DOI: 10.5061/dryad.3tx95x6hk
title: Pahrump Poolfish antipredator behavior

description: Predator naïveté 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 species that
 evolved in isolation since the end of the Pleistocene. We tested poolfish
 for anti-predator responses to conspecific chemical alarm cues released
 from damaged epithelial tissue versus responses to distilled water as a
 control. Poolfish did not respond to conspecific alarm cues in terms of
 two well-documented components of behavioural alarm reactions: reduction
 in fish activity and movement out of the water column. Epidermal club
 cells are the presumptive source of alarm cues. We found a low density of
 club cells in Pahrump Poolfish relative to the well-studied Fathead
 Minnow, Pimephales promelas, used here as a positive control. 
 Therefore, anti-predator competence mediated by conspecific alarm cues
 does not seem to be a component of the ecology of Pahrump Poolfish. The
 phylogenetic context of our findings suggests that this is the first
 reported example of secondary loss of olfactory assessment of predation
 risk. These findings provide a proximate mechanism for the vulnerability
 of Pahrump Poolfish to non-native predators.
description: We conducted
 behavioural trials in 2017 with lab-reared F<sub>2</sub>
 generation poolfish during which activity and vertical position of
 poolfish was recorded before and after the introduction of conspecific
 alarm cue. Alarm cue was produced by euthanizing individual fish in a
 solution of 500 mg/L of tricaine methanesulfonate (MS-222) (NDSU
 Institutional Animal Care and Use Committee protocols #A15072), and
 filleting skin from both sides of the carcass. The 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 by a hand blender
 for 30s, and further diluted with distilled water to a final concentration
 of 1.0 cm<sup>2</sup> skin in 10 mL of water. In Fathead
 Minnows, 1 cm<sup>2</sup> of skin activates 58,000 L of water
 (Lawrence and Smith 1989, Wisenden 2008). Thus, this amount of skin
 extract concentrate (1 cm2 / 37.85 L) should produce a strong behavioural
 response in poolfish if they have a behavioural response to alarm cues.
 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 using 37.85-L glass aquaria under broad-spectrum
 fluorescent lights and maintained on a photoperiod of 12 h light: 12 h
 dark. Each tank contained an air-powered sponge filter with an additional
 2.5 m length of airline tubing inserted into the outflow of the filter
 used to deliver test cues surreptitiously. A grid of 5 x 5 cm cells was
 drawn on the outside of the front-facing panel of each test
 tank. Each test fish
 was acclimated for 24 h to an experimental tank and randomly assigned to
 either alarm cue or control treatment. Experimental fish were fed
 commercial 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. A Canon®
 camcorder (model VIZIA HF
 R700) was placed 1.0 to 1.5 m directly across from each test
 tank. The camcorder recorded fish behaviour for a 5-min pre-stimulus
 observation period. Once completed, either control water or conspecific
 chemical alarm cue was introduced to the tank through the delivery tube,
 followed by the 50 mL flush of the previously retained tank water.
 Immediately after injection of stimulus, the camcorder recorded a 5-min
 post-stimulus observation period. Experimental tanks were drained, rinsed,
 and refilled with fresh water and cue injection tubes were replaced
 between trials. Behavioural
 measures were vertical distribution and activity of the test fish,
 following standard operating protocols (Wisenden 2011). Vertical
 distribution was determined as the vertical location of test fish relative
 to the grid every 15 s and then averaged over each 5-min observational
 period. Using video recordings, activity was measured as the number of
 lines crossed (using the fish’s eye to determine its position) per minute
 during each 5-min observational period. 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. We ran 84 trials
 (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 analyses sets, and thus we report
 the analyses based on the reduced data set (full data set analyses are
 available from the authors upon request). <strong><em>Histological Examination</em></strong> Twenty-nine Pahrump Poolfish and 7 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 (Wisenden and Smith 1998; Chivers et al. 2007). Thin sectioned histological samples were stained and mounted on slides and then digitally scanned using a MoticEasyScan Slide Scanner ® using Plan Apochromatic objective (20X/0.75) with image detail equivalent to 40X lens. Using Image-Pro Premier®, the area of epithelial tissue was calculated with smart segment tool and the number of visible club cells recorded for each sample. These data were used to estimate club cell density per mm<sup>2</sup> 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 to test for differences in club cell density (club cells per mm<sup>2</sup>) 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 (Manly 2007). For each random 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. Using the absolute value of mean difference was analogous to a two-tailed t-test.
description: For the behavioral dataset, we ran 84 trials (42 control water
 and 42 alarm cue). In one trial the fish displayed unusual swimming
 movements and in another 25 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.