Aggregation Status and Cue Type Modify Tadpole Response to Chemical Cues

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1 Notes Aggregation Status and Cue Type Modify Tadpole Response to Chemical Cues Devin B. Preston,* M. R. J. Forstner Department of Biology, Texas State University, San Marcos, Texas Abstract Many anuran larvae exhibit an antipredator response to chemical cues released by potential predators. The genus Bufo is no exception, as many bufonids exhibit an antipredator response (e.g., reduction in activity) to the presence (recent and current) of predators. Using a mesocosm experiment in a field laboratory setting, we tested solo and groups of Bufo (Incilius) nebulifer tadpoles for an antipredator response to chemical cues produced by 1) the presence of anisopteran nymphs (kairomone cue) and 2) the predation of conspecifics by anisopteran nymphs (a combination of diet and alarm cues, which we termed predation cue). We quantified the magnitude of the response by calculating response strength. We analyzed data with a blocked ANOVA followed by a Tukey s honestly significant difference analysis. We found that chemical cue type (kairomone vs. predation) affected response strength, but aggregation status (solo vs. group) did not. Furthermore, solo tadpoles and groups of tadpoles reduced their activity in response to predation cues, whereas only solo tadpoles reduced their activity in response to kairomone cues, a heretofore unobserved phenomenon. Our results suggest that B. nebulifer tadpoles modulate their response to specific types of chemical cues depending on their aggregation status. As reduced activity comes at a cost to resource acquisition and growth, aggregation status may indirectly affect the life history of B. nebulifer. The elucidation of these potential life history effects may aid managers in estimating anuran population viability. Keywords: amphibian; behavior; Bufo nebulifer; chemical cues; predation Received: April 10, 2014; Accepted: October 30, 2014; Published Online Early: December 2014; Published: June 2015 Citation: Preston DB, Forstner MRJ Aggregation status and cue type modify tadpole response to chemical cues. Journal of Fish and Wildlife Management 6(1): ; e x. doi: / JFWM-028 Copyright: All material appearing in the Journal of Fish and Wildlife Management is in the public domain and may be reproduced or copied without permission unless specifically noted with the copyright symbol ß. Citation of the source, as given above, is requested. The findings and conclusions in this article are those of the author(s) and do not necessarily represent the views of the U.S. Fish and Wildlife Service. * Corresponding author: dpreston@bio.tamu.edu Introduction A stable predator prey system depends on a reproductively viable prey population. One of the ways this is accomplished is through a substantial portion of the prey population avoiding death by predation. To avoid predation, many potential prey species utilize detection of various predator-produced stimuli. In aquatic systems, prey employ the detection of visual, hydrodynamic, and chemical stimuli (Tikkanen et al. 1994). The detection by prey of predation-related chemical cues is especially useful, as chemical cues can be more reliable than visual cues (Hickman et al. 2004; Ferrari et al. 2010). Unlike visual cues, chemical cues are not affected by turbidity, and do not require the source of the cue to be immediately present. Predators release different types of chemical cues, thought to be a blend of metabolic wastes and hormones. Detection of these cues by prey is a common method of avoiding predation (Kats and Dill 1998; Petranka and Hayes 1998). Chemicals produced via normal metabolic activity can be used for intraspecific communication (pheromones), but can also be detected by prey (Kats and Dill 1998; Turner et al. 1999; Relyea 2001; Mathis et al. 2003). When prey utilize these chemicals for predator avoidance, they are referred to as kairomones, or kairomone cues. Another type of cue results when tissue is shed from sympatric organisms via predation or injury; these are known as alarm cues. Prey organisms producing alarm cues are often (but not necessarily) conspecific or congeneric to the receiver (Chivers and Smith 1998; Chivers and Mirza 2001; Sullivan et al. 2005). Similar to alarm cues are diet cues, a combination of predator waste products and digested prey. Diet cues can be sensed by prey when produced by Journal of Fish and Wildlife Management June 2015 Volume 6 Issue 1 199

