Zoological Science

The ascidians occupy a crucial phylogenetic position between invertebrates and vertebrates. Advances in molecular biological techniques and molecular cloning have enabled studies of molecular mechanisms to be more and more accessible in ascidians. Recently, an increasing number of ascidian genes have been cloned. In this review, we summarize the molecular features and expression profiles of all ascidian genes published to date and describe how and why they were cloned. Such information not only is valuable for understanding the recent advancements in ascidian molecular biology but also to bring out the importance of ascidians as an experimental system for a variety of fields in biology.

The direct effect of K⁺ on xanthophores of the medaka, Oryzias latipes, was studied using denervated scale preparations and cultured cells. At concentrations of 30 mM or higher, K⁺ ions caused almost complete aggregation of pigment in denervated xanthophores. The withdrawal of divalent cations from the perfusing solution, or the addition of Ca²⁺ channel blocker to the medium, made the cells unresponsive to K⁺. When Fluo 3-loaded xanthophores were stimulated by K⁺, a temporary increase in the fluorescence intensity was observed. Experimental elevation of cytosolic Ca²⁺ concentration induced aggregation of pigment in denervated xanthophores pretreated with A23187. Forskolin, an activator of adenylate cyclase, inhibited the K⁺-induced aggregation of pigment in a dose-dependent manner, whereas W-7, a calmodulin antagonist, did not. From these results, it is likely that membrane depolarization caused by elevated K⁺concentration in the perfusing medium may be accompanied by Ca²⁺ entry into the cytoplasm through voltage-dependent Ca²⁺ channels present in the xanthophore membrane. This would result in inhibition of adenylate cyclase activity, since it is well established that decreases in intracellular cAMP level induce pigment aggregation in fish chromatophores.

Drosophila rhodopsin consists of an apoprotein, opsin, and a chromophore, 11-cis-3-hydroxyretinal. When flies are raised in carotenoid-free medium, rhodopsin transport is blocked between the rough endoplasmic reticulum (rER) and the Golgi body, and immature opsin accumulates in the rER. To elucidate how carotenoid controls the protein transport between these organelles, we investigated the morphological and molecular-biological defects caused by carotenoid deprivation. The results demonstrated that this deprivation causes rER elongation and a shortening of the rhabdomeric microvilli, whereas the density and morphological features of the Golgi body are not significantly affected. Moreover, the content of rhabdomeric proteins other than rhodopsin is not decreased by the deprivation. These results indicate that carotenoid deprivation does not affect the overall traffic of vesicle transport, but selectively suppresses rhodopsin transport between the rER and the Golgi body. We further investigated the gene and protein expression of Rab proteins that control the vesicle transport between cell organelles. The results demonstrated that neither gene nor protein expression of RAB1 and RAB2 is influenced by carotenoid deprivation, which also supports the above conclusion.

The responses of cultured antennal neurons of blowflies to odorants, ethyl acetate and isovaleric acid, were measured using fura-2, a calcium-sensitive fluorescent dye. Both odorants induced increases in cytosolic free calcium concentration ([Car²⁺]_i) in the neurons. The peak [Ca²⁺]_i was 1.1 to 7.5 times as high as [Ca²⁺]_i of the resting neurons. The increase in [Ca²⁺]_i occurred within 10 sec after exposure to the odorants in most neurons. It, however, occurred up to 110 sec after the odorant exposure in some neurons. As [Ca²⁺]_i increase was diminished upon removing Ca²⁺ from saline, [Ca²⁺]_i increase was assumed to be due to Ca²⁺influx. [Ca²⁺]_i increase was also induced by introducing forskolin and membrane-permeable analogues of cyclic AMP and GMP.

