Acta Herpetologica 17(1): 13-20, 2022 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.36253/a_h-11453 Influence of tail injury on the development of Neotropical elegant treefrog tadpoles Ana Glaucia da Silva Martins1,#, Raoni Rebouças2,3,*,#, Isaias Santos1, Adão Henrique Rosa Domingos1, Luís Felipe Toledo2 1 IPBio – Instituto de Pesquisas da Biodiversidade, Reserva Betary, Iporanga, São Paulo, Brazil 2 Laboratório de História Natural de Anfíbios Brasileiros (LaHNAB), Departamento de Biologia Animal, Instituto de Biologia, Universi- dade Estadual de Campinas, Cidade Universitária Zeferino Vaz, 13083-970, Campinas, São Paulo, Brazil 3 Programa de Pós-Graduação em Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz, 13083-970, Campinas, São Paulo, Brazil *Corresponding author. E-mail: raonisreboucas@gmail.com # These authors contributed equally to this work Submitted on: 2021, 5th July; revised on: 2021, 1st November; accepted on: 2021, 8th November Editor: Simon Baeckens Abstract. Anuran larvae in aquatic environments are important prey items for several vertebrate and invertebrate spe- cies. Besides avoiding predation, there are some strategies that may reduce the physical damage in those tadpoles that survive the predation attempt. For example, the injured tadpole tail can regrow after a predator bite, but few studies have examined the consequences of such injury. We examined the consequences of three levels of injury to the tail and how this influenced development and feeding behavior of tadpoles of the Neotropical elegant treefrog, Dendrop- sophus elegans. We collected spawns and kept them in the laboratory until tadpoles reached Gosner’s stages 28 to 35. Then, they were separated in four experimental groups: individuals with tail trimmed in 30, 50 or 70 % of its length, and a control group, with no tail removing. We counted the days until metamorphosis, calculated the Scaled Mass Index (SMI) through weight and length of newly-metamorphosed, and evaluated the feeding frequency to evaluate the influence of tail amputation on them. We found that the time until metamorphosis was positively related to the extent of the amputation, but SMI and feeding behavior were not influenced. As the time to metamorphose is related to the survivorship chances of individuals: i.e., if the aquatic environment is with high density of predators, it would be advantageous to rapidly metamorphose out of the water. However, tail injury delays the metamorphose process, which could influence the survival of the individual. Keywords. Anuran larvae, Dendropsophus elegans, Atlantic rainforest, tail loss, development, feeding. INTRODUCTION Most anurans present aquatic larval stages and terres- trial post-metamorphic (adult) life stages, and are suscep- tible to predators of both environments. In this context, several defensive strategies were already reported for tad- poles in face of predators’ attack. For example, tadpoles of Pelophylax lessonae can alter their behavior in the pres- ence of dragonfly larvae (van Buskirk and Arioli, 2002), and tadpoles of Dryophites crysoscelis can change the morphology of their tails in order to increase swimming speed, which consequently promotes a higher probability of escaping in a possible attack of predators (McCollum and Leimberger, 1997). Also, Other species rely on visu- al aspects to avoid predation, such as tadpoles of Scinax machadoi, which select background colors to improve 14 Ana Glaucia da Silva Martins et alii their camouflage (Eterovick et al., 2018; Gontijo et al., 2018), Pseudacris regilla, which alter their tail color to avoid predator attacks (Benard, 2006), and Boana semi- lineata, which uses aposematic coloration to avoid pre- dation (D’Heursel and Haddad, 1999). Hence, other spe- cies, such as Bufo bufo, rely on chemical defenses to avoid attacks of predators (Üveges et al., 2019). Anurans are well known to be centralized in trophic webs (Blanco-Torres et al., 2020) since they are both prey and predators (Rebouças et al., 2013; Rebouças and Solé, 2015). In this way, they evolved several strategies to avoid predation (e.g., Lourenço-de-Moraes et al., 2016; Toledo et al., 2007). In larvae, one of the possible sublethal con- sequences of a predation attempt is the partial tail loss or injury (Morin, 1985; Touchon and Wojdak, 2014; Wilbur and Semlitsch, 1990), but the consequences of it to indi- vidual survival are very variable. For some species, past evidence suggest that it incurs little cost for tadpoles, since they, after escaping the predation, can regenerate the tail completely (Wilbur and Semlitsch, 1990). For example, van Buskirk et al. (2003) observed that tails may play a role as a lure, in which larger tail fins reduced predations in 16 % of the observations. Indeed, although firstly reported that enlarged tail fins enables predator escaping by enabling faster swimming (Smith and van Buskirk, 1995), posterior studies showed that tadpoles with injured tails did not lost speed in relation to those with an intact tails (van Buskirk and McCollum, 2000a). The effect on speed was significant only if large portions of the tails were removed (Hoff and Wassersug, 2000; van Buskirk and McCollum, 2000b). However, for some spe- cies tail injuries result in less swimming performance, and consequently a higher predation risk. In Dryophytes chrysoscelis, for example, tadpoles with no tail injury pre- sented a survival almost twice as high as those with 75 % of tail loss (Semlitsch, 1990). Also, in Bombina orienta- lis tadpoles presented less survivorship and longer larval period (Parichy and Kaplan, 1992). Beyond the ecological consequences, tail loss in tadpoles can also present feeding activity modification. Theoretically, if individuals need no regenerate tails after a predation attempt, they should acquire more energy through feeding to reach the maximum of tail length as less time as possible, and consequently reach the full swim performance, which is related to tail shape (van Buskirk and McCollum, 2000b). However, although modification of feeding behavior is already observed in presence of predators (e.g., Feminella and Hawkins, 1994; Pueta et al., 2016), the effects of tail loss on it, which is the most common consequence of predation attempt, still were not observed. Hence, while regenerating the tail, tadpoles are in continuous growth, which per se requires a constant food intake until reach the metamorphosis stage. Thus, the tail injury, and an extra acquisition of nutrients during its regeneration, must affect the feeding- growth-time until metamorphosis balance. It is relevant because tail injuries may impact on the population sur- vivorship coupled with the fact that this species occurs in Atlantic rainforest, one of the most diverse and vulner- able environments of the world, where pandemic diseases (Carvalho et al., 2017) climatic changes (Moura-Campos et al., 2021; Rebouças et al., 2021), habitat fragmentation (Becker et al., 2010; Dixo et al., 2009), and introduced predators (da Silva et al., 2009; de Oliveira et al., 2016; Forti et al., 2017) are threatening endemic anurans. Therefore, this study evaluates the consequences of tadpole’s tail injuries in a Neotropical anuran species, Dendropsophus elegans (Anura; Hylidae), testing the fol- lowing hypotheses: i) different levels of tail injury result in less healthy newly-metamophosed; ii) different levels of tail injury increase the time to complete metamorpho- sis; and iii) tail injury reduces foraging activity of tad- poles. MATERIALS AND METHODS Tadpoles of Dendropsophus elegans (Fig. 1) were obtained through the maintenance of egg masses collect- ed at Reserva Betary, Iporanga, São Paulo, Brazil. After hatching, each tadpole was kept in an individual aquar- ium (40 x 45 x 30 cm), to avoid pseudo-replicates and the influence of one individual in another, maintained at room temperature (25 ºC), and half of the water was replaced twice a week after tadpoles reach the stage 28. We used tadpoles between Gosner’s (1960) stages 28 and 36 for the experiments. These stages were chosen because they comprehend most of growth and development of anuran larvae (Pfab et al., 2020). Environmental condi- tions of laboratory were constantly