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Printed in the Unitated States of America, 2014
ISBN : 978-1-63315-205-2
Short Views on Insect Biochemistry and Molecular Biology Vol.(2), October 2014
2014
Section VII
Insect Pest Management
through Biochemical and
Molecular approaches

ISBN : 978-1-63315-205-2
Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

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Short Views on Insect Biochemistry and Molecular Biology

Vol. (1), 00 00, 2009
Vol. (2) 575 594, 2014
Review
Review
Chapter 26
Mosquito control using biological larvicides: Current Scenario
Subbiah Poopathi1*, C. Mani1 and Raman Chandrasekar2
1
Unit of Microbiology and Immunology, Vector Control Research Centre

(Indian Council of Medical Research), Medical complex, Indira Nagar, Puducherry 60 5006, India.
2
Department of Biochemistry and Molecular Biophysics, Kansas State University,
Manhattan 66506, KS, USA.
Abstract
Emerging infectious diseases are defined as new, reemerging or drug-resistant infections whose
incidence in humans has increased within the past two decades or whose incidence threatens to
increase in the near future. The causes of resurgence of infectious diseases include changes in
human industrial practices, economic development, and changes in land use, increase in
international travel and commerce and adaptation of the microbes including development of
resistance to antimicrobial agents. Mosquitoes cause great nuisance to human beings and pose
threats to public health as vectors of diseases like malaria, filariasis, dengue, Japanese encephalitis,
and West Nile fever. Annually 300 million people are estimated to be affected by malaria,
transmitted by Anopheles mosquitoes with more than one million deaths. The world burden of
lymphatic filariasis is estimated to be 250 million people. Approximately 20 million people are
infected every year by dengue viruses transmitted by Aedes mosquitoes with ~24,000 deaths.
Several strategies have been adopted to control these dipteran pests and to reduce vector-borne
diseases. Synthetic insecticides have been effectively used during the past several decades for
mosquito control operations. But the chemical approach has demerits, such as the development
of insecticide resistance, environmental pollution, bioamplification of contamination of food
chain and harmful effects to beneficial insects. Hence, there has been an increased interest in
recent years in the use of biological control agents (Bacillus sphaericus, Bs and Bacillus thuringiensis
subsp. israelensis, Bti) for mosquito control. Though the high efficacy and specificity of these
biological agents are useful in controlling mosquitoes, the cost to grow the bacteria through a
highly refined laboratory bacterial culture medium is exorbitant. Hence, efforts are under process
to develop an inexpensive culture media globally. Considering the abundant supply of these
bioorganic wastes, scientists from all over world have now utilized these bio-organic waste
materials for the production of biopesticides to control disease transmitting mosquitoes. In the
present study, we focus the current scenario of biopesticide production from cost-effective
culture media for the control of mosquito vectors.
Key words: Insecticide, Insect growth regulators, Bacillus thuringiensis, mosquitoes, malaria, Japanese
encephalitis
*For Correspondence (email: Subbiahpoopathi@rediffmail.com)

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Overview
1. Introduction
2.Method of Vector Control
2.1. Synthetic chemicals
a) Insecticide impregnated bednets
2.2. Non-synthetic chemicals
b) Expanded polystyrene beads (EPS)
c) Insect growth regulators (IGRs)
d) Genetic manipulation (SIT)
2.3. Biological control
a) Synergism
b) Toxin mode of action
c) Other mosquitocidal Bt strains
d) Slow release formulations
2.4. Other control measures
a) Mosquito larvivorous fishes
b) Dragonfly
c) Predatory mosquitoes
d) Entomopathogenic Fungus
2.5. Precedence of future research
a) Genetic Engineering with Bacterial agents
b) Biopesticide production from
environmental waste
4. Conclusion
5. Acknowledgement
6. Reference
1. Introduction
Vector-borne diseases form a major
component of communicable diseases
(filariasis, malaria, dengue and
Japanese encephalitis) in India and in
other Asian countries. Every year
about 300 million people are estimated
to be affected by malaria, transmitted
by Anopheles species of mosquitoes
with about 1 million deaths (1,2). The
world burden of lymphatic filariasis is
estimated to be 250 million people
infected primarily by filarial parasites
(Wuchereria bancrofti, Brugia malayi
and Brugia timori). About 20 million
people are infected every year by
dengue viruses transmitted by Aedes
species of mosquitoes with about
24,000 deaths (3). Therefore, control of

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mosquitoes is an essential step towards

the reduction of mosquito borne
diseases. Several strategies have been
adopted to control the various diseases
transmitted by mosquitoes. Synthetic
insecticides have been effectively used
during the past several decades to
control these dipteran pests and to
reduce vector-borne diseases. But the
use of chemical insecticides has
become problematic because of a
multiplicity of factors including
physiological resistance in the vectors,
environmental pollution resulting in
bio-amplification of food chain
contamination and harmful effects on
beneficial insects. Hence, there has
been an increased interest in recent
years in the use of biological vector
control agents. The discovery of
bacteria like Bacillus sphaericus (Bs)
and Bacillus thuringiensis subsp.
israelensis (Bti), highly toxic to
dipteran larvae, opened up the
possibility of the use of these
biolarvicides in mosquito eradication
programmes (4). Mosquito pathogenic
bacilli have some advantages over
conventional insecticides in mosquito
control operations, because, they have
a broader host spectrum, are safer to
non-target
organisms
(including
humans) and are more environment
friendly. Bti synthesizes intracellular
crystal inclusions by sporulation that
contains multiple protein components
of 134, 125, 67, and 27 kilo Daltons
(5,6). These proteins have been cloned
individually and are toxic to mosquito
larvae (7,8). On the other hand, Bs
synthesizes proteins that assemble into
crystals, which are composed of two
major polypeptides, with molecular
masses of 51 and 42 kilo Daltons.
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Though the high efficacy and specificity of Bs and Bti are useful in controlling
mosquitoes, the cost to grow and produce Bs or Bti formulations, through a highly
refined laboratory bacterial culture medium, is exorbitant. The cost of Bs/Bti
biopesticide production depends on many factors; however, the raw material cost is
one of the most important criteria, which comprises > 70 % of the overall production
cost (9,10). Therefore, selection of growth medium or raw material is critical for
commercial production of these biopesticides. In order to encourage the commercial
production of biopesticides, utilization of less expensive raw material is advisable
(11,12). Several raw materials (industrial and agricultural by-products) have been
tested successfully in mosquito control program, as alternative culture media, for the
production of Bs/Bti (13,14,15). The authors of the present paper have also studied
extensively on different biological waste materials on the production of biopesticides.
This aspect will be discussed separately in different sections.
Fig.1a. Different approaches of mostquito control methods
2. Method of vector control

2.1. Synthetic chemicals
Insecticide-impregnated fabrics have been used effectively against
mosquito-bites (16). The impregnated synthetic pyrethroid insecticide (deltamethrin)
ropes, bed nets and curtains in the human dwellings were found to be promising
against Anopheles and Culex species (17). Mosquito nets treated with a
water-dispersible tablet formulation of deltamethrin was evaluated against malaria
vectors and found to be effective (18). Olyset nets are permethrin insecticide
impregnated bednets, recommended by WHO, and are currently in use in rural malaria
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endemic areas (19). The method of mosquito control has been illustrated as schematic
diagram in Figure 1.
Fig.1b. 3-D molecular structure of Cry5 (PBD code: 4D8M) generated by using PyMol program.
The three domains of the protein are represented with different colours with domainI coloured (sky blue),
domain II coloured (green) and domain III is coloured (pink) from Hui et al. (47).
2.2. Non-synthetic chemicals

a) Impregnated paint
This technology has been in use for decades in developed countries. The
Vernacide is an example of insecticide-impregnated paint effective against mosquitoes
(Culex quinquefasciatus) (81). 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.
b) Expanded polystyrene beads (EPS)
A synthetic non-biodegradable product of very light thermocol beads can remain
effective on treated water surface have shown tremendous potential of the EPS in
abating breeding of Cx. quinquefasciatus and An.stephensi, in latrine pits in Tanzania
and in overhead tanks in India, respectively (20). These beads are non-polluting and
socially acceptable. Its long-life, non-hazardous nature and one time application
renders it cost-effective and feasible.

