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GREEN SYNTHESIS AND CHARACTERIZATION OF
SILVER NANOPARTICLES USING SELECTED INDIAN
PLANT SPECIES AGAINST MOSQUITO VECTORS OF
PUBLIC HEALTH IMPORTANCE
Thesis submitted to
BHARATHIDASAN UNIVERSITY
In partial fulfillment of the requirement
For the award of degree of
DOCTOR OF PHILOSOPHY IN ZOOLOGY
Submitted By
Mr. R. THAMEEM AZARUDEEN, M.Sc., M.Phil.,
Reg. No. 31453/Ph.D. k7/Zoology /Full-Time/October 2013/Dt. 07.10.2013.
Under the Guidance of
DR. A. AMSATH, M. Sc., M. Phil., Ph. D.,
P. G. AND RESEARCH DEPARTMENT OF ZOOLOGY
KHADIR MOHIDEEN COLLEGE
ADIRAMPATTINAM- 614 701
APRIL 2017
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Dr. A. AMSATH, M. Sc., M. Phil., Ph. D.,
Associate Professor and Research Advisor
P.G. and Research Department of Zoology,
Khadir Mohideen College,
Adirampattinam – 614 701.
Tamil Nadu, India
Date:………..……………
CERTIFICATE
This is to certify that the thesis entitled, “Green synthesis and
characterization of silver nanoparticles using selected Indian plant species
against mosquito vectors of public health importance” is a record of research
work done by the candidate Mr. R. THAMEEM AZARUDEEN, during the
year 2013-2017 submitted to Bharathidasan University, Tiruchirappalli in
partial fulfillment of the requirements for the award of Degree of Doctor of
Philosophy in Zoology under my supervision and that the thesis has not been
submitted earlier for the award of any Degree anywhere.
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DECLARATION
I hereby declare that the thesis entitled “Green synthesis and
characterization of silver nanoparticles using selected Indian plant species
against mosquito vectors of public health importance” is a record of
research work done by me in the Post Graduate and Research
Department of Zoology, Khadir Mohideen College, Adirampattinam
under the supervision of Dr. A. AMSATH, during the year 2013-2017
submitted to Bharathidasan University, Tiruchirappalli in partial
fulfillment of the requirements for the award of Degree of Doctor of
Philosophy in Zoology and that the thesis has not been submitted earlier
for the award of any Degree anywhere.
Station: Adirampattinam.
Date: (R. THAMEEM AZARUDEEN)
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ACKNOWLEDGEMENTS
First and foremost, I find great pleasure in expressing my deep sense
of gratitude and heartful thanks to my research advisor Dr. A. AMSATH,
Associate Professor, P.G. and Research Department of Zoology, Khadir
Mohideen College, Adirampattinam-614 701, Thanjavur District, Tamil
Nadu, India for suggesting the problem, sincere guidance, critical correction
and encouragement for the successful completion of this work.
I express my deep sense of gratitude and heartfelt thanks to
Dr. A. UDUMAN MOHIDEEN, Principal and Dr. P. KUMARASAMY,
HOD of Zoology, Khadir Mohideen College, Adirampattinam for having
permitted me to pursue my research to complete this work successfully.
I sincerely express my heart full thanks to Dr. M. GOVINDARAJAN,
Assistant Professor, Unit of Vector Control, Phytochemistry and
Nanotechnology, Department of Zoology, Annamalai University,
Annamalainagar for his assistance in the extraction of green nanoparticles and
the staff members of the Vector Control Research Centre (ICMR-VCRC),
Pondicherry for facilities provided to carry out the experimental part of this
works.
I express my heartfelt thanks to Mr. U. MUTHUKUMAR, Research
Scholar, Department of Zoology, Annamalai University, Annamalainagar for
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his assistance in the extraction of green nanoparticles and to carry out the
experimental part of this works.
I wish to extend my sincere thanks to Doctoral Committee Members,
Dr. S. RAVEENDRAN, Associate Professor and Dr. K. MUTHUKUMARA-
VEL, Assistant Professor, and all the staff members of zoology, Khadir
Mohideen College, Adirampattinam for their moral support and
encouragement during the course of this investigation.
Last, but not the least, I am very much grateful to my parents and
sister without whom this study would not have been completed.
R. THAMEEM AZARUDEEN
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CONTENTS
Chapter
No. TITLE
Page
No.
1 INTRODUCTION 1
Objectives of the present study 15
2 REVIEW OF LITERATURE 16
2.1 Larvicidal activity 17
2.2 Ovicidal activity 36
2.3 Adulticidal activity 43
2.4 Non-target effects against aquatic organism 47
2.5 Plant description 50
3 MATERIALS AND METHODS 53
3.1 Collection and identification of plants 53
3.2 Preparation of the plant aqueous leaf extracts 53
3.3 Synthesis of silver nanoparticles 56
3.4 Characterization silver nanoparticles 57
3.5 Laboratory colonization of mosquitoes 59
3.6 Bioassay 62
3.6.1 Larvicidal activity 62
3.6.2 Ovicidal activity 62
3.6.3 Adulticidal activity 63
3.6.4 Biotoxicity on Non-target aquatic organism 64
3.7 Statistical analysis 64
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4 RESULTS 66
4.1 Characterization of silver nanoparticles 66
4.2 Larvicidal activity against mosquito vectors 79
4.3 Ovicidal activity against mosquito vectors 84
4.4 Adulticidal activity against mosquito vectors 90
4.5 Biotoxicity of non-target organism 95
5 DISCUSSION 175
5.1 Plant-mediated synthesis and characterization of
mosquitocidal nanoparticles 176
5.1.1 UV–vis analysis of Phyto-synthesized Ag NPs 176
5.1.2 FTIR analysis of synthesized silver nanoparticles 179
5.1.3 SEM and EDX analysis of synthesized silver
nanoparticles 181
5.1.4 TEM analysis of synthesized silver nanoparticles 183
5.1.5 X-ray diffraction (XRD) of synthesized silver
nanoparticles 185
5.2 Larvicidal activity 187
5.3 Ovicidal activity 205
5.4 Adulticidal activity 211
5.5 Non-target aquatic organism 218
6 SUMMARY AND CONCLUSIONS 222
7 REFERENCES 225
Appendix: List of Paper Publications
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ABBREVIATIONS
AgNO3 - Silver nitrate
AgNPs - Silver nanoparticles
ANOVA - Analysis of variance
cm-1
- Centimeter
EDX - Energy dispersive X-ray spectroscopy
FTIR - Fourier transform infrared spectroscopy
hr - Hours
ICMR - Indian medical council research
IMM - Integrated mosquito management
keV - Kiloelectron volt
LC50 - Lethal concentration killing 50% population
LC90 - Lethal concentration killing 90% population
LCL - Lower confidence limits
mg - Milligrams
Mmol - Millimoles
nm - Nanometer
ºC - Centigrade
SD - Standard deviation
SEM - Scanning electron microscope
TEM - Transmission electron microscope
UCL - Upper confidence limits
UV - Ultraviolet
VCRC - Vector control research centre
WHO - World health organization
XRD - X-ray diffraction
μg - Microgram
μL - Micro liter
χ2 - Chi-square
% - Percentage
*p<0.05 - Level of significance
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1. INTRODUCTION
Mosquitoes are well known as annoying pests and as carriers of
disease-causing agents to humans and animals. Their rapid wing movement
produces a distinctive high-pitched hum, and their bites cause red, itchy welts.
Mosquitoes are small, slender flies that are members of the family Culicidae.
They generally look alike to the naked eye, but the different species vary
greatly, with various patterns of bands, stripes, and spots that adorn their
bodies. Knowing which mosquito species lives in and around your home is
important because they vary in biology and behavior and have different
impacts on humans and wildlife. Understanding their biology will help you
make more informed decisions about dealing with the health risks they pose,
whether to attempt mosquito control, and what methods to use to control
them.
Vector-borne infectious diseases are emerging or resurging as a result
of changes in public health policy, insecticide and drug resistance, shift in
emphasis from prevention to emergency response, demographic and societal
changes, and genetic changes in pathogens. Effective prevention strategies
can reverse this trend. Research on vaccines, environmentally safe
insecticides, alternative approaches to vector control, and training programs
for health-care workers are needed.
Mosquitos are important vectors of several tropical diseases, including
malaria, filariases, and numerous viral diseases, such as dengue, Japanese
encephalitis and yellow fever. In countries with a temperate climate they are
more important as nuisance pests than as vectors. There are about 3500
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species of mosquito, of which about 100 are vectors of human diseases.
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.
Vector control is an essential requirement in control of epidemic
diseases such as malaria, filariasis, dengue that are transmitted by different
species of mosquitoes. Emergence of insecticide resistance and their harmful
effects on non-target organisms and environment has necessitated an urgent
search for development of new and improved mosquito control methods that
are economical and effective as well as safe for non-target organisms and the
environment. Insecticides synthesized from natural products, such as silver,
gold or silicon nanoparticles of herbal origin have become a priority in this
search.
Nanoparticles are defined as particulate dispersions or solid particles
with a size of 10-1000 nm. The word “nano” is derived from a Greek word
meaning “dwarf”. In technical terms, the word “nano” means 10-9, or one
billionth of a meter. Naturally, the word nanotechnology evolved due to use
of nanometer size particles. Targeted nanoparticles exhibit many novel
characteristic features, such as extra ordinary strength, more chemical
reactivity, magnetic properties and or high electrical conductivity.
“Nanotechnology” deals with application of such particles in biological,
physical, chemical, environmental, agricultural, industrial or pharmaceutical
science. Depending upon the method of preparation, nanoparticles,
nanospheres or nanocapsules can be obtained. Although physical and
chemical methods are more popular and widely used for synthesis of
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nanoparticles, the related environmental toxicity and nonbiodegradable nature
of the products limited their applications. So, the “green” route for synthesis
of nanoparticles from herbal origin is of great interest due to eco-friendliness,
economic prospects, feasibility and wide range of applications.
1.1 Vector mosquitoes
1.1.1 Anopheles stephensi
About 380 species of Anopheles occur around the world. Some 60
species are sufficiently attracted to humans to act as vectors of malaria. A
number of Anopheles species are also vectors of filariasis and viral diseases.
Life cycle
Larval habitats vary from species to species, but are frequently exposed
to sunlight and commonly found in association with emergent vegetation,
such as grass or mats of floating vegetation or algae. The most preferred
breeding sites are pools, seepages, quiet places in slow-running streams, rice
fields, leaf axils of certain epiphytic plants and puddles of rainwater. Artificial
containers, such as pots, tubs, cisterns and overhead tanks are not usually
suitable, except in the case of Anopheles stephensi in south-west Asia. The
eggs, laid singly on the water surface where they float until hatching, are
elongated, have a pair of lateral floats, and are about 1 mm in length.
Hatching occurs in 2–3 days. The larvae float in a horizontal position at the
surface, where they feed on small organic particles. In the tropics the duration
of development from egg to adult is 11–13 days.
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Behaviour
Anopheles mosquitos are active between sunset and sunrise. Each
species has specific peak biting hours, and there are also variations in their
preference for biting indoors or outdoors. The anophelines that enter houses to
feed often rest indoors for a few hours after feeding. They may then leave for
outdoor sheltered resting sites, among them vegetation, rodent burrows,
cracks and crevices in trees or in the ground, caves and the undersides of
bridges. Alternatively, they may stay indoors for the whole period needed to
digest the blood-meal and produce eggs. Indoor resting is most common in
dry or windy areas where safe outdoor resting sites are scarce. Once the eggs
are fully developed the gravid mosquitos leave their resting sites and try to
find a suitable breeding habitat. Many Anopheles species feed on both humans
and animals. They differ, however, in the degree to which they prefer one
over the other. Some species feed mostly on animals while others feed almost
entirely on humans. The latter species are the more dangerous as vectors of
malaria.
1.1.2 Aedes aegypti
Aedes mosquitos occur around the world and there are over 950
species. They can cause a serious biting nuisance to people and animals, both
in the tropics and in cooler climates. In tropical countries Aedes aegypti is an
important vector of dengue, dengue haemorrhagic fever, yellow fever and
other viral diseases. A closely related species, A. albopictus, can also transmit
dengue. In some areas Aedes species transmit filariasis, bancroftian filariasis
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and arboviral diseases, such as Japanese encephalitis. In some areas they are a
considerable nuisance.
Life cycleoften found resting in dark corners of rooms, shelters and
culverts. They also rest outdoors on vegetation and in holes in trees in
forested areas.
1.1.3 Culex quinquefasciatus
About 550 species of Culex have been described, most of them from
tropical and subtropical regions. Some species are important as vectors of
bancroftian filariasis and arboviral diseases, such as Japanese encephalitis. In
some areas they are a considerable nuisance.
Life cycle
Rafts of 100 or more eggs are laid on the water surface. The rafts
remain afloat until hatching occurs 2–3 days later. Culex species breed in a
large variety of still waters, ranging from artificial containers and catchment
basins of drainage systems to large bodies of permanent water. The most
common species, C. quinquefasciatus, a major nuisance and vector of
bancroftian filariasis, breeds especially in water polluted with organic
material, such as refuse and excreta or rotting plants. Examples of such
breeding sites are soak away pits, septic tanks, pit latrines, blocked drains,
canals and abandoned wells. In many developing countries
C. quinquefasciatus is common in rapidly expanding urban areas where
drainage and sanitation are inadequate. C. tritaeniorhynchus, the vector of
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Japanese encephalitis in Asia, prefers cleaner water. It is most commonly
found in irrigated rice fields and in ditches.
Behaviour
C. quinquefasciatus is a markedly domestic species. The adult females
bite people and animals throughout the night, indoors and outdoors. During
the day they are inactive and are often found resting in dark corners of rooms,
shelters and culverts. They also rest outdoors on vegetation and in holes in
trees in forested areas.
1.2 Mosquito-borne diseases
1.2.1 Malaria
Malaria is caused by the protozoan parasite Plasmodium which spends
part of its life-cycle in humans and part in certain species of mosquitoes. Five
species of Plasmodium cause malaria in humans: Plasmodium falciparum, P.
vivax, P. malariae, P. ovale, and the monkey malaria parasite P. knowlesi. Of
these, P. falciparum is the most important in most parts of the tropics and is
responsible for most severe illnesses and deaths due to malaria. Malaria
parasites are transmied by female mosquitoes belonging to the genus
Anopheles. Male Anopheles mosquitoes only feed on plant juices and nectar
and cannot transmit malaria. The life-cycle of the malaria parasite is divided
into three different phases: one in the mosquito, the sporogonic cycle; and two
in the human host – the erythrocytic cycle (in human red blood cells) and the
exo-erythrocytic cycle.
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1.2.2 Dengue
Dengue is a mosquito-borne disease caused by any one of four closely
related dengue viruses (DENV-1, -2, -3, and -4). Infection with one serotype
of DENV provides immunity to that serotype for life, but provides no long-
term immunity to other serotypes. Thus, a person can be infected as many as
four times, once with each serotype. Dengue viruses are transmitted from
person to person by Aedes mosquitoes (most often A. aegypti) in the domestic
environment. Epidemics have occurred periodically in the Western
Hemisphere for more than 200 years. In the past 30 years, dengue
transmission and the frequency of dengue epidemics have increased greatly in
most tropical countries in the American region.
Symptoms of dengue include fever, severe headache, pain behind the
eyes, muscle and joint pain, swollen glands and rash. There is no vaccine or
any specific medicine to treat dengue. People who have dengue fever should
rest, drink plenty of fluids and reduce the fever using paracetamol.
1.2.3 Yellow fever
Yellow fever is an acute viral haemorrhagic disease transmitted by
Aedes mosquitoes. The “yellow” in the name refers to the jaundice that
affects some patients. There are an estimated 200 000 cases of yellow fever
causing 30 000 deaths worldwide each year. The virus that causes yellow
fever is endemic in tropical areas of Africa and Latin America where a
combined population of over 900 million people lives. Small numbers of
imported cases occur in countries free of yellow fever.
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Symptoms of the disease include fever, muscle pain with prominent
backache, headache, shivers, loss of appetite, and nausea or vomiting. Most
patients improve and their symptoms disappear after 3 to 4 days. However,
15% of patients enter a second, more toxic phase within 24 hours of the initial
remission. High fever returns and several body systems are affected. The
patient rapidly develops jaundice and complains of abdominal pain with
vomiting and internal bleeding. Half of these patients die within 10 to 14
days. There is no specific treatment for yellow fever, only supportive care to
treat dehydration, respiratory failure and fever. Vaccination is the most
important preventive measure against yellow fever. The vaccine is safe,
affordable and highly effective. A single dose of yellow fever vaccine is
sufficient to provide life-long protection against the disease. For more
information, see the factsheet on yellow fever.
1.2.4 Chikungunya
Chikungunya is a viral tropical disease transmitted also by Aedes
mosquitoes. It is relatively uncommon and poorly documented. The disease
has been found in Africa, Asia, and on islands in the Caribbean, Indian and
Pacific Oceans. Typical symptoms are an acute illness with fever, skin rash
and incapacitating joint pains that can last for weeks. The latter distinguishes
chikungunya virus from dengue, which otherwise shares the same vectors,
symptoms and geographical distribution. There is no cure or commercial
vaccine for the disease. Most patients recover fully but, in some cases, joint
pain may persist for several months or even years. As with dengue, the only
method to reduce transmission of chikungunya virus is to control vector
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mosquitoes and protect against mosquitoes bites. For more information, see
the factsheet on chikungunya.
Typical symptoms are an acute illness with fever, skin rash and
incapacitating joint pains that can last for weeks. The latter distinguishes
chikungunya virus from dengue, which otherwise shares the same vectors,
symptoms and geographical distribution. There is no cure or commercial
vaccine for the disease. Most patients recover fully but, in some cases, joint
pain may persist for several months or even years. As with dengue, the only
method to reduce transmission of chikungunya virus is to control vector
mosquitoes and protect against mosquitoes bites. For more information, see
the factsheet on chikungunya.
1.2.5 Lymphatic filariasis
Infection with lymphatic filariasis, commonly known as elephantiasis,
occurs when thread-like, filarial parasites are transmitted to humans through
mosquitoes. Lymphatic filariasis is transmitted by different types of
mosquitoes, for example by the Culex mosquito, widespread across urban and
semi-urban areas; Anopheles, mainly in rural areas; and Aedes, mainly in the
Pacific Islands and parts of the Philippines. It is also transmitted by 3 types of
parasite (Wuchereria bancrofti, responsible for 90% of cases, Brugia malayi
and B. timori). Microscopic parasitic worms lodge in the lymphatic system
and disrupt the immune system. They live for 6–8 years and, during their
lifetime, produce millions of microfilariae (tiny larvae) that circulate in the
blood. More than 120 million people are currently infected with lymphatic
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filariasis, about 40 million of them are disfigured and incapacitated by the
disease. Lymphatic filariasis afflicts more than 25 million men with genital
disease and more than 15 million people with lymphoedema. The majority of
infections have no symptoms but silently cause damage to the lymphatic
system and the kidneys as well as alter the body’s immune system. Acute
episodes of local inflammation involving skin, lymph nodes and lymphatic
vessels often accompany chronic lymphoedema (tissue swelling).
Approximately 65% of those infected live in the WHO South-East Asia
Region, 30% in the African Region, and the rest in other tropical areas.
Recommended treatment to clear the parasites from the bloodstream is a
single dose of albendazole given together with either diethylcarbamazine or
ivermectin. Interruption of transmission of infection can be achieved if at least
65% of the population at risk is treated over 5 years. For more information,
see the factsheet on lymphatic filariasis.
1.3 Control of mosquitoes
Environmental modification and manipulation for the control of
mosquito vectors of disease almost disappeared with the development of
chemical insecticides. After the Second World War, the use of such
insecticides, especially as residual house sprays, was so efficacious in
controlling mosquitos and mosquito-borne diseases that little or no use was
made of biological and physical methods of mosquito control (Amer and
Mehlhorn, 2006).
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1.4 Disadvantages of Chemical control
The use of insecticides for the control of malaria and other vector-
borne diseases acquired great impetus with the advent of DDT and other
organochlorine compounds in the late 1940s. Their use in public health
increased in extent and intensity with the worldwide programme of malaria
eradication which was initiated in 1956/57. The residual insecticidal effect of
some of these chemicals made it possible to sustain an attack on the malaria
vectors by means of the periodic indoor spraying of houses (see section 3.2.1
below). The development of mosquito resistance to some residual
insecticides, and the elusive behaviour of certain mosquito vectors have
diminished the effectiveness of residual spraying and hence the extent of its
application. Faced with this problem and in line with the universal acceptance
of the principles and advantages of an integrated approach, planners and
operators of vector control programmes have directed their attention to other
methods of mosquito control silch as larviciding, the space application of
pesticides using ultra-lowvolume (ULV) techniques or thermal fogging, and
the reintroduction of environmental management and biological control
measures to supplement residual spraying. In the control of certain mosquito-
borne diseases such as dengue haemorrhagic fever and the encephalitides,
reliance is at present placed mainly on aerosol application of pesticides (ULV,
fog, mist, etc.). For others, e.g., urban yellow fever, larviciding also has a
prominent place. In antimalaria programmes, there is still heavy reliance on
residual insecticide applications indoors, while larviciding is increasingly
being used, particularly in urban environments.
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1.5 Advantages of biological control
Biological control, particularly using larvivorous fish, was important to
malaria control programmes in the 20th century, particularly in urban and
periurban areas for immediate use in developed and developing countries. It
has a very positive role to play in the integrated control methodologies in
which both pesticides and fish or other biotic agents have their own roles.
Biological control refers to the introduction or manipulation of organisms to
suppress vector populations. A wide range of organisms helps to regulate
mosquito populations naturally through predation, parasitism and
competition. As biological mosquito control agents, larvivorous fish (i.e.,
those that feed on immature stages of mosquitoes) are being used extensively
all over the world since the early 1900s (pre DDT era) (Raghavendra and
Subbarao 2002). During the pre DDT era, control of mosquitoes and mosquito
vectors of different mosquito borne diseases was undertaken mainly by
environmental management, pyrethrum space spraying, use of Paris green,
oiling with petrol products and introduction of larvivorous fish. Recognizing
the high larvivorous potential of Gambusia affinis, this fish species was
purposely introduced from its native Texas (Southern USA) to the Hawaiin
Islands in 1905. In 1921, it was introduced in Spain; then from there in Italy
during 1920s and later to 60 other countries. Beginning in 1908, another
larvivorous fish, Poecilia reticulata, a native of South America, was
introduced for malaria control into British India and many other countries.
The introduction of the use of DDT in indoor residual spraying for malaria
control around the mid - 1940s led to the gradual decline in the use of
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concepts of environmental management and biological control methods,
except in a few programmes in Russia. In the fifties, attention was directed to
eradicate mosquitoes using synthetic insecticides until insecticide resistance
began to assume prominence. In 1969, the WHO changed its strategy of
malaria eradication by spraying houses with synthetic insecticides in favour of
the more realistic one for the control of mosquito populations in the larval
stages (post DDT era) (Raghavendra and Subbarao 2002) . The selection of
biological control agents should be based on their potential for unintended
impacts, self replicating capacity, climatic compatibility, and their capability
to maintain very close interactions with target prey populations (Waage and
Greathead 1988). They eliminate certain prey and sustain in such
environments (i.e., they eat the prey, when introduced) for long periods
thereafter (Marten et al., 1994). However, this will only be possible if the
predator possesses extraordinary search efficiency irrespective of the
illuminated situation in response to the emergence of prey. It is important to
have a sound knowledge of predator’s prey selective patterns and particularly
of its mosquito larval selection in the presence of alternate natural prey.
1.6 Advantages of silver nanoparticles by plants
The major advantage of using plant extracts for silver nanoparticle
synthesis is that they are easily available, safe, and nontoxic in most cases,
have a broad variety of metabolites that can aid in the reduction of silver ions,
and are quicker than microbes in the synthesis. The main mechanism
considered for the process is plant-assisted reduction due to phytochemicals.
The main phytochemicals involved are terpenoids, flavones, ketones,
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aldehydes, amides, and carboxylic acids. Flavones, organic acids, and
quinones are water-soluble phytochemicals that are responsible for the
immediate reduction of the ions. Studies have revealed that xerophytes
contain emodin, an anthraquinone that undergoes tautomerization, leading to
the formation of the silver nanoparticles. In the case of mesophytes, it was
found that they contain three types of benzoquinones: cyperoquinone,
dietchequinone, and remirin. It was suggested that the phytochemicals are
involved directly in the reduction of the ions and formation of silver
nanoparticles.
1.7 Objectives of the present study
In this research, the mosquitocidal activity of synthesized silver
nanoparticles using five plants Merremia emarginata, Naregamia alata,
Hedyotis puberula, Aglaia elaeagnoidea, and Ventilago madrasapatna
against three important mosquito vectors Anopheles stephensi, Aedes aegypti
and Culex quinquefasciatus were analyzed. The detailed objectives of this
study were,
1. To collect, identify, prepare the aqueous extract and synthesis of silver
nanoparticles from the selected plant leaves of M. emarginata, N.alata,
H.puberula, A. elaeagnoidea, and V.madrasapatna from Nilgiris, Western
Ghats, Tamil Nadu, India.
2. To characterize the AgNPs using UV-vis spectrophotometry, Fourier
transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD),
atomic force microscopy (AFM), scanning electron microscopy (SEM)
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with energy dispersive X-ray spectroscopy (EDX) and transmission
electron microscopy (TEM).
3. To evaluate the efficacy of aqueous leaf extract and synthesized AgNPs
against larvicidal, ovicidal and adulticidal activities of Anopheles
stephensi, Aedes aegypti and Culex quinquefasciatus.
4. To test the effect of aqueous leaf extract and AgNPs on non-target
organism.
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2. REVIEW OF LITERATURE
Mosquitoes are principal vector of many vector borne diseases
affecting human beings and animals. These vectors are responsible for
transmitting several diseases such as malaria, dengue fever, dengue
hemorrhagic fever, dengue shock syndrome, chikungunya, lymphatic
filariasis, Japanese encephalitis and leishmaniasis etc. and cause thousands of
deaths every year (Benelli, 2015a,b). There is an urgent need to check the
proliferation of the population of vector mosquitoes in order to reduce vector
borne diseases by appropriate control methods (Kuppusamy and Murugan,
2009). As mosquitoes are water breeders, their larval stages are attractive
targets of pesticides, and it is easy to deal with them in this habitat (Dhiman et
al., 2010; Benelli et al., 2015; Benelli, 2016). Use of plant extract for the
synthesis of nanoparticles could be advantageous over other environmentally
benign biological processes by eliminating the elaborate process of
maintaining cell cultures.
The problem has a complex face and it has to be handled carefully. It is
essential to control mosquito population so that people can be protected from
mosquito borne diseases. These diseases can be controlled by targeting the
causative parasites and pathogens. It is easier to control vectors than parasites.
The chemical control was one of the most widely used conventional methods
for mosquito control since chemical pesticides are relatively inexpensive
usually produces immediate control. Generally, the chemical control is carried
out by the indoor residual spraying of insecticides such as dichloro diphenyl
trichloro ethane, hexa chlorocyclo hexane, benzene hexa chloride, melathion
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and synthetic pyrothroid. But, the development of resistance against these
chemicals in various mosquito populations has been reported. Therefore,
biological control can thus provide and effective and environmental friendly
approach, which can be used as an alternative to minimize the mosquito
population. Fungi and fungus derived products are highly toxic to mosquitoes,
yet have low toxicity to non-target organisms (Govindarajan et al., 2005).
Silver nanoparticles are emerging as one of the fastest growing
materials due to their unique physical, chemical and biological properties;
small size and high specific surface area. Biological synthesis of nanoparticles
has received increased attention due to a growing need to develop
environmentally benign technologies in material synthesis. Several plant
species have been utilized in this regard.
The use of environmentally benign materials such as silver
nanoparticles offers numerous benefits of eco-friendliness and compatibility
for larvicidal application. In these circumstances, an improvised method using
the biologically synthesised silver nanoparticles were evaluated for the
destruction of the mosquito larvae of An. stephensi, Ae. aegypti and Cx.
quinquefasciatus. .
2.1 Larvicidal activity
Larvicidal activity of green synthesized silver nanoparticles from the
aqueous leaf extract of Carissa spinarum showed against larvae of An.
subpictus, Ae. albopictus and Cx. tritaeniorhynchus (Govindarajan et al.,
2016a). Mahesh Kumar et al. (2016) reported that the silver nanoparticles
synthesized using Berberis tinctoria leaf extract was toxic against larvae of
Ae. albopictus. Chandramohan et al. (2016) studied that the biosynthesized
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silver nano particles using neem cake extract of Azadirachta indica against
larvae and pupae of Ae.aegypti. Govindarajan et al. (2016a) investigated the
synthesized silver nanoparticles from Bauhinia variegata aqueous leaf extract
were toxic to third instar larvae of An. subpictus, Ae. albopictus, and Cx.
tritaeniorhynchus.
The mosquitocidal properties of synthesized silver nanoparticles using
Pteridium aquilinum leaf extract against An. stephensi (Panneerselvam et al.,
2016). Govindarajan and Benelli (2016a) evaluated the biosynthesized silver
nanoparticles using Barleria cristata leaf extract against larvae of Anopheles
subpictus, Aedes albopictus, and Culex tritaeniorhynchus. Murugan et al.
(2016a) determined that the carbon and silver nanoparticles synthesized from
Moringa oleifera seed extract were toxic against larvae and pupae of Cx.
quinquefasciatus. Govindarajan et al. (2016c) investigated the biosynthesized
silver nanoparticles with Clerodendrum chinense leaf extract against larvae of
An. subpictus, Ae. albopictus and Cx. tritaeniorhynchus. Green synthesized of
silver nanoparticles using Hybanthus enneaspermus aqueous extract against
the fourth instar larvae of An. subpictus and Cx. quinquefasciatus (Suman
et al., 2016).
Govindarajan et al. (2016d) reported that the acute toxicity of essential
oil from Plectranthus barbatus against third instar larvae of An. subpictus Ae.
albopictus and Cx. tritaeniorhynchus. Mosquito larvicidal and pupicidal
activity of synthesized silver nanoparticles using Centroceras clavulatum leaf
extract was toxic against dengue vector Ae. aegypti (Murugan et al., 2016).
The larvicidal and repellent activity of Zingiber nimmonii essential oil was
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evaluated against the malaria vector An. stephensi, dengue vector Ae. aegypti,
and the lymphatic filariasis vector Cx. quinquefasciatus (Govindarajan et al.,
2016b).
Murugan et al. (2015) evaluated that the mosquitocidal properties of
the Toddalia asiatica aqueous extract and synthesized silver nanoparticles
against the filariasis vector Cx. quinquefasciatus and predatory effectiveness
of guppy Poecilia reticulata. Velu et al. (2015) observed the maximum
larvicidal activity in synthesized silver nanoparticles from Arachis hypogaea
peels against the fourth instar larvae of Ae. aegypti and An. stephensi.
Synthesized silver nanoparticles from Aloe vera extract were highly effective
against malarial vector An. stephensi (Dinesh et al., 2015).
Ramesh Kumar et al. (2015) have reported that the larvicidal activity
of synthesized silver nanoparticles from Morinda tinctoria leaf extract against
the third instar larvae of Cx. quinquefasciatus. The larvicidal activity of
biosynthesized silver nanoparticles from A. indica aqueous leaf extract
against larvae of Ae. aegypti and Cx. quinquefasciatus (Poopathi et al., 2015).
Rajasekharreddy and Rani (2015) have reported that the fabricated silver
nanoparticles using the seed extract of Sterculia foetida showed
mosquitocidal activity against fourth instar larvae of Ae. aegypti, An.
stephensi and Cx. quinquefasciatus. Murugan et al. (2015a) investigated the
silver nanoparticles fabricated with Caulerpa scalpelliformis were effective
against larvae and pupae of Cx. quinquefasciatus. Murugan et al. (2015b)
reported that the synthesis of gold nanoparticles from Cymbopogon citratus
were toxic against An. stephensi and Ae. aegypti. Synthesized silver
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nanoparticles using Artemisia vulgaris aqueous leaf extract against larvae and
pupae of Ae. aegypti (Murugan et al., 2015c).
Suresh et al. (2015) determined that the silver nanoparticles fabricated
using the aqueous extract of Phyllanthus niruri was highly effective against
larvae and pupae of Ae. aegypti. The high mortality rate was showed the
fabricated silver nanoparticles with Crotalaria verrucosa leaf extract against
larvae and pupae of Ae. aegypti (Murugan et al., 2015e). Murugan et al.
(2015f) performed the larvicidal activity of synthesized silver nanoparticles
from Aristolochia indica against young instars larvae of An. stephensi.
Murugan et al. (2015g) investigated the synthesized silver nanoparticles using
Bruguiera cylindrical aqueous leaf extract were highly toxic against larvae
and pupae of Ae. aegypti. Silver nanoparticles fabricated with Datura metel
aqueous leaf extract against larvae and pupae of malarial vector An. stephensi
(Murugan et al., 2015h).
Balakrishnan et al. (2015) determined that the larvicidal activity of
synthesized silver nanoparticles with Avicennia marina leaf extract were toxic
against larvae of An. stephensi and Ae. aegypti. Green synthesized silver
nanoparticles produced using the Annona muricata leaf extract against third
instar larvae of Ae. aegypti, An. stephensi and Cx. quinquefasciatus (Santhosh
et al., 2015). Madhiyazhagan et al. (2015) have reported that the silver
nanoparticles synthesized using the aqueous extract of the seaweed
Sargassum muticum against An. stephensi, Ae. aegypti and Cx.
quinquefasciatus. Low doses of Mimusops elengi synthesized silver
nanoparticles showed larvicidal and pupicidal toxicity against the malaria
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vector An. stephensi and the arbovirus vector Ae. albopictus (Subramaniam et
al., 2015).
Vimala et al. (2015) evaluated the larvicidal activity of synthesized
silver nanoparticles using leaf and fruit extracts from C. guianensis against
fourth instar larvae of Ae. aegypti. Biosynthesized silver nanoparticles using
2, 7.bis [2-[diethylamino]-ethoxy]fluorence isolate from Melia azedarach
leaves have been tested against third instar larvae of Ae. aegypti and
Cx. quinquefasciatus (Ramanibai and Velayutham, 2015). Extremely stable
silver nanoparticles synthesized using the leaf aqueous extract of Mukia
maderaspatana against fourth instar larvae of Ae. aegypti and Cx.
quinquefasciatus (Chitra et al., 2015).
Arokiyaraj et al. (2015) reported that the synthesized silver
nanoparticles using Chrysanthemum indicum were toxic against An. stephensi.
Silver nanoparticles synthesized from the seed extract of Moringa oleifera
have been reported as toxic towards young instars Ae. aegypti (Sujitha et al.,
2015). Najitha Banu and Balasubramanian, (2015) investigated that the silver
nanoparticles synthesized extracellular method using Bacillus megaterium
against first, second, third, and fourth instar larvae of Cx. quinquefasciatus
and Ae.aegypti. Lallawmawma et al. (2015) evaluated the fabricated silver
nanoparticles and gold nanoparticles with Jasminum nervosum were toxic
against Cx. quinquefasciatus. The larvicidal activity of synthesized silver
nanoparticles using Mangifera indica aqueous leaf extracts against fourth
larvae of An. subpictus and Cx. quinquefasciatus (Rajakumar et al., 2015).
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The synthesized silver nanoparticles using bacterial strains of Listeria
monocytogenes, Bacillus subtilius and Streptomyces anulatus against the
larvae, pupae and adults of An. stephensi and Cx. quinquefasciatus (Soni and
Prakash, 2015). Larvicidal activity of silver nanoparticles synthesized using
Pseudomonas mandelii against larvae of An.subpictus and Cx.
tritaeniorhynchus (Mageswari et al., 2015). Amerasan et al. (2015)
determined that the lower dosages of myco-synthesized silver nanoparticles
using Metarhizium anisopliae were effects on larvae and pupae of An.
culicifacies. The biosynthesized silver nanoparticles using Artemisia vulgaris
leaves extract were toxic against dengue vector larvae and pupae of Ae.
aegypti (Murugan et al., 2015). Mosquito larvicidal and pupicidal potential of
synthesized silver nanoparticles using seaweed extract of Ulva lactuca against
larvae and pupae of malaria vector An. stephensi (Murugan et al., 2015).
Murugan et al. (2015) determined that the larvicidal, pupicidal, and
smoke toxicity of Senna occidentalis and Ocimum basilicum leaf extracts
against the malaria vector An. stephensi. The larvicidal potential of hexane,
choloroform, ethyl acetate, acetone, and methanol extracts of seven aromatic
plants, viz., Blumea mollis, Chloroxylon swietenia, Clausena anisata, Feronia
limnonia, Lantana camara, Plectranthus amboinicus and Tagetes erecta were
screened against Cx. quinquefasciatus, Ae.aegypti and An. stephensi
(Jayaraman et al., 2015). Escaline et al. (2015) evaluated the mosquito
larvicidal activity of Piper nigrum crude extract was toxic against the dengue
vector Ae. aegypti. Ragavendran et al. (2015) examined the mosquito
larvicidal and pupicidal potential of fungus mycelia using ethyl acetate and
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methanol solvent extracts produced by Aspergillus terreus against An.
stephensi, Cx. quinquefasciatus, and Ae. aegypti.
