Analyses of mating process in intact WT zebrafish pairs. (A) A schematic illustration showing the entire process of mating. The male fish is shown in grey. The expanded panel represents processes from quivering to spawning. In hooking, one pectoral fin of the male is positioned under the female belly (arrow head). Arrows in illustrations represent directions of fish movements. (B) Photographs from a video showing the process from freezing to spawning. The male fish is behind the female in the initial frame. Released eggs are encircled with a white line in the last frame. Arrows indicate temporal sequence. (C) Photographs of freezing (upper) and hooking behavior (lower). In freezing, the female bends her body in S-shape in response to quivering. In hooking, the male holds the female using the female dorsal fin as an anchor for its contorted trunk. (D) Representative time course displaying the number of mating events (left) and eggs released (right) in 1 min during a 60 min pairing session. (E) Total egg numbers were divided by the numbers of spawning in a single pairing session (n = 10 pairs). Dots represent values of individual pairs. A box represents the accumulated data. (F, G) The number of fertilized eggs and fertilization rate were plotted against the total number of eggs. The number of fertilized eggs strongly correlated with the total egg number (r = 0.81, p = 0.0047). Fertilization rate was independent from the total egg number. Regression lines are shown in (D), (F) and (G).

Functional significance of hooking behavior. (A) The numbers of quivering plotted against those of hooking. (r = 0.85, p = 0.00010). (B) The numbers of spawning plotted against those of hooking (r = 0.68, p = 0.0069). (C) Photographs showing mating behavior of an intact male and a female with its dorsal fin removed. The female is behind the male in the initial frame. The trunk of the male slips rostrally without switching to squeezing. (D) Illustrations showing typical hooking behavior of a WT intact pair (upper blue box), and the WT pair with the female fin removal (lower red box). (E) Identified episodes of spawning before and after the fin removal (p = 0.00088, t-score = 3.98, n = 10). (F) Total number of released eggs before and after the fin removal (p = 0.0029, t-score = 3.44). (G) Fertilization rate before and after the amputation. Fertilization rate was significantly decreased after dorsal fin removal (p = 0.027, t-score = 2.45). (H) Episodes of quivering before and after the fin removal (p = 0.29, t-score =  − 1.08). (I) The percentage of pairs proceeding from quivering to hooking (Note all hooking bouts finished incompletely after the fin removal, p = 0.27, t-score = 1.15). Regression lines are shown in (A) and (B).

Analyses of quivering using εKO males (A) Photographs showing the quivering behavior of the WT male x WT female pair (upper) and the εKO male x WT female pair (lower). A cropped image of the male provided below each photograph for clarity. (B) Kinematics for representative traces of head turns in the WT male and the εKO male. Schematic illustrations in the upper panel shows measurement of head angles. Lines represent outlines of a fish, and the head angle θ is obtained from broken lines. 0° corresponds to a straight body. In the lower panels, each trace represents a different male (n=5 each). Head angles are shown in degrees, with 0 indicating a straight body, and positive and negative values indicating body bends in opposite directions. Scale bars: 10 ms. (C) Traces shown in (B) were averaged. (D, E) The amplitude of head turns (p = 0.00060) (D) and the total number of released egg (E) (p = 0.00086) compared between the WT male x WT female and the εKO male x WT female pairs.

Summary. Mating of a typical zebrafish pair (intact, WT; Fig. 1) are compared with a pair that includes a female with the dorsal fin removal (without dorsal fin; Fig. 2) and a pair that includes the εKO male (εKO male; Fig. 3).