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Cuad. herpetol., 2121 (1):(1): 4545–52,–52, 20072007 45

BEHAVIOURAL RESPONSES OF TADPOLES (ANURA: LEPTODACTYLIDAE) TO FISH CHEMICAL CUES

SCARABOTTI, PABLO A.; JAVIER A. LÓPEZ & ROMINA GHIRARDI Instituto Nacional de Limnología, José Maciá 1933, (3016) Santo Tomé, Santa Fe, Argentina. [email protected].

R E S U M E N. — Las larvas de muchas especies de anfibios viven en ambientes con vegetación y de aguas turbias, donde la visibilidad es muy limitada. En estos ambientes, la utilización de señales químicas para la detección de depredadores podría ser más confiable que la utilización de señales visuales. Las larvas de Odontophrynus americanus habitan en las lagunas del valle aluvial del río Paraná Medio, Argentina, donde normalmente se dan condiciones de baja visibilidad. La hipótesis de este trabajo fue que los renacuajos de esta especie pueden detectar a los depredadores y responder subsecuentemente con conducta antipredador ante la percepción de señales químicas de los depredadores. En el presente trabajo se llevaron a cabo tres experimentos de laboratorio para evaluar si los renacuajos de O. americanus responden conductualmente al estímulo químico del pez Astyanax fasciatus. Las respuestas conductuales fueron evaluadas observando cambios en actividad, utilización de refugio y distribución espacial de los renacuajos. En presencia del estímulo químico del pez, los renacuajos redujeron su actividad en un 27%, incrementaron cinco veces la utilización de refugios, pero no exhibieron una respuesta de elusión espacial al estímulo del depredador. Las respuestas conductuales de los renacuajos de esta especie podrían reducir las probabilidades de encuentro y ataque por parte de los depredadores. Palabras claves: Mecanismos antipredador, larvas de anfibios, Odontophrynus americanus, Astyanax fasciatus.

A B S T R A C T. — Many larvae live in turbid and vegetated habitats with very limited visibility. In these habitats, the use of chemical cues for predator detection may be more reliable than visual ones. Odontophrynus americanus tadpoles inhabit floodplain ponds of the middle Paraná River, Argentina, where they frequently encounter low visibility conditions. We hypothesize that tadpoles of this species can detect predators and subsequently respond with antipredator behaviour by means of chemical cues from predators. In the present work, we ran three separate laboratory experiments to evaluate whether O. americanus tadpoles respond behaviourally to a chemical stimulus of the characid fish Astyanax fasciatus. We evaluated behavioural responses by observing changes in activity level, refuge use and spatial distribution of the tadpoles. In the presence of the fish chemical stimulus, tadpoles reduced their activity by 27% and increased refuge use approximately five-fold, but they did not exhibit spatial avoidance from the predator stimulus. The behavioural responses of the tadpoles of this species may reduce the rate of encounter and attack by predators. Keywords: Antipredator mechanisms, anuran larvae, Odontophrynus americanus, Astyanax fasciatus.