Shawky et al. (2014) evaluated the synthesized silver nanoparticles
with the aqueous leaf extract of Citrullus colocynthis were toxic to third instar
larvae of Cx. pipiens. Morinda tinctoria acetone leaf extract has been used to
produce silver nanoparticles against third instar larvae of Cx. quinquefasciatus
(Kumar et al., 2014).Veerakumar et al. (2014a) reported that the synthesized
silver nanoparticles using Feronia elephantum aqueous leaf extract against
larvae of An. stephensi, Ae. aegypti and Cx. quinquefasciatus. Larvicidal
potential of silver nanoparticles produced using the aqueous leaf extract of
Leucas aspera against fouth instar larvae of Ae. aegypti (Suganya et al.,
2014).
Silver nanoparticles fabricated with the leaf extract of Heliotropium
indicum have been tested against third instar larvae of An. stephensi, Ae.
aegypti and Cx. quinquefasciatus (Veerakumar et al., 2014b). Suresh et al.
(2014) found that the silver nanoparticles synthesized using the aqueous root
extract of Delphinium denudatum exhibited toxic activity towards second
instar larvae of Ae. aegypti. Najitha banu et al. (2014) evaluated the larvicidal
efficacy of synthesized silver nanoparticles using Bacillus thuringiensis
against dengue vector Ae. aegypti. The aqueous leaf extracts of neem has been
employed to produce silver nanoparticles active as larvicides and pupicides
against An. stephensi and Cx. quinquefasciatus (Soni and Prakash, 2014a).
Karthikeyan et al. (2014) performed that the larvicidal potential of
synthesized silver nanoparticles using Melia dubia were toxic to fourth instar
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larvae of Cx. quinquefasciatus. The aqueous leaf extracts of Aegle marmelos
have been used to synthesize nickel nanoparticles toxic to Ae. aegypti, An.
stephensi and Cx. quinquefasciatus (Angajala et al., 2014).
The larvicidal efficacy of silver nanoparticles synthesized using fungus
Beauveria bassiana was assessed against the larval instars of dengue vector
Ae. aegypti (Najitha Banu and Balasubramanian, 2014a). Sundaravadivelan
and Padmanabhan, (2014) investigated the larvicidal and pupicidal effect of
mycosynthesized silver nanoparticles using an entomopathogenic fungi
Trichoderma harzianum against the dengue vector Ae. aegypti. A green
process for the extracellular production of silver (Ag) and gold (Au)
nanoparticles (NP) synthesized using the soil fungi Chrysosporium
keratinophilum and Verticillium lecanii against the larvae and pupae of An.
stephensi , Cx. quinquefasciatus and Ae. aegypti (Soni and Prakash, 2014b).
The larvicidal efficacy of synthesized silver nanoparticles using fungi of
Isaria fumosorosea against first, second, third and fourth instar larvae of Cx.
quinquefasciatus and Ae. aegypti (Najitha Banu and Balasubramanian,
2014b).
Sharma et al. (2014) reported that the chloroform extract of Artemisia
annua leaf has shown a strong larvicidal activity against An. stephensi and Ae.
aegypti. Nayak, (2014) studied the larvicidal potential of crude extracts from
Annona reticulata and Pongamia pinnata were assessed against early fourth
instar larvae of Cx. quinquefasciatus. The compounds, ecbolin A and ecbolin
B isolated from the ethyl acetate extract of Ecbolium viride root showed
larvicidal activity against the third instar larvae and pupae of Cx.
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quinquefasciatus (Cecilia et al., 2014). Maheswaran and Ignacimuthu, (2014)
evaluated the essential oil and an isolated compound from the leaves of
Polygonum hydropiper against dengue vector Ae. albopictus.
The larvicidal properties of methonal, chloroform, petroleum ether
extracts of Monstera adansonii against early fourth instar larva of Culex
quinequefaciatus (Gomathi et al., 2014). Larvicidal potential of Lawsonia
inermis and Murraya exotica leaves extract were assessed against third and
fourth instar larvae and pupae of Cx. quinquefasciatus (Dass and Mariappan,
2014). Liu et al. (2014) investigated that the larvicidal activity of the essential
oil from Allium macrostemon was found to possess against larvae of Ae.
albopictus. Raveen et al. (2014) showed that the crude hexane and aqueous
extract of Nerium oleander flowers were assessed for larvicidal activity
against the filarial vector Cx. quinquefasciatus.
Velayutham et al. (2013) determined the larvicidal potential of
synthesized silver nanoparticles using the aqueous bark extract of Ficus
racemosa was successfully studied against fourth larvae of the filariasis
vector Cx. quinquefasciatus and the Japanese encephalitis vector Culex
gelidus. The coir extract of Cocos nucifera has been employed to produce
silver nanoparticles toxic to fourth instar larvae of An. stephensi Cx.
quinquefasciatus (Roopan et al., 2013). Veerakumar et al. (2013) have
reported that the silver nanoparticles fabricated with Sida acuta leaf extract
was assessed against third instar larvae of An. stephensi, Ae. aegypti and
Cx. quinquefasciatus. Silver nanoparticles were produced using the leaf and
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berry extracts of Solanum nigrum and tested against second and third instar
larvae of An. stephensi and Cx. quinquefasciatus (Rawani et al., 2013).
Silver nanoparticles fabricated with the Murraya koenigii leaf extract
were toxic to An. stephensi and Ae. aegypti (Suganya et al., 2013). Naresh
Kumar et al. (2013) studied the larvicidal properties of Anthocephalus
cadamba synthesized gold nanoparticles (AuNP) has been ascertained against
third instar larvae of Cx. quinquefasciatus. The silver nanoparticles produced
using Nerium oleander leaf extract were toxic to An. stephensi larvae and
pupae (Roni et al., 2013). Suman et al. (2013) evaluated synthesized silver
nanoparticles using the aqueous aerial extract of Ammannia baccifera as
reducing agent showed toxic effects against fourth instar larvae of An.
subpictus and Cx. quinquefasciatus.
Sundaravadivelan et al. (2013) determined that the synthesized silver
nanoparticles using Pedilanthus tithymaloides aqueous leaf extract showed
anti-developmental activity and acute toxicity towards larval instar of Ae.
aegypti. Synthesized silver nanoparticles using aqueous fruit extract of
Drypetes roxburghii were assessed against larvae of An. stephensi and
Cx. quinquefasciatus (Haldar et al., 2013). Subarani et al. (2013) investigated
the silver nanoparticles performed with the aqueous leaf extract of Vinca
rosea were toxic to fourth instar larvae of An. stephensi and Cx.
quinquefasciatus. Marimuthu et al. (2013) investigated the larvicidal activities
of synthesized cobalt nanoparticles (Co NP) using bio control agent of
Bacillus thuringiensis against malaria vector An. subpictus and dengue vector
Ae. aegypti.
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The larvicidal and growth inhibitory activities of one formulation
comprising eight plant volatile oils of Acorus calamus, Cinnamomum veerum,
Cymbopogon nardus, Myrtus caryophyllus, Eucalyptus globulus, Mentha
piperita, Citrus limon, Citrus sinensis, with natural camphor were studied
against An. stephensi, Ae.aegypti and Cx. quinquefasciatus (Manimaran et al.,
2013). Govindarajan et al. (2013c) observed that the larvicidal activity of
essential oil from Coleus aromaticus and its pure isolated constituent thymol
against larvae of Cx. tritaeniorhynchus, Ae. albopictus and An. subpictus.
Essential oils and acetone extracts from Lavandula gibsoni and
Plectranthus mollis were investigated for their mosquito larvicidal activity
against the fourth instar larvae of Ae. aegypti, An. stephensi, and Cx.
quinquefasciatus (Kulkarni et al., 2013). Cheng et al. (2013) investigated the
mosquito larvicidal activities of the wood and leaf essential oils and ethanol
extracts from Cunninghamia konishii against Ae. aegypti and Ae. albopictus.
The toxicity of mosquito larvicidal activity of leaf essential oil and their major
chemical constituents from Ocimum basilicum were evaluated against Cx.
tritaeniorhynchus, Ae. albopictus and An. subpictus (Govindarajan et al.,
2013a). Edriss et al. (2013) showed the extracts prepared from two
asclepiadaceous plants, viz., Solenostemma argel and Calotropis procera, as
natural larvicides against An. arabiensis. Liu et al. (2013) determined that the
larvicidal activity of essential oil derived from roots of T. asiatica against
the larvae of Ae. albopictus.
Silver nanoparticles synthesized using the aqueous leaf extract of
Pithecellobium dulce showed toxicity against fourth instar larvae of Cx.
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quinquefasciatus (Raman et al., 2012). Arjunan et al. (2012) determined that
the synthesized silver nanoparticles using Annona squamosa aqueous leaf
extract was assessed against fourth instar larvae of Ae. aegypti, Cx.
quinquefasciatus, and An. stephensi. Synthesized silver nanoparticles using
Euphorbia hirta leaf extract against larvae and pupae of An. stephensi
(Priyadarshini et al., 2012). Patil et al. (2012a) observed that the synthesized
silver nanoparticles using Plumeria rubra plant latex were toxic against
second and fourth instar larvae of Ae. aegypti and An. stephensi. Silver
nanoparticles produced with Pergularia daemia latex were toxic to Ae.
aegypti and An. stephensi larvae (Patil et al., 2012b).
The leaf extract of Acalypha alnifolia with different solvents hexane,
chloroform, ethyl acetate, acetone and methanol were tested for larvicidal
activity against three important mosquitoes such as malarial vector An.
stephensi, dengue vector Ae. aegypti and Bancroftian filariasis vector Cx.
quinquefasciatus (Kovendan et al., 2012b). The essential oil extracted from
fresh leaves of Hyptis suaveolens, and its main constituents were tested for
larvicidal and repellent activity against the Asian tiger mosquito, Ae.
albopictus (Conti et al., 2012). Liu et al. (2012) observed that the essential oil
derived from roots of Saussurea lappa and the isolated constituents against
the larvae of the mosquito Ae. albopictus. The water extract of Moringa
oleifera seeds were tested the larvicidal, pupicidal and repellent activity
against the Cx. quinquefasciatus (Ashfaq and Ashfaq, 2012).
Kimbaris et al. (2012) investigated the larvicidal effect exhibited by
essential oils of Dianthus caryophyllus, Lepidium sativum, Pimpinella
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anisum, and Illicium verum against late third to early fourth instar mosquito
larvae of Cx. pipiens. Marimuthu et al. (2011) have reported that the efficacy
of synthesized silver nanoparticles with the aqueous leaf extract of Mimosa
pudica against fourth instar larvae of An. subpictus and Cx. quinquefasciatus.
Rajakumar and Rahuman, (2011) evaluated the toxicity of silver nanoparticles
synthesized using the aqueous leaf extract from Eclipta prostrata towards
fourth instar larvae of Cx. quinquefasciatus and An. subpictus. Silver
nanoparticles fabricated using Rhizophora mucronata leaf extract have been
tested on fourth instar larvae of Ae. aegypti and Cx. quinquefasciatus
(Gnanadesigan et al., 2011).
Salunkhe et al. (2011) observed that the mosquito larvicidal activities
of mycosynthesized silver nanoparticles against Ae. aegypti and An. stephensi
responsible for diseases of public health importance. The maximum efficacy
was observed in crude methanol, aqueous and synthesized silver nanoparticles
using the aqueous leaf extract of Nelumbo nucifera were toxic to fourth instar
larvae of An. subpictus and Cx. quinquefasciatus (Santhoshkumar et al.,
2011). Synthesized gold nanoparticles from the aqueous leaf extract of
Hibiscus rosasinensis showed potent activity against the larvae of Ae.
albopictus (Sareen et al., 2011). Jayaseelan et al. (2011) investigated the
synthesized silver nanoparticles with the leaf aqueous extract of Tinospora
cordifolia were toxic against fourth instar larvae of An. subpictus and Cx.
quinquefasciatus.
The larvicidal and repellent activities of ethyl acetate and methanol
extracts of Acacia concinna, Cuminum cyminum, Lantana camara, Nelumbo
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nucifera, Phyllanthus amarus, Piper nigrum and Trachyspermum ammi
against An. stephensi and Cx. quinquefasciatus (Kamaraj et al., 2011).
Prathibha et al. (2011) observed that the larvicidal potential of Euodia ridleyi
leaf extract was assessed against filariasis vector Cx. quinquefasciatus.
Kovendan et al. (2011) have reported the leaf extract of methanol
Jatropha curcas against Cx. quinquefasciatus. The toxicity of mosquito
larvicidal activity of leaf essential oil and their major chemical constituents
from Mentha spicata were toxic against Cx. quinquefasciatus, Ae. aegypti and
An. stephensi (Govindarajan et al., 2011a). Khanna et al. (2011) investigated
that the larvicidal properties of crude extract of Gymnema sylvestre against
the larvae An. subpictus, Cx. quinquefasciatus.
The larvicidal and repellent properties of essential oils is from various
parts off our plant species Cymbopogon citratus, Cinnamomum zeylanicum,
Rosmarinus officinalis and Zingiber officinale against Cx. tritaeniorhynchus
and An. subpictus (Govindarajan, 2011b). Raghavendra et al. (2011)
examined the larvicidal efficacy of Eugenia jambolana leaf extract against
dengue vector Ae. aegypti. Patil et al. (2011) evaluated the larvicidal activity
of extracts of medicinal plants Plumbago zeylanica and Cestrum nocturnum
against fourth instar larvae of Ae. aegypti.
Aivazi and Vijayan, (2010) evaluated the larvicidal activity of Ruta
graveolens extract was assessed against malaria vector An. stephensi.
Mathivanan et al. (2010) investigated the larvicidal efficacy of Ervatamia
coronaria leaves extract against Cx. quinquefasciatus, Ae.aegypti and An.
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stephensi. The larvicidal efficacy of the crude leaf extract of Ficus
benghalensis with three different solvents like methanol, benzene, and
acetone were tested against the early second, third and fourth instar larvae of
Cx. quinquefasciatus, Ae. aegypti and An. stephensi (Govindarajan, 2010c).
The larvicidal activity of essential oils extracted from Achillea
millefolium, Lavandula angustifolia, Helichrysum italicum, Foeniculum
vulgare, Myrtus communis, and Rosmarinus officinalis were carried out
against the larvae of Ae. albopictus (Conti et al., 2010). Kumar et al. (2010)
investigated that the larvicidal potential of ethanolic extracts of dried fruits of
Piper nigrum against early fourth instar larvae of Ae. aegypti. Larvicidal and
repellent activities of S. acuta leaf extract were assessed against Cx.
quinquefasciatus, Ae. aegypti and An. stephensi (Govindarajan, 2010a).
Rawani et al, (2010) determined that the larvicidal activities of crude and
solvent extracts of Solanum nigrum leaves were assessed against Cx.
quinquefasciatus. Govindarajan (2010b) investigated that the mosquito
larvicidal activity of leaf essential oil and their chemical constituents from
Clausena anisata against Cx. quinquefasciatus, Ae. aegypti and An. stephensi.
The ethanolic leaf extract of Cassia obtusifolia and the larvicidal
efficacy of the crude leaf extract of Ficus benghalensis with three different
solvents like methanol, benzene, and acetone was tested against larvae of Cx.
quinquefasciatus, Ae. aegypti, and An. stephensi (Rajkumar and Jebanesan,
2009). Govindarajan, (2009) reported that the leaf methanol, benzene, and
acetone extracts of Cassia fistula were studied for the larvicidal, ovicidal, and
repellent activities against Ae. aegypti. Mathew et al. (2009) evaluated the
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leaf chloroform extracts of Nyctanthes arbortristis were toxic against Ae.
aegypti and An. stephensi. Larvicidal potential of ethyl acetate extract from
the leaves of Ocimum canum and O. sanctum showed good larvicidal activity
against the larvae of An. subpictus and Cx. tritaeniorhynchus (Bagavan et al.,
2009a). Kamaraj et al. (2009) investigated that the leaf petroleum ether and
flower methanol extracts of Cassia auriculata against the larvae of An.
subpictus and Cx. tritaeniorhynchus.
Samidurai et al. (2009) observed that the leaf extracts of Pemphis
acidula were evaluated for larvicidal, ovicidal, and repellent activities against
Cx. quinquefasciatus and Ae. aegypti. Elimam et al. (2009) determined that
the aqueous leaves extract of Calotropis procera were toxic against second,
third and fourth larvae of Anopheles arabiensis Cx. quinquefasciatus. The
larvicidal and adulticidal activities of ethanolic and water mixture of plant
extracts Eucalyptus globulus, Cymbopogan citratus, Artemisia annua, Justicia
gendarussa, Myristica fragrans, Annona squamosa, and Centella asiatica
were against An. stephensi (Senthilkumar et al., 2009). Karunamoorthi et al.
(2008) have reported that the petroleum ether extracts of the leaves of Vitex
negundo were evaluated for larvicidal activity against larvae of
Cx. tritaeniorhynchus.
The bioactive compounds from medicinal plants such as Ocimum
canum, Ocimum sanctum, and Rhinacanthus nasutus were extracted with
acetone, chloroform, ethyl acetate, hexane and methanol for evaluating the
larvicidal activity against Ae.aegypti and Cx. quinquefasciatus (Kamaraj et
al., 2008). Govindarajan et al. (2008a) showed the methanolic leaf extract of
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Cassia fistula was tested for larvicidal and ovicidal activity against
Cx. quinquefasciatus and An. stephensi. The acetone, chloroform, ethyl
acetate, hexane, and methanol leaf extracts of Acalypha indica, Achyranthes
aspera, Leucas aspera, Morinda tinctoria, and Ocimum sanctum were studied
against the early fourth instar larvae of Ae. aegypti and Cx. quinquefasciatus
(Bagavan et al., 2008). Nathan et al. (2008) evaluated that the fourth instar
larvae of An. stephensi are highly sensitive to the ethyl acetate extract of the
leaves of Dysoxylum malabaricum. The ethyl acetate extract of leaves of
Ocimum sanctum produced significant mortality against Ae. aegypti and Cx.
quinquefasciatus (Anees, 2008).
Matasyoh et al. (2008) investigated the ethyl acetate leaves extract of
Aloe turkanensis were toxic against larvae of Anopheles gambiae. The leaf
extract of Acalypha indica with different solvents, viz., benzene, chloroform,
ethyl acetate, and methanol, was tested for larvicidal, ovicidal activity, and
oviposition attractancy against An.stephensi (Govindarajan et al., 2008a).
Mullai et al. (2008) determined that the leaf extract of Citrullus vulgaris with
different solvents, viz., benzene, petroleum ether, ethyl acetate, and methanol,
was tested for larvicidal, ovicidal, repellent, and insect growth regulatory
activities against An. stephensi. Mullai et al. (2008) have reported that the leaf
extract of Citrullus vulgaris with different solvents, viz., benzene, petroleum
ether, ethyl acetate, and methanol, were tested for larvicidal, ovicidal,
repellent, and insect growth regulatory activities against An. stephensi.
The bioactive compounds from Solanum villosum berry using distilled
water and five different organic solvents and detected the higher mortality
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against the third instar larvae of Ae. aegypti (Chowdhury et al., 2008).
Rahuman et al. (2008a) reported that petroleum ether extracts of Jatropha
curcas, Pedilanthus tithymaloides, Phyllanthus amarus, Euphorbia hirta, and
Euphorbia tirucalli were against Ae. aegypti against Cx. quinquefasciatus.
Larvicidal activity of crude hexane, ethyl acetate, petroleum ether, acetone,
and methanol extracts of the leaf of five species of cucurbitaceous plants,
Citrullus colocynthis, Coccinia indica, Cucumis sativus, Momordica
charantia, and Trichosanthes anguina, were tested against early fourth instar
larvae of Ae. aegypti and Cx. quinquefasciatus (Rahuman et al., 2008b).
Larvicidal and repellent activity of essential oil from Zingiber officinalis was
assessed against the filarial vector Cx. quinquefasciatus (Pushpanathan et al.,
2008).
Govindarajan et al. (2007) evaluated the larvicidal activity of
extracellular secondary metabolites of the actinomycetes isolates were toxic
against dengue vector Ae. aegypti. The petroleum ether extracts of Citrullus
colocynthis and the methanol extract Momordica charantia were highly
effective against the larvae of Ae. aegypti and against Cx. quinquefasciatus
(Kashiwagi et al., 2007). Mullai and Jebanesan, (2007) studied the ethyl
acetate, petroleum ether, and methanol leaf extracts of Citrullus colocynthis
and Cucurbita maxima against Cx. quinquefasciatus larvae. Kannathasan et
al. (2007) reported that the methanol leaf extracts of Vitex negundo, Vitex
trifolia, Vitex peduncularis and Vitex altissima were used for larvicidal assay
against the early fourth instar larvae of Cx. quinquefasciatus. Khanna and
Kannabiran, (2007) investigated three different plants has demonstrated the
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highest lethal effect of aqueous root extract of Hemidesmus indicus on the
larvae of Cx. quinquefasciatus.
Larvicidal properties of hexane crude extract of Momordica charantia
against larvae of An. stephensi, Cx. quinquefasciatus and Ae. aegypti (Singh
et al., 2006). Amer and Mehlhorn, (2006) demonstrated the potent larvicidal
effects of essential oils from 13 out of 41 plants tested against larvae of Ae.
aegypti, An. stephensi and Cx. quinquefasciatus. Singhi et al. (2006a) have
reported that the larvicidal efficacy of Calotropis procera were toxic against
Ae. aegypti, An. stephensi, and Cx. quinquefasciatus. Dua et al. (2006)
evaluated the aqueous extract from the roots of Hibiscus abelmoschus against
the larvae of An. culicifacies, An. stephensi, and Cx. quinquefasciatus.
Govindarajan et al. (2005) investigated the culture filtrates of five
different soil fungi viz., Aspergillus flavus, Aspergillus parasiticus,
Penicillium falicum, Fusarium vasinfectum and Trichoderma viride were
tested for the larvicidal activity against third instar larvae of mosquito vector
Cx. quinquefasciatus. The petroleum ether extract of Solanum xanthocarpum
fruits was observed as most toxic against Cx. quinquefasciatus (Mohan et al.,
2005). Sharma et al. (2005) reported that the acetone extract of Nerium
indicum and Thuja orientelis have been studied against third instar larvae of
An. stephensi and Cx. quinquefasciatus.
Larvicidal activity of the acetone extracts of Murraya koenigii,
Coriandrum sativum, Ferula asafoetida, and Toenum foenum graceum were
tested out using different concentrations of each plant against larvae of Ae.
aegypti (Harve and Kamath, 2004). Singh et al. (2005) reported that the
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larvicidal properties of leaf extract of Calotropis procera against mosquito
larvae of An. stephensi, Cx. quinquefasciatus, and Ae. aegypti. Chansang et al.
(2005) screened nine different plants and reported that the aqueous extract of
the fruits from Piper retrofractum showed the highest larvicidal effect on the
third and fourth instar larvae of Ae. aegypti and Cx. quinquefasciatus.
Larvicidal efficacies of methanol extracts of Momordica charantia,
Trichosanthes anguina, Luffa acutangula, Benincasa cerifera, and Citrullus
vulgaris tested against the late third larval age group of Cx. quinquefasciatus
(Prabakar and Jebanesan, 2004). Sivagnaname and Kalyanasundaram, (2004)
investigated the methanolic extracts of the leaves of Atlantia monophylla
(Rutaceae) for mosquitocidal activity against immature stages of three
mosquito species, Cx. quinquefasciatus, An. stephensi and Ae. aegypti in the
laboratory Phukan and Kalita, (2005) attempted to extract the bioactive
compound with antimosquito activity from the leaf powder of Litsea
salicifolia using polar (water) and various nonpolar solvents (hexane, toluene,
chloroform, acetone, and methanol) among which the aqueous extract,
exhibited more effective larvicidal activity against the fourth instar larvae of
Ae. aegypti.
2.2 Ovicidal activity
Mosquito ovicidal and adulticidal properties of hexane, benzene,
chloroform, ethyl acetate, and methanol extracts of leaf and seed of Albizia
lebbeck were investigated against Cx. quinquefasciatus, Ae. aegypti, and An.
stephensi (Govindarajan and Rajeswary, 2015). Reegan et al. (2015) reported
that the ovicidal and oviposition deterrent activities of Limonia acidissima
hexane leaf extract against Ae. aegypti and Cx. quinquefasciatus.
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Tennyson et al. (2015) studied the ovicidal efficacy of ethyl acetate
and methanol extract of Ageratum houstonianum leaf extract were toxic
against An. stephensi, Ae. aegypti and Cx. quinquefasciatus. Ovicidal and
oviposition deterrence activity of synthesized silver nanoparticles using
Sargassum muticum aqueous extract were evaluated against mosquito vectors
of Ae. aegypti, An. stephensi and Cx. quinquefasciatus (Madhiyazhagan et
al., 2015).
Benelli et al. (2015) investigated the ovicidal, larvicidal, pupicidal and
oviposition deterrence activities of neem cake A. indica were assessed against
Aedes, Anopheles and Culex mosquito vectors.The essential oil of Cananga
odorata flowers was investigated for ovicidal, oviposition-deterrent,
insecticidal, and repellent activities against Ae. aegypti, An. dirus, and Cx.
quinquefasciatus (Soonwera, 2015). Maheswaran and Ignacimuthu, (2015)
reported that the ovicidal, oviposition-deterrents and larvicidal activities of A.
indica and Pongamia glabra were tested against An. stephensi and Cx.
quinquefasciatus.
To determine the ovicidal and repellent activities of benzene and
methanol extract of Polygala arvensis against Ae. aegypti, An. stephensi and
Cx. quinquefasciatus (Deepa et al., 2014). Govindarajan and Sivakumar,
(2014) evaluated the ovicidal, larvicidal and adulticidal properties of crude
hexane, ethyl acetate, benzene, chloroform and methanol extracts of root of
Asparagus racemosus were tested against Cx. quinquefasciatus, Ae. aegypti
and An. stephensi. The ovicidal and oviposition deterrent activities of
petroleum ether and ethyl acetate extracts of the leaves of Eugenia jambolana,
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Solidago canadensis, Euodia ridleyi and Spilanthes mauritiana were
investigated against An. stephensi, Ae.aegypti, and Cx. quinquefasciatus
(Prathibha et al., 2014).
Maheswaran and Ignacimuthu, (2014) determined that the ovicidal,
larvicidal, repellent oviposition deterrent and adulticidal activities of
Polygonum hydropiper were assessed against dengue vector Ae. albopictus.
Mosquito ovicidal, larvicidal, and adulticidal potential of the crude hexane,
benzene, chloroform, ethyl acetate, and methanol solvent extracts from
Erythrina indica were evaluated against An. stephensi, Ae. aegypti and Cx
quinquefasciatus (Govindarajan and Sivakumar, 2014a). Baranitharan and
Dhanasekaran (2014) observed that the larvicidal, ovicidal and repellent
activities of diethyl ether, hexane, benzene and acetone extract of Coleus
aromaticus was tested against Ae. aegypti. Cheah et al. (2013) reported that
the larvicidal, oviposition, and ovicidal activities of crude extract of Artemisia
annua was assessed against Ae. aegypti, An. sinensis, and Cx.
quinquefasciatus.
The ovicidal potential of the crude hexane, benzene, chloroform, ethyl
acetate, and methanol solvent extracts from Ageratina adenophora was
assessed against filariasis vector Cx. quinquefasciatus (Rajeswary et al.,
2014). Ovicidal, repellent, adulticidal activities of crude hexane, benzene,
ethyl acetate, acetone and methanol leaf extracts of Acalypha alnifolia were
evaluated toxicity against Ae. aegypti, An. stephensi and Cx. quinquefasciatus
(Kovendan et al., 2013). The ovicidal potential of the crude chloroform, ethyl
acetate and methanol solvent extracts from the medicinal, Pithecellobium
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dulce against filariasis vector mosquito, Cx. quinquefasciatus (Govindarajan
et al., 2013b).
The larvicidal, ovicidal, and repellent activities of marine
sponge Cliona celata extracts was investigated against Cx. quinquefasciatus
and Ae. aegypti (Reegan et al., 2013). Karthika Devi et al. (2013) observed
that the larvicidal, pupicidal, ovicidal, and ovipositional deterrent activity of
methanol leaf extract of Spathodea campanulata against Ae. aegypti.
Krishnappa et al. (2013) determined that the larvicidal and ovicidal activities
of acetone, benzene, ethyl acetate and methanol leaf extract of Cissus
quadrangularis and Combretum ovalifolium against An. stephensi. The
adulticidal, repellent, and ovicidal potential of the crude hexane, ethyl acetate,
benzene, aqueous, and methanol solvent extracts from the medicinal plants
Andrographis paniculata, Cassia occidentalis and Euphorbia hirta were
investigated against An. stephensi (Panneerselvam and Murugan, 2013).
The ovicidal properties of leaf and seed extracts of Delonix elata
against malaria An.stephensi and dengue Ae.aegypti vector mosquitoes
(Govindarajan et al., 2012). Maheswaran and Ignacimuthu, (2012) revealed
the larvicidal, ovicidal and oviposition deterrent activities of A. indica and
Pongamia glabra extracts were tested against Ae. aegypti and Ae. albopictus.
Samidurai, (2012) evaluated that the larvicidal and ovicidal potential of the
crude methanol, benzene and acetone solvent extracts from the medicinal
plant Pemphis acidula against the medically important mosquito vectors Cx.
tritaeniorhynchus and An. subpictus. Gokulakrishnan et al. (2012) studied the
larvicidal and ovicidal efficacy of acetone, benzene, chloroform, hexane and
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methanol extracts of Ariitolochia indica against An. stephensi. Barik et al.
(2012) investigated the oviposition behavior of three mosquito species in the
presence of different types of nanosilica and complete ovideterrence activity
of hydrophobic nanosilica was observed against Ae. aegypti, An. stephensi
and Cx. quinquefasciatus.
Ovicidal and repellent activities of methanol leaf extract of Ervatamia
coronaria and Caesalpinia pulcherrima were evaluated against Cx.
quinquefasciatus, Ae. aegypti, and An. stephensi (Govindarajan et al., 2011).
Karthik et al. (2011) investigated the larvicidal, repellent, and ovicidal
activities of marine actinobacterial Saccharomonospora, Streptomyces
oseiscleroticus and Streptomyces gedanensis crude extracts were assessed
against Cx. tritaeniorhynchus and Culex gelidus. The oviposition deterrence
and ovicidal potential of five different essential oils, peppermint oil (Mentha
piperita), basil oil (Ocimum basilicum), rosemary oil (Rosemarinus
officinalis), citronella oil (Cymbopogon nardus), and celery seed oil (Apium
graveolens), were assessed against dengue vector Ae. aegypti (Warikoo et al.,
2011).
The leaf ethyl acetate, acetone, and methanol extracts of Andrographis
lineata (Acanthaceae) and Andrographis paniculata showed ovicidal and
oviposition deterrent activities against Cx. tritaeniorhynchus (Elango et al.,
2010). The antimosquito activity of the kernel extract of Sapindus
emarginatus exerted ovicidal, larvicidal, and pupicidal activity against An.
stephensi and Cx. quinquefasciatus (Koodalingam et al., 2009). Elango et al.
(2009) showed that indigenous plant extracts such as Aegle marmelos,
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Andrographis lineate, Andrographis paniculata, Cocculus hirsutus, Eclipta
prostrata, and Tagetes erecta have oviposition-deterrent, ovicidal, and
repellent activity against An. subpictus.
The larvicidal, pupicidal, adulticidal, and ovicidal properties of
Andrographis paniculata ethanolic extract were evaluated against malaria
vector An. stephensi (Kuppusamy and Murugan 2009). Samidurai et al.
(2009) determined that the leaf extracts of Pemphis acidula were evaluated
for larvicidal, ovicidal, and repellent activities against Cx. quinquefasciatus
and Ae. aegypti. Govindarajan (2009) investigated that the methanol,
benzene, and acetone extracts of Cassia fistula were assessed for the
larvicidal, ovicidal, and repellent activities against Ae. aegypti.
The leaf extract of Acalypha indica with different solvents benzene,
chloroform, ethyl acetate, and methanol has been tested for larvicidal,
ovicidal activity, and oviposition attractancy against An. stephensi
(Govindarajan et al., 2008b). The isolated flavonoid compounds of poncirin,
rhoifolin, naringin and marmesin from Poncirus trifoliate showed strongest
ovicidal, oviposition deterrent and repellent activity against Ae. aegypti
(Rajkumar and Jebanesan 2008). Mullai, et al. (2008) have reported that the
leaf extract of Citrullus vulgaris with different solvents, viz., benzene,
petroleum ether, ethyl acetate, and methanol were tested for larvicidal,
ovicidal, repellent, and insect growth regulatory activities against An.
stephensi.
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Luz et al. (2007) reported that the ovicidal activity of hyphomycetes
fungi species were Paecilomyces carneus, Paecilomyces marquandii, Isaria
fumosorosea, Metarhizium anisopliae, Penicillium sp., Paecilomyces
lilacinus, Beauveria bassiana, and Evlachovaea kintrischica against Ae.
aegypti. Govindarajan et al. (2006) evaluated the secondary metabolites of
Streptomyces spp. were examined for oviposition attractancy of the filariasis
vector Cx. quinquefasciatus. The toxicity of dichloromethane, petroleum
ether, and methanol extracts from Vitex negundo seed and leaf to the second
and fourth instar larvae showed oviposition deterrent effects on Plutella
xylostella against Ae.aegypti (Yuan et al., 2006). Mullai and Jebanesan (2006)
reported that the complete ovicidal activity of ethanol, benzene, petroleum
ether, and ethyl acetate extracts of Citrullus pubescens was toxic against Cx.
quinquefasciatus. Tawatsin et al. (2006) have observed the relatively high
oviposition-deterrent activity was obtained by essential oils of Curcuma
longa, Zingiber officinale, V. trifolia, Melaleuca cajuputi, Manglietia
garrettii, and Houttuynia cordata against Ae.aegypti. Pushpanathan et al.
(2006) revealed the larvicidal, ovicidal and repellent activities of essential oil
from Cymbopogan citratus against Cx. quinquefasciatus.
Essential oil of Cinnamomum zeylanicum, Zingiber officinale, and
Rosmarinus officinalis showed oviposition deterrent, ovicidal, and repellent
activities against An. stephensi, Ae. aegypti, and Cx. quinquefasciatus
(Prajapati et al., 2005). The toxicity of mosquito ovicidal activity of essential
oil from Ocimum basilicum was evaluated against Cx. quinquefasciatus, Ae.
aegypti and An.stephensi (Prajapati et al., 2005). Prajapati et al. (2005) have
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revealed the oviposition deterrent, ovicidal and repellent activities of the
essential oils of Cinnamomum zeylanicum, Zingiber officinale and
Rosemarinus officinalis against An. stephensi, Ae. aegypti and Cx.
quinquefasciatus.
Rajkumar and Jebanesan (2004) studied the ovicidal activity of
Moschosma polystachyum leaf extract was tested against Cx.
quinquefasciatus. The methanol extracts of Pelargonium citrosa leaf were
tested for their biological, larvicidal, pupicidal, adulticidal, antiovipositional
activity, repellency, and biting deterrence against An. stephensi (Jeyabalan et
al., 2003). Mehra and Hiradhar, (2002) evaluated the crude acetone extract of
Cuscuta hyalina was an effective oviposition deterrent against Cx.
quinquefasciatus. Su and Mulla (1998) investigated the ovicidal activity of
the Neem product azadirachtin against the mosquitoes Culex tarsalis and Cx.
quinquefasciatus. Ovicidal activity of the seed extract of Atriplex canescens
was evaluated against Cx. quinquefasciatus (Ouda et al. 1998).
2.3 Adulticidal activity
Mukandiwa et al. (2016) reported that the repellency and adulticidal
activities of Clausena anisata leaf extracts were assessed against the yellow
fever mosquito Ae. aegypti. Synthesized gold nanoparticles from Couroupita
guianensis flower extract were investigated against the larvae, pupae, and
adults of malaria vector An. stephensi (Subramaniam et al., 2016).
Govindarajan and Rajeswary (2015) examined the adulticidal activity of
hexane, benzene, chloroform, ethyl acetate, and methanol extracts of leaf and
seed of Albizia lebbeck were assayed for their toxicity against Cx.
quinquefasciatus, Ae. aegypti and An. Stephensi. Suresh et al. (2015)
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evaluated the adulticidal activity of synthesized silver nanoparticles using
Phyllanthus niruri were toxic against Ae. aegypti. Adulticidal activity of
synthesized silver nanoparticles from Mimusops elengi was highly effective
against An. stephensi and Ae. albopictus (Subramaniam et al., 2015). Roni et
al. (2015) reported that fabricated silver nanoparticles using Hypnea
musciformis was evaluated against female adult of Ae. aegypti.
