Physiological and metabolic responses to rising temperature in Gammarus pulex (Crustacea) populations living under continental or Mediterranean climates.

Latitudinal thermal gradients offer the possibility of comparing the current performance of populations of a single species living in contrasting thermal conditions. The Rhône River Valley (France) presents a 5°C thermal gradient corresponding to the increase in temperature predicted by climatic models (IPCC, 2007). We studied the thermal tolerance to rising temperature (from 15 to 30°C) of five populations of the key species Gammarus pulex living either in the North (i.e. the cold part) or in the South (i.e. the warm part) of the river Valley. Individuals were acclimated at 18, 21, 24, 27 or 30°C during 10days. After this period, we here measured experimentally the populations' survival, ventilatory rate, oxygen consumption, and glycogen and triglyceride contents. Southern populations have a higher survival rate and higher oxygen consumption at higher temperatures (27 and 30°C) in comparison with northern populations. Southern individuals also presented a hyperventilation, and higher energy stores compared to northern individuals whatever the acclimation temperature considered. In a global change context, the rising temperatures during the next decades may differently impair the metabolism and the survival of populations of G. pulex from different geographical origins. These differences in ecophysiological responses of organisms must be taken into account to predict the consequences of climate change.

Preventive antioxidant responses to extreme oxygen level fluctuation in a subterranean crustacean.

The principal aim of this work was to explore the responses of the groundwater crustacean Niphargus rhenorhodanensis to oxidative stress caused by short- and long-term drastic variations in oxygen level. To this end, we investigated thiobarbituric acid reactive substances (TBARS) levels and anti-oxidative enzyme (SOD and GPx) activities during 24 h anoxia and post-anoxia recovery, and during 10 days of severe hypoxia and post-hypoxia recovery. We observed a decrease in TBARS amounts during recovery from severe hypoxia. Parallel to these results, we observed an overactivation of SOD activity after a 24 h anoxic stress. GPx activity measured at the end of anoxia or severe hypoxia and in the early hours of post-stress recovery also showed an overactivation compared to the control group. We can hypothesize that this overproduction of GPx corresponded to an anticipatory mechanism coping with the overproduction of reactive oxygen species (ROS) during the recovery phase in subterranean animals. This response could be considered as a major asset for life in alternately normoxic and hypoxic conditions, and therefore in extreme biotopes such as groundwaters.

Ecophysiological responses to temperature of the "killer shrimp" Dikerogammarus villosus: is the invader really stronger than the native Gammarus pulex?

With global climate changes, biological invasions are considered to be one of the main causes of the decline of freshwater biodiversity. In this context, predicted increases in global temperature may alter the geographical distributions of native and invasive species. The purpose of our study was to examine the metabolic, behavioral and physiological responses to short-term temperature acclimation of two widely distributed species (the most successful European invader, Dikerogammarus villosus, and its main victim, Gammarus pulex), in order to estimate the potential effect of global warming on its invasion of freshwater ecosystems. Our results show that D. villosus is more vulnerable to high temperatures than G. pulex. The native species seems to be best adapted to intermediate temperatures (10-20°C) with a possibility of adjustment to "extreme" temperatures (5-27°C), whereas the "killer shrimp" D. villosus seems best adapted to lower temperatures (5-10°C) with a limited possibility of adjustment above 20°C. In the light of our results, global warming is likely to be less favorable to the invasive species. However, D. villosus showed reduced metabolic and activity rates, associated with higher glycogen content. This adaptive strategy was interpreted as having functional advantages, allowing D. villosus to successfully invade harsh and/or unpredictable biotopes. In addition, our results show that glycogen stores may be used as a powerful indicator of the optimal thermal window for aquatic ectotherms.

Metabolic rate and oxidative stress in insects exposed to low temperature thermal fluctuations.

Fluctuating temperatures are a predominant feature of the natural environment but their effects on ectotherm physiology are not well-understood. The warm periods of fluctuating thermal regimes (FTRs) provide opportunities for repair leading to increased survival, but there are also indications of negative effects of warm exposure. In this study, we examined respiration and oxidative stress in adult Alphitobius diaperinus exposed to FTRs and to constant low temperatures. We hypothesized that cold exposure will cause oxidative stress and that FTRs would reduce the amount of chill injuries, via activation of the antioxidant system. We measured V˙CO2, activities of super oxide dismutase (SOD), amounts of total (GSHt) and oxidized glutathione (GSSG) during cold and warm periods of FTRs. Increased severity of cold exposure caused a decrease in the glutathione pool. SOD levels increased during the recovery period in the more severe FTR. The antioxidant response was sufficient to counter the reactive oxygen species production, as the GSH:GSSG ratio increased. We conclude that cold stress causes oxidative damage in these beetles, and that a warm recovery period activates the antioxidant system allowing repair of cold-induced damage, leading to the increased survival previously noted in beetles exposed to fluctuating versus constant temperatures.

