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2014
2014

Section III

Insect Immunity

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NAL B OO

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TERNA
IN
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SION
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Short
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Invited Review
Review
Invited

Chapter 11

Key biochemical markers in silkworms challenged with immuno

elicitors and their association in genetic resistance for survival

Somasundaram, P.1*, Raman Chandrasekar2, Ashok Kumar, K.1, Manjula, A.1

1

Central Sericultural Germplasm Resources Centre, P.B.No.44, Thally Road,

Hosur-635109, Tamilnadu, India.
2
Department of Biochemistry and Molecular Biophysics, 238, Burt Hall, Kansas State University,
Manhattan KS 66506, USA.

Abstract

Sericigenous insects like silkworm possess an effective immune system against pathogens. The
knowledge on the immune system of these insects implicating the mechanism of resistance to
diseases as mainly attributable to the presence of key biomolecules such as an inducible proteins
viz., cecropin, attacin, lebocin ,moricin and isoforms of enzymes viz., carboxyl esterase and
prophenoloxidase, which are reproducible qualitative biochemical markers that would favor us in
enhancing our theoretical level on the mechanism of immunity. Studies on these key biomolecules
in silkworms challenged with immuno elicitor viz., lipopolysaccharide (LPS) as a function of
immunity and survival ability of the silkworms revealed the mode of cellular and humoral functions
combating the diseases in nature. These factors can be used by researchers or breeders to identify
the appropriate hardy genetic material from the germplasm stock for their further breeding work. In
this review an attempt has been made to delineate those key biomolelcules which are qualitatively
reproducible and that support survival of the silkworms of Bombyx mori (L). These factors may be
considered as key factors to identify the genetic resistance of various silkworm races/breeds of B.
mori and documentation of hardy silkworm breeds for field exploitation in different climatic
conditions of the tropical zones of the country.
Key words: silkworm, resistance, inducible proteins, biomolecules, germplasm, survival
*For Correspondence Email: drpsunder@gmail.com

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Over view
1. Introduction
2. Mechanisms of insect immunity
2.1. Proteins markers for silkworm disease resistance
2.2. Enzyme markers associated with disease resistance
3. Role of antimicrobial proteins on immunity
4. Conclusion
5. Acknowledgements
6. References

1. Introduction
Conservation of economically important sericigenous insects from varied
agro-calimate conditions are imperative to preserve their genetic potential and for
survival. It ensures, their maintenance of the genetic resources and increasing of the
yield potentials for the commercial exploiters in the silk industry. The survival of
sericigenous insect Bombyx mori (L) varies because several silkworm breeds were
developed by the breeders suiting the specific need of varied environmental conditions
prevailing in the tropical zones of India for their better survival and higher crop
productivity. There are many as much as 450 different breeds which are being
conserved and maintained at Central Sericulture Germplasm Resources Centre (Fig.1),
India for utility by the researchers and breeders in the country (1,2).
Conservation biologists are interested to maintain these breeds for harnessing
their genetic potentials for higher silk production. However, the breeders always look
for high surviving /yielding breeds apart from the good economic characters because
the effective of rearing of these breeds fetch higher cocoon yield and in turn better
market value. India is a vast country with varying geo-physical and climatic
conditions in different agro-climate zones. Introduction of polyvoltine breeds, which
is genetically hardy as far as disease resistance is concerned into less resistant bivoltine
breeds for developing hardy bivoltine breeds, is practiced extensively in China (3,4,5).
To know the genetic potential for survival of these breeds, an approach of biochemical
analysis of immuno competence of them would throw a better light on the role of key
biomolecules associated with survival factors which support their ability to resist
diseases in the prevailing atmospheric conditions. The biochemical analysis involves
identification of reproducible qualitative markers that have prospective utility in
identification of hardy breeds. Among those are the isozymes and total induced
proteins whose banding patterns were used to identify silkworm breeds, thermo
tolerance and disease resistance (6).
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Fig.1 Central Sericulture Germplasm Resources Centre, Banglore, India.

In this context, it is focused on the key biochemical factors that play a vital role in
successful attempt of any breeding programme resulting in the selection of suitable
parents for choosing them as suitable breeding germplasm stocks (7). The most
important character that determines the commercial success of any silkworm breed is
its tolerance to diseases under adverse conditions. Tolerance or susceptibility to
diseases is characterized by inherent immune responses of the breed. The insect
haemolymph is responsible for eliciting immune responses and protecting the insects
from the infection of pathogenic bacteria. Swiftness of clearance of ingested bacteria is
determined by the combined action of cellular and humoral defenses of insects (8).
Numerous studies were carried out to know the biochemical mechanisms viz.,
antibacterial proteins and induced isoforms of enzymes that are playing key role in
imparting immunity in insects (9-11). To date, many antibacterial proteins have been
isolated from different species of insects (12) and can be classified into 5 major groups
such as cecropins, insects defensins, and attacins like proteins, proline rich peptides
and lysozymes (13,14).
Cecropins are thought to be primarily responsible for the antibacterial activity in
some insects since they show antibacterial activity against many kinds of Gram+ve and
Gram-ve bacteria. Antibacterial proteins are important factors involved in humoral
defense reaction in insect immunity. Insect antibacterial proteins are rapidly
synthesized in specific tissues and secreted into the hemolymph after bacterial
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infection or injury. In B. mori an immune mechanisms such as phagocytosis,

