FishResp: R package and GUI application for analysis of aquatic respirometry data.
ABSTRACT: Intermittent-flow respirometry is widely used to measure oxygen uptake rates and subsequently estimate aerobic metabolic rates of aquatic animals. However, the lack of a standard quality-control software to detect technical problems represents a potential impediment to comparisons across studies in the field of evolutionary and conservation physiology. Here, we introduce 'FishResp', a versatile R package and its graphical implementation for quality-control and filtering of raw respirometry data. Our goal is to provide a straightforward, cross-platform and free software to help improve the quality and comparability of metabolic rate estimates for reducing methodological fragmentation in the field of aquatic respirometry. FishResp accepts data from various respirometry systems, allows users to detect potential mechanical problems which can occur during oxygen uptake measurements (e.g. chamber leaking, poor water circulation), and offers six options to correct raw data for microbial oxygen consumption. The software performs filtering of raw data based on user criteria, and produces accurate and unbiased estimates of absolute and mass-specific metabolic rates. Using data from three-spined sticklebacks (Gasterosteus aculeatus) and Trinidadian guppies (Poecilia reticulata), we demonstrate the virtues of FishResp, highlighting the importance of detecting mechanical problems and correcting measurements for background respiration.
Project description:Interest in the measurement of metabolic rates is growing rapidly, because of the importance of metabolism in advancing our understanding of organismal physiology, behaviour, evolution and responses to environmental change. The study of metabolism in aquatic animals is undergoing an especially pronounced expansion, with more researchers utilising intermittent-flow respirometry as a research tool than ever before. Aquatic respirometry measures the rate of oxygen uptake as a proxy for metabolic rate, and the intermittent-flow technique has numerous strengths for use with aquatic animals, allowing metabolic rate to be repeatedly estimated on individual animals over several hours or days and during exposure to various conditions or stimuli. There are, however, no published guidelines for the reporting of methodological details when using this method. Here, we provide the first guidelines for reporting intermittent-flow respirometry methods, in the form of a checklist of criteria that we consider to be the minimum required for the interpretation, evaluation and replication of experiments using intermittent-flow respirometry. Furthermore, using a survey of the existing literature, we show that there has been incomplete and inconsistent reporting of methods for intermittent-flow respirometry over the past few decades. Use of the provided checklist of required criteria by researchers when publishing their work should increase consistency of the reporting of methods for studies that use intermittent-flow respirometry. With the steep increase in studies using intermittent-flow respirometry, now is the ideal time to standardise reporting of methods, so that - in the future - data can be properly assessed by other scientists and conservationists.
Project description:Respirometry is frequently used to estimate metabolic rates and examine organismal responses to environmental change. Although a range of methodologies exists, it remains unclear whether differences in chamber design and exercise (type and duration) produce comparable results within individuals and whether the most appropriate method differs across taxa. We used a repeated-measures design to compare estimates of maximal and standard metabolic rates (MMR and SMR) in four coral reef fish species using the following three methods: (i) prolonged swimming in a traditional swimming respirometer; (ii) short-duration exhaustive chase with air exposure followed by resting respirometry; and (iii) short-duration exhaustive swimming in a circular chamber. We chose species that are steady/prolonged swimmers, using either a body-caudal fin or a median-paired fin swimming mode during routine swimming. Individual MMR estimates differed significantly depending on the method used. Swimming respirometry consistently provided the best (i.e. highest) estimate of MMR in all four species irrespective of swimming mode. Both short-duration protocols (exhaustive chase and swimming in a circular chamber) produced similar MMR estimates, which were up to 38% lower than those obtained during prolonged swimming. Furthermore, underestimates were not consistent across swimming modes or species, indicating that a general correction factor cannot be used. However, SMR estimates (upon recovery from both of the exhausting swimming methods) were consistent across both short-duration methods. Given the increasing use of metabolic data to assess organismal responses to environmental stressors, we recommend carefully considering respirometry protocols before experimentation. Specifically, results should not readily be compared across methods; discrepancies could result in misinterpretation of MMR and aerobic scope.
Project description:Oxygen consumption is a key metric of metabolism in aerobic organisms. Current respirometric methods led to seminal discoveries despite limitations such as high sample demand, exchange with atmospheric O2, and cumulative titration protocols leading to limited choice of useable tissue, complex data interpretation, and restricted experimental design. We developed a sensitive and customizable method of measuring O2 consumption rates by a variety of biological samples in microliter volumes without interference from the aerobic environment. We demonstrate that O2 permeability of the photopolymer, VeroClear, is comparable to that of polyetheretherketone (0.125 vs. 0.143 barrer, respectively) providing an efficient barrier to oxygen ingress. Optical transparency of VeroClear, combined with high resolution 3D printing, allows for optode-based oxygen detection in enclosed samples. These properties yield a microrespirometer with over 100× dynamic range for O2 consumption rates. Importantly, the enclosed respirometer configuration and very low oxygen permeability of materials makes it suitable, with resin pre-conditioning, for quantitative assessment of O2 consumption rates at any desired [O2], including hyperbaric, physiological or hypoxic conditions as necessary for each cell type. We characterized two configurations to study soluble enzymes, isolated mitochondria, cells in suspension, and adherent cells cultured on-chip. Improved sensitivity allows for routine quantitative detection of respiration by as few as several hundred cells. Specific activity of cell suspensions in the microrespirometer was in close agreement with that obtained by high-resolution polarographic respirometry. Adherent cell protocols allowed for physiologically relevant assessment of respiration in retinal pigment epithelial cells, ARPE-19, which displayed lower metabolic rates compared with those in suspension. By exchanging medium composition, we demonstrate that cells can be transiently inhibited by cyanide and that 99.6% of basal O2 uptake is recovered upon its removal. This approach is amenable to new experimental designs and precision measurements on limited sample quantities across basic research and applied fields.
