Data Set (knb-lter-sbc.86.10)

Data from: Competition among eggs shifts to cooperation along a sperm supply gradient in an external fertilizer

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These methods, instrumentation and/or protocols apply to all data in this dataset:

Protocols and/or Procedures
Description:

Laboratory experiments

I conducted eight laboratory trials of an experiment the purple sea urchin Strongylocentrotus purpuratus between February 23 and March 8, 2014 during their spawning season. Each trial included a factorially crossed gradient of four egg concentrations(approx 1, 0.25, 0.0625 and 0.0126 per microliter) with six sperm concentrations (approx 10^3, 10^4, 10^5, 10^6, 10^7 and 10^8 per microliter), using gametes from one male and one female per trial. The experiment took place in a climate-controlled room at 13 degrees C, and each experimental unit consisted of a 20 ml vial with 10 ml seawater. For each trial I first added 8 ml seawater to the 24 experimental vials, followed by 1 ml appropriate egg solution and finally providing each vial with 1 ml freshly diluted sperm solution. Vials were gently agitated by hand and placed on an orbital shaker table at approx 1.5 revolutions per second and left for 120 minutes to allow fertilization and first cleavage to occur. Following the 120 minute incubation period, vials were agitated by hand and emptied into shallow petri dishes for examination under an inverted compound microscope where 100 random, undamaged eggs were scored for fertilization, cleavage and polyspermy. Fertilization criteria included the presence of a raised vitelline membrane or cell division; cleavage criteria included only cells that divided normally (radial division) at least once; positive recognition of polyspermy required tetrahedral or further abnormal division of the cell. Very few fertilized eggs did not divide after 120 minutes and were scored as viable; as such, polyspermy may be underestimated in those few cases.

Urchins were collected in collaboration with the Santa Barbara Coastal Long Term Ecological Research (SBC LTER) program at a depth of approximately 7 m below mean low water at the Mohawk Reef (119.7296 W, 34.3941 N) near Santa Barbara, California in late February, 2014. I maintained urchins in flow through seawater tables until needed (generally 3-5 days). To obtain eggs, I induced females to spawn by injecting 1 ml 0.55M KCl adjacent to the Aristotle’s lantern, gently agitated, and placed them upright into a small container with seawater. Once a female began profusely extruding eggs, I extracted 1 ml of concentrated egg material in 200 μl batches directly from the gonopores and placed into 50 ml seawater. I then added appropriate dilutions of this solution to each vial such that vials had final concentrations of approximately 1, 0.25, 0.0625, or 0.0126 eggs μl^−1. To obtain sperm, I injected males with KCl in the same manner as females but extracted 100 μl “dry” sperm directly from the gonopore without submerging the animal. Sperm were immediately diluted 100× in seawater, directly followed by six serial 10× dilutions. Sperm solutions were then added in 1 ml aliquots to the vials prepared with eggs (final concentrations approx 10^-3, 10^−4, 10^−5, 10^−6, 10^−7 and 10^−8 μl−1). Actual sperm concentrations in vials varied slightly because of small variability in sperm counts. Note that the highest egg concentration was chosen such that eggs would not form a densely packed layer on the bottom of the vial. Specifically, at 1 egg μl^−1, even if eggs were to completely sink to the bottom of the vial they would cover approximately 15% of the vial bottom surface area, assuming an average 0.084 mm egg diameter (Levitan 1993) and an internal vial diameter of 22 mm.

To estimate sperm concentrations used in each trial, I preserved the 10^−3 microliter^−1 sperm solution with 2 % buffered formalin and conducted five replicate sperm cell counts on a hemocytometer. I estimated egg concentrations for each trial by counting the number of eggs in five replicate 100 microliter subsamples (agitated and homogeneous) using an inverted microscope.