The microbiome at the interface between environmental stress and animal health: an example from the most threatened vertebrate group.

Nitrate pollution and global warming are ubiquitous stressors likely to interact and affect the health and survival of wildlife, particularly aquatic ectotherms. Animal health is largely influenced by its microbiome (commensal/symbiotic microorganisms), which responds to such stressors. We used a crossed experimental design including three nitrate levels and five temperature regimes to investigate their interactive and individual effects on an aquatic ectotherm, the European common frog. We associated health biomarkers in larvae with changes in gut bacteria diversity and composition. Larvae experienced higher stress levels and lower body condition under high temperatures and nitrate exposure. Developmental rate increased with temperature but decreased with nitrate pollution. Alterations in bacteria composition but not diversity are likely to correlate with the observed outcomes in larvae health. Leucine degradation decreased at higher temperatures corroborating accelerated development, nitrate degradation increased with nitrate level corroborating reduced body condition and an increase in lysine biosynthesis may have helped larvae deal with the combined effects of both stressors. These results reinforce the importance of associating traditional health biomarkers with underlying microbiome changes. Therefore, we urge studies to investigate the effects of environmental stressors on microbiome composition and consequences for host health in a world threatened by biodiversity loss.

Hypoxia inhibits the regulatory volume decrease in red blood cells of common frog (Rana temporaria).

Red blood cells of vertebrates can restore their cellular volume after hyposmotic swelling. The process strictly depends on oxygen availability in the environment. However, the role of hemoglobin in regulation of cell volume recovery is not clear yet. Little is known about the osmotic reactions and regulatory volume decrease of amphibian red blood cells. We investigated volume recovery process in oxygenated (oxyhemoglobin concentration 97 ±â€¯3% of total hemoglobin) deoxygenated (96 ±â€¯2% of deoxyhemolobin) and oxidized (47 ±â€¯2% of methemoglobin, 41 ±â€¯3% of deoxyhemoglobin) red blood cells of common frog (Rana temporaria) after hyposmotic swelling. Using the low-angle light scattering method we demonstrated the regulatory volume decrease in oxygenated cells and showed that the process was eliminated in hypoxic conditions. Reoxygenation of hypoxic cells restored the regulatory volume decrease. Oxidation of cellular hemoglobin to methemoglobin inhibited the volume recovery response in hyposmotically swollen oxygenated and reoxygenated hypoxic cells.

Widespread distribution of histamine in the nervous system of a trematode flatworm.

In general, most flatworms contain very little histamine (HA) and their nervous systems often lack, or contain very few, histaminergic elements. However, preliminary studies in our laboratory have revealed that the frog lung parasite, Haplometra cylindracea (Trematoda: Digenea), contains HA in a very high concentration. For this reason, the present study was undertaken to study the localization and synthesis of HA in this worm by using immunocytochemistry and high-pressure liquid chromatography (HPLC). Essentially all parts of the nervous system of H. cylindracea showed HA-like immunoreactivity. The paired cerebral ganglia and nerves emanating from these, including the longitudinal nerve cords, were intensely immunoreactive. The musculature of the pharynx, oral and ventral suckers, and those of the reproductive organs were all innervated by HA-immunoreactive fibers. Fiber plexuses beneath the tegument and throughout the parenchyma also showed HA-like immunoreactivity. HPLC studies revealed one of the highest HA concentrations in the animal kingdom, 6.49 +/- 1.36 nmole/mg protein, in the worm. The frog lung and blood contained very low concentrations of HA and could be excluded as sources for HA, while an enzyme assay revealed that the worm produces HA by decarboxylation of histidine. Thus, it is likely that H. cylindracea uses HA as a neurotransmitter or modulator.

Oxygen binding and aggregation of hemoglobin from the common European frog, Rana temporaria.

