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Chapter 4
Copyright: © 2017 Subbiah Poopathi, et al.
Control of Mosquito Vectors using
Biological Pesticides: An Integrative
Approach
Subbiah Poopathi1*, Lourduraj John De Britto1, Chinnasamy Mani1 and Somaiah Sundarapandian2
Vector Control Research Centre (Indian Council of
Medical Research), Department of Health Research,
Ministry of Health and Family Welfare, India
2
Department of Ecology and Environmental Sciences,
School of Life-Sciences, Pondicherry University, India
1
Corresponding Author: Subbiah Poopathi, Vector
Control Research Centre (Indian Council of Medical
Research), Department of Health Research, Ministry
of Health and Family Welfare, Indira Nagar, Puducherry-605 006, India, Tel: +91-413-2272475, 2272397
& 2272948; Fax: 91-413-2272041; Email: [email protected]
*
First Published February 15, 2017
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This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted use, distribution, and reproduction
in any medium, provided you give appropriate credit to
the original author(s) and the source.
Abstract
The mosquitoes are the most diverse group of insect
in environment with about 3500 species and which found
almost in every habitat, with the exception of the sea.
Mosquitoes are the most important single group of insects
in term of public health concern. These insect successfully
adaptable insect and coexist with man by feeding on him
and his domesticated animals and able to transmit most
important life threatening or disabling disease like malaria, lymphatic filariasis, west nile fever, dengue, Japanese
encephalitis, chikungunya in human beings. Hence, the
mosquito vectors are medically important phenomenon
and a series of dramatic discoveries during the late 1930s
led to the production of new synthetic insecticides, which
had enormous potential and which further reinforced
the use of chemical approach towards vector control. The
mosquito started to develop resistant against synthetic insecticides and therefore, since 1977, biological control of
mosquito has been carried out by biolarvicides, particularly the well known biocontrol agents such as B. thuringiensis and B. sphaericus etc.
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Introduction
Insects are the most diverse group of animals in environment with more than a million different species found
almost in every habitat, with the exception of the sea. At
the end of the nineteenth century, researchers discovered
that certain species of insect were accountable for the
transmission of some serious diseases to humans. Since
effective vaccines or drugs were not available for these diseases, the only means of prevention was to destroy these
insects, also known as ‘vector’, to prevent the diseases
from spreading. Due to their wide distribution, insects are
inevitably associated with an enormously large variety of
microscopic life forms, including viruses, bacteria, fungi,
protozoa, nematodes and other multi-cellular parasites.
Mosquitoes are the major vector capable of transmitting
most important life threatening or disabling disease like
malaria, lymphatic filariasis, west nile fever, dengue, Japanese encephalitis, chikungunya in human beings. Mosquitoes are found throughout the world except in places
that are permanently frozen. There are about 3,500 species
of which nearly three – quarters are native to the humid
tropics and subtropics [1]. Therefore, mosquito-borne
diseases are leading causes of illness and death among the
developing countries.
Mosquito Ecology
Mosquitoes are the most important single group of insects in terms of public health concern. These insects suc4
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cessfully adapt and coexist with man by feeding on him
and his domesticated animals. Mosquito belong to the
family Culicidae of the order Diptera (Two winged flies)
and the adults differ from other flies in having an elongated mouth or proboscis and scales on the wing veins
and wing margins. There are about 3500 mosquito species
belonging to 34 genera though out the world. Of these,
only about 300 species effectively transmit infection to human and animals. Mosquitoes belonging to three genera,
Culex, Anopheles and Aedes are well known for transmitting major mosquito borne diseases. The breeding habitats
of these mosquitoes are wide that include stable collection
of water, like marshes, rice fields, fresh water swamps and
burrow pits to smaller collections of temporary water such
as drains puddles, small pools ditches, gullies and variety of natural habitats water filled tree hole, rock pools,
water filled bamboo stumps, leaf axles, water filled coconut husks, grinding stone etc. While natural habitats provide ideal breeding grounds for mosquitoes in rural areas,
man-made habitats are the major concerns in urban areas
[2].
Mosquito Borne Diseases
Vector-borne diseases form a major component of
communicable diseases (filariasis, malaria, dengue and
Japanese encephalitis) in India and in other Asian countries. There are three main mosquito vectors namely Culex
quinquefasciatus, Aedes aegypti, and Anopheles stephensi,
which are responsible for millions of deaths worldwide.
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These diseases pose a major problem in disease-prevalent
countries and cause extensive morbidity and mortality
and are a major economic burden within diseases endemic countries [3]. These diseases are rampant owing to
amplified globalization, urbanization, and global warming [4], especially in many tropical and subtropical countries [5]. Malaria (derived from Italian word that means
bad air) is most deadly vector borne disease, transmitted
by mosquitoes of the genus Anopheles which kills more
than 1.2 million people annually, mainly African children
below the age of five. Poorly planned irrigation and water systems, inadequate housing, deforestation, and loss
of biodiversity, poor waste disposal and water storage are
the major contributing factors to the most common vector borne diseases including malaria. Malaria is a global emergency and also a public health problem today in
more than 90 countries, inhibited by a total of some 2400
million people, 40% of the world’s population. Malaria affected by four species of the genus Plasmodium (Plasmodium falciparum, P. vivax, P. ovale, and P. malariae) infect
by inoculation via the bite of infected blood-feeding female mosquitoes of the genus Anopheles, which transfer
parasites from human to humans. Plasmodium falciparum
is the main cause of severe clinical malaria and death.
Vectors of Lymphatic Filariasis
Otto Wucherer 1866, discovered microfilariae parasite
in the chylous urine of patients. In 1870, Tumothy Richard
Lew detected filariae in the blood of a patient, and later
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in 1876, Bancrofti in Australia could remove adult worm
from an abscess on the arm of a Chinese immigrant. It
was Sir Patrick Manson, in 1878, made the discovery that
mosquitoes transmit the nematodes, which causes filariasis.
