Chapter 2 Review of Literature
2. REVIEW OF LITERATURE
There are about three thousand five hundred species of mosquitoes found throughout the
world. Mosquitoes are foremost in man's war against insects. The blood sucking arthropod
not only cause nuisance by their irritating bites but also create health menace, as they are
responsible for spreading serious diseases like Malaria, Dengue, Yellow fever, Japanese
Encephalitis, Chikungunya etc. Several synthetic chemicals have been used for many years
to control vector population in order to prevent people from mosquito borne diseases. A
growing global awareness towards environmental hazards, caused by chemical insecticides
development of resistance in vectors against these chemicals necessitate the search for new
and effective alternative approaches for an effective and environmental friendly mosquito
control. Therefore, fungal control can thus provide an effective and environmental friendly
and green approach, which can be used as an alternative to chemical insecticides in order
to minimize the mosquito population. More importantly, the incidences of resistance to
larvicide of mosquito larvae have been reported also. Thus, attempt to develop novel
materials as mosquito larvicides are still necessary. Metal nanoparticles are emerging as
one of the fastest growing effective materials for controlling diseases due to their unique
physical and chemical properties, small size and high specific surface area and geometry
which needs integration with larvicides and adulticides.
2.1 ENTOMOPATHOGENIC FUNGI
2.1.1 Phylum Oomycota
Several species of entomopathogenic fungi have been investigated so far for many years.
Amongst them, Oomycetes fungi have proved their potential as biocontrol agents for
mosquitoes. Most of the Oomycetes produce two distinct types of spores. The main
dispersive spores are asexual, self-motile spores called zoospores, which are also capable
of chemotaxis. Asexual reproduction by means of biflagellate zoospores is the
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Chapter 2 Review of Literature
characteristic feature of this phylum. There are about sixty-five genera of Oomycetes and
some 500-800 species (Dick 1990).
2.1.1.1 Genus Lagenidium
Only one species of the genus Lagenidium is known to be a facultative parasite of
mosquito larvae, namely Lagenidium giganteum. It consists of too stages oospores
(sexual), and zoospores (asexual). L. giganteum is one of the most promising oomycetous
fungus, which was originally described in 1935 (Couch 1935).
L. giganteum is a
facultative parasite of mosquito larvae (McCray et al. 1973). It infects the mosquito larvae
by mechanical and enzymatic activity of encysted biflagellate motile zoospores. However,
it has some restrictive environmental limitations, such as a low tolerance for organic water
pollution (Jaronski and Axtell 1982) and salinity (Marriam and Axtell 1982). It has also the
ability to recycle in nature (Lasko and Washino 1983, Jaronski and Axtell 1983b). A study
was conducted to determine the effects of dilution in deionized water and in weak
solutions of organic compounds on the durations of activity of L. giganteum zoospore
(Lord and Roberts 1985).
A cytological description of the zoospores was provided with electron
microphotographs (Domnas et al. 1986). Some experiments were carried out in India to
ascertain the ability of an indigenous Lagenidium strain to produce zoospores in vitro
under different cultural conditions (Balaraman and Hoti 1986). In that experiment, the
effects of nutrition, growth time, H-ion concentration, salinity and two insecticides were
studied on the zoosporogenesis. While the procedures for encapsulation of sexual and
asexual stages of L. giganteum cultures were described on sunflower seed extracts (Axtell
and Guzman 1987). Consequently, the effects of L. giganteum were reported on nonmammalian non-target organisms, including green plants, algae, aquatic and terrestrial
insects, copepods, fishes and ducks (Kerwin et al. 1988). Whereas the safety of L.
giganteum was confirmed to mice following interperitoneal and intravenous injection and
no discernible effects were found on these animals (Kerwin et al. 1990). The compatibility
of Bacillus thuringienesis var. israelensis and Bacillus sphaericus with the fungal
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Chapter 2 Review of Literature
pathogen Coelomomyces giganteum (Oomycetes: Lagenidiales) have been evaluated
(Orduz and Axtell 1991). They reported high virulence for 1-2 day old larvae, intermediate
mortality in 3-day old larvae and low mortality in 4-5 day old larvae. Effects of rice field
water quality and host age on the infectivity of L. giganteum to Cx. tarsalis were
investigated in the laboratory (Woodring and Kaya 1992). Whereas the taxis and cuticle
recognition phenomenon of L. giganteum were studied in Ae. aegypti, An. gambiae and Cx.
pipiens (Golkar et al. 1993). L. giganteum is the only biological control agent approaching
operational use in mosquito control (Kerwin et al. 1994). Effects of concentrations,
movement, light and temperature on infection of mosquito larvae by the L. giganteum
zoospores have been studied (Suh and Axtell 1999). They found maximum virulence of L.
giganteum against Cx. quinquefasciatus at the concentration of >150 zoospores ml-1 of
water, at water temperatures between 20 and 30 oC. Further, information has been provided
on the cultivation of L. giganteum in various media for production of zoospores (Sur et al
2002).
