Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Diagrams of catheters for (A) visualizing urine release and (B) collecting and preventing urine release. (A) To visualize urine release, tube A (inner diameter 3.2 mm, outer diameter 4.7 mm, wall thickness 0.79 mm, length 1 cm) was attached to the area around the nephropores to provide a base for the more flexible tube B (i.d. 1.6 mm, o.d. 3.2 mm, w.t. 0.79 mm, length 2−3 cm). Tube B was attached to the larger tube C (i.d. 3.2 mm, o.d. 4.7 mm, w.t. 0.79 mm, length 3−6 cm) which contains a cartridge (silastic tube o.d. 1.96 mm, i.d. 1.47mm) filled with concentrated fluorescein (acid yellow 73, Sigma F-6377, concentration 50 mg ml⁻¹ water). The cartridge was enclosed by alumina-silicate beads at both ends (Davison, Fisher Scientific) so it can leak small amounts of concentrated dye continuously into tube C to color the released urine. The fluorescent urine was prevented from passively leaking out of tube C by a one-way sealing rubber valve (valve diameter 3 mm, Da/Pro Rubber, Inc.) attached at the 3-way plastic connector, because this valve opened only when urine was released. (B) To collect urine or to prevent urine release into the aquarium, lobsters were catheterized in a manner similar to A but with two modifications: (1) tube B was longer (6−8 cm) and looped around the animal and attached to the 3-way plastic connector; (2) instead of the one-way valve, a long and flexible tube (tube C: i.d. 3.2 mm, o.d. 4.7 mm, w.t. 0.79 mm, length 40−45 cm) was attached to the output end of the 3-way connector, which allowed collection of urine outside the aquarium. Tubes were attached to nephropores and to each other with cyanoacrylate glue (Pacer Technology, Superglue gel).

• Supplemental Figure S2 -

  Fig. S2. Behavioral assay of stimuli influencing urine release. Lobsters catheterized to visualize urine release were presented with 10 ml of sea water as a control and urine release (UR) was measured for 5 min. Lobsters were then presented with one of the stimuli − conspecific urine, conspecific hemolymph or shrimp juice (each 10 ml and diluted 100× with sea water) − and UR was again measured for 5 min. Then, 30−60 min later, food was offered and UR was again measured, as a positive control because lobsters reliably release urine during feeding. This procedure was first performed on solitary VU lobsters; then, 5 days later, each VU lobster was paired with another lobster for 2 days and was tested again using the same procedure.

• Supplemental Figure S3 -

  Fig. S3. Behavioral assay of urine release during social interactions. Animals (catheterized for visualization of urine, or VU lobsters) were isolated for 1 week prior to pairing with another VU lobster. The first hour of interaction was video recorded and behaviors and urine release scored. At the end of the first hour, food (shrimp or squid) was offered and ingestion and urine release were scored for each animal.

• Supplemental Figure S4 -

  Fig. S4. Behavioral assay of role of urine release in communicating social status. Three groups of animals were studied: two groups of catheterized lobsters and one group of uncatheterized lobsters (as a control). Animals (catheterized as indicated in Fig. S1B) were isolated for 1 week prior to pairing. After pairing, the first hour of interaction was video recorded and behaviors and urine release (UR) were scored as indicated on the figure. At the end of the first hour, food (shrimp or squid) was offered and ingestion and UR were scored for each animal; 24 h later, food was again offered and ingestion and UR were scored. This diagram of behavioral design refers to Figs 2 and 3.

• Supplemental Figure S5 -

  Fig. S5. Behavioral assay of responses to urine. This diagram of behavioral design refers to Figures 4, 5 and 6.

• Movie 1 -

  Movie 1. A spiny lobster is eating shrimp and releasing a long pulse of urine. Note that urine is normally released from the nephropores, located at the base of the antennae. The catheterization technique redirects the urine to be released from a single tube at the dorsal carapace. The fan organs create currents that move the urine, similar to other crustaceans (Atema, 1985; Breithaupt and Eger, 2002). Based on our behavioral results, spiny lobsters can disperse urine signals several body lengths away.

• Movie 2 -

  Movie 2. Spiny lobsters in the laboratory displaying stereotypical avoidance responses to urine: retreat by backwards walking into the shelter.

• Movie 3 -

  Movie 3. Spiny lobsters engaged in ‘antenna-lock’ and releasing short pulses of urine.

• Movie 4 -

  Movie 4. The dominant spiny lobster (left) engages in aggressive behavior by ‘leg grabbing’ and ‘antenna grabbing’ of the subordinate (right). The subordinate spiny lobster retreats in lowered posture and tail flips.

• Movie 5 -

  Movie 5. The dominant spiny lobster (right) ‘abdomen pokes’ the subordinate (left), which responds with tail flip.

• Movie 6 -

  Movie 6. The dominant spiny lobster (left) engages in ‘leg-grabbing’ and releases a long pulse of urine, followed by a release of urine from the subordinate (right). The subordinate spiny lobster stops the release of urine earlier than the dominant spiny lobster and retreats slightly against the wall. The total event is much longer than shown here, and only the important parts of the sequence are shown.

• Movie 7 -

  Movie 7. The dominant spiny lobster (right), which is eating food, moves closer to the subordinate and releases a long pulse of urine.

• Movie 8 -

  Movie 8. Two spiny lobsters in their natural habitat in the Florida Keys displaying avoidance responses to conspecific urine within seconds of its delivery. The avoidance response is the stereotypical retreat followed by rapid backward or sideways walking to another nearby den.