WHO [6] described the mosquito species, viz: Cx.
quinquefasciatus, Culex pipiens molestus, Culex pipiens
pipiens, Anopheles sinensis, Anopheles gambiae, Anopheles melas, Anopheles merus, and Anopheles maculates
are natural vectors of periodic W. bancrofti. On the other
hand, sub-periodic W. bancrofti is transmitted by Anopheles niveus, Anopheles oceanicus, Aedes polynesiensis, and
Aedes pseudoscutellaris. In the case of B. malayi periodic
form, Mansonia annulifera, Mansonia uniformis, Anopheles nigerrimus, Anopheles umbrosis, Anopheles barbirostris
and Anopheles melas are known as natural vectors. Mansonia bonnese, Mansonia annulata and Mansonia dives are
the known vectors of B. malayi sub-periodic form. Anopheles barbirostris is the vector of B. timori. Filarial parasites
require two different host systems to complete their life
cycle. The definitive host is the man or some other vertebrate animal, depending upon the species of the parasite.
The intermediate host for extrinsic life cycle is a blood
sucking arthropod such as mosquitoes. Culex quinquesfasciatus is principal vector for transmitting Filariasis.
WHO [7] reported that Culex quinquefasciatus is a
principal house-resting mosquito in many tropical countries. It is imperative as a vector of Filariasis in some counwww.avidscience.com
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tries as well as an annoyance mosquito. Mosquitoes breed
in polluted waters such as blocked drains, damaged septic
tanks, or soak age pools close to human habitations. Lymphatic Filariasis is conceivably a wide spreading and disabling insect-borne disease of humans in the tropics, afflicts
about 146 million people (Figure 1). Culex quinquefasciatus is the most commonly distributed mosquito in India,
generally found in urban and suburban areas. The most
efficient approach to control the vector is to target the juvenile stages of the life cycle (Figure 2). Lymphatic Filariasis is a mosquito-borne disease caused by transmitted filarial nematodes, including Brugia malayi and Wuchereria
bancrofti. The infected people carry the nocturnally periodic W. bancrofti, which has Culex quinquefasciatus as
the key mosquito vector. Culex quinquefasciatus is a vector of lymphatic Filariasis, which is a widely distributed
tropical disease with around 120 million people infected
worldwide and 44 million people have common constant
manifestation [8]. The Indian subcontinent that comprise
Bangladesh, India, Maldives, Nepal and Sri Lanka harbours 50% of the world’s lymphatic filarial disease encumber (Figure 3). As per current epidemiological estimates
on prevalence of W. bancrofti and B.malayi about 428 million people are at risk, with 28 million microfilaria carriers
and 21 million clinical cases spread out in 13 States and 5
Union Territories of India [9]. In India alone, over 25 million people are infected with micro-filaria and 19 million
people endure from filarial disease manifestation.
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Figure 1: Global scenario of lymphatic filariasis transmitted by Culex
quinquefasciatus.
Figure 2: Life cycle of Wuchereria bancrofti.
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been reported in many parts of the country. The incidence
has been reported to be high among pediatric age group
with high mortality.
Figure 3: Symptom of Lymphatic Filariasis infected person
Source: Filarial clinic VCRC, Puducherry
Diseases of Epidemic Potential
Dengue is a fast expanding health problem in developed countries also. About two-fifths of the world population is at risk of acquiring dengue with 50-100 million cases of acute febrile illness yearly including about 5, 00,000
cases of DHF/DSS [10].
Chikungunya virus that shares the environment and
vector with dengue, re-emerged in 2005 and many countries in India Ocean suffered from outbreak. This outbreak
appears to be the most severe and one of the biggest outbreaks caused by this virus (Figure 4). India, where this
virus was last reported in 1973, is also one of the most affected countries. Japanese encephalitis (JE)-epidemics has
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Figure 4: Dengue risk map, 2013.
Mosquito Control Measures
History of Mosquito Control
WHO [11] stated that the vector control is an important element of the strategies used to control major vector
borne diseases globally and chemical remained the mostly
widely used approach in the past several decades. The first
recorded use of a synthetic organic insecticide, dinitro-ocresol was in 1892, and by the year 1930, a range of such
compounds had been discovered and they have found
limited use [12]. A series of dramatic discoveries during the late 1930s led to the production of new synthetic
insecticides, which had enormous potential and which
further reinforced the use of chemical approach towards
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vector control. In 1939, the most popular insecticide dichloro-diphenyl tricholoethane (DDT), an insecticide of
the chlorinated hydrocarbon group was introduced which
was later followed by the organophosphate insecticides.
Since World War II, disease control methods have relied
heavily on broad spectrum synthetic chemical insecticides
to reduce vector population.
Vector Control Strategies
1. Chemical control measures
2. Non chemical vector control measures
3. Biological control
Chemical Control Measures
Insecticide-Impregnated Paint
Several developed countries adopted this technology
during last decade. The vernacide is instance of insecticide-impregnated paint successful against mosquitoes
(Cules quinquefasciatus) [13]. This paint is safer than conventional water dispersible powder (WDP) formulation of
adulticides and can be conveniently employed in public
places where pest free conditions are desirable for considerably longer time.
Insecticide Impregnated Bednets
The impregnated synthetic pyrethroid insecticide
(deltamethrin) ropes, bed nets and curtains in the human
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dwellings were found to be promising against Anopheles
and Culex species. Mosquito nets treated with a waterdispersible tablet formulation of deltamethrin (K-O TAB)
was evaluated against malaria vectors and found to be
effective [14]. Olyset nets are permethrin insecticide impregnated betnets, recommended by WHO, and are currently in use in rural malaria endemic areas.
