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2014
2014

Section III

Insect Immunity

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Short Views on Insect Biochemistry and Molecular Biology

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Vol. (1) 253 270, 2014

Invited Review
Invited Review

Chapter 10

Immune Pathways in Anopheles gambiae

Maria Lusa Simes1*, and Raman Chandrasekar 2

1

UEI Parasitologia Mdica, Centro de Malria e Outras Doenas Tropicais, Instituto de Higiene e
Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal.
2
Department of Biochemistry and Molecular Biophysics, 238 Burt Hall, Kansas State University,
Manhattan 66502, KS, USA.

Abstract

Several experimental studies have demonstrated that Anopheles mosquitoes are able to mount an
efficient immune response against Plasmodium infection, and that this results in major losses for
the parasite during its development inside the vector. The mosquito immune defence involves
complex mechanisms of action, which have been intensively researched during the last decade,
and have led to the development of novel interventions against malaria based on vector-parasite
interactions. In this review, we describe the main components of the innate immune system in
Anopheles gambiae and how they impact Plasmodium development. We focus our attention on the
two major immune signalling pathways, Toll and Imd, and their role in controlling the
transcription and expression of genes described as anti-Plasmodium effectors for their activity in
fighting the pathogen responsible for one of the worlds deadliest diseases.
Key words: Anopheles, Plasmodium, malaria, innate immunity, PAMPs, PRRs, Toll and Imd pathways, gene
expression, AMPs, transgenic mosquitoes.
*For Correspondence (email:

maria.luisa.simoes@ihmt.unl.pt)

1. Introduction
Malaria is a worldwide infectious disease caused by Plasmodium parasites and
transmitted by female Anopheles mosquitoes. As the host immune system is unable to

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Overview
1. Introduction
2. Pattern Recognition Receptors (PRRs)
3. Serine Proteases
4. Antimicrobial Peptides (AMPs)
5. Immune Signalling Pathways
5.1 The Toll Pathway
5.2 The Imd Pathway
5.3 Regulatory Pathways of anti-Plasmodium Genes
5.4 The Jak/Stat Pathway
6. Role of Bacteria in the anti-Plasmodium Response
7. Transmission Blocking Strategies for the Control of the Malaria Vector
8. Conclusion
9. Acknolwedgement
10. References

control parasite replication and the challenge of producing an effective vaccine has
yet to be met, malaria devastates the tropics and subtropics, causing an estimated two
million deaths per year. Further hurdles to disease control arise from the growing
resistance in parasites to anti-malarial drugs, as well as in mosquitoes to insecticides.
New strategies to control malaria are desperately needed, and it is therefore becoming
important to concentrate research efforts on the interactions between the parasite and
its vector, in the hopes of enhancing the anti-parasitic properties of the mosquitos
immune system.
Plasmodium parasites are transmitted to the vertebrate host as part of a complex
life cycle. Within the host, Plasmodium completes the asexual stages of its life cycle,
while the sexual sporogonic stage takes place inside the mosquito vector and is
marked by several developmental transitions for the parasite (Fig.1A). The
sporogonic cycle starts when the mosquito ingests infected blood containing
Plasmodium gametocytes. These differentiate into male and female gametes and
fertilization occurs within the midgut lumen of the mosquito, resulting in the
formation of zygotes. Zygotes develop into motile ookinetes, which penetrate the
peritrophic membrane and invade the midgut epithelial cells. Ookinetes settle under
the basal lamina surrounding the mosquitos midgut, differentiating into oocysts.
During oocyst maturation, the parasite undergoes several mitotic divisions to form
thousands of sporozoites, that are then released into the hemolymph. Sporozoites
migrate through the hemocoel and invade the salivary glands (Fig.1A), until they are
transmitted to the vertebrate host upon another blood meal, thus starting the asexual
part of the Plasmodium life cycle.

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Fig. 1. Plasmodium sporogonic cycle inside the Anopheles mosquito. (A) The approximate
developmental time for each stage is indicated. (B) 1-3 indicate the three main bottlenecks. hr (hours),
pbm (post-blood meal). Note: figures not drawn to scale.

