Acta Herpetologica 14(2): 129-133, 2019 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/a_h-7751 Visible Implant Alphanumeric (VIA) as a marking method in the lesser snouted treefrog Scinax nasicus Andrea Caballero-Gini1,2,3,*, Diego Bueno Villafañe2,3, Lía Romero2, Marcela Ferreira2,3, Lucas Cañete4, Rafaela Laino2, Karim Musalem2,5 1 Insituto de Biología Subtropical, Universidad Nacional de Misiones, Félix de Azara 1552, CP 3300, Posadas, Misiones, Argentina. *Corresponding author. Email: ancgini@gmail.com 2 Centro de Investigación del Chaco Americano, Fundación Manuel Gondra, San José 365, Asunción, Paraguay 3 Instituto de Investigación Biológica del Paraguay, Del Escudo, CP, Asunción, Paraguay 4 Universidad Nacional de Asunción, Facultad de Ciencias Exactas y Naturales (FACEN), Campus Universitario, San Lorenzo, Paraguay 5 World Wildlife Fund, Bernardino Caballero 191, Asunción, Paraguay Submitted on: 2019, February 22nd; Revised on: 2019, June 3th; Accepted on: 2019, June 3th Editor: Daniele Pellitteri-Rosa Abstract. In this study we assessed the efficacy of Visible Implant Alphanumeric (VIA) for marking adults and juve- niles of the Neotropical treefrog Scinax nasicus. We evaluated the success of this technique in the identification of individuals and the prevalence of tags in the field. As a control, we marked the same individuals through toe-clipping. Of 196 marked individuals, 57 were recaptured in a 7-month study period. Only one mark was unreadable because it was located too deep in the skin. We found one case of tag expulsion and two inverted tags. Almost complete regen- eration of the adhesive disk was observed by the fifth month of the study in all recaptured frogs. We suggest VIA tagging method as suitable for S. nasicus over long term studies. Even though, a hybrid method for marking (VIA + toe-clipping) is recommended for species with dark and/or loose skin, or large frogs. Keywords. Amphibian, mark-recapture, fluorescent elastomer, toe-clipping. The recognition of individuals in a population is a key aspect in many amphibian ecology and conserva- tion studies (Seber, 1982; Campbell et al., 2009; Clemas et al., 2009). Their identification over time and space helps researchers to estimate individual and demographic parameters such as growth and mortality rates, dispersal, population size and habitat use (Osbourne et al., 2011). In cases where individuals of a species do not possess characteristics that distinguish them from each other, like coloration and color patterns, and photo-identification is not suitable, it is necessary to use marking techniques (Donelly et al., 1994). For amphibians, the most com- monly used are toe-clipping, Passive Integrated Tran- sponder (PIT), Visible Implant Elastomer (VIE) tags, and more recently Visible Implant Alphanumeric (VIA) tags, the last two manufactured by North-west Marine Tech- nology, Shaw Island, USA (Heard et al., 2008; Branelly et al., 2014). Toe-clipping is the most widely used technique for marking anurans and salamanders (Ferner, 2010). How- ever, this method is highly criticized because a decrease in the probability of recapture has been detected in some amphibian species (Clarke, 1972; Mccarthy and Par- ris, 2004; Waddle et al., 2008). Another disadvantage of toe-clipping in long term studies is the possibility of tis- sue regeneration, although this has been observed only in a few species (Ferner, 2007; Ursprung et al., 2011). PIT tags consist of an electromagnetic capsule with an alphanumeric code, which is read by a scanner. Inser- tion is done subcutaneously or in a body cavity (Ferner, 130 Andrea Caballero-Gini et alii 2010). However, loss of tags and deleterious effects relat- ed to survival have been detected using this technique (Scherer et al., 2005; Guimaraes et al., 2014). VIE tagging is done through the subcutaneous injection of an elas- tomer mixed with a curing agent. This technique allows the individualization of many animals by creating differ- ent fluorescent color codes as well as placing the marks in various body locations (Moosman and Moosman, 2006). When comparing these three techniques, Branelly et al. (2014) mention that the least efficient is the VIE tagging, due to the migration, darkening and in some cases expul- sion of the tag. VIA tags are compacted elastomers in rectangular shape with an alphanumeric code in fluorescent colors visible under UV light (Heard et al., 2008). The insertion is done with an injector provided by the manufacturer, but in some cases the results are better making a previous incision (Buchan et al., 2005; Gower et al., 2006; Heard et al., 2008; Clemas