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c) Insect growth regulators (IGRs)

Insect growth regulators (IGRs), the third generation insecticides, are diverse
groups of chemical compounds that are highly active against larvae of mosquitoes and
other insects. The IGRs in general have a good margin of safety to most non-target
biota including invertebrates, fish, birds and other wildlife. They are also relatively safe
to man and domestic animals. The IGR compounds do not induce quick mortality in the
pre-imaginal stages treated and occur many days post treatment. This is indeed a
desirable feature of a control agent because larvae of mosquitoes and other vectors are
an important source of food for fish and wildlife. On account of these advantages of
IGRs and the high level of activity against target species, it is likely that IGRs could
play an important role in vector control programs in the future. They are more specific
for mosquitoes than conventional insecticides. The IGRs interfere with the hormonal
mechanisms of target organisms and result in various kinds of morphological,
anatomical and physiological abnormalities so that the target species does not reach the
final stage of development (21). There is no likelihood of resistance development
against these IGRs. A large number of IGRs, both juvenoids and chitin synthesis
inhibitors, have been evaluated for the vector control but only very few of these are
found effective and commercially feasible, e.g., diflubenzuron, methoprene, fenoxycarb (37). The IGRs have an added advantage of being used at a relatively very low
dose compared to the conventional insecticides. Ecdysone agonists are hormonally
active insect growth regulators that disrupt development of larvae and are found to be
active against Aedes aegypti, Anopheles stephensi, and Cx.quinquefasciatus (4).
d) Genetic manipulation (SIT)
Potential applications for reducing transmission of mosquito-borne diseases by
releasing genetically modified mosquitoes have undergone extensive research and
mosquitoes are being created with such an application in mind in several laboratories
(21, 22). The use of the sterile insect technique (SIT) provides a safe programme in
which production; release and mating competitiveness questions related to mass-reared
genetically modified mosquitoes could be answered. It also provides a reversible effect
that would be difficult to accomplish with gene introgression approaches. SIT using
transgenic material would provide an essentially safe and efficacious foundation for
other possible approaches that are more ambitious. Apart from the use of SIT, other
approaches of developing genetically modified mosquitoes that are refractory to
malaria parasite development include mapping of QTLs associated with refractoriness,
use of RNAi technology to identify genes and gene pathways that can be perturbed to
reduce vectors potential in pathogen transmission etc.
2.3. Biological control

Bacillus thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus (Bs) are
entomopathogenic bacteria. They are spore forming bacteria producing parasporal
crystal toxin (23). The parasporal inclusion body is composed of proteins or
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-endotoxins, varying in quantity and type depending on the strain. Each type of crystal
protein is characterized by a specific host range, and based upon differences in
sequence and specificity; insecticidal crystal -endotoxins have classified into several
groups of proteins designated Cry (24, 25). Bti is toxic to both mosquito and blackfly
whereas Bs only to mosquitoes. Extensive field trials have been conducted with Bti
using powder/ liquid formulation and comparatively fewer number of field-tests with
Bs (26). During sporulation, both produce crystals that are toxic to dipteran larvae after
ingestion. Since the discovery of Bti in 1977 (27), other bacteria have also been found
to be highly toxic to mosquito larvae, including Bs (28) and more recently, Clostridium
bifermentans serovar malaysia (29). Among the various species of Bt, few strains are
as toxic as Bti (30).
a) Synergism
B.thuringiensis strains are pathogenic to insects which produce two distinct types
of toxin proteins, Cry and Cyt proteins (14). Generally, the genes encoding these
proteins are located on large plasmids, and the proteins are synthesized and form
crystalline inclusions during sporulation. More than 100 different Cry genes have been
identified and sequenced, and significant homologies among the amino acid sequences
of this group, in combination with experimental studies, suggest they have a common
mode of action, colloid-osmotic lysis (13). Cyt toxin, however have only been found in
Bt strains that are mosquitocidal, have amino acid sequences that are unrelated to those
of the Cry toxins, and can lyse a variety of cell types in vitro (12). The mode of action
of Cry toxins has not been fully elucidated. However, research suggests that Cyt toxins
are also involved in colloid-osmotic lysis (20), but may differ in the mechanism by
which lesions are formed in the cell membrane (19). One of the most interesting
features of the Cry and Cyt toxin combination that is found in Bti, the susceptible upon
which many commercial mosquito larvicides are based, is the effect of this
combination on toxicity. This subspecies produces a crystalline parasporal inclusion
containing four major toxic proteins, Cry4A (134 kDa), Cry4B (128 kDa), Cry11A (66
kDa), and CytA (27 kDa). These four proteins are assembled into separate inclusions
that are enveloped together to form parasporal body. The proportion of each protein in
the parasporal body, based on SDS-PAGE and electron microscopy (15, 28) is
approximately 40 % Cyt A and 20 % for each of the three Cyt proteins. Studies of these
proteins revealed that the toxicity of individual proteins in Bti was much less than that
of the intact parasporal crystal. Subsequently, it was shown that the Cry toxins in Bti
interact synergistically with the Cyt1A toxin, as well with each other, to produce this
high level of activity (29). This synergism has also been shown to be important in the
relatively low rate of resistance development toward Bti in Culex mosquitoes and can
suppress high levels of resistance to Cry4 and Cyt11 toxins. The mechanism of this
synergism is not understood, but we have postulated that Cyt1A aids these toxins in
binding to or inserting into the mosquito microvillar membrane. The synergistic
capacity of Cyt1A was extended by the recent observation that sublethal concentrations
of Cyt1A combined with Bs, an unrelated mosquitocidal bacterium which does not
have Cry type toxins, were synergistic and toxic towards highly resistant
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Cx. quinquefasciatus (14). Because, the combination was synergistic against resistant
mosquitoes that have lost the capacity to bind Bs toxins (16). This same mixture might
be synergistic toward a mosquito species that is naturally insensitive to Bs.
b) Evaluation strategies
Both Bti and Bs respectively, have shown great promise, particularly those
breeding in highly polluted water. The formulations of Bs effective against mosquito
larvae are DP1593, 2362; Dulmage 2362, 2297; Abbot: DP1593: Strains B6, B64:
Vectolex (G); Microgel 1593M; Biocide-SABG 6185; Vectolex (G); Spherix etc. The
formulations of Bti effective against vector mosquitoes are Deltox (briquettes);
Bactimos (WP); Teknar (4 formulations); dust preparation Bactimos (flowable
concentrate), Vectobac (granule), Bti (ultra low volume), Vectobac 12 aqueous
solution etc. These formulations would lose efficacy in cold climatic conditions and is
useful in warm months or tropical climatic conditions for most of the year. Efficacy of
various other formulations of Bti in the form of tablet, granule and wettable powder
against different disease vector species in urban and rural areas were tested and found
to be convincing.
While Bti is now commercially available as Deltox, Bactimos and Teknar in
different formulations (e.g., liquid concentrate and water dispersible powder), Bs is still
undergoing the marketing process and is expected to be effective against all larval
stages of An. stephensi, Cx. quinquefasciatus and Aed. aegypti mosquitoes. These
bacterial species although pathogenic to the target vector species, are non-hazardous to
other beneficial and non-target organisms.
Microbial mosquito larvicides may also suffer the same fate in inducing resistance
development as synthetic insecticides. For resistance management, Bti would be a good
candidate to manage Bs resistance, because there is a complete lack of cross-resistance
between Bti and Bs toxins (31, 32, 33, 34). It is a well known fact that Bti and Bs toxins
bind to different classes of specific receptors on the surface of midgut brush border
membranes (35) which could explain the lack of cross-resistance between Bti and Bs.
Using Bti alone or in rotation or mixture with Bs would be a logical choice to restore Bs
susceptibility in Bs resistant mosquitoes.
c) Toxin mode of action
Bti produces a proteinaceous parasporal crystalline inclusion during sporulation.
Upon ingestion by insects, this crystalline inclusion is solubilized in the midgut,
releasing proteins called delta-endotoxins. These proteins (protoxins) are activated by
midgut protease and the activated toxins interact with the larval midgut epithelium
causing a disruption in membrane integrity and ultimately leading to insect death (36).
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Exposure of Cx. quinquefaciatus to Bti over several generations will likely result in the
selection of populations with lower sensitivity to the crystal mosquitocial toxin.
Exposure of insect populations to signle toxins, after prior exposure to complex
mistures of toxins can result in the rapid increase of resistance. To delay onset of
resistance, Bti formaulations must contain toxins that interact either with multiple
receptor sites or that have different mode of action. Ingestion of Bs protein causes
destruction of gut epithelial cells in thelarvae of certain mosquito species, resulting in
the death of insect. In general, the larvae of genus Cx. quinquefaciatus are especially
susceptible to this toxin, those of An. stephensi are moderately susceptible and larvae
of Aed.aegypti are quite resistant. The larvicial properties of the crystal have made Bs a
useful agent for the biological control mosquitoes. But there are some reports of
resistance of Bs from field in France (125) and India (25, 32).
The nature of change involved in resistance to Bs is unknown but a mutation
affecting the synthesis of the target receptor molecule or inducing a change in its
conformation, leading to non-functionally may be responsible. However, mosquito
population resistance to Bs remain susceptible to Bti toxins indicating that different
receptors are involved. Some laboratory seleced resistance to Bt to Indian meal moth
and also cases of field selected resistance to diamondback moth in Philippines and
Hawaii has been reported. Laboratory and field-selected resistance may be due
different factors. Laboratory development of resistance is more likely to involve
polygenes since they are prone to be selected under conditions where biological and
environmental stress factors are minimized (32). In contrast, development of resistance
in the field is more likely to involve single major genes, which can be selected from a
much wider genetic under stressfull condition (37).
The mechanism of resistance to Bs is not yet defined but more than one
mechanism seems to be involved. Bs is at high risk for selecting resistance in mosquito
populations, because its binary toxin apparently binds to single receptor type on midgut
microvilli (37). A potential key strategy for delaying resistance to insecticidal proteins
is to use mistures of toxins that act at different targets within the insect, especially
mistrues that interact synergistically (38). Resistance to Bti has resulted from reduced
binding of the toxin to the brush border in the lumen of the insect gut (39) or by
enhanced digestion of toxin by gut proteases (40). The six different toxin types in the
Bti strain used for vector control were expected to retard ro prevent development of a
comprehensive resistance mechanism; however multi toxin resistance to Bti has
already appread.
e) Slow release formulations
The formulation of bacterial agents has two important problems. One is that, they
do not remain in the larval feeding zone for a longer period and thus are not effective
for long duration. Also, the spores do not replicate to sufficient extent, thus, powder
/liquid formulations have to be applied frequently. Hence slow release formulations
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were developed and were found to be active for a longer duration. But larger quantities
of larvicidal material are required for use in operational programmes, which affects
logistics and cost.
These formulations now commercially available in different forms have
invariably a biodegradable base carrier, which is imbibed with a relatively high
concentration of some larvicide so that when placed in the mosquito breeding habitat
the base carrier spontaneously biodegrades releasing the larvicide slowly in desired
low, yet toxic, concentration. Such a method is particularly very desirably useful in
highly polluted media where repeated spraying of the conventional larvicide (e.g.,
fenthion) is highly cost prohibitive and time consuming Cx.quinquefasciatus and
Cx.tritaeniorhynchus can be significantly brought under check by this methodology.
These formulations are required to be sprayed on weekly basis to bring about good
reduction in the larval density.
VectoBac, a tablet formulation of Bti and zeolite granules of Temephos (ZG) were
found to be effective as larvicides of Ae. aegypti in Thailand (26). Ice granules
containing endotoxins of microbial agents for the control of mosquito larvae was found
to be a new application technique against Aedes spp. Solutions containing powder
formulations of Bti or Bs were transformed into ice pellets using a special ice-making
machine (44). This new technique was demonstrated to have the following advantages
over Bti sand granules: (1) the Bti ice pellets melted on the water surface and released
the microbial crystals there, (2) the control agent remained inside the ice pellets during
the application and were not lost by friction in the spraying equipment, and (3) the ice
formulation resulted in increased swath widths, significantly reducing the cost of
application. The efficacy of new water-dispersible granular (WDG) formulations of Bti
(VectoBac) and Bs (VectoLex) was tried against malaria vectors and also found to be
good. The effectiveness of larvicidal treatment by AQUABAC Biolarvicide (Bti),
Biological Larvicide Aqueous Suspension against Cx. quinquefasciatus in slowflowing water ditches has been evaluated in USA and found to be effective. The bioefficacy and residual activity of Bti H-14 (water-dispersible granules of VectoBac
ABG 6511 and liquid formulations of VectoBac 12AS) and pyriproxyfen (insect
growth regulator) for control of larvae of Ae. aegypti and Ae. albopictus were found to
be effective in Malaysia (32).
2.4. Other control measures