2 predator consumption of certain conspecific or heterospecific sympatric organisms (Hazlett 1990; Petranka and Hayes 1998; Anholt et al. 2000; Ferrari et al. 2010). Kairomone, alarm, and diet cues can allow earlier detection of the predator by the prey, resulting in a negative effect (missed predation opportunity) for the predator and a positive effect (greater chance of avoiding predation) for the prey. Larval anurans exhibit a variety of behavioral responses when exposed to chemical cues, including spatial avoidance of the cue (Nunes et al. 2013), increase in refuge use (Scarabotti et al. 2007), and swimming in bursts (Takahara et al. 2008). The most commonly documented response is the reduction of activity, and has been demonstrated using bullfrogs (Rana catesbeiana), green frogs (Rana clamitans), leopard frogs (Rana pipiens), wood frogs, (Rana sylvatica), American toads (Bufo americanus), and others using cues produced from nymphal dragonflies and fish (Skelly and Werner 1990; Petranka and Hayes 1998; Anholt et al. 2000; Eklöv 2000; Relyea 2004; Fraker 2010; Takahara et al. 2013). Activity reduction helps protect larval anurans from predation by nymphal anisopterans such as the dragonfly Anax junius, which are attracted to active prey (Folsom and Collins 1984). Tadpoles respond behaviorally to both kairomone and diet cues released from these predators (Petranka and Hayes 1998; Anholt et al. 2000; Eklöv 2000; Fraker 2010). Repeated behavioral responses to chemical cues can lead to life history responses, such as reduced metamorph size (Skelly and Werner 1990; Hagman et al. 2009), reduced growth rate (Eklöv 2000; Relyea 2004), altered morphology (Buskirk andand Arioli 2002; Relyea 2004), and increased energy investment in predator defense (Hagman et al. 2009). The management implications of these life history responses have heretofore been overlooked by researchers, as these responses have mainly been considered in evolutionary and ethological contexts. However, our results (discussed below) and the results of the aforementioned studies may be incorporated into management plans to provide more contextsensitive estimates of population viability. We collected our experimental organisms from an ephemeral pond on the Griffith League Ranch near Bastrop, Texas. The Griffith League Ranch is a Boy Scout owned 1,967-ha tract of land characterized by loblolly pine (Pinus taeda) forest with deep sandy soils and multiple periodic water bodies as close to each other as 0.5 km. Bufo nebulifer is a toad of least concern commonly found throughout southern Texas, Louisiana, and Mississippi, as well as northeastern Mexico (Hammerson and Canseco-Márquez 2004). Tadpoles of B. nebulifer are sympatric with anisopteran nymphs at our study site (D. B. Preston, personal observation). We used nymphs of the species A. junius (Aeshnidae), the common green darner. A. junius is globally widespread (Paulson 2009), has been previously identified both in nymphal and adult form in central Texas (Lasswell and Mitchell 1997), and occurs locally within the Lost Pines of Bastrop County, Texas. It is attracted to active over inactive prey (Folsom and Collins 1984), making actively moving tadpoles more vulnerable to predation. A. junius nymphs are only known to prey upon the larval stage of anurans, and tadpole risk to nymph predation decreases as the tadpole grows larger (Anholt et al. 2000; Bennett et al. 2013). In an experiment in which bullfrog (Rana catesbeiana) tadpoles were exposed to a fish predator (Lepomis macrochirus) anda. junius nymphs one at a time and in combination, tadpoles experienced the highest mortality in the presence of the nymphs alone (Eklöv 2000).Because anuran breeding at our study site occurs in fishless ponds, this implies that A. junius nymphs can have a large impact on the population from which our experimental organisms originate. Additionally, the nymphal stage constitutes the majority of an anisopteran s lifespan, as they can take up to 5 y to reach adulthood, whereas the adults typically live from a few weeks to 6 mo. Since many anurans breed one or more times per year, one generation of nymphs has the potential to influence multiple generations of larval anurans. Multiple factors influence the degree of a tadpole s response to dragonfly chemical cues. Researchers have shown that tadpoles exhibit a continuous dosage response when exposed to chemical cues, so responses are generally graded rather than binary (Buskirk andand Arioli 2002; Relyea 2004). In general, tadpoles will show a greater degree of behavioral alteration when 1) tadpoles are small (Anholt et al. 2000), 2) the observed species has a long developmental period (Laurila et al. 1997; Fraker 2010), 3) cue concentration is high (Petranka and Hayes 1998; Buskirk andand Arioli 2002), 4) tadpoles are satiated (i.e., maximally fed) (Anholt et al. 2000; Fraker 2010), 5) phylogenetically proximal organisms are consumed (Laurila et al. 1997), 6) certain contaminants are present (Reeves et al. 2011), and 7) the predator associated with the cue commonly co-occurs with the tadpoles (Garcia et al. 2012). However, few studies have focused on the effects of aggregation status or differing chemical cues on larvae belonging to a single anuran species, and none that we know of incorporate both of these parameters (Table 1). Other studies that have used variable numbers of tadpoles have shown that response to chemical