encapsulation, carboxylesterase and prophenoloxidase cascades and their synthesis as
antibacterial proteins were already investigated and documented (15). Apart from
earlier reports on antibacterial proteins like cecropins, attacins and lysozymes three
novel proteins such as lebocin, moricin and haemocytein have also been isolated from
B. mori (16-18). Invasion of bacteria induces a variety of antibacterial polypeptides and
triggers protenaceous interactions (19). Atttacin, cecropin, lebocin and moricin are
antibacterial polypeptides found in the silkworm, B. mori upon infection. These
polypeptides are induced when the silkworm larvae are injected with LPS from the
bacteria Escherichia coli (9).
Immune responses of insects can be studied by following the pattern of
colonization of bacteria in the haemolymph in vivo. Studies with Hylophora cecropia
showed that the injected E. coli could be cleared 48 h after post injection (20-23).
Induction kinetics of antibacterial activity reveal that after the initial log period,
immune system is activated the bacterial multiplication in check and leading to the
clearance of inoculated bacteria (23). Further studies are required to measure other
immune response parameters like changes in prophenol oxidase activity, cecropin and
lysozyme units to ascertain the immune dynamics of these breeds (8,24,25).
Cellular immune reactions involve phagocytosis, nodule formation and
encapsulation by plasmatocytes and granulocytes. Antimicrobial proteins appear to be
ubiquitous and multi-components of the innate immune mechanisms existing in B.
mori. Several such immune proteins have been isolated from B. mori and their amino
acid sequences determined. Apart from the earlier reported antibacterial proteins like
cecropin, attacin and lysozyme, three novel proteins such as lebocin, moricin and
hemocytin were isolated from B. mori. The antibacterial mechanisms of lebocin and
moricin have been analyzed and their ability to form ion channels in bacterial
membranes shows their important role in defense against bacterial infection.
Interestingly, hemocytin plays a dual role in immune mechanism against bacterial
infection as well as in metamorphosis (11,26-28).
This review describes the recent findings on antibacterial proteins, inducible
isoforms of enzymes primarily involved in resistance in silkworm strain of B. mori
against immuno elicitor LPS and their use as reliable and informative biochemical
markers for identification of genetically hardy and immunologically resistant silkworm
breeds for selection by breeders and for exploitation in the field as parental stock and in
combination as hybrids for augmenting higher silk productivity by assured higher
effective rate of rearing performance of the silkworm breeds. Insects possess a complex
and efficient system of biological defense against pathogens and parasites. This
involves the following: (a) the integument and gut as physical barrier to infection, (b)
activation of prophenol oxidase cascade and co-ordinated responses of several
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subpopulations of hemocytes when these barriers are breached, and (c) the induced
synthesis of antimicrobial proteins, primarily by the fat body within hours following a
septic injury (26,29,30).
In general, the insect immunity consists of cellular and humoral reactions. The
biochemical study that is needed to link the immuno competence through reproducible
qualitative markers involve the investigation of humoral reactions such as activation of
enzyme cascades like prophenol oxidase and stress related enzyme esterase cascades
and induction of immune proteins such as lysozymes, lectins, antibacterial proteins and
antifungal proteins (11,30). The acquisition of the updated silkworm genome has
enabled to identified 218 immune-related genes from 21gene families are associated
with recognition, signaling modulation, effectors and oxidative defense (Table 1).
Both immune reactions work in concert to prevent insects from acquiring infections
from microorganisms. Various mechanisms that operate in the insect system to combat
the diseases by producing antibacterial proteins and their gene regulation in producing
various other proteins to counteract the invasion of pathogens have been documented
for many insects and silkworms (27,30). However, there is a paucity of literature to
show the mechanism of humoral responses to LPS in silkworms and especially in
various breeds of B. mori. Hence studies are need to understand the biochemical factors
involved in the immune competence of the silkworms that can be used as a dependable
biochemical makers to estimate the degree of resistance against infections. Hence, this
article reviewed various studies pertaining to humoral and biochemical mechanisms by
which the various breeds of the silkworm of B. mori develop their required resistance
to the pathogen for better survival and higher yield.
2. Mechanisms of insect immunity
This section, is delineates briefly to pinpoint the role of key biomolecules viz.,
induced proteins and isoenzymes that are known to have functions in insect immunity.
Insect innate immunity is based on the recognition of microbial molecules, such as
lipopolysaccharide (LPS) by specific receptors with the subsequent activation of
immune effector responses. In this process, the inducible proteins and enzyme isoforms
are expressed in response to bacterial ligand LPS and are reported to have associated
with silkworm immunity. Hence, a detailed study on the mechanism of immuno
competence in silkworm breeds based on the key biomolecules elicited or expressed in
response to LPS treatment is of paramount importance to know the survival mechanism
in silkworms (7). So far, the studies carried out in the silkworm immunity link cellular
and humoral responses as the major immune responses. However, studies on the
reproducible key biomolecules that play a major role on immuno competence
attributing to the survival ability have not yet been fully studied in the different
silkworm breeds of the species B. mori. Hence, a review focusing the role of proteins
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and enzymes as outlined below needs to be brought to light to know the genetic
resistance of silkworm that breeders to choose a better parental genetic material for
further evolution of disease resistant breeds for befitting varied agro climatic
conditions of the tropical zones.
2.1. Proteins markers for silkworm disease resistance
Insects produce a battery of proteins when stimulated by microbial infection (32).
Antibacterial molecules such as cecropin, which destroys the bacterial membrane by
disturbing its structure (33) and lysozyme, that degrades the bacterial cell wall were the
proteins identified (34). Later other type of microbe inducible proteins, e.g., nonself
recognition molecules such as hemolin (35) and proteases (36) were identified. Many
bacterially inducible proteins with as yet unknown functions have also been reported
(37,38). These findings indicate that the immune response induced by bacterial
infection involves the production of both antibacterial molecules and proteins with
functions not yet fully elucidated. Some proteins involved in detoxification may be
inducible by microbial infection.
Lipophorin, for instance helps detoxify LPS originating from bacteria in the
hemolymph of the silkworm, B. mori (39). Single cecropin molecules aggregate and
build clusters, which form pores on the membranes of the target bacterial cell and
destabilize the cell (40,41). Further, the moricin and cecropins are simultaneously
induced upon bacterial infection, and can effectively protect from a wide variety of
invading bacteria pathogens (12). Such defence proteins are aplenty in silkworm
hemolymph (42). Hence, a study focusing on the investigations of induced proteins in
the hemolymph of different breeds of silkworm challenged with LPS would be of great
use to assign them as biochemical markers associated with protection from infections.
The innate immunity of invertebrates (especially insects) consists of humoral and
cellular components. The humoral components include antimicrobial peptides, lectins
and melanin. Significant antimicrobial proteins like, lebocin, moricin (12), cecropins,
lysozyme like proteins (LLPs) with a molecular mass ranging from 35 KDa were
noticed in Kolar gold, CSR2 and in Pure Mysore strains of B. mori. The cecropins are
peptides which have a broad spectrum of activity against both gram +ve and gram ve
bacteria (43,44) .
Antibacterial proteins are induced, mainly in the fat body and hemocytes upon
bacterial infection. Insect antibacterial proteins are heat stable and have a broad
antibacterial spectrum. Cecropin consists of about 40 amino acid residues (Fig.2) and is
heat stable. Three subtypes of cecropin (A, B and D) have been reported from B. mori.
Cecropins are active mainly against Gram -ve bacteria. It was demonstrated that
cecropin induces the formation of ion channels in bacterial membranes and, as a result,
bacteria are exterminated. Attacin is a glycine rich antibacterial protein has a molecular
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mass of 20 kDa. Attacin acts against growing Gram -ve bacteria and was shown to
inhibit synthesis of the bacterial outer membrane.

