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There is growing recognition that the hypoxic regions of the ocean are also becoming more acidic due to increasing levels of global carbon dioxide emissions. The impact of water acidification on marine life is largely unknown, as most previous studies have not taken into account the effects of hypoxia, which may affect how organisms respond to low pH levels. In this study, we experimentally examined the consequences of water acidification in combination with normoxic or hypoxic conditions on cellular immune parameters in Mediterranean mussels. We measured total hemocyte counts in hemolymph, the cellular composition of hemolymph, phagocytosis, reactive oxygen species (ROS) production. General response of the organism was evaluated on the basis of the activity of antioxidant enzymes in the hepatopancreas, as well as respiratory rates over an 8-day exposure period. The mussels were exposed to low pH conditions (7.3), either under normoxic conditions (dissolved oxygen concentration of 8 mg/L) or hypoxic conditions (dissolved oxygen concentration of 2 mg/L). The parameters were assessed at days 1, 3, 6, and 8 of the experiment. Experimental acidification under normoxic conditions reduced THC and ROS production by hemocytes during later stages of exposure, but phagocytic activity (PA) only decreased at day 3 and then recovered. Combined acidification and hypoxia suppressed PA in hemocytes at the beginning of exposure, while hemocyte ROS production and THC decreased by the end of the experiment. The hemolymph cellular composition and activity of antioxidant enzymes were unaffected by acidified conditions under different oxygen regimes, but mussel respiratory rate (RR) decreased with a more significant reduction in oxygen consumption under hypoxia. Mussels showed a relatively high tolerance to acidification in combination with various dissolved oxygen levels, although prolonged acidification exposure led to increased detrimental effects on immunity and metabolism., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The effect of water acidification in combination with normoxia or hypoxia on the antioxidant capacity and oxidative stress markers in gills and hemolymph of the Mediterranean mussel (Mytilus galloprovincialis), as well as on gill microstructure, has been evaluated through an in vivo experiment. Mussels were exposed to a low pH (7.3) under normal dissolved oxygen (DO) conditions (8 mg/L), and hypoxia (2 mg/L) for 8 days, and samples were collected on days 1, 3, 6, and 8 to evaluate dynamic changes of physiological responses. Cytoplasmic concentrations of reactive oxygen species (ROS) and levels of DNA damage were measured in hemocytes, while the activity of catalase (CAT) and superoxide dismutase (SOD) and histopathological changes were assessed in gills. The results revealed that while water acidification did not significantly affect the activity of SOD and CAT in gills under normoxic and hypoxic conditions, there was a trend towards suppression of CAT activity at the end of the experimental period (day 8). Similarly, we did not observe increased formation of ROS in hemocytes or changes in the levels of DNA damage during the experimental period. These results strongly suggest that the oxidative stress response system in mussels is relatively stable to experimental conditions of acidification and hypoxia. Experimental acidification under normoxia and hypoxia caused changes to the structure of the gills, leading to various histopathological alterations, including dilation, hemocyte infiltration into the hemal sinuses, intercellular edema, vacuolization of epithelial cells in gill filaments, lipofuscin accumulation, changes in the shape and adjacent gill filaments, hyperplasia, exfoliation of the epithelial layer, necrosis, swelling, and destruction of chitinous layers (chitinous rods). Most of these alterations were reversible, non-specific changes that represent a general inflammatory response and changes in the morphology of the gill filaments. The dynamics of histopathological alterations suggests an active adaptive response of gills to environmental stresses. Taken together, our data indicate that Mediterranean mussels have a relative tolerance to water acidification and hypoxia at tissue and cellular levels., Competing Interests: Declaration of competing interest Aleksandra Andreyeva reports financial support was provided by Russian scientific foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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 ZnTi 2 O 4 -TiO 2 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, ZnTi 2 O 4 -TiO 2 NPs (1 mg/L) significantly reduced the production of ROS by hemocytes. These findings suggest that Ag-TiO 2 and ZnTi 2 O 4 -TiO 2 NPs have low acute toxicity for marine bivalves., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Semi-anadromous animals experience salinity fluctuations during their life-span period. Alterations of environmental conditions induce stress response where catecholamines (CA) play a central role. Physiological stress and changes in external and internal osmolarity are frequently associated with increased production of reactive oxygen species (ROS). In this work, we studied the involvement