Climate change stands as a principal concern for conservationists, posing a significant threat to biodiversity. Rising temperatures are reshaping environmental conditions and imposing novel selection pressures on organisms (Crozier, Hutchings, 2014). Consequently, understanding the capacity of species and populations to respond and adapt to climate change, particularly warming, is more pressing than ever. This understanding is crucial for predicting the consequences of climate change and enhancing our management and conservation efforts (Donelson et al., 2011; Sinclair et al., 2016; Campbell et al., 2017). Ectothermic animals are especially vulnerable to ambient temperature variations due to their reliance on external temperatures to regulate body temperature and metabolism (Schulte, 2015).

Rising temperatures exert a significant influence on ectothermic metabolism, with the ability to survive in warm environments being closely linked to an individual’s capacity to meet its energetic requirements (Pörtner, Gutt, 2011; Pörtner et al., 2014). Lactate dehydrogenase (LDH) is a key metabolic enzyme involved in the final stages of glycolytic metabolism. It facilitates the interconversion of pyruvate and lactate while concurrently interconverting NADH and NAD+, thus playing a crucial role in energy metabolism (Driedzic, Almeida-Val, 1996).

While the effects of temperature on the kinetics of LDH in the forward reaction (pyruvate reduction) have been extensively studied, relatively few studies have focused on the lactate oxidation reaction (Fields, Houseman, 2004; Fields et al., 2015). The utilization of lactate as an aerobic fuel has been associated with enhanced aerobic and swimming performance in fish (Omlin et al., 2014; Ferreira et al., 2018), suggesting its potential importance when fish are exposed to high temperatures. Therefore, a comprehensive understanding of LDH kinetics in both the forward and reverse reactions in fish acclimatized to different temperatures would provide valuable insights into the modulation of glycolytic metabolism to cope with thermal stress.

The ability of organisms to respond and survive increases in temperature hinges on their ability to supply energy and manage internal components and processes to maintain physiological homeostasis (Somero, 2010). As oxygen consumption rises to meet energy demands, there is a concomitant increase in the production of reactive oxygen species (ROS) due to warming. The excessive production of ROS can inflict severe damage to cells, leading to lipid peroxidation, protein carbonylation, and DNA damage – collectively known as oxidative stress.

Campos et al. (2019) reported a heightened vulnerability of the characid fish Hyphessobrycon melazonatus acclimated to a climate change scenario characterized by elevated temperatures (4°C) and atmospheric CO2 levels (900 ppm above current levels), with a notable link between increased mortality and oxidative damage. However, cells possess mechanisms to counteract the deleterious effects of ROS through the activation of ROS scavengers, including superoxide dismutase, catalase, glutathione-S-transferase, and others. This activation serves to reduce the flux of ROS generated during oxidative metabolism, thereby mitigating oxidative stress (Madeira et al., 2016a). Lopes et al. (2018) demonstrated that the activation of antioxidant enzymes provides an effective defense mechanism to cope with ROS formation in Chiloscyllium plagiosum exposed to a climate change scenario.

The vast majority of studies examining the effects of temperature on physiological and biochemical plasticity have focused on acclimating individuals to different temperature regimes (Campos et al., 2017, 2019). However, only a few studies have investigated the effects of natural populations inhabiting distinct thermal habitats. Phenotypic variation can arise from both environmental and genetic influences, underscoring the importance of understanding the relative contributions of local adaptation and phenotypic plasticity. This understanding is crucial for elucidating the impact of environmental variation on populations.

In this study, we investigate the physiological and biochemical adaptation of Bryconops giacopinii (Fernández-Yépez, 1950), a rheophilic species, residing in different thermal habitats. While B. giacopinii typically inhabits natural forested streams in the Amazon, the construction of roads through the Amazon Forest has altered its habitat. Deforestation of Amazonian streams has resulted in temperature increases of up to 6°C (Ilha et al., 2018) a rise warmer than projections for the year 2100 due to climate change. Consequently, our aim is to explore the physiology and biochemistry of B. giacopinii populations living in diverse thermal habitats. We hypothesize that: i) individuals from roadside streams will exhibit different LDH kinetics parameters compared to those from forested streams, and ii) individuals from roadside streams will display higher levels of antioxidants and oxidative stress. Therefore, we expect changes in LDH kinetics and redox balance related to the increase in energy demand to live in warm waters. Changes in glycolytic and redox balance will inform us about the physiological adjustment of this species to survive in a climate change world.