a) Mosquito 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), Aplocheilus 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
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larvivorous potential has been studied. In different countries the local fishes available
have been explored to exploit their use against Anopheline and Culicine larvae. The
ability of 2 freshwater fishes, eastern rainbow fish Melanotaenia splendida and flies
pecked hardy head Craterocephalus stercusmuscarum, native to North Queensland to
prey on immature Ae. aegypti was evaluated (43). Larvivorous fish Oreochromis
spilurus was found to be effective against malaria vectors in Somalia (44).
b) Dragonfly
Biocontrol potential of dragonfly nymph, Brachythemis contaminata against the
larvae of An. stephensi, Cx. quinquefasciatus and Ae. aegypti was conducted and found
that they had good predatory potential and can be used as a biological control agent for
control of mosquito breeding (45).
c) Predatory mosquitoes
Toxorhyncites 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 (46). The predatory efficacy of this mosquito was also proved in field evaluation
studies against thirteen species of mosquitoes sp., Culex sp., and Aedes spp., conducted
in Japan. Aed. aegypti populations were suppressed by Toxorhyncites splendens larvae
in household water storage containers in Jakarta (29).
d) Entomopathogenic Fungus
Many fungi such as Coelomomyces, Lagenidium, Metarrhizium, Culinomyces and
Tolypocladium have been isolated and tested (37). 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 on 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 (38). The other fungi, viz.,
Metarrhizium, Culicinomyces and Tolypocladium could be cultured on artificial media
and formulated easily but to achieve a substantial reduction in the larval population
their spores have to be applied in very high doses which would be uneconomical. Also,
they do not recycle in aquatic habitats. 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 Hypocrella/
Aschersonia group might be useful as an agent for pest control. Fungal pathogens such
as Lagenidium, Coelomomyces and Culicinomyces are known to affect mosquito
populations, and have been studied extensively (18). Lagenidium giganteum was
highly pathogenic to immatures of Cx. quinquefasciatus, Cx. tritaeniorhynchus,
An. culicifacies, An. stephensi and An. subpictus. There are, however, many other
fungi that infect and kill mosquitoes at the larval and/or adult stage. Several isolates of
deuteromycetes fungi such as Metarhizium, Beauveria tenella, Fusarium oxysporum
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were isolated indigenously and were found pathogenic to Cx. fatigans and
An. stephensi. Some water molds such as Leptolegnia caudate and Aphanomyces laevis
were found to be naturally occurring parasites of mosquito larvae. 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 vertebrates was
found to be effective against Anopheline and Culicine mosquitoes (39). Efficacy of
fungal metabolites of Chrysosporium tropicum was evaluated against
Cx. quinquefasciatus larvae in the laboratory was conducted and found to have
promising effects (11). Trichophyton ajelloi, a fungus isolated from soil, caused high
larval mortality in An. stephensi and Cx. quinquefasciatus (72). Residual sprays of
fungal bio-pesticides might replace or supplement chemical insecticides for malaria
control, particularly in areas of high insecticide resistance (40).
2.5. Precedence of future research

