Species restricted to living in flooded caves (stygofauna) possess adaptations, known as troglomorphisms, that distinguish them from aquatic species living in surface water bodies (Christiansen, 1962; Pipan and Culver, 2012). Morphological differences, such as the reduction or loss of eyes and pigments, and extension of appendages are some of the most obvious. However, physiological traits of stygobionts in response to the distinct conditions of anchialine caves are determinant for the persistence of their populations. In the adaptation process, individuals in a population respond with a set of behavioral, physiological, biochemical, and metabolic mechanisms to environmental change. This responsiveness to different stimuli can be considered phenotypic plasticity, which can be fixed in a population through natural selection over evolutionary time scales, ultimately resulting in a population with greater fitness to the novel environment (Pigliucci et al., 2006; Norin and Metcalfe, 2019). The study of physiological plasticity and its relation to the adaptive capacity of species to their environment has allowed to establish tolerance limits to different environmental factors, such as temperature, oxygen or salinity (Guderley and Pörtner, 2010; Pörtner, 2010; Sokolova et al., 2012).

The response to environmental change, whether of biotic or abiotic origin, necessarily involves changes in energy production and allocation (Sokolova et al., 2012). Metabolism is the process that produces energy (ATP) at the cellular level, and the metabolic rate (the speed at which it is produced) measures the amount of energy required (and supplied) by the organism. The energy required for the maintenance of essential functions, such as respiration, blood circulation, and excretion, is known as the basal metabolic rate (BMR). Due to methodological difficulties in measuring BMR, the standard metabolic rate (SMR) has been used as the practical measure of BMR (Chabot et al., 2016a).

Once the energy requirements for the maintenance of homeostasis have been met, organisms can generate a surplus of energy for growth, reproduction, food digestion, physical activity, and response to environmental variations (Pörtner and Farrell, 2008; Sokolova et al., 2012). One way to assess that capacity is by measuring the maximum metabolic rate (MMR), which has been recognized as the physiological limit at which an individual can produce ATP via the aerobic pathway. The difference between the MMR and SMR was first defined as the aerobic scope of metabolic activity (Fry, 1947) and is now called the aerobic scope (AS). The AS is the energy that an individual can produce beyond the metabolic cost of basal processes and is intended to perform ecological functions (Sokolova et al., 2012; Norin and Metcalfe, 2019). Since basal maintenance always prioritizes energy resource allocation over any other process, when energy production is limited or the cost of basal maintenance increases, AS is reduced (Sokolova et al., 2012). Therefore, higher AS has been linked to greater fitness, both in long-term processes, such as adaptive potential (Farrell et al., 2008; Schulte 2015; Norin and Metcalfe, 2019), growth and reproduction (Meza-Buendía et al., 2021), as well as in the short-term processes, such as predator evasion, foraging and response to immediate environmental disturbances (Pörtner and Knust, 2007; Pörtner and Farrell, 2008; Clark et al., 2013). Likewise, changes in AS may distinguish an optimal situation from one of moderate or even critical stress (Pörtner, 2002; Pörtner, 2010; Sokolova et al., 2012; Pörtner et al., 2017).

One way to quantify stress is by assessing the energetic-metabolic costs that environmental conditions produce in an organism (Pörtner, 2010; Lushchak, 2011; Sokolova et al., 2012; Paschke et al., 2018). It is possible to establish how environmental changes modulate the physiological mechanisms that enable the response of individuals to the environment (Sokolova et al., 2012). This is how the study of metabolism, and its response to environmental change allows the evaluation of the capacity to obtain energy under a variety of stress conditions (Sokolova et al., 2012; Paschke et al., 2018). Moreover, physiological response to environmental heterogeneity contributes to the definition of some dimensions of the fundamental ecological niche (Hutchinson, 1957; Elton and Charles, 2001; Soberón and Peterson, 2005; Ángeles-González et al., 2020; Ángeles-González et al., 2021).

