The Lethal Impact of Ixora coccinea L. Extracts on Anopheles and culex Larvae and some of their Aquatic Predators
Sabah Abd Alla Birama Omer
B.Sc. (Biology), University of the Holy Quran (2008)
Post Graduate Diploma in Biosciences and Biotechnology, University of Gezira (2010)
M.Sc. in Biosciences and Biotechnology, University of Gezira (2012)
A Thesis
Submitted to the University of Gezira in Fulfillment of the Requirements for the Award of the Degree of
Doctor of Philosophy
in
Biosciences and Biotechnology (Applied Entomology)
Center of Biosciences and Biotechnology
Faculty of Engineering and Technology
February, 2017
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The Lethal Impact of Ixora coccinea L. Extracts on Anopheles and culex Larvae and some of their Aquatic Predators
Sabah Abd Alla Birama Omer
Supervision Committee
Name Position Signature
Dr. Mutaman Ali A. Kehail Main supervisor ………………….
Dr. Siddig Gabriel Rahoud Co-supervisor ………………….
Data: February, 2017
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The Lethal Impact of Ixora coccinea L. Extracts on Anopheles and culex Larvae and some of their Aquatic Predators
Sabah Abd Alla Birama Omer
Examination Committee
Name Position Signature
Dr. Mutaman Ali A. Kehail Chairperson ………………….
Prof. Elamin Mohamed Elamin External Examiner ………………….
Dr. Faiza Elgaili Elhassan Salah Internal Examiner ………………….
Date : February, 2017
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DEDICATION
To my Family
Friends
Teachers
Sabah
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ACKNOWLEDGEMENTS
My praises and thanks to (Allah) the most gracious the most merciful who granted me the health and patience to conclude this study.
Special thanks and gratitude to my main supervisor Dr. Mutaman A. A. Kehial for their valuable guidance and help throughout the work and writing of dissertation.
Also thanks extended to the technicians in the University of the Holy Quran.
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The Lethal Impact of Ixora coccinea L. Extracts on Anopheles and culex Larvae and some of their Aquatic Predators
Sabah Abd Alla Birama Omer
Ph.D in Biosciences and Biotechnology (Applied Entomology) February 2017
Abstract
The use of insecticides in Gezira Scheme resulted in many environmental problems (specially on aquatic predators that naturally control mosquitoes larvae) in addition to that, many strains of mosquitoes developed resistance to these insecticides. This study aimed to investigate the impact of Ixora leaves and flowerss aqueous and ethanol extracts on Anopheles and culex larva and some of their of their aquatic predators during three seasons (2013 – 2015). The work involved also the determination of the phytochemical composition and natural extract of Ixora leavess and flowerss. Anopheles and Culex larvae were collected from temporary pooled water at Wad Medani City, Gezira State. The selected aquatic predators: Hemipteran boatmen, great diving beetle adults and larvae, dragonfly naiads were collected during the collection of mosquitoes, whereas, Gambusia fish were brought from the Blue Nile National institu te for Training and Research, University of Gezira. Mosquitoes larvae were provided during the bioassay test to feed the aquatic predators. The tested concentrations of aqueous leaves extracts ranged between 11.13 and 64.90 (mg/l) and of Leaves ethanol extracts between 5.91 and 45.53 (mg/l), while, thoses of aqueous flowerss extracts between 15.15 and 85.38 (mg/l), and of flowerss ethanol extracts between 5.18 and 36.76 (mg/l). Malathion was used as positive control at concentrations ranged between
0.0912 and 0.3648 (mg/L). The results showed that respective LC50 and LC95 for Hemipteran boatman were 43.22 and 261.66, for swimming beetle larvae were 29.47 and 202.36, for dragonfly naiad were 25.27 and 84.43,and for Gambusia fish were 70.95 and 545.13. The
LC50 and LC95 ‘s of the aquatic predators seemed to be relatively high compared to thoses of mosquitoes' larvae (LC50 between 14.24 and 36.96 for Anopheles larvae; 46.04 and 333.89 mg/L for Culex larvae). The ethanol extracts of Ixora leaves’s and flowerss were more potent than the aqueous extracts and both can play an important role in Anopheles and Culex control with slight effects on those aquatic predators. The study recommended running further researches to be sure of no environmental problems result’s from using the extracts of Ixora leavess and flowers for mosquitoes larval control.
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دالمردو القاتل للمستخلصات اإلكسورا على يرقات الكيلوكس واالنفوليس وبعض مفترساتها المائية
صباح عبد اهلل بريمة عمر
دكتوراه الفلسفة في العلوم والتقنية البيولوجية )حشرات تطبيقي( فبراير 2017 م
ملخص الدراسة
أدى استخدام المبيدات في مشروع الجزيرة إلي العديد من المشاكل البيئية )خصوصاً علي المفترسات المائية
والتي تقضي بصورة طبيعية علي يرقات البعوض( باإلضافة لذلك, طورت العديد من سالالت البعوض مقاومة لهذه
المبيدات. هدفت هذه الدراسة للتحقق من المردود القاتل للمستخلصات المائية واإليثانولية ألوراق وأزهار اإلكسورا علي
بعض المفترسات المائية ليرقات البعوض خالل )2013 إلي 2015(. شمل العمل تحديد التركيب الكيميائي النباتي,
وأيضاً التأثير القاتل لمستخلصات أوراق وأزهار اإلكسورا علي بعض المفترسات المائية. تم جمع يرقات األنوفلس
والكيولكس من المياه المؤقتة المتجمعة في مدينة ود مدني, والية الجزيرة. تم جمع المفترسات المائية التي اختبرت:
نصفيات األجنحة المائية, بالغات ويرقات الخنافس المائية الكبيرة, حوريات الرعاشات إثناء جمع يرقات البعوض, في حين
احضرت أسماك القمبوزيا من معهد النيل األزرق القومي للتدريب والبحوث, جامعة الجزيرة. تم إضافة يرقات البعوض
خالل تطبيق التجربة لمنع المفترسات المائية من الموت بسبب الجوع. التراكيز المختبرة للمستخلص المائي لألوراق تراوحت
بين 11.13 و 64.9 )ملجم/لتر/ماء( وللمستخلص اإليثانولي لألوراق بين 5.91 و 45.53 )ملجم/لتر(, بينما
للمستخلص المائي لألزهار تراوح بين 15.15 و 85.38 )ملجم/لتر( وللمستخلص اإليثانولي لألزهار بين 5.18 و
36.76 )ملجم/لتر(. تم إستخدام مبيد المالثيون كشاهد موجب عند تراكيز تتراوح بين 0.0912 و 0.3648
)ملجم/لتر/ماء(. أوضحت النتائج أن قيم LC50 و LC95 علي التوالي لنصفيات األجنحة المائية هي 43.22 و
261.66, وللخنافس المائية الكبيرة كانت 29.47 و 202.36, ولحوريات الرعاشات كانت 25.27 و 8.43, وألسماك
القمبوزيا كانت 70.95 و 545.13. يبدو أن قيم التراكيز الكافية للقضاء علي 50% و95% من المفترسات المائية نسبياً
عالية مقارنة مع يرقات البعوض )تراوحت LC50 بين 14.24 و 36.96 ليرقات األنوفلس و 46.04 و 333.89
)ملجم/لتر/ماء( ليرقات الكيولكس. كانت لمستخلصات االيثانول ألوراق وأزهار نبات اإلكسورا أكثر فعالية من
المستخلصات المائية وهما معاً يمكنهما لعب دور في مكافحة بعوض األنوفلس والكيولكس مع تأثير طفيف علي هذه
المفترسات المائية. توصي الدراسة بإجراءالمزيد من األبحاث الالحقة للتأكد من عدم حدوث مشاكل بيئية يمكن أن تنتج
من إستخدام مستخلصات أوراق وأزهار نبات اإلكسورا لمكافحة يرقات البعوض.
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Table of Contents
Page Dedication …………………………………………………………………… Iii Acknowledgements …………………………………………………………. Iv Abstract ……………………………………………………………………… V Arabic Abstract ……………………………………………………………... Vi Table of Contents …………………………………………………………. Vii List of Tables ………………………………………………………………… Xi List of Plates …………………………………………………………………. Xiii Chapter One : Introduction ………………………………………………. 1 Chapter Two : Literature Review ………………………………………… 3 2.1 Mosquitoes 3 2.1.1 Scientific classification 4 2.1.2 Life cycle 4 2.1.2.1 Eggs and oviposition 5 2.1.2.2 Larva 7 2.1.2.3 Pupa 8 2.1.2.4 Adult 8 2.1.3Mosquito as vector 12 2.1.4 Genetic resistance 15 2.2 Prevention 16 2.2.1 Vector control 16
2.2.3 Other methods 17 2.2.4 chemical control 18 2.2.5 Natural products 19
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Page 2.2.5.1 Oil drip 19 2.2.5.2 Soaps and oils 22
2.2.5.2.1 Insecticidal soap 22 2.2.5.3 Mineral Insecticides 23 2.2.5.1 Sulfur 23 2.2.6 Biological control of mosquitoes 23 2.2.6.1 Avian predators 23 2.2.6.2 Mammalian predators 24 2.2.6.3 Amphibian and reptilian predators 25 2.2.6.4 Insect predators 25 2.3 Aquatic predators 25 2.3.1 Dragonfly 25 2.3.1.1 Classification 26 2.3.1.2 Life cycle 27 2.3.2 Hemipteran boatman 27 2.3.2.1 Classification 27 2.3.2.2. Habitat and food 28 2.3.3 Mosquito-fish 28 2.3.3.1 Classification 29 2.3.3.2 Habitat and food 29 2.4 Ixora 30 2.4.1 Classification 31 2.4.2 Traditional use and plant parts used 32 2.4.3 Chemical composition 32 2.4.3.1 Leavess 32 2.4.3.2 Flowerss 32 2.4.3.3 Above-ground parts 32 2.4.3.4 Roots 33
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Page 2.4.4 Bioactivity 33
2.4.5 Toxicity 34 2.5 Malathion insecticide 35 2.5.1 Malathion used in mosquito control 35 Chapter Three : Materials and Methods ………………………………….. 36 3.1 The study area 36 3.2 Materials 36 3.2.1 Samples 36 3.2.2 Preparation of aqueous and ethanolic plant extracts 37 3.2.3 Preparation and dilution of Malathion 37 3.3 Methods 38 3.3.1 Toxicity test 38 3.3.2 Phytochemical screening 38 3.3.2.1 Test for Glycosides 38 3.3.2.2 Test for flavonoinds 38 3.3.2.3 Test for saponins 39 3.3.2.4 Test for tannins 39 3.3.2.5 Test for sterols and/or triterpenes 39 3.3.2.6 Test for alkaloids and/or nitrogenous bases 39 3.3.2.7 Test for coumarins 40 3.4 Proximate analysis 40 3.4.1 Moisture content 40 3.4.2 Ash content 40 3.4.3 Protein content 41 3.4.4 Crude fiber content 41 3.5 Statistical analysis 42 Chapter Four : Results and Discussion ……………………………………. 43 4.1 Toxicity test on mosquito larvae and some aquatic predators 43 4.1.1 Effect of ethanol extract of Ixora leaves (season 2013) 43
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Page 4.1.2 Effect of aqueous extract of Ixora leaves (season 2013) 45 4.3 Effect of aqueous extract of Ixora flowers (season 2013) 47 4.4 Effect of ethanol extract of Ixora flowers (season 2013) 49 4.5 Effect of Malathion insecticide (season 2013) 51 4.6 Effect of aqueous extract of Ixora leaves (season 2014) 53 4.7 Effect of aqueous extract of Ixora flowers (season 2014) 55 4.8 Effect of ethanol extract of Ixora flowers (season 2014) 57 4.9 Effect of ethanol extract of Ixora leaves (season 2014) 59 4.10 Effect of Malathion insecticide on Anopheles and Culex larvae 61 and some aquatic predators in the season 2014 4.11 Effect of ethanol extract of Ixora leaves (season 2015) 63 4.12 Effect of aqueous extract of Ixora leaves (season 2015) 65 4.13 Effect of aqueous extract of Ixora flowers (season 2014) 67 4.14 Effect of ethanol extract of Ixora flowers (season 2015) 69 4.15 Effect of Malathion insecticide on Anopheles and Culex larvae 71 and some aquatic predators in the season 2015 4.16 Samary of the LC50 OF lexora ectract on mosquito anopheles and 74 culex and some of their aquatic predators) 2013-2015( 4.17 Phytochemical composition of Ixora leaf and flowers 75 4.18 Proximate composition of Ixora leaves and flowers 75 Chapter Five : Conclusion and Recommendations 78 5.1 Conclusion 78 5.2 Recommendations . 78 References 79
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List of Tables
Table Page number 4.1 Percentage mortality of different Alcoholic extract of Ixora Leaves on Anopheles and Culex Larvae and some of their aquatic predators 44 ………………………………………………….…. 4.2 percentage mortality of different doses of Aqueose extract of Ixora Leaves on Anopheles and Culex Larvae and some of their aquatic 46 predators ……………………………………………… 4.3 percentage mortality of different doses of Aqueous extract of Ixora flowers on Anopheles and Culex larvae and some of their aquatic 48 predators 4.4 percentage mortality of different doses of Aqueous extract of Ixora flowers on Anopheles and Culex larvae and some of their aquatic 50 predators 4.5 percentage mortality of different doses of Malathion insecticides n 52 Anopheles and Culex Larvae and some of their aquatic predators 4.6 percentage mortality of different doses of Aqueous extract of Ixora Leaves on anopheles and culex larva and some of their aquatic predators. 54 4.7 percentage mortality of different doses of Aqueous extract of Ixora 56 flowers on Anopheles and Culex Larvae and some aquatic predators 4.8 percentage mortality of different doses of ethanol extract of Ixora flowers on Anopheles and Culex Larvae and some of their aquatic 58 predators 4.9 percentage mortality of different doses of ethanol extract of Ixora Leaves on Anopheles and Culex Larvae and some of their aquatic 60 predators 4.10 percentage mortality of different doses of Malathion insecticide on [ Anopheles and Culex Larvae and some of their aquatic predators 62 4.11 percentage mortality of different doses of ethanol extract of Ixora Leaves on Anopheles and Culex Larvae and some of their aquatic 64 predators 4.12 percentage mortality of different doses of Aqueose extract of Ixora Leaves on Anopheles and Culex Larvae and some of their aquatic 66 predators 4.13 percentage mortality of different doses of Aqueous extract of Ixora flowers on Anopheles and Culex Larvae and some of their aquatic 68 predators
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Table Page number 4.14 percentage mortality of different doses of ethanol extract of Ixora flowers on Anopheles and Culex Larvae and some of their aquatic 70 predators 4.15 Percentage mortality of different doses of Malathion insecticide on Anopheles and Culex Larvae and some of their aquatic predators 72 4.16 Qualitative phytochemical screening ,presence (+) and absence (-) of 74 Ixora Leaves and flowers 4.17 The nutrient contents (%) of Ixora Leaves and flowers 76 4.18 The proximate contents (%) of Ixora leaves and flowers 77
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List of Plates
Plate Page number An egg raft of a Culex species, partly broken, showing 1 6 individual egg shapes ………………………………………. 2 Mosquito’s larva and pupa …………………………………. 9 Adults of the yellow fever mosquito Aedes aegypti, a typical 3 member of the subfamily Culicinae, the male is on the left, 10 and females are on the right ………………………………… 4 Ixora coccinea L……………………………………………. 31
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CHAPTER ONE INTRODUCTION
Mosquitoes are important vectors of several tropical diseases; including malaria, filariasis, and numerous viral diseases, such as dengue, Japanese encephalitis and yellow fever, in countries with a temperate climate they are more important as nuisance pests than as vectors. There are about 3000 species of mosquitoes, of which about 100 are vectors of human diseases. Human malaria is transmitted only by females of the genus Anopheles. Of the approximately 430 Anopheles species, only 30-40 transmit malaria (vectors) in nature (Abdullah and Merdan, 1995). Since it was first shown, 100 years ago, that the malaria parasite transmitted through the bite of mosquito, it has been recognized that knowledge of the mosquito of major importance in malaria control. Mosquitoes are estimated to transmit disease in South America, Central America, Mexico, much of Asia resulting in millions of deaths, and more than 70 million people infected annually in Africa (WHO, 2005). The burden of malaria in sub-Saharan Africa remains intolerable with more than 20% of all deaths of children younger than 5 years attributed to malaria and up to 11.9 deaths per 10.000 children living in malaria endemic areas. In Sudan malaria remains a common problem and leads to over 7 million cases per year. In Gezira state malaria also is a common problem according to the ministry of health (Sid Ahmed, 2006). The introduction of irrigation in the arid areas of Gezira state produced drastic changes in the ecology. As a result of these changes, Gezira is currently the largest area in Sudan with the highest number of permanent mosquitoes breeding sites. Thirty one Anopheles species were identified from different areas of Sudan. Anopheles arabiensis is the main vector of malaria throughout the Gezira State. Insecticides were used in Gezira state for many years for controlling mosquitoes and other agricultural pests. The use of insecticides resulted in many ecological and environmental problems (Abdel Karim et al., 1985) in addition to that, many strains of mosquitoes developed resistance to these insecticides. Efforts are directed towards finding some natural products alternatives to the use of conventional insecticides. The use of some natural products against mosquitoes has been discussed by several scientists (e.g. Abd Aldafae, 2009; Kehail, 2004; Elsayed, 1992; Zarrough, et al., 1990, and Koul, 1988).
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Some garden plants naturally repel mosquitoes. Rose-scented geraniums contain the natural insect repellents citronellal and geraniol. Some gardeners report swishing their hands through the leaves is enough to deter mosquitoes. Lemon balm (Melissa officinalis), which is easy to grow from seed, contains the repellents citronellal, geraniol and geranial. The essential oil in catnip (Nepeta cataria), nepetalactone, was found to be about 10 times more effective in repelling mosquitoes than DEET (Beaty and Miller, 1999).