Cave colonization without fasting capacities: an example with the fish Astyanax fasciatus mexicanus.

Subterranean animals have commonly evolved hypoactivity, hypometabolism and/or the sequential use of energetic reserves to tolerate long fasting periods imposed by the low food levels found in subterranean environments. However, some tropical caves are characterized by a potential high level of nutriments. By using the tropical fish Astyanax fasciatus that presents both populations subterranean (Astyanaxfasciatus mexicanus) and epigean (Astyanaxfasciatus fasciatus) populations, we described behavioral, metabolic and biochemical responses during a long-term fasting period followed by a refeeding period. The results demonstrated that fed hypogean fishes exhibited different energy stores together with a hypometabolism. But, despite drastic decreases in locomotory activity and oxygen consumption during fasting, hypogean fishes consumed significantly more glycogen, triglycerides and proteins during the starvation period than epigean fishes. This lower fasting capacity showed by hypogean fishes is confirmed by the higher activation of the compensatory metabolic pathways (ketogenesis and gluconeogenesis). After the refeeding period, cave fishes did not recover from the "food deprivation" stress, and resume fed levels in glycogen, triglyceride reserves and proteins, in contrast to epigean ones. This study thus demonstrates that starvation adaptations are not necessary for cave life, but are rather correlated to the "energetic state" of each ecosystem, and that troglomorphism is not linked to starvation capacities and thus not to the impoverished food availability.

Effects of temperature on biological and biochemical indicators of the life-history strategy of bullhead Cottus gobio.

The biological and biochemical effects of temperature on life-history strategy of female bullhead Cottus gobio were investigated. Fish from two populations (Bez Basin, south-east France) experiencing contrasted thermal environments (i.e. more or less stable) were reared during 4 months at three distinct temperatures (7, 9 or 12 degrees C). Both somatic (soma fresh mass and muscle triglyceride content) and reproductive (gonad fresh mass, fecundity, mean diameter of eggs and gonad triglyceride content) indicators were examined. Mixed models indicated that an increasing temperature had significant negative effects on all life-history indicators except for soma fresh mass. Differences in life-history strategy with regard to muscle and gonad triglyceride contents, however, suggest that populations experiencing more variable thermal environments may be better adapted than others to cope with an increasing temperature. These findings may have important implications for C. gobio populations, within the context of climate warming.

Urban pollution of sediments: Impact on the physiology and burrowing activity of tubificid worms and consequences on biogeochemical processes.

In urban areas, infiltration basins are designed to manage stormwater runoff from impervious surfaces and allow the settling of associated pollutants. The sedimentary layer deposited at the surface of these structures is highly organic and multicontaminated (mainly heavy metals and hydrocarbons). Only few aquatic species are able to maintain permanent populations in such an extreme environment, including the oligochaete Limnodrilus hoffmeisteri. Nevertheless, the impact of urban pollutants on these organisms and the resulting influence on infiltration basin functioning remain poorly studied. Thus, the aim of this study was to determine how polluted sediments could impact the survival, the physiology and the bioturbation activity of L. hoffmeisteri and thereby modify biogeochemical processes occurring at the water-sediment interface. To this end, we conducted laboratory incubations of worms, in polluted sediments from infiltration basins or slightly polluted sediments from a stream. Analyses were performed to evaluate physiological state and burrowing activity (X-ray micro-tomography) of worms and their influences on biogeochemical processes (nutrient fluxes, CO2 and CH4 degassing rates) during 30-day long experiments. Our results showed that worms exhibited physiological responses to cope with high pollution levels, including a strong ability to withstand the oxidative stress linked to contamination with heavy metals. We also showed that the presence of urban pollutants significantly increased the burrowing activity of L. hoffmeisteri, demonstrating the sensitivity and the relevance of such a behavioural response as biomarker of sediment toxicity. In addition, we showed that X-ray micro-tomography was an adequate technique for accurate and non-invasive three-dimensional investigations of biogenic structures formed by bioturbators. The presence of worms induced stimulations of nutrient fluxes and organic matter recycling (between +100% and 200% of CO2 degassing rate). Nevertheless, these stimulations were comparable within the three sediments, suggesting a low influence of urban contaminants on bioturbation-driven biogeochemical processes under our experimental conditions.

