DOI: 10.5281/zenodo.10284220
title: Data on heat tolerance from: Field and laboratory studies reveal interacting effects of stream oxygenation and warming on aquatic ectotherms

description: Heat tolerance data on two widespread Eurasian mayflies, Ephemera danica, Müller 1764 and Serratella ignita (Poda 1761).
Methods thermal tolerance experiments
Mayfly nymphs for laboratory experiments were collected in spring (early May) from Torrington River, Devon, UK, ranging in fresh weight between 15 and 128 mg (E. danica) and between 2.0 and 11.6 mg (S. ignita). Nymphs were maintained in the laboratory at 10 ± 1 °C in a 12 L:12 D regime in aquaria containing artificial pond water, buffered and diluted to reflect the pH and conductivity of the field site. Before recording critical temperatures, all species were acclimated for at least 7 days to laboratory conditions.
To assess critical thermal maxima (CTmax), we employed previously described methods (Verberk & Bilton, 2011; Verberk & Calosi, 2012). Individual nymphs (n = 18 for E. danica and n = 27 for S. ignita) were placed in flow-through chambers and water was supplied to these chambers from a header tank after having passed through a tubular counter-current heat exchanger. Water in the header tank was of the same composition as that used to maintain animals and was bubbled with a mixture of 20% oxygen and 80% nitrogen, obtained using a gas-mixing pump (Wösthoff, Bochum, Germany). Individuals were left resting for 1 h at the equilibration temperature of 10 °C, after which temperature in the experimental chambers was increased by 0.25 °C min−1, using a Grant R5 water bath with a GP200 pump unit (Grant Instrument Ltd, Cambridge, UK), connected to the heat exchanger. Temperatures were logged using a HH806AU digital thermometer (Omega Engineering Inc., Stamford, CT, USA). Different sized flow-through chambers were used for each species. E. danica was placed in larger chambers (70 × 70 × 30 mm) and provided with sand as burrowing substrate, which they readily used. S. ignita was placed in smaller cylindrical chambers (6 mm in diameter, 20 mm long) and their behaviour was observed under a magnifying glass. The amount of water passing through these flow-through chambers was matched to their size. For the larger chambers containing E. danica, water was supplied to five chambers (total volume of 0.735 l) at a flow rates of 0.031–0.033 l per second, resulting in a refresh rate of 22–24 s. For the smaller chambers with S. ignita, water was supplied to each chamber individually at 0.21–0.22 ml per second, resulting in a refresh rate of 10–11 s.
CTmax is defined as the point at which an animal loses its ability to escape from conditions that will lead to its death (Lutterschmidt & Hutchison, 1997). During progressive warming, nymphs of E. danica first emerged from their burrowed position and began swimming (at about 6 °C below CTmax). Loss of equilibrium occurred next as nymphs fell upon their backs, which was followed by the onset of spasms. After that, gill movement was no longer coordinated and faltered and this endpoint could be most reliably determined and is here taken as CTmax. Similarly, S. ignita stopped ventilation and movement at CTmax. Below CTmax, larvae were inactive, until near the end of the trials, when they began to crawl, lose equilibrium and gill beating became intermittent shortly before stopping altogether at CTmax.
CTmax was assessed at hypoxic (5 kPa), normoxic (20 kPa) and hyperoxic (60 kPa) conditions. Different levels of oxygenation were achieved by changing the oxygen–nitrogen gas mixture obtained using the gas-mixing pump (Wösthoff). The gas mixture was adjusted 10 min after placing the animals in the small flow-through chambers, to allow for gradual exposure to hypoxic and hyperoxic conditions during the 1 h resting period. To prevent equilibration with the atmosphere, the header tank was sealed using an 18 mm thick expanded polystyrene sheeting and other openings were closed off with plastic material. During the 1 h resting period, oxygen levels in the outflow water from the chambers were measured approximately every 15 min, to verify that the oxygen levels had stabilized to hypoxic, normoxic and hyperoxic conditions at the onset of warming. Because some equilibration with the atmosphere could not be prevented, nominal output values from the gas mixer were slightly more extreme (3 kPa for hypoxia and 65 kPa for hyperoxia) in order to achieve the desired oxygen conditions in the test chambers.