Predator naiveté has been invoked to explain the impacts of non-native predators on isolated populations that evolved with limited predation. Such impacts have been repeatedly observed for the endangered Pahrump poolfish, Empetrichthys latos, a desert fish species that evolved in isolation since the end of the Pleistocene. We tested Pahrump poolfish anti-predator responses to conspecific chemical alarm cues released from damaged epidermal tissue in terms of fish activity and water column position. Pahrump poolfish behavioural responses to conspecific alarm cues did not differ from responses to a dechlorinated tap water control. As a positive control, the well-studied fathead minnow, Pimephales promelas, showed significant alarm cue responses in terms of reduced activity and lowered water column position. The density of epidermal club cells, the presumptive source of alarm cues, was significantly lower in Pahrump poolfish relative to fathead minnows. Therefore, anti-predator competence mediated by conspecific alarm cues does not seem to be a component of the ecology of Pahrump poolfish. These findings provide a proximate mechanism for the vulnerability of Pahrump poolfish to non-native predators, with implications for conservation and management of insular species.

Pahrump poolfish were obtained from two of the three extant refuge populations: Spring Mountain Ranch near Las Vegas, NV, USA (36°04’16.9”; N 115°27’13.7” W) in 2014 and Shoshone Ponds in South Spring Valley near Ely, NV, USA (38°56'21.8"N; 114°25'04.6"W) in 2017. Behavioural assays were conducted during spring 2017, using descendants of fish from the 2014 sample (F1-F3). Histological analyses are based on 29 Pahrump poolfish fish from the 2014 and 2017 collections. These two collections were previously mixed together to provide sufficient sample sizes for mesocosm experiment conducted during the summer of 2017 [23, 24]. Lab-reared fathead minnows were acquired from EMR Inc., a commercial supplier of research-grade animals subcontracted with the US Environmental Protection Agency, Duluth, MN. 

Behavioural trials

We conducted behavioural trials with lab-reared Pahrump poolfish and recorded activity and vertical position of Pahrump poolfish before and after the introduction of a stimulus, either conspecific alarm cue or dechlorinated tap water as a control. Alarm cue was produced by euthanizing individual fish in a solution of 500 mg/L of tricaine methanesulfonate (MS-222) [30], and filleting skin from both sides of the carcass. Fillets were laid flat on a piece of wet glass to measure skin area before transfer to a beaker of 50 mL dechlorinated tap water resting on crushed ice. For each species, the combined skin from all individuals was homogenized with a hand blender for 30s, and further diluted with dechlorinated tap water to a final concentration of 1.0 cm2 skin in 10 mL of water. Previous work with fathead minnows has shown that 1.0 cm2 of skin activates 58,000 L of water [10,11]. Thus, this amount of skin extract concentrate (1.0 cm2 / 37.85 L) should illicit a strong behavioural response in both species. Control cue was prepared from dechlorinated tap water. Both alarm and control cue solutions were aliquoted into 10-mL replicates and frozen at -18 oC until needed.

Behavioural trials were conducted on single subjects that were individually placed in 37.85-L glass aquaria (Figure S-1) under broad-spectrum fluorescent lights and maintained on a photoperiod of 12 h light: 12 h dark and maintained at room temperature (~25 oC). Each tank received oxygen pumped through an air-powered sponge filter with an additional 2.5 m length of airline tubing inserted into the lift tube of the filter through which test cues could be introduced surreptitiously. A grid of 5 x 5 cm cells was drawn on the outside of the front-facing panel of each test tank. For Pahrump poolfish trials, the large pane of the aquarium faced forward, while for fathead trials the small pane of the aquarium faced forward. Test fish were acclimated for 24 h to their experimental tank and randomly assigned to either alarm cue or control treatment. Experimental fish were fed Tetra-min® flake food 60-75 min before trials began. For each trial, 50 mL of tank water was withdrawn through the delivery tube with a 60 mL syringe and discarded to rinse any residues from the delivery tube. An additional 50 mL of tank water was drawn and retained to be used later to flush test stimuli from the delivery tube into the tank.

