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During their development, amphibians undergo various physiological processes that may affect their susceptibility to environmental pollutants. Naturally occurring fluctuations caused by developmental events are often overlooked in ecotoxicological studies. Our aim is to investigate how biomarkers of oxidative stress are modulated at different stages of larval development in the Amazonian amphibian species, Physalaemus ephippifer. The premetamorphosis, prometamorphosis and metamorphic climax stages were used to analyze total antioxidant capacity (ACAP), glutathione S-transferase (GST) activity, lipid peroxidation (LPO) levels and the expression of genes nrf2, gst, gsr (glutathione reductase) and gclc (glycine-cysteine ligase, catalytic subunit). Although there was no difference in ACAP and the genes expression among the studied stages, individuals from the premetamorphosis and prometamorphosis showed higher GST activity than ones under the climax. LPO levels were highest in individuals from the metamorphic climax. The present study suggests that the oxidative status changes during ontogeny of P. ephippifer tadpoles, especially during the metamorphic climax, the most demanding developmental phase. Variations in the redox balance at different developmental stages may lead to a divergent response to pollution. Therefore, we recommend that studies using anuran larvae as biomonitors consider possible physiological differences during ontogeny in their respective analyses., (© 2024. The Author(s).)

Amphibian larvae inhabiting temporary ponds often exhibit the capacity to accelerate development and undergo metamorphosis in challenging conditions like desiccation. However, not all species exhibit this ability, the yellow-bellied toad (Bombina variegata) is one such example. The underlying mechanisms behind the inability to accelerate development under desiccation remain largely unexplored. The hypothalamic-pituitary-interrenal (HPI) axis and corticosterone (CORT), which act synergistically with thyroid hormone, are thought to facilitate metamorphosis in response to desiccation stress. In this study, we aimed to investigate whether modification in the HPI axis, particularly CORT levels, contributes to the absence of adaptive plasticity in B. variegata under desiccation stress. The study design included four treatments: high water level, high water level with exogenous CORT, low water level, and low water level with metyrapone (a CORT synthesis inhibitor). The main objective was to evaluate the effects of these treatments on whole-body corticosterone levels, life history, morphological traits, and oxidative stress parameters during the prometamorphic and metamorphic climax developmental stages. While low water level had no effect on total corticosterone levels, larval period, body condition index, and metamorphic body shape, it negatively affected metamorph size, mass, and growth rate. Our findings suggest that constant exposure to desiccation stress over generations may have led to modifications in the HPI axis activity in B. variegata, resulting in adaptation to changes in water level, evident through the absence of stress response. Consequently, CORT may not be a relevant stress indicator in desiccation conditions for this species., (© 2024 The Authors. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology published by Wiley Periodicals LLC.)

The effect of anesthesia/euthanasia with ethyl 3-aminobenzoate methanesulfonate (MS-222) on the oxidative status of Hyla arborea tadpoles was examined to determine whether the use of the anesthetic can confound the experimental results of the oxidative stress-based investigation. The experiment was conducted on two groups of tadpoles reared at different temperatures to produce differences in antioxidant capacity between the groups. After development at different temperatures (20 °C and 25 °C), the animals were exposed to different concentrations of MS-222 (0, 0.1, 1, and 5 g/L) for 15 min. The higher temperature decreased catalase activity, glutathione and protein carbonyl levels and increased glutathione reductase activity. The glutathione level and glutathione/thiol-related parameters were significantly changed after MS-222 exposure. However, individuals from the different temperature groups responded differently to the tested anesthetic, pointing to the possible influence of the initial levels of antioxidant capacity. The analysis of the interaction between the factors (temperature and MS-222) confirmed that the anesthetic can confound the results regarding the effects of temperature on the oxidative status parameters. The concentration of 0.1 g/L MS-222 had the lowest influence on the alterations in oxidative status and the results of the effect of temperature. A brief review of the current literature on the use of MS-222 in tadpoles made clear the absence of precise information on anesthetic concentration and exposure time. Similar studies should be repeated and extended to other amphibian species and other factors of interest to provide better guidance on tadpole anesthesia/euthanasia for future experiments that consider oxidative status parameters., 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.)

Dynamic changes of tissues, organs and growth that occur in fish larvae during the transition to the juvenile stage are accompanied by differences in metabolic, locomotor and feeding activities that can reflect on fish's oxidative status. In this study, we examine how body growth, antioxidant system (superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and total antioxidant capacity (TAC) and oxidative damage (malondialdehyde-MDA) parameters change in larvae of yellowfin seabream larvae (Acanthopagrus latus) during early development (0, 7, 15, 22 and 30 day after hatching-DAH). Body growth (length and weight) starts to intensify from 15 DAH. We observed general increase in the antioxidant system (AOS) with the age of larvae from newly hatched and 7 DAH up to 15 and 22 DAH individuals. 15 and 22 DAH larvae had the greatest levels of TAC, SOD and GR activity, while 30 DAH larvae had higher CAT activity from 0, 7 and 15 DAH and MDA concentration in comparison to 15 DAH individuals. Several developmental events can be linked with observed results: lower AOS in 0 and 7 DAH individuals with low locomotor activity, growth, endo-exogenous feeding phase and cell differentiation; 15 and 22 DAH larvae are under pressure of fast growth, enhanced swimming and foraging capacity; while higher MDA production in 30 DAH larvae can be a result of shifts in muscle metabolism, changes in both quality and quantity of food and a significant increase in weight. The present study provides insight into the changes in redox status during the ontogeny of A. latus, fish species about which physiology is still little known but with a potential for use in marine culture. Ability to lower oxidative stress during critical developmental periods can enhance that potential., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