of the cAMP/PKA pathway in mediating catecholamine-dependent effects on osmoregulatory responses, intracellular production of ROS, and mitochondrial membrane potential of the river lamprey (Lampetra fluviatilis, Linnaeus, 1758) red blood cells (RBCs). We also investigated the role of hypoosmotic shock in the process of ROS production and mitochondrial respiration of RBCs. For this, osmotic stability and the dynamics of the regulatory volume decrease (RVD) following hypoosmotic swelling, intracellular ROS levels, and changes in mitochondrial membrane potential were assessed in RBCs treated with epinephrine (Epi, 25 μM) and forskolin (Forsk, 20 μM). Epi and Forsk markedly reduced the osmotic stability of the lamprey RBCs whereas did not affect the dynamics of the RVD response in a hypoosmotic environment. Activation of PKA with Epi and Forsk increased ROS levels and decreased mitochondrial membrane potential of the lamprey RBCs. In contrast, upon hypoosmotic shock enhanced ROS production in RBCs was accompanied by increased mitochondrial membrane potential. Overall, a decrease in RBC osmotic stability and the enhancement of ROS formation induced by β-adrenergic stimulation raises concerns about stress-associated changes in RBC functions in agnathans. Increased ROS production in RBCs under hypoosmotic shock indicates that a decrease in blood osmolarity may be associated with oxidative damage of RBCs during lamprey migration., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Boring sponge infection affects growth, development and reduces the soft tissue weight of oysters. In this study, we investigated the effects of boring sponge on the activity of three antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GP)) in the mantle, and the production of reactive oxygen species (ROS) and potential genotoxicity in hemocytes of the Pacific oyster Magallana gigas. Our results showed a significant increase in ROS production and DNA damage in hemocytes. Notably, the activity of SOD, CAT, and GP in the mantle was not significantly affected by boring sponge infection. Collectively, these results suggest that sponge invasion may cause oxidative stress in Pacific oyster hemocytes through ROS overproduction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bivalve mollusks that inhabit low-depth coastal and estuarine areas frequently experience osmotic stress that may be also associated with alterations of antioxidant enzyme activities and markers of oxidative stress. Mitochondria are a major source of reactive oxygen species (ROS) in eucaryotic cells. Overpoduction of ROS induces oxidative stress leading to a damage of intracellular compartments and cell death. In euryhaline bivalves, information concerning cellular ROS production upon osmotic stress and changes in mitochondrial membrane potential is scarce. The present study investigates osmotic stability and hemocytes` regulatory volume decrease (RVD) of Mediterranean mussel (Mytilus galloprovincialis) and the Pacific oyster (Magallana gigas). We also studied dynamic changes in intracellular ROS levels and mitochondrial membrane potential in hemocytes undergoing the RVD response following hypoosmotic swelling. Our data revealed that osmotic stability of mussel and oyster hemocytes did not significantly differ. Loss of environmental osmolarity from 460.0 ± 2.0 mOsm l -1 to 216.0 ± 4.0 mOsm l -1 resulted in an increase of hemocyte volume by 60% of the initial cellular volume in mussels and by 28% in oysters. After rapid hypoosmotic swelling hemocytes of both species demonstrated the RVD response. At the end of 60 min exposure to hypoosmotic environment, hemocyte volume significantly decreased in both species by 10-12% compared to the maximal hemocyte volume. Hypoosmotic shock induced an increase of mitochondrial membrane potential in hemocytes of mussels and oysters. In mussels, increased mitochondrial membrane potential was accompanied with decreased ROS levels in hemocytes, whereas oyster hemocytes showed enhanced ROS production., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Bivalve mollusks as typical osmoconformers are unable to maintain a constant level of internal osmolarity in conditions of salinity stress. Adaptation to fluctuations of environmental salinity is achieved through cellular osmoregulatory responses, which are accompanied with a substantial shift in functional state of cells. In the present work we investigated the effect of hypersalinity stress on hemolymph cellular composition and morphology of the ark clam (Anadara kagoshimensis) hemocytes. Ark clams were subjected to a gradual increase of environmental salinity from 18‰ to 35‰ and 45‰ and maintained at those conditions for two days. Exposure to hypersalinity 35‰ induced changes in erythrocyte morphology and led to a decrease of their diameter. At salinity 45‰ a substantial increase of hemocyte average diameter was observed, whereas the shape of cells did not change (18‰). Hyperosmotic stress was not associated with changes in hemocyte viability as well as changes in hemolymph cellular composition. The results of the present work demonstrate high tolerance of A. kagoshimensis to short-time exposure to hypersalinic conditions., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Mussels were