Bioassay of the secondary metabolites of L. gigenteum on mosquito larvae for vector
control has recommended diversity of fungi to be used (Vyas et al. 2006a). Laboratory
efficacy of metabolites of L. gigenteum on An. stephensi after filtration by column
chromatography has been evaluated (Vyas et al. 2006b). They observed the high virulence
in three instars and very lower value in fourth instars. Further, efficacy of L. giganteum
metabolites on mosquito larvae with reference to non target organisms have been evaluated
(Vyas et al. 2007). Recently, new isolate media of the mosquito pathogenic fungus L.
gigenteum (Oomycetes: Lagenidiales) for fungal maintenance and zoospores release have
been provided (Shathele 2009). Efficacy of L. gigenteum metabolites for control of An.
stephensi a malaria vector has been evaluated (Singh and Prakash 2010).
2.1.1.2 Genus Leptolegnia
Leptolegnia is another fungus of Oomycetes that is pathogenic to mosquitoes. Le. Ornate
is an Oomycetes fungus, which has been isolated from a mosquito larva of Ae. triseristus
(Seymour 1976). Another species, Le. Chapmanii was isolated from insects (McInnis and
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Chapter 2 Review of Literature
Zattau 1982). Additionally, the South Carolina isolate was studied to test the susceptibility
of various mosquito larvae, including Ae. aegypti, An. gambiae, Cx. fatigans and Cx.
pipiens (Nnakumusana 1986). Later on, a species of Leptolegnia was isolated from larvae
of Mansonia titillans (Lord and Fukuda 1990). The infection was found on Mansonia dyari
and Anopheles species. Further, the presence of Leptolegnia sp., pathogenic to Ae.
albopictus was reported from discarded tyres and artificial containers in North Central
Florida (Fukuda 1992). An isolate from Argentina of the fungal mosquito pathogen
Leptolegnia chapmanii (ARSEF 5499), was tested against twelve species of mosquito
larvae and on species of non-target aquatic invertebrates and vertebrates. The mosquito
species tested were Ae. aegypti, Anopheles sp., Cx. apicinus, Cx. castroi, Cx. dolosus, Cx.
pipiens, Cx. renatoi, Isostomyia paranensis, Ochlerotatus albifasciatus, Oc. crinifer,
Psorophora cyanescens, and P. ferox (Lastra et al. 2004). The effects of water volume,
container surface area and the density of hosts and fungal zoospores on the infectivity of
the oomycete fungus Le. chapmanii to Ae. aegypti were investigated in the laboratory
(Pelizza et al. 2007). Late third or early fourth instars from a laboratory colony have been
used as hosts in all assays. Further, the effect of temperature on the production, survival
and infectivity of zoospores of an Argentinean isolate of Le. chapmanii was determined
under laboratory conditions. Production of zoospores of Le. chapmanii in vitro and in vivo
upon first and fourth instars larvae of the mosquito Ae. aegypti was studied at three
different temperatures (Pelizza et al. 2008). Larvicidal effects of interaction between
Bacillus thuringiensis var. israelensis (Bti), temephos and Le. chapmanii zoospores on
larvae of Ae. aegypti were determined under laboratory and semi natural conditions
(Pelizza et al. 2010). Further, production of Oogonia and Oospores of Le. chapmanii in Ae.
aegypti larvae at different temperatures have been studied (Pelizza et al. 2010).
2.1.1.3 Genus Crypticola
Crypticola is the another fungus of Oomycetes that is also pathogenic to mosquitoes. Cr.
Clavulifera Humber, Frances and Sweeney have been isolated from the midge
Forcipomyia marksae ITokunaga (Ceratopogonidae) in Queenslands, Australia, in 1984
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(Frances et al 1989). Its biology is similar of L. gigenteum. In the laboratory the fungus
successfully infected Ae. notoscriptus (Skuse), An. farauti Laveran, Cx. annulirostris
Skuse, Cx. quinquefasciatus and Ae. aegypti (Frances 1991).