General objective: This study aimed to investigate the impact of Ixora leaves and flowers aqueous and ethanol extracts on the aquatic predators of mosquitoes during three seasons (2013 – 2015).
Specific objective: 1- To determine the chemical and phytochemical composition of Ixora Leaves and flowers. 2- To determine the lethal effect of aqueous and ethanol of leaves and flowers extracts of Ixora against Anopheles and Culex larvae and some of their aquatic predators . 3- To evaluate the lethal effect of Malathion insecticide (as standard) against Anopheles and Culex larvae and the selected aquatic predators.
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CHAPTER TWO LITERATURE REVIEW 2.1 Mosquitoes Mosquitoes are members of a family of nematoceran’s flies: the Culicidae (from the Latin meaning midge or gnat). The word mosquito is from the Spanish for "little fly". Mosquitoes resemble crane flies (family: Tipulidae) and chironomid flies (family: Chironomidae); as a result, casual observers seldom realize the important differences between the members of the respective families. In particular, the females of many species of mosquitoes are blood sucking pests and dangerous vectors of diseases, whereas members of the similar-looking Chironomidae and Tipulidae are not. Many species of mosquitoes are not blood eaters, and many of those that do create a "high to low pressure" in the blood to obtain it do not transmit disease. Also, in the bloodsucking species, only the females suck blood. Furthermore, even among mosquitoes that do carry important diseases, neither all species of mosquitoes, nor all strains of a given species transmit the same kinds of diseases, nor do they all transmit the diseases under the same circumstances; their habits differ. For example, some species attack people in houses, and others prefer to attack people walking in forests. Accordingly, in managing public health, knowing which species, even which strains, of mosquitoes with which one is dealing is important (Ralph, 2008). Over 3,500 species of mosquitoes have already been described from various parts of the world. Some mosquitoes that bite humans routinely act as vectors for a number of infectious diseases affecting millions of people per year. Others that do not routinely bite humans, but are the vectors for animal diseases, may become disastrous agents for zoonosis of new diseases when their habitat is disturbed, for instance by sudden deforestation (mimosq.org, 2012). Several scientists have suggested complete eradication of mosquitoes would not have serious ecological consequences. As a generalization, though, this cannot be sustained. The roles of various species in different ecologies differ greatly and many are active agents in recycling aquatic detritus or competing with other aquatic pests, for example. In practice, control measures focus on mosquito species that are vectors of human or livestock disease, or that are seriously irritant pests. Some, such as members of the genus Toxorhynchites, actually are beneficial predators of other mosquitoes (Wayne, 1989). 2.1.1 Scientific classification
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Kingdom: Animalia Phylum: Arthropoda Class: Insecta Order: Diptera Suborder: Nematocera Infraorder: Culicomorpha Superfamily: Culicoidea Family: Culicidae (Wayne, 1989).
2.1.2 Life cycle Like all flies, mosquitoes go through four stages in their life cycle: egg, larva, pupa and adult . In most species, adult females lay their eggs in stagnant water; some lay eggs near the water's edge; others attach their eggs to aquatic plants. Each species select the situation of the water into which it lays its eggs and does so according to its own ecological adaptations. Some are generalists and are not very fussy. Some breed in lakes, some in temporary puddles. Some breed in marshes, some in salt-marshes. Among those that breed in salt water, some are equally at home in fresh and salt water up to about one third the concentration of seawater, whereas others must acclimatise themselves to the saltiness. Such differences are important because certain ecological preferences keep mosquitoes away from most humans, whereas Some species of mosquitoes prefer to breed in phytotelmata (natural reservoirs on plants) such as rainwater accumulated in holes in tree trunks, or in the leaf-axils of bromeliads. Some specialize in the liquid in pitchers of particular species of pitcher plants, their larvae feeding on decaying insects that had drowned there or on the associated bacteria; the harmless genus Wyeomyia provides (W. smithii) breeds only in the pitchers of Sarracenia purpurea (Wayne, 1989). In contrast, artificial water containers, such as the odd plastic bucket, flowerspot "saucer", or discarded bottle or tire, are important breeding places for some of the most serious disease vectors, such as species of Aedes that transmit dengue and yellow fever. Some of these are disproportionately important vectors because they are well placed to pick up pathogens from humans, and to pass them on to other humans. In contrast, no matter how voracious, mosquitoes that breed and feed mainly in remote wetlands and salt marshes may well remain uninfected and seldom encounter humans to infect in turn anyway.
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The first three stages: egg, larva and pupa, are largely aquatic. These stages typically last 5–14 days, depending on the species and the ambient temperature, but there are important exceptions. Mosquitoes living in regions where some seasons are freezing or waterless spend part of the year in diapause; they delay their development, typically for months, and carry on with life only when there is enough water or warmth for their needs. For instance, Wyeomyia larvae typically get frozen into solid lumps of ice during winter and only complete their development in spring. The eggs of some species of Aedes remain unharmed in diapause if they dry out, and hatch later when they are covered by water. Eggs hatch to become larvae, which grow until they are able to change into pupae. The adult mosquito emerges from the mature pupa as it floats at the water surface. Bloodsucking mosquitoes, depending on species, gender, and weather conditions, have potential adult lifespan ranging from as little as a week to as long as several months. Some species can overwinter as adults in diapause (Ralph, 2008).
2.1.2.1 Eggs and oviposition Mosquito habits of oviposition, the ways in which they lay their eggs, vary considerably between species, and the morphologies of the eggs vary accordingly. The simplest procedure is that followed by many species of Anopheles: like many other gracile species of aquatic insects, females just fly over the water, bobbing up and down to the water surface and dropping eggs more or less singly. The bobbing behavior occurs among some other aquatic insects, as well, for example mayflies and dragonflies; it is sometimes called "dapping". The eggs of Anopheles species are roughly cigar-shaped and have floats down the sides. Females of many common species of mosquito can lay 100-200 eggs during the course of the adult phase of their lifecycle. Even with high egg and intergenerational mortality, over a period of several weeks a single successful breeding pair can create a population of thousands (mimosq.org, 2012). Some other species, for example members of the genus Mansonia, lay their eggs in arrays, attached usually to the under-surfaces of water-lily pads. Their close
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Plate (1): An egg raft of a Culex species, partly broken, showing individual egg shapes
relatives, the genus Coquillettidia, lay their eggs similarly, but not attached to plants. Instead, the eggs form layers called "rafts" that float on the water. This is a common mode of oviposition, and most species of Culex are known for the habit, which also occurs in some other genera, such as Culiseta and Uranotaenia. Anopheles eggs may on occasion cluster together on the water, too, but the clusters do not generally look much like compactly glued rafts of eggs (Ralph, 2008).
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In species that lay their eggs in rafts, rafts do not form adventitiously; the female Culex settles carefully on still water with her hind legs crossed, and as she lays the eggs one by one, she twitches to arrange them into a head-down array that sticks together to form the raft (mimosq.org, 2012). Aedes females generally drop their eggs singly, much as Anopheles do, but not as a rule into water. Instead, they lay their eggs on damp mud or other surfaces near the water's edge. Such an oviposition site commonly is the wall of a cavity such as a hollow stump or a container such as a bucket or a discarded vehicle tire. The eggs generally do not hatch until they are flooded, and they may have to withstand considerable desiccation before that happens. They are not resistant to desiccation straight after oviposition, but must develop to a suitable degree first. Once they have achieved that, however, they can enter diapause for several months if they dry out. Clutches of eggs of the majority of mosquito species hatch as soon as possible, and all the eggs in the clutch hatch at much the same time. In contrast, a batch of Aedes eggs in diapause tends to hatch irregularly over an extended period of time. This makes it much more difficult to control such species than those mosquitoes whose larvae can be killed all together as they hatch. Some Anopheles species do also behave in such a manner, though not to the same degree of sophistication (mimosq.org, 2012).
2.1.2.2 Larva The mosquito larva has a well-developed head with mouth brushes used for feeding, a large thorax with no legs, and a segmented abdomen. Larvae breathe through spiracles located on the eighth abdominal segment, or through a siphon, and therefore must come to the surface frequently. The larvae spend most of their time feeding on algae, bacteria, and other microbes in the surface microlayer. They dive below the surface only when disturbed. Larvae swim either through propulsion with their mouth brushes, or by jerky movements of their entire bodies, giving them the common name of "wigglers" or "wrigglers". Larvae develop through four stages (instars), after which they metamorphose into pupae. At the end of each instar, the larvae molt, shedding their skins to allow for further growth (mimosq.org, 2012).
2.1.2.3 Pupa The mosquito pupa is comma-shaped. The head and thorax are merged into a cephalothorax, with the abdomen curving around underneath. The pupa can swim actively by flipping its abdomen, and it is commonly called a "tumbler" because of its swimming action.
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The pupae of most species must come to the surface frequently to breathe, which they do through a pair of respiratory trumpets on the cephalothorax. However, pupae do not feed during this stage; typically they pass their time hanging from the surface of the water by their respiratory trumpets. If alarmed, they nimbly swim downwards by flipping their abdomens in much the same way as the larvae do. If undisturbed they soon float up again. After a few days or longer, depending on the temperature and other circumstances, the pupa rises to the water surface, the dorsal surface of its cephalothorax splits, and the adult mosquito emerges. The lower activity of the pupa is as compared to the larva is understandable, bearing in mind that it does not feed, whereas the larva feeds constantly (Ralph, 2008).
2.1.2.4 Adult The period of development from egg to adult varies among species and is strongly influenced by ambient temperature. Some species of mosquitoes can develop from egg to adult in as little as five days, but a more typical period of development in tropical conditions would be some 40 days or more for most species. The variation of the body size in adult mosquitoes depends on the density of the larval population and food supply within the breeding water (mimosq.org, 2012). Adult mosquitoes usually mate within a few days after emerging from the pupal stage. In most species, the males form large swarms, usually around dusk, and the females fly into the swarms to mate. Males typically live for about a week, feeding on nectar and other sources of sugar. After obtaining a full blood meal, the female will rest for a few days while the blood is digested and eggs are developed. This process depends on the temperature, but usually takes two to three days in tropical conditions. Once the eggs are fully developed, the female lays them and resumes host-seeking.
(a)
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(b)
(c)
Plate (2) : Mosquito’s Larva and Pupa: a- Mosquito larvae b- Culex larvae c- Culex larva and pupa
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Plate (3) : Adults of the yellow fever mosquito Aedes aegypti, a typical member of the subfamily Culicinae, the male is on the left, and females are on the right.
The cycle repeats itself until the female dies. While females can live longer than a month in captivity, most do not live longer than one to two weeks in nature. Their life spans depend on temperature, humidity, and their ability to successfully obtain a blood meal while avoiding host defenses and predators. Length of the adult varies, but is rarely greater than 16 mm (0.6 in and weight up to 2.5 mg (0.04 grains). All mosquitoes have slender bodies with three segments: head, thorax and abdomen (Ralph, 2008).
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The head is specialized for receiving sensory information and for feeding. It has eyes and a pair of long, many-segmented antennae. The antennae are important for detecting host odors, as well as odors of breeding sites where females lay eggs. In all mosquito species, the antennae of the males in comparison to the females are noticeably bushier and contain auditory receptors to detect the characteristic whine of the female. The compound eyes are distinctly separated from one another. Their larvae only possess a pit-eye ocellus. The compound eyes of adults develop in a separate region of the head. New ommatidia are added in semicircular rows at the rear of the eye. During the first phase of growth, this leads to individual ommatidia being square, but later in development they become hexagonal. The hexagonal pattern will only become visible when the carapace of the stage with square eyes is molted. The head also has an elongated, forward-projecting "stinger-like" proboscis used for feeding, and two sensory palps. The maxillary palps of the males are longer than their proboscises, whereas the females’ maxillary palps are much shorter. In typical bloodsucking species, the female has an elongated proboscis (mimosq.org, 2012). The thorax is specialized for locomotion. Three pairs of legs and a pair of wings are attached to the thorax. The insect wing is an outgrowth of the exoskeleton. The Anopheles mosquito can fly for up to four hours continuously at 1 to 2 km per hour (0.6–1 mph), travelling up to 12 km (7.5 mile) in a night. Males beat their wings between 450 and 600 times per second. The abdomen is specialized for food digestion and egg development; the abdomen of a mosquito can hold three times its own weight in blood. This segment expands considerably when a female takes a blood meal. The blood is digested over time, serving as a source of protein for the production of eggs, which gradually fill the abdomen (mimosq.org, 2012).
2.1.3 Mosquito as vector Mosquitoes carrying viruses stay healthy while carrying them because their immune system recognizes them as bad and "chops off" the virus's genetic coding, rendering it harmless. It is currently unknown how they handle parasites so they can safely carry them. Infection of humans occurs when a mosquito bites someone while its immune system is still in the process of destroying the virus's harmful coding. Female mosquitoes suck blood from people and other animals as part of their eating and breeding habits.
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When a mosquito bites, it also injects saliva and anti-coagulants into the blood which may also contain disease-causing viruses or other parasites. This cycle can be interrupted by killing the mosquitoes, isolating infected people from all mosquitoes while they are infectious or vaccinating the exposed population. All three techniques have been used, often in combination, to control mosquito transmitted diseases. Window screens, introduced in the 1880s, were called "the most humane contribution the 19th century made to the preservation of sanity and good temper (Susannah, 2008) Mosquitoes are estimated to transmit disease to more than 700 million people annually in Africa, South America, Central America, Mexico and much of Asia with millions of resulting deaths. In Europe, Russia, Greenland, Canada, the United States, Australia, New Zealand, Japan and other temperate and developed countries, mosquito bites are now mostly an irritating nuisance; but still cause some deaths each year (Annals of Internal Medicine, 1998) Historically, before mosquito transmitted diseases were brought under control, they caused tens of thousands of deaths in these countries and hundreds of thousands of infections. Mosquitoes were shown to be the method by which yellow fever and malaria were transmitted from person to person by Walter Reed, William C. Gorgas and associates in the U.S. Army Medical Corps first in Cuba and then around the Panama Canal in the early 1900s. Since then other diseases have been shown to be transmitted the same way. Mosquitoes are a perfect example of one of the many organisms that can host diseases. Of the known 14,000 infectious microorganisms, 600 are shared between animals and humans. Mosquitoes are known to carry many infectious diseases from several different classes of microorganisms, including viruses and parasites. Mosquito-borne illnesses include Malaria, West Nile Virus, Elephantiasis, Dengue Fever, Yellow Fever etc. These infections are normally rare to certain geographic areas. For instance Dengue Hemorrhagic Fever is a viral, mosquito borne illness usually regarded only as a risk in the tropics. However, cases of Dengue Fever have been popping up in the U.S. along the Texas-Mexican border where it has never been seen before (David, 1977). The mosquito genus Anopheles carries the malaria parasite (Plasmodium). Worldwide, malaria is a leading cause of premature mortality, particularly in children under the age of five, with around 2 million deaths annually, according to the Centers for Disease Control (Annals of Internal Medicine, 1998) Some species of mosquito can carry the filariasis worm, a parasite that causes a disfiguring condition (often referred to as elephantiasis) characterized by a great swelling of
xxvi several parts of the body; worldwide, around 40 million people are living with a filariasis disability (David, 1977). The viral diseases yellow fever and dengue fever are transmitted mostly by Aedes aegypti mosquitoes. Other viral diseases like epidemic polyarthritis, Rift Valley fever, Ross River Fever, St. Louis encephalitis, West Nile virus (WNV), Japanese encephalitis, La Crosse encephalitis and several other encephalitis type diseases are carried by several different mosquitoes. Eastern equine encephalitis (EEE) and Western Equine Encephalitis (WEE) occur in the United States where it causes disease in humans, horses, and some bird species (David, 1977). Because of the high mortality rate, EEE and WEE are regarded as two of the most serious mosquito-borne diseases in the United States. Symptoms range from mild flu- like illness to encephalitis, coma and death. Viruses carried by arthropods such as mosquitoes or ticks are known collectively as arboviruses. West Nile virus was accidentally introduced into the United States in 1999 and by 2003 had spread to almost every state with over 3,000 cases in 2006. Culex and Culiseta are also involved in the transmission of disease (Fairhurst and Wellems, 2010). A mosquito's period of feeding is often undetected; the bite only becomes apparent because of the immune reaction it provokes. When a mosquito bites a human, it injects saliva and anti-coagulants. For any given individual, with the initial bite there is no reaction but with subsequent bites the body's immune system develops antibodies and a bite becomes inflamed and itchy within 24 hours. This is the usual reaction in young children. With more bites, the sensitivity of the human immune system increases, and an itchy red hive appears in minutes where the immune response has broken capillary blood vessels and fluid has collected under the skin. This type of reaction is common in older children and adults. Some adults can become desensitized to mosquitoes and have little or no reaction to their bites, while others can become hyper-sensitive with bites causing blistering, bruising, and large inflammatory reactions, a response known as Skeeter Syndrome (Berkley Books, 2005). Malaria is a mosquito-borne infectious disease of humans and other animals caused by protists (a type of microorganism) of the genus Plasmodium. It begins with a bite from an infected female mosquito (Anopheles Mosquito), which introduces the protists via its saliva into the circulatory system, and ultimately to the liver where they mature and reproduce. The disease causes symptoms that typically include fever and headache, which in severe cases can progress to coma or death. Malaria is widespread in tropical and subtropical regions in a broad band around the equator, including much of Sub-Saharan Africa, Asia, and the Americas (Berkley, 2005).
