For each female, eggs were then gently poured into a Petri dish c

For each female, eggs were then gently poured into a Petri dish containing a small volume of RNAlater, and forceps cleaned with RNase AWAY (Molecular BioProducts, San Diego, CA) and sterile transfer pipettes were used to carefully transfer 3 sets of 25 eggs to RNase-free 1.5 mL tubes. The RNAlater was then removed by pipette, and the eggs were stored at − 80 °C until RNA extraction. Controlled/timed egg fertilizations were conducted as follows. Eggs were transferred from plastic collection beakers into 1.5 L graduated glass “fertilization beakers” by gentle pouring, and sperm (2 mL selleck compound sperm per 100 mL of eggs) was added using a plastic transfer pipette (note: each of the 15 females involved in the study

was represented by a separate 1.5 L fertilization beaker). The egg and sperm mixture was gently stirred using the pipette, 100 mL of UV-treated filtered seawater was added, and the mixture was again stirred. After incubating for 1 minute, 500 mL of UV-treated filtered seawater was added and the mixture incubated for an additional 5 minutes. Each fertilization beaker was then filled to 1.4 L with UV-treated filtered seawater, placed in a walk-in cold room at 6 °C, and left undisturbed until 7 hours post-fertilization (hpf) (~ 2-cell stage). Prior to the distribution of eggs from each female into incubation beakers at 7 hpf, a subsample of eggs was placed into a Petri

dish and photographed using a dissecting microscope and video camera. These images were transferred into ImageJ (http://imagej.nih.gov/ij), and the diameter of a number of eggs per female (approx. 15–30) was measured relative to a 2 mm GSK-3 inhibitor micrometer that was included in the image. At 7 hpf, ID-8 a sterile pipette was used to transfer approximately 0.25 mL of floating (fertilized) eggs from each fertilization beaker into each of three 1.5 mL RNase-free tubes. Seawater was removed by pipette, and the samples were flash-frozen

in liquid nitrogen and stored at − 80 °C until RNA extraction. In addition, sixty 600 mL beakers containing 500 mL of UV-treated filtered seawater were each stocked with ~ 1000 fertilized eggs (4 replicate beakers per female). Total percent fertilization (i.e. floating volume) was also determined at this time for each of the 1.5 L fertilization beakers. The number of eggs was determined by collecting 200 μL of eggs using a wide bore pipette, counting the eggs, and then extrapolating to the volume required for 1000 eggs; this was performed twice and averaged for each female. Replicate “incubation beakers” (4 per female) were randomly placed on the bench top of a walk-in cold room (~ 6 °C), whose fluorescent lights and reflective metal surfaces were covered with shade cloth and black garbage bags to achieve a light intensity range of 107–179 LUX at the top of the beakers. Water temperature was maintained at 6.2–6.4 °C until 100% hatch (i.e. for 17 days).

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