A long-standing hypothesis in subterranean biology posits that organisms living in poor resource subsurface habitats can withstand long periods of bioenergetic shortages due to an innate reduced metabolic rate when compared to their epigean counterparts. However, previous studies have proposed that caves with ample energy resources may not evolve organisms with reduced metabolic rate. The equivocal nature of previous findings suggests that there is a need to compare food deprivation responses of subterranean and surface species in order to elucidate whether there are widespread adaptations to low energy systems in subterranean taxa. The purpose of the study was to examine patterns in basal metabolism and the effects of food deprivation in closely related subterranean- and epigean- amphipods, Stygobromus pecki and Synurella sp. from central and east Texas, USA, respectively. Basal metabolic rates (measured as O₂ consumption) differed between species, with S. pecki having substantially lower rates than Synurella. Individuals of both species were food deprived for a pre-determined time interval and changes in total body protein, lipids, and carbohydrates were measured throughout food deprivation experiments. Stygobromus pecki had larger initial energy stores than Synurella and were more conservative in the use of energetic reserves over a prolonged period of food deprivation. Thus, it appears that although S. pecki are currently found in shallow phreatic and spring opening environments, they have maintained more efficient metabolic adaptations to deal with prolonged periods of food deprivation.

biochemical composition, hypogean, karst, metabolic rate, physiological adaptation, Stygobromus pecki, Synurella

Subterranean habitats are thought to be energy-limited ecosystems characterized by spatiotemporal patchiness of food resources (Culver et al. 1995; Juan et al. 2010). This is due to a lack of in situ autotrophic production and intermittent allochthonous (surface-generated organic matter) inputs of organic matter (OM) (Poulson 1964; Hüppop 1985; Venarsky et al. 2014; but see Hutchins et al. 2016). It is widely reported that hypogean (subterranean) organisms living in such low energy habitats have evolved a variety of behavioral, physiological, and metabolic adaptations that allow them to withstand long periods of resource shortage and show high starvation resistance (Hervant et al. 1997, 1999; Hervant and Renault 2002; Issartel et al. 2010). For example, some hypogean organisms have greater energetic reserves (e.g., greater glycogen, triglycerides, and protein content) and utilize these reserves at slower rates than related epigean taxa (Hervant et al. 1997, 1999; Hervant and Renault 2002; Mezek et al. 2010). Several studies have demonstrated that hypogean invertebrates exhibit physiological responses to prolonged food deprivation that differ from related epigean species. Hypogean amphipods and isopods respond to prolonged food deprivation in successive phases, with an immediate but slower rate of glycogen depletion, followed by utilization of proteins, and then use of lipids (Hervant et al. 1999; Hervant and Renault 2002). In contrast, epigean amphipods and isopods exhibit immediate, linear, and simultaneous declines in all energy reserve forms when exposed to periods of prolonged food deprivation (Hervant et al. 1999; Hervant and Renault 2002). However, it is unclear how conserved these metabolic responses to prolonged food deprivation is across hypogean fauna from different subterranean conditions.

Although organisms in many subterranean systems face low-energy conditions and rely heavily on infrequent inputs of surface-generated OM, there is growing recognition that some systems do not align with this paradigm. Some subterranean systems are relatively open and receive frequent and/or sustained inputs of allochthonous terrestrial OM (Culver and Poulson 1971; Schneider et al. 2011) or receive large inputs of high quality OM in the form of guano (Ferreira et al. 2000; Gnaspini 2005). In addition, some subterranean systems exhibit in situ chemoautotrophic production by microbial communities (Engel 2007; Porter et al. 2009), which can increase overall resource availability for organisms and lead to increased organismal diversity and greater food web complexity (Hutchins et al. 2016). It has been hypothesized that selection pressures to exhibit reduced metabolic rates or other adaptations to low energy conditions would be relaxed in these relatively energy-rich subterranean environments (Culver and Poulson 1971; Spicer 1998; Riesch et al. 2011). Thus, there is potential for subterranean obligate fauna to exhibit metabolism and physiological responses to prolonged food deprivation similar to those found in related epigean taxa, but there has been limited experimental examination of this hypothesis.