As previously explained, Plasmodium parasites live within three main

compartments inside the mosquito vector: the midgut, the hemocoel and the salivary
glands. All of these compartments constitute physical barriers against invasive agents.
Moreover, the cells within these compartments produce immune factors with
antimicrobial activity, as we will later discuss in further detail. During the sporogonic
cycle, the parasite experiences three main bottlenecks, when its numbers are largely
reduced: 1) the transition between gametocytes and ookinetes, 2) between ookinetes
and mature oocysts and 3) between midgut sporozoites and salivary gland sporozoites
(Fig.1B). Indeed, it is said that Anopheles gambiae mosquitoes can kill around 80%
of invading Plasmodium ookinetes (1).
The extreme parasite losses are certainly due to efficient mosquito immune
defences; the malaria vector mosquito Anopheles can trigger effective mechanisms to
control completion of Plasmodium lifecycle. A wealth of knowledge on mosquito
immunity to Plasmodium has been building up in the last decade. These studies are
mostly based on the major African vector of malaria, Anopheles gambiae
(A. gambiae), a suitable model for studying the cellular and molecular interactions
between vector and parasite, and for which genome sequencing offers a fundamental
tool (2). In vertebrates, the fight against invasive pathogens is a result of the
interaction between adaptive and innate immunity. Insects, however, lack an adaptive
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immune system, and thus rely solely on innate immunity for their defence. Insect
blood cells (hemocytes), play a prominent role in their immune response, through
phagocytosis and encapsulation of foreign invaders (cellular response), as well as
their melanization (humoral response). Hemocytes, together with the fat body (similar
to the mammalian liver), are also able to release immune effectors into the insects
circulatory fluid (hemolymph). The production of these antimicrobial effector
molecules is regulated by intricate intracellular immune signalling pathways, which
have been extensively described in Drosophila. Although the majority of intracellular
components are conserved, several differences exist between the immune pathways of
the fruit fly and the malaria vector, as we will further explore. In this review, we
describe the immune responses of A. gambiae to Plasmodium infection, focusing on
the immune signalling pathways and the activation of anti-Plasmodium effectors.

2. Pattern Recognition Receptors (PRRs)

Pathogen elimination in both vertebrates and invertebrates is initiated by pattern
recognition receptors (PRRs), which sense, recognize and bind to pathogen associated
molecular patterns (PAMPs). Insects fight microbial infections using a limited
number of PRRs. In A. gambiae, about 150 putative PRRs have been identified (3),
which are mostly members of large gene families, such as the thioester-containing
proteins (TEPs), leucine-rich repeat proteins (LRRs), C-type lectins (CTLs) and
fibrinogen-related proteins (FREPs or FBNs), the latest being the largest PRR family
in this vector (4). Among these families are some of the most potent anti-Plasmodium
immune factors identified to date.
The most studied member of the TEPs family is TEP1, a hemocyte-specific
protein first described as being involved in bacterial opsonization (5) and later as
mediator of Plasmodium berghei (P. berghei) ookinete destruction in A. gambiae (6).
TEP1 activation and stabilization is achieved by interaction with LRIM1 and APL1C
(PRR proteins of the LRR family) to form a complex prior to binding to ookinetes in
the midgut, mediating their degeneration. The composition of this complex (TEP1, a
complement-like protein with structural similarities to vertebrate complement
component C3 (7), and two LRR proteins), suggests that mosquitoes have a
complement-like system comparable to the one in mammals, which is able to
effectively fight Plasmodium infection. Independent silencing of these three genes
has shown that each one is individually relevant to control the development of
P. berghei oocysts, besides playing an important role in parasite melanization (8), an
effective immune killing mechanism involving the deposition of melanin on the
parasite surface. Another key anti-Plasmodium protein of the LRR family is LRRD7
(also designated as APL2) (9).
Acting in an opposing manner, A. gambiae CTL4 and CTLMA2 seem to be
agonists of Plasmodium infection in the midgut, where their silencing results in strong
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ookinete melanization and reduction in the number of oocysts (10). Recently, these
two CTLs have been identified as essential for mosquito defence against
Gram-negative, but not Gram-positive bacteria (11). The mechanism by which some
PRRs fight the parasite in the vector, while other seem to protect it from the mosquito
immune responses, is under study, together with the possible relation between CTLs
antibacterial role and ookinete melanization.
Fibrinogen-related proteins (FREPs) constitute a large PRR family in
mosquitoes, with 59 genes identified in A. gambiae and 37 genes in Aedes aegypti.
This family seems particularly broad in the mosquito, as only 14 such genes have
been reported in Drosophila melanogaster (4,12,13). The majority of FREPs in
mosquitoes are upregulated upon immune challenge and several have
anti-Plasmodium activity. FBN9, an important member of the family mediating
Plasmodium killing, co-localizes with the ookinete stage of both P. berghei and
P. falciparum parasites in the midgut (4). Evidence from observations in vertebrates
and co-localization at parasite surface led Garver et al. (14) to speculate that a
mechanism similar to the lectin complement pathway, in which TEP1 and FBN9
cooperate to destroy pathogens, may also exist in mosquitoes. Another important PRR
family within the mosquito, the peptidoglycan recognition proteins (PGRPs), with 7
genes described in the A. gambiae genome (15), is also involved in the activation of
various immune reactions in the Anopheles mosquito.

