Permits: All protocols and experiments, described below, followed institutional guidelines and were approved by the University of Hawaii Institutional Animal Care & Use Committee (IACUC protocol number 2099).

Experimental design: We set three larval fish age-classes of the clownfish, Amphiprion ocellaris (early: 1 to 5 days post-hatch [dph]; mid: 6 to 9 dph; and late: 11 to 14 dph) against three developmental stages of the copepod prey, Bestiolina similis (nauplii: NIII-NIV stages; copepodites: CII-CIII stages; and adults: CVI stage). The choice of developmental stages provided a range in prey size (length: ~100 to 500 µm; McKinnon et al., 2003), mechanosensitivity, and escape performance (Buskey et al., 2017). We designed the experiment to quantify how strike posture changed through larval development (and size: ~4 to 8 mm total length; Jackson and Lenz, 2016), while also assessing the effect of a prey’s stage on its predator’s posture.

Larval fish-rearing conditions: The experiments used larval clownfish lab-reared over their two-week planktonic phase. Their rearing, the culturing of copepod prey, the experimental apparatus and protocols, and the high-speed video recording and analysis software have been described previously (Robinson et al., accepted). Briefly, up to 200 recently hatched larvae were raised in a 30-L seawater aquarium kept at 24-26° C on a 12:12 L:D light cycle. They were fed twice daily on a mixed diet of rotifers (Brachionus plicatilis) and different developmental stages of another calanoid copepod (Parvocalanus crassirostris). Different prey were used for daily feeding than for experiments so that fish were exposed to a novel prey type during their trial, thus avoiding complications arising from learned feeding behavior and laboratory acclimation.

Behavioral observations and video set-up: For the experiments, two larvae that had been kept without food for 4 to 6 hours were placed into a circular observation chamber of 20 cm diameter, filled with seawater to a depth of 2 cm containing copepods at a density of 0.2 to 0.7 individuals ml-1. Experimental trials lasted for one hour or less and no fish larvae were used in more than one trial. Interactions between fish larvae and copepods were recorded at 500 frames per second (fps) using a Photron FastCAM SA4 video camera mounted above the observation chamber with dark-field illumination. The field-of-view of the camera was 35 x 35 mm with an image resolution of 1024 x 1024 pixels.

Data analysis: Predatory attacks were analyzed to characterize the temporal sequence up to and through the strike of fish larvae on different copepod developmental stages. We quantified the duration (in sec) of the approach phase of the fish, defined and described in Robinson et al. (accepted) as beginning when the tail stopped beating and started to bend to the left or right, and ending at t0. Thirty-seven successful captures (n = 37) by fish larvae were analyzed using the Fiji software package built on ImageJ (v1.51) (Schindelin et al., 2012) to digitize the changing body shape of the fish as it prepared for and initiated the strike. For each interaction, we established a temporal reference (t0) as the image just prior to the opening of the fish’s mouth. Twenty-five frames, including 12 frames before and 12 frames after t0, were then extracted and the posture of the fish larva was characterized frame-by-frame. Twelve frames (24 ms) before t0, labeled as t-24, was chosen as a standardized interval that captured the final approach phase of fish in our trials, which ranged from 28 to 1130 ms preceding t0. The median speed during the final frame of approach (t-2) was 0 mm s-1. Therefore, fish were most often motionless at this time interval, meaning that all motion involved in the acceleration of the strike occurred after t0. Twelve frames after t0, labelled as t+24, was chosen as a standardized interval as it always included peak strike speed, copepod capture, and subsequent deceleration of the fish. Starting with t-24 and continuing every other frame up to and including t+24, we digitized the x,y coordinates of 12 points along the central axis of the larva. Because its position with respect to the fish did not change, we used a small pigment spot located at the narrowest point between the eyes as a spatial reference for each frame (origin at x=0, y=0). The position of the copepod in each frame was also digitized to obtain the change in distance over time between fish and copepod. 

We quantified the relative curvature of the body and caudal fin during the larva’s initial lunge, frame-by-frame from t0 to t+8 (8 ms, i.e., 4 frames after t0). To do so, we used the oval tool in Fiji to place a circle within the curl of the tail. The “fit circle” and “measure” commands were then used to calculate the area (A) of the circle. From the circle’s area, we derived the reciprocal radius, r -1 = √(π / A) as a metric of relative curvature of the larva’s caudal fin, with greater values being more curved and lesser values being less curved. The measurement of reciprocal radius became less reliable as an estimate of curvature after t+8 because the tails of most fish began to curve in the opposing direction, making the inscribed circle too large to measure. We therefore employed another relative measure of curvature, the straight-line distance between the tip of the tail and the narrowest point between the eyes (chord length), divided by the length of the fish when its body was straight (fish length), also measured between the tip of the tail and origin/pigment. These normalized distances (chord length-to-fish length ratio, or CVF) approached 1 when the fish was straight and were decreasing fractions of 1 when the fish was increasingly bent into a J-like posture. To measure speed of the fish during its predatory lunge (in mm s-1), we tracked the changing position of the pigment between its eyes from t-4 to t+20 (4 ms, i.e., 2 frames before t0 and 20 ms, i.e., 10 frames after t0, respectively) and divided the frame-by-frame distance travelled by the time elapsed between frames. In addition, the distance from the spot between the eyes and the tip of the mouth was measured at t0 and t+8 to determine the contribution of jaw extension to prey capture.