Acta Herpetologica 15(2): 119-123, 2020 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/a_h-10016 Does chronic exposure to ammonium during the pre-metamorphic stages promote hindlimb abnormality in anuran metamorphs? A comparison between natural-habitat and agrosystem frogs Sonia Zambrano-Fernández1, Francisco Javier Zamora-Camacho2,3,*, Pedro Aragón2,4 1 Universidad de Málaga, Avda. de Cervantes 2, 29016, Málaga, Spain 2 Museo Nacional de Ciencias Naturales, (MNCN-CSIC), C/ José Gutiérrez Abascal 2, 28006, Madrid, Spain 3 Department of Biological Sciences, Dartmouth College, 78 College Street, 03755 Hanover, New Hampshire, USA 4Universidad Complutense de Madrid, C/José Antonio Novais 2, 2804, Madrid, Spain *Corresponding author. E-mail: zamcam@ugr.es Submitted on: 2019, 19th December; revised on: 2020, 6th March; accepted on: 2020, 26th May Editor: Raoul Manenti Abstract. Despite their detrimental effects on locomotion, prevalence of hindlimb abnormalities is increasing in anu- ran populations worldwide. Among others, agrochemical pollution during the larval stage is a potential cause. How- ever, populations exposed to such a strong selective pressure could evolve resistance. In this work, we examine the potential effects of chronic exposure to ammonium during the pre-metamorphic stages of Pelophylax perezi frogs on metamorph hindlimb abnormality prevalence, as compared with unpolluted-water-reared conspecifics. We conducted the experiment on tadpoles either from natural-habitat or from agrosystem parents. We detected no effect of chronic exposure to ammonium on hindlimb abnormality prevalence in frogs from either habitat, which suggests that the lack of effect detected is not related to resistance evolved in agrosystem frogs. Keywords. Amphibian, anuran, anomaly, malformation, Pelophylax perezi. Functioning appendages are fundamental for whole- organism performance of most anurans (Johansson et al., 2010; Zamora-Camacho et al., 2019). Hind limb morphology is directly responsible for locomotion of metamorph (Zamora-Camacho and Aragón, 2019a) and adult anurans (Zamora-Camacho, 2018), either from terrestrial (Gomes et al., 2009) or aquatic environ- ments (Herrel et al., 2012), regardless of their locomo- tion mode (Enriquez-Urzelai et al., 2015). Therefore, hindlimb abnormality in this group is likely eradicated by natural selection due to its severe negative effects on locomotion (Zamora-Camacho and Aragón, 2019b). Consistently, prevalence of anuran appendage abnor- mality appears generally below 5% (Ouellet, 2000; Mester et al., 2015). Nonetheless, limb abnormality rates are increasing in anurans worldwide (Johnson and Bowerman, 2010; Laurentino et al., 2016). These include diverse malforma- tions, such as lacking and extra limbs and digits, as well as fused or misshaped limbs (Johnson and Bowerman, 2010; Reeves et al., 2013). Limb abnormalities are par- ticularly common in metamorphs (Kiesecker, 2002; Piha et al., 2006), seemingly because reduced locomotor per- formance (Zamora-Camacho and Aragón, 2019b) might cause their death shortly after metamorphosis. Besides a genetic origin (Droin and Fischberg, 1980), hindlimb abnormalities in anurans have been related to biotic interactions such as predatory pressure (Johnson and Bowerman, 2010) or parasite infections (Roberts and Dickinson, 2012), as well as abiotic factors such as ultra- 120 Sonia Zambrano-Fernández, Francisco Javier Zamora-Camacho, Pedro Aragón violet-B radiation (Pahkala et al., 2001). However, human perturbance frequently provokes these malformations (Blaustein and Johnson, 2003), which are more com- mon next to roads (Reeves et al., 2008) or in agrosystems (Ouellet et al., 1997; Spolyarich et al., 2011). Agrochemi- cals such as fungicides (Bernabo et al., 2016), pesticides (Jayawardena et al., 2010), and fertilizers (Xu and Old- ham, 1997) increase limb abnormality prevalence