Research Article |
Corresponding author: Parvathi Nair ( parvathinair@utexas.edu ) Academic editor: Oana Teodora Moldovan
© 2020 Parvathi Nair, Mar Huertas, Weston H. Nowlin.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Nair P, Huertas M, Nowlin WH (2020) Metabolic responses to long-term food deprivation in subterranean and surface amphipods. Subterranean Biology 33: 1-15. https://doi.org/10.3897/subtbiol.33.48483
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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 (
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 (
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 (
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) (
Live individuals of S. pecki were collected from spring openings at Comal Springs (29°43.0887'N, 98°7.8823'W). Comal Springs (city of New Braunfels, Comal County) is the largest spring complex in Texas and is located along the eastern edge of the Edwards Plateau. The Comal Springs system discharges groundwater from the Edwards Aquifer from more than 400 spring openings and is the principle location for S. pecki. Live individuals of this species were collected from the immediate vicinity of spring openings by a combination of hand picking and sweeps of small aquarium nets. Synurella were collected using the same techniques from perennially flowing surface streams (30°35.8967'N, 95°7.71'W) near the city of Coldspring, Texas (San Jacinto County). Coldspring is located ~300 km to the northeast of Comal Springs. All known Synurella species in the southeastern United States are epigean (
Animals were acclimated to laboratory conditions in species-specific large plastic flow-through chambers with untreated Edwards Aquifer groundwater approximating the conditions found at collection sites [water temperature = 23 °C , dissolved oxygen (DO) concentration > 6 mg /L]. Synurella were exposed to a 12:12 light: dark cycle during housing, but S. pecki individuals were maintained in 24h darkness (
We estimated basal metabolic rates of both species by measuring O2 consumption. Oxygen consumption rates of well-fed individuals were estimated using Qubit systems OX1LP-30 DO cuvettes with Clark cell type polarographic oxygen sensor (Qubit Systems, Kingston, ON, Canada). The respirometric cuvette chamber had a small magnetic stirring bar positioned at the bottom of the chamber (but physically separated from the experimental animal) to continuously mix chamber water and ensure accurate O2 concentrations in the chamber. The stirring rate was set to minimal speed in order to adequately mix chamber water but not induce stress on experimental animals. An individual test subject of either species was placed in a cuvette filled with 5mL of Edwards Aquifer water and was allowed to acclimate to the chamber for 30 minutes prior to recording O2 changes (in mg/L) of the chamber. After acclimation, DO concentration was recorded at 30-second intervals for 15 minutes. Cuvettes are externally jacketed with a water flow through system in order to maintain thermal stability at 23 °C. All experiments were carried out in a dark room. Per capita O2 consumption rates were calculated by the dividing the change in DO concentration by 15 min. Mass-specific O2 consumption rates (µmol O2/g/h) were calculated by dividing O2 consumption by wet weight of each individual (g). Oxygen consumption rates were estimated for n = 5 fed individuals of each species.
To assess biochemical changes and use of potential energy reserves in amphipods during extended periods of food deprivation, we experimentally examined change in whole-body metabolites of both species. Changes in metabolites during food deprivation was estimated at regular sampling intervals during food deprivation over 90 days for S. pecki (metabolites measured on days 0, 15, 30, 60, and 90) and over 30 days for Synurella (metabolites measured on days 0, 15, and 30). These species-specific food deprivation time periods were based on the literature and our own pilot experiments. Previous studies (
Prior the start of food deprivation experiments, animals were acclimated to laboratory conditions, maintained, and fed as above for ~1 month. At the start of experiments, individuals of each species were separated into two treatments: fed (receiving weekly dense culture fish food pellets) and unfed (food deprived). For both species, each treatment contained n = 75 individuals. Individual animals were housed in 4 cm long and 1.91 cm diameter PVC flow-through holding chambers. Amphipods were segregated from each other during experiments in order to prevent cannibalism (
Whole-body metabolites were estimated on each sampling date (Day 0, 15, 30, 60 and 90 days for S. pecki and day 0, 15, and 30 for Synurella). In order to minimize the inclusion of food materials in the guts of animals in biochemical analyses, animals were removed from chambers prior to weekly feedings; no leftover food was observed in holding chambers when animals were removed for analysis. In addition, once animals were removed, they were held for ~2 hours to clear gut contents and then frozen at - 80 °C in clean 2 mL microcentrifuge tubes. Before metabolites were assayed, animals were thawed and wet weight of each individual was determined (mg). For each sample (composed of n = 3 individuals), tissue was homogenized and one-third of the tissue was further homogenized in phosphate buffer solution (pH = 7.4) for protein and total carbohydrate analysis. Proteins were analyzed using a Coomassie (Bradford) protein assay kit (Thermo Fisher Scientific) and total carbohydrates were analyzed using a total carbohydrate assay kit (Cell Biolabs, STA-682). Remaining tissue for each sample was homogenized in 2:1 chloroform-methanol (v/v) and lipids were extracted using a modified procedure by
For analysis of basal metabolic rates, difference in mass-specific O2 consumption rates between species was assessed using one-way ANOVA. For the food deprivation experiment, body composition (protein, carbohydrate, and lipid content) between species was compared on Day 0 (immediately prior to the start of food deprivation) using one-way ANOVA. The effect of food deprivation on body composition within each species was then assessed by comparing treatments (fed versus unfed) with repeated measures ANOVA, which provides the treatment effect (fed versus unfed), time effect (Days 15 and Day 30 dates for Synurella, Days, 15, 30, 60, and 90 for S. pecki), and the treatment x time interaction. Prior to analyses, data were examined for normality, homoscedasticity, and sphericity (for the repeated measures ANOVA). If data did not meet assumptions, they were either ln- or square root-transformed. Significance for all tests was inferred at P < 0.05 and analyses were performed in R (version 3.5.0,
Basal metabolic rates differed between S. pecki and Synurella (F1, 8 = 15.99, P < 0.004; Fig.
Prior to the start of experiments (Day 0), the content of some metabolites differed between the two species (Fig.
Changes in the levels of body metabolites in Stygobromus pecki and Synurella sp. A Carbohydrates B proteins C lipids concentrations during long-term food deprivation at 23 °C in darkness. Values are means ± SEM for n = 5 replicates. (*) indicates significance at P < 0.05 for the main effects of Treatment, Time and the Time × Treatment interaction.
During the period of food deprivation, total carbohydrate content of S. pecki did not differ between fed and unfed treatments (F1, 4 = 0.356, P = 0.583; Fig.
Protein content of S. pecki differed between fed and unfed treatments, with fed animals having higher protein content (F1, 8 = 19.28, P = 0.023; Fig.
Lipid content of S. pecki did not differ between treatments during the food deprivation period (F1, 8 = 2.51, P = 0.152; Fig.
Over the entire experimental period, mortality of Synurella was 24% for fed and 38% for unfed treatments. S. pecki mortality was 18% for fed and 37% for unfed treatments.
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 (
In the present study, lipid reserves were substantially higher in Synurella, when compared to S. pecki. In contrast,
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 (
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 (
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;
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 (
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.