The maximum efficacy was observed the synthesized silver
nanoparticles using Feronia elephantum leaf extract against adults of An.
stephensi, Ae. aegypti and Cx. quinquefasciatus (Veerakumar and
Govindarajan, 2014). The adulticidal activity of silver nanoparticles
fabricated using Heliotropium indicum leaf extract has been evaluated against
adults of An. stephensi, Ae. aegypti and Cx. quinquefasciatus (Veerakumar et
al., 2014b). Soni and Prakash (2014b) determined that the synthesized silver
nanoparticles using the neem leaf extract were assessed against adult of Cx.
quinquefasciatus. Mavundza et al. (2014) investigated that the adulticidal
activity of Clausena anisata was toxic against Anopheles arabiensis.
Ramkumar et al. (2014) determined that the adulticidal and smoke
toxicity activity of acetone, ethyl acetate, benzene, chloroform and methanol
leaf extracts of Cipadessa baccifera against Cx. quinquefasciatus, Ae. aegypti
and An. stephensi.
Kovendan et al. (2013) studied the adulticidal activity of methanol
extract of Acalypha alinifolia leaf extract against three mosquito species, Ae.
aegypti, An. stephensi and Cx. quinquefasciatus. Panneerselvam and
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Murugan, (2013) determined that the Adulticidal, repellent and ovicidal
activity of the crude hexane, ethyl acetate, benzene, aqueous and methanol
solvent extracts from the medicinal plants Andrographis paniculata, Cassia
occidentalis and Euphorbia hirta leaf extract against the malarial vector An.
stephensi. Panneerselvam et al. (2012) evaluated the larvicidal, pupicidal,
repellent, and adulticidal activities of methanol crude extract of Artemisia
nilagirica were assayed for their toxicity against two important vector
mosquitoes, viz., An. stephensi and Ae. aegypti.
Murugan et al. (2012) studied the effects of orange peel ethanol extract
of Citrus sinensis on larvicidal, pupicidal, repellent and adulticidal activity
against An. stephensi, Ae. aegypti and Cx. quinquefasciatus. The adulticidal
activity of methanol extracts of indigenous plant Andrographis paniculata
against the adults of Cx. quinquefasciatus and Ae. aegypti (Govindarajan and
Sivakumar, 2012). The adulticidal and repellent activities of crude hexane,
chloroform, benzene, acetone and methanol extracts of leaf of Cassia tora
were assayed for their toxicity against three important vector mosquitoes, viz.,
Cx. quinquefasciatus, Ae. aegypti and An. stephensi (Amerasan et al., 2012).
Lalrotluanga et al. (2012) showed the larvicidal, adulticidal, and repellent
activities of acetone root bark extract of Hiptage benghalensis were tested
against the larvae and adults of Anopheles barbirostris,Cx. quinquefasciatus
and Ae. Albopictus.
The adulticidal activity and adult emergence inhibition of leaf hexane,
chloroform, ethyl acetate, acetone, and methanol extracts of Aegle marmelos
Andrographis lineate, Andrographis paniculata, Cocculus hirsutus, Eclipta
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prostrata, and Tagetes erecta, tested against malarial vector An.subpictus
(Elango et al., 2011). The essential oil of Lantana camara was tested against
adulticidal activity of Ae. aegypti, Cx. quinquefasciatus, Anopheles
culicifacies, Anopheles fluviatilis, and An. stephensi mosquitoes (Dua et al.,
2010). Kamaraj et al, (2010) evaluated the adulticidal, repellent and larvicidal
activity of crude hexane, ethyl acetate, and methanol extracts of eight plants,
viz., Aristolochia indica, Cassia angustifolia, Diospyros melanoxylon,
Dolichos biflorus, Gymnema sylvestre, Justicia procumbens, Mimosa pudica,
and Zingiber zerumbet, were tested against adult and early fourth instar larvae
of Cx. gelidus and Cx. quinquefasciatus.
Adulticidal efficacy of Chrysosporium tropicum was performed against
a mixed population of adult mosquitoes Cx. quinquefasciatus, An. stephensi,
Ae. aegypti (Verma and Prakash, 2010). Dua et al. (2010) observed that the
adulticidal activities of essential oil from Lantana camara leaves were toxic
against Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluvialitis and
An. stephensi. The adulticidal and larvicidal effect of leaf hexane, chloroform,
ethyl acetate, acetone and methanol extracts of Annona squamosa, Centella
asiatica, Gloriosa superba Mukia maderaspatensis, Pergularia daemia, and
Phyllanthus emblica were exposed against An. subpictus and Cx.
tritaeniorhynchus (Bagavan et al., 2009). The larvicidal and adulticidal
activities of ethanolic and water mixture (50:50) of plant extracts Eucalyptus
globulus, Cymbopogan citratus, Artemisia annua, Justicia gendarussa,
Myristica fragrans, Annona squamosa, and Centella asiatica were
investigated against An. stephensi (Senthilkumar et al., 2009).
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Kamalakannan et al. (2008) proved that the adulticidal activity of
entomopathogenic fungus Metarhizium anisopliae was tested against malarial
vector An. stephensi. Adulticidal activity of the essential oil isolated from
Mentha longifolia was screened fumigant toxicity against the house mosquito
Cx. pipiens (Oz et al., 2007). Nathan et al. (2005) investigated that the pure
limonoids of neem seed, testing for biological, larvicidal, pupicidal,
adulticidal, and antiovipositional activity against An. stephensi. The
adulticidal activity of methanol extracts from three Malaysian plants namely
Acorus calamus, Litsea elliptica, and Piper aduncum against adult of Ae.
aegypti were studied (Hidayatulfathi et al., 2004). The methanol extracts of
Pelargonium citrosa leaf were tested for their biological, adulticidal,
antiovipositional activity; repellency and biting deterrency against An.
stephensi (Jeyabalan et al., 2003).
Zaridah et al. (2001) have reported that the aqueous extract from the
dried leaves of Andrographis paniculata showed the strongest activity against
adult worms of Brugia malayi. Murugan and Jeyabalan, (1999) investigated
that Leucas aspera, Ocimum santum, Azadirachta indica, Allium sativum, and
Curcuma longa had a strong larvicidal, antiemergence, adult repellency, and
antireproductive activity against An. stephensi.
2.4 Non-target effects against aquatic organisms
The biosynthesized Ag NP were found safer to non-target organisms
Diplonychu indicus, Anisops bouvieri, and Gambusia affinis, and higher
toxicity against An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus
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(Govindarajan and Benelli, 2016a). Govindarajan et al. (2016b) investigated
the Z. nimmonii essential oil was safer towards two non-target aquatic
organisms, D. indicus and G. affinis, and against larvae of An. stephensi, Ae.
aegypti and Cx. quinquefasciatus.
The predation efficiency of a Carassius auratus was boosted by
biosynthesized silver nano particles using neem cake extract of A. indica, and
against larvae and pupae of Ae. aegypti (Chandramohan et al., 2016). The
silver nanoparticles synthesized using B. tinctoria leaf extract showed reduced
toxicity against the mosquito natural enemies Mesocyclops thermocyclopoides
and Toxorhynchites splendens (Mahesh Kumar et al., 2016). Biosynthesized
Ag NP using C. spinarum was found safer to non-target organisms D. indicus,
A. bouvieri and G. affinis, and toxicity against A. subpictus, A. albopictus, and
C. tritaeniorhynchus (Govindarajan et al., 2016a). Subramaniam et al. (2015)
observed the M. elengi-synthesized silver nanoparticles did not negatively
impact predation rates of the mosquitofish G. affinis against An. stephensi and
Ae. albopictus. Murugan et al. (2015d) showed evidence that P. reticulata
predation against Cx. quinquefasciatus larvae is not impacted by sub lethal
doses of T. asiatica synthesized Ag NP.
Biosynthesized silver nanoparticles using the 2, 7.bis [2-
[diethylamino]-ethoxy] fluorence isolate from the Melia azedarach leaves did
not show acute toxicity against Mesocyclops pehpeiensis copepods
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(Ramanibai and Velayutham, 2015). Chobu et al. (2015) reported that the
G. affinis is more efficient as a predator of An. gambiae third instar larvae
than the Cyprinidae goldfish Carassius auratus. A single treatment with 1
ppm of Aristolochia indica synthesized silver nanoparticles enhanced the
predation of D. indicus water bugs against An. stephensi larvae (Murugan et
al., 2015f). Goldfish (C. auratus) predation efficiency was higher if Ae.
aegypti larvae were exposed to 1 ppm of Bruguiera cylindrical fabricated Ag
NP (Murugan et al., 2015g).
The exposure to sub-lethal doses of Datura metel fabricated Ag NP
magnified predation of dragonfly (Anax immaculifrons) nymphs against An.
stephensi larvae (Murugan et al., 2015i). Haldar et al. (2013) screened did not
detect toxicity of Ag NP produced using dried green fruits of Drypetes
roxburghii against P. reticulata, after 48 h exposure to LC50 of IV instar
larvae of An. stephensi and Cx. quinquefasciatus.
Rawani et al. (2013) showed that mosquitocidal Ag NP synthesized
using S. nigrum berry extracts were not toxic against two mosquito predators,
Toxorhynchites larvae and Diplonychus annulatum, and Chironomus
circumdatus larvae, exposed to lethal concentrations of dry nanoparticles
calculated on An. stephensi and Cx. quinquefasciatus larvae. Subarani et al.
(2013) did not report toxicity effects of V. rosea-synthesized Ag NP against
P. reticulata, after 72 h of exposure to dosages toxic against An. stephensi and
Cx. quinquefasciatus. P. rubra and P. Daemia synthesized Ag NP did not
exhibit any evident toxicity effect against Poecilia reticulata fishes after 48 h
of exposure to LC50 and LC90 values calculated on fourth instar larvae of Ae.
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aegypti and An. stephensi (Patil et al., 2012a). G. affinis has been reported as
a more efficient predator of mosquito young instars than other aquatic
organisms such as Belostomatidae and odonate nymphs (Kweka et al., 2011).
2.5 Plant description
2.5.1 Merremia emerginata (Burm.f) (Family: Convolvulaceae)
Merremia emarginata is a perennial, much branched herb (creeper). It
is found widely distributed all over the India, especially in damp places in
upper gangetic plain, Gujarat, Bihar, West Bengal, Western‐ Ghats, ascending
up to 900m in the hills, Goa, Karnataka in India, Ceylon and Tropical Africa
(Ghosh, 1991). M. emarginata is also known as Ipomoea reniformis. It is
reported to have many important medicinal properties. In the Indigenous
system of Medicine, I. reniformis has been claimed to be useful for cough,
headache, neuralgia, rheumatism, diuretic, inflammation, troubles of nose
amd fever due to enlargement of liver and also in kidney diseases.
2.5.2 Naregamia alata (Weight & Arn) (Family: Meliaceae)
Naregamia alata is belonging to the family Meliaceae, under shrub
with pungent, aromatic roots. Distributed throughtout South India in all
districts upto 900 m. The plant is acid, sweet, cooling, aromatic, alexeteric,
vulnerary, emetic, cholagogue, expectorant, depurative and antipyretic. It is
useful in the treatments of wounds, ulcers, vitiated conditions of pitta and
vata, halitosis, cough, asthma, bronchitis, splenomegaly, scabies, pruritus,
dysentery, dyspepsia, catarrh, anaemia and malarial fevers. It is reported to be
used as antioxidant (Sonu et al., 2012).
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2.5.3 Hedyotis puberula (George Don) (Family: Rubiaceae)
Hedyotis puberula is an annual or biennial herb which is commonly
known as ‘‘Imbural”, is naturalized throughout the sea-coast regions of
Tamilnadu, Orissa and West Bengal in India. In folklore medicine this plant is
widely used in the treatment of various ailments. The decoction of the plant is
widely used as an expectorant and febrifuge. It is also used in treatment of
cancer, asthma, tuberculosis and cold. The leaves and roots are considered as
expectorant and used in bronchitis and consumption. Medicinally the decotion
of the leaves is used as a wash for poisonous bites (Kirtikar and Basu, 1975).
The root bark contains alizarin, rubichloric acid and ruberythric acid as
anthraquinone derivatives (Khare, 2007).
2.5.4 Aglaia elaeagnoidea (JUSS) (Family: Meliaceae)
Aglaia elaeagnoidea is an endemic medicinal tree of peninsular India
commonly called as “Chokkala”. It is distributed in the red soil of Western
Ghats of Tamil Nadu, India. The trees grow up to 10 m tall, leaves are
alternate to sub- opposite, elliptic or oblongelliptic, 6-12 cm long, 2.5-5.5cm
wide. Flowers are roundish yellow. The medicinal use of this plant includes
anti-tumor, anti-inflammatory, anesthetic, analgesic, antioxidant and
antibacterial activity (Jia et al. 2007). The aerial part of the bark is grayish
brown and the leaves are used by the rural folk for curing various ailments
like skin diseases, fever, dysentery, cooling, astringent, abdominal pain and
hemorrhages (King et al. 1982). In traditional Chinese medicine, extracts
from Aglaia are also used for treatment of inflammatory diseases (Kann,
1979).
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2.5.5 Ventilago madrasapatna (Joseph Gaertner) (family: Rhamnaceae)
Ventilago madrasapatna is a woody liana belonging to the family
Rhamnaceae. It is commonly known as Red creeper. This plant is a woody
climber. Traditionally, the root bark of V. madraspatana is used as a
carminative, stomachic, vitiated conditions of kapha, dyspepsia, colic
flatulence, erysipelas, leprosy, scabies, and pruritis. The powdered stem bark
mixed with gingelly oil is applied externally to treat skin diseases and itch
(Chopra et al., 1956). It has been reported to possess antioxidant, anti-
inflammatory, hepatoprotective and antibacterial activities.
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3. MATERIALS AND METHODS
3.1 Collection and identification of plants
The plants, Merremia emarginata, Naregamia alata, Hedyotis
puberula, Aglaia elaeagnoidea, and Ventilago madrasapatna were collected
from Nilgiris, Western Ghats, Tamil Nadu State, India (Plate 1). The identity
was confirmed at the Department of Botany, Annamalai University,
Annamalai Nagar, Tamil Nadu. Voucher specimens were numbered and kept
in our research laboratory for further reference (Plate 2). Table 1 shows the
list of plants species vernacular name and plants part used.
3.2 Preparation of the plant aqueous leaf extracts
Leaves of M. emarginata, N. alata, H. puberula, A. elaeagnoidea, and
V. madrasapatna were dried in the shade and ground to fine powder in an
electric grinder. Aqueous extract was prepared by mixing 50 g of dried leaf
powder with 500 mL of water (boiled and cooled distilled water) with
constant stirring on a magnetic stirrer. The suspension of dried leaf powder in
water was left for 3 h and filtered through Whatman no. 1 filter paper and the
aqueous filtrate were stored in an amber-colored airtight bottle at 10°C
temperature until testing.
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Plate 1. Geographical area of the present study.
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Plate 2. Plants used in the study.
Merremia
emerginata
Naregania alata
Hedyotis puberula
Aglaia elaeagnoidea
Ventilago madrasapatna
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Table 1. Plants used in the present study.
S.
No.
Plant species Common
name
Vernacular
name
Plant
parts used
Collection
area
1 Merremia
emarginata
Kidney Leaf
Morning Glory
Elikkadhu-
keerai
Leaf Nilgiris
2
Naregamia
alata
Goanese
ipecacuanh Nelanaringu
Leaf Nilgiris
3 Hedyotis
puberula
Imbural Chaaya ver Leaf Nilgiris
4 Aglaia
elaeagnoidea
Priyangu Chokkala Leaf Nilgiris
5 Ventilago
madrasapatna
Red Creeper Surulbattaikkoti Leaf Nilgiris
3.3 Synthesis of silver nanoparticles
Ten grams of thoroughly washed and finely cut leaves were added in a
300-mL Erlenmeyer flask along with 100 mL of sterilized double-distilled
water, the mixture was boiled for 5 min before finally decanting it. The
colloidal extract was filtered with Whatman filter paper n. 1, stored at −15 °C
and tested within a week. The filtrate was treated with aqueous 1mM AgNO3
(21.2 mg of AgNO3 powder in 125 mL of Milli-Q water) solution in an
Erlenmeyer flask and incubated at room temperature. Eighty-eight milliliters
of an aqueous solution of 1 mM silver nitrate was reduced using 12 mL of (M.
emarginata, N. alata, H. puberula, A. elaeagnoidea, and V. madrasapatna)
leaf extract at room temperature for 10 min, resulting in a brown–yellow
solution indicating the formation of Ag NPs.
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3.4 Characterization silver nanoparticles
3.4.1 UV–vis spectroscopy (UV-vis)
The UV–Visible absorption involves the spectroscopy of photons in
the UV- Visible region. This means it is light in the visible, near ultraviolet
(UV) range. The absorption in the visible ranges directly affects the color of
the chemicals involved. In this region of the electromagnetic spectrum,
molecules undergo electronic transitions. This technique is complementary to
fluorescence spectroscopy. UV-visible spectra of these aliquots were
monitored as a function of time of reaction on a Shimadzu 160v
spectrophotometer in the 300–800-nm range operated at a resolution of 1 nm.
3.4.2 Fourier Transforms Infrared Spectroscopy (FTIR)
The surface groups of the nanoparticles were qualitatively confirmed
using FTIR spectroscopy. The aliquot of this filtrate containing silver
nanoparticles was used for Fourier transform infrared (FTIR) analysis. FTIR
spectra (Thermo Scientific Nicolet 380 FT-IR Spectrometer) of the samples
were measured using a Perkin Elmer spectrum one instrument in the diffuse
reflectance mode at a resolution of 4 cm−1
in KBr pellets.
3.4.3 Scanning electron microscope with energy dispersive X-ray
spectroscopy (SEM - EDX)
Scanning electron microscopy (SEM) is a technique whereby a beam
of energetically well-defined and highly focused electrons are scanned across
a material (sample). The microscope uses a lanthanum hexaboride (La B6)
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source and is pumped using turbo and ion pumps to maintain the highest
possible vacuum. For electron microscopic studies, 25 μL of sample was
sputter-coated on a copper stub, and the images of nanoparticles were studied
using scanning electron microscopy (SEM; JEOL, Model JFC-1600, (Hitachi
S3000 H SEM). Energy dispersive X-ray spectroscopy (EDX) is an analytical
technique used for the chemical analysis or chemical characterization of a
sample. As a type of spectroscopy, it relies on the investigation of a sample
through interaction between electromagnetic radiation and matter, analyzing
X- rays emitted from a specimen can be by an energy dispersive spectrometer.
3.4.4 Transmission electron microscope (TEM)
The transmission electron microscopy (TEM) is an imaging technique
whereby a beam of electron is transmitted through a specimen, and then the
image is formatted, TEM (JEOL, model 1200EX, (TEM Technite 10 Philips)
measurements were operated at an accelerating voltage of 120 kV and later
with an XDL 3000 powder.
3.4.5 X-ray diffraction analysis (XRD)
The silver nanoparticles solution thus obtained was purified by
repeated centrifugation 5000 rpm for 20 min followed by redispersion of the
pellet of silver nano particles into 10 mL of deionized water. After freeze
drying of the purified silver particles, the structure and composition were
analyzed by XRD. The dried mixture of silver nanoparticles was collected for
the determination of size of Ag nanoparticles. Pro X-ray diffract, meter
operated at a voltage of 40 kv and a current of 30 mA with Cu kα radiation in
a 0-20 configuration. The crystallite domain size was calculated from the
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XRD peak. The aliquot of this filtrate containing silver nanoparticles was
used for X-ray diffraction (XRD) analysis.
3.4.6 Atomic Force Microscopy (AFM)
The analysis of size, morphology, agglomeration pattern and dispersed
nature of Ag NPs were performed using atomic force microscopy (Agilent
Technologies AFM- 5500).
3.5 Laboratory colonization of mosquitoes
For the different bioassays enormous amount of different stage of
mosquito colony needed. Laboratory-bred pathogen-free strains of
mosquitoes were reared in the vector control laboratory, Department of
Zoology, Annamalai University. At the time of adult feeding, these
mosquitoes were 3–4 days old after emergences (maintained on raisins and
water) and were starved for 12 h before feeding. Each time, 500 mosquitoes
per cage were fed on blood using a feeding unit fitted with Parafilm as
membrane for 4 h. Ae. aegypti feeding was done from 12 noon to 4.00 p.m.
and An. stephensi, and Cx. quinquefasciatus were fed during 6.00 p.m. to
10.00 p.m. A membrane feeder with the bottom end fitted with Parafilm was
placed with 2.0 ml of the blood sample (obtained from a slaughter house by
collecting in a heparinized vial and stored at 4 °C) and kept over a netted cage
of mosquitoes. The blood was stirred continuously using an automated
stirring device, and a constant temperature of 37 °C were maintained using a
water jacket circulating system. After feeding, the fully engorged females
were separated and maintained on raisins. Mosquitoes were held at 28±2 °C,
70–85 % relative humidity, with a photo period of 12-h light and 12-h dark
(Plate 3 and 4).
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Plate 3. Life cycles of vector mosquitoes.
Egg rafts
Larva
a
Pupa
Adult Adult
Egg
Larva
Pupa
Adult
Egg
Larva
Pupa
Anopheles stephensi Aedes aegypti Culex quinquefasciatus
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Plate 4. Non-target organisms.
Anisops bouvieri Diplonychus indicus
Gambusia affinis
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3.6 Bioassay
3.6.1 Larvicidal activity
Larvicidal activity of the aqueous crude extract and Ag NPs from M.
emarginata, N. alata, H. puberula, A. elaeagnoidea, and V. madrasapatna
was evaluated according to WHO protocol (2005). The aqueous extract and
Ag NPs was tested at various concentrations ranging from 70 to 600 µg/mL
and 4 to 60 µg/ mL, respectively. Twenty numbers of late III instar larvae
were introduced into a 500-mL glass beaker containing 250 mL of
dechlorinated water, plus the desired concentrations of leaf extract or Ag NPs.
For each concentration, five replicates were performed. Larval mortality was
recorded at 24 h after exposure, during which no food was given to the larvae.
Each test included a set control groups (silver nitrate and distilled water) with
five replicates for each individual concentration.
3.6.2 Ovicidal activity
For ovicidal slightly modified method of Su and Mulla (1998) was
performed. Eggs were collected from vector control laboratory, Department of
Zoology, Annamalai University. The different aqueous leaf extracts and silver
nanoparticle were to achieve various concentrations ranging from 50 to 600
µg/mL and 10 to 180 µg/ mL, respectively. Eggs of these mosquito species
(100 no. of 0-6, 6-12 and 12-18hr old eggs) were exposed to each
concentration of leaf aqueous extracts and Ag NPs. After treatment, the eggs
from each concentration were individually transferred to distilled water cups
for hatching assessment after counting the eggs under microscope. Each
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experiment was replicated six times along with appropriate control. The hatch
rates were assessed 48 hr post-treatment by the following formula.
% of egg hatchability =
Number of hatched larvae 100
Total number of eggs
3.6.3 Adulticidal activity
Adulticidal bioassay was performed by slightly modified method of
WHO (1981). Based on the wide range and narrow range tests, aqueous crude
extract was tested at different concentrations, and Ag NPs were tested
different concentrations. Aqueous crude extract and Ag NPs were applied on
Whatman no. 1 filter papers (size 12×15 cm). Control papers were treated
with silver nitrate and distilled water. Twenty female mosquitoes were
collected and gently transferred into a plastic holding tube. The mosquitoes
were allowed to acclimatize in the holding tube for 1 hr and then exposed to
test paper for 1 hr. At the end of exposure period, the mosquitoes were
transferred back to the holding tube and kept for 24-hr recovery period. A pad
of cotton soaked with 10 % glucose solution was placed on the mesh screen.
Each test was replicated five times equal number of controls was set up
simultaneously using tap water (silver nitrate and distilled water). The above
procedure was carried out in triplicate for each plants extract concentration.
The data were subjected to probit analysis (Finney, 1971) in order to estimate
the LD50, LD90, 95 percent confidence limit of lower confidence limit (LCL)
and Upper confidence limit (UCL) and chi-square values.
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3.6.4 Biotoxicity on Non-target aquatic organism
The effect of non-target organisms was assessed following the method
by Sivagnaname and Kalyanasundaram (2004). The effect of aqueous extract
and Ag NP of the potential plant was tested against non-target organism’s A.
bouvieri, D. indicus, and G. affinis. The species were field collected and
separately maintained in cement tanks (85-cm diameter and 30-cm depth)
containing water at 27±3 ° C and relative humidity 85 %.
The aqueous extracts and Ag NP of M. emarginata, N. alata, H.
puberula, A. elaeagnoidea, and V. madrasapatna were evaluated at a
concentration of 50 times higher the lethal concentration (LC50) dose for
mosquito larvae. Ten replicates will be performed for each concentration
along with four replicates of untreated controls. The non-target organisms
were observed for mortality and other abnormalities such as sluggishness and
reduced swimming activity after 48-h exposure. The exposed non-target
organisms were also observed continuously for 10 days to understand the post
treatment effect of this extract on survival and swimming activity.
3.7 Statistical analysis
The average larval and adult mortality data were subjected to probit
analysis for calculating LC50, LC90, LD50, LD90, and other statistics at 95 %
confidence limit of upper confidence limit and lower confidence limit, and
chi-square values were calculated using the statistical package. In experiments
evaluating biotoxicity on non-target organisms, the Suitability Index (SI) was
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calculated for each non-target species using the following formula (Deo et al.,
1988).
SI = LC50 of non‐target organisms
LC50 of target vector species
All data were analyzed using the Statistical Package of Social Sciences
(SPSS) version 16.0. A probability level of P<0.05 was used for the
significance of differences between values.
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4. RESULTS
4.1 CHARACTERIZATION OF SILVER NANOPARTICLES
4.1.1 UV-visible spectroscopy
After adding the plant leaf (M. emarginata, N. alata, H. puberula, A.
elaeagnoidea, and V. madrasapatna) extract to the silver nitrate solution, the
formation of Ag NPs occurred and they exhibited a color change, probably
due to surface plasmon resonance (SPR). The intensity of color was directly
proportional to the formation of Ag NPs. The color change was rapid, when
the two solutions were mixed the color turned brown within 10 min, and by 6
hr the solution turned dark brown. This color change was linked to the
reduction of Ag+ to Ag
0 by various biomolecules present in the leaf extract.
The surface Plasmon resonance bands were influenced by size, shape,
morphology, composition and dielectric environment of prepared Ag NPs.
The Ag NPs was characterized using UV–visible spectroscopy and an intense,
broad absorption peak was observed. This SPR peak was sensitive to the size
and shape of the nanoparticles, amount of extract, silver nitrate concentration
and the type of biomolecules present in the leaf extract.
4.1.1.1 Merremia emerginata
The mixture of M. emerginata -AgNO3 solution color was noted by
visual observation. The plant leaf extract without Ag NO3 solution did not
show any change in color. The synthesis of Ag NPs was confirmed within 3 h
after that the M. emerginata leaf extract was added to AgNO3 solution. The
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color of the extract changed from yellowish to dark brown (Fig. 1a), and this
can be due to excitation of surface plasmon resonance in M. emerginata -
synthesized Ag NPs. The formation of Ag NPs was confirmed by an
absorption peak at 440 nm (Fig. 1b).
4.1.1.2 Naregamia alata
The mixture of N. alata -AgNO3 solution color was noted by visual
observation. The plant leaf extract without Ag NO3 solution did not show any
change in color. The synthesis of Ag NPs was confirmed within 3 h after that
the N. alata leaf extract was added to AgNO3 solution. The color of the
extract changed from light brown to dark brown (Fig. 2a), and this can be due
to excitation of surface plasmon resonance in N. alata -synthesized Ag NPs.
The formation of Ag NPs was confirmed by an absorption peak at 473 nm
(Fig. 2b).
4.1.1.3 Hedyotis puberula
The mixture of H. puberula -AgNO3 solution color was noted by visual
observation. The plant leaf extract without Ag NO3 solution did not show any
change in color. The synthesis of Ag NPs was confirmed within 3 h after that
the H. puberula leaf extract was added to AgNO3 solution. The color of the
extract changed from light brown to dark brown (Fig. 3a), and this can be due
to excitation of surface plasmon resonance in H. puberula -synthesized Ag
NPs. The formation of Ag NPs was confirmed by an absorption peak at 445
nm (Fig. 3b).
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4.1.1.4 Aglaia elaeagnoidea
The color change in the mixture of A. elaeagnoidea -AgNO3 solution
color was noted by visual observation. The plant leaf extract without Ag NO3
solution did not show any change in color. The synthesis of Ag NPs was
confirmed within 3 h after that the A. elaeagnoidea leaf extract was added to
AgNO3 solution. The color of the extract changed from light brown to dark
brown (Fig. 4a), and this can be due to excitation of surface plasmon
resonance in A. elaeagnoidea -synthesized Ag NPs. The formation of Ag NPs
was confirmed by an absorption peak at 440.5 nm (Fig. 4b).
4.1.1.5 Ventilago madrasapatna
The mixture of V. madrasapatna-AgNO3 solution color was noted by
visual observation. The plant leaf extract without Ag NO3 solution did not
show any change in color. The synthesis of Ag NPs was confirmed within 3 h
after that the V. madrasapatna leaf extract was added to AgNO3 solution. The
color of the extract changed from light brown to dark brown (Fig. 5a), and
this can be due to excitation of surface plasmon resonance in V.
madrasapatna-synthesized Ag NPs. The formation of Ag NPs was confirmed
by an absorption peak at 411 nm (Fig. 5b).
4.1.2 Fourier transforms infrared spectroscopy (FTIR)
FTIR measurements of the freeze-dried samples were carried out to
identify the possible interactions between silver and bioactive molecules,
which may be responsible for synthesis and stabilization (capping material) of
silver nanoparticles. The amide linkages between amino acid residues in
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proteins give rise to well known signatures in the infrared region of the
electromagnetic spectrum. The result of this FTIR spectroscopic study
confirmed that the plant extract has the ability to perform dual functions of
reduction and stabilization of silver nanoparticles.
4.1.2.1 Merremia emerginata
The FTIR spectrum of Ag NPs biosynthesized using M. emarginata
plant extract is shown in Figure 4.6. In the three replicates, prominent bands
of absorbance were always detected at 616.66, 823.79, 872.67, 1021.62,
1108.68, 1383.31, 2917.72 and 3286.37 cm−1
. The observed peaks denote C-
N stretch (aromatic amines), C-C stretch (aromatics), N-H bend (1° amines),
C-O stretch (carboxylic group), and O-H stretch, H- bonded (alcohols,
phenol), respectively. These bands denote stretching vibrational bands
responsible for compounds like flavonoids and terpenoids and may be held
responsible for efficient capping and stabilization of obtained Ag NPs.
Overall, the rapid reduction of silver ions might be linked with the presence of
water-soluble phytochemicals such as flavones, quinones, and organic acids
present in the leaf extract of M. emarginata.
4.1.2.2 Naregamia alata
The FTIR spectrum of biosynthesized AgNPs by using N. alata
leaf extract is shown in Fig. 7. In order to identify the biomolecules
responsible for reduction and efficient stabilization of the metal nanoparticles,
it was highlighted that the band at 3148 cm-1
may correspond to O–H, as well
as to H-bonded alcohols and phenols. The peak at 2201 cm-1
may be lined
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with the presence of carboxylic acids. Shoulder peaks at 1554 cm-1
probably
indicate that the amide I and amide II arisen, due to carbonyl and –NH stretch
vibrations in the amide linkages of the proteins, respectively. The band at
1467 and1383 cm-1
may correspond to C–C stretching of aromatic amine. The
band at 1111 and 1013 cm-1
probably indicate the presence of C–O stretching
alcohols, carboxylic acids, esters and ethers. The peak near 663 cm-1
may be
assigned to CH out of plane bending vibrations of substituted ethylene
systems –CH=CH. The immediate reduction of silver ions in the present
investigation might be linked with the presence of water-soluble
phytochemicals such as flavones, quinones, and organic acids present in the
leaves of N. alata.
4.1.2.3 Hedyotis puberula
FTIR analysis was carried out, to identify the functional groups of the
synthesized AgNPs. FTIR spectrum indicated the clear peaks with (3417.98,
2922.58, 2853.13, 2358.43, 1746.75, 1730.54, 1574.44, 1538.98, 1470.68,
1384.41, 1114.35, 869.29, 791.31, 661.15 and 617.59 cm¯1) different values
(Fig. 8). Above the peak values they corresponded to functional groups like,
amide group (N–H stretching 3417.98 cm¯1), an aliphatic group (cyclic CH2 –
2922.58 cm¯1), a methyl group (bend CH2–CH3 stretching 1384.41 cm¯1),
aliphatic amine group (C–N stretching 1114.35 cm¯1), alkyl halides group
(C–Cl stretching 869.29 and 717.07 cm¯1) and alkyl halides (C–Br stretching
661.15 cm¯1and 617.59 cm¯1). The functional groups such as alcohol, amines,
amides, alkanes, methyl, aliphatic and halides were denoted as possible
stabilizing, capping and reducing agents of the AgNPs.
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4.1.2.4 Aglaia elaeagnoidea
The FTIR spectrum of AgNPs synthesized from A.
elaeagnoidea leaf extract is given in Fig. 9. The slight shift in peak position
from 3382, 2922, 2359, 1556, 1470, 1415, 1065, 864, 777, 668 and 616 cm-1
,
respectively indicate that the different phytochemicals present in the A.
elaeagnoidea leaf extract are involved in reducing and capping the AgNPs.
The peak at 3380 cm-1
is due to the N–H stretch vibrations of the peptide
linkages. The peaks at 1556, 1065, 777 and 616 cm-1
could be assigned to
amide I, II, III, and N–H bending of peptide linkages of proteins, respectively.
The peaks at 668 and 1415 cm-1
may be due to the presence of C–S stretch
(CH2–S) of thiol or thioether and absorption peaks of –C–O–C bonds,
respectively. The peaks at 1556 and 11470 cm-1
can be linked to carbonyl
stretching vibration and germinal methyl group, respectively. The FTIR data
suggest the proteins involved in capping and stabilization of the synthesized
AgNPs.
4.1.2.5 Ventilago madrasapatna
The FTIR spectrum of the cell-free extract alone showed five
distinct peaks at the range of 3626, 3381, 1567, 1384, and 1077 cm−1
(Fig.
10). The band at 3626, 3381 cm−1
was referred as the strong stretching
vibrations of the OH– functional group. The peak at 1567 cm−1
was due to the
symmetric stretching vibrations of the COO–functional group, and 1384
cm−1
was assigned to the amide functional groups present in the cell free
supernatant. The band at 1077 cm−1
was attributed to the C–O– stretching
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vibrations. To compare the IR spectrum of the cell free supernatant and
synthesized AgNPs, the major shifts were observed in the hydroxyl and
carboxyl functional groups of protein which may be responsible for synthesis
of AgNPs.
4.1.3 Scanning electron microscopy (SEM) with energy dispersive X-ray
spectroscopy (EDX)
The synthesized silver nanoparticles morphology was characterized by
scanning electron microscopy. Scanning electron micrographs enabled
visualization of the size and shape of silver nanoparticles. The plant-
fabricated silver nanoparticles formed were predominantly spherical, cubic,
triangular and uniform shape. The scanning electron microscopy (SEM)
results indicate that the silver powder consists of a nano metrical
conglomerate which contains 89% silver, 8% carbon, and 3% oxygen. The
EDX attachment with the SEM was known to provide information on the
chemical analysis of the fields that are being investigated or the composition
at specific locations. The EDX profile shows a strong silver signal along with
weak oxygen and carbon peaks, which may have originated from the
biomolecules bound to the surface of the silver nanoparticles.
4.1.3.1 Merremia emerginata
SEM micrographs of the synthesized Ag NPs of M. emerginata
magnified at 50,000X are shown in Fig. 11a. The spherical or with cubic
structures are clear. EDX proves the chemical purity of the synthesized Ag
NPs (Fig. 11b).
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4.1.3.2 Naregamia alata
SEM micrographs of the synthesized Ag NPs of N. alata magnified at
60,000X are shown in Fig. 12a. The triangular, pentagonal, and hexagonal
structures are clear. EDX proves the chemical purity of the synthesized Ag
NPs (Fig. 12b).
4.1.3.3 Hedyotis puberula
SEM micrographs of the synthesized Ag NPs of H. puberula
magnified at 60,000X are shown in Fig. 13a. The spherical, triangular,
pentagonal, and hexagonal structures are clear. EDX proves the chemical
purity of the synthesized Ag NPs (Fig. 13b).
4.1.3.4 Aglaia elaeagnoidea
SEM micrographs of the synthesized Ag NPs of A. elaeagnoidea
magnified at 60,000X are shown in Fig. 14a. The triangular, pentagonal, and
hexagonal structures are clear. EDX proves the chemical purity of the
synthesized Ag NPs (Fig. 14b).
4.1.3.5 Ventilago madrasapatna
SEM micrographs of the synthesized Ag NPs of V. madrasapatna
magnified at 50,000X are shown in Fig. 15a. The spherical or with cubic
structures are clear. EDX proves the chemical purity of the synthesized Ag
NPs (Fig. 15b).
