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Printed in the Unitated States of America, 2014
ISBN : 978-1-63315-205-2
Short Views on Insect Biochemistry and Molecular Biology Vol.(1), October 2014
2014
Section IV
Insect Molecular Genetics

ISBN : 978-1-63315-205-2
NAL B OO
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TERNA
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SION
MIS
Short
on Insect
Biochemistry
and Molecular
Biology and
Vol.(1),
2014 Biology
Views Short
Views
on Insect
Biochemistry
Molecular
Vol. (1), 00 00, 2009
Vol. (1) 291 316, 2014
Invited Review
Review
Invited
Chapter 12
The recent progress of the W and Z chromosome studies

of the silkworm, Bombyx mori
*
Hiroaki Abe1 , Tsuguru Fujii2 and Raman Chandrasekar3

1
Department of Biological Production, Faculty of Agriculture, Tokyo University of Agriculture and

Technology, Saiwai-cho 3-5-8, 183-8509, Fuchu, Tokyo, Japan.
2
Institute of Genetic Resources, Graduate School of Bio Resources and Bioenvironmental Science,
Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan.
3
Department of Biochemistry and Molecular Biophysics, 238 Burt Hall, Kansas State University,
Manhattan 66506, KS, USA.
.
Abstract
Sex chromosomes of the silkworm, Bombyx mori, are designated as ZW (XY) for females and ZZ
(XX) for males. The female mode of development is determined by the presence of a single W
chromosome. Therefore, it is presumed that the female-determining gene (Fem) is present on the
W chromosome. Until date, 12 W-specific random amplified polymorphic DNA (RAPD) markers
have been identified on the normal W chromosome. We have compared the W-translocation
chromosomes with normal W chromosome. The T(W;3)Ze (the sex-limited Zebra strain)
chromosome lacked 2 W-specific RAPD markers, indicating that the region containing these two
W-specific RAPD markers has been deleted. Additionally, we have investigated T(W;2)Y (the
sex-limited Yellow-cocoon strain). The T(W;2)Y chromosome lacked 11 such markers. These
results indicate that the W chromosomes of the sex-limited strains are the products of reciprocal
translocation accompanied by deletion, and an extremely limited region is required to determine
femaleness. No genes for morphological traits have been mapped to the W chromosome. Contrary,
Z chromosome is rich in genes. Many long terminal repeat (LTR) and non-LTR retrotransposons,
retroposons, DNA transposons, and their derivatives, had accumulated as strata on the W
chromosome. It is notable that some of these transposable elements contained the Bombyx short
interspersed element (Bm1) sequences in the elements. On the other hand, the transposable
elements on the Z chromosome were excluded by unequal crossing over or intra-element
homologous recombination between LTRs.
Key words: Bombyx mori, female-determining gene, transposable elements, single nucleotide, polymorphism, random amplified polymorphic DNA, W chromosomes, Z chromosomes
*For Correspondence (email: wfem@cc.tuat.ac.jp)
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Invited Review
Over view
1. Overview of W chromosome in Bombyx mori
2. The beginning of the molecular biological studies of the W chromosome
3. Presence of the W-markers in the silkworm strains maintained in Japan
4. Mapping of the W-markers on the W chromosome
5. Nested structure of transposable elements on the W chromosome
6. The structure of W chromosome and novel transposable elements
6.1. Structure of W chromosome
6.2. Structure of transposable elements the W chromosome
6.2a. BMC1
6.2b. Kendo
6.2c. Bm1 group
6.2d. Supermite-BMC1
6.2e. Other transposable elements
7. Recognition of W chromosome by FISH
8. piRNA derived from the W chromosome
9. The relations of the Z chromosome with W chromosome
9.1. Z-linked genes involved in the sexual differentiation
9.2. Transposable elements on the Z chromosome
10. Analyses of W chromosome of Lepidopteran insects except B. mori
11. Conclusion
12. References
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1. Overview of W chromosome in Bombyx mori

Initially, we have to introduce the history of the W chromosome studies of the
silkworm Bombyx mori. Therefore, in this section, at first we look back on the history
of the W chromosome studies and then report recent molecular biological studies.
About 100 years ago, Tanaka (1) determined the chromosomal make up of the
silkworm in terms of sex linked (Z-linked os gene) inheritance namely that females
were ZW (equivalent to XY) and males were ZZ (XX). Afterwards, Hashimoto (2)
obtained very interesting results by using polyploidy related to the W chromosome.
He found that the polyploidy individuals such as ZZW and ZZWW were
phenotypically normal females and postulated that the female mode of development
was determined by the presence of a single W chromosome regardless of the number
of autosome or Z chromosomes present. However, there were no methods at that time
to identify the W chromosome cytology because the chromosomes of B. mori are
small, holocentric, and numerous (2n=56). Moreover, it was very difficult to prove the
existence of the W chromosome by genetic experimentation because many
morphological gene loci had been found on the Z chromosome and, none had been
found on the W chromosome. Furthermore, recombination is restricted to males in
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B. mori. Thus, based on genetic studies, the existence of the W chromosome was
almost certain, but the true characteristic of the W chromosome was unclear.
In Japan, sericulture is stable in the spring season. However, it becomes
unstable in summer and autumn because the quality of mulberry leaves deteriorates
after spring and because of unfavorable weather conditions, namely high temperature
and humidity. Therefore, establishment of more vigorous silkworm strains was
desired. In 1906, Toyama (3), one of the founders of silkworm genetics, rediscovered
Mendels laws in a study of inheritance of cocoon color. He recognized heterosis in
hybrids between Japanese and Thai strains (3,4). Following his advice, the Japanese
government decided to encourage the rearing of hybrid silkworms for general
sericultural farms and began distributing hybrid eggs in 1914. Thereafter, almost all
the silkworm strains reared on Japanese sericultural farms have been F1 hybrids. At
the same time, for the production of F1 eggs, specialized farms for F1 eggs were
necessary, and males and females must be separated at or before the cocoon stage. In
the absence of separation, just after emergence, sibling moths copulate mutually and
females cannot be used for the production of F1 hybrid eggs. Therefore, determining
the sex of silkworms for hybridization became very critical to silkworm egg
producers. The traditional separation method was inspecting Ishiwatas germal discs
in the female and Herolds gland in the male on the post-ventral surface of the final
(5th) instar larvae. This method was generally performed by a professional
discriminator, but it was inconvenient. Therefore, development of a convenient
method (visible marker) of discriminating males from females that even an amateur
was capable of was desired. Tazima, during the investigations of chromosomal
aberrations induced by X-rays, proposed, If we can translocate artificially any one
autosome that carries dominant genes for noticeable traits to the W chromosome, we
will be able to discriminate females and males based on these traits easily. For these
reasons, Tazima (5) attempted to achieve translocation between the W chromosome
and the 2nd chromosome, which carries the pSa gene (sable marking) for larval trait
by X-ray irradiation and achieved wonderful success at last! Stimulated by Tazimas
successful experiment, several researchers undertook the challenge of inducing
translocation between the W chromosome and an autosome carrying the dominant
genes for noticeable traits (egg color, larval marking, and cocoon color Fig. 1), and
were also successful (5-9). These traits were inherited from mother to daughter
(sex-limited inheritance). These translocations were also used to verify and conclude
that the W chromosome does exist and femaleness is determined solely based on the
presence of the W chromosome in B. mori. Tazima (6) also demonstrated that the
determiner of femaleness (female determining gene(s), Fem) is localized in a
particular region of the W chromosome not diffusely distributed.
Molecular biological techniques have been introduced to the biological studies of
B. mori, various genes have been cloned as the DNA nucleotide sequence data. In
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parallel with molecular biological progress, so-called classical genetics of B. mori

have progressed too, and linkage maps of B. mori have been filled up steadily.
Fig. 1. Visible markers translocated from autosomes or Z chromosome to the W chromosome.