INTRODUCTION The ability of some amphibian larvae cies that co-occur with fishes (Kats et to respond behaviourally to waterborne al., 1988; Pearl et al., 2003; Hickman et chemical cues from predators is well al., 2004). Common behavioural respons- documented and represents a valuable es of tadpoles to predators are: reduced strategy to reduce predation risk in spe- activity, increased refuge use and spa-

Recibido: 05/09/06 — Aceptado: 23/02/07 Ed. asoc.: Marissa Fabrezi 46 P. A. SCARABOTTI et al.: Tadpole Behavioural Responses tial avoidance (Petranka et al., 1987; from the main channel into intermittent Semilitsch and Reyer, 1992; Schemidt drainages and reach the floodplain ponds and Amézquita, 2001). These behav- (Bonetto et al., 1969; Lowe-McConnell, ioural changes can diminish predation 1987; Junk et al.; PAS, unpubl.). Asty- rates but also generally decrease feed- anax fasciatus is a widespread characid ing and reduce growth and size at fish that occurs frequently in these hab- metamorphosis (Skelly and Werner, itats (Cordiviola de Yuan, 1992). Al- 1990). This trade-off between growth though individuals seldom reach 10 cm rate and predation risk is the central in length, its voracity and abundance component of the current theoretical (Ringuelet et al., 1967) make this fish approaches that explain the plasticity of an effective potential predator. antipredator responses (Van Buskirk, In this study, we examine whether 2000). tadpoles of O. americanus, change activ- In larval , chemical cues ity level, refuge use, and spatial distri- often permit discrimination of predator bution when presented with chemical and non-predator heterospecifics more cues from this common fish predator. effectively than visual cues, favouring We predicted that O. americanus tad- more precise antipredator responses poles, which frequently inhabit turbid (Kiesecker et al., 1996; Hickman et waters where visual cues are not avail- al., 2004). In turbid water habitats, the able, should respond behaviourally to use of chemical cues can allow earlier the chemical cues of A. fasciatus by de- detection of the predator, allowing creasing activity, increasing refuge use more successful evasion of predators and exhibiting spatial avoidance from (Lima and Dill, 1990; Hickmann et al., predator chemical cues. 2004). Mathis and Vincent (2000) sug- gested that the priority use of chemical over visual cues in larval salamanders MATERIAL AND METHODS could be explained by the difficulty of detecting cryptic predators, the low vis- Recently hatched tadpoles of Odon- ibility in turbid or vegetated habitats, tophrynus americanus were collected and the poor development of visual with a dip net at a roadside pond in the systems (myopia). west suburbs of Santa Fe city in April Odontophrynus americanus is a 2004 (Santa Fe province, Argentina) South American leptodactylid with (31º39’52” S, 60º44’00” W). Tadpoles markedly terrestrial habits that spawns were transported to a laboratory for in puddles and streams (Cei, 1980). Tad- testing. Historically this site belonged to poles are benthic-nektonic and inhabit the floodplains of the Paraná and Salado temporary and semipermanent ponds in rivers, but currently the pond is isolat- the floodplain of the middle Paraná Riv- ed from them by water retaining walls. er, east-central Argentina, where they In spite of this, the proximity to the frequently co-occur with fishes (Peltzer floodplains (less than 1 km) and the and Lajmanovich, 2004). Tadpoles of O. nature of the retaining walls allow free americanus are also found in roadside migration of adult O. americanus. Tad- temporary ponds where they rarely en- poles were maintained in cylindrical 25 counter predatory fishes. These flood- cm diameter glass aquaria, filled with 6 plain habitats are characterized by very cm of aged dechlorinated tap water and turbid water and vegetated margins were fed ad libitum with boiled lettuce. (Ludwigia sp.) where the tadpoles of O. Water was replaced every other day. americanus are commonly encountered Specimens of Astyanax fasciatus (Lajmanovich, 2000). During high water (mean ± SD standard length = 6.26 ± periods (flood pulse) many fishes move 0,62 cm; n = 10) were collected from a Cuad. herpetol., 21 (1): 45–52, 2007 47 permanent pond on Los Sapos Island tifiable, we counted the number of (Santa Fe Province, 2 km west of Santa times any tadpole made a swimming Fe City, 31º39’58” S, 60º45’09” W), with- movement during a period of 5 min. in the flood plain of the Salado River, Once the test finished, we removed the with a 10 M wide haul. Fishes were tadpoles carefully with a dip net and kept in a continuously aerated 10 l thoroughly