Zoological Science

Prothoracicotropic hormone (PTTH) is an insect brain neuropeptide that is a primary factor regulating an insect development. Curtailment of its release is thought to be responsible for the pupal diapause of tobacco hornworm, Manduca sexta. The cell synthesizing and secreting the PTTH has been identified as a pair of neurosecretory cells in the pars lateralis on each brain hemisphere. Using intracellular recording techniques, we have demonstrated electrical properties of the PTTH cells in different physiological status, i.e., diapausing and developing pupae. In diapausing pupae, they showed threshold value increasing and input resistance decreasing with the progress of diapausing state, indicating that they were getting unexcitable. Spontaneous action potentials and excitatory postsynaptic potentials (EPSPs) were rarely observed in deeply diapausing state. Non-diapausing PTTH cells were almost silent except day-2, showing rather constant values of electrical properties. On day-2, a significant proportion of the cells had spontaneous action potentials, showing less negative membrane potential values than inactive cells. Exclusively inhibitory postsynaptic potentials (IPSPs) were observed in significant numbers of the cells during the period from day-2 to day-5. On the basis of the results obtained, we proposed a working hypothesis that electrical activities of the PTTH cell may be primarily regulated by its membrane properties which are further modulated by the synaptic mediation.

Incorporation of ¹⁵N-ammonia into five nitrogenous components (Ammonia-N, Amide-N, Amino-N, Urea-N and Protein-N) in tissue was compared among the blood, brain, liver, gill, skin and muscle of the mudskipper, Periophthalmus modestus, following immersion in 15 mM ¹⁵N-ammonium sulfate (99.7 atom%) dissolved in diluted sea water for 24 to 168 hr. Total net ¹⁵N-uptake (μmol-¹⁵N/g wet mass) into tissue was greater in the order of the brain, liver, gill, muscle, skin and blood. Among the components in each tissue, amount of ¹⁵N in the form of Amino-N was the highest during the first 24 hr. Subsequently, that of Protein-N greatly increased, particularly in the liver, gill and brain. Amount of ¹⁵N in the form of Urea-N was negligible throughout the experimental period. In addition to having the highest ammonia content, the muscle showed the highest ¹⁵N-Concentrations (36–38 atom% excess) in Ammonia-N and Amide-N during the course of immersion. Amount of ¹⁵N in the form of Ammonia-N in the blood remained at the lowest levels among the tissues examined until 96 hr at which the nitrogen in the muscle reached equilibrium with ¹⁵N. It may be concluded that under ammonia loading conditions, the muscle plays the most important role not only in trapping a large amount of ammonia, but also in producing glutamine and other amino acids.

Tentacle ball formation may be a component of sequential feeding behavior in Hydra. This behavioral response is elicited by reduced glutathione after exposure to trypsin for 5 min at concentrations ranging from 0.1 fg/ml to 1 μg/ml. Trypsin and thrombin potentiated this response more effectively than the other proteases examined. Trypsin significantly promoted the ingestion of dead, fixed shrimp attached to their tentacles in the presence of glutathione. In an actual feeding situation, a trypsin-like protease, released from living wounded prey, may potentiate tentacle ball formation, and as a result, the ingestion of prey would be promoted in co-operation with reduced glutathione. We found that an immunoreactive protein for the monoclonal antibodies J245 and J5 was reduced in size in animals treated with trypsin; >300 kDa in animals without trypsin vs. 250 kDa or 110 kDa depending on the extent of trypsin treatment. Thus, this protein that is immunoreactive with J245 and J5 is likely to be involved in the trypsin-dependent potentiation of tentacle ball formation and the promotion of ingestion.

The nautilus retina contains retinochrome in addition to rhodopsin, just like the retinas of squid and octopus. Those photopigments of Nautilus pompilium were extracted to examine their chemical properties. The absorption maximum (λ_max) of rhodopsin is very short in wavelength, being at 465 nm. On irradiation with blue light, rhodopsin is changed to a photoequilibrium mixture with metarhodopsin (λ_max = 510 nm), which is photoregenerated back to rhodopsin on reirradiation with orange light. Rhodopsin remains stable in the presence of 100 mM hydroxylamine (NH₂OH), whereas metarhodopsin is gradually decomposed forming retinaloxime. The molecular weight of nautilus rhodopsin is estimated to be 84,000 by SDS-PAGE, larger than that of squid rhodopsin. In the nautilus retina, retinochrome is present at a level of only about 4% of the rhodopsin content. When exposed to orange light, retinochrome (λ_max = 510 nm) is readily bleached to metaretinochrome, which again yields retinochrome on addition of all-trans-retinal. Consequently, retinochrome catalyzes the isomerization of all-trans-retinal to the 11-cis form in the light. It is stable in 20 mM NH₂OH, but metaretinochrome is rapidly destroyed. The molecular weight of nautilus retinochrome is 26,000, similar to that of squid retinochrome. It was also suggested that the nautilus retina is provided with the same rhodopsin-retinochrome system as established in the squid retina.