In order to increase aerobic metabolism, an increase of oxygen influx into the mitochondria is needed. During this process, the monovalent reduction of oxygen generates a series of free radicals (reactive oxygen species, ROS), which can be harmful to organisms as they oxidize other molecules to reach ionic equilibrium (Martínez-Álvarez et al., 2005). Oxidative stress occurs when the production of ROS is greater than the capacity of the antioxidant system to eliminate them (Martínez-Álvarez et al., 2005; Díaz-Acosta and Membrillo-Hernández, 2006). Organisms rely on a series of oxidation-reduction reactions (the antioxidant system, AOS) that continuously maintain cells in a highly dynamic equilibrium to prevent cellular damage from ROS. Antioxidants yield electrons to ROS, transforming compounds into less reactive or detoxified molecules (Regoli et al., 2011; Gumulec et al., 2013). An increase in the concentration of antioxidants such as superoxide dismutase (SOD), Catalase (CAT), Glutathione (GSH) and Glutathione S-transferase (GST) indicate an increase in ROS production, whereas lipid peroxidation (LPO) and protein carbonylation (PO) are biomarkers of oxidative stress showing cellular damage.

Typhlatya mitchelli, Typhlatya pearsei and Typhlatya dzilamensis occupy distinct habitats in the cenotes (local term for dolines/sinkholes) and caves of the Yucatan Peninsula (Chávez-Solís et al., 2020). These anchialine habitats are characterized by vertically stratified water bodies, where temperature exhibits slight variations (~1°C) across this gradient, but salinity may change abruptly with depth, frequently accompanied by changes in dissolved oxygen, oxidation-reduction (redox) potential and pH (Pohlman, 2011; Brankovits et al., 2017; Benítez et al., 2019). Such systems, therefore, present unique abiotic characteristics that generally keep fresh and marine groundwater species spatially separated. Here, T. mitchelli, and T. pearsei inhabit strictly freshwater environments, while T. dzilamensis inhabit environments with higher salinity levels (Espinasa et al., 2019; Chávez-Solís et al., 2020).

The thermal window, the metabolic capacities and the antioxidant mechanism have been used to understand how the environment can influence the adaptive capacity of aquatic organisms (Vinagre et al., 2016; Vinagre et al., 2021). These findings have been key to understanding species’ adaptation from an eco-physiological point of view (Niklitschek and Secor, 2009; Paschke et al., 2013; Sumaila et al., 2020; Eddy et al., 2021) and help to predict changes in future warming scenarios (Cheung et al., 2008; Cheung et al., 2010; Maharaj et al., 2018). If there is a strong association between the physiology of an organism and its habitat (Huey, 1991), it is reasonable to hypothesize that differences in the distribution of these Typhlatya species correspond to certain physiological traits, which were selected by the constraints of past environmental conditions and fixed through evolution. Thus, the comparison of metabolic, behavioral, and biochemical indicators among these closely related species presents a sublime opportunity to evaluate their physiological capacities and discuss them from an adaptive perspective.

This work aimed to characterize the physiology of three Typhlatya species through its thermal window, respiratory metabolism and biochemical indicators reflecting their aerobic capacity under the conditions of their natural habitats in the anchialine ecosystems of the Yucatan Peninsula. For this purpose, in situ experiments to measure thermal window and oxygen consumption rates under routine conditions and in response to swimming activity were carried out. In addition, samples were taken to examine the response of the antioxidant system (enzymes and metabolites indicators of oxidative damage) and anaerobic metabolism derived from the swimming activity at different times throughout the experiments.

The present study for the first time compares metabolic, thermal and biochemical indicators of three closely related species of Typhlatya, and shows striking differences related to the characteristics of the subterranean systems of the Yucatan Peninsula in which they occur. Results indicate that T. dzilamensis, the only species found in marine groundwater, has a higher AS and thermal tolerance than T. mitchelli and T. pearsei, which occur only in fresh groundwater habitats. This difference is noteworthy because all three species showed a similar RMR, indicating that the energetic cost of routine activities is independent of the environments they inhabit. It also shows that the difference in AS between species is mainly due to variations in the MMR attained after being induced to the same type, intensity, and duration of physical exercise. Considering the strong effect of environmental conditions on the metabolism of aquatic animals (Pörtner, 2002; Levin, 2003; Pörtner, 2010; Lushchak, 2012; Fang et al., 2019), results herein are discussed in the light of the physiological traits characterizing each species and how these relate to their distribution patterns in the anchialine systems of the Yucatan Peninsula.

