Microplastics in Freshwater Sediments Impact the Role of a Main Bioturbator in Ecosystem Functioning.

While microplastic transport, fate, and effects have been a focus of studies globally, the consequences of their presence on ecosystem functioning have not received the same attention. With increasing evidence of the accumulation of microplastics at sediment-water interfaces there is a need to assess their impacts on ecosystem engineers, also known as bioturbators, which have direct and indirect effects on ecosystem health. This study investigated the impact of microplastics on the bioturbator Tubifex tubifex alongside any effects on the biogeochemical processes at the sediment-water interface. Bioturbators were exposed to four sediment microplastic concentrations: 0, 700, 7000, and 70000 particles kg-1 sediment dry weight. Though no mortality was present, a significant response to oxidative stress was detected in tubificid worms after exposure to medium microplastic concentration (7000 particles kg-1 sediment dry weight). This was accompanied by a reduction in worm bioturbation activities assessed by their ability to rework sediment and to stimulate exchange water fluxes at the sediment-water interface. Consequently, the contributions of tubificid worms on organic matter mineralization and nutrient fluxes were significantly reduced in the presence of microplastics. This study demonstrated that environmentally realistic microplastic concentrations had an impact on biogeochemical processes at the sediment-water interface by reducing the bioturbation activities of tubificid worms.

Effects of bioturbation by tubificid worms on biogeochemical processes, bacterial community structure and diversity in heterotrophic wetland sediments.

Bioturbation activity of tubificid worms has been recognized as a key process influencing organic matter processing and nutrient cycling in benthic aquatic ecosystems. This activity is expected to modify benthic microbial communities by affecting the physical and chemical environment in sediments. Nevertheless, quantifications of bacterial community changes associated with bioturbation in freshwater ecosystems are still lacking. The present study aimed at evaluating the impact of tubificid worms on bacterial community structure using NGS approach (16S metabarcoding) and long (6 months) laboratory experiments on four heterotrophic wetland sediments. Worm bioturbation activity significantly stimulated biogeochemical processes at the water-sediment interface but only had a marginally significant effect on bacterial community structures. Yet, bacterial diversity was consistently reduced in presence of worms. Such decrease could be associated with the stimulation of organic matter mineralization by worms, leading to a reduction of the diversity of trophic niches available for bacterial species. The slight changes in bacterial community structures induced by bioturbation did not appear to control biogeochemical processes. Thus, the stimulation of biogeochemical processes by worm bioturbation was more associated with a stimulation of the initial bacterial community than with a drastic change in bacterial communities induced by worms.

Anaerobic end-products and mitochondrial parameters as physiological biomarkers to assess the impact of urban pollutants on a key bioturbator.

The impact of long-term exposure (6 months) to highly or slightly polluted sediments on the energy metabolism of an ecosystem engineer (the oligochaete Limnodrilus hoffmeisteri) was investigated in laboratory conditions. We evaluated some mitochondrial parameters (respiratory chain activity and ATP production rate) and the accumulation of anaerobic end-products (lactate, alanine, succinate, and propionate). The sediments were collected from stormwater infiltration basins and presented high levels of heavy metals and polycyclic aromatic hydrocarbons (PAHs). These compounds had been drained by the runoff water on impervious surfaces of urban areas during rainfall events. A decrease in the activity of the mitochondrial electron transport chain was observed in worms exposed to the most polluted sediment. Urban contaminants disrupted both aerobic metabolism and mitochondrial functioning, forcing organisms to shift from aerobic to anaerobic metabolism (which is characteristic of a situation of functional hypoxia). Although L. hoffmeisteri is very tolerant to urban pollutants, long-term exposure to high concentrations can cause disruption in mitochondrial activity and therefore energy production. Finally, this study demonstrated that anaerobic end-products could be used as biomarkers to evaluate the impact of a mixture of urban pollutants on invertebrates.

Mitochondrial oxidative phosphorylation efficiency is upregulated during fasting in two major oxidative tissues of ducklings.

