A method for studying the metabolic activity of individual tardigrades by measuring oxygen uptake using microrespirometry.

Studies of tardigrade biology have been severely limited by the sparsity of appropriate quantitative techniques, informative on a single-organism level. Therefore, many studies rely on motility-based survival scoring and quantifying reproductive success. Measurements of O2 respiration rates, as an integrating expression of the metabolic activity of single tardigrades, would provide a more comprehensive insight into how an individual tardigrade is responding to specific environmental factors or changes in life stages. Here, we present and validate a new method for determining the O2 respiration rate (nmol O2 mg-1 h-1) of single tardigrades under steady state, using O2 microsensors. As an example, we show that the O2 respiration rate determined in MilliQ water for individuals of Richtersius coronifer and of Macrobiotus macrocalix at 22°C was 10.8±1.84 and 13.1±2.19 nmol O2 mg-1 h-1, respectively.

Respiration Measurements of Individual Tardigrades of the Species Richtersius cf coronifer as a Function of Temperature and Salinity and Termination of Anhydrobiosis.

Numerous studies have demonstrated that tardigrades in a resting state (tun state) are very resistant to exceptional stress levels in comparison with the resistance observed in multicellular organisms in general. The types of stress include desiccation and radiation, which are also relevant in astrobiological research, and therefore, tardigrades are used as multicellular model organisms. For example, tardigrades have been investigated in the TARSE, TARDIS, RoTaRad, and TARDIKISS projects; their survival has been evaluated according to stressful conditions that prevail in low earth orbit, including the effects of cosmic radiation and microgravity. Despite this interest, the study of tardigrade biology has been severely hampered by the sparsity of appropriate quantitative techniques that inform at the single-organism level. In this study, we present results on mass-specific respiration rates as a function of termination of anhydrobiosis and variations in temperature and salinity, including Mars-analog perchlorate solutions, by using microsensor technology to measure respiration. Based on our results for Richtersius cf coronifer, we estimated the activation energy (50.8 kJ/mole O2) for its metabolism as well as Q10 for selected temperature intervals. Q10 was constant-∼1.5-between 2°C and 33°C, except for the interval 11-16°C, where Q10 was 5.5. The steady-state mass-specific respiration rate of individuals of Richtersius cf coronifer increased with increasing salinity below the lethal limit, likely representing the energy requirements of its osmoregulatory response. We report the first quantitative data of a tardigrade's metabolic dynamics during the termination of anhydrobiosis, revealing significant variation between individuals. However, we observed a general trend, that is, a high initial metabolic rate after exposure to water. Our approach would allow us to carry out quantitative physiological studies of tardigrades on board of the International Space Station, and thus significantly extend the possibility of studying the response of multicellular organisms in space. Summary statement This article reports on first measurements of mass-specific respiration rates of individual tardigrades of the species Richtersius cf coronifer during termination of anhydrobiosis as well as measurements of the impact of temperature and salinity on oxygen uptake rates.

High-throughput dilution-based growth method enables time-resolved exo-metabolomics of Pseudomonas putida and Pseudomonas aeruginosa.

Understanding metabolism is fundamental to access and harness bacterial physiology. In most bacteria, nutrient utilization is hierarchically optimized according to their energetic potential and their availability in the environment to maximise growth rates. Low-throughput methods have been largely used to characterize bacterial metabolic profiles. However, in-depth analysis of large collections of strains across several conditions is challenging since high-throughput approaches are still limited - especially for non-traditional hosts. Here, we developed a high-throughput dilution-resolved cultivation method for metabolic footprinting of Pseudomonas putida and Pseudomonas aeruginosa. This method was benchmarked against a conventional low-throughput time-resolved cultivation approach using either a synthetic culture medium (where a single carbon source is present) for P. putida or a complex nutrient mixture for P. aeruginosa. Dynamic metabolic footprinting, either by sugar quantification or by targeted exo-metabolomic analyses, revealed overlaps between the bacterial metabolic profiles irrespective of the cultivation strategy, suggesting a certain level of robustness and flexibility of the high-throughput dilution-resolved method. Cultivation of P. putida in microtiter plates imposed a metabolic constraint, dependent on oxygen availability, which altered the pattern of secreted metabolites at the level of sugar oxidation. Deep-well plates, however, constituted an optimal cultivation set-up yielding consistent and comparable metabolic profiles across conditions and strains. Altogether, the results illustrate the usefulness of this technological advance for high-throughput analyses of bacterial metabolism for both biotechnological applications and automation purposes.