According to several authors (Sokolova et al., 2012; Farrell, 2016; Pörtner et al., 2017; Rodríguez-Fuentes et al., 2017) the AS is an indicator of the metabolic energy available once the basal physiological requirements have been satisfied. Therefore, differences in the ability to adjust metabolism in the face of the same stressor – in this case induced physical activity – can be interpreted as differences in physiological plasticity amongst species. The greater AS as a result of MMR in T. dzilamensis suggests that the metabolic increase involves physical capacities to deliver oxygen to the cells, as well as the onset of biochemical mechanisms for energy production and pathways to control ROS, amongst others. Physiological plasticity has been linked to an increased resilience (Tepolt and Somero, 2014; Magozzi and Calosi, 2015; Seebacher et al., 2015; Vinagre et al., 2016; Norin and Metcalfe, 2019) and greater adaptive capacity (Gorban et al., 2016; Norin and Metcalfe, 2019). Furthermore, it has been proposed that a limited antioxidant capacity implies a lower acclimation potential, leading to reduced fitness and, ultimately, an inability to respond to environmental stress (Madeira et al., 2013; Pallarés et al., 2020). In our study, T. dzilamensis was the species with the greatest metabolic response to physical activity, coupled with a primed and responsive AOS and a greater CTmax. Whilst research directed to compare physiological tolerance to salinity is needed, it is possible that the greater physiological plasticity and resilience found in T. dzilamensis relate to its occurrence in saline underground environments beyond the limits of those where T. mitchelli and T. pearsei can endure.

Considering that osmoregulation in euryhaline crustaceans requires, among other things, an increase of metabolic rate (Ramaglia et al., 2018), it is likely that those individuals with a broad AS could meet the energy expenditure that a change in environmental salinity demands. T. dzilamensis is the only species recorded in both fresh and marine groundwater bodies of the Yucatan Peninsula (Benítez et al., 2019), and shrimp have been seen crossing the halocline in a time span of a few minutes (Chavez.Solís pers. obs.). Vertical migrations in search of food or escaping from predators could drive T. dzilamensis to an abrupt and extreme environmental change that requires an instantaneous increase in energy production. Whilst such an euryhaline nature still remains to be investigated, results herein suggest that an increased AS could be one of the key differences allowing this species to survive on either side of the halocline.

According to our results, such physiological adjustments are realistic and have been previously described in Halocaridina rubra, an Atyid from Hawaiian anchialine systems, where salinity fluctuates abruptly and frequently (~20‰ in 24-hour periods). This species presents structural modifications, an increase in mitochondria-rich cells (involved in osmoregulation) in the gills (Havird et al., 2014a), and maintains active the mechanisms and expression of genes involved in osmoregulation (Henry, 2001; Jayasundara et al., 2007; Rahi et al., 2018; Havird et al., 2019). Although the presence of these adaptations has not been studied in any Typhlatya species, results herein may help to explain the presence of T. dzilamensis on either side of the halocline.

Food availability and primary production in anchialine systems are heterogeneous. Cenotes receive a greater input of allochthonous organic matter, and in some instances, there can be photosynthesis (Pohlman et al., 1997; Chávez-Solís et al., 2020). In contrast, caves are considered oligotrophic due to a severe and continuous lack of food availability (Brankovits et al., 2017; Hershey and Barton, 2018). In environments where food is scarce, the survival of a population may be determined by extreme physiological modifications. Such is the case of cavefish Astyanax mexicanus that have developed insulin resistance, allowing them to survive for long periods without feeding (Riddle et al., 2018). Auer et al. (2018) observed that juvenile wild salmons with greater MMR had higher survival rates in environments with lower food availability, suggesting that higher MMR confers greater competitive ability in an environment where food is the limiting resource. Along with the differences in the dietary carbon apportionment amongst the studied Typhlatya species, (Chávez-Solís et al., 2020), it seems that the higher MMR observed in T. dzilamensis could constitute an advantage in the oligotrophic cave environment below the halocline. Under the strong selective pressure exerted by food scarcity, a higher AS could represent an expansion in the availability of potential energy for foraging or migrating in search of food.

One noteworthy aspect of the exercise-derived metabolic responses measured herein is the high intraspecific heterogeneity observed in T. dzilamensis. In addition to the inherent variability of measuring physiological indicators in the laboratory, it is necessary to keep in mind that the experiments were conducted in situ, with wild organisms that had not been subject to previous conditions standardizing their experience. Although we do not have enough information to explain such variability, sources of variation in metabolic descriptors of individuals of the same species are usually related to size, reproductive state, feeding, sex, and molting stage in the case of crustaceans (Alter et al., 2015; Chabot et al., 2016b). While individual differences were methodologically unfeasible to control in the field experiments, it can be assumed that these sources of variation were common to the three species studied. Nevertheless, it is interesting to note that individuals of T. mitchelli and T. pearsei were more homogeneous in their responses compared to T. dzilamensis, something that could be explained if physiological responses to stress that are smaller in their mean magnitude also tend to be more homogeneous than those with larger mean magnitudes.

