Kinetic properties of sodium transport pathways in the river lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> erythrocytes

To activate Na⁺/H⁺ exchange, intracellular pH (pH_i) of erythrocytes of the river lamprey Lampetra fluviatilis were changed from 6 and 8 using nigericin. The Na⁺/H⁺ exchanger activity was estimated from the values of amiloride-sensitive components of Na⁺ (²²Na) inflow or of H⁺ outflow from erythrocytes. Kinetic parameters of the carrier functioning were determined by using Hill equation. Dependence of Na⁺ and H⁺ transport on pH_i value is described by hyperbolic function with the Hill coefficient value (n) close to 1. Maximal rate of ion transport was within the limits of 9–10 mmol/l cells/min, and the H⁺ concentration producing the exchanger 50% activation amounted to 0.6–1.0 μM. Stimulation of H⁺ outcome from acidified erythrocytes (pH_i 5.9) with increase of H⁺ concentration in the incubation medium is described by Hill equation with n value of 1.6. Concentration Na⁺ for the semimaximal stimulation of H⁺ outcome amounted to 10 mM. The obtained results indicate the presence in lamprey erythrocytes of only binding site for H⁺ from the cytoplasm side and the presence of positive cooperativity in Na⁺-binding from the extracellular side of the Na⁺/H⁺ exchanger. Na⁺ efflux from cells in the Na⁺-free medium did not change at a 10-fold increase of H⁺ concentration in the incubation medium. The presented data indicate differences of kinetic properties of the lamprey erythrocyte Na⁺/H⁺ exchanger and of this carrier isoforms in mammalian cells. In intact erythrocytes the dependence of the amiloride-sensitive Na⁺ inflow on its concentration in the medium is described by Hill equitation with n 1.6. The Na⁺ concentration producing the 50% transport activation amounted to 39 mM and was essentially higher as compared with that in acidified erythrocytes. These data confirm conception of the presence of two amiloride-sensitive pathways of Na+ transport in lamprey erythrocytes.     

Larval growth in the river lamprey, Lampetra fluviatilis

A series of 987 ammocoetes from the rivers Towy, Teme, and Taw have been identified as mainly L. fluviatilis (L.) on the basis of oocyte counts on female ammocoetes. The length frequency distributions for this material differs from either L. planeri or P. marinus in showing only three modes in addition to the young of the year and the length distribution of the final mode coincides with the length range for 119 metamorphosing and macrophthalmia stages of L. fluviatilis that have been found at the same sites. These animals measured from 80–117 mm in length and weights varied from 0.76–2.28 g. Metamorphosis is believed to take place in late summer and early autumn when in the majority of cases, the ammocoetes are four and a half years old. The evidence that the non-parasitic L. planeri has a longer larval life than the closely related parasitic L. fluviatilis is thought to have some significance in relation to the evolution of the brook lamprey species.     

Accumulation of sodium and potassium ions in oocytes of the river lamprey Lampetra fluviatilis during prespawning period

Accumulation of Na+ and K+ ions in oocytes of the river lamprey Lampetra fluviatilis and their transport across the plasma membrane is realized by two main mechanisms--Na,K-pump and Na,K,Cl-cotransport. At the prespawning period from December to May the intracellular Na+ concentration was observed to increase from 10 to 25 mM and the K+ concentration--from 28 to 45 mM. Results obtained on isolated oocytes with aid of 204Tl radioactive label have shown that contributions of the Na,K-pump and Na,K,Cl-cotransport to potassium accumulations were close until March. In spring, the total K+ inflow almost doubled owing to activation of the Na,K-pump, whereas contribution of Na,K,Cl-cotransport did not change. It seems that an increase of the intracellular content of the main inorganic cations in oocytes resulted in parallel activation of the Na,K-pump and probably of Na/H-exchange. The biological significance of activation of these mechanisms of ion transport at the prespawning period might be due to a necessity of accumulation of Na+ and K+ ions in concentrations optimal for subsequent embryonic development.     

D-glucose permeability in river lamprey (Lampetra fluviatilis) and carp (Cyprinus carpio) erythrocytes.

1. The transport of 3-O-methyl-D-glucose (3-OMG) in lamprey and carp erythrocytes was studied. 2. In lamprey erythrocytes the half-time for the equilibration of 3-OMG was fast, approx. 8 min. In contrast, the erythrocytes of carp were almost impermeable to 3-OMG, with a half-time for equilibration of 14.2 hr. 3. 3-OMG was taken up by lamprey erythrocytes both by facilitated diffusion and simple diffusion. The presence of carrier-mediated transport was indicated by saturation kinetics and by inhibition by phloretin. The Km and Vm of the saturable component of 3-OMG transport were 1.6 mmol/l and 12.4 mmol/kg packed cells/hr. 4. Since the 3-OMG transport in carp erythrocytes showed no sign of saturation kinetics, it appears to proceed by simple diffusion only. 5. These results suggest that, as for most other teleost fish so far studied, low glucose permeability is insufficient to maintain normal energy metabolism in carp erythrocytes. In contrast, in agnathans facilitated glucose transport seems to be quite effective.     