3. Serine Proteases
Serine proteases are proteolytic enzymes present in the hemolymph, where they
can rapidly activate immune pathways in response to pathogen detection by PRRs,
amplifying the signal and triggering downstream effector responses to eradicate the
pathogen invader. The most important components of the serine protease cascade are
the CLIP domain serine proteases, 41 of which have been identified in A. gambiae so
far. Many CLIPs are involved in the melanization of P. berghei ookinetes, where they
have roles as both positive and negative regulators of this anti-parasitic process.
CLIPA2 and CLIPA7 are inhibitors of parasite melanization (10), hence playing a
role in parasite protection, while CLIPA8 and CLIB17 seem to be essential to activate
the enzyme prophenoloxidase (PPO), responsible for melanin synthesis (16) and thus
melanization. Moreover, other CLIPs such as CLIPB14 and CLIPB15, are involved
in P. berghei elimination in A. gambiae, in a melanization-independent way (15).
Serpins (serine protease inhibitors) are regulators of the serine protease cascade
and, similarly to CLIPs, modulate melanization responses. SRPN2 inhibits
melanization of P. berghei ookinetes in A. gambiae (17). It is thought that this protein
may interact with CLIPs, CTLs and LRIM1, as they all are molecules known for
interfering in the melanization process.

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4. Antimicrobial Peptides (AMPs)

Besides melanization, the humoral responses to pathogen invasion culminate
with the production of antimicrobial peptides (AMPs), which are synthesized by the
fat body and hemocytes and secreted into the hemolymph upon immune challenge.
Smaller contributions to AMP production in the malaria mosquito as well as other
insects occur as local secretion by different tissues, such as the barrier epithelia. AMP
production constitutes the ultimate step of the defence mechanisms employed by
Anopheles mosquitoes to fight Plasmodium. In A. gambiae, four AMP families have
been identified to date, comprising four defensins (DEFs), four cecropins (CECs),
one attacin and one gambicin (GAM1) (15). Their antimicrobial action comprehends
responses against Gram-negative (mainly CEC1 and GAM1) and Gram-positive
(mainly DEF1) bacteria. Some are known to have an anti-Plasmodium role in
A. gambiae as well. CEC1 was one of the first anti-Plasmodium factors to be
identified (18). This gene was significantly upregulated following Plasmodium
infection (15) and its ectopic overexpression was shown to increase A. gambiae
resistance to P. berghei (19). In a more recent study, silencing of GAM1 resulted in
increased P. berghei infection (9).
Defensins are the most extensive insect family of AMPs, occurring in every
insect species investigated so far, from primitive to more recent insect orders, as well
as in other arthropods. This family of small, cationic peptides, plays an essential role
in the innate immunity of virtually almost all life forms from plants, to insects,
amphibians and mammals. Anopheles DEFs 2-4 are clustered together in the genome
and their sequences are very alike, suggesting that they represent a branch of
divergent defensins that may be the result of recent gene duplication (15). The
Anopheles Defensin 1 (Ag DEF1) is 102 amino acids (aa) long, including a 27-aa
putative signal sequence (MKCATIVCTIAVVLAATLLNGSVQAAP) for secretion
(Fig.2A), and has a molecular mass of 10kDa. Sequence comparison showed that Ag
DEF1 has a significant degree of similarity to other insect defensins, with six cysteine
residues at the conserved place as other defensin-like peptides
(CCXXXCCCXC) (Fig.2A). These findings agree with previous studies
(20-22). The three-dimensional structure of Ag DEF1, based on its secondary
structure in aqueous solution or by sequence homology, shows the peptide with
triple-stranded anti-parallel -sheets (Fig.2B). Interestingly, this motif is also
common to several peptides (gallerimycin, heliomicin, sapecin, termicin, royalism,
drosomycin), and appears to be a frequent organization for several families of small
proteins with toxic properties. Fig.3 shows the phylogenetic relationship between
A. gambiae and other arthropods based on the amino acid sequence of DEF1. As
previously mentioned, transcriptional regulation of AMPS is controlled by immune
signalling pathways in both Drosophila and Anopheles, which are explained in detail
below.