Aerobic metabolism in the mitochondria generates about 90% of ROS even during basal metabolism (Sies, 1993; Balaban et al., 2005), and ROS increase with increasing metabolic rate (Storey, 1996; Rosa et al., 2008; Rodríguez-Fuentes et al., 2017). An increased production of reactive molecules can damage proteins such as DNA and lipid membranes and, if the propagation of these ROS is not prevented, the organism may undergo severe cell stress, lose functionality, and ultimately die. The AOS response in all three Typhlatya species was in close correspondence with the levels of aerobic metabolism following physical activity. As T. dzilamensis increased oxygen consumption, the antioxidant system was activated accordingly to the increase in ROS production, thus preventing cellular damage. In contrast, T. mitchelli and T. pearsei did not significantly increase oxygen consumption and consequently did not show an increase in AOS indicators or cell damage. As expected, T. mitchelli and T. pearsei display an efficient regulation of ROS even under induced metabolic activity, demonstrating that the energetic and biochemical mechanisms triggered by maximum activity are highly efficient in the caves where they inhabit.

A striking difference from the antioxidant analysis was the high quantity of GSH in BL individuals of T. dzilamensis compared to T. mitchelli and T. pearsei. Storey (1996) was the first to propose that animals that frequently face oxidative stress maintain the AOS constitutively activated. This strategy has been termed “defensive priming” and was first postulated in species with high thermal tolerance (Dong et al., 2008), and a similar mechanism has been suggested in sulfur-tolerant populations (Joyner-Matos and Julian, 2012). An alternative strategy to defensive priming is a quick activation of the AOS when ROS increase and the activation of oxidative stress repair mechanisms is needed (Joyner-Matos and Julian, 2012). The high concentration of GSH observed in T. dzilamensis could be indicative of a defensive priming strategy that would bring advantages to individuals that inhabit a more heterogeneous environment where frequent interactions with the halocline and hypoxia conditions are likely to occur. In contrast, T. mitchelli and T. pearsei may have a more conventional stress response strategy, as they exclusively inhabit freshwater environments, where there is little or no interactions with haloclines (Chávez-Solís et al., 2020).

Compared to surface inland water bodies, temperature in anchialine systems is remarkably constant both on daily and annual time scales (Chávez-Solís, 2015; Mejía-Ortíz et al., 2020). Whilst this led to expect similar thermal tolerance in the three Typhlatya species, T. dzilamensis presented a higher CTmax than T. mitchelli and T. pearsei. A greater physiological plasticity has been linked to higher thermal tolerance and the potential to invade and colonize diverse habitats (Sokolova and Pörtner, 2003; Lande, 2009; Mascaró et al., 2016). Tepolt and Somero (2014) suggested that due to its thermal tolerance (measured as critical thermal maxima and cardiac function), Carcinus maenas could colonize large subpolar areas of the European continent, as well as temperate and subtropical areas of the American continent. Vinagre et al. (2016) and Cumillaf et al. (2016) found a direct relationship between the size of the thermal window and tolerance to warming in 20 species of crustaceans and fish, showing that species from temperate environments and wider thermal windows have greater tolerance to warming than the tropical species. A wider thermal window in T. dzilamensis further emphasizes the relatively greater tolerance of this species to environmental heterogeneity.

The critical temperature is a metric that integrates cellular, biochemical and tissue scales which allows a direct comparison of temperature sensitivity among populations, species and communities (Bates and Morley, 2020). The similarity of the biochemical indicators from BL individuals of all species and those who underwent the critical thermal limits procedures is evidence that the method complies with the theoretical expectations when applied to Typhlatya, and that there is a difference in thermal tolerance among these species.