Non-Chemical Vector Control Measures
Surface Active Agent (SAA)
In this technology, non-ionic biological degradable
chemicals such as Arosurf which was used successfully
to control Culex quinquefasciatus, Anopheles stephensi
and Aedes aegypti in different breeding habitats (Cesspits,
cesspools, drains and wells) [15]. Arosurf act as a SAA
on the water surface to form a monomolecular film and
exhibits mortality of mosquito larvae. Arosurf in combination with fast acting and residual larvicide (fenthion)
enables better coverage of breeding habitats and long effective life of the film in inaccessible habitats like marshes
and lagoons.
Biological Control
During last decade, the bacilli based mosquito larvicides commonly recognized as biocides or biolarvicides
are becoming popular in vector control [16]. Nowadays,
lots of commercial formulations are available and can be
used in extensive mosquito control operations (Figure
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5). Several organisms have been investigated as possible
agents for vector mosquito control including viruses, fungi, bacteria, protozoa, nematodes, invertebrate predators
and fish. However, many of these agents were shown to be
of little operational use, largely because of the difficulties
in multiplying them in large quantities. Microbial control
agents as alternatives to chemical insecticides have been
successfully demonstrated [17]. Apart from chemotherapy, the mosquito borne disease can be controlled by implementing vector control measures by means of applying
insecticides thereby reducing the breeding potential
Figure 5: Different types of mosquito control
of the mosquitoes. There was success in vector control between 1950 and 1970 but worldwide resistance followed it to synthetic insecticides where they were used
intensively. Insect resistance to one or more categories
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of insecticides has limited the effectiveness of these compounds, and their non-selective mode of action adversely
affects non-target organisms [18]. For instance, global
DDT spraying to control mosquito populations succeeded
for only 8 years, as mosquito resistance appeared thereafter. As a result, synthetic chemical insecticides are being phased out in many countries and furthermore, many
governments restrict chemical insecticide use owing to
concerns over their environmental effects on non-target
beneficial insects and especially on vertebrates through
contamination of food and water supplies [19].
As a result, WHO facilitated the replacement of
these chemicals with bacterial insecticides through the
development of standards for their registration and use.
The current interest in the development of biological
agents for the control of vectors, especially mosquitoes
is an indication of concern and sheer helplessness faced
by the scientific community in the recrudescence of mosquito borne diseases like malaria, and dengue in epidemic
proportions which was under control during fifties. The
simplest definition for biological control is, “direct and indirect manipulation of natural enemies (pathogens, parasites and predators) to increase the incidence of mortality
in the mosquito population under attack”. The different
biological control agents being studied in different parts
of the world for the control of vector mosquitoes includes
many naturally occurring predators, parasites, and pathogens of vector insects including fungi, and bacteria.
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Bacterial Larvicidal Agents
Bacillus Thuringiensis
Bacillus thuringiensis (Bt) is a gram-positive bacterium that produces insecticidal crystal protein toxins during sporulation. B. thuringiensis was first discovered in diseased silkworms in 1901 [20]. In 1977, the first Bt strain,
ONR-60A demonstrating a high level of larvicidal activity,
was isolated from a mosquito-breeding pond in the Negev
Desert of Israel and was found to be highly active towards
dipteran larvae. This strain was later acknowledged as
a new H antigenic type H-14 of B. thuringiensis and assigned the name subspecies israelensis [21]. This ubiquitous spore forming bacterium was an eye-opener for the
biologists to explore the potential of these crystalliferous
bacteria in biological control of vector mosquitoes. Ever
since, the discovery of the first Bt strain capable of killing mosquito larvae, several subspecies of Bt have been
isolated from a range of environments, including insects,
soil, dust from stored grain, and leaves of coniferous and
deciduous trees. There are several reports from different
parts of the world about the occurrence of mosquitocidal
strains belonging to different subspecies/serotypes. Bti
contains four major proteins – CytIA (27.3 kDa), Cry4A
(128 kDa), Cry4B (134 kDa) and CryIIA (72 kDa) in three
different inclusion types assembled into a spherical parasporal body held together by lamellar envelope.
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Bacillus Sphaericus
Bacillus sphaericus (Bs) is widespread in soil and aquatic environments [22]. The mosquitocidal strain studied
extensively belongs to serotype H5a5b (e.g., Strains 1593
and 2362), which is highly toxic. In general, members of
highly toxic groups have certain positive characteristics
which are relevant to their use as microbial insecticides:
their toxic crystals are protected within the exosporium,
they are stable over a range of temperature, and they may
remain insecticidal in polluted water [23]. The major
components of the crystal are two proteins i.e. the binary
of Bin toxin 51 and 42 kDa. Either protein alone is toxic
to larvae or are both required for toxicity [24]. In addition
to the binary toxin, many strains of Bs produce other mos
quitocidal toxins during vegetative growth that are referred to as Mtx toxins. But these are not as toxic as the
Bin toxin [25]. The target spectrum of Bs is restricted to
mosquitoes, and its highest activity is against Culex and
certain Anopheles species [26]. Resistance to Bs has already been reported in field populations of
Culex mosquitoes [26].
Bacillus Alvei and Bacillus Brevis
From rice fields of Pondicherry, India two indigenous
bacterial pathogens of mosquitoes viz. Bacillus alvei and
Bacillus brevis were isolated from dead culicine larvae. The
cell mass of these bacteria was highly effective against the
larvae of Culex fatigans, Anopheles stephensi and Aedes aewww.avidscience.com
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gypti. Recently Khyami Horani et al. [27] isolated B.brevis
toxic to Culuseta longiareolata (Diptera: Culicidae) from
soil and water samples collected from Jordan.
Bacillus Circulans
A new strain of Bacillus circulans isolated from a larva of Cx. quinquefasciatus showed larvicidal activity on 3
mosquitoes of medical importance. Compared to B. sphaericus 2362, this B. circulans isolate proved less toxic to
Cx. quinquefasciatus and Anopheles gambiae but was 107
times more toxic to Ae. aegypti. The tests have showed
that the toxicity of the bacterial culture of B. circulans resulted from its spores and not from the insecticidal effect
of chitinases or exotoxins.