These markers are very useful to distinguish B. mori males from females in larval or pupal stages. (A)
Fifth instar larvae of the sex-limited strain carrying +od pSa +p W chromosome. The female has normal
and sable skin due to the genotype od/+od pSa +p W, p/p, whereas the male has translucent and plain skin
due to the genotype od/od, p/p. (B) Sex-limited W-B strain. The female larva has black skin (Z/T(W;2)pB,
+p/+p), whereas the male larva has white normal larval marking skin (Z/Z, +p/+p). (C) Sex-limited
Zebra-W strain. The female larva has zebra marking (Z/T(W;3)Ze, p/p), whereas the male larva lacks
marking (Z/Z, p/p). (D) Cocoons spun by the sex-limited yellow cocoon strain. The yellow cocoon is
female (Z/T(W;2)Y, +Y/+Y), whereas the white cocoon is male (Z/Z, +Y/+Y). Adapted from Abe and Fujii
(2009).
However, although 400 or more visible mutations have been placed on linkage
maps in B. mori (10-12), no gene for morphological character has been found on the
normal W chromosome. Thus, we were interested in the following question: What is
the nature of the nucleotide sequence of the nonrecombinant W chromosome, which
contains no gene except for the Fem gene? Therefore, the research group led by Abe,
who was one of the authors, tried to find a DNA marker as a beginning of the W
chromosome analysis. We continue analyzing the W and Z chromosomes of B. mori
now.
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2. The beginning of the molecular biological studies of the W chromosome

The main focus of the W chromosome of B. mori was for the practical use of the
resulting strains. Therefore, a molecular biological study of the W chromosome was
not performed for many years even after it was used in practice. Promboon et al. (13)
constructed a linkage map of random amplified polymorphic DNAs (RAPDs).
Subsequently, Yasukochi (14) created a dense genetic map based on 1018 molecular
markers. Still later, an amplified fragment length polymorphism (AFLP) map (15), a
simple sequence repeat-based consensus linkage map (SSR) (16), and a single
nucleotide polymorphism (SNP) linkage map (17) were constructed. However, no
W-specific markers of B. mori were reported in these studies. A draft sequence of the
genome of the p50T strain (standard strain) was constructed by 3-fold whole-genome
shotgun (WGS) sequencing by Japanese group (18). In addition, a draft sequence of
the p50T strain was constructed by 5.9-fold WGS sequencing by a Chinese group
(19). However, these nucleotide sequence data were obtained using only the male
(ZZ) genome.
To begin the molecular biological analysis of the W chromosome, Abe et al. (20)
felt that the acquisition of a W-specific nucleotide sequence was very important as a
first step. Initially, they attempted to obtain a RAPD marker on the W chromosome.
This method is relatively simple by using PCR with a large set of arbitrary 10-mer
primers. Template DNAs for PCR are genomic DNAs extracted from highly inbred
strains that have been maintained by sister-brother matings. The PCR amplification
patterns between male and female are then compared. If the PCR product amplified
only from a female is obtained, it is derived from the W chromosome. Initially, Abe et
al. (20) used the T(W;2)+p chromosome of the sex-limited C137 (Chinese 137) strain,
and not the normal W chromosome, as template DNA for PCR aimed at detection of
W-specific RAPD markers for two reasons. First, if there are polymorphisms in the
normal W chromosome at the molecular level, the differences based on larval
appearance cannot be discriminated. Second, even if a given W-specific RAPD
marker is derived from the chromosome 2 and not the W chromosome region, it
should be possible to access the W chromosome region via a chromosome walk. For
these reasons, Abe et al. (20) used genomic DNA from males and females of the C137
strain with the T(W;2)+p chromosome as a PCR template and screened approximately
400 arbitrary 10-mer primers. Fortunately, they obtained one female-specific RAPD
marker, Female-218 (20, 21). The Female-218 RAPD marker resulted in the
amplification of a product from the T(W;2)pSa and T(W;2)pB chromosomes, but not
from the normal W chromosome (22). At that time, whether the Female-218 RAPD
marker was derived from the W region or the chromosome 2 fragment region was not
known. Afterwards, it was clarified that the Female-218 RAPD marker is localized to
the chromosome 2 fragment region of the translocated W chromosome (T(W;2)+p,
T(W;2)pSa and T(W;2)pB) according to the results of detachment experiments (23).
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In subsequent analyses, Abe et al. (24, 25) used genomic DNA from several
strains with the normal W chromosomes (for example p50T strain) as templates.
Fortunately, they obtained 12 W-specific RAPD markers from the normal W
chromosome (24, 25). Then, the conversion of these female-specific RAPD markers
(W-markers) into Sequence Characterized Amplified Region (SCAR) markers using
longer primers was successful. Thus, the existence of the W chromosome of B. mori
became clear a molecular biological level.
3. Presence of the W-markers in the silkworm strains maintained in Japan

As mentioned above, Abe et al. (20) were at a loss before beginning the analyses
of the W chromosome which strain should have chosen because whether there is
polymorphism of the W chromosome every strains at the molecular level or not.
However, Abe et al. (24, 26) obtained 12 W-markers in the p50T standard strain.
Then, Abe et al. (25) examined the possession of the W-markers of the silkworm
strains maintained in Japan. They screened the randomly selected twenty-five
silkworm strains maintained at The National Institute Agrobiological Science.
Surprisingly, almost all W chromosomes of these strains contained 12 W-markers
(25). As mentioned below in detail, almost all of the W-marker sequences contained
the border region of retrotransposable elements, namely portions of nested
retrotransposable elements. There is little possibility that a specific retrotransposable
element was inserted into an identical position of another element in different
individuals, but it is more likely that this occurred very early in a single individual.
Therefore, these results strongly indicate that the W chromosomes of the strains
(Japanese-W-Eve) maintained in Japan are of an almost identical type (25). Although
there are many silkworm strains, it is thought that the Japanese silkworms have been
bred from an extremely limited group derived from a single female ancestor. To
investigate the effect of the W chromosome for the character of economic value,
Takasaki and Aratake (27) brood the many strains that are congenic to the standard
strain for the W chromosome, starting with the females of various strains and males of
standard strain. Backcrossing of females were continued to eliminate autosomes of
various strains except the W chromosomes. As results, there are no effects of the W
chromosomes from various strains for the characters of economic values. At that time,
there was no method to detect polymorphism of the W chromosomes. It is felt the
effort that was going to discover the role or polymorphism of the W chromosome
some how.
4. Mapping of the W-markers on the W chromosome