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4.1.4 Transmission electron microscopy (TEM)
Transmission electron microscopy has been found to be an excellent
tool for characterizing the size of nanoparticles. The size, shape, and
morphology of the silver nanoparticles were studied by the transmission
electron microscopy images. The grid used in the TEM was prepared by
placing a drop of the bioreduced diluted solution on a carbon-coated copper
grid and further dried it under a lamp. This assay protocol outlines procedures
for sample preparation and the determination of mean nanoparticle size using
TEM. Although the projected particle size is the primary determinant of the
measured particle diameter, other parameters can impact these measurements
and influence the measured size. Therefore, guidelines for making successful
size measurements in the nanometer-size range are provided, as well as a
discussion of relevant standards.
4.1.4.1 Merremia emerginata
TEM micrograph indicated the M. emerginata -synthesized Ag NPs
with 50 nm scales and most of the Ag NPs was spherical in structure and few
of the Ag NPs were agglomerated. The particle sizes vary from 25 to 30 nm
and the average size of the Ag NPs was 28 nm (Fig. 16).
4.1.4.2 Naregamia alata
TEM micrograph indicated the N. alata -synthesized Ag NPs with 100
nm scales and most of the Ag NPs was spherical in structure and few of the
Ag NPs were agglomerated. The particle sizes vary from 25 to 50 nm and the
average size of the Ag NPs was 35 nm (Fig. 17).
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4.1.4.3 Hedyotis puberula
TEM micrograph indicated the H. puberula -synthesized Ag NPs with
100 nm scales and most of the Ag NPs was spherical in structure and few of
the Ag NPs were agglomerated. The particle sizes vary from 15 to 22 nm and
the average size of the Ag NPs was 20 nm (Fig. 18).
4.1.4.4 Aglaia elaeagnoidea
TEM micrograph indicated the A. elaeagnoidea -synthesized Ag NPs
with 100 nm scales and most of the Ag NPs was spherical in structure and
few of the Ag NPs were agglomerated. The particle sizes vary from 8 to 34
nm and the average size of the Ag NPs was 20 nm (Fig. 19).
4.1.4.5 Ventilago madrasapatna
TEM micrograph indicated the V. madrasapatna -synthesized Ag NPs
with 100 nm scales and most of the Ag NPs was spherical in structure and
few of the Ag NPs were agglomerated. The particle sizes vary from 15 to 22
nm and the average size of the Ag NPs was 18 nm (Fig.4.20).
4.1.5 X-ray diffraction (XRD) analysis
Biosynthesized silver nanoparticles from five plants (M. emarginata,
N. alata, H. puberula, A. elaeagnoidea, and V. madrasapatna) were further
confirmed by the characteristic peaks observed in XRD spectrum. The XRD
is carried out to study the crystalline nature of synthesized Ag NPs. The
diffract meter was operating at 40 kV and 30 mA, with a step size of 0.02°
(2θ). The scanning was done in the region of 10˚to 80˚ for 2θ. The XRD
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pattern showed numbers of Bragg reflections that may be indexed on the basis
of the face-centered cubic structure of metallic silver.
4.1.5.1 Merremia emerginata
The crystalline nature of M.erremia emerginata -synthesized silver
nanoparticles was studied by XRD analysis. The XRD pattern confirmed the
crystalline nature of synthesized Ag NPs. Four diffraction peaks were
observed at 38.07, 44.27, 64.41 and 77.34 which represent the (111), (200),
(220) and (311) reflections and the face-centered cubic structure of metallic
silver, respectively (Fig. 21).
4.1.5.2 Naregamia alata
The crystalline nature of N. alata -synthesized silver nanoparticles was
studied by XRD analysis. The XRD pattern confirmed the crystalline nature
of synthesized Ag NPs. Four diffraction peaks were observed at 38.02, 44.20,
64.38 and 77.34 which represent the (111), (200), (220) and (311) reflections
and the face-centered cubic structure of metallic silver, respectively
(Fig.4.22).
4.1.5.3 Hedyotis puberula
The crystalline nature of H. puberula -synthesized silver nanoparticles
was studied by XRD analysis. The XRD pattern confirmed the crystalline
nature of synthesized Ag NPs. Four diffraction peaks were observed at 38.00,
44.19, 64.34 and 77.28 which represent the (111), (200), (220) and (311)
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reflections and the face-centered cubic structure of metallic silver,
respectively (Fig. 23).
4.1.5.4 Aglaia elaeagnoidea
The crystalline nature of A. elaeagnoidea -synthesized silver
nanoparticles was studied by XRD analysis. The XRD pattern confirmed the
crystalline nature of synthesized Ag NPs. Four diffraction peaks were
observed at 38.13, 44.32, 64.12 and 77.38 which represent the (111), (200),
(220) and (311) reflections and the face-centered cubic structure of metallic
silver, respectively (Fig.4.24).
4.1.5.5 Ventilago madrasapatna
The crystalline nature of V. madrasapatna -synthesized silver
nanoparticles was studied by XRD analysis. The XRD pattern confirmed the
crystalline nature of synthesized Ag NPs. Four diffraction peaks were
observed at 38.20, 44.50, 64.43 and 77.40 which represent the (111), (200),
(220) and (311) reflections and the face-centered cubic structure of metallic
silver, respectively (Fig. 25).
4.1.6 Atomic Force Microscopy
AFM is a primary tool for analyzing size, shape, agglomeration pattern
and offers visualizations of three-dimensional views of the nanoparticles
unlike the electron microscopes. It has an advantage over combination of high
resolution, samples does not have to be conductive and does not require the
high-pressure vacuum conditions.
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4.1.6.1 Merremia emerginata
2.5 μm resolution studies of biologically synthesized Ag NPs with AFM
reveal the particles are polydispersed, spherical in shape, having the size
ranging from 30 to 44 nm and there is no agglomeration observed between the
particles (Fig. 26a). Raw data obtained from this AFM microscope is treated
with a specially designed image processing software (NOVA-TX) to further
exploit the 3D image of nanoparticles (Fig. 26b). The average particle size
obtained from the corresponding diameter distribution was about 38 nm (Fig.
26c and d).
4.1.6.2 Naregamia alata
2.5 μm resolution studies of green-synthesized AgNPs with AFM
reveal the particles are poly-dispersed, spherical in shape, having the size
range from 0 to 5.5 nm and there is no agglomeration observed between the
particles (Fig. 27a). Raw data obtained from AFM microscope were treated
with a specially designed image processing software (NOVA-TX) to further
exploit the 3D image of nanoparticles (Fig. 27b). The average particle size
obtained from the corresponding diameter distribution was about 5 nm (Fig.
27c and d).
4.1.6.3 Hedyotis puberula
As outlined in Fig. 28a, the biosynthesized AgNPs are spherical, poly-
dispersed, and sized between 3.59 and 7.18 nm, as shown via 2.5 μm
resolution studies with AFM. We treated the raw data produced by the AFM
microscope with a specially-designed image processing software (NOVA-
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TX) to further investigate the AgNPs 3D image (Fig. 28b). The corresponding
diameter distribution revealed a mean particle size of approximately 6.46 nm
(Fig. 28c and d).
4.1.6.4 Aglaia elaeagnoidea
2.5 μm resolution studies of biologically fabricated AgNPs with AFM
reveal the particles are poly-dispersed, spherical in shape, having the size
range from 1 to 4 nm and there is no agglomeration observed between the
particles (Fig. 29a). Raw data obtained from AFM were treated with a
specially designed image processing software (NOVA-TX) to further exploit
the 3D image of AgNPs (Fig. 29b). The average particle size obtained from
the corresponding diameter distribution was about 4.91 nm (Fig. 29c and d).
4.1.6.5 Ventilago madrasapatna
2.5 μm resolution studies of biologically synthesized AgNPs with
AFM reveal the particles are polydispersed, spherical in shape, having the
main size ranging from 1 to 6 nm and there is no agglomeration observed
between the particles (Fig. 30a). Raw data obtained from this AFM
microscope is treated with a specially designed image processing software
(NOVA-TX) to further exploit the 3D image of nanoparticles (Fig. 30b). The
average particle size obtained from the corresponding diameter distribution
was 4.6-5 nm (Fig. 30c and d).
4.2 LARVICIDAL ACTIVITY AGAINST MOSQUITO VECTORS
In laboratory conditions, the larvicidal potential of five plants (M.
emarginata, N. alata, H. puberula, A. elaeagnoidea, and V. madrasapatna)
aqueous leaf extract and synthesized silver nanoparticles (Ag NPs) were
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tested against the larvae of three important mosquito vectors An. stephensi,
Ae. aegypti and Cx. quinquefasciatus and the results are presented in tables 2
to 11.
4.2.1 Merremia emerginata
The larvicidal response of M. emarginataaqueous leaf extract and
synthesized silver nanoparticles against three mosquito larvae are presented in
table 2 and 3.
4.2.1.1 Larvicidal activity of M. emarginata leaf extract against vector
mosquitoes
The larvicidal activity of M. emarginata aqueous leaf extract against
An. stephensi, Ae. aegypti and Cx. quinquefasciatus are presented table 2. The
results revealed that the aqueous extract had the significant larvicidal activity
with the LC50 and LC90 values of 146.91, 157.87, 169.24 and 286.29, 301.63,
315.81 µg/mL, respectively.
4.2.1.2 Larvicidal activity of synthesized silver nanoparticles against
vector mosquitoes
The larvicidal activity of synthesized Ag NPs against An. stephensi,
Ae. aegypti and Cx. quinquefasciatus are presented in table 3. The results
revealed that the silver nanoparticles had the most significant larvicidal
activity with the LC50 and LC90 values of 8.36, 9.20, 10.02 and 16.33, 17.86,
18.62 µg/mL, respectively.
4.2.2 Naregamia alata
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The larvicidal response of N. alata aqueous leaf extract and
synthesized silver nanoparticles against three mosquito larvae are presented in
table 4 and 5.
4.2.2.1 Larvicidal activity of N. alata leaf extract against vector
mosquitoes
The larvicidal activity of N. alata aqueous leaf extract against An.
stephensi, Ae. aegypti and Cx. quinquefasciatus are presented table 4. The
results revealed that the aqueous extract had the significant larvicidal activity
with the LC50 and LC90 values of 165.15, 179.17, 196.48 and 321.56, 342.21,
371.50 µg/mL, respectively.
4.2.2.2 Larvicidal activity of synthesized silver nanoparticles against
vector mosquitoes
The larvicidal activity of synthesized Ag NPs against An. stephensi,
Ae. aegypti and Cx. quinquefasciatus are presented table 5. The results
revealed that the silver nanoparticles had the most significant larvicidal
activity with the LC50 and LC90 values of 12.40, 13.57, 14.84 and 24.20,
25.71, 27.49 µg/mL, respectively.
4.2.3 Hedyotis puberula
The larvicidal response of H. puberula aqueous leaf extract and
synthesized silver nanoparticles against three mosquito larvae are presented in
table 6 and 7.
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4.2.3.1 Larvicidal activity of H. puberula leaf extract against vector
mosquitoes
The larvicidal activity of H. puberula aqueous leaf extract against An.
stephensi, Ae. aegypti and Cx. quinquefasciatus are presented table 6. The
results revealed that the aqueous extract had the significant larvicidal activity
with the LC50 and LC90 values of 182.67, 199.14, 217.67 and 369.97, 385.34,
404.10 µg/mL, respectively.
4.2.3.2 Larvicidal activity of synthesized silver nanoparticles against
vector mosquitoes
The larvicidal activity of synthesized Ag NPs against An. stephensi,
Ae. aegypti and Cx. quinquefasciatus are presented table 7. The results
revealed that the silver nanoparticles had the most significant larvicidal
activity with the LC50 and LC90 values of 16.58, 18.05, 19.52 and 32.11,
34.04, 36.07 µg/mL, respectively.
4.2.4 Aglaia elaeagnoidea
The larvicidal response of A. elaeagnoidea aqueous leaf extract and
synthesized silver nanoparticles against three mosquito larvae are presented in
table 8 and 9.
4.2.4.1 Larvicidal activity of A. elaeagnoidea leaf extract against vector
mosquitoes
The larvicidal activity of A. elaeagnoidea aqueous leaf extract against
An. stephensi, Ae. aegypti and Cx. quinquefasciatus are presented table 8.
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The results revealed that the aqueous extract had the significant larvicidal
activity with the LC50 and LC90 values of 207.06, 229.79, 246.43 and 408.46,
442.71, 462.09 µg/mL, respectively.
4.2.4.2 Larvicidal activity of synthesized silver nanoparticles against
vector mosquitoes
The larvicidal activity of synthesized Ag NPs against An. stephensi,
Ae. aegypti and Cx. quinquefasciatus are presented table 9. The results
revealed that the silver nanoparticles had the most significant larvicidal
activity with the LC50 and LC90 values of 20.66, 22.80, 24.91 and 39.94,
43.23, 45.96 µg/mL, respectively.
4.2.5 Ventilago madrasapatna
The larvicidal response of V. madrasapatna aqueous leaf extract and
synthesized silver nanoparticles against three mosquito larvae are presented in
table 10 and 11.
4.2.5.1 Larvicidal activity of V. madrasapatna leaf extract against vector
mosquitoes
The larvicidal activity of V. madrasapatna aqueous leaf extract against
An. stephensi, Ae. aegypti and Cx. quinquefasciatus are presented table 10.
The results revealed that the aqueous extract had the significant larvicidal
activity with the LC50 and LC90 values of 245.46, 267.27, 289.86 and 481.55,
507.89, 538.91 µg/mL, respectively.
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4.2.5.2 Larvicidal activity of synthesized silver nanoparticles against
vector mosquitoes
The larvicidal activity of synthesized Ag NPs against An. stephensi,
Ae. aegypti and Cx. quinquefasciatus are presented table 11. The results
revealed that the silver nanoparticles had the most significant larvicidal
activity with the LC50 and LC90 values of 24.89, 26.92, 29.24 and 48.03,
51.26, 54.89 µg/mL, respectively.
4.3 OVICIDAL ACTIVITY AGAINST MOSQUITO VECTORS
In laboratory conditions, the result of ovicidal activity of five plants
(M. emarginata, N. alata, H. puberula, A. elaeagnoidea, and V.
madrasapatna) aqueous leaf extract and synthesized silver nanoparticles
against the eggs of three important vector mosquitoes viz., An. stephensi, Ae.
aegypti and Cx. quinquefasciatus are presented in table 12 to 21.
4.3.1 Merremia emerginata
The percentage of egg hatchability of An. stephensi, Ae. aegypti and
Cx. quinquefasciatus with the aqueous leaf extract and synthesized Ag NPs
of M. emarginataare presented in table 12 and 13. The rate of hatchability
was higher in lower concentration and when concentration increased the
hatchability rate decreased these results clearly revealed that the toxicity of
aqueous leaf extract and Ag NPs were dependent on its concentration and
which will determine the egg hatchability. The treatment of eggs of 0 to 6 hr
old was more effective, including higher rates of mortality as compared to
eggs of 6 to12 h old and treated for 12 to 18 h.
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4.3.1.1 Ovicidal activity of M. emarginata aqueous extract against vector
mosquitoes
The toxicity of M. emarginata aqueous leaf extract was dependent on
its concentration and the age of the eggs treated. Eggs aged 0 to 6, 6 to 12,
and 12 to18 hr suffered incomplete ovicidal activity even at 160, 200 and
240µg/mL against An. stephensi, respectively. An. stephensi eggs were
slightly more susceptible to the aqueous leaf extract than those of Ae. aegypti
and Cx. quinquefasciatus. Control eggs showed 100% hatchability in the all
age groups.
4.3.1.2 Ovicidal activity of synthesized silver nanoparticles against vector
mosquitoes
The toxicity of synthesized Ag NPs was dependent on its concentration
and the age of the eggs treated. Eggs aged 0 to 6, 6 to 12, and 12 to18 h
suffered incomplete ovicidal activity even at 32, 40 and 48µg/mL against An.
stephensi, respectively. An. stephensi eggs were slightly more susceptible to
the synthesized Ag NPs than those of Ae. aegypti and Cx. quinquefasciatus.
Control eggs showed 100% hatchability in the all age groups.
4.3.2 Naregamia alata
The percentage of egg hatchability of An. stephensi, Ae. aegypti and
Cx. quinquefasciatus with the aqueous leaf extract and synthesized Ag NPs of
N. alataare presented in table 14 and 15. The rate of hatchability was higher
in lower concentration and when concentration increased the hatchability rate
decreased these results clearly revealed that the toxicity of aqueous leaf
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extract and Ag NPs were dependent on its concentration and which will
determine the egg hatchability. The treatment of eggs of 0 to 6 h old was more
effective, including higher rates of mortality as compared to eggs of 6 to12 h
old and treated for 12 to 18 h.
4.3.2.1 Ovicidal activity of N. alataaqueous extract against vector
mosquitoes
The toxicity of N. alataaqueous leaf extract was dependent on its
concentration and the age of the eggs treated. Eggs aged 0 to 6, 6 to 12, and
12 to18 h suffered incomplete ovicidal activity even at 200, 250 and 300
µg/mL against An. stephensi, respectively. An. stephensi eggs were slightly
more susceptible to the aqueous leaf extract than those of Ae. aegypti and Cx.
quinquefasciatus. Control eggs showed 100% hatchability in the all age
groups.
4.3.2.2 Ovicidal activity of synthesized silver nanoparticles against vector
mosquitoes
The toxicity of synthesized Ag NPs was dependent on its concentration
and the age of the eggs treated. Eggs aged 0 to 6, 6 to 12, and 12 to18 h
suffered incomplete ovicidal activity even at 40, 50 and 60 µg/mL against An.
stephensi, respectively. An. stephensi eggs were slightly more susceptible to
the synthesized Ag NPs than those of Ae. aegypti and Cx. quinquefasciatus.
Control eggs showed 100% hatchability in the all age groups.
4.3.3 Hedyotis puberula
The percentage of egg hatchability of An. stephensi, Ae. aegypti and
Cx. quinquefasciatus with the aqueous leaf extract and synthesized Ag NPs
of H. puberulaare presented in table 16 and 17. The rate of hatchability was
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higher in lower concentration and when concentration increased the
hatchability rate decreased these results clearly revealed that the toxicity of
aqueous leaf extract and Ag NPs were dependent on its concentration and
which will determine the egg hatchability. The treatment of eggs of 0 to 6 h
old was more effective, including higher rates of mortality as compared to
eggs of 6 to 12 h old and treated for 12 to 18 h.
4.3.3.1 Ovicidal activity of H. puberulaaqueous extract against vector
mosquitoes
The toxicity of H. puberulaaqueous leaf extract was dependent on its
concentration and the age of the eggs treated. Eggs aged 0 to 6, 6 to 12, and
12 to 18 h suffered incomplete ovicidal activity even at 240, 300 and
360µg/mL against An. stephensi, respectively. An. stephensi eggs were
slightly more susceptible to the aqueous leaf extract than those of Ae. aegypti
and Cx. quinquefasciatus. Control eggs showed 100% hatchability in the all
age groups.
4.3.3.2 Ovicidal activity of synthesized silver nanoparticles against vector
mosquitoes
The toxicity of synthesized Ag NPs was dependent on its concentration
and the age of the eggs treated. Eggs aged 0 to 6, 6 to 12, and 12 to18 hr
suffered incomplete ovicidal activity even at 60, 75 and 90µg/mL against An.
stephensi, respectively. An. stephensi eggs were slightly more susceptible to
the synthesized Ag NPs than those of Ae. aegypti and Cx. quinquefasciatus.
Control eggs showed 100% hatchability in the all age groups.
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4.3.4 Aglaia elaeagnoidea
The percentage of egg hatchability of An. stephensi, Ae. aegypti and
Cx. quinquefasciatus with the aqueous leaf extract and synthesized Ag NPs of
A. elaeagnoideaare presented in table 18 and 19. The rate of hatchability was
higher in lower concentration and when concentration increased the
hatchability rate decreased these results clearly revealed that the toxicity of
aqueous leaf extract and Ag NPs s were dependent on its concentration and
which will determine the egg hatchability. The treatment of eggs of 0 to 6 h
old was more effective, including higher rates of mortality as compared to
eggs of 6 to12 h old and treated for 12 to 18 h.
4.3.4.1 Ovicidal activity of A. elaeagnoideaaqueous extract against vector
mosquitoes
The toxicity of A. elaeagnoideaaqueous leaf extract was dependent on
its concentration and the age of the eggs treated. Eggs aged 0 to 6, 6 to 12,
and 12 to18 hr suffered incomplete ovicidal activity even at 320, 400 and
480µg/mL against An. stephensi, respectively. An. stephensi eggs were
slightly more susceptible to the aqueous leaf extract than those of Ae. aegypti
and Cx. quinquefasciatus. Control eggs showed 100% hatchability in the all
age groups.
4.3.4.2 Ovicidal activity of synthesized silver nanoparticles against vector
mosquitoes
The toxicity of synthesized Ag NPs s was dependent on its
concentration and the age of the eggs treated. Eggs aged 0 to 6, 6 to 12, and
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12 to18 hr suffered incomplete ovicidal activity even at 80, 100 and
120µg/mL against An. stephensi, respectively. An. stephensi eggs were
slightly more susceptible to the synthesized Ag NPs s than those of Ae.
aegypti and Cx. quinquefasciatus. Control eggs showed 100% hatchability in
the all age groups.
4.3.5 Ventilago madrasapatna
The percentage of egg hatchability of An. stephensi, Ae. aegypti and
Cx. quinquefasciatus with the aqueous leaf extract and synthesized Ag NPs s
of V. madrasapatna are presented in table 20 and 21. The rate of hatchability
was higher in lower concentration and when concentration increased the
hatchability rate decreased these results clearly revealed that the toxicity of
aqueous leaf extract and Ag NPs s were dependent on its concentration and
which will determine the egg hatchability. The treatment of eggs of 0 to 6 h
old was more effective, including higher rates of mortality as compared to
eggs of 6 to12 h old and treated for 12 to 18 h.
4.3.5.1 Ovicidal activity of V. madrasapatna aqueous extract against
vector mosquitoes
The toxicity of V. madrasapatna aqueous leaf extract was dependent
on its concentration and the age of the eggs treated. Eggs aged 0 to 6, 6 to 12,
and 12 to18 hr suffered incomplete ovicidal activity even at 360, 450 and
540µg/mL against An. stephensi, respectively. An. stephensi eggs were
slightly more susceptible to the aqueous leaf extract than those of Ae. aegypti
and Cx. quinquefasciatus. Control eggs showed 100% hatchability in the all
age groups.
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4.3.5.2 Ovicidal activity of synthesized silver nanoparticles against vector
mosquitoes
The toxicity of synthesized Ag NPs was dependent on its concentration
and the age of the eggs treated. Eggs aged 0 to 6, 6 to 12, and 12 to18 h
suffered incomplete ovicidal activity even at 100, 125 and 150 µg/mL against
An. stephensi, respectively. An. stephensi eggs were slightly more susceptible
to the synthesized Ag NPs than those of Ae. aegypti and Cx. quinquefasciatus.
Control eggs showed 100% hatchability in the all age groups.
4.4 ADULTICIDAL ACTIVITY AGAINST MOSQUITO VECTORS
In laboratory conditions, the adulticidal activity of five plants
(M. emarginata, N. alata, H. puberula, A. elaeagnoidea, and V.
madrasapatna) aqueous leaf extract and synthesized silver nanoparticles were
tested against the adults of three important vector mosquitoes viz., An.
stephensi, Ae. aegypti and Cx. quinquefasciatus and the results are presented
in tables 22 to 31.
4.4.1 Merremia emerginata
The adulticidal response of M. emarginata aqueous leaf extract and
synthesized silver nanoparticles against the three mosquito species adults are
presented in table 22 and 23.
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4.4.1.1 Adulticidal activity of M. emarginata aqueous leaf extract against
vector mosquitoes
The adulticidal activity of M. emarginata aqueous leaf extract against
An. stephensi, Ae. aegypti and Cx. quinquefasciatus are presented in table 22.
The results revealed that the aqueous leaf extract had the significant
adulticidal activity with the LD50 and LD90 values of 204.82, 224.44, 244.52
and 400.39, 428.65, 457.27 µg/mL, respectively.
4.4.1.2 Adulticidal activity of synthesized silver nanoparticles against
vector mosquitoes
The adulticidal activity of synthesized Ag NPs against An. stephensi,
Ae. aegypti and Cx. quinquefasciatus are presented in table 23. The results
revealed that the Ag NPs had the most significant adulticidal activity with the
LD50 and LD90 values of 24.73, 27.24, 30.02 and 48.24, 51.65, 54.40 µg/mL,
respectively.
4.4.2 Naregamia alata
The adulticidal response of N. alata aqueous leaf extract and
synthesized silver nanoparticles against the three mosquito species adults are
presented in table 24 and 25.
4.4.2.1 Adulticidal activity of N. alata aqueous leaf extract against vector
mosquitoes
The adulticidal activity of N. alata aqueous leaf extract against An.
stephensi, Ae. aegypti and Cx. quinquefasciatus are presented in table 24. The
results revealed that the aqueous leaf extract had the significant adulticidal
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activity with the LD50 and LD90 values of 247.59, 271.25, 296.43 and 477.69,
508.78, 542.55 µg/mL, respectively.
4.4.2.2 Adulticidal activity of synthesized silver nanoparticles against
vector mosquitoes
The adulticidal activity of synthesized Ag NPs against An. stephensi,
Ae. aegypti and Cx. quinquefasciatus are presented in table 25. The results
revealed that the Ag NPs had the most significant adulticidal activity with the
LD50 and LD90 values of 31.60, 34.31, 37.52 and 60.94, 65.89, 70.06µg/mL,
respectively.
4.4.3 Hedyotis puberula
The adulticidal response of H. puberula aqueous leaf extract and
synthesized silver nanoparticles against the three mosquito species adults are
presented in table 26 and 27.
4.4.3.1 Adulticidal activity of H. puberula aqueous leaf extract against
vector mosquitoes
The adulticidal activity of H. puberula aqueous leaf extract against An.
stephensi, Ae. aegypti and Cx. quinquefasciatus are presented in table 26. The
results revealed that the aqueous leaf extract had the significant adulticidal
activity with the LD50 and LD90 values of 269.40, 295.65, 321.29 and 526.61,
554.42, 585.42 µg/mL, respectively.
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4.4.3.2 Adulticidal activity of synthesized silver nanoparticles against
vector mosquitoes
The adulticidal activity of synthesized Ag NPs against An. stephensi,
Ae. aegypti and Cx. quinquefasciatus are presented in table 27. The results
revealed that the Ag NPs had the most significant adulticidal activity with the
LD50 and LD90 values of 33.11, 36.34, 39.56 and 64.17, 69.35, 74.08 µg/mL,
respectively.
4.4.4 Aglaia elaeagnoidea
The adulticidal response of A. elaeagnoide aaqueous leaf extract and
synthesized silver nanoparticles against the three mosquito species adults are
presented in table 28 and 29.
4.4.4.1 Adulticidal activity of A. elaeagnoide aaqueous leaf extract against
vector mosquitoes
The adulticidal activity of A. elaeagnoide aaqueous leaf extract against
An. stephensi, Ae. aegypti and Cx. quinquefasciatus are presented in table 28.
The results revealed that the aqueous leaf extract had the significant
adulticidal activity with the LD50 and LD90 values of 285.48, 312.93, 343.96
and 569.66, 612.39, 648.16 µg/mL, respectively.
4.4.4.2 Adulticidal activity of synthesized silver nanoparticles against
vector mosquitoes
The adulticidal activity of synthesized Ag NPs against An. stephensi,
Ae. aegypti and Cx. quinquefasciatus are presented in table 29. The results
revealed that the Ag NPs had the most significant adulticidal activity with the
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LD50 and LD90 values of 37.52, 40.74, 44.55 and 73.46, 76.99, 81.25 µg/mL,
respectively.
4.4.5 Ventilago madrasapatna
The adulticidal response of V. madrasapatna aqueous leaf extract and
synthesized silver nanoparticles against the three mosquito species adults are
presented in table 30 and 31.
4.4.5.1 Adulticidal activity of V. madrasapatna aqueous leaf extract
against vector mosquitoes
The adulticidal activity of V. madrasapatna aqueous leaf extract
against An. stephensi, Ae. aegypti and Cx. quinquefasciatus are presented in
table 30. The results revealed that the aqueous leaf extract had the significant
adulticidal activity with the LD50 and LD90 values of 307.24, 334.46, 363.82
and 600.45, 644.34, 681.56 µg/mL, respectively.
4.4.5.2 Adulticidal activity of synthesized silver nanoparticles against
vector mosquitoes
The adulticidal activity of synthesized Ag NPs s against An. stephensi,
Ae. aegypti and Cx. quinquefasciatus are presented in table 31. The results
revealed that the Ag NPs had the most significant adulticidal activity with the
LD50 and LD90 values of 41.19, 44.85, 48.94 and 80.39, 85.79, 90.99 µg/mL,
respectively.
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4.5 BIOTOXICITY OF NON-TARGET ORGANISM
Under laboratory conditions, the biotoxicity of five plants (M.
emarginata, N. alata, H. puberula, A. elaeagnoidea, and V. madrasapatna)
aqueous leaf extract and synthesized silver nanoparticles were evaluated on
the three important mosquito predators viz., A. bouvieri, D. indicus and G.
affinis and the results are presented in tables 32 to 46.
4.5.1 Merremia emerginata
The biotoxicity of M. emarginata aqueous leaf extract and synthesized
silver nanoparticles against several non-target organisms sharing the same
ecological niche of Anopheles, Aedes and Culex mosquito vectors are
presented in table 32 to 34.
4.5.1.1 Biotoxicity of M. emarginata aqueous leaf extract and Ag NPs
against non-target organism
The biotoxicity of M. emarginata aqueous leaf extract and green-
synthesized Ag NPs was evaluated on non-target organism A. bouvieri, D.
indicus and G. affinis are presented in table 32 and 33. The toxicity treatments
achieved negligible toxicity against A. bouvieri, D. indicus and G. affinis with
LC50 values ranging from 8317.45 to 1056.04 μg/mL, respectively. Focal
observations highlighted that longevity and swimming activity of the study
species were not altered for a week after testing. Significantly, it has been
elucidated that green-synthesized Ag NPs showed little or no toxicity against
a number of aquatic mosquito predators.
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4.5.1.2 Suitability index (SI)
Suitability index of different non-target organism over young instars of
An. stephensi, Ae. aegypti and Cx. quinquefasciatus exposed to M.
emarginata aqueous leaf extract and green-synthesized silver nanoparticles
are presented in table 34. SI indicated that M. emarginataaqueous leaf extract
and synthesized Ag NPs were less toxic to the non-target organism tested if
compared to the targeted mosquito larval populations.
4.5.2 Naregamia alata
The biotoxicity of N. alata aqueous leaf extract and synthesized silver
nanoparticles against several non-target organisms sharing the same
ecological niche of Anopheles, Aedes and Culex mosquito vectors are
presented in table 35 to 37.
4.5.2.1 Biotoxicity of N. alata aqueous leaf extract and Ag NPs against
non-target organism
The biotoxicity of N. alata aqueous leaf extract and green-synthesized
Ag NPs was evaluated on non-target organism A. bouvieri, D. indicus and G.
affinis are presented in table 35 and 36. The toxicity treatments achieved
negligible toxicity against A. bouvieri, D. indicus and G. affinis with LC50
values ranging from 10409.26 to 2111.34 μg/mL, respectively. Focal
observations highlighted that longevity and swimming activity of the study
species were not altered for a week after testing. Significantly, it has been
elucidated that green-synthesized Ag NPs showed little or no toxicity against
a number of aquatic mosquito predators.
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4.5.2.2 Suitability index (SI)
Suitability index of different non-target organism over young instars of
An. stephensi, Ae. aegypti and Cx. quinquefasciatus exposed to N.
alataaqueous leaf extract and green-synthesized silver nanoparticles are
presented in table 37. SI indicated that N. alata aqueous leaf extract and
synthesized Ag NPs were less toxic to the non-target organism tested if
compared to the targeted mosquito larval populations.
4.5.3 Hedyotis puberula
The biotoxicity of H. puberula aqueous leaf extract and synthesized
silver nanoparticles against several non-target organisms sharing the same
ecological niche of Anopheles, Aedes and Culex mosquito vectors are
presented in table 38 to 40.
4.5.3.1 Biotoxicity of H. puberula aqueous leaf extract and Ag NPs against
non-target organism
The biotoxicity of H. puberula aqueous leaf extract and green-
synthesized Ag NPs was evaluated on non-target organism A. bouvieri, D.
indicus and G. affinis are presented in table 38 and 39. The toxicity treatments
achieved negligible toxicity against A. bouvieri, D. indicus and G. affinis with
LC50 values ranging from 12364.98 to 2704.29 μg/mL, respectively. Focal
observations highlighted that longevity and swimming activity of the study
species were not altered for a week after testing. Significantly, it has been
elucidated that green-synthesized Ag NPs showed little or no toxicity against
a number of aquatic mosquito predators.
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4.5.3.2 Suitability index (SI)
Suitability index of three non-target organism over young instars of An.
stephensi, Ae. aegypti and Cx. quinquefasciatus exposed to H.
puberulaaqueous leaf extract and green-synthesized silver nanoparticles are
presented in table 40. SI indicated that H. puberulaaqueous leaf extract and
synthesized Ag NPs were less toxic to the non-target organism tested if
compared to the targeted mosquito larval populations.
4.5.4 Aglaia elaeagnoidea
The biotoxicity of A. elaeagnoideaaqueous leaf extract and synthesized
silver nanoparticles against several non-target organisms sharing the same
ecological niche of Anopheles, Aedes and Culex mosquito vectors are
presented in table 41 to 43.
4.5.4.1 Biotoxicity of A. elaeagnoideaaqueous leaf extract and Ag NPs
against non-target organism
The biotoxicity of A. elaeagnoideaaqueous leaf extract and green-
synthesized Ag NPs was evaluated on non-target organism A. bouvieri, D.
indicus and G. affinis are presented in table 41 and 42. The toxicity treatments
achieved negligible toxicity against A. bouvieri, D. indicus and G. affinis with
LC50 values ranging from 14654.69 to 3342.23μg/mL, respectively. Focal
observations highlighted that longevity and swimming activity of the study
species were not altered for a week after testing. Significantly, it has been
elucidated that green-synthesized Ag NPs showed little or no toxicity against
a number of aquatic mosquito predators.
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4.5.4.2 Suitability index (SI)
Suitability index of different non-target organism over young instars of
An. stephensi, Ae. aegypti and Cx. quinquefasciatus exposed to A.
elaeagnoideaaqueous leaf extract and green-synthesized silver nanoparticles
are presented in table 43. SI indicated that A. elaeagnoideaaqueous leaf
extract and synthesized Ag NPs were less toxic to the non-target organism
tested if compared to the targeted mosquito larval populations.
4.5.5 Ventilago madrasapatna
The biotoxicity of V. madrasapatna aqueous leaf extract and
synthesized silver nanoparticles against several non-target organisms sharing
the same ecological niche of Anopheles, Aedes and Culex mosquito vectors
are presented in table 44 to 46.
4.5.5.1 Biotoxicity of A. elaeagnoideaaqueous leaf extract and Ag NPs
against non-target organism
The biotoxicity of V. madrasapatna aqueous leaf extract and green-
synthesized Ag NPs was evaluated on non-target organism A. bouvieri, D.
indicus and G. affinis are presented in table 44 and 45. The toxicity treatments
achieved negligible toxicity against A. bouvieri, D. indicus and G. affinis with
LC50 values ranging from 18776.91 to 3757.73 μg/mL, respectively. Focal
observations highlighted that longevity and swimming activity of the study
species were not altered for a week after testing. Significantly, it has been
elucidated that green-synthesized Ag NPs showed little or no toxicity against
a number of aquatic mosquito predators.
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4.5.5.2 Suitability index (SI)
Suitability index of different non-target organism over young instars of
An. stephensi, Ae. aegypti and Cx. quinquefasciatus exposed to V.
madrasapatna aqueous leaf extract and green-synthesized silver nanoparticles
are presented in table 46. SI indicated that V. madrasapatna aqueous leaf
extract and synthesized Ag NPs were less toxic to the non-target organism
tested if compared to the targeted mosquito larval populations.
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Table 2. Larvicidal activity of Merremia emarginata aqueous leaf extract against Anopheles stephensi, Aedes aegypti
and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
70
140
210
280
350
27.6±0.8
45.8±1.2
68.3±0.4
86.5±0.6
100.0±0.0
146.91
(130.70-
161.33)
286.29
(265.65-
313.37)
3.02 y= 9.99+0.265x 5.278
(4)
n.s.
Ae. aegypti
70
140
210
280
350
24.2±0.4
42.8±0.6
65.3±1.2
83.6±0.8
98.4±0.6
157.87
(141.89-
172.36)
301.63
(280.09-
329.93)
2.76 y= 6.1+0.27x 3.084
(4)
n.s.
Cx.
quinquefasciatus
70
140
210
280
350
21.8±1.2
38.5±0.8
62.2±0.6
80.4±0.4
97.1±0.8
169.24
(153.59-
183.72)
315.81
(293.42-
345.28)
2.51 y= 2.25+0.275x 2.710
(4)
n.s.
a Values are mean±SD of five replicates, No mortality was observed in the control, SD = standard deviation.