Fish in wild are often faced with various types of xenobiotics, that may display synergistic or antagonistic effects. In this study, we aim to examine how exposure to agrochemical compound (Bacilar) and cadmium (CdCl 2 ) alone and in combination affect biochemical parameters (lactate dehydrogenase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, alanine aminotransferase; creatine phosphokinase (CKP), cholinesterase) and oxidative stress (total antioxidant capacity, catalase, malondialdehyde and protein carbonyl concentrations) of freshwater fish Alburnus mossulensis. Fish were exposed to two concentrations of Bacilar (0.3, and 0.6 mL L -1 ) and to 1 mg L -1 cadmium chloride alone and in combination for 21 days. Results showed that fish accumulate Cd in their body, with the highest rate in individuals exposed to Cd in combination with Bacilar. Both xenobiotics in fish liver induced the activation of liver enzymes suggesting hepatotoxic effects, with the greatest impact in co-exposed groups. A significant decrease in the hepatocyte's total antioxidant capacity indicates the collapse of the antioxidant defense in fish exposed to Cd and Bacilar. A decrease in the antioxidant biomarkers was followed by increased oxidative damage of lipids and proteins. We also reported altered function in the muscle of individuals exposed to Bacilar and Cd seen as decreased activities in CKP and butyrylcholinesterase. Overall, our results point to the toxicity of both Bacilar and Cd on fish but also to their synergistic effects on Cd bioaccumulation, oxidative stress, and liver and muscle damage. This study highlights the need for evaluating the use of agrochemicals and their possible additive effects on non-target organisms., 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.)

Ectotherms are particularly sensitive to global warming due to their limited capacity to thermoregulate, which can impact their performance and fitness. From a physiological standpoint, higher temperatures often enhance biological processes that can induce the production of reactive oxygen species and result in a state of cellular oxidative stress. Temperature alters interspecific interactions, including species hybridization. Hybridization under different thermal conditions could amplify parental (genetic) incompatibilities, thus affecting a hybrid's development and distribution. Understanding the impact of global warming on the physiology of hybrids and particularly their oxidative status could help in predicting future scenarios in ecosystems and in hybrids. In the present study, we investigated the effect of water temperature on the development, growth and oxidative stress of two crested newt species and their reciprocal hybrids. Larvae of Triturus macedonicus and T. ivanbureschi, and their T. macedonicus-mothered and T. ivanbureschi-mothered hybrids were exposed for 30 days to temperatures of 19°C and 24°C. Under the higher temperature, the hybrids experienced increases in both growth and developmental rates, while parental species exhibited accelerated growth (T. macedonicus) or development (T. ivanbureschi). Warm conditions also had different effects on the oxidative status of hybrid and parental species. Parental species had enhanced antioxidant responses (catalase, glutathione peroxidase, glutathione S-transferase and SH groups), which allowed them to alleviate temperature-induced stress (revealed by the absence of oxidative damage). However, warming induced an antioxidant response in the hybrids, including oxidative damage in the form of lipid peroxidation. These findings point to a greater disruption of redox regulation and metabolic machinery in hybrid newts, which can be interpreted as the cost of hybridization that is likely linked to parental incompatibilities expressed under a higher temperature. Our study aims to improve mechanistic understanding of the resilience and distribution of hybrid species that cope with climate-driven 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 © 2023 Elsevier Ltd. All rights reserved.)

Background: Urban development results in habitat destruction, affecting populations of amphibians, the most fragile group of vertebrates. With changes in the environment, these animals become more exposed to light and predators. To enhance their chances of survival, they display plasticity of body coloration. Aside from adaptive benefits, animals exhibiting background matching meet the energetic costs and restrictions of changing body tones. To study the physiological consequences of Hyla arborea tadpole adaptation to background color, we followed oxidative stress parameters after rearing larvae on a constant background (black/white) and after changing the background color., Results: Larvae cultivated for 20 days on constant substrate color exhibited differences in body coloration but without differences in lipid peroxidation (LPO) concentration between dark and pale individuals, suggesting that coloration investment during this period did not induce higher oxidative damage in darker tadpoles. Prolonged exposure of larvae (37 days) to a dark habitat increased antioxidative system defense and LPO concentrations, compared to animals reared permanently in the white surroundings. The positive correlation of oxidative damage with color intensity of individuals points to the physiological consequences of higher investment in the number of pigment cells necessary for dark pigmentation. In individuals faced with non-matching background and change in body coloration, defense system declined and LPO occurred relative to individuals cultivated in white habitat., Conclusion: Here, we have pointed to consequences related to background matching and stress that amphibians experienced during chromatic adaptations. Background color change causes a complex physiological response affecting the antioxidative defense parameters. This investigation elucidates the accompanying cost of amphibians' adjustment to an altered environment., (© 2023. The Author(s).)

Industrial chemical solutions are widely used as a method to disinfection of aquaculture water and environments. The aim of the present study was to evaluate the toxicity effect of Betadine (10% solution of povidone-iodine) as a disinfectant solution on the survival and gill tissue of Oranda goldfish (Carassius auratus). For these purposes, 225 fingerling Oranda goldfish with an average weight 5 ± 0.67 g were divided into 15 groups with 3 replications. Fish were exposed to series of concentrations (0, 10, 20, 40, 60, 80, 90, 100, 120, 140, 160, 180, 200, 220 and 240 mg/L) of Betadine for 96 h. The mortality of fish and the samples of gill were observed at 6, 12, 18, 24, 48, 72 and 96 h after exposure. The results of the present study showed that the half-life of Betadine was less than 24 h and mortality were not record after 24 h. The highest mortality rate was at 240 mg/L, and LC 50 24 h of Betadine was 158.800 mg/L. Histopathological results showed that lethal concentrations of Betadine lead to hyperemia, hypertrophy, hyperplasia and adhesion of secondary lamellar of the gill. Moreover, fish that were exposed to these concentrations displayed clinical signs such as anxiety, darkening of the skin. Overall results showed that Betadine have short half-life in the aquatic environment and are toxic to fish at very high concentrations, therefore it can be considered as practically non-toxic and useful for disinfection of the aquatic environment., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