In addition to variation in resource availability among subterranean systems, many groundwater systems also exhibit vertical gradients in resource availability. Specifically, shallow phreatic habitats within aquifers, such as spring opening ecotones (i.e., transition zones between groundwater and surface–water habitats) are more likely to have greater access to terrestrial OM sources and therefore greater resource availability for faunal assemblages (Nair et al., in review). Spring ecotones often contain unique and diverse assemblages composed of surface, crenic (spring obligates) and hypogean taxa (Cantonati et al. 2012). Indeed, spring ecotone environments are considered “windows” to the subterranean world and present unique environments to investigate the ecology and evolution of groundwater organisms (Galassi et al. 2014). Although it has been hypothesized that subterranean fauna living in more energy-rich subterranean environments do not experience strong selection pressures to exhibit stygomorphic adaptations and should exhibit metabolic responses more akin to epigean species, this question has not been assessed for subterranean fauna that occupy shallow phreatic zones and spring opening ecotones.

The purpose of this study was to examine metabolic and food deprivation responses of a subterranean adapted organism that exists in a more energy-rich environment (i.e., shallow phreatic habitats and spring openings) in order to assess the hypothesis that subterranean organisms in more energy-rich environments experience relaxation of selective pressures on stygomorphic metabolic adaptations. These responses to prolonged food deprivation were then compared to food deprivation responses of a related surface species. Specifically, we examined the energy utilization (use of proteins, carbohydrates and lipids) and metabolic responses (O₂ consumption rates) to long-term food deprivation of two crangonyctid amphipods: the subterranean amphipod Stygobromus pecki and the largely epigean amphipod Synurella sp. Stygobromus pecki (Peck’s cave amphipod) (Holsinger, 1967) is a federally endangered species endemic to two spring systems in the Edwards Aquifer of central Texas (i.e., Comal and Hueco springs) (United States Fish and Wildlife Service 1997; Gibson et al. 2008). Stygobromus pecki is found in shallow phreatic areas and at spring ecotones. It feeds largely on surface derived OM sources at its occurrence sites (Nair et al., in review), but is morphologically adapted for subterranean existence (e.g., eyeless, long appendages) (Holsinger 1967). In contrast, Synurella is widely distributed mostly epigean genus found in a variety of habitat types across the southeastern United States (including portions of Texas) and Europe (Holsinger 1977). The examination of metabolic responses of S. pecki to prolonged food deprivation and the comparison of these responses to a known epigean close relative presents a unique opportunity to examine the physiological responses and potential adaptations of a subterranean organism living in a more energy-rich environment.