LC50= lethal concentration that kills 50% of the exposed organisms.
LC90= lethal concentration that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit.
LCL= 95% lower confidence limit.
χ2= chi square, d.f. = degrees of freedom, n.s. = not significant (α=0.05).
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Table 3. Larvicidal activity of silver nanoparticles synthesized using Merremia emarginata leaf extract against
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
4
8
12
16
20
26.8±0.6
49.4±0.4
65.2±1.2
87.6±0.8
100.0±0.0
8.36
(7.43-9.18)
16.33
(15.15-17.88)
3.08 y=
10.42+4.615x
6.217
(4)
n.s.
Ae. aegypti
4
8
12
16
20
23.5±1.2
44.7±0.8
61.4±0.4
82.3±1.2
97.2±0.6
9.20
(8.26-10.06)
17.86
(16.55-19.59)
2.96 y= 6.32+4.625x 2.915
(4)
n.s.
Cx.
quinquefasciatus
4
8
12
16
20
19.4±0.8
40.8±1.2
57.5±0.4
78.3±0.6
96.2±1.2
10.02
(9.11-10.86)
18.62
(17.29-20.39)
2.48 y= 1.11+4.778x 3.130
(4)
n.s.
a Values are mean±SD of five replicates, No mortality was observed in the control, SD = standard deviation.
LC50= lethal concentration that kills 50% of the exposed organisms.
LC90= lethal concentration that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit.
LCL= 95% lower confidence limit.
χ2= chi square, d.f. = degrees of freedom, n.s. = not significant (α=0.05).
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Table 4. Larvicidal activity of Naregamia alata aqueous leaf extract against Anopheles stephensi, Aedes aegypti and
Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
80
160
240
320
400
26.9±0.6
47.5±0.8
69.7±1.2
88.3±0.4
99.5±0.8
165.15
(146.72-
181.50)
321.56
(298.52-
351.71)
3.01 y=10.58+0.233x 3.056
(4)
n.s.
Ae. aegypti
80
160
240
320
400
23.4±1.2
44.6±0.8
65.3±0.6
84.5±0.4
98.2±0.6
179.17
(160.93-
195.66)
342.21
(317.86-
374.16)
2.75 y=6.35+0.237x 2.349
(4)
n.s.
Cx. quinquefasciatus
80
160
240
320
400
20.6±0.4
41.2±1.2
59.8±0.8
78.3±0.6
96.10±0.4
196.48
(178.01-
213.56)
371.50
(344.48-
407.39)
2.64 y=2.77+0.235x 2.602
(4)
n.s.
a Values are mean±SD of five replicates, No mortality was observed in the control, SD = standard deviation.
LC50= lethal concentration that kills 50% of the exposed organisms.
LC90= lethal concentration that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit.
LCL= 95% lower confidence limit.
χ2= chi square, d.f. = degrees of freedom, n.s. = not significant (α=0.05).
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Table 5. Larvicidal activity of silver nanoparticles synthesized using Naregamia alata leaf extract against
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
6
12
18
24
30
28.5±1.2
46.3±0.6
67.8±0.8
88.5±0.4
100.0±0.0
12.40
(11.02-13.63)
24.20
(22.46-26.49)
3.04 y=10.66+3.087x 5.387
(4)
n.s.
Ae. aegypti
6
12
18
24
30
24.1±0.4
42.8±0.8
63.4±0.6
85.6±1.2
98.2±0.4
13.57
(12.22-14.79)
25.71
(23.89-28.09)
2.67 y=5.52+3.183x 2.929
(4)
n.s.
Cx. quinquefasciatus
6
12
18
24
30
21.3±0.6
38.6±0.4
60.2±1.2
81.4±0.8
96.5±0.6
14.84
(13.51-16.08)
27.49
(25.54-30.06)
2.45 y=1.64+3.22x 2.571
(4)
n.s.
a Values are mean±SD of five replicates, No mortality was observed in the control, SD = standard deviation.
LC50= lethal concentration that kills 50% of the exposed organisms.
LC90= lethal concentration that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit.
LCL= 95% lower confidence limit.
χ2= chi square, d.f. = degrees of freedom, n.s. = not significant (α=0.05).
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Table 6. Larvicidal activity of Hedyotis puberula aqueous leaf extract against Anopheles stephensi, Aedes aegypti and
Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
90
180
270
360
450
29.6±0.4
47.2±1.2
68.5±0.6
89.3±0.8
98.1±1.2
182.67
(160.41-
202.13)
369.97
(342.55-
406.15)
3.83 y=12.81+0.199x 2.225
(4)
n.s.
Ae. aegypti
90
180
270
360
450
24.8±1.2
43.5±0.6
66.4±0.8
85.9±0.4
97.3±1.2
199.14
(178.18-
217.99)
385.34
(357.65-
421.73)
2.92 y=7.36+0.208x 1.190
(4)
n.s.
Cx. quinquefasciatus
90
180
270
360
450
20.6±0.6
39.2±0.8
62.4±1.2
81.9±0.4
96.3±0.8
217.67
(197.73-
236.15)
404.10
(375.73-
441.33)
2.47 y=1.85+0.216x 1.140
(4)
n.s.
a Values are mean±SD of five replicates, No mortality was observed in the control, SD = standard deviation.
LC50= lethal concentration that kills 50% of the exposed organisms.
LC90= lethal concentration that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit.
LCL= 95% lower confidence limit.
χ2= chi square, d.f. = degrees of freedom, n.s. = not significant (α=0.05).
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106
Table 7. Larvicidal activity of silver nanoparticles synthesized using Hedyotis puberula leaf extract against Anopheles
stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
8
16
24
32
40
26.2±0.8
49.5±1.2
67.3±0.6
88.4±0.4
100.0±0.0
16.58
(14.75-18.19)
32.11
(29.81-35.11)
2.94 y=10.33+2.331x 4.962
(4)
n.s.
Ae. aegypti
8
16
24
32
40
22.8±0.8
45.2±0.6
63.5±1.2
84.3±0.4
99.1±0.6
18.05
(16.27-19.67)
34.04
(31.64-37.18)
2.62 y=5.47+2.396x 4.372
(4)
n.s.
Cx. quinquefasciatus
8
16
24
32
40
19.5±0.6
41.9±0.4
59.3±1.2
80.6±0.8
97.2±0.4
19.52
(17.75-21.15)
36.07
(33.54-39.40)
2.43 y=1.47+2.426x 3.170
(4)
n.s.
a Values are mean±SD of five replicates, No mortality was observed in the control, SD = standard deviation.
LC50= lethal concentration that kills 50% of the exposed organisms.
LC90= lethal concentration that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit.
LCL= 95% lower confidence limit.
χ2= chi square, d.f. = degrees of freedom, n.s. = not significant (α=0.05).
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107
Table 8. Larvicidal activity of Aglaia elaeagnoidea aqueous leaf extract against Anopheles stephensi, Aedes aegypti and
Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
100
200
300
400
500
27.9±1.2
48.6±0.8
66.4±0.6
87.3±0.4
100.0±0.0
207.06
(183.43-
227.94)
408.46
(378.71-
447.63)
3.23 y=11.17+0.183x 5.776
(4)
n.s.
Ae. aegypti
100
200
300
400
500
23.2±0.8
44.7±0.6
62.5±0.4
81.3±1.2
98.1±0.6
229.79
(206.42-
250.99)
442.71
(410.55-
485.27)
2.86 y=6.04+0.186x 4.038
(4)
n.s.
Cx. quinquefasciatus
100
200
300
400
500
20.7±0.4
41.5±0.6
58.3±0.8
77.2±1.2
97.6±0.8
246.43
(223.72-
267.52)
462.09
(428.71-
506.38)
2.56 y=2.21+0.19x 5.490
(4)
n.s.
a Values are mean±SD of five replicates, No mortality was observed in the control, SD = standard deviation.
LC50= lethal concentration that kills 50% of the exposed organisms.
LC90= lethal concentration that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit.
LCL= 95% lower confidence limit.
χ2= chi square, d.f. = degrees of freedom, n.s. = not significant (α=0.05).
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108
Table 9. Larvicidal activity of silver nanoparticles synthesized using Aglaia elaeagnoidea leaf extract against
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
10
20
30
40
50
26.9±0.6
47.3±0.4
69.7±1.2
88.2±0.8
100.0±0.0
20.66
(18.39-22.68)
39.94
(37.09-43.66)
2.91 y=10.29+1.871x 4.184
(4)
n.s.
Ae. aegypti
10
20
30
40
50
23.6±0.8
42.2±0.4
64.7±1.2
83.5±0.4
98.3±0.6
22.80
(20.55-24.86)
43.23
(40.16-47.26)
2.67 y=5.25+1.907x 2.978
(4)
n.s.
Cx. quinquefasciatus
10
20
30
40
50
19.7±1.2
38.2±0.8
60.5±0.6
79.3±0.4
96.1±1.2
24.91
(22.70-26.97)
45.96
(42.72-50.23)
2.42 y=0.59+1.939x 1.795
(4)
n.s.
a Values are mean±SD of five replicates, No mortality was observed in the control, SD = standard deviation.
LC50= lethal concentration that kills 50% of the exposed organisms.
LC90= lethal concentration that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit.
LCL= 95% lower confidence limit.
χ2= chi square, d.f. = degrees of freedom, n.s. = not significant (α=0.05).
Page 118
109
Table 10. Larvicidal activity of Ventilago madrasapatna aqueous leaf extract against Anopheles stephensi, Aedes
aegypti and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
120
240
360
480
600
28.2±1.2
47.5±0.8
69.3±0.6
88.1±1.2
100.0±0.0
245.46
(217.53-
270.17)
481.55
(446.76-
527.18)
3.13 y=11.36+0.154x 4.634
(4)
n.s.
Ae. aegypti
120
240
360
480
600
24.8±0.8
43.6±1.2
64.2±0.4
85.4±0.6
99.1±0.8
267.27
(240.31-
291.71)
507.89
(471.87-
555.09)
2.69 y=6.3+0.159x 4.238
(4)
n.s.
Cx. quinquefasciatus
120
240
360
480
600
21.6±0.6
39.2±0.8
61.3±1.2
80.7±0.4
97.5±1.2
289.86
(263.25-
314.51)
538.91
(500.86-
588.93)
2.48 y=2.07+0.161x 3.131(4)
n.s.
a Values are mean±SD of five replicates, No mortality was observed in the control, SD = standard deviation.
LC50= lethal concentration that kills 50% of the exposed organisms.
LC90= lethal concentration that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit.
LCL= 95% lower confidence limit.
χ2= chi square, d.f. = degrees of freedom, n.s. = not significant (α=0.05).
Page 119
110
Table 11. Larvicidal activity of silver nanoparticles synthesized using Ventilago madrasapatna leaf extract against
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
12
24
36
48
60
26.7±0.4
48.2±0.8
67.5±1.2
88.9±0.6
100.0±0.0
24.89
(22.17-27.31)
48.03
(44.61-52.51)
2.89 y=10.07+1.561x 4.849
(4)
n.s.
Ae. aegypti
12
24
36
48
60
23.9±1.2
44.5±0.8
63.2±0.6
85.7±0.4
98.3±1.2
26.92
(24.20-29.38)
51.26
(47.62-56.04)
2.72 y=6.12+1.583x 3.084
(4)
n.s.
Cx. quinquefasciatus
12
24
36
48
60
20.5±0.6
41.3±1.2
59.7±0.4
80.4±0.8
96.2±1.2
29.24
(26.52-31.76)
54.89
(50.96-60.09)
2.57 y=2.47+1.588x 2.042
(4)
n.s.
a Values are mean±SD of five replicates, No mortality was observed in the control, SD = standard deviation.
LC50= lethal concentration that kills 50% of the exposed organisms.
LC90= lethal concentration that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit.
LCL= 95% lower confidence limit.
χ2= chi square, d.f. = degrees of freedom, n.s. = not significant (α=0.05).
Page 120
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Table 12. Ovicidal activity of Merremia emarginata aqueous leaf extract against Anopheles stephensi, Aedes aegypti
and Culex quinquefasciatus.
Mosquito species
Age of the
egg raft/
eggs (h)
Egg hatchability (%)
Control 50
(µg/mL)
100
(µg/mL)
150
(µg/mL)
200
(µg/mL)
250
(µg/mL)
300
(µg/mL)
An. stephensi
0-6 100±0.0 28.6±1.5 16.3±1.8 NH NH NH NH
6-12 100±0.0 37.3±1.2 21.2±1.5 NH NH NH NH
12-18 100±0.0 45.4±1.8 36.4±1.6 17.6±1.2 NH NH NH
Ae. aegypti
0-6 100±0.0 47.7±1.2 39.2±1.5 20.4±1.3 NH NH NH
6-12 100±0.0 56.3±1.4 44.6±1.7 24.2±1.5 NH NH NH
12-18 100±0.0 67.3±1.3 53.8±1.5 33.6±1.2 19.5±1.6 NH NH
Cx. quinquefasciatus
0-6 100±0.0 68.7±1.5 44.6±1.4 27.3±1.6 16.5±1.7 NH NH
6-12 100±0.0 74.2±1.2 58.3±1.6 36.5±1.4 22.3±1.5 NH NH
12-18 100±0.0 85.2±1.5 74.6±1.8 52.8±1.3 35.4±1.7 19.6±1.4 NH
a Values are mean ± SD of five replicates
NH = no hatchability
Page 121
112
Table 13. Ovicidal activity of silver nanoparticles synthesized using the Merremia emarginata leaf extract against
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species
Age of the
egg raft/
eggs (h)
Egg hatchability (%)
Control 10
µg/mL
20
µg/mL
30
µg/mL
40
µg/mL
50
µg/mL
60
µg/mL
An. stephensi
0-6 100±0.0 27.6±1.6 16.4±1.2 NH NH NH NH
6-12 100±0.0 39.2±1.8 20.7±1.3 NH NH NH NH
12-18 100±0.0 47.2±1.3 28.3±1.5 16.4±1.9 NH NH NH
Ae. aegypti
0-6 100±0.0 39.9±1.2 28.5±1.4 19.7±1.8 NH NH NH
6-12 100±0.0 52.6±1.6 37.3±0.6 26.8±1.3 NH NH NH
12-18 100±0.0 68.7±1.5 51.3±1.7 36.2±0.4 17.4±1.6 NH NH
Cx. quinquefasciatus
0-6 100±0.0 69.4±1.4 44.2±1.2 27.7±1.6 16.6±1.2 NH NH
6-12 100±0.0 78.4±0.7 59.2±1.6 38.4±1.2 20.2±1.8 NH NH
12-18 100±0.0 84.7±1.3 72.3±0.7 52.2±1.8 32.6±1.4 19.4±1.6 NH
a Values are mean ± SD of five replicates
NH = no hatchability
Page 122
113
Table 14. Ovicidal activity of Naregamia alata aqueous leaf extract against Anopheles stephensi, Aedes aegypti and
Culex quinquefasciatus.
Mosquito species
Age of the
egg raft/
eggs (h)
Egg hatchability (%)
Control 60
µg/mL
120
µg/mL
180
µg/mL
240
µg/mL
300
µg/mL
360
µg/mL
An. stephensi
0-6 100±0.0 27.4±1.5 18.6±0.7 NH NH NH NH
6-12 100±0.0 37.4±1.9 22.8±1.2 NH NH NH NH
12-18 100±0.0 49.6±1.7 38.3±1.5 20.6±0.3 NH NH NH
Ae. aegypti
0-6 100±0.0 42.7±1.4 28.5±1.9 19.5±0.4 NH NH NH
6-12 100±0.0 56.3±1.8 39.4±1.5 22.5±1.7 NH NH NH
12-18 100±0.0 68.4±0.2 56.8±1.6 39.7±0.4 20.6±1.9 NH NH
Cx. quinquefasciatus
0-6 100±0.0 69.8±1.9 47.4±0.7 32.2±1.5 18.5±1.3 NH NH
6-12 100±0.0 75.3±1.5 62.8±0.3 45.6±1.8 27.2±0.6 NH NH
12-18 100±0.0 87.6±0.2 75.2±1.6 57.6±1.9 42.8±1.4 23.5±0.7 NH
a Values are mean ± SD of five replicates.
NH = no hatchability.
Page 123
114
Table 15. Ovicidal activity of silver nanoparticles synthesized using the Naregamia alata leaf extract against
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species
Age of the
egg raft/
eggs (h)
Egg hatchability (%)
Control 15
µg/mL
30
µg/mL
45
µg/mL
60
µg/mL
75
µg/mL
90
µg/mL
An. stephensi
0-6 100±0.0 28.5±1.6 16.7±1.9 NH NH NH NH
6-12 100±0.0 34.7±1.7 22.4±1.6 NH NH NH NH
12-18 100±0.0 46.1±0.2 34.8±0.3 20.6±1.5 NH NH NH
Ae. aegypti
0-6 100±0.0 41.3±1.7 27.4±1.6 18.7±1.8 NH NH NH
6-12 100±0.0 49.7±1.3 39.6±0.8 23.5±1.2 NH NH NH
12-18 100±0.0 64.6±1.8 53.9±1.4 38.4±1.9 21.4±0.6 NH NH
Cx. quinquefasciatus
0-6 100±0.0 65.8±1.9 48.3±1.6 26.9±1.4 19.2±1.8 NH NH
6-12 100±0.0 75.5±1.4 56.4±0.5 39.6±1.7 24.3±1.2 NH NH
12-18 100±0.0 87.9±1.2 69.6±1.3 58.2±0.5 37.5±1.6 23.7±1.4 NH
a Values are mean ± SD of five replicates.
NH = no hatchability.
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115
Table 16. Ovicidal activity of Hedyotis puberula aqueous leaf extract against Anopheles stephensi, Aedes aegypti and
Culex quinquefasciatus.
Mosquito species
Age of the
egg raft/
eggs (h)
Egg hatchability (%)
Control 70
µg/mL
140
µg/mL
210
µg/mL
280
µg/mL
350
µg/mL
420
µg/mL
An. stephensi
0-6 100±0.0 29.2±1.9 18.4±1.5 NH NH NH NH
6-12 100±0.0 36.7±1.3 20.6±1.7 NH NH NH NH
12-18 100±0.0 48.9±1.8 37.5±1.4 21.7±0.8 NH NH NH
Ae. aegypti
0-6 100±0.0 47.5±1.4 36.3±1.2 18.4±1.7 NH NH NH
6-12 100±0.0 58.3±1.6 45.8±1.3 21.7±1.9 NH NH NH
12-18 100±0.0 66.2±1.9 53.6±1.6 36.3±1.5 23.4±1.2 NH NH
Cx. quinquefasciatus
0-6 100±0.0 68.7±0.4 55.9±1.5 38.2±1.3 19.7±1.6 NH NH
6-12 100±0.0 76.9±1.2 67.8±1.4 43.5±0.6 23.8±1.3 NH NH
12-18 100±0.0 86.1±0.3 76.2±1.3 57.4±1.9 37.5±1.4 22.9±1.6 NH
a Values are mean ± SD of five replicates.
NH = no hatchability.
Page 125
116
Table 17. Ovicidal activity of silver nanoparticles synthesized using the Hedyotis puberula leaf extract against
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species
Age of the
egg raft/eggs
(h)
Egg hatchability (%)
Control 20
(µg/mL)
40
(µg/mL)
60
(µg/mL)
80
(µg/mL)
100
(µg/mL)
120
(µg/mL)
An. stephensi
0-6 100±0.0 28.4±1.6 16.3±1.7 NH NH NH NH
6-12 100±0.0 35.7±1.2 20.4±1.6 NH NH NH NH
12-18 100±0.0 44.8±0.5 29.3±1.9 18.4±1.8 NH NH NH
Ae. aegypti
0-6 100±0.0 49.2±1.3 28.7±1.5 19.5±1.7 NH NH NH
6-12 100±0.0 54.8±1.7 36.9±0.3 22.8±1.6 NH NH NH
12-18 100±0.0 59.4±1.8 45.6±1.5 32.6±1.9 21.4±0.8 NH NH
Cx. quinquefasciatus
0-6 100±0.0 56.7±1.3 48.9±1.8 27.9±1.3 19.6±1.7 NH NH
6-12 100±0.0 74.8±1.5 59.3±1.4 38.5±1.4 22.4±1.2 NH NH
12-18 100±0.0 86.6±1.2 69.4±1.2 56.3±1.5 34.8±0.6 19.5±1.3 NH
a Values are mean ± SD of five replicates.
NH = no hatchability.
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117
Table 18. Ovicidal activity of Aglaia elaeagnoidea aqueous leaf extract against Anopheles stephensi, Aedes aegypti and
Culex quinquefasciatus.
Mosquito species
Age of the
egg raft/
eggs (h)
Egg hatchability (%)
Control 80
(µg/mL)
160
(µg/mL)
240
(µg/mL)
320
(µg/mL)
400
(µg/mL)
480
(µg/mL)
An. stephensi
0-6 100±0.0 26.4±1.7 17.8±1.2 NH NH NH NH
6-12 100±0.0 35.7±1.9 20.4±1.5 NH NH NH NH
12-18 100±0.0 48.4±1.7 33.6±0.5 15.2±1.2 NH NH NH
Ae. aegypti
0-6 100±0.0 46.6±0.4 27.9±1.8 17.8±1.3 NH NH NH
6-12 100±0.0 57.8±1.2 38.2±1.4 20.4±0.5 NH NH NH
12-18 100±0.0 65.7±1.7 45.6±1.2 26.3±1.6 16.3±1.5 NH NH
Cx. quinquefasciatus
0-6 100±0.0 68.4±1.2 47.3±0.9 38.5±1.8 19.6±1.4 NH NH
6-12 100±0.0 75.3±1.8 58.6±1.7 45.9±1.4 23.8±1.6 NH NH
12-18 100±0.0 86.9±0.5 66.4±1.9 48.2±1.7 27.5±1.3 19.6±0.4 NH
a Values are mean ± SD of five replicates.
NH = no hatchability.
Page 127
118
Table 19. Ovicidal activity of silver nanoparticles synthesized using the Aglaia elaeagnoidea leaf extract against
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species
Age of the
egg raft/
eggs (h)
Egg hatchability (%)
Control 25
(µg/mL)
50
(µg/mL)
75
(µg/mL)
100
(µg/mL)
125
(µg/mL)
150
(µg/mL)
An. stephensi
0-6 100±0.0 27.3±1.7 15.4±1.8 NH NH NH NH
6-12 100±0.0 36.2±0.3 18.6±1.7 NH NH NH NH
12-18 100±0.0 48.5±1.9 32.9±1.3 19.5±0.6 NH NH NH
Ae. aegypti
0-6 100±0.0 44.5±1.9 27.7±1.9 16.8±1.2 NH NH NH
6-12 100±0.0 55.8±1.2 37.4±0.3 18.4±1.9 NH NH NH
12-18 100±0.0 68.9±1.4 59.6±1.7 42.7±1.4 20.8±0.5 NH NH
Cx. quinquefasciatus
0-6 100±0.0 67.5±1.3 47.5±1.4 26.8±1.6 17.3±1.8 NH NH
6-12 100±0.0 76.8±1.8 56.9±0.5 37.5±1.9 21.4±1.2 NH NH
12-18 100±0.0 87.3±14 67.9±0.2 56.8±1.5 38.4±0.6 20.3±1.9 NH
a Values are mean ± SD of five replicates.
NH = no hatchability.
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119
Table 20. Ovicidal activity of Ventilago madrasapatna aqueous leaf extract against Anopheles stephensi, Aedes aegypti
and Culex quinquefasciatus.
Mosquito species
Age of the
egg raft/
eggs (h)
Egg hatchability (%)
Control 100
(µg/mL)
200
(µg/mL)
300
(µg/mL)
400
(µg/mL)
500
(µg/mL)
600
(µg/mL)
An. stephensi
0-6 100±0.0 29.2±1.8 18.6±1.9 NH NH NH NH
6-12 100±0.0 38.7±0.2 21.5±1.3 NH NH NH NH
12-18 100±0.0 47.5±1.7 30.7±1.8 19.3±1.6 NH NH NH
Ae. aegypti
0-6 100±0.0 45.9±1.2 26.4±0.4 18.5±1.8 NH NH NH
6-12 100±0.0 56.2±1.3 39.4±1.6 21.2±0.5 NH NH NH
12-18 100±0.0 64.8±1.6 53.6±0.4 35.9±1.8 19.6±1.5 NH NH
Cx. quinquefasciatus
0-6 100±0.0 66.4±1.8 45.3±1.9 27.6±1.4 18.5±1.7 NH NH
6-12 100±0.0 78.5±0.7 59.4±1.8 35.7±1.6 20.4±1.3 NH NH
12-18 100±0.0 87.2±1.9 65.7±1.6 47.3±1.5 35.9±1.8 19.7±1.2 NH
a Values are mean ± SD of five replicates.
NH = no hatchability.
Page 129
120
Table 21. Ovicidal activity of silver nanoparticles synthesized using the Ventilago madrasapatna leaf extract against
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species
Age of the
egg raft/
eggs (h)
Egg hatchability (%)
Control 30
(µg/mL)
60
(µg/mL)
90
(µg/mL)
120
(µg/mL)
150
(µg/mL)
180
(µg/mL)
An. stephensi
0-6 100±0.0 27.8±1.9 17.6±1.2 NH NH NH NH
6-12 100±0.0 39.4±0.8 21.4±1.3 NH NH NH NH
12-18 100±0.0 44.2±0.3 32.8±1.9 19.4±1.7 NH NH NH
Ae. aegypti
0-6 100±0.0 45.5±1.8 27.7±1.2 17.3±1.9 NH NH NH
6-12 100±0.0 57.4±1.3 36.2±1.8 21.6±1.4 NH NH NH
12-18 100±0.0 64.9±1.8 54.3±0.5 34.8±1.6 19.5±1.2 NH NH
Cx. quinquefasciatus
0-6 100±0.0 66.2±1.7 462.8±1.3 25.4±1.2 18.6±1.9 NH NH
6-12 100±0.0 77.3±0.5 59.6±1.5 38.7±1.9 22.8±1.2 NH NH
12-18 100±0.0 86.9±1.4 68.2±0.9 47.3±1.5 36.4±1.8 20.6±0.7 NH
a Values are mean ± SD of five replicates
NH = no hatchability
Page 130
121
Table 22. Adulticidal activity of Merremia emarginata aqueous leaf extract against Anopheles stephensi, Aedes aegypti
and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LD50 (µg/ml)
(LCL-UCL)
LD90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
100
200
300
400
500
28.4±0.6
46.8±1.2
69.3±0.8
88.5±0.4
100.0±0.0
204.82
(181.71-
225.29)
400.39
(371.54-
438.20)
3.07 y=11.13+0.185x 4.729
(4)
n.s.
Ae. aegypti
100
200
300
400
500
24.9±0.6
42.6±1.2
65.4±0.8
83.1±0.6
99.2±0.4
224.44
(201.69-
245.06)
428.65
(398.02-
468.89)
2.75 y=6.31+0.189x 4.857
(4)
n.s.
Cx. quinquefasciatus
100
200
300
400
500
21.9±0.4
38.4±1.2
60.6±0.8
79.2±0.6
97.1±0.4
244.52
(222.00-
265.42)
457.27
(424.55-
500.50)
2.53 y=2.08+0.191x 3.424
(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
Page 131
122
Table 23. Adulticidal activity of silver nanoparticles synthesized using Merremia emarginata leaf extract against
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LD50 (µg/ml)
(LCL-UCL)
LD90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
12
24
36
48
60
28.4±0.8
47.6±0.6
66.2±1.2
89.5±0.4
100.0±0.0
24.73
(21.97-27.19)
48.24
(44.76-52.80)
3.04 y=10.81+1.543x 6.095
(4)
n.s.
Ae. aegypti
12
24
36
48
60
24.2±1.2
43.9±0.8
61.5±0.6
84.3±1.2
99.1±0.4
27.24
(24.54-29.70)
51.65
(47.98-56.49)
2.67 y=5.54+1.585x 5.419
(4)
n.s.
Cx. quinquefasciatus
12
24
36
48
60
19.6±0.8
38.2±0.6
56.8±1.2
81.4±0.4
97.1±0.8
30.02
(27.46-32.43)
54.40
(50.64-59.32)
2.28 y=-0.84+1.652x 3.367
(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
Page 132
123
Table 24. Adulticidal activity of Naregamia alata aqueous leaf extract against Anopheles stephensi, Aedes aegypti and
Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LD50 (µg/ml)
(LCL-UCL)
LD90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
120
240
360
480
600
27.6±1.2
46.8±0.8
68.5±0.4
89.4±0.6
100.0±0.0
247.59
(220.51-
271.71)
477.69
(443.64-
522.21)
2.88 y=10.24+0.156x 4.732
(4)
n.s.
Ae. aegypti
120
240
360
480
600
23.9±0.6
42.5±0.8
63.2±1.2
86.4±0.4
98.6±0.8
271.25
(244.83-
295.35)
508.78
(473.14-
555.34)
2.56 y=4.93+0.161x 3.502
(4)
n.s.
Cx. quinquefasciatus
120
240
360
480
600
19.8±0.4
38.2±1.2
59.6±0.8
82.3±0.6
96.1±1.2
296.43
(270.37-
320.78)
542.55
(504.85-
591.92)
2.35 y=0.19+0.164x 1.372
(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
Page 133
124
Table 25. Adulticidal activity of silver nanoparticles synthesized using Naregamia alata leaf extract against
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LD50 (µg/ml)
(LCL-UCL)
LD90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
15
30
45
60
75
26.4±1.2
48.2±0.8
65.1±0.6
88.3±0.4
100.0±0.0
31.60
(28.19-34.65)
60.94
(56.59-66.65)
2.88 y=9.41+1.249x 6.038
(4)
n.s.
Ae. aegypti
15
30
45
60
75
23.7±0.4
45.3±0.8
60.1±1.2
83.5±0.6
98.2±0.8
34.31
(30.84-37.46)
65.89
(61.12-72.19)
2.822 y=6+1.248x 4.767
(4)
n.s.
Cx. quinquefasciatus
15
30
45
60
75
19.6±0.6
41.8±0.4
56.3±1.2
78.1±0.8
96.4±0.6
37.52
(34.11-40.69)
70.06
(64.99-76.77)
2.52 y=1.47+1.266x 4.132
(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
Page 134
125
Table 26. Adulticidal activity of Hedyotis puberula aqueous leaf extract against Anopheles stephensi, Aedes aegypti and
Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LD50 (µg/ml)
(LCL-UCL)
LD90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
130
260
390
520
650
28.4±1.2
46.6±0.8
67.3±0.6
88.2±0.4
100.0±0.0
269.40
(239.27-
296.15)
526.61
(488.57-
576.56)
3.07 y=10.66+0.142x 5.528
(4)
n.s.
Ae. aegypti
130
260
390
520
650
23.8±0.8
42.5±0.6
63.1±1.2
84.6±0.4
99.2±0.6
295.65
(266.99-
321.82)
554.42
(515.48-
605.37)
2.56 y=4.77+0.148x 4.734
(4)
n.s.
Cx. quinquefasciatus
130
260
390
520
650
20.5±0.4
37.2±0.6
59.4±1.2
81.6±0.8
97.1±0.6
321.29
(293.34-
347.47)
585.42
(544.88-
638.46)
2.32 y=-0.12+0.152x 2.703
(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
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Table 27. Adulticidal activity of silver nanoparticles synthesized using Hedyotis puberula leaf extract against
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LD50 (µg/ml)
(LCL-UCL)
LD90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
16
32
48
64
80
27.3±0.8
48.5±0.6
66.2±1.2
89.4±0.4
100.0±0.0
33.11
(29.46-36.35)
64.17
(59.58-70.19)
2.94 y=10.39+1.164x 5.728
(4)
n.s.
Ae. aegypti
16
32
48
64
80
23.4±0.6
44.8±1.2
62.1±0.8
84.3±0.4
98.2±1.2
36.34
(32.69-39.66)
69.35
(64.39-75.87)
2.74 y=5.83+1.182x 3.517
(4)
n.s.
Cx.
quinquefasciatus
16
32
48
64
80
20.1±0.4
41.6±0.6
57.4±0.8
79.5±1.2
96.2±0.6
39.56
(35.93-42.93)
74.08
(68.74-81.14)
2.54 y=1.93+1.188x 3.054
(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
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127
Table 28. Adulticidal activity of Aglaia elaeagnoidea aqueous leaf extract against Anopheles stephensi, Aedes aegypti
and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LD50 (µg/ml)
(LCL-UCL)
LD90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
140
280
420
560
700
29.6±1.2
47.2±0.8
68.4±0.4
87.3±0.6
100.0±0.0
285.48
(251.84-
315.05)
569.66
(527.81-
624.83)
3.46 y=12.23+0.129x 5.509
(4)
n.s.
Ae. aegypti
140
280
420
560
700
25.3±0.8
43.9±1.2
65.4±0.6
82.1±0.4
98.2±0.8
312.93
(279.50-
342.92)
612.39
(567.50-
671.86)
3.09 y=7.78+0.131x 3.518
(4)
n.s.
Cx.
quinquefasciatus
140
280
420
560
700
21.6±0.6
39.1±0.4
60.4±1.2
78.3±0.8
96.5±0.4
343.96
(311.88-
373.68)
648.16
(601.19-
710.45)
2.61 y=2.48+0.135x 2.960
(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
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128
Table 29. Adulticidal activity of silver nanoparticles synthesized using Aglaia elaeagnoidea leaf extract against
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LD50 (µg/ml)
(LCL-UCL)
LD90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. stephensi
18
36
54
72
90
27.4±0.8
48.1±1.2
66.5±0.4
87.3±0.6
100.0±0.0
37.52
(33.33-41.25)
73.46
(68.15-80.45)
3.10 y=10.54+1.024x 5.591
(4)
n.s.
Ae. aegypti
18
36
54
72
90
23.7±0.6
44.3±0.8
62.2±1.2
84.6±0.4
99.1±0.8
40.74
(36.72-44.40)
76.99
(71.55-84.15)
2.64 y=5.45+1.062x 4.927
(4)
n.s.
Cx. quinquefasciatus
18
36
54
72
90
19.6±1.2
40.2±0.8
57.3±0.6
80.5±0.4
97.8±1.2
44.55
(40.67-48.18)
81.25
(75.60-88.65)
2.32 y=0.07+1.093x 4.462
(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
Page 138
129
Table 30. Adulticidal activity of Ventilago madrasapatna aqueous leaf extract against Anopheles stephensi, Aedes
aegypti and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LD50 (µg/ml)
(LCL-UCL)
LD90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. Stephensi
150
300
450
600
750
28.6±0.8
46.2±1.2
69.8±0.6
88.4±0.4
100.0±0.0
307.24
(272.59-
337.93)
600.45
(557.19-
657.12)
3.07 y=11.1+0.123x 4.766
(4)
n.s.
Ae. Aegypti
150
300
450
600
750
25.4±0.6
42.8±0.4
65.3±1.2
84.2±0.8
98.5±0.6
334.46
(299.80-
365.73)
644.34
(597.98-
705.35)
2.86 y=6.96+0.125x 3.392
(4)
n.s.
Cx.
quinquefasciatus
150
300
450
600
750
21.9±0.8
38.3±1.2
62.6±0.6
79.2±0.4
97.1±0.8
363.82
(330.00-
395.11)
681.56
(632.89-
745.75)
2.55 y=2.43+0.128x 3.210
(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
Page 139
130
Table 31. Adulticidal activity of silver nanoparticles synthesized using Ventilago madrasapatna leaf extract against
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus.
Mosquito species Concentration
(µg/ml)
Mortality
(%)± SDa
LD50 (µg/ml)
(LCL-UCL)
LD90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
An. Stephensi
20
40
60
80
100
28.9±0.6
46.3±0.4
67.4±1.2
89.2±0.6
100.0±0.0
41.19
(36.58-45.28)
80.39
(74.59-87.99)
3.05 y=10.83+0.926x 5.744
(4)
n.s.
Ae. Aegypti
20
40
60
80
100
25.6±1.2
42.9±0.8
63.2±0.6
84.1±0.4
99.3±0.8
44.85
(40.29-48.98)
85.79
(79.64-93.89)
2.77 y=6.44+0.943x 5.573
(4)
n.s.
Cx. quinquefasciatus
20
40
60
80
100
21.8±0.6
38.5±0.4
59.2±1.2
80.6±0.8
97.1±0.4
48.94
(44.49-53.08)
90.99
(84.52-99.50)
2.48 y=1.63+0.964x 3.298
(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
Page 140
131
Table 32. Biotoxicity of Merremia emarginata aqueous leaf extract against several non-target organisms sharing the
same ecological niche of Anopheles, Aedes and Culex mosquito vectors.
Non-
target
organism
Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
A.bouvieri
4000
8000
12000
16000
20000
27.5±0.6
48.3±0.4
65.8±1.2
88.4±0.8
100.0±0.0
8317.45
(7392.91-9138.72)
16221.12
(15051.07-17757.04)
3.03 y=
10.47+0.005x
5.929
(4)
n.s.
