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We recently purified a peptide with structural features similar to vertebrate gonadotropin-releasing hormone (GnRH) from the brain of Octopus vulgaris, cloned a cDNA encoding the precursor protein, and named it oct-GnRH. In the current study, we investigated the expression and distribution of oct-GnRH throughout the central nervous system (CNS) and peripheral organs of Octopus by in situ hybridization on the basis of the cDNA sequence and by immunohistochemistry using a specific antiserum against oct-GnRH. Oct-GnRH mRNA-expressing cell bodies were located in 10 of 19 lobes in the supraesophageal and subesophageal parts of the CNS. Several oct-GnRH-like immunoreactive fibers were seen in all the neuropils of the CNS lobes. The sites of oct-GnRH mRNA expression and the mature peptide distribution were consistent with each other as judged by in situ hybridization and immunohistochemistry. In addition, many immunoreactive fibers were distributed in peripheral organs such as the heart, the oviduct, and the oviducal gland. Modulatory effects of oct-GnRH on the contractions of the heart and the oviduct were demonstrated. The results suggested that, in the context of reproduction, oct-GnRH is a key peptide in the subpedunculate lobe and/or posterior olfactory lobe-optic gland-gonadal axis, an octopus analogue of the hypothalamo-hypophysial-gonadal axis. It may also act as a modulatory factor in controlling higher brain functions such as feeding, memory, movement, maturation, and autonomic functions

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The deadly box jellyˆsh (Sea Wasp, Habu-kurage in Japanese)Chiropsalmus quadrigatus Haeckel (Cubozoa) is distributed widely in the tropical Paciˆc region. In Japan, three fatal cases due to stings from this species have been reported o‹cially. We successfully isolated C. quadrigatus toxin-A (CqTX-A, 44 kDa), a major proteinaceous toxin, for theˆrst time, from the nematocysts of C. quadrigatus. CqTX-A showed lethal toxicity to crayˆsh when administered via intraperitoneal injection (LD 50 ＝80 mg W kg) and hemolytic activity toward 0.8% sheep red blood cells (ED 50 ＝ 160 ng W ml). Furthermore, we sequenced the cDNA encoding CqTX-A. The deduced amino acid sequence of CqTX-A (462 amino acids) showed 25.2% and 21.6% sequence similarity to Carybdea rastoni toxins (CrTXs) and Carybdea alata toxin-A (CrTX-A), respectively, which are Cubozoan jellyˆsh toxins.

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We reported that the common octopus, Octopus vulgaris, in common with vertebrates, possesses two members of the oxytocin/vasopressin superfamily: octopressin (OP) and cephalotocin (CT). This was the first observation of its kind in invertebrates. As OP and CT have different biological activities, the presence of specific receptors has been proposed. We cloned the cDNA of an orphan receptor from Octopus brain and found it to encode a polypeptide of 397 amino acids that displays sequences characteristic of G-protein coupled receptors. The orphan receptor showed high homology to receptors of the oxytocin/vasopressin superfamily and seemed to conserve the agonist-binding pocket common to the oxytocin and vasopressin receptors. Xenopus oocytes that express the orphan receptor responded to the application of CT by an induction of membrane Cl currents coupled to the inositol phosphate/Ca 2+ pathway. OP and the other members of the oxytocin/vasopressin superfamily did not activate this receptor. HPLC fractionation of the Octopus brain extract combined with an oocyte assay yielded a single substance that was identical to CT. On the basis of these results, we conclude that the cloned receptor is the CT receptor (CTR). Expression of CTR mRNA in Octopus was detected in the central and the peripheral nervous systems, the pancreas, the oviduct and the ovary. This receptor may mediate physiological functions of CT in Octopus such as neurotransmission, reproduction and metabolism.

The tachykinin (TK) and tachykinin‐related peptide (TKRP) family represent one of the largest peptide families in the animal kingdom and exert their actions via a subfamily of structurally related G‐protein‐coupled receptors. In this study, we have identified a novel TKRP receptor from the Octopus heart, oct‐TKRPR. oct‐TKRPR includes domains and motifs typical of G‐protein‐coupled receptors. Xenopus oocytes that expressed oct‐TKRPR, like TK and TKRP receptors, elicited an induction of membrane chloride currents coupled to the inositol phosphate/calcium pathway in response to Octopus TKRPs (oct‐TKRP I–VII) with moderate ligand selectivity. Substance P and Octopus salivary gland‐specific TK, oct‐TK‐I, completely failed to activate oct‐TKRPR, whereas a Substance P analog containing a C‐terminal Arg‐NH2 exhibited equipotent activation of oct‐TKRPs. These functional analyses prove that oct‐TKRPs, but not oct‐TK‐I, serve as endogenous functional ligands through oct‐TKRPR, although both of the family peptides were identified in a single species, and the importance of C‐terminal Arg‐NH2 in the specific recognition of TKRPs by TKRPR is conserved through evolutionary lineages of Octopus. Southern blotting of RT‐PCR products revealed that the oct‐TKRPR mRNA was widely distributed in the central and peripheral nervous systems plus several peripheral tissues. These results suggest multiple physiologic functions of oct‐TKRPs as neuropeptides both in the Octopus central nervous system and in peripheral tissues. This is the first report on functional discrimination between invertebrate TKRPs and salivary gland‐specific TKs.