In large-scale aquaculture, the fast growth rate of fish is positively influenced by feed additives such as medicinal plants. This is however; infectious disease may reduce fish growth and cause devastating economic loss. The present study investigated in vitro antibacterial efficacy of Mooseer (Allium hirtifolium) extract against Streptococcus iniae and its in vivo effects on growth, biochemical parameters, innate immunity of rainbow trout (Oncorhynchus mykiss). Therefore, six experimental diets were designed to include different levels of Mooseer from zero (as control), 5, 10, 15, 20, and 25 g per kg diet respectively referred to as M1 to M5. Results from the antibacterial evaluation showed that Mooseer extract inhibits S. iniae growth with MIC and MBC values of 128 and 256 μg ml -1 . Appreciable results were obtained in the groups supplemented with Mooseer. Mooseer enhanced growth performance, and modulated serum biochemical and immunological parameters (total protein, albumin, triglyceride, glucose, cortisol, cholesterol, lysozyme, Ig, ACH50, ALP, and protease activity), and liver enzymes (ALT, AST and ALP). The greatest effects were found for higher doses of Mooseer supplementation (M4 and M5). Meanwhile, results from the survival rate of fish challenged with S. iniae showed higher survival in M2 and M4 treatments. The present findings suggest the beneficial use of Mooseer in rainbow trout diet, with 20 g kg -1 inclusion as the recommended dose., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Amphibians are sensitive to deteriorating environmental conditions, especially during transition to a terrestrial environment which is full of uncertainties. Harsh conditions, such as desiccation during earlier stages, affect different larval traits with possible carry-over effects on juvenile and adult life histories. The first consequences of the effects can be seen in juveniles in the challenges to find food and the ability to survive without it in a terrestrial habitat. Body size and the internal energy reserves acquired during the larval phase play an important role in this period. Herein, we tested how different water regimes (low water availability, desiccation and constant high-water availability) during larval development reflect on the oxidative status and ability of yellow belly toad ( Bombina variegata ) juveniles to endure short-term fasting. The desiccation regime significantly reduced the body size of metamorphs. The same was observed after 2 weeks of fasting, while the feeding treatment reduced differences mostly in the body mass of individuals from different water regimes. This was the result of a greater gain in mass in juveniles pre-exposed to desiccation. Pre-exposure to desiccation also modified the parameters of the antioxidant system (AOS) under feeding conditions, leading to higher values of superoxide dismutase, glutathione reductase and glutathione S-transferase, glutathione and sulfhydryl group concentrations, and lower glutathione peroxidase in comparison to juveniles reared under constant water. The increase in the AOS of juveniles can be considered as a physiological carry-over effect of desiccation, probably as the result of compensatory growth and/or earlier exposure to chronic stress. However, water levels during larval development did not exert significant effects on the oxidative status of juveniles subjected to food unavailability. Fasting juveniles, both control and desiccated, were exposed to oxidative stress, significantly higher lipid peroxide concentrations, lower superoxide dismutase, glutathione peroxidase, glutathione S-transferase, glutathione and sulfhydryl group values in comparison to feeding individuals. The lack of food in juvenile anurans activated the AOS response in the same manner, regardless of body size and stress pre-exposure, suggesting that the generally accepted hypothesis about the influence of metamorphic body size on the fitness of the postmetamorphic stage should be tested further., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Prokić, Petrović, Gavrilović, Despotović, Gavrić, Kijanović, Tomašević Kolarov, Vukov and Radovanović.)

This study investigates acute toxicity and histological effects of Lindane insecticide on intestinal tissues of immature grass carp (Ctenopharyngodon idella). For these purposes, 105 fish were exposed to different concentrations of Lindane for 96 h in 5 groups with 3 replications. Samples of intestinal tissues were prepared in both control and exposure groups during and after the toxicity test. Vaculation, necrosis, bleeding and epithelial degeneration were apparent in the intestine tissue of treated individuals. The LC 50 96 h of Lindane was 0.788 ppm in the present study, also the results showed besides pesticide-induced lesions, symptoms of abnormal swimming, anxiety, tendency to swim near the surface, and death due to terminal toxicity with mouth widely open. The exposed fish were pale in color, showed oxygen deficit and severe respiratory problems. Overall, our results confirm the toxicity of different concentrations of Lindane insecticide affecting behavior, intestinal function and inducing tissue lesions of immature grass carp., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Global warming represents a severe threat to existing ecosystems, especially for anuran tadpoles who encounter significant fluctuations in their habitats. Decreasing water levels in permanent and temporary water bodies is a significant risk for larval survival or fitness. On the other hand, the natural environment of amphibians is extremely polluted by various xenobiotics. This study evaluated how pre-exposure of Bombina variegata tadpoles to chronic environmental stress (desiccation) modulates the biochemical response of juvenile individuals to following acute chemical stressor (pesticide deltamethrin). Our results demonstrated that individually applied pesticide changed the thiol and lipid status of the treated juveniles but animals subjected solely to desiccation pressure were more tolerant to free radicals and showed no induction of lipid peroxidation. Comparison of juveniles exposed to deltamethrin revealed that desiccation pretreatment during the larval stage of development modified cellular protection in the juveniles. Higher activities of CAT, GSH-Px and GR were recorded in the pre-exposed group, as well as a lower degree of lipid peroxidation relative to the group that was not pre-exposed to low water stress. Pre-desiccated groups displayed the greatest range of coordination of investigated antioxidant parameters, supported by Pearson's correlations. Activation of the GSH-redox system is a significant marker in juveniles against stress caused by desiccation and a chemical stressor. The stressful environment experienced during tadpole development produced an adaptive reaction to subsequent exposure to another stressor in juveniles. To develop relevant management and conservation strategies, more studies of the interactive effects of environmental and chemical stressors are necessary., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In this trial, the interaction between two different light resource and different levels of two pigments was investigated in order to determine their effects on growth and reproductive performance of the ornamental fish swordtail ( Xiphophorus helleri ) broodstock. Fish were fed with different levels of 50, 100 and 200 mg · kg-1 of astaxanthin or canthaxanthin, under two different light conditions (natural and artificial). The obtained results were more evident in animals fed with astaxanthin in comparison to canthaxanthin. Furthermore, swordtails reared under artificial light in comparison to the natural light source showed better performance. Overall the greatest effect was for the fish treated with medium dose of astaxanthin (100 mg.kg-1), under the artificial light conditions of rearing. Results indicated the important effects of all three factors (light source, dosage and carotenoids type), with significant interactions between the carotenoid dosage with carotenoid type and light source.