in anu- rans. The aetiology of these malformations is often mul- tiple (Meteyer et al., 2000): trematode infections (Haas et al., 2018) and predator attacks (Reeves et al., 2010) boost the effects of agrochemicals. Albeit, greater selective pres- sures could also drive the appearance of resistance to the environmental stressors (Miaud and Merilä, 2001), which could eventually reduce the prevalence of abnormalities in agrosystem populations. Metamorph morphology is often related to tadpole growth history (Tejedo et al., 2000). In this work, we compare the prevalence of hindlimb abnormality in met- amorphs of Pelophylax perezi (López Seoane, 1885) frogs resulting from tadpoles chronically exposed to ammo- nium contamination with unpolluted-water-reared con- specifics. Ammonium is among the most common com- pounds derived from agricultural fertilizers, with several negative effects on amphibian populations (Ortiz et al., 2004). We checked any possible resistance to contamina- tion evolved in agrosystems by applying this treatment to tadpoles from natural habitats and from agrosystems. We expected higher prevalence of hindlimb abnormalities in metamorphs from the ammonium treatment. However, if agrosystem populations have evolved resistance, this effect would be greater in natural-habitat tadpoles. Pelophylax perezi is a Ranid that occurs naturally throughout the Iberian Peninsula and southern France (Egea-Serrano, 2014), in a wide variety of habitats, but always in or not far from waterbodies, either pristine or polluted (Egea-Serrano, 2014). Indeed, it often inhab- its human-altered habitats, such as urban or agricultural environments (Egea-Serrano, 2014). Fieldwork was conducted in pristine Pinares de Car- taya Pinus pinea grove and surrounding agrosystems (SW Spain, 37º20’ N, 7º09’ W). Agrosystems are about 6 km away from pine grove, and consist of a traditional exten- sive vegetable crop area that has lately transitioned into intensive plantations regularly added fertilizers and at owners’ discretion. In April 2018, 10 adult males and 10 adult females were randomly caught from each habitat. Capture was manual, and males were recognized for their greyish forelimb nuptial pads and their vocal sacs in the mouth commissures (Egea-Serrano, 2014). Frogs were pooled separately according to their provenance in two adjacent outdoor semi-natural enclosures with ponds (Fig. S1 in Supplementary Material). Ponds were daily checked for the presence of egg masses, which we transferred to the laboratory within 12 hours after they had been laid. In the laboratory, we immediately separated eggs randomly in groups of 15. Each group was placed in an aquarium (Length×Width×Height: 38×27×19 cm) with 6 L of untreated spring water. In half of the aquariums, randomly chosen for each egg mass, we added 178.87 mg of 99.7% pure NH4Cl, so we obtained a concentra- tion of 10 mg NH4+/L. In a previous study on this spe- cies, a concentration of 13.5 mg NH4+/L caused circa 70% mortality rate in a mid-term experiment on larvae of this species from natural habitats (Egea-Serrano et al., 2009). We chose a concentration slightly lower in order to avoid such mortality rates, while triggering sublethal effects. The other aquaria contained untreated spring water, as a control. Thus, we had 15 aquaria with eggs from frogs from each habitat and treatment, totalling 60 aquaria, in a 2 × 2 factorial experimental design. Aquaria were kept in shelves in the laboratory until larvae finished metamorphosis. Water was completely replaced twice a week, and each time we maintained the treatment and randomly changed the position of each aquaria within the shelves. A window let natural daylight in, permitting adjustment of circadian rhythms. Because tadpole diet can affect limb abnormality rates in this spe- cies (Martínez et al., 1992), all specimens were standardly fed boiled spinach ad libitum. In