find ways to reduce the burden these diseases impose. (Schneider et al., 2006). Mosquito- Transmitted diseases are strongly influenced by the abundance, longevity, human contact, and feeding frequency of competent vectors, an ensemble of characteristics embodied in the measure of Victorial capacity (Dye, 1992) The size of adult mosquitoes may be one determinant of these factors, thereby contributing to the efficiency of pathogen transmission. Although body size alone does not account for all of the variation in biting rate (Scott et al., 2000). 2-3-1. Morphology :

Male and female mosquitoes differ in a number of ways that are useful in distinguishing between them. Sexual dimorphism is the term used to describe situations where characters differ between males and females (Bruce et al., 2008). The mosquito has a cylindrical body divided into head, thorax and abdomen, which thorax and abdomen are continuous, a head is spherical: most of it is occupied by scales. Several appendages project from the head, the median unpaired appendage is proboscis, the medial pair of appendages lying just lateral to the proboscis are the palps and lateral pair are antennae. The proboscis is a flexible gutter- like tube that encloses the

16 mouth parts of the mosquito. The mouth part comprises a pair of maxillae, a pair of mandibles, an epipharynx and a hypopharynx. The epipharynx and a hypopharynx together form a complete tube through which the mosquito sucks liquid food material. The mouth parts of mosquito are adapted for piercing and sucking. The palps which project from the base of the proboscis, are long in the male and short in the female mosquitoes, with the exception of Anopheles female, which also possesses long palps. The antennae are multisegmented, carrying whorls of coarse hair round the base of segments. The antennae of the male mosquitoes are described as plumose as they are densely hairy. The antennae of the female mosquitoes are described as pilose as they sparsely hairy (Dheer et al., 2008). The thorax is cylindrical and covered with scales. It is formed by the fusion of the tree segments, namely, prothorax, mesothorax and metathorax. Mesothorax provides attachment to a pair of wings and metathorax to a pair of halters, additionally, each segments of thorax provides attachment to one pair of legs, in all six in number. The wing have a distinct shape and characteristic venation: all wing veins are covered with scales. The wings are held out at right angles to the body during flight. In resting position the wings are folded, one over the other, along the long axis of the body. In this position, wings cover the dorsal aspect of the abdomen completely. Halters are drumstick-like outgrowths that arise from the sides of metathorax. They represent rudimentary wings and vibrate rapidly when the mosquito is in flight. Buzzing, balancing and hearing of mosquitoes have been ascribed to halters. The legs are multi-jointed and thickly covered with scales: the last segment of each leg ends in a pair of claws (Dheer et al., 2008).

The abdomen is longer than the thorax and is composed of nine to ten segments. The last two segments are modified to meet reproductive requirements and are represented by a pair of claspers in the male and a pair of cerci in the female mosquitoes. The claspers are long and curved, whereas the cerci are small and rounded (Dheer et al., 2008).

Small larvae that are rarely invisible to the naked eye hatch from eggs, larvae molt three times to become a fourth stage larva, several days later, this larval from molts again to become a pupa. All four of the larval stages of Culicine and Aedine mosquitoes are easily to recognize, because of the presence of elongate air tube used for breathing (William et al., 2005).

The pupa has superficial similarity to that of other mosquitoes although it differs from them in several details, the pupa is comma- shaped and differs generally from the larvae appearance. The front part consists of the head and thorax which are combined into a large cephalothorax, on the upper surface of which is a pair of respiratory trumpets. The abdomen comprises eight freely movable segments with a pair of paddles at the tip. Pupae do not feed during their aquatic existence, but come to the water surface to breathe (Gill et al., 1996).

2-3-2. Life cycle

Mosquitoes have a complete metamorphosis; eggs are laid, either singly or in bunch, on a water surface or on a place where they will become wet when flooding occurs. Mosquito larvae and pupae live in water. Various species have become adapted to living in practically all kinds of water except fast flowing streams and the open portions of large

18 bodies of water, where there is considerable wave action. The choice of type of water for larvae is generally quite specific (Dheer et al., 2008).

Larvae have a well defined head, thorax and abdomen. Most species move about actively in the water and come to the surface frequently to breathe. They assume characteristic positions when at the surface of the water. Larvae go through four instars, usually in a period of 4 to 10 days, to form the pupa. (Truman, 1997).

Mosquito larvae drink water and most species eat particulate food (diatoms, micro crustaceans, etc.) present in their aquatic habitats. These larvae have mandibles adapted for chewing and scraping, and they feed by creating a current of water with the movement of brush-like structures on the mouthparts (Eldridge, 2008). The pupa lives in water and like the larva, is quite active. The head and thorax are greatly enlarged and enclosed in sheath, and a pair of respiratory tubes project from its upper surface. Most species are lighter than water, so that when they are not actively swimming, they rise to the surface, where the respiratory tubes contact the open air for breathing.(Truman, 1997).The digestive tract is changed from that of a larva to that of an adult during the pupal stage, and consequently, pupae do not feed (Eldridge, 2008).

The pupal stage lasts from a day to as long as a few weeks. When ready to hatch, the pupa rises to the surface, and the pupal skin breaks. The adult works its way out of the pupal skin and uses the skin for support until the body wall has hardened to the extent that the adult is capable of flight. Males usually emerge first and wait near the hatching point to mate with the female soon after they emerge. Most female mosquitoes must have a blood meal before they can lay fertile eggs. The

19 adult female is the only stage that feeds on blood. The mouthparts of the adult male are not suitable for blood sucking. Therefore, their nourishment is normally derived from nectar and plant juices (Truman, 1997). Mosquitoes utilize sexual reproduction to produce new generations. Sperm from male mosquitoes is deposited in structures called spermathecae located at the tip of the abdomen of females. Fertilization of eggs takes place at the time of oviposition, not during mating. The numbers of spermathecae vary from one to three. Culex females have three, Anopheles females have only one. A single female usually deposits from 100 to 150 eggs at a time. Over the course of her life she may deposit 3 to 4 batches of eggs. Mating between males and females nearly always takes place in midair swarms. Once a female is inseminated, she remains for life and her spermathecae contain sperm (Eldridge, 2008).

2.4. Culex quinquefasciatus

2.4.1. Characteristic Features

Female: Medium-sized mosquito of brownish appearance; proboscis dark but often with some pale scaling midway on the underside; scutum with golden and bronzy narrow scales; wings all dark scaled; hind legs with femur pale almost to the tip except for dark scales along length dorsally, remainder of legs all dark scaled except for pale patch at tibial-tarsal joint; abdominal tergites dark scaled with pale basal bands constricted laterally and not merging with lateral patches except perhaps on terminal segments, sternites generally pale scaled but with a few to more dark scales scattered medially (Russell,1996).

Adult females may be confused with Cx. australicus (usually more pale scaling on underside of proboscis and more dark scaling on sternites but it will not be possible to separate some specimens), Cx. molestus and Cx. globocoxitus (tergal bands not constricted laterally), Cx. cylindricus and Cx. orbostiensis (smaller species with sparser wing scaling; Russell, 1996).

A female blood fed Cx. quinquefasciatus. This is a common species which is often a pest around the home and tends breeding in polluted waters (ICPMR, 2002).

The larvae feed on biotic material in the water and require between five to eight days to complete their development at 30°C (Gerberg et al., 1994). The larvae progress through four larval instars, and towards the end of the fourth instar they stop eating and molt to the pupal stage. Following 36 hours at 27°C the adults emerge from the pupal stage (Gerberg et al., 1994). The time of development under natural conditions for all stages is variable and dependant on temperature.

Both males and females take sugar meals from plants. Following mating, the female seeks a blood meal. Cx. quinquefasciatus are opportunistic

21 feeders, feeding on mammals and/or birds throughout the night. Males survive only on sugar meals, while the female will take multiple blood meals. After a female mosquito digests the blood meal and the eggs develop, she finds a suitable place to lay her eggs, and the cycle begins again. A single female can lay up to five rafts of eggs in a lifetime (Gerberg et al., 1994). The number of eggs per raft varies with climatic conditions.

