Larvicidal Activity of the Crude Extracts of Some Local Plants on Housefly Musca Domestica Vicina Macq

Larvicidal Activity of the Crude Extracts of some Local Plants on Housefly Musca domestica vicina Macq. (Dipetra: Muscidae)

Misaab Mohammed Hanai Tamim

B.Sc. (Hon.) in Biology/Chemistry, Faculty of Education, University of Dalanj, (2006)

Postgraduate Diploma in Zoology, Faculty of Science, University of Kurdofan, (2008)

M.Sc. in Biosciences and Biotechnology, Faculty of Engineering and Technology, University of Gezira, (2010)

A Thesis

Submitted to the University of Gezira in Fulfilment of the Requirements for the Award of Doctor of Philosophy Degree

Biosciences and Biotechnology (Applied Entomology)

Centre of Biosciences and Biotechnology

Faculty of Engineering and Technology

October 2015

Larvicidal Activity of the Crude Extracts of some Local Plants on Housefly Musca domestica vicina Macq. (Dipetra: Muscidae)

Misaab Mohammed Hanai Tamim

Supervision Committee:

Position Name

Dr.: Mutaman Ali Abdalgadir Kehail Main Supervisor

Prof: El-Nour El-Amin A. Rahman Co-Supervisor

Date: October, 2015

Larvicidal Activity of the Crude Extracts of some Local Plants on Housefly Musca domestica vicina Macq. (Dipetra: Muscidae)

Tamim Misaab Mohammed Hanai

Examination Committee:

Name Position

Dr. Mutaman Ali Abdalgadir Kehail Chairperson

Elamin Mohamed Elamin Elfaki External Examiner Prof.

Dr. Faiza Elgaili Elhassan Salah Internal Examiner

Date of Examination: 8 /October/2015

Dedication

To my Mather,

My wife (Maryam)

My children

My family

My teachers and my colleagues

I Dedicate this work

ACKNOWLEDGEMENTS

First, I render Plentiful thanks and praise to Allah, the Most Merciful, for having given me patience, strength and health to accomplish this work. I send specific dept of gratitude to my main Supervisor Dr. Mutaman Ali Abdelgadir Kehail, for his keen supervision, sound advises; also plentiful thanks are due to my Co- Supervisor Prof: Elnour El-Amin for his remarkable support and continuous help. Thanks are due to my wife, Maryam abu hawaa. I am also indented to my friends for their continuous encouragement, creation of most suitable conditions, invariable assistance and moral support which encouraged me for accomplishing this study.

I am also indeted to my all colleagues in Centre of Biosciences and Biotechnology, Faculty of Engineering and Technology, University of Gezira.

Larvicidal Activity of the Crude Extracts of some Local Plants on Housefly Musca domestica vicina Macq. (Dipetra: Muscidae) Misaab Mohammed Hanai Tamim PhD. in Biosciences and Biotechnology (Applied Entomology) October, 2015 Center of Biosciences and Biotechnology Faculty of Engineering and Technology University of Gezira Abstract

The housefly (Musca d. vicina) is a subspecies of dipteral flies that become vector to many serious diseases. The control of housefly depended mainly on environmental control and chemical insecticides against adult and larvae. In the present study, the larvicidal activity of the crude extracts (aqueous, methanol and hexane) of some local plants (neem; Azadirachta indica, Aweer; Ipomoea helderbranditti and Sunut; Acacia nilotica) on housefly larval stage was investigated in the faculty of Engineering and Technology during three seasons (2013, 2014 and 2015). The plants used in this study were brought from Alnishishiba area, Wad Madani City, Gezira State, Sudan. The leaves of these plants were cut-off at early morning, cleaned and dried under shade in the room temperature then crushed and extracted with water, methanol and hexane for 24 hours. Housefly larvae were collected from Al Andalus area and were reared in special cages (35x35x35 cm). The standard larval feed (control) was prepared by mixing wheat bran, milk powder, sugar and yeast. The treated feed was made by adding each of the prepared crude extract to the previously prepared control feed and was used for 6 days. Each test was triplicated. The results of this study showed that, in the first season, the aqueous extract of Ipomoea leaves at (1%) resulted in 95% mortality followed by methanol extract of Sunut leaves (93.67), while Hexane extract of Ipomoea showed the lowest effect (49%). The concentration 5% of aqueous extract of neem leaves and of methanol extract of Ipomoea in season one and two resulted in 100% mortality of housefly larvae, while that of hexane extract of Ipomoea resulted in 32.5% mortality (the lowest). Varies toxic and deterrent effects of these crude extracts beside an evidence of development deteriorations were recorded. ANOVA proved no significant difference in the toxicity of the used crude extracts. This study recommends continuing investigation in these plants to be used in housefly control programs.

LIST OF CONTENTS

Subject Page Dedication iii Acknowledgement iv Abstract v Arabic Abstract vi List of Contents vii List of Tables xi List of Figures xii List of appendices xiii Chapter One: Introduction 1 Chapter Two: Literature Review 3 2.1 The House Flies 3 2.1.1 Classification 4 2.1.2. Molecular Taxonomy 4

2.1.3 Morphology and General Descriptions 5 2.1.4 Life Cycle 6 2.1.4.1 The Eggs 7 2.1.4.2 The Larvae 7 2.1.4.3 The Pupa 7 2.1.4.4 The Adult 8 2.1.5 Some Factors Affecting the Life of Musca domestica 8 2.1.5.1 Temperature 8 2.1.5.2 Humidity 8 2.1.5.3 Light 8 2.1.6 Control 8 2.1.6.1 Sanitation 9 2.1.6.2. Exclusion 9 2.1.6.3. Physical Control (Using Traps) 9

2.1.6.4: Biological Control 10 2.1.6.5. Chemical Control 10 2.1.6.6. Integrated Fly Control (IFC) 10 2.1.6.7 Natural Product Extracts 11 2.2 The Neem Tree (Azadirachta indica A. Juss) 14 2.2.1 Classification and General Description 14 2.2.2 Propagation and Distribution 14 2.2.3 Uses for Pests Control 14 2.2.4. The Active Ingredients 16 2.3 Ipomoea helderbranditti (Elaweer) 17 2.3.1Medicinal Uses 17 2.3.2: The Uses as Control Agent 17 2.4 Acacia nilotica (L.) 19 2.4.1 Description 20 2.4.2 Distribution 20 2.4.3 Ecology 22 2.4.4 Economic Importance 22 2.4.5 Allelopathic Effect of A. nilotica 23 2.4.6 Phytochemistry 24 2.4.7 Medicinal Uses and Pharmacological Effects 24 Chapter Three: Materials and Methods 3.1. Collection and Breading of House Flies and Plant Samples: 26 3.2 Collection of Plant Samples and Preparation of their Extracts 26 3.3. Phytochemical screening tests 27 3.3.1 Test for the presence of alkaloids 27 3.3.2 Test for the presence of flavonoids and flavonones 27 3.3.3 Test for the presence of glycosides 27 3.3.4 Test for the presence of Saponins 27 3.3.5 Test for the presence of steroids 27 3.3.6 Test for the presence of tannins 27

3.3.7 Test for the presence of terpenoids 27 3.4. Bioassay 28 3.5 Data Analysis 28 Chapter Four: Results and Discussion 4.1. The Photochemical analysis of A. indica, A. nilotica and I. helderbranditti Leaves 29

4.2. Larvicidal Activity of Neem Leaves Extracts on M. d. vicina (Season 1) 31

4.2.1. The aqueous extract 31

4.2.2 Methanol extract 31

4.2.3 Hexane extract 31

4.3 Larvicidal Activity of I. helderbranditti Leaves on M. d. vicina (Season 1) 33 4.3.1 Aqueous extract 33

4.3.2 Methanol extract 33

4.3.3 Hexane extract 33

4.4 Larvicidal Activity of A. nilotica Leave Extracts on M. d. vicina (Season 1) 35 4.4.1. Aqueous extract 35

4.4.2 Methanol extract 35

4.4.3 Hexane extract 35

4.5. Larvicidal Activity of Neem Leaves Extracts on M. d. vicina (Season 2) 37 4.5.1. Aqueous extract 37 4.5.2 Methanol extract 37 4.5.3 Hexane extract 37 4.6 Larvicidal Activity of I. helderbranditti Leaves on M.d. vicina (Season 2) 39 4.6.1. Aqueous extract 39

4.6.2 Methanol extract 39

4.6.3: Hexane extract 39

4.7 Larvicidal Activity of A. nilotica Leaves Extracts on M. d. vicina (Season 2) 41 4.7.1 Aqueous extract 41

4.7.2 Methanol extract 41 4.7.3 Hexane extract 41 4.8. Larvicidal Activity of Neem Leaves Extracts on M.d. vicina (Season 3) 43 4.8.1 Aqueous extract 43 4.8.2 Methanol extract 43 4.8.3 Hexane extract 43 4.9 Larvicidal Activity of I. helderbranditti Leaves on M.d. vicina (Season 3) 45 4.9.1. Aqueous extract 45 4.9.2 Methanol extract 45 4.9.3 Hexane extract 45 4.10 Larvicidal Activity of A. nilotica Leaves Extracts on M.d. vicina (Season 3) 47 4.10.1. Aqueous extract 47

4.10.2 Methanol extract 47

4.10.3 Hexane extract 47

CHAPTER FIVE: CONCULSIONS AND RECOMMENDATIONS 5.1 Conclusions 64 5.2 Recommendations 64 References 65

LIST OF TABLES

Table Title Page No. 4.1 Qualitative Phytochemical Analysis of A. indica, A. nilotica and I. 30 helderbranditti Leaves. 2.4 Effect of neem leaves extracts on mortality % of M. d.vicina larvae (S1: 24 2013) 4.3 Effect of I. helderbranditti leaves extracts on the mortality of M. vicina 34 larvae (S1: 2013) 4.4 Effect of A. nilotica leaves extracts on the mortality % of M.d. vicina 36 larvae (S1: 2013). 4.5 Effect of neem leaves extracts on the mortality % of M. d. vicina larvae 38 (S2: 2014). 4.6 Effect of I. helderbranditti leaves extracts on the mortality % of M.d. 40 vicina larvae (S2: 2014). 4.7 Effect of Acacia nilotica leaves extracts on the mortality % of M.d. vicina 42 larvae (S2: 2014). 4.8 Effect of neem leaves extracts on the mortality % of M. d. vicina larvae 44 (S3: 2015). 4.9 Effect of I. helderbranditti leaves extracts on the mortality % of M. d. 46 vicina larvae (S3: 2015). 4.10 Effect of A. nilotica leaves extracts on the mortality % of M.d. vicina 48 larvae (S3: 2015) 4.11 The Overall Effect of the Concentration of (1%) of each Extract on the 50 Mortality % of M. d. vicina larvae in Season 1, 2 and 3 at the 6th day

LIST OF FIGURES

Figure Title Page No. 2.1 The Neem Tree leaves 15 2.2 The Ipomoea helderbranditti (El Aweer) Plant 18 2.3 The Acacia nilotica leaves 21 4.1 Mortalities (%) of Housefly Larvae Fed on neem (AZ), Ipomoea (IP) 51 and Acaccia (Ac) Leaves Aqueous (Aq), Methanol (Me) and Hexane (He) Extracts for 6 days at concentration of (1%).

LIST OF PLATES

Plate No. Title Page 1 The Normal Shape of house fly 58 2 Abnormal Formation larvae, pupa and adult of M. d.vicina treated 59 with Neem extracts. 3 Abnormal Formation Larvae, Pupa and Adult of M.d. vicina treated 60 with A. nilotica extracts. 4 Abnormal Formation Larvae, Pupa and Adult of M.d. vicina treated 61 by Ipomoea extracts.

CHAPTER ONE INTRODUCTION

The houseflies have been implicated as vectors or transporters of various human pathogens, including Vibrio cholerae, Enterobacteriaceae pathogens, Staphylococcus aureus, and Pseudomonas spp. Transmission takes place when the fly makes contact with people or their food. As many as 500000 microorganisms may swarm over its body and legs. There are four different subspecies of house flies: Musca domestica domestica Linnaeus, M. d. vicina Macqvart, M. d. nebulo Fabricius, and M. d. curviforceps Sacca and Rivosecchi. The first three subspecies are found in temperate zones all over the world including subarctic and subtropical areas where as the fourth subspecies is limited to Africa (Keiding 1986). Most of houseflies in Sudan are Musca vicina Macq., while Musca domestica are restricted to certain humid and shade places (Kehail and Abdalla, 2012). Saccà (1967) recognized three forms (calleva, vicina, and nebulo sensu stricto) as constituents of a geographic clime of M. domestica. based on the ratio of fronts to head width of males and on abdominal coloration. Generally, four different subspecies were distinguished: M. d. domestica Linnaeus is found in temperate zones all over the world. M. d. nebulo Fabricius, is found only in tropical Asia, M. d. curviforceps Sacca and Rivosecchi, is restricted to Africa M. d. vicina Macqvart, is found in subtropical and tropical zones (Sacca, 1964). The common subspecies found in Sudan is M. d. vicina Macqvart (Omer, 2011). Flies can spread diseases because they feed freely on human food and dirty matter alike. The fly picks up disease-causing organisms while crawling and feeding. The diseases that flies can transmit include enteric infections, eye infections, poliomyelitis and certain skin infections. Thus, houseflies are widely recognized as potential reservoirs and vectors of food borne pathogens (Batish et al., 2008). Houseflies have been suspected to be reservoirs and vectors for pathogens. The harmful effects of the housefly on the human beings and some animals are indicated by the carrying of the diseases-causing agents and the difficulties of controlling the adults. Insecticide treatments include residual surface sprays, space or area sprays, wet sprays, baits, feed additives and pest strips (Tripathi and O’Brien, 1973).

The baits should be distributed along walls, window sills, or other areas where flies congregate inside or outside buildings. Flies congregate because of the high reproductive rate short generation time and genetic diversity. The house flies have the innate ability to develop resistance to pesticides rather quickly; it is therefore advisable to rotate insecticides periodically at least seasonally, ideally the rotation would replace an insecticide of one chemical class with one in a completely different class. A chlorinated hydrocarbon (methoxychlor) would be replaced with a phosphate (rabon) which in turn would be replaced with a synthetic pyrethroid (permethrin) (Campbell, 1997). Potentially active plant products are safer than synthetic insecticides for controlling house fly larvae and adult. Acacia nilotica, Ipomoea helderbranditti and Azadirachta indica are the botanical candidates that oriented to be used to control M. d. vicina. Objectives of the Study: General: This study aims at introducing and scanning a new method for controlling Musca vicina in their larval stages using some plants such as Acacia nilotica, Ipomea helderbranditti and Azadirachta indica by testing the efficacy of these Sudanese plants leaves extracts against larvae and adults of housefly. Specific: To study the effect of Acacia nilotica, Ipomea helderbranditti and Azadirachta indica leaves aqueous, methanol and hexane extracts on housefly M. vicina for three seasons as: - Larvicides on 2nd, 3rd and 4th insters. - Antifeedants - Inducer of Morphological abnormality on the dead larvae and on the further emerged pupae and adults from the survived larvae In addition to run a phytochemical screening for the main chemical classes for the leaves of these plants.

