Chapter 1 Introduction
1. INTRODUCTION
Mosquitoes are foremost in man's war against insects. These blood sucking creatures not
only cause nuisance by their irritating bites but also create health menace. These are
responsible for spreading serious diseases like Malaria, Dengue, Yellow fever, Japanese
Encephalitis, Chikungunya etc.
Mosquitoes belong to the order Diptera. There are about three thousand five hundred
species of mosquitoes existing in the world, out of which the following three species are of
medical importance in India. Mosquito borne diseases are prevalent in more than hundred
countries, infecting three hundred-five hundred million people and causing about one
million deaths every year. In India, more than forty million people suffer from mosquito
diseases annually. India spends hundred million dollars on malaria. In spite of spending so
much, the diseases continue to explode from time to time. The reason is that these
mosquitoes develop resistance to medicines and chemicals.
1.1 EPIDEMIOLOGY OF MOSQUITOES
Anopheles mosquitoes breed in clean water collections. Therefore, breeding increases
dramatically in the rainy season because many artificial water collections occur. Anopheles
species are the most important species as they are capable vector for malaria parasites.
Malaria parasite alone can kill more than a million people every year (Daily et al. 2007).
The estimated five hundred fifteen million cases of human malaria each year are generally
caused by four species, including Plasmodium falciparum, P. ovale, P. vivax and P.
malariae, are transmitted by the bites of female Anopheles mosquitoes (Snow et al.
2008). Globally, malaria remains a leading cause of ill health, causing an estimated two
hundred forty three million cases of clinical malaria and eight hundred sixty three thousand
deaths (WHO 2009). More than 85% of malaria cases and 90% of malaria deaths occur in
Africa, south of Sahara. In Africa, the vast majority of cases and deaths occur in young
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Chapter 1 Introduction
children. Approximately half of the world's population is at risk of malaria, particularly
those living in lower-income countries. It infects more than five hundred million people
per year and kills more than one million (WHO 2011).
Culex mosquitoes are painful and persistent biters and are responsible for causing
filariasis to man. These mosquitoes are very common in Indian sub-continent. Lymphatic
Filariasis, commonly known as elephantiasis, is a painful and profoundly disfiguring
disease. The disease is caused by three species of nematode thread-like worms known as
Wuchereria Bancrofti, Brugia malayi and Brugia timori. An estimated one hundred twenty
million people in tropical and subtropical areas of the world are infected with lymphatic
filariasis; of these, almost twenty five million men have genital disease (most commonly
hydrocele) and almost fifteen million, mostly women, have lymphoedema or elephantiasis
of the leg. Approximately 66% of those at risk of infection live in the WHO South-East
Asia Region and 33% in the African Region (WHO 2010).
Aedes mosquitoes on the other hand are also persistent biters. Aedes aegypti is
responsible for spreading Dengue and Chikungunya. Dengue is prevalent throughout the
tropics and subtropics. The World Health Organization estimates that around 2.5 billion
people are at risk of dengue. Infections have dramatically increased in recent decades due
to increased urbanization, trade and travel. No effective drug or vaccine is available so far.
Only solution is to prevent the disease-carrying mosquito from breeding and biting
humans. Dengue is the most important mosquito spread viral disease and a major
international public health concern. It is a self limiting disease found in tropical and subtropical regions around the world, predominantly in urban and semi-urban areas. DF/DHF
is caused by dengue virus which belongs to genus Flavivirus, family Flaviviridae and
includes serotypes 1, 2, 3 and 4 (Den-1, Den-2, Den-3 and Den-4) (WHO 2010).
Relatively, new, Chikungunya is a rare form of viral fever caused by an alpha virus
that is also spread by mosquito bites from the Aedes aegypti mosquito. In recent decades
mosquito vectors of chikungunya have spread to Europe and the Americas (WHO 2008),
creating global health problem. Mosquito control manages the population of mosquitoes to
reduce their damage to human health, economies, and enjoyment. Mosquito control is a
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Chapter 1 Introduction
vital public-health practice throughout the world and especially in the tropics where
maximum mortalities happens.
1.2 MOSQUITO CONTROL MEASURES
The best way of mosquito control is to control the larval stage in water. Mosquito-control
operations are targeted against three different problems:
1. Nuisance mosquitoes bother people around homes or in parks and recreational
areas.
2. Economically important mosquitoes reduce real estate values, adversely affect
tourism and related business interests, or negatively impact livestock or poultry
production.
3. Public health is the focus when mosquitoes are vectors, or transmitters, of
infectious disease.
Vector control can be achieved mainly by four ways:1. Environmental control (Mechanical)
2. Chemical control
3. Genetic control
4. Biological control
1.2.1 Environmental Control (Mechanical)
Identify the water logging areas and do filling, leveling or making way for draining the
water which is appropriate. Put salt if the water collections are small. Burn household
wastes in pits or dispose appropriately. Look for any space in between septic tank slabs
and it should be sealed with cement. Mosquito netting of the went of the pipe from septic
tanks. Bush clearance in the premises of houses and institutions.