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Five species of Plasmodium can infect and be transmitted by humans. The vast majority of deaths are caused by P. falciparum and P. vivax, while P. ovale, and P. malariae cause a generally milder form of malaria that is rarely fatal. The zoonotic species P. knowlesi, prevalent in Southeast Asia, causes malaria in macaques but can also cause severe infections in humans. Malaria is prevalent in tropical and subtropical regions because rainfall, warm temperatures, and stagnant waters provide habitats ideal for mosquito larvae. Disease transmission can be reduced by preventing mosquito bites by distribution of mosquito nets and insect repellents, or with mosquito-control measures such as spraying insecticides and draining standing water (Fairhurst and Wellems, 2010). Malaria is typically diagnosed by the microscopic examination of blood using blood films, or with antigen-based rapid diagnostic tests. Modern techniques that use the polymerase chain reaction to detect the parasite's DNA have also been developed, but these are not widely used in malaria-endemic areas due to their cost and complexity. The World Health Organization has estimated that in 2010, there were 219 million documented cases of malaria. That year, between 660,000 and 1.2 million people died from the disease (roughly 2000–3000 per day), many of whom were children in Africa. The actual number of deaths is not known with certainty, as accurate data is unavailable in many rural areas, and many cases are undocumented. Malaria is commonly associated with poverty and may also be a major hindrance to economic development. Despite a need, no effective vaccine currently exists, although efforts to develop one are ongoing. Several medications are available to prevent malaria in travellers to malaria-endemic countries (prophylaxis). A variety of antimalarial medications are available. Severe malaria is treated with intravenous or intramuscular quinine or, since the mid-2000s, the artemisinin derivative artesunate, which is superior to quinine in both children and adults and is given in combination with a second anti-malarial such as mefloquine. Resistance has developed to several antimalarial drugs; for example, chloroquine-resistant P. falciparum has spread to most malarial areas, and emerging resistance to artemisinin has become a problem in some parts of Southeast Asia (Nadjm and Behrens, 2012). The signs and symptoms of malaria are reviewed by Bartoloni and Zammarchi. (2012); Nadjm and Behrens (2012), while, the serious complications of malaria are reviewed Bartoloni and Zammarchi (2012); Taylor et al., (2012); Korenromp et al., (2005).
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2.1.4 Genetic resistance
Due to the high levels of mortality and morbidity caused by malaria, especially the P. falciparum species, it has placed the greatest selective pressure on the human genome in recent history. Several genetic factors provide some resistance to it including sickle cell trait, thalassaemia traits, glucose-6-phosphate dehydrogenase deficiency, and the absence of Duffy antigens on red blood cells (Bartoloni and Zammarchi, 2012). Sickle cell trait causes a defect in the hemoglobin molecule in the blood. Instead of retaining the biconcave shape of a normal red blood cell, the modified hemoglobin S molecule causes the cell to sickle or distort into a curved shape. Due to the sickle shape, the molecule is not as effective in taking or releasing oxygen. Infection causes red cells to sickle more, and so they are removed from circulation sooner. This reduces the frequency with which malaria parasites complete their life cycle in the cell. Individuals who are homozygous have sickle-cell anaemia, while those who are heterozygous experience resistance to malaria. Although the shorter life expectancy for those with the homozygous condition seems to be unfavourable to the trait's survival, the trait is preserved because of the benefits provided by the heterozygous form (Hartman et al., 2010). 2.2 Prevention
Methods used to prevent malaria include medications, mosquito elimination and the prevention of bites. The presence of malaria in an area requires a combination of high human population density, high mosquito population density and high rates of transmission from humans to mosquitoes and from mosquitoes to humans. If any of these is lowered sufficiently, the parasite will eventually disappear from that area, as happened in North America, Europe and much of the Middle East. However, unless the parasite is eliminated from the whole world, it could become re-established if conditions revert to a combination that favours the parasite's reproduction (Hartman et al., 2010). Many researchers argue that prevention of malaria may be more cost-effective than treatment of the disease in the long run, but the capital costs required are out of reach of many of the world's poorest people. There is a wide disparity in the costs of control (i.e. maintenance of low endemicity) and elimination programs between countries. For example, in China, whose government announced a strategy to pursue malaria elimination in the Chinese provinces, the required investment is a small proportion of public expenditure on health. In contrast, a similar program in Tanzania would cost an estimated one-fifth of the public health budget (Sarkar et al., 2009).
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2.2.1 Vector control
Vector control refers to preventative methods used to decrease malaria and morbidity and mortality by reducing the levels of transmission. For individual protection, the most effective chemical insect repellents to reduce human-mosquito contact are those based on DEET and picaridin.http://en.wikipedia.org/wiki/Malaria - cite_note-Kajfasz_2009-44 Insecticide-treated mosquito nets (ITNs) and indoor residual spraying (IRS) have been shown to be highly effective vector control interventions in preventing malaria morbidity and mortality among children in malaria-endemic settings. IRS is the practice of spraying insecticides on the interior walls of homes in malaria-affected areas. After feeding, many mosquito species rest on a nearby surface while digesting the blood meal, so if the walls of dwellings have been coated with insecticides, the resting mosquitoes can be killed before they can bite another victim and transfer the malaria parasite. As of 2006, the World Health Organization advises the use of 12 insecticides in IRS operations, including DDT and the pyrethroids cyfluthrin and deltamethrin; (Sarkar et al., 2009). This public health use of small amounts of DDT is permitted under the Stockholm Convention on Persistent Organic Pollutants (POPs), which prohibits the agricultural use of DDT (Gillies , 1988). One problem with all forms of IRS is insecticide resistance via evolution. Mosquitoes that are affected by IRS tend to rest and live indoors, and due to the irritation caused by spraying, their descendants tend to rest and live outdoors, meaning that they are not as affected if affected at all by the IRS, which greatly reduces its effectiveness as a defense mechanism (Baird, 2013). Mosquito nets help keep mosquitoes away from people and significantly reduce infection rates and transmission of malaria. The nets are not a perfect barrier and they are often treated with an insecticide designed to kill the mosquito before it has time to search for a way past the net. Insecticide-treated nets are estimated to be twice as effective as untreated nets and offer greater than 70% protection compared with no net. Between 2000 and 2008, the use of ITNs saved the lives of an estimated 250,000 infants in Sub-Saharan Africa. Although ITNs prevent malaria, only about 13% of households in Sub-Saharan countries own them. A recommended practice for usage is to hang a large "bed net" above the center of a bed to drape over it completely with the edges tucked in. Pyrethroid-treated nets and long- lasting insecticide-treated nets offer the best personal protection, and are most effective when used from dusk to dawn (Gillies , 1967).
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2.2.3 Other methods Community participation and health education strategies promoting awareness of malaria and the importance of control measures have been successfully used to reduce the incidence of malaria in some areas of the developing world. Recognizing the disease in the early stages can stop the disease from becoming fatal. Education can also inform people to cover over areas of stagnant, still water, such as water tanks that are ideal breeding grounds for the parasite and mosquito, thus cutting down the risk of the transmission between people. This is generally used in urban areas where there are large centers of population in a confined space and transmission would be most likely in these areas. Intermittent preventive therapy is another intervention that has been used successfully to control malaria in pregnant women and infants, and in preschool children where transmission is seasonal (Meremikwu et al., 2012). 2.2.4 Chemical control
Chemical control of mosquitoes primarily targets the adult. Outdoor foggers will keep mosquitoes away for several hours, but once the chemical dissipates, mosquitoes may return to the area. Spraying thickets or shrubs along the perimeter of your yard helps reduce the population of mosquitoes that rest in these areas. However, some species of mosquitoes may move readily back into these areas from surrounding untreated places. Consult the NC Agricultural Chemicals Manual or your county Cooperative Extension Center for more information on selecting appropriate pesticides for use against mosquitoes (Wayne, 1989). Insecticides are available for controlling larvae, but their application in either large bodies of water or small artificial breeding sites can be difficult and expensive, particularly for an individual homeowner. Control programs targeting mosquito larvae are best left to trained individuals in county or local government agencies. Most of these chemicals are not selective and some may even harm beneficial insects and other non-target organisms. Furthermore, use of these chemicals will provide only temporary reduction in mosquito populations. Modifying or eliminating breeding sites is the only long-term solution to severe mosquito problems (Chevillon et al., 1999). Homeowners wanting to treat small areas, such as bird baths, garden pools, etc, might want to try bacterial insecticides that are available at many retail stores, garden centers and on-line garden suppliers. There are several products formulated as "donuts" (dunks) or as granules that contain the bacterium Bacillus thuringiensis israelensis or "Bti". This bacterium kills mosquitoes, but does not harm fish, birds or other wildlife. The "dunk" versions are
xxxi well-suited for small breeding sites (100 sq. ft. or less) and will control mosquito larvae for about 30 days. Before using Bti products, you need information on the life cycle and habitat requirements of mosquitoes in your area. Simply treating all areas of standing water without knowing if they are actually sources of the problem is a waste of time (Florida Coordinating Council on Mosquito Control, 1998).
2.2.5 Natural products Some garden plants naturally repel mosquitoes. Rose-scented geraniums contain the natural insect repellents citronellal and geraniol—some gardeners report swishing their hands through the leaves is enough to deter mosquitoes. Lemon balm (Melissa officinalis), which is easy to grow from seed, contains the repellents citronellal, geraniol and geranial. researchers at Iowa State University found. That the essential oil in catnip (Nepeta cataria), nepetalactone, was found to be about 10 times more effective at repelling mosquitoes than DEET, (Beaty and Miller, 1999). Gardeners also report anecdotally that crushing handfuls of basil (Ocimum basilicum), lemon thyme (Thymus citriodorus) and lemongrass (Cymbopogon citratus) can repel mosquitoes for short periods—usually less than 30 minutes. To try these plants, just crush a handful of leaves in your hand and rub them on exposed skin. (Use any herb with caution until you know how your skin will react). Another natural solution may be soybean oil. In a study by the New England Journal of Medicine, a soybean oil-based repellent offered protection from mosquito bites for 1.5 hours (Beaty and Miller, 1999). A number of smart yard management techniques can help reduce the number of mosquitoes in an area. First, eliminate breeding places: any objects that can hold as little as a few tablespoons of water for seven to 10 days—the time it takes for eggs to hatch and larvae to mature. Commonly overlooked breeding spots include old tires, clogged gutters and abandoned tubs or buckets. Change the water weekly in bird baths, wading pools, outdoor pet bowls or anywhere else you might find standing water around your property (Louis, 1948).
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2.2.5.1 Oil drip
An oil drip can or oil drip barrel was a common and nontoxic anti-mosquito measure. The thin layer of oil on top of the water prevents mosquito breeding in two ways: mosquito larvae in the water cannot penetrate the oil film with their breathing tube, and so drown and die; also adult mosquitoes do not lay eggs on the oiled water. Citrus oils are extracted from oranges and other citrus fruit peels and refined to make the insecticidal compounds d-limonene and linalool. Both natural compounds are generally regarded as safe for mammals by the United States Food and Drug Administration, and are used extensively as flavorings and scents in foods, cosmetics, soaps, and perfumes. Limonene and linalool are contact poisons (nerve toxins) that may be synergized by piperonyl butoxide (PBO). They have low oral and dermal toxicities. Both compounds evaporate readily from treated surfaces and have no residual. They have been registered for use against fleas, aphids and mites, but also kill fire ants, several types of flies, paper wasps and house crickets. Commercial products (usually called “d-Limonene”) are available as liquids, aerosols, shampoos, and dips for pets. Topical application can irritate the skin and eyes of some animals, and although symptoms are usually temporary, use these products cautiously and sparingly (Pedigo, 1999). Neem or neem oil is extracted from the seeds of the neem tree, Azadirachta indica, a native of India. The neem tree supplies at least two compounds with insecticidal activity (azadirachtin and salannin), and other unknown compounds with fungicidal activity. Azadirachtin acts as an insect feeding deterrent and growth regulator. The treated insect usually cannot molt to its next life stage and dies. It acts as a repellent when applied to a plant and does not produce a quick knockdown and kill. It has low mammalian toxicity and does not cause skin irritation in most formulations. Neem has some systemic activity in plants. Currently registered products for ornamental pest control claim activity against a variety of sucking and chewing insects. Neem is most effective against actively growing immature insects. Neem oil is used to control powdery mildew (Carr et al., 1991). Pyrethrins are highly concentrated active compounds which are extracted from the daisy-like flowers of Chrysanthemum cinerariaefolium, commercially grown in Kenya. When the flowers is ground into a powder, the product is called a pyrethrum. Pyrethrum is the most widely used botanical insecticide in the United States. Synthetic insecticides that mimic the action of the pyrethrins are known as pyrethroids (e.g., bifenthrin, cyfluthrin, and permethrin). Most insects are highly susceptible to low concentrations of pyrethrins. The
xxxiii toxins cause immediate knockdown or paralysis on contact, but insects often metabolize them and recover. Pyrethrins break down quickly, have a short residual, and low mammalian toxicity, making them among the safest insecticides in use. However, people may have allergic skin reactions and cats are highly susceptible to poisoning (e.g., flea drops and powder). Pyrethrins may be used against a broad range of pests including ants, aphids, roaches, fleas, flies, and ticks. They are available in dusts, sprays, and aerosol “bombs,” and may be mixed with synthetic pesticides or other botanicals (Pedigo, 1999). Rotenone is a broad-spectrum contact and stomach poison that is used against leaf- feeding insects, such as aphids, certain beetles (asparagus beetle, bean leaf beetle, Colorado potato beetle, cucumber beetle, flea beetle, strawberry leaf beetle, and others) and caterpillars, as well as fleas and lice on animals. Rotenone is extracted from the roots of two tropical legumes, Lonchocarpus and Derris, and is commonly formulated as a dust or wettable powder. Insects quickly stop feeding and death occurs several hours to a few days after exposure. Rotenone degrades rapidly when exposed to air and sunlight. It is not phytotoxic, but is extremely toxic to fish, and moderately toxic to mammals. Protective clothing and a mask should be worn to protect skin and the respiratory tract. It may be mixed with pyrethrins or piperonyl butoxide to improve its effectiveness (Carr et al., 1991). Ryania is extracted from the stems of a woody South American plant, Ryania speciosa. Although a slow-acting stomach poison, it causes insects to stop feeding soon after ingestion. It works well in hot weather. Ryania is moderate in acute or chronic oral toxicity in mammals. It is generally not harmful to most natural enemies, but may be toxic to certain predatory mites. Ryania has longer residual activity than most other botanicals. It has been used commercially in fruit and vegetable production against caterpillars (European corn borer, corn earworm, and others) and thrips. Ryania may be difficult to find in stores but may be available from online vendors alone or mixed with rotenone and pyrethrin (Carr et al., 1991). Sabadilla comes from the ripe seeds of the tropical lily Schoenocaulon officinale. Sabadilla is a broad-spectrum contact poison, but has some activity as a stomach poison. Baits, dusts or sprays may be used in organic fruit and vegetable production against squash bugs, harlequin bugs, thrips, caterpillars, leaf hoppers, and stink bugs. The alkaloids in Sabadilla affect insect nerve cells, causing loss of nerve function, paralysis, and insect death. The dust formulation of sabadilla is the least toxic to mammals of all registered botanical insecticides, but protective clothing and a mask should still be worn to protect skin and the respiratory tract. Sabadilla breaks down rapidly in sunlight and air, leaving no harmful
xxxiv residues. However, it is highly toxic to honeybees, and should only be used when bees are not present (e.g., in the evening, after bees return to their hives) (Pedigo, 1999).
2.2.5.2 Soaps and oils
Various oils can be used to manage some pest insects and mites. Horticultural oils used to be called either “dormant” or “summer” oils. Dormant oils originally referred to heavier weight, less well-refined oils that were unsafe to use on plants after they broke dormancy. However, these older oils have been replaced with more refined, light-weight oils that may be applied to plant foliage (summer or foliar oils). A dormant or summer oil now indicates the time of application rather than any particular type of oil. Dormant applications are ideal for treating the overwintering life stages of pests that are more difficult to control during the growing season. Oils may affect the target pests in several ways. Petroleum oils and vegetable oils may block the insects air or breathing holes (spiracles), so the insect dies by suffocation. Oils prevent gas exchange through egg membranes, so eggs are often targets of control with oils. The fatty acids in oils may disrupt cell membranes and interfere with normal metabolism. Other oils may also have anti-feedant properties or may clog stylets (stylet oils), which may help prevent insects, like aphids and leafhoppers, from transmitting viruses to plants. In general, oils are most effective against small, immobile or slow-moving, soft-bodied insects (e.g., aphids, scales, leafhopper nymphs, whiteflies) and mites that are thoroughly coated by an oil spray. Because oils lack residual activity, they do not provide control of insects moving into a treated area (Pedigo, 1999).
2.2.5.2.1 Insecticidal soap
Insecticidal soaps are made from plant oils (cottonseed, olive, palm, or coconut) or animal fat (lard, fish oil), but are generally not considered botanicals. They are made from the salts of fatty acids, which are in the fats and oils of animals and plants. The mode of action is still unclear, despite years of use. Soaps are thought to physically disrupt the insect cuticle (outer skin), but additional toxic action is suspected. Soaps act on contact and must be applied directly to the insect to be effective. No residues remain on plants. They are effective against soft-bodied insects like aphids, some scales, psyllids, whiteflies, mealybugs, thrips, and spider mites. Hard-bodied insects (e.g., adult beetles or wasps) are not harmed because of their tough, chitinous bodies. Some plants may be sensitive to soaps, resulting in leaf burn.
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Plants that have hairy leaves may be more susceptible to soap injury than smooth-leavesd plants. Consult the label to see which plants are listed. Apply the soap spray on a small area of the plant to check for phytotoxicity. Commercial soaps are less likely to be phytotoxic (Carr et al., 1991). 2.2.5.3 Mineral insecticides
Diatomaceous earth is a nontoxic insecticide mined from the fossilized silica shell remains of diatoms (single-celled or colonial algae). It absorbs the waxy layer on insect bodies, abrades the skin, and causes the insect to dry out. Diatomaceous earth is sold as a dust, and is sometimes combined with pyrethrin. It may control slugs, millipedes and sow bugs, as well as soft-bodied insects like aphids. It has low mammalian toxicity. Two kinds of diatomaceous earth are available, a "natural grade" and a filtering agent in swimming pools, but the "natural grade" is the one used as an insecticide (Carr et al., 1991).