a) Genetic Engineering with Bacterial Agents
The cloning of mosquitocidal toxin genes from Bti and different Bs strains has
allowed the re-expression of a combination of toxins from both species in one
recombinant cell in an attempt to broaden the host range of the toxins and obtain
beneficial synergistic effects (39,40). Two sequences 100 % and 65 % identical to the
left inverted repeat of the insertion sequence IS 240A of Bti were found starting at
nucleotides 2756 and 3104, respectively. These insertion sequences are located in the
same transcription direction as that of gene Cry 11 Bb1 downstream from the second
inverted repeat begins an open reading frame starting at nucleotide 3194 and encoding
the first 27 amino acids of a protein which shares 62.9% identity with the protein
encoded by the IS240 of Bti. These types of sequences were reported initially in Bti
flanking the Cry4Aal gene (5), later the presence of IS like sequences were described in
several mosquito and non-mosquito active strains (29). It is believed that these
elements could be involved in crystal protein genes. Dispersion among the
Homologous recombination can be applied to Bti or Bs (or other bacteria) to integrate
novel combinations of mosquitocidal toxin genes into the bacterial chromosome and
obtain their expression during the vegetative or sporulation phase of cell growth.
Bacterial spores are used to control mosquito larvae but spores generally
sediment more rapidly from the larval feeding zone than vegetative cells. A novel
approach to controlling pests utilizes heat killed, encapsulated vegetative cells of
Pseudomonas fluorescens that are genetically engineered to express high levels of
Bt toxins. Much effort has been put into evaluating engineered vegetative bacteria such
as cyanobacteria, Caulobacter and Ancylobacter aquaticus as delivery vehicles form
mosquitocidal toxins. Being surface dwellers, cyanobacteria, caulobacters and
A.aquaticus are able to resist rapid inactivation by UV light. These bacteria are
non-pathogenic to animals and grow in simple, inexpensive media. Moreover, in the
reproduction of these bacteria occurs naturally in environments that are low in nutrient
levels, unlike the spores of bacilli, which depend on protein rich larval cadavers for
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their germination.Various species of cyanobacteria have been investigated as vehicles

for prolonged delivery of insecticidal toxins to the larval feeding zone (3, 23, 44).
Various toxin genes from Bti and Bs have been cloned into different cyanobacteria,
Caulobacter crescentus or A.aquaticus, and the recombinant cells have been shown to
be significantly toxic to Culex and Aedes larvae. Although toxin expression levels
remain disappointingly low, the characteristic buoyancy of the cells may prolong their
larvicidal action. By 1993, approximately 27 recombinant micro-organisms had been
approved for release into the environment in several countries but so far no genetically
modified mosquitocidal bacteria have been released. Before seeking approval to
release engineered bacteria, a number of issues must be raised and experiments
performed in order to determine, as far as possible, that the modified organism would
kill only mosquito larvae and not disturb the ecosystem. There are powerful arguments
both for and against the release of genetically manipulated microorganisms.
Protagonists point out that there is an urgent need for a new approach to control vectors
of parasitic and viral diseases. The decision whether or not to release a genetically
modified organism must be taken on a case by case basis and made after very careful
consideration.
Currently 19 distinct mosquitocidal toxin genes are known, and novel genes will
undoubtedly be found and cloned. The toxins appear to vary in their species specificity
and mode of action, making it likely that particular combinations cloned in
recombinant microorganisms can be chosen so as to enlarge insect host range and delay
or prevent the development of resistance. The mosquito host range of the toxins is
rather narrow and difficult to predict. The spores do not multiply significantly outside
the larval cadaver; the spore-crystal complex is sensitive to UV light and the spores
settle rapidly, limiting the duration of control. Insecticidal bacteria are costlier to
produce than chemical insecticides. However, a combination of novel genetic
manipulation approaches (high level expression of toxin combinations; chromosomal
integration of toxin genes), coupled with existing formulation technology or the use of
engineered vegetative bacteria which can exist in the upper layer of water, may well
overcome these problems. The phasing out of chemical insecticides, together with the
emergence of pesticide resistant mosquitoes and drug resistant parasites, will act as a
strong incentive to develop effective mosquitocidal bacteria.
Biological control of mosquito larvae mainly relies on the use of two
entomopathogenic bacteria, Bacillus sphaericus (Bs) and B.thuringiensis serovar
israelensis (Bti). Both bacteria synthesize during sporulation, proteins that assemble
into crystals which are toxic for the larvae upon ingestion. Bti crystals are composed of
four major polypeptides with molecular weights of 125, 135, 68 and 28 kDa, now
referred to as Cry IVA, Cry IVB, Cry IVD and Cyt A, respectively (45). Genes
encoding these polypeptides are located on a 72 kDa resident plasmid (3) and have all
been cloned and expressed in various hosts. Sufficient means exist to detect and
manage resistance at a higher level and with greater effectiveness. While initial
detection and field surveillance for resistance will likely continue to be based upon
simple bioassay, biochemical and molecular tools, the deeper understanding of how
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resistance arises and maintains itself in populations requires molecular genetics

studies. Resistance detection should be made an integral part of all control
programmes. The resources for vector control, even under emergency situations, are
limited and must be used as effectively as possible. Any attempt to devise management
strategies for delaying or forestalling the evolution of pesticide resistance requires a
thorough understanding of the parameters influencing the selection process. More than
447 species of arthropods have now developed resistance to insecticides (43). The
main weapon for countering this resistance has been the use of alternative chemicals
with structures that are unaffected by cross-resistance. As resistance is now developed
against many chemicals, there is need to maximize the useful life of new chemicals
through their application under conditions that delay or prevent the development of
resistance. Hence, an understanding of the parameters influencing the selection process
is essential.
Fig 2. a) Clarified butter sediment waste from dairy industries; b)Clarified butter sediment (air
dried) waste; c) Culture medium CBW and NYSM.
b) Biopesticide Production from Environmental Waste

There are several cost-effective culture media were standardized by the author
using different kinds of raw materials such as, coconut oil cake (COC), neem oil cake
(NOC), groundnut oil cake (GOC), wheat bran (WB), chickpea husk (CPH), corncob
(CC), chicken feather waste (CFW), slaughter house waste (SHW), coffee husk waste
(CHW), clarified butter waste (CBW) and water melon fruit waste (WMW). The Fig.
2a-c shows the dairy waste product (clarified butter waste) and the extraction of the
nutrients for medium preparation. These raw materials were used to yield superior
level of bacterial production, than the conventional media (LB, NYSM). These new
experimental media were found to have a higher yield, in terms of, biomass and toxin
production. Biochemical analysis showed that, COC, NOC, GOC, WB, CPH, CC
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comprised with rich fatty acids, and the CFW, SHW and WMFP comprised with rich
carbohydrates which are the key components for efficient bacterial production. Thus,
the authors concluded that these materials can be utilized as a suitable substrate for
the biopesticide production. Figure 3,4 depicted the growth pattern of bacteria (Bs/Bti
spore/crystals) and the mosquitocidal toxin production from culture medium prepared
from clarified butter waste (CBW) collected from dairy industries. As seen from the
Figures, the material is as good as that of conventional medium for biopesticide
production. Further, cost wise, the production of Bti from CBW is much more
economical than conventional medium.
Fig.3 Growth pattern of B.thuringiensis subsp. Israelensis.

