Materials and Methods

Collection of animals Individuals of Lybia leptochelis and their symbiotic sea anemones Alicia sp. were collected from the shallow infra littoral zone at two separated beaches in Eilat, Israel during 2007–08 and again during 2013. The sites were approximately 3 km apart, Tur-Yam (29°31′49.69N; 34°55′36.39″E) and Red Rock Beach (29°31′01.40″N; 34°55′13.34″E). Only intact crabs were collected. Oviparous females were not collected. Female crabs were observed carrying eggs from >4 mm carapace width (CW; Y Schnytzer, 2008, unpublished data), and therefore female crabs at least this size were defined as adults. The collected crabs had a CW between 5 to 11 mm. The sea anemones held by the crabs were ≤2.5 mm pedal disc diameter (PDD). Using a small hand-held net, the crabs were collected and then individually placed in 0.5 L bottles filled with fresh sea water from the collection site, kept in a thermally insulated box and transported to Bar-Ilan University, Ramat Gan, Israel. The animals were collected and maintained within the guidelines of the Israel Nature and National Parks Authority (Permit no. 26103/2006/7/13).

Sea anemone removal For the splitting experiment, each of the crabs had one sea anemone removed. For the theft experiment both sea anemones from half of the crabs were removed. The removal process was based on the protocol presented by Karplus, Fiedler & Ramcharan (1998). The crab was held in a glass Petri dish with enough sea water to cover it. The crab was then placed under a binocular microscope for constant monitoring. A solution of 7.5% MgCl 2 in distilled water was used to relax the sea anemones and prevent their contraction during removal. The solution was pipetted into the Petri dish in 500-µL increments every 2 min. Removal of the sea anemones took between 50 and 80 min. On some rare occasions, it was possible to remove the sea anemone from the crab’s claws without MgCl 2 sedation. All the crabs, including those that did not have their sea anemones removed, were treated equally by the crab handler (i.e., sedation and contact with delicate forceps) to control for possible effects of crab “harassment.”

Animal measurement The crabs with and without sea anemones and the lone sea anemones were photographed in small Petri dishes half filled with water placed on millimeter paper. The sea anemones were photographed after settling on the bottom of the dish. The CW of each crab was measured from the two furthest points on each side of the carapace (anterolateral lobes), and the PDD of each sea anemone was measured using Image J (NIH freeware) software.

Experimental set up-general All the crabs used for the behavioral experiments were individually maintained in the laboratory in small seven liter aquaria. Each aquarium was provided with a standard corner filter and a 5 cm long black PVC pipe lengthwise cut, which served as a shelter. The crabs and their sea anemones were fed every two days ad libitum with frozen adult Artemia. For further details of the general setup, day/night lighting regime, temperature and water quality in the aquaria see Schnytzer et al. (2013).

Crab sea anemone field data Over the course of three years we documented the size of 54 L. leptochelis, 22 male and 32 females, which sea anemones they held and their size. We measured the crab and sea anemone sizes (as detailed above) right after collection from the sea.

Sea anemone splitting experiment To empirically test the hypothesis that when left with one sea anemone, L. leptochelis will split the other, we conducted the following experiment: twenty two L. leptochelis (14 males and eight females) had one sea anemone removed (as detailed above). We performed this for both left (10 trials) and right (12 trials) held sea anemones. Upon removal of one sea anemone, the crab was placed in a small aquarium (18 × 10 × 10 cm) and monitored with a video camera (VHS HI8; Sony or Lumix TS2; Panasonic) for a period of 2–3 h. The trials were conducted in a closed room, behind a black curtain in order to minimize human interference. In the event that the crab split the sea anemone within this time frame, the trial was terminated and the crab was returned to its normal holding aquarium. In the event that the crab did nothing, the video recording was terminated after three hours and the crab was returned to its normal holding aquarium. However, the crabs that did not split the sea anemone in the initial monitoring period were examined twice a day for a period of two weeks. In any event of splitting, the crabs and their sea anemones were measured 10–14 days after the splitting and their morphology was assessed for regeneration (base, column, mouth and tentacles). See above section for measurement details.

