DOI: 10.5061/dryad.ngf1vhhxz
title: A test of the predator naiveté hypothesis using desert pupfishes: History of isolation does not matter

description: 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.
description: <strong><em>Population
 selection and fish collection</em></strong> 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 (<em>Micropterus
 salmoides</em>), orange spotted sunfish (<em>Lepomis
 humilis</em>), western mosquitofish, and red shiners
 (<em>Cyprinella lutrensis</em>). 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.<em> </em> <strong><em>Preparation
 of alarm cues and evaluation of fish
 behavior</em></strong> 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 cm<sup>2</sup> 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. <strong><em>Behavioral
 responses of five populations of Red River pupfish to alarm
 cue</em></strong> 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 (&gt;
 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.