Invertebrate Neuroscience, 3,199-204 (1997) cDNA cloning of a mandibular organ inhibiting hormone from the spider crab Libinia emarginata LEI LIUt, HANS LAUFER*, PETER J. GOGARTEN and MINHUA WANG Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06268, USA t Present address: Department of Computer Science and Engineering, University of Connecticut, Storrs, CT 06268, USA ABSTRACT Mandibular organs (MO) produce a crustacean juvenile hormone, methyl famesoate (MF). MO activ- ity is negatively regulated by factors, called mandibular organ inhibiting hormones (MOIHs), from the crustacean sinus gland X-organ complex in the eyestalks. Three MOIHs have been isolated previously from the spider crab Libinia emar- gmata and are characterized as members of the crustacean hyperglycemic hormone (CHH) neuropeptide family. In the research reported here, a full length cDNA sequence of 972 bp of a MOIH was isolated by screening a cDNA library constructed from the eyestalks of Libinia emarginata. This cDNA sequence encodes a preprohormone peptide with 137 amino acid residues, including a 26-amino acid long signal peptide, a 34-amino acid long precursor peptide, a dibasic peptide, the full length of 72-amino acid long MOIH, and a tri-peptide Gly-Lys-Lys which designates the potential ami- dation site at the C-terminus of the mature peptide. KEY WORDS: crustacea; MOIH; mandibular organ; cDNA cloning; sinus gland; neuropeptide Introduction The crustacean mandibular organ (MO) produces methyl farnesoate (MF), which is the unepoxidated form of the insect juvenile hormone III (Laufer et al., 1987). It is involved in the regulation of crustacean morphogenesis and metamorphosis (Laufer et al., 1997, 1993b, 1993c, 1992; Sagi, et al., 1993).It has been shown that MF synthesis by the MO and the concen- tration of MF in the blood will increase after eyestalks are removed (Laufer et al., 1987). These results indi- cated that factors exist in eyestalks that negatively reg- ulate MF production which were then named mandibular organ inhibiting hormones (MOIHs). The X-organ sinus gland complex located in the eyestalk regulates and modulates various physiological processes in decapod Crustacea. This neuroendocrine system consists of neurons whose axon endings form a neurohemal organ, the sinus gland (SG). This gland secretes a variety of neurohormones including a family of large peptides, the CHH/MIH/VIH family (Keller, 1992). The crustacean hyperglycemic hormones (CHHs) are mainly involved in the regulation of car- bohydrate metabolism (Keller and Sedlmeier, 1987). The molt inhibiting hormones (MIHs) inhibit ecdysone production by Y-organs (Chang et al., 1990; Webster, 1991). Laufer et al. (1993a) also found that CHH and MIH inhibit MF production by MO. The vitellogenesis inhibiting hormones (VIHs) or gonad inhibiting hormones (GIHs) regulate the reproduction of crustaceans (Soyez et al., 1991). Recent studies on MOIHs have shown that MOIHs are new members of the CHH/MIH/VIH peptide family (Liu and Laufer, 1996; Liu et al., 1997; Wainwright et al., 1996). In previous studies (Liu and Laufer, 1996), using a two-step HPLC procedure, three peptides with MOIH activity were isolated from SG extracts of Libinia emar- g/nata. The complete amino acid sequence of one of the peptides has been elucidated (Liu et al., 1997). These peptides appear to be members of the family of CHH peptides. Wainwright et al. (1996) also reported two MOIH amino acid sequences from the crab Cancer pagurus which have high similarity to the MIH from the crab Carcinus maenas. Several cDNA cloning stud- ies on the members of CHH/MIH/VIH peptide family have revealed the preprohormonal structure of these peptides (Weidemann et al., 1989; Tensen et al., 1991a; Klein, 1992; De Kleijn, 1992, 1994a, 1994b, 1995; Sun, 1994). No cDNA data has previously been available for preproMOIH. The cDNA cloning for preproMOIH would give supplemental information about the signifi- cance of a precursor-related peptide in folding or pro- cessing and the expression pattern of MOIHs in crustaceans. In this paper, we report for the first time a full-length cDNA encoding the preprohormonal struc- ture of a MOIH. The structure is from the crab Libinia emar~nata. Included also is a study of the specificity of MOIH mRNA expression in several tissues of the crab. Corresponding author: Author for correspondence: H. Laufer. e-mail: laufer@uconnvm.uconn.edu