CHAPTER TWO LITERATURE REVIEW 2.1 The Houseflies: Musca d. vicina Macq. (Diptera: Muscidae) is one of the most common insects associated with man. Flies are mechanical vectors of several human and animal diseases (Malik et al., 2007). Not only they are a nuisance, the insects also have high efficiency of mechanical transmission of microorganisms which cause serious diseases for man particularly that affect elementary tract such as tuberculosis, dysentery, diarrhea, typhoid and some other diseases thus large population of the flies is a potential threat to the health of animal and man (Axtell, 1980). Musca d. vicina and M. d. domestica were vectors of hook worm and Ascaris lumbricoides (Dipeolu, 1982; Bhuiyan and Shafiq, 1959). 2.1.1 Classification: Kingdom Animalia Phylum Arthropoda Class Insecta Order Diptera Family Muscidae Genus Musca Species Musca domestica, 1758 – housefly, Subspecies Musca domestica domestica Linnaeus, 1758 Subspecies Musca domestica vicina Macquart (Hogsette, 1996). 2.1.2. Molecular Taxonomy

Genetic code: The Standard Code (transl_table=1).

AAs= FLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG

Base1 = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG

Base2 = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG

Base3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG

Initiation Codon: AUG

Mitochondrial genetic code: The Invertebrate Mitochondrial Code (transl_table = 5)

AAs = FFLLSSSSYY**CCWWLLLLPPPPHHQQRRRRIIMMTTTTNNKKSSSSVVVVAAAADDEEGGGG Starts = ---M------MMMM------M------Base1 = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG Base2 = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG Base3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG Differences from the Standard Code: Code 5 Standard AGA Ser S Arg R AGG Ser S Arg R AUA Met M Ile I UGA Trp W Ter , (NCBI, 2015) 2.1.3 Morphology and General Descriptions: The order Diptera includes many common pest house flies, mosquitoes, deer flies, and gnats. M. d.vicina is commonly found around houses and human activity area. M. d. vicina has three body parts: head, thorax and abdomen. The adult has one pair of fully developed wings the hind wings are reduced to halters (small knob-like structures) used to maintain equilibrium. Adult mouthparts are lapping, or piercing. All adults look like flies, but some may have a metallic color (blue bottle fly). Antennae may be difficult to see (Jerry, 2003). Adult males and females are hard to distinguish; females are usually larger and can extend the tip of the abdomen to form an ovipositor which is used for egg laying. Often males have enlarged eyes, which meet on top of the head. The house flies are dark grey in color; adult is approximately 1/8-1/4 inch (inch=2.52 cm) i.e. 4.6 mm long with four dark longitudinal black stripes behind the head on grayish-black thorax, and grey to yellow black abdomen with dark workings on the sides, reddish compound eyes, feathery antennae, and transparent wings. Mouthparts are expanded (Dennis, 2003). The eggs are pearly cylindrical and about 1.2 mm long, they resemble minute pine kernels or rice grains; each egg is elongate with bluntly rounded ends. The larva or (maggot) is white, legless and fully grown, about 13mm long. The body consists of thirteen segments arranged to form a shallow cone. The maggot has a pointed head and no appendages, two slit- tike spiracles, which in early instars resemble a pair of eyes in amateur larva they occur in the

18 tail end –second and third instars larvae also have spiracles near the head each with six or seven tiny dots. The pupa develops within dark brown puparium has round end and increases slightly in diameter from front to rear (Rosen, 2003). Detailed morphological descriptions form M. d. domestica, M. d. vicina and M. d. nebula are given by Patton (1932, 1937). The main differentiating characters are the width of male frons (half width of an eye in domestica, one-third in vicina, one seventh to one-eighth in nebula), and in abdomen pigmentation which is mainly bright orange in nebula, much darker in domestica and intermediate in vicina. These morphological variations are modified by ecological conditions and crosses showed that· there was gene flow between these three forms (Sacca, 1967; Milani, 1975). However, there is a clear-cut separation between catleva and curviforceps from the three other forms, proved by morphological, geographical and chromosomal data as well as by hybrid studies (Milani, 1975). After the establishment of detailed polytene chromosome map of M. d. domestica (El Agoze et al., 1992) (until recently cytogenetic studies of muscoid flies encountered technical and interpretational difficulties unknown in lower diptera) we were interested to discover if the three forms of domestica complex could be separated on the basis of banding patterns in the salivary gland chromosomes, if they had characteristic patterns of inversion and other rearrangements. In the present study a comparison of the overall aspect of the polytene chromosomes was made between a chromosomic map taken from an Egyptian population of M. d. vicina and that of the standard European WHO strain M. d. domestica (El Agoze et al., 1992). El Agoze et al., (2014) showed that the two forms of domestica complex, M. d. domestica and M. d. vicina are not clearly distinguishable by cytogenetic analysis of their banding pattern. These results suggest that the two forms are closely related at the phylogenetic level. However it is possible that differences in the synthesis of DNA may exist between these two forms as it has already been demonstrated in other species of Anophilinae that in spite of having practically identical chromosomal complement there was a difference in the timing of DNA duplication in some structurally identical chromosome segments in an inter specific hybrid. 2.1.4 Life Cycle: The house fly has complete metamorphosis; egg, larval instars, pupa and adult. The life cycle from egg to adult can be completed in 7 to 10 days, during warm summer weather (Lynsk, 1993). It breeds in a variety of organic materials such as manure, garbage, sewage; food wastes

19 lawn clippings, septic tanks and silage, over winters in either the larval or pupal stage under manure piles or other protected location. In worm summer, conditions are generally suitable for the development of the house fly and as many as 10-12 generations may occur in one summer (Sanchez, 1998). The females, usually found in group's 20-50 insects, can be seen depositing their eggs on suitable material, such as decaying organic matter, garbage, human and animal excrement. But manure is the preferred generation medium (Caron, 1999). Life tables for the imago of M. d. vicina and Calliphora erythrocephala were compiled from flies kept under controlled conditions. Musca flies were fed on diluted milk and Calliphora on 10% sugar solution and chicken liver. In Musca females are slightly longer lived than males and the contrary is true for Calliphora. The survivorship curves, and more strikingly the death eurves, exhibit a relatively high mortality during the early days of imaginal life. For the purpose of graduation, the life tables were broken up into two portions and the first one, containing the period of “premature imaginal mortality,” fitted by a fifth order parabola, and the second one by the Gompertz-Makeham formula. For Musca, a complete life table, covering all stages of development, has been compiled and compared to the human life table including miscarriages (Feildman-Muhsam and Muhsam, 1946). The nervous system of this housefly had been also studied (Sakural, 1977). 2.1.4.1 The Eggs: Eggs are white, about 1.2 mm long in length, laid singly, but pile up in small masses (Morgan et al., 1981). Each female can lay up to 500 eggs, in several batches, of about 75 to 150 eggs, over a three to four days period, and during her life time, it may lay five or sex batches at intervals of several days between each batch. One female has been known to deposit or lay 2387 in 21 batches (Durn, 1922). Usually, the house fly commences to lay her eggs from 4 to 12 days after emerging from the pupal case, and favor moist material for egg deposition. Egg that become too dry during the incubation period, will fail to hatch (Ananth et al., 1992). 2.1.4.2 The Larva: The mature larvae are 3 to 9 mm long, worm-like creatures called maggots that lack definite heads, eyes, antennae and legs, typical creamy whitish in color, cylindrical, but tapering towards the head. The head contains one of dark hooks. The legless maggots emerge from the eggs in the material where the eggs were laid. The full-grown maggots have a greasy, cream-

21 colored appearance, and are 8 to 12 mm long. The larvae go through three instars when the maggots are full-grown, they crawl up to 50 feet to a dry; cool place in generation area, and transform to the pupal stage. High manure moisture favors the survival of the house fly larvae (Sanchez, 1998). 2.1.4.3 The Pupa: The pupae are dark brown, and 8 mm long. The Pupal stage is passed in a pupal case formed from the last larval skin, which varies in color from yellow, red, brown to black as the pupa ages. The emerging fly escapes from the pupal case through the use of an alternately swelling and shrinking sac, called the ptilinum, on front of its head, which it uses like a pneumatic hammer (Rosen, 2003). 2.1.4.4 The Adult: The adult is 6 to 7 mm long and female is usually larger than male. The eyes are reddish and mouthparts are spongy. The thorax bears four narrow black stripes, and there is a sharp over bend in fourth longitudinal wing vein. The abdomen is gray or yellowish with dark midline, and irregular dark marking on the side. The underside of male is yellowish. The sex can be readily separated by noting the space between the eyes, which in female is almost twice as broad as in male (Rosen, 2003). 2.1.5 Some Factors Affecting Housefly Life: The weather may affect the distribution of insects i.e. the change in temperature, humidity and sun light etc. Generally insects are cold blooded, their body temperature changes directly with change in surrounding environment temperature (Holy et al., 1983). 2.1.5.1 Temperature: The response of house fly to the enzyme activity increased with increase in temperature to high rate, then decreased due to decrease in temperature. The high rate of house fly activity is found in February, March, August and September (the optimum temperature) (Holy et al., 1983). 2.1.5.2 Humidity: Relative humidity affects the water loss or gain in house fly this effect showed clearly in eggs and pupal stages which are dormant stages that have low ability to gain additional water. Thus the larva transmits to pupal stage and then pupation occurs by mining or digging on the humid soil to complete pupal stage and reduce water loss (Holy et al., 1983).

2.1.5.3 Light: Light play important role in house fly orientation, time –occurs on life cycle, the behavior response to light and light-dark effect to house fly behavior and activity. The adults emerge from the pupae in the morning, also attracted to feeding and reproduction. But, they lay eggs during the early morning or before the sunrise, and the pupation occurs at night (Holy et al., 1983). 2.1.6 Control The flies commonly develop in large number in poultry manure under cage hens and this is serious problem requiring control. The control of the house fly is vital to humans comfort in many areas of the world. The most important damage related with this insect is annoyance and the direct damage produced by potential transmission of more than 100 pathogens associated with this fly. The threshold density determining when to control flies depends on the area where the control measure will be taken. More common control measures involved with control of house fly are the sanitation use of traps and insecticides (Caron, 1999). Integrated pest management programs (IPM) seem to be a good alternative for the control of house flies. These programs combine different control methods that include the use of botanical insecticides. Among the botanical insecticides, plant essential oils (or their components) have been evaluated as they show a broad spectrum of biological activities including toxicity, repellant, oviposition and feeding deterrence (Isman and Machial, 2006; Rosell et al., 2008). The technique for stabilizing the microflora and density of larvae breeding in the medium has been described. A short summary of the technique is given here. The housefly larvae were reared in round glass containers, 20 cm. in height and 15 cm. in diameter. The breeding media consisted of the following thoroughly mixed ingredients: 300 g. of wheat bran, 40 g. straw and 300 ml. water. The jars were covered with a double layer of cloth toweling which was held in place by a metal rim. They were sterilized by autoclave and then allowed to cool to room temperature before seeding with eggs. Each jar was seeded with 1000-1250 eggs in 20 ml (Levinson and Silverman, 1954). The housefly M. d. vicina Macq., can also be reared in the laboratory according to the methods proposed by Chang and Wang (1958). Coconut poonac, the cake left over after the extraction of oil from coconut pulp, has been used very successfully for breeding M. d. vicina Macq. in the laboratory in Ceylon. The fly lays

22 its eggs in the poonac and the larvae live and pupate in the same medium (Tharumarajah and Thevasagayam, 2009). 2.1.6.1 Sanitation: Good sanitation is the basic step in all fly management. Food and materials on which the flies can lay their eggs must be removed, destroyed as breeding medium or isolated from the flies' egg laying adult (Broce and Urban, 1998). For control at waste disposal sites, refuse should be deposited to the same area as in organic wastes to deteriorate the capacity of breeding resources or the disposed refuse should be covered with the soil or other inorganic waste (15 cm thickness) on every weekend or other weekend. Removal of wet manure at least twice a week is necessary to break the breeding cycle. Wet straw should not be allowed to pile up in or near buildings. Since straw is one of the best fly breading material, it's not recommended as bedding. Spilled feed should not be allowed to accumulate but should be cleaned up two times a week. Ordinarily, fly control from 1 to 2 km around a municipality will prevent increase of house fly into the municipality. Killing adult flies may reduce the infestation, but elimination of breeding areas is necessary for good management. Garbage cans and dumpsters should have tight-fitting lids and be cleaned regularly. Dry garbage and trash should be placed in plastic garbage bags and sealed up. All garbage receptacles should be located as far from building entrances as possible (Caron, 1999). 2.1.6.2. Exclusion: Flies can be kept outside of homes by the use of window and door screens. Make sure screens are light fitting and without holes. Keep doors closed and make sure there are no openings at the top or bottom. Check for openings around water or gas pipes or electrical conductivity that feed into the building. Caulk or plug any openings. Ventilation holes should be screened as they can serve as entry ways for flies as well (Caron, 1999). 2.1.6.3. Physical Control (Using Traps): The fly traps may be useful in some fly control programs if enough traps are used when they are placed correctly and if they are used both indoors and outdoors. House fly is attracted to white surface and to bait that gives off odors. Indoors ultraviolet light traps collect the flies inside an inverted cone or kill them with an electro cutting grid. One trap should be placed for every 30 feet of wall inside buildings but not placed over or within five feet of food preparation area (Frick and Tallmy, 1996). Recommended placement area outdoors include near building

23 entrances in always beneath trees and around animal sleeping area and manure piles to building should be tightly screened with standard window screen thereby denying entrance to flies (Broce and Urban, 1998). 2.1.6.4 Biological Control: The housefly has many natural enemies and among the more important in poultry facilities the wasps (hymenoptera: pteromalidae). Leaving a layer of old manure in the pits when manure is removed might enhance or stabilize the suppression of the house fly densities by parasitoids and predators. Periodic release of parasitoids during winter and spring and following manure removal might effectively suppress densities in poultry facilities (Merchant et al., 1987). 2.1.6.5. Chemical Control: Insecticide treatments include residual surface sprays, space or area sprays, wet sprays, baits, feed additives and pest strips. Insecticide baits can be used to aid in house fly control. The baits should be distributed along walls, window sills, or other areas where flies congregate inside or outside buildings. Flies congregate because of the high reproductive rate short generation time and genetic diversity. The house flies have the innate ability to develop resistance to pesticides rather quickly; it is therefore advisable to rotate insecticides periodically at least seasonally, ideally the rotation would replace an insecticide of one chemical class with one in a completely different class. A chlorinated hydrocarbon (methoxychlor) would be replaced with a phosphate which in turn would be replaced with a synthetic pyrethroid (permethrin) (Campbell, 1997). When the house fly is a major pest, the control of this insect is by the application of adulticides or larvicides to directly or indirectly suppress adult densities. Residual wall sprays can be applied where the flies congregate (Campbell, 1997). 2.1.6.6. Integrated Fly Control (IFC): The (IFC) programs for caged-poultry houses are based on the following strategy: (1) Selective application of insecticides against the adult. (2) Start insecticides control measures early in the spring before appeared and repeated as frequently as needs through the warm month (Sanchez, 1998). 2.1.6.7 Natural Products Extracts Control measure against this insect in the short-term is the use of conventional insecticides (Cao et al., 2006; Malik et al., 2007). The indiscriminate use of chemical

