Effect of Cathedral Cactus Aqueous Extract Euphorbia ...

1

Effect of Cathedral Cactus Aqueous Extract (Euphorbia

trigona Mill)on Anopheles larvae

Hufsa Ebrahim Musa Abdelah

B.Sc. Chemistry /Biology, Faculty of Education, University of

Gezira,2001

Postgraduate Diploma in Biosciences and Biotechnology, Faculty of

Engineering and Technology, University of Gezira,2014

A Dissertation

Submitted to the University of Gezira in Partial Fulfillment of the

Requirements for the Award of the Degree of Master of Science

in

Biosciences and Biotechnology(Biosciences)

Center of Biosciences and Bio technology

Faculty of Engineering and technology

October, 201

2

Effect of Cathedral Cactus Aqueous Extract (Euphorbia

trigona Mill) on Anopheles Larvae

Hufsa Ebrahim Musa Abdelah

Supervision Committee;

Name Position Signature

Dr. Mutaman Ali Kehail Main supervisor …………..

Dr. Abdalla Ibrahim Abdalla Co-supervisor ……………

Date, October 2015

3

Effect of Cathedral Cactus Aqueous Extract (Euphorbia

Trigona Mill) on Anopheles Larvae

Hufsa Ebrahim Musa Abdelah

Examination Committee

Name Position Signature

Dr. Mutaman Ali Kehail Chairperson …………

Prof. Elamin Mohamed Elamin External Examiner …………

Dr. Awadallah Belal Dafaallah Internal Examiner …………

Date of Examination:6, October, 2015

4

DEDICATION

To the soul of my parents

and

to my children

5

ACKNOWLEDGEMENTS

I Would like to thank My God and also those who contributed

one way or another to the realization of this work: Dr. Mutaman

Ali Kehail and The staff of Center of Bio sciences and Bio

technology, Faculty of Engineering and Technology, University

of Gezira

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Effect of Cathedral Cactus Aqueous Extract of (Euphorbia trigona

Mill) on Anopheles larvae

Hufsa Ebrahim Musa Abdelah`

M.Sc.in Biosciences and Biotechnology (Biosciences), October, 2015

Center of Biosciences and Biotechnology

Faculty of Engineering and Technology

University of Gezira

Abstract

Mosquitoes are considered as vector of malaria disease and some other

endemic diseases in the world. There are some methods already been used for

controlling mosquito; of which is using natural products. This study was conducted in

February 2015, at Basic Sciences Laboratory, Faculty of Engineering and

Technology, University of Gezira. to evaluate the effect of cortex, spine and pith parts

of cactus (Euphorbia trigona) on Anopheles mosquito larvae. The plant parts were

collected from Wad Medani City, whereas, the mosquito larvae were collected from

the breeding sites at Tayba, Gezira State, Sudan. The plant parts (cortex, spines and

pith) were shade dried away from the direct sunlight, ground and then kept separately

in small plastic sacks. From each plant part, a concentration of 1200 ml/ L was

prepared. The standards of WHO for testing toxicity of the toxic compound against

mosquito larvae was followed. Anopheles larvae (mortality was: 48%, 37% and 62%,

for cactus trigona ( cortex, spine and pith), the results also showed that, the three used

parts have a varied great impact on the survived larval morphology. Changes in skin

color was of 82%, loosening in digestive system was of 48%, and dislocation of some

body part was of 32% after 48 hours of applying them. The study recommends adding

cactus parts as potential natural products for Anopheles larval control, and also

running more tests to measure the environmental impact of these products.

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على يرقات بعوض الانوفلس)ايفوربيا ترايقونا ( نبات الصبار تأثير

حفصة إبراهيم موسى عبد الله

ا2015كتوبرا( بيولوجية ) العلوم البيولوجية ماجستير العلوم في العلوم والتقنية ال

مركز العلوم والتقنية البيولوجية

كلية الهندسة والتكنولوجيا

جامعة الجزيرة

ملخص الدراسة

هنالك عدة طرق يمكن .يعتبر البعوض ناقل لمرض الملا ريا وبعض الامراض المستوطنة الاخرى في العالم

هدفت الدراسة إلى تقييم الأثر القاتل والتغير .استعمالها لمكافحة البعوض منها استخدام المنتجات الطبيعية

.قونا على يرقات بعوض الانوفلسياشوك ولب )نخاع( نبات صبار تر و الظاهري المحدث بواسطة أجزاء قشرة

تم جمع أجزاء نبات صبار الترايقونا من مدينة ود مدني، بينما تم جمع عينات يرقات البعوض من مواقع للتوالد

بمعمل العلوم الأساسية, كلية 2015فبراير خلال أجريت هذه الدراسة .في منطقة طيبة, ولاية الجزيرة, السودان

تم استبعاد يرقات بعوض الكيولكس والمفترسات المائية التي جمعت .والتكنولوجيا, جامعة الجزيرةالهندسة

تم تجفيف الأجزاء النباتية )القشرة, الاشواك واللب( في الظل بعيدا عن .عرضيا مع يرقات بعوض الانوفلس

م وزن عينة من كل جزء ت.سحنت وحفظت بصورة منفصلة في أوعية بلاستيكية صغيرة ,ضوء الشمس المباشر

