Staining with paraldehyde-thionin-paraldehyde-fuchsin labeled 10–14 median neurosecretory cells (MNC) in each hemisphere of the brain of diapause and nondiapause adults of the blow fly, Protophormia terraenovae (Diptera: Calliphoridae). Various surgical operations were performed on the brain-retrocerebral complex of female adults to investigate the role of these cells in ovarian development and diapause. Under the diapause-averting conditions of LD 18:6 (18 h light and 6 h darkness) and 25°C, most of the intact and sham-operated females had vitellogenic ovaries, whereas all of the females with the MNC completely removed had immature ovaries. The corpus allatum (CA) was significantly smaller in the MNC-removed females than in the intact and sham-operated ones. After severance of the nervi corporis cardiaci, most of the females had immature ovaries. Even after severance of the cardiac recurrent nerve, there were vitellogenic ovaries in about 60 % of the females. However, after removal of the corpus cardiacum and hypocerebral ganglion complex (CCHG), no females had vitellogenic ovaries. Under the diapause-inducing conditions of LD 12:12 and 20°C, most females had immature ovaries and the CA was small irrespective of the surgical procedures. The results indicate that the MNC may secrete an allatotropic factor to stimulate vitellogenesis. The factor seems to be released to the hemolymph from the CCHG and a part of the aorta.

Anatomical locations of the photoreceptor and circadian pacemaker for the locomotor activity rhythm were investigated by removal of the external photoreceptors, the compound eyes and ocelli, or the optic lobes in the adult cricket, Dianemobius nigrofasciatus (Orthoptera: Gryllidae). The activity rhythm of intact crickets freeran under DD with a freerunning period (τ) of 24.6 ± 1.0 h (mean ± SD) and entrained to light-dark cycles of LD 13:13 and LD 12:12. When both compound eyes were removed, some crickets entrained both to LD 13:13 and LD 12:12. Some of the others entrained only to LD 12:12 or showed a complex pattern of the activity. When both compound eyes and all ocelli were removed, some crickets still entrained both to LD 13:13 and LD 12:12. The activity pattern of the crickets receiving a sham operation or unilateral removal of the compound eye was not different from that in the intact crickets. Bilateral removal of the optic lobes caused arrhythmicity both under LD 13:13 and DD, although the activity rhythm in crickets of which the optic lobe was unilaterally removed entrained to LD 13:13 and freeran under DD. These results suggest that D. nigrofasciatus possesses its circadian pacemaker in the optic lobe, and uses both extraretinal photoreceptors and compound eyes for its entrainment.

A calling female of Bombyx mori every several seconds lifts the abdomen to extrude pheromone glands. The amplitude of the abdominal movement changes with alternation in the flow of haemolymph due to rhythmic heartbeat reversal. The first forward beating pulse usually occurs at the lowered position of the abdomen and subsequent forward beating pulses are often inhibited by elevation of the abdomen. During a calling period in the photophase or in the early scotophase, the duration of backward beat phases is significantly longer than that of forward beat ones. When a female terminates calling behavior in the late photophase and the late scotophase, backward phases become shorter and forward phases become longer. Similar changes in length of the forward and backward beat phases occur after elimination of calling behavior by mating and by transection of the ventral nerve cord. These observations suggest peripheral modulation of heartbeat reversal rhythm. The longer duration of the backward beat phases may be suitable to maintain the calling posture and for efficient transport of metabolites and tracheal ventilation in the abdomen with a high rate of metabolism during calling behavior.

Copulation duration differed between black and brown morphs of Drosophila elegans: longer in the former than in the latter. Experiments on copulation between these two morphs revealed that copulation duration was determined by both sexes. The genetic analyses using F₁ hybrids and recombinant inbred lines suggest that two or more loci were responsible for the differences in both of male and female properties for the determination of copulation duration between the black and brown morphs and at least one of the loci governing the male property was probably located on the X chromosome. It also appeared that loci responsible for the difference in copulation duration of males between the brown morph and black morph strains differed from those responsible for the difference in copulation duration of females between them. Genes controlling male copulation duration are at least partly linked with a gene controlling body coloration.