cues can be modified by both group size and predation risk, but have not reached a consensus about the effects of group size on behavioral responses to chemical cues (Relyea 2004; Awan andand Smith 2007). We tested solo tadpoles and groups of five tadpoles for a behavioral antipredator response (measured by reduction in activity) to the following: 1) a kairomone cue produced from the presence of A. junius nymphs, hereafter termed kairomone cue and 2) a combination of alarm and diet cues produced from the consumption of conspecific tadpoles by A. junius nymphs, hereafter termed predation cue. We hypothesized that B. nebulifer tadpoles would reduce their activity when exposed to either non-control cue, as bufonids have been shown to display antipredator behavior when exposed to chemical cues produced from syntopic predators (Laurila et al 1997; Maag et al. 2012; Nunes et al. 2013) and anisopterans occur at the site from which our organisms were collected. We also hypothesized that tadpoles were more likely to respond to either chemical cue when alone, as tadpoles may aggregate as a predator Journal of Fish and Wildlife Management June 2015 Volume 6 Issue 1 200

3 Table 1. Publications describing larval anuran response to chemical cues emphasizing the shortage of comparison between differing chemical cues and aggregation status, and lack of publications incorporating both at once. Publication Cue Group size Skelly and Werner 1990 Conspecific and heterospecific diet (uncontrolled) 10 Laurila et al Conspecific and heterospecific diet 1 Petranka and Hayes 1998 Kairomone and conspecific diet 10 Anholt et al Conspecific diet 10 Eklöv 2000 Kairomone 22 Chivers and Mirza 2001 Diet 1 Gallie et al Kairomone 5 Buskirk and Arioli 2002 Conspecific diet 2 Relyea 2004 Conspecific diet 20,40,80,160 Awan and Smith 2007 Kairomone 1,2,4 Fraker 2008 Conspecific diet 10 Fraker 2009 Conspecific and heterospecific diet 10 Fraker 2010 Conspecific diet 10 Garcia et al Conspecific and heterospecific diet 1 avoidance strategy (Watt et al. 1997), and tadpoles already aggregated have a smaller chance of being preyed upon than solo tadpoles (Hamilton 1971; Olson et al. 2013). Methods We reared experimental organisms from 17 May 2011 to 1 June 2011 and ran trials from 2 June 2011 to 15 June 2011 on the Welsh tract, a property owned by Bastrop County and managed by the Texas State University s Department of Biology. We collected four amplectant B. nebulifer pairs from the adjacent Griffith League Ranch, a ha tract of land owned by the Boy Scouts of America, and placed them into a 3.12-kL outdoor plastic tub, hereafter referred to as a brooding tank. We oriented the brooding tank on a decline so that the downhill half accumulated rainwater while the uphill half was filled with sand. Standpipes kept the water from overflowing onto the sand in the event of heavy rainfall. Only one amplectant pair laid eggs, providing the tadpoles used in this study. At time of cue exposure, tadpoles were d old and were in Gosner stages (Gosner 1960). After we acquired eggs, we returned all adult toads to the ponds from which we collected them. We collected A. junius nymphs from the Griffith League Ranch for use in cue production and maintained them in 37.9-L aquaria with aquatic vegetation. After the study was concluded, we returned remaining tadpoles to the same ponds from which we collected their parents and dragonfly nymphs to ponds from which we collected them. The experimental design was a three by two factorial arrangement. The factors were 1) cue, with three levels, and 2) aggregation status, with two levels. Cue levels were control, kairomone cue, and predation cue. Aggregation status levels were solo and group. We maintained organisms with the approval of Texas State University San Marcos (IACUC 1105_0323_05) and Texas Parks and Wildlife (TPWD SPR ). Cue production The control consisted of well water drawn on site. We produced the kairomone cue in the following manner: we fasted 15 nymphs in 22uC well water for 24 h, after which we separated them and transferred them to individual plastic containers, each with 1,050 ml of 22uC well water. We left them undisturbed for 1 h and then removed them from their containers. The resulting solution provided kairomone cue. We produced the predation cue by placing 13 of the same nymphs (two had died in the interim between cue productions) into a single aquarium containing 1,050 ml of 22uC water per individual (13.65 L total), then we deposited 39 tadpoles (three per nymph into the aquarium) and left them alone for 24 h, after which we removed the nymphs and surviving tadpoles. Twelve of 39 tadpoles remained after production of the predation cue, indicating that 27 had been consumed by nymphs in the 24-h feeding period. This provided a diet cue component in the predation cue. Residual tissue from consumed tadpoles also remained in the aquarium, which we did not filter out. This provided the alarm cue component in the predation