تم تطبيق مواصفة منظمة الصحة العالمية لاختبار حساسية المركبات السامة ضد .جرام 0.3نباتي مقدارها

48أظهرت الأجزاء الثلاثة نسبة سمية مختلفة ضد يرقات بعوض الانوفلس )نسبة القتل كانت .يرقات البعوض

كما أظهرت النتائج أيضا أن .قونا(يبات صبار التراأشواك ولب ن و ، علي التوالي لقشرة69%، 37%، %

الأجزاء المستخدمة الثلاثة كان لها تأثير كبير متباين علي مظهر يرقات بعوض الانوفلس الناجي )متمثلة في

( وتفكك في بعض أجزاء الجسم %48(، اهتراء في الجهاز الهضمي )بمتوسط %82تغير لون الجلد )بمتوسط

توصي هذه الدراسة بإضافة هذه الأجزاء للمنتجات 0ساعة من تطبيق التجربة 48بعد ( وذلك% 32)بمتوسط

اتاس المردود البيئي لهذه المنتجالطبيعية الفعالة في مكافحة يرقات الانوفلس وبإجراء المزيد من الاختبارات لقي

8

Table of Contents

Subject Page

Dedication Iii

Acknowledgements Iv

Abstract V

Arabic Abstract Vi

Table of Contents Vii

List of Tables Ix

List of Plates X

CHAPTER ONE: INTRODUCTION 1

CHAPTER TWO: LITERATURE REVIEW 3

2.1. Mosquito 3

2.1.1 Mosquito control 5

2.2 . Natural products 6

2.3. Cactus 7

2 .3 .1Euphorbia 8

2.3. 2 Euphorbia trigona 10

2.3.3 Morphological effect of natural products on mosquito larvae 11

C HAPTER THREE: MATERIALS AND METHODS

14

3.1 The study area 14

3. 2. Maintenance of mosquitoes and preparation of plant parts 14

9

3.3 Tests procedures 14

3.4. Statistical analysis 14

CHAPTER FOUR: RESULTS AND DISSCUSION 15

4.1 Toxicity of Euphorbia trigona parts on Anopheles larvae 15

4.2. The morphological changes in Anopheles larvae after 24 hours 17

4.3. The morphological changes in Anopheles larvae after 48 hours 20

CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATION 24

5.1 Conclusions 24

5.2 Recommendations 24

REFERENCES 25

10

List of Tables

Table

No.

Title Page

4.1 Percentage mortality of Anopheles larvae towards cortex, spines

and pith (at 1200 mg/L) of Euphorbia trigona after 24 hours

16

4.2 Morphological changes (%) observed in Anopheles larvae

towards cortex, spines and pith (at 1200 mg/L) of Euphorbia

trigona after 24 hours

18

4.3 Morphological changes (%) observed in Anopheles larvae

towards cortex, spines and pith (at 1200 mg/L) of Euphorbia

trigona after 48 hours

21

11

List of Plates

Plate No. Title Page

1 Euphorbia trigona 11

4.1 The morphological change in color in the survived Anopheles

larvae

19

4.2 Some morphological changes in the survived larvae 22

4.3 The larva that failed to pupate 23

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CHAPTER ONE

INTRODUCTION

Mosquitoes can transmit more diseases pathogens than any other group of

arthropods and affect millions of people throughout the world. WHO has declared the

mosquitoes as “public enemy number one” (WHO,1996). Mosquito borne diseases are

prevalent in more than 100 countries across the world, infecting over 700 million

people every year globally and 40 million of the Indian population. They act as a

vector for most of the life threatening diseases like malaria, yellow fever, dengue

fever, chikungunya ferver, filariasis, encephalitis, West Nile virus infection, etc., in

almost all tropical and subtropical countries and many other parts of the world.

To prevent proliferation of mosquito borne diseases and to improve quality of

environment and public health, mosquito control is essential. The major tool in

mosquito control operation is the application of synthetic insecticides such as

organochlorine and organophosphate compounds. But this has not been very

successful due to human, technical, operational, ecological, and economic factors. In

recent years, use of many of the former synthetic insecticides in mosquito control

programme has been limited. It is due to lack of novel insecticides, high cost of

synthetic insecticides, concern for environmental sustainability, harmful effect on

human health, and other non-target populations, their non biodegradable nature,

higher rate of biological magnification through ecosystem, and increasing insecticide

resistance on a global scale (Brown, 1986 and Russell et al., 2009). Thus, the

Environmental Protection Act in 1969 has framed a number of rules and regulations

to check the application of chemical control agents in nature (Bhatt and Khanal,

2009). It has prompted researchers to look for alternative approaches ranging from

provision of or promoting the adoption of effective and transparent mosquito

management strategies that focus on public education, monitoring and surveillance,

source reduction and environment friendly least-toxic larval control. These factors

have resulted in an urge to look for environment friendly, cost-effective,

biodegradable and target specific insecticides against mosquito species. Considering

these, the application of eco-friendly alternatives such as biological control of vectors

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has become the central focus of the control programmme in lieu of the chemical

insecticides.