The spinning behavior of the silkworm, Bombyx mori, was recorded on videotapes from two angles and analysed by three dimentional computer graphics using the Japanese (J. 124), Chinese (C.124) and their hybrid (J.124 × C.124) strains. These strains constructed typical peanut-shaped, spherical and ellipsoidal cocoons, respectively. Linear representation of the spinning posture revealed that larvae fixed the posterior half of the larval body (6th to 13th segment) and spun silk moving their anterior half (1st to 5th segment) for the most spinning period in all strains used. Little difference was observed in the average spinning speed among them. The Japanese strain spun primarily in a S-letter posture and changed its direction frequently. The larva of Chinese strain often assumed a C-letter posture and showed directionchanging behavior with comparatively lower frequency. The hybrid larva threw the head back largely in an U-letter shape during most of the spinning period and showed cocoon expansion behaviors most frequently. The cocoon expansion behavior occurred mainly at both ends of the peanut-shaped cocoon (J.124), at the center part of the spherical cocoon (C.124) and at both shoulders in the ellipsoidal cocoon of the hybrid strain. Thus, there exist strain-specific features in the spinning behavior, and it is suggested that the main behavioral factors affecting cocoon shape formation are the spinning posture and the cocoon expansion behavior during spinning.

In the normal embryo of Xenopus laevis, the central nervous system (CNS) and epidermis, a pair of main ectodermal tissues, are derived mainly from the animal dorsal (AD) and animal ventral (AV) blastomeres, respectively. The defect embryo, from which all AD blastomeres have been removed at the 16-cell stage, can develop into a normally proportioned embryo, i.e., a regulated embryo, despite the striking deficiency of the prospective CNS. To compare the contribution of the AV blastomeres to the CNS and epidermis between the normal and regulated embryos, each of the AV blastomeres was labelled by a tracer injection at the 16-cell stage, and a clonal domain originating from the labelled blastomere in these ectodermal tissues was examined. By the removal of the AD blastomeres, the clonal domains of the each AV blastomeres were expanded in a dorsal direction, and covered the regions not only in the epidermis just as in normal embryos but also in the CNS extending from the anterior to the posterior end, respectively. Most of the lost prospective regions of the AD blastomeres in the ectodermal tissues were compensated not by the descendants of all the remained blastomeres in the defect embryo, but by the progeny of the AV blastomeres extending dorsally. These facts suggest that compensation for the lost prospective CNS owes mainly to the regulation in the prospective ectoderm, and spreading of the prospective ectoderm is progressively directed after the 16-cell stage by interaction with other parts of the embryo.

In sperm of echiuroid, oyster and sea urchin, which had been incubated in sea water at 20°C for 15 hr, the respiratory rate, which was lower than in fresh sperm, was enhanced by light at 430, 530 and 570 nm more strongly than at the other examined wavelengths. In fresh sperm, respiration was not affected by light. This incubation resulted in marked decrease in the activity of NADH cytochrome c reductase and the amount of cytochrome b in these sperm, more evidently than appreciable decrease in the cytochrome c oxidase activity and the amount of cytochrome aa₃. NADH cytochrome c reductase was activated by light with peaks of photo-activation at 430, 530 and 570 nm in fresh and incubated sperm. A marked decrease in the cytochrome b amount during incubation probably makes the reactions, such as catalyzed by NADH cytochrome c reductase, rate-limiting in mitochondrial respiratory chain, to reduce the respiratory rate in incubated sperm. Photo-activation of these enzyme reactions, in which cytochrome b is involved, seems to enhance the respiratory rate, only when they become rate-limiting. Tunicate sperm, which NADH cytochrome c reductase was insensitive to light, did not exhibit photo-activation of respiration, even after a long time incubation.

When unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus were treated with the extracts from the sea algae Laminaria diabolica, fertilization envelope formation (FEF) was observed in 1.5 to 2 min without subsequent cleavage. The molecular mass of the FEF-inducing protein purified from the Laminaria extracts was estimated by gel-filtration to be 120 kDa, and it induced a 100% FEF at a concentration of 525 ng/ml (4.4 nM). The FEF activity of the purified protein was completely inhibited by GlcNAc, less effectively by GlcN, and not at all by various other sugars and amino-sugars, suggesting its nature as a lectin. Normal FEF and cleavage were observed when eggs were inseminated in the presence of GlcNAc. Immunohistochemical observations using antibodies against purified protein revealed its specific localization in the cells lining the wall of mucilaginous lacunae and in secretory products in the lumen. The purified protein did not activate the pretreated with verapamil or diltiazem, while ionomycin, a Ca² -ionophore, did induce activation of the similarly pretreated eggs. These results indicate that the Laminaria lectin triggers a very initial step in the cascade of the egg cortical reactions leading to FEF, independently of those induced by sperm.

Adrenal corticoids accelerate metamorphosis of amphibians by potentiating the action of thyroid hormone. Adrenal corticoid secretion is considered to be controlled mainly by adrenocorticotropic hormone generated from proopiomelanocortin (POMC) in the anterior lobe of the pituitary. In order to assess the changes in POMC mRNA levels during metamorphosis, a cDNA for POMC was isolated from a cDNA library constructed from bullfrog (Rana catesbeiana) pituitary polyadenylated RNA. Northern blot analysis using the POMC cDNA as a probe revealed that POMC mRNA levels in the anterior lobe were relatively low during premetamorphosis, rose during prometamorphosis, reached the maximum at the end of prometamorphosis and remained very high during climax. The POMC mRNA levels of the intermediate lobe, where α-melanophore-stimulating hormone is generated from POMC, were also determined in metamorphosing tadpoles. The POMC mRNA levels of the intermediate lobe increased as metamorphosis progressed and were maximal at mid-climax. High POMC mRNA levels were observed even in larvae that had adapted to a white background. The significance of these findings and their relationships to the hormonal requirements during metamorphosis are discussed.

The pupae of Papilio xuthus show green, brown, and orange color polymorphism. The color of the pupal body is determined by a hormone that produces brown coloration, a hormone called pupal-cuticlemelanizing hormone (PCMH) which is secreted from brain-suboesophageal ganglion and prothoracic ganglion (Br-SG-PG) complexes during the pharate pupal stage. PCMH was extracted with 2% NaCl from Br-SG-PG complexes of P. xuthus green pupae. When pharate pupae producing brown pupae (brown-pupa-producers) were ligatured between the thorax and abdomen with cotton thread, the head-thorax complex developed into the brown type and the abdomens into the green type. Ligatured abdomens treated with crude PCMH produced intermediates of green- and brown-pupae (melanization degree of grades 0–3), which was used to assay PCMH-activity. Extracts of Br-SG complexes from Bombyx mori adults also shifted the color of the pupal cuticle toward the brown type in ligatured P. xuthus abdomens of brown-pupa-producers. The molecular weight of the B. mori factor showing PCMH activity (PCMH-active factor) was estimated to be 3,000–4,000 Da by gel filtration. The PCMH-active factor is a hydrophobic peptide(s) that binds to a cation exchange resin at pH 6.9.

To investigate post-dormant regulation of trehalose metabolism in the brine shrimp, we cloned and characterized two trehalase cDNAs from embryos of Artemia franciscana using a PCR probe corresponding to a highly conserved region among trehalases. The cDNAs consisted of 2496 and 2485 nucleotides, and had almost the same open reading frame encoding 703 amino acids which showed 46.6–42.6% similarities to trehalases of animals. The calculated molecular mass of the trehalase was 79,995 Da. The deduced sequence had a cleavable signal peptide, a cell adhesion motif, four potential N-glycosylation sites, trehalase signatures and a unique, long carboxyl terminal polypeptide containing a predicted transmembrane region and a potential cAMP-dependent phosphorylation site. Phylogenetic analyses showed a large divergence among trehalases of arthropods. Northern blots revealed the presence of two mRNAs. One of them, a 2.6 kb mRNA, was abundant in the dormant cysts and prenauplii. The other 5.0 kb mRNA was newly synthesized during post-dormant development. Possible mechanisms of trehalase regulation are presented on the basis of the results shown by the Northern blots and developmental changes of trehalase activity.

