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Repulsion from Chemical Cues in Bufo marinus (Cane Toad) Tadpole s Brittany Kolehmaine n Department of Biology, Central Michigan University ABSTRACT Predator prey relationships have been studied relentlessly throughout all different taxa and systems. Larval anurans use the mode of chemosensory to detect and avoid predation. By exposing Bufo marinus tadpoles to a variety of natural chemicals (crushed conspecific larvae and metamorphs, and crushed annelid prey) a strong repulsion was demonstrated. Avoi dance of the treatments verified that Bufo marinus tadpoles are sensitive to changes in the chemical make up of their environment that could be caused by a predator in close proximity. RESUMEN L as relaciones de la Despredador presa se han estudiado implac ablemente a travs de todas las diversos taxus y sistemas. Los anurans larvales utilizan el modo de chemosensory para detectar y para evitar la depredacin. Exponiendo los tadpoles de Bufo m arinus a una variedad de productos qumicos naturales (las larvas y los metamorphs conspecific machacados, y presa anlida machacada) una repulsin fuerte fue demostrada. La evitacin de los tratamientos verific que los tadpoles de Bufo m arinus sean sensibles a los cambios en el maquillaje qumico de su ambiente que se podra causar por un depredador en gran proximidad. INTRODUCTION Being able to detect and avoid predators is vital to survival in nature ( Lefcort 1998; Petranka & Hayes 1998; Summey & Mathis 1998; Laurila 2000; Laurila et al 2002 ) There are numerous mod es of detection such as sight, sound, and even thermosensory. The common mode in which many anuran larvae use to perceive predators is chemosensory ( Laurila, Kujasalo, & Ranta 1998; Lefcort 1998; Petranka & Hayes 1998; Summey & Mathis 1998; Chivers et al 1999; Gallie, Mumme, & Wissinnger 2001; Hagman & Shine 2 008; Pearl et al 2003 ) This ability to receive and interpret chemical cues is not only used for predation avoidance; it is also used for species identification (Gallie, Mumme, & Wissinnger 2001) a nd prey recognition by some tadpoles ( Chivers et al 1999; Hagman & Shine 2008 ) But when it comes to evading predators chemosensory is especially useful since chemicals can be perceived while still in hiding, thus not having to reveal ones self, or wit h enough of a warning to allow an individual to take hiding or flee before the predator is able to locate the individual being pursuit ( Lefcort 1998; Petranka & Hayes 1998; Summey & Mathis 1998 ; B uskirk & A rioli 2002 ) It has been shown in many prior stu dies that chemical cues induce plastic behavior responses in many aquatic larvae ( Werner & Anholt 1996; Laurila, Kujasalo, & Ranta 1998; Laurila 2000; Laurila et al 2002; Relyea 2002 ) Such responses vary greatly; some behaviors are aggregation, dispersio n, decreased movement, and increased movement ( Anholt, Skelly, & Werner 1996; Lefcort 1998; Petranka & Hayes 1998;
Summey & Mathis 1998; Chivers et al 1999; Eterovick & Sazima 199 9; Laurila 2000; Relyea 2002; Pearl et al 2003; Hagman & Shine 2008 ) In the case of Bufo marinus tadpoles, it has been shown that they react in a repulsive manner to the chemicals releas ed from injured conspecifics and that several other stimulus do not trigger an avo idance response (Hagman & Shine 2008). Although Bufo marinus tadpoles do have poisons in their skin (Crossland & Azevedo Ramos 1999), it is unknown whether it is the release of poisons or the release of some other "alarm pheromone" that triggers this repulsion response ( Laurila, Kujasalo, & Ranta 1998; Summey & Math is 1998) It is curious though since in the aforementioned experiment (Hagman & Shine 2008) one of the stimulus treatments consisted of lettuce extract as a prey item. Although tadpoles have an extensive list of possible prey items (Savage 2002) lettuce is not one of which they would encounter naturall y. The following experiment further investigates the reaction to different chemicals encountered naturally by Bufo marinus tadpoles. Each chemical treatment indicates either a prey item or predator in cl ose proximity feeding on a conspecific. Predator cues released from injured conspecifics, both in the larval and metamorph stage, are expected to cause a repulsion response. Prey cues released from injured annelids are expected to cause a congregating, a ttraction effect. METHODS Natural History Bufo marinus g estation within the eggs can last between 36 hours and 4 days (Lampa & Leo 1998; Savage 2002) The larvae are not extremely large especially considering the massive adults they will become; in th eir largest tadpoles stage they have a length of only 24mm While in their larval form, B. marinus are very social and can be found in large ag gregations (Savage 2002) Collection Collection of the B. marinus tadpoles occurred during the late dry season in Monteverde, Costa Rica from an artificial pond l ocated behind the