One of the most effective alternative approaches under the biological control

programme is to explore the floral biodiversity and enter the field of using safer

insecticides of botanical origin as a simple and sustainable method of mosquito

control. Further, unlike conventional insecticides which are based on a single active

ingredient, plant derived insecticides comprise botanical blends of chemical

compounds which act concertedly on both behavourial and physiological processes.

Thus there is very little chance of pests developing resistance to such substances.

Identifying bio-insecticides that are efficient, as well as being suitable and adaptive to

ecological conditions, is imperative for continued effective vector control

management. Botanicals have widespread insecticidal properties and will obviously

work as a new weapon in the arsenal of synthetic insecticides and in future may act as

suitable alternative product to fight against mosquito borne diseases.( Roark 1947)

described approximately 1,200 plant species having potential insecticidal value, while

(Sukumar et al., 1991) listed and discussed 344 plant species that only exhibited

mosquitocidal activity (Shallan et al., 2005) reviewed the current state of knowledge

on larvicidal plant species, extraction processes, growth and reproduction inhibiting

phytochemicals, botanical ovicides, synergistic, additive and antagonistic joint action

effects of mixtures, residual capacity, effects on non-target organisms, resistance and

screening methodologies, and discussed some promising advances made in

phytochemical research. Summarized the mosquitocidal activities of various herbal

products from edible crops, ornamental plants, trees, shrubs, herbs, grasses and

marine plants according to the extraction procedure developed in eleven different

solvent systems and the nature of mosquitocidal activities against different life ages

of different vector species as a ready reference for further studies.

Objectives

This study aimed to assess the larvaicidal activities and morphological

effects exerted by the cactus plant (Euphorbia trigona Mill) on the mosquito larvae

(Anopheles) and to know which of the three parts of the plant (cortex, spines and pith)

is more effective on mosquito larvae

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CHAPTER TWO

LITERATURE REVIEW

2 .1. Mosquito

Mosquitoes are members of a family of nematocerid flies: the Culicidae

Superficially, In particular, the females of many species of mosquitoes are blood-

sucking pests and dangerous vectors of diseases. Many species of mosquitoes are not

blood suckers and of those that are, many create a "high to low pressure" in the blood

to obtain it and 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. (Paul Leisnham 2010)

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 habitats are disturbed, for instance by sudden

deforestation (Wilcox and Ellis 2006).

Eggs of some species of Aedes remain unharmed in diapause if they dry out,

and hatch later when they are covered by water. 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 their eighth

abdominal segments, or through a siphon, so must come to the surface frequently. The

larvae spend most of their time feeding on algae, bacteria, and other microbes in the

surface micro-layer. 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, or instars, after which

they metamorphose into pupae. At the end of each instar, the larvae molt, shedding

their skins to allow for further growth.

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.

15

Their lifespans depend on temperature, humidity, and their ability to successfully

obtain a blood meal while avoiding host defenses and predators. The length of the

adult varies, but is rarely greater than 16 mm (0.6 in), and it weighs up to 2.5

milligrams (0.04 grains). All mosquitoes have slender bodies with three segments: a

head, a thorax and an abdomen. Typically, both male and female mosquitoes feed on

nectar and plant juices, but in many species the mouthparts of the females are adapted

for piercing the skin of animal hosts and sucking their blood as ectoparasites. In many

species, the female needs to obtain nutrients from a blood meal before she can

produce eggs, whereas in many other species, she can produce more eggs after a

blood meal. The feeding preferences of mosquitoes include those with type O blood,

heavy breathers, those with a lot of skin bacteria, people with a lot of body heat, and

the pregnant (Shirai et al., 2004 and Bill, 2013).

Both plant materials and blood are useful sources of energy in the form of

sugars, and blood also supplies more concentrated nutrients, such as lipids, but the

most important function of blood meals is to obtain proteins as materials for egg

production. Mosquitoes of the genus Toxorhynchites never suck blood (Jones and

Schreiber 1994).

Female mosquitoes use two very different food sources. They need sugar for

energy, which is taken from sources such as nectar, and they need blood as a source of

protein for egg development. Because biting is risky and hosts may be difficult to

find, mosquitoes take as much blood as possible when they have the opportunity.

Worldwide introduction of various mosquito species over large distances into regions

where they are not indigenous has occurred through human agencies, primarily on sea

routes, in which the eggs, larvae, and pupae inhabiting water-filled used tires and cut

flowers are transported. However, apart from sea transport, mosquitoes have been

effectively carried by personal vehicles, delivery trucks, trains, and aircraft. Man-

made areas such as storm water retention basins, or storm drains also provide

sprawling sanctuaries. Sufficient quarantine measures have proven difficult to

implement. In addition, outdoor pool areas make a perfect place for them to grow.

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2.1.1 Mosquito control

Many methods are used for mosquito control. Depending on the situation, the

most Source important usually include:

Source reduction

Trapping and/or insecticides to kill larvae or adults

Exclusion (mosquito nets and window screening

Source reduction means elimination of breeding places of mosquitoes. It

includes engineering measures such as filling, leveling and drainage of breeding

places, and water management (such as intermittent irrigation). Source reduction can

also be done by making water unsuitable for mosquitoes to breed in (such as changing

the salinity of the water if ecologically viable). Details of the biology of different

species of mosquitoes differ too widely for any limited set of rules to be sufficient in

all circumstances. However, the foregoing are the most economical/ecological and

practical measures for most purposes. The importance of peridomestic control arises

largely because most species of mosquitoes rarely travel more than a few hundred

meters unless the wind is favorable (Marten and Reid, 2007).