Although 12 W-markers have been identified (24, 25), the genetic mapping of
these W-markers on the W chromosome is impossible by conventional recombination
experiments because crossing over is restricted to males in B. mori. Fortunately, there
are several sex-limited (W-translocation) strains in which the autosomal fragment
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containing the dominant genes for visible traits have been translocated to the
W chromosome by using X-ray or gamma-ray irradiation (5-7, 9), resulting in the
strains having mutated W chromosomes (28 and 29). Abe et al (26) investigated the
presence or absence of the W-markers in these strains and cleared that there were
differences at each W chromosome. The T(W;2)+p chromosome of larval-marking-W
strain contained all 12 W-markers, same as the normal W chromosome. The
T(W;10)+w-2 chromoosme of the Black-egg-W strain (7) did not contain the W-Mikan
marker. The T(W;3)Ze chromosome of the Zebra-W strain (8) did not contain the
W-Samurai and W-Mikan markers. Moreover, in the newly established strains of the
W-translocation strains, a part of the W chromosome inherited to male (25-29).
Furthermore, the T(W;2)Y-Abe chromosome (sex-limited yellow cocoon strain)
contained only the W-Rikishi marker (26). From these results, it is possible to map the
positions of the W-markers relative to the putative Fem gene. The order of W-Kabuki,
W-Kamikaze, W-Musashi, W-Sasuke, W-Sakura, and BMC1-Kabuki markers could
not be determined, but Abe et al. (26) concluded that the W-Rikishi marker is distal to
the end of the W chromosome (Fig. 2). This positional information should be of
significant help towards the goal of cloning the putative Fem gene.
In B. mori, efficient construction methods for transgenic silkworm have been
developed (30,31). Uchino et al. (32) constructed 105 enhancer trap lines in which the
BmA3-GAL4 gene is inserted into different positions of the chromosomes. However,
no line shows W-linkage of the transgene. Recently, insertion of the transgene into the
W chromosome has been reported (33) One of 160 newly established transgenic line
shows W-linkage of the transgene. In the strain, EGFP is expressed stably under the
control of the 3 x P3 promoter. Interestingly, insertion position of sex-limited EGPF
strain is mapped to the BAC clone containing W-Rikishi marker. This result suggests
that a small region of the W chromosome containing the W-Rikishi marker is
transcrptionally active compared to the rest of the heterochromatic W chromosome.
In addition, these W-translocation results indicate that the W chromosomes of the
sex-limited strains are not the result of simple fusion of the autosomal fragment to the
end of the unaltered W chromosome but, rather, the product of reciprocal
translocation accompanied by the breakage of the W chromosome. The intention was
to isolate translocations of autosomal fragments to the W chromosome, rather than to
delete the W chromosome. However, in the process of generating these strains,
deletions of the W chromosome were unconsciously selected. From these strains, we
can conclude that even large deletions of the W chromosome or large translocations to
W do not affect the female sex determination.
In addition, the pSa+pW chromosome, in which two 2nd chromosome fragments
(pSa and +p, respectively) were contained, was generated by translocation of a
fragment of chromosome 2 (pSa connected with +p) to the left end of the W
chromosome (4, 6). Subsequently, the pSa+pW+od chromosome was produced by
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connecting the short fragment of the Z chromosome having the +od gene with the
pSa+pW chromosome (4, 34). Through a dissociation experiment on the pSa+pW+od
chromosome, Tazima reached the conclusion that the +od gene was translocated to the
right end of the pSa+pW chromosome as a result of exceptional crossover between the
Z chromosome and the pSa+pW chromosome (right-end model). However, based on
the results of the presence or absence of W-markers on the W chromosome variants,
left-end model could be supported over the right-end model (26,28). The generation
of the Df(pSa+pW+od)Fem chromosome connected with the deleted Z chromosome can
be easily explained by the translocation between the pSa+pW+od chromosome and the
Z chromosome. Therefore, Fujii et al. (28,35) designated the pSa+pW+od
chromosome and the Df(pSa+pW+od)Fem chromosome as +odpSa+pW and
Df(+odpSa+pW)Fem, respectively.
Fig. 2. Higher-resolution mapping of W-specific RAPD markers and the putative Fem gene on the
W chromosome using W chromosome variants. The order of six W-specific RAPD markers
(W-Kabuki, W-Kamikaze, W-Musashi, W-Sasuke, W-Sakura and BMC1-Kabuki) and two W-specific
RAPD markers (W-Yukemuri-L and W-Yukemuri-S) could not be determined. Mapping of the other
markers is shown. For the T(W;3)Ze, T(W;10)+w-2 and DfZ-DfW chromosomes, see Abe et al. (25) and
Fujii et al. (28). Adapted from Abe and Fujii (74).
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5. Nested structure of transposable elements on the W chromosome
Fig. 3. Nested transposable elements in the BAC clone that includes the W-Kabuki RAPD marker
sequence. See Abe et al. (38). This figure is adapted based on the recent DNA sequence information
from Abe and Fujii (74).
Based on the BLASTX search, except for the W-Musashi, all of the deduced
amino acid sequences of W-markers show similar to previously reported amino acid
sequences of retrotransposable elements. Moreover, almost all amino acid sequences
of W-markers contain boundaries of two or three retrotransposable elements (24, 25).
To analyze the W chromosome in detail, genomic DNA lambda phage libraries have
been constructed (36). Then, two lambda phage clones were obtained containing the
W-Kabuki and W-Samurai RAPD sequences. The lambda clone containing the
W-Kabuki RAPD sequence (18.1 kb) comprises a nested structure of at least six
transposable elements (36). The lambda clone containing the W-Samurai RAPD
sequence (14.7 kb) comprises a nested structure of six transposable elements (25).
Three of these six transposable elements are non-LTR retrotransposon BMC1
(37, 41). Moreover, to analyze the flanking regions of W-Kabuki RAPD marker in
detail, Abe et al. (25, 38) obtained a BAC (bacterial artificial chromosome) clone
containing a W-Kabuki RAPD marker and subjected this clone to shotgun sequencing.
The resulting assembly of sequences did not produce a single contiguous sequence
due to the presence of many repetitive DNA elements, in particular the non-LTR
retrotransposon BMC1 (37) and Kendo (39). As shown in Fig. 3, the structural
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features of the W chromosome of B. mori are quite different from structures typical of
other chromosomes. The W chromosome comprises the nested structures of many
transposable elements (38-40).
Thus, in the process of the W chromosome analysis, its become clear that it is
very difficult to use the normal molecular biological method for analysis of the W
chromosome due to the presence of many interspersed transposable elements
(repetitive sequences). For example in the case of non-LTR BMC1, there are many
copies on the autosomes but almost all open reading frames (ORFs) of BMC1 copies
are incomplete, and the full-length and complete ORF BMC1 are not found (41).
However, almost all BMC1 copies on the W chromosome are full-length and have
complete ORFs (37). When there are multiple BMC1 elements in one phage or BAC
clone of the W chromosome, the shotgun sequence analysis is very difficult.
Moreover, when a computer performs an assembly operation, often a false result is
given. It seems to be an extremely difficult jigsaw puzzle that includes a lot of
resembling pieces. There is not a completely connected, single contig BAC clone of
W chromosome so far.
6. The structure of W chromosome and novel transposable elements

6.1. Structure of W chromosome
As mentioned above, the flanking regions of W-Kabuki RAPD marker comprise
the nested structure of many transposable elements (Fig.3). The flanking regions of
W-markers analyzed so far exhibit the nested structure of many transposable elements
similar to the W-Kabuki flanking regions. Since RAPD markers are well interspersed
in the genome and unbiased with regard to linkage group (13, 43), it is likely that the
12 W-markers are randomly distributed on the W chromosome. Under this
assumption, the nested structure of transposable elements (especially retrotransposable elements) is representative of the overall organization of the W
chromosome.
When the molecular biological analysis was begun, there was very little
retrotransposable element where the molecular structure was determined. Therefore,
the figure of the nested structure of transposable elements was drawn at the same time
the structure of the new transposable element was determined. Even if it became clear
that the W-marker was a part of the retrotransposable element based on the BLASTX,
the full-length of sequence was not able to be determined readily. In B. mori, whole
genome shotgun sequence (WGS) data was generated using the p50 strain (18, 19).
However only the male genome was used in this shotgun analysis because it is
predicted that the repetitive nature of the DNA sequence of the W chromosome makes
it very difficult to assemble. Although it has been predicted that almost all
transposable elements on the autosomes and the Z chromosome have degenerated
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because of recombination or rearrangement, genome studies in B. mori using male