Gosner stage 42, preced- ing tail resorption (Gosner, 1960), tadpoles were trans- ferred to tilted aquaria to allow them to exit the water as metamorphosis ended. Some metamorphs presented an abnormality in one of their hindlimbs (Fig. 1). Abnormal limbs were aber- rantly inserted in the pelvis with an approximate angle of 270º with respect to the body axis (Fig. 1). Moreover, the knee-joints were unable to fold normally in rest- ing position (Fig. 1). In all cases, only one hindlimb was abnormal in each individual affected, either the right or the left appendage. We calculated the proportion (num- ber of abnormal metamorphs divided by number of total surviving metamorphs) of abnormal-limbed metamorphs from each aquarium. Data met the criteria of homoscedasticity and residu- al normality (Quinn and Keough, 2002), so we conducted a two-way ANOVA to test the effects of habitat, treat- ment, and their interaction on the proportion of abnor- mal-limbed metamorphs, using the software Statistica 8.0. The total numbers and proportions of abnormal- limbed metamorphs from each habitat and treatment are in Table S1 in Supplementary Material. The effects of habitat (F1, 56 = 0.026; P = 0.874; Fig. 2), treatment (F1, 56 121Tadpole exposure to ammonium and froglet hindlimb abnormality = 0.007; P = 0.932: Fig. 2), and their interaction (F1, 56 = 0.914; P = 0.343; Fig. 2) on the proportion of abnormal- limbed metamorphs obtained in each aquarium were non-significant. At the concentration used, chronic exposure to ammonium during the larval stage does not increase the prevalence of hindlimb abnormality in these frogs. How- ever, a subchronic exposure to even lower concentrations of this compound reduces survivorship (Egea-Serrano et al., 2009) and affects behaviour of P. perezi larvae (Egea- Serrano et al., 2011). Prevalence of limb abnormality in Bufo bufo toad metamorphs were higher following an acute exposure to 100 mg/L ammonium nitrate during the larval stage than following a subchronic exposure to 50 and 100 mg/L ammonium nitrate (Xu and Oldham, 1997). Those results could be a consequence of greater mortality of larvae in the subchronic exposure treatment (Xu and Oldham, 1997), which could mask potential limb abnormalities if future bearers die. Chronic expo- sure to other pollutants, such as mercury in Rana sphe- nocephala frogs (Unrine et al., 2004), and nickel, cobalt, or cadmium chlorides in Xenopus laevis frogs (Plowman et al., 1994), causes malformations in metamorphs. Also, subchronic exposure to carbamate and organophosphate pesticides causes malformations in P. perezi (Alvarez et al., 1995). Juvenile P. perezi from agrosystems are smaller, and show increased limb fluctuant asymmetry, than conspe- cifics from natural habitats (Burghelea et al., 2013). How- ever, we detected no effect of habitat on hindlimb abnor- mality prevalence on either treatment. Aligned with our results, prevalence of limb abnormality was not greater in Rana temporaria frogs from agrosystems than from natu- ral habitats (Piha et al., 2006). Nevertheless, these find- ings contrast with others that detected increased preva- lence of limb abnormality close to agrosystems in several anurans (Kiesecker, 2002; Guerra and Aráoz, 2016). Our results do not support the hypothesis of resistance in agrosystem frogs. We obtained an overall prevalence of hindlimb abnormality notably below the 5% detected in other wild amphibian populations (Ouellet, 2000; Mester et al., 2015). Low prevalence in both habitats could be a consequence of the capability of this species to thrive in polluted waters (Egea-Serrano et al., 2008). ACKNOWLEDGEMENTS FJZ-C was partly supported by a Juan de la Cier- va-Formación contract by the Spanish Ministerio de Economía, Industria y Competitividad (MINECO). PA was supported by a “Ramón y Cajal” contract (RYC- 2011-07670, MINECO). This study was in part funded