Zoological Science

The olfactory placode and its derivative, the olfactory pit, give rise to several different populations of migrating cells, which contribute to drive the organization of the prosencephalon, but also to form a part of the central neuroendocrine compartments. Some cell types are seemingly transient and can play a role in the establishment of the final connections. The understanding of the mechanisms involved in the migration and differentiation of these cell populations can give an insight on the interplay between peripheral structures and central nervous system and on the mechanisms of commitment, phenotype selection and control for neuroendocrine cells able to selectively “colonize” the brain.

The anterior lateral (AL) eyes of the orb weaving spiders, Argiope amoena and A. bruennichii, have a single type of receptor cell with a maximum sensitivity at about 520–540 nm. Efferent optic nerve signals control the photoreceptor response in the AL eyes. Specifically, efferent signals decrease the threshold for light responses and change the waveform of responses. Similar changes in the threshold and waveform could be produced by exogenous application of octopamine, suggesting that it is an efferent neurotransmitter.

In our studies on mechanisms of larval settlement and metamorphosis in barnacles, we examined the effects of various agents involved in signal transduction on the cyprid larvae of Balanus amphitrite. When exposed to 10⁻⁵ and 10⁻⁶M phorbol esters, protein kinase C (PKC) activators, cyprid larvae metamorphosed normally but without settling to substrata. In contrast, α-type phorbol esters, the inactivated phorbol esters, showed no effects. The metamorphosis-inducing effects of phorbol esters were diminished by PKC inhibitors such as staurosporine and H7, whereas kinase inhibitors such as H8, H9 and HA1004 had no effect on cyprids. These results suggest that PKC may play an important role in the metamorphosis of barnacle cyprid larvae.

The mechanisms of beat coordination in the heart of the oyster Crassostrea gigas were analyzed by examining phase-response characteristics of both the auricle and ventricle to brief stretches. Brief stretching of an isolated auricle or ventricle produced phase delay or phase advance in the following beats, in accordance with the stimulation phase. Therefore, the phase-response curve (PRC) was always biphasic and the phase shift was larger with a larger stretch. As predicted from the PRC, the beat frequency of an isolated auricle or ventricle was entrained by repeated brief stretches when the stretch frequency was in a limited frequency range (range of entrainment) around the free-running beat frequency. The range of entrainment was wider with a larger stretch. When the stretch frequency was outside the range, the beat frequency was not entrained. Instead, it changed cyclically as a process of repeated discrete phase shifts. These results suggest that the beat coordination of the auricle and ventricle is achieved by reciprocal stretching between them.

Beat coordination between auricle and ventricle in the oyster heart was analyzed in situ. When the heart was exposed in the pericardial cavity, auricular and ventricular beats were not synchronized and the heart beat was irregular. In that state, the phase relationship between the auricular and ventricular beats changed successively and the beat frequency of both the auricle and ventricle changed cyclically. The relationship between stretch phase and beat interval change in the auricle and ventricle was identical with the phase response curve (PRC) of auricle and ventricle to brief stretches. By perfusing the pericardial cavity with sea water, the magnitude of reciprocal stretching between auricle and ventricle increased gradually. Then the auricle and ventricle began to beat synchronously with a fixed phase relation and coordinated beating of the heart was achieved. These results strongly support the idea that a coordinated heart beat of the oyster is achieved as a result of mutual entrainment of the auricle and ventricle mediated by reciprocal stretching.

Coelomocytes (blood cells) of the solitary ascidian Halocynthia roretzi were placed in culture to which sheep red blood cells (SRBCs) were introduced. Their reactions against SRBCs were observed by phase-contrast, time-lapse video and scanning electron microscopy. Three cell types, the phago-amoebocyte, the vacuolated cell and the fusogenic phagocyte, reacted to the SRBCs by phagocytosis. The vacuolated cell occasionally discharged the material of their vacuoles at the time of or shortly after ingesting the SRBC. The lymphoid cell captured SRBCs, but did not ingest them. Three other cell types, the fibroblastic cell, the giant cell and the multipolar cell, showed no reaction to the SRBC.

The SRBCs captured by the lymphoid cells were transferred either to phago-amoebocytes or fusogenic phagocytes, probably to be ingested. Such collaboration between multifunctional cells is considered to characterize the cellular defense mechanism of H. roretzi.

Survival of sympathetic neurons of chick embryo depends on nerve growth factor (NGF), and removal of NGF from culture medium caused apoptosis. We found that puromycin, an analogue of aminoacyl-tRNA, also induced apoptosis in a manner quite similar to that caused by NGF-deprivation. First, transcriptional inhibitors such as actinomycin D and α-amanitin effectively prevented both apoptoses, suggesting a requirement of gene expression. Second, the apoptoses were effectively prevented by translational inhibitors acting on peptidyltransfer reaction, such as anisomycin and blasticidin S. On the other hand, emetine, an inhibitor of translocation of peptidyl-tRNA, was not effective. Finally, NGF-deprivation and puromycin-addition affected the same three phosphorylated proteins to undergo dephosphorylation or to be eliminated. Besides, these changes were suppressed by anisomycin and blasticidin S, but not by emetine. Based on these findings, we discuss the mechanism to induce apoptosis of chick sympathetic neurons.

