Sampling of yolk sac larvae

Embryos and larvae were collected using a 1 m long, 4 cm diameter open plastic tube inserted vertically into each aerated incubator. The top of the tube was sealed and a 250 mL sample was collected daily to determine the proportion of larvae to embryos during the period where both stages were observed simultaneously in the incubators. On days 28 and 50 in the warm and cold groups incubators, respectively, the sampling by tube method was repeated until 30 larvae were collected from each incubator. Larvae were subsequently sedated and photographed at 1.6× magnification. Total length (to the nearest 0.01 mm), yolk sac area (freeform traced from picture, in mm²), and presence of yolk sac edema (Table ^S4) were assessed from pictures for each individual larva using ImageJ⁹⁸.

Newly hatched yolk sac larvae were sampled from each incubator at 29–30 days for 2.8 °C and 47–48 days for 0.5 °C (n = 3/incubator; 12/treatment). Unsedated larvae were placed laterally on the left side in a watch glass with 500µL of seawater set on a stage thermally controlled by a circulating cooling bath calibrated to the respective temperature group at 2.8 °C or 0.5 °C. Each larva was video recorded for one minute at 4× magnification under a stereomicroscope. Heart rate was determined by counting heartbeats within a 30 s time window when the larvae was not moving using a manual counter. Arrhythmia, a measure of the irregularity in the heartbeats, was calculated using the number of frames between beats for a 20 s period and the standard deviation between the first seven beats recorded³⁶. All analysis was done blindly in reference to crude oil treatment and all recordings were analyzed by the same observer.