Guanylyl cyclase (GC) converts GTP into cGMP, an intracellular second messenger involved in a wide variety of cellular, developmental, and neuronal processes. Medaka fish, a small teleost, Oryzias latipes has been used to study organization and transcriptional regulation of the guanylyl cyclase gene family. Medaka fish expresses virtually all types of GCs found in mammals. Eight membrane GCs (OlGC1-7 and OlGC-R2) have been identified in medaka fish. OlGC1, OlGC2, and OlGC7 belong to the natriuretic peptide receptor subfamily. OlGC6 is a homologue of the mammalian GC-C, an enterotoxin/guanylin receptor, expressed predominantly in the intestine. OlGC3, OlGC4, OlGC5, and OlGC-R2 are members of the sensory organ-specific GC subfamily where they are differentially expressed in rods and cones of the retina and in the pineal organ. Complete genomic DNA sequences have been determined for the OlGC1 and OlGC6 genes. Their exon-intron organization is highly conserved between fish and mammals. The medaka fish genome also contains genes encoding α and β subunits of the cytoplasmic form of GC (soluble GC), which is activated by nitric oxide. The two subunit genes are closely linked in tandem in the order of α and β. Function of cis-regulatory regions of medaka fish GC genes have been investigated in transgenic medaka fish embryos and in mammalian cell lines. The upstream region of the α subunit gene of soluble GC appears to regulate expression of both α and β subunit genes, suggesting a mechanism of coordinated transcription of the two subunit genes. The upstream regions sufficient for the tissue-specific expression of sensory organ GCs also have been determined by transgenic analysis. Readiness for genetics and genetic manipulations in medaka fish would make this small fish a useful experimental system for studying the regulation of gene expression and roles of the guanylyl cyclase family in vertebrates.

The pond snail, Lymnaea stagnalis, is a useful preparation for analyzing the commonality between development and learning. To promote this analysis, the anatomical substrate should be provided upon which learning is superposed during development. Because we previously demonstrated that L. stagnalis change their ability of conditioned taste aversion (CTA) as a long-term memory from veliconcha embryos to immatures, we examined in the present study the numbers of cells and the volume of the buccal and cerebral ganglia in the snails at the critical developmental stages. The buccal and cerebral ganglia include the majority of neurons involved in the CTA. We found that the numbers of cells in these ganglia are almost saturated in the immatures, but the volumes of these ganglia still increase from the immatures to the adults. These results suggested that most of the cells indispensable to the CTA emerge at the immature stage, but that individual cells in the ganglia continue to enlarge even in adulthood. Furthermore, the key neuron for the CTA was found to mature at the immature stage. The present study provided the anatomical substrate upon the long-term CTA, by which snails can eat safe food in a wide territory.

Changes in morphology and cellular activity of the chloride cells in branchial and opercular epithelia were examined in tilapia, Oreochromis mossambicus, adapted to fresh water (FW), seawater (SW) and concentrated SW (180% SW). The tilapia are adaptable to a wide range of salinity, maintaining the plasma osmolality within physiological levels. Gill Na , K -ATPase activity was remarkably increased in response to elevated environmental salinity. Using immunocytochemical staining with an antiserum specific for α-subunit of Na , K -ATPase, chloride cells were detected on the afferent half of the filament epithelia. The size of immunoreactive chloride cells was twice larger in SW and four times larger in 180% SW than in FW. Confocal laser scanning microscopic observations revealed the frequent occurrence of chloride cell complexes under hypersaline conditions. By electron microscopy, a deeply invaginated apical crypt and well-developed tubular network were observed in chloride cells of SW- and 180% SW-adapted fish, indicating enhanced cellular activity. Chloride cells present in the opercular membrane were also developed in response to increased salinity. These findings suggest that highly activated chloride cells in branchial and opercular epithelia may be responsible for salt secretion in hyperosmotic environments. The excellent salinity tolerance of tilapia appears to be attributed to their ability to develop chloride cells in response to increased environmental salinity.

