A novel technique for the precise measurement of CO2 production rate in small aquatic organisms as validated on Aeshnid dragonfly nymphs

Abstract

The present study describes and validates a novel yet simple system for simultaneous in vivo measurements of rates of aquatic CO 2 production (Ṁ CO2 ) and oxygen consumption (Ṁ O2 ), thus allowing the calculation of respiratory exchange ratios (RER). Diffusion of CO 2 from the aquatic phase into a gas phase, across a hollow fibre membrane, enabled aquatic Ṁ CO2 measurements with a highprecision infrared gas CO 2 analyser. Ṁ O2 was measured with a P O2 optode using a stop-flow approach. Injections of known amounts of CO 2 into the apparatus yielded accurate and highly reproducible measurements of CO 2 content (R 2 =0.997, P<0.001). The viability of in vivo measurements was demonstrated on aquatic dragonfly nymphs (Aeshnidae; wet mass 2.17 mg-1.46 g, n=15) and the apparatus produced precise Ṁ CO2 (R 2 =0.967, P<0.001) and Ṁ O2 (R 2 =0.957, P<0.001) measurements; average RER was 0.73± 0.06. The described system is scalable, offering great potential for the study of a wide range of aquatic species, including fish.

Metabolic fuel use after feeding in the zebrafish (Danio rerio): a respirometric analysis

“…This approach allowed us to quantify how much food the group ate, and thus we estimated the individual feed intake by dividing the total ingested food mass (g) by three. Immediately after feeding stopped, one fish was randomly captured and transferred to a modified glass scintillation vial (22.5 ml), which was part of the novel respirometry system developed by Harter et al (2017). Fasting fish (N=6) were treated identically to feeding fish (N=6), with the same feeding process being simulated, but with no food added to the water.…”

“…The respirometer (see detailed description and diagrams in Harter et al, 2017) allows simultaneous measurements of ṀC O2 and ṀO 2 , and water sampling for measurements of ṀN in small aquatic organisms. The novel aspect of this apparatus is the use of a hollow fibre membrane (HFM) as the interface between the aqueous phase and a gas phase, providing a large ratio of gas exchange surface area to system volume.…”

“…ṀO 2 and ṀC O2 were recorded using a PowerLab 4/35 data acquisition unit (ADInstruments, Dunedin, New Zealand) and LabChart v8.1.5 software. Calibration of the system for P O2 and [CO 2 ] was performed exactly as described by Harter et al (2017). Ammonia-N (total of NH 3 and NH 4 + ) and urea-N were measured by the colorimetric assays described by Verdouw et al (1978) and Rahmatullah and Boyde (1980), respectively.…”

“…However, a recently published article has described a new apparatus which allows easy and accurate measurement of total CO 2 , directly from a small volume of water. This method for ṀC O2 determination, developed by Harter et al (2017), was combined with ṀO 2 measurements to accurately calculate respiratory quotients in dragonfly nymphs. The apparatus, described below, employs a hollow fibre membrane for CO 2 collection into a gas phase, coupled to CO 2 measurement by an infrared analyser.…”

“…Our objective was to apply the fuel use calculations for instantaneous quantification of substrate usage of Lauff and Wood (1996a) in conjunction with the simultaneous ṀO 2 /ṀC O2 respirometry system of Harter et al (2017) to calculate, for the first time, the energy substrates used during the SDA of a fish: D. rerio. We tested the hypothesis that lipid and carbohydrate are the major fuels used during fasting, whereas endogenous carbohydrate, which can be readily mobilized, becomes more important early in the absorptive stage when SDA is already occurring, but the nutrients from food have not yet fully reached the bloodstream.…”