It has been believed that clonal propagation by asexual reproduction has serious disadvantages for long-term survival, because asexual reproduction seems not to remove harmful mutations, it seems not to give rise to genetic variations upon which evolution depends and it seems not to reset cell aging. In this article, we re-consider those arguments, by reviewing asexual reproduction of the tunicate, Polyandrocarpa misakiensis. Tracer experiments of bud formation and growth using morphological and chimeric phenotypes showed that the parental epithelial tissues surrounding the bud primordium do not enter the growing bud. It is possible, therefore, to assume that budding involves the purge of a large number of parental somatic cells and tissues. Unlike sexuals, asexuals do not carry out meiotic recombination nor gene shuffling that are two major sources of genetic variation, but we can show that in P. misakiensis at least two genes have significant redundancy and genetic variation even in a clonal colony. Telomerase expressed in germlines is thought to reset the molecular clock executed by telomere shortening. In our Polyandrocarpa cDNA projects, four out of about 2,000 cDNAs examined were matched with retroviral reverse transcriptase that is the catalytic subunit of telomerase, suggesting that telomerase might work in asexual reproduction. In P. misakiensis, dedifferentiation system is used to make new asexual generations. TC14 lectin plays an important role in the maintenance of multipotent but differentiated state of the formative tissue. It is antagonized by tunicate serine protease (TRAMP) that has striking mitogenic and dedifferentiation-inducing activities on the multipotent cells. This system would serve to delay aging of somatic cells. In conclusion, empirical arguments that asexual reproduction is disadvantageous to long-term life do not appear to be tenable to budding of P. misakiensis.

Japanese toad (Bufo japonicus) tracks the route to and from the breeding sites using the olfactory cues from the migration route and not from the destination (Ishii et al., 1995). We recorded a slow extracellular potential change (electro-olfactogram or EOG) evoked on the olfactory epithelium by applying an olfactory stimulus with an air stream. In September toads, only a simple typical EOG that is common in various vertebrate species was observed. Oscillatory potential changes (OSC) superimposed on the typical EOG were observed in the breeding season when studied throughout a year. There were no sexual differences in the occurrence and the amplitude of the OSC. Oscillatory potentials were observed also from the olfactory nerve of the brain. The OSC in the olfactory epithelium remained even after denervation. In addition, it was suggested that there are multiple sites of OSC initiation in the olfactory epithelium. These results suggest an intimate relationship between OSC appearance and the breeding migration in the toad.

Since highly concentrated NaCl is suspected to enter into the heart of the seawater eels, effects of high NaCl concentration on the atrial beating was examined, and plasma ion concentrations and osmolality were measured simultaneously in the blood collected from the bulbus arteriosus and from the caudal vessels. When 100 mmole l⁻¹ NaCl was added to the incubation medium, atrial contraction was enhanced significantly. Similar enhancement in the atrial contractility was also observed after addition of NaCH₃SO₄ (100 mmole l⁻¹) or Tris HCl (100 mmole l⁻¹), indicating that Na and Cl⁻ are not indispensable for the positive inotropic effect. Furthermore, an addition of sucrose (200 mmole l⁻¹) also enhanced the contraction. Inversely, hypoosmotic solution reduced the atrial contraction. These results indicate that the eel atrium is sensitive to environmental osmolarity. The eel atrium responses even at 20 mmole l⁻¹ sucrose. Such an inotropic effect of sucrose was not depressed after blocking adrenoceptor with betaxolol, a β₁-adrenoceptor antagonist, indicating that the effect is not due to adrenaline release from nerve endings. Plasma osmolality and Na concentration were higher in bulbus arteriosus than in caudal vessels, indicating that the eel heart is really exposed to hyperosmotic blood in sea water. The osmotically enhanced atrial contraction may increase the cardiac outflow into the gill. Such property of the atrium would have clear advantages for seawater teleosts, since the concentrated NaCl from the esophagus can be excreted immediately through the gill, without circulating their body, and blood homeostasis can be maintained efficiently.

Guanabenz, an I₂-imidazoline-related compound with high affinity for intestinal membrane of the eel (Kim et al., 1998), enhanced the transepithelial potential difference (PD) and short-circuit current (Isc) from serosa to mucosa after pretreatment with isobutylmethylxanthine (IBMX), serotonin (5-HT) and methacholine (MCh). The mucosal effect of guanabenz was not mimicked by adrenaline, indicating that the mucosal guanabenz binding site is not adrenoceptors. The mucosal guanabenz enhanced the Isc in a concentration-dependent manner. Similar enhancement in the Isc was also obtained after addition of other imidazoline derivatives such as ST93, clonidine, ST91, naphazoline and UK14,304 into the mucosal fluid. On the other hand, the effect of guanabenz was completely blocked by mucosal RX821002 or efaroxan, another imidazoline derivatives. Since some imidazoline derivatives act as agonists and others as antagonist, there must exist imidazoline receptor on the mucosal side of the eel intestine. Accompanied by an increase in the PD, NaCl and water absorption across the intestine was also enhanced by mucosal guanabenz. To search for endogenous ligands for the imidazoline receptor, luminal fluid in the intestine of the seawater eels was collected. However, most luminal fluid was ineffective. Only one among 10 samples showed guanabenz-like activity, suggesting that the endogenous ligands is secreted into the lumen under restricted condition alone.