Brevibacillus Laterosporus
Brevibacillus laterosporus previously classified as Bacillus laterosporus [28] and aerobic spore-forming bacterium that is characterized by its ability to produce a
parasporal inclusion adjacent to the spore. Some strains
produce crystalline inclusion of various shaped and sizes,
which are related separately from spores during lysis of
the sporangium. B. laterosporus has the potential to be
used as a biological control agent which, in comparison
with strains of B. thuringiensis and B. sphaericus, demonstrates a very wide spectrum of biological activities. Toxicity of these bacteria towards larvae of the mosquitoes Cx.
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quinquefasciatus and Ae. aegypti has been reported.
Despite showing such wide-ranging biological activities, B. laterosporus has not been seriously considered
for use in biological control, most probably because the
observed mosquitocidal activity is generally much weaker
than that of B. thuringiesis subsp. israelensis. Yet Orlove et
al. [29] demonstrated that crystalliferous strains B. laterosporus presented LC50 values similar to those attained with
B. thuringiesis subsp. israelensis in bioassays employing
larvae of three species of mosquitoes, with the larvicidal
activity of B. laterosporus being associated with spores and
crystalline inclusions.
Bacillus Subtilis
Bacillus subtilis is a ubiquitous bacterium commonly
recovered from water, soil, air and decomposing plant residue. The bacterium produced an endospore that allows
it to endure extreme conditions of heat and desiccation
in the environment. Bacillus subtilis produces a variety of
proteases and other enzymes that enable it to degrade a
variety of natural substrates and contribute to nutrient cycling. However, under unfavourable conditions the organism is not biologically active but exists in the spore form
[30].
As early as in 1980, Gupta and Vyas reported a strain
of B. subtilis capable of infecting and causing mortality of
larvae of Anopheles culicifacies, the primary vector of malaria in central India. Recently, Das and Mukherjee [31]
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have reported the mosquito larvicidal activity of B. subtilis (DM-03 and DM-04) strains. The mosquito larvicidal
activity is by the cyclic lipopeptides (CLPs) secreted by
B. subtilis strains. The LC50 of the crude CLPs secreted by
B. subtilis DM-03 and DM-04 strains against third instars
larvae of Cx. quinquefasciatus was 120.0+/-8.0 mg/l respectively post 24 hour of treatment. Also, physic chemical factors such as pH of water, incubation, temperature,
heating and exposure to sunlight hardly influenced the
larvicidal potency of these CLPs and were safe to Indian
major carp Labeo rohita, a non-target aquatic organism.
Clostridium Bifermentans
A nationwide screening program in Malaysia for microbial control agents of mosquitoes resulted in the isolation of Clostridium bifermentans, an anaerobe, by Lee and
Seleena [32]. This new strain of Clostridium bifermentans,
individualized as serovar Malaysia (C.b.m.) according to
its specific H antigen is toxic to mosquito and blackfly larvae when given orally. The toxicity exhibited in sporulated
cells, which contain, in addition to spores, heat labile proteinaceous parasporal inclusion bodies and feather-like
appendages. The amino acid content of the inclusion bodies is similar to that of B. thuringiesis ssp. israelensis and
B. sphaericus crystals. Cell of C. bifermentans ssp.malaysia
are safe for laboratory mammals and goldfish [33].
Another strain of C. bifermentans toxic to mosquito larvae on ingestion was isolated from a soil sample
collected from secondary forest floor [34]. This strain was
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designated as serovar Paraiba (C. bifermentans paraiba)
according to its specific H antigen. C. bifermentans araiba
is most toxic to Anopheles maculates Theo bald larvae and
its activity is as high as that of B. thuringiesis ssp. israelensis.
Bacterial Pupicidal Agent
Pseudomonas Fluorescens
Since 1977, biological control of mosquito was carried
out by biolarvicides and till 2002 none of the biological
control agents were reported to be mosquito pupicidal i.e.
the ability to kill the pupal stages of mosquitoes. The first
bacterium known to exhibit mosquito pupicidal activity is
a gram-negative bacterium Pseudomonas fluorescens. The
metabolites were toxic to larvae and pupae of mosquitoes
[35].
A formulation was developed from the metabolites(s)
of P. fluorescens Migula strain (VCRC B426) and tested
against 4th-instar larvae and pupae of three species of vector mosquitoes, An. stephensi liston, Cx. quinquefasciatus
Say and Aed. aegypti (L). The larvae and pupae of An. stephensi were the most susceptible followed by those of Cx.
quinquefasciatus and Ae. aegypti and the dosage requirement for pupal mortality was less than that required for
larval mortality. The LC50 dosage requirements for larvae
of these mosquito species were, respectively, 70.4, 511.5
and 757.3 µl protein ml (-1), whereas for pupae they were,
respectively, 2.0, 9.4 and 19.2 µl protein ml (-1). The lethal
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fraction was purified from the culture broth and its molecular mass, as determined by high performance liquid
chromatography (HPLC), was 44 kDa [35]. Further, an
emulsifiable concentrate (EC) formulation developed
from a metabolite of P. fluorescens was tested for efficacy
against Cx. quinquefasciatus larvae and pupae under field
conditions. At application rates of 100, 200, 300 ml/m2,
the formulation caused 100% elimination of larvae and
pupae at day 1 after treatments and >80% reduction in pupal density for periods of 7, 12 and 11 days in cesspits and
5, 9 and 10 days in U-shaped drains [36].