Fig.4 SDS-PAGE of Bs and Bti.
Lane M: Protein M.wt.marker, Lane 1: NYSM (Bs), Lane 2: CBW (Bs),
Lane 3: NYSM (Bti), Lane 4: CBW (Bti)
The methods reviewed that a known quantity of dried raw materials (50 gm/L)
were boiled separately in ordinary tap water for 15 minutes. After cooling, the
extracts were removed and the pH was adjusted (7.5). The extracts were dispensed
separately into three Erlenmeyer flasks (2 liter capacity each) for culturing Bs and Bti
and plain medium (control, without Bs and Bti) respectively. Similarly, the extracts of
combination media (3:1) were dispensed separately into three Erlenmeyer flasks for
culturing Bs/Bti and plain medium. The conventional medium (NYSM) was also
maintained along with. All the bacterial culture media were autoclaved (at 120 0C / 20
lb / in2 / 20 min). A small amount of Bs and Bti lyophilized primary powder was
inoculated separately in 2 ml each NYSM medium and allowed to grow for 12 h at
32 0C as pre-culture. A small volume of these pre-cultures (50l each) was inoculated
into culture media. The cultures were allowed to grow under constant agitation (120
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rev / min) under room temperature (32 0C) in an orbital shaker. Culture samples
(2 ml) were drawn from each culture medium at 6 hr intervals from 0 to 72 hours.
The pH and culture turbidity were measured using a digital pH meter and UV-VIS
spectrophotometer. These were also examined microscopically for detection of
sporulation and production of crystalline inclusions.
After complete sporulation of Bs/Bti from the media, the spores/crystals were
harvested by centrifugation (10,000 x g / 30 min / 4 0C), by discarding the culture
supernatants. The pellets containing cell mass were washed three times each with
0.1 M NaCl and sterile double distilled water (10,000 x g / 15 min / 4 0C) and finally
these were treated with protease inhibitor (phenyl methyl sulphonyl fluoride,
1mM,Sigma), resuspended in sterile water and stored at 200C, until further use. The
protein concentration in samples is an indication of toxin production from Bs/Bti and
toxicity against mosquito species (Cx. quinquefasciatus, An. stephensi and
Aed. aegypti) were performed. From the above preparation, it was observed that the
toxins produced from experimental media are very useful for the control of mosquito
larvae.
Every day, large quantity of clarified butter waste (CBW) is discarded, by dairy
industries as a waste byproduct, which is also an environmental menace. Many
methods have been adopted to dispose CBW, like burning (103). The basic idea of
the study was to utilize the entire clarified butter waste (CBW), as an economical
substrate, for biopesticide production. The underlining principle was based on the
complete bio-degradation of nutrients from CBW, by the bacterial strains (Bti and
Bs). As it is the bi-product of milk industries, it does not need other nutrients for
culturing the degrading bacteria. This fermentation technology facilitated the
complete utilization of CBW, by avoiding any kind of resultant residual loss or
wastage, resulting in an enhanced production of biopesticides and maintaining a
cleaner environment. This study is, of much relevance, as it upholds the dual benefits
of complete utilization of clarified butter waste from the environment and enabling
the production of mosquitocidal biopesticides.
In the present study, the yield of bacterial cells/crystals, an indicator of biomass
production was estimated by bacterial growth in different hours of culture time. This
was measured by OD values of the bacterial samples (at 650 nm). In both the culture
media (CBW and NYSM), after a lag phase of an hour, there was a rapid
multiplication of bacterial cells and spore maturation till 48 hours. The culture density
showed a corresponding increase and reached a plateau (range 2 to 2.5). This was
followed by cell lysis, releasing the endotoxins into the medium, reaching a peak at
72 hours. Thus, the overall growth and production of Bti and Bs from CBW, was
comparable, to that of NYSM and the mean values were not significantly different
(P>0.05) (Fig. 3). Figure 4 showed the major polypeptides of Bti and Bs spore/crystal
complex produced from CBW and NYSM (Bti: 134, 125, 67 and 27 kDa and Bs: 51
and 42 kDa proteins). There was no variation in the protein pattern, between the
toxins, produced from these two culture media. The microscopic observation of Bti
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spores / crystal complex, obtained from NYSM and CBW, after 72 hours growth,
indicated that, the sporulation in the latter medium was as good as that of the former.
The comparative toxicities (bioassays) of Bti and Bs toxins produced from
CBW and NYSM were shown in Table 1. The LC50 and LC90 values against
Cx. quinquefasciatus for Bti (CBW) were 0.0036 and 0.01mg /l respectively and for
Bti (NYSM) were 0.0033 and 0.011 mg/l respectively, which were statistically
similar (fiducial limits overlapping). The other two mosquito species, also exhibited
similar toxicity effects. Table 1 also presents data on the efficacy of Bs toxins
produced from CBW, in comparison with that of NYSM. The Bs produced from
CBW was also effective against all the three mosquito species tested and was found
to be equally comparable to the toxins produced from NYSM. It is worth mentioning
here that, the Bti toxins produced from both the culture media exhibited a higher
lethal effect on the larvae than the Bs toxins, obviously, due to the presence of
multiple toxins in Bti.
Table 1. Toxic effect of Bti and B.sphaericus against mosquito vectors.
Cost-effective analysis showed that the quantity of clarified butter waste

required to prepare one liter of culture was only 30 gm/L, which is cost free, being a
milk waste. On the contrary, preparation of one liter of NYSM involved a cost of US
$ 3.6 (Table 2). This shows that CBW was cheaper than NYSM, nevertheless, the
bacterial growth, toxin production and toxicity levels of test (CBW) and conventional
(NYSM) media was similar. These results permit us to conclude that, the use of
CBW, in the dried powder form, had manifold benefits, like complete degradation of
feather waste, to avoid environmental contamination, increased shelf-life, easy
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preservation, transportation and convenience in handling and application. This

method is highly economical, as it required comparatively lesser quantity of raw
material (CBW) for culture media preparation, showing high efficacy of bacterial
growth and toxicity, over vector mosquitoes.
Table 2. Comparative costing for producing new culture medium (CBW)
4. Conclusion
Vector-borne diseases like, filariasis, malaria, dengue and Japanese encephalitis,
remain a serious public health problem, all over the world. The control of these
important mosquito borne diseases relies heavily on the extensive use of chemical
insecticides, although, they are very expensive and toxic to non-target organisms.
Owing to these constraints, biological control agents like, Bacillus sphaericus (Bs)
and B. thuringiensis serovar israelensis (Bti) have been found to apply, widely and
effectively, in mosquito control program. But, for operational purposes, there is an
urgent need to produce these bacteria, utilizing cheap and commonly available
biological waste materials, through simple fermentation biotechnology.
From the foregoing studies, it can be concluded that: 1. The use of several
biological waste materials, as an effective bacterial culture medium, is highly
economical for the industrial production of these mosquito pathogenic bacilli, in
terms of easy availability, cost-effectiveness, efficacy, environmental safety, and
bio-waste remediation. 2. The mosquitocidal spore/crystal toxins produced from the
experimental culture medium (CBW) are higher than that of conventional medium
(NYSM). 3. The protein profiles of Bs and Bti spore/crystal toxins produced from
CBW are similar to that of NYSM, by biochemical studies. 4. The entomotoxicity
studies with different mosquito species showed that the efficacy of Bs and Bti toxins
produced from experimental and conventional media were comparable. 5. The
cost-effective analysis indicated that the use of CBW culture medium is highly
economical for the industrial production of these mosquito pathogenic bacilli. 6. This
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study is, therefore, important from the point of recycling of environmental waste, as it
possesses the dual benefits of effective utilization of environmental bio-organic waste
and efficient production of mosquitocidal pathogenic bacilli.
5. Acknowledgements
To Department of Science and Technology, Govt of India, New Delhi
(Ref: SR/FTP/LS-A-86/2001) for funding, Indian Council of Medical Research, New
Delhi for patent (Ref: Indian Patent No. 1106 / Del/ 2008), Director, VCRC,
Pondicherry for permission, and Smt. R. Sundarammal, Senior Library Information
Officer, VCRC, Pondicherry for supply of literatures.
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Article History:
Reviewer:
Review
Received 22nd January, 2013; Revised 20th July, 2013 and Accepted 5th May, 2014; and
Published 30th October 2014.
Philip samuel, ICMR, India,
Raman Chandrasekar, Kansas State University, USA.
BK. Tyagi, ICMR, India.

