Zoological Science

Actin is a ubiquitous protein in eukaryotic cells and plays an important role in cell structure, cell motility, and the generation of contractile force in both muscle and nonmuscle cells. Multiple genes encoding muscle or nonmuscle actins have been isolated from several species of ascidians and their expression patterns have been investigated. Sequence and expression analyses of muscle actin genes have shown that ascidians have at least two distinct isoforms of muscle actin, the larval muscle and body-wall isoforms. In the ascidian Halocynthia roretzi, two clusters of actin genes are expressed in the larval muscle cells. The HrMA2/4 cluster contains at least five actin genes and the HrMA1 cluster contains a pair of actin genes whose expression is regulated by a single bidirectional promoter. cis-Regulatory elements essential for muscle-specific expression of a larval muscle actin gene HrMA4a have been identified. The adult body-wall muscle actin is clearly distinguished from the larval muscle actin by diagnostic amino acids. The adult muscle actin genes may be useful tools to investigate the mechanisms of muscle development in ascidian adults. The evolution of chordate actin genes has been inferred by comparing the organization and sequences of actin genes and performing molecular phylogenetic analysis. The results suggest a close relationship between ascidian and vertebrate actins. The chordate ancestor seems to have evolved the “chordate-type” cytoplasmic and muscle actins before its divergence into vertebrates and urochordates. The phylogenetic analysis also suggests that the vertebrate muscle actin isoforms evolved after the separation of the vertebrates and urochordates. Muscle actin genes have been used to investigate the mechanism of muscle cell regression during the evolution of anural development. The results suggest that the regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in the trans-acting regulatory factors required for their expression. Actin genes have provided a unique system to study developmental and evolutionary mechanisms in chordates.

Circadian locomotor rhythm and its underlying mechanism were investigated in the cricket, Gryllodes sigiilatus. Adult male crickets showed a nocturnal locomotor rhythm peaking early in the dark phase of a light to dark cycle. This rhythm persisted under constant darkness (DD) with a free-running period averaging 23.1 ± 0.3 hr. Although constant bright light made most animals arrhythmic, about 40% of the animals showed free-running rhythms with a period longer than 24 hr under constant dim light condition. On transfer to DD, all arrhythmic animals restored the locomotor rhythm. Bilateral optic nerve severance resulted in free-running of the rhythm even under light-dark cycles. The free-running period of the optic nerve severed animals was significantly longer than sham operated crickets in DD, suggesting that the compound eye plays some role in determining the free-running period. Removal of bilateral lamina-medulla portion of the optic lobe abolished the rhythm under DD. These results demonstrate that the photoreceptor for entrain-ment is the compound eye and the optic lobe is indispensable for persistence of the rhythm. However, 75% and 54% of the optic lobeless animals showed aberrant rhythms with a period very close to 24 hr under light and temperature cycles, respectively, suggesting that there are neural and/or humoral mechanisms for the aberrant rhythms outside of the optic lobe. Since ocelli removal did not affect the photoperiodically induced rhythm, it is likely that the photoreception for the rhythm is performed through an extraretinal photoreceptor.

The optic lobe is essential for circadian locomotor rhythms in the cricket, Gryllodes sigillatus. We examined potential interactions between the bilaterally paired optic lobes in circadian rhythm generation. When one optic lobe was removed, the free-running period of the locomotor rhythm slightly but significantly lengthened. When exposed to light-dark cycles (LD) with 26 hr period, intact and sham operated animals were clearly entrained to the light cycle, but a large number of animals receiving unilateral optic nerve severance showed rhythm dissociation. In the dissociation, two rhythmic components appeared; one was readily entrained to the given LD and the other free-ran with a period shorter than 24 hr, and activity was expressed only when they were inphase. The period of the free-running component was significantly longer than that of the animals with a single blinded pacemaker kept in LD13:13, suggesting that the pacemaker on the intact side had some influence on the blinded pacemaker even in the dissociated state. The ratio of animals with rhythm dissociation was greater with the lower light intensity of the LD. The results suggest that the bilaterally distributed pacemakers are only weakly coupled to one another but strongly suppress the activity driven by the partner pacemaker during their subjective day. The strong suppression of activity would be advantageous to keep a stable nocturnality for this cricket living indoors.

