Background choice and immobility as context dependent tadpole responses to perceived predation risk

Study site

Experiments were conducted in the Reserva Particular do Patrimônio Natural (RPPN) Santuário do Caraça, a private conservation unit in the southern portion of the Espinhaço Mountain range, Minas Gerais state, Brazil. The climate is seasonal, with a rainy period from October to March, and a dry period from April to September. Mean air temperatures vary between 13 and 29^oC²⁶.

The focal species of this study was Ololygon machadoi. The tadpoles of this treefrog have been previously shown to react to both visual and chemical predator cues (from Belostoma testaceopallidum; Melo et al.¹⁸) by positioning themselves preferentially on yellow backgrounds where they are disruptive^17,18. Ololygon machadoi breeds year-round in many streams in the RPPN, and we used one of these streams (20^o05′37″S, 43^o29′59″W; 1293 m above sea level), where its tadpoles are abundant, to conduct the experiments. It is a small first order stream²⁷ with sandy or rocky bottoms. Stream bank vegetation is dense, composed of herbs, shrubs and trees. In the vicinities of the point where we conducted the experiments (and up to 150 m upstream) stream width ranges from 2 to 6 m, and stream depth, from just a few centimeters to about 1 m²⁸.

Response to predator cues in the natural habitat

We built three enclosures measuring 40 × 35 cm with a plastic mesh around a metal frame that limited two compartments, one measuring 35 × 35 cm, and another contiguous to it measuring 5 × 35 cm. Both compartments were 15 cm high and were open both below and above. We set the enclosures at a stream section of shallow water and flat rocky bottom, where water filled the enclosures up to about 5 cm (Fig. 4). We placed the enclosures with the small compartment upstream, so that the tadpole in the larger compartment would be exposed to both visual and chemical cues of the predator to be introduced in the smaller compartment. We collected three water bugs (Belostoma testaceopallidum) to be used in the experiments in a nearby stream from the same water basin (20^o06′40″S, 43^o28′48″W; 1,254 m above sea level). We collected the tadpoles very close to the enclosures (up to 5 m upstream).

Before we started each trial, we inspected the area covered by the enclosures to make sure no tadpoles or other animals remained inside. We then sealed the bottom carefully with sand from the same stream and placed one tadpole within each cage, in the larger compartment. We waited 3 min, sufficient for the tadpole to return to normal activity levels after translocation to the enclosures¹⁷, and then we recorded whether each tadpole was moving or standing still in 30 s intervals, during 15 min for a total of 30 observations. When tadpoles moved, they always moved on the bottom, never through the water column. After this, we waited another 3 min and repeated the 30 movement records for the next 15 min. We then removed the water bug and repeated another observation turn (waited 3 min, then made 30 movement observations separated by 30 s intervals). After each individual tadpole was tested, we released it downstream, to avoid using the same individual more than once. Tadpoles were all in developmental stage 25²⁹ and measured 20.2 ± 2.4 mm (n = 33 tadpoles measured). We maintained the water bugs in individual recipients with clean stream water and used them randomly in the three enclosures. After all the experiments we collected them for identification.

We tested 3 tadpoles simultaneously, then restarted the whole experiment with other 3 tadpoles, and so on, until we tested 54 tadpoles during three consecutive days (3–5 October, 2018). The weather was sunny with some clouds and short periods of light rain, during which we did not conduct experiments.

Defensive responses based on previous experiences

For this experiment, we collected stage 25 tadpoles at the same stream section and kept them for no more than 2 h in polystyrene boxes with stream water by the nearby (about 1.3 km) lodging of the RPPN Santuário do Caraça, where we conducted the experiments to test the influence of previous experience on tadpole background choice and immobility. We placed individual tadpoles in plastic trays measuring 43 cm length, 30 cm width, 9 cm height, with half the bottom covered by a picture of a natural yellow background (rocks in its natural habitat). The other half was covered with the same picture manipulated digitally to match the hues and luminance of natural dark backgrounds, as in¹⁷. We filled the trays with tap water that comes straight from the main stream at the reserve, replacing the water at every trial. For each trial, we waited 3 min. after tadpole placement in the center of the tray, then we observed tadpoles for 30 min, recording their background every minute. After that, we applied one of three treatments during a 5-min interval: (1) an aversive stimulus was applied to the tadpole every time it positioned itself on the yellow background, or (2) on the dark background or (3) no stimulus was applied (control). The aversive stimulus consisted in one person approaching a wood stick slowly towards the tadpole until it reacted fleeing. After the treatments, we conducted another 30 min of observations recording tadpole background every minute. The tadpoles were all returned to their original stream after the experiments. We tested 2 tadpoles in each treatment simultaneously and then repeated the trials until tests of 30 tadpoles for each treatment (total 90 tadpoles) were completed. Experiments were performed from 4 to 6 October 2016. Experiments were conducted in the shade with natural light, and all days were sunny.

All the procedures were performed in accordance with relevant guidelines/regulations adopted by the responsible institutions: Sisbio/ICMBio (45302-1, 62316-1) authorized animal manipulation in situ and the Ethical Committee of the Pontifícia Universidade Católica de Minas Gerais (032/2016, 003/2018) approved the experimental procedures in accordance with animal welfare guidelines. The water bugs were identified as Belostoma testaceopallidum Latreille, 1807, and the collected specimens were deposited in the collection of aquatic insects of the Parasitology Department of the Institute of Biological Sciences (DPIC) of the Federal University of Minas Gerais, Belo Horizonte, Minas Gerais state, Brazil, under the accession number 9498.

Statistical analyses

We compared the level of activity of tadpoles (given by the number of instant positive records of movement) before, during, and after the presence of the water bug in the enclosures in the stream. We also evaluated a possible effect of direct sun incidence or shade on the enclosures³⁰, and its interaction with tadpole activity levels. We built Generalized Linear Mixed Models (GLMM) with the packages “car”³¹ and “MASS”³² in R³³. We considered number of movement records + 1 (to adjust to distributions that must be non-zero) as the dependent variable, phase (before, during, or after the presence of the water bug) and light (sun or shade) as explanatory variables, and individual tadpole as a random variable. Tadpoles might present different reactions to predators based on their previous experiences³⁴. Considering individual as a random variable would also account for possible differences among times of the day and cages on individual behaviour. We built models including each one or both explanatory variables, with or without their interaction. We compared these models with a null model that included only the random variable, in order to identify the variable(s) with the strongest explanatory power.

We also used GLMMs to test for the ability of tadpoles to avoid a background colour after an aversive experience on it. Since tadpoles had to choose between dark and yellow, we arbitrarily used the number of records on dark backgrounds as dependent variable, because the records on yellow would represent the alternative situation (not on dark). We used treatment and phase (before and after the treatments were applied) as fixed variables and tadpole as a random variable. In order to test for the expression of immobility after the aversive stimuli, we considered the number of times tadpoles changed background colour in consecutive observations as a surrogate for tadpole movement (our dependent variable), treatment and phase as fixed variables and tadpole as a random variable.

We used the package MuMIn³⁵ for R³³ to select the best models, a procedure recommended to control the overall type I error rate³⁶. We conducted Tukey post hoc tests with the package emmeans³⁷.

Source: Ecology - nature.com