THE EFFECT OF HONEY BEE VENOM ON MOSQUITO Anopheles
THE EFFECT OF HONEY BEE VENOM ON MOSQUITO Anopheles arabiensis Diptera - Culicideae By Haytham Awad Abdelgadir B.Sc (Honours) (1999). University of Khartoum A thesis submitted in partial fulfillment of the requirements for the degree of M. Sc. (Crop Protection) Supervisor: Prof. Mohamed Saeed Ali Elsarrag Department of Crop Protection Faculty of Agriculture University of Khartoum January - 2006 DEDICATION For malaria infected patients in my beloved country Sudan ACKNOWLEDGMENTS Praise be to Allah the Almighty, who gave me the strength, health and patience to accomplish this work. I would like to thank my supervisor Prof. Mohamed. S. A. ElSarrag. University of Khartoum. Faculty of Agriculture, for his supervision, guidance, continuous support, valuable comments and advice through out the period of the study and research. My sincere thanks and gratitude to my colleagues in Aaselat Investment Co. for their unlimited help and encouragement I would like to express my deep thanks and sincere gratitude to the technologists Salah Gomaa, Ihsan M. Babiker and their staff (Soaad, Rehab and Rasha), for their unlimited help and donation in laboratory work. Thanks also extended to staff members of Sudanese Atomic Energy Research Center, and Modern Medicine Center Laboratory in Khartoum, for their regarded help in laboratory work. Special thanks to Prof. Ahmed. K. Bolad. University of AlNelein, Faculty of Medicine, for his advice and stimulating discussion. Thanks are also due to Mr. Abdel-Hameed. A. M. Ahmed, for typing this thesis and unlimited assistance. Finally, special thanks to my family and friends, for their support and continuous encouragement. Abstract This experiment was designed in the University of Khartoum, Faculty of Agriculture to study the effect of honey bee venom introduced into the mosquito to diminish their ability to transmit the malaria parasite, which will lead to new hope towards the control of the disease. Mosquito Anopheles spp. were reared under lab conditions separated into two groups: Group (A) mosquitoes fed on sheep blood meal mixed with bee venom, dissected at different times after blood meal, sectional and mounted onto glass slides and examined under the microscope. Group (B) mosquitoes fed on sheep blood meal free of bee venom, sectioned, mounted and examined as the previous one. This study demonstrated that bee venom lined the mid gut lumen and that limited the diffusion and the invasion of the Ookinate and inhibit Oocyst formation, the initial stage of the malaria parasite development. ﺧﻼﺻﺔ ﺻﻤﻤﺖ اﻟﺘﺠﺮﺑﺔ ﻓﻲ ﺟﺎﻣﻌﺔ اﻟﺨﺮﻃﻮم – آﻠﻴﺔ اﻟﺰراﻋﺔ ﻟﺪراﺳﺔ أﺛﺮ ﺳﻢ اﻟﻨﺤﻞ ﻋﻠﻰ اﻟﺒﻌﻮﺿﺔ اﻟﻨﺎﻗﻠﺔ ﻟﻠﻤﻼرﻳﺎ ﻟﻠﺤﺪ ﻣﻦ ﻣﻘﺪرﺗﻬﺎ ﻓﻲ ﻧﻘﻞ ﻃﻔﻴﻞ اﻟﻤﺮض ،اﻟﺸﺊ اﻟﺬي ﻗﺪ ﻳﻘﻮد ﻷﻣﻞ ﺟﺪﻳﺪ ﻓﻲ ﻣﻜﺎﻓﺤﺔ اﻟﻤﻼرﻳﺎ. ﺗﻤﺖ ﺗﺮﺑﻴﺔ اﻟﺒﻌﻮض ﻣﻦ ﻧﻮع اﻷﻧﻮﻓﻠﻴﺲ ﺗﺤﺖ اﻟﻈﺮوف اﻟﻤﻌﻤﻠﻴﺔ وﻣﻦ ﺛﻢ ﺗﻢ ﺗﻘﺴﻴﻤﻬﺎ ﻟﻤﺠﻤﻮﻋﺘﻴﻦ: اﻟﻤﺠﻤﻮﻋﺔ )أ( ﻏﺬى اﻟﺒﻌﻮض ﻓﻲ هﺬﻩ اﻟﻤﺠﻤﻮﻋﺔ ﻋﻠﻰ دم أﻏﻨﺎم ﻣﺨﻠﻮط ﺑﺴﻢ اﻟﻨﺤﻞ ،وﻣﻦ ﺛﻢ ﺗﻢ ﺗﺸﺮﻳﺢ اﻟﺤﺸﺮات ﻓﻲ ﻓﺘﺮات ﻋﻠﻰ اﻟﺸﺮاﺋﺢ اﻟﺰﺟﺎﺟﻴﺔ وﻣﻦ ﺛﻢ ﻓﺤﺼﻬﺎ ﺗﺤﺖ اﻟﻤﺠﻬﺮ. اﻟﻤﺠﻤﻮﻋﺔ )ب( ﻏﺬى اﻟﺒﻌﻮض ﻓﻲ هﺬﻩ اﻟﻤﺠﻤﻮﻋﺔ ﻋﻠﻰ دم أﻏﻨﺎم ﺧﺎل ﻣﻦ ﺳﻢ اﻟﻨﺤﻞ وﻣﻦ ﺛﻢ أﺟﺮﻳﺖ ﻋﻠﻴﻬﺎ ﻧﻔﺲ اﻹﺧﺘﺒﺎرات ﻟﻠﻤﺠﻤﻮﻋﺔ اﻟﺴﺎﺑﻘﺔ. أوﺿﺤﺖ هﺬﻩ اﻟﺪراﺳﺔ أن ﺳﻢ اﻟﻨﺤﻞ ﻳﺆدي ﻟﺘﻐﻠﻴﻆ وﺗﻘﻠﻴﻞ ﻧﻔﺎذﻳﺔ ﺟﺪار ﻣﻌﺪة اﻟﺒﻌﻮض وﺑﺎﻟﺘﺎﻟﻲ ﻳﺤﺪ ﻣﻦ اﺧﺘﺮاق ﻃﻮر اﻷوآﻴﻨﻴﺖ ﻣﻤﺎ ﻳﺆدي ﻟﻤﻨﻊ ﺗﺸﻜﻴﻞ اﻟﻄﻮر اﻟﺘﺎﻟﻲ اﻷوﺳﺴﺖ ،أﺣﺪ اﻷﻃﻮار اﻷﺳﺎﺳﻴﺔ ﻓﻲ ﺗﻄﻮر ﻃﻔﻴﻞ اﻟﻤﻼرﻳﺎ. Table of contents contents Page Dedication………….…….………..……………………………………………………………… i Acknowledgements………….…..…………..………..……………………………………… ii English abstract………….…….………………….……..……………………………………… iii Arabic abstract………….…….………………………..……………………………………… iv Table of contents ………….………………….………..……………………………………… v List of tables………….……………………….….………..……………………………………… vi List of plates ……………………………...…….………..……………………………………… vii CHAPTER ONE: INTRODUCTION……………………………………………… 1 CHAPTER TWO: LITERATURE REVIEW………….…….………………… 4 2.1. Bee venom………….…….……………………..…..……………………………………… 4 2.2. Bee venom chemical composition………….…….…………………………… 6 2.3. Bee venom therapy…………..….…….………..