by MINECO (project number CGL2014-56416-P). Frog capture and management was conducted according to a research permit issued to the authors by the Consejería de Medio Ambiente of the Junta de Andalucía (reference Fig. 2. Effects (mean ± 1SE) of treatment and habitat on the pro- portion of abnormal-limbed metamorphs obtained from each aquarium, calculated as the number of abnormal metamorphs divided by the total number of surviving metamorphs. Sample sizes indicated represent the number of aquaria in each treatment. Fig. 1. Pelophylax perezi metamorph affected by the hindlimb abnormality described, with a measuring tape in cm. 122 Sonia Zambrano-Fernández, Francisco Javier Zamora-Camacho, Pedro Aragón SGMN/AWG/MGD/MGM GB-270-18) and the Bioeth- ics Committee of the Spanish National Research Council (reference 720/2018). SUPPLEMENTARY MATERIAL Supplementary material associated with this article can be found at < http://www.unipv.it/webshi/appendix > Manuscript number 10016. REFERENCES Alvarez, R., Honrubia, M.P., Herráez, M.P. (1995): Skel- etal malformations induced by the insecticides ZZ- Aphox® and Folidol® during larval development of Rana perezi. Arch. Environ. Contam. Toxicol. 28: 349- 356. Bernabo, I., Guardia, A., Macirella, R., Sesti, S., Crescente, A., Brunelli, E. (2016): Effects of long-term exposure to two fungicides, pyrimethanil and tebuconazole, on survival and life-history traits of Italian tree frog (Hyla intermedia). Aquat. Toxicol. 172: 56-66. Blaustein, A.R., Johnson, P.T.J. (2003): The complexity of deformed amphibians. Front. Ecol. Environ. 1: 87-94. Burghelea, C., Zaharescum D., Palanca, A. (2013): Phe- notypic indicators of developmental instability in an endemic amphibian from an altered landscape (Mon- egros, NE Spain). Amphib-Reptil. 34: 505-516. Droin, A., Fischberg, M. (1980): Abnormal limbs (abl), a recessive mutation affecting the tadpoles of Xenopus l. laevis. Experientia 36: 1286-1288. Egea-Serrano, A. (2014): Rana común – Pelophylax pere- zi. In: Enciclopedia Virtual de los Vertebrados Espa- ñoles. Salvador, A., Martínez-Solano, I. Eds. Museo Nacional de Ciencias Naturales, Madrid. http://verte- bradosibericos.org Egea-Serrano, A., Tejedo, M., Torralva, M. (2008): Analy- sis of the avoidance of nitrogen fertilizers in the water column by juvenile Iberian water frog, Pelophylax perezi (Seoane, 1885), in laboratory conditions. Bull. Environ. Contam. Toxicol. 80: 178-183. Egea-Serrano, A., Tejedo, M., Torralva, M. (2009): Popu- lational divergence in the impact of three nitrogenous compounds and their combination on larvae of the frog Pelophylax perezi. Chemosphere 76: 869-877. Egea-Serrano, A., Tejedo, M., Torralva, M. (2011): Behav- ioral responses of the Iberian waterfrog, Pelophylax perezi (Seoane, 1885), to three nitrogenous com- pounds in laboratory conditions. Ecotoxicology 20: 1246-1257. Enriquez-Urzelai, U., Montori, A., Llorente, G.A., Kaliontzopoulou, A. (2015): Locomotor mode and the evolution of the hindlimb in Western Mediterranean anurans. Evol. Biol. 42: 199-209. Gomes, F.R., Rezende, E.L., Grizante, M.B., Navas, C.A. (2009): The evolution of jumping performance in anu- rans: morphological correlates and ecological implica- tions. J. Evol. Biol. 22: 1088-1097. Gosner, K.L. (1960): A simplified table for staging anuran embryos and larvae with notes on identification. Her- petologica 16: 183-190. Guerra, C., Aráoz, E. (2016): Amphibian malformations and body condition across an agricultural landscape of northwest Argentina. Dis. Aquat. Organ. 