Upload
others
View
3
Download
0
Embed Size (px)
Citation preview
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
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
6
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.
7
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
12
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
13
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
14
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.
16
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)
17
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).
18
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.
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
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
REFERENCES
Alwan, H. A. A. (2015). Qualitative phytochemical screening, thin layer
chromatography and Toxicity tests for the stems of tow cactus gerera:
Eupharbia trigona and E. abyssinica. M.Sc. thesis, University of Gezira.
Anderson (2001), pp. 15–37
Ben Amor, T. and Jori, G. (2000). Sunlight-activated insecticides: historical
background and mechanisms of phototoxic activity. Insect Biochem. Mole.
Biol., 30, 915-925.
Bhat, S. V.; Nagasampagi, B. A. and Sivakumar, M. (2005). Chemistry of Natural
Products. Berlin ; New York: Springer. ISBN 81-7319-481-5
Bhatt, R. P. and Khanal, S. N. (2009). Environmental impact assessment system in
Nepal - an overview of policy, legal instruments and process. Kathmandu
Univ. J. Sci. Enginnering Tech., 5:160–170.
Bill, C. ( 2013). A Mosquito's Idea Of A Delicious Human""5 Stars:
Brown, A.W.(1986). Insecticide resistance in mosquitoes: a pragmatic review. J. Am.
Mosquito Control Assoc., 123–40.
Canyon, D.V. and Hii, J. L. (1997). "The gecko: An environmentally friendly
biological agent for mosquito control". Medical and Veterinary Entomology,
11 (4): 319–323.
Callaham; M. F. Lewis, L. R. Poe, W. E. and Heitz, J. R. (1977). Time dependence
of light-independent biochemical changes in the boll weevil, Anthonomous
grandis, caused by dietary rose bengal. Environ. Entomol., 6 668-673.
Chaithong, U; Choochote W; Kamsuk, K; Jitpakdi, A; Tippawangkosol, P;
Chaiyasit, D; Champakaew, D; Tuetun, B; and Pitasawat, B; (2006).
Larvicidal effect of pepper plants on Aedes aegypti(L.) (Diptera: Culicidae). J.
Vector Ecol., 31, 138-143
Crozier, A; Clifford, M.N. and Ashihara, H .(2006). Plant secondary metabolites:
occurrence, structure and role in the human diet. "Chapters 1, 3 and 4".
Oxford, UK: Blackwell Publishing Ltd. pp. 1–24, 47–136. ISBN 978-1-4051-
38
2509-3.
Dewick, P. M. (2009). Medicinal natural products: a biosynthetic approach (3rd
edn.). Chichester: Wiley. ISBN 978-0-470-74167-2
Dijoux, N.; Guingand, Y.; Bourgeois, C.; Durand, S.; Fromageot, C.; Combe, C.
and Ferret, P. J. (2006). Assessment of the phototoxic hazard of some
essential oils using modified 3T3 neutral red uptake assay. Toxicol. in vitro.,
480-489.
Dossey, A. T. (2010). "Insects and their chemical weaponry: new potential for drug
discovery". Nat. Prod. Rep., 27 (12): 1737–57.
Downum, K. R.; Rosenthal, G. A. and Towers, G. H. N. (1984). Phototoxicity of
the allelochemical, a-terthienyl, to larvae of Manduca sexta (L.) (Sphingidae).
Pesticide Biochem. Physiol.
Dave's Grade (2011) Euphorbia "Flowers," an introduction to the amazing Cyathia,
Geoff Stein, 4-22-
El-Barky, N. M. (1993). Effect of some insect growth regulators on Culex pipiens in
Qalyubia 104-109
Fairbrother, T. E. Essig, H. W. Combs, R. L. and Heitz, J. R. (1981). Toxic
effects of rose bengal and erythrosin B on three life stages of the face fly.
Environ. Entomol., 506-510.
Fang, J. (2010). "Ecology: A world without mosquitoes". Nature, 466 (7305): 432–4.