24 insecticides has given rise to many well-known and serious problems, such as the risk of developing insect resistance and insecticidal residues for humans and the environment. Insecticide resistance in house fly is a global problem and several surveys have shown that such resistance is wide spread and increasing (Georghiou and Mellon, 1983; Scott et al., 2000). House fly has been successfully controlled by the application of various insecticides, but reports of insecticide resistance in this insect have been amply found (Kaufman et al., 2001; Shono and Scott, 2003). For this reason, alternative house fly control strategies, including the use of botanical insecticides have been studied (Wang-Jian and Lei, 2005; Ghoneim et al., 2007; Pavela, 2008). These problems coupled with the high cost of chemical pesticides have stimulated the search for biologically based alternatives. Accordingly, botanical insecticides based on natural compounds from plants, are expected to be a possible alternative. They tend to have broad-spectrum activity, relative specificity in their mode of action, and easy to process and use. They also tend to be safe for animals and the environment. Several studies have shown the possibility of using plant extracts in the control of housefly eggs, larvae, pupae and adults (Issakul et al., 2004; Malik et al., 2007). Plants products are considered alternatives to conventional insect-control agents as they constitute a rich source of bioactive chemicals, against number of species including specific target insects, and are often biodegradable to non-toxic products (Hashem and Youssef, 1991). The successful use of plant products in the control of certain insect species depends on contained substances that inhibit the developmental process of those insects. In larvicidal tests in vitro against the housefly (Diptera: Muscidae), in which early-stage larvae were submerged for 1 min in acetone and essential oil extracts at concentrations of 100–300 ppm (0.01–0.03%), the most effective essential oil, peppermint (Mentha piperita), was found to have an LC50 of 104 ppm. Peppermint essential oil also demonstrated a repellent effect when newly emerged adults were placed into cages containing two conical flask traps, of which one contained 1% essential oil in milk and the other contained only milk; 96.8% of the flies were found in the milk- only trap. In the same study, peppermint essential oil also deterred oviposition (Morey and Khandagle, 2012). Similarly, essential oils of both peppermint and eucalyptus were shown to be effective repellents at doses of approximately 70 µg/cm2, with repellencies of 86% and 76%, respectively. 2 These oils also demonstrated high larvicidal efficacy: LC50 values were 5 µg/cm for peppermint

25 essential oil and 7 µg/cm2 for eucalyptus essential oil. Mortality of 100% was achieved when pupae were exposed to 10% formulations of both oils. However, the same authors found that essential oil of peppermint was effective at repelling housefly on cattle in vivo only at concentrations of 100%; 10% formulations applied to cattle had no effect. On barn surfaces, a 10% peppermint oil formulation was effective at deterring housefly from landing (Kumar et al., 2011). However, the control used in the in vivo studies was water, whereas the excipient for the peppermint formulation contained 45% xylene, 3% butane, castor oil ethoxylate and nonylphenol. No excipient-only control was used and hence the contribution of the excipient to any repellent effect cannot be determined. This finding of relatively poor efficacy of peppermint oil as a deterrent in the field does not agree with earlier observations made in water buffalo. Numbers of three species of nuisance fly, Stomoxy scalcitrans (Linnaeus) (Diptera: Muscidae), M. domestica and Hippobosca equina (Linnaeus) (Diptera: Hippoboscidae), were found to decrease significantly on cattle treated for lice infestations with essential oils of camphor (Cinnamomum camphora), peppermint and chamomile (Matricaria chamomilla) until 6 days post-treatment; however, this experiment used only an untreated control and thus did not account for any possible repellent effects of a hydrophobic solution (Khater et al., 2009). The preliminary toxicity screening of 13 plant extracts against M. domestica L. adult at 300 and 1000 ppm, revealed excluding both Opuntia vulgaris and Saccharum spp. which showed very low toxicity even at the higher concentration. Based on the obtained LD50 values for the eleven ethanolic extracts applied topically to the house fly adult, the extract of Piper nigrum showed the highest toxicity (LD50 = 0.115 ug/insect), while Punica granatum induced the lowest toxicity (LD50= 0.278 ug/insect). Toxicity values of the other tested extracts ranged between the above mentioned values. For the tested insecticides, the LD50 values ranged between 0.00026ug/insect for methomyl and 0.0013 ug/insect for flufenoxuron. Combining of 11 botanical extracts with 4 insecticides has resulted in 44 binary mixtures; all of them showed potentiating effects with different degrees. Moreover, mixing the insecticides at LC0 (a concentration level causing no observed mortality) with the LC50 of each of the plant extracts have resulted in 44 paired combinations of high synergistic factor (S.F.). Based on the obtained RC50 values (repellent concentration for 50% of the tested housefly population), the bioassayed extracts could be arranged with respect to

26 their efficacy as follows: Salix safsaf (0.24 mg/cm2) Conyzaa egyptiaca (0.25 mg/cm2)> Azadirachta indica (0.28 mg/cm2); followed by 5 extracts of the same RC50 value; 0.29 mg/cm2 (Cichorium intybus, Citrusaur antifolia, Piper nigrum, Sonchuso leracues and Zea mays).

The results of toxicity against adult stage of housefly by sugar bait method revealed that the most potent plant extract was C. aegyptiaca which showed LC50 value of 4.8 ppm, and the lowest one was P. granatum (LC50 =10.4 ppm). Compared to the plant extracts, the tested insecticides showed very high toxicity; where they obtained LC50 squealed to 0.60, 0.64, 0.66 and 0.74 ppm, respectively for deltamethrin, chlorpyrifos, methomyl, and flufenoxuron. The residual toxicity of the tested plant extracts and insecticides against the adult stage of M. domestica indicated that C. aegyptiaca possessed the highest t50 and t20 values (10.6 and 24.8 days, respectively) (Sameh et al., 2012). In the other study, crude extracts of Aloe vera have been screened for their larvicidal activity against M. domestica. All the three instars larvae of house fly were treated with the different concentrations by dipping method for 24 and 48hrs. The LC50 values of Aloevera extract were found to be 32.67, 36 and 38.67 ppm in 24 hours; 24, 25.67 and 28.33 ppm in 48 hours on1st, 2nd and 3rd instars, respectively. The crude extract of Aloe vera was found to be more active in terms of larvicidal potential (Jesikha, 2012). 2.2 The Neem Tree (Azadirachta indica A. Juss) 2.2.1 Classification and General Description: The neem tree (Azadirachta indica) belongs to the family; Meliaceae (Mahogany family). The neem or margosa tree also is called Indian lilac (Schmutterer, 1990). The neem is small to medium-sized tree, 5-20 m height, evergreen, sheds its leaves (Figure, 2.1) only under extremes of heat and drought for a short time (Jorgen, 1990).With its widely extended branches the tree forms an ovate to round crown on a straight smooth stem. The bark is of medium thickness, and longitudinally and obliquely fissured, brown-grey reddish brown. Leaves imparipinnate, alternate 20-40 cm long on long stem petioles, 7 to 17 pairs of leaflets, alternate or opposite, 6-10 cm long. Flowers white, yellowish or cream colored. The fruit is an ellipsoidal drupe with one rarely two seeds. The tree tolerates temperature up to 500C and poor shallow soil in addition to saline soil (Koul, 1990 and Ascher, 1993).

2.2.2 Propagation and Distribution: Neem reproduce by seeds, grow very fast, with high production of seeds, resistance to drought and grows randomly or to decorate cities (Siddig, 1991) readily from cuttings, stumps, tissue culture or seeds. Seed propagation in nurseries, followed by direct planting into the field. The accepted method to produce planting stands fast and efficiently (Jacobson, 1989). The neem tree center of origin lies in southern and southeastern Asia. Today, A. indica also occurs in tropical and subtropical areas of Africa, America and Australia. Neem tree has been introduced in many countries. Mainly for a forestation and fuel wood production in dry areas, but also for other purposes including use as an avenue or shade tree and as a producer of natural pesticides (Schmuterer, 1990, and Ascher, 1993). Moreover, neem tree grows rapidly; 4- 7 meters in its first five years of growth, and 5-11 meters for the following five years. The tree will bear fruits within three years and reaches the maximum fruiting yield of 50 kg/yr, ten years after planting (Jacobson, 1989; Koul, 1990; Ascher, 1993). 2.2.3 Uses for Pests Control: Singh and Kataria (1985) reported that plants produce various types of chemicals to defend themselves against insects and other organisms. Such chemicals are wide spread among plants and have been reported to effect insects by poisoning, inhibition of feeding and disturbing hormonal balance. They found that derivatives of four plant part, one of this neem (A. indica) seed kernel, were found to possess biologically active substances when screened for their toxicity against mosquito larvae. According to Meinwal, et al. (1978), neem leaves extract had produced pronounced morphological changes in the coffee bug, Antestiopsis sp. upon topical application. Singh and Kataria (1986), studied the effect of de-oiled neem kernel powder extract, mixed with wheat grains at 0.06, 0.125, 0.5, 1.0 and 2.0 %, on the development of Trogoderma granarium. The result showed that eggs hatched in all variant and all larvae in the treated variant died in the first instar before the 11th day after hatching, while all those in the untreated variant had reached the 3rd instar by the 11th day. Field trials were conducted in India to determine the efficacy of six plant extracts and two insecticides for the control of Aphis gossypii and Amras cadevestans on okras one of these extracts A. indica 5%, gave a similar level of control compared with endosulfan at 0.06 % and monocrotophos at 0.05 % (Kulat et al., 1998).

Figure (2.1): The Neem Tree leaves

(Source: website: http//www.aos.org/AM/Template.cfm)

Kulkarni and Joshi (1997) reported that, the efficacy of methanolic extracts from seeds of A. indica was evaluated against larvae of Rhesala imparata in laboratory. Laboratory studies were carried out to evaluate some medicinal and aromatic plants against cotton bacterial blight infection. They showed that plant extracts from A. indica and I. helderbranditti were effective in reducing the incidence and intensity of the disease which is caused by Xanthomonas campestris (Patil and Ghoderao, 1997). 2.2.4. The Active Ingredients Many biologically active compounds can be extracted from neem; including triterpenoids, phenolic compounds, carotenoid, steroids and ketones. The tetranortriterpenoid azadirachtin (AZ) has received the most attention as pesticide because it is relatively abundant in neem tree and has shown biological activity on a wide range of insects and considered as the most important active in neem seed kernels from the commercial and biological points of view. AZ is actually a mixture of seven isomeric compounds labeled as AZ-A to AZ-G with AZ-A being present in the highest quantity (Verkerk and Wright, 1993). The impetus towards the investigation of neem was given by the AZ as the most interesting triterpenoid from neem tree, which was discovered in 1967 by Morgan (Jones et al., 1989). 2.3 Ipomoea helderbranditti (Elaweer): Abdelhadi (1987); Tartour et al., (1974) and Adam et al., (1973) reported that the plant was highly toxic and causes many losses in animals every year; Where in Hag Abdalla Town alone about 300 goats died during the 1983 drought as the result of eating the fresh leaves of Ipomoea. The chemical composition of Ipomoea had been studied by many workers. Taber et al., (1963) found that the seed, leaves and the stem of the mature plant of Morning glory (I. carnea) contain clavinet and lysergic acid alkaloids. Tirkey et al. (1988) analyzed the extract of I. carnea leaves and found that it contain alkaloids, reducing sugars, glycosides and tannins. 2.3.1 Medicinal Uses It is reported to have stimulatory allelopathic effects. Roots are boiled to use as laxative and to provoke menstruation. Traditional healers for treatment of skin diseases have used it. The milky juice of plant has been used for the treatment of leucoderma and other related skin diseases. Only external applications have been recommended due to poisonous nature. The plant is also of medicinal value.

It contains a component identical to marsilin, a sedative and anti-covulsant. A saponin has also been purified from I. helderbranditti (Figure, 2.2) with anti-carcenogenic and oxytoxic properiteies (Navin, 2005). 2.3.2 Uses as Control Agent: I. helderbranditti plant and its constituents and their derivatives are used an insecticide (Sasmal, 1992). Kulkarni and Joshi (1997) reported that the efficacy of methanolic extracts from seed of I. carnea fistulosa Jacq were evaluated against larvae of Rhesala imparata in the laboratory. Kulat et al., (1998), carried out field trial in India to test plant extracts for the control of Aphids on sunflower. The leaves extract of I. carnea were equally effective as the insecticides. Laboratory studies were carried out to evaluate some medicinal and aromatic plants against cotton bacterial blight infection. They showed that the plant extracts from I. carnea were effective in reducing the incidence and intensity of the disease which is caused by Xanthomonas campestris (Patil and Ghoderao, 1997). Kehail (2004) found that, the aqueous extract of I. helderbranditti leaves was lethal for Anopheles arabiensis and Culex quinquefasciatus larvae.