cue. We stirred both cues with a sterile glass stir rod to standardize them before use. All cues were kept in a cool area to keep their temperatures constant and used within 12 h of production. Cue exposure In all treatments, we did not introduce cues into aquaria at the beginning of the sampling period. In the solo treatments, we placed an individual tadpole into a 37.9-L capacity aquarium containing 8.4 L of 22uC well water and allowed the tadpole to acclimate for 15 min. We introduced approximately 1 g of rabbit chow pellets to provide a foraging incentive, started a stopwatch, and began recording the activity level. We quantified the activity level by summing activity events (hereafter AEs) in 5-s intervals over a period of 15 min. We defined an AE as a tadpole swimming at least one body length (tail Journal of Fish and Wildlife Management June 2015 Volume 6 Issue 1 201

4 included), or a tadpole ingesting a piece of rabbit chow. We used 5-s observation intervals with a 3-s recovery period. If an individual performed an AE during an observation interval, we tallied a mark for that interval. Subsequent AEs performed by that individual were not counted for the remainder of the interval. After the interval, we allowed a 3-s recovery period to pass without recording regardless of whether there was an AE or not. This served to standardize the time it took for the observer to record data after every interval. We recorded activity levels for 15 min, at which point we introduced a 1,050-mL aliquot of the respective experimental cue by pouring it gently into the front and center (relative to the observer s view) of the aquarium. Tadpole activity did not appear to be affected by the physical disturbance. We continued observations for 15 more minutes. We summed AEs for each 15-min (pre- and postexposure) period. For the group treatments, our methods were identical to those of the solo treatments except that five tadpoles were used at once in an aquarium instead of one. We used the average AEs per tadpole per aquarium to quantify activity instead of using total AEs. Whereas we directly observed the solo treatments, we filmed group treatments with a Sony DCR-HC62 Handycam and quantified activity afterward. In the group treatments, we filmed from the same angle as the observer s point of view in the solo treatments for standardization purposes. Each treatment had 12 repetitions (n = 12), with the exception of the group predation cue treatment, which only had 11 repetitions (n = 11) due to a paucity of cue available. In between repetitions, we emptied each aquarium, cleaned it with hydrogen peroxide, and triple-rinsed it with 22uC well water. We conducted the solo kairomone, solo predation, and solo control treatments on 3 June 2011, 4 June 2011, and 5 June 2011, respectively. We conducted the group control, group predation, and group kairomone treatments on 9 June 2011, 10 June 2011, and 11 June 2011, respectively. We began all treatments at approximately 0600 hours and ended at approximately 2100 hours. Though this was a wide temporal range throughout the day, the temporal consistency in sampling times among treatments controlled for diel effects. Statistical analyses We calculated response strength (RS) as the proportional reduction in AEs between the first (preexposure) 15-min period and the second (postexposure) 15-min period ([pre-exposure AEs 2 postexposure AEs]/ pre-exposure AEs). A higher RS indicated a stronger reduction in activity. We evaluated normality in the data set using a Shapiro Wilk test and normal Quantile Quantile plot (Figure S1, Supplemental Material). We confirmed homoscedasticity with a residuals versus fits plot (Figure S2), Supplemental Material, then applied a 3- way blocked ANOVA with aggregation status, cue, and block (aquarium) as factors, then followed with a Tukey s honestly significant difference test to examine differences between specific treatments. We performed calculations in R (R Core Team 2012). Results We observed an unexpected negative RS (increase in activity) in the solo control treatment (Figure 1) which was significantly different from zero (Welch t-test: t 11 = 3.587, p = 0.004). However, in the group control treatment, RS did not differ significantly from zero (Welch t-test: t 11 = , p = 0.739). Cue was a significant factor in Response Strength (RS) (ANOVA: F 2,64 = p, 0.001; g 2 = 0.571), but aggregation status (F 1,64 = 2.139, p = 0.15; g 2 = 0.011) and block (F 1,64 = p = 0.71; g 2 = 0.001) were not. However, the interaction effect between aggregation status and cue was significant (F 2,64 = 7.362, p = 0.001; g 2 = 0.078; Table S1). In the solo treatments (Figure 2), RS was significantly lower in the control treatments than it was in the kairomone (Tukey s honestly significant difference: P, 0.001, df = 65) and predation (P, 0.001, df = 65) cue treatments, whereas RS of the kairomone and predation cue treatments did not differ from one another (P, 0.98, df = 65). In the