Pigment-dispersing factor (PDF), an 18-amino acid neuropeptide, is a principal circadian neuromodulator functioning downstream of the insect brain's circadian clock, modulating daily rhythms of locomotor activity. Recently, we found that PDF precursors of the cricket Gryllus bimaculatus comprise a nuclear localization signal (NLS). Moreover, the nuclear localization of PDF immunoreactivity and the trans-location of GFP-fused PDF precursor into the nucleus have both been demonstrated. These suggest a fundamental role for PDF peptide in the circadian clock system within the nucleus, in addition to its role in downstream neural events. In the present study, we carried out the cDNA cloning of PDF from adult brains of the last-summer cicada Meimuna opalifera, and found that an isolated clone (545 bp) encodes an ordinary PDF precursor protein. PDF peptide itself shows a high sequence identity (78–94%) and similarity (89–100%) to insect PDFs and also to the crustacean β-PDH peptides. The computer-assisted sequence analysis of PDF precursor revealed a possible translocation into the nucleus, despite the lack of a definite NLS-like sequence. Using immunocytochemistry, the optic lobes of M. opalifera revealed PDF-immunore-active neurons in both the medulla and lamina neuropiles. All these PDF cells exhibited prominent immunolabeling of both their perikarya and axons, but not their nuclei. Our results provide the first structural and immunocytochemical identification of PDF neurons in Hemiptera.

An increase in the concentration of Ca² ions in the external medium ([Ca² ]_o) induced pigment aggregation in melanophores of three species of freshwater teleosts examined. Denervated melanophores were refractory to elevations of [Ca² ]_o. The pigment-aggregating action was inhibited by the sympathetic blocking agents, phentolamine, prazosin and yohimbine. Bretylium, an agent known to block the release of the neurotransmitter, interfered with the response effectively. Ca² blockers, such as Mn² , verapamil and gallopamil, also inhibited the response, possibly by inhibiting Ca² entry into the presynaptic elements of melanosome-aggregating fibers. The conclusion is that the increase in [Ca² ]_o may induce membrane depolarization of presynaptic nervous elements around the melanophores, which open the voltage-dependent Ca² channels there. The liberation of adrenergic neurotransmitter follows, which induces the aggregation of pigment in melanophores.

The circadian clock of Drosophila melanogaster is thought to include rhythmic expression of period gene. Recent studies suggested, however, that a per-less oscillation is also involved in the regulation of circadian locomotor rhythms. In the present study, we examined the existence and the property of the possible per-less oscillation using arrhythmic clock mutant flies carrying per⁰¹, tim⁰¹, dClk^Jrk or cyc⁰¹, which lack rhythmic per expression. When temperature cycles consisting of 25°C and 30°C with various periods (T=8∼32 hr) were given, wild-type (Canton-S) flies showed locomotor rhythms entrained to temperature cycles over a wide range of period (T=8∼32 hr) in constant light (LL) while only to T=24 hr in constant darkness (DD). The mutant flies showed rhythms synchronizing with the given cycle both under LL and DD. In per⁰¹ and tim⁰¹ flies, the phase of a major peak slightly changed dependent on Ts in DD, while it did not in dClk^Jrk and cyc⁰¹ flies. When they were transferred from a constant temperature to a temperature cycle under DD, several cycles were necessary to establish a clear temperature entrainment in per⁰¹ and tim⁰¹ flies. These results suggest that per⁰¹ and tim⁰¹ flies have a temperature-entrainable weak oscillatory mechanism and that the per-less oscillatory mechanism may require dClk and cyc. In addition, per⁰¹ and tim⁰¹ flies changed from thermoactive in DD to cryoactive in LL, while dClk^Jrk and cyc⁰¹ flies did not. It is thus suggested that dClk and cyc are also involved in determining the light-associated temperature preference in per⁰¹ and tim⁰¹ flies.

The mating behavior of the domesticated house musk shrew was observed in detail under laboratory conditions. The observations revealed that the house musk shrew has a series of behaviors before copulation. Tactile, auditory and chemical senses appear to function as flags for the recognition of conspecifics and to promote the development of an interaction between the sexes. The tactile senses and the use of the snout were particularly important in the mating sequence, and mutual contact appeared to give rise to driving the sequence to completion. The two sexes contacted each other ‘politely’, came to mounting by continuous following, and the male finished with a series of post-ejaculatory offensive behaviors and scent markings. The variation in the contact reduced once the female commenced tail-wagging. The ratio of the time spent in front-and-behind contact to that spent in multi-lateral contact increased when both sexes commenced following formation. This ratio was maintained until the male's post-ejaculatory offensive behavior finished. The series of mating behavior was completed by the continuous touching of both sexes and by changes in the manner of contact.