Comparative study on the metabolic responses of subterranean and surface-dwelling amphipods to long-term starvation and subsequent refeeding

The effects of long-term starvation and subsequent refeeding on intermediary and energy metabolism were investigated in two subterranean aquatic crustaceans, Niphargus rhenorhodanensis and Niphargus virei, and in a morphologically similar surface-dwelling species, Gammarus fossarum. The metabolic response to prolonged food deprivation was monophasic in G. fossarum, showing an immediate, linear and large decline in all of the energy reserves. In contrast, both subterranean organisms displayed successive periods of glucidic, proteo-glucidic then lipidic-dominant catabolism during food deprivation. In both subterranean species, lipids (51 % of the energy consumed during a 180-day starvation period) and proteins (44 %) were the most metabolized substrates in terms of total energy, whereas glycogen (5 %) contributed little energy. G. fossarum displayed a different energetic strategy: proteins comprised 56 % of the energy losses during a 28-day starvation period, total lipids some 39 % and glycogen reserves only 5 %. We propose an energy strategy for food-limited subterranean crustaceans involving the possession of (1) higher amounts of stored arginine phosphate, triglycerides and glycogen and (2) lower utilization rates of stored metabolites than G. fossarum and numerous other surface-dwelling crustaceans, making the fueling of food deprivation possible for a longer time. In addition, these species had a faster and more efficient assimilation of available nutrients during recovery from food deprivation, enabling preparation for a new nutritional stress. These specific adaptive responses might be considered, for N. virei and N. rhenorhodanensis, as an efficient energy-saving strategy for an environment where extended starvation periods alternate with sporadic feeding events, therefore improving their competitive advantages.

Lactate metabolism and glucose turnover in the subterranean crustacean niphargus virei during post-hypoxic recovery

Glucose and lactate metabolism were studied in a hypoxia-resistant subterranean crustacean, Niphargus virei, using an injection of l-[U-14C]lactate and tracer d-[6-3H]glucose either in normoxic conditions or after a 24 h exposure to severe hypoxic. Post-hypoxic animals (H animals) were compared with two treatment groups of normoxic animals. In the first normoxic group (NLL animals), animals were simultaneously injected with labelled and unlabelled lactate to obtain a lactate load similar to that of H animals. In the second normoxic group (N, control animals), animals were only injected with labelled lactate. During a 24 h recovery period, the incorporation of 14C and 3H into glycogen, lactate, glucose, amino acids, lipids and CO2 was measured. During recovery, glucose turnover rate was enhanced in H and depressed in NLL compared with N animals. However, when energy expenditure was taken into account, the changes were due only to a reduction of glucose turnover rate by lactate load. It was concluded that gluconeogenesis was not the main source of glyconeogenesis. Equivalent lactate loading in NLL and H animals resulted in an equivalent enhancement (fivefold) of lactate utilization in both groups when energy expenditure was taken into account. Lactate label incorporation appeared later in glycogen than in glucose, but remained high 24 h after the injection. Since glucose is mainly an extracellular metabolite, this observation may be consistent with the hypothesis of two distinct sites for glycogen restoration in hypogean crustaceans: a gluconeogenic organ (a liver equivalent) and a glyconeogenic organ (a muscle equivalent). The oxidative pathways of glucose and lactate were depressed in post-hypoxic N. virei and to a lesser extent in the NLL group. Since there is no evidence of marked protein utilization, it is postulated that, during recovery, repayment of the O2 debt relies on an increase in lipid utilization. During recovery from severe hypoxia or after a lactate load, the subterranean N. virei appeared to implement a strategy of lactate removal quite different from that observed in epigean crustaceans, favouring lactate-supported gluco- and glyconeogenesis and rapid glycogen replenishment instead of rapid lactate removal via oxidative pathways.

Behavioural and physiological effects of the trophically transmitted cestode parasite, Cyathocephalus truncatus, on its intermediate host, Gammarus pulex.