2.2.5.1 Sulfur
Sulfur is probably the oldest known pesticide in current use. It can be used as a dust, wettable powder, paste or liquid, primarily for disease control (e.g., powdery mi ldews, rusts, leaf blights, and fruit rots). However, mites, psyllids and thrips also are susceptible to sulfur. Most pesticidal sulfur is labeled for vegetables (e.g., beans, potatoes, tomatoes, and peas) and fruit crops (e.g., grapes, apples, pears, cherries, peaches, plums, and prunes). Sulfur is nontoxic to mammals, but may irritate skin or especially eyes. Sulfur has the potential to damage plants in hot (90°F and above), dry weather. It is also incompatible with other pesticides. Do not use sulfur within 20 to 30 days on plants where spray oils have been applied; it reacts with the oils to make a more phytotoxic combination (Pedigo, 1999). 2.2.6 Biological mosquito control There are no effective avian, mammalian, amphibian, reptilian, or insect predators that will provide natural or biological control of mosquito populations. There are species of these predators that will feed on mosquito larvae and adults but not to the extent that they will control the population (Michigan.gov, 2013).
2.2.6.1 Avian predators There are no bird species that are effective at controlling mosquito populations; Tree swallows are the most effective and adult and juvenile waterfowl species and migratory
xxxvi songbirds will eat larvae and may be dependent on them at certain times of the year. Purple martins eat insignificant numbers of mosquitoes (Michigan.gov, 2013). 2.2.6.2 Mammalian predators Bats play an important role in the environment and can serve as agricultural pest control agents but they are not effective as mosquito control agents. They will eat 500 to 1200 insects/hour. There are no bat species that specialize in eating mosquitoes but there are bat species that have a portion of their diet consisting of mosquitoes (big brown Bat-nearly 0%, little brown Bat-much more likely to feed on mosquitoes). Due to their high metabolic rate, big brown Bats don't select mosquitoes as a food source. Mosquitoes don't swarm, they provide small amounts of energy, and they are more likely to be found in vegetation, not in areas where the bats will be feeding. The big brown Bat is the most common bat in Michigan and their diet primarily consists of June beetles, moths, ground beetles, and stink bugs. The little brown Bat, which commonly occurs In the northern part of the state, has a diet that primarily consists of spiders, mosquitoes, and dipterous insects. Inhabitants of bat houses in residential areas will have minimal impact on mosquito populations, especially in the southern portion of the state where the big brown Bat is the most common species to utilize a bat house. little brown Bats (much less common in the southern portion of the state) will have a minor impact on the mosquito population as mosquitoes make up a small percentage of their diet. If bat houses are placed in residential areas, they likely will provide an alternative roosting area of bats already present. It is important to erect the house in a proper location (15 feet in the air, in direct sunlight for no more than 4 to 6 hours, and not attached to a residence). Big brown Bats are the main species in Michigan to be diagnosed with rabies (it is estimated that less than 1% of the bats in the state are rabid). Because of this, it is important to avoid contact with bats that are found on the ground, outside of a bat house, or during daylight hours. If dead bats are found and there has been no known pet or human exposure, they should be collected by picking them up with a shovel, placing them in a plastic bag, and disposing of them properly (Michigan.gov, 2013).
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2.2.6.3 Amphibian and reptilian predators There are no amphibians or reptiles that are effective at mosquito control. The cricket frog, Chorus frog, and Spring peeper will eat mosquitoes but won't impact a population (R0mi,etal. 2006).
2.2.6.4 Insect predators Dragonfly larvae and aquatic beetles will feed on mosquito larvae, but won't control the population. Mosquito populations are limited by predation, but the intensity of predation and its significance should vary greatly among habitat types. Larval habitats of mosquitoes are diverse. At one extreme, small containers that hold water such as tree holes, bamboo stumps, plant pitchers, etc. are the typical larval habitats of some groups. At the other extreme, edges of lakes and ponds also can be colonized by certain kinds of mosquitoes. In general, it is considered that predation regulates mosquitoes in pool-type habitats whereas container mosquitoes are limited by resource rather than predation . Many kinds of predators have been reported from pool habitats, while small containers often lack predators, although some predators such as Toxorhynchites larvae have specialized to these habitats (Abdalla,et al., 2008). The small size of container habitats may be an important characteristic that determines community structure of mosquitoes and other aquatic insects including predators . Island biogeography theory as an explanation of poorer species richness in containers than in pools. Even within a narrower range, metazoan diversity is positively correlated with capacity of tree holes and bamboo stumps . It is expected that the significance of predation in mosquito populations depends on the habitat size. Extensive studies have been carried out for container mosquitoes from phytotelmata such as tree holes, bamboo stumps, pitcher plants, and artificial containers (Kathleen,2002). However, most of the previous studies on container mosquitoes treated habitats of various sizes over small ranges. Differences in regulatory factors of mosquitoes between container type and pool-type habitats suggested the importance of habitat size in relation to mosquito larvae . 2.3 Aquatic predators 2.3.1 Dragonfly
A dragonfly is an insect belonging to the order Odonata. It is characterized by large multifaceted eyes, two pairs of strong transparent wings, and an elongated body. Dragonflies can sometimes be mistaken for damselflies, which are morphologically similar; however,
xxxviii adults can be differentiated by the fact that the wings of most dragonflies are held away from, and perpendicular to, the body when at rest. Dragonflies possess six legs (like any other insect), but most of them cannot walk well. Dragonflies are among the fastest flying insects in the world. Dragonflies are important predators that feed on mosquitoes, and other small insects like flies, bees, ants, wasps, and very rarely butterflies. They are usually found around marshes, lakes, ponds, streams, and wetlands because their larvae, known as "nymphs", are aquatic. Some 5680 different species of dragonflies (Odonata) are known in the world today (Kalkman et al., 2008). Though dragonflies are predators, they themselves are subject to predation by birds, lizards, frogs, spiders, fish, water bugs, and even other large dragonflies.
2.3.1.1 Classification
Kingdom: Animalia
Phylum: Arthropoda
Class: Insecta
Order: Odonata
(unranked): Epiprocta
Suborder: Anisoptera,(Kalkma et al.,2008). Formerly, the Anisoptera were given suborder rank beside the "ancient dragonflies" (Anisozygoptera), which were believed to contain the two living species of the genus Epiophlebia and numerous fossil ones. More recently it turned out that the "Anisozygopterans" form a paraphyletic assemblage of morphologically primitive relatives of the Anisoptera. Thus, the Anisoptera (true dragonflies) are reduced to an infraorder in the new suborder Epiprocta (dragonflies in general). The artificial grouping Anisozygoptera is disbanded, its members recognized as extinct offshoots at various stages of dragonfly evolution. The two living species formerly placed there-the Asian relict dragonflies-form the infraorder Epiophlebioptera alongside Anisoptera (Kalkma, 2008).
2.3.1.2 Life cycle
Female dragonflies lay eggs in or near water, often on floating or emergent plants. When laying eggs, some species will submerge themselves completely in order to lay their
xxxix eggs on a good surface. The eggs then hatch into naiads. Most of a dragonfly's life is spent in the naiad form, beneath the water's surface, using extendable jaws to catch other invertebrates (often mosquito larvae) or even vertebrates such as tadpoles and fish. They breathe through gills in their rectum, and can rapidly propel themselves by suddenly expelling water through the anus (Mill and Pickard, 1975). Some naiads even hunt on land (Grzimeck, and Bernard,1975). The larval stage of large dragonflies may last as long as five years. In smaller species, this stage may last between two months and three years. When the naiad is ready to metamorphose into an adult, it climbs up a reed or other emergent plant. Exposure to air causes the naiad to begin breathing. The skin splits at a weak spot behind the head and the adult dragonfly crawls out of its larval skin, pumps up its wings, and flies off to feed on midges and flies. In flight the adult dragonfly can propel itself in six directions; upward, downward, forward, back, and side to side (Waldbauer, 2006). The adult stage of larger species of dragonfly can last as long as five or six months.
2.3.2 Hemipteran boatman Water boatmen are somewhat flattened and elongate in shape. They have the hind two pairs of legs fitted with hairs and the tarsi of the hind legs is scoop or oar-shaped which allows them to swim. Adults range in length from 3/16 to 3/8 inch (3 to 11 mm) long and are usually dull colored and often mottled. Water boatmen are the largest group of aquatic true bugs. They are sometimes confused with backswimmers (Hemiptera: Notonectidae) because they have the same general shape. However, backswimmers swim upside down in the water and are colored with the wings lighter than the leg area. Adults are relatively small and soft bodied so they do not preserve well on insect pins (Drees and John 1999). 2.3.2.1 Classification Kingdom: Animalia Phylum : Arthropoda Subphylum: Hexapoda Class: Insecta Order: Hemiptera Suborder: Heteroptera Infraorder: Nepomorpha Family : Corixidae
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Scientific Name: Corixa spp (Ross, 2000).
2.3.2.2. Habitat and food Common in ponds, and also found in birdbaths. A few species live in streams, and others are found in brackish pools along the seashore above the high tidemark. Water boatmen feed on: Algae, detritus, other aquatic organisms (mosquito larvae, brine shrimp); a few (uniquely among Hemiptera) consume small particles of solid food (Ross, 2000). 2.3.3 Mosquito-fish
The western mosquito-fish (Gambusia affinis) is a species of freshwater fish, also known commonly, as simply mosquito-fish or by its generic name, Gambusia, or by the name gambezi. There is also an eastern mosquito-fish (Masterson, 2011). (Mosquito-fish are small in comparison to other fish, with females reaching an overall length of 7 centimeters (2.8 in) and males at a length of 4 centimeters (1.6 in). Females can be distinguished from males by their size and a gravid spot at the posterior of their abdomen). The name "mosquitofish" was given because the diet of this fish sometimes consists of large amounts of mosquito larvae, relative to body size. Gambusia typically eat zooplankton, beetles, mayflies, caddis flies, mites and other invertebrates; mosquito larvae make up only a small portion of their diet (Mark, 2008) Mosquito-fish were introduced directly into ecosystems in many parts of the world as a biocontrol to lower mosquito populations which in turn negatively affected by many other species in each distinct bioregion. Mosquito-fish in Australia are classified as a noxious pest and may have exacerbated the mosquito problem in many areas by outcompeting native invertebrate predators of mosquito larvae. Several counties in California distribute mosquito fish at no charge to residents with man-made fishponds and pools as part of their Mosquito Abatement programs. The fish are made available to residents only and are to be used only on their own property, not introduced into natural habitat. Fertilization is internal, the male secreting milt into the genital aperture of the female through his gonopodium. Within 16 to 28 days after mating, the female will give birth to about 60 young’s. The males reach sexual maturity within 43 to 62 days. The females, if born early in the reproductive season, reach sexual maturity within 21 to 28 days; females born later in the season reach sexual maturity in six to seven months (Anon, 2011).
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Mosquito-fish are small, dull grey, with a large abdomen, and have rounded dorsal and caudal fins and an upturned mouth towards the surface. Sexual dimorphism is pronounced; mature females reach a maximum overall length of 7 centimeters (2.8 in), while males reach only 4 centimeters (1.6 in). Sexual dimorphism is also seen in the physiological structures of the body (Anon,2012). The anal fins on adult females resemble the dorsal fins, while the anal fins of adult males are pointed. This pointed fin, referred to as a gonopodium, is used to deposit milt inside the female. Adult female mosquitofish can be identified by a gravid spot they possess on the posterior of their abdomen. Other species considered similar to G. affinis include Poecilia latipinna, Poecilia reticulata, and Xiphophorus maculatus; which is commonly misidentified as the eastern mosquitofish (Dionne, 1985).
2.3.3.1 Classification
Kingdom: Animalia
Phylum: Chordata
Class: Actinopterygii
Order: Cyprinodontiformes Family: Poeciliidae Genus: Gambusia Species: Affinis 2.3.3.2 Habitat and food
The native range of the mosquito-fish is from southern parts of Illinois and Indiana, throughout the Mississippi River and its tributary waters, to as far south as the Gulf Coast in the northeastern parts of Mexico. They are found most abundantly in shallow water protected from larger fish. Mosquito-fish can survive relatively inhospitable environments, and are resilient to low oxygen concentrations, high salt concentrations (up to twice that of sea water), and temperatures up to 42 °C (108 °F) for short periods. Because of their notable adaptability to harsh conditions and their global introduction into many habitats for mosquito control they have been described as the most widespread freshwater fish in the world (Kitching, 1986). Based on diet, mosquito-fish are classified as larvivorous fish. Their diet consists of zooplankton, small insects and insect larvae, and detritus material. Mosquito-fish feed on mosquito larvae at all stages of life. Adult females can consume in one day hundreds of
xlii mosquito larvae. Maximum consumption rate in a day by one mosquito-fish has been observed to be from 42%–167% of its own body weight. Studies have shown, however, that mosquito-fish can suffer mortalities if fed only on mosquito larvae, and survivors show poor growth and maturation. Mosquito-fish have also shown cannibalistic behavior in laboratory experiments; however, whether these traits are hereditary is unknown (Krumholz, 1944) 2.4 Ixora Ixora coccinea (Jungle geranium; Plate, 1) is a perennial shrub 0.6–0.9 m in height, widely grown in gardens as an ornamental plant. The flowers are bright scarlet red, sometimes yellow, pink or orange-yellow. The bush has small globular fruits which are purple when ripe. The shrub is native to tropical Asia. However, it is cultivated for ornamental purposes in tropical and subtropical areas in other continents; as well Ixora coccinea is a species of flowersing plant in the Rubiaceae family. It is a common flowersing shrub native to Southern India and Sri Lanka. It has become one of the most popular flowersing shrubs in South Florida gardens and landscapes. Its name is derived from an Indian deity (Baliga and kurian, 2012).
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Plate (1) : Ixora coccinea Source : Baliga and Kurian (2012)
2.4.1 Classification
Kingdom: Plantae
Phylum Angiospers
Class Eudicots
Order: Gentianales
Family: Rubiaceae
Genus: Ixora
Species: Coccinea
2.4.2 Traditional use and plant parts used
The roots, bark, leaves and flowers are used in traditional medicine in South East Asia from India to the Philippines. The roots of I. coccinea are used to treat hiccoughs, nausea, fever, ulcers, gonorrhea, and loss of appetite. The flowers of I. coccinea are used against reddened eyes, eruptions, catarrhal bronchitis, dysentery, and as an anti-inflammatory agent. The leaves have been utilized in the treatment of diarrhoea. A paste from the root of an unspecified Ixora species is used against diarrhoea in children. The ethnomedical uses and pharmacology of this plant have been reviewed previously (Latha,et.al, 2001).
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2.4.3 Chemical composition 2.4.3.1 Leaves
Triterpenoid: lupeol, Proanthocyanidins: ixoratannin A-2 (a trimeric A-type proanthocyanidin), procyanidin A2, cinnamtannin B-1; Flavonoids: epicatechin, kaempferol- and quercetin-rhamnosides (Zachariah, et.al, 1994). 2.4.3.2 Flowers Triterpenoids: ursolic acid, cycloartenol esters, lupeol esters, lupeol, oleanolic acid; Sterols: sitosterol; Flavonoids: biochanin A, myricetin, quercetin, rutin, daidzein formononetin; monoglycosides of cyanidin and delphinidin, rutin, kaempferol-3- rutinoside;traces of leucocyanidin glycoside (Sumathy, et.al, 2011). 2.4.3.3 Above-ground parts Triterpenoids: lupeol, acetylbetulic acid, betunolic acid, α-amyrin, β-amyrin, ursolic acid, 3-acetylursolic acid, oleanonic acid; Sterols: 6β-hydroxystigmast-4-en-3-one, sitosteryl- 3-O-β-d-glucoside, β-sitosterol, stigmasterol; Flavonoids: kaempferol, kaempferol-7-O-α- rhamnoside, kaempferitrin, luteolin, (−)-epicatechin, (+)-catechin; Proanthocyanidin: epicatechin-4β->8, 2β->O->7-ent-epicatechin; Coumarins: scopoletin, coumarin, erythro- 1′,2′-albiflorin; Diterpenoids: 16α-hydro-19-acetoxy-(−) kauran-17-oic acid, 16α-hydro-19- ol-(−)-kauran-17-oic acid; Quinones: 1,4-dihydroxy -3-methylanthraquinone, tocopherylquinone; Peptides: ixorapeptides I and II (Lee et al., 2010).