……………………………………… 10 2.4. Bee venom as therapeutic agent against malaria……………………… 12 2.5. Human being malaria………….…….………..……………………………………… 15 2.6. Mosquito- Life cycle………….…….………..…..…………………………………… 22 CHAPTER THREE: MATERIALS AND METHODS…..…….…….… 25 CHAPTER FOUR: RESULTS &: DISCUSSION………….…….………… 36 REFERENCES………….…….………..………………………..……………………………… 39 LIST OF TABLES Table 2.1 Page Bee venom chemical composition…………………………….. 9 LIST OF PLATES Plate Page 1 The life cycle of the plasmodium in human and mosquito 17 2 Eggs and larvae rearing plates………………….……………………… 32 3 Pupae stage cups……………………………………….……………………… 33 4 Mosquitoes rearing cages………………………………………………… 33 5 Larvae transfer pipette…………………………………..…………………… 34 6 Disectioning microscope……………………………….…………………… 34 7 Mosquitoes different stages slides…………………………..………… 35 CHAPTER ONE INTRODUCTION With the resurgence of malaria in many countries, we are again encountering an increase in the numbers of people developing severe or complicated manifestations of the disease. In recent years our understanding of the pathological processes involved in producing these serious symptoms has increased gauntly and, as a result, new methods of treatments have been introduced. The first requirement of the revised strategy for the control of malaria is that deaths from this disease should be prevented through carry and adequate treatment. Despite the problems that have developed in recent years in controlling malaria, it is not only possible but essential that this initial objective be attained, because time may be short and life-sawing measures often have to be provided at the local level, a choice of method must be provided which is appropriate to differing levels of clinical skill and facilities and in a movement towards healthier life styles and in the reaction to the dangers of modern drugs and their side effects, people are now turning to more natural ways of treating their conditions. Thus the use of bee products, once again, gaining popularity and the Apitherapy as an alternative therapy found its way in curing various chronic diseases including malaria, by using bee venom. The venom of Apis mellifera (honey bee) has been used to treat various inflammatory diseases for over 2000 years and many identified components of bee venom contain strong antiinflammatory properties (Broad man; 1962, Naum Iyorish,1974 and Kim,1989). Bee venom is composed of 78 different components, the main anti-inflammatory, pharmacological components, are peptides, melittin, apamin, adolapin, Mast-cell degranulating peptide (MCDP) and protease inhibitors. Melittin stimulates the hypophyseal adrenal system and produces cortisone (Vick and Brooks., 1972). It has also been reported that bee venom is strong immunological agent and stimulates the body’s protective mechanism against diseases, but there are only a few reports on this substance for clinical use. The principal objective of the present work is to study the effect of bee venom administration on mosquito Anopheles arabiensis. CHAPTER TWO REVIEW OF LITERATURE 2.1. Bee venom: Among many species of insects, very few have the capability of defending themselves with a sting and venom injection during stinging. All insects that can sting are members of the order Hymenoptera, which include ants, wasps and bees. Since sting is believed to have evolved from the egg-laying apparatus of an ancestor of the order Hymenoptera, only females can sting. The sting is always at or near the abdominal end, rather than the head (Krell, 1996). Two glands associated with the sting apparatus of worker bees produce venom, their production increases during the first two weeks of the adult workers life and reaches maximum when worker bee becomes involved in hive defense and foraging. It diminishes as the bee gets older (Krell, 1996). Bee venom is a clear liquid with a sharp bitter taste, aromatic odor, acidic reaction, of specific gravity 1.1313 (Beck, 1935). It dries quickly at room temperature, to 30-40% of the original liquid