genomic DNA would allow us to examine numerous fragments of transposable
elements and repetitive sequences. Therefore, the WGS data helped to analyze the
uncharacterized nucleotide sequence of the W chromosome very much. For example,
when a certain transposable element A turned into B halfway in the nucleotide
sequence of the W chromosome, the remaining sequence of A from the boundary
between A and B could not be determined until WGS data was reported.
However, after the WGS data was reported, the sequence that it is thought to be an
approximately full-length sequence of A could be obtained. Further, through these
analyses, the nucleotide sequences that do not show the characteristics of typical
transposable elements were found on the W chromosome (38). However, almost all of
these uncharacterized sequences were determined as non-autonomous transposable
elements that do not fit into the conventional classification (42), by using the WGS
data (49).
6.2. Structure of transposable elements on the W chromosome
Transposable elements are divided into main two groups. One are DNA
transposon which reinsert into a new DNA site without an RNA intermediate. The
other groups are retrotransposons which are first transcribed into RNA and then
retrotranscribed by an RNA-dependent DNA polymerase into DNA (44). Many
retrotransposons have accumulated on the W chromosome of B. mori. In general,
retrotransposons are divided into two major classes: one class is the long terminal
repeat (LTR) type, and the second class is the non-LTR type which have poly(A) or a
poly(T) tail. LTR retrotransposons themselves have been divided into three major
groups (or families) based on the characteristics of ORFs, namely Ty1-copia,
gyspy-Ty3, and BEL-Pao (not isolated from plants) groups (45, 46). All types of
retrotransposons (non-LTR, Ty1-copia, gypsy-Ty3, and BEL-Pao) exist both as
fragments and full-length on the W chromosome of B. mori. There are many cases
that the nucleotide sequence of full-length retrotransposon before another transposable
element was inserted could be reconstructed by eliminating the inserted transposable
element sequence from the sequence data.
6.2a. BMC1
In B. mori, the BMC1 is considered a LINE-like element because it is dispersed
throughout the genome. The number of BMC1 elements is estimated to be
approximately 3500 copies per haploid genome (41). Although the full-length BMC1
elements are dispersed throughout the genome, most numbers are preferentially
truncated to varying extents at their 5 end. Moreover, long ORFs could not be
detected within the BMC1 full unit (41). However, the BMC1 inserted into Kabuki on
the W chromosome is a complete full-length element (GenBank Accession No.
AB018558). This BMC1 is 5091 bp long and has a 5 untranslated region (5-UTR),
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two intact ORFs, and a 3-UTR sequence, which terminates in a poly(A) tail. The
ORF1 of BMC1 encodes three putative cysteine histidine (Cys) motifs, while ORF2
encodes a protein containing an endonuclease (EN) domain and a reverse transcriptase
(RT) domain (37). Why does a complete full-length BMC1 element exist on the
W chromosome? One possible explanation is that because crossing over is restricted
to males in B. mori and the W chromosome is recombinationally isolated from the
Z chromosome, the BMC1 elements inserted into the W chromosome would be
expected to change more slowly than those inserted into other chromosomes.
Moreover, higher mutation rates are predicted for Z chromosomes or autosomes
relative to the W chromosome because of the greater number of cell divisions required
for spermatogenesis relative to oogenesis. Unfortunately, the presence of many
complete full-length BMC1 elements on the W chromosome is a main cause making
the assembly of the shotgun clone sequence extremely difficult.
Fig. 4. Structure of six retrotransposons and Supermite-BMC1 of Bombyx mori. Each box with an
arrow except in Supermite-BMC1 indicates one LTR (long terminal repeat). Each box with an arrow in
Supermite-BMC1 indicates an IR (inverted repeat). PBS, primer-binding site; PPT, polypurine tract;
Pro, protease domain; RT, reverse transcriptase domain; RH, RNase H domain; Int, integrase domain;
EN, endonuclease domain; 5-UTR, 5untranslated region; 3-UTR, 3untranslated region; poly(A),
poly(A) tail. Cys, cysteine and histidine motif are indicated solid bar in the box of ORF.
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6.2b. Kendo
Kendo is the most abundant non-LTR retrotransposon next to the BMC1 in B.
mori genome (39). Despite numerous sequences of B. mori, a complete full-length
Kendo had not been found. As mentioned below, the longest Kendo has been found in
the Z-specific BAC clone (47). However, in the WGS sequence data (18), we could
not find longer Kendo than that in the Z chromosome. Therefore, we think that the
Kendo in the Z chromosome (47) is the complete full-length element. The
full-length Kendo is about 3670 bp long and contains a single ORF encoding an EN
and an RT domains (Fig.4f).
6.2c. Bm1 group
The Bm1 element is a major short interspersed element (SINE) in the genome of
B. mori (48). Therefore, the Bm1 element is thought to be analogous to the human Alu
element. The longest Bm1 element is about 450 bp containing no coding region and
having a poly(A) tail at the 3 end (48). It is thought that the enzyme required for
mobility of the Bm1 element is supplied by other elements. The Bm1 element is often
found on the W chromosome of B. mori (36). We thought that if Bm1 element
insertions occurred randomly in the genome, the Bm1 elements on the W chromosome
would be useful for the analysis of the W chromosome as landmarks. In other words,
even if there are identical nucleotide sequences before the Bm1 element was inserted
on the autosomes, it will be possible to discriminate the PCR product amplified from
the W chromosome based on the size. However, when we carried out such a PCR
experiment, the same size PCR products were amplified from both the male genome
and female genome. We were not able to understand the meaning of these results.
Then, when the same sequences containing Bm1 elements on the W chromosome
were found in the WGS data of males, we suspected that these sequences resulted
from contamination of male genomic DNA female DNA during WGS analyses. If
this were so, we would have been able to find the W chromosome-specific sequences
(25,26,38) in the WGS data. However, no W-specific sequences, for example, the
nested structure of retrotransposable elements, were found in the WGS data.
Moreover, we analyzed the WGS data to find out if there were ancestral states of these
sequences that did not contain the Bm1 sequence. However, the ancestral states were
not found in the WGS data. Therefore, we could not but change a thought
fundamentally as follows. The Bm1 sequences found in these sequences were not
inserted, but the flanking sequences of Bm1 sequence and Bm1 are one unit of
transposition! We got possible to explain the relationship of the Bm1 sequence data of
the W chromosome and the WGS data without contradiction. We designated these
transposable elements as secondary-Bm1 transposable elements (SBTEs) (49).
There are several types of SBTEs as shown in Fig. 5, long SBTEs more than 5kb
long (ChoBm1), short SBTEs about 1kb long (Bm1modoki), and the LTR types
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(Yaocho, Rinne-L and Rinne-S). We initially thought that Bm1 sequences of SBTEs
were carried on each SBTE merely as a package. For example, in ChoBm1, we
thought that the Bm1 sequence was inserted fortuitously into this position of ChoBm1.
If this were so, the putative ancestral ChoBm1 not containing the Bm1 sequence would
be found in the WGS data. However, we could not find the ancestral ChoBm1
sequence in the WGS data. These results strongly indicate that the ChoBm1 acquired
the ability to propagate after this structure containing the Bm1 sequence had been
constructed. This applies to all SBTEs. The generation processes of SBTEs in the
genome of B. mori are unknown. The role of Bm1 sequences in SBTEs for
transcription or translation is also unknown. It is likely that the Bm1 sequence has a
specific role for transcription or transposition because the sequences of the flanking
regions of Bm1 sequences in each SBTE are not homologous to each other.
In the case of human Alu elements, there is no report of a transposable element
containing an Alu sequence [International Human Genome Sequencing Consortium
(52)]. In Drosophila, there is no SINE in the genome (51). A unified classification
system for eukaryotic transposable elements was proposed (42). However, SBTEs
cannot be classified into the known groups. Therefore, SBTEs should be classified as
a new group of transposable elements. It seems likely that the SBTEs are unique
transposable elements found in the Bombyx genome. However, our idea was not
accepted at first. It took a long time until our idea was understood and accepted.
Because mysterious sequences will appear in the data of the W chromosome in the
future, we have to analyze the data without having a preconception.
6.2d. Supermite-BMC1
Previously, we found an amino acid coding sequence that is a part of the
non-LTR retrotransposon BMC1 in 45J23 BAC clone (Accession no. AB126052; 38).
Moreover, this coding sequence was flanked with a relatively longer inverted repeat
(500 bp). Initially, we thought that this sequence was the result of the insertion of
5-truncated BMC1 into the DNA transposon-like sequence or into MITE (miniature
inverted-repeat transposable element) like sequence. If this were so, the putative
ancestral DNA transposon-like sequence would be found in the WGS and Buil2 data.
However, we could not find the putative ancestral DNA transposon-like sequence.
Therefore, we consider that this sequence is the unit of transposition. We designated
this sequence (nucleotide position 13270-15260 in AB126052) as Supermite-BMC1
(Fig.5g).
6.2e. Other transposable elements
In the processes of the analyses of the W chromosome, typical LTR and
non-LTR retrotransposons, and DNA transposons have been found. Additionally,
many SINE-like sequences have been found. For these include Nagisa (390 bp),
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Sayama (952 bp), Shirigonomi (279 bp), Kohichi (1302 bp), Kusanagi (421 bp),
Chikuri (712 bp), Minichikuri (413 bp), and Amanoya (834 bp). The characteristics of
these transposable elements can be seen in Abe et al. (49).
Fig. 5. Schematic diagrams of SBTEs (secondary-Bm1 transposable elements). (a) ChoBm1 (5412
bp). (b) Neet (4046 bp). (c) Bm1modoki (1178 bp). (d) Ins11 (1088 bp). (e) Tenshi (1360 bp). (f)
Hikikomori (1740 bp). (g) Yaocho (5851 bp). (h) Rinne-L (3695 bp). (i) Rinne-S (3437 bp). Each box
with an arrow in Yaocho, Rinne-L and Rinne-S indicates one LTR or LTR-like sequence. PBS,
primer-binding site. Adapted from Abe et al. (49).
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7. Recognition of W chromosome by FISH