Some parasites with complex life-cycles are able to manipulate the behaviour of their intermediate hosts in a way that increases their transmission to the next host. Gammarids infected by the tapeworm Cyathocephalus truncatus (Cestoda: Spathebothriidea) are known to be more predated by fish than uninfected ones, but potential behavioural manipulation by the parasite has never been investigated. In this study, we tested the hypothesis that C. truncatus is able to manipulate the behaviour of one of its intermediate hosts, Gammarus pulex (Crustacea: Amphipoda). To assess if any behavioural change was linked to other phenotypic alterations, we also measured the immunity of infected and uninfected individuals and investigated the pathogenic effects of the parasite. Infected gammarids were significantly less photophobic than uninfected ones, but no effect of infection on the level of immune defence was found. The results on survival, swimming activity and oxygen consumption suggest that the parasite also has various pathogenic effects. However, the alteration in host phototaxis was not correlated to some of these pathogenic effects. Therefore, we propose that the modification in host reaction to light is a behavioural manipulation, explaining the previously observed increase of gammarid predation rate.

Changes in free amino acids in Alphitobius diaperinus (Coleoptera: Tenebrionidae) during thermal and food stress.

Temporal changes in free amino acid pools are examined in starved and cold-exposed (10 degrees C) beetles (Alphitobius diaperinus) (control beetles; 4, 7, 14, 28 and 56 days of cold-exposure). The range of individual responses and the effect of gender on survival and free amino acid levels are evaluated. Females survived significantly longer (Lt50 and Lt90) than males under stressing conditions. Pro, Gln, Ala, Arg and Thr are the major components of the free amino acid pool in the whole body of adult beetles. Multivariate analysis indicates that the duration of exposure explains most of the observed variation in amino acid levels, while sex effect is not significant. Moreover, amino acid levels fluctuate strongly within each sampled date, revealing a high inter-individual heterogeneity. However, this heterogeneity decreases after four weeks of cold-exposure and starvation. The increase in Ala level and the concomitant decrease of Pro after four weeks suggest that Pro might be an important fuel for metabolism when fat reserves are reduced. We conclude that changes in free amino acid pools are due to a combination of reduced individual heterogeneity, cold-acclimation and amino acid degradation for energetic purposes.

Are hybridogenetic complexes structured by habitat in water frogs?

The success and the evolutionary fate of hybridogenetic lineages are explained by both a generalistic heterosis hypothesis and an alternative hypothesis, the habitat segregation hypothesis. Because such hypotheses have rarely been tested at the level of whole habitats, our aim was to compare performances of two taxa within a hybridogenetic complex across diverse natural habitats. We took advantage of the waterfrog hybridogenetic complex (Rana esculenta and R. lessonae) by rearing tadpoles in natural contrasted habitats by means of enclosure experiments. We also monitored the frequency of each taxon in the waterfrog assemblages that naturally breed in the studied ponds. The hybridogenenetic taxon showed no evidence of broader tolerance as growth, development and physiology strongly varied in response to environmental heterogeneity. Our study reveals a differential success of the hybridogenetic taxon and its sexual host among environments. Moreover, hybridogenetic taxa rarely dominated the sexual species in natural assemblages. Consequently, our results show that the generalistic model does not explain the success of hybridogenetic lineages, but rather support the habitat segregation, among other alternative concepts.

The activities of enzymes associated with the intermediary and energy metabolism in hypogean and epigean crustaceans.

The activities of 18 enzymes involved in the intermediary and energy metabolism were measured in certain widely-spread peracarid crustaceans: 3 hypogean (Niphargus virei, Niphargus rhenorhodanensis and Stenasellus virei) and 2 epigean (Gammarus fossarum and Asellus aquaticus) ones. The activities of numerous enzymes were correlated with the known metabolic rates of the 5 species. Such rates are reduced in hypogean organisms: levels of enzymatic activity in subterranean species were 1.2 to 8.6 times lower than in epigean species for the main key regulatory enzymes involved in the Krebs cycle and glycolysis (phosphofructokinase, pyruvate kinase, hexokinase and citrate synthetase). The relative activities of phosphofructokinase, glycogen phosphorylase and hexokinase clearly indicated that glycogen was the main fuel oxidized in both epigean and hypogean organisms. A higher glycogen phosphorylase/hexokinase ratio in hypogean than in epigean crustaceans showed that subterranean species had a greater ability to function anaerobically. The presence of high activities of glutamate-pyruvate transaminase and lactate dehydrogenase in all species (and of malate dehydrogenase and fumarase in hypogean species) was indicative of a coupled fermentation of glycogen and glutamate during anaerobiosis, with lactate and alanine as end-products (as well as succinate in hypogean species). A low fructose-1,6-bisphosphatase/phosphofructokinase ratio, associated with a low level of phosphoenolpyruvate carboxykinase activity, indicated that the glycolytic pathway was active and that gluconeogenic ability was limited in epigean crustaceans. In contrast, in hypogean species, association of a higher ratio and a high level of phosphoenolpyruvate carboxykinase activity suggested a low glycolytic activity and a high gluconeogenic ability.