In order to study the olfactory discriminating ability of lacustrine sockeye salmon (Oncorhynchus nerka) and masu salmon (O. masou), the integrated olfactory nerve response to various freshwaters was recorded by electrophysiological techniques. In both species independent of sex and gonadal maturity, each freshwater caused a different olfactory response. Source and effluent waters of the culture pond at Toya Lake Station (the source and culture pond waters) evoked the minimum and maximum response magnitude, respectively. In cross-adaptation experiments, the culture pond water abolished all secondary responses to other freshwaters, and no freshwater abolished the secondary response to the culture pond water. The concentration response study revealed that the minimum concentration (threshold) to induce response to the culture pond water after adaptation to Lake Toya water was between 0.1 and 1.0%. The present study indicates that the olfactory organ of lacustrine salmonids may discriminate different intensities of various freshwater odors.

Siberian chipmunks are known to gnaw snake-related objects such as skin, urine, feces and anal sac excretion, and apply the gnawed bits to their body fur repeatedly (SSA behavior). After SSA, chipmunks often rub their bodies on nearby wood stumps and branches. The SSA behavior is surmised to have function of information-spreading on snakes to neighboring chipmunks. In the present study, responses of the other chipmunks towards SSA individuals were experimentally analyzed to examine the possibility of this hypothesis. In the experiments, subject chipmunks were presented with 2 kinds of objects; (1) SSA-performed chipmunks in boxes and non-performed chipmunks in boxes, (2) wood boards to which the scent of SSA-performed chipmunks adhered and wood boards to which the scent of non-performed chipmunks adhered. In both cases of (1) and (2), subject chipmunks nosed significantly longer the scent from SSA-performed chipmunks and that they performed SSA only in the contact with the former.

Individually marked males of Pieris rapae crucivora, were observed to determine how they allocate time to reproduction and feeding. Males were found to alternately feed and search for females. This manner of time allocation persisted throughout the day. The total times that males allocated to the two behaviors were positively correlated, i.e. those males that spent longer searching for females, also feed for longer periods. Males, however, tended to allocate more time to the female-searching in the morning than in the afternoon, while time allocated to feeding throughout the day. Older males spent more time searching for females in the morning. The body weight of male butterflies also changed as they aged. The results are discussed in terms of both proximal and ultimate aspects of female-search.

The present study is designed to clarify the mechanism by which the circadian pacemaker controls the locomotor activity of the hagfish and also to estimate the role of brain and spinal cord in the swimming behavior of the animal. We examined the effect of cutting the spinal cord at the 6 different positions on the circadian rhythm and the locomotor behavior of the animal. The most frontal cut was located between the brain and spinal cord, and the other 5 cuts were given to every 1/6 the length of the spinal cord. The relation between the locomotor activity and the cut position of spinal cord was summarized as follows. (1) When the ratio of frontal part before the cut was 0/6–1/6, the animal locomoted under initiative of caudal part, in random direction at the bottom and showed neither nocturnal rhythm in LD nor circadian rhythm in DD. (2) When the ratio of the frontal part before the cut was 4/6–5/6, the animal swam up to the surface under initiative of frontal part, and showed both nocturnal rhythm in LD and circadian rhythm in DD. (3) When the frontal ratio of spinal cord was 2/6 or 3/6, the animal showed both kinds of swimming behavior of (1) and (2). These results suggest that the descending system from the brain enable the hagfish to swim up to the surface and to express the rhythmicity of locomotor activity under control of the circadian pacemaker when at least frontal 2/6 of the spinal cord is connected to the brain by neuronal networks not by humoral factors.

The usefulness of microsatellite markers in pedigree analysis of the sika deer (Cervus nippon) was tested in a herd in which the maternal lineages were recorded. Eighteen sets of microsatellite primers originally designed for bovine, ovine, and cervine loci successfully amplified polymorphic DNA in the deer. The numbers of alleles per locus ranged from two to seven, and the observed heterozygosity ranged from 0.350 to 0.900. The resolution power of the markers in paternity testing was then determined by calculating exclusion probabilities and paternity indices. Parentages of the study population were efficiently discriminated by genotyping 17 microsatellite loci. The microsatellite data were also used to calculate the genetic relatedness between individuals, which significantly correlated with coancestry coefficients for the pairs. Our results demonstrate that the microsatellite markers are efficient tools in studying the social structure and behavior of the sika deer, as well as in monitoring the inbreeding status.