Translation elongation factor 1α (EF-1α) catalyzes the GTP-dependent binding of aminoacyl-tRNA to the ribosome. We previously reported that Tetrahymena EF-1α induced bundles of rabbit skeletal muscle F-actin as well as Tetrahymena F-actin (Kurasawa et al., (1996) Zool. Sci. 13: 371–375), and that Ca² /calmodulin (CaM) regulated the F-actin-bundling activity of EF-1α without inhibition of the binding between EF-1α and F-actin (Kurasawa et al., (1996) J. Biochem. 119: 791–798). In this study, we investigated EF-1α-binding proteins in Tetrahymena using a Tetrahymena EF-1α affinity column. Tetrahymena EF-1α bound directly to 74 kDa, 77 kDa, and 78 kDa proteins, in addition to CaM. The bindings of 74 kDa, 77 kDa, and 78 kDa proteins to Tetrahymena EF-1α were Ca² -independent and ATP-sensitive. The N-terminal amino acid sequence of the 74 kDa protein was similar to those of 70 kDa heat shock protein (hsp70) family.

α₂-Macroglobulin is a high-molecular-weight glycoprotein that inhibits a variety of endoproteases. Proteolytic enzymes associated with this inhibitor are thought to be unable to act on protein substrates. This paper reports that the α₂-macroglobulin fraction isolated from the follicular fluid of human ovaries is capable of proteolytically activating human single-chain tissue-type plasminogen activator. We demonstrated that a bound protease unlike plasma kallikrein was involved in the activation. This activity was maximally detected at pH values in the ranges 6–9, and was strongly inhibited by diisopropylfluorophosphate and aprotinin, indicating that the enzyme responsible for the activation is a serine protease. In summary, this paper describes for the first time that a protease complexed with α₂-macroglobulin exhibits detectable proteolytic activity toward the protein substrate single-chain tissue-type plasminogen activator.

An assay system has been established for the sexual induction in the OH strain, an exclusively fissiparous (asexual) strain, of Dugesia ryukyuensis by feeding them with sexually matured worms of Bdellocephala brunnea, an exclusively oviparous (sexual) species. In this assay system, asexual worms gradually differentiated sexual organs, namely the ovary, testis, genital pore and yolk gland in this order, and eventually mated and laid cocoons filled with fertilized eggs. Although the OH strain worms were believed not to have any sexual organs, a pair of undeveloped ovaries with a few oogonia were detected by an intensive histological search. Along with the progression of sexualization, five distinct stages were histologically recognized: In the first stage, the ovaries became larger enough to be externally apparent; oocytes appeared first at stage 2; the primordial testes emerged at stage 3; a genital pore opened, yolk gland primordia developed and spermatocytes appeared at stage 4; and finally at stage 5 matured spermatozoa and yolk glands were formed. Worms in stages 1 and 2 but not in later stages returned asexual if feeding on B. brunnea was interrupted. Furthermore, when the worms at stage 3 onwards were cut posterior to the ovaries, all the tail regenerants developed eventually into fully sexualized worms. Taking these results in account, we have concluded that the process of sexualization has a point-of-no-return between stages 2 and 3. It is likely also that the testes, even the primordia, play an important role in the maintenance and development of sexuality.

Increased levels of gonadotropins in the perinatal and prepubertal period may be responsible for the rapid phase of concurrent follicular atresia. This study tests the hypothesis that follicular atresia during this period can be reduced by suppressing gonadotropin release with a GnRH antagonist. Female rat litter mates were randomized to receive daily injections of GnRHi (100 μg Detirelix® [Syntex, Palo Alto, CA] from the day of birth and were sacrificed at 5, 15, or 26 days of life. Follicular atresia was assessed by measuring number and size distribution of ovarian follicles. Serum FSH levels were assayed. GnRHi treatment significantly depressed serum FSH and decreased the number of large antral follicles in 26 day rats, while body weight, reproductive tract weight and total follicle number per representative section were not significantly altered. Age-related changes were significant for all variables. The loss of primordial follicles is likely the result of another mechanism or combination of mechanisms. Gonadotropins do not appear to play a major role in follicular atresia in the neonatal and infant rat.