A transparent wing of a hawkmoth, Cephonodes hylas, has a highly ordered array of nanosized protuberances in its surface. This protuberance array has almost the same morphology as that of the “corneal nipple array”, which is suggested to function as an antireflective device to light, using a scaled model experiment, a theoretical calculation, and a comparative way. For the direct study of the function of this protuberance array, we prepared an artificial wing without protuberances and compared it with an intact one with protuberances. Directly measuring the light reflectance spectra of the intact wing and the artificial one, we demonstrated that the nanocomposite in the Cephopnodes wing decreases the light reflectance by the wing into 29-48% in the broad wavelength range. This anti reflective effect is also showed by the color difference between the wings, coated by gold, with and without protuberances.

The effects of vitamin D₃, 24,25(OH)₂ vitamin D₃, 25(OH) vitamin D₃ and 1,25(OH)₂ vitamin D₃ were investigated on the serum calcium and phosphate levels of freshwater catfish, Heteropneustes fossilis. The fish were injected daily intraperitonealy with these secosteroids for 10 days. Blood samples were collected at day 1, 3, 5 and 10. Serum calcium and inorganic phosphate levels were elevated by all of the treatments except for 24,25(OH)₂ vitamin D₃.

Paramecium tetraurelia cells age with repeated cell cycles and finally die after a specific number of cell divisions. In addition to this clonal aging, starvation results in cultural aging in division-arrested cells. They are reset to time zero to initiate a new clonal life cycle, if autogamy occurs when starved. Increased UV sensitivity is one of the representative markers of clonal aging in P. tetraurelia. We studied changes in UV sensitivity with the advance of cell cycle, clonal age, and cultural age. In this study, we made the following observations: 1) cells of any clonal age became less sensitive to UV as the cell cycle advanced; 2) cells became more sensitive to UV as clonal age advanced; 3) cells became temporarily hypersensitive to UV through an unknown mechanism at the clonal ages of less than 80 fissions; 4) cells at the stationary phase were more sensitive to UV than those at the log-phase; and 5) both autogamy-mature and immature cells became more sensitive to UV as cultural age advanced and became less sensitive with similar kinetics when transferred to culture medium. The fact that UV sensitivity increased with cultural age irrespective of the occurrence of autogamy suggests that this is caused by a different mechanism from that associated with clonal age because the latter could be canceled by autogamy.

The endoskeleton of echinoderms consists of a meshwork of calcite. Using light and electron microscopy, this study investigates a cortex covering the arms of crinoids. In Metacrinus rotundus, it consists of massive calcite and has a regular pattern of ridges and holes. The cortex is covered by thin extensions of epidermal cells whose cell bodies are located in the holes. The cells carry intracuticular cilia and seem to have contact with axons connecting to the central nervous system. The cilia probably have sensory function. We compared three other species of living stalked crinoids and two species of stalkless crinoids and found that they have a similar cortex with varying surface patterns, possibly due to various modes of life. The cortex of arms with its pattern seems to be a species-specific characteristic of crinoids. The ridges of the cortex might influence drag caused by currents or serve to facilitate current detection.