Pesticides can induce changes in behavior and reduce the survival chance of aquatic organisms. In this study, the toxic effects of glyphosate suspension (Glyphosate Aria 41% SL, Tehran Iran) on behavior and tissues of common carp ( Cyprinus carpio ) were assessed. For this purpose, a 96 h LC 50 of glyphosate suspension (68.788 mL·L -1 ) was used in the toxicity test. All individuals were divided into control and treatment groups with four replicates. Exposure operations were performed under two conditions: increasing concentration of suspension from 0 to 68.788 mL·L -1 ; then, decreasing to the first level. The swimming pattern was recorded by digital cameras during the test and tissue samples were collected at the end of the test. There were significant differences between the swimming pattern of treated individuals and control ones during both steps. The sublethal concentration of glyphosate led to hypertrophy, hyperplasia and hyperemia in the gill of fish. However, changes were obvious only after sampling. The exposed fish also displayed clinical signs such as darkening of the skin and increasing movement of the operculum. Moreover, glyphosate suspension affected swimming patterns of fish suggest that the swimming behavior test can indicate the potential toxicity of environmental pollutants and be used as a noninvasive, useful method for managing environmental changes and assessing fish health conditions by video monitoring.

Thiourea is an established disruptor of thyroid hormone synthesis and is frequently used as an inhibitor of metamorphosis. The changes caused by thiourea can affect processes associated with the oxidative status of individuals (metabolic rate, the HPI axis, antioxidant system). We investigated the parameters of oxidative stress in crested newt ( Triturus spp.) larvae during normal development in late larval stage 62 and newly metamorphosed individuals, and during thiourea-stimulated metamorphosis arrest in individuals exposed to low (0.05%) and high (0.1%) concentrations of thiourea. Both groups of crested newts exposed to thiourea retained their larval characteristics until the end of the experiment. The low activities of antioxidant enzymes and the high lipid peroxidation level pointed to increased oxidative stress in larvae at the beginning of stage 62 as compared to fully metamorphosed individuals. The activities of catalase (CAT) and glutathione-S-transferase (GST) and the concentration of sulfhydryl (SH) groups were significantly lower in larvae reared in aqueous solutions containing thiourea than in newly metamorphosed individuals. The high thiourea concentration (0.1%) affected the antioxidative parameters to the extent that oxidative damage could not be avoided, contrary to a lower concentration. Our results provide a first insight into the physiological adaptations of crested newts during normal development and simulated metamorphosis arrest.

In the study, we assessed how acute changes in water temperature and dissolved ammonia concentration can affect the swimming behavior pattern of rainbow trout (Oncorhynchus mykiss). The behavior was analyzed in three different stages: 1) increase stage, (where temperature and ammonia concentration increase during this step, respectively); 2) unchanged stage, (where levels of both factors do not change during this stage); 3) reduction stage, (where ammonia concentration and temperature during this stage are reduced), respectively. The results showed that both factors significantly changed the swimming pattern of the rainbow trout. There were significant differences in swimming parameters (distance from the center, swimming speed, total movement and the average of angular changes of movement) of treated fish in the comparison between treatments, and with the control group. The changes in the swimming pattern of fish in response to physicochemical parameters of water were confirmed to be a good tool in ecotoxicological studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Although we are witnesses of an increase in the number of studies examining the exposure/effects of microplastics (MPs) on different organisms, there are many unknowns. This review aims to: (i) analyze current studies devoted to investigating the exposure/effects of MPs on animals; (ii) provide some basic knowledge about different model organisms and experimental approaches used in studying MPs; and to (iii) convey directions for future studies. We have summarized data from 500 studies published from January 2011 to May 2020, about different aspects of model organisms (taxonomic group of organisms, type of ecosystem they inhabit, life-stage, sex, tissue and/or organ) and experimental design (laboratory/field, ingestion/bioaccumulation/effect). We also discuss and try to encourage investigation of some less studied organisms (terrestrial and freshwater species, among groups including Annelida, Nematoda, Echinodermata, Cnidaria, Rotifera, birds, amphibians, reptiles), and aspects of MP pollution (long-term field studies, comparative studies examining life stages, sexes, laboratory and field work). We hope that the information presented in this review will serve as a good starting point and will provide useful guidelines for researchers during the process of deciding on the model organism and study designs for investigating MPs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In this paper, we examined how the oxidative status (antioxidant system and oxidative damage) of Bombina variegata larvae changed during the metamorphic climax (Gosner stages: 42-beginning, 44-middle and 46-end) and compared the patterns and levels of oxidative stress parameters between individuals developing under constant water availability (control) and those developing under decreasing water availability (desiccation group). Our results revealed that larvae developing under decreasing water availability exhibited increased oxidative damage in the middle and end stages. This was followed by lower levels of glutathione in stages 44 and 46, as well as lower values of catalase, glutathione peroxidase, glutathione S-transferase and sulfhydryl groups in stage 46 (all in relation to control animals). Comparison between stages 42, 44 and 46 within treatments showed that individuals in the last stage demonstrated the highest intensities of lipid oxidative damage in both the control and desiccation groups. As for the parameters of the antioxidant system, control individuals displayed greater variety in response to changes induced by metamorphic climax than individuals exposed to desiccation treatment. The overall decrease in water availability during development led to increased oxidative stress and modifications in the pattern of AOS response to changes induced by metamorphic climax in larvae of B. variegata .