A novel bioactive peptide was isolated from the brain of the Japanese crucian carp, Carassius auratus langsdorfii, by using the intestine of the fish as the bioassay system. The primary structure of the peptide was determined to be KPRPHQFIGLMamide. The sequence was found to be highly homologous to the fish substance P-related peptides isolated from Atlantic cod and rainbow trout. We designated the peptide Carassius Substance P (Caa-SP). Caa-SP was identical with the peptide whose presence was inferred from a cDNA analysis by other investigators. Caa-SP exerted an excitatory effect on visceral muscle tissues of fish with the threshold at about 3 × 10⁻⁹ M. The peptide enhanced the ERG b-wave, depolarized the dark membrane potential and slightly decreased the amplitude of S-potentials of the L-type horizontal cells in the carp retina.

The effects of Joro spider toxin (JSTX), a specific glutamate antagonist, on the adult heart of the isopod crustacean Ligia exotica were examined. By application of JSTX, excitatory junctional potentials (EJPs) caused by the cardiac ganglion activity in the myocardium were gradually abolished. Subsequently, the cardiac ganglion and myocardium exhibited independent activities with their respective rhythms. In saline containing JSTX, no changes were observed in the muscle activity when the ganglionic activity was changed by current injection into the cardiac ganglion neuron. These results indicate that two pacemaker sites, the cardiac ganglion and cardiac muscle, are present in the adult heart of Ligia exotica and suggest glutamatergic neuromuscular transmission between them.

A new system has been developed for continuous measurement of drinking rate in eels in which drunk water exteriorized via an esophageal fistula was reintroduced into the stomach by a pulse injector synchronized with a drop counter. Using intact fish (controls), esophagus-cannulated fish whose drunk water was drained (drained fish), and esophagus and stomach-cannulated fish whose drunk water was reintroduced into the stomach (reintroduced fish), the validity of this system was examined by monitoring the changes in drinking rate and hydromineral balance after exposure to seawater (SW).

In reintroduced fish, the SW exposure was followed by an immediate burst of drinking and a subsequent cyclic pattern of drinking. The drained fish exhibited a similar pattern of drinking but at much higher rate. The plasma Na concentration and osmolality increased linearly for one day and then decreased gradually to a steady SW level in 5–6 days in control and reintroduced fish. However, both parameters increased linearly for 4–5 days in drained fish until they died at plasma osmolality of ca. 500 mOsm. The initial increase in plasma Na and osmolality was steeper for a day in control and reintroduced fish than in drained fish. Hematocrit scarcely changed for one week in control and reintroduced fish, but it increased abruptly from the second day in drained fish, suggesting severe hypovolemia.

These results show that the water and electrolyte balance of reintroduced fish were normal as in intact fish after exposure to SW. Thus, the drinking rate measured by the current system may represent actual drinking. The present study also provides first direct evidence to show that drinking plays a key role in SW adaptation in fish.

Operant conditioning that the pond snail, Lymnaea stagnalis, suppressed its naturally occurring behavior of escape from a water tank was examined by using a negative reinforcement (i.e. an aversive stimulus) prepared outside the tank. During the training period, the number of escapes from a tank was strongly suppressed. One of behavioral factors for this suppression was confirmed as the elongation of latency to the first escape after training. The effects on the memory retention were examined in the massed and spaced training procedures. The latter procedure interposes a rest interval between three sets of 20-min training sessions, whereas the former has the same number of training sessions with no rest interval within 60 min. The memory retention by the massed training was observed within 20 min after training. By the spaced training, the learning acquisition was found to be stronger, which was observed as the slower latency to the first escape, than by the massed training, but the longer-lasting memory retention, which had been expected first, was not formed. These results suggest that once Lymnaea recognize the external environment is safe after training, they may extinguish their memory of the past situation quickly, resulting in no or very little difference in the memory retention by two different training procedures in this operant conditioning. Together with the facts that classical conditioning and its neuronal mechanisms in Lymnaea were previously clarified, the present findings may help to address not only the neuronal basis of operant conditioning but also the relation between classical and operant conditioning.