group treatments (Figure 3), the control RS did not differ from that of the kairomone cue treatment (Tukey s honestly significant difference: P = 0.314, df = 65), but was significantly lower than the RS of the predation cue treatment (P, 0.001, df = 65). The RS of the kairomone cue treatment was also significantly lower than that of the predation cue treatment (P, 0.001, df = 65). All raw data is included in Data S1, and summarized in Table S1, Supplemental Material. Discussion The unexpected increase in activity in the solo control treatment (Figure 1) may have been due to an increase in foraging activity as tadpoles acclimated in a situation of no predation threat and available food. However, this result may not be reproducible, as the group control treatment showed no significant increase in activity. Because we measured the average activity level per five tadpoles in the group control treatment instead of the activity level of an individual tadpole as in the solo control treatment, it is more likely that the group control treatment results are more representative of B. nebulifer tadpole activity as a whole. By itself, aggregation status was not a significant factor on the strength of the antipredator response. This is consistent with results from previous ethological research on anurans (Awan and Smith 2007) and implies that B. nebulifer response to chemical cues is not always affected directly by density. However, as indicated by the significant interaction effect between aggregation status and cue in the ANOVA and the Tukey s honestly significant difference results, aggregation status mediated tadpole response in the kairomone cue treatments. This result is consistent with a study using R. sylvatica (wood frog) tadpoles, which showed a negative correlation between antipredator response and competitor density (Relyea 2004). As kairomone cues simulate the presence of predators, but not predation, we hypothesize that a factor driving the lack of response to kairomone cues when in a group could be a relatively Journal of Fish and Wildlife Management June 2015 Volume 6 Issue 1 202

5 Figure 1. Mean activity events (6SE) of solo and groups of Bufo (Incilius) nebulifer tadpoles per aquarium per tadpole during experimental trials in Bastrop, Texas, in June 2011 before (Pre) and after (Post) exposure to three chemical cues: 1) a control cue consisting of well water, 2) a kairomone cue consisting of well water that had held fasted Anax junius (dragonfly) nymphs for 1 h, and 3) a predation cue consisting of well water in which B. nebulifer tadpoles had been fed to A. junius nymphs. We defined an activity event as a tadpole swimming at least one body length (tail included), or a tadpole ingesting a piece of rabbit chow. Both pre- and postexposure periods were 15 min long. low perceived degree of predation risk. When we observed B. nebulifer tadpoles at the collection site, they were found in groups of more than five individuals without exception, suggesting that B. nebulifer aggregation is a common behavior in the population we sampled. Bufo tadpoles may aggregate as a predator avoidance strategy (Watt et al. 1997; Spieler and Lisenmair 1999), which should lower their risk of being preyed upon (Hamilton 1971; Olson et al. 2013). Thus, tadpoles in the group treatments may have already been incidentally displaying a degree of antipredator behavior (aggregation), resulting indirectly in less investment by tadpoles in another antipredator behavior (reduction in activity). Another possible factor driving the weaker response in the group kairomone treatment as compared to the solo Figure 2. Mean response strength of solo Bufo (Incilius) nebulifer tadpoles (6SE) during experimental trials in Bastrop, Texas, in June 2011 in response to three chemical cues: 1) a control cue consisting of well water, 2) a kairomone cue consisting of well water that had held fasted Anax junius (dragonfly) nymphs for 1 h, and 3) a predation cue consisting of well water in which B. nebulifer tadpoles had been fed to A. junius nymphs. We measured response strength in proportional reduction in activity events from the pre-exposure period to the postexposure period. We defined an activity event as a tadpole swimming at least one body length (tail included), or a tadpole ingesting a piece of rabbit chow. Letters in figure indicate groupings with no significant difference from each other as determined by Tukey s honestly significant difference means comparisons. The control treatment response strength differed from the kairomone (P, ) and the predation (P, ) cue treatments, but the response strength of the kairomone cue treatment did not differ from that of the predation cue treatment (P = 0.982). Journal of Fish and Wildlife Management June 2015 Volume 6 Issue 1 203