A family of highly repetitive DNAs (Hi-a) in the genome from a population of the crucian carp, tentatively identified as the ginbuna (Carassius auratus langsdorfi), was isolated from the Hindlll digests and characterized. The Hi-a monomer (268 or 269 bp) was AT-rich (64.9%) with internal repetitive oligomers. The nucleotide similarity among monomers within the same individual was 88-98%, whose sequence alterations occurred mostly at restricted sites. Hybridization analyses revealed that the Hi-a family was organized into tandem array(s) representing satellite DNAs, and that its variants that share certain restriction sites of the repeat unit were dispersed into the tandem array(s) of Hi-a DNAs at varying periodicities. The lack of sequences homologous to the ginbuna Hi-a in the genomes from cyprinid fishes other than gengorobuna and goldfish suggests that Hi-a DNAs are specifically present in the species C. auratus. The genome of goldfish differed somewhat in the quantity or the genomic organization of Hi-a DNAs from that of the ginbuna.

14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that possess diverse biochemical activities such as regulation of gene transcription, cell proliferation and activation of protein kinase C. At least 7 subtypes (α to θ) of 14-3-3 protein are known, but the ζ subtype of this protein has been cloned only in mammals. We cloned the ζ subtype of 14-3-3 protein (14-3-3ζ from the frog, Rana rugosa. The sequence encoded 245 amino acids that share 92% identity with rat and bovine 14-3-3ζs, and 92% with human phospholipase A2 (PLA2; 14-3-3ζ). Northern blot analysis revealed a single band of about 1.8 kb in tadpoles at stage 25. The 14-3-3ζ mRNA level was high in the brain, lung, spleen and kidney, and low in the heart and testis, as opposed to the mRNA level, which was only faintly detected in the liver, pancreas, ovary and muscle. Furthermore, high similarity in the 3′-untranslated region (3′-UTR) was observed between frog and human 14-3-3ζ cDNA. The results suggest that 14-3-3ζ is highly conserved throughout eukaryotic evolution, and that the homologous sequence in the 3′-UTR of 14-3-3ζ cDNA may be conserved in frogs and humans.

Blue biliprotein, insecticyanin (INS), has been purified from the hemolymph of the sweet potato hornworm, Agrius convolvuli. The protein was efficiently isolated from the hemolymph of fifth instar larvae using three successive column chromatographic techniques: hydrophobic interaction chromatography, ion exchange chromatography and gel-filtration. The purified INS showed a native molecular weight of approximately 59,000 by gel-filtration. SDS-PAGE revealed a single band with Mr of approximately 26,000. Moreover, the molecular mass of INS was 21, 213 by MALDI-TOF/MS. Thus, the native A convolvuli INS molecule was assumed to be a trimer in solution. The blue coloration of A. convolvuli IMS from the hemolymph was attributed to the presence of biliverdin IXγ, due to the absorbance maxima at 360 and 695 nm, which was non-covalently bound with the apoprotein. Amino acid composition and N-terminal sequence of A. convolvuli INS is similar to M. sexta INS. A. convolvuli INS represents one of the biliverdin-binding proteins in lepidopteran insects.

The crustacean molt-inhibiting hormone (MIH) is released from the X-organ sinus gland complex and suppresses ecdysteroid synthesis by the Y-organ. In the present study, we have isolated a cDNA which encodes a MiH (Pej-SGP-IV) of the kuruma prawn Penaeus japonicus in order to study its expression and characterize the structure of its precursor. A cDNA fragment was isolated using RT-PCR with two degenerate oligonucleotide primers that were designed based on the peptide sequence of Pej-SGP-IV, and this fragment was used as a probe to screen an eyestalk cDNA library. In a positive cDNA clone (814 base pairs (bp)), an open reading frame of 315 bp was found; the conceptually translated protein consists of a putative signal peptide (28 residues) and Pej-SGP-IV (77 residues). In Northern blot analysis using a cDNA probe, specific hybridization to a transcript of 0.95 kb was seen in RNA extracted from the eyestalk but not from hepatopancreas, abdominal muscle, brain, thoracic ganglia or abdominal ganglia. The level of the Pej-SGP-IV mRNA in the eyestalk did not change significantly during the molt cycle.