Study area and fish collection. The individuals’ collection was performed on February 2020 (Amazon wet season) in two road-side streams (BR#1 - 02°34’45”S 60°01’44”W; BR#2 - 02°44’51”S 60°01’35”W) and one forested stream (Ducke - 02°58’07”S 60°00’20”W). The two roadside streams are located along the Manaus-Boa Vista road, the BR-174 (Fig. 1). The highway BR-174 construction beginning in the 1970’s years and its paving only finished in 1997. The construction and paving of the highways represent the main vectors of deforestation in the Amazon, and according to Rodrigues, Pinheiro (2012), there was a total of 2618, 58 km2 of deforested areas near the BR-174. Deforestation has impacted the physical structure of the small aquatic ecosystems, what once was a forested lotic system, turned into a semi-lentic system without riparian forest, leading to the increase in the mean and fluctuation of the temperature. The forested stream is located inside the Reserva Adolpho Ducke (02°53’S 59°58’W), a protected area located in the central Brazilian Amazon near the confluence of the Negro and Solimões rivers and bordering Manaus. The Ducke Reserve encompasses an area of 100 km2 and comprises two distinct watersheds: Puraquequara basin in the Tarumã basin. The animals were collected specifically from the Tarumã Basin (Acará streams). The distance between Ducke and the roadside streams was approximately 40 km in a straight line, with a distance of around 10 km between the roadside streams. Importantly, there is no connection between these streams.

In both sites, ten individuals were collected using a seine net. Captured fish were maintained in a container with stream water and aeration. The animals were collected over two consecutive days. On the first day, between 9 and 11 am, we gathered populations from the roadside. The following day, at the same time frame of 9 to 10 am, we collected animals from the Ducke reserve. After collection, fish were anesthetized and dissected, the muscle and gills were removed and immediately frozen in liquid N2. The samples were stored in the ultra-freezer at -80°C until the enzymatic analyses were performed. Besides, temperature and oxygen were measured by YSI55 probe, and 5 mL of water was collected to measure pH in the laboratory with Ohaus ST2100-F Starter 2100 pHmeter. The species are included in the ichthyological collection of the Instituto Nacional de Pesquisas da Amazônia (INPA) under voucher number INPA-ICT 044364.

LDH Kinetics. Enzyme assays were realized according to protocols described in Driedzic, Almeida-Val (1996). The absolute activity of LDH was measured in the white muscle of fish that was homogenized according to Driedzic and Almeida-Val’s method. Briefly, the portions were homogenized manually with a conic pistil and microtubes in a 1:10 (w:v) ice-cold buffer (150mM imidazole, 1mM EDTA, 1% triton X-100, pH 7.4). Homogenates were centrifuged during 15 min at 13.000g at 4oC to separate particulate material. Assays were performed in 300 μL microplate in 288 μL reaction buffer (NADH 0.15mM for pyruvate reductase or NAD+ 0.15mM for lactate oxidase, KCN 1mM, and imidazole 50mM), pH 7.4 at 25oC, 2μL homogenate, and reaction was started adding 10μL pyruvate (0.0, 0.05, 0.10, 0.25, 0.50, 0.75, 1.00, 5.00 mM) or lactate (0.0, 0.5, 1.0, 2.0, 4.0, 8.0 and 16mM). In a spectrophotometer spectramax M5 plate reader the reaction was read at 340 nm. Km and Vmax for each substrate were calculated after assays and expressed as mM and μmol pyruvate/minute/g protein, respectively. Km and Vmax were calculated with a nonlinear least square regression fit to the one site saturation ligand biding equation using SigmaPlot 3.0 (Systat Software Inc).

Antioxidant and reactive oxygen species analyses. Muscle and Gills were homogenized in buffer (pH 7.6) containing (in mM): Tris base 20, EDTA 1.0, dithiothreitol 1.0, sucrose 50, KCl 150. Homogenates were centrifuged at 15,000 g for 20 min at 4°C and used to determine GST, SOD and CAT activities, and LPO levels (1:10 w/v for GST and SOD and 1:4 w/v for CAT and LPO). The glutathione-S-transferase (GST) activity was determined using 1-chloro 2,4-dinitrobenzene (CDNB) as substrate, according to Keen et al. (1976). Changes in absorbance were recorded at 340 nm. The enzyme activity was calculated as nmol CDNB conjugate formed per min per mg protein. To determine the catalase (CAT) activity, the inhibition rate of H2O2 decomposition was monitored at 240 nm (Beutler, 1975), and expressed as μM H2O2 min−1 mg protein− 1. Superoxide dismutase (SOD) activity was quantified based on the inhibition of cytochrome c reduction rate by the superoxide radical at 550 nm and 25°C (Turrens, 1997). Enzyme activity is expressed as U SOD mg protein−1, where 1 U of SOD corresponds to the quantity of enzyme that promoted the inhibition of 50% of cytochrome c. The lipid peroxidation (LPO) levels were quantified based on the oxidation of the Fe+2 to Fe+3 by hydroperoxides in the acid medium in the presence of ferrous oxidation-xylenol orange, at 560 nm, according to the method described by Jiang et al. (1991).

