Plate‐Based Respirometry to Assess Zebrafish Embryo Bioenergetics in situ in Changing Environmental Conditions

Abstract

All species require bioenergetic adaptation to maintain life functions, especially in changing environmental conditions. Global warming and deteriorating water quality may pose threats to aquatic species by interfering with bioenergetic mechanisms. Mitochondrial oxidative metabolism is a key component for bioenergetic responses as it provides the major proportion of cellular energy in the form of adenosine triphosphate (ATP). In our studies, we aim to establish the assessment of zebrafish mitochondrial bioenergetics and investigate the mechanisms, adaptation, and plasticity to temperature, pollutants, and the combination of both to gain insights into the upcoming threats for their energy homeostasis and survival. Using plate‐based respirometry of the Seahorse XF96 analyzer, we measured zebrafish (Danio rerio) embryo oxygen consumption rates in situ. To understand how temperature alters oxidative phosphorylation, we exposed 48 hours post‐fertilization zebrafish embryos to five different water temperatures before measuring oxygen consumption at various assay temperatures. The oxygen consumption rates were challenged using respiratory chain inhibitors and chemical uncouplers to reveal bioenergetic mechanisms and adjustments of mitochondrial efficiency. We show that at 18°C assay temperature, basal oxygen consumption of the embryo is low due to decreased ATP‐linked respiration and not limited by maximum substrate oxidation. Basal oxygen consumption rates increase with assay temperature and remain stable up to 37°C, before rapidly deteriorating at 41°C and above. The pre‐exposure of embryos to 37°C, however, expands the thermal window to higher limits, indicated by stable basal oxygen consumption rates at 41°C. Proton leak respiration, which wastes nutrient energy in parallel to ATP production, increases at assay temperatures above 28°C, thereby reducing the efficiency of ATP synthesis. This is seen as a decrease in coupling efficiency, which is defined as the fraction of oxygen consumption that drives ATP synthesis. Collectively, temperature affects the performance of zebrafish embryos by reducing cellular ATP turnover at lower temperatures and by increasing proton leak at higher temperatures, resulting in an optimum energy conversion temperature that can be shifted by thermal adaptation. In current experiments, we measure how pollutants impact zebrafish bioenergetics and its relation to thermal adaptation.