Larvae: The larval head is short and stout becoming darker toward the base. The mouth brushes have long yellow filaments that are used for filtering organic materials. The abdomen consists of eight segments, the siphon, and the saddle. Each segment has a unique setae pattern (Sirivanakarn and White, 1978). The siphon is on the dorsal side of the abdomen, and in Cx quinquefasciatus the siphon is four times longer than it is wide with multiple setae tufts (Darsie and Morris, 2000). The saddle is barrel shaped and located on the ventral side of the abdomen with four long anal papillae protruding from the posterior end (Sirivanakarn and White, 1978).

Culex quinquefasciatus larvae 4th instar (ICPMR 2002).

Pupae: Similar to other mosquito species, Cx quinquefasciatus pupae are comma shaped and consist of a fused head and thorax (cephalothorax and an abdomen). The cephalothorax color varies with habitat and darkens on the posterior side. The trumpet, which is used for breathing, is a tube that widens and becomes lighter in color as it extends away from the body. The abdomen has eight segments. The first four segments are the darkest, and the color lightens towards the posterior. The paddle, at the apex of the abdomen, is translucent and robust with two small setae on the posterior end (Sirivanakarn and White 1978).

Adults: Adult Cx quinquefasciatus vary from 3.96 to 4.25 mm in length (Lima et al., 2003). The mosquito is brown with the proboscis, thorax, wings, and tarsi darker than the rest of the body. The head is light brown with the lightest portion in the center. The antennae and the proboscis are about the same length, but in some cases the antennae are slightly shorter than the proboscis. The flagellum has thirteen segments that have few to no scales (Sirivanakarn et al., 1987). The scales of the thorax are narrow and curved. The abdomen has pale, narrow, rounded bands on the basal side of each tergite. The bands barely touch the basolateral spots taking on a half-moon shape (Darsie and Ward 2005).

2.5. INSECTICIDES 2.5.1. Organophosphates (Ops) OPs were first recognized in 1854, but their general toxicity was not established until the 1930s. Tetraethyl pyrophosphate (TEPP) was the first OP insecticide, which was developed in Germany during 1945 as a by-product of nerve gas development (Minton et al., 1988).

Chronic fatigue is common amongst those who consider their health is affected by pesticides and research from 2003 suggested there was an association between exposure to Ops and chronic fatigue symptoms. (Tahmaz, et al., 2003). A 2010 study has found that OP exposure is associated with an increased risk of Alzheimer's disease (Hayden, et al., 2010).

2.5.2. Toxicology Ops poison insects and mammals primarily by phosphorylation of the acetyl cholinesterase (AchE) enzyme (AchE) at nerve endings. The result is a loss of available AChE so that the effector organ becomes over stimulated by the excess acetylcholine (Ach, the impulse-transmitting substance) in the nerve ending. The enzyme is critical to normal control of nerve impulse transmission from nerve fibers to smooth skeletal muscle cells, glandular cells, and autonomic ganglia, as well as within the central nervous system (CNS). Some critical proportion of the tissue enzyme mass must be inactivated by phosphorylation before symptoms and signs of poisoning become manifest (DuBois, 1971).

At sufficient dosage, loss of enzyme function allows accumulation of Ach peripherally at cholinergic neuroeffector junctions (muscarinic effects) skeletal nerve–muscle junctions and autonomic ganglia (nicotinic effects) as well as centrally. At cholinergic nerve junctions with smooth muscle and gland cells, high Ach concentration causes muscle contraction and secretion, respectively. At skeletal muscle junctions excess Ach may be excitatory (cause muscle twitching) but may also weaken or paralyze the cell by depolarizing the end- plate. In the CNS, high Ach concentration cause sensory and behavioral disturbances, in coordination, depressed motor function, and respiratory depression. Increased

24 pulmonary secretions coupled with respiratory failure are the usual causes of death from OP poisoning. Recovery depends ultimately on generation of new enzyme in all critical tissues (DuBois, 1971).

2-5-3. Resistance Pesticide resistance is the adaptation of pest population targeted by a pesticide resulting in decreased susceptibility to that chemical. In other words, pests develop a resistance to a chemical through natural selection: the most resistant organisms are the ones to survive and pass on their genetic traits to their offspring, also Insecticide resistance is a heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product to achieve the expected level of control when used according to the label recommendation for that pest species (IRAC, 2007).

Insecticide resistance has become a major concern in vector control with resistance now present in mosquitoes to all major classes of chemical insecticides. This can lead to product failure and a rise in disease transmission (Invest et al., 2008).

Resistance to OPs first reported 14 years after their introduction, numbers 260 insect and mite species. Resistance to carbamate insecticides has appeared after five years, partly due to conditioning by previous OP exposure (Green et al., 1990).

Multiple resistances are the phenomenon in which a pest is resistant to more than one class of pesticides. This can happen if one pesticide is used until pests display a resistance and then another is used until them are resistant to that one, and so on. Cross resistance, a related phenomenon, occurs when the genetic mutation that made the pest

25 resistant to one pesticide also makes it resistant to other pesticides,

STATUS: ISO 1750 (published)

especially ones with similar mechanisms of action (Doyen and Purcell, 1998).

Not all mosquito control failures are the result of resistance. Improper control practices may be at fault. However, if the material was timed and applied properly at the recommended rate and no other important factors (such as unfavorable weather) have interfered with the insecticide application, resistance should be considered (Eldridge, 2008).

2.6. Temephos “temefos”:

IUPAC: O,O,O′,O′-tetramethylO,O′-thiodi-p- phenylenebis(phosphorothioate) or O,O,O′,O′-tetramethylO,O′-thiodi-p- phenylenediphosphorothioate CAS: O,O′-(thiodi-4,1-phenylene) bis(O,O-dimethyl phosphorothioate) REG. NO.: 3383-96-8 FORMULA C16H20O6P2S3 : ACTIVITY: insecticides phenyl organothiophosphate insecticides NOTES: The name “Temefos®” is approved by the WHO

STRUCTU RE:

Common Name: temephos, Chemical Name: Phosphorothioic acid, Chemical Family: Organophosphate

Temephos is a phenyl OP insecticide that is mainly used as a larvicide to control mosquitoes, midge, blackly and other insects. Its toxic action against the target species is by inhibition of ChE in the nervous system. Temephos has been used for the control of mosquito larvae in potable water since the early 1970s (WHO, 1991), and has been recommended for treatment of potable water at a dose not exceeding 1 mg/l (WHO, 1984).

Temephos is a non-systemic OP insecticide, mainly used as a larvicide to control mosquitoes, including domestic water containers use. Two formulations of temephos have been evaluated by the WHO

Pesticide Evaluation Scheme (WHOPES) for mosquito larviciding (WHO, 2006). WHO specifications for quality control and international trade have been published for the two formulations: (EC) and (GR) formulations (WHO, 2008). Only the granule formulation is used for mosquito larviciding in potable water at a dosage not exceeding 1 mg/l of the active ingredient (WHO, 2009).

Temephos is one of a few organophosphates registered to control mosquito larvae, and is the only organophosphate with any appreciable larvicidal use. It is an important resistance management tool for mosquito abatement programs. Temephos residues in food and drinking water do not pose risk concerns since Temephos has no food uses and, because of its limited use pattern, is not expected to be found in drinking water. Residential risk is not of concern since temephos has no residential use and its use in mosquito abatement programs does not result in residential exposure. With mitigation measures, Temephos’ worker and ecological risks will be eligible for reregistration (EPA, 2001).