2.4.3.4 Roots Fatty acids: palmitic, stearic, oleic and linoleic acid; Essential oil: (main constituent β-sesquiphellandrene (Yadava and Asian, et.al, 1989). 2.4.4 Bioactivity
The anti-diarrhoeal effect of I. coccinea has been investigated. An aqueous extract of the flowers showed significant inhibition of castor oil induced diarrhoea in rats as determined by weight and volume of intestinal content and by gastrointestinal motility.(Atiq Ur Rahman,et al,2012) I. coccinea has been investigated for antimicrobial effects. In a study by Annapurna et al. ether and methanol extracts of the leaves were tested against a selection of bacteria and fungi. The ether extract was found to have higher activity than the methanol extract, and both
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Gram-negative and Gram-positive bacteria were inhibited. The activity against fungi was not significant. Srinivasan reported that the essential oil from I. coccinea roots exhibited antimicrobial activity towards Gram-positive and Gram-negative bacteria. Sasidharan .found that an alcoholic extract of I. coccinea (plant part not specified) was active against S. aureus and E. coli, the aqueous extract was active against E. coli but not S. aureus, and neither the aqueous nor the alcoholic extract were active against the fungi Aspergillus niger. Leaf constituents were active against E. coli, S. aureus, P. aeruginosa and B. subtilis. In these studies the disc-diffusion method was employed. Ethanolic and aqueous root extracts inhibited bacterial growth of Enterococcus faecalis, E. coli, Salmonella typhi and several other bacteria (S. aureus, B. pumilus, P. aeruginosa) with MIC values of 12.5–100 μg/ml. The extracts were, however, inactive against fungi. Interestingly, these extracts were also reported to have wound-healing properties (Srinivasan, et.al, 2010). An ethanol extract of I. coccinea roots protected rats from aflatoxin B1-induced liver damage. This was suggested to be due to the potent antioxidant activity of the extract. Antioxidant properties (as radical scavenging, total antioxidant capacity, and xanthine oxidase inhibition) of methanol extracts from flowers, leaves and stems of I. coccinea were reported .The antioxidant activity seemed to be correlated to the phenolic content. The antioxidant properties of the methanolic extract of I. coccinea was also believed to be important for its ability to counteract doxorubicin induced cardiotoxicity in rats (Ratnasooriya, et.al, 2005). The antioxidant and cytotoxic activity of petroleum ether, ethyl acetate and methanol extracts of I. coccinea flowers has been investigated, finding that, the ethyl acetate extract was the most active one. Ixora peptide was found to have selective cytotoxicity towards Hep3B liver cancer cells relative to normal cells. An aqueous extract of the flowers of I. coccinea had antimutagenic activity (Lee, et.al, 2010). An aqueous extract of the leaves exhibited hypoglycaemic and hypolipidaemic activity in diabetic rats. A methanolic flowers extract had anti-inflammatory and analgesic properties (Ratnasooriya, et.al, 2005). The methanol extract of I. coccinea leaves has been reported to be without larvicidal activity towards Anopheles mosquitoes (Khare, 2007). 2.4.5 Toxicity
In a mice toxicity test it was found that the petroleum ether extract of I. coccinea root, up to an oral dose of 1.5 g/kg body weight, did not show any toxic effects. In another study, the active fraction (AF) (the cytotoxic fraction from a flowers hexane extract) up to 400
xlvi mg/kg was given i.p. to mice. No deaths were observed in 24 hour. The test animals did not show any changes in general behavior during the study. Chronic administration of AF (200 mg/kg i.p.) did not produce any significant differences in the food or water consumption and body weight of the mice either. However, a methanolic extract of I. coccinea flowers and fractions there from were cardiotoxic to the perfused guinea pig heart and might lead to heart failure. Whether this is relevant for in vivo conditions appears unknown (Ratnasooriya, et.al, 2005). The leaves of I. coccinea have been used in traditional medicine against diarrhoea. This activity has recently been documented in animal experiments. Numerous biological activities have been reported for different parts of the plant, although most of these were in vitro. It would appear that some of these effects were related to the antioxidant activity of the plant, which again has been suggested to be correlated to its content of phenolic compounds such as flavonoids and A-type proanthocyanidins. In this connection, it might be mentioned that A-type proanthocyanidins from cranberries have been reported to be partly responsible for the putative effects of cranberries against urinary bladder infections. Investigation of proanthocyanidins from I. coccinea for this condition might seem interesting (Khare, 2007).
2.5 Malathion insecticide Malathion is a pesticide that is widely used in agriculture, residential landscaping, public recreation areas, and in public health pest control programs such as mosquito eradication. In the USA, it is the most commonly used organophosphate insecticide. Malathion is an organophosphate parasympathomimetic which binds irreversibly to cholinesterase. Malathion is an insecticide of relatively low human toxicity. In the former USSR, it was known as carbophos, in New Zealand and Australia as maldison and in South Africa as mercaptothion (Hansch and Leo, 1995). 2.5.1 Malathion used in mosquito control
The mosquito goes through four distinct stages during its life cycle: egg, larva, pupa, and adult. Malathion is an adulticide, used to kill adult mosquitoes. In mosquito control programs conducted by state or local authorities, malathion is applied by truck-mounted or aircraft-mounted sprayers. Malathion is applied as an ultra-low volume spray. UL sprayers dispense very fine aerosol droplets that stay aloft and kill mosquitoes on contact. UL applications involve small quantities of pesticide active ingredient in relation to the size of the area treated. For mosquito control, malathion is applied at a maximum rate of 0.23 pounds
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(less than 4 ounces) of active ingredient per acre per day, which minimizes exposure and risks to people and the environment. Malathion used in mosquito control programs does not pose unreasonable risks to wildlife or the environment. Malathion degrades rapidly in the environment, especially in moist soil. Malathion is highly toxic to insects, including beneficial insects such as honeybees, it is toxic to aquatic organisms, including fish and invertebrates. For that reason, we have established specific precautions on the label to reduce such risks. Additionally, previously completed screening-level risk assessments and exceeds of agency levels of concern indicated a need for further examine and potentially refine estimates of acute and chronic risk to terrestrial and aquatic animals during registration review (Anon, 2006).
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CHAPTER THREE MATERIALS AND METHODS
3.1 The study area Gezira State is located in the eastern central region of Sudan. It lies between latitudes (13 – 32 and 12 -30) North and longitudes (22 -32 and 20-34) East. The introduction of irrigation in the arid areas of Gezira state produced drastic considerable changes in ecology. As a result of these changes; Gezira state is considered as the largest area in Sudan with the highest number of permanent mosquito breeding sites and the malaria transmission changed from seasonal to perennial and malaria endemicity from mesoendemic to hyperendemic. The Gezira irrigation scheme is the main economic activity in the state and a major challenge for malaria control as it contributes to the accumulation of water resulting in both permanent and temporary breeding sites for mosquitoes and other vectors that cause water-borne diseases. Different localities within and around Wad Medani (the capital of Gezira State) were selected for sampling mosquitoes and their aquatic predators. 3.2 Materials 3.2.1. Samples Anopheles and Culex larvae were collected from temporary pooled water around Alkaraiba neighborhood, Wad Medani, Gezira State, using network made of special cloth and long iron stick; the samples were then placed in labeled dishes containing 200 ml of water. Hemipteran boatmen (Corixa) great diving beetle adults and larvae (Coleoptera) and Mayfly naiads (Odonata) were caught during the collection of mosquitoes using the same procedures, the collection was carried out during autumn 2013, 2014 and 2015. Each Toxicity test was repeated 2 times. Gambusia affinis, fish were brought from the Blue Nile National institute for Training and Research, University of Gezira. Rearing and maintenance of mosquito larvae followed the instructions of WHO (1980). The collected samples were immediately transferred to the Biology Laboratory, University of Holy Quran, Wad Medani, where the phytochemical and toxicity tests were done.
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3.2.2. Preparation of Ixora aqueous and ethanolic extracts Ixora leaves and flowers were collected from Alnishaishiba area, Wad Medani, Gezira State; the collected samples were then dried in the shade under the room temperature away from the direct sun light. Ten grams of each dried product was placed in a 500 ml beaker, and 500 ml of tap water or 500 ml of alcohol were added. The beaker was then covered and kept under the room temperature. After 24 hours the mixtures were filtered in a clean, 500 ml conical flask, using filter papers, the components of the beaker were filtered. The filter paper with its un-dissolved components was dried in the oven under 160oC for two hours, and then weighed so as to calculate the actual quantity of each plant product that was dissolved in the final volume of each solvent, the stock solvents. From each Ixora aqueous or alcoholic leaves and flowers extracts: 0.5, 1, 1.5, 2, 2.5 ml extract/ 250 ml tap water were added. For season 2013, the tested concentrations of aqueous leaves extracts were (12.71, 25.42,38.13,50.84 and 63.55 mg/l) and the ethanolic leaves extracts were (6.69, 13.38,20.07,26.76 and 33.45 mg/l), while the tested concentrations for aqueous flowers extracts were (15.15, 30.10,45.45,60.60 and 75.75 mg/l), and the flowers ethanolic extracts were (5.18, 10.36,15.54, 20.72 and 25.89 mg/l). For season 2014, the tested concentrations of aqueous leaves extracts were (11.13, 22.26, 33.39, 44.52 and 55.65 mg/l) and The ethanolic leaves extracts were (9.12, 18.21, 27.32, 36.42 and 45.53 mg/l), while those of aqueous flowers extracts were (17.10, 34.15, 51.23, 68.30 and 85.38 mg/l), and the flowers ethanolic extracts were (7.35, 14.70, 22.06, 29.41 and 36.76 mg/l). In the season 2015, the tested concentrations of aqueous leaves extracts were (12.98, 25.96, 38.94, 51.92 and 64.90 mg/l) and the ethanolic leaves extracts were (5.91, 11.81, 17.72, 23.62 and 29.53 mg/l), while the tested concentration for aqueous flowers extracts were (16.11, 32.22, 48.33, 64.45 and 80.56 mg/l), and the ethanolic flowers extracts were (6.45, 12.90, 19.35, 25.80 and 32.25 mg/l). 3.2.3. Preparation and dilution of Malathion Malathion EC insecticide (production date: September 2012; Eias Industrial Groups of Company-Germany; Ex date: September 2016; Concentration (57%) 57 g/L; 57000 mg/L) was used as standard. One ml of the original concentration was dissolved in 250 ml tap water to form the stock solution (concentration = 228 mg/L). Each of 0.2, 0.4, 0.6, and 0.8 ml from this stock solution was added to 500 ml tap water and accordingly the corresponding
l concentrations were 0.0912, 0.1824, 0.2736, and 0.3648 mg/L, respectively. These concentrations were used to test the susceptibility of Anopheles and Culex larvae and some of their aquatic predators. 3.3 Methods 3.3.1. Toxicity test Experiments were started by preparing a number of (45) plastic cups (250 and 500 ml), filled with tap water. Randomly 20 individuals of each of the Anopheles or Culex larvae or of the selected aquatic predators were gently added to those cups. A series of the prepared concentrations were applied to test their toxicity against Anopheles and Culex larvae and the aquatic predators. Three replications were used to conduct the experiments, and 24 hours was the submission period. Each plastic cup of the aquatic predators was supplied with some of the Anopheles and Culex larvae during the toxicity test procedure, so as to exclude the death resulted of starvation. Control batches were also designed. The mortalities were ccounted accordingly. These tests were done during the seasons 2013, 2014 and 2015, except for Malathion insecticide, which was tested once during 2013. 3.3.2. Phytochemical screening 3.3.2.1. Test for glycosides A known weight (3.0 g) of the dried powder of each part of the Ixora plant, was boiled with an aliquot of distilled water (10 ml) and filtered. Aliquots (2 ml each) of the filtrate were tested for glycosides as described by Harborne (1973). The filtrate was dissolved in 2 ml of glacial acetic acid. To this solution two drops of ferric chloride solution were added and mixed. The mixture was transferred to a narrow test tube. About 1-2 ml of conc.
H2SO4 was added carefully on the side of the tube using a pipette to form a layer. In presence of glycosides, a reddish brown layer at the interface was formed which gradually acquired a bluish-green colour that darkened on standing. 3.3.2..2 Test for flavonoids A know weight (2.0 g) of the dried powder of each part of the Ixora plant, was measured in 1% of hydrochloric acid (50 ml) over night, filtered and the filtrate was subjected to the following tests : a) A known (10 ml) from each filtrate was rendered alkaline with sodium hydroxide (10%, w/v); if a yellow colour was formed, that might indicate the presence of flavonoids.
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b) Shindoa test : 5 ml of each filtrate was mixed with conc. HCl (1 ml) and magnesium turning was added. The formation of red colour indicate the presence of flavonoids, flaonones, and/or flavonols (Harbone, 1998). 3.3.2.3 Test for saponins A known weight (5 g) of the dried powder of each part of the Ixora plant, was extracted with 20 ml ethanol (50%) and filtrated. Aliquots of the alcoholic extracts (10 ml each) were evaporated to dryness under reduced pressure. The residue was dissolved in distilled water (4 ml) and filtered. The filtrate was vigorously shaken; if a voluminous forth was developed and persisted for almost one hour, this indicates the presence of saponins (Harbone, 1973). 3.3.2.4 Test for tannins The dried powder of each part of the Loxra plant (5 g), was extracted with ethanol (50%) and filtered. Ferric chloride reagent (5%, w/v in methanol) was added, the appearance of green colour which changes to a bluish black colour or precipitate, indicate the presence of tannins (Harbone, 1974). 3.3.2.5 Test for sterols and/or triterpenes The dried powder of each part of the Loxra plant (1 g), was extracted with petroleum ether (10 ml each) and filtered. The filtrate was evaporated to dryness and the residue was dissolved in chloroform (10 ml), of which (3 ml) were mixed with concentrated acetic acid an hydride (3 ml), and a few drops of H2SO4 were added. The formation of a reddish violet ring at the junction of the two layers, indicates the presence of unsaturated sterols and/or triterpenes (Harbone, 1973). 3.3.2.6 Test for alkaloids and/or nitrogenous bases The dried powder of each part of the Ixora plant (5 g), was extracted with ethanol and filtered. Aliquots from the ethanolic extract (10 ml each) were mixed with hydrochloric acid (20 ml 10% v/v), and filtered. The filtrate was rendered alkaline with ammonium hydroxide and extracted with successive portions of chloroform. The acid (2 ml 10% v/v) and tested with Mayer’s reagent, and Dragendorff’s reagent combined chloroformic extract was evaporated to dryness, the residue was dissolved in hydrochloric, respectively. If a precipitate was formed, it indicates the presence of alkaloids and/or nitrogenous bases (Harbone, 1974). 3.3.2.7 Test for coumarins The dried powder of each part of the Ixora plant (5 mg), One ml of the plant extract were taken in a small test tube and covered with filter paper moistened with 1N NaOH . The
lii test tube was placed for few minutes in boiling water. Then the filter paper was removed and examined in UV light for yellow fluorescence to indicate the presence of Coumarins (Harbone, 1973). .3.4 Proximate analysis 3.4.1 Moisture content Moisture content of each sample was determined according to the method described by AOAC (1984). Three gram of samples was pulverized in blender. The powder samples were taken in a pre-heated crucible of know weight, and then dried in an air oven at 105C0 over night, then transferred to desiccators, allowed to cool at room temperature and were then reweighed the moisture content of sample was calculated according to the following equation : Moisture content % = (A – B ) ×100 Weight of sample Where : A = weight of crucible + sample (before drying) B = weight of crucible + sample (after drying). 3.4.2 Ash content Determination of ash content of samples was estimated according to AOAC (1984). Two grams of the sample were put in a clean dry crucible of known weight and then placed in muffle furnace at 550C0 for 3 hours. The crucible was then cooled in a desiccators and reweighed at room temperature. The ash content of the sample was calculated using the following equation :
Ash content % = (W1-W2) x 100
W3 Where :
W1 = weight of crucible (g) + ash
W2 = weight of empty crucible (g)
W3 = weight of sample (g) 3.4.3 Protein content Protein content of each tested sample was determined by the semi-micro Kjedahl method as described by AOCS (1985) in which 0.2 g sample were digested using one gram of catalyst sodium sulphate: cupric sulphate; 20 : 1 by weight and 10 ml of concentrated sulphuric acid and the content was heated for 6 hours till a clear solution. Then the digested
liii sample was transferred to 100 ml volumetric flask. The clean digested sample was pipette at into distillation unit after adding few pieces of granulated pumice. Ten ml of 20% boric acid were transferred to 50 ml conical flask and a few drops of indicator methyl red were added, then the flask was placed under the condenser of the distillation unit, with the tip of delivery tube below the level of liquid. Ten ml of 40% NaOH were poured carefully down the neck of the distillation flask. The flask was attached to distillation unit and mixed by swirling gently. Moderate heat was applied. Delivery tube washed down and ammonia was titrated against 0.1 N hydrochloric acid, a faint pink color was taken as the end point. The total nitrogen percentage was determined by the following equation : Total nitrogen % = V x N x 14 x 100 W x 1000 Where : V = volume (ml) of hydrochloric acid. N = Normality of hydrochloric acid. W = weight of original sample (in gram). Total nitrogen was multiplied by the factor (6.25) to obtain crude protein as shown by the following equation : Crude protein = 6.25 x total nitrogen. 3.4.4 Crude fiber content Crude fiber contents were determined for the various samples according to AOCS (1985). Three grams of the defatted samples was weighed in 600 ml beaker. Then 200 ml of boiling 1.25% sulphuric acid and one drop of diluted anti foam agent were added. The contents were boiled under reflex for 30 minutes and filtrated through Buchner funnel. The residue was then transferred back into the beaker using 200 ml of 1.25% boiling sodium hydroxide, and boiled under reflux for 30 minutes. The contents were again filtered and transferred to pre-dried and weighed dish. They were then dried at 100C0 to constant weight. The contents were then reweighed and ignited in muffle furnace at 550C0 for 5 hours. The crude fiber content was calculated as follow : Crude fiber % = (a-b) x 100 W Where : a= weight of dish content before ashing b= weight of dish content after ashing
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W = weight of sample 3.5 Statistical analysis Mortality of each of Anopheles larvae, Culex larvae and the tested aquatic predators were plotted in special tables designed to present the data and its corresponding analysis. The obtained data were submitted to a simple regression analysis by using Excel program 2007. The used concentrations were transformed to as log, whereas the resulted mortalities were transformed to as probit by using probit transformation table. The Log-concentration and the probit data were used to run a regression analysis so as to calculate the LC50 and LC95 from the resulted intercept (a) and x-coefficient (b); the element of the regression equation. The correlation (R2) was also obtained to detect the level of homogeneity in any single test.