Project description:Flow-through respirometry systems provide accurate measurement of gas exchange over long periods of time. However, these systems have limitations in tracking rapid changes. When an animal infuses a metabolic gas into the respirometry chamber in a short burst, diffusion and airflow in the chamber gradually alter the original signal before it arrives at the gas analyzer. For single or multiple bursts, the recorded signal is smeared or mixed, which may result in dramatically altered recordings compared to the emitted signal. Recovering the original metabolic signal is a difficult task because of the inherent ill conditioning problem. Here, we present two new methods to recover the fast dynamics of metabolic patterns from recorded data. We first re-derive the equations of the well-known Z-transform method (ZT method) to show the source of imprecision in this method. Then, we develop a new model of analysis for respirometry systems based on the experimentally determined impulse response, which is the response of the system to a very short unit input. As a result, we present a major modification of the ZT method (dubbed the 'EZT method') by using a new model for the impulse response, enhancing its precision to recover the true metabolic signals. The second method, the generalized Z-transform (GZT) method, was then developed by generalizing the EZT method; it can be applied to any flow-through respirometry system with any arbitrary impulse response. Experiments verified that the accuracy of recovering the true metabolic signals is significantly improved by the new methods. These new methods can be used more broadly for input estimation in variety of physiological systems.
Project description:Platelet activation plays a key role in normal haemostasis and pathological thrombosis. Platelet activation is rapid; within minutes of stimulation, platelets generate bioactive phospholipids, secrete their granule contents, activate integrins and aggregate together to form a haemostatic plug. These events are dependent on ATP synthesis. Mitochondrial function in platelets from healthy volunteers and patients with a range of diseases indicate an important role for oxygen consumption in oxidative phosphorylation in normal and pathological function. Platelets also consume oxygen during oxidation reactions, such as cyclooxygenase-dependent thromboxane A<sub>2</sub> synthesis. In this study, we used high-resolution respirometry to investigate rapid changes in oxygen consumption during platelet activation. We demonstrated a rapid, transient increase in oxygen consumption rate within minutes of platelet stimulation by the physiological activator, thrombin. This was partly inhibited by aspirin and by oligomycin. This shows that high resolution respirometry can provide information regarding rapid and dynamic changes in oxygen consumption during platelet activation.
Project description:Oxygen consumption allows measuring the metabolic activity of organisms. Here, we adopted the multi-well plate-based respirometry of the extracellular flux analyzer (Seahorse XF96) to investigate the effect of temperature on the bioenergetics of zebrafish embryos (<i>Danio rerio</i>) <i>in situ</i>. We show that the removal of the embryonic chorion is beneficial for oxygen consumption rates (OCR) and penetration of various mitochondrial inhibitors, and confirm that sedation reduces the variability of OCR. At 48h post-fertilization, embryos (maintained at a routine temperature of 28°C) were exposed to different medium temperatures ranging from 18°C to 37°C for 20h prior OCR measurement. Measurement temperatures from 18°C to 45°C in the XF96 were achieved by lowering the room temperature and active in-built heating. At 18°C assay temperature, basal OCR was low due to decreased ATP-linked respiration, which was not limited by mitochondrial power, as seen in substantial spare respiratory capacity. Basal OCR of the embryos increased with assay temperature and were stable up to 37°C assay temperature, with pre-exposure of 37°C resulting in more thermo-resistant basal OCR measured at 41°C. Adverse effects of the mitochondrial inhibitor oligomycin were seen at 37°C and chemical uncouplers disrupted substrate oxidation gradually with increasing assay temperature. Proton leak respiration increased at assay temperatures above 28°C and compromised the efficiency of ATP production, calculated as coupling efficiency. Thus, temperature impacts mitochondrial respiration by reduced cellular ATP turnover at lower temperatures and by increased proton leak at higher temperatures. This conclusion is coherent with the assessment of heart rate, an independent indicator of systemic metabolic rate, which increased with exposure temperature, peaking at 28°C, and decreased at higher temperatures. Collectively, plate-based respirometry allows assessing distinct parts of mitochondrial energy transduction in zebrafish embryos and investigating the effect of temperature and temperature acclimation on mitochondrial bioenergetics <i>in situ</i>.