monitored and indi- viduals were observed until metamorphosis. Thus, our experiment began before hatching and finished after met- amorphosis. After the experiment, all individuals were released in the original sampling locality. Tadpole development. To evaluate the influence of tail loss in the size and growth of individuals, we selected tadpoles that measured 25 mm of total length. Individu- als were measured with a digital caliper (to the nearest 0.01 mm) and weighted with a digital scale (to the near- est 0.01 g). We then arranged these tadpoles into four groups, following Semlitsch (1990) and Figiel Jr and Sem- litsch (1991), representing each of the treatments: i) tad- poles with 30 % of the tail clipped; ii) tadpoles with 50 % of tail clipped; iii) tadpoles with 70 % of tail clipped; 15Influence of tail injury on the development of Neotropical elegant treefrog tadpoles and iv) tadpoles with intact tails, which was the control group (Fig 1). Each group contained between 8 and 10 individuals (Table 1), which were isolated in each aquar- ium. Tail modifications were performed using a sterilized scalpel blade. Individuals in all treatments were equally fed with a standard fish food (extruded AquaLine), with 0.1 g every day. Individuals were observed until the meta- morphosis was completed (complete tail absorption), and snout-vent length (SVL) of newly-metamorphosed indi- viduals was measured with the digital caliper and body mass was weighted with the digital scale. Body mass and weight were used to calculated the Scale Mass Index (SMI), which is and index that can be used as a proxy of animals’ health and fitness (Peig and Green, 2009). Foraging. In order to evaluate the influence of par- tial tail loss in tadpoles foraging, we performed a second experiment also using 10 individuals measuring 25 mm in total length and between Gosner’s (1960) stages 28 and 36. These individuals were separated in two treatments: i) individuals with 70 % of tail amputated; and ii) indi- viduals with intact tails, treated as the control. Tadpoles were kept individually in glass jars measuring 6.5 cm in diameter and 6 cm height, with 120 ml of water and 0.1 g of fish food. After two min of acclimation, tadpoles were observed for 12 min. During this time, the feeding fre- quency was observed in intervals of 20 s, and during each observation we evaluated if were feeding or not. Statistical analyses. Firstly, we used an Analysis of Variance (ANOVA) and a Student’s t test to evaluate of SMI present difference between treatments (tail amputa- tions of 30 %, 50 % and 70 %; and tail amputation per se, respectively). To evaluate the influence of tail injury on SMI and on time until metamorphosis, we ran two Generalized Linear Models analyses (GLM), both using treatment (30 %, 50 % and 70 % of tail amputations and control, coded as 1, 2, 3 and 0, respectively) as predic- tive variable, the first one with SMI of newly metamor- phosed individual as response, and the second with days until metamorphosis as response. Both analyses were performed using gaussian family and identity link. Addi- tionally, we ran other two GLM’s, with the same param- eters, to evaluate if SMI or days until metamorphosis were influenced by amputation per se (all treatments were classified as “amputated”, for treatments which the tail was clipped, coded as 1, and “intact” for the control treatment, coded as 0). Finally, in order to evaluate the Fig. 1. An adult individual of Dendropsophus elegans (A), tadpoles of control (B) and 50 % of the tail clipped treatments (C), and with regenerated tail (D). 