Fig.2 Superimpose 3D homology of Cecropin (Yellow) and Moricin (Sky blue). Highly conserved
residues from Pro26 Ala46.

Another novel antibacterial protein designated moricin was isolated from the
hemolymph of B. mori and it showed antibacterial activity against Staphylococcus
aureus (15). Moricin consists of 42 amino acid residues, highly basic, and had no
sequence similarity with other antibacterial proteins. Moricin has antibacterial activity
against several Gram -ve and Gram +ve bacteria, with a higher activity against
Gram-positive bacteria than cecropin B and is inducible upon bacterial infection. These
results suggest that the protein is responsible for antibacterial activity against Gram -ve
bacteria in B. mori (45) The effects of the protein on bacterial liposomal membranes
indicate the target of the protein is the bacterial cytoplasmic membrane. Two lectins
have also been reported from B. mori; one has a molecular mass of 260 kDa and
another designated hemocytin 280 kDa (41).
In India, the indigenous tropical polyvoltine races showed more resistance to
diseases than temperate bivoltine races (46). In order to understand the differential
response at bio-molecular level, it may be an ideal approach to compare the expression
level of antibacterial genes in hardy polyvoltine races like Pure Mysore and Nistari
with temperate races, which are less hardy, and at the same time highly productive.
Studies in this directions on bivoltine and polyvoltine silkworm breeds showed that the
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presence of induced proteins viz., attacins, a from 20-23 kDa protein (13) was
noticed in CSR2 silkworm breed (47). Attacins has drastic effect on the permeability
properties of the outer membrane of gram -ve bacterial (48) and the protein Lysozyme
of 15-16 kDa size as noticed in other silkworm breeds such as Kolar Gold and Pure
Mysore (7). Hence, a study focusing on the investigations of induced proteins in the
hemolymph of silkworm breeds challenged with LPS would be of great use to make
them as potential biochemical markers of immunity in silkworm races.
2.2 Enzyme markers associated with disease resistance
The melanin is synthesized by the activation of the prophenoloxidase activating
system (49) comprising a complex cascade of serine proteases that allows the
conversion of proPO to phenoloxide (PO), which, in turn, acts on substrates such as
tyrosine and its derivatives (DOPA and dopamine) to form melanin. This process was
extensively studied for the first time in crustacean Astacus astacus (49) and in B. mori
(50,51). The proPO system has also been seen as a recognition system activated by
different foreign materials, such as LPS and peptidoglycans from microbial cell walls
(52). Cuticular PO is normally considered to be injury PO, however, other two types of
PO are present in the cuticle of insects: granular PO involved in the body colour and
laccase-type PO involved in sclerotization of a newly ecdysed cuticle. Studies on
inducible proteins revealed that some degrading enzymes, such as esterase may also
help eliminate toxic molecules generated during microbial infection (14,25).
The carboxyl esterase act as immune defense molecules against bacteria and
continuous exposure to insecticides over several generations has resulted in the
selection of carboxylesterase that specialize in insecticidal degradation. Carboxyl
esterase isozymes have been reported in mammal macrophages and monocytes, cells
involved in the immune system (53-55).Studies on induction of carboxylesterase
isozymes in B. mori by E. coli infection showed an inducible proteins or isoforms of
enzymes viz., carboxyl esterase (CEs) Est-1 and Est-2 induced by the injection of
E.coli or LPS. They were found with a similar other known bacterially inducible
proteins and CEs clearly differed from noninducible CEs (Est-3,4 and 5 which were
visualized in the haemoloymps of silkworm in the native gel) in migration on analytical
native PAGE and inhibitor sensitivity studies. These results suggest that Est-1 and
Est-2 are novel CEs that are inducible either by bacterial or bacterial ligands (LPS)
injections into the silkworms (15).
3. Role of antimicrobial proteins on immunity
Silkworm have developed an efficient host defense against invading
microorganisms viz., bacteria, virus, fungi and/or microbial components viz., LPS,
-1,3 glucan (Fig.3) which includes major components of antimicrobial proteins and
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immune enzymes (Table 1; Fig.3 & 4). Cecropin, a small family of lytic peptide,
strongly basic, heat stable comprises of five major subtypes A, B, C, D and E.
Molecular weight of cecropin is 6.7, and pI 11.10. The known sequences for the
major cecropins show that the N-terminal (MNFVRILSFVFALVLALGAVSAA) parts
are strongly basic while the C-terminal regions are neutral and contain long
hydrophobic stretches (Fig.2). Cecropins exhibit a broad spectrum of antibacterial
activity against Gram -ve and Gram +ve bacteria by adapting -helical structure on
interaction with bacterial membranes resulting in the formation of ion channels to
neutralize the foreign components. Cecropins has weak activity against fungi. When
cecropin gene transcripts induced by microbes, the proteins have 72 hrs stability. In
three dimensional views, the positively charged N-terminal helices are supposed to
bind the negatively charged head groups on the membrane surface, while the terminal
helices insert into the membrane core. Subsequently, the application of a positive
potential is believed to push the positively charged N-terminal helices into the
membrane and a channel would be formed by the association of multiple
transmembrane N-terminal, such that the hydrophilic residues form the aqueous pore
and the hydrophobic residues are in contact with the aliphatic phase membranes
(56-58).