weight. When coming into contact with mucous membrane or eyes, it causes considerable burning and irritation. Dried venom has a light yellow color, however, some commercial preparations are brown, and this may be due to oxidation of some of the venom proteins (Krell, 1996). Bee venom is a colourless clear liquid with sweet taste and a little bitter. It is soluble in water insoluble in alcohol and ammonium sulphate. If it comes in contact with air, it forms opaque or grayish-white crystals. When a bee stings, it does not normally inject all of the 0.15-0.3 mg of venom held in a full venom sac (Schumacher et al., 1989 and Crane, 1990). Only when it stings an animal with skin as tough as ours will it lose its sting- and with it the whole sting apparatus, including the venom sac, muscles and the nerve center. These nerves and muscles keep injecting venom for a while, or until the venom sac is empty. The median lethal dose (LD50) for an adult human is 2.5mg of venom per kg (2.8mg/kg) of body weight (Schumacher et al, 1989). Assuming each bee injects all its venom and no sting are quickly removed at maximum of 0.3mg venom per sting. 600 stings could well be lethal for such a person. However, most human deaths result from one or few bee sting due to allergic reaction (Schumacher et al, 1989). Use in small doses however, bee venom can be benefit in treating a large number of ailments. This therapeutic value was already known to many ancient civilizations. Today, the only use of bee is in human and veterinary medicine. 2.2. Bee venom chemical composition: Many investigations were carried out regarding composition of honey bee venom. The basic information dealing with venom constituent (their fraction and their pharmacological effects were done in the 1950`s and 1960`s (Krell, 1996). There are some comprehensive summaries in Pieck (1986), which cover the morphology of the venom apparatus, the collection of venom, the pharmacological effects of bee venom and allergies of the Hymenoptera venom of bees, wasps and ants. Water constitutes 88% of bee venom. The glucose, fructose and phospholipids contents of venom are similar to those in bee’s blood (Crane, 1990). At least 18 pharmacologically active components have been described, including various enzymes, peptides and amines. Bee venom is composed of 78 different components: (Table 2.1). The main anti- inflammatory pharmacological components are peptides, melittin, apamin, adolapin, mast-cell degranulating peptide (M.C.D.P) and protease inhibitors. Melittin stimulates hypophyseal adrenal system and produce cortisone, it is 100 times more potent than hydrocortisone (Vick and Brooks1972). Bee venom contained several biochemical or pharmacological active substances, including at least histamine, dopamine, melittin, apamin mast cells degranulating peptides (M.C.D.P), minimine and the enzymes phosopholipaseA2 and hyaluronidase (Hodgson, 1955; Beard, 1963 and Haberman, 1972). Venom contains a number of very volatile compounds which are easily disappeared during collection, Banks et al., (1976) and Shipolini (1984), Dotimas and Hider (1987), Crane (1990), Krell, (1996) gave a good review of its composition, effects, harvesting and use. Table (2-1) Bee venom chemical composition Class and molecules Enzymes Component % of dry venom Phospholipase A2 10 – 12 Hyaluronidase 1–3 phosphomonesterase Lysophospholipase α - glucosidase Other proteins peptides and Melittin apamine 50 1-3 Mast cell degranulating peptide 1-2 (MCDP) 0.5-2.0 Secapin 1-2 Procamine 1.0 Adolapin 0.8 Protease inhibitor 0.1 Tertiapin Small peptides (with 13-15 less that 5 amino acids) Physiologically amines Amino acids active Histamine 0.5 – 2.0 Dopamine 0.2 – 1.0 Poradrenaline 0.1 – 0.5 T. aminobutyric acid 0.5 α-amino acids 1 Sugars Glucose & fructose 2 Phospholipids 5 Volatile compounds 4–8 (Shipolini, 1984; Dotimas and Hider, 1987). 