Sea anemone theft experiment To assess the stealing behavior of L. leptochelis, 44 specimens of L. leptochelis were grouped into 22 pairs, comprising of crabs of similar size and same gender (14 male pairs and eight female pairs; new cohort, not crabs used in previous splitting experiments). The crabs ranged in size from 4–10 mm CW, with a maximal difference of 0.3 mm between each pair. Male–female pair trials were conducted during the preliminary stages of the study. Their behavior was identical to same sex pairs. However, following sea anemone theft/attempts, the fight was often followed by mating. Thus, to avoid confounding behavioral factors, only same sex trials were conducted. Each crab was only tested once. Each pair consisted of one crab holding both of its sea anemones, and the other had both removed. The crabs without sea anemones had them removed between two to five days prior to the contest. All the crabs were handled in the same manner, even if sea anemones were not removed, to control for the harassment effect. White Styrofoam boards placed between each crab aquarium prevented the crabs from coming into visual contact with their conspecifics. A black canvas sheet was hung over the experimental setup, minimizing the visual contact between the observer and the animals. The rest of the holding conditions were as mentioned above. The contests were conducted in part under daylight conditions (14 trials), and in part under night conditions (eight trials). The night trials were conducted under a dim red light, as it does not appear to have an effect on their behavior (Schnytzer et al., 2013). In general, Lybia crabs are more active at night (Karplus, Fiedler & Ramcharan, 1998; Y Schnytzer, 2008, unpublished data). However, during the preliminary stages of this study we observed that the crabs were equally active when placed into the same small aquarium, so the trials were conducted under both light regimens. In the trials conducted under daylight conditions, identification of the individual crabs was conducted based on observable differences in their coloration. For the night trials, the crabs were marked with a small piece of plastic affixed to the dorsal surface of their carapace with a cyano-acrylate ester based adhesive (Super Glue). In each trial, two crabs were introduced into an aquarium (23 × 23 × 20 cm), each inside a separate transparent glass cylinder on opposing sides of the aquarium. After 10 min of acclimation, the cylinders were slowly and simultaneously removed. In the event that no contact was made between the crabs after a period of 45 min the trial was terminated. The behavioral interactions between the crabs were recorded with a digital video camera (VHS HI8; Sony or Lumix TS2; Panasonic). Typically, during the preliminary trials, we observed that after coming into contact, whether theft occurred or not, each crab would retreat into a corner of the aquarium and no longer approached the other, thus the trials were terminated at this stage. At the end of each trial, the crabs were returned to their original aquaria for a period of two weeks. During this period, daily observations were made for the monitoring of sea anemone splitting activity.

DNA extraction For AFLP analysis, DNA was extracted from fresh material. Genomic DNA was extracted using a High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany) according to the manufacturer’s protocol. Due to their small size, DNA was extracted from the entire sea anemone. DNA concentration was determined by a NanoDrop ND1000 (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 260 nm.

Amplification and “fingerprinting” Eight pairs of sea anemones removed from L. leptochelis from the two above mentioned Eilat beaches were analyzed (specimens 1–5 from Tur-Yam; 6–8 from Red-Rock Beach). We employed AFLP genotyping (Vos et al., 1995) with modifications according to Huys & Swings (1999) and Amar et al. (2008), in which radioactive labeling was replaced with fluorescent dyes. Restriction enzyme digests were performed on 250 ng of genomic DNA for 3 h at 37 °C using two restriction enzymes (MseI and EcoRI), followed by the ligation of respective double strand adapters (EcoRI adaptor E1-CTCGTAGACTGCGTACC and E2-AATTGGTACGCAGTCTAC, and MseI adaptors M1-GACGATGAGTCCTGAG and M2-TACTCAGGACTCAT). The E1 and M1 oligonucleotides were used as primers for pre-selective PCR amplification using 1 µl of ligation products for the second selective amplification. The PCR product was diluted 1:50, and 5 µl was used for the second amplification. We used three pairs of fluorescent labeled primers (VIC, FAM, and NED; Applied Biosystems, Foster City, CA, USA) as follows: (E=GACTGCGTACCAATTC+XXX and M=GATGAGTCCTGAGTAA+XXX): VIC—E+ACC: 5′ 3′with M+CTC: 5′ 3′; NED—E+ACA: 5′ 3′with M+CTC: 5′ 3′; and FAM—E+AGC: 5′ 3′with M+CTT: 5′ 3′. The process was repeated twice (duplicates) for each sample to attain maximum accuracy.

AFLP analysis DNA sequencing was performed at the Instrumentation and Service Center of the George S. Wise Faculty of Life Sciences, Tel-Aviv University. The samples were analyzed using a Genetic Analyzer 3100 (ABI PRISMA; Applied Biosystems). The samples were diluted and 0.3–0.5 µl of size standard Lis 600 was added to the PCR product in the presence of formamide. Fluorescent-labeled PCR products appear as peaks and were first analyzed using GeneScan ABI PRISM 3.7 software (PE Biosystems; Oda et al., 1997) to determine peak sizes in base pairs, according to the size marker. Each PCR peak obtained from the samples was then aligned and converted into a binary system. The results were transferred to binary scores (0, 1) using AFLP Macro2 software. Nei’s genetic distance (Nei, 1978) was calculated using POPGENE version 1.31 (http://www.ualberta.ca/ fyeh). The binary results were then converted to NEXUS format and the maximum parsimony option of PAUP was used to build a dendrogram of the sea anemone population.