cDNA encoding maternal cyclin E (HpCycE) has been cloned from the oocyte cDNA library of the sea urchin, Hernicentrotus pulcherrimus, by differential screening with a cDNA probe covering the total poly(A)⁺ RNAs of 16 cell-stage embryos and gastrulae. In this communication we describe similarity of amino acid sequences between HpCycE and those of cyclin E from other organisms and maternal origin of HpCycE. The amino acid sequence deduced from the nucleotide sequence of HpCycE cDNA is highly similar to those of human, rat, chicken, Xenopus, zebrafish and Drosophila, while its similarity to other cyclins is much lower. A gene for HpCycE exists as a single copy in the genome of H. pulcherrimus. Northern blotting revealed that the mRNA for HpCycE is maintained at a high level up to the morula stage and thereafter declines.

In the retina of the medaka four kinds of retinal cone photoreceptor cells are arranged in a simple, repeating organizational pattern known as a square mosaic. We found that the distribution of cones in the retina could be easily detected by autofluorescence-emission from the photoreceptor cells without any staining. In tangential sections of the retina, cones were located at a specific position in a crystalline lattice as follows: Double cone pairs display a zigzagging appearance, oriented roughly 90-120 degrees to one another, and single cones were in the center of the square consisting of four double cone pairs. In order to determine the continuity of this regular arrangement on the spherical surface, the distribution of this cone mosaic pattern was examined in central, dorsal, ventral, nasal and caudal areas of the retina. The regular arrangement of cones was confirmed in the whole retina. Double cones and single cones are in their respective lines and these lines form a lattice-work. As a result of reconstructing these arrangements on the retinal hemisphere, the lines of this lattice-work of cones were found to be orthogonal to the retinal margin, radiating from the center of the retina-like meridians, and parallel to the retinal margin forming concentric circles that is reminiscent of a longitudinal and a latitudinal lines of a terrestrial globe. This construction of the cone arrangement in whole retina of the medaka was consistent with maintaining a rectangular mosaic in growing retina with newly produced cells only in a marginal cell proliferating zone.

Transforming growth factor-a (TGFα) and epidermal growth factor receptor (EGF-R) mRNAs were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) in the normal and neoplastic mammary glands of four strains of mice with different mammary tumor potentials (from highest to lowest potential): SHN, GR/A, SLN and C3H/He. At 2 months of age, when the mammary glands of these strains consisted mostly of normal tissue, the samples examined showed the positive expressions of both TGFα and EGF-R mRNAs in all strains (4-6 mice per group), except for EGF-R mRNA in the SLN mice, expressed in only 2 of 4 samples associated with no end-bud formation in the mammary glands. At 10 months, all of the samples from all four strains had a positive expression of TGFα mRNA. The EGF-R mRNA expression paralleled the degree of the formation of preneoplastic hyperplastic alveolar nodules (HAN) in all strains. These findings indicate that TGFα and EGF-R participate in the growth of the mammary glands, and that EGF-R especially contributes to the formation of end-buds at younger ages and to that of preneoplastic HAN at later ages. All of the samples of mammary tumors from four strains had positive expressions of both TGFα and EGF-R mRNAs.

Calcitonin-producing cells (C-cells) are endocrine cells derived from the neural crest. We examined the effects of three types of protein kinase inhibitors on the induction of neuronal phenotypes in the rat thyroid C-cells in vitro. In a primary culture of 16-day-old fetal rat thyroid glands, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 25-75 μM) induced both process extrusion and expression of highly polysialylated neural cell adhesion molecule (NCAM) in the C-cells. These effects of H-7 were completely prevented by okadaic acid, a potent protein phosphatase inhibitor. In contrast to H-7, selective inhibitors for cyclic nucleotide-dependent protein kinases such as N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004, 25-200 μM) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89, 0.25-20 μM) failed to induce process extrusion or the expression of highly polysialylated NCAM in fetal rat C-cells. In cultured C-cells of adult origin, H-7 failed to induce marked process elongation or the expression of highly polysialylated NCAM. These results suggest that the morphological plasticity of the fetal C-cells depends upon the degree of phosphorylation of some proteins, and that the plasticity of adult C-cells are more restricted than that of fetal origin.

