Many desert fishes, which evolved in isolated aquatic “islands” with limited predation pressure, have been severely impacted by non-native predators. These impacts have been attributed to the evolutionary loss of antipredator competence, known as the predator naiveté hypothesis. Recent work provided support for this hypothesis for one desert fish species. We sought to examine the generality of predator naiveté hypothesis by evaluating antipredator competence in five populations of Red River pupfish (Cyprinodon rubrofluviatilis), a species that occupies habitats that vary in the degree of isolation and levels of fish species richness. Fish were exposed to conspecific chemical alarm cues released from damaged epidermal tissue as a general assay of antipredator response. We found that pupfish from all five populations exhibited antipredator behavior in response to alarm cues, regardless of isolation duration or exposure to predation risk. These data demonstrate that antipredator responses to alarm cues are conserved in pupfishes even after prolonged periods of isolation from piscivorous species.

Population selection and fish collection

Female Red River pupfish were collected from five different locations on the Red River. Populations were selected based on fish community composition outlined in Ruppel (2019) to test the effects of duration of isolation and community complexity on antipredator response intensity (Anderson 2022). Red River pupfish in populations 1 and 2 co-occurred with only one other fish species, plains killifish, and these populations were ranked as having low predation risk. Red River pupfish in populations 3 and 4 co-occurred with two to three other species but no large predators and were ranked as having moderate predation risk. Red River pupfish population 5 was classified as having high predation risk due to complex community structure including piscivorous fishes including largemouth bass (Micropterus salmoides), orange spotted sunfish (Lepomis humilis), western mosquitofish, and red shiners (Cyprinella lutrensis).

Female Red River pupfish were collected and shipped to NDSU overnight on the day of capture. Upon arrival, fish were transported to a field site on the North Dakota Agricultural Experiment Station where fish were acclimated to 1135-L population-specific holding tanks that had been set up two weeks prior with salinity set at 10 ppt. Salinity was set to 10 ppt to reflect a moderate salinity level relative to the salinities measured at sampling sites and to standardize salinity across all trials. 

Preparation of alarm cues and evaluation of fish behavior

Chemical alarm cues were prepared following the protocol provided by Wisenden (2011). Donor fish were euthanized with MS-222 (tricaine mesthanesulfonate, 500mg/L) and cervical dislocation before the epidermis of each fish was removed (NDSU IACUC Protocols A18054 and A21042; MSUM protocol 19-R/T-BIO-018-N-Y-C). Skin fillets from each side of the fish were laid on a flat surface and measured for total skin area, then placed in a beaker of deionized water resting on a bed of crushed ice. Once skin fillets were removed from all donor fish, the skin was blended with a handheld blender for 3 min and diluted to a final concentration of 1 cm² of skin per 10 mL concentration. Chemical alarm cue was then aliquoted into 10-mL doses in individual 10-mL mailing tubes, then stored at -20 °C until needed for trials.

Trials were conducted by testing single focal fish in 37-L glass aquaria with a 5 x 5 cm grid drawn on the short side of the tank. Opaque dividers were placed between adjacent aquaria to visually isolate the focal fish. An air stone supplied oxygen to the trial tanks and a separate stimulus delivery tube secured to the air stone housing apparatus for stability was used to deliver test stimuli to the tank (alarm cue or water as a control). Focal fish were acclimated for a minimum acclimation period of 20 h. Each fish was fed at least 20 min before the start of the trial to reduce overall stress.

All observations were recorded using a Canon VIZIA HF R700 video camera positioned in front of the test tank. For each trial, activity was measured by counting the total number of lines crossed by the eye of the focal fish during 5-min pre- and 5-min post-stimulus observation periods (Wisenden, 2011). Vertical position was recorded every 10 s for both pre- and post-stimulus periods by noting the horizontal row in the grid occupied by the test subject, where 1 was the row at the tank bottom and 5 was the surface row. For each trial, the test stimulus (water or alarm cue) was delivered to the tank after the pre-observation period via aquarium tubing that exited near the rising bubbles produced by the air stone.

Behavioral responses of five populations of Red River pupfish to alarm cue

Trials were conducted using the technique outlined in the previous section. Dechlorinated tap water was used for all trials and salinity level was kept consistent at 10 ppt using Instant Ocean aquarium salt (Spectrum Brand, Blacksburg, VA) across all trials. All trials were completed at 24 ºC as experiments occurred from June to August 2020. Lighting was set for a 16h:8h light:dark setting in the trial room to match lighting conditions of the outdoor holding tanks.

A randomized block design was used to standardize the evaluation of all five populations across time for the duration of the experiment, with each block composed of 10 aquaria. Within each block, two females from each of the five populations were randomly assigned the two treatments. A randomized block design also allowed each block of populations to be tested within a single day, which controlled for any effect of time spent in captivity. Fish that did not exhibit normal behavior and were not consistently active in the pre-stimulus observation period and therefore would not provide a valid assessment of the effect of injected test stimuli were excluded from analysis. Thus, we excluded all trials where fish did not move within at least one of the five 1-min intervals during the pre-stimulus observation period. We also excluded one hyperactive fish that crossed more than 1300 lines during the pre-stimulus period (> 4  SD from the mean). Data were analyzed using analysis of covariance (ANCOVA; JMP Pro 15.0 software), with a categorical predictor of Treatment (alarm cue or water) and Population (1, 2, 3, 4, or 5) as categorical predictors and Pre-stimulus behavior (activity or vertical position) as a covariate. Block effects were included in the initial models but were not significant (Anderson 2022) and were not included in the results presented here. Significant effects of alarm cue could be revealed either by a significant interaction of Cue Type X Pre-Stimulus or by a significant treatment effect of Cue Type.