Bacterial Diversity of
Swamps and Sediments
Mangrove
Estuarine, mangrove and coral reef environs in Gulf
of Mannar was studied by Kannapiran et al. [37] for the
isolation of magnetotactic bacteria. Totally 37 strains were
isolated with predominance of Bacillus spp. followed by
Pseudomonas spp. Spirillium spp., and Vibrio spp. Based
on the fatty acid profile, a few of the strains were identified as Pseudomonas mesophilico and Bacillus cereusi. The
sediments of the mangrove swamps of Cochin showed the
presence of Aeromonas sp., Alcaligenes sp., Bacillus sp.,
Flavobacterium sp. Micrococcus sp., Pseudomonas sp. and
Vibrio sp [38].
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Bacterial Diversity of Mangroves of
Andaman & Nicobar Islands
The mangrove of Andaman and Nicobar Islands constitute 9.4% of land area and -10.95% of the forest cover
of these islands. The fauna and flora of these islands was
studied.
Shome et al. [39] investigated the bacterial flora of
mangrove litter fall and underneath sediments from South
Andaman. Thirty-eight bacterial isolates were obtained
from Rhizophora, Avicenia and Nypa species inhabited areas. The cultural, morphological and biochemical features
revealed that most of the isolates belong to Bacillus spp
(50%). In addition, Aeromonas, Vibrio, Escherichia, Enterobacter, Corynebacterium, Kurthia, Staphylococcus, Micrococcus and Listeria were also present. Most of the isolated
were gram positive (76.3%), motile (87%) and fermentative bacteria.
Serpentine soils collected from Saddle Hills,
Chidyatapu and Rutland of Andaman islands, India were
analysed for physic-chemical and microbiological characteristics and compared with those from adjacent nonserpentine localities. The serpentine soils contained high
levels of nickel (1740.9-8033.4 mg/kg dry soil), cobalt
(93.2-533.4 mg/kg dry soil) and chromium (302.9-4437.4
mg/kg dry soil), in addition to 62-152 g of iron and 3760 g of magnesium per kg dry soil. Characteristically the
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serpentine soils showed low microbial density (6.2-11.3
106 colony forming unit/g soil) and activity (1.7-3.5 µg
fluorescein/g dry soil/h) than non-serpentine outcrops.
Serpentine microbial population was dominated by bacteria, which represented 5.12 to 9.5 106cfu/g of soil, while
the fungal population ranged from 0.17 to 3.21 106 cfu/g of
soil [40].
Mosquitocidal Bacteria from Mangrove
C. bifermentans and B. thuringiensis subsp. israelesis/
tochigiensis were isolated from mangrove swamps and
mangrove sediments of Malaysia and Japan [41]. In the
light of this, the mangrove forests of Andaman-Nicobar
Islands were explored for the microbial diversity looking
for the mosquitocidal bacterial flora of these islands.
Biology of B. subtilis
Taxonomy and Characterization
The genus Bacillus is a large and heterogeneous collection of aerobic or facultative anaerobic, rod shaped,
endospore-forming bacteria that are widely distributed
in the environment. Many kinds of species belong to this
genus. They are known to have acidophilic, alkalophilic,
thermophilic or other properties [42]. The genus Bacillus
encompasses 203 validly described species (http://www.
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bacterio.cict.fr/bacillus.html) exhibiting a wide range of
nutritional requirements, physiological and metabolic diversity and DNA base composition. B. subtilis is a ubiquitous soil microorganism that contributes to nutrient cycling due to the various enzymes produced by members
of the species. This bacterium occurs at population levels
of 106 to 107 per gram of soil [30]. However, unless a soil
has been recently amended with organic matter providing readily utilizable nutrients, the bacteria exist in the
endospore stage. About 60 to 100% of soil bacilli populations exist in the inactive spore state [3].
Historically, prior to the monographs of Smith
in1946 and 1952, B.subtilis was a term given to all aerobic
endospore-forming bacilli. The Bacillus species subtilis, licheniformis and pumilus are closely related and there has
been difficulty in distinguishing among the three species
that historically were grouped together as the subtilisgroup or subtilis-spectrum. These three species clustered
together (78%) in the “subtilis” group in a numerical classification based on 118 unit characteristics of 368 strains
of Bacillus. The reclassification of genus Bacillus, which
began in 1991, revealed at least eight genera: Alicyclobacillus, Aneurinibacillus, Bacillus, Brevibacillus [28], Gracilibacillus, Paenibacillus, Salibacillus and Virgibacillus. Since
these eight genera consist of more than 100 species that
have similar characteristics, identifying them is difficult.
The identification of Bacillus species has been performed
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mainly with morphological and physiological criteria, and
this method is widely employed in various fields. However, the process used requires skilful techniques and is
very complex and time-consuming. Hence, the reliance
on only biochemical-based identification could lead to inaccurate identification of the genus Bacillus.
Molecular Taxonomy
With the advance of genetic engineering, the randomly amplified polymorphic DNA (RAPD) method
[43], the hybridization method and restriction mapping
were adapted for the identification of Bacillus species. Recently MALDI-TOP-MS and oligonucleotide microarrays
are also being employed for the identification [44].
The utility of the rRNA sequence as a taxonomic
tool has been amply demonstrated in bacteria, where 16S
rRNA sequence analyses have completely redefined phylogenetic relationships previously too dependent on cellular metabolism [45]. In addition to highly conserved areas
that have been used to study the relationships among distant taxa, the 16S sequence contains more variable regions
that have been useful in the differentiation of genera and
species [46]. This differentiation has been accomplished
through the use of probes which have been generated by
using conserved 16S sequences as universal primers for
polymerase chain reaction (PCR) amplification of certain
variable 16S regions [47].16S rDNA probes have been
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used for the identification of campylobacters, gram positive cocci, Klebsiella, Nocardia, Leuconostoc, Streptomyces,
Clostridia, Vibrio.
DNA base (GC) composition of species within a
genus should not differ by more than 10 to 12% mol GC.