In combination with scrupulous attention to control of breeding areas, window

screens and mosquito nets are the most effective measures for residential areas.

Insecticide-impregnated mosquito nets are particularly effective because they

selectively kill those insects that attack humans, without affecting the general ecology

of the area. Biological control or "biocontrol" is the use of natural enemies to manage

mosquito populations. There are several types of biological control methods including

the direct introduction of non-ecologically invasive parasites, pathogens, vegetation,

and predators (aquatic and non-aquatic) to target mosquitoes. Experimental genetic

methods including cytoplasmic incompatibility, chromosomal translocations, sex

distortion and gene replacement have been explored. They are cheaper and not subject

to vector resistance (Canyon and Hii, 1997). Insect repellents are applied on skin and

give short-term protection against mosquito bites. There are also electronic insect

repellent devices which produce ultrasounds that were developed to keep away insects

(and mosquitoes). However, no scientific research based on the EPA's and many

universities' studies has ever sought evidence that these devices prevent a human from

being bitten by a mosquito. Many scientists have suggested that complete eradication

of mosquitoes would not have serious ecological consequences (Fang, 2010)

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2 .2 Natural products

A natural product is a chemical compound or substance produced by a living

organism—that is, found in nature. Natural products can also be prepared by chemical

synthesis (both semisynthesis and total synthesis) and have played a central role in the

development of the field of organic chemistry by providing challenging synthetic

targets. The term natural product has also been extended for commercial purposes to

refer to cosmetics, dietary supplements, and foods produced from natural sources

without added artificial ingredients (Hanson, 2003).

Within the field of organic chemistry, the definition of natural products is usually

restricted to mean purified organic compounds isolated from natural sources that are

produced by the pathways of primary or secondary metabolism. Within the field of

medicinal chemistry, the definition is often further restricted to secondary metabolites

(Williams and Lemke 2002). Secondary metabolites are not essential for survival, but

nevertheless provide organisms that produce them an evolutionary advantage (Hunter,

2008). Many secondary metabolites are selected and optimized through evolution for

use as "chemical warfare" agents against prey, predators, and competing organisms

(Bhat et al., 2005).

The broadest definition of natural product is anything that is produced by life and

includes the likes of biotic materials (e.g. wood, silk), bio-based materials (e.g.

bioplastics, cornstarch), bodily fluids (e.g. milk, plant exudates), and other natural

materials (e.g. soil and coal).

Natural products are often divided into two major classes, the primary and

secondary metabolites (Karlovsky, 2008 and Kliebenstein, 2004). Primary metabolites

have an intrinsic function that is essential to the survival of the organism that

produces them. Secondary metabolites in contrast have an extrinsic function that

mainly affects other organisms. Secondary metabolites are not essential to survival

but do increase the competitiveness of the organism within its environment. Because

of their ability to modulate biochemical and signal transduction pathways, some

secondary metabolites have useful medicinal properties. Natural products especially

within the field of organic chemistry are often defined as primary and secondary

metabolites. A more restrictive definition limiting natural products to secondary

metabolites is commonly used within the fields of medicinal chemistry and

pharmacognosy (Bhat et al., 2005).

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Plants are a major source of complex and highly structurally diverse chemical

compounds (phytochemicals), this structural diversity attributed in part to the natural

selection of organisms producing potent compounds to deter herbivory (feeding

deterrents). Major classes of phytochemical include phenols, polyphenols, tannins,

terpenes, and alkaloids (Crozier et al., 2006). Though the number of plants that have

been extensively studied is relatively small, many pharmacologically active natural

productTaxus brevifolia and Cephalotaxus harringtonii, respectively (Kittakoop et al.,

2014) have already been identified. Clinically useful examples include the anticancer

agents paclitaxel and omacetaxine mepesuccinate. The antimalarial agent artemisinin

(from Artemisia annua) (Kano, 2014) and the acetylcholinesterase inhibitor

galantamine (from Galanthus spp.), used to treat Alzheimer's disease. Other plant-

derived drugs, used medicinally and/or recreationally include morphine, cocaine,

quinine, tubocurarine, muscarine, and nicotine (Dewick, 2009).

Animals also represent a source of bioactive natural products. In particular,

venomous animals such as snakes, spiders, scorpions, caterpillars, bees, wasps

centipedes, ants, toads, and frogs have attracted much attention. This is because

venom constituents (peptides, enzymes, nucleotides, lipids, biogenic amines etc.)often

have very specific interactions with a macromolecular target in the body (e.g. α-

bungarotoxin from cobras) (Dossey, 2010 and Fernandes etal, 2013) of killing or

paralyzing their prey and/or defending themselves against predators being more likely

to survive and reproduce.