Capacity to withstand periods of low to no food supply depends on the presence of endogenous reserves and metabolic responses that ensure efficient utilization of stored metabolites (Hervant et al. 1997, 1999, 2001; Fuglei et al. 2000; Issartel et al. 2010). Previous studies have reported species-specific differences in storage and utilization of metabolites in epigean and hypogean crustaceans during periods of food deprivation (Hervant et al. 1999, 2001; Hervant and Renault 2002; Sacristán et al. 2016). In the current study, related hypogean and epigean amphipods exhibited markedly different physiological strategies to deal with food deprivation. Lower overall metabolic rates observed in S. pecki (in comparison to Synurella) are in accordance with studies that have found that hypogean organisms have lower metabolic activity when compared to surface relatives (Hervant et al. 1997, 1998; Spicer 1998; Hervant and Renault 2002; Simčič et al. 2005; Mezek et al. 2010). Stygobromus pecki used in experiments were substantially larger than Synurella (S. pecki mean wet mass = 13.4 mg, Synurella mean wet mass = 7.1 mg) and metabolic scaling indicates that mass-specific metabolic rates decline with increasing body size (Brown and Sibly 2012). However, when we express O₂ consumption rates on a per capita basis (µmol O₂ consumed/h per individual), Synurella still exhibits a substantially greater basal metabolic rate than S. pecki (~5× greater), further indicating that S. pecki has lower basal metabolic rates even when body size differences are considered. In addition, presence of greater total carbohydrate stores in S. pecki (relative to Synurella) on Day 0 prior to start of food deprivation is in line with previous studies that have compared metabolic responses of epigean and hypogean taxa to food deprivation (Hervant et al. 1999, 2001; Hervant and Renault 2002). Presence of greater energy stores in hypogean species is thought to be indicative of adaptation to low energy subterranean conditions so that individuals can continuously fuel metabolic needs for longer periods of low food availability. Thus, results from this study indicate that despite the shallow subterranean existence of S. pecki (and its presumed greater access to food resources), basal metabolism and initial energy reserves in this species seem to follow the paradigm that subterranean species have relatively depressed metabolic rates and different energy reserve concentrations when compared to epigean fauna.

In the present study, lipid reserves were substantially higher in Synurella, when compared to S. pecki. In contrast, Hervant et al. (1999) found that that the subterranean amphipods, Niphargus virei and N. rhenorhodanensis had higher stored lipids and carbohydrates (as glycogen) than surface amphipods (Gammarus fossarum). Contrasting results from Hervant et al. (1999) and this study could be due to differences in species-specific adaptations to food limitation resulting from different ways of life and metabolic needs (Danielopol and Rouch 1991). In addition, species-specific differences in storage and utilization of body reserves may occur due to differences in thermal adaptation, type and quality of food consumed, feeding history and life-cycle strategy, as well as their biosynthesis from either dietary fatty acids or dietary proteins, carbohydrates or lipids (Lahdes et al. 2010; Pond 2012). The two species which Hervant et al. (1999) studied (N. virei and N. rhenorhodanensis) are typically found in deep phreatic habitats, whereas S. pecki is a shallow phreatic zone specialist. Thus, it is possible that differences between the findings of this study and that of Hervant et al. (1999) could come from the evolutionary history, specific habitat associations, and access to food resources of the specific fauna used in experiments.

Although S. pecki did not have higher lipid stores than Synurella, the present study found differences between epigean and hypogean species in terms of utilization and depletion of various energy reserves during starvation. Starvation leads to changes in the body composition (Gibert and Mathieu 1980; Barclay et al. 1983), and the relative importance of different metabolite reserves depends on the duration of starvation as well as species-specific differences in metabolism and regulation (Hervant et al. 1999; Caruso et al. 2008). During starvation, crustaceans meet energy demands by regulating enzymatic activities to access energy reserves (i.e., hydrolysis of proteins to amino acids, glycogen to glucose, and triglycerides to free fatty acids), while ensuring cell integrity (Sánchez-Paz et al. 2006). In the present study, Synurella demonstrated a monophasic response to food deprivation, characterized by an immediate linear decrease in all energy reserves, including lipids. In contrast, the only metabolite that differed between fed and unfed treatments during starvation of S. pecki were proteins. Thus, it appears that S. pecki accessed proteins as an energy reserve during the 90-day starvation period. Hervant et al. (1999) observed that glycogen stores were first utilized by food deprived hypogean amphipods (Niphargus sp.) and that protein reserves were sparingly utilized after 30 days of food deprivation. Thus, initial utilization of protein energy reserves during periods of starvation may not be a cosmopolitan feature of hypogean amphipod metabolism, but further comparative studies are required.

