DOI: 10.5061/dryad.z8w9ghxdt
title: Ignorance is not bliss: Evolutionary naïveté in an endangered desert fish and implications for conservation

description: 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.
description: 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 (&lt; 50 lines, n = 22) or very high activity
 (&gt; 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 (&gt; 50 lines and &lt; 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.
description: 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.