2.3. Cactus

Cactus (plural: cacti, cactuses), ",( Merriam-Webster's Online Dictionary) is a

member of the plant family Cactaceae within the order Caryophyllales. The word

"cactus" derived, through Latin, from the Ancient Greek κάκτος, kaktos, a name

originally used by Theophrastus for a spiny plant whose identity is not certain

(Johnson and smith1972).

Cacti occur in a wide range of shapes and sizes. Most cacti live in habitats subject

to at least some drought. Many live in extremely dry environments, even being found

in the Atacama Desert, one of the driest places on earth. Cacti show many adaptations

to conserve water. Almost all cacti are succulents. Unlike many other succulents, the

stem is the only part of most cacti where this vital process takes place. Cactus stems

19

store water. Most species of cacti have lost true leaves, retaining only spines, which

are highly modified leaves. As well as defending against herbivores, spines help

prevent water loss by reducing air flow close to the cactus and providing some shade.

In the absence of leaves, enlarged stems carry out photosynthesis. (Anderson 2001).

Cacti are native to the Americas, ranging from Patagonia in the south to parts of

western Canada in the north—except for Rhipsalis baccifera, which also grows in

Africa and Sri Lanka. Cactus spines are produced from specialized structures called

areoles, a kind of highly reduced branch. Areoles are an identifying feature of cacti.

As well as spines, areoles give rise to flowers, which are usually tubular and

multipetaled. Many cacti have short growing seasons and long dormancies, and are

able to react quickly to any rainfall, helped by an extensive but relatively shallow root

system that quickly absorb any water reaching the ground surface. Cactus stems are

often ribbed or fluted, which allows them to expand and contract easily for quick

water absorption after rain, followed by long drought periods. Like other succulent

plants, most cacti employ a special mechanism called "crassulacean acid metabolism"

(CAM) as part of photosynthesis. Transpiration, during which carbon dioxide enters

the plant and water escapes, does not take place during the day at the same time as

photosynthesis, but instead occurs at night. The plant stores the carbon dioxide it

takes in as malic acid, retaining it until daylight returns, and only then using it in

photosynthesis. Because transpiration takes place during the cooler, more humid night

hours, water loss is significantly reduced. Many smaller cacti have globe-shaped

stems, combining the highest possible volume for water storage, with the lowest

possible surface area for water loss from transpiration. The tallest free-standing cactus

is Pachycereus pringlei, with a maximum recorded height of 19.2 m (Salak, 2000),

and the smallest is Blossfeldia liliputiana, only about 1 cm (Mauseth and James,

2012).

2.3.1 Euphorbia

Euphorbia (spurge) is a very large and diverse genus of flowering plants in the

spurge family (Euphorbiaceae). Sometimes in ordinary English, "euphorbia" is used

to refer to the entire Euphorbiaceae family (as the type genus), not just to members of

the genus . ",( Merriam-Webster's Online Dictionary)

20

Euphorbia flowers are tiny, and the variation attracting different pollinators (and

the human eye), with different forms and colors occur, in the cyathium, involucre,

cyathophyll, or additional parts such as glands that attached to these. The collection of

many flowers may be shaped and arranged to appear collectively as a single

individual flower, sometimes called a pseudanthium in Asteraceae, and also in

Euphorbia. The majority of species are monoecious (bearing male and female flowers

on the same plant), although some are dioecious with male and female flowers

occurring on different plants. (Dave's Grade 2011) In the genus Euphorbia,

succulence in the species has often evolved divergently and to differing degrees.

Sometimes it is difficult to decide, and it is a question of interpretation, whether or not

a species is really succulent or "only" xerophytic. In some cases, especially with

geophytes, plants closely related to the succulents are normal herbs. About 850

species are succulent in the strictest sense. If one includes slightly succulent and

xerophytic species, this figure rises to about 1000, representing about 45% of all

Euphorbia species.The milky sap of spurges (called "latex") evolved as a deterrent to

herbivores. It is white and colorless when dry, except in E. abdelkuri, where it is

yellow. The pressurized sap seeps from the slightest wound and congeals after a few

minutes in air. The skin irritating and caustic effects are largely caused by varying

amounts of diterpenes. Triterpenes such as betulin and corresponding esters are other

major components of the latex (Richard, 2013).

In contact with mucous membranes (eyes, nose, mouth), the latex can produce

extremely painful inflammation. Therefore, spurges should be handled with caution

and kept away from children and pets. Latex on skin should be washed off

immediately and thoroughly. Congealed latex is insoluble in water, but can be

removed with an emulsifier like milk or soap. A physician should be consulted if

inflammation occurs, as severe eye damage including permanent blindness may result

from exposure to the sap. When large succulent spurges in a greenhouse are cut,

vapours can cause irritation to the eyes and throat several meters away. Precautions,

including sufficient ventilation, are required. Commercialized to treat actinic

keratosis, a precancerous skin condition. It is produced by the Euphorbia plants (Tom

et al., 2000).

21

2.3.2 Euphorbia trigona

Euphorbia trigona (Plate 1) also known as African milk tree, cathedral cactus

(Timothy et al., 1991) and high chaparallis a perennial plant that originally comes

from Central Africa. It has an upright stem that is branched into three or four sides.

