Study site and species
Field observations were conducted in a coral reef protected area of approximately 100 m × 350 m (127°52' E, 26°38' N) , in front of the Tropical Biosphere Research Center, University of the Ryukyus, Sesoko Island, Okinawa, Japan, between April and October 2019. The study site was a fringing reef with a sandy bottom off the reef edge at a depth of 5 m (during high tide). The reef was species-rich with benthic animals (tubeworms, 4.8 individuals per 5 m2; boring clams, 14.5 individuals per 5 m2 ), small fishes as targets for fin-biting, and fish eggs, which are seasonal resources. All the damselfish and triggerfish species observed in this study spawned during the warm season from April to October (Table 1).
All individuals of the false cleanerfish were identified by tagging. Fish were captured using a screen net and a hand net, immediately brought back to the laboratory, anesthetized with clove oil, measured for total length, sexed by external genitalia, photographed on both sides of the body, and injected subcutaneously with fluorescent elastomer tag (Northwest Marine Technology, Shaw Island, USA). As soon as the marking was completed, they were recovered with fresh seawater and returned to the captured point.
For testing the two hypotheses, we examined the raiding behavior of the false cleanerfish against the scissors-tail sergeant, A. sexfasciatus, which was the most frequently observed in this study (see Table 1). The scissors-tail sergeant is one of the damselfishes commonly observed in the study reef . Parental male fish care eggs laid on rock surfaces until they hatch , and on Sesoko Island, egg-guarding males formed breeding colonies. During egg-guarding, they showed defensive behavior chasing all the fish that approached their nests. We used 19 cases of raiding behavior against the scissors-tail sergeant for which video data were obtained (recording time: median 52 s, range 18–201 s) to quantitatively test our hypothesis.
All field work was conducted by snorkeling. Between 07:00 and 18:00, a randomly chosen individual in the study area was tracked from behind for at least 30 min, and the following items were visually recorded on the waterproof datasheet: time spent swimming with other individuals per 30 min as a group activity; group size; the number of feeding occasions (bites on fish-fins, tubeworms, and boring clams); the number of raids on fish nests and invasion time; and the number of attacks from other fishes. The target species for egg-eating were photographed with an underwater camera (TG5 Tough; Olympus, Tokyo, Japan) to identify the species.
As for the nest-raiding behavior toward the scissors-tail sergeant, the entire bouts were captured by videos. Based on the video data, we counted the number of times the false cleanerfish were chased (as damselfish attacks), jumped into the nest of the scissors-tail sergeant, and whether it succeeded or not. We defined one "nest-raiding" event as the time from when they started targeting the eggs until they moved away from the nest, regardless of the success or failure. In the case of no eggs in the nest (3/19 bouts), the false cleanerfish had mistakenly raided the nest; however, the scissors-tail sergeant always defended their nest; therefore, we treated them as nest-raiding data. The false cleanerfish were sometimes attacked by other egg-guarding males that were not the target damselfish because the males always formed colonies (13/19 bouts). Since these attacks can also be assumed as a cost of egg-eating, they were included in the data analysis.
Non-parametric tests for statistical analyses were used, and all data characteristics were presented as medians and ranges. All the correlation analyses were performed using Spearman's rank correlation coefficient test. As group activities of individuals, we calculated the percentage of the swimming time spent with other individuals in 30 min tracking, to examine the relationship between group activity and total length. The frequency of damselfish attacks per 10 s was calculated (10 * [total number of attacks/nest-raiding time]), and the correlation with group size was analyzed from the video data. The success rate of nest invasion of the false cleanerfish was calculated (100 * [success trials/total trials]), and the correlation with group size was analyzed from the video data. All analyses were performed using the free statistical software, R ver. 4.01 (R Development Core Team, 2020) .
All procedures performed in this study followed the Guidelines for the Proper Conduct of Animal Experiments and related activities laid down by the Hiroshima University Animal Research Committee (No. 020A170410 certified on April 10th, 2017), the ASAB/ABS Guidelines for the Use of Animals in Research (Guidelines for the Treatment of Animals in Behavioral Research and Teaching; https://doi.org/10.1016/j.anbehav.2019.11.002), the Guidelines for the Use of Fishes in Research by the Ichthyological Society of Japan (http://www.fish-isj.jp/english/guidelines.html), and the Guideline for Ethological Studies by the Japan Ethological Society(http://www.ethology.jp/guideline.pdf).