2.1.2 Phylum Chytridiomycota
This phylum consists of approximately one thousand species (Barr 1990), most of which
are saprophytic in nature. They have flagellate zoospores and chitinous hyphae. The
phylum consists of five orders, of which the Blastocladiales contains the only mosquitopathogenic genus of this group: Coelomomyces.
2.1.2.1 Genus Coelomomyces
Coelomomyces is a chrytridiaceous genus of aquatic fungi that is parasitic on mosquito
larvae. This genus consist of more than seventy species of obligate parasitic fungi that
under go a complex life cycle involving alternating sexual (gametophytic) and asexual
(sporophytic) generations (Couch and Bland 1985). In India, Co. indicus was found to
infect larvae of An. culicifacies, An. subpictus and An. vagus (Achuthan 1988). Another
species, Co. neotropicus was found pathogenic to the larvae of Aedes and Culex species
(Lichtwardt and Gomez 1994). Co. stegomyiae usually kills its definitive mosquito host,
Ae. aegypti, in its fourth and final larval instar. Infected larvae that survive through the
pupal stage produce infected adults (Shoulkamy et al. 1997). The results here demonstrate
that production of infected adults is significantly affected by larval instar and inoculum
(fungal zygotes) at the time of infection and rearing temperature following infection. These
findings suggest that Coelomomyces can be used as prime candidate to mosquitoes control.
2.1.3 Phylum Deuteromycota
Deuteromycota is a group of about seventeen thousand species in which sexual
reproduction is not favoured.
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2.1.3.1 Genus Tolypocladium
Tolypocladium cylindrosporum Games is the Deuteromycetes fungus, which is a parasitic
to mosquito larvae. It was isolated from Europe as a soil saprobe (Gams 1971) one strain
of this fungus was isolated from Ae. sirrensis Ludlow in Nothern California (Soares et al.
1979). Later on, the identity of another isolate of T. cylindrosporum was confirmed that
was isolated from Ae. australis Erichson in New Zealand (Weiser and Pillai 1982). A
comparative bioassay of T. cylindrosporum was conducted against third instar larvae of
four species of mosquitoes, Ae. aegypti, An. balabacensis, Cx. quinquefasciatus, and
Mansonia uniformis (Serit and Yap 1984). Eleven strains of this fungus were tested for
pathogenicity to Ae. aegypti and An. stephensi larvae (Soares et al. 1985). It was found that
second instar larvae of Ae. aegypti were more susceptible to T. cylindrosporum than An.
stephensi larvae during that study. Strains of T. niveum, another member of this genus
killed mosquito larvae with LT50 after 8-10 days and mortalities of 88-90% after 13 days
(Weiser 1986).
In a study, two strains of T. cylindrosporum and one strain of T. niveum were tested
as mosquito pathogens (Weiser 1987). The LD50 value was 3 X 105 conidia/ml in T.
cylindrosporum and 2 X 105 conidia/ml in T. niveum. The infection process of T.
cylindrosporum was described in Ae. albopictus (Ravallec et al 1989). A new species, T.
terricola from soil typical features of intoxication in fourth instar of Cx. pipiens
autogenicus (Weiser et al. 1991). Furthermore, the pathogenicity of T. cylindrosporum has
been evaluated against Ae. triseriatus and all larval instars were found to be susceptible at
18-250C (Nadeau and Biosvert 1994).
2.1.3.2 Genus Culicinomyces
Two separate isolations of mosquito-pathogenic fungi were obtained from laboratoryreared Anophelines, one from An. hilli Woodhill and Lee in Sydney, Australia (Sweeney et
al. 1973), and the other from An. quadrimaculatus in North Carolina, USA (Couch et al.
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1974). The Australian fungus was not identified at the time of its discovery but a new
taxonomic status was established for the American fungus, which was described as
Culicinomyces clavosporus Couch. The Canadian isolate was compared with isolates from
the U.S. and Australia with regard to growth rate on solid media, colonial morphology and
pigmentation, and growth in a liquid medium (Mark et al. 1984).
In laboratory
experiments Chironomus species larvae were exposed to the Australian strain of the
mosquito pathogenic fungus C. clavisporus Couch. Romney and Rao, were infected in the
absence of a substrate but were not infected when given a substrate in which to tunnel and
construct their cases (Robert 1984). Preliminary field trials with C. clavosporus against
some Egyptian mosquitoes in selected habitats have been studied (Amal and Fatma 2003).