A sex chromatin body (SB) observed in the nuclei of lepidopteran females, has
been deduced to be composed of condensed W chromosomes (52, 53). The SB is
detected in the nuclei of sucking stomachs in females by staining with acetic orcein
(54). However, in the case of the translocation or fusion of the W chromosome with
an autosome or with the Z chromosome, the number of SBs may not accord with the
number of actual W chromosome (55, 56). Moreover, no SB is detected in the nuclei
of female moths having an extremely short W chromosome (ex. T(W;2)Y-Abe type
chromosome) (26).
Cytogenetic identification of the W chromosome of B. mori had been considered
impossible for a long time because in the chromosomes are small and numerous
(2n=56). However, Sahara et al. (57) succeeded the identification of the W
chromosome of B. mori by using four W chromosome-derived BAC clones as probes
for fluorescence in situ hybridization (FISH). Surprisingly, all four W-BAC probes
highlighted the W chromosome as a whole of one chromosome in a paint-like
manner. These results indicate that the four W-BAC clones contain many repetitive
sequences that are dispersed through the entire length of the W chromosome.
8. piRNA derived from the W chromosome

Many transposable elements have accumulated on the W chromosome of B. mori
as strata. Recently, RNAs transcribed from the W chromosome and female-enriched
PIWI-interacting RNAs (piRNA) derived from the W chromosome have been
recognized (58, 59). Although the role of these piRNA for the sex determination is
unknown, the progress of the study of piRNA is expected.
9. The relations of the Z chromosome with W chromosome

As described above, femaleness of the B. mori is determined by the presence of
the W chromosome irrespective of the number of the Z chromosome. However, no
functional gene was ever cloned from W chromosomes. Contrary, the Z chromosome
is rich in genes. The Z chromosome consists of a 20.4 Mb sequence and includes 655
predicted genes (The International Silkworm Genome Consortium, 60). Thus far,
responsible genes for the 6 loci of Z-linked mutants have been reported (Table 1).
9.1. Z-linked genes involved in the sexual differentiation
Three important genes (Or1, Or2, and Bmacj6) involved in the perception of sex
pheromone are linked to the Z chromosome. B. mori females secrete an 11:1 mixture
of bombykol and bombykal (61). Bombykol alone elicits full male courtship behavior,
while bombykal alone shows no apparent activity.
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Table 1: Molecular information about the Z-linked mutants
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In the antenna of moth, two genes encoding sex pheromone receptors, Or1 and
Or2, are expressed specifically in males, which are tuned to bombykol and bombykal
respectively (62,63). Both of the genes are linked to the Z chromosome (The
International Silkworm Genome Consortium, 60). The neurons expressing pheromone
receptors are projected into a macroglomerular complex (MGC) in the male brain
where pheromone information is further integrated to elicit the mating behavior (64).
spli (soft and pliable) is a Z-linked mutant showing an extremely soft and pliable
larval body (65). Moreover, sex-pheromone preference of spli male moths is different
from normal male moths. In the spli strain, the behavioral responsiveness of male
moths to bombykol was markedly reduced, whereas bombykal alone evoked full
courtship behavior (66).
In the spli strain, the Z chromosome is broken into two pieces. A Bombyx
homolog of Drosophila acj6 is broken by the breakage event of the Z chromosome
(67). Drosophila acj6 is a transcriptional factor involved in the olfactory receptor
gene choice and axon targeting of olfactory receptor neurons (ORNs) (68, 69).
Quantitative RT-PCR analysis of olfactory receptors in male moth antenna revealed
that expression levels of Or3 in the spli males was almost the same as those in normal
males. However, about 1000-fold reduction in Or1 expression was observed in the
spli males. (1) These results suggest that expression of the Or1 depends on Bmacj6
while that of the Or3 is independent of Bmacj6 (2). The reduced response of spli
males to bombykol was caused by the paucity of bombykol receptors on the male
antennae.
The MGC of B. mori consists of three subdivisions: the toroid, cumulus, and
horseshoe. ORNs responding to bombykol and bombykal send their axons to the
toroid and cumulus, respectively (70). It was also found that in the spli males, neurons
projecting to the toroid responded strongly to bombykal. A possible explanation for
this phenomenon is mistargeting of the neuron expressing Or1 caused by the lack of
Bmacj6 (67).
9.2. Transposable elements on the Z chromosome
A 320-kb region of the Z chromosome of B. mori containing a kettin gene has
been analyzed (47). This 320-kb sequence contains many transposable-like sequences,
47 non-LTR retrotransposons, 50 retroposons, 10 DNA-type transposons, and other
uncharacterized repetitive sequences. The non-LTR retrotransposons BMC1, the
DNA-type transposon mariner (71,72), and the retroposon Bm1 (48) are dispersed
throughout the region of 320-kb on the Z chromosome. These transposable elements
were found to be incomplete. However, as mentioned above, we think that the kendo
in this region (AB090307, nucleotide positions: 89135-85464 from 5 to 3; Koike et
al.(47) is the complete full-length element (74). The nested structure of many
transposable elements found on the W chromosome is not found in this 320-kb region
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of the Z chromosome. It should be noted that at that time, no LTR retrotransposons

has been found in this 320-kb region. However, we found a part of an LTR
retrotransposon in this region (AB090307, nucleotide positions: 34714-34358 from 5
to 3). We determined that this sequence was a solo LTR of the LTR retrotransposon
Suzuka (74). Solo LTRs may result from removal of the internal domain of LTR
retrotransposons by unequal crossing over or from intra-element recombination
between LTRs. Similarly, in the Z chromosome of B. mori, the exclusion of a region
between two Bm1 elements that inserted into some extent remote with the same
direction may occur. The od (distinct oily) mutant gene was generated by the
retroposon-mediated deletion through intra-chromosomal recombination between two
Bm1 elements (35). We could not conclude whether solo LTRs on the Z chromosome
had been generated by unequal crossing over or from an intra-element homologous
recombination between LTRs. Nonetheless, unequal crossing over and intra-element
recombination between LTRs are likely to be mechanisms for deletions, mutations,
and decreases in the Z chromosome size.
10. Analyses of W chromosome of lepidopteran insects except B. mori