Participation of protein phosphatases in regulation of sodium transport across the erythrocyte membrane of the lamprey Lampetra fluviatilis

Erythrocytes of lamprey Lampetra fluviatilis were incubated in standard isotonic medium at 20°C with ²²Na to determine the unidirectional Na⁺ influx. Cell incubation in the presence of various protein phosphatase inhibitors (NaF, cantharidin, calyculin A) led to a considerable increase of Na⁺ transport into erythrocytes. The stimulation of Na⁺ influx into erythrocytes rose with increase of concentration of calyculin A within the range of 10–100 nM. The calyculin A concentration producing a 50% activation of Na⁺ transport amounted to 41.5 nM. Under optimal experimental conditions, the Na⁺ influx increased from control level of 5–8 to 20–40 mmol/l cells/h under effect of protein phosphatase blockers. The Na⁺ transport induced by these inhibitors was completely suppressed on addition of amiloride to the incubation medium. The treatment of lamprey erythrocytes with protein phosphatase inhibitors was accompanied by a small (～12%), but statistically significant decrease of intracellular Na⁺ content. A small decrease of intracellular K⁺ content in erythrocyte was observed only under the effect of NaF. The obtained data allow making the conclusion that protein phosphatases of the PP1 and PP2A types play a significant role in regulation of Na⁺ transport across the lamprey erythrocyte membrane in both directions.     

Ripening of the oocyte of the river lamprey (Lampetra fluviatilis L.) after river entry

River lampreys (Lampetra fluviatilis L.) enter rivers for spawning with their gonads in the final stages of maturation, however the oocytes continue to develop until the spawn. This study was undertaken to depict detailed changes in the oocytes in the period approaching the spawn, using metric analysis and to look for atresia to determine if the time period influences the final fecundity. The study was performed on 37 females caught between October and May in the Rega River, north‐western Poland. Mid‐part sections of fixed ovaries were stained with Heidenhain haematoxylin and PAS. Ten cell structures of the oocytes were measured under light microscope with the aid of a computer image analysis programme. In the autumn, when the first lampreys entered the river, the nuclei of the oocytes were in the polar position. With the approaching mating season many oocyte structures changed significantly. Statistically significant (P < 0.001, Mann–Whitney U‐test) was the increase in oocyte size (up to 1.06 × 0.78 mm in morphometric measurement), enlargement of the yolk platelets (f.a. 8.0 × 5.1 μm), elevation of theca over the chorion followed by the accumulation of glycoconjugates (f. a. 29.2 μm), growth of the zona granulosa at the vegetative pole (f. a. 24.2 μm), and increase in the thickness of the chorion at the animal pole (f. a. 11.1 μm). Other statistically significant changes (P < 0.05 and P < 0.01, Mann–Whitney U‐test) included a decrease in the width of the cortical cytoplasm band (f. a. 20.5 μm), decrease in the ratio of the distance between the nucleus and cell membrane and the long axis of the oocyte (f. a. 7.6 μm), and increase in the thickness of the chorion at the vegetative pole (f. a. 6.0 μm). No statistically significant changes in the area of the nucleus and diameter of the cortical alveoles were noted. Beginning with the lamprey entering the river until the spawn, the oocytes undergo significant growth and maturation. In this period the fecundity of the lamprey decreased only slightly, as atresia was observed sporadically in the ovaries.     

Locomotor activity of river lamprey Lampetra fluviatilis (L.) during the spawning season

At high latitudes (62–64°N) the river lamprey Lampetra fluviatilis (L.) is shown to have a 24-hr locomotor activity during its spawning period just before midsummer. By keeping lampreys in cold water, the spawning was delayed about one month. Analysis of event recorder graphs indicate that the 24-hr activity is caused by addition of diurnal spawning activity to the basic nocturnal activity and is not caused by weak Zeitgeber conditions during midsummer.Water temperature might indirectly determine time of spawning of the river lamprey.     