Postnatal development and differentiation of the masseter muscles consisting only of fast-twitch oxidative (FO) fibers in the adult Japanese field vole Microtus montebelli were studied using his-tochemical and electron microscopic techniques. The masseter muscles were composed of myotubes and muscle fibers at day 0 (birth day). Most muscle cells showed the strong reaction for myosin ATPase after both alkaline and acid preincubations. For NADH-dehydrogenase (NADH-DH), small granular diformazan deposits were recognized in the sarcoplasm. Afterwards, the masseter muscles consisted of myofibers and satellite cells at day 5. For myosin ATPase, weakly-reactive fibers after acid preincubation (fast-twitch fibers) increased in number. For NADH-DH, granular diformazan deposits in all the myofibers increased in size. Since all the myofibers had numerous sarcoplasmic reticula, and they reacted strongly after alkaline preincubation and weakly after acid preincubation for myosin ATPase at day 10 when the young start to take solid food, it seems that the masseter muscles become contractive fast. At day 15 (before weaning), all the myofibers showed the adult-like strong reaction for NADH-DH and had numerous well-developed mitochondria, thus they acquired the ability of the fast and sustained contraction. It is accordingly considered that the masseter muscles of the vole mature in a short time after birth because of adaptation for herbivorous food habit.

Labeling and immunoprecipitation experiments confirmed that three proopiomelanocortin (POMC)-derived peptides, the N-terminal peptide of POMC (NPP), joining peptide (JP) and adrenocorticotropic hormone (ACTH), were released by the bullfrog (Rana catesbeiana) anterior pituitary. The effects of these three peptides on luteinizing hormone (LH) release by bullfrog dispersed anterior pituitary cells were studied. NPP and ACTH, but not JP, enhanced LH release concentration-dependently. Approximately 6 hr elapsed before the gonadotrophs responded to NPP and ACTH by releasing LH, whereas their response to human GnRH (hGnRH) was faster, suggesting that the modes of action of these two peptides and hGnRH differ. These results raise the possibility that NPP and ACTH act as paracrine factors in the bullfrog pituitary to enhance LH release either directly or indirectly.

The calcitonin genes of four species of reptiles (Reeve's turtle, rat snake, grass lizard, and spectacled caiman) were amplified from the genomic DNA, as well as from the mRNA of the ultimobranchial glands of the former three species, by the polymerase chain reaction (PCR) method, and were sequenced. Among several primer sets, only one primer set synthesized from the chicken calcitonin gene was compatible with those of the reptiles. The nucleotide sequences of the reptile calcitonin genes were highly homologous with that of chicken calcitonin (100% for turtle, 99% for caiman, 96% for lizard and 93% for snake). The products amplified from mRNA by the RT-PCR method matched completely those from genomic DNA in the turtle, snake and lizard.

The androgenic gland hormone (AGH) is known to control sex differentiation in crustaceans. AGH was purified from isolated androgenic glands (AGs) of the male isopod Armadillidium vulgare by three steps of reverse-phase high performance liquid chromatography (RP-HPLC) and its chemical properties were examined. The purified AGH-active fraction showed masculinizing activity when 38 pg of this preparation was injected into a young female of the same species. Only 160 ng of the material was obtainable from 2000 animals at about an 11% rate of recovery. The elution of AGH activity by molecular sieve HPLC indicated that molecular weight of AGH was 11,000 ∼ 13,000. AGH was inactivated by treatment with trypsin or by reductive carboxymethylation. The AGH activity was not affected by heat treatment at 100°C for 3 min. These results indicated that AGH was a heat-stable protein with disulfide bond(s).