Based on the observation of SB in nuclei, many lepidopteran species (butterfly
and moth) thought to be have the W chromosome have been reported (55). As an
example of the molecular biological analysis of the W chromosome except for B. mori
is the wild silkworm Bombyx mandarina. Abe et al. (24) collected the larvae of wild
B. mandarina from the field and paired the female moth with the male of B. mori by
compulsory handpairing. The W chromosome of B. mandarina has been maintained
by repeatedly backcrossing the females to B. mori males. This strain containing the W
chromosome of B. mandarina is named as the WILD-W strain (24). Then, a
female-specific RAPD marker, designated W-Yamato, from the W chromosome of
B. mandarina was obtained (24). This W-Yamato RAPD marker was not amplified
from the W chromosome of B. mori. Additionally, eleven of twelve females-specific
RAPD markers found in B. mori were not found on the W chromosome of B.
mandarina (25). These results indicate that the W chromosome of B. mandarina
differs from that of B. mori at the DNA sequence level (39). In the meiosis of the
ZZWW tetraploid female, the frequency of pairing of the W chromosome of
B. mandarina and the Z chromosome of B. mori is lower than that of the pairing of the
W and Z chromosomes of B. mori (75). The W chromosome of B. mandarina is a
valuable chromosome that can be compared with the W chromosome of B. mori.
In other insects, the nucleotide sequences of the W chromosome of codling moth,
Cydia pomonella, have been obtained by the method of microdissection of sex
chromatin (76, 77). Recently, the nucleotide sequence analysis of the W chromosome
of Biston betularia, famous for industrial melanism, has begun (78).
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11. Conclusion
The W chromosome of B. mori has been artificially modified for practical use by
irradiation with X-rays of gamma ray (5-9, 34). In these processes, the intention was
to isolate translocations of autosomal fragments to the W chromosome, rather than to
delete the W chromosome. However, deletions of the W chromosome were selected
unconsciously in the processes of generating sex-limited strains, and these modified
W chromosomes became very useful materials for molecular biological analysis.
From our results using the W chromosomes of these W-translocation strains, we can
conclude that even large deletions of the W chromosome or large translocations to W
do not affect the female sex determination. Therefore, we think that a full-length
normal W chromosome is not essential for femaleness. Instead, we conclude from
translocation tests and deletion strains that putative Fem genes is not distributed
evenly over the W chromosome. Provably, a putative Fem gene exists in an extremely
limited region around the W-Rikishi RAPD marker (26). Although the
W chromosome continues to be analyzed, nobody has successfully identified the Fem
gene.
In the genomic sequence of Drosophila melanogaster, there are 1572 different
transposable elements belonging to many families (51). In contrast, although the
accurate number is not determined, there are many more transposable elements in the
genomic sequence of B. mori. It is surprising that the numerous copy numbers of
transposable elements in addition to a great many kinds including unpublished
elements. It is very likely that the SBTEs containing the Bm1 sequence that cannot be
classified into the conventional classification are unique transposable elements only in
the genome of B. mori (49). In the nucleotide sequences of the Z chromosome, a trace
of the W chromosome could not be found. It is widely accepted that mammal
(including human) X and Y chromosomes originated from the same ancestral
autosomes (non-sex chromosomes) during the evolution of sex determination (79).
However in B. mori, it is hard to think that the W chromosome was originated from
the ancestral Z chromosome degenerated by insertion of numerous transposable
elements. It is thought that the origin of the W chromosome of B. mori is different
from the Z chromosome.
In Drosophila genome projects, it was impossible to assemble Y chromosome
sequence data into a single contig due to the presence of many repetitive sequences
(73). Therefore, to identify new genes on the Y chromosome of D. melanogaster,
another strategy, TBLAST search, was developed (77, 80). In the Africa malaria
mosquito Anopheles gambiae, numerous Y-linked repetitive sequences in the Y
chromosome have been obtained (81, 82). However, no genes on the Y chromosome
were identified based on a TBLASTN search (83). Thus, both in Drosophila and
Anopheles, molecular biological analyses of the Y chromosome are made very
difficult by the presence of unusual chromosomal features. However, the W
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chromosome of B. mori has been constructed by insertion of many transposable