Mitochondrial and lysosomal pathways of lamprey (Lampetra fluviatilis L.) hepatocyte death

Mechanisms of mitochondrial and lysosomal pathways of natural death of lamprey hepatocytes are described at the spring period of the prespawn migration. The mitochondrial pathway (release of cytochrome c from mitochondria into cytosol and activation of caspases) is realized by the classic scheme of apoptosis. Comparatively recently, the lysosomal pathway of cell death associated with cathepsin B activation has been revealed in cells in pathologies, specifically in obstruction of gallbladder and bile ducts. A peculiarity of lamprey hepatocytes consists in that in the adult animal liver there takes place biliary atresia (the absence of gallbladder and bile ducts. Thereby, the lamprey hepatocytes are an excellent object for study of this new pathway of cell death. We have revealed development of the mitochondrial and the lysosomal pathways of cell death of lamprey hepatocytes.     

Oxidative enzymes and myosin-ATPase in the trunk musculature of the river lamprey (Lampetra fluviatilis).

Synopsis The distribution and activities of several oxidative enzymes and of myosin-ATPase have been studied in the trunk musculature of the river lamprey. The strongest activity of oxidative enzymes and of myosin-ATPase was shown by the parietal fibre bundles of the myotomal subunits, whilst the central fibres reacted to a lesser extent.Pre-incubation of sections in media containing formaldehyde and glycine or with buffers of different pH, followed by incubation for myosin-ATPase, also differentiated the muscle fibres into a specific distribution pattern within the myotomes, resembling that of the oxidative enzymes to some extent.The parietal fibres were found to be of the slow type (type I) and the central fibres to be fast (type II A).     

Parasite fauna of the European river lamprey Lampetra fluviatilis (L.) from Lake Onega

Data on the parasite fauna of the adult European river lamprey Lampetra fluviatilis (L.) from Lake Onega are reported. Ten parasite species are found, including trematodes Diplostomum petromyzifluviatilis and D. spathaceum (metacercariae), cestode Proteocephalus longicollis, nematodes Cucullanus truttae and Raphidascaris acus, acanthocephalan Echinorhynchus salmonis, ectoparasitic infusoria Chilodonella hexastica, Trichodina tenuidens, and Trichodinella epizootica, and fungus Saprolegnia parasitica. Three species are found to be dominate, namely D. petromyzifluviatilis, Cucullanus truttae, and P. longicollis. Comparative analysis of the parasite faunas of the lampreys from other basins is carried out. Some similarity in the parasite faunas of lampreys and salmonids is discovered.     

Population genetics of a cyclostome species pair, river lamprey (Lampetra fluviatilis L.) and brook lamprey (Lampetra planeri Bloch)

Horizontal agarose gel electrophoresis of 24 allozyme loci in four species of Central European lampreys (321 Lampetra planeri , 83 L. fluviatilis , 11 Eudontomyzon mariae and nine Petromyzon marinus ) was used to study the 'paired species' L. fluviatilis and L. planeri . The genetic differentiation of the anadromous river lamprey ( L. fluviatilis ) from the stationary brook lamprey ( L. planeri ) was within the range of ingroup differentiation of the latter, but L. fluviatilis exhibited much greater population cohesion over a more extended geographic range: G _ST = 0.0537 versus G _ST = 0.3398, N _em = 4.402 versus N _em = 0.4856, mean genetic among-stock distances D = 0.0047 versus D = 0.0257. L. planeri populations coexisting geographically with L. fluviatilis in the Rhine and Elbe river systems were genetically more cohesive than L. planeri stocks from the Danubian basin where L. fluviatilis is absent. Danubian L. planeri populations exhibit a lower degree of heterozygosity than brook lampreys from the Rhine river system, but comprise deeper genetic lineages ( G _ST = 0.4629 versus G _ST = 0.2434), despite being sampled from a much more restricted area. Isolation-by-distance is observed for L. planeri from the Danubian but not from the Atlantic drainage basins. Transspecific gene flow between L. planeri from Atlantic drainage basins and the long-distant migrating L. fluviatilis is inferred, raising doubt on the validity of two separate biospecies. E. mariae and P. marinus are clearly differentiated from Lampetra spp. at several allozyme loci.     

Phylogeography of the European brook lamprey (Lampetra planeri) and the European river lamprey (Lampetra fluviatilis) species pair based on mitochondrial data

The European river lamprey Lampetra fluviatilis and the European brook lamprey Lampetra planeri (Block 1784) are classified as a paired species, characterized by notably different life histories but morphological similarities. Previous work has further shown limited genetic differentiation between these two species at the mitochondrial DNA level. Here, we expand on this previous work, which focused on lamprey species from the Iberian Peninsula in the south and mainland Europe in the north, by sequencing three mitochondrial marker regions of Lampetra individuals from five river systems in Ireland and five in southern Italy. Our results corroborate the previously identified pattern of genetic diversity for the species pair. We also show significant genetic differentiation between Irish and mainland European lamprey populations, suggesting another ichthyogeographic district distinct from those previously defined. Finally, our results stress the importance of southern Italian L. planeri populations, which maintain several private alleles and notable genetic diversity.