Reactive oxygen species (ROS) were measured according to the method of Amado et al. (2009). In the homogenates, as described above, were added the reaction buffer (30 mM HEPES, 200 mM KCl, and 1 mM MgCl2, pH 7.2) and the fluorescent dye 2′,7′ dichlorofluorescein diacetate (H2DCF-DA). The acetate groups of H2DCF-DA are cleaved by intracellular esterase present in sample supernatants. After that, the non-fluorescent compound H2DCF was oxidized by ROS to the fluorescent compound, DCF, which was detected in the wavelengths of 488 (excitation) and 525 (emission) nm. Total fluorescence production was calculated by integrating the fluorescence units (FU) over the measurement time and expressed as the area of fluorescence. The results were expressed as area difference of FU × min in the sample.

Statistics. The data are presented as mean ± SEM (n = 10). All statistical analyses were carried out in SigmaStat 3.1 using a significance level of 5%. Parametric Anova or ANOVA on ranks was performed to detect significant differences in the biomarker’s response among different populations followed by the Tukey post hoc analysis.

The temperature and pH were higher, oxygen was lower in both roadside streams compared to the forested stream (see Tab. 1). The maximum activity (Vmax) of LDH for lactate formation surpasses that for pyruvate formation, indicating that this enzyme is more effective in catalyzing lactate formation. Significant differences were observed among collection sites for LDH kinetic parameters. Specifically, the maximum activity (Vmax) of LDH for lactate formation is higher in fish from the Ducke compared to those from BR#1. Conversely, the maximum activity (Vmax) of LDH for pyruvate formation is higher in fish from the BR#1 population than in those from the Ducke population (Fig. 2). This suggests that higher temperatures enhance the ability for pyruvate formation, which can be used to enter in citric acid cycle and improve oxidative phosphorilation. Furthermore, LDH Kmpyr does not exhibit significant differences among populations. However, LDH Kmlac is lower in the Ducke population compared to BR#1, indicating a higher affinity for lactate in the Ducke population (Fig. 3). Taken together, the kinetics of LDH suggest that populations inhabiting higher temperatures have adapted to increase lactate oxidation.

Regarding the antioxidant enzymes, there were significant differences among the collection sites in gills (Tab. 2). The SOD activity was lower in BR#1 compare to BR#2 and Ducke. The CAT was lower in BR#1 compare to BR#2, the GSH was higher in BR#1 and BR#2 compare to Ducke, and the GST activity was lower in BR#1 compare to Ducke. There were no differences in LPO and ROS among treatments, although BR#1 and BR#2 showed higher ROS mean values.

Also, there were significant differences in the muscle among the collection sites (Tab. 3). SOD activity was lower in BR#1 and BR#2 compare to Ducke, CAT activity was higher in BR#1 compare to Ducke, the GST activity was lower in BR#1 compare to Ducke and ROS was higher in BR#1 and BR#2 compare to Ducke. There were no differences in the GSH activity and LPO.

In our study, we observed distinct environmental conditions among collection sites, particularly with roadside streams exhibiting higher temperatures and pH levels, along with lower oxygen concentrations. These changes are closely associated with the deforestation that has occurred along the BR-174 highway. While our data represents a single measurement and may not capture the entire thermal dynamics of the environments, other studies conducted in Amazonian streams have also documented warming trends linked to deforestation (see Ilha et al., 2018). Although data specific to the Tarumã-Açu basin are limited, a recent report indicated that temperatures in roadside streams ranged from 29.31°C to 36.07°C between September 2019 and July 2020 (Costa, 2020). Interestingly, this corresponds to the timeframe of our study, suggesting consistency with our observations and highlighting the impact of environmental changes on stream temperatures.