We previously described two different water absorption systems in the ventral skin of the Japanese tree-frog, Hyla arborea japonica: i.e., a rapid enhanced flow, which is observed in dehydrated tree-frogs or those stimulated by adrenaline β-agonists or vasotocin, and a slow basal flow, which is observed in normally hydrated frogs during the non-breeding season. The rapid flow is completely blocked by ouabain, which has no effects on the slow basal flow. In the present experiment, we show that the vaso-constrictive hormone angiotensin II completely inhibits basal water absorption, but has no effect on rapid water absorption. These results confirm our previous finding that the two water absorption systems in the ventral skin of the Japanese tree-frog are independent of each other.

Interneurons in the antennal lobe of the worker honeybee brain were stained with Lucifer Yellow and their responses to the Nasonov pheromone were examined. The antennae were stimulated with a synthetic blend of Nasonov pheromone at 5 dose levels, which elicited both behavioral and electroantennogram responses dose-dependently. Morphologically characterized neurons were categorized into 4 classes: projection neurons with axons in the median antenno-glomerular tract and uniglomerular dendritic arborizations in the antennal lobe, Pm(u); projection neurons with axons in the median lateral antenno-glomerular tract and multiglomerular arborizations, Pml(m); projection neurons with axons in the antennal lobe commissure, Pc(m); local interneurons, L(m). All classes of neurons exhibited changes of spike activity in response to the pheromone blend. The temporal patterns of the responses tended to be correlated with these morphological classes: Pm(u) showed mainly phasic-tonic excitation, and in some cases on-off excitation or inhibition; Pc(m) showed slight excitation; Pml(m) and L(m) showed mainly phasic excitation. The response intensity of most cells increased dose-dependently, and the response pattern of some Pm(u) changed with the dose of the blend. These results suggest that signals induced by the Nasonov pheromone are distributed in multiple morphological classes of antennal lobe neurons, each having various response characteristics. The architecture of the bee antennal lobe relating to Nasonov pheromone processing thus appears to be complex.

The species of Paramecium are sexually isolated primarily by the specificity of mating reaction. By this reason, it has been thought that interspecific pair formation does not occur when cells of different species are mixed. However we discovered that interspecific pair formation can be induced simply by mixing the mating reactive cells of two species. Among four species of Paramecium, P. tetraurelia, P. multimicronucleatum, P. caudatum, and P. bursaria, the interspecific pairs were observed in the former three species which belong to the “aurelia” group, but those three species did not mate with P. bursaria which belongs to the “bursaria” group. The percentage of interspecific pair formation was less than 10% in all positive cases. Macronuclear fragmentation, one of the remarkable nuclear changes in normal conjugation, was also observed in the interspecific pairs. The time course of the pair formation and macronuclear changes were similar to those of intraspecific conjugation.

Three humoral defense activities (antibacterial, bacteriolytic and agglutinating) were detected in the body fluid of the nematode Ascaris suum. Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) were more sensitive to the antibacterial activity than the Gram-negative bacteria (Escherichia coli). The antibacterial activity was heat stable and was lost by trypsin digestion. The molecular mass of the factor responsible for antibacterial activity was estimated as 6 kDa. The bacteriolytic activity against dried Micrococcus luteus was also detected. The bacteriolytic factor was 6–9 kDa in molecular mass, heat sensitive and trypsin sensitive. Both E. coli and glutaraldehyde-fixed trypsin-treated human A-type red blood cells were agglutinated in the body fluid. An analytical gel permeation HPLC revealed the agglutinating activity consists of at least two factors. Activities of both agglutinating factors were lost by heat treatment or trypsin digestion. The molecular masses estimated for the two agglutinating factors were 500 kDa and 25 kDa. Under experimental conditions, microbe-injection was not a prerequisite for the appearance of these defense activities.