2.3. Bee venom therapy: Bee venom has long been used in traditional medicine for the treatment of various kinds of rheumatoid. The list of benefits to human beings as well as to animals is very long (Sharma and Sighn, 1983). Reported clinical tests were often conducted in countries with less rigorous method than the standard. Despite this consideration, many patients did report positive results and many of the successful treatments occurred after established medical or surgical procedures had failed. However, there is a very real resistance in Western countries either to accept these results or to test bee venom treatments according to Western medical standard. (Krell, 1996). Bee venom composed of several biochemically or pharmcologically active substances including at least histamine, dopamine, melittin, apamine, mast cells degranulating peptides (MCDP), minimine and enzymes phospholipase A2 and hyaluronidase (Hodgson, 1955; Beard, 1963 and Haberman, 1972). Bee venom is haemorrhagic, differing from snake (viper) venom which is a coagulant. As well as containing apamine, melittin, phospholipase A2. hyaluronidase, which have the opposing action of inhibiting, the nervous system, and stimulating the heart and the adrenal glands, the venom also contains mineral substances, volatile, organic acids i,e formic acid, hydrochloric acid, ortho-phosphoric acid. Also contains some antibiotics, as well as two amino acids, rich in sulpher methionine and cystine sulpher is the main element in inducing the release of cortisone from the adrenal glands, and in protecting the body against infections (Palos 1985). 2.4. Bee venom as therapeutic agent against malaria Anti bacterial, anti-parasitical and anti-viral properties have recently been attributed to members of secreted phospholipases A2 (PLA2) super family. Seven PLA2 from group; A1,A2,A3and B1 were tested here in different culture conditions for inhibition of the in vitro intraerythrocytic development of Plasmodium falciparum, the causative agent of the most sever form of human malaria. In the presence of human serum, all PLA2s were inhibitory, with three out of seven exhibiting inhibitory concentrations (IC). In all cases, inhibition could be induced by enzymatic pre-treatment of the serum. By contrast, no effect was observed when parasites were grown in semi-defined medium devoid of lipoproteins and containing 10 times less phospholipids than the medium with human serum, strongly suggesting that hydrolysis of serum generating toxic lipid by-products, rather than a direct interaction of the PLA2s with the infected erythrocyte, is general feature of the antiPlasmodium properties of PLA2s. Further more in serum, six out of seven PLA2s were toxic against both trophozoite and schizont stages of parasite development, contrasting with the trophozoite- selective bee venom enzyme toxicity. Deciphering the molecular mechanisms in the phenotypic singularity of bee venom enzyme toxicity might offer new prospects in anti malaria fight (Guillaume et al., 2004). Secreted phospholipaseA2 (PLA2s) from snake and insect venom and from mammalian pancreas are structurally related enzymes that have been associated with several toxic pathological, or physiological processes. The issue at whether toxic PLA2s might exert specific effects on the Plasmodium falciparum intraerythrocyte development. Both toxic and nontoxic PLA2s are lethal to Plasmodium falciparum grown in vitro, with large discrepancies between respective inhibitor concentration IC(50) values IC(50) values from toxic PLA2s ranged from 1.1 to 200 ppm, and IC(50) volumes from non toxic PAL(2)s ranged from 0.14 to 1 micron. Analysis of the molecular mechanism responsible for cytotoxicity of bee venom PLA2s (non-toxic) demonstrated that in both cases, enzymatic hydrolysis of serum phospholipids present in the culture medium was responsible for parasite growth arrest. However, bee PLA2–lipolyzed serum induced stage-specific inhibition of Plasmodium falciparum development, where as hog PLA2- lipolyzed serum killed parasites at either stage. Sensitivity to