D.indicus
6000
12000
18000
24000
30000
25.3±1.2
47.8±0.8
63.4±0.6
86.2±0.4
96.5±0.8
13081.08
(11599.57-
14395.18)
26130.69
(24190.39-38705.80)
3.48 y= 9.6+0.003x 1.841
(4)
n.s.
G.affinis
12000
24000
36000
48000
60000
27.9±0.6
46.3±0.4
68.2±1.2
85.4±0.8
100.0±0.0
25153.46
(22314.96-
27668.87)
49552.80
(45943.75-54305.78)
3.21 y=
10.57+0.002x
5.668
(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
Page 141
132
Table 33 Biotoxicity of green-synthesized silver nanoparticles using the Merremia emarginata leaf extract against
several non-target organisms sharing the same ecological niche of Anopheles, Aedes and Culex mosquito vectors.
Non-
target
organism
Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
A.bouvieri
200
400
600
800
1000
28.6±0.8
45.3±0.6
67.9±1.2
88.4±0.4
100.0±0.0
415.61
(369.71-456.46)
808.24
(750.09-884.45)
2.99 y= 10.27+0.093x 5.571(4)
n.s.
D.indicus
300
600
900
1200
1500
27.5±1.2
48.3±0.8
64.7±0.6
86.4±1.2
98.2±0.4
633.51
(559.29-698.87)
1274.56
(1179.75-1400.29)
3.65 y= 11.17+0.06x 3.249(4)
n.s.
G.affinis
500
1000
1500
2000
2500
25.9±0.6
47.3±0.8
66.5±1.2
88.4±0.4
100.0±0.0
1056.04
(944.44-1156.09)
2017.83
(1874.99-2204.43)
2.76 y= 8.83+0.038x 5.117(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
Page 142
133
Table 34. Biotoxicity of Naregamia alata aqueous leaf extract against several non-target organisms sharing the same
ecological niche of Anopheles, Aedes and Culex mosquito vectors.
Non-
target
organism
Concentratio
n (µg/ml)
Mortalit
y (%)±
SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
A.bouvieri
5000
10000
15000
20000
25000
28.3±1.2
46.4±0.8
67.2±0.6
87.6±0.4
100.0±0.0
10409.26
(9246.17-
11441.93)
20368.43
(18894.54-
22304.61)
3.09 y=10.52+0.004x 5.593(4)
n.s.
D.indicus
7000
14000
21000
28000
35000
26.9±0.6
48.3±1.2
65.4±0.8
88.6±0.6
100.0±0.0
14644.29
(13046.01-
16068.81)
28355.31
(26322.43-
31019.99)
2.93 y=9.89+0.003x 6.010(4)
n.s.
G.affinis
14000
28000
42000
56000
70000
27.4±0.4
46.2±0.6
65.7±1.2
87.3±0.8
100.0±0.0
29633.74
(26419.26-
32504.24)
57454.95
(53322.36-
62879.81)
2.95 y=9.43+0.001x 5.991(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
Page 143
134
Table 35. Biotoxicity of green-synthesized silver nanoparticles using the Naregamia alata leaf extract against several
non-target organisms sharing the same ecological niche of Anopheles, Aedes and Culex mosquito vectors.
Non-
target
organism
Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
A.bouvieri
300
600
900
1200
1500
26.5±0.4
48.2±1.2
65.7±0.6
88.4±0.8
100.0±0.0
629.58
(561.45-690.39)
1214.95
(1128.14-1328.66)
2.89 y=9.6+0.062x 5.722(4)
n.s.
D.indicus
500
1000
1500
2000
2500
27.4±0.4
43.8±0.6
67.3±1.2
89.2±0.8
100.0±0.0
1058.31
(948.12-1157.48)
2011.14
(1869.25-2196.24)
2.69 y=8.36+0.038x 5.692(4)
n.s.
G.affinis
1000
2000
3000
4000
5000
25.7±0.6
48.5±0.8
66.3±1.2
87.2±0.4
100.0±0.0
2111.34
(1883.65-2314.64)
4073.07
(3782.26-4454.01)
2.88 y=9.35+0.019x 5.376(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
Page 144
135
Table 36. Biotoxicity of Hedyotis puberula aqueous leaf extract against several non-target organisms sharing the
same ecological niche of Anopheles, Aedes and Culex mosquito vectors.
Non-
target
organism
Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL)
Slope Regression
equation
χ2
(d.f.)
A.bouvieri
6000
12000
18000
24000
30000
29.6±1.2
45.8±0.8
67.4±0.6
88.2±0.4
100.0±0.0
12364.98
(10955.09-
13612.47)
24372.65
(22597.61-
26707.44)
3.21 y=11.24+0.003x 6.068(4)
n.s.
D.indicus
9000
18000
27000
36000
45000
27.2±0.8
48.3±1.2
66.9±0.6
87.1±0.4
100.0±0.0
18747.76
(16647.76-
20610.21)
36708.68
(34053.35-
40197.65)
3.10 y=10.58+0.002x 5.406(4)
n.s.
G.affinis
16000
32000
48000
64000
80000
26.4±0.4
47.2±0.8
68.9±1.2
86.3±0.6
100.0±0.0
33552.24
(29871.31-
36828.76)
65127.06
(60459.27-
71243.83)
2.97 y=9.87+0.001x 4.805(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
Page 145
136
Table 37. Biotoxicity of green-synthesized silver nanoparticles using the Hedyotis puberula leaf extract against
several non-target organisms sharing the same ecological niche of Anopheles, Aedes and Culex mosquito vectors.
Non-
target
organism
Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
A.bouvieri
500
1000
1500
2000
2500
26.4±1.2
48.2±0.8
66.5±0.6
87.9±0.4
100.0±0.0
1048.08
(934.09-1149.72)
2026.20
(1881.29-2216.01)
2.91 y=9.73+0.037x 5.303(4)
n.s.
D.indicus
800
1600
2400
3200
4000
28.5±0.6
45.8±0.8
67.3±1.2
89.2±0.6
100.0±0.0
1658.82
(1476.45-1821.24)
3216.53
(2985.72-3518.85)
2.95 y=10.24+0.023x 5.722(4)
n.s.
G.affinis
1300
2600
3900
5200
6500
29.3±0.8
46.9±1.2
65.1±0.6
87.4±0.4
100.0±0.0
2704.29
(2394.52-2978.18)
5360.04
(4966.61-5879.30)
3.32 y=11.17+0.014x 6.906(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
Page 146
137
Table 38. Biotoxicity of Aglaia elaeagnoidea aqueous leaf extract against several non-target organisms sharing the
same ecological niche of Anopheles, Aedes and Culex mosquito vectors.
Non-target
organism
Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
A.bouvieri
7000
14000
21000
28000
35000
27.5±0.6
45.9±1.2
68.2±0.8
87.4±0.4
100.0±0.0
14654.69
(13051.59-
16083.37)
28415.43
(26377.81-31085.26)
2.95 y=9.85+0.003x 5.069(4)
n.s.
D.indicus
10000
20000
30000
40000
50000
26.7±0.8
48.1±0.4
65.3±1.2
89.6±0.4
100.0±0.0
20915.77
(18669.86-
22923.53)
40172.88
(37313.66-43912.42)
2.82 y=9.51+0.002x 6.130(4)
n.s.
G.affinis
18000
36000
54000
72000
90000
28.2±0.4
46.7±0.6
68.3±0.8
87.5±1.2
100.0±0.0
37254.45
(33060.53-
40971.79)
73001.03
(67720.66-79933.92)
3.11 y=10.82+0.001x 5.136(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
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Table 39. Biotoxicity of green-synthesized silver nanoparticles using the Aglaia elaeagnoidea leaf extract against
several non-target organisms sharing the same ecological niche of Anopheles, Aedes and Culex mosquito vectors.
Non-
target
organism
Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
A.bouvieri
600
1200
1800
2400
3000
27.6±0.4
46.9±0.6
67.5±1.2
88.3±0.8
100.0±0.0
1247.71
(1110.33-1369.97)
2421.84
(2248.06-2649.53)
2.97 y=10.2+0.031
x
5.150(4)
n.s.
D.indicus
900
1800
2700
3600
4500
25.3±1.2
47.9±0.8
68.2±0.6
87.4±0.4
100.0±0.0
1895.03
(1693.02-2075.74)
3629.89
(3372.92-3965.49)
2.79 y=9.09+0.021
x
4.483(4)
n.s.
G.affinis
1600
3200
4800
6400
8000
28.5±0.6
46.9±1.2
64.3±0.8
89.2±0.4
100.0±0.0
3342.23
(2973.66-3670.37)
6509.16
(6038.94-7126.82)
3.01 y=10.19+0.01
2x
7.321(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
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Table 40. Biotoxicity of Ventilago madrasapatna aqueous leaf extract against several non-target organisms sharing the
same ecological niche of Anopheles, Aedes and Culex mosquito vectors.
Non-
target
organism
Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
A.bouvieri
9000
18000
27000
36000
45000
26.8±1.2
47.3±0.8
68.2±0.6
87.6±0.4
100.0±0.0
18776.91
(16720.89-20607.89)
36364.11
(33762.61-
39770.21)
2.93 y=9.97+0.002x 4.728(4)
n.s.
D.indicus
12000
24000
36000
48000
60000
28.4±0.6
46.9±1.2
65.3±0.8
88.2±0.4
100.0±0.0
25056.82
(22266.55-27536.31)
49015.44
(45463.26-
53685.73)
3.09 y=10.41+0.002x 6.501(4)
n.s.
G.affinis
20000
40000
60000
80000
100000
27.9±0.8
46.2±0.6
65.7±1.2
89.4±0.8
100.0±0.0
41854.07
(37332.15-45894.81)
80708.99
(74937.82-
88265.61)
2.88 y=9.62+0.001x 6.301(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
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Table 41. Biotoxicity of green-synthesized silver nanoparticles using the Ventilago madrasapatna leaf extract against
several non-target organisms sharing the same ecological niche of Anopheles, Aedes and Culex mosquito vectors.
Non-
target
organism
Concentration
(µg/ml)
Mortality
(%)± SDa
LC50 (µg/ml)
(LCL-UCL)
LC90 (µg/ml)
(LCL-UCL) Slope
Regression
equation χ
2 (d.f.)
A.bouvieri
800
1600
2400
3200
4000
25.8±0.8
47.3±0.6
68.2±1.2
89.1±0.4
100.0±0.0
1673.72
(1497.06-1832.07)
3184.99
(2960.78-3477.19)
2.71 y=9.02+0.024x 4.317(4)
n.s.
D.indicus
1000
2000
3000
4000
5000
28.4±0.6
46.3±0.8
65.7±1.2
87.2±0.4
100.0±0.0
2099.08
(1865.15-2306.98)
4114.97
(3815.98-4508.51)
3.12 y=10.29+0.018x 6.328(4)
n.s.
G.affinis
1800
3600
5400
7200
9000
26.5±0.4
48.3±0.6
66.7±1.2
88.4±0.8
100.0±0.0
3757.73
(3348.76-4122.31)
7257.47
(6739.16-7935.93)
2.90 y=9.85+0.01x 5.204(4)
n.s.
a Values are mean±SD of five replicates. No mortality was observed in the control. SD = standard deviation.
LD50= lethal dose that kills 50% of the exposed organisms.
LD90= lethal dose that kills 90% of the exposed organisms.
UCL= 95% upper confidence limit. LCL= 95% lower confidence limit.
χ2= chi square. d.f. = degrees of freedom. n.s. = not significant (α=0.05).
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Table 42. Suitability index of different non-target organism over young instars of Anopheles stephensi, Aedes aegypti
and Culex quinquefasciatus exposed to Merremia emarginata aqueous leaf extract and green-synthesized silver
nanoparticles.
Treatment Non-target organism An.stephensi Ae.aegypti Cx.quinquefasciatus
A.bouvieri 56.61 52.68 49.14
Aqueous leaf extract D.indicus 89.04 82.85 77.29
G.affinis 171.21 159.33 148.62
A.bouvieri 49.71 45.14 41.47
Silver nanoparticles D.indicus 75.77 68.80 63.22
G.affinis 126.32 114.69 105.39
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Table 43. Suitability index of different non-target organism over young instars of Anopheles stephensi, Aedes aegypti
and Culex quinquefasciatus exposed to Naregamia alata aqueous leaf extract and green-synthesized silver
nanoparticles.
Treatment Non-target organism An.stephensi Ae.aegypti Cx.quinquefasciatus
A.bouvieri 63.02 58.09 52.97
Aqueous leaf extract D.indicus 88.67 81.73 74.53
G.affinis 179.43 165.39 150.82
A.bouvieri 50.77 46.39 42.42
Silver nanoparticles D.indicus 85.34 77.98 71.31
G.affinis 170.26 155.58 142.27
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Table 44. Suitability index of different non-target organism over young instars of Anopheles stephensi, Aedes aegypti
and Culex quinquefasciatus exposed to Hedyotis puberula aqueous leaf extract and green-synthesized silver
nanoparticles.
Treatment Non-target organism An.stephensi Ae.aegypti Cx.quinquefasciatus
A.bouvieri 67.69 62.09 56.80
Aqueous leaf extract D.indicus 102.63 94.14 86.12
G.affinis 183.67 168.48 154.14
A.bouvieri 63.21 58.06 53.69
Silver nanoparticles D.indicus 100.04 91.90 84.98
G.affinis 163.10 149.82 138.53
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Table 45. Suitability index of different non-target organism over young instars of Anopheles stephensi, Aedes aegypti
and Culex quinquefasciatus exposed to Aglaia elaeagnoidea aqueous leaf extract and green-synthesized silver
nanoparticles.
Treatment Non-target organism An.stephensi Ae.aegypti Cx.quinquefasciatus
A.bouvieri 70.77 63.77 59.46
Aqueous leaf extract D.indicus 101.01 91.02 84.87
G.affinis 179.92 162.12 151.17
A.bouvieri 60.39 54.72 50.08
Silver nanoparticles D.indicus 91.72 83.11 76.07
G.affinis 161.77 146.58 134.17
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Table 46. Suitability index of different non-target organism over young instars of Anopheles stephensi, Aedes aegypti
and Culex quinquefasciatus exposed to Ventilago madrasapatna aqueous leaf extract and green-synthesized silver
nanoparticles.
Treatment Non-target organism An.stephensi Ae.aegypti Cx.quinquefasciatus
A.bouvieri 76.49 70.25 64.77
Aqueous leaf extract D.indicus 102.08 93.75 86.44
G.affinis 170.51 156.59 144.39
A.bouvieri 67.24 62.17 57.24
Silver nanoparticles D.indicus 84.33 77.97 71.78
G.affinis 150.97 139.58 128.51
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Fig. 1. (a) Color intensity of Merremia emarginata aqueous extract before and after
the reduction of silver nitrate (1mM). The color change indicates Ag+ reduction to
elemental nanosilver (Ag NPs). (b) UV-visible spectrum of silver nanoparticles after
180 min from the reaction.
(a)
Ag NPs Ag NO3 Merremia
emarginata extract
( b )
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Fig. 2. (a) Color intensity of Naregamia alata aqueous extract before and after the
reduction of silver nitrate (1mM). The color change indicates Ag+ reduction to
elemental nanosilver. (b) UV-visible spectrum of silver nanoparticles after 180 min
from the reaction.
(a)
( b ) A
g N
Ps
Ag
NO
3
Nare
ga
mia
ala
ta
extr
act
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Fig. 3. (a) Color intensity of the Hedyotis puberula aqueous extract before and after
the reduction of silver nitrate (1mM). The color change indicates Ag+ reduction to
elemental nanosilver. (b) UV-visible spectrum of silver nanoparticles after 180 min
from the reaction.
(a
)
AgNPs
AgNO3 Hedyotis
puberula extract
( b )
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Fig. 4. (a) Color intensity of the Aglaia elaeagnoidea aqueous extract before and
after the reduction of silver nitrate (1mM). The color change indicates Ag+ reduction
to elemental nanosilver (Ag NPs). (b) UV-visible spectrum of silver nanoparticles
after 180 min from the reaction.
(a
)
AgNPs
AgNO3 Aglaia
elaeagnoidea
extract
( b
)
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Fig. 5. (a) Color intensity of Ventilago maderaspatana aqueous extract before and
after the reduction of silver nitrate (1mM). The color change indicates Ag+ reduction
to elemental nanosilver (Ag NPs). (b) UV-visible spectrum of silver nanoparticles
after 180 min from the reaction.
( b )
(a)
AgN
Ps
AgN
O3
V. m
ader
asp
ata
na
extr
act
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Fig. 6. FTIR spectrum of silver nanoparticles biofabricated using the Merremia
emarginata aqueous leaf extract.
Fig. 7. FTIR spectrum of silver nanoparticles fabricated using the Naregamia alata
aqueous leaf extract.
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A1
Thu Aug 11 14:58:27 2016 (GMT+05:30)
41
7.2
447
0.5
0
61
7.5
9
66
7.1
5
79
1.3
1
86
9.2
9
11
14
.35
13
84
.41
14
55
.14
14
70
.68
15
05
.23
15
38
.98
15
56
.21
15
74
.44
17
30
.54
17
46
.75
23
58
.43
28
53
.13
29
22
.58
34
17
.98
36
26
.10
36
46
.26
36
67
.83
36
86
.65
37
08
.35
37
31
.96
37
42
.91
37
66
.23
37
98
.83
38
14
.49
38
34
.75
38
50
.60
38
61
.53
38
98
.43
8
9
10
11
12
13
14
15
16
17
18
19
%T
500 1000 1500 2000 2500 3000 3500 4000
Wavenumbers (cm-1)
A2
Thu Aug 11 15:05:55 2016 (GMT+05:30)
40
5.1
941
6.4
242
7.5
543
7.7
846
3.8
2
61
6.0
366
8.4
9
77
7.8
8
86
4.2
4
10
65
.65
14
15
.41
14
70
.73
15
56
.42
23
59
.33
29
22
.65
33
82
.37
36
26
.51
36
87
.07
37
08
.68
37
32
.35
37
43
.14
37
99
.01
38
15
.15
38
35
.51
38
50
.70
38
97
.85
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
%T
500 1000 1500 2000 2500 3000 3500 4000
Wavenumbers (cm-1)
Fig. 8. FTIR spectrum of silver nanoparticles biofabricated using the Hedyotis
puberula aqueous leaf extract.
Fig. 9. FTIR spectrum of silver nanoparticles biofabricated using the Aglaia
elaeagnoidea aqueous leaf extract.
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A3
Thu Aug 11 14:56:28 2016 (GMT+05:30)
40
2.9
441
8.1
942
5.8
546
9.0
7
61
5.3
4
10
77
.88
13
84
.16
15
67
.61
33
81
.65
36
26
.68
36
67
.56
37
08
.49
37
31
.44
37
43
.30
38
50
.62
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
%T
500 1000 1500 2000 2500 3000 3500 4000
Wavenumbers (cm-1)
Fig. 10. FTIR spectrum of silver nanoparticles biofabricated using the Ventilago
maderaspatana aqueous leaf extract.
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Fig. 11a. Scanning electron microscopy (SEM) of Merremia emarginata-fabricated
silver nanoparticles.
Fig. 11b. Energy dispersive X-ray (EDX) spectrum of Merremia emarginata-synthesized
silver nanoparticles showing presence of different phyto-elements as capping agents.
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Fig. 12a. Scanning electron microscopy (SEM) of Naregamia alata-fabricated
silver nanoparticles.
Fig. 4.12b. Energy dispersive X-ray (EDX) spectrum of Naregamia alata-synthesized
silver nanoparticles showing presence of different phyto-elements as capping agents.
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Fig. 13a. Scanning electron microscopy (SEM) of Hedyotis puberula-fabricated
silver nanoparticles.
Fig. 13b. Energy dispersive X-ray (EDX) spectrum of Hedyotis puberula-synthesized
silver nanoparticles showing the presence of different phyto-elements as capping agents.
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Fig. 14a. Scanning electron microscopy (SEM) of Aglaia elaeagnoidea-fabricated
silver nanoparticles.
Fig. 14b. Energy dispersive X-ray (EDX) spectrum of Aglaia elaeagnoidea -synthesized
silver nanoparticles showing presence of different phyto-elements as capping agents.
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Fig. 15a. Scanning electron microscopy (SEM) of Ventilago maderaspatana-
fabricated silver nanoparticles.
Fig. 15b. Energy dispersive X-ray (EDX) spectrum of Ventilago maderaspatana-
synthesized silver nanoparticles showing presence of different phyto-elements as capping
agents.
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Fig. 16. Trasmission electron microscopy (TEM) of silver nanoparticles
biofabricated using the Merremia emarginata aqueous leaf extract.
Fig. 17. Trasmission electron microscopy (TEM) of silver nanoparticles fabricated
using the Naregamia alata aqueous leaf extract.
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Fig. 18. Trasmission electron microscopy (TEM) of silver nanoparticles
biofabricated using the Hedyotis puberula aqueous leaf extract.
Fig. 19. Trasmission electron microscopy (TEM) of silver nanoparticles
biofabricated using the Aglaia elaeagnoidea aqueous leaf extract.
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Fig. 20. Trasmission electron microscopy (TEM) of silver nanoparticles
biofabricated using the Ventilago maderaspatana aqueous leaf extract.
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Fig. 21. XRD pattern of silver nanoparticles biofabricated using the Merremia
emarginata aqueous leaf extract.
Fig.22. XRD pattern of silver nanoparticles fabricated using the Naregamia alata
aqueous extract.
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Fig. 23. XRD pattern of silver nanoparticles biofabricated using the Hedyotis
puberula aqueous leaf extract.
Fig. 24. XRD pattern of silver nanoparticles biofabricated using the Aglaia
elaeagnoidea aqueous leaf extract.
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Fig. 25. XRD pattern of silver nanoparticles biofabricated using the Ventilago
maderaspatana aqueous leaf extract.
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Fig. 26. AFM micrograph of silver nanoparticles biofabricated using the Merremia
emarginata extract (a) 2.5 μm resolution studies 0 to 40 nm size, spherical shaped,
polydispersed particles, (b) 3D image of Ag nanoparticles analyzed by NOVA-TX
software.
(a)
(b)
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Fig. 26. AFM micrograph of silver nanoparticles biofabricated using the Merremia
emarginata extract (c) histogram showing the particle size distribution, (d) line
graph showing the size distribution of green-synthesized Ag nanoparticles.
(c)
(d)
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Fig. 27. AFM micrograph of synthesized silver nanoparticles biofabricated using the
Naregamia alata extract (a) 2.5 μm resolution studies 0 to 5.5 nm size, spherical
shaped, polydispersed particles, (b) 3D image of AgNPs analyzed by NOVA-TX
software.
( a )
( b )
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Fig. 27. AFM micrograph of synthesized silver nanoparticles biofabricated using the
Naregamia alata extract (c) histogram showing the particle size distribution, (d) line
graph showing the size distribution of green-synthesized silver nanoparticles.
( c )
( d )
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Fig. 28. AFM micrograph of silver nanoparticles biofabricated using the Hedyotis
puberula extract (a) 2.5 μm resolution studies 0 to 7 nm size, spherical shaped, poly-
dispersed particles, (b) 3D image of Ag nanoparticles analyzed by NOVA-TX
software,
(a)
(b)
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Fig. 28. AFM micrograph of silver nanoparticles biofabricated using the Hedyotis
puberula extract (c) histogram showing the particle size distribution, (d) line graph
showing the size distribution of green-synthesized Ag nanoparticles.
(c)
(d)
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Fig. 29. AFM micrograph of silver nanoparticles biofabricated using the Aglaia
elaeagnoidea extract (a) 2.5 μm resolution studies 0 to 6 nm size, spherical shaped,
poly-dispersed particles, (b) 3D image of Ag nanoparticles analyzed by NOVA-TX
software.
(a)
(b)
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Fig. 29. AFM micrograph of silver nanoparticles biofabricated using the Aglaia
elaeagnoidea extract (c) histogram showing the particle size distribution, (d) line
graph showing the size distribution of green-synthesized Ag nanoparticles.
(c)
(d)
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Fig. 30. AFM micrograph of silver nanoparticles biofabricated using the Ventilago
maderaspatana extract (a) 2.5 μm resolution studies 0 to 6 nm size, spherical
shaped, polydispersed particles, (b) 3D image of Ag nanoparticles analyzed by
NOVA-TX software.
(a)
(b)
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Fig. 30. AFM micrograph of silver nanoparticles biofabricated using the Ventilago
maderaspatana extract (c) histogram showing the particle size distribution, (d) line
graph showing the size distribution of green-synthesized Ag nanoparticles.
(c)
(d)
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5. DISCUSSION
Mosquito borne diseases have an economic impact, including loss in
commercial and labor outputs, particularly in countries with tropical and
subtropical climates; however, no part of the world is free from vector-borne
diseases (Benelli, 2016). Mosquitoes are the most important single group of
insects in terms of public health importance, which transmit a number of
diseases, such as malaria, filariasis, dengue, Japanese encephalitis, etc.
causing millions of deaths every year.
Repeated use of synthetic insecticides for mosquito control has
disrupted natural biological control systems and led to resurgences in
mosquito populations. It has also resulted in the development of resistance,
undesirable effects on non-target organisms, and fostered environmental and
human health concern. Chemical control methods using synthetic insecticides
are in practice due to their speedy action and ease of application. Use of
chemical agents however results in environmental degradation in addition to
accumulation of toxicants as residual deposits in non-target species. The Ag
NPs which are less like lyto cause ecological damage have been identified as
potential replacement of synthetic chemical insecticides, hence the need to use
green synthesized silver nanoparticles for the control of disease vectors
(Benelli, 2015a). Use of plant extract for the synthesis of nanoparticles could
be advantageous over other environmentally benign biological processes by
eliminating the elaborate process of maintaining cell cultures. Biological
synthesis of nanoparticles has received increased attention due to a growing
need to develop environmentally benign technologies in material synthesis.
Several plant species have been utilized in this regard.
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In our observation, the plant-mediated synthesis of silver nanoparticles
using Merremia emarginata, Naregamia alata, Hedyotis puberula, Aglaia
elaeagnoidea, and Ventilago madrasapatna against Anopheles stephensi,
Aedes aegypti, and Culex quinquefasciatus. Furthermore, we evaluated the
biotoxicity of M. emarginata, N. alata, H. puberula, A. elaeagnoidea, and V.
madrasapatna aqueous extract and biosynthesized Ag NPs on three non-
target aquatic organisms sharing the same ecological niche of Anopheles,
Aedes and Culex mosquitoes, Diplonychus indicus, Anisops bouvieri, and
Gambusia affinis.
5.1 PLANT-MEDIATED SYNTHESIS AND CHARACTERIZATION
OF MOSQUITOCIDAL NANOPARTICLES
Different analyses were carried out to characterize the biosynthesized
silver nanoparticles. The biosynthesis of metal nanoparticles often exploits the
reducing and stabilizing potential of plant extracts and metabolites. Two main
factors influence the size, shape, and stability of nanoparticles, namely, the
concentration of the plant extract/metabolite and the substrate (metal ions)
concentration (Rajan et al., 2015).
5.1.1 UV–vis analysis of Phyto-synthesized Ag NPs
The leaf extract from five plant (M. emarginata, N. alata, H. puberula,
A. elaeagnoidea, and V. madrasapatna) under study showed rapid conversion
of silver nitrate into silver nanoparticles indicated by distinct color changes
from yellowish to dark brownish within 6 hours of plant extract addition in
AgNO3 (1 mM). The UV absorption spectrum of silver nanoparticles as a
function of reaction time is shown. There was maximum absorption between
430 to 460 nm and this absorption is the unique property of metal
nanoparticles, namely, surface plasmon resonance; it arises as a result of the
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conduction of electrons on the surface of Ag NPs. With an increase in gum
concentration, there is an enhancement in the nanoparticle synthesis. After
adding leaves extract in AgNO3 solution, the biomolecules are stabilized in a
medium. The biomolecules interact with each other and with silver salt; after
the initial interaction, silver salt is consumed, and the process of nucleation,
reduction, and capping starts, leading nanoparticles synthesis. The presence of
broad resonance indicated an aggregated structure of the Ag NPs in the
solution.
For a large number of mosquitocidal nanoparticles synthesized using
plant extracts, it has been showed that the color intensity of the plant extract
incubated with the aqueous solution of metal ions usually changed from
yellowish/pale brown to reddish/dark brown. In the majority of cases, a
maximum absorption peak is observed between 350 and 450 nm, after 60 min
or more. The absorption peak varies as the function of reaction time and
concentration of metal ions. As the size of ultrafine particles decreases, the
energy gap is widened; hence, the absorption peaks shifted towards a higher
energy (Suresh et al., 2015).
The surface plasmon peak of Ag NPs at 420 nm increases steadily as
the reaction time increases, and the peak gets saturated after 120 min,
indicating that silver nitrate is completely reduced (Madhiyazhagan et al.,
2015). Lallawmawma et al. (2015) showed that the Jasminum nervosum-
mediated biosynthesis of Au NPs evoked a maximum absorbance peak at 550
nm. Similarly, silver nanoparticles synthesized using leaf extract of A. indica
showed an average size of 20–30 nm (Krishnaraj et al., 2010). Forough and
Farhadi, (2010) noticed that the absorption spectra of Acanthe phylum
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bracteatum-synthesized Ag NPs obtained by UV–vis spectroscopy showed an
intense peak at 438 nm, which progressively decreased while nanometer
increased.
SPR peak is sensitive to the size and shape of the nanoparticles,
amount of extract, silver nitrate concentration, and the type of biomolecules
present in the leaf extract (Singh et al., 2010). The color change may be
attributed to the excitation of surface plasmon resonance (SPR) in metal
nanoparticles (Natarajan et al., 2010). The control Ag NO3 solution (without
peel extract) showed no change in colour and these results indicates the
abiotic reduction of AgNO3 did not occur under the reduction conditions. It is
generally recognized that UV–vis spectroscopy could be used to examine size
and shape nano particles in aqueous suspensions (Srivastava et al., 2010). In
particular, Ag NPs display an optical absorption band peaking at 3 keV (due
to surface plasmon resonance), which is typical of the absorption of metallic
silver nanocrystallites (Shahverdi et al., 2007).
Biosynthesized metal nanoparticles have free electrons, which give rise
to a SPR absorption band, due to the combined vibration of electrons of metal
nanoparticles in resonance with the light wave (Noginov et al., 2006). As a
general trend, the shape of plant-mediated silver nanoparticles has been
described as spherical, with the exception of those synthesized using neem
plant material, which leads to nanoparticles with spherical or flat morphology
(size 5–35 nm) (Shankar et al., 2004). The Ag NPs were observed as stable in
the aqueous medium and also showed little aggregation. Plasmon bands were
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broadened with an absorption tail at longer wavelengths, and this could be
related to the size distribution of Ag NPs (Ahmad et al., 2003).
5.1.2 FTIR analysis of synthesized silver nanoparticles
The Ag NPs results of FTIR studies (M. emarginata, N. alata, H.
puberula, A. elaeagnoidea, and V. madrasapatna) showed that the functional
groups of the diverse metabolites react with metal ions and reduced their size
into nano range. The FTIR spectrum of S. muticum-synthesized Ag NPs
showed peaks at 3408.02 cm−1
(amine and/or amides N–H stretch), 2272.01
cm−1
(acid C–O stretch), 1637.47 cm−1
(alkene and/ or aromatic ring C=C
stretch), 1095.50 cm−1
(ester C–O stretch), and 601.75 cm−1
(alkyl halide C–
Cl stretch) (Madhiyazhagan et al., 2016). Moreover, it has been elucidated
that the mentioned functional groups acted as capping agents around the
biosynthesized metal nanoparticles, providing stability as well as
biocompatibility (Rajan et al., 2015).
For instance, the FTIR spectrum of Ag NPs fabricated using the P.
niruri leaf extract showed transmittance peaks at 3327.63, 2125.87, 1637.89,
644.35, 597.41, and 554.63 cm-1
, indicating that the carbonyl groups from
amino acid residues probably acted as capping agents on nanoparticles,
preventing agglomeration, thereby stabilizing the medium (Suresh et al.,
2015). A further example is the synthesis of Au NPs using the lemongrass leaf
extract; Au NPs showed FTIR peaks at 1448.54 cm-1
(C=C stretch nitro
groups of aromatics), 1643.35 cm-1
(C=O stretch amides), 2360.87 cm-1
(P–H
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stretch phosphines), and 2856.58 cm-1
and 2918.30 cm-1
(bending carboxylic
acids) (Murugan et al., 2015b).
FTIR spectra of Ag NPs exhibited prominent peaks between 1384.58
and 1640.17 cm−1
that show the presence of alkane, alkene, and alkyl groups
in the sample which is similar to the earlier report of Veerakumar et al.
(2014). Peaks close to 1381 cm−1
may be related to C–N stretching of the
aromatic amine groups (Mahitha et al., 2011). FTIR spectroscopy was used to
shed light on the different functional groups from plant-borne molecules
(flavonoids, triterpenoids and polyphenols) that may act as reducing and
capping agents of the bio-fabricated Ag NPs (Asmathunisha et al., 2010).
The peaks at 1,620–1,636 cm−1
represent carbonyl groups from
polyphenols such as catechin gallate, epicatechin gallate, epigallocatechin,
epigallocatechin gallate, gallocatechin gallate, and theaflavin; the results
suggest that molecules attached with Ag NPs have free and bound amide
groups. These amide groups may also be in the aromatic rings. This concludes
that the compounds attached with the Ag NPs could be polyphenols with an
aromatic ring and bound amide region (Kumar et al., 2010b). Furthermore,
the peaks at 1027–1092 cm-1
corresponded to the C–N stretching vibration of
aliphatic amines or to alcohols/phenols, representing the presence of
polyphenols (Song et al., 2009). In detail, peaks close to 3402 cm−1
may be
related with N–H stretching frequency arising from peptide linkages from
proteins of the extract (Mukherjee et al., 2008).
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In addition, the absorbance peaks at 802 cm−1
, which represent the C–
H vibration in the aromatic ring, probably indicate the involvement of free
catechins (Krishnan and Maru, 2006). FTIR highlighted the presence of
different functional groups, including alkane, alkene, methylene, amine, and
carboxylic acid, previously reported as reducing agents in the biosynthesis of
Ag NPs (Cho et al., 2005).
5.1.3 SEM and EDX analysis of synthesized silver nanoparticles
The scanning electron microscope was employed to analyse the
structure of the silver nanoparticles. The SEM image of five plants (M.
emarginata, N. alata, H. puberula, A. elaeagnoidea, and V. madrasapatna)
with the synthesized silver nanoparticles confirmed to the formation of
nanoparticles capped with its biomoieties. The SEM shows that the Ag NPs
biosynthesized using the leaf extract of P. aquilinum were mostly spherical in
shape with the size measured at 35–65 nm (Panneerselvam et al., 2016). The
SEM image of C. scalpelliformis-synthesized Ag NPs was mono-dispersed
with spherical and cubic structures and mean size of 20–35 nm (Murugan
et al., 2015a).
Madhiyazhagan et al. (2015) studied the S. muticum-mediated
synthesis of Ag NPs, indicating that they were well dispersed and with a size
range of 43–79 nm. Murugan et al. (2015d) reported that A. indica leaf extract
can be used for effective biosynthesis of Ag NPs with 20–35 nm in size. The
Ag NPs synthesized using plant extracts are often surrounded by a thin layer
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of “capping” organic material from the plant leaf extract that play a key role
in stabilizing Ag NPs over time (Dinesh et al., 2015).
C. citratus-synthesized gold nanoparticles showed spherical,
triangular, hexagonal, and rod shapes, with size ranging from 20 to 50 nm
(Murugan et al., 2015b). Comparable morphological characteristics of metal
nanoparticles employed for different purposes have been obtained via plant-
mediated synthesis with aqueous extracts from different plant species
(Benelli, 2015a). Vivek et al. (2011) observed that the SEM analysis of silver
nanoparticles synthesized by the help of Gelidiella acerosa extract having
average mean size of the silver nanoparticles and seems to be spherical in
morphology.
The SEM analysis by Khandelwal et al. (2010) showed the particles
between 25 and 50 nm as well as the cubic structure of the nanoparticles.
Ankanna et al. (2010) reported the biogenic silver nanoparticles produced
using Boswellia ovalifoliolata stem bark were well-dispersed and with a mean
size of 30–40 nm. Chandran et al. (2006) reported that the SEM image
showed relatively spherical shape nanoparticles formed with diameter range
48–67 nm. Furthermore, SEM images of Ag NPs fabricated using Emblica
officinalis were also predominantly spherical with an average size of 16.8 nm
ranging from 7.5 to 25 nm (Ankamwar et al., 2005). A. indica synthesized
nanoparticles were reported to be spherical and plate like in morphology
ranging in size from 5 to 35 nm in size (Shankar et al., 2004).
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The EDX pattern showed the chemical composition of synthesized Ag
NPs. It shows the presence of pure silver and other elements confirming the
biosynthesis of Ag NPs. The sharp optical absorption band peak at 3keV is a
typical absorption range of metallic silver nano-crystallites (Kaviya et al.,
2011). The EDX attachments present in the SEM were known to provide
information on the chemical analysis of fields being investigated (Fayaz et al.
