Directory UMM :Data Elmu:jurnal:I:Insect Biochemistry and Molecular Biology:Vol30.Issue5.2000:

Insect Biochemistry and Molecular Biology 30 (2000) 355–361
www.elsevier.com/locate/ibmb

Isolation of a cDNA encoding a CHH-family peptide from the
silkworm Bombyx mori
Hirotoshi Endo a, Hiromichi Nagasawa b, Toshiki Watanabe
a

a,*

Laboratory of Molecular Biology of Marine Organisms, Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo
164-8639, Japan
b
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657,
Japan
Received 30 August 1999; received in revised form 29 November 1999; accepted 13 December 1999

Abstract
The crustacean hyperglycemic hormone (CHH) peptide family includes four types of neuropeptide in decapod and isopod crustaceans, and the ion-transport peptide in orthopteran insects. To identify a new member of this family in Insecta, a PCR-based
search for cDNAs encoding CHH-family peptides was carried out in the silkworm Bombyx mori. A cDNA, named BmCHHL
(Bombyx mori CHH-like protein), with an open reading frame of 110 amino acids was isolated. Sequence analyses suggested that

the conceptual protein was a precursor of a peptide of 72 amino acids which was amidated at the carboxy terminus. The BmCHHL
sequence exhibited significant similarities to members of the CHH family including the orthopteran ion-transport peptide. BmCHHL
expression was detected in five or six cells (per hemisphere) in the frontal area of the brain in day 4 fifth instar larvae.  2000
Elsevier Science Ltd. All rights reserved.
Keywords: Silkworm; CHH family; Ion-transport peptide; Neuropeptide; cDNA sequence

1. Introduction
The CHH peptide family comprises peptides that have
been isolated from arthropods including crustaceans,
orthopteran insects and an arachnid, and is named after
the first member of the family, the crustacean hyperglycemic hormone (Keller, 1992). CHH-family peptides
consist of 69–78 amino acids including six conserved
cysteine residues which form three intramolecular disulfide bonds.
The following four neuropeptides of this family have
been identified in crustaceans: CHHs, molt-inhibiting
hormones (MIHs), vitellogenesis-inhibiting hormones
(VIHs) and mandibular organ-inhibiting hormones
(MOIHs) (Keller, 1992; Wainwright et al., 1996; Liu et
al., 1997) These crustacean peptides are released from
the X-organ sinus gland complex which is located in the

medulla terminalis of the eyestalk. CHHs regulate the

* Corresponding author. Tel.: +81-3-5351-6534; fax: +81-3-53516488.
E-mail address: toshi@ori.u-tokyo.ac.jp (T. Watanabe).

level of glucose in the hemolymph. MIHs and MOIHs
inhibit the release of ecdysteroids from the Y-organ and
methyl farnesoate from the mandibular organ, respectively, and VIHs inhibit vitellogenesis. All of the above
peptides have been identified in decapod crustaceans,
except for one CHH isolated from the isopod Armadillidium vulgae (Martin et al., 1993).
In Arachnida, a CHH-family peptide of unknown
function was isolated from the venom of the black
widow spider Latrodectus mactans tredecimguttatus
(Gasparini et al., 1994).
In Insecta, CHH-family peptides have been identified
only in orthopteran insects. Ion-transport peptide (ITP),
which stimulates salt and water reabsorption and inhibits
acid secretion in the ileum, was isolated from the corpus
cardiacum (CC) of the locust Schistocerca gregaria, and
its cDNA cloned (Audsley et al., 1992; Meredith et al.,

1996). Another cDNA, presumably an alternative splicing product of the ITP gene, potentially encoding ITPlike peptide (ITP-L) was also cloned (Meredith et al.,
1996). Expression of ITP-L has not been detected at the
protein level, although transcripts were detected in a
wide range of tissues, and thus the biological role of ITP-

0965-1748/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 6 5 - 1 7 4 8 ( 9 9 ) 0 0 1 2 9 - 0

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H. Endo et al. / Insect Biochemistry and Molecular Biology 30 (2000) 355–361

L has not been elucidated (Macins et al., 1999). cDNAs
encoding ITP and ITP-L were also cloned in Locusta
migratoria (Macins et al., 1999).
It is an intriguing question whether insects, like crustaceans, make use of multiple CHH-family peptides to
regulate physiology, development and/or reproduction.
It also remains unknown whether the ITP is unique to
orthopterans, or common to other insect orders. To
address these questions, the authors set out to search for

cDNAs encoding CHH-family peptides in the silkworm
Bombyx mori (order Lepidoptera). Here we report the
first isolation of a cDNA encoding a CHH-family peptide in non-orthopteran insects.

