Nitric oxide (NO) acts as a signalling molecule by activating soluble guanylate cyclase and causing accumulation of the second messenger cyclic guanosine 3′,5′-monophosphate (cGMP) in target cells. In order to detect the presence of NO-cGMP signalling pathway in the crayfish abdominal nervous system, accumulation of NO-induced cGMP was investigated by anti-cGMP immunochemistry. Some preparations were incubated in a high-K saline containing an inhibitor of cGMP-degrading phosphodiesterase, 3-isobutyl-1-methyxanthine (IBMX), to activate NO generating neurones, which could release NO in the ganglion, and then immunohistochemistry using an anti-cGMP antibody was performed. The other preparations were incubated in NO donor, sodium nitroprusside (SNP) saline containing IBMX before anti-cGMP immunohistochemistry was performed. The distribution of cGMP-like immunoreactive neurones in high-K treated preparations was similar to that of cGMP-like immunoreactive neurones in NO donor treated preparations. About 70-80 cell bodies and many neuronal branches in the neuropilar area of the ganglion were stained, although no neurones showed immunoreactivity unless preparations were activated by either high-K or the NO donor. Some of them were identical neurones, and they were intersegmental ascending interneurones and motor neurones. Sensory afferents that innervates hind gut showed strong cGMP-like immunoreactivity, although no mechanosensory afferents showed any immunoreactivity. These results strongly suggest the presence of an NO-cGMP signalling pathway that regulates neuronal events in the abdominal nervous system of the crayfish.

Rab proteins of the small G-protein superfamily are known to be involved in intracellular vesicle transport. Here, we describe the unique characteristics of a novel Rab protein, RABRP1 (Rab-Related Protein 1). The Drosophila RabRP1 gene is mainly transcribed in the eyes and testes, where the 3-kb and 1.5-kb mRNAs, respectively, are the predominant gene products. The amino-acid sequence deduced from the longer cDNA indicated that the C-terminal 1/3 of the sequence shares homology with Rab proteins, whereas the rest of the peptide shows no significant homology with any other proteins. Immunoblot analysis using antiserum against the Rab-domain indicated that the multiple translates (94 k, 53 k, 30 k, 29 k and 27 k) were expressed in the eyes. In contrast, only smaller peptides (30 k, 29 k and 27 k) were identified in the testes. Molecular phylogenetic analysis revealed that RABRP1 forms a subgroup with Dictiostelium RabE and mammalian Rab29, Rab32, Rab38 proteins, whose functions have not been identified yet. RABRP1 and its relatives were characterized by the amino acid substitution occurring in the conserved GTP-binding motifs. Immunohistochemical studies demonstrated that RABRP1 was localized on the subrhabdomeric cisternae of photoreceptor cells and on the pigment granules in photoreceptor and pigment cells in the retina. The expression of the dominant negative RABRP1 caused the abnormal accumulation of autophagosome-like vesicles. These data suggest that RABRP1 is involved in the lysosomal vesicle transport pathway, including the biogenesis or degradation of pigment granules.

Oxygenation function of annelid erythrocruorin (Er) is affected by Mg and Ca concentration in the blood. Four classes of responces may be encountered in different species: 1) Mg=Ca (equal effects), 2) Mg>Ca, 3) Mg<Ca and 4) no effect. In the marine polychaete Arenicola marina, at physiological pH and 20°C, Mg and Ca exerted almost equivalent effects in increasing oxygen affinity in the range of 1–200 mM. As measured from the slope of Δlog P_1/2 /Δlog [Cation] the effect of Mg was larger than that of Ca at the physiological concentration of respective ion (55 mM Mg; 10 mM Ca). The n_1/2 value was similar in the presence of both cations (pH 7.0) or higher for Mg (pH 7.6). In the terrestrial oligochaete Lumbricus terrestris, at the same condition, Ca was more effective than Mg, in raising oxygen affinity at both pHs, also at the physiological concentration (2–4 mM Mg; 8 mM Ca), and n_1/2 was similar for Mg and Ca (pH 7.0) or higher for Ca (pH 7.6). The Bohr factor, -[Δlog P_1/2 /ΔpH], is maintained its maximum value within the span of the physiological concentration of Mg in Arenicola. In Lumbricus, Ca can contribute to the increase of the Bohr factor at the physiological concentration, but Mg cannot contribute to it. These results reveal that Arenicola and Lumbricus belong to classes 1) and 3), respectively, and that the oxygenation function of both Ers may be controlled by effective utilization of the more dominant of the divalent cations Mg and Ca.