Fasted endothermic vertebrates must develop physiological responses to maximize energy conservation and survival. The aim of this study was to determine the effect of 1-wk. fasting in 5-wk. old ducklings (Cairina moschata) from whole-body resting metabolic rate and body temperature to metabolic phenotype of tissues and mitochondrial coupling efficiency. At the level of whole organism, the mass-specific metabolic rate of ducklings was decreased by 40% after 1-wk. of fasting, which was associated with nocturnal Tb declines and shallow diurnal hypothermia during fasting. At the cellular level, fasting induced a large reduction in liver, gastrocnemius (oxidative) and pectoralis (glycolytic) muscle masses together with a fuel selection towards lipid oxidation and ketone body production in liver and a lower glycolytic phenotype in skeletal muscles. At the level of mitochondria, fasting induced a reduction of oxidative phosphorylation activities and an up-regulation of coupling efficiency (+30% on average) in liver and skeletal muscles. The present integrative study shows that energy conservation in fasted ducklings is mainly achieved by an overall reduction in mitochondrial activity and an increase in mitochondrial coupling efficiency, which would, in association with shallow hypothermia, increase the conservation of endogenous fuel stores during fasting.

Skeletal muscle phenotype affects fasting-induced mitochondrial oxidative phosphorylation flexibility in cold-acclimated ducklings.

Starvation is particularly challenging for endotherms that remain active in cold environments or during winter. The aim of this study was to determine whether fasting-induced mitochondrial coupling flexibility depends upon the phenotype of skeletal muscles. The rates of oxidative phosphorylation and mitochondrial efficiency were measured in pectoralis (glycolytic) and gastrocnemius (oxidative) muscles from cold-acclimated ducklings (Cairina moschata). Pyruvate and palmitoyl-l-carnitine were used in the presence of malate as respiratory substrates. Plasma metabolites, skeletal muscle concentrations of triglycerides, glycogen and total protein and mitochondrial levels of oxidative phosphorylation complexes were also quantified. Results from ad libitum fed ducklings were compared with those from ducklings that were fasted for 4 days. During the 4 days of nutritional treatment, birds remained in the cold, at 4°C. The 4 days of starvation preferentially affected the pectoralis muscles, inducing an up-regulation of mitochondrial efficiency, which was associated with a reduction of both total muscle and mitochondrial oxidative phosphorylation protein, and with an increase of intramuscular lipid concentration. By contrast, fasting decreased the activity of oxidative phosphorylation but did not alter the coupling efficiency and protein expression of mitochondria isolated from the gastrocnemius muscles. Hence, the adjustment of mitochondrial efficiency to fasting depends upon the muscle phenotype of cold-acclimated birds. Furthermore, these results suggest that the reduced cost of mitochondrial ATP production in pectoralis muscles may trigger lipid storage within this tissue and help to sustain an important metabolic homeostatic function of skeletal muscles, which is to maintain levels of amino acids in the circulation during the fast.

Differential regulation of hsp70 genes in the freshwater key species Gammarus pulex (Crustacea, Amphipoda) exposed to thermal stress: effects of latitude and ontogeny.

Temperature is one of the main abiotic factors influencing the distribution and abundance of organisms. In the Rhône River Valley, populations of the crustacean Gammarus pulex are distributed along a 5 °C thermal gradient from the North to the South of the valley. In this present work, we investigated the heat shock response of G. pulex according to latitudinal distribution (northern vs. southern populations) and ontogeny (adults vs. embryos from early stages). We isolated two isoforms (one constitutive hsc70 and one inducible hsp70) of heat shock proteins 70 (HSP70) and quantitatively compared their amounts of mRNA after heat shocks, using real-time PCR. Whereas the hsc70 (constitutive) gene did not vary between the two populations, a significant effect of the population was observed on the expression of the hsp70 (inducible) gene in adult specimens. The northern population of amphipods showed a greater magnitude of induction and a 2 °C lower onset temperature when compared to the southern population, suggesting that the northern population is more affected by elevated temperature than the southern one. We demonstrated that the expression of hsp70 may play a crucial role in the persistence of biogeographical patterns of G. pulex, since it reflects the natural distribution of this species along the latitudinal thermal gradient. A differential regulation of hsc70 gene was also observed according to the ontogenetic stage, with a switch from heat inducible in early life stages to constitutively and highly expressed in adults. These findings demonstrate the importance of considering the entire life cycle to better understand the adaptive response to thermal stress.

Effect of climate-related change in vegetation on leaf litter consumption and energy storage by Gammarus pulex from Continental or Mediterranean populations.