The drumming calls of two perlodid species from Southern Spain (Sierra Morena, Jaén) are analyzed and described. Nineteen calls from one male of Guadalgenus franzi were recorded, showing a simple call pattern composed of two beats with an average duration between them of 184.32 msec (range: 165–227). This is the simplest call pattern yet discovered in the Plecoptera. The female of this species also presented a simple call consisting of only one beat. The male of Isoperla pallida showed an ancestral call pattern, more simple than the other Iberian Isoperla species (I. grammatica, I. nevada or I. curtata, all of them with multiphasic drumming calls or I. bipartita with a notably different model consisting of rubbing). Calls from six different males (the female did not drum) consisted of an average of 65.94 beats (range: 25–83) with a duration average of 1462.69 msec (range: 548–1835), regularly repeated with intervals with a duration average of 22.27 msec (range: 17–30). With this study, the vibrational calls of all the Perlodid species from the Southern Iberian Peninsula, except Perlodes microcephalus and Besdolus bicolor, are known. Moreover, it presents the first record and description of the call of the monospecific genus Guadalgenus. An evolutionary approach to the vibrational communication diversification in the perlodids of the Southern Iberian Peninsula is presented, showing a complexity graduation from the more simple drumming calls (shown by Hemimelaena flaviventris and G. franzi) to the rubbing call of I. bipartita and intermediate drumming calls of other Isoperla species, consistent with the drumming evolution paradigm of Stewart (2001).

A large number of autotetraploids of Rana nigromaculata were produced in order to assess their developmental capacity and chromosome number in their offspring. An original autotetraploid male was first produced by transplanting a nucleus from an embryo (triploid) into an unfertilized egg. Next, eggs were inseminated with sperm of the autotetraploid male, then cold-treated to obtain first-, second-, and third-generation offspring. According to an investigation of the chromosome numbers of the tadpoles by the tail-tip squash method, the three generations of offspring included many tetraploids (50–80%), as well as some diploids, triploids, hexaploids and mosaics at the early tadpole stage. In addition, several percent of the second- and third-generation offspring were found to be aneuploids. Evidently, a complete set of diploid chromosomes was not precisely transferred to all of the next-generation offspring from the sperm of the artificially produced autotetraploid males. These observations suggest that there were some abnormalities in the course of spermatogenesis in the male autotetraploid frogs.

Ascidians are lower chordates that possess a possible prototype of the vertebrate nervous system. The central and peripheral nervous systems of ascidian larvae are composed of only a few hundred cells (Nicol and Meinertzhagen, 1991). To investigate how these ascidian nervous systems develop, dissection at the molecular level using subset-specific markers is essential. Here we describe four new genes zygotically expressed in subsets of the ascidian neural cells. The spatial expression domains of these genes overlap in some parts but not in other parts of the nervous systems. Our results suggest that there are functionally different regions in the nervous systems owing to the gene expression differences. Further analyses of these genes will enable us to determine the molecular neuro-developmental characteristics of various clusters of neural cells.

Circadian changes in serum concentrations of testosterone (T), 11-ketotestosterone (11KT), estradiol-17β (E2), 17α,20β-dihydroxy-4-pregnen-3-one (DHP), 17α-hydroxyprogesterone (OHP), cortisol (F) and progesterone (P) were investigated in the spermiated/ovulated Japanese char Salvelinus leucomaenis for over three days using newly developed time-resolved fluoroimmunoassays. Testosterone and DHP in both sex and 11KT in male showed significantly (P<0.05) higher serum levels just before/after onset of darkness (15:00 or 18:00), and the levels during night and daytime were significantly (P<0.05) lower than those of the peak levels. Serum F levels in both sex during dark phase were significantly (P<0.05) higher than those levels during daytime. A surge of serum OHP concentrations in both sexes was observed at the time of twilight (03:00). The peak time of serum T, 11KT and DHP levels were approximately 6 hours prior to those of serum F and OHP levels. Serum E2 in female and P in both sex fluctuated intensely during sampling period, and did not show remarkable changes. These results strongly suggest the existence of circadian-like diel changes in serum T, DHP, F and OHP levels in both sex and 11KT in male, and no variations in serum E2 in female and P in both sex in spermiated/ovulated Japanese char under the stage of final maturation. Furthermore, relationship between circadian rhythms of steroid hormones and spawning behaviors are discussed in the present study.

We investigated the mechanism of egg transport in the newt not only by inserting various conditioned eggs into the recipient's body but also by placing them on the coelomic epithelia of the opened body cavity in the adult female newt. Most of the inserted coelomic eggs were oviposited, while 4 of 14 inserted de-jellied uterine eggs and 3 of 10 inserted de-jellied fertilized eggs were oviposited. The coelomic eggs placed on the coelomic epithelia were transported toward the ostium and entered the ostium. The de-jellied uterine eggs and the de-jellied fertilized eggs were transported to the ostium as well. Of all the eggs examined, the coelomic egg was transported the fastest. The transport speeds of coelomic eggs treated with periodic acid and the speed of boiled coelomic eggs were less than those of untreated coelomic eggs. In contrast, the transport speeds of coelomic eggs treated with trypsin and the speed of coelomic eggs removed from their vitelline envelopes (naked eggs) were faster than those of untreated coelomic eggs. Other experiments were carried out in order to ascertain the dependence of sexual activity on egg transport. The speed of coelomic egg transport in artificially sexually activated females was faster than in sexually inactive females, although the ciliary movement could always be observed in both sexually active females and sexually inactive females. This suggests that the speed of egg transport on the coelomic epithelia is controlled by the sexual activity of the female.