In Kanagawa Prefecture and its surrounding area, two types of body color populations (DA: dark and BL: blue) of Japanese freshwater crab, Geothelphusa dehaani, are distributed parapatrically, suggesting some extent of genetic differentiation between them. In this study we examined the genetic variation of G. dehaani in Kanagawa Prefecture and Tokyo by means of electrophoretic analysis on 5 enzyme loci. The results clearly show the existence of substantial genetic differentiation among different color type populations. In addition, some extent of differentiation is also observed within the BL populations.

There is little information about colony specificity (allorecognition) in bryozoans. We examined the presence of colony specificity in the bryozoan Dakaria subovoidea, a common species on the Japanese coast. When two colonies made contact with each other at their growing edges, four types of reactions were observed: (1) overgrowth onto the opposing colony, (2) bilaminar erect growth, (3) nonfusion reaction, and (4) fusion reaction. When one of the growing edges was in poor condition, the first type of reaction was observed in both allogeneic and autogeneic combinations. The colony that was in poor condition was always overgrown with the good one. When both of the growing edges were in good condition, one of the other types of reactions was observed. In the second type of reaction, the colonies recognized the opposite colony as if it were a substratum and grew so as to form a standing wall. The third type of reaction exhibited the degeneration of zooids at the contact area. In this case, there were no fused zooids at the contact area. The fourth type of reaction was the fusion of colonies leading to the formation of contact border pores between zooids of the two colonies and sometimes to the formation of fused zooids at the contact area. The contact border pores exhibited unique morphology, which has not been reported previously. In this case, both colonies continued to grow harmoniously in direction in the same plane. These results suggest that colony specificity exists in D. subovoidea, as found in other sessile colonial marine organisms.

Earthworm coelomocytes may act as effector cells which destroy targets in vitro. In a ⁵¹Cr release assay, Lumbricus coelomocyte effectors showed lytic activities of 3–14% against K562 human tumor cells when incubated 1–4 hr at 23°C or 37°C. Cytotoxicity was correlated with effector: target ratio. However, targets were not killed by incubating them in cell-free, 0.2 μm filtered coelomic fluid. The supernatant from coelomocytes cultured alone failed to kill K562 targets but coelomocyte lysates were toxic to target cells in a concentration-dependent manner. Coelomocytes were examined using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). When effectors and targets were examined under TEM, we found close apposition of effector granulocytic coelomocytes and target cell membranes but not with coelomocytes nor eleocytes at up to 15 min incubation. By SEM, effector cells appeared not only to be in close contact with targets, but instances of target lysis were observed. These results suggest that effector cell/target cell contact is essential for cytotoxicity to occur.

The blood clam Barbatia virescens expresses a unique heterodimeric hemoglobin consisting of chains I and II in erythrocytes. This is in sharp contrast to the tetrameric (α₂β₂) and polymeric two-domain hemoglobins of the congeneric species Barbatia reeveana and Barbatia lima. The 3′ and 5′ parts of the cDNA of B. virescens chain II have been amplified separately by polymerase chain reaction (PCR), and the complete nucleotide sequence of 690 bp was determined. The open reading frame is 477 nucleotides in length and encodes a protein with 158 amino acid residues, of which 120 amino acids were identified directly by the protein sequencing of the peptides obtained from digestions with trypsin, S. aureus V8 protease and pepsin. The mature protein begins with the blocked Ser, and thus the N-terminal Met is cleaved away. The molecular mass for the protein was calculated to be 17605 Da. The cDNA-derived amino acid sequence of B. virescens heterodimeric chain II shows the highest homology (42%) with that of B. virescens chain I, but shows lower homology (32–35%) with those of tetrameric α and β chains of B. lima. This indicates that B. virescens chains I and II do not correspond to B. lima α and β chains, namely the heterodimeric hemoglobin is a unique gene product expressed only in B. virecens.

The Harderian gland (HG) of the frog Rana esculenta contains mast cells in the interstitial tissue. The mast cell number (MCN) is influenced by sex hormones. Gonadectomy in both sexes provoked a decrease in MCN in January, while no effect was observed in September. Sex hormone-replacement therapy gave different results; estradiol treatment in castrated males and females always increased MCN, while testosterone did not. Acute estradiol treatment provoked an increase in MCN on days 2 and 4 of treatment and the morphology of the glandular compartment appeared normal. On days 8, 10 and 12 of treatment the MCN drastically decreased. The majority of glandular acini appeared strongly disorganized and the interstitial tissue became hypertrophic in concomitance with an increased vascularization. Our results suggest that estradiol acts by stimulating mast cells and acute estradiol treatment provokes proliferation of interstitial connective tissue together with glandular cells damage.