16 Ana Glaucia da Silva Martins et alii influence of tail loss in foraging we also used a GLM, but with quasipoisson family and logit link, considering “treatment” as predictive variable (control, coded as 0, or amputation, coded as 1), and the feeding frequency as response variable. All models were checked through residuals deviance, and models with more than one predictive variable and collinearity was checked through Variance Inflation Factor (VIF) through the “vif ” function of “car” package (Fox & Weisberg, 2019). We considered levels higher than 4 as an indicator of multicollinearity (Hair et al., 2010). Hence, as pos hoc tests, we used estimated marginal means to com- pare groups of tail-trimmed individuals with the control group through the “emmeans” package (Lenth, 2020). All analyses were carried out in R 4.1.0 (R Core Team, 2021) considering a significance level of 5 %. RESULTS During the experiment about tadpole development, we recorded the death of four individuals: one from the control group, one from the 50 % amputation group, and two from the 70 % amputation group. All individu- als from the treatment groups presented the tail totally regenerated within 12 days after the beginning of the experiment (Table 1). We observed tail regeneration in all individuals that had their tail clipped (Fig. 2). The average time until metamorphosis (from eggs until newly-metamorphosed) was 87.5 days for the con- trol group (room mean temperature of 26.5 ºC; Table 1). We observed no difference between treatment groups (F = 0.91, P = 0.44) or between individuals with tail ampu- tation or not (t = -0.06, P = 0.95). Newly metamorphosed individuals presented an average SMI of 0.148 ± 0.012, with control group presenting 0.148 ± 0.011, 30 % group presenting 0.143 ± 0.013, 50 % group presenting 0.153 ± 0.012, and 70 % group presenting 0.149 ± 0.014. During foraging experiment, individuals with injured tail were observed feeding in an average of 36.6 ± 25.9 times, while individuals with no tail injuring were observed feeding in an average of 55.1 ± 22.9 times. In our analysis, none of the variables presented VIF higher than 4 (SVL = 3.02, weight = 3.35, days until met- amorphosis = 1.2). We observed no influence of treat- ment on SMI or in weight, but treatment presented a sig- nificant influence on time until metamorphosis. Amputa- tion per se showed no influence in any of our variables. Regarding to foraging, we observed no influence of tail injury on feeding frequency. All model outputs are in Table 2 and estimated marginal means in Table 3. Table 1. Time until metamorphosis, snout-vent length (SVL) and body mass of newly metamorphosed individuals during experimentation. Values presented as mean ± standard deviation (minimum – maximum; number of individuals tested; standard error). Treatment Time until metamorphosis (days) SVL (mm) Weight (g) Control 29.8 ± 11.5 (18.3 – 41.3; 9; 3.83) 12.06 ± 0.44 (11.62 – 12.49; 9; 0.14) 0.15 ± 0.02 (0.13 – 0.17; 9; 0.007) 30 % 35 ± 10.8 (24.2 – 45.8; 10; 2.98) 11.86 ± 0.46 (11.40 – 12.32; 10; 0.14) 0.14 ± 0.02 (0.12 – 0.16; 10; 0.006) 50 % 32.8 ± 7.1 (25.7 – 39.9; 9; 2.36) 12.13 ± 0.24 (11.90 – 12.37; 9; 0.08) 0.16 ± 0.02 (0.14 – 0.18; 9; 0.007) 70 % 42.0 ± 15.1 (26.9 – 57.1; 8; 5.34) 12.01 ± 0.44 (11.57 – 12.46; 8; 0.14) 0.15 ± 0.02 (0.13 – 0.17; 10; 0.006) Fig. 2. Days until metamorphosis of Dendropsophus elegans tad- poles subjected to four treatments: control (intact tail), and 30, 50 and 70% of tail removal. The top and bottom of the boxes repre- sent the first and last quartiles, the horizontal line within the box represents the median, the whiskers represent the tenth and 90th percentiles. Asterisks represent the category of tail amputation that showed significant reduction of time until metamorphosis. 17Influence of tail injury on the development of Neotropical elegant treefrog tadpoles DISCUSSION We showed that although tadpoles reach metamor- phosis with the same weight and size in all classes, the time spent until the end of the metamorphosis tends to increase, and it was significantly longer when 70 % of tail is removed. It means that individuals with a severe dam- age in tail tend to spend more time under larval stage, which can submit individuals that were already threat- ened by a predator under aquatic predation pressure for a longer time. Also, it delays the development of adult life stage, and consequently reproduction can be retarded. Therefore, a high predation pressure can influence other life stages of individuals, and in a larger scale, can impair the permanence of a population. We also observed that the feeding frequency was not significantly