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CHAPTER FOUR RESULTS AND DISCUSSION
4.1 Toxicity test on mosquito larvae and some aquatic predators 4.1.1 Effect of ethanol extract of Ixora leaves (season 2013) The present investigations involved an attempt to determine the potential role of Ixora extracts as natural products to control mosquito larvae and also to assess their effects on their aquatic predators. Five different concentrations of ethanol extract of Ixora leaves were applied against the tested organisms. The tested concentrations ranged between 6.69 to 33.45 mg/L. These concentrations resulted in 20% to 95% mortality in Anopheles larvae, 7.5% to 45.7% mortality in Culex larvae, 5.9% to 45.5 mortality in Hemipteran boatman,12.5% to 60% mortality in swimming beetle larvae, 7.5% to 69.5% mortality in Dragonfly naiad, 5% to 40% mortality in Gumbosia fish (Table, 4.1)
The LC50 and LC95 were 14.24 and 36.96 mg/L, for Anopheles larvae, and 46.04 and 333.89, for Culex larvae, respectively. Culex larvae seemed to be considerably high compared
to that of Anopheles larvae. The LC50 and LC95 for the aquatic predators were 43.22 and 261.66 for Hemipteran boatman, 29.47 and 202.36 for swimming beetle larvae, 25.27 and 84.43 for Dragonfly naiads, 70.95 and 545.13 for Gambusia fish, following the same order.
The LC50 and LC95 ‘s of the aquatic predators seemed to be relatively high compared to that of mosquitoes' larvae. This finding indicated that, the ethanol extract of Ixora leaves can play an important role in Anopheles control with slight effects on the aquatic predators. The Ixora leaves contain the lupeol, ursolic acid, oleanlic acid, sitosterol, rutin, lecocyanadin, anthocyanins, proanthocyanidins, and glycosides of kaempferol and quercetiin (Baliga and Kurian, 2012). The regression analysis reflected that, the R2‘s were 0.75 for Gumbosia fish, 0.79 for Dragonfly, 0.93 for swimming beetle larvae, 0.93 for Hemipteran boatman, while it was 0.88 in Anopheles and Culex larvae. In a similar research, El Mahi (2014) found that, Ixora extracts were toxic against mosquito larvae, also the Anopheles larvae were more susceptible to ethanol extract of Ixora leaves than Culex larvae. Table (4.1) Percentage mortality of ethanolic extract of Ixora leaves on Anopheles and Culex Larvae and some of their aquatic predators (season 2013)
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Concentration Mortality (%) (mg/L) Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia Boatman beetle Naiad Fish larvae 6.69 20 7.5 5.9 12.5 5.7 5 13.38 25 10 10 20 5 5 20.07 65 20 20 40 40 10 26.76 85 35 35 40 60 20 33.45 95 45.7 45.5 60 69.5 40 Control 0 0 0 0 0 0 Log- Probit transformation concentration 0.825 4.16 3.59 3.45 3.82 3.59 3.36 1.126 4.33 3.72 3.72 4.16 3.36 3.36 1.303 5.39 4.16 4.16 4.75 4.75 3.72 1.427 6.08 4.61 4.61 4.75 5.25 4.16 1.524 6.64 4.90 4.90 5.25 5.52 4.75 Regression analysis R2 0.88 0.88 0.93 0.93 0.79 0.75 A 0.78 1.83 1.57 2.12 0.61 1.572 B 3.66 1.906 2.097 1.96 3.13 1.852 SE-Y 0.996 0.52 0.43 0.39 1.19 0.789 SE-X 0.787 0.412 0.34 0.31 0.94 0.624 LC50 (mg/L) 14.24 46.04 43.22 29.47 25.27 70.95 LC95 (mg/L) 39.96 333.89 261.66 202.36 84.43 545.13
4.1.2 Effect of aqueous extract of Ixora leaves (season 2013)
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The aqueous extract of Ixora leaves was tested at concentrations ranged between 12.71 to 63.55 mg/L. These concentrations resulted in 7.5% to 95.7% mortality in Anopheles larvae, 10% to 85.5% mortality in Culex larvae, 20% to 70.5% mortality in Hemipteran boatman,7.5% to 70.5% mortality in swimming beetle larvae, 10% to 80% mortality in Dragon fly naiads, and 5.5% to 65.5% mortality in Gumbosia fish (Table, 4.2)
The LC50 and LC95 were 12.9 and 33.0 (mg/L) for Anopheles larvae, 30.34 and 93.98, respectively, for Culex larvae which seemed to be very high compared to that of Anopheles
larvae. The LC50 and LC95 for the aquatic predators were 36.92 and 226.87 for Hemipteran boatman, 39.51 and 139.10 for swimming beetle larvae, 32.48 and 113.89 for Dragon fly
naiads, 119.84 and 703.33 for Gumbosia fish. The LC50 and LC95 of the aquatic predators seemed to be very high compared to that of mosquitoes' larvae. This finding indicated that, the aqueous extract of Ixora leaves can play an important role in mosquito control with slight effects on the aquatic predators. The Ixora leaves contain the Flavonoids, Saponins and Steroids. The regression analysis revealed that, the R2‘s were 0.99 for Gumbosia fish, 0.99 for Dragonfly 0.94 for swimming beetle larvae, 0.94 for Hemipteran boatman, while in Culex larvae it was 0.82 and 0.987 in Anopheles larvae. According to the obtained LC50 and LC95 values (Table 4.1 and Table 4.2), the ethanolic leaves extract was more potent compared to that of aqueous extract. The value was found in between (12.9 -33) mg/L for LC50 and LC95 receptively, These results compared to the aqueous were in agreement with Yousif (2013), and Culex larvae extracts. i.e. the Anopheles and Culex larvae and the tested aquatic predators were susceptible to wards different doses of aqueous extract of Ixora leaves.
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Table (4.2) Percentage mortality of aqueous extract of Ixora leaves on Anopheles and Culex Larvae and some aquatic predators (season 2013) Concentration Mortality (%) (mg/L) Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia Boatman beetle Naiad Fish larvae 12.71 7.5 10 20 7.5 10 5.5 25.42 20 30 30 20 40 20 38.13 35 65 45 60 60 40 50.84 65 70 65.5 62.5 70.5 60 63.55 95.7 85.5 70.5 70.5 80 65.5 Control 0 0 0 0 0 0 Log-concentration Probit transformation 1.104 3.59 3.72 4.16 3.59 3.72 3.45 1.405 4.16 4.48 4.48 4.16 4.75 4.16 1.580 4.61 5.39 4.87 5.25 5.25 4.75 1.706 5.39 5.52 5.41 5.33 5.55 5.25 1.803 6.75 6.08 5.55 5.55 5.84 5.41 Regression analysis R2 0.82 0.98 0.94 0.94 0.99 0.99 A 0.52 0.05 1.74 0.21 0.45 0.12 B 4.03 3.34 2.08 3.00 3.01 2.29 SE-Y 0.52 0.48 0.49 0.66 0.18 0.27 SE-X 0.05 0.31 0.32 0.43 0.12 0.17 LC50 (mg/L) 12.9 30.34 36.92 39.51 32.48 119.84 LC95 (mg/L) 33.0 93.98 226.87 139.10 113.89 703.33
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4.3 Effect of aqueous extract of Ixora flowers (season 2013) The aqueous extract of Ixora flowers was tested at concentrations ranged between 15.15 to 75.75 mg/L. These concentrations resulted in 30.5% to 95.5% mortality in Anopheles larvae, 40% to 90.5% mortality in Culex larvae, 10% to 67.5% mortality in Hemipteran boatman,7.5% to 45.7% mortality in swimming beetle larvae, 5% to 65% mortality in Dragonfly naiads and 5% to 60% mortality in Gumbosia fish (Table, 4.3)
The LC50 and LC95 were 17.88 and 104.10 (mg/L) for Anopheles larvae, 30.57 and 122.53, respectively, for Culex larvae which seemed to be very low compared to that of
Anopheles larvae. The LC50 and LC95 for the aquatic predators were 138.71 and 1012.19 for Hemipteran boatman, 106.39 and 810.22 for swimming beetle larvae, 53.49 and 210.12 for
Dragonfly naiads 61.28 and 250.76 for Gumbosia fish. The LC’s of the aquatic predators seemed to be high compared to that of mosquitoes' larvae. This finding indicated that, the aqueous extract of Ixora flowers can play an important role in mosquito control with effects on the aquatic predators. The Ixora flowers contain the Alkaloids, Saponins and Steroids. The regression analysis revealed that, the R2‘s were 0.88 for Gumbosia fish, 0.88 for Dragonfly 0.89 for swimming beetle larvae, 0.75 for Hemipteran boatman, while in Culex larvae it was 0.96 and 0.71 in Anopheles larvae.
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Table (4.3) Percentage mortality of aqueous extract of Ixora flowers on Anopheles and Culex Larvae and some of their aquatic predators (season 2013)
Concentration Mortality (%) (mg/L) Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia Boatman beetle Naiad Fish larvae 15.15 30.5 40 10 7.5 5 5 30.298 40 75 15 10 10 7.5 45.45 50 80 20 25 20 20 60.597 70 85 35 30 40 40 75.75 95.5 90.5 67.5 45.7 65 60 Control 0 0 0 0 0 0 Log-concentration Probit transformation
1.18 4.50 4.75 3.72 3.59 3.36 3.36 1.48 4.75 5.67 3.96 3.72 3.72 3.59 1.66 5.00 5.84 4.16 4.33 4.16 4.16 1.78 5.52 6.04 4.61 4.48 4.75 4.75 1.88 6.75 6.34 5.47 4.90 5.39 5.25
Regression analysis
R2 0.71 0.96 0.75 0.89 0.88 0.88 A 0.96 2.32 0.93 1.23 0.23 0.21 B 2.72 2.14 1.90 1.86 2.76 2.68 SE-Y 1.6 0.38 1.15 0.61 0.93 0.92 SE-X 1.0 0.24 0.72 038 0.58 0.57 LC50 (mg/L) 17.88 30.57 138.71 106.39 53.49 61.28 LC95 (mg/L) 104.10 122.53 1012.19 810.22 210.12 250.76
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4.4 Effect of ethanol extract of Ixora flowers (season 2013) The ethanol extract of Ixora flowers was tested at concentrations ranged between 5.18 to 25.89 mg/L. These concentrations resulted in 40.5% to 95% mortality in Anopheles larvae, 45% to 85.5% mortality in Culex larvae, 10.8% to 60% mortality in Hemipteran boatman, 20.5% to 65% mortality in swimming beetle larvae,10.5% to 65.7% mortality in Dragonfly naiads and 7.5% to 70% mortality in Gumbosia fish (Table, 4.4)
The LC50 and LC95 were 6.63 and 32.37 mg/L, for Anopheles larvae, 6.93 and 58.13, respectively, for Culex larvae which seemed to be relatively high compared to that of
Anopheles larvae. The LC50 and LC95 for the aquatic predators were 23.43 and 167.48 for Hemipteran boatman, 18.03 and 170.66 for swimming beetle larvae, 17.65 and 88.04 for
Dragonfly naiads, 17.10 and 66.13 for Gumbosia fish. The LC95 of the aquatic predators seemed to be high compared to that of mosquitoes' larvae. This finding indicated that, the ethanol extract of Ixora flowers can play an important role in mosquito control with slight effects on the aquatic predators. The regression analysis revealed that, the R2‘s were 0.88 for Gumbosia fish, 0.99 for Dragonfly, 0.93 for swimming beetle larvae, 0.95 for Hemipteran boatman, while in Culex larvae it was 0.98 and 0.94 in Anopheles larvae.
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Table (4.4) Percentage mortality of ethanol extract of Ixora flowers on Anopheles and Culex Larvae and some of their aquatic predators (season 2013) Concentration Mortality (%) (mg/L) Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia Boatman beetle Naiad Fish Larvae 5.18 40.5 45 10.8 20.5 10.5 7.5 10.36 65 60 25 30 30 20 15.54 75 75 35 40 40 45 20.72 85 80.7 40 55 60 60 25.89 95 85.5 60 65 65.7 70 Control 0 0 0 0 0 0 Log-concentration Probit transformation
0.71 4.77 4.87 3.77 4.19 3.77 3.59 1.02 5.39 5.25 4.33 4.48 4.48 4.16 1.19 5.67 5.62 4.61 4.75 4.75 4.87 1.32 6.04 5.88 4.75 5.13 5.25 5.25 1.41 6.64 6.08 5.25 5.39 5.41 5.52
Regression analysis
R2 0.94 0.98 0.95 0.93 0.99 0.98 A 2.94 3.57 2.37 2.89 2.07 1.47 B 2.45 1.74 1.92 1.68 2.35 2.84 SE-Y 0.42 0.16 0.29 0.30 0.18 0.29 SE-X 0.37 0.14 0.25 0.26 0.15 0.25 LC50 (mg/L) 6.63 6.93 23.43 18.03 17.65 17.10 LC95 (mg/L) 32.37 58.13 167.48 170.66 88.04 66.13
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4.5 Effect of Malathion insecticide (season 2013) The Malathion insecticide was tested at concentrations ranged between 0.046 to 0.3648 mg/L. These concentrations resulted in 60% to 95% mortality in Anopheles larvae, 60% to 90% mortality in Culex larvae, 10.7% to 90% mortality in Hemipteran boatman,25% to 80% mortality in swimming beetle larvae,40 % to 92% mortality in Dragon fly naiads and 10% to 80% mortality in Gumbosia fish (Table, 4.5)
The LC50 and LC95 were 0.79 and 40.26 mg/L, for Anopheles larvae,0.49 and 85.78, respectively, for Culex larvae which seemed to be higher compared to that of Anopheles larvae. The LC50 and LC95 for the aquatic predators were 11.94 and 49.04 for Hemipteran boatman, 11.21 and 94.43 for swimming beetle larvae, 3.47 and 41.04 for Dragon fly naiads,
16.41 and 90.43 for Gumbosia fish The LC50 and LC95 seemed to be very high compared to that of mosquitoes' larvae. This finding indicated that, the Malathion insecticide can play an important role in mosquito control with more effects on the aquatic predators. The regression analysis revealed that, the R2 were 0.98 for Gumbosia fish, 0.98 for Dragonfly 0.91 for swimming beetle larvae, 0.97 for Hemipteran boatman, while in Culex larvae it was 0.98 and 0.98 in Anopheles larvae. Results of this study showed that, Malathion was more toxic to Anopheles larvae and the aquatic predators compared to Ixora extracts. This result was in agreement with Kehail (1995) who found that, Malathion was more toxic against Anopheles than Culex. The United States Environmental Protection Agency (2006) found that Malathion is highly toxic to insects, including beneficial insects such as honeybees, and it is toxic to aquatic organisms, including fish and invertebrates.
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Table (4.5) Percentage mortality of Malathion insecticide on Anopheles and Culex Larvae and some of their aquatic predators (season 2013) Concentration Mortality (%) (mg/L) Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia Boatman beetle Naiad Fish larvae 0.0456 60 60 10.7 25 40 10 0.0912 80 75 20 25 70 20 0.1824 85 82 65 65 85 45 0.2736 90 85 80 75 90 65 0.3648 95 90 90 80 92 80 Control 0 0 0 0 0 0 Log-concentration Probit transformation -1.34 5.25 5.25 3.77 4.33 4.75 3.72 -1.04 5.84 5.67 4.16 4.33 5.52 4.16 -0.74 6.04 5.92 5.39 5.39 6.04 4.87 -0.56 6.28 6.04 5.84 5.67 6.28 5.39 -0.44 6.64 6.28 6.28 5.84 6.41 5.84
Regression analysis
R2 0.98 0.98 0.97 0.91 0.98 0.98 a 4.38 4.58 1.67 2.87 3.65 2.04 b 1.39 1.06 2.91 1.9 1.83 2.34 SE-Y 0.203 0.11 0.35 0.42 0.19 0.26 SE-X 0.17 0.088 0.29 0.35 0.16 0.21 LC50 (mg/L) 0.79 0.49 11.94 11.21 3.47 16.41 LC95 (mg/L) 40.26 85.78 49.04 94.43 41.04 90.43
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4.6 Effect of aqueous extract of Ixora leaves (season 2014) The aqueous extract of Ixora leaves was tested at concentrations ranged between 11.13 to 55.65 mg/L. These concentrations resulted in 15.7% to 92.5% mortality in Anopheles larvae, 12.5% to 75.7% mortality in Culex larvae, 10% to 45% mortality in Hemipteran boatman, 20.5% to 60% mortality in swimming beetle larvae, 7.5% to 72% mortality in Dragonfly naiads and 5.7% to 42.5% mortality in Gumbosia fish (Table, 4.6)
The LC50 and LC95 were 26.19 and 82.25 mg/L, for Anopheles larvae, 36.97 and 183.11, respectively, for Culex larvae which seemed to be high compared to that of
Anopheles larvae. The LC50 and LC95 for the aquatic predators were 83.13 and 855.26 for Hemipteran boatman, 49.18 and 653.23 for swimming beetle larvae, 38.51 and 128.70 for
Dragonfly naiads, 96.28 and 766.68 for Gumbosia fish. The LC50 and LC95 of the aquatic predators seemed to be high compared to that of mosquitoes' larvae. This finding confirmed the result of season 2013 that, the aqueous extract of Ixora leaves can play an important role in mosquito control with slight effects on the aquatic predators. The regression analysis revealed that, the R2‘s were 0.88 for Gumbosia fish, 0.88 for Dragonfly, 0.89 for swimming beetle larvae, 0.89 for Hemipteran boatman, while in Culex larvae it was 0.92 and 0.90 in Anopheles larvae. According to the LC50 and LC95 values (Table 4.6), the aqueous leaves extract in season 2013 was more effective compared to that of this season. This result was in agreement with Yousif (2013), who observed that, the ethanolic extract of both flowers and leaves , showed promising results against Anopheles and Culex larvae compared to the aqueous extracts. i.e. the Anopheles and Culex larvae and the tested aquatic predators were susceptible towards different doses of aqueous extract of Ixora leaves.