6 Figure 3. Mean response strength of groups of five Bufo (Incilius) nebulifer tadpoles (6SE) during experimental trials in Bastrop, Texas, in June 2011 in response to three chemical cues: 1) a control cue consisting of well water, 2) a kairomone cue consisting of well water that had held fasted Anax junius (dragonfly) nymphs for 1 h, and 3) a predation cue consisting of well water in which B. nebulifer tadpoles had been fed to A. junius nymphs. We measured response strength in proportional reduction in activity events from the pre-exposure period to the post-exposure period. We defined an activity event as a tadpole swimming at least one body length (tail included), or a tadpole ingesting a piece of rabbit chow. Letters in figure indicate groupings with no significant difference from each other as determined by Tukey s honestly significant difference means comparisons. The control treatment response strength did not differ from the kairomone cue treatment (P = 0.314), but did differ from the predation cue treatment (P, ) The kairomone cue treatment differed from that of the predation cue treatment (P, ). kairomone treatment could be an adjustment in activity to increase the likelihood of metamorphosing in a timely manner, as B. nebulifer can breed in temporary water bodies (Santos-Barrera et al. 2010). Tadpoles developing in ephemeral ponds depend on a steady intake of nutrients in order to metamorphose before their natal pond dries, and length of time to metamorphose in bufonids can increase with increasing densities (Brockelman 1969). Additionally, Newman (1987), using Couch s spadefoot (Scaphiopus couchi) tadpoles, observed that high conspecific densities cause increased tadpole mortality via dessication. Thus, B. nebulifer tadpoles, when in higher densities, could be taking a greater risk of being preyed upon for the tradeoff of more rapid resource acquisition, as the mortality rate by dessication is effectively 100% on B. nebulifer tadpoles that fail to metamorphose before an ephemeral pond (e.g., the pond from which we collected our parent toads) dries. A final hypothesis for the lack of response in the group kairomone treatment may have been weak cue concentration. Tadpoles have been shown to respond more strongly to increasing concentrations of chemical cues (Buskirk andand Arioli 2002), and the concentration of predation-related cues that B. nebulifer tadpoles are typically exposed to in the wild remains unknown. Because our experimental setup kept tadpoles intimately associated with the cue instead of letting the cue disperse as would have occurred with the much higher-volume water body from which we collected their parents, this hypothesis may not be ecologically relevant. However, chemical cue concentrations in the wild may approach the levels that we used in our experiment by repeated release of a stationary predator, such as A. junius. Tadpoles have been shown to respond more strongly to chemical cues when they overlap, even when they are weak (Fraker 2009a), so the tadpoles in our experimental environment may have behaved similarly to those in the wild. One of the major limitations in this study was the experimental organisms shared parentage. Although it is possible that our results are representative of general B. nebulifer tadpole antipredator response, as some anuran phenotypic plasticity is likely driven by environment rather than genetics (Alho et al. 2010), the genetic diversity in our sample was too low to make any definitive conclusions. Our findings would have been more widely applicable and conclusive if we had been able to use multiple breeding pairs. Amphibian populations are in global decline (Wake and Vredenburg 2008), and proper management of species requires an understanding of their life history and behavior (Pearson and Healey 2003; Pecl et al. 2004; Mangel et al. 2006). Our results add to a growing body of literature suggesting that activity reduction in response to predation-related chemical cues may be a characteristic behavioral response in Anura (Skelly and Werner 1990; Laurila et al. 1997; Petranka and Hayes 1998; Anholt et al. 2000; Eklöv 2000; Chivers and Mirza 2001; Relyea 2001, 2004; Awan andand Smith 2007; Fraker 2009b, 2010; Nunes et al. 2013). Though advantageous in the short term, this behavioral antipredator response may slow B. nebulifer growth or development rates by impeding resource consumption, as has been demonstrated in other anurans (Skelly and Werner 1990; Eklöv 2000; Buskirk andand Arioli 2002; Relyea 2004; Hagman et al. 2009), though the duration of this experiment was too short to test this here. Anuran metamorph size affects Journal of Fish and Wildlife Management June 2015 Volume 6 Issue 1 204