Figure (2.2): The Ipomoea helderbranditti (El Aweer) Plant (Source: website: http//www.desert-tropicals.com)

2.4 Acacia nilotica (L.) Natural medicinal plants promote self healing, good health and durability in ayurvedic medicine practices and have acknowledged that A. nilotica can provide the nutrients and therapeutic ingredients to prevent, mitigate or treat many diseases or conditions). It also serves as a source of polyphenols. The role of these polyphenols to the plant itself is not well implicit, but for the human kind they can be of prime strategies (Singh et al., 2009a). The phytochemicals contribute chemically to a number of groups among which are alkaloids, volatile essential oils, phenols and phenolic glycosides, resins oleosins, steroids, tannins and terpenes (Banso, 2009). This plant contains a profile of a variety of bioactive components such as gallic acid, ellagic acid, isoquercitin, leucocyanadin, kaempferol-7-diglucoside, glucopyranoside, rutin, derivatives of catechin-5-gallate, apigenin-6,8-bis-C-glucopyranoside, m-catechol and their derivatives (Singh et al., 2009a). It has been reported that different parts of the plant are prosperous in tannins (ellagic acid, gallic acid and tannic acid), stearic acid, vitamin-C (ascorbic acid), carotene, crude protein, crude fiber, arabin, calcium, magnesium and selenium. A number of medicinal properties have been ascribed to various parts of this highly esteemed plant. Traditionally the bark, leaves, pods and flowers are used against cancer, cold, congestion, cough, diarrhea, dysentery, fever, gall bladder, hemorrhoid, ophthalmia, sclerosis, tuberculosis and small pox, leprosy, bleeding piles, leucoderma and menstrual problems. They have spasmogenic, vasoconstrictor, antihypertensive, -mutagenic, carcinogenic, -spasmodic, - inflammatory,-oxidant and platelet aggregatory properties (Singh et al., 2009b). A. nilotica has antiplasmodial, molluscicidal, anti-fungal, anti-microbial activity, inhibitory activity against HCV and HIV-I (Sultana et al., 2007). The bark of the plant is used as astringent, acrid, cooling, styptic, emollient, anthelmintic, aphrodisiac, diuretic, expectorant, emetic and nutritive, in hemorrhage, wound ulcers, leprosy, leucoderma, skin diseases and seminal weakness. Gum is used as astringent, emollient, liver tonic, antipyretic and antiasthmatic (Baravkar et al., 2008). The bark is used extensively for colds, bronchitis, biliousness, diarrhoea, dysentery, bleeding piles and leucoderma (Del, 2009). It is used by traditional healers of different regions of Chattisgarh in treatment of various cancer types of mouth, bone and skin. In West Africa, the bark and gum are used against cancers and/or tumors (of ear, eye, or testicles) and indurations of

33 liver and spleen, the root for tuberculosis, the wood for smallpox and the leaves for ulcers (Kalaivani and Methew, 2010). Pods and tender leaves are given to treat diarrhoea and are also considered very useful in folk medicine to treat diabetes mellitus. The tender twings are used as toothbrushes. So far no comprehensive review has been compiled encircling the efficacy of this plant in all proportions from the literature. Its stretchy utility as a medicine forced us to bridge the information gap in this area and to write a comprehensive review on the medicinal, phytochemical and pharmacological traits of this plant of high economic value (Gilani et al., 1999). 2.4.1 Description Acacia nilotica (family: Leguminosae, subfamily: Mimosoideae) grows to 15-18 m in height and 2-3 m in diameter. The bark is generally slight green in young trees or nearly black in mature trees with deep longitudinal fissures exposing the inner grey-pinkish slash, exuding a reddish low quality gum. The leaves (Figure, 2.3) are bipinnate, pinnae 3-10 pairs, 1.3- 3.8 cm long, leaflets 10-20 pairs, and 2-5mm long. Thin, straight, light grey spines present in axillary pairs, usually 3-12 pairs, 5-7.5 cm long in young trees, and mature trees commonly without thorns. Flowers in globulous heads, 1.2-1.5 cm in diameter of a bright golden yellow colour, born either axillary or whorly on peduncles 2-3 cm long located at the end of branches. Pods7-15 cm long, green and tomentose when immature and greenish black when mature, indehiscent, deeply constricted between the seed giving a necklace appearance. Seeds 8-12 per pod, compressed, ovoid, and dark brown shining with hard testa (Puri and Khybri, 1975). 2.4.2 Distribution: A. nilotica is naturally wide spread in the drier areas of Africa, from Senegal to Egypt and down to South Africa, and in Asia from Arabia eastward to India, Burma and Sri Lanka. The largest tracts are found in Sind. It is distributed throughout the greater part of India in forest areas, roadsides, farmlands, tank foreshores, agricultural fields, village grazing lands, wastelands, bunds, along the national highways and railway lines. Mostly it occurs as an isolated tree and rarely found in patches to a limited extent in forests. It has been widely planted on farms throughout the plains of the Indian subcontinent. It is a species of Southern Tropical dry deciduous forests and Southern Tropical thorn forests as distinguished by Champion and Seth (1968).

Figure (2.3): The Acacia nilotica leaves (Source: website: http//www.tropicalforages)

2.4.3 Ecology There is some evidence that A. nilotica is a weed in its native habitat e.g. South Africa (Holm et al., 1979), but in other areas it is planted for forestry or reclamation of degraded land. The ecological implication of using A. nilotica as a browse source while maintaining in appropriate stocking rates is land degradation. It grows well in two types of soils i.e. riverian alluvial soil and black cotton soil. This species grow on saline, alkaline soils and those with calcareous pans. A. nilotica grows under climatic conditions ranging from sub-tropical to tropical. It can withstand extremes of temperature (> 50 o C) and conditions of drought however; adequate moisture is needed for full growth and development. It is frost tender when young and trees of all age classes are adversely affected by conditions of severe frost. It is fire tender and both seedlings and saplings are adversely affected by fire. The average annual rainfall varies from 250-1500 mm (Shetty, 1979). 2.4.4 Economic Importance: Acacia's are established as very important economic plants since early times as source of tannins, gums, timber, fuel and fodder. They have significant pharmacological and toxicological effects In Africa and the Indian subcontinent; A. nilotica is extensively used as a browse, timber and firewood species (Gupta, 1970; Mahgoub, 1979; New, 1980). The bark and seeds are used as a source of tannins (Shetty, 1979).The species is also used for medicinal purposes. Bark of A. nilotica has been used for treating hemorrhages, colds, diarrhea tuberculosis and leprosy while the roots have been used as an aphrodisiac and the flowers for treating syphilis lesions (New, 1980). The gum of A. nilotica is sometimes used as a substitute for gum Arabic (obtained from A. senegal) although the quality is inferior (Gupta, 1970). Indian Gum is sweeter in taste than that of the other varieties and is used in paints and medicine. The species is suitable for the production of paper and has similar pulping properties to a range of other tropical timbers (Nasroun, 1979). The dark brown wood is strong, durable, nearly twice as hard as teak, very shock resistant and is used for construction, tool handles and carts. It has a high calorific value of 4950 kcal/kg, making excellent fuel wood and quality charcoal. It burns slow with little smoke when dry. It has a 25% more shock resisting ability than teak. At the time of tree felling total wood production was estimated 167 Mg ha-1 that included 45 m3 marketable timbers. Survey of local timber market revealed that farmers fetch Rs 1000 from one tree (>15

36 years) and Rs 30 to 90 thousand from 1 ha land, depending upon the stocking rate that makes the system economically viable. A. nilotica leaf is very digestible and has high levels of protein. Micronutrients, with the exception of sodium, are adequate for animal requirements leaves and pods contain 8% digestive protein (12.4% crude protein), 7.2 MJ/ kg energy and are rich in minerals and generally used for feeding sheep and goats in certain parts of India and also very popular with cattle. Pods are best fed dry as a supplement not as a green fodder. The bark contains high levels of tannin (12-20%) that is used for tanning leathers. Deseeded pods from ssp. indica have 18-27 tannin levels, whereas ssp. nilotica reached up to 50%. The relative tannin levels in A. nilotica from least to most are pods (5.4%), leaves (7.6%), bark (13.5%) and twigs (15.8%). The tannin also contributes to its medicinal use as a powerful astringent. It is also a powerful molluscicide and algaecide. Sub species indica and cupressiformis are commonly used in agro-forestry. These subspecies make an ideal windbreak surrounding fields. In India this species is used extensively on degraded saline/alkaline soils, growing on soils up to pH 9, with a soluble salt content below3%. It also grows well when irrigated with tannery effluent; and colonizes waste heaps from coalmines. Over 50, 000 hectares of the Indian Chambal ravines have been rehabilitated with A. nilotica (Kalaivani and Methew, 2010). 2.4.5 Allelopathic Effect of A. nilotica: El-khawas and Shehata (2005) reported that the leaf of A. nilotica inhibited the germination and growth of Zea mays and Phaseolus vulgaris. Duhan and Lakshinarayana (1995) found that the growth of Cyamopsis tetragonoloba and Pennisetum growing at distance of 1-2 and 7.5 m from tree of A. nilotica was inhibited. Velu et al., (1999) reported that the Acacia spp. Have phytotoxic effects on the tree crops of legumes. These results suggested that the inhibitory effect of A. nilotica on seed germination and seedling growth is related to the presence of allelochemicals including tannins, flavonoids and phenolic acids. Moreover, the toxicity is caused due to synergistic effect rather than single one (Fag and Stewart 1994). According to Stratmann and Ryan (1997) and El- Khawas and Shehata (2005) allelopathic effect of Acacia spp. induced the formation of stress proteins. These proteins are responsible for folding, assembling, translocation and degradation in a broad array of normal cellular processes such as improvement of plant growth, physiological and molecular characteristics (Wang et al. 2004). This allelopathic ability of A. nilotica may have the potential as herbicide and can be used in biological control of weeds (Li And Wang, 1998).

2.4.6 Phytochemistry Plant compounds have interest as a source of safer or more valuable substitutes than synthetically created agents. Phytochemical progress has been aided extremely by the development of rapid and accurate methods of screening plants for particular chemicals. The phytochemicals are divided chemically into a number of groups among which are alkaloids, volatile essential oils, phenols and phenolic glycosides, resins, oleosins, steroids, tannins and terpenes (Banso, 2009). Phytochemistry confirmed that all the tested extracts contain physterols, fixed oils, fats, phenolic compounds, flavanoids and saponins (Kalaivani et al., 2010). The phytochemicals alkaloids and glycosides detected in the crude extracts of A. nilotica roots are indicated below. Phytochemical screening of the stem bark of A. nilotica exposed that the plant contain terpenoids, alkaloids, saponins and glycosides. Negative results were recorded for steroids and flavonoids which authenticate the absence of these phytochemicals (Banso, 2009). This plant recommends a variety of phytochemical such as gallic acid, ellagic acid, isoquercitin, leucocyanadin, kaempferol-7-diglucoside glucopyranoside, rutin, derivatives of catechin-5-gallate, apigenin-6,8-bis-C-glucopyranoside, m-catechol and their derivatives. A. nilotica contains gallic acid, m-digallic acid, catechin, chlorogenic acid, gallolyated flavan-3,4- diol, robidandiol (7,3,4,5-tetrahydroxyflavan-3-4-diol), androstene steroid, D-pinitol carbohydrate and catechin-5-galloyl ester. The bark is prosperous in phenolics viz. condensed tannin and phlobatannin, gallic acid, protocatechuic acid pyrocatechol, catechin, epigallocatechin-7-gallate, and epigallocatechin-5,7- digallate (Singh et al., 2009a). The bark is also reported to contain epicatechin, dicatechin, quercetin, gallic acid, leucocyanidin gallate, sucrose and catechin-5-gallate (Mitra and Sundaram, 2007). The umbelliferone and the polyphenolic compounds kaempferol has been reported in A. nilotica (Singh et al., 2010b). 2.4.7 Medicinal Uses and Pharmacological Effects A. nilotica also has numerous medicinal uses. A decrease in arterial blood pressure is reported by use of methanolic extract of A. nilotica pods and provides evidence of anti hypertensive activities independent of muscarinic receptor stimulation. In the in vitro studies, A. nilotica has inhibitory effect on force and rate of spontaneous contractions in guinea-pig paired atria and rabbit jejunum. A. nilotica also inhibits K+ induced contractions in rabbit jejunum advocating the antispasmodic action of A. nilotica which is mediated through calcium channel

38 blockade and this may also be responsible for the blood pressure lowering effect of A. nilotica, observed in the in vivo studies (Gilani et al., 1999). An aqueous extract of the seed of A. nilotica is also investigated on the isolated guinea-pig ileum which exposed the sustained dose- related contractile activity. A dose-related significant elevation of blood pressure is produced by intravenous administration of the extract (Amos et al., 1999). The assays of the stem bark extracts confirm the antimicrobial activity against Streptococcus viridans, Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Shigella sonnei using the agar diffusion method. A. nilotica could be a potential source of antimicrobial agents (Banso, 2009). A. nilotica demonstrates highest activity against three bacterial (E. coli, S. aureus and Salmonella typhi) and two fungal strains (Candida albicans and Aspergillus niger) (Kalaivani and Methew, 2010). The ethyl acetate extract holds the highest activity on Plasmodium falciparum. Phytochemical analysis indicated that the most active phase contained terpenoids and tannins and was devoid of alkaloids and saponins (El-Tahir et al., 1999). Crude methanolic root extracts of A. nilotica reveals significant activity against chloroquine sensitive strain of Plasmodium berghei in mice. Water extract fractions of A. nilotica (L.) in lipid peroxidation assay possess the peroxyl radical scavenging capacity and results prove the anti-oxidant activity of plant. The bark powder of the plant extracts with different solvents found the scavenging activity using maceration extraction (Del, 2009). Another study reveals that A. nilotica is easily accessible source of natural antioxidants, which can be used as supplement to aid the therapy of free radical mediated diseases such as cancer, diabetes, inflammation, etc (Amos et al., 1999). Furthermore, the high scavenging property of A. nilotica may be due to hydroxyl groups existing in the phenolic compounds that can scavenge the free radicals (Kalaivani and Mathew, 2010). The extract of A. nilotica is found to stimulate the synthesis and release of prolactin in the female and may give a better result for lactating women (Lompo et al., 2004), for tanning, dyeing of leather, gastrointestinal disorders, syphilitic ulcers and toothache (Amos et al., 1999), while pods inhibited HIV-1 induced cytopathogenicity (Asres et al., 2005). Fresh roots extract used as narcotic, local bear, gum is used as aphrodisiac with water; branches used for cleaning teeth (Badshah and Hussain, 2011). Methanolic extract of bark has significant inhibitory effects on HCV protease (Hussein et al., 1999a) and against HIV-1 (Hussein et al., 2000b).

CHAPTER THREE MATERIALS AND METHODS

3.1. Collection and Rearing of Houseflies: Samples of housefly (Musca d.vicina) larval stage were collected from Wad Kanan area, west of Wad Medani Town (throughout the study; 2013-2015). The collected larvae were kept in special container in the Basic Science Laboratory, University of Gezira under room temperature. The collected larvae were reared in the laboratory till they became adults, then M. d.vicina (According to Patton 1932 and 1937) adults were separated for the further study whereas, other Musca species were discarded (whenever found). Larvae of the third generation (to ensure homogeneity) were used for the study tests and were fed by horse feces till they became pupae (the non-feeding stage) in ordinary mosquito's cages. Two cages were prepared for rearing of housefly larvae. Each cage was 35x35x35 cm and has one designed opening through which food and larvae were passed. Adult flies were provided with water and fed by 1:1 (v/v) mixture of granulated sugar and powdered milk. Special meal was prepared by adding horse feces to the mixture at ratio of 1:3 for attracting female house flies to lay eggs on. All houseflies larvae used later were picked from the first generation which was emerged and reared in the laboratory. 3.2 Collection of Plant Samples and Preparation of their Extracts: The neem (A. indica) leaves were collected from within Alnishishiba area, while El- Aweer (I. helderbranditti) leaves were collected from Kereba area (west of Wad Medani), whereas, Garad (A. nilotica) leaves were collected from Um Sunt forest (south of Wad Medani). Clean and freshly collected leaves samples of these plants were dried under shade in the room temperature away from the direct sunlight. The dried samples were kept in clean and well labeled small plastic bags. Each sample was ground to fine powder by using an electrical grinder. 50 g of each powder was soaked into 500 ml of distilled water, methanol or hexane in 500 ml conical flasks for 24 hours. Each solution was filtrated by using a filter paper. Each solvent was then evaporated to dryness under vacuum using a rotary evaporator with a water bath adjusted to 80°C. The dry crude residues (except that of hexane) were then weighed and kept as stock. Concentrations of 7%, 5% and 1% were prepared and used at the first and the second seasons, while in the third season, other diluted concentrations were used (0.25%, 0.5% and 2.5%).