As a consequence of global warming, it is important to characterise the potential changes occurring for some functional processes through the intra-specific study of key species. Changes in species distribution, particularly when key or engineer species are affected, should contribute to global changes in ecosystem functioning. In this study, we examined the potential consequences induced by global warming on ecosystem functioning in term of organic matter recycling. We compared consumption of leaf litter by some shredder populations (Gammarus pulex) between five tree species inhabiting continental (i.e., the northern region of the Rhône River Valley) and/or Mediterranean (i.e., the southern region of the Rhône River Valley) conditions. To consider any potential adaptation of the gammarid population to vegetation in the same climate conditions, three populations of the key shredder Gammarus pulex from the northern region and three from the southern region of the Rhône River Valley were used. We experimentally compared the effects of the geographical origin of both the gammarid populations and the leaf litter species on the shredding activity and the physiological state of animals (through body triglyceride content). This study demonstrated that leaf toughness is more important than geographical origin for determining shredder leaf litter consumption. The overall consumption rate of the gammarid populations from the southern region of Rhône Valley was much higher than that of the populations from the northern region, but no clear differences between the origins of the leaf litter (i.e., continental vs. Mediterranean) were observed. The northwards shift of G. pulex populations adapted to warmer conditions might significantly modify organic matter recycling in continental streams. As gammarid populations can demonstrate local adaptations to certain leaf species as a trophic resource, changes in riparian vegetation associated with climate change might locally affect the leaf litter degradation process by this shredder.

Disentangling the effects of local and regional factors on the thermal tolerance of freshwater crustaceans.

In the global warming context, we compared the thermal tolerance of several populations of the crustacean Gammarus pulex (Amphipoda: Gammaridae) along a latitudinal thermal gradient in the Rhône Valley. To disentangle the effect of regional (North vs. South) and local (site-specific) factors, the ecophysiological responses of populations were investigated at two levels of biological organisation: whole organism level considering body size [critical thermal maximum (CTmax), mean speed of locomotion (MS), time mobile (TM)] and organelle function level [mitochondrial respiratory control ratios (RCRs)]. CTmax and RCRs, but not MS and TM, revealed a significantly higher thermal tolerance in southern populations compared to northern ones. Nevertheless, temperatures ≥ 30°C were deleterious for all populations, suggesting that populations located in the warmer limit of the species distribution will be more threatened by climate change as they live closer to their upper thermal limits. The strong differences observed between populations indicate that the species-level thermal tolerance used in predictive models may not be informative enough to study the impact of global warming on species distributions. This work also reveals that an appropriate choice of indicators is essential to study the consequences of global warming since the response of organisms at the whole body level can be influenced by local conditions.

First cellular approach of the effects of global warming on groundwater organisms: a study of the HSP70 gene expression.

Whereas the consequences of global warming at population or community levels are well documented, studies at the cellular level are still scarce. The study of the physiological or metabolic effects of such small increases in temperature (between +2 degrees C and +6 degrees C) is difficult because they are below the amplitude of the daily or seasonal thermal variations occurring in most environments. In contrast, subterranean biotopes are highly thermally buffered (+/-1 degrees C within a year), and underground water organisms could thus be particularly well suited to characterise cellular responses of global warming. To this purpose, we studied genes encoding chaperone proteins of the HSP70 family in amphipod crustaceans belonging to the ubiquitous subterranean genus Niphargus. An HSP70 sequence was identified in eight populations of two complexes of species of the Niphargus genus (Niphargus rhenorhodanensis and Niphargus virei complexes). Expression profiles were determined for one of these by reverse transcription and quantitative polymerase chain reaction, confirming the inducible nature of this gene. An increase in temperature of 2 degrees C seemed to be without effect on N. rhenorhodanensis physiology, whereas a heat shock of +6 degrees C represented an important thermal stress for these individuals. Thus, this study shows that although Niphargus individuals do not undergo any daily or seasonal thermal variations in underground water, they display an inducible HSP70 heat shock response. This controlled laboratory-based physiological experiment constitutes a first step towards field investigations of the cellular consequences of global warming on subterranean organisms.

Selection of physiological and metabolic adaptations to food deprivation in the Pyrenean newt Calotriton asper during cave colonisation.

Food restriction is one of the major and most common constraints that subterranean animals face in their biotope. Cave-dwelling organisms thus have to cope with fasting periods that can extend from a month to a year. However, adaptive fasting resistance previously found in subterranean fauna has only been highlighted by direct comparisons with phylogenetically distant epigean organisms, which could severely impact conclusions. Here we report physiological and metabolic responses to 42 days of fasting followed by 10 days of refeeding in two populations (one subterranean and one epigean) of Calotriton asper. In the fed state (control), the hypogean population exhibited a hypometabolism together with higher glycogen (+25% in liver and muscles) and triglyceride stores (+50% in muscles). During the fasting period, cave individuals exhibited a 20% decrease in VO(2) whereas epigean individuals experienced no significant change. In addition, the energetic reserves always remained higher in the hypogean population. According to phylogenic and biogeographic data, cave colonization by this species dates back to less than 10,000 years, suggesting a rapid selection of adaptive traits related to fasting. This study strongly suggests that cave colonization induces a decrease in metabolism together with a higher capacity to accumulate energy reserves and therefore to withstand unpredictable fasting periods.