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Chapter 1 Introduction
1.2.2 Chemical Control
I. Using oils
Apply diesel / crude oil / kerosene / kitchen waste oil into stagnant water collections
around houses and institutions. The larvae will die because it cannot breathe due to oil.
II. Impregnates
Clothing and bed nets can be impregnated, clothing with benzyl benzoate and bed nets
with pyrethroids which can kill mosquitoes.
III. Repellents
Repellents like lemon grass oil, eucalypts oil, odomos, etc. can be applied on the exposed
parts of the body that can keep the mosquitoes away. Mosquito coils, mates and vaporizers
can also be used.
IV. Insecticides
a. Residual treatments: Chlorinated hydrocarbons like D.D.T. (Dichloro Diphenyl
Trichloro ethane), B.H.C. (Benzene Hexa Chloride) and Organo phosphates like Melathion
solutions can be sprayed on the walls of houses and other places where the mosquitoes
commonly sit. This should be done once a month and mosquitoes can be killed.
b. Interior space treatment: Applied in the form of small droplets or powder inside
rooms of houses. Chemicals used are lime sulfur, synthetic D.D.T. and B.H.C and plan
products like pyrethrum, ocimum (Thulasi) and neem (Vepu). This is sprayed using hand
pump. The disadvantage of this is that the action is short lived and it has to be done daily.
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Chapter 1 Introduction
But lime sulfur DDT and BHC are toxic and can not be used daily. But the plan product
can be used daily. This is very useful during outbreaks of epidemics.
c. Larvicidal spray: Larvicides like Melathion, Fenthion, Temethos, and Pyrethrum etc.
can be sprayed in water loggings which can kill the larvae. This should be done once a
week and it is very effective in controlling mosquitoes.
V. Fumigants
Fumigation with camphor in the evenings can be done in the houses. Fumigation with
Hydrogen Cyanide and Sulphur dioxide can be done in institutions. Application of space
spray (Fogging) using Melathion and pyrethrum should be done in public places.
1.2.3 Genetic Control
I. Chemosterilants
Mosquitoes can be effectively controlled by using some chemosterilants especially
alcalating agence like tepa 0.1% and metapa 0.06% and alpholate 0.6%.
II. Irradiating males
Another method of sterilization is the radio sterilization of males with gamma irradiation
causing a distortion of chromosomes and there by making them sterile. Large number of
such sterile mails is introduced into high density mosquito population areas thereby
producing unfertile eggs.
III. Cytoplasmic incompatibility
Normal offspring was prevented in crosses between alien strains (of different geographical
origin) owing to the incompatible factors in the egg cytoplasm. The sperms from the males
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Chapter 1 Introduction
enter the egg cell and even induce embryo formation but the sperm nucleus is prevented
from uniting with the egg nucleolus by plasma genes in the cytoplasm.
1.2.4 Biological Control
Biological control is the use of natural enemies to manage mosquito populations. There are
several types of biological control including the direct introduction of parasites, pathogens
and predators to target mosquitoes. Effective biocontrol agents include predatory fish that
feed on mosquito larvae such as mosquito fish (Gambusia affinis) and some cyprinids
(carps and minnows) and killifish. Tilapia will also consume mosquito larvae. Like all
animals, mosquitoes have their own set of diseases. Invertebrate pathologists study these
diseases in the hope that some of them can be utilized for mosquito management.
Microbial pathogens of mosquitoes include viruses, bacteria, protozoa, nematodes, and
microsproidia also used as biological control agent are the dead spores of varieties of the
natural soil bacterium Bacillus thuringienesis, especially Bt israelensis (Bti). Bti is used to
interfere in the digestion systems of larvae. It can be dispersed by hand or dropped by
helicopter in large areas. BTI is no longer effective after the larvae turn into pupae,
because they stop eating.
But the resistance has been developed by the mosquito against above mentioned
controlling methods. Therefore, biological control can thus provide an effective and
environmental friendly approach, which can be used as an alternative to minimize the
mosquito population. Fungi and their metabolites are being used in recent years to control
mosquito vectors, which have potentials to control the diseases. Fungal control of
mosquitoes recently has been preferred for their specificity. Many of the species of fungus
like Lagenidium giganteum (Couch 1935), Metarhizium anisopolie (Balaraman et al.
1979), Chrysosporium tropicum (Bendek 1962, Priyanka and Prakash 2003) etc., have so
far been used for controlling mosquito in different countries. The extracellular metabolites
of fungi can also be used as larvicides and adulticides, if found suitable.
Presently, fungi are also been used in nanotechnology for producing nanoparticles.