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Fig. 2. Sequence alignment of Anopheles gambiae Defensin 1 with other insects and its 3D
structure. (A) Ag DEF1 sequence, including a 27-aa putative signal sequence for secretion. The
significant sequence similarities with other insect defensins and the six cysteine residues at the
conserved position are marked. (B) Schematic representation of the secondary structure of leader
peptide, mature peptide and C-terminal tail. Residues involved in helix are cylindrical and residues
involved in the strands of -sheet are arrows as indicated.

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Fig. 3. Phylogenetic tree based on the amino acid sequence of Anopheles gambiae Defensin 1 and
comparison to other arthropods sequences registered at NCBI. (A) Phylogenetic tree was
constructed using the aligned sequences with a neighbour-joining distance method of Phylip software
Version 3.2 for Windows. The lengths of the lines are proportional to the minimum number of aa
differences required to join nodes. (B) Short sequences containing the 59-RAKRAT-64 or RVRR
motif region variations across insect taxa.

5. Immune Signalling Pathways

In both mammals and insects, recognition of PAMPs by PAMP-associated PRRs
is followed by signal transduction through immune signalling pathways that activate
NF-kB/Rel transcription factors. These factors translocate to the nucleus to initiate
the transcription of an array of effector genes, including AMPs. In Drosophila, two
immune signalling pathways, the Toll and Immune deficiency (Imd) pathways, have
been extensively studied. The Toll pathway is triggered by fungi and Gram-positive
bacterial infections, leading to the nuclear translocation of NF-kB transcription factor
Dif (as well as Dorsal, which is involved in early developmental processes), whilst
the Imd pathway signalling results in nuclear translocation of Relish, the third NF-kB
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protein in Drosophila, following Gram-negative bacterial infection. Both factors

induce the transcription of all the AMPs encoded in the Drosophila genome (23).

Fig. 4. Toll and Imd signalling pathways components involved in anti-Plasmodium defence.
Gram +, Gram (Gram-positive, Gram-negative bacteria), AMPs (antimicrobial peptides).

Sequencing of the A. gambiae genome allowed for the comparison of the

putative immunity genes between Anopheles and Drosophila (2), supporting the
conclusion that most intracellular components of these pathways are conserved in the
mosquito (Fig.4). A remarkable difference between the two evolutionarily related
insects is the lack of Dif in Anopheles; therefore, the malaria mosquito genome
encodes two NF-kB transcription factors only: REL1 (the orthologue of Dorsal,
previously called Gambif1 (24) and REL2, Drosophilas Relish orthologue.
Both the mosquitos REL proteins are directly involved in the immune response
against invaders. They translocate to the nucleus to initiate the transcription of many
effector genes and REL-dependent transcription has been shown to be particularly
critical to the mosquitos ability to manage infection with P. berghei and
P. falciparum (14, 25-29). Therefore, REL1, REL2 and their associated pathways are
critical targets for the development of anti-malaria strategies based on vector-parasite
interactions (30).
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5.1 The Toll Pathway