Cannibalism is common in high density larval populations of Hynobius retardatus. Because cannibals are gape-limited, possessing a wider head (mouth) may be advantageous in these populations. Field observations showed that the pre-feeding stage larvae were more vulnerable to cannibalism than feeding stage larvae in a high-density larval population. The data also showed that larvae with proportionally smaller head widths are more vulnerable to cannibalism than those with larger heads. Therefore, faster growth of head size during pre-feeding stage is predicted to be favored in a population with frequent cannibalism. A laboratory comparison revealed that head growth (proportionate change in head width to body length) during the pre-feeding stage was greater in the larvae of a cannibalistic population than in those of a non-cannibalistic population. These results support the hypothesis that a wide head is an adaptation against frequent cannibalism in larval H. retardatus.

A periwinkle, Littorina sitkana Philippi, 1846, does direct development and exhibits three types of shell sculpture. Lacking pelagic larval stages, this species is expected to be genetically differentiated among populations. In the present study, genetic variation of 19 populations along northern Japanese coasts was examined using protein electrophoresis. The relative abundance of shell types was also investigated at each locality. The analyzed populations were significantly differentiated genetically from one another. However, no significant genetic difference was detected between shell types within localities where two types were nearly equally frequent. When clustered genetically using UPGMA, the populations were divided into four geographic groups. The UPGMA tree also showed that the Japanese population of L. sitkana is clearly divided into two groups, which may have been derived from two mother populations with different genetic structures. On the other hand, allozymic and anatomical analyses of the present samples have refuted possible occurrence of sibling species of L. sitkana on the coasts of northern Japan.

The Bengalese finch is a domesticated strain of the white-backed munia. The process of domestication began some 250 years ago in Japan and several modifications in coloration and behavior occurred. We recently found that songs of Bengalese finches are much more complex in their temporal organizations than songs of related species such as zebra finches. We hypothesized that this complexity occurred during domestication. To explore this hypothesis, we compared syntactical and acoustical parameters of songs between the wild and domesticated strains of white-backed munia. Acoustical morphologies of the song elements were strain-specific: similarities among song elements were higher within individuals of each strain, but the degrees of morphological variations were comparable between the strains. In the time domain, white-backed munias sang a highly stereotyped song: a song element was always followed by one of certain song elements in a deterministic way. Bengalese finches, on the other hand, sang complex song with one song note followed by several possible song notes. Male songs should evolve largely under two different pressures: female preference and risk of predation. The low degree of complexity found in wild white-backed munias may be the result of compensating these two factors. In Bengalese finches, because of the domestication, predation is no longer a selection pressure. Thus, it is likely that Bengalese finch songs had undergone changes that were favored by females.

Hydra can regenerate its complete adult form from aggregates of dissociated cells. Aggregates of hydra cells are made by dissociating hydra tissue into a suspension of cells and then re-aggregating the cells by centrifugation. An aggregate formed in this way is a disorganized mass of individual cells and does not possess any regeneration polarity. In this study, we analyzed the development of motion in cell aggregates during the regeneration stages in which a new body axis was being established. Two perpendicular diameters (widths) of binalized projection images of an aggregate were continuously measured in order to detect changes in form, i.e., motion. Between 30–35 hr, when the aggregates still appeared spherical, slight motion along a distinct axis was detected along with a simple expansion in the size of the mass. After that, quick twitches along a distinct axis, also seen in intact hydra, began to develop. The axis of the motion corresponded to the future body axis of the regenerated animal, and the future head-end of the body axis showed a larger degree motion than the foot-end. Motion in the aggregates made of cells from hydroxyurea-treated animals in which the stem cells of nerve cells has been eliminated, suggested that the slow one-directional motion observed was due to the epithelial cells, while the twitches were controlled by nerve cells. These results show that the development of motion could provide a useful index to the recovery of organization in the cell aggregates.