establishment on the property of Marvin Hidalgo Tadpoles were collected early in the morning of each testing day and released the same day in the afternoon after testing was complete. The larvae were extremely abundant with the estimated population over 1 500 (estimations made by observation). The pond ha d two gentle sloping shorelines; one on the north shore and one on the south shore. The easily accessible shorelines not only made c apture relatively simple but they also allowed capture to alternate daily between each shore to reduce the chances of recapture. This was significant because in theory each tadpole was only to be used once. A handled sieve was used to aid in capture as w ell as a small cooler for transportation from the pond up to the Biological Station. Once at the Biological Station tadpoles were then divided into groups of 50 individuals and each set was contained in a separate plastic bowls. Set Up The testing arena consisted of a l arge glass tank (70x70x30cm ; Fig. 1 ) with a grid of 100 squares (7x7cm) divided into 10 zones underneath it Zone 1 was determined by the origin of the treatment being introduced. Thus, Zone 1 had the highest concentration
in the gradient of the treatment. Zone 2 was determined as all of the squares that touched Zone 1, therefore, having the second highest concentration. Zone 3 then was named those squares that touched Zone 2. Each zone followed this pattern sequentially, hence, making Zone 10 those squares that are the very furthest from the point of origin and consequently then having the lowest concentration of the treatment in the gradient. FIGURE 1 Depiction of the Zone layout of the arena grid Zone 1 had the strongest concentration of treatment; it was the origin point for treatment distribution. Zone 10 had the weakest concentration of treatment in the gradient. The numbers were not written on the grid during any of the trials. For each trial the tank was emptied, cleaned and filled 6cm deep (approximately 30 liters) with fresh water. Each trial required a new set of 50 tadpoles which were added into the center of the tank and allowed a 5 minute adjusting period. The treatment was introduced in one corne r via a 50 ml burette ; the tip of which was slightly under the surface of the water to reduce the effect ripples/surface dist urbance could have on the results Eac h treatment was diluted into 1 l iter of water for each trial. The 1 liter of treatment was completely added to the tank over the first 30 minutes of the trial (approximately 160ml every 5 minutes). Three trials were ran per each treatment and each trial lasted for an hour. During that hour pictures were taken every 5 minutes to capture the mov ement of the B. marinus larvae. As a visual aid of the gradient 3 drops of red food coloring were added to each liter of treatment. Treatments Test 1 Larval Conspecific Chemical Cues To test the effect of chemical cues released from injured conspeci fics 2g of B. marinus tadpoles were macerated and diluted into 1 liter of water for each of the 3 trials. The individuals that were crushed were casualties from another peers experiment. So, no tadpoles were injured for this experiment.
Test 2 Dye Co ntrol The first control was to test the effect the food coloring had on the larvae. This was accomplished by adding 3 drops of the dye to 1 liter of fresh water for each of the 3 trials. Test 3 Annelid Prey Chemical Cues This stimulus was found and collected as it was being preyed upon by tadpoles while in the pond. Two individuals were found, they were distributed betwee n 3 trials. T hus making it so that each trial had approximately 0.7g of annelid that was macerated into 1 liter of water added to it. Test 4 Human Control To ensure that taking pictures had no human induced effect on the movement of the tadpoles this treatment actually consisted of nothing. No actual treatment was added to the tank. Only pictures were taken each 5 minute increm ent. Test 5 Metamorph Conspecific Chemical Cues This stimulus tested the effect that injured metamorphs of B. marinus had on the larvae. To ensure that the mortality rate remained at 0 for this experiment a search was carried out around the ponds edge to find metamo rphs that had already drowned; 3 individuals were found. Each trial consisted of approximately 0.33g of crushed metamorphs diluted into 1 liter of water. Data Analysis The number of tadpoles per zone was then tallied up for each 5 minute increment. Calculations were made to determine the average number of tadpoles per square per zone to take a way the influence the area of each zone has on the results. Next, a linear regression was run for each of the 5 minute intervals to determine the s lope between time passed and the average number of individuals per square. This allowed the direction of movement to be determined; a positive slope indicates repulsion from the origin and a negative slope proposes attraction or a lack of