3.3. Phytochemical Screening Tests Phytochemical screening for the presence of the main classes in the leaves of the selected plant-leaves were done according to Yusha’u et al., (2009): 3.3.1 Test for the Presence of Alkaloids To about 0.1 ml of the extract and fractions in a test tube, 2 – 3 drops of Dragendoff’s reagent were added. An orange red precipitate with turbidity denoted the presence of alkaloids. 3.3.2 Test for the Presence of Flavonoids and Flavonones To about 4 mg/ml of the extracts and fractions a piece of magnesium ribbon was added followed by drop-wise addition of concentrated HCl. A color change from orange to red indicated the presence of flavonones; red to crimson indicated the presence of flavonoids. 3.3.3 Test for the Presence of Glycosides

Ten ml of 50% H2SO4 was added to 1 ml of the filtrate in separate test tubes and the mixtures heated for 15mins followed by addition of 10 ml of Fehling’s solution and boiled. A brick red precipitate indicated the presence of glycosides. 3.3.4 Test for the Presence of Saponins To about 0.5 g of the powdered leaf was dispensed in a test-tube and 5.0 ml of distilled water was added and shaken vigorously. A persistent froth that lasted for about 15 minutes indicated the presence of saponins. 3.3.5 Test for the Presence of Steroids Two ml of the extracts were evaporated to dryness in separate test tubes and the residues dissolved in acetic anhydride followed by addition of chloroform. Concentrated H2SO4 was added by means of a pipette via the side of the test tubes. Formation of brown ring at the interface of the two liquids and violet color in the supernatant layer denoted the presence of steroids. 3.3.6 Test for the Presence of Tannins Two ml of the extract/fraction was diluted with distilled water in separate test tubes; 2 – 3 drops of 5% ferric chloride (FeCl3) solution was added. A green – black or blue coloration indicated tannins. 3.3.7 Test for the Presence of Terpenoids This test depended on the volatility and scent (physical) emitted by the plant part-oils.

3.4. Bioassay: Bioassay was conducted to study the effect of each plant extract on the mortality of M. d. vicina larvae by using feeding medium-treatment method. Each test was triplicate and run for three successive seasons (S1: 2013, S2: 2014 and S3: 2015). The normal larval feed (NLF) was composed of 15 g of wheat bran, 3 g of milk powder, 1.5 g sugar, 0.5 g yeast (total was 20 g) and 20 ml of water. Certain weight of each of the stock extracts were mixed with NFL to prepare treated larval feed at concentrations of 1%, 5% and 7% (at the first and the second seasons) and three diluted concentrations of 0.25%, 0.5%, 2.5% in addition to 1% (at the third season due to the high mortality produced by the concentrations that used in the first and second seasons). A set of 20 larvae were picked randomly and placed separately in special containers (5X7X5 cm) containing only one concentration of the treated larval feed (for testing their toxicity against housefly larvae). The percentage larval mortalities were recorded after 1, 2, 3, 4, 5 and 6 days (before it pupate). For control, the larvae were fed on NFL which was not mixed with any amount of stock extract, until pupation. 3.5 Data Analysis: Microsoft Office, Excel 2007, was used to analyze the data obtained. Simple descriptive statistics (count, sum, average (mean) and variance), regression analysis (R-square (the homogeneity factor), the X-coefficient (b; the constant rate of increase in mortality in respect to each one day late), the intercept (a; the hypothetical value of mortality at day 0) and the standard error of X (SE-X) and Y (SE-Y) variables) and ANOVA analysis used to describe the observed variations in the toxicities exerted by any plant-extract used. The difference will be significant whenever the f-stat value is bigger than that of f-crit at certain P value.

CHAPTER FOUR RESULTS AND DISCUSSION 4.1. The Qualitative Phytochemical Screening in the Selected Leaves:

The results obtained from the qualitative phytochemical screening (for the main classes of phytochemicals) of A. indica (neem), A. nilotica (Sunut) and I. helderbranditti (Aweer) leaves were presented in Table (4.1). The results revealed that, flavonoids was detected in the neem, aweer and Garad leaves, while steroids and saponins were found just in neem, whereas tannins were only detected in the Garad, alkaloids were detected in the Aweer and steroid showed only in neem. The detection of the main phytochemical classes in plant materials usually depended on several chemical factors including the methods and the solvents used. In a similar study conducted by Sameh et al., (2012), tannins, saponins, flavonoids, phytosterols, coumarins and alkaloids were detected in both neem seeds and leaves. The results of qualitative Phytochemical screening for neem-leaves in this study agreed with the findings of Sameh et al., (2012). The phytochemical analysis of the water and organic (ethanol and petroleum ether) extracts of A. nilotica (leaves and fruits) were done through chemical approach as described by Harbone (1973, 1983). Accordingly, alkaloids, amino acids, flavonoids, tannins and sterols/triterpenoids, were detected. The findings of this study on A. nilotica leaves did not match completely with the work of Harbone (1973, 1983), and this may be referred to environmental factors and the quality of the materials used for these tests. Taber et al., (1963) found that the seed, leaves and the stem of the mature plant of Morning glory (I. carnea) contain clavinet and lysergic acid alkaloids. Analyzed the extract of I. carnea leaves and found that it contain alkaloids, reducing sugars, glycoside, phenolic compounds, saponins, xanthoproteins and tannins (Tirkey et al., 1988; Sahayara et al., 2008). The findings of this study on I. helderbranditti leaves did not match completely with the work of Tirkey et al., (1988), and this may also be referred to the environmental factors and the quality of the materials used for these tests.

Table (4.1): Qualitative Phytochemical screening for neem, Sunut and Elaweer Leaves:

Neem Sunut Elaweer Sample A. indica A. nilotica I. helderbranditti

Flavonoids + + + + Tannins - + - Alkaloids - - + Steroids + - - Saponins + + - -

+ indicated the presence of the class. ++ indicated the presence of the class in relatively high concentration. - indicated not detection class.

4.2. Larvicidal Activity of Neem Leaves Extracts on M. d. vicina (Season 1) 4.2.1. The Aqueous Extract The results obtained from the first season (S1; 2013) exhibited the toxicity of A. indica leaves extracts against the first, second and third instars larvae of M. vicina, after 1, 2, 3, 4, 5 and 6 days at different concentrations. Table (4.2) showed that, the neem aqueous extract resulted in no mortality (0%) in the control group, while in the lower concentration (1%) mortality was increased from 15% after the first day to 100% at the 4th day with constant rate of mortality of 17.5%/day, as same as, the mortality in the concentration of (5%) that increased to 100% at the 4th day, with constant rate of mortality of 19.2% / day, whereas the higher concentration (7%) showed an increase in mortality to 100% at the third day. 4.2.2 Methanol Extract Table (4.2) also showed that, the mortality in the control was (0%). In the lower concentration (1%), neem methanol extract resulted in mortality which reached 85% at the 6th day with constant rate of mortality of 15.2%/day, while the mortality in the concentration of (5%) increased to 60% at the end of the 6th day with constant rate of mortality of 13%/day, whereas in the higher concentration (7%), an increase in mortality from 15% at the first day to 90% at the sixth day with constant rate of mortality of 15.4%/day was observed. All methanol- extract concentrations of neem did not score 100% mortality against larvae. 4.2.3 Hexane Extract Table (4.2) also showed that, the mortality in control was (0%). The lower concentration (1%) of neem hexane-extract resulted in 50% mortality at the 6th day with constant rate of mortality of 9.57%/day, while the mortality corresponding to the concentration of (5%) increased to 95% at the end of the 6th day with constant rate of mortality of 13.8%/day, whereas in the higher concentration (7%), an increase in mortality to 35% at the 6th day. It was clear that, neem aqueous extract exerted a high toxic effect against housefly larvae (score 100% mortality) more than methanol extract (score between 60%-90% mortality) and hexane extract (score between 35%-95% mortality) at the first season.

Table (4.2): Effect of neem leaves extracts on mortality % of M. d. vicina larvae (S1: 2013)

Aqueous extract Methanol extract Hexane extract

Control Concentrations % Day 1% 5% 7% 1% 5% 7% 1% 5% 7% 1 0 15 5 5 0 0 15 0 5 10 2 0 40 35 50 20 10 40 5 70 15 3 0 90 95 100 50 25 75 15 75 20 4 0 100 100 100 55 45 90 25 80 25 5 0 100 100 100 55 55 90 30 80 25 6 0 100 100 100 85 60 90 50 95 35 Regression Analysis R2 1 0.78 0.74 0.69 0.91 0.97 0.81 0.95 0.66 0.95 A 0 12.6 5 13.3 -9.33 -13 12.6 -12.6 19 5.66 B 0 17.5 19.2 17.8 15.2 13 15.4 9.57 13.8 4.57 SE-Y 0 18.1 21.9 22.9 9.09 4.16 14.1 3.82 19.0 1.95 SE-X 0 4.65 5.64 5.90 2.33 1.06 3.62 0.98 4.89 0.50

4.3 Larvicidal Activity of I. helderbranditti Leaves on M. d. vicina (Season 1) 4.3.1 Aqueous extract Table (4.3) showed that, at the lower concentration (1%) mortality reached 100% at the 6th day with constant rate of mortality of 19.5% / day, whereas, the mortality in the concentration of (5%) increased to 50% at the end of the test period, while in the higher concentration (7%), mortality reached 60% at the 6th day with constant rate of mortality of 4.71%/day. It was noticed that, the highest mortality (100%) corresponded to the lower concentration (1%) followed by the higher concentration (7%) which resulted in 60% mortality, while the medium concentration (5%) resulted in 50% mortality. 4.3.2 Methanol extract Table (4.3) showed that, at the lower concentration (1%) mortality was increased from 0% after the first day to 95%, whereas, the mortality in the concentration of (5%) increased to 100% at the end of the 6th days, while in the higher concentration of (7%), an increase in mortality from 10% at the first day to 85% at the 6th day was observed. In comparison, the highest mortality (100%) at the end of the test period corresponded to the medium concentration (5%) followed by the lower concentration (1%) which resulted in 95% mortality, while the higher concentration of (7%) resulted in 85% mortality. 4.3.3 Hexane extract Table (4.3) showed that, the mortality resulted from the lower concentration (1%) was 0% till the 6th day, whereas, the mortality in the concentration of (5%) increased to 65% at the end of the test period (six days), while the higher concentration (7%), showed an increase in mortality from 5% at the first day to only 15% at the 6th day. In comparison, the highest mortality (65%) produced by the hexane extract at the end of the test period was corresponded to the medium concentration (5%) followed by the concentration of (7%) which resulted in 15% mortality, while the concentration of (1%) and the control resulted in 0% mortality. The inconsistency of concentrations and mortalities of the I. helderbranditti leaves extracts (aqueous, methanol and hexane) in the housefly larvae, may be due to the presence of resins that may act as a deterant material, hence this case may be taken as an anti-feedant evidence. It was also clear that, the aqueous extract exerted a toxic effect against housefly larvae (between 50%-100% mortality), the methanol extract (between 85%-100% mortality) and the hexane extract (between 0%-65% mortality) at the first season.

Table (4.3): Effect of I. helderbranditti leaves extracts on the mortality of M. d. vicina larvae (S1: 2013)

Aqueous extract Methanol extract Hexane extract

Control Concentrations % Day 1% 5% 7% 1% 5% 7% 1% 5% 7% 1 0 10 30 30 0 65 10 0 50 5 2 0 20 30 45 25 75 55 0 50 10 3 0 30 30 50 35 80 75 0 55 10 4 0 55 35 50 60 90 75 0 65 10 5 0 90 45 50 65 90 80 0 65 10 6 0 100 50 60 95 100 85 0 65 15

Regression Analysis

R2 1 0.95 0.83 0.79 1 0.97 0.96 1 0.85 0.71 A 0 -17.6 21.6 31 0 -15.3 60.3 0 45.3 5 B 0 19.5 4.28 4.71 0 17.7 6.57 0 3.71 1.42 SE-Y 0 8.29 3.66 4.62 0 5.54 2.44 0 3.01 1.75 SE-X 0 2.13 0.94 1.18 0 1.42 0.62 0 0.77 0.45

4.4 Larvicidal Activity of A. nilotica Leave Extracts on M. d. vicina (Season 1) 4.4.1. Aqueous Extract Table (4.4) showed that, in the lower concentration (1%) mortality increased to 70% at the 6th day, whereas, the mortality in the medium concentration (5%) increased to 90% at the end of the 6th day, while the higher concentration (7%), showed an increase in mortality from 50% at the first day to 95% at the 6th day. In comparison, the highest mortality (95%) at the end of the test period corresponded to the higher concentration (7%) followed by the concentration of (5%) and (1%) which resulted in 90% and 70% mortalities, respectively. 4.4.2 Methanol Extract Table (4.4) showed that, in the lower concentration (1%) mortality reached 100% at the 6th day, whereas, 100% mortality was caused by the medium concentration (5%) at the 3rd day, while the higher concentration (7%), produced 100% mortality at the 5th day. It was clear that, the highest mortality (100%) was caused by all concentrations but with different periods. 4.4.3 Hexane extract Table (4.4) showed that, in the lower concentration (1%) mortality was increased from 0% after the first day to 45% at the 6th day, whereas, the mortality in the medium concentration (5%) increased from 5% to 45% at the end of the test period, while in the higher concentration (7%), an increase in mortality was recorded from 10% at the first day to 45% at the 6th day. It was clear that, all hexane extract concentrations did not cause more than 45% mortality at the end of the test period. It was also clear that, the aqueous extract of A. nilotica leaves exerted a toxic effect against housefly larvae (between 70%-95% mortality), the methanol extract (100% mortality) and the hexane extract (45% mortality) at the first season.