2. Materials and methods
2.1. Experimental animals
Fourth instar B. mori larvae were purchased from
Kanebo Silk Elegance, Co., and grown at 25°C until sacrificed during fifth instar.
2.2. PCR amplification of B. mori genomic DNA
B. mori genomic DNA was generously provided by
Dr Haruhiko Fujiwara. The polymerase chain reaction
(PCR) reaction with degenerate oligonucleotide primers
was carried out as previously reported (Watanabe et al.,
1996), except that denatured genomic DNA was added
to 1 ng/µl.
2.3. Preparation of RNA
The brain [to which the CC and corpus allatum (CA)
were attached] and the suboesophageal, prothoracic and
mesothoracic ganglia, were collected from 200 fifth
instar larvae. The mesothoracic ganglion was only

occasionally included. The mixture of tissues was homogenized in 6 ml of Isogen (Wako), and total RNA was
prepared according to instructions from the manufacturer. Poly(A)+ RNA was prepared using Oligotex-dT30
super (Roche Japan).
2.4. 59 Rapid amplification of cDNA ends (RACE)
To 100 ng of the total RNA, 100 pmol of primer
CHHR (Fig. 1) was annealed, and cDNA was synthesized and poly(A)-tailed. The cDNA was used as a template in a PCR reaction with the following three primers:
a 39 primer (59-GCAGATTCTGTCGAGACG-39), and
two 59 primers (59-GAGTCGACTCGAGAATTCT17-39
and 59-GAGTCGACTCGAGAATTC-39). The product
of this reaction was subcloned in pCR2.1 (Invitrogen),
and analyzed for the nucleotide sequence.

2.5. Construction and screening of a cDNA library
cDNA was synthesized with 1 µg of the poly(A)+
RNA using a Time Saver cDNA synthesis kit
(Pharmacia Biotech), and ligated to the λZAPII vector
(Stratagene). The ligation product was packaged using
Gigapack Gold Packaging Extract (Stratagene).
The 59 RACE product was radio-labeled to be used
as a probe to screen the library. Procedures for library

screening have previously been described (Watanabe et
al., 1996).
2.6. Nucleotide sequence analysis
Nucleotide sequences of cDNA clones were determined, and conceptual translation performed as has been
described (Watanabe et al., 1996). In order to reveal
similarities between the BmCHHL protein sequence and
previously reported protein sequences, a homology
search was performed in ‘Protein All’ (version 3.0t84)
databases (including PIR and SWISS-PROT) of DNA
Databank of Japan using the FASTA program (Pearson
and Lipman, 1988).
2.7. Northern hybridization and reverse transcription
(RT)-PCR
Poly(A)+ RNA (0.5 µg) prepared as described above
was electrophoresed and blotted on to a nylon membrane, and hybridization was performed as previously
described (Watanabe et al., 1996). A HindIII fragment
of 515 bp [A622–A1137 nucleotide positions based on Fig.
2(A)] was used as the hybridization probe. After
hybridization the filter was washed under a low stringency condition (2×SSPE, 37°C).
For RT-PCR analysis, total RNA was isolated from

staged fifth instar larvae. An anti-sense probe, the
sequence of which was inverse-complementary to A247–
C264 [nucleotide positions based on Fig. 2(A)], was
annealed to the total RNA samples and cDNA was synthesized as previously described (Watanabe et al., 1996).
The cDNA samples were used as templates in a PCR
reaction (24 or 27 cycles of 94°C for 30 s, 55°C for 30 s
and 72°C for 30 s) using the following two primers: the
59 primer corresponding to C75–A95 [Fig. 2(A)] and the
39 primer inverse-complementary to A247–C264. PCR
products were run on 6% polyacrylamide gels, and
detected with ethidium bromide. A control experiment
in which the reverse transcriptase step was omitted was
also performed.
2.8. In situ hybridization
The above HindIII fragment was subcloned in the
pBluescript II SK vector (Stratagene). The digoxigenin11-dUTP (Boehringer-Mannheim) labeled anti-sense and

H. Endo et al. / Insect Biochemistry and Molecular Biology 30 (2000) 355–361

357

sense riboprobes were generated with T7 and T3 RNA
polymerases (PROMEGA), respectively. The latter
probe was used as a negative control. The whole mount
in situ hybridization was performed on the brain (with
attached CC and CA), and suboesophageal and prothoracic ganglia, from day 4 fifth instar larvae as previously
reported (Tsuzuki et al., 1997), except that non-fat dry
milk was used as the blocking agent instead of sheep
serum and that the methyl salicylate clarification step
was omitted.