et al., 2009; Kaiser et al., 2009). Despite being considered a reliable technique due to the capacity of individual identification, time of marking, handling of the individuals, durability of the marks and relatively low cost (Haw et al., 1990; Buchan et al., 2005; Gower et al., 2006), this technique also has disadvantages when used in amphibians: difficulty of tag insertion in species with loose skin, variation on tag retention among species and darkening of the tag when marking heavily pigmented species (Kaiser et al., 2009). Additionally, tag reten- tion was low in studies of mark-recapture of tadpoles (Courtois et al., 2013). We tested the VIA tags in the lesser snouted treefrog Scinax nasicus (Cope, 1862) in order to 1) evaluate the effectiveness of this technique in the identification of individuals, 2) determine the prevalence of tags in mark- recapture studies, and 3) provide some recommendations for further studies upon this species and others with sim- ilar morphology. S. nasicus is a small sized hylid frog distributed in northern and central Argentina, Paraguay, Uruguay, eastern Bolivia, and central and southern Brazil (Frost, 2019). The species is commonly found in open areas of the Atlantic Forest, Cerrado, Chaco, Pampa, and Pantanal domains (Dalmolin et al., 2017). We captured individuals of S. nasicus using 173 PVC pipes as a refuge in an area of approximately 3 km2 of wetland and associated ripar- ian forest in Benjamín Aceval, Presidente Hayes depart- ment in Paraguay (-24.960522S, -57.359425W). Field work was done during the months of November 2017, January, March and May 2018. The individuals were tagged both with VIA tags and toe-clipping. Additionally, measurements of the snout- vent length (SVL) were taken with a digital caliper Mitu- toyo Absolute AOS Digimatic. The VIA tags used were of standard size (1.2 × 2.7 mm) in fluorescent green with black letters. The insertion site was the ventral region of the right thigh following Buchan et al. (2005) (Fig. 1). The implantation site was sterilized with ethanol 90%, then the tag was inserted under the skin using the injec- tor provided by NMT following the manufacturer proto- col and no veterinary glue was used. All marking equip- ment was sterilized between frogs by immersion in 90% ethanol for several minutes. Toe-clipping was carried out as control following the method of Martof (1953). As suggested by Kinkead et al. (2006), no anesthesia was used in either both procedures. Frogs showed no signs of discomfort, e.g., emitting distress calls or abnormal movements of the affected limb and/or foot. Frogs were put under observation for 24 hours in plastic bags filled with air to observe presence of redness, edema or bleed- ing on the treated foot or at the injection site. Subse- quently they were released on the same PVC pipe where they were captured. We marked 196 individuals of S. nasicus (55 females, 46 males, and 95 juveniles). Frogs averaged 25.9 mm (SD = 5.34 mm) of SVL. We obtained a total of 57 (29%) recaptures, of which 2 individuals were recaptured three times, 14 individuals twice, and 41 individuals only once. The recapture rates in juve- niles was 47%, males 16% and females 37% (Fig. 2). We did not find significant differences in the recapture rates of juveniles and adults (t = 0.3487, d.f. = 4, P = 0.7449); therefore, it can be suggested that the VIA implants did not have negative effects on the survival of juveniles. Every time the label was observed, the code could be identified without ambiguity with the help of the UV light lantern provided by the kit, in 98% of the times. In one case, the label was located too deep under the skin and could not be read properly. At the same time, by Jan- uary 2018, we observed tissue regeneration in recaptured Fig. 1. Location of the VIA tag in the inner thigh of Scinax nasicus. VIA tag was injected just below the skin. 131Evaluating VIA tags in Scinax nasicus frogs where the beginning of the growth of the adhesive discs was noticeable, and by March 2018 the discs had similar diameters to those that were not clipped (Fig. 3). In general, discs presented similar shape and colora- tion to those not clipped and no cases of aberrant growth were observed, such as those described by Hoffman et al. (2008). Kaiser et al. (2009) studied the possibility of using VIA tags as a marking method that does not require the recapture of animals (by placing the tags on the back of a small frog species). They concluded that it is not pos- sible to read the codes on the labels without having the animal in hand. When comparing the handling times between VIA tags and toe-clipping, Clemas