Establishment of a novel behavioral test to quantify visual familiarization
To examine whether dominance of persistent mate-guarding enhances familiarization with females while at the same time blocking the female’s familiarization with rival males, we modified the previous behavioral tests [14, 19] and established a novel behavioral test using a tank divided into three zones with two walls (Fig. 1a). First, we placed a female in the larger zone on one side and examined whether the female could become visually familiarized with the wild-type (WT) male in the “far” or “near” zones. To quantify visual familiarization, we performed a female mating receptivity test by calculating the latency to mate with the male of interest, which negatively correlates with female receptivity toward the male. We previously reported that the latency to mate with visually familiarized males is significantly shorter than that with unfamiliar males [19]. One male was placed in either the “far” or the “near” zone, separated by transparent or opaque walls (Fig. 1b-d) in the evening before mating. In this setup, the latency to mate in the opaque wall group (“Near: WT, Wall: opaque”) was significantly longer than that in the transparent wall groups (“Near: WT, Wall: transparent” and “Far: WT, Wall: transparent”; Kruskal-Wallis: chi-squared = 14.931, df = 2, P = 0.0005. post-hoc Steel test: “Near: WT, Wall: opaque” VS “Near: WT, Wall: transparent”, P = 0.0008; “Near: WT, Wall: opaque” VS “Far: WT, Wall: transparent”, P = 0.033; Fig. 1e). Additionally, in the opaque wall group, the number of courtship behaviors (male mating activity) did not significantly decrease, suggesting that low female receptivity toward the male in this group was not derived from decreased male activity (Additional file 1a). These findings indicated that females could become familiarized with males in either the near or the far zone, and confirmed that the female and male could become visually familiarized under this setup.
Effect of mate-guarding on female visual familiarization
Next, we placed the two males and one female in the three zones, which allowed the male in the near zone (near male) to maintain closer proximity to the female than the male in the far zone (far male), and performed a mate-guarding test (Fig. 2a-b). The WT male in the near zone exhibited mate-guarding over the WT male in the far zone (Mann–Whitney U test: “WT experimental group” VS “WT negative control”, U = 3.50, N
1 = N
2 = 11, P < 0.0001; Fig. 2d, Additional file 2). On the following morning, we calculated the latency to mate with the far male in dyadic relationships (Fig. 2a) and found that the presence of the WT male in the near zone significantly decreased female receptivity toward the far male (Kruskal-Wallis: chi-squared = 6.806, df = 2, P = 0.0333. post-hoc Steel test: “Far: WT, Wall: transparent” VS “Far: WT (focal), Near: WT, Wall: transparent”, P = 0.047; Fig. 2e). In this experimental group, the number of courtship displays was not significantly less than that in control group (Kruskal-Wallis: chi-squared = 1.0216, df = 2, P = 0.6. Additional file 1b), confirming that low female receptivity toward the far male was not derived from decreased male activity. These findings indicated that the presence of the near male blocked visual familiarization of the far males. We also investigated whether mate-guarding behavior of the near male was required to block visual familiarization with the far male.
To clarify this issue, we used arginine-vasotocin receptor 2 (V1a2) knockout (KO) males generated by TALEN (Transcription Activator-Like Effector Nucleases) methods [25, 26] as the near male (Fig. 2c). Previously, we reported that V1a2 KO males exhibit defective mate-guarding behavior under free-swimming conditions [14]. The mate-guarding test (Fig. 2a) confirmed that V1a2 KO near males did not exhibit mate-guarding behavior (Mann–Whitney U test: “V1a2 KO experimental group” VS “V1a2 KO negative control”, U = 55.5, N
1 = N
2 = 11, P = 0.759; Fig. 2d, Additional file 3). The V1a2 KO near male did not maintain its position between the female and the far male, although there was no significant difference in the proximity to the female between the WT near male and the V1a2 KO near males (Mann–Whitney U test: U = 57, N
1 = N
2 = 11, P = 0.832; Additional file 4). The placement of a V1a2 KO in the near zone did not affect the latency to mate with the far male (Kruskal-Wallis: chi-squared = 6.806, df = 2, P = 0.0333. post-hoc Steel test: “Far: WT, Wall: transparent” VS “Far: WT (focal), Near: V1a2 KO, Wall: transparent”, P = 0.924; Fig. 2e). Additionally, there was no significant difference between the number of courtship display in this group (“Far: WT (focal), Near: V1a2 KO, Wall: transparent”) and that in control group (“Far: WT, Wall: transparent”) (Kruskal-Wallis: chi-squared = 1.0216, df = 2, P = 0.6. Additional file 1b). Furthermore, we confirmed that the free swimming velocity (Mann–Whitney U test: U = 7, N
1 = N
2 = 5, P = 0.310; Additional file 5b) and visual response and locomotion ability (Mann–Whitney U test: U = 9, N
1 = N
2 = 5, P = 0.532; Additional file 5c-d) were normal in mutant males, suggesting that the high female receptivity toward WT far males was not due to abnormal movement of the V1a2 KO near males. These results demonstrated the necessity of mate-guarding, rather than mere spatial proximity, for inhibiting the formation of familiarity between the female and the rival male. Taken together, our findings indicate that mate-guarding enhanced visual familiarization with the dominant male and blocked the female’s familiarization with the subordinate male, at least under this experimental condition. Furthermore, the presence of the far male did not affect female receptivity to the near male (Mann–Whitney U test: U = 50, N
1 = N
2 = 11, P = 0.507; Additional file 6b). As V1a2 KO males showed courtship behaviors less frequently than WT males (Mann–Whitney U test: U = 32.5, N
1 = N
2 = 11, P = 0.02; Additional file 7), we could not investigate female receptivity toward the V1a2 KO males.
Requirement of persistent mate-guarding for high male mating success
We previously reported that mate-guarding positively correlates with male reproductive success [14]. Here we examined whether visual familiarization is required for the male reproductive success. On the evening before mating, we performed the dominance test and judged which male was dominant (6 successive days; Fig. 3a). The duration of the mate-guarding was significantly different in 5 of 9 groups, while not in 4 groups (the two males were considered to be equivalent). After the dominance test, for 3 of the 6 days, the three fish were allowed to freely swim, whereas on the other 3 days, we added a separation procedure in which the females were visually familiarized with non-dominant males (subordinate males and equivalent males). The next morning, we performed the paternity test and calculated the effect of the separation procedure on the mating success rate (Fig. 3a). The separation procedure significantly decreased the mating success rate of the dominant (N = 5) and equivalent (N = 4) males (Wilcoxon signed-ranks test: T = 3, N = 9, P = 0.036; Fig. 3b and c). These findings suggest that persistent mate-guarding increased male reproductive success by blocking the familiarization of the female and the rival male, and confirmed the importance of recent familiarization in the development of mating preference in females.