phageal, prothoracic and mesothoracic ganglia. Two
positive clones were isolated. One of them [1796 bp
excluding the putative poly(A) tail] was analyzed for the
nucleotide sequence [Fig. 2(A)]. The clone contained a
sequence of 31 bp [T 231–C261 in Fig. 2(A)] that was
identical to the PCR product, and another [C1–C236 in
Fig. 2(A)] identical to a 39 part of the RACE product
(data not shown). As described below, this cDNA contained an open reading frame (ORF) for a CHH-family
peptide.

3. Results

3.2. Inferred structure of the encoded protein

3.1. Isolation of a cDNA encoding a CHH-family
peptide
As the first step in isolating a cDNA encoding a CHHfamily peptide, degenerate oligonucleotide PCR primers
were designed based on amino acid sequences of regions
conserved among CHH-family peptides [Fig. 1(A)]. The
amino acid sequences of six CHH-family peptides were
aligned to find highly conserved regions. Two regions
(Cys7–Asp12 and Cys23–Asn28 in the locust ITP) were
thereby selected, and two degenerate oligonucleotide primers (CHHF and CHHR) were designed by reverse
translation [Fig. 1(B)].
Genomic DNA prepared from B. mori was used as the
template in a PCR reaction using CHHF and CHHR.
Multiple bands were observed when the PCR product
was separated by polyacrylamide gel electrophoresis and
detected with ethidium bromide (data not shown). A

band of about 65 bp, the size expected for a CHH-family
peptide, was excised from the gel, and DNA eluted from
the gel was subjected to another round of PCR amplification. The product of the second PCR was subcloned
and analyzed for the nucleotide sequence. cDNA prepared from the brain (with CC and CA attached), and
the suboesophageal, prothoracic and mesothoracic ganglia, was also used as the PCR template, but a product of
the expected size was not detected.
One of the subclones analyzed contained a PCR product of 65 bp, including 34 bp derived from the two primers [Fig. 1(C)]. Its amino acid sequence, when conceptually translated in one of the reading frames, exhibited
significant similarities to CHH-family peptides such as
the ITP of the locust, S. gregaria (Meredith et al., 1996),
and a CHH of the crayfish, Orconectes limosus (Kegel
et al., 1991) [Fig. 1(C)]. Thus, the PCR product was
likely to be derived from mRNA encoding a CHH-family peptide.
Next, the nucleotide sequence (253 bp) of a 59 portion
of the cDNA was obtained by 59 RACE (data not
shown), and this RACE product was used as a probe to
screen a cDNA library (about 200,000 clones) of the
brain (with attached CC and CA), and the suboeso-

In the clone analyzed, an ORF of 110 amino acids
was found near the 59 end [Fig. 2(A)]. This conceptual

protein was named BmCHHL due to its sequence similarities to CHH-famaily peptides. In order to reveal similarities between the conceptual BmCHHL sequence and
previously reported protein sequences, a computer-aided
homology search was performed. Five proteins with the
highest similarity score were the ITP and ITP-L of the
locusts S. gregaria and L. migratoria (Meredith et al.,
1996; Macins et al., 1999), and the MOIH of the spider
crab Libinia emarginata (Liu et al., 1997).
A computer-aided analysis (Nielsen et al., 1997) suggests that the conceptual BmCHHL protein contains a
signal peptide with the likeliest signal cleavage site
between Ala23 and Leu24. The protein is likely to be
further processed between Arg35 and Ser36, since this
putative processing site is preceded by a dibasic
sequence (Arg34–Arg35), and the ITP precursor of S. gregaria is processed at the corresponding site (Meredith et
al., 1996). The three amino acids at the C-terminus of
the ORF (Gly108–Lys109–Arg110) are likely to constitute
the site of cleavage and amidation, as many CHH-family
peptides are amidated at the C-terminus (Keller, 1992).
Based on these results, we presume that the conceptual BmCHHL protein is a prepropeptide consisting of
a signal peptide (Met1–Ala23), a decapeptide (Leu24–
Glu33), a dibasic cleavage site (Arg34–Arg35), a peptide
of 72 amino acids (Ser36–Val107), and an amidation signal (Gly108–Arg110).
The putative mature BmCHHL (Ser36–Val107) displayed 63% identity to the S. gregaria and L. migratoria
ITP at the amino acid level [Fig. 2(B)]. Sequence similarities to the ITP-L of S. gregaria and L. migratoria
were slightly lower (60 and 61% identity, respectively).
The level of amino acid identity to crustacean CHH-family peptides was much lower (about 40% or less). In the
putative mature BmCHHL peptide, six cysteine residues
were found at positions conserved in the CHH family
[Fig. 2(B)], indicating that BmCHHL is a member of the
CHH family.