The carbon dioxide receptor in the temporal organ of the Japanese house centipede Thereuonema hilgendorfi shows spontaneous discharges in carbon dioxide-free air. These responses are decreased or totally suppressed accompanied by a hyperpolarizing receptor potential induced by carbon dioxide stimulation. In the present study the ionic basis of the CO₂ reception has been examined by impulse analysis based upon a known linear relationship between receptor potential and impulse frequency. Even when the temporal organ was perfused with CO₂-free distilled water, receptor cells showed spontaneous discharges, and these discharges were also decreased due to perfusion of CO₂-containing solution. The spontaneous impulse frequency in the CO₂-free solution increased with increasing Na⁺ and K⁺ concentration, and decreased with increasing Ca²⁺ concentration. Other ions such as Mg²⁺, Li⁺, choline⁺ and Cl⁻ had little effect on the receptor cell discharges. In the CO₂ containing perfusate, the effects of increasing Na⁺ or Ca²⁺concentration on the receptor cell discharges disappeared, whereas that of increasing K⁺ concentration remained. Response amplitudes to CO₂ stimulation depended largely on Na⁺ and Ca²⁺ concentration, but less on K⁺ concentration in the perfusate. These results suggest that Na⁺ ions are major current carriers for the generation of receptor potential in response to CO₂ stimulation.

Partial sequences (454 bases) of the mitochondrial DNA (mtDNA) cytochrome b gene (cyt b) were determined for 94 dogs including 73 Japanese native animals. Thirteen nucleotide positions of this region showed nucleotide substitutions, which were all transitions. Three of 13 nucleotide substitutions were nonsynonymous. A total of 14 cyt b haplotypes were found, but the Japanese native dog breeds could not be differentiated as distinct clusters in phylogenetic trees. These results support the previous view that genetic variations observed among Japanese native dog breeds could have resulted from interbreeding and/or intrabreeding.

The hemoglobin types of mouse strains can be distinguished according to patterns observed on cellulose acetate electrophoresis. The two common mouse hemoglobin patterns are single and diffuse. The differences in the patterns result from differences in the β-globin chains of the hemoglobin molecules. Mice with the single hemoglobin pattern have one β-globin type identified as β-single (Hbb^s ), whereas mice with the diffuse hemoglobin pattern have two different β-globin types identified as β-major (Hbb^maj ) and β-minor (Hbb^min ). We examined the functional and stability properties in these mouse hemoglobins, and the oxygen binding properties of red blood cells obtained from mice with four different hemoglobin phenotypes: Hbb^s /Hbb^s , Hbb^s /Hbb^min , Hbb^min /Hbb^min and Hbb^maj /Hbb^min . The P₅₀, the partial pressure of oxygen at which hemoglobin is half-saturated, of purified forms of Hbb^s , Hbb^min and Hbb^maj was 14.8 ± 0.4 mm Hg, 13.3 ± 0.6 mm Hg and 13.6 ± 0.5 mm Hg, respectively. The n value, determined from the slope of the Hill plot was 2.45 to 2.59 for the mouse hemoglobins. The alkaline Bohr effects of purified Hbb^S , Hbb^min and Hbb^maj were 0.69, 0.61 and 0.60, respectively. The mechanical stability of Hbb^s , Hbb^min and Hbb^maj , expressed by the first order kinetic constant, were 0.098 ± 0.01/min, 0.027 ± 0.013/min and 0.27/min, respectively. The P₅₀ of red blood cell suspensions from lines of mice expressing Hbb^s /Hbb^s , Hbb^min /Hbb^min , Hbb^s /Hbb^min and Hbb^maj /Hbb^min were 40.2 ± 1.8 mm Hg, 40.4 ± 1.5 mm Hg, 38.9 ± 1.4 mm Hg, and 38.7 ± 0.9 mm Hg, respectively.