The hemoglobin from the European frog, Rana temporaria, consists of one major and three minor components. The tetramers aggregate upon deoxygenation notably at pH 7:3. Aggregation due to formation of disulphide bridges, as occurs in related species, was observed only in polyacrylamide gels. The hemolysate showed a pronounced Bohr effect. Oxygen affinity decreased with increasing hemoglobin concentration, indicating that aggregation affects the functional properties of the hemolysate. Oxygen binding equilibria of unfractionated hemolysate are insensitive to chloride and show low sensitivity to ATP. Analysis of oxygen equilibrium curves in terms of the two-state allosteric model (MWC) showed that pH change exerted a greater effect on the association constant of the oxygenated state (KR) than that of the deoxy state (KT). The number of interacting binding sites (q) increased with hemoglobin concentration. Cooperativity of oxygen binding, evaluated as Hill coefficient n, never exceeded the value of 3.0. Earlier studies on hemoglobin and blood from this and related species, report significantly higher n values at high O2 saturation. Molecular adaptive aspects are discussed.

[Comparative analysis of seasonal changes in the hematopoietic processes in the spleen and peripheral blood of the common frog. II. Thrombocyte and neutrophil series]. / Sravnitel'nyi analiz sezonnykh izmenenii protsessov krovetvoreniia v selezenke i perifericheskoi krovi travianoi liagushki, II. Trombotsitarnyi i neitrofil'nyi riady.

Dynamics of the appearance of immature forms of the thrombocytic and neutrophilic cell lineages in the blood of the grass frog was studied during the forced cessation of hibernation. The thrombocytic lineage cells were classified into three maturation stages, their morphology being described. In winter, immature cells of both the lineages are present in hemopoietic tissue and occasionally in blood. Under the action of warm, they rapidly increase in number, within the next 1-2.5 days. The number of cells being at various stages of maturation in the blood of each animal showed, during the experiment, synchronous and similarly directed changes. A proposal is made that the maturing thrombocytes and neutrophils may pass easily the blood-spleen barrier without making any limited population in the blood. By this they differ from the erythroid cells which develop within the blood flow in summer as an independent synchronous population. Seasonal changes in migration rates of the newly formed thrombocytes and neutrophils into the blood were studied using thymidine autoradiography. The entry of these cells into the blood proved to be limited in winter and in spring. It means that a kind of seasonal regulation exists in the functioning barrier between the hemopoietic tissue and the blood.

[The seasonal dynamics of erythropoiesis in the frog Rana temporaria]. / Sezonnaia dinamika éritropoéza liagushki Rana temporaria.

Studies have been made on the dynamics of erythropoietic activity of the bone marrow in mature female frogs. Evident monocyclic pattern of erythroid blood production was found, its maximum activity being closely related to seasonal changes in physiological reparation of the main bulk of functional systems of frog's organism. The data obtained indicate that the bone marrow is the main site of erythropoiesis in the frog.

[The interaction of organosilicon bisammonium compounds with the cholinesterases of Far-Eastern squids and vertebrates. The comparative enzymological, kinetic and conformational aspects]. / Vzaimodeistvie kremniiorganicheskikh bisammonievykh soedinenii s kholinésterazami dal'nevostochnykh kalmarov i nekotorykh pozvonochnykh. Sravnitel'no-énzimologicheskie, kineticheskie i konformatsionnye aspekty.

The research of interaction of cholinesterases from human erythrocytes, horse blood serum, brain of the frog Rana temporaria, optical ganglia of the Pacific squid Todarodes pacificus, as well as optical ganglia of specimens of the Comandor squid Berryteuthis magister from different areas in the Bering sea with 13 organosilicon bis (trimethylammonium) compounds (silane and siloxane rows) as reversible cholinesterase inhibitors have been first studied and generalized. Calculations of the effector structure using the method of theoretical conformational analysis showed higher rigidity of interonium chain of siloxane derivatives and gave grounds to accept the asymmetry of hydrate envelope for these compounds. The data of conformational and kinetic analysis allowed to propose that specificity of antienzymic effect of organosilicon compounds was mainly due to conformational flexibility of interonium chain and the nature of hydration of effector molecule. The new information on the mechanism of catalytic reactions effected by cholinesterases from animals at different evolutionary stages was obtained.

Cytochemistry of blood cells in two West African amphibians.