This study was conducted to compare the effects of commercially available (C) and green synthesized (GS) Zinc oxide nanoparticles (ZnO-NPs) on immunological responses of common carp ( Cyprinus carpio ) skin mucus. GS ZnO-NPs were generated using Thymus pubescent and characterized by UV-vis diffuse reflectance spectroscopy (DRS), Fourier-transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), scanning electron microscope (SEM), and energy-dispersive X-ray spectroscopy (EDX). Fish ( n = 150) were randomly allocated into five groups in triplicate and received a waterborne concentration of 0% (control), 25%, and 50% of LC50 96 h of commercially available (C1 and C2) and green synthesized ZnO-NPs (GS1 and GS2) for 21 days. Results from XRD displayed ZnO-NPs with 58 nm in size and UV-vis DRS, EDX, and FT-IR analysis showed that some functional groups from plant extract bonded to the surface of NPs. The SEM images showed that ZnO-NPs have conical morphology. Acute toxicity study showed a higher dose of LC50 96h for green synthesized ZnO-NPs (78.9 mg.L -1 ) compared to the commercial source (59.95 mg.L -1 ). The highest activity of lysozyme and alternative complement activity (ACH50) were found in control and GS1 groups. A significant decrease in alkaline phosphatase activity (ALP) was found in C1 and C2 groups compared to other treatments. Protease activity (P) was significantly decreased in the C2 group compared to the control and GS groups. Total immunoglobulin (total Ig) content was the highest in the control. In addition, total Ig in the GS1 group was higher than GS2. The exposure to ZnO-NPs lowered total protein content in all experimental groups when compared to control. Present findings revealed lower induced immunosuppressive effects by green synthesized ZnO-NPs on key parameters of fish skin mucus.

This study evaluated the dietary effects of an ultrasound-assisted extract of Origanum vulgare on the growth, antioxidant and immune responses (serum and mucosal) and resistance of zebrafish ( Danio rerio ) against Aeromonas hydrophila . Four hundred and forty adult zebrafish were distributed into 12 tanks and fed 4 experimental diets including 0% (control), 0.5% (M1), 1% (M2) and 2% (M3) of the extract per kg -1 diet for eight weeks. Fish were then challenged with A. hydrophila and mortality was recorded for 10 days. Results revealed that the extract exerted potent effects on growth parameters of weight gain and specific growth rate. The feed conversion ratio was significantly lower in fish fed extract-incorporated diets. O. vulgare extract improved antioxidant and immune responses, resulting in less sensitivity to oxidative stress and a higher survival rate when challenged with A. hydrophila . Overall, the greatest effects were observed in individuals with 1% dietary inclusion of the extract. These results suggest that the extract from the plant Origanum vulgare possesses a great potential to be used in the aquaculture industry and that zebrafish is an appropriate model for nutrition studies.

In nature, animals often face periods without food caused by seasonal fluctuations and/or prey scarcity. An organism's physiological response to imposed energetic limitations is followed by changes in mitochondrial functioning (adjustment of energy metabolism) and a reduction of non-essential processes. However, this energy-saving strategy can have its costs. In this study, we examined oxidative stress as one of the possible physiological costs of short-term, two-week-long food deprivation on developing amphibian larvae of the crested newts Triturus macedonicus and Triturus ivanbureschi and their hybrids. We investigated whether this exogenous factor additionally affected the oxidative status (fitness-related trait) of hybrid individuals. The fasting treatment led to lower growth and a lower body mass and body condition index of individuals. The results revealed that the antioxidant system (AOS) of food-deprived larvae could not cope in a proper manner with reactive oxygen species production under limited energy availability, leading to higher lipid oxidative damage. The lowest AOS response was observed for H 2 O 2 scavenging parameters (catalase, glutathione peroxidase, and total glutathione), which together with the elevated activity of superoxide dismutase suggested increased H 2 O 2 concentrations. Comparison between parental species and their hybrids showed that hybrid individuals suffered greater oxidative damage (as demonstrated by higher concentrations of lipid peroxides), indicating that they were more susceptible to fasting-induced oxidative stress. Overall, this study illustrates that: (i) an oxidative event is one of the costs amphibian larvae face during short-term periods of fasting, (ii) hybrids are less capable of dealing with this stressful condition, which can lower their chances of survival in a changing environment., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Although the long-term exposure of aquatic organisms to cyanobacterial blooms is a regular occurrence in the environment, the prooxidant and neurotoxic effects of such conditions are still insufficiently investigated in situ. We examined the temporal dynamics of the biochemical parameters in the liver of Pelophylax kl. esculentus frogs that inhabit the northern (N) side of Lake Ludaš (Serbia) with microcystins (MCs) produced in a cyanobacterial bloom over three summer months. The obtained data were compared with data on frogs that live on the southern (S), MC-free side of the same lake. Our results showed that the MC-producing bloom induced oxidative damage to proteins and lipids, observed as a decrease in the concentration of protein -SH groups and increased lipid peroxidation (LPO) in the liver of N frogs in comparison to S frogs. Glutathione (GSH) played a key role in the transient defense against the MC-induced development of LPO. The low glutathione peroxidase (GPx) activity detected in all groups of frogs from the N site was crucial for the observed prooxidant consequences. The bloom impaired cholinergic homeostasis as a result of a decrease in ChE activity. A delayed neurotoxic effect in relation to the prooxidant outcomes was observed. Our results also showed that even though the integrated biomarker response (IBR) of the antioxidant biomarkers increased during exposure, the individual biochemical parameters did not exhibit a well-defined time-dependent pattern because of specific adaptation dynamics and/or additional effects of the physicochemical parameters of the water. This comprehensive environmental ecotoxicological evaluation of the cyanobacterial bloom-induced biochemical alterations in the liver of frogs provides a new basis for further investigations of the prolonged, real-life ecotoxicity of the blooms., 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 © 2020 Elsevier B.V. All rights reserved.)