Retinal proteins in the cerebral ganglion of the ascidian, Halocynthia roretzi, were successfully visualized and their localization was determined by the time-resolved fluorescence difference imaging method. This visualizes retinal proteins in the tissue even though the concentration of the retinal proteins is low and there is considerable endogenous fluorescence. Heterogeneous retinal proteins in the same tissues could be distinguished by differential sensitivities to photobleaching, standardized by rhodopsin and retinochrome in octopus retina. Retinal proteins in the ocellus of ascidian larva, which is composed of only about 20 photoreceptor cells, were successfully visualized.

Retinal proteins in the cerebral ganglion of adult Halocynthia roretzi, localized mainly at the surface of the anterodorsal root and the posterodorsal root. In the cross sections along the anteroposterio axis of the cerebral ganglion, the cells bearing retinal proteins were found in the peripheral cellular cortex mainly at the dorsal surface. Close localization of retinal protein and GnRH bearing cells suggests that retinal protein may trigger the biological clock for spawning in this ascidian.

Intravenous administration of eel angiotensin II (eANG II), histamine (HA), serotonin (5-HT), acetylcholine (ACh) or carbachol (CCh), mammalian substance P (mSP) and isoproterenol (β-adrenoceptor agonist) enhanced drinking rate in the seawater eels. The dipsogenic effects of HA and 5-HT seem to be due to ANG II synthesis, because these effects were completely blocked by captopril, an inhibitor of angiotensin converting enzyme (ACE). Captopril blocked eANG I effect, but not eANG II effect, suggesting existence of ACE in seawater eels. 800 μl Hemorrhage also enhanced water intake, and this effect was completely blocked by captopril. Therefore, it is likely that blood withdrawal stimulates renin-angiotensin system (RAS) in seawater eels. Effects of ACh, CCh and mSP were not inhibited by captopril, suggesting separate action of these regulators from ANG II synthesis. Isoproterenol action was partially inhibited by captopril, suggesting existence of some β-adrenoceptors other than the RAS. On the other hand, intravenous eel atrial natriuretic peptide (eANP), arginine vasotocin (AVT), human vasoactive intestinal peptide (hVIP), mammalian bradykinin (mBK), eel intestinal pentapeptide (EIPP), cholecystokinin (CCK-8), and phenylephrine (α-adrenoceptor agonist) depressed the drinking rate. In the presence of mBK, HA and 5-HT enhanced water intake similarly as in the absence of mBK. Plasma hyperosmolarity also reduced drinking. Although the in vivo system is so complicated and many regulators are involved in the drinking behavior, a possible regulatory mechanisms are proposed. Compared to mammalian results, eels seem to be a suitable model for anlayzing drinking mechanisms in vertebrates.

Gastrolith matrix protein (GAMP) is a novel protein purified from gastroliths of the crayfish, Procambarus clarkii, and has been suggested to be associated with calcium carbonate deposition. In the present study, a specific antibody against GAMP was raised and distribution of GAMP immunoreactivity was studied in crayfish gastrolith and exoskeleton. Localization of calcium carbonate in the exoskeleton, determined by silver nitrate staining and energy dispersive X-ray microanalysis, was compared with that of GAMP immunoreactivity. Crystalline forms of calcium carbonate were also determined. SDS-PAGE and Western blotting revealed that the gastrolith extract contained a major band which was GAMP-immunopositive and showed the same mobility as that of purified GAMP. The exoskeleton extract showed smear bands, but no immunoreaction was detected. By using immunohistochemistry, the anti-GAMP antiserum reacted almost uniformly with gastrolith matrix irrespective of gastrolith size. Epithelial cells of the gastrolith disc were also immunopositive. In the exoskeleton, exocuticle was strongly GAMP-immunopositive, whereas the endocuticle and membrane layer was slightly positive. The epicuticle was immunonegative. Calcium carbonate was detected in exocuticle, endocuticle and a part of the membrane layer, but not in the epicuticle. Thus, the distribution of GAMP immunoreactivity roughly corresponded with that of calcium carbonate. X-ray diffraction study showed that calcium carbonate in the gastrolith was amorphous, whereas that in the exoskeleton consisted of calcite crystals. These data indicate that a GAMP-immunoreactive substance is commonly distributed in the mineralized tissues of the crayfish, but may exist in a chemically different form in other tissues.

