REVIEW: Evidence supporting the 'preparation for oxidative stress' (POS) strategy in animals in their natural environment.

Hypometabolism is a common strategy employed by resilient species to withstand environmental stressors that would be life-threatening for other organisms. Under conditions such as hypoxia/anoxia, temperature and salinity stress, or seasonal changes (e.g. hibernation, estivation), stress-tolerant species down-regulate pathways to decrease energy expenditures until the return of less challenging conditions. However, it is with the return of these more favorable conditions and the reactivation of basal metabolic rates that a strong increase of reactive oxygen and nitrogen species (RONS) occurs, leading to oxidative stress. Over the last few decades, cases of species capable of enhancing antioxidant defenses during hypometabolic states have been reported across taxa and in response to a variety of stressors. Interpreted as an adaptive mechanism to counteract RONS formation during tissue hypometabolism and reactivation, this strategy was coined "Preparation for Oxidative Stress" (POS). Laboratory experiments have confirmed that over 100 species, spanning 9 animal phyla, apply this strategy to endure harsh environments. However, the challenge remains to confirm its occurrence in the natural environment and its wide applicability as a key survival element, through controlled experimentation in field and in natural conditions. Under such conditions, numerous confounding factors may complicate data interpretation, but this remains the only approach to provide an integrative look at the evolutionary aspects of ecophysiological adaptations. In this review, we provide an overview of representative cases where the POS strategy has been demonstrated among diverse species in natural environmental conditions, discussing the strengths and weaknesses of these results and conclusions.

Cellular composition of the black scorpionfish (Scorpaena porcus, L 1758) blood and head kidney under short-time acute exposure to hypoxia.

In the present work, we studied the effect of short-term acute hypoxia on the cellular composition of the blood and the head kidney of the black scorpionfish. Dissolved oxygen concentration was decreased from 8.5-8.7 mg O2 l-1 (normoxia) to 3-5 mg O2 l-1 (relative normoxia), 1-3 mg O2 l-1 (moderate hypoxia), and 0-1 mg O2 l-1 (acute hypoxia) within 1.5-2 h by bubbling of water with N2. Exposure period was 4 h, water temperature was adjusted to 14-16 °C, and photoperiod was 12 h (light). Short-time acute hypoxia induced a rapid release of blast and immature cells from the head kidney into the circulating blood of the black scorpionfish, which was associated with reduction in erythropoietic reserves in 2.5 times. The number of immature erythroid cells (pronormoblasts, basophilic and polychromatophilic normoblasts) significantly increased in blood, and the simultaneously relative decrease of the number of abnormal red blood cell (RBC) and the increase of the number of RBC ghosts (lysed RBCs) in circulating blood were observed. The significant correlation between methemoglobin concentration and the number of RBC ghosts was shown (R2 = 0.640 or r = 0.800). Hypoxia induced RBC swelling on 5-6% compared to control. The number of RBC ghosts in the blood is likely involved in the stimulation of erythropoietin production under hypoxia.

Protein kinase A activity and NO are involved in the regulation of crucian carp (Carassius carassius) red blood cell osmotic fragility.

Activation of the cAMP pathway by ß-adrenergic stimulation and cGMP pathway by activation of guanylate cyclase substantially affects red blood cell (RBC) membrane properties in mammals. However, whether similar mechanisms are involved in RBC regulation of lower vertebrates, especially teleosts, is not elucidated yet. In this study, we evaluated the effects of adenylate cyclase activation by epinephrine and forskolin, guanylate cyclase activation by sodium nitroprusside, and the role of Na+/H+-exchanger in the changes of osmotic fragility and regulatory volume decrease (RVD) response in crucian carp RBCs. Western blot analysis of protein kinase A and protein kinase G substrate phosphorylation revealed that changes in osmotic fragility were regulated via the protein kinase A, but not protein kinase G signaling pathway. At the same time, the RVD response in crucian carp RBCs was not affected either by activation of adenylate or guanylate cyclase. Adenylate cyclase/protein kinase A activation significantly decreased RBC osmotic fragility, i.e., increased cell rigidity. Inhibition of Na+/H+-exchanger by amiloride had no effect on the epinephrine-mediated decrease of RBC osmotic fragility. NO donor SNP did not activate guanylate cyclase, however affected RBCs osmotic fragility by protein kinase G-independent mechanisms. Taken together, our data demonstrated that the cAMP/PKA signaling pathway and NO are involved in the regulation of crucian carp RBC osmotic fragility, but not in RVD response. The authors confirm that the study has no clinical trial.