Autogamy-immaturity is the period during which autogamy can not be induced by natural starvation; in Paramecium tetraurelia, autogamy first becomes induceable at about 7 fissions after the previous autogamy, and thereafter the percent of cells undergoing autogamy increases gradually to almost 100% at the clonal age of about 20 fissions and remains at 100% thereafter. The length of autogamy-immaturity (LAI), determined by plotting the percent of cells in autogamy versus the number of fissions, was found to be similar in two cultures grown at different fission rates at 25°C and 30°C. This indicates that paramecia count LAI by the number of fissions, not by the calendar time. LAI estimated from the peaks of percent autogamy through successive autogamous generations was also similar in two continuous cultures grown with different cycles of growth and starvation at 25°C and 30°C, indicating stability of LAI under ordinary laboratory conditions. However, LAI was affected by the cultural age of paramecia from which the new autogamous generation was derived: advanced cultural age shortened the LAI in the following generation.

DIF-1 is a putative morphogen that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum. We have previously discovered that DIF-1 suppresses cell growth and induces cell differentiation in vitro in some tumor cells, and also that relatively low concentrations of DIF-1 promote retinoic acid-induced cell differentiation in the human myeloid leukemia HL-60 cells. In this study, to verify cell biological and therapeutic potential of DIF-1, we have examined whether and how DIF-1 affects normal mammalian cells in vitro, using rat leptomeningeal (RLM) cells and rat gastric mucosal RGM-1 cells. In growing phase of both cells, DIF-1 at 5–40 μM suppressed cell growth in a dose-dependent manner. High concentrations (15–40 μM) of DIF-1 were toxic to the growing cells so that the cells showed unusual morphology, but many of them were still alive even at Day 3–4. Withdrawal of DIF-1 allowed the cells to grow. In confluent phase of the cells, DIF-1 at more than 15 μM promoted medium acidification that resulted in cell death, but DIF-1 itself did not markedly affect cell viability for 3 days. DIF-1 increased [Ca²⁺]_i in RLM cells but did not affect β-trace secretion (an index of cell function in RLM cells). A low concentration (5 μM) of DIF-1 in combination with retinoic acid (0.1 μM) showed no marked effects on cell growth, cell viability, cell morphology and β-trace secretion in RLM cells. The present results indicate that DIF-1 may be utilized as a tool for cell biology. Also, since these concentrations of DIF-1 kill some tumor cells within 2–3 days, the present results suggest that DIF-1 might be utilized in the treatment of some sorts of cancer with a bearable degree of side-effects.

We describe here rapid gamete growth and artificial fertilization method of species of the larvaceans (appendicularians), Oikopleura dioica and O. longicauda (Family Oikopleuridae: Class Appendicularia: Subphylum Urochordata). In these species, oocytes grew very rapidly from about 40 μm in diameter to about 75 μm (O. dioica) and 110 μm (O. longicauda), respectively within a few hr. Moreover, cutting off the gonads at the last phase of the growth stage yielded matured gametes. The eggs and sperm obtained by the dissection of gonads could be fertilized when they were mixed together.

To examine whether the expression pattern of muscle-specific protein in embryonic muscle tissues at the different developmental stages depends on the types of myogenic cells, chicken breast muscle (pectoralis major) tissues of 10-day, 14-day, and 18-day old embryos (E10, E14, and E18, respectively) were grafted on chorio-allantoic membrane (CAM) of 9-day-old chicken embryos and cultured for 12 days at the longest, since the chorio-allantoic grafting is useful in pursuing muscle-cell lineage during muscle differentiation. The muscle fiber formation and expression of troponin T (TnT) isoforms in grafts were investigated by histological and immunohistochemical methods with anti-fast-muscle-type and anti-slow-muscle-type TnT (rabbit sera). In grafts of E10 breast muscle, most muscle fibers continued to develop without degeneration and TnT isoform expression of the fibers changed from the concomitant expression of fast-muscle-type (Ftype) and slow-muscle-type (S-type) to the expression of F-type only. In grafts of E14 and E18 breast muscles, the muscle fibers first degenerated with pyknotic nuclei and hyaline cytoplasm, and then new muscle fibers expressing F-type TnT isoforms were formed by the fusion of basophilic cells. The new muscle fibers in grafts of E14 muscles were different from those of E18 ones in that the former also expressed S-type TnT isoforms. In this paper, developmental stage-dependent TnT isoform expression of the embryonic breast muscles grafted on CAM is discussed in connection with cell-type difference of grafted muscle cells.