Medicinal plants play an important role in aquaculture as feed additives. This study aimed to investigate effects of alcoholic extract of acorn on growth performance, body composition, digestive enzymes activity and blood biochemical parameters of rainbow trout ( O. mykiss ) as a commercially important fish. Five dietary treatments were supplemented: 100, 200, 400 and 600 mg.kg -1 of the extract. Fishes were fed twice per day for 8 weeks, and results showed that acorn extract positively affected all investigated parameters in rainbow trout fishes. Digestive enzymes activity and growth performance were increased, while activity of liver enzymes and cortisol were lowed in comparison to control individuals. Body composition of treated animals was also enhanced. Comparison between treated groups together with integrative biomarker response (IBR) values indicated greatest effects in animals fed with 400 and 600 mg.kg -1 of the extract. Positive effects of the acorn represent promising start point for further studies.

Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this special issue.

Shelters are important for animal survival. Provision of adequate hiding places allow animals to express their natural sheltering behavior and it can have different positive effects on cortisol levels, physiological processes and mental performance. Although the absence of a refuge activates some stress response, its effect on oxidative stress has not been adequately examined. This study investigated whether the presence/absence of a shelter modifies the oxidative status (the antioxidant system and oxidative damage) and aggressive behavior of crested newt larvae (Triturus macedonicus and its hybrid with T. ivanbureschi ). Our results show that individuals reared with shelters had lower values of the tested antioxidant parameters (catalase, glutathione peroxidase, glutathione S-transferase and glutathione), indicating a lower production of reactive species than individuals reared without shelter. The same pattern was observed in both T. macedonicus and its hybrid. Contrary to the activation of some physiological pathways, shelter availability did not significantly affect the rate of intraspecific aggressive behavior. The physiological benefits of shelter use can be manifested as a lower requirement for investment in the energy necessary for the maintenance of the upregulated antioxidant defenses, activation of repair systems and synthesis of endogenous antioxidants. This study highlights the importance of shelter provision, which may be valuable in habitat restoration and animal conservation studies., Competing Interests: The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The contents of the manuscript have not been published previously, they have not been submitted elsewhere for consideration, nor are they in press. All of the authors have seen and approved the manuscript. There are no competing interests, neither financial, personal or other relationships with other persons or organizations.

There is little information in scientific literature as to how conditions created by a microcystin (MC) producing cyanobacterial bloom affect the oxidant/antioxidant, biotransformation and neurotoxicity parameters in adult frogs in situ. We investigated biochemical parameters in the skin and muscle of Pelophylax kl. esculentus from Lake Ludaš (Serbia) by comparing frogs that live on the northern bloom side (BS) of the lake with those that inhabit the southern no-bloom side (NBS). A higher protein carbonylation level and lower antioxidant defense system capability in the skin of frogs living in conditions of the cyanobacterial bloom were observed. Inhibition of glutathione-dependent machinery was the major mechanism responsible for the induction of cyanobacterial bloom-mediated oxidative stress in frog skin. On the other hand, the detected higher ability of muscle to overcome bloom prooxidant toxicity was linked to a higher efficiency of the biotransformation system through glutathione-S-transferase activity and/or was the consequence of indirect exposure of the tissue to the bloom. Our results have also revealed that the cyanobacterial bloom conditions induced the cholinergic neurotransmitter system in both tissues. This study provides a better understanding of the ecotoxicological impact of the MC producing cyanobacterial bloom on frogs in situ. However, further investigations of the complex mechanism involved in cyanobacterial bloom toxicity in real environmental conditions are required., 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 © 2019 Elsevier B.V. All rights reserved.)

The dietary effects of a native medicinal plant from Iran, common mallow (Malvae sylvestris), was evaluated on growth performance, innate immune parameters, mucosal immune parameters, and resistance of rainbow trout (Oncorhynchus mykiss) against Yersinia ruckeri. Therefore, 360 fish (initial weight 10.42 ± 0.09 g) were randomly distributed into 12 fiberglass tanks. Experimental diets supplemented with 0 (as control- C), 1% (M1), 3% (M2) and 5% (M3) levels of M. sylvestris flowers extract were fed to the fish based on 3% of body weight for 8 weeks. At the terminal sampling, growth performance, liver and digestive enzymes activities, blood and mucosal immune responses were determined. Results showed that M2 and M3 had greater final weight, weight gain, SGR, survival rate and lower FCR; higher levels of total protein, albumin, globulin, and lower cortisol levels in comparison to control; 5% extract also lowered cholesterol and glucose levels as well as Lactate Dehydrogenase (LDH) activity. We reported higher values of hematocrit, hemoglobin, Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV), White Blood Cell (WBC), Red Blood Cell (RBC) and lymphocytes for treated groups. Innate immune responses (Alternative complement activity (ACH50) in M2 and M3 group, total Immunoglobulin (Ig) and lysozyme in M3), mucosal immune parameters (ACH50, total Ig for M2 and M3 group and lysozyme in all treated groups) were enhanced. Activities of digestive enzymes (protease in all treated groups, amylase for 3 and 5%, while lipase only for 5%) and lower activity of liver ALT enzyme in individuals treated with highest dose was observed. Overall results indicated that the extract can positively affect growth performance and immune responses of rainbow trout., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