In humans and mice, respectively ten and twelve Toll-like receptors (TLRs) have
been characterized thus far. TLRs play a critical role in the early innate immunity
against foreign pathogens. Their name derives from the firstly identified Drosophila
Toll protein, which is stimulated by the binding of the proteolytically activated
extracellular ligand, Sptzle (31), following fungal and Gram-positive bacterial
infection. Upon activation by Sptzle, the intracytoplasmatic domain of the transmembrane Toll receptor (named TIR), recruits three death-domain proteins, MyD88,
Tube and Pelle. Thus forms the Toll receptor complex, which causes the degradation
of Cactus, an ankyrin-repeat protein, hence allowing the nuclear translocation of the
above mentioned NF-kB transcription factors Dorsal and Dif.
In A. gambiae, the intracellular machinery of the Toll pathway is conserved
(Fig.4). Orthologues of MyD88, Tube and Pelle, as well as negative regulator Cactus,
have been identified in the malaria mosquito (15). As mentioned, REL1 is the NF-kB
factor controlling the transcription of genes regulated by the Toll pathway in
Anopheles. The importance of REL1-pathway stimulation was made clear when
knocking down of Cactus (by dsRNA injection) dramatically decreased the parasite
load of P. berghei in A. gambiae. Further, simultaneous co-silencing of REL1 and
Cactus reversed the dsCactus phenotype (26). Similar results were obtained by
Garver et al., where a significant reduction in the infection levels of P. berghei was
observed after Cactus depletion (14). The same authors have also shown that Cactus
silencing altered the expression of several genes from other functional groups besides
immunity, suggesting that the Toll pathway is a ubiquitous signalling pathway, with a
wide-ranging action, this probably being the reason for the marked reduction in both
longevity and fecundity shown in the dsCactus mosquitoes in this study (14).
Importantly, in the above mentioned study (26), it has been said that the mosquitos
basal immunity level (before the mosquito encounters the parasite) is a key factor for
parasite control, i.e., it is significantly more influent in Plasmodium development than
the induction of immune responses upon parasite infection.
5.2 The Imd Pathway
Activation of the immune deficiency (Imd) pathway starts when a pathogen
(classically, a Gram-negative bacteria) is detected by a transmembrane PRR from the
PGRPs family, a family of proteins conserved from insects to mammals. This
pathway is similar to that of the mammalian tumor-necrosis factor (TNF) (32, 20).
Intracellular signalling begins with the recruitment of IMD, a death domain adaptor
located downstream of the receptor and upstream of a series of caspase-like proteins
and kinases. This process culminates with caspase Dredd-dependent cleavage of
Relish (33, 34), and the consequent release of its Rel-homology domain (RHD) from
the inhibitory ankyrin domain. This cascade results in RHDs nuclear translocation
and the subsequent transcriptional induction of a battery of AMPs (25). Caspar has
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been identified as a specific negative regulator of the Imd pathway, preventing the
nuclear migration of Relish (34).
All components of the Imd pathway are conserved in the Anopheles mosquito
(15) (Fig.4). Through alternative splicing, two isoforms of REL2, the Relish
orthologue, are present in Anopheles: REL2-F (a full-length form) and REL2-S
(a short-length form), the latter lacking the inhibitory ankyrin repeats and death
domain present in REL2-F. Both transcripts are expressed constitutively throughout
A. gambiae, as well as in cultured cell lines (25, 27). In the absence of immune
stimulation, REL2 exists in the two variants: REL2-S, that is constitutively active, and
REL2-F, that is inactive until immune stimulus. Imd pathway activation stimulates
cleavage of the inhibitory ankyrin terminal domain of REL2-F, exposing it to nuclear
translocation and subsequent transcription initiation (35). Besides their role against
Plasmodium, in A. gambiae REL2-F was shown to be involved in the defence against
Gram-positive Staphylococcus aureus, and REL2-S against Gram-negative
Escherichia coli (25), a contrast to the Gram-negative specificity of the Imd pathway
in Drosophila.
Elucidation of the role of REL2 in fighting Plasmodium was obtained through
observation that its knockdown increased the number of P. berghei oocysts in
A. gambiae (25, 26). In a more recent study (14), depletion of Caspar reduced the
number of P. berghei oocysts, in addition to almost abrogating P. falciparum infection
in three Anopheles species: A. gambiae (the major African vector), A. stephensi (the
major Asian vector) and A. albimanus (the major South American vector). In contrast
to Toll, the Imd pathway is thought to be immunity-specific (14). Overall, its
properties suggest that this pathway could be used in the development of malaria
control approaches, through interference in the parasite sporogonic cycle. Indeed,
target expression of REL2 has been recently used to create transgenic lines of
A. stephensi mosquitoes with particularly reinforced immunity against Plasmodium
and microbial infection (30).
5.3 Regulatory Pathways of anti-Plasmodium Genes
The process of regulation for the expression of immunity-specific genes is
dependent on the instigating PAMP, as well as the selectively activated pathway.
PAMPs that are effective elicitors of the mosquitos immune response are
lipopolysaccharides (LPS), peptidoglycans (PGNs) and -1, 3-glucans. Current
studies are being developed to shed light on Plasmodium-specific PAMPs that
activate the Anopheles immune system. One molecule that has already been identified
as an inducer of AMPs production in A. gambiae is glycosylphosphatidylinositol
(GPI), labelled as the prominent toxin that contributes to malaria pathogenesis in
mammals, through the anchoring of parasite proteins on cellular plasma membranes
(36). In our laboratory, we found that the Plasmodium metabolite hemozoin acts as a
stimulator of A. gambiaes immune response against P. berghei (Simes et al.,
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unpublished). Both GPI and hemozoin induce the transcription of the enzyme nitric
oxide synthase (NOS) (37, 38) and the resulting nitric oxide (NO) production prompts
the killing of Plasmodium ookinetes in the midgut (39, 40). Inhibition of NOS was
shown to increase the parasite numbers in A. stephensi mosquitoes (39). Together
with reactive nitrogen species, the Plasmodium blood meal also activates the
production of reactive oxygen species (ROS), to help containing parasite infection
(41, 42).
In A. gambiae, both Toll/REL1 and Imd/REL2 pathways control the expression
of important anti-Plasmodium factors. Among the most relevant genes described as
having anti-malaria activity, are those of the AMPs DEF1, CEC1, CEC3 and GAM1,
which appear to be controlled by both pathways/transcription factors. Silencing of the
negative regulators of the Toll and Imd pathways (Cactus and Caspar, respectively)
has upregulated the levels of these four AMPs (14). Further, co-silencing of the
negative regulator together with the correspondent transcription factor reversed this
phenotype, indicating that both immune pathways control the expression of these
important anti-Plasmodium genes (14). These results contradict a previous
A. gambiae study, where Frolet et al. (26) had also knocked down the Toll negative
regulator Cactus, but instead did not observe a distinct upregulation of any of these
four AMPs. Luna and colleagues (27) have shown in vitro (using mosquito cell lines)
that both pathways regulate DEF1 and GAM1 expression. Double regulation of
AMPs by the Toll and Imd pathways is not a particular Anopheles feature, as it has
also been described in Drosophila (34, 43). Meister and co-workers (25) classified
CEC1, CEC3 and GAM1, among other immune genes, as REL2-regulated, when they
assessed gene expression profiles by DNA microarray following REL2 silencing in
A. gambiae hemocyte-like cells; similarly, Hoa & Zheng (28) showed a
REL2-dependent upregulation of CEC1 in vitro.
Alongside AMPs, some key Anopheles anti-malaria effectors belonging to the
PRRs group, seem to be regulated by either of the immune pathways. TEP1 has been
reported as REL1- and REL2-dependent. In the above mentioned study, Frolet et al.
(26) indicated that before Plasmodium invasion, TEP1 basal expression is controlled
by both REL factors, but Plasmodium-dependent upregulation of TEP1 is
independent of REL1 and REL2. Subsequent studies (14, 30) have shown that TEP1
expression is REL2-dependent. The combined outcomes of these studies suggest that
this potent anti-Plasmodium factor regulation may not be pathway-specific. APL1C, a
TEP1-interacting PRR in antimalarial defence, seems to be Toll pathway-dependent
(29), although in (26) the authors have not noticed any effect on this genes
expression profile upon REL1, REL2 nor Cactus silencing. As for LRIM1, another
key gene in the Anopheles immune response, interacting with TEP1 and APL1C,
although it was shown to be Imd-regulated in A. gambiae cells (25), a subsequent
study (44) suggested a REL1/Caspar-dependency. Other very important PRRs that act
as Plasmodium-fighting genes, FBN9 and LRRD7, are thought to be regulated by the
Imd-REL2 pathway (30). Moreover, another two immunity factors involved in the
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Anopheles anti-malaria response, TEP3 and TEP4, are likely to be REL2-regulated