2-6-1. TOXICOLOGY

Temephos containing products are moderately toxic compounds that carry the signal word WARNING on their labels despite the relatively high LD50 of the technical compound. This is due to the high toxicity of xylene, one of the components (or carriers) found in many of the trade products. This Pesticide Information Profile is oriented to the toxicity of the technical product temephos and not the different trade products unless specifically noted. Typical of other organophosphate insecticides, temephos inhibits the action of the group of enzymes called cholin esterases. Specific types

28 of these enzymes are found throughout the body including the nervous system, the brain, and the blood stream. Symptoms of acute exposure are also similar to other organophosphates and may include nausea, salivation, headache, loss of muscle coordination, and difficulty breathing (Bathesda, 1993). Temephos is recommended by WHO for addition to potable water as larvicide treatment at an application rate not exceeding 1 mg/L. Assuming that an adult weighing 60 kg would consume 2 L/day of drinking-water containing temephos at 1 mg/l, this would be equal to an oral exposure of 0.033 mg/kg body weight. However, given the limited solubility of temephos in water, incomplete dissolution in drinking-water would be expected, and this could result in actual exposures being appreciably less than this estimate. Consequently, 0.033 mg/kg body weight per day was regarded as a worst-case upper limit of exposure. Some of the studies that were critical to the assessment were of poor quality. The Meeting considered that the database was insufficiently robust to serve as the basis for establishing an ADI or an ARfD for Temephos (WHO, 2009). In a study in mice, groups of five females were given single doses of temephos at 1250, 2500 or 5000 mg/kg bw in corn oil by oral gavage. The treatment was followed by a 14-day observation period. Body weights were measured at the beginning and end of the study. Necropsies were not performed. All animals at 5000 mg/kg bw died before the end of the observation period. At all doses, ataxia, decreased activity, diarrhea and prostration were observed. Salivation and diuresis were also observed at 1250 and 2500 mg/kg bw. The oral median lethal dose (LD50) for female mice in this study was estimated to be 2062 mg/kg bw (Fischer et al., 1986).

2-7. Fenitrothion

STATUS: ISO 1750 (published) IUPAC: O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate CAS: O,O-dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate REG. NO.: 122-14-5 FORMULA: C9H12NO5PS ACTIVITY: insecticides phenyl organothiophosphate insecticides NOTES: The name “MEP” is approved by the Japanese Ministry of Agriculture, Forestry and Fisheries.

STRUCTURE:

Product Name: fenitrothion 50% EC, Trade Name SUMITHION® 50 EC, Chemical Family: OP. Fenitrothion is a cholinesterase inhibiting insecticide / acaricide registered for use on ornamentals, including trees, and in ant and roach baits. Sumitomo Chemical Company requested voluntary cancellation of the malaria control uses of fenitrothion on February 28, 1995. Fenitrothion was first registered in the United States in 1975. The registration was for control of spruce budworm in forests under the supervision of federal and state officials responsible for insect control

31 programs in forest areas. In November of 1992, at the request of the registrant, all of the fenitrothion products bearing forestry uses were cancelled. Fenitrothion is used in the United States. for commercial ornamental pest control. The registrant has requested voluntary cancellation of the mosquito control products (i.e., malaria control). Technical grade fenitrothion is highly toxic to birds and aquatic invertebrates, and chronic effects were observed in an avian reproduction study. Fenitrothion is also moderately toxic to mammals. Outdoor uses of fenitrothion exceed the hazard limits established for restricted-use classification. Therefore, the Agency will require all fenitrothion products labeled for outdoor use to be labeled for restricted use only (EPA, 1995).

2.8. Insect Growth Regulators (IGRs) An IGR, may be defined in terms of its mechanism of action, as substance which acts within an insect to accelerate or inhibit a physiological regulatory process essential to the normal development of the insect o its progeny, in such a way that the action of the substance is necessarily dependent on the life stage of the insect.(Siddall, 1976).

The origin of IGRs was entirely different. Their discovery was based on knowledge of how insects grow, develop, function and behave. They have been discovered in two ways. One way was to expose an insect to IGRs and observe abnormalities in how it develops, functions or behaves. Chemicals that produce desired effects were developed. Another was to find out what processes in the insects' development involves hormones and to use those hormones as models to synthesize chemical analogs that will interfere with normal insect growth and development. Insect Growth Regulators (IGRs), also called third-generation insecticides, are pesticides that disrupt the normal activity of the

31 endocrine or hormone system of insects, affecting the development, reproduction, or metamorphosis of the target insect. They have a much slower mode of action than synthetic chemical insecticides. IGRs include juvenile hormone (JH) mimics and chitin synthesis inhibitors (CSIs). There is a large concern about the effect of IGRs on non-target species and many IGRs are lacking full health and safety data. (Krysan and Dunley, 1993).

2-8-1. Juvenile Hormones (JH)

Most currently registered IGRs, such as hydroprene and methoprene, fall into the juvenile hormone mimic category. These pesticides mimic the JH produced in the insect brain, which forces the insect to remain in a juvenile state. Normally, the production of the JH would ebb as the insect progressed through the nymphal stages until the final molt into the adult stage, when JH production would cease. When exposed to a JH mimic, the insect remains in an immature state, and is rendered unable to successfully molt into the adult stage or become reproductively viable. JH mimics are not be specific to the pest under attack, as the molecules based on the JH system are more or less common to all insect species.(Krysan and Dunley, 1993).

2-8-2. Anti-juvenile hormone agents

Anti-juvenile hormone agents cancel the effect of juvenile hormone by blocking juvenile hormone production. For example, an early instar treated with an anti-juvenile hormone agent molts prematurely into a nonfunctional adult. A disadvantage of these chemicals is that they are so selective that they may not be economic for a manufacturer to develop (Krysan and Dunley, 1993).

2-8-3. Chitin Synthesis Inhibitors (CSIs)

CSIs, such as hexaflumuron, lufenuron and diflubenzuron, inhibit the production of chitin, a major component of the insect exoskeleton. Insects treated with CSIs become unable to synthesize new cuticle, and therefore unable to successfully molt into the next stage. CSIs may be toxic to other arthropods, and IGR metabolites may have adverse effects on vertebrates due to their ability to bind to certain members of the nuclear hormone receptor family. (Hoffman and Lorenz, 1998).

Several features of insect growth regulators (IGRs) make them attractive as alternatives to broad-spectrum insecticides. Because they are more selective, they are less harmful to the environment and more compatible with pest management systems that include biological controls (Krysan and Dunley, 1993).

2-8-4. Pest Resistance to IGRs

It was originally thought that insects would be unable to develop resistance to molecules that mimic their own hormones, but there is already evidence of developing resistance to several IGRs, including methoprene, hydroprene, kinoprene, pyriproxyfen, and diflubenzuron. Resistance seems to result from decreased penetration and increased metabolism of the compound. (Hoffman and Lorenz, 1998)

Pyriproxyfen is a broad-spectrum insect growth regulator with insecticidal activity against public health insect pests: houseflies, mosquitoes and cockroaches. In agriculture and horticulture, pyriproxyfen has registered uses for the control of scale insects, whitefly, bollworm, aphids and cutworms. (FAO/WHO, 1999).

The mimic (JH) has been developed comprehensively recently. Some JH mimics have been used as insecticides, with selective action on different insect pest species. These compounds are toxic during the last larval, embryonic and reproductive stages of insects (Aribi et al., 2006).

CHAPTER THREE 3. MATERIAL AND METHODS

3.1. STUDY AREA This study was conducted in Qatar state. Qatar is a peninsula situated on the western coast of the Arabian Gulf. The country covers 11,525 Km², including a number of islands. The length of the peninsula from south to extreme north is about 187 Km. .The peninsula of Qatar is located between °24 ′27 and °26 ′10 north latitudes and °50 ′45 and °51 ′40 east longitudes. The population of Qatar is 1,699,435 persons distributed in seven municipalities. The Rayyan municipality population is 26.8% of the whole population).

