Multiple occurrences of spiggin genes in sticklebacks
Introduction
The threespine stickleback (Gasterosteus aculeatus) is known to have a unique reproductive mode with various interesting behaviors (Tinbergen, 1951). For evolutionary biologists, one of the most intriguing stickleback reproductive behaviors is nest-building since diverse reproductive modes are observed in closely related species (Limbaugh, 1962, Wootton, 1976, Akagawa and Okiyama, 1993, Akagawa et al., 2004). To build nests from plant materials, male threespine sticklebacks synthesize and secrete a glue-like protein (Wootton, 1976). This protein, named “spiggin”, is produced in the hypertrophied kidney only during the breeding season (De Ruiter and Mein, 1982, Jakobsson et al., 1999).
Spiggin can be considered to be a “key character” for understanding the molecular mechanism that underlies the evolution of nest-building behavior because the glue-like protein is essential for nest-building. In addition, spiggin is reported to be induced by some kinds of androgen, such as 11-ketotestosterone, which has earned it attention as an important biomarker for the field of environmental science (Katsiadaki et al., 2002).
Recently, it has been demonstrated that this glue-like protein is a protein complex assembled from three distinct subunits, which arise by alternative splicing from a single gene (Jones et al., 2001). More recently, however, the existence of two different types of spiggin cDNAs was suggested although the details are not clear (Kawasaki et al., 2003). These reports are in conflict as to whether the gene encoding spiggin exists as a single copy or multiple copies, although it seems plausible that spiggin is encoded by multiple copies since the glue-like protein is required in large quantities to make the nest in the breeding season.
To resolve the contradiction in the understanding of this gene, we isolated the gene from a mature male of the “Pacific Ocean group” (Higuchi and Goto, 1996) of the threespine stickleback (G. aculeatus) collected in Hokkaido, Japan, and consequently cloned many more than two types of spiggin cDNAs. A detailed inspection of the results revealed that the spiggin genes constituted a multi-gene family and extensive alternative splicing occurred in these genes. To examine the evolutionary relationships of spiggin genes, the cDNA sequences were subjected to a phylogenetic analysis with related sequences including spiggin genes of ninespine stickleback (Pungitius pungitius) and putative homologues of the spiggin gene isolated from the genome databases of zebrafish (Danio rerio), torafugu (Takifugu rubripes), and spotted green pufferfish (Tetraodon nigroviridis).
Section snippets
Animals
Adults of the Pacific Ocean group of G. aculeatus were collected in eastern Hokkaido, Japan, transferred to the laboratory, and then kept in an aquarium (60 × 30 × 36 cm). They were maintained at 17 °C with a 12 L:12 D cycle and fed once a day with frozen bloodworms (larval chironomids). Water salinity was maintained at 1–3‰. Mature males with nuptial coloration that had begun to make nests were anaesthetized with ice, and at autopsy, kidneys were dissected and fixed in RNAlater ™ (Takara Bio Inc.,
Cloning of the spiggin gene
Seven types of full-length spiggin cDNA sequences were obtained by 3′ and 5′-RACE (spg1.1, 1.2, 1.3, 1.4, 2, 3, and 4; DDBJ/EMBL/GenBank accession numbers, AB221477–AB221483AB221477AB221478AB221479AB221480AB221481AB221482AB221483; Fig. 1). These full-length cDNAs ranged from 2252 to 3734 base pairs (bp); the lengths in the 5′ untranslated region (UTR) were the same (31 bp), but different in the rest (Table 2).
Nucleotide BLAST searches with these sequences found the highest sequence identity
Conclusions
In this study, we cloned four genes, including three new genes, most probably encoding the glue-like protein spiggin in threespine sticklebacks. This demonstrates that this protein is encoded by a multi-gene family. The multiple copies of the gene may contribute to the effective synthesis of large amounts of glue-like protein to make nests in the breeding season. We also found four splicing variants of one of the genes. In addition, considerable diversity was observed in the obtained sequences,
Acknowledgements
We thank H. Takahashi and T. Kitamura for samples, Y. Hashiguchi, H. Takeshima, and T. Mizuta for their valuable discussion and suggestions, and S. DeVaney for reviewing the manuscript. The manuscript benefited from the comments of the associate editor and two anonymous reviewers. This study was supported in part by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists (11450) and Grants-in-Aid from the same society (12NP0201 and 16057201).
References (34)
- et al.
Testosterone-dependent transformation of nephronic tubule cells into serous and mucous gland cells in stickleback kidneys in vivo and in vitro
Gen. Comp. Endocrinol.
(1982) - et al.
Molecular cloning of human intestinal mucin (MUC2) cDNA
J. Biol. Chem.
(1994) - et al.
Similarities of integumentary mucin B.1 from Xenopus laevis and prepro-von Willebrand factor at their amino-terminal regions
J. Biol. Chem.
(1997) Molecular cloning and characterization of spiggin
J. Biol. Chem.
(2001)- et al.
The potential of the three-spined stickleback (Gasterosteus aculeatus L.) as a combined biomarker for oestrogens and androgens in European waters
Mar. Environ. Res.
(2002) Cloning and expression of the gene of hemocytin, an insect humoral lectin which is homologous with the mammalian von Willebrand factor
Biochim. Biophys. Acta
(1995)Molecular cloning of the amino-terminal region of a rat MUC 2 mucin gene homologue
J. Biol. Chem.
(1994)- et al.
Identification of the half-cystine residues in porcine submaxillary mucin critical for multimerization through the D-domains
J. Biol. Chem.
(1998) - et al.
Alternative pre-mRNA splicing: the logic of combinatorial control
Trends Biochem. Sci.
(2000) - et al.
Alternative male mating tactics in Hypoptychus dybowskii (Gasterosteiformes): territoriality, body size and nuptial colouration
Jpn. J. Ichthyol.
(1993)
Reproductive behaviour of Japanese tubesnout, Aulichthys japonicus (Gasterosteiformes), in the natural habitat compared with relatives
Environ. Biol. Fisches
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs
Nucleic Acids Res.
Mucin biophysics
Annu. Rev. Physiol.
Improved prediction of signal peptides: signalp 3.0
J. Mol. Biol.
GeneWise and Genomewise
Genome Res.
The role of N-glycosylation in the stability, trafficking and GABA-uptake of GABA-transporter 1
FEBS J.
Otogelin: a glycoprotein specific to the acellular membranes of the inner ear
Proc. Natl. Acad. Sci. U. S. A.
Cited by (14)
2.23 Recombinant proteins as emerging biomaterials
2017, Comprehensive Biomaterials IIFish toxicogenomics
2008, Advances in Experimental BiologyCitation Excerpt :Spiggin production is under the control of endogenous androgens and spiggin has also shown to be sensitive for detecting a range of (anti)androgenic xenobiotics (Hahlbeck et al., 2004; Katsiadaki et al., 2002a, 2002b, 2006). Multiple spiggin gene transcripts have been cloned (Jones et al., 2001b; Kawahara and Nishida, 2006; Kawasaki et al., 2003) and, so far, have been shown to be highly responsive to androgens (Nagae et al., 2007). A wide range of other genes representing central nodes in endocrine networks have been employed to measure exposure to environmental oestrogens, androgens, thyroid disruptors and other EACs in fish.
A new ELISA for the three-spined stickleback (Gasterosteus aculeatus L.) spiggin, using antibodies against synthetic peptide
2008, Comparative Biochemistry and Physiology - C Toxicology and PharmacologyThe molecular evolution of spiggin nesting glue in sticklebacks
2015, Molecular Ecology