Adult acorn barnacles, Semibalanus balanoides, were collected from the beach at the end of Park Rd, Woods Hole, Massachusetts, USA (41.532° N, 70.671° W) on December 6, 2012. Individuals were collected on cobbles and transported to the University of South Carolina at Columbia where they were placed within environmental chambers.

Treatment containers were placed within environmental chambers (0.6 m3 interior volume) with individual temperature and lighting controls. Barnacles were exposed to a simulated semi-diurnal tide cycle with the timing of both the tide and daylight schedule at Woods Hole. Seawater was pumped from the experimental chamber to a head tank and the barnacles were exposed to the air for 6 h to simulate low tide and then submerged for 6 h to simulate high tide. Seawater was constantly aerated. Every two weeks the timing of the tide and light cycle was adjusted to match that of Woods Hole. Treatment containers were covered with transparent Plexiglas to prevent evaporation but allowed the barnacles to be exposed to the light cycle. Barnacles were liberally fed every 2 days throughout the experiment by addition of Spat Formula (Innovative Aquaculture Products LTD), a commercial aquaculture product containing the diatoms Chaetoceros-B and Phaeodactylum tricornutum and the flagellate Nannochloropsis oculata, at a final concentration of 108 cells L−1. The seawater in the experimental chambers was maintained at 35 ppt and was replaced every two weeks. Feces and settled algal particles were removed weekly. The water changes occurred during low tide (when the water was in the head tank), allowing it to equilibrate to the treatment temperature before the animals were submerged.

In three of the treatments, barnacles were exposed to the same air and water temperature of 7°, 10°, and 13 °C to represent a range of environmentally relevant temperatures spanning the temperature threshold proposed in the literature (Barnes, 1963; Crisp, 1959; Crisp and Patel, 1969). The temperatures were maintained using the internal temperature controls of the incubators and monitored, which indicated that the mean temperature of each treatment over the duration of the experiment was within 0.5 °C of the intended temperature. Two additional, mixed-temperature treatments were also conducted, one with 7 °C air temperature and 13 °C water temperature, the other with 13 °C air temperature and 7 °C water temperature. These treatments were used to differentiate among the effects of air temperature, water temperature, and mean temperature on reproduction.

All experimental treatments were conducted simultaneously over a total of 63 days and chambers were sampled at 8 time-points, 2, 9, 15, 27, 36, 46, 55, and 63 days after the start of the experiment. At each time-point, 10–20 individuals were selected from randomly chosen cobbles and carefully removed from the cobbles using a razor blade. Efforts were made to sample a representation of available sizes of adults (5–13 mm basal diameter). Basal diameter at the widest point, operculum length at longest point, and height at tallest point were measured for each individual (±0.1 mm). The developmental stage of the embryos was assessed under a dissecting microscope (50×) using the developmental scale described in Table 1 (J Pineda and VR Starczak, personal communication). Somatic and gonad tissue were dissected, dried at 55 °C for at least 48 h, and then weighed with a microbalance. At the final time-point (Day 63), a subsample of embryos (200–800) was taken from each individual and counted using a Sedgwick Rafting Counter, then dried and weighed to determine the average mass per embryo for each treatment.

Table 1: Description of developmental traits used to stage barnacle embryos (J Pineda and V Starczak, Woods Hole Oceanographic Institution, personal communication).
Stage Description
0 - White tissue.
1 - Yellow tissue, gonad is stringy and not yet egg-shaped.
2 - Eggs are visible and ovoid, but not developed.
3 - Eggs are translucent and yellow. They have brown eyespots, but no body structure
4 - Visible naupliar body structure, including caudal spines and distinctive eyespot, developing gut sometimes visible. Eggs beginning to change color from yellow to white.
4b - Fully-developed nauplii with appendages and two brown spots, embryos contained within egg case.
5 - Free-swimming or loose nauplii with appendages free from the membrane.