Bacterized plant infusion is a popular culture medium for Paramecium, using Klebsiella pneumoniae for the bacterium and Wheat Grass Powder (WGP) for the plant. It has been thought that WGP plays a role in the growth of bacteria, which in turn serve as the direct food for paramecia. However, we found that bacteria suspended in saline solution were unable to support the growth of paramecia. WGP including no bacteria was able to support neither the growth nor the survival of paramecia; instead, it killed paramecia. The killing effect of the WGP-derived substance(s), estimated to be of molecular weight less than 1,000, was abolished when bacteria were once grown in the WGP and then eliminated, suggesting that bacteria might change the toxic substance into an inactive form. This inactivation of the toxic substance may be caused either by metabolization inside of the bacteria or by neutralization by means of bacteria-derived substance outside of the bacteria. The second alternative is likely, because paramecia were able to survive and grow in the WGP medium containing a sufficient amount of dead bacteria killed by formalin or kanamycin. Dead bacteria killed by autoclaving were ineffective, probably because bacterial contents were lost. These findings revealed an ectosymbiotic role of bacteria; they confer benefits upon paramecia not only as food but also as machinery to detoxicate a plant toxin.

Earlier, it has been demonstrated that wild populations of a Japanese harvestman Metagagrella tenuipes (Arachnida: Opiliones) are polymorphic for B chromosomes. In this paper, we present results of a study of the morphology and mitotic and meiotic behavior of the Bs. The B chromosomes varied considerably in size and proportion of eu- and heterochromatin. The single nucleolus organizing region, found in males, was located on a chromosome of the A complement. Some intercell variation in number of Bs may be explained by accidental chromosome losses during chromosome preparation. We also found no intertissue variation in number of Bs. There were also no differences in mean number of B chromosomes per individual among males and females, adult and subadult harvestmen. Segregation of Bs in mitotic and meiotic divisions was nonrandom; B chromosomes tended to segregate equally between daughter cells. The results obtained provide no support for the hypothesis of existence of B accumulation mechanism in this species.

The population structure of genus Carassius in Lake Koyama, southeast Japan, was analyzed by genetic markers as microsatellite DNA, mtDNA RFLP and isozymes. Based on the ploidy level and morphological analysis, four Carassius groups were detected. The triploid group was identified as Ginbuna (C. langsdorfii). In the diploid group, Nagabuna (C. burugeri sp) and Gengoroubuna, (C. cuvieri) were identified. Remaining diploid individuals had morphological traits that were intermediate between Nagabuna and Gengoroubuna. These were considered as hybrids and their descendants. From the results of mtDNA RFLP and isozyme patterns, the triploid population was considered to be independent from the gene pools of diploid. The hybrids had the mtDNA haplotypes which were common to Gengoroubuna and Nagabuna populations. Based on the three microsatellite loci, Ginbuna was classified into six clonal lines. In the diploid population, substitution of the major alleles of Nagabuna and Gengoroubuna were found. The hybrids had alleles that were common in Nagabuna and Gengoroubuna. The values of the hybrid index (I_H) which are ranged from 0.771 to 0.964 in Nagabuna, from 0.102 to 0.806 in the hybrids and from 0.068 to 0.157 in Gengoroubuna. The hybrid population was verified to be derived from crossbreeding between the Gengoroubuna and Nagabuna populations. Evidence of backcrossing in nature by microsatellite DNA markers was also obtained in the diploid populations.

The Cdc2-cyclin B complex (named the M-phase-promoting factor, MPF) is well known to be a key regulator of G2-M transition in both mitosis and meiosis. However, MPF may have functions other than the cell cycle regulation, since its activity is detectable in post-mitotic (or post-meiotic) non-dividing cells. Cyclin B comprises several subtypes, but their functional differences are still unknown. Despite the established function of MPF during oocyte maturation, its role during spermatogenesis, where spermatogenic cells undergo drastic morphological changes after meiosis, remains to be elucidated. To address these issues, we have isolated cDNA clones encoding cyclins B1 and B2 from medaka testis and raised polyclonal antibodies against their products. Using these as probes, we examined the expression patterns of cyclins B1 and B2 in medaka testis at both mRNA and protein levels. Cyclin B1 and B2 mRNAs were expressed in all stages of spermatogenic cells except for spermatozoa, although the expression levels varied according to the spermatogenic stages. Cyclin B1 protein was expressed only in spermatogonia and spermatocytes at prophase and metaphase with a transient disappearance at anaphase. On the other hand, cyclin B2 protein was continuously expressed throughout spermatogenesis, even in spermatogonia and spermatocytes at anaphase and in post-meiotic spermatids and spermatozoa. The difference in their expression patterns suggests that cyclins B1 and B2 have distinct roles in medaka spermatogenesis; i.e., cyclin B1 controls the meiotic cell cycle, whereas cyclin B2 is involved in process(es) other than meiosis.