Table (4.4) Effect of A. nilotica leaves extracts on the mortality of M. d. vicina larvae (S1: 2013)

Aqueous extract Methanol extract Hexane extract

Control Concentrations % Day 1% 5% 7% 1% 5% 7% 1% 5% 7% 1 0 30 80 50 5 60 50 0 5 10 2 0 35 90 95 55 90 50 25 15 25 3 0 50 90 95 60 100 90 40 25 35 4 0 60 90 95 75 100 95 40 30 35 5 0 70 90 95 85 100 100 40 30 35 6 0 70 90 95 100 100 100 45 45 45

Regression Analysis

R2 1 0.95 0.42 0.42 0.88 0.58 0.80 0.72 0.93 0.83 A 0 21 83.3 65 5.33 68.6 40.3 4.66 0 10.3 B 0 9 1.42 6.42 16.5 6.57 11.5 7.71 7.14 5.85 SE-Y 0 3.89 3.21 14.4 11.6 10.6 11.1 8 9.21 3.51 5.10 SE-X 0 1 0.82 3.71 2.99 2.74 2.87 2.36 0.90 1.31

4.5. Larvicidal Activity of Neem Leaves Extracts on M. d. vicina (Season 2) 4.5.1. Aqueous Extract The results of the second season (S2: 2014) showed the toxicity of A. indica leaves extract (at three concentrations; 1%, 5% and 7%) against the first, second and 3rd instars larvae of M. vicina, after 1, 2, 3, 4, 5 and 6 days (Table, 4.5). The mortality in the control increased from 0% to 5% at the 6th day. In the lower concentration (1%) mortality increased from 85% after the first day to 100% at the 6th day, whereas, the mortality caused by the medium concentration (5%) was 100% at the first day, as same as that of the higher concentration (7%). It was clear that, all concentrations lead to cause 100% mortality. 4.5.2 Methanol Extract Table (4.5) showed that, in the lower concentration (1%) mortality increased from 50% to 90% at the 6th day, whereas, the mortality caused by the medium concentration (5%) increased from 50% to 95% at the end of the test period, while the higher concentration (7%), produced mortality from 60% to 95% at the 6th day. It was clear that, the highest mortality (95%) at the end of the test period was caused by the higher (7%) and medium concentrations (5%), while the concentration of (1%) resulted in 90% mortality. 4.5.3 Hexane Extract Table (4.5) showed that, in the lower concentration (1%), mortality increased from 10% to 65% at the 6th day, whereas, the mortality in the medium concentration (5%) increased from 40% to 50% at the 6th day, while the higher concentration (7%), produced mortalities from 45% to 75% at the 6th day. In comparison, the highest mortality (75%) at the end of the test period was caused by the concentration (7%), followed by the concentration (1%) which resulted in 65% mortality, while the concentration (5%) resulted in 50% mortality. The control resulted in 5% mortality. The aqueous extract of neem leaves exerted the maximum toxic effect against housefly larvae (100% mortality), whereas methanol extract produced 95% mortality, while the hexane extract produced from 50% to 75% mortality, at the second season (2014). At the first season (S1), neem aqueous extract scored 100% mortality on housefly larvae, whereas the methanol extract scored between 60%-90% mortality and the hexane extract score between 35%-95% mortality.

Table (4.5) Effect of neem leaves extracts on the mortality of M. d. vicina larvae (S2: 2014)

Aqueous extract Methanol extract Hexane extract

Control Concentrations % Day 1% 5% 7% 1% 5% 7% 1% 5% 7% 1 0 85 100 100 50 50 60 10 40 45 2 0 100 100 100 90 60 65 10 40 75 3 0 100 100 100 90 85 70 45 45 75 4 5 100 100 100 90 95 95 65 45 75 5 5 100 100 100 90 95 95 65 50 75 6 5 100 100 100 90 95 95 65 50 75

Regression Analysis

R2 0.77 0.42 1 1 0.42 0.82 0.85 0.83 0.91 0.42 A -2 90 100 100 63.3 46 51 -2.66 37 55 B 1.28 2.14 0 0 5.71 9.71 8.28 13.14 2.28 4.28 SE-Y 1.36 4.81 0 0 12.8 8.69 6.55 11.50 1.36 9.63 SE-X 0.34 1.2 0 0 3.29 2.23 1.68 2.95 0.34 2.47

4.6 Larvicidal Activity of I. helderbranditti Leaves on M. d. vicina (Season 2) 4.6.1. Aqueous Extract Table (4.6) showed that, in the lower concentration (1%) mortality increased from 15% to 85% at the 6th day, whereas, the mortality in the medium concentration (5%) increased from 45% to 90% at the end of the test period, while in the higher concentration (7%), the resulted mortality was 90%. In comparison, the highest mortality (95%) at the end of the test period was caused by the concentration of (5%) followed by the concentration of (7%) which resulted in 90% mortality and the concentration of (1%) which resulted in 85% mortality (the difference was not more than 2 larvae; 10% mortality). 4.6.2 Methanol extract In the lower concentration (1%) mortality was increased from 10% to 100% at the 4th day, whereas, the mortality caused by the concentration of (5%) increased from 60% to 100% at the end of the test period, while in the higher concentration (7%), an increase in mortality from 95% at the first day to 100% at the sixth day was noticed (Table 4.5). It was clear that, the highest mortality (100%) was caused by all concentrations. 4.6.3 Hexane extract Table (4.6) showed that, in the lower concentration (1%) mortality was increased from 35% to 65% at the 6th day, whereas, the mortality in the concentration of (5%) increased from 10% to 65% at the end of the test period, while in the higher concentration (7%), an increase in mortality from 25% to 90% at the 6th day was recorded. It was observed that, the highest mortality (90%) at the end of the test period was caused by the concentration of (7%) followed by the concentrations of (1%) and (5%) which resulted in 65%. It was also clear that, the aqueous extract exerted between 85%-95% mortality in housefly larvae, whereas, the methanol extract caused 100% mortality, while the hexane extract (between 65%-90% mortality) at the second season (S2). The inconsistence of concentrations and mortalities of the I. helderbranditti leaves extracts (aqueous, methanol and hexane) in the housefly larvae, that observed in S1 was also observed in S2. Also, the aqueous and the methanol extracts exerted the highest toxic effect against housefly larvae than the hexane extract at the first and the second seasons.

Table (4.6) Effect of I. helderbranditti leaves extracts on the mortality of M. d. vicina larvae (S2: 2014)

Aqueous extract Methanol extract Hexane extract

Control Concentrations % Day 1% 5% 7% 1% 5% 7% 1% 5% 7% 1 0 15 45 90 10 60 95 35 10 25 2 0 80 90 90 50 75 100 40 20 60 3 0 80 90 90 60 85 100 60 20 65 4 5 80 90 90 100 100 100 60 20 85 5 5 85 90 90 100 100 100 65 50 90 6 5 85 95 90 100 100 100 65 65 90

Regression Analysis

R2 0.77 0.50 0.50 1 0.86 0.86 0.42 0.83 0.82 0.85 a -2 34.3 58.3 90 6 57.6 96.6 31.66 -5.66 25.66 b 1.28 10.42 7.14 0 18.28 8.28 0.71 6.42 10.42 12.42 SE-Y 1.36 20.1 13.89 0 14.33 6.27 1.60 5.65 9.51 10.06 SE-X 0.34 5.16 3.56 0 3.68 1.61 0.41 1.45 2.44 2.58

4.7 Larvicidal Activity of A. nilotica Leaves Extracts on M. d. vicina (Season 2) 4.7.1 Aqueous Extract Table (4.7) showed that, in the lower concentration (1%) mortality increased from 15% to 80% at the 6th day, whereas, the mortality in the medium concentration (5%) increased from 30% to 65% at the 6th day, while the higher concentration (7%), caused an increase in mortality from 15% to 65% at the 6th day. In comparison, the highest mortality (80%) at the end of the test period was caused by the concentration of (1%) followed by the concentration of (7%) and (5%) which resulted in 65% mortality, while the control resulted in 5% mortality. 4.7.2 Methanol Extract The mortality increased to 5% at the end of the sixth days in the control. In the lower concentration (1%) mortality increased from 10% to 85% at the 6th day, whereas, the mortality caused by the concentration of (5%) increased from 10% to 60% at the 6th day, while in the higher concentration (7%), slight increase in mortality from 70% to 75% at the 6th day was recorded. In comparison, the highest mortality (85%) was caused by the concentration of (1%) followed by that of (7%) which resulted in 75% mortality, while the concentration of (5%) resulted in 60% mortality (Table 4.7). 4.7.3 Hexane Extract Table (4.7) showed that, in the lower concentration (1%) mortality increased from 5% to 80% at the 6th day, whereas, the mortality caused by the concentration (5%) increased from 25% to 85% at the 6th day, while the higher concentration (7%), resulted in an increase in mortality from 15% to 55% at the 6th day. In comparison, the highest mortality (85%) at the end of the test period was caused by the concentration (5%) followed by the concentration of (1%) which resulted in 80%, while the concentration (7%) resulted in 55% mortality. In the second season (S2), the aqueous extract of A. nilotica leaves exerted a toxic effect against housefly larvae (between 65%-80% mortality), the methanol extract (60%-85% mortality) and the hexane extract (55%-85% mortality), compared to 70%-95%, 100% and 45%, following the same order, in the S1.

Table (4.7) Effect of Acacia nilotica leaves extracts on the mortality of M. d. vicina larvae (S2: 2014)

Aqueous extract Methanol extract Hexane extract

Control Concentrations % Day 1% 5% 7% 1% 5% 7% 1% 5% 7% 1 0 15 30 15 10 10 70 5 25 15 2 0 40 35 25 60 20 75 20 55 35 3 0 55 55 35 75 45 75 60 85 55 4 5 60 65 60 80 55 75 80 85 55 5 5 75 65 65 85 60 75 80 85 55 6 5 80 65 65 85 60 75 80 85 55

Regression Analysis

R2 0.77 0.94 0.83 0.92 0.70 0.88 0.42 0.84 0.68 0.68 a -2 10.6 25 4.66 20.3 3.66 71.6 -3.33 31 19 b 1.28 12.4 7.85 11.28 13 10.8 0.71 16.42 11.14 7.42 SE-Y 1.36 6.02 6.69 6.44 16.2 7.65 1.60 13.94 14.55 9.70 SE-X 0.34 1.54 1.72 1.65 4.16 1.96 0.41 3.58 3.73 2.49

4.8. Larvicidal Activity of Neem Leaves Extracts on M. d. vicina (Season 3) 4.8.1 Aqueous Extract The results of the larvicidal activity of neem leaves extracts against housefly at the third season (S3; 2015) was presented in (Table, 4.8). The mortality was 6% at the end of the 6th day in control. In the lower concentration (0.25%) mortality was increased from 6% to 80%, whereas the concentration (1%) resulted in 48% mortality, while the concentrations of (0.5%) and of (2.5%) resulted in less than 40% mortality at the end of the test period. 4.8.2 Methanol extract Table (4.8), showed that, in the lower concentration (0.25%) mortality increased from 0% to 96% at the 6th day, whereas, the mortality in the concentration of (0.5%) increased to 40% at the end of the test period, while the concentration of (1%) caused 42% mortality at the 6th day. The higher concentration (2.5%) resulted in 44% mortality at the 6th day. The concentrations 2.5%, 1.0% and 0.5% resulted in less than 50% mortality (44%, 42% and 40%, respectively). 4.8.3 Hexane extract Table (4.8), showed that, in the lower concentration (0.25%) mortality increased from 0% to 52% at the 6th day, whereas, the mortality at the concentration (0.5%) increased from 42% to 96% at the end of the test period. The concentration (1%) resulted in 52% mortality at the 6th day, while the higher concentration (2.5%) caused 94% mortality at the end of the test period. In comparison, the higher mortality (96%) at the end of the test period was caused by the concentration of (0.5%), followed by the concentration (2.5%) which resulted in 94% mortality; the concentration (0.25%) and (1%) resulted in 52% mortality, while the control resulted in 6% mortality. At the third season (S3), neem aqueous extract scored a maximum of 80% mortality on housefly larvae, whereas the methanol extract scored a maximum of 92% mortality and the hexane extract scored a maximum of 96% mortality. The results of S1 and S2 did not differ greatly from these of S3, with the consideration of diluting the concentrations at S3. Also, it was observed that, there were no consistency between concentrations and mortalities on housefly larvae at these lower concentrations.

Table (4.8) Effect of neem leaves extracts on the mortality of M. d. vicina larvae (S3: 2015)

Aqueous extract Methanol extract Hexane extract Day C Concentrations % 0.25 0.5 1 2.5 0.25 0.5 1 2.5 0.25 0.5 1 2.5 1 0 6 6 14 14 0 6 30 4 0 42 18 18 2 2 56 26 34 14 40 22 40 4 12 76 18 76 3 2 76 34 40 14 60 36 42 8 42 96 34 92 4 6 80 38 42 14 76 40 42 8 50 96 38 94 5 6 80 38 48 14 88 40 42 18 52 96 50 94 6 6 80 38 48 20 92 40 42 44 52 96 52 94

Regression Analysis

R2 0.86 0.66 0.71 0.80 0.43 0.87 0.68 0.47 0.81 0.82 0.65 0.94 0.59 A 0.86 18.4 10 16.26 12 4.57 11.7 34.2 -11.42 -4.13 50.66 8 34.4 B 1.31 12.74 5.71 6.11 0.85 15 5.07 1.42 7.5 11.08 9.42 7.71 12.46 SEY 1.05 17.98 7.04 5.83 1.92 11.42 6.84 2.99 7.24 9.92 13.55 3.62 20.03 SEX 0.27 4.61 1.81 1.50 0.49 2.55 1.53 0.67 1.62 2.55 3.48 0.93 5.14

4.9 Larvicidal Activity of I. helderbranditti Leaves on M. d. vicina (Season 3) 4.9.1. Aqueous Extract The mortality in the lower concentration (0.25%) increased from 14% to 16% at the 6th day, whereas, the mortality in the concentration (0.5%) increased to 100% as same as the concentration (1.0%) at the end of the test period. In the higher concentration (2.5%) the mortality reached 100% at the first day of the test period (Table 4.9). 4.9.2 Methanol extract Table (4.9) also showed that, the mortality was 6% at the 6th day in the control, while in the lower concentration (0.25%) mortality increased to 88% at the 6th day, whereas, the mortality in the concentration (0.5%) increased to 100% at the end of the test period, while the concentration (1%), caused an increase in mortality to 90% at the 6th day. In the higher concentration (2.5%), the mortality increased to 14% at the end of the period. It was obvious that, the highest mortality (100%) at the end of the test period was caused by the concentration of (0.5%) and of (1%) resulted in almost 90%, the concentration (0.25%) resulted in 88%, and the concentration (2.5%) resulted in 14% mortality. 4.9.3 Hexane extract Table (4.9), showed that, the mortality was 6% at the 6th days in the control. In the lower concentration (0.25%) mortality increased from 30% to 70% at the 6th day, whereas, the mortality at the concentration (0.5%) increased to 54%, the concentration of (1%), increased to 82% and the concentration (2.5%) increase to 98% at the end of the test period. It was clear that, the highest mortality (98%) resulted from the concentration (2.5%), followed by the concentration of (1%) which resulted in 82% mortality, the concentration (0.25%) resulted in 70% mortality, the concentration (0.5%) resulted in 54% mortality. In S2, the aqueous extract exerted a maximum of 95% mortality in housefly larvae, whereas, the methanol extract caused 100% mortality, while and the hexane extract showed a maximum of 90% mortality (the aqueous and the methanol extracts exerted relatively higher toxic effect against housefly larvae than the hexane extract at both the S1 and the S2 seasons). The inconsistence of concentrations and mortalities of the I. helderbranditti leaves extracts (aqueous, methanol and hexane) in the housefly larvae, observed in S1 and S2 was also observed in S3.

