Zoological Science

Effects of 15 min light pulses given at various intervals (every 1, 2, 4, 6, 8 and 12 hr) under constant darkness on the locomotor rhythm were investigated in the adult male cricket, Gryllus bimaculatus. A single pulse per 24 hr induced period modulation in a circadian phase dependent manner, yielding a period modulation curve (PMC): the 15 min light pulse lengthened the period in the early subjective night (CT11-16) and shortened it during the late subjective night to the early subjective day (CT20-5). Frequent light pulses modulated the freerunning period of the rhythm dependent on the interval of the pulses: when compared with the freerunning period in DD (23.74 ± 0.03 hr) the period was significantly shorter in intervals of 2 and 4 hr, but lengthened when the interval was 1 and 12 hr. Frequent light pulses also resulted in entrainment of the rhythm to run with the period of 24 hr and the ratio of the entrained animals varied from 12% to 72% depending on the interval of the light pulses. The period modulation and the entrainment by the repetitive light pulses could be interpreted according to the PMC. In about 15% of animals, the light pulses induced a rhythm dissociation, suggesting that the bilaterally paired circadian pacemakers have their own sensitivity to the entraining photic information. The light pulse caused a masking effect, i.e., an intense burst of activity. The magnitude of the light induced responses was dependent on the circadian phase. The strongest masking effect was observed in the subjective night. The phase of the prominent period modulation and of the marked masking effects well coincides with the previously reported sensitive phase of the photoreceptive system.

The effect of direct 5,7-dihydroxytryptamine (5,7-DHT) injection into the medulla region of the optic lobe on the locomotor activity was investigated in the adult male cricket, Gryllus bimaculatus. After a 6 hr phase advance of a light-dark cycle, the 5,7-DHT injected animals needed significantly longer time for resynchronization to the new cycle (6.55 ± 0.62 days) than the control, Ringer's solution injected animals (3.17 ± 0.15 days; P < 0.001, t-test). Light induced a bout of activity (i.e., masking effect) when light-dark cycle was phase advanced by 6 hr and the duration of the masking effect was significantly longer in 5,7-DHT injected animals. An initial bout of the nocturnal activity was significantly greater in the 5,7-DHT injected animal. Under constant darkness, the freerunning periods of both groups were not significantly different. Under constant light, a significantly higher percentage of 5,7-DHT injected animals showed arrhythmicity compared with the control group. An analysis carried by high-pressure liquid chromatography with electro-chemical detection (HPLC-ECD) revealed that the serotonin content in the optic lobe was significantly reduced to less than 50% in the 5,7-DHT injected animals, even one month after the injection. These results suggest that serotonin plays important roles in the regulation of circadian locomotor rhythms of the cricket mainly by regulating the sensitivity of the photoreceptive system.

Measurements of spectral reflectance from the sky-blue portion of skin from the common surgeonfish, Paracanthurus hepatus, showed a relatively steep peak at around 490 nm. We consider that a multilayer thin-film interference phenomenon of the non-ideal type, which occurs in stacks of very thin light-reflecting platelets in iridophores of that region, is primarily responsible for the revelation of that hue. The structural organization of the iridophore closely resembles that of bluish damselfish species, although one difference is the presence of iridophores in a monolayer in the damselfish compared to the double layer of iridophores in the uppermost part of the dermis of surgeonfish. If compared with the vivid cobalt blue tone of the damselfish, the purity of the blue hue of the surgeonfish is rather low. This may be ascribable mainly to the double layer of iridophores in the latter since incident lightrays are complicatedly reflected and scattered in the strata. The dark-blue hue of the characteristic scissors-shaped pattern on the trunk of surgeonfish is mainly due to the dense population of melanophores, because iridophores are only present there in a scattered fashion. Photographic and spectral reflectance studies in vivo, as well as photomicrographic, photo-electric, and spectrometric examinations of the state of chromatophores in skin specimens in vitro, indicate that both melanophores and iridophores are motile. Physiological analyses disclosed that melanophores are under the control of the sympathetic nervous and the endocrine systems, while iridophores are regulated mainly by nerves. The body color of surgeonfish shows circadian changes, and becomes paler at night; this effect may be mediated by the pineal hormone, melatonin, which aggregates pigment in melanophores.