C. clavisporus, a fungal pathogen of a wide range of mosquito species, was investigated in
relation to potential pathogenicity against Culicoides nubeculosus biting midge larvae
(Unkles et al. 2004). Seven different C. clavisporus strains were assayed. Each showed
some degree of activity against C. nubeculosus larvae with LC50 values of between 3.2 X
105 and 1.1 X 106 spores/ mL. These effects occurred in dose-dependent manners and
tended to be delayed until 72-96 h post treatment. Fungi can play significant role in
reducing not only malaria vector in Africa but the cases of malaria also (Scholte et al.
2004, 2005).
2.1.3.3 Genus Metarhizium
Metarhizium is one of the most common entomopathogenic fungi, with a world wide
distribution. The species is soil-borne and infects predominantly soil-dwelling insects. First
time, Metarhizium anisopliae was isolated from mosquito larva of Cx. fatigans in India and
LC50 values were determined for all larval instars of An. stephensi and Cx. fatigans
(Balaraman et al. 1979). The potential of some isolates of M. anisopliae and Beauveria
bassiana for use in the integrated management of Cx. quinquefasciatus was evaluated
(Alves et al. 2002). This fungus caused higher mosquito larva mortality when applied as a
conidial suspension to the surface of the water than as dry conidia, with a time to 50%
lethal (LT50) of 1 day compared with 3.6 days for the dry conidial application. Infection of
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the malaria and filariasis vector with the entomopathogenic fungus M. anisopliae has been
tested (Scholte et al. 2003). The experiments clearly showed that the malaria vector An.
gambiae s.s., and the filariasis vector Cx quinquefasciatus are susceptible to M. anisopliae.
Their lifespan is greatly reduced if contaminated with an appropriate dose of conidia.
Furthermore, autodissemination of fungal inoculum between An. gambiae s.s. mosquitoes
during mating activity is possible under laboratory conditions (Scholt et al. 2004). They
observed that the males had acquired fungal infection after mating indicate that passive
transfer of the pathogen from infected females does occur, with mean male infection rates
between 10.7 ± 3.2% and 33.3 ± 3.8%. Later on, a study on avoidance and repellency of
the African malaria vector An. gambiae upon exposure to the entomopathogenic fungus M.
anisopliae was observed (Scholte et al. 2005). The experiments showed that dry conidia of
the fungus M. anisopliae have a moderate repellent effect on female An. gambiae.
Infection of the malaria mosquito A. gambiae with the entomopathogenic fungus M.
anisopliae reduces blood feeding and fecundity (Scholte et al. 2006). Similarly, infection
of adult Ae. aegypti and Ae. albopictus mosquitoes with the entomopathogenic fungus M.
anisopliae have been reported and found that at a dosage of 1.6 × 1010 conidia/m2, applied
on material that served as a mosquito resting site, an average of 87.1 ± 2.65% of Ae.
aegypti and 89.3 ± 2.2% of Ae. albopictus became infected with the fungus (Scholte et al.
2007).
The efficacy of virulent strain M. anisopliae 892 obtained from Pyrausta nubilalis was
evaluated against mosquito larvae and it was compared with the cuticle degrading enzyme
chymoelastase (Pr1) and trypsin like protease (Pr2) in the presence of inducers (Mohanty
et al. 2008). Further, efficacy of culture filtrates of five strains of M. anisopliae isolated
from insects were evaluated against An. stephensi and Cx. quinquefasciatus (Mohanty et
al. 2008). Entomopathogenic fungi M. anisopliae and B. bassiana isolates have been
shown to infect and reduce the survival of mosquito vectors (Ladslaus et al. 2009). They
found that the both fungus isolates are effective and persistent at low concentrations and
short exposure times.
Synergy in the efficacy study of fungal entomopathogens have been studied with
permethrin against West African insecticide-resistant An. gambiae mosquitoes has been
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observed (Farenhorst et al. 2010). The observed synergism in efficacy shows the potential
for integrated fungus-insecticide control measures to dramatically reduce malaria
transmission and enable control at more moderate levels of coverage even in areas where
insecticide resistance has rendered pyrethroids essentially ineffective. Recently, first time
the combination of the entomopathogenic fungus M. anisopliae with the insecticide
Imidacloprid increases virulence against the dengue vector Ae. aegypti has been tested
(Paula et al. 2011). They observed that adult Ae. aegypti could be controlled by surface
application of entomopathogenic fungi and that the efficiency of these fungi could be
increased by combining the fungi with ultra-low concentrations of insecticides, resulting in
higher mortality following relatively short exposure times.