Table (4.9) Effect of I. helderbranditti leaves extracts on the mortality of M. d. vicina larvae (S3: 2015)

Aqueous extract Methanol extract Hexane extract Day C Concentrations % 0.25 0.5 1 2.5 0.25 0.5 1 2.5 0.25 0.5 1 2.5 1 0 14 44 50 100 24 8 44 10 30 20 54 22 2 2 16 64 68 100 84 38 60 10 36 28 76 58 3 2 16 70 74 100 84 54 60 14 46 34 78 68 4 6 16 82 88 100 84 72 64 14 60 44 78 74 5 6 16 98 90 100 88 100 78 14 70 52 78 90 6 6 16 100 96 100 88 100 88 14 70 54 80 94

Regression Analysis

R2 0.80 0.37 0.96 0.93 1 0.45 0.91 0.95 0.62 0.90 0.94 0.56 0.88 a -0.28 14.86 36.93 49.142 100 48 3.71 38.8 10 24.28 16.28 62 26 b 1.071 0.21 11.26 7.93 0 7.28 15.93 7.57 0.71 7.57 6.14 3.28 11.5 SEY 1.060 0.55 4.21 4.39 0 16.1s 9.84 3.58 1.10 4.79 3.17 5.86 8.43 SEX 0.236 0.124 1.08 0.98 0 3.62 2.20 0.80 0.25 1.07 0.71 1.31 1.88

4.10 Larvicidal Activity of A. nilotica Leaves Extracts on M. d. vicina (Season 3) 4.10.1. Aqueous Extract Table (4.10), showed that the mortality was 6% at the 6th day in the control. In the lower concentration (0.25%) mortality increased to 90% at the 6th day, whereas, the mortality in the concentration (0.5%) increased to 100% at the 6th, the concentration (1%), resulted in an increase in mortality to 98%, while the higher concentration (2.5%) resulted in 88% mortality. The highest mortality (100%) caused by the concentration (0.5%), followed by the concentration (1%) which resulted in 98% and the concentration (0.25%) resulted in 90%, while the concentration (2.5%) resulted in 88% mortality. 4.10.2 Methanol extract Table (4.10), showed that, in the lower concentration (0.25%) mortality increased to 30% at the 6th day, whereas, the mortality at the concentration (0.5%) increased to 80% at the end of the 6th day, while the concentration (1%) showed an increase in mortality to 70%. The higher concentration (2.5%) resulted in 96% mortality. 4.10.3 Hexane extract Table, (4.10), also showed that, the mortality produced by the lower concentration (0.25%) of hexane extract of A. nilotica leaves increased to 72%. The lower concentrations (0.5%) resulted in 64% mortality, while the concentration (1%) showed an increase in mortality from 26% to 70% at the 6th day. The higher concentration (2.5%) resulted in 100% mortality. In the S3, the aqueous extract of A. nilotica leaves exerted a maximum of 100% mortality in housefly larvae, The methanol extract caused a maximum of 92% mortality, while the hexane extract resulted in a maximum of 100% mortality. In the second season (S2), the aqueous extract of A. nilotica leaves exerted a maximum toxic effect against housefly larvae of 80% mortality, the methanol and the hexane extracts a maximum of 85% mortality, compared to 95%, 100% and 45%, following the same order, in the S1. An inconsistence in concentrations and their corresponding mortalities of the tested leaves extracts (aqueous, methanol and hexane) in the housefly larvae, was observed clearly.

Table (4.10) Effect of A. nilotica leaves extracts on the mortality of M. d. vicina larvae (S3: 2015) Aqueous extract Methanol extract Hexane extract Day C Concentrations % 0.25 0.5 1 2.5 0.25 0.5 1 2.5 0.25 0.5 1 2.5 1 0 10 4 0 34 6 2 6 46 30 24 26 80 2 2 46 46 2 56 18 14 6 66 66 36 44 80 3 2 82 98 48 86 22 58 82 92 68 56 60 82 4 6 100 98 68 90 24 80 88 92 68 64 68 84 5 6 100 98 76 90 24 80 88 92 68 64 68 92 6 6 100 98 84 90 26 80 92 92 68 64 70 100

Regression Analysis

R2 0.80 0.74 0.63 0.89 0.67 0.80 0.77 0.69 0.68 0.47 0.74 0.77 0.89 a -0.28 20.28 22 -12.86 42.28 8.57 0.86 2.57 53.43 44.28 26.86 30.57 72.57 b 1.071 14.14 13.79 16.28 8.57 3.21 13.86 15.57 7.21 4.64 6.57 6.86 3.93 SEY 1.06 16.86 21.29 11.62 11.88 3.13 15.17 20.62 9.93 9.82 7.69 7.51 2.70 SEX 0.25 3.77 4.76 2.50 2.66 0.70 3.39 4.61 2.22 2.20 1.72 1.68 0.60

Concerning the concentration of (1%) as a common concentration in all extracts at the three seasons, Table (4.11) and Figure (4.1) showed the effect of aqueous, methanol and hexane leaves extracts of the selected plant-leaves on the mortality % of Musca d. vicina larvae in three seasons (S1: 2013, S2:2014, and S3: 2015). Neem and Ipomoea (Ip) leaves aqueous-extract (Aq-Extr), and Acacia (Ac) methanol- extract (Me-Extr) produced 100% mortality in housefly larvae, followed by the methanol-extract of Ipomoea (95%), neem (Az) methanol-extract (85%), Acacia aqueous extract (70%) at the first season (S1). The rest (all hexane extracts; He-Extr.) of the botanical preparations resulted in 50% mortality or less. At the second season (S2), relatively similar finding was noticed (the difference did not exceed 3 larvae anywhere; a mortality of 15%), except in the hexane-extract that showed a lethal effect ranged between 65%-80% (and this finding may be attributed to the active ingredients produced in those plants at that period). At S3, the toxicity of the polar (aqueous and methanol) extract of neem leaves decreased by about half, than S2, but the toxicity of the apolar extract (hexane) did not affected greatly (the difference was about 13%). On the other hand, Ipomoea leaves exerted more toxicity in the polar and in the apolar extracts than S2, while in Acacia the toxicity did not differ greatly in the all extracts (not more than 11% than S2) The statistical analysis revealed that, the average mortality during S1 was 71.67%, during S2 was 83.33, while it was 67.11% during S3. Although, there were clear differences between mortalities resulted during the different seasons, but ANOVA (two factors) showed a non-significant difference in this level (f=0.86, f- crit= 3.63), i.e. the obvious differences in the average mortality during S1, S2 and S3 in respect to the concentration of (1%) of the leaves extract (aqueous, methanol and hexane) of the selected plants were statistically not significant.

Table (4.11) The Overall Effect of the Concentration (1%) of each Extract on the Mortality of M. d. vicina larvae in Seasons 1, 2 and 3 at the 6th day: % Mortality Season Aq-Extr Me-Extr He-Extr AZ Ac Ip AZ Ac Ip AZ Ac Ip S1 100 70 100 85 100 95 50 45 0 S2 100 80 85 90 85 100 65 80 65 S3 48 84 96 42 92 88 52 70 80

SUMMARY Count Sum Average Variance S1 9 645 71.67 1181.25 S2 9 750 83.33 162.5 S3 9 652 72.44 881.11

Aqueous -AZ 3 248 82.67 901.33 Aqueous-Ac 3 234 78 201.33 Aqueous-Ip 3 281 93.67 75 Methanol-AZ 3 217 72.33 637 Methanol-Ac 3 277 92.33 60.33 Methanol-Ip 3 283 94.33 2330.33 Hexane-AZ 3 167 55.67 66.33 Hexane-Ac 3 195 65 325 Hexane –Ip 3 145 48.33 1873

Source SS Df MS F P-value F crit Rows 1260.07 2 630.04 0.86 0.44 3.63 Columns 6119.63 8 764.95 1.05 0.44 2.59 Error 11679.26 16 729.95 Total 19058.96 26

100

90 S3, 80

40 Mortality percentage Mortality 30

0 IpHe Ac-He AZ-He Ip-Me Ac-Me AZ-Me Ip-Aq Ac-Aq AZ-Aq

Figure (4.1) Mortalities (%) of Housefly Larvae Fed on neem (AZ), Ipomoea (IP) and Acaccia (Ac) Leaves Aqueous (Aq), Methanol (Me) and Hexane (He) Extracts after 6 days at concentration (1%).

The same Table (4.11) also showed that, the methanol-extract of Ipomoea resulted in the highest average mortality (94.33%) among all other plant preparations, followed by the aqueous –extract of Ipomoea (93.67%), the methanol-extract of Acacia (92.33%), the aqueous-extract of neem (82.67%), the aqueous-extract of Acacia (78%), the methanol-extract of neem (72.33), the hexane-extract of Acacia (65%), the hexane-extract of neem (55.67%) and at last the hexane extract of Ipomoea (48.33). Although, there were clear differences between mortalities resulted from the different plant- extracts, but ANOVA (two factors) showed a non-significant difference in this level (f=1.05, f- crit= 2.59), i.e. the obvious differences in the average mortality of different plant preparations in respect to the concentration of (1%) were statistically not significant (each are statistically similar in their toxicities). It was clear that, the polar extracts (aqueous and methanol) exerted more toxicity than the apolar (hexane-extract) towards housefly larvae, during the three seasons irrespective of the special habitat of the plants or the housefly larvae during the periods of 2013 to 2015. The reason of the larval death may be due to the poisoning of the larvae or of hunger as a result of not feeding (antifeedant effect). According to Pankaj and Anita, (2010) and Mustafa and Al Khazraji (2008) Azadirachta excels Jack showed excellent larvicidal properties at low concentrations against Culex pipiens molestus. The present research shows that Neem extract kills insects in its immature stage of development and if the larvae succeed to pupate, then insecticide convert some of the pupae in deformed conditions which become unable to produce adult insects. If the pupae succeeded to produce the adult insect, then adults laid limited number of eggs and the eggs later failed to hatch. This result is similar and typically to Jahan and Syed (2011) Many reports proved that Neem have strong efficacy against the insect emergence, reproduction and many biological and biochemical activities. There is no doubt to say that Neem and its products is a best replacement of synthetic conventional pesticides. Many plants have been reported about their potential insecticidal actions on larvae and/or adults of houseflies via crude extracts or extracted active compounds Schmutterer (1990), extracts from the Azadirachta indica (Meliaceae) seeds (Isman et al., 1990). Jahan et al, (1990) reported that, preimaginal stages of houseflies failed to emerge after treatment with Margosan-O (0.03% Azadirachtin).

Our finding is in accordance with the previous report (Jahan et al., 1990) as larvae, treated with Neem extract, were failed to come out of the pupal case. Matemu and Mosha (1986) reported that methanolic neem extract at 100 ppm caused 80% mortality when used against adults of both Anopheles gambiae and Culex quinquefasciatus. Chavan et al., (1979) found that neem fruit extract at 200 ppm resulted in 73 % mortality when used against the adult of C. guingue fatigans mosquitoes. Abdaldafae (2009) found that the neem leaves powder has a lethal effect on the Anopheles larvae. Breama (2005) reported that neem seed powder extract proved to be effective against adult and larval stages of house fly and also the study showed that the larvae were also capable of taking the active ingredients from the treated surfaces and die within 24 hrs. All parts of neem tree contain bitter compounds that often have an antifeedant effect and can interfere with hormonal processes in insects. Extracts from the neem are often mixed with seeds to protect it against insects upon storage Boeke et al (1990). The neem based pesticides do not rapidly knockdown insects, it might be an advantage to be used as an alternative pesticides for control of vector-borne diseases without any side effects and are environmentally safe. The future repellents mark an addition departure because they could be incorporated into time release system that put active ingredients into the air. The main action of crude neem leaf extract was found to prevent the host seeking vectors from landing on or biting animals (Arunpandiyan, 2011). In the present study neem leaf extract showed promising insecticidal activity against house fly M. d. vicina. Repellent properties of natural oils and various plant extracts on M. domestica have been documented by Braverman and Hogsette (2001). There was a considerable variation in the repellent action of the different botanicals used in the present study and this may reflect the complexity of the phytochemical composition of the materials tested. For instance, were found to induce high repellency to the house fly larvae in season three aqueous extract at highest concentration 2.5% (20% mortality) compared with low concentration and methanolic extract also at high concentration 2.5% (44%mortality), and the repellent action showed at season one in hexane extract at high concentration 7% (35% mortality).this result agreed with Sameh et al (2012). The chloroform extract of Curcuma longa was reported to act as repellent to Tribolium castaneum (Herbst) adults (Abida et al., 2010).