Project description:MPP+ is the active metabolite of MPTP, a molecule structurally similar to the herbicide Paraquat, known to injure the dopaminergic neurons of the nigrostriatal system in Parkinson's disease models. Within the cells, MPP+ accumulates in mitochondria where it inhibits complex I of the electron transport chain, resulting in ATP depletion and neuronal impairment/death. So far, MPP+ is recognized as a valuable tool to mimic dopaminergic degeneration in various cell lines. However, despite a large number of studies, a detailed characterization of mitochondrial respiration in neuronal cells upon MPP+ treatment is still missing. By using high-resolution respirometry, we deeply investigated oxygen consumption related to each respiratory state in differentiated neuroblastoma cells exposed to the neurotoxin. Our results indicated the presence of extended mitochondrial damage at the inner membrane level, supported by increased LEAK respiration, and a drastic drop in oxygen flow devoted to ADP phosphorylation in respirometry measurements. Furthermore, prior to complex I inhibition, an enhancement of complex II activity was observed, suggesting the occurrence of some compensatory effect. Overall our findings provide a mechanistic insight on the mitochondrial toxicity mediated by MPP+, relevant for the standardization of studies that employ this neurotoxin as a disease model.
Project description:Respirometry using modified cell culture microplates offers an increase in throughput and a decrease in biological material required for each assay. Plate based respirometers are susceptible to a range of diffusion phenomena; as O(2) is consumed by the specimen, atmospheric O(2) leaks into the measurement volume. Oxygen also dissolves in and diffuses passively through the polystyrene commonly used as a microplate material. Consequently the walls of such respirometer chambers are not just permeable to O(2) but also store substantial amounts of gas. O(2) flux between the walls and the measurement volume biases the measured oxygen consumption rate depending on the actual [O(2)] gradient. We describe a compartment model-based correction algorithm to deconvolute the biological oxygen consumption rate from the measured [O(2)]. We optimize the algorithm to work with the Seahorse XF24 extracellular flux analyzer. The correction algorithm is biologically validated using mouse cortical synaptosomes and liver mitochondria attached to XF24 V7 cell culture microplates, and by comparison to classical Clark electrode oxygraph measurements. The algorithm increases the useful range of oxygen consumption rates, the temporal resolution, and durations of measurements. The algorithm is presented in a general format and is therefore applicable to other respirometer systems.
Project description:Resting metabolic rate (RMR) studies frequently involve genetically-manipulated mice and high fat diets (HFD). We hypothesize that the use of inadequate methods impedes the identification of novel regulators of RMR. This idea was tested by simultaneously measuring RMR by direct calorimetry and respirometry in C57BL/6J mice fed chow, 45% HFD, and then returned to chow. Comparing results during chow feeding uncovered an underestimation of RMR by respirometry (0.010 ± 0.001 kcal/h, P < 0.05), which is equivalent in magnitude to ?2% of total daily caloric turnover. RMR during 45% HFD feeding was increased by respirometry (+0.013 ± 0.003 kcal/h, P < 0.05), but not direct calorimetry (+0.001 ± 0.002 kcal/h). Both methods indicated that return to chow reduced RMR compared to HFD, though direct calorimetry indicated a reduction below the initial chow fed state (-0.019 ± 0.004 kcal/h versus baseline, P < 0.05) that was not detected by respirometry (-0.003 ± 0.002 kcal/h versus baseline). These results highlight method-specific interpretations of the effects of dietary interventions upon RMR in mice, and prompt the reevaluation of preclinical screening methods used to identify novel RMR modulators.
Project description:The human microbiota composition is associated with a number of diseases including obesity, inflammatory bowel disease, and bacterial vaginosis. Thus, microbiome research has the potential to reshape clinical and therapeutic approaches. However, raw microbiome count data require careful pre-processing steps that take into account both the sparsity of counts and the large number of taxa that are being measured. Filtering is defined as removing taxa that are present in a small number of samples and have small counts in the samples where they are observed. Despite progress in the number and quality of filtering approaches, there is no consensus on filtering standards and quality assessment. This can adversely affect downstream analyses and reproducibility of results across platforms and software. We introduce PERFect, a novel permutation filtering approach designed to address two unsolved problems in microbiome data processing: (i) define and quantify loss due to filtering by implementing thresholds and (ii) introduce and evaluate a permutation test for filtering loss to provide a measure of excessive filtering. Methods are assessed on three "mock experiment" data sets, where the true taxa compositions are known, and are applied to two publicly available real microbiome data sets. The method correctly removes contaminant taxa in "mock" data sets, quantifies and visualizes the corresponding filtering loss, providing a uniform data-driven filtering criteria for real microbiome data sets. In real data analyses PERFect tends to remove more taxa than existing approaches; this likely happens because the method is based on an explicit loss function, uses statistically principled testing, and takes into account correlation between taxa. The PERFect software is freely available at https://github.com/katiasmirn/PERFect.