Our study found that the use of bulk energy reserves differed between the two crangonyctid species we examined. Synurella exhibited monophasic declines in all bulk energy reserves during food deprivation, whereas S. pecki only exhibited differences in protein content in fed and food deprived animals. However, it is likely that our analysis of bulk energy reserves (i.e., total lipids and carbohydrates) may have obscured differences between the two study species in the utilization of specific energy reserve constituents during periods of food deprivation. Within crustaceans, neutral lipids (mainly triglycerides) are preferentially catabolized during food deprivation, but polar lipids (i.e., phospholipids and cholesterol) are conserved because of they serve as structural components of cell membranes (Hervant et al. 1999). Similarly, crustaceans access carbohydrate reserves stored mainly as glycogen when they are food deprived (Hervant et al. 1999; Hervant and Renault 2002; Sánchez-Paz et al. 2006). Given the differential use of energy reserve constituents (i.e., triglycerides versus phospholipids, glycogen versus glucose) during food deprivation periods, future studies should attempt to examine these constituents in order to better elucidate patterns of energy use within and among our study species.

In this study, carbohydrate reserves in S. pecki were depleted in both fed and unfed populations in the lab throughout the experimental period. Stygobromus pecki in the fed treatment were supplied food at similar rates to other S. pecki individuals we have maintained in the laboratory and it appeared as though they were consuming the added food items (P. Nair, personal observation). The reason or mechanisms for carbohydrate depletion in fed S. pecki in the current experiments is not known, but it may be due to stress associated with being held in captivity in a for a relatively long period of time. Some hypogean species can be sensitive to being held in captivity for extended time periods; Gibert and Mathieu (1980) found that individuals of the hypogean amphipod N. rhenorhodanensis that were fed and kept under conditions close to their natural environment in the lab showed significant reduction in carbohydrates reserves within a month of being held in captivity. These authors hypothesized that this was due to captive stress, but this hypothesis requires further study. The USFWS maintains captive populations of S. pecki at the San Marcos Aquatic Resource Center facility (SMARC; San Marcos, Texas), but it is unclear if these individuals similarly experience carbohydrate reduction or any stress associated with being held in a captive setting. Clearly, there is a need to further explore these results from an applied and captive breeding perspective as it applies to S. pecki and its endangered conservation status.

Our study shows that S. pecki has lower energetic requirements (i.e., basal metabolic rates), greater total carbohydrate reserves, and lower rates of lipid use during starvation when compared to a surface relative. Cumulatively, these findings suggest that S. pecki maintains a stygomorphic metabolic strategy for survival in environments with low or sporadic food availability, despite its occurrence in shallow phreatic and spring opening environments. S. pecki is closely related to amphipod species which occur in deeper phreatic environments (Ethridge et al. 2013) and it is hypothesized that ancestors of these taxa invaded freshwater subterranean systems during the late Cretaceous or early Cenozoic (Holsinger 1967). Thus, present-day metabolic adaptations to persistence in a deeper phreatic low-energy environments observed in S. pecki is consistent with their longer-term evolutionary history, rather than their current distribution in and around surface spring opening ecotones. Stygobromus pecki serves as the top invertebrate predator in spring opening ecotones, feeding on surface invertebrates, including the epigean hyalellid amphipod Hyalella azteca (Nair et al., in review) and S. pecki has the ability to detect and actively avoid light, presumably to avoid predation (Nowlin et al. 2015; Worsham et al., in preparation). The juxtaposition of some surface-adapted traits (i.e., surface foraging, light avoidance) and subterranean metabolic characteristics in S. pecki suggests that metabolic traits are not easily modified even though S. pecki now lives in shallow groundwater – surface water interfaces. However, future studies focused on comparing metabolic requirements and response of S. pecki with their deep phreatic relatives are needed to further evaluate the hypothesis that S. pecki exhibits deeper phreatic metabolic characteristics despite its current shallow phreatic distribution.

We would like to thank Randy Gibson and Nina Noreika for their valuable assistance with field collections. The funding for this project was provided by the Doctoral Research Support Fellowship Award. Endangered species used in this study were collected under Texas State University Permit No. SPR-0116-011.