Crude plant extracts contain several phytochemicals of different biopesticidal activity. The active ingredients responsible for potency are usually present in very little concentrations compared to those of traditional synthetic pesticides. Such difference has to be taken into account when comparing biocidal activity of botanicals with chemical pesticides (Sameh et al, 2012). The Seeds and leaves of A. nilotica were reported to contain various active ingredients of multiuse (Pande et al., 1981; Chaubal et al., 2005). Therefore, leaves and fruits extracts of A. nilotica have proved to be effective as fungicides, bactericides, molluscicides and insecticides (Umalkar et al., 1976; Fagg and Greaves, 1990; Chaubal et al., 2005). The results of Ali (1987) on the insecticidal effects of A. nilotica fruits (Garad) water extract against some Culex and Anopheles species. Chaubal et al. (2005) recorded chronic larval toxicity from A. nilotica acetone extracts against Aedes aegypti and Culex quinquefaciatus. More recently, comparable results were achieved in Saudi Arabia by Zaitoun et al. (2012), who showed acute (212.1 ppm) and chronic (144.2ppm) effects of A. nilotica acetone extract against Culex pipienis, which induced 93.33% larval mortality plus reduction of egg hatchability and suppression of adult emergence. Moreover, some studies proved the potential activities of A. nilotica fruit pods in controlling some aquatic pests such as schistosomiasis transmitting snails, which were found to be affected mainly by saponins, tannins and terpenoids (Ayoub and Yankov, 1984; Fagg and Greaves, 1990). In our results the c r u d e extract was found to be quite effective against M. d. vicina larvae in aqueous extract as 95% mortality was recorded at the concentration 7%, and 90%, 70% mortality at concentrations 5% and 1% respectively in all the three larval instars at season one, while at season two high mortality was 80% recorded at concentration 1% and 65% mortality at 5% and 7%. While in season three the highest mortalities were 100%, 98%, 90% and 88% at the concentrations 0.5%, 1%, 0.25% and 2.5% respectively Table (4).Whereas methanolic and hexane extracts at season one, two and three resulted different mortalities between (100% – 30% mortality/day) at different concentrations (Table 5 and 6).Thus, the reason of the larval death may be due to the poisoning of the larvae or of hunger as a result of not feeding (antifeedant effect). The same mentioned result of Neem was shown by crude extract with different solvents of Acaccia nilotica kill insects in their immature stage of development and if the larvae succeed to pupate, then insecticide convert some of the pupae in deformed conditions which

68 become unable to produce adult insects. If the pupae succeeded to produce the adult insects then adults laid limited number of eggs and the eggs later failed to hatch. This result similar and typically to Jahan and Syed (2011). Azam et al (2002) tested leaf and seed extracts of Acacia nilotica, Annona squamosa, Azadirachta indica, Boswellia sacra, Crotolaria juncea, Jatropa dhofarica, Myrtus communis, and Suaeda aegyptiaca to determine their efficacy against nymphs of whitefly Bemisia tabaci. The plant extracts were tested at 1.0, 1.5, 2.0, and 2.5% concentrations. The mortalities of whitefly nymphs at different concentrations were significant at <0.05. The highest mortality (41%) was caused by J. dhofarica but was at par with Acacia nilotica (40%), Annona squamosa (31.63%), and Azadirachta indica (30.85%). J. dhofarica and Acacia nilotica caused the highest mortality at 1.5% followed by Annona squamosa and Azadirachta indica at 2%. Al-Lawati et al. (2002) tested eight plants local to Oman, namely Qarat (Acacia nilotica), Mustafal (Annona squamosa), Shereesh (Azadirachta indica), Luban (Boswellia sacra), Kheshkhash (Crotolaria juncea [Crotalaria juncea]), Zebrot (Jatropha dhofarica), Yas (Myrtus communis) and Suwwad (Suaeda aegyptiaca) were prepared by steeping shaded dried leaf or seed powder of each plant in water and solvent (methanol or ethanol). The extracts were tested for their insecticidal and repellent properties against the pulse beetle, Callosobruchus chinensis. The extracts from the seeds of Annona squamosa recorded 100% mortality of beetles within twenty and four hours of their exposure to methanol and ethanol extracts, respectively. The other extracts that caused high mortality were from Acacia nilotica, Crotalaria juncea, Myrtus communis and Suaeda aegyptiaca in methanol and Boswellia sacra, Jatropha dhofarica, Suaeda aegyptiaca and commercial neem in ethanol. The extracts o f Myrtus communis in methanol were highly repellent to the beetles compared to other extracts. Legume seeds treated with extracts of Annona squamosa were not repellent; rather the beetles were attracted to them. Dwivedi et al. (2000) examined the acetone extracts of leaves of four plants, Jasminum arbores, Eucalyptus rudis, Bignomia carpreolata and Acacia nilotica against A. aegypti. The extracts proved to be potent larvicides causing complete developmental arrest of the fourth instar larvae. Acetone, ethyl acetate and petroleum ether extracts of Acacia nilotica showed high larvicidal effects against different mosquito species (Chaubal et al., 2005 ) .

The petroleum ether extracts of Acacia nilotica, Nerium oleander and Artemisia annua were also reported to exert superior mortality effects as mosquito larvicides (Sharma et al., 2006; Tonk et al., 2006; Sakthivadivel and Daniel, 2008 and Abdul Rahuman et al., 2008). Nabawy et al. (2011) reported the LC50 of A. nilotica caused a highly significant reduction in the average larval period compared to controls and the treatment by A. nilotica of house fly the pupation was decreased as the concentration of plant leaves powder was incresed. All LC's of P. zonale, C. rotundus, A. nilotica and C. macrocarpa, L. camara caused a highly significant decrease in fecundity of adult females and the tested plant powders showed a highly significant decrease in the egg hatching (Nabawy et al 2011). Nabawy et al (2011) reported that A. nilotica powders are harmful to M. domestica, not only reducing longevity of adults but also decreasing their reproductive potential. A. nilotica show effective IGR-like activities and exhibit great promise in suppressing populations of M. domestica (Nabawy et al., 2011) Repellent properties of natural oils and various plant extracts on M. d. vicina have been documented by Braverman and Hogsette (2001). There was a considerable variation in the repellent action of the different botanicals used in the present study and this may reflect the complexity of the phytochemical composition of the materials tested. For instance, were found to induce high repellency to the house fly larvae in season one hexane extract at all concentration (45% mortality) compared with second and third season, this result was similar to Sameh., et al (2012). The c r u d e extract was found to be quite effective against M. d. vicina larvae as 100% mortality was observed at the concentration 1% at season one and at the concentration 1% and 2.5% in all the three larval instars at season three. While another total mortality between (16% to 90%) at the different concentrations and different seasons at aqueous extract .Whereas methanolic and hexane extracts at season one, two and three resulted in different mortality between (100% to 0% mortality/day) at different concentrations. Thus, the reason of the larval death may be due to the poisoning the larvae or of hunger as a result of not feeding (antifeedant effect) according to Pankaj and Anita (2010) Kulkami (1998) reported that the methanolic leaf extracts of Ipomoea carnea subsp against larvae of the poplar pest Clostera cupreata under laboratory conditions, with Feeding method. The results indicated that leaf extracts were the most effective deterrent.

Saxena and Sumithra (1985) noted that the leaf extracts of I. carnea fistulosa significantly increased the average larval and pupal development periods of the mosquito, C. quinquefasciatus and also resulted in increased larval and pupal mortality. acetone extracts of I. carnea fistula exhibited excellent IGR activity against An. stephensi with average larval, pupal, developmental periods and growth index of 18.93, 6.36; 25.29 (days) and 0.94, compared to control of 14.44, 3.58, 18.02 (days) and 5.54 (Saxene and Sumithra, 1985). Elija et al (2014) reported that the crude extracts and pure compound of I. carnea stem showed potent larvicidal activity against C. quinquefasciatus and A. aegypti. The isolated compounds as well as extracts were highly effective at lower concentration against the mosquitoes (Elija et al 2014). Saifuddin et al (2006) reported the aqueous extract of I. carnea (500 mg/kg) exhibited significant anti- inflammatory activity at 3rd hour against carrageenan-induced rat paw edema. The result showed that the highest repellency to the house fly larvae in season three aqueous extract at all concentrations in season one (0% mortality) at hexane extract compared with other treatment. Also the repellent action showed at season three in methanolic extract at high concentration 2.5% (14% mortality).and this result agreed with Sameh et al (2012). In the present study, the application of all concentrations of Neem, Ipomea and A. nilotica leaves extract against M. d. vicina induced different morphological abnormalities in the dead larvae and on the pupae and adults that emerged from the survived larvae. Considerable number of larvae, pupae and adults showed obvious malformations after the treatment of larvae with plant crude extracts. Malformations include completed darkened larvae, curved larvae, irregular-shaped larvae, swelling larvae, larvae with patches of cuticle melanization, larval-pupal intermediates, compressed and shrinkage pupae, dry and darkened pupae, C-shaped pupa, peanut shaped pupa, and small sized pupae. Many adults could not emerge completely and remained concealed in the puparia. Other adults with defective wings, and deformed abdomen were also observed (Plates 1 (normal shapes), 2, 3 and 4 (abnormal shapes). The morphological aberrations induced by plant crude extracts were concentration dependant, in almost all cases, the higher concentration the more morphogenetic aberrations. Adamski et al. (2005) observed that the degree of malformation was directly proportional to the concentration of pesticides. Khalaf et al. (2009) reported that, the essential oil of C. macrocarpa had been reported to produced clear morphological abonrmalities in S. nudiseta.

Plate (1): The Normal Shape of house fly:

a- Larva

b- Pupa

c- Adult

A B C

D E F

G H I

Plate (2): Abnormal larvae, pupa and adult of M. d. vicina treated with Neem extracts:- A: pyramid shape larvae ( He-extr); B: stretched shape larvae (aq-extr); C: C-shaped larvae (Me-extr), D: compressed shape pupa (He-extr); E: twisted shape pupa (aq-extr); F: abnormal shape pupa (Me-extr), G: legs lost Adult (He-extr); H: stretched wing Adult (aq-extr); I: shrinkaged legs Adult (Me-extr).

A B C

D E F

G H I

Plate (3): Abnormal Larvae, Pupa and Adult of M. d. vicina treated with A. nilotica extracts:- A: swelled shape larvae (He-extr); B: Twisted larvae (Aq-extr); C: carved larvae (Me-extr), D: pupal-adult (He-extr); E: compressed pupa (Aq-extr); F: transparent normal shape pupa (Me-extr), G: head lost Adult (He-extr); H: shrinkaged Adult (aq-extr); I: legs stretched Adult (Me-extr).

A B C

D E F

G H I

Plate (4): Abnormal Larvae, Pupa and Adult of M. d.vicina treated by Ipomoea extracts: A: Twisted shape larvae (He-extr); B: partial shrinkaged shape larvae (Aq-extr); C: fossiled larvae (Me-extr), D: larval-pupa (He-extr); E: stretched pupa (Aq-extr); F: swelled fossiled pupa (Me-extr), G: short right wing and stretched left wing Adult (He-extr); H: blackened back Adult (Aq-extr); I: abdomen shrinkage Adult (Me-extr).

Some deformed larvae were pigmented and larval-pupal intermediate, the resultant some individuals showed C-shaped pupae, elongated pupae and balloon shaped pupae, most of the pupae failed to reach adults, however, some emerged adults have various degrees of morphological abnormalities. Similar abnormalities were reported by Hashem and Youssef

(1991), they observed dark inter segmental pigments on the 3rd larvae of M. domestica and fully formed pupa but with a constricted puparium after treatment of the 1st instar larvae with methanolic extraction of leaves and flowers of M. azedarach. Bakr et,al. (2003) found larval pupal intermediate as a result of treatment of M. domestica larvae with A. monosperma, C. inerme and C. antiqorum. El-Domiaty et al. (2003) found shrinkage of the pupae and folding of the wing of adults as a result of treatment of 3rd instar larvae of M. domestica with P. nigra volatile oil. Sripongpun (2008) observed small sized pupae (1 mm wide x 3 mm long) after treated M. domestica larvae with the extract of Chinese star anise fruits, while the size of the control ones was 2 mm wide x 5 mm long. In addition, the number of small pupae developed to adults was less than that of normal one. Emerging of adults with malformed wings may be attributed to the failure of the wings to expand and flatten after adult emergence (Saxena et al., 1981). Aly et al. (2010) attributed to to the adult malformation of S. gregaria to the intervening of F. bruguieri extracts with the hormonally controlled program of morphogenesis. This may be due to the modification of the ecdysteroid titer, which in turn leads to changes in lysosomal enzyme activity causing overt morphological abnormalities (Josephrajkumar et al., 1999). Sexena et al. (1981) reported developmental abnormalities in larvae of Cnaphalocrocis medinalis after treatment with 50% neem oil. Various morphological abnormalities on larvae, pupae, and adult stages induced by using essential oils against Culex pipiens, Lucilia sericata and M. domestica were detected by Khater and Shalaby (2008), Khater and Khater (2009) and Mansour et al. (2011), respectively. The abnormalities could be attributed to the metamorphosis inhibiting effect of the essential oils, as a result of the disturbance of hormonal control. Khater and Khater (2009) suggested that the larviform puparia could be caused by the failure of larvae to contract to the pupal form, as a result of muscle paralysis, but their ability to acquire melanization of the pupal cuticle is attributed to the continuation of the enzymatic process of tanning.

The common house fly, M. d. vicina is a well known, cosmopolitan insect, which shares man his environment and is considered as one of the major insect vectors which transmits and disseminates different human pathogens, particularly in temperate and tropical countries. Accordingly, this insect may pause public health problems. In addition to their role in disease transmission (Khatter, 2013), flies are usually regarded as indicator organisms, symptomatic of disposal problems and reflecting the sanitary level of the community. In the absence of valid statistical data, bacteriological information about an essential health situation. We know almost nothing about the danger level of a fly population nor do we have well-tested criteria, as there are for Anopheles and malaria, for evaluating the vector role of the fly. The complexities and uniqueness of enzootic and endemic situations involving flies make generalization hazardous and often of limited value. The biggest gap in the logical development of incriminating evidence against flies is that which exists between our knowledge of the ability of flies to transmit and the actuality that they do (Greenberg, 1973).

CHAPTER FIVE CONCULSIONS AND RECOMMENDATION 5.1 Conclusions: The phytochemical screening revealed the presence of flavonoids in the neem, El aweer and Sunut leaves, while steroids and saponins were found only in neem, tannins was detected in the Sunut, alkaloids in El aweer and steroid only in neem. The neem-leaves aqueous extract exerted a high toxic effect against housefly larvae than methanol and hexane extracts, while the Ipomoae –leaves aqueous and methanol extracts exerted a toxic effect against housefly larvae more than the hexane extract, whereas the methanol and aqueous extracts of A. nilotica leaves exerted a toxic effect against housefly larvae more than the hexane extract in the first season. The larvicidal effects observed in Season (S2) and Season (S3) was not far greatly from Season (S1), the odd cases may be referred to the seasonality (environmental factors) and phytochemical presents in each season. The cases inconsistently of concentrations and mortalities of the neem and I. helderbranditti leaves extracts (aqueous, methanol and hexane) in the housefly larvae that observed in S1, S2 and in S3, may be due to antifeedant properties of this leaves. The larvicidal effects observed in S1 and S2 did not differ greatly than these of S3, with the consideration of diluting the concentrations at S3. Also it was observed that, there were no consistency between concentrations and mortalities on housefly larvae at these diluted concentrations. The different extracts led to abnormalities in the dead larvae (e.g: stretched shape, C-shaped) and on the formed pupae (e.g: twisted, compressed shape) and also on the emerged adults (e.g: shrinkaged, blackened back). The aqueous leaves extract of Neem, Elaweer and Sunut resulted in high toxic effects against housefly larvae. In addition to that, the aqueous extract easily to be prepared, available, applicable, safer and economically feasible compared to other solvents. 5.2 Recommendations: 1- The tested plant products could be used for controlling the housefly and should be exploited as a component of IPM strategies assuming that they tend to be safe for the environment. 2- Specifying the optimum concentration of each extract to achieve 100% mortality must be run. 3- Other plant products should be tested similarly. 4- Aqueous extracts can be applied to control of house flies at home garbage.

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