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Table Contents
SION
MIS
TERNA
IN
T
N AL B OO
IO
Page No.
Preface
Forward message
Contributors
Reviewers
Acknolwedgement
i
ii
iii
iv
v
Volume1
Section I: Insect Biochemical approaches
1. Introduction to Insect Molecular Biology.
Raman Chandrasekar, P.G., Brintha, Enoch Y.Park, Paolo Pelsoi, Fei Liu,
Marian Goldsmith, Anthony Ejiofor, B.R., Pittendrigh, Y.S., Han,
Fernando G. Noriega, Manickam Sugumaran, B.K., Tyagi, Zhong Zheng Gui,
Fang Zhu, Bharath Bhusan Patnaik, and P. Michailova
2.
Modulation of Botanicals on pests biochemistry.
57
Sahayaraj, K.
3.
Detoxication, stress and immune responses in insect antenna:

new insights from transcriptomics.
75
David Siaussat, Thomas Chertemps and Martine Maibeche
4.
Application of isotopically labeled compounds and tandem mass

spectrometry for studying metabolic pathways in mosquitoes.
99
Stacy Mazzalupo and PatriciaY.Scaraffia
5.
Field Response of Dendroctonus armandi Tsai & Li (Coleoptera:

Scolytinae) to Synthetic Semiochemicals in Shaanxi, China.
127
Shou-An Xie, Shu-Jie L.V., Hui-Chen, Raman Chandrasekar
xvii
Section II: Insect Growth
6. Insect Cuticular SclerotizationHardening Mechanisms and Enzymes.
149
Manickam Sugumaran
7. New Approaches to Study Juvenile Hormone Biosynthesis in Insects.
185
Crisalejandra Rivera-Perez, Marcela Nouzova and Fernando G. Noriega
8. The regulatory biosynthetic pathway of juvenile hormone.
217
Zhentao Sheng and Raman Chandrasekar

Section III:
Insect Immunity
9. The innate immune network in a hemimetabolous insect, the brown

planthopper, Nilaparvata lugens.
233
Yanyuan Bao, Raman Chandrasekar, Chuan-Xi Zhang
10. Immune Pathways in Anopheles gambiae.
253
Maria L. Simes and Raman Chandrasekar
11. Key biochemical markers in silkworms challenged with immuno-
271
elicitors and their association in genetic resistance for survival.
Somasundaram, P., Chandraskear, R., Kumar,K.A., and Manjula, A.

Section IV:
Insect Molecular Genetics
12. The recent progress of the W and Z chromosome studies of the
291
silkworm, Bombyx mori
Hiroaki Abe, Tsuguru Fujii and Raman Chandrasekar
13. Molecular characterization and DNA barcoding for identification of
317
agriculturally important insects.
Rakshit Ojha, Jalali, S.K., and Venkatesan, T.
14. Polytene chromosomes and their significance for Taxonomy,
331
Speciation and Genotoxicology
Paraskeva V. Michailova
15. Insect exuvium extracted DNA marker: a good complementary

molecular taxonomic characteristics with special reference
to mosquitoes.
355
Dhanenjeyan, K. J., Paramasivam, R., Thanmozhi, V., Chandrasekar,R., and Tyagi, B.K.
Index
363
xviii
Volume2
Section V:
Molecular Biology of Insect Pheromones
16. Understanding the functions of sex-peptide receptors?
373
Orly Hanin, Ada Rafaeli
17. Current views on the function and evolution of olfactory receptors
385
in Lepidoptera.
Arthur de Fouchier, Nicolas Montagn, Olivier Mirabeau, Emmanuelle Jacquin-Joly
18. Molecular architecture, phylogeny and biogeography of pheromone
409
biosynthesis and reception genes / proteins in Lepidoptera.
Jian-Cheng Chang, P. Malini, R. Srinivasan
Section VI:
Insect Molecular Biology
19. Application of Nanoparticles in sustainable Agriculture :
429
Its Current Status.
Atanu Bhattacharyya , Raman Chandrasekar, Asit Kumar Chandra,

Timothy T. Epidi and Prakasham, R.S.
20. Mosquito Ribonucleotide Reductase: A Site for Control.
449
Daphne Q.-D. Pham, Victor H. Perez, Lissette Velasquez, Dharty Bhakta,

Erica L. Berzin, Guoli Zhou, and Joy. J. Winzerling.
21. Green protocol for synthesis of metal nanoparticles

to control insect pests.
473
Murugan, K., Chandrasekar, R., Panneerselvam, C., Naresh Kumar, A.,

Madhiyazhagan, P., Mahesh Kumar, P., Jiang-Shiou Hwang, Jiang Wei
22. Aquaporins in Blood-Feeding Arthropods.
497
Lisa L. Drake, Hitoshi Tsujimoto, Immo A. Hansen
23. Mimetic analogs of three insect neuropeptide classes
509
for pest management.
Ronald J. Nachman
xix
Section VII:
Insect Pest Management through

Biochemical and Molecular approaches
24. Induced resistance in plants against insect pests and
533
counter-adaptation by insect pests.
Abdul Rashid War and Hari C Sharma
25. Insect Chemical communication - an important component of
549
novel approaches to insect pest management.
Usha Rani, P.
26. Mosquito control using biological larvicides: Current Scenario.
575
Subbiah Poopathi, C. Mani and R. Chandrasekar
27. Application of RNAi toward insecticide resistance management.
595
Fang Zhu, Yingjun Cui, Douglas B. Walsh, Laura C. Lavine
Section VIII:
Insect Bioinformatics
28. Entomo-informatics: A prelude to the concepts in Bioinformatics.
621
Habeeb, S.K.M. and Raman Chandrasekar
29. Molecular expression and structure-function relationships of
633
apolipophorin III in insects with special reference to innate immunity.
Bharat Bhusan Patnaik, Raman Chandrasekar, Yeon Soo Han
30. Computer-aided pesticide design: A short view
685
Jitrayut Jitonnom
Index
709
xx
ISBN No. 978-1-63315-205-2 (USA)
First Edition: Volume 1, 2 October 2014

Total No. Pages: 398 + 372 = 770
Edited by Raman Chandrasekar

B.K. Tyagi
Zhong Zheng Gui
Gerald R. Reeck
Copyright Reserved
Published by International Book Mission, Academic Publisher, South India.
Printed in the K-State Union, Copy and Printing services,

Kansas State University, Manhattan 66506, KS, USA.
This publication is considered to provide accurate and authoritative information with regards to the
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Book Mission Project # 2: Initiated on June 2010; Completed on March 2014 and Published on Oct. 2014.
Volume 1 & 2, October 2014
Short Views on Insect Biochemistry

and Molecular Biology
PREFACE
Entomology as a science of inter-depended branches like biochemistry, molecular entomology, insect
biotechnology; has made rapid progress in its attributes in the light of modern discoveries. This also
implies that there is an urgent need to manage the available resources scientifically for the good of man.
In the past five decades, entomology in the world/country has taken giant steps ahead. Continued
research has evolved better pest management through molecular approaches. The aim of the Short
Views on Insect Biochemistry and Molecular Biology book is to integrate perspectives across
biochemistry and molecular biology, physiology, immunology, molecular evolution, genetics,
developmental biology and reproduction of insects. This century is proclaimed as the Era of
Biotechnology and its consists of all types of Mol-Bio applications, which is an essential component for
a through understanding of the Insect Biology. This volume 1 & 2 (8 section with 30 chapters)
establishes a thorough understanding of physiological and biochemical functions of proteins, genes in
insects life processes; the topics dealt with in the individual chapters include chemistry of the insect
cuticle, hormone and growth regulators; biochemical defenses of insects; the biochemistry of the toxic
and detoxification action; modern molecular genetics and evolution; inter- and intra-specific chemical
communication and behavior; insect pheromone and molecular architecture, phylogeny and chemical
control of insect by using insect pheromones biotechnology; insect modern biology and novel plant
chemical and microbial insecticides for insect control, followed by a discussion of the various
mechanisms of resistance (both behavioral and physiological) and resistance management; modern insect
pest management through biochemical and molecular approaches; Mimetic analogs of insect
neuropeptide for pest management; entomo-informatics and computer-aided pesticide designing. In short
this book provides comprehensive reviews of recent research from various geographic areas around the
world and contributing authors area recognized experts (leading entomologist/scientist) in their
respective filed of molecular entomology. We will miss this collaboration now it has ended, but will feel
rewarded if this book is appreciated by our team/colleagues and remarkable mile stone in entomology
field.
This book emphasizes upon the need for and relevance of studying molecular aspects of entomology in
Universities, Agricultural Universities and other centers of molecular research. To encompass this
knowledge and, particularly disseminate it to the scientific community free of cost, was the major
inspiring force behind the launch of Short Views on Insect Biochemistry and Molecular Biology.
Editors
Raman Chandrasekar
Brij Kishore Tyagi
ii
iii
iv
vi
ShortViewson
InsectBiochemistryand
MolecularBiology
Editedby
Raman Chandrasekar, Ph.D.,
Kansas State University, USA.
B.K.Tyagi, Ph.D.,
Centre for Research in Medical Entomology (ICMR), India.
Zhong Zheng Gui, Ph.D.,
Jiangsu University of Science and Technology,
Sericultural Research Institute, Chinese Academy of
Agricultural Sciences, China.
Gerald R. Reeck, Ph.D.,
Kansas State University, USA.
vii
viii
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Contributing Authors
Dr. B.K.Tyagi
Prof.Fernando G. Noriega
Centre for Research in Medical Entomology,