Nonetheless, values within the Bacillus genus ranged from
33 to 65% mol GC in 1993, although many of the species
did cluster at 40 to 50% mol G_C [48]. Subsequently, recent phylogenetic analyses have reclassified some of the
Bacillus species into new genera, including Paenibacillus,
Geobacillus and Brevibacillus [49]. It has been reported
that there are two subspecies within B. subtilis viz: B. subtilis subsp. subtilis and B. subtilis subsp. spizizenii, which
share phenotypic profile but are segregated based on DNA
re-association values of 58 to 69%, in addition to minor
polymorphisms in the 16S rRNA gene between the type
strains [50]. Due to these recent advances, it has become
increasingly difficult to classify species within the Bacillus
genus, as many share similar physiology metabolism and
morphology as well as highly conserved 16S rRNA genes.
Other Biological Control Agents
Cyclopoid Copepods
Cyclopoid copepods like Macrocyclops distictus, Mesocyclops pehpeiensis, and Megacyclops viridis are used as
control agents against dengue vector Aedes albopictus
in japan [51]. Mesocyclops spp., aided by the corixid bug
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Micronecta quadristrigata was also utilised in Vietnam
against Aedes aegypti [52]. Mesocyclops spp., a predacious
copepod, was found to be an effective larvicide against Aedes aegypti in Vietnam. Use of predacious copepods of the
genus Mesocyclops as a biological control agent; delivered
by community activities of health volunteers, schools and
public was found to be effective against Aed. aegypti in Vietnam [53].
Larvivorous Fishes
Larvivorous fishes were the first biocontrol agents employed to control mosquito vectors. Fish have been used
in many countries for malaria control by controlling vectors. Of these, the common varieties utilized as biocontrol
agents are the mosquito fish (Gambusia affinis), Guppies
(Poecilia reticulata), Aplicheilus blochii, Macropodus and
a variety of other local and indigenous fishes as per their
availability in the local habitat. Many indigenous varieties
of fishes are available and their larvivorous potential has
been studied. In different countries the local fishes available have been explored to exploit their use against Anopheles and Culicine larvae. The ability of 2 freshwater fishes, eastern rainbow fish Melanotaenia splendid and flies
pecked hardy head Craterocephalus stercusmuscarum, native to North Queensland to prey on immature Ae. aegypti
was evaluated. Larvivorous fish Oreochromis spilurus was
found to be effective against malaria vectors in Somalia.
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Mermithid Nematodes
Mermithids like Romanomermis iyengari, R. Culicivorax and Octomyomermis muspratti show very high specificity to mosquito larvae. They must undergo part of their
development within mosquito larvae and they recycle and
infect mosquito larvae season in nature. However since
they cannot tolerate extreme pH and pollution they are
yet to be developed for use in polluted habitats. R. iyengari and R. culicivorax have a broad host range and are
promising bio control agents of various species of mosquitoes. Mermithid nematodes were found parasitizing Cx.
quinquefasciatus as early as 1906 and subsequent records
show that larvae of several species of Anophelines were
found infected [54]. The mermithid nematode R. iyengari
found parasitizing mosquito larvae in paddy fields [55],
has been successfully mass cultured in the laboratory,
found safe against non-target organisms and has been
tested in the field also [56]. But it is suitable for fresh water
habitats alone as habitats with high pH and salinity is detrimental. The nematode Strelkovimermis spiculatus was
also found to be a promising biological control against Cx.
quinquefasciatus in Cuba [57].
Dragonfly Nymphs
Biocontrol potential of dragonfly nymph Brachythemis
contaminate against the larvae of An. stephensi, Cx.
quinquefasciatus and Ae. aegypti was conducted and
found that they had good predatory potential and can be
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used as a biological control agent for control of mosquito
breeding [58].
Protozoa
Microsporidians such as Nosema, Thelohania, Parathelohania, Amblyospora and Vavraia have been studied in
detail for mosquito control efficacy. Selective infection of
Anopheles larvae with some ciliates belonging to the genus
Lamborella was first reported from forest areas of Assam.
Natural infection was found in the immature of An. barbirostris, An. hyrcanus and An. philippinesis. However, none
of these agents are yet ready for field application. Anopheline larvae are parasitized by Thelohania spp., Nosema algerae were infective to Cx. quinquefasciatus, Aed. aegypti,
An. stephensi and Armigeres subalbatus [59].
Predatory Mosquitoes
Toxorhyncities splendens is a non-blood sucking predatory mosquito whose larvae were found to be effective
in controlling Anopheline and Culicine larvae by feeding
on them [60]. The predatory efficacy of this mosquito was
also proved in field evaluation studies against thirteen species of mosquito’s sp. Cules spp., and Aedes spp., conducted
in Japan [61]. Ae. aegypti populations were suppressed by
Toxorhyncites splendens larvae in household water storage
containers in Jakarta [62].
Viruses
Several viruses such as Iridescnt virus, Densonucleosis
virus, Cytoplasmic polydedrosis virus, Nuclear polyhedro30
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sis virus etc. have been evaluated in the past for mosquito
control [63]. These viruses attack a wide range of tissues
and although they are highly lethal to their hosts, and are
not very infectious. While the problems of insufficient infectivity or virulence handicap the development of viruses
as biocontrol agents, the most serious obstacle to their development and use is the non-availability of an efficient
method for their mass production as the viruses are highly
specific obligate pathogens.
Entomopathogenic Fungus
Many fungi such as Coelomomyces, Lagenidium have
been isolated and tested [63]. Coelomomyces is an obligate
parasite with a complex lifecycle in which an alternate
crustacean host is required to complete the life cycle. Lagenidium can be grown artificial media and can maintain
itself in a habitat without the presence of a host. But it is
yet to reach large scale testing because its infective propagules pose certain problems such as fragility of zoospores
and asynchronous and poor germination of oospores [64].