bee PLA2 treated serum appeared restricted to the 19–26h period of the 48h parasite cycle. Analysis the respective role of the different lipoprotein classes as substrates of bee (PLA2 should the enzyme treatment at high density lipoproteins, low density lipoproteins and very low density lipoprotein. In conclusion, the results demonstrate that toxic and non – toxic PLA2s: Are cytotoxic to Plasmodium falciparum via hydrolysis of lipoprotein phospholipids. Display different killing processes presumably involving lipoprotein by-product recognizing different targets on the infected red blood cells. (Deregnaucourt and Schrevel 2000). 2.5. Human being malaria: Human malaria is caused by one or more of the four species of plasmodium (P. vivax, P. malaria, P. ovale, and P. falciparum) account for more than 95 percent of cases of malaria in the world (WHO, 1990). All the above species of plasmodium have a life cycle both in man and in some species of anopheles mosquitoes Plate (1). When the female vector mosquito takes an infective blood meal it ingests both asexual and sexual forms of the parasite (Perlman, 2002). Asexual forms are digested in the mosquito stomach but the mature sexual forms gametocytes, survive. The male and female gametocytes undergo further development and form micro (male) and macro (female) gametes. A male gamete fertilizes a female gamete and the resultant structure (a zygote, which later develops into an Oökinete) penetrates the stomach wall. There it develops into Oocyst which forms the infective forms named sporozoites. Sporozoites are released into the haemocoel of the mosquitoes and can eventually be found in its salivary glands. When such an infected mosquito bites man, sporozoites injected together with saliva into the skin and circulate in the blood stream for up to one hour, during which time some of them invade liver cells (Hepatocytes) and develop into erythrocytic forms Plate (1). These normally rupture in 6 – 15 days and release thousands (5000 – 30000) merozoites, the number varies with the parasite species (about 10000 in P. vivax and 30000 in P.falciparum .Some of the merozoites are phagocytosed, other enter erythrocytes. Parasite life-cycle continues until death of the host or parasites or immunity of the host prevent further development of the parasite (WHO, 1990). Plate 1. The life cycle of the plasmodium in human and mosquito Mosquito: Mosquitoes belong to the phylum Arthropoda. Arthropods include (among many others). Spiders beetles, ticks, butterflies, house flies and mosquitoes. They can be recognized by the following characteristics: • The body is composed of several parts or segments. • The body is covered with a tough skin called exoskeleton. • The body normally has paired jointed legs and antennae. With in Arthropoda, there are several classes, including the class insecta – mosquitoes are members of this group. Insecta have the following characteristics: • The body is divided into three sections – head, thorax and abdomen. • The head has one pair of antenna, and a pair of compound eyes. • The thorax has three pairs of legs. Class insecta includes several orders, mosquitoes belong to the orders Diptera. Insects in this order have the following characteristics: • The thorax has one pair of visible wings. • The hind wings which are vestigial are small movable filaments known as halters which are mainly used for balance (WHO, 1997). Distinguishing characteristic of anophelines and culicines, eggs clump together in a raft (Culex) or float separately (Aedes), anopheline eggs, float separately and each of them has floats. The culicine larva has abreathing tube (Siphon) which it also used to hang down from the water surface whereas the anopheline larva has no siphon and rest parallel to and immediately below the surface. Pupae of both anopheline and culicines are comma–shaped and hang just below the water surface. They swim when disturbed. The breathing trumpet at the anopheline pupa is short and