Our study revealed that Bryconops giacopinii inhabiting road-side streams exhibit alterations in LDH kinetics and antioxidant response systems. Through cellular analyses, we were able to identify physiological mechanisms in response to increased temperatures. Overall, individuals from roadside habitats, which experience higher temperatures, displayed elevated LDH activity for pyruvate formation in muscle, increased levels of reactive oxygen species (ROS) production, and heightened glutathione (GSH) activity. Conversely, these populations exhibited decreased LDH activity for lactate formation in muscle, as well as reduced superoxide dismutase (SOD) and glutathione-S-transferase (GST) activity in both gills and muscle tissues.

The absence of oxidative stress, indicated by the lack of changes in lipid peroxidation (LPO), suggests that the antioxidant response exhibited by Bryconops giacopinii effectively detoxifies reactive oxygen species. These findings suggest that the species possesses physiological adaptations enabling it to thrive in warmer environments.

It has been established that the kinetic parameters of fish LDH enzymes are influenced by their environment (Almeida-Val et al., 2006). However, there is limited evidence regarding alterations in the kinetic properties of glycolytic enzymes in Amazonian natural populations inhabiting different temperature regimes. LDH, being a pivotal enzyme in glycolysis, has garnered attention for its kinetic parameters in the context of thermal adaptations (Fields et al., 2015).

Our current study revealed that LDH in the forward reaction from Ducke exhibits higher Vmax compared to BR#1, while the Kmpyr of LDH among sites collection did not differ significantly. The elevation of LDH Vmax in Ducke, despite a constant Km, could be attributed to increased anaerobic activity in populations inhabiting colder environments. Consequently, LDH activity for pyruvate formation is lower in Ducke compared to BR#1. These findings suggest that populations residing in high temperatures (BR#1; 29.3°C) enhance lactate oxidation, potentially reflecting their utilization of lactate as an aerobic fuel.

Indeed, during periods of high aerobic demand, such as elevated temperatures or exercise, fish tend to increase muscle lactate oxidation to sustain aerobic metabolism. Ferreira et al. (2018) demonstrated that the Amazon characid Brycon amazonicus, known for its high aerobic and swimming capabilities, enhances muscle lactate oxidation to meet the demands of exercise. Therefore, heightened metabolic demand may enable fishes to utilize lactate as an aerobic substrate, consequently improving swimming performance.

Consistent with this notion, the higher Kmlac (indicative of lower affinity) observed in the BR#1 population suggests that under high temperature conditions, this species may accumulate lactate in muscle to be utilized when needed. Bryconops giacopinii, being a rheophilic species inhabiting Amazonian streams, consistently swims against the flow, necessitating significant muscle work to sustain its lifestyle.

In vertebrates, the LDH reaction is widely recognized to be in equilibrium (Spriet et al., 2000; Quistorff, Grunnet, 2011), indicating that the enzyme’s activity is regulated by concentrations of substrates and products. Consequently, alterations in equilibrium may be associated with changes in one of these compartments. Our finding shows that when there are changes that reduce the affinity for lactate, it can result in more lactate accumulating in tissues. As a result, there is an increase in LDH Vmax to speed up the rate at which lactate is utilized, especially in individuals living in high-temperature environments. This suggests that lactate plays a vital role as a metabolic fuel in such conditions. Indeed, a preference for lactate oxidation has been observed in skeletal muscle (Donovan, Pagliassotti, 2000) and cardiac muscle (Chatham et al., 2001) during exercised mammals.

Warming conditions elevate oxygen consumption in ectotherms to cope with heightened physiological demands. However, as a by-product of respiration, increased oxygen consumption is also associated with the generation of potentially harmful reactive oxygen species (ROS) (Madeira et al., 2018). The production of ROS in mitochondria is directly linked to oxygen availability (Pelster et al., 2020). Therefore, the elevation of oxygen consumption to meet energy demands under warming conditions may incur cellular costs (Madeira et al., 2016a,b). Our findings revealed higher ROS production in the BR#1 population, which inhabits warmer streams. This suggests an upsurge in cellular respiration, supporting the hypothesis that these individuals enhance lactate oxidation to improve aerobic metabolism.

Warming-related oxidative stress occurs from the inability of the antioxidant defense system to avoid damage caused by ROS (Kültz, 2020; Somero, 2020). Herein, we showed that the population living in a high-temperature environment presents different levels of antioxidant enzymes to detoxify ROS production. In general, road-side populations showed lower levels of SOD and GST, while increased CAT and GSH in both muscle and gills. Therefore, we showed that to detoxify the increased levels of ROS at warming they increased the activity of specific enzymes, while decreased others. Theses physiological mechanism seems efficient to deal with oxidative stress since no difference was observed increase in LPO levels in both tissues.