Fig.3 Schematic diagram shows the families of anti-bacterial proteins in Bombyx mori. Most of the
anti-bacterial peptides which one expressed constitutively, these peptides are up regulated in infection
(bacteria/Fungi) silkworm. All these families have several members, which are encoded typically by small
cluster of related genes. Only few peptides, which have studied in detail and others underway.

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Table 1. Immuno-related genes and pathway of Bombxy mori.

* Immuno pathways including Toll, Imd, JNK and JAKSTAT pathway;
** Anti-microbial peptides (see more detail in the table No.2)

GeneFamily
Recognition
PGRP
GNBP
Galectin
Ctypelectin
Fibrinogendomain
Scavenger
Signalling
Spz
CLIPSP
Serpin
Toll
REL
Components*
Effectors
Prophenoloxidase
TEP
LYS
AMP**
Others
Caspase
Catalase
Inhibitorsofapoptosis
Peroxidase
Superoidasedismutatse
Total

Numbers
11
4
2
22
3
12
6
15
15
13
2
7
3
6
3
39
4
7
5
23
6
218

Moricin is a novel antibacterial peptide, MW 5.1 kDa, pI 10.89, number of

aminoacids are 42. The moricin antibacterial peptides of moricin were present in 2
forms viz., moricin 1 and 2. There was 97.2% of sequence similarity is observed in
both the forms except one aminoacid residule (Phe) of moricin and the putative signal
peptide is replaced in moricin 2. Hemmia et al., (2002) determined the
three-dimensional structure of moricin by using two-dimensional (2D) 1H-nuclear
magnetic resonance (NMR) spectroscopy. The results also suggested that the
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N-terminal half of the peptide is a nearly perfect amphipathic -helix with equally large
hydrophilic and hydrophobic faces (Fig.2).The C-terminal region is more hydrophobic.
Moricin gene expression in B. mori larve injected with E.coli was present is present in
hemolymph, fat body and malpighian tubule. Accumulation of moricin gene transcripts
induced by microbes reached a maximum level after 8 hrs of injection and persisted up
to 48 hrs (31,59,60).
Gloverin is a lytic peptide with a molecular weight of 19.1kDa, pI 6.97. The
N-terminal region of Gloverin peptides (MYSKVLLSAALLVCVNAQVSMPPG
YAE) start from 1- 27. The number of amino acids is 178. Gloverins existed as four
major subytpes and exhibit a broad spectrum of antibacterial activity. The gloverin
family showed slow evolutionary rates. By induced transcriptional activity, genes
encoding active antimicrobial peptides were up regulated at different levels (61). The
gloverin familys proteins are positively correlated with cecropin and moricin families.
It is strongly suggest that representative Bmcec B6, BmcecD and Bmmor as the major
effectors genes have broad spectrum activities against invading microbes (62).
Defensins were found in every insect species investigated to date. More than 60
defensins have been isolated from insects belonging to phylogenetically recent orders
(Diptera, Lepidoptera, Coleoptera, Hymenoptera and Odonata). In B. mori, eleven
defensins (or isoforms) are cationic, 34- to 43-residue peptides (with the exception of
royalisin), all containing six cysteines engaged in three intramolecular disulfide
bridges. Three domains are apparent: 1) a N-terminal loop, formed by residues 1-13,
which has a certain degree of flexibility; 2) a central amphipathic a-helix consisting of
residues 14-24; and 3) a C-terminal antiparallel -sheet comprising residues 27- 40,
with a turn involving residues 31-34. The -helix is stabilized via two disulfide bridges
to one of the strands of the -sheet and the N-terminal loop is linked via one disulfide
bridge to the other -strand.
Lebocin is a linear proline rich antimicrobial peptides with 20kDa and pI 7.95 and
made of 179 aminoacids. N-terminal region starts from 1-21 (MYKFLVFSSVLV
LFFAQASCQ). Lebocin have two subfamilies: Shortchain (>20 residues) and long
chain peptides (<20aminoacids). Proline residue frequently associated with arginine /
histidine. Long chain lebocins are active against both Gram -ve and +ve bacteria and
fungi (59). For lebocins, the activity is strongly salt dependent as it is significantly
reduced even at physiological salt concentrations. Lebocins allow synergitic activity
with cecropin and when mixed together may improve the efficacy of unrelated
antimicrobial peptides by minimum inhibitory concentration.
Attacins are inducible bacteriostatic polypeptides with 179 amino acids and
molecular masses higher than 22 kDa, strongly basic (pI 9.85) and glycine rich.
N-terminal region consists of 1-18 (MSKSVALLLLCACLASGR) and glutamine
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residue involves a partial loss of one positive charge. Subsequent studies revealed as
many as six attacins (184-186 residues) were isolated from the immune blood of the
lepidopteran. Four of which are basic and the other two are either neutral or slightly
acidic. The sequence homology between basic and acidic attacins is close to 80%.
Attacins have two 60-residue G domains which show a certain degree of sequence
similarity with the G domain of diptericin. Attacin inhibits growth of Gram -ve bacteria
and increases the permeability of the outer membrane - a structure unique to Gram -ve
cells serving as a permeability barrier to the passage of hydrophobic and large (> 800
Da) hydrophilic substances.
Phenoloxidase (PO) is a key enzyme in the melanization response, which is
effective against pathogens, particularly parasites and parasitoid eggs (63,64). PO is
activated from its pro-enzyme (zymogen), prophenoloxidase (proPO), and the
activation process involves a cascade of serine proteinases with similarities to the
mammalian complement cascade. ProPO is cleaved by proPO-activating proteinases
(PAPs) that are also present as pro-enzymes in hemolymph.