has a wide opening, whereas that of the culicine pupa is long and slender with a narrow opening. However, it is difficulties to distinguish anopheline from culicine pupae in the field (WHO, 1997). With live mosquitoes, you can distinguish between adult anopheline and culicine mosquitoes by observing their resting postures. Anopheline rest at an angle between 50° and 90° to surface when as culicines rest more or less parallet to the surface (WHO, 1997). Some anopheline species are similar in external morphology, while they are actually different species. These species are genetically related and are known as sibling species, and are morphologically grouped under the same complex. For example in the Anopheles gambiae complex (also known as Anopheles gambiae sensus lato or S. I.), there are seven different species: A. gambiae sensus stricto (S.S.), A. arabiensis, A. quadriannulatus, A. bwambae, A. merus, and A. melas. It is not possible to differentiate between these species by using an identification key that is based on external morphology (WHO, 1997). Mosquitoes differ from the other biting Diptera in having along slender body, long legs and long needle-shaped mouth parts( WHO 1997). The adult insects measure between 2mm and 12.5mm in length. Some species bite in the morning or evening or at night, other feed during the day. Species may bite indoors or out the doors (WHO 1997). Mosquitoes are important vectors of several tropical diseases, including malaria, filariases and numerous viral diseases. In countries with temperate climate they are more important as nuisance pests than as vectors (WHO 1997). There are about 3000 species of mosquitoes, of which about 100 are vector of human diseases (Botha 1947). Control measures are generally directed against only one or a few of the most important species and can be aimed at the adults or the larvae( WHO 1997). 2.6. Mosquito - Life cycle: Mosquitoes have four distinct stages in their life cycle: eggs, larva, pupa and adult. The female usually mate only once but produce eggs at intervals throughout their life. In order to be able to do so most female mosquitoes require a blood-meal. Males do not suck blood but feed on plant juices. The digestion of a blood-meal and the simultaneous development of eggs takes 2-3 days in the tropics but longer in temperate zones. Females search for suitable places to deposit their eggs, after which another blood- meal is taken and another batch of eggs is laid. This process is repeated until the mosquito dies (WHO 1997). Depending on the species, a female lays between 30 and 300 eggs at a time. Many species lay their eggs directly on the surface of water, either single (Anopheles) or stuck together in floating rafts( Culex) ( Botha 1947). In the tropics, the eggs usually hatch within 2-3 days. Some species (e.g. Ades) lay their eggs just above the water line or on wet mud; these eggs hatch only when flooded with water. If left dry they can remain viable for many weeks (Botha 1947). Once hatched, the larvae do not grow continuously but in four different stages (instars). The first instar measures about 1.5mm in length, the fourth about 8-10mm. Although they have no legs, they have a well developed head and body covered with hairs, and swim with sweeping movements of the body. They feed on yeasts, bacteria and small aquatic organisms (Botha 1947). Most mosquito larvae a siphon located at the tip of the abdomen through which air is taken in and come to water surface to breath. In warm climates the larval period lasts about 4-7 days, or longer if there is a shortage of food. The fully grown larva then changes into comma-shaped pupa which does not feed and spends most of its time at water surface. If disturbed it dives swiftly to the bottom. When mature, the pupal skin splits at one end and a fully developed adult mosquito emerges (Botha 1947). In the tropics the pupal period lasts in 1-3 days. The entire period from egg to adult takes about 7-13 days under good