concern toward t he stimulus (Hagman & Shine 2008) Lastly, a repeated measures ANOVA. RESULTS The preliminary linear regressions displayed an interesting trend (Fig. 2 ). In all of the tests it was observed that as the time progressed any response that had initially oc curred became weaker, as in the slopes moved closer to 0. During actual trials it was observed that the integrity of the gradient declined. Meaning that, as time progressed the stimulus became more uniform throughout the tank instead of maintaining a dec reasing gradient of concentrations from the point of origin. Congruently with the unification of the gradient the number of tadpoles per zone became more unified. Note that Zone 1 was not calculated into the r esults or depicted in the Fig. 2 All but on e of the stimulus clearly resulted in repulsion (Fig 3 ) The conspecific chemical cues, dye control, annelid prey chemical cues, and metamorph conspecific chemical cues all had a positive slope which showed that as the zones move further away from the st imulus point of origin the higher the density of larvae gets. A repeated measures analysis showed that no one stimulus caused a greater repulsion than the others (F (4,9) =1.5087, p=0.2788 ; Fig. 3 )
FIGURE 2 The progression of Bufo marinus tadpole s' movement over a gradient from high concentration (Zone 2) to low concentration (Zone 10) of larval conspecific chemical cues (Test 1). Each linear regression plots the number of tadpoles per zone per 5 minute increments. FIGURE 3 Th is index of repulsion for Bufo marinus larvae from 5 different treatments (Test 1 Larval Conspecific Chemical Cues; Test 2 Dye Control; Test 3 Annelid Prey Chemical Cues; Test 4 Human Control; Test 5 Metamorph Conspecific Chemical Cues ) depicts the sl opes calculated from linear regressions (fig. 3). A positive slope indicates repulsion from the origin and a negative slope proposes attraction or a lack of concern toward the stimulus.
DISCUSSION Repulsion is an effect of natural chemical cues for Bufo marinus tadpoles. It was expected that both the injured larval and metamorph conspecifics would induce a repulsive effect but, it was interesting to see that the injured prey also provoked repulsion. From this prey repulsion it is possible that the same or similar chemical that is released by the conspecifics is universal and also released by the annelids. Another possible explanation is that B. marinus is extremely sensitive to any change in the chemical make up of their surrounding no matter what chem ical is being introduced; this would help to explain their response to the food coloring and is, also, backed up with the fact that no one chemical induced a greater response than the rest. Another factor to consider is that the gradient deteriorated aft er approximately half the time had passed. This could have had an effect on the magnitude of the response. It would be interesting in future studies to devise a way to continue the gradient completely through the trial. Suggestions to accomplish this ar e a larger tank or less individuals per trial since the unification of the gradient appeared to be propelled by the movement of the tadpoles. The final thoughts for a continuation of this project would be to test the effect corners had on the aggregation o f the tadpoles. It has been suggested in previous studies that once predator reception has occurred, tadpoles move into smaller spaces or crannies and then reduce all movement ( Eterovick & Sazima 1999). This was exhibited in Zone 1, thus is the reason Zo ne 1 was not calculated into the results. Predation avoidance via chemosensory is an intricate and interesting behavior that effects anuran larvae movement drastically. Development and further investigation in the understanding of the chemical cues receiv ed by larval anurans will give a clearer picture as to what behavior is to be elicited and why. ACKNOWLEDGMENTS My sincerest gratitude goes to Pablo Allen Monge and the rest of the CIEE staff for allowing me draw upon their knowledge and experience which made this project possible. Also, to Marvin Hidalgo for allowing me to collect tadpoles from the pond found on his property. And lastly my thanks go to Brenna Levine for sharing her study site and knowledge LITERATURE CITED Anholt, B. R., Skelly, D. K., and Werner, E. E. 1996. Factors Modifying Antipredator Behavior in Larval Toads. Herpetologica. 52:3. 301 313 Buskirk, J. and Arioli, M. 2002. Dosage Response of an Induced Defense: How Sensitive Are Tadpoles to Predation Risk? Ecology 83:6 1580 1585 Chivers, D. P., Kiesecker, J. M., Wildy, E. L., Belden, L. K., Kats, L. B., and Blaustein, A. R. 1999. Avoidance Response of Post Metamorphic Anurans to Cues of Injured Conspecifics and Predators. Journal of Herpetology. 33:3. 472 476 Cr ossland, M.R. and Azevedo Ramos, C. 1999. Effects of Bufo (Anura: Bufonidae) Toxins on Tadpoles from Native and Exotic Bufo Habitats. Herpetologica 55:2.