None of the species studied showed accumulation of LAC derived from either exercise or short-term exposure to critical temperatures, which indicates that these conditions or procedures did not require resorting to anaerobiosis to satisfy the demand for metabolic energy. It is noteworthy, however, the comparatively higher baseline concentration of lactate in T. dzilamensis. Records of Typhlatya swimming in sulfur clouds (Pakes et al., 2014), hypoxic zones (Brankovits et al., 2017; Chávez-Solís et al., 2020), as well as lower levels of dissolved oxygen below the halocline in Ponderosa (Chávez-Solís et al., 2020), suggest that T. dzilamensis may have to resort to anaerobic metabolism frequently. It is possible that these behaviors together with habitat conditions common to T. dzilamensis result in chronic lactate accumulation. An effect such as this has been reported in Niphargus virei, a hypoxia-resistant amphipod (Hervant et al., 1999), where lactate accumulated during periods of hypoxia can be de novo synthesized to glycogen once re-oxygenation is possible. Similarly, experiments with H. rubra (Atyidae) have revealed their capacity to survive up to 7 days under anoxia conditions without any oxygen consumption (Havird et al., 2014b). Although lactate concentration was not measured in their study, the authors concluded that this species must rely on anaerobic metabolism to meet metabolic energy demands. These and other questions on lactate fluctuations and the role of anaerobiosis in supplying energy makes it necessary to further investigate hypotheses on physiological capacities in species that inhabit environments where hypoxic or anoxic conditions are common.

According to Pigliucci et al. (2006), phenotypic plasticity can be defined as the attribute of an individual’s genotype to produce different phenotypes in response to environmental stimuli. Physiological plasticity, therefore, is a component of the response repertoire of the expressed phenotypes and is related to the organic functioning of systems at various levels of organization. It has been also postulated that physiological plasticity allows the emergence of a new environmentally induced phenotype, the expression of which can be fixed through natural selection (Pigliucci et al., 2006). Recent ancestral reconstructions support the hypothesis of a low salinity common ancestor for the Yucatan Typhlatya (Ballou et al., 2022), which in turn suggests that the salinity tolerance trait observed in T. dzilamensis is an independent secondary transition. In their study, Ballou et al. (2022) recorded at least two unknown Typhlatya species in the Yucatan Peninsula that were genetically identified, but have not yet been morphologically described. Whilst cryptic speciation amongst Typhlatya is possible, COI barcoding allowed to confirm that all of the specimens in the present study were correctly identified. Results herein, thus, provide evidence in support of a physiological plasticity observed in T. dzilamensis compared to its sister species that closely relates to the heterogeneous environment they inhabit.

The species of the genus Typhlatya in the present study possess contrasting physiological traits that correspond to the environmental characteristics of the microhabitat in which each species is distributed. T. dzilamensis, commonly found in the subterranean saltwater environment, displayed the greatest physiological plasticity compared to T. mitchelli and T. pearsei, hence has a greater resilience to environmental fluctuations. The activity of GSH and lactate concentration in T. dzilamensis further suggest that this species has a permanent “defensive priming” strategy that keeps the antioxidant system constitutively prepared for the eventuality of physiological stress, a recurrent condition in the relatively heterogeneous environment they inhabit.

In contrast to T. dzilamensis, the freshwater species T. mitchelli and T. pearsei, were very similar in exhibiting low aerobic scope, low lactate and an overall low reactivity to imposed physical activity. This suggests that T. mitchelli and T. pearsei have less physiological capacity for an immediate response to intensive exercise, which indicates a reduced plasticity, and thus expected to be more vulnerable to abrupt environmental fluctuations.

Current anthropogenic pressures on the aquifer may pose an increased risk to these species, all the more knowing that freshwater species may be particularly susceptible to environmental shifts. Future research should focus on evaluating physiological limits and resilience of species to changes in salinity, dissolved oxygen, and the presence of contaminants in the aquifer for the purpose of prevention and restoration of subterranean aquatic ecosystems.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

EC-S, KP, CR, and MM contributed to the conception and design of the study. EC and MM performed the statistical analysis and prepared de figures. EC-S, FD, MM, KP, DR, CR, and NT performed field experiments, analysed metabolic response and thermal tolerance. EC and GR-F obtained and analysed the biochemical indicators. EC wrote the first draft of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.

EC-S was awarded a CONACYT scholarship for PhD studies (CVU/Becario: 545211/294499). Field work was partially funded by project IN228319 (PAPIIT-DGAPA-UNAM).

EC-S acknowledges the CONACYT grant for PhD studies (545211/294499), as this manuscript is part of the requirements for the doctoral program of the Posgrado en Ciencias Biológicas UNAM. Field work was supported by the Cenoteando organization. Authors are grateful to Quetzalli Hernández, Dorottya Angyal, Michel Vázquez, and Arturo Mora for their cavediving support, and Parque Ecológico Chikin Ha for their support in our field experiments. Authors are grateful to Claudia Caamal, Ariadna Sanchez Arteaga and Karen Ortega for their support in obtaining biochemical indicators in the laboratory.

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.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.