conditions (WHO 1997). Female mosquitoes feed on animals or humans. Most species show preference for certain animals or for humans. They are attracted by the body odours, carbon dioxide and heat emitted from the animal or person (WHO 1997). Feeding usually take place during the night but daytime biting also occurs. Species that prefer to feeding on animals are usually not very effective in transmitting diseases from person to person. Those that bite in the early evening may be more difficult to avoid than species that feed at night (WHO 1997). CHAPTER THREE MATERIAL AND METHOD Mosquitoes collection: Many of the anopheline species that are malaria vectors rest indoors. Hand collection provides information about usual resting places, resting density and for any purposes such as research rearing. Equipments: • Sucking tube. • Flash light. • Cups with covering net (Plate 3) • Mosquitoes cages (Plate 4). Procedure: • Mouth piece at the sucking tube held in mouth gently neared to the mosquito and sucked. • Finger was placed over the tube to prevent the mosquito from escaping and with this position neared the hole of the tube to the cup and removed quickly into the hole. • Mosquitoes blowed gently into the cup. Mosquitoes were kept in the field and during transport, some precautions were taken to keep them in good conditions: • Pieces of cotton soaked in 5 – 8% sugar solution, any excess sugar squeezed out, cotton were placed over the tops of the cups. • Cups holding mosquitoes placed uprights in an insulated picnic box. • Mosquitoes kept in places free from insecticides contamination and away from ants. Some mosquitoes were collected from outdoors such as vegetation or on solid surfaces such as the banks of streams and ditches, holes in rocks, on the trunks of larger trees and holes in rocks. The equipments required for outdoors collection is the same as that listed under hand collection of indoor – resting mosquitoes. In addition a hand net may be used. Larvae and pupae collection: It is important to know the preferred breeding sites of the anopheline mosquitoes in the area, and the densities of larvae and pupae at these sites. Collection of different types of breeding site in an area allowed to: • Determine the species present. • Determine the preferred breeding sites of each vector species. • Make an assessment of the effectiveness of vector control programme. Equipment: • Dipper. • Larval net. • Large trays (Plate 2). • Pipette (Plate 5). • Safety match or lighter (Plate 2). The mixture of bee venom and the blood was composed of 1µg/1ml respectively. Mosquito dissection and examination techniques: Determining the abd50ominal and midgut condition or blood digestion stages of mosquito is important component. Many times you need to know when a mosquito takes blood and how long it takes to digest the blood, develop eggs and lay eggs and return to take blood meal again. It is one of the important components needed to calculate a vectors capacity to transmit malaria (WHO, 1997). Dissection and examination of midgut is required in order to study longevity, viability and age of vector and it’s often to transmit the disease. Before dissecting it is essential to know the position of the different organs within its body. The position of the various structures are: • The salivary glands lie inside the thorax, but are joined to the head by salivary ducts. • The stomach or midgut lies in the abdomen, and the malpighian tubules are at the bottom end of the midgut • The ovaries lie on either side of the gut in the posterior part of the abdomen and join at the ampulla to form a common oviduct. • A single spermatheca where the male sperm is stored, is attached to the common oviduct. Equipments: Equipments needed to dissect midgut: • Dissecting or stereoscopic microscope (Plate 6). • Dissecting needles. • Fine forceps. • Slides. • Dropper and distilled water. Procedure • Female mosquito killed, legs and wings removed. • Placed on slide and drop of distilled water were added. • While holding one needle on the thorax tip of the abdomen sectioned longitudinally with another needle held on the right hand. • Cut through the midgut and separated from the rest of the specimen. • Midgut which sectioned transferred to another slide to dry. • Dried midgut examined under the microscope using 10 x objectives and confirmed using 40 x objectives. 