genes (14, 27, 28).
In conclusion, REL1 and REL2 control the A. gambiae immune response against
Plasmodium by regulating the expression of anti-parasitic genes. This regulation can
be mediated by each transcription factor independently, or, as seen in many cases,
both factors can be involved in the transcription of the same gene. The disclosure of
the specific regulation for each anti-Plasmodium effector is an essential question that
awaits further investigation.
5.4 The Jak/Stat Pathway
In addition to the Toll and Imd, there is a further mechanism of immune
signalling, the Janus kinase-Signal Transducer and activator of Transcription
(Jak/Stat) pathway. This pathway has been intensively researched in Drosophila but
still rather under-explored in the context of Anopheles-Plasmodium interaction.
Nevertheless, two Stat transcription factors, Stat-A and Stat-B, have been identified in
the A. gambiae genome (15), with the first playing a role in the mosquito defence
against Plasmodium. In fact, Gupta et al. (45) observed an increase in P. berghei
oocysts following silencing of this transcription factor, while knockdown of SOCS,
the pathways negative regulator, reduced the levels of P. berghei infection, by
increasing NOS expression. In a more recent study, the Jak/Stat pathway was shown
to be activated in A. aquasilis (a major Brazilian malaria vector) in response to
P. vivax challenge, and an increase in P. vivax oocysts number was observed after
depletion of the Stat transcription factor (46).
The role of the Jak/Stat pathway in the activation of Anopheles immunity against
the malaria parasite is subject of a growing interest and requires additional
investigation. Together with the Imd/REL2 and Toll/REL1 pathways, this immune
pathway can be seen as a potential target for development of malaria control
strategies.