higher in the group with tail trimmed. Some similar results were observed in other experiments involving artificial tail removing in tadpoles of Aquarana catesbeiana, where individuals also had a delay in growth and development (Wilbur and Semlitsch, 1990). A possi- ble explanation for these observed results is that a preda- tion attempt does not result in increasing of uptake but in reallocation of energy, since feeding presented no increas- ing, and it consequently could cause a delay in develop- ment. Additional studies are necessary to further eluci- date the physiology of this possible energy reallocation and verify this hypothesis. We did not observe influence of tail removal on the SMI of newly-metamorphosed individuals, similarly to what was reported for size in Osteopilus septentrionalis (Koch and Wilcoxen, 2019) and Hoplobatrachus rugulo- sus (Ding et al., 2014). However, opposing results were found for other species. For example, in Bombina ori- entalis, for which the time until metamorphosis was the same independently of the tail injury extent, newly meta- morphosed individuals were smaller than those without tail injury (Parichy and Kaplan, 1992). Likewise, tadpoles with 55 % of the tail removed resulted in smaller newly- metamorphosed individuals in Pelobates cultripes (Zamo- ra-Camacho et al., 2019). Besides, such effect lead to a reduction in the jumping performance of post-metamor- phic individuals (Zamora-Camacho and Aragón, 2019), which could expose them to higher risk of predation on land. So, these cases highlight a trade-off: tadpoles will either stay longer in the water, exposed for a longer time to aquatic predators but with newly metamorphosed with an ‘ideal’ size, with less exposure to terrestrial preda- tors (Semlitsch, 1990; Wilbur and Semlitsch, 1990), or they could leave the water smaller and with some mobil- ity handicaps, which could limit the exposure to aquatic predators but exposing them more to terrestrial predation Table 2. Coefficients of Generalized Linear Model analysis, which considers the percentage of tail injury as a predictor of (1) Scaled Mass Index (SMI) and (2) days until metamorphosis; tail injury per se as a predictor of (3) SMI and (4) days until metamorphosis; and (5) tail injury as a predictor of feeding frequency. All models pre- sent degrees of freedom = 35 and significant values are in bold. Estimate Std Error t value P (1) SMI ~ % tail injury Intercept 0.15 0.004 34.42 <0.001 30% -0.005 0.006 0.79 0.44 50% 0.005 0.006 -0.81 0.42 70% 0.001 0.006 0.25 0.81 (2) Days until metamorphosis ~ % tail injury Intercept 29.78 2.99 9.95 <0.001 30% 5.22 4.13 1.27 0.21 50% 3 4.23 0.71 0.48 70% 12.22 4.36 2.8 0.008 (3) SMI ~ tail injury Intercept 0.15 0.004 34.05 <0.001 tail loss 0.0003 0.005 0.05 0.96 (4) Days until metamorphosis ~ tail injury Intercept 29.79 3.15 9.56 <0.001 tail loss 6.56 3.6 1.82 0.08 (5) Feeding frequency ~ tail injury Intercept 4.01 0.18 22.56 <0.001 tail loss -0.41 0.28 -1.45 0.17 Table 3. Summary contrasts of Estimated Marginal Means, used as a pos hoc test to compare groups of different levels of tail injury with the control group. Significant value is in bold. Estimate Std. Error P SMI ~ % tail injury 30% - control -0.005 0.006 0.73 50% - control 0.005 0.006 0.75 70% - control 0.001 0.006 0.98 Days until metamorphosis ~ % tail injury 30% - control 5.2 4.13 0.44 50% - control 3 4.23 0.79 70% - control 12.22 4.36 0.01 SMI ~ tail injury injuried - control 0.0003 0.005 0.96 Days until metamorphosis ~ tail injury injuried - control 6.56 3.6 0.07 Feeding frequency ~ tail injury injuried - control -0.41 0.28 0.15 18 Ana Glaucia da Silva Martins et alii in the developmental stage that they are most suscep- tible to predation (Toledo et al. 2007). In D. elegans we observed that the strategy adopted is the first one. Tad- poles threatened by a predator spend more time under larval stage, i.e., reduce the growth rhythm, but reach the same size after metamorphosis, and consequently the same SMI, than unharmed individuals. We also did not observe change in feeding frequency as a result of tail injury. It probably