exposed to di-(2-ethylhexyl) phthalate (DEHP) (0.4 mg L -1 and 4.0 mg L -1 ) for 24 h and 48 h and its effect on hemocyte cellular composition and spontaneous reactive oxygen production (ROS) levels in hemocytes were evaluated. Exposure to DEHP induced a loss in spontaneous ROS production levels in hemocytes and decreased agranulocyte number in hemolymph. DEHP was found to accumulate in hepatopancreas of mussels and this process was associated with an increase of catalase (CAT) activity after 24 h incubation. At the end of the experimental period (48 h) CAT activity recovered up to control levels. Superoxide dismutase (SOD) activity in hepatopancreas increased following 48 h exposure to DEHP. The results indicated that DEHP could affect hemocyte immune properties, and also cause non-specific general stress-response of the antioxidant complex, which, in turn, was not associated with pronounced oxidative stress., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ekaterina Kladchenko reports financial support was provided by A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS., (Copyright © 2023. Published by Elsevier Ltd.)

This study describes the analysis of antioxidant enzymatic activities (catalase and superoxide dismutase) in gills and functional state of hemocytes (osmotic stability, mitochondrial membrane potential) of ark clams (Anadara kagoshimensis) from the Black Sea basin exposed to salinity stress. For this, the effects of 48 h periods of exposure to low (8 ‰, 14 ‰) and high (35 ‰, 45 ‰) salinity were assessed. Our results showed that ark clams, A. kagoshimensis, possessed pronounced tolerance to hypersalinity stress and are sensitive to a short-time hyposalinity treatment. Salinity 35 ‰ inhibited production of reactive oxygen species (ROS) by hemocytes and did not affect their levels of mitochondrial membrane potential. Acclimation to 45 ‰ salinity caused significant increase in mitochondrial membrane potential accompanied with recovery of intracellular ROS levels up to controls levels. Acclimation to low salinity (8 ‰) induced an increase in both ROS and mitochondrial membrane potential levels in hemocytes. Catalase and superoxide dismutase activity in gills decreased following acclimation to low (8 ‰) and high (35 ‰) salinity. Exposure to the highest salinity levels (45 ‰) led to a decrease of superoxide dismutase activity levels, but did not influence the levels of catalase activity. Acclimation to low and high salinity was not accompanied with changes in osmotic fragility of hemocytes despite osmotic fragility curve according to changes in hemolymph osmolarity. Based upon these results, we postulate the involvement of cellular osmoregulatory mechanisms in the adaptation of the ark clam to short-term fluctuations of environmental salinity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Environmental hypoxia naturally occurs in coastal ecosystems and bivalve mollusks have to frequently face fluctuations of dissolved oxygen concentrations. Exposure to hypoxia is often associated with the change of the antioxidant and functional status in bivalves, and restoration of the normal oxygen supply is considered to induce oxidative stress in tissues of mollusks. The study investigates changes in the activity of two antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD), as well as the expression level of SOD and CAT genes in gills of the Mediterranean mussel, Mytilus galloprovincialis, under exposure to low dissolved oxygen concentration (2.2 mg L -1 ) for 24 h and 72 h, and 24 h reoxygenation period. We also evaluated the intracellular level of reactive oxygen species (ROS), mortality and changes in mitochondrial membrane potential in hemocytes following hypoxia-reoxygenation cycle. 24 h exposure to hypoxia significantly decreased activity of both enzymes, which then recovered up to control levels at the end of 72 h experimental period for SOD and after reoxygenation for CAT. Expression of antioxidant enzyme genes was up-regulated following the 72 h hypoxic exposure period and returned to the basal normoxic level after 24 h reoxygenation. Hypoxia demonstrated a time-dependent effect on the functional state of hemocytes. The 24 h exposure period did not influence aerobic respiration of hemocytes, but prolonged hypoxia (72 h) was associated with a substantial decrease in mitochondrial membrane potential of hemocytes. The intracellular ROS level and mortality of hemocytes did not change under hypoxia. Reoxygenation period was accompanied with a significant decrease of intracellular ROS level. This study indicated that hypoxia did not induce the pronounced oxidative stress in gills and the changes in the antioxidant status were reversible within 24 h of reoxygenation. Hemolymph demonstrated a stable functional state indicating the tolerance of mussels to short-time hypoxia., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 O 2 l -1 (normoxia) to 3-5 mg O 2 l -1 (relative normoxia), 1-3 mg O 2 l -1 (moderate hypoxia), and 0-1 mg O 2 l -1 (acute hypoxia) within 1.5-2 h by bubbling of water with N 2 . 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 (R 2  = 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., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (© 2022 Wiley Periodicals LLC.)