The complete mitochondrial (mt) genome of the gynogenetic triploid ginbuna (Carassius auratus langsdorfi, AZ3 line) has been cloned and sequenced. The genome consisted of 16,578 bp and encoded the same set of genes (13 proteins, 2 rRNAs and 22 tRNAs) in addition to a D-loop region, as described for other vertebrate mtDNAs. Comparison with other teleost mtDNAs demonstrated that the protein/rRNA-coding regions of the ginbuna were highly homologous both in length and nucleotide composition to those of the carp, indicating fairly close relationship between the triploid ginbuna and the carp. Although the size of the ginbuna D-loop was almost the same as that of the carp, the nucleotide sequence showed a moderate variation. More comprehensive sequence data of the D-loop regions will lead to the elucidation of phylogenetic relationships among Carassius auratus subspecies.

Symbiosis is observed between zooxanthellae, symbiotic dinoflagellates, and giant clams and related clams which belong to the families Tridacnidae and Cardiidae. We have previously shown that a photo-symbiotic clam Tridacna crocea has three types of hemocytes, the eosinophilic granular hemocyte with phagocytic activity, the agranular cell with electron lucent granules, and the morula-like cell with large (ca. 2 μm in diameter) colorless granules. The function of the morula-like cell is not clear, but it has not been reported in any other bivalves except photo-symbiotic clams T. crocea and Tridacna maxima. In order to clarify whether it is specific to photo-symbiotic clams or not, we studied hemocytes in the photo-symbiotic clams Tridacna derasa (Tridacnidae), Hippopus hippopus (Tridacnidae) and Corculum cardissa (Cardiidae), and a closely related non-symbiotic clam Fulvia mutica (Cardiidae). The eosinophilic granular hemocytes and the agranular cells were found in all of the clams examined. However, the morula-like cells which were packed with many large electron dense granules (ca. 2 μm in diameter), were observed only in the photo-symbiotic clams. In F. mutica, a closely related non-symbiotic clam, this type of hemocyte was not found. Instead a hemocyte with vacuoles and a few large granules containing peroxidase activity was observed. The large granules of F. mutica varied in size from ca. 1–9 μm in diameter. Present data suggests that the presence of morula-like cells is restricted to photo-symbiotic clams and that the hemocytes associated with the morula-like cells may have some functional relationship to symbiosis with zooxanthellae.

Melatonin synthesis in the retina as well as in the pineal gland exhibits daily variations with higher levels during the dark phase of light-dark cycles. To analyze the molecular mechanism of melatonin synthesis in the retina, we have cloned, sequenced and characterized a putative cDNA for arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87), a rate-limiting enzyme in melatonin production, from the retina of the rainbow trout (Oncorhynchus mykiss). The trout AANAT cDNA (1,585 bp) contains an open reading frame encoding 240 amino acid protein (predicted molecular weight, 27,420) that is 51–65% identical to avian and mammalian AANAT. The trout retinal AANAT protein contains motifs A and B that are conserved among the N-acetyltransferase superfamily and eight potential phosphorylation sites. Southern blot analysis demonstrated that the protein is expressed by a single copy gene. A single AANAT transcript (1.6 kb) was detected in the retina but not in the liver by Northern blot analysis. The levels of AANAT mRNA in the retina exhibited day-night changes with 3.3-fold increase at night. These results indicate that in the rainbow trout retina, the activity of AANAT and thus melatonin synthesis are regulated at least in part at the transcriptional level.

The 20S proteasome of eukaryotic cells has at least three distinct peptidase activities (trypsin-like, chymotrypsin-like and peptidylglutamylpeptide (PGP) hydrolase activities). These peptidases are latent and require appropriate activators. SDS has been widely used as an activator of these peptidases, but the mechanism of its activation remains unresolved. In this study, we investigated the kinetics of the SDS-activated hydrolysis of the above three types of peptidase of the 20S proteasome purified from Xenopus oocytes. When the reaction was started by simultaneous adding both SDS and substrate, maximal rates of hydrolysis were reached after appreciable lag phases with the trypsin-type substrate [t-butyloxycarbonylLeu-Arg-Arg-4-methylcoumaryl-7-amide (Boc-LRR-MCA)], but no such lag phases were observed with the chymotrypsin-type and PGP hydrolase-type substrates [succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (Suc-LLVY-MCA), and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide (Cbz-LLE-2NA), respectively]. Similarly, changes in the hydrolysis rate to a reduced level upon dilution of SDS occurred after an appreciable lag phase again in the trypsin-like peptidase, but not in the other types. The lag phase characteristic of the trypsin-like peptidase was dependent on the substrate concentration. Thus, the lag phase was less discernible at very low concentrations of the substrate (e.g. at concentrations in the order of 1/100 of the Km value), but became more conspicuous with the increases in the substrate concentration. This lag phase also vanished upon preincubation of the activator (SDS) for a short period of 5 sec. These results suggest that the formation of the enzyme-substrate complex in the trypsin-like reaction induces a conformational change in the enzyme which makes the SDS activator site(s) in an occluded form, reducing the rates of SDS binding and dissociation.