40 mosquitoes were collected and kept into tow groups. Group A: Mosquitoes that blood fed for two days were separated and kept at 25C with 10% sugar solution for 24hour. After feeding mosquitoes were cold immobilized. Mosquito guts were dissected at different time, after blood meal 50% ethanol and opened longitudinally to make a sheet. The gut sheet was treated with methanol series fixed over night in4% formaldehyde and washed three times with xylenes and incubated in the dark for 2hours. The gut sheets were mounted into glass slides at room temperature followed by rehydration in graded ethanol. Nuclei were visualized by staining with phenylindole and stained gut wall was observed by microscope at x40 magnification Plate 2: Eggs and larvae rearing plates Plate 3: Pupae stage cups Plate 4: Mosquitoes rearing cages Plate 5: Larvae transfer pipette Plate 6: Disectioning microscope Plate 7: Mosquitoes different stages slides CHAPTER FOUR RESULTS & DISCUSSION Blood digestion stage refers to the appearance of the midgut of the female Anopheles spp. As the result of the blood digestion and changing in the appearance of the midgut occur at the same time based on their type of the blood meal taken (with bee venom and free of bee venom), mosquitoes can be grouped as fed and unfed: • Freshly fed: the abdomen appeared bright or dark red from the blood in the midgut. • Fed: (with b.v.) the blood was dark in colour – almost black and occupied three to four segments on the ventral surface and six to seven on the dorsal surface of the midgut. • Unfed (with b.v.): the blood was reduced to a small black patch on the ventral surface or may be completely digested. Plasmodium, the causative agent of malaria have to complete a complex development in the mosquito for transmission to occur. The first interaction between the parasite and the mosquito occur in the mid gut lumen, where the parasite has to traverse tow barriers, the peritrophic matrix and the mid gut epithelium. Because the gut is closed compartment that limit, anti malarial compounds that secreted into the mid gut lumen are expected to efficiently target the initial stage of parasite development (Ghosh and et al, 2002). Catteruccia and et al, (2000) expressed that as in different types of mosquitoes the PLA2 has been secreted in mid gut of mosquito is most likely responsible for inhibition of ookinete mid gut invasion ,as observed into experiment . The binding of phospholipase to their substrate, such as aggregated phospholipids and surface membrane is independent on their enzymatic activity (Lambeau and Lazdunski, 1999). Indeed, it has been shown that bee venom inhibit plasmodium development even when its enzymatic activity was inhibited suggesting that bee venom acts primarily via its binding to exposed membrane lipids, moreover, bee venom had no effect on exflaglation and zygote formation and did not affect normal ookinet motility on glass slides, suggesting that this substrate does not kill the parasite (Zieler et al, 2001) These consideration in the B.V support the hypothesis that B.V may be acting by interacting with the interaction between the plasmodium and the mid gut surface. In summary, these experiments demonstrated, that mid gut binding peptide, effectively inhibits development and transmission in the parasite (Ghosha and et al., 2001). However, the result indicate that (B.V) may be uses as additional effector to block the development of the malaria parasite in the mosquito. REFERENCES Banks, B. E. C., Hanson, N., and Jenifer, N. M. (1976). 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