elements in turn. Moreover, the W chromosome evolves more slowly than other
chromosomes because crossing-over is restricted to males in B. mori and the W
chromosome is recombinationally isolated from the Z chromosome and autosomes.
Therefore, it is possible to clarify the history of the construction of the W
chromosome precisely by analyzing the insertion order of transposable elements
carefully.
Acknowledgment
This work was supported by the Grants-in-Aid for Scientific Research, JSPS/MEXT
(No. 26450464).
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and Tamura, T. (2008) Construction of a piggyBac-based enhancer trap system for the analysis of gene
function in silkworm Bombyx mori. Insect Biochem. Mol. Biol. 38: 1165-1173.
33. Ma, S., Wang, X., Fei, J., Liu, Y., Duan, J., Wang, F., Xu, H., Zhao, P., and Xia, Q. (2013) Genetic
marking of sex using a W chromosome-linked transgene. Insect Biochem. Mol. Biol. 43(12): 1079-1086.
34. Tazima, Y. (1948) Translocation of the Z chromosome to the W chromosome of the silkworm, Bombyx
mori. In Oguma Commemoration Volume on Cytology and Genetics (in Japanese). pp.88-99.
35. Fujii, T., Abe, H., Katsuma, S., Mita, K., and Shimada, T. (2008) Mapping of sex-linked genes onto the
genome sequence using various aberrations of the Z chromosome in Bombyx mori. Insect Biochem. Mol.
Biol. 38: 1072-1079.
36. Abe, H., Ohbayashi, F., Shimada, T., Sugasaki, T., Kawai, S., Mita, K., and Oshiki, T. (2000) Molecular
structure of a novel gypsy-Ty3-like retrotransposon (Kabuki) and nested retrotransposable elements on
the W chromosome of the silkworm Bombyx mori. Mol. Gen. Genet. 263: 916-924.
37. Abe, H., Ohbayashi, F., Shimada, T., Sugasaki, T., Kawai, S., and Oshiki, T. (1998) A complete
full-length non-LTR retrotransposon, BMC1, on the W chromosome of the silkworm, Bombyx mori.
Genes Genet. Syst. 73: 353-358.
38. Abe, H., Mita, K., Yasukochi, Y., Oshiki, T., and Shimada, T. (2005) Retrotransposable elements on the
W chromosome of the silkworm Bombyx mori. Cytogenet. Genome Res. 110: 144-151.
39. Abe, H., Sugasaki, T., Terada, T., Kanehara, M., Ohbayashi, F., Shimada, T., Kawai, S., Mita, K., and
Oshiki, T. (2002) Nested retrotransposons on the W chromosome of the wild silkworm Bombyx
mandarina. Insect Mol Biol. 11: 307-314.
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40. Abe, H., Fujii, T., and Shimada, T. (2010) Sex chromosomes and sex determination in Bombyx mori. In
Molecular Biology and Genetics of the Lepidoptera 65-87. Edited by MR. Goldsmith and F. Marec.
CRC Press, Taylor and Francis Group. (ISBN 978-1-4200-6014-0).
41. Ogura, T., Okano, K., Tsuchida, K., Miyajima, N., Tanaka, H., Takada, N., Izumi, S., Tomino, S., and
Maekawa, H. (1994) A defective non-LTR retrotransposon is dispersed throughout the genome of the
silkworm, Bombyx mori. Chromosoma. 103: 311-323.
42. Wicker, T., Sabot, F., Hua-Van, A., Bennetzen, JL., Capy, P., Chalhoub, B., Flavell, A., Leroy, P.,
Morgante, M., Panaud, O., Paux, E., SanMiguel, P., and Schulman, A.H. (2007) A unified classification
system for eukaryotic transposable elements. Nature Review Genetics. 8: 973-982.
43. Liu, C., Yamamoto, K., Cheng, T.C., Kadono-Okuda, K., Narukawa, J., Liu, S.P., Han, Y., Futahashi, R.,
Kidokoro, K., Noda, H., Kobayashi, I., Tamura, T., Ohnuma, A., Banno, Y., Dai F.Y., Xiang, Z.H.,
Goldsmith, M.R., Mita, K., and Xia, Q.Y. (2010) Repression of tyrosine hydroxylase is responsible for the
sex-linked chocolate mutation of the silkworm, Bombyx mori. Proc. Natl. Acad. Sci. USA. 107:
12980-12985.
44. Galun, E. (2003) Transposable Elements, A Guide to the Perplexed and the Novice With Appendices on
RNAi, Chromatin Remodeling and Gene Tagging. Kluwer Academic Publishers, Dordrecht.
45. Xiong, Y., Burke, W.D., and Eickbush, T.H. (1993) Pao, a highly divergent retrotransposable element
from Bombyx mori containing long terminal repeats with tandem copies of the putative R region. Nucleic
Acids Res. 21: 2117-2123.
46. Abe, H., Ohbayashi, F., Sugasaki, T., Kanehara, M., Terada, T., Shimada, T., Kawai, S., Mita, K.,
Kanamori, Y., Yamamoto, M.T., and Oshiki, T. (2001) Two novel Pao-like retrotransposons (Kamikaze
and Yamato) from the silkworm species Bombyx mori and B. mandarina: common structural features of
Pao-like elements. Mol. Genet. Genomics, 265: 375-385.
47. Koike, Y., Mita, K., Suzuki, M.G., Maeda, S., Abe, H., Osoegawa, K., deJong, P.J., and Shimad,a T.
(2003) Genomic sequence of a 320-kb segment of the Z chromosome of Bombyx mori containing a kettin
ortholog. Mol. Genet. Genomics, 269: 137-149.
48. Adams, D.S., Eickbush, T.H., Herrera, R.J., and Lizardi, P.M. (1986) A highly reiterated family of
transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Bombyx mori. J. Mol. Biol.
187: 465-478.
49. Abe, H., Fujii, T., Shimada, T., and Mita, K. (2010b) Novel non-autonomous transposable elements on W
chromosome of the silkworm, Bombyx mori. J. Genetics. 89: 375-387.
50. International Human Genome Sequencing Consortium (2001) Initial sequencing and analysis of the
human genome. Nature 409: 860-921.
51. Ashburner, M., Golic, K.G., and Hawley, R.S. (2005) Drosophila, a laboratory handbook (2nd edition).
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. (ISBN 0-87969-706-7).
52. Ennis, T.J. (1976) Sex chromatin and chromosome numbers in Lepidoptera. Can. J. Cytol. 18: 119-130
53. Traut, W., and Marec, F. (1996) Sex chromatin in Lepidoptera. Quart. Rev. Biol. 71: 239-256.
54. Pan, Q.Z., Sugai, E., and Oshiki, T. (1987) Sex-chromatin observation in cell nuclei of tissues of the
silkworm, Bombyx mori. J. Seric. Sci. Jpn. 56: 436-439. (in Japanese with English summary).
55. Traut, W., Weith, A., and Traut, G. (1986) Structural mutants of the W chromosome in Ephestia (Insecta,
Lepidoptera). Genetica, 70: 69-79.
56. Tanaka, N., Yokoyama, Y., Irobe, Y., Abe, H., Ninagi, O., and Oshiki, T. (2000) Breakage and
subsequent return for W chromosome-pB fragment-fifth linkage group fusion chromosome in the
sex-limited pB silkworm strain (TWPB), Bombyx mori. J. Seric. Sci. Jpn. 69: 327-330.
314
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57. Sahara, K., Yoshido, A., Kawamura, N., Ohnuma, A., Abe, H., Mita, K., Oshiki, T., Shimada, T., Asano,
SI., Bando, H., and Yasukochi, Y. (2003) W-derived BAC probes as a new tool for identification of the W
chromosome and its aberrations in Bombyx mori. Chromosoma, 112: 48-55.
58. Kawaoka, S., Hayashi, N., Suzuki, Y., Abe, H., Sugano, S., Tomari, Y., Shimada, T., and Katsuma, S.
(2009) The Bombyx ovary-derived cell line endogenously expresses PIWI/PIWI-interacting RNA
complexes. RNA 15: 1258-1264.
59. Kawaoka, S., Kadota, K., Arai, Y., Suzuki, Y., Fujii, T., Abe, H., Yasukochi, Y., Mita, K., Sugano, S.,
Shimizu, K., Tomari, Y., Shimada, T., and Katsuma, S. (2011) The silkworm W chromosome is a source
of female-enriched piRNAs. RNA, 17: 2144-2152.
60. The International Silkworm Genome Consortium. (2008) The genome of a lepidopteran model insect, the
silkworm Bombyx mori. Insect Biochem. Mol. Biol. 38: 1036-1045.
61. Kaissling, K.E., Kasang, G., Bestmann, H.J., Stransky, W., and Vostrowsky, O. (1978) A new pheromone
of the silkworm moth Bombyx mori. Naturwissenschaften 65: 382-384.
62. Sakurai, T., Nakagawa, T., Mitsuno, H., Mori, H., Endo, Y., Tanoue, S., Yasukochi, Y., Touhara, K., and
Nishioka, T. (2004) Identification and functional characterization of a sex pheromone receptor in the
silkmoth Bombyx mori. Proc. Natl. Acad. Sci. USA. 101: 16653-16658.
63. Nakagawa, T., Sakurai, T., Nishioka, T., and Touhara, K. (2005) Insect sex pheromone signals mediated
by specific combinations of olfactory receptors. Science 307: 1638-1642.
64. Kanzaki, R., and Shibuya, T. (1986) Descending protocerebral neurons related to the mating dance of the
male silkworm moth. Brain Res. 377: 378-382.
65. Murakami, A., and Ohnuma, A. (1978) Genetic analysis of a mutant showing abnormal behavior in the
silkworm. Ann. Rep. Nati. Inst. Genet. Jpn. 28: 99-100.
66. Fujii, T., Fujii, T., Namiki, S., Abe, H., Sakurai, T., Ohnuma, A., Kanzaki, R., Katsuma, S., Ishikawa, Y.,
and Shimada, T. (2011b) Sex-linked transcription factor involved in a shift of sex-pheromone preference
in the silkworm Bombyx mori. Proc. Natl. Acad. Sci. USA, 108: 18038-18043.
67. Fujii, T., Daimon, T., Uchino, K., Banno, Y., Katsuma, S., Sezutsu, H., Tamura, T., and Shimada, T.
(2010) Transgenic analysis of the BmBLOS2 gene that governs the translucency of the larval integument
of the silkworm, Bombyx mori. Insect Mol Biol. 19: 659-667.
68. Clyne, P.J., Certel, S.J., de Bruyne, M., Zaslavsky, L., Johnson, W.A., and Carlson, J.R. (1999) The odor
specificities of a subset of olfactory receptor neurons are governed by Acj6, a POU-domain transcription
factor. Neuron 22: 339-347.
69. Komiyama, T., Calson, J.R., and Luo, L. (2004) Olfactory receptor neuron axon targeting: intrinsic
transcriptional control and hierarchical interactions. Nat. Neurosci. 7: 819-825.
70. Kanzaki, R., Soo, K., Seki, Y., and Wada, S. (2003) Projections to higher olfactory centers from
subdivisions of the antennal lobe macroglomerular complex of the male silkmoth. Chem. Senses. 28:
113-130.
71. Robertson, H.N., and Asplund, M.L. (1996) Bmmar1: a basal lineage of the mariner family of
transposable elements in the silkworm moth, Bombyx mori. Insect Biochem. Mol. Biol. 26: 945-954.
72. Tomita, S., Sohn, B.H., and Tamura, T. (1997) Cloning and characterization of a mariner-like element in
the silkworm, Bombyx mori. Genes Genet. Syst. 72: 219-228.
73. Adams, M.D., Celniker, S.E., Holt, R.A., et al. (2000) The genome sequence of Drosophila melanogaster.
Science, 287: 2185-2195.
74. Abe H., and Fujii T. (2009) Molecular characteristics of the chromosome, Z chromosome and
retrotransposable elements of the silkworm, Bombyx mori. In Short Views on Insect Molecular Biology
Vol.1, Edited by Raman Chandrasekar, Academic Publisher, South India. (ISBN 81-7813-364-010).
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75. Tanaka, N., Yokoyama, T., Abe, H., Ninagi, O., and Oshiki, T. (2002) The effect of W chromosome
origin on sex-chromosome pairing in ZZWW tetraploid females of the domesticated silkworm, Bombyx
mori, and the congenic wild silkmoth, Bombyx mandarina. Genetica, 114: 89-94.
76. Traut, W., Vogel, H., Glockner, G., Hartmann, E., and Heckel, D.G. (2013) High-throughput sequencing
of a single chromosome: a moth W chromosome. Chromosome Res. 21: 491-505.
77. Carvalho, A.B., and Clark, A.G. (2005) Y chromosome D. psseudoobscura is not homologous to the
ancestral Drosophila Y. Science, 307: 108-110.
78. Vant Hof, A.E., Nguyen, P., Dalikova, M., Edmonds, N., Marec, F., and Saccheri, I.J. (2013) Linkage
map of the peppered moth, Biston betularia (Lepidoptera, Geometridae): a model of industrial melanism.
Heredity, 110: 283-295.
79. Ohno, S. (1967) Sex Chromosomes and Sex-linked Genes. (Springer-Verlag, Berlin).
80. Carvalho, A.B., Dobo, B.A., Vibranovski, M.D., and Clark, A.G. (2001) Identification of five new genes
on the Y chromosome of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA, 98: 13225-13230.
81. Krzywinski, J., Nusskern, D.R., Kern, M.K., and Besansky, N.J. (2004) Isolation and characterization of
Y chromosome sequences from the African malaria mosquito Anopheles gambiae. Genetics, 166:
1291-1302.
82. Krzywinski, J., Sangara, D., and Besansky, N.J. (2005) Satellite DNA from the Y chromosome of malaria
vector Anopheles gambiae. Genetics, 169: 185-196.
83. Krzywinski, J., Chrystal, M.A., and Besansky, N.J. (2006) Gene finding on the Y: fruitful strategy in
Drosophila does not deliver in Anopheles. Genetica, 126: 369-375.
84. Kiuchi, T., Banno, Y., Katsuma, S., and Shimada, T. (2011) Mutations in an amino acid transporter gene
are responsible for sex-linked translucent larval skin of the silkworm, Bombyx mori. Insect Biochem.
Mol.Biol. 41: 680-687.
85. Fujii, T., Abe, H., Katsuma, S., and Shimada, T. (2011) Identification and characterization of the fusion
transcript, composed of the apterous homology and putative protein phosphatase gene, generated by 1.5
Mb interstitial deletion in the vestigial (Vg) mutant of Bombyx mori. Insect Biochem. Mol. Biol. 41:
306-312.
86. Fujii, T., Daimon, T., Uchino, K., Banno, Y., Katsuma, S., Sezutsu, H., Tamura, T., and Shimada, T.
(2010) Transgenic analysis of the BmBLOS2 gene that governs the translucency of the larval integument
of the silkworm, Bombyx mori. Insect Mol. Biol. 19: 659-667.
87. Futahashi, R., Banno, Y., and Fujiwara, H. (2010) Caterpillar color patterns are determined by a
two-phase melanin gene pre-patterning process: new evidence from tan and laccase2. Evol.Dev. 12:
157-167.
88. Fujii, T., Kuwazaki, S., Yamamoto, K., Abe, H., Ohnuma, A., Katsuma, S., Mita, K., and Shimada, T.
(2010) Identification and molecular characterization of a sex chromosome rearrangement causing a soft
and pliable (spli) larval body phenotype in the silkworm, Bombyx mori. Genome, 53: 45-54.
_____________________________________________________________________________________
Article History: Received on 15th December 2013; Revised on 15th Feburary 2014; Accepted on 2nd May 2014;
Publsihed 30th October 2014.
316
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Table Contents
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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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Book Mission Project # 2: Initiated on June 2010; Completed on March 2014 and Published on Oct. 2014.
Volume 1 & 2, October 2014
Short Views on Insect Biochemistry