Table 2. Bombyx moi antibacterial proteins

Name of
anti-microbacteri
al protein / gene

Accession No.

MW

pI

(kDa)

Total
No.
amino
acid

N-termin
al Signal
peptide
region

Ref.

Bm_Cecropin

D84396.1

6.7

11.10

63

1-23

(65)

Bm_ Moricin

NM_001043364.2

5.1

10.89

42

1-23

(66)

Bm_Attacin

NM_001043541.1

22.0

9.85

179

1-18

(67)

Bm_Lebocin

NM_001044003.1

20.0

7.95

179

1-21

(68)

Bm_Hemolin

AB115084.1

44.9

5.12

410

1-19

(69)

Bm_Carboxyl/

NM_001171920.1

60.1

8.63

535

1-16

(70)

Bm_Phenoloxidase

NM_001044069.1

80.1

5.62

693

1-19

(71)

Bm_gloverin

AB190863.1

19.1

6.97

178

1-27

(72)

Bm_lyzosome

NM_001043983.1

15.6

8.98

137

1-19

(71)

Bm_enbocin

AAC02238.1

6.3

11.0

59

1-28

(65,73)

cholinesterase

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In general, the electrostatic potential of small molecules has provided insight into
intermolecular association, molecular properties of small molecules as drug,
biological functions as antibacterial proteins, and enzyme catalysis. Here, we can
made attempt for the review qualitative electrostatic representation (Fig.5 & Table 2),
amount of approximate potential energies computed between partial charges located
atomic centers and that the molecular surface is located in the region where artifacts
and noise in PB calculation would be greatest due to discrete discontinuities between
the low-dielectric interior and the high dielectric exterior (solvent region) of
antibacterial protein from silkworm (Fig.5).

Fig. 5. Comparison of the electrostatic surface features of silkworm antibacterial proteins family.
The surface is colored according to electrostatic potential, ranging from blue (the most positive region) to
red (the most negative region) and white (hydrophobic region). Figure was produced using the PyMOL
program.

4. Conclusion
The present chapter discusses the use of biochemical markers that affords immuno
competence in silkworms, which qualitative and is reproducible. These markers
enables any researcher or breeders to select the better silkworm breed possessing
genetic resistance to any diseases that can be better exploited in varied agro climatic
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conditions for raising disease free crop and thus promote quality and quantity of silk
production.
In short, the practical utility of the studies on protein and enzyme markers reveal
that different type of proteins induced upon LPS treatment and different isoenzymes of
carboxyl esterase and phenoxidases are considered as the key biomolecules which are
of utmost importance in associating immune competence of the silkworm breeds as
they are associated with ceullular and/or humoral defense system of silk worms in
either neutralizing or degrading by bacterial derived LPS molecule.
The majority of the research carried out in the various aspects of the experimental
investigations such as physiological, immunological and genetic in many insects in
general and in silkworms in particular. These studies revealed that inducible proteins
viz., cecrobin, lebonin etc and inducible isoforms of enzymes viz., carboxyl esterase
and phenoloxidase under the effect of LPS in the system are considered as the reliable
and potential biochemical markers and that be used to estimate the degree of resistance
and genetic hardiness of individual breeds of silkworm by various recent advance
biochemical techniques. Inducible protein viz., cecropin, attacin, lebocin and moricin
and isoforms of enzymes via., carboxy esterase and prophenol oxidase isoforms in
insects system are reproducible and inducible molecular biomarkers that could be used
to study the degree of immunity in silkworm. These biochemical markers related to
immunity will tag silkworm races which as stronger in immune competence. These
values would form a yard stick to screen resistant breeds befitting varied agro climatic
conditions for silkworm crop improvement in the tropical country for better silk
production. Such protein markers as considered as dependable markers for screening
large number of germplasm materials to list out genetically resistant or hardy breed to
promote the silk industry.
Key points:
1. Immunity is complex and vary among species and/or among the breeds.
2. Profiling of isozyme and antimicrobial peptides has contributed to the
understanding of silkworm immunity.
3. They will also contribute to the sericulture field by establishing transgenic nonsusceptible strains of silkworm and to agriculture for better control of
lepidopteran pests.
4. The use of MS provides several advantages to study the immune mechanism of
silkworm, including: Sensitivity: only individual silkworm races per assay is
required; Selectivity: allelic forms of different races can be identified in a single
simple isozyme techniques; Efficiency: the elicitor induced level can be identified
at primary level and can give a basic insight as far as individual/breed variation is
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Invited Review

concerned. Temporal resolution: metabolic changes can be monitored under

biotic stressful environment.
5. Acknowledgement
The authors express sincere thanks to the competent authority of Central Silk
Board, Ministry of Textiles, Madivala, Bangalore, Karnataka state, India for constant
encouragement and perseverance in the preparation of this review article.