Lipid, glycogen, peroxidase, acid and alkaline phosphatases were studied in the cells of blood smears in the toad and frog. While lipid and glycogen were largely confined to granulocytes and monocytes, peroxidase activity was intense in all cell types except lymphocytes in both species.

Glucose and free amino acids in the blood of lampreys (Lampetra fluviatilis L.) and frogs (Rana temporaria L.) under prolonged starvation.

The content variation dynamics of glucose and free amino acids in blood plasma was followed for lampreys and frogs from autumn till spring, when the exogenous feeding is switched off. In October, the glucose level is rather high (8-10 mM) in blood plasma of both lampreys and frogs. It falls by 50% during winter and falls to a critically low level (1-2 mM) during spring. The lamprey plasma amino acid levels increase by 74% from November to April and thus reach the lower limit known for mammals. The amount of free amino acids in frog plasma decreases by 40% by spring in comparison with the values in autumn. More intensive proteolysis in lamprey tissues compared with that in frog tissues has been confirmed by quantitatively determining leucine, isoleucine, and valine in the blood of these animals. Besides these three amino acids, alanine, glycine, lysine, threonine and, in certain periods, tyrosine have been found to be quantitatively significant in the plasma of both animals.

Changes in thyroid hormones in the serum and the thyroid gland of hibernating frogs, Rana temporaria L.

The hypothalamo-pituitary-gonadal axis spontaneously activates in hibernating frogs, Rana temporaria, under constant conditions (0-4 degrees, darkness). The hypothesis that the spontaneous hibernatory activation involves other regulatory processes preparing the frogs for breeding and posthibernatory activity was tested. The serum concentrations and glandular contents of thyroid hormones (THs) were determined during hibernation. It was shown that (i) in both sexes, serum thyroxine and triiodothyronine levels significantly increased in the middle of January (week 13/14, between day 92 and 98 of hibernation); (ii) the peak of THs blood concentration was accompanied by a slow decrease of free forms of THs and the bound forms of both hormones dropped rapidly over this period; (iii) after a decrease on day 111, the low level of serum THs (but higher than before the peak) was sustained to the end of hibernation; (iv) the thyroid content of free THs significantly rose after their "surge" into the blood, reached maximal values in the middle of February, and remained at this level to the end of hibernation (last week of March); (v) in spring, after spawning, the THs levels in glands and in serum were much lower than those at the end of hibernation or were not detectable; and (vi) the results were confirmed during the second season, when the material in the period of expected elevation of serum THs was collected every other day.

The protective effects of metabolic rate depression in hypoxic cold submerged frogs.

Aerobic metabolic rates (MO2) and respiratory quotients (RQ = MCO2/MO2) were measured in hypoxic frogs, hibernating underwater for up to 90 days at 3 degrees C. After 3 months of submergence at a PO2 of 50 mmHg, MO2 levels were 25% of those seen prior to hibernation. In progressive hypoxia, a gradual shift in RQ from 0.85 to 1.0 signaled an increasing reliance upon carbohydrate metabolism. Moreover, the glycogen concentrations of skeletal muscle, liver and heart of hypoxic frogs were more rapidly depleted than in their normoxic counterparts. A plasma lactacidosis revealed that the hypoxic animals recruited anaerobiosis to fuel a large 'Pasteur effect'. Throughout all stages of hypoxia, cellular ATP levels were maintained homeostatic. The ability to depress metabolic rate such that ATP demands can be met by oxidative phosphorylation in an oxygen limited environment is the key to the frogs' overwintering survival.

Toxicity of tetrachlorvinphos to Rana temporaria L.

1. Changes in the erythrocyte system of frogs poisoned with tetrachlorwinfos depend on the sex of the animals and the dose of pesticide applied. They are a result of the pathomorphological changes due to translocation of fluids from the tissues to the circulation and swelling of the blood cells. 2. Changes in the leucocyte system of frogs are caused by several mechanisms: lytic action of the pesticide on the blood cell membrane, the stressogenic effect of the agent and enhanced activity of the reticuloendothelial system. 3. The appearance of typical changes due to stress, after even the lowest dose of tetrachlorwinfos, and low LD50 values indicate that this pesticide is highly toxic for frogs. 4. The relatively high susceptibility of frogs to intoxication with tetrachlorwinfos is probably the result of a high affinity of cholinesterase to this pesticide, because of the presence of the P = O bond in its molecule.