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

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

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

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

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

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

Zhejiang University, China.
UEI Parasitologia Mdica,

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

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

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

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

Japan.
National Bureau of Agriculturally Important

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Printed in the Unitated States of America, 2014

Printed in the Unitated States of America, 2014

Vol. (1) 291 316, 2014

The recent progress of the W and Z chromosome studies

Hiroaki Abe1 , Tsuguru Fujii2 and Raman Chandrasekar3

Department of Biological Production, Faculty of Agriculture, Tokyo University of Agriculture and

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

1. Overview of W chromosome in Bombyx mori

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

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

parallel with molecular biological progress, so-called classical genetics of B. mori

Fig. 1. Visible markers translocated from autosomes or Z chromosome to the W chromosome.

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

2. The beginning of the molecular biological studies of the W chromosome

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

3. Presence of the W-markers in the silkworm strains maintained in Japan

4. Mapping of the W-markers on the W chromosome

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

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

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

5. Nested structure of transposable elements on the W chromosome

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

6. The structure of W chromosome and novel transposable elements

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

because of recombination or rearrangement, genome studies in B. mori using male

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

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

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

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

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

7. Recognition of W chromosome by FISH

8. piRNA derived from the W chromosome

9. The relations of the Z chromosome with W chromosome

Table 1: Molecular information about the Z-linked mutants

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

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

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

of the Z chromosome. It should be noted that at that time, no LTR retrotransposons

10. Analyses of W chromosome of lepidopteran insects except B. mori

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

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

chromosome of B. mori has been constructed by insertion of many transposable

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

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

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

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

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

1. Introduction to Insect Molecular Biology.

Modulation of Botanicals on pests biochemistry.

Detoxication, stress and immune responses in insect antenna:

David Siaussat, Thomas Chertemps and Martine Maibeche

Application of isotopically labeled compounds and tandem mass

Stacy Mazzalupo and PatriciaY.Scaraffia

Field Response of Dendroctonus armandi Tsai & Li (Coleoptera:

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

Section II: Insect Growth

6. Insect Cuticular SclerotizationHardening Mechanisms and Enzymes.

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

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

8. The regulatory biosynthetic pathway of juvenile hormone.

Zhentao Sheng and Raman Chandrasekar

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