Fernandes-P. M.F; Félix-Silva, J. and Menezes, Y.A.S (2013). An Integrated View
of the Molecular Recognition and Toxinology: From Analytical Procedures to
Biomedical Applications. In Tech. Open. p. 23-72.
Garrett, M. and Bradley, T. J. (1984). The pattern of osmotic regulation in larvae of
the mosquito Culiseta inornata. J. Exp. Biol. 113., 133-141.
Green, M. M. Singer, J. M. Sutherland, D. J. and Hibben. C. R. (1991). Larvicidal
activity of T agetes minuta (Marigold) toward Aedes aegypti. J. Am. Mosq.
Contr. Assoc.,7.282-286.
Gusmão, D. S; Páscoa, V ;Mathias, L Derris Lonchocarp usurucu; Vieira, I. J. C;
Braz- Filho, R. and Lemos, F. J. A. (2002). (Leguminosae) extract modifies
the peritrophic matrix structure of Aedes aegypti (Diptera: Culicidae). Mem.
Inst. Oswaldo. Cruz., 371-375.
39
Hanson, J,R. (2003). Natural products : the secondary metabolite. Cambridge: Royal
Society of Chemistry. ISBN 0-85404-490-6.
Hunter, P. (2008). "Harnessing Nature's wisdom. Turning to Nature for inspiration
and avoiding her follies". EMBO Rep., 9 (9): 838–40.
Insun, D.; Choochote, W.; Jitpakdi, A.; Chaithong, U.; Tippawangkosol, P. and
Pitasawat, B. (1999). Possible site of action of Kaempferia galangain killing
Culex quinquefasciatus larvae. Southeast Asian J. Trop. Med. Publ. Hlth., 195-
199.
James, C.; Sabina, G.; Knees, H. and Suzanne, C. (2011). The European Garden
Flora Flowering Plants: A Manual for the Identification of Plants Cultivated in
Europe, Both Out-of-Doors and Under Glass. Cambridge University Press,
2011. p. 498. ISBN 9780521761550
Johnson, A. T. and Smith, H. A. (1972). Plant Names Simplified: Their
Pronunciation Derivation and Meaning, Buckenhill, Herefordshire: Landsmans
Bookshop, ISBN 978-0-900513-04-6, p. 19
Jones, C. and Schreiber, E. (1994). The carnivores, Toxorhynchites. Wing Beats, 5
(4): 4.
Kano, S. (2014). "Artemisinin-based combination therapies and their introduction in
Japan". Kansenshogaku Zasshi, 88 (3 Suppl 9-10): 18–25.
Karlovsky, P. (2008). "Secondary metabolites in soil ecology". Soil Biology, 14: 1–
19.
Khater, H. F. and Khater, D. F. ( 2009). The insecticidal activity of four medicinal
plants against the blowfly Lucilia sericata (Diptera: Calliphoridae). Int. J.
Dermatol., 48, 492-497.
Kittakoop, P.; Mahidol, C. and Ruchirawat, S. (2014). "Alkaloids as important
scaffolds in therapeutic drugs for the treatments of cancer, tuberculosis, and
smoking cessation". Curr. Top. Med. Chem., 14 (2): 239–252.
Kliebenstein, D .J. (2004). "Secondary metabolites and plant/environment
interactions: a view through Arabidopsis thaliana tinged glasses". Plant Cell
and Environment, 27 (6): 675–684.
Koch, H. J. (1938). The absorption of chloride ions by the anal papillae of Diptera
larvae. J. Exp. Biol. 15, 152-160.
Marten, G. G. and Reid, J. W. (2007). "Cyclopoid copepods". Journal of the
40
American Mosquito Control Association, 23 (2 Suppl): 65–92.
Merriam-Webster's Online Dictionary,( 2012)
Mauseth, A. and James D. (2012). Mauseth Cactus research: Blossfeldia liliputiana,
retrieved. Medicinal Plants, Vol.1. PROTA, p. 260. ISBN 9789057822049
Peters, W.; Bradshaw, S. D.; Burggren, W.; Heller, C.; Ishii, S.; Langer, H.;
Neuweiler, G.; Randall, D. J.(eds.);(1992) Peritrophic membranes.