2.1.3.4 Genus Beauveria
Beauveria tenella was reported for its larvicidal activity against all instars of An. stephensi
and Cx. fatigans (Balaraman et al. 1979). Another species of this genus is B. bassiana that
is an insect pathogenic Deuteromycetes fungus. It is found naturally on some plants and
saprophytically in the soil.
In our laboratory, the efficacy of extracellular metabolites of Deuteromycetes
fungus, Trichophyton ajelloi Vanbreuseghem has been tested against An. stephensi and Cx.
quinquefasciatus (Mohanty and Prakash 2004).
First report of pathogenicity of B. bassiana against malaria vector has been reported
(Achonduh and Tondje 2008). This study indicates that dry conidia of B. bassiana
RBL1034 are pathogenic to adult A. gambiae and could be a potential biological control
agent for these mosquitoes. Entomopathogenic fungi M. anisopliae and B. bassiana
isolates have been shown to infect and reduce the survival of mosquito vectors (Ladslaus et
al. 2009). They found that the both fungus isolates are effective and persistent at low
concentrations and short exposure times.
Further in our laboratory, the biolarvicidal activity of B. bassiana metabolites on
larvae of An. stephensi Liston and Cx. quinquefasciatus Say has been tested in laboratory
conditions (Singh and Prakash 2009). The infectivity of the entomopathogenic fungus B.
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bassiana to insecticide-resistant and susceptible to An. arabiensis mosquitoes at two
different temperatures has been observed (Kikankie et al. 2010). Survival data showed no
relationship between insecticide susceptibility and susceptibility to B. bassiana.
2.1.4 Phylum Zygomycota
2.1.4.1 Genus Smittium
The phylum Zygomycota found to be existing in two classes, the Trichomycetes and
Zygomycetes. Zygomycetes are characterized by the presence of a coenocytic mycelium,
by the absence of flagellate spores, and by the sexual reproduction through the formation
of zygospores. Smittium species differentially colonize particular species of black fly
(Diptera: Simuliidae) hosts as measured by differences in prevalence, abundance and
fecundity (Nelder and McCreadie 2005). Reasons for this differential occurrence and
fecundity in hosts are unclear but might include fungal responses to variations in host
morphology, physiology, distribution or behavior. Recently, the growth and development
of the trichomycete S. culisetae (Harpellales: Legeriomycetaceae) in the larval hosts
Simulium vittatum Zetterstedt (Diptera: Simuliidae) and Ae. aegypti (L.) (Diptera:
Culicidae) at three temperatures, 17, 22 and 300C has also been observed (Vojvodic and
McCreadie 2007).
2.1.5 Phylum Ascomycota
The Ascomycetes fungi are known as sac fungi. It comprises some thirty thousand three
hundred species, including both saprophytic and parasitic fungi.
2.1.5.1 Genus Chrysosporium
Chrysosporium is a keratinophilic filamentous fungus commonly isolated from soil, plant
material, dung, and birds. It lives on remains of hairs and feathers in soil. The
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keratinophilic soil fungus Chrysosporium tropicum was evaluated as a biological control
agent against Anopheles stephensi larvae in the laboratory (Priyanka et al. 2001). Culture
filtrates of C. tropicum were found to be toxic to An. stephensi larvae at various
concentrations. The ranges of the 50% lethal concentration (LC50) values of fungal filtrates
were 16.60-17.78, 12.02-12.88, and 34.67-35.48 micro l/ml against first, second, and third
stage larvae, respectively. The ranges of LC99 values were 38.90-63.10, 12.02-213.80, and
74.13-109.65 micro l/ml against first, second, and third stage larvae, respectively.