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Table (4.6) Percentage mortality of aqueous extract of Ixora leaves on Anopheles and Culex Larvae and some aquatic predators (2014)
Concentration Mortality (%) (mg/L) Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia Boatman beetle Naiad Fish larvae 11.13 15.7 12.5 10 20.5 7.5 5.7 22.26 30 30 15 25 10 10 33.39 60 40 20 40 45 15 44.52 70 50 35 45 65 25 55.65 92.5 75.7 45 60 72 42.5 Control 0 0 0 0 0 0 Log-concentration Probit transformation
1.05 4.01 3.87 3.72 4.19 3.59 3.45 1.35 4.48 4.48 3.96 4.33 3.72 3.72 1.52 5.25 4.75 4.16 4.75 4.87 3.96 1.65 5.52 5.00 4.61 4.87 5.39 4.33 1.75 6.48 5.71 4.87 5.25 5.55 4.82
Regression analysis R2 0.90 0.92 0.89 0.89 0.88 0.88 a 0.32 1.3 1.89 2.53 0.037 1.39 b 3.30 2.36 1.62 1.46 3.13 1.82 SE-Y 0.92 0.59 0.47 0.43 0.99 0.58 SE-X 0.62 0.39 0.32 0.29 0.67 0.39 LC50 (mg/L) 26.19 36.97 83.13 49.18 38.51 96.28 LC95 (mg/L) 82.25 183.11 855.26 653.23 128.70 766.68
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4.7 Effect of aqueous extract of Ixora flowers (season 2014) The aqueous extract of Ixora flowers was tested at concentrations ranged between 17.08 to 85.38 (mg/L). These concentrations resulted in 35% to 92% mortality in Anopheles larvae, 20.5% to 80% mortality in Culex larvae, 15% to 62.5% mortality in Hemipteran boatman, 10.5% to 65% mortality in swimming beetle larvae, 7.5% to 67.5% mortality in Dragonfly naiads and 5% to 65.5% mortality in Gumbosia fish (Table, 4.7)
The LC50 and LC95 were 32.68 and 152.64 (mg/L), for Anopheles larvae, and 52.39 and 336.58 for Culex larvae, respectively. LC’s for Culex seemed to be high compared to that of Anopheles larvae. The LC50 and LC95 for the aquatic predators were 90.79 and 871.20 for Hemipteran boatman, 82.01 and 499.53 for swimming beetle larvae, 33.61 and 117.36 for Dragonfly naiads, 43.32 and 160.01 for Gumbosia fish. The LC’s of aquatic predators seemed to be high compared to those of mosquitoes' larvae. This finding indicated that, the aqueous extract of Ixora flowers can play an important role in mosquito control with some effects on the aquatic predators (as was indicated in season 2013). The regression analysis revealed that, the R2’s were 0.92 for Gumbosia fish, 0.84 for Dragonfly 0.83 for swimming beetle larvae, 0.75 for Hemipteran boatman, while in Culex larvae it was 0.79 and 0.76 in Anopheles larvae. According to the LC50 and LC95 values (Table 4.3 and Table4.7), the aqueous flowers extract in the season 2013 was more effective compared to that of aqueous flowers extract in this season. i.e. the same doses resulted in significantly different mortalities, and also, there was a significant difference in the rows level (T=0.5 to 0.55), i.e. Anopheles and Culex larvae were more susceptible to wards different doses of t the aqueous extract of Ixora flowers.
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Table (4.7) Percentage mortality of aqueous extract of Ixora flowers on Anopheles and Culex Larvae and some of their aquatic predators (2014)
Concentratio Mortality (%) n Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia (mg/L) Boatman beetle Naiad Fish Larvae 17.08 35 20.5 15 10.5 7.5 5 34.15 40 30 20 15 10 10 51.23 50 40 25 30 30 25 68.3 80 50 35 35 50 45 85.38 92 80 62.5 65 67.5 65.5 Control 0 0 0 0 0 0 Log- concentration Probit transformation
1.23 4.61 4.19 3.96 3.77 3.59 3.36 1.53 4.75 4.48 4.16 3.96 3.72 3.72 1.71 5.00 4.75 4.33 4.48 4.48 4.33 1.83 5.84 5.00 4.61 4.61 5.00 4.87 1.93 6.41 5.84 5.33 5.39 5.77 5.41
Regression analysis
R2 0.76 0.79 0.75 0.83 0.84 0.92 a 1.29 1.51 1.73 1.00 0.39 0.27 b 2.45 2.03 1.67 2.09 3.02 2.89 SE-Y 1.31 0.99 0.93 0.92 1.26 0.84 SE-X 0.78 0.59 0.56 0.55 0.75 0.50 LC50 (mg/L) 32.68 52.39 90.79 82.01 33.61 43.32 LC95 (mg/L) 152.64 336.58 871.20 499.53 117.36 160.01
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4.8 Effect of ethanol extract of Ixora flowers (season 2014)
The ethanol extract of Ixora flowers was tested at concentrations ranged between 7.35 to 36.76 (mg/L). These concentrations resulted in 10% to 90% mortality in Anopheles larvae, 20% to 80.5% mortality in Culex larvae, 7.5% to 80% mortality in Hemipteran boatman, 12.5% to 82% mortality in swimming beetle larvae, 10. 5% to 78% mortality in Dragonfly naiads and 10% to 75% mortality in Gumbosia fish (Table, 4.8)
The LC50 and LC95 were 16.87 and 48.02 (mg/L) for Anopheles larvae, 17.20 and 81.89, respectively, for Culex larvae which seemed to be relatively higher compared to that of Anopheles larvae. The LC50 and LC95 for the aquatic predators were: 22.79 and 66.03 for Hemipteran boatman, 15.91and 48.80 for swimming beetle larvae, 18.22 and 68.87 for
Dragonfly naiads, 27.41 and 118.48 for Gumbosia fish. In aquatic predators, the LC50 and
LC95 seemed to be higher compared to that of mosquitoes' larvae. This finding indicated that, the ethanol extract of Ixora flowers extracts can play an important role in mosquito control with slight effects on the aquatic predators. The regression analysis revealed that, the R2‘s were 0.89 for Gumbosia fish, 0.97 for Dragonfly, 0.99 for swimming beetle larvae, 0.87 for Hemipteran boatman, while in Culex and Anopheles larvae it was 0.99. According to the LC50 and LC95 values (Table 4.4 and Table 4.8), the ethanol flowers extract in the season 2013 was relatively more effective compared to that of ethanol flowers extract in this season (2014).
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Table (4.8) Percentage mortality of ethanol extract of Ixora flowers on Anopheles and Culex Larvae and some of their aquatic predators (2014)
Concentration Mortality (%) (mg/L)
Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia Boatman beetle Naiad Fish Larvae 7.35 10 20 7.5 12.5 10.5 10 14.70 40 40 10 45 45 20 22.06 65 60 45 72 65 30 29.41 80 70 75 79 70.5 50 36.76 90 80.5 80 82 78 75 Control 0 0 0 0 0 0 Log- concentration Probit transformation 0.87 3.72 4.16 3.59 3.87 3.77 3.72 1.17 4.75 4.75 3.72 4.87 4.87 4.16 1.34 5.39 5.25 4.87 5.55 5.39 4.48 1.47 5.84 5.52 5.67 5.81 5.55 5.00 1.57 6.28 5.88 5.84 6.28 5.77 5.67
Regression analysis R2 0.99 0.99 0.87 0.99 0.97 0.89 A 0.57 2.01 0.18 0.95 1.42 1.29 B 3.61 2.42 3.55 3.37 2.84 2.58 SE-Y 0.10 0.18 1.03 0.18 0.36 0.68 SE-X 0.08 0.14 0.79 0.14 0.27 0.52 LC50 (mg/L) 16.87 17.20 22.79 15.91 18.22 27.41 LC95 (mg/L) 48.02 81.89 66.03 48.80 68.87 118.48
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4.9 Effect of ethanol extract of Ixora leaves (season 2014) The tested concentrations ranged between 9.12 to 45.53 mg/L. These concentrations resulted in 20% to 95% mortality in Anopheles larvae, 10% to 75% mortality in Culex larvae, 15.5% to 72 mortality in Hemipteran boatman,10% to 75% mortality in swimming beetle larvae, 20.5% to 75% mortality in Dragon fly naiads, and 10.5% to 70% mortality in Gumbosia fish (Table, 4.9)
The LC50 and LC95 were 19.29 and 58.72 mg/L, for Anopheles larvae, 35.61 and 159.36, respectively, for Culex larvae which seemed to be high compared to that of
Anopheles larvae. The LC50 and LC95 for the aquatic predators were 24.92 and 118.68 for Hemipteran boatman ,32.19 and 139.13 for swimming beetle larvae, 21.47 and 125.35 for
Dragon fly naiads, 29.85 and 128.26 for Gumbosia fish The LC50 and LC95seemed to be very high compared to that of mosquitoes' larvae. This finding indicated that, the ethanol extract of Ixora can play an important role in mosquito control with slight effects on the aquatic predators. The Ixora leaves contain the lupeol, ursolic acid, oleanlic acid, sitosterol, rutin, lecocyanadin, anthocyanins, proanthocyanidins, and glycosides of kaempferol, and quercetiin (Baliga and Kurian, 2012). In a similar research, El Mahi (2014) found that, Ixora extracts were toxic against mosquito larvae, and Anopheles larvae were more susceptible towards ethanol extract of Ixora leaves than Culex larvae.
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Table (4.9) Percentage mortality of ethanol extract of Ixora leaves on Anopheles and Culex Larvae and some of their aquatic predators (2014)
Concentration Mortality (%) (mg/L) Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia Boatman beetle Naiad Fish larvae 9.12 20 10 15.5 10 20.5 10.5 18.21 40 35 30.7 20 45 25 27.32 60 50 55 40.5 60 40 36.42 80 65 70 55 70 65 45.53 95 75 72 70 75 70 Control 0 0 0 0 0 0 Log- concentration Probit transformation
0.96 4.16 3.72 4.01 3.72 4.19 3.77 1.26 4.75 4.61 4.50 4.16 4.87 4.33 1.44 5.25 5.00 5.13 4.77 5.25 4.75 1.56 5.84 5.39 5.52 5.13 5.52 5.39 1.66 6.64 5.67 5.58 5.52 5.67 5.52
Regression analysis
R2 0.92 0.99 0.97 0.96 0.99 0.96 A 0.75 1.09 1.62 1.11 2.15 1.18 B 3.33 2.52 2.42 2.58 2.14 2.59 SE-Y 0.82 0.11 0.33 0.41 0.08 0.41 SE-X 0.58 0.08 0.24 0.29 0.06 0.29 LC50 (mg/L) 19.29 35.61 24.92 32.19 21.47 29.85 LC95 (mg/L) 58.72 159.36 118.68 139.13 125.35 128.26
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4.10 Effect of Malathion insecticide on Anopheles and Culex larvae and some aquatic predators in the season 2014 The Malathion insecticide showed promising results against Anopheles The tested concentrations ranged between 0.046 to 0.3648 mg/L. These concentrations resulted in 45.5% to 97% mortality in Anopheles larvae, 40.5% to 95.7% mortality in Culex larvae, 20% to 90% mortality in Hemipteran boatman,10% to 85% mortality in swimming beetle larvae,20 % to 85.5% mortality in Dragon fly naiads and 7.5% to 65% mortality in Gumbosia fish (Table, 4.10)
The LC50 and LC95 were 2.53 and 31.40 mg/L, for Anopheles larvae, 2.89 and 32.58,
respectively, for Culex larvae. The LC50 and LC95 for the aquatic predators were 10.43 and 52.97 for Hemipteran boatman ,12.79 and 59.78 for swimming beetle larvae, 9.75 and 69.18
for Dragon fly naiads, 32.65 and 253.5 for Gumbosia fish The LC50 and LC95 seemed to be very high compared to that of mosquitoes' larvae. This finding indicated that, the Malathion insecticide can play an important role in mosquito control with more effects on the aquatic predators. The regression analysis revealed that, the R2 were 0.86 for Gumbosia fish, 0.998 for Dragonfly 0.99 for swimming beetle larvae, 0.93 for Hemipteran boatman , while in Culex larvae it was 0.92 and 0.92 in Anopheles larvae According to the LC50 and LC95 values (Table 4.5 and Table 4.10), The Malathion insecticide in the season 2013 was very converged effective compared to that of The Malathion insecticide in this season. Results of this study showed that, Malathion was more toxic to Anopheles larvae and the aquatic predators compared to Ixora extracts. This result was in agreement with Kehail (1995) who found that, Malathion was more toxic against Anopheles. The US Environmental Protection Agency (2006) found that Malathion is highly toxic to insects, including beneficial insects such as honeybees, and it is toxic to aquatic organisms, including fish and invertebrates.
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Table (4.10) Percentage mortality of Malathion insecticide on Anopheles and Culex Larvae and some of their aquatic predators (2014)
Concentration Mortality (%) (mg/L) Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia Boatman beetle Naiad Fish larvae 0.046 45.5 40.5 20 10 20 7.5 0.0912 80 80 25 25 40 10 0.1824 85 85 60 65 65 20 0.2736 90 90 85 75 77 40 0.3648 97 95.7 90 85 85.5 65 Control 0 0 0 0 0 0 Log-concentration Probit transformation -1.34 4.90 4.77 4.16 3.72 4.16 3.59 -1.04 5.84 5.84 4.33 4.33 4.75 3.72 -0.74 6.04 6.04 5.25 5.39 5.39 4.16 -0.56 6.28 6.28 6.04 5.67 5.74 4.75 -0.44 6.88 6.75 6.28 6.04 6.08 5.39
Regression analysis R2 0.92 0.92 0.93 0.99 0.99 0.86 A 3.76 3.67 2.22 1.93 2.75 2.09 B 1.89 1.93 2.54 2.63 2.10 1.89 SE-Y 0.38 0.39 0.47 0.197 0.067 0.55 SE-X 0.32 0.32 0.38 0.16 0.05 0.45 LC50 (mg/L) 2.53 2.89 10.43 12.79 9.75 32.65 LC95 (mg/L) 31.40 32.58 52.97 59.78 69.18 253.5
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4.11 Effect of ethanol extract of Ixora leaves (season 2015) The tested concentrations ranged between 5.91 to 29.53 mg/L. These concentrations resulted in 25% to 97% mortality in Anopheles larvae, 20% to 95% mortality in Culex larvae, 10.5% to 90 mortality in Hemipteran boatman,12.5% to 85% mortality in swimming beetle larvae, 15% to 80% mortality in Dragonfly naiads, and 5.7% to 65.7% mortality in Gumbosia fish (Table, 4.11).
The LC50 and LC95 were 12.20 and 39.14 mg/L, for Anopheles larvae, 10.88 and
31.52, respectively, for Culex larvae. The LC50 and LC95, respectively, for the aquatic predators were 13.47 and 39.86 for Hemipteran boatman, 16.33 and 56.34 for swimming beetle larvae, 15.49 and 63.42 for Dragonfly naiads,and 21.544 and 79.94 for Gumbosia fish. This finding indicated that, the ethanol extract of Ixora leaves extracts can play an important role in mosquito control with slight effects on the aquatic predators. The regression analysis revealed that, the R2‘s were 0.98 for Gumbosia fish, for Dragonfly and for swimming beetle larvae, 0.99 for Hemipteran boatman, while in Culex larvae it was 0.97 and 0.79 in Anopheles larvae. According to the LC50 and LC95 values (Table 4.1 and Table 4.9), the ethanolic leaves extract in the season 2013 and 2014 was more effective compared to that of aqueous leaves extract in this season.
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Table (4.11) Percentage mortality of ethanol extract of Ixora leaves on Anopheles and Culex Larvae and some of their aquatic predators (2015)
Concentration Mortality (%) (mg/L) Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia Boatman beetle Naiad Fish larvae 5.91 25 20 10.5 12.5 15 5.7 11.81 35 50 40 20 35 20 17.72 55 70 70 60 50 35 23.62 75 90 75 60.5 70.5 60 29.53 97 95 90 85 80 65.7 Control 0 0 0 0 0 0 Log- concentration Probit transformation 0.77 4.33 4.16 3.77 3.87 3.96 3.45 1.07 4.61 5.00 4.75 4.16 4.61 4.16 1.25 5.13 5.52 5.52 5.25 5.00 4.61 1.37 5.67 6.28 5.67 5.28 5.55 5.25 1.47 6.88 6.64 6.28 6.04 5.84 5.41
Regression analysis
R2 0.79 0.97 0.99 0.89 0.98 0.98 A 1.48 1.32 1.07 1.30 1.81 1.16 B 3.24 3.55 3.48 3.05 2.68 2.88 SE-Y 1.18 0.40 0.26 0.73 0.28 0.29 SE-X 0.97 0.33 0.23 0.60 0.23 0.25 LC50 (mg/L) 12.20 10.88 13.47 16.33 15.49 21.54 LC95 (mg/L) 39.14 31.52 39.86 56.34 63.42 79.94
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4.12 Effect of aqueous extract of Ixora leaves (season 2015) The tested concentrations ranged between 12.98 to 64.90 mg/L. These concentrations resulted in 20% to 90% mortality in Anopheles larvae, 30% to 87.5% mortality in Culex larvae, 15% to 70.7% mortality in Hemipteran boatman, 20% to 72% mortality in swimming beetle larvae, 20% to 75% mortality in Dragonfly naiads, and 15% to 62.7 % mortality in Gumbosia fish (Table, 4.12).
The respective LC50 and LC95 were 29.22 and 114.23 mg/L for Anopheles larvae, 25.17 and 128.16 for Culex larvae which seemed to be equivalent to that of Anopheles larvae.
The LC50 and LC95 for the aquatic predators were: 44.83 and247.55 for Hemipteran boatman, 37.21 and 268.73 for swimming beetle larvae, 31.79 and 181.16 for Dragonfly naiads and 61.17 and 510.35 for Gumbosia fish.