The entomopathogenic fungus, Metarhizium anisopliae,
was found to be effective against Anopheles gambiae (malaria vector) and Cx. quinquefasciatus (filariasis vector)
insect pathogenic fungi of the Hypocrellal aschersonia
group might be useful as an agent for pest control. Coelomomyces and Culicinomyces are known to affect mosquito
populations and have been studied extensively [55]. The
fungal mosquito pathogen Leptolegnia chapmanii (ARSEF 5499) was tested against 12 species of mosquito larvae and on species of non-target aquatic invertebrates and
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vertebrates was found to be effective against Anopheline
and Culicine mosquitoes. Efficacy of fungal metabolites
of Chrysosporium tropicum was evaluated against Cx.
quinquefasciatus larvae in the laboratory and found to
have promising effects [65].
Poopathi et al, [22] reported for the biosynthesis of silver
nanoparticles from neem extract (A. indica) as an agent
with the most potential for the control of mosquito vectors.
Nanoparticles Used for Mosquito
Control Operation
It was thought that development of insect resistance
against Bs and Bti would not appear, due to possible multisite interactions between the pathogens and their targets.
Indeed, no records of field resistance have been found to
Bti, because of the presence of the four different toxins
with putative different modes of action. For Bs, the Bin
toxin has to be considered as one site-acting molecule, because of the single receptor interaction with Bin B component (at least in C. pipiens). For Bs cases of resistance
have been recorded during the last four years, in Brazil (10
fold-resistance [70], in India (150 fold [71] and in France
on C. pipiens (10,000 fold). More recently, two other reports from China (25,000 fold) and Tunisia (2,000 fold;
Yuan and Sinègre, pers. commun.), confirmed that resistance to Bs may develop in the field when this bacteria is
used intensively. Before records of field resistance to Bs,
active laboratory selections for resistance had been done
in two different laboratories in California (>100,000 fold
[72]).
Nanoparticle-based methods, particularly silver nanoparticles, which are reported to possess antifungal, antiinflammatory, and anti-viral activity [66], have attracted
greater attention owing to their wide applications. These
nanoparticles are emerging as one of the fastest growing
materials because of their unique physical, chemical, and
biological properties. Small size and high specific surface
area have led to the development of newer biocidal agents
and alternatives to synthetic and microbial biopesticides
[67]. However, the synthesis of nanoparticles by chemical
and physical methods requires high pressure, energy, temperature, and toxic chemicals. Therefore, plant extracts
are suitably scaled up for large scale biosynthesis of silver
nanoparticles in a controlled manner according to their
size, shape, and sensitivity. In this manner, several plant
species have been utilized for synthesis of silver nanoparticles [68]. A green approach for the production of stable,
bioactive silver nanoparticles using Pseudomonas stutzeri,
Verticilium spp., Fusarium oxysporum, Thermomonospora
spp, Medicago sativa, and A. indica has been reported [69].
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Resistance Phenomena
Mechanisms of Resistance to Bs
In vitro binding investigations between the toxin and
midgut BBMF from three resistant Culex populations gave
some knowledge about the mechanisms of resistance. For
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the high-level resistant lab-selected colony, no binding
was found, meaning that the receptor was not functional
[73]. For the high-level resistant population from France
and the low-level resistant population from Brazil (both
field-selected), no changes were found in binding kinetics
[74]. Furthermore, the gut juice proteases from this colony were able to proteolyse the protoxins to the activated
forms. Then, if the Bs crystal toxin has selected highly resistant individuals possessing a mutation influencing the
initial toxin-binding in one case, in the other case the same
toxin selected highly resistant individuals expressing their
resistance at another level of the intoxication process.
Cross-Resistance to Bs[MH]
In the treated areas, only three different Bs were used,
2362, 1593 and C3- 41, all belonging to serotype H5a5b,
which express the same crystal toxin (identical amino acid
compositions. These are used in most commercial Bs formulations. Investigations on the level of cross-resistance
among natural Bs have been done by testing the toxicity of
several highly active Bs on some of the Bs-resistant Culex
colonies. For the laboratory-selected low-level resistant
colony from California, cross resistance was found to
strain 2297 [75]. This was also the case for the field-selected population from India. However, among five other Bs
isolated from Ghana and Singapore, we have found at least
two, which seem to confer only a very low level of crossresistance. These are presently under investigation [73].
There is no cross resistance to Bti within the populations
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resistant to Bs, and there is even evidence for an increased
susceptibility to Bti. This is in agreement with the finding
that the crystal toxin from Bs and the crystal toxins from
Bti do not compete for the same binding sites.
Implementation of Cost Effective
Technology
The use of a conserved, housekeeping gene necessary
for the survival of the organism was reported as the desirable alternative in molecular taxonomy. Protein coding genes exhibit much higher genetic variation than 16S
rRNA gene and can be used for classification and identification of closely related taxa [76]. Chun and Bae [77]
demonstrated the use of gyrA sequences (coding for DNA
gyrase submit A) for accurate classification of Bacillus subtilis and related taxa, including bacillus Amyloliquefaciens,
Bacillus vallismortis, Worldwide 24 billion chickens are
killed annually and around 8.5 billion tonnes of poultry
feather are produced. According to a recent report in leading newspaper India’s contribution alone is 350 million
tonnes. The poultry feathers are dumped, used for land
filling, incinerated or buried, which involves problems
in storage, handling, emissions control and ash disposal.
Discarded feather also causes various human ailments including chlorosis, mycoplasmosis and fowl cholera [78].