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.

Toxic effect of mussel Mytilus galloprovincialis exposed to Ag-TiO2 and ZnTi2O4-TiO2 bicomponent nanoparticles.

Nanoparticles (NPs) are widely used in various fields, including antifouling paints for ships and industrial structures submerged in water. The potential impact of NPs on aquatic organisms, particularly their potential toxicity, is a significant concern, as their negative impact has been relatively poorly studied. In this study, we evaluated the effect of different concentrations of bimetallic Ag-TiO2 and ZnTi2O4-TiO2 NPs, which could potentially be used in antifouling coatings, on the hemocytes of the Mediterranean mussel Mytilus galloprovincialis. Hemocytes were exposed to NPs at concentrations of 0.1-1 mg/L for 1 and 2 h, and the production of reactive oxygen species (ROS), levels of DNA damage, and number of dead cells were measured. Exposure to Ag-TiO2 NPs at 1 mg/L concentration for 1 h suppressed ROS production in hemocytes and reduced the relative number of agranulocytes in cell suspensions, without inducing DNA damage or cell death. Exposure to ZnTi2O4-TiO2 NPs did not cause changes in the ratio of granulocytes to agranulocytes in suspensions, nor did it affect other functional parameters of hemocytes. However, after a 2 h exposure period, ZnTi2O4-TiO2 NPs (1 mg/L) significantly reduced the production of ROS by hemocytes. These findings suggest that Ag-TiO2 and ZnTi2O4-TiO2 NPs have low acute toxicity for marine bivalves.

Shift in hemocyte immune parameters of marine bivalve Mytilus galloprovincialis (Lamarck, 1819) after exposure to methane.

Methane are widely used in industry as an emerge source may be released significantly higher aquatic ecosystems due to gas seepages. In this study, short-term (90 min) methane effects on bivalve hemocytes were investigated using flow cytometry. Hemocyte parameters including hemolymph cellular composition, phagocytosis activity, mitochondrial membrane potential and reactive oxygen species (ROS) content were evaluated in the mussel Mytilus galloprovincialis (Lamarck, 1819) exposed to hypoxia (control group), pure methane and industrial methane (industrial hydrocarbon mixture). Comparison of biomarkers showed that the mussel was more sensitive to methane than to low oxygen concentration, supporting the effects of methane on the mussel's immune system. After exposure to pure and industrial methane, the number of granulocytes decreased dramatically and the levels of reactive oxygen species, mitochondrial membrane potential and phagocytosis capacity increased significantly. It was shown that the methane type-dependent effect was pronounced, with industrial methane leading to more pronounced changes.

Cellular osmoregulation of the ark clam (Anadara kagoshimensis) hemocytes to hyposmotic media.

Many bivalve species are considered to be euryhaline organisms due to effective adaptation to fluctuations of environmental salinity. Cellular mechanisms responsible for tolerance to salinity changes remain unclear for bivalves despite this question being critically important for commercially cultured species frequently introduced into regions differing from natural habitat by salinity regime. In the present work laser diffraction method was used for the analysis of volume changes in hemoglobin-containing ark clam (Anadara kagoshimensis) hemocytes following hyposmotic stimulation. Hemocytes responded to hyposmotic shock (decrease of media osmolarity from 461 to 216 mОsm/L) by a rapid swelling up to 171.5 ± 15.2% of control level. At normal osmotic conditions (osmolarity 461 mOsm/L), hemocyte mean cellular volume (MCV) was 354.0 ± 24.4 fl and maximum MCV of hyposmotically swollen cells prior lysis was 555.5 ± 57.4 fl (at the osmolarity 194 mOsm/L). Ark clam hemocytes demonstrated volume recovery response following hyposmotic swelling. Regulatory volume decrease (RVD) reaction did not depend on hemoglobin confirmation status. Final MCV of swollen hemocytes at the end of experimental period of RVD in oxygenated and deoxygenated suspensions did not significantly differ.