Rana tadpoles are known to have a left-handed turning bias whereas Xenopus larvae lack such a preference in turning direction. Since Rana tadpoles have a single, external, left-handed spiracle, we previously suggested that a turning bias in tadpoles may be obligatorily linked to that external asymmetry. We have tested this idea by examining turning bias in the startle response of Microhyla ornata tadpoles. Microhyla tadpoles are, like Xenopus, externally symmetrical, but phylogenetically they are more closely related to Rana.

Individual Microhyla tadpoles were startled by a solenoid-driven plunger that sent a shock wave up through the bottom of a container holding each tadpole. High speed videography (250 frames per second) was used to witness the tadpole's response.

Microhyla tadpoles show no turning bias during the first few days post-hatching, when they are very small. However, they develop a left-handed turning bias while still in the earliest free-swimming stage (Gosner stage 25) and that bias persists through stage 41. At stage 42, after forelimbs emerge, the laterality in startle responses fades away.

Since Microhyla larvae are externally symmetrical, yet preferentially turn to the left, we can reject the hypothesis that a turning bias in tadpoles is obligated by external morphological asymmetry. An alternative working hypothesis, given the limited taxa that have been examined to date, is that handedness in tadpoles is phylogenetically conserved and independent of spiracle position in tadpoles.

Low molecular signalling molecules such as cAMP and cGMP are expected to have important functions in early morphogenetic processes in animal development. We examined the effect of 8-bromo-cyclic GMP (Br-cGMP) on Xenopus embryogenesis, using 8-bromo-cyclic AMP (Br-cAMP) as a reference. When Xenopus gastrulae were cultured in the medium which contained these analogues, their development was affected in specific and dosage-dependent manners: While Br-cAMP induced anomaly only in head part (swelling of myelencephalon with enlarged ventricle), Br-cGMP induced shortening in body length often accompanied by bending of the cephalo-caudal axis. In embryos treated with Br-cGMP at a high dose, cellular movement was inhibited as revealed by SEM and this resulted in the formation of tadpoles with unclosed yolk plug. Br-cGMP at lower doses induced severe inhibition of the development of notochord and muscles. Since HPLC analyses revealed that both analogues were uptaken into embryonic cells, we assumed that the morphological effects observed were induced by the interference of the normal functioning of cGMP and cAMP, respectively, by Br-cGMP and Br-cAMP. Based on the results obtained, we assume that while cGMP is involved mainly in the differentiation of mesodermal structures, especially in formation of notochord and muscles, cAMP is involved mainly in the differentiation of neural structures.

The development of serum-free and chemically defined media remains an important mission in the in vitro research of cells. The present study describes an improved serum-free medium that supports the proliferation and differentiation of chick embryonic primary skeletal myogenic cells, a cell type which has long been utilized for the study of cell differentiation.

We show that serum can be replaced with transferrin, insulin, serum albumin, and fibroblast growth factor-2 as supplements to Dulbecco's modified Eagle minimum essential medium, with no loss in the ability of the medium to support proliferation and differentiation of myogenic cells. This medium has several additional advantages over serum-supplemented medium in that it also suppresses the proliferation of contaminating fibroblasts, and may allow the sensitive evaluation of the effects of various humoral factors on myogenic cells. We believe this medium will prove useful to the primary culture of chick myogenic cells.