et al. (2009) found that toe-clipping took slightly less amount of initial time of handling and marking than time with the VIA tags, but the last one took less time during identification. Despite results showed by Clemas et al. (2009), by using VIA tags in adults and juveniles, we had success identify- ing individuals of S. nasicus through this methodology. Regarding the second objective of this work, we observed the retention of tags on 97% of the cases. Only on one occasion the label was expelled and, in another opportunity, as a result of an inflammation caused in a frog’s leg, the tag had to be removed when the animal was recaptured. Also, in two cases the tags were invert- ed but could be easily rotated by prodding with a finger, and on one occasion a small piece of the tag broke when removed from the tag block but was not discarded. When marking individuals at the caudal end of the dorsum, Kaiser et al. (2009) noted that very often the labels were turned over and migrated ventrally, but they do not mention if this affects frog’s survivor or movement capacity. They also mention that when tags were not eas- ily detected they remarked animals, and this constituted an extra effort and a waste of resources. Contrarily, Heart et al. (2008) compared tag migration placing them in the thigh and dorsolateral region of the thorax, finding that tag retention was higher in the latter site. Although, movement of tags in dorsal regions of the body is less common, this area tends to be more pigmented, making reading difficult (Moosman and Moosman, 2006). We opted for the ventral thigh location because this area is not heavily pigmented in S. nasicus, being almost trans- lucid, thus we did not observe any noticeable migra- tion, which may arise when the target species is large and allows movements of the tag in the interfemoral sac (Clemas et al., 2009). We also consider that the use of veterinary glue is not necessary since only in one case we observed the expulsion of the tag. When we purchased the product, manufacturers mentioned that the injection needle was re-designed and that it should not be neces- sary to make an initial incision. However, for marking large or thick-skinned species as well as when marking a large number of individuals this may be necessary, in order to maintain the instrument sharp and in shape to not cause discomfort to the animals. The estimation of population and demographic parameters in ecological studies are based on assump- tions that depend on the marking technique; these are: (1) no loss of marks; (2) no misidentification of marks; and (3) marking procedures do not alter survival or cap- ture probabilities (Seber, 1986; Pollock et al., 1990). This last assumption is the most controversial because in most techniques’ negative effects on both survival and recap- ture probability have been observed, being toe-clipping the most deleterious technique and VIE and VIA tagging the least, although this last one has been scarcely stud- ied (Heard et al., 2008; Schmidt and Schwarzkopf, 2010; Sapsford et al., 2014, 2015). VIA tags are an interesting method to test in amphibians due to its relatively low cost, lower invasive- ness when compared to other techniques such as toe- clipping, and straightforward code interpretation. In our study, the rate of success of the VIA tag method sug- gests that it is suitable for S. nasicus, as it was easy, safe, rapid and effective to carry out, as well as easy to detect. Moreover, the method is well advised for the species Fig. 3. Toe pad regeneration in Scinax nasicus. A: after a week of clipping, B: after two months of clipping. Fig. 2. Number of capture and recapture males, females and juve- niles of Scinax nasicus. 132 Andrea Caballero-Gini et alii if long term studies are made, due to the pad regenera- tion observed in clipped toes. Still, we suggest keeping a hybrid marking method (i.e., VIA tags + toe-clipping) when working with other species than S. nasicus, since VIA tags can have a distinct rate of success depending on several factors, such as the degree of stretch in the skin, size of the leg or other body part, transparency. We also advise to take time to inspect carefully the animals to detect VIA tag migration or loss, so there is no resource waste and unnecessary animal stress. ACKNOWLEDGEMENTS The permits to perform this study were obtained from the Ministerio del Ambiente y Desarrollo Sosteni- ble (Scientific collection permit N° 173-2017). This work is part of the project PINV15-143 financed by the Con- sejo Nacional de Ciencia y Tecnología (CONACYT). 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