6. References
Thangavelu, K., Mukherjee, P., Sinha, R.K., Mahadevamurthy, M., Sunita, M., Sashani,
N.K., Kumersan, P., Rajaraja, A., Mohan, B., Sekar, S. (1997) Catalogue on silkworm
(Bombyx mori L), germplasm, silkworm and mulberry germplasm station, Hosur, Tamil Nadu,
India 1: 1-138.
2. Thangavelu, K., Sinha, R.K., Mahadevamurthy, M., Kumersan, P., Mohan, B., Rayaraddar,
F.R., Sekar, S. (2001) Catalogue on silkworm (Bombyx mori L), germplasm, Central
Sericultural Germplasm Resources Centre, Hosur, Tamil Nadu, India 2: 1-138.
3. Das, S.K. (2001) Techniques of breeding for evolving improved breeds of bivoltine mulberry
silkworm Bombyx mori L., in Perspective in cytology and genetics. 10: 129-34.
4. Das, S.K., Chattopadhyay, G.K., Verma, A.K., Sengupta, Sarkar, A. (2005a) Development of
high yielding silkworm breeds of Bombyx mori L for Eastern India through congenic line
breeding approach: in 20th Congress of the International Sericultural Commission. Bangalore
1: pp. 268-72.
5. Das, S.K., Moorthy, S.M., Chattopadhyay, G.K., Verma, A.K., Ghosh, B., Rao, PR., Sengupta,
A.K., Sarkar, A. (2005b) Silkworm breeding for tropical conditions of Eastern India: in
Proceedings of 12th AICCG, Bombay, India.pp. 4-5.
6. Somasundaram, P., Ashok Kumar, K., Babu, G.K.S., Kamble, C.K. (2009) Heat stable esterase
a biochemical marker for evolution of thermo tolerant breeds of Bombyx mori (L.) J. Adv.
Biotechnol. 1(2): 20-21.
7. Ashok Kumar, K., Somasundaram, P., Rajesh, G.K. Chandrasekar, R., Balachandran, N., and
Sivaprasad, V. (2013) Protein markers as a potential tool for biochemical characterization of
silkworm genetic resources. Silkwormmori.blogspot.com/2013/09/.
8. Thangamalar, A., Subramanian, S., Muthuswami, M. and Mahalingam, C.A. (2010) Fate of
bacteria and on set of immune response in silkworm, Bombyx mori L. J. Biopesticides 3(1) 47
50.
9. Yamakawa, M., Tanaka, H. (1999) Immune proteins and their gene expression in the silkworm,
Bombyx mori. Dev. Comp. Immunol. 23: 281-289.
10. Altincicek, B., Linder, M., Linder, D., Preissner, K.T., and Vilcinskas, A. (2007) Microbial
Metallo- proteinases Mediate Sensing of Invading Pathogens and Activate Innate Immune
1.

285
Printed in the Unitated States of America, 2014
ISBN: 978-1-63315-205-2

Short Views on Insect Biochemistry and Molecular Biology Vol.(1), 2014

11.

12.
13.
14.
15.
16.
17.
18.
19.
20.

21.
22.

23.
24.

25.
26.
27.

Invited Review

Responses in the Lepidopteran Model Host Galleria mellonella. Infect. Immun. 75(1):
175183.
Chang, T., Zhao, P., Liu, C., Xu, P., Gao, Z., Xia, Q., Xiang, Z. (2006) Structure, regulatory
regions, and inductive expression pattern of antimicrobial peptide genes in the silkworm
Bombyx mori. Genomics, 87:356-365.
Seiichi Hara, Yamakawa, M. (1995) A novel antibacterial peptide family isolated from the
silkworm, Bombyx mori. Biochem. J. 31: 651-656.
Boman, H.G., Hultmark, D. (1987) Cell-free immunity in insects. Annu. Rev. Microbiol.
41:103126.
Hultmark, D. (1993) Trends Genet. 9:178183.
Shiotsuki, T., Kato, Y. (1999) Induction of carboxyl esterase isozymes in Bombyx mori by
E.coli infection. Insect Biochem. Mol. 29: 731-736.
Russell, V., Dunn, P.E. (1996) Antibacterial proteins in the midgut of Manduca sexta during
metamorphosis. J. Insect Physiol. 42: 6571.
Boman, H.G.(2000) Innate immunity and the normal microflora. Immunol Rev. 173:5-16.
Zasloff, M. (2002) Antimicrobial peptides of multicellular organisms. Nature 415: (6870):
389-395.
Gillepe, J.P., Kanost, M.R., Trenczeck, T. (1997) Biological mediators of insect immunity.
Annu. Rev. Entomol. 42: 61143.
Faye, I, Pye, A., Rasmuson, T., Boman, H.G., Boman, I.A. (1975) Simultaneous induction of
antibacterial activity and selection synthesis of some hemolymph proteins in diapausing pupae
of Hyalophora cecropia and Samia cynthia. Infect. Immun.12 (6):142638
Flyg, C., Dalhammar, G., Rasmuson, B., Boman, H.G. (1987) Insect immunity. Inducible
antibacterial activity in Drosophilla. Insect Biochem. 17: 153-60.
Kaaya, G.P., Flyg, C., Boman, H.G. (1987) Insect immunity: Induction of cecropin and attacin
like antibacterial factors in the haemolymph of Glossina morsitans. Insect Biochem. 17:
309-315.
Faye, I. (1978) Insect immunity: Early fate of bacteria injected in saturniid pupae. J.
Invertebrate Pathol. 31(1): 19 26.
Ochiai, M., Ashida, M. (1988) Purification of a beta-1, 3-glucan recognition protein in the
prophenoloxidase activating system from hemolymph of the silkworm, Bombyx mori. J. Bio.
Chem. 263: 12056 12602.
Engstrome, A., Engstrom, P., Tao, Z.-J., Carlsson, A., Bennich, H. (1984) Amino acid and
cDNA synthesis of lysozyme from Hyalophora cecropia. EMBO J. 4:2119 -2122.
Ponnuvel, K.M., Yamakawa, M. (2002) Immuno responses against bacterial infection in
Bombyx mori and regulation of host gene expression. Current Sci. 83 (4): 447-454.
Tign-Cai, C, Qing-You, X., Ping-Zhen, X., Xiang, T., Ting, F. and Zhong-Huai, X. (2009)
Identification and comparative analysis of immune-related genes and signaling pathways in the
silkworm, Bomboxy mori. Acta Entomologica Sinica, 53(3): 235-245.

286
Printed in the Unitated States of America, 2014
ISBN: 978-1-63315-205-2

Short Views on Insect Biochemistry and Molecular Biology Vol.(1), 2014

Invited Review

28. Zhang, S., Gunaratna, R.T., Zhang, X., Fares Najar, Wang, Y., Roe, B. and Jiang, H. (2011)

29.
30.
31.