Do current environmental conditions explain physiological and metabolic responses of subterranean crustaceans to cold?

Subterranean environments are characterized by the quasi absence of thermal variations (+/-1 degrees C within a year), and organisms living in these biotopes for several millions of years, such as hypogean crustaceans, can be expected to have adapted to this very stable habitat. As hypogean organisms experience minimal thermal variation in their native biotopes, they should not be able to develop any particular cold adaptations to cope with thermal fluctuations. Indeed, physiological responses of organisms to an environmental stress are proportional to the amplitude of the stress they endure in their habitats. Surprisingly, previous studies have shown that a population of an aquatic hypogean crustacean, Niphargus rhenorhodanensis, exhibited a high level of cold hardiness. Subterranean environments thus appeared not to be following the classical above-mentioned theory. To confirm this counter-example, we studied seven karstic populations of N. rhenorhodanensis living in aquifers at approximately 10 degrees C all year round and we analysed their behavioural, metabolic and biochemical responses during cold exposure (3 degrees C). These seven populations showed reduced activities, and some cryoprotective molecules were accumulated. More surprisingly, the amplitude of the response varied greatly among the seven populations, despite their exposure to similar thermal conditions. Thus, the overall relationship that can be established between the amplitude of thermal variations and cold-hardiness abilities of ectotherm species may be more complex in subterranean crustaceans than in other arthropods.

High anoxia tolerance in the subterranean salamander Proteus anguinus without oxidative stress nor activation of antioxidant defenses during reoxygenation.

The present study describes a high anoxia tolerance in an amphibian at high temperature. Indeed, the subterranean salamander Proteus anguinus survived 12 h under anoxia at 12 degrees C. Surprisingly, such experimental conditions did not affect P. anguinus oxidative status while muscles and liver antioxidant enzymes activities decreased under 8 h anoxia and only return to basal level during reoxygenation. To test if such adaptation is common in Urodels, equivalent experimentations have been conducted on another newt: the stream-dwelling Calotriton asper. This latter species exhibited only 1.5 h survival under anoxia in spite of higher antioxidant enzymes activities than P. anguinus. Furthermore, aerobic recovery after 1 h anoxia induced a 30% increase of oxidative damage partly explained by SOD and CAT activities that did not return to control values during reoxygenation, demonstrating a lower capacity to counteract ROS overproduction than P. anguinus. In addition, uncoupling protein (UCP) transcript was for the first time detected, partly sequenced and quantified in amphibian muscles and liver. UCP may be considered as a ROS production attenuator by mediating a discharge of the proton gradient generated by the respiratory chain. The putative role of UCP in post-anoxic oxidative status of both species is discussed.

Freezing or supercooling: how does an aquatic subterranean crustacean survive exposures at subzero temperatures?

Crystallization temperature (T(c)), resistance to inoculative freezing (IF), ice contents, bound water, protein and glycogen body contents were measured in the aquatic subterranean crustacean Niphargus rhenorhodanensis and in the morphologically close surface-dwelling aquatic crustacean Gammarus fossarum, both acclimated at 12 degrees C, 3 degrees C and -2 degrees C. Cold acclimation induced an increase in the T(c) values in both species but no survival was observed after thawing. However, after inoculation at high sub-zero temperatures, cold-acclimated N. rhenorhodanensis survived whereas all others, including the 3 degrees C and -2 degrees C acclimated G. fossarum died. In its aquatic environment, N. rhenorhodanensis is likely to encounter inoculative freezing before reaching the T(c) and IF tolerance appears as a highly adaptive trait in this species. Bound water and glycogen were found to increase in the 3 degrees C and -2 degrees C acclimated N. rhenorhodanensis, whereas no variation was observed in G. fossarum. Considering the hydrophilic properties of glycogen, such a rise may be correlated with the increased bound water measured in cold-acclimated N. rhenorhodanensis, and may be linked to the survival of this species when it was inoculated. The ecological significance of the survival of the aquatic subterranean crustacean to inoculative freezing is paradoxical, as temperature is currently highly buffered in its habitat. However, we assume that past geographical distribution and resulting life history traits of N. rhenorhodanensis are key parameters in the current cold-hardiness of the species.

