Insect Biochemistry and Molecular Biology 30 2000 355–361 www.elsevier.comlocateibmb
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 crus- taceans, 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 hypergly-
cemic hormone Keller, 1992. CHH-family peptides consist of 69–78 amino acids including six conserved
cysteine residues which form three intramolecular disul- fide 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: +
81-3-5351- 6488.
E-mail address: toshiori.u-tokyo.ac.jp T. Watanabe.
0965-174800 - 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
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, respect- ively, and VIHs inhibit vitellogenesis. All of the above
peptides have been identified in decapod crustaceans, except for one CHH isolated from the isopod Armadillid-
ium 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 splic- ing product of the ITP gene, potentially encoding ITP-
like 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-
356 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 crus-
taceans, make use of multiple CHH-family peptides to regulate physiology, development andor 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 pep-
tide in non-orthopteran insects.
2. Materials and methods
