Katsuya Iwata

Nitrogen metabolism in the mudskipper, Periophthalmus cantonens: Changes in free amino acids and related compounds in various tissues under conditions of ammonia loading, with special reference to its high ammonia tolerance

1. 1. When the fish was kept out of water, the levels of ammonia increased greatly in the liver, skin and muscle, whereas those in the blood and brain increased to a lesser extent. 2. 2. A marked increase in free amino acids (FAA), particularly non-essential ones was found in various tissues during exposure to air; the level of glutamine in the brain of the fish kept out of water for 48 hr increased up to 10-fold that of the fish kept in 20% SW (control). 3. 3. P. cantonensis has a great tolerance to ammonia; it could survive for more than 7 days in 15 mM NH4C1 solution, while the water breathing gobid fishes died within 24 hr at 10 mM NH4C1. 4. 4. In the fish immersed in 15 mM NH4C1 solution for 3–7 days, changes in ammonia and FAA content in various tissues were essentially the same as those found in the fish kept out of water. 5. 5. By either treatment, urea contents in various tissues were not different from those of the control. 6. 6. Glutamic dehydrogenase and glutamine synthetase levels in the brain of the mudskipper were about 10- and 2-fold higher than those of several water-breathing gobid fishes, respectively. 7. 7. It appears that the mudskipper does not modify the mode of nitrogen metabolism toward ureotelism, but develops the ability to protect the central nervous system against ammonia toxicity in relation to the amphibious mode of life.

Nitrogen metabolism in the mudskipper, Periophthalmus cantonensis: A role of free amino acids in detoxication of ammonia produced during its terrestrial life

Abstract 1. 1. The mudskipper, Periophthalmus cantonesis kept out of water had lower rates of ammonia and urea excretion than those adapted in 20% seawater (S.W.). 2. 2. The muscle ammonia level showed a great increase in fish kept out of water, while urea increased to a lesser extent. 3. 3. Fish adapted to 80% S.W. also retained a higher level of muscle ammonia than fish adapted in 20% S.W. 4. 4. Fish out of water and in 80% S.W. possessed an increased level of FAA in the muscle. 5. 5. In addition, both the fish showed a great increase in GDH levels of the muscle as well as the liver. 6. 6. The muscle enzyme has kinetic properties which function in favor of removing ammonia from the tissue to produce FAA. 7. 7. FAA but not urea, as shown by these findings, play a central role in ammonia detoxication as well as in cell volume regulation.

Intra-specific variations of egg size, clutch size and larval survival relatedto maternal size in amphidromous Rhinogobius goby

SynopsisNewly hatched amphidromous Rhinogobius sp. CB (cross band type) larvae drift downstream to the sea to grow and develop before returning upstream as juveniles. Since larger and older individuals usually inhabit the upper reaches of rivers, larvae from larger females are more likely to suffer higher risks of starvation or predation during their longer migration to the sea. We examined the relationship between reproductive parameters (egg volume and clutch size) and maternal length. We collected adult Rhinogobius sp. CB along the course of the Aizu River (Wakayama Prefecture, Japan) and spawned them under laboratory conditions. We measured egg volumes and clutch sizes, as well as larval starvation tolerance. Both egg volume and clutch size increased with standard length or age of the maternal fish, while egg density in these clusters did not correlate with standard length. Gonad-somatic index (GSI) also tended to increase with maternal standard length. There was a significant positive correlation between egg size and the 72-h survival rate of unfed hatchlings. Intra-specific variation of egg volumes and clutch sizes in this species seems to be an adaptation for enhancing offspring survivorship during migration to the sea. Some females spawned a second time. Second spawned egg sizes were smaller than first spawned egg sizes, although there was no difference in clutch size between the two. Egg size variation between subsequent spawns may be an adjustment to the changes in seasonal environmental conditions.

Inhibition of ammonia excretion and production in rainbow trout during severe alkaline exposure

Exposure of fish to alkaline conditions clearly inhibits the rate of ammonia excretion (JAMM) leading to problems of ammonia accumulation and toxicity. There is some evidence that higher environmental calcium levels may ameliorate the negative effects of alkaline exposure. In the present study, we investigated the effects of elevated environmental calcium on the recovery of ammonia excretion during acute (6 h) and chronic (15 day) exposures to high pH (pH 10), and the levels of nitrogenous products (ammonia, urea, and glutamine) in tissues and plasma (1–15 days). Acclimation to alkaline conditions results in a chronically depressed ammonia excretion rate. Glutamine as well as urea do not appear to be important in ammonia detoxification and storage. Calculations of the production rates from storage and excretion data indicate a reduction in the rate of ammonia production. Elevating environmental calcium during acute exposure to alkaline conditions does not affect the recovery of JAMM but may reduce stress. We conclude that rainbow trout survive alkaline exposure by reducing ammonia production and that the role of calcium may be in the amelioration of the stress of alkaline exposure.