In teleosts, prolactin (PRL) cells are localized in rostral region of the pars distalis (PD) forming clusters or follicles. The present study was undertaken to examine whether interactions resulting from the arrangement of PRL cells might be involved in the regulation of PRL release. To this end, the release of PRL from the rainbow trout (Oncorhynchus mykiss) and tilapia (Oreochromis mossambicus) was compared under three different conditions of incubation: 1) the organ culture of PRL cells in intact PD; 2) the incubation of individual PD cells that were dispersed and subsequently attached to the culture plate; 3) the incubation of PD cells that had been allowed to aggregate after dispersion. For the trout, PRL release from the dispersed cells was greater than that from either the organ-cultured PD or the cell aggregates. For the tilapia, by contrast, the release of PRL from dispersed cells was similar to that observed during the incubation of either the organ-cultured PD or the cell aggregates. In both the trout and tilapia, growth hormone (GH) cells form clusters in the proximal PD. For both species, the release of GH from the dispersed cells was similar to that from either the organ-cultured PD or the cell aggregates. For the trout, but not the tilapia, it would appear that the close association of PRL cells in pituitary follicles has considerable importance in establishing basal hormone release. The release of newly synthesized (pulse-labeled) PRL from dispersed trout PRL cells was reduced when the incubation medium was conditioned by previous incubation of the trout PD. Nevertheless, the release of newly synthesized PRL was not diminished by the addition of salmon PRL to the incubation medium. Taken together, our findings suggest that, for the trout, the suppression of PRL release from the PD and cell aggregates was mediated through (an) inhibitory factor(s) within the PD other than PRL itself.

Effects of ultimobranchialectomy (UBX) on plasma and bile minerals were examined in the stingray Dasyatis akajei (Elasmobranchii). Plasma urea and glucose concentrations were also measured as references. At 1 week after the operation, plasma CT level in the UBX group was significantly lower than that in the sham-operated (SHAM) group. However, there was no significant difference in plasma Ca level between UBX and SHAM groups at 1 week. On the other hand, bile Ca concentration in the UBX group was significantly lower than that in the SHAM group. Bile K and Cl levels, and bile volumes in the UBX group were significantly higher than those in the SHAM group. These results suggests that in the stingray, CT may function to control bile mineral concentrations.

We studied the immunohistochemical localization of androgen receptor in the mouse submandibular gland, and developmental profiles of its expression using polyclonal human androgen receptor antibody. In the submandibular glands of both sexes, specific immunoreactivity appeared only in cell nuclei of the acini, the intercalated ducts, the granular convoluted tubules (GCT) and the excretory striated ducts. The percentage of immunoreactive cells in each region gradually declined with age during the first 90 days of postnatal development studied. The sexual difference in the percentage of immunoreactive cells was oberserved in the acini on days 20 and 30 and in the GCT on day 30. Incidence of immunoreactive cells in the female was significantly smaller than that in the male. On day 60, the percentage of immunoreactive cells of these two regions turned to increase slightly in the female but continued to decline in the male, and then it became higher in the female than in the male. In addition, one-week castration did not cause any changes in the intracellular distribution of androgen receptor and the percentage of immunoreactive cells in each region of the adult gland.

These results suggest that androgen receptor is localized primarily in cell nuclei in all four regions of the mouse submandibular gland in situ, and that its expression in acini and GCT is superior in the male around days 20 to 30, when sex difference of the gland becomes evident.

The status of insulin-like growth factor I (IGF-I) mRNA in the liver and gill of coho salmon (Oncorhynchus kisutch) during the parr-smolt transformation (smoltification) was examined in relation to changes in plasma levels of thyroxine, cortisol, growth hormone and somatolactin. Smoltification coincided with silvering and an increase in gill Na⁺/K⁺-ATPase activity occurred during mid-April and May [22]. The levels of IGF-I mRNA in the liver rose steadily from the first sampling date in February to maximum levels in April. Increases in IGF-I mRNA in the gill occurred later in smoltication. Plasma cortisol, thyroxine and growth hormone increased during the latter half of April and May. Plasma somatolactin declined in April. These results demonstrate that IGF-I gene expression in both liver and gill increased during smoltification and suggest that IGF-I expression in both organs may be involved in salmonid smoltification.