rinsed the aquarium with aquarium and were fed with Hypsiboas tap water. We filled the aquarium with pulchellus tadpoles collected from the fish conditioned water and gently placed same pond. Throughout the experimen- the same group into the aquarium. After tal period, were kept in con- a new 10 min acclimation period we trolled lab conditions, at 25 ± 2 ºC and again measured the activity level. We on a 14:10 h light: dark photoperiod considered each group of ten tadpoles with fluorescent overhead lamps. as a single replicate and had 10 repli- We measured three behavioural re- cates (N = 100 tadpoles). sponses of tadpoles to fish chemical In the second experiment, we tested stimuli in successive experiments: (1) whether groups of tadpoles responded activity level, (2) refuge use, and (3) to chemical fish stimulus by increasing spatial avoidance from the predator the use of artificial refuges. Testing chemical stimulus. In all experiments took place in a manner identical to ex- we used dechlorinated tap water as con- periment 1, except that we employed a trol and fish conditioned water as stim- 35 x 17 x 20 cm aquarium and before ulus. Water conditioning involved plac- introducing the tadpoles, we placed ing 10 fishes in a continuously aerated eight refuges (uniformly distributed) on 15 l aquarium filled with dechlorinated the aquarium bottom. The refuges were tap water 24 hours before tests started. semi-cylindrical tunnels, each 5 cm long Fishes were fed just prior to placement and 3 cm wide, made from longitudinal- into the stimulus collection aquaria but ly divided plastic tubes. For each test, were not fed during the water condi- we recorded the number of tadpoles in- tioning period. Tadpoles used in all ex- side refuges at 1 min intervals for 10 periments were between Gosner (1960) min. The groups of tadpoles were differ- developmental stage 28 and 36. ent from those used in experiment 1. In the first experiment, we tested Again, we had 10 replicates (10 groups whether O. americanus tadpoles re- of ten tadpoles, N = 100 tadpoles). duced their activity level when exposed In the third experiment we mea- to chemical cues from A. fasciatus. In sured the spatial avoidance of groups of this experiment we formed 10 groups of tadpoles in response to a fish chemical 10 tadpoles choosing each one randomly stimulus gradient. We used an aquarium from the stock available. For each of the same size as in experiment 2 di- group we measured the activity level vided longitudinally into 4 equal quad- first in dechlorinated tap water and sub- rants via lines drawn on the bottom sequently in fish stimulus water. Tad- and walls of the tank. To generate the poles were tested in a cylindrical 25 cm gradient of fish stimulus, we placed diameter glass aquarium filled to a level dropping systems 1 cm away from the of 8 cm with the stimulus water. We wall at both ends of the aquarium. In placed a group of 10 tadpoles in the each test we placed one dropping sys- aquarium that were allowed to acclimate tem containing fish conditioned water at for 10 min. Odontophrynus americanus one end, and another containing de- tadpoles are characterized by moderate chlorinated tap water at the other end activity with rapid short movements sep- of the aquarium. Each dropping system arated in time by inactive periods. consisted of a 50 ml buret regulated to Since these movements are easily quan- release 1 drop/second. The buret was 48 P. A. SCARABOTTI et al.: Tadpole Behavioural Responses placed just above the water surface to To analyse the spatial distribution reduce the disturbance caused by the data from experiment 3, we compared drop. To determine the spatial distribu- the data to the null value of 2.5 by tion of the tadpoles we scored the num- means of a one-sample t test. ber of larvae in each quadrant at 1 min intervals for 10 min. After each test we changed the position of the aquarium RESULTS or swapped the end corresponding to fish stimulus water with the control Tadpoles showed a mean decrease in stimulus to avoid positional bias. The activity level of 27% in the fish condi- spatial affinity index S (Fishwild et al., tioned water treatment (57.8 ± 15.4) 1990) was adapted to estimate the mean relative to the control (81.3 ± 18.7) quadrant position of the group of tad- (paired t-test, n = 10, t = 3.64, P < poles, and is calculated as follows: 0.01). The activity in the fish stimulus treatment decreased by 23.5 ± 20.44 S = 1(A) + 2(B) + 3(C) + 4(D) movements relative to the control (Fig- N ure 1A). Tadpoles used refuges significantly where A, B, C and D represent the to- more often in the presence of a fish tal number of tadpoles scored in each