The climate of Qatar is tropical and conspicuously hot during the summer, and pleasant with little rain in winter. Summer, however, lasts from June to September. Other seasons in Qatar are pleasant and fine.

3.1.1. Climate Data for Qatar (temp):

Season winter spring Summer Autumn

Ma Month Jan Feb Mar Apr Jun Jul Aug Sep Oct Nov Dec y

Average 22 23 27 32 38 41 46 44 41 35 29 24 high (°C)

Source: Qatar meteorology department, 2012.

3.2. STUDY DESIGN

The study was conducted in Rayyan municipality, the biggest municipality in Qatar state and has the most breeding sites of mosquitoes. The selected breeding locations of mosquitoes are considered as sample unit using simple random sampling techniques.

This research is designed and developed to test the susceptibility of Culex quinquefasciatus to the two larvicides. The study includes inspection and collection of mosquito larvae in different breeding sites, such as stagnant water, marshes, sewage water and manholes.

3.3. SAMPLE SIZE: The final sample size will be calculated from the whole number of mosquito breeding sites (only 28, locations) for the purpose of this study, then 20% is quite enough of the whole breeding sites as sample size. 3.4. EXPERIMENTAL MATERIAL:

1. Synthetic larvicides: two Ops, fenitrothion 50% EC and temephos 10% EC 3.5. METHODOLOGY: Mosquito larval bioassays, regarding the WHO Guidelines for laboratory and field testing of mosquito larvicides, 2005)

3.5.1. Collection of Larvae

Culex quinquefasciatus larvae were collected directly from the field for each test. The collection tools were nets, dipping technique without changing the environmental water habitat.

3.5.2. Preparation of stock solutions

1 ml of larvicide in 100 ml solvent to produce stock solution (10000 ppm)

Then, according to the larvicide concentration, preparing the stock solutions.(S.S.1, S.S.2 and S.S.3 ), from these dilutions making a series of concentrations.

3.5.3. Larval Treatment

(20) late third (L3) and early 4th instars (L4) of mosquito larvae species of Culex quinquefasciatus (Say) were used in all tests. Then exposed to the larvicides for 24 and 48 hours (Fenitrothion 50% and Temephos 10%) at various concentrations. Mortality was recorded for the two larvicides in both winter and summer.

Disposable plastic cups were used as mosquito breeding containers in this study. Each cup with capacity of 120 to 250 ml (holding 100 ml), considering the depth of water remain 10 cm. The tests were held at 25– 28 °C, and photoperiod of (12: 12 L: D) with relative humidity (R.H.) 80±10%.

Three - Four replicates (3 + 1) were set up for each concentration (five concentrations or more). As follow:

For temephos 10%: 0.02, 0.05, 0.08, 0.1 and 0.2 ppm .

For Fenitrothion 50%: 0.02, 0.05, 0.08, 0.1, 0.2 –, 0.25 and 0.3 ppm.

Each test was done three times on different days in each season (winter and summer) repeated to determine LC50, LC90 values.

Fenitrothion test was done another repeated three times in both season (winter and summer) in order to confirm the first findings results of these larvicide.

3.5.3.1 Diagrammatic illustration of the experimental design of the test:

0.00 0.02 0.05 0.08 0.1 0.2 0.25 0.3

The control mortalities of treated groups should be corrected according to Abbott’s formula (3) (WHO, 2005):

X – Y

Mortality (% ) = ———— 100 , (for synthetic larvicides)

X ( late 3rd& early 4th stage larvae)

Where X = percentage survival in the untreated control and,

Y = percentage survival in the treated sample.

3.6. DATA ANALYSIS:

Data was analyzed using the Probit analysis, the most common outcome of a dose-response experiment which used in the LC50/LD50. Computer software programs will be use in all the analysis procedure.

CHAPTER FOUR RESULTS

20 16.6

15 13 11

10 8 6 winter 5 5 5 5 summer 5

mortality % mortality 3 خطي )(winter 1 1 خطي )(summer 0 0 0 0 0.0 0.02 0.05 0.08 0.1 0.2 0.25 0.3

-5 concentrations

Figure (1) Efficacy of Fenitrothion 50% against Culex quinquefasciatus larvae - test No. (1) showing winter & summer curve (Jan&July2012)

Figure (1) Shows the efficacy of the organophosphate larvicide fenitrothion 50%, against mosquito larvae Culex quinquefasciatus.The test was done in January 2012 and repeated in July2012, which represents the first months in both seasons winter, summer respectivly. The average temperature started at the range of (15 °C - 22°C) in winter and (33 °C - 43°C) in summe, the maximum value of the mortyality in summer was (16.6%) and (11%) in winter.

16 13.3 14

12 10 10 10

8.3 summer

8 6.6 6.6 6 6 winter 6 5 خطي )(winter 3.3

4 mortality % mortality خطي )(summer 1.6 1.6 2 0 0 0 0 0.0 0.02 0.05 0.08 0.1 0.2 0.25 0.3 -2

-4 concentrations

Figure (2) Efficacy of Feniitrothion 50% against Culex quinquefasciatus larvae -test No. (2) showing winter & summer curve

(Feb&Aug2012)

Figure (2) Shows the efficacy of the fenitrothion 50%, the 2nd and 5th tests against Culex quinquefasciatus larvae, which done in February 2012 and repeated in August2012, which represents the second months in both seasons winter, summer respectivly. The average temperature started at the range of (15 °C - 23°C) in winter and (33 °C - 42°C) in summer. The study result show that the two curves rise parallel from the begening . the maximum value of the mortyality in summer was (13.3%) and (8%) in winter.

18 16.6 16

12 winter 11.6 11.6

10 10 10 summer

خطي 8.3 8.3 8.3 8 6.6 6.6 winter)( خطي 6

mortality% 5 5 summer)( 4 3.3 3.3 3.3 2

0 0.0 0.02 0.05 0.08 0.1 0.2 0.25 0.3 concentrations

Figure (3) Efficacy of Fenitrothion 50% against Culex quinquefasciatus larvae - test No. (3 ) showing winter & summer curves March & September 2012

Figure (3) Showing the efficacy of the fenitrothion 50%, the 3rd and 6th tests against Culex quinquefasciatus larvae, which done in March 2012 and repeated in September 2012, which represents the last months in both seasons winter, summer respectivly. The average temperature started at the range of (18 °C - 26°C) in winter and (30 °C - 39°C) in summer. The result show that the two curves rise parallel from the begening. the maximum value of the mortyality in summer was (16.6%) and (10%) in winter.

110

100 98.3 93.3 90 85 85 winter 80 73.3 73.3 70 summer 65 60 خطي )(winter 50 53.3 خطي 40

mortality % mortality summer)( 30 31.6 20 18.3 10 8.3 3.3 0 1.63.3 -10 0.0 0.01 0.02 0.05 0.08 0.1 0.2 concentrations Figure (4) Efficacy of temephos 10% against Culex quinquefasciatus larvae - test (1) showing winter & summer curve (Jan&July2012)

Figure (4) Shows the efficacy of the larvicide temephos 10%, against Culex quinquefasciatus larvae, the test was done in January 2012 and repeated in July2012, which represents the first months in both seasons winter, summer respectivly. The average temperature started at the range of (15 °C - 22°C) in winter and (33 °C - 43°C, appendix,7) in summer. The study result show that the LC50 in between the concentration value (0.05 – 0.08 mg/l). the maximum value of the mortyality in summer was (98.3%) and (85%) in winter.