4Sarojini Street, Chinna Chokkikulam,
Madurai 625002 (TN), India
Department of Biological Sciences

HLS 227, Florida International University
11200 SW 8th St, Miami, FL 33199, USA.
Prof. Gui Zhongzheng
Dr. Zhentao Sheng
Sericultural Research Institute,

Chinese Academy of Agricultural
Sciences, Zhenjiang, 212018,
Jiangsu, P. R. China.
Chicogo University, Chicogo, USA.
Prof. K. Sahayaraj
Prof.Yanyuan Bao
Institute of Insect Science,
Zhejiang University, China.
Dept. of Advanced Zoology and Biotechnology,

St. Xavier's College
Palayamkottai 627 002, Tamil Nadu, India.
Prof. Chuan-Xi Zhang,
Prof. David Siaussat
Dr. Maria L. Simes
Universit Pierre et Marie Curie (Paris 6/UPMC),

UMR 1272A Physiologie de l'Insecte:
Signalisation et Communication (PISC),
7 Quai Saint Bernard, Batiment A - 4me tage bureau 410, 75252 Paris Cedex 05, France.
Prof. PatriciaY.Scaraffia
Department of Tropical Medicine,
Tulane University, New Orleans,
LA 70112, USA.
Prof. Shou-An Xie
Institute of Insect Science,

Zhejiang University, China.
UEI Parasitologia Mdica,

Centro de Malria e Outras Doenas Tropicais,
Instituto de Higiene e Medicina Tropical,
Rua da Junqueira 96, 1300 Lisboa,
Portugal.
Dr. P. Somasundaram
Central Sericultural Germplasm Resources Centre,
P.B.No.44, Thally Road,
Hosur-635109,
Tamilnadu, India.
College of Forestry,
Northwest A & F University
Yangling, Shaanxi 712100, China
Dr. Hiroaki Abe
Dr. Raman Chandrasekar
Dr. S.K. Jalali
Department of Biochemistry and Molecular

Biophysics, Kanas State University,
Manhattan, 66506, KS, USA.
Prof. Gerald R. Reeck

Department of Biochem. and Molecular
Biophyscis, Kansas State University, KS, USA.
Prof. Manickam Sugumaran

Department of Biology
University of Massachusetts Boston
100 Morrissey Blvd,
Boston, MA 02125, USA.
Tokyo University of Agriculture and Technology,

Japan.
National Bureau of Agriculturally Important

Insects, ICAR, India.
Prof. Paraskeva V. Michailova

Institute of Biodiversity and
Ecosystem Research,
1 Tzar Osvoboditel boulv
Bulgarian Academy of Sciences
Sofia 1000, Bulgaria.
Prof. Ada Rafaeli

Associate Director for Academic Affairs &
International Cooperation
Agricultural Research Organization,
The Volcani Center, P. O. Box 6,
Bet Dagan 50250, Iseral.
ix
Prof. Emmanuelle Jacquin-Joly
Dr. Fei Liu
UMR PISC Physiologie de l'insecte

INRA, Route de Saint-Cyr
78026 Versailles cedex, France..
Department of Biological Science & Technol.,

Shaanxi Xueqian Normal University,
Shaanxi, China.
Dr. R. Srinivasan
Prof. Marian Goldsmith
Entomologist and Head of Entomology Group

AVRDC-The World Vegetable Center
60 Yi Ming Liao, Shanhua
Tainan 74151, Taiwan.
Biological Sciences Department,

University of Rhode Island,
Kingston, RI 02881, USA
Prof. Atanu Bhattacharyya
Prof. Anthony Ejiofor
Vidyasagar College for Women,

Post Graduate Department of Environmental
Science,
University of Kolkata, India.
Department of Biological Sciences,

College of Agriculture, Human & Natural
Sciences, Tennessee State University,
3500 John A Merritt Blvd., Nashville,
Tennessee 37209, USA.
Prof. Daphne Q.-D. Pham
Dr. Bharath Bhusan Patnaik
Dept of Biological Sciences,

University of Wisconsin-Parkside,
900 Wood Road, Kensoha,
WI 53144, USA.
School of Biotechnology,
Trident Academy of Creative Technology
(TACT), Bhubaneswar 751013 Odisha, India.
Prof. Jitrayut Jitonnom
Prof. B.R. Pittendrigh
School of Science
University of Phayao, Thailand.
Department of Entomology,
University of Illinois, Urbana-Champaign, IL,
61801, USA
.
Prof. K. Murugan
Dr. Subbiah Poopathi
Department of Zoology, School of Life Sciences,

Bharathiar University,
Coimbatore - 641 046, India.
Prof. Immo A. Hansen

Department of Biology,
New Mexico State University,
Las Cruces, NM, USA.
Dr. Ronald J. Nachman

USDA-ARS,
Food Animal Protection Research Laboratory,
USA.
Dr. Hari C Sharma

International Crops Research Institute for the
Semi-Arid Tropics (ICRISAT), Patancheru502324,
Andhra Pradesh, India.
Prof. Paolo Pelsoi

State Key Laboratory for Biology Plant Diseases
and Insect Pests, Institute of Plant Protection,
Chinease Academy of Agricultural Sciences,
Bejing, China.
Unit of Microbiology and Immunology,

Vector Control Research Centre
(Indian Council of Medical Research),
Medical complex, Indira Nagar,
Puducherry 60 5006, India.
Dr. P.Usha Rani

Biology and Biotechnology Division
Indian Institute of Chemical Technology
(CSIR)Taranaka,
Hyderabad - 500 007 (AP), India.
Dr. Fang Zhu

Irrigated Agriculture Research and Extension
Center, Dept.of Entomology,
Washington State University,
Prosser, WA, USA.
Prof. S.K.M. Habeeb

Department of Bioinformatics,
Faculty of Engineering & Technology,
SRM University, Kattankulathur,
Chennai 603203, Tamilnadu, India.
Prof. Yeon Soo Han

Division of Plant Biotechnology,
College of Agriculture & Life Science,
Chonnam National University,
Gwangju 500-757, South Korea
SION
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Reviewer & External supportive members
Prof. Michael Riehle, Department of Entomology, University of Arizona, USA.