Furthermore, efficacy of fungal metabolites of C. tropicum was evaluated against Cx.
quinquefasciatus larvae in the laboratory to determine their larvicidal activity at six
concentrations, with a mortality range of 10-95% (Priyanka and Prakash 2003). Whereas,
the role of the secondary metabolites of C. lobatum as a biological control agent for
mosquitoes, effects of culture media on the larvicidal property of secondary metabolites
have been evaluated (Mohanty and Prakash 2008). Later on, efficacy of C. tropicum
metabolite against mixed population of adult mosquito (Cx. quinquefasciatus, An.
stephensi, and Ae. aegypti) after purification with flash chromatography has been tested
(Verma and Prakash 2010). They observed that metabolites of C. tropicum could be
utilized as alternative biological control agents for adult mosquitoes. Recently, effect of C.
keratinophilum
metabolites
against
Cx.
quinquefasciatus
after
chromatographic
purification has been evaluated (Soni and Prakash 2010). They found that the extracellular
metabolites of C. keratinophilum could be a fungal based larvicides resource for the
control of Cx. quinquefasciatus larvae.
2.1.5.2 Genus Aspergillus
Most of the species of Aspergillus grow saprophytically. However, a few species are
parasitic on plants and animals. Isolates of Aspergillus were found in the gut of Cx.
quinquefasciatus, which inhibited the emergence of adult mosquitoes completely (Vasanthi
and Hoti 1992). Eleven strains, of Aspergillus species were used for bioassays in second
instar larvae of Ae. fluviatilis and Cx. quinquefasciatus that caused mortality in two tested
mosquito species (Moraes and Costa 2001). Where as, the culture filtrates of five different
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soil fungi viz., A. flavus, A. parasiticus, Penicillium falicum, Fusarium vasinfectum and
Trichoderma viride have been tested for the larvicidal activity against third instar larvae of
Cx. quinquefasciatus (Govindarajan et al. 2005). Later on, pathogenicity of the fungus, A.
clavatus, isolated from the locust, Oedaleus senegalensis, against the larvae of Ae. aegypti,
An. gambiae and Cx. quinquefasciatus have been reported (Seye et al. 2009). Recently,
comparative efficacy and pathogenicity of keratinophilic soil fungi against Cx.
quinquefasciatus larvae have been found (Mohanty and Prakash 2010).
2.1.5.3 Genus Fusarium
Fusarium a filamentous fungus is widely distributed on plants and in the soil. As well as
being plant pathogens, Fusarium species are causative agent of superficial and systemic
infections in humans. Fusarium oxysporum is an asexual fungus that produces three types
of spores: microconidia, macroconidia and chlamydospores. The culture filtrates of five
different soil fungi viz., Aspergillus flavus, A. parasiticus, Penicillium falicum, F.
vasinfectum and Trichoderma viride were tested for the larvicidal activity against third
instar larvae of mosquito vector Cx. quinquefasciatus (Govindrajan et al. 2005). Later on,
the efficacy of entomopathogenic fungi F. pallidoroseum against female Cx.
quinquefasciatus has been tested (Mohanty et al. 2008). They found that the F.
pallidoroseum is one of the alternative biological control agents of adult mosquitoes.
Recently, pathogenicity of F. oxysporum against the larvae of Cx. quinquefasciatus and
An. stephensi have been tested (Prakash et al. 2010). They observed that the metabolites
have more pathogenicity after exposure of 48h.
2.1.5.4 Genus Verticillium
Verticillium is a genus of fungi in the division Ascomycota. Within the genus, diverse
groups are formed comprising saprotrophs and parasites of higher plants, insects and
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nematodes. This fungus species was first described in 1861. It has a worldwide distribution
found on insects. In horticulture and agriculture it is sometimes used as an
entomopathogen for controlling insect pests.
2.2 METALS NANOPARTICLES
Nanoscience has emerged as an integration of various disciplines for more new particles.
Nanoparticles are defined as particulate dispersions or solid particles with a size in the
range of 10-1000nm. Nanoparticles have been reported from a wide range of organisms,
including bacteria, actinomycetes, yeast, fungi and plants. Pseudomonas stutzeri AG259
isolated from silver mines has been shown to produce silver nanoparticles (Klaus et al.
1999). Also, the synthesis of magnetic nanoparticles has been reported by using
magnetotactic bacteria (Roh et al. 2001). Magnetotactic bacteria such as Magnetospirillum
magneticum produce two types of particles; some produce magnetic (Fe 3O4) nanoparticles
in chains and some produce gregite (Fe3S4) nanoparticles, while some other produce both
types of particles. Similarly, in the presence of exogenous electron donor, sulphatereducing bacterium Desulfovibrio desulfuricans NCIMB 8307 has been shown
synthesizing palladium nanoparticles (Yong et al. 2002). It has been observed that the
extremophilic actinomycetes, Thermomonospora species when exposed to gold ions
reduced the metal ions extracellularly, yielding gold nanoparticles with a much improved
polydispersity (Sastry et al. 2003). However, in an effort towards elucidating mechanism
or conditions favouring the formation of nanoparticles with desired features, carried out the
reduction of AuCl4 - ions by using an extremophilic Thermomonospora species biomass
that has resulted in efficient synthesis of monodispersed gold nanoparticles (Ahmad et al.