110 100 96.6 90 91.6 86.6 81.6 80 78.3 winter

70 63.3 summer 60 56.6 خطي )(winter 50

خطي 38.3 40 mortality % mortality 33.3 30 summer)(

20 18.3 10 10 6.6 3.3 0 1.6 -10 0.0 0.01 0.02 0.05 0.08 0.1 0.2 concentrations

Figure (5) Efficacy of temephos 10% against Culex quinquefasciatus larvae - test No. (2) Showing winter & summer curve (Feb & Aug 2012)

Figure (5) Shows the efficacy of the larvicide temephos 10%, against Culex quinquefasciatus larvae, the 2nd test which was done in February 2012 and repeated in August 2012, The average temperature started at the range of (15 °C - 22°C) in winter and (33 °C - 43°C) in summer. the maximum value of the mortyality in summer was (96.6%) and (86.6%) in winter

110 100 95 90 86.6 81.6 80 78.3 73.3 winter

70 63.3 60 60 summer 50 45 40 خطي )(winter 30% mortality 31.6 20 خطي 15 13.3 10 summer)( 3.3 5 0 1.6 -10 0.0 0.01 0.02 0.05 0.08 0.1 0.2 concentrations

Figure (6) Efficacy of temephos 10% against Culex quinquefasciatus larvae - test (3) Showing winter & summer curve (March & Seb 2012)

Figure (6) Shows the efficacy of the larvicide temephos 10%, against Culex quinquefasciatus larvae, the 3rd and 6th test was done in March 2012 and repeated in September 2012, which represents the last months in both seasons winter, summer respectivly. The average temperature started at the range of (18 °C - 26°C) in winter and (30 °C - 39°C) in summer. the maximum value of the mortyality in summer was (95%) and (81.6%) in winter

120 110 fenitrothion 100 winter 90 80 81.7 73.3 temephos

70 winter

60 63 50 خطي 45 40

mortality% fenitrothion ( 30 winter ) 20 خطي ) 15 10 8.3 8.3 10 10 temephos 3.3 53.3 3.3 5 winter) 0 1.7 -10 0.0 0.02 0.05 0.08 0.1 0.2 0.25 0.3 -20 concentrations

Figure (7) Comparing the efficacy of fenitrothion and temephos against Culex quinquefasciatus in winter season March 2012

Figure (7) shows comparing the efficacy of the two larvicides, fenitrothion 50% and temephos 10%, against Culex quinquefasciatus larvae, the test was done in winter, 2012. The average temperature started at the range of (18 °C - 26°C, appendix,7). It is clear from the above figure that temephos is showing a remarkable sensitivity against mosquito larvae, while fenitrothion is resistant.

120

110 fenitrothion summer 100 95 90 86.7 temephos 80 78.3 summer 70

خطي 60 60 fenitrothion (

mortality% 50 summer) 40

خطي 31.7 30 temephos ( 20 summer) 16.6 13.3 10 11.6 11.6 6.6 6.6 8.3 8.3 53.3 0 0.0 0.01 0.02 0.05 0.08 0.1 0.2 0.25 0.3 ooncentrations

Figure (8) Comparing the efficacy of fenitrothion and Temephos against Culex quinquefasciatus in summer season sep 2012

Figure (8) Shows comparing the efficacy of the two larvicides, fenitrothion 50% and temephos 10%, against Culex quinquefasciatus larvae, the test was done in summer, 2012. The average temperature started at the range of (18 °C - 26°C appendix ,7).

CHAPTER FIVE DISCUSSION

Figure (4, 5 and 6) shows the efficacy of the other tested larvicide, Temephos 10% which used before the organophosphate Fenitrothion 50% in vector control of the aquatic stages of mosquito in municipality during (2010 -2011). In general the efficacy of this larvicide is more than the fenitrothion, i.e. The LC50 of the Temephos 10% at the range of 0.039 – 0.047 in summer (and, (0.071 – 0.08) in winter. On the other hand, the

LC50 of the fenitrothion 50% at the range of (0.51 – 0.61) in summer and, (0.42 – 0.70) in winter, referring to appendix (5).

With regard to figure (1) which Showed the efficacy of fenitrothion 50% against mosquito larvae test. (1) during January and July 2012. The mortality of mosquito larvae was more, the LC50 of the test in summer, which maximum temperature are 47°C is 0.421 compared with the LD50 in winter, temperature (20 – 28°C), appendix (7), which means that the mortality of mosquito larvae in summer is more than the mortality in winter season, And this is due to the high temperature in these months. larval mortality was highest at the upper rang (30 – 32°C) with death rather than the adult emergence, the optimal survival temperature were lower than the temperature at which the development was quickest, suggesting a critical relationship between temperature and the life cycle of the insect.(Bayoh and Lindsay, 2003). Mosquito larvae that survive at high temperature produce smaller and less successful adults, (Shelton, 1973). As the body temperature of an insect rises, the rats of both metabolism and respiration increase up to critical thermal limit. It has been reported that death occurs soon after the

48 respiration rate begins to drop. Even the insect returned to normal temperatures indicative of systematic cell deaths at the high temperature, (Neven, 2000). Temperature is one of the major factors that affect the survival of aquatic stages of mosquitoes, which requires an understanding of seasonal temperatures, and also in recognition of how the impact of climate on mosquito survival and reproduction, and advantage from it in the process of control. Vector control in Rayyan municipality facing a serious threat due to the acquiring of resistance to Culex quinquefasciatus larvae against larvicide fenitrothion 50% and this showed in figures. (1, 2 and 3). Also the mortality in the present study of fenitrothion showed in appendix (6) which was 59.9% after 48 hours. Therefore Culex quinquefasciatus facing a threat of resistance to larvicide fenitrothion, because the mortality values lower than 80%, which considered by WHO resistant to the insecticide (WHO, 1992).

Resistance to insecticides has appeared in the major insect vectors from every genus and has developed to every chemical class of insecticides (Brodon and Allistor, 1998). However, continuous use of various insecticides has resulted in development of resistance in mosquitoes larvae. (Tikar et al., 2008), have recorded development of resistance in Ae. aegypti larvae against Temephos, Fenithion, Malathion and DDT from different locations in India.

In such situation Cx. quinquefasciatus co-exist in the same breeding sites with the Anopheles spp. In spite of the continued exposure to temephos. Cx. quinquefasciatus larvae in both areas were found susceptible to temephos with LC50 LC90 values being 0.0157 and 0.0480 ppm in India (Baruah, 2004), and 0.031 and 0.075 ppm in Khartoum

(Jamal, 2009). With refer to figures (4, 5 and 6) temephos tests, the LC50 of Temephos of the present study at the range of (0.036 to 0.071) in both season winter and summer, therefore accepting these results, with mortality ranged between 81.6% and 98.3% after 24 hours.

Tentative diagnostic doses of temephos for larval mosquitoes have been recommended for Anophelenes, Culex quinquefasciatus and Ae. aegypti by WHO since 1986. The diagnostic concentration (DC) is referred to as a double the concentration at LC 99 that kills all individuals or produces ≥ 98% mortality in 24 hr, if the insect strain is susceptible. Mortality values between 80% and 98% in calculated in the verification of response category or suggest at incipient alteration in susceptibility to the insecticide (Hany, 2009). And this confirms the previous mentioned point of the present study.

In Bahry locality, Khartoum state, Sudan is highly susceptible to temephos LC50 and LC90 of 0.00271 and 0.025 ppm (Ahmed, 2007). Also larvae from Ouagadougou, Burkina Faso west Africa showed complete susceptibility to organophosphours and carbamates with temephos scoring LC50 of 0.0038 mg/l (Majori et al., 1968).

Operational factors are related to the use of insecticides, and may appear as a result of selective pressure or failures in control operations. Resistance gens are rare, and appear after prolonged periods (De Carvalho et al., 2004). This is relatively comparable to the situation in Rayyan municipality, in addition to the conclusion of Hamdan, The speed and degree of development of resistance depends on the frequency of resistance gen(s) in the population, the type of gene which responsible for resistance, the insecticide dosage applied and frequency of application (Hamdan et al., 2005).

CONCLUSION

It is important to detect and characterize developing resistance problem so that future control strategies can be developed by optimizing current insecticides usage specially larvicides.