Metabolic Fate and Distribution of 15N-Ammonia in an Ammonotelic Amphibious Fish, Periophthalmus modestus, Following Immersion in 15N-Ammonium Sulfate: a Long Term Experiment

Abstract 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.

Ion transport across the skin of the mudskipper Periophthalmus modestus

Abstract An amphibious teleost, the mudskipper (Periophthalmus modestus), showed a serosa-positive transepithelial potential difference (PD) across the skin isolated from several areas of the body. However, the highest PD was observed in skins isolated from the pectoral fin. In skin isolated from the pectoral fin of mudskipper, the PD was due to active Cl− secretion, because net 36Cl− flux was identical with the simultaneously measured short-circuit current (Isc). No net Na+ flux was observed under the short-circuited condition. The Isc in skins isolated from pectoral fin was diminished by omitting Cl− or Na+ bilaterally from the bathing media. The Isc was also diminished by serosal ouabain, a specific blocker of Na + K + -ATPase , and by serosal bumetanide, a specific inhibitor of the Na+-K+-Cl− cotransporter. K+ channel blockers, Ba2+ and Cs+, applied to the serosal surface and the Cl− channel blocker, diphenylamine-2-carboxilic acid, applied to the apical membrane reduced Isc and increased tissue resistance, Rt. The Na+ channel blocker, amiloride, had no effect on either Isc or Rt. From these data we proposed a current model for ion transport across the skin of the mudskipper; Na + K + -ATPase , Na+-K+-Cl− cotransporter and K+ channels are localized to the basolateral membrane and CI channels are located in the apical membrane of the epithelium. A morphological study demonstrated that skins isolated from the pectoral fin contained a large number of mitochondrion-rich cells (MRC) and many pits on the surface. Beneath the pits, relatively large cells were observed on light micrograph, which contained many cytoplasmic granules, presumably mitochondria. A good correlation was obtained between the area occupied by the MRC and the Isc. These characteristics were similar to those of the operculum, which is considered as a model for gills in seawater teleosts but different from those of frog skins.

Diurnal nitrogen excretion rhythm of the functionally ureogenic gobiid fish Mugilogobius abei

This study was performed to determine the daily periodicity of urea excretion in the ureogenic gobiid fish Mugilogobius abei. In 20% seawater, urea excretion of all the fish examined showed daily periodic changes under a 12-h light-dark cycle, and some showed a free-running rhythm under constant darkness. This is the first report of a circadian rhythm in urea excretion in fishes. Daily variations in urea excretion under light-dark cycles were also observed under various conditions, i.e. exposure to water ammonia, confinement/non-confinement and solitary/group. Due to the daily variations in urea excretion, urea contents in tissues changed periodically, whereas enzyme activities related to urea synthesis did not change significantly. The index of urea permeability as determined by changes in body urea contents after 2-h immersion of 25 mM urea solution was high during the peak of daily variation in urea excretion. Locomotor activity and urea excretion showed clear daily variations under light-dark cycles, both of which were diurnal. Furthermore, daily variations in urea excretion were maintained even when the diurnal pattern in the locomotor activity was disturbed. These results suggest that periodic urea excretion was mediated by periodic enhancement of permeability for urea at excretion sites.

Evolution of a Ureagenic Ability of Japanese Mugilogobius Species (Pisces: Gobiidae)

Abstract Interspecific differences in the ability to excrete urea in four gobiid fishes, Mugilogobius abei, M. chulae, M. sp. 1 and M. sp. 2, in response to elevated external ammonia, were compared and set against the phylogenetic relationships of species, following molecular phylogenetic analyses using a portion of mitochondrially-encoded 12S ribosomal RNA and tRNA-Val genes, which were sequenced from five species of Mugilogobius. The resulting tree, based on 710 base pairs of aligned sequences, was statistically robust and indicated two major clades, (M. abei, M. sp. 1 and M. chulae) and (M. parvus and M. sp. 2), respectively. High ureagenic ability was demonstrated in M. abei and M. sp. 1, being sister species according to the phylogenetic analysis, while the remaining species having a low level of ability. The two former species are restricted to temperate and subtropical Japan, whereas the others are widely distributed throughout tropical Asia. The most parsimonious reconstruction of these features suggested that ancestral Mugilogobius, probably widely distributed in tropical Asia, may had low ureagenic ability. The high ureagenic ability of the temperate species may have evolved concurrently with the northward spread of Mugilogobius from the tropical zone.