Mouse preimplantation embryonic cleavage rate is dependent upon the presence or absence of the Preimplantation-embryo-development (Ped) gene; which is linked to the Qa-2 subregion of the H-2 complex. Expression of Qa-2 antigens by fast developing mouse embryos correlates with Ped gene pheno-type: Qa-2^a. It is not known if the Ped gene (Qa-2^a) participates in cell differentiation in the preimplantation mouse blastocyst. Therefore, the study objective was to determine the differentiation of cells to the inner cell mass (ICM) and trophectoderm (TE) in Qa-2^a positive (Ped +) and Qa-2^a negative (Ped −) mouse blastocysts. One-cell stage embryos were recovered from the excised oviducts of PMSG (5 IU) and hCG (5 IU) primed virgin female (3–4 weeks) BALB/cByJ (Qa-2^a: Ped −) and BALB/cJ (Qa-2^a: Ped +) mice mated to fertile males (12+ weeks). Embryos were collected, 14 hr after hCG, and cultured in modified α-MEM, to the hatched blastocyst stage in an atmosphere of 5% CO₂ in air, 95% relative humidity at 37°C. Cell differentiation was determined by differential staining (bis-benzimide and propidium iodide) and fluorescence microscopy. Data were analyzed by Students t-test. There was no significant difference in total cell number between BALB/cJ (mean 139) and BALB/cByJ (mean 143) embryos. A significant difference (p < 0.001) was found in the number of cells differentiating to the ICM between BALB/cJ (mean 59.0) and BALB/cByJ (mean 29.0) mouse embryos. The number of cells differentiating to the TE, between BALB/cJ (mean 80.0) and BALB/cByJ (mean 114) embryos, approached significance (p = 0.062). The results suggest that the Ped gene (Qa-2^a) may have an influential role in preimplantation blastocyst cell differentiation. Additional studies are warranted to further elucidate the role of the Ped gene in preimplantation embryo development and blastocyst formation.

The swimming larvae of most solitary ascidians belonging to the Ascidiidae family bear three anterior, simple conic adhesive papillae. They secrete adhesive substances that are used to effect transitory settlement at the beginning of the metamorphosis.

The adhesive papillae of newly hatched Phallusia mamillata larvae examined by the SEM are covered by the tunic. When the larvae are about to settle, the tunic becomes fenestrated over the central part of the papilla and bulb-ended microvilli protrude through the holes. These papillae have two types of elongated cells: many peripheral cells and few larger central cells with microvilli and bundles of microtubules oriented along the major axis of the cells.

We have done immunofluorescence experiments with an anti-β-tubulin monoclonal antibody (clone 2-28-33) reacting with axonal microtubules. Only the central cells of the papillae were stained and the axons appeared to arise from the proximal ends of these cells. These axons form a long nerve that reaches the brain vesicle. Branches of the same nerve appear to connect to the basal ends of the peripheral cells. By confocal laser microscopy we were able to follow the course of the papillary nerve. The two nerves connecting the dorsal papillae fuse together into a single nerve that runs posteriorly. The nerve connecting the ventral papilla runs posteriorly for a long tract before fusing with the nerve of the dorsal papillae just near the brain.

The reported observations raise the hypothesis that the central cells of the adhesive papillae might be primary sensory neurons and that they may have chemosensory function.

Bombyx mori L. (Lepidoptera: Bombycidae) larva was investigated with a transmission electron microscopy to determine hemocytic differentiation in the hemopoietic organ located in the prothorax. Three and/or four types of stem cells in compact islets of the organ were observed. Immatured hemocytes in loose islets of the organ were more differentiated and developed than in compact islets. Four types of hemocytes such as prohemocyte, plasmatocyte, granulocyte and oenocytoid were observed in loose islets. Each type of hemocyte was differentiated from each type of stem cell. However, none of spherulocyte was observed. Each type of hemocytes matured in loose islets was discharged into hemolymph by the tearing of acellular membrane covering the islets. These observation strongly suggests that the four kinds of hemocytes except for spherulocytes first appeared in islets and then moved to the region of loose islets in matured form. The more detailed pathway of hemocytic differentiation in B. mori was represented here.