The effects of warming on antioxidant and oxidative stress in Amazon species have attracted attention in recent years, and the physiological mechanism to detoxify ROS production seems specie, temperature, and time-specific (Campos et al., 2019; Baldissera et al., 2020). As we observed in the present work the decrease in some enzyme activity in fish exposed to warming has been reported by many authors. For instance, SOD activity and GPx activity were inhibited in Brycon amazonicus exposed to 34°C, compared to 28°C (Baldissera et al., 2020). Souza et al. (2018) reported significant decreases in muscle SOD activity of Notothenia rossii and N. coriiceps exposed 14 days to 8°C compared to 0°C, and Madeira et al. (2016b) showed a decrease in muscle SOD activity of Amphiprion ocellaris acclimated 28 days to 30°C compare to 26°C. In contrast, others antioxidant enzymes increased when fish are exposed to heat, especially CAT. Exposure to high temperatures can increase the formation of H2O2 as showed by Iftikar et al. (2014). CAT plays the primary role in the elimination of H2O2 (López-Cruz et al., 2010) converting it back to H2O and O2, decreasing the flow of ROS and preventing oxidative stress. Indeed, Campos et al., 2019 showed that acclimation for 30 days to the projected climate future (4°C and 900 ppm above current levels) increased CAT activity in the muscle of Pyrrhulina brevis and Hyphessobrycon melazonatus, while Apistogramma agassizii increased GST. In accordance, we found an increase in the CAT activity and GSH levels in road-side individuals, suggesting an activation of this antioxidant system to deal with the increase of H2O2 production in warm living individuals.

The ability to regulate ROS levels and prevent oxidative damage is a crucial mechanism for maintaining cellular homeostasis (Madeira et al., 2016a,b; Rosa et al., 2016). Lipids play a vital role in preserving the integrity of cellular membranes, regulating ion gradients, membrane fluidity, and permeability (Gaschler, Stockwell, 2017). The absence of elevated levels of lipid peroxidation (LPO) in populations inhabiting warm environments suggests that the antioxidant systems of this species effectively counteract ROS production, thereby preserving cellular homeostasis. This indicates that the species is capable of physiological adaptation to warming conditions.

The disparities observed between the roadside streams (BR#1 and BR#2) may stem from differences in intrapopulational responses. Factors such as the duration of exposure to high temperatures and the duration of species separation could contribute to these differences. Additionally, the degree of exposure to temperature variation can impact antioxidant enzyme levels. For instance, research by Madeira et al. (2016b) demonstrated that mild temperature exposure can enhance antioxidant enzyme activity, whereas extreme temperatures lead to a decrease in enzyme activities. The decline in SOD and CAT activity observed at BR#1 compared to BR#2 could be linked to enzyme denaturation or impaired synthesis within the cells. Enzymes are proteins held together by hydrogen bonds, which can weaken when temperatures exceed a certain threshold, resulting in denaturation and reduced catalytic activity (Rosa et al., 2016). Tropical species are particularly susceptible to temperature changes as they often live near their thermal limits. Therefore, even slight increases in temperature can significantly impact animal physiology (Campos et al., 2021).

Although our work presents important findings, it is not possible to identify if these responses are physiological plasticity or result of local adaptation among populations, since differences in phenotype are both genetic and environmental induced (Allendorf, Luikart, 2007). Phenotypic divergence can be caused by genetic differences or phenotypic plasticity (Via, Lande, 1985). Therefore, the differentiation between roadside and Ducke individuals does not necessarily indicate local adaptation (Conover, Schultz, 1995). Typically, the causes of phenotypic variation are assessed by removing the effect of environment via common garden and/or reciprocal transplant experiments (Hansen et al., 2012). To understand this evolutionary question, the next step is to acclimate these populations to different temperatures to identify possible mechanisms of local adaptation or physiological plasticity, which will be important to understand in light of climate change.

In summary, our findings indicate that roadside populations of Bryconops giacopinii inhabiting high-temperature environments exhibit modifications in muscle LDH kinetics parameters, enhancing lactate oxidation. This adaptation likely corresponds to increased energetic demands. Furthermore, these populations demonstrate elevated levels of CAT and GSH in both gills and muscle, aiding in the management of heightened ROS production. The robust antioxidant system effectively mitigates oxidative stress, suggesting that this population has adapted to survive in warmer environments. Therefore, our study reveals that B. giacopinii living in a warming world adjust their glycolytic metabolism and bolster antioxidant enzymes to meet energy demands without experiencing heightened oxidative stress.