Dietary data were ascertained for 229 T. stejnegeri (snout vent length >300mm) from 36 localities throughout the main island of Taiwan and the outlying Orchid (Lanyu) and Green (Ludau) Islands. Twenty nine percent of the snakes were devoid of any prey, and of the snakes containing prey, 43% of the cases were unidentifiable. This relatively large proportion of unidentifiable prey items (observed in the hindgut) may reflect either rapid digestion of amphibian prey and/or rapid venting of feces as an evolutionary adaptation to arboreal life. Trimeresurus stejnegeri appears euryphagous, taking primarily amphibians, but additionally reptilian, mammalian and insect prey. There was no discrepancy in prey composition based on comparisons of where the prey item was recorded in the digestive tract. No sexual variation in diet composition was evident, although males were more likely to contain prey than females, indicating the utilisation of different foraging strategies on similar sympatric prey items. Variation in diet composition was observed between mainland Taiwan and offshore islands, which is most likely the result of differences in prey availability.

A preliminary study was conducted on phylogenetic relationships among some selected genera of the Australian Spilomelini, focusing on relationships among the Australian Glyphodes group (Glyphodes Guenée, 1854, Dysallacta Lederer, 1863, Talanga Moore, 1885 and Agrioglypta Meyrick, 1932) and the 17 genera which are morphologically similar to it. Representatives of three genera of the Pyarustini were used as outgroups. Cladistic analysis of morphological data from the adult moths produced 10 equally MP trees (length = 221, CI=0.294, and RI=0.608). The clade formed by the 21 selected genera of the Australian Spilomelini had low bootstrap support even though a good apomorphy supported the monophyly of this group, namely, a strong, bilobed praecinctorium of abdominal tympanal organs. The analysis showed that the Glyphodes group is not monophyletic because the genus Chrysothyridia Snellen appears within it in the 10 MP trees. The concept of the Glyphodes group should be expanded to include Chrysothyridia and also the Synclera Zeller and Didymostoma (Walker) since the Synclera Didymostoma clade, as the hypothesised sister group of the Glyphodes group, is not sufficiently supported as a separate monophyletic group. The analysis also showed that genus Glyphodes is not a monophyletic group, while Metallarcha Meyrick is a monophyletic group.

Vorticellid ciliates generally dwell in freshwater. In nature, the species have up until now been identified by comparison with previous descriptions. It is difficult to identify between species of the genus Vorticella, because the morphological markers of vorticellid ciliates described in reports are limited and variable. Unfortunately, culturing them has only succeeded with certain species such as Vorticella convallaria, but many others have been impossible to culture. To find out whether the sequence of a small subunit rRNA gene was an appropriate marker to identify vorticellid ciliates, the gene was aligned and compared. Finding a new convenient method will contribute to research on vorticellid ciliates. In strains of V. convallaria, classified morphologically, some varieties of the SSrRNA gene sequences were recognized, but there were large variations within the same species. According to the phylogenetic tree, these strains are closely related. However, the difference was not as big as between Vorticella and Carchesium. In addition, Carchesium constructed a distinct clade from the genus Vorticella and Epistylis. These results show the possibility that the SSrRNA gene is one of the important markers to identify species of Vorticella. This study is first to approach and clarify the complicated taxa in the genus Vorticella.

Three new species of the genus Gammarus are described from Lijiang, Yunnan Province, South China. Gammarus elevatus sp. nov. is characterized by mid-dorsal keel on pleonites 1-3 and compressed elevation on urosomites 1-2; G. denticulatus sp. nov. by many small spinules and setae on pleonites 1-3; G. stagnarius sp. nov. by lack of calceoli on antenna 2 and shorter inner ramus of uropod 3. These amphipods are compared with other known Gammarus species from China.

Extant crinoids can be divided into two groups, stalked sea lilies and stalkless feather stars. Feather stars are considered to have evolved from stalked ancestors by losing most of the stalk, but other differences are present between the two groups. The unsegmented centrodorsal, long and curved cirri near the crown, small calyx, and the ability to swim are all feather star features not found in the sea lilies. To figure out which of the above features evolved directly correlating with loss of the stalk in feather stars, we cut off the stalk from the sea lily Metacrinus rotundus and kept them alive in an aquarium. The specimens with shortened stalks were able to stand and crawl with their arms without the support of their stalks, but swimming was not observed for any of the animals. Morphologically, neither fusion of the remaining segments nor the reduction of the size of the calyx were observed, but the cirri became long and curved near the crown. Therefore, the extant sea lilies possess a potential to adapt to incidents of stalk loss. Specimens autotomizing most of their stalks were observed, suggesting that the potential is actually employed in nature. This mechanism linking the reduction of the stalk and the changes in the morphology of cirri may have played an important role in the evolution of the feather stars, if the stalked ancestors of feather stars also possessed this potential. Experimental zoological approaches as this study may provide new insights to the questions of evolution.