Using the syringe method, oxygen saturation was
recorded with an oxygen micro-optode sensor (needle
type, fibre-optic microsensor, flat broken tip, diameter:
140 lm) introduced through the top of the syringe by a
hole in the gasket. The optode was placed at a distance of
approximately 5 mm from the experimental egg clutch
and connected to a Microx TX2 (PreSens GmbH, Regensburg,
Germany). Oxygen consumption was recorded
in the eight micro-chambers using the electrode microrespiration
system, MRS-8 (Unisense, Aarhus, Denmark).
All trials were conducted during daytime and ended before
oxygen saturation dropped below 70 %, in both media, in
order to avoid exposure of the embryos to hypoxic
conditions.
Two-point calibration of both sensor types was conducted
with air-equilibrated (100 %) and oxygen-free
(0 %) sea water, the latter obtained with a solution of 1 %
sodium dithionite (Na2S2O4). For each test, small bunches
of eggs (100–200 for all species) were removed from
ovigerous females, and they were assigned to one of the
five embryo stages as described in Simoni et al. (2011):
stage I, yolk represents almost 100 % of the total volume;
stage II, gastrulation; stage III, appearance of the first
chromatophores and eye spots; stage IV, appearance of a
regular heartbeat and further developed eyes and stage V,
embryo filling almost the entire egg volume. Eggs from
each female were then placed into separate closed experimental
chambers. During the measurements, the respiration
chambers were submersed in a water bath at ambient
temperature; average temperature measured during the
experimental trials was 30.5 ± 2 C. In order to correct
for possible confounding factors of oxygen consumption
(e.g. bacterial respiration), controls with no eggs were
tested after each trial.