The stem itself is dark green with V-shaped light green patterns. The about 5mm long

thorns are placed in pairs of two on the stem's ridges. The drop shaped leaves grow

from between the two thorns on each ridge. The plant has never been known to flower

(James et al., 2011)

2.3.3 Morphological effect of natural products on mosquito larvae

Detection of the morphological growth disruption affects due to the

photoactivated cytotoxicity of some compounds in the extracts on treated fourth instar

larvae which possibly generated from the neuro-muscular disturbance and subsequent

cytological degenerations in the electrolytes control mechanisms located in the anal

papillae of the mosquito larvae, probably further led to an interruption of the osmotic

and ionic regulation and may be this phenomena intrinsically associated with the

death of mosquito larvae. This positively correlated in our behavioral and also

morphology observations on the treated mosquito larvae. Although the lower limits of

ion concentrations that permit survival of mosquito larvae have not yet been

established, ionic imbalance resulting from the interruption of ionic regulation is also

a harmful condition. Their findings corresponded to those of earlier works that

investigated the effect of plant natural products on some species of mosquitoes.

Chaithong et al., (2006) reported that pepper extract when tested to the Aedes larvae

extensively damage and shrunken cuticle of the anal papillae.

Similarly alpha-terthienyl, once introduced into the water medium containing

mosquito larvae, enters into the anal gills and subsequently made halide leakage,

releasing all the electrolytes into the medium leading to death of the larvae Downum

et al., (1984). observed an increase in the Superoxide dismutase activity from 1st

instar to 4th instar Aedes larval stage. This increase seems to be a protective

mechanism against hazardous oxygen derivatives generated by the actin of the

phototoxin alpha-terthienyl . superoxide dismutase is found in the entire gill, except in

the tracheal network. Further studies conducted by Insun et al., (1999) revealed the

severely morphological disruption of anal papillae observed in dead Culex

.quinquefasciatus larvae.

22

Plate(1)Euphorbia trigona

23

After treatment with ethanolic extract of K. galanga, damaged anal papillae,

with a shrunken cuticle border and destroyed surface with loss of ridge-like reticulum

were found under light and scanning electron icroscopes, respectively. Green et al.,

(1991), reported distinct features of alteration such as highly swollen anal papillae of

A. aegypti larvae after treatment with whole oil of Tagetes minuta. The two pairs of

anal papillae are flexible, sac-like structures consisting of an epithelium covered by

cuticle and situated on an extension of the terminal segment of mosquito larvae. In the

fresh-water mosquito larvae, uptake and elimination of most ions occur via the anal

papillae, while the process of ion conservation is mainly located in the digestive tract

(Garrett and Radley, 1984). The capacity to take up sodium, potassium, chloride, and

phosphate ions from the medium was markedly reduced or lost in papilla-less larvae

reported from other lipoidal membranes (Koch, 1938), in particular the neuromuscular

sheath, become involved in the photoprocess as the photosensitizer diffuses to other

sites (Robinson, 1983). This is confirmed by the early photoinactivation of enzymes

such as acetylcholinesterase (Ben Amor and Jori, 2000) which represents the

neurotransmitter enzyme.

A generalized oxidative modification of the membranes takes place, as

suggested by ultrastructural studies (Callaham et al., 1977). Changes in membrane

permeability are also demonstrated by the presence of altered potassium levels in the

hemolymph (Weaver et al., 1976). The hemolymph volumes decrease significantly

upon photosensitization and the hemocoel fluids undergo a rapid transfer from the

body cavity to the alimentary canal with a consequent increase in crop volume.

Result presented in this paper also observed that Most of the larvae reating on

celery seed extract having no PM lines in the midgut lumen compared to controls,

indicated that toxic compounds in the extract can also act on peritrophic membrane

degeneration after entering orally and possibly act through the cytotoxic mechanisms

as well. Dijoux et al., (2006) have shown that Citrus aurantium dulcisand

Cymbopogon citratus essential oils were phototoxic and cytotoxic.

In other words, cytotoxicity seems rather antagonistic to phototoxicity. In the

case of cytotoxicity, essential oils damage the cellular and organelle membranes and

can act as prooxidants on proteins and DNA with production of reactive oxygen

species (ROS), and light exposures do not add much to the overall Reaction.

Obviously, cytotoxicity or phototoxicity depends on the type of molecules present in

the extracts and their compartmentation in the cell, producing different types of

24

radicals with or without light exposure. However, such an antagonism is not quite a

strict rule. Thus, when studying extracts or essential oils it may be of interest to

determine systematically its cytotoxic as well as its possible phototoxic capacity.

Similarly it has been documented that the root extract Derris urucu affected peritropic

matrix structure of Aedes aegypti larvae causing damage to the midgut epithelium

(Gusmao et al., 2002).

The midgut lumen is lined by non-cellular membranous structure, “peritrophic

membrane”, which protects the mid gut, cells from toxic substances and pathogens

that enter the midgut through food (Peters, et al; 1992). Gut disruption by the activity

of phototoxic Alpha-terthienyl was also observed before in other insects

the toxic effect of ethanolic-extracted Magonia ubescens on Ae. aegypti larvae was

mainly in the midgut, showing partial or total cell destruction, high citoplasmatic

vacuolization, increased subperitrophic space and cell hypertrophy, and the

epithelium did not maintain its monolayerappearance (Arruda et al., 2003).