2009). The EDX profile showed a strong elemental signal along with oxygen,
which may have originated from the biomolecules bound to the surface of the
nanoparticles (Jea and Beom, 2009). EDX analysis confirmed the presence of
silver, while the oxygen signal indicated that the extracellular organic
material was probably adsorbed on the Ag NPs surface. The two peaks
located at about 3 kV were typical of the absorption of metallic silver nano
crystallites (Shahverdi et al., 2007).
5.1.4 TEM analysis of synthesized silver nanoparticles
The size and morphology of Ag NPs were determined by transmission
electron microscopy (TEM) images. The TEM analysis was done to analyse
the size and the shape of the biogenically stabilized Ag nano clusters shows
that most of the nano-crystals formed from five plants (M. emarginata, N.
alata, H. puberula, A. elaeagnoidea, and V. madrasapatna) extract are
spherical in shape, largely uniform with a moderate variation in particle size.
According to size distribution, most of nanoparticles ranged from 20 to 60
nm.
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The TEM of Ag NPs synthesized using B. cristata leaf extract among
shapes, spheres, triangle, truncated triangles, and decahedral morphologies
dominate and ranged from 38 to 41 nm with an average size of 39 nm
(Govindarajan and Benelli, 2016b). TEM analysis showed different shapes of
M. elengi-synthesized Ag NPs, including spherical, round and hexagonal
ones, with a mean size ranging from 25 to 40 nm (Subramaniam et al., 2016).
TEM micrograph of Au NPs displays both triangle and spherical NPs with
size ranging from 2–20 nm (Lallawmawma et al., 2015). More generally,
similar morphological features have been obtained from green synthesis
protocols using several terrestrial and marine plants, with mean nanoparticle
size ranging from 20 to 60 nm (Roni et al. 2013).
Naresh kumar et al. (2013) reported that the gold NPs mostly exist in
spherical, hexagonal, triangular, and rod shape. Higher concentration of
extract offers to more number of spherical particles with sizes from 10 to 60
nm with an average particle size of 20 to 30 nm. Thirunavokkarasu et al.
(2013) reported spherical nanoparticles with size ranging from 8 to 90 nm
using Desmodium gangeticum as a reducing agent. HR-TEM were utilized to
characterize the particle size, shape, and distribution by taking micrograph
from drop coated films of the Ag NPs that shows most of them are spherical
in shape with the average size of 20 to 53 nm similar to Haldar et al. (2013).
Ag NPs from A. squamosa leaf extract was spherical in shape with an
average size ranging from 20 to 100 nm (Vivek et al., 2012). Soni and
Prakash, (2012) shows typical TEM micrographs of C. tropicum silver
nanoparticles at 20–50 nm. Silver nanoparticles synthesized using C. lunatus
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biomass was filtered (0.22 μm filter assembly) and centrifuged at 20,000 rpm
for 10 min and further characterized by TEM. The bright field TEM
micrograph showed spherical silver nanoparticles with size ranging between 3
and 21 nm (Salunkhe et al., 2011). Nanoparticles mostly exist in the
hexagonal shape structure when concentration of plant extract is low. When
the concentration of the plant extract is medium, the conversion of gold
nanoparticles from hexagonal to triangular forms increased (Kasthuri et al.,
2009). TEM images of silver nanoparticles from E. officinalis were also
predominantly spherical with an average size of 16.8 nm ranging from 7.5 to
25 nm (Ankamwar et al., 2005). The shape of plant-mediated Ag NPs was
mostly spherical with the exception of A. indica which yielded both spherical
and flat plate-like morphology (Shankar et al., 2004).
5.1.5 X-ray diffraction (XRD) of synthesized silver nanoparticles
The XRD pattern is carried out to study the crystalline nature of
biosynthesized mosquitocidal nanoparticles of Ag NPs. The XRD patterns of
Ag NPs synthesized of Bragg reflections with 2θ values of five plants (M.
emarginata, N. alata, H. puberula, A. elaeagnoidea, and V. madrasapatna)
were assigned to the (111), (200), (220) and (311) sets of crystalline planes
are observed which may be indexed as the band for face centered cubic
structures of silver. The XRD analysis showed intense peaks at 2θ values of
28.42°, 38.21°, 44.38°, 64.51°, and 77.49° corresponding to (38), (117), (46),
(25), and (37) Bragg’s reflection based on the face centered cubic structure of
Ag NPs (Govindarajan et al., 2016a). The XRD pattern exhibited size
dependent features, with a number of Bragg reflections corresponding to the
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(111), (200), (220), (311), and (222) sets of lattice planes. On this basis, it has
been pointed out that the Ag NPs formed by the reduction of AgNO3 by P.
niruri leaf extract were crystalline in nature, and the sharp Bragg’s peaks
were probably due to the capping agent stabilizing the MNP (Suresh et al.,
2015).
With regards to gold nanoparticles (Au NPs), the XRD pattern of C.
citratus-synthesized Au nanostructures showed peaks corresponding to (111),
(200), and (220) Bragg’s reflection based on the face-centered cubic structure
of Au NPs. Again, XRD highlighted that the nanoparticles formed by the
reduction of HAuCl4 with C. citratus leaf extract were crystalline in nature
(Murugan et al., 2015b). Krishnaraj et al. (2012) reported that the XRD
patterns of vacuum-dried Ag NPs synthesized using on hydroponically grown
Bacopa monnieri and the numbers of Bragg reflections with 2θ values were
38.1° (111), 44.3° (200), and 64.4° (220). XRD patterns of Ag NPs
synthesized from seed extract of Medicago sativa showed distinct diffraction
peaks at 38.32°, 44.48°, 64.68°, and 77.64° corresponding to the respective
(111), (200), (220) and (31 1) crystalline planes (Lukman et al., 2011).
The XRD patterns of Ag NPs synthesized using leaf extract of E.
prostrata and the number of Bragg reflections with 2θ values of 38.06, 44.35,
64.51, and 77.36° sets of lattice planes are observed which may be indexed to
the (111), (200), (220), and (311) facts of silver, respectively (Rajakumar and
Rahuman, 2011). Sathyavathi et al. (2010) reported diffraction peaks at
38.10°, 28.00°, and 24.40° 2θ, which correspond to the (111), (85), and (84)
facets of the face centered cubic crystal structure. Bar et al. (2009) reported
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that the XRD analysis of synthesized Ag NPs using latex of J. curcas extract
showed four distinct diffractions 38.03, 46.18, 63.43, and 77.18° which
indexed to the (111), (200), (220), and (311) facets of silver, respectively.
The XRD pattern showed that the green-synthesized Ag NPs formed
by the reduction of AgNO3 using B. cylindrica leaf extract were crystalline in
nature. The sharp Bragg’s peaks reported above might have resulted due to
the capping agent stabilizing the Ag NPs. Therefore, XRD results suggest that
crystallization of the bioorganic phase occurred on the surface of the Ag NPs
(Bar et al., 2009). Also, Dubey et al. (2009) noted that the size of silver
nanocrystals as estimated from the full width at half-maximum of (111) peak
of silver using the Scherrer’s formula was 20– 60 nm. The XRD pattern of
pure silver ions is known to display peaks at 2θ = 7.9°, 11.4°, 17.8°, 30.38°
and 44° (Gong et al., 2007).
5.2 Larvicidal activity
From the results of the present study, the plants which were tested
against three important mosquito species for the larvicidal activity were
grouped into two categories based on their LC50 values. The plant species M.
emarginata showed the LC50 values 170 µg/mL (<170) (Ag NPs 10 µg/mL),
the plant species N. alata and H. puberula, exhibited the LC50 values below
250µg/mL (<250) (Ag NPs 20 µg/mL). Based on these LC50 values with
Anopheles, Aedes and Culex, species, M. emarginata are group under the
categories of more effective plants species, N. alata and H. puberula are
categorized under moderately effective plants species, A. elaeagnoidea, and
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V. madrasapatna plants grouped under less effective plants species. Among
the five aqueous leaf extracts and Ag NPs tested the larvae of An.stephensi,
Ae.aegypti and Cx.quinquefasciatus, larvae of An.stephensi were more
susceptible than the larvae of Ae.aegypti and Cx.quinquefasciatus. The
highest larvicidal efficacy was noticed in Ag NPs of M. emarginata (8.36
µg/mL) followed by N. alata (12.40 µg/mL), H. puberula (16.58 µg/mL), A.
elaeagnoidea (20.66 µg/mL) and V. madrasapatna (24.89 µg/mL).
In latest years, a growing number of plant-synthesized Ag NPs has
been studied for their excellent larvicidal activity against important mosquito
vectors. Govindarajan et al. (2016e) reported that the acute toxicity of Malva
sylvestris leaf extract and green-synthesized Ag NPs were effective against
larvae of the malaria vector An. stephensi, the dengue vector Ae. aegypti and
the filariasis vector Cx. quinquefasciatus. Compared to the leaf aqueous
extract, Ag NPs showed higher toxicity against An. stephensi, Ae. aegypti, and
Cx. quinquefasciatus with LC50 values of 10.33, 11.23, and 12.19 μg/mL,
respectively. The B. cristata-synthesized Ag NPs and aqueous leaf extract
showed larvicidal properties against third instar larvae of the mosquito vectors
An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus; LC50 values of
synthesized Ag NP were 12.46, 13.49, and 15.01 μg/mL, respectively and
aqueous leaf extract LC50 values were 124.27, 135.32, and 146.31 μg/mL,
respectively (Govindarajan and Benelli, 2016b).
The neem cake (A. indica) extract and the biosynthesized Ag NPs were
tested for acute toxicity against larvae and pupae of the dengue vector Ae.
aegypti. LC50 values of aqueous extract were 106.53 ppm (I instar), 121.95
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ppm (II), 148.50 ppm (III), 189.16 ppm (IV), and 235.36 ppm (pupae)
respectively, and LC50 of Ag NPs were 3.969 ppm (I), 4.528 ppm (II), 5.425
ppm (III), 6.711 ppm (IV), and 8.308 ppm (pupae) respectively
(Chandramohan et al., 2016). The LC50 and LC90 values of B. variegata
aqueous leaf extract appeared to be effective against An. subpictus (LC50
132.78 μg/mL and LC90 261.25 μg/mL), followed by Ae. albopictus (LC50
145.08 μg/mL and LC90 278.36 μg/mL), and Cx. tritaeniorhynchus (LC50
157.24 μg/mL and LC90 299.87 μg/mL). Ag NPs against the vector
mosquitoes of An. subpictus LC50 and LC90 values were 41.96 and 82.93
μg/mL; Ae. albopictus LC50 and LC90 values were 46.16 and 89.42 μg/mL;
Cx. tritaeniorhynchus LC50 and LC90 values were 51.92 and 97.12 μg/mL
(Govindarajan et al., 2016a).
Murugan et al. (2016b) documented the synthesized Ag NPs with C.
clavulatum against dengue vector Ae. aegypti; the LC50 values of C.
clavulatum extract against A. aegypti larvae and pupae were 269.361 ppm
(larva I), 309.698 ppm (larva II), 348.325 ppm (larva III), 387.637 ppm (larva
IV), and 446.262 ppm (pupa) respectively. LC50 values of C. clavulatum-
synthesized Ag NPs were 21.460 ppm (larva I), 23.579 ppm (larva II), 25.912
ppm (larva III), 29.155 ppm (larva IV), and 33.877 ppm (pupa) respectively.
The acute toxicity of biosynthesized Ag NPs with C. spinarum leaf extract
was evaluated against larvae of the malaria vector An. subpictus, the dengue
vector Ae. albopictus and the Japanese encephalitis vector Cx.
tritaeniorhynchus; Ag NPs showed higher toxicity against An. subpictus, Ae.
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albopictus, and Cx. tritaeniorhynchus with LC50 values of 8.37, 9.01 and
10.04 μg/mL, respectively (Govindarajan et al., 2016c).
Mahesh Kumar et al. (2016) reported that the leaf extract of B.
tinctoria was toxic against larval instars (I–IV) and pupae of the arbovirus
vector Ae. albopictus. LC50 values were 182.72 ppm (I instar larvae), 230.99
ppm (II), 269.65 ppm (III), 321.75 ppm (IV), and 359.71 ppm (pupae),
respectively. Ag NPs synthesized from the leaf extract of B. tinctoria was
highly effective against Ae. albopictus young instars, with LC50 of 4.97 ppm
(I), 5.97 ppm (II), 7.60 ppm (III), 9.65 ppm (IV), and 14.87 ppm (pupae). In
mosquitocidal assays, LC50 of P. aquilinum leaf extract against An. stephensi
larvae and pupae were 220.44 ppm (larva I), 254.12 ppm (II), 302.32 ppm
(III), 395.12 ppm (IV), and 502.20 ppm (pupa). LC50 of P. aquilinum-
synthesized Ag NPs were 7.48 ppm (I), 10.68 ppm (II), 13.77 ppm (III), 18.45
ppm (IV), and 31.51 ppm (pupa) (Panneerselvam et al., 2016).
Govindarajan et al. (2016c) evaluated the acute toxicity of essential oil
from P. barbatus and its major constituents, against larvae of An. subpictus,
Ae. albopictus and Cx. tritaeniorhynchus, with 50 % lethal concentration
(LC50) values of 84.20, 87.25 and 94.34 μg/ml and 90% lethal concentration
(LC90) values of 165.25, 170.56 and 179.58μg/mL, respectively. Concerning
major constituents, eugenol, α- pinene and β-caryophyllene appeared to be
most effective against An. subpictus (LC50=25.45, 32.09 and 41.66 μg/mL,
respectively), followed by Ae. albopictus (LC50=28.14, 34.09 and
44.77μg/mL, respectively) and Cx. tritaeniorhynchus (LC50=30.80, 36.75 and
48.17μg/mL, respectively).
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Rajasekharreddy and Rani (2015) determined that the Ag NPs
produced using the seed extract of S. foetida showed mosquitocidal activity
against IV instar larvae of Ae. aegypti (LC50=67.75 mg/ml), An. stephensi
(LC50= 57.36 mg/ml), and Cx. quinquefasciatus (LC50=71.54 mg/ml). Besides
Aedes and Anopheles mosquitoes, low doses of Ag NPs were also toxic
against other species, such as the filariasis mosquito Cx. quinquefasciatus. A
good example is the toxic activity of C. scalpelliformis-synthesized Ag NP,
which had LC50 values from 3.08 ppm (I) to 7.33 ppm (pupae) against Cx.
quinquefasciatus (Murugan et al., 2015a). Low doses of M. elengi-
synthesized Ag NPs showed larvicidal and pupicidal toxicity against the
malaria vector An. stephensi and the arbovirus vector Ae. albopictus. The
LC50 value ranged from synthesized Ag NPs against An. stephensi, 12.53 (I)
to 23.55 ppm (pupa) and LC50 against Ae. albopictus ranged from 11.72 ppm
(I) to 21.46 ppm (pupa) (Subramaniam et al., 2015).
Madhiyazhagan et al. (2015) reported that the Ag NPs synthesized
using the aqueous extract of the seaweed S. muticum were tested against An.
stephensi, Ae. aegypti, and Cx. quinquefasciatus. Maximum acute toxicity
was observed against An. stephensi young instars, with LC50 ranging from
16.156 ppm (I) to 28.881 ppm (pupa). Arokiyaraj et al. (2015) studied the
larvicidal activity of Ag NPs synthesized from the floral extract of C. indicum
against An. stephensi, with LC50 values of 5.07 ppm (I), 10.35 ppm (II), 14.19
ppm (III), 22.81 ppm (IV) and 35.05 ppm (pupae). It should be noted that
other green-synthesized Ag NPs were effective at lower dosages than those in
the present study.
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Suresh et al. (2015) documented that Ag NPs synthesized from the
aqueous extract of P. niruri were highly effective against larvae and pupae of
the dengue vector Ae. aegypti, with LC50 values ranging from 3.90 ppm (I) to
13.04 ppm (pupae). Ag NPs synthesized from the seed extract of M. oleifera
have been reported as toxic towards Ae. aegypti young instars, with LC50 of
10.24 ppm (I), 11.81 ppm (II), 13.84 ppm (III), 16.73 ppm (IV), and 21.17
ppm (pupae). In addition, Ag NPs was able to inhibit the growth of dengue
virus, serotype DEN-2 (Sujitha et al., 2015).
Low doses of C. scalpelliformis-synthesized Ag NPs are highly toxic
also against the filariasis vector Cx. quinquefasciatus, with LC50 values
ranging from 3.08 ppm (I) to 7.33 ppm (pupae) (Murugan et al., 2015).
Balakrishnan et al. (2015) observed the synthesized Ag NP with A. marina
leaf extract have been tested against I-IV larvae of An. stephensi and Ae.
aegypti, with LC50 values of 4.374 and 7.406 mg/l, respectively. Recently, C.
scalpelliformis-synthesized Ag NPs have been tested against Cx.
quinquefasciatus, with LC50 values of 3.08 ppm (I instar), 3.49 ppm (II
instar), 4.64 ppm (III instar), 5.86 ppm (IV instar), and 7.33 ppm (pupae)
(Murugan et al., 2015). The aqueous leaf extract of neem, A. indica, has been
tested against III instar larvae of Ae. aegypti and Cx. quinquefasciatus, LC50
were 0.006 and 0.047 mg/l, respectively (Poopathi et al., 2015).
In acute toxicity experiments, the aqueous extract of H. musciformis
was effective against larvae and pupae of Ae. aegypti. LC50 were 246.59 ppm
(I), 269.05 ppm (II), 301.66 ppm (III), 319.30 ppm (IV), and 342.43 ppm
(pupa) (Roni et al., 2015). Santhosh et al. (2015) observed that the
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synthesized Ag NPs using A. muricata, achieved good LC50 against several
mosquito vectors, including Ae. aegypti (LC50=12.58 μg/ml), An. stephensi
(LC50=15.28 μg/ml), and Cx. quinquefasciatus (LC50=18.77 μg/ml). The LC50
values of Plectranthus amboinicus-synthesized ZnO nanoparticles were 3.1,
3.1, and 4.2 mg/l, against An. stephensi, Cx. quinquefasciatus, and Cx.
tritaeniorhynchus III instar larvae, respectively (Vijayakumar et al., 2015).
Dinesh et al. (2015) showed the Ag NPs fabricated with A. vera was effective
against An. stephensi; LC50 values were 3.825 ppm (I), 4.119 ppm (II), 4.982
ppm (III), 5.711 ppm (IV), and 6.113 ppm (pupa).
Ag NPs were successfully synthesized from aqueous silver nitrate
using the extracts of A. hypogaea peels and achieved LC50 against IV instar
larvae of Ae. aegypti of 1.85 mg/l and against An. stephensi of 3.13 mg/l
(Velu et al., 2015). Ag NPs fabricated using leaf and fruit extracts from C.
guianensis were toxic to IV instar larvae of Ae. aegypti, with LC50 of 2.1 ppm
(leaf extract) and 2.09 ppm (fruit extract) (Vimala et al., 2015). Extremely
stable Ag NPs synthesized using the leaf aqueous extract of M.
maderaspatana; LC50 values against Ae. aegypti and Cx. quinquefasciatus IV
instar larvae were 0.211 and 0.094 ppm, respectively (Chitra et al., 2015).
The larvicidal potential of hexane, choloroform, ethyl acetate, acetone,
and methanol extracts of seven aromatic plants, viz., B. mollis, C. swietenia,
C. anisata, F. limnonia, L. camera, P. amboinicus, and T. erecta were
screened against Cx. quinquefasciatus, Ae. aegypti, and An. stephensi.
However, the ethyl acetate extract of C. swietenia showed the remarkable
larvicidal activity against Cx. quinquefasciatus, Ae. aegypti, and An.
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stephensi. After 12 hr of exposure period, the larvicidal activity was
LC50=194.22 and LC90=458.83ppm (Cx. quinquefasciatus), LC50=173.04 and
LC90=442.73 ppm (Ae. aegypti), and LC50=167.28 and LC90=433.07 ppm (An.
stephensi), and the larvicidal activity after 24-hr exposure period was LC50 =
94.12 and LC90 = 249.83 ppm (Cx. quinquefasciatus), LC50=80.58 and
LC90=200.96 ppm (Ae. aegypti), and LC50=76.24 and LC90=194.51 ppm (An.
stephensi) (Jayaraman et al., 2015).
M. tinctoria acetone leaf extract has been used to produce Ag NPs that
achieved an LC50 of 1.442 ppm towards III instar larvae of Cx.
quinquefasciatus (Kumar et al., 2014). Suganya et al. (2014) studied the Ag
NPs produced using the aqueous leaf extract of L. aspera were toxic against
IV instar larvae of Ae. aegypti, with LC50 of 8.563 mg/l. Silver nanoparticles
fabricated with the aqueous extract of C. colocynthis were toxic to III instar
larvae of Cx. pipiens, with LD50 of 0.5mg/ml (Shawky et al., 2014).
Synthesized silver nanoparticles with the leaves of M. dubia were effective
against IV instar larvae of Cx. quinquefasciatus (LC50= 11.27 ppm), and it has
been showed that the larvicidal effect of these Ag NPs was probably due to
the different phyto-constituents coating Ag NP (Karthikeyan et al., 2014).
Soni and Prakash (2014a) reported that the aqueous leaf extracts of
neem has been employed to produce Ag NPs active as larvicides and
pupicides against An. stephensi and Cx. quinquefasciatus. After exposure
times shorter than 24 hr, LC50 values against Cx. quinquefasciatus were 6
(II), 10 (III), and 1 ppm (pupa). No values have been calculated for I and IV
instar larvae. LC50 values against An. stephensi were 2 (I), 2 (II), 2 (III), and 1
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(IV). No values have been calculated for pupae. Synthesized Ag NPs with the
leaf extract of H. indicum had been tested against III instar larvae of An.
stephensi (LC50= 18.40 μg/mL), Ae. aegypti (LC50=20.10 μg/mL), and Cx.
quinquefasciatus (LC50=21.84 μg/mL) (Veerakumar et al., 2014b).
The aqueous leaf extracts of A. marmelos have been used to synthesize
nickel nanoparticles toxic to Ae. aegypti, An. stephensi, and Cx.
quinquefasciatus; LC50 were 534.83, 595.23, and 520.83 ppm, respectively
(Angajala et al., 2014).
Veerakumar et al. (2014b) investigated the F. elephantum synthesized
silver nanoparticles were toxic against An. stephensi, Ae. aegypti, and Cx.
quinquefasciatus; An. stephensi has LC50 of 11.56 μg/mL; Ae. aegypti has
LC50 of 13.13 μg/mL; and Cx. quinquefasciatus has LC50 of 14.19 μg/mL.
The synthesized silver nanoparticles have been tested against the different
larval instars of Ae. aegypti at different concentrations for a period of 24 hr.
The LC50 and LC90 values are 0.79 and 1.09 ppm with respect to the Ae.
aegypti treated Ag NPs with B. bassiana. The first and second instar larvae of
Ae.aegypti have shown cent percent mortality while third and fourth instars
found 50.0, 56.6, 70.0, 80.0, and 86.6 and 52.4, 60.0, 68.5, 76.0, and
83.3%mortality at 24 hr of exposure in 0.06 and 1.00 ppm, respectively
(Najitha Banu and Balasubramanian, 2014a).
Synthesized Ag NPs using the leaf and berry extracts of S. nigrum
were tested against II and III instar larvae of An. stephensi and Cx.
quinquefasciatus. Concerning II instar larvae, An. stephensi LC50 values were
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2.12, 2.04, and 1.67 ppm for dry leaves, fresh leaves, and berries,
respectively. Cx.quinquefasciatus LC50 values were 2.62, 2.20, and 2.88 ppm
for dry leaves, fresh leaves, and berries, respectively. Concerning III instar
larvae, An. stephensi LC50 values were 1.33, 1.59, and 1.54 ppm for dry
leaves, fresh leaves, and berries, respectively. Cx. quinquefasciatus LC50
values were 1.26, 1.33, and 2.44 ppm for dry leaves, fresh leaves, and berries,
respectively (Rawani et al., 2013).
Soni and Prakash, (2013) reported on the potentiality of Ag NPs
synthesized by a fungus F. oxysporum and found LC50 and LC90 values of 8,
6, 4; 12.30, 12.58, 11.48 against first, second and fourth instar larvae of Cx.
quinquefasciatus, An. stephensi and Ae. aegypti, respectively, which are much
higher to the concentration in the present investigation. The LC50 and LC90
values are 0.240 to 0.652 and 1.219 to 2.916, respectively, in all larval instars
(I–IV) of Cx. quinquefasciatus and for Ae.aegypti 0.065 to 0.137 and 0.558 to
1.278 ppm. Larvicidal activities of synthesized Ag NPs using aqueous leaf
extract of V. rosea against the larvae of An. stephensi and Cx.
quinquefasciatus were also determined. In larvicidal activity, their results
showed the maximum efficacy in Ag NPs against fourth instar larvae of An.
stephensi (LC50 12.47 and 16.84 mg/mL and LC90 36.33 and 68.62 mg/mL) on
48 and 72 h of exposure. The efficacy against Cx. quinquefasciatus (LC50
43.80 mg/mL and LC90 120.54 mg/mL) was on 72-hr exposure, and aqueous
extract showed 100% mortality against An. stephensi and Cx.
quinquefasciatus (LC50 78.62 and 55.21 mg/mL and LC90 184.85 and 112.72
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mg/mL) on 72h exposure at concentrations of 50 mg/mL, respectively
(Subarani et al., 2013).
Velayutham et al. (2013) documented the larvicidal activity of Ag NPs
synthesized using the aqueous bark extract of F. racemosa was successfully
tested against IV larvae of the filariasis vector Cx. quinquefasciatus and the
Japanese encephalitis vector Cx. gelidus (LC50=12.00 and 11.21 mg/l,
respectively).
Ag NPs fabricated with the S. acuta leaf extract was tested against III
instar larvae of An. stephensi, Ae.aegypti, and Cx. quinquefasciatus, with
LC50 values of 21.92, 23.96, and 26.13 μg/ml, respectively (Veerakumar et
al., 2013). The mesocarp layer extract of C. nucifera has been employed to
produce Ag NPs toxic to IV instar larvae of An. stephensi and
Cx. quinquefasciatus; after 72 hr of exposure, LC50 was 87.24 mg/l for An.
stephensi and 84.85 mg/l for Cx. quinquefasciatus (Roopan et al., 2013).
Suman et al. (2013) investigated the Ag NPs fabricated using the aqueous
aerial extract of A. baccifera was toxic effects against IV instar larvae of An.
subpictus (LC50=29.54 ppm) and Cx. quinquefasciatus (LC50=22.32 ppm).
Suganya et al. (2013) determined that the fabricated Ag NPs with the
M. koenigii leaf extract was effective against An. stephensi with LC50 values
were 10.82 (I), 14.67 (II), 19.13 (III), 24.35 (IV), and 32.09 ppm (pupa),
while Ae.aegypti with LC50 were 13.34 (I), 17.19 (II), 22.03 (III), 27.57 (IV),
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and 34.84 ppm (pupa). The larvicidal activity of A. cadamba synthesized Au
NPs has been ascertained against III instar larvae of Cx. quinquefasciatus,
with LC50 of 1.08 ppm (Naresh Kumar et al., 2013).
Ag NPs synthesized using dried green fruits of D. roxburghii have
been found toxic against An. stephensi and Cx. quinquefasciatus; LC50 for II,
III, and IV larval instars were 0.863, 1.162, and 1.281 ppm against Cx.
quinquefasciatus and 0.7329, 0.8397, and 0.9848 ppm against An. stephensi,
respectively (Haldar et al., 2013). After 48 h of exposure, Ag NPs synthesized
with the aqueous leaf extract of V. rosea were toxic to IV instar larvae of An.
stephensi and Cx. quinquefasciatus, with LC50 values of 12.47 and 43.80 mg
ml, respectively (Subarani et al., 2013).
Sundaravadivelan and Nalini (2012) exemplified the effect of P.
tithymaloides leaf synthesized silver nanoparticles against the dengue vector
Ae. aegypti and has been observed their LC50 values 0.046, 0.051, 0.046,
0.167, and 0.054 % (I–IV instars and pupa) have been observed at 0.25 %
concentration level, which has the lowest concentration compare to aqueous
stem extract alone, and its LC50 values 1.529, 1.282, 1.450, 2.210, and
1.455% have also been noticed after 24hr exposure. The higher mortality
rates was showed in the Ag NPs produced by plant N. nucifera leaf extracts
(LC50= 0.69 ppm, LC90=2.15 ppm) against An. subpictus and Cx.
quinquefasciatus (LC50=1.10 ppm, LC90=3.59 ppm) values were 575.77,
539.31, 513.99, 497.06, and 476.03 ppm; for Ae. aegypti, LC50 values were
329.82, 307.3, and 252.03 ppm and LC90 values were 563.24, 528.33, 496.92,
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477.61, and 448.05 ppm; and for An. stephensi, LC50 values were 317.28,
300.30, 277.51, 263.35, and 251.43 ppm and LC90 values were 538.22,
512.90, 483.78, 461.08, and 430.70 ppm, respectively (Amerasan et al.,
2012).
Sundaravadivelan et al. (2013) reported that the Ag NPs produced
using P. tithymaloides aqueous leaf extract showed anti-developmental
activity and acute toxicity towards Ae. aegypti, with LC50 values of 0.029 (I),
0.027 (II), 0.047 (III), 0.086 (IV), and 0.018 % (pupa). Ag NPs fabricated
using N. oleander leaf extract were effective against An. stephensi larvae and
pupae, with LC50 values of 20.60 (I), 24.90 (II), 28.22 (III), 33.99 (IV), and
39.55 ppm (pupa) (Roni et al., 2013). Panneerselvam et al. (2013) reported
that the leaf extract of E. hirta was highly effective in field trials against An.
stephensi, as it led to larval density reductions of 13.17, 37.64 and 84.00 %
after 24, 48 and 72 h, respectively.
Ag NPs synthesized using E. hirta plant leaf extract against malarial
vector An. stephensi was determined; the highest larval mortality was found in
synthesized Ag NPs against the first to fourth instar larvae and pupae with the
following values: LC50 (10.14, 16.82, 21.51, and 27.89 ppm, respectively),
LC90 (31.98, 50.38, 60.09, and 69.94 ppm, respectively), and LC50 and LC90 of
pupae (34.52 and 79.76 ppm, respectively) (Priyadarshini et al., 2012). Ag
NPs produced using P. rubra plant latex was toxic to II and IV instar larvae of
Ae. aegypti and An. stephensi; LC50 values were 1.49 (II) and 1.82 ppm (IV)
for Ae. aegypti and 1.10 (II) and 1.74 ppm (IV) for An. stephensi (Patil et al.,
2012a).
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Patil et al. (2012b) investigated the Ag NPs synthesized with the P.
daemia latex were toxic to Ae. aegypti and An. stephensi larvae; LC50 values
were 4.39 (I), 5.12 (II), 5.66 (III), and 6.18 ppm (IV) for Ae. aegypti, and 4.41
(I), 5.35 (II), 5.91 (III), and 6.47 ppm (IV) for An. stephensi. Synthesized Ag
NPs using the aqueous leaf extract of P. dulce showed toxicity against IV
instar larvae of Cx. quinquefasciatus (LC50=21.56 mg l) (Raman et al., 2012).
Panneerselvam et al. (2012) studied which focused on the larval and
pupal mortality of An. stephensi after the treatment of methanolic extract of A.
nilagirica leaf extract showed 41 % mortality at first instar larvae as a result
of treatment at 200 ppm, whereas at 600-ppm concentration, it was increased
to 94 %. In pupal mortality at 200-ppm concentration, it was 23 % increased
to 63 % at 600 ppm. The LC50 and LC90 values were represented as follows:
the LC50 value of the first instar was 272.50 ppm, second instar 311.40 ppm,
third instar 361.51 ppm, and that of the fourth instar was 442.51 ppm; the
LC90 value of the first instar was 590.07 ppm, second instar 688.81 ppm, third
instar 789.34 ppm, and that of the fourth instar was 901.59 ppm, and the LC50
and LC90 values of pupae were 477.23 and 959.30 ppm, respectively. The
maximum efficacy was observed in crude aqueous and synthesized Ag NPs
against Cx. quinquefasciatus (LC50 27.49 and 4.56 mg/L; LC90 70.38 and
13.14 mg/L) and against An. subpictus (LC50 27.85 and 5.14 mg/L; LC90
71.45 and 25.68 mg/L), respectively. A biological method has been used to
synthesize stable silver nanoparticles that were tested as mosquito larvicides
against Ae. aegypti, An. stephensi, and Cx. quinquefasciatus (Arjunan et al.,
2012).
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Santhoshkumar et al. (2011) also obtained LC50 and LC90 values of
0.69 and 1.10 ppm as well as 2.15 and 3.59 ppm of Ag NPs synthesized by
leaf extract of N. nucifera against Cx. quinquefasciatus and An. subpictus
which were analogous to the results obtained in the present study. Similarly,
synthesized Ag NPs using T. cordifolia extract tested against the larvae of An.
subpictus (LC50 06.43 mg/l) and against the larvae of Cx. quinquefasciatus
(LC50 06.96 mg/l) (Jayaseelan et al., 2011). Marimuthu et al. (2011) reported
the larvicidal effect of aqueous crude leaf extracts and synthesized Ag NPs
using Mimosa pudica showed highest mortality in synthesized Ag NPs against
the larvae of An. subpictus (LC50=8.89, 11.82, and 0.69 ppm) and against the
larvae of Cx. quinquefasciatus (LC50=9.51, 13.65, and 1.10 ppm),
respectively. Biosynthesized silver nanoparticles using the fungus
Cochliobolus lunatus was used for the control of Ae. aegypti and An.
stephensi, and it was reported that the nanoparticles are effective against the
second, third, and fourth-instar larvae of Ae. aegypti (LC50 1.29, 1.48, and
1.58 ppm; LC90 3.08, 3.33, and 3.41 ppm) and against An. stephensi (LC50
1.17, 1.30, and 1.41 ppm; LC90 2.99, 3.13, and 3.29 ppm) (Salunkhe et al.,
2011). Relevant literature shows a high dosage of nanoparticles are required
to achieve 50 % and 90 % larval mortality from plant species N. nucifera
against An. subpictus (LC50= 0.69 ppm, LC90=2.15 ppm) and
Cx. quinquefasciatus (LC50=1.10 ppm, LC90 = 3.59 ppm), respectively
(Santhoshkumar et al. 2011).
Soni and Prakash (2011) reported on the potentiality of Ag NPs
synthesized by a fungus C. tropicum and found LC50 and LC90 values of 4 and
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8.91 ppm against third-instar larvae of Ae.aegypti, which were much higher
to the concentration recorded in the present investigation. Gnanadesigan et al.
(2011) determined that the synthesized Ag NPs using R. mucronata leaf
extract were tested on IV instar larvae of Ae. aegypti and Cx.
quinquefasciatus, with LC50 of 0.585 and 0.891 mg/l, respectively. The higher
mortality rates was found in the Ag NPs produced by plant N. nucifera leaf
extracts (LC50 00.69 ppm, LC90 02.15 ppm) against An. subpictus and Cx.
quinquefasciatus (LC50 01.10 ppm, LC90 03.59 ppm) (Thirunavukkarasu et al.,
2010).
The larvicidal efficacy of benzene, hexane, ethyl acetate, methanol,
and chloroform leaf extract of Cardiospermum halicacabum against Cx.
quinquefasciatus and Ae. aegypti. The LC50 values were 174.24, 193.31,
183.36, 150.44, and 154.95 ppm, and 182.51, 200.02, 192.31, 156.80, and
164.54 ppm, respectively (Govindarajan, 2011). The larvicidal efficacy of
benzene, hexane, ethyl acetate, methanol, and chloroform leaf extract of
Cardiospermum halicacabum against Cx. quinquefasciatus and Ae. aegypti.
The LC50 values were 174.24, 193.31, 183.36, 150.44, and 154.95 ppm, and
182.51, 200.02, 192.31, 156.80, and 164.54 ppm, respectively (Govindarajan,
2011).
Larvicidal activity of synthesized Ag NPs utilizing an aqueous extract
from E. prostrata was observed in crude aqueous and synthesized Ag NPs
against Cx. quinquefasciatus (LC50=27.49 and 4.56 mg/L; LC90=70.38 and
13.14 mg/L) and against An. subpictus (LC50=27.85 and 5.14 mg/L;
LC90=71.45 and 25.68 mg/L), respectively (Rajakumar and Rahuman, 2011).
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Potential antiplasmodial activity of synthesized silver nanoparticle using A.
paniculata with the inhibitory concentration (LC50) values were 26±0.2 % at
25 μg/ml, 83±0.5 % at 100 μg/ml (Panneerselvam et al., 2011).
Mathivanan et al. (2010) determine that the LC50 and LC90 values of
crude methanol extract of leaves of E. coronaria on Cx. quinquefasciatus, Ae.
aegypti, and An. stephensi larvae in 24 hr were 72.41, 65.67, and 62.08 and
136.55, 127.24, and 120.86 mg/L, respectively. The essential oil from the
leaves of C. anisata exhibited significant larvicidal activity, with 24 h LC50
values of 140.96, 130.19, and 119.59 ppm, respectively (Govindarajan
2010b). The highest larval mortality was found in leaf ethyl acetate of A.
marmelos and E. prostrata, hexane, and methanol of A. paniculata and C.
hirsutus showing LC50 values of 167.00, 78.28, 67.24, and 142.83 ppm and
LC90 values of 588.31, 360.75, 371.91, and 830.01 ppm, respectively (Elango
et al., 2009).