Zoophysiology. In Springer Berlin,Germany. pp. 1-238
Phiiogene; B. J. R. and Morand, P. (1985) Insecticides of Plant Origin. American
Chemical Society Symposium Series 387, Washington, DC, pp. 164-172.
Paul Leisnham( 2010)Taking a bite out of mosquito research, , University of
Maryland. Enst.umd.edu
Richard, J. H. (2013) Research into Euphorbia latex and irritant ingredients.
Retrieved, 2013. Collected by Dr. Tom, Eke, Sahar, Al-Husainy Mathew, K.
Roark, R .C . (1947) .Some promising insecticidal plants. Econ Bot., 37–45.
Robinson, J. R. (1983). Photodynamic insecticides: a review of studies on
photosensitizing dyes as insect control agents, their practical application,
hazards and residues. In: Heitz, J. R. (Ed.) Residue Reviews 88. Springer
Verlag, Berlin, pp. 69-100
Russell T. L; Kay, B.H. and Skilleter, G.A (2009) Environmental effects of
mosquito insecticides on saltmarsh invertebrate fauna. Aquat Biol., 77–90.
Sakharov, I. Y., Makarova, I. E., and Ermolin, G. A. (1989). Chemical
modification and composition of tetrameric isozyme K of alkaline phosphatase
from harp seal intestinal mucosa. Comp. Biochem. Physiol. Comp. Biochem.,
92: 119–122.
Salak, M (2000). "In search of the tallest cactus" Cactus and Succulent Journal, 72
Saxena, R. C.; Dixit, O. P. and Sukumaran, P. (1992). Laboratory assessment of
indigenous plant extracts for anti-juvenile hormone activity in Culex
quinquefasciatus. Indian J. Med. Res., 95:199–204.
Shallan, E. A. S.; Canyonb, D.; Younesc, M. W.; Abdel-Wahaba, H. and
41
Mansoura, A. H. (2005) A review of botanical phytochemicals with
mosquitocidal potential. Environ. Int., 49–66.
Shirai, Y.; Funada, H.; Seki, T.; Morohashi, M. and Kamimura, K. (2004).
"Landing preference of Aedes albopictus (Diptera: Culicidae) on human skin
among ABO blood groups, secretors or nonsecretors, and ABH antigens".
Journal of Medical Entomology, 41 (4): 796–799.
Sukumar, K. Perich, MJ andBoobar, LR (1991) Botanical derivatives in mosquito
control: a review. J Am Mosq Control Assoc. 10–37.
Timothy, K. Broschat and Alan W.(1991) Betrock Information Systems Meerow,
Betrock's Reference Guide to Florida Landscape Plants, p. 123. ISBN
9780962976100
Tom, E, S. A.; Mathew, K . and Raynor, K. (2000). "The spectrum of ocular
inflammation caused by Euphorbia plant sap "Research into Euphorbia latex
and irritant ingredients Richard J. Hodgkiss.
Weaver, J. E. Butler, L. and Yoho, T. P. (1976). Photodynamic action in insects:
volumetric change in the hemolymph and crop contents of dye-treated, light-
exposed cockroaches. Environ. Entomol., 5: 840-843.
WHO, World Health Organization, (1996) Report of the WHO informal
consultation on the evaluation on the testing of insecticides, CTD/WHO PES/IC/96.1.
Geneva: WHO;. p. 69.
Wilcox, B. A. and Ellis, B. (2006). "Forests and emerging infectious diseases of
humans". Unasylva, 57. ISSN 0041-6436.
Williams, D. A. and Lemke, T. L. (2002). Principles of Medicinal Chemistry
"Chapter 1: Natural Products". (5th ed.) Philadelphia: Lippincott Williams
Wilkins. p. 25. ISBN 0-683-30737-1