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Figures
- Fig. 1.
Larval settlement assays for visual signal involvement using adult barnacles. (A,B) Three UV-transparent polycarbonate boxes were placed in a PVC container, with one of the boxes housing five to 10 adult barnacles. Then, 200 cyprids were incubated in the container under a broad-spectrum white light. The container was covered with a UV-transparent polycarbonate sheet. (C) After 72 h, cyprid settlement sites in the container were observed. The number of settlers in each area (A, B or C) was plotted. Data are means ± s.e.m. of four replicate trials using four different batches of larvae (N=4). (D) Three UV-transparent polycarbonate boxes were placed in a large glass Petri dish covered with a black sheet outside the bottom and side walls, with one of the boxes housing five to 10 adult barnacles and another box housing five to 10 stones. Then, 200 cyprids were placed in the container under white light. The Petri dish was covered with a UV-transparent sheet. (E) After 72 h, cyprid settlement sites in the container were observed. The settlement in each area is plotted. Data are means ± s.e.m. of five replicate trials using five different batches of larvae (N=5). Asterisks indicate significant differences between numbers of settlers (Mann–Whitney U-test, *P<0.05, **P<0.01).
- Fig. 2.
Larval settlement assays for color discrimination. (A) Mean (±s.e.m.) numbers of settled cyprids on red (R), green (G), blue (B) and yellow (Y) surfaces in each Petri dish. (B) Mean (±s.e.m.) numbers of settled cyprids on gray surfaces with different levels of brightness: DD, darkest; D, second darkest; L, second lightest; LL, lightest. Three different batches of cyprids with five replicates in each batch were used (N=15). Asterisks indicate significant differences between numbers of settlers (Mann–Whitney U-test, ***P<0.001). In the color discrimination assay (A), the number of settlers on red surfaces was significantly higher than that on the other colors. In the gray assay (B), no significant differences between numbers of settlers on surfaces of different brightness were detected.
- Fig. 3.
Adult Balanus amphitrite has red auto-fluorescence. (A) A photograph of adult B. amphitrite under a stereo microscope with normal light. (B) A photograph of the same barnacles under a fluorescence stereo microscope with an SZX-FG filter (excitation: 510–550 nm, emission: 590 nm). Strong red fluorescence was detected on the surface of the barnacle shell plates.
- Fig. 4.
Fluorescence intensity in barnacle extracts. Crude extracts (3.5 mg ml−1) were prepared from the adult barnacles, B. amphitrite, in 50 mmol l−1 Tris-HCl pH 7.5 containing 1 mmol l−1 dithiothreitol (=TD buffer). Fluorescence intensity of the crude extracts was measured with a fluorometer (CytoFluor II Microplate Reader, PerSeptive Biosystems). (A) Relative fluorescence intensity of the crude extracts at each excitation/emission wavelength (nm). (B) Relative fluorescence intensity of crude extracts, heat-treated supernatant, <30 kDa fraction, shell extracts and soft tissue extracts at 530/590 nm excitation/emission wavelength. Each sample was prepared from the same mass (20 g) of barnacles in the same volume (20 ml) of TD buffer.
- Fig. 5.
Larval settlement assay using adult extracts with red fluorescence. (A) Three UV-transparent polystyrene boxes were placed in a polystyrene Petri dish, with one of the boxes containing barnacle extracts. Then, 50 cyprids were incubated in the Petri dish under white light. After 48 h, cyprid settlement sites in the Petri dish were observed. (B–F) Mean (±s.e.m.) numbers of settled cyprids in each area (adult extracts, none 1 and none 2). Barnacle extracts varied as follows: under light conditions, one box contained (B) a high concentration of crude extracts (2 mg ml−1), (C) a low concentration of crude extracts (0.2 mg ml−1), (D) a high concentration of heat-treated supernatant or (E) a low concentration of heat-treated supernatant; (F) under dark conditions, one box contained a high concentration of crude extracts (2 mg ml−1). Three different batches of cyprids with three replicates in each batch were used for the assays (N=9). Asterisks indicate significant differences between numbers of settlers (Mann–Whitney U-test, **P<0.01).
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