7 multiple life history aspects of the subsequent adult, including parasite resistance (Goater et al. 1993) and survival (Boone 2005), and may affect foraging ability (McCallum and McCallum 2012). In the absence of empirical data for a particular anuran species, our results, taken with the results of the above studies, suggest that an anuran population will experience life history shifts when developing in an environment with high predator densities. This information may increase the efficacy of a management plans projecting anuran population viability, as accurate tadpole-to-adult survival rates are critical when using population estimates such as spawn counts. Additionally, though B. nebulifer is a widespread species of least concern, it is syntopic with the endangered Houston toad (Bufo [Anaxyrus] houstonensis) to the degree that hybrid individuals are produced (Kennedy 1962; Brown 1971; Hillis et al. 1984) and B. houstonensis larvae respond to chemical cues produced from the predation of B. nebulifer larvae (Preston and Forstner, in press). If B. nebulifer are growing at a slower rate to a smaller metamorph size in a situation of higher predator density (and therefore higher chemical cue density), this may have implications for syntopic individuals of B. houstonensis during postmetamorphic stages, such as reduced intraguild predation, resource competition, and other ecological factors. Studies examining the interactions between behavioral effects of chemical cues at the larval stage and life history effects at the postmetamorphic stage in these syntopic species would be useful in predicting how both species might fare in situations of high predator density in natal ponds. We have concluded from our study that, like many larval anurans, B. nebulifer tadpoles sense and respond to predation-related chemical cues. Furthermore, both cue type and the interaction between cue type and aggregation status modify the tadpoles response to these cues, which has heretofore been undocumented. Future studies should use a wide spectrum of cue concentrations and group sizes rather than treating them as binary variables. This may help to disentangle the relationship of cue type and group size and their effects on larval B. nebulifer and other anurans. In addition, future researchers may opt for a nonproportional response variable. Though using the proportional measure of RS allowed us to eliminate the effects of extremely high and low baseline (pre-exposure) activity levels, it may have eliminated much natural variance that we would have seen otherwise. Also, we highly recommend that future researchers use more than one breeding pair, as more pairs will lead to less chance of obtaining data from genetic outliers. Supplemental Material Please note: The Journal of Fish and Wildlife Management is not responsible for the content or functionality of any supplemental material. Queries should be directed to the corresponding author for the article. Table S1. Activity data from experimental trials in Bastrop, Texas, in June 2011 on Bufo (Incilius) nebulifer tadpoles. The variables were as follows: 1) Aggregation status, with two levels. This was either solo (tadpole was exposed by itself to chemical cues) or group (tadpole was exposed to chemical cues alongside four of its fellows). 2) Chemical cue type. This was control (well water), kairomone (unfed predator cues), or diet (fed predator cues). For each repetition of each treatment, we reported the number of activity events in the preexposure period, the number of activity events in the postexposure period, and the response strength. We defined an activity event as a tadpole swimming at least one body length (tail included), or a tadpole ingesting a piece of rabbit chow. We calculated response strength as the proportional reduction in activity events between the first (pre-exposure) 15-min period and the second (postexposure) 15-min period ([pre-exposure activity events 2 post exposure activity events]/pre-exposure activity events). Found at DOI: / JFWM-028.S1 (10 KB XLSX). Figure S1. Quantile Quantile plot constructed from activity data acquired during experimental trials in Bastrop, Texas, in June 2011 on Bufo (Incilius) nebulifer tadpoles. Model residuals from an ANOVA are plotted on the Y axis and theoretical quantiles based on a normal distribution are plotted on the X axis. Found at DOI: / JFWM-028.S2 (31 KB DOCX). Figure S2. Residuals vs. fits plot constructed from activity data acquired during experimental trials in Bastrop, Texas, in June 2011 on Bufo (Incilius) nebulifer tadpoles. Model residuals from an ANOVA are plotted on the Y axis and fitted values are plotted on the X axis. Found at DOI: / JFWM-028.S3 (33 KB DOCX). Data S1. Raw data acquired during experimental trials in Bastrop, Texas, in June 2011 on Bufo (Incilius) nebulifer tadpoles. Found at DOI: / JFWM-028.S4 (14 KB XLSX). Reference S1. Preston DP, Forstner MRJ. In Press. Houston toad (Bufo [Anaxyrus] houstonensis) tadpoles decrease their activity in response to chemical cues produced from the predation of conspecifics and congeneric (Bufo [Incilius] nebulifer) tadpoles. Journal of Herpetology. Found at DOI: JFWM-028.S5 (54 KB DOCX). Acknowledgments We thank Amanda Moore, Caitlin Gabor, the evolution, ecology and behavior discussion group at Texas State, the Environmental Defense Fund, the County of Bastrop, the editors at the Journal of Fish and Wildlife Management, the manuscript reviewers, Donald Brown, Emrah Ozel, Floyd Weckerly, the Forstner lab, Joseph Veech, the Texas Department of Transportation, the Texas Forest Journal of Fish and Wildlife Management June 2015 Volume 6 Issue 1 205