192 199 Eterovick, P. C. and Sazima, I. 1999. Description of the Tadpole of Bufo rufus with Notes on Aggregative Behavior. Journal of Herpetology. 33:4. 711 713 Gallie, J. A., Mumme, R. L., and Wissinger, S. A. 2001. Experience Has no Effect on the Development of Chemosensory Recognition of Predators by Tadpoles of the American Toad, Buf o americanus. Herpetologica 57:3. 376 383 Hagman, M. and Shine, R. 2008. Understanding the toad code: Behavioural responses of cane toad ( Chaunus marinus ) larvae and metamorphs to chemical cues. Austral Ecology 33. 37 44 Lampo, M. and Leo, G. A 1998. The Invasion Ecology of the Toad Bufo marinus : From South America to Australia. Ecological Applications. 8:2. 388 396 Laurila, A. 2000. Behavioural Responses to Predator Chemical Cues and Local Variation in Antipredator Performance in Ran a temporaria Tadpoles. Oikos 88:1. 159 168 Laurila, A., Kujasalo, J., and Ranta, E. 1998. Predator Induced Changes in Life History in Two Anuran Tadpoles: Effects of Predator Diet. Oikos 83:2. 307 317 Laurila, A., Pakkasmaa, S., Crochet, P. A., and Merila, J. 2002. Predator Induced Plasticity in Early Life History and Morphology in Two Anuran Amphibians. Oecologia 132:4. 524 530 Lefcort, H. 1998. Chemically Mediated Fright Response in Southern Toad ( Bufo terrestris ) Tadpoles. Cope ia. 1998:2. 445 450 Pearl, C. A., Adams, M. J., Schuytema, G. S., and Nebeker, A. V. 2003. Behavioral Responses of Anuran Larvae to Chemical Cues of Native and Introduced Predators in the Pacific Northwestern United States. Journal of Herpetology 37:3. 572 576 Petranka, J. and Hayes, L. 1998. Chemically Mediated Avoidance of a Predatory Odonate ( Anax junius ) by American Toad ( Bufo americanus ) and Wood Frog ( Rana sylvatica ) Tadpoles. Behavioral Ecology and Sociobiology 42:4. 263 271 R elyea, R. A. 2002. Competitor Induced Plasticity in Tadpoles: Consequences, Cues, and Connections to Predator Induced Plasticity. Ecological Monographs 72:4. 523 540 Savage, J. M. 2002. The Amphibians and Reptiles of Costa Rica: A Herpetofauna between Two Continents, between Two Seas. The University of Chicago Press 199 202 Summey, M. R. and Mathis, A. 1998. Alarm Responses to Chemical Stimuli from Damaged Conspecifics by Larval Anurans: Tests of Three Neotropical Species. Herpetol ogica. 54:3. 402 408 Werner, E. E. and Anholt, B. R. 1996. Predator Induced Behavioral Indirect Effects: Consequences to Competitive Interactions in Anuran Larvae. Ecology 77:1. 157 169
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Repulsin de seales qumicas en renacuajos Bufo marinus (sapo de caa)
Repulsion from chemical cues in Bufo marinus (Cane toad) tadpoles
Predator-prey relationships have been studied relentlessly throughout all different taxa and systems. Larval anurans use the mode of chemosensory to detect and avoid predation. By exposing Bufo marinus tadpoles to a variety of natural chemicals (crushed conspecific larvae and metamorphs, and crushed annelid prey) a strong repulsion was demonstrated. Avoidance of the treatments verified that Bufo marinus tadpoles are sensitive to changes in the chemical make-up of their environment that could be caused by a predator in close proximity.
Las relaciones de depredador-presa se han estudiado implacablemente a travs de todos los diversos taxones y sistemas. Los anurans larvales utilizan el modo de quimiosensoriales para detectar y para evitar la depredacin. Exponiendo a los renacuajos Bufo marinus a una variedad de productos qumicos naturales (las larvas y los metamorphs aplastados y la presa anlida aplastada) se demostr una repulsin fuerte.
Text in English.
Tropical Ecology 2009
Ecologa Tropical 2009
Sapo de cao
t Monteverde Institute : Tropical Ecology