2003a). Further more, the reduction of metal ions and stabilization of the gold
nanoparticles were believed to occur by an enzymatic process (Ahmad et al. 2003b).
Adding further to the mechanism, a sulphate-reducing bacterial enrichment was used to
destabilized gold (I)-thiosulfate complex to elemental gold and proposed that this could
occur by three possible mechanisms involving iron sulfide, localized reducing conditions
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Chapter 2 Review of Literature
and metabolism (Lengke and Southam 2006). Recently, bacterial cell supernatant of P.
aeruginosa has been used for the reduction of gold ions resulting in extracellular
biosynthesis of gold nanoparticles (Husseiny et al. 2007).
Amorphous iron phosphate nanoparticles mineralized in yeast cells have been
studied by transmission electron microscopy (He et al. 2009). Whereas, the synthesis of
gold nanoparticles by the non-conventional yeast Yarrowia lipolytica has been described
(Agnihotri et al. 2009). Moreover, silver nanoparticles has been successfully synthesized
from AgNO3 through a simple green route using the latex of Jatropha curcas as reducing
as well as capping agent (Bar et al. 2009). Later on, the synthesis of metal oxide
nanoparticles by a Streptomyces species isolated from a site Pichavaram mangrove in India
has been carried out (Usha et al. 2010). They found that copper sulphate and zinc nitrate
when exposed to Streptomyces species are reduced in solution, thereby leading to the
formation of metal oxide nanoparticle. The metal oxide nanoparticles were in the range of
100-150 nm. The possibility of the reduction of metal ions may be by reductase enzyme.
Where as, the biosynthesis of silver nanoparticles using plant extracts has been done
(Gilaki 2010). Recently, the extra cellular synthesis of gold and silver nanoparticles by
using the yeast Candida guilliermondii has been described (Mishra et al. 2011).
2.2.1 Fungus role in nanoparticles formation
Extracellularly produced nanoparticles were stabilized by the proteins and reducing agents
secreted by the fungus. A minimum of four high molecular weight proteins released by the
fungal biomass have been found in association with nanoparticles. One of these was strain
specific NADH-dependent reductase. However, emission band produced by fluorescence
spectra indicate the native form of these proteins present in the solution as well as bound to
the surface of nanoparticles (Mcdonald and Smith 1996, Kumar and McLendon 1997).
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Chapter 2 Review of Literature
Later on, in addition to good monodispersity, nanoparticles with well defined dimensions
can be obtained by using fungi. This has been shown with an experiment where
bioreduction of aqueous AuCl4- ions was carried out using the fungus Verticillium species
that led to the formation of gold nanoparticles with fairly well-defined dimension and good
monodispersity (Mukherjee et al. 2001). These results have documented that the trapping
of AuCl4- ions on the surface of fungal cells could occur by electrostatic interaction with
positively charged groups (such as, lysine residues) in the enzymes that are present in the
wall of the mycelia. Towards elucidating mechanism of nanoparticles formation, an in
vitro approach was followed where species specific NADH dependent reductase, released
by the Fusarium oxysporum, were successfully used to carry out the reduction of AuCl4ions to gold nanoparticles. This has first time opened up a novel fungal/enzyme-based in
vitro approach for nanomaterials synthesis (Mukherjee et al. 2002). The acidophilic fungus
Verticillium species has capability of producing gold as well as silver nanoparticles upon
their incubation with Ag+ and AuCl4- (Sastry et al. 2003). However, a novel biological
method for the intra- and extra-cellular synthesis of silver nanoparticles using the fungus,
Verticillium and F. oxysporum respectively has been documented. This has opened up an
exciting possibility wherein the nanoparticles may be entrapped in the biomass in the form
of a film or produced in solution, both having interesting commercial potential (Senapati et
al. 2004).
An important role during growth plays the production of nanoparticles while using
the fungi cultures. When gold ions were incubated with the Trichithecium species biomass
under stationary conditions led to the formation of extracellular nanoparticles. While under
shaking conditions, this has been resulted in the formation of intracellular gold
nanoparticles. The possible reason for this could be the enzymes and proteins responsible
for the synthesis of nanoparticles. These proteins were released into the medium under
stationary conditions and have not release under shaking conditions (Ahmad et al. 2005).
