Shoaling is an evolved behavior in fishes that has several adaptive advantages, including allowing individuals to avoid predation through risk dilution. However, factors such as size disparity and the presence of heterospecifics may influence the behavior of individual fish within shoals following exposure to elevated predation risk. Using bluegill Lepomis macrochirus as a model species, we measured changes in area use, shoaling index, and movement of a focal individual in isolation, in single-species shoals with two conspecifics, or in mixed-species shoals with two congeneric pumpkinseed L. gibbosus. The experimental shoals were exposed to one of three chemical cues selected to present graded levels of risk: lakewater controls (lowest risk), Northern pike Esox lucius predator odor (kairomones; intermediate risk), or conspecific chemical alarm cues (highest risk). Within the individual bluegill, we found that the multivariate response of area use and post-stimulus activity (line crosses) of the focal fish was significantly influenced by pre-stimulus activity, but not by cue type or fish size. As univariate responses, post-stimulus activity varied positively with pre-stimulus activity. Post-stimulus activity was greater in single-species shoals compared to mixed-species shoals, and again varied positively with pre-stimulus activity. Contrary to predictions, bluegill did not demonstrate graded antipredator responses to the chemical cues. Our findings suggest that prey fish may alter their risk-aversive behaviors in response to chemical stimuli based on shoal composition and provide further insight into the role of intra-prey guild interactions in response to predators in co-occurring prey species.
Study site and specimen collection
We conducted the study at the Queen’s University Biological Station (QUBS) on Lake Opinicon (44°35′06″N, − 76°17′47″W) in Elgin, Ontario, Canada. From 7 to 11 May 2017, we collected a total of 155 bluegill (mean total length + SE = 114 + 17.6 mm) and 60 pumpkinseed (mean total length + SE = 133 + 34.4 mm), by either beach seine netting or angling with size 8 barbless J-hooks baited with earthworm Umbrica spp. Any fish that were deeply hooked, bleeding, or showing any sign of impairment were not suitable for our study. Because they were considered viable, we immediately released them back into the lake as stipulated in the scientific collection permit issued by the Ontario Ministry of Natural Resources and Forestry. We transferred the bluegill and pumpkinseed via aerated coolers into separate floating net pens (~ 3.5m3) in Lake Opinicon. Approximately 1 h before each block of trials, we moved test fish for that block from the net pens into the QUBS wet lab and introduced them into three glass aquaria. Upon completion of each block of trials, we released the test fish back into the lake.
Preparation of alarm cue and predator odor
We euthanized five bluegill (mean total length + SE = 129 + 11.5 cm) via cerebral percussion to generate the alarm cues used in this experiment. We removed skin fillets from both sides of the fish and mechanically homogenized and diluted them with lake water to a final concentration of 0.1 cm2 of skin/ml. This method and concentration have previously been shown to elicit predictable alarm responses in shoals under laboratory and field conditions (Brown and Godin 1999; Wisenden et al. 2003; Brown et al. 2009). We kept one adult Northern pike (total length = 45 cm, weight = 650 g) captured via angling from the same area of the lake as the bluegill and pumpkinseed in an outdoor holding tank filled with ~ 200 L of water, food-deprived, for 24 h to generate the predator odor. We froze the alarm cues and predator odor in 60 mL aliquots and stored them at − 20 °C until needed.
Experimental apparatus and design
Each aquarium measured 60 cm (length) × 30 cm (width) × 30 cm (height) and was filled with lake water to a depth of 18 cm (~ 32.54 L volume). To reduce exterior interference, we covered three of the walls on each aquarium with white paper. We taped green construction paper on the outside of the bottom surface to mimic natural substrate and left the fourth wall uncovered to allow visual observations (Fig. S1). We surrounded the test aquaria with a curtain to minimize disturbance and monitored each of them with two remotely controlled GoPro™ cameras (GoPro, San Mateo, California, USA) placed in front of and above the aquaria to record the responses of focal individuals. We positioned the cameras to capture the entire width of the aquarium in their fields of view.
We used three shoal scenarios for the trials, consisting of a solitary bluegill, a single-species shoal of three bluegill, and a mixed-species shoal of one bluegill and two pumpkinseed. In each scenario, observations focused on a single bluegill, which was haphazardly chosen and identified on video prior to each experimental trial. Each focal bluegill, as well as any shoalmates, were tested only once, measured (total length), and then released back into the lake. Trials consisted of 5 min pre-stimulus observations followed by the injection of 20 ml of lake water (control), alarm cues, or predator odor via plastic syringes and 1.5 m pieces of standard aquarium airline tubing, ~ 3 cm below the water surface in the back-right corner of each aquaria (Fig. S1), and an additional 5-min post-stimulus observation. We used a random number generator to assign treatments. Due to logistical constraints, we placed an upper limit of 12 trials per treatment per shoal type (Table 1). To avoid any contamination from previous trials, we emptied and cleaned each tank before starting the next trial. The mean temperature of the lake water used in the aquaria, creation of alarm cue, and predator odor, and in the floating pens was 14 °C.