The present results showed that the mortality of Culex quinquefasciatus larvae in summer was more than the mortality in winter season, and this was most probably due to the high temperature during these months.

The study showed the appearance of resistance in Culex quinquefasciatus larvae against larvicide Fenitrothion 50%, because the highest mortality value of Fenitrothion in study was 59.9% after 48 hours which was lower than 80%, the value identified by WHO. On the other hand the results showed that the larvicide temephos is good for use in larval control in Qatar (98.3%).

RECOMMENDATOINS

1. An initial comprehensive entomological survey is recommended to be conducted in addition to continuing field surveillance for the development of larvicides resistance.

2. Taking into account the seasonal changes in control strategy against the larval stages of mosquitoes in Rayyan municipality, because climate is an important determinant of insect distribution.

REFERENCES

Ahmed, B. A. (2007) Field evaluation of temephos and Agnique® MMF against immature stages of Anopheles arabiensis Patton (Diptera: Culicidae) the vector of malaria in Khartoum, Sudan. M.sc. Thesis University of sains, Malaysia.

Aribi, N., G. Smagghe, S. Lakbar, N. Soltani-Mazouni, and N. Soltani. (2006.) Effects of Pyriproxyfen, a Juvenile Hormone Analog, on Development of the Mealworm, Tenebriomolitor Pesticide Biochemistry and Physiology 84:55-62

Bathesda, MD, (1993) Hazardous Substance Data Base. TOXNET. US National Library of Medicine, National institute of Health).

Brodon, W.G and McAllister, J.C (1998), insecticide resistance and vector control, CDC and prevention Atlanta, Georgia, USA.

Bruce F. Eldridge (2008). Biology and control of mosquitoes in California, Department of Public Health, California. Carvalho M, Caldasb do, S.L, Degallierc E.D.M, Vilarinhosa P.R du Souzed, L.C.K.R Yoshizawaa M.A.C and Du Oliveiraa,(2004) . susceptibility of Ae. aegypti larvae to insecticide temephos in the federal district, Brazil, Rev sade pblica 38 (5).

Chen, C.D. and Lee, H.L. (2006) Laboratory bioefficacy of CREEK 1.0G (Temephos) against Aedes (Stegomyia) aegypti (Linnaeus) larvae.

Daly H, Doyen JT, and Purcell AH III (1998), Introduction to insect biology and diversity, 2nd edition. Oxford University Press. New York. Chapter 14, Pages 279-300.

Darsie Jr RF, Ward RA. (2005). Identification and Geographical Distribution of the Mosquitoes of North America, North of Mexico. University of Florida Press. Gainesville, FL. 300 pp.

Darsie Jr RF, Morris CD. (2000). Keys to the adult females and fourth- instar larvae of the mosquitoes of Florida (Diptera: Culicidae). Technical Bulletin of the Florida Mosquito Control Association 1: 148-155.

DuBois KP, (1971). The toxicity of oganophosphorous compounds to mammals. Bull WHO 1971, 44:233-40.)

Dye, C. (1992.) The analysis of parasite transmission by bloodsucking, insects. Annul. Rev. Entomology. 37: 1-19. El-Shazly and Refaie (2002) Larvicidal Effect Of The Juvenile Hormone Mimics Pyriproxyfen On Culex Pipiens Journal o f the American Mosquito Control Association.

EPA, (1995). Office of Prevention, Pesticides and Toxic Substances, Fenitrothion, EPA, 738-R-95-018, July 1995). EPA, (2001). Temephos Facts, Office of Prevention, Pesticides and Toxic Substances – EPA, 738-F-00-08, July 2001). FAO/WHO, (1999).

Fischer, J.E. (1986) Toxicity data report: 0, 0, 0’, 0’ tetramethyl-0, 0’- thiodi-p-phenylene phosphothioate. Unpublished report No. A86-7 (BASF Registration Document No. TM-410-002) from American Cyanomid Company, Princetown, New Jersey, USA. Submitted to WHO by BASF. Nielsson, R.J. (1990) Abate – acute toxicity. Letter dated 6

February 1990 from Cyanomid to C. von Malzahn, Brussels. Company reference No. RJN: mdj90m1 AF3 (BASF Registration Document No. TM-A5.2.1-007, incorporated into TM-410-002). Gerberg EJ, Barnard DR, Ward RA. (1994). Manual for Mosquito Rearing and Experimental Techniques. American Mosquito Control Association Bulletin No. 5: 61-62 Gill SS, Singh GJ, and Hornung JM (1987) Cell Membrane interaction of Bacillus thuringiensis subsp. Israelensis cytolytic toxins. Infect immune 55:1300-1308. Dheer. I Robbani, (2008.) Foundations of Community Medicine -2nd edition.

Green., M., et. al., (1990). Managing Resistance to Agrochemicals, ACS Series 421, US, , 496pp).

Hamdan H; Sofian Aziron M, Ahmed N.W and Lim L.H, (2005). insecticide resistance development in Culex quinquefasciatus (say), Aedes aegypti (L) and Aedes albopictus (skuse) larvae against malathion, permethrin and temephos. Tropical Biomedicine, 22 (1): 45-52,.

Hany elsayed Alamohanadi, (2009). Susceptibility of Anopheles arabiensis larvae to the insecticide Temephos in Wad Medani district, M.sc. thesis (BNNICD).

Harbach, R.E, (2007). The Culicidae (Diptera): a review of taxonomy, classification and phylogeny. Pp. 591-638In: Zhang, Z.-Q. and Shear, W.A.

Hayden, K. .; Norton, M. .; Darcey, D. .; Ostbye, T. .; Zandi, P. .; Breitner, J. .; Welsh-Bohmer, K. .; Cache County Study, (2010).

"Occupational exposure to pesticides increases the risk of incident AD: the Cache County study". Neurology 74 (19): 1524–1530.

H.M.Gill & D.A Warell, (1996). Bruce Chwatt's Essential Malariology, 3rd edition. Hoffman, K.H., Lorenz, M.W, (1998). Recent Advances in Hormones in Insect Pest Control.

IRAC, (2007). I definition: Insecticide Resistance Action Committee, Resistance Definition. Retrieved on September 15.

Jamal Khalifa, (2009). susceptibility of Culex quinquefasciatus say 1823 (Diptera: Culicidae), to Malathion, temephos, lambada-cyhalothrin and permethrin insecticides in Khartoum locality, Sudan, M.sc. thesis (BNNICD).

James L. Krysan and John Dunley, (1993). Insect growth regulators: , Washington State University.

J B Siddall, (1976). Insect growth regulators and insect control: a critical appraisal Environ Health Perspective. J.F. Invest And J.R. Lucas, (2008). Pyriproxyfen As A Mosquito Larvicide. Lee C. Truman, (1997). Truman's Scientific Guide to Pest Control Operations, 5th edition. Lieutenant Colonel Leon L. Robert, Jr, (2001). Guide to Entomological Surveillance during Contingency Operations. U.S. Army. Lima CA, Almeida WR, Hurd H, Albuquerque CM. (2003). Reproductive aspects of the mosquito Culex quinquefasciatus (Diptera: Culicidae) infected with Wuchereria bancrofti (Spirurida: Onchocercidae). Memories do Institute Oswald Cruz. 98: 217-222.

Majori E; Salatinelli G; Villani F and Petrarca V, (1986). studies on insecticide susceptibility of Anopheles gambiea and Culex quinquefasciatus in the area of Ouagadougou, Burkina Faso (west Africa), J.AM mosquito control assoc. 2(3): 305-309.

Mathieu Tilquin1,2, Margot Paris2, Stephaney Reynaud2, Laurence Des pres2, Patrick Ravanel2, Roberto A. Geremia2* Je´roˆme Gury2, (2008). Long Lasting Persistence of Bacillus thuringiensis Subsp. israelensis (Bti) in Mosquito Natural Habitats. Minton, N.A. and Murray, V.S.G., A review of Organophosphate Poisoning, Medical Toxicology, 3: 350-375.)