Reproductive strategies, growth rates, survival, and their correlates with life-history traits are studied in the Savi's pine vole (Microtus savii) under both natural and laboratory conditions. 119 reproductive events were monitored, 50 out of which came from wild-born females and 69 came from captive-born females. Fertility rates were nearly identical in both males and females. Mean age at first parturition of females averaged 72.6±12.1 days, and the mean time elapsed between the constitution of a given pair and the first partum was 31.5±11.5 days. The litter size of captive-bred females (2.49± 0.7; range: 1 to 4) was nearly identical to that of the free-ranging females (2.5±1.1). The newborn sex ratio did not significantly deviate from equality. Litter size was independent on season and on number of times that a given pair reproduced. Nevertheless, litter size tended to be inversely correlated with the length of the interpartum time, and was positively correlated with parental age. The size of the litter from which the mother and the father of a given litter were generated tended to be positively correlated with the litter size produced by them, but only the relationship relative to males attained statistical significance. Females first gave birth to young at an age between 65 and 99 days. Female puberty was attained at 50.2±13.1 days, almost the same as male puberty (x = 47.2 ± 11.1 days). Females gave birth to young on average once every 29.4 days. The interpartum time was not influenced by either female age or male age, but was shorter in spring and winter (wet seasons in the Mediterranean bioclimate) than in summer and autumn (dry seasons). Mean weight of the juveniles was negatively correlated with the litter size. The facts that litter size was constituted by a small number of young and that the mean gestation time was relatively prolonged suggest that Microtus savii is a K strategist within the Microtinae, whereas these rodents are well known to be usually extreme r-strategists. In general terms, our data are consistent with those coming from other fossorial voles, and suggest that fossorial voles (i) give birth to a lesser number of young per litter than above-ground active voles, and (ii) have an age at puberty delayed in comparison with that of above-ground active voles.

The seasonal migration of the hagfish, Eptatretus burgeri, was studied in the sea near the Misaki Marine Biological Station of University of Tokyo during the period from October 1970 to October 1975. The hagfish was caught by a cylindrical trap (80 cm in length, 15 cm in diameter) with a funnel-shaped one way entry at both ends. Sardines were used for baits. The hagfish were found in water of 6 to 10 m depths in Koajiro Bay and Moroiso Bay near the Station from mid October to mid July of the following year, but they were absent from the bays at other times. On the other hand, they were found throughout the year in offshore water of 50 m depth, about 1,600 m west of the Station. Two hundred and nine hagfish caught in water of 50 m depth in October and November were marked by making a small triangle cut on the tail fin and releasing them at the original place of capture. Sixty five marked hagfish in total (31.1%) were recaptured in Koajiro Bay and Moroiso Bay. Among 1,323 hagfish marked in Koajiro Bay in April, May and June, 23 marked hagfish (1.7%) were recaptured in water of 50 and 100 m depths. The mean total lengths of the hagfish collected in Koajiro Bay and water of 50 m depth during November through April were 43.3± 0.7 cm (± SEM, n=1,796), and 34.3± 0.9 cm (± SEM, n=689), respectively. Data on monthly collections of the hagfish in both Koajiro Bay and water of 50 m depth indicated that when they migrate from shallow to deep water and the reverse, larger hagfish more than 39 cm in total length move first being followed by smaller ones, but many hagfish less than 35 cm in length remain in water of 50 m depth throughout the year. We failed to collect either fertilized eggs or juveniles by net sweeping of the bottom of water of 40 to 110 m depth. Furthermore, we could not find hagfish less than 20 cm in either shallow water or water of 50 m depth. It seems that the spawning ground of E. burgeri may not be at the 50 m depth but at a deeper place than 100 m depth. Finally, the present study demonstrated directly the seasonal migration of the hagfish, E. burgeri, between shallow and deeper waters of Sagami Bay by the marking experiments.

Seasonal development of gonads was studied in the hagfish, Eptatretus burgeri, caught near the Misaki Marine Biological Station facing the Sagami Bay of the Pacific coast of Japan, during periods from October 1971 to March 1973 and from November 1977 to December 1978. The hagfish were collected at water depths of 6 to 10 m (shallow water) in Koajiro Bay close to the Station or water depths of 50 and 100 m (deep water), about 1,600 and 4.800 m west to the Station. They were collected monthly from both shallow and deep waters, but they could not be collected from shallow water during warmer summer months (July-September) due to seasonal migration. The mean total lengths of the females collected in both shallow and deep waters during January to April were 40.7± 0.4 cm (± SEM; n=317) and 34.5± 0.3 cm (± SEM; n=556), respectively. In both locations, developing eggs longer than 5 mm were found in females larger than 39 cm in total length. There was no difference in the annual growth curves of developing eggs between shallow and deep water: the sizes of the developing eggs were the smallest in October, and was the largest in September. There was no apparent difference in testicular development between two locations, so these data were combined. Testicular development occurs in males larger than 38 cm in total length. The testis weight was the heaviest in July, and was the lightest in September. In autumn and winter, most testicular follicles contained only spermatogonia. In spring, follicles containing spermatocytes increased. Follicles with spermatids or maturing sperms were relatively abundant in summer. Although egg deposition is supposed to occur in September and/or early October somewhere in deep water, both testis weight and spermatogenesis were minimal at this time. Why testis development is lowest at the time when females presumably deposit eggs is discussed in relation to the question of the location of the spawning ground.