32.
33.
34.

35.
36.

37.
38.
39.

40.
41.
42.

43.
44.

Pyrosequencing-based expression profiling and identification of differentially regulated genes

from Manduca sexta, a lepidopteran model insect. Insect Biochem. Mol. Biol. 41(9): 733746.
Gillespie, J.P. and Kanost, M.R. (1997) Biological mediators of insect immunity. Annu. Rev.
Entomol. 42:611643.
Tanaka, H., Yamakawa, M. (2011) Regulation of the innate immune responses in the
silkworm, Bombyx mori. Intl. Silkworm J. 8: 59-69.
Qu, X.M., Wu, K.-Z, Qiu, X.-Z., Zhang, S.-Q., Lee, S.Y. (1986) Isolation and characterization
of six antibacterial peptides from the hemolymph of immunized Bombyx mori pupae by
injection of poly I:C. Acta Biochim. Biophys. Sin.18: 284-291.
Boman, H.G. (1991) Cell-free immunity in Cecropia, a model system for antibacterial proteins.
Eur. J. Biochem. 201:23-31
Boman, H.G. (1995) Peptide antibiotics and their role in innate immunity. Annu. Rev.
Immunol. 13:6192
Daffre, S., Kylsten, P., Samakovlis, C., Hultmark, D.(1994) The lysozyme locus in Drosophila
melanogaster: an expanded gene family adapted for expression in the digestive tract. Mol. Gen.
Genetics 24: 152-162.
Bettencourt, R., Lanz-Mendoza, H., Lindquist, K.R., Faye, I. (1997) Cell adhesion properties
of hemolin, an insect immune protein in the Ig superfamily. Eur. J. Biochem. 250: 630-637.
Dimopoulos, G., Richman, A., Mller, H.M., Kafatos, F.C. (1997) Molecular immune
responses of the mosquito Anopheles gambiae to bacteria and malaria parasites. Proc. Natl.
Acad. Sci. USA 94: 11508-11513.
Kang, D., Liu, G., Gunne, H., Steiner, H. (1996) PCR differential display of immune gene
expression in Trichoplusia ni. Insect Biochem. Mol. Biol. 26:177-184.
Hughes, J.A., Hurlbert, R.E., Rupp, R.A., Spence, K.D. (1983) Bacteria induced haemolymph
proteins of Manduca sexta pupae and larvae. J. Insect Physiol. 29:625632.
Kato, Y., Moto, M., Taniai, K., Kadono-Okuda, K., Yamamoto, M., et al., (1994)
Lipopolysaccharide lipophorin complex formation in insect haemolymph. A common
pathway of lipopolysaccharide detoxification both in insects and in mammals. Insect Biochem.
Mole. Biol. 24: 547-555.
Steiner, H.D., Andreu, R., Merrifield, B.(1988) Binding and action of cecropin and cecropin
analogues: antibacterial peptides from insects. Biochimica. et Biophysica. Acta 939: 260266.
Jaynes, J. (1989) Peptides to the rescue. New Scientist 124 (1695): 42-44.
Kumar, D., Pandey, J.P., Jain, J., Mishra, P.K., Prasad, B.C. (2011) Qualitative and
quantitative changes in protein profile of various tissue of tropical tasar silkworm Antheraea
mylitta drury. Int. J. Zool. Res.7: 147-155.
Ganz, T. (2007) The role of antimicrobial peptides in innate immunity. Intergr. Comp. Biol.
43: 300- 304
Gotz, P., Boman, H.G. (1985) Insect immunity. In: Comp Biochem. Physiol. and Biochem.
Mole. Biol. (G. A. Kerkut and L. I. Gilbert, eds.), Pergamon, Oxford, NewYork, 453-485.
287

Printed in the Unitated States of America, 2014

ISBN: 978-1-63315-205-2

Short Views on Insect Biochemistry and Molecular Biology Vol.(1), 2014

Invited Review

45. Lee, W.J., Lee, J.D., Kravchenko, V., Ulevitch, R.J., Brey, P.T. (1996) Purification and

46.

47.
48.

49.
50.
51.

52.
53.

54.
55.
56.
57.

58.

59.
60.

molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm,

Bombyx mori. Proc. Natl. Acad. Sci. USA, 93: 7888-7893.
Moorthy, S.M., Das, S.K., Rao, P.R.T., Raje, Urs S., Sarkar, A. (2007) Evaluation and
selection potential parents based on selection indices and isozyme variability in silkworm,
Bombyx mori L. Int. J. Entomol. 14: 1-7.
Suparna, M.K., Mallikarjun, S.S., Ingalhalli, V., Shyam Kumar, V., Hooli, A. (2011) Role of
antibacterial proteins in different silkworm strains against flacherie. The Bioscan 6: 365-369.
Carlsson, A., Engstrom, P., Palva, E.T., Bennich, H.(1998) Attacin-an insect immune
protein-binds LPS and triggers the specific inhibition of bacterial outer membrane protein
synthesis. 144: 2179-2188.
Soderhall, K.(1982) Prophenoloxidase activating system and melanization a recognition
mechansism of arthropods ? A review. Dev. Comp. Immunol. 6: 601-611.
Ashida, M., Onishi, E. (1967) Activation of pre-phenol oxidase in haemolymph of the
silkworm, Bombyx mori. Arch. Biochem. Biophy. 122: 411-6
Ashida, M., Ishizaki, Y., Iwahana, H. (1983) Activation of prophenolxidase by bacterial cell
walls or [beta] -1,3 Glucan in plasma of the silkworm. Biochem. Biophys. Res. Commun.
113:562-568.
Soderhall, K., Cerenius, L. (1998) Role of the prophenolxidase activating system in
invertebrate immunity. Current Opinion in Immunol. 10: 23-28.
Munger, J.S, Shi, G.P., Mark, E.A., Chin, D.T., Gerard, C., Chapman, H.A. (1991) A serine
protease released by human alveolar macrophages is closely related to liver microsomal
carboxylesterase. J. Biol. Chem. 266: 18832- 18838.
Kawaoka, S., et al. (2008) Functional analysis of four Gloverin-like genes in the silkworm,
Bombyx mori. Arch. Insect Biochem. Physiol. 67 (2): 87-96.
Suzuki, T., Natori, S. (1983) Identification of a protein having hemagglutinating activity in the
hemolymph of the silkworm, Bombyx mori. J. Biochem. 93: 583-590.
Boman, H.G., and Hultmark, D. (1987) Cell-free immunity in insects. Ann. Rev. Microbiol.
41:103-126.
Qu, X.-M., Steiner, H., Engstrom, A., Bennich, H., Boman, H.G. (1982) Insect immunity:
Isolation and structure of cecropins B and D from pupae of the Chinese oak silk moth,
Antheraea pernyi. Eur. J. Biochem. 127: 219-224.
Morishma, I., Suginaka, S., Ueno, T., Hirano, H. (1990) Isolation and structure of cecropins,
inducible antibacterial peptides, from the silkworm, Bombyx mori. Comp. Biochem. Physiol. B,
95: 551-554.
Bulet, P. and Stcklin, R. (2005) Insect Antimicrobial Peptides: Structures, Properties and
Gene Regulation. Protein and Peptide letters, 12: 3 -11.
Huang, L., Cheng, T., Xu, P., Cheng, D., Fang, T. and Xia, Q. (2009) A Genome-Wide Survey
for Host Response of Silkworm, Bombyx mori during Pathogen Bacillus bombyseptieus
Infection. PLoS ONE 4(12): e8098.
288

