THE EFFECT OF HONEY BEE VENOM ON MOSQUITO Anopheles

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