Annetocin is an egg-laying-inducing oxytocin-related peptide which we have previously isolated from the earthworm, Eisenia foetida. Here we report the results of immunohistochemical and ultrastructural studies on annetocin-secretory cells in the earthworm. Annetocin-immunoreactive (IR) cell-somata were located mainly at the ventro-lateral side of the subesophageal ganglion. Only four annetocin-IR cells were seen in the cerebral ganglion. Some annetocin-IR cells displayed unipolar-like structure with a process directing to the core region (the neuropile) of the ganglion. Annetocin-IR fibers were also observed in the neuropile of the ventral ganglia and the ventral nerve cord between the 4th and the 30th segments including the clitellum, but not in the posterior segments (31-55th). The number of annetocin-IR fibers decreased from the 4th to the 30th segment. The annetocin-secretory cells were identified by the immunogold staining, and filled with gold-labeled vesicles, 200-250 nm in diameter, which included moderately electron dense material. The annetocin-secretory cells possessed a euchromatic nucleus, well-developed rough endoplasmic reticulum and Golgi apparatus. Some of the annetocin-secretory cells were found to form a neurohemal-like structure, where somata or fibers with loose glial investment came in contact with the coelomic space at the ventral side of the subesophageal ganglion. The results suggest that annetocin is a neuropeptide produced and secreted by the neuron in the cerebral and subesophageal ganglia of the earthworm.

This study deals with the localization of crustacean hyperglycemic hormone (CHH, Pej-SGPIII) and molt-inhibiting hormone (MIH, Pej-SGP-IV) in the eyestalk of the kuruma prawn Penaeus japonicus using immunohistochemistry. High-titer and highly specific antisera were raised in rabbits against synthetic Pej-SGP-III C-terminal peptide (Glu-Glu-His-Met-Ala-Ala-Met-Gln-Thr-Val-NH₂) and Pej-SGP-IV C-terminal peptide (Val-Trp-Ile-Ser-Ile-Leu-Asn-Ala-Gly-Gln-OH), both of which were conjugated with bovine serum albumin by a cross linker. Eyestalks were removed from mature male prawns at the intermolt stage of the molting cycle and fixed in Bouin's solution. Serial sections stained immunohistochemically showed that neurosecretory cells of Pej-SGP-III and Pej-SGP-IV were located in the same cluster of the medulla terminalis ganglionic X-organ (MTGX), and that three kinds of neurosecretory cells, which were stained with anti-PejSGP-III antiserum and/or anti-Pej-SGP-IV antiserum were present. The number of neurosecretory cells which stained with both antisera was much fewer than that of neurosecretory cells which stained with one of the antisera only. The axon and axon terminals in the sinus gland were also stained and the staining density of the sinus gland was always deeper than that of the neurosecretory cells.

Atrial natriuretic peptide (ANP) is known as a potent natriuretic/diuretic hormone in vertebrates. However, eel ANP infused at doses that did not alter arterial blood pressure (0.3-3.0 pmol/kg/min) decreased urine volume and increased urinary Na concentration in seawater (SW)-adapted eels but not in freshwater (FW)-adapted eels. The renal effects were dose-dependent and disappeared after infusate was switched back to a vehicle (0.9% NaCl). Urinary Na excretion (volume × Na concentration) did not change during ANP infusion. ANP infusion increased plasma ANP concentration, but the increase at the highest dose was still within those observed endogenously after injection of hypertonic saline. Urinary Mg and Ca concentrations increased during ANP infusion in SW eels, but urinary Ca excretion decreased in FW eels. Plasma Na concentration profoundly decreased during ANP infusion only in SW eels, suggesting that ANP stimulates Na extrusion via non-renal routes. These results indicate that ANP is a hormone which specifically extrudes Na ions and thereby promotes SW adaptation in the eel. This is in sharp contrast with mammals where ANP is a volume regulating hormone that extrudes both Na and water.

Direction of mouth-opening of a small herbivorous Tanganyikan cichlid, Telmatochromis temporalis, was studied. Each sample fish opened its mouth either rightward or leftward in some degree. The directions of mouth-opening were independent of the body curve directions, and the asymmetry will be due to asymmetric mouth morph individually specific. The degree of the mouth asymmetry was not related to body size, suggesting the asymmetry being not acquired characters. No fish opened the mouth in lateral symmetry, indicating that the asymmetry is different from “fluctuating asymmetry”. This fish took algae on rock surface usually using right or left side of its mouth. However, strong relations between directions of mouth-openings and frequencies of mouth side used in foraging were not found, and the biological role of the asymmetry, if any, is not clear now.