A cannibalism in the larvae of a salamander (Hynobius retardatus) and a resultant dimorphism were firstly described in laboratory-raised population. The cannibalistic larva develops to a “broad-headed” morph which has a wider head and grows faster compared with a non-cannibalistic “typical” morph. Laboratory experiments were conducted to examine a possible relationship between the dimorphism and cannibalism. Level of food supply and kinship of larvae affected the frequency of cannibalism. Low level of the food supply resulted in higher incidence of the cannibalism. The frequency of cannibalism in siblings was significantly lower than in the mixed-sibling groups. Possible biological significance of the cannibalism in this species was discussed.

A variety of orientation cues has been suggested for the migration to the breeding site in adult amphibians. We categorized the cues into the following 3 groups: 1) cues from the breeding pond such as male calling and pond odors, 2) celestial cues such as the sun light and the magnetic field of the earth and 3) cues from the area or route of the migration which compose a local map such as a visual and olfactory maps. To determine which of these is used by the toad, Bufo japonicus, we designed and conducted a displacement experiment in which migrating toads from one direction were transported to the ground in the opposite side of the pond. The displaced toads were completely disoriented and moved to random directions. We conclude that the toad uses a local map to orient to the breeding pond and cues from celestial bodies and the pond are not used. We also found that adult toads tracked the same route on both trips from and to the pond. This suggests that the local map was memorized by newly metamorphosed toads at their first terrestrial trip from the pond. The next step of our study was to determine what sense is used to receive the cue. We found blind toads, whose upper and lower eye-lids were stitched together, could reach the pond at a similar rate with the sham-operated and intact toads. However, anosmic toads, whose olfactory mucosa were damaged by the treatment with a 5% silver nitrate solution, rarely reached the pond. Tracking experiments with blind toads and toads bearing a bar magnet on their heads revealed that neither blindness nor the magnet had any effect on either the direction or distance of the migration to the pond. In contrast, anosmic toads could not orient properly and moved in random directions. We speculate that newly metamorphosed toadlets remember the route or area of their first terrestrial migration by using the olfactory sense. In the spring, two to three years later, they start their breeding migration to the home pond by using a local olfactory map and reverse tracking of the memorized route.

Post-copulatory isolation and the difference in morphological traits between populations of the melon fly, Bactrocera (Dacus) cucurbitae on Okinawa and Isigaki Islands in the Ryukyu Archipelago, which shared the common ancestor in 1929 or later, were studied. The hatchabilities of eggs from inter-population crosses were lower than those from intra-population crosses. Of four morphological traits studied, two measures of wing length and width between eyes of females in the Okinawa population were larger than those of the Isigaki population. Measures of wing length and midleg length of males in the Okinawa population were larger than those of the Isigaki population. The degrees of sexual dimorphism in two measures of wing length and width between eyes in Okinawa population were larger than in the Isigaki population. There were differences between the populations in allometry of several pairs of the morphological traits.

A new tubificid oligochaete, Rhyacodrilus hiemalis sp. nov., is described from four lakes in central Japan. This species resembles R. coccineus (Vejdovsky), R. sodalis (Eisen) and their unclear relatives (the Far East Russian R. sokolskajae Semernoi and R. sibiricus Semernoi, the Chinese R. sinicus (Chen)), but is distinguished from these congeners by having many long hair chaetae, many long intermediate teeth on the dorsal pectinate chaetae, ventral pectinate chaetae, and masses of gland cells around the male pores. This species is an annual that breeds during the cold season but aestivates in deeper sediment in summer.

Innervation of the ovary is known to be affected by neurotrophic factors; mRNA for some of these molecules and their low-affinity receptors have been identified in ovarian follicular tissue. We attempted to determine whether neurotrophin (NT-3) exerts a non-neuronal impact on the ovary, affecting follicular estradiol secretion via a possible humoral action. Ovaries were excised from mature Golden Syrian Hamsters (Mesocricetus auratus) and large preovulatory follicles were removed by microdissection. Follicles were incubated for 72 hr in control medium, or medium containing 10 ng, 100 ng, or 1000 ng/ml NT-3. After culture, medium samples were assayed for estradiol content. NT-3 exerted dichotomous effects, depending on concentration: NT-3 augmented mean estradiol output two-fold at 100 ng/ml, while 1000 ng/ml NT-3 decreased estradiol secretion back to control levels. By 48–72 hr in culture, all groups exhibited a significant decline in mean estradiol secretion due presumably to diminished viability, independent of neurotrophin action. These data suggest a humoral role for NT-3 in estradiol secretion of preovulatory follicles in culture.

We recorded vocalizations of megachiropteran bats, Cynopterus brachyotis, C. horsfieldi, Megaerops ecaudatus and Macroglossus sobrinus as they were held in our hands, in a hanging position or moved up and down, simulating flight. All four species produced regular audible tone pulses with a frequency spectrum of less than 9 kHz (peak at 4–6 kHz). The duration of a single pulse varied from 14.9 to 23.6 msec on average among these species. The interpulse interval was between 108.3 and 127.7 msec. In M. ecaudatus and M. sobrinus, double pulses were occasionally emitted, with interpulse intervals averaging 39 msec and 31 msec, respectively. The regular pulse emission may indicate that the bats studied use sounds to probe space.