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H. Endo et al. / Insect Biochemistry and Molecular Biology 30 (2000) 355–361

Fig. 1. PCR amplification of a fragment of a cDNA encoding a CHH-family peptide in B. mori. (A) Designing PCR primers. Alignments of the
following CHH-family peptides are shown: a crab (Carcinus maenas) CHH (Kegel et al., 1989), a crayfish (Orconectes limosus) CHH (Kegel et
al., 1991), a lobster (Homarus americanus) CHH (Chang et al., 1990), a woodlice (Armadillium vulgae) CHH (Martin et al., 1993), a prawn
(Penaeus japonicus) CHH (Yang et al., 1995; Ohira et al., 1997) and a locust (Schistocerca gregaria) ITP (Meredith et al., 1996). These peptides
belong to ‘Type I’ subgroup of the CHH family (see Discussion). Amino acids that are identical to those in the ITP sequence are indicated by
dots, and conserved cysteine residues by asterisks. The two PCR primers (CHHF and CHHR) are derived from highly conserved regions (underlined).
(B) Nucleotide sequences of the two degenerate oligonucleotide primers. (C) Nucleotide sequence of a PCR product using B. mori genomic DNA
as the template. Sequences corresponding to the primers are underlined. Conceptual amino acid sequence in one reading frame is shown in the
one-letter representation above the respective codons. The derived sequence of 10 amino acids is aligned against the corresponding regions of the
locust ITP and the crayfish CHH. The amino acids that are identical to those in the B. mori sequence are indicated by dots.

3.3. Expression of BmCHHL
BmCHHL transcripts were detected using RT-PCR in
total RNA samples prepared from the brain (with CC
and CA attached), and the suboesophageal, prothoracic
and mesothoracic ganglia, of fifth instar larvae. After 27
cycles of amplification, PCR products were clearly
detected, and no significant change in the level of
BmCHHL was observed during the first eight days of
the fifth larval instar [Fig. 3(a)]. When the cycle was
reduced to 24, the signals observed were much weaker
and barely above the detection level, and even then no
significant difference in the signal level was seen among
staged samples (data not shown). BmCHHL transcripts
could not be detected in Northern analysis, presumably
due to the small number of cells expressing BmCHHL.
Cells expressing BmCHHL were identified by whole
mount in situ hybridization on the brain (with CA and
CC attached), and the suboesophageal and prothoracic

ganglia, dissected from day 4 fifth instar larvae (N=2).
BmCHHL expression could be detected only in a small
number of cells in the frontal area of the brain. The number of cells expressing BmCHHL was five per brain
hemisphere in one individual [Fig. 3(b)] and six in the
other (data not shown). In a control experiment with a
sense probe, these cells were not stained (data not
shown). Strong signals were also found in tracheal debris
attached to the brain and the ganglia. They are not due
to BmCHHL expression, but to non-specific hybridization of the probe, because similar signals were detected
in a control experiment using the sense probe (data
not shown).

4. Discussion
In the present study, a cDNA encoding a CHH-family
peptide was isolated in B. mori. BmCHHL exhibits

H. Endo et al. / Insect Biochemistry and Molecular Biology 30 (2000) 355–361

359

Fig. 2. Nucleotide sequence of the cDNA clone and deduced amino acid sequence of BmCHHL. (A) Nucleotide sequence of the cDNA clone
and conceptually translated sequence of an open reading frame of 110 amino acids. The amino acid sequence is shown in the one-letter representation
below the respective codons. The positions of the nucleotides and amino acids are indicated on the left of the sequences. The nucleotide sequence
identical to the PCR product (T 231–C261) is underlined. ‘u’ indicates the presumptive signal cleavage site (between Ala23 and Leu24). The putative
dibasic cleavage site (Arg34–Arg35) and amidation signal (Gly108–Arg110) are indicated in shadowed fonts, and the putative mature BmCHHL peptide
in bold fonts (Ser36–Val107). An asterisk indicates a termination codon. A sequence (A1776–A1781) similar to the consensus polyadenylation signal
(AATAAA) is underlined. The accession number of this sequence in the DDBJ/EMBL/GenBank nucleotide sequence databases is AB031074. (B)
FASTA alignment of the Schistocerca gregaria ITP (the upper sequence) and the putative mature BmCHHL peptide (lower). The numbers on the
left of the sequences indicate positions of amino acids in the ORFs. An asterisk indicates amino acid identity between the two sequences, and the
six conserved cysteine residues are underlined.