The embryos of Diplosoma similis are brooded within the thick walled tunic of the colony in isolation from the symbiotic algae Prochloron sp., which are in the cloacal cavity of the parent colony. Prior to the spawning, the plant rake, a tassel-like structure, protrudes from the postero-dorsal end of the larval trunk and extends into the cloacal cavity. The algal cells in the cloacal cavity adhere to the plant rake. When the larvae are spawned, the trunk tunic extends posteriorly and forms a pouch entirely covering the plant rake. The algal cells are packed in the pouch (algal pouch) enveloping the basal part of the tail. The cell density of the algae in the pouch is much higher than that in the colony, suggesting that the plant rake functions for gathering and concentrating the symbionts into the algal pouch. In the course of metamorphosis, the algal pouch expands and turns into the cloacal cavity of the young colony. The high density of algal cells in the pouch would ensure that the young colony possesses the symbiotic algae of appropriate cell density in the cloacal cavity, and the colony can sufficiently receive benefits from the symbionts just after the settlement.

The subtidal zones near the shore are inhabited by many small invertebrates, including benthos and plankton. To characterize their emergence in the water column with regards to day/night, tidal, and lunar cycles, field investigations were carried out at the subtropical island (Iriomote-jima), Okinawa Prefecture. By use of two impeller pumps installed in both surface and bottom waters, invertebrates were sampled continuously for 23 days. Although most patterns were much the same between the surface and bottom waters, the abundance of animals was different between the two depths. A notable feature was that nocturnal patterns were very dominant. More than half of these patterns were not affected by the tidal cycle at all. In contrast, the pattern of Ericthonius sp. (Amphipoda) showed a clear synchrony with the nocturnal tide. Other patterns were weakly modified by the nocturnal tide (e.g. Propallene longiceps; Pantopoda). A pattern coincided with the lunar phase was only seen in Vargula hilgendorfii (Myodocopida). Most arthropods would hide in the bottom substrate, or would swarm under or near the lower pump in the daytime, and they would disperse in the water column at night. A variety in the synchrony with nocturnal tides strongly supports a notion that the tidal rhythm is only a variation of the day/night rhythm, rather than the hypothesis that both rhythms are present simultaneously in an animal. Statistical methods (autocorrelogram and periodogram) are used to demonstrate the tide-correlated component of the activity. However, these methods are not sufficient for this purpose; visual inspection of the pattern is very important.

Age in 96 individuals of a tropical frog Rana nigrovittata from Thailand was skeleto-chronologically investigated. Hematoxylinophilic lines interpreted as lines of arrested growth (LAGs) were observed clearly in the periosteal bone suggesting that this technique can be used to estimate the age of frogs from tropical Asia. The maximum age observed was nine years in males and six in females, but age structure did not differ significantly between the sexes. In the same age class, males were significantly larger than females.

A new semiterrestrial tardigrade species of the genus Calcarobiotus is described from the Imperial Palace of Japan as the third species of the genus which has been previously known only from South Africa. The new species, Calcarobiotus imperialis, is closely related to C. occultus Dastych, but can be distinguished from the latter mainly by the absence of a spine on the tip of each egg process, and by having larger pores on egg surface, a narrower buccal tube, and larger claws.

Nemertellina minuta Friedrich, 1935, sensu Yamaoka, 1940, from Akkeshi Bay, Japan, is fully redescribed as a new species, Nemertellina yamaokai. The main characters distinguishing this taxon from N. minuta are the presence of two pairs of cephalic furrows and cephalic glands, and the absence of dorsoventral muscles from the intestinal region.