The LC50 and LC95 values for the tested aquatic predators seemed to be very high compared to that of mosquitoes' larvae. This finding indicated that, the aqueous extract of Ixora leaves can play an important role in mosquito control with slight effects on the aquatic predators. The regression analysis revealed that, the R2‘s were: 0.84 for Gumbosia fish, 0.99 for Dragonfly, 0.96 for swimming beetle larvae, 0.91 for Hemipteran boatman, while in Culex larvae it was 0.92 and 0.92 in Anopheles larvae. According to the LC50 and LC95 values (Table 4.2 and Table 4.6), the aqueous leaves extract (at 2013) was more effective compared to that of aqueous leaves extract in this season.
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Table (4.12) Percentage mortality of aqueous extract of Ixora leaves on Anopheles and Culex Larvae and some of their aquatic predators (2015)
Concentrati Mortality (%) on Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia (mg/L) Boatman beetle Naiad Fish larvae 12.98 20 30 15 20 20 15 25.96 40 45 25 40 40 20 38.94 55 60 35 45 60 30 51.92 70.5 75.7 57.5 60.5 67 40.5 64.90 90 87.5 70.7 72 75 62.7 Control 0 0 0 0 0 0 Log- concentrati Probit transformation on 1.11 4.16 4.48 3.96 4.16 4.16 3.96 1.41 4.75 4.87 4.33 4.75 4.75 4.16 1.59 5.13 5.25 4.61 4.87 5.25 4.48 1.72 5.55 5.71 5.20 5.28 5.44 4.77 1.81 6.28 6.18 5.55 5.58 5.67 5.33
Regression analysis
R2 0.92 0.92 0.91 0.96 0.99 0.84 A 0.94 1.75 1.35 2.00 1.74 1.82 B 2.77 2.32 2.21 1.91 2.17 1.78 SE-Y 0.73 0.59 0.62 0.35 0.14 0.68 SE-X 0.47 0.38 0.40 0.23 0.09 0.44 LC50 29.22 25.17 44.83 37.21 31.79 61.17 (mg/L) LC95 114.23 128.16 247.55 268.73 181.16 510.35 (mg/L)
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4.13 Effect of aqueous extract of Ixora flowers (season 2014) The aqueous extract of Ixora flowers was tested at concentrations ranged between 16.11 to 80.55 mg/L. These concentrations resulted in 30.5% to 95% mortality in Anopheles larvae, 20% to 90.5% mortality in Culex larvae, 7.5% to 65% mortality in Hemipteran boatman, 5% to 65.5% mortality in swimming beetle larvae, 10.5% to 62% mortality in Dragonfly naiads, and 10% to 60.5% mortality in Gumbosia fish (Table, 4.13)
The respective LC50 and LC95 were: 31.23 and 122.08 mg/L for Anopheles larvae, 38.71 and 149.84 for Culex larvae (which seemed to be relatively more compared to that of
Anopheles larvae). The respective LC50 and LC95 for the aquatic predators were: 83.96 and 413.08 for Hemipteran boatman, 64.11 and 275.52 for swimming beetle larvae, 75.31 and 483.87 for Dragonfly naiads, 83.84 and 510.66 for Gumbosia fish (which seemed to be very high compared to that of mosquitoes' larvae). This finding indicated that, the aqueous extract of Ixora flowers can play an important role in mosquito control with slight effects on the aquatic predators. The regression analysis revealed that, the R2‘s were: 0.82 for Gumbosia fish, 0.92 for Dragonfly, 0.80 for swimming beetle larvae and for Hemipteran boatman, while in Culex larvae it was 0.86 and 0.82 in Anopheles larvae. According to the LC50 and LC95 values (Table 4.3 and Table 4.7), the aqueous flowers extract in the season 2013 was more effective compared to that of aqueous flowers extract in this season.
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Table (4.13) Percentage mortality of aqueous extract of Ixora flowers on Anopheles and Culex Larvae and some of their aquatic predators (2015) Concentration Mortality (%) (mg/L) Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia Boatman beetle Naiad Fish larvae 16.11 30.5 20 7.5 5 10.5 10 32.22 40 35 10 10 20 15 48.33 60 50 20 15 30 20 64.44 75 65 35 30 40 40 80.55 95 90.5 65 65.5 62 60.5 Control 0 0 0 0 0 0 Log- concentration Probit transformation 1.21 4.50 4.16 3.59 3.36 3.77 3.72 1.51 4.75 4.61 3.72 3.72 4.16 3.96 1.68 5.25 5.00 4.16 3.96 4.48 4.16 1.81 5.67 5.39 4.61 4.48 4.75 4.75 1.91 6.64 6.34 5.39 5.41 5.31 5.28
Regression analysis
R2 0.82 0.86 0.80 0.80 0.92 0.82 A 0.86 0.57 0.44 0.32 1.19 0.98 B 2.77 2.79 2.37 2.59 2.03 2.09 SE-Y 1.24 1.07 1.13 1.21 0.57 0.91 SE-X 0.75 0.65 0.69 0.74 0.35 0.55 LC50 (mg/L) 31.23 38.71 83.96 64.11 75.31 83.84 LC95 (mg/L) 122.08 149.84 413.08 275.52 483.87 510.66
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4.14 Effect of ethanol extract of Ixora flowers (season 2015) The ethanol extract of Ixora flowers was tested at concentrations ranged between 6.45 to 32.25 mg/L. These concentrations resulted in 10.7% to 95% mortality in Anopheles larvae, 20.5% to 85% mortality in Culex larvae, 10% to 75.7% mortality in Hemipteran boatman, 10.7% to 65% mortality in swimming beetle larvae, 20% to 80% mortality in Dragonfly naiads, and 7.5% to 70% mortality in Gumbosia fish (Table, 4.14)
The respective LC50 and LC95 were 14.07 and 37.63 mg/L for Anopheles larvae, 15.33
and 70.27 for Culex larvae. The LC50 and LC95 for the aquatic predators were: 19.80 and 78.97 for Hemipteran boatman, 24.11 and 129.15 for swimming beetle larvae, 14.61 and 69.10 for Dragonfly naiads, and 20.32 and 75.06 for Gumbosia fish.
The LC50 and LC95‘s of the tested aquatic predators seemed to be high compared to that of mosquitoes' larvae. This finding indicated that, the ethanol extract of Ixora flowers can play an important role in mosquito control with slight effects on the aquatic predators. The regression analysis revealed that, the R2‘s were 0.99 for Gumbosia fish, and for Dragonfly, 0.98 for swimming beetle larvae, and for Hemipteran boatman, while in Culex larvae it was 0.93 and 0.97 in Anopheles larvae.
According to the LC50 and LC95 values (Table 4.4 and Table 4.8), the ethanol extract of Ixora flowers in this season was more effective compared to that of 2014 and less effective than in the season 2013. In comparison with other natural products used to control mosquitoes, and according
to the LC50 values, the ethanolic extracts of Ixora leaves and flowers were better than Ipoemea helderbranditi flowers (377.12 and 1760.41 mg/L) and Punica granatum pericarps (1266.97 and 2310.13 mg/L; Kehail, 2004), but they were less efficient than Ushar latex (0.028 and 1.903 mg/L; Masaad, 2010), Ricinus communis seeds (74.14 and 84.81 mg/L) and Ocimum Basilicum flowers (93.98 and 771.79 mg/L; Kehail, 2004), respectively, against Anopheles and Culex larvae.
Table (4.14) Percentage mortality of ethanol extract of Ixora flowers on Anopheles and Culex Larvae and some aquatic predators (2015)
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Concentration Mortality (%) (mg/L) Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia Boatman beetle Naiad Fish larvae 6.45 10.7 20.5 10 10.7 20 7.5 12.90 45 40 30 25 45 25 19.35 65 50 45 37.5 60 50 25.80 80 70 60 50.7 75 65 32.25 95 85 75.7 65 80 70 Control 0 0 0 0 0 0 Log- concentration Probit transformation
0.81 3.77 4.19 3.72 3.77 4.16 3.59 1.11 4.87 4.75 4.48 4.33 4.87 4.33 1.29 5.39 5.00 4.87 4.69 5.25 5.00 1.41 5.84 5.52 5.25 5.03 5.67 5.39 1.51 6.64 6.04 5.71 5.39 5.84 5.52
Regression analysis
R2 0.97 0.93 0.98 0.98 0.99 0.99 A 0.59 2.06 1.46 1.89 2.17 1.22 B 3.84 2.48 2.73 2.25 2.43 2.89 SE-Y 0.45 0.49 0.24 0.22 0.12 0.21 SE-X 0.36 0.39 0.19 0.18 0.09 0.17 LC50 (mg/L) 14.07 15.33 19.80 24.11 14.61 20.32 LC95 (mg/L) 37.63 70.27 78.97 129.15 69.10 75.06
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4.15 Effect of Malathion insecticide on Anopheles and Culex larvae and some aquatic predators in the season 2015 The tested concentrations ranged between 0.046 to 0.3648 mg/L. These concentrations resulted in 30.5% to 95% mortality in Anopheles larvae, 45.5% to 90% mortality in Culex larvae, 17.5% to 85% mortality in Hemipteran boatman,15.7% to 87% mortality in swimming beetle larvae, 20 % to 86% mortality in Dragon fly naiads, and 5.7% to 25% mortality in Gumbosia fish (Table, 4.15)
The LC50 and LC95 were 5.11 and 46.85 mg/L, for Anopheles larvae, 3.598 and 62.04, respectively, for Culex larvae which seemed to be approximately compared to that of
Anopheles larvae. The LC50 and LC95 for the aquatic predators were 11.09 and 73.86 for Hemipteran boatman ,11.74 and 67.95 for swimming beetle larvae, 8.61 and 73.596 for
Dragon fly naiad, 26.84 and 355.55 for Gumbosia fish The LC50 and LC95 seemed to be same compared to that of mosquitoes' larvae. This finding indicated that, the Malathion insecticide can play an important role in mosquito control with more effects on the aquatic predators. The regression analysis revealed that, the R2 were 0.99 for Gumbosia fish, 0.95 for Dragonfly 0.99 for swimming beetle larvae, 0.99 for Hemipteran boatman , while in Culex larvae it was 0.99 and 0.88 in Anopheles larvae According to the LC50 and LC95 values (Table 4.5 and Table 4.10), The Malathion insecticide in the season 2013 and 2014 was very converged effective compared to that of The Malathion insecticide in this season. Results of this study showed that, Malathion was more toxic to Anopheles larvae and the aquatic predators compared to Ixora extracts. This result was in agreement with Kehail 1995 who found that, Malathion was more toxic against Anopheles. The United States Environmental Protection Agency (2006) found that Malathion is highly toxic to insects, including beneficial insects such as honeybees, and it is toxic to aquatic organisms, including fish and invertebrates.
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Table (4.15) Percentage mortality of Malathion insecticide on Anopheles and Culex Larvae and some of their aquatic predators (2015)
Concentration Mortality (%) (mg/L) Anopheles Culex Hemipteran Swimming Dragonfly Gumbosia Boatman beetle Naiad Fish larvae 0.046 30.5 45.5 17.5 15.7 20 5.7 0.0912 70 60 35 30 35 10 0.1824 75 80 60 55 52 15 0.2736 80 85 75 75 69 20 0.3648 95 90 85 87 86 25 Control 0 0 0 0 0 0 Log- Probit transformation concentration -1.34 4.50 4.90 4.08 4.01 4.16 3.45 -1.04 5.52 5.25 4.61 4.48 4.61 3.72 -0.74 5.67 5.84 5.25 5.13 5.05 3.96 -0.56 5.84 6.04 5.67 5.67 5.81 4.16 -0.44 6.64 6.28 6.04 6.13 6.08 4.33
Regression analysis
R2 0.88 0.99 0.99 0.99 0.95 0.99 A 3.33 3.84 2.598 2.36 2.62 2.81 B 1.96 1.55 2.15 2.32 2.14 1.5 SE-Y 0.52 0.10 0.13 0.26 0.33 0.06 SE-X 0.42 0.08 0.11 0.21 0.37 0.05 LC50 (mg/L) 5.11 3.598 11.09 11.74 8.61 26.84 LC95 (mg/L) 46.85 62.04 73.86 67.95 73.596 355.55
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Table (4.16) Samary of the LC50 OF lexora ectract on mosquito anopheles and culex and some of their aquatic predators) 2013-2015(
Season LC50 M /L Anophel Culex Hemipteran Swimming Dragonfly Gumbosia es Boatman beetle Naiad Fish larvae 2013 Table (4.1) 14.24 46.04 43.22 29.47 25.27 70.95 table(4.2) 12.9 30.34 36.92 39.51 32.48 119.84 table(4.3) 17.88 30.57 138.71 106.39 53.49 61.28 table(4.4) 6.63 6.93 23.43 18.03 17.65 17.10 table(4.5) 0.79 0.49 11.94 11.21 3.47 16.41 2014 table(4.6) 26.19 36.97 83.13 49.18 38.51 96.28 table(4.7) 32.68 52.39 90.79 82.01 33.61 43.32 table(4.8) 16.87 17.20 22.79 15.91 18.22 27.41 table(4.9) 19.29 35.61 24.92 32.19 21.47 29.85 table(4.10) 2.53 2.89 10.43 12.79 9.75 32.65 2015 table(4.11) 12.20 10.88 13.47 16.33 15.49 21.54 Table(4.12) 29.22 25.17 44.83 37.21 31.79 61.17 Table(4.13) 31.23 38.71 83.96 64.11 75.31 83.84 Table(4.14) 14.07 15.33 19.80 24.11 14.61 20.32 Table(4.15) 5.11 3.598 11.09 11.74 8.61 26.84
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4.16 Samary of the LC50 OF lexora ectract on mosquito anopheles and culex and some of their aquatic predators) 2013-2015(
According to the obtained LC50 values (Table 4.1 and Table 4.2), the ethanolic leaves extract was more potent compared to that of aqueous extract. According to the LC50 values (Table 4.6), the aqueous leaves extract in season 2013 was more effective compared to that of this season(2014). According to the LC50 and LC95 values (Table 4.3 and Table4.7), the aqueous flowers extract in the season 2013 was more effective compared to that of aqueous flowers extract in this season(2014). According to the LC50 values (T able 4.4 and Table 4.8), the ethanol flowers extract in the season 2013 was relatively more effective compared to that of ethanol flowers extract in this season (2014). According to the LC50 values (Table 4.5 and Table 4.10), The Malathion insecticide in the season 2013 was very converged effective compared to that of The Malathion insecticide in this season(2014). According to the LC50 values (Table 4.1 and Table 4.9), the ethanolic leaves extract in the season 2013 and 2014 was more effective compared to that of aqueous leaves extract in this season(2015). According to the LC50 values (Table 4.2 and Table 4.6), the aqueous leaves extract (at 2013) was more effective compared to that of aqueous leaves extract in this season(2015). According to the LC50 values (Table 4.3 and Table 4.7), the aqueous flowers extract in the season 2013 was more effective compared to that of aqueous flowers extract in this season(2015).
According to the LC50 values (Table 4.4 and Table 4.8), the ethanol extract of Ixora flowers in this season was more effective compared to that of 2014 and less effective than in the season (2015). According to the LC50 values (Table 4.5 and Table 4.10), The Malathion insecticide in the season 2013 and 2014 was very converged effective compared to that of The Malathion insecticide in this season(2015).
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4.17 Phytochemical composition of Ixora leaves and flowers The qualitative phytochemical screening (in terms of presence (+) and absence (-) of the main class) of Ixora leaves and flowers samples were presented in Table (4.16). The leaves containes flavonids, saponins and steroids, while tannins, alkaloids and glycosides were not detected, whereas flowers contain saponins, steroids and alkaloids and the rest were not detected.
4.18 Proximate composition of Ixora leaves and flowers The comparative proximate composition (moisture, ash, protein and fiber) of Ixora leaves and flowers were shown in Table (4.17). The results indicated that, the leaves contained ash of 5.59%, moisture 21.8%, protein of 1.22% and fiber of 19.85%, while those of Ixora flowers were: ash content of 6.27%, moisture of 61.3%, protein of 7.0% and fiber of 5.3%.
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Table (4.17): Qualitative phytochemical screening (presence (+) and absence (-) of the main classes) of Ixora leaves and flowers
Main class Leaves Flowers Flavonoids + - Saponins + + Tannins - - Steroids + + Alkaloids - + Glycosides - -
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Table (4.18): The proximate contents (%) of Ixora leaves and flowers
Parameters (%) Leaves Flowers Ash 5.91 6.27 Moisture 21.8 61.3 Protein 1.22 7.01 Fiber 19.85 5.30
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CHAPTER FIVE CONCLUSIONS AND RECOMMENDATIONS
5.1 Conclusion 1- The ethanolic and aqueous extracts of Ixora leaves and flowers have a considerable lethal effect on Anopheles arabiensis and Culex larvae but with varying effects on the aquatic predators. 2- Anopheles and Culex larvae and the tested aquatic predators different in their susceptibilities towards different doses of the ethanol and aqueous extract of Ixora flowers. 3- Dragon fly naiads showed more susceptibility to ethanolic and aqueous extracts of Ixora leaves and flowers than Gumbosia fish, hemipteran boatman, and swimming beetle larvae. 4-Gumbosia fish was the most resilient predators to both extracts. 5- The Malathion insecticide was more toxic than ethanolic and aqueous extracts of Ixora leaves and flowers.
5.2 Recommendations 1- According to the lethal effect exerted by the extracts of Ixora leaves and flowers on Anopheles larvae, they can be recommended for use as methods to control mosquitoes 2- Malathion was founds to be more toxic to both mosquito larvae and its predators and it should be for their tested in very low concentrations. 3- Small scale field triad could be carried out to test the efficacy of Ixora leaves and flowers aqueous extracts in controlling A. arabiensis larvae under field conditions with observer of ions on the aquatic predators.
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REFRENCES
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