higher sequence similarities to the orthopteran ITPs and
ITP-Ls than the crustacean peptides of the CHH family.
This observation suggests that BmCHHL is a lepidopteran ortholog of ITP or ITP-L. BmCHHL is more likely
to be an ortholog of the former, because the locust ITP
precursors, like the BmCHHL precursors, contain putative amidation signals at the C-termini, whereas the ITP-

L precursors do not (Meredith et al., 1996; Macins et
al., 1999). Thus, the sequence analysis suggests the
possibility that BmCHHL is a lepidopteran ITP.
Identification of highly similar protein sequences in
two distant insect orders suggests that ITP or its homolog may be universally found in Insecta. The degenerate
oligonucleotide primer set used in this study, however,

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H. Endo et al. / Insect Biochemistry and Molecular Biology 30 (2000) 355–361

Fig. 3. Expression of BmCHHL transcripts. (a) Amplification of
BmCHHL transcripts by RT-PCR (27 cycles). A product of 190 bp
(arrowhead) was detected in RNA samples from staged fifth instar larvae [days 0–7 (lanes 1–8) after ecdysis], but not in a control sample
(lane C). (b) Identification of BmCHHL-expressing cells by whole
mount in situ hybridization. Expression was detected in five cells (per
hemisphere) in the frontal area of the brain (arrows) from a fifth instar
larva. In the hemisphere shown on the right, three expressing cells form
a tight cluster (arrowhead). Non-specific hybridization of the probe was
seen to tracheal debris (asterisk). Scale bar, 500 µm.

may not be universally applicable in Insecta, because a
search carried out in the fruitfly Drosophila melanogaster (order Diptera) was not successful.
CHH-family peptides in Crustacea are grouped into
two subgroups (Types I and II) based on the primary
structure of the mature peptide and the precursor (De
Kleijn and Van Herp, 1995; Yang et al., 1995). Type II
peptides have an insertion of a Gly residue near the Nterminus, and the precursors consist only of a signal peptide and a mature peptide. By contrast, the precursors of
Type I peptides comprise a signal peptide, a peptide of
unknown function (called CHH precursor-related
peptide) followed by a dibasic cleavage site, and the
mature hormone, which is often followed by an amidation signal. BmCHHL belongs to Type I, since it lacks
the Gly insertion, and its precursor structure resembles
that of Type I peptides. The locust ITP and ITP-L also
belong to Type I, based on the lack of the Gly insertion
and presence of a dibasic cleavage site preceding the
mature peptide, although the precursors do not contain
an apparent signal sequence.

In the present study we designed a set of primers for
cDNAs encoding Type II peptides, and conducted
searches in B. mori and D. melanogaster, but no such
cDNAs could be isolated. Thus, divergence of the CHH
family into the two subgroups has been observed only
in Crustacea.
The ITP in S. gregaria is synthesized in the brain and
released from the CC (Meredith et al., 1996; Macins et
al., 1999). In the present study, expression of BmCHHL
was also detected in the brain of B. mori larvae. Comparison of expression patterns of BmCHHL and the
locust ITP in the brain is not currently feasible, because
in situ hybridization or immunodetection analysis to
identify cells expressing the ITPs has not been reported.
BmCHHL expression was detected in a small number
(five or six per hemisphere) of cells in the frontal region
of the brain. The difference in the number of expressing
cells may be due to a slight difference in the developmental stage. In the brain of B. mori larvae, neurosecretory cells expressing peptide hormones such as prothoracicotropic hormone (Kawakami et al., 1990),
bombyxin (Mizoguchi et al., 1987; Iwami, 1990) and
eclosion hormone (Kono et al., 1990; Kamito et al.,
1992) have been identified. The locations of these cells
in the brain are distinct from that of the BmCHHL
expressing cells.

Acknowledgements
The authors are grateful to Dr Haruhiko Fujiwara for
provision of B. mori genomic DNA, and Dr Yoshiaki
Tanaka for provision of B. mori larvae. This work was
in part supported by a Grant-in-Aid for Scientific
Research (No. 08276204) to TW from the Ministry of
Education, Science, Sports and Culture of Japan.

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