Cellular Composition and Proliferation Levels in the Hematopoietic Tissue of Black Scorpionfish (Scorpaena porcus L.) Head Kidney and Spleen During the Spawning and Wintering Periods.

To identify cells and analyze proliferative activity of hematopoietic tissue, black scorpionfish head kidney and spleen cells were characterized by light microscopy and flow cytometry. Hematopoiesis of black scorpionfish head kidney was formed by the following series: erythropoietic, granulopoietic, lymphopoietic, and thrombopoietic. Flow cytometric analysis allowed dividing blood cells in hematopoietic organs into subpopulations differing by size, granularity, and proliferative activity. Three distinct subpopulations were observed during the wintering period. The number of small low-granulated cells, identified as lymphocytes and thrombocytes, was 41% ± 4% in both wintering and spawning fish. Proliferating subpopulation of blast (high-granulated) cells amounted to about 36% of the total cell count with 50% ± 5% of proliferating cells; the largest low-granulated cells (10% of total cells) comprised maturing white blood cells, monocytes, and macrophages. The spawning period was accompanied with an increase of maturing neutrophils and enhancement of blast cell proliferation. In the spleen three distinct subpopulations were observed. The subpopulation of small low-granulated cells comprised lymphocytes and thrombocytes similar to the head kidney and amounted 33% ± 4%. Other cells with larger diameter were identified as red blood cells. No proliferation was observed during the wintering period in the spleen, however, spawning induced cell proliferation of erythroblasts (small granulated cells) with the number of dividing cells 84% ± 1%. Anat Rec, 302:1136-1143, 2019. © 2018 Wiley Periodicals, Inc.

Black Scorpionfish (Scorpaena porcus) Hemopoiesis: Analysis by Flow Cytometry and Light Microscopy.

Cell suspensions of head kidney and spleen of black scorpionfish (Scorpaena porcus L.) have been studied using flow cytometry and light microscopy. On the basis of forward scatter (FS) and side scatter (SS) distribution and light microscopy, two main types of cells in the hemopoietic organs were identified: "small cells" (5.1-8.9 µm) and "large cells" (10.8-15.3 µm). Subpopulation of small cells was formed by thrombocytes, lymphocytes, and elements with functionally inactive nuclei. Euchromatin level in their nuclei was low and acidophilic cytoplasm corresponded to negligible nucleic acids content. No proliferative activity was observed using SYBR Green I fluorescence analysis. Morphological characteristics of these cells coincided with colony forming units of mammals. Large cells in head kidney consisted of two subpopulations of cells differing in granularity and DNA content. Proliferating blast cells and differentiating cells of all hemopoietic lines were identified among them. Macrophages and apoptotic cells were also detected in head kidney. In spleen large cells cluster mainly included aged red blood cells with extended lengthwise axis. Blast cells and differentiating elements in spleen were not observed. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1993-1999, 2017. © 2017 Wiley Periodicals, Inc.

The influence of acute hypoxia on the functional and morphological state of the black scorpionfish red blood cells.

The investigation of the mechanisms of red blood cell steadiness to the oxygen lack in tolerant teleosts is of current scientific interest. Black scorpionfish, Scorpaena porcus L., is a widespread benthal species in the Black Sea and is highly resistant to hypoxic influence. The morphological state of black scorpionfish red blood cells under acute hypoxia was assessed using DNA-binding dye SYBR Green I and fluorescent microscopy. Changes in membrane potential of mitochondria and functional activity of cells were determined by rhodamine 123 (R123) and fluorescein diacetate (FDA) fluorescence. Oxygen deficiency leads to bidirectional changes in volume of erythrocytes and their nuclei. Between 0.57 and 1.76 mg О2 l-1, both parameters increased on 3-12 and 7-21%, respectively. At 1.76-4.03, cells shrank on 1.5-6.0% and nucleus size decreased on 1.5-3%. Acute hypoxia induced a significant increase of R123 (12-60%) and FDA (30-184%) fluorescence. These reactions are caused by a probable decrease in erythrocyte membrane permeability.