Queen number, mating frequency and nest kin-structure of the ant Formica japonica were studied in the field and the laboratory. Nest excavation in the study site, the east slope of Mt. Fuji, Gotenba, Japan, revealed that F. japonica is weakly polygynous all year round and the queen number increases after the nuptial flight season, suggesting the adoption of newly mated queens by established nests. Dissection and laboratory rearing demonstrated that nearly all queens in polygynous nests had mated and were fertile with mature oocytes in their ovaries. Multilocus DNA fingerprinting was used to examine kin relationships among ants found in the same nests. The fingerprint band patterns were apparently governed by a simple genetic rule and suggested monoandry (single mating per queen). The mean band sharing score of DNA fingerprints among full sisters was 0.90, and the mean value between queens and their daughters was 0.75. Comparison of DNA fingerprints of adult and pupal workers with pupal gynes suggested that multiple queens in a nest may contribute unequally to gyne (new queen) production.

Platycopia compacta new species (Copepoda: Platycopioida) is described from the hyperbenthic zone in the Tokara Islands, southern Japan. This is the second species of the order in the Indo-Pacific region, and the eighth species of the genus. The present new species is distinguishable from the closely related P. inornata from the Bahamas by (1) the formation of allobasis in the antenna; (2) two unequal setae on the basal exite of the maxillule; (3) the intermaxillipedal process with only 3 pairs of prominences at tip; (4) the fusion of the coxa and basis in leg 3; and (5) the fusion of the second and third endopod segments in female legs 3–5. Copepodid stages III, V, and VI are compared with those of P. orientalis collected from Okinawa, and differ in the developmental patterns of the antennule and legs. The presence of an intermaxillipedal process and gut content analysis revealed that Platycopia is carnivorous. The armature of legs 2–5 of the hyperbenthic genus Platycopia is stouter than that of cavernicolous platycopioids, which indicates that Platycopia may penetrate into loose sediment, whereas the latter group might be more adapted to pelagic life. The horizontal distribution of the genus Platycopia apparently exhibits a full Tethyan track.

Transparent-scaled variant (TSV) of the rosy bitterling Rhodeus ocellatus ocellatus (Kner) was observed on both morphology and heredity. Compared with the normal-scaled type (NST), TSV is characterized by the blackish coloration in both eyes and peritoneum, and the luminescent one over the whole body. Histologically, the density of guanophores containing reflecting platelets was conspicuously low, especially in scale, iris, choroid and peritoneum, while the increase in the number of goblet cells (mucous cells) was recognized all over the dermal/epidermal regions. The heredity of TSV was recessive and supposed to be controlled by a single pair of genes unrelated to sex, judging from the result of crossbreeding experiments between NST and TSV. In growth and reproduction, no difference was seen between these two phenotypes. Transparent-scaled variant of the rosy bitterling can be competent for a genetic marker in experimental and developmental biology.

Planarians are considered to be among the most primitive animals which developed the central nervous system (CNS). To understand the origin and evolution of the CNS, we have isolated a neural marker gene from a planarian, Dugesia japonica, and analyzed the structure of the planarian CNS by in situ hybridization. The planarian CNS is located on the ventral side of the body, and composed of a mass of cephalic ganglions in the head region and a pair of ventral nerve cords (VNC). Cephalic ganglions cluster independently from VNC, are more dorsal than VNC, and form an inverted U-shaped brain-like structure with nine branches on each outer side. Two eyes are located on the dorsal side of the 3^rd branch and visual axons form optic chiasma on the dorsal-inside region of the inverted U-shaped brain. The 6^th–9^th branches cluster more closely and form auricles on the surface which may function as the sensory organ of taste. We found that the gross structure of the planarian CNS along the anterior-posterior (A–P) axis is strikingly similar to the distribution pattern of the “primary” neurons of vertebrate embryos which differentiate at the neural plate stage to provide a fundamental nervous system, although the vertebrate CNS is located on the dorsal side. These data suggest that the basic plan for the CNS development along the A–P axis might have been acquired at an early stage of evolution before conversion of the location of the CNS from the ventral to the dorsal side.