The leaf petroleum ether, flower methanol extracts of C. auriculata,
flower methanol extracts of L. aspera and R. nasutus, leaf and seed methanol
extracts of Solanum torvum, and leaf hexane extract of V. negundo were
evaluated for larvicidal activity with LC50 values of 44.21, 44.69, 53.16,
41.07, 35.32, 28.90, and 44.40 ppm, respectively (Kamaraj et al., 2009). The
leaf ethyl acetate extract of A. aspera leaf chloroform extract of Anisomeles
malabarica, flower methanol of G. superba, and leaf methanol extract of
Ricinus communis exhibited LC50 values of 48.83, 135.36, 106.77, and 102.71
ppm and LC90 of 225.36, 527.24, 471.90, and 483.04 ppm, respectively (Zahir
et al., 2009).
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Bagavan et al. (2009b) investigated that the ethyl acetate extract from
the leaves of O. canum and O. sanctum showed good larvicidal activity
against the larvae of An. subpictus (LC50=88.15 and 21.67 ppm and
LC90=528.70 and 98.34 ppm, respectively) and ethyl acetate extracts of O.
sanctum against the larvae of Cx. tritaeniorhynchus (LC50=109.12 ppm and
LC90=646.62 ppm). The ethyl acetate extract of leaves of O. sanctum
produced significant mortality against Ae. aegypti and Cx. quinquefasciatus,
with LC50 values of 425.94 and 592.60 ppm, respectively (Anees, 2008).
Matasyoh et al. (2008) reported 100 % mortality at a concentration of 0.2
mg/l with a LC50 value of 0.11 mg/ml to the ethyl acetate leaves extract of A.
turkanensis against An. gambiae.
Earlier authors reported that the methanol leaf extracts of V. negundo,
V. trifolia, V. peduncularis, and V. altissima were used for larvicidal assay
with LC50 values of 212.57, 41.41, 76.28, and 128.04 ppm, respectively,
against the early fourth-instar larvae of Cx. quinquefasciatus (Kannathasan et
al., 2007). Mullai and Jebanesan (2007) determined the ethyl acetate leaf
extract of C. colocynthis and C. maxima had larvicidal activity. The extracts
showed the LC50 values of 47.58 and 75.91 ppm, respectively, against Cx.
quinquefasciatus larvae. The larvicidal activity of the essential oil aqueous
solutions of the stalks and leaves of Croton argyrophylloides, Croton
nepetaefolius, Croton sonderianus, and Croton zehntneri showed 100%
mortality at 50 ml against Ae. aegypti (Lima et al., 2006).
Dua et al. (2006) examined he mean median lethal concentration
values of the aqueous extract from the roots of H. abelmoschus against the
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larvae of An. culicifacies, An. stephensi, and Cx. quinquefasciatus were 52.3,
52.6, and 43.8 ppm, respectively. Amer and Mehlhorn (2006) evaluated that
the Lippia citriodora essential oil having LC50 value of 101.4 ppm was
effective against third instar larvae of An. stephensi. Sharma et al. (2005)
reported that the acetone extract of N. indicum and T. orientalis have been
studied with LC50 values of 200.87, 127.53, 209.00, and 155.97 ppm against
III instar larvae of An. stephensi and Cx. quinquefasciatus, respectively.
5.3 OVICIDAL ACTIVITY
Ovicides of botanical origin are a promising eco-friendly tool to be
used against mosquito vectors of medical and veterinary importance. Indeed,
some of them are cheap and effective against different species, even when
tested at low doses. In this scenario, employing of single-step formulations
from local plants as mosquito ovicides may be helpful for marginalized
populations living in rural areas of the world (Benelli 2015b).
The ovicidal activities of five different plants leaf extract were tested
against three clinically important mosquito species namely An.stephensi,
Ae.aegypti, and Cx.quinquefasciatus. The concentration of the leaf extracts
vary with the plant species used. Among the five plants species investigated
for ovicidal activity against the vector mosquitoes viz., An.stephensi,
Ae.aegypti, and Cx.quinquefasciatus. The M. emarginata exerted highest
mortality (100 percent mortality) at 200 µg/mL against An.stephensi, followed
by Ae.aegypti (250 µg/mL) and Cx.quinquefasciatus (300 µg/mL). The Ag
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NPs showed zero hatchability at 40 µg/mL against An.stephensi, followed by
Ae.aegypti (50 µg/mL) and Cx.quinquefasciatus (60 µg/mL).
The ovicidal activity of C. odorata against Ae.aegypti eggs exposed to
1, 5, and 10 % were not different (P>0.05) at 77.6, 83.2, and 83.2 %,
respectively. However, the ovicidal activity of extracted oil on An. dirus eggs
increased from 71.2 % at 1 % concentration to 73.6 % at 5 % and to 94.4 % at
10 % concentration, respectively. The result also showed that the ovicidal
effect at 1, 5, and 10 % of C. odorata was highly significant against
Cx. quinquefasciatus (P<0.05), producing 86.7, 96.1, and 98.9 %, respectively
(Soonwera, 2015). Reegan et al., (2015) reported that the 500 ppm of the
hexane leaf extract of L. acidissima led to egg mortality of 60 and 79.2 %
against Ae.aegypti and Cx. quinquefasciatus, respectively.
The ovicidal activity of Cereus hildmannianus extracts on Ae.aegypti
eggs are present the moderate ovicidal activity was noted only in the
petroleum ether extract on the eggs of Ae.aegypti with 52.8% EMR at 1000
mg/L at 96 h post treatment period. The lowest concentration (62.5 mg/L) of
petroleum ether extract caused 28.8% egg mortality against the eggs of Ae.
aegypti, the carbon tetrachloride extract showed 38.4% and hexane, ethyl
acetate and aqueous extracts recorded egg mortality of 21.6%, 24.8% and
20.0% respectively, at 1000 mg/L concentration against Ae.aegypti
(Kamakshi et al., 2015).
Ovicidal activity of green-synthesized nanoparticles with S. muticum
aqueous extract was toxic against An. stephensi, Ae. aegypti, and Cx.
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quinquefasciatus; the egg hatchability was reduced by 100 % after a single
exposure to 30 ppm (Madhiyazhagan et al., 2015). Govindarajan and
Rajeswary (2015) investigated the ovicidal activity of crude hexane, benzene,
chloroform, ethyl acetate, and methanol solvent extracts of leaf and seed of A.
lebbeck against the eggs of three important vector mosquitoes viz., Cx.
quinquefasciatus, Ae. aegypti, and An. stephensi. One hundred percent
mortality was observed at 250, 200, and 150 ppm for leaf methanol extract
and 375, 300, and 225 ppm for seed methanol extract of A. lebbeck against
Cx. quinquefasciatus, Ae. aegypti, and An. stephensi, respectively.
Govindarajan and Sivakumar (2014a) evaluated the ovicidal potential
of the crude hexane, benzene, chloroform, ethyl acetate, and methanol solvent
extracts from the medicinal plant E. indica against the medically important
mosquito vectors, An. stephensi, Ae. aegypti, and Cx. quinquefasciatus;
however, the methanol extract showed the highest ovicidal activity. The
methanol extract of E. indica against An. stephensi, Ae. aegypti, and Cx.
quinquefasciatus exerted 100 % mortality (zero hatchability) at 150, 200, and
250 ppm, respectively. Prathibha et al. (2014) showed the ethyl acetate
extracts of S. mauritiana S. canadensis, E. ridleyi, and E. jambolana were
tested for ovicidal activity against An. stephensi, Ae. aegypti, and Cx.
quinquefasciatus, exerted 100%mortality (zero hatchability) at 100 ppm.
Maheswaran and Ignacimuthu (2014) determined that the essential oil
and an isolated compound from the leaves of P. hydropiper against dengue
vector mosquito Ae. albopictus. The results of ovicidal activity were 100 % at
10 ppm of confertifolin at 0- to 6-h-old eggs of Ae. albopictus. The result of
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the ovicidal activity of crude hexane, benzene, chloroform, ethyl acetate, and
methanol solvent extracts of leaf of A. adenophora against the vector
mosquito Cx. quinquefasciatus. Among the extracts tested for ovicidal
activity against Cx. quinquefasciatus the leaf methanol extract of A.
adenophora exerted 100% mortality (zero hatchability) at 375 mg/L
(Rajeswary et al., 2014). Govindarajan and Sivakumar (2014b) reported that
the methanol extract of A. racemosus against Cx. quinquefasciatus, Ae.
aegypti and An. stephensi exerted 100 % mortality (zero hatchability) at 375,
300 and 225 ppm, respectively. Control eggs showed 99–100 % hatchability.
The percent hatchability was inversely proportional to the
concentration of extract and directly proportional to the eggs. Among the
extracts tested for ovicidal activity against An. stephensi, the leaf methanol
extract of A. paniculata exerted 100 % mortality (zero hatchability) at 150
and 300 ppm, respectively. The leaf extract of A. paniculata was found to be
most effective than C. occidentalis and E. hirta against larvae and eggs of
vector mosquitoes (Panneerselvam and Murugan, 2013). The mean percent
hatchability of the eggs was observed after 48 hr post treatment. All the five
solvent extracts showed moderate ovicidal activity; however, the methanol
extract showed the highest ovicidal activity. One hundred percent mortality
was observed at 300 ppm for leaf methanol extract and 500 ppm for seed
methanol extract of D. elata against An. stephensi and Ae. aegypti,
respectively (Govindarajan et al., 2012).
Samidurai (2012) reported that the mean percent of egg hatchability of
Cx. tritaeniorhynchus and An. subpictus were tested with three different
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solvents at different concentrations of P. acidula leaves extracts, and the
percent hatchability was inversely proportional to the eggs. Among the
extracts tested for ovicidal activity against Cx. tritaeniorhynchus and An.
subpictus, the methanol extract of P. acidula exerted 100% mortality (zero
hatchability) at 350 and 400 respectively. Govindarajan et al. (2011a)
observed the leaf extract of E. coronaria and C. pulcherrima showed
moderate ovicidal and larvicidal activities against An. stephensi, Ae. aegypti
and Cx. quinquefasciatus, respectively.
Govindarajan (2011) reported that the leaf extracts of Cardiospermum
halicacabum showed ovicidal activity ranging from 100– 600 ppm against Ae.
aegypti. Souza et al. (2011) observed that none of the seed ethanolic extract
of 21 Brazilian plants was able to exert 100 % mortality against Ae.aegypti
eggs, the 500-ppm treated cups received a mean number of 34.1.01and
24.1.21 eggs per cup while the control cups received a mean number of
460.1.71 and 500.1.48 eggs per cup tested the leaf acetone and methanol
extracts of A. marmelos, respectively. Elango et al. (2010) reported that the
complete ovicidal activity (100%mortality) was attained at 1000 ppm hexane
and chloroform extracts of A. lineate against An. subpictus and
Cx. tritaeniorhynchus.
The leaf extract of C. fistula with different solvents viz., methanol,
benzene, and acetone was studied for the ovicidal activity against Ae. aegypti,
the mean percent hatchability of the eggs was observed after 120.00 h post
treatment, 100% mortality (zero hatchability) at 160 mg/ml (Govindarajan
2009). Mullai et al. (2008) reported that the benzene extracts of C. vulgaris
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exerted 100 % mortality (zero hatchability) at 250 ppm, a very low
hatchability (11.8 %) at 200 ppm, complete ovicidal activity at 300 ppm and
the fraction I at 80 ppm exerted a very low hatchability rate of 3.2 % followed
by fraction II (6.9 %), and fraction III and fraction IV which afforded 4.9 and
5.3 % hatchability recorded against An. stephensi and Ae. aegypti,
respectively.
The methanol containing water that served as a control showed 94%
hatchability in 0-3hr old egg rafts/eggs, but the 100% hatchability was noted
in egg rafts/eggs beyond the age of 0-3 h old in leaf methanol (90%) extract
of C. fistula against egg raft of Cx. quinquefasciatus (Govindarajan et al.,
2008a). Mullai and Jebanesan (2007) investigated the mean percent
hatchability of Cx. quinquefasciatus with C. colocynthis and C. maxima, the
toxicity of leaf extracts was dependent on its concentration. Zero hatchability
(100% mortality) was attained at the concentration of 450 ppm for C.
colocynthis and 600 ppm for C. maxima. Control eggs (water with respective
solvents) shows the hatchability ranged from 97.4 to 100% with
C. colocynthis and C. maxima exhibited 100% hatchability for all the extracts.
The 100 % ovicidal efficacy of the C. citratus oil against the filariasis vector
Cx. quinquefasciatus and dengue vector Ae. aegypti has been revealed by
Pushpanathan et al. (2006) at 300 ppm.
Mullai and Jebanesan (2006) evaluated that the complete ovicidal
activity (100%mortality) was attained at 300 ppm for methanol, benzene,
petroleum ether and ethyl acetate extracts of C. pubescens against C.
quinquefasciatus. The leaf extract of Solanum trilobatum reduced egg laying
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by gravid females of An. stephensi from 18 to 99%comparedwith ethanol-
treated controls at 0.01, 0.025, 0.05, 0.075 and 0.1 % (Rajkumar and
Jebanesan 2005). Rajkumar and Jebanesan (2004) studied ovicidal activity of
M. polystachyum leaf extract against Cx. quinquefasciatus and observed 100
% egg mortality at 100 ml/L.
The ovicidal effect of S. argel was low; however, concentrations of
0.05 and 0.1 % exhibited significant effects (p<0.05), producing 65 and 75 %,
and 62.9 and 62.9 %, respectively, on the first and second day after treatment,
respectively; the 0.1 % concentration reduced egg hatch by 33.7 %, compared
with the control, and 100 % mortality values were evident in concentrations
as low as 0.025 % at 2 days post hatching against C. pipiens (Al-Doghairi et
al. 2004). Assis et al. (2003) reported that the egg hatching inhibition of ethyl
acetate and methanol extracts of Spigelia anthelmia, which showed 97.4% to
100% at 50.0 mg ml−1, respectively. The bioactive compound Azadirachtin
(A. indica) showed complete ovicidal activity in the eggs of Cx. tarsalis and
Cx.quinquefasciatus exposed to 10 ppm concentration (Su and Mulla, 1998).
5.4 ADULTICIDAL ACTIVITY
From the results of the present study, the plants which were tested
against three important mosquito species for the adulticidal activity were
grouped into two categories based on their LD50 values. The plant species M.
emarginata showed the LD50 values 250 µg/mL (<250) (Ag NPs 30 µg/mL),
the plant species N. alata and H. puberula, exhibited the LD50 values below
325µg/mL (<325) (Ag NPs 40µg/mL). Based on these LD50 values with
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Anopheles, Aedes and Culex, species, M. emarginata are group under the
categories of more effective plants species, N. alata and H. puberula are
categorized under moderately effective plants species, and A. elaeagnoidea,
and V. madrasapatna plants grouped under less effective plants species.
Synthesized gold nanoparticles (Au NPs) using C. guianensis flower
extract were investigated against the adults of malaria vector An. stephensi;
the LC50 and LC90 values of flower extract were 133.96 and 287.65 ppm, LC50
and LC90 values of Au NPs were 11.23 and 24.61 ppm, respectively
(Subramaniam et al., 2016). The C. anisata acetone extract and its hexane
fraction caused mosquito knockdown and eventually death when nebulised
into the testing chamber, with an EC50 of 78.9 mg/ml (7.89 %) and 71.6
mg/ml (7.16 %) in the first 15 min after spraying. C. anisata leaf extracts
have potential to be included in protection products against mosquitoes due to
the adulticidal compounds contained therein (Mukandiwa et al., 2016).
Suresh et al. (2015) reported that the synthesized Ag NPs with P.
niruri tested against adults of Ae. aegypti, achieved LC50 and LC90 values of
6.68 and 23.58 ppm, respectively. Using other plant species for nano
synthesis, adulticidal toxicity may variate consistently. For instance, M.
elengi-synthesized Ag NPs showed LC50 of 13.7 ppm against adult of An.
stephensi and 14.7 ppm against adult of Ae. albopictus (Subramaniam et al.,
2015). Notably, exposure to doses ranging from 100 to 500 ppm of H.
musciformis-fabricated Ag NPs strongly reduced Ae. aegypti longevity in both
sexes, as well as female fecundity (Roni et al., 2015). Madhiyazhagan et al.
(2015) showed that 10 ppm of Ag NPs synthesized using S. muticum reduced
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oviposition rates of more than 70 % in Ae. aegypti, An. stephensi, and Cx.
quinquefasciatus (OAI= −0.61, −0.63, and −0.58, respectively).
Soni and Prakash (2014b) evaluated the synthesized silver
nanoparticles using the neem leaf extract were effective for Cx.
quinquefasciatus adults, with LC50 of 0.53 ppm calculated after 4 hr of
exposure. The adulticidal activity of Ag NPs synthesized using H. indicum
leaf extract has been evaluated against adults of An. stephensi, Ae. aegypti,
and Cx. quinquefasciatus; the maximum efficacy has been observed against
the adults of An. stephensi (LD50=26.712 and LD90= 49.061 μg/mL), followed
by Ae. aegypti (LD50=29.626 and LD90= 54.269 μg/mL), and Cx.
quinquefasciatus (LD50= 32.077 and LD90=58.426 μg/mL) (Veerakumar et
al., 2014b).
The adulticidal activity of D. elata leaf and seed extracts showed
moderate toxic effect on the adult mosquitoes after 24 hr of exposure period.
However, the highest adulticidal activity was observed in the leaf methanol
extract of D. elata against Ae.aegypti with the LC50 and LC90 values 162.87
and 309.32 ppm, respectively (Rajeswary and Govindarajan 2014).
Govindarajan and Sivakumar (2014b) reported that the LD50 and LD90 values
of A. racemosus root extracts against adulticidal activity of (hexane, benzene,
chloroform, ethyl acetate and methanol) Cx. quinquefasciatus, Ae.aegypti and
An. stephensi were the following: An. stephensi, LD50 values 157.16, 143.49,
154.68, 133.43 and 120.44 ppm, and LD90 values were 282.57, 254.28,
272.87, 241.19 and 214.65 ppm; Ae. aegypti, LD50 values were 177.69,
162.52, 172.92, 143.14 and 135.60 ppm, and LD90 values were 322.30,
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296.84, 315.30, 257.76 and 248.35 ppm; and Cx. quinquefasciatus LD50
values were 208.46, 186.22, 196.36, 166.57 and 157.71 ppm, and LD90 values
were 368.22, 333.71, 350.12, 303.44 and 290.95 ppm, respectively.
Kovendan et al. (2013) evaluated that the Amelanchier alnifolia leaf
extracts against adulticidal activity of (hexane, benzene, ethyl acetate,
acetone, and methanol) Ae. aegypti, An. stephensi, and Cx. quinquefasciatus
were the following: Ae. aegypti LC50 values were 371.87, 342.97, 320.17,
300.86, and 279.75 ppm; An. stephensi LC50 values were 358.35, 336.64,
306.10, 293.01, and 274.76 ppm; Cx. quinquefasciatus LC50 values were
383.59, 354.13, 327.74, 314.33, and 291.71 ppm, respectively.
Panneerselvam and Murugan (2013) observed that the adult mortality
was found in methanol extract of A. paniculata followed by C. occidentalis
and E. hirta against the adults of An. stephensi with LC50 and LC90 values of
210.30, 225.91, and 263.91 ppm and 527.31, 586.36, and 621.91 ppm,
respectively. The LC50 values of A. alnifolia plant leaf extracts against
adulticidal activity of (hexane, benzene, ethyl acetate, acetone and methanol)
Ae. aegypti, An. stephensi and Cx. quinquefasciatus were the following: Ae.
aegypti values were 371.87, 342.97, 320.17, 300.86 and 279.75 ppm; An.
stephensi values were 358.35, 336.64, 306.10, 293.01 and 274.76 ppm; Cx.
quinquefasciatus values were 383.59, 354.13, 327.74, 314.33 and 291.71 ppm
(Kovendan et al., 2013).
The effect of silver nanoparticles synthesized with Chrysosporium
keratinophilum and Verticillium lecanii has been evaluated against the adult
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mosquito of filariasis vector Cx. quinquefasciatus, with LC50 values 0.19 and
0.4 μL/cm2; LC90 values 2.4 and 3.2 μL/cm
2; after 22 hr of exposure period
(Soni and Prakash, 2012). The adult mortality was found in methanol extract
of A. paniculata against the adults of Cx. quinquefasciatus and Ae. aegypti
with the LC50 and LC90 values 149.81, 172.37 ppm and 288.12, 321.01 ppm,
respectively (Govindarajan and Sivakumar, 2012).
Barik et al. (2012) investigated the oviposition behavior of three
mosquito species in the presence of different types of nanosilica. Complete
ovideterrence activity of hydrophobic nanosilica was observed at 112.5 ppm
in Ae. aegypti, An. stephensi, and Cx. quinquefasciatus, while there was no
effect of lipophilic nanosilica on oviposition behavior of the three vectors.
The highest adult mortality was found in methanol extract of A. paniculata
followed by C. occidentalis and E. hirta against the adults of An. stephensi
with LC50 and LC90 values of 210.30, 225.91 and 263.91 ppm and 527.31,
586.36 and 621.91 ppm, respectively (Panneerselvam and Murugan, 2012).
The LC50 and LC90 values of C. tora leaf extracts against adulticidal
activity of hexane, chloroform benzene, acetone, and methanol (Cx.
quinquefasciatus, Ae. aegypti, and An. stephensi) were the following: for Cx.
quinquefasciatus, LC50 values were 338.81, 315.73, 296.13, 279.23, and
261.03 ppm and LC90 values were 575.77, 539.31, 513.99, 497.06, and 476.03
ppm; for Ae aegypti, LC50 values were 329.82, 307.3, and 252.03 ppm and
LC90 values were 563.24, 528.33, 496.92, 477.61, and 448.05 ppm; and for
An. stephensi, LC50 values were 317.28, 300.30, 277.51, 263.35, and 251.43
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ppm and LC90 values were 538.22, 512.90, 483.78, 461.08, and 430.70 ppm,
respectively (Amerasan et al., 2012).
The adult mortality was found in ethanol extract of C. sinensis with the
LC50 and LC90 values of 272.19 and 457.14 ppm, An. stephensi of 289.62 and
494.88 ppm, and Ae. aegypti of 320.38 and 524.57 ppm, respectively
(Murugan et al., 2012). The adult mortality was found in methanol extract of
A. nilagirica, with the LC50 and LC90 values of 205.78 and 459.51 ppm for
An. stephensi, and 242.52 and 523.73 ppm for Ae. aegypti, respectively
(Panneerselvam et al., 2012). The maximum adulticidal activity observed in
ethyl acetate extract of the A. lineata, chloroform extract of A. paniculata,
acetone extract of C. hirsutus, and methanol extract of T. erecta
(LD50=126.92, 95.82, 109.40, and 89.83 ppm; LD90 = 542.95, 720.82, 459.03,
and 607.85 ppm); and EI of leaf acetone extract of the A. marmelos, ethyl
acetate extract of A. lineata, methanol extract of the C. hirsutus, and T.
erecta (EI50=128.14, 79.39, 143.97, and 92.82 ppm; EI90=713.53, 293.70,
682.72 and 582.59 ppm), respectively against An. subpictus (Elango et al.,
2011).
The adulticidal activity of tested plant extracts showed moderate toxic
effect on the adult mosquitoes after 24hr of exposure at 500 µg/ mL; however,
the highest mortality was found in methanol, extract of M. charantia, M.
oleifera and ethyl acetate extract of O. gratissiumum, ethyl acetate and
methanol extract of O. tenuiflorum and P. granatum against adult of Cx.
gelidus with LD50 values of 27.21, 68.03, 72.56, 47.60, 82.66, and 34.26 µg/
mL; LD90 values of 146.23, 285.83, 319.14, 264.58, 299.23 and 356.14 µg/
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mL; and methanol extract of M. charantia, ethyl acetate extract of M. oleifera,
O. gratissimum, O. tenuiflorum and P. granatum against adult of
Cx.quinquefasciatus with LD50 values of 43.06, 59.34, 62.42,58.89 and 54.15
µg/mL; LD90 values of 248.18, 250.46, 450.72, 326.13 and 423.38 µg/mL,
respectively (Kamaraj et al., 2010).
Dua et al. (2010) observed that the adulticidal activity of the essential
oil of L. camara was evaluated against different mosquito species on 0.208
mg/cm2 impregnated papers, and the KDT50 and KDT90 values of the essential
oil were 20, 18, 15, 12 and 14 min, and 35, 28, 25, 18 and 23 min against Ae.
aegypti, Cx. quinquefasciatus, An. culicifacies, Anopheles fluvialitis and An.
stephensi with their percent mortality of 93.3 %, 95.2 %, 100 %, 100 % and
100 %, respectively. The larvicidal and adulticidal activities of ethanolic and
water mixture (50:50) of plant extracts E. globulus, C. citratus, A. annua, J.
gendarussa, M. fragrans, A. squamosa, and C. asiatica were tested against
An. stephensi, and the most effective between 80 and 100 % was observed in
all extracts (Senthilkumar et al. 2009).
Similar result was obtained in the root extract of Valeriana jatamansi
which exhibited adulticidal activity of 90 % lethal concentration against adult
An. stephensi, An. culicifacies, Ae. aegypti, Ae. albopictus and Cx.
quinquefasciatus and were 0.14, 0.16, 0.09, 0.08 and 0.17, and 0.24, 0.34,
0.25, 0.21 and 0.28 mg/cm2, respectively (Dua et al. 2008). The adulticidal
activity of the essential oil isolated from Mentha longifolia was screened by
fumigant toxicity assay against the house mosquito, Cx. pipiens by Oz et al.
(2007). The highest adulticidal effect was established from Piper
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sarmentosum, followed by Piper ribesoides and Piper longum, with LD50
values of 0.14, 0.15 and 0.26 μg/female, respectively (Choochote et al.,
2006).
Nathan et al. (2005) considered pure limonoids of neem seed, testing
for biological, larvicidal, pupicidal, adulticidal and anti-ovipositional activity,
An. stephensi and the larval mortality was dose-dependent with the highest
dose of 1 ppm azadirachtin evoking almost 100 % mortality, affecting
pupicidal and adulticidal activity and significantly decreased fecundity and
longevity of An. stephensi. Jeyabalan et al. (2003) also have reported the
adulticidal effect of P. citrosa on An. stephensi, with LC50 and LC90 value of
1.56 and 5.22 %, respectively. However, it is worth to note that their LC50 and
LC90 values were much higher than the extracts, which were tested in this
study.
5.5 NON-TARGET AQUATIC ORGANISM
The effect on a non-target organism revealed that the EO of
Zanthoxylum monophyllum and its major chemical compounds are safe for the
predatory fish, G. affinis. Given that the LC50 for G. affinis was estimated at
4234.07μg/mL, the EO can be considered completely safe for this fish
species, which was manifested in a high suitability index/predator safety
factor, ranging from 86.39 for the least sensitive larvae of Cx.
tritaeniorhynchus to 102.02 for the most sensitive larvae of An. subpictus
(Pavela and Govindarajan, 2016). Govindarajan et al. (2016e) reported that
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the biotoxicity of M. sylvestris aqueous extract and green-synthesized Ag NPs
was evaluated on non-target organisms D. indicus and G. affinis.
Govindarajan et al. (2016f) investigated the biotoxicity of aqueous
extract and green-synthesized Ag NP on non-target organisms D. indicus, A.
bouvieri and G. affinis. Toxicity treatments achieved negligible toxicity
against D. indicus, A. bouvieri and G. affinis, with LC50 values ranging from
424.09 to 6402.68μg/mL. Similarly, G. affinis showed high predation rates
against both An. stephensi and Ae. albopictus larvae. After 24 hr, predation of
III instars larvae of An. stephensi and Ae. albopictus were 60.90 and 57.42 %,
respectively (Subramaniam et al., 2015). Chobu et al. (2015) observed that G.
affinis is more efficient as a predator of An. gambiae third instar larvae than
the Cyprinidae goldfish Carassius auratus.
Toxicity treatments achieved negligible toxicity against D. indicus and
G. affinis, with LC50 values ranging from 813.16 to 10,459.13μg/mL. The
biotoxicity of B. cristata aqueous extract and green-synthesized Ag NP was
evaluated on non-target organisms D. indicus, A. bouvieri, and G. affinis with
LC50 values ranging from 633.26 to 8595.89 μg/mL, respectively
(Govindarajan and Benelli, 2016b). B. tinctoria was tested against the non-
target mosquito predators T. splendens and M. thermocyclopoids, with LC50
values of 552.28 and 480.92 ppm, respectively. Experiments conducted
testing Ag NPs on T. splendens and M. thermocyclopoids lead to LC50 values
of 234.48 and 218.16 ppm, respectively. The Safety Index/Predator Safety
Factor calculated for the leaf extract of B. tinctoria was 3.02 and 2.63 for T.
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splendens and M. thermocyclopoids, respectively, while for Ag NPs, it was
47.1 and 43.8, respectively (Mahesh Kumar et al., 2016).
Ag NPs biosynthesized using the 2, 7.bis [2-(diethylamino)-ethoxy]
fluorence isolate from the Melia azedarach leaves did not show acute toxicity
against Mesocyclops pehpeiensis copepods (Ramanibai and Velayutham,
2015). Moreover, goldfish (C. auratus) predation efficiency was higher if Ae.
aegypti larvae were exposed to 1 ppm of B. cylindrica-fabricated Ag NPs
(Murugan et al., 2015g). Haldar et al. (2013) did not detected toxicity of
Ag NPs produced using dried green fruits of Drypetes roxburghii against P.
reticulata, after 48 hr of exposure to LC50 of IV instar larvae of An. stephensi
and Cx. quinquefasciatus.
Subarani et al. (2013) reported that the V. rosea-synthesized silver
nanoparticles did not exhibit any noticeable toxicity on P. reticulata after 24,
48, and 72 hr of exposure. These results suggest that the synthesized Ag NPs
have the potential to be used as an ideal eco-friendly approach for the control
of the Ae. aegypti larvae. Rawani et al. (2013) showed that mosquitocidal Ag
NPs synthesized using S. nigrum berry extracts were not toxic against two
mosquito predators, Toxorhynchites larvae and Diplonychus annulatum, and
Chironomus circumdatus larvae, exposed to lethal concentrations of dry
nanoparticles calculated on An. stephensi and Cx. quinquefasciatus larvae. P.
rubra and P. daemia-synthesized Ag NPs did not exhibit any evident toxicity
effect against P. reticulata fishes, after 48 hr of exposure to LC50 and LC90
values calculated on IV instar larvae of Ae. aegypti and An. stephensi (Patil et
al., 2012a).
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Patil et al. (2012b) investigated the acute toxicity of green-synthesized
metal nanoparticles against fishes, showing that mosquitocidal Ag NPs
synthesized from P. daemia latex were not toxic to the non-target guppy fish
P. reticulata. In laboratory conditions, G. affinis showed high predation rates
against both An. stephensi and Ae. albopictus larvae. After 24 hr, predations
of III instar larvae of An. stephensi and Ae. albopictus were 60.90 and 57.42
%, respectively. The western mosquito-fish is widely known as one of the
more efficient biocontrol agents against Culicidae larvae (Griffin and Knight,
2012).
G. affinis have reported as a more efficient predator of mosquito young
instars than other aquatic organisms such as Belostomatidae and odonate
nymphs (Kweka et al., 2011). The effect of S. emarginatus extract against two
nontarget aquatic insects, namely, Chironomus costatus and Diplonychus
rusticus were tested. This extract did not cause mortality of fourth-instar
larvae of C. costatus at a concentration of 2 mg dry weight per milliliter up to
48 hr, whereas no mortality of the first-instar nymphs of the aquatic bug
D. rusticus was observed up to a concentration of 6 mg dry weight per
milliliter within 48 hr of exposure (Koodalingam et al., 2009). In a similar
study, Sivagnaname and Kalyanasundaram, (2004) have tested the toxicity of
A. monophylla extract on five nontarget mosquito predators and demonstrated
that this extract was highly toxic to the back-swimming hemipteran water
bug, A. bouvieri, and moderately toxic to another water bug D. indicus
(D. rusticus). Kreutzweiser, (1997) reported the deleterious effects of neem
extract and a commercial neem formulation (Azatin) on a variety of eight
species of nontarget aquatic invertebrates with the highest lethal effect on the
larvae of mayfly (Isonychia bicolor/rufa).
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SUMMARY CONCLUSIONS
In the present study, 5 medicinal plants viz., Merremia emarginata,
Naregamia alata, Hedyotis puberula, Aglaia elaeagnoidea, and Ventilago
madrasapatna were collected from the Nilgiris, Western Ghats, Tamil Nadu
State, India.
Healthy and fresh leaves were collected and washed with tap water,
dried in shade at room temperature. They were grounded to fine powder in an
electric grinder. Aqueous extract was prepared by mixing 50 g of dried leaf
powder with 500 mL of water (boiled and cooled distilled water) with
constant stirring on a magnetic stirrer. The suspension of dried leaf powder in
water was left for 3 hr, filtered through Whatman no. 1 filter paper, and the
filtrate was stored in amber-colored airtight bottle at 10 °C temperature
until use.
Green synthesis of silver nanoparticles (Ag NPs) was performed using
the aqueous leaf extracts of five plants. The synthesized Ag NPs were
characterized using the techniques of UV–vis spectroscopy, Fourier transform
infrared spectroscopy (FT-IR), Scanning electron microscopy (SEM) with
EDX analysis, Transmission electron microscopy (TEM), Atomic force
microscopy (AFM) and X-ray diffraction (XRD) analysis.
The egg rafts/eggs of three mosquito vectors, An. stephensi, Ae.
aegypti and Cx. quinquefasciatus, were obtained from vector control
laboratory, Department of Zoology, Annamalai University. The laboratory
colony was maintained at 70–85 % RH, 28±2 °C temperature, with a
photoperiod of 12-h light and 12-h dark.
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The larvicidal activity of these five plants aqueous leaf extracts and Ag
NPs was tested against Aedes, Anopheles and Culex mosquitoes. The larval
mortality was calculated after 24 hr exposure period. The LC50 and LC90, 95%
confidence limit of lower confidence limit (LCL) and upper confidence limit
(UCL) and chi-square values calculated by using probit analysis.
Among the five plants aqueous leaf extract tested, the maximum
larvicidal activity was observed in M. emarginata against An. stephensi,
Ae.aegypti and Cx. quinquefasciatus, LC50 and LC90 values were
146.91,157.87,169.24 and 286.29,301.63,315.81µg/mL, respectively.
The maximum larvicidal activity was reported with the M. emarginata
mediated synthesized Ag NPs against An. stephensi followed by Ae.aegypti
and Cx. quinquefasciatus with the LC50 and LC90 values were 8.36,9.20,10.02
and 16.33,17.86,18.62/mL, respectively.
Among the five plants species evaluated for ovicidal activity against
An. stephensi, Ae. aegypti and Cx. quinquefasciatus, the aqueous leaf extract
of M. emarginata exerted 100% mortality (12-18 hr old eggs/egg raft) at 200
µg/mL against An. stephensi, followed by Ae. aegypti (250 µg/mL) and Cx.
quinquefasciatus (300 µg/mL).
M. emarginata mediated synthesized Ag NPs showed (12-18 hrs old
eggs/egg raft) zero hatchability at 40 µg/mL against An. stephensi, followed
by Ae. aegypti (50µg/mL) and Cx. quinquefasciatus (60µg/mL).
The adulticidal activity of five plants aqueous leaf extract and Ag NPs
was evaluated against the vector mosquitoes An. stephensi, Ae. aegypti and
Cx. quinquefasciatus. The LD50 and LD90 95 percent confidence limit of
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lower confidence limit (LCL) and (UCL) and Chi-square value were
calculated using by probit analysis.
CONCLUSION
Among the five plants aqueous leaf extract tested, the maximum
adulticidal activity was observed in M. emarginata against An. stephensi, Ae.
aegypti and Cx. quinquefasciatus. The maximum adulticidal activity was
reported with the M. emarginata mediated synthesized Ag NPs against An.
stephensi followed by Ae.aegypti and Cx. quinquefasciatus.
The biotoxicity of five plant aqueous extract and green-synthesized Ag
NPs was evaluated on non-target organisms Anisops bouvieri, Diplonychus
indicus, Gambusia affinis. Synthesized Ag NPs with M. emarginata was
tested against the non-target organism, the toxicity treatments achieved
negligible toxicity against A. bouvieri, D. indicus, and G. affinis
Focal observations highlighted that longevity and swimming activity of
the study species were not altered for a week after testing. SI indicated that
M. emarginata fabricated Ag NPs were less toxic to the non-target organism
tested if compared to the targeted mosquito larval populations. Therefore,
M. emarginata fabricated Ag NPs could be used to control the vectors of
mosquitoes in the fields.
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