Some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, which corresponds to about 10⁷ times that found in seawater. The vanadium ions are stored in vacuoles located within vanadium-containing blood cells, vanadocytes. To investigate the phenomenon, an expressed sequence tag analysis (EST) of a cDNA library of Ascidia sydneiensis samea blood cells was carried out. Three hundred clones were obtained and sequenced by EST analysis. A similarity search revealed that 158 of the clones (52.7%) were known genes, and 142 of the clones (47.3%) did not have any similarity to genes registered in the SwissProt database. According to the functions of their genes the identified EST clones were categorized into eight types of clones; these consisted of genes; metal-related proteins (29 clones), signal transduction (22 clones), protein synthesis (17 clones), nuclear proteins (17 clones), cytoskeleton and motility (14 clones), energy conversion (3 clones), hypothetical proteins (11 clones), and others (45 clones). The ferritin homologue has a high degree of similarity to that of mammals; the iron-binding sites of ferritin are well conserved including His-118 which is important for capturing Fe² , also works as a ligand for VO² .

Recent findings have indicated that the Gr genes for putative gustatory receptors of Drosophila melanogaster are expressed in a spatially restricted pattern among chemosensilla on the labellum. However, evidence for a functional segregation among the chemosensilla is lacking. In this work, labellar chemosensilla were classified and numbered into three groups, L-, I- and S-type, based on their morphology. Electrophysiological responses to sugars and salt were recorded from all the accessible labellar chemosensilla by the tip-recording method. All the L-type sensilla gave good responses to sugars in terms of action potential firing rates, while the probability for successful recordings from the I-type and S-type sensilla was lower. No differences were found in the responses to sugars between chemosensilla belonging to the same type; however, dose-response curves for several different sugars varied among the sensilla types. The L-type sensilla gave the highest frequency of nerve responses to all the sugars. The I-type sensilla also responded to all the sugars but with a lower magnitude of firing rate than the L-type sensilla. The S-type sensilla gave a good response to sucrose, and lower responses to the other sugars. These results suggest that there might be variations in the expression level or pattern of multiple receptors for sugars among the three types of chemosensilla. The expression pattern of six Gr genes was examined using the Gal4/UAS-GFP system, and sensilla were identified according to the innervation pattern of each GFP-expressing taste cell. None of the spatial expression patterns of the six Gr genes corresponded to the sugar sensitivity differences we observed.

The queen discrimination abilities of laboratory-reared Camponotus japonicus workers were examined individually by allowing them to carry their nestmate larvae toward either the mother queen or an alien queen. Source colonies had been reared under controlled conditions from founding queens and maintained at small size (<=10 workers each). Fifty-two of fifty-four workers raised in these eight different colonies carried nestmate larvae to the mother queen, and never carried them to the alien queen. Most of them attended nestmate larvae but never alien larvae. These results clearly demonstrate that the tested workers discriminate the nestmate queen and larvae from non-nestmate conspecifics. The assay used in this study is novel and sensitive, and may be suitable for neuroethological and molecular studies of social discrimination mechanisms.

Shiro-uo (ice goby; teleost fish), Leucopsarion petersii, shows a unique cleavage pattern characterized by two tires of blastomeres at 8-cell stage, like that of echinoderm and amphibian embryo. Such a pattern is suitable to isolation and cell lineage experiments. In this study, cell lineage of germ-line was traced by histological observation and cell labelling experiment at the 8-cell stage. Primordial germ cells (PGCs) were first detected histologically at the 10-somite stage, and migrated to gonadal anlage at 10 days post-fertilization, through usual way described in other teleost species. When a single blastomere was labelled with tracer dye at 8-cell stage, both upper and lower tires generated labelled PGCs at gonadal anlage although upper tires occasionally. This result suggests that all blastomeres at the 8-cell stage have potential to produce PGCs in shiro-uo.

A study was conducted to investigate the serum progesterone (SP₄) profiles and duration of estrous cycles in the farmed Formosan sika deer (FSD; Cervus nippon taiouanus) during the major breeding season. Five parous, open and non-milking hinds were allotted to collect peripheral blood samples twice weekly for P₄ measurement by radioimmunoassay beginning at the initiation of the rutting season indicated by rutting behaviors of the sexually mature stags. The hinds were polyestrous as proved by cyclic changes of SP₄ levels. After the presumptive estrus shown by the lowest concentration of SP₄ (0.20±0.01 ng/ml), this ovarian hormone markedly elevated on day 7 of the cycle (1.67±0.11 ng/ml), reached plateau (3.15±0.16 ng/ml, P<0.01) during days 11 to 18, and then declined to the basal levels in the subsequent estrus. It is concluded that mean duration of the estrous cycle in FSD during the major rutting season is 19.3 days with a range of 17 to 21 days, and that the patterns of circulating progesterone profiles during the estrous cycles of the FSD are similar to those of other deer species so far investigated.