Vertical distribution was determined as the horizontal row in the grid occupied by the test fish every 15s for Pahrump poolfish or 10s for fathead minnows, averaged over each 5-min observational period. For both species, activity of individual fish was measured as the number of lines crossed over 5 min, using the fish’s eye to determine its position. Once the pre-stimulus observations were completed, either conspecific chemical alarm cue or dechlorinated tap water (control) was introduced to the tank through the delivery tube, followed by 50 mL flush of previously retained tank water. Immediately after injection of the test stimulus, we recorded fish activity and vertical position for a 5-min post-stimulus observation period.

For both species, vertical position was recorded by two observers in real-time. Activity was recorded in real time for trials with fathead minnows; however, activity for trials with Pahrump poolfish was scored from videos recorded with a Canon® camcorder (model VIZIA HF R700) placed 1.0 to 1.5 m directly across from each test tank. The observer effects were minimized by turning off ceiling lights so that the only illumination came from above the test tanks. Observers were positioned 1.5 m away from the tank on an elevated shelf so that observers were not looming above test subjects. Observers moved slowly, calmly and spoke in hushed tones, and the fish were habituated to the presence of people in the lab. For both species, experimental tanks were drained, rinsed, and refilled with fresh water and cue injection tubes were replaced after each trial.

For Pahrump poolfish, we tested 84 fish (42 control water and 42 alarm cue). In one trial the fish displayed unusual swimming movements and in another 26 trials fish had either very low pre-stimulus activity (< 50 lines, n = 22) or very high activity (> 400 lines, n = 3). In such cases, responses during the post-stimulus period would be inherently limited to a one-sided response, i.e., speeding up for the slow fish, and slowing down for the fast fish. We ran analyses both with the full data set and with a reduced data set of 58 fish (29 control trials and 29 alarm-cue trials) limited to fish with pre-stimulus movement in the range of 50-400 lines. There was no difference in the outcomes from the two analysis sets, and thus we report the analyses based on the reduced data set (full data set analyses are available from the authors upon request). For fathead minnows, we ran 30 trials (15 control and 15 treatment), all of which met the activity criteria outlined above (> 50 lines and < 400 lines). These data were previously reported as a positive control in an experiment evaluating the effects of hypoxia on alarm cue response of fathead minnows [31].

Post-stimulus response data were analysed using ANCOVA in JMP Pro 15 ® software (type III sums of squares, 0.05 alpha level). Treatment type (control water or alarm cue) was treated as the categorical predictor, with the pre-stimulus behaviour as a covariate.

Histological Examination

Twenty-nine Pahrump poolfish and seven fathead minnows were sacrificed using a lethal dosage of MS-222 (~500 mg/L) and a 3-4 mm section of skin was taken from the nape region [6,32]. Thin-sectioned histological samples were stained and mounted on slides and then digitally scanned using a MoticEasyScan Slide Scanner ® using Plane Apochromatic objective (20X/0.75) with image detail equivalent to 40X lens. The number of visible club cells was counted for each slide and normalized using the estimated area of epithelial tissue to club cell density per mm² of skin using Image-Pro Premier®. These data were used to estimate club cell density (club cells per mm2 of skin). 

Data were analysed with JMP Pro 15® software. We used a likelihood chi square to test for inter-species differences in club cell prevalence. Due to small sample sizes, we used a permutation procedure [33] to test for differences in club cell density (club cells per mm2) where we performed a t-test to obtain the empirical difference in club cell densities between the two species and then conducted a permutation test. For each permutation, the observed club cell density estimates were randomized between the two species and the inter-species difference was calculated. This procedure was repeated 9,999 times, along with the observed empirical difference, to create a distribution of 10,000 inter-species club cell differences expected by chance. The p-value was calculated as the proportion of random inter-species differences (absolute value of the difference between means) greater than or equal to the absolute observed difference, making it analogous to a two-tailed t-test.

For poolfish behavioral data, the analyses were conducted on a) full data set and b) on a reduced dataset that included only fish with pre-stimulus activities levels that were greater than 50 lines and less than 400 lines.  Also, one fish that showed jerky movements was not included in the reduced data set.

For fathead behavioral data, all 30 trials met the criteria described above.

For the histology data, we used a permutation procedure to analyse data as described in Methods.