stimulus (2.33 ± 1.20 sheltered individu- of the four numbered quadrants during als) than in control water (0.45 ± 0.27 a 10 min test and N represents the to- sheltered individuals) (paired t-test, n = tal number of tadpoles scored in all 10, t = –4.84, P < 0.001). On average, quadrants. This index ranged from four for each group of tadpoles, the fish con- (maximum avoidance to fish) to one ditioned treatment showed an increase (maximum affinity to fish). Values high- of 1.88 ± 1.23 sheltered individuals er or lower than 2.5 indicate spatial af- (Figure 1B). Although this difference finity, whereas values around 2.5 indi- was small in relation to the number of cate no preference for either stimulus. tadpoles, it represents a five-fold in- This kind of index is recommended be- crease in the number of sheltered indi- cause it includes information from all viduals. four quadrants in the aquarium, provid- Tadpoles did not show spatial avoid- ing a finer estimate of the mean posi- ance from the aquarium side with tion of the test tadpoles (Fishwild et chemical fish stimulus (2.53 ± 0.37; one al., 1990). sample t-test, n = 10, t = 0.15, P = 0.8830) (Figure 1C). In six groups the Statistical Analysis.— For experi- spatial affinity index was greater than ment 1, we calculated the mean num- 2.5, and in the remaining four groups it ber of movements during each test as was lower than 2.5. the response variable. In experiment 2, we calculated the average number of tadpoles inside the refuges during each DISCUSSION test. Normality of the data for the three experiments was confirmed with Our results show that O. americanus Kolmogorov-Smirnov tests. Since data tadpoles respond behaviourally to a for the control and fish conditioned wa- chemical fish stimulus by changing activ- ter treatments from the same group of ity level and microhabitat use. Since tad- tadpoles were not independent, we de- poles used in the experiments had no termined the significance of the differ- previous exposure to fish stimuli, the ences between these treatments by us- antipredator behaviour observed is pre- ing paired t-tests. sumed to be a genetically determined Cuad. herpetol., 21 (1): 45–52, 2007 49 trait, and thus an evolutionary mecha- (Petranka et al., 1987, Kats et al., nism in response to predation risk. 1988, Pearl et al., 2003). The kind of Some studies have shown that tadpoles microhabitats available can strongly in- raised from eggs in the laboratory were fluence tadpoles’ susceptibility to fish able to respond behaviourally to fish predation (Hews, 1995). Hero et al. predators (Kats et al., 1988) and to dis- (2001) suggested that the use of a ref- criminate between predatory and non- uge (e. g. leaf litter) could be the only predatory fishes (Pearl et al., 2003). Al- defense against predators by palatable though our data strongly suggest that O. species that frequently coexist with fish- americanus tadpoles are able to respond es. In laboratory aquaria, A. fasciatus to unfamiliar predators (since tadpoles preyed on tadpoles of O. americanus were obtained from a fishless pond) the (unpublished data), suggesting that potential instinctive nature of this re- these tadpoles are palatable at least to sponse should be further examined by A. fasciatus. In this case, the change in the collection of fresh eggs in the field refuge use by tadpoles could reduce and subsequent raising of the tadpoles in their exposure to predators, thus dimin- a neutral laboratory environment. ishing predation risk. The use of chemical cues to detect The lack of spatial avoidance in re- predators in O. americanus could allow sponse to the chemical stimulus indi- this species to respond behaviourally to cates that tadpoles do not respond to fish presence in the turbid and highly the gradient generated in the laborato- vegetated habitats in the Paraná River ry. Presumably, in the habitats where floodplain. Chemical stimuli may provide O. americanus occur, chemical gradients aquatic animals with critical informa- tion, especially at night, in turbid or in highly vegetated habitats where visual information is not available. This could be essential for detecting cryptic ambush predators (Kiesecker et al., 1996). Reduction in activity level in larval amphibians has been suggested to be re- lated with decreased predation risk (Skelly, 1994; Schemidt and Amézquita, 2001). This disminution in predation risk can be attributed to a lower encounter rate with predators (Abrams, 1984) and, in cryptic prey, to a reduction in the rates of detection and attack (Skelly, Figure 1. Mean responses (± 1 SD) of tadpoles 1994). Fishes are generally thought to in each of the three behavioural