In eels, a CaCl₂ solution was infused into the pneumatic duct vein. Plasma Ca levels were significantly increased during 3 hr and were followed by significant raises in plasma calcitonin levels. These results strongly suggest that, in eels, direct raises in blood Ca levels by infusion of a high-Ca solution via blood vessels can accelerate the secretion of calcitonin from the ultimobranchial gland.

The present study examined diurnal cycles of oocyte development and maturation in the kyusen wrasse, Halichoeres poecilopterus, and investigated the sensitivity of oocytes to maturation-inducing hormone (MIH) and gonadotropic hormone (GTH). Female fish were sampled at fixed intervals throughout the day, revealing that final oocyte maturation and ovulation were completed by 6:00 hr, and that spawning occurred daily between 6:00 and 9:00 hr. In vitro experiments showed that the steroids 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) were equally potent and highly effective inducers of germinal vesicle breakdown (GVBD) in kyusen wrasse oocytes. Additionally, circulating levels of 17,20β-P and 20β-S increased around the time of GVBD and ovulation, suggesting that 17,20β-P and 20β-S act as MIHs in the kyusen wrasse. Moreover, In vitro experiments clearly showed that kyusen wrasse oocytes had a daily developmental cycle of GTH and MIH sensitivity, and that oocytes that completed vitellogenesis acquired GTH-induced maturational competence. An endogenous GTH surge likely occurs between 12:00 and 15:00 hr, and this daily pre-maturational GTH surge probably controls the diurnal maturation cycles of kyusen wrasse oocytes.

Growth hormone (GH), prolactin (PRL) and somatolactin (SL) are members of a pituitary hormone family that are believed to have evolved from a common ancestral gene by duplication and subsequent divergence. Since these hormones are found both in bony fish and cartilaginous fish, their ancestral form(s) should be present in the Agnatha. Thus, although there is no convincing evidence that the lamprey pituitary secretes GH or PRL, GH- and/or PRL-like immunoreactivity was examined in the pituitary of adult sea lampreys (Petromyzon marinus), using antibodies to GHs, PRLs and SL of mammalian and/or fish origins. Our initial attempt with ordinary immunohistochemical procedures failed to detect any positive reactions in the lamprey pituitary. Following the hydrated autoclave pretreatment of the sections, anti-salmon GH, anti-salmon PRL and anti-blue shark GH gave positive reactions in most cells distributed in the dorsal half of the proximal pars distalis. These results suggest that the material immunoreactive to those antibodies is related, to some extent, to GH/PRL, but enhancement of immunoreactivity to reveal this by the hydrated autoclave pretreatment of sections is needed due to low crossreactivity. The similarity of the topographic distributions within the pituitary between lampreys and teleosts suggests that lamprey GH/PRL-like cells are GH cells of the lamprey.

Immunohistochemical studies on the neural complex (neural gland, dorsal strand, and cerebral ganglion) of an ascidian, Halocynthia roretzi, were performed by using an antiserum against porcine ACTH. The antiserum recognized a considerable number of the cells scattered along the tubular structure of the dorsal strand and a few cells in the cerebral ganglion. Immunoelectron microscopic studies revealed that the ACTH-like substance resided within secretory granules with diameter of 300–500 nm. Furthermore, those ACTH-immunoreactive cells were demonstrated to be different from PRL-immunoreactive cells, the presence of which had previously been reported.

Mating behavior and the processes of insemination and sperm transfer in the ground beetle Carabus insulicola were analyzed. C. insulicola has elaborate genitalia, in which the strongly sclerotized male copulatory piece is inserted into the female vaginal appendix in copula. During mating, I observed pre-copulatory struggles of males and females, as well as delays in ejaculation, suggesting the presence of intersexual conflicts. Insemination was achieved with a spermatophore, which strongly adhered to the openings of the spermatheca, common oviduct, and vaginal appendix. The spermatophore dissolved after copulation, and sperm were transferred into the spermatheca within three hours after copulation. Sperm bundles were contained within the testes and spermatophores, but free spermatozoa were found in the spermatheca.