6. Role of Bacteria in the anti-Plasmodium Response

Analysis of the Anopheles immune responses against Plasmodium has brought
attention to the fact that these mechanisms are effective not just in fighting
Plasmodium infection, but also in contending bacterial challenge. Reversely, immune
responses mounted by the mosquito against invading- or midgut endogenousbacteria, incite the activation of genes associated with the anti-Plasmodium immune
response and the consequent elimination of a large number of parasites (47-49).
Indeed, a bacterial colonization of the midgut following a blood meal coincides with
Plasmodium invasion and an immune response mounted by the vector would target
both types of invaders. The Anopheles immune system activation following bacterial
challenge is thought to be Imd pathway-regulated (47, 48), a hypothesis that had
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Invited Review

already been proposed for Drosophila (23). Results from a microarray screen in which
the gene expression between Plasmodium-infected and bacteria-injected A. gambiae
mosquitoes was compared, revealed an overlap in Plasmodium- and bacteriastimulated gene regulation. In fact, important anti-Plasmodium factors such as PRRs
TEP1, FBN9, LRRD7 and CTL4, among others, have all been found to control
resistance to bacterial infection (9). In a more recent study, the same authors
compared the susceptibility to P. falciparum between septic and aseptic
(antibiotic-treated) A. gambiae mosquitoes and concluded that the bacteria-deprived
group was significantly more susceptible to malaria infection. Alternatively,
co-feeding bacteria with parasites has decreased the number of P. falciparum oocysts
in the midgut (47). Future studies are needed to reveal the molecular mechanisms by
which gut microbiota are able to modulate the vectorial capacity of the Anopheles
mosquito.

