DOI: 10.5061/dryad.51c59zw8z
title: Suction feeding by predators limits direct release of alarm cues in fishes

description: Chemical alarm cues alert aquatic prey to the presence of an actively
 foraging predator. There is a large literature based upon responses to
 alarm cues derived from skin extract, because it is anticipated that prey
 skin is damaged when prey are attacked by a predator. However, many
 predators feed by suction feeding whereby prey are quickly drawn into the
 buccal cavity and swallowed whole with little, if any, direct contact
 between the teeth of the predator and the prey. Here, we test if predation
 by suction feeding releases chemical information in sufficient quantity to
 elicit an antipredator response in conspecific prey. In tests of
 individual zebrafish Danio rerio, we found that odor of crushed zebrafish
 produced a clear antipredator behavioral response, but water collected
 immediately adjacent to staged predation events between a largemouth bass
 Micropterus salmoides (122-145 mm TL) and adult zebrafish (39 mm TL) did
 not elicit alarm behavior, and did not differ from behavioral responses to
 blank water or bass odor (on a diet of earthworms). In a second
 experiment, zebrafish swallowed by largemouth bass, then retrieved seconds
 later through gastric lavage, produced zebrafish that were alive and
 completely intact with minimal epidermal damage. Published relationships
 between bass length, gape size and the geometry of suction feeding suggest
 that in a hypothetical population of largemouth bass feeding on adult
 zebrafish, or fathead minnows, the majority of predation events by
 piscivorous fish probably would not release detectable levels of prey
 alarm cue. Accounting for the role of feeding mechanics by fish predators
 requires a recalibration of the literature on risk assessment by small
 prey fishes. Chemically-mediated antipredator behaviors against
 suction-feeding predators may occur primarily via post-ingestion dietary
 cues, or disturbance cues released near the moment of attack.
description: <strong>Experimental
 design</strong><br>There were four treatment groups: (1) alarm
 cue (positive control), (2) deionized blank water (negative control), (3)
 water collected from a holding tank containing bass kept on a diet of
 earthworms <em>Lumbricus terrestris </em>to control for the
 possibility of pre-existing recognition of bass odor and (4) water from
 where a bass had eaten an adult zebrafish 15 s beforehand that should
 contain detectable levels of alarm cue. <strong>Cue
 preparation</strong><br>To create alarm cue we euthanized 20
 zebrafish (TL = 39.0 ± 0.89 mm) by an overdose of tricaine methanesulfonate
 (MS-222), added them to 210 ml of deionized water and
 homogenized them for 60 s using a blender, then filtered the solution
 through a loose wad of polyester fiber and aliquoted the filtrate into 21,
 10-ml doses and froze them at -20° C until needed. The same number of
 doses of blank deionized water cue was aliquoted into 10-ml doses and
 frozen at -20° C until needed.  Bass-earthworm-diet
 odor was collected from a 75-L aquarium filled with 32 L of dechlorinated
 water that held three largemouth bass (122, 127 and 145 mm TL) that had
 been maintained on a diet of earthworms. Fish were held for 24 h then tank
 water was collected and aliquoted into 60-ml doses in whirlpac® bags and
 frozen at -20° C until needed. Bass-zebrafish alarm cue
 was collected by moving each bass to separate 37-L aquaria. Each bass was
 restricted to one half of the aquarium using a coroplast® barrier to limit
 diffusion and dilution of any alarm cue released during predation.
 Filtration was turned off during collection of alarm cue to cease all
 water flow in the aquarium. A live zebrafish from the same stock from
 which the alarm cue was prepared (ca. 39 mm TL) was dropped into the side
 of the tank containing the bass. The bass struck and ingested the
 zebrafish. We waited 15 s to allow time for alarm cue to diffuse from the
 bass, then we immersed a 1-L beaker with a gloved hand to collect water
 from the site of predation adjacent to the bass. We repeated this
 procedure for each of the three bass. The three 1-L water samples were
 combined together in a 20-L liter pail, from which 20, 60-ml doses were
 aliquoted into whirlpac® bags and frozen at -20° C until needed.
 <strong>Experimental
 apparatus</strong><br>Twelve 37-L tanks were arranged on a
 shelf in the laboratory with the short side facing the aisle. Tanks were
 filled with dechlorinated tap water. Each tank was fitted with an
 air-powered sponge filter and a shelter object (8 × 8 cm ceramic tile with
 a supporting leg on each corner approximately 6 cm tall). Black coroplast®
 dividers were placed between tanks to visually isolate test subjects.
 Airline tubing approximately 2 m in length was inserted into the lift tube
 of the sponge filter to allow for surreptitious injection and rapid
 dispersal of test cues. Gridlines approximately 5 x 5 cm were marked on
 the front pane of each tank for scoring activity (number of lines crossed)
 and vertical distribution (horizontal rows numbered 1 (bottom) through 5
 (surface) (Wisenden 2011). <strong>Experimental
 protocol</strong><br>Individual zebrafish were arbitrarily
 selected from stock tanks and placed into each 37-L tank and allowed to
 acclimate for 24 h. Prior to the start of each trial, 60 ml of tank water
 was withdrawn through the injection tubing and discarded to rinse the
 tubing. A second 60 ml was withdrawn and retained to be used as a flush to
 fully dispense cue treatments into test aquaria. Trials were completed in
 groups of four representing one of each treatment type, with the order of
 the treatment type and the order of tanks being tested both randomized. A
 dose of each treatment type was thawed in a cup of warm deionized water
 and drawn into a 60 ml syringe. Overhead ceiling room lights were shut off
 leaving only the above-tank lights on to decrease the visibility of
 observers to test subjects. Each zebrafish was observed for 5 min before
 cue was introduced (pre-treatment observation period). We recorded (1)
 Activity, as the total number of grid lines crossed by the fish over each
 5-min observation period, (2) Vertical Distribution as the horizontal row
 occupied by the fish every 10 s, and (3) Time in Shelter as time (s) spent
 under the shelter object or hidden behind the sponge filter. When the
 pre-stimulus observation period was over, one of the four test cues was
 gently injected into the aquarium followed by a flush of 60 ml of
 previously-retained tank water. Post-stimulus behavioral observation
 commenced immediately for another 5 min, recording activity, vertical
 distribution, and time in shelter as before. When each trial was finished,
 the zebrafish was moved to a separate 190 L holding tank so that each fish
 was tested only once. Test tanks were drained, rinsed, and refilled with
 dechlorinated water between subsequent trials. Injection tubes were
 replaced after each trial. We conducted 15 replicates of each treatment,
 for a total of 60 trials.