Ventilatory and locomotory activities in anoxia and subsequent recovery of epigean and hypogean crustaceans.

Locomotory and ventilatory responses to severe hypoxia and subsequent recovery were investigated in 3 amphipod crustaceans: 2 hypogean species (1 interstitial species Niphargus rhenorhodanensis and 1 karstic species N. virei) and 1 epigean species (Gammarus fossarum), and in an epigean population of 1 isopod crustacean (Asellus aquaticus). These species displayed respectively 46.7 h, 52.1 h, 6.3 h and 19.7 h lethal times for 50% of the population (LT50) values for anoxic survival. The aim of this study was to determine why the hypogean species displayed a survival time during severe hypoxia longer than that of Gammarus, Asellus and most other epigean crustaceans, and to better understand the ecological problems concerning Niphargus survival and perennation modalities in subterranean habitats which very often present hypoxic conditions during a hydrological cycle. The high resistance to severe hypoxia of hypogean animals partly results from an adaptation to the limitation of energetic expenditure linked to locomotion and ventilation in anaerobiosis, and from a decrease of general metabolism in severe hypoxia.

Behavioural, physiological and metabolic responses to long-term starvation and refeeding in a blind cave-dwelling (Proteus anguinus) and a surface-dwelling (Euproctus asper) salamander.

The effects of long-term starvation and subsequent refeeding on haematological variables, behaviour, rates of oxygen consumption and intermediary and energy metabolism were studied in morphologically similar surface- and cave-dwelling salamanders. To provide a hypothetical general model representing the responses of amphibians to food stress, a sequential energy strategy has been proposed, suggesting that four successive phases (termed stress, transition, adaptation and recovery) can be distinguished. The metabolic response to prolonged food deprivation was monophasic in the epigean Euproctus asper (Salamandridae), showing an immediate, linear and large decrease in all the energy reserves. In contrast, the hypogean Proteus anguinus (Proteidae) displayed successive periods of glucidic, lipidic and finally lipido-proteic-dominant catabolism during the course of food deprivation. The remarkable resistance to long-term fasting and the very quick recovery from nutritional stress of this cave organism may be explained partly by its ability to remain in an extremely prolonged state of protein sparing and temporary torpor. Proteus anguinus had reduced metabolic and activity rates (considerably lower than those of most surface-dwelling amphibians). These results are interpreted as adaptations to a subterranean existence in which poor and discontinuous food supplies and/or intermittent hypoxia may occur for long periods. Therefore, P. anguinus appears to be a good example of a low-energy-system vertebrate.

Locomotory, ventilatory and metabolic responses of the subterranean Stenasellus virei (Crustacea, Isopoda) to severe hypoxia and subsequent recovery.

The locomotory and ventilatory activities and the intermediary and energy metabolism modifications of the hypogean aquatic isopod crustacean Stenasellus virei were investigated in severe hypoxia (PO2 < 0.03 kPa) and subsequent recovery. The aims of this study were i) to determine why the subterranean species displayed a greater tolerance of hypoxia than numerous other epigean crustaceans, ii) to confirm previous results obtained with four hypogean and epigean crustaceans, iii) to compare the responses to severe hypoxia in hypogean amphipods and isopods, and iv) to better understand the ecological problems of the hypogean organisms survival in subterranean habitats. S. virei responded to experimental long-term, severe hypoxia with classical anaerobic metabolism mainly characterized by a decrease in adenosine triphosphate (ATP) and phosphagen, utilization of glycogen and glutamate, and accumulation of lactate and alanine. Lactate was also largely excreted by this organism, which is unusual for crustaceans in general. Compared to most other epigean crustaceans, the isopod S. virei showed high amounts of stored glycogen and arginine phosphate. These differences in glycogen and phosphagen stores, and the ability to reduce energetic expenditures linked to locomotion and ventilation, extended the survival of S. virei under experimental anaerobiosis. During recovery, the isopod S. virei showed a higher capacity for glyconeogenesis from lactate and a faster and total replenishment of ATP and arginine phosphate levels than epigean crustaceans. Data concerning responses to hypoxia and subsequent recovery in S. virei are similar to those previously obtained with two other hypogean amphipods, except that this isopod did not synthesize succinate in anaerobiosis.