UE8 is a uterine epithelial cell line established from p53-deficient fetal female mice. UE8 exhibits a typical epithelial morphology in culture and is strongly positive for cytokeratin, but negative for vimentin in immunocytochemistry. UE8 shows an active growth in a phenol red free 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12 supplemented with 3% heat-inactivated and dextran-coated charcoal-treated fetal calf serum. Both immunocytochemistry and immunoblot analyses confirmed that UE8 was negative for the estrogen receptor. Diethylstilbestrol added to the medium at concentrations between 10⁻⁶ and 10⁻⁸ M had no significant effect on the proliferative rate, and estradiol-17β at 10⁻⁶ M was slightly inhibitory. Unexpectedly, however, the“pure anti-estrogen”ICI 182,780 at 10⁻⁶ M significantly enhanced cell proliferation. This is the first observation that the“pure anti-estrogen”ICI 182,780 stimulates the growth of uterine epithelial cells without estrogen receptors.

Primary structure of a newt prolactin receptor (nPRL-R) was deduced from a cDNA clone isolated from a kidney cDNA library as well as from a polymerase chain reaction (PCR) product. The predicted nPRL-R protein was composed of 626 amino acids (aa), and contained a signal sequence and a transmembrane region. The extracellular domain had two pairs of cysteine residues and a WSXWS motif. The cytoplasmic domain comprised 368 residues and contained both box 1 and box 2 sequences which are considered to be required for the signal transduction of the cytokine/growth hormone (GH)/PRL-R family in mammals. The nPRL-R shares 50-52% protein sequence identity with mammalian PRL-Rs. When nPRL-R was expressed in COS-7 cells, specific binding of [¹²⁵I] rat prolactin (PRL) was observed. Northern blot analysis revealed the existence of a single transcript, more than 10 kb in length, which was expressed in the kidney and brain. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the expression of PRL-R mRNA in the testis, ovary, liver and bladder of the newt. This is, as far as we know, the first report of cloning on amphibian PRL-R.

In general, both larval and adult amphibians are known to change their skin color depending on the background color. However, in the Japanese toad Bufo japonicus, the larvae were found to be devoid of the ability of responding to the background color. Melanin granules in the dermal melanophores were in a state of constant dispersion independently of the color of the background to which they were subjected. Pharmacological and histochemical studies revealed that in the toad tadpoles, melanophore-stimulating hormone is being released without inhibition, presumably because innervation of catecholaminergic fibers in the intermediate lobe is incomplete until they finish metamorphosis. Biological significance of the dark color of Bufo tadpoles is discussed in conjunction with another characteristic that they have a tendency to aggregate to form a dense black mass.

We only have little information on expression of gonadotropin (GTH) subunit genes during spawning migration in salmonids. Changes in the levels of mRNAs for GTH subunits (GTH α2, Iβ and IIβ) were therefore analyzed in the pituitaries of chum salmon (Oncorhynchus keta) during the final stages of spawning migration to the Ishikari river. The fish were caught at Atsuta, a fisherman's village facing the Ishikari bay, and at Chitose, a tributary of the Ishikari river, in 1993 and 1994. The former is referred to as seawater (SW) fish, and the latter as freshwater (FW) fish. The levels of GTH subunit mRNAs in the pituitaries were determined by a quantitative dot blot analysis, using single-stranded sense DNA as the standard. The sense DNAs have the same nucleic acid sequences of mRNAs. The level of GTH α2 mRNA in the FW males was higher than that in the SW ones. Similar tendency was seen in the females. No significant changes were observed in the levels of GTH Iβ mRNA in both the males and females. Whereas, the level of GTH IIβ mRNA in the FW fish was higher than that in the SW fish regardless of sexes in 1994. Although not statistically significant in the males, similar tendency was seen in the 1993 fish. The present study thus showed that the level of GTH IIβ mRNA was increased concomitantly with that of GTH a2 mRNA during the final stages of spawning migration.