During life, anuran individuals undergo drastic changes in the course of transition from aquatic to terrestrial habitat, when they are faced with metabolically demanding processes (growth, responses to developmental pressures), which result in increased production of reactive oxygen species (ROS), signaling molecules involved in development that can induce oxidative damage and stress. This situation can be further complicated by environmental influences. The aim of this study was to investigate oxidative stress parameters in naturally developing Pelophylax esculentus complex frogs during four developmental periods: premetamorphosis, prometamorphosis, metamorphic climax and juvenile stage, in order to examine changes in the response of the antioxidative system (AOS) and oxidative damage during the transition from aquatic to terrestrial life. Results show that ontogenetic shifts in anurans are accompanied by different levels of damage and AOS responses, which vary from the increased first-line enzymatic activities during the early period of development (premetamorphosis), through increased changes in the non-enzymatic complement during the metamorphic climax, to changes in both the enzymatic and non-enzymatic components observed in juvenile individuals. Premetamorphic individuals and individuals in metamorphosis displayed higher levels of lipid peroxidation, indicating that direct exposure to the environment for the first time and the modulation of organs are the most susceptible stages for oxidative damage. On the other hand, lower oxidative damage in juveniles points to the ability of their AOS to efficiently respond to challenges of the terrestrial environment. This study highlights the importance of ROS and the AOS of anurans in response to different developmental and/or environmental pressures that individuals face., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Oxidative stress has most recently been suggested as one of the possible mechanisms responsible for reduced fitness of hybrids. To explore possible oxidative cost of hybridization, we examined anti-oxidant defence system parameters (superoxide dismutase, catalase, glutathione peroxidase, glutathione S -transferase, glutathione reductase, glutathione, SH groups), their interconnectedness (index of integration) and levels of oxidative damage [concentrations of lipid peroxides, TBARS (thiobarbituric acid reactive substances)] in laboratory-reared newt species, Triturus macedonicus and T. ivanbureschi , and their hybrid. Our results showed that parental species differed in anti-oxidant defence system parameters, but not in the levels of integration of the whole system and oxidative damage. Individuals of T. ivanbureschi had higher activities of superoxide dismutase, glutathione S -transferase and concentrations of glutathione. Hybrid individuals of crested newts displayed higher levels of the anti-oxidant defence system (higher superoxide dismutase, catalase, glutathione peroxidase activities and concentrations of SH groups), and a lower overall correlation of anti-oxidant system (lower index of integration) in comparison with both parental species, suggesting that they may possess a less efficient anti-oxidant defence system and a higher investment in maintaining oxidative balance. The higher investment in the anti-oxidant system could divert limited resources away from other functions and affect further hybrid fitness. The presented findings contribute to a better understanding of the anti-oxidant defence system of crested newts and their interspecies differences, and support the hypothesis that oxidative stress is one of the costs of interspecific hybridization., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

During their complex life cycle, anurans are faced with various physiological and biochemical demands that can result in increased free radical production. The antioxidative system (AOS), assumes a central role in protection from oxidative stress, and increased knowledge of its response would allow us to identify and quantify underlying costs of free radical production. In this study, we compared the patterns and levels of integration of the AOS during two life stages of Bufotes viridis toads from natural populations: young, fully developed, reproductively inactive subadults and reproductively active adults. All examined parameters (SOD, CAT, GSH-Px, GR, GST, GSH and SH groups) were measured in the liver, skin and muscle. The obtained results showed that: (1) subadult individuals possessed a more integrated AOS (a higher index of integration and number of significant correlations), based mostly on the coordinated action of SOD-CAT; (2) adult individuals used the GSH-Px/GSH system; (3) all examined tissues exhibited similar patterns of variation of AOS parameters; (4) significant correlations between the AOS and biometric parameters. Our results indicate that adult and subadult toads differed in the way they processed free radicals, implying that adults may be more susceptible to oxidative damage. The knowledge from this study will contribute to a better understanding of the AOS and how its activity is modulated in natural populations of anurans throughout their life., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Pesticide-induced oxidative stress, as one of mechanism of toxicity, has been a focus of toxicological research. However, there is a lack of data for certain pesticides-oxidative stress effects especially on terrestrial amphibians. This study evaluates the prooxidative effects of orally administered insecticide deltamethrin (DM) in some tissues of the terrestrial toad Bufotes viridis. Toads were randomly divided and assigned to a control group and a test group that was orally exposed to the pesticide (5 mg/kg of body weight/daily) for 21 days. Animals were euthanized from each group on days 7, 14, and 21, and the liver, leg muscle, ventral skin, and gastrointestinal tissue (GIT) were dissected and used for analysis. From battery of investigated antioxidant components, superoxide dismutase (SOD) was the most differentiate parameter in all examined tissues. For the period of prolonged exposure to pesticide, antioxidative strategy of Bufotes viridis was based on SOD utilization in attempt to maintain the oxidative disbalance at acceptable level. The integrated biomarker response (IBR) as the measure of the overall biochemical response to DM exposure revealed that the group exposed for 21 days had the highest response. Our work has offered valuable data ensuring evidence that toads exposed to deltamethrin developed adaptive reactions that were tissue-specific and that DM-generated systemic toxicity was time-dependent. The present work showed that oxidative stress has significant role in pesticide-induced toxicity and contributes to better understanding of ecotoxicological risk in the terrestrial amphibians exposed to DM.

In spring, frogs from temperate regions are faced with arousal-induced oxidative stress and exposure to various xenobiotics from the environment. The question is whether pollutants can significantly modify the antioxidative defense system (AOS) response of hibernators during recovery from hibernation. If this assumption is true, we would then expect different patterns of seasonal variations in the AOS between individuals exposed to different levels of pollution. To examine this assumption, we determined the relationship between seasonal variations of accumulated metals and AOS parameters in the skin and muscle of two frog species from the Pelophylax esculentus complex (P. ridibundus and P. esculentus) inhabiting two localities (the Danube-Tisza-Danube canal and the Ponjavica River) with different levels of pollution during pre- and post-hibernation periods, respectively autumn and spring. Our results showed that even though there were differences in the concentrations of accumulated metals and AOS parameters between localities and species, the frogs displayed almost the same patterns of AOS variations during seasons, with a higher AOS response observed in spring. The parameters SH groups, GSH, GR and SOD had been contributed most rather than others. Our findings indicate that oxidative stress during the post-hibernation period was mainly caused by the organisms' recovery from hibernation, as the result of natural selection acting on the AOS, and that the accumulated metals did not significantly modify the AOS response. The present study provides new insight into the biological and physiological cellular responses of frogs to arousal stress., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pyrethroids are synthetic insecticides widely used in agriculture, public health, and veterinary medicine. Deltamethrin, a type II pyrethroid insecticide, has attracted particular attention because of its frequent use. The mechanisms of the toxicity of most pesticides (including pyrethroids) in nontarget organisms is linked to the production of free radicals, oxidative stress induction, increased lipid peroxidation, and disruption of the total antioxidant potential. The aim of the present study was to investigate the effects of acute toxicity of 3 different concentrations (8, 16, and 32 mg/kg body wt) of orally applied deltamethrin after 96 h of treatment. Some of the front-line oxidative stress parameters as well as cholinesterase (ChE) activity and cytochrome P4501A (CYP1A) expression in the liver, muscle, skin, and gastrointestinal tissue of the adult green toad Bufotes viridis were determined. Compared with the control group, the activity of catalase and glutathione reductase was increased in the liver and skin, while the concentration of sulfhydryl groups was reduced. In the liver and muscle, concentrations of thiobarbituric reactive substances were increased, as well as liver CYP1A expression. In the muscle and skin, glutathione-S-transferase activity was higher in treated toads. The oxidative stress parameters examined were affected by different deltamethrin concentrations. We conclude that the assessed parameters represent good biomarkers of pesticide-induced oxidative stress. Environ Toxicol Chem 2017;36:2814-2822. © 2017 SETAC., (© 2017 SETAC.)