Monthly Averages for Doha, Qatar, (2009). Weather.com. (The Weather Channel..

Mulla MS, Thavara U, Tawatsin A, Chompoosri J, Zaim M, Su T. (2003). Laboratory and field evaluation of novaluron, a new acylurea insect growth regulator, against Aedes aegypti (Diptera: Culicidae). Journal of Vector Ecology.

Russell, R.C. (1996). A color photo atlas of mosquitoes of Southeastern Australia. Medical Entomology, West mead Hospital.

Schneider DS, James AA, (2006). Bridging the gaps in vector biology. Workshop on the molecular and population biology of mosquitoes and other disease vectors. EMBO Rep 7:259–262. Schnepf E, Crickmore N ,Van Rie J, Lereclus D, Baum G,et al, (1998). Bacillus thuringiensis and its pesticidal crystal proteins, Microbial MolBiol Rev 62:775-806.

Sirivanakarn S, White GB. (1978). Neotype designation of Culex quinquefasciatus Say (Diptera: Culicidae). Proceedings of the Entomological Society of Washington 80: 360-372.

Scott, T. W., P. H. Amerasinghe, A. C. Morrison, L. H. Lorenz, G. G. Clark, D. Strickman, P. Kittaypong, and J. D. Edman. (2000). Longitudinal studies of Aede saegypti (Diptera: Culicidae) in Thailand and Puerto Rico: blood feeding frequency. J. Med. Entomology. 37: 89- 101. Tahmaz, Soutar and Cherrie, (2003). chronic fatigue and organophosphate pesticides in sheep farming: a retrospective study amongst people reporting to a UK pharmacovigilance scheme. The Annals of Occupational Hygiene vol. 47 (4) pp. 261-7.)

WHO, (2009). Temephos in Drinking-water: Use for Vector Control in Drinking-water Sources and Containers Background document for development of WHO Guidelines for Drinking-water Quality, World Health Organization.

Thomas WE, Ellar DJ (1983). Mechanism of action of Bacillus thuringiensis var israelensis insecticidal delta-endotoxin. FEBS Let 154: 362-368. WHO, 1984; Rozendaal, (1997).Rozendaal, J. (1997) Vector control: methods for use by individuals and communities. World Health Organization, Geneva, Switzerland

WHO, (1986). Resistance of vector and reservoirs of disease to pesticide, tenth report of WHO expert committee on vector biology and control. WHO tech. report series 737.

WHO (2003). (Pyriproxyfen As A Mosquito Larvicide , J.F. Invest And J.R. Lucas, Sumitomo Chemical (UK) Plc. Horatio House, 77-85 Fulham

Palace Road, London W6 8JA/ Proceedings of the Sixth International Conference on Urban Pests William H Robinson and Dániel Bajomi (editors), 2008).

WHO, (2005). Guidelines for Laboratory and Field testing of mosquito larvicides WHO/CDS/WHOPES/GCDPP.13. World Health Organization. 2003. (Pyriproxyfen in drinking water. WHO/SDE/WSH/03.04/113).

William C. Kondratieff, William C. Black iv, Chester G. Moore, Anthony A. Games, Jeromo E. Freier, Janet Hemingway, Henry H. Hadedorn, Stephen Higgs, (2005). Biology of Disease vector, 2nd Eddition.

Temephos 10% winter test {LC50 = 0.08, LC 90 =0.18}

7.0

6.5

6.0

5.5

5.0 y=23.75x +3.449

Probit slope = 23.8 4.5

4.0

3.5

3.0 0 0.025 0.05 0.075 0.1 0.125 0.15 0.175 0.2 0.225 0.25 Dose stimulus

Appendix (1) Effect of Temephos 10% against Culex quinquefasciatus larvae (Winter test)

Temephos 10% summer test {LC50 = 0.05, LC 90 =0.12}

8.0 7.5 7.0 6.5

6.0

5.5 Probit 5.0 y=27.03x +3.742 slope = 27.0 4.5 4.0 3.5 3.0 0 0.025 0.05 0.075 0.1 0.125 0.15 0.175 0.2 0.225 0.25 Dose stimulus

Appendix (2) Effect of Temephos 10% against Culex quinquefasciatus larvae (Summer test)

Fenitrothion 50% EC {LC50=.47, LC90=0.71}

4.5

4.0

3.5

Probit 3.0 y = 13.33x + 2.143 2.5 slope = 13.3

2.0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Dose stimulus

Appendix (3) Effect of Fenitrothion 50% against Culex quinquefasciatus larvae (Winter test)

Fenitrothion 50% EC {LC50=0.60, LC90=1.10}

4.4

4.2

4.0

3.8

Probit 3.6

3.4 y=5.715x +3.391 slope = 5.72 3.2

3.0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Dose stimulus

Appendix (4) Effect of Fenitrothion 50% against Culex quinquefasciatus larvae (Summer test)

Appendix (5) Table (1) the Results of the efficacy test of each larvicides, Fenitrothion 50% and Temephos 10%

Fenitrothion 50% seasons Winter summer months January February March July August September

LC50 0.518 0.603 0.700 0.421 0.474 0.618

LC50 SE 0.015 0.018 0.036 0.023 0.029 0.031

LC50 LCL 0.472 0.546 0.628 0.391 0.437 0.566

LC50 UCL 0.564 0.661 0.772 0.452 0.511 0.681 Temephos 10% seasons Winter summer months January February March July August September

LC50 0.071 0.076 0.080 0.039 0.046 0.047 LC50 SE 0.006 0.004 0.006 0.004 0.007 0.005 LC50 LCL 0.058 0.063 0.065 0.030 0.036 0.036 LC50 UCL 0.084 0.089 0.094 0.048 0.055 0.057

Table (1) Shows the commulative results of the efficacy tests of larvicides. Laboratory test of collected Field larvae of Culex quinquefasciatus exposed in different climatic condition according to the seasons. The efficacy during the summer is more than the winter season,

(LC50, 0.518 mg/l in winter and LC50, 0.421 mg/l in summer) and these results was same condition and variation only in seasonal temperature.

Appendix (6), Table (2) the Results of Culex quinquefasciatus larvae mortality for each larvicide according to the exposure time

Season Month Sumithion 50% Temephos 10% 24 h 48 h 24 h 48 h

January 11% 24.3% 85% 86.6%

Winter February 8.3% 21.9% 86.6% 89.9%

2012 March 10% 46.6% 81.6% 88.6%

July 16.6% 31.6% 98.3% 99.9%

Summer August 13.3% 28.3% 96.6% 99.9%

2012 September 16.6% 59.9% 95% 98.3%

Table (2) Shows the commulative results of the larvae mortality for each larvicides. For the organophosphate Fenitrothion 50% against Culex quinquefasciatus larvae in Rayyan municipality showed that the maximum value is (16.6%) after 24 hours and (59.9%) after 48 hours of the remaining larvae. With regard to other larvicide temephos 10%, the table showed that the maximum values ranged from 81% to 98.3% after only 24 hours in both winter and summer season.