Our previous study suggested that, in the pituitaries of pre-spawning chum salmon, salmon gonadotropin-releasing hormone (sGnRH) stimulates expression of genes for gonadotropin (GTH) IIβ but not for Iβ, since the levels of mRNAs encoding sGnRH and GTH II but not I were increased during the final stages of spawning migration. In the present study, a capsule of GnRH analog (GnRHa) was implanted into the dorsal muscle of maturing sockeye salmon to clarify function of GnRH on expression of GTH subunit genes in pre-spawning homing salmonids. The amounts of GTH subunit mRNAs in the individual pituitaries were analyzed by a quantitative dot blot analysis using single-stranded sense DNA as the standard. The levels of GTH α and IIβ mRNAs in the GnRHa-implanted fish were significantly higher than those in the control fish in both the males and females, whereas the levels of GTH Iβ mRNA did not show any significant differences in both sexes. These results indicate that GnRH elevates expression of GTH subunit genes which encode the components of GTH II, α and IIβ chains, in the pituitary of maturing sockeye salmon, and then accelerates final maturation.

Gonadotropin-releasing hormone analog (GnRHa), testosterone (T) or 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) was implanted in lacustrine sockeye salmon (Oncorhynchus nerka) to examine their effects on homing behavior prior to spawning. Maturing adult fish in Lake Shikotsu were captured by a set net adjacent to their natal hatchery in September and October, 1997. They were tagged, implanted with hormones and released at the center of lake. More than 70% of released fish returned to the hatchery. In September, GnRHa-implanted fish returned significantly earlier than the controls regardless of sexes. DHP also shortened homing duration in the females but not in the males. T tended to reduce homing duration in the males, however it did not have any significant effect in the females. In October, fish in all groups quickly returned within a few days after the release. Hence, the shortening effect of GnRHa on homing duration was not seen. The October fish may be already well primed by endogenous hormones. The present study showed that, in September, GnRHa consistently shortened homing duration, whereas sex steroid actions varied depending on sex in lacustrine sockeye salmon.

Proteasomes are large, multisubunit particles that act as the proteolytic machinery for most regulated intracellular protein breakdown in eukaryotic cells. The core proteinase of this complex, the 20S proteasome, is comprised of four stacked rings with seven subunits each. The outer two rings are made up of seven, distinct α-type subunits, while the two inner rings are composed of seven, different β-type subunits. Here we present the cloning, sequencing and expression analysis of Carassius auratus, α2_ca, which encodes one of the proteasome α subunits from goldfish ovary. The cloned cDNA is 838 bp long and encodes 234 amino acids. The deduced amino acid sequence is highly homologous to α2 subunits from other vertebrates. The expression of mRNA for α2_ca occurs at very high levels in ovary and muscle and moderately high levels in testis, brain and gill. It was also shown that protein content was different from mRNA expression levels.

Unique reproductive phenomena, such as delayed fertilization in females and asynchrony between spermatogenesis and mating behavior in males, are known in hibernating bats. The present study was undertaken to examine sex differences and seasonal changes in the gonadotropin-releasing hormone (GnRH)-immunoreactive (ir) neuronal system of Japanese bats, Rhinolophus ferrumequinum. GnRH-ir neurons were preferentially distributed in the medial preoptic area (POA) and medial basal hypothalamus (MBH). The number and immunoreactivity of GnRH-ir neurons decreased in summer (lactation period in females and spermatogenic period in males), whereas both increased dramatically in winter (hibernation period). The number and immunoreactivity of GnRH-ir neurons varied more in the MBH than in the POA throughout the annual reproductive cycle of both sexes. The changes in GnRH neurons in the MBH closely paralleled those of GnRH-ir fibers in the median eminence (ME), which is the release site for GnRH. In contrast, there was no sex difference in the number and immunoreactivity of GnRH-ir neuronal perikarya in either region except for the number of GnRH-ir neurons in the POA in spring and summer. These findings suggest that GnRH neurons in the MBH supply the major GnRH innervation to the ME and play a central role in seasonal regulation of gonadotropin secretion in this bat.