tests. (A-B) preferentially attack active prey, detect- Mean differences between the control and stim- ed either visually or via the lateral line ulus treatments in activity level and refuge use (Bleckman, 1993; Fitzgerald and Wooton, respectively. Negative differences indicate a re- 1993). By lowering activity, O. america- duction in the variable in the fish stimulus nus tadpoles could reduce encounter treatment in relation to the control, and posi- rate with fishes. Also, due to their cryp- tive differences indicate an increment in the values of the variable. The null value of 0 tic coloration, they might reduce the (dashed line) indicates no differences between rates of detection and attack, thus reduc- control and stimulus treatment. (C) Mean val- ing mortality risk. ues of the spatial distribution index. Dashed Several studies have documented a line represents the null value of 2.5. Variables change in microhabitat use by larval used in each behavioural test are described in amphibians in response to predators the text. 50 P. A. SCARABOTTI et al.: Tadpole Behavioural Responses

are either not a good indicator of the ACKNOWLEDGEMENTS position of a fast moving predator such as A. fasciatus, or simply these gradi- This study was supported by a doc- ents are never generated. In laboratory toral fellowship from the Consejo Nacio- observations, tadpoles remain inactive nal de Investigaciones Científicas y Téc- in the bottom of the aquarium until a nicas de Argentina (CONICET) to P. A. fish predator is close enough to make a Scarabotti and J. A. López. We thank strike. We observed that when this hap- Michelle Brett and Gabriela Perotti for pens, tadpoles flee rapidly to another comments and suggestions on the position. Possibly, at short distances, manuscript. Cristián De Bonis helped in the combination of mechanical and visu- preparation of figure 1. al stimuli is a much more efficient indi- cator of the position of fish predators to trigger spatial avoidance behaviour. LITERATURE CITED Hickman et al. (2004) observed that Eurycea multiplicata griseogaster re- ABRAMS, P. A. 1984. Foraging time op- sponded to banded sculpins, Cottus caro- timization and interaction in food linae, in the field by showing flight and webs. The American Naturalist burrowing behaviour. However these re- 124: 80-96. sponses were not observed in the labora- BLECKMAN, H. 1993. Role of the lateral tory. They suggested that these respons- line in fish behaviour: 201-246. es were not practical in simplified labo- En: PITCHER, T. J. (ed.), Behav- ratory chambers. Although we did not iour of teleost fishes. Chapman perform experiments in the field, we ob- and Hall. London. XX + 715 pp served that in natural habitats, O. amer- BONETTO, A. A.; E. CORDIVIOLA DE icanus tadpoles flee away and take ref- YUAN; C. PIGNALBERI & O. B. OL- uge in the leaf litter or in rooted vegeta- IVEROS. 1969. Ciclos hidrológicos tion in response to any physical distur- del río Paraná y las poblaciones de bance like vibrations and sudden chang- peces contenidas en las cuencas es in light intensity. Consequently, al- temporarias de su valle de inun- though we did not observe a spatial dación. Physis 29 (78): 213-223. avoidance response in the laboratory, CEI, J. M. 1980. Amphibians of Argenti- tadpoles could exhibit this antipredator na. Monitore Zoologico Italiano behaviour in natural habitats, where N.S. Monografia 2. XII + 609 pp spatial and refuge possibilities make this CORDIVIOLA DE YUAN, E. 1992. Fish response more practical. populations of lentic environments The geographic range of O. america- of the Parana River. Hydrobiolo- nus reaches western regions of Argenti- gia 237 (3): 159-173. na where A. fasciatus and most of the FISHWILD, T. G.; R. A. SCHEMIDT; K. fishes found in the floodplains of the M. JANKENS; K. A. BERVEN; G. Paraná River are absent. In these envi- J. GAMBOA & C. M. RICHARDS. ronments tadpoles rarely encounter 1990. Sibling recognition by larval fish, and the biotic and abiotic charac- (Rana pipiens, Rana sylvati- teristics (e. g. turbidity, vegetation) of ca, and Pseudacris crucifer). Jour- the habitats are very different. Compar- nal of Herpetology 24 (11): 40-44. isons of the antipredator responses be- FITZGERALD, G. J. & R. J. WOOTON. tween populations that vary in their 1993. The behavioural ecology of encounter rates with fish may reveal sticklebacks: 537-572. En: PITCH- interesting patterns of local adaptation ER, T. J. (ed.), Behaviour of teleo- to the selective pressures in the river st fishes. Chapman and Hall. Lon- floodplains. don. XX + 715 pp. Cuad. herpetol., 21 (1): 45–52, 2007 51

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