The result suggest that ethanol extract containg growth regulatory compounds

which possibly generated on the disturbance of hormonal control. The most important

deformities, pupal-adult intermediates and ecdysal failure, seemed to be the second

cause of the mortalities. Likewise, such abnormalities were noted following treatment

of immature mosquitoes with juvenile hormone (JH) analogues and chitin synthesis

inhibitors( El-Barky, 1993). The plant natural products that detrimentally affect

insectgrowth development offer a continual source of inspiration and challenge. Insect

growth regulation properties of plant extracts are very interesting and unique in

nature, since insect growth regulator works on juvenile hormone.

In particular, there often appears to be an incomplete extrication of the pupal

stage from the larval cuticle, while several adults are stuck to the chitin inner lining of

the puparium (Fairbrother etal., 1981). Similarly treatment with phototoxin alpha

terthienyl on herbivorous insects shows dense sclerotization on pupae( Downum et

al., 1984). Our results made also clear co-relation with the recent findings reported

from( Khater and Khater, 2009) where the essential oil of Apium graveolens has been

reported not only to cause blowfly, Lucilia sericata larval mortality but also produced

clear morphological abnormalities in larvae, pupae and adults. Similar observations

were obtained by other plant extracts againstdifferent mosquito species in earlier

studies. (Saxena et al., 1992) who had noticed similar morphological deformities,

including darkening of the larval cuticle, during moulting and development of C.

25

quinquefasciatus induced by Ageratum conyzoides extract. (Sakharov et al.,1989)

reported that the acetone fraction of the petroleum ether extract of A. mexicana seeds

exhibited larvicidal activity, formation of larval-pupal intermediates, formation of

pupal-adult intermediates. The crude ethanol extract of the seed of A. graveolens

which reportedly possess phototoxic compounds offers potentials against Ae. aegypti,

particularly through its toxic and growth disruption activities. Its promising toxicity to

mosquitoes makes it a promising candidate for commercial ioinsecticide development.

The photoactivatable insecticides, which act through photodynamic pathways, clearly

appear from many studies to possess several favorable features and a broad scope of

applications. The main advantage of light-activatable phototoxins is certainly

represented by their lack of toxicity towards most biological systems in the dark,

which minimizes their impact on the environment.

However, its vertebrate toxicology and its effects non-target organisms need

further study before it is seriously be considered alternative to conventional

mosquitocides.

In fact, while most phototoxins and acridines are fairly photostable, both

porphyrins and xanthenes undergo a fast degradation of the aromatic macrocycle upon

illumination with sunlight or equivalent artificial light sources, with a consequent loss

of absorption in the near- UV/visible range (Phiiognc and Morand 1985).

26

C HAPTER THREE

MATERIALS AND METHODS

3.1 The Study Area

Wad Medani City is located in the central parts of the Gezira state. Different

Localities around Wad Medani were selected for sampling mosquitoes (Tayba

village), whereas, cactus plant (Euphorbia trigona) was brought from within Wad

Medani to the Faculty of Engineering and Technology, where the study tests were

run.

3. 2. Maintenance of Mosquitoes and preparation of plant parts

Larvae of Anopheles were collected with sufficient amounts of breeding water.

Rearing and maintenance of mosquito larvae followed. The cactus plant was divided

into three parts (cortex, spines and pith). Each part was shade dried at room

temperature. The dried parts were grounded separately, using mortar and pestle and

were then kept in small bags for the further experiments.

3.3 Tests Procedures

Experiments were started by preparing sufficient number of plastic cups (size

of more than 250 ml). Random samples (20 individuals) of Anopheles larvae of the

third or early fourth instars were introduced to these cups which were filled with 250

ml ordinary water. About 0.3 g of individual ground of trigona plant part was added

in one cup containing 20 Anopheles larvae and 250 ml water. This experiment was

triplicate for each plant part (cortex, spines and pith). A control batch was included

for comparison .These experiments were run in the Faculty of Engineering and

Technology, at the room temperature (26+ 3oC). After 24 hours, in each test cup, the

dead larvae were counted, while the survived larvae were left for another 24 hours for

monitoring the morphological changes (color, digestive tract and separation of some

body parts) and in the rate of the movement. A digital microscope provided with

camera was used for documentation of these observed changes.

3.4. Statistical Analysis

Data were collected and subjected to descriptive statistics and Anova analysis to

clear the toxicity and the morphological impact of E. trigona different parts on

Anopheles larvae.

27

CHAPTER FOUR

RESULTS AND DISSCUSION

4.1 Toxicity of Euphorbia trigona parts on Anopheles larvae

The toxicity of E. trigona cortex, spines and pith parts (at the concentration of

0.3 g/250ml water = 1200 mg/L) on Anopheles larvae in terms of % mortality, was

represented in Table (4.1).

At this concentration, and after 24 hours, the cortex powder produced 43.33%

mortality on Anopheles larvae, while the mortality was 35.67% when spine powder

was used, whereas the pith powder caused 54.6% mortality.