Dr. Dawn L.Geiser, College of Agriculture and Life Sciences, University of Arizona, USA.
Prof. Young Jung Kwon, School of Applied Biosci., Kyungpook National University, South Korea.
Dr. Kaliappandar Nellaiappan, CuriRx Inc. USA.
Prof. Patricia Y. Scaraffia, Department of Tropical Medicine, Tulane University, USA.
Prof. Richard Newcomb, Plant & Food Research, University of Auckland, New Zealand.
Dr. S. Krishnaswamy, School of Biotechnology, Madurai Kamaraj University, South India.
Dr. Mary-Anne Hartley, University of Lausanne, Switzerland.
Dr. Igor F. Zhimulev, Institute of Molecular and Cellular Biology, Novosibirsk, Russia.
Dr. S. Subramanin, Indian Agricultural Research Institute. India.
Prof. Gustavo F. Martins, Departament de Biologia Geral, Universidade Federal de Vicosa, Brazil.
Prof. Helena Janols, Infektionsklinien, Skanes Universitetsisjukhus, Sweden.
Prof. Donald R.Barnard, USDA, Agricultural Research Service, CMAVE, USA.
Dr. Keith White, Faculty of Life Science, University of Manchester, UK.
Prof. Marten J.Edwards, Biology Department, Muhlenberg College, USA.
Prof. E. Warchalowska-Sliwa, Polish Academy of Sciences, Poland.
Dr. K. Balakrishnan, Department of Immunology, Madurai Kamaraj University, India.
Dr. J.Joe Hull, USAD-ARS, Arid Land Agricultural Research Centre, USA.
Dr. Neil Audsley, The Food & Environment Research Agency, UK.
Dr. Raman Chandrasekar, Kansas State University, USA.
Dr. B.K. Tyagi, Centre for Research in Medical Entomology (ICMR), Madurai, TN, India.
Prof. Zhongzheng Gui, Sericulture Research Institute, Chinese Academy of Agricultural Sci., China.
Dr. Fang Zhu, Irrigated Agril. Research and Extension Center, Washington State University, USA.
Prof. K. Murugan, Department of Zoology, Bharathiar University, Coimbatore, India.
Dr. Xiao-Wei Wang, Institute of Insect Science, Zhejiang University, China.
Dr. Haijun Xu, Institute of Insect Science, Zhejiang University, China.
Dr. Alisha Anderson, CSIRO Ecosystem Sciences, Australia.
Prof. Eric D.Dodds, Department of Chemistry, University of Nebraska-Lincoln, USA.
Prof. P. Mosae Selvakumar, Department of Chemistry, Karnaya University, Coimbatore, India.
Prof. A.K.Dikshit, Indian Agriculture Research Institute, New Delhi.
Prof. K.R.S. Sambasiva Rao, Dept. of Biotech. & Zoology, Acharya Nagarjuna University, India
Dr. R. Rangeshwaran, National Bureau of Agriculturally Important Insects, Banglore, India.
Dr. V. Selvanarayanan, Faculty of Agriculture, Annamalai University, Tamil Nadu, India.
Prof. Fernando G. Noriega, Florida International University, Miami, USA.
Prof. Ada Rafaeli, Department of Food Quality and Safety, A.R.O., Israel.
Prof. Daphne Q.-D. Pham, Dept. of Biological Sciences, University of Wisconsin-Parkside, USA.
Prof. Emmanuelle Jacquin-Joly, INRA, UMR 1272 Physiologie de lInsecte, Versailles, France.
Prof. Manickam Sugumaran, University of Massachusetts Boston, USA.
Prof. Nannan Liu, Auburn University, USA.
Prof. Michihiro Kobyashi, Nagoya University, Japan.
Prof. Enoch Y.Park, Innovative Joint Research Center, Shizuoka University, Japan.
Prof. Luiz Paulo Moura ANDRIOLI, Universidade de So Paulo, SP - Brazil
Prof. SHIMADA Toru, The University of Tokyo, Japan.
Prof. Erjun Ling, Institute of Plant Physiology and Ecology, China.
xi
xii
Acknowledgements
Writing and publishing a book requires the assistance of individuals who are
creative, talented, and hard-working. All of these qualities were present in the
individuals assembled to produce this book volume. I would like to express my
heartfelt gratitude to my former teacher Prof. Seo Sook Jae, (GSNU, South Korea),
Prof. Subba Reddy Palli (University of Kentucky, USA), and other external mentors
Prof. Marian R. Goldsmith (University of Rhode Island, USA), Prof. Enoch Y. Park
(Shizuoka University, Japan), Prof. M. Kobayashi (Nagoya University, Japan), Prof.
CHU Jang Hann (National University of Singapore, Singapore), Prof. Thomas W.
Sappington (USDA-ARS, USA), Prof. Fernando G. Noriega (Florida International
University, USA), Dr. Srinivasan Ramasamy, AVRDC, The World Vegetable
Center, Taiwan), Dr. H.C. Sharam (ICRISAT, India), who inspiration and
supported me at many ways for the commencement of this International Book
Mission Program. The book mission program was initiated on May 2010,
completed on March 2014 and published on October 2014. I have no words to
express my feeling for all those who provided valuable contributions from USA,
South Korea, Japan, China, India, Thailand, Taiwan, Bulgaria, France, Iseral, and
Portugal (Contributors name list, see page no. v) and made the completion of this
book possible. We express our appreciation to the following people (Reviewer
name list, see page no. vii) who reviewed various part of the manuscript as it was
being developed and improved quality of each chapter. I thank the ICMR, New
Delhi, and Chinese Academy of Agricultural, China, and Kansas State University for
support from several aspects. Many others (scientists and publishers) have also
allowed us to use their materials in the various chapters, their color image have then
been converted to gray color/BW. Iam especially indebted to International Book
Mission Organization, Academic Publishing Services for the production of book. I
thank my Co-Editors for their continuous vigilance over the book project and for
always giving advance notice of the editing and proofreading schedules. I thank also
my Brintha, P.G., (my wife), who in all possible way, encouragement helped
transform our original efforts into an acceptable final form. I apologize to those
whose work could not be cited owing to space considerations limitation. Further, I
wish to recognize the moral support extended by colleagues and friends. I hope that
this volume will inspire interest on the diverse aspects of insect biochemistry and
molecular biology in aspiring and established scientists.
Raman Chandrasekar
xiii
xiv
A Note from the Publisher

Dear Readers,
This edition represents the first number of the Short Views on Insect
Biochemistry and Molecular Biology book series published by International
Book Mission. It serves to show the public how important entomology field in
expanding basic knowledge or in the development of new technologies nowadays,
in virtually all fields of knowledge. We called for piece of work falling into two
volumes (Basic and Advance aspects).
Far from being complete, the 30 chapters clearly structured and simply explained
experts contributions may provide an overview about current and prominent
advances in insect biochemistry and molecular biology which will help students and
researchers to broaden their knowledge and to gain an understanding of both the
challenges and the opportunities behind each approach.
We look forward to receiving new proposals for the new edition 2015 - 2017.
International Book Mission
Academic Publisher
Manager
xv
Book Series
xvi
xvii
xviii

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Printed in the Unitated States of America, 2014

Printed in the Unitated States of America, 2014

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

Short Views on Insect Biochemistry and Molecular Biology

Unit of Microbiology and Immunology, Vector Control Research Centre

Printed in the Unitated States of America, 2014

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

Printed in the Unitated States of America, 2014

mosquitoes is an essential step towards

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

Fig.1a. Different approaches of mostquito control methods

2. Method of vector control

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

2.2. Non-synthetic chemicals

Printed in the Unitated States of America, 2014

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

c) Insect growth regulators (IGRs)

2.3. Biological control

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

2.4. Other control measures

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

2.5. Precedence of future research

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

their germination.Various species of cyanobacteria have been investigated as vehicles

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

resistance arises and maintains itself in populations requires molecular genetics

b) Biopesticide Production from Environmental Waste

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

Fig.3 Growth pattern of B.thuringiensis subsp. Israelensis.

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

Cost-effective analysis showed that the quantity of clarified butter waste

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

preservation, transportation and convenience in handling and application. This

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

Sharma,V.P. (1999) Fighting malaria in India. Ind. J.Med.Res. 75:1127-1140.

Printed in the Unitated States of America, 2014

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

Short Views on Insect Biochemistry and Molecular Biology Vol.(2), 2014

Printed in the Unitated States of America, 2014

1. Introduction to Insect Molecular Biology.

Modulation of Botanicals on pests biochemistry.

Detoxication, stress and immune responses in insect antenna:

David Siaussat, Thomas Chertemps and Martine Maibeche

Application of isotopically labeled compounds and tandem mass

Stacy Mazzalupo and PatriciaY.Scaraffia

Field Response of Dendroctonus armandi Tsai & Li (Coleoptera:

Shou-An Xie, Shu-Jie L.V., Hui-Chen, Raman Chandrasekar

Section II: Insect Growth

6. Insect Cuticular SclerotizationHardening Mechanisms and Enzymes.

7. New Approaches to Study Juvenile Hormone Biosynthesis in Insects.

Crisalejandra Rivera-Perez, Marcela Nouzova and Fernando G. Noriega

8. The regulatory biosynthetic pathway of juvenile hormone.

Zhentao Sheng and Raman Chandrasekar

9. The innate immune network in a hemimetabolous insect, the brown

Yanyuan Bao, Raman Chandrasekar, Chuan-Xi Zhang

10. Immune Pathways in Anopheles gambiae.

Maria L. Simes and Raman Chandrasekar