Therefore, present green synthesis has shown that the environmentally benign and
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Chapter 1 Introduction
renewable source of fungi used as an effective reducing agent for the synthesis of silver
and gold nanoparticles. This biological reduction of metal would be boon for the
development of clean, nontoxic and environmentally acceptable “green approach” to
produce metal nanoparticles. The formed silver and gold nanoparticles are highly stable
and have significant mosquito larvicidal activity. This reveals high efficacy of AgNPs and
AuNPs as a strong nano larvicidal agents. Nanogold and silver are not only can be used in
mosquito control but also in Cancer treatment, Medicine, Diagnosis etc. Many of the
species of fungi like Fusarium oxysporum (Ahmad et al. 2003), A. fumigates (Bhainsa and
D’Souza 2006) and Verticillium species (Mukherjee et al. 2001) used in nanotechnology
for nanoparticles production. In addition to good monodispersity, nanoparticles with well
defined dimensions can be obtained by using fungi.
1.3 OBJECTIVES AND RATIONALES
The following objectives have been selected during present investigation
1. To study the efficacies of fungal metabolites to develop new control strategy of the
selected fungi on the mosquito species
a. To study the efficacy of fungal metabolites of Chrysosporium tropicum on mosquito
larvae of the selected species, then is to compare control with other fungus.
b. To study the efficacy of fungal metabolites of Chrysosporium keratinophilum on
mosquito larvae of the selected species, then its to compare efficacies with other
fungus.
c. To study the efficacy of fungal metabolites of Aspergillus niger on mosquito larvae of
the selected species and to compare efficacies with other fungus.
d. To study the efficacy of fungal metabolites of Fusarium oxysporum on mosquito
larvae of the selected species, then to compare with other fungus.
e. To study the efficacy of fungal metabolites of Verticillium lecanii on mosquito larvae
of the selected species and to compare it with other fungus.
2. To ascertain the role of selected fungi in biosynthesis of nanoparticles of metals likeAu, Ag, etc.
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Chapter 1 Introduction
3. To formulate a new larvicide of mosquito from the selected fungal species with the help
of nanoparticles of different geometries and compare their efficacies with and without
nanogold and nanosilver particles.
Targeted Experimental Objectives
 The maintenance of different larval stages of Cx. quinquefasciatus, An. stephensi
and Ae. aegypti.
 The maintenance and culture of five fungus species C. tropicum, C.
keratinophilum, F. oxysporum, A. niger, and V. lecanii in the specific culture under
laboratory conditions.
 The bioefficacy study of C. keratinophilum, F. oxysporum, A. niger, and V. lecanii
metabolites against Cx. quinquefasciatus, An. stephensi and Ae. aegypti with C.
tropicum using as standard fungus.
 The bioefficacy study of C. tropicum, C. keratinophilum, F. oxysporum, A. niger,
and V. lecanii metabolites after purification through column chromatography and
flash chromatography at different fractions (ethanol: metabolites) against Cx.
quinquefasciatus, An. stephensi and Ae. aegypti.
 The study of synthesis of silver and gold nanoparticles using C. tropicum, C.
keratinophilum, F. oxysporum, A. niger, and V. lecanii (fungal liquids).
 The characterization of silver and gold nanoparticles by Micro-scan reader, X-Rays
diffractometer, Furiour transform infrared spectrophotometer, Transmission
electron microscope, Scanning electron microscope, Auto lab Analyzer.
 The study of physio-chemical parameters (growth parameters) pH, concentrations,
time, and temperature on silver and gold nanoparticles formation.
 The study of efficacy of C. tropicum, C. keratinophilum, F. oxysporum, A. niger,
and V. lecanii synthesized silver and gold nanoparticles against the larvae of Cx.
quinquefasciatus, An. stephensi and Ae. aegypti.
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Chapter 1 Introduction
 The formulations of different geometrical silver and gold nanoparticles with C.
tropicum, C. keratinophilum, F. oxysporum, A. niger, and V. lecanii for mosquito
larval control.
1.4 SIGNIFICANCE AND FUTURE APPLICATION OF THE PRESENT
INVESTIGATION
Several species of entomopathogenic fungi have been reported, but only few of them have
developed for insect control. Therefore, more research is needed to explore vector control
potential of the fungi. The testing of keratinophilic fungi producing lethal effects against
mosquito larvae in present investigation may provide an effective, environmental friendly
and alternative candidate for future vector control program. Furthermore, the synthesis of
silver and gold nanoparticles, their effects and their formulations in the current study can
thus provide a potential green approach keratinophilic fungus for targeting the mosquitoes.
Presently, there is no evidence for a role of silver and gold nanoparticles synthesized with
fungus in controlling mosquito larvae to our knowledge. Therefore, it can be a useful green
exercise to invent and discover new fungal nanolarvicide for respective ecology and
environmental management system.
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