Conditions which affected resting sites of laboratory mice (Mus musculus) were examined. Pairs of mice which encountered one another after living in separate home cages for 2 days, established dominant-subordinate relationships. About half of the dominants observed rested in their home cages solitarily and the remainder rested in the home cage of the subordinate gregariously. The subordinates always rested in their home cages. Removing or enclosing one of the paired males after physical contact had no effect on the resting site of the remaining mouse. When one male was removed before physical contact, the other always rested in its home cage. When one male was enclosed before physical contact, the other rested in its home cage or in the cage of the enclosed male: the former being referred to as an active male, and the latter being to as an inactive male. Active males were apt to become dominant and inactive males were apt to become subordinate. The preferences of active and inactive males changed after physical contacts. I conclude that resting sites after physical contact show the social relations between the males, and resting sites before physical contacts show the aggressiveness and cautiousness of each mouse.

One of the two colour variations in Facelinella quadrilineata (Baba, 1930) is found to be an undescribed species, which has been commonly collected from the Pacific coast of Boso Peninsula, central Japan. Although both species are similar enough morphologically to be considered cryptic species, there is a marked difference in the penial structure. The new species possesses an ejaculatory penis, while Facelinella quadrilineata does not. Considering close affinity between the two species and the reasons for separating the two genera of Facelinella and Facelina, invalidity of Facelinella becomes obvious and it is proposed to merge Facelinella into Facelina. The new species is herein described and illustrated as Facelina bilineata and compared with Facelina quadrilineata comb. nov.

The amphioxus, Branchiostoma belcheri, is currently listed in the registry of “Endangered Animals of Japanese Marine and Fresh Water Organisms” issued by the Japan Fisheries Resource Conservation Association. We surveyed a new population of this species in an area near the Irago Channel and the Enshu-Nada Sea in Aichi Prefecture, Japan. This population was originally discovered by a research group of the Aichi Fisheries Research Institute when they were collecting Japanese sand lances (Ammodytes personatus) in this area. Every month from June to November of 1995 with the exception of October, we collected amphioxi and sand lances in an area of about 1 km² to study the population profile of the amphioxus and the relation between these two animals. The newly discovered population of the amphioxus was suspected to be larger than any other Japanese populations of this species. Animals of this population could be divided into two groups according to body size distribution: those of approximately 20 mm long on the average (termed small) and those of approximately 50 mm long on the average (termed large). No small amphioxus was found in June; 10% or fewer of the animals were of small size in July and August; and about 40% of the animals were of small size in September and November. The breeding season was estimated to last from June (or earlier) to August based on observation of the gonadal size. Amphioxi share a habitat with sand lances, which stay in the sand during the summer dormant period. The newly discovered amphioxus population may provide an important source of material for modern biological researches on Cephalochordata in Japan, although special care for protection of this population should be taken.

Nine of ten gorgonian extracts from all four families tested showed antimicrobial activity. Inhibition of Gram positive bacteria was more widespread than that of Gram negative bacteria. Inhibition of the yeast Saccharomyces cerevisiae was confined to (and widespread within) the family Ellisellidae. No antifungal activity against Aspergillus sp. was found in all the extracts tested. Acute (6 hr) and chronic (24 hr) extract toxicity towards Artemia salina varied between extracts and showed no taxonomic correlations; compared to potassium dichromate, extract toxicity was low. Chronic exposure to the extracts was more effective in killing A. salina; chronic LD₅₀ values ranged from 3.2 to 125.9 mg/ml. All ten extracts failed to extend survival, lower percentage parasitemia or affect parasite morphology in Plasmodium berghei-infected mice.