Potassium transport in red blood cells of frog Rana temporaria: demonstration of a K-Cl cotransport.

Pathways of K+ movement across the erythrocyte membrane of frog Rana temporaria were studied using 86Rb as a tracer. The K+ influx was significantly blocked by 0.1 mmol.l-1 ouabain (by 30%) and 1 mmol.l-1 furosemide (by 56%) in the red cells incubated in saline at physiological K+ concentration (2.7 mmol.l-1). Ouabain and furosemide had an additive effect on K+ transport in frog red cells. The ouabain-sensitive and furosemide-sensitive components of K+ influx saturated as f(K+)e with apparent Km values for external Ke+ concentration of 0.96 +/- 0.11 and 4.6 +/- 0.5 mmol.l-1 and Vmax of 0.89 +/- 0.04 and 2.8 +/- 0.4 mmol.l cells-1.h-1, respectively. The residual ouabain-furosemide-resistant component was also a saturable function of Ke+ medium concentration. Total K+ influx was significantly reduced when frog erythrocytes were incubated in NO3- medium. Furosemide did not affect K+ transport in frog red cells in NO3- media. At the same Ke+ concentration the ouabain-furosemide-insensitive K+ influx in Cl- medium was significantly greater than that in NO3- medium. We found no inhibitory effect of 1 mmol.l-1 furosemide on Na+ influx in frog red cells in Cl- medium. K+ loss from the frog erythrocytes in a K(+)-free medium was significantly reduced (mean 58%) after replacement of Cl- with NO3-. Furosemide (0.5 mmol.l-1) did not produce any significant reduction in the K+ loss in both media. The Cl(-)-dependent component of K+ loss from frog red cells was 5.7 +/- 1.2 mmol.l-1.h-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Temperature effects on ion transport across the erythrocyte membrane of the frog Rana temporaria.

Unidirectional K+ and Na+ influxes in the frog erythrocytes incubated in Cl- or NO(3)- media with 2.7 mM K+ were measured using 86Rb and 22Na as tracers. K+ influx was inhibited by 35-55% in the presence of 0.2-1.0 mM furosemide but it was unaffected by 0.1-0.2 mM bumetanide. Furosemide at a concentration of 0.5 mM had no effect on K+ loss from the frog red cells incubated in a nominally K(+)-free medium. Together with our previous studies the data support the existence of K-Cl cotransport and the absence of Na-K-2Cl cotransport in the frog erythrocyte membrane. Cell cooling from 20 to 5 degrees C caused a decrease in K+ influx and K+ efflux via the K-Cl cotransporter (3.2- and 3.7-fold, respectively) giving an apparent energy of activation (EA) of about 60 kJ/mol and Q10 value of 2.5. Only small decline (approximately 30%) in the ouabain-sensitive K+ influx was found as temperature was changed from 20 to 5-10 degrees C. Low values of Q10 (approximately 1.5) and EA (27.3 kJ/mol) were obtained for passive K+ influx in the frog erythrocytes (ouabain-insensitive in NO(3)- medium) at temperature within 5-20 degrees C. However, the temperature coefficients were greater for passive Na+ influx and passive K+ efflux (Q10 approximately 2.4-2.5 and EA approximately 56-58 kJ/mol). The temperature dependence of all ion transport components displayed discontinuities showing no changes at temperature between 5 and 10 degrees C. Thus, cooling of the frog red cells is associated with a greater decrease of Na+ influx and K+ efflux than passive and active K+ influx. These data indicate that the preservation of a relative high activity of the Na,K-pump during cell cooling and also the temperature-induced changes in the K-Cl cotransport activity and ion passive diffusion contribute to maintenance of ion concentration gradients in the frog erythrocytes at decreased temperature.