Printed in the Unitated States of America, 2014

ISBN: 978-1-63315-205-2

Short Views on Insect Biochemistry and Molecular Biology Vol.(1), 2014

Invited Review

61. Axen, A., Carlsson, A., Engstrom, A., Bennich, H. (1997) Gloverin, an antibacterial protein

from the immune hemolymph of Hyalophora pupae. Eur. J. Biochem. 247: 614-619.

62. Yang, W., Cheng, T., Ye. M., Deng, X., Yi, H., et al. (2011) Functional Divergence among

63.
64.

65.

66.
67.

68.

69.
70.

71.
72.

73.

Silkworm Antimicrobial Peptide Paralogs by the Activities of Recombinant Proteins and the
Induced Expression Profiles. PLoS ONE 6(3): e18109.
Ashida, M., Dohke, K. (1980) Activation of pro-phenoloxidase by the activating enzyme of the
silkworm, Bombyx mori. Insect Bicohem. 10:37-47.
Yoshida, H., Ashida, M. (1986) Microbial activation of two serine enzymes and
prophenoloxidase in the plasma fraction of hemolymph of the silkworm, Bombyx mori. Insect
Biochem. 16: 539-545.
Yamano,Y., Matsumoto,M., Sasahara,K., Sakamoto,E. and Morishima, I. (1998) Structure of
genes for cecropin A and an inducible nuclear protein that binds to the promoter region of the
genes from the silkworm, Bombyx mori.Biosci. Biotechnol. Biochem. 62 (2): 237-241.
Hemmi, H., Ishibashi, J., Hara, S. and Yamakawa, M. (2002) Solution structure of moricin, an
antibacterial peptide, isolated from the silkworm Bombyx mori. FEBS Lett. 518 (1-3): 33-38.
Tanaka, H., Ishibashi, J., Fujita, K., Nakajima,Y., Sagisaka, A.,Tomimoto, K., Suzuki, N.,
Yoshiyama, M., Kaneko,Y., Iwasaki, T.,Sunagawa, T.,Yamaji, K., Asaoka, A., Mita, K. and
Yamakawa, M. (2008) A genome-wide analysis of genes and gene families involved in innate
immunity of Bombyx mori. Insect Biochem. Mol. Biol. 38 (12): 1087-1110.
Tanaka, H., Sagisaka, A., Fujita, K., Furukawa, S., Ishibashi, J. andYamakawa, M. (2012)
BmEts upregulates promoter activity of lebocin in Bombyx mori. Insect Biochem. Mol. Biol. 42
(7): 474 - 481.
Shimada, T., Iwanaga, M., Omuro, N. and Mita, K. (2003) Bombyx mori mRNA for hemolin,
complete cds Published Only in Database 2013.
Tsubota, T., Shimomura, M., Ogura, T., Seino, A., Nakakura, T., Mita, K., Shinoda, T. and
Shiotsuki, T. (2010) Molecular characterization and functional analysis of novel
carboxyl/cholinesterases with GQSAG motif in the silkworm Bombyx mori. Insect Biochem.
Mol. Biol. 40 (2): 100-112.
Suetsugu Y., et al. (2013) Large scale full-length cDNA sequencing reveals a unique genomic
landscape in a lepidopteran model insect, Bombyx mori. G3 (Bethesda) 3 (9): 1481-1492.
Kawaoka, S., Katsuma, S., Daimon, T., Isono, R., Omuro, N., Mita, K. and Shimada, T. (2008)
Functional analysis of four Gloverin-like genes in the silkworm, Bombyx mori. Arch. Insect
Biochem. Physiol. 67 (2): 87-96.
Kim, S.H., Je, Y.H., Kim, K.Y. and Kang, S.K. (1998) Molecular characterization of a new
antibacterial peptide gene, Enbocin, from Bombyx mori. (NCBI Accession NO. AAC02238).

Article History:
Reviewed by:

Received on May 14th 2013, Revised on November 10th 2013 and Accepted on July 15th 2014;
Published 30th October, 2014.
Gustavo Ferreira Martins, Universidade Federal de Viosa, Brazil.
K.Balakrishnan, Madurai Kamaraj University, India.

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2014

Section IV
Insect Molecular Genetics

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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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Published by International Book Mission, Academic Publisher, South India.

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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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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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