The cardiovascular effects of homologous natriuretic peptides (fNPs) were examined in the bullfrog, Rana catesbeiana. Synthetic bullfrog atrial natriuretic (fANP), brain natriuretic (fBNP) and C-type natriuretic peptides (fCNP I and fCNP II), were tested in vitro and compared under the same experimental conditions. All frog NPs produced a significant, and concentration-dependent, reduction in tension (relaxant effect) in the isolated dorsal aorta. Frog CNP II exhibited similar vasorelaxation profile as that of fANP, while fBNP and fCNP I had lower activity than fANP. Frog NPs inhibited norepinephrine induced contraction and fCNP II was most potent. In isolated preparations of atrium and ventricle, fCNP I and II produced a significant, and concentration-dependent, reduction in tension, but neither fANP nor fBNP produced any significant effects. Frog CNP II is most potent among fNPs in relation to reduce the cardiac output. Chronotropic responses of the heart to administrations of NPs were insignificant. The present results for the first time showed that fNPs play roles in the control of cardiovascular system, both blood vessels and heart, in the bullfrog.

Cold tolerance of overwintering nymphs of a cockroach, Periplaneta japonica, was examined in terms of the supercooling capacity and lower lethal temperature. The supercooling point of overwintering nymphs fell in a relatively narrow range of temperature from -6 to -9°C and no correlation was observed between the supercooling point and body size. In the temperature range from -5 to -8°C, a significant proportion of cockroaches could tolerate a 12 hr period of tissue freezing. The freeze tolerance capacity differed between nymphal instars, but the supercooling capacity was similar for all nymphs. In a freezing trial at -6 and -7°C, none of the first instar nymphs recovered after tissue freezing, whereas many mid (from 3rd to 5th) and final (8th) instar nymphs survived freezing. Glucose, myo-inositol, scyllo-inositol and trehalose were found in overwintering nymphs, but neither the array nor the content except for trehalose differed among the nymphal instars. Unexpectedly, the concentration of trehalose was negatively correlated to freeze tolerance. Winter survival of this cockroach may be based on both the freeze tolerance and microhabitat selection.

The karyotypes of the Drosophila robusta species-group, especially its species-subgroup okadai, were studied by using brain cell-Giemsa staining method. Drosophila gani possessed a diploid number of 12 chromosomes, comprising 4 pairs of metacentric and 2 pairs of acrocentric chromosomes, and both D. okadai and D. neokadai did 2n=12, with 3 pairs of metacentrics, 2 pairs of acrocentrics and 1 pair of microchromosomes. The okadai subgroup characteristically retained a middle-sized acrocentric × chromosome, which implies that this subgroup may occupy an ancestral position for the robusta group.

Genetic variation among populations of Gekko hokouensis from Kyushu, and the northern and the central Ryukyus was investigated by use of protein electrophoresis. From the results, the following three groups were recognized: Group 1, consisting of populations of Kyushu except for that of the southwestern part; Group 2, consisting of populations of southwestern Kyushu and the northern Ryukyus; and Group 3, consisting of populations of the central Ryukyus. Samples belonging to the Group 2 were genetically fairly uniform with low heterozygosities despite their wide geographic range that includes both the Oriental and Palearctic faunal regions delimited by the Tokara Tectonic Straits. This strongly suggests that the populations composing this group have spread over their current range recently. In addition, the degree of genetic differentiation between populations of the Groups 1 and 2 and the Group 3 (Nei's D: 0.083-0.175, x̄ = 0.129) was too small to have resulted merely from the deduced geographic isolation between the northern Ryukyu-Kyushu region and the central Ryukyus. This suggests that at least one of those two groups has colonized after formation of the Tokara Tectonic Straits by secondary dispersals. Circumstantial evidences seem to favor the secondary dispersals of the northern Ryukyu-Kyushu groups. Data for geographic patterns of allelic distributions and of intrapopulational genetic variabilities suggest that the southern Kyushu populations have received gene flow also from an extralimital source as well.