8 Service, the Texas Parks and Wildlife Department, the U.S. Fish and Wildlife Service, and the U.S. Geological Survey for making this project possible. We collected and maintained experimental organisms with the approval of Texas State University-San Marcos (IACUC 1105_0323_05) and Texas Parks and Wildlife (TPWD SPR ). Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government. References Alho JS, Herczeg G, Söderman F, Laurila A, Jönsson KI, Merilä J Increasing melanism along a latitudinal gradient in a widespread amphibian: local adaptation, ontogenic or environmental plasticity? Evolutionary Biology 10:317. Anholt BR, Werner E, Skelly DK Effect of food and predators on the activity of four larval ranid frogs. Ecology 81: Awan, AR, Smith GR The effect of group size on the responses of wood frog tadpoles to fish. American Midland Naturalist 158: Bennett AM, Periera D, Murray DL Investment into defensive traits by anuran prey (Lithobates pipiens) is mediated by the starvation-predation risk trade-off. PLoS ONE 8:e doi: /journal.pone Boone MD Juvenile frogs compensate for small metamorph size with terrestrial growth: overcoming the effects of larval density and insecticide exposure. Journal of Herpetology 39: Brockelman WY An analysis of density effects and predation in Bufo americanus tadpoles. Ecology 50: Brown LE Natural hybridization and trend toward extinction in some relict Texas toad populations. The Southwestern Naturalist 16: Buskirk JV, Arioli M Dosage response of an induced defense: how sensitive are tadpoles to predation risk? Ecology 83: Chivers DP, Mirza RS Importance of predator diet cues in responses of larval wood frogs to fish and invertebrate predators. Journal of Chemical Ecology 27: Chivers DP, Smith RJF Chemical alarm signalling in aquatic predator prey systems: a review and prospectus. Ecoscience 5: Eklöv P Chemical cues from multiple predator prey interactions induce changes in behavior and growth of anuran larvae. Oecologia 123: Ferrari CO, Wisenden BD, Chivers DP Chemical ecology of predator prey interactions in aquatic ecosystems: a review and prospectus. Canadian Journal of Zoology 88: Folsom TC, Collins NC The diet and foraging behavior of the larval dragonfly Anax junius (Aeshnidae), with an assessment of the role of refuges and prey activity. Oikos 42: Fraker ME The dynamics of predation risk assessment: responses of anuran larvae to chemical cues of predators. Journal of Animal Ecology 77: Fraker ME. 2009a. The effect of prior experience on a prey s current perceived risk. Behavioral Ecology 158: Fraker ME. 2009b. Predation risk assessment by green frog (Rana clamitans) tadpoles through chemical cues produced by multiple prey. Behavioral Ecology and Sociobiology 63: Fraker ME Risk assessment and anti-predator behavior of wood frog (Rana sylvatica) tadpoles: a comparison with green frog (Rana clamitans) tadpoles. Journal of Herpetology 44: Gallie JA, Mumme RL, Wissinger SA Experience has no effect on the development of chemosensory recognition of predators by tadpoles of the American toad, Bufo americanus. Herpetologica 57: Garcia TS, Thurman LL, Rowe JC, Selego SM Antipredator behavior of American bullfrogs (Lithobates catebeianus) in a novel environment. Ethology 118:1 9. Goater CP, Semlitsch RD, Bernasconi MV Effects of body size and parasite infection on the locomotory performance of juvenile toads, Bufo bufo. Oikos 66: Gosner KL A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16: Hagman M, Hayes RA, Capon RJ, Shine R Alarm cues experienced by cane toad tadpoles affect postmetamorphic morphology and chemical defences. Functional Ecology 23: Hamilton WD Geometry for the selfish herd. Journal of Theoretical Biology 31: Hammerson G, Canseco-Márquez L Incilius nebulifer. In: IUCN IUCN red list of threatened species. Version Available: (September 2014). Hazlett BA Source and nature of disturbancechemical system in crayfish. Journal of Chemical Ecology 16: Hickman CR, Stone MD, Mathis A Priority use of chemical over visual cues for detection of predators by graybelly salamanders, Eurycea multiplicata griseogaster. Herpetologica 60: Hillis DM, Ann MH, Martin RF Reproductive ecology and hybridization of the endangered Houston toad (Bufo houstonensis). Herpetology 18: Kats LB, Dill LM The scent of death: chemosensory assessment of predation risk by prey animals. Ecoscience 5: Kennedy JP Spawning season and experimental hybridization of the Houston toad, Bufo houstonensis. Herpetologica 17: Lasswell JL, Mitchell FL Survey of dragonflies (Odonata: Anisoptera) in ponds of central Texas. Journal of the Kansas Entomological Society 70: Journal of Fish and Wildlife Management June 2015 Volume 6 Issue 1 206

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