121: 105-116. Haas, S.E., Reeves, M.K., Pinkney, A.E., Johnson, P.T.J. (2018): Continental-extent patterns in amphibian malformations linked to parasites, chemical contami- nants, and their interactions. Glob. Chang. Biol. 24: E275-E288. Herrel, A., Gonwouo, L.N., Fokam, E.B., Ngundu, W.I., Bonneaud, C. (2012): Intersexual differences in body shape and locomotor performance in the aquatic frog, Xenopus tropicalis. J. Zool. 287: 311-316. Jayawardena, U.A., Rajakaruna, R.S., Navaratne, A.N., Amerasinghe, P.H. (2010): Toxicity of agrochemicals to common hourglass tree frog (Polypedates cruciger) in acute and chronic exposure. Int. J. Agric. Biol. 12: 641-648. Johansson, F., Lederer, B., Lind, M.I. (2010): Trait perfor- mance correlations across life stages under environ- mental stress conditions in the common frog, Rana temporaria. PLOS One 5: e11680. Johnson, P.T.J., Bowerman, J. (2010): Do predators cause frog deformities? The need for an eco-epidemiological approach. J. Exp. Zool. B: Mol. Dev. Evol. 314: 515- 518. Kiesecker, J.M. (2002): Synergism between trematode infection and pesticide exposure: a link to amphibian limb deformities in nature? PNAS 99: 9900-9904. Laurentino, T.G., Pais, M.P., Rosa, G.M. (2016): From a local observation to an European-wide phenomenon: amphibian deformities at Serra da Estrela Natural Park, Portugal. Basic Appl. Herpetol. 30: 7-23. Martínez, I., Álvarez, R., Herráez, I., Herráez, P. (1992): Skeletal malformations in hatchery reared Rana perezi tadpoles. Anat. Rec. 233: 314-320. Mester, B., Lengyel, S., Puky, M. (2015): Low frequency of amphibian morphological anomalies in a large pro- tected wetland and grassland complex in Hungary. Herpetol. Conserv. Biol. 10: 679-687. Meteyer, C.U., Loeffler, I.K., Fallon, J.F., Converse, K.A., Green, E., Helgen, J.C., Kersten, S., Levey, R., Eaton- 123Tadpole exposure to ammonium and froglet hindlimb abnormality Poole, L., Burkhart, J.G. (2010): Hind limb malforma- tions in free-living northern leopard frogs (Rana pipi- ens) from Maine, Minnesota, and Vermont suggest multiple etiologies. Teratology 62: 151-171. Miaud, C., Merilä, J. (2001): Local adaptation or environ- mental induction? Causes of population differentia- tion in alpine amphibians. Biota 2: 31-50. Ortiz, M.E., Marco, A., Saiz, N., Lizana, M. (2004): Impact of ammonium nitrate on growth and survival of six European amphibians. Arch. Environ. Contam. Toxicol. 47: 234-239. Ouellet, M. (2000): Amphibian deformities: current state of knowledge. In: Ecotoxicology of amphibians and reptiles. Sparling, D., Linder, G., Bishop, C. Eds. SETAC Press, Pensacola. Ouellet, M., Bonin, J., Rodrigue, J., DesGranges, J.L., Lair, S. (1997): Hindlimb deformities (ectromelia, ectro- dactyly) in free-living anurans from agricultural habi- tats. J. Wildl. Dis. 33: 95-104. Pahkala, M., Laurila, A., Merilä, J. (2001): Carry-over effects of ultraviolet-B radiation of larval fitness in Rana temporaria. Proc. R. Soc. Lond. B 268: 1699- 1706. Piha, H., Pekkonen, M., Merilä, J. (2006): Morphological abnormalities in amphibians in agricultural habitats: a case study of the common frog Rana temporaria. Copeia 2006: 810-817. Plowman, M.C., Grbac-Ivankovic, S., Martin, J., Hopfer, S.M., Sunderman, F.W. (1994): Malformations per- sist after metamorphosis of Xenopus laevis tadpoles exposed to Ni2+, Co2+, or Cd2+ in FETAX assays. Tera- tog. Carcinog. Mutag. 14: 135-144. Quinn, G.P., Keough, M.J. (2002): Experimental design and data analysis for biologists. Cambridge: Cam- bridge University Press. Reeves, M.K., Dolph, C.L., Zimmer, H., Tjeerdema, R.S., Trust, K.A. (2008): Road proximity increases risk of skeletal abnormalities in wood frogs from national wildlife refuges in Alaska. Environ. Health Perspect. 116: 1009-1014. Reeves, M.K., Jensen, P., Dolph, C.L., Holyoak, M., Trust, K.A. (2010): Multiple stressors and the cause of amphibian abnormalities. Ecol. Monogr. 80: 423-440. Reeves, M.K., Medley, K.A., Pinkney, A.E., Holyoak, M., Johnson, P.T.J., Lannoo, M.J. (2013): Localized hotspots drive continental geography of abnormal amphibians on U.S. wildlife refuges. PLoS ONE 8: e77467. Roberts, C., Dickinson, T. (2012): Ribeiroia ondatrae causes limb abnormalities in a Canadian amphibian community. Can. J. Zool. 90: 808-814. Spolyarich, N., Hyne, R.V., Wilson, S.P., Palmer, C.G., Byrne, M. (2011): Morphological abnormalities in frogs from a rice-growing region in NSW, Australia, with investigations into pesticide exposure. Environ. Monit. Assess. 