implies that the tail regeneration was not provided by an extra acquisition of energy – expected by a more frequent feeding. Although these stages (stages 28 until 36) are those when generally tadpoles present the most significant growth and energy uptake (Pfab et al., 2020), we did not observe any differ- ence when the tail was lost. Considering that for some spe- cies locomotion is more important than feeding, such as in Pleurodema thaul (Pueta et al., 2016) and Pelophylax lesso- nae (Steiner, 2007), and that tail fins enable fast swimming (Smith and van Buskirk, 1995), perhaps for D. elegans the regeneration of tail is energetically more important than time until metamorphosis. Consequently, there is not an increase in feeding to regenerate the tail, but a realloca- tion of the energy that otherwise would be used to growth. Thus, such observation supports the hypothesis of a prob- able reallocation of the energy from the regular develop- ment/metamorphosis process directed to tail regeneration. However, different results were reported for other species. For example, in Ascaphus truei, a simple clue of preda- tors’ presence was enough to modify the foraging in tad- poles, which reduced up to six-folds its foraging activity (Feminella and Hawkins, 1994). Also, similar results were observed for Rana sylvatica (Fraker, 2010) and Rana clami- tans (Fraker, 2008, 2009). It efforts that more studies are necessary to elucidate this process of energy reallocation during larval stage until metamorphosis. Our experiments showed consequences of predatory events in D. elegans tadpoles. Tail injury caused by preda- tors can result in several consequences for the individu- als, decreasing their survivorship, affecting tadpole mor- phology (Nunes et al., 2010), and swimming speed (Figiel Jr and Semlitsch, 1991). 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XI International Symposium on the Mediterranean Lacertid Lizards Marco Mangiacotti1, Pietro Lo Cascio2, Claudia Corti2, Marta Biaggini2, Miguel Angel Carretero2, Petros Lymberakis2 The directional testes asymmetry increases with temperature in seven plateau brown frog (Rana kukunoris) populations Hai Ying Li1, Man Jun Shang2, Jie Guo2, Bo Jun Chen2, Peng Zhen Chen2, Tong Lei Yu1,* Influence of tail injury on the development of Neotropical elegant treefrog tadpoles Ana Glaucia da Silva Martins1,#, Raoni Rebouças2,3,*,#, Isaias Santos1, Adão Henrique Rosa Domingos1, Luís Felipe Toledo2 The effect of weight and prey species on gut passage time in an endemic gecko Quedenfeldtia moerens (Chabanaud, 1916) from Morocco Jalal Mouadi1,*, Panayiotis Pafilis2, Abderrafea Elbahi3, zahra Okba3, Hassan ElOuizgani3, El Hassan El Mouden4, Mohamed Aourir1 A contribution to the knowledge on the diet and food preferences of Darevskia praticola (Reptilia: Lacertidae)§ Emiliya Vacheva*, Borislav Naumov First report on two loggerhead turtle (Caretta caretta) nests in the Aeolian Archipelago (Southern Italy) Monica Francesca Blasi1,*, Sandra Hochscheid2, Roberta Bardelli3, Chiara Bruno1, Carolina Melodia1, Perla Salzeri1, Paolo De Rosa4 and Paolo Madonia5 Threatened and extinct amphibians and reptiles in Italian natural history collections are useful conservation tools Franco Andreone1,*, Ivano Ansaloni2, Enrico Bellia3, Andrea Benocci4, Carlotta Betto5, Gabriella Bianchi6, Giovanni Boano7, Antonio Borzatti de Loewenstern8, Rino Brancato9, Nicola Bressi10, Stefano Bulla11, Massimo Capula12, Vincenzo Caputo Barucchi13, P Re-description of external morphology and factors affecting body and tail shape of the stone frog tadpoles’ Brena da Silva Gonçalves1,*, Carla. D. Hendges2, Bruno Madalozzo2, Tiago G. Santos2,3 Preliminary data on the diet of Chalcides chalcides (Squamata: Scincidae) from Northern Italy Andrea Ciracì1, Edoardo Razzetti2, Maurizio Pavesi3, Daniele Pellitteri-Rosa4,* The high diversity and phylogenetic signal of antipredator mechanisms of the horned frog species of Proceratophrys Miranda-Ribeiro, 1920 (Amphibia: Anura: Odontophrynidae) Cássio Zocca1,2,*, Ricardo Lourenço-de-Moraes3, Felipe S. Campos4, Rodrigo B. Ferreira1,2,5