Metals are involved in the formation of reactive oxygen species and can induce oxidative stress. The aim of this study was to assess the effects of several metals on oxidative stress in the skin and muscle of the Pelophylax esculentus "complex" frogs (parental species Pelophylax ridibundus, Pelophylax lessonae, and their hybrid Pelophylax esculentus) that inhabit the wetland Obedska Bara in Serbia, and the potential use of these species as bioindicator organisms in biomonitoring studies. The biomarkers of oxidative stress (SOD, CAT, GSH-Px, GR, GST activities and GSH, SH concentrations) and cholinesterase activity were investigated. The concentrations of nine metals (Fe, Cu, Zn, As, Cd, Cr, Hg, Ni, and Pb) were measured in the water and tissues. Correlations were established between metals and biomarkers in the tissues. The results of metal accumulation distinguished the skin of P. lessonae and muscle of P. ridibundus from other P. esculentus complex species. The oxidative stress biomarkers observed in P. ridibundus and P. esculentus had greater similarity than in P. lessonae. The P. lessonae displayed the highest number of correlations between biomarkers and metals. The results of tissue responses revealed that skin was more susceptible to metal-induced oxidative stress, with only exception of As. In the light of these findings, we can suggest the use of P. esculentus complex species as a biomonitoring species in studies of metal accumulation and metal-induced oxidative stress, but with special emphasis on P. lessonae.

Heavy metal pollution of the aquatic environment is of great concern worldwide. Heavy metals are capable of inducing oxidative stress by increasing the formation of reactive oxygen species (ROS), and directly affecting the antioxidant defense system (AOS) in living organisms. The frog Pelophylax kl. esculentus is a semiaquatic species with semipermeable skin and a complex lifecycle, and represents a potentially useful bioindicator organism. The aim of this study was to investigate the accumulation of several heavy metals (Cd, Co, Cr, Cu, Fe, Hg, Ni, Pb and Zn), and their effects on selected parameters of the AOS, including the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), phase II biotransformation enzyme glutathione-S-transferase (GST), the total glutathione (GSH) contents and sulfhydryl (SH) group concentrations, as well as cholinesterases (ChEs) activities in the liver, skin and muscle of P. kl. esculentus. Frog samples were collected at two sites (the Danube-Tisza-Danube canal (DTDC) and the river Ponjavica) in Serbia, which are characterized by different levels of metal pollution. Differences between the metal contents in different tissues showed that the skin of frogs from the DTDC accumulated statistically higher concentrations of Cd, Cu, Pb and Zn, while only the Fe concentration was lower. No significant differences between metal concentrations in muscle tissues of frogs from the DTDC and Ponjavica were observed. Examination of the parameters of the AOS revealed that frogs from the DTDC had higher concentrations of GSH in the liver and of SH groups in the skin and muscle, whereas the activities of the antioxidative enzymes SOD, GHS-Px and GR in the liver and of GR in the skin were lower than in frogs from the Ponjavica. The relationship between metal concentrations and AOS parameters showed the highest number of correlations with GSH, GR and CAT, and with Ni, Zn, Hg, Cr and Cd. Based on the results in this study, we concluded that increased concentrations of heavy metals in frog tissues can alter the AOS, which leads to higher concentrations of GSH and SH groups and lower activities of antioxidative enzymes. The response of the AOS to metal pollutants allowed us to make a distinction between different frog tissues, and to conclude that the liver and skin are more suitable for assessing metal-induced oxidative stress in frogs than muscle., (Copyright © 2016 Elsevier Inc. All rights reserved.)

To understand the effect of metals on the marsh frog Pelophylax ridibundus and the possible environment-induced changes in oxidative stress enzymes, we determined the concentrations of 18 metals: Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, Ga, Hg, In, Li, Mn, Ni, Pb, Sr, and Zn, in the tissues (liver, skin, and muscle) and water samples collected from different locations in Serbia. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione S-transferase (GST), acetylcholinesterase (AChE), and changes in concentrations of reduced glutathione (GSH) and sulfhydryl groups (SH) were analyzed in the tissues of the sampled frogs. The concentrations of Al, Cd, Co, Cr, Cu, Fe, Ga, Hg, and Ni were highest in the liver, whereas those of Ba, Ca, Li, Mn, Pb, Sr, and Zn were highest in the skin. Hg correlated positively with liver SOD (in frogs from Danube-Tisza-Danube Canal (DTD)), muscle CAT (DTD), and muscle GST Ponjavica River (PO); Pb demonstrated a strong positive correlation with liver GR in frogs from Mt. Fruška Gora (FG); Cd only exhibited a positive correlation with AChE in the skin of frogs from DTD. In the skin, Zn correlated positively with AChE (DTD), SH groups (PO), and CAT (FG), and negatively with CAT, GST, and SH in the liver of frogs from DTD. Examination of these oxidative stress biomarkers, together with analysis of metal accumulation in the liver and skin of marsh frogs, provides a powerful tool for the assessment of metal pollution.