173: 397-407. Tejedo, M., Semlitsch, R.D., Hotz, A. (2000): Covariation of morphology and jumping performance in newly metamorphosed water frogs: effects of larval growth history. Copeia 2000: 448-458. Unrine, J.M., Jagoe, C.H., Hopkins, W.A., Brant, H.A. (2004): Adverse effects of ecologically relevant dietary mercury exposure in southern leopard frog (Rana sphenocephala) larvae. Environ. Toxicol. Chem. 23: 2964-2970. Xu, Q., Oldham, R.S. (1997): Lethal and sublethal effects of nitrogen fertilizer ammonium nitrate on common toad (Bufo bufo) tadpoles. Arch. Environ. Contam. Toxicol. 32: 298-303. Zamora-Camacho, F.J. (2018): Locomotor performance in a running toad: roles of morphology, sex, and agrosystem versus natural habitat. Biol. J. Linn. Soc. 123: 411-421. Zamora-Camacho, F.J., Aragón, P. (2019a): Failed preda- tor attacks have detrimental effects on antipredatory capabilities through developmental plasticity in Pelo- bates cultripes toads. Funct. Ecol. 33: 846-854. Zamora-Camacho, F.J., Aragón, P. (2019b): Hindlimb abnormality reduces locomotor performance in Pelo- bates cultripes metamorphs but is not predicted by larva morphometrics. Herpetozoa 32: 125-131. Zamora-Camacho, F.J., Zambrano-Fernández, S., Medi- na-Gálvez, L. (2019): The roles of sex and morphol- ogy on burrowing depth of Iberian spadefoot toads in different biotic and abiotic environments. J. Zool. 309: 224-230. Acta Herpetologica Vol. 15, n. 2 - December 2020 Firenze University Press Estimating abundance and habitat suitability in a micro-endemic snake: the Walser viper Gentile Francesco Ficetola1,2,*, Mauro Fanelli3, Lorenzo Garizio3, Mattia Falaschi1, Simone Tenan4, Samuele Ghielmi5, Lorenzo Laddaga6, Michele Menegon7,8, Massimo Delfino3,9. Potential effects of climate change on the distribution of invasive bullfrogs Lithobates catesbeianus in China Li Qing Peng1, Min Tang1, Jia Hong Liao1, Hai Fen Qing1, Zhen Kun Zhao1, David A. Pike2, Wei Chen1,* A bibliometric-mapping approach to identifying patterns and trends in amphibian decline research Claudio Angelini1,*, Jon Bielby2, Corrado Costa3 Food composition of a breeding population the endemic Anatolia newt, Neurergus strauchii (Steindachner, 1887) (Caudata: Salamandridae), from Bingöl, Eastern Turkey Kerim Çiçek1,*, Mustafa Koyun2, Ahmet Mermer1, Cemal Varol Tok3 Stomach histology of Crocodylus siamensis and Gavialis gangeticus reveals analogy of archosaur “gizzards”, with implication on crocodylian gastroliths function Ryuji Takasaki1,2,*, Yoshitsugu Kobayashi3 Does chronic exposure to ammonium during the pre-metamorphic stages promote hindlimb abnormality in anuran metamorphs? A comparison between natural-habitat and agrosystem frogs Sonia Zambrano-Fernández1, Francisco Javier Zamora-Camacho2,3,*, Pedro Aragón2,4 Confirming Lessona’s brown frogs distribution sketch: Rana temporaria is present on Turin Hills (Piedmont, NW Italy) Davide Marino1, Angelica Crottini2, Franco Andreone3,* Phylogenetic relationships of the Italian populations of Horseshoe Whip Snake Hemorrhois hippocrepis (Serpentes, Colubridae) Francesco Paolo Faraone1, Raffaella Melfi2, Matteo Riccardo Di Nicola3, Gabriele Giacalone4, Mario Lo Valvo5* First karyological analysis of the endemic Malagasy phantom gecko Matoatoa brevipes (Squamata: Gekkonidae) Marcello Mezzasalma1,2,*, Fabio M. Guarino3, Simon P. Loader1, Gaetano Odierna3, Jeffrey W. Streicher1, Natalie Cooper1 Notes on sexual dimorphism, diet and reproduction of the false coral snake Oxyrhopus rhombifer Duméril, Bibron & Duméril, 1854 (Dipsadidae: Pseudoboini) from coastal plains of Subtropical Brazil Fernando M. Quintela1,*, Felipe Caseiro¹, Daniel Loebmann¹