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Ignorance is not bliss: Evolutionary naïveté in an endangered desert fish and implications for conservation

Citation

Stockwell, Craig et al. (2022), Ignorance is not bliss: Evolutionary naïveté in an endangered desert fish and implications for conservation, Dryad, Dataset, https://doi.org/10.5061/dryad.z8w9ghxdt

Abstract

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 anti-predator responses to conspecific chemical alarm cues released from damaged epithelial tissue in terms of reduction in fish activity and movement out of the water column. Poolfish anti-predator behavioural responses to conspecific alarm cues did not differ from poolfish responses to distilled water as a control. As a positive control, the well-studied Fathead Minnow, Pimephales promelas, showed significant alarm cue responses in terms of activity and water column position. The density of epidermal club cells, the presumptive source of alarm cues, were 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. 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.

Methods

Pahrump Poolfish were obtained from two populations: Spring Mountain Ranch near Las Vegas, NV, USA (36°04’16.9” N 115°27’13.7” W) in 2014 and Shoshone Spring near Ely, NV, USA (38°56'21.8"N 114°25'04.6"W) in 2017. 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 F2 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 cm2 skin in 10 mL of water. Previous work with Fathead Minnows has shown that 1 cm2 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 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 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. For the poolfish trials, the large pane of the aquarium faced forward, while for the fathead trials the small pane of the aquarium faced forward.  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.

Vertical distribution was determined as the vertical location of test fish relative to the grid at fixed intervals of time, every 15s or 10s for poolfish and fathead minnows, respectively, and then averaged over each 5-min observational period.  For both species, fish activity was measured as the number of lines crossed using the fish’s eye to determine its position. Once the pre-stimulus observation was 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, 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. Fathead Minnow activity was also recorded in real time.  However, poolfish activity was subsequently scored from each entire trial from a video that was recorded with a Canon® camcorder (model VIZIA HF R700) which had been placed 1.0 to 1.5m directly across from each test tank. For both species, experimental tanks were drained, rinsed, and refilled with fresh water and cue injection tubes were replaced between trials.

For Pahrump Poolfish 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 (< 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 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).

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 also used as a positive control to test for the effects of hypoxia on alarm cue response of Fathead Minnows (Strand et al. 2022).  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 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 Plane 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 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 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 (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 created 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.

Usage Notes

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.