Appendix (7) Average temperature, Qatar climate, 2012

Month o Average o Maximum temp(C ) Minimum temp(C ) January 2012 Max Temperature 28 °C 22 °C 16 °C

Mean Temperature 24 °C 18 °C 13 °C

Min Temperature 20 °C 15 °C 9 °C

July 2012 Max Temperature 47 °C 43 °C 40 °C

Mean Temperature 41 °C 38 °C 36 °C

Min Temperature 36 °C 33 °C 30 °C

Month o Average o Maximum temp(C ) Minimum temp(C ) February 2012 Max Temperature 30 °C 23 °C 19 °C

Mean Temperature 26 °C 19 °C 16 °C

Min Temperature 21 °C 15 °C 12 °C

August 2012 Max Temperature 46 °C 42 °C 38 °C

Mean Temperature 40 °C 37 °C 33 °C

Min Temperature 35 °C 33 °C 28 °C

Month o Average o Maximum temp(C ) Minimum temp(C ) Marsh 2012 Max Temperature 37 °C 26 °C 19 °C

Mean Temperature 29 °C 22 °C 17 °C

Min Temperature 23 °C 18 °C 13 °C

September 2012 Max Temperature 43 °C 39 °C 35 °C

Mean Temperature 38 °C 35 °C 30 °C

Min Temperature 34 °C 30 °C 26 °C

Appendix (8 ) Probit analysis data, Fenitrothion 50%

Probit Analysis - Least squares [Normal Distribution] Fenitrothion summer test 1 Dose N (Stimulus) Actual Percent (%) Probit (Y) Weight (Z) 0.02 0.08 60 3.62 2.35 0.05 0.08 60 3.62 2.35 0.08 0.10 60 3.72 2.65 0.1 0.08 60 3.62 2.35 0.2 0.17 60 4.03 3.57 0.25 0.22 60 4.22 3.93 0.3 0.27 60 4.38 4.25

Probit Analysis - Least squares [Normal Distribution] Fenitrothion, summer test 2 Dose (Stimulus) Actual Percent (%) N Probit (Y) Weight (Z) 0.02 0.05 60 3.35 1.71 0.05 0.08 60 3.62 2.35 0.08 0.10 60 3.72 2.65 0.1 0.10 60 3.72 2.65 0.2 0.17 60 4.03 3.57 0.25 0.17 60 4.03 3.57 0.3 0.22 60 4.22 3.93

Probit Analysis - Least squares [Normal Distribution] Fenitrothion summer test 3 Dose (Stimulus) Actual Percent (%) N Probit (Y) Weight (Z) 0.02 0.1 60 3.72 2.65 0.05 0.1 60 3.72 2.65 0.08 0.13 60 3.89 3.17 0.1 0.13 60 3.89 3.17 0.2 0.18 60 4.10 3.69 0.25 0.18 60 4.10 3.69 0.3 0.27 60 4.38 4.25

Source: Murtada 2013.

Appendix (8-1) Probit analysis data, Fenitrothion 50%

Probit Analysis - Least squares [Normal Distribution] Fenitrothion winter test 1 Dose N Probit (Y) (Stimulus) Actual Percent (%) Weight (Z) 0.02 0.004 60 2.36 1.00 0.05 0.004 60 2.36 1.00 0.08 0.017 60 2.87 1.00 0.1 0.004 60 2.36 1.00 0.2 0.083 60 3.62 2.35 0.25 0.133 60 3.89 3.17 0.3 0.183 60 4.10 3.69

Probit Analysis - Least squares [Normal Distribution] Fenitrothion winter test 2 Dose Actual Percent (%) N Probit (Y) Weight (Z) (Stimulus) 0.02 0.004 60 2.36 1.00 0.05 0.004 60 2.36 1.00 0.08 0.017 60 2.87 1.00 0.1 0.017 60 2.87 1.00 0.2 0.100 60 3.72 2.65 0.25 0.100 60 3.72 2.65 0.3 0.133 60 3.89 3.17

Probit Analysis - Least squares [Normal Distribution] Fenitrothion winter test 3 Dose Actual (Stimulus) Percent (%) N Probit (Y) Weight (Z) 0.02 0.05 60 3.35 1.71 0.05 0.05 60 3.35 1.71 0.08 0.08 60 3.62 2.35 0.10 0.13 60 3.89 3.17 0.20 0.13 60 3.89 3.17 0.25 0.17 60 4.03 3.57 0.30 0.17 60 4.03 3.57

Source: Murtada 2013.

Appendix (9) Probit analysis data, Temephos 10%

Probit Analysis - Least squares [Normal Distribution] Temephos winter test 1 Dose Actual (Stimulus) Percent (%) N Probit (Y) Weight (Z) 0.01 0.083 60 3.62 2.35 0.02 0.183 60 4.10 3.69 0.05 0.533 60 5.08 4.92 0.08 0.650 60 5.38 4.62 0.1 0.733 60 5.62 4.25 0.2 0.850 60 6.04 3.39

Probit Analysis - Least squares [Normal Distribution] Temephos winter test 2 Actual Dose Percent (Stimulus) (%) N Probit (Y) Weight (Z) 0.01 0.067 60 3.499 1.997 0.02 0.183 60 4.097 3.695 0.05 0.383 60 4.704 4.704 0.08 0.633 60 5.340 4.660 0.1 0.783 60 5.783 3.933 0.2 0.867 60 6.111 3.168

Probit Analysis - Least squares [Normal Distribution] Temephos winter test 3

Actual Dose (Stimulus) Percent (%) N Probit (Y) Weight (Z) 0.01 0.05 60 3.355 1.710 0.02 0.15 60 3.964 3.391 0.05 0.45 60 4.875 4.875 0.08 0.633 60 5.340 4.660 0.1 0.733 60 5.623 4.255 0.2 0.817 60 5.903 3.695

Source: Murtada 2013.

Appendix (9-1) Probit analysis data, Temephos10%

Probit Analysis - Least squares [Normal Distribution] Temephos summer test 1 Actual Probit Dose (Stimulus) Percent (%) N (Y) Weight (Z) 0.01 0.100 60 3.718 2.655 0.02 0.317 60 4.523 4.523 0.05 0.733 60 5.623 4.255 0.08 0.850 60 6.036 3.391 0.1 0.933 60 6.501 1.997 0.2 0.983 60 7.128 1.000

Probit Analysis - Least squares [Normal Distribution] Temephos summer test 2 Actual Probit Dose (Stimulus) Percent (%) N (Y) Weight (Z)

0.01 0.100 60 3.72 2.655 0.02 0.333 60 4.57 4.570 0.05 0.567 60 5.17 4.832 0.08 0.817 60 5.90 3.695 0.1 0.917 60 6.38 2.350 0.2 0.967 60 6.83 1.331

Probit Analysis - Least squares [Normal Distribution] Temephos summer test 3 Actual Probit Dose (Stimulus) Percent (%) N (Y) Weight (Z) 0.01 0.133 60 3.89 3.17 0.02 0.317 60 4.52 4.52 0.05 0.600 60 5.25 4.75 0.08 0.783 60 5.78 3.93 0.1 0.867 60 6.11 3.17 0.2 0.950 60 6.65 1.71

Source: Murtada 2013

Appendix (10) Notable Arthropod-Borne Diseases

Nature of Disease Nature of Vector Agent

Viral Yellow fever Mosquito Dengue fever Mosquito Dengue haemorrhagic fever Mosquito Phlebotomus fever Sand fly Chikunguna fever Mosquito West Nile fever Mosquito Rift valley fever Mosquito Kyasnur forest disease Tick Japanese B encephalitis Mosquito Equine encephalitis Mosquito Louping ill encephalitis Tick

Reckettsial Epidemic typhus Louse Murine typhus Rat flea

Scrup typhus Mite Tick typhus Tick Reckettsialpox Mite Rocky mountain spotted fever Tick Trench fever Louse/tick Q fever Tick

Appendix (10-1) Notable Arthropod-Borne Diseases

Nature of disease Nature of vector agent

Bacterial Plague Rat flea Tularaemia Deerfly Relapsing fever Louse Shigellosis Housefly Salmonellosis Housefly Cholera Housefly Typhoid/paratyphoid Housefly Protozoal Malaria Mosquito Leishmaniasis Sand fly African trypanosomiasis Tsetse fly Brazilian trypanosomiasis Reduvid bug Nematodal Filariasis Mosquito Drancunculiasis Water flea

(Cyclops) Onchocerciasis Black fly Loaisis Deer fly

Source: Foundations of Community Medicine - 2nd edition (GM Dheer. I Robbani, 2008).