Planarians can propagate asexually by fission and successive regeneration. During head regeneration, they again form a new pair of eyes, and sometimes supernumerary eyes. The positions of normal and supernumerary eyes and their regeneration abilities are expected to be highly relevant to the question of where and how the field to regenerate eyes is determined. In this study, spontaneously generated supernumerary eyes were classified into various types. In all cases, they were formed in the anterior part of the head. Enucleation of a normal eye elicited regeneration of a new eye; however, enucleation of a supernumerary eye did not. The supernumerary eyes were morphologically and functionally indistinguishable from the normal eyes, revealed by the studies of immunohistology and photophobic response, respectively. From the obtained results, we proposed a model of the eye regeneration field that changes its distribution spatiotemporally during regeneration. Immunohistological studies also showed that the optic nerves from the normal and supernumerary eyes ran independently, which might have implication about the nature of guidance cues for the optic nerves.

A cDNA clone for a sea cucumber T-box gene was isolated and characterized. Based on molecular phylogenetic analysis it is concluded that the putative gene Hl-Tbr, encoded by the cDNA clone, is a T-box gene of the T-brain subfamily and hence a homolog of the mouse T-brain-1 (Tbr-1) as well as of the Xenopus Eomesodermin. In situ hybridization analysis of whole mount specimens showed that Hl-Tbr was expressed in the invaginated cells at the early gastrula stage and the expression of the gene was scarcely detectable by the end of the late gastrula stage.

In Drosophila ananassae, male remating was studied using ten mass culture stocks which were initiated from flies collected from different geographic localities. Male remating occurs at a high frequency and varies within narrow limits (84–96 percent) in different strains. Interestingly, male remating time (in min) varies from 7.41 (Bhutan) to 21.59 (PAT) in different strains and the variation is highly significant. Further, the results also show that males copulate for shorter duration during second mating. This is the first report in the genus Drosophila which provides evidence for interstrain variations for male remating time as well as for shorter duration of copulation during second mating as compared to first mating in D. ananassae.

A heterologous radioimmunoassay was developed for measuring gonadotrophin-II (GTH-II) in the catfish Clarias batrachus. Serum and/or pituitary levels of GTH-II showed significant annual/seasonal variations in male and female catfish, which could be correlated with both gonadosomatic index and/or serum testosterone level. GTH-II was not detected in resting phase, increased during gonadal recrudescence to peak values in late prespawning /spawning phases, and declined to low values in postspawning phase. During gonadal recrudescence, the pituitary and serum levels of GTH-II maintained positive or inverse relationships implying differential rates of hormone release and synthesis/storage. Gonadectomy resulted in increased release of GTH-II; the release pattern varied in females and hemi-castrated or completely castrated males. In females, the GTH-II increase followed a distinct biphasic pattern with the peak rise at week 4 of ovariectomy. In males, castration resulted in significant rise of serum GTH-II levels at all duration except week 5, but the magnitude of the rise was higher in completely castrated fish (weeks 1, 2 and 3). Testosterone replacement in 3-week hemi-castrated fish restored the GTH-II level to that of the sham control vehicle group. In intact fish, administration of testosterone elicited an increase in serum GTH-II levels in the low dose (0.25 and 0.5 μg / g BW) groups and no change in the high dose (1.0 μg / g BW) group. Methallibure treatment inhibited GTH-II levels in a dose-dependent manner. The reduction was greater in males. Withdrawal of the drug treatment restored the GTH-II and testosterone levels after 15 days in the low dose group (2 μg / g BW). The results indicate that there exists a dynamic positive or negative feedback relationship between gonadal steroids and GTH-II, which is essential to control the release and availability of circulating GTH-II.

To investigate the phylogenetic relationships between the New World Sciurus and the Old World Sciurus and their biogeographic history, the partial mitochondrial cytochrome b gene sequences (1,040 base pairs) were analyzed on six Sciurus species: S. aberti, S. carolinensis, S. lis, S. niger, S. stramineus, and S. vulgaris. Phylogenetic trees (maximum parsimony, neighbor-joining, and maximum likelihood methods) commonly showed two groups with high bootstrap values (73–100%): one consisting of the New World Sciurus and the other consisting of the Old World Sciurus. Genetic distances among the New World Sciurus species were remarkably larger than that between two Sciurus species of the Old World, suggesting the earlier radiation of the New World Sciurus than the Old World Sciurus.