7. Transmission Blocking Strategies for the Control of the Malaria Vector

Blocking the transmission of Plasmodium using the transgenic manipulation of
its mosquito vector has been achieved in a number of malaria transmitting species,
such as A. stephensi (30, 50-53), A. gambiae (19, 54) and Aedes aegypti (55, 56). It
constitutes a promising malaria-control strategy, presently being applied in several
vector-studying laboratories. In theory, these Plasmodium refractory mosquitoes
should be capable of replacing the wild populations and some semi-field studies are
currently taking place to validate the implementation of this technology.
Identification of promoters to drive transgene expression in a stage- and
tissue-specific manner is a critical step in engineering transgenic lines for the
mosquito. In such technology, a genes coding region, driven by a tissue- and
time-specific promoter, is transformed into the mosquito vector. In the case of
Anopheles transgenic manipulation, this could result in the overproduction of an
anti-Plasmodium factor or silencing of a Plasmodium positive regulator, culminating
in a decline in the overall number of parasites, therefore interfering with the
Plasmodium cycle through reduction of the transmission to the vertebrate host. In
Anopheles, the most commonly used midgut-specific promoter is the A. gambiae
carboxypeptidase A (30, 50-54). The use of another midgut-specific promoter, G12,
has been recently validated in A. stephensi (57). Concerning the fat body, the most
frequent specific promoters for transgenic studies are the Anopheles vitellogenin
genes 1 (30, 53) and 2 (58). All mentioned promoters are blood meal induced. Both
the carboxypeptidase and vitellogenin promoters have been extensively used to target
the malaria parasite at its early stages of development.
In a very promising paper, Dong and colleagues (30) were able to create
transgenic lines of A. stephensi mosquitoes with increased expression of REL2
transcription factor in the midgut and fat body tissues. The engineered mosquitoes
showed higher resistance to Plasmodium and bacterial infections, probably due to the
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Invited Review

action of overexpressed anti-Plasmodium effector genes (TEP1, APL1, LRRD7) in

these REL2-enhanced transgenic lines. Although most transgenic studies in malaria
vectors have been performed in A. stephensi, the creation of transgenic A. gambiae
lines is essential, as this is the major vector for malaria.
Another approach to malaria transmission blocking consists of the development
of vaccines that would fight the disease at the vector level, thus interrupting its
transmission to the human host. Presently, research involving transmission-blocking
vaccines (TBVs), molecules that elicit the production of human antibodies interfering
with the parasite development inside the mosquito, aims to identify smart candidate
antigens to be used as transmission blocking targets.

8. Conclusion
The dynamic immune interaction between the vector host and the malaria
pathogen determines the success of Plasmodium development and continuation of the
subsequent disease transmission cycle. The failure to produce an effective malaria
vaccine has generated a growing interest in controlling malaria dissemination at the
vector level. An understanding of the interactions between the vector and its parasite
is essential, in order to effectively harness the mosquitos innate anti-Plasmodium
immune pathways as a means for transmission control. In this context, the future
challenge to fight this life-threatening disease includes detailed dissection of all the
Anopheles genes and proteins involved in these interactions, alongside the parasite
molecules targeting the activation of the mosquito immune response. A better
understanding of how mosquitoes kill microbial pathogens will be fundamental to
develop new control strategies, namely the improvement of transmission-blocking
vaccines and the creation of mosquito transgenic lines with a reinforced
anti-Plasmodium activity, which could eventually eradicate this devastating parasitic
infection.

9. Acknowledgements
This work was supported by Fundao para a Cincia e a Tecnologia Grant
SFRH/BD/70110/2010 and Project PTDC/SAU-MII/102596/2008.

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____________________________________________________________________________________________________
Article History: Received 10th May 2013; Revised 25th September 2013 and Accepted 15th February 2014;
Pubished 30th October 2014.
Reviewed by: Marry-Anne Haerteley, University Lausanne, Switzerland.
Helena Janoels, Skanes Universitetssjukhus, Sweden.
Stefanie Meese, Charit Universitatsmedizin, Berlin, Germany.

GFP-tagged Plasmodium berghei oocysts inside the

midgut of the Anopheles gambiae mosquito.
Scale bar 15m. Photo Credit: Maria Lusia Simoes
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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
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Kansas State University, Manhattan 66506, KS, USA.
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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

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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.

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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.

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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

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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.

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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

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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

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