Activation of the cAMP pathway by β-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., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Bivalve mollusks are frequently subjected to fluctuations of dissolved oxygen concentration in the environment which can represent a significant threat to bivalve antioxidant status. In this work the effects of hypoxia on hemocyte reactive oxygen species (ROS) production and level of mitochondrial potential as well as the activity and expression level of catalase (CAT) and superoxide dismutase (SOD) in gills of Crassostrea gigas were investigated after 24 h and 72 h exposure. 24 h hypoxia promoted an increase of mitochondrial membrane potential in agranulocytes and induced ROS accumulation in granulocytes. 72 h exposure substantially decreased hemocyte mitochondrial potential and intracellular ROS level in all hemocyte types. No significant changes in the activity of CAT in gills were observed following both 24 h and 72 h exposure periods compared to control. SOD activity in gills decreased after 72 h exposure to hypoxia but did not change under 24 h hypoxia. Significant up-regulation of SOD gene and no changes in expression level of CAT were observed in all experimental groups. The results indicate an overall shift in antioxidant status in gills and hemocytes of the Pacific oyster that may act as compensatory mechanisms to maintain redox homeostasis after a short-term (24 h) exposure and represent the occurrence of oxidative stress conditions at the end of 72 h hypoxia., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The influence of hypothermia on erythrocyte profile of thermophile teleost species round goby, Neogobius melanostomus (Pallas, 1814), has been studied. Fish were acclimated to temperature 1-2 о С, 15-16 о С and 19-20 о С (control group) and held at given conditions for 5 days. The number of red blood cell precursors (pronormoblasts, basophilic and polychromatophilic normoblasts) in circulating blood has been estimated. Also, the number of abnormal erythrocytes, i.e. cells with micronuclei, nuclei invaginations, red blood cell shades, dacryocytes and cells undergoing amitosis has been determined on smears. The number of immature erythrocytes increased more than two times (p < 0,001) at 1-2 о С. The number of low-differentiated precursors, pronormoblasts and early basophilic normoblasts, increased for the most part. The number of abnormal erythrocytes did not change substantially, The changes in cellular blood composition were accompanied with the increase of plasma lactate concentration, indicating hypoxic state of fish. The results of the present work indicate that hematopoietic tissue remains sensitive to controlling factors at hypothermia, such as hypoxia, and may enhance proliferation and differentiation of erythroid cells., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Circulating hemocytes of ark clam, Anadara kagoshimensis, were investigated using light microscopy and flow cytometry. Hemolymph contained 3 morphotype of cells, amebocytes, erythrocytes and intermediate type cells, which formed two distinct subpopulations on flow cytometric dot plots. Large cells (intermediate morphotype and erythrocytes) amounted 85.6 ± 2.8% total cells in hemolymph. Erythrocytes were hemoglobin-containing cells with up to 40 granules presented in the cytoplasm. All hemocyte types, observed in the ark clam hemolymph demonstrated equal capacity to spontaneous production of reactive oxygen species., (Copyright © 2019 Elsevier Ltd. All rights reserved.)