The role of phenotypic plasticity and corticosterone in coping with pond drying conditions in yellow-bellied toad (Bombina variegata, Linnaeus 1758) tadpoles.

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.

Does the anesthetic tricaine methanesulfonate (MS-222) distort oxidative status parameters in tadpoles?

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.

Changes of oxidative status in yellowfin seabream larvae (Acanthopagrus latus) during development.

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.

Effects of cadmium chloride and biofertilizer (Bacilar) on biochemical parameters of freshwater fish, Alburnus mossulensis.

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 (CdCl2) 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.

Higher temperature induces oxidative stress in hybrids but not in parental species: A case study of crested newts.

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.

What coloration brings: Implications of background adaptation to oxidative stress in anurans.

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.

Carry-Over Effects of Desiccation Stress on the Oxidative Status of Fasting Anuran Juveniles.

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.

Oxidative Stress Parameters in Goitrogen-Exposed Crested Newt Larvae (Triturus spp.): Arrested Metamorphosis.

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.

Impact of desiccation pre-exposure on deltamethrin-induced oxidative stress in Bombina variegata juveniles.

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.

Effects of Desiccation on Metamorphic Climax in Bombina variegata: Changes in Levels and Patterns of Oxidative Stress Parameters.

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.

Studying microplastics: Lessons from evaluated literature on animal model organisms and experimental approaches.

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.

Patterns of correlations and locomotor specialization in anuran limbs: association with phylogeny and ecology.

As anuran saltatory locomotion has specific functional requirements achieved through certain intra- and inter-limb proportions, we analyzed pattern and degree of morphological integration in limbs of ten anuran species to reveal the relationship of shared developmental programs of serially homologous structures and locomotor specialization. Our main objectives were (1) to examine if morphological and functional differences in forelimb and hindlimb were associated with reduced covariation between limbs, (2) and to reveal patterns of correlation between species and the roles played by evolutionary history (phylogeny) and ecology (lifestyle and habitat use). Species with different locomotor behaviours (walking, jumping, hopping, running, climbing, swimming and burrowing) were used. Partial correlations showed that species shared similar patterns of functionally based morphological integration, with increased correlations in elements within limbs and reduced correlations between limbs. This was mainly based on strong correlations between proximal elements, humerus-radioulna and femur-tibiofibula. To test the influence of phylogenetic relationships and ecological demands we used different matrices (correlation similarity matrix, ecological similarity matrix, matrices of phylogenetic distance and morphological distance). The changes in correlation patterns are shown to be dissociated from phylogeny. On the other hand, they are to some extent shaped by habitat use and locomotion, as the species with similar locomotor behaviour also tend to have stronger similarity in integration patterns. The results from this study provide insight into the processes underlying the evolutionary change of anuran limbs, highlighting function as the main factor that shaped morphological integration of the examined species.

Hepatic oxidative stress and neurotoxicity in Pelophylax kl. esculentus frogs: Influence of long-term exposure to a cyanobacterial bloom.

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 Ludas (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.

The effect of short-term fasting on the oxidative status of larvae of crested newt species and their hybrids.

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 H2O2 scavenging parameters (catalase, glutathione peroxidase, and total glutathione), which together with the elevated activity of superoxide dismutase suggested increased H2O2 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.

The Effect of Shelter on Oxidative Stress and Aggressive Behavior in Crested Newt Larvae (Triturus spp.).

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.

Biochemical parameters in skin and muscle of Pelophylax kl. esculentus frogs: Influence of a cyanobacterial bloom in situ.

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 Ludas (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.

Oxidative stress in Pelophylax esculentus complex frogs in the wild during transition from aquatic to terrestrial life.

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.

Comparative assessment of the antioxidative defense system in subadult and adult anurans: A lesson from the Bufotes viridis toad.

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.

Oxidative cost of interspecific hybridization: a case study of two Triturus species and their hybrids.

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.

Morphometric ratio analyses: Locomotor mode in anurans.

Current studies of anuran morphology use sacral vertebrae, hindlimbs as main predictor for locomotor modes, while forelimbs and their segments were not analysed in this context. Due the fact that the complex interplay of numerous morphological traits determines the locomotion ability of anurans, we used ratios for both fore- and hindlimbs to obtain useful information of the structural and functional properties of locomotor system. We examined patterns of variation in total length of fore-, and hindlimbs as well as sacral vertebrae width and diapophyseal expansion in order to position two locomotor modes (jumpers and walkers) in a morphological shape space. Further, we investigated patterns of variability in limbs proximal, medial and distal elements to highlight differences in intra- and inter-limb relations in two locomotor modes. Our results showed that the ratios between total hindlimb length and sacral width/diapophyseal expansion, as well as ratio between humerus and tibiofibula relate the most to locomotor modes. This implies that functional specialization in different locomotor modes is attained through modifications of ratios, not only within hindlimbs, but also through modifications of the whole integrated system, which consists of forelimb, hindlimb, and girdle elements.