Feather is pure keratin protein and is insoluble and hard
to degrade due to highly rigid structure rendered by extensive disulphide bond and cross-linkages. The keratin
chain is insoluble, high stable structure tightly packed in
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the “α helix (“-Keratin) and β-sheets (β-keratin) into super coiled polypeptide chain. 90% of the feather contain
$-keratin by mass [79] and $-keratin are extensively cross
linked. Cross-linking of protein chains by cysteine bridges
confers high mechanical stability and resistance to proteolytic degradation by pepsin, trypsin and papain. The disulphide bonds of β-keratin can be reduced by the enzyme
disulphide reductase followed by proteolyitc keratinases
[80]. Feather can be utilized so that it can be used as animal feed, this can prevent accumulation of feather in the
environment and decrease the development of pathogenic
strains. Biotechnological processing of feathers for the
production of feather meal, instead of chemical processing is preferred as it preserves the essential amino acids
(Methionine, Lysine, Histidine) [81]. Innovative solution
for waste disposal along with biotechnological alternative for recycling of such wastes is of utmost importance.
Structural keratin can be degraded by some proteolytic
micro-organisms as reported by [79]. Keratinase are specific proteases that degrade keratin specifically. It is produced by saprophytic and dermatophytic Fungi and some
Bacillus species. Feather degrading bacteria are physiologically diverse and approximately 99% of Bacterial
species are uncultivable because of their ability to enter
non cultivable state or because no culture methods have
been established. A number of keratinolytic microorganisms have been reported, including some species of fungi
such as Microsporum, Trichophyton and from the bacteria
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Bacillus and Streptomyces and Actinomycetes. Increase in
keratinolytic activity is also found to be associated with
thermophilic organisms, which require high energy inputs to achieve maximum growth and the decomposition
of keratin wastes [82]. Till date, most of purified keratinizes known cannot completely solubilize native keratin,
their exact nature and uniqueness for keratinolysis is still
not clear. There is always a requirement of isolation of enzymes from new sources to meet the industrial and environmental demand. Keratinolytic enzymes have found
important utilities in biotechnological processes involving keratin-containing wastes from poultry and leather
industries, through the development of non-polluting
processes. After hydrolysis, the feathers can be converted to feedstuffs, fertilizers, glues, films and as the source
of rare amino acids, such as serine, cysteine and proline.
In this study, we report the isolation of three mesophilic
bacteria that produce keratinolytic enzymes. This can efficiently degrade chicken and pigeon feather within 120
hrs of incubation. Earlier studies from our lab involving
screening of micro-organism from same soil sample of
dumping site of Gazipur poultry processing plant, we have
reported isolation of Pseudomonas thermaerum GW1,
GenBank accession GU95151, this bacteria showed proteolytic activity but not keratinolytic activity [83]. Poultry
farms produce enormous feather waste in all over world.
This is a reflection of the properties of the ecological niche
occupied by the insects and the associated microbes, the
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needs of the insect or the microorganism, and the genetic
mechanisms used by the microorganism to establish the
interaction.
Priorities for Current Research
Emerging consequence of microbial bio-pesticides
in insect control activities has encouraged many research
programs aiming to discover new bacterial strains as alternative to existing bio-pesticides. Further, it is specific to
mention that with the increasing problem of resistance to
available mosquitocidal agents (Bs and Bti), increasing attention has been devoted to search for alternative mosquitocidal agents globally. Recently, we have reported for the
first time that a marine B. cereus (Bc) isolated from the gut
content of the marine fish (Lutjanus sanguineous) in the
East coastal zone of Bay of Bengal at Pondicherry (India)
has been shown to be a promising agent in controlling the
mosquito vectors (Cx. quinquefasciatus, An. stephensi, and
Ae. aegypti). We further report that this mosquitocidal activity is due to a single insecticidal protein (51.7 kDa) recognized from B. cereus as “Surface layer protein (SLP)”.
Recent study in our laboratory on biopesticide production using cost-effective culture medium has revealed
the potential of utilizing the chicken feather waste (CFW)
from poultry industries/shops for Bti production. In continuation of this study, we now report for the first time,
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the purification and characterization of the enzyme, produced by Bti, responsible for the biodegradation of feather
waste. Eventually, the outcome of this study will not only
highlight the potential use of the bird feather waste for the
production of mosquitocidal Bti, but also the management of solid waste of this kind.
Mosquito Control Benefits and Their
Risks
In mosquito control, the current modern mosquito
control method poses some ecological problem; however,
it evidently provides reimbursements. The public health
concern, enhanced human comport from mosquito problem including mosquito biting, annoyance and socio-economic problems are the most evidently benefits whereas
risk on the some other non-target organisms such as fish,
wildlife, non-target arthropods and also growing risk on
the human being during the exposure to pesticides in
commonly. Potential impacts on human as well as other
natural sources need to be critically analysed for specific
bio-control application of these organisms. The purpose
of this review is to present an overview of the diversity of
associations between insects and bacteria and their potential and current applications. In the first part, we describe
different types of interactions between these groups of
organisms and their characteristics, highlighting several
examples and focusing in the molecular mechanisms unwww.avidscience.com
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derlying these interactions. In the second part, we review
the features of these interactions with potential for insect
control, including insecticidal toxins, and summarise several strategies and recent developments with agricultural
and epidemiological implications.
The entire bacterial groups produce parasporal endotoxin crystal during sporulation and these crystal proteins
are supposed to be responsible for mosquitocidal activity.
Among mosquitocidal bacteria identified, B. thuringiensis
serovar israelensis (Bti) is the most potent, effective and
produces foremost mosquitocidal toxins of Cry4A, Cry4B,
Cry11A, and Cyt1A in a parasporal body. Followed by this
bacterium, the B. sphaericus (Bs) ranked next on its potency and produces two major endotoxins of 51 and 42
kDa proteins. Inspite of its advantages, a high level of resistance to Bs has been reported from several countries.
A recent report on the development of resistance to Bti is
inevitable.
Acknowledgements
The authors acknowledge Dr. P. Jamulingam, Director VCRC, Pondicherry-605006, for providing the institutional facilities. They also acknowledge Mr. S. Kandasamy,
Technical Assistant VCRC, Pondicherry for providing resources for references.
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