Administration of exogenous estradiol between stages 50 and 52 completely feminized the developing gonads of Xenopus laevis. However, when tadpoles were injected or cultured during the critical period with an inhibitor (CGS 16949A) of aromatase that prevents synthesis of estradiol from androgen, there were no detectable effects on the sexual differentiation of the gonads. Aromatase transcription in Xenopus gonads was then studied by the reverse-transcription polymerase chain reaction (PCR) method. In embryos at the beginning of the estradiol-sensitive period (stages 49 and 50), expression of the aromatase gene was not detected in the gonad. These results show that the period between stages 50 and 52 is the time when Xenopus is sensitive to sex reversal by estradiol and critical for sex determination, although estradiol synthesis may not be naturally involved in the gonad at this step.

The number of cells in the gut of a sea urchin embryo was counted to clarify the cellular mechanisms of gut formation during development. It has been determined that in the gut of a normal embryo, the number of cells amounted to at least 49, 100, 181, 175 and 169 at the early gastrula, mid-gastrula, late gastrula, prism and pluteus stage, respectively. The percentage of the number of cells in the gut to the total number of the cells in the whole embryo was the highest at the late gastrula stage.

The seminal vesicle (SV) and testis of Clarias batrachus (L) exhibit significant annual variations in SVSI and GSI, and in the levels of total proteins, fructose, hexosamines and sialic acid with low values in resting phase and high levels in spawning phase. Histologically, the SV is composed of numerous lobules lined by epithelial cells enclosing a lumen and an interstitial matrix containing interstitial cells (homologous to Leydig cells) and fibroblasts. In the preparatory phase, the SV undergoes extensive growth and proliferation of the lobules (April) with numerous buds of differentiating lobules. With the progress of secretory activity, the epithelial cells which were tall and columnar initially become cubical and squamous. The secretions filled the lumen resulting in distension and enlargement of the follicles and spermatozoa appear in the lumina. After voiding the contents, the SV lobules collapse and degenerate. New lobules are formed from the interstitium by proliferation. They showed transient secretory activity and remained inactive during the resting phase. The increases in total proteins, fructose, hexosamines and sialic acid levels in the SV are directly correlated with the histological changes and secretory activity. The levels of phospholipids and free fatty acids which showed significant variations are low during the spawning phase. The decline in the concentrations of esterified and free cholesterol (EC and FC) in both the testis and SV during the breeding season suggests their utilization as precursors in the synthesis of steroid hormones. The two, however, maintained an inverse relation during the non-breeding season with EC levels high and FC levels low. Scattered spermatogenic cysts in different stages of development are present in the SV epithelium. The similarities in the annual distribution of various biochemical correlates, and the presence of interstitial (Leydig) cells and scattered spermatogenic cysts suggest that the SV has a common origin with the testis and is specialized for secreting a glyco-lipoprotein-rich substance for temporary storage of the sperm.

We locate two tetranucleotide repeat sequences (AT3 and T2C2) between the markers sY44 and sY46 within the Xq21.3/Yp block of homology that has been created since the separation of the chimpanzee and human lineages, and trace their origin in primate evolution. The T2C2 repeat is present only in hominoid primates. The sequence AT3 is present in Old World monkeys but not in New World monkeys, and has been lost in some gibbon species. In the bonobo, the AT3 repeat is the site of a new Alu insertion. These findings and their relationship to the conservation of other markers in this region cast light on the structure of a genomic region that has been subject to change in the course of primate evolution and may include one or more sites of instability.

Partial sequences of the cytochrome b gene (740 bp) and restriction fragment length polymorphisms in the mitochondrial DNA were used to examine inter-and intraspecific relationships among nine species of bagrid catfishes (family Bagridae) in Japan and Korea. Several opinions have been expressed regarding the kinship among Japanese and Korean bagrid catfishes, based on external morphological similarities. Almost all of them, however, were rejected by the current data sets. For instance, it has been considered that the Korean species, Pseudobagrus brevicorpus and P. fulvidraco, were closely-related to P. ichikawai and P. nudiceps, respectively, found in Japan. Resulting trees indicated that P. ichikawai branched off separately from all of the remaining Pseudobagrus species. Similarly, P. nudiceps and P. fulvidraco were represented by distantly separated branches. The intraspecific genetic divergence of bagrid catfishes was relatively small, even among geographically distant populations.