Anova analysis revealed that, the difference observed in %mortalities was

significant (f-stat= 10.08; f-crit= 5.14), i.e. the toxicities of the three parts were not

similar against Anopheles larvae (the pith part was more toxic against Anopheles

larvae than the other parts, whereas, the spine parts has the lowest effect).

Alwan (2015) found that, the mortalities produced by E. trigona cortex and

pith at the concentration of 1200 mg/L were 42% and 47%, respectively (which were

not far from the obtained data of this study). Also, Alwan study found that, cortex and

pith of Wad Medani sample were rich in flavonoids, alkaloids, triterpenes, saponins,

glycosides and steroids, but steroids are present in relatively more concentration in

pith than in cortex (tannins were not detected in both parts).

28

Table (4.1) Percentage mortality of Anopheles larvae towards cortex, spine and pith

(at 1200 mg/L of water) of Euphorbia trigona after 24 hours

Rep. Cortex Spines Pith

1 48 37 62

2 42 35 47

3 40 35 53

Descriptive statistics

Mean 43.33 35.67 54.0

SE 2.40 0.66 4.36

Min 40 35 47

Max 48 37 62

Anova analysis

f-stat 10.08

f-crit 5.14

P 0.0121

29

4.2. The morphological changes in Anopheles larvae after 24 hours

After 24 hours of applying each of the three cactus parts (cortex, spines and pith),

some morphological changes (in color and in digestive tract) in addition to the rate of

the movement were monitored by using digital microscope provided with camera on

the survived larvae (Table 4.2 and Plate 4.1). The change in the larval color was high

in the larvae subjected to cortex part (55%), followed by those subjected to pith part

(43%) and spine part (35%)approximately .

There were considerable number of larvae with non-homogeneous digestive tract

(13%, 5% and 10%, in those subjected to cortex, spine and pith parts respectively),.

The reason may be referred to the fact that, mosquito larvae usually feed on plant

parts scattered within the breeding water, and as consequently, these parts containing

different toxic materials (phytochemicals; as was stated by (Alwan, 2015) that already

caused death to some of these larvae, while the survived ones are suffering from the

presence of these phytochemicals in their digestive tracts and in their skins.

In addition to that, the movement of the survived larvae was noticed to be slow.

"relatively"

30

Table (4.2) Morphological changes (%) observed in Anopheles larvae towards cortex,

spines and pith (at 1200 mg/L of water) of Euphorbia trigona after 24 hours

Morphological

Change

Cortex Spines Pith

Color 55 35 43

Digestive system 13 5 10

31

Larva with a changed color

Control larva

Plate (4.1) The morphological change in color in the survived Anopheles larvae after

24 hour

32

4.3. The morphological changes in Anopheles larvae after 48 hours

After 48 hours of applying each of the three cactus parts (cortex, spines and pith),

some morphological changes (in color, digestive tract and separation of some body

parts) in addition to the rate of the movement were monitored by using digital

microscope provided with camera on the survived larvae (Table 4.3 and Plate 4.2).

The change in the larval color was high in the larvae subjected to cortex part (83%),

followed by those subjected to pith part (65%) and spines part (55%).

The survived larvae with non-homogeneous digestive tract (cut) were 52%, 35%

and 45%, respectively, in those subjected to cortex, spine and pith parts. It was also

noticed that, after 48 hours, some of the body parts of the larvae were cut dawn

(disconnected), of which 17% of those subjected to cortex part, 5% of those subjected

to spine part and 12% of those subjected to pith part). It was also noticed that, the

larvae subjected to pith part were obviously swollen in comparison to the others,

oldest larvae failed to pupate (Plate, 4.3) and all the survived larvae died.

It was clear that, E. trigona parts killed some the Anopheles larvae after 24 hours

and caused some morphological changes, and its effect extended after that period to

hinder pupation and kill the rest of the survived larvae.

33

Table (4.3) Morphological changes (%) observed in Anopheles larvae towards cortex,

spines and pith (at 1200 mg/L of water) of Euphorbia trigona after 48 hours

Change Cortex Spines Pith

Color 83 55 65

Digestive tract 52 35 45

Disconnection 17 5 12

34

Larva with a cut digestive tract

Swelling larva treated with pith part

Larva with disconnected head

Larva with disconnected paddles

Plate (4.2) Some morphological changes in the survived larvae after 48 hours

35

Larva which failed to pupate

Control pupa

Plate (4.3) The larva which failed to pupate

36

Chapter Five

Conclusions and Recommendations

5.1 Conclusions

1- At 1200 mg/L of water concentration, and after 24 hours of experiment the cortex,

Spines and pith produce of Euphorbia trigona produced 43.3, 35.6 and 54.6 % mean

mortalities of Anopheles larvae

2- The movement of the survived larvae was noticed to become "relatively" slow.

3- It was clear that, E. trigona parts killed some the Anopheles larvae after 24 hours

and caused some morphological changes, and its effect extended after that period to

hinder pupation and kill the rest of the survived larvae.

5.2 Recommendations

The study recommends adding these parts to the potential natural products in

Anopheles larval control, and also running more tests to measure the environmental

impact of these products. Specially on the aquatic predators.

37

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