Metabolic responses to cold in subterranean crustaceans.

Changes in polyol, sugar and free amino acid (FAA) body contents were investigated in the aquatic, subterranean (i.e. hypogean) crustaceans Niphargus rhenorhodanensis and Niphargus virei and in a morphologically close aquatic, surface-dwelling (i.e. epigean) crustacean Gammarus fossarum acclimated to 12 degrees C, 3 degrees C and -2 degrees C. With decreasing temperature, G. fossarum significantly increased its alanine and glutamine levels, while trehalose body content was found to increase above control levels only at -2 degrees C. N. virei showed moderate increases of alanine and glycine, and no change in trehalose level was observed in this species. By contrast, N. rhenorhodanensis was the only species showing a significant rise in its total FAA pool, mainly explained by alanine, glycine, arginine and glutamine accumulations. This species also gradually increased its trehalose body content with decreasing temperature. Several cold-hardy ectotherms show metabolic responses to cold that are identical to those observed in N. rhenorhodanensis. A previous comparative study showed that the hypogean N. rhenorhodanensis exhibited a survival time (Lt50) at -2 degrees C that was 26.3 times and 2.6 times higher than the hypogean N. virei and the epigean G. fossarum, respectively. Thus, crustacean levels of FAA and trehalose were correlated with their respective cold tolerances. Such differences in metabolic responses to cold in both hypogean organisms were unexpected since they both live in thermally buffered biotopes. Considering the current distribution areas of the two subterranean crustaceans studied, we assume that the cold hardiness found in the hypogean N. rhenorhodanensis could be correlated with its biogeography history during the quaternary glaciations.

Behavioural, ventilatory and respiratory responses of epigean and hypogean crustaceans to different temperatures.

Impact of temperature (from -2 to 28 degrees C) on survival, oxygen consumption, locomotory and ventilatory activities was measured in two aquatic subterranean crustaceans (Niphargus rhenorhodanensis and Niphargus virei) and in a morphologically close surface-dwelling crustacean (Gammarus fossarum). The hypogean N. virei presented all characteristics of a stenothermal organism: it showed small thermal plasticity and optimised its performance on a narrow range of temperature. In contrast, the epigean G. fossarum and more surprisingly the hypogean N. rhenorodanensis can be both characterized as eurythermal organisms: they exhibited important survival times and conserved their performance optimum throughout a large range of temperature. Such differences of survival and performance patterns in two hypogean organisms were unexpected since they both live in very thermally buffered biotopes. Our data suggest fresh hypotheses about the role of glaciations in the history and adaptation of hypogean crustaceans.

Long-term fasting and realimentation in hypogean and epigean isopods: a proposed adaptive strategy for groundwater organisms.

The effects of long-term fasting and subsequent refeeding on digestive physiology and energy metabolism were investigated in a subterranean aquatic crustacean, Stenasellus virei, and in a morphologically similar surface-dwelling species, Asellus aquaticus. Metabolic response to food deprivation was monophasic in A. aquaticus, with an immediate, large decrease in all energy reserves. In contrast, S. virei displayed three successive periods of phosphageno-glucidic, lipidic and, finally, proteo-lipidic-dominant catabolism over the course of the nutritional stress. To represent the responses of subterranean crustaceans to food stress and renutrition, a sequential energy strategy was hypothesized, suggesting that four successive phases (called stress, transition, adaptation and recovery) can be distinguished. Based on these results, a general adaptive strategy for groundwater organisms was proposed. Their remarkable resistance to long-term fasting may be partly explained by (1) a depressed metabolism, during which they mainly subsist on lipid stores, (2) a prolonged state of glycogen- and protein-sparing, (3) low energetic requirements and (4) large body stores. In addition, these groundwater species displayed high recovery abilities during refeeding, showing an optimal utilization of available food and a rapid restoration of their body reserves. These adaptive responses might be considered for numerous subterranean organisms as an efficient energy-saving strategy in a harsh and unpredictable environment where fasting (and/or hypoxic) periods of variable duration alternate with sporadic feeding events (and/or normoxic periods). Therefore, food-limited and/or hypoxia-tolerant groundwater species appear to be good examples of animals representing a low-energy system.