Whereas, the extracellular biosynthesis of silver nanoparticles using Aspergillus fumigates
has been done (Bhainsa and D’Souza 2006). In this effort they could get fairly
monodispersed silver nanoparticles within 10 min. Further, the fungus A. flavus has also
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Chapter 2 Review of Literature
been resulted in the accumulation of silver nanoparticles on the surface of its cell wall
when incubated with silver nitrate solution (Vigneshwran et al. 2007).
Crystallized and spherical-shaped Au and Au–Ag alloy nanoparticles have been
synthesized and stabilized using a fungus, F. semitectum in an aqueous system (Sawle et
al. 2008). They have analyzed of the feasibility of the biosynthesized nanoparticles and a
core–shell alloy nanoparticle from fungal strains is particularly significant. Whereas, the
silver nanoparticles have been synthesized within 10 min from potato plant pathogenic
fungus Phytophthora infestans and their anti-bacterial activity were investigated by disc
diffusion method and MIC (Thirumurugan et al. 2009). They have showed that silver
nanoparticles exhibited discrete antibacterial activity against clinically isolated seven
pathogenic bacteria at a concentration of 5μg/ml. Further on, antiparasitic activities to
determine the efficacies of synthesized silver nanoparticles using aqueous leaf extract of
Mimosa pudica against the larvae of malaria vector, An. subpictus, filariasis vector Cx.
quinquefasciatus and Rhipicephalus microplus have been evaluated (Marimuthu et al.
2010). Later on, the larvicidal potential of the hexane, chloroform, ethyl acetate, acetone,
methanol, and aqueous leaf extracts of Nelumbo nucifera and synthesized silver
nanoparticles using aqueous leaf extract against fourth instar larvae of An. subpictus and
Cx. quinquefasciatus have been observed (Santhoshkumar et al. 2011). Recently, the
Trichosporon beigelii NCIM 3326 has been used in aqueous silver ions to study its
potential to reduce silver ions to stable silver nanoparticles (Ghodake et al. 2011). The
exposure of T. beigelii NCIM 3326 to aqueous silver ions resulted in synthesis of silver
nanoparticles. Silver nanoparticles by fungus Trichoderma ressei have been synthesized
(Vahabi et al. 2011).
2.3 THE FORMULATIONS
A number of fungi and nanoparticles have been reported as biocontrol agents for
mosquitoes, and have antimicrobial activity. The nanoparticles have therefore been
formulated from poly(l) lactide by a modified nanopre-cipitation method and effect of
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Chapter 2 Review of Literature
cosolvent on the shape, size, formation efficiency, degree of crystallinity, x-ray diffraction
re-flection pattern, and zeta potential value of the particles (Peltonen et al. 2002). During
artificial culture of some lichens fungi (mycobiont) in synthetic medium for bioactive
metabolite synthesis, the Usnea longissima lichen (Ascomycetes fungi) found to be
synthesized bioactive nanoparticles (usnic acid) in specified medium under additional
conditions. These bioactive nanoparticles have been tested against human pathogenic
fungi, Epidermophyton floccosum, T. rubrum, T. tonsurance, T.violaceum (Shashi and
Patra 2003). Whereas, the preparation and evaluation of polymethacrylic acid nanoparticles
containing lamivudine in different drug to polymer ratio by nanoprecipitation method has
been studied (Tamizhrasi et al. 2009). Later on, the polyethylene glycol (PEG) coated
nanoparticles loaded with garlic essential oil have been evaluated insecticidal activity
against adult Tribolium castaneum (Yang et al. 2009).
The efficiency and durability of the nanosilver particles-based antibacterial finish has
been determined (El-Rafie et al. 2010). Efficiency of the antibacterial finish on the cotton
fabric, expressed as bacterial reduction %, amounts to 97% and 91% for Staphylococcus
aureus and Escherichia coli, respectively. Method has been developed for the preparation
of nanoparticles of curcumin with a view to improve its aqueous-phase solubility and
examine the effect on its antimicrobial properties (Bhawana et al. 2011). We therefore,
investigated the five selected fungal strains screened against the mosquito